Nucleotide analogs, methods for their preparation and uses

Abstract

The present invention relates to a lipophilic nucleotide analogue represented by formula (I), its preparation method and use. The lipophilic nucleotide analogue of the present invention can enhance the in vivo delivery of nucleic acid drugs. [Formula 1] TIFF2026503329000039.tif42170 Formula I

Description

[Technical Field]

[0001] The present invention belongs to the field of medicine, and specifically relates to nucleotide analogues, their preparation and use. [Background technology]

[0002] With the development of nucleic acid chemical synthesis technology, nucleic acids and their modified analogs have been widely applied in chemistry, biology, and medicine. Chemical modifications of nucleotides include modifications to the sugar ring, base moiety, phosphodiester backbone, or substituting chemical structures with specific functions for natural nucleosides. Non-natural nucleotides include peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), glycerol nucleic acids (GNAs), threose nucleic acids (TNAs), and unlocked nucleic acids (UNAs).

[0003] In vivo delivery of small interfering RNA (siRNA) to cells requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. siRNA therapy holds promise for treating liver-related disorders. However, obstacles to delivering siRNA to extrahepatic tissues limit its use. Adding lipophilic moieties to nucleic acid molecules is one of the fastest ways to improve cellular uptake and deliver antisense oligonucleotides (ASOs) and siRNA to the liver and various other organ systems. Lipophilic conjugates can enhance siRNA delivery and nucleic acid drug uptake into alveolar and bronchiolar epithelia (Brown, KM, Nair, JK, Janas, MM et al. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates. Nat Biotechnol (2022)). Currently, extrahepatic delivery strategies employ lipid nanoparticles and N-acetylgalactosamine (GalNAc) conjugates. However, new and improved methods for delivering siRNA molecules in vivo are still needed to achieve and enhance the therapeutic potential of siRNA.

[0004] The present invention provides lipophilic nucleotide analogs for the purpose of improving the delivery efficiency of siRNA in the body, and a method for synthesizing the nucleotide analogs with simple, convenient procedures and high yields. Summary of the Invention

[0005] The present invention provides a compound represented by Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [ka] Formula I wherein Y is a hydroxyl protecting group; R 1 is selected from C1-6 alkyl and C1-6 alkyl substituted with halogen; R 2 is C 1~6 alkyl, C1-6 alkyl substituted with halogen, L is a lipophilic group, Base is a nucleotide base.

[0006] Further, the compound of formula I is represented by formula II: [ka] Formula II In the formula, Y, R 1 , R 2 , L, and Base have the same meanings as above.

[0007] In some embodiments of the invention, L is a cholesterol group, C6-C 20 Alkyl, C6-C 20 Alkenyl, C6-C 20 alkynyl.

[0008] moreover, R 1 is selected from isopropyl, R 2 is selected from isopropyl, L is the cholesterol group, C6-C 20 alkyl.

[0009] Preferably, L is C-C 20 More preferably, L is selected from C6 straight chain alkyl, C7 straight chain alkyl, C8 straight chain alkyl, C9 straight chain alkyl, C 10 Straight chain alkyl, C 11 Straight chain alkyl, C 12 Straight chain alkyl, C 13 Straight chain alkyl, C 14 Straight chain alkyl, C 15 Straight chain alkyl, C 16 Straight chain alkyl, C 17 Straight chain alkyl, C 18 Straight chain alkyl, C 19 Straight chain alkyl, C 20straight chain alkyl.

[0010] moreover, L is [ka] is selected from.

[0011] moreover, Base is, [ka] is selected from.

[0012] Furthermore, Y is selected from 4,4'-dimethoxytriphenylmethyl, 4-monomethoxytrityl, trityl, trimethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, triethylsilyl, phenyldimethylsilyl, benzyloxycarbonyl or 2-bromo-benzyloxycarbonyl.

[0013] In some specific embodiments of the present invention, the compound of formula I is specifically [ka] is.

[0014] The present invention further provides the use of the nucleotide analogues in nucleic acid delivery.

[0015] The present invention further provides the use of said nucleotide analogues in siRNA and ASO delivery.

[0016] The present invention further provides the use of said nucleotide analogues as intermediates in the preparation of nucleic acid molecules.

[0017] The present invention further provides the use of said nucleotide analogues as intermediates in the preparation of siRNA and ASO molecules.

[0018] Preferably, the present invention provides a use of the nucleotide analogue as an intermediate in the preparation of an siRNA sense strand. More preferably, the present invention provides a use of the nucleotide analogue as an intermediate in the preparation of nucleotides at positions 2 to 9 of the 5'-end of an siRNA sense strand. More preferably, the present invention provides a use of the nucleotide analogue as an intermediate in the preparation of nucleotides at positions 2, 3, 4, 5, 6, 7, or 8 of the 5'-end of an siRNA sense strand.

[0019] The present invention provides a method for synthesizing the nucleotide analogues, which comprises the steps of: [ka] wherein Y is a hydroxyl protecting group, L is a lipophilic group, and Base is a nucleotide base; Step 1: Compound A1 is dissolved in dichloromethane, and the Dess-Martin oxidant, NaHCO3, is added. The reaction is allowed to proceed at room temperature for 5 to 16 hours to obtain compound A2. Step 2: Ethoxyformylmethylenetriphenylphosphine is dissolved in dichloromethane, and the dichloromethane solution of compound A2 is added with stirring. The mixture is allowed to react at room temperature for 5 to 16 hours to obtain compound A3. Step 3: Compound A3 is dissolved in dichloromethane, diisobutylaluminum hydride is added, and the mixture is reacted at -10 to 5°C for 1 to 3 hours to obtain compound A4. Step 4: Dissolve tetraisopropyl titanate in dichloromethane, add D-(-)-diethyl tartrate and stir at -30 to 5°C, then add a solution of compound A4 in dichloromethane and tert-butyl hydroperoxide. Continue reacting for 10 to 24 hours to obtain compound A5. Step 5: Dissolve compound A5 in pyridine, slowly add a hydroxyl protecting group reagent, and react at room temperature with stirring for 5-16 h to obtain compound A6. Step 6: In a microwave reaction tube, compound A6, a nucleotide base reagent, and 1,8-diazabicyclo[5.4.0]undec-7-ene are dissolved in a solvent and reacted at 90-120°C for 5-12 hours with stirring, and then compound A7 is added. Step 7: Compound A7 is dissolved in dichloromethane, and 4,5-dicyanoimidazole and bis(diisopropylamino)(2-cyanoethoxy)phosphine are added. The mixture is reacted at 20 to 40° C. for 20 minutes to 3 hours to obtain compound A8.

[0020] In some specific embodiments of the invention, Y is 4,4'-dimethoxytriphenylmethyl and L is C 16 It is a straight chain alkyl and the base is a uracil base.

