Novel ionizable lipid as well as preparation method and application thereof

Abstract

The invention provides novel ionizable lipid as well as a preparation method and application thereof. The structural formula is shown as a formula (I). The ionizable lipid compound contains urea and a bioisostere structure thereof, the encapsulation effect of the ionizable lipid is improved through hydrogen bonds formed by squaramide, urea and thiourea structures and nucleic acid, and the immunogenicity of the ionizable lipid is reduced. The nanoparticles prepared from the ionizable lipid are high in encapsulation efficiency, low in PDI and wide and stable in process adaptability. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a novel ionizable lipid and a preparation method thereof and an application thereof in a delivery system. Background Art

[0002] Lipid nanoparticles (LNP) are the most advanced nucleic acid molecule delivery system in clinical practice. As a non-viral delivery vector, they have been approved for in vivo delivery of mRNA. LNP is a multi-component system, usually composed of ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol-lipid conjugates.

[0003] As the most important component of LNP, ionizable lipids can promote cell uptake and help drug molecules escape from endosomes. At the same time, they affect the encapsulation rate of nucleic acid drugs, the delivery efficiency of nucleic acid drugs in the body, and cytotoxicity, which determines the efficacy of LNP.

[0004] The US FDA has fully approved two LNP-based COVID-19 vaccines, Pfizer-BioNTech (BNT162b2, using ionizable lipid ALC-0315) and Moderna (mRNA-1273, using ionizable lipid SM-102). The great success of mRNA vaccines in fighting COVID-19 has promoted the research and rapid transformation of LNPs, but LNPs still have safety issues in preclinical development, mainly due to the strong inflammatory response caused by ionizable lipids. This problem hinders the long-term administration of LNPs and limits their dosage. Therefore, ensuring that LNPs have high transfection efficiency while maximizing their immunogenicity has become a major issue in the design of ionizable lipids. Summary of the invention

[0005] The object of the present invention is to provide an ionizable lipid compound, which contains the structure of urea and its bioisostere, and increases the encapsulation effect of the ionizable lipid through hydrogen bonds formed between the amide, urea and thiourea structures and nucleic acids, thereby reducing the immunogenicity of the ionizable lipid.

[0006] In a first aspect, the present invention provides an ionizable lipid compound, the structural formula of which is shown in formula (I):

[0007]

[0008] In formula (I), L1, L2, L3 and L4 are the same or different and are independently selected from: -O(C=O)-, -(C=O)O-, -O(C=O)O-, -(C=O)S-, -S(C=O)-, -O(C=S)-, -(C=S)O-, -O-, -SS-, -S-, -(C=S)S-, -S(C=S)-;

[0009] R1 and R2 are the same or different and are independently selected from: C1-C 18 Straight or branched alkyl, C3-C 18 alkenyl;

[0010] M1 and M5 are the same or different and are independently selected from: C1-C 15 Straight or branched alkylene, M7-SS-M7, M7 is selected from C1-C6 alkylene;

[0011] M2 and M4 are the same or different and are independently selected from: C1-C 15 Alkylene, C2-C 15 Alkenylene, Wherein M6 is selected from C2-C6 alkylene, and two M6 are connected to L2 and N atom in formula (I) respectively;

[0012] M3 is selected from C1-C6 alkylene;

[0013] G is selected from H, -OH and at least one of the following structures:

[0014]

[0015] Wherein A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl.

[0016] Specifically, L1, L2, L3 and L4 are the same or different, and are each independently selected from: -O(C=O)-, -(C=O)O-, -O(C=O)O-, -(C=O)S-, -S(C=O)-, -O-, -SS-, -S-.

[0017] More specifically, L1 and L4 are the same, L2 and L3 are the same, and are each independently selected from: -O(C=O)-, -(C=O)O-, -(C=O)S-, -S(C=O)-, -SS-.

[0018] Specifically, R1 and R2 are the same or different and are independently selected from: C2-C 18 Straight chain or branched chain alkyl.

[0019] Specifically, M1 and M5 are the same or different and are independently selected from: C2-C 15Straight chain or branched alkylene, M7-SS-M7, M7 is selected from C1-C3 straight chain alkylene.

[0020] Specifically, M2 and M4 are the same and are each independently selected from C2-C 11 Alkylene;

[0021] More specifically, -M2-L2-M1-L1-R1 and -M4-L3-M5-L4-R2 are the same.

[0022] Specifically, G is selected from H, -OH and at least one of the following structures:

[0023]

[0024] Wherein A is selected from the following:

[0025]

[0026] Furthermore, the above-mentioned ionizable lipid compound is selected from any one of the following compounds:

[0027]

[0028]

[0029]

[0030]

[0031] In a second aspect, the present invention provides a method for preparing the ionizable lipid compound represented by the above formula (I).

[0032] G is The ionizable lipid compound represented by formula (I) is prepared by a method comprising the following steps: under alkaline conditions, a compound represented by formula (A) and a compound represented by formula (B) undergo a substitution reaction to obtain an ionizable lipid compound represented by formula (I);

[0033]

[0034] In formula (A), L1, L2, L3, L4, M1, M2, M3, M4, M5, R1, and R2 are defined the same as L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 in formula (I);

[0035] In formula (B), the definition of A is the same as that of A in formula (I)G, that is, A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl.

[0036] G is The ionizable lipid compound represented by formula (I) is prepared by a method comprising the following steps: allowing the compound represented by formula (C) to undergo an addition reaction with the compound represented by formula (D) to obtain the ionizable lipid compound represented by formula (I);

[0037]

[0038] In formula (C), L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 are defined the same as L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 in formula (I);

[0039] In formula (D), the definition of A is the same as that of A in formula (I)G, that is, A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl.

[0040] Or, G is The ionizable lipid compound represented by formula (I) is prepared by a method comprising the following steps: allowing the compound represented by formula (A) to undergo an addition reaction with the compound represented by formula (E) to obtain the ionizable lipid compound represented by formula (I);

[0041]

[0042] In formula (E), the definition of A is the same as that of A in formula (I) G, that is, A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl.

[0043] G is H or -OH, and the ionizable lipid compound of formula (I) is prepared by a method comprising the following steps: subjecting a compound of formula (F) and a compound of formula (H) to a substitution reaction with a compound of formula (I) to obtain an ionizable lipid compound of formula (I);

[0044]

[0045] In formula (F), L1, L2, M1, M2, and R1 are defined the same as L1, L2, M1, M2, and R1 in formula (I);

[0046] In formula (H), L3, L4, M4, M5, and R2 are defined the same as L3, L4, M4, M5, and R2 in formula (I);

[0047] In formula (I), M3 is the same as the definition of M3 in formula (I); and G is H or -OH.

[0048] In a third aspect, the present invention provides a lipid carrier comprising an ionizable lipid compound represented by the above formula (I).

[0049] The lipid carrier provided by the present invention comprises the following components in its raw materials: an ionizable lipid compound represented by formula (I), a steroid, a neutral lipid and / or a polymer-bound lipid;

[0050] The polymer-bound lipid has a chemical formula of PYL, wherein P is a hydrophilic polymer portion, Y is an optional linker, and L is a lipid portion;

[0051] In the lipid nanoparticles, the molar ratio of the ionizable lipid compound represented by formula (I), the steroid, the neutral lipid and the polymer-bound lipid is preferably 30-70:30-65:0-30:0.2-5, and more preferably 30-60:35-60:0-20:0.3-3;

[0052] More preferably, the molar ratio of the ionizable lipid compound represented by formula (I), the steroid, the neutral lipid and the polymer-bound lipid is 40-50:40-45:10-15:0.5-2.

[0053] The polymer-bound lipids include one or more PEG-modified lipids;

[0054] Preferably, the PEG-modified lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphate, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; preferably, one or more of PEG-diacylglycerol (PEG-DAG), PEG-dialkoxypropyl (PEG-DAA), PEG-dimyristylglycerol (DMG-PEG), PEG-ceramide (PEG-Cer) and PEG-distearoylphosphatidylethanolamine (DSPE-PEG); more preferably, the relative molecular mass of the PEG-modified lipid is 500 to 5000, most preferably 1000, 2000, 3000, 4000 or 5000; the polymer-bound lipid is preferably DMG-PEG2000;

[0055] The steroid is selected from one or more of cholesterol, cholesterol ester, steroid hormone, steroid vitamin and phytosterol, more preferably one or more of cholesterol, cholesterol ester and phytosterol, most preferably cholesterol;

[0056] The neutral lipid is selected from phospholipids, more preferably at least one of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1, ... Glycerol-3-phospho-(1'-rac-glycerol) (DOPG), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylserine (sodium salt) (DOPS), oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), more preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0057] In a fourth aspect, the present invention provides a lipid nanoparticle loaded with nucleic acid.

[0058] The nucleic acid-loaded lipid nanoparticles provided by the present invention include the lipid carrier described in the third aspect of the present invention and the nucleic acid.

[0059] Preferably, the nucleic acid is a nucleic acid compound selected from the group consisting of artificial mRNA, chemically modified or unmodified messenger RNA comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA and replicon RNA; or any combination thereof;

[0060] Further preferably, the nucleic acid is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof;

[0061] More preferably, the nucleic acid is mRNA or an mRNA compound;

[0062] The mRNA includes one or more of a stem-loop, a chain-terminating nucleoside, a polyA sequence, a polyadenylation signal and / or a 5' cap structure.

[0063] In some embodiments, the nucleic acid is luciferase mRNA (Luc mRNA) or chicken ovalbumin (OVA) mRNA.

[0064] The "chicken ovalbumin (OVA) mRNA" in the present invention may be an mRNA encoding a known OVA protein sequence, wherein the OVA protein may be derived from the OVA protein existing in an existing biological organism, or from the OVA protein sequence in an existing gene database.

