Double-stranded sirna and preparation method therefor, pharmaceutical composition and use thereof

Abstract

Disclosed in the present invention is a double-stranded siRNA having a lipophilic modification. The modified siRNA molecule, via intrathecal injection, intradermal injection and other administration modes, achieves an excellent in-vivo distribution performance and delivery efficiency, and has a good tolerability and safety while effectively knocking down the expression of a target gene. Further disclosed in the present invention are a method for preparing the siRNA having a lipophilic modification, a pharmaceutical composition, and the use thereof in the preparation of a drug for treating a TNF-α-mediated disease or condition.

Description

Double-stranded siRNA, preparation method thereof, pharmaceutical composition and use thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular relates to a double-stranded siRNA, a preparation method thereof, a pharmaceutical composition and use thereof in preparing a medicament for treating a TNF-alpha mediated disease or disorder. BACKGROUND

[0002] Among the marketed RNAi drugs, lipid nanoparticles and N-acetylgalactosamine modification are used to target liver tissue. Due to the hydrophilic and negatively charged properties of the siRNA molecule, it is difficult for the cell to uptake. If the RNAi technology is to be applied to other tissues (such as the central nervous system, the eye, the lung), new technologies need to be developed to improve the delivery and distribution performance of siRNA in extrahepatic tissues.

[0003] Currently, different types of siRNA carriers have been reported, including polymers and polypeptides. For example, US patent No. US9061995 B2 describes a peptide conjugate for delivering nucleic acids (such as siRNA), which contains a cell penetrating peptide part, aiming to improve the intracellular delivery efficiency of nucleic acid drugs. Although these peptides can show good results in laboratory conditions, there are still many difficulties in the process of converting them into clinical applications. The cell penetrating peptide has poor stability in vivo and is easily degraded by enzymes, resulting in reduced drug efficacy. In addition, large-scale synthesis and purification are complex and costly. With the progress of technology, polymeric materials are also being developed. PEI is a cationic polymer that can form nanoscale complexes by binding to negatively charged nucleic acids. These complexes can enter cells and release nucleic acids through the endocytosis pathway. PLGA is a biodegradable copolymer with good biocompatibility and controllable degradation rate. It can encapsulate small nucleic acid drugs and deliver them to target tissues or cells through passive or active targeting mechanisms. However, some polymers may cause immune reactions or cytotoxicity, leading to adverse side effects.

[0004] More extrahepatic delivery of drugs is under development. In pulmonary targeting, ALN-HPN-19 developed by Alnylam Pharmaceuticals is used to treat pulmonary fibrosis, which delivers small nucleic acid drugs to the lungs by inhalation. In ocular targeting, RGX-314 developed by Regeneron Pharmaceuticals is used to treat wet age-related macular degeneration, which delivers small nucleic acid drugs to the eye by eye drops, which may also be used to treat glaucoma, diabetic retinopathy, etc. in the future. In tumor targeting, AMG-160 developed by Arrowhead Pharmaceuticals is used to treat solid tumors, which uses the characteristics of the tumor microenvironment (such as pH, redox state, etc.) to design a responsive delivery system to specifically deliver small nucleic acid drugs to tumor tissue. In cardiac targeting, CARDIO-RNA-001 developed by Cardior Pharmaceuticals is used to treat heart failure. In kidney targeting, nanoparticles are used to encapsulate small nucleic acid drugs, which are delivered to kidney tissue by intravenous injection for the treatment of nephrotic syndrome and chronic kidney disease, such as SLN201 developed by Silence Therapeutics for the treatment of IgA nephropathy.

[0005] Delivery of nucleic acid drugs to the nervous system is a very challenging task because the presence of the blood-brain barrier makes it difficult for many conventional delivery methods to effectively deliver drugs, but nucleic acid drugs have shown great potential in treating nervous system diseases, and many companies are increasing research and development. VY-HTT01 of Voyager Therapeutics uses AAV (adeno-associated virus) vector to deliver siRNA, targeting the huntingtin gene (HTT), reducing the expression of mutant HTT protein, which is in clinical trials, and the preliminary results show safety and potential efficacy. Biogen's Spinraza delivers antisense oligonucleotides (ASO) through intrathecal injection to change the splicing of the SMN2 gene and increase the production of functional SMN protein, which has been approved by the FDA and is widely used in the treatment of SMA patients. ACU-193 of Acuitas Therapeutics uses lipid nanoparticles (LNP) to deliver siRNA, targeting the beta-amyloid precursor gene (APP), reducing the production of beta-amyloid, which is in the early stage of research and development. AMT-130 of UniQure uses AAV vector to deliver miRNA, targeting the synuclein gene (SNCA), reducing the expression of alpha-synuclein, which is in clinical trials to evaluate its safety and effectiveness.

[0006] The dependence on delivery vectors is very high for effective delivery of small nucleic acids, and the development of many small nucleic acid drugs needs to be based on the development of suitable delivery vectors, which increases the limitations of small nucleic acid drugs. Therefore, the development of an effective carrier-free siRNA extrahepatic delivery technology is the current focus of siRNA drug research and development. SUMMARY

[0007] Improving the lipophilicity of drugs is an effective strategy to improve the cellular uptake of siRNA drugs and achieve extrahepatic targeting. In order to improve the carrier-free delivery and distribution performance of siRNA in extrahepatic tissues, the present application enhances the lipophilicity of siRNA molecules through fine structural design, thereby improving their distribution performance and delivery effect.

