Novel ionizable lipid, preparation method therefor, and use thereof

Abstract

Provided in the present invention are a novel ionizable lipid, a preparation method therefor, and the use thereof. The structural formula of the novel ionizable lipid is as shown in formula (I). An ionizable lipid compound of the present invention contains structures of urea and a bioisostere thereof. By virtue of hydrogen bonds which squaramide, urea, and thiourea structures form with nucleic acid, the encapsulation effect of the ionizable lipid is enhanced and the immunogenicity of the ionizable lipid is reduced. Nanoparticles prepared using the ionizable lipid of the present invention have a high encapsulation efficiency, a low PDI, and a broadly adaptable and stable process.

Description

A novel ionizable lipid, its preparation method and application

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 2024115393276, filed on October 31, 2024, entitled “A Novel Ionizable Lipid and Its Preparation Method and Application,” the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This invention belongs to the pharmaceutical field, specifically relating to novel ionizable lipids, their preparation methods, and their applications in delivery systems. Background Technology

[0004] Lipid nanoparticles (LNPs) represent the cutting-edge nucleic acid delivery system currently available for clinical use. As a non-viral delivery vector, they have been approved for in vivo mRNA delivery. LNPs are multi-component systems, typically composed of ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol-lipid conjugates.

[0005] Ionizable lipids, as the most important component of LNPs, can promote cellular uptake and help drug molecules escape from endosomes. At the same time, they also affect the encapsulation efficiency of nucleic acid drugs, the delivery efficiency of nucleic acid drugs in vivo, and cytotoxicity, thus determining the efficacy of LNPs.

[0006] 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 tremendous success of mRNA vaccines in combating COVID-19 has spurred research and rapid translation of LNPs. However, safety concerns remain in preclinical development, primarily stemming from the strong inflammatory response induced by ionizable lipids. This issue hinders long-term administration of LNPs and limits their dosage. Therefore, ensuring high transfection efficiency while maximizing immunogenicity has become a key challenge in the design of ionizable lipids. Summary of the Invention

[0007] The purpose of this invention is to provide an ionizable lipid compound containing urea and its bioisosteres. The compound enhances the encapsulation effect of ionizable lipids and reduces their immunogenicity by forming hydrogen bonds between the squaramide, urea, and thiourea structures and nucleic acids.

[0008] In a first aspect, the present invention provides an ionizable lipid compound having the structural formula shown in formula (I):

[0009] In formula (I), L1, L2, L3 and L4 may be the same or different, and each is 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)-;

[0010] R1 and R2 may be the same or different, and each is independently selected from: C1-C 18 Straight-chain or branched alkyl groups, C3-C 18 alkenyl;

[0011] M1 and M5 may be the same or different, and each is independently selected from: C1-C 15 Straight-chain or branched alkylene compounds, M7-SS-M7, where M7 is selected from C1-C6 alkylene compounds;

[0012] M2 and M4 may be the same or different, and each is independently selected from: C1-C 15 Alkylene, C2-C 15 sub-alkenyl, M6 is selected from C2-C6 alkylene groups, and the two M6 atoms are respectively attached to L2 and N atoms in formula (I);

[0013] M3 is selected from C1-C6 alkylene groups;

[0014] G is selected from H, -OH and at least one of the following structures:

[0015] Where A is selected from C1-C6 alkyl or hydroxylated C2-C6 alkyl.

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

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

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

[0020] Specifically, M2 and M4 are identical, 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] A is selected from the following:

[0024] Furthermore, the aforementioned ionizable lipid compound is selected from any one of the following compounds:

[0025] Secondly, the present invention provides a method for preparing the ionizable lipid compound shown in formula (I) above.

[0026] G is The ionizable lipid compound shown in formula (I) is prepared by a method comprising the following steps: under alkaline conditions, the compound shown in formula (A) undergoes a substitution reaction with the compound shown in formula (B) to obtain the ionizable lipid compound shown in formula (I).

[0027] In equation (A), the definitions of L1, L2, L3, L4, M1, M2, M3, M4, M5, R1, and R2 are the same as the definitions of L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 in equation (I);

[0028] In formula (B), A is defined in the same way as A in formula (I)G, that is, A is selected from C1-C6 alkyl or hydroxylated C2-C6 alkyl.

[0029] G is The ionizable lipid compound shown in formula (I) is prepared by a method comprising the following steps: subjecting the compound shown in formula (C) to the compound shown in formula (D) by an addition reaction to obtain the ionizable lipid compound shown in formula (I);

[0030] In equation (C), the definitions of L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 are the same as the definitions of L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 in equation (I);

[0031] In formula (D), A is defined in the same way as A in formula (I)G, that is, A is selected from C1-C6 alkyl or hydroxylated C2-C6 alkyl.

[0032] Or, G is The ionizable lipid compound shown in formula (I) is prepared by a method comprising the following steps: subjecting the compound shown in formula (A) to the compound shown in formula (E) by an addition reaction to obtain the ionizable lipid compound shown in formula (I);

[0033] In formula (E), A is defined in the same way as A in formula (I)G, that is, A is selected from C1-C6 alkyl or hydroxylated C2-C6 alkyl.

[0034] G is H or -OH. The ionizable lipid compound shown in formula (I) is prepared by a method including the following steps: the compound shown in formula (F), the compound shown in formula (H) and the compound shown in formula (I) undergo a substitution reaction to obtain the ionizable lipid compound shown in formula (I).

[0035] In equation (F), the definitions of L1, L2, M1, M2, and R1 are the same as those in equation (I);

[0036] In equation (H), the definitions of L3, L4, M4, M5, and R2 are the same as those in equation (I);

[0037] In formula (I), M3 is defined as M3 in formula (I); G is H or -OH.

[0038] Thirdly, the present invention provides a lipid carrier containing the ionizable lipid compound shown in formula (I) above.

[0039] The lipid carrier provided by the present invention comprises the following components: ionizable lipid compounds of formula (I), steroids, neutral lipids and / or polymer-bound lipids;

[0040] The polymer-bound lipid has the chemical formula PYL, where P is the hydrophilic polymer moiety, Y is an optional linker, and L is the lipid moiety.

[0041] In the lipid nanoparticles, the molar ratio of the ionizable lipid compound, steroid, neutral lipid and polymer-bound lipid shown in formula (I) is preferably 30-70:30-65:0-30:0.2-5, and more preferably 30-60:35-60:0-20:0.3-3.

[0042] More preferably, the molar ratio of the ionizable lipid compound, steroid, neutral lipid and polymer-bound lipid shown in formula (I) is 40-50:40-45:10-15:0.5-2.

[0043] The lipids bound to the polymer include one or more PEG-modified lipids;

[0044] Preferably, the PEG-modified lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphoric acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; preferably, it is selected from one or more of PEG-diacylglycerol (PEG-DAG), PEG-dialkoxypropyl (PEG-DAA), PEG-dimyristylglycerol (DMG-PEG), PEG-ceramide (PEG-Cer), and PEG-distearatephosphatidylethanolamine (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;

[0045] The steroid is selected from one or more of cholesterol, cholesterol esters, steroid hormones, steroid vitamins and phytosterols, more preferably one or more of cholesterol, cholesterol esters and phytosterols, and most preferably cholesterol;

[0046] The neutral lipid is selected from phospholipids, more preferably at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 2-dioleoyl-sn- Glyceryl-3-phosphate-(1'-rac-glycerol) (DOPG), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylserine (sodium salt) (DOPS), oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), more preferably 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC) or 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

[0047] Fourthly, the present invention provides a lipid nanoparticle loaded with nucleic acid.

[0048] The nucleic acid-loaded lipid nanoparticles provided by this invention include the lipid carrier and nucleic acid described in the third aspect of this invention.

[0049] Preferably, the nucleic acid is a nucleic acid compound selected from the group consisting of artificial mRNA, chemically modified or unmodified messenger RNA containing at least one coding sequence, self-replicating RNA, circular RNA, viral RNA and replicon RNA; or any combination thereof;

[0050] More preferably, the nucleic acid is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof;

[0051] More preferably, the nucleic acid is mRNA or an mRNA compound;

[0052] The mRNA includes one or more of stem-loop, chain-terminating nucleoside, polyA sequence, polyadenylation signal and / or 5' cap structure.

[0053] In some implementations, the nucleic acid is luciferase mRNA (Luc mRNA) or ovalbumin (OVA) mRNA.

