WO2023217237A1 - Lipid compound, and composition, preparation and use thereof - Google Patents

Lipid compound, and composition, preparation and use thereof Download PDF

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Publication number
WO2023217237A1
WO2023217237A1 PCT/CN2023/093558 CN2023093558W WO2023217237A1 WO 2023217237 A1 WO2023217237 A1 WO 2023217237A1 CN 2023093558 W CN2023093558 W CN 2023093558W WO 2023217237 A1 WO2023217237 A1 WO 2023217237A1
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Prior art keywords
acid
lipid
compound
dcm
mmol
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PCT/CN2023/093558
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French (fr)
Chinese (zh)
Inventor
陈重
钟天翼
黄从报
胡洪鹏
郏侃
翁天伟
路青青
葛友祯
李晓明
耿亦程
张丽霞
蒋剑豪
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苏州慧疗生物医药科技有限公司
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Publication of WO2023217237A1 publication Critical patent/WO2023217237A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

Definitions

  • the present invention specifically relates to a type of lipid compound and its composition, preparation and use, especially its application in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
  • Nucleic acid drugs refer to artificially designed DNA or RNA with disease prevention or treatment functions. They act on disease-causing target genes or target mRNA to fundamentally regulate the expression of disease-causing genes to achieve the purpose of disease prevention or treatment. Nucleic acid drugs mainly include antisense oligonucleotide (ASO), small interference RNA (siRNA), microRNA (miRNA), messenger RNA (message, mRNA), etc. As of the end of 2021, the FDA has approved more than 10 nucleic acid drugs, and multiple drug candidates are in clinical trials or preclinical trials.
  • ASO antisense oligonucleotide
  • siRNA small interference RNA
  • miRNA microRNA
  • messenger RNA messenger RNA
  • RNA vaccines BNT162b2 Pfizer/BioNTech
  • mRNA-1273 Moderna
  • my country also has multiple new coronavirus vaccines based on mRNA technology in clinical trials or preclinical trials.
  • mRNA technology has proven its unique advantages over traditional biopharmaceutical and vaccine technologies.
  • Therapeutic nucleic acids have the potential to revolutionize vaccinations, gene therapies, protein replacement therapies and the treatment of other genetic diseases.
  • RNA degradation determines the need for a high-quality delivery system to deliver it into the body.
  • the present invention focuses on providing a new delivery carrier for RNA drugs.
  • the present invention provides a new class of lipid compounds for delivering therapeutic or preventive drugs, namely lipids based on cholic acid or its derivatives, which enriches the types of lipid compounds and can be used for nucleic acid drugs, gene vaccines, polypeptides, and proteins.
  • lipids based on cholic acid or its derivatives which enriches the types of lipid compounds and can be used for nucleic acid drugs, gene vaccines, polypeptides, and proteins.
  • the delivery of antibodies, small molecule drugs, etc. provides more options.
  • the present invention provides a lipid compound represented by the following general formula 1, general formula 2, or a pharmaceutically acceptable salt thereof, including stereoisomers, tautomers, solvates, chelates, and non-covalent compounds.
  • a pharmaceutically acceptable salt thereof refers to an acid addition salt or a base addition salt.
  • the acid described in the present invention includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzene
  • Base addition salts refer to salts prepared by adding an inorganic base or an organic base to a free base compound.
  • Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, etc.; the organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, etc.
  • the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • the core of the lipid compound is cholic acid or a derivative thereof; wherein the linker includes one or more of an ester group, an amide group, a carbamate group, a carbonate group or a urea group. kind.
  • the lipid compound, wherein cholic acid or its derivatives is optionally selected from cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3 ⁇ -hydroxy-D5-cholenoic acid, Chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholic acid, 5 ⁇ -cholic acid, dehydrocholic acid, hyocholic acid, cholic acid, glycinechenodeoxycholic acid, tauroursodeoxycholic acid Acid, taurochenodeoxycholic acid, glycocholic acid, hyodeoxycholic acid, hyodeoxycholic acid methyl ester, taurohodeoxycholic acid sodium, sodium dehydrocholate, sodium cholate, sodium deoxyglycholate , sodium taurodeoxycholate, sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate diso
  • the lipid compound is one or more lipid compounds selected from ursodeoxycholic acid derivatives, or one or more obeticholic acid derivative lipid compounds. kind.
  • the lipid compound is also selected from one or more cholic acid lipid compounds, or one or more hyodeoxycholic acid derivative lipid compounds, or chenodeoxycholic acid derivative lipids.
  • One or more chemical compounds are also selected from one or more cholic acid lipid compounds, or one or more hyodeoxycholic acid derivative lipid compounds, or chenodeoxycholic acid derivative lipids.
  • One or more chemical compounds are also selected from one or more cholic acid lipid compounds, or one or more hyodeoxycholic acid derivative lipid compounds, or chenodeoxycholic acid derivative lipids.
  • One or more chemical compounds are also selected from one or more cholic acid lipid compounds, or one or more hyodeoxycholic acid derivative lipid compounds, or chenodeoxycholic acid derivative lipids.
  • One or more chemical compounds are also selected from one or more cholic acid lipid compounds, or one or more hyodeoxycholic acid derivative lipid compounds, or
  • the lipid compound may be an ionizable lipid, or a cationic lipid, or an anionic lipid, or a neutral lipid, or a polyethylene glycol derivative lipid, or a polysarcosine derivative lipid. substance, or chitosan derivative lipid, or hyaluronic acid derivative lipid.
  • the lipid compound can also be a conjugate of a lipid compound, and the conjugate is mainly composed of three parts: a therapeutic or preventive drug, a linker, and a lipid compound.
  • the therapeutic or preventive drugs include one or more of nucleic acids, polypeptides, proteins, antibodies, carbohydrates, polyethylene glycol and its derivatives, and small molecules.
  • the nucleic acid includes any form of nucleic acid molecule.
  • DNA can be non-coding DNA or coding DNA
  • RNA can be selected from one or more of ASO, mRNA, siRNA, micRNA, etc.
  • linker is independently selected from the group consisting of non-existent, linear compounds or containing one or more cyclic compounds, which are connected to both ends through ester groups, amide groups, carbamate groups, Carbonate group, ether group or urea group, etc. are connected.
  • the linker is independently selected from linear saturated or unsaturated hydrocarbons with 2-25 C, and its two ends are one of carboxyl group, hydroxyl group, amino group, sulfate group, sulfonic acid group, and phosphate group, or Various.
  • the linker is independently selected from saturated or unsaturated hydrocarbons containing 3-8 membered rings, and its two ends are one or more of carboxyl groups, hydroxyl groups, amino groups, sulfate groups, sulfonic acid groups, and phosphate groups. kind.
  • the present invention also provides a lipid compound that is a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable salt of a lipid based on cholic acid or a derivative thereof, Prodrug or stereoisomer, the lipid compound has the structure of the following general formula 1 or general formula 2:
  • the core of the lipid compound is cholic acid or a derivative thereof; wherein the linker includes one or more of an ester group, an amide group, a carbamate group, a carbonate group or a urea group. kind.
  • the cholic acid or its derivative is optionally selected from the group consisting of cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3 ⁇ -hydroxy-D5-cholic acid, chenodeoxycholic acid, lithophore Cholic acid, deoxycholic acid, taurocholic acid, 5 ⁇ -cholic acid, dehydrocholic acid, hyocholic acid, cholancholic acid, glycochenodeoxycholic acid, tauroursodeoxycholic acid, taurochenodeoxycholic acid Cholic acid, glycocholic acid, hyodeoxycholic acid, methyl hyodeoxycholate, sodium taurodeoxycholate, sodium dehydrocholate, sodium cholate, sodium deoxyglycholate, sodium taurodeoxycholate , sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate disodium salt, sodium
  • the lipid compound is one or more lipid compounds selected from ursodeoxycholic acid derivatives. species, or one or more obeticholic acid derivative lipid compounds, one or more cholic acid lipid compounds, one or more hyodeoxycholic acid derivative lipid compounds, chenodeoxy Cholic acid derivatives are one or more lipid compounds.
  • the lipid compound may be an ionizable lipid, or a cationic lipid, or an anionic lipid, or a neutral lipid, or a polyethylene glycol derivative lipid, or a polysarcosine derivative lipid. substance, or chitosan derivative lipid, or hyaluronic acid derivative lipid.
  • the lipid compound can also be a conjugate of a lipid compound, and the conjugate is mainly composed of three parts: a therapeutic or preventive drug, a linker, and a lipid compound.
  • the therapeutic or preventive drugs include one or more of nucleic acids, antibodies, polypeptides, proteins, carbohydrates, polyethylene glycol and its derivatives, and small molecules.
  • the nucleic acid includes any form of nucleic acid molecule.
  • DNA can be non-coding DNA or coding DNA
  • RNA can be selected from one or more of ASO, mRNA, siRNA, micRNA, etc.
  • linker is independently selected from the group consisting of non-existent, linear compounds or containing one or more cyclic compounds, which are connected to both ends through ester groups, amide groups, carbamate groups, Carbonate group, ether group or urea group, etc. are connected.
  • the linker is independently selected from linear saturated or unsaturated hydrocarbons with 2-25 C, and its two ends are one of carboxyl group, hydroxyl group, amino group, sulfate group, sulfonic acid group, and phosphate group, or Various.
  • the linker is independently selected from saturated or unsaturated hydrocarbons containing 3-8 membered rings, and its two ends are one or more of carboxyl groups, hydroxyl groups, amino groups, sulfate groups, sulfonic acid groups, and phosphate groups. kind.
  • a composition of lipid compounds includes a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier includes one or more of the aforementioned lipid compounds.
  • the composition, therapeutic or preventive agent includes one or more of nucleic acid molecules, polypeptides, proteins, antibodies and small molecule drugs.
  • the mass ratio of the carrier to the therapeutic or preventive agent is 1:1 to 100:1.
  • the composition is a nanoparticle preparation
  • the average size of the nanoparticle preparation is 20 nm to 1000 nm
  • the polydispersity coefficient of the nanoparticle preparation is ⁇ 0.5.
  • the composition contains three different lipid components in the carrier, one of which Lipids are lipids based on cholic acid or its derivatives.
  • the composition is characterized in that the carrier further includes a charge-assisted lipid with neutral charge, negative charge or bipolar charge.
  • the composition is characterized in that the charge-auxiliary lipid is one or more of the following: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) , dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE- mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphati
  • the carrier further includes a structurally modified lipid.
  • the structurally modified lipid includes polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, One or more dialkylglycerols.
  • the composition and the carrier also include but are not limited to lipids of cholic acid or its derivatives, charge-assisted lipids, and structurally modified lipids.
  • the cholic acid lipids, the charge-assisted lipids And the molar ratio of the structurally modified lipid is (30-80): (5-50): (0.5-10).
  • the composition further includes one or more pharmaceutically acceptable excipients or diluents.
  • the carrier also includes lipids of cholic acid or its derivatives, charge-assisted lipids, cholesterol or derivatives thereof, and structurally modified lipids.
  • the cholic acid lipid, the charge-assisted lipid, the The molar ratio of cholesterol or its derivatives and the structurally modified lipid is (30-80): (0.5-10): (5-50): (0.5-2.5).
  • the lipid compound or composition of the present invention can be used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
  • the lipid compound or composition of the present invention is used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, wherein the lipid nanoparticles have a concentration of 20 to 1000 nm particle size.
  • compositions for preparing nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs which include nucleic acids and lipid nanoparticles encapsulating the nucleic acids, wherein each individual lipid nanoparticle contains a variety of lipids A lipid component, wherein one of the lipid components is a cholic acid-based lipid compound, including compounds thereof or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non- A covalent compound or prodrug, and wherein the lipid nanoparticle has a nucleic acid encapsulation ratio of at least 70%.
  • the lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution of any lipid compound described in this patent, wherein the medium of the mRNA solution is HEPES (hydroxyethyl ethyl alcohol). piperazine ethyl sulfate buffer), sodium phosphate, sodium acetate, ammonium sulfate, sodium bicarbonate or sodium citrate; the medium of the lipid solution is ethanol, isopropanol or dimethyl sulfoxide; wherein the lipid nanoparticles The particles are further purified by dialysis or ultrafiltration.
  • HEPES hydroxyethyl ethyl alcohol
  • piperazine ethyl sulfate buffer sodium phosphate, sodium acetate, ammonium sulfate, sodium bicarbonate or sodium citrate
  • the medium of the lipid solution is ethanol, isopropanol or dimethyl sulfoxide
  • the lipid nanoparticles The particles are further purified by
  • composition described in this patent also contains one or more of buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids, antioxidants, bacteriostatic agents, chelating agents, and adjuvants.
  • the acid described in the present invention includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzene
  • Base addition salts refer to salts prepared by adding an inorganic base or an organic base to a free base compound.
  • Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, etc.
  • the organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, Diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, Procaine, hydrazine, choline, betaine, benethamine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine pyridine, piperazine, piperidine, N-ethylpiperidine, and polyamine resin.
  • the present invention provides a composition comprising a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable agent thereof.
  • a composition comprising a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable agent thereof.
  • the carrier comprising a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable agent thereof.
  • the therapeutic or preventive agent is encapsulated in or associated with a carrier.
  • the therapeutic or preventive agent includes one or more of nucleic acid molecules, genetic vaccines, polypeptides, proteins, antibodies and small molecule drugs.
  • the nucleic acid includes any form of nucleic acid molecule, including but not limited to single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA ( aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and other forms of RNA molecules known in the art, or locked nucleic acids Nucleic acid mimics such as (LNA), peptide nucleic acid (PNA) and morpholino cyclic oligonucleotides.
  • LNA small interfering RNA
  • aiRNA asymmetric interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • tRNA transfer RNA
  • mRNA messenger RNA
  • the therapeutic or preventive agent comprises at least one mRNA encoding an antigen or a protein or a peptide or a fragment or epitope thereof.
  • the mRNA is monocistronic or polycistronic.
  • the antigen is a pathogenic antigen.
  • the mRNA contains one or more functional nucleotide analogs, including but not limited to pseudouridine, 1-methyl-pseudouridine and one or more of 5-methylcytosine.
  • the small molecule compounds include, but are not limited to, the active ingredients of therapeutic and/or preventive agents.
  • the therapeutic and/or preventive agents are currently known drugs, such as anti-tumor drugs, anti-infective drugs, and local anesthetics. , antidepressants, anticonvulsants, antibiotics/antimicrobials, antifungals, antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, anesthetics, or contrast media.
  • the lipids comprise three different lipid components, one of which is a cholic acid or derivative thereof based lipid.
  • the lipid further includes an auxiliary lipid with a neutral charge, a negative charge, or a bipolar charge.
  • the lipid includes one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and derivatives thereof.
  • the lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine base (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 11,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-(((2,3- Bis(oleoyloxy)propyl)dimethylammonium phosphate)ethylhydrogen (DOCP), sphingomyelin (SM), ceramide and its derivatives.
  • the lipids may be synthetic or derived from (isolated or modified) natural sources or compounds.
  • the carrier further includes a structurally modified lipid.
  • the structurally modified lipids mainly include disclosed or undisclosed lipid compounds, which can improve the stability of liposomes and reduce protein absorption of liposomes, such as polyethylene glycol, dextran, polyethylene glycol, etc.
  • lipid compounds which can improve the stability of liposomes and reduce protein absorption of liposomes, such as polyethylene glycol, dextran, polyethylene glycol, etc.
  • the structurally modified lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, Chol-PEG2000, 1-(monomethyl Oxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), 4-O-(2',3'-bis( Tetradecanoyloxy)propyl-1-O-( ⁇ -methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), polyglycolated ceramide (PEG- cer), ⁇ -methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate, or 2,3-di(tetradecyloxy) methyl)propyl-
  • the mass ratio of the carrier to the therapeutic or preventive agent is 5:1 to 50:1, more preferably 5:1 to 35:1, and more preferably 10:1 to 30:1.
  • composition according to the preceding claims wherein the carrier further includes, but is not limited to, lipids of cholic acid or its derivatives, charge-assisted lipids and structurally modified lipids.
  • the molar ratio of the cholic acid lipid, the charge-assisted lipid, and the structurally modified lipid is (30-80): (5-50): (0.5-10).
  • lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution. In some embodiments, lipid nanoparticles are further purified by tangential flow filtration.
  • lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution.
  • the medium of the mRNA solution is HEPES, phosphate, acetate, ammonium sulfate, sodium bicarbonate or citrate.
  • the medium of lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide.
  • the pharmaceutical composition is a nanoparticle preparation, and the average size of the nanoparticle preparation is 20 nm to 1000 nm, preferably 40 nm to 150 nm, further preferably 50 nm to 100 nm, and more preferably 70 nm to 100 nm.
  • the polydispersity index of the nanoparticle preparation is ⁇ 0.5, further preferably ⁇ 0.3, and more preferably ⁇ 0.25.
  • compositions of the present invention also typically include one or more buffers (eg neutral buffered saline or phosphate buffered water), carbohydrates (eg glucose, mannitol, sucrose, trehalose, dextrose or dextran). sugar), mannitol, proteins, peptides or amino acids (such as glycine and lysine), antioxidants (vitamin E and butylated hydroxytoluene), bacteriostatic agents, chelating agents (such as EDTA and glutathione), adjuvants (e.g. aluminum hydroxide), suspending agents/thickening agents/preservatives, etc. that make the formulation isotonic with the recipient's blood, or the composition of the invention can be formulated as a lyophilisate.
  • buffers eg neutral buffered saline or phosphate buffered water
  • carbohydrates eg glucose, mannitol, sucrose, trehalose, dextrose or dextran. sugar
  • the administration methods of the composition include but are not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, ocular administration, ear administration, nasal administration, and oral administration. medicine, transanal administration, transvaginal administration, etc.
  • the administration objects of the composition include but are not limited to mammals such as cattle, horses, mules, donkeys, camels, pigs, sheep, dogs, foxes, rabbits, etc., and poultry such as chickens, ducks, geese, pigeons, etc., Fish, non-human primates, humans.
  • the technical solution of the present invention provides a new class of lipid compounds for delivering therapeutic or preventive drugs, namely lipids based on cholic acid or its derivatives.
  • the technical solution of the present invention is different from the existing patented technical solutions of foreign pharmaceutical companies. , enriches the types of lipid compounds, provides more options for the delivery of nucleic acid drugs, gene vaccines, peptides, proteins, antibodies and small molecule drugs, and can be significantly different from the technical routes of foreign companies such as Pfizer and Moderna. .
  • the pharmaceutical composition of the present invention relates to the field of lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • the preparation method of the nanoparticles is simple, has good repeatability, simplifies the production process and reduces costs; at the same time, it can avoid the patent blockade of the four-component LNP and is conducive to the promotion of nucleic acids. Domestic production of drugs.
  • the prepared lipid nanoparticles encapsulating siRNA have better cell transfection effect; whether the nanoparticles are injected into small cells through intravenous injection Whether in mice or via intramuscular injection, LNP has a good in vivo delivery effect.
  • the nanoparticles encapsulating the mRNA were injected into the tumor, and pictures were taken 6 hours after the injection. There was fluorescence expression in the tumor of the mice, indicating that the lipid nanoparticles can be administered by intratumoral injection. . Based on the in vivo and in vitro evaluation results, the nanoparticles have good delivery effects and application scenarios.
  • Figure 1 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 1 (compound 1).
  • Figure 2 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 2 (compound 2).
  • Figure 3 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 7 (compound 7).
  • Figure 4 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 9 (compound 9).
  • Figure 5 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 10 (compound 10).
  • Figure 6 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 13 (compound 13).
  • Figure 7 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 16 (compound 16).
  • Figure 8 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 17 (compound 17).
  • Figure 9 is a proton nuclear magnetic resonance spectrum of obeticholic acid derivative 18 (compound 18).
  • Figure 10 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 42 (compound 42).
  • Figure 11 is a proton nuclear magnetic resonance spectrum of lithocholic acid derivative 58 (compound 58).
  • Figure 12 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 62 (compound 62).
  • Figure 13 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 63 (compound 63).
  • Figure 14 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 66 (compound 66).
  • Figure 15 is a hydrogen nuclear magnetic resonance spectrum of chenodeoxycholic acid derivative 69 (compound 69).
  • Figure 16 is a hydrogen nuclear magnetic resonance spectrum of hyodeoxycholic acid derivative 72 (compound 72).
  • Figure 17 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 75 (compound 75).
  • Figure 18 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 85 (compound 85).
  • Figure 19 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 87 (compound 87).
  • Figure 20 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 89 (compound 89).
  • Figure 21 shows the experimental results of cells transfected with LNP-encapsulated cy3-siRNA, including a) bright field of fluorescence microscope, b) dark field of fluorescence microscope, c) cell flow cytometry.
  • Figure 22 shows the experimental results of cells transfected with LNP-encapsulated EGFP mRNA, including a) bright field of fluorescence microscope, b) dark field of fluorescence microscope, c) cell flow cytometry.
  • Figure 23 shows the fluorescence imaging results of LNP encapsulating Luciferase mRNA 12 hours after intravenous injection in mice.
  • Figure 24 shows the fluorescence imaging results of LNP encapsulating Luciferase mRNA 12 hours after intramuscular injection in mice.
  • Figure 25 is a diagram of the killing effect of tumor cells A549.
  • Figure 26 shows the results of fluorescence imaging after intramuscular injection in mice.
  • Figure 27 shows the results of fluorescence imaging after intravenous injection into mice.
  • Figure 28 shows the results of fluorescence imaging after intratumoral injection in mice.
  • Cholic acid is a naturally ubiquitous steroid molecule in humans and mammals, synthesized in the liver from cholesterol. After eating, bile acid is actively secreted by liver cells, enters the gallbladder with bile, and then enters the intestine from the gallbladder to perform its digestive function. Cholic acid enters the small intestine in the form of sodium salt to help digest and absorb lipids, and then passes through the terminal ileum. It is returned to the liver through the portal vein through active absorption or passive transport, processed and transformed in liver cells, and then secreted into the small intestine together with newly synthesized bile acids.
  • This EHC (enterohepatic circulation) process of bile acids circulates 4 to 12 times a day, and about 95% of the bile acids are reabsorbed and utilized. If the EHC of bile acids is destroyed, it will not only affect the digestion and absorption of lipids in the body, but also cause the body to form cholesterol stones. Therefore, the biggest advantage of compound delivery carriers designed based on bile acid is that it has high enterohepatic circulation efficiency and participates in the enterohepatic circulation of bile acid, thereby improving drug absorption in the liver and gallbladder.
  • the structural formula of cholic acid is as follows.
  • the three six-membered rings and one five-membered ring on the steroid skeleton of the cholic acid molecule are on the same plane, and ring A and ring B are connected in reverse, making the molecule form a cave-like structure.
  • three methyl groups are distributed on one side of the plane where the steroid ring is located, forming the hydrophobic part of the molecule.
  • Three hydroxyl groups are distributed on the other side of the plane where the steroid ring is located, and together with the C24 carboxyl group, form the hydrophilic part of the molecule.
  • cholic acid The special structure of the cholic acid molecule determines that it is amphiphilic, acid-base, and easy to undergo chemical modification. Therefore, this patent uses cholic acid as a building block to prepare polymers or oligomers. These cholic acid-based products Functional molecules have good technical effects in drug delivery.
  • Cholic acid drugs have been on the market in China or the United States for many years, including ursodeoxycholic acid, obeticholic acid, chenodeoxycholic acid, and tauroursodeoxycholic acid.
  • ursodeoxycholic acid is used to treat cholesterol gallstones and bile reflux gastritis
  • obeticholic acid is used to treat primary biliary cirrhosis (PBC), and there is no adequate response to ursodeoxycholic acid or Patients who cannot tolerate it.
  • PBC primary biliary cirrhosis
  • Years of clinical application have shown that cholic acid compounds have good safety.
  • cholic acid compounds have hydrophilic and lipophilic amphiphilic properties, and can be encapsulated or The conjugation method can improve the bioavailability of small molecule chemical drugs.
  • Cholic acid compounds and cholesterol are both steroidal compounds with similar structures and more sites that can be chemically modified. Structural modification of cholic acid compounds to prepare new lipid components may potentially replace the ionizable lipids and cholesterol in the original four-component LNP to achieve similar or better mRNA delivery effects.
  • This patented study constructs lipid nanoparticle carriers based on bile acid analogs and explores their application in mRNA drug delivery.
  • the LNP of the present invention can simplify the production process and reduce costs; at the same time, it can avoid the patent blockade of four-component LNP, which is beneficial to promoting the localization of nucleic acid drugs.
  • cholic acid or its derivatives are composed of a rigid steroid ring and an aliphatic side chain
  • the steroid ring includes three six-membered rings and one five-membered ring.
  • the side chain structure of cholic acid, the conformation of the steroid ring, the number of hydroxyl groups and the orientation of the steroid ring will be different.
  • the common hydroxyl group of cholic acid or its derivatives and the carboxyl group of the fatty side chain are good chemical modification sites. Therefore, we believe that cholic acid or its derivatives can achieve the desired purpose through some common modifications.
  • Cholic acid or its derivatives in this patent is optionally selected from the group consisting of cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3 ⁇ -hydroxy-D5-cholic acid, chenodeoxycholic acid, and stone.
  • the lipid compound described in this patent is one or more compounds selected from the following structures;
  • Ursodeoxycholic acid derivative lipid 1 Ursodeoxycholic acid derivative lipid:
  • Ursodeoxycholic acid (393 mg, 1.0 mmol) was dissolved in DMF (8 mL), and HBTU (569 mg, 1.5 mmol), DIEA (194 mg, 1.5 mmol), and 4-pyrrole-1-butylamine (213 mg, 1.5 mmol) were added in sequence. mmol), under nitrogen protection, and stirred at room temperature overnight. TLC detection, after the reaction is completed, add Add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product.
  • Ursodeoxycholic acid (393 mg, 1.0 mmol) was dissolved in acetonitrile (10 mL), K 2 CO 3 (415 mg, 3.0 mmol), BnBr (850 mg, 5.0 mmol) were added in sequence, protected by nitrogen, and the reaction was stirred at 80°C for 5 h. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with PE/EA (1:1), gave compound 6 (390 mg, 81%) as a white solid product.
  • Ursodeoxycholic acid (785mg, 1.0mmol) was dissolved in DMF (20mL), K 2 CO 3 (829mg, 6.0mmol), CH 3 I (852mg, 6.0mmol) were added in sequence, under nitrogen protection, and the reaction was stirred at room temperature overnight. .
  • TLC detection after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product.
  • Silica gel column chromatography eluting with PE/EA (1:1), gave compound 8 (700 mg, 86%) as a white solid product.
  • Linoleic acid (476 mg, 1.7 mmol) was dissolved in anhydrous DCM (5 mL), oxalyl chloride (0.30 mL) was added, and the mixture was stirred at room temperature for 5 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (2mL), and set aside. Compound 2 (240 mg, 0.34 mmol) and TEA (69 mg, 0.68 mmol) were dissolved in anhydrous DCM (5 mL). The above standby product was dropped into the reaction solution and allowed to react at room temperature overnight. Add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude product.
  • Example 10 The synthetic route of ursodeoxycholic acid derivative (compound 19) is as follows
  • Example 11 The synthetic route of ursodeoxycholic acid derivative (compound 20) is as follows
  • Example 12 The synthetic route of ursodeoxycholic acid derivative (compound 21) is as follows
  • Example 13 The synthetic route of ursodeoxycholic acid derivative (compound 22) is as follows
  • Example 14 The synthetic route of ursodeoxycholic acid derivative (compound 23) is as follows
  • Example 15 The synthetic route of ursodeoxycholic acid derivative (compound 24) is as follows
  • Example 16 The synthetic route of ursodeoxycholic acid derivative (compound 25) is as follows
  • Example 17 The synthetic route of ursodeoxycholic acid derivative (compound 26) is as follows
  • Example 18 The synthetic route of ursodeoxycholic acid derivative (compound 27) is as follows
  • Example 19 The synthetic route of ursodeoxycholic acid derivative (compound 28) is as follows
  • Example 20 The synthetic route of ursodeoxycholic acid derivative (compound 29) is as follows
  • Example 21 The synthetic route of ursodeoxycholic acid derivative (compound 30) is as follows
  • Example 22 The synthesis route of ursodeoxycholic acid derivative (compound 31) is as follows
  • Example 23 The synthesis route of lithocholic acid derivative (compound 32) is as follows
  • Example 24 The synthesis route of ursodeoxycholic acid derivative (compound 33) is as follows
  • Example 25 The synthetic route of cholesterol derivative (compound 34) is as follows
  • Example 26 The synthetic route of ursodeoxycholic acid derivative (compound 35) is as follows
  • Example 27 The synthetic route of cholic acid derivative (compound 36) is as follows
  • Example 28 The synthetic route of ursodeoxycholic acid derivative (compound 37) is as follows
  • Example 29 The synthetic route of ursodeoxycholic acid derivative (compound 38) is as follows
  • Example 30 The synthetic route of ursodeoxycholic acid derivative (compound 39) is as follows
  • Example 31 The synthetic route of ursodeoxycholic acid derivative (compound 40) is as follows
  • intermediate 40-2 Combine intermediate 40-1 (1.60g, 8.37mmol), stearic acid (5.00g, 17.57mmol), EDCI (10.00g, 52.16mmol), DMAP (0.50g, 4.09mmol) Dissolve in DCM and stir at room temperature for 16h. After TLC detection, after the reaction was completed, the reaction mixture was concentrated to obtain white viscous liquid intermediate 40-2 (5.31 g, 87%). Crude products are directly transferred to the next step.
  • intermediate 40-4 Dissolve lithocholic acid (500mg, 1.33mmol), intermediate 40-3 (1.17g, 1.59mmol), HATU (0.76g, 2.00mmol), and DIPEA (0.74mL, 4.00mmol) Stir in DMF at room temperature for 20h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (50:1), gave off-white solid intermediate 40-4 (750 mg, 57.4%).
  • Example 32 The synthesis route of ursodeoxycholic acid derivative (compound 41) is as follows
  • Example 33 The synthetic route of ursodeoxycholic acid derivative (compound 42) is as follows
  • Example 34 The synthesis route of ursodeoxycholic acid derivative (compound 43) is as follows
  • Example 35 The synthetic route of ursodeoxycholic acid derivative (compound 44) is as follows
  • Example 36 The synthetic route of ursodeoxycholic acid derivative (compound 45) is as follows
  • Example 37 The synthetic route of ursodeoxycholic acid derivative (compound 46) is as follows
  • Example 38 The synthetic route of ursodeoxycholic acid derivative (compound 47) is as follows
  • Example 39 The synthetic route of ursodeoxycholic acid derivative (compound 48) is as follows
  • Example 40 The synthetic route of ursodeoxycholic acid derivative (compound 49) is as follows
  • Example 41 The synthetic route of ursodeoxycholic acid derivative (compound 50) is as follows
  • Example 42 The synthetic route of obeticholic acid derivative (compound 51) is as follows
  • Example 43 The synthesis route of chenodeoxycholic acid derivative (compound 52) is as follows
  • Example 44 The synthetic route of ursodeoxycholic acid derivative (compound 53) is as follows
  • Example 45 The synthetic route of obeticholic acid derivative (compound 54) is as follows
  • Example 46 The synthetic route of obeticholic acid derivative (compound 55) is as follows
  • Example 47 The synthetic route of obeticholic acid derivative (compound 56) is as follows
  • Example 48 The synthetic route of ursodeoxycholic acid derivative (compound 57) is as follows
  • Example 49 The synthesis route of lithocholic acid derivative (compound 58) is as follows
  • Example 50 The synthetic route of ursodeoxycholic acid derivative (compound 59) is as follows
  • Example 51 The synthetic route of ursodeoxycholic acid derivative (compound 60) is as follows
  • intermediate 60-2 Dissolve intermediate 59-7 (200mg, 0.22mmol), intermediate 60-1 (134mg, 0.27mmol), HATU (126mg, 0.33mmol), TEA (68mg, 0.66mmol) in In DMF, stir at room temperature for 20h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (50:1), gave light yellow oily intermediate 60-2 (140 mg, 46.6%).
  • Example 52 The synthetic route of ursodeoxycholic acid derivative (compound 61) is as follows
  • Example 53 The synthetic route of ursodeoxycholic acid derivative (compound 62) is as follows
  • Dissolve intermediate 59-7 (100mg, 0.115mmol) in ACN, add raw materials 62-1 (24mg, 0.138mmol), NMI (24mg, 0.288mmol), TCFH (39mg, 0.138mmol) in sequence, stir and react at room temperature for 16h . After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 62 (55 mg, 47%) as a light yellow oil.
  • Example 54 The synthetic route of ursodeoxycholic acid derivative (compound 63) is as follows
  • Example 55 The synthetic route of ursodeoxycholic acid derivative (compound 64) is as follows
  • 59-7 is a carboxylic acid, an intermediate in the preparation of compound 59.
  • Example 56 The synthetic route of ursodeoxycholic acid derivative (compound 65) is as follows
  • Example 57 The synthetic route of ursodeoxycholic acid derivative (compound 66) is as follows
  • Example 58 The synthetic route of ursodeoxycholic acid derivative (compound 67) is as follows
  • Example 59 The synthetic route of ursodeoxycholic acid derivative (compound 68) is as follows
  • Example 60 The synthesis route of chenodeoxycholic acid derivative (compound 69) is as follows
  • Example 61 The synthesis route of chenodeoxycholic acid derivative (compound 70) is as follows
  • Example 62 The synthesis route of chenodeoxycholic acid derivative (compound 71) is as follows
  • intermediate 71-3 Dissolve intermediate 71-1 (650 mg, 1.27 mmol) in pyridine, then add fresh acid chloride 71-2 (2.80 g, 10.18 mmol), and stir at room temperature for 3 hours. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (5:1), gave colorless oil 71-3 (495 mg, 52%).
  • intermediate 71-4 Dissolve intermediate 71-3 (395 mg, 0.527 mmol) in MeOH, add 10% Pd/C (59 mg, 15% wt), and stir for 16 hours under a hydrogen atmosphere. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluted with DCM/MeOH (30:1), gave colorless oily substance 71-4 (270 mg, 78%).
  • Example 63 The synthesis route of hyodeoxycholic acid derivative (compound 72) is as follows
  • Example 64 The synthetic route of ursodeoxycholic acid derivative (compound 73) is as follows
  • Example 65 The synthetic route of ursodeoxycholic acid derivative (compound 74) is as follows
  • Example 66 The synthetic route of ursodeoxycholic acid derivative (compound 75) is as follows
  • intermediate 75-2 Dissolve intermediate 75-1 (180 mg, 0.12 mmol), Pd/C (20 mg) in methanol, and stir at 25°C for 16 hours under a hydrogen atmosphere. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluted with DCM/MeOH (20:1), gave colorless oily intermediate 75-2 (110 mg, 67.4%).
  • Example 67 The synthetic route of obeticholic acid derivative (compound 76) is as follows
  • Example 68 The synthetic route of obeticholic acid derivative (compound 77) is as follows
  • Example 69 The synthetic route of obeticholic acid derivative (compound 78) is as follows
  • Example 70 The synthetic route of obeticholic acid derivative (compound 79) is as follows
  • Example 71 The synthetic route of ursodeoxycholic acid derivative (compound 80) is as follows
  • Ursodeoxycholic acid (4.0g, 10.2mmol), amine (597mg, 5.1mmol), HBTU (4.63g, 12.21mmol), DIEA (2.06g, 15.9mmol) and THF were added to the reaction bottle in sequence. Stir at room temperature overnight. TLC showed that the starting material reacted completely. The reaction solution was concentrated and subjected to silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain compound 80 (1.8 g, 40%) as a white solid.
  • Example 72 The synthetic route of ursodeoxycholic acid derivative (compound 81) is as follows
  • Example 73 The synthetic route of ursodeoxycholic acid derivative (compound 82) is as follows
  • Example 74 The synthetic route of ursodeoxycholic acid derivative (compound 83) is as follows
  • Example 75 The synthetic route of ursodeoxycholic acid derivative (compound 84) is as follows
  • Example 76 The synthetic route of ursodeoxycholic acid derivative (compound 85) is as follows
  • Example 77 The synthetic route of ursodeoxycholic acid derivative (compound 86) is as follows
  • intermediate 86-3 Dissolve intermediate 86-2 (1.40g, 1.44mmol) in DCM, add TFA (1mL), and stir at room temperature for 20h. TLC detection, after the reaction is completed, the solvent is directly evaporated from the reaction solution to obtain the crude product. Silica gel column chromatography, eluting with DCM, gave colorless oily intermediate 86-3 (0.98g, 76.6%).
  • Example 78 The synthetic route of ursodeoxycholic acid derivative (compound 87) is as follows
  • Example 79 The synthetic route of ursodeoxycholic acid derivative (compound 88) is as follows
  • Example 80 The synthesis route of the conjugate of ursodeoxycholic acid and paclitaxel (compound 89) is as follows
  • Example 081 The synthesis route of the conjugate of ursodeoxycholic acid and small nucleic acid (compound 90) is as follows
  • Example 082 The synthesis route of the conjugate of ursodeoxycholic acid and polypeptide (compound 91) is as follows
  • Compound 91 is a solid-phase synthetic peptide with 30 amino acids, and the synthesis process adopts the Fmoc (9-fluorenylmethoxycarbonyl) solid-phase synthesis method. Connect the first alanine (Ala) at the C-terminus to the Wang resin, and then synthesize it sequentially from the C-terminus to the N-terminus according to the peptide sequence until the last Ursodeoxycholic Acid (Ursodeoxycholic Acid) is completed to obtain the compound 91 peptide resin. The compound 91 peptide resin was cleaved, precipitated with anhydrous ether, and washed to obtain the crude peptide of compound 91. The crude peptide of compound 91 is coarsely filtered, purified, salted and freeze-dried to obtain the finished product. The specific steps are as follows:
  • nanoparticle compositions for delivering therapeutic and/or prophylactic agents to cells
  • a series of formulations were prepared and tested. Specifically, specific ingredients and their ratios in the lipid component of the nanoparticle composition are optimized.
  • Nanoparticles can be produced by mixing two fluid streams, one of which contains therapeutic and/or prophylactic agents and the other of which has a lipid component, by mixing methods such as microfluidization and T-junctions.
  • phospholipids such as DOPE or DSPC, available from Avanti Polar Lipids (Alabaster, AL)
  • PEG lipids such as 1,2-dimyristoyl -sn-Glycerylmethoxypolyethylene glycol, also known as PEG-DMG, available from Avanti Polar Lipids (Alabaster, AL)
  • structural lipids such as cholesterol, available from Sigma-Aldrich (Tauf Wegn, Germany)
  • corticosteroids such as prednisolone, dexamethasone, prednisolone and hydrocortisone
  • a combination thereof to prepare a lipid composition at a concentration of about 50mM.
  • corticosteroids such as prednisolone, dexamethasone, pred
  • a formulation containing a therapeutic agent is prepared by combining a lipid solution with a therapeutic agent and/or prophylactic agent in a lipid component to therapeutic agent and/or prophylactic agent wt:wt ratio of between about 5:1 and about 50:1.
  • Nanoparticle compositions of agents and/or prophylactic agents and lipid components are rapidly injected into the therapeutic agent and/or preventive agent solution at a flow rate between about 10 mL/min and about 18 mL/min to create a dispersion. , wherein the water to ethanol ratio is between about 2:1 and about 5:1.
  • Zetasizer NanoZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, polydispersity index (PDI) and ⁇ potential of the nanoparticle composition.
  • the particle size is measured in 1 ⁇ PBS and ⁇ Potentials were measured in 15mM PBS.
  • RNA For nanoparticle compositions containing RNA, the QUANT-ITTM RNA (Invitrogen Corporation Carlsbad, CA) assay can be used to evaluate the encapsulation of RNA by the nanoparticle composition. Samples were diluted in TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 7.5) to a concentration of approximately 5 ⁇ g/mL. Transfer 50 ⁇ L of the diluted sample to a polystyrene 96-well plate and add 50 ⁇ L of TE buffer or 50 ⁇ L of 2% Triton X-100 solution to each well. Incubate the plate at 37°C for 15 minutes.
