Aminocholesterol derivative, nanoparticles containing aminocholesterol derivative and use thereof

Abstract

Disclosed are an aminocholesterol derivative and a composition comprising same. Nanoparticles containing the aminocholesterol derivative are used for delivering nucleic acid drugs to improve the delivery efficiency for the nucleic acid drugs, thereby enhancing the targeting and therapeutic effect of the nucleic acid drugs. Thus, the nanoparticles are of great significance for the development and application of nucleic acid prophylactic and therapeutic agents.

Description

Aminocholesterol derivatives, nanoparticles containing the same and applications thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to and the benefit of the following Chinese patent application, filed with the State Intellectual Property Office of China: Chinese Patent Application No. 202311419461.8, filed on October 30, 2023, entitled “Aminocholesterol Derivatives, Nanoparticles Containing Same, and Uses Thereof.” The entirety of the aforementioned patent application is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of medical technology, and in particular to an aminocholesterol derivative, lipid nanoparticles containing the aminocholesterol derivative, and applications of the same in the delivery of nucleic acid drugs. Background Art

[0004] Nucleic acid drugs use DNA or RNA to express proteins in the body, producing therapeutic effects. Alternatively, they utilize siRNA to interfere with protein expression in the body, thereby producing therapeutic effects. Nucleic acid drugs have shown therapeutic potential in viral vaccines, protein replacement therapy, tumor immunotherapy, cell therapy, and gene editing. To achieve these therapeutic effects, nucleic acid drugs must first enter target cells and produce sufficient target proteins. However, developing safe and effective nucleic acid delivery vectors is crucial for targeted delivery of nucleic acid drugs and achieving endosomal escape.

[0005] Researchers have developed a series of delivery systems for mRNA delivery, including lipid-based delivery systems and polymer-based delivery systems. Among them, lipid-based lipid nanoparticles (LNPs) have become the most promising mRNA delivery system due to their good biosafety and delivery efficiency. Many other lipid nanoparticle preparations have entered the clinic for the treatment of viral infections, tumors, and genetic diseases. The protein expression level of lipid nanoparticle preparations is an important evaluation indicator for clinical research on nucleic acid drugs. Therefore, how to construct a highly transfective delivery material has become the key to the successful development of nucleic acid drugs.

[0006] Summary of the Invention

[0007] The present invention aims to provide an aminocholesterol derivative and nanoparticles containing the aminocholesterol derivative. The aminocholesterol lipid disclosed herein is used to deliver nucleic acids, thereby increasing the delivery efficiency of nucleic acid drugs and thereby enhancing the targeting and therapeutic effect of nucleic acid drugs.

[0008] The present disclosure provides a compound represented by formula (I), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0009] in,

[0010] X is selected from a bond, C(=O), (CH2) p , S, O, CH2CH2O or OC(=O)NH;

[0011] Y is selected from a bond, C(=O), (CH2) k , S, O, CH2CH2S, CH2CH2S-S, CH2CH2O or CH2CH2NH;

[0012] Z is selected from a bond, C(=O), (CH2) q , S or O;

[0013] R 1 Selected from C1-C 14 Alkyl or C2-C 14 Alkenyl, the C1-C 14 Alkyl or C2-C 14 Alkenyl is optionally substituted with OH, NH2 or halogen;

[0014] Every R 2 are independently selected from H or C1-C6 alkyl, the C1-C6 alkyl being optionally replaced by R a replace;

[0015] Every R 3 are independently selected from H or C1-C6 alkyl, the C1-C6 alkyl being optionally replaced by R a replace;

[0016] R a Selected from OH, NH2 or halogen, wherein the OH, NH2 is optionally substituted by C1-C6 alkyl or C1-C6 alkyl-NH2;

[0017] p, k, q are independently selected from 1, 2, 3 or 4;

[0018] n is selected from 0, 1, 2 or 3;

[0019] m is selected from 1, 2, 3 or 4.

[0020] In some embodiments, X is selected from C(=O), (CH2) p , S, O, CH2CH2O or OC(=O)NH.

[0021] In some embodiments, X is selected from C(═O), CH 2 , S, O, CH 2 CH 2 O, or OC(═O)NH.

[0022] In some embodiments, Y is selected from a bond, C(=O), (CH2) k, S, CH2CH2S-S or CH2CH2O.

[0023] In some embodiments, Y is selected from a bond, C(═O), CH 2 CH 2 , S, CH 2 CH 2 S—S, or CH 2 CH 2 O.

[0024] In some embodiments, Z is selected from a bond, C(=O) or (CH2) q .

[0025] In some embodiments, Z is selected from a bond, C(=O), CH2CH2, or CH2.

[0026] In some embodiments, Selected from the following structures: C(=O)O, CH2, C(=O), CH2CH2O, CH2CH2S-S, C(=O)CH2CH2O, C(=O)CH2CH2S-SO, C(=O)(CH2)4O, C(=O)CH2CH2OCH2CH2O or CH2CH2NHCH2CH2NH C(=O)O.

[0027] In some embodiments, Selected from the following structures: C(=O)O, CH2, C(=O) or CH2CH2O.

[0028] In some embodiments, It is C(=O)O.

[0029] In some embodiments, R 1 Selected from C1-C 10 Alkyl, the C1-C 10 The alkyl group is optionally substituted with OH, NH2 or halogen.

[0030] In some embodiments, R 1 Selected from

[0031] In some embodiments, R 1 Selected from

[0032] In some embodiments, R a is selected from NH2, wherein the NH2 is optionally substituted by C1-C6 alkyl or C1-C6 alkyl-NH2.

[0033] In some embodiments, R a Selected from NH2, -NHCH2CH2NH2 or -N(CH2CH2NH2)2.

[0034] In some embodiments, R aSelected from NH2 or -NHCH2CH2NH2.

[0035] In some embodiments, each R 2 Independently selected from H, CH2CH2NH2 or CH2CH2NHCH2CH2NH2.

[0036] In some embodiments, R 2 For H.

[0037] In some embodiments, each R 3 Independently selected from H, CH2CH2NH2 or CH2CH2NHCH2CH2NH2.

[0038] In some embodiments, R 3 For H.

[0039] In some embodiments, p, k, q are independently selected from 1 or 2.

[0040] In some embodiments, n is selected from 0 or 1.

[0041] In some embodiments, m is selected from 1, 2, or 3.

[0042] In some embodiments, n is selected from 0, and m is selected from 1, 2, or 3; or if n is 1, then m is 1 or 2.

[0043] In some embodiments, the compound represented by formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is selected from the compound represented by formula (II), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0044] Among them, X, Y, Z, R 2 、R 3 , m, n are as defined above.