[0054] The "egg white albumin (OVA) mRNA" described in this invention can be an mRNA encoding a known OVA protein sequence. The OVA protein can be derived from OVA proteins present in existing organisms or from OVA protein sequences in existing gene databases.

[0055] Preferably, the mass ratio of lipid carrier to nucleic acid in the nucleic acid-loaded lipid nanoparticles is 5:1-60:1.

[0056] More preferably, the mass ratio of lipid carrier to nucleic acid in the nucleic acid-loaded lipid nanoparticles is 30:1-50:1.

[0057] 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.

[0058] Preferably, the particle size of the nucleic acid-loaded lipid nanoparticles is 40–160 nm.

[0059] More preferably, the particle size of the nucleic acid-loaded lipid nanoparticles is 70–100 nm.

[0060] The optimal particle size is 75nm to 95nm.

[0061] Preferably, the encapsulation efficiency of the nucleic acid in the nucleic acid-loaded lipid nanoparticles is greater than 80%. Exemplarily, the encapsulation efficiency can be 82%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.

[0062] Fifthly, the present invention provides a method for preparing the nucleic acid-loaded lipid nanoparticles described in the fourth aspect of the present invention.

[0063] This invention provides a method for preparing nucleic acid-loaded lipid nanoparticles, comprising the following steps:

[0064] (A1) Mix the ionizable lipid compound, steroid, neutral lipid and / or polymer-bound lipid shown in formula (I) in the proportions described above, and dissolve in a solvent to obtain an organic phase liposome solution.

[0065] (A2) Dissolve the nucleic acid in a buffer solution with an appropriate pH value to obtain an aqueous nucleic acid solution;

[0066] (A3) The organic phase liposome solution and the aqueous phase nucleic acid solution are mixed uniformly with a microfluidic device at a certain volume ratio to obtain a nucleic acid-loaded lipid nanoparticle solution.

[0067] Preferably, the solvent used to dissolve lipid molecules in step (A1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, or N,N-dimethylformamide, specifically anhydrous ethanol.

[0068] Preferably, the buffer solution in step (A2) is an acetic acid / sodium acetate solution (acetic acid buffer) or a citric acid / sodium citrate solution.

[0069] More preferably, the buffer solution in step (A2) is an acetic acid / sodium acetate solution.

[0070] Preferably, the pH of the buffer solution in step (A2) is 4-6.

[0071] More preferably, the pH of the buffer solution in step (A2) is 5.

[0072] Preferably, the concentration of the buffer solution in step (A2) is 1 mM-100 mM.

[0073] More preferably, the concentration of the buffer solution in step (A2) is 10 mM-50 mM.

[0074] Most preferably, the concentration of the buffer solution in step (A2) is 25 mM.

[0075] Preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:1 to 1:10.

[0076] More preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:1 to 1:5.

[0077] Most preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid solution in step (A3) is 1:3.

[0078] The above method further includes performing ultrafiltration and microporous membrane filtration sequentially on the obtained nucleic acid-loaded lipid nanoparticle solution.

[0079] In a sixth aspect, the present invention provides a lipid nanoparticle formulation loaded with nucleic acids.

[0080] The formulations provided by this invention include the nucleic acid-loaded lipid nanoparticles and pharmaceutically acceptable carriers described in the fourth aspect of this invention.

[0081] Pharmaceutically acceptable carriers include, but are not limited to: diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0082] In a seventh aspect, the present invention provides the use of the ionizable lipid compound of formula (I) described in the first aspect above or the lipid carrier described in the second aspect in the preparation of a medicament for delivering nucleic acids.

[0083] 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.

[0084] In some implementations, the nucleic acid is mRNA, including mRNAs of different sequences and lengths.

[0085] In some implementations, the nucleic acid is firefly luciferase mRNA or ovalbumin (OVA) mRNA.

[0086] In some implementations, the drug is used to treat and / or prevent diseases including, but not limited to: infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0087] The aforementioned infectious diseases, especially viral infections;

[0088] The autoimmune diseases mentioned include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, and sensitized kidney transplantation.

[0089] The cancers mentioned include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia, and prostate cancer.

[0090] 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 above or the nucleic acid-loaded lipid nanoparticle formulations described in the sixth aspect above.

[0091] Preferably, the infectious disease is a viral infection;

[0092] The autoimmune diseases mentioned include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, and sensitized kidney transplantation.

[0093] The cancers mentioned include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia, and prostate cancer.

[0094] 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 formulation described in the sixth aspect in the preparation of drugs for the treatment or prevention of infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0095] Preferably, the infectious disease is a viral infection;

[0096] The autoimmune diseases mentioned include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, and sensitized kidney transplantation.

[0097] The cancers mentioned include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia, and prostate cancer.

[0098] In a tenth aspect, the present invention provides a method for treating and / or preventing diseases or conditions in a subject using the nucleic acid-loaded lipid nanoparticles described in the fourth aspect or the nucleic acid-loaded lipid nanoparticle formulation described in the sixth aspect, the method comprising administering the nucleic acid-loaded lipid nanoparticles or the nucleic acid-loaded lipid nanoparticle formulation to a subject in need.

[0099] In this invention, the term "inflammatory disease" includes autoimmune diseases, allergic diseases, and inflammatory conditions, such as those 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 graft rejection, Bechtel's disease, uveitis, psoriasis, dermatitis, atopic dermatitis, dermatomyositis, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, Sjögren's syndrome, and blistering diseases (e.g., pemphigus vulgaris), antibody-mediated vasculitis syndromes including ANCA-associated vasculitis, purpura, and immune complex vasculitis (stage I or II cancer or infection). Allergic diseases may be selected from contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, and beryllium poisoning.

[0100] In this invention, the term "viral infection" includes, but is not limited to, retroviral infection, hepatitis virus infection, COVID-19 SARS-CoV-2 infection, Zika virus infection, dengue virus infection, etc.

[0101] In this 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.

[0102] Compared with the prior art, the present invention has the following advantages: the nanoparticles prepared by the ionizable lipids of the present invention have high encapsulation efficiency, low PDI, and wide and stable process adaptability. Attached Figure Description

[0103] Figure 1 shows the experimental results of the lipid nanoparticles prepared from the lipid compounds of the present invention in Example 70 of the present invention inhibiting tumor growth.

[0104] Figure 2 shows the effect of lipid nanoparticles prepared by the lipid compound of the present invention in Example 70 of the present invention on extending the survival rate of mice after tumor inoculation. Detailed Implementation

[0105] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0106] definition

[0107] For clarity and readability, the following scientific background information and definitions are provided. Any technical features mentioned herein or disclosed herein may be part of, or read in, each embodiment of the invention. Other definitions and interpretations may be provided in the context of the invention. Unless otherwise defined, or unless required by a particular context, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0108] The expressions "one embodiment," "implementation," "a particular embodiment," etc., mean that a specific feature, property, or characteristic exists in at least one embodiment of the invention, or a specific group or combination of features, properties, or characteristics as described in conjunction with the corresponding expression. These expressions appearing in different places throughout the description do not necessarily refer to the same embodiment. Furthermore, specific features, properties, or characteristics can be combined in any suitable manner in one or more embodiments.

[0109] When the term "neutral" is applied to compounds such as lipids or steroids, or groups or parts thereof, it means that it is neither a cation nor an anion, such as compounds that do not have ionizable functional groups under physiological conditions, such as hydrocarbons; or it means that it is both a cation and an anion under typical physiological conditions, i.e., a zwitterion, such as typical natural phosphatidylcholine.

[0110] 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 classified into at least three categories: (1) “simple lipids,” including fats, oils, and waxes; (2) “complex lipids,” including phospholipids and glycolipids; and (3) “derived lipids,” such as steroids. Regarding glycolipids, in some embodiments, LNPs comprise glycolipids (e.g., monosialotetrahexosylganglioside GM1).

[0111] 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 a single sequence (not naturally occurring) and / or due to other modifications, such as structural modifications of non-natural nucleotides. Typically, artificial mRNA molecules can be designed and / or generated using genetic engineering methods to correspond to a desired artificial nucleotide sequence (heterologous sequence). In this context, an artificial sequence is usually a non-natural sequence, meaning it differs from the wild-type sequence by at least one nucleotide.

[0112] The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to the administered organism and does not diminish the compound's biological activity and properties.

[0113] Amino lipids

[0114] Amino lipids are preferably cationic, meaning that when the pH value drops below the pKa of the ionizable group of the lipid, the amino lipid will protonate. When it is positively charged, the lipid can bind to the negatively charged nucleic acid.

