An ionizable lipid compound and its application

Abstract

The present invention relates to the field of drug delivery technology, and in particular to ionizable lipid compounds and applications thereof. The present invention provides an ionizable lipid compound containing #imgabs0#-(C=O)O- or -O(C=O)- and a hydroxyl fragment, and its chemical structure is shown in formula (I). The mRNA-LNP containing the ionizable lipid compound provided by the present invention can improve the biocompatibility and mRNA transfection efficiency of LNP as a whole, while having extremely high safety and having a wider range of clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an ionizable lipid compound and application thereof. Background Art

[0002] Compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs are theoretically not limited by the drugability of target proteins. With the advancement of clinical trials and the maturity of related technologies, they have a wider range of application scenarios.

[0003] As an exogenous drug, nucleic acid drugs need to overcome multiple obstacles before they can take effect in the body: instability, immunogenicity, low cellular uptake efficiency, difficulty in endosome escape, etc. An efficient and safe delivery system is an important guarantee for nucleic acid drugs to overcome the above defects and exert stable targeted efficacy.

[0004] At present, lipid nanoparticles (LNP) have become one of the mainstream delivery systems for nucleic acid drugs due to their unique structure and physicochemical properties, showing high delivery efficiency and good safety in the body. They have been verified in nucleic acid COVID-19 vaccines and have gradually been used in tumor vaccines, cell therapy, gene editing and other fields.

[0005] Ionizable lipid compounds are critical core components in LNP delivery systems, which have the functions of binding to negatively charged nucleic acids, promoting cellular uptake and endosomal escape, and enhancing the in vivo transfection of nucleic acid drugs.

[0006] Although the prior art has disclosed a variety of ionizable lipid compounds, most of these ionizable lipid compounds have problems such as low transfection efficiency, poor stability, and low safety. Especially for the field that needs to carry large doses of nucleic acid drugs, in addition to improving transfection efficiency, safety is an important factor that needs to be considered. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a novel ionizable lipid compound, which contains -(C=O)O- or -O(C=O)- and hydroxyl fragments, this specific structural feature can improve the biocompatibility, mRNA transfection efficiency and safety of LNP as a whole.

[0008] To achieve the above-mentioned and other related purposes, the present invention provides an ionizable lipid compound in a first aspect, the chemical structure of which is shown in the following formula (I):

[0009]

[0010] wherein a and b are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8; G1 and G2 are each independently selected from C2 to C10 alkylene groups;

[0011] M is CH or N;

[0012] G3 is selected from C1-C10 alkylene; or G3 is selected from (CH2) d -O-(CH2) e , wherein d and e are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9, and d+e is an integer from 2 to 10; or G3 is selected from wherein g is selected from 1, 2, 3 or 4;

[0013] L1 is selected from -(C=O)O-, -O(C=O)-, -(C=O)S-, -S(C=O)-, -(C=O)NR-, -NR(C=O)-, -NR(C=O)O-, -O(C=O)NR-, R is independently selected from H, C1-C12 hydrocarbon group;

[0014] L2 is selected from -(C=O)O-, -O(C=O)-;

[0015] R1 and R2 are each independently selected from a C1-C20 straight chain saturated hydrocarbon group;

[0016] R3 and R4 are each independently selected from C4-C10 straight-chain saturated hydrocarbon groups.

[0017] The second aspect of the present invention provides a lipid nanoparticle, which comprises the above-mentioned ionizable lipid compound, its stereoisomers, its tautomers or a combination of one or more thereof in a pharmaceutically acceptable salt.

[0018] The third aspect of the present invention provides the use of the lipid nanoparticles in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition further comprises the carried drug and / or a pharmaceutically acceptable excipient.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The ionizable lipid compound provided by the present invention has an N atom as an ionizable center, a hydrophilic hydroxyl group as a head, two hydrophobic alkane chains as tails, and a -(C=O)O- or -O(C=O)- fragment is introduced into one tail to achieve a certain asymmetry of the two tails, so that the compound has a moderate length (i.e., giving the ionizable lipid compound appropriate hydrophilic and hydrophobic properties); this type of innovatively designed ionizable lipid compound can simulate biological phospholipid molecules, promote membrane fusion, and improve the endosomal escape ability of nucleic acids. Compared with the prior art, the nanocarrier formed by the self-assembly of the ionizable lipid compound with this type of structural characteristics can not only improve the intracellular delivery efficiency and transfection efficiency, but also have better safety. In particular, compared with the commercial lipid compound ALC0315, its safety in vivo and in vitro has been greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a particle size distribution diagram of mRNA-LNP prepared from the ionizable lipid compound J-03 provided by the present invention. DETAILED DESCRIPTION

[0022] In order to solve the deficiencies of the prior art, the present invention aims to provide a -(C=O)O- or -O(C=O)- and hydroxyl fragments, lipid nanoparticle compositions and uses. The mRNA-LNP prepared by the ionizable lipid compound with a new structure of the present invention can improve the overall biocompatibility of LNP and the mRNA transfection efficiency, while having extremely high safety. It can be applied to tumor vaccines, cell therapy, gene editing and other fields, and has a wider clinical application value.

[0023] In order to achieve the above object, the present invention adopts the following technical solution:

[0024] An ionizable lipid compound, the chemical structure of which is shown in the following formula (I):

[0025]

[0026] wherein a and b are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8; G1 and G2 are each independently selected from C2 to C10 alkylene groups;

[0027] M is CH or N;

[0028] G3 is selected from C1-C10 alkylene; or G3 is selected from (CH2) d -O-(CH2) e , wherein d and e are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9, and d+e is an integer from 2 to 10; or G3 is selected from wherein g is selected from 1, 2, 3 or 4;

[0029] L1 is selected from -(C=O)O-, -O(C=O)-, -(C=O)S-, -S(C=O)-, -(C=O)NR-, -NR(C=O)-, -NR(C=O)O-, -O(C=O)NR-, R is independently selected from H, C1-C12 hydrocarbon group;

[0030] L2 is selected from -(C=O)O-, -O(C=O)-;

[0031] R1 and R2 are each independently selected from a C1-C20 straight chain saturated hydrocarbon group;

[0032] R3 and R4 are each independently selected from C4-C10 straight-chain saturated hydrocarbon groups.

[0033] The "hydrocarbon group" includes saturated hydrocarbon groups and unsaturated hydrocarbon groups. The preferred saturated hydrocarbon group may be a C1-C20 (i.e., carbon atom number is 1-20) hydrocarbon group, further preferred is a C4-C10 hydrocarbon group, and the most preferred is a C5-C8 hydrocarbon group; the preferred unsaturated hydrocarbon group may be a C2-C20 (i.e., carbon atom number is 2-20) olefin group and a C2-C20 (i.e., carbon atom number is 2-20) alkynyl group. The unsaturated hydrocarbon group may be an unsaturated polyhydrocarbon group or an unsaturated monohydrocarbon group.

[0034] The "hydrocarbon group" can be a branched or straight chain structure, preferably a straight chain structure;

[0035] The "hydrocarbon group" may be substituted or unsubstituted, preferably unsubstituted.