[0065] Preferably, the mass ratio of lipid carrier to nucleic acid in the nucleic acid-loaded lipid nanoparticles is 5:1-60:1.

[0066] More preferably, the mass ratio of lipid carrier to nucleic acid in the nucleic acid-loaded lipid nanoparticles is 30:1-50:1.

[0067] Most preferably, the nucleic acid is mRNA, and the mass ratio of lipid carrier to nucleic acid in the nucleic acid-loaded lipid nanoparticles is 40:1.

[0068] Preferably, the particle size of the nucleic acid-loaded lipid nanoparticles is 40 to 160 nm.

[0069] More preferably, the particle size of the nucleic acid-loaded lipid nanoparticles is 70 to 100 nm.

[0070] Most preferably, the particle size may be 75 nm to 95 nm.

[0071] Preferably, the encapsulation efficiency of nucleic acid in the lipid nanoparticles loaded with nucleic acid is greater than 80%. Exemplarily, the encapsulation efficiency can be 82%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.

[0072] In a fifth aspect, the present invention provides a method for preparing the nucleic acid-loaded lipid nanoparticles described in the fourth aspect of the present invention.

[0073] The present invention provides a method for preparing nucleic acid-loaded lipid nanoparticles, which comprises the following steps:

[0074] (A1) mixing the ionizable lipid compound represented by formula (I), steroid, neutral lipid and / or polymer-bound lipid in the above-described proportions, dissolving them in a solvent, and obtaining an organic phase liposome solution;

[0075] (A2) dissolving the nucleic acid in a buffer solution of appropriate pH value to obtain an aqueous nucleic acid solution;

[0076] (A3) The organic phase liposome solution and the aqueous phase nucleic acid solution are uniformly mixed using a microfluidic device at a certain volume ratio to obtain a nucleic acid-loaded lipid nanoparticle solution.

[0077] Preferably, the solvent used to dissolve the lipid molecules in step (A1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide or N,N-dimethylformamide, specifically anhydrous ethanol.

[0078] Preferably, the buffer solution in step (A2) is acetic acid / sodium acetate solution (acetate buffer) or citric acid / sodium citrate solution.

[0079] More preferably, the buffer solution in step (A2) is acetic acid / sodium acetate solution.

[0080] Preferably, the pH of the buffer solution in step (A2) is 4-6.

[0081] More preferably, the pH of the buffer solution in step (A2) is 5.

[0082] Preferably, the concentration of the buffer solution in step (A2) is 1 mM-100 mM.

[0083] More preferably, the concentration of the buffer solution in step (A2) is 10 mM-50 mM.

[0084] Most preferably, the concentration of the buffer solution in step (A2) is 25 mM.

[0085] Preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:1-1:10.

[0086] More preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:1-1:5.

[0087] Most preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:3.

[0088] The method further comprises the operations of sequentially performing ultrafiltration and microporous membrane filtration on the obtained nucleic acid-loaded lipid nanoparticle solution.

[0089] In a sixth aspect, the present invention provides a lipid nanoparticle preparation loaded with nucleic acid.

[0090] The preparation provided by the present invention comprises the nucleic acid-loaded lipid nanoparticles described in the fourth aspect of the present invention and a pharmaceutically acceptable carrier.

[0091] The pharmaceutically acceptable carrier includes, but is not limited to, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0092] In a seventh aspect, the present invention provides use of the ionizable lipid compound represented by formula (I) described in the first aspect or the lipid carrier described in the second aspect in the preparation of a drug for delivering nucleic acids.

[0093] In some embodiments, the nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, and circular RNA.

[0094] In some embodiments, the nucleic acid is mRNA, including mRNAs of different sequences and lengths.

[0095] In some embodiments, the nucleic acid is Firefly Luciferase mRNA or Chicken Ovalbumin (OVA) mRNA.

[0096] In some embodiments, the drugs are used to treat and / or prevent diseases including, but not limited to, infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0097] The infectious disease is especially a viral infection;

[0098] The autoimmune diseases include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetase syndrome, antineutrophil-associated vasculitis, and sensitized renal transplantation;

[0099] The cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer.

[0100] In an eighth aspect, the present invention provides a method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases using the nucleic acid-loaded lipid nanoparticles described in the fourth aspect or the nucleic acid-loaded lipid nanoparticle preparations described in the sixth aspect;

[0101] Preferably, the infectious disease is particularly a viral infection;

[0102] The autoimmune diseases include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetase syndrome, antineutrophil-associated vasculitis, and sensitized renal transplantation;

[0103] The cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer.

[0104] In a ninth aspect, the present invention provides the use of the nucleic acid-loaded lipid nanoparticles described in the fourth aspect or the nucleic acid-loaded lipid nanoparticle preparations described in the sixth aspect in the preparation of drugs for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases;

[0105] Preferably, the infectious disease is particularly a viral infection;

[0106] The autoimmune diseases include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetase syndrome, antineutrophil-associated vasculitis, and sensitized renal transplantation;

[0107] The cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer.

[0108] In the tenth aspect, the present invention provides a method for treating and / or preventing a disease or condition in a subject using the nucleic acid-encapsulated lipid nanoparticles described in the fourth aspect or the nucleic acid-encapsulated lipid nanoparticle preparation described in the sixth aspect, the method comprising administering the nucleic acid-loaded lipid nanoparticles or nucleic acid-loaded lipid nanoparticle preparation to a subject in need.

[0109] In the present invention, the term "inflammatory disease" includes autoimmune disorders, allergic disorders and inflammatory disorders, for example, selected from arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis, Crohn's disease, celiac disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, rheumatic fever, gout, organ or transplant rejection, acute or chronic graft-versus-host disease, chronic allogeneic transplant rejection, Behcet's disease, uveitis, psoriasis, dermatitis, atopic dermatitis, dermatomyositis, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, Sjogren's syndrome, and blistering disorders (e.g. pemphigus vulgaris), antibody-mediated vasculitis syndromes, including ANCA-associated vasculitis, purpura, and immune complex vasculitis (cancer or infection primary or secondary). The allergic disorder can be selected from contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, berylliosis.

[0110] In the present invention, the term "viral infection" includes but is not limited to retroviral infection, hepatitis virus infection, COVID-19 novel coronavirus infection, Zika virus infection, dengue virus infection, etc.

[0111] In the present invention, the term "cancer" includes but is not limited to melanoma, primary lung cancer (including non-small cell lung cancer and small cell lung cancer), metastatic lung cancer (lung metastasis of liver cancer, lung metastasis of breast cancer, lung metastasis of colon cancer, lung metastasis of melanoma, etc.) and cancers in other parts of the body.

[0112] Compared with the prior art, the present invention has the following beneficial effects: the nanoparticles prepared by using the ionizable lipids of the present invention have high encapsulation efficiency, low PDI, and wide and stable process adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 This is a graph showing the experimental results of the inhibition of tumor growth by lipid nanoparticles prepared from the lipid compounds of the present invention in Example 70 of the present invention.

[0114] Figure 2 This is a graph showing the effect of lipid nanoparticles prepared from the lipid compounds of the present invention in Example 70 of the present invention on prolonging the survival rate of mice after tumor inoculation. DETAILED DESCRIPTION

[0115] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0116] definition

[0117] For clarity and readability, the following scientific background information and definitions are provided. Any technical features mentioned herein or disclosed thereby may be part of or read on each embodiment of the present invention. Other definitions and explanations may be provided in the context of the present invention. Unless otherwise defined, or unless otherwise required by a specific context, the meanings of all technical terms used herein are the same as those commonly understood by those skilled in the relevant art.

[0118] The expressions "one embodiment," "an embodiment," "a specific embodiment," and the like mean that a particular characteristic, property, or feature, or a particular group or combination of characteristics, properties, or features as described in conjunction with the corresponding expression, is present in at least one embodiment of the invention. The appearances of these expressions in various places throughout the description do not necessarily refer to the same embodiment. Furthermore, the particular characteristics, properties, or features may be combined in any suitable manner in one or more embodiments.

[0119] The expression "neutral" when applied to a compound such as a lipid or steroid, or a group or moiety, means that it is neither a cationic nor anionic, e.g. a compound that does not have ionizable functional groups under physiological conditions, such as hydrocarbons; or that it is both cationic and anionic under typical physiological conditions, i.e. a zwitterionic, such as the typical natural phosphatidylcholine.

[0120] As used herein, "lipid" refers to a group of organic compounds that are derivatives of fatty acids (such as esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three categories: (1) "simple lipids", including fats, oils, and waxes; (2) "complex lipids", including phospholipids and glycolipids; and (3) "derivatized lipids", such as steroids. With respect to glycolipids, in certain embodiments, LNPs comprise glycolipids (e.g., monosialoganglioside GM1).

[0121] The term "artificial mRNA" (sequence) can generally be understood as an mRNA molecule that is not naturally occurring. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such an mRNA molecule may be non-natural due to its single sequence (which is not naturally occurring) and / or due to other modifications, such as structural modifications of non-naturally occurring nucleotides. Generally, artificial mRNA molecules can be designed and / or generated by genetic engineering methods to correspond to the desired artificial nucleotide sequence (heterologous sequence). In this context, an artificial sequence is generally a non-naturally occurring sequence, i.e., it differs from the wild-type sequence by at least one nucleotide.

[0122] The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound.

[0123] Amino lipids

[0124] The amino lipid is preferably cationizable, that is, when the pH is lowered below the pKa of the lipid's ionizable group, the amino lipid will be protonated and, when positively charged, the lipid will be able to bind to negatively charged nucleic acids.