[0115] steroids

[0116] "Steroid" is an organic compound having four rings arranged in a specific molecular configuration. Steroids and neutral steroids include naturally occurring steroids and their analogues (such as amphiphilic lipid cholesterol hemisuccinate (CHEMS), which is composed of succinate-esterified β-hydroxy groups of cholesterol as a cholesterol derivative). Neutral steroids can be steroids that do not have ionizable atoms or groups under physiological conditions, or they can be zwitterionic steroids. In a preferred embodiment, neutral steroids do not contain atoms or groups that are ionizable under physiological conditions. In some preferred embodiments, the steroid or steroid analogue is cholesterol. The terms "steroid" and "neutral steroid" are used interchangeably herein.

[0117] neutral lipids

[0118] The "neutral lipids" of this invention, also known as "auxiliary lipids," are preferably phospholipids or neutral phospholipids. As used herein, a "neutral phospholipid" is an amphiphilic compound consisting of a molecule typically having 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 are abundant in nature. In this invention, "phospholipids" or "neutral phospholipids" include both natural and synthetic phospholipids.

[0119] Polymer-bound lipids

[0120] The term "polymer-bound lipid" refers to a molecule that simultaneously comprises a lipid moiety and a polymer moiety. Preferably, the polymer-bound lipid is a polyethylene glycol-modified lipid or a PEG-lipid. The term "polyethylene glycol-modified lipid" or "PEG-lipid" refers to a molecule that simultaneously comprises a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-modified lipids are known in the art and include PEG-DMG, etc.

[0121] In a specific embodiment, the polymer-bound lipid has the chemical formula PYL, where P is the hydrophilic polymer portion, Y is an optional linker, and L is the lipid portion.

[0122] Specifically, the hydrophilic polymer portion P can be polyethylene glycol (PEG). In specific embodiments, the average molecular weight of the PEG portion is between 1 kDa and 3 kDa, for example, between 1.5 kDa and 2.5 kDa, between 1.7 kDa and 2.3 kDa, between 1.8 kDa and 2.2 kDa, between 1.9 kDa and 2.1 kDa, or 2 kDa. Therefore, the PEG can be the PEG commonly referred to as "PEG 2000".

[0123] In another embodiment, the hydrophilic polymer portion P in the polymer-bound lipid can also be a substantially hydrophilic polymer that is different from the hydrophilic polymer portion described above. That is, the hydrophilic polymer portion P in the polymer-bound lipid can be based on poly(propylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), poly-N-(2-hydroxypropyl)methacrylamide, hydroxyethyl starchization (HESylation) process (according to PMID 24681396), PASylation method (i.e., proline-alanine-serine), XTEN method (i.e., PEG-based peptide) known in the art, polysarcosin, or poly(vinyl acetate).

[0124] Specifically, the optional linker Y can be any useful spacer structure, such as those spacers commonly found in polyethylene glycol-modified lipids, for example, but not limited to succinimide, amine, ether, ester, acid anhydride, aldehyde, ketone, amide, carbamate linker or combinations thereof.

[0125] Specifically, the lipid moiety L can be derived from phospholipids, sphingolipids, or ceramides. As used herein, the term "derived from phospholipids or ceramides" includes free radicals of phospholipids and ceramides. Examples are lipids that contain polymer-bound phosphatidylethanolamine or phosphatidylglycerol moieties.

[0126] In a preferred embodiment, the polymer-bound lipid is a polyethylene glycol-modified lipid. The polyethylene glycol-modified lipid includes, but is not limited to, the following: polyethylene glycol-modified diacylglycerol lipids (PEG-DAG); polyethylene glycol-modified ceramide lipids (PEG-Cer); polyethylene glycol-modified phosphatidylethanolamine lipids (PEG-PE); polyethylene glycol-modified succinate diacylglycerol lipids (PEG-S-DAG); polyethylene glycol-modified dialkoxypropyl carbamate lipids; and 1,2-dimyristyl-rac-glycerol-3-methoxy polyethylene glycol (“PEG-DMG” or “DMG-PEG”). In a more preferred embodiment, the polymer-bound lipid is DMG-PEG2000.

[0127] 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.

[0128] The lipid nanoparticles of the present invention are not limited to any particular form and should be interpreted to include any form produced when aminolipids are combined with optional one or more other lipids, for example in an aqueous environment and / or in the presence of nucleic acid compounds. For example, liposomes, lipid complexes, lipoplexes, etc., are within the scope of lipid nanoparticles.

[0129] 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 tablet. Alternatively, the composition can be in liquid form, and each excipient can be added independently in a dissolved or dispersed (e.g., suspension or emulsion) form. In a preferred embodiment, the composition is formulated as a sterile solid composition, such as a powder or lyophilized form, for recombination with an aqueous liquid carrier. Such formulations are also preferred for compositions containing bioactive components as described further in detail below.

[0130] As used herein, “nanoparticle” refers to a submicron particle having any structure or morphology. Submicron particles may also be referred to as colloids or gels. Nanoparticles can be classified in relation to the material they are based on, as well as their structure or morphology, for example, as nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polymers, mixtures, or mixed complexes; only a few possible names for specific types of nanoparticles are mentioned. “Lipid nanoparticles” (LNPs) are nanoparticles formed from lipids, typically containing at least one amphiphilic, film-forming lipid and optionally other lipids, further optionally containing a loading material such as a nucleic acid compound. As used herein, the expression “lipid nanoparticle” or “LNP” includes any subtype and morphology of nanoparticles formed or co-formed by lipids, such as liposomes and lipoplexes.

[0131] 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 lipid ensemble comprising at least one amphiphilic, vesicle-forming lipid. Liposomes and lipoplexes are examples of lipid nanoparticles.

[0132] application

[0133] This invention provides a method for treating or preventing infectious diseases, cancer, tumors, genetic diseases, allergies, toxicities, and autoimmune diseases using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0134] 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, cancer, tumors, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0135] The infectious diseases mentioned include viral, bacterial, or protozoan infectious diseases. The viruses include, but are 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, extraintestinal 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, and Zika virus.

[0136] The cancers mentioned include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia, and prostate cancer.

[0137] The present invention also provides methods for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer, or therapy via interfering RNA using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0138] Accordingly, the present invention also provides the use of the above-mentioned amino lipids or lipid nanoparticles in the preparation of medicaments for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or therapy by interfering RNA.

[0139] The present invention also provides a method for delivering a drug to a subject, comprising: administering to the subject a drug formulated in the above-mentioned lipid nanoparticles.

[0140] When administering a drug, the route of administration is determined based on the drug's dosage form. The dosage form is related to the excipients and / or carrier. Common routes of systemic administration include transdermal, oral, and parenteral routes, including subcutaneous, intravenous, intramuscular, intra-arterial, intradermal, and intraperitoneal injections and / or intranasal administration. Routes of local administration generally include intradermal, transdermal, subcutaneous, or intramuscular injections, or intralesional, intracranial, intrapulmonary, intracardiac, intratumoral, and sublingual injections. When the drug is in vaccine form, intramuscular and intradermal injections are preferred routes of administration.

[0141] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0142] Example 1

[0143] Synthetic route of compound A1

[0144] Step 1: Synthesis of compound A1-1

[0145] 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 sequentially to a 25 mL round-bottom flask. The mixture was stirred overnight at room temperature. After the reaction was completed, the mixture was purified by column chromatography (n-heptane:ethyl acetate = 50:1 to 10:1) to give 1.02 g of intermediate A1-1 (white solid), yield 85%.

[0146] Step 2, Synthesis of Compound A1-2

[0147] In a 25 mL round-bottom flask, 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, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (n-heptane:ethyl acetate = 100:1 to 20:1) to give 1.08 g of intermediate A1-2 (colorless oily liquid), yield 76%.

[0148] Step 3, Synthesis of Compound A1-3

[0149] In a 25 mL round-bottom flask, 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-propanediamine (174.24 g / mol, 139 mg, 0.8 mmol), and acetonitrile (5 mL) were added, and the mixture was stirred overnight at 75 °C. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 100:1 to 20:1) to give 511 mg of intermediate A1-3 (yellow oily liquid), yield 66%.

