Piperazine-containing cationic lipid compound as well as nano composition and application thereof

Abstract

The invention discloses a cationic lipid compound containing piperazine as well as a nano composition and application thereof. The invention discloses a piperazine-containing cationic lipid compound with a structure as shown in a formula I and pharmaceutically acceptable salts thereof, and further discloses a nano composition containing the compound or the pharmaceutically acceptable salts thereof, and the nano composition disclosed by the invention can realize high-efficiency delivery of mRNA (messenger Ribonucleic Acid), and can be applied to the fields of delivery of nucleic acid drugs, treatment of tumor cells and the like. And particularly, the application prospect in the aspect of delivery of mRNA drugs is extremely high. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a cationic lipid compound containing piperazine, its nano-composition and application. Background Art

[0002] Nucleic acid therapy is based on the intervention of disease genes by molecules such as DNA and RNA, covering the fields of gene therapy, tumors and genetic diseases. Since the first clinical study of therapeutic nucleic acids at the beginning of the 21st century, significant progress has been made in the design of nucleic acid molecules and their delivery systems.

[0003] Among many delivery systems, lipid-based RNA delivery systems, especially lipid nanoparticles (LNPs), have become the most widely used delivery system in the field of RNA therapy due to their excellent performance in clinical applications. The core function of lipid nanoparticles is to efficiently encapsulate and target the delivery of active ingredients such as nucleic acids and small molecule drugs. LNPs usually consist of ionizable amino lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. Ionizable cationic lipids play a key role in mRNA encapsulation through pH-responsive properties. Through pH-dependent charge switching, the balance between delivery efficiency and safety is achieved, becoming the core technical support for mRNA vaccines. In 2018, the first LNP-based siRNA drug, Patisiran (Onpattro), was approved for the treatment of hereditary transthyretin amyloidosis, marking the further maturity of LNPs in nucleic acid delivery. The large-scale application of mRNA vaccines using LNP delivery technology by Pfizer-BioNtech and Moderna through the selection of appropriate cationic ionizable liposomes has achieved efficient immune responses and made important contributions to the prevention and control of COVID-19. The cationic ionizable liposomes used are MC3 and SM102 (structural formulas are shown below).

[0004]

[0005] Although the prior art has reported the structures and applications of various cationic lipids, it is still necessary to further optimize the structures of cationic lipids to improve their delivery efficiency and safety. Therefore, it is very necessary to develop new delivery molecules. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a cationic lipid compound containing piperazine, its nano-composition and application.

[0007] To solve the above problems, the technical solutions adopted by the present invention are as follows:

[0008] Technical Theme 1

[0009] A cationic lipid compound containing piperazine or a pharmaceutically acceptable salt thereof, the structure of which is shown in Formula I:

[0010]

[0011] Wherein, X is an alkylene group of C1-C8;

[0012] G is selected from an alkylene group of C1-C12;

[0013] L is -OC(=O)- or -C(=O)O-;

[0014] R1 is an alkyl group of C1-C15;

[0015] R2 is hydrogen or the same as R1.

[0016] As a further improvement of the present invention, the R1 is

[0017]

[0018] As a further improvement of the present invention, the X is a straight-chain alkylene group of C1-C5;

[0019] G is selected from a straight-chain alkylene group of C3-C10

[0020] L is -OC(=O)- or -C(=O)O-;

[0021] R1 is a straight-chain alkyl group of C6-C12;

[0022] R2 is hydrogen or the same as R1.

[0023] As a further improvement of the present invention, the X is a straight-chain alkylene group of C2-C3;

[0024] G is selected from a straight-chain alkylene group of C5-C7;

[0025] L is -OC(=O)- or -C(=O)O-;

[0026] R1 is a straight-chain alkyl group of C8-C10;

[0027] R2 is hydrogen or the same as R1.

[0028] As a further improvement of the present invention, the specific structure of the compound is as follows:

[0029]

[0030] Technical Subject Two

[0031] A lipid nano - composition comprising a lipid component, said lipid component comprising the compound described in any one of Technical Subject One or a pharmaceutically acceptable salt thereof.

