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

Abstract

The present invention discloses a double-stranded siRNA with lipophilic modification, and the modified siRNA molecules achieve excellent in vivo distribution performance and delivery efficiency through intrathecal injection, intradermal injection and other administration methods, and have good tolerability and safety while effectively knocking down the expression of the target gene. The present invention also discloses a preparation method of the siRNA with lipophilic modification, a pharmaceutical composition, and its use in the preparation of a drug for treating TNF-α-mediated diseases or conditions.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a double-stranded siRNA, a preparation method thereof, a pharmaceutical composition and use thereof in preparing a drug for treating TNF-α-mediated diseases or conditions. Background Art

[0002] Most of the RNAi drugs that have been marketed are modified with lipid nanoparticles and N-acetylgalactosamine to target liver tissue. Due to the hydrophilicity and negative charge of siRNA molecules, it is difficult for them to be taken up by cells. If RNAi technology is to be applied to other tissues (such as the central nervous system, eyes, and lungs), new technologies need to be developed to improve the delivery and distribution performance of siRNA in extrahepatic tissues.

[0003] At present, studies have reported different types of siRNA carriers, including polymers and peptides. 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 effects under laboratory conditions, there are still many difficulties in converting them into clinical applications. Cell-penetrating peptides have poor stability in the body and are easily degraded by enzymes, resulting in reduced efficacy. In addition, large-scale synthesis and purification are relatively complex and costly. With the advancement of technology, polymer materials are also being developed. PEI is a cationic polymer that forms nanoscale complexes by binding to negatively charged nucleic acids. These complexes can enter cells and release nucleic acids through endocytosis. 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 responses or cytotoxicity, leading to adverse side effects.

[0004] More drugs for extrahepatic delivery have been under development. In lung targeting, ALN-HPN-19 developed by Alnylam Pharmaceuticals is used to treat pulmonary fibrosis, and small nucleic acid drugs are delivered to the lungs by inhalation. In eye targeting, RGX-314 developed by Regeneron Pharmaceuticals is used to treat wet age-related macular degeneration, and small nucleic acid drugs are delivered to the eyes by eye drops. This method may also treat diseases such as glaucoma and diabetic retinopathy in the future. In tumor targeting, AMG-160 developed by Arrowhead Pharmaceuticals is used to treat solid tumors. The characteristics of the tumor microenvironment (such as pH value, redox state, etc.) are used 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, small nucleic acid drugs are encapsulated in nanoparticles and 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] Delivering nucleic acid drugs to the nervous system is a very challenging task, because the existence 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 the treatment of neurological diseases, and many companies are increasing their research and development. Voyager Therapeutics' VY-HTT01 uses AAV (adeno-associated virus) vectors to deliver siRNA, targeting the Huntington gene (HTT) and reducing the expression of mutant HTT protein. It is undergoing clinical trials, and preliminary results show safety and potential efficacy. Biogen's Spinraza, which delivers antisense oligonucleotides (ASO) by intrathecal injection, changes the splicing of the SMN2 gene and increases the production of functional SMN protein. It has been approved by the FDA and is widely used in the treatment of SMA patients. Acuitas Therapeutics' ACU-193 uses lipid nanoparticles (LNP) to deliver siRNA, targeting the β-amyloid precursor gene (APP) and reducing the production of β-amyloid protein. It is in the early stages of research and development. UniQure's AMT-130 uses AAV vectors to deliver miRNA, targeting the synuclein gene (SNCA) and reducing the expression of α-synuclein. Clinical trials are ongoing to evaluate its safety and efficacy.

[0006] The effective delivery of small nucleic acids is highly dependent on the delivery vector. The development of many small nucleic acid drugs requires 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 research and development focus of siRNA drugs. Summary of the invention

[0007] Improving the lipophilicity of drugs is an effective strategy to increase 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 invention enhances the lipophilicity of siRNA molecules through refined structural design, thereby improving its distribution performance and delivery effect.

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

[0009] (I)

[0010] R1 is selected from a nitrogenous base or a nitrogenous base analog;

[0011] R2 is selected from the following groups:

[0012] ,

[0013] ,

[0014] ,

[0015] ,or

[0016] ;

[0017] 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-diisopropylaminophosphine 1,1',3,3'-tetraisopropyl disiloxanyl (TEA), trifluoroacetyl, triisopropylsilyloxymethyl (TOM), 4-isopropylphenoxyacetyl (iPrPAC), acetal levulinyl ester (ALE), phenylacetyl, or 1,1',3,3'-tetraisopropyl disiloxanyl.

[0018] Another aspect of the present invention provides a double-stranded siRNA comprising a sense strand and an antisense strand, wherein the nucleotides are specially modified, wherein the specially modified nucleotides comprise R1 and lipophilic R2, and have a structure as shown in formula (II):

[0019] (II)

[0020] R1 is selected from a nitrogenous base or a nitrogenous base analog;

[0021] R2 is selected from the following groups:

[0022] ,

[0023] ,

[0024] ,

[0025] ,or

[0026] .

[0027] In one or more embodiments, the specifically modified nucleotide is located at any one or more positions in the 5' to 3' direction of the sense strand or the antisense strand.

[0028] In one or more embodiments, the specifically modified nucleotide is located 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.

[0029] In one or more embodiments, the specifically modified nucleotide is 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.

