2′-O-substituted nucleosides with high stability and affinity and their applications

Abstract

The present invention relates to a compound of formula I, #imgabs0# wherein R 1 R is selected from hydrogen, C1-C8 alkyl or halogen; 2 The compound of formula I is selected from a substituted or unsubstituted C1-C8 alkyl group, a C1-C8 alkoxy group, a C3-C6 cycloalkyl group, an aromatic ring or a heterocyclic group; the preparation method of the compound of formula I comprises the following steps: placing the compound of formula II and the compound of formula III in a solvent, reacting in the presence of a boron catalyst and a base to obtain the compound of formula I. The method of the present invention is simple to operate, low in cost, high in yield, high in product purity, environmentally friendly and safe, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to 2'-O-substituted nucleosides with high stability and affinity, a preparation method thereof, and applications thereof. Background Art

[0002] Antisense oligonucleotides are oligonucleotide molecules that are complementary to specific sequences in the target gene's pre-mRNA or mRNA. They primarily regulate target gene expression at the levels of gene replication, transcription, splicing, and translation, primarily through the principles of complementary base pairing and nucleic acid hybridization. This allows them to modulate the transmission of genetic information from nucleic acid to protein, thereby achieving their intended target gene regulation. Antisense oligonucleotides are typically composed of dozens of nucleosides linked by 3',5'-phosphodiester bonds. Unmodified antisense oligonucleotides are generally poorly absorbed by cells and are easily degraded by ubiquitous nucleases. To overcome these shortcomings and improve drugability, scientists have applied various chemical modifications to their backbones: phosphate groups, sugar groups, and bases. Chemical modifications of sugar groups are primarily intended to increase stability and affinity. Modification of the 2' position of the sugar group has been a hot topic in nucleic acid chemical modification. Numerous studies have shown that 2'-O-alkoxy-modified nucleosides can enhance RNA binding and enhance the oligonucleotide's resistance to nucleases. For example, antisense oligonucleotides synthesized from nucleosides modified with ethylene glycol monomethyl ether (2'-O-MOE) at the 2' position have higher affinity for RNA, better nuclease resistance, less toxicity and immunostimulation, and are widely used in clinical antisense oligonucleotide drugs. Summary of the Invention

[0003] The object of the present invention is to provide a compound of formula I having the following structure:

[0004]

[0005] Among them, R 1 Selected from hydrogen, C1-C8 alkyl or halogen;

[0006] R 2 It is selected from substituted or unsubstituted C1-C8 alkyl, C1-C8 alkoxy, C3-C6 cycloalkyl, aromatic ring or heterocyclic group.

[0007] In the preferred technical solution of the present invention, R 1 is selected from hydrogen, methyl, bromine or chlorine.

[0008] In the preferred technical solution of the present invention, the R 2 Selected from

[0009]

[0010] In the preferred technical solution of the present invention, the compound of formula I is selected from

[0011]

[0012] The object of the present invention is to provide a method for preparing a compound of formula I, comprising the following steps:

[0013] The compound of formula II and the compound of formula III are placed in a solvent and reacted in the presence of a boron catalyst and a base to obtain the compound of formula I. The synthesis route is as follows:

[0014]

[0015] Among them, R 1 Selected from hydrogen, C1-C8 alkyl or halogen;

[0016] R 2 It is selected from substituted or unsubstituted C1-C8 alkyl, C1-C8 alkoxy, C3-C6 cycloalkyl, aromatic ring or heterocyclic group.

[0017] In the preferred technical solution of the present invention, R 1 is selected from hydrogen, methyl, bromine or chlorine.

[0018] In the preferred technical solution of the present invention, the R 2 Selected from

[0019]

[0020] In a preferred technical solution of the present invention, the boron catalyst is selected from any one of boric acid, boron trioxide, trimethyl borate, triethyl borate, or a combination thereof.

[0021] In a preferred technical solution of the present invention, the base is selected from any one of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, or a combination thereof.

[0022] In the preferred technical solution of the present invention, the compound of formula III is selected from

[0023]

[0024] In a preferred technical solution of the present invention, the solvent is selected from an organic solvent that can form an azeotrope with water.

[0025] In a preferred technical solution of the present invention, the organic solvent capable of forming an azeotrope with water is selected from any one of a benzene solvent and an ester solvent, or a combination thereof.

[0026] In a preferred technical solution of the present invention, the benzene solvent is selected from toluene and / or xylene.

[0027] In a preferred technical solution of the present invention, the ester solvent is selected from any one of ethyl acetate, methyl acetate, ethyl formate, n-butyl acetate, and isobutyl acetate, or a combination thereof.

[0028] In a preferred technical solution of the present invention, the molar ratio of the compound of formula II to the compound of formula III is 1:3-20, preferably 1:5-15, and more preferably 1:6-13.

[0029] In a preferred technical solution of the present invention, the mass volume ratio of the compound of formula II to the solvent is 1:1-5 g / ml, preferably 1:1.5-3 g / ml.

[0030] In a preferred technical solution of the present invention, the molar ratio of the compound of formula II to the boron catalyst is 1:0.1-0.5, preferably 1:0.1-0.2.

[0031] In a preferred technical solution of the present invention, the molar ratio of the compound of formula II to the base is 1:0.1-0.5, preferably 1:0.1-0.2.

[0032] In the preferred technical solution of the present invention, the reaction temperature is 120-200°C, preferably 150-160°C.

[0033] In the preferred technical solution of the present invention, the reaction time is 8-26 hours, preferably 10-18 hours.

[0034] In a preferred technical solution of the present invention, the reaction is carried out in a water separator or a thorn-shaped distillation column.

[0035] In a preferred technical solution of the present invention, the compound of formula I can also be purified by column chromatography or recrystallization.