[0021] Furthermore, in Step 1, the molar ratio of compound A1, Dess-Martin oxidant, and NaHCO3 is 1:1.0-1.5:2-6. In the step 2, the molar ratio of compound A2 to ethoxyformylmethylenetriphenylphosphine is 1:1.0 to 1.5. In the above step 3, the molar ratio of compound A3 to diisobutylaluminum hydride is 1:2.0-3.0. In the step 4, the molar ratio of compound A4, tetraisopropyl titanate, and D-(-)-diethyl tartrate is 1:1.0-1.5:1.0-1.5. In the above step 5, the molar ratio of compound A5 to the hydroxyl-protecting group reagent is 1:1.0-1.5. In the step 6, the molar ratio of compound A6, nucleotide base reagent, and 1,8-diazabicyclo[5.4.0]undec-7-ene is 1:1.0-2.0:1.0-2.0. In the above Step 7, the molar ratio of compound A7, 4,5-dicyanoimidazole, and bis(diisopropylamino)(2-cyanoethoxy)phosphine is 1:0.5-2.0:1.0-2.5.

[0022] Furthermore, in Step 1, the molar equivalent of A1 is 1, the molar equivalent of the Dess-Martin oxidant is 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5, and the molar equivalent of NAHCO3 is 2, 3, 4, 5, or 6, and preferably, the molar equivalent of the Dess-Martin oxidant is 1.2 and the molar equivalent of NAHCO3 is 5. In the step 2, the molar equivalent of compound A2 is 1, and the molar equivalent of ethoxyformylmethylenetriphenylphosphine is 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5, preferably the molar equivalent of ethoxyformylmethylenetriphenylphosphine is 1.3. In the above step 3, the molar equivalent of compound A3 is 1, and the molar equivalent of diisobutylaluminum hydride is 2.1, 2.2, 2.4, 2.6, 2.7, 2.8, 3.0, preferably the molar equivalent of diisobutylaluminum hydride is 2.6. In step 4, the molar equivalent of compound A4 is 1, the molar equivalents of tetraisopropyl titanate are 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, and the molar equivalents of D-(-)-diethyl tartrate are 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, preferably the molar equivalent of tetraisopropyl titanate is 1.4; the molar equivalent of D-(-)-diethyl tartrate is 1.3. In the above step 5, the molar equivalent of compound A5 is 1, and the molar equivalent of the hydroxyl protecting group reagent is 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5, preferably the molar equivalent of the hydroxyl protecting group reagent is 1.2. In step 6, the molar equivalent of compound A6 is 1, the molar equivalents of the nucleotide base reagent are 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8, and the molar equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene are 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8, preferably, the molar equivalent of the nucleotide base reagent is 1.5, and the molar equivalent of 1,8-diazabicyclo[5.4.0]undec-7-ene is 1.6. In step 7, the molar equivalent of compound A7 is 1, the molar equivalents of 4,5-dicyanoimidazole are 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0, and the molar equivalents of bis(diisopropylamino)(2-cyanoethoxy)phosphine are 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, and 2.2. Preferably, the molar equivalent of 4,5-dicyanoimidazole is 0.8, and the molar equivalent of bis(diisopropylamino)(2-cyanoethoxy)phosphine is 1.9.

[0023] Furthermore, in Step 5, the hydroxyl protecting group reagent is any one of tert-butyldimethylchlorosilane, trimethylchlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, trityl chloride, 4-methoxytrityl chloride, 4,4′,4″-trimethoxytrityl chloride, and 4,4′-bismethoxytrityl chloride. Preferably, the hydroxyl protecting group reagent is 4,4′-bismethoxytrityl chloride. In step 6, the nucleotide base reagent is any one of adenine, guanine, thymine, cytosine, uracil, purine, xanthine, and diaminopurine. Preferably, the nucleotide base reagent in step 6 is uracil.

[0024] Furthermore, the reaction time of step 1 is 5 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours, and preferably, the reaction time of step 1 is 16 hours. The reaction time of step 2 is 5 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours, and preferably, the reaction time of step 2 is 16 hours. The reaction time of step 3 is 1 hour, 2 hours, or 3 hours, and the reaction temperature of step 3 is -10°C, -5°C, 0°C, or 5°C. Preferably, the reaction time of step 3 is 1 hour, and the reaction temperature of step 3 is -5°C. The reaction time of step 4 is 10 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, and the reaction temperature of step 4 is -30°C, -25°C, -10°C, 0°C, or 5°C. Preferably, the reaction time of step 4 is 24 hours and the reaction temperature of step 4 is 0°C. The reaction time of step 5 is 5 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours, and preferably, the reaction time of step 5 is 16 hours. The reaction time of step 6 is 5 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours, and the reaction temperature of step 4 is 90°C, 100°C, 110°C, or 120°C. Preferably, the reaction time of step 6 is 5 hours and the reaction temperature of step 6 is 110°C. The reaction time of step 7 is 20 min, 0.5 h, 1 h, or 2 h, and the reaction temperature of step 7 is 20°C, 25°C, 30°C, or 35°C. Preferably, the reaction time of step 7 is 0.5 h; and the reaction temperature of step 6 is 25°C.

[0025] The present invention provides an intermediate compound for synthesizing the nucleotide analogues of the present invention, which has the structure represented by Formula III. [ka] Formula III wherein Y is a hydroxyl protecting group; L is a lipophilic group, Base is a nucleotide base.

[0026] Further, the intermediate compound of formula III is represented by formula IV. [ka] Formula IV wherein Y is selected from 4,4'-dimethoxytriphenylmethyl, 4-methoxytrityl, trityl, trimethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, triethylsilyl, phenyldimethylsilyl, benzyloxycarbonyl or 2-bromobenzyloxycarbonyl; L is C6 linear alkyl, C7 linear alkyl, C8 linear alkyl, C9 linear alkyl, C 10 Straight chain alkyl, C 11 Straight chain alkyl, C 12 Straight chain alkyl, C 13 Straight chain alkyl, C 14 Straight chain alkyl, C 15 Straight chain alkyl, C 16 Straight chain alkyl, C 17 Straight chain alkyl, C 18 Straight chain alkyl, C 19 Straight chain alkyl, C 20 straight chain alkyl; Base is, [ka] is selected from.

[0027] Preferably, Y is 4,4'-dimethoxytriphenylmethyl and L is C 16 It is a straight chain alkyl and the base is a uracil base.

[0028] In some embodiments of the present invention, the intermediate compound is specifically [ka] is.

[0029] The present invention further provides an siRNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand each comprise 15 to 45 modified or unmodified nucleotides, the sense strand and the antisense strand are partially complementary to form a double-stranded region, and the sense strand has at least one nucleotide having a structure represented by Formula V: [ka] The nucleotide sequence is covalently linked to the remainder of the siRNA at this position. [ka] Formula V wherein X is selected from O or S; L is a lipophilic group, Base is a nucleotide base.

[0030] In some embodiments of the present invention, the structure of formula V is represented by formula Va: [ka] Formula Va In the formula, X, L, and Base have the same meanings as in claim 10.

[0031] Furthermore, L is a cholesterol group, C6 20 alkyl.

[0032] Furthermore, L is [ka] is selected from.