[0150] Step 4: Synthesis of compound A1-4

[0151] In a 10 mL reaction tube, A1-3 (967.47 g / mol, 967 mg, 1.0 mmol) and dichloromethane (5 mL) were added and stirred at 0 °C for 5 minutes. Then, 1 mL of trifluoroacetic acid was added to the reaction mixture. The reaction was brought to room temperature and stirred for another 2 hours. After the reaction was complete, the pH of the reaction mixture was adjusted to 8–9 with 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 give 780 mg of intermediate A1-4 (yellow oily liquid), with a yield of 90%.

[0152] Step 5: Synthesis of compound A1

[0153] In a 10 mL reaction tube, A1-4 (867.35 g / mol, 434 mg, 0.5 mmol), 3-methoxy-4-(methylamino)cyclobuten-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 and stirred overnight at room temperature. After the reaction was complete, the product A1 (yellow oily liquid) was purified by column chromatography (dichloromethane:methanol = 100:1 to 20:1) to give 369 mg of product A1. The yield was 76%. ESI-MS C 56 H 102 N3O 10 + [M+H] + Calculated value: 976.7560; Measured value: 976.7568.

[0154] Example 2

[0155] Synthetic route of compound A2

[0156] Step 1: Synthesis of compound A2-1

[0157] A2-1 was synthesized according to steps 1 to 4 of Example 1.

[0158] Step 2, Synthesis of Compound A2-2

[0159] In a 10 mL reaction tube, 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 and stirred at room temperature for 3 hours. Then, p-toluenesulfonyl chloride (190.65 g / mol, 286 mg, 1.5 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was purified by column chromatography (n-heptane:ethyl acetate = 50:1 to 5:1) to give 655 mg of intermediate A2-2 (yellow oily liquid), with a yield of 72%.

[0160] Step 3, Synthesis of Compound A2

[0161] In a 10 mL reaction tube, 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 and stirred at room temperature for 2 hours. After the reaction was complete, the product A2 (yellow oily liquid) was purified by column chromatography (dichloromethane:methanol = 100:1 to 40:1) to give 656 mg of product A2. The yield was 66%. ESI-MS C 55 H 106 N3O 10 + [M+H] + Calculated value: 1000.7593; Measured value: 1000.7599.

[0162] Example 3

[0163] Synthetic route of compound A3

[0164] Following the synthesis method of Example 2, A3 was prepared, yielding 442 mg of a yellow oily liquid. ESI-MS C 53 H 102 N3O8 + [M+H] + Calculated value: 940.7382; Measured value: 940.7354.

[0165] Example 4

[0166] Synthetic route of compound A4

[0167] Step 1: Synthesis of compound A4-1

[0168] In a 50 mL round-bottom flask, 3,4-dimethoxy-3-cyclobutene-1,2-dione (142.11 g / mol, 853 mg, 6 mmol), 3-amino-1,2-propanediol (91.11 g / mol, 273 mg, 3 mmol), triethylamine (101.19 g / mol, 455 mg, 4.5 mmol), and ethanol (25 mL) were added and stirred overnight at room temperature. After the reaction was complete, the mixture was purified by column chromatography (dichloromethane:methanol = 100:1 to 10:1) to give 300 mg of intermediate A4-1 (pale yellow solid), yield 50%.

[0169] Step 2, Synthesis of Compound A4

[0170] In a 10 mL reaction tube, 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 and stirred overnight at room temperature. After the reaction was complete, the product was purified by column chromatography (dichloromethane:methanol = 100:1 to 20:1) to give 651 mg of product A4 (pale yellow solid), yield 63%. ESI-MS C 58 H 106 N3O 12 + [M+H] + Calculated value: 1036.7771; Measured value: 1036.7771.

[0171] Example 5

[0172] Synthetic route of compound A5

[0173] Step 1: Synthesis of compound A5-1

[0174] In a 25 mL round-bottom flask, 3,3'-dithiodipropionic acid (210.27 g / mol, 8.41 g, 40 mmol) and 9-heptadecyl alcohol (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. The mixture was stirred at room temperature for 2 hours, then cooled to 0 °C. DCC (206.33 g / mol, 2.48 g, 12 mmol) was added, and the mixture was stirred overnight. After the reaction was complete, the mixture was filtered to remove the byproduct DCU. The filter cake was washed with a small amount of ethyl acetate, and the filtrate was collected. The solvent in the filtrate was evaporated to dryness, and the residual solid was dissolved in 50 mL of dichloromethane. The mixture was filtered again to remove unreacted 3,3'-dithiodipropionic acid. The filter cake was washed with a small amount of dichloromethane, and the filtrate was collected. The filtrate obtained after two filtrations was concentrated by rotary evaporation and purified by column chromatography (petroleum ether: ethyl acetate = 10:1 to 2:1) to give 1.92 g of intermediate A5-1 (yellow oily liquid), with a yield of 43%.

[0175] Step 2, Synthesis of Compound A5-2

[0176] In a 50 mL round-bottom flask, 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, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (heptane:ethyl acetate = 100:1 to 50:1) to give 1.71 g of intermediates A5-2 and A1-2 (colorless oily liquid), yield 56%.

[0177] Step 3, Synthesis of Compound A5

[0178] In a 25 mL round-bottom flask, 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, and the mixture was stirred overnight at 75 °C. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 100:1 to 20:1) to give 704 mg of product A5 (yellow oily liquid), yield 62%. ESI-MS C 61 H 118 N9 + [M+H] + Calculated value: 1136.7684; Measured value: 1136.7681.

[0179] Example 6

[0180] Synthetic route of compound A6

[0181] Step 1: Synthesis of compound A6-1

[0182] In a 50 mL round-bottom flask, 6-mercaptohexane-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, and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction solution was diluted with dichloromethane, washed three times with water, dried over anhydrous sodium sulfate, and purified by column chromatography (n-heptane:ethyl acetate = 10:1 to 2:1) to give 2.36 g of intermediate A6-1 (colorless oily liquid), yield 86%.

[0183] Step 2, Synthesis of Compound A6-2

[0184] In a 25 mL round-bottom flask, 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, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (n-heptane:ethyl acetate = 100:1 to 40:1) to give 1.71 g of intermediate A6-2 (colorless oily liquid), yield 42%.

[0185] Step 3, Synthesis of Compound A6

[0186] In a 25 mL round-bottom flask, 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, and the mixture was stirred overnight at 75 °C. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 100:1 to 20:1) to give 694 mg of product A6 (yellow oily liquid), yield 85%. ESI-MS C 47 H 94 NO5S2 + [M+H] + Calculated value: 816.6568; Measured value: 816.6574.

[0187] Example 7

[0188] Synthetic route of compound A7

[0189] Step 1: Synthesis of compound A7-1

[0190] Potassium methoxide (70.13 g / mol, 70 mg, 1 mmol) and 1-hexamethylenetetramine (118.24 g / mol, 1.18 g, 10 mmol) were added to a 25 mL round-bottom flask. The mixture was heated to 100 °C, and 1,2-epoxydodecane (184.18 g / mol, 1.84 g, 10 mmol) was added dropwise over 15 minutes. After the addition was complete, the reaction was continued for 20 minutes. After the reaction was completed, the mixture was purified by column chromatography (n-heptane:ethyl acetate = 50:1 to 10:1) to give 2.54 g of intermediate A7-1 (colorless oily liquid), with a yield of 84%.

[0191] Step 2, Synthesis of Compound A7-2

[0192] In a 25 mL round-bottom flask, 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, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (n-heptane:ethyl acetate = 100:1 to 20:1) to give 835 mg of intermediate A7-2 (colorless oily liquid), yield 58%.

[0193] Step 3, Synthesis of Compound A7

[0194] In a 10 mL reaction tube, 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, and the mixture was stirred overnight at 75 °C. After the reaction was complete, the reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane:methanol = 100:1 to 20:1) to give 636 mg of product A6 (yellow oily liquid), yield 73%. ESI-MS C 51H 102 NO5S2 + [M+H] + Calculated value: 872.7194; Measured value: 872.7195.