[0036] In a preferred technical solution of the present invention, the eluent for column chromatography is dichloromethane:methanol in a volume ratio of 100:1 to 20:1.

[0037] In a preferred technical solution of the present invention, the recrystallization solvent is any one of ethyl acetate and methanol or a combination thereof.

[0038] Another object of the present invention is to provide a method for preparing 5'-DMT-2'-O-substituted nucleosides, comprising the following steps: placing the compound of formula I in pyridine, and reacting with DMTrCl at a temperature of 0-5°C to obtain 5'-DMT-2'-O-substituted nucleosides.

[0039] In the preferred technical solution of the present invention, the reaction time is 1-10 hours, preferably 3-5 hours.

[0040] In a preferred technical solution of the present invention, the molar ratio of the compound of formula I to DMTrCl is 1:1.05-1.2, preferably 1:1.1.

[0041] In a preferred technical solution of the present invention, the reaction is followed by separation and purification by column chromatography.

[0042] In a preferred technical solution of the present invention, the eluent for column chromatography is dichloromethane:methanol in a volume ratio of 100:1 to 20:1.

[0043] Another object of the present invention is to provide the use of the compound of formula I in preparing clinical antisense oligonucleotide drugs.

[0044] In this article,

[0045] MOE is methoxyethyl;

[0046] DMTrCl refers to 4,4'-dimethoxytriphenylmethane;

[0047] DMT refers to 4,4'-dimethoxytrityl.

[0048] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentages are volume / volume percentages; when the present invention relates to the percentage between liquids and solids, the percentages are volume / weight percentages; when the present invention relates to the percentage between solids and liquids, the percentages are weight / volume percentages; and the rest are weight / weight percentages.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1. The compound of formula I of the present invention has good stability, high affinity, better nuclease resistance, less toxicity and immunostimulation, and can be widely used in clinical antisense oligonucleotide drugs.

[0051] 2. The preparation method of the present invention can achieve the preparation of 2'-O-substituted nucleosides in one step through the combination of boron catalyst and base, without the need to prepare borate ester first, avoiding the use of flammable and explosive borane or corrosive boron trichloride, making the method of the present invention simple to operate, low-cost, and applicable to large-scale industrial production.

[0052] 3. The method of the present invention has high yield and high product purity. Furthermore, the method of the present invention is rapid and complete, saving at least 50% of the reaction time compared to the prior art. It has significant economic benefits and is suitable for industrial application. It is also inexpensive, and the solvent used in the reaction can be recycled, which is in line with the concept of green chemistry.

[0053] 4. The 2'-O-substituted nucleoside prepared by the method of the present invention can be used in the preparation of the antisense oligonucleotide drug intermediate 5'-DMT-2'-O-substituted nucleoside without separation and purification, and the two-step yield is high. DETAILED DESCRIPTION

[0054] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0055] Example 1 Preparation of 2'-O-substituted-5-methyluridine (Compound C)

[0056] To a 50 mL round-bottom flask, add 2,2'-anhydro-5-methyluridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.168 g, 0.002 mol, 0.20 equiv.), 10 mL of compound 7 (Formula III), and 4-5 mL of toluene. The mixture was then loaded onto a spiked column and heated at 150°C for 21 h. The reaction was monitored by TLC until complete. The mixture was then concentrated to dryness on a rotary evaporator under reduced pressure to yield compound c.

[0057] The obtained compound c was purified by column chromatography using dichloromethane:methanol=20:1 as the eluent.

[0058] Compound c (2.89 g, yield 84%, purity >95%) was obtained by column chromatography separation and purification.

[0059] The structural characterization data of compound c are as follows:

[0060] 1 H NMR (400MHz, CDCl3) δ8.98(1H,s),7.31(1H,d,J=1.3Hz),5.54(1H,d,J=5.8Hz),5.30(1H,s),4.45(1H,dd,J=5.8,4.9Hz),4.37(1H,dd,J=5.0,3.0Hz),4 .24(1H,s),4.15(1H,q,J=2.4Hz),4.03–3.89(2H,m),3.82–3.73(1H,m),3.7 7–3.47(3H,m),3.45–3.38(1H,m),1.92(3H,d,J=1.3Hz),1.30–1.14(6H,m). 13CNMR (101MHz, CDCl3) δ163.7,150.5,139.1,111.0,92.5,85.9,80.5,72.5,70.2,69.6,67.0,62.5,21.9,21.8,12.4.

[0061] MS (ESI) 345.1 [M+H] + .

[0062] Example 2 Preparation of 2'-O-substituted-5-methyluridine (Compound d)

[0063] To a 50 mL round-bottom flask, add 2,2'-anhydro-5-methyluridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 6 (Formula III), and 4-5 mL of toluene. The mixture was loaded onto a spiked column and heated at 150°C for 13 h. Monitor by TLC until the reaction is complete. The mixture was concentrated to dryness on a rotary evaporator under reduced pressure to yield compound d.

[0064] The obtained compound d was purified by column chromatography. The column chromatography eluent was dichloromethane:methanol = 20:1.

[0065] Compound d (2.75 g, yield 80%, purity >95%) was obtained by column chromatography separation and purification.

[0066] The structural characterization data of compound d are as follows:

[0067] 1 H NMR (400MHz, CDCl3) δ9.62 (1H, s), 7.44–7.39 (1H, m), 5.61 (1H, d, J = 4.5Hz), 5.31 (1H,s),4.37(2H,q,J=3.9,2.9Hz),4.12(2H,t,J=6.1Hz),4.03–3.85(2H,m),3.7 9(1H,d,J=12.5Hz),3.76–3.48(2H,m),3.46(2H,td,J=6.8,1.9Hz),1.91(3H,s), 1.63(2H,dt,J=14.3,7.1Hz), 1.22(1H,dt,J=11.0,6.9Hz), 0.92(3H,t,J=7.4Hz).