[0045] In some embodiments, the compound of formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is selected from the following compounds, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0046] Another object of the present disclosure is to provide a composition comprising a nucleic acid and a compound represented by the above formula (I) or formula (II), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0047] In some embodiments, the composition further comprises one or more of a cationic lipid, a neutral helper lipid, cholesterol, and a PEG-modified lipid, preferably two or more.

[0048] In some embodiments, the composition comprises:

[0049] a) cationic lipid; b) neutral auxiliary lipid; c) cholesterol; d) PEG-modified lipid; and e) the compound represented by the above formula (I) or formula (II), or its stereoisomer or pharmaceutically acceptable salt.

[0050] In some embodiments, in the composition, based on the total molar amount of lipids as 100%, the molar ratio of each lipid component is:

[0051] a) cationic lipid 45% to 50%;

[0052] b) neutral helper lipids 5% to 10%;

[0053] c) Cholesterol 35% to 48%;

[0054] d) PEG-modified lipids 0-3%;

[0055] and e) 2-10% of the compound represented by formula (I) or formula (II) as described above, or its stereoisomers, or its pharmaceutically acceptable salts.

[0056] Another object of the present disclosure is to provide a nanoparticle, wherein the nanoparticle comprises the compound represented by the above formula (I) or formula (II), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0057] In some embodiments, the nanoparticles further comprise one or more of a cationic lipid, a neutral helper lipid, cholesterol, and a PEG-modified lipid, preferably two or more thereof.

[0058] In some embodiments, the nanoparticles comprise:

[0059] a) cationic lipids;

[0060] b) neutral helper lipids;

[0061] c) cholesterol;

[0062] d) PEG-modified lipids; and

[0063] e) The compound represented by the above formula (I) or (II), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0064] In some embodiments, the cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethanoloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315, or any one or more thereof.

[0065] In some embodiments, the neutral helper lipid is selected from any one or more of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE).

[0066] In some embodiments, the PEG-modified lipid is selected from any one or more of methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), DMG-PEG2000, DMG-PEG5000, DSPE-PEG5000, and DSPE-PEG2000.

[0067] In some embodiments, in the nanoparticles, based on the total molar amount of lipids as 100%, the molar ratio of each lipid component is:

[0068] a) cationic lipid 45% to 50%;

[0069] b) neutral helper lipids 5% to 10%;

[0070] c) Cholesterol 35% to 48%;

[0071] d) PEG-modified lipids 0-3%;

[0072] and e) 2-10% of the compound represented by formula (I) or formula (II) as described above, or its stereoisomers, or its pharmaceutically acceptable salts.

[0073] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the cationic lipid is 45% to 48%.

[0074] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the neutral helper lipid is 6% to 10%.

[0075] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the neutral helper lipid is 8% to 10%.

[0076] In some embodiments, the molar ratio of the PEG-modified lipid is 1% to 3%, based on the total molar amount of the lipid being 100%.

[0077] In some embodiments, the molar ratio of the PEG-modified lipid is 1% to 2%, based on the total molar amount of the lipid being 100%.

[0078] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of cholesterol is 35% to 42%.

[0079] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of cholesterol is 36% to 40%.

[0080] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the compound represented by formula (I) or formula (II), or its stereoisomer, or its pharmaceutically acceptable salt is 4% to 8%.

[0081] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the compound represented by formula (I) or formula (II), or its stereoisomer, or its pharmaceutically acceptable salt is 4% to 6%.

[0082] In some embodiments, the nanoparticle further comprises a nucleic acid.

[0083] In some embodiments, the nucleic acid comprises RNA or DNA.

[0084] In some embodiments, the RNA includes one or more of non-self-replicating RNA (mRNA), self-replicating RNA (repRNA), anti-replicating RNA (taRNA), circular RNA (circRNA), microRNA (miRNA), small (short) interfering (siRNA), lncRNA, saRNA, piRNA, sgRNA, and tsRNA.

[0085] In some embodiments, the DNA comprises one or more of linear DNA, circular DNA, complementary DNA (cDNA), plasmid DNA, oligonucleotide, and antisense oligonucleotide.

[0086] In some embodiments, the nanoparticles can be in liquid or solid formulations.

[0087] In some embodiments, the nanoparticles are suitable for administration by injection.

[0088] In some embodiments, the nanoparticles are suitable for administration by inhalation.

[0089] Another object of the present disclosure is to provide the use of a compound represented by formula (I) or formula (II), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a composition thereof, or a nanoparticle in the preparation of a nucleic acid drug.

[0090] In another aspect, the present disclosure provides a method for targeted delivery of nucleic acids, comprising administering to a subject in need thereof a therapeutically effective amount of the composition or nanoparticles, wherein the composition or nanoparticles comprises the compound represented by formula (I) or formula (II) described above, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0091] In another aspect, the present disclosure provides methods of modulating gene expression comprising delivering a nucleic acid to a cell, the method comprising contacting the cell with a nanoparticle or composition described herein under conditions sufficient to cause uptake of the nucleic acid into the cell.

[0092] In some embodiments, the cells are contacted in vitro or ex vivo.

[0093] In some embodiments, the cells are contacted in vivo.

[0094] In some embodiments, the modulation of gene expression is sufficient to treat or prevent a disease or disorder.

[0095] In another aspect, the present disclosure provides a nanoparticle for delivering non-self-replicating RNA, wherein the nanoparticle comprises an aminocholesterol derivative, and the cell transfection efficiency of the nanoparticle is above 90%.

[0096] In another aspect, the present disclosure provides a nanoparticle for delivering self-replicating RNA, wherein the nanoparticle comprises an aminocholesterol derivative, and the cell transfection efficiency of the nanoparticle is above 50%.

[0097] In another aspect, the present disclosure provides a nanoparticle for delivering anti-replicative RNA, wherein the nanoparticle comprises an aminocholesterol derivative, and the cell transfection efficiency of the nanoparticle is above 20%.

[0098] In another aspect, the present disclosure provides a nanoparticle for delivering circular RNA, wherein the nanoparticle comprises an aminocholesterol derivative, and the cell transfection efficiency of the nanoparticle is above 50%.

[0099] In some embodiments, the aminocholesterol derivative is a compound represented by formula (I) or formula (II) as described above, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0100] In some embodiments, the cell is a HeLa cell.

[0101] Any embodiment of any aspect of the present disclosure may be combined with other embodiments, provided that no contradiction occurs. In addition, in any embodiment of any aspect of the present disclosure, any technical feature may be applicable to the technical feature in other embodiments, provided that no contradiction occurs.