[0195] Example 8

[0196] Following the synthesis method of Example 3, A8 was prepared, yielding 612 mg of a yellow oily liquid. ESI-MS C 52 H 100 N3O8S + [M+H] + The calculated value is 926.7226, and the measured value is 926.7207. Its structural formula is as follows:

[0197] Example 9

[0198] Following the synthesis method of Example 3, A9 was prepared, yielding 485 mg of a yellow oily liquid. ESI-MS C 54 H 105 N4O7S + [M+H] + The calculated value is 953.7698, and the measured value is 953.7742. Its structural formula is as follows:

[0199] Example 10

[0200] Following the synthesis method of Example 3, A10 was prepared, yielding 504 mg of a yellow oily liquid. ESI-MS C 57 H 110 N3O8S + [M+H] + The calculated value is 996.8008, and the measured value is 996.8101. Its structural formula is as follows:

[0201] Example 11

[0202] Following the synthesis method of Example 3, A11 was prepared, yielding 361 mg of a yellow oily liquid. ESI-MS C 58 H 112 N3O8S + [M+H] + The calculated value is 1010.8165, and the measured value is 1010.8198. Its structural formula is as follows:

[0203] Example 12

[0204] Following the synthesis method of Example 1, A12 was prepared, yielding 428 mg of a yellow oily liquid. ESI-MS C 57H 104 N3O 10 + [M+H] + The calculated value is 990.7716, and the measured value is 990.7328. Its structural formula is as follows:

[0205] Example 13

[0206] Following the synthesis method of Example 1, A13 was prepared, yielding 291 mg of a yellow oily liquid. ESI-MS C 55 H 100 N3O 10 + [M+H] + The calculated value is 962.7403, and the measured value is 962.7608. Its structural formula is as follows:

[0207] Example 14

[0208] Following the synthesis method of Example 1, A14 was prepared, yielding 352 mg of a yellow oily liquid. (ESI-MSC) 60 H 110 N3O 10 + [M+H] + The calculated value is 1032.8191, and the measured value is 1032.8425. Its structural formula is as follows:

[0209] Example 15

[0210] Following the synthesis method of Example 1, A15 was prepared, yielding 376 mg of a yellow oily liquid. ESI-MS C 54 H 98 N3O 10 + [M+H] + The calculated value is 948.7247, and the measured value is also 948.7247. Its structural formula is as follows:

[0211] Example 16

[0212] Following the synthesis method of Example 1, A16 was prepared, yielding 402 mg of a yellow oily liquid. ESI-MS C 61 H 112 N3O 10 + [M+H] + The calculated value is 1046.8342, and the measured value is 1046.8344. Its structural formula is as follows:

[0213] Example 17

[0214] Following the synthesis method of Example 1, A17 was prepared, yielding 649 mg of a yellow oily liquid. ESI-MS C 52 H 94 N3O 10 + [M+H] + The calculated value is 920.6934, and the measured value is 920.6938. Its structural formula is as follows:

[0215] Example 18

[0216] Following the synthesis method of Example 4, A18 was prepared, yielding 322 mg of a yellow oily liquid. ESI-MS C 58 H 106 N3O 11 + [M+H] + The calculated value is 1020.7822, and the measured value is 1020.7844. Its structural formula is as follows:

[0217] Example 19

[0218] Following the synthesis method of Example 4, A19 was prepared, yielding 495 mg of a yellow oily liquid. ESI-MS C 57 H 104 N3O 11 + [M+H] + The calculated value is 1006.7665, and the measured value is 1006.7676. Its structural formula is as follows:

[0219] Example 20

[0220] Following the synthesis method of Example 4, A20 was prepared, yielding 387 mg of a yellow oily liquid. ESI-MS C 57 H 104 N3O 12 + [M+H] + The calculated value is 1022.7615, and the measured value is 1022.7626. Its structural formula is as follows:

[0221] Example 21

[0222] Following the synthesis method of Example 4, A21 was prepared, yielding 568 mg of a yellow oily liquid. ESI-MS C 59 H 109 N4O 11 +[M+H] + The calculated value is 1049.8087, and the measured value is 1049.8085. Its structural formula is as follows:

[0223] Example 22

[0224] Following the synthesis method of Example 4, A22 was prepared, yielding 486 mg of a yellow oily liquid. ESI-MS C 59 H 109 N4O 10 + [M+H] + The calculated value is 1033.8138, and the measured value is 1033.8149. Its structural formula is as follows:

[0225] Example 23

[0226] Following the synthesis method of Example 4, A23 was prepared, yielding 233 mg of a yellow oily liquid. ESI-MS C 57 H 104 N3O 11 + [M+H] + The calculated value is 1006.7665, and the measured value is 1006.7667. Its structural formula is as follows:

[0227] Example 24

[0228] Following the synthesis method of Example 4, A24 was prepared, yielding 350 mg of a yellow oily liquid. ESI-MS C 62 H 114 N3O 12 + [M+H] + The calculated value is 1092.8397, and the measured value is 1092.8399. Its structural formula is as follows:

[0229] Example 25

[0230] Following the synthesis method of Example 4, A25 was prepared, yielding 618 mg of a yellow oily liquid. ESI-MS C 62 H 114 N3O 11 + [M+H] + The calculated value is 1076.8448, and the measured value is 1076.8453. Its structural formula is as follows:

[0231] Example 26

[0232] Following the synthesis method of Example 1, A26 was prepared, yielding 589 mg of a yellow oily liquid. ESI-MS C 56 H 102 N3O 10 + [M+H] + The calculated value is 976.7560, and the measured value is 976.7572. Its structural formula is as follows:

[0233] Example 27

[0234] Following the synthesis method of Example 2, A27 was prepared, yielding 588 mg of a yellow oily liquid. ESI-MS C 56 H 108 N3O9S + [M+H] + The calculated value is 998.7806, and the measured value is 998.7815. Its structural formula is as follows:

[0235] Example 28

[0236] Following the synthesis method of Example 2, A28 was prepared, yielding 344 mg of a yellow oily liquid. ESI-MS C 55 H 106 N3O9S + [M+H] + The calculated value is 984.7644, and the measured value is 984.7632. Its structural formula is as follows:

[0237] Example 29

[0238] The synthetic route for compound A29 is as follows:

[0239] Step 1: Synthesis of compound A29-1

[0240] In a 200 mL round-bottom flask, 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. The mixture was stirred in an ice bath for 5 minutes. Over 30 minutes, a solution of 2-hexyl-1-decyl alcohol (242.44 g / mol, 2.42 g, 10 mmol) and triethylamine (101.19 g / mol, 2.02 g, 20 mmol) in DCM (50 mL) was added dropwise. After the addition was complete, the temperature was raised to room temperature and the reaction was stirred overnight. After the reaction was completed, the mixture was purified by column chromatography (n-heptane:ethyl acetate = 50:1 to 20:1) to give 3.75 g of intermediate A29-1 (colorless oily liquid), with a yield of 94%.

[0241] Step 2, Synthesis of Compound A29-2

[0242] In a 100 mL round-bottom flask, 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 and stirred overnight at 50 °C. After the reaction was completed, the mixture was purified by column chromatography (to heptane) to give 1.4 g of intermediate A29-2 (colorless oily liquid), with a yield of 60%.

[0243] Step 3, Synthesis of Compound A29-3

[0244] In a 100 mL round-bottom flask, 2,2'-dithiodipyridine (220.31 g / mol, 440 mg, 20 mmol), acetic acid (2 mL), and EtOH (20 mL) were added. The mixture was stirred at room temperature for 5 minutes. Over 30 minutes, a solution of 3-mercapto-1-propanol (92.16 g / mol, 922 mg, 10 mmol) in EtOH (20 mL) was added dropwise. After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, the solution was purified by column chromatography (n-heptane:ethyl acetate = 50:1 to 4:1) to give 1.79 g of intermediate A29-3 (yellow-green viscous liquid), with a yield of 89%.

[0245] Step 4: Synthesis of compound A29-4

[0246] In a 100 mL round-bottom flask, A29-2 (259.51 g / mol, 2.08 g, 8 mmol), acetic acid (1.6 mL), and MeOH (20 mL) were added. The mixture was stirred at room temperature for 5 minutes. Over 30 minutes, a solution of A29-3 (201.30 g / mol, 1.61 g, 8 mmol) in MeOH (20 mL) was added dropwise. After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was purified by column chromatography (n-heptane:ethyl acetate = 100:1 to 5:1) to give 2.01 g of intermediate A29-4 (pale yellow oily liquid), with a yield of 72%.

[0247] Step 5: Synthesis of compound A29-5

[0248] Add A29-4 (348.65 g / mol, 348 mg, 1 mmol) and acrylic acid (72.06 g / mol) to a 50 ml round-bottom flask.

[0249] 86 mg (1.2 mmol), EDCI (191.70 g / mol, 288 mg, 1.5 mmol), DMAP (122.17 g / mol, 12 mg, 0.1 mmol), DIPEA (129.24 g / mol, 258 mg, 2 mmol), and DCM (10 mg) were added and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (n-heptane:ethyl acetate = 100:1 to 20:1) to give 242 mg of intermediate A29-5 (colorless oily liquid), yield 60%.