[0030] In one or more embodiments, the specifically modified nucleotide is 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.

[0031] In one or more embodiments, the specifically modified nucleotides are located at:

[0032] position 1 in the 5' to 3' direction of the sense strand;

[0033] positions 1 and 19 in the 5′ to 3′ direction of the sense strand;

[0034] positions 1 and 2 in the 5′ to 3′ direction of the sense strand;

[0035] Positions 1, 2, 18 and 19 in the 5' to 3' direction of the sense strand; or

[0036] Positions 1, 5, 8, 14 and 17 in the 5' to 3' direction of the sense strand.

[0037] In one or more embodiments, the specifically modified nucleotides are located at:

[0038] 1st position in the 5' to 3' direction of the antisense strand;

[0039] positions 1 and 19 in the 5′ to 3′ direction of the antisense strand;

[0040] positions 1 and 2 in the 5′ to 3′ direction of the antisense strand;

[0041] Positions 1, 2, 18 and 19 in the 5' to 3' direction of the antisense strand; or

[0042] Positions 9, 10, 11, 17 and 18 in the 5' to 3' direction of the antisense strand.

[0043] In one or more embodiments, the specifically modified nucleotides are located at:

[0044] position 1 in the 5' to 3' direction of the sense strand, and position 1 in the 5' to 3' direction of the antisense strand;

[0045] positions 1 and 19 in the 5′ to 3′ direction of the sense strand, and positions 1 and 19 in the 5′ to 3′ direction of the antisense strand;

[0046] positions 1 and 2 in the 5' to 3' direction of the sense strand, and positions 1 and 2 in the 5' to 3' direction of the antisense strand;

[0047] positions 1, 2, 18 and 19 in the 5' to 3' direction of the sense strand, and positions 1, 2, 18 and 19 in the 5' to 3' direction of the antisense strand;

[0048] Positions 1, 5, 8, 14 and 17 in the 5' to 3' direction of the sense strand; or

[0049] positions 1, 5, 8, 14 and 17 in the 5' to 3' direction of the sense strand, and positions 9, 10, 11, 17 and 18 in the 5' to 3' direction of the antisense strand.

[0050] In one or more embodiments,

[0051] The sequence of the sense strand is 5'- GCCUGUAGCCCAUGUUGUATT - 3', and the sequence of the antisense strand is 5'- UACAACAUGGGCUACAGGCTT - 3'.

[0052] In one or more embodiments, a non-bridging oxygen atom in a phosphate group attached to 3'-C in any one or more nucleotide monomers of the sense strand or antisense strand is replaced by a sulfur atom.

[0053] In one or more embodiments, one of the non-bridging oxygen atoms in the phosphate group attached to 3'-C in nucleomonomers 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 15, 16, 18 and 19 of the sense strand is replaced by a sulfur atom.

[0054] Another aspect of the present invention provides a method for preparing a nucleoside as described in any embodiment of the present invention, the method comprising the synthesis of a lipophilic chain and reacting the lipophilic chain with a dehydrated nucleoside to obtain a nucleoside as described in any embodiment of the present invention, wherein the dehydrated nucleoside is selected from dehydrated adenosine or an analog thereof, dehydrated uracil or an analog thereof, dehydrated cytosine or an analog thereof, dehydrated thymine or an analog thereof, or dehydrated guanine or an analog thereof.

[0055] Another aspect of the present invention provides a method for preparing a double-stranded siRNA as described in any embodiment of the present invention, the method comprising synthesizing RNA using the nucleoside described in any embodiment of the present invention as a raw material.

[0056] In one or more embodiments, the method is a phosphoramidite triester method.

[0057] Another aspect of the present invention provides 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.

[0058] Another aspect of the present invention provides a method for preparing a pharmaceutical composition as described in any embodiment herein, the method comprising adding double-stranded siRNA and a pharmaceutically acceptable carrier or excipient to a solvent.

[0059] In one or more embodiments, the method comprises adding 0.2-20 mg / ml double-stranded siRNA, 0.02-1 mg / ml calcium chloride dihydrate, 0.01-1 mg / ml sodium dihydrogen phosphate, 0.01-1 mg / ml magnesium chloride hexahydrate, 0.01-1 mg / ml sodium phosphate, and 0.01-1 mg / ml potassium chloride to a portion of the solvent at a mass concentration calculated by the final volume, and after sufficient dissolution, diluting the solution with a solvent to a near final volume, wherein the solvent is sterile water for injection, and finally adjusting the pH to 6.5-7.5 using 1 mg / ml HCl or NaOH solution.

[0060] In one or more embodiments, the method comprises adding 2 mg / ml double-stranded 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, and 0.22 mg / ml potassium chloride at a mass concentration calculated by the final volume to a portion of the solvent, and after sufficient dissolution, diluting the solution with a solvent to a near final volume, wherein the solvent is sterile water for injection, and finally adjusting the pH to 6.9-7.1 using 1 mg / ml HCl or NaOH solution.

[0061] 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 drug 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, pain sensitivity 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.

[0062] 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

[0063] Figure 1 It is lipophilic chain a 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 1 A is the lipophilic chain a 1 H-NMR spectrum, Figure 1 B is the lipophilic chain a 13 C-NMR spectrum.