[0036] When describing a "C2-C10 alkylene group", it means that the group can be an alkylene group (such as an alkylene group, an alkenylene group, an alkynylene group) having 2-10 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), and the alkylene group can be a straight chain or branched structure.

[0037] In some embodiments of the present invention, a and b are each independently selected from 0, 1, 2, 3, 4 or 5; preferably 0, 1, 2 or 3; further preferably, a is selected from 2 or 3, and b is selected from 0 or 1.

[0038] In some embodiments of the present invention, G1 and G2 are each independently selected from C5-C10 alkylene groups; preferably, G1 is C5-C9 alkylene group, and G2 is C5-C8 alkylene group.

[0039] In some embodiments of the present invention, G3 is selected from C1 to C5 alkylene groups, preferably C2 to C4 alkylene groups, and further preferably C4 alkylene groups; or G3 is selected from (CH2)2-O-(CH2)2.

[0040] In some embodiments of the present invention, R1 and R2 are each independently selected from a C6-C10 straight chain saturated hydrocarbon group, preferably a C8 straight chain saturated hydrocarbon group.

[0041] In some embodiments of the present invention, R3 and R4 are each independently selected from a C5-C10 straight chain saturated hydrocarbon group, preferably a C5-C8 straight chain saturated hydrocarbon group.

[0042] In some embodiments of the present invention, R is independently selected from H, C1-C4 hydrocarbon group, preferably H, -CH3.

[0043] In some embodiments of the present invention, the ionizable lipid compound is selected from the group consisting of:

[0044]

[0045]

[0046]

[0047]

[0048] The second aspect of the present invention provides a lipid nanoparticle, which comprises the above-mentioned ionizable lipid compound, its stereoisomers, its tautomers or a combination of one or more thereof in a pharmaceutically acceptable salt.

[0049] The term "stereoisomer" refers to isomers that have the same sequence of atoms connected but different arrangements of atoms in space.

[0050] The term "tautomer" refers to a phenomenon in which the structure of a compound undergoes equilibrium interconversion between two functional group isomers, and the corresponding isomers are called tautomers.

[0051] The "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the present invention that exist in the form of free base or free acid can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of compounds of the present invention can be converted into their free bases or acids by standard techniques.

[0052] Pharmaceutically acceptable salts of the compounds of the invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods such as ion exchange used in the art. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate heptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and aryl sulfonates. Other pharmaceutically acceptable salts include salts formed by quaternization of amines, which are carried out using suitable electrophilic reagents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0053] The lipid nanoparticles provided by the present invention further comprise a combination of one or more of structural lipids, auxiliary lipids, PEG-lipids, and polymers.

[0054] In some specific embodiments of the present invention, in the lipid nanoparticles, the molar percentage of the ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid is (20-65): (0-60): (0-60): (0-10), and at least any one of the auxiliary lipid, structural lipid, and PEG-lipid is not 0. Preferably, the molar percentage of the ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid is (20-65): (3-50): (15-60): (0.1-10). In other specific embodiments of the present invention, in the lipid nanoparticles, the molar ratio of the ionizable lipid compound to the polymer is 0.5:1-100:1, preferably 10:1-80:1, and more preferably 40:1-80:1.

[0055] In some specific embodiments of the present invention, the lipid nanoparticles comprise 20% to 65% ionizable lipid compounds, 3% to 40% auxiliary lipids, 20% to 60% structural lipids, and 0.1% to 10% PEG-lipids, where % refers to molar percentage.

[0056] In some specific embodiments of the present invention, the lipid nanoparticles comprise 35% to 49% ionizable lipid compounds, 5% to 20% auxiliary lipids, 35% to 50% structural lipids, and 1% to 2% PEG-lipids, where % refers to molar percentage.

[0057] The "structured lipids" mentioned above refer to lipids containing structures that can stabilize the composition, including but not limited to a combination of one or more of sterols and their derivatives and non-sterols and their derivatives.

[0058] In some specific embodiments, the structural lipids include, but are not limited to, a combination of one or more of sterols and their derivatives, non-sterols, sitosterols, ergosterols, cholestanones, cholestenones, campesterols, stigmasterols, brassicasterols, tomatine, ursolic acid, coprosterols, α-tocopherols, or corticosteroids. Sterols are preferably cholesterol and its derivatives; non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholestanol, 5α-coprosterol, cholesteryl-(2'-hydroxy)ethyl ether, cholesteryl-(4'-hydroxy)butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholesterenone, 5α-cholestenone, and decanoic acid cholesterol ester; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)butyl ether. This is not exhaustive, the selection of structural lipids is not limited, and any structural lipid can be applied to the present invention.

[0059] In some embodiments, the structured lipid is a combination of one or more of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives.

[0060] In some embodiments, the structured lipid is cholesterol.

[0061] The type of the "auxiliary lipid" is not limited, and phospholipids are preferred, including but not limited to: a combination of one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and dimyristoylphosphatidylglycerol.

[0062] In some specific embodiments, the helper lipid can be selected from: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC ), 1,2-heneicosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dipalmitoylphosphatidylethanolamine (DPPE), 1- Oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexanoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl A combination of one or more of acyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, and sphingomyelin.

[0063] In some specific embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, and POPC.

[0064] In some specific embodiments, the helper lipid is phosphatidylcholine, specifically DSPC.

[0065] In some specific embodiments, the helper lipid is phosphatidylethanolamine, specifically DOPE.

[0066] In some specific embodiments, the auxiliary lipid is selected from a combination of one or more of DOTAP ((1,2-dioleyloxypropyl)trimethylammonium chloride), DODAP (1,2-dioleoyl-3-dimethylammonium-propane), 18:1PA (1,2-DI (cis-9-octadecenoyl)-SN-glycerol 3-phosphate sodium salt), HS15 (polyethylene glycol (15)-hydroxystearate), and GL67 (N4-arginine cholesterol carbonylamide).

[0067] The "PEG-lipid" described in the present invention generally refers to a conjugate formed by linking PEG (polyethylene glycol) and lipid molecules through chemical bonds, including but not limited to PEG-modified phospholipids and derived lipids, such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, methoxypolyethylene glycol ditetradecyl acetamide, or a combination of one or more thereof.

[0068] In some embodiments, the PEG-lipids include but are not limited to PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, polyethylene glycol A combination of one or more of PEG-DMA, PEG-distearylglycerol, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide, PEG-dipalmitoylphosphatidylethanolamine, PEG-phosphatidylethanol, PEG-phosphatidylethylmyristyloxypropyl-3-amine, PEG-oxypropyl alcoholamine, 1,2-distearoyloxypropyl-3-amine-N[methoxy(polyethylene glycol)](PEG-DSA), methoxypolyethylene glycol lauric acid, and methoxypolyethylene glycol ditetradecylacetamide (ALC0159).

[0069] In some embodiments, the PEG-lipid is PEG-DMG.