[0068] 13C NMR (101MHz, CDCl3) δ164.2,150.7,138.6,110.9,91.6,85.6,80.9,73.2,70.0,69.6,69.4,62.1,22.7,12.4,10.4.

[0069] MS (ESI) 345.2 [M+H] + . .

[0070] Example 3 Preparation of 2'-O-substituted-5-methyluridine (Compound g)

[0071] To a 50 mL round-bottom flask, add 2,2'-anhydro-5-methyluridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 3 (Formula III), and 4-5 mL of toluene. The mixture was then loaded onto a spiked column and heated at 150°C for 13 h. The reaction was monitored by TLC until complete. The mixture was concentrated to dryness under reduced pressure on a rotary evaporator to yield compound g.

[0072] The obtained compound g was purified by column chromatography, and the column chromatography eluent was dichloromethane:methanol=30:1.

[0073] Compound g (2.94 g, yield 79%, purity >95%) was obtained by column chromatography separation and purification.

[0074] The structural characterization data of compound g are as follows:

[0075] 1 H NMR(400MHz, CDCl3) δ9.85(s,1H),7.50(d,J=1.3Hz,1H),5.67(d,J=4.7Hz,1H),4.31(dt,J=19.9,5.3Hz,2H),4 .10(dt,J=4.4,2.2Hz,1H),4.00–3.89(m,3H),3.83–3.47(m,10H),1.89(d,J=1.3Hz,3H),1.20(t,J=7.1Hz,3H).

[0076] 13 C NMR (101MHz, CDCl3) δ164.3,150.7,138.3,110.8,90.9,85.4,81.3,70.5,70.2,70.1,69.5,69.2,66.7,61.8,15.1,12.4.

[0077] MS (ESI) 374.3 [M+H] + .

[0078] Example 4 Preparation of 2'-O-substituted-5-methyluridine (Compound i)

[0079] To a 50 mL round-bottom flask was added 2,2'-anhydro-5-methyluridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 10 of formula III, and 4-5 mL of toluene. The mixture was then loaded onto a spiked column and heated at 150°C for 14 h. The reaction was monitored by TLC until complete. The mixture was then concentrated to dryness on a rotary evaporator under reduced pressure to afford compound I.

[0080] The obtained compound i was purified by column chromatography, and the column chromatography eluent was dichloromethane:methanol=20:1.

[0081] Compound i (2.2 g, yield 65%, purity >95%) was obtained by column chromatography separation and purification.

[0082] The structural characterization data of compound i are as follows:

[0083] 1 H NMR(400MHz,D2O)δ7.35–7.29(m,2H),6.33(d,J=3.2Hz,1H),6.25(dd,J=3.3,1.9Hz,1H),5.80(d,J=6.6Hz,1H),4.61(d,J=13.7Hz,1H), 4.49–4.37(m,1H),4.22(dd,J=5.6,3.3Hz,1H),4.12(dd,J=6.6,5.6Hz,1H),4.02(q,J=3.5Hz,1H),3.77–3.63(m,2H),1.81–1.70(m,3H).

[0084] 13 C NMR (101MHz, D2O) δ166.3,151.5,150.4,144.1,137.1,111.7,111.1,110.3,86.6,85.3,79.0,68.7,64.2,61.1,11.4.

[0085] MS (ESI) 339.2 [M+H] + .

[0086] Example 5 Preparation of 2'-O-substituted-5-methyluridine (Compound j)

[0087] To a 50 mL round-bottom flask was added 2,2'-anhydro-5-methyluridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 11 (Formula III), and 4-5 mL of toluene. The mixture was then loaded onto a spiked column and heated at 150°C for 12 h. The reaction was monitored by TLC until complete. The mixture was then concentrated to dryness on a rotary evaporator under reduced pressure to obtain compound j.

[0088] The obtained compound j was purified by column chromatography. The column chromatography eluent was dichloromethane:methanol = 20:1.

[0089] Compound j (3.47 g, yield 98%, purity >95%) was obtained by column chromatography separation and purification.

[0090] The structural characterization data of compound j are as follows:

[0091] 1 H NMR(400MHz,DMSO-d6)δ11.32(1H,s),7.64–7.59(1H,m),7.47(1H,dd,J=5.0,1.3Hz) ,7.06(1H,ddd,J=11.9,3.4,1.2Hz),6.96(1H,ddd,J=6.6,5.1,3.4Hz),5.90(1H,d,J= 5.7Hz),5.24(1H,d,J=5.6Hz),5.17(1H,t,J=5.0Hz),4.83(1H,d,J=12.7Hz),4.70(1H ,d,J=12.7Hz), 4.15(1H,td,J=5.4,3.9Hz), 4.08–3.51(4H,m), 1.71(3H,d,J=1.2Hz).

[0092] 13 C NMR (101MHz, DMSO-d6) δ164.2,151.1,141.0,136.3,127.5,109.8,86.0,85.7,80.0,75.7,68.8,66.1,61.2,13.4,12.7.

[0093] MS (ESI) 355.2 [M+H] + .

[0094] Example 6 Preparation of 2'-O-substituted-5-methyluridine (Compound k)

[0095] To a 50 mL round-bottom flask, add 2,2'-anhydro-5-methyluridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.924 g, 0.011 mol, 1.10 equiv.), 10 mL of compound 9 (Formula III), and 4-5 mL of toluene. The mixture was then loaded onto a spiked column and heated at 150°C for 15 h. TLC monitoring was performed until the reaction was complete. The mixture was then concentrated to dryness under reduced pressure on a rotary evaporator to yield compound K.