  • TE buffer solution 10mM Tris-HCl, 1mM EDTA, pH 7.5
  • a fluorescent plate reader can be used ( Nivo TM Multimode Plate Readers, PerkinElmer, GER) measure fluorescence intensity at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm.
  • the fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample and determined by dividing the fluorescence intensity of the intact sample (without the addition of Triton X-100) by the fluorescence of the disrupted sample (caused by the addition of Triton X-100). value to determine the percentage of free RNA. See Table 1 for specific data.
  • ONE-GlO+TOX Luciferase Reporter and Cell Viability Assay can be used to evaluate its transfection effect and cytotoxicity. Calculate the volume of the required nanoparticle assembly based on the RNA concentration measured in the encapsulation efficiency determination, dilute the nanoparticle assembly to 20ng/ ⁇ L, and add it according to the cell density of 2 ⁇ 10 5 cells/well in a polystyrene 96-well plate. Add 5 ⁇ L of diluent to each well for transfection. A fluorescent plate reader can be used ( Nivo TM Multimode Plate Readers, PerkinElmer, GER) were used to measure chemiluminescence intensity. See Table 2 for specific data.
  • Transfection effect can provide multiples of MC3.
  • Example 84 Preparation and detection of lipid nanoparticles (to verify the ability to deliver siRNA in vitro)
  • Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the lipid nanoparticles was measured to be 82nm, the PDI (polydispersity index) was 0.13, and the encapsulation rate It's 96.5%.
  • Example 85 Preparation and detection of lipid nanoparticles (to verify the ability to deliver mRNA in vitro)
  • the lipid nanoparticles are then filtered with a 0.22 ⁇ m sterile filter to obtain a preparation encapsulating EGFP mRNA.
  • Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit.
  • the particle size of the LNP preparation was measured to be 95nm, PDI 0.19, and the encapsulation rate was 93.1%.
  • Example 86 Preparation and detection of lipid nanoparticles (to verify the in vivo delivery effect of mRNA)
  • the weight ratio of total lipid to mRNA is Prepare liposomes at 20:1, remove ethanol using dialysis or tangential flow filtration, and replace with PBS solution.
  • the lipid nanoparticles are then filtered with a 0.22 ⁇ m sterile filter to obtain a preparation that encapsulates mRNA.
  • Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the lipid nanoparticles was measured to be 98 nm, the PDI was 0.22, and the encapsulation rate was 88.9%.
  • the synthesized lipid compound-1, DOPE and DMG-PEG2000 were dissolved in absolute ethanol at a molar ratio of 89.9:10:0.1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was dissolved in 50mM lemon respectively.
  • Dilute the ethanol lipid solution and the mRNA solution in acid salt buffer (pH 4) using a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3 to prepare the lipid with a weight ratio of total lipid to mRNA of 20:1.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 1249nm, the PDI was 0.67, and the encapsulation rate was 31%.
  • Prepare liposomes Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 467 nm, the PDI was 0.40, and the encapsulation rate was 78%.
  • Use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 20: 1.
  • Prepare liposomes Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 347nm, the PDI was 0.41, and the encapsulation rate was 81%.
  • Use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 20: 1.
  • Prepare liposomes Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 94nm, the PDI was 0.50, and the encapsulation rate was 85%.
  • Use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 20: 1.
  • Prepare liposomes Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 82nm, the PDI was 0.11, and the encapsulation rate was 96%.
  • Prepare liposomes Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 145nm, the PDI was 0.23, and the encapsulation rate was 76%.
  • Prepare liposomes Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 302nm, the PDI was 0.38, and the encapsulation rate was 73%.
  • Prepare liposomes Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 302nm, the PDI was 0.38, and the encapsulation rate was 73%.
  • the particle size (Size), polydispersity index (PDI) and electrokinetic potential (Zeta P) of the particles were determined by dynamic light scattering using the Malvern Zetasizer NanoZS (Malvern Nanoparticle Size Testing Instrument).
  • the encapsulation efficiency of lipid nanoparticles was determined using QUANT-ITTM RNA (Invitrogen Corporation Carlsbad, CA). According to the characterization method of the nanoparticle assembly in Example 083, as shown in Table 3: the size of the lipid nanoparticle preparation is mostly around 100 nm, the potential is between ⁇ 20mv, the polydispersity index varies around 0.1-0.3, and the encapsulation The rate part is around 80%-99%.
  • Example 96 In vitro cell transfection experiment of lipid nanoparticles
  • 293T cells were seeded in a 96-well cell culture plate at a density of 3X10 ⁇ 4 cells/well and grown for 24 hours to adhere.
  • Table 4 shows that the lipid nanoformulation can carry mRNA and be transfected into 293T cells and express the target fluorescent protein.
  • A, B, C, and D respectively represent the compound and the commercial MC3-LNP luminous flux after normalization.
  • the luminescence flux of 31 compounds was higher than that of the MC3-LNP group, indicating that the lipid nanoparticle preparation can effectively carry nucleic acids into cells.
  • A, B, C, and D respectively represent the normalized results after multiplying the luminous flux of the compound and MC3-LNP.
  • the magnification range is as follows:
  • Example 97 Compound 46 encapsulates paclitaxel to kill cells
  • Compound 46, DSPC, cholesterol, and PEG2000 were prepared according to the method in Example 83 to remove alcohol, and then 0.2, 1, 5, 10, and 20 mg/ml paclitaxel solutions were added to evaluate the killing effect of paclitaxel-loaded LNP on tumor cells A549, as follows: The results in Figure 25 show that paclitaxel-loaded lipid nanoparticles have a better tumor cell killing effect than free paclitaxel at high concentrations.
  • Compound 27 was used for prescription screening, and the formula was proportioned according to the prescription composition in the following table. It was prepared by the method described in Example 83. The results are shown in Table 5. The size of the lipid nanoparticle preparation was between 70nm and 170nm, and the potential was within ⁇ Between 20mv, the polydispersity index varies between 0.1-0.3, the encapsulation rate is around 80%-90%, and the luminous flux values of the cell evaluation results are mostly higher than those of the MC3-LNP group, indicating that lipid nanoparticle preparations can Change the composition of the prescription to carry nucleic acids into cells.
  • Example 99 Fluorescent protein expression experiment of lipid nanoparticle preparation in mice
  • Samples were prepared according to the preparation method of the nanoparticle assembly in Example 83, using MC3-LNP as a positive reference.
  • the preparations were prepared by intramuscular injection at a dose of 100 ⁇ L (0.05 ⁇ g/ ⁇ L), using the preparation method of the preparation in Example 83, and MC3-LNP was used in the control group for comparison of in vivo effects. 6 hours after administration, 200ul of D-luciferin potassium salt with a concentration of 15 mg/ml was injected intraperitoneally, and in vivo imaging was performed 5 minutes after the injection of D-luciferin potassium salt.
  • A, B, C, and D respectively represent the normalized results of compound and commercial MC3-LNP fluorescence expression values after fold ratio.
  • Table 6 there are compounds 27, 37, 39, 42, 43, 46, 50, 66, 69, 74, 82, 86, and 87 all reached the MC3-LNP level when administered locally, and compounds 42, 43, 50, and 69 exceeded the MC3-LNP level, indicating that lipid nanoparticle formulations can be effective Delivers mRNA and efficiently expresses the target gene protein locally.
  • Table 7 and Figure 26 are the comparison results of the abdominal expression of compounds and commercial MC3-LNP, as shown in Table 7: among them, the fluorescence level of compounds 52, 67, 75, 81, 82, and 83 in the abdomen exceeds the level of MC3-LNP. , indicating that cholic acid series lipid compounds can efficiently express the target gene protein.
  • a and B respectively represent the normalized results after multiplying the luminescence values of the compound and MC3-LNP.
  • magnification range is as follows:
  • Example 100 Fluorescent protein expression by intravenous injection of lipid nanoparticle formulations
  • Compound 52, compound 67, and compound 75 were prepared using the preparation method of the preparation in Example 83, and administered intravenously according to Example 099.
  • the control group used MC3-LNP for in vivo effect comparison.
  • compound 52, Compound 67 and compound 75 are weaker than MC3-LNP, but both have fluorescent expression.
  • Example 101 Expression of lipid nanoparticle preparation after intratumoral administration
  • Compound 27 was formulated for intratumoral administration and prepared according to the method described in Example 83.
  • the EG7-OVA model was established using C57 mice. The tumor was grown to 500mm3 before drug administration and intratumoral injection. Photographs were taken 6 hours after injection to observe the expression and distribution. As shown in Figure 28, there is fluorescence expression in the intratumoral site of mice, indicating that lipid nanoparticles can be administered via intratumoral injection.
  • Example 102 Selecting compound 42 and compound 58 for LNP formulation screening
  • Compound 42 was used for prescription screening, and the formula was proportioned according to the prescription composition in the following table. It was prepared by the method described in Example 83. The results are shown in Table 8. The size of the lipid nanoparticle preparation was between 50 nm and 200 nm, and the potential was within ⁇ 5 mv, the polydispersity index is around 0.1, the encapsulation rate is mostly above 90%, and the luminous flux values of the cell evaluation results are mostly higher than the MC3-LNP group, indicating that lipid nanoparticle preparations can change the composition of the prescription and carry nucleic acids Enter the cell.
  • Example 103 Fluorescent protein expression experiment of lipid nanoparticle preparation in mice
  • Example 102 The nanoparticle assembly obtained in Example 102 was used.
  • the preparations were prepared by intramuscular injection at a dose of 100 ⁇ L (0.05 ⁇ g/ ⁇ L), using the preparation method of the preparation in Example 83, and MC3-LNP was used in the control group for comparison of in vivo effects. 6 hours after administration, 200ul of D-luciferin potassium salt with a concentration of 15 mg/ml was injected intraperitoneally, and in vivo imaging was performed 5 minutes after the injection of D-luciferin potassium salt.
  • a and B respectively represent the normalized results of the fluorescence expression values of each group of formulas and the classic formula No. 13.
  • Table 9 there are 7 formulas that have reached the level of the classic formula when administered locally in the muscle, indicating that lipid Plasma nanoparticle preparations can effectively deliver mRNA through formula changes and efficiently express the target gene protein locally in the muscle.
  • the fluorescence expression levels of formulas 1, 8, and 9 in the spleen far exceeded the level of classic formulas, indicating that bile acid series lipid compounds can efficiently express gene proteins in the spleen by changing the proportion of the formula.
  • Formula 3 is a three-component formula without DSPC. The results show that the local fluorescence expression value in the muscle is higher than the classic formula, indicating that the three-component formula can form nanoparticles and effectively express fluorescent proteins in the body.
  • a and B respectively represent the luminescence values of each group of formulas and the classic formula No. 13 after being doubled.
  • the result of normalization, the magnification range is as follows:

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Abstract

Disclosed are a lipid compound having a general formula as shown, and a composition, preparation and use thereof. The lipid compound is a compound based on cholic acid or a derivative thereof, or a pharmaceutically acceptable salt thereof. The lipid compound and the composition thereof can provide more carrier options for the delivery of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies, small molecule drugs etc.

Description

脂质化合物及其组合物、制备和用途Lipid compounds and compositions, preparation and uses thereof 技术领域Technical field
本发明具体涉及一类脂类化合物及其组合物、制备和用途，尤其在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用。The present invention specifically relates to a type of lipid compound and its composition, preparation and use, especially its application in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
背景技术Background technique
核酸药物是指人为设计的具有疾病预防或治疗功能的DNA或RNA，其通过作用于致病靶基因或者靶mRNA，从根源上调控致病基因的表达，达到疾病预防或治疗的目的。核酸药物主要有反义核酸(antisense oligonucleotide,ASO)、小干扰RNA(small interference RNA，siRNA)、微小RNA(micro RNA,miRNA)、信使RNA(message,mRNA)等。截至2021年底，FDA已经批准了10多种核酸药物，多种候选药物处于临床试验或临床前试验阶段。Nucleic acid drugs refer to artificially designed DNA or RNA with disease prevention or treatment functions. They act on disease-causing target genes or target mRNA to fundamentally regulate the expression of disease-causing genes to achieve the purpose of disease prevention or treatment. Nucleic acid drugs mainly include antisense oligonucleotide (ASO), small interference RNA (siRNA), microRNA (miRNA), messenger RNA (message, mRNA), etc. As of the end of 2021, the FDA has approved more than 10 nucleic acid drugs, and multiple drug candidates are in clinical trials or preclinical trials.
FDA已经批准预防新冠病毒COVID-2019的RNA疫苗BNT162b2(辉瑞/BioNTech公司)和mRNA-1273(Moderna公司)上市，我国也有多个基于mRNA技术的新冠疫苗处于临床试验或临床前试验阶段。在新冠疫情战斗中，mRNA技术证明了它相比传统生物制药和疫苗技术的独特优势。治疗性核酸具有彻底改变疫苗接种、基因疗法、蛋白质替代疗法和其它遗传疾病疗法的潜力。The FDA has approved the RNA vaccines BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna) to prevent the new coronavirus COVID-2019. my country also has multiple new coronavirus vaccines based on mRNA technology in clinical trials or preclinical trials. In the fight against the COVID-19 epidemic, mRNA technology has proven its unique advantages over traditional biopharmaceutical and vaccine technologies. Therapeutic nucleic acids have the potential to revolutionize vaccinations, gene therapies, protein replacement therapies and the treatment of other genetic diseases.
RNA本身极容易降解的特性，决定了需要有一个优质的递送系统将其递送到体内。本发明侧重为RNA药物提供一种新的递送载体。The very easy degradation of RNA determines the need for a high-quality delivery system to deliver it into the body. The present invention focuses on providing a new delivery carrier for RNA drugs.
发明内容Contents of the invention
本发明提供了一类新的用于递送治疗或预防用药物的脂质化合物，即基于胆酸或其衍生物的脂质，丰富了脂质化合物种类，为核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物等的递送提供了更多选择。The present invention provides a new class of lipid compounds for delivering therapeutic or preventive drugs, namely lipids based on cholic acid or its derivatives, which enriches the types of lipid compounds and can be used for nucleic acid drugs, gene vaccines, polypeptides, and proteins. The delivery of antibodies, small molecule drugs, etc. provides more options.
本发明提供一种如下通式1，通式2所示的脂质化合物，或其药物可用的盐，包括立体异构体、互变异构体、溶剂化物、螯合物、非共价化合物或前体药物，具体地，所述“其药物可用的盐”是指酸加成盐或碱加成盐。The present invention provides a lipid compound represented by the following general formula 1, general formula 2, or a pharmaceutically acceptable salt thereof, including stereoisomers, tautomers, solvates, chelates, and non-covalent compounds. Or prodrug, specifically, the "pharmaceutically acceptable salt thereof" refers to an acid addition salt or a base addition salt.
通式1：
General formula 1:
或；通式2：
Or; general formula 2:
本发明所述酸包括但不限于盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、2,2-二氯乙酸、己二酸、海藻酸、抗坏血酸、天冬氨酸、苯磺酸、苯甲酸、4-乙酰氨基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸，柠檬酸、环酰胺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙磺酸、甲酸、富马酸、半乳糖酸、龙胆酸、葡庚酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲磺酸、、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、棕榈酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、癸二酸、硬脂酸、琥珀酸、酒石酸、对甲苯磺酸、三氟乙酸、以及十一碳烯酸。The acid described in the present invention includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzene Formic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cycloamic acid, dodecyl sulfate, ethane-1, 2-Disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, gluconic acid, gluconic acid, glucuronic acid, glutamic acid, pentanoic acid Diacid, 2-oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, , Naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, palmitic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecenoic acid.
本发明所述的碱加成盐指通过将无机碱或有机碱加成至游离碱化合物而制备的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐等；所述有机碱包括但不限于氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、二乙醇胺、乙醇胺、脱醇、2-二甲基氨基乙醇、2-二乙基氨基乙醇、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、肼苯胺、胆碱、甜菜碱、苯那敏(benethamine)、乙二胺、葡糖胺、甲基葡糖胺、可可碱、三乙醇胺、嘌呤、哌嗪、哌啶、N-乙基哌啶、以及聚胺树脂。优选地，有机碱是异丙胺、二乙胺、乙醇胺、三甲胺、二环己胺、胆碱和咖啡因。Base addition salts according to the present invention refer to salts prepared by adding an inorganic base or an organic base to a free base compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, etc.; the organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, etc. Amine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, purulin Caine, hydrazine, choline, betaine, benethamine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylamine piperidine, and polyamine resin. Preferably, the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
优选的，所述脂质化合物母核为胆酸或其衍生物；其中所述连接键(Linker)包含酯基、酰胺基、氨基甲酸酯基、碳酸酯基或脲基的一种或多种。Preferably, the core of the lipid compound is cholic acid or a derivative thereof; wherein the linker includes one or more of an ester group, an amide group, a carbamate group, a carbonate group or a urea group. kind.
优选的，所述的脂质化合物，其中胆酸或其衍生物是任选自胆酸、奥贝胆酸、熊去氧胆酸、熊果胆酸、3β-羟基-D5-胆烯酸、鹅去氧胆酸、石胆酸、脱氧胆酸、牛磺胆酸、5β-胆酸、去氢胆酸、猪胆酸、络胆酸、甘氨鹅脱氧胆酸、牛磺熊去氧胆酸、牛磺鹅去氧胆酸、甘氨胆酸、猪脱氧胆酸、猪去氧胆酸甲酯、牛磺猪去氧胆酸钠、去氢胆酸钠、胆酸钠、脱氧甘胆酸钠、牛磺脱氧胆酸钠、牛磺胆酸钠、牛磺鹅去氧胆酸钠、甘氨胆酸钠盐、牛磺胆酸-3-硫酸酯二钠盐、牛磺熊去氧胆酸钠和牛磺石胆酸钠的一种或多种。Preferably, the lipid compound, wherein cholic acid or its derivatives is optionally selected from cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3β-hydroxy-D5-cholenoic acid, Chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholic acid, 5β-cholic acid, dehydrocholic acid, hyocholic acid, cholic acid, glycinechenodeoxycholic acid, tauroursodeoxycholic acid Acid, taurochenodeoxycholic acid, glycocholic acid, hyodeoxycholic acid, hyodeoxycholic acid methyl ester, taurohodeoxycholic acid sodium, sodium dehydrocholate, sodium cholate, sodium deoxyglycholate , sodium taurodeoxycholate, sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate disodium salt, tauroursodeoxycholic acid Sodium and one or more of sodium taurite cholate.
优选的，所述的脂质化合物，所述脂质化合物为选自熊去氧胆酸衍生物脂质化合物的一种或多种，或奥贝胆酸衍生物脂质化合物的一种或多种。 Preferably, the lipid compound is one or more lipid compounds selected from ursodeoxycholic acid derivatives, or one or more obeticholic acid derivative lipid compounds. kind.
优选的，所述脂质化合物还为选自胆酸脂质化合物的一种或多种、或猪去氧胆酸衍生物脂质化合物的一种或多种、或鹅去氧胆酸衍生物脂质化合物的一种或多种。Preferably, the lipid compound is also selected from one or more cholic acid lipid compounds, or one or more hyodeoxycholic acid derivative lipid compounds, or chenodeoxycholic acid derivative lipids. One or more chemical compounds.
具体地，所述脂质化合物可以为可离子化脂质、或阳离子脂质、或阴离子脂质、或中性脂质、或聚乙二醇衍生物脂质、或聚肌氨酸衍生物脂质、或壳聚糖衍生物脂质、或透明质酸衍生物脂质。Specifically, the lipid compound may be an ionizable lipid, or a cationic lipid, or an anionic lipid, or a neutral lipid, or a polyethylene glycol derivative lipid, or a polysarcosine derivative lipid. substance, or chitosan derivative lipid, or hyaluronic acid derivative lipid.
具体地，所述脂质化合物还可以为一种脂质化合物的偶联物，所述偶联物主要由治疗或预防性药物、连接子(linker)与脂质化合物三个部分构成。所述治疗或预防性药物包括核酸、多肽、蛋白、抗体、糖类、聚乙二醇及其衍生物、小分子中的一种或多种。Specifically, the lipid compound can also be a conjugate of a lipid compound, and the conjugate is mainly composed of three parts: a therapeutic or preventive drug, a linker, and a lipid compound. The therapeutic or preventive drugs include one or more of nucleic acids, polypeptides, proteins, antibodies, carbohydrates, polyethylene glycol and its derivatives, and small molecules.
具体地，所述核酸包括任何形式的核酸分子，例如，DNA可以为非编码DNA或编码DNA，RNA可以选自：ASO、mRNA、siRNA、micRNA等中的一种或多种。Specifically, the nucleic acid includes any form of nucleic acid molecule. For example, DNA can be non-coding DNA or coding DNA, and RNA can be selected from one or more of ASO, mRNA, siRNA, micRNA, etc.
具体地，所述连接子(linker)独立地选自不存在，直链状化合物或者含有环状化合物其中一种或多种，其与两端通过酯基、酰胺基、氨基甲酸酯基、碳酸酯基、醚基或脲基等相连。Specifically, the linker (linker) is independently selected from the group consisting of non-existent, linear compounds or containing one or more cyclic compounds, which are connected to both ends through ester groups, amide groups, carbamate groups, Carbonate group, ether group or urea group, etc. are connected.
具体地，所述连接子独立地选自2-25个C的直链饱和烃或不饱和烃，其两端是羧基、羟基、氨基、硫酸基、磺酸基、磷酸基中的一种或多种。Specifically, the linker is independently selected from linear saturated or unsaturated hydrocarbons with 2-25 C, and its two ends are one of carboxyl group, hydroxyl group, amino group, sulfate group, sulfonic acid group, and phosphate group, or Various.
具体地，所述连接子独立地选自含有3-8元环的饱和烃或不饱和烃，其两端是羧基、羟基、氨基、硫酸基、磺酸基、磷酸基中的一种或多种。Specifically, the linker is independently selected from saturated or unsaturated hydrocarbons containing 3-8 membered rings, and its two ends are one or more of carboxyl groups, hydroxyl groups, amino groups, sulfate groups, sulfonic acid groups, and phosphate groups. kind.
本发明还提供一种脂质化合物，所述脂质化合物为基于胆酸或其衍生物的脂质，或所述脂质化合物为基于胆酸或其衍生物的脂质的药物可用的盐、前药或立体异构体，所述脂质化合物具有如下通式1或通式2的结构：The present invention also provides a lipid compound that is a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable salt of a lipid based on cholic acid or a derivative thereof, Prodrug or stereoisomer, the lipid compound has the structure of the following general formula 1 or general formula 2:
通式1：
General formula 1:
通式2：
General formula 2:
具体地，所述脂质化合物母核为胆酸或其衍生物；其中所述连接键(Linker)包含酯基、酰胺基、氨基甲酸酯基、碳酸酯基或脲基的一种或多种。Specifically, the core of the lipid compound is cholic acid or a derivative thereof; wherein the linker includes one or more of an ester group, an amide group, a carbamate group, a carbonate group or a urea group. kind.
具体地，其中胆酸或其衍生物是任选自胆酸、奥贝胆酸、熊去氧胆酸、熊果胆酸、3β-羟基-D5-胆烯酸、鹅去氧胆酸、石胆酸、脱氧胆酸、牛磺胆酸、5β-胆酸、去氢胆酸、猪胆酸、络胆酸、甘氨鹅脱氧胆酸、牛磺熊去氧胆酸、牛磺鹅去氧胆酸、甘氨胆酸、猪脱氧胆酸、猪去氧胆酸甲酯、牛磺猪去氧胆酸钠、去氢胆酸钠、胆酸钠、脱氧甘胆酸钠、牛磺脱氧胆酸钠、牛磺胆酸钠、牛磺鹅去氧胆酸钠、甘氨胆酸钠盐、牛磺胆酸-3-硫酸酯二钠盐、牛磺熊去氧胆酸钠和牛磺石胆酸钠的一种或多种。Specifically, wherein the cholic acid or its derivative is optionally selected from the group consisting of cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3β-hydroxy-D5-cholic acid, chenodeoxycholic acid, lithophore Cholic acid, deoxycholic acid, taurocholic acid, 5β-cholic acid, dehydrocholic acid, hyocholic acid, cholancholic acid, glycochenodeoxycholic acid, tauroursodeoxycholic acid, taurochenodeoxycholic acid Cholic acid, glycocholic acid, hyodeoxycholic acid, methyl hyodeoxycholate, sodium taurodeoxycholate, sodium dehydrocholate, sodium cholate, sodium deoxyglycholate, sodium taurodeoxycholate , sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate disodium salt, sodium tauroursodeoxycholate and sodium taurolithocholic acid of one or more.
具体地，所述脂质化合物为选自熊去氧胆酸衍生物脂质化合物的一种或多 种，或奥贝胆酸衍生物脂质化合物的一种或多种，胆酸脂质化合物的一种或多种、猪去氧胆酸衍生物脂质化合物的一种或多种、鹅去氧胆酸衍生物脂质化合物的一种或多种。Specifically, the lipid compound is one or more lipid compounds selected from ursodeoxycholic acid derivatives. species, or one or more obeticholic acid derivative lipid compounds, one or more cholic acid lipid compounds, one or more hyodeoxycholic acid derivative lipid compounds, chenodeoxy Cholic acid derivatives are one or more lipid compounds.
具体地，所述脂质化合物可以为可离子化脂质、或阳离子脂质、或阴离子脂质、或中性脂质、或聚乙二醇衍生物脂质、或聚肌氨酸衍生物脂质、或壳聚糖衍生物脂质、或透明质酸衍生物脂质。Specifically, the lipid compound may be an ionizable lipid, or a cationic lipid, or an anionic lipid, or a neutral lipid, or a polyethylene glycol derivative lipid, or a polysarcosine derivative lipid. substance, or chitosan derivative lipid, or hyaluronic acid derivative lipid.
具体地，所述脂质化合物还可以为一种脂质化合物的偶联物，所述偶联物主要由治疗或预防性药物、连接子(linker)与脂质化合物三个部分构成。所述治疗或预防性药物包括核酸、抗体、多肽、蛋白、糖类、聚乙二醇及其衍生物、小分子中的一种或多种。Specifically, the lipid compound can also be a conjugate of a lipid compound, and the conjugate is mainly composed of three parts: a therapeutic or preventive drug, a linker, and a lipid compound. The therapeutic or preventive drugs include one or more of nucleic acids, antibodies, polypeptides, proteins, carbohydrates, polyethylene glycol and its derivatives, and small molecules.
具体地，所述核酸包括任何形式的核酸分子，例如，DNA可以为非编码DNA或编码DNA，RNA可以选自：ASO、mRNA、siRNA、micRNA等中的一种或多种。Specifically, the nucleic acid includes any form of nucleic acid molecule. For example, DNA can be non-coding DNA or coding DNA, and RNA can be selected from one or more of ASO, mRNA, siRNA, micRNA, etc.
具体地，所述连接子(linker)独立地选自不存在，直链状化合物或者含有环状化合物其中一种或多种，其与两端通过酯基、酰胺基、氨基甲酸酯基、碳酸酯基、醚基或脲基等相连。Specifically, the linker (linker) is independently selected from the group consisting of non-existent, linear compounds or containing one or more cyclic compounds, which are connected to both ends through ester groups, amide groups, carbamate groups, Carbonate group, ether group or urea group, etc. are connected.
具体地，所述连接子独立地选自2-25个C的直链饱和烃或不饱和烃，其两端是羧基、羟基、氨基、硫酸基、磺酸基、磷酸基中的一种或多种。Specifically, the linker is independently selected from linear saturated or unsaturated hydrocarbons with 2-25 C, and its two ends are one of carboxyl group, hydroxyl group, amino group, sulfate group, sulfonic acid group, and phosphate group, or Various.
具体地，所述连接子独立地选自含有3-8元环的饱和烃或不饱和烃，其两端是羧基、羟基、氨基、硫酸基、磺酸基、磷酸基中的一种或多种。Specifically, the linker is independently selected from saturated or unsaturated hydrocarbons containing 3-8 membered rings, and its two ends are one or more of carboxyl groups, hydroxyl groups, amino groups, sulfate groups, sulfonic acid groups, and phosphate groups. kind.
一种脂质化合物的组合物，所述组合物包括治疗或预防剂和用于递送所述治疗或预防剂的载体，所述载体包括前述脂质化合物的一种或多种。A composition of lipid compounds, the composition includes a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier includes one or more of the aforementioned lipid compounds.
优选的，所述的组合物，所述治疗或预防剂包括核酸分子、多肽、蛋白、抗体及小分子药物中的一种或多种。Preferably, the composition, therapeutic or preventive agent includes one or more of nucleic acid molecules, polypeptides, proteins, antibodies and small molecule drugs.
优选的，所述的组合物，所述载体与所述治疗或预防剂的质量比为1:1～100:1。Preferably, in the composition, the mass ratio of the carrier to the therapeutic or preventive agent is 1:1 to 100:1.
优选的，所述的组合物，所述组合物为纳米颗粒制剂，所述纳米颗粒制剂的平均尺寸为20nm～1000nm；所述纳米颗粒制剂的多分散系数≤0.5。Preferably, the composition is a nanoparticle preparation, the average size of the nanoparticle preparation is 20 nm to 1000 nm; the polydispersity coefficient of the nanoparticle preparation is ≤ 0.5.
优选的，所述的组合物，所述载体中包含三种不同的脂质组分，其中一种 脂质是基于胆酸或其衍生物的脂质。Preferably, the composition contains three different lipid components in the carrier, one of which Lipids are lipids based on cholic acid or its derivatives.
优选的，所述的组合物，其特征在于，所述载体中还包括中性电荷、阴性电荷或双性电荷的电荷辅助脂质。Preferably, the composition is characterized in that the carrier further includes a charge-assisted lipid with neutral charge, negative charge or bipolar charge.
优选的，所述的组合物，其特征在于，所述电荷辅助脂质为如下的一种或多种：二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基磷脂酰胆碱(DOPC)、二棕榈酰基磷脂酰胆碱(DPPC)、二油酰基磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二油酰基磷脂酰乙醇胺(DOPE)、棕榈酰油酰基磷脂酰胆碱(POPC)、棕榈酰油酰基-磷脂酰乙醇胺(POPE)、二油酰基-磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-甲酸酯(DOPE-mal)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷脂酰乙醇胺(DMPE)、二硬脂酰基-磷脂酰基-乙醇胺(DSPE)、16-O-单甲基PE,16-O-二甲基PE,18-1-反式PE,1-硬脂酰基-2-油酰基-磷脂酰乙醇胺(SOPE)或其混合物。Preferably, the composition is characterized in that the charge-auxiliary lipid is one or more of the following: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) , dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE- mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O- Dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or mixtures thereof.
优选的，所述的组合物，所述载体中还包括结构修饰脂质。Preferably, in the composition, the carrier further includes a structurally modified lipid.
优选的，所述的组合物，所述结构修饰脂质包括聚乙二醇、葡聚糖、聚乳酸或氨基酸修饰的磷脂酰乙醇胺、磷脂酸、神经酰胺、二烷基胺、二酰基甘油、二烷基甘油中的一种或多种。Preferably, in the composition, the structurally modified lipid includes polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, One or more dialkylglycerols.
优选的，所述的组合物，所述载体还包括但不限于胆酸或其衍生物的脂质、电荷辅助脂质以及结构修饰脂质，所述胆酸脂质、所述电荷辅助脂质以及所述结构修饰脂质的摩尔比为(30～80)：(5-50)：(0.5～10)。Preferably, the composition and the carrier also include but are not limited to lipids of cholic acid or its derivatives, charge-assisted lipids, and structurally modified lipids. The cholic acid lipids, the charge-assisted lipids And the molar ratio of the structurally modified lipid is (30-80): (5-50): (0.5-10).
优选的，所述的组合物，所述组合物还包括药物可用的赋形剂或稀释剂中的一种或多种。Preferably, the composition further includes one or more pharmaceutically acceptable excipients or diluents.
优选的，所述载体还包括胆酸或其衍生物的脂质、电荷辅助脂质、胆固醇或其衍生物以及结构修饰脂质，所述胆酸脂质、所述电荷辅助脂质、所述胆固醇或其衍生物、所述结构修饰脂质的摩尔比为(30～80)：(0.5～10)：(5-50)：(0.5～2.5)。Preferably, the carrier also includes lipids of cholic acid or its derivatives, charge-assisted lipids, cholesterol or derivatives thereof, and structurally modified lipids. The cholic acid lipid, the charge-assisted lipid, the The molar ratio of cholesterol or its derivatives and the structurally modified lipid is (30-80): (0.5-10): (5-50): (0.5-2.5).
本发明所述脂质化合物或组合物可用于在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用。The lipid compound or composition of the present invention can be used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
本发明所述脂质化合物或组合物用于在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用中，其中所述脂质纳米颗粒具有20～1000 nm粒径。The lipid compound or composition of the present invention is used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, wherein the lipid nanoparticles have a concentration of 20 to 1000 nm particle size.
用于制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物的组合物，其包含核酸和包载所述核酸的脂质纳米颗粒，其中每个单独的脂质纳米颗粒包含多种脂质组分，其中一种脂质组分是基于胆酸的脂质化合物，包括其化合物或其药学上可用的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价化合物或前体药物，并且其中所述脂质纳米颗粒具有至少70％的核酸包封比例。Compositions for preparing nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, which include nucleic acids and lipid nanoparticles encapsulating the nucleic acids, wherein each individual lipid nanoparticle contains a variety of lipids A lipid component, wherein one of the lipid components is a cholic acid-based lipid compound, including compounds thereof or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non- A covalent compound or prodrug, and wherein the lipid nanoparticle has a nucleic acid encapsulation ratio of at least 70%.
制备本专利所述组合物的方法，其中所述脂质纳米颗粒通过将mRNA溶液和本专利所述任一种脂质化合物的脂质溶液混合而形成，其中mRNA溶液的介质为HEPES(羟乙基哌嗪乙硫磺酸缓冲液)、磷酸钠、乙酸钠、硫酸铵、碳酸氢钠或柠檬酸钠；脂质溶液的介质为乙醇、异丙醇或二甲亚砜；其中所述脂质纳米颗粒通过透析或超滤进一步纯化。Method for preparing the composition described in this patent, wherein the lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution of any lipid compound described in this patent, wherein the medium of the mRNA solution is HEPES (hydroxyethyl ethyl alcohol). piperazine ethyl sulfate buffer), sodium phosphate, sodium acetate, ammonium sulfate, sodium bicarbonate or sodium citrate; the medium of the lipid solution is ethanol, isopropanol or dimethyl sulfoxide; wherein the lipid nanoparticles The particles are further purified by dialysis or ultrafiltration.
本专利所述的组合物，还包含缓冲剂、碳水化合物、甘露醇、蛋白质、多肽或者氨基酸、抗氧化剂、抑菌剂、螯合剂、佐剂中的一种或多种。The composition described in this patent also contains one or more of buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids, antioxidants, bacteriostatic agents, chelating agents, and adjuvants.
本发明所述酸包括但不限于盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、2,2-二氯乙酸、己二酸、海藻酸、抗坏血酸、天冬氨酸、苯磺酸、苯甲酸、4-乙酰氨基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸，柠檬酸、环酰胺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙磺酸、甲酸、富马酸、半乳糖酸、龙胆酸、葡庚酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲磺酸、、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、棕榈酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、癸二酸、硬脂酸、琥珀酸、酒石酸、对甲苯磺酸、三氟乙酸、以及十一碳烯酸。The acid described in the present invention includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzene Formic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cycloamic acid, dodecyl sulfate, ethane-1, 2-Disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, gluconic acid, gluconic acid, glucuronic acid, glutamic acid, pentanoic acid Diacid, 2-oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, , Naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, palmitic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecenoic acid.
本发明所述的碱加成盐指通过将无机碱或有机碱加成至游离碱化合物而制备的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐，、等；所述有机碱包括但不限于氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、二乙醇胺、乙醇胺、脱醇、2-二甲基氨基乙醇、2-二乙基氨基乙醇、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、肼苯胺、胆碱、甜菜碱、苯那敏(benethamine)、、乙二胺、葡糖胺、甲基葡糖胺、可可碱、三乙醇胺、嘌 呤、哌嗪、哌啶、N-乙基哌啶、以及聚胺树脂。优选地，有机碱是异丙胺、二乙胺、乙醇胺、三甲胺、二环己胺、胆碱和咖啡因。Base addition salts according to the present invention refer to salts prepared by adding an inorganic base or an organic base to a free base compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, etc.; the organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, Diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, Procaine, hydrazine, choline, betaine, benethamine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine pyridine, piperazine, piperidine, N-ethylpiperidine, and polyamine resin. Preferably, the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
本发明提供一种组合物，所述组合物包括治疗或预防剂和用于递送所述治疗或预防剂的载体，所述载体包括基于胆酸或其衍生物的脂质，或其药物可用的盐中的一种或多种。The present invention provides a composition comprising a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable agent thereof. One or more of the salts.
具体的，所述治疗或者预防剂被包封在载体内或与载体缔合。Specifically, the therapeutic or preventive agent is encapsulated in or associated with a carrier.
具体的，所述治疗或者预防剂包括核酸分子、基因疫苗、多肽、蛋白、抗体及小分子药物中的一种或多种。Specifically, the therapeutic or preventive agent includes one or more of nucleic acid molecules, genetic vaccines, polypeptides, proteins, antibodies and small molecule drugs.
具体的，所述核酸包括任何形式的核酸分子，包括但不限于单链DNA、双链DNA、短异构体、agomir、antagomir、反义分子、小干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、microRNA(miRNA)、Dicer-substrate RNA(dsRNA)、小发夹RNA(shRNA)、转移RNA(tRNA)、信使RNA(mRNA)和本领域已知的其他形式的RNA分子，或锁核酸(LNA)、肽核酸(PNA)和吗啉环寡聚核苷酸等核酸模拟物。Specifically, the nucleic acid includes any form of nucleic acid molecule, including but not limited to single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA ( aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and other forms of RNA molecules known in the art, or locked nucleic acids Nucleic acid mimics such as (LNA), peptide nucleic acid (PNA) and morpholino cyclic oligonucleotides.
根据一些具体地实施方式，所述治疗或预防剂包含至少一种编码抗原或蛋白或肽的mRNA或其片段或表位。According to some specific embodiments, the therapeutic or preventive agent comprises at least one mRNA encoding an antigen or a protein or a peptide or a fragment or epitope thereof.
更具体地，所述mRNA是单顺反子mRNA或多顺反子mRNA。More specifically, the mRNA is monocistronic or polycistronic.
更具体地，所述抗原是病原性抗原。More specifically, the antigen is a pathogenic antigen.
更具体地，所述mRNA包含一种或多种功能性核苷酸类似物，所述功能性核苷酸类似物包括但不限于假尿嘧啶核苷、1-甲基-假尿嘧啶核苷和5-甲基胞嘧啶中的一种或多种。More specifically, the mRNA contains one or more functional nucleotide analogs, including but not limited to pseudouridine, 1-methyl-pseudouridine and one or more of 5-methylcytosine.
具体地，所述小分子化合物包括但不限于治疗和/或预防剂的有效成分，所述治疗和/或预防剂为现有已知的药物，例如抗肿瘤药、抗感染药、局部麻醉药、抗抑郁药、抗惊厥药、抗生素/抗菌剂、抗真菌药、抗寄生虫药、激素、激素拮抗剂、免疫调节剂、神经递质拮抗剂、抗青光眼剂、麻醉剂、或造影剂。Specifically, the small molecule compounds include, but are not limited to, the active ingredients of therapeutic and/or preventive agents. The therapeutic and/or preventive agents are currently known drugs, such as anti-tumor drugs, anti-infective drugs, and local anesthetics. , antidepressants, anticonvulsants, antibiotics/antimicrobials, antifungals, antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, anesthetics, or contrast media.
优选地，所述脂质包含三种不同的脂质组分，其中一种脂质是基于胆酸或其衍生物的脂质。优选地，所述脂质还包括带中性电荷、阴性电荷或双性电荷的辅助脂质。 Preferably, the lipids comprise three different lipid components, one of which is a cholic acid or derivative thereof based lipid. Preferably, the lipid further includes an auxiliary lipid with a neutral charge, a negative charge, or a bipolar charge.
更具体地，所述脂质包括磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂、神经酰胺、甾醇及其衍生物中的一种或多种。More specifically, the lipid includes one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and derivatives thereof.