[0064] Figure 2 It is lipophilic chain b 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 2 A is the lipophilic chain b 1 H-NMR spectrum, Figure 2 B is the lipophilic chain b 13 C-NMR spectrum.

[0065] Figure 3 It is lipophilic chain c 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 3 A is the lipophilic chain c 1 H-NMR spectrum, Figure 3 B is the lipophilic chain c 13 C-NMR spectrum.

[0066] Figure 4 It is lipophilic chain d 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 4 A is a lipophilic chain d 1 H-NMR spectrum, Figure 4 B is the lipophilic chain d 13 C-NMR spectrum.

[0067] Figure 5 It is lipophilic chain e 1 H-NMR spectrum and 13 C-NMR spectrum. Figure 5 A is the lipophilic chain e 1 H-NMR spectrum, Figure 5 B is the lipophilic chain e 13 C-NMR spectrum.

[0068] Figure 6 FIG. 1 is an exemplary synthetic route of nucleoside A. FIG.

[0069] Figure 7 is the structural diagram of nucleoside A-nucleoside E. Figure 7 A is the structural diagram of nucleoside A, Figure 7 B is the structural diagram of nucleoside B, Figure 7 C is the structural diagram of nucleoside C, Figure 7 D is the structural diagram of nucleoside D, Figure 7 E is a diagram of the structure of nucleoside E.

[0070] Figure 8 It is the change in the expression level of target gene mRNA in mouse brain cells after transfection of siRNA. Figure 8 A is the change in target gene mRNA expression level in mouse brain cells after transfection 1#~6#, Figure 8 B is the change in target gene mRNA expression level in mouse brain cells after transfection with 5#B~5#E.

[0071] Fig. 9 It is the change of target gene mRNA expression level in various organs of mice after intrathecal injection of siRNA. Fig. 9 A is the change of target gene mRNA expression level in various organs of mice after intrathecal injection of 5#, among which Fig. 9 B is the change of target gene mRNA expression level in various organs of mice after intrathecal injection of 5#A. Fig. 9 C is the change of target gene mRNA expression level in various organs of mice after intrathecal injection of 5#B, among which Fig. 9 D is the change of target gene mRNA expression level in various organs of mice after intrathecal injection of 5#C, among which Fig. 9 E is the change of target gene mRNA expression level in various organs of mice after intrathecal injection of 5#D, among which Fig. 9 F is the changes in target gene mRNA expression levels in various organs of mice after intrathecal injection of 5#E.

[0072] Fig.10 It is the change in target gene mRNA expression level in mouse skin tissue after intradermal injection of siRNA.

[0073] Fig.11 It is the change of cell viability of HaCat cells after transfection of siRNA.

[0074] Fig.12 This is the fluorescence imaging of various tissues of mice after intrathecal injection of 5#A. Fig.12 A is the fluorescence imaging of various tissues of mice after intrathecal injection of 5#A. Fig.12 B is the corresponding color scale. DETAILED DESCRIPTION

[0075] Nucleosides

[0076] The present invention provides a nucleoside having a structure as shown in formula (I), comprising R1, R3 and a lipophilic R2, wherein:

[0077] (I)

[0078] R1 is selected from a nitrogenous base or a nitrogenous base analog;

[0079] Preferably, R1 is selected from adenyl, uracil, cytosine or guanyl. Preferably, R1 is selected from adenyl, uracil, cytosine or guanyl modified by one or more of thio, halogenation, hydroxylation, amination, carboxylation or methylation. Preferably, R1 is selected from adenine methyl, uracil methyl, cytosine methyl or guanine methyl modified by one or more of thio, halogenation, hydroxylation, amination, carboxylation or methylation. Preferably, R1 is selected from adenine formyl, uracil formyl, cytosine formyl or guanine formyl modified by one or more of thio, halogenation, hydroxylation, amination, carboxylation or methylation.

[0080] R2 is selected from H, C 12 -C 50 Alkyl, or C having one or more modifications selected from hydroxyl, ether bond, carbonyl, amide bond, carboxyl, ester bond, amino, imino, tertiary amino, halogen, thio, thioether bond, mercapto, sulfonic acid, phosphorus and phosphoric acid groups. 12 -C 50 alkyl.

[0081] Preferably, R2 is selected from the following groups:

[0082] ,

[0083] ,

[0084] ,

[0085] ,or

[0086] ;

[0087] 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-diisopropylaminophosphine R3 is 4,4'-dimethoxytrityl (DMTr).

[0088] ds-siRNA

[0089] The present invention provides a double-stranded siRNA, 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):

[0090] (II)

[0091] R1 is selected from a nitrogenous base or a nitrogenous base analog; preferably, R1 is selected from adenine, uracil, cytosine or guanine.

[0092] Preferably, R1 is selected from adenine, uracil, cytosine or guanine radicals comprising one or more modifications of thio, halogenation, hydroxylation, amination, carboxylation or methylation. Preferably, R1 is selected from adenine methyl, uracil methyl, cytosine methyl or guanine methyl radicals comprising one or more modifications of thio, halogenation, hydroxylation, amination, carboxylation or methylation. Preferably, R1 is selected from adenine formyl, uracil formyl, cytosine formyl or guanine formyl radicals comprising one or more modifications of thio, halogenation, hydroxylation, amination, carboxylation or methylation.