[0096] The obtained compound k was purified by column chromatography. The column chromatography eluent was dichloromethane:methanol = 30:1.

[0097] Compound k (2.80 g, yield 79%, purity >95%) was obtained by column chromatography separation and purification.

[0098] The structural characterization data of compound K are as follows:

[0099] 1 H NMR (400MHz, CDCl3) δ9.62 (s, 1H), 7.46 (d, J = 1.4Hz, 1H), 7.14–7.01 (m, 2H), 6 .95–6.84(m,2H),5.92(d,J=4.9Hz,1H),4.88(t,J=5.0Hz,1H),4.40(t,J=4.6H z,1H),4.22(dt,J=4.3,2.2Hz,1H),3.97(dd,J=12.5,2.2Hz,1H),3.84(s,3H), 3.81(dd,J=12.5,2.3Hz,1H),3.56(s,1H),1.89(d,J=1.4Hz,3H),1.26(s,1H).

[0100] 13 C NMR (101MHz, CDCl3) δ164.1,150.6,146.8,138.5,125.0,121.7,120.8,112.2,111.0,91.6,85.4,83.9,69.5,61.8,55.8,12.4.

[0101] MS (ESI) 365.1 [M+H] + .

[0102] Example 7 Preparation of 2'-O-substituted-5-methyluridine (Compound 1)

[0103] To a 50 mL round-bottom flask was added 2,2'-anhydro-5-methyluridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 12 (Formula III), and 4-5 mL of toluene. The mixture was then placed on a spiked column and heated at 150°C for 26 h. The reaction was monitored by TLC until complete. The mixture was then concentrated to dryness under reduced pressure on a rotary evaporator to afford compound 1.

[0104] The obtained compound 1 was purified by column chromatography using dichloromethane:methanol = 20:1 as the eluent.

[0105] Compound 1 (0.98 g, yield 30%, purity >95%) was obtained by column chromatography separation and purification.

[0106] The structural characterization data of compound 1 are as follows:

[0107] 1 H NMR(400MHz,D2O)δ7.70(d,J=1.3Hz,1H),5.87(d,J=3.7Hz,1H),4.46(ddt,J=5.9,4.3,2.1Hz,1H),4.23(dd,J=6.3,5.4Hz,1H),4. 14(dd,J=5.4,3.8Hz,1H), 4.04(ddd,J=6.6,4.1,2.8Hz,1H), 3.92–3.66(m,6H), 2.03(td,J=7.9,4.4Hz,2H), 1.82(d,J=1.3Hz,3H).

[0108] 13 C NMR (101MHz, D2O) δ166.4,151.4,137.0,111.3,87.8,84.0,79.7,79.4,72.5,68.0,66.7,60.1,31.7,11.5.

[0109] MS (ESI) 329.2 [M+H] + .

[0110] Example 8 Preparation of 2'-O-substituted uridine (Compound o)

[0111] To a 50 mL round-bottom flask, add 2,2'-anhydrouridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 3 (Formula III), and 4-5 mL of toluene. Add the mixture to a thorn-shaped column and heat at 150°C for 14 h. Monitor by TLC until the reaction is complete. Concentrate the mixture to dryness under reduced pressure on a rotary evaporator to obtain compound o.

[0112] The obtained compound o was purified by column chromatography, and the column chromatography eluent was dichloromethane:methanol=20:1.

[0113] Compound o (2.70 g, yield 75%, purity>95%) was obtained by column chromatography separation and purification.

[0114] The structural characterization data of compound o are as follows:

[0115] 1 H NMR (400MHz, CDCl3) δ10.07(s,1H),7.83(d,J=8.1Hz,1H),5.77(d,J=4.2Hz,1H),5.75–5.71(m,1H),4.30(q,J=4.6Hz,1H),4.17(t,J=4 .6Hz,1H),4.08(dd,J=4.8,2.8Hz,1H),4.02–3.90(m,2H),3.84–3.56(m,8H),3.51(q,J=7.0Hz,2H),2.17(s,1H),1.19(t,J=7.0Hz,3H).

[0116] 13 C NMR (101MHz, CDCl3) δ164.1,150.6,142.0,102.3,89.9,85.1,81.9,70.4,70.2,69.5,68.9,66.7,61.3,31.0,15.0.

[0117] MS (ESI) 361.1 [M+H] + .

[0118] Example 9 Preparation of 2'-O-substituted uridine (Compound p)

[0119] To a 50 mL round-bottom flask was added 2,2'-anhydrouridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 4 of formula III, and 4-5 mL of toluene. The mixture was then loaded onto a spiked column and heated at 150°C for 10 h. The reaction was monitored by TLC until complete. The mixture was then concentrated to dryness under reduced pressure on a rotary evaporator to yield compound p.

[0120] The obtained compound p was purified by column chromatography, and the column chromatography eluent was dichloromethane:methanol=20:1.

[0121] Compound p (3.31 g, yield 85%, purity >95%) was obtained by column chromatography separation and purification.

[0122] The structural characterization data of compound p are as follows:

[0123] 1 H NMR(400MHz,D2O)δ7.78(dd,J=8.1,1.4Hz,1H),5.85(dd,J=4.3,1.6Hz,1H),5.76(dd,J=8.0,1.7Hz,1H),4.18(td,J=5.7, 1.6Hz,1H),4.10–4.04(m,1H),4.00(qd,J=4.2,1.9Hz,1H),3.82–3.64(m,5H),3.62–3.46(m,11H),3.24(d,J=2.1Hz,3H).

[0124] 13 C NMR (101MHz, D2O) δ166.2,151.4,141.7,102.1,87.8,84.2,81.4,70.9,69.7,69.6,69.5,69.4,68.3,60.4,58.0.