[0033] In some embodiments of the present invention, further comprising: Base is, [ka] is selected from.

[0034] In some specific embodiments of the present invention, the structure represented by formula V is specifically [ka] is.

[0035] In some specific embodiments of the present invention, the siRNA sense strand contains one, two, three, four or five nucleotides having a structure represented by formula V, preferably the siRNA sense strand contains one or two nucleotides having a structure represented by formula V, more preferably the siRNA sense strand contains one nucleotide having a structure represented by formula V.

[0036] In some specific embodiments of the present invention, the structure represented by formula V is the second to tenth nucleotide at the 5' end of the sense strand, preferably the structure represented by formula V is the second, third, fourth, fifth, sixth, seventh, or eighth nucleotide at the 5' end of the sense strand, and more preferably the structure represented by formula V is the sixth nucleotide at the 5' end of the sense strand.

[0037] In some specific embodiments of the present invention, the length of the antisense strand of the siRNA is 19 to 27 nucleotides, and the length of the sense strand is 19 to 25 nucleotides. Preferably, the length of the antisense strand is 19 to 23 nucleotides, and the length of the sense strand is 19 to 21 nucleotides. More preferably, the length of the antisense strand is 23 nucleotides, and the length of the sense strand is 21 nucleotides.

[0038] In some embodiments of the present invention, the siRNA comprises one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotide overhangs. In some embodiments of the present invention, the overhangs may be located on the sense strand, the antisense strand, or a combination thereof. In some embodiments of the present invention, the overhangs are located at the 5'-end, the 3'-end, or both ends of the siRNA antisense strand or sense strand.

[0039] In some embodiments of the present invention, the 3' end of the antisense strand of the siRNA has an overhang of two nucleotides.

[0040] In some embodiments of the present invention, the siRNA has blunt ends. In some embodiments of the present invention, the siRNA has at least one blunt end, located at the 5' end of the antisense strand (or the 3' end of the sense strand).

[0041] In some embodiments of the invention, the siRNA has two blunt ends.

[0042] In some embodiments of the present invention, the 3' end of the antisense strand of the siRNA has an overhang of two nucleotides.

[0043] In some specific embodiments of the present invention, the siRNA comprises at least one modified nucleotide. Preferably, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

[0044] In some specific embodiments of the invention, the modified nucleotide or nucleotide analog is selected from a 2'-methoxy nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2',3'-open-ring nucleotide analog, a 2'-fluoroarabino nucleotide, a 2'-methoxyethyl nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a 3'-methoxy nucleotide, a 2'-allyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate ester, a nucleotide containing a 5'-phosphate ester mimic, a diol-modified nucleotide, an abasic nucleotide, a morpholino nucleotide, a locked nucleotide, an unlocked nucleotide, or a glycerol nucleotide.

[0045] In some embodiments of the present invention, the nucleotide sequence modified in the siRNA comprises: 1) the nucleotides at positions 7, 9, 10, and 11 of the 5'-end of the sense strand are 2'-fluoronucleotides, and / or 2) The nucleotides at positions 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoronucleotides.

[0046] In some embodiments of the present invention, the nucleotide sequence modified in the siRNA comprises: 1) the nucleotides at positions 2, 14, and 16 of the 5'-end of the antisense strand are 2'-fluoronucleotides, and / or 2) The nucleotides at positions 2, 6, 14, and 16 of the 5' end of the antisense strand are 2'-fluoronucleotides.

[0047] In some embodiments of the present invention, for the siRNA-modified nucleotide sequence, the antisense strand further comprises at least one glycerol nucleotide. Preferably, the glycerol nucleotide is located at the second to ninth nucleotides of the 5'-end of the antisense strand. More preferably, the glycerol nucleotide is located at the third, fourth, fifth, sixth, seventh, or eighth nucleotide of the 5'-end of the antisense strand.

[0048] In some specific embodiments of the present invention, one or two phosphorothioate groups are independently linked to the 5'-end and 3'-end of the sense strand of the siRNA, and / or one or two phosphorothioate groups are independently linked to the 5'-end and 3'-end of the antisense strand.

[0049] In some embodiments of the present invention, at least one of the following nucleotides is linked by a phosphorothioate group: between the first and second nucleotides at the 5' end of the sense strand, between the second and third nucleotides at the 5' end of the sense strand, between the first and second nucleotides at the 3' end of the sense strand, between the second and third nucleotides at the 3' end of the sense strand, between the first and second nucleotides at the 3' end of the antisense strand, between the second and third nucleotides at the 3' end of the antisense strand, between the first and second nucleotides at the 5' end of the antisense strand, and between the second and third nucleotides at the 5' end of the antisense strand; and preferably at least four of these nucleotides are linked by phosphorothioate groups. In some embodiments of the present invention, at least six of these nucleotides are linked by phosphorothioate groups. In some embodiments of the present invention, all eight of these nucleotides are linked by phosphorothioate groups.

[0050] In some embodiments of the present invention, the first and second nucleotides and the second and third nucleotides at the 5' end of the sense strand are linked by phosphorothioate groups.

[0051] In some embodiments of the present invention, the first and second nucleotides and the second and third nucleotides at the 5' end of the sense strand are linked by phosphorothioate groups, and the first and second nucleotides and the second and third nucleotides at the 3' end are linked by phosphorothioate groups.

[0052] In some embodiments of the present invention, the first and second nucleotides and the second and third nucleotides at the 3' end of the antisense strand are linked by phosphorothioate groups, and the first and second nucleotides and the second and third nucleotides at the 5' end are linked by phosphorothioate groups.

[0053] In some embodiments of the present invention, the first and second nucleotides at the 5' end of the sense strand, the second and third nucleotides at the 5' end of the sense strand, the first and second nucleotides at the 3' end of the sense strand, the second and third nucleotides at the 3' end of the sense strand, the first and second nucleotides at the 3' end of the antisense strand, the second and third nucleotides at the 3' end of the antisense strand, the first and second nucleotides at the 5' end of the antisense strand, and the second and third nucleotides at the 5' end of the antisense strand are all linked by phosphorothioate groups.

[0054] In some embodiments of the invention, the first nucleotide at the 5' end of the antisense strand is an (E)-vinyl phosphate modified nucleotide.

[0055] The present invention further provides a pharmaceutical composition comprising the siRNA and a pharmaceutically acceptable carrier.

[0056] The present invention further provides a use of said siRNA and / or said pharmaceutical composition in the preparation of a medicament.

[0057] Regarding the definitions of terms used in the present invention, unless otherwise specified, the initial definition provided for a group or term in this specification shall apply to that group or term throughout the specification, and terms not specifically defined in this specification shall be given the meaning that a person skilled in the art can give them based on the disclosure and context.

[0058] "DMTr" in the structural formula of the present invention is 4,4'-dimethoxytriphenylmethyl.