[0032] As a further improvement of the present invention, the lipid component further comprises neutral phospholipids, cholesterol, PEG - lipids;

[0033] The neutral phospholipids are selected from one or more of the following compounds: distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), soy lecithin (SL), egg yolk lecithin (EPC), hydrogenated soy lecithin (HSPC), hydrogenated egg yolk lecithin (HEPC), phosphatidic acid, sphingomyelin;

[0034] The PEG - lipids are selected from one or more of the following compounds: PEG - modified distearoyl phosphatidylethanolamine (DSPE - PEG), PEG - modified dimyristoyl glycerol (DMG - PEG), PEG - modified dipalmitoyl phosphatidylethanolamine (DPPE - PEG), PEG - modified dimyristoyl phosphatidylethanolamine (DMPE - PEG), PEG - modified dilauroyl phosphatidylethanolamine (DLPE - PEG), PEG - modified ceramide (Ceramide - PEG), mPEG - modified ditetradecylacetamide (mPEG - DTA);

[0035] As a further improvement of the present invention, the molecular weight of the PEG or mPEG is 1000 - 5000 Da.

[0036] As a further improvement of the present invention, the PEG - lipid is DMG - PEG 2000.

[0037] As a further improvement of the present invention, the nano - composition further comprises a therapeutic agent and / or a prophylactic agent, and the therapeutic agent and / or the prophylactic agent is one or more of nucleic acid molecules, small molecule compounds, proteins or polypeptides.

[0038] As a further improvement of the present invention, the nucleic acid molecule is selected from single-stranded DNA, double-stranded DNA, agomir, antagomir, antisense molecule, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), small nuclear RNA (snRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA (circRNA), self-replicating RNA (saRNA), long non-coding RNA (lncRNA), messenger RNA (mRNA), nucleic acid aptamer, ribozyme, locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholino oligonucleotide, or more than one of them;

[0039] As a further improvement of the present invention, the nucleic acid drug is mRNA;

[0040] As a further improvement of the present invention, the small molecule compound can be selected from one of anti-tumor drugs, anti-infective drugs, antidepressants, anti-convulsants, antibiotics / antibacterials, anti-fungal drugs, anti-parasitic drugs, immunomodulatory drugs, or anesthetics.

[0041] As a further improvement of the present invention, the therapeutic or prophylactic agent is a vaccine.

[0042] Technical theme three

[0043] A pharmaceutical composition comprising the lipid nano-composition described in any one of Technical theme two and a pharmaceutically acceptable carrier.

[0044] Technical theme four

[0045] Use of the compound described in Technical theme one in the preparation of nucleic acid drugs, vaccines, protein or polypeptide drugs, and small molecule drugs.

[0046] The beneficial effects produced by adopting the above technical solutions are as follows:

[0047] The present invention provides a new class of cationic lipid compounds containing piperazine. Such compounds can be combined with neutral phospholipids, cholesterol, and PEG lipids to form a lipid nano-composition. It has been experimentally verified that the lipid nano-composition has a high encapsulation rate and good delivery effect, providing a new way for the delivery of therapeutic and / or prophylactic agents. Description of the drawings

[0048] Figure 1 In vivo expression levels after intramuscular injection of mLuci-loaded LNP in mice, where PBS is the PBS group in Example 6, MC3 is the MC3 positive control group in Example 6, and Examples 1-4 are the formulation 1-4 groups in Example 6 in sequence. Detailed implementation manners

[0049] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0050] Terms and definitions

[0051] It should be noted that for the description of structural formulas and chemical formulas in the embodiments or implementation manners of the present invention, the present invention intends to cover all alternative, modified and equivalent technical solutions, and all should be regarded as being included within the scope of the present invention. Those skilled in the art should understand that many similar or equivalent methods and materials can be used to implement the present invention, so the present invention is not limited to the specific methods and materials described. In the case where the cited literature, patents or similar materials are different or contradictory to the present application (including but not limited to term definitions, term applications, technologies described, etc.), the content of the present invention shall prevail.