[0093] R2 is selected from H, C 12 -C 50Alkyl, or C having one or more modifications selected from hydroxyl, ether bond, carbonyl, amide bond, carboxyl, ester bond, amino, imino, tertiary amino, halogen, thio, thioether bond, mercapto, sulfonic acid, phosphorus and phosphoric acid groups. 12 -C 50 alkyl.

[0094] Preferably, R2 is selected from the following groups:

[0095] ,

[0096] ,

[0097] ,

[0098] ,or

[0099] .

[0100] Example

[0101] Example 1: Synthesis of Nucleosides

[0102] Synthetic route of lipophilic chain a

[0103] S1: Raw material 1 (CAS No.: 55182-74-6) was used to obtain intermediate product 1 according to the method of reference (CN110028405A), and intermediate product 1 was used as raw material to obtain intermediate product 2 according to the method of reference (Journal of Chemical Ecology; vol. 27; 4;(2001); p. 791-806).

[0104] S2: Starting material 2 (CAS No.: 112-31-2) and intermediate 2 were used to obtain intermediate 3 according to the method of reference European Journal of Organic Chemistry; 9; (2000); p. 1821-1826.

[0105] S3: Intermediate product 3 is prepared according to the method of reference (Vanka, Kumar; vol. 10; 14; (2020); p. 4586-4592) to obtain lipophilic chain a, whose structure is shown in formula (III).

[0106] (III)

[0107] Get lipophilic chain a 1 H-NMR spectrum Figure 1 As shown in A, 13 C-NMR spectrum Figure 1 As shown in B.

[0108] Synthetic route of lipophilic chain b

[0109] S1: Mix 3-buten-1-ol (200 mg, 2.77 mmol) and N,N-dimethylformamide (DMF, 2.0 mL, purity >99%).

[0110] S2: 1-Bromononane (355 μL, 1.85 mmol) and sodium hydride (NaH, 112 mg, 2.77 mmol) were added to the mixture of S1 at room temperature and stirred continuously until the starting material was completely consumed (monitored by thin layer chromatography). After stirring, the reaction was quenched with water.

[0111] S3: After the stirring of S2 was completed, the reaction mixture was extracted with ethyl acetate / hexane (volume ratio 10:1).

[0112] S4: Take the organic layer extracted in S3, wash it once with water and once with brine, and separate and extract the organic layer after each washing. Then dry the washed organic layer with anhydrous sodium sulfate, and filter it after drying. Concentrate the filtrate under vacuum to obtain a crude product.

[0113] 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).

[0114] S6: According to reference (Liebigs Annalen der Chemie; 1; (1981); p. 92 - 98), intermediate 2 was obtained.

[0115] S7: According to reference (Journal of the American Chemical Society; vol. 123;50; (2001); p. 12504 - 12509), intermediate 1 and intermediate 2 are reacted to obtain a lipophilic chain b, whose structure is shown in formula (IV).

[0116] (IV)

[0117] Get lipophilic chain b 1 H-NMR spectrum Figure 2 As shown in A, 13 C-NMR spectrum Figure 2 As shown in B.

[0118] Synthetic route of lipophilic chain c

[0119] S1: According to the following references, intermediate 1 was obtained:

[0120] Chemistry and Industry (London, United Kingdom); (1959); p. 1288;Journal of the Chemical Society; (1961); p. 2779,2786;

[0121] Bioorganic and Medicinal Chemistry; vol. 15; 2; (2007); p. 854 – 867;

[0122] Journal of Organic Chemistry; vol. 63; 11; (1998); p. 3741 – 3744;

[0123] Journal of Organic Chemistry; vol. 54; 23; (1989); p. 5522 – 5527.

[0124] S2: According to the following reference, compound (CAS No. 2566-89-4) and compound (CAS No. 2134-29-4) were reacted to obtain intermediate 2:

[0125] Angewandte Chemie - International Edition; vol. 54; 13; (2015); p.4023 – 4027;

[0126] Angew. Chem.; vol. 127; 13; (2015); p. 5.

[0127] S3: According to reference (WO2019010414A1), intermediate 1 and intermediate 2 are reacted to obtain intermediate 3.

[0128] S4: According to the following reference, intermediate 4 was obtained.

[0129] Liebigs Annalen der Chemie; 11; (1992); p. 1113 – 1124

[0130] Monatshefte fuer Chemie; vol. 112; (1981); p. 825 – 840

[0131] S5: According to the reference (Journal of Medicinal Chemistry; vol. 40; 22; (1997); p. 3626 – 3634), intermediate 3 and intermediate 4 are reacted to obtain a lipophilic chain c, the structure of which is shown in formula (V).

[0132] (V)

[0133] Get the lipophilic chain c 1 H-NMR spectrum Figure 3 As shown in A, 13 C-NMR spectrum Figure 3 As shown in B.

[0134] Synthetic route of lipophilic chain d

[0135] S1: According to the reference (Tetrahedron Letters; vol. 48; 42; (2007); p. 7456 –7459), starting material 1 (CAS No.: 542-78-9) and starting material 2 (CAS No.: 1120-16-7) were reacted to obtain intermediate 1.

[0136] S2: According to reference (Molecules; vol. 27; 9; (2022)), the intermediate product 1 is reacted to obtain a lipophilic chain d, whose structure is shown in formula (VI).

