Multi-glycol modified lipid compound as well as preparation method and application thereof

Abstract

The invention discloses a multi-glycol modified lipid compound as well as a preparation method and application thereof. The lipid compound and the lipid nano-particles prepared from the lipid compound can effectively deliver bioactive substances to target cells and parts in a targeted mode, the pharmacological action of the bioactive substances is efficiently achieved, the molecular weight of the lipid compound is single, batch-to-batch difference can be controlled, patent medicine stability can be improved, immunogenicity can be reduced, and the lipid nano-particles can be applied to preparation of drugs. The compound is expected to be used for development and application of related drugs.

Description

[0001] This application is a divisional application of a Chinese patent application with the application number 202210873087.8, the application date of July 22, 2022, and the invention title of "A Lipid Compound Modified with Polyhydric Glycol, Its Preparation Method and Application". Technical Field

[0002] The present invention relates to the field of pharmaceutical technology, and specifically relates to a lipid compound modified with polyhydric glycol, its preparation method and application. Background Art

[0003] Gene therapy refers to using molecular biology methods to introduce a target gene into a patient's body for expression to correct or compensate for diseases caused by gene defects and abnormalities, or to introduce nucleic acids into cells to inhibit the expression of a target gene (gene silencing) or increase the expression of a target gene (gene activation) to achieve the purpose of treating diseases. In recent years, with the progress of biotechnology, gene therapy drugs have developed rapidly, bringing great development prospects to the field of biopharmaceuticals. Alnylam Company in the United States has developed a series of gene drugs using RNAi technology and successfully launched four RNAi drugs: Onpattro (patisiran), Givlaari (givosiran), Oxlumo (lumasiran), and Leavio (inclisiran). Among them, Leavio (inclisiran) is a biopharmaceutical for treating hyperlipidemia. The successful launch of this drug has broadened the field of gene therapy drugs from the traditional treatment of rare genetic diseases to the treatment of common diseases, bringing new opportunities for RNAi drugs.

[0004] Due to the nature of nucleic acid drugs themselves, such as negative charge and easy degradation by nucleases, they cannot effectively penetrate cell membranes and enter cells and are quickly degraded in the body. Therefore, a suitable delivery system is needed to stably deliver nucleic acids to the target location and take effect. The common difficulty in the development of mRNA and siRNA is how to effectively deliver them into the cells at the target site. During the delivery process, issues such as how to avoid rapid clearance, avoid degradation by nucleases, and improve escape after endocytosis also need to be considered.

[0005] Currently, most nucleic acid delivery systems adopt different types of liposome delivery methods, such as lipid nanoparticles (LNP), GalNac, LPP (lipopolyplexes), etc. LNP can be prepared from four lipids in a certain proportion. Usually, these four lipids include cationic lipids, neutral lipids, steroidal lipids, and polymer-conjugated lipids, where the polymer-conjugated lipid refers to polyethylene glycol (PEG) lipid.

[0006] Some polyethylene glycol lipids have been reported in the prior art. For example, patent documents CN110352071A and CN1882693A disclose polyethylene glycol lipids and lipid nanoparticles prepared by using polyethylene glycol lipids for delivering bioactive substances into the body. However, polyethylene glycol is a mixture as a polymer and is prone to batch differences, which has an adverse impact on the quality and efficacy stability of the drugs formed therefrom. Summary of the Invention

[0007] To overcome the deficiencies of the prior art, the present invention provides a compound, a preparation method thereof, and an application thereof.

[0008] In a first aspect of the present invention, there is provided a compound having the following structure:

[0009]

[0010]

[0011] Wherein,

[0012] R1 and R2 are each independently a hydrocarbon group containing 6 to 30 carbon atoms;

[0013] L1, L2, and L3 are independent linking groups;

[0014] X is N or CR4, wherein R4 is selected from: H, alkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic group, heterocyclic group alkyl, halogen, -CN, -NO2, -COR A , -C(O)OR A , -OCOR A , -C(O)NR A R B , -CH=NR A , -OR A , -OC(O)R A , -S(O) t -R A , -S(O) t , -S(O) A R B , -NR A R B , -NR A C(O)R B ; wherein t is an integer from 0 to 2 (such as 0, 1, 2), and each R A and R B are independently selected from: H, alkyl, cycloalkyl, aryl, heterocyclic group, and halogen;

[0015] n is an integer selected from 30 to 90;

[0016] Y is a terminal group.

[0017] The compound has a single molecular weight and is not a mixture composed of substances with different molecular weights (such as polymers, which are usually characterized by an average molecular weight).

[0018] Specifically, L1 and L2 can independently be selected from: a single bond, an alkylene group, -OC(O)(CH2) i -, -C(O)O(CH2) i -, -C(O)(CH2) i -, -O(CH2) i -, -S(O) x (CH2) i -, -S-S-(CH2) i -, -C(O)S(CH2) i -, -SC(O)(CH2) i -, -NR a -(CH2)i-, -C(O)NR a -(CH2)i-, -OC(O)NR a -(CH2)i-, -NR a -C(O)(CH2)i-, -NR a -C(O)O(CH2) i -, -S(O) x NR a -(CH2) i -, -OC(O)O(CH2) i -; where x is an integer from 0 to 2 (such as 0, 1, 2), i is an integer from 0 to 10, and each R a is independently selected from: H, alkyl, cycloalkyl, aryl, heterocyclic group, and halogen.

[0019] Specifically, i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, especially 0 or 1.

[0020] Specifically, each R a is independently selected from: H, C1-6 alkyl; in some embodiments of the present invention, R a is H.

[0021] More specifically, L1 and L2 can be independently selected from: a single bond, C1-6 alkylene (such as -CH2-, -CH2CH2-, -CH2CH2CH2-), -OC(O)(CH2)-, -OC(O)-, -C(O)O(CH2)-, -C(O)O-, -C(O)(CH2)-, -C(O)-, -C(O)S(CH2)-, -C(O)S-, -C(O)NH-(CH2)-, -C(O)NH-, -OC(O)O(CH2)-, -OC(O)O-.

[0022] In some embodiments of the present invention, both L1 and L2 are single bonds.

[0023] In some other embodiments of the present invention, L1 is -C(O)O(CH2)- and L2 is -C(O)O-.

[0024] Specifically, L3 can be selected from: a single bond, alkylene, -(CH2) h OC(O)(CH2) j -, -(CH2) h C(O)O(CH2) j -, -(CH2)hC(O)(CH2)j-, -(CH2)hO(CH2)j-, -(CH2)hS(O) y (CH2)j-, -(CH2)hS-S-(CH2)j-, -(CH2) h C(O)S(CH2) j -, -(CH2) h SC(O)(CH2) j -, -(CH2) h NR b -(CH2) j -, -(CH2) h C(O)NR b -(CH2) j -, -(CH2) h OC(O)NR b -(CH2) j -, -(CH2) h NR b -C(O)(CH2) j -, -(CH2) h NR b -C(O)O(CH2) j -, -(CH2) h S(O) y NR b -(CH2) j -, -(CH2) hOC(O)O(CH2) j -; wherein y is an integer from 0 to 2 (such as 0, 1, 2), h and j are independently selected from integers from 0 to 10, and each R b is independently selected from: H, alkyl, cycloalkyl, aryl, heterocyclic group, and halogen.

[0025] Specifically, h and j are independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, especially 0 and 2; in some embodiments of the present invention, h is 0.

[0026] Specifically, each R b is independently selected from: H, C1-6 alkyl; in some embodiments of the present invention, R b is H.