[0125] Steroids

[0126] "Steroid" is an organic compound having four rings arranged in a specific molecular configuration. Steroids and neutral steroids include naturally occurring steroids and their analogs (such as the amphipathic lipid cholesterol hemisuccinate (CHEMS), composed of succinate esterified to the β-hydroxyl group of cholesterol as a cholesterol derivative). Neutral steroids can be steroids that do not have ionizable atoms or groups under physiological conditions, or can be zwitterionic steroids. In a preferred embodiment, the neutral steroid does not contain ionizable atoms or groups under physiological conditions. In some preferred embodiments, the steroid or steroid analog is cholesterol. The terms "steroid" and "neutral steroid" are used interchangeably herein.

[0127] Neutral lipids

[0128] The "neutral lipids" of the present invention, also referred to as "helper lipids", are preferably phospholipids or neutral phospholipids. As used herein, "neutral phospholipids" are amphiphilic compounds consisting of molecules that typically have two hydrophobic fatty acid "tails" and a hydrophilic "head" containing a phosphate group. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine or serine. Phospholipids exist in large quantities in nature. In the present invention, "phospholipids" or "neutral phospholipids" include both natural phospholipids and synthetic phospholipids.

[0129] Polymer-bound lipids

[0130] The term "polymer-bound lipid" refers to a molecule that comprises both a lipid portion and a polymer portion. Preferably, the polymer-bound lipid is a pegylated lipid or a PEG-lipid. The term "pegylated lipid" or "PEG-lipid" refers to a molecule that comprises both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include PEG-DMG and the like.

[0131] In a specific embodiment, the polymer-bound lipid has the formula PYL, wherein P is a hydrophilic polymer moiety, Y is an optional linker, and L is a lipid moiety.

[0132] Specifically, the hydrophilic polymer part P can be polyethylene glycol PEG. In a specific embodiment, the average molecular weight of the PEG part is between 1kDa and 3kDa, such as between 1.5kDa and 2.5kDa, between 1.7kDa and 2.3kDa, between 1.8kDa and 2.2kDa, between 1.9kDa and 2.1kDa, or 2kDa. Therefore, PEG can be the PEG commonly referred to as "PEG2000".

[0133] In another embodiment, the hydrophilic polymer moiety P in the polymer-bound lipid can also be a substantially hydrophilic polymer different from the above-mentioned hydrophilic polymer moieties, i.e., the hydrophilic polymer moiety P in the polymer-bound lipid can be based on poly(propylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), poly-N-(2-hydroxypropyl)methacrylamide, HESylation process (according to PMID 24681396), PASylation method (i.e., proline-alanine-serine), XTEN method known in the art (i.e., PEG-based peptides), polysarcosin or poly(vinyl acetate).

[0134] Specifically, the optional linker Y can be any useful spacer structure, for example selected from those commonly found useful in PEGylated lipids, such as, but not limited to, succinimide, amine, ether, ester, anhydride, aldehyde, ketone, amide, carbamate linkers or combinations thereof.

[0135] In particular, the lipid moiety L may be derived from a phospholipid, a sphingolipid or a ceramide. As used herein, the term "derived from a phospholipid or a ceramide" includes free radicals of phospholipids and ceramides. Examples are polymer-bound lipids comprising a phosphatidylethanolamine or phosphatidylglycerol moiety.

[0136] In a preferred embodiment, the polymer-bound lipid is a pegylated lipid. The pegylated lipid includes, but is not limited to, the following pegylated lipids: pegylated diacylglycerol lipids (PEG-DAG); pegylated ceramide lipids (PEG-Cer); pegylated phosphatidylethanolamine lipids (PEG-PE); pegylated succinic diacylglycerol lipids (PEG-S-DAG); pegylated dialkoxypropylcarbamate lipids; 1,2-dimyristyl-rac-glycerol-3-methoxypolyethylene glycol ("PEG-DMG" or "DMG-PEG"). In a more preferred embodiment, the polymer-bound lipid is DMG-PEG2000.

[0137] Preferably, as used in the art, "DMG-PEG 2000" is considered to be a mixture of 1,2-DMG PEG2000 and 1,3-DMG PEG2000 in a ratio of about 97:3.

[0138] The lipid nanoparticles of the present invention are not limited to any particular morphology, and should be interpreted as including any morphology produced when an amino lipid and optionally one or more other lipids are combined, such as in an aqueous environment and / or in the presence of a nucleic acid compound. For example, liposomes, lipid complexes, lipoplexes, etc. are within the scope of lipid nanoparticles.

[0139] The lipid nanoparticles of the present invention can be combined with at least one pharmaceutically acceptable carrier or excipient to obtain a pharmaceutical composition. The composition can be a dry composition, such as a powder or granules, or a solid unit, such as a lyophilized form or a tablet. Alternatively, the composition can be in liquid form, and each excipient can be added independently in a dissolved or dispersed (such as suspended or emulsified) form. In a preferred embodiment, the composition is formulated into a sterile solid composition, such as a powder or lyophilized form, for reorganization with an aqueous liquid carrier. For compositions comprising bioactive components described in further detail below, this preparation is also preferred.

[0140] As used herein, "nanoparticle" is a submicron particle with any structure or morphology. Submicron particles may also be referred to as colloids or colloids. Submicron particles may also be referred to as colloids or colloids. About the material on which the nanoparticle is based, and structure or morphology, the nanoparticle can be classified, for example, as nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polymers, mixing or mixed complexes, only mentioning several possible naming of specific types of nanoparticles. "Lipid nanoparticle" (LNP) is a nanoparticle formed by lipids, typically comprising at least one amphipathic, film-forming lipids and optional other lipids, further optionally comprising a load material such as a nucleic acid compound. As used herein, statement "lipid nanoparticle" or "LNP" includes any subtype and morphology of the nanoparticle formed or formed together by lipids, for example liposomes and lipoplexes.

[0141] As defined above, lipid nanoparticles include any type of nanoparticles formed or co-formed by lipids. In particular, lipid nanoparticles can be co-formed by a combination of lipids comprising at least one amphipathic, vesicle-forming lipid. Liposomes and lipoplexes are examples of lipid nanoparticles.

[0142] application

[0143] The present invention provides a method for treating or preventing infectious diseases, cancer, tumor diseases, genetic diseases, allergies, toxicity, and autoimmune diseases by using the amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0144] Accordingly, the present invention provides the use of the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions in the preparation of drugs for treating or preventing infectious diseases, cancers, tumor diseases, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0145] Wherein, the infectious disease includes viral, bacterial or protozoan infectious diseases. The virus includes but is not limited to SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), bunyavirus, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, flavivirus, hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human metapneumovirus (hMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), influenza virus, extra-intestinal pathogenic Escherichia coli, Lassa virus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, Zika virus.

[0146] The cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer.

[0147] The present invention also provides a method for using the amino lipid or lipid nanoparticle or pharmaceutical composition to perform gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA.

[0148] Accordingly, the present invention also provides the use of the above amino lipids or lipid nanoparticles in the preparation of drugs for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA.

[0149] The present invention also provides a method for delivering a drug to a subject, comprising: administering the drug formulated in the above-mentioned lipid nanoparticles to the subject.

[0150] When the drug is administered, the mode of administration is determined according to the dosage form of the drug. The dosage form of the drug is related to the excipient and / or the carrier. The general route of systemic administration includes: transdermal, oral, parenteral, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injection and / or intranasal administration. The route of local administration generally includes intradermal, transdermal, subcutaneous or intramuscular injection, or intralesional, intracranial, intrapulmonary, intracardiac, intratumoral and sublingual injection. When the drug is in the form of a vaccine, the preferred route of administration is intramuscular injection and intradermal injection.

[0151] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0152] Example 1

[0153] Synthesis route of compound A1

[0154]

[0155] Step 1, Synthesis of Compound A1-1

[0156] In a 25 mL round-bottom flask, ferric chloride (16.2 mg, 2.5 mol%), pyridine (4 mg, 1.25 mmol%), n-hexanoic acid (116.16 g / mol, 464 mg, 4.0 mmol) and 1,2-epoxydodecane (184.18 g / mol, 1.47 g, 8 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction, 1.02 g of intermediate A1-1 (white solid) was obtained by column chromatography separation and purification (n-heptane: ethyl acetate = 50:1 to 10:1) with a yield of 85%.

[0157] Step 2, Synthesis of Compound A1-2

[0158] A1-1 (300.48 g / mol, 901 mg, 3.0 mmol), 6-bromohexanoic acid (195.06 g / mol, 703 mg, 3.6 mmol), EDCI (191.70 g / mol, 2.3 g, 12.0 mmol), DMAP (122.17 g / mol, 37 mg, 0.3 mmol), DIPEA (129.24 g / mol, 2.33 g, 18 mmol) and DCM (10 mL) were added to a 25 mL round-bottom flask, and the reaction was stirred overnight at room temperature. After the reaction was completed, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (n-heptane: ethyl acetate = 100: 1 to 20: 1) to obtain 1.08 g of intermediate A1-2 (colorless oily liquid), with a yield of 76%.

[0159] Step 3, Synthesis of Compound A1-3

[0160] A1-2 (477.52 g / mol, 956 mg, 2.0 mmol), potassium carbonate (138.21 g / mol, 221 mg, 1.6 mmol), sodium iodide (149.89 g / mol, 120 mg, 0.8 mmol), N-tert-butyloxycarbonyl-1,3-propylenediamine (174.24 g / mol, 139 mg, 0.8 mmol) and acetonitrile (5 mL) were added to a 25 mL round-bottom flask, and the reaction was stirred at 75 ° C overnight. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (dichloromethane: methanol = 100: 1 to 20: 1) to obtain 511 mg of intermediate A1-3 (yellow oily liquid), with a yield of 66%.