更具体地，所述脂质包括但不限于1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱(DMPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、11,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、2-(((2,3-双(油酰氧基)丙基))磷酸二甲基铵)乙基氢(DOCP)、鞘磷脂(SM)、神经酰胺及其衍生物。所述的脂质为可为合成的或者衍生自天然来源或化合物(自其分离或改性)。More specifically, the lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine base (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 11,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-(((2,3- Bis(oleoyloxy)propyl)dimethylammonium phosphate)ethylhydrogen (DOCP), sphingomyelin (SM), ceramide and its derivatives. The lipids may be synthetic or derived from (isolated or modified) natural sources or compounds.
根据一些具体实施方式，所述载体还包括结构修饰脂质。According to some embodiments, the carrier further includes a structurally modified lipid.
具体地，所述结构修饰脂质主要包括已公开的或未公开的脂质化合物，可以改善脂质体的稳定性并减少脂质体的蛋白质吸收，例如聚乙二醇、葡聚糖、聚乳酸或氨基酸修饰的磷脂酰乙醇胺、磷脂酸、神经酰胺、二烷基胺、二酰基甘油、二烷基甘油中的一种或多种。Specifically, the structurally modified lipids mainly include disclosed or undisclosed lipid compounds, which can improve the stability of liposomes and reduce protein absorption of liposomes, such as polyethylene glycol, dextran, polyethylene glycol, etc. One or more of lactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, and dialkylglycerol.
更具体地，所述结构修饰脂质可以是PEG-c-DOMG、PEG-DMG、PEG-DLPE、PEGDMPE、PEG-DPPC、PEG-DSPE、神经酰胺-PEG2000、Chol-PEG2000、1-(单甲氧基-聚乙二醇)-2,3-二肉豆蔻基甘油(PEG-DMG)、聚乙二醇化磷脂酰乙醇胺(PEG-PE)、4-O-(2'，3'-二(十四烷酰氧基)丙基-1-O-(ω-甲氧基(聚乙氧基)乙基)丁二酸酯(PEG-S-DMG)、聚乙二醇化神经酰胺(PEG-cer)、ω-甲氧基(聚乙氧基)乙基-N-(2,3-二(十四烷氧基)丙基)氨基甲酸酯、或2,3-二(四癸氧基)丙基-N-(ω-甲氧基)(聚乙氧基)乙基)氨基甲酸酯。More specifically, the structurally modified lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, Chol-PEG2000, 1-(monomethyl Oxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), 4-O-(2',3'-bis( Tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), polyglycolated ceramide (PEG- cer), ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate, or 2,3-di(tetradecyloxy) methyl)propyl-N-(ω-methoxy)(polyethoxy)ethyl)carbamate.
优选地，所述载体与所述有治疗或预防剂的质量比为5:1～50:1，进一步优选为5:1～35:1，更优选为10:1～30:1。Preferably, the mass ratio of the carrier to the therapeutic or preventive agent is 5:1 to 50:1, more preferably 5:1 to 35:1, and more preferably 10:1 to 30:1.
根据前述权利要求所述的组合物，其特征在于，所述载体还包括但不限于胆酸或其衍生物的脂质、电荷辅助脂质以及结构修饰脂质。所述胆酸脂质、所述电荷辅助脂质，以及所述结构修饰脂质的摩尔比为(30～80)：(5～50)：(0.5～10)。The composition according to the preceding claims, wherein the carrier further includes, but is not limited to, lipids of cholic acid or its derivatives, charge-assisted lipids and structurally modified lipids. The molar ratio of the cholic acid lipid, the charge-assisted lipid, and the structurally modified lipid is (30-80): (5-50): (0.5-10).
在一些实施例中，脂质纳米颗粒通过将mRNA溶液和脂质溶液混合而形成。在一些实施例中，脂质纳米颗粒通过切向流过滤进一步纯化。 In some embodiments, lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution. In some embodiments, lipid nanoparticles are further purified by tangential flow filtration.
根据前述权利要求的方法，其中所述脂质纳米颗粒通过将mRNA溶液和脂质溶液混合而形成。其中mRNA溶液的介质为HEPES、磷酸盐、乙酸盐、硫酸铵、碳酸氢钠或柠檬酸盐。脂质溶液的介质为乙醇、异丙醇或二甲亚砜。Method according to the preceding claim, wherein said lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution. The medium of the mRNA solution is HEPES, phosphate, acetate, ammonium sulfate, sodium bicarbonate or citrate. The medium of lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide.
优选地，所述药物组合物为纳米颗粒制剂，所述纳米颗粒制剂的平均尺寸为20nm～1000nm，优选为40nm～150nm，进一步优选为50nm～100nm，更优选为70nm～100nm。Preferably, the pharmaceutical composition is a nanoparticle preparation, and the average size of the nanoparticle preparation is 20 nm to 1000 nm, preferably 40 nm to 150 nm, further preferably 50 nm to 100 nm, and more preferably 70 nm to 100 nm.
进一步优选地，所述纳米颗粒制剂的多分散指数≤0.5，进一步优选≤0.3，更优选≤0.25。Further preferably, the polydispersity index of the nanoparticle preparation is ≤0.5, further preferably ≤0.3, and more preferably ≤0.25.
本发明的药物组合物通常还包含一种或多种缓冲剂(例如中性缓冲盐水或磷酸盐缓冲液水)、碳水化合物(例如葡萄糖、甘露醇、蔗糖、海藻糖、右旋糖或葡聚糖)、甘露醇、蛋白质、多肽或者氨基酸(例如甘氨酸和赖氨酸)、抗氧化剂(维生素E和丁基羟基甲苯)、抑菌剂、螯合剂(例如EDTA和谷胱甘肽)、佐剂(例如氢氧化铝)、使制剂与接受者的血液等渗的助悬剂/增稠剂/防腐剂等，或者可以将本发明的组合物配制成冻干物。Pharmaceutical compositions of the present invention also typically include one or more buffers (eg neutral buffered saline or phosphate buffered water), carbohydrates (eg glucose, mannitol, sucrose, trehalose, dextrose or dextran). sugar), mannitol, proteins, peptides or amino acids (such as glycine and lysine), antioxidants (vitamin E and butylated hydroxytoluene), bacteriostatic agents, chelating agents (such as EDTA and glutathione), adjuvants (e.g. aluminum hydroxide), suspending agents/thickening agents/preservatives, etc. that make the formulation isotonic with the recipient's blood, or the composition of the invention can be formulated as a lyophilisate.
具体地，所述组合物的给药方式包括但不限于静脉注射、肌肉注射、皮下注射、皮内注射、瘤内注射、眼部给药、耳部给药、经鼻给药、经口腔给药、经肛门给药、经阴道给药等。Specifically, the administration methods of the composition include but are not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, ocular administration, ear administration, nasal administration, and oral administration. medicine, transanal administration, transvaginal administration, etc.
具体地，所述组合物的给药对象包括但不限于哺乳动物如牛、马、骡、驴、骆驼、猪、羊、犬、狐、兔等，禽类如鸡、鸭、鹅、鸽子等，鱼类，非人灵长类，人。Specifically, the administration objects of the composition include but are not limited to mammals such as cattle, horses, mules, donkeys, camels, pigs, sheep, dogs, foxes, rabbits, etc., and poultry such as chickens, ducks, geese, pigeons, etc., Fish, non-human primates, humans.
本发明技术方案提供了一类全新的用于递送治疗或预防用药物的脂质化合物，即基于胆酸或其衍生物的脂质，本发明技术方案不同于国外医药公司的现有专利技术方案，丰富了脂质化合物种类，为核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物等的递送提供了更多选择，并且能够与国外辉瑞公司、莫德纳公司的技术路线具有明显区别。The technical solution of the present invention provides a new class of lipid compounds for delivering therapeutic or preventive drugs, namely lipids based on cholic acid or its derivatives. The technical solution of the present invention is different from the existing patented technical solutions of foreign pharmaceutical companies. , enriches the types of lipid compounds, provides more options for the delivery of nucleic acid drugs, gene vaccines, peptides, proteins, antibodies and small molecule drugs, and can be significantly different from the technical routes of foreign companies such as Pfizer and Moderna. .
本发明的药物组合物涉及脂质纳米颗粒(LNP)领域，该纳米颗粒制备方法简单，重复性好，简化生产流程，降低成本；同时可以避开四组分LNP的专利封锁，有利于推动核酸药物的国产化。在具体实施例中，制备的包载siRNA的脂质纳米颗粒具有较好的细胞转染效果；该纳米颗粒无论通过静脉注射进小 鼠体内还是通过肌肉注射进小鼠体内，LNP均具有较好的体内递送效果。另外，在具体实施例中，将该由包载mRNA的纳米颗粒进行瘤内注射，在注射后6h拍照，小鼠瘤内部位有荧光表达，说明脂质纳米颗粒可以采用瘤内注射方式给药。综合体内和体外评价结果，该纳米颗粒具备很好的递送效果和应用场景。The pharmaceutical composition of the present invention relates to the field of lipid nanoparticles (LNP). The preparation method of the nanoparticles is simple, has good repeatability, simplifies the production process and reduces costs; at the same time, it can avoid the patent blockade of the four-component LNP and is conducive to the promotion of nucleic acids. Domestic production of drugs. In specific embodiments, the prepared lipid nanoparticles encapsulating siRNA have better cell transfection effect; whether the nanoparticles are injected into small cells through intravenous injection Whether in mice or via intramuscular injection, LNP has a good in vivo delivery effect. In addition, in a specific embodiment, the nanoparticles encapsulating the mRNA were injected into the tumor, and pictures were taken 6 hours after the injection. There was fluorescence expression in the tumor of the mice, indicating that the lipid nanoparticles can be administered by intratumoral injection. . Based on the in vivo and in vitro evaluation results, the nanoparticles have good delivery effects and application scenarios.
附图说明Description of the drawings
图1为熊去氧胆酸衍生物1(化合物1)核磁共振氢谱图。Figure 1 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 1 (compound 1).
图2为熊去氧胆酸衍生物2(化合物2)核磁共振氢谱图。Figure 2 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 2 (compound 2).
图3为熊去氧胆酸衍生物7(化合物7)核磁共振氢谱图。Figure 3 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 7 (compound 7).
图4为熊去氧胆酸衍生物9(化合物9)核磁共振氢谱图。Figure 4 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 9 (compound 9).
图5为熊去氧胆酸衍生物10(化合物10)核磁共振氢谱图。Figure 5 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 10 (compound 10).
图6为熊去氧胆酸衍生物13(化合物13)核磁共振氢谱图。Figure 6 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 13 (compound 13).
图7为熊去氧胆酸衍生物16(化合物16)核磁共振氢谱图。Figure 7 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 16 (compound 16).
图8为熊去氧胆酸衍生物17(化合物17)核磁共振氢谱图。Figure 8 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 17 (compound 17).
图9为奥贝胆酸衍生物18(化合物18)核磁共振氢谱图。Figure 9 is a proton nuclear magnetic resonance spectrum of obeticholic acid derivative 18 (compound 18).
图10为熊去氧胆酸衍生物42(化合物42)核磁共振氢谱图。Figure 10 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 42 (compound 42).
图11为石胆酸衍生物58(化合物58)核磁共振氢谱图。Figure 11 is a proton nuclear magnetic resonance spectrum of lithocholic acid derivative 58 (compound 58).
图12为熊去氧胆酸衍生物62(化合物62)核磁共振氢谱图。Figure 12 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 62 (compound 62).
图13为熊去氧胆酸衍生物63(化合物63)核磁共振氢谱图。Figure 13 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 63 (compound 63).
图14为熊去氧胆酸衍生物66(化合物66)核磁共振氢谱图。Figure 14 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 66 (compound 66).
图15为鹅去氧胆酸衍生物69(化合物69)核磁共振氢谱图。Figure 15 is a hydrogen nuclear magnetic resonance spectrum of chenodeoxycholic acid derivative 69 (compound 69).
图16为猪去氧胆酸衍生物72(化合物72)核磁共振氢谱图。Figure 16 is a hydrogen nuclear magnetic resonance spectrum of hyodeoxycholic acid derivative 72 (compound 72).
图17为熊去氧胆酸衍生物75(化合物75)核磁共振氢谱图。Figure 17 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 75 (compound 75).
图18为熊去氧胆酸衍生物85(化合物85)核磁共振氢谱图。Figure 18 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 85 (compound 85).
图19为熊去氧胆酸衍生物87(化合物87)核磁共振氢谱图。Figure 19 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 87 (compound 87).
图20为熊去氧胆酸衍生物89(化合物89)核磁共振氢谱图。Figure 20 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 89 (compound 89).
图21为LNP包载cy3-siRNA转染细胞实验结果，其中，a)荧光显微镜明场,b)荧光显微镜暗场，c)细胞流式图。Figure 21 shows the experimental results of cells transfected with LNP-encapsulated cy3-siRNA, including a) bright field of fluorescence microscope, b) dark field of fluorescence microscope, c) cell flow cytometry.
图22为LNP包载EGFP mRNA转染细胞实验结果，其中，a)荧光显微镜明场,b)荧光显微镜暗场，c)细胞流式图。Figure 22 shows the experimental results of cells transfected with LNP-encapsulated EGFP mRNA, including a) bright field of fluorescence microscope, b) dark field of fluorescence microscope, c) cell flow cytometry.
图23为包载Luciferase mRNA的LNP,小鼠静脉注射后12h荧光成像结果。Figure 23 shows the fluorescence imaging results of LNP encapsulating Luciferase mRNA 12 hours after intravenous injection in mice.
图24为包载Luciferase mRNA的LNP,小鼠肌肉注射后12h荧光成像结果。Figure 24 shows the fluorescence imaging results of LNP encapsulating Luciferase mRNA 12 hours after intramuscular injection in mice.
图25为肿瘤细胞A549杀伤效果图。Figure 25 is a diagram of the killing effect of tumor cells A549.
图26为小鼠肌肉注射后荧光成像结果图。Figure 26 shows the results of fluorescence imaging after intramuscular injection in mice.
图27为小鼠静脉注射后荧光成像结果图。Figure 27 shows the results of fluorescence imaging after intravenous injection into mice.
图28为小鼠瘤内注射后荧光成像结果图。 Figure 28 shows the results of fluorescence imaging after intratumoral injection in mice.
具体实施方式Detailed ways
胆酸是人和哺乳动物体内天然普遍存在的甾类分子，由胆固醇在肝脏中合成。进食后，胆酸通过肝细胞的主动分泌，随胆汁进入胆囊，再由胆囊进入肠道发挥它的消化功能，胆酸以钠盐形式进入小肠，帮助脂类的消化吸收，随后在回肠末端通过主动吸收或被动运输的方式经门静脉回到肝脏，在肝细胞内进行加工转化，然后同新合成的胆汁酸一起又被分泌入小肠。胆汁酸的这种EHC(肝肠循环)过程每天会循环4～12次，大约有95％的胆汁酸被回吸利用。如果胆汁酸的EHC遭到破坏，不仅会影响体内脂类的消化吸收，也会使机体形成胆固醇结石。因此基于胆酸设计的化合物递送载体最大的优势就是肝肠循环效率高，参与胆酸的肝肠循环，从而提高药物在肝脏和胆囊中的吸收。胆酸结构式如下。
Cholic acid is a naturally ubiquitous steroid molecule in humans and mammals, synthesized in the liver from cholesterol. After eating, bile acid is actively secreted by liver cells, enters the gallbladder with bile, and then enters the intestine from the gallbladder to perform its digestive function. Cholic acid enters the small intestine in the form of sodium salt to help digest and absorb lipids, and then passes through the terminal ileum. It is returned to the liver through the portal vein through active absorption or passive transport, processed and transformed in liver cells, and then secreted into the small intestine together with newly synthesized bile acids. This EHC (enterohepatic circulation) process of bile acids circulates 4 to 12 times a day, and about 95% of the bile acids are reabsorbed and utilized. If the EHC of bile acids is destroyed, it will not only affect the digestion and absorption of lipids in the body, but also cause the body to form cholesterol stones. Therefore, the biggest advantage of compound delivery carriers designed based on bile acid is that it has high enterohepatic circulation efficiency and participates in the enterohepatic circulation of bile acid, thereby improving drug absorption in the liver and gallbladder. The structural formula of cholic acid is as follows.
胆酸分子中甾体骨架上的3个六元环和1个五元环在同一平面上,其中环A和环B反向连接,使分子形成1个穴状结构。分子中3个甲基分布在甾环所在平面的一边,形成分子憎水部分,3个羟基分布在甾环所在平面的另一边,同C24位羧基一起形成分子的亲水部分。The three six-membered rings and one five-membered ring on the steroid skeleton of the cholic acid molecule are on the same plane, and ring A and ring B are connected in reverse, making the molecule form a cave-like structure. In the molecule, three methyl groups are distributed on one side of the plane where the steroid ring is located, forming the hydrophobic part of the molecule. Three hydroxyl groups are distributed on the other side of the plane where the steroid ring is located, and together with the C24 carboxyl group, form the hydrophilic part of the molecule.
胆酸分子的特殊结构,决定了它具有双亲性、酸碱性,并容易进行化学修饰，因此，本专利利用胆酸作为构筑基元用来制备高分子或者低聚物，这些基于胆酸的功能性分子在药物传输具有良好的技术效果。The special structure of the cholic acid molecule determines that it is amphiphilic, acid-base, and easy to undergo chemical modification. Therefore, this patent uses cholic acid as a building block to prepare polymers or oligomers. These cholic acid-based products Functional molecules have good technical effects in drug delivery.
胆酸类药物已经在中国或美国市场上市多年，包括熊去氧胆酸、奥贝胆酸、鹅去氧胆酸、牛磺熊去氧胆酸。如熊去氧胆酸，用于治疗胆固醇型胆结石及胆汁反流性胃炎；奥贝胆酸，用于治疗原发性胆汁性肝硬化(PBC)，针对熊去氧胆酸没有充分应答或不能耐受的患者。多年的临床应用表明，胆酸类化合物具有较好的安全性。另外，胆酸类化合物具有亲水亲油的两亲性质，通过包裹或 偶联的方式可以提高小分子化学药物的生物利用度。Cholic acid drugs have been on the market in China or the United States for many years, including ursodeoxycholic acid, obeticholic acid, chenodeoxycholic acid, and tauroursodeoxycholic acid. For example, ursodeoxycholic acid is used to treat cholesterol gallstones and bile reflux gastritis; obeticholic acid is used to treat primary biliary cirrhosis (PBC), and there is no adequate response to ursodeoxycholic acid or Patients who cannot tolerate it. Years of clinical application have shown that cholic acid compounds have good safety. In addition, cholic acid compounds have hydrophilic and lipophilic amphiphilic properties, and can be encapsulated or The conjugation method can improve the bioavailability of small molecule chemical drugs.
胆酸类化合物与胆固醇同属于甾体类化合物，结构相似，并且可进行化学修饰的位点更多。将胆酸类化合物进行结构修饰制备新的脂质成分，有可能替代原四组分LNP中的可离子化脂和胆固醇，达到相似或更优的递送mRNA效果。本专利研究基于胆酸类似物构建脂质纳米颗粒载体，并探索其在mRNA药物递送中的应用。本发明的LNP可以简化生产流程，降低成本；同时可以避开四组分LNP的专利封锁，有利于推动核酸药物的国产化。Cholic acid compounds and cholesterol are both steroidal compounds with similar structures and more sites that can be chemically modified. Structural modification of cholic acid compounds to prepare new lipid components may potentially replace the ionizable lipids and cholesterol in the original four-component LNP to achieve similar or better mRNA delivery effects. This patented study constructs lipid nanoparticle carriers based on bile acid analogs and explores their application in mRNA drug delivery. The LNP of the present invention can simplify the production process and reduce costs; at the same time, it can avoid the patent blockade of four-component LNP, which is beneficial to promoting the localization of nucleic acid drugs.
因为胆酸或其衍生物均由一个刚性甾环和一个脂肪侧链组成，其中甾环包括三个六元环和一个五元环。只是根据来源的不同，胆酸的侧链结构、甾环构象、羟基的数目及在甾环上的朝向会有所不同。胆酸或其衍生物的共有的羟基和脂肪侧链的羧基，均是很好的化学修饰位点。因此我们认为胆酸或其衍生物均可以通过一些共同的修饰，达到预期目的。Because cholic acid or its derivatives are composed of a rigid steroid ring and an aliphatic side chain, the steroid ring includes three six-membered rings and one five-membered ring. However, depending on the source, the side chain structure of cholic acid, the conformation of the steroid ring, the number of hydroxyl groups and the orientation of the steroid ring will be different. The common hydroxyl group of cholic acid or its derivatives and the carboxyl group of the fatty side chain are good chemical modification sites. Therefore, we believe that cholic acid or its derivatives can achieve the desired purpose through some common modifications.
本专利中的胆酸或其衍生物是任选自胆酸、奥贝胆酸、熊去氧胆酸、熊果胆酸、3β-羟基-D5-胆烯酸、鹅去氧胆酸、石胆酸、脱氧胆酸、牛磺胆酸、5β-胆酸、去氢胆酸、猪胆酸、络胆酸、甘氨鹅脱氧胆酸、牛磺熊去氧胆酸、牛磺鹅去氧胆酸、甘氨胆酸、猪脱氧胆酸、猪去氧胆酸甲酯、牛磺猪去氧胆酸钠、去氢胆酸钠、胆酸钠、脱氧甘胆酸钠、牛磺脱氧胆酸钠、牛磺胆酸钠、牛磺鹅去氧胆酸钠、甘氨胆酸钠盐、牛磺胆酸-3-硫酸酯二钠盐、牛磺熊去氧胆酸钠和牛磺石胆酸钠，具体结构如下。


Cholic acid or its derivatives in this patent is optionally selected from the group consisting of cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3β-hydroxy-D5-cholic acid, chenodeoxycholic acid, and stone. Cholic acid, deoxycholic acid, taurocholic acid, 5β-cholic acid, dehydrocholic acid, hyocholic acid, cholancholic acid, glycochenodeoxycholic acid, tauroursodeoxycholic acid, taurochenodeoxycholic acid Cholic acid, glycocholic acid, hyodeoxycholic acid, methyl hyodeoxycholate, sodium taurodeoxycholate, sodium dehydrocholate, sodium cholate, sodium deoxyglycholate, sodium taurodeoxycholate , sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate disodium salt, sodium tauroursodeoxycholate and sodium taurolithocholic acid , the specific structure is as follows.


本专利所述的脂质化合物为选自如下结构所示化合物的一种或多种；The lipid compound described in this patent is one or more compounds selected from the following structures;
①熊去氧胆酸衍生物脂质：









① Ursodeoxycholic acid derivative lipid:









②奥贝胆酸衍生物脂质：









② Obeticholic acid derivative lipids:









下面结合实施例对本发明作进一步描述，但本发明并不限于以下实施例。实施例中采用的实施条件可以根据具体使用的不同要求做进一步调整，未做注明的实施条件为本行业的常规条件。本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互结合。The present invention will be further described below with reference to examples, but the present invention is not limited to the following examples. The implementation conditions used in the embodiments can be further adjusted according to different requirements for specific use. Implementation conditions that are not noted are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.
实施例1：熊去氧胆酸衍生物1(化合物1)的合成
Example 1: Synthesis of Ursodeoxycholic Acid Derivative 1 (Compound 1)
将熊去氧胆酸(393mg,1.0mmol)溶解于DMF(8mL)中,依次加入HBTU(569mg,1.5mmol),DIEA(194mg,1.5mmol),N,N-二甲基乙二胺(132mg,1.5mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体化合物1(333mg,72％)。1HNMR(400MHz,CD3OD,)δ3.29-3.44(m,3H),2.89(t,J＝8.0Hz,2H),2.64(s,6H,CH3×2),0.95-0.97(m,6H,CH3×2),0.70(s,3H,CH3).ESI-MS m/z Calc.C28H51N2O3[M+H]+463.72,Found 464.15.化合物1的核磁共振氢谱图见说明书附图中的图1。Dissolve ursodeoxycholic acid (393mg, 1.0mmol) in DMF (8mL), then add HBTU (569mg, 1.5mmol), DIEA (194mg, 1.5mmol), N,N-dimethylethylenediamine (132mg ,1.5mmol), under nitrogen protection, the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 1 as a white solid (333 mg, 72%). 1 HNMR (400MHz, CD 3 OD,) δ3.29-3.44 (m, 3H), 2.89 (t, J = 8.0Hz, 2H), 2.64 (s, 6H, CH 3 × 2), 0.95-0.97 (m ,6H,CH 3 ×2),0.70(s,3H,CH 3 ).ESI-MS m/z Calc.C 28 H 51 N 2 O 3 [M+H] + 463.72, Found 464.15. NMR of compound 1 The resonance hydrogen spectrum is shown in Figure 1 in the accompanying drawings of the specification.
实施例2：熊去氧胆酸衍生物2(化合物2)的合成
Example 2: Synthesis of Ursodeoxycholic Acid Derivative 2 (Compound 2)
将熊去氧胆酸(393mg,1.0mmol)溶解于DMF(8mL)中,依次加入HBTU(569mg,1.5mmol),DIEA(194mg,1.5mmol),4-吡咯-1-丁胺(213mg,1.5mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加 入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体产品化合物2(403mg,78％)。1HNMR(400MHz,CD3OD)δ5.52(s,1H,CONH),3.50-3.54(m,2H),3.22-3.26(m,4H),2.64(s,6H,CH3×2),1.00-1.02(m,6H,CH3×2),0.75(s,3H CH3).ESI-MS m/z Calc.C33H57N2O5[M+HCOO]-561.43,Found 561.35。化合物2的核磁共振氢谱图见说明书附图中的图2.Ursodeoxycholic acid (393 mg, 1.0 mmol) was dissolved in DMF (8 mL), and HBTU (569 mg, 1.5 mmol), DIEA (194 mg, 1.5 mmol), and 4-pyrrole-1-butylamine (213 mg, 1.5 mmol) were added in sequence. mmol), under nitrogen protection, and stirred at room temperature overnight. TLC detection, after the reaction is completed, add Add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 2 as a white solid product (403 mg, 78%). 1 HNMR (400MHz, CD 3 OD) δ5.52 (s, 1H, CONH), 3.50-3.54 (m, 2H), 3.22-3.26 (m, 4H), 2.64 (s, 6H, CH 3 × 2), 1.00-1.02(m,6H,CH 3 ×2),0.75(s,3H CH3).ESI-MS m/z Calc.C 33 H 57 N 2 O 5 [M+HCOO] - 561.43, Found 561.35. The proton nuclear magnetic resonance spectrum of compound 2 is shown in Figure 2 in the accompanying drawings of the description.
实施例3：熊去氧胆酸衍生物7(化合物7)的合成
Example 3: Synthesis of Ursodeoxycholic Acid Derivative 7 (Compound 7)
将熊去氧胆酸(393mg,1.0mmol)溶解于乙腈(10mL)中,依次加入K2CO3(415mg,3.0mmol),BnBr(850mg,5.0mmol),氮气保护，80℃搅拌反应5h。TLC检测，反应结束后，滤过，浓缩溶剂得粗品。硅胶柱层析，PE/EA(1:1)洗脱，得白色固体产品化合物6(390mg,81％)。1HNMR(400MHz,CDCl3)δ7.32-7.36(m,5H),5.08-5.15(m,2H),3.55-3.62(m,2H),0.94(s,3H,CH3),0.91(d,J＝4Hz,3H,CH3),0.65(s,3H,CH3)。Ursodeoxycholic acid (393 mg, 1.0 mmol) was dissolved in acetonitrile (10 mL), K 2 CO 3 (415 mg, 3.0 mmol), BnBr (850 mg, 5.0 mmol) were added in sequence, protected by nitrogen, and the reaction was stirred at 80°C for 5 h. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with PE/EA (1:1), gave compound 6 (390 mg, 81%) as a white solid product. 1 HNMR (400MHz, CDCl 3 ) δ7.32-7.36(m,5H),5.08-5.15(m,2H),3.55-3.62(m,2H),0.94(s,3H,CH 3 ),0.91(d ,J＝4Hz,3H,CH 3 ),0.65(s,3H,CH 3 ).
4-二甲基氨基丁酸盐酸盐(336mg,2.0mmol)溶解于无水DCM(8mL)中，加入草酰氯(1mL)，室温搅拌4h。真空浓缩至无溶剂,用无水DCM(3mL)溶解,备用。把化合物6(241mg,0.5mmol)，TEA(202mg,2.0mmol)溶解于无水DCM(3mL)中，将上述备用产物滴入反应液中，室温反应过夜。加入适量水， 用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体产品化合物7(213mg,60％)。1HNMR(400MHz,d6-DMSO)δ7.33-7.37(m,5H),5.04-5.11(nm,2H),4.59-4.68(m,2H),2.92-2.98(m,4H),2.66(s,6H),2.65(s,6H),0.93(s,3H,CH3),0.87(d,J＝8Hz,3H,CH3),0.60(s,3H,CH3)；ESI-MS m/z Calc.C43H69N2O6[M+H]+709.52,Found 709.85.化合物7的核磁共振氢谱图见说明书附图中的图3。4-Dimethylaminobutyric hydrochloride (336 mg, 2.0 mmol) was dissolved in anhydrous DCM (8 mL), oxalyl chloride (1 mL) was added, and stirred at room temperature for 4 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (3mL), and set aside. Dissolve compound 6 (241 mg, 0.5 mmol) and TEA (202 mg, 2.0 mmol) in anhydrous DCM (3 mL). Add the above standby product dropwise into the reaction solution and react at room temperature overnight. Add appropriate amount of water, Extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 7 as a white solid product (213 mg, 60%). 1 HNMR (400MHz, d 6 -DMSO) δ7.33-7.37(m,5H),5.04-5.11(nm,2H),4.59-4.68(m,2H),2.92-2.98(m,4H),2.66( s,6H),2.65(s,6H),0.93(s,3H,CH 3 ),0.87(d,J＝8Hz,3H,CH 3 ),0.60(s,3H,CH 3 ); ESI-MS m /z Calc.C 43 H 69 N 2 O 6 [M+H] + 709.52, Found 709.85. The hydrogen nuclear magnetic resonance spectrum of compound 7 is shown in Figure 3 in the appendix of the specification.
实施例4：熊去氧胆酸衍生物9(化合物9)的合成
Example 4: Synthesis of Ursodeoxycholic Acid Derivative 9 (Compound 9)
将熊去氧胆酸(785mg,1.0mmol)溶解于DMF(20mL)中,依次加入K2CO3(829mg,6.0mmol)，CH3I(852mg,6.0mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，PE/EA(1:1)洗脱，得白色固体产品化合物8(700mg,86％)。Ursodeoxycholic acid (785mg, 1.0mmol) was dissolved in DMF (20mL), K 2 CO 3 (829mg, 6.0mmol), CH 3 I (852mg, 6.0mmol) were added in sequence, under nitrogen protection, and the reaction was stirred at room temperature overnight. . TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with PE/EA (1:1), gave compound 8 (700 mg, 86%) as a white solid product.
4-二甲基氨基丁酸盐酸盐(336mg,2.0mmol)溶解于无水DCM(8mL)中，加入草酰氯(0.5mL)，室温搅拌4h。真空浓缩至无溶剂,用无水DCM(3mL)溶解,备用。把化合物8(203mg,0.5mmol)，TEA(202mg,2.0mmol)溶解于无水DCM(3mL)中，将上述备用产物滴入反应液中，室温反应过夜。加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗 品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体产品化合物9(221mg,70％)。1HNMR(400MHz,d6-DMSO)δ4.58-4.70(m,2H),3.57(s,3H),2.63(s,6H),2.61(s,6H),0.93(s,3H,CH3),0.88(d,J＝4Hz,3H,CH3),0.63(s,3H,CH3)；ESI-MS m/z Calc.C37H65N2O6[M+H]+633.48,Found 634.15.化合物9的核磁共振氢谱图见说明书附图中的图4。4-Dimethylaminobutyric hydrochloride (336 mg, 2.0 mmol) was dissolved in anhydrous DCM (8 mL), oxalyl chloride (0.5 mL) was added, and stirred at room temperature for 4 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (3mL), and set aside. Dissolve compound 8 (203 mg, 0.5 mmol) and TEA (202 mg, 2.0 mmol) in anhydrous DCM (3 mL). Add the above standby product dropwise into the reaction solution and react at room temperature overnight. Add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude Taste. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 9 as a white solid product (221 mg, 70%). 1 HNMR (400MHz, d 6 -DMSO) δ4.58-4.70 (m, 2H), 3.57 (s, 3H), 2.63 (s, 6H), 2.61 (s, 6H), 0.93 (s, 3H, CH 3 ),0.88(d,J＝4Hz,3H,CH 3 ),0.63(s,3H,CH 3 ); ESI-MS m/z Calc.C 37 H 65 N 2 O 6 [M+H] + 633.48, Found 634.15. The hydrogen nuclear magnetic resonance spectrum of compound 9 is shown in Figure 4 in the accompanying drawings of the description.
实施例5：熊去氧胆酸衍生物10(化合物10)的合成
Example 5: Synthesis of Ursodeoxycholic Acid Derivative 10 (Compound 10)
亚油酸(476mg,1.7mmol)溶解于无水DCM(5mL)中，加入草酰氯(0.30mL)，室温搅拌5h。真空浓缩至无溶剂,用无水DCM(2mL)溶解,备用。把化合物2(240mg,0.34mmol)，TEA(69mg,0.68mmol)溶解于无水DCM(5mL)中，将上述备用产物滴入反应液中，室温反应过夜。加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色半固体产品化合物10(212mg,60％)。1HNMR(400MHz,CD3OD)δ5.34-5.36(m,8H),1.01(s,3H,CH3),0.98(d,J＝4Hz,3H,CH3),0.72(s,3H,CH3)；ESI-MS m/z Calc.C68H119N2O6[M+H2O+H]+1059.91,Found 1060.50.化合物10的核磁共振氢谱图见说明书附图中的图5。Linoleic acid (476 mg, 1.7 mmol) was dissolved in anhydrous DCM (5 mL), oxalyl chloride (0.30 mL) was added, and the mixture was stirred at room temperature for 5 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (2mL), and set aside. Compound 2 (240 mg, 0.34 mmol) and TEA (69 mg, 0.68 mmol) were dissolved in anhydrous DCM (5 mL). The above standby product was dropped into the reaction solution and allowed to react at room temperature overnight. Add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 10, a light yellow semi-solid product (212 mg, 60%). 1 HNMR (400MHz, CD 3 OD) δ5.34-5.36 (m, 8H), 1.01 (s, 3H, CH 3 ), 0.98 (d, J = 4Hz, 3H, CH 3 ), 0.72 (s, 3H, CH 3 ); ESI-MS m/z Calc.C 68 H 119 N 2 O 6 [M+H 2 O+H] + 1059.91, Found 1060.50. The hydrogen nuclear magnetic resonance spectrum of compound 10 is shown in the figure in the appendix of the description. 5.
实施例6：熊去氧胆酸衍生物13(化合物13)的合成
Example 6: Synthesis of Ursodeoxycholic Acid Derivative 13 (Compound 13)
中间体化合物11的合成：将化合物6(3.0g,6.2mmol)溶解于干燥的吡啶(20mL)中,依次加入DMAP(159mg,1.3mmol),乙酸酐(3.2g,31.0mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，PE/EA(2:1)洗脱，得白色固体产品化合物11(3.0g,86％)。1HNMR(400MHz,CDCl3)δ7.33-7.37(m,5H),5.08-5.15(m,2H),4.74-4.79(m,1H),4.64-4.70(m,1H)，2.03(s,3H),1.90(s,3H),0.97(s,3H),0.91(d,J＝8.0Hz,3H),0.75(s,3H).Synthesis of intermediate compound 11: Dissolve compound 6 (3.0g, 6.2mmol) in dry pyridine (20mL), add DMAP (159mg, 1.3mmol), acetic anhydride (3.2g, 31.0mmol) in sequence, and protect with nitrogen. The reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with PE/EA (2:1), gave compound 11 (3.0 g, 86%) as a white solid product. 1 HNMR (400MHz, CDCl 3 ) δ7.33-7.37(m,5H),5.08-5.15(m,2H),4.74-4.79(m,1H),4.64-4.70(m,1H), 2.03(s, 3H),1.90(s,3H),0.97(s,3H),0.91(d,J=8.0Hz,3H),0.75(s,3H).
中间体化合物12的合成：将化合物11(1.7g,3.0mmol)溶解于干燥的甲醇(40mL)中,加入K2CO3(0.83g,6.0mmol),室温搅拌反应2h。TLC检测，反应结束后，过滤，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，PE/EA(2:1)洗脱，得白色固体产品化合物12(1.1g,70％)。1HNMR(400MHz,CDCl3)δ4.74-4.81(m,1H),3.66(s,3H),3.54-3.62(m,1H),1.98(s,3H)，0.96(s,3H),0.91(d,J＝4.0Hz,3H),0.68(s,3H). Synthesis of intermediate compound 12: Dissolve compound 11 (1.7g, 3.0mmol) in dry methanol (40mL), add K 2 CO 3 (0.83g, 6.0mmol), and stir for 2 hours at room temperature. TLC detection, after the reaction is completed, filter, add an appropriate amount of water, extract three times with ethyl acetate, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with PE/EA (2:1), gave compound 12 (1.1 g, 70%) as a white solid product. 1 HNMR (400MHz, CDCl 3 ) δ4.74-4.81(m,1H),3.66(s,3H),3.54-3.62(m,1H),1.98(s,3H),0.96(s,3H),0.91 (d,J＝4.0Hz,3H),0.68(s,3H).
将化合物12(1.0g,2.2mmol)溶解于DMF(8mL)中,依次加入HBTU(1.15g,3.0mmol),DIEA(786mg,6.1mmol),4-二甲基氨基丁酸盐酸盐(509mg,3.0mmol)氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体产品化合物13(900mg,73％)。1HNMR(400MHz,d6-DMSO)δ4.56-4.68(m,2H),3.57(s,3H,OCH3),3.02-3.06(m,2H),2.77(s,6H),2.37(t,J＝8.0Hz,2H),1.94(s,3H),0.93(s,3H,CH3),0.87(d,J＝4Hz,3H,CH3),0.63(s,3H,CH3)；ESI-MS m/z Calc.C33H56N2O6[M+H]+562.41,Found 562.95.化合物13的核磁共振氢谱图见说明书附图中的图6。Compound 12 (1.0g, 2.2mmol) was dissolved in DMF (8mL), and HBTU (1.15g, 3.0mmol), DIEA (786mg, 6.1mmol), and 4-dimethylaminobutyric hydrochloride (509mg) were added in sequence. , 3.0 mmol) under nitrogen protection, and the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 13 (900 mg, 73%) as a white solid product. 1 HNMR (400MHz, d 6 -DMSO) δ4.56-4.68(m,2H),3.57(s,3H,OCH 3 ),3.02-3.06(m,2H),2.77(s,6H),2.37(t ,J＝8.0Hz,2H),1.94(s,3H),0.93(s,3H,CH 3 ),0.87(d,J＝4Hz,3H,CH 3 ),0.63(s,3H,CH 3 ); ESI-MS m/z Calc.C 33 H 56 N 2 O 6 [M+H] + 562.41, Found 562.95. The hydrogen nuclear magnetic resonance spectrum of compound 13 is shown in Figure 6 in the appendix of the description.