[0125] MS (ESI) 391.2 [M+H] + .

[0126] Example 10 Preparation of 2'-O-substituted uridine (Compound q)

[0127] To a 50 mL round-bottom flask, add 2,2'-anhydrouridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 5 (Formula III), and 4-5 mL of toluene. The mixture was then loaded onto a spiked column and heated at 150°C for 12 h. TLC monitoring was performed until the reaction was complete. The mixture was then concentrated to dryness under reduced pressure on a rotary evaporator to yield compound q.

[0128] The obtained compound q was purified by column chromatography using dichloromethane:methanol=20:1 as the eluent.

[0129] Compound q (2.54 g, yield 80%, purity>95%) was obtained by column chromatography separation and purification.

[0130] The structural characterization data of compound q are as follows:

[0131] 1 H NMR (400MHz, CDCl3) δ9.32(s,1H),7.65(d,J=8.1Hz,1H),5.74(d,J=8.0Hz,1H),5.67(d,J=4.7Hz,1H),4.37–4.27(m,2H),4.14(dt,J= 4.2, 2.3Hz, 1H), 4.04–3.92 (m, 2H), 3.81 (d, J = 12.3Hz, 1H), 3.74–3.60 (m, 2H), 3.60–3.49 (m, 3H), 3.30 (s, 1H), 1.23 (t, J = 7.0Hz, 4H).

[0132] 13 C NMR (101MHz, CDCl3) δ163.4,150.4,142.4,102.5,91.3,85.5,81.3,70.1,69.5,69.2,66.8,61.9,14.9.

[0133] MS (ESI) 317.1 [M+H] + .

[0134] Example 11 Preparation of 2'-O-substituted uridine (Compound r)

[0135] To a 50 mL round-bottom flask, add 2,2'-anhydrouridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 6 (Formula III), and 4-5 mL of toluene. The mixture was loaded onto a spiked column and heated at 150°C for 12 h. TLC monitoring was performed until the reaction was complete. The mixture was concentrated to dryness under reduced pressure on a rotary evaporator to yield compound r.

[0136] The obtained compound r was purified by column chromatography, and the column chromatography eluent was dichloromethane:methanol=20:1.

[0137] Compound r (2.61 g, yield 79%, purity >95%) was obtained by column chromatography separation and purification.

[0138] The structural characterization data of compound r are as follows:

[0139] 1 H NMR (400MHz, CDCl3) δ9.54(s,1H),7.67(d,J=8.1Hz,1H),5.74(d,J=8.1Hz,1H),5.68(d,J=4.9Hz,1H),4.32(dq,J=9.7,4.7Hz,2H),4.14(d t,J=4.1,2.2Hz,1H),4.04–3.91(m,2H),3.80(d,J=13.6Hz,1H),3.74–3.58(m,3H),3.58–3.49(m,1H),3.43(s,1H),1.19(t,J=6.1Hz,6H).

[0140] 13 C NMR (101MHz, CDCl3) δ163.6,150.5,142.5,102.4,91.3,85.6,81.3,72.5,70.3,69.2,67.0,61.9,21.9,21.8.

[0141] MS (ESI) 331.2 [M+H] + .

[0142] Example 12 Preparation of 2'-O-substituted-5-bromo-uridine (Compound S)

[0143] To a 50 mL round-bottom flask, add 2,2'-anhydro-5-bromouridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 1 (Formula III), and 4-5 mL of toluene. The mixture was loaded onto a spiked column and heated at 150°C for 14 h. TLC monitoring was performed until the reaction was complete. The mixture was concentrated to dryness under reduced pressure on a rotary evaporator to obtain compound s.

[0144] The obtained compound d was purified by column chromatography. The column chromatography eluent was dichloromethane:methanol = 20:1.

[0145] Compound s (2.85 g, yield 75%, purity >95%) was obtained by column chromatography separation and purification.

[0146] The structural characterization data of compound s are as follows:

[0147] 1 H NMR (400MHz, D2O) δ8.27(d,J=1.8Hz,1H),5.80(t,J=2.6Hz,1H),4.17(ddd,J=6.6,5.0,1.8Hz,1H),4.00(ddt,J=8.9,6.3,2.6H z,2H),3.82(dt,J=13.0,2.3Hz,1H),3.79–3.64(m,4H),3.55–3.46(m,3H),3.23(d,J=2.7Hz,2H),1.04(td,J=7.4,2.0Hz,1H).

[0148] 13 C NMR (101MHz, D2O) δ161.8,150.7,141.0,96.3,87.9,84.0,81.7,71.1,69.5,67.8,59.7,58.0.

[0149] MS (ESI) 403.1 [M+Na] + .

[0150] Example 13 Preparation of 2'-O-substituted-5-chloro-uridine (Compound t)

[0151] To a 50 mL round-bottom flask, add 2,2'-anhydro-5-chlorouridine (2.4 g, 0.010 mol, 1.0 equiv.), boric acid (0.124 g, 0.002 mol, 0.20 equiv.), sodium bicarbonate (0.084 g, 0.001 mol, 0.10 equiv.), 10 mL of compound 1 (Formula III), and 4-5 mL of toluene. The mixture was then loaded onto a spiked column and heated at 150°C for 10 h. The reaction was monitored by TLC until complete. The mixture was concentrated to dryness under reduced pressure on a rotary evaporator to yield compound t.

[0152] The obtained compound d was purified by column chromatography with a column chromatography eluent of dichloromethane:methanol = 40:1.

[0153] Compound t (2.45 g, yield 73%, purity >95%) was obtained by column chromatography separation and purification.