[0008] In one aspect of the present application, a nucleoside having a structure as shown in formula (I) is provided, which comprises R1, R3 and lipophilic R2, wherein:

[0009] R1 is selected from a nitrogen-containing base or a nitrogen-containing base analogue;

[0010] R2 is selected from the following groups:

[0011] R3 is selected from 4,4'-dimethoxytrityl (DMTr), 4-monomethoxytrityl (MMTr), 4,4',4"-trimethoxytrityl (TMTr), pivaloyloxymethyl (PIVOM), tert-butyldimethylsilyl (TBDMS), 9-fluorenylmethoxycarbonyl (Fmoc), phenoxyacetyl (PAC), 4-tert-butylphenoxyacetyl (tBPAC), 2-cyanoethoxy-N,N-diisopropylaminophosphino (CEP), 3-levulinyl, acetyl, benzoyl, 2-cyanoethyl, N,N-dibutylaminocarbonyl (DBF), N,N-dimethylaminocarbonyl (DMF), isobutyryl, 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC), triethylammonium (TEA), trifluoroacetyl, triisopropylsilyloxymethyl (TOM), p-isopropylphenoxyacetyl (iPrPAC), O-acetal levulinyl ester (ALE), phenylacetyl, or 1,1',3,3'-tetraisopropyl disiloxanyl.

[0012] In another aspect of the present application, a double-stranded siRNA is provided, which comprises a sense strand and an antisense strand, wherein the nucleotides are specially modified, and the specially modified nucleotides comprise R1 and lipophilic R2, and have a structure as shown in formula (II):

[0013] R1is selected from a nitrogenous base or a nitrogenous base analog;

[0014] R2is selected from the following groups:

[0015] In one or more embodiments, the specially modified nucleotide is at one or more positions in the 5' to 3' direction of the sense strand or the antisense strand.

[0016] In one or more embodiments, the specially modified nucleotide is at one or more of positions 1, 2, 5, 8, 9, 10, 11, 14, 17, 18, or 19 in the 5' to 3' direction of the sense strand or the antisense strand.

[0017] In one or more embodiments, the specially modified nucleotide is at one or more of positions 1, 2, 5, 8, 14, 17, 18, or 19 in the 5' to 3' direction of the sense strand.

[0018] In one or more embodiments, the specially modified nucleotide is at one or more of positions 1, 2, 9, 10, 11, 17, 18, or 19 in the 5' to 3' direction of the antisense strand.

[0019] In one or more embodiments, the specially modified nucleotide is at:

[0020] position 1 in the 5' to 3' direction of the sense strand;

[0021] positions 1 and 19 in the 5' to 3' direction of the sense strand;

[0022] positions 1 and 2 in the 5' to 3' direction of the sense strand;

[0023] positions 1, 2, 18, and 19 in the 5' to 3' direction of the sense strand; or

[0024] positions 1, 5, 8, 14, and 17 in the 5' to 3' direction of the sense strand.

[0025] In one or more embodiments, the specially modified nucleotide is at:

[0026] position 1 in the 5' to 3' direction of the antisense strand;

[0027] positions 1 and 19 in the 5' to 3' direction of the antisense strand;

[0028] positions 1 and 2 in the 5' to 3' direction of the antisense strand;

[0029] positions 1, 2, 18, and 19 of the 5' to 3' direction of the antisense strand; or

[0030] positions 9, 10, 11, 17, and 18 of the 5' to 3' direction of the antisense strand.

[0031] In one or more embodiments, the specially modified nucleotide is located at:

[0032] position 1 of the 5' to 3' direction of the sense strand, and position 1 of the 5' to 3' direction of the antisense strand;

[0033] positions 1 and 19 of the 5' to 3' direction of the sense strand, and positions 1 and 19 of the 5' to 3' direction of the antisense strand;

[0034] positions 1 and 2 of the 5' to 3' direction of the sense strand, and positions 1 and 2 of the 5' to 3' direction of the antisense strand;

[0035] positions 1, 2, 18, and 19 of the 5' to 3' direction of the sense strand, and positions 1, 2, 18, and 19 of the 5' to 3' direction of the antisense strand;

[0036] positions 1, 5, 8, 14, and 17 of the 5' to 3' direction of the sense strand; or

[0037] positions 1, 5, 8, 14, and 17 of the 5' to 3' direction of the sense strand, and positions 9, 10, 11, 17, and 18 of the 5' to 3' direction of the antisense strand.

[0038] In one or more embodiments,

[0039] the sequence of the sense strand is 5'-GCCUGUAGCCCAUGUUGUATT-3' (SEQ ID NO. 1), and the sequence of the antisense strand is 5'-UACAACAUGGGCUACAGGCTT-3' (SEQ ID NO. 2).

[0040] In one or more embodiments, one or more of the non-bridging oxygen atoms in the phosphate groups attached to the 3'-C of the nucleotide monomers of the sense strand or the antisense strand is replaced by a sulfur atom.

[0041] In one or more embodiments, one or more of the non-bridging oxygen atoms in the phosphate groups attached to the 3'-C of the nucleotide monomers of the sense strand is replaced by a sulfur atom.

[0042] In another aspect of the present application, a method for preparing the nucleoside as described in any of the embodiments herein is provided, which comprises synthesis of the lipophilic chain and reacting the lipophilic chain with a dehydrated nucleoside selected from the group consisting of dehydrated adenosine or its analog, dehydrated uracil or its analog, dehydrated cytosine or its analog, dehydrated thymine or its analog, or dehydrated guanine or its analog to obtain the nucleoside as described in any of the embodiments herein.

[0043] In another aspect of the present application, a method for preparing the double-stranded siRNA as described in any of the embodiments herein is provided, which comprises RNA synthesis using the nucleoside as described in any of the embodiments herein as a raw material.

[0044] In one or more embodiments, the method is a phosphoramidite trister method.

[0045] In another aspect of the present application, a pharmaceutical composition comprising a therapeutically effective amount of the double-stranded siRNA as described in any of the embodiments herein, and a pharmaceutically acceptable carrier, solvent or excipient is provided.

[0046] In another aspect of the present application, a method for preparing the pharmaceutical composition as described in any of the embodiments herein is provided, which comprises adding the double-stranded siRNA to a solvent, and a pharmaceutically acceptable carrier or excipient.