[0137] (VI)

[0138] Get the lipophilic chain d 1 H-NMR spectrum Figure 4 As shown in A, 13 C-NMR spectrum Figure 4 As shown in B.

[0139] Synthetic route of lipophilic chain e

[0140] Using the raw material (CAS No. 156-60-5), the reaction was carried out according to the reference (Journal of Organic Chemistry; vol. 34; (1969); p. 1130 – 1133) to obtain a lipophilic chain e, whose structure is shown in formula (VII).

[0141] (VII)

[0142] Get the lipophilic chain e 1 H-NMR spectrum Figure 5 As shown in A, 13C-NMR spectrum Figure 5 As shown in B.

[0143] Synthesis of nucleosides containing special modifications

[0144] Synthesis of Nucleoside A

[0145] Nucleoside A can be synthesized using lipophilic chain a and anhydronucleoside as raw materials. The anhydronucleoside is selected from anhydroadenosine, anhydrouracil, anhydrocytosine, anhydrothymine, anhydroguanine, or analogs thereof.

[0146] For example, a nucleoside A, i.e. uridine containing lipophilic chain a, is synthesized from lipophilic chain a and dehydrated uridine. The process is as follows: Figure 6 As shown:

[0147] S1: Add 0.06~0.1M compound ① (2,2'-dehydrouridine, CAS No. 3736-77-4) to 2ml tetrahydrofuran (THF), slowly drop 100 μl tert-butyldiphenylsilyl chloride (TBDPSCl, CAS No. 58479-61-1), and stir to mix evenly. Slowly drop 100 μl of basic catalyst triethylamine into the reaction system, continue to stir to mix evenly, and keep at room temperature overnight to generate a solution containing compound ②.

[0148] S2: Slowly add 10 μl of 0.1M trimethylaluminum (CAS No. 75-24-1) to the solution containing compound ② in S1 while stirring to mix evenly to generate a solution containing compound ③.

[0149] S3: Slowly add 10 μl of pyridine hydrofluoride (CAS No. 62778-11-4) to the solution containing compound ③ in S2 while stirring to mix evenly to generate a solution containing compound ④.

[0150] S4: Slowly add 100 μl of DMTrCl (CAS No. 40615-36-9) to the solution containing compound ④ in S3, and add a small amount of pyridine (CAS No. 110-86-1) and DMAP (CAS No. 1122-58-3) as catalysts to obtain a solution containing compound ⑤.

[0151] S5: Add 50 μl of bis(diisopropylamino)(2-cyanoethoxy)phosphine (CAS No. 102691-36-1) to the solution containing compound ⑤ in S4, and stir to mix evenly to obtain nucleoside A.

[0152] Synthesis of Nucleoside BE

[0153] The synthesis conditions of nucleoside BE are basically the same as those of nucleoside A, except that the lipophilic chain a in the raw material is replaced by one of the lipophilic chains be.

[0154] Table 1: Some synthetic raw materials for nucleoside AE

[0155]

[0156] Some of the synthetic raw materials for nucleoside AE ​​are shown in Table 1.

[0157] Nucleosides AE with different special modification types are synthesized by the above method, and their general structural formula is as follows: Figure 7 shown.

[0158] Example 2: siRNA sequence design and synthesis

[0159] siRNA sequence design

[0160] siRNA-T1 (target gene GenBank: MH180383.1), the sequence of its sense strand is 5'- GCCUG UAGCCCAUGU UGUA TT-3', and the sequence of its antisense strand is 5'- UACAA CAUGG GCUAC AGGC TT -3'. From the 5' end of its sense strand, a non-bridging oxygen atom of the α-phosphate group in the 2nd, 3rd, 4th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 16th, 18th and 19th base nucleotides is replaced by a sulfur atom, that is, a thio modification is used. In addition, different types of lipophilic chain modifications are made to each siRNA-T1 sequence. See Table 2 for sequence information.

[0161] Table 2: siRNA-T1 modified sequence information

[0162]

[0163] siRNA sequence synthesis

[0164] Using 5 commercially available nucleosides of AGCUT and nucleosides AE with different nitrogenous bases obtained in Example 1, the siRNA sequence was synthesized by the phosphoramidite triester method, that is, by repeating the four procedures of "deprotection-coupling-oxidation-capping", and the oligonucleotide chain was extended by one base after each repetition. The deprotection reagent was a trichloroethane / dichloromethane mixture, the activator was 5-benzylthiotetrazole, the oxidant was iodine solution or (E)-N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithiazol-5-yl)formamidine, the thio reagent used 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 synthetic product, deprotection and annealing were performed.

[0165] The annealing products were purified by HPLC to obtain different siRNA sequences.

[0166] Purification materials and parameters: NanoQ-15L anion exchange column was used, balance solution: 100 mmol / L tris (pH 7.5), 10 mmol / L ethylenediaminetetraacetic acid (pH 8.0), 300 mmol / L NaCl, pH 9.0, conductivity 32 mS / cm. Eluent: 100 mmol / L tris (pH 7.5), 10 mmol / L ethylenediaminetetraacetic acid (pH 8.0), 700 mmol / L NaCl, pH 9.0, conductivity 65.2 ms / cm, flow rate 10 mL / min.