[0154] The structural characterization data of compound t are as follows:

[0155] 1 H NMR(400MHz,D2O)δ8.18(s,1H),5.86(d,J=3.5Hz,1H),4.21(dd,J=6.5,5.2Hz,1H),4.10–4.00(m,2 H), 3.86 (dd, J = 12.9, 2.8 Hz, 1H), 3.84–3.69 (m, 4H), 3.56 (t, J = 4.4 Hz, 2H), 3.27 (d, J = 10.0 Hz, 3H).

[0156] 13 C NMR (101MHz, D2O) δ161.8,150.5,138.4,108.7,87.9,84.1,81.7,71.2,69.5,67.9,59.9,58.0.

[0157] MS (ESI) 337.1 [M+H] + .

[0158] Example 14 Preparation of 5'-DMT-2'-O-propoxyethyl-5-methyluridine phosphoramidite monomer

[0159] Compound d (600 mg, 1.74 mmol) was dissolved in pyridine (30 mL) solution, DMTrCl (884 mg, 2.16 mmol) was added at 0°C, and the reaction was heated to 50°C and stirred for 16 hours. TLC monitoring was performed until the reaction was completed, and the reaction solution was diluted with saturated NaHCO3 solution (50 mL), and then extracted with ethyl acetate (50 mL×3). The organic layer was washed with saturated brine (30 mL) and dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH=100:1) to obtain 5'-DMT-2'-O-propoxyethyl-5-methyluridine (460 mg, yield 41%).

[0160] 5'-DMT-2'-O-propoxyethyl-5-methyluridine (350 mg, 0.54 mmol) was dissolved in acetonitrile (15 mL), and tetrazole (45.5 mg, 0.65 mmol) was added. The reaction mixture was stirred at room temperature for 10 minutes under N2 protection. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (325 mg, 1.08 mmol) was then added to the reaction mixture, and the reaction was stirred at 30°C for 16 hours. After completion of the reaction, the reaction mixture was filtered and concentrated to obtain the crude product. The crude product was purified by column chromatography (PE / TEA (1000:1):EA / TEA (1000:1) = 50:50) to obtain 5'-DMT-2'-O-propoxyethyl-5-methyluridine phosphorodiamidite monomer (130 mg, 29% yield).

[0161] The structural characterization data of the 5'-DMT-2'-O-propoxyethyl-5-methyluridine phosphoramidite monomer are as follows:

[0162] 1 H NMR (400MHz, DMSO-d6) δ11.42(s,1H),7.52(s,1H),7.39(d,J=7.2Hz,2H),7.27(m,8H),6.88(m,4H),5.86(d,1H),4.03(m,3H),3.73(d,6H),3.72 –3.67(m,2H),3.52–3.47(m,2H),3.30(d,4H),2.78(s,1H),1.99(s,4H) ,1.41(m,5H),1.17(m,4H),1.12(m,4H),0.96(d,3H),0.87–0.73(m,4H).

[0163] Example 15 Preparation of 5'-DMT-2'-O-(thiophene-2-methyl)-5-methyluridine phosphoramidite monomer

[0164] Compound j (900 mg, 2.54 mmol) was dissolved in pyridine (30 mL), and DMTrCl (1033 mg, 3.04 mmol) was added. The reaction was heated to 50°C and stirred for 16 hours. After the reaction was completed by TLC, the reaction solution was diluted with saturated NaHCO3 (50 mL) and then extracted with ethyl acetate (50 mL × 3). The organic layer was washed with saturated NaCl (30 mL) and dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH = 98:2) to obtain 5'-DMT-2'-O-(thiophene-2-methyl)-5-methyluridine (600 mg, yield 36%).

[0165] 5'-DMT-2'-O-(thiophene-2-methyl)-5-methyluridine (400 mg, 0.609 mmol) was dissolved in acetonitrile (15 mL), and tetrazole (52 mg, 0.73 mmol) was added. The reaction mixture was stirred at room temperature for 10 minutes under N2 protection. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (365.4 mg, 1.21 mmol) was then added to the reaction mixture, and the reaction was stirred at 30°C for 16 hours. After completion of the reaction, the reaction mixture was filtered and concentrated to obtain the crude product. The crude product was purified by column chromatography (PE / TEA (1000:1):EA / TEA (1000:1) = 50:50) to obtain 5'-DMT-2'-O-(thiophene-2-methyl)-5-methyluridine phosphoramidite monomer (200 mg, 39% yield).

[0166] The structural characterization data of the 5'-DMT-2'-O-(thiophene-2-methyl)-5-methyluridine phosphoramidite monomer are as follows:

[0167] 1 H NMR(400MHz,DMSO-d6)δ11.42(s,1H),7.52(s,1H),7.39(d,2H),7.27(m,8H),6.88(m,4H),5.86(d,1H),4.03(m,3H),3.73(d,6H),3.72–3.6 7(m,2H),3.52–3.47(m,2H),3.30(d,4H),2.78(s,1H),1.99(s,4H),1 .41(m,5H),1.17(m,4H),1.12(m,4H),0.96(m,3H),0.87–0.73(m,4H).

[0168] Example 16 Preparation of 5'-DMT-2'-O-(o-methoxyphenyl)-5-methyluridine phosphoramidite monomer

[0169] Compound k (800 mg, 2.20 mmol) was dissolved in pyridine (30 mL), and then DMTrCl (1.12 g, 3.30 mmol) was added. The reaction was heated to 50°C and stirred for 16 hours. After the reaction was completed, the reaction solution was diluted with saturated NaHCO3 (50 mL) and then extracted with ethyl acetate (50 mL × 3). The organic layer was washed with saturated NaCl (30 mL) and dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH=98:2) to obtain 5'-DMT-2'-O-(o-methoxyphenyl)-5-methyluridine (450 mg, yield 31%).