[0047] In one or more embodiments, the method comprises adding 0.2-20 mg / ml of the double-stranded siRNA, 0.02-1 mg / ml of calcium chloride dihydrate, 0.01-1 mg / ml of sodium dihydrogen phosphate, 0.01-1 mg / ml of magnesium chloride hexahydrate, 0.01-1 mg / ml of sodium phosphate, and 0.01-1 mg / ml of potassium chloride in terms of mass concentration of the final volume to a partial solvent, dissolving the solution sufficiently, diluting the solution to near the final volume with a solvent which is sterile water for injection, and finally adjusting the pH to 6.5-7.5 using 1 mg / ml of HCl or NaOH solution.

[0048] In one or more embodiments, the method comprises adding 2 mg / ml of the double-stranded siRNA, 0.2 mg / ml of calcium chloride dihydrate, 0.1 mg / ml of sodium dihydrogen phosphate, 0.16 mg / ml of magnesium chloride hexahydrate, 0.03 mg / ml of sodium phosphate, and 0.22 mg / ml of potassium chloride in terms of mass concentration of the final volume to a partial solvent, dissolving the solution sufficiently, diluting the solution to near the final volume with a solvent which is sterile water for injection, and finally adjusting the pH to 6.9-7.1 using 1 mg / ml of HCl or NaOH solution.

[0049] Another aspect of the present invention provides the use of a double-stranded siRNA as described in any embodiment herein in the preparation of a medicament for treating a disease or condition mediated by TNF-α. In one or more embodiments, the disease or condition mediated by TNF-α is selected from inflammation, autoimmune disease, hyperalgesia and cancer. More preferably, the disease or condition mediated by TNF-α is selected from psoriasis, psoriatic arthritis, spondylitis, myelitis, encephalitis, systemic lupus erythematosus, arthritis, inflammatory bowel disease, central nervous system hypersensitivity, peripheral nervous system hypersensitivity, leukemia, lymphoma, melanoma, gastric cancer, liver cancer, gallbladder cancer, kidney cancer, lung cancer, myeloma, reproductive system cancer, breast cancer, pancreatic cancer, bone cancer or head and neck tumors.

[0050] The present invention connects a lipophilic carbon chain to the 2'-C terminus of the pentose in the nucleotide monomer in the siRNA to increase the lipophilicity of the siRNA sequence, thereby improving its carrier-free delivery effect and distribution performance without negatively affecting its efficacy and safety. The siRNA modified with a lipophilic chain can more effectively knock down the expression of the target gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is the lipophilic chain a 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 1A is the lipophilic chain a 1 H-NMR spectrum, Figure 1B is the lipophilic chain a 13 C-NMR spectrum.

[0052] Figure 2 is the lipophilic chain b 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 2A is the lipophilic chain b 1 H-NMR spectrum, Figure 2B is the lipophilic chain b 13 C-NMR spectrum.

[0053] Figure 3 is the lipophilic chain c 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 3A is the lipophilic chain c 1 H-NMR spectrum, Figure 3B is the lipophilic chain c 13 C-NMR spectrum.

[0054] Figure 4 is the lipophilic chain d 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 4A is the lipophilic chain d 1 H-NMR spectrum, Figure 4B is the lipophilic chain d 13 C-NMR spectrum.

[0055] Figure 5 is the lipophilic chain e 1 H-NMR spectrum and13 C-NMR spectrum. Wherein Fig. 5A is the C-NMR spectrum of the lipophilic chain e 1 H-NMR spectrum, Fig. 5B is the H-NMR spectrum of the lipophilic chain e 13 C-NMR spectrum.

[0056] Fig. 6 is a schematic diagram of the synthesis route of a nucleoside A.

[0057] Fig. 7 is a structural diagram of nucleosides A-E. Wherein Fig. 7A is a structural diagram of nucleoside A, Fig. 7B is a structural diagram of nucleoside B, Fig. 7C is a structural diagram of nucleoside C, Fig. 7D is a structural diagram of nucleoside D, and Fig. 7E is a structural diagram of nucleoside E.

[0058] Fig. 8 is the change of the mRNA expression level of the target gene in the mouse brain cells after transfection of siRNA. Wherein Fig. 8A is the change of the mRNA expression level of the target gene in the mouse brain cells after transfection of 1#-6#, Fig. 8B is the change of the mRNA expression level of the target gene in the mouse brain cells after transfection of 5#B-5#E.

[0059] Fig. 9 is the change of the mRNA expression level of the target gene in the mouse organs after intrathecal injection of siRNA. Wherein Fig. 9A is the change of the mRNA expression level of the target gene in the mouse organs after intrathecal injection of 5#, Fig. 9B is the change of the mRNA expression level of the target gene in the mouse organs after intrathecal injection of 5#A, Fig. 9C is the change of the mRNA expression level of the target gene in the mouse organs after intrathecal injection of 5#B, Fig. 9D is the change of the mRNA expression level of the target gene in the mouse organs after intrathecal injection of 5#C, Fig. 9E is the change of the mRNA expression level of the target gene in the mouse organs after intrathecal injection of 5#D, and Fig. 9F is the change of the mRNA expression level of the target gene in the mouse organs after intrathecal injection of 5#E.

[0060] Fig. 10 is the change of the mRNA expression level of the target gene in the skin tissue of the mouse after intradermal injection of siRNA.

[0061] Fig. 11 is the change of the cell viability of HaCat cells after transfection of siRNA.

[0062] Fig. 12 is a fluorescence imaging diagram of the mouse tissues after intrathecal injection of 5#A. Wherein Fig. 12A is a fluorescence imaging diagram of the mouse tissues after intrathecal injection of 5#A, and Fig. 12B is a corresponding color scale. DETAILED DESCRIPTION

[0063] Nucleoside

[0064] The present application provides a nucleoside having a structure as shown in formula (I), comprising R1, R3 and a lipophilic R2, wherein:

[0065] R1 is selected from a nitrogen-containing base or a nitrogen-containing base analogue;

[0066] Preferably, R1 is selected from adenine, uracil, cytosine or guanine. Preferably, R1 is selected from adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated. Preferably, R1 is selected from adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated. Preferably, R1 is selected from adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated. Preferably, R1 is selected from adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated.