[0250] Step 6, Synthesis of Compound A29

[0251] A29-4 (402.69 g / mol, 242 mg, 0.6 mmol) and N-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 was completed, the product A29 (pale yellow oily liquid) was purified by column chromatography (dichloromethane:methanol = 100:1 to 20:1) to give 97 mg of product A29. The yield was 56%. The calculated value of C93H183N3O8S8+[M+H]+ by ESI-MS was 1727.1804, and the measured value was 1727.1805.

[0252] Example 30

[0253] Following the synthesis methods of Examples 1 and 3, A30 was prepared, yielding 326 mg of a yellow oily liquid. ESI-MS C 56 H 108 N3O8S + [M+H] + The calculated value is 982.7852, and the measured value is 982.7780. Its structural formula is as follows:

[0254] Example 31

[0255] Following the synthesis methods of Examples 1 and 3, A31 was prepared, yielding 225 mg of a yellow oily liquid. ESI-MS C 57 H 110 N3O8S + [M+H] + The calculated value is 996.8008, and the measured value is 996.8041. Its structural formula is as follows:

[0256] Example 32

[0257] Following the synthesis methods of Examples 1 and 3, A32 was prepared, yielding 283 mg of a yellow oily liquid. ESI-MS C 54 H 104 N3O8S + [M+H] + The calculated value is 954.7539, and the measured value is 954.7573. Its structural formula is as follows:

[0258] Example 33

[0259] Following the synthesis methods of Examples 1 and 3, A33 was prepared, yielding 289 mg of a yellow oily liquid. ESI-MS C 55 H 106 N3O8S + [M+H] + The calculated value is 968.7695, and the measured value is 968.7877. Its structural formula is as follows:

[0260] Example 34

[0261] Following the synthesis methods of Examples 1 and 3, A34 was prepared, yielding 241 mg of a yellow oily liquid. ESI-MS C 56 H 108 N3O8S + [M+H] + The calculated value is 982.7852, and the measured value is 982.7884. Its structural formula is as follows:

[0262] Example 35

[0263] Following the synthesis methods of Examples 1 and 3, A35 was prepared, yielding 411 mg of a yellow oily liquid. ESI-MS C 55 H 106 N3O8S + [M+H] + The calculated value is 968.7695, and the measured value is 968.7740. Its structural formula is as follows:

[0264] Example 36

[0265] Following the synthesis methods of Examples 1 and 4, A36 was prepared, yielding 453 mg of a yellow oily liquid. ESI-MS C 60 H 110 N3O 10 + [M+H] + The calculated value is 1032.8186, and the measured value is 1032.8092. Its structural formula is as follows:

[0266] Example 37

[0267] Following the synthesis methods of Examples 1 and 4, A37 was prepared, yielding 469 mg of a yellow oily liquid. ESI-MS C 59 H 108 N3O 10 + [M+H] + The calculated value is 1018.8029, and the measured value is 1018.8226. Its structural formula is as follows:

[0268] Example 38

[0269] Following the synthesis methods of Examples 1 and 4, A38 was prepared, yielding 138 mg of a yellow oily liquid. ESI-MS C 59 H 108 N3O 10 + [M+H] + The calculated value is 1018.8029, and the measured value is 1018.8223. Its structural formula is as follows:

[0270] Example 39

[0271] Following the synthesis methods of Examples 1 and 4, A39 was prepared, yielding 149 mg of a yellow oily liquid. ESI-MS C 60 H 110 N3O 10 + [M+H] + The calculated value is 1032.8186, and the measured value is 1032.8205. Its structural formula is as follows:

[0272] Example 40

[0273] Following the synthesis methods of Examples 1 and 4, A40 was prepared, yielding 358 mg of a yellow oily liquid. ESI-MS C 57 H 104 N3O 10 + [M+H] + The calculated value is 990.7716, and the measured value is 990.7735. Its structural formula is as follows:

[0274] Example 41

[0275] Following the synthesis methods of Examples 1 and 4, A41 was prepared, yielding 355 mg of a yellow oily liquid. ESI-MS C 58 H 106 N3O 10 + [M+H] + The calculated value is 1004.7873, and the measured value is 1004.7889. Its structural formula is as follows:

[0276] Example 42

[0277] Following the synthesis method of Example 5, A42 was prepared, yielding 343 mg of a yellow oily liquid. ESI-MS C 55 H 106 NO9S4 + [M+H] + The calculated value is 1052.6745, and the measured value is 1052.6924. Its structural formula is as follows:

[0278] Example 43

[0279] Following the synthesis method of Example 5, A43 was prepared, yielding 258 mg of a yellow oily liquid. ESI-MS C 59 H 114 NO9S4 + [M+H] + The calculated value is 1108.7371, and the measured value is 1108.7398. Its structural formula is as follows:

[0280] Example 44

[0281] Following the synthesis method of Example 5, A44 was prepared, yielding 422 mg of a yellow oily liquid. ESI-MS C 63 H 122 NO9S4 + [M+H] + The calculated value is 1164.7997, and the measured value is 1164.8053. Its structural formula is as follows:

[0282] Example 45

[0283] Following the synthesis methods of Examples 1 and 7, A45 was prepared, yielding 432 mg of a yellow oily liquid. ESI-MS C 56 H 106 N3O6S2 + [M+H] + The calculated value is 980.7518, and the measured value is 980.7675. Its structural formula is as follows:

[0284] Example 46

[0285] Following the synthesis methods of Examples 1 and 5, A46 was prepared, yielding 314 mg of a yellow oily liquid. ESI-MS C 60 H 110 N3O 10 S4 + [M+H] + The calculated value is 1160.7069, and the measured value is 1160.7250. Its structural formula is as follows:

[0286] Example 47

[0287] Following the synthesis methods of Examples 1 and 7, A47 was prepared, yielding 132 mg of a yellow oily liquid. ESI-MS C 64 H 118 N3O 10 S4 + [M+H] + The calculated value is 1216.7695, and the measured value is 1216.7804. Its structural formula is as follows:

[0288] Example 48

[0289] Following the synthesis methods of Examples 1 and 7, A48 was prepared, yielding 564 mg of a yellow oily liquid. (ESI-MSC) 64 H 126 N3O 10 S4 + [M+H] + The calculated value is 1272.8321, and the measured value is 1272.8023. Its structural formula is as follows:

[0290] Example 49

[0291] Following the synthesis methods of Examples 3 and 7, A49 was prepared, yielding 328 mg of a yellow oily liquid. (ESI-MSC) 61 H 118 N3O8S5 + [M+H] + The calculated value is 1180.7517, and the measured value is 1180.7458. Its structural formula is as follows:

[0292] Example 50

[0293] Following the synthesis methods of Examples 3 and 7, A50 was prepared, yielding 248 mg of a yellow oily liquid. (ESI-MSC) 65H 126 N3O8S5 + [M+H] + The calculated value is 1236.8143, and the measured value is 1236.8459. Its structural formula is as follows:

[0294] Example 51

[0295] Following the synthesis methods of Examples 3 and 7, A51 was prepared, yielding 395 mg of a yellow oily liquid. (ESI-MSC) 62 H 120 N3O8S5 + [M+H] + The calculated value is 1194.7673, and the measured value is 1194.7824. Its structural formula is as follows:

[0296] Example 52

[0297] Following the synthesis methods of Examples 3 and 7, A52 was prepared, yielding 395 mg of a yellow oily liquid. (ESI-MSC) 63 H 122 N3O8S5 + [M+H] + The calculated value is 1208.7830, and the measured value is 1208.7329. Its structural formula is as follows:

[0298] Example 53

[0299] Following the synthesis methods of Examples 1 and 7, A53 was prepared, yielding 256 mg of a yellow oily liquid. (ESI-MSC) 65 H 120 N3O 10 S4 + [M+H] + The calculated value is 1230.7851, and the measured value is 1230.7859. Its structural formula is as follows:

[0300] Example 54

[0301] Following the synthesis methods of Examples 1 and 5, A54 was prepared, yielding 210 mg of a yellow oily liquid. (ESI-MSC) 60 H 110 N3O 10 S4 + [M+H] + The calculated value is 1160.7074, and the measured value is 1160.7076. Its structural formula is as follows:

[0302] Example 55

[0303] Following the synthesis methods of Examples 1 and 5, A55 was prepared, yielding 110 mg of a yellow oily liquid. ESI-MS C 53 H 96 N3O 10 S4 + [M+H] + The calculated value is 1062.5979, and the measured value is 1062.5983. The synthesis route and structural formula are as follows:

[0304] Example 56

[0305] Following the synthesis methods of Examples 1 and 5, A56 was prepared, yielding 163 mg of a yellow oily liquid. ESI-MS C 54 H 98 N3O 10 S2 + [M+H] + The calculated value is 1012.6694, and the measured value is 1012.6692. The synthesis route and structural formula are as follows:

[0306] Example 57

[0307] Following the synthesis methods of Examples 1 and 5, A57 was prepared, yielding 185 mg of a yellow oily liquid. ESI-MS C 61 H 112 N3O 10 S2 + [M+H] + The calculated value is 1110.7789, and the measured value is 1110.7795. The synthesis route and structural formula are as follows:

[0308] Example 58

[0309] Following the synthesis methods of Examples 1 and 5, A58 was prepared, yielding 185 mg of a yellow oily liquid. ESI-MS C 58 H 106 N3O 10 S2 + [M+H] + The calculated value is 1068.7320, and the measured value is 1068.7316. The synthesis route and structural formula are as follows:

[0310] Example 59

[0311] Following the synthesis methods of Examples 1 and 5, A59 was prepared, yielding 126 mg of a yellow oily liquid. ESI-MS C56 H 102 N3O 10 S2 + [M+H] + The calculated value is 1104.6448, and the measured value is 1104.6449. The synthesis route and structural formula are as follows:

[0312] Example 60

[0313] Following the synthesis methods of Examples 1 and 5, A60 was prepared, yielding 130 mg of a yellow oily liquid. ESI-MS C 53 H 102 N3O9S2 + [M+H] + The calculated value is 960.6996, and the measured value is 960.6992. The synthesis route and structural formula are as follows:

[0314] Example 61

[0315] Following the synthesis methods of Examples 1 and 5, A61 was prepared, yielding 98 mg of a yellow oily liquid. ESI-MS C 53 H 102 N3O9S2 + [M+H] + The calculated value is 1032.7142, and the measured value is 1032.7149. The synthesis route and structural formula are as follows:

[0316] Example 62

[0317] Following the synthesis method of Example 5, A62 was prepared, yielding 165 mg of a yellow oily liquid. ESI-MS C 53 H 106 NO5S4 + [M+H] + The calculated value is 964.6954, and the measured value is 964.6948. The synthetic route and structural formula are as follows:

[0318] Example 63

[0319] Following the synthesis methods of Examples 1 and 5, A63 was prepared, yielding 182 mg of a yellow oily liquid. ESI-MS C 58 H 110 N3O6S4 + [M+H] + The calculated value is 1072.7227, and the measured value is 1072.72283. The synthesis route and structural formula are as follows:

[0320] Example 64

[0321] Following the synthesis methods of Examples 1 and 5, A64 was prepared, yielding 102 mg of a yellow oily liquid. ESI-MS C 56 H 108 NO9S2 + [M+H] + The calculated value is 1002.7466, and the measured value is 1002.7458. The synthesis route and structural formula are as follows:

[0322] Example 65

[0323] Following the synthesis methods of Examples 1 and 5, A65 was prepared, yielding 141 mg of a yellow oily liquid. ESI-MS C 58 H 110 N3O6S4 + [M+H] + The calculated value is 904.6370, and the measured value is 904.6362. The synthesis route and structural formula are as follows:

[0324] Example 66

[0325] Following the synthesis methods of Examples 1 and 5, A66 was prepared, yielding 130 mg of a yellow oily liquid. ESI-MS C 51 H 98 N3O8S3 + [M+H] + The calculated value is 976.6516, and the measured value is 976.6523. The synthesis route and structural formula are as follows:

[0326] Example 67: Preparation of lipid nanoparticles

[0327] The ionizable lipid compound, phospholipid (DSPC), cholesterol, and polyethylene glycol-modified lipid (DMG-PEG2000) prepared in Examples 1-66 of this invention were mixed in a molar ratio of 47.5:10:41:1.5 and dissolved in anhydrous ethanol. Using a microfluidic preparation system, the resulting ethanol solution and acetate buffer (25 mM, pH = 5.0) containing Luc-mRNA were mixed at a volume ratio of 1:3 in a microfluidic chip to prepare a crude solution of lipid nanoparticles. This solution was then ultrafiltered using a 100 kDa ultrafiltration tube (Millipore, FTT500500) at 4°C. Before use, it was further filtered through a 0.22 μm microporous membrane. The mass ratio of the lipid compound to luciferase mRNA (Luc mRNA) was approximately 40:1.

[0328] The obtained lipid nanoparticles were characterized, and the results are shown in Table 1.

[0329] The particle size and PDI (polydispersity index) of the prepared lipid nanoparticles were determined using a Nano-ZSZEN3600 (Malvern) micrometer. 20 μL of LNP solution was used for particle size measurement, with three cycles of 30 s each.

[0330] Encapsulation efficiency was determined according to the standard procedure of the Quant-iT RiboGreen RNA kit.

[0331] Table 1 Characterization data of LNPs prepared using representative aminolipid compounds

[0332] The results above show that the encapsulation efficiency of the lipid nanoparticles of the present invention is higher than that of DLin-MC3-DMA (MC3).

[0333] Example 68: Transfection of lipid nanoparticles prepared from amino lipid compounds into primary BMDC cells.

[0334] Animal preparation: Six-week-old female C57BL / 6 mice, weighing approximately 20g, were selected and housed in an SPF-grade enclosure. Animal experiments were conducted strictly in accordance with the guidelines of national health agencies and animal ethics requirements.

[0335] Cell Acquisition: C57BL / 6 mice were euthanized by cervical dislocation and disinfected by immersion in 75% alcohol for 5 minutes. The thigh and tibia were dissected, and the attached muscles were removed to expose the bone. The bone marrow was then expelled from the tibia using a syringe filled with 1 mL of PBS. The expelled bone marrow was filtered through a 50 μm filter to remove impurities. Red blood cell lysis buffer (3–4 mL) was added to the filtrate, and after standing for 5 minutes, the mixture was centrifuged at 800 g for 5 minutes to remove the supernatant. The resulting cells were then cultured in 1640 medium (containing 10% fetal bovine serum, 20 ng / mL GMCSF, and 10 ng / mL...). Resuspend the cells in IL-4 and seed them into 6-well plates at a density of 100,000 cells / mL of culture medium. Incubate the plates at 37°C in a cell culture incubator containing 5% CO2. Perform half-medium medium changes every 2 days. On the seventh day, collect the suspended cells and loosely adherent cells and seed them into 96-well pure white microplates at a density of 20,000 cells per well. The culture medium volume is 100 μL.

[0336] Cell transfection: Lipid nanoparticles encapsulating luciferase mRNA were added to 96-well pure white microplates containing primary cells, with the volume of mRNA-lipid nanoparticles added to each well controlled at 10 μL. The plates were then incubated at 37°C in a 5% CO2 incubator for 12 hours.

[0337] Transfection efficiency assay: 20 μL of substrate ONE-Glo™ Luciferase was added to each well of a 96-well all-white microplate, and the result was measured using a Biorek Synergy H1 multi-functional microplate reader after 1 min. The expression intensity of Luc mRNA transfected onto BMDC by lipid nanoparticles (LNPs) prepared from representative aminolipid compounds according to the method in Example 54 is shown in Table 2, with MC3 as a control.

[0338] Table 2. Expression intensity of LNPs with representative amino lipid compositions transfected into BMDC.

[0339] It can be seen that the aminolipidase provided by this invention has a better expression intensity than MC3 after cell transfection.

[0340] Example 69: Evaluation of in vivo delivery performance of luciferase mRNA from lipid nanoparticles

[0341] Preparation of lipid nanoparticles: Same as in Example 67.

[0342] animal experiments

[0343] Animal preparation: Six-week-old female C57BL / 6 mice weighing approximately 20g were selected and housed in an SPF-grade enclosure. Animal experiments were conducted strictly in accordance with national health agency guidelines and animal ethics requirements.

[0344] In vivo delivery: Five C57BL / 6 mice were randomly selected from each group and injected with lipid nanoparticle solution via intramuscular injection at a dose of 0.1 mg / kg mRNA. Six hours later, 200 μL of 10 mg / mL D-luciferin potassium salt was injected intraperitoneally into each mouse. Five minutes later, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen) to observe and record the total fluorescence intensity of each mouse. The expression intensity of Luc mRNA delivered via intramuscular injection of representative aminolipid compounds is shown in Table 3, with MC3 as a control.