[0052] In this article, the minimum and maximum values of the carbon atom content in a hydrocarbon group are represented by prefixes. For example, the prefix Ca-Cb refers to a carbon chain containing "a" to "b" carbon atoms. Exemplarily, "C1-Cn" refers to a straight-chain or branched-chain saturated / unsaturated carbon chain containing 1, 2, 3, 4, 5, …… or n carbon atoms; further understanding, "C1-Cn" should be interpreted as any sub-range included therein, for example, C1-C15, C1-C12, C6-C12, C5-C7, C8-C10, C1-C8, C1-C5, C2-C3, etc.

[0053] As used herein, "alkyl" refers to a straight-chain or branched-chain hydrocarbon chain group that is completely saturated (without double bonds or triple bonds). The alkyl group can have 1 to 15 carbon atoms (whenever it appears herein, for example, the numerical range of "1 to 15" refers to each integer within the given range; for example, "1 to 15 carbon atoms" means that the alkyl group can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 15 carbon atoms, although this definition also covers the appearance of the term "alkyl" without specifying a numerical range). The alkyl group can also be a medium-sized alkyl group having 1 to 10 carbon atoms, such as "C1-6". The alkyl group can also be a lower alkyl group having 1 to 4 carbon atoms. The alkyl group of a compound can be designated as "C1-C4 alkyl", "C1-4 alkyl" or a similar name. Only as an example, "C1-C4 alkyl" or "C1-4 alkyl" means that there are one to four carbon atoms in the alkyl chain, that is, the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, etc.

[0054] The term "treatment" generally refers to the use of drugs to improve, relieve or cure a disease state, and refers to partial or complete remission, improvement, delaying its onset, inhibiting its progression, reducing its severity and / or reducing the incidence of one or more of its symptoms or characteristics. For a particular infection, disease, disorder and / or condition. For example, "treating" cancer can refer to inhibiting the survival, growth and / or spread of a tumor. To reduce the risk, subjects who do not exhibit a disease, disorder and / or condition and / or subjects who only exhibit early signs of a disease, disorder and / or condition can be treated. The pathological development status associated with a disease, disorder and / or condition.

[0055] The term "therapeutic agent" or "preventive agent" refers to any agent that has a therapeutic, diagnostic and / or preventive effect and / or causes the desired biological and / or pharmacological effect when administered to a subject. A therapeutic agent is also referred to as an "active agent" or "active ingredient". In the present invention, the therapeutic agent can be one or more of a nucleic acid molecule, a polypeptide, a protein or a small molecule compound.

[0056] In the present invention, the term "pharmaceutically acceptable" means that a substance or composition does not produce unreasonable toxicity, irritation, allergic reaction or other adverse side effects after contact with a mammal.

[0057] In the present invention, the term "pharmaceutically acceptable salt" refers to the organic salts and inorganic salts of the compounds of the present invention. These salts not only retain the pharmacological activity of the compound, but also have good pharmaceutical properties, such as enhanced solubility, stability or bioavailability, etc. These salts include, but are not limited to, organic acid salts such as acetate, citrate, fumarate, maleate, oxalate, malate, citrate, succinate, tartrate, lactate, camphorsulfonate, benzenesulfonate, p-toluenesulfonate, mesylate, trifluoroacetate, trifluoromethanesulfonate, etc.; inorganic acid salts such as hydrohalide, sulfate, phosphate, nitrate, etc. In addition, the drug active ingredient can also form salts with amino acids such as glutamic acid or aspartic acid, namely glutamate or aspartate.

[0058] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving the active compound) and that is substantially non-toxic and non-inflammatory to the patient. Excipients can include, for example: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, taste masking agents, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, thickening agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, cross-linked carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0059] The compounds in the present invention can be formulated into conventional formulations, or can also be formulated into sustained-release formulations, controlled-release formulations, targeted formulations, and various particulate drug delivery systems.