[0161] Step 4, Synthesis of Compound A1-4

[0162] A1-3 (967.47 g / mol, 967 mg, 1.0 mmol) and dichloromethane (5 mL) were added to a 10 mL reaction tube and stirred at 0°C for 5 minutes, and then 1 mL of trifluoroacetic acid was added to the reaction. The reaction was warmed to room temperature and continued to stir at room temperature for 2 hours. After the reaction was completed, the reaction solution was adjusted to pH 8-9 with a saturated sodium bicarbonate solution, then extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 780 mg of intermediate A1-4 (yellow oily liquid), with a yield of 90%.

[0163] Step 5, Synthesis of Compound A1

[0164] A1-4 (867.35 g / mol, 434 mg, 0.5 mmol), 3-methoxy-4-(methylamino)cyclobutene-3-ene-1,2-dione (141.12 g / mol, 71 mg, 0.5 mmol), triethylamine (141.12 g / mol, 71 mg, 0.5 mmol) and ethanol (3 mL) were added to a 10 mL reaction tube and stirred overnight at room temperature. After the reaction was completed, 369 mg of product A1 (yellow oily liquid) was obtained by column chromatography separation and purification (dichloromethane: methanol = 100: 1 to 20: 1), with a yield of 76%. ESI-MS C 56 H 102 N3O 10 + [M+H] + Calculated value: 976.7560, measured value: 976.7568.

[0165] Example 2

[0166] Synthesis route of compound A2

[0167]

[0168] Step 1, Synthesis of Compound A2-1

[0169] According to steps 1 to 4 of Example 1, A2-1 was synthesized.

[0170] Step 2, Synthesis of Compound A2-2

[0171] A2-1 (867.35 g / mol, 867 mg, 1 mmol), carbon disulfide (76.14 g / mol, 152 mg, 2 mmol), triethylamine (101.19 g / mol, 152 mg, 1.5 mmol) and anhydrous dichloromethane (5 mL) were added to a 10 mL reaction tube and stirred at room temperature for 3 hours, then p-toluenesulfonyl chloride (190.65 g / mol, 286 mg, 1.5 mmol) was added and stirred at room temperature overnight. After the reaction was completed, 655 mg of intermediate A2-2 (yellow oily liquid) was obtained by column chromatography separation and purification (n-heptane: ethyl acetate = 50: 1 to 5: 1), with a yield of 72%.

[0172] Step 3, Synthesis of Compound A2

[0173] A2-2 (909.41 g / mol, 909 mg, 1 mmol), 3-amino-1,2-propanediol (91.11 g / mol, 182 mg, 2 mmol) and anhydrous tetrahydrofuran (5 mL) were added to a 10 mL reaction tube and stirred at room temperature for 2 hours. After the reaction was completed, 656 mg of product A2 (yellow oily liquid) was obtained by column chromatography separation and purification (dichloromethane: methanol = 100: 1 to 40: 1), with a yield of 66%. ESI-MS C 55 H 106 N3O 10 + [M+H] + Calculated value 1000.7593, measured value 1000.7599.

[0174] Example 3

[0175] Synthesis route of compound A3

[0176]

[0177] Referring to the synthesis method of Example 2, A3 was prepared to obtain 442 mg of yellow oily liquid. 53 H 102 N3O8 + [M+H] + Calculated value: 940.7382, measured value: 940.7354.

[0178] Example 4

[0179] Synthesis route of compound A4

[0180]

[0181] Step 1, Synthesis of Compound A4-1

[0182] 3,4-dimethoxy-3-cyclobutene-1,2-dione (142.11 g / mol, 853 mg, 6 mmol), 3-amino-1,2-propylene glycol (91.11 g / mol, 273 mg, 3 mmol), triethylamine (101.19 g / mol, 455 mg, 4.5 mmol) and ethanol (25 mL) were added to a 50 mL round-bottom flask and stirred at room temperature overnight. After the reaction was completed, column chromatography was used for separation and purification (dichloromethane: methanol = 100: 1 to 10: 1) to obtain 300 mg of intermediate A4-1 (light yellow solid), with a yield of 50%.

[0183] Step 2, Synthesis of Compound A4

[0184] A4-1 (201.18 g / mol, 302 mg, 1.5 mmol), amine (867.35 g / mol, 867 mg, 1 mmol), triethylamine (101.19 g / mol, 152 mg, 1.5 mmol) and ethanol (5 mL) were added to a 10 mL reaction tube and stirred at room temperature overnight. After the reaction was completed, column chromatography was performed for separation and purification (dichloromethane: methanol = 100: 1 to 20: 1) to obtain 651 mg of product A4 (light yellow solid) with a yield of 63%. ESI-MS C 58 H 106 N3O 12 + [M+H] + Calculated value 1036.7771, measured value 1036.7771.

[0185] Example 5

[0186] Synthesis route of compound A5

[0187]

[0188] Step 1, Synthesis of Compound A5-1

[0189] 3,3'-dithiodipropionic acid (210.27 g / mol, 8.41 g, 40 mmol) and 9-heptadecanol (256.47 g / mol, 2.56 g, 10 mmol), DMAP (122.17 g / mol, 122.2 mg, 1 mmol) and tetrahydrofuran (75 mL) were added to a 25 mL round-bottom flask and stirred at room temperature for 2 hours, then the reaction temperature was cooled to 0 ° C, DCC (206.33 g / mol, 2.48 g, 12 mmol) was added, and stirred overnight. After the reaction was completed, the by-product DCU was removed, the filter cake was rinsed with a small amount of ethyl acetate, and the filtrate was collected. The filtrate was dried with solvent, the residual solid was dissolved with 50 mL of dichloromethane, and filtered again to remove the unreacted 3,3'-dithiodipropionic acid, the filter cake was rinsed with a small amount of dichloromethane, and the filtrate was collected. The filtrate obtained after two filtrations was concentrated by rotary drying, and purified by column chromatography (petroleum ether:ethyl acetate=10:1 to 2:1) to obtain 1.92 g of intermediate A5-1 (yellow oily liquid) with a yield of 43%.

[0190] Step 2, Synthesis of Compound A5-2

[0191] A5-1 (448.72 g / mol, 2.24 g, 5 mmol), 6-bromohexanol (181.07 g / mol, 1.09 g, 6 mmol), EDCI (191.70 g / mol, 2.30 g, 12 mmol), DMAP (122.17 g / mol, 61 mg, 0.5 mmol), DIPEA (129.24 g / mol, 2.58 g, 20 mmol) and DCM (25 mL) were added to a 50 mL round-bottom flask and stirred overnight at room temperature. After the reaction was completed, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (n-heptane: ethyl acetate = 100: 1 to 50: 1) to obtain 1.71 g of intermediate A5-2, A1-2 (colorless oily liquid), with a yield of 56%.

[0192] Step 3, Synthesis of Compound A5

[0193] A5-2 (611.78 g / mol, 612 mg, 1.0 mmol), potassium carbonate (138.21 g / mol, 276 mg, 2 mmol), sodium iodide (149.89 g / mol, 150 mg, 1 mmol), 3-aminopropanol (75.11 g / mol, 30 mg, 0.4 mmol) and acetonitrile (5 mL) were added to a 25 mL round-bottom flask and stirred at 75 ° C overnight. After the reaction, the reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (dichloromethane: methanol = 100: 1 to 20: 1) to obtain 704 mg of product A5 (yellow oily liquid), with a yield of 62%. ESI-MS C 61 H 118 N9 + [M+H] + Calculated value 1136.7684, measured value 1136.7681.

[0194] Example 6

[0195] Synthesis route of compound A6

[0196]

[0197] Step 1, Synthesis of Compound A6-1

[0198] 6-mercaptohexan-1-ol (134.24 g / mol, 1.34 g, 10 mmol), 1-bromodecane (221.18 g / mol, 2.21 g, 10 mmol), potassium carbonate (138.21 g / mol, 1.38 g, 10 mmol) and DMF (30 mL) were added to a 50 mL round-bottom flask and stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was diluted with dichloromethane, washed three times with water, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (n-heptane: ethyl acetate = 10: 1 to 2: 1) to obtain 2.36 g of intermediate A6-1 (colorless oily liquid), with a yield of 86%.

[0199] Step 2, Synthesis of Compound A6-2

[0200] A6-1 (274.51 g / mol, 1.37 g, 5 mmol), 6-bromohexanoic acid (195.06 g / mol, 1.17 g, 6 mmol), EDCI (191.70 g / mol, 1.92 g, 10 mmol), DMAP (122.17 g / mol, 122 mg, 1 mmol), DIPEA (129.24 g / mol, 2.58 g, 20 mmol) and DCM (25 mL) were added to a 25 mL round-bottom flask and stirred overnight at room temperature. After the reaction was completed, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (n-heptane: ethyl acetate = 100: 1 to 40: 1) to obtain 1.71 g of intermediate A6-2 (colorless oily liquid) with a yield of 42%.

[0201] Step 3, Synthesis of Compound A6

[0202] A6-2 (451.55 g / mol, 452 mg, 1.0 mmol), potassium carbonate (138.21 g / mol, 276 mg, 2 mmol), sodium iodide (149.89 g / mol, 150 mg, 1 mmol), 3-aminopropanol (75.11 g / mol, 30 mg, 0.4 mmol) and acetonitrile (5 mL) were added to a 25 mL round-bottom flask and stirred at 75 ° C overnight. After the reaction, the reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (dichloromethane: methanol = 100: 1 to 20: 1) to obtain 694 mg of product A6 (yellow oily liquid), with a yield of 85%. ESI-MS C 47 H 94 NO5S2 + [M+H] + Calculated value 816.6568, measured value 816.6574.