[0027] More specifically, L3 can be selected from: single bond, C1-6 alkylene (such as -CH2-, -CH2CH2-, -CH2CH2CH2-), -OC(O)(CH2)2-, -OC(O)-, -C(O)O(CH2)2-, -C(O)O-, -C(O)(CH2)2-, -C(O)-, -C(O)S(CH2)2-, -C(O)S-, -C(O)NH-(CH2)2-, -C(O)NH-, -OC(O)O(CH2)2-, -OC(O)O-.

[0028] In some embodiments of the present invention, L3 is C1-6 alkylene, such as -CH2-, -CH2CH2-, -CH2CH2CH2-.

[0029] In some other embodiments of the present invention, L3 is -C(O)CH2CH2-.

[0030] In some embodiments of the present invention, L1, L2, and L3 can be uncleavable linkers, which are relatively stable in the in vivo environment and are not easily cleaved, for example, thioether linkers, alkylene linkers, or directly connected to R1, R2 or connected as a single bond, especially C1-10 (linear or branched) alkylene, such as C2-8 alkylene, C2-5 alkylene, -CH2-, -CH2CH2-, -CH2CH2CH2-.

[0031] Specifically, each of R1 and R2 is independently a straight-chain or branched-chain, saturated or unsaturated hydrocarbon group containing 6 to 30 carbon atoms (such as 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30 carbon atoms), especially a straight-chain hydrocarbon group containing 6 to 30 carbon atoms; more specifically, R1 and R2 can each independently be an alkyl group containing 6 to 30 carbon atoms, especially a straight-chain alkyl group containing 6 to 30 carbon atoms, especially a straight-chain alkyl group containing 10 to 24 carbon atoms. For example, a C12 straight-chain alkyl group (lauryl), a C14 straight-chain alkyl group (myristyl), a C16 straight-chain alkyl group (palmityl), a C18 straight-chain alkyl group (stearyl), a C20 straight-chain alkyl group (eicosyl).

[0032] In some embodiments of the present invention, R1 and R2 are the same. For example, both R1 and R2 are straight-chain alkyl groups containing 6 to 24 carbon atoms. For example, both R1 and R2 are C14 straight-chain alkyl groups (myristyl).

[0033] Specifically, R4 is selected from: H, C1-6 alkyl, halogen; in some embodiments of the present invention, R4 is H.

[0034] Specifically, n can be 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 60, 65, 70, 75, 80, 85, 90; more specifically, n can be an integer from 30 to 60, such as an integer from 40 to 50, an integer from 44 to 48; in some embodiments of the present invention, n is 46 or 47.

[0035] Specifically, Y can be selected from: H, alkyl, alkoxy, cycloalkyl, aralkyl, monosaccharide and oligosaccharide groups; more specifically, Y can be selected from: methyl, ethyl, isopropyl, methoxy, ethoxy, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, especially methyl, ethyl, isopropyl, methoxy, ethoxy, cyclopropyl, cyclobutyl, cyclohexyl or benzyl; in some embodiments of the present invention, Y is methoxy.

[0036] Specifically, Y can also be selected from: hydroxyl, carboxyl, ester group, amino, mercapto, maleimide group, succinimide group, succinimide ester group, alkynyl, azide group, aldehyde group, nitrobenzene carbonate group, acrylate group, methacrylate group, dibenzocyclooctyne group, isocyanate group, vinyl sulfone group, dithiopyridine group, glutaric acid group, hydrazide group, p-nitro carbonate group, silyl group, epoxy group, etc.

[0037] Specifically, the compound can have the following structure:

[0038]

[0039] Among them, L1, L2, L3, R1, R2, n, and Y have the corresponding definitions as described above in the present invention.

[0040] More specifically, the compound may be selected from the following structures:

[0041]

[0042] Among them, R1, R2, n, and Y have the corresponding definitions as described above in the present invention.

[0043] In some embodiments of the present invention, the compound has the following structure:

[0044]

[0045] In the second aspect of the present invention, there is provided a pharmaceutically acceptable salt, ester, isomer, prodrug, and solvate of the compound described in the first aspect.

[0046] In the third aspect of the present invention, there is provided a method for preparing the compound described in the first aspect, which may include using to carry out the reaction step, wherein n and Y have the corresponding definitions in the first aspect of the present invention, and R c is a reactive group, for example, hydroxyl, carboxyl, amino, mercapto, maleimide, succinimide, alkynyl, azide, aldehyde, epoxy group, etc.

[0047] In the fourth aspect of the present invention, there is provided a lipid composition, which comprises the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug, and solvate.

[0048] Specifically, the lipid composition may further comprise a cationic lipid, such as stearamide (SA), lauryl trimethyl ammonium bromide, cetyl trimethyl ammonium bromide, myristyl trimethyl ammonium bromide, dimethyldioctadecylammonium bromide (DDAB), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-cholesterol), 1,2-di-tetradecanoyl-3-trimethylammonium-propane (DMTAP), 1,2-di-octadecanoyl-3-trimethylammonium-propane (DOTAP), and DOTAP derivatives such as 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and 1,2-di-hexadecanoyl-3-trimethylammonium-propane, 1,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane (DODAP), and DODAP derivatives such as 1,2-di-tetradecanoyl-3-dimethylammonium-propane, 1,2-di-hexadecanoyl-3-dimethylammonium-propane, and 1,2-di-octadecanoyl-3-dimethylammonium-propane, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-c-(4'-trimethylammonium)-butyryl-sn-glycerol (DOTB), di-stearamide-propionyl spermine, SAINT-2, polycationic lipid 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA), ((4-hydroxybutyl)aza-dialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), etc., and one or more of them, especially ALC-0315.

[0049] Specifically, in the lipid composition, the molar ratio of the compound or its pharmaceutically acceptable salt, ester, isomer, prodrug, and solvate described in the first aspect to the cationic lipid may be 1:0.01 - 0.1 (such as 1:0.01, 1:0.02, 1:0.025, 1:0.03, 1:0.035, 1:0.04, 1:0.045, 1:0.05, 1:0.06, 1:0.08, 1:0.1), especially 1:0.03 - 0.05.

[0050] Specifically, the lipid composition may further comprise neutral lipids, such as one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (DOPG), palmitoyl-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), etc., especially DSPC.

[0051] Specifically, in the lipid composition, the molar ratio of the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug and solvate to the neutral lipid may be 1:0.1 - 0.5 (such as 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.5), especially 1:0.1 - 0.3.

[0052] Specifically, the lipid composition may further comprise sterol lipids, such as one or more of avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, chondrillasterol, epicholesterol, ergosterol, fucosterol, hexahydro-lumisterol, hydroxycholesterol; lanosterol, lumisterol, poriferasterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholalic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, etc., especially cholesterol.

[0053] Specifically, in the lipid composition, the molar ratio of the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug and solvate to the sterol lipid may be 1:0.5 - 1.5 (1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.4, 1:1.5), especially 1:0.8 - 1.2.

[0054] More specifically, the lipid composition comprises the compound described in the first aspect, as well as cationic lipids, neutral lipids and sterol lipids; in some embodiments of the present invention, the composition comprises the compound described in the first aspect, as well as ALC-0315, DSPC and cholesterol.

[0055] Specifically, the lipid composition may further comprise one or more pharmaceutically acceptable excipients, such as carriers, adjuvants, diluents, etc.