[0060] As used herein, the term "cationic lipid" refers to a lipid that exhibits a positive charge under specific pH conditions and that can bind to a negatively charged nucleic acid molecule through electrostatic interaction.

[0061] As used herein, "lipid nano-composition" is a composition comprising one or more lipids. The particle size of the lipid nano-composition is typically on the order of microns or smaller and can include a lipid bilayer. Lipid nano-compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes.

[0062] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that contains a polyethylene glycol moiety.

[0063] As used herein, "phospholipid" is a lipid that includes a phosphate ester moiety and one or more carbon chains, such as unsaturated fatty acid chains. The phospholipid may contain one or more bonds (such as one or more unsaturated bonds) (e.g., double or triple bonds). Certain phospholipids can facilitate fusion with a membrane. For example, cationic phospholipids can interact with one or more negatively charged phospholipids of a membrane (such as a cell membrane or an intracellular membrane). The fusion of the phospholipid with the membrane can allow one or more elements of the lipid-containing composition to cross the membrane, thereby allowing, for example, delivery of one or more elements to a cell.

[0064] As used herein, "encapsulation efficiency" refers to the amount of a therapeutic agent and / or prophylactic agent that becomes part of a lipid nano-composition, relative to the total amount of the therapeutic agent or prophylactic agent used in the preparation of the lipid nano-composition. For example, if 97 mg of a therapeutic and / or prophylactic agent is encapsulated in a lipid nano-composition out of a total of 100 mg of the therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency can be 97%. As used herein, "encapsulation" can refer to complete, substantial or partial encapsulation, enclosure, surrounding or envelopment.

[0065] As used herein, in the context of a lipid nano-composition, "particle size" or "average particle size" refers to the average diameter of the lipid nano-composition.

[0066] The term "nucleic acid molecule" refers to a biopolymer composed of nucleotide units, and there are mainly two types: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They play a crucial role in the storage, transmission and expression of genetic information. It includes: single-stranded DNA, double-stranded DNA, agomir, antagomir, antisense molecule, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), small nuclear RNA (snRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA (circRNA), self-replicating RNA (saRNA), long non-coding RNA (lncRNA), messenger RNA (mRNA), nucleic acid aptamer, ribozyme, locked nucleic acid (LNA), peptide nucleic acid (PNA) or morpholino oligonucleotide. In certain treatment regimens, the RNA is mRNA.

[0067] In one embodiment, the lipid nanoparticles are administered by intramuscular injection.

[0068] The term "targeting" generally refers to the ability of a carrier system to deliver a drug to a predetermined target area to enhance the therapeutic or diagnostic effect.

[0069] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than events that occur within a living organism (e.g., an animal, a plant, or a microorganism).

[0070] As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, a plant, or a microorganism or its cells or tissues).

[0071] In the examples of this patent, all reagents and instruments used in the tests are conventional items in the art, and their functions and uses are known. The technical means involved are conventional operating methods commonly mastered by those skilled in the art. Experiments without special instructions are carried out under normal temperature and pressure conditions. For the operating steps without special requirements, they are all carried out according to the standard procedures in the product manuals of the reagents or instruments to ensure the repeatability and reliability of the results.

[0072] The synthetic routes of the compounds in the examples of this application are as follows:

[0073]

[0074] In the route, the definitions of X, G, L, R1, and R2 are the same as those in the foregoing general formula.

[0075] Example 1

[0076]

[0077] At room temperature, to a solution of compound 1 (10.5 g, 46.8 mmol) in dichloromethane (100 ml) was added compound 2 (10.0 g, 58.0 mmol), EDCI (14.5 g, 75.4 mmol) and DMAP (2.8 g, 23.2 mmol). The mixture was stirred at room temperature for 7 hours, and the reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated sodium bicarbonate solution was added to the reaction solution for dilution. After liquid separation, the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (petroleum ether:ethyl acetate = 10:1) gave the colorless oily product compound 3 (20.0 g, 98.7%).