[0102] Compared with the prior art, the present disclosure has the following beneficial effects:

[0103] The present disclosure provides a new class of aminocholesterol lipids, which are added to nanoparticles composed of any one or more components of cationic lipids, neutral lipids, cholesterol and PEG lipids for the delivery of nucleic acid drugs. They can significantly improve the transfection efficiency of nucleic acid drugs (such as anti-replicating RNA, circular RNA, self-replicating RNA, and non-self-replicating RNA), thereby increasing the targeting and therapeutic effect of nucleic acid drugs.

[0104] Definitions and Explanations of Terms

[0105] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The groups and term definitions recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A specific term should not be considered as uncertain or unclear in the absence of a special definition, but should be understood according to its common meaning in the art. Scientific and technical terms related to this disclosure should have the meanings understood by those of ordinary skill in the art. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0106] As used herein, the term "nucleic acid" includes in its broadest sense any compound and / or substance comprising a nucleotide polymer. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof.

[0107] As used herein, the term "mRNA" refers to messenger ribonucleic acid. mRNA can be naturally occurring or non-naturally occurring or synthetic. For example, mRNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. mRNA can include a cap structure, a 5' transcription leader, a 5' untranslated region, a start codon, an open reading frame, a stop codon, a chain terminating nucleoside, a stem-loop, a hairpin, polyadenylic acid (polyA), a polyadenylation signal, and / or one or more cis-regulatory elements. mRNA can have a nucleotide sequence encoding a polypeptide. Translation of mRNA, such as in vivo translation of mRNA in mammalian cells, can produce a polypeptide. Traditionally, the basic components of a natural mRNA molecule include at least one coding region, a 5'-untranslated region (5'UTR), a 3'UTR, a 5' cap, and a polyA sequence.

[0108] As used herein, the term "untranslated region" or "UTR" refers to the portion of the mRNA upstream of the start codon and downstream of the stop codon that is not translated and is therefore referred to as the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR), respectively. These regions are transcribed with the coding region and are therefore exonic when present in the mature mRNA.

[0109] As used herein, the term "5' untranslated region, 5'UTR" generally refers to a portion of an mRNA that is located 5' (i.e., "upstream") of the open reading frame and is not translated into protein. The 5'UTR is generally understood to be a specific segment of a messenger RNA (mRNA) that is located at the 5' end of the open reading frame of the mRNA. Typically, the 5'UTR starts at the transcription start site and terminates at one nucleotide before the start codon of the open reading frame. Preferably, the 5'UTR has a length of more than 20, 30, 40, or 50 nucleotides. The 5'UTR may contain elements for controlling gene expression, also referred to as regulatory elements. The regulatory elements may be, for example, ribosome binding sites. The 5'UTR may be modified post-transcriptionally, for example, by adding a 5'-cap. The 5'UTR of an mRNA is not translated into an amino acid sequence. The 5'UTR sequence is generally encoded by the gene that is transcribed into each mRNA during gene expression. The genomic sequence is first transcribed into pre-mRNA, which includes optional introns. The pre-mRNA is then further processed into mature mRNA during the maturation process. The maturation process comprises the following steps: 5' capping, splicing of the pre-mature mRNA to remove optional introns and 3' end modification (such as polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / or exonuclease cleavage, etc.). Within the scope of the present disclosure, the 5'UTR corresponds to the mature mRNA sequence located between the start codon and, for example, the 5'-cap. Preferably, the 5'UTR corresponds to a sequence extending from the nucleotide located at the 3' side of the 5' cap, more preferably from the 3' side nucleotide immediately adjacent to the 5' cap, to the nucleotide located at the 5' side of the start codon of the protein coding region, preferably to the nucleotide immediately adjacent to the 5' side of the start codon of the protein coding region. The nucleotide immediately adjacent to the 3' side of the mature mRNA 5' cap typically corresponds to the transcription start site. The term "corresponding to" means that the 5'UTR sequence can be an RNA sequence in the mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to this RNA sequence.

[0110] As used herein, the term "3' untranslated region, 3'UTR" generally refers to a part of mRNA that is located 3' (i.e., "downstream") of the open reading frame and is not translated into protein. Typically, 3'UTR is a part of the mRNA between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of the mRNA. In the context of the present disclosure, the term 3'UTR can also include elements that are not encoded in the template from which the RNA is transcribed, but are added after transcription during maturation, such as poly(A) sequences. The 3'UTR of mRNA is not translated into an amino acid sequence. The 3'UTR sequence is typically encoded by a gene that is transcribed into a respective mRNA during gene expression. The genomic sequence is first transcribed into a pre-mature mRNA comprising optional introns. The pre-mature mRNA is then further processed into a mature mRNA during maturation. The maturation process comprises the following steps: 5' capping, splicing of the pre-mature mRNA to remove optional introns and 3' end modification (such as polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / or exonuclease cleavage, etc.). Within the scope of the present disclosure, the 3'-UTR corresponds to the stop codon of the protein coding region, preferably between the 3' end of the stop codon of the protein coding region and the poly (A) sequence of the mRNA. The term "corresponding to" means that the 3'-UTR sequence can be an RNA sequence in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to this RNA sequence. Preferably, the 3'UTR has a length of more than 20, 30, 40 or 50 nucleotides.

[0111] The term "RNA" as used herein includes but is not limited to non-self-replicating RNA (mRNA), self-replicating RNA (repRNA), anti-replicating RNA (taRNA), circular RNA (circRNA), microRNA (miRNA), small (short) interfering (siRNA), lncRNA, saRNA, piRNA, sgRNA, and tsRNA.

[0112] The term "non-self-replicating RNA" as used herein refers to conventional mRNA in the art, and its exemplary sequence is as follows:

[0113] As used herein, the term "self-replicating RNA (repRNA)" refers to self-replicating RNA with an open reading frame at its 5' end that encodes four nonstructural proteins (nsPs). Upon entry into host cells, repRNA is first translated into four nonstructural proteins (nsP1, nsP2, nsP3, and nsP4), which then assemble into an RNA-dependent RNA polymerase complex. The RNA polymerase complex then synthesizes a complementary negative-strand RNA intermediate from the positive-strand RNA, which then serves as a template for the synthesis of positive-strand RNA, leading to protein expression within the cell.

[0114] As used herein, the term "anti-replicating RNA (taRNA)" refers to the process of splitting self-replicating RNA into two sequences: a replicase (nsPs) and an anti-replicating RNA (TaRNA). After mixing the two sequences, cells are transfected. The nsPs segment translates into an RNA replicase complex, which then synthesizes the TaRNA into a complementary negative-strand RNA intermediate. The negative-strand RNA is then used as a template to synthesize positive-strand RNA, which then expresses protein in the cell.