[0203] Example 7

[0204] Synthesis route of compound A7

[0205]

[0206] Step 1, Synthesis of Compound A7-1

[0207] Potassium methoxide (70.13 g / mol, 70 mg, 1 mmol) and 1-hexanethiol (118.24 g / mol, 1.18 g, 10 mmol) were added to a 25 mL round-bottom flask, heated to 100 ° C, and 1,2-epoxydodecane (184.18 g / mol, 1.84 g, 10 mmol) was added dropwise within 15 minutes. After the addition was completed, the reaction was continued for 20 minutes. After the reaction was completed, column chromatography was used for separation and purification (n-heptane: ethyl acetate = 50: 1 to 10: 1) to obtain 2.54 g of intermediate A7-1 (colorless oily liquid), with a yield of 84%.

[0208] Step 2, Synthesis of Compound A7-2

[0209] A7-1 (302.56 g / mol, 908 mg, 3 mmol), 6-bromohexanoic acid (195.06 g / mol, 703 mg, 3.6 mmol), EDCI (191.70 g / mol, 2.3 g, 12.0 mmol), DMAP (122.17 g / mol, 37 mg, 0.3 mmol), DIPEA (129.24 g / mol, 2.33 g, 18 mmol) and DCM (10 mL) were added to a 25 mL round-bottom flask and stirred at room temperature overnight. After the reaction was completed, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (n-heptane: ethyl acetate = 100: 1 to 20: 1) to obtain 835 mg of intermediate A7-2 (colorless oily liquid) with a yield of 58%.

[0210] Step 3, Synthesis of Compound A7

[0211] A7-2 (479.60 g / mol, 480 mg, 1 mmol), potassium carbonate (138.21 g / mol, 276 mg, 2 mmol), sodium iodide (149.89 g / mol, 150 mg, 1 mmol), 3-aminopropanol (75.11 g / mol, 30 mg, 0.4 mmol) and acetonitrile (5 mL) were added to a 10 mL reaction tube and stirred at 75 ° C for overnight reaction. After the reaction, the reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried with anhydrous sodium sulfate, and separated and purified by column chromatography (dichloromethane: methanol = 100: 1 to 20: 1) to obtain 636 mg of product A6 (yellow oily liquid), with a yield of 73%. ESI-MS C 51 H 102 NO5S2 + [M+H] + Calculated value 872.7194, measured value 872.7195.

[0212] Example 8

[0213] Referring to the synthesis method of Example 3, A8 was prepared to obtain 612 mg of yellow oily liquid. 52 H 100 N3O8S + [M+H] + Calculated value: 926.7226, measured value: 926.7207. Its structural formula is as follows:

[0214]

[0215] Example 9

[0216] Referring to the synthesis method of Example 3, A9 was prepared to obtain 485 mg of yellow oily liquid. 54 H 105 N4O7S + [M+H] + Calculated value 953.7698, measured value 953.7742. Its structural formula is as follows:

[0217]

[0218] Example 10

[0219] Referring to the synthesis method of Example 3, A10 was prepared to obtain 504 mg of yellow oily liquid. 57 H 110 N3O8S + [M+H] + Calculated value: 996.8008, measured value: 996.8101. Its structural formula is as follows:

[0220]

[0221] Embodiment 11

[0222] Referring to the synthesis method of Example 3, A11 was prepared to obtain 361 mg of yellow oily liquid. 58 H 112 N3O8S + [M+H] + Calculated value is 1010.8165, measured value is 1010.8198. Its structural formula is as follows:

[0223]

[0224] Example 12

[0225] Referring to the synthesis method of Example 1, A12 was prepared to obtain 428 mg of yellow oily liquid. 57 H 104 N3O10 + [M+H] + Calculated value 990.7716, measured value 990.7328. Its structural formula is as follows:

[0226]

[0227] Example 13

[0228] A13 was prepared by referring to the synthesis method of Example 1 to obtain 291 mg of yellow oily liquid. 55 H 100 N3O 10 + [M+H] + Calculated value 962.7403, measured value 962.7608. Its structural formula is as follows:

[0229]

[0230] Embodiment 14

[0231] Referring to the synthesis method of Example 1, A14 was prepared to obtain 352 mg of yellow oily liquid. 60 H 110 N3O 10 + [M+H] + Calculated value is 1032.8191, measured value is 1032.8425. Its structural formula is as follows:

[0232]

[0233] Embodiment 15

[0234] A15 was prepared by referring to the synthesis method of Example 1 to obtain 376 mg of yellow oily liquid. 54 H 98 N3O 10 + [M+H] + Calculated value 948.7247, measured value 948.7247. Its structural formula is as follows:

[0235]

[0236] Example 16

[0237] Referring to the synthesis method of Example 1, A16 was prepared to obtain 402 mg of yellow oily liquid. 61 H 112 N3O 10 + [M+H]+ Calculated value is 1046.8342, measured value is 1046.8344. Its structural formula is as follows:

[0238]

[0239] Embodiment 17

[0240] A17 was prepared by referring to the synthesis method of Example 1 to obtain 649 mg of yellow oily liquid. 52 H 94 N3O 10 + [M+H] + Calculated value: 920.6934, measured value: 920.6938. Its structural formula is as follows:

[0241]

[0242] Embodiment 18

[0243] Referring to the synthesis method of Example 4, A18 was prepared to obtain 322 mg of yellow oily liquid. 58 H 106 N3O 11 + [M+H] + Calculated value is 1020.7822, measured value is 1020.7844. Its structural formula is as follows:

[0244]

[0245] Embodiment 19

[0246] A19 was prepared by referring to the synthesis method of Example 4 to obtain 495 mg of yellow oily liquid. 57 H 104 N3O 11 + [M+H] + Calculated value is 1006.7665, measured value is 1006.7676. Its structural formula is as follows:

[0247]

[0248] Embodiment 20

[0249] A20 was prepared by referring to the synthesis method of Example 4 to obtain 387 mg of yellow oily liquid. 57 H 104 N3O 12 + [M+H] +Calculated value is 1022.7615, measured value is 1022.7626. Its structural formula is as follows:

[0250]

[0251] Embodiment 21

[0252] Referring to the synthesis method of Example 4, A21 was prepared to obtain 568 mg of yellow oily liquid. 59 H 109 N4O 11 + [M+H] + Calculated value is 1049.8087, measured value is 1049.8085. Its structural formula is as follows:

[0253]

[0254] Embodiment 22

[0255] A22 was prepared by referring to the synthesis method of Example 4 to obtain 486 mg of yellow oily liquid. 59 H 109 N4O 10 + [M+H] + Calculated value is 1033.8138, measured value is 1033.8149. Its structural formula is as follows:

[0256]

[0257] Embodiment 23

[0258] A23 was prepared by referring to the synthesis method of Example 4 to obtain 233 mg of yellow oily liquid. 57 H 104 N3O 11 + [M+H] + Calculated value is 1006.7665, measured value is 1006.7667. Its structural formula is as follows:

[0259]

[0260] Embodiment 24

[0261] A24 was prepared by referring to the synthesis method of Example 4 to obtain 350 mg of yellow oily liquid. 62 H 114 N3O 12 + [M+H] +Calculated value 1092.8397, measured value 1092.8399. Its structural formula is as follows:

[0262]

[0263] Embodiment 25

[0264] A25 was prepared by referring to the synthesis method of Example 4 to obtain 618 mg of yellow oily liquid. 62 H 114 N3O 11 + [M+H] + Calculated value is 1076.8448, measured value is 1076.8453. Its structural formula is as follows:

[0265]

[0266] Embodiment 26

[0267] A26 was prepared by referring to the synthesis method of Example 1 to obtain 589 mg of yellow oily liquid. 56 H 102 N3O 10 + [M+H] + Calculated value 976.7560, measured value 976.7572. Its structural formula is as follows:

[0268]

[0269] Embodiment 27

[0270] A27 was prepared by referring to the synthesis method of Example 2 to obtain 588 mg of yellow oily liquid. 56 H 108 N3O9S + [M+H] + Calculated value 998.7806, measured value 998.7815. Its structural formula is as follows:

[0271]

[0272] Embodiment 28

[0273] A28 was prepared by referring to the synthesis method of Example 2 to obtain 344 mg of yellow oily liquid. 55 H 106 N3O9S + [M+H] + Calculated value 984.7644, measured value 984.7632. Its structural formula is as follows:

[0274]

[0275] Embodiment 29

[0276] The synthetic route of compound A29 is as follows:

[0277]

[0278] Step 1. Synthesis of Compound A29-1

[0279] TsCl (190.64 g / mol, 5.72 g, 30 mmol), DMAP (122.17 g / mol, 122 mg, 1 mmol) and DCM (50 ml) were added to a 200 ml round-bottom flask, stirred for 5 minutes under an ice bath, and a DCM (50 ml) solution of 2-hexyl-1-decanol (242.44 g / mol, 2.42 g, 10 mmol) and triethylamine (101.19 g / mol, 2.02 g, 20 mmol) was added dropwise over 30 minutes. After the addition was complete, the temperature was raised to room temperature and stirred to react overnight. After the reaction was completed, 3.75 g of intermediate A29-1 (colorless oily liquid) was obtained by column chromatography separation and purification (n-heptane: ethyl acetate = 50:1 to 20:1), with a yield of 94%.

[0280] Step 2, Synthesis of Compound A29-2

[0281] A29-1 (396.63 g / mol, 3.57 g, 9 mmol), NaSH (56.06 g / mol, 1.01 g, 18 mmol) and EtOH (30 ml) were added to a 100 ml round-bottom flask and stirred at 50°C overnight. After the reaction, 1.4 g of intermediate A29-2 (colorless oily liquid) was obtained by column chromatography purification (n-heptane) with a yield of 60%.