[0059] The minimum and maximum carbon atom content of the hydrocarbon group is indicated by a prefix, e.g., the prefix C a~b Alkyl refers to an alkyl containing "a" to "b" carbon atoms. Thus, for example, C 6~20 Alkyl refers to straight or branched chain alkyl having 6 to 20 carbon atoms.

[0060] Alkyl refers to a straight or branched chain hydrocarbon group in an alkane molecule, such as methyl-CH3, ethyl-CH2CH3, or methylene-CH2-. Alkyl may be part of another group. Such other groups include, for example, C1-C6 alkoxy and C1-C6 alkylamino.

[0061] "Alkenyl" means a straight or branched chain hydrocarbon group having at least two carbon atoms and at least one site of vinyl unsaturation (>C=C<). For example, C a-b Alkenyl means an alkenyl having a to b carbon atoms, and examples include vinyl, propenyl, isopropenyl, 1,3-butadienyl, and the like.

[0062] "Alkynyl" means a linear or branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" is intended to further include hydrocarbon radicals having one triple bond and one double bond. For example, C 2-6 Alkynyl includes ethynyl, propynyl and the like.

[0063] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0064] The term "nucleotide base" includes naturally occurring and non-naturally occurring nucleotide bases. This will be apparent to those skilled in the art, i.e., various nucleotide bases previously considered "non-naturally occurring" have been discovered in nature. Thus, "nucleotide base" includes not only the known purine and pyrimidine heterocycles, but also their heterocyclic analogs and tautomers. Examples of nucleotide bases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-diazaxanthine, 7-diazaguanine, N4,N4-bridged ethylenecytosine, N6,N6-bridged ethylene-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleotide bases described in U.S. Patent No. 5,432,272 to Benner et al. The term "nucleotide base" includes each and every one of these examples, as well as analogs and tautomers thereof. Nucleotide bases of particular importance include adenine, guanine, thymine, cytosine and uracil, which are considered the naturally occurring nucleotide bases relevant to human therapeutic and diagnostic applications.

[0065] As used herein, the terms "protecting group" and "protecting group" refer to reactive groups (e.g., hydroxyl, amino, carboxyl, and mercapto) known in the art for protecting unstable chemical moieties from undesired reaction during synthesis. Protecting groups are typically used selectively and / or orthogonally to protect other reactive sites in a reaction, and are subsequently removed to release the unprotected group for further reaction. In some embodiments, a "substituted" group or substituent comprises a protecting group.

[0066] Illustrative examples of the term "hydroxy protecting group" are optionally substituted trityl, such as 4,4'-dimethoxytrityl (DMTr), 4-monomethoxytrityl (MMT) and trityl, optionally substituted 9-(9-phenyl)xanthene (pixyl), optionally substituted ethoxycarbonyloxy, p-phenylazonyloxycarbonyloxy, tetrahydropyranyl group (thp), 9-fluorenylmethoxycarbonyl (Fmoc), methoxytetrahydropyranyl (mthp), silyloxy, such as trimethylsilyl (TMS), triisopropyl Silyl (TIPS), tert-butyldimethylsilyl (TBDMS), triethylsilyl and phenyldimethylsilyl, benzyloxycarbonyl or substituted benzyloxycarbonyl ethers such as 2-bromobenzyloxycarbonyl, tert-butyl ether, alkyl ethers such as methyl ether, acetals (containing two hydroxyls), acyloxy such as chloroacetyl or fluoroacetyl, isobutyryl, pivaloyl, benzoyl and substituted benzoyl, methoxymethyl (MOM), benzyl ether or substituted benzyl ether.

[0067] The term "lipophilic moiety" refers to cholesterol, C6- 20 Contains alkyl, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl or phenoxazine.

[0068] Lipophilic nucleotide analogs containing a lipophilic moiety can be attached to siRNA via functional groups, such as hydroxyl (e.g., -CO-CH-OH), present in the nucleotide analog or introduced into the siRNA. Functional groups present in the nucleotide analog or introduced into the siRNA include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0069] Conjugates of siRNA and lipophilic nucleotide analogs can be formed, for example, by forming an ether, carboxy, or carbamoyl ester bond between hydroxy and alkyl R-, alkanoyl RCO-, or substituted carbamoyl RNHCO-. Alkyl R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., linear or branched; and saturated or unsaturated). Alkyl R can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, or the like.

[0070] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a polymer of linked nucleosides, each of which may be independently modified or unmodified, comprising an oligonucleotide sequence of approximately 10-50 single-stranded nucleotides or double-stranded nucleotide base pairs. In some embodiments, the oligonucleotide has a nucleobase sequence that is at least partially complementary to a target gene core sequence expressed in a cell. In some embodiments, the oligonucleotide can regulate the expression of the corresponding target gene after being delivered to a cell expressing the gene. Target gene expression can be controlled in vitro or in vivo. "Nucleic acid" and "nucleic acid molecule" include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0071] As used herein, "siRNA" refers to an oligonucleotide molecule comprising RNA or RNAs (eg, chemically modified RNA) that can reduce or suppress messenger RNA (mRNA) translation in a sequence-specific manner.

[0072] siRNA can act via an RNA interference mechanism (e.g., by inducing mRNA degradation by interacting with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) in mammalian cells), or any other mechanism or pathway. The term "siRNA drug" as used in the present invention is believed to act primarily via the RNA interference mechanism, but the siRNA drug is not limited to any particular pathway or mechanism of action. siRNA drugs include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The siRNA drug of the present invention has an oligonucleotide strand complementary to at least a portion of the target mRNA. In some embodiments, the siRNA drug of the present invention is double-stranded, consisting of an antisense strand and a sense strand at least partially complementary to the antisense strand.

[0073] The terms "silencing," "reduction," "suppression," "downregulation," or "knockdown" mean that when a given gene is expressed, administration of an siRNA drug molecule described in the present invention directly into a cell, tissue, organ, or animal body results in a decrease or reduction in the expression level of the gene compared to administration into a cell, tissue, organ, or animal body that has not been so treated.

[0074] The term "sequence" or "nucleotide sequence" refers to the order or ordering of nucleic acid bases or nucleotides, presented in alphabetical order using standard nucleotide nomenclature, usually from the 5' to the 3' end.

[0075] The term "complementary" is used to describe the relationship between a first nucleotide sequence (e.g., the sense strand of an siRNA drug or a target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or the antisense strand of a double-stranded siRNA drug). It refers to the ability of an oligonucleotide or oligonucleotides comprising a first nucleotide sequence to hybridize with an oligonucleotide or oligonucleotides comprising a second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions) to form base pairs and a double-stranded or double-helical structure. Complementary sequences may include Watson-Crick base pairs or non-Watson-Crick base pairs and may contain natural or modified nucleotides or nucleotide analogs, to the extent that they can at least meet the hybridization requirements described above. For example, for purposes of determining identity or complementarity, the monomers a and Af are complementary to U (or T) and correspond to A.

[0076] The term "sense strand" refers to the strand in an RNA molecule that has a nucleotide sequence that encodes the amino acid information of a protein, and is also called the coding strand, sense strand, or positive strand, and the other nucleotide sequence that is complementary to it is the antisense strand.