[0067] R2 is selected from H, C 12 -C 50 alkyl, or C 12 -C 50 alkyl.

[0068] Preferably, R2 is selected from the following groups:

[0069] R3 is selected from 4,4'-dimethoxytrityl (DMTr), 4-monomethoxytrityl (MMTr), 4,4',4"-trimethoxytrityl (TMTr), pivaloyloxymethyl (PIVOM), tert-butyldimethylsilyl (TBDMS), 9-fluorenylmethoxycarbonyl (Fmoc), phenoxyacetyl (PAC), 4-tert-butylphenoxyacetyl (tBPAC), 2-cyanoethoxy-N,N-diisopropylaminophosphino (CEP), 3- levulinyl, acetyl, benzoyl, 2-cyanoethyl, N,N-dibutylaminocarbonyl (DBF), N,N- dimethylaminocarbonyl (DMF), isobutyryl, 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC), triethylammonium (TEA), trifluoroacetyl, triisopropylsilyloxymethyl (TOM), p- isopropylphenoxyacetyl (iPrPAC), O-acetal levulinyl ester (ALE), phenylacetyl, or 1,1',3,3'-tetraisopropyl disiloxanyl. Preferably, R3 is 4,4'-dimethoxytrityl (DMTr).

[0070] Double-stranded siRNA

[0071] The present application provides a double-stranded siRNA comprising a sense strand and an antisense strand, wherein the nucleotides are specially modified, the specially modified nucleotides comprise R1 and a lipophilic R2, and have a structure as shown in formula (II):

[0072] R1 is selected from a nitrogen-containing base or a nitrogen-containing base analogue; preferably, R1 is selected from an adenine group, a uracil group, a cytosine group or a guanine group.

[0073] Preferably, R1 is selected from an adenine group, a uracil group, a cytosine group or a guanine group comprising one or more modifications selected from thio, halo, hydroxylation, amination, carboxylation or methylation. Preferably, R1 is selected from an adenine methyl group, a uracil methyl group, a cytosine methyl group or a guanine methyl group comprising one or more modifications selected from thio, halo, hydroxylation, amination, carboxylation or methylation. Preferably, R1 is selected from an adenine formyl group, a uracil formyl group, a cytosine formyl group or a guanine formyl group comprising one or more modifications selected from thio, halo, hydroxylation, amination, carboxylation or methylation.

[0074] R2 is selected from H, C 12 -C 50 alkyl, or C 12 -C 50 alkyl having one or more modifications selected from hydroxyl, ether bond, carbonyl, amide bond, carboxyl, ester bond, amino, imino, tertiary amino, halo, thio, thioether bond, sulfhydryl, sulfonic acid group, phosphorus and phosphonic acid group.

[0075] Preferably, R2 is selected from the following groups:

[0076] Embodiment

[0077] Embodiment 1: Synthesis of nucleosides

[0078] Synthetic route of lipophilic chain a

[0079] S1: Starting material 1 (CAS No.: 55182-74-6) was used to obtain intermediate 1 according to the method of the reference (CN110028405A), and intermediate 1 was used as the starting material to obtain intermediate 2 according to the method of the reference (Journal of Chemical Ecology; vol. 27; 4; (2001); p. 791-806).

[0080] S2: Starting material 2 (CAS No.: 112-31-2) and intermediate 2 were used to obtain intermediate 3 according to the method of the reference (European Journal of Organic Chemistry; 9; (2000); p. 1821-1826).

[0081] S3: Lipophilic chain a was obtained from the method according to the reference (Vanka, Kumar; vol. 10; 14; (2020); p. 4586-4592).

[0082] Lipophilic chain a was obtained from the method according to the reference (Vanka, Kumar; vol. 10; 14; (2020); p. 4586-4592). 1 H-NMR spectrum is shown in Figure 1A, 13 C-NMR spectrum is shown in Figure IB.

[0083] Synthetic route of lipophilic chain b

[0084] S1 : 3-Buten-1-ol (200 mg, 2.77 mmol) was mixed with N,N-dimethylformamide (DMF, 2.0 mL, purity > 99%).

[0085] S2: To the mixture of S1, 1-bromononane (355 μL, 1.85 mmol) and sodium hydride (NaH, 112 mg, 2.77 mmol) were added at room temperature and stirring was continued until complete consumption of the starting material (monitored by thin layer chromatography). After completion of stirring, the reaction was quenched with water.

[0086] S3: The reaction mixture after completion of stirring of S2 was extracted with ethyl acetate / hexane (volume ratio 10:1).

[0087] S4: The organic layer after extraction of S3 was washed with water and brine once each and the organic layer was separated after each washing. The washed organic layer was then dried over anhydrous sodium sulfate and after completion of drying, it was filtered. The filtrate was concentrated under vacuum to obtain the crude product.

[0088] S5: The crude product was purified by silica gel column chromatography using hexane / ethyl acetate (volume ratio 20:1) as eluent to obtain a colorless oil (intermediate 1).

[0089] S6: Intermediate 2 was obtained according to the reference (Liebigs Annalen der Chemie; 1 ; (1981); p. 92-98).

[0090] S7: Intermediate 1 and intermediate 2 were reacted according to the reference (Journal of the American Chemical Society; vol. 123; 50; (2001); p. 12504-12509) to obtain lipophilic chain b, the structure of which is shown in formula (IV).

[0091] Lipophilic chain b was obtained from the method according to the reference (Journal of the American Chemical Society; vol. 123; 50; (2001); p. 12504-12509). 1The H-NMR spectrum is shown in Figure 2A, 13 The C-NMR spectrum is shown in Figure 2B.