[0078] At room temperature, to a solution of compound 4 (1.41 g, 10.8 mmol) in acetonitrile (10 ml) was added compound 5 (2.4 g, 10.8 mmol) and K2CO3 (3.2 g, 21.6 mmol). The mixture was stirred at 75 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, the acetonitrile was removed by concentration. 50 ml of water was added, and the mixture was extracted with ethyl acetate (50 ml × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (dichloromethane:methanol = 15:1) gave compound 6 (1.29 g, 43.7%).

[0079] To a solution of Compound 6 (1.29 g, 4.7 mmol) in dichloromethane (20 mL) was slowly added 10 mL of 12 N HCl. The mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC on a plate. After completion of the reaction, dichloromethane and hydrochloric acid were removed by concentration to obtain Compound 7 (1.11 g).

[0080] To a solution of Compound 7 (0.5 g, 2.9 mmol) in acetonitrile (20 mL) were added potassium carbonate (1.19 g, 8.6 mmol), potassium iodide (48.0 mg, 0.29 mmol) and Compound 3 (3.0 g, 8.6 mmol). The mixture was stirred at 75 °C overnight. The reaction was monitored by TLC. After completion of the reaction, ethanol was removed by concentration. 20 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Column chromatography on silica gel (dichloromethane:methanol = 20:1) gave Compound A (321.0 mg, 15.6%). 1 H NMR (400 MHz, Chloroform-d) δ 3.99 (t, J = 6.8 Hz, 4H), 3.59 (t, J = 5.3 Hz, 2H), 3.38 (s, 1H), 2.66 (t, J = 6.9 Hz, 2H), 2.62–2.44 (m, 14H), 2.24 (t, J = 7.4 Hz, 4H), 1.64–1.41 (m, 12H), 1.31 -1.12 (m, 38H), 0.81 (t, J = 6.6 Hz, 6H); 13 C NMR (101 MHz, Chloroform-d) δ 173.70, 64.49, 59.45, 57.68, 55.35, 53.98, 53.20, 52.68, 50.70, 34.17, 31.87, 29.67, 29.56, 29.55, 29.49, 29.30, 29.23, 28.62, 26.86, 25.90, 25.74, 24.75, 22.65, 14.08; MS-ESI (m / z): 710.9 (M+H)+.

[0081] Example 2

[0082]

[0083] At room temperature, to a solution of compound 8 (10.0 g, 55.2 mmol) in dichloromethane (100 ml) was added compound 9 (11.3 g, 60.8 mmol), EDCI (15.9 g, 82.8 mmol) and DMAP (2.7 g, 22.1 mmol). The mixture was stirred at room temperature for 7 hours and monitored by TLC. After the reaction was complete, an equal volume of saturated sodium bicarbonate solution was added to dilute the reaction mixture. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) gave the colorless oily product compound 10 (15.2 g, 78.7%).

[0084] To a solution of compound 10 (0.4 g, 2.3 mmol) in acetonitrile (30 ml) were added potassium carbonate (0.90 g, 6.9 mmol), potassium iodide (38.2 mg, 0.23 mmol) and compound 7 (1.6 g, 4.6 mmol). The mixture was stirred at 75 °C overnight and monitored by TLC. After the reaction was complete, ethanol was removed by concentration. 20 ml of water was added, and the mixture was extracted with ethyl acetate (30 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (dichloromethane:methanol = 20:1) gave the colorless oily compound B (379 mg, 23.1%). 1 H NMR (400 MHz, Chloroform-d) δ 4.00 (t, J = 6.7 Hz, 4H), 3.59 (t, J = 5.4 Hz, 2H), 2.71 (t, J = 6.8 Hz, 2H), 2.64–2.44 (m, 15H), 2.23 (t, J = 7.6 Hz, 4H), 1.62–1.45 (m, 12H), 1.37–1.15 (m, 38H), 0.83 (t, J = 6.6 Hz, 6H); 13 C NMR (101 MHz, Chloroform-d) δ 173.98, 64.10, 59.41, 57.69, 55.13, 54.02, 53.24, 52.70, 50.59, 34.35, 31.86, 29.68, 29.53, 29.44, 29.27, 29.24, 29.14, 28.58, 26.96, 25.77, 25.71, 24.98, 22.64, 14.09; MS-ESI (m / z): 710.9 (M + H)+.