实施例7：熊去氧胆酸衍生物16(化合物16)的合成
Example 7: Synthesis of Ursodeoxycholic Acid Derivative 16 (Compound 16)
将化合物6(965mg,2.0mmol)溶解于DMF(30mL)中,依次加入HBTU(1.15g,3.0mmol),DIEA(517mg,4.0mmol),4-二甲基氨基丁酸盐酸盐(335mg,2.0mmol)氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体产品化合物16(400mg,33％).1HNMR(400MHz,d6-DMSO)δ7.33-7.38(m,5H),5.05-5.12(m,2H),4.55-4.63(m,1H),3.92(d,J＝8.0Hz,1H),2.98-3.02(m,2H),2.74(s,6H),2.23-2.43(m,4H),0.91(s,3H,CH3),0.87(d,J＝4Hz,3H,CH3),0.59(s,3H,CH3)；ESI-MS m/z Calc.C37H58NO5[M+H]+596.4,Found 597.3.化合物16的核磁共振氢谱图见说明书附图中的图7。Compound 6 (965mg, 2.0mmol) was dissolved in DMF (30mL), and HBTU (1.15g, 3.0mmol), DIEA (517mg, 4.0mmol), 4-dimethylaminobutyric hydrochloride (335mg, 2.0 mmol) under nitrogen protection, and the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave a white solid product compound 16 (400 mg, 33%). 1 HNMR (400MHz, d 6 -DMSO) δ7.33-7.38 (m, 5H) ),5.05-5.12(m,2H),4.55-4.63(m,1H),3.92(d,J＝8.0Hz,1H),2.98-3.02(m,2H),2.74(s,6H),2.23- 2.43(m,4H),0.91(s,3H,CH 3 ),0.87(d,J＝4Hz,3H,CH 3 ),0.59(s,3H,CH 3 ); ESI-MS m/z Calc.C 37 H 58 NO 5 [M+H] + 596.4, Found 597.3. The hydrogen nuclear magnetic resonance spectrum of compound 16 is shown in Figure 7 in the appendix of the description.
实施例8：熊去氧胆酸衍生物17(化合物17)的合成
Example 8: Synthesis of Ursodeoxycholic Acid Derivative 17 (Compound 17)
将化合物4(200mg,0.39mmol)溶解于干燥的吡啶(4mL)中,依次加入DMAP(19mg,0.16mmol),乙酸酐(0.5mL)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体产品化合物17(100mg,44％)。1HNMR(400MHz,d6-DMSO)δ4.52-4.60(m,1H),4.63-4.68(m,2H),3.08-3.12(m,2H),3.02-3.07(m,2H),1.98(s,3H),1.94(s,3H),0.93(s,3H),0.89(d,J＝8.0Hz,3H),0.63(s,3H).化合物17的核磁共振氢谱图见说明书附图中的图8。Compound 4 (200 mg, 0.39 mmol) was dissolved in dry pyridine (4 mL), DMAP (19 mg, 0.16 mmol) and acetic anhydride (0.5 mL) were added in sequence, under nitrogen protection, and the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 17 (100 mg, 44%) as a white solid product. 1 HNMR (400MHz, d 6 -DMSO) δ4.52-4.60(m,1H),4.63-4.68(m,2H),3.08-3.12(m,2H),3.02-3.07(m,2H),1.98( s, 3H), 1.94 (s, 3H), 0.93 (s, 3H), 0.89 (d, J = 8.0Hz, 3H), 0.63 (s, 3H). The hydrogen nuclear magnetic resonance spectrum of compound 17 is shown in the attached figure of the description. Figure 8 in.
实施例9：奥贝胆酸衍生物(化合物18)的合成
Example 9: Synthesis of Obeticholic Acid Derivative (Compound 18)
将奥贝胆酸(421mg,1mmol)溶解于DMF(8mL)中,依次加入HBTU(569mg,1.5mmol),DIEA(194mg,1.5mmol),N,N-二甲基乙二胺(132mg,1.5mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体化合物18(343mg,70％)。1HNMR(400MHz,d6-DMSO)δ7.83(brs,1H),4.31(brs,1H),4.05(d,J＝4.0Hz,1H),3.49(s,2H),2.36(s 2H),2.36(s,6H),0.88(d,J＝4.0Hz,3H),0.81-0.83(m,6H),0.60(s,3H).ESI-MS m/z Calc.C30H56N2O3[M+H]+491.42,Found  492.20.化合物18的核磁共振氢谱图见说明书附图中的图9。Dissolve obeticholic acid (421mg, 1mmol) in DMF (8mL), then add HBTU (569mg, 1.5mmol), DIEA (194mg, 1.5mmol), N,N-dimethylethylenediamine (132mg, 1.5 mmol), under nitrogen protection, and stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 18 as a white solid (343 mg, 70%). 1 HNMR (400MHz, d 6 -DMSO) δ7.83 (brs, 1H), 4.31 (brs, 1H), 4.05 (d, J = 4.0Hz, 1H), 3.49 (s, 2H), 2.36 (s 2H) ,2.36(s,6H),0.88(d,J＝4.0Hz,3H),0.81-0.83(m,6H),0.60(s,3H).ESI-MS m/z Calc.C 30 H 56 N 2 O 3 [M+H] + 491.42,Found 492.20. The hydrogen nuclear magnetic resonance spectrum of compound 18 is shown in Figure 9 in the appendix of the description.
实施例10：熊去氧胆酸衍生物(化合物19)的合成路线如下
Example 10: The synthetic route of ursodeoxycholic acid derivative (compound 19) is as follows
中间体19-1的合成：将化合物11(10.2g，18.0mmol)，钯碳(0.5g，5wt％)和甲醇依次加入反应瓶中，氢气氛围下，室温反应16个小时。TLC检测，反应结束后，直接过滤，有机相浓缩后，粗品中间体19-1(7.3g，85％)直接用于下一步。Synthesis of intermediate 19-1: Compound 11 (10.2g, 18.0mmol), palladium on carbon (0.5g, 5wt%) and methanol were added to the reaction bottle in sequence, and the reaction was carried out at room temperature for 16 hours under a hydrogen atmosphere. After TLC detection, the reaction was directly filtered. After the organic phase was concentrated, the crude intermediate 19-1 (7.3g, 85%) was directly used in the next step.
中间体19-2的合成：将中间体19-1(7.3g,15.0mmol)溶解于DMF中，依次加入HBTU(8.7g,23mmol)，DIEA(3.0g,23.0mmol)，N,N-二甲基丙二胺(2.4g,23.0mmol)，室温搅拌反应16小时。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体19-2(6.0g,69.5％)。1HNMR(400MHz,DMSO-d6)δ4.66(m,1H),4.55(m,1H),3.08(m,2H),3.00(m,2H),2.76(d,6H),2.11(m,1H),1.96(d,6H,CH3×2),0.92(s,3H,CH3),0.89(d,3H,CH3),0.63(s,3H,CH3).ESI-MS m/z Calc.C33H56N2O5[M+H]+561.42,Found 562.25.HPLC:71.8％。Synthesis of intermediate 19-2: Dissolve intermediate 19-1 (7.3g, 15.0mmol) in DMF, add HBTU (8.7g, 23mmol), DIEA (3.0g, 23.0mmol), N, N-di Methylpropanediamine (2.4g, 23.0mmol) was stirred at room temperature for 16 hours. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 19-2 (6.0 g, 69.5%). 1 HNMR (400MHz, DMSO-d 6 ) δ4.66(m,1H),4.55(m,1H),3.08(m,2H),3.00(m,2H),2.76(d,6H),2.11(m ,1H),1.96(d,6H,CH 3 ×2),0.92(s,3H,CH 3 ),0.89(d,3H,CH 3 ),0.63(s,3H,CH 3 ).ESI-MS m /z Calc.C 33 H 56 N 2 O 5 [M+H] + 561.42, Found 562.25. HPLC: 71.8%.
中间体19-3/19-4的合成：将中间体19-2(5.4g,10.0mmol)溶解于THF/MeOH(3：1)中,将NaOH(2.3g,58.0mmol)溶于H2O中，加入到上述反应液中，室温搅拌反应过夜。TLC检测，反应结束后，直接浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体19-3(2.7g,54.0％)和白色固体中间体19-4(1.8g,41.3％)。19-3:1HNMR(400MHz,DMSO-d6)δ7.85(t,1H),4.65(m,1H),4.50(s,1H),3.04(q,2H),2.69(t,2H),2.52(s,6H),2.08(m,1H), 1.93-2.0(s,5H),0.89-0.85(m,6H,CH3×2),0.62(s,3H,CH3).ESI-MS m/z Calc.C31H54N2O4[M+H]+519.41,Found 520.20.HPLC:74.9％。19-4:1HNMR(400MHz,DMSO-d6)δ7.75(s,1H),3.01(m,2H),2.16(t,2H),2.09(s,6H),1.90-1.97(m,3H),0.88-0.84(m,6H,CH3×2),0.59(s,3H,CH3).ESI-MS m/z Calc.C29H52N2O3[M+H]+477.40,Found 478.20.HPLC:76.4％。Synthesis of intermediate 19-3/19-4: Dissolve intermediate 19-2 (5.4g, 10.0mmol) in THF/MeOH (3:1), and dissolve NaOH (2.3g, 58.0mmol) in H 2 O, was added to the above reaction solution, and the reaction was stirred at room temperature overnight. After TLC detection, after the reaction is completed, the crude product is directly concentrated. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 19-3 (2.7g, 54.0%) and white solid intermediate 19-4 (1.8g, 41.3%). 19-3: 1 HNMR(400MHz, DMSO-d 6 )δ7.85(t,1H),4.65(m,1H),4.50(s,1H),3.04(q,2H),2.69(t,2H) ,2.52(s,6H),2.08(m,1H), 1.93-2.0(s,5H),0.89-0.85(m,6H,CH 3 ×2),0.62(s,3H,CH 3 ).ESI-MS m/z Calc.C 31 H 54 N 2 O 4 [ M+H] + 519.41, Found 520.20. HPLC: 74.9%. 19-4: 1 HNMR(400MHz, DMSO-d 6 )δ7.75(s,1H),3.01(m,2H),2.16(t,2H),2.09(s,6H),1.90-1.97(m, 3H),0.88-0.84(m,6H,CH 3 ×2),0.59(s,3H,CH 3 ).ESI-MS m/z Calc.C 29 H 52 N 2 O 3 [M+H] + 477.40 , Found 478.20. HPLC: 76.4%.
化合物19的合成：将亚油酸(1.4g,5.0mmol)溶解于DCM中，加入DMF(1滴)，滴加入草酰氯(1.2g,10.0mmol),室温搅拌4h。反应液直接浓缩拉干。将中间体19-3(500mg,1mmol)溶解于DCM中，加入Et3N(195mg,2.0mmol)，加入上述制备的酰氯，室温搅拌反应过夜。TLC检测，直接浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物19(97.7mg，12.5％)。1HNMR(400MHz,Methanol-d4)δ5.40-5.34(m,4H),4.65-4.60(m,2H),3.08-3.01(m,1H),2.80-2.77(m,2H),1.97(s,3H),1.94(s,6H),1.00-0.97(m,6H,CH3x 2),0.93-0.89(m,3H,CH3),0.72(s,3H,CH3)。Synthesis of compound 19: Dissolve linoleic acid (1.4g, 5.0mmol) in DCM, add DMF (1 drop), add oxalyl chloride (1.2g, 10.0mmol) dropwise, and stir at room temperature for 4 hours. The reaction solution was directly concentrated and drained. Intermediate 19-3 (500 mg, 1 mmol) was dissolved in DCM, Et 3 N (195 mg, 2.0 mmol) was added, the acid chloride prepared above was added, and the reaction was stirred at room temperature overnight. TLC detection, direct concentration to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 19 (97.7 mg, 12.5%) as a yellow oil. 1 HNMR (400MHz, Methanol-d4) δ5.40-5.34(m,4H),4.65-4.60(m,2H),3.08-3.01(m,1H),2.80-2.77(m,2H),1.97(s ,3H),1.94(s,6H),1.00-0.97(m,6H,CH 3 x 2),0.93-0.89(m,3H,CH 3 ),0.72(s,3H,CH 3 ).
实施例11：熊去氧胆酸衍生物(化合物20)的合成路线如下
Example 11: The synthetic route of ursodeoxycholic acid derivative (compound 20) is as follows
中间体20-1的合成：将化合物17(1.8g,3.0mmol)溶解于THF/MeOH(3:1)中,再加入NaOH(719mg,18mmol)的水溶液，室温搅拌过夜。TLC检测，反应结束后，直接浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色固体中间体20-1(720mg,43.1％)。1HNMR(400MHz,DMSO-d6)δ7.77(t,1H),4.66(m,1H),4.50(s,1H),3.17(s,1H),3.01(q,2H),2.58(d,2H),2.07(m,1H),1.97(m,2H),1.93(s,3H,CH3),0.92-0.84(m,6H,CH3×2),0.62(s,3H,CH3).HPLC:75.2％。ESI-MS m/z Calc.C34H58N2O4[M+H]+559.44,Found 560.30。Synthesis of intermediate 20-1: Dissolve compound 17 (1.8g, 3.0mmol) in THF/MeOH (3:1), add an aqueous solution of NaOH (719mg, 18mmol), and stir at room temperature overnight. After TLC detection, after the reaction is completed, the crude product is directly concentrated. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave light yellow solid intermediate 20-1 (720 mg, 43.1%). 1 HNMR (400MHz, DMSO-d 6 ) δ7.77(t,1H),4.66(m,1H),4.50(s,1H),3.17(s,1H),3.01(q,2H),2.58(d ,2H),2.07(m,1H),1.97(m,2H),1.93(s,3H,CH 3 ),0.92-0.84(m,6H,CH 3 ×2),0.62(s,3H,CH 3 ).HPLC:75.2%. ESI-MS m/z Calc.C 34 H 58 N 2 O 4 [M+H] + 559.44, Found 560.30.
化合物20的合成：将亚油酸(1.6g,6mmol)溶解于DCM中，加入草酰氯 (1.5g,12mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将中间体20-1(600mg,1.2mmol)溶解于DCM中，加入Et3N(235mg,2.3mmol)，加入上述制备的酰氯，室温搅拌反应过夜。TLC检测，反应结束后，直接浓缩粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物20(194.4mg,22.0％)。1HNMR(400MHz,Methanol-d4)δ7.86(s，1H)5.51–5.20(m,4H),4.60-4.67(m,1H),4.51–4.58(m,1H),2.70-2.74(t,2H),2.20-2.24(t,2H),1.92-2.01–2.00(m,14H),0.91(s,3H,CH3),0.84-0.87(t,6H,CH3×2),0.61(s,3H,CH3).HPLC:71.6％。Synthesis of compound 20: Dissolve linoleic acid (1.6g, 6mmol) in DCM, add oxalyl chloride (1.5g, 12mmol) and add DMF (1 drop) and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Intermediate 20-1 (600 mg, 1.2 mmol) was dissolved in DCM, Et 3 N (235 mg, 2.3 mmol) was added, the acid chloride prepared above was added, and the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, the crude product is directly concentrated. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 20 (194.4 mg, 22.0%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ7.86(s,1H)5.51–5.20(m,4H),4.60-4.67(m,1H),4.51–4.58(m,1H),2.70-2.74(t, 2H),2.20-2.24(t,2H),1.92-2.01–2.00(m,14H),0.91(s,3H,CH 3 ),0.84-0.87(t,6H,CH 3 ×2),0.61(s ,3H,CH 3 ).HPLC: 71.6%.
实施例12：熊去氧胆酸衍生物(化合物21)的合成路线如下
Example 12: The synthetic route of ursodeoxycholic acid derivative (compound 21) is as follows
将亚油酸(1.5g,5.3mmol)溶解于DCM中，滴加入草酰氯(1.3g,11.0mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将中间体19-4(500mg,1.1mmol)溶解于DCM中，加入Et3N(212mg,2.1mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，直接浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物21(197mg，18％)。1HNMR(400MHz,Methanol-d4)δ5.34-5.36(m,8H),4.78-4.80(m,1H),4.62-4.68(m,1H),3.23(t,2H),2.77-2.82(m,6H),2.62(s,6H),2.31-2.18(m,6H),2.05-2.10(m,11H),1.01(s，3H,CH3)0.97-0.99(d,3H,CH3),0.90-0.93(m,6H,2x CH3),0.72(s,3H,CH3).HPLC:80.76％。Dissolve linoleic acid (1.5g, 5.3mmol) in DCM, add oxalyl chloride (1.3g, 11.0mmol) dropwise, add DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Intermediate 19-4 (500 mg, 1.1 mmol) was dissolved in DCM, Et 3 N (212 mg, 2.1 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. After TLC detection, after the reaction is completed, the crude product is directly concentrated. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 21 (197 mg, 18%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ5.34-5.36(m,8H),4.78-4.80(m,1H),4.62-4.68(m,1H),3.23(t,2H),2.77-2.82(m ,6H),2.62(s,6H),2.31-2.18(m,6H),2.05-2.10(m,11H),1.01(s,3H,CH 3 )0.97-0.99(d,3H,CH 3 ), 0.90-0.93 (m, 6H, 2x CH 3 ), 0.72 (s, 3H, CH 3 ). HPLC: 80.76%.
实施例13：熊去氧胆酸衍生物(化合物22)的合成路线如下
Example 13: The synthetic route of ursodeoxycholic acid derivative (compound 22) is as follows
将油酸(1.4g,5.0mmol)溶解于DCM中，加入草酰氯(1.3g,10.0mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将中间体19-4(480mg,1.0mmol)溶解于DCM中，加入Et3N(204mg,2.0mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，直接浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物22(98.5mg,9.8％)。1HNMR(400MHz,Methanol-d4)δ5.38-5.30(m,4H),4.80-4.73(m,1H),4.70-4.61(m,1H),3.34(s,2H),3.14-3.08(m,2H),2.88(s,6H),2.34-2.18(m,6H),2.12-2.00(m,9H),1.00(s,3H,CH3),0.97-0.98(d,3H,CH3),0.89-0.92(m,6H,2x CH3),0.72(s,3H,CH3)。Dissolve oleic acid (1.4g, 5.0mmol) in DCM, add oxalyl chloride (1.3g, 10.0mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Intermediate 19-4 (480 mg, 1.0 mmol) was dissolved in DCM, Et 3 N (204 mg, 2.0 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, direct concentration to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 22 (98.5 mg, 9.8%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ5.38-5.30(m,4H),4.80-4.73(m,1H),4.70-4.61(m,1H),3.34(s,2H),3.14-3.08(m ,2H),2.88(s,6H),2.34-2.18(m,6H),2.12-2.00(m,9H),1.00(s,3H,CH 3 ),0.97-0.98(d,3H,CH 3 ) ,0.89-0.92(m,6H,2x CH 3 ),0.72(s,3H,CH 3 ).
实施例14：熊去氧胆酸衍生物(化合物23)的合成路线如下
Example 14: The synthetic route of ursodeoxycholic acid derivative (compound 23) is as follows
将硬脂酸(1.4g,5.0mmol)溶解于DCM中，加入草酰氯(1.3g,10mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将中间体19-4(480mg,1.0mmol)溶解于DCM中，加入Et3N(204mg,2.0mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，直接浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状物23(290mg，28.7％)。1HNMR(400MHz,Methanol-d4)δ4.78(m,1H),4.71-4.59(m,1H),3.17-3.20(t,2H),2.43-2.47(t,2H),2.33(s,6H),2.19-2.31(m,5H),2.04-2.11(m,2H),1.01(s,3H,CH3),0.97-0.98(d,3H,CH3),0.88-0.92(t,6H,2x CH3),0.72(s,3H,CH3)。Dissolve stearic acid (1.4g, 5.0mmol) in DCM, add oxalyl chloride (1.3g, 10mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Intermediate 19-4 (480 mg, 1.0 mmol) was dissolved in DCM, Et 3 N (204 mg, 2.0 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. After TLC detection, after the reaction is completed, the crude product is directly concentrated. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave light yellow oil 23 (290 mg, 28.7%). 1 HNMR(400MHz,Methanol-d4)δ4.78(m,1H),4.71-4.59(m,1H),3.17-3.20(t,2H),2.43-2.47(t,2H),2.33(s,6H ),2.19-2.31(m,5H),2.04-2.11(m,2H),1.01(s,3H,CH 3 ),0.97-0.98(d,3H,CH 3 ),0.88-0.92(t,6H, 2x CH 3 ),0.72(s,3H,CH 3 ).
实施例15：熊去氧胆酸衍生物(化合物24)的合成路线如下
Example 15: The synthetic route of ursodeoxycholic acid derivative (compound 24) is as follows
将油酸(2.2g,7.7mmol)溶解于DCM中，加入草酰氯(4.9g,38.7mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将化合物2(500mg,1.0mmol)溶解于DCM中，加入Et3N(196mg,2.0mmol)，加入上述制备的酰氯，室温搅拌反应过夜。TLC检测，反应结束后，反应液浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物24(247.3mg,23.6％)。1HNMR(400MHz,Methanol-d4)δ5.34-5.36(m,4H),4.76-4.80(m,1H),4.65-4.67(m,1H),3.34(s,2H),3.17-3.22(m,4H),2.23–2.29(m,6H),2.04-2.09(m,11H),1.00(s,3H,CH3),0.96-0.98(d,3H,CH3),0.90-0.92(m,6H,CH3x 2),0.72(s,3H,CH3)。Dissolve oleic acid (2.2g, 7.7mmol) in DCM, add oxalyl chloride (4.9g, 38.7mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (500 mg, 1.0 mmol) was dissolved in DCM, Et 3 N (196 mg, 2.0 mmol) was added, the acid chloride prepared above was added, and the reaction was stirred at room temperature overnight. After TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 24 (247.3 mg, 23.6%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ5.34-5.36(m,4H),4.76-4.80(m,1H),4.65-4.67(m,1H),3.34(s,2H),3.17-3.22(m ,4H),2.23–2.29(m,6H),2.04-2.09(m,11H),1.00(s,3H,CH 3 ),0.96-0.98(d,3H,CH 3 ),0.90-0.92(m, 6H,CH 3 x 2),0.72(s,3H,CH 3 ).
实施例16：熊去氧胆酸衍生物(化合物25)的合成路线如下
Example 16: The synthetic route of ursodeoxycholic acid derivative (compound 25) is as follows
将硬脂酸(2.2g,7.7mmol)溶解于DCM中，加入草酰氯(4.9g,38.7mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将化合物2(500mg,1.0mmol)溶解于DCM中，加入Et3N(196mg,2.0mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，反应液浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物25(323.8mg,30.9％)。1HNMR(400MHz,Methanol-d4)δ4.58-4.66(m,2H),3.13-3.23(m,7H),2.21-2.31(m,5H),2.05-2.14(m,6H),1.01(s,3H,CH3),0.97-0.99(d,3H,CH3),0.89-0.92(m,6H,CH3x2),0.72(s,3H,CH3)。Dissolve stearic acid (2.2g, 7.7mmol) in DCM, add oxalyl chloride (4.9g, 38.7mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (500 mg, 1.0 mmol) was dissolved in DCM, Et 3 N (196 mg, 2.0 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. After TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 25 (323.8 mg, 30.9%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ4.58-4.66(m,2H),3.13-3.23(m,7H),2.21-2.31(m,5H),2.05-2.14(m,6H),1.01(s ,3H,CH 3 ),0.97-0.99(d,3H,CH 3 ),0.89-0.92(m,6H,CH 3 x2),0.72(s,3H,CH 3 ).
实施例17：熊去氧胆酸衍生物(化合物26)的合成路线如下
Example 17: The synthetic route of ursodeoxycholic acid derivative (compound 26) is as follows
将肉桂酸(1.29g,8.7mmol)溶解于DCM中，加入草酰氯(11.1g,87mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将化合物2(450mg,0.9mmol)溶解于DCM中，加入Et3N(180mg,1.8mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，反应液浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物26(116mg,16.6％)。1HNMR(400MHz,Methanol-d4)δ7.55-7.72(m,7H),7.37-7.43(m,7H),6.45-6.54(m,2H),4.91-4.95(m，2H),2.20-2.26(m,1H),2.02-2.09(m,6H),1.06(s,3H,CH3),0.97-0.98(d,3H,CH3),0.75(s,3H,CH3)。Dissolve cinnamic acid (1.29g, 8.7mmol) in DCM, add oxalyl chloride (11.1g, 87mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (450 mg, 0.9 mmol) was dissolved in DCM, Et 3 N (180 mg, 1.8 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. After TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 26 (116 mg, 16.6%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ7.55-7.72(m,7H),7.37-7.43(m,7H),6.45-6.54(m,2H),4.91-4.95(m,2H),2.20-2.26 (m,1H),2.02-2.09(m,6H),1.06(s,3H,CH 3 ),0.97-0.98(d,3H,CH 3 ),0.75(s,3H,CH 3 ).
实施例18：熊去氧胆酸衍生物(化合物27)的合成路线如下
Example 18: The synthetic route of ursodeoxycholic acid derivative (compound 27) is as follows
将2-辛基癸酸(0.9g,3.1mmol)溶解于DCM中，加入草酰氯(2.0g,16mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将化合物2(200mg,0.4mmol)溶解于DCM中，加入Et3N(78mg,0.8mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应液浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物27(54.1mg，12.9％)。1HNMR(400MHz,Methanol-d4)δ4.75(s,1H),4.73-4.64(m,1H),4.58(s,2H),3.17(m,4H),2.37-2.17(m,3H),2.14-2.01(m,6H),1.93-1.19(m,87H),1.19-1.09(m,3H),1.02(s,3H,CH3),0.98(d,3H,CH3),0.94-0.86(m,12H,CH3x 4),0.74(s,3H,CH3)。HPLC:92.42％。Dissolve 2-octyldecanoic acid (0.9g, 3.1mmol) in DCM, add oxalyl chloride (2.0g, 16mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (200 mg, 0.4 mmol) was dissolved in DCM, Et 3 N (78 mg, 0.8 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 27 (54.1 mg, 12.9%) as a yellow oil. 1 HNMR(400MHz,Methanol-d4)δ4.75(s,1H),4.73-4.64(m,1H),4.58(s,2H),3.17(m,4H),2.37-2.17(m,3H), 2.14-2.01(m,6H),1.93-1.19(m,87H),1.19-1.09(m,3H),1.02(s,3H,CH 3 ),0.98(d,3H,CH 3 ),0.94-0.86 (m,12H,CH 3 x 4),0.74(s,3H,CH 3 ). HPLC: 92.42%.
实施例19：熊去氧胆酸衍生物(化合物28)的合成路线如下
Example 19: The synthetic route of ursodeoxycholic acid derivative (compound 28) is as follows
将正辛酸(0.4g,3.1mmol)溶解于DCM中，加入草酰氯(2.0g,16mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将化合物2(200mg,0.4mmol)溶解于DCM中，加入Et3N(78mg,0.8mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物28(58.7mg,19％)。1HNMR(400MHz,Methanol-d4)δ4.80-4.76(m,1H),4.65-4.69(m,1H),4.58(s,2H),3.14-3.23(m,4H),2.24-2.31(m,5H),2.05-2.09(m,6H),1.86-1.90(m,1H),1.01(s,3H,CH3),0.97-0.99(d,3H,CH3),0.89-0.93(m,6H,CH3x2),0.73(s,3H,CH3).HPLC:88.4％。Dissolve n-octanoic acid (0.4g, 3.1mmol) in DCM, add oxalyl chloride (2.0g, 16mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (200 mg, 0.4 mmol) was dissolved in DCM, Et 3 N (78 mg, 0.8 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 28 (58.7 mg, 19%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ4.80-4.76(m,1H),4.65-4.69(m,1H),4.58(s,2H),3.14-3.23(m,4H),2.24-2.31(m ,5H),2.05-2.09(m,6H),1.86-1.90(m,1H),1.01(s,3H,CH 3 ),0.97-0.99(d,3H,CH 3 ),0.89-0.93(m, 6H,CH 3 x2), 0.73 (s, 3H, CH 3 ). HPLC: 88.4%.
实施例20：熊去氧胆酸衍生物(化合物29)的合成路线如下
Example 20: The synthetic route of ursodeoxycholic acid derivative (compound 29) is as follows
将亚油酸(1.72g,6.12mmol)溶解于DCM中,加入1滴DMF,加入草酰氯(0.78g,6.12mmol)，室温搅拌5小时。TLC检测，反应液直接浓缩干。将中间体37-1(0.50g,1.02mmol)，三乙胺(3.1g,30.60mmol)溶解于DCM中,将上述亚油酰氯加入该反应液中。室温搅拌16小时。TLC检测，反应完全后，浓缩溶剂得粗品，硅胶柱层析，DCM/CH3OH(20:1)洗脱，得淡黄色油状化合物29(130mg,17％)。1HNMR(400MHz,CDCl3,)δ5.41-5.29(m,4H),4.71-4.63(m,1H),4.08(t,2H),3.60-3.55(m,1H),2.75-2.68(t,2H),2.43-2.40(t,2H,CH2),2.32(s,6H,CH3×2),0.95(s,3H,CH3),0.93-0.91(d,3H,CH3),0.90-0.87(m,3H, CH3),0.67(s,3H,CH3)。HPLC:85.63％。Dissolve linoleic acid (1.72g, 6.12mmol) in DCM, add 1 drop of DMF, add oxalyl chloride (0.78g, 6.12mmol), and stir at room temperature for 5 hours. For TLC detection, the reaction solution was directly concentrated to dryness. Intermediate 37-1 (0.50g, 1.02mmol) and triethylamine (3.1g, 30.60mmol) were dissolved in DCM, and the above-mentioned linoleoyl chloride was added to the reaction solution. Stir at room temperature for 16 hours. TLC detection showed that after the reaction was complete, the solvent was concentrated to obtain a crude product, which was then subjected to silica gel column chromatography and eluted with DCM/CH 3 OH (20:1) to obtain light yellow oily compound 29 (130 mg, 17%). 1 HNMR (400MHz, CDCl 3 ,)δ5.41-5.29(m,4H),4.71-4.63(m,1H),4.08(t,2H),3.60-3.55(m,1H),2.75-2.68(t ,2H),2.43-2.40(t,2H,CH 2 ),2.32(s,6H,CH 3 ×2),0.95(s,3H,CH 3 ),0.93-0.91(d,3H,CH 3 ), 0.90-0.87(m,3H, CH 3 ),0.67(s,3H,CH 3 ). HPLC: 85.63%.
实施例21：熊去氧胆酸衍生物(化合物30)的合成路线如下
Example 21: The synthetic route of ursodeoxycholic acid derivative (compound 30) is as follows
中间体30-1的合成：将石胆酸(2.0g,5.3mmol)溶解于THF中，依次加入TEA(1.1g,11mmol)、HBTU(2.4g,6.4mmol)和4-吡咯烷丁胺(0.9g,6.4mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩反应液，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状中间体30-1(1.9g,72％)。1HNMR(400MHz,DMSO-d6)δ4.46(d,1H),3.49(s,2H),3.16-2.86(m,8H),0.90-0.85(m,6H,CH3x2),0.60(s,3H,CH3).Synthesis of intermediate 30-1: Dissolve lithocholic acid (2.0g, 5.3mmol) in THF, add TEA (1.1g, 11mmol), HBTU (2.4g, 6.4mmol) and 4-pyrrolidinebutylamine ( 0.9g, 6.4mmol), stir at room temperature overnight. After TLC detection, the reaction solution was concentrated and subjected to silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain yellow oily intermediate 30-1 (1.9 g, 72%). 1 HNMR(400MHz,DMSO-d6)δ4.46(d,1H),3.49(s,2H),3.16-2.86(m,8H),0.90-0.85(m,6H,CH 3 x2),0.60(s ,3H,CH 3 ).
化合物30的合成：将亚油酸(0.56g,2.0mmol)溶解于DCM中，滴加入草酰氯(1.27g,10mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将中间体30-1(250mg,0.5mmol)溶解于DCM中，加入Et3N(101mg,1.0mmol)，缓慢滴加上述制备的酰氯的DCM溶液，室温搅拌反应过夜。TLC检测，反应液浓缩得粗品。经硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物30(122mg，32％)。1HNMR(400MHz,Methanol-d4)δ5.42-5.31(m,4H),4.75-4.67(m,1H),4.59(s,4H),3.27-3.14(m,4H),2.82-2.79(m,2H),2.30(t,3H),1.00-0.96(m,6H,CH3x2),0.93-0.91(m,3H,CH3),0.71(s,3H,CH3).HPLC:67.1％。Synthesis of compound 30: Dissolve linoleic acid (0.56g, 2.0mmol) in DCM, add oxalyl chloride (1.27g, 10mmol) dropwise, add DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Intermediate 30-1 (250 mg, 0.5 mmol) was dissolved in DCM, Et 3 N (101 mg, 1.0 mmol) was added, the acid chloride solution in DCM prepared above was slowly added dropwise, and the reaction was stirred at room temperature overnight. TLC detection, the reaction solution was concentrated to obtain crude product. After silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), compound 30 (122 mg, 32%) was obtained as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ5.42-5.31(m,4H),4.75-4.67(m,1H),4.59(s,4H),3.27-3.14(m,4H),2.82-2.79(m ,2H),2.30(t,3H),1.00-0.96(m,6H,CH 3 x2),0.93-0.91(m,3H,CH 3 ),0.71(s,3H,CH 3 ).HPLC:67.1% .
实施例22：熊去氧胆酸衍生物(化合物31)的合成路线如下
Example 22: The synthesis route of ursodeoxycholic acid derivative (compound 31) is as follows
中间体31-1的合成：将石胆酸(2.0g,5.3mmol)溶解于THF中，依次加入TEA(1.1g,11mmol)、HBTU(2.4g,6.4mmol)和3-二甲胺基丙胺(0.7g,6.4mmol)，室温搅拌过夜。TLC检测，反应结束后，反应液浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物31-1(1.7g,70％)。1HNMR(400MHz,DMSO-d6)δ2.89-2.86(m，2H)2.63-2.60(m,2H),2.45(s,6H),2.70(s,4H),2.63(d,2H),2.45(d,6H),0.91-0.86(m,6H,CH3x2),0.61(s,3H,CH3).Synthesis of intermediate 31-1: Dissolve lithocholic acid (2.0g, 5.3mmol) in THF, add TEA (1.1g, 11mmol), HBTU (2.4g, 6.4mmol) and 3-dimethylaminopropylamine in sequence (0.7g, 6.4mmol), stirred at room temperature overnight. After TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 31-1 (1.7 g, 70%) as a yellow oil. 1 HNMR(400MHz,DMSO-d6)δ2.89-2.86(m,2H)2.63-2.60(m,2H),2.45(s,6H),2.70(s,4H),2.63(d,2H),2.45 (d,6H),0.91-0.86(m,6H,CH 3 x2),0.61(s,3H,CH 3 ).
化合物31的合成：将亚油酸(0.61g,2.2mmol)溶解于DCM中，加入草酰氯(1.38g,11mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将中间体31-1(250mg,0.5mmol)溶解于DCM中，加入Et3N(110mg,1.1mmol)，加入上述制备的酰氯，室温搅拌反应过夜。TLC检测，反应结束后，反应液浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物31(109mg,30％)。1HNMR(400MHz,Methanol-d4)δ5.43-5.31(m,4H),4.76-4.68m,1H),4.59(s,3H),3.29-3.26(t,2H),3.07-3.03(m,2H),2.84(s,6H),2.82-2.79(m,2H),2.35-2.26(m,3H),1.00-0.96(m,6H,CH3x 2),0.95-0.92(m,3H,CH3),0.71(s,3H,CH3).HPLC:69.6％。Synthesis of compound 31: Dissolve linoleic acid (0.61g, 2.2mmol) in DCM, add oxalyl chloride (1.38g, 11mmol) and DMF (1 drop), and stir at room temperature for 5 hours. The reaction solution was directly concentrated to dryness. Intermediate 31-1 (250 mg, 0.5 mmol) was dissolved in DCM, Et 3 N (110 mg, 1.1 mmol) was added, the acid chloride prepared above was added, and the reaction was stirred at room temperature overnight. After TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 31 (109 mg, 30%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ5.43-5.31(m,4H),4.76-4.68m,1H),4.59(s,3H),3.29-3.26(t,2H),3.07-3.03(m, 2H),2.84(s,6H),2.82-2.79(m,2H),2.35-2.26(m,3H),1.00-0.96(m,6H,CH 3 x 2),0.95-0.92(m,3H, CH 3 ), 0.71 (s, 3H, CH 3 ). HPLC: 69.6%.
实施例23：石胆酸衍生物(化合物32)的合成路线如下
Example 23: The synthesis route of lithocholic acid derivative (compound 32) is as follows
中间体32-1的合成：将石胆酸(1.00g,2.6mmol)，溴代十八烷(0.97g,2.9mmol)，碳酸钾(1.87g,13.5mmol)溶解于DMSO中,室温搅拌24小时。TLC检测，反应液乙酸乙酯稀释，再用水洗三次，无水硫酸钠干燥，过滤，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(15:1)洗脱，得到白色固体化合物32-1(1.48g,88％)。1HNMR(400MHz,CD3OD,)δ4.05(t,2H),3.66-3.60(m,1H),2.38-2.30(m,1H),2.25-2.17(m,1H),0.92-0.86(m,9H,CH3×3),0.64(s,3H,CH3).Synthesis of intermediate 32-1: Dissolve lithocholic acid (1.00g, 2.6mmol), bromooctadecane (0.97g, 2.9mmol), and potassium carbonate (1.87g, 13.5mmol) in DMSO, stir at room temperature for 24 Hour. For TLC detection, the reaction solution was diluted with ethyl acetate, washed three times with water, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (15:1), gave compound 32-1 (1.48g, 88%) as a white solid. 1 HNMR (400MHz, CD 3 OD,) δ4.05(t,2H),3.66-3.60(m,1H),2.38-2.30(m,1H),2.25-2.17(m,1H),0.92-0.86( m,9H,CH 3 ×3),0.64(s,3H,CH 3 ).
化合物32的合成：将二甲氨基丁酸盐酸盐(0.40g,2.40mmol)溶解于DCM中,加入1滴DMF和草酰氯(0.31g,2.40mmol)，室温搅拌4小时。直接浓缩得酰氯粗品。将中间体32-1(0.40g,0.63mmol)，三乙胺(1.20g,12.00mmol)溶解于THF中,加入上述酰氯粗品，室温搅拌16小时。TLC检测，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体化合物32(410mg,87％)。1HNMR(400MHz,CD3OD,)δ4.76-4.70(m,1H),4.05(t,2H),2.45(t,2H),2.34(s,6H,CH3×2),0.92-0.86(m,9H,CH3×3),0.64(s,3H,CH3).Synthesis of compound 32: Dissolve dimethylaminobutyric hydrochloride (0.40g, 2.40mmol) in DCM, add 1 drop of DMF and oxalyl chloride (0.31g, 2.40mmol), and stir at room temperature for 4 hours. Concentrate directly to obtain crude acid chloride. Dissolve intermediate 32-1 (0.40g, 0.63mmol) and triethylamine (1.20g, 12.00mmol) in THF, add the above crude acid chloride, and stir at room temperature for 16 hours. After TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 32 as a white solid (410 mg, 87%). 1 HNMR (400MHz, CD 3 OD,) δ4.76-4.70 (m, 1H), 4.05 (t, 2H), 2.45 (t, 2H), 2.34 (s, 6H, CH 3 × 2), 0.92-0.86 (m,9H,CH 3 ×3),0.64(s,3H,CH 3 ).
实施例24：熊去氧胆酸衍生物(化合物33)的合成路线如下
Example 24: The synthesis route of ursodeoxycholic acid derivative (compound 33) is as follows
将软脂酸(1.0g,3.8mmol)溶解于DCM中，加入草酰氯(2.5g,19mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将化合物2(250mg,0.5mmol)溶解于DCM中，加入Et3N(98mg,1.0mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，反应液浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物33(77.5mg，15％)。1HNMR(400MHz,Methanol-d4)δ4.66(m,1H),4.53(s,1H),3.26(s,3H),3.20(t,2H),3.16-3.09(m,2H),2.26(m,5H),2.06(m,5H),1.01(s,3H,CH3),0.97-0.98(d,3H),0.88-0.92(t,6H,2x CH3),0.72(s,3H,CH3).HPLC:97.08％。 Dissolve palmitic acid (1.0g, 3.8mmol) in DCM, add oxalyl chloride (2.5g, 19mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (250 mg, 0.5 mmol) was dissolved in DCM, Et 3 N (98 mg, 1.0 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. After TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 33 (77.5 mg, 15%) as a yellow oil. 1 HNMR(400MHz,Methanol-d4)δ4.66(m,1H),4.53(s,1H),3.26(s,3H),3.20(t,2H),3.16-3.09(m,2H),2.26( m,5H),2.06(m,5H),1.01(s,3H,CH 3 ),0.97-0.98(d,3H),0.88-0.92(t,6H,2x CH 3 ),0.72(s,3H, CH 3 ). HPLC: 97.08%.