[0070] The type of the "polymer" is not limited, and the polymer may include but is not limited to amphiphilic block copolymers, which are block copolymers composed of hydrophobic polymers and hydrophilic compounds, including but not limited to polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), glycolide-lactide copolymer (PLCG), polycaprolactone (PCL), polyorthoester, polyanhydride (PAH), polyphosphazene, poly-β-amino ester (PBAE), poly(α-hydroxy acid), lactide / glycolide copolymer (PLGA or PLG) ( It includes lactide / glycolide copolymers, D-lactide / glycolide copolymers, L-lactide / glycolide copolymers and D,L-lactide / glycolide copolymers), polyglycolide (PGA), polyorthoesters (POE), linear or branched polyethylene glycol (PEG), conjugates of poly(α-hydroxy acids), polyaspirins, polyphosphazenes, D-lactide, D,L-lactide-caprolactone, D,L-lactide-glycolide-caprolactone, dextran, vinylpyrrolidone , polyvinyl alcohol (PVA), methacrylate, poly N-isopropylacrylamide), SAIB (sucrose acetate isoate), hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxymethyl cellulose or its salt, carboxyvinyl, poly (hydroxyethyl methacrylate), poly (methoxyethyl methacrylate), poly (methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), methyl methacrylate (MMA), PVA-g-PLGA, PEGT-PBT copolymer, PEO-PPO-PEO (pluronics), PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, PEG-PLGA, PLA-PLGA, PEG-PLA, PEG-PCL, poloxamer 407, PEG-PLGA-PEG triblock copolymer, PEG-PLA-PEG triblock copolymer, PEG-PCL-PEG triblock copolymer or their block copolymers with polyethylene glycol (PEG), or a combination of one or more of the above polymers or copolymers.

[0071] In some embodiments of the present invention, the weight average molecular weight of PEG in the PEG-lipid is 1000-10000, for example, 1000-2000, 2000-4000, 4000-6000, 6000-8000, 8000-10000, preferably 2000.

[0072] The third aspect of the present invention provides the use of the lipid nanoparticles in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition further comprises the carried drug and / or a pharmaceutically acceptable excipient.

[0073] The "carried drug" described in the present invention includes but is not limited to any one of nucleic acids, small molecules, proteins or a combination of multiple thereof.

[0074] The "nucleic acid" of the present invention can be a nucleotide polymer of any length. Including but not limited to single-stranded DNA, double-stranded DNA, plasmid DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), micro non-coding RNA (miRNA and siRNA), telomerase RNA (Telomerase RNA), small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotide (ASO), ribozyme, asymmetric interfering RNA (aiRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotide, morpholino oligonucleotide or biological custom oligonucleotide one or more combinations thereof.

[0075] In certain embodiments of the present invention, the nucleic acid is mRNA. The mRNA is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template and carries genetic information and can guide protein synthesis. The mRNA can be a monocistronic mRNA or a polycistronic mRNA.

[0076] The "small molecule" of the present invention refers to a compound that is not a protein or nucleic acid molecule. The small molecule can be a small molecule of a therapeutic agent and / or a preventive agent, such as an antibiotic, an anti-inflammatory drug, an anticancer drug, an antiviral drug, an immunosuppressant, an analgesic, an antifungal drug, an antiparasitic drug, an anticonvulsant, an antidepressant, an anxiolytic, an antipsychotic, etc.

[0077] "Protein" as used herein refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary and / or quaternary structures. The secondary, tertiary and / or quaternary structures of proteins are typically stabilized using non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions and / or van der Waals interactions. Additionally or alternatively, proteins may include disulfide bonds, for example between thiol groups of cysteine ​​residues. Exemplary proteins include, but are not limited to, antibodies, antigens or fragments thereof, fusion proteins, recombinant proteins, polypeptides, short peptides, enzymes, and the like.

[0078] The pharmaceutical composition of the present invention also includes pharmaceutically acceptable excipients. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-7, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, intracavity injection, inhalation administration, implantation administration, etc.

[0079] The term "pharmaceutically acceptable" as used herein means that when the drugs are properly administered to animals or humans, they will not produce adverse, allergic or other untoward reactions.

[0080] A "pharmaceutically acceptable excipient" should be compatible with the active ingredient, that is, it should be able to be mixed with it without significantly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable excipients can be sugars, such as glucose, mannitol, sucrose, lactose, trehalose, maltose, etc.; starches, such as corn starch and potato starch, etc.; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose, etc.; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate, etc.; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter, etc.; alcohols, such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol, etc.; alginic acid; emulsifiers, such as Tween, etc.; wetting agents, such as sodium lauryl sulfate, etc.; surfactants; freeze-drying protectants; colorants; flavorings; tablets; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; buffer solution, etc., and combinations thereof. These materials are used as needed to improve the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0081] The pharmaceutical composition of the present invention can be prepared into an inhalation atomized preparation (such as a dry powder preparation, an aerosol preparation, an inhalation mist droplet preparation, etc.), an implantable gel preparation, a microneedle preparation, and can also be prepared into an injection form, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day.

[0082] In some embodiments of the present invention, the pharmaceutical composition contains lipid nanoparticles (LNP), and the average particle size of the lipid nanoparticles is 60-120 nm, which can be 60-70 nm, 70-80 nm, 80-90 nm, 90-100 nm, 100-110 nm, 110-120 nm, or 60-80 nm, 80-100 nm, 100-120 nm.

[0083] In some embodiments of the present invention, the pharmaceutical composition comprises nucleic acid lipid nanoparticles, and the encapsulation efficiency of the nucleic acid in the nanolipid particles is greater than 80%, and can be 80% to 85%, 85% to 90%, 90% to 95%, 95% to 97%, 97% to 99% or more.

[0084] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0085] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0086] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. The materials and equipment used in the present invention are all commercially available products.

[0087] Example 1: Synthesis of ionizable lipid compounds

[0088] 1. Synthesis of ionizable lipid compound J-03

[0089] The synthetic route is as follows:

[0090]

[0091] (1) Synthesis of Compound J-I01

[0092] 4,4-dimethoxybutyronitrile (10.00 g, 77.42 mmol), n-octanol (30.25 g, 232.27 mmol) and PTSA (p-toluenesulfonic acid, 1.33 g, 7.74 mmol) were added to the reaction bottle, heated to 100°C, and reacted for 24 hours under nitrogen. After TLC monitoring, 300 mL of ethyl acetate was added to the reaction solution to dilute the reaction solution, and the reaction solution was washed three times with an equal volume of saturated sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate. The reaction solution was filtered, the filtrate was collected, and concentrated, and purified by column separation (silica gel column, eluent PE:EA=100:1) to obtain 10.78 g of the product, with a yield of 43%.

[0093] (2) Synthesis of Compound J-I02

[0094] J-I01 (10.00 g, 30.72 mmol) was dissolved in 50 mL of ethanol, and then 25 mL of an aqueous solution containing potassium hydroxide (3.45 g, 61.44 mmol) was added, and the temperature was raised to 100°C. The reaction was carried out under nitrogen protection for 24 hours. After the reaction was completed as monitored by TLC, 200 mL of ethyl acetate was added to the reaction solution to dilute the reaction solution, and the reaction solution was washed three times with an equal volume of saturated sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate. The reaction solution was filtered to collect the filtrate, and concentrated, and purified by column separation (silica gel column, eluent PE:EA=10:1) to obtain 8.56 g of the product, with a yield of 81%.