[0056] In the fifth aspect of the present invention, there is also provided a pharmaceutical composition, which comprises a bioactive substance, and the compound described in the first aspect or the lipid composition described in the third aspect.

[0057] Specifically, the bioactive substance can be a small molecule compound, nucleic acid, peptide, protein, etc.

[0058] Specifically, the bioactive substance is a nucleic acid, such as DNA or RNA. Among them, DNA can be non-coding DNA (antisense DNA) or coding DNA, and RNA can be selected from one or more of the following: antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, etc., especially mRNA, siRNA.

[0059] In one embodiment of the present invention, the bioactive substance is siRNA that inhibits the expression of a target gene, such as anti-KDR siRNA.

[0060] Specifically, the bioactive substance can be used for preventing and / or treating one or more diseases selected from the following: cancer, inflammation, fibrotic diseases, autoimmune diseases, diseases caused by pathogen infections, mental disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic disorders, oral diseases, musculoskeletal diseases, nutritional diseases, skin diseases, etc.

[0061] Specifically, in the pharmaceutical composition, the molar ratio of the bioactive substance to the compound described in the first aspect or the lipid composition described in the third aspect is 0.01-0.5:1 (such as 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1), especially 0.01-0.1.

[0062] Specifically, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients, such as carriers, adjuvants, diluents, etc.

[0063] In some embodiments of the present invention, the above-mentioned pharmaceutically acceptable excipients are pharmaceutically acceptable injection excipients, such as isotonic sterile salt (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., or a mixture of the above salts) solutions.

[0064] Specifically, the above-mentioned pharmaceutical composition can be administered by any suitable route, such as enteral administration or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, infusion, intracerebral, rectal, etc.); the above-mentioned drug can be in any suitable dosage form, such as enteral dosage forms, for example, including, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, etc.; parenteral dosage forms, for example, injection dosage forms: such as injections (e.g., for subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), respiratory dosage forms: such as sprays, aerosols, powder aerosols, etc., skin dosage forms, such as topical solutions, lotions, ointments, plasters, pastes, patches, etc., mucosal dosage forms: such as eye drops, ophthalmic ointments, nasal drops, gargles, sublingual tablets, etc., cavity dosage forms: such as suppositories, aerosols, effervescent tablets, drops, dripping pills, etc., for rectum, vagina, urethra, nasal cavity, ear canal, etc. Especially injection dosage forms.

[0065] Specifically, the various dosage forms of the above-mentioned pharmaceutical composition can be prepared according to the conventional production methods in the pharmaceutical field.

[0066] In the sixth aspect of the present invention, there is provided the use of the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug and solvate, and the lipid composition described in the fourth aspect in the preparation of a delivery system for bioactive substances and in the delivery of bioactive substances.

[0067] Specifically, in this application, the bioactive substance has the definition described in the fourth aspect of the present invention.

[0068] Specifically, the delivery system of the above-mentioned bioactive substance is a lipid nanoparticle (LNP).

[0069] In the seventh aspect of the present invention, there is provided the use of the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug and solvate, the lipid composition described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect in the preparation of a drug for preventing and / or treating diseases and in the prevention and / or treatment of diseases.

[0070] Specifically, the disease can be selected from one or more of the following: cancer, inflammation, fibrotic diseases, autoimmune diseases, diseases caused by pathogen infections, mental disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic disorders, oral diseases, musculoskeletal diseases, nutritional diseases, skin diseases, etc.

[0071] Specifically, the pathogen can be a microorganism, a parasite (protozoa, worm, etc.) or other vectors; among them, the microorganism can be selected from one or more of the following: virus, chlamydia, rickettsia, mycoplasma, bacterium, spirochete, fungus, etc.

[0072] Specifically, viruses such as, but not limited to, Adenoviridae (such as adenovirus), Herpesviridae (such as HSV1 (oral herpes), HSV2 (genital herpes), VZV (varicella), EBV (Epstein - Barr virus), CMV (cytomegalovirus)), Poxviridae (such as smallpox virus, vaccinia virus), Papovaviridae (such as papillomavirus (HPV)), Parvoviridae (such as B19 virus), Hepadnaviridae (such as hepatitis B virus), Polyomaviridae (such as polyomavirus), Reoviridae (such as reovirus, rotavirus), Picornaviridae (such as enterovirus, foot - and - mouth disease virus), Caliciviridae (such as Norwalk virus, hepatitis E virus), Togaviridae (such as rubella virus), Arenaviridae (such as lymphocytic choriomeningitis virus), Retroviridae (HIV - 1, HIV - 2, HTLV - 1), Flaviviridae (such as dengue virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.), Orthomyxoviridae (such as influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.)), Paramyxoviridae (such as human parainfluenza virus type 1 (HPIV), HPIV type 2, HPIV type 3, HPIV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus, etc.), Bunyaviridae (such as California encephalitis virus, hantavirus), Rhabdoviridae (such as rabies virus), Filoviridae (such as Ebola virus, Marburg virus), Coronaviridae (such as HCoV - 229E, HCoV - OC43, HCoV - NL63, HCoV - HKU1, SARS - CoV, MERS - CoV, SARS - CoV - 2, etc.), Astroviridae (such as astrovirus), Bornaviridae (such as Borna virus).

[0073] In some embodiments of the present invention, the virus is a coronavirus, specifically such as HCoV - 229E, HCoV - OC43, HCoV - NL63, HCoV - HKU1, SARS - CoV, MERS - CoV, SARS - CoV - 2, etc.

[0074] In some embodiments of the present invention, the virus is an orthomyxovirus, such as influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.).

[0075] In some embodiments of the present invention, the virus is a paramyxovirus, such as HPIV type 1, HPIV type 2, HPIV type 3, HPIV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, etc.

[0076] In some embodiments of the present invention, the virus is a flavivirus, such as dengue virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.

[0077] In some embodiments of the present invention, the virus is a papillomavirus (HPV).

[0078] Specifically, diseases caused by viral infections include but are not limited to influenza, SARS, COVID-19, viral hepatitis (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), mumps, measles, dengue fever, Zika virus disease, Japanese encephalitis, chikungunya disease, yellow fever, West Nile virus disease, cervical cancer, etc.

[0079] In some embodiments of the present invention, the above-mentioned drug is a drug for preventing diseases, such as a vaccine.

[0080] In other embodiments of the present invention, the above-mentioned drug is a therapeutic agent for treating diseases.

[0081] In the eighth aspect of the present invention, a method for delivering a bioactive substance is provided, which includes preparing a delivery system for the bioactive substance with the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug and solvate, or the lipid composition described in the fourth aspect, and then delivering the bioactive substance to a subject in need thereof through the delivery system.

[0082] Specifically, in this method, the bioactive substance has the definition described in the fourth aspect of the present invention.

[0083] Specifically, the bioactive substance is encapsulated in the delivery system.

[0084] Specifically, in this method, the subject can be any animal or its cells (in vitro or in situ) that receives this method; more specifically, the subject is a mammal, such as a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey or a human, especially a human.

[0085] In a ninth aspect of the present invention, there is provided a method for preventing and / or treating a disease, which comprises the step of administering to a subject in need thereof the pharmaceutical composition described in the fifth aspect, or the step of preparing a delivery system for a bioactive substance using the compound described in the first aspect or a pharmaceutically acceptable salt, ester, isomer, prodrug and solvate thereof, or the lipid composition described in the fourth aspect, and then delivering the bioactive substance to the subject in need thereof through the delivery system, or the step of adopting the method for delivering a bioactive substance described in the eighth aspect.