[0115] As used herein, the term "circular RNA (circRNA)" refers to a closed RNA molecule formed by reverse splicing of a linear RNA. CircRNAs lack a 5' cap and a 3' poly(A) tail, forming a complete circular structure by linking the 5' and 3' ends. After circRNAs enter cells, the internal ribosome entry site (IRES) sequence they contain mediates RNA translation, leading to intracellular protein expression.

[0116] The RNA described herein can be linearized using BsaI or BspQI restriction endonucleases according to conventional plasmid linearization and purification methods in the art. An in vitro transcription system is prepared according to conventional systems in the art. mRNA is transcribed in vitro and purified for subsequent LNP preparation.

[0117] As used herein, the term "deoxyribonucleic acid (DNA)" can be naturally occurring or non-naturally occurring, including but not limited to single-stranded DNA, double-stranded DNA, circular DNA. The DNA can comprise one or more modified (e.g., altered or substituted) nucleobases, nucleosides, nucleotides, or combinations thereof. The DNA in the nanoparticle can comprise any useful modification or alteration, such as modifications or alterations to nucleobases, sugars, or internucleoside linkages (e.g., to phosphate linkages, to phosphodiester linkages, to phosphodiester backbones).

[0118] In some embodiments, DNA includes, but is not limited to, linear DNA, circular DNA, complementary DNA (cDNA), plasmid DNA, oligonucleotides, and antisense oligonucleotides. As used herein, the term "nucleic acid drug" or "nucleic acid-based drug" refers to a drug containing nucleic acid for the prevention or treatment of a specific disease, condition, or disorder.

[0119] As used herein, a "nanoparticle" is a particle comprising one or more lipids. Nanoparticles are typically on the order of microns or smaller in size.

[0120] As used herein, the term "cationic lipid" is ionizable, and the cationic ionizable lipid contains one or more groups that are protonated at physiological pH but can be deprotonated and are uncharged at a pH above 8, 9, 10, 11 or 12. The ionizable cationic group can contain one or more protonatable amines that can form cationic groups at physiological pH. Other examples of cationic lipids include, but are not limited to, N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium bromide. ammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethanoloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315.

[0121] The structural compound of DLin-MC3-DMA has a CAS number of 1224606-06-7 and a structural formula as follows:

[0122] The structural compound of SM-102 has a CAS number of 2089251-47-6 and a structural formula as follows:

[0123] The structural compound of ALC-0315 has the CAS number 2036272-55-4 and the structural formula is as follows:

[0124] Other examples of the term "neutral lipid" as used herein include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), or phosphatidylethanolamine (DLPE).

[0125] Other examples of the term "PEG-modified lipids" as used herein include, but are not limited to, methoxypolyethylene glycol ditetradecylacetamide (ALC-0159), DMG-PEG2000, DMG-PEG5000, DSPE-PEG5000, DSPE-PEG2000.

[0126] As used herein, the term "w / v%" represents mass volume concentration. For example, a mass volume concentration (w / v%) of about 4% to about 25% may represent about 4 to 25 g / 100 mL.

[0127] In this article Indicates the attachment site.

[0128] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge and dotted wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0129] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom in a molecule between two positions. The compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist as two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present disclosure encompasses all tautomeric forms of the compounds.

[0130] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0131] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so that the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present invention and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of which are within the definition of the compounds of the present invention and mixtures thereof. Compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.

[0132] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0133] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes that the event or situation occurs and that the event or situation does not occur. For example, an ethyl group is "optionally" substituted with halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any sterically impossible and / or incomposable replacement or substitution pattern will not be introduced.

[0134] When any variable (such as R a 、R b ) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is represented by two R b is replaced, then each R b There are independent options.

[0135] When one of the variables is selected from a chemical bond or does not exist, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0136] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a bond.

[0137] When the linking group mentioned in this article does not specify its connection direction, its connection direction is arbitrary. When Y is selected from "CH2CH2O", Y can be connected to Z and X from left to right to form "Z-CH2CH2O-X", or Z and X can be connected from right to left to form "Z-OCH2CH2-X".

[0138] In this article, C m -C n It means having an integer number of carbon atoms in the range mn. For example, "C1-C 10 ” means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0139] The term "alkyl" refers to a group of the formula C n H 2n+1 The term "C1-C 14The term "alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon radical having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms. Specific examples of the alkyl radical include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2 -dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C1-C6 alkyl" may be understood to mean an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms, specific examples of which include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc. The term "C1-C3 alkyl" may be understood to mean a straight-chain or branched saturated alkyl group having 1, 2 or 3 carbon atoms. The "C1-C 14 "Alkyl" may contain "C1-C 10 "C1-C6 alkyl" or "C1-C3 alkyl" and the like, wherein the "C1-C6 alkyl" may further include "C1-C3 alkyl".

[0140] The term "alkenyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. 14 "Alkenyl" is understood to mean a linear or branched unsaturated hydrocarbon radical containing one or more double bonds and having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms, "C2-C 14 "Alkenyl" is preferably "C2-C 10 Preferably, the alkenyl group is a C2-C6 alkenyl group, more preferably a C2-C4 alkenyl group, and even more preferably a C2 or C3 alkenyl group. It will be understood that when the alkenyl group contains more than one double bond, the double bonds may be separated from each other or conjugated. Specific examples of the alkenyl group include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, or (Z)-1-methylprop-1-enyl.

[0141] The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine.

[0142] The term "hydroxy" refers to an -OH group.

[0143] The term "amino" refers to a -NH2 group.

[0144] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0145] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0146] The term "therapeutically effective amount" means

[0147] An amount of a compound of the present disclosure that (i) treats a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein.

[0148] The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0149] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0150] The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable acid addition salts or base addition salts, including salts formed between a compound and an inorganic acid or organic acid, and salts formed between a compound and an inorganic base or an organic base.

[0151] The term "composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0152] The term "pharmaceutically acceptable carrier" refers to those excipients that have no significant irritation to organisms and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art.

[0153] The term "comprise" or "comprises" and its English variations such as comprises or comprising are to be understood in an open, non-exclusive sense, ie, "including but not limited to".