[0095] (3) Synthesis of Compound J-I03

[0096] J-I02 (5.00 g, 14.51 mmol), 6-bromohexanol (3.15 g, 17.41 mmol), 4-dimethylaminopyridine (0.53 g, 4.35 mmol) were dissolved in 50 mL of dichloromethane, cooled to 0°C, N'N-dicyclohexyldiimide (DCC, 3.59 g, 17.41 mmol) was added, and then the mixture was heated to room temperature and stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column separation (silica gel column, eluent PE:EA=10:1) to obtain 6.31 g of the product, with a yield of 90%.

[0097] (4) Synthesis of Compound J-I04

[0098] Dissolve 6-bromohexanoic acid (5.00 g, 25.63 mmol), 2-hexyldecanol (6.21 g, 25.63 mmol) and DMAP (0.94 g, 7.69 mmol) in 50 mL of dichloromethane, cool to 0°C, add DCC (6.35 g, 30.76 mmol), gradually warm to room temperature, and stir to react for 12 hours. After TLC monitoring of the reaction is complete, filter the reaction solution, collect the filtrate and concentrate it, and purify it by column separation (silica gel column, eluent PE:EA=50:1) to obtain 8.31 g of the product, with a yield of 77%.

[0099] (5) Synthesis of Compound J-I05

[0100] J-I04 (2.48 g, 5.9 mmol), 4-amino-1-butanol (5.26 g, 59.0 mmol) and 5.0 mL of ethanol were added to the reaction flask, the reaction solution was heated to 90 ° C, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated, then poured into 200 mL of ethyl acetate / water (1:1), and extracted, the organic phase was collected, and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 2.52 g of the product, with a yield of 89%.

[0101] (6) Synthesis of ionizable lipid compound J-03

[0102] J-I03 (0.24 g, 0.47 mmol), J-I05 (0.2 g, 0.47 mmol), DIPEA (73 mg, 0.56 mmol) and 2.0 mL of acetonitrile were added to the reaction flask, the reaction solution was heated to 80 ° C, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated, then poured into 50 mL of ethyl acetate / water (1:1), and extracted. The organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE:EA=1:1) to obtain 271.0 mg of the product, with a yield of 68%.

[0103] By using the above method and replacing the corresponding starting materials, compounds J-01 to J-02, J-04, J-06 to J-11, J-18 to J-20, J-27, J-28, J-30, J-31, J-37 to J-41, J-58 to J-61, and J-85 can be prepared. For example, replacing 4-amino-1-butanol with diglycolamine can produce J-04; replacing 2-hexyldecanoic acid with 2-hexyldecylamine can produce J-37.

[0104] 2. Synthesis of ionizable lipid compound J-46

[0105]

[0106] (1) Synthesis of Compound J-I29

[0107] Take a certain amount of compound J-I02 (10.0g, 29.02mmol) and dissolve it in 200mL of anhydrous tetrahydrofuran. Stir it in an ice bath for 10min under nitrogen protection, then add lithium aluminum hydride tetrahydrofuran solution (11.6mL, 2.5M, 29.02mmol) dropwise. After the addition is complete, gradually return to room temperature and stir overnight. After TLC monitoring, add 1.1mL of water, add 1.1mL of 15% NaOH aqueous solution, and then add 3.3mL of water, filter, and remove the solvent by vacuum distillation. Add an appropriate volume of ethyl acetate, wash with an equal volume of saturated brine 3 times, dry with anhydrous sodium sulfate, and concentrate. Column separation and purification (silica gel column, eluent PE: EA = 10: 1 (volume ratio)) to obtain 8.31g of colorless oily liquid with a yield of 86%.

[0108] (2) Synthesis of Compound J-I30

[0109] Referring to the synthesis method of compound J-I03 in (3), compound J-I27 can be synthesized.

[0110] (3) Synthesis of ionizable lipid compound J-46

[0111] Referring to (6) the synthesis method of ionizable lipid compound J-03, compound J-46 can be synthesized.

[0112] By using the above method and replacing the corresponding starting materials, compounds J-47 to J-57 can be prepared. For example, by replacing 6-bromohexanoic acid with 8-bromooctanoic acid, J-47 can be prepared.

[0113] 3. Synthesis of ionizable lipid compound J-05

[0114] The synthetic route is as follows:

[0115]

[0116] (1) Synthesis of Compound J-I06

[0117] 9-heptadecanol (3.85 g, 15.0 mmol), N,N-disuccinimidyl carbonate (5.84 g, 22.8 mmol) and DMAP (2.75 g, 22.5 mmol) were added to DMF (60 mL), heated to 75 ° C, and stirred for 12 h. The reaction solution was poured into 300 mL of water and extracted with ethyl acetate twice, using 150 mL of ethyl acetate each time. The organic phase was collected and washed three times with an equal volume of saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (silica gel column, eluent PE: EA = 50: 1) to obtain 1.8 g of the product, with a yield of 42%. .

[0118] (2) Synthesis of Compound J-I07

[0119] J-I06 (1.8 g, 4.53 mmol), 6-amino-1-hexanol (636 mg, 5.43 mmol) and DMAP (554 mg, 4.53 mmol) were added to DMF (30 mL), heated to 60 ° C, and stirred for 12 h. The reaction solution was poured into 300 mL of water and extracted with ethyl acetate twice, using 150 mL of ethyl acetate each time. The organic phase was collected and washed three times with an equal volume of saturated sodium chloride aqueous solution. The phase was dried over anhydrous sodium sulfate and concentrated to obtain 1.84 g of the product with a yield of 100%, which was directly used in the next step.

[0120] (3) Synthesis of Compound J-I08

[0121] At 0°C, a solution of Ms2O (1.57 g, 9 mmol) in DCM (9 mL) was slowly added to a solution of J-I07 (1.84 g, 4.53 mmol), DMAP (61 mg, 0.5 mmol) and TEA (triethylamine, 2.5 mL, 18 mmol) in DCM (60 mL). After stirring the reaction for 30 min at 0°C, water (100 mL) was added to quench the reaction. The organic phase was separated and washed once with an equal volume of pure water, once with an equal volume of 10% citric acid and once with a saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain 2.05 g of crude product, which was directly used in the next step.

[0122] (4) Synthesis of Compound J-I09

[0123] J-I08 (2.05 g, 4.29 mmol) and LiBr (1.2 g, 13.6 mmol) were added to THF (tetrahydrofuran, 25 mL), heated to 45°C and stirred for 12 h. Filtered through a pad of silica gel (10 g), the filtrate was collected and concentrated, and purified by silica gel column chromatography (silica gel column, eluent PE:EA=10:1) to obtain 1.84 g of the product, with a yield of 93%.

[0124] (5) Synthesis of Compound J-I10

[0125] J-I09 (5.00 g, 10.81 mmol), 4-amino-1-butanol (9.64 g, 108.09 mmol) and 10.0 mL of ethanol were added to the reaction flask, the reaction solution was heated to 80 ° C, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated, then poured into 300 mL of ethyl acetate / water (1:1), and extracted, the organic phase was collected, and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 4.72 g of the product, with a yield of 93%.