[0086] Specifically, the subject is a mammal, such as a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey or a human, especially a human.

[0087] After the anti-KDR siRNA (siRNA that inhibits the expression of VEGFR2 mRNA) is delivered into cells by the lipid nanoparticles (LNP) prepared from the lipid compound of the present invention, there is an obvious and continuous inhibitory effect on KDR mRNA, indicating that the lipid compound and the lipid nanoparticles prepared therefrom can effectively target and deliver the bioactive substance to the target cells and sites, efficiently achieve the pharmacological effect of the bioactive substance, and the lipid compound of the present invention has a single molecular weight, which is beneficial to controlling the differences between batches, improving the stability of the drug product, reducing the immunogenicity, and is expected to be used in the development and application of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 The mass spectrum of Compound 1 prepared in Example 1 of the present invention is shown.

[0089] Figure 2 The mass spectrum of Compound 2 prepared in Example 2 of the present invention is shown.

[0090] Figure 3 The mass spectrum of Compound 3 prepared in Example 3 of the present invention is shown. DETAILED DESCRIPTION OF THE INVENTION

[0091] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0092] In the present invention, the terms "VEGFR2" and "KDR" both represent Vascular Endothelial Growth Factor Receptor 2 and can be used interchangeably.

[0093] In the present invention, the term "alkyl" refers to a straight-chain or branched-chain hydrocarbon group without unsaturated bonds, and the hydrocarbon group is connected to other parts of the molecule by a single bond. The alkyl group used herein usually contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkyl), preferably 1 to 6 carbon atoms (i.e., C1-6 alkyl). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc. If the alkyl group is substituted by a cycloalkyl group, it is correspondingly "cycloalkylalkyl", such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. If the alkyl group is substituted by an aryl group, then it is correspondingly "arylalkyl", such as benzyl, diphenylmethyl or phenethyl. If the alkyl group is substituted by a heterocyclic group, then it is correspondingly "heterocyclic alkyl".

[0094] In the present invention, the term "alkylene" refers to a hydrocarbon group (divalent alkyl) formed by removing two hydrogen atoms from an alkane molecule, which can be straight-chain or branched-chain and is connected to other parts of the molecule by a single bond. The alkylene group used herein usually contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkylene), especially 1 to 6 carbon atoms (i.e., C1-6 alkylene). Examples of alkylene groups include methylene (-CH2-), ethylene (-CH2CH2-), propylene, butylene, etc.

[0095] In the present invention, the term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group containing at least two carbon atoms and at least one unsaturated bond, and the hydrocarbon group is connected to other parts of the molecule by a single bond. The alkenyl group used herein usually contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkenyl), preferably 1 to 6 carbon atoms (i.e., C1-6 alkenyl). Examples of alkenyl groups include, but are not limited to, vinyl, 1-methyl-vinyl, 1-propenyl, 2-propenyl or butenyl, etc.

[0096] In the present invention, the term "cycloalkyl" refers to an alicyclic hydrocarbon. The cycloalkyl group used herein usually contains 1 to 4 single rings and / or fused rings, contains 3 - 18 carbon atoms, preferably 3 - 10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (e.g., C3-10 cycloalkyl, C3-6 cycloalkyl), such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or adamantyl, etc.

[0097] In the present invention, the term "aryl" refers to any functional group or substituent derived from a simple aromatic ring, including monocyclic aryl groups and / or fused-ring aryl groups, such as monocyclic or fused-ring aryl groups containing 1-3 rings and having 6-18 (e.g., 6, 8, 10, 12, 14, 16, 18) carbon ring atoms. The aryl used herein is generally an aryl containing 1-2 rings, which is monocyclic or fused-ring and has 6-12 carbon ring atoms (i.e., C6-12 aryl), wherein the H on the carbon atom can be substituted, for example, by groups such as alkyl, halogen, etc. Examples of the aryl include, but are not limited to, phenyl, p-methylphenyl, naphthyl, biphenyl, indenyl, etc.

[0098] In the present invention, the term "halogen" refers to bromine, chlorine, iodine or fluorine.

[0099] In the present invention, the term "heterocyclic group" refers to a 3- to 18-membered non-aromatic ring group, which contains 2 to 17 carbon atoms and 1 to 10 heteroatoms. The heterocyclic group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can contain a fused, spiro, or bridged ring system. The heterocyclic group can be partially saturated (heteroaryl) or fully saturated (heterocycloalkyl). Suitable heteroaryls in the compounds of the present invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms, and the heteroaryls include, for example, coumarin, including 8-coumarin, quinolinyl, including 8-quinolinyl, isoquinolinyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indazinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furanopyridyl. Suitable heterocycloalkyls in the compounds of the present invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms, and the heterocycloalkyls include, for example, pyrrolidinyl, tetrahydrofuryl, dihydrofuran, tetrahydrothienyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxathiane, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxiranyl, thiiranyl, azepanyl, oxazepanyl, diazepanyl, triazepanyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxane, 1,3-dioxolanyl, pyrazolinyl, dithiane, dithiolanyl, dihydropyranyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl and quinuclidinyl.

[0100] The pharmaceutically acceptable salts of the present invention include acid addition salts and base addition salts.

[0101] The acid addition salts include, but are not limited to, salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphonic acid, and salts derived from organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Thus, these salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, iodide, acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, and mesylate, and also include salts of amino acids such as arginine salts, gluconate, galacturonate, etc. The acid addition salts can be prepared by contacting the free base form in a conventional manner with a sufficient amount of the desired acid to form the salt. The free base form can be regenerated by contacting the salt form with a base and isolating the free base in a conventional manner.

[0102] The base addition salts of the present invention refer to salts formed with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or formed with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Examples of suitable amines include, but are not limited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1,2-diamine), N-methylglucamine, and procaine. The base addition salts can be prepared by contacting the free acid form in a conventional manner with a sufficient amount of the desired base to form the salt. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner.

[0103] In the present invention, stereoisomers exist in the forms of enantiomers, diastereomers, and geometric isomers. Some compounds of the present invention have cycloalkyl groups which can be substituted on more than one carbon atom, in which case all geometric forms, including cis and trans, and their mixtures, are within the scope of the present invention. The cycloalkyl groups include cycloaliphatic groups and aryl groups, where the cycloaliphatic group can be a non-aromatic monocyclic, fused-ring, bridged-ring, or spiro-ring saturated or unsaturated cyclic hydrocarbon group, and aryl groups such as phenyl, naphthyl, phenanthryl, biphenyl, etc.

[0104] In the present invention, a solvate refers to the physical combination of a compound of the present invention with one or more solvent molecules. This physical combination includes various degrees of ionic and covalent bonding, including hydrogen bonding. In certain cases, solvates can be isolated, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Solvates include solution-phase and separable solvates. Representative solvates include ethanolates, methanolates, etc.

[0105] In the present invention, a prodrug refers to a form of a compound of formula I that is suitable for administration to a patient, has no excessive toxicity, irritation, allergic reaction, etc., and is effective for its intended purpose, including acetal, ester, and zwitterionic forms. The prodrug is converted in vivo, such as by hydrolysis in the blood, to obtain the parent compound.

[0106] In the present invention, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and their hybrids.

[0107] In the present invention, the term "lipid" refers to a group of organic compounds that includes, but is not limited to, esters of fatty acids, and is typically characterized by being sparingly soluble in water but soluble in many organic solvents.

[0108] In the present invention, the term "cationic lipid" refers to a lipid molecule that can carry a positive charge.