[0154] Typical routes of administration of the compositions of the present disclosure include, but are not limited to, inhalation, intraperitoneal, mucosal, intramuscular, subcutaneous, and intravenous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] FIG1 is a diagram showing the in vivo transfection effects of the LNPs prepared in the example and the control group after 24 h of administration;

[0156] FIG2 is a diagram showing the in vivo transfection effects of the LNPs prepared in the example and the control group after administration for 72 h. DETAILED DESCRIPTION

[0157] The present disclosure is further described below with reference to specific embodiments and accompanying drawings. The following embodiments are intended only to illustrate the present disclosure and are not intended to limit the scope of application of the present disclosure. Modifications or substitutions made to the methods, steps, or conditions of the present disclosure without departing from the spirit and essence of the present disclosure are within the scope of the present disclosure.

[0158] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0159] The compounds and English abbreviations involved in the examples are as follows:

[0160] ALC-0315, CAS No.: 2036272-55-4, structural formula is as follows:

[0161] SM-102, CAS No.: 2089251-47-6, structural formula is as follows:

[0162] ALC-0159, CAS number: 1849616-42-7, structural formula is as follows:

[0163] DMG-PEG2000, CAS No.: 160743-62-4, structural formula is as follows:

[0164] DSPC, CAS No.: 816-94-4, structural formula is as follows:

[0165] DC Cholesterol, CAS No.: 166023-21-8, has the following structural formula:

[0166] GL67, the structural formula is as follows:

[0167] Cholesterol, CAS number: 57-88-5.

[0168] (1) Equipment and consumables

[0169] (2) Reagents

[0170] Unless otherwise specified, the ratios of mixed solvents are by volume. For example, "0-60% ethyl acetate / petroleum ether gradient elution" means that the volume ratio of ethyl acetate to petroleum ether during the gradient elution process is 0:100-60:40. Alternatively, "petroleum ether / ethyl acetate = 10 / 1 to 1 / 1" means that the volume ratio of petroleum ether to ethyl acetate during the elution process is 10 / 1 to 1 / 1.

[0171] Unless otherwise stated, % refers to wt%.

[0172] Compounds are manually or The software named the commercially available compounds using the supplier's catalog name.

[0173] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6 The solvents for NMR measurements are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and tetramethylsilane (TMS) is the internal standard.

[0174] Abbreviations:

[0175] MeOH: methanol; Boc2O: di-tert-butyl dicarbonate; DMF: N,N-dimethylformamide; THF: tetrahydrofuran; TEA: triethylamine; TFAA: trifluoroacetic anhydride; DCM: dichloromethane; TFA: trifluoroacetic acid.

[0176] Example 1 Synthesis of Compound 1 (SHA)

[0177] Compound 1 (SHA): (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl(2-aminoethyl)(2-((2-((2-aminoethyl)amino)ethyl)amino)ethyl)amino)ethyl)formate trifluoroacetate was synthesized as follows:

[0178] Step 1: Synthesis of compound 1-2

[0179] Compound 1-1 (50.0 g, 264 mmol, 1.00 eq) was dissolved in methanol (2.50 L) at -70°C, and a methanol solution of CF3COOEt (37.5 g, 264 mmol, 1.00 eq) (250 mL) was added. The mixture was stirred at -70°C for 1 hour, then at 0°C for 1 hour. LCMS showed that compound 1-1 was completely reacted to give compound 1-2, which was dissolved in 2.75 L of methanol and used directly in the next reaction.

[0180] Step 2: Synthesis of Compounds 1-3

[0181] To a solution of compound 1-2 (75.4 g, 264 mmol, 1.00 eq) in methanol (2.75 L) was added a solution of Boc2O (288 g, 1.32 mol, 5.00 eq) in methanol (250 mL) at 0°C. The mixture was stirred at 20°C for 16 hours. LCMS indicated that compound 1-2 had reacted completely. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (Silica Flash Column, 0-50% ethyl acetate / petroleum ether gradient elution) to afford compound 1-3 (80.0 g, 117 mmol, 44.2% yield) as a white solid.

[0182] 1 HNMR(CDCl3): δ3.48(s,4H),3.33(s,12H),1.43–1.66(m,36H).

[0183] Step 3: Synthesis of Compounds 1-4

[0184] Compound 1-3 (60.0 g, 87.5 mmol, 1.00 eq) was dissolved in methanol (1.5 L) and aqueous ammonia (197 g, 1.40 mol, 25% purity) was added to adjust the pH to 11. The mixture was stirred at 35°C for 40 hours. The reaction mixture was concentrated under reduced pressure to remove the methanol. The residue was diluted with water (300 mL), extracted twice with dichloromethane (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Silica Flash Column, 0-10% methanol / dichloromethane gradient elution) to afford compound 1-4 (35.0 g, 59.4 mmol, 67.8% yield) as a pale yellow solid.

[0185] 1 HNMR(CDCl3): δ3.26–3.32(m,14H),1.42–1.46(m,38H).

[0186] Step 4: Synthesis of Compounds 1-5

[0187] Compound 1-4 (15.0 g, 25.4 mmol, 1.00 eq) was dissolved in N,N-dimethylformamide (70.0 mL), and NaHCO₃ (6.41 g, 76.3 mmol, 3.00 eq) was added. 2-Bromoacetonitrile (6.10 g, 50.9 mmol, 2.00 eq) was dissolved in DMF (20.0 mL) and added dropwise to the reaction mixture. The mixture was stirred at 20°C for 16 hours. The mixture was poured into water (1.00 L), the precipitate was filtered, and the filter cake was concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to afford compound 1-5 (9.00 g, 14.3 mmol, 56.3% yield).

[0188] Step 5: Synthesis of Compounds 1-6

[0189] Compound 1-5 (15.0 g, 23.9 mmol, 1.00 eq) was dissolved in tetrahydrofuran (100 mL), and triethylamine (4.83 g, 47.7 mmol, 6.64 mL, 2.00 eq) and trifluoroacetic anhydride (6.01 g, 28.6 mmol, 1.20 eq) were added. The mixture was stirred at 20°C for 1 hour and then concentrated under reduced pressure. The crude product was purified by column chromatography (Silica Flash Column, 0-60% ethyl acetate / petroleum ether gradient elution) to obtain compound 1-6 (10.6 g, 14.6 mmol, 61.3% yield).

[0190] 1 HNMR (d6-DMSO): δ4.56 (s, 1H), 3.65 (s, 2H), 3.23 (s, 8H), 3.10-3.17 (m, 2H), 3.01 (s, 2H), 1.37 (d, J = 6.11Hz, 36H).

[0191] Step 6: Synthesis of Compounds 1-7

[0192] Compound 1-6 (10.6 g, 14.6 mmol, 1.00 eq) and Boc2O (6.97 g, 31.9 mmol, 2.18 eq) were dissolved in THF (60 mL). Pd / C (1.00 g, 10% purity) was then added. Under argon, the suspension was vacuum degassed, filled with hydrogen, and stirred at 50°C under a hydrogen atmosphere (50 psi) for 16 hours. The precipitate was filtered off, and the filtrate was concentrated under reduced pressure. The concentrate was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to obtain compound 1-7 (9.10 g, 11.0 mmol, 75% yield).