[0282] Step 3, Synthesis of Compound A29-3

[0283] 2,2'-disulfide dipyridine (220.31 g / mol, 440 mg, 20 mmol), acetic acid (2 ml) and EtOH (20 ml) were added to a 100 ml round-bottom flask, stirred at room temperature for 5 minutes, and a solution of 3-mercapto-1-propanol (92.16 g / mol, 922 mg, 10 mmol) in EtOH (20 ml) was added dropwise over 30 minutes. After the addition was complete, stirring was continued at room temperature overnight. After the reaction was completed, 1.79 g of intermediate A29-3 (yellow-green viscous liquid) was obtained by column chromatography separation and purification (n-heptane: ethyl acetate = 50:1 to 4:1), with a yield of 89%.

[0284] Step 4, Synthesis of Compound A29-4

[0285] A29-2 (259.51 g / mol, 2.08 g, 8 mmol), acetic acid (1.6 ml) and MeOH (20 ml) were added to a 100 ml round-bottom flask, stirred at room temperature for 5 minutes, and a solution of A29-3 (201.30 g / mol, 1.61 g, 8 mmol) in MeOH (20 ml) was added dropwise over 30 minutes. After the addition was complete, stirring was continued at room temperature overnight. After the reaction was completed, 2.01 g of intermediate A29-4 (light yellow oily liquid) was obtained by column chromatography separation and purification (n-heptane: ethyl acetate = 100:1 to 5:1), with a yield of 72%.

[0286] Step 5, Synthesis of Compound A29-5

[0287] A29-4 (348.65 g / mol, 348 mg, 1 mmol), acrylic acid (72.06 g / mol,

[0288] 86mg, 1.2mmol), EDCI (191.70g / mol, 288mg, 1.5mmol), DMAP (122.17g / mol, 12mg, 0.1mmol), DIPEA (129.24g / mol, 258mg, 2mmol) and DCM (10m), stirred at room temperature overnight. After the reaction, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and separated and purified by column chromatography (n-heptane: ethyl acetate = 100: 1 to 20: 1) to obtain 242mg intermediate A29-5 (colorless oily liquid), with a yield of 60%.

[0289] Step 6. Synthesis of Compound A29

[0290] A29-4 (402.69 g / mol, 242 mg, 0.6 mmol) and nitrogen-methyl-2,2'-diaminodiethylamine (117.19 g / mol, 11.7 mg, 0.1 mmol) were added to a 5 ml reaction tube and stirred at 90°C for 2 days. After the reaction, 97 mg of product A29 (light yellow oily liquid) was obtained by column chromatography separation and purification (dichloromethane: methanol = 100: 1 to 20: 1), with a yield of 56%. ESI-MSC93H183N3O8S8+[M+H]+ calculated value 1727.1804, found value 1727.1805.

[0291] Embodiment 30

[0292] Referring to the synthesis method of Example 1 and Example 3, A30 was prepared to obtain 326 mg of yellow oily liquid. 56 H 108 N3O8S+ [M+H] + Calculated value 982.7852, measured value 982.7780. Its structural formula is as follows:

[0293]

[0294] Embodiment 31

[0295] Referring to the synthesis methods of Examples 1 and 3, A31 was prepared to obtain 225 mg of a yellow oily liquid. 57 H 110 N3O8S + [M+H] + Calculated value: 996.8008, measured value: 996.8041. Its structural formula is as follows:

[0296]

[0297] Embodiment 32

[0298] Referring to the synthesis methods of Examples 1 and 3, A32 was prepared to obtain 283 mg of a yellow oily liquid. 54 H 104 N3O8S + [M+H] + Calculated value: 954.7539, measured value: 954.7573. Its structural formula is as follows:

[0299]

[0300] Embodiment 33

[0301] A33 was prepared by referring to the synthesis methods of Examples 1 and 3 to obtain 289 mg of a yellow oily liquid. 55 H 106 N3O8S + [M+H] + Calculated value 968.7695, measured value 968.7877. Its structural formula is as follows:

[0302]

[0303] Embodiment 34

[0304] Referring to the synthesis methods of Examples 1 and 3, A34 was prepared to obtain 241 mg of a yellow oily liquid. 56 H 108 N3O8S + [M+H] + Calculated value: 982.7852, measured value: 982.7884. Its structural formula is as follows:

[0305]

[0306] Embodiment 35

[0307] A35 was prepared by referring to the synthesis methods of Examples 1 and 3 to obtain 411 mg of a yellow oily liquid. 55 H 106 N3O8S + [M+H] + Calculated value 968.7695, measured value 968.7740. Its structural formula is as follows:

[0308]

[0309] Embodiment 36

[0310] Referring to the synthesis methods of Examples 1 and 4, A36 was prepared to obtain 453 mg of a yellow oily liquid. 60 H 110 N3O 10 + [M+H] + Calculated value is 1032.8186, measured value is 1032.8092. Its structural formula is as follows:

[0311]

[0312] Embodiment 37

[0313] A37 was prepared by referring to the synthesis methods of Examples 1 and 4 to obtain 469 mg of a yellow oily liquid. 59 H 108 N3O 10 + [M+H] + Calculated value is 1018.8029, measured value is 1018.8226. Its structural formula is as follows:

[0314]

[0315] Embodiment 38

[0316] A38 was prepared by referring to the synthesis methods of Examples 1 and 4 to obtain 138 mg of a yellow oily liquid. 59 H 108 N3O 10 + [M+H] + Calculated value is 1018.8029, measured value is 1018.8223. Its structural formula is as follows:

[0317]

[0318] Embodiment 39

[0319] A39 was prepared by referring to the synthesis methods of Examples 1 and 4 to obtain 149 mg of a yellow oily liquid. 60 H 110 N3O 10 + [M+H] + Calculated value is 1032.8186, measured value is 1032.8205. Its structural formula is as follows:

[0320]

[0321] Embodiment 40

[0322] Referring to the synthesis methods of Examples 1 and 4, A40 was prepared to obtain 358 mg of a yellow oily liquid. 57 H 104 N3O 10 + [M+H] + Calculated value 990.7716, measured value 990.7735. Its structural formula is as follows:

[0323]

[0324] Embodiment 41

[0325] Referring to the synthesis methods of Examples 1 and 4, A41 was prepared to obtain 355 mg of a yellow oily liquid. 58 H 106 N3O 10 + [M+H] + Calculated value is 1004.7873, measured value is 1004.7889. Its structural formula is as follows:

[0326]

[0327] Embodiment 42

[0328] Referring to the synthesis method of Example 5, A42 was prepared to obtain 343 mg of yellow oily liquid. 55 H 106 NO9S4 + [M+H] + Calculated value 1052.6745, measured value 1052.6924. Its structural formula is as follows:

[0329]

[0330] Embodiment 43

[0331] A43 was prepared by referring to the synthesis method of Example 5 to obtain 258 mg of yellow oily liquid. 59 H 114 NO9S4 + [M+H] + Calculated value 1108.7371, measured value 1108.7398. Its structural formula is as follows:

[0332]

[0333] Embodiment 44

[0334] A44 was prepared by referring to the synthesis method of Example 5 to obtain 422 mg of yellow oily liquid. 63 H 122 NO9S4 + [M+H] + Calculated value is 1164.7997, measured value is 1164.8053. Its structural formula is as follows:

[0335]

[0336] Embodiment 45

[0337] Referring to the synthesis methods of Examples 1 and 7, A45 was prepared to obtain 432 mg of a yellow oily liquid. 56 H 106 N3O6S2 + [M+H] + Calculated value is 980.7518, measured value is 980.7675. Its structural formula is as follows:

[0338]

[0339] Embodiment 46

[0340] Referring to the synthesis methods of Examples 1 and 5, A46 was prepared to obtain 314 mg of a yellow oily liquid. 60 H 110 N3O 10 S4 + [M+H] + Calculated value 1160.7069, measured value 1160.7250. Its structural formula is as follows:

[0341]

[0342] Embodiment 47

[0343] Referring to the synthesis methods of Examples 1 and 7, A47 was prepared to obtain 132 mg of a yellow oily liquid. 64 H 118 N3O 10 S4 + [M+H] + Calculated value 1216.7695, measured value 1216.7804. Its structural formula is as follows:

[0344]

[0345] Embodiment 48

[0346] A48 was prepared by referring to the synthesis methods of Examples 1 and 7 to obtain 564 mg of a yellow oily liquid. 64 H 126 N3O 10 S4 + [M+H] + Calculated value 1272.8321, measured value 1272.8023. Its structural formula is as follows:

[0347]

[0348] Embodiment 49

[0349] A49 was prepared by referring to the synthesis methods of Examples 3 and 7 to obtain 328 mg of a yellow oily liquid. 61 H 118 N3O8S5 + [M+H] + Calculated value is 1180.7517, measured value is 1180.7458. Its structural formula is as follows:

[0350]

[0351] Embodiment 50

[0352] A50 was prepared by referring to the synthesis methods of Examples 3 and 7 to obtain 248 mg of a yellow oily liquid. 65 H 126 N3O8S5 + [M+H] + Calculated value 1236.8143, measured value 1236.8459. Its structural formula is as follows:

[0353]

[0354] Embodiment 51

[0355] Referring to the synthesis methods of Examples 3 and 7, A51 was prepared to obtain 395 mg of a yellow oily liquid. 62 H 120 N3O8S5 + [M+H] + Calculated value is 1194.7673, measured value is 1194.7824. Its structural formula is as follows:

[0356]