[0109] In the present invention, the term "neutral lipid" refers to a lipid molecule that is uncharged and non-phosphoglyceride.

[0110] In the present invention, the term "lipid nanoparticle" refers to a particle having at least one dimension in the nanometer range, which contains at least one lipid.

[0111] In the present invention, the term "vaccine" refers to a composition suitable for administration to an animal (especially a mammal, such as a human), which induces an immune response after administration, the intensity of which is sufficient to minimally help prevent, ameliorate, or cure a clinical disease caused by a microbial infection.

[0112] In the present invention, the term "delivery system" refers to a formulation or composition that regulates the spatial, temporal, and dosage distribution of a bioactive ingredient in an organism.

[0113] In the present invention, the terms "patient" or "subject", etc. are used interchangeably herein, and refer to any animal or its cells treated according to the methods described herein, which can be in vitro or in situ. Specifically, the aforementioned animals include mammals, such as rats, mice, guinea pigs, rabbits, dogs, monkeys, or humans, especially humans.

[0114] In the present invention, the term "treatment" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing, arresting, and / or stopping one or more clinical symptoms of a disease after the onset of the disease.

[0115] In the present invention, the term "prevention" refers to, prior to the onset of a disease, treating to avoid, minimize, or make it difficult for the disease to occur or develop.

[0116] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0117] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0118] Example 1: Synthesis of Compound 1

[0119]

[0120] 1. Synthesis of Compound 1a

[0121]

[0122] Dissolve tetradecylamine (3.0 g, 14 mmol) and tetradecyl bromide (3.9 g, 14 mmol) in 20 ml of acetonitrile, add potassium carbonate (1.9 g, 14 mmol), and stir the mixture at 80 °C for 12 hours. After the raw materials have completely reacted, cool to room temperature, filter by suction, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the filtrate, extract with dichloromethane twice, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain Compound 1a (3.9 g, white solid), with a yield of 68%. MS m / z (ESI): 410.40 [M+1].

[0123] 2. Synthesis of Compound 1

[0124]

[0125] Dissolve Compound 1a (0.576 g, 1.4 mmol) and (2g, 1 mmol) was added to a 100 ml single-necked flask containing dichloromethane (20 ml), stirred and dissolved. Then DIEA (260 mg, 2 mmol) and HATU (760 mg, 2 mmol) were added successively, and the mixture was stirred at room temperature for 5 hours. The reaction system was filtered, and the mother liquor was concentrated under vacuum. After concentration, 40 ml of purified water was added, and the mixture was stirred and dissolved, then washed with ethyl acetate three times, 40 ml each time (emulsification occurred during the washing process, and a small amount of ethanol was added to break the emulsion). After washing, 6 g of sodium chloride was added to the aqueous phase and stirred to dissolve. The aqueous phase was extracted with dichloromethane twice, 20 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, filtered by suction, and the filtrate was concentrated under vacuum. 30 ml of isopropanol and 10 ml of ethyl acetate were added, and the mixture was heated to 40 °C to dissolve, then cooled to -15 °C for crystallization, and filtered by suction. The filter cake was dried under vacuum to obtain product compound 1 (1.5 g, white solid), with a yield of 62.5%. MS m / z (ESI): 2582.76 [M + NH4] + ;

[0126] 1H-NMR (300 MHz, DMSO) δ: 2.96 (2H, s), 3.1 - 3.8 (180H, m), 1.46 (4H, m), 1.24 (44H, m), 0.86 (6H, t).

[0127] Example 2: Synthesis of Compound 2

[0128]

[0129] Under a nitrogen atmosphere, LiAlH4 (380 mg, 10 mmol) was slowly added to a three-necked flask containing 30 ml of tetrahydrofuran in an ice-water bath. Compound 1 (2 g, 1 mmol) was dissolved in 10 ml of tetrahydrofuran and slowly added dropwise to the reaction flask. After 3 hours, 10 ml of purified water was added dropwise to the reaction system. The system was filtered using diatomaceous earth, and the filtrate was concentrated under vacuum. 20 ml of isopropanol and 6 ml of ethyl acetate were added, and the mixture was heated to 40 °C to dissolve, then cooled in an ice-water bath for crystallization, and filtered by suction. The filter cake was dried under vacuum to obtain product compound 2 (1.3 g, white solid), with a yield of 64%. MS m / z (ESI): 2551.76 [M + H] + ;

[0130] 1H-NMR (300 MHz, DMSO) δ: 3.2 - 3.8 (180H, m), 2.36 (4H, t), 2.26 (4H, m), 1.53 (2H, m), 1.22 (44H, m), 0.84 (6H, t).

[0131] Example 3: Synthesis of Compound 3

[0132]

[0133] 1. Synthesis of Compound 3a

[0134]

[0135] Add (40 g, 20 mmol) and purified water (0.4 ml) into a 1 L three-necked flask containing THF (400 ml), stir to dissolve, then successively add NaOH (6.4 g, 160 mmol) and epichlorohydrin (37 g, 400 mmol), and reflux under heating for 18 hours. Supplement NaOH (3.2 g, 80 mmol) and epichlorohydrin (18.5 g, 200 mmol) and continue the reaction for 18 hours. Cool the reaction system to room temperature, filter with diatomaceous earth, add phosphate buffer solution with pH = 7 (100 ml) to the mother liquor, concentrate under vacuum to remove THF, add purified water (200 ml) after concentration, wash with ethyl acetate twice, 100 ml each time. After washing, add 45 g of sodium chloride to the aqueous phase and stir to dissolve, extract the aqueous phase with dichloromethane twice, 100 ml each time. Combine the organic phases, dry over anhydrous sodium sulfate, filter by suction, concentrate the filtrate under vacuum, recrystallize with 400 ml of isopropanol, filter by suction, and vacuum-dry the filter cake to obtain the product Compound 3a (34 g, light yellow solid), with a yield of 85%. MS m / z (ESI): 2114.41 [M + H] + ;

[0136] 1H-NMR (300 MHz, DMSO) δ: 3.3~3.85 (184H, m), 3.28 (1H, t), 3.28 (3H, s), 3.1 (1H, m), 2.7 (1H, t), 2.55 (1H, m).

[0137] 2. Synthesis of Compound 3b

[0138]

[0139] Dissolve Compound 3a (30 g, 15 mmol) in 2 mol / L potassium hydroxide solution (300 ml) and stir at room temperature for 12 hours, add 45 g of sodium chloride and stir to dissolve, extract the aqueous phase with dichloromethane twice, 150 ml each time. Combine the organic phases, dry over anhydrous sodium sulfate, filter by suction, concentrate the filtrate under vacuum, add 450 ml of methyl tert-butyl ether to precipitate the solid, filter by suction, and vacuum-dry the filter cake to obtain the product Compound 3b (28 g, light yellow solid), with a yield of 93.3%. MS m / z (ESI): 2132.43 [M + H] + ;

[0140] 1H-NMR (300 MHz, DMSO) δ: 4.6 (1H, s), 4.45 (1H, s), 3.3 - 3.8 (189H, m), 3.25 (3H, s).