[0126] (6) Synthesis of ionizable lipid compound J-05

[0127] Referring to the synthesis method of ionizable lipid compound J-03 (6), compound J-05 can be synthesized.

[0128] By using the above method and replacing the corresponding starting materials, compounds J-12 to J-14, J-22 to J-23, J-29, J-32, J-44, J-45, J-62, J-63, J-82, and J-86 can be prepared. For example, replacing 9-heptadecanol with 2-hexyldecanol can produce J-12.

[0129] 4. Synthesis of ionizable lipid compound J-15

[0130] The synthetic route is as follows:

[0131]

[0132] (1) Synthesis of Compound J-I11

[0133] Dissolve 1,6-dibromohexane (85.45 g, 350.25 mmol) in 400 mL of tetrahydrofuran, cool to 0°C, add potassium thioacetate (20.00 g, 175.13 mmol) in six batches within 1 hour, warm to room temperature, and stir to react for 12 hours. After TLC monitoring, filter the reaction solution after the reaction is complete, collect the filtrate and concentrate it, and purify it by column separation (silica gel column, eluent PE:EA=500:1) to obtain 28.15 g of the product, with a yield of 67%.

[0134] (2) Synthesis of Compound J-I12

[0135] J-I06 (20.00 g, 83.62 mmol) and 13.94 mL of concentrated hydrochloric acid (12 M, 167.24 mmol) were dissolved in 200 mL of methanol, heated to 60 ° C, and stirred for 12 h. After the reaction was completed by TLC monitoring, the reaction solution was poured into 600 mL of ethyl acetate / water (1:1) and extracted, and the organic phase was collected and washed once with an equal volume of pure water and an equal volume of saturated sodium bicarbonate aqueous solution. The organic phase was collected, dried with anhydrous sodium sulfate, concentrated, and purified by column separation (silica gel column, eluent PE: EA = 20:1) to obtain 13.26 g of the product, with a yield of 80%.

[0136] (3) Synthesis of Compound J-I13

[0137] J-I12 (10.00 g, 50.73 mmol), 2-hexyldecanoic acid (13.01 g, 50.73 mmol) and DMAP (1.86 g, 15.22 mmol) were dissolved in 200 mL of dichloromethane, cooled to 0°C, DCC (12.56 g, 60.87 mmol) was added, the temperature was raised to room temperature, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column separation (silica gel column, eluent PE:EA=100:1) to obtain 17.51 ​​g of the product, with a yield of 79%.

[0138] (4) Synthesis of Compound J-I14

[0139] J-I13 (10.00 g, 22.96 mmol), 4-amino-1-butanol (20.47 g, 229.60 mmol) and 20.0 mL of ethanol were added to the reaction flask, the reaction solution was heated to 80 ° C, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated, then poured into 600 mL of ethyl acetate / water (1:1), and extracted, the organic phase was collected, and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 8.94 g of the product, with a yield of 88%.

[0140] (5) Synthesis of ionizable lipid compound J-15

[0141] Referring to the synthesis method of ionizable lipid compound J-03 (6), compound J-15 can be synthesized.

[0142] By using the above method and replacing the corresponding starting materials, compounds J-16 to J-17, J-33, J-64 to J-67, J-83, and J-87 can be prepared. For example, replacing 4-amino-1-butanol with diglycolamine can produce J-17; replacing 1,6-dibromohexane with 1-bromo-2-hexyldecane and replacing 2-hexyldecanoic acid with 6-bromo-hexanoic acid can produce J-64.

[0143] 5. Synthesis of ionizable lipid compound J-21

[0144]

[0145] (1) Synthesis of Compound J-I15

[0146] At 0°C, J-I09 (1.00 g, 2.16 mmol) was added to a suspension of NaH (purity 60%, 173 mg, 4.32 mmol) and MeI (614 mg, 4.32 mmol) in THF (80 mL). The mixture was heated to room temperature and stirred for 48 h. After concentration, sodium bicarbonate solution (100 mL) was added and extracted three times with ethyl acetate / petroleum ether (20:1). The organic phase was dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (10% EA in PE) to obtain 1.01 g of the product with a yield of 98%.

[0147] (2) Synthesis of Compound J-I16

[0148] J-I15 (1.00 g, 2.10 mmol), 4-amino-1-butanol (1.87 g, 20.98 mmol) and 2.0 mL of ethanol were added to the reaction flask, the reaction solution was heated to 80 ° C, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated, then poured into 60 mL of ethyl acetate / water (1:1), and extracted, the organic phase was collected, and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 0.89 g of the product, with a yield of 87%.

[0149] (3) Synthesis of ionizable lipid compound J-21

[0150] Referring to (6) the synthesis method of ionizable lipid compound J-03, compound J-21 can be synthesized.

[0151] By using the above method and replacing the corresponding starting materials, compounds J-68 to J-72 can be prepared. For example, by replacing 6-bromohexanol with 8-bromooctanol, J-72 can be prepared.

[0152] 6. Synthesis of ionizable lipid compound J-24

[0153]

[0154] (1) Synthesis of Compound J-I17

[0155] Dissolve n-bromohexane (33.00 g, 375.59 mmol) in DMSO (330 mL), add K2CO3 (51.91 g, 375.59 mmol), n-octylamine (48.54 g, 375.59 mmol), and react at 80 ° C for 36 h. Cool to room temperature, add ethyl acetate (2000 mL), wash three times with an equal volume of saturated sodium chloride aqueous solution, dry the organic phase over anhydrous sodium sulfate, and concentrate to obtain a crude product. The crude product is purified by column chromatography (silica gel column, DCM: EtOH = 100: 1-40: 1) to obtain 33.05 g of the product, with a yield of 41%.

[0156] (2) Synthesis of Compound J-I18

[0157] Triphosgene (202.8 mg, 683.3 μmol) was dissolved in DCM (4 mL), and a DCM (2 mL) solution containing DMAP (758.9 mg, 6.2 mmol) was added dropwise under an ice-salt bath, and the reaction was continued for 30 min. 6-bromohexanol (404.0 mg, 2.1 mmol) was added dropwise, and the reaction was continued for 30 min. J-I17 (500.0 mg, 2.1 mmol) was added dropwise, and the reaction was continued for 1.5 h at room temperature. Water (6 mL) was added to the reaction solution, and the aqueous phase was washed with dichloromethane (6 mL×3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by column chromatography (PE:EA=100:1) to obtain 930.9 mg of the product, with a yield of 97.2%.

[0158] (3) Synthesis of Compound J-I19

[0159] J-I18 (5.00 g, 11.51 mmol), 4-amino-1-butanol (5.13 g, 57.54 mmol) and 10.0 mL of ethanol were added to the reaction flask, the reaction solution was heated to 80 ° C, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated, then poured into 200 mL of ethyl acetate / water (1:1), and extracted, the organic phase was collected, and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 3.96 g of the product, with a yield of 80%.

[0160] (4) Synthesis of ionizable lipid compound J-24

[0161] Referring to (6) the synthesis method of ionizable lipid compound J-03, compound J-24 can be synthesized.

[0162] By using the above method and replacing the corresponding starting materials, compounds J-25, J-26, J-73 to J-77, and J-84 can be prepared. For example, replacing 4-amino-1-butanol with diglycolamine can prepare J-26.