[0357] Embodiment 52

[0358] A52 was prepared by referring to the synthesis methods of Examples 3 and 7 to obtain 395 mg of a yellow oily liquid. 63 H 122 N3O8S5 + [M+H] + Calculated value 1208.7830, measured value 1208.7329. Its structural formula is as follows:

[0359]

[0360] Embodiment 53

[0361] A53 was prepared by referring to the synthesis methods of Examples 1 and 7 to obtain 256 mg of a yellow oily liquid. 65 H 120 N3O 10 S4 + [M+H] + Calculated value 1230.7851, measured value 1230.7859. Its structural formula is as follows:

[0362]

[0363] Embodiment 54

[0364] Referring to the synthesis methods of Examples 1 and 5, A54 was prepared to obtain 210 mg of a yellow oily liquid. 60 H 110 N3O 10 S4 + [M+H] + Calculated value is 1160.7074, measured value is 1160.7076. Its structural formula is as follows:

[0365]

[0366] Embodiment 55

[0367] A55 was prepared by referring to the synthesis methods of Examples 1 and 5 to obtain 110 mg of a yellow oily liquid. 53 H 96 N3O 10 S4 + [M+H] + Calculated value 1062.5979, measured value 1062.5983. Its synthesis route and structural formula are as follows:

[0368]

[0369] Embodiment 56

[0370] A56 was prepared by referring to the synthesis methods of Examples 1 and 5 to obtain 163 mg of a yellow oily liquid. 54 H 98 N3O 10 S2 + [M+H] + Calculated value 1012.6694, measured value 1012.6692. Its synthesis route and structural formula are as follows:

[0371]

[0372] Embodiment 57

[0373] A57 was prepared by referring to the synthesis methods of Examples 1 and 5 to obtain 185 mg of a yellow oily liquid. 61 H 112 N3O 10 S2 + [M+H] + Calculated value 1110.7789, measured value 1110.7795. Its synthetic route and structural formula are as follows:

[0374]

[0375] Embodiment 58

[0376] A58 was prepared by referring to the synthesis methods of Examples 1 and 5 to obtain 185 mg of a yellow oily liquid. 58 H 106 N3O 10 S2 + [M+H] + Calculated value 1068.7320, measured value 1068.7316. Its synthesis route and structural formula are as follows:

[0377]

[0378] Embodiment 59

[0379] A59 was prepared by referring to the synthesis methods of Examples 1 and 5 to obtain 126 mg of a yellow oily liquid. 56 H 102 N3O 10 S2 + [M+H] + Calculated value 1104.6448, measured value 1104.6449. Its synthesis route and structural formula are as follows:

[0380]

[0381] Embodiment 60

[0382] Referring to the synthesis methods of Examples 1 and 5, A60 was prepared to obtain 130 mg of a yellow oily liquid. 53 H 102 N3O9S2 + [M+H] + Calculated value: 960.6996, measured value: 960.6992. Its synthetic route and structural formula are as follows

[0383]

[0384] Embodiment 61

[0385] Referring to the synthesis methods of Example 1 and Example 5, A61 was prepared to obtain 98 mg of yellow oily liquid. 53 H 102 N3O9S2 + [M+H] + Calculated value 1032.7142, measured value 1032.7149. Its synthesis route and structural formula are as follows

[0386]

[0387] Embodiment 62

[0388] A62 was prepared by referring to the synthesis method of Example 5 to obtain 165 mg of yellow oily liquid. 53 H 106 NO5S4 + [M+H] + Calculated value: 964.6954, measured value: 964.6948. Its synthetic route and structural formula are as follows

[0389]

[0390] Embodiment 63

[0391] Referring to the synthesis methods of Examples 1 and 5, A63 was prepared to obtain 182 mg of a yellow oily liquid. 58 H 110 N3O6S4 + [M+H] + Calculated value 1072.7227, measured value 1072.72283. Its synthesis route and structural formula are as follows

[0392]

[0393] Embodiment 64

[0394] Referring to the synthesis methods of Examples 1 and 5, A64 was prepared to obtain 102 mg of a yellow oily liquid. 56 H 108 NO9S2 + [M+H] + Calculated value 1002.7466, measured value 1002.7458. Its synthesis route and structural formula are as follows

[0395]

[0396] Embodiment 65

[0397] A65 was prepared by referring to the synthesis methods of Examples 1 and 5 to obtain 141 mg of a yellow oily liquid. 58 H 110 N3O6S4 + [M+H] + Calculated value 904.6370, measured value 904.6362. Its synthesis route and structural formula are as follows

[0398]

[0399] Embodiment 66

[0400] Referring to the synthesis methods of Examples 1 and 5, A66 was prepared to obtain 130 mg of a yellow oily liquid. 51 H 98 N3O8S3 + [M+H] + Calculated value: 976.6516, measured value: 976.6523. Its synthetic route and structural formula are as follows

[0401]

[0402] Example 67. Preparation of lipid nanoparticles

[0403] The ionizable lipid compound, phospholipid lipid (DSPC), cholesterol, and pegylated lipid (DMG-PEG2000) prepared in Example 1-66 of the present invention were mixed and dissolved in anhydrous ethanol at a molar ratio of 47.5:10:41:1.5. The obtained ethanol solution and acetate buffer (25mM, pH=5.0) dissolved with Luc-mRNA were mixed in a microfluidic chip at a volume ratio of 1:3 using a microfluidic preparation system to obtain a crude solution of lipid nanoparticles, which was then ultrafiltered at 4°C using a 100KD ultrafiltration tube (Millipore, FTT500500). Filtered again through a 0.22μm microporous filter membrane before use. The mass ratio of lipid compound to luciferase mRNA (Luc mRNA) was about 40:1.

[0404] The obtained lipid nanoparticles were characterized and the results are shown in Table 1.

[0405] The particle size and PDI (polydispersity index) of the prepared lipid nanoparticles were measured by Nano-ZSZEN3600 (Malvern). 20 μL of LNP solution was taken for particle size measurement, and the cycle was repeated three times, with each cycle lasting 30 seconds.

[0406] The encapsulation efficiency was determined according to the standard procedure of the Quant-iT RiboGreen RNA Kit.

[0407] Table 1 Characterization data of LNPs prepared using representative amino lipid compounds

[0408]

[0409]

[0410] From the above results, it can be seen that the encapsulation efficiency of the lipid nanoparticles of the present invention is higher than that of DLin-MC3-DMA (MC3).

[0411] Example 68: Transfection of lipid nanoparticles prepared from amino lipid compounds on primary BMDC cells

[0412] Animal preparation: Six-week-old female C57BL / 6 mice weighing about 20 g were selected and kept in an SPF-grade breeding room. Animal experiments were conducted in strict accordance with the guidelines of the National Health Agency and animal ethics requirements.

[0413] Cell acquisition: C57BL / 6 mice were killed by cervical dislocation and immersed in 75% alcohol for 5 minutes for disinfection. The thigh and tibia of the mice were dissected and the attached muscles were removed to expose the bone. The bone marrow in the tibia was blown out with a 1 mL syringe filled with PBS. The obtained bone marrow was blown out and filtered through a 50 μm filter to remove impurities. Red blood cell lysis solution (3-4 mL) was added to the obtained filtrate and allowed to stand for 5 minutes. After that, the supernatant was removed by centrifugation at 800 g for 5 minutes. The obtained cells were placed in 1640 culture medium (containing 10% fetal bovine serum, 20 ng / mL GMCSF, 10 ng / mL The cells were resuspended in IL4 and inoculated into 6-well plates at a density of 100,000 cells / mL of culture medium. The plates were placed in a cell culture incubator at 37°C and 5% CO2. The medium was half-changed every 2 days. On the seventh day, the suspended cells and loosely attached cells were collected and inoculated into 96-well all-white ELISA plates at a density of 20,000 cells per well in a culture medium volume of 100 μL.

[0414] Cell transfection: Add lipid nanoparticles encapsulating luciferase mRNA to a 96-well all-white ELISA plate containing primary cells, and control the volume of mRNA lipid nanoparticles added to each well to be 10 μL. Then place in an incubator at 37°C and 5% CO2 for 12 hours.

[0415] Transfection efficiency detection: 20 μL of substrate ONE-GloTM Luciferase was added to each well of a 96-well all-white ELISA plate, and the detection was performed using a multifunctional ELISA reader (Biorek SynergyH1) after 1 min. The expression intensity of Luc mRNA transfected on BMDC by lipid nanoparticles (LNPs) prepared by representative amino lipid compounds according to the method of Example 54 is shown in Table 2, with MC3 as a control.

[0416] Table 2 Expression intensity of LNPs composed of representative amino lipids after transfection on BMDCs

[0417]

[0418]

[0419] It can be seen that the expression intensity of the amino lipid provided by the present invention after cell transfection is better than that of MC3.

[0420] Example 69: Evaluation of the in vivo delivery performance of lipid nanoparticles for luciferase mRNA

[0421] Preparation of lipid nanoparticles: same as Example 67.

[0422] Animal experiments

[0423] Animal preparation: Six-week-old female C57BL / 6 mice weighing approximately 20 g were selected and raised in an SPF-grade breeding room. Animal experiments were conducted in strict accordance with the guidelines of the National Health Agency and animal ethics requirements.

[0424] In vivo delivery: Five C57BL / 6 mice were randomly selected from each group, and lipid nanoparticle solution was injected by intramuscular injection at a dosage of 0.1 mg / kg mRNA. After 6 hours, 200 μL of 10 mg / mL D-luciferin potassium salt was injected into the peritoneal cavity of each mouse. After 5 minutes, the mice were placed under a living imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and photographed. The expression intensity of LucmRNA delivered by LNP intramuscular injection composed of representative amino lipid compounds is shown in Table 3, with MC3 as a control.