[0085] Example 3

[0086]

[0087] At room temperature, to a solution of Compound 11 (10.5 g, 46.8 mmol) in dichloromethane (100 ml) was added Compound 12 (10.0 g, 39.0 mmol), EDCI (9.7 g, 50.7 mmol) and DMAP (1.9 g, 15.6 mmol). The mixture was stirred at room temperature for 7 hours, and the reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated sodium bicarbonate solution was added to dilute the reaction solution. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) gave the colorless oily product Compound 13 (15.2 g, 78.7%).

[0088] To a solution of Compound 13 (0.4 g, 2.3 mmol) in acetonitrile (30 ml) were added potassium carbonate (0.90 g, 6.9 mmol), potassium iodide (38.2 mg, 0.23 mmol) and Compound 7 (1.6 g, 4.6 mmol). The mixture was stirred at 75 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, ethanol was removed by concentration. 20 ml of water was added, and the mixture was extracted with ethyl acetate (30 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (dichloromethane: methanol = 20:1) gave the colorless oily Compound C (281 mg, 13.1%). 1 H NMR (400 MHz, Chloroform-d) δ 4.88–4.79 (m, 2H), 3.60 (t, J = 5.3 Hz, 2H), 2.67 (t, J = 7.0 Hz, 2H), 2.60–2.43 (m, 14H), 2.25 (t, J = 7.6 Hz, 4H), 1.59 (t, J = 7.2 Hz, 4H), 1.47 (t, J = 6.4 Hz, 11H), 1.34–1.16 (m, 62H), 0.85 (t, 12H). 13 C NMR (101 MHz, Chloroform-d) δ 173.56, 74.10, 59.23, 57.67, 54.30, 53.53, 52.71, 50.89, 34.61, 34.09, 31.81, 29.47, 29.44, 29.18, 29.15, 29.11, 27.24, 26.14, 25.26, 25.02, 22.60, 14.04; MS-ESI (m / z): 935.2 (M+H)+.

[0089] Example 4

[0090]

[0091] At room temperature, to a solution of compound 4 (1.5 g, 11.5 mmol) in acetonitrile (10 ml) was added compound 14 (2.7 g, 11.5 mmol) and K2CO3 (3.2 g, 23.0 mmol). The mixture was stirred at 75 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, the acetonitrile was removed by concentration. 50 ml of water was added, and the mixture was extracted with ethyl acetate (50 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (dichloromethane:methanol = 15:1) gave compound 15 (1.49 g, 45.2%).

[0092] To a solution of compound 15 (1.49 g, 5.2 mmol) in dichloromethane (20 ml) was slowly added 10 ml of 12N HCl. The mixture was stirred at room temperature for 2 h, and the reaction was monitored by TLC spotting. After the solvent was removed by concentration, compound 16 (0.9 g) was obtained.