[0193] 1 HNMR(d6-DMSO): δ3.51(s,2H),3.38-3.43(m,2H),3.19-3.27(m,10H),3.13(s,3H),3.01(s,2H),1.37–1.39(m,45H).

[0194] Step 7: Synthesis of Compounds 1-8

[0195] Compound 1-7 (10.8 g, 13.0 mmol, 1.00 eq) was dissolved in methanol (500 mL) and K2CO3 (9.00 g, 65.1 mmol, 5.00 eq) was added. The mixture was stirred at 30°C for 16 hours. The precipitate was filtered off, and the filtrate was concentrated under reduced pressure. The concentrate was purified by column chromatography (Silica Flash Column, elution with 0-10% methanol / dichloromethane) to obtain compound 1-8 (3.90 g, 5.32 mmol, 40.8% yield).

[0196] 1 HNMR(d6-DMSO): δ4.10(s,2H),3.08-3.27(m,23H),3.01(s,3H),2.61-2.71(m,2H),1.37–1.39(m,45H)

[0197] Step 8: Synthesis of Compounds 1-10

[0198] Compound 1-8 (3.90 g, 5.32 mmol, 1.00 eq) and compound 1-9 (5.87 g, 10.6 mmol, 2.00 eq) were dissolved in dichloromethane (40.0 mL), and triethylamine (1.37 g, 13.6 mmol, 2.55 eq) was added. The mixture was stirred at 40°C for 16 hours and then concentrated under reduced pressure. The concentrate was purified by column chromatography (Silica Flash Column, 0-50% ethyl acetate / petroleum ether gradient elution) to obtain compound 1-10 (4.5 g crude product).

[0199] 1 HNMR (d6-DMSO): δ8.12 (d, J=9.17 Hz, 0.5H), 7.01-7.15 (m, 1H), 6.90-6.95 (m, 0.5H), 6.72-6.87 (m, 1H), 5.32 (s, 1H), 4.22-4.38 (m, 1H), 2.93-3.28 (m, 21H), 2.14-2.36 (m, 2H), 1.72-1.97 (m, 5H), 1.46-1.63 (m, 6H), 1.28-1.45 (m, 49H), 0.81-1.15 (m, 22H), 0.65 (s, 3H). Step 9: Synthesis of Compounds 1-11

[0200] Compound 1-10 (3.60 g, 3.14 mmol, 1.00 eq) was dissolved in methanolic hydrochloric acid (2 M, 100 mL, 63.6 eq). The mixture was stirred at 30°C for 16 hours. The mixture was concentrated under reduced pressure to afford compound 1-11 (2.35 g, 2.84 mmol, 90.4% yield).

[0201] 1 HNMR(d6-DMSO): δ5.29(s,1H),2.98-3.39(m,21H),2.22(s,2H),1.66-1.98(m,5H),0.72-1.59(m,36H),0.58(s,3H).

[0202] Step 10: Synthesis of Compound 1

[0203] Using HPLC (column: Phenomenex luna C 18 150*25mm*10um; mobile phase A: water (containing 0.1% TFA), mobile phase B: acetonitrile; gradient elution: mobile phase B 34%-64%, elution 10 min). Compound 1 (abbreviated as SHA) (2.30 g, 1.89 mmol, yield 62.6%) was obtained.

[0204] m / z(ESI):645.6[M+H] + .

[0205] 1 HNMR(d6-DMSO): δ5.28-5.36(m,1H),4.25-4.38(m,1H),2.97-3.34(m,20H ),2.16-2.30(m,2H),1.66-2.00(m,5H),0.73-1.58(m,34H),0.62(s,3H).

[0206] In addition to compound 1 synthesized in this example, the synthesis of other specific compounds involved in the present invention can be obtained by referring to the synthesis route and source materials of this example.

[0207] Example 2 Preparation of lipid nanoparticles (Green Fluorescent Protein (GFP) non-self-replicating RNA)

[0208] Different lipids and green fluorescent protein (GFP) non-self-replicating RNA were added to lipid nanoparticles to prepare different lipid nanoparticles. Green fluorescent protein (GFP) non-self-replicating RNA was prepared using conventional methods in the art and used to prepare different lipid nanoparticles.

[0209] Table 1 Formulation of lipid nanoparticles

[0210] Preparation process:

[0211] (1) Aqueous phase preparation: Dilute GFP non-self-replicating RNA to 0.2 mg / mL with citrate buffer;

[0212] (2) Preparation of organic phase: Dissolve lipids (ALC0315, ALC0159, DSPC, cholesterol, aminocholesterol and its derivatives; or SM102, DMG PEG2K, DSPC, cholesterol, aminocholesterol and its derivatives) in anhydrous ethanol. The specific formula is shown in Table 1.

[0213] (3) The aqueous phase and the organic phase were subjected to microfluidics to prepare lipid nanoparticles, which were diluted with PBS buffer and concentrated by ultrafiltration centrifuge tube. The particles were then diluted again with PBS buffer and concentrated by ultrafiltration centrifuge tube. Sucrose (cryoprotectant) mother solution was added to make its content reach 10% (w / v%), and the final product was frozen at -80°C for storage.

[0214] Example 3 Preparation of lipid nanoparticles (GFP repRNA)

[0215] Different lipids and GFP self-replicating RNA were added to lipid nanoparticles to prepare different lipid nanoparticles. GFP self-replicating RNA was prepared using conventional methods in the art and used to prepare different lipid nanoparticles.

[0216] The prescription LNP9 of this embodiment is the same as the prescription LNP1 of Example 2, the prescription LNP10 of this embodiment is the same as the prescription LNP2 of Example 2, the prescription LNP11 of this embodiment is the same as the prescription LNP3 of Example 2, the prescription LNP12 of this embodiment is the same as the prescription LNP4 of Example 2, the prescription LNP13 of this embodiment is the same as the prescription LNP5 of Example 2, the prescription LNP14 of this embodiment is the same as the prescription LNP6 of Example 2, the prescription LNP15 of this embodiment is the same as the prescription LNP7 of Example 2, and the prescription LNP16 of this embodiment is the same as the prescription LNP8 of Example 2; the preparation process is the same as that of Example 2, except that the GFP non-self-replicating RNA of Example 2 is replaced by GFP self-replicating RNA (GFP repRNA), and the corresponding prescriptions prepared are LNP 9-LNP16.