[0167] Example 3: siRNA transfection of mouse brain cells

[0168] Mouse brain cell preparation

[0169] Take the gray matter of the brain from the brain of B6 mice (3-4 months old, female, purchased from Shanghai Shengchang Biotechnology Co., Ltd.), carefully remove the meninges, blood vessels and fiber components, and rinse them in Hanks solution once or twice. Place the rinsed gray matter tissue in 30-50 times the volume of Hanks solution, and repeatedly blow and beat to make a cell suspension. Pour the suspension into a centrifuge tube, and after the centrifuge tube is left upright at room temperature for 5-10 minutes, the cells or cell clumps will naturally sink, and fat and other debris will float easily. Carefully remove the upper liquid, and repeat two or three times. Add an appropriate amount of culture medium to the remaining sediment, filter it through a gauze mesh, count the cells of the filtered cell fluid, and adjust the cell density. Inoculate the mouse brain cells into a culture medium bottle or dish and culture them in a 5% CO2 incubator. Subculture an appropriate amount of cells to a 12-well plate to ensure that the cells can grow to a density of about 50% per well the next day.

[0170] Type A modified siRNA transfection

[0171] During transfection, one of the six siRNA molecules 1#A~6#A was added to each well of cells. The six siRNA molecules were set up in high and low dose groups. The siRNA transfection concentration of the high dose group was 100nM, and the siRNA transfection concentration of the low dose group was 30nM. 48h after transfection, the RNA genome was extracted using the Tiangen kit RNA prep Pure Cell / Bacteria Kit (Cat. No. DP430), and then reverse transcribed to obtain cDNA.

[0172] The mRNA expression of TNF-α was detected by Q-PCR. Figure 8As shown in A. When the transfection concentration was 30nM or 100nM, all six siRNAs could effectively knock down the expression of mRNA. At low and high transfection concentrations, the knockdown effect of mRNA expression of 5#A molecule was the best, and the expression levels of the remaining mRNA after knockdown were about 36% and about 18.3%, respectively. Since 5#A molecule had the best effect in specifically knocking down TNF-α mRNA, 5#A molecule was selected for subsequent verification.

[0173] B~E type modified siRNA transfection

[0174] Referring to the transfection results of type A modified siRNA, 5#B~5#E siRNAs were used for transfection experiments.

[0175] During transfection, one siRNA from 5#B, 5#C, 5#D or 5#E was added to each well of cells. Each siRNA was set up in high and low dose groups. The siRNA transfection concentration of the high dose group was 100nM, and the siRNA transfection concentration of the low dose group was 30nM. At 48h, RNA genome was extracted according to the Tiangen kit RNA prep Pure Cell / Bacteria Kit (Cat. No. DP430), and then reverse transcribed to obtain cDNA.

[0176] The mRNA expression of TNF-α was detected by Q-PCR. Figure 8 B. When the transfection concentration was 30nM or 100nM, the four siRNAs could effectively knock down the expression of mRNA. At low and high transfection concentrations, 5#C had the best effect, and the expression levels of the remaining mRNA after knockdown were about 29% and about 15.5%, respectively. The knockdown effect from high to low was: 5#C>5#B>5#D>5#E.

[0177] Example 4: Effect of intrathecal injection of siRNA molecules on target gene mRNA expression in mice

[0178] siRNA 5# (without special modification), 5#A, 5#B, 5#C, 5#D and 5#E were prepared into sterile preparations, and the solvent was sterile water for injection. During the preparation, 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, and 0.22 mg / ml potassium chloride were added to part of the solvent at a mass concentration calculated according to the final volume. After fully dissolved, the solution was diluted with the solvent to a near final volume, and finally the pH was adjusted to 6.9-7.1 using 1 mg / ml HCl or NaOH solution. 50 μL of siRNA preparation was injected intrathecally into B6 mice. The mice were euthanized 24 hours after the injection, and the hearts, livers, spleens, lungs, kidneys, spinal cords and cerebral white matter of the mice were taken, and the organs of a blank mouse were taken as controls and immediately frozen in liquid nitrogen.

[0179] Use 75% ethanol solution to disinfect tweezers and scissors to eliminate RNase as much as possible. Place a 2 ml centrifuge tube on ice and add an appropriate amount of PBS buffer to prepare tissue homogenate. The mass volume ratio of tissue homogenate required for the experiment is about 10%. Take the tissue sample to be ground from the refrigerator at minus 80°C, place it in a centrifuge tube on ice, and use sterilized tweezers and scissors to cut the sample into small pieces. Put the sterilized zirconium oxide grinding balls into the centrifuge tube according to the number of one per centrifuge tube. Place the centrifuge tube in a grinder for grinding, set the vibration frequency to 1000 times / minute, the vibration time to 5 minutes, and the temperature to -20°C. After grinding, place the centrifuge tube in a precooled high-speed centrifuge and centrifuge at 4000 rpm for 30 min. Take out the supernatant with a pipette and transfer it to a new centrifuge tube. Use Tiangen RNA prep Pure Cell / Bacteria Kit (Cat. No. DP430) to extract the RNA genome, and use Q-PCR to detect the expression of TNF-α mRNA in each organ.