[0092] Synthesis route of the lipophilic chain c

[0093] S1 : The intermediate 1 was obtained according to the following reference:

[0094] Chemistry and Industry (London, United Kingdom); (1959); p. 1288; Journal of the Chemical Society; (1961); p. 2779, 2786;

[0095] Bioorganic and Medicinal Chemistry; vol. 15; 2; (2007); p. 854-867;

[0096] Journal of Organic Chemistry; vol. 63; 11 ; (1998); p. 3741-3744;

[0097] Journal of Organic Chemistry; vol. 54; 23; (1989); p. 5522-5527.

[0098] S2: The intermediate 2 was obtained by reacting compound (CAS No. 2566-89-4) and compound (CAS No. 2134-29-4) according to the following reference:

[0099] Angewandte Chemie - International Edition; vol. 54; 13; (2015); p. 4023-4027;

[0100] Angew. Chem.; vol. 127; 13; (2015); p. 5.

[0101] S3: The intermediate 3 was obtained by reacting intermediate 1 and intermediate 2 according to the reference (WO2019010414A1).

[0102] S4: The intermediate 4 was obtained according to the following reference:

[0103] Liebigs Annalen der Chemie; 11 ; (1992); p. 1113-1124

[0104] Monatshefte fuer Chemie; vol. 112; (1981); p. 825-840

[0105] S5: According to the reference (Journal of Medicinal Chemistry; vol. 40; 22; (1997); p. 3626-3634), reaction with intermediate 3 and intermediate 4 was carried out to obtain the lipophilic chain c, whose structure is shown in formula (V).

[0106] obtained from the lipophilic chain c 1 The H-NMR spectrum is shown in Figure 3A, 13 The C-NMR spectrum is shown in Figure 3B.

[0107] Synthesis route of the lipophilic chain d

[0108] S1 : According to the reference (Tetrahedron Letters; vol. 48; 42; (2007); p. 7456-7459), reaction with starting material 1 (CAS number: 542-78-9) and starting material 2 (CAS number: 1120-16-7) was carried out to obtain intermediate 1.

[0109] S2: According to the reference (Molecules; vol. 27; 9; (2022)), reaction with intermediate 1 was carried out to obtain the lipophilic chain d, whose structure is shown in formula (VI).

[0110] obtained from the lipophilic chain d 1 The H-NMR spectrum is shown in Figure 4A, 13 The C-NMR spectrum is shown in Figure 4B.

[0111] Synthesis route of the lipophilic chain e

[0112] Reaction with starting material (CAS number 156-60-5) was carried out according to the reference (Journal of Organic Chemistry; vol. 34; (1969); p. 1130-1133) to obtain the lipophilic chain e, whose structure is shown in formula (VII).

[0113] obtained from the lipophilic chain e 1 The H-NMR spectrum is shown in Figure 5A, 13 The C-NMR spectrum is shown in Figure 5B.

[0114] Synthesis of nucleosides containing special modifications

[0115] Synthesis of nucleoside A

[0116] Nucleoside A can be synthesized from lipophilic chain a and an anhydro nucleoside. The anhydro nucleoside is selected from an anhydro adenosine, an anhydro uracil, an anhydro cytosine, an anhydro thymine, an anhydro guanine, or an analog thereof.

[0117] For example, a nucleoside A, i.e., a uridine containing lipophilic chain a, is synthesized from lipophilic chain a and an anhydro uridine, as shown in the flow chart of Figure 6:

[0118] S1 : To 2 ml of tetrahydrofuran (THF) is added 0.06-0.1 M of compound 1 (2,2'-anhydro uridine, CAS No. 3736-77-4), and 100 μl of t-butyldiphenylsilyl chloride (TBDPSCl, CAS No. 58479-61-1) is slowly added dropwise while stirring to mix well. To the reaction system is slowly added dropwise 100 μl of a basic catalyst, triethylamine, and stirring is continued to mix well, and the mixture is left overnight at room temperature to form a solution containing compound 2.

[0119] S2: To the solution containing compound 2 in S1 is slowly added dropwise 10 μl of 0.1 M trimethylaluminum (CAS No. 75-24-1) while stirring to mix well, and a solution containing compound 3 is formed.

[0120] S3: To the solution containing compound 3 in S2 is slowly added dropwise 10 μl of pyridine hydrofluoride (CAS No. 62778-11-4) while stirring to mix well, and a solution containing compound 4 is formed.

[0121] S4: To the solution containing compound 4 in S3 is slowly added dropwise 100 μl of DMTrCl (CAS No. 40615-36-9) while adding a small amount of pyridine (CAS No. 110-86-1) and DMAP (CAS No. 1122-58-3) as catalysts, and a solution containing compound 5 is formed.

[0122] S5: To the solution containing compound 5 in S4 is added 50 μl of bis(diisopropylamino)(2-cyanoethoxy) phosphine (CAS No. 102691-36-1) while stirring to mix well, and nucleoside A is formed.

[0123] Synthesis of nucleosides B-E

[0124] The synthesis conditions of nucleosides B-E are basically the same as those of nucleoside A, except that the lipophilic chain a in the starting material is replaced by one of lipophilic chains b-e.

[0125] Table 1: Partial synthesis materials of nucleosides A-E

[0126] The partial synthesis materials of nucleosides A-E are shown in Table 1.

[0127] Nucleosides A-E with different special modification types were synthesized by the above method, and the general structure thereof is shown in Figure 7.

[0128] Example 2: siRNA sequence design and synthesis

[0129] siRNA sequence design

[0130] siRNA-T1 (target gene GenBank: MH180383.1), the sequence of the sense strand thereof is 5'-GCCUG UAGCC CAUGU UGUA TT-3', and the sequence of the antisense strand thereof is 5'-UACAA CAUGG GCUAC AGGC TT-3'. One non-bridging oxygen atom of the phosphate group at the a position of the 2nd, 3rd, 4th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 16th, 18th and 19th base nucleotide from the 5' end of the sense strand is replaced by a sulfur atom, i.e., a thio modification is used. In addition, the siRNA-T1 sequence is modified with different types of lipophilic chains. The sequence information is shown in Table 2.