[0163] 7. Synthesis of ionizable lipid compound J-42

[0164]

[0165] (1) Synthesis of Compound J-I22

[0166] 2-Hexyldecanoic acid (4.38 g, 17.07 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.34 g, 20.48 mmol) were dissolved in 100 mL of dichloromethane (DCM), and stirred at room temperature for 10 min under nitrogen protection. Then, N-hydroxysuccinimide (2.37 g, 20.48 mmol) was added. After stirring at room temperature for 6 h, the reaction solution was washed three times with an equal volume of saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate for 1 h, and then 6-bromohexanol (2.00 g, 17.07 mmol) was added under stirring. The reaction was stirred at room temperature for 6 h. After the reaction was complete as monitored by TLC, the solvent was removed by reduced pressure distillation using a rotary evaporator. Add 100 mL of ethyl acetate, wash twice with an equal volume of saturated sodium bicarbonate solution, wash once with an equal volume of saturated sodium chloride solution, dry over anhydrous sodium sulfate for 30 min, use a rotary evaporator to distill off the solvent under reduced pressure, and purify by column separation (silica gel column, eluent PE:EA=10:1 (volume ratio)) to obtain 5.16 g of colorless liquid, with a yield of 85%.

[0167] (2) Synthesis of Compound J-I23

[0168] Referring to the synthesis method of compound J-I08 in (3), compound J-I23 can be synthesized.

[0169] (3) Synthesis of Compound J-I24

[0170] Referring to the synthesis method of compound J-I09 in (4), compound J-I24 can be synthesized.

[0171] (4) Synthesis of Compound J-I25

[0172] Referring to the synthesis method of compound J-I10 in (5), compound J-I25 can be synthesized.

[0173] (5) Synthesis of ionizable lipid compound J-42

[0174] Referring to (6) the synthesis method of ionizable lipid compound J-03, compound J-42 can be synthesized.

[0175] By using the above method and replacing the corresponding starting materials, compound J-78 can be prepared. J-78 can be prepared by replacing 6-bromohexanol with 8-bromooctanol.

[0176] 8. Synthesis of ionizable lipid compound J-43

[0177]

[0178] (1) Synthesis of Compound J-I26

[0179] Referring to the synthesis method of compound J-I04 in (4), compound J-I26 can be synthesized.

[0180] (2) Synthesis of Compound J-I27

[0181] Referring to the synthesis method of compound J-I15 in (1), compound J-I27 can be synthesized.

[0182] (3) Synthesis of Compound J-I28

[0183] Referring to the synthesis method of compound J-I16 in (2), compound J-I28 can be synthesized.

[0184] (4) Synthesis of ionizable lipid compound J-43

[0185] Referring to (6) the synthesis method of ionizable lipid compound J-03, compound J-43 can be synthesized.

[0186] By using the above method and replacing the corresponding starting materials, compounds J-79 to J-81 can be prepared. J-79 can be prepared by replacing 8-bromooctanoic acid with 6-bromohexanoic acid.

[0187] 9. Synthesis and comparison of ionizable lipid compound j-01

[0188]

[0189] (1) Synthesis of ionizable lipid compound j-01

[0190] J-I03 (0.50 g, 0.99 mmol), 4-amino-1-butanol (35.12 mg, 0.39 mmol), DIPEA (0.13 g, 0.99 mmol) and 2.0 mL of ethanol were added to the reaction flask, the reaction solution was added to reflux, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated, then poured into 50 mL of ethyl acetate / water (1:1), and extracted, the organic phase was collected, and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE:EA=2:1) ​​to obtain 265.0 mg of the product, with a yield of 71%.

[0191] 10. Synthesis and comparison of ionizable lipid compound j-02

[0192] Referring to the synthetic route of J-03, the corresponding raw materials were replaced to prepare j-02.

[0193] The structure is as follows:

[0194]

[0195] 11. Synthesis and comparison of ionizable lipid compound j-03

[0196]

[0197] (1) Synthesis of compound j-I03

[0198] J-I10 (5.00 g, 11.69 mmol), N-Boc-6-bromohexylamine (3.28 g, 11.69 mmol), DIPEA (1.81 g, 14.03 mmol) and 50.0 mL of ethanol were added to the reaction flask, the reaction solution was heated to 80 ° C, and the reaction was stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated, then poured into 100 mL of ethyl acetate / water (1:1), and extracted, the organic phase was collected, and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE:EA=3:1) to obtain 5.12 g of the product, with a yield of 70%.

[0199] (2) Synthesis of compound j-I04

[0200] j-I03 (1.00 g, 1.59 mmol), 5 mL of dichloromethane and 5 mL of trifluoroacetic acid were added to the reaction flask and reacted at room temperature for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated and then poured into 50 mL of ethyl acetate / water (1:1) and extracted. The organic phase was collected and washed twice with an equal volume of saturated sodium bicarbonate aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 0.76 g of the product with a yield of 91%.

[0201] (3) Synthesis of compound 3-(FMOC-amino)-1-propanal

[0202] 3-(FMOC-amino)-1-propanol (10.00 g, 33.63 mmol), PCC (pyridinium chlorochromate, 8.70 g, 40.36 mmol) and 100 mL of dichloromethane were added to the reaction flask and stirred at room temperature for 12 h. After the reaction was completed by TLC monitoring, the reaction solution was filtered through silica gel and concentrated to obtain a crude product. The crude product was purified by column chromatography (PE:EA=5:1) to obtain 4.65 g of the product with a yield of 47%.

[0203] (4) Synthesis of compound j-I05

[0204] 3-(FMOC-amino)-1-propanal (4.00 g, 13.44 mmol), n-octanol (5.29 g, 40.63 mmol), p-toluenesulfonic acid (0.23 g, 1.35 mmol), anhydrous sodium sulfate (5.77 g, 40.63 mmol) and 100 mL of dichloromethane were added to the reaction flask and stirred at 40°C for 12 h. After the reaction was completed as monitored by TLC, the mixture was filtered through a pad of silica gel and the reaction solution was concentrated to obtain a crude product. The crude product was purified by column chromatography (PE:EA=50:1) to obtain 4.65 g of the product with a yield of 62%.

[0205] (5) Synthesis of Compound j-I06

[0206] Add j-I05 (4.00 g, 13.44 mmol), palladium carbon (0.40 g), and 40 mL of ethanol into a reaction flask, pass hydrogen, and stir at room temperature for 12 h. After the reaction is complete as monitored by TLC, filter with silica gel, and concentrate the reaction solution to obtain a crude product. The crude product is purified by column chromatography (PE:EA=1:5) to obtain 2.15 g of the product, with a yield of 90%.