[0077] The term "antisense strand" refers to a sequence that is substantially reverse complementary or substantially reverse complementary to a nucleotide sequence of the same length as the antisense strand in the mRNA expressed by the target gene.

[0078] The compounds and compositions of the present invention may have certain atoms (e.g., N, O, or S atoms) that are in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Thus, as used herein, the structures described herein contemplate that certain functional groups, such as OH, SH, or NH, can be protonated or deprotonated. The disclosure of the present invention is intended to cover the above compounds and compositions, regardless of their protonation state based on the pH of their environment, as would be readily apparent to one skilled in the art.

[0079] The term "stereoisomer" includes enantiomers and diastereomers or mixtures thereof.

[0080] The term "pharmaceutically acceptable" refers to a carrier, carrier, diluent, adjuvant, and / or formed salt that is usually chemically or physically compatible with the other ingredients that make up a drug dosage form and physiologically compatible with the receptor.

[0081] The terms "salt" and "pharmaceutically acceptable salt" refer to acidic and / or basic salts formed from the above-mentioned compound or its stereoisomer with an inorganic and / or organic acid and a base, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final isolation and purification of the compound. They may also be obtained by mixing the above-mentioned compound or its stereoisomer with a certain number of acids or bases, appropriately (e.g., in equal amounts). These salts can be produced by forming a precipitate in a solution and collecting it by filtration, by recovering it after evaporating the solvent, or by reacting it in an aqueous medium and then lyophilizing it.

[0082] The above content of the present invention will be described in more detail below through specific embodiments of the examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Any technology realized based on the above content of the present invention belongs to the scope of the present invention. [Brief explanation of the drawings]

[0083] [Figure 1] 1 is a HNMR spectrum of compound a2. [Figure 2] 1 is a HNMR spectrum of compound a3. [Figure 3] 1 is a HNMR spectrum of compound a4. [Figure 4] 1 is a HNMR spectrum of compound a5. [Figure 5] 1 is a HNMR spectrum of compound a6. [Figure 6] 1 is a HNMR spectrum of compound a7. [Figure 7] 1 is a HNMR spectrum of compound a8. [Figure 8] 1 is a PNMR spectrum of compound a8. [Figure 9] FIG. 1 shows an in vitro activity test of the siRNA synthesized in Example 2 of the present invention. [Figure 10] FIG. 1 shows the expression level of SOD1 mRNA on DAY7 in rat brain tissues using siRNA synthesized in Example 2 of the present invention. [Figure 11] FIG. 1 shows the expression levels of SOD1 mRNA on day 14 in rat brain tissues using siRNA synthesized in Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0084] Based on the above content of the present invention, it is apparent that various other modifications, substitutions or alterations can be made in accordance with common technical knowledge and conventional means in the art without departing from the above basic technical idea of ​​the present invention.

[0085] The above content of the present invention will be described in more detail below through specific embodiments of the examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Any technology realized based on the above content of the present invention belongs to the scope of the present invention.

[0086] The raw materials and equipment used in the present invention are all known products and can be obtained by purchasing them commercially.

[0087] DMP: Dess-Martin oxidant; DIBAL-H: diisobutylaluminum hydride; DMTrCL: 4,4'-bismethoxytrityl chloride; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; DCI: 4,5-dicyanoimidazole; CEP: bis(diisopropylamino)(2-cyanoethoxy)phosphine.

[0088] Unless otherwise specified in the examples, room temperature (20°C to 30°C) is the most suitable reaction temperature. M is the number of moles per liter.

[0089] Example 1: Synthesis of compound a8 1) Synthesis of compound a2 [ka]

[0090] A 2000 mL round-bottom flask purged with nitrogen was charged with DMP (174.50 g, 411.55 mmol) and NaHCO (144.00 g, 1714.29 mmol), followed by dichloromethane (400 mL). The resulting solution was stirred, and a dichloromethane solution (500 mL) of 1-heptadecanol (88.00 g, 343.08 mmol) was added dropwise. After the addition, the mixture was stirred at room temperature for 16 hours.

[0091] After 16 h, analysis by TLC indicated complete reaction of a1. The reaction mixture was cooled in an ice-salt bath, stirred for 10 min, and then filtered through a bed of diatomaceous earth into a clean 2000 mL round-bottom flask. The filtration was repeated 3-4 times, and the solution was concentrated to give a crude white powder a2 (110.00 g, 79.0%).

[0092] 2) Synthesis of compound a3 [ka]

[0093] Ethoxyformylmethylenetriphenylphosphine (155.7 g, 446.90 mmol) was added to a 2000 mL round-bottom flask, followed by dichloromethane (500 mL). The resulting solution was stirred, and a dichloromethane solution (500 mL) of a2 (110 g, 79%, 343.70 mmol) was added dropwise. After the dropwise addition, the mixture was stirred at room temperature for 16 hours.

[0094] After 16 hours, analysis by TLC showed that the starting materials had reacted completely. The reaction mixture was concentrated. The product was separated by column chromatography, and the product-containing fraction was collected using an eluent (PE:DCM = 8:1). The product-containing fraction was concentrated to give a white powder a3 (64 g, 80.0%).

[0095] 3) Synthesis of compound a4 [ka]

[0096] A 2000 mL three-necked round-bottom flask equipped with a thermometer and blanketed with nitrogen gas was charged with a3 (64 g, 80%, 158.02 mmol) and dichloromethane (700 mL). The solution was cooled to -5 °C in an ice-salt bath, and diisobutylaluminum hydride (414.00 mL, 1 M in hexanes) was added dropwise, maintaining the temperature below 0 °C. After the addition, the mixture was stirred at -5 °C for 1 hour.

[0097] After 1 hour, analysis by TLC indicated that a3 had reacted completely. At -5°C, the nitrogen gas plug was removed, an exhaust port was connected, and saturated potassium sodium tartrate solution (50 mL) was slowly added dropwise to the solution, followed by stirring at -5°C for 1 hour. The solution was filtered through a diatomaceous earth bed and washed with purified water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated to give a white powder, which was purified by column chromatography. The product-containing fraction was collected using an eluent (PE:DCM = 2:1) and concentrated to give a white powder a4 (42 g, 85%).

[0098] 4) Synthesis of compound a5 [ka]

[0099] Tetraisopropyl titanate (4.374 g, 15.40 mmol) was added to a 250 mL three-necked round-bottom flask equipped with a thermometer and blanketed with nitrogen gas, followed by the addition of dichloromethane (30 mL). The reaction mixture was cooled to -25 °C by circulating the reactor at low temperature. Then, D-(-)-diethyl tartrate (4.23 g, 20.53 mmol) was added dropwise and stirred for 20 minutes. A solution of a4 (3.62 g, 85%, 10.9 mmol) in dichloromethane (30 mL) was then added dropwise and stirred for an additional 20 minutes. Then, tert-butyl hydroperoxide (6.87 mL, 5.6 M in decane) was added dropwise. After the addition, the solution was stored in a refrigerator for 24 hours.