[0170] 5'-DMT-2'-O-(o-methoxyphenyl)-5-methyluridine (450 mg, 0.68 mmol) was dissolved in acetonitrile (40 mL), and tetrazole (61 mg, 0.88 mmol) was added. The reaction mixture was stirred at room temperature for 10 minutes under N2 protection. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (594 mg, 1.35 mmol) was then added to the reaction mixture, and the reaction was stirred at 30°C for 16 hours. After completion of the reaction, the reaction mixture was filtered and concentrated to obtain the crude product. The crude product was purified by column chromatography (PE / TEA (1000:1):EA / TEA (1000:1) = 50:50) to obtain 5'-DMT-2'-O-(o-methoxyphenyl)-5-methyluridine phosphoramidite monomer (204 mg, 23% yield).

[0171] Example 17 Preparation of 5'-DMT-2'-O-ethoxyethoxyethyl-5-methyluridine phosphoramidite monomer

[0172] Compound g (700 mg, 1.94 mmol) was dissolved in pyridine (30 mL) solution, and then DMTrCl (789 mg, 2.32 mmol) was added. The reaction was heated to 50 ° C and stirred for 16 hours. After the reaction was completed, the reaction solution was diluted with saturated NaHCO3 (50 mL) and then extracted with ethyl acetate (50 mL × 3). The organic layer was washed with saturated NaCl (30 mL) and dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by column chromatography (DCM: MeOH = 98: 2) to obtain 5'-DMT-2'-O-ethoxyethoxyethyl-5-methyluridine (500 mg, yield 39%).

[0173] 5'-DMT-2'-O-ethoxyethoxyethyl-5-methyluridine (500 mg, 0.755 mmol) was dissolved in acetonitrile (30 mL), and tetrazole (63 mg, 0.906 mmol) was added. The reaction mixture was stirred at room temperature for 10 minutes under N2 protection. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (453 mg, 1.51 mmol) was then added to the reaction mixture, and the reaction was stirred at 30°C for 16 hours. After completion of the reaction, the reaction mixture was filtered and concentrated to obtain the crude product. The crude product was purified by column chromatography (PE / TEA (1000:1):EA / TEA (1000:1) = 50:50) to obtain 5'-DMT-2'-O-ethoxyethoxyethyl-5-methyluridine phosphoramidite monomer (200 mg, 16% yield).

[0174] The structural characterization data of 5'-DMT-2'-O-ethoxyethoxyethyl-5-methyluridine phosphoramidite monomer are as follows:

[0175] 1 H NMR(400MHz,DMSO-d6)δ11.42(s,1H),7.52(s,1H),7.39(d,2H),7.27(m,8H),6.88(m,4H),5.86(d,1H),4.03(m,3H),3.73(d,6H),3.72–3.6 7(m,2H),3.52–3.47(m,4H),3.30(d,1H),2.78(s,1H),1.99(s,4H),1 .41(m,5H),1.17(m,4H),1.12(m,4H),0.96(d,3H),0.87–0.73(m,7H).

[0176] Example 18 Preparation of 5'-DMT-2'-O-(3"-S-tetrahydrofuranyl)-5-methyluridine phosphoramidite monomer

[0177] Compound 1 (600 mg, 1.83 mmol) was dissolved in pyridine (30 mL) solution, and then DMTrCl (743 mg, 2.19 mmol) was added, and the reaction was heated to 50°C and stirred for 16 hours. After the reaction was completed, the reaction solution was diluted with saturated NaHCO3 (50 mL) and then extracted with ethyl acetate (50 mL×3). The organic layer was washed with saturated NaCl (30 mL) and dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH=98:2) to obtain 5'-DMT-2'-O-(3"-S-tetrahydrofuranyl)-5-methyluridine (600 mg).

[0178] 5'-DMT-2'-O-(3"-S-tetrahydrofuryl)-5-methyluridine (600 mg, 0.807 mmol) was dissolved in acetonitrile (30 mL), and tetrazole (68 mg, 0.969 mmol) was added. The reaction solution was stirred at room temperature for 10 minutes under N2 protection. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (461 mg, 1.61 mmol) was then added to the reaction solution, and the reaction was stirred at 30°C for 16 hours. After completion of the reaction, the reaction solution was filtered and concentrated to obtain a crude product. The crude product was purified by column chromatography (PE / TEA (1000:1):EA / TEA (1000:1) = 50:50) to obtain 5'-DMT-2'-O-(3"-S-tetrahydrofuryl)-5-methyluridine phosphoramidite monomer (330 mg, 49% yield).

[0179] The structural characterization data of the 5'-DMT-2'-O-(3"-S-tetrahydrofuranyl)-5-methyluridine phosphoramidite monomer are as follows:

[0180] 1 H NMR(400MHz,DMSO-d6)δ11.32(s,1H),7.42(s,1H),7.39(d,2H),7.28(m,7H) ,6.89(m,4H),5.86(d,1H),4.03(m,3H),4.00–3.85(m,2H),3.82–3.70(m,2H ),3.72–3.67(m,3H),,3.30(d,1H),2.78(s,1H),2.10–1.85(m,2H),1.99(s, 4H),1.41(m,5H),1.17(m,4H),1.12(m,4H),0.96(d,3H),0.87–0.73(m,4H).

[0181] Example 19 General Method for Preparing Oligonucleotide Sequences by Solid Phase Techniques

[0182] (Preparation of SEQ 1-4)

[0183] Unless otherwise noted, all reagents and solutions used to synthesize oligomeric compounds were purchased from commercial sources. Anhydrous acetonitrile was used to dissolve standard phosphoramidite monomers, including, for example, dT, dA, dG, and dC, as well as designated phosphoramidite monomers described in this patent. The phosphoramidite monomer solution in anhydrous acetonitrile was used at a concentration of 0.06 M.