[0131] Table 2: siRNA-T1 modified sequence information

[0132] siRNA sequence synthesis

[0133] The siRNA sequence was synthesized by the phosphoramidite triester method using five commercially available nucleosides AGCUT and the nucleosides A-E with different nitrogen-containing bases obtained in Example 1, i.e., four procedures of "deprotection - coupling - oxidation - capping" were repeated, and the oligonucleotide chain was lengthened by one base after the end of each repetition. The deprotection reagent was a mixture of trichloroethane / dichloromethane, the activating agent was 5-benzylthio tetrazole, the oxidizing agent was iodine solution or (E)-N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithiazol-5-yl)formamidine, the thio reagent was a compound with CAS No. 56950-66-4, and the capping reagent was a mixture of acetic anhydride / tetrahydrofuran and N-methylimidazole. After obtaining the synthesis product, deprotection and annealing were performed.

[0134] The annealing product was purified by HPLC to obtain different siRNA sequences.

[0135] Purification material and parameters: NanoQ-15L anion exchange column was selected, and the equilibration liquid was 100 mmol / L Tris-Hydroxymethyl Amino Methane (pH 7.5), 10 mmol / L Ethylenediamine Tetraacetic Acid (pH 8.0), 300 mmol / L NaCl, pH 9.0, and the conductivity was 32 mS / cm. The elution liquid was 100 mmol / L Tris-Hydroxymethyl Amino Methane (pH 7.5), 10 mmol / L Ethylenediamine Tetraacetic Acid (pH 8.0), 700 mmol / L NaCl, pH 9.0, and the conductivity was 65.2 ms / cm, and the flow rate was 10 mL / min.

[0136] Example 3: siRNA transfection of mouse brain cells

[0137] Preparation of mouse brain cells

[0138] The brain gray matter was taken from the brain of B6 mice (3-4 months old, female, purchased from Shanghai Shengchang Biotechnology Co., Ltd.), and the dura mater, blood vessels and fiber components were carefully removed and rinsed in Hanks solution for one or two times. The rinsed gray matter tissue was placed in 30-50 times the volume of Hanks solution, and a cell suspension was prepared by repeatedly blowing. The suspension was injected into a centrifuge tube, and after the centrifuge tube was vertically placed at room temperature for 5-10 minutes, the cells or cell clumps naturally sank, and the fat and other impurities floated easily. The upper liquid was carefully aspirated, and the process was repeated two or three times. An appropriate amount of culture medium was added to the remaining sediment, and the cell suspension was filtered through a gauze cloth. The filtered cell suspension was counted and the cell density was adjusted. The mouse brain cells were inoculated into culture medium bottles or dishes and cultured in a 5% CO2 incubator. An appropriate amount of cells were passaged into a 12-well plate to ensure that the cells grew to about 50% density per well the next day.

[0139] Transfection of siRNA of type A modification

[0140] During transfection, one of the six siRNA molecules (1#A-6#A) was added to each well, and the six siRNA molecules were divided into high and low dose groups. The siRNA transfection concentration of the high dose group was 100 nM, and the siRNA transfection concentration of the low dose group was 30 nM. 48 hours after transfection, the RNA genome was extracted using the Tengen kit RNA prep Pure Cell / Bacteria Kit (item number DP430), and the cDNA was obtained by reverse transcription.

[0141] The mRNA expression of TNF-α was detected by Q-PCR, and the results are shown in Figure 8A. When the transfection concentration was 30 nM or 100 nM, the six siRNAs could effectively knock down the mRNA expression. The mRNA expression knockdown effect of the molecule 5#A was optimal at both low and high transfection concentrations, and the remaining mRNA expression levels were about 36% and about 18.3%, respectively. Since the molecule 5#A had the optimal effect of specifically knocking down the mRNA of TNF-α, the molecule 5#A was selected for subsequent verification.

[0142] Transfection of siRNAs with B-E type modification

[0143] Based on the transfection results of siRNAs with A type modification, siRNAs 5#B-5#E were used for transfection experiments.

[0144] During transfection, one of the siRNAs 5#B, 5#C, 5#D or 5#E was added to each well of cells, and each siRNA was set as a high-dose group and a low-dose group. The transfection concentration of siRNA in the high-dose group was 100 nM, and the transfection concentration of siRNA in the low-dose group was 30 nM. At 48 h, the RNA genome was extracted according to the RNA prep Pure Cell / Bacteria Kit (DP430) kit, and then cDNA was obtained by reverse transcription.

[0145] The mRNA expression of TNF-α was detected by Q-PCR, and the results are shown in Figure 8B. When the transfection concentration was 30 nM or 100 nM, the four siRNAs could effectively knock down the mRNA expression. The mRNA expression knockdown effect of 5#C was optimal at both low and high transfection concentrations, and the remaining mRNA expression levels were about 29% and about 15.5%, respectively. The knockdown effect from high to low was 5#C > 5#B > 5#D > 5#E.

[0146] Example 4: Effect of intrathecal injection of siRNA molecules on the mRNA expression of target genes in mice in vivo

[0147] The siRNAs 5# (without special modification), 5#A, 5#B, 5#C, 5#D and 5#E were prepared into sterile preparations respectively, and the solvent was sterile water for injection. When preparing, 2 mg / ml siRNA, 0.2 mg / ml calcium chloride dihydrate, 0.1 mg / ml sodium dihydrogen phosphate, 0.16 mg / ml magnesium chloride hexahydrate, 0.03 mg / ml sodium phosphate, 0.22 mg / ml potassium chloride were added into part of the solvent according to the mass concentration of the final volume, and then the solution was dissolved thoroughly, diluted with the solvent to the final volume, and finally the pH was adjusted to 6.9-7.1 using 1 mg / ml HCl or NaOH solution. The B6 mice were injected intrathecally with 50 μL of siRNA preparation, and then the mice were euthanized 24 h after the injection. The heart, liver, spleen, lung, kidney, spinal cord and brain white matter of the mice were taken respectively, and the organs of a blank mouse were taken as control, and then the samples were quickly frozen in liquid nitrogen.