[0141] Synthesis of Compound 3

[0142]

[0143] Compound 3b (20 g, 10 mmol) was added to a 500 mL single-necked flask containing DCM (200 mL), stirred until dissolved, and then myristic acid (6.4 g, 28 mmol) and DMAP (489 mg, 4 mmol) were added successively, and the mixture was placed in an ice-water bath. DCC (5.8 g, 28 mmol) was dissolved in 40 mL of DCM and then added dropwise to the reaction system. After 16 hours, the reaction system was filtered, the mother liquor was concentrated under vacuum to remove dichloromethane, 300 mL of isopropanol and 100 mL of ethyl acetate were added, and the mixture was heated to 40 °C to dissolve, then cooled to -10 °C for crystallization, and filtered by suction. The filter cake was dried under vacuum to obtain the product compound 3 (17 g, light yellow solid), with a yield of 85%. MS m / z (ESI): 2569.70 [M + NH4] + ;

[0144] 1H-NMR (300 MHz, DMSO) δ: 5.1 (1H, s), 4.28 (1H, m), 4.1 (1H, m), 3.3 - 3.8 (183H, m), 3.24 (3H, s), 2.25 (4H, t), 1.5 (4H, m), 1.24 (44H, m), 0.86 (6H, t).

[0145] Example 4: Gene silencing effect after cellular delivery of siRNA: polyethylene glycol lipid (PEG-lipid) particle composition in ARPE-19 cells

[0146] 1. Cell culture and transfection

[0147] Cell name: ARPE-19

[0148] (1) ARPE-19 cells were cultured in a medium of DMEM / F12 + 12% FBS + double antibiotics (containing 100 U / ml Penicillin, 100 μg / ml Streptomycin) in a 37 °C, 5% CO2 saturated humidity incubator. 24 h before the experiment, 1.2×10 5 cells were seeded in a 12-well plate and cultured overnight.

[0149] (2) Take 50 μL of OPTI-MEM medium and dilute 2.5 mL of anti-KDR siRNA: Compound 1 = 1:10, anti-KDR siRNA: Compound 2 = 1:10, anti-KDR siRNA: Compound 3 = 1:10 (the above ratios are molar ratios, and the siRNA concentration is 20 μM). For the positive control, take 50 μL of OPTI-MEM medium and dilute 3 μL of Lipofectamine 3000 TM Transfection reagent, mix the two gently and shake well, then let it stand for 15 min. In addition, set up a blank group, an NC group, and an NC-RL group.

[0150] Compound 1, Compound 2, and Compound 3 are prepared according to Examples 1-3 respectively.

[0151] Anti-KDR siRNA refers to the siRNA that inhibits the expression of VEGFR2 mRNA, and its preparation method is described in the patent document CN202010229195.2, and the sequence is:

[0152] Sense strand: 5′-GGAGUGAGAUGAAGAAAUU-3′;

[0153] Antisense strand: 5′-AAUUUCUUCAUCUCACUCC-3′.

[0154] (3) Change the medium for the cells in each well of the cell plate, add antibiotic-free medium, with a volume of 900 μL per well, and then add 100 μL of the above mixed solution to each well. The final total volume per well is 1000 μL. The transfection concentration of siRNA (or siRNA NC or siR-NC RL) is 50 nM.

[0155] (4) After 24 h and 48 h of transfection, take out the 12-well plate from the incubator at 37 °C and 5% CO2, collect the cells for RNA extraction for subsequent detection.

[0156] 2. RNA Extraction

[0157] (1) Extract RNA using the instructions of the RNA extraction kit from Promega. Briefly, after washing the cells with PBS, add 300 μL of lysis buffer, pipette to lyse the cells, add 300 μL of dilution buffer after sufficient lysis, and then incubate in a water bath at 70 °C for 3 min. Centrifuge at 14000 rpm for 10 min, transfer the supernatant to a new 1.5 mL Ep tube, add 300 μL of absolute ethanol, mix well, and then add it to the adsorption column. Centrifuge at 14000 rpm for 1 min, discard the filtrate, add 600 μL of washing buffer, and centrifuge again for 1 min. Discard the filtrate, add 50 μL of freshly prepared DNAse reaction solution to each well, let it stand at room temperature for 15 min; add 600 μL of washing buffer and centrifuge for 1 min; after discarding the filtrate, add 600 μL of washing buffer again and centrifuge for 1 min; discard the filtrate, centrifuge for 2 min, add 50 μL of nuclease-free water to each well, let it stand at room temperature for 5 min, and then centrifuge to collect the eluted RNA.

[0158] (2) RNA quality control, detect the RNA content and purity using Nanodrop, and detect the integrity of RNA by 1% agarose gel electrophoresis.

[0159] 3. Q-PCR detection procedure

[0160] (1) RNA reverse transcription

[0161] Using the total RNA extracted from the sample as a template, use the Promega reverse transcription kit to establish the following reaction system:

[0162] Table 1 RNA reverse transcription system

[0163]

[0164] Mix the above system well, centrifuge to collect the liquid to the bottom of the tube, incubate at 42 °C for 60 min and 72 °C for 10 min; the product is the cDNA template.

[0165] (2) Fluorescent quantitative PCR detection

[0166] Use the TB green Premix Ex Taq II (Tli RNaseH Plus) (Promega) reagent to establish the following reaction system:

[0167] Table 2 Fluorescent quantitative PCR reaction system

[0168]

[0169] Perform PCR amplification according to the following procedure

[0170] Pre-denature at 95 °C for 10 min, and then enter the following cycle

[0171] *95℃ for 10 s

[0172] 60℃ for 20 s

[0173] 70℃ for 10 s

[0174] Read the plate

[0175] Return * A total of 40 cycles are performed.

[0176] Construct the melting curve: Read the plate every 0.5℃ and pause for 5 s between 65℃ and 95℃.

[0177] 4. Gene inhibition effect

[0178] Using GAPDH as the internal reference gene, calculate the relative expression level of KDR mRNA by the ΔΔCt method; normalize the mRNA expression levels of each group with the expression level of the blank group as 100%.

[0179] The results show that the relative expression levels of KDR mRNA in the cells treated with the siRNA of the present invention are significantly decreased at 24 h compared with the blank group, siNC group and siNC RL group, and further decreased at 48 h after transfection, indicating that the particles composed of the compounds 1 - 3 and siRNA of the present invention have a significant and continuous inhibitory effect on KDR mRNA.

[0180] Example 5: Gene inhibition effect after cell delivery of siRNA:PEG - lipid particle composition in A375 cells

[0181] Except that the culture medium of A375 cells is DMEM medium containing 10% FBS (containing 100 U / ml Penicillin, 100 μg / ml Streptomycin), the remaining operations are similar to those in Example 4. The results show that the relative expression levels of KDR mRNA in the cells treated with the siRNA of the present invention are significantly decreased at 24 h compared with the blank group, siNC group and siNC RL group, and further decreased at 48 h after transfection, indicating that the particles (compound 1, compound 2, compound 3) made of the PEG - lipid and siRNA of the present invention have a significant and continuous inhibitory effect on KDR mRNA.

[0182] Example 6: Gene inhibition effect after cell delivery of PEG - lipid / cationic lipid / neutral lipid / steroidal lipid - siRNA nanoparticles (LNP) in ARPE - 19 cells

[0183] The formulations and preparation methods of each group of PEG - lipid / cationic lipid / neutral lipid / steroidal lipid - siRNA nanoparticles (LNP) are as follows:

[0184] Compound 1 / ALC-0315 / DSPC / cholesterol was dissolved in absolute ethanol at a molar ratio of 46.3% / 1.7% / 9.4% / 42.6% to prepare a 10 mmol / L mixed solution. Then, citric acid buffer solution with pH = 4 was added to prepare a 30% ethanol-citric acid solution containing four lipids, which was filtered through a 0.1 μm filter membrane for standby. Anti-KDR siRNA was dissolved in a 30% ethanol-citric acid solution without lipids at a concentration of 2 mg / ml, and then mixed with the above 30% ethanol-citric acid solution containing four lipids at a mass ratio of anti-KDR siRNA:lipids of 0.06:1, incubated for 30 minutes, dialyzed with PBS at pH = 7.4 for more than 16 hours, and freeze-dried to obtain lipid nanoparticles containing anti-KDR siRNA. The preparation methods of other groups of different PEG-lipid ionic lipid nanoparticles (Compound 1 was replaced by Compound 2 or Compound 3) were the same as that of Compound 1.