[0184] A 2 μmol synthesis column made of Universal CPG solid support was loaded on LK-48E (1-2 μmol scale) and the specified sequence was synthesized using the phosphoramidite coupling method. For the coupling step, the phosphoramidite monomer was delivered in an amount exceeding 4 times the loading on the solid support and the phosphoramidite condensation was continued for 10 min. All other steps were in accordance with the standard protocol supplied by the manufacturer. A solution of 3% trichloroacetic acid in dichloromethane was used to remove dimethoxytrityl (DMT) from the 5'-hydroxyl of the nucleotide. BTT (0.35 M) in anhydrous CH3CN was used as an activator during the coupling step. The phosphite bond was introduced by oxidation with a 0.05 M solution of I2 in THF / Pyridine for a contact time of 2 minutes.

[0185] After synthesizing the desired sequence, the solid support-bound sequence is suspended in aqueous ammonia (25-30 wt%) and heated at 85°C for 2 hours. The solid support is then filtered off and the ammonia is boiled off. The residue is purified by high-pressure liquid chromatography.

[0186] The prepared oligonucleotide sequence is: 5'-d(GCGTTTTTTGCT)-3', T is a DNA or modified RNA monomer, and other sites are DNA monomers.

[0187] Serial number T site modification Calculate molecular weight Observed molecular weight SEQ 1 The corresponding monomer of compound d 3719.5 3719.6 SEQ 2 The corresponding monomer of compound 1 3747.5 3747.4 SEQ 3 The corresponding monomer of compound j 3729.5 3729.2 SEQ 4 The corresponding monomer of compound g 3749.5 3749.4

[0188] Example 20 Tm test of oligonucleotide sequences

[0189] The test oligonucleotide sequence (5'-d(GCGTTTTTTGCT)-3', T is a DNA or modified RNA monomer, and all other sites are DNA monomers; 4uM PBS solution) and the complementary RNA sequence (5'-r(AGCAAAAAACGC)-3'; 4uM PBS solution) were mixed in a 1:1 ratio (total sample volume 3mL) and added to a cuvette. The cuvette was placed in a UV-visible spectrophotometer (ThermoScientific Evolution One, equipped with a Peltier 8-strip sample cell), heated at 95°C for 5 minutes, and then slowly cooled to 25°C at a rate of 2°C / min. The melting curve was obtained by heating the sample (25-85°C, 0.5°C / min) and measuring the absorption at 260nm. The Tm value was calculated by Insight Pro software. The modification of the compound shows a stronger ability to bind to the complementary RNA sequence, which can be used as a direction for drug optimization.

[0190]

[0191]

[0192] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

Claims

1. A 2'-O-substituted nucleoside compound with high stability and affinity: 。 2. A method for preparing a compound of formula I, comprising the steps of: reacting a compound of formula II with R 2 OH is placed in a solvent and reacted in the presence of a boron catalyst and a base to obtain a compound of formula I. The synthesis route is as follows: ； in, R 1 selected from methyl; The compound of formula I is selected from: or , R 2 Selected from or ; The boron catalyst is selected from boric acid; The base is selected from sodium bicarbonate; The solvent is selected from toluene; The molar ratio of the compound of formula II to the base is 1:0.1-0.5; The reaction time is 8-26 hours.

3. The preparation method according to claim 2, characterized in that The mass volume ratio of the compound of formula II to the solvent is 1:1-5 g / ml.

4. The preparation method according to claim 3, characterized in that The mass volume ratio of the compound of formula II to the solvent is 1:1.5-3 g / ml.

5. The preparation method according to claim 2, characterized in that The molar ratio of the compound of formula II to the boron catalyst is 1:0.1-0.

5.

6. The preparation method according to claim 5, characterized in that The molar ratio of the compound of formula II to the boron catalyst is 1:0.1-0.

2.

7. The preparation method according to claim 2, characterized in that The molar ratio of the compound of formula II to the base is 1:0.1-0.

2.

8. The preparation method according to claim 2, characterized in that The reaction temperature is 150-160°C.

9. The preparation method according to claim 2, characterized in that The reaction time is 10-18 hours.

10. The preparation method according to claim 2, characterized in that The reaction is carried out in a water separator or a thorn-shaped distillation column.

11. The preparation method according to claim 2, characterized in that The compound of formula I is purified by column chromatography or recrystallization.

12. The preparation method according to claim 11, characterized in that The eluent for the column chromatography is dichloromethane:methanol, with a volume ratio of 100:1-20:

1.

13. The preparation method according to claim 11, characterized in that The recrystallization solvent is any one of ethyl acetate and methanol or a combination thereof.

14. A method for preparing a 5'-DMT-2'-O-substituted nucleoside, comprising the following steps: The compound of claim 1 is placed in pyridine, reacted with DMTrCl, and then separated and purified to obtain 5'-DMT-2'-O-substituted nucleoside.

15. The preparation method according to claim 14, characterized in that The reaction time is 1-10h.

16. The preparation method according to claim 15, characterized in that The reaction time is 3-5h.

17. The preparation method according to claim 14, characterized in that The molar ratio of the compound of formula I to DMTrCl is 1:1.05-1.

2.

18. The preparation method according to claim 17, characterized in that The molar ratio of the compound of formula I to DMTrCl is 1:1.

1.

19. The preparation method according to claim 14, characterized in that After the reaction, the product is separated and purified by column chromatography.

20. The preparation method according to claim 19, characterized in that The eluent for the column chromatography is dichloromethane:methanol, with a volume ratio of 100:1 to 20:

1.

21. Use of the compound according to claim 1 in preparing clinical antisense oligonucleotide drugs.

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