[0207] (6) Synthesis of Compound j-I07

[0208] j-I06 (1.00 g, 3.03 mmol), triethylamine (0.46 g, 4.55 mmol), 10 mL tetrahydrofuran were added to the reaction bottle, cooled to 0 ° C, carbon disulfide (0.28 g, 3.64 mmol) was added dropwise under stirring, and the reaction was carried out at 0 ° C for 4 h, and then Boc anhydride (0.79 g, 3.64 mmol) was added, and the reaction was gradually restored to room temperature and reacted for 12 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated, added to 100 mL ethyl acetate / water (1:1), and extracted, the organic phase was collected, and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography (PE:EA=50:1) to obtain 0.73 g of the product, with a yield of 65%

[0209] (7) Synthesis of ionizable lipid compound j-03

[0210] Add j-I07 (0.50 g, 1.35 mmol) and 5 mL DMF to the reaction flask, add compound j-I04 (0.71 g, 1.35 mmol) under stirring at 0 ° C, and stir at room temperature for 12 h. After the reaction is complete, add 50 mL of ethyl acetate / water (1:1) and extract, collect the organic phase, and wash once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. Collect the organic phase, dry it with anhydrous sodium sulfate, filter and concentrate, and the crude product is purified by column chromatography (PE:EA=2:1) to obtain 0.81 g of the product, with a yield of 67%.

[0211] Table 1 Comparison table of H NMR spectra of exemplary ionizable lipid compounds J-01 to J-33 in Example 1

[0212]

[0213]

[0214]

[0215]

[0216] Example 2: Preparation and screening of nucleic acid lipid nanoparticles (mRNA-LNP)

[0217] 1. Preparation of Nucleic Acid-Lipid Nanoparticles

[0218] (1) The ionizable lipid compounds (Lipid) J-01 to J-87, DSPC, cholesterol, and PEG-lipid (PEG2000-DMG) prepared in Example 1 were dissolved in ethanol according to the molar ratio of 46.3 / 9.4 / 42.7 / 1.6 (molar ratio) to prepare different lipid ethanol solutions (Lipid concentration 20 mg / mL).

[0219] (2) mRNA was prepared at a lipid nanoparticle (LNP) to mRNA mass ratio of 10:1 to 30:1 (the ratio used in this example was 15:1), and the mRNA was diluted to 0.2 mg / mL using citrate or sodium acetate buffer (pH = 3 or 5) to obtain an mRNA solution.

[0220] (3) The lipid ethanol solution and the mRNA solution were fully mixed at a volume ratio of 1:5 to 1:1 (the ratio used in this embodiment was 1:3). The obtained lipid nanoparticles were purified by ultrafiltration and dialysis, and after filtration and sterilization, the average particle size and PDI (polydispersity index) of mRNA-LNP (lipid nanoparticles encapsulating mRNA) were characterized using Malvern Zetasizer Nano ZS, and the mRNA encapsulation efficiency was determined using Ribogreen RNA quantitative determination kit. The results are shown in Table 2.

[0221] Table 2 Characterization data of each group of mRNA-LNP

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] The results show that each ionizable lipid compound J-01 to J-87 provided in Example 2 of the present invention can form a stable nanostructure, and the size distribution of the obtained mRNA-LNP is relatively narrow, and the size varies with the structure of different mRNA-LNPs, within the range of 60 to 110 nm. Under the same other conditions, the comparison samples shown in Table 3, commercially available ALC0315, comparison samples j-01 (prepared in the aforementioned Example 1), j-02 (prepared in the aforementioned Example 1) and j-03 (prepared in the aforementioned Example 1) are used to replace the above-mentioned ionizable lipid compounds J-01 to J-87 to prepare mRNA-LNP, and the average particle size, PDI and encapsulation efficiency are tested. The structural formulas of ALC0315, j-01, j-02 and j-03 are shown in Table 3. The test results are shown in Table 4.

[0228] Table 3 Structural formula of comparative samples

[0229]

[0230] Table 4 Characterization data of comparative sample mRNA-LNP

[0231]

[0232] From the comparison of the data in Table 2 and Table 4, it can be seen that the mRNA-LNP provided in the embodiments of the present invention can form a stable nanostructure with a narrow size distribution. The size varies with the structure of different mRNA-LNPs. In the range of 60-100 nm, the encapsulation rate is greater than 95%, and it has relatively excellent physical and chemical properties.

[0233] 2. Low temperature storage stability

[0234] The mRNA-LNP provided by J-03 prepared in Example 2 was stored in a dry environment at 4°C. The average particle size, PDI, and mRNA encapsulation efficiency of the mRNA-LNP were tested at different time points (0 days, 15 days, 30 days, 60 days, and 120 days). The measurement results are shown in Table 5.

[0235] Table 5 Comparison of effects of low temperature storage at different time

[0236] Retention time (days) Average particle size (nm) PDI Encapsulation rate (%) 0 67.4 0.039 97.2 15 67.1 0.040 97.5 30 67.8 0.043 96.5 60 68.6 0.046 96.9 120 68.8 0.048 96.2

[0237] As shown in Table 5, the average particle size, PDI and encapsulation efficiency of the mRNA-LNP provided by the Example Group J-03 remained basically unchanged within 120 days, and had good low-temperature storage stability, which facilitated the storage and transportation of the product.

[0238] According to the aforementioned method, mRNA-LNPs prepared from other compounds in this example group were selected, and the measurement results showed that the average particle size, PDI and encapsulation efficiency did not change substantially within 120 days.

[0239] Example 3: mRNA-LNP transfection efficiency and biocompatibility

[0240] 1. Cell transfection efficiency

[0241] HEK293 cells (100 μL, cell density 6×10 4 The suspension (100 μg / ml) was added to a 96-well plate and incubated in a cell culture incubator for 24 hours. The complete medium was replaced with 60 μL DMEM serum-free medium, and 0.4 μg or 0.2 μg of the prepared Luciferase mRNA LNP preparation was added to each well. Three replicates of each concentration were placed in a 37°C, 6% CO2 incubator for 4 hours. Then 60 μL of complete medium was added and cultured for another 24 hours. The culture medium was removed, 100 μL PBS was added to wash gently once, and then 30 μL / well cell lysis solution was added, and the cells were shaken on a micro-oscillator at room temperature for 16 minutes to fully lyse the cells. The lysate was collected, centrifuged at 12000 rpm for 6 minutes, and the supernatant was collected. 20 μL of the supernatant was added to a new black opaque 96-well plate, the cell lysate was used as the blank control, 100 μL of firefly luciferase detection solution was added, and after incubation in the dark for 6 minutes, the chemiluminescence intensity RLU was detected using a multifunctional microplate reader, and the RLU of the blank control was deducted to take the average value. In Table 6, E represents the power of 10 in scientific notation. For example, "6.41E+04" means 6.41×10 4 .

[0242] Table 6 Transfection efficiency comparison table HEK293 cells

[0243]

[0244]

[0245]

[0246] 2. Animal transfection efficiency

[0247] Male ICR mice (6-8 weeks, Shanghai Jiesijie Experimental Animal Co., Ltd.) were housed under experimental conditions of 22±2℃ and relative humidity of 45–75%, with a light / dark cycle of 12h. Luciferase mRNA was used as a reporter gene. Luciferase catalyzes luciferin to produce bioluminescence, and the transfection efficiency of LNP is reflected by detecting the intensity of bioluminescence per unit time. Taking luciferase mRNA (purchased from ApexBio Technology) as an example, mRNA-LNP samples J-01~J-87 obtained in Experiment 1, commercially available comparison sample ALC0315, comparison sample j-01, comparison sample j-02, and comparison sample j-03 were prepared; the above samples were administered by intramuscular injection at a dose of 100μg / kg mRNA, two mice per group of samples, two legs. At a specific time point, fluorescein (20 μg / mL) was injected into the mouse peritoneum. Five minutes later, the mouse was placed in a small animal in vivo imaging instrument to measure the fluorescence intensity. The final result was expressed as the average fluorescence intensity. The experimental results of fluorescence intensity after intraperitoneal injection of the mouse are shown in Table 7.