[0185] The results showed that after delivering anti-KDR siRNA into cells by using the lipid nanoparticles prepared from PEG-lipid / ALC-0315 / DSPC / cholesterol and siRNA of the present invention, there was an obvious and continuous inhibitory effect on KDR mRNA.

[0186] Example 7: Gene expression effect after cell delivery of PEG-lipid / cationic lipid / neutral lipid / steroid lipid-mRNA nanoparticles in 293T cells

[0187] The formulations and preparation methods of each group of PEG-lipid / cationic lipid / neutral lipid / steroid lipid-siRNA nanoparticles (LNP) were as follows:

[0188] Compound 1 / ALC-0315 / DSPC / cholesterol was dissolved in absolute ethanol at a molar ratio of 46.3% / 1.7% / 9.4% / 42.6% to prepare a 10 mmol / L mixed solution. Then, citric acid buffer solution with pH = 4 was added to prepare a 30% ethanol-citric acid solution containing four lipids, which was filtered through a 0.1 μm filter membrane for standby. GFP mRNA was dissolved in a 30% ethanol-citric acid solution without lipids at a concentration of 2 mg / ml, and then mixed with the above 30% ethanol-citric acid solution containing four lipids at a mass ratio of GFP mRNA:lipids of 0.06:1, incubated for 30 minutes, dialyzed with PBS at pH = 7.4 for more than 16 hours, and freeze-dried to obtain lipid nanoparticles containing GFP mRNA. The preparation methods of other groups of different cationic lipid nanoparticles (Compound 1 was replaced by Compound 2, Compound 3) were the same as that of Compound 1.

[0189] The transfection efficiency of LNP was determined by detecting the number of cells expressing green fluorescent protein (GFP) using a fluorescence microscope. The results showed that the LNP groups with compounds 1-3 as PEG lipids all had good delivery effects.

[0190] Example 8: Study on the allergic reactions of PEG-lipids with different molecular weights in guinea pigs

[0191] 1. Animal grouping

[0192] Forty-eight healthy guinea pigs that had been normally raised for 5 days were randomly divided into 6 groups, with 8 animals in each group. Group A: negative control group (0.9% sodium chloride injection); Group B: positive control group (0.15 mg / mL ovalbumin solution, ovalbumin was purchased from Sigma, USA, batch number DHO15-4); Group C: M-PEG47-DTDPAM (i.e., compound 1, prepared in Example 1), Group D: M-PEG47-DTDPA (i.e., compound 2, prepared in Example 2), Group E: M-DTDPAM-2000 (provided by Tianjin KeyGen Biotech Co., Ltd., batch number ZZ409P002), Group F: M-DTDPA-2000 (provided by Tianjin KeyGen Biotech Co., Ltd., batch number ZZ409P004).

[0193] 2. Administration method

[0194] The animals were sensitized by intraperitoneal injection, with a dosing volume of 1 mL / animal, once every other day for a total of 4 times. On the 2nd day after the last sensitization, the guinea pigs in each group were injected with the corresponding drug solution through the toe vein for challenge, and the challenge dose was 2 mL / animal. During the sensitization period, the animal status was observed daily. Fifteen minutes before the intravenous injection challenge and continuously for 40 minutes after the injection, the reactions of the guinea pigs were observed, and the onset time and disappearance time of the symptoms were recorded. Blood was collected 40 minutes after the challenge stop, anticoagulated with heparin, and plasma was prepared.

[0195] 3. Observation indicators

[0196] During the sensitization period and at the time of challenge administration, the reaction symptoms and duration of the animals were observed and recorded, such as nose scratching, sneezing, restlessness, jumping, wheezing, purpura, etc., and the scores and grades of the reaction symptoms were judged according to the systemic sensitization reaction symptom grading standard (Table 3).

[0197] Table 3 Systemic sensitization reaction symptom grading standard

[0198]

[0199] 4. Experimental results:

[0200] (1) Results of the systemic allergy experiment

[0201] Guinea pig reaction symptoms: None of the animals in each group showed abnormal symptoms during the first sensitization. During the sensitization period, there were no abnormalities in diet, drinking water, or behavior, and the body weights increased normally. None of the animals in group A showed allergic reactions after challenge administration; animals in group B showed strong allergic reactions after challenge administration, manifested as convulsions, purpura, unsteady gait, wheezing, jumping, tearing, and listlessness; in group C, 3 guinea pigs showed occasional scratching and restlessness 10 - 35 minutes after challenge administration, and 2 animals in group D had the above symptoms. 4 guinea pigs in group E showed symptoms such as sneezing, coughing, and rapid breathing, and 1 showed symptoms such as piloerection and shivering; 3 guinea pigs in group F showed symptoms such as sneezing, coughing, and rapid breathing, and 2 guinea pigs showed symptoms of shivering and nose scratching; the other guinea pigs showed no abnormalities. According to Table 3, the reaction symptom levels of guinea pigs in each group were determined. Group B was extremely strongly positive, groups C and D were weakly positive, and groups E and F were positive. However, both the number of allergic reactions and the degree of occurrence in groups C and D were lower than those in groups E and F. It is suggested that the sensitization of guinea pigs by PEG-lipids with a single molecular weight is lower than that of PEG-lipids with non-single molecular weights. The results of the reaction symptom grading of guinea pigs in each group after challenge administration are shown in Table 4.

[0202] Table 4 Results of allergic reactions in guinea pigs caused by PEG-lipids with different molecular weights

[0203] Code Group Average Score Allergic Symptoms Group A Negative Control Group 0 Negative Group B Positive Control Group 2.8 Strongly Positive Group C M-PEG47-DTDPAM 0.375 Weakly Positive Group D M-PEG47-DTDPA 0.25 Weakly Positive Group E M-DTDPAM-2000 1.125 Positive Group F M-DTDPA-2000 1 Positive

[0204] (2) Comparison of plasma IgE and histamine contents

[0205] After challenge administration to the animals in each group, the plasma IgE content in each group was higher than that in group A, and the increase was less than 100%. The plasma IgE content in animals in groups C and D was significantly lower than that in groups E and F.

[0206] The plasma histamine content in each drug-treated group was higher than that in group A. Among them, there was a significant difference in the histamine content between group B and group A (P < 0.05); the plasma histamine content in groups E and F was significantly higher than that in group A, with statistical differences. Although the plasma histamine content in groups C and D increased to some extent, it was much lower than that in groups E and F, and there was no statistical difference compared with group A.