实施例25：胆固醇衍生物(化合物34)的合成路线如下
Example 25: The synthetic route of cholesterol derivative (compound 34) is as follows
将3-(咪唑-4-基)丙酸(0.27g,1.9mmol)溶解于DCM中，加入草酰氯(0.74g,5.8mmol)并加入DMF(1滴)，室温搅拌5h。反应液直接浓缩干。将胆固醇(250mg,0.65mmol)溶解于DCM中，加入Et3N(131mg,1.3mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，反应液浓缩得粗品。硅胶柱层析，PE/EA(15:1)洗脱，得白色固体化合物34(65mg,19.7％)。1HNMR(400MHz,Chloroform-d)δ7.54(d,1H),6.80(d,1H),5.37-5.38(d,1H),4.59-4.67(m,1H),2.90-2.93(t,2H),2.62-2.65(t,2H),2.29-2.31(m,2H),1.94-2.04(m,2H),1.79–1.88(m,3H),1.01(s,3H,CH3),0.91-0.92(d,3H,CH3),0.85-0.88(m,6H,CH3x 2),0.68(s,3H,CH3).HPLC:81.8％。Dissolve 3-(imidazol-4-yl)propionic acid (0.27g, 1.9mmol) in DCM, add oxalyl chloride (0.74g, 5.8mmol) and DMF (1 drop), and stir at room temperature for 5h. The reaction solution was directly concentrated to dryness. Dissolve cholesterol (250 mg, 0.65 mmol) in DCM, add Et 3 N (131 mg, 1.3 mmol), add the acid chloride prepared above, and stir at room temperature overnight. After TLC detection, the reaction solution was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (15:1), gave compound 34 (65 mg, 19.7%) as a white solid. 1 HNMR(400MHz,Chloroform-d)δ7.54(d,1H),6.80(d,1H),5.37-5.38(d,1H),4.59-4.67(m,1H),2.90-2.93(t,2H ),2.62-2.65(t,2H),2.29-2.31(m,2H),1.94-2.04(m,2H),1.79–1.88(m,3H),1.01(s,3H,CH 3 ),0.91- 0.92(d,3H,CH 3 ), 0.85-0.88(m,6H,CH 3 x 2), 0.68(s,3H,CH 3 ). HPLC: 81.8%.
实施例26：熊去氧胆酸衍生物(化合物35)的合成路线如下
Example 26: The synthetic route of ursodeoxycholic acid derivative (compound 35) is as follows
中间体35-1的合成：将石胆酸(5.0g,13mmol)溶解于DMF中，依次加入K2CO3(1.8g,13mmol)和碘甲烷(1.9g,13mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩反应液，硅胶柱层析，PE/EA(1:1)洗脱，得白色固体中间体35-1(3.8g,75％)。Synthesis of intermediate 35-1: Dissolve lithocholic acid (5.0g, 13mmol) in DMF, add K 2 CO 3 (1.8g, 13mmol) and methyl iodide (1.9g, 13mmol) in sequence, and stir at room temperature overnight. After TLC detection, the reaction solution was concentrated and subjected to silica gel column chromatography and PE/EA (1:1) elution to obtain white solid intermediate 35-1 (3.8 g, 75%).
化合物35的合成：将中间体35-1(300mg,0.77mmol)溶解于DMF中，依次加入3-(咪唑-4-基)丙酸(129mg,0.92mmol)、HBTU(437mg,1.2mmol)和DIEA(298mg,2.3mmol)，室温搅拌过夜。TLC检测，反应结束后，反应液浓缩得粗品,硅胶柱层析，DCM/MeOH(20:1)洗脱，得白色固体化合物35(200mg，51％)。1HNMR(400MHz,Chloroform-d)δ7.85(s,1H),6.90(s,1H),4.79-4.71(m,1H),3.66(s,3H,CH3),2.96-2.93(t,2H),2.66-2.63(t,2H),2.35-2.33(m,1H),2.24-2.22(m,1H),0.93-0.90(m,6H,CH3x 2),0.64(s,3H,CH3).Synthesis of compound 35: Dissolve intermediate 35-1 (300 mg, 0.77 mmol) in DMF, and add 3-(imidazol-4-yl)propionic acid (129 mg, 0.92 mmol), HBTU (437 mg, 1.2 mmol) and DIEA (298 mg, 2.3 mmol), stirred at room temperature overnight. After TLC detection, the reaction solution was concentrated to obtain a crude product, which was subjected to silica gel column chromatography and eluted with DCM/MeOH (20:1) to obtain compound 35 as a white solid (200 mg, 51%). 1 HNMR(400MHz,Chloroform-d)δ7.85(s,1H),6.90(s,1H),4.79-4.71(m,1H),3.66(s,3H,CH 3 ),2.96-2.93(t, 2H),2.66-2.63(t,2H),2.35-2.33(m,1H),2.24-2.22(m,1H),0.93-0.90(m,6H,CH 3 x 2),0.64(s,3H, CH 3 ).
实施例27：胆酸衍生物(化合物36)的合成路线如下
Example 27: The synthetic route of cholic acid derivative (compound 36) is as follows
中间体36-1的合成：将胆酸(2.00g,4.89mmol),碘甲烷(0.73g,5.13mmol),碳酸钾(1.01g,7.34mmol)溶解在DMF中，室温搅拌16小时。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得到白色固体中间体36-1(1.75g,84％)。1HNMR(400MHz,CD3OD,)δ3.98-3.96(m,1H),3.86-3.84(m,1H),3.66(s,3H,CH3),3.47-3.41(m,1H),2.41-2.34(m,1H),2.28-2.16(m,4H),0.99-0.97(d,3H,CH3),0.89(s,3H,CH3),0.68(s,3H,CH3).HPLC：94.00％。Synthesis of intermediate 36-1: Dissolve cholic acid (2.00g, 4.89mmol), methyl iodide (0.73g, 5.13mmol), and potassium carbonate (1.01g, 7.34mmol) in DMF, and stir at room temperature for 16 hours. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (20:1), gave white solid intermediate 36-1 (1.75 g, 84%). 1 HNMR (400MHz, CD 3 OD,) δ3.98-3.96(m,1H),3.86-3.84(m,1H),3.66(s,3H,CH3),3.47-3.41(m,1H),2.41- 2.34(m,1H),2.28-2.16(m,4H),0.99-0.97(d,3H,CH3),0.89(s,3H,CH3),0.68(s,3H,CH3). HPLC: 94.00%.
中间体36-2的合成：将中间体36-1(1.20g,2.84mmol),4-二甲基胺基丁酸盐酸盐(0.48g,2.84mmol),HBTU(1.62g,4.26mmol)溶解在DMF中，再加入DIPEA(1.6mL,8.52mmol),室温搅拌16小时。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得到白色固体中间体36-2(0.82g,54％)。1HNMR(400MHz,CD3OD,)δ4.62-4.54(m,1H),3.99-3.97(m,1H),3.85-3.84(m,1H),3.66(s,3H,CH3),3.37-3.29(m,6H),2.26(s,6H,CH3×2),0.97-0.99(d,3H,CH3),0.90(s,3H,CH3),0.69(s,3H,CH3).ESI-MS m/z Calc.C31H54NO6[M+H]+536.39,Found 536.85.Synthesis of intermediate 36-2: Combine intermediate 36-1 (1.20g, 2.84mmol), 4-dimethylaminobutyrate hydrochloride (0.48g, 2.84mmol), HBTU (1.62g, 4.26mmol) Dissolve in DMF, add DIPEA (1.6 mL, 8.52 mmol), and stir at room temperature for 16 hours. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (20:1), gave white solid intermediate 36-2 (0.82g, 54%). 1HNMR (400MHz, CD 3 OD,) δ4.62-4.54(m,1H),3.99-3.97(m,1H),3.85-3.84(m,1H),3.66(s,3H,CH3),3.37-3.29 (m,6H),2.26(s,6H,CH3×2),0.97-0.99(d,3H,CH3),0.90(s,3H,CH3),0.69(s,3H,CH3).ESI-MS m /z Calc.C 31 H 54 NO 6 [M+H] + 536.39,Found 536.85.
化合物36的合成：将硬脂酸(0.77g,3.36mmol)溶解于DCM中,加入1滴DMF,加入草酰氯(2.13g,16.80mmol)，室温搅拌4小时。TLC检测，反应结束后，反应液浓缩去除溶剂得酰氯粗品。将中间体36-2(0.30g,0.56mmol)溶解于DCM中,加入氢化钠(0.23g,5.60mmol)，搅拌20分钟。加入上述硬脂酰氯，室温搅拌16小时。TLC检测，反应结束后，加适量水淬灭，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(25:1)洗脱，得淡黄色油状化合物36(350 mg,58％)。1HNMR(400MHz,CD3OD,)δ5.11-5.09(m,1H),4.94-4.92(m,1H),4.61-4.54(m,1H),3.65(s,3H,CH3),2.59(m,4H),2.46-2.42(m,2H),2.32(s,6H,CH3×2),0.92-0.86(m,9H,CH3×3),0.82-0.81(d,3H,CH3),0.73(s,3H,CH3).Synthesis of compound 36: Dissolve stearic acid (0.77g, 3.36mmol) in DCM, add 1 drop of DMF, add oxalyl chloride (2.13g, 16.80mmol), and stir at room temperature for 4 hours. TLC detection, after the reaction is completed, the reaction solution is concentrated to remove the solvent to obtain crude acid chloride. Dissolve intermediate 36-2 (0.30g, 0.56mmol) in DCM, add sodium hydride (0.23g, 5.60mmol), and stir for 20 minutes. Add the above stearyl chloride and stir at room temperature for 16 hours. After TLC detection, after the reaction is completed, add an appropriate amount of water to quench, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (25:1), gave light yellow oily compound 36 (350 mg, 58%). 1 HNMR (400MHz, CD 3 OD,) δ5.11-5.09(m,1H),4.94-4.92(m,1H),4.61-4.54(m,1H),3.65(s,3H,CH3),2.59( m,4H),2.46-2.42(m,2H),2.32(s,6H,CH3×2),0.92-0.86(m,9H,CH3×3),0.82-0.81(d,3H,CH3),0.73 (s,3H,CH3).
实施例28：熊去氧胆酸衍生物(化合物37)的合成路线如下
Example 28: The synthetic route of ursodeoxycholic acid derivative (compound 37) is as follows
中间体37-1的合成：将熊去氧胆酸(2.00g,5.1mmol)，二甲氨基丁醇(0.72g,6.1mmol)，EDCI(1.17g,6.1mmol)，DIPEA(1.98g,15.3mmol)，DMAP(0.32g,2.6mmol)溶解于DMF中,室温搅拌16小时。TLC检测原料反应完，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(15:1)洗脱，得到白色固体中间体37-1(1.08g,43％)。ESI-MS m/z Calc.C30H53NO4[M+H]+491.76,Found 492.15.Synthesis of intermediate 37-1: Combine ursodeoxycholic acid (2.00g, 5.1mmol), dimethylaminobutanol (0.72g, 6.1mmol), EDCI (1.17g, 6.1mmol), DIPEA (1.98g, 15.3 mmol), DMAP (0.32g, 2.6mmol) was dissolved in DMF, and stirred at room temperature for 16 hours. TLC detects that the reaction of the raw materials is complete, and the solvent is concentrated to obtain the crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (15:1), gave white solid intermediate 37-1 (1.08 g, 43%). ESI-MS m/z Calc.C 30 H 53 NO 4 [M+H] + 491.76, Found 492.15.
化合物37的合成:将亚油酸(1.72g,6.12mmol)溶解于DCM中,加入1滴DMF,加入草酰氯(0.78g,6.12mmol)，室温搅拌4小时。TLC检测，反应结束后，反应液浓缩去除溶剂得酰氯粗品。将中间体37-1(0.50g,1.02mmol)溶解于DCM中,加入氢化钠(0.41g,10.20mmol)，搅拌20分钟。再加入上述亚油酰氯，室温搅拌16小时。TLC检测，反应结束后，加适量水淬灭，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得淡黄色油状化合物37(160mg,18％)。1H NMR(400MHz,Chloroform-d)δ5.43-5.28(m,8H),4.82-4.75(m,1H),4.72-4.64(m,1H),4.09-4.06(t,2H),2.79-2.76(t,4H),2.40-2.31(m,1H),2.30-2.24(m,4H),2.22(s,7H),2.21-2.14(m,2H),2.08-1.98(m,9H),0.97(s,3H),0.92-0.87(m,9H),0.67(s,3H).HPLC:95.11。ESI-MS m/z Calc.C66H114NO6[M+H]+1016.86,Found 1016.90。Synthesis of compound 37: Dissolve linoleic acid (1.72g, 6.12mmol) in DCM, add 1 drop of DMF, add oxalyl chloride (0.78g, 6.12mmol), and stir at room temperature for 4 hours. TLC detection, after the reaction is completed, the reaction solution is concentrated to remove the solvent to obtain crude acid chloride. Dissolve intermediate 37-1 (0.50g, 1.02mmol) in DCM, add sodium hydride (0.41g, 10.20mmol), and stir for 20 minutes. Then add the above linoleoyl chloride and stir at room temperature for 16 hours. After TLC detection, after the reaction is completed, add an appropriate amount of water to quench, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (20:1), gave compound 37 (160 mg, 18%) as light yellow oil. 1 H NMR(400MHz,Chloroform-d)δ5.43-5.28(m,8H),4.82-4.75(m,1H),4.72-4.64(m,1H),4.09-4.06(t,2H),2.79- 2.76(t,4H),2.40-2.31(m,1H),2.30-2.24(m,4H),2.22(s,7H),2.21-2.14(m,2H),2.08-1.98(m,9H), 0.97(s,3H),0.92-0.87(m,9H),0.67(s,3H).HPLC:95.11. ESI-MS m/z Calc.C 66 H 114 NO 6 [M+H] + 1016.86, Found 1016.90.
实施例29：熊去氧胆酸衍生物(化合物38)的合成路线如下
Example 29: The synthetic route of ursodeoxycholic acid derivative (compound 38) is as follows
将2-辛基癸酸(1.4g,4.9mmol)溶解于DCM中，加入草酰氯(3.1g,24mmol)并加入DMF(1滴)，室温搅拌4h。反应液直接浓缩。将中间体37-1(300mg,0.6mmol)溶解于DCM中，加入Et3N(124mg,1.2mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物38(129mg,21％)。1HNMR(400MHz,CDCl3)δ4.82-4.76(m,1H),4.72-4.67(m,1H),4.09-4.06(t,2H),2.49(m,2H),2.38(s,6H,CH3),0.97(s,3H,CH3),0.92-0.91(d,3H,CH3),0.89-0.86(m,12H,CH3x 4),0.68(s,3H,CH3).HPLC:96.54％。Dissolve 2-octyldecanoic acid (1.4g, 4.9mmol) in DCM, add oxalyl chloride (3.1g, 24mmol) and DMF (1 drop), and stir at room temperature for 4h. The reaction solution was directly concentrated. Intermediate 37-1 (300 mg, 0.6 mmol) was dissolved in DCM, Et 3 N (124 mg, 1.2 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 38 (129 mg, 21%) as a yellow oil. 1 HNMR (400MHz, CDCl 3 ) δ4.82-4.76(m,1H),4.72-4.67(m,1H),4.09-4.06(t,2H),2.49(m,2H),2.38(s,6H, CH 3 ),0.97(s,3H,CH 3 ),0.92-0.91(d,3H,CH 3 ),0.89-0.86(m,12H,CH 3 x 4),0.68(s,3H,CH 3 ). HPLC: 96.54%.
实施例30：熊去氧胆酸衍生物(化合物39)的合成路线如下
Example 30: The synthetic route of ursodeoxycholic acid derivative (compound 39) is as follows
将2-己基癸酸(0.7g,2.9mmol)溶解于DCM中，加入DMF(1滴)，加入草酰氯(1.8g,15mmol)，室温搅拌4h。反应液直接浓缩。将化合物2(250mg,0.5mmol)溶解于DCM中，加入Et3N(98mg,1.0mmol)，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物39(129mg,26％)。1HNMR(400MHz,CDCl3)δ6.39(s，1H)，4.82-4.75(m,1H),4.72-4.67(m,1H),3.32-3.28(m,2H),2.27-2.06(m,8H),0.97(s,3H,CH3),0.93-0.92(d,3H,CH3),0.89-0.86(m,12H,CH3x 4),0.68(s,3H,CH3).HPLC:97.7％。 Dissolve 2-hexyldecanoic acid (0.7g, 2.9mmol) in DCM, add DMF (1 drop), add oxalyl chloride (1.8g, 15mmol), and stir at room temperature for 4 hours. The reaction solution was directly concentrated. Compound 2 (250 mg, 0.5 mmol) was dissolved in DCM, Et 3 N (98 mg, 1.0 mmol) was added, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 39 (129 mg, 26%) as a yellow oil. 1 HNMR (400MHz, CDCl 3 ) δ6.39 (s, 1H), 4.82-4.75 (m, 1H), 4.72-4.67 (m, 1H), 3.32-3.28 (m, 2H), 2.27-2.06 (m, 8H),0.97(s,3H,CH 3 ),0.93-0.92(d,3H,CH 3 ),0.89-0.86(m,12H,CH 3 x 4),0.68(s,3H,CH 3 ).HPLC :97.7%.
实施例31：熊去氧胆酸衍生物(化合物40)的合成路线如下
Example 31: The synthetic route of ursodeoxycholic acid derivative (compound 40) is as follows
中间体40-1的合成：将化合物3-氨基-1,2-丙二醇(5.00g,54.88mmol)溶解于乙醇中，再加入Boc酸酐(13.18g,60.37mmol),室温搅拌20h。TLC检测，反应结束后，反应液直接浓缩得粗品。硅胶柱层析，DCM/CH3OH(100:1-19:1)洗脱，得无色油状中间体40-1(9.80g,93.3％)。1HNMR(400MHz,CDCl3)δ5.12(s,1H),3.77-3.70(m,1H),3.63-3.52(m,2H),3.29-3.19(m,2H)，1.43(s，9H)。Synthesis of intermediate 40-1: Dissolve compound 3-amino-1,2-propanediol (5.00g, 54.88mmol) in ethanol, add Boc acid anhydride (13.18g, 60.37mmol), and stir at room temperature for 20h. After TLC detection, the reaction solution was directly concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (100:1-19:1), gave colorless oily intermediate 40-1 (9.80g, 93.3%). 1 HNMR (400MHz, CDCl 3 ) δ5.12 (s, 1H), 3.77-3.70 (m, 1H), 3.63-3.52 (m, 2H), 3.29-3.19 (m, 2H), 1.43 (s, 9H) .
中间体40-2的合成：将中间体40-1(1.60g,8.37mmol),硬脂酸(5.00g,17.57mmol),EDCI(10.00g,52.16mmol),DMAP(0.50g,4.09mmol)溶解于DCM中，室温搅拌16h。TLC检测，反应结束后，浓缩得白色粘稠状液体中间体40-2(5.31g，87％)。粗品直接投下一步。Synthesis of intermediate 40-2: Combine intermediate 40-1 (1.60g, 8.37mmol), stearic acid (5.00g, 17.57mmol), EDCI (10.00g, 52.16mmol), DMAP (0.50g, 4.09mmol) Dissolve in DCM and stir at room temperature for 16h. After TLC detection, after the reaction was completed, the reaction mixture was concentrated to obtain white viscous liquid intermediate 40-2 (5.31 g, 87%). Crude products are directly transferred to the next step.
中间体40-3的合成：将中间体40-2(3.20g,4.41mmol),溶解于DCM中，加入TFA，室温搅拌7h。反应液浓缩，得到的中间体40-3粗品直接用于一步。Synthesis of intermediate 40-3: Dissolve intermediate 40-2 (3.20g, 4.41mmol) in DCM, add TFA, and stir at room temperature for 7 hours. The reaction solution was concentrated, and the crude intermediate 40-3 obtained was directly used in the first step.
中间体40-4的合成：将石胆酸(500mg,1.33mmol)，中间体40-3(1.17g,1.59mmol)，HATU(0.76g,2.00mmol)，DIPEA(0.74mL,4.00mmol)溶解于DMF中，室温搅拌20h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(50:1)洗脱，得类白色固体中间体40-4(750mg,57.4％)。1HNMR(400MHz,CDCl3)δ5.75-5.72(m,1H),5.11-5.08(m,1H),4.27-4.23(m,1H),4.15-4.11(m,1H),3.65-3.59(m,1H),3.52-3.43(m,2H),2.95(s,1H),2.88(s,1H),2.34-2.29(m,4H),2.27-2.19(m,1H),2.10-2.02(m,1H),1.97-1.93(m,1H),0.91-0.86(m,12H,CH3x 4),0.64(s,3H,CH3)。Synthesis of intermediate 40-4: Dissolve lithocholic acid (500mg, 1.33mmol), intermediate 40-3 (1.17g, 1.59mmol), HATU (0.76g, 2.00mmol), and DIPEA (0.74mL, 4.00mmol) Stir in DMF at room temperature for 20h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (50:1), gave off-white solid intermediate 40-4 (750 mg, 57.4%). 1 HNMR (400MHz, CDCl 3 ) δ5.75-5.72(m,1H),5.11-5.08(m,1H),4.27-4.23(m,1H),4.15-4.11(m,1H),3.65-3.59( m,1H),3.52-3.43(m,2H),2.95(s,1H),2.88(s,1H),2.34-2.29(m,4H),2.27-2.19(m,1H),2.10-2.02( m,1H),1.97-1.93(m,1H),0.91-0.86(m,12H,CH 3 x 4),0.64(s,3H,CH 3 ).
化合物40的合成：将中间体40-4(300mg,0.31mmol)，4-N,N-二甲基丁酸盐酸盐(102mg,0.61mmol)，Py-BOP(476mg,0.91mmol)，TEA(0.22 mL,1.52mmol)溶解于DMF中，室温搅拌16h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得类白色固体化合物40(90mg,26.1％)。1HNMR(400MHz,CDCl3)δ5.78-5.75(m,1H),5.11-5.08(m,1H),4.76-4.70(m,1H),4.27-4.23(m,1H),4.16-4.11(m,1H),3.49-3.45(m,2H),3.24-3.20(t,2H),2.97(s,6H,CH3×2),2.53-2.50(m,2H),2.34-2.29(m,4H),2.27-2.20(m,2H),0.93-0.86(m,12H x 4),0.64(s,3H,CH3)。Synthesis of compound 40: Combine intermediate 40-4 (300 mg, 0.31 mmol), 4-N, N-dimethylbutyrate hydrochloride (102 mg, 0.61 mmol), Py-BOP (476 mg, 0.91 mmol), TEA (0.22 mL, 1.52 mmol) was dissolved in DMF and stirred at room temperature for 16 h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (20:1), gave off-white solid compound 40 (90 mg, 26.1%). 1 HNMR (400MHz, CDCl 3 ) δ5.78-5.75(m,1H),5.11-5.08(m,1H),4.76-4.70(m,1H),4.27-4.23(m,1H),4.16-4.11( m,1H),3.49-3.45(m,2H),3.24-3.20(t,2H),2.97(s,6H,CH 3 ×2),2.53-2.50(m,2H),2.34-2.29(m, 4H),2.27-2.20(m,2H),0.93-0.86(m,12H x 4),0.64(s,3H,CH 3 ).
实施例32：熊去氧胆酸衍生物(化合物41)的合成路线如下
Example 32: The synthesis route of ursodeoxycholic acid derivative (compound 41) is as follows
将化合物2-己基癸酸(1.22g,4.79mmol)溶解于DCM中,加入1滴DMF,加入草酰氯(2mL,23.63mmol)，搅拌反应3h。TLC检测，反应结束后，直接浓缩。将中间体37-1(400mg,0.81mmol)溶解于DCM中，加入氢化钠(320mg,8.10mmol),搅拌30分钟。加入上述酰氯，室温搅拌20h。TLC检测，反应结束后，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得淡黄色油状化合物41(120mg,20.5％)。1HNMR(400MHz,CDCl3)δ4.74-4.66(m,1H,C3),4.09-4.06(t,2H),3.61-3.55(m,1H,C7),2.38-2.25(m,4H),2.23(s,6H,CH3×2),2.02-1.98(m,1H),0.96(s,3H,CH3),0.93(d,CH3),0.89-0.86(m,6H),0.68(s,3H,CH3).HPLC:86.2％。Dissolve compound 2-hexyldecanoic acid (1.22g, 4.79mmol) in DCM, add 1 drop of DMF, add oxalyl chloride (2mL, 23.63mmol), and stir for 3 hours. TLC detection, after the reaction is completed, concentrate directly. Intermediate 37-1 (400 mg, 0.81 mmol) was dissolved in DCM, sodium hydride (320 mg, 8.10 mmol) was added, and stirred for 30 minutes. Add the above acid chloride and stir at room temperature for 20h. After TLC detection, after the reaction is completed, add an appropriate amount of water and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (20:1), gave compound 41 (120 mg, 20.5%) as light yellow oil. 1 HNMR(400MHz, CDCl 3 )δ4.74-4.66(m,1H,C3),4.09-4.06(t,2H),3.61-3.55(m,1H,C7),2.38-2.25(m,4H), 2.23(s,6H,CH 3 ×2),2.02-1.98(m,1H),0.96(s,3H,CH 3 ),0.93(d,CH 3 ),0.89-0.86(m,6H),0.68( s,3H,CH 3 ). HPLC: 86.2%.
实施例33：熊去氧胆酸衍生物(化合物42)的合成路线如下
Example 33: The synthetic route of ursodeoxycholic acid derivative (compound 42) is as follows
将化合物2-己基癸酸(1.23g,4.79mmol)溶解于DCM中,加入1滴DMF,加入草酰氯(2mL,23.63mmol)，搅拌反应3h。TLC检测，反应结束后，直接浓缩干。将中间体37-1(400mg,0.81mmol),(320mg,8.10mmol)溶解于DCM中，加入上述酰氯，室温搅拌20h。TLC检测，浓缩溶剂得粗品。硅 胶柱层析，DCM/CH3OH(20:1)洗脱，得淡黄色油状化合物42(40mg,5.2％)。1HNMR(400MHz,CDCl3)δ4.82-4.76(m,1H,C7),4.73-4.66(m,1H,C3),4.09-4.05(t,2H),2.37-2.29(m,3H),2.25(s,6H,N-CH3×2),2.23-2.16(m,2H),2.01-1.98(m,2H),0.97(s,3H,CH3),0.92-0.86(m,15H),0.68(s,3H,CH3).HPLC:80.6％。Dissolve compound 2-hexyldecanoic acid (1.23g, 4.79mmol) in DCM, add 1 drop of DMF, add oxalyl chloride (2mL, 23.63mmol), and stir for 3 hours. TLC detection, after the reaction is completed, directly concentrate to dryness. Dissolve intermediate 37-1 (400 mg, 0.81 mmol), (320 mg, 8.10 mmol) in DCM, add the above acid chloride, and stir at room temperature for 20 h. TLC detection, concentrated solvent to obtain crude product. silicon Gel column chromatography, eluted with DCM/CH 3 OH (20:1), gave compound 42 (40 mg, 5.2%) as light yellow oil. 1 HNMR(400MHz, CDCl 3 )δ4.82-4.76(m,1H,C7),4.73-4.66(m,1H,C3),4.09-4.05(t,2H),2.37-2.29(m,3H), 2.25(s,6H,N-CH 3 ×2),2.23-2.16(m,2H),2.01-1.98(m,2H),0.97(s,3H,CH 3 ),0.92-0.86(m,15H) ,0.68(s,3H,CH 3 ). HPLC: 80.6%.
实施例34：熊去氧胆酸衍生物(化合物43)的合成路线如下
Example 34: The synthesis route of ursodeoxycholic acid derivative (compound 43) is as follows
中间体43-1的合成：将熊去氧胆酸(786mg,2.0mmol)溶解于DMF中,依次加入HBTU(950mg,2.5mmol),DIEA(400mg,3.0mmol),4-二甲基氨基丁胺(290mg,2.5mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体43-1(735mg，75％)。Synthesis of intermediate 43-1: Dissolve ursodeoxycholic acid (786mg, 2.0mmol) in DMF, add HBTU (950mg, 2.5mmol), DIEA (400mg, 3.0mmol), and 4-dimethylaminobutyl in sequence Amine (290 mg, 2.5 mmol), stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 43-1 (735 mg, 75%).
将2-辛基癸酸(0.9g,3.1mmol)溶解于DCM中，加入DMF(1滴)，滴加草酰氯(1.9g,15mmol)，室温搅拌4h。反应液浓缩。将中间体43-1(250mg,0.5mmol)溶解于DCM中，加入Et3N(103mg,1.0mmol)，再加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物43(115mg,23％)。1HNMR(400MHz,Methanol-d4)δ4.80-4.73(m,1H),4.71-4.66(m,1H),3.20-3.18(t,2H),3.01-2.97(m,2H),2.76(s,6H,CH3x2),2.33-2.21(m,3H),2.12-2.05(m,2H),1.01(s,3H,CH3),0.98-0.97(d,3H,CH3),0.92-0.88(t,12H,CH3x4),0.73(s,3H,CH3).HPLC:93.53％。Dissolve 2-octyldecanoic acid (0.9g, 3.1mmol) in DCM, add DMF (1 drop), add oxalyl chloride (1.9g, 15mmol) dropwise, and stir at room temperature for 4 hours. The reaction solution was concentrated. Intermediate 43-1 (250 mg, 0.5 mmol) was dissolved in DCM, Et 3 N (103 mg, 1.0 mmol) was added, then the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 43 (115 mg, 23%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ4.80-4.73(m,1H),4.71-4.66(m,1H),3.20-3.18(t,2H),3.01-2.97(m,2H),2.76(s , 6H , CH 3 0.88(t,12H,CH 3 x4), 0.73(s, 3H, CH 3 ). HPLC: 93.53%.
实施例35：熊去氧胆酸衍生物(化合物44)的合成路线如下
Example 35: The synthetic route of ursodeoxycholic acid derivative (compound 44) is as follows
将2-己基癸酸(0.8g,3.1mmol)溶解于DCM中，加入草酰氯(1.9g,15mmol)并加入DMF(1滴)，室温搅拌4h。反应液浓缩。将中间体43-1(250mg,0.5mmol)溶解于DCM中，加入Et3N(103mg,1.0mmol)，再加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物44(116mg，24％)。1HNMR(400MHz,Methanol-d4)δ4.78-4.74(s,1H),4.71-4.66(m,1H),3.21-3.18(t,2H),3.02-2.98(m,2H),2.77(s,6H,CH3x2),2.29(m,3H),2.07(m,2H),1.01(s,3H,CH3),0.98-0.97(d,3H,CH3),0.92-0.88(m,12H,CH3x 4),0.73(s,3H,CH3).HPLC:95.01％。Dissolve 2-hexyldecanoic acid (0.8g, 3.1mmol) in DCM, add oxalyl chloride (1.9g, 15mmol) and DMF (1 drop), and stir at room temperature for 4 hours. The reaction solution was concentrated. Intermediate 43-1 (250 mg, 0.5 mmol) was dissolved in DCM, Et 3 N (103 mg, 1.0 mmol) was added, then the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 44 (116 mg, 24%) as a yellow oil. 1 HNMR (400MHz, Methanol-d4) δ4.78-4.74(s,1H),4.71-4.66(m,1H),3.21-3.18(t,2H),3.02-2.98(m,2H),2.77(s ,6H,CH 3 x2),2.29(m,3H),2.07(m,2H),1.01(s,3H,CH 3 ),0.98-0.97(d,3H,CH 3 ),0.92-0.88(m, 12H, CH 3 x 4), 0.73 (s, 3H, CH 3 ). HPLC: 95.01%.
实施例36：熊去氧胆酸衍生物(化合物45)的合成路线如下
Example 36: The synthetic route of ursodeoxycholic acid derivative (compound 45) is as follows
中间体45-1的合成：将奥贝胆酸(840mg,2.0mmol)溶解于DMF中,依次加入HBTU(948mg,2.5mmol),DIEA(400mg,3.0mmol),4-二甲基氨基丁胺(290mg,2.5mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体45-1(746mg，72％)。1HNMR(400MHz,Methanol-d4)δ3.66-3.65(m,1H),3.35(s,1H),3.19-3.16(t,2H),2.44-2.40(t,2H),2.31(s,6H,CH3x2),2.27-2.20(m,1H),2.13-2.06(m,1H),0.98-0.97(d,3H，CH3),0.92-0.89(s,6H，CH3x 2),0.69(s,3H,CH3)。Synthesis of intermediate 45-1: Dissolve obeticholic acid (840mg, 2.0mmol) in DMF, add HBTU (948mg, 2.5mmol), DIEA (400mg, 3.0mmol), and 4-dimethylaminobutylamine in sequence (290 mg, 2.5 mmol), stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 45-1 (746 mg, 72%). 1 HNMR(400MHz,Methanol-d4)δ3.66-3.65(m,1H),3.35(s,1H),3.19-3.16(t,2H),2.44-2.40(t,2H),2.31(s,6H) ,CH 3 x2),2.27-2.20(m,1H),2.13-2.06(m,1H),0.98-0.97(d,3H,CH 3 ),0.92-0.89(s,6H,CH 3 x 2), 0.69(s,3H,CH 3 ).
化合物45的合成：将2-辛基癸酸(0.7g,2.9mmol)溶解于DCM中，加入草 酰氯(1.8g,15mmol)并加入DMF(1滴)，室温搅拌4h。反应液浓缩。将中间体45-1(250mg,0.5mmol)溶解于DCM中，加入Et3N(98mg,1.0mmol)，再加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物45(60mg,12％)。1HNMR(400MHz,Methanol-d4)δ4.52-4.49(m,1H),3.71-3.66(m,3H),2.47-2.44(t,2H),2.33(s,6H,CH3x 2),2.29-2.27(m,1H),0.99-0.97(d,3H),0.94(s,3H),0.92-0.90(m,12H,CH3x 4),0.71(s,3H,CH3).HPLC:84.6％。ESI-MS m/z Calc.C64H119N2O5[M+H]+995.90,Found 996.00.Synthesis of compound 45: Dissolve 2-octyldecanoic acid (0.7g, 2.9mmol) in DCM, add grass Acid chloride (1.8g, 15mmol) was added and DMF (1 drop) was added, and stirred at room temperature for 4h. The reaction solution was concentrated. Intermediate 45-1 (250 mg, 0.5 mmol) was dissolved in DCM, Et 3 N (98 mg, 1.0 mmol) was added, then the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 45 (60 mg, 12%) as a yellow oil. 1 HNMR(400MHz,Methanol-d4)δ4.52-4.49(m,1H),3.71-3.66(m,3H),2.47-2.44(t,2H),2.33(s,6H,CH 3 x 2), 2.29-2.27(m,1H),0.99-0.97(d,3H),0.94(s,3H),0.92-0.90(m,12H,CH 3 x 4),0.71(s,3H,CH 3 ).HPLC :84.6%. ESI-MS m/z Calc.C 64 H 119 N 2 O 5 [M+H] + 995.90,Found 996.00.
实施例37：熊去氧胆酸衍生物(化合物46)的合成路线如下
Example 37: The synthetic route of ursodeoxycholic acid derivative (compound 46) is as follows
中间体46-1的合成：将熊去氧胆酸(3.92g,10.0mmol)溶解于DMF中,依次加入HBTU(4.56g,12.0mmol),DIEA(3.87g,30.0mmol),4-吡咯烷-1-丁醇(1.72g,12.0mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体46-1(3.9g，75％)。Synthesis of intermediate 46-1: Dissolve ursodeoxycholic acid (3.92g, 10.0mmol) in DMF, add HBTU (4.56g, 12.0mmol), DIEA (3.87g, 30.0mmol), and 4-pyrrolidine in sequence -1-Butanol (1.72g, 12.0mmol), stir at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 46-1 (3.9 g, 75%).
化合物46的合成：将2-己基癸酸(0.7g,2.9mmol)溶解于DCM中，加入草酰氯(1.8g,14mmol)并加入DMF(1滴)，室温搅拌4h。反应液直接浓缩干。将中间体46-1(250mg,0.5mmol)溶解于DCM中，加入Et3N(97mg,1.0mmol)，再加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色油状化合物46(105mg，21.1％)。1HNMR(400MHz,Methanol-d4)δ4.82-4.75(m,1H),4.73-4.65(m,1H),4.06-4.09(t,2H),2.97-2.79(m,6H),2.37-2.16(m,5H),0.97(s,3H,CH3),0.92-0.90(d,3H,CH3),0.89-0.85(t,12H,CH3x 4)，0.68(s,3H,CH3).HPLC:94.58％。 Synthesis of compound 46: Dissolve 2-hexyldecanoic acid (0.7g, 2.9mmol) in DCM, add oxalyl chloride (1.8g, 14mmol) and DMF (1 drop), and stir at room temperature for 4 hours. The reaction solution was directly concentrated to dryness. Intermediate 46-1 (250 mg, 0.5 mmol) was dissolved in DCM, Et 3 N (97 mg, 1.0 mmol) was added, then the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 46 (105 mg, 21.1%) as a yellow oil. 1 HNMR(400MHz, Methanol-d4)δ4.82-4.75(m,1H),4.73-4.65(m,1H),4.06-4.09(t,2H),2.97-2.79(m,6H),2.37-2.16 (m,5H),0.97(s,3H,CH 3 ),0.92-0.90(d,3H,CH 3 ),0.89-0.85(t,12H,CH 3 x 4),0.68(s,3H,CH 3 ).HPLC:94.58%.
实施例38：熊去氧胆酸衍生物(化合物47)的合成路线如下
Example 38: The synthetic route of ursodeoxycholic acid derivative (compound 47) is as follows
中间体47-1的合成：将胆酸(2.00g,5.1mmol)溶解于DMF中,依次加入HATU(2.89g,7.6mmol),DIEA(3.8mL,20.4mmol),4-四氢吡咯丁胺(0.87g,6.1mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得到黄色固体中间体47-1(1.65g，60％)。1HNMR(400MHz,CD3OD)δ3.96-3.94(m,1H,C12),3.81-3.79(m,1H,C7),3.41-3.33(m,1H,C3),3.21-3.14(m,2H),2.60-2.57(m,4H),2.53-2.49(m,2H),2.33-2.18(m,3H),2.14-1.85(m,5H),1.84-1.71(m,7H),1.68-1.47(m,10H),1.45-1.06(m,8H),1.03(d,J＝8.0Hz,3H,CH3),0.99-0.94(m,1H),0.92(s,3H,CH3),0.71(s,3H,CH3)。Synthesis of intermediate 47-1: Dissolve cholic acid (2.00g, 5.1mmol) in DMF, and add HATU (2.89g, 7.6mmol), DIEA (3.8mL, 20.4mmol), and 4-tetrahydropyrrolidine in sequence. (0.87g, 6.1mmol), stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave yellow solid intermediate 47-1 (1.65 g, 60%). 1 HNMR (400MHz, CD 3 OD) δ3.96-3.94(m,1H,C 12 ),3.81-3.79(m,1H,C 7 ),3.41-3.33(m,1H,C 3 ),3.21-3.14 (m,2H),2.60-2.57(m,4H),2.53-2.49(m,2H),2.33-2.18(m,3H),2.14-1.85(m,5H),1.84-1.71(m,7H) ,1.68-1.47(m,10H),1.45-1.06(m,8H),1.03(d,J＝8.0Hz,3H,CH 3 ),0.99-0.94(m,1H),0.92(s,3H,CH 3 ),0.71(s,3H,CH 3 ).