[0148] The tweezers and scissors were sterilized using 75% ethanol solution to eliminate RNAse as much as possible. The 2 ml centrifuge tubes were placed on ice blocks, and appropriate amount of PBS buffer was added for preparing tissue homogenate. The mass volume ratio of the tissue homogenate required by the experiment was about 10%. The tissue sample to be ground was taken out from the freezer at minus 80°C, and was placed in the centrifuge tube on ice. The sample was cut into small pieces using the sterilized tweezers and scissors. The sterilized zirconium oxide grinding ball was placed in the centrifuge tube at one ball per tube. The centrifuge tube was placed in the grinder, and the vibration frequency was set to 1000 times / min, the vibration time was 5 min, and the temperature was minus 20°C. After grinding, the centrifuge tube was placed in the pre-cooled high-speed centrifuge, and was centrifuged at 4000 rpm for 30 min. The supernatant was taken out using a pipette, and was transferred into a new centrifuge tube. The RNA genome was extracted using the TIANGEN kit RNA prep Pure Cell / Bacteria Kit (product number DP430), and the expression of TNF-α mRNA in each organ was detected by Q-PCR.

[0149] The effect of intrathecal injection of 5# siRNA molecule on the expression of target gene mRNA in the mouse body is shown in FIG. 9. The results show that 5# has no knockdown effect on the target gene in the heart, spleen and lung, and has a certain knockdown effect in the liver, kidney, spinal cord and brain white matter, but the knockdown effect is less than 50%. 5#A-5#E have almost no knockdown effect in the heart, and have different degrees of knockdown effect in the liver, spleen, lung, kidney, spinal cord and brain white matter, and are generally better than 5#. Among them, 5#C is relatively optimal, and the knockdown effect in the liver, spleen, lung, kidney, spinal cord and brain white matter is about 45%, 71%, 65%, 52.5%, 21% and 28.5% respectively (FIG. 9D).

[0150] In summary, the siRNA molecule with special modification of the application can achieve the effect of knocking down the mRNA expression of target genes in liver, spleen, lung, spinal cord and brain white matter by intrathecal injection, and the effect is significantly improved compared with unmodified siRNA, and has excellent lipophilicity and distribution performance.

[0151] Example 5: Effect of intradermal injection of siRNA molecules on mRNA expression of target genes in mice

[0152] 5#A, 5#B, 5#C, 5#D, 5#E and 5# molecules without modification structure were prepared into sterile preparations, and the preparation method was referred to Example 4. B6 mice were intradermally injected with 50 μg siRNA-containing preparations, and the mice were euthanized 24 hours after injection. The skin at the injection site was removed, and the skin (including epidermis and dermis) of a blank mouse was taken as a control, and then immediately frozen in liquid nitrogen.

[0153] The tweezers and scissors were sterilized with 75% ethanol solution to eliminate RNAse as much as possible. 2 ml centrifuge tubes were placed on ice blocks, and appropriate amount of PBS buffer was added for preparing tissue homogenate. The mass-volume ratio of the tissue homogenate required for the experiment was about 10%. The tissue sample to be ground was taken out from the freezer at minus 80°C, and placed in the centrifuge tube on ice. The sterilized zirconium oxide grinding balls were placed in the centrifuge tube at one ball per tube. The centrifuge tube was placed in the grinder, and the vibration frequency was set to 1000 times / min, the vibration time was 5 minutes, and the temperature was minus 20°C. After grinding, the centrifuge tube was placed in a pre-cooled high-speed centrifuge at a speed of 4000 rpm for 30 min. The supernatant was taken out with a pipette and transferred to a new centrifuge tube. The RNA genome was extracted using a Tengen kit RNA prep Pure Cell / Bacteria Kit (item number DP430), and the expression of TNF-α mRNA in each organ was detected by Q-PCR.

[0154] The effect of intradermal injection of siRNA molecules on mRNA expression in mouse skin is shown in Figure 10. The results show that the expression level of mRNA in the skin tissue is knocked down to about 67.5% by 5#. The five special modified siRNA molecules can effectively knock down the expression of mRNA, and the effect is better than that of 5#. The remaining mRNA expression levels after knocking down 5#A-5#E are about 19.5%, 27.5%, 3.5%, 52.5%, and 55.0%, respectively. It can be seen that the effect of 5#C is the best, and the knocking down effect from high to low is: 5#C>5#A>5#B>5#D>5#E.

[0155] The experimental results show that the siRNA with special modification has the effect of knocking down the mRNA expression of the target gene in the skin tissue by the intradermal injection mode, and the effect is significantly improved compared with the unmodified siRNA, and the siRNA has excellent lipophilicity and transdermal property.

[0156] Example 6: Cytotoxicity of siRNA molecules

[0157] The 5#A, 5#B, 5#C, 5#D and 5#E molecules are respectively transfected into HaCat cells, and then the cytotoxicity is detected by using a kit, and the specific operation steps are as follows:

[0158] S1: On the first day, a proper amount of cells are passaged into a 96-well plate, so as to ensure that the cells can grow to about 50% density per well on the second day.

[0159] S2: On the second day, when the density reaches about 50%, the siRNA preparation (the preparation method is referred to Example 4) of 5#A-5#E is added into each well for transfection, and the transfection concentration is 100 pmol siRNA.

[0160] S3: The cells after transfection are incubated overnight in a 5% CO2, 37℃ environment.

[0161] S4: At different time points, the absorbance value of each well is measured at 450 nm by using an enzyme marker.

[0162] As shown in FIG. 11, the five siRNA molecules show slight toxicity at 4-8 h after transfection, and then the cell viability gradually recovers to normal, which shows that the five siRNA molecules have small cytotoxicity.