[0093] To a solution of compound 16 (0.2 g, 1.1 mmol) in acetonitrile (20 ml) were added K2CO3 (0.46 g, 3.3 mmol), potassium iodide (18.3 mg, 0.11 mmol) and compound 3 (0.77 g, 2.2 mmol). The mixture was stirred at 75 °C overnight, and the reaction was monitored by TLC. After the reaction was complete, the acetonitrile was removed by concentration. 20 ml of water was added, and the mixture was extracted with ethyl acetate (200 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Silica gel column chromatography (dichloromethane:methanol = 20:1) gave colorless oily compound D (151 mg, 19.0%). 1 HNMR (400 MHz, Chloroform-d) δ 4.04 (t, J = 6.8 Hz, 4H), 3.62 (t, J = 5.3 Hz, 2H), 2.76–2.46 (m, 15H), 2.40 (t, J = 7.1 Hz, 2H), 2.29 (t, J = 7.4 Hz, 4H), 1.68–1.53 (m, 12H), 1.38–1.18 (m, 39H), 0.86 (t, J = 6.7 Hz, 6H); 13 C NMR (101 MHz, Chloroform-d) δ 173.62, 64.59, 59.34, 57.70, 53.42, 53.21, 52.97, 52.70, 51.36, 50.73, 34.10, 31.90, 29.69, 29.59, 29.53, 29.32, 29.26, 28.65, 26.76, 25.93, 24.63, 22.67, 14.10; MS-ESI (m / z): 724.9 (M+H)+.

[0094] Example 5 Preparation and Characterization of LNP Formulations

[0095] Disperse mLuci-RNA (Luciferase mRNA) in 20 mM acetic acid solution (pH = 5.0) to make the final concentration of mRNA 200 μg / mL (aqueous phase). Mix the cationic lipids (Compound A-D, MC3): cholesterol: DSPC: DMG-PEG2000 at a molar ratio of 50:38.5:10:1.5 to form a mixed lipid (oil phase). Control the total flow rate of the aqueous phase and the oil phase to be 12 ml / min, and mix the mRNA and the lipid mixture at a volume ratio of 3:1 by the T-mixing method to obtain LNP encapsulating mRNA. Dilute the encapsulated LNP 10-fold with 0.01 M phosphate buffer, then concentrate it by ultrafiltration and replace the 0.01 M phosphate buffer. Finally, concentrate the LNP to a mRNA concentration of 200 μg / mL and adjust the pH of the LNP suspension to 7-8.

[0096] Use the Ribogreen kit to detect the total and free content of mRNA in LNP and calculate the encapsulation efficiency of LNP.

[0097] Dilute the final product of LNP 10-fold with 0.01 M phosphate buffer, add 1 ml to the particle size cell, and place it on a Malvern ZetaSizer instrument to detect the particle size and PDI of LNP.

[0098] The particle size, PDI, and encapsulation efficiency results of each preparation are shown in Table 1.

[0099] Table 1: LNP characterization data of compounds in the examples

[0100]

[0101]

[0102] The results show that the LNP prepared using Compounds A-E in the examples of this application has a particle size below 100 nm, a PDI less than 0.3, and an encapsulation efficiency greater than 98%, with excellent encapsulation performance and particle homogeneity.

[0103] Example 6 Detection of reporter gene expression in mice

[0104] Randomly divide female BALB / c mice (6-8 weeks old) weighing about 20 g into 6 groups, with 5 mice in each group, namely the PBS group, the MC3 positive control group, the preparation 1 group, the preparation 2 group, the preparation 3 group, and the preparation 4 group. Inject the injection drug into the quadriceps femoris of the mice using an insulin syringe by intramuscular injection, with an injection volume of 50 μl for each group. The settings for each group are as follows:

[0105] PBS group: Inject PBS buffer containing mLuci-RNA, where the mRNA concentration is 100 μg / ml;

[0106] MC3 positive control group: Inject PBS buffer containing MC3 preparation, where the mRNA concentration is 100 μg / ml;

[0107] Preparation 1 group: Inject PBS buffer containing Preparation 1, where the mRNA concentration is 100 μg / ml;

[0108] Preparation 2 group: Inject PBS buffer containing Preparation 2, where the mRNA concentration is 100 μg / ml;

[0109] Preparation 3 group: Inject PBS buffer containing Preparation 3, where the mRNA concentration is 100 μg / ml;

[0110] Preparation 4 group: Inject PBS buffer containing Preparation 4, where the mRNA concentration is 100 μg / ml.

[0111] The preparations used in each group were prepared as described in Example 5.