[0180] Effects of intrathecal injection of 5# siRNA on target gene mRNA expression in mice Fig. 9 As shown. The results showed that 5# had basically no knockdown effect on the target gene in the heart, spleen, and lung, and had a certain knockdown effect in the liver, kidney, spinal cord, and brain white matter, but the knockdown effect was less than 50%. 5#A~5#E had almost no knockdown effect in the heart, and had different degrees of knockdown effects in the liver, spleen, lung, kidney, spinal cord, and brain white matter, and were generally better than 5#. Among them, 5#C had the best effect, with a knockdown of about 45% in the liver, about 71% in the spleen, about 65% in the lung, about 52.5% in the kidney, about 21% in the spinal cord, and about 28.5% in the brain white matter ( Fig. 9 D).

[0181] In summary, the specially modified siRNA molecules of the present invention can achieve the effect of knocking down the expression of target gene mRNA in the liver, spleen, lung, spinal cord, and brain white matter through intrathecal injection. The effect is significantly improved compared with unmodified siRNA, and has excellent lipophilicity and distribution properties.

[0182] Example 5: Effect of intradermal injection of siRNA molecules on target gene mRNA expression in mice

[0183] 5#A, 5#B, 5#C, 5#D, 5#E and 5#molecule without modified structure were prepared into sterile preparations respectively, and the preparation method was referred to Example 4. The preparation containing 50 μg siRNA was intradermally injected into B6 mice, and the mice were euthanized 24 hours after the 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 immediately quickly frozen in liquid nitrogen.

[0184] Use 75% ethanol solution to disinfect tweezers and scissors to eliminate RNase as much as possible. Place a 2 ml centrifuge tube on ice and add an appropriate amount of PBS buffer to prepare tissue homogenate. The mass volume ratio of tissue homogenate required for the experiment is about 10%. Take the tissue sample to be ground from the refrigerator at minus 80°C, place it in a centrifuge tube on ice, and use sterilized tweezers and scissors to cut the sample into small pieces. Put the sterilized zirconium oxide grinding balls into the centrifuge tube according to the number of one per centrifuge tube. Place the centrifuge tube in a grinder for grinding, set the vibration frequency to 1000 times / minute, the vibration time to 5 minutes, and the temperature to -20°C. After grinding, place the centrifuge tube in a precooled high-speed centrifuge and centrifuge at 4000 rpm for 30 min. Take out the supernatant with a pipette and transfer it to a new centrifuge tube. Use Tiangen RNA prep Pure Cell / Bacteria Kit (Cat. No. DP430) to extract the RNA genome, and use Q-PCR to detect the expression of TNF-α mRNA in each organ.

[0185] Effects of intradermal injection of siRNA on target gene mRNA expression in mouse skin Fig.10As shown. The results showed that 5# knocked down the expression level of mRNA in skin tissue to about 67.5%. The five specially modified siRNA molecules can effectively knock down the expression of mRNA, and the effect is better than 5#. The expression levels of the remaining mRNA after knockdown of 5#A~5#E were approximately 19.5%, 27.5%, 3.5%, 52.5%, and 55.0%, respectively. It can be seen that 5#C has the best effect, and the knockdown effect from high to low is: 5#C>5#A>5#B>5#D>5#E.

[0186] The experimental results show that the specially modified siRNA of the present invention can achieve the effect of knocking down the expression of target gene mRNA in skin tissue by intradermal injection. The effect is significantly improved compared with unmodified siRNA, and it has excellent lipophilicity and transdermal permeability.

[0187] Example 6: Cytotoxicity of siRNA molecules

[0188] 5# A, 5# B, 5# C, 5# D, and 5# E molecules were transfected into HaCat cells respectively, and then the cytotoxicity was detected using a kit. The specific steps are as follows:

[0189] S1: On the first day, passage an appropriate amount of cells into a 96-well plate to ensure that the cells can grow to about 50% density in each well on the second day.

[0190] S2: On the second day, when the density reached about 50%, siRNA preparations 5#A to 5#E (the preparation method refers to Example 4) were added to each well for transfection, and the transfection concentration was 100 pmol siRNA.

[0191] S3: The transfected cells were incubated overnight at 37°C with 5% CO2.

[0192] S4: At different time points, measure the absorbance of each well at 450 nm using an enzyme reader.

[0193] Depend on Fig.11 It was found that the five siRNA molecules showed slight toxicity 4-8 hours after transfection, and then the cell viability gradually returned to normal, which shows that the five siRNA molecules have little toxicity to cells.

[0194] Example 7: Evaluation of in vivo delivery distribution of siRNA molecules

[0195] The 5#A siRNA molecule with FAM linked to the 3' end of the sense strand was synthesized using a CPG (Controlled Pore Glass) column linked to FAM, and then prepared into a sterile preparation (preparation method refers to Example 4). 50 μL of siRNA preparation was injected intrathecally into B6 mice, and the mice were euthanized 3 hours after injection. The heart, liver, spleen, lung, kidney, spinal cord and brain white matter of the mice were taken for tissue fluorescence imaging.

[0196] Depend on Fig.12 The fluorescence signals are shown in descending order: spinal cord, brain, liver, kidney, while there is basically no signal detected in the heart, spleen, and lung. 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.

[0197] In summary, the present invention increases the lipophilicity of the siRNA sequence through lipophilic chain modification. The modified siRNA molecules achieve excellent in vivo carrier-free delivery efficiency and distribution performance through intrathecal injection, intradermal injection and other administration methods, and have good tolerability and safety while effectively knocking down the expression of the target gene.