[0425] Table 3 Expression intensity of Luc mRNA delivered by intramuscular injection of LNPs composed of representative amino lipids

[0426]

[0427]

[0428] It can be seen that the expression intensity of the amino lipid provided by the present invention in intramuscular administration and delivery is significantly better than that of MC3.

[0429] Example 70 Evaluation of the in vivo immune and tumor therapeutic effects of lipid nanoparticles prepared from amino lipid compounds

[0430] Preparation of lipid nanoparticles: same as Example 67.

[0431] Animal experiments

[0432] Animal preparation: Female C57BL / 6 mice aged 5-6 weeks, weighing about 18-20 g, were selected and kept in an SPF-grade breeding room. Animal experiments were conducted in strict accordance with the guidelines of the National Health Agency and animal ethics requirements.

[0433] In vivo delivery: B16-OVA melanoma cells (1.5×105) were injected subcutaneously into the outer thigh of mice. When the tumor size grew to 50 mm 3 Vaccination was started at 1:00 pm (approximately 6 or 7 days after tumor inoculation). Animals were immunized twice by intramuscular injection of LNP formulation containing 1 μg OVA-mRNA, with the second injection 7 days apart. Tumor growth was measured 3 times a week using a digital caliper, calculated as 0.5 × length × width × width. When the tumor volume reached 1500 mm 3 The mice were euthanized at 4 ℃ and 1 ℃. The tumor growth rates of the A12 and A48 groups were significantly slower than those of the MC3 group ( Figure 1 ), and 100% (A12 group) and 80% (A48 group) of mice achieved complete remission, which was significantly better than that of the MC3 group (as shown in Figure 2 shown).

[0434] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An ionizable lipid compound, the structural formula of which is shown in formula (I): In formula (I), L1, L2, L3 and L4 are the same or different and are independently selected from: -O(C=O)-, -(C=O)O-, -O(C=O)O-, -(C=O)S-, -S(C=O)-, -O(C=S)-, -(C=S)O-, -O-, -SS-, -S-, -(C=S)S-, -S(C=S)-; R1 and R2 are the same or different and are independently selected from: C1-C 18 Straight or branched alkyl, C3-C 18 alkenyl; M1 and M5 are the same or different and are independently selected from: C1-C 15 Straight or branched alkylene, M7-SS-M7, M7 is selected from C1-C6 alkylene; M2 and M4 are the same or different and are independently selected from: C1-C 15 Alkylene, C2-C 15 Alkenylene, Wherein M6 is selected from C2-C6 alkylene, and two M6 are connected to L2 and N atom in formula (I) respectively; M3 is selected from C1-C6 alkylene; G is selected from H, -OH and at least one of the following structures: Wherein A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl.

2. The ionizable lipid compound according to claim 1, characterized in that L1, L2, L3 and L4 are the same or different and are independently selected from: -O(C=O)-, -(C=O)O-, -O(C=O)O-, -(C=O)S-, -S(C=O)-, -O-, -SS-, -S-; Further, L1 and L4 are the same, L2 and L3 are the same, and are independently selected from: -O(C=O)-, -(C=O)O-, -(C=O)S-, -S(C=O)-, -SS-; Further, R1 and R2 are the same or different and are independently selected from: C2-C 18 Straight or branched chain alkyl; Further, M1 and M5 are the same or different and are independently selected from: C2-C 15 Straight or branched alkylene, M7-SS-M7, M7 is selected from C1-C3 straight chain alkylene; Further, M2 and M4 are the same and are independently selected from C2-C 11 Alkylene; Furthermore, G is selected from at least one of H, -OH and the following structures: Wherein A is selected from the following: Furthermore, the ionizable lipid compound is selected from any one of the following compounds:

3. A method for preparing the ionizable lipid compound of formula (I) according to claim 1 or 2, G is The method comprises the following steps: under alkaline conditions, allowing the compound represented by formula (A) to undergo a substitution reaction with the compound represented by formula (B) to obtain an ionizable lipid compound represented by formula (I); In formula (A), L1, L2, L3, L4, M1, M2, M3, M4, M5, R1, and R2 are defined the same as L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 in formula (I); In formula (B), the definition of A is the same as that of A in formula (I) G, i.e., A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl; G is The method comprises the following steps: allowing the compound represented by formula (C) to undergo an addition reaction with the compound represented by formula (D) to obtain an ionizable lipid compound represented by formula (I); In formula (C), L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 are defined the same as L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 in formula (I); In formula (D), the definition of A is the same as that of A in formula (I) G, i.e., A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl; Or, G is The method comprises: Allowing the compound represented by formula (A) to undergo an addition reaction with the compound represented by formula (E) to obtain an ionizable lipid compound represented by formula (I); In formula (E), the definition of A is the same as that of A in formula (I) G, i.e., A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl; G is H or -OH, and the method comprises: subjecting the compound represented by formula (F), the compound represented by formula (H) and the compound represented by formula (I) to a substitution reaction to obtain an ionizable lipid compound represented by formula (I); In formula (F), L1, L2, M1, M2, and R1 are defined the same as L1, L2, M1, M2, and R1 in formula (I); In formula (H), L3, L4, M4, M5, and R2 are defined the same as L3, L4, M4, M5, and R2 in formula (I); In formula (I), M3 is the same as the definition of M3 in formula (I); and G is H or -OH.

4. A lipid carrier containing the ionizable lipid compound of formula (I) according to claim 1 or 2, wherein the raw materials of the lipid carrier include the following components: the ionizable lipid compound of formula (I), a steroid, a neutral lipid and / or a polymer-bound lipid; Preferably, the polymer-bound lipid has a chemical formula of PYL, wherein P is a hydrophilic polymer moiety, Y is an optional linker, and L is a lipid moiety; Preferably, in the lipid nanoparticles, the molar ratio of the ionizable lipid compound represented by formula (I), steroid, neutral lipid and polymer-bound lipid is preferably 30-70:30-65:0-30:0.2-5.

5. The lipid carrier according to claim 4, characterized in that The polymer-bound lipids include one or more PEG-modified lipids; preferably, the PEG-modified lipids are selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphate, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol; preferably, one or more of PEG-diacylglycerol (PEG-DAG), PEG-dialkoxypropyl (PEG-DAA), PEG-dimyristylglycerol (DMG-PEG), PEG-ceramide (PEG-Cer) and PEG-distearoylphosphatidylethanolamine (DSPE-PEG); more preferably, the relative molecular mass of the PEG-modified lipid is 500-5000, most preferably 1000, 2000, 3000, 4000 or 5000; the polymer-bound lipid is preferably DMG-PEG2000; The steroid is selected from one or more of cholesterol, cholesterol ester, steroid hormone, steroid vitamin and phytosterol, more preferably one or more of cholesterol, cholesterol ester and phytosterol, most preferably cholesterol; The neutral lipid is selected from phospholipids, more preferably at least one of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1, ... Glycerol-3-phospho-(1'-rac-glycerol) (DOPG), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylserine (sodium salt) (DOPS), oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), more preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

6. A nucleic acid-loaded lipid nanoparticle comprising the lipid carrier according to claim 4 or 5 and a nucleic acid, wherein: The mass ratio of the lipid carrier to the nucleic acid in the nucleic acid-loaded lipid nanoparticles is 5:1-60:1; Preferably, the nucleic acid is a nucleic acid compound selected from the group consisting of artificial mRNA, chemically modified or unmodified messenger RNA comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA and replicon RNA, or any combination thereof; Further preferably, the nucleic acid is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof; More preferably, the nucleic acid is mRNA or an mRNA compound; The mRNA includes one or more of a stem-loop, a chain-terminating nucleoside, a polyA sequence, a polyadenylation signal and / or a 5' cap structure.

7. A method for preparing the nucleic acid-loaded lipid nanoparticles of claim 6, comprising the steps of: (A1) mixing the ionizable lipid compound represented by formula (I), steroid, neutral lipid and / or polymer-bound lipid in the above-described proportions, dissolving them in a solvent, and obtaining an organic phase liposome solution; (A2) dissolving the nucleic acid in a buffer solution of appropriate pH value to obtain an aqueous nucleic acid solution; (A3) The organic phase liposome solution and the aqueous phase nucleic acid solution are uniformly mixed using a microfluidic device at a certain volume ratio to obtain a nucleic acid-loaded lipid nanoparticle solution.

8. A nucleic acid-loaded lipid nanoparticle preparation, comprising the nucleic acid-loaded lipid nanoparticle of claim 6 and a pharmaceutically acceptable carrier.

9. Use of the ionizable lipid compound of formula (I) according to claim 1 or 2 or the lipid carrier according to claim 4 or 5 in the preparation of a drug for delivering nucleic acids.

10. A method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases using the nucleic acid-loaded lipid nanoparticles of claim 6 or the nucleic acid-loaded lipid nanoparticle preparation of claim 8; Preferably, the infectious disease is particularly a viral infection; The cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer; The autoimmune diseases include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetase syndrome, antineutrophil-associated vasculitis, and sensitized renal transplantation.

11. Use of the nucleic acid-loaded lipid nanoparticles of claim 6 or the nucleic acid-loaded lipid nanoparticle preparation of claim 8 in the preparation of drugs for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases; Preferably, the infectious disease is particularly a viral infection; The cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer; The autoimmune diseases include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetase syndrome, antineutrophil-associated vasculitis, and sensitized renal transplantation.

12. A method for delivering a nucleic acid to a cell in a subject, the method comprising administering the nucleic acid-loaded lipid nanoparticle of claim 6 or the nucleic acid-loaded lipid nanoparticle formulation of claim 8 to a subject in need thereof.

Images