化合物47的合成：将中间体47-1(200mg,0.37mmol),TEA(0.8mL,5.62mmol)溶解于DCM中，将2-己基癸酰氯(1031mg,0.37mmol)加入上述反应液中，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(30:1)洗脱，得淡黄色油状化合物47(150mg,39.7％)。1HNMR(400MHz,CDCl3)δ4.73-4.66(m,1H，C12),4.64-4.55(m,1H,C3),4.01-3.98(m,1H),3.87-3.83(m,1H,C7),3.69-3,65(m,2H),3.08-3.01(m,3H),2.70-2.57(m,2H),2.39-2.29(m,1H),2.27-2.20(m,2H),2.17-2.06(m,4H),2.04-1.76(m,12H),1.02(d,J＝8.0Hz,3H,CH3),0.91-0.95(m,15H),0.71(s,3H,CH3).ESI-MS m/z Calc.C64H116N2O6[M+H]+1009.64,Found 1010.04.HPLC：91.8％。Synthesis of compound 47: Dissolve intermediate 47-1 (200mg, 0.37mmol) and TEA (0.8mL, 5.62mmol) in DCM, add 2-hexyldecanoyl chloride (1031mg, 0.37mmol) to the above reaction solution, keep at room temperature Stir overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (30:1), gave compound 47 (150 mg, 39.7%) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ) δ4.73-4.66(m,1H,C12),4.64-4.55(m,1H,C3),4.01-3.98(m,1H),3.87-3.83(m,1H,C7 ),3.69-3,65(m,2H),3.08-3.01(m,3H),2.70-2.57(m,2H),2.39-2.29(m,1H),2.27-2.20(m,2H),2.17 -2.06(m,4H),2.04-1.76(m,12H),1.02(d,J＝8.0Hz,3H,CH 3 ),0.91-0.95(m,15H),0.71(s,3H,CH 3 ) .ESI-MS m/z Calc.C 64 H 116 N 2 O 6 [M+H] + 1009.64, Found 1010.04. HPLC: 91.8%.
实施例39：熊去氧胆酸衍生物(化合物48)的合成路线如下
Example 39: The synthetic route of ursodeoxycholic acid derivative (compound 48) is as follows
中间体48-1的合成：将中间体37-1(400mg,0.8mmol),NaH(320mg,8.0mmol)溶解于DCM中，将现制的2-己基癸酰氯(1319mg,4.8mmol)加入上述反应液中，室温搅拌过夜。TLC检测，反应结束后，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状中间体48-1(152mg，26％)。1HNMR(400MHz,CDCl3)δ4.73-4.66(m,1H,C3),4.07(t,2H),3.61-3.55(m,1H,C7),2.38-2.25(m,4H),2.23(s,6H,N-CH3×2),2.02-1.98(m,1H),1.92-1.87(m,1H),1.12-0.99(m,3H),0.96(s,3H,CH3),0.93(d,3H,CH3),0.89-0.86(m,6H),0.68(s,3H,CH3)。Synthesis of intermediate 48-1: Dissolve intermediate 37-1 (400mg, 0.8mmol) and NaH (320mg, 8.0mmol) in DCM, and add the freshly prepared 2-hexyldecanoyl chloride (1319mg, 4.8mmol) to the above The reaction solution was stirred at room temperature overnight. After TLC detection, after the reaction is completed, add an appropriate amount of water and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave light yellow oily intermediate 48-1 (152 mg, 26%). 1 HNMR(400MHz, CDCl 3 )δ4.73-4.66(m,1H,C 3 ),4.07(t,2H),3.61-3.55(m,1H,C 7 ),2.38-2.25(m,4H), 2.23(s,6H,N-CH 3 ×2),2.02-1.98(m,1H),1.92-1.87(m,1H),1.12-0.99(m,3H),0.96(s,3H,CH 3 ) ,0.93(d,3H,CH 3 ),0.89-0.86(m,6H),0.68(s,3H,CH 3 ).
化合物48的合成：将中间体48-1(120mg,0.16mmol),NaH(131mg,3.28mmol)溶解于DCM中，将现制的硬脂酰氯(530mg,1.75mmol)加入上述反应液中，室温搅拌过夜。TLC检测，反应结束后，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(30:1)洗脱，得无色油状化合物48(40mg,24.5％)。1HNMR(400MHz,CDCl3)δ4.95-4.88(m,1H，C7),4.74-4.66(m,1H,C3),4.07(t,2H,,O-CH2),2.37-2.29(m,3H),2.25(s,6H,,N-CH3×2),2.22-2.16(m,1H),2.08-1.96(m,2H),1.86-1.01(m,65H),0.98(s,3H,CH3)，0.92-0.83(m,12H),0.68(s,3H,CH3).HPLC：92.8％。Synthesis of compound 48: Dissolve intermediate 48-1 (120 mg, 0.16 mmol) and NaH (131 mg, 3.28 mmol) in DCM, add fresh stearyl chloride (530 mg, 1.75 mmol) to the above reaction solution, keep at room temperature Stir overnight. After TLC detection, after the reaction is completed, add an appropriate amount of water and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (30:1), gave compound 48 (40 mg, 24.5%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ4.95-4.88(m,1H,C7),4.74-4.66(m,1H,C3),4.07(t,2H,,O-CH 2 ),2.37-2.29(m ,3H),2.25(s,6H,,N-CH 3 ×2),2.22-2.16(m,1H),2.08-1.96(m,2H),1.86-1.01(m,65H),0.98(s, 3H,CH 3 ), 0.92-0.83 (m, 12H), 0.68 (s, 3H, CH 3 ). HPLC: 92.8%.
实施例40：熊去氧胆酸衍生物(化合物49)的合成路线如下
Example 40: The synthetic route of ursodeoxycholic acid derivative (compound 49) is as follows
中间体49-1的合成：将化合物2(300mg,0.58mmol),TEA(0.8mL,5.62mmol)溶解于DCM中，将现制的2-己基癸酰氯(956mg,3.48mmol)加入到上述反应液中，室温搅拌过夜。TLC检测，反应结束后，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(30:1)洗脱，得淡黄色油状中间体49-1(155mg，35％)。1HNMR(400MHz,CDCl3)δ4.73-4.68(m,1H,C3),3.69-3.63(m,2H),3.61-3.57(m,1H,C7),2.97-2.90(m,1H),2.78-2.70(m,1H),2.63-2.57(m,6H),2.29-2.20(m,1H),2.03-1.99(m,2H),1.86-1.60(m,20H),1.47-1.33(m,12H),1.29-1.25(m,49H),1.19-1.05(m,4H),0.97-0.93(m,6H,CH3×2),0.89-0.86(m,12H),0.68(s,3H,CH3)。Synthesis of intermediate 49-1: Dissolve compound 2 (300mg, 0.58mmol) and TEA (0.8mL, 5.62mmol) in DCM, and add freshly prepared 2-hexyldecanoyl chloride (956mg, 3.48mmol) to the above reaction solution and stirred at room temperature overnight. After TLC detection, after the reaction is completed, add an appropriate amount of water and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (30:1), gave light yellow oily intermediate 49-1 (155 mg, 35%). 1 HNMR (400MHz, CDCl 3 ) δ4.73-4.68(m,1H,C 3 ),3.69-3.63(m,2H),3.61-3.57(m,1H,C 7 ),2.97-2.90(m,1H ),2.78-2.70(m,1H),2.63-2.57(m,6H),2.29-2.20(m,1H),2.03-1.99(m,2H),1.86-1.60(m,20H),1.47-1.33 (m,12H),1.29-1.25(m,49H),1.19-1.05(m,4H),0.97-0.93(m,6H,CH 3 ×2),0.89-0.86(m,12H),0.68(s ,3H,CH 3 ).
化合物49的合成：将中间体49-1(110mg,0.14mmol),NaH(117mg,2.92mmol)溶解于DCM中，将现制的月桂酰氯(319mg,1.46mmol)加入到上述反应液中，室温搅拌过夜。TLC检测，反应结束后，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(30:1)洗脱，得淡黄色油状化合物49(23mg,16.8％)。1HNMR(400MHz,CDCl3)δ4.95-4.88(m,1H，C7),4.74-4.67(m,1H,C3),3.67-3.63(m,2H),2.98-2.90(m,4H),2.75-2.53(m,3H),2.31-2.20(m,5H),2.07-2.00(m,8H),1.13-1.03(m,4H),0.98(s,3H,CH3)，0.93(d,3H,CH3),0.89-0.83(m,16H),0.69(s,3H,CH3).HPLC：88.3％。Synthesis of compound 49: Dissolve intermediate 49-1 (110 mg, 0.14 mmol) and NaH (117 mg, 2.92 mmol) in DCM, add freshly prepared lauroyl chloride (319 mg, 1.46 mmol) to the above reaction solution, keep at room temperature Stir overnight. After TLC detection, after the reaction is completed, add an appropriate amount of water and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (30:1), gave compound 49 (23 mg, 16.8%) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ) δ4.95-4.88(m,1H,C7),4.74-4.67(m,1H,C3),3.67-3.63(m,2H),2.98-2.90(m,4H), 2.75-2.53(m,3H),2.31-2.20(m,5H),2.07-2.00(m,8H),1.13-1.03(m,4H),0.98(s,3H,CH 3 ),0.93(d, 3H,CH 3 ), 0.89-0.83 (m, 16H), 0.69 (s, 3H, CH 3 ). HPLC: 88.3%.
实施例41：熊去氧胆酸衍生物(化合物50)的合成路线如下
Example 41: The synthetic route of ursodeoxycholic acid derivative (compound 50) is as follows
将2-辛基癸酸(0.82g，2.89mmol)溶解于DCM中，加入1滴DMF后， 再加入草酰氯(1.83g，14.4mmol)，室温反应4个小时，反应液直接浓缩，将所得酰氯粗品加入到装有熊去氧胆酸中间体46-1(250mg，0.48mmol)、三乙胺(0.96g，9.64mmol)和DCM的反应瓶中，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得浅黄色油状化合物50(152mg,30.4％)。1HNMR(400MHz,CDCl3,)δ4.82-4.76(m,1H),4.71-4.67(m,1H),4.10-4.07(t，3H),2.96-2.90(m，2H)，2.25-2.19(m，4H),2.13-2.05(m，3H),0.98(s,3H,CH3),0.93-0.91(d,3H,CH3)0.89-0.86(t,12H,CH3×4),0.69(s,3H,CH3).ESI-MS m/z Calc.C68H124NO6[M+H]+1050.73,Found 1050.70。HPLC：83.6％。Dissolve 2-octyldecanoic acid (0.82g, 2.89mmol) in DCM, add 1 drop of DMF, Then add oxalyl chloride (1.83g, 14.4mmol) and react at room temperature for 4 hours. The reaction solution is directly concentrated. The crude acid chloride obtained is added to a container containing ursodeoxycholic acid intermediate 46-1 (250mg, 0.48mmol) and triethyl. Amine (0.96g, 9.64mmol) and DCM were placed in a reaction flask and stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 50 (152 mg, 30.4%) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ,)δ4.82-4.76(m,1H),4.71-4.67(m,1H),4.10-4.07(t,3H),2.96-2.90(m,2H),2.25-2.19 (m, 4H), 2.13-2.05 (m, 3H), 0.98 (s, 3H, CH 3 ), 0.93-0.91 (d, 3H, CH 3 ) 0.89-0.86 (t, 12H, CH 3 × 4), 0.69(s,3H,CH 3 ).ESI-MS m/z Calc.C 68 H 124 NO 6 [M+H] + 1050.73, Found 1050.70. HPLC: 83.6%.
实施例42：奥贝胆酸衍生物(化合物51)的合成路线如下
Example 42: The synthetic route of obeticholic acid derivative (compound 51) is as follows
将2-辛基癸酸(0.82g，2.89mmol)溶解于DCM中，加入1滴DMF后，再加入草酰氯(1.83g，14.4mmol)，室温反应4个小时，反应液直接浓缩，将所得酰氯粗品加入到装有奥贝胆酸中间体45-1(250mg，0.48mmol)、三乙胺(0.96g，9.64mmol)和DCM的反应瓶中，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得浅黄色油状化合物51(60mg,12％)。1HNMR(400MHz,CDCl3,)δ5.36-5.34(m，1H),4.61-4.53(m,1H),3.72-3.73(m,1H),3.68-3.65(m，1H),2.97-2.84(m，5H),2.68(s,6H),0.95-0.94(d,3H),0.86-0.92(m,44H),0.67(s,3H,CH3).ESI-MS m/z Calc.C68H127N2O5[M+H]+1051.77,Found 1051.8.HPLC：85.3％。Dissolve 2-octyldecanoic acid (0.82g, 2.89mmol) in DCM, add 1 drop of DMF, then add oxalyl chloride (1.83g, 14.4mmol), react at room temperature for 4 hours, and concentrate the reaction solution directly. The crude acid chloride was added to a reaction flask containing obeticholic acid intermediate 45-1 (250 mg, 0.48 mmol), triethylamine (0.96 g, 9.64 mmol) and DCM, and stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 51 (60 mg, 12%) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ,)δ5.36-5.34(m,1H),4.61-4.53(m,1H),3.72-3.73(m,1H),3.68-3.65(m,1H),2.97-2.84 (m,5H),2.68(s,6H),0.95-0.94(d,3H),0.86-0.92(m,44H),0.67(s,3H,CH 3 ).ESI-MS m/z Calc.C 68 H 127 N 2 O 5 [M+H] + 1051.77, Found 1051.8. HPLC: 85.3%.
实施例43：鹅去氧胆酸衍生物(化合物52)的合成路线如下
Example 43: The synthesis route of chenodeoxycholic acid derivative (compound 52) is as follows
中间体52-1的合成：将鹅去氧胆酸(3.92g,10.0mmol)溶解于DMF中，依次加入TEA(3.1g,30mmol)、HBTU(4.55g,12.0mmol)和4-吡咯烷丁胺(1.7g,12.0mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色固体中间体52-1(4.0g,78％)。Synthesis of intermediate 52-1: Dissolve chenodeoxycholic acid (3.92g, 10.0mmol) in DMF, then add TEA (3.1g, 30mmol), HBTU (4.55g, 12.0mmol) and 4-pyrrolidine Amine (1.7g, 12.0mmol), stirred at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave yellow solid intermediate 52-1 (4.0 g, 78%).
化合物52的合成：将2-辛基癸酸(0.82g，2.89mmol)溶解于DCM中，加入1滴DMF后，再加入草酰氯(1.83g，14.4mmol)，室温反应4个小时，反应液直接浓缩，将所得酰氯粗品加入到装有中间体52-1(250mg，0.48mmol)、三乙胺(0.96g，9.64mmol)和DCM的反应瓶中，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得浅黄色油状化合物52(118mg,23.6％)。1HNMR(400MHz,CDCl3,)δ5.36-5.34(m，1H),4.58-4.61(m,1H),3.85(m,1H),3.66-3.62(m，2H),2.76-2.70(m，1H),2.64-2.52(m，7H)，0.95-0.93(d，3H),0.92(s，3H),0.89-0.86(t,12H),0.67(s,3H,CH3).HPLC：89.2％。Synthesis of compound 52: Dissolve 2-octyldecanoic acid (0.82g, 2.89mmol) in DCM, add 1 drop of DMF, then add oxalyl chloride (1.83g, 14.4mmol), react at room temperature for 4 hours, the reaction solution Concentrate directly, add the crude acid chloride obtained into a reaction flask containing intermediate 52-1 (250 mg, 0.48 mmol), triethylamine (0.96 g, 9.64 mmol) and DCM, and stir at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 52 (118 mg, 23.6%) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ,)δ5.36-5.34(m,1H),4.58-4.61(m,1H),3.85(m,1H),3.66-3.62(m,2H),2.76-2.70(m , 1H), 2.64-2.52 (m, 7H), 0.95-0.93 (d, 3H), 0.92 (s, 3H), 0.89-0.86 (t, 12H), 0.67 (s, 3H, CH 3 ). HPLC: 89.2%.
实施例44：熊去氧胆酸衍生物(化合物53)的合成路线如下
Example 44: The synthetic route of ursodeoxycholic acid derivative (compound 53) is as follows
将化合物2(100mg,0.19mmol)溶解于THF中,加入NaH(46mg,1.15mmol)，室温搅拌20分钟，将碘辛烷(186mg,0.77mmol)加入上述反应液中。室温搅拌过夜。TLC检测，反应结束后，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(15:1)洗脱，得白色固体化合物53(30mg,18.2％)。1HNMR(400MHz,CDCl3,)δ3.58-3.52(m,3H),3.48-3.40(m,4H),3.39-3.31(m,3H),3.24-3.14(m,4H),2.39-2.31(m,1H),2.24-2.20(m,4H),1.12-0.97(m,4H), 0.98-0.94(m,6H,CH3×2),0.90-0.83(m,11H),0.67(s,3H,CH3).ESI-MS m/z Calc.C56H105N2O3 +[M+H]+854.47,Found 853.8.HPLC：81.0％。Dissolve compound 2 (100 mg, 0.19 mmol) in THF, add NaH (46 mg, 1.15 mmol), stir at room temperature for 20 minutes, and add iodooctane (186 mg, 0.77 mmol) to the above reaction solution. Stir at room temperature overnight. After TLC detection, after the reaction is completed, add an appropriate amount of water and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (15:1), gave compound 53 (30 mg, 18.2%) as a white solid. 1 HNMR (400MHz, CDCl 3 ,)δ3.58-3.52(m,3H),3.48-3.40(m,4H),3.39-3.31(m,3H),3.24-3.14(m,4H),2.39-2.31 (m,1H),2.24-2.20(m,4H),1.12-0.97(m,4H), 0.98-0.94(m,6H,CH 3 ×2),0.90-0.83(m,11H),0.67(s,3H,CH 3 ).ESI-MS m/z Calc.C 56 H 105 N 2 O 3 + [M+H] + 854.47, Found 853.8. HPLC: 81.0%.
实施例45：奥贝胆酸衍生物(化合物54)的合成路线如下
Example 45: The synthetic route of obeticholic acid derivative (compound 54) is as follows
将2-辛基癸酸(0.82g，2.89mmol)溶解于DCM中，加入1滴DMF后，再加入草酰氯(1.83g，14.4mmol)，室温反应4个小时，反应液直接浓缩，将所得酰氯粗品加入到装有中间体54-1(250mg，0.46mmol)、三乙胺(0.47g，4.6mmol)和DCM的反应瓶中，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(30:1)洗脱，得淡黄色油状化合物54(65mg,13％)。1HNMR(400MHz,CD3OD,)δ5.37-5.32(m,1H),4.78-4.71(m,1H),4.61-4.53(m,1H),3.75-3.60(m,4H),3.00-2.81(m,4H),2.77-2.67(m,2H),2.66-2.49(m,2H),2.25-2.20(m,2H),0.95-0.93(m,3H),0.90-0.86(m,18H),0.67(s,3H).ESI-MS m/z Calc.C70H128N2O5[M+H]+1077.98,Found 1078.8.HPLC：88.0％。Dissolve 2-octyldecanoic acid (0.82g, 2.89mmol) in DCM, add 1 drop of DMF, then add oxalyl chloride (1.83g, 14.4mmol), react at room temperature for 4 hours, and concentrate the reaction solution directly. The crude acid chloride was added to a reaction flask containing intermediate 54-1 (250 mg, 0.46 mmol), triethylamine (0.47 g, 4.6 mmol) and DCM, and stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (30:1), gave compound 54 (65 mg, 13%) as a light yellow oil. 1 HNMR (400MHz, CD 3 OD,) δ5.37-5.32(m,1H),4.78-4.71(m,1H),4.61-4.53(m,1H),3.75-3.60(m,4H),3.00- 2.81(m,4H),2.77-2.67(m,2H),2.66-2.49(m,2H),2.25-2.20(m,2H),0.95-0.93(m,3H),0.90-0.86(m,18H ), 0.67 (s, 3H). ESI-MS m/z Calc. C 70 H 128 N 2 O 5 [M+H] + 1077.98, Found 1078.8. HPLC: 88.0%.
实施例46：奥贝胆酸衍生物(化合物55)的合成路线如下
Example 46: The synthetic route of obeticholic acid derivative (compound 55) is as follows
中间体55-1的合成：将奥贝胆酸(1.5g,3.57mmol)溶解于DMF中,依次加入EDCI(1.0g,5.35mmol),DIEA(1.38g,10.7mmol)，DMAP(0.22g 1.78mmol),醇(0.56g 3.9mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体55-1(1.0g,52.5％)。Synthesis of intermediate 55-1: Dissolve obeticholic acid (1.5g, 3.57mmol) in DMF, then add EDCI (1.0g, 5.35mmol), DIEA (1.38g, 10.7mmol), and DMAP (0.22g 1.78 mmol), alcohol (0.56g 3.9mmol), stir at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 55-1 (1.0 g, 52.5%).
化合物55的合成：将化合物2-辛基癸酸(62 5mg,2.2mmol)溶解于DCM中,加入1滴DMF,加入草酰氯(1.4g,11.0mmol)，搅拌反应4h。TLC 检测，反应结束后，浓缩除去草酰氯。将中间体55-1(200mg,0.37mmol)溶于DCM，加入上述制备的酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状化合物55(130mg,33％)。1HNMR(400MHz,CDCl3)δ4.61-4.53(m，1H),4.10-4.07(t，2H),3.75-3.70(m,2H),2.63-2.56(m,6H),2.34-2.30(m，1H)，2.26-2.20(m，3H),0.94-0.86(m,15H,CH3×5),0.66(s,3H,CH3).ESI-MS m/z Calc.C52H94NO5[M+H]+812.71,Found 812.70。HPLC：81.1％。Synthesis of compound 55: Dissolve compound 2-octyldecanoic acid (62 5 mg, 2.2 mmol) in DCM, add 1 drop of DMF, add oxalyl chloride (1.4 g, 11.0 mmol), and stir for 4 hours. TLC Detection, after the reaction is completed, concentrate to remove oxalyl chloride. Intermediate 55-1 (200 mg, 0.37 mmol) was dissolved in DCM, the acid chloride prepared above was added, and the mixture was stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 55 (130 mg, 33%) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ) δ4.61-4.53 (m, 1H), 4.10-4.07 (t, 2H), 3.75-3.70 (m, 2H), 2.63-2.56 (m, 6H), 2.34-2.30 ( m, 1H), 2.26-2.20 (m, 3H), 0.94-0.86 (m, 15H, CH 3 × 5), 0.66 (s, 3H, CH 3 ).ESI-MS m/z Calc.C 52 H 94 NO 5 [M+H] + 812.71,Found 812.70. HPLC: 81.1%.
实施例47：奥贝胆酸衍生物(化合物56)的合成路线如下
Example 47: The synthetic route of obeticholic acid derivative (compound 56) is as follows
中间体56-1的合成：将奥贝胆酸(1.7g,4.04mmol)溶解于DMF中,依次加入EDCI(1.17g,6.06mmol),DIEA(1.58g,12.12mmol),DMAP(0.25g,2.02mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体56-1(1.5g,71.5％)。1HNMR(400MHz,CDCl3)δ4.09-4.06(t，2H),3.70-3.69(m，1H)，3.44-3.36(m,1H),2.34-2.26(m,2H),2.22(s,6H),0.97-0.88(m,9H,CH3×3),0.67(s,3H,CH3)。Synthesis of intermediate 56-1: Dissolve obeticholic acid (1.7g, 4.04mmol) in DMF, add EDCI (1.17g, 6.06mmol), DIEA (1.58g, 12.12mmol), DMAP (0.25g, 2.02 mmol), stir at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 56-1 (1.5 g, 71.5%). 1 HNMR (400MHz, CDCl 3 ) δ4.09-4.06 (t, 2H), 3.70-3.69 (m, 1H), 3.44-3.36 (m, 1H), 2.34-2.26 (m, 2H), 2.22 (s, 6H), 0.97-0.88(m,9H,CH 3 ×3), 0.67(s,3H,CH 3 ).
化合物56的合成：将中间体56-1(230mg,0.44mmol)溶于DCM，加入现制的酰氯，室温搅拌过夜。TLC检测，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状化合物56(130mg,28.8％)。1HNMR(400MHz,CDCl3)δ4.60-4.54(m，1H)，4.10-4.07(t，2H),3.72(s，1H),2.44-2.42(m，2H),2.38-2.31(m,7H),0.94-0.86(m,15H,CH3×5),0.66(s,3H,CH3).ESI-MS m/z Calc.C50H92NO5[M+H]+786.69,Found 786.70.HPLC：95.5％。Synthesis of compound 56: Dissolve intermediate 56-1 (230 mg, 0.44 mmol) in DCM, add fresh acid chloride, and stir at room temperature overnight. TLC detection, concentrated solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 56 (130 mg, 28.8%) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ) δ4.60-4.54 (m, 1H), 4.10-4.07 (t, 2H), 3.72 (s, 1H), 2.44-2.42 (m, 2H), 2.38-2.31 (m, 7H),0.94-0.86(m,15H,CH 3 ×5),0.66(s,3H,CH 3 ).ESI-MS m/z Calc.C 50 H 92 NO 5 [M+H] + 786.69,Found 786.70. HPLC: 95.5%.
实施例48：熊去氧胆酸衍生物(化合物57)的合成路线如下
Example 48: The synthetic route of ursodeoxycholic acid derivative (compound 57) is as follows
中间体57-1的合成：将熊去氧胆酸(1.5g,3.82mmol)，HBTU(2.17g,5.73mmol),DIEA(1.48g,11.46mmol)，胺(0.66g,4.2mmol)和DMF依次加入反应瓶中,室温搅拌过夜。TLC检测，反应结束后，浓缩得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体57-1(1.5g,71.5％)。1H NMR(400MHz,Chloroform-d)δ6.39(s,1H),3.64-3.52(m,2H),3.24(q,2H),2.42(s,3H),2.35(t,2H),2.22(m,1H),2.09–1.96(m,4H),1.90(m,1H),0.93(d,6H),0.67(s,3H).Synthesis of intermediate 57-1: ursodeoxycholic acid (1.5g, 3.82mmol), HBTU (2.17g, 5.73mmol), DIEA (1.48g, 11.46mmol), amine (0.66g, 4.2mmol) and DMF Add to the reaction flask one by one and stir at room temperature overnight. TLC detection, after the reaction is completed, concentrate to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 57-1 (1.5 g, 71.5%). 1 H NMR(400MHz,Chloroform-d)δ6.39(s,1H),3.64-3.52(m,2H),3.24(q,2H),2.42(s,3H),2.35(t,2H),2.22 (m,1H),2.09–1.96(m,4H),1.90(m,1H),0.93(d,6H),0.67(s,3H).
将中间体57-1(250mg,0.47mmol)溶于DCM，加入现制的酰氯，室温搅拌过夜。TLC检测，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状化合物57(180mg,36.0％)。HPLC：82.1％。ESI-MS m/z Calc.C69H127N2O5[M+H]+1063.97,Found 1064.10.Dissolve intermediate 57-1 (250 mg, 0.47 mmol) in DCM, add fresh acid chloride, and stir at room temperature overnight. TLC detection, concentrated solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 57 (180 mg, 36.0%) as a light yellow oil. HPLC: 82.1%. ESI-MS m/z Calc.C 69 H 127 N 2 O 5 [M+H] + 1063.97, Found 1064.10.
实施例49：石胆酸衍生物(化合物58)的合成路线如下
Example 49: The synthesis route of lithocholic acid derivative (compound 58) is as follows
中间体58-1的合成：向反应瓶中依次加入石胆酸(10g,2.66mmol)，溴化苄(5.9g，3.45mmol)，碳酸钾(5.5g，3.98mmol)和乙腈。升温至40℃，反应过夜。TLC显示反应完全。反应液过滤，滤液浓缩拉干，硅胶柱层析，DCM/CH3OH(20:1)洗脱，得类白色固体58-1(10g,80％)。1HNMR(400MHz,CDCl3)δ7.37-7.31(m,5H,ArH),5.14-5.08(m,2H,O-CH2-Ph),3.66-3.58(m,1H,C3),2.44-2.36(m,1H),2.31-2.23(m,1H),1.96-1.92(m,1H),0.91-0.89(m,6H,CH3×2),0.62(s,3H,CH3).HPLC：98.41％。Synthesis of intermediate 58-1: Add lithocholic acid (10g, 2.66mmol), benzyl bromide (5.9g, 3.45mmol), potassium carbonate (5.5g, 3.98mmol) and acetonitrile to the reaction bottle in sequence. The temperature was raised to 40°C and the reaction was carried out overnight. TLC showed the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated and pulled to dryness, and then subjected to silica gel column chromatography with DCM/CH 3 OH (20:1) as eluent to obtain an off-white solid 58-1 (10 g, 80%). 1 HNMR (400MHz, CDCl 3 ) δ7.37-7.31(m,5H,ArH),5.14-5.08(m,2H,O-CH 2 -Ph),3.66-3.58(m,1H,C3),2.44- 2.36(m,1H),2.31-2.23(m,1H),1.96-1.92(m,1H),0.91-0.89(m,6H,CH 3 ×2),0.62(s,3H,CH 3 ).HPLC :98.41%.
中间体58-2的合成：将中间体58-1(450mg,0.96mmol)溶解于吡啶中，将肉豆蔻酰氯(1mL)加入反应液中，室温搅拌反应16h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE:DCM(3:1-3:2)洗脱，得白色固体58-2(230mg,35.4％)1HNMR(400MHz,CDCl3)δ7.37-7.31(m,5H),5.14-5.07(m,2H),4.76-4.68(m,1H，C3),2.44-2.36(m,1H),2.31-2.22(m,3H),1.96-1.92(m, 1H),1.86-1.77(m,5H),1.68-1.56(m,3H),1.54-0.98(m,43H),0.92(s,3H,CH3),0.91-0.86(m,6H),0.62(s,3H,CH3).HPLC:82.2％。Synthesis of intermediate 58-2: Dissolve intermediate 58-1 (450 mg, 0.96 mmol) in pyridine, add myristoyl chloride (1 mL) to the reaction solution, and stir the reaction at room temperature for 16 hours. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE:DCM (3:1-3:2), gave white solid 58-2 (230mg, 35.4%) 1 HNMR (400MHz, CDCl 3 ) δ7.37-7.31 (m, 5H) ,5.14-5.07(m,2H),4.76-4.68(m,1H,C3),2.44-2.36(m,1H),2.31-2.22(m,3H),1.96-1.92(m, 1H),1.86-1.77(m,5H),1.68-1.56(m,3H),1.54-0.98(m,43H),0.92(s,3H,CH 3 ),0.91-0.86(m,6H),0.62 (s,3H,CH 3 ). HPLC: 82.2%.
中间体58-3的合成：将中间体58-2(220mg,0.33mmol),Pd/C(30mg)和甲醇加入反应瓶中，氢气氛围下25℃搅拌1h。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/MeOH(50:1-30:1)洗脱，得白色固体58-3(160mg,84.6％)。1HNMR(400MHz,CDCl3)δ4.77-4.69(m,1H),2.44-2.36(m,1H),2.30-2.22(m,3H),1.98-1.95(m,1H),1.86-1.77(m,5H),1.68-1.57(m,3H),1.53-0.98(m,42H),0.92-0.86(m,9H),0.64(s,3H,CH3).HPLC:78.8％。Synthesis of intermediate 58-3: Add intermediate 58-2 (220 mg, 0.33 mmol), Pd/C (30 mg) and methanol into the reaction bottle, and stir at 25°C for 1 hour under a hydrogen atmosphere. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluting with DCM/MeOH (50:1-30:1), gave white solid 58-3 (160 mg, 84.6%). 1 HNMR (400MHz, CDCl 3 ) δ4.77-4.69(m,1H),2.44-2.36(m,1H),2.30-2.22(m,3H),1.98-1.95(m,1H),1.86-1.77( m, 5H), 1.68-1.57 (m, 3H), 1.53-0.98 (m, 42H), 0.92-0.86 (m, 9H), 0.64 (s, 3H, CH 3 ). HPLC: 78.8%.
化合物58的合成：将中间体58-3(80mg,0.14mmol),mPEG2000-NH2(342mg,0.17mmol),HBTU(70mg,0.18mmol),DIPEA(46mg,0.35mmol)溶解于DMF中，室温搅拌16h。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(15:1)洗脱，得白色蜡状固体58(210mg,27.8％)。1HNMR(400MHz,CDCl3)δ6.19-6.14(m,1H，N-H),4.75-4.67(m,1H),3.81-3.79(m,1H),3.64-3.62(m,160H,PEG-chain),3.55-3.52(m,3H),3.45-3.41(m,2H),3.37(s,3H，O-CH3),2.27-2.21(m,3H),2.07-2.02(m,1H),1.96-1.93(m,1H),1.85-1.77(m,5H),1.61-1.01(m,48H),0.91-0.85(m,9H)，0.62(s,3H,CH3).HPLC：95.31％。Synthesis of compound 58: Dissolve intermediate 58-3 (80mg, 0.14mmol), mPEG2000-NH 2 (342mg, 0.17mmol), HBTU (70mg, 0.18mmol), DIPEA (46mg, 0.35mmol) in DMF, room temperature Stir for 16h. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (15:1), gave 58 (210 mg, 27.8%) as a white waxy solid. 1 HNMR (400MHz, CDCl 3 ) δ6.19-6.14(m,1H,NH),4.75-4.67(m,1H),3.81-3.79(m,1H),3.64-3.62(m,160H,PEG-chain ),3.55-3.52(m,3H),3.45-3.41(m,2H),3.37(s,3H,O-CH 3 ),2.27-2.21(m,3H),2.07-2.02(m,1H), 1.96-1.93(m,1H),1.85-1.77(m,5H),1.61-1.01(m,48H),0.91-0.85(m,9H),0.62(s,3H,CH 3 ).HPLC: 95.31% .
实施例50.熊去氧胆酸衍生物(化合物59)的合成路线如下
Example 50. The synthetic route of ursodeoxycholic acid derivative (compound 59) is as follows
中间体59-2的合成：将甲胺盐酸盐(675mg，10mmol)，中间体59-1(4.26g，30mmol)，TEA(2.0g，20mmol)和甲醇依次加入反应瓶中，室温搅拌16h。TLC检测,反应液直接浓缩干得粗品，硅胶柱层析，DCM/CH3OH(30:1)洗脱，得黄色固体中间体59-2(1.0g,71％)。1HNMR(400MHz,CD3OD,)δ4.37-4.39(d,3H),3.31-3.25(d,3H)。Synthesis of intermediate 59-2: Add methylamine hydrochloride (675 mg, 10 mmol), intermediate 59-1 (4.26 g, 30 mmol), TEA (2.0 g, 20 mmol) and methanol into the reaction bottle in sequence, and stir at room temperature for 16 hours. . After TLC detection, the reaction solution was directly concentrated to dryness to obtain a crude product, which was then subjected to silica gel column chromatography and eluted with DCM/CH 3 OH (30:1) to obtain yellow solid intermediate 59-2 (1.0 g, 71%). 1 HNMR (400MHz, CD 3 OD,) δ4.37-4.39(d,3H), 3.31-3.25(d,3H).
中间体59-4的合成：将中间体59-2(1.0g，7.08mmol)，中间体59-3(2.4g，14.1mmol)，TEA(1.4g，14.1mmol)和甲醇依次加入反应瓶中，室温搅拌16h。TLC检测。反应液直接浓缩得粗品，硅胶柱层析，DCM/CH3OH(20:1)洗脱，得白色固体中间体59-4(1.05g,52.5％)。1HNMR(400MHz,CD3OD,)δ3.64(s,2H),3.27(s,3H)，3.13-3.16(t，2H),1.76-1.79(t,2H),1.45(s,9H)。Synthesis of intermediate 59-4: Add intermediate 59-2 (1.0g, 7.08mmol), intermediate 59-3 (2.4g, 14.1mmol), TEA (1.4g, 14.1mmol) and methanol into the reaction bottle in sequence , stirred at room temperature for 16h. TLC detection. The reaction solution was directly concentrated to obtain a crude product, which was subjected to silica gel column chromatography and eluted with DCM/CH 3 OH (20:1) to obtain white solid intermediate 59-4 (1.05 g, 52.5%). 1 HNMR (400MHz, CD 3 OD,) δ3.64 (s, 2H), 3.27 (s, 3H), 3.13-3.16 (t, 2H), 1.76-1.79 (t, 2H), 1.45 (s, 9H) .
中间体59-5的合成：将中间体59-4(210mg，0.74mmol)溶解于二氯甲烷中，再加入0.1mL三氟乙酸，室温搅拌16h，TLC检测，反应液浓缩干，得到的粗品中间体59-5(150mg，110％)直接用于下一步。Synthesis of intermediate 59-5: Dissolve intermediate 59-4 (210 mg, 0.74 mmol) in dichloromethane, then add 0.1 mL trifluoroacetic acid, stir at room temperature for 16 hours, detect by TLC, and concentrate the reaction solution to dryness to obtain the crude product Intermediate 59-5 (150 mg, 110%) was used directly in the next step.
中间体59-6的合成：2-己基癸酸(6.4g,25mmol)溶解于DCM中，加入1滴DMF,滴加草酰氯(6.4g,50mmol)，室温搅拌2h。直接浓缩得酰氯粗品。将化合物6(2.4g,5.0mmol)，TEA(1.5g,15.0mmol)溶解于DCM中，将上述酰氯加入反应液中，室温反应过夜。TLC检测，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得淡黄色油状中间体59-6(2.0g,42％)。Synthesis of intermediate 59-6: Dissolve 2-hexyldecanoic acid (6.4g, 25mmol) in DCM, add 1 drop of DMF, add oxalyl chloride (6.4g, 50mmol) dropwise, and stir at room temperature for 2 hours. Concentrate directly to obtain crude acid chloride. Dissolve compound 6 (2.4g, 5.0mmol) and TEA (1.5g, 15.0mmol) in DCM, add the above acid chloride to the reaction solution, and react at room temperature overnight. TLC detection, concentrated solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (20:1), gave light yellow oily intermediate 59-6 (2.0 g, 42%).
中间体59-7的合成：单口烧瓶中加入中间体59-6(2.0g，2.08mmol)，Pd/C(100mg，5wt％)和甲醇，于H2氛围下室温反应16h。TLC显示原料反应完全。反应液过滤，浓缩，硅胶柱层析。DCM/CH3OH(10:1)洗脱，得无色油状中间体59-7(1.43g,79％)。1HNMR(400MHz,CDCl3)δ4.83-4.76(m，1H),4.72-4.67(m，1H),2.43-2.35(m,1H),2.29-2.19(m,3H),2.02-1.98(m,1H),0.98(s，3H,CH3)，0.94-0.92(d,3H,CH3),0.89-0.86(t,12H,CH3x 4),0.69(s,3H,CH3).HPLC：96.27％。Synthesis of intermediate 59-7: Add intermediate 59-6 (2.0g, 2.08mmol), Pd/C (100mg, 5wt%) and methanol into a single-neck flask, and react at room temperature for 16h under H2 atmosphere. TLC showed that the starting material reacted completely. The reaction solution was filtered, concentrated, and subjected to silica gel column chromatography. DCM/CH 3 OH (10:1) was eluted to obtain colorless oily intermediate 59-7 (1.43g, 79%). 1 HNMR (400MHz, CDCl 3 ) δ4.83-4.76 (m, 1H), 4.72-4.67 (m, 1H), 2.43-2.35 (m, 1H), 2.29-2.19 (m, 3H), 2.02-1.98 ( m,1H),0.98(s,3H,CH 3 ),0.94-0.92(d,3H,CH 3 ),0.89-0.86(t,12H,CH 3 x 4),0.69(s,3H,CH 3 ) .HPLC: 96.27%.
化合物59的合成：将中间体59-7(200mg,0.223mmol)，HATU(85mg,0.268mmol),DIEA(90mg,0.690mmol)，中间体59-5(49mg,0.268mmol)，和DMF依次加入反应瓶中，室温搅拌过夜。TLC检测，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体化合物59(45.8mg,22.1％)。 1HNMR(400MHz,CDCl3)δ7.04(s，1H),6.18(s，1H),6.03(s，1H),4.76-4.80(m，1H)，4.67-4.72(m，1H)，3.61-3.68(m，2H),3.36-3.38(m，2H),3.30-3.32(d,3H),0.98(s,3H),0.93-0.95(d，3H)，0.86-0.89(m，12H),0.68(s,3H).HPLC：96.24％。Synthesis of compound 59: Intermediate 59-7 (200mg, 0.223mmol), HATU (85mg, 0.268mmol), DIEA (90mg, 0.690mmol), intermediate 59-5 (49mg, 0.268mmol), and DMF were added in sequence In the reaction bottle, stir at room temperature overnight. TLC detection, concentrated solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 59 as a white solid (45.8 mg, 22.1%). 1 HNMR (400MHz, CDCl 3 ) δ7.04 (s, 1H), 6.18 (s, 1H), 6.03 (s, 1H), 4.76-4.80 (m, 1H), 4.67-4.72 (m, 1H), 3.61 -3.68(m,2H),3.36-3.38(m,2H),3.30-3.32(d,3H),0.98(s,3H),0.93-0.95(d,3H),0.86-0.89(m,12H) ,0.68(s,3H).HPLC: 96.24%.