[0217] Example 4 Preparation of lipid nanoparticles (GFP TaRNA)

[0218] Different lipids and GFP anti-replicating RNA were added to the lipid nanoparticles to prepare different lipid nanoparticles. GFP anti-replicating RNA was prepared using conventional methods in the art and used to prepare different lipid nanoparticles.

[0219] The prescription LNP17 of this embodiment is the same as the prescription LNP1 of Example 2, the prescription LNP18 of this embodiment is the same as the prescription LNP2 of Example 2, the prescription LNP19 of this embodiment is the same as the prescription LNP3 of Example 2, the prescription LNP20 of this embodiment is the same as the prescription LNP4 of Example 2, the prescription LNP21 of this embodiment is the same as the prescription LNP5 of Example 2, the prescription LNP22 of this embodiment is the same as the prescription LNP6 of Example 2, the prescription LNP23 of this embodiment is the same as the prescription LNP7 of Example 2, and the prescription LNP24 of this embodiment is the same as the prescription LNP8 of Example 2; the preparation process is the same as that of Example 2, except that the GFP non-self-replicating RNA of Example 2 is replaced by a mixed solution of GFP anti-replicating RNA (GFP taRNA) and nsPs RNA (molar ratio 2.6:1), and the corresponding prescriptions are prepared as LNP 17-LNP24.

[0220] Example 5 Preparation of lipid nanoparticles (GFP CircRNA)

[0221] Different lipids and GFP circular RNA were added to lipid nanoparticles to prepare different lipid nanoparticles. GFP circular RNA was prepared using conventional methods in the art and used to prepare different lipid nanoparticles.

[0222] The prescription LNP25 of this embodiment is the same as the prescription LNP1 of Example 2, the prescription LNP26 of this embodiment is the same as the prescription LNP2 of Example 2, the prescription LNP27 of this embodiment is the same as the prescription LNP3 of Example 2, the prescription LNP28 of this embodiment is the same as the prescription LNP4 of Example 2, the prescription LN29 of this embodiment is the same as the prescription LNP5 of Example 2, the prescription LNP30 of this embodiment is the same as the prescription LNP6 of Example 2, the prescription LNP31 of this embodiment is the same as the prescription LNP7 of Example 2, and the prescription LNP32 of this embodiment is the same as the prescription LNP8 of Example 2; the preparation process is the same as that of Example 2, except that the GFP non-self-replicating RNA of Example 2 is replaced with GFP circular RNA (GFPCircRNA), and the corresponding prescriptions prepared are LNP 25-LNP32.

[0223] Test Example 1: Lipid Nanoparticle Size and Potential Detection

[0224] The lipid nanoparticles were measured for particle size and potential using a Malvern Zetasizer ultra. 10 μL of the lipid nanoparticles prepared in this example was diluted to 1 mL with water for injection and placed in a test cell. Particle size, PDI, and zeta potential were measured in triplicate for each sample. The results are shown in Table 2.

[0225] Test Example 2: Encapsulation Efficiency Detection

[0226] The total RNA concentration in the LNP preparation was determined using the RiboGreen assay. The mRNA-LNP preparation solution was demulsified with Triton X100. 100 μL of the demulsified solution was added to a 96-well plate. Then, 100 μL of the RiboGreen dye solution was added. The plate was shaken on a plate shaker for 5 minutes at 600 rpm. Detection was performed using a SpectraMax iD3 multi-function microplate reader. Total RNA concentration was calculated using a standard curve.

[0227] Take 10 μL of the mRNA-LNP preparation solution, add 990 μL of buffer, mix thoroughly, add 100 μL of RiboGreen dye solution, and place on a plate shaker for 5 minutes at 600 rpm. Detect using a SpectraMax iD3 multi-function microplate reader. Calculate free RNA concentration using a standard curve.

[0228] Encapsulation efficiency (%) = 100% - (free mRNA concentration / total mRNA concentration) x 100%

[0229] The test results are shown in Table 2.

[0230] Test Example 3: Detection of in vitro transfection efficiency

[0231] HeLa cells were cultured at 1×10 5 The cells were seeded into 24-well plates at a density of 100 cells / well and incubated overnight in a 37°C cell culture incubator until the cells covered approximately 80% of the bottom area of ​​the wells. The LNPs prepared in the example were transfected into Hela cells at a final concentration of 1 μg / ml.

[0232] The specific operations are as follows:

[0233] Add 50 μl of opti-MEM medium to a nuclease-free 1.5 mL centrifuge tube, then add 0.5 μg of the LNP prepared in the example, mix gently and incubate at room temperature for 2-5 minutes, then add dropwise to the wells of a 24-well plate. Gently shake the culture medium to evenly distribute the transfection complex in the culture medium. After incubation in a 37°C cell culture incubator for 20-24 hours, the expression of the target protein GFP was detected by flow cytometry. Transfection efficiency (%) = (number of cells expressing fluorescence / total number of cells) x 100%. The test results are shown in Table 2.

[0234] Table 2 Physicochemical properties of lipid nanoparticles and cell transfection results NA: indicates no aminocholesterol added

[0235] Results showed that LNPs containing cationic lipids, phospholipids, neutral lipids, and cholesterol had low or no transfection efficiency for RNA. However, the addition of aminocholesterol significantly improved transfection efficiency in HeLa cells, particularly for self-replicating RNA, anti-replicating RNA, and circular RNA.

[0236] Test Example 4: In vivo transfection experiment

[0237] C57BL / 6 mice aged 6-8 weeks and weighing over 16 g (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were acclimated and housed for one week. On day 0, they were randomly divided into groups of three. Each group received LNP2 (30 μg, Group G1); LNP2 (10 μg, Group G2); LNP1 (10 μg, Group G3); and saline (Group G4) via the right hind leg. Live imaging was performed 6, 24, and 72 hours after administration. At 24 hours, one mouse from each group was euthanized, and the heart, liver, spleen, lung, and kidney tissues were dissected and imaged. At 72 hours, all mice were euthanized, and the heart, liver, spleen, lung, and kidney tissues were dissected and imaged. Fluorescence intensity was measured. Statistical results are shown in Figures 1 and 2.

[0238] The results showed that the LNP formulation containing aminocholesterol significantly increased lung expression compared to the formulation without aminocholesterol. In particular, the LNP formulation prepared by adding SHA aminocholesterol to the LNP had a more pronounced lung targeting effect.