[0345] Table 3. Expression intensity of Luc mRNA delivered via intramuscular administration of LNPs with representative aminolipid compositions.

[0346] It can be seen that the expression intensity of the amino lipids provided by the present invention is significantly better than that of MC3 in intramuscular injection delivery.

[0347] Example 70: Evaluation of the in vivo immunotherapy and tumor treatment effects of lipid nanoparticles prepared from amino lipid compounds.

[0348] Preparation of lipid nanoparticles: Same as in Example 67.

[0349] animal experiments

[0350] Animal preparation: 5-6 week old female C57BL / 6 mice, weighing about 18-20g, were selected and housed in an SPF-grade enclosure. Animal experiments were conducted in strict accordance with the guidelines of national health agencies and animal ethics requirements.

[0351] In vivo delivery: B16-OVA melanoma cells (1.5 × 10⁵) were subcutaneously injected into the lateral thigh of mice. When the tumor size reached 50 mm... 3 Vaccination began around day 6 or 7 after tumor inoculation. Animals were immunized twice via intramuscular injection of an LNP formulation containing 1 μg of OVA-mRNA, with the second injection 7 days later. Tumor growth was measured three times weekly using a digital caliper, calculated as 0.5 × length × width × width. When the tumor volume reached 1500 mm², [the vaccination was initiated]. 3 The mice were euthanized. The tumor growth rate in the A12 and A48 groups was significantly slower than that in the MC3 group (as shown in Figure 1), and 100% (A12 group) and 80% (A48 group) of the mice achieved complete remission, which was significantly better than that in the MC3 group (as shown in Figure 2).

[0352] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Industrial applicability

[0353] This invention provides a novel ionizable lipid, its preparation method, and its applications. Its structural formula is shown in Formula (I). The ionizable lipid compound of this invention contains the structure of urea and its bioisosteres. Hydrogen bonds formed between the squaramide, urea, and thiourea structures and nucleic acids enhance the encapsulation effect of the ionizable lipid and reduce its immunogenicity. Nanoparticles prepared using the ionizable lipid of this invention exhibit high encapsulation efficiency, low PDI, wide process adaptability, and stability, demonstrating good economic value and application prospects.

Claims

1. An ionizable lipid compound, the structural formula of which is shown in formula (I): In formula (I), L1, L2, L3 and L4 may be the same or different, and each is 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 may be the same or different, and each is independently selected from: C1-C 18 Straight-chain or branched alkyl groups, C3-C 18 alkenyl; M1 and M5 may be the same or different, and each is independently selected from: C1-C 15 Straight-chain or branched alkylene compounds, M7-SS-M7, where M7 is selected from C1-C6 alkylene compounds; M2 and M4 may be the same or different, and each is independently selected from: C1-C 15 Alkylene, C2-C 15 sub-alkenyl, M6 is selected from C2-C6 alkylene groups, and the two M6 atoms are respectively attached to L2 and N atoms in formula (I); M3 is selected from C1-C6 alkylene groups; G is selected from H, -OH and at least one of the following structures: Where A is selected from C1-C6 alkyl or hydroxylated C2-C6 alkyl.

2. The ionizable lipid compound according to claim 1, characterized in that, L1, L2, L3 and L4 may be the same or different, and each is independently selected from: -O(C=O)-, -(C=O)O-, -O(C=O)O-, -(C=O)S-, -S(C=O)-, -O-, -SS-, -S-; Furthermore, L1 and L4 are the same, L2 and L3 are the same, and each is independently selected from: -O(C=O)-, -(C=O)O-, -(C=O)S-, -S(C=O)-, -SS-; Furthermore, R1 and R2 may be the same or different, and each is independently selected from: C2-C 18 Straight-chain or branched alkyl groups; Furthermore, M1 and M5 may be the same or different, and each is independently selected from: C2-C 15 Straight-chain or branched alkylene, M7-SS-M7, where M7 is selected from C1-C3 straight-chain alkylene; Furthermore, M2 and M4 are identical, each independently selected from C2-C. 11 Alkylene; Furthermore, G is selected from H, -OH, and at least one of the following structures: 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) as described in claim 1 or 2, G is The method includes the following steps: under alkaline conditions, the compound shown in formula (A) undergoes a substitution reaction with the compound shown in formula (B) to obtain the ionizable lipid compound shown in formula (I); In equation (A), the definitions of L1, L2, L3, L4, M1, M2, M3, M4, M5, R1, and R2 are the same as the definitions of L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 in equation (I); In formula (B), A is defined in the same way as A in formula (I)G, that is, A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl. G is The method includes the following steps: reacting the compound shown in formula (C) with the compound shown in formula (D) in an addition reaction to obtain the ionizable lipid compound shown in formula (I); In equation (C), the definitions of L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 are the same as the definitions of L1, L2, L3, L4, M1, M2, M3, M4, R1, and R2 in equation (I); In formula (D), A is defined in the same way as A in formula (I)G, that is, A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl. Or, G is The method includes: The compound shown in formula (A) is subjected to an addition reaction with the compound shown in formula (E) to obtain the ionizable lipid compound shown in formula (I); In formula (E), A is defined in the same way as A in formula (I)G, that is, A is selected from C1-C6 alkyl or hydroxy-substituted C2-C6 alkyl. G is H or -OH, and the method includes: subjecting the compound shown in formula (F), the compound shown in formula (H) to the compound shown in formula (I) by a substitution reaction to obtain the ionizable lipid compound shown in formula (I); In equation (F), the definitions of L1, L2, M1, M2, and R1 are the same as those in equation (I); In equation (H), the definitions of L3, L4, M4, M5, and R2 are the same as those in equation (I); In formula (I), M3 is defined as M3 in formula (I); G is H or -OH.

4. A lipid carrier containing an ionizable lipid compound of formula (I) as described in claim 1 or 2, wherein the lipid carrier is made from the following components: an ionizable lipid compound of formula (I), a steroid, a neutral lipid, and / or a polymer-bound lipid. Preferably, the polymer-bound lipid has the chemical formula PYL, where P is the hydrophilic polymer moiety, Y is an optional linker, and L is the lipid moiety. Preferably, in the lipid nanoparticles, the molar ratio of the ionizable lipid compound, steroid, neutral lipid and polymer-bound lipid shown in formula (I) 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 phosphoric acid, 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-distearate phosphatidylethanolamine (DSPE-PEG); more preferably, the relative molecular mass of the PEG-modified lipids is 500 to 5000, most preferably 1000, 2000, 3000, 4000, or 5000; the polymer-bound lipids are preferably DMG-PEG2000; The steroid is selected from one or more of cholesterol, cholesterol esters, steroid hormones, steroid vitamins and phytosterols, more preferably one or more of cholesterol, cholesterol esters and phytosterols, and most preferably cholesterol; The neutral lipid is selected from phospholipids, more preferably at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 2-dioleoyl-sn- Glyceryl-3-phosphate-(1'-rac-glycerol) (DOPG), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylserine (sodium salt) (DOPS), oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), more preferably 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC) or 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

6. A lipid nanoparticle loaded with nucleic acid, comprising the lipid carrier and nucleic acid as described in claim 4 or 5, wherein, The mass ratio of lipid carrier to 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 containing at least one coding sequence, self-replicating RNA, circular RNA, viral RNA and replicon RNA, or any combination thereof; More preferably, the nucleic acid is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (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 stem-loop, chain-terminating nucleoside, polyA sequence, polyadenylation signal and / or 5' cap structure.

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

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

9. Use of the ionizable lipid compound of formula (I) as described in claim 1 or 2, or the lipid carrier as described in claim 4 or 5, in the preparation of a medicament 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 formulation of claim 8; Preferably, the infectious disease is a viral infection; The cancers mentioned include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia, and prostate cancer; The autoimmune diseases mentioned include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, and sensitized kidney transplantation.

11. The use of the nucleic acid-loaded lipid nanoparticles of claim 6 or the nucleic acid-loaded lipid nanoparticle formulation of claim 8 in the preparation of drugs for the treatment or prevention of infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases; Preferably, The aforementioned infectious diseases, especially viral infections; The cancers mentioned include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia, and prostate cancer; The autoimmune diseases mentioned include systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, and sensitized kidney transplantation.

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

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