实施例51：熊去氧胆酸衍生物(化合物60)的合成路线如下
Example 51: The synthetic route of ursodeoxycholic acid derivative (compound 60) is as follows
中间体60-2的合成：将中间体59-7(200mg,0.22mmol)，中间体60-1(134mg,0.27mmol)，HATU(126mg,0.33mmol)，TEA(68mg,0.66mmol)溶解于DMF中，室温搅拌20h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(50:1)洗脱，得淡黄色油状中间体60-2(140mg,46.6％)。1HNMR(400MHz,CDCl3)δ4.81-4.75(m,1H，C7),4.73-4.65(m,1H，C3),2.26-2.20(m,3H),2.06-1.98(m,3H),0.97(s,3H,CH3),0.92(d,3H,CH3),0.88-0.85(m,12H),0.67(s,3H,CH3).HPLC：63.0％。Synthesis of intermediate 60-2: Dissolve intermediate 59-7 (200mg, 0.22mmol), intermediate 60-1 (134mg, 0.27mmol), HATU (126mg, 0.33mmol), TEA (68mg, 0.66mmol) in In DMF, stir at room temperature for 20h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (50:1), gave light yellow oily intermediate 60-2 (140 mg, 46.6%). 1 HNMR (400MHz, CDCl 3 ) δ4.81-4.75 (m, 1H, C7), 4.73-4.65 (m, 1H, C3), 2.26-2.20 (m, 3H), 2.06-1.98 (m, 3H), 0.97(s,3H,CH 3 ), 0.92(d,3H,CH 3 ), 0.88-0.85(m,12H), 0.67(s,3H,CH 3 ). HPLC: 63.0%.
化合物60的合成：将中间体60-2(100mg,0.07mmol)溶解于氯化氢的乙酸乙酯溶液中，室温搅拌16h。TLC检测，反应结束后，反应液浓缩，加入二氯甲烷溶解，再用饱和碳酸钠水溶液洗，无水硫酸钠干燥，过滤，浓缩。粗品反相HPLC纯化，得白色蜡状固体化合物60(160mg,19.3％)。1HNMR(400MHz,CDCl3)δ6.65-6.63(m,1H，N-H),4.82-4.75(m,1H,C7),4.71-4.67(m,1H,C3),3.38-3.30(m,3H),2.77(t,3H),2.71-2.66(m,5H),0.97(s,3H,CH3)，0.92(d,3H,CH3),0.89-0.85(m,12H),0.68(s,3H,CH3).HPLC：82.0％。Synthesis of compound 60: Intermediate 60-2 (100 mg, 0.07 mmol) was dissolved in hydrogen chloride in ethyl acetate solution, and stirred at room temperature for 16 h. After TLC detection, the reaction solution was concentrated, dichloromethane was added to dissolve, washed with saturated sodium carbonate aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by reverse-phase HPLC to obtain compound 60 (160 mg, 19.3%) as a white waxy solid. 1 HNMR (400MHz, CDCl 3 ) δ6.65-6.63(m,1H,NH),4.82-4.75(m,1H,C7),4.71-4.67(m,1H,C3),3.38-3.30(m,3H ),2.77(t,3H),2.71-2.66(m,5H),0.97(s,3H,CH 3 ),0.92(d,3H,CH 3 ),0.89-0.85(m,12H),0.68(s ,3H,CH 3 ).HPLC: 82.0%.
实施例52：熊去氧胆酸衍生物(化合物61)的合成路线如下
Example 52: The synthetic route of ursodeoxycholic acid derivative (compound 61) is as follows
中间体61-2的合成：将中间体61-1(5.0g，56.2mmol)，TEA(17g，168.6mmol)，DCM分别加入反应瓶中，再加入TBSCl(12.7g，84.3mmol)，室温反应16个小时。TLC检测，浓缩得粗品，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得到黄色油状中间体61-2(9.3g，82％)。1HNMR(400MHz,CDCl3,)δ3.61-3.64(t,2H),2.69-2.72(t,2H),1.51-1.57(m,4H),0.89(s,9H),0.05(s,6H)。Synthesis of intermediate 61-2: Add intermediate 61-1 (5.0g, 56.2mmol), TEA (17g, 168.6mmol), and DCM into the reaction flask respectively, then add TBSCl (12.7g, 84.3mmol), and react at room temperature 16 hours. After TLC detection, the crude product was obtained by concentration. It was subjected to silica gel column chromatography and eluted with DCM/CH 3 OH (10:1) to obtain yellow oily intermediate 61-2 (9.3 g, 82%). 1 HNMR (400MHz, CDCl 3 ,)δ3.61-3.64(t,2H),2.69-2.72(t,2H),1.51-1.57(m,4H),0.89(s,9H),0.05(s,6H) ).
中间体61-3的合成：将熊去氧胆酸(5.0g,12.7mmol)溶解于DMF中,依次加入HBTU(5.8g,15.2mmol),DIEA(4.9g,38.1mmol)和中间体61-2(3.1g,15.2mmol)，室温搅拌过夜。TLC检测，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体61-3(5.5g,75％)。1HNMR(400MHz,CDCl3)δ5.68-5.79(m，1H),3.54-3.63(m，4H),3.23-3.27(m，2H),2.17-2.25(m，1H),1.96-2.07(m,2H),0.93(s,3H),0.91-0.92(d，3H)，0.88(s，9H),0.66(s,3H)，0.04(s，6H)。Synthesis of intermediate 61-3: Dissolve ursodeoxycholic acid (5.0g, 12.7mmol) in DMF, add HBTU (5.8g, 15.2mmol), DIEA (4.9g, 38.1mmol) and intermediate 61- in sequence 2 (3.1g, 15.2mmol), stirred at room temperature overnight. TLC detection, concentrated solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 61-3 (5.5 g, 75%). 1 HNMR (400MHz, CDCl3) δ5.68-5.79 (m, 1H), 3.54-3.63 (m, 4H), 3.23-3.27 (m, 2H), 2.17-2.25 (m, 1H), 1.96-2.07 (m ,2H),0.93(s,3H),0.91-0.92(d,3H),0.88(s,9H),0.66(s,3H),0.04(s,6H).
中间体61-5的合成：将中间体61-3(1.15g,2.0mmol)溶解于DMF中,加入NaH(240mg，6.0mmol)，室温搅拌半个小时，再加入中间体61-4(2.0g，6.0mmol)，室温搅拌反应过夜。TLC检测，加入适量水淬灭，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体61-5(756mg,35％)。1HNMR(400MHz,CDCl3)δ3.59-3.65(m，2H),3.14-3.44(m，8H),2.29-2.36(m，1H),2.16-2.24(m，1H)，1.97-2.0(d，1H)，0.92-0.94(m，6H)，0.86-0.89(m，15H),0.67(s,3H)，0.04(d，6H)。 Synthesis of intermediate 61-5: Dissolve intermediate 61-3 (1.15g, 2.0mmol) in DMF, add NaH (240mg, 6.0mmol), stir at room temperature for half an hour, and then add intermediate 61-4 (2.0 g, 6.0 mmol), and the reaction was stirred at room temperature overnight. TLC detection, add appropriate amount of water to quench, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid intermediate 61-5 (756 mg, 35%). 1 HNMR (400MHz, CDCl 3 ) δ3.59-3.65 (m, 2H), 3.14-3.44 (m, 8H), 2.29-2.36 (m, 1H), 2.16-2.24 (m, 1H), 1.97-2.0 ( d, 1H), 0.92-0.94 (m, 6H), 0.86-0.89 (m, 15H), 0.67 (s, 3H), 0.04 (d, 6H).
中间体61-6的合成：将中间体61-5(720mg,0.665mmol)溶解于THF中,加入TBAF-3H2O(631mg，2.0mmol)，室温搅拌16个小时。TLC检测，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得无色油状中间体61-6(142mg,72％)。1HNMR(400MHz,CDCl3)δ3.59-3.70(m，3H),3.16-3.44(m，7H),2.30-2.36(m，1H),2.15-2.24(m，1H)，1.97-2.0(d，1H)，0.93-0.95(m，6H)，0.86-0.89(m，6H),0.67(s,3H)。Synthesis of intermediate 61-6: Dissolve intermediate 61-5 (720 mg, 0.665 mmol) in THF, add TBAF-3H 2 O (631 mg, 2.0 mmol), and stir at room temperature for 16 hours. TLC detection, add appropriate amount of water, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (20:1), gave colorless oily intermediate 61-6 (142 mg, 72%). 1 HNMR (400MHz, CDCl3) δ3.59-3.70 (m, 3H), 3.16-3.44 (m, 7H), 2.30-2.36 (m, 1H), 2.15-2.24 (m, 1H), 1.97-2.0 (d , 1H), 0.93-0.95 (m, 6H), 0.86-0.89 (m, 6H), 0.67 (s, 3H).
中间体61-7的合成：将中间体61-6(130mg,0.134mmol)溶解于DCM中,加入Dess-Martin试剂(85mg，0.2mmol)，室温搅拌16个小时。TLC检测，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(30:1)洗脱，得无色油状中间体61-7(87mg,67％)。1HNMR(400MHz,CDCl3)δ9.77(s，1H),3.17-3.49(m，7H),2.81-2.86(m，1H),2.44-2.48(m，1H)，2.28-2.39(m，2H)，2.15-2.24(m，2H),1.92-1.96(m，1H),0.86-0.95(m，12H)，0.65(s,3H)。Synthesis of intermediate 61-7: Dissolve intermediate 61-6 (130 mg, 0.134 mmol) in DCM, add Dess-Martin reagent (85 mg, 0.2 mmol), and stir at room temperature for 16 hours. TLC detection, add appropriate amount of water, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (30:1), gave colorless oily intermediate 61-7 (87 mg, 67%). 1 HNMR (400MHz, CDCl3) δ9.77 (s, 1H), 3.17-3.49 (m, 7H), 2.81-2.86 (m, 1H), 2.44-2.48 (m, 1H), 2.28-2.39 (m, 2H ), 2.15-2.24(m, 2H), 1.92-1.96(m, 1H), 0.86-0.95(m, 12H), 0.65(s, 3H).
化合物61的合成：将中间体61-7(87mg,0.09mmol)，中间体61-8(13mg，0.18mmol)和DCM依次加入到反应瓶中,加入一滴醋酸，室温搅拌1个小时后，加入NaHB(OAc)3(29mg，0.145mmol)后，室温反应过夜。TLC检测，加入适量水，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(15:1)洗脱，得无色油状化合物61(31mg,33％)。1HNMR(400MHz,CDCl3)δ3.16-3.45(m，7H),2.81-2.86(m，1H),2.44-2.50(m，6H)，2.27-2.38(m，2H)，2.14-2.21(m，2H),1.91-1.95(m，1H),0.85-0.93(m，12H)，0.64(s,3H)。ESI-MS m/z Calc.C68H128N2O3[M+H]+1021.99,Found 1021.9.HPLC：97.01％。Synthesis of compound 61: Add intermediate 61-7 (87 mg, 0.09 mmol), intermediate 61-8 (13 mg, 0.18 mmol) and DCM to the reaction bottle in sequence, add a drop of acetic acid, stir at room temperature for 1 hour, and then add After adding NaHB(OAc) 3 (29 mg, 0.145 mmol), the reaction was carried out at room temperature overnight. TLC detection, add appropriate amount of water, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (15:1), gave compound 61 (31 mg, 33%) as a colorless oil. 1 HNMR (400MHz, CDCl3) δ3.16-3.45 (m, 7H), 2.81-2.86 (m, 1H), 2.44-2.50 (m, 6H), 2.27-2.38 (m, 2H), 2.14-2.21 (m , 2H), 1.91-1.95 (m, 1H), 0.85-0.93 (m, 12H), 0.64 (s, 3H). ESI-MS m/z Calc.C 68 H 128 N 2 O 3 [M+H] + 1021.99, Found 1021.9. HPLC: 97.01%.
实施例53：熊去氧胆酸衍生物(化合物62)的合成路线如下
Example 53: The synthetic route of ursodeoxycholic acid derivative (compound 62) is as follows
将中间体59-7(100mg,0.115mmol)溶解于ACN中，依次加入原料62-1(24mg,0.138mmol)，NMI(24mg,0.288mmol)，TCFH(39mg,0.138mmol)，室温搅拌反应16h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状化合物62(55mg,47％)。1HNMR(400MHz,CDCl3,)δ4.83-4.75(m,1H,C7),4.74-4.65(m,1H,C3),4.19(t,2H),3.63(t, 2H),2.67-2.44(m,12H),2.39-2.30(m,1H),2.26-2.19(m,3H),2.01-1.98(m,1H),0.97(s,3H,CH3),0.92-0.90(m,3H,CH3),0.89-0.85(m,12H,CH3×4),0.68(s,3H,CH3).HPLC：92.05％。Dissolve intermediate 59-7 (100mg, 0.115mmol) in ACN, add raw materials 62-1 (24mg, 0.138mmol), NMI (24mg, 0.288mmol), TCFH (39mg, 0.138mmol) in sequence, stir and react at room temperature for 16h . After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 62 (55 mg, 47%) as a light yellow oil. 1 HNMR(400MHz, CDCl 3 ,)δ4.83-4.75(m,1H,C7),4.74-4.65(m,1H,C3),4.19(t,2H),3.63(t, 2H),2.67-2.44(m,12H),2.39-2.30(m,1H),2.26-2.19(m,3H),2.01-1.98(m,1H),0.97(s,3H,CH 3 ),0.92 -0.90(m,3H,CH 3 ), 0.89-0.85(m,12H,CH 3 ×4), 0.68(s,3H,CH 3 ). HPLC: 92.05%.
实施例54：熊去氧胆酸衍生物(化合物63)的合成路线如下
Example 54: The synthetic route of ursodeoxycholic acid derivative (compound 63) is as follows
将中间体59-7(220mg,0.253mmol)溶解于ACN中，依次加入二醇(20mg,0.115mmol)，NMI(47mg,0.574mmol)，TCFH(71mg,0.253mmol)，室温搅拌反应16h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状化合物63(135mg,63％)。1HNMR(400MHz,CDCl3,)δ4.83-4.75(m,2H,C7),4.74-4.64(m,2H,C3),4.18(t,4H),2.67-2.47(m,12H),2.39-2.30(m,2H),2.27-2.18(m,6H),2.02-1.98(m,2H),0.97(s,6H,CH3×2),0.92-0.90(m,6H,CH3×2),0.89-0.85(m,24H,CH3×8),0.68(s,6H,CH3×2)。Intermediate 59-7 (220 mg, 0.253 mmol) was dissolved in ACN, diol (20 mg, 0.115 mmol), NMI (47 mg, 0.574 mmol), TCFH (71 mg, 0.253 mmol) were added in sequence, and the reaction was stirred at room temperature for 16 h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 63 (135 mg, 63%) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ,)δ4.83-4.75(m,2H,C7),4.74-4.64(m,2H,C3),4.18(t,4H),2.67-2.47(m,12H),2.39 -2.30(m,2H),2.27-2.18(m,6H),2.02-1.98(m,2H),0.97(s,6H,CH 3 ×2),0.92-0.90(m,6H,CH 3 ×2 ),0.89-0.85(m,24H,CH 3 ×8),0.68(s,6H,CH 3 ×2).
实施例55：熊去氧胆酸衍生物(化合物64)的合成路线如下
Example 55: The synthetic route of ursodeoxycholic acid derivative (compound 64) is as follows
将中间体59-7(100mg,0.115mmol)，HBTU(52mg，0.14mmol)，DMF(3mL)，DIEA(44mg，0.34mmol)，胺(70mg，0.6mmol)和DMF依次加入反应瓶中，室温搅拌16h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状化合物64(10mg)。1HNMR(400MHz,CDCl3,)δ4.67-4.72(m,1H),4.75-4.82(m,1H),3.35-3.37(m,2H),3.05-3.06(m,2H),2.74-2.75(m,2H),2.56-2.59(m,2H),2.30(s，3H)，2.20-2.25(m,3H),2.01-1.98(m,2H),0.97(s,3H,CH3),0.92-0.93(m,3H,CH3),0.86-0.89(m,12H,CH3×4),0.67(s,3H,CH3).HPLC：86.0％。Intermediate 59-7 (100 mg, 0.115 mmol), HBTU (52 mg, 0.14 mmol), DMF (3 mL), DIEA (44 mg, 0.34 mmol), amine (70 mg, 0.6 mmol) and DMF were added to the reaction bottle in sequence, at room temperature. Stir for 16h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 64 (10 mg) as a light yellow oil. 1 HNMR (400MHz, CDCl 3 ,)δ4.67-4.72(m,1H),4.75-4.82(m,1H),3.35-3.37(m,2H),3.05-3.06(m,2H),2.74-2.75 (m,2H),2.56-2.59(m,2H),2.30(s,3H),2.20-2.25(m,3H),2.01-1.98(m,2H),0.97(s,3H,CH 3 ), 0.92-0.93(m,3H,CH 3 ), 0.86-0.89(m,12H,CH 3 ×4), 0.67(s,3H,CH 3 ). HPLC: 86.0%.
其中，59-7是一种羧酸，在制备化合物59过程中的中间体。 Among them, 59-7 is a carboxylic acid, an intermediate in the preparation of compound 59.
实施例56:熊去氧胆酸衍生物(化合物65)的合成路线如下
Example 56: The synthetic route of ursodeoxycholic acid derivative (compound 65) is as follows
单口烧瓶中依次加入中间体59-7(200mg，0.23mmol)，TCFH(77.5mg，0.28mmol)，NMI(57mg，0.69mmol)，中间体65-1(13.5mg，0.12mmol)和DMF。室温搅拌16h。TLC检测，反应液加适量水，浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体化合物65(131mg,31.5％)。1HNMR(400MHz,CDCl3)δ4.82-4.75(m,2H),4.73-4.67(m,2H)，3.37-3.20(m，4H),2.57(m，3H)，2.28-2.98(m,8H),0.97(s,6H,CH3×2),0.93-0.92(d,6H,CH3×2),0.68(s,6H,CH3×2)。Intermediate 59-7 (200 mg, 0.23 mmol), TCFH (77.5 mg, 0.28 mmol), NMI (57 mg, 0.69 mmol), intermediate 65-1 (13.5 mg, 0.12 mmol) and DMF were added in sequence to the single-neck flask. Stir at room temperature for 16h. For TLC detection, the reaction solution was added with an appropriate amount of water, concentrated, and subjected to silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain compound 65 (131 mg, 31.5%) as a white solid. 1 HNMR (400MHz, CDCl 3 ) δ4.82-4.75 (m, 2H), 4.73-4.67 (m, 2H), 3.37-3.20 (m, 4H), 2.57 (m, 3H), 2.28-2.98 (m, 8H), 0.97 (s, 6H, CH 3 × 2), 0.93-0.92 (d, 6H, CH 3 × 2), 0.68 (s, 6H, CH 3 × 2).
实施例57:熊去氧胆酸衍生物(化合物66)的合成路线如下
Example 57: The synthetic route of ursodeoxycholic acid derivative (compound 66) is as follows
中间体66-1的合成：将熊去氧胆酸(4.00g,10.00mmol)，TFAA(11.55g,55.00mmol)和THF依次加入反应瓶中,再加入叔丁醇(23.25g,314.00mmol)，室温搅拌20h。TLC检测反应，浓缩溶剂得粗品。硅胶柱层析，DCM/MeOH(20:1)洗脱，得白色发泡状固体中间体66-1(4.30g,95.8％)。1HNMR(400MHz,CDCl3)δ3.63-3.54(m,2H),2.29-2.22(m,1H),2.16-2.08(m,1H),2.02-2.00(m,1H),1.95-1.86(m,1H),1.44(m,9H,CH3×3),0.94(s,3H,CH3),0.92(d,3H,CH3),0.67(s,3H,CH3).HPLC:86.8％。 Synthesis of intermediate 66-1: Add ursodeoxycholic acid (4.00g, 10.00mmol), TFAA (11.55g, 55.00mmol) and THF into the reaction bottle in sequence, and then add tert-butyl alcohol (23.25g, 314.00mmol) , stirred at room temperature for 20h. The reaction was detected by TLC, and the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/MeOH (20:1), gave white foamy solid intermediate 66-1 (4.30 g, 95.8%). 1 HNMR (400MHz, CDCl 3 ) δ3.63-3.54(m,2H),2.29-2.22(m,1H),2.16-2.08(m,1H),2.02-2.00(m,1H),1.95-1.86( m,1H),1.44(m,9H,CH 3 ×3),0.94(s,3H,CH 3 ),0.92(d,3H,CH 3 ),0.67(s,3H,CH 3 ).HPLC:86.8 %.
中间体66-2的合成：将亚油酸(23.11g,82.40mmol)溶解于DCM中,加入1滴DMF,加入草酰氯(20.92g,164.80mmol)，室温搅拌4h。直接浓缩得酰氯粗品。将中间体66-1(3.70g,8.24mmol)溶解于吡啶中，加入上述酰氯，室温搅拌20h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE/EA(1:1)洗脱，得无色油状中间体66-2(2.51g,31.1％)。1HNMR(400MHz,CDCl3)δ5.41-5.30(m,8H),4.81-4.75(m,1H,C7),4.71-4.63(m,1H,C3),2.78-2.75(m,4H),2.28-2.18(m,5H),2.15-2.09(m,1H),2.07-2.02(m,9H),1.44(s,9H,CH3×3),0.97(s,3H,CH3),0.91-0.87(m,9H),0.67(s,3H,CH3).HPLC:90.1％。Synthesis of intermediate 66-2: Dissolve linoleic acid (23.11g, 82.40mmol) in DCM, add 1 drop of DMF, add oxalyl chloride (20.92g, 164.80mmol), and stir at room temperature for 4 hours. Concentrate directly to obtain crude acid chloride. Intermediate 66-1 (3.70g, 8.24mmol) was dissolved in pyridine, the above acid chloride was added, and stirred at room temperature for 20 h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (1:1), gave colorless oily intermediate 66-2 (2.51g, 31.1%). 1 HNMR(400MHz, CDCl 3 )δ5.41-5.30(m,8H),4.81-4.75(m,1H,C7),4.71-4.63(m,1H,C3),2.78-2.75(m,4H), 2.28-2.18(m,5H),2.15-2.09(m,1H),2.07-2.02(m,9H),1.44(s,9H,CH 3 ×3),0.97(s,3H,CH 3 ),0.91 -0.87(m,9H),0.67(s,3H,CH 3 ). HPLC: 90.1%.
中间体66-3的合成：将中间体66-2(2.50g,2.57mmol)溶解在DCM中，再加入TFA(2.5mL),室温搅拌16h。TLC检测，反应结束后，反应液直接蒸掉溶剂得粗品。硅胶柱层析，DCM/MeOH(20:1)洗脱，得无色油状中间体66-3(1.79g,75.8％)。1HNMR(400MHz,CDCl3)δ5.41-5.30(m,8H),4.82-4.75(m,1H,C7),4.71-4.63(m,1H,C3),2.78-2.75(m,4H),2.43-2.35(m,1H),2.29-2.16(m,5H),2.07-1.97(m,9H),0.97(s,3H,CH3),0.93-0.87(m,9H),0.68(s,3H,CH3).HPLC:93.1％。Synthesis of intermediate 66-3: Dissolve intermediate 66-2 (2.50g, 2.57mmol) in DCM, add TFA (2.5mL), and stir at room temperature for 16h. TLC detection, after the reaction is completed, the solvent is directly evaporated from the reaction solution to obtain the crude product. Silica gel column chromatography, eluted with DCM/MeOH (20:1), gave colorless oily intermediate 66-3 (1.79g, 75.8%). 1 HNMR (400MHz, CDCl 3 ) δ5.41-5.30(m,8H),4.82-4.75(m,1H,C7),4.71-4.63(m,1H,C3),2.78-2.75(m,4H), 2.43-2.35(m,1H),2.29-2.16(m,5H),2.07-1.97(m,9H),0.97(s,3H,CH 3 ),0.93-0.87(m,9H),0.68(s, 3H,CH 3 ). HPLC: 93.1%.
化合物66的合成：将中间体66-3(250mg,0.27mmol),4-吡咯烷-1基丁-1-醇(58mg,0.41mmol),EDCI(68mg,0.35mmol),DIPEA(105mg,0.82mmol)溶解于DMF中，室温搅拌20h。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得无色油状化合物66(170mg,59.8％)。1HNMR(400MHz,CDCl3)δ5.41-5.29(m,8H),4.81-4.75(m,1H,C7),4.71-4.63(m,1H,C3),4.07(t,2H,O-CH2),2.78-2.75(m,4H),2.51-2.43(m,6H),2.36-2.28(m,1H),2.27-2.15(m,5H),2.07-1.96(m,9H),0.97(s,3H,CH3)，0.92-0.87(m,9H),0.67(s,3H,CH3).HPLC:95.3％.ESI-MS m/z Calc.C68H116NO6[M+H]+1042.67,Found 1042.67。Synthesis of compound 66: Combine intermediate 66-3 (250mg, 0.27mmol), 4-pyrrolidin-1-ylbutan-1-ol (58mg, 0.41mmol), EDCI (68mg, 0.35mmol), DIPEA (105mg, 0.82 mmol) was dissolved in DMF and stirred at room temperature for 20 h. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 66 (170 mg, 59.8%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ5.41-5.29(m,8H),4.81-4.75(m,1H,C7),4.71-4.63(m,1H,C3),4.07(t,2H,O-CH 2 ),2.78-2.75(m,4H),2.51-2.43(m,6H),2.36-2.28(m,1H),2.27-2.15(m,5H),2.07-1.96(m,9H),0.97( s,3H,CH 3 ), 0.92-0.87(m,9H),0.67(s,3H,CH 3 ).HPLC:95.3%.ESI-MS m/z Calc.C 68 H 116 NO 6 [M+H ] + 1042.67, Found 1042.67.
实施例58：熊去氧胆酸衍生物(化合物67)的合成路线如下
Example 58: The synthetic route of ursodeoxycholic acid derivative (compound 67) is as follows
将中间体66-3(100mg,0.11mmol),3-二甲氨基-1丙醇(17mg,0.16 mmol),EDCI(27mg,0.14mmol),DIPEA(42mg,0.33mmol)溶解于DMF中，室温搅拌20h。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得无色油状化合物67(73mg,67.0％)。1HNMR(400MHz,CDCl3)δ5.41-5.29(m,8H),4.81-4.75(m,1H,C7),4.70-4.65(m,1H,C3),4.10(t,2H,O-CH2),2.78-2.75(m,4H),2.38-2.27(m,4H),2.24(s,6H,N-CH3×2),2.23-2.16(m,4H),2.07-1.98(m,10H),0.97(s,3H,CH3)，0.92-0.87(m,9H),0.67(s,3H,CH3).HPLC:95.0％.ESI-MS m/z Calc.C65H112NO6[M+H]+1002.60,Found 1002.9。Intermediate 66-3 (100 mg, 0.11 mmol), 3-dimethylamino-1 propanol (17 mg, 0.16 mmol), EDCI (27 mg, 0.14 mmol), and DIPEA (42 mg, 0.33 mmol) were dissolved in DMF and stirred at room temperature for 20 h. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 67 (73 mg, 67.0%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ5.41-5.29(m,8H),4.81-4.75(m,1H,C7),4.70-4.65(m,1H,C3),4.10(t,2H,O-CH 2 ),2.78-2.75(m,4H),2.38-2.27(m,4H),2.24(s,6H,N-CH 3 ×2),2.23-2.16(m,4H),2.07-1.98(m, 10H),0.97(s,3H,CH 3 ), 0.92-0.87(m,9H),0.67(s,3H,CH 3 ).HPLC:95.0%.ESI-MS m/z Calc.C 65 H 112 NO 6 [M+H] + 1002.60, Found 1002.9.
实施例59：熊去氧胆酸衍生物(化合物68)的合成路线如下
Example 59: The synthetic route of ursodeoxycholic acid derivative (compound 68) is as follows
化合物68的合成：将中间体66-3(200mg,0.22mmol),N,N-二甲基乙醇胺(29mg,0.33mmol),EDCI(54mg,0.28mmol),DMAP(41mg,0.33mmol)溶解于DMF中，室温搅拌20h。TLC检测反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得无色油状化合物68(96mg,44.6％)。1HNMR(400MHz,CDCl3)δ5.41-5.29(m,8H),4.81-4.75(m,1H,C7),4.72-4.63(m,1H,C3),4.16(t,2H,O-CH2),2.79-2.75(m,4H),2.55(t,2H,N-CH2),2.40-2.33(m,1H),2.28(s,6H,N-CH3×2),2.27-2.18(m,4H),2.07-2.02(m,8H),0.96(s,3H,CH3)，0.91-0.87(m,9H),0.67(s,3H,CH3).HPLC:95.8％.ESI-MS m/z Calc.C64H110NO6[M+H]+988.83,Found 988.90。Synthesis of compound 68: Dissolve intermediate 66-3 (200mg, 0.22mmol), N,N-dimethylethanolamine (29mg, 0.33mmol), EDCI (54mg, 0.28mmol), DMAP (41mg, 0.33mmol) in In DMF, stir at room temperature for 20h. After the reaction was detected by TLC, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 68 (96 mg, 44.6%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ5.41-5.29(m,8H),4.81-4.75(m,1H,C7),4.72-4.63(m,1H,C3),4.16(t,2H,O-CH 2 ),2.79-2.75(m,4H),2.55(t,2H,N-CH 2 ),2.40-2.33(m,1H),2.28(s,6H,N-CH 3 ×2),2.27-2.18 (m,4H),2.07-2.02(m,8H),0.96(s,3H,CH 3 ), 0.91-0.87(m,9H),0.67(s,3H,CH 3 ).HPLC:95.8%.ESI -MS m/z Calc.C 64 H 110 NO 6 [M+H] + 988.83, Found 988.90.
实施例60：鹅去氧胆酸衍生物(化合物69)的合成路线如下
Example 60: The synthesis route of chenodeoxycholic acid derivative (compound 69) is as follows
中间体69-1的合成：单口烧瓶中依次加入鹅去氧胆酸(3.0g，7.64mmol)，K2CO3(1.6g 11.46mmol)，KI(130mg，0.77mmol)，溴化苄(1.5g，9.17mmol)和乙腈，随后反应液升温至40℃，搅拌反应过夜。TLC显示原料反应完全。反应液浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体69-1(1.9g,52.8％)。1H NMR(400MHz,Chloroform-d)δ7.37–7.29(m,5H),5.10(d,2H),3.58(m,2H),2.40(m,1H),2.27(m,1H),1.98(m,1H),0.94(s,3H),0.91(d,3H),0.64(s,3H).HPLC：90.7％。Synthesis of intermediate 69-1: Add chenodeoxycholic acid (3.0g, 7.64mmol), K 2 CO 3 (1.6g 11.46mmol), KI (130mg, 0.77mmol), and benzyl bromide (1.5 g, 9.17 mmol) and acetonitrile, then the reaction solution was heated to 40°C, and the reaction was stirred overnight. TLC showed that the starting material reacted completely. The reaction solution was concentrated and subjected to silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain white solid intermediate 69-1 (1.9 g, 52.8%). 1H NMR(400MHz,Chloroform-d)δ7.37–7.29(m,5H),5.10(d,2H),3.58(m,2H),2.40(m,1H),2.27(m,1H),1.98( m, 1H), 0.94 (s, 3H), 0.91 (d, 3H), 0.64 (s, 3H). HPLC: 90.7%.
中间体69-3的合成：将2-辛基癸酸(4.25g，16.6mmol)溶于DCM中，加入DMF(1滴)和草酰氯(10.5g 82.9mmol)，室温搅拌4h。反应液直接浓缩除去溶剂得酰氯粗品。将此酰氯加入到装有中间体69-1(1.0m，2.07mmol)和吡啶的反应瓶中，室温搅拌16h。TLC显示原料反应完全。浓缩，硅胶柱层析。DCM/CH3OH(20:1)洗脱，得无色油状中间体69-3(530mg,27.5％)。1H NMR(400MHz,Chloroform-d)δ7.37-7.30(m,5H),5.10(d,2H),5.00(q,1H),4.60(m,1H),2.41-2.22(m,4H),2.10(t,1H),2.02-1.94(m,2H),0.94(s,3H),0.91-0.86(m,15H),0.62(s,3H).HPLC：88.27％。Synthesis of intermediate 69-3: Dissolve 2-octyldecanoic acid (4.25g, 16.6mmol) in DCM, add DMF (1 drop) and oxalyl chloride (10.5g 82.9mmol), and stir at room temperature for 4 hours. The reaction solution is directly concentrated to remove the solvent to obtain crude acid chloride. This acid chloride was added to the reaction flask containing intermediate 69-1 (1.0 m, 2.07 mmol) and pyridine, and stirred at room temperature for 16 h. TLC showed that the starting material reacted completely. Concentrate and perform silica gel column chromatography. DCM/CH 3 OH (20:1) was eluted to obtain colorless oily intermediate 69-3 (530 mg, 27.5%). 1H NMR(400MHz,Chloroform-d)δ7.37-7.30(m,5H),5.10(d,2H),5.00(q,1H),4.60(m,1H),2.41-2.22(m,4H), 2.10(t,1H),2.02-1.94(m,2H),0.94(s,3H),0.91-0.86(m,15H),0.62(s,3H). HPLC: 88.27%.
中间体69-4的合成：单口烧瓶中依次加入中间体69-3(530mg，0.552mmol)，MeOH，Pd/C(27mg，5wt％)。于H2氛围下室温反应16h。TLC显示原料反应完全。反应液过滤，浓缩，硅胶柱层析。DCM/CH3OH(20:1)洗脱，得无色油状中间体69-4(330mg,69％)。1H NMR(400MHz,Chloroform-d)δ5.00(d,1H),4.60(m,1H),2.39(m,1H),2.32-2.20(m,3H),2.12(q,1H),2.03-1.95(m,2H),0.93(d,6H),0.88(m,12H),0.65(s,3H).HPLC：96.27％。 Synthesis of intermediate 69-4: Add intermediate 69-3 (530 mg, 0.552 mmol), MeOH, and Pd/C (27 mg, 5 wt%) in sequence to a one-neck flask. React at room temperature for 16h under H2 atmosphere. TLC showed that the starting material reacted completely. The reaction solution was filtered, concentrated, and subjected to silica gel column chromatography. DCM/CH 3 OH (20:1) was eluted to obtain colorless oily intermediate 69-4 (330 mg, 69%). 1 H NMR(400MHz,Chloroform-d)δ5.00(d,1H),4.60(m,1H),2.39(m,1H),2.32-2.20(m,3H),2.12(q,1H),2.03 -1.95(m,2H),0.93(d,6H),0.88(m,12H),0.65(s,3H). HPLC: 96.27%.
化合物69的合成：单口烧瓶中依次加入中间体69-4(160mg，0.184mmol)，4-四氢吡咯烷-1丁醇(34.3mg，0.221mmol)，EDCI(45.9mg，0.239mmol)，DIEA(59.5mg，0.460mmol)和DMF。室温搅拌反应16h。TLC显示原料反应完全。反应液浓缩，硅胶柱层析。DCM/CH3OH(10:1)洗脱，得无色油状化合物69(111.8mg,61.1％)。1H NMR(400MHz,Chloroform-d)δ5.01(s,1H),4.66-4.55(m,1H),4.10(t,2H),3.20-3.11(m,2H),2.38-2.10(m,9H),1.99(d,3H),0.94(s,3H),0.92(d,3H),0.90-0.86(m,12H),0.65(s,3H).ESI-MS m/z Calc.C64H115O6[M+H]+994.87Found 994.5。HPLC：95.00％。Synthesis of compound 69: Add intermediate 69-4 (160 mg, 0.184 mmol), 4-tetrahydropyrrolidine-1 butanol (34.3 mg, 0.221 mmol), EDCI (45.9 mg, 0.239 mmol), and DIEA to a single-necked flask in sequence. (59.5 mg, 0.460 mmol) and DMF. The reaction was stirred at room temperature for 16 h. TLC showed that the starting material reacted completely. The reaction solution was concentrated and subjected to silica gel column chromatography. Elution with DCM/CH 3 OH (10:1) gave compound 69 (111.8 mg, 61.1%) as colorless oil. 1 H NMR(400MHz,Chloroform-d)δ5.01(s,1H),4.66-4.55(m,1H),4.10(t,2H),3.20-3.11(m,2H),2.38-2.10(m, 9H),1.99(d,3H),0.94(s,3H),0.92(d,3H),0.90-0.86(m,12H),0.65(s,3H).ESI-MS m/z Calc.C 64 H 115 O 6 [M+H] + 994.87Found 994.5. HPLC: 95.00%.
实施例61：鹅去氧胆酸衍生物(化合物70)的合成路线如下
Example 61: The synthesis route of chenodeoxycholic acid derivative (compound 70) is as follows
向单口烧瓶中依次加入中间体69-4(160mg，0.184mmol)，二甲氨丁醇(28mg，0.221mmol)，EDCI(45.9mg，0.239mmol)，DIEA(59.5mg，0.460mmol)和DMF。室温搅拌反应16h。TLC显示原料反应完全。浓缩，硅胶柱层析。DCM/CH3OH(10:1)洗脱，得无色油状化合物70(160.9mg,89.9％)。1H NMR(400MHz,Chloroform-d)δ5.00(s,1H),4.66-4.53(m,1H),4.07(t,2H),2.46(s,2H),2.35(d,6H),2.33-2.07(m,6H),1.99(d,2H),0.94(s,3H),0.91(d,3H),0.89-0.85(m,12H),0.65(s,3H).ESI-MS m/z Calc.C62H113O6[M+H]+968.86Found 967.86。HPLC：95.68％。Intermediate 69-4 (160 mg, 0.184 mmol), dimethylaminobutanol (28 mg, 0.221 mmol), EDCI (45.9 mg, 0.239 mmol), DIEA (59.5 mg, 0.460 mmol) and DMF were added sequentially to the one-neck flask. The reaction was stirred at room temperature for 16 h. TLC showed that the starting material reacted completely. Concentrate and perform silica gel column chromatography. Elution with DCM/CH 3 OH (10:1) gave compound 70 (160.9 mg, 89.9%) as colorless oil. 1 H NMR(400MHz,Chloroform-d)δ5.00(s,1H),4.66-4.53(m,1H),4.07(t,2H),2.46(s,2H),2.35(d,6H),2.33 -2.07(m,6H),1.99(d,2H),0.94(s,3H),0.91(d,3H),0.89-0.85(m,12H),0.65(s,3H).ESI-MS m/ z Calc.C 62 H 113 O 6 [M+H] + 968.86Found 967.86. HPLC: 95.68%.
实施例62：鹅去氧胆酸衍生物(化合物71)的合成路线如下
Example 62: The synthesis route of chenodeoxycholic acid derivative (compound 71) is as follows
中间体71-1的合成：将奥贝胆酸(2.0g,4.75mmol)溶解于ACN中，再加入苄溴(1.63g,9.51mmol)，K2CO3(2.63g,19.02mmol)，40℃下搅拌过夜。TLC检测，反应结束后，过滤，浓缩溶剂得粗品。硅胶柱层析，PE/EA(3:1)洗脱，得白色固体71-1(1.7g,70％)。1HNMR(400MHz,CDCl3)δ7.39-7.30(m,5H),5.11(d,2H),3.69(s,1H,C7),3.45-3.36(m,1H,C3),2.45-2.35(m,1H),2.32-2.23(m,1H),0.93-0.88(m,9H,CH3×3),0.63(s,3H,CH3).HPLC：98.61％。Synthesis of intermediate 71-1: Dissolve obeticholic acid (2.0g, 4.75mmol) in ACN, then add benzyl bromide (1.63g, 9.51mmol), K 2 CO 3 (2.63g, 19.02mmol), 40 Stir overnight at ℃. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with PE/EA (3:1), gave white solid 71-1 (1.7g, 70%). 1 HNMR (400MHz, CDCl 3 ) δ7.39-7.30(m,5H),5.11(d,2H),3.69(s,1H,C7),3.45-3.36(m,1H,C3),2.45-2.35( m, 1H), 2.32-2.23 (m, 1H), 0.93-0.88 (m, 9H, CH 3 × 3), 0.63 (s, 3H, CH 3 ). HPLC: 98.61%.