[0163] Example 7: Evaluation of in vivo delivery distribution of siRNA molecules

[0164] The 5#A siRNA molecule with FAM (Carboxyfluorescein) linked to the 3' end of the sense strand is synthesized by using a CPG (Controlled Pore Glass) column with FAM, and then a sterile preparation is prepared (the preparation method is referred to Example 4). The B6 mouse is intrathecally injected with 50 μL of the siRNA preparation, and the mouse is euthanized after 3 h of injection, and the heart, liver, spleen, lung, kidney, spinal cord and brain white matter of the mouse are taken for tissue fluorescence imaging.

[0165] As shown in FIG. 12, the fluorescence signal from strong to weak is in the order of spinal cord, brain, liver and kidney, and at this time, the heart, spleen and lung have no signal detection. It can be seen that the 5#A siRNA molecule can be rapidly distributed in the spinal cord and spinal tissue after intrathecal injection, and has excellent distribution performance.

[0166] In summary, the present application increases the lipophilicity of the siRNA sequence by lipophilic chain modification, and the modified siRNA molecule achieves excellent in vivo carrier-free delivery efficiency and distribution performance through intrathecal injection and intradermal injection and other administration methods, effectively knocks down the expression of target genes while having good tolerance and safety.

[0167] The above examples are only preferred examples for fully illustrating the technical solutions and effects of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. The equivalent substitutions or transformations made by those skilled in the art on the basis of the present application are within the protection scope of the present application.

Claims

1. A nucleoside having a structure as shown in formula (I), characterized in that: which comprises R1, R3 and a lipophilic R2, wherein: R1 is selected from adenine, uracil, cytosine or guanine, adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, R2is selected from the group consisting of: adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, 2. A double stranded siRNA comprising a sense strand and an antisense strand, the sequence of the sense strand is 5'-GCCUGUAGCCCAUGUUGUATT-3', the sequence of the antisense strand is 5'-UACAACAUGGGCUACAGGCTT-3', characterized in that, wherein the nucleotides have special modifications, the specially modified nucleotides comprising R1and a lipophilic R2, having a structure as shown in formula (II): adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, R3 is selected from 4,4'-dimethoxytrityl, 4-monomethoxytrityl, 4,4',4"-trimethoxytrityl, pivaloyloxymethyl, t-butyldimethylsilyl, 9-fluorenylmethyloxycarbonyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, 2-cyanoethoxy-N,N-diisopropylaminophosphino, 3-acetylenyl, acetyl, benzoyl, 2-cyanoethyl, N,N-dibutylaminocarbonyl, N,N-dimethylaminocarbonyl, isobutyryl, 2-(2-nitrophenyl)-propyloxycarbonyl, triethylammonium, trifluoroacetyl, triisopropylsilyloxymethyl, p-isopropylphenoxyacetyl, O-acetal acetylenyl ester, phenylacetyl, or 1,1',3,3'-tetraisopropyldisiloxanyl. R1 is selected from adenine, uracil, cytosine or guanine, adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, R2is selected from the group consisting of:

3. The double-stranded siRNA of claim 2, wherein adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, 4. The double-stranded siRNA of claim 2, wherein adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, 5. The double-stranded siRNA of claim 3, wherein adenine, uracil, cytosine or guanine comprising one or more modifications selected from thio, halo, hydroxylated, aminated, carboxylated or methylated, the specially modified nucleotides are located at one or more of positions 1, 2, 5, 8, 14, 17, 18 or 19 in the 5' to 3' direction of the sense strand. the specially modified nucleotides are located at one or more of positions 1, 2, 9, 10, 11, 17, 18 or 19 in the 5' to 3' direction of the antisense strand. the specially modified nucleotides are located at: position 1 in the 5' to 3' direction of the sense strand; positions 1 and 19 in the 5' to 3' direction of the sense strand; 6. The double-stranded siRNA of claim 4, wherein positions 1 and 2 in the 5' to 3' direction of the sense strand; positions 1, 2, 18 and 19 in the 5' to 3' direction of the sense strand; or positions 1, 5, 8, 14 and 17 in the 5' to 3' direction of the sense strand. the specially modified nucleotides are located at: position 1 in the 5' to 3' direction of the antisense strand; positions 1 and 19 in the 5' to 3' direction of the antisense strand; positions 1 and 2 in the 5' to 3' direction of the antisense strand; positions 1, 2, 18 and 19 in the 5' to 3' direction of the antisense strand; or positions 9, 10, 11, 17, and 18 of the 5' to 3' direction of the antisense strand.

7. The double-stranded siRNA of claim 2, wherein the 2nd, 3rd, 4th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 16th, 18th, and 19th positions of the sense strand are modified to have a phosphorothioate backbone linkage at the 3'-C position. the 1st position of the 5' to 3' direction of the sense strand, and the 1st position of the 5' to 3' direction of the antisense strand; the 1st and 19th positions of the 5' to 3' direction of the sense strand, and the 1st and 19th positions of the 5' to 3' direction of the antisense strand; the 1st and 2nd positions of the 5' to 3' direction of the sense strand, and the 1st and 2nd positions of the 5' to 3' direction of the antisense strand; the 1st, 2nd, 18th, and 19th positions of the 5' to 3' direction of the sense strand, and the 1st, 2nd, 18th, and 19th positions of the 5' to 3' direction of the antisense strand; the 1st, 5th, 8th, 14th, and 17th positions of the 5' to 3' direction of the sense strand; or the 1st, 5th, 8th, 14th, and 17th positions of the 5' to 3' direction of the sense strand, and the 9th, 10th, 11th, 17th, and 18th positions of the 5' to 3' direction of the antisense strand.

8. The double-stranded siRNA of any one of claims 2-7, wherein, the 2nd, 3rd, 4th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 16th, 18th, and 19th positions of the sense strand are modified to have a phosphorothioate backbone linkage at the 3'-C position.

9. A pharmaceutical composition comprising a therapeutically effective amount of the double-stranded siRNA of any one of claims 2-8, and a pharmaceutically acceptable carrier, solvent, or excipient.

Images