[0198] The above embodiments are only preferred embodiments for fully illustrating the technical solutions and effects of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Equivalent substitutions or changes made by technicians in this field on the basis of the present invention are all within the protection scope of the present invention.

Claims

1. A nucleoside having a structure as shown in formula (I), characterized in that: It comprises R1, R3 and lipophilic R2, wherein: R1 is selected from adenine, uracil, cytosine or guanine, An adenine group, a uracil group, a cytosine group or a guanine group containing one or more modifications selected from the group consisting of thiolation, halogenation, hydroxylation, amination, carboxylation or methylation, an adenine methyl group, a uracil methyl group, a cytosine methyl group or a guanine methyl group containing one or more modifications selected from the group consisting of thiolation, halogenation, hydroxylation, amination, carboxylation or methylation, or An adenine carboxyl group, a uracil carboxyl group, a cytosine carboxyl group or a guanine carboxyl group containing one or more modifications selected from the group consisting of thiolation, halogenation, hydroxylation, amination, carboxylation or methylation; R2 is selected from the following groups: R3 is selected from 4,4'-dimethoxytrityl, 4-monomethoxytrityl, 4,4',4"-trimethoxytrityl, pivaloyloxymethyl, tert-butyldimethylsilyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, 2-cyanoethoxy-N,N-diisopropylaminophosphino, 3-levulinyl, acetyl, benzoyl, 2-cyanoethyl, N,N-dibutylaminocarbonyl, N,N-dimethylaminocarbonyl, isobutyryl, 2-(2-nitrophenyl)-propoxycarbonyl, triethylammonium, trifluoroacetyl, triisopropylsiloxymethyl, p-isopropylphenoxyacetyl, O-acetal levulinyl ester, phenylacetyl, or 1,1',3,3'-tetraisopropyldisiloxane.

2. A double-stranded siRNA comprising a sense strand and an antisense strand, wherein the sequence of the sense strand is 5'-GCCUGUAGCCCAUGUUGUATT-3', and the sequence of the antisense strand is 5'-UACAACAUGGGCUACAGGCTT-3', characterized in that: The nucleotides are specially modified, and the specially modified nucleotides contain R1 and lipophilic R2, and have a structure as shown in formula (II): R1 is selected from adenine, uracil, cytosine or guanine, An adenine group, a uracil group, a cytosine group or a guanine group containing one or more modifications selected from the group consisting of thiolation, halogenation, hydroxylation, amination, carboxylation or methylation, an adenine methyl group, a uracil methyl group, a cytosine methyl group or a guanine methyl group containing one or more modifications selected from the group consisting of thiolation, halogenation, hydroxylation, amination, carboxylation or methylation, or An adenine carboxyl group, a uracil carboxyl group, a cytosine carboxyl group or a guanine carboxyl group containing one or more modifications selected from the group consisting of thiolation, halogenation, hydroxylation, amination, carboxylation or methylation; R2 is selected from the following groups:

3. The double-stranded siRNA according to claim 2, characterized in that The specially modified nucleotide is 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.

4. The double-stranded siRNA according to claim 2, characterized in that 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.

5. The double-stranded siRNA according to claim 3, characterized in that 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; 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.

6. The double-stranded siRNA according to claim 4, characterized in that The specially modified nucleotides are located at: 1st position 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 in the 5' to 3' direction of the antisense strand.

7. The double-stranded siRNA according to claim 2, characterized in that The specially modified nucleotides are located at: position 1 in the 5' to 3' direction of the sense strand, and position 1 in the 5' to 3' direction of the antisense strand; positions 1 and 19 in the 5′ to 3′ direction of the sense strand, and 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 sense strand, and 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 sense strand, and positions 1, 2, 18 and 19 in the 5' to 3' direction of the antisense strand; Positions 1, 5, 8, 14 and 17 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, and positions 9, 10, 11, 17 and 18 in the 5' to 3' direction of the antisense strand.

8. The double-stranded siRNA according to any one of claims 2 to 7, characterized in that One of the non-bridging oxygen atoms in the phosphate group attached to 3'-C in the 2nd, 3rd, 4th, 6th, 7th, 9th, 10th, 11th, 12th, 13th, 15th, 16th, 18th and 19th nucleomonomers of the sense strand is replaced by a sulfur atom.

9. A method for preparing the nucleoside according to claim 1, characterized in that: The method comprises the synthesis of a lipophilic chain and reacting the lipophilic chain with a dehydrated nucleoside to obtain the nucleoside as claimed in claim 1, wherein the dehydrated nucleoside is selected from dehydrated adenosine or an analog thereof, dehydrated uracil or an analog thereof, dehydrated cytosine or an analog thereof, dehydrated thymine or an analog thereof, or dehydrated guanine or an analog thereof.

10. A method for preparing a double-stranded siRNA according to any one of claims 2 to 8, characterized in that: The method comprises synthesizing RNA using the nucleoside described in claim 1 as a raw material.

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

12. A method for preparing the pharmaceutical composition according to claim 11, characterized in that: The method comprises adding double-stranded siRNA and a pharmaceutically acceptable carrier or excipient into a solvent.

13. Use of the double-stranded siRNA according to any one of claims 2 to 8 in the preparation of a medicament for treating a disease or condition mediated by TNF-α.

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