[0112] At 24 h after administration, the in vivo expression of Luciferase was detected by IVIS of Perkinelmer. The substrate was D-luciferin Sodium salt (GOLDBIO, LUCNA-1G), prepared into a concentration of 15 mg / ml with physiological saline, filtered and sterilized with a 0.22 μm filter membrane, and stored in the dark at -20 °C in aliquots. Before imaging, each mouse was intraperitoneally injected with 200 μl of the substrate solution, allowed to act for 10 - 20 minutes, and then the mouse was anesthetized with isoflurane gas and placed on the imaging plate to detect the fluorescence of the live animal.

[0113] The results are as Figure 1 shown. The expression of luciferase transfected by the LNP of the lipid in Example 3 in vivo was significantly higher than that of other groups.

[0114] The experiment proves that the cationic lipid compound of Example 3 of the present invention can efficiently deliver mRNA in vivo and ensure the protein expression level. It provides a reliable way to use the cationic lipid of the present invention to deliver nucleic acid molecules, small molecule compounds, polypeptides or proteins.

[0115] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cationic lipid compound containing piperazine or a pharmaceutically acceptable salt thereof, characterized in that: Its structure is shown in Formula I: Wherein, X is a C1-C8 alkylene group; G is selected from C1-C12 alkylene; L is -OC(=O)- or -C(=O)O-; R1 is a C1-C15 alkyl group; R2 is hydrogen or the same as R1.

2. The cationic lipid compound containing piperazine or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Said X is a C1-C5 straight chain alkylene group; G is selected from a C3-C10 straight chain alkylene group; L is -OC(=O)- or -C(=O)O-; R1 is a C6-C12 straight chain alkyl group; R2 is hydrogen or the same as R1.

3. The cationic lipid compound containing piperazine or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Said X is a C2-C3 straight chain alkylene group; G is selected from a C5-C7 straight chain alkylene group; L is -OC(=O)- or -C(=O)O-; R1 is a C8-C10 straight chain alkyl group; R2 is hydrogen or the same as R1.

4. The cationic lipid compound containing piperazine or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Its specific structure is as follows:

5. A lipid nanocomposition, characterized in that: Comprising a lipid component, wherein the lipid component comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

6. The lipid nanocomposition according to claim 5, characterized in that wherein the lipid component further comprises neutral phospholipids, cholesterol, and PEG lipids; Preferably, the neutral phospholipid is selected from one or more of the following compounds: distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, phosphatidic acid, sphingomyelin, most preferably, distearoylphosphatidylcholine; The PEG lipid is selected from one or more of the following compounds: PEG-modified distearoylphosphatidylethanolamine, PEG-modified dimyristoylglycerol, PEG-modified dipalmitoylphosphatidylethanolamine, PEG-modified dimyristoylphosphatidylethanolamine, PEG-modified dilauroylphosphatidylethanolamine, PEG-modified ceramide, and mPEG-modified ditetradecanoylacetamide; further preferably, the molecular weight of the PEG or mPEG is 1000-5000Da, and most preferably, DMG-PEG2000.

7. The lipid nanocomposition according to any one of claims 5-6, characterized in that It further includes therapeutic agents and / or preventive agents, which are one or more of nucleic acid molecules, small molecule compounds, proteins or polypeptides.

8. The lipid nanocomposition according to claim 7, characterized in that The nucleic acid molecule is selected from one or more of single-stranded DNA, double-stranded DNA, agomir, antagomir, antisense molecule, siRNA, aiRNA, miRNA, dsRNA, shRNA, snRNA, tRNA, rRNA, circRNA, saRNA, lncRNA, mRNA, nucleic acid aptamer, ribozyme, LNA, PNA or morpholino oligonucleotide, preferably mRNA.

9. A pharmaceutical composition, characterized in that It comprises the lipid nanocomposition according to any one of claims 5 to 8 and a pharmaceutically acceptable carrier.

10. Use of the compound according to any one of claims 1 to 4 in the preparation of nucleic acid drugs, vaccines, protein or polypeptide drugs, and small molecule drugs.

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