[0100] After 24 hours, analysis by TLC indicated that a4 had completely reacted. Dimethyl sulfide was added dropwise to the solution at -9°C, stirred for 30 minutes, and filtered through a diatomaceous earth bed. Saturated sodium sulfate solution was then added, and the mixture was washed three times with ether. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography. The fraction containing a5 was collected using an eluent (PE:DCM = 1:4) and concentrated to give a5 (1.42 g, 98%) as a clear oil.

[0101] 5) Synthesis of compound a6 [ka]

[0102] A 100 mL round-bottom flask was charged with nitrogen gas and charged with a5 (1.64 g, 5.50 mmol), followed by pyridine (30 mL). The resulting solution was stirred, and 4,4'-bismethoxytrityl chloride (2.23 g, 6.59 mmol) was slowly added. The mixture was stirred at room temperature for 16 hours.

[0103] After 16 hours, TLC analysis showed that a5 had reacted completely. The reaction mixture was concentrated and purified by medium-low pressure preparative chromatography using a C18 column to give a6 (2.31 g, 98.0%) as a pale yellow oil.

[0104] 6) Synthesis of compound a7 [ka]

[0105] A 50 mL microwave reaction tube was charged with a6 (1.5 g, 2.49 mmol), uracil (0.42 g, 3.75 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.6 g, 3.94 mmol), and anhydrous ethanol (24 mL) was added, followed by stirring at 110 °C for 8 h.

[0106] After 8 hours, LCMS showed that 65% of the starting material had been converted to the product. The reaction mixture was diluted with dichloromethane, washed with purified water and saturated brine, and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography. The product-containing fraction was collected with an eluent (DCM:EA=1:1, 1% TEA) and concentrated to give 1.27 g of a white foamy solid. SFC chiral separation afforded compound a7 (480 mg, 97%) as a white foamy solid.

[0107] 7) Synthesis of compound a8 [ka]

[0108] A 25 mL round-bottom flask blanketed with nitrogen gas was charged with a7 (0.37 g, 0.52 mmol) and dichloromethane (6 mL). The resulting solution was stirred, and 4,5-dicyanoimidazole (0.05 g, 0.42 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.3 g, 0.99 mmol) were added sequentially. The mixture was then stirred at room temperature for 30 minutes.

[0109] After 30 minutes, LCMS detection confirmed that 90% of the starting material was converted to the product. The solution was concentrated in vacuo and purified by medium-low pressure preparative chromatography using a C18 column to give compound a8 (320 mg, 98%) as a white foam solid.

[0110] Example 2: siRNA synthesis For the sense and antisense strands of the siRNA sequences of the present invention, deoxynucleoside CPG was used as a solid support, and the sense strand was synthesized using the solid support, while the antisense strand was synthesized using a general-purpose CPG.

[0111] The sequence was synthesized on a 0.2 μmol scale using a 48-channel synthesizer, with the concentrations of phosphoramidite monomers and the compound a8 monomer of the present invention at 0.05 M, and 0.3 M BTT as an activator.

[0112] The sequences were cleaved and deprotected in 1.5 ml tubes, with the 2-position protecting group removed using AMA in the first step and triethylamine trifluoride in the second step. For sequences containing all modifications at the 2-position, ammonolysis with aqueous ammonia was required. The cleaved and deprotected sequences were precipitated using a mixture of acetone and ethanol (80:20) and dissolved in RNase-free water. Each sequence was analyzed by LC-MS for sequence accuracy, quantified by spectrophotometry, and confirmed for purity by HPLC.

[0113] After HPLC purification, freeze-drying, and quality inspection, the salt was precipitated with sodium acetate alcohol and desalted using a 3KD ultrafiltration tube. After desalting, the sense and antisense strands were quantified and determined using a spectrophotometer, and then mixed in a 1:1 ratio and annealed to form siRNA duplexes.

[0114] The present inventors synthesized the following siRNA duplexes. [Table 1]

[0115] To illustrate the beneficial effects of the present invention, the present invention provides the following test examples.

[0116] Test Example 1: In vitro activity detection cell culture 1) After receiving primary mouse hepatocytes (PMH), they were stained with 0.04% trypan blue and the cell viability and number were analyzed. 2) Based on the viable cell density and the number of plates required, the required volume of cell suspension and medium were calculated and mixed uniformly to prepare a cell suspension. Using a 24-well plate, 1 x 10 cells were inoculated with 500 μL of inoculation medium (DMEM medium supplemented with 5% fetal bovine serum, 1% penicillin-streptomycin solution, 5 μg / mL dexamethasone, and 0.1% ITSA (insulin-transferrin-selenium-sodium pyruvate)). 5 Cells were seeded onto well plates coated with rat tail collagen. After incubation at 37°C for 3 hours, cell adhesion was observed under a microscope. If cells were not yet attached, the incubation time could be extended appropriately. 3) After the cells adhered, the culture was replaced with 450 μL of complete medium (Hepato ZYME-SFM medium supplemented with 5 μg / mL dexamethasone and 0.1% ITSA (insulin-transferrin-selenium-sodium pyruvate)).

[0117] Cellular free uptake 1) Double-stranded siRNA was diluted to a predetermined concentration in complete cell culture medium. 2) The double-stranded siRNA medium prepared above was added to a 24-well plate for culturing primary hepatocytes (50 μL per well, final siRNA concentrations in the medium were 10 nM, 100 nM, and 500 nM), and the cells were placed in a carbon dioxide incubator and cultured for the specified time.

[0118] RNA extraction RNA was extracted from the cultured cells using the Trizol method, and finally, 20 μL of nuclease-free water was added to each sample to dissolve the RNA.

[0119] Real-time quantitative PCR ABI PowerUp TMqPCR was performed using the SYBR Green Premix Kit (Cat: A25742) according to the manufacturer's instructions. ΔΔCt measurements were performed using the ABI QuantStudio. TM Real-time fluorescent PCR was performed using a real-time fluorescent PCR system with 6. Three independent transfection tests were performed for each duplex, and measurements were performed in triplicate for each transfection. The results of Hitgen-C16 assay of the siRNA duplex of the present invention are shown in Figure 9. The experimental data demonstrate that the siRNA synthesized using the nucleotide analogues of the present invention has a good effect of reducing the expression of SOD1 mRNA.

[0120] Test Example 2: siRNA stability test Brain homogenate stability test 1) 190 μL of brain homogenate was mixed uniformly with 10 μL of 20 μM siRNA duplex and incubated at 37°C for 24 hours. Three groups were repeated to prepare 24-hour samples. 2) Another 190 μL of brain homogenate was taken, and three groups were repeated. The homogenate was incubated with the step for 24 hours, after which 10 μL of 20 μM siRNA duplex was added to prepare the 0-hour sample. 3) 150 μL of each sample from steps 1 and 2 was taken, and the diluent and internal standard were added. The mixture was mixed uniformly by vortexing and allowed to stand for 30 minutes. 4) SPE trabecular activation, equilibration, loading, elution, loading, nitrogen blow. 5) The system was sampled using HFIP+DIEA+EDTA ion. 6) Based on the analysis results, the amount of siRNA sample remaining after 24 hours of incubation was calculated.