[0248] Table 7 Fluorescence intensity comparison table

[0249]

[0250]

[0251] Compared with commercial products, the mRNA-LNP prepared by the ionizable lipid compound provided by the present invention can improve the transfection effect of various cell levels by 1 to 2 orders of magnitude, which is far beyond the expected range, has excellent cell transfection efficiency, and greatly expands its application in the field of cell therapy. In addition, the transfection efficiency in animals is comparable to that of commercial products.

[0252] 3. Biocompatibility

[0253] The cell viability was determined using the CCK-8 kit. HEK 293 cell suspension (100 μL, cell density 2×10 4 / mL) was added to a 96-well plate and incubated in a cell culture incubator for 24h. Then the cell culture medium was removed, and 100μL of the newly prepared cell culture medium containing 100μg / mL of mRNA obtained in Example 2 for each mRNA-LNP sample was added and incubated with the cells for 24h. Subsequently, the cell supernatant was removed, fresh cell culture medium was added, and the incubation continued for 24h. The supernatant was removed, and 100μL of fresh cell culture medium containing CCK-8 working solution (10μL / mL) was added and incubated for 2h. At the same time, a blank group was set: an equal amount of CCK-8 working solution was used to replace mRNA-LNP, and the other conditions were exactly the same. The absorbance of each well at 450nm was detected using a multifunctional microplate detector (no bubbles should appear in the well plate during the detection process), and the viability of cells without any treatment (control group) was set to 100%. The formula for calculating the cell viability of each group is as follows: Cell viability (%) = [A1-A0] / [A2-A0]×100. Among them, A1 is the absorbance of the drug-added group, A0 is the absorbance of the blank group, and A2 is the absorbance of the control group. The experimental results are shown in Table 8.

[0254] Table 8 Cell viability effect comparison table

[0255]

[0256]

[0257]

[0258] The experimental results show that the LNP prepared from the lipid compound of the present invention has little effect on cell viability, and the cells maintain more than 80% viability, while the cell viability of the commercially available comparison sample ALC0315 is only 55% at the same high concentration, and its in vitro safety is far inferior to the lipid compound of the present invention.

[0259] Example 4: In vivo safety experiment

[0260] In order to further verify the safety of the lipid compounds of the present invention, we exemplarily selected some of the compounds prepared in Example 1 to carry out relevant animal experiments.

[0261] Material preparation: 110 six-week-old Balb / c mice, half female and half male, weighing 15-20g, were raised in an experimental environment with a temperature of 22±2℃, a relative humidity of 45-75%, and a light / dark cycle of 12h. After the mice were purchased, they were first adapted to the animal room for one week before formal animal experiments. The 110 mice were randomly divided into 11 groups, with half male and half female mice in each group. The first group was intravenously injected with an equal volume of PBS (negative control group), the second group was intravenously injected with the commercially available comparison sample ALC-0315 (260μg of mRNA), the third group was intravenously injected with the commercially available comparison sample ALC-0315 (520μg of mRNA), the fourth group was intravenously injected with the comparison sample j-01 (260μg of mRNA), the fifth group was intravenously injected with the comparison sample j-01 (520μg of mRNA), and the sixth group was intravenously injected with the comparison sample j-02 (260μg of mRNA). RNA), group 7 intravenously injected comparison sample j-02 (520 μg of mRNA), group 8 intravenously injected comparison sample j-03 (260 μg of mRNA), group 9 intravenously injected comparison sample j-03 (520 μg of mRNA), group 10 intravenously injected comparison sample J-03 (260 μg of mRNA), group 11 intravenously injected comparison sample J-03 (520 μg of mRNA); the above mRNA is synthesized by in vitro transcription based on the independently designed template and can express the full-length Luciferase.

[0262] The experimental process is as follows: at 0h, 24h, 48h and 72h, the number of surviving mice in each group was counted respectively.

[0263] Table 10 Statistics of the number of mice surviving

[0264]

[0265]

[0266] The experimental results show that when ALC0315 and the control sample j-03 are injected intravenously at large doses, the mRNA-LNPs will directly cause different degrees of death in mice (260μg mRNA) or even total death (520μg mRNA). However, when the mRNA-LNPs composed of J-03 are injected at large doses, no mice are observed to die.

[0267] The other compounds in this example also have similar safety effects as J-03. For example, almost all mice survived at higher doses of compounds J-01, J-05, J-09, and J-48.

[0268] In summary, it can be proved that the ionizable lipid prepared by the present invention is extremely safe and has a wider clinical application value compared with the marketed product ALC0315 and other compounds with similar structures.

[0269] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. An ionizable lipid compound, characterized in that The ionizable lipid compound is selected from the group consisting of:

2. The ionizable lipid compound according to claim 1, characterized in that The ionizable lipid compound is selected from the group consisting of:

3. A lipid nanoparticle, characterized in that: A combination comprising an ionizable lipid compound according to any one of claims 1 to 2, its stereoisomers, its tautomers or one or more of their pharmaceutically acceptable salts.

4. The lipid nanoparticle according to claim 3, characterized in that It also includes any one or more combinations of structural lipids, auxiliary lipids, PEG-lipids, and polymers.

5. The lipid nanoparticle according to claim 4, characterized in that The structural lipid is selected from a combination of one or more of sterols, non-sterols or their respective derivatives; and / or the auxiliary lipid is selected from a combination of one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dimyristoylphosphatidylglycerol, DOTAP, DODAP, 18:1PA, HS15, and GL67; and / or the PEG-lipid is selected from a combination of one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxypolyethylene glycol ditetradecylacetamide.

6. The lipid nanoparticle according to claim 4, characterized in that The molar percentage of the ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid is (20-65): (0-60): (0-60): (0-10), and at least any one of the auxiliary lipid, structural lipid and PEG-lipid is not zero.

7. The lipid nanoparticle according to claim 4, characterized in that The molar percentage of the ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid is (20-65): (3-50): (15-60): (0.1-10).

8. The lipid nanoparticle according to claim 4, characterized in that The molar ratio of the ionizable lipid compound to the polymer is 0.5:1-100:

1.

9. The lipid nanoparticle according to claim 4, characterized in that The molar ratio of the ionizable lipid compound to the polymer is 10:1-80:

1.

10. The lipid nanoparticle according to claim 4, characterized in that The molar ratio of the ionizable lipid compound to the polymer is 40:1-80:

1.

11. Use of the lipid nanoparticles according to any one of claims 3 to 10 in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition further comprises the drug and / or a pharmaceutically acceptable excipient.

12. The use according to claim 11, characterized in that: The drugs carried include one or more of nucleic acid molecules, small molecule compounds, and proteins.

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