[0207] Table 5 Plasma IgE and histamine contents (ng / ml) in guinea pigs in each group after challenge administration

[0208] Code Group IgE Content (ng / ml) Histamine Content (ng / ml) Group A Negative Control Group 204.83±18.65 4.92±0.98 Group B Positive Control Group 286.33±34.67 12.21±4.25 Group C M-PEG47-DTDPAM 235±26.79 5.23±1.21 Group D M-PEG47-DTDPA 225±31.44 6.71±2.10 Group E M-DTDPAM-2000 255±25.78 8.89±2.54 Group F M-DTDPA-2000 248±34.11 8.23±2.23

[0209] Conclusion: The results of the guinea pig systemic allergic experiment showed that PEG-lipids with non-single molecular weights showed positive results, while PEG-lipids with a single molecular weight showed weak positive results, suggesting that compared with PEG-lipids with non-single molecular weights, the immunogenicity of PEG-lipids with a single molecular weight was reduced. The detection results of plasma IgE and histamine contents also supported this conclusion.

[0210] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0211] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0212] Listing the steps of the method in a certain order in the present invention does not constitute any limitation on the order of the method steps.

Claims

1. A compound of a single molecular weight having the following structure: Wherein, R1 and R2 are each independently a hydrocarbon group containing 6 to 30 carbon atoms; L1, L2 and L3 are independent linking groups; X is N or CR4; n is an integer selected from 30 - 90; Y is a terminal group; L1 and L2 are independently selected from: single bond, C1-6 alkylene (such as -CH2-, -CH2CH2-, -CH2CH2CH2-), -OC(O)(CH2)-, -OC(O)-, -C(O)O(CH2)-, -C(O)O-, -C(O)(CH2)-, -C(O)-, -C(O)S(CH2)-, -C(O)S-, -C(O)NH-(CH2)-, -C(O)NH-, -OC(O)O(CH2)-, -OC(O)O-; Preferably, both L1 and L2 are single bonds, or, L1 is -C(O)O(CH2)- and L2 is -C(O)O-; L3 is selected from: single bond, C1-6 alkylene (such as -CH2-, -CH2CH2-, -CH2CH2CH2-), -OC(O)(CH2)2-, -OC(O)-, -C(O)O(CH2)2-, -C(O)O-, -C(O)(CH2)2-, -C(O)-, -C(O)S(CH2)2-, -C(O)S-, -C(O)NH-(CH2)2-, -C(O)NH-, -OC(O)O(CH2)2-, -OC(O)O-; Preferably, L3 is C1-6 alkylene, such as -CH2-, -CH2CH2-, -CH2CH2CH2-; or, L3 is -C(O)CH2CH2-; R4 is selected from: H, C1-6 alkyl, halogen; preferably, R4 is H.

2. The compound according to claim 1, wherein R1 and R2 are each independently an alkyl group containing 6 to 30 carbon atoms, especially a straight-chain alkyl group containing 6 to 30 carbon atoms, especially a straight-chain alkyl group containing 10 to 24 carbon atoms; Preferably, R1 and R2 are independently selected from: C12 straight-chain alkyl, C13 straight-chain alkyl, C14 straight-chain alkyl, C16 straight-chain alkyl, C18 straight-chain alkyl, C20 straight-chain alkyl; More preferably, both R1 and R2 are C14 straight-chain alkyl, or, both R1 and R2 are C13 straight-chain alkyl.

3. The compound according to claim 1, wherein, n is an integer from 30 - 60; preferably, n is selected from: 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 60.

4. The compound according to claim 1, wherein Y is selected from: H, alkyl, alkoxy, cycloalkyl, aralkyl, monosaccharide and oligosaccharide groups; preferably, Y is selected from: methyl, ethyl, isopropyl, methoxy, ethoxy, cyclopropyl, cyclobutyl, cyclohexyl, benzyl; Alternatively, Y is selected from: hydroxyl, carboxyl, ester, amino, mercapto, maleimide, succinimide, succinimidyl ester, alkynyl, azide, aldehyde, nitrobenzyl carbonate, acrylate, methacrylate, dibenzocyclooctyne, isocyanate, vinyl sulfone, dithiolpyridyl, glutaric acid, hydrazide, p-nitrophenyl carbonate, silyl, epoxy group.

5. The compound according to any one of claims 2 - 4, characterized in that, The compound is selected from the following structures:

6. The compound according to claim 1, wherein The compound has the following structure:

7. A lipid composition comprising the compound according to any one of claims 1-6 or a pharmaceutically acceptable salt, ester, isomer, prodrug and solvate thereof.

8. The composition according to claim 7, wherein The composition further comprises a cationic lipid; Preferably, the cationic lipid is selected from: octadecanamide (SA), lauryltrimethylammonium bromide, cetyltrimethylammonium bromide, myristyltrimethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-cholesterol), 1,2-di-tetradecanoyl-3-trimethylammonium-propane (DMTAP), 1,2-di-octadecanoyl-3-trimethylammonium-propane (DOTAP) and DOTAP derivatives such as 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and 1,2-di-hexadecanoyl-3-trimethylammonium-propane, 1,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane (DODAP) and DODAP derivatives such as 1,2-di-tetradecanoyl-3-dimethylammonium-propane, 1,2-di-hexadecanoyl-3-dimethylammonium-propane and 1,2-di-octadecanoyl-3-dimethylammonium-propane, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-c-(4'-trimethylammonium)-butyryl-sn-glycerol (DOTB), dioctadecanamide-propionyl spermine, SAINT-2, polycationic lipid 2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA), ((4-hydroxybutyl)aza-dialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), one or more of them, especially ALC-0315; Preferably, the molar ratio of the compound to the cationic lipid is 1:0.01-0.

1.

9. The composition according to claim 7 or 8, characterized in that, The composition further comprises a neutral lipid; Preferably, the neutral lipid is selected from one or more of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), palmitoyl-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), especially DSPC; Preferably, the molar ratio of the compound to the neutral lipid is 1:0.1-0.

5.

10. The composition according to any one of claims 7-9, characterized in that, The composition further comprises a sterol lipid; Preferably, the sterol lipid is selected from one or more of: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, chondrillasterol, epicholesterol, ergosterol, fucosterol, hexahydroprovitamin D3, hydroxycholesterol; lanosterol, provitamin D3, fucosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholanic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid, especially cholesterol; Preferably, the molar ratio of the compound to the sterol lipid is 1:0.5-1.

5.

11. The composition according to claim 7, which comprises the compound, and ALC-0315, DSPC and cholesterol.

12. A pharmaceutical composition, which comprises a bioactive substance, and the compound according to any one of claims 1-6 or the lipid composition according to any one of claims 7-11.

13. The composition according to claim 12, wherein The bioactive substance is a small molecule compound, nucleic acid, peptide or protein; Preferably, the bioactive substance is a nucleic acid, such as DNA or RNA; Preferably, the RNA is selected from one or more of: antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, especially mRNA, siRNA.

14. The composition according to claim 12 or 13, characterized in that, The molar ratio of the bioactive substance to the compound or the lipid composition is 0.01-0.5:1, especially 0.01-0.

1.

15. Use of the compound according to any one of claims 1-6 or its pharmaceutically acceptable salts, esters, isomers, prodrugs and solvates, and the lipid composition according to any one of claims 7-11 in the preparation of a delivery system for a bioactive substance; Preferably, the bioactive substance is a nucleic acid, such as DNA or RNA; Preferably, the RNA is selected from one or more of: antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, especially mRNA, siRNA; Preferably, the delivery system of the bioactive substance is a lipid nanoparticle (LNP).

16. Use of a compound according to any one of claims 1-6 or a pharmaceutically acceptable salt, ester, isomer, prodrug and solvate thereof, a lipid composition according to any one of claims 7-11, and a pharmaceutical composition according to any one of claims 12-14 in the preparation of a medicament for preventing and / or treating a disease.

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