[0121] Cerebrospinal fluid stability test 1) 38 μL of cerebrospinal fluid was mixed uniformly with 2 μL of 20 μM siRNA duplex and incubated at 37°C for 24 hours. Three groups were repeated to prepare 24-hour samples. 2) Another 38 μL of cerebrospinal fluid was taken, and the three groups were repeated. The samples were incubated with the step for 24 hours, after which 2 μL of 20 μM siRNA duplex was added to serve as the 0-hour sample. 3) 30 μL of each sample from steps 1 and 2 was taken, and the diluent and internal standard were added, mixed uniformly by vortexing, and phenol chloroform was added and vortexed. 4) The sample in step 3 was centrifuged and the supernatant was taken for measurement. 5) The system was sampled using HFIP+DIEA+EDTA ion. 6) Based on the analysis results, the amount of siRNA sample remaining after 24 hours of incubation was calculated.

[0122] The siRNA test data of the present invention is as follows: Table 1: Cerebrospinal fluid stability test data [Table 2]

[0123] Table 2: Brain homogenate stability test data [Table 3]

[0124] As can be seen from the test data, both the sense strand and the antisense strand of the siRNA synthesized using the nucleotide analogues of the present invention have good stability.

[0125] Test Example 3: In vivo activity test of siRNA Drug efficacy evaluation experiment in the brain of SD rats 1) Model: Male SD rat (D000017), 6 weeks old, approximately 250 g, provided by Shusui Pharmaceutical Co., Ltd. 2) Grouping: After the rats were adapted for 7 days, they were randomly divided into groups according to their body weight, and the day of grouping was defined as D0. 3) Administration: Administration began on Day 1. Administration volume: 30 μL / animal, administration route: lumbar intraspinal injection, administration frequency: once. (Administration was performed in a safety cabinet. Before administration, the related equipment was sprayed and wiped with 75% alcohol, then disinfected with ultraviolet light for 30 minutes. After administration, the related equipment was disinfected with RNase Away. TM Spray and wipe with a cleaning agent, then soak the surgical instruments in 75% alcohol for 30 minutes, followed by RNase Away. TM The rats were immersed in a stain removal reagent and wiped. During the administration process, care was taken to maintain an RNase-free environment as much as possible. Before and after each administration, the rats, related equipment, and the body parts of the experimenter who entered the safety cabinet were cleaned with RNase-free detergent. TM The rats were sprayed and wiped appropriately with the soiling removal reagent, weighed before administration, and observed before and after administration. 4) Test endpoint a) Seven days after administration, half of the rats in each group were euthanized, and tissues from the cortex, cerebellum, brainstem, hippocampus, striatum, and spinal cord (divided into cervical, thoracic, and lumbar vertebrae) were isolated. All of the above tissues were divided into two aliquots, rapidly cooled in liquid nitrogen, and then frozen at -80°C. b) 14 days after administration, the remaining rats in each group were euthanized, and tissues from the cortex, cerebellum, brainstem, hippocampus, striatum, and spinal cord (divided into cervical, thoracic, and lumbar vertebrae) were isolated. All of the above tissues were divided into two aliquots, rapidly cooled in liquid nitrogen, and then frozen at -80°C. 5) RNA extraction: RNA was extracted from the isolated tissues using the Trizol method, and finally, 200 μL of nuclease-free water was added to each sample to dissolve the RNA. 6) Real-time fluorescent quantitative PCR: ABI PowerUp TM SYBR TM qPCR was performed using the ABI QuantStudio Green Premix Kit (Cat: A25742) according to the manufacturer's instructions. ΔΔCt measurements were performed using the ABI QuantStudio. TM Real-time fluorescent PCR was performed using a real-time fluorescent PCR system.

[0126] The test results of the siRNA duplex Hitgen-C16 of the present invention are shown in Figures 10 and 11. As can be seen from the experimental data, the siRNA synthesized with the nucleotide analogue of the present invention can enter brain tissue and has an excellent effect of reducing SOD1 mRNA expression.

[0127] In summary, the nucleotide analogues provided by the present invention enhance the delivery of nucleic acid drugs (especially siRNA) in vivo, have particularly good delivery effects to brain tissue, and produce corresponding biological functions.

Claims

1. A compound of formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 Formula I wherein Y is a hydroxyl protecting group; R １ is C １ ~ ６ Alkyl, halogen-substituted C １ ~ ６ alkyl, R ２ is C １～６ Alkyl, halogen-substituted C １ ~ ６ alkyl, L is a lipophilic group, Base is a nucleotide base.

2. R １ is selected from isopropyl, R ２ is selected from isopropyl, L is a cholesterol group, C ６ ~ ２０ 2. The compound according to claim 1, characterized in that it is selected from alkyl.

3. L is, 【Chemistry 2】 2. The compound according to claim 1, characterized in that it is selected from:

4. Base is, 【Transformation 3】 2. The compound according to claim 1, characterized in that it is selected from:

5. 2. The compound according to claim 1, wherein Y is selected from 4,4'-dimethoxytriphenylmethyl, 4-methoxytrityl, trityl, trimethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, triethylsilyl, phenyldimethylsilyl, benzyloxycarbonyl or 2-bromo-benzyloxycarbonyl.

6. The compounds of formula I are specifically 【Chemistry 4】 6. The compound according to claim 1, wherein

7. A method for synthesizing a nucleotide analogue, comprising the following steps 1 to 7: 【Transformation 5】 Step 1: Compound A1 is dissolved in dichloromethane and treated with a Dess-Martin oxidant, NaHCO ３ The mixture is reacted at room temperature for 5 to 16 hours to obtain compound A2. Step 2: Ethoxyformylmethylenetriphenylphosphine is dissolved in dichloromethane, and the dichloromethane solution of compound A2 is added with stirring. The reaction is allowed to proceed at room temperature for 5 to 16 hours to obtain compound A3. Step 3: Compound A3 is dissolved in dichloromethane, diisobutylaluminum hydride is added, and the mixture is reacted at −10 to 5° C. for 1 to 3 hours to obtain Compound A4. Step 4: Tetraisopropyl titanate is dissolved in dichloromethane, and D-(-)-diethyl tartrate is added at −30 to 5° C. and stirred. Then, a solution of compound A4 in dichloromethane and tert-butyl hydroperoxide are added, and the mixture is reacted for 10 to 24 hours to obtain compound A5. Step 5: Compound A5 is dissolved in pyridine, a hydroxyl protecting group reagent is slowly added, and the reaction is allowed to proceed at room temperature with stirring for 5 to 16 hours to obtain Compound A6. Step 6: In a microwave reaction tube, compound A6, a nucleotide base reagent, and 1,8-diazabicyclo[5.4.0]undec-7-ene are dissolved in a solvent, and the mixture is reacted at 90 to 120°C for 5 to 12 hours with stirring to obtain compound A7. Step 7: Compound A7 is dissolved in dichloromethane, and 4,5-dicyanoimidazole and bis(diisopropylamino)(2-cyanoethoxy)phosphine are added. The reaction is carried out at 20 to 40°C for 20 minutes to 3 hours to obtain Compound A8.