[0239] Although the embodiments of the present disclosure have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A compound represented by formula (I), or a stereoisomer thereof or a pharmaceutically acceptable salt thereof: in, X is selected from a bond, C(=O), (CH2) p , S, O, CH2CH2O or OC(=O)NH; Y is selected from a bond, C(=O), (CH2) k , S, O, CH2CH2S, CH2CH2S-S, CH2CH2O or CH2CH2NH; Z is selected from a bond, C(=O), (CH2) q , S or O; p, k, q are independently selected from 1, 2, 3 or 4; R 1 Selected from C1-C 14 Alkyl or C2-C 14 Alkenyl, the C1-C 14 Alkyl or C2-C 14 The alkenyl group is optionally substituted with OH, NH2 or halogen; Every R 2 independently selected from H or C1-C6 alkyl, the C1-C6 alkyl being optionally substituted by R a replace; n is selected from 0, 1, 2 or 3; Every R 3 independently selected from H or C1-C6 alkyl, the C1-C6 alkyl being optionally substituted by R a replace; m is selected from 1, 2, 3 or 4; R a Selected from OH, NH2 or halogen, wherein the OH, NH2 is optionally substituted by C1-C6 alkyl or C1-C6 alkyl-NH2.

2. The compound of formula (I) according to claim 1, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: X is selected from C(=O), (CH2) p , S, O, CH2CH2O or OC(=O)NH; Alternatively, X is selected from C(═O), CH 2 , S, O, CH 2 CH 2 O or OC(═O)NH.

3. The compound of formula (I) according to claim 1 or 2, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: Y is selected from a bond, C(=O), (CH2) k , S, CH2CH2S-S or CH2CH2O; Alternatively, Y is selected from a bond, C(=O), CH2CH2, S, CH2CH2S-S or CH2CH2O.

4. The compound of formula (I) according to any one of claims 1 to 3, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: Z is selected from a bond, C(=O) or (CH2) q ; Alternatively, Z is selected from a bond, C(=O), CH2CH2 or CH2.

5. The compound of formula (I) according to any one of claims 1 to 4, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: Selected from the following structures: C(=O)O, CH2, C(=O), CH2CH2O, CH2CH2S-S, C(=O)CH2CH2O, C(=O)CH2CH2S-SO, C(=O)(CH2)4O, C(=O)CH2CH2OCH2CH2O or CH2CH2NHCH2CH2NHC(=O)O; or, Selected from the following structures: C(=O)O, CH2, C(=O) or CH2CH2O; or, It is C(=O)O.

6. The compound of formula (I) according to any one of claims 1 to 5, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R 1 Selected from C1-C 10 Alkyl, the C1-C 10 The alkyl group is optionally substituted with OH, NH2 or halogen; Or, R 1 Selected from Or, R 1 Selected from 7. The compound of formula (I) according to any one of claims 1 to 6, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R a is selected from NH2, wherein the NH2 is optionally substituted by C1-C6 alkyl or C1-C6 alkyl-NH2; or, R a is selected from NH2, -NHCH2CH2NH2 or -N(CH2CH2NH2)2; or, R a Selected from NH2 or -NHCH2CH2NH2.

8. The compound of formula (I) according to any one of claims 1 to 7, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: Every R 2 is independently selected from H, CH2CH2NH2 or CH2CH2NHCH2CH2NH2; or, R 2 For H.

9. The compound of formula (I) according to any one of claims 1 to 8, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: Every R 3 is independently selected from H, CH2CH2NH2 or CH2CH2NHCH2CH2NH2; or, R 3 For H.

10. The compound of formula (I) according to any one of claims 1 to 9, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: n is selected from 0 or 1.

11. The compound of formula (I) according to any one of claims 1 to 10, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: m is selected from 1, 2 or 3.

12. The compound of formula (I) according to any one of claims 1 to 11, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: The compound represented by formula (I), or its stereoisomer or its pharmaceutically acceptable salt is selected from the compound represented by formula (II), or its stereoisomer or its pharmaceutically acceptable salt:

13. The compound of formula (I) according to claim 1, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: The compound represented by formula (I), or its stereoisomer or its pharmaceutically acceptable salt is selected from the following compounds, or its stereoisomer or its pharmaceutically acceptable salt:

14. A composition, wherein The composition comprises a nucleic acid and a compound of formula (I) as described in any one of claims 1 to 13, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

15. A nanoparticle, wherein: The nanoparticles include the compound of formula (I) as described in any one of claims 1 to 13, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

16. The nanoparticle according to claim 15, wherein The nanoparticles further include one or more of cationic lipids, neutral auxiliary lipids, cholesterol and PEG-modified lipids.

17. The nanoparticle according to claim 15 or 16, wherein The nanoparticles include: a) cationic lipids; b) neutral helper lipids; c) cholesterol; d) PEG-modified lipids; and e) A compound of formula (I) according to any one of claims 1 to 13, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

18. The nanoparticle according to claim 17, wherein The cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOT AP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethoxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315, any one or more thereof; The neutral auxiliary lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatid ylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl- Any one or more of phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE); The PEG-modified lipid is selected from any one or more of methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), DMG-PEG2000, DMG-PEG5000, DSPE-PEG2000, and DSPE-PEG5000.

19. The nanoparticle according to claim 17 or 18, wherein In the nanoparticles, the molar ratio of each lipid component is as follows, based on the total molar amount of lipids as 100%: a) cationic lipid 45% to 50%; b) neutral auxiliary lipid 5% to 10%; c) cholesterol 35% to 48%; d) PEG-modified lipid 0 to 3%; and e) 2-10% of the compound of formula (I) according to any one of claims 1 to 13, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

20. The nanoparticle according to any one of claims 15 to 19, wherein The nanoparticles also include nucleic acids.

21. The nanoparticle according to claim 20, wherein The nucleic acid includes RNA or DNA.

22. The nanoparticle according to claim 21, wherein The RNA includes one or more of non-self-replicating RNA (mRNA), self-replicating RNA (repRNA), anti-replicating RNA (taRNA), circular RNA (circRNA), micro RNA (miRNA), siRNA, lncRNA, saRNA, piRNA, sgRNA, and tsRNA.

23. The nanoparticle according to claim 21, wherein The DNA includes one or more of linear DNA, circular DNA, complementary DNA (cDNA), plasmid DNA, oligonucleotide, and antisense oligonucleotide.

24. Use of the compound of formula (I) as described in any one of claims 1 to 13, or its stereoisomer or pharmaceutically acceptable salt, or the composition as described in claim 14, or the nanoparticles as described in any one of claims 15 to 23 in the preparation of nucleic acid drugs.

25. A method for targeted delivery of nucleic acids, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 14 or the nanoparticles of any one of claims 15-23, wherein the composition or nanoparticles comprises a compound of formula (I) as described in any one of claims 1-13, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

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