8. In the step 1, compound A1, a Dess-Martin oxidizing agent, and NAHCO ３ The molar ratio of In the step 2, the molar ratio of compound A2 to ethoxyformylmethylenetriphenylphosphine is 1:1.0-1.5; In the step 3, the molar ratio of compound A3 to diisobutylaluminum hydride is 1:2.0-3.0; In the step 4, the molar ratio of compound A4, tetraisopropyl titanate, and diethyl D-(-)-tartrate is 1:1.0-1.5:1.0-1.5; In the step 5, the molar ratio of compound A5 to the hydroxyl protecting group reagent is 1:1.0-1.5; In the step 6, the molar ratio of compound A6, nucleotide base reagent, and 1,8-diazabicyclo[5.4.0]undec-7-ene is 1:1.0-2.0:1.0-2.0; 8. The method of claim 7, wherein in step 7, the molar ratio of compound A7, 4,5-dicyanoimidazole, and bis(diisopropylamino)(2-cyanoethoxy)phosphine is 1:0.5-2.0:1.0-2.

5.

9. In step 1, the molar equivalent of compound A1 is 1, the molar equivalents of the Dess-Martin oxidizing agent are 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5, and NAHCO ３ The target molar equivalents are 2, 3, 4, 5, and 6, In step 2, the molar equivalent of compound A2 is 1, and the molar equivalents of ethoxyformylmethylenetriphenylphosphine are 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5; In the step 3, the molar equivalent of compound A3 is 1, and the molar equivalents of diisobutylaluminum hydride are 2.1, 2.2, 2.4, 2.6, 2.7, 2.8, and 3.0; In step 4, the molar equivalent of compound A4 is 1, the molar equivalents of tetraisopropyl titanate are 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5, and the molar equivalents of D-(-)-diethyl tartrate are 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5; In step 5, the molar equivalent of compound A5 is 1, and the molar equivalents of the hydroxyl protecting group reagent are 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5; In step 6, the molar equivalent of compound A6 is 1, the molar equivalents of the nucleotide base reagent are 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, and 1.8, and the molar equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene are 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, and 1.8; 8. The method of claim 7, wherein in step 7, the molar equivalent of compound A7 is 1, the molar equivalents of 4,5-dicyanoimidazole are 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0, and the molar equivalents of bis(diisopropylamino)(2-cyanoethoxy)phosphine are 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, and 2.

2.

10. In step 5, the hydroxyl protecting group reagent is 4,4'-bismethoxytrityl chloride; 8. The synthesis method according to claim 7, wherein in step 6, the nucleotide base reagent is uracil.

11. The reaction time of step 1 is 5 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours. The reaction time of step 2 is 5 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours; The reaction time of step 3 is 1 hour, 2 hours, or 3 hours. The reaction temperatures in step 3 are −10° C., −5° C., 0° C., and 5° C. The reaction time of step 4 is 10 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours; The reaction temperatures in step 4 are −30° C., −25° C., −10° C., 0° C., and 5° C. The reaction time of step 5 is 5 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours; The reaction time of step 6 is 5 hours, 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours; The reaction temperatures in step 4 are 90°C, 100°C, 110°C, and 120°C; The reaction time of step 7 is 20 min, 0.5 h, 1 h, or 2 h. The synthesis method according to claim 7, wherein the reaction temperature in step 7 is 20°C, 25°C, 30°C, or 35°C.

12. An siRNA comprising a sense strand and an antisense strand, The sense strand and the antisense strand each contain 15 to 45 modified or unmodified nucleotides, and the sense strand and the antisense strand are partially complementary to form a double-stranded region, wherein the sense strand has a structure represented by Formula V 【Transformation 6】 Formula V wherein X is selected from O or S; L is a lipophilic group, Base is a nucleotide base. At least one nucleotide having a structure represented by 【Transformation 7】 The siRNA is covalently linked to the remainder of the siRNA at position

13. The siRNA according to claim 12, wherein the structure represented by formula V is represented by formula Va. 【Transformation 8】 Formula Va (In the formula, X, L, and Base have the same meanings as in claim 12.)

14. L is a cholesterol group, C ６ ~ ２０ 14. The siRNA according to claim 12 or 13, characterized in that the aryl group is selected from alkyl.

15. L is, 【Chemistry 9】 The siRNA according to claim 14, characterized in that it is selected from the following:

16. Base is, 【Chemistry 10】 The siRNA according to claim 12 or 13, characterized in that it is selected from the group consisting of:

17. The structure represented by formula V is specifically 【Chemistry 11】 The siRNA according to any one of claims 12 to 16, characterized in that it is

18. 18. The siRNA of any one of claims 12 to 17, wherein the structure represented by formula V is the second to tenth nucleotide at the 5' end of the sense strand, preferably the structure represented by formula V is the second, third, fourth, fifth, sixth, seventh, or eighth nucleotide at the 5' end of the sense strand, and more preferably the structure represented by formula V is the sixth nucleotide at the 5' end of the strand.

19. The siRNA according to any one of claims 12 to 18, wherein the length of the antisense strand is 19 to 27 nucleotides and the length of the sense strand is 19 to 25 nucleotides, preferably the length of the antisense strand is 19 to 23 nucleotides and the length of the sense strand is 19 to 21 nucleotides, and more preferably the length of the antisense strand is 23 nucleotides and the length of the sense strand is 21 nucleotides.

20. 19. The siRNA of any one of claims 12 to 18, characterized in that the siRNA comprises at least one modified nucleotide, and preferably all nucleotides in the sense and / or antisense strands of the siRNA are modified nucleotides or nucleotide analogues.

21. 21. The siRNA of claim 20, wherein the modified nucleotide or nucleotide analog is selected from a 2'-methoxy nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2',3'-open-ring nucleotide analog, a 2'-fluoroarabino nucleotide, a 2'-methoxyethyl nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a 3'-methoxy nucleotide, a 2'-allyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate ester, a nucleotide containing a 5'-phosphate ester mimic, a diol-modified nucleotide, an abasic nucleotide, a morpholino nucleotide, a locked nucleotide, an unlocked nucleotide, or a glycerol nucleotide.

22. The siRNA of claim 20, wherein the 5'-end and 3'-end of the sense strand are each independently bound to one or two phosphorothioate groups, and / or the 5'-end and 3'-end of the antisense strand are each independently bound to one or two phosphorothioate groups.

23. A pharmaceutical composition comprising the siRNA of any one of claims 12 to 22 and a pharmaceutically acceptable carrier.

24. Use of the siRNA of any one of claims 12 to 22 and / or the pharmaceutical composition of claim 23 in the manufacture of a medicament.

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