中间体71-3的合成：将中间体71-1(650mg,1.27mmol)溶解于吡啶中，再加入现制的酰氯71-2(2.80g,10.18mmol)，室温下搅拌3h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE/EA(5:1)洗脱，得无色油状物71-3(495mg,52％)。1HNMR(400MHz,CDCl3)δ7.39-7.32(m,5H),5.11(d,2H),4.63-4.52(m,1H,C3),3.71(s,1H,C7),2.45-2.35(m,1H),2.33-2.20(m,2H),0.92-0.89(m,9H,CH3×3),0.88-0.85(m,6H,CH3×2),0.64(s,3H).HPLC：93.69％。Synthesis of intermediate 71-3: Dissolve intermediate 71-1 (650 mg, 1.27 mmol) in pyridine, then add fresh acid chloride 71-2 (2.80 g, 10.18 mmol), and stir at room temperature for 3 hours. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (5:1), gave colorless oil 71-3 (495 mg, 52%). 1 HNMR (400MHz, CDCl 3 ) δ7.39-7.32(m,5H),5.11(d,2H),4.63-4.52(m,1H,C3),3.71(s,1H,C7),2.45-2.35( m,1H),2.33-2.20(m,2H),0.92-0.89(m,9H,CH 3 ×3),0.88-0.85(m,6H,CH 3 ×2),0.64(s,3H).HPLC :93.69%.
中间体71-4的合成：将中间体71-3(395mg,0.527mmol)溶解于MeOH中,加入10％Pd/C(59mg,15％wt)，氢气氛围下搅拌反应16h。TLC检测，反应结束后，过滤，浓缩溶剂得粗品。硅胶柱层析，DCM/MeOH(30:1)洗脱，得无色油状物71-4(270mg,78％)。1HNMR(400MHz,CDCl3,)δ4.62-4.51(m,1H,C3),3.72(s,1H,C7),2.44-2.35(m,1H),2.30-2.21(m,2H),0.95-0.89(m,9H,CH3×3),0.89-0.85(m,6H,CH3×2),0.66(s,3H,CH3).HPLC：92.50％。Synthesis of intermediate 71-4: Dissolve intermediate 71-3 (395 mg, 0.527 mmol) in MeOH, add 10% Pd/C (59 mg, 15% wt), and stir for 16 hours under a hydrogen atmosphere. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluted with DCM/MeOH (30:1), gave colorless oily substance 71-4 (270 mg, 78%). 1 HNMR (400MHz, CDCl 3 ,)δ4.62-4.51(m,1H,C3),3.72(s,1H,C7),2.44-2.35(m,1H),2.30-2.21(m,2H),0.95 -0.89(m,9H,CH 3 ×3), 0.89-0.85(m,6H,CH 3 ×2), 0.66(s,3H,CH 3 ). HPLC: 92.50%.
化合物71的合成：将中间体71-4(140mg,0.212mmol)溶解于DMF中，依次加入4-吡咯烷-1-基丁-1-醇(46mg,0.319mmol)，EDCI(89mg,0.319mmol)，DMAP(59mg，0.48mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/MeOH(25:1)洗脱，得无色油状物71(120mg,72％)。1HNMR(400MHz,CDCl3,)δ4.62-4.51(m,1H,C3),4.11(t,2H),3.72(s,1H,C7),3.23-3.16(m,2H),2.42-2.33(m,1H),2.31-2.14(m,6H),0.95-0.89(m,9H,CH3×3),0.89-0.85(m,6H,CH3×2),0.66(s,3H,CH3).HPLC：86.36％。ESI-MS m/z Calc.C50H89NO5[M+H]+783.67,Found 784.8。Synthesis of compound 71: Dissolve intermediate 71-4 (140mg, 0.212mmol) in DMF, add 4-pyrrolidin-1-ylbutan-1-ol (46mg, 0.319mmol), EDCI (89mg, 0.319mmol) in sequence ), DMAP (59 mg, 0.48 mmol), stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluted with DCM/MeOH (25:1), gave colorless oil 71 (120 mg, 72%). 1 HNMR (400MHz, CDCl 3 ,)δ4.62-4.51(m,1H,C3),4.11(t,2H),3.72(s,1H,C7),3.23-3.16(m,2H),2.42-2.33 (m,1H),2.31-2.14(m,6H),0.95-0.89(m,9H,CH 3 ×3),0.89-0.85(m,6H,CH 3 ×2),0.66(s,3H,CH 3 ).HPLC: 86.36%. ESI-MS m/z Calc.C 50 H 89 NO 5 [M+H] + 783.67, Found 784.8.
实施例63：猪去氧胆酸衍生物(化合物72)的合成路线如下
Example 63: The synthesis route of hyodeoxycholic acid derivative (compound 72) is as follows
中间体72-1的合成：单口烧瓶中依次加入猪去氧胆酸(8.0g，20.4mmol)，KHCO3(4.1g,40.8mmol)，溴化苄(4.2g，24.5mmol)和乙腈。随后反应液升温至40℃，搅拌反应过夜。TLC显示原料反应完全。浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体中间体72-1(9.1g,92％)。1HNMR(400MHz,CDCl3)δ7.39-7.31(m,5H),5.15-5.08(m,2H),4.08-4.04(m,1H，C7),3.67-3.58(m,1H，C3),2.45-2.37(m,1H),2.31-2.20(m,1H),0.91-0.88(m,6H,CH3×2),0.61(s,3H,CH3).HPLC:80.3％。Synthesis of intermediate 72-1: Add hyodeoxycholic acid (8.0g, 20.4mmol), KHCO 3 (4.1g, 40.8mmol), benzyl bromide (4.2g, 24.5mmol) and acetonitrile in sequence to a single-neck flask. Then the temperature of the reaction solution was raised to 40°C, and the reaction was stirred overnight. TLC showed that the starting material reacted completely. Concentrate and perform silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain white solid intermediate 72-1 (9.1 g, 92%). 1 HNMR (400MHz, CDCl 3 ) δ7.39-7.31(m,5H),5.15-5.08(m,2H),4.08-4.04(m,1H,C7),3.67-3.58(m,1H,C3), 2.45-2.37(m,1H), 2.31-2.20(m,1H), 0.91-0.88(m,6H,CH 3 ×2), 0.61(s,3H,CH 3 ). HPLC: 80.3%.
中间体72-2的合成：将2-己基癸酸(11.10g,43.28mmol)溶解于DCM中,加入1滴DMF和草酰氯(10.99g,86.57mmol)，室温搅拌4h。直接浓缩得酰氯粗品。将中间体72-1(2.10g,4.35mmol)溶解于吡啶中，加入上述酰氯，室温搅拌20h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE/EA(20:1-5:1)洗脱，得无色油状中间体72-2(2.85g,68.3％)。1HNMR(400MHz,CDCl3)δ7.37-7.32(m,5H),5.21-5.15(m,1H，C7),5.15-5.07(m,2H),4.76-4.70(m,1H,C3),2.30-2.22(m,3H),1.98-1.95(m,1H),0.98(s,3H,CH3),0.93(d,J＝4.0,3H,CH3),0.91-0.86(m,15H),0.62(s,3H,CH3).HPLC:73.8％。Synthesis of intermediate 72-2: Dissolve 2-hexyldecanoic acid (11.10g, 43.28mmol) in DCM, add 1 drop of DMF and oxalyl chloride (10.99g, 86.57mmol), and stir at room temperature for 4 hours. Concentrate directly to obtain crude acid chloride. Intermediate 72-1 (2.10g, 4.35mmol) was dissolved in pyridine, the above acid chloride was added, and stirred at room temperature for 20h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (20:1-5:1), gave colorless oily intermediate 72-2 (2.85g, 68.3%). 1 HNMR (400MHz, CDCl 3 ) δ7.37-7.32(m,5H),5.21-5.15(m,1H,C7),5.15-5.07(m,2H),4.76-4.70(m,1H,C3), 2.30-2.22(m,3H),1.98-1.95(m,1H),0.98(s,3H,CH 3 ),0.93(d,J＝4.0,3H,CH 3 ),0.91-0.86(m,15H) ,0.62(s,3H,CH 3 ). HPLC: 73.8%.
中间体72-3的合成：将中间体72-2(1.20g,1.25mmol),Pd/C(100mg)溶解于甲醇中，氢气氛围下25℃搅拌16h。TLC检测，反应结束后，过滤，浓缩溶剂得粗品。硅胶柱层析，DCM/MeOH(15:1)洗脱，得无色油状中间体72-3(1.02g,93.6％)。1HNMR(400MHz,CDCl3)δ5.21-5.15(m,1H，C7),4.74-4.70 (m,1H,C3),2.44-2.36(m,1H),2.31-2.22(m,3H),2.00-1.97(m,1H),0.98(s,3H,CH3),0.93(d,J＝4.0,3H,CH3),0.89-0.85(m,12H CH3×4),0.65(s,3H,CH3).HPLC:95.73％。Synthesis of intermediate 72-3: Dissolve intermediate 72-2 (1.20g, 1.25mmol), Pd/C (100mg) in methanol, and stir at 25°C for 16 hours under a hydrogen atmosphere. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluted with DCM/MeOH (15:1), gave colorless oily intermediate 72-3 (1.02g, 93.6%). 1 HNMR (400MHz, CDCl 3 ) δ5.21-5.15 (m, 1H, C7), 4.74-4.70 (m,1H,C3),2.44-2.36(m,1H),2.31-2.22(m,3H),2.00-1.97(m,1H),0.98(s,3H,CH 3 ),0.93(d,J =4.0, 3H, CH 3 ), 0.89-0.85 (m, 12H CH 3 × 4), 0.65 (s, 3H, CH 3 ). HPLC: 95.73%.
化合物72的合成：将中间体72-3(300mg,0.34mmol),4-吡咯烷-1丁基-1-醇(74mg,0.52mmol),EDCI(86mg,0.45mmol),DIPEA(134mg,1.04mmol)溶解于DMF中，室温搅拌16h。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得无色油状化合物72(94mg,27.4％)。1HNMR(400MHz,CDCl3)δ5.21-5.15(m,1H,C7),4.75-4.69(m,1H,C3),4.07(t,2H,O-CH2),2.51-2.44(m,5H),2.37-2.16(m,4H),1.99-1.97(m,1H),0.98(s,3H,CH3)，0.92-0.82(m,15H),0.64(s,3H,CH3).ESI-MS m/z Calc.C64H116NO6[M+H]+994.87,Found 955.0.HPLC:95.1％。Synthesis of compound 72: Combine intermediate 72-3 (300mg, 0.34mmol), 4-pyrrolidin-1butyl-1-ol (74mg, 0.52mmol), EDCI (86mg, 0.45mmol), DIPEA (134mg, 1.04 mmol) was dissolved in DMF and stirred at room temperature for 16 h. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 72 (94 mg, 27.4%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ5.21-5.15(m,1H,C7),4.75-4.69(m,1H,C3),4.07(t,2H,O-CH 2 ),2.51-2.44(m, 5H),2.37-2.16(m,4H),1.99-1.97(m,1H),0.98(s,3H,CH 3 ), 0.92-0.82(m,15H),0.64(s,3H,CH 3 ). ESI-MS m/z Calc. C 64 H 116 NO 6 [M+H] + 994.87, Found 955.0. HPLC: 95.1%.
实施例64：熊去氧胆酸衍生物(化合物73)的合成路线如下
Example 64: The synthetic route of ursodeoxycholic acid derivative (compound 73) is as follows
将中间体72-3(300mg,0.34mmol),4-二甲氨基-1-丁醇(61mg,0.52mmol),EDCI(86mg,0.45mmol),DIPEA(134mg,1.04mmol)溶解于DMF中，室温搅拌16h。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得无色油状化合物73(175mg,52.4％)。1HNMR(400MHz,CDCl3)δ5.20-5.15(m,1H,C7),4.75-4.69(m,1H,C3),4.07(t,2H,O-CH2),2.37-2.27(m,4H),2.24(s,6H,N-CH3×2),2.22-2.16(m,1H),1.99-1.96(m,1H),0.98(s,3H,CH3)，0.92-0.85(m,15H),0.64(s,3H,CH3).ESI-MS m/z Calc.C62H114NO6[M+H]+968.86,Found 969.0。HPLC:96.2％。Dissolve intermediate 72-3 (300mg, 0.34mmol), 4-dimethylamino-1-butanol (61mg, 0.52mmol), EDCI (86mg, 0.45mmol), DIPEA (134mg, 1.04mmol) in DMF, Stir at room temperature for 16h. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 73 (175 mg, 52.4%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ5.20-5.15(m,1H,C7),4.75-4.69(m,1H,C3),4.07(t,2H,O-CH 2 ),2.37-2.27(m, 4H),2.24(s,6H,N-CH 3 ×2),2.22-2.16(m,1H),1.99-1.96(m,1H),0.98(s,3H,CH 3 ),0.92-0.85(m ,15H),0.64(s,3H,CH 3 ).ESI-MS m/z Calc.C 62 H 114 NO 6 [M+H] + 968.86, Found 969.0. HPLC: 96.2%.
实施例65：熊去氧胆酸衍生物(化合物74)的合成路线如下
Example 65: The synthetic route of ursodeoxycholic acid derivative (compound 74) is as follows
中间体74-1的合成：将中间体59-2(1.0g，7.09mmol),胺(1.9g，8.5mmol)，TEA(860mg，8.50mmol)和MeOH依次加入反应瓶中。室温搅拌16h。TLC检测，反应结束后，直接浓缩除去溶剂，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得灰白色固体中间体74-1(2.5g，40％)。HPLC：99.42％。Synthesis of intermediate 74-1: Intermediate 59-2 (1.0g, 7.09mmol), amine (1.9g, 8.5mmol), TEA (860mg, 8.50mmol) and MeOH were added to the reaction bottle in sequence. Stir at room temperature for 16h. After TLC detection, after the reaction, the solvent was directly concentrated to remove, followed by silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain off-white solid intermediate 74-1 (2.5 g, 40%). HPLC: 99.42%.
中间体74-2的合成：将中间体74-1(500mg，1.41mmol)溶于DCM中，加入化合物HCl-EA(9.5mL，7.05mmol)。室温反应4h。TLC检测反应结束后，直接浓缩，得灰白色固体中间体74-2(400mg，97.5％)。Synthesis of intermediate 74-2: Intermediate 74-1 (500 mg, 1.41 mmol) was dissolved in DCM, and compound HCl-EA (9.5 mL, 7.05 mmol) was added. React at room temperature for 4 hours. After TLC detection, the reaction was directly concentrated to obtain off-white solid intermediate 74-2 (400 mg, 97.5%).
化合物74的合成：将中间体59-7(200mg，0.23mmol)，中间体74-2(82.0mg，0.28mmol)，EDCI(85mg，0.44mmol)，DIEA(130mg，1.00mmol)和DMF依次加入反应瓶中。室温反应16h。TLC检测，反应结束后，反应液浓缩，硅胶柱层析。DCM/CH3OH(10:1)洗脱，得黄色固体化合物74(183.1mg，72.1％)。1H NMR(400MHz,Chloroform-d)δ7.59-7.36(m,2H),5.87(t,1H),4.79(m,1H),4.70(m,1H),3.79(d,2H),3.36(q,2H),3.30(d,3H),2.38(t,2H),2.31(t,2H),2.24(m,3H),2.10(s,4H),1.99(d,1H),0.98(s,3H),0.95(d,3H),0.89-0.85(m,12H),0.69(s,3H).HPLC：95.26％。Synthesis of compound 74: Intermediate 59-7 (200mg, 0.23mmol), intermediate 74-2 (82.0mg, 0.28mmol), EDCI (85mg, 0.44mmol), DIEA (130mg, 1.00mmol) and DMF were added in sequence in the reaction bottle. React at room temperature for 16 hours. After TLC detection, the reaction solution was concentrated and subjected to silica gel column chromatography. Elution with DCM/CH 3 OH (10:1) gave compound 74 (183.1 mg, 72.1%) as a yellow solid. 1 H NMR(400MHz,Chloroform-d)δ7.59-7.36(m,2H),5.87(t,1H),4.79(m,1H),4.70(m,1H),3.79(d,2H),3.36 (q,2H),3.30(d,3H),2.38(t,2H),2.31(t,2H),2.24(m,3H),2.10(s,4H),1.99(d,1H),0.98( s, 3H), 0.95 (d, 3H), 0.89-0.85 (m, 12H), 0.69 (s, 3H). HPLC: 95.26%.
实施例66：熊去氧胆酸衍生物(化合物75)的合成路线如下
Example 66: The synthetic route of ursodeoxycholic acid derivative (compound 75) is as follows
中间体75-1的合成：将亚油酸(2.90g,10.36mmol)溶解于DCM中,加入1滴DMF和草酰氯(2.63g,20.72mmol)，室温搅拌反应4h。TLC检测，反应结束后，浓缩除去草酰氯。将化合物6(0.50g,1.03mmol)溶解于吡啶中，加入上述酰氯，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE/EA(30:1-1:1)洗脱，得无色油状中间体75-1(180mg,17.3％)。1HNMR(400MHz,CDCl3)δ7.39-7.31(m,5H),5.41-5.29(m,8H),5.14-5.07(m,2H),4.81-4.74(m,1H，C7),4.71-4.63(m,1H，C3),2.79-2.75(m,4H),2.43-2.35(m,1H),2.07-2.02(m,8H),1.99-1.96(m,1H),0.96(s,3H,CH3),0.91-0.85(m,9H),0.65(s,3H,CH3).HPLC:84.9％。Synthesis of intermediate 75-1: Dissolve linoleic acid (2.90g, 10.36mmol) in DCM, add 1 drop of DMF and oxalyl chloride (2.63g, 20.72mmol), stir and react at room temperature for 4 hours. TLC detection, after the reaction is completed, concentrate to remove oxalyl chloride. Compound 6 (0.50g, 1.03mmol) was dissolved in pyridine, the above acid chloride was added, and the mixture was stirred at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (30:1-1:1), gave colorless oily intermediate 75-1 (180 mg, 17.3%). 1 HNMR (400MHz, CDCl 3 ) δ7.39-7.31(m,5H),5.41-5.29(m,8H),5.14-5.07(m,2H),4.81-4.74(m,1H,C7),4.71- 4.63(m,1H,C3),2.79-2.75(m,4H),2.43-2.35(m,1H),2.07-2.02(m,8H),1.99-1.96(m,1H),0.96(s,3H , CH 3 ), 0.91-0.85 (m, 9H), 0.65 (s, 3H, CH 3 ). HPLC: 84.9%.
中间体75-2的合成：将中间体75-1(180mg,0.12mmol),Pd/C(20mg)溶解于甲醇中，氢气氛围下25℃搅拌16h。TLC检测，反应结束后，过滤，浓缩溶剂得粗品。硅胶柱层析，DCM/MeOH(20:1)洗脱，得无色油状中间体75-2(110mg,67.4％)。1HNMR(400MHz,CDCl3)δ4.82-4.75(m,1H，C7),4.71-4.64(m,1H,C3),2.43-2.35(m,1H),2.29-2.18(m,5H),2.01-1.98(m,1H),0.97(s,3H,CH3),0.93(d,3H,CH3),0.89-0.86(m,6H CH3×2),0.68(s,3H,CH3).HPLC:88.9％。Synthesis of intermediate 75-2: Dissolve intermediate 75-1 (180 mg, 0.12 mmol), Pd/C (20 mg) in methanol, and stir at 25°C for 16 hours under a hydrogen atmosphere. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluted with DCM/MeOH (20:1), gave colorless oily intermediate 75-2 (110 mg, 67.4%). 1 HNMR (400MHz, CDCl 3 ) δ4.82-4.75(m,1H,C7),4.71-4.64(m,1H,C3),2.43-2.35(m,1H),2.29-2.18(m,5H), 2.01-1.98(m,1H),0.97(s,3H,CH 3 ),0.93(d,3H,CH 3 ),0.89-0.86(m,6H CH 3 ×2),0.68(s,3H,CH 3 ). HPLC: 88.9%.
化合物75的合成：将中间体75-2(105mg,0.11mmol),4-(N,N二甲基)氨基丁醇(16mg,0.14mmol),HATU(56mg,0.15mmol),DIPEA(37mg,0.29mmol)溶解于DMF中，室温搅拌16h。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得淡黄色油状物75(40mg,34.4％)。1HNMR(400MHz,CDCl3)δ4.81-4.75(m,1H,C7),4.72-4.63(m,1H,C3),4.07(t,2H,O-CH2),2.41-2.35(m,2H),2.31(s,6H，N-CH3×2),2.27-2.18(m,5H),2.00-1.97(m,1H),0.97(s,3H,CH3)，0.92-0.86(m,9H),0.67(s,3H,CH3).ESI-MS m/z Calc.C66H122NO6[M+H]+1024.92,Found 1025.0。HPLC:95.3％。Synthesis of compound 75: Combine intermediate 75-2 (105mg, 0.11mmol), 4-(N,N dimethyl)aminobutanol (16mg, 0.14mmol), HATU (56mg, 0.15mmol), DIPEA (37mg, 0.29mmol) was dissolved in DMF and stirred at room temperature for 16h. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave 75 as a light yellow oil (40 mg, 34.4%). 1 HNMR(400MHz, CDCl 3 )δ4.81-4.75(m,1H,C7),4.72-4.63(m,1H,C3),4.07(t,2H,O-CH 2 ),2.41-2.35(m, 2H),2.31(s,6H,N-CH 3 ×2),2.27-2.18(m,5H),2.00-1.97(m,1H),0.97(s,3H,CH 3 ),0.92-0.86(m ,9H),0.67(s,3H,CH 3 ).ESI-MS m/z Calc.C 66 H 122 NO 6 [M+H] + 1024.92,Found 1025.0. HPLC: 95.3%.
实施例67：奥贝胆酸衍生物(化合物76)的合成路线如下
Example 67: The synthetic route of obeticholic acid derivative (compound 76) is as follows
单口烧瓶中依次加入奥贝胆酸(2.0g，4.75mmol)，二胺(279mg，2.38mmol)，HBTU(2.35g，6.18mmol)，DIEA(1.85g，14.26mmol)和THF，室温搅拌过夜。TLC显示原料反应完全。反应液浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体化合物76(800m g,18.6％)。1H NMR(400MHz,Chloroform-d)δ6.11(t,1H),4.17(t,2H),3.70(d,2H),3.59(t,2H),3.48(s,3H),3.40(m，2H),3.28(q,2H),2.74(t,2H),2.65(t,2H),2.57(t,2H),2.36(m,1H),2.22(m,2H),2.05(m,1H),1.95(d,3H),0.94-0.91(m,8H),0.89(d,11H),0.65(s,6H).ESI-MS m/z Calc.C56H98N3O6[M+H]+908.74Found 908.80。HPLC：95.39％。Obeticholic acid (2.0g, 4.75mmol), diamine (279mg, 2.38mmol), HBTU (2.35g, 6.18mmol), DIEA (1.85g, 14.26mmol) and THF were added in sequence to the one-neck flask, and stirred at room temperature overnight. TLC showed that the starting material reacted completely. The reaction solution was concentrated and subjected to silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain compound 76 (800 mg, 18.6%) as a white solid. 1 H NMR(400MHz,Chloroform-d)δ6.11(t,1H),4.17(t,2H),3.70(d,2H),3.59(t,2H),3.48(s,3H),3.40(m ,2H),3.28(q,2H),2.74(t,2H),2.65(t,2H),2.57(t,2H),2.36(m,1H),2.22(m,2H),2.05(m, 1H),1.95(d,3H),0.94-0.91(m,8H),0.89(d,11H),0.65(s,6H).ESI-MS m/z Calc.C 56 H 98 N 3 O 6 [ M+H] + 908.74Found 908.80. HPLC: 95.39%.
实施例68：奥贝胆酸衍生物(化合物77)的合成路线如下
Example 68: The synthetic route of obeticholic acid derivative (compound 77) is as follows
向反应瓶中依次加入化合物79(300mg，0.37mmol)，DMAP(4.5mg，0.04mmol)，吡啶和醋酸酐(560mg，5.49mmol)，室温搅拌过夜。TLC显示原料反应完全。反应液浓缩，硅胶柱层析。DCM/CH3OH(20:1)洗脱，得白色固体化合物77(115mg,49.1％)。1H NMR(400MHz,Chloroform-d)δ4.71(m,2H),3.60(t,4H),3.13(d,4H),2.75(s,3H),2.20(m,2H),2.03(s,8H),1.91(d,2H),0.91(s,6H),0.82(d,6H),0.59(s,6H).ESI-MS m/z Calc.C57H96N3O6[M+H]+918.72Found 918.80.HPLC：95.90％。Compound 79 (300 mg, 0.37 mmol), DMAP (4.5 mg, 0.04 mmol), pyridine and acetic anhydride (560 mg, 5.49 mmol) were added to the reaction flask in sequence, and the mixture was stirred at room temperature overnight. TLC showed that the starting material reacted completely. The reaction solution was concentrated and subjected to silica gel column chromatography. Elution with DCM/CH 3 OH (20:1) gave compound 77 as a white solid (115 mg, 49.1%). 1 H NMR(400MHz,Chloroform-d)δ4.71(m,2H),3.60(t,4H),3.13(d,4H),2.75(s,3H),2.20(m,2H),2.03(s ,8H),1.91(d,2H),0.91(s,6H),0.82(d,6H),0.59(s,6H).ESI-MS m/z Calc.C 57 H 96 N 3 O 6 [M +H] + 918.72Found 918.80.HPLC: 95.90%.
实施例69：奥贝胆酸衍生物(化合物78)的合成路线如下
Example 69: The synthetic route of obeticholic acid derivative (compound 78) is as follows
向反应瓶中依次加入化合物80(300mg，0.37mmol)，DMAP(4.5mg，0.04mmol)，吡啶和醋酸酐(560mg，5.49mmol)，室温搅拌过夜。TLC显示原料反应完全。反应液浓缩，硅胶柱层析，DCM/CH3OH(20:1)洗脱，得白色固体化合物78(115mg,49.1％)。1H NMR(400MHz,Chloroform-d)δ5.96(s,2H),4.76(m,2H),4.67(m,2H),3.34(s,4H),2.50(s,3H),2.25(m,5H),2.09(m,2H),2.03(s,6H),1.98(s,6H),1.82(d,6H),0.97(s,6H),0.93(d,6H),0.68(s,6H).ESI-MS m/z Calc.C61H100N3O10[M+H]+1034.73Found 1034.80.HPLC：96.86％。Compound 80 (300 mg, 0.37 mmol), DMAP (4.5 mg, 0.04 mmol), pyridine and acetic anhydride (560 mg, 5.49 mmol) were added sequentially to the reaction flask, and the mixture was stirred at room temperature overnight. TLC showed that the starting material reacted completely. The reaction solution was concentrated and subjected to silica gel column chromatography, eluting with DCM/CH 3 OH (20:1) to obtain compound 78 (115 mg, 49.1%) as a white solid. 1 H NMR(400MHz,Chloroform-d)δ5.96(s,2H),4.76(m,2H),4.67(m,2H),3.34(s,4H),2.50(s,3H),2.25(m ,5H),2.09(m,2H),2.03(s,6H),1.98(s,6H),1.82(d,6H),0.97(s,6H),0.93(d,6H),0.68(s, 6H).ESI-MS m/z Calc.C 61 H 100 N 3 O 10 [M+H] + 1034.73 Found 1034.80. HPLC: 96.86%.
实施例70：奥贝胆酸衍生物(化合物79)的合成路线如下
Example 70: The synthetic route of obeticholic acid derivative (compound 79) is as follows
向反应瓶中依次加入石胆酸(2.0g，5.3mmol)，胺(610mg，2.66mmol)，HBTU(2.42g，6.37mmol),DIEA(1.08g，10.62mmol)和THF，室温搅拌过夜。TLC显示原料反应完全。反应液浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体化合物79(2.3g,46.94％)。1H NMR(400MHz,Chloroform-d)δ5.95(t,2H),3.68-3.56(m,2H),3.33(q,4H),2.57-2.50(m,3H),2.49(t,4H),2.31-2.24(m,2H),2.23(s,4H),2.14-2.03(m,2H),1.96(d,2H),0.92(d,12H),0.64(s,6H).ESI-MS m/z Calc.C53H92N3O4[M+H]+834.70Found 834.80.HPLC：98.13％。Lithocholic acid (2.0g, 5.3mmol), amine (610mg, 2.66mmol), HBTU (2.42g, 6.37mmol), DIEA (1.08g, 10.62mmol) and THF were added in sequence to the reaction flask, and stirred at room temperature overnight. TLC showed that the starting material reacted completely. The reaction solution was concentrated and subjected to silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain compound 79 (2.3 g, 46.94%) as a white solid. 1 H NMR(400MHz,Chloroform-d)δ5.95(t,2H),3.68-3.56(m,2H),3.33(q,4H),2.57-2.50(m,3H),2.49(t,4H) ,2.31-2.24(m,2H),2.23(s,4H),2.14-2.03(m,2H),1.96(d,2H),0.92(d,12H),0.64(s,6H).ESI-MS m/z Calc. C 53 H 92 N 3 O 4 [M+H] + 834.70 Found 834.80. HPLC: 98.13%.
实施例71：熊去氧胆酸衍生物(化合物80)的合成路线如下
Example 71: The synthetic route of ursodeoxycholic acid derivative (compound 80) is as follows
向反应瓶中依次加入熊去氧胆酸(4.0g，10.2mmol)，胺(597mg，5.1mmol)，HBTU(4.63g，12.21mmol)，DIEA(2.06g，15.9mmol)和THF。室温搅拌过夜。TLC显示原料反应完全。反应液浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体化合物80(1.8g,40％)。1H NMR(400MHz,Chloroform-d)δ6.32(s,2H),3.68-3.51(m,4H),3.49(s,3H),3.41(m,2H),3.27-3.15(m,2H),2.60-2.49(m,2H),2.43(d,2H),2.33-2.08(m,10H),2.03(d,2H),0.95(d,12H),0.68(s,6H).ESI-MS m/z Calc.C53H92N3O6[M+H]+866.69Found 866.80.HPLC：95.68％。Ursodeoxycholic acid (4.0g, 10.2mmol), amine (597mg, 5.1mmol), HBTU (4.63g, 12.21mmol), DIEA (2.06g, 15.9mmol) and THF were added to the reaction bottle in sequence. Stir at room temperature overnight. TLC showed that the starting material reacted completely. The reaction solution was concentrated and subjected to silica gel column chromatography, eluting with DCM/CH 3 OH (10:1) to obtain compound 80 (1.8 g, 40%) as a white solid. 1 H NMR(400MHz,Chloroform-d)δ6.32(s,2H),3.68-3.51(m,4H),3.49(s,3H),3.41(m,2H),3.27-3.15(m,2H) ,2.60-2.49(m,2H),2.43(d,2H),2.33-2.08(m,10H),2.03(d,2H),0.95(d,12H),0.68(s,6H).ESI-MS m/z Calc.C 53 H 92 N 3 O 6 [M+H] + 866.69 Found 866.80. HPLC: 95.68%.
实施例72：熊去氧胆酸衍生物(化合物81)的合成路线如下
Example 72: The synthetic route of ursodeoxycholic acid derivative (compound 81) is as follows
将中间体59-7(85mg,0.1mmol),4-(N,N二甲基)氨基丙醇(16mg,0.15mmol),HATU(56mg,0.15mmol),DIPEA(37mg,0.29mmol)溶解于DMF中，室温搅拌16h。TLC检测，反应结束后，反应液浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得淡黄色油状化合物81(50mg,52％)。1HNMR(400MHz,CDCl3)δ4.82-4.75(m,1H,C7),4.73-4.65(m,1H,C3),4.12-4.09(t,2H),2.46-2.42(t,2H),2.31(s,6H),2.25-2.20(m,3H),2.01-1.97(m,1H),0.97(s,3H,CH3)，0.92-0.85(m,15H，CH3×5),0.68(s,3H,CH3).ESI-MS m/z Calc.C61H112NO6[M+H]+954.84,Found 955.0。HPLC:95.07％。Intermediate 59-7 (85 mg, 0.1 mmol), 4-(N,N dimethyl)aminopropanol (16 mg, 0.15 mmol), HATU (56 mg, 0.15 mmol), and DIPEA (37 mg, 0.29 mmol) were dissolved in In DMF, stir at room temperature for 16 h. After TLC detection, after the reaction is completed, the reaction solution is concentrated to obtain a crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (20:1), gave compound 81 (50 mg, 52%) as light yellow oil. 1 HNMR (400MHz, CDCl 3 ) δ4.82-4.75(m,1H,C7),4.73-4.65(m,1H,C3),4.12-4.09(t,2H),2.46-2.42(t,2H), 2.31(s,6H),2.25-2.20(m,3H),2.01-1.97(m,1H),0.97(s,3H,CH 3 ),0.92-0.85(m,15H,CH 3 ×5),0.68 (s,3H,CH 3 ).ESI-MS m/z Calc.C 61 H 112 NO 6 [M+H] + 954.84, Found 955.0. HPLC: 95.07%.
实施例73：熊去氧胆酸衍生物(化合物82)的合成路线如下
Example 73: The synthetic route of ursodeoxycholic acid derivative (compound 82) is as follows
化合物82的合成：将中间体66-3(200mg,0.22mmol),4-吡咯烷-1-丙醇(42mg,0.33mmol),EDCI(54mg,0.28mmol),DIPEA(85mg,0.65mmol)溶解于DMF中，室温搅拌20h。TLC检测反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得无色油状化合物82(60mg,26.8％)。1HNMR(400MHz,CDCl3)δ5.41-5.29(m,8H),4.82-4.75(m,1H,C7),4.72-4.62(m,1H,C3),4.12(t,2H,O-CH2),2.78-2.69(m,9H),2.37-2.29(m,1H),2.27-2.16(m,5H),2.07-2.01(m,8H),0.97(s,3H,CH3)，0.92-0.87(m,9H),0.67(s,3H,CH3).HPLC:96.01％.ESI-MS m/z Calc.C67H114NO6[M+H]+1028.86,Found 1028.90。Synthesis of compound 82: Dissolve intermediate 66-3 (200mg, 0.22mmol), 4-pyrrolidine-1-propanol (42mg, 0.33mmol), EDCI (54mg, 0.28mmol), DIPEA (85mg, 0.65mmol) Stir in DMF at room temperature for 20h. After the reaction was detected by TLC, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (20:1), gave compound 82 (60 mg, 26.8%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ5.41-5.29(m,8H),4.82-4.75(m,1H,C7),4.72-4.62(m,1H,C3),4.12(t,2H,O-CH 2 ),2.78-2.69(m,9H),2.37-2.29(m,1H),2.27-2.16(m,5H),2.07-2.01(m,8H),0.97(s,3H,CH 3 ),0.92 -0.87 (m, 9H), 0.67 (s, 3H, CH 3 ). HPLC: 96.01%. ESI-MS m/z Calc. C 67 H 114 NO 6 [M+H] + 1028.86, Found 1028.90.
实施例74：熊去氧胆酸衍生物(化合物83)的合成路线如下
Example 74: The synthetic route of ursodeoxycholic acid derivative (compound 83) is as follows
化合物83的合成：将中间体66-3(200mg,0.22mmol),4-吡咯烷-1-乙醇(38mg,0.33mmol),EDCI(54mg,0.28mmol),DIPEA(85mg,0.65mmol)溶解于DMF中，室温搅拌20h。TLC检测反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得无色油状化合物83(55mg,24.9％)。1HNMR(400MHz,CDCl3)δ5.41-5.29(m,8H),4.81-4.75(m,1H,C7),4.71-4.63(m,1H,C3),4.21(t,2H,O-CH2),2.78-2.74(m,6H),2.59(s,4H),2.40-2.32(m,1H),2.27-2.16(m,5H),2.07-2.02(m,8H),0.97(s,3H,CH3)，0.92-0.86(m,9H),0.67(s,3H,CH3).HPLC:95.10％.ESI-MS m/z Calc.C66H112NO6[M+H]+1014.84,Found 1014.90。Synthesis of compound 83: Dissolve intermediate 66-3 (200mg, 0.22mmol), 4-pyrrolidine-1-ethanol (38mg, 0.33mmol), EDCI (54mg, 0.28mmol), DIPEA (85mg, 0.65mmol) in In DMF, stir at room temperature for 20h. After the reaction was detected by TLC, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (20:1), gave compound 83 (55 mg, 24.9%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ5.41-5.29(m,8H),4.81-4.75(m,1H,C7),4.71-4.63(m,1H,C3),4.21(t,2H,O-CH 2 ),2.78-2.74(m,6H),2.59(s,4H),2.40-2.32(m,1H),2.27-2.16(m,5H),2.07-2.02(m,8H),0.97(s, 3H,CH 3 ), 0.92-0.86(m,9H),0.67(s,3H,CH 3 ).HPLC:95.10%.ESI-MS m/z Calc.C 66 H 112 NO 6 [M+H] + 1014.84, Found 1014.90.
实施例75：熊去氧胆酸衍生物(化合物84)的合成路线如下
Example 75: The synthetic route of ursodeoxycholic acid derivative (compound 84) is as follows
将化合物66-3(160mg,0.17mmol),74-2(76mg,0.26mmol),EDCI(43 mg,0.23mmol),DIPEA(112mg,0.87mmol)溶解于DMF中，室温搅拌20h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(20:1)洗脱，得无色油状物84(100mg,50.0％)。1HNMR(400MHz,CDCl3)δ7.57-7.52(m,2H,N-H×2),6.05-6.01(m,2H,N-H),5.41-5.29(m,8H),4.82-4.75(m,1H,C7),4.71-4.63(m,1H,C3),3.82-3.76(m,2H),3.38-3.33(m,2H),3.31(d,3H,NH-CH3),2.78-2.75(m,4H),2.44-2.41(m,2H),2.36-2.33(m,2H),2.30-2.18(m,5H),2.13(s,3H,N-CH3),2.12-2.09(m,1H),2.07-2.02(m,8H),0.97-0.94(m,6H),0.90-0.87(m,6H),0.68(s,3H,CH3).HPLC:96.20％。ESI-MS m/z Calc.C72H121N4O7[M+H]+1053.92,Found 1153.9。Compound 66-3 (160mg, 0.17mmol), 74-2 (76mg, 0.26mmol), EDCI (43 mg, 0.23mmol), DIPEA (112mg, 0.87mmol) were dissolved in DMF, and stirred at room temperature for 20h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (20:1), gave colorless oil 84 (100 mg, 50.0%). 1 HNMR (400MHz, CDCl 3 ) δ7.57-7.52(m,2H,NH×2),6.05-6.01(m,2H,NH),5.41-5.29(m,8H),4.82-4.75(m,1H ,C7),4.71-4.63(m,1H,C3),3.82-3.76(m,2H),3.38-3.33(m,2H),3.31(d,3H,NH-CH 3 ),2.78-2.75(m ,4H),2.44-2.41(m,2H),2.36-2.33(m,2H),2.30-2.18(m,5H),2.13(s,3H,N-CH 3 ),2.12-2.09(m,1H ), 2.07-2.02(m,8H), 0.97-0.94(m,6H), 0.90-0.87(m,6H), 0.68(s,3H,CH 3 ). HPLC: 96.20%. ESI-MS m/z Calc.C 72 H 121 N 4 O 7 [M+H] + 1053.92, Found 1153.9.
实施例76：熊去氧胆酸衍生物(化合物85)的合成路线如下
Example 76: The synthetic route of ursodeoxycholic acid derivative (compound 85) is as follows
中间体85-1的合成：将化合物3-溴-1丙醇(5.0g,35.72mmol)和TEA(10.8g,107.16mmol)溶解于DCM中，加入TBSCl(8.1g,53.58mmol)，室温搅拌16小时。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE/EA(10:1)洗脱，得无色油状物(7.3g,81.1％)。Synthesis of intermediate 85-1: Dissolve compound 3-bromo-1 propanol (5.0g, 35.72mmol) and TEA (10.8g, 107.16mmol) in DCM, add TBSCl (8.1g, 53.58mmol), and stir at room temperature 16 hours. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (10:1), gave colorless oil (7.3g, 81.1%).
中间体85-2的合成：将中间体85-1(1.6g，6.38mmol)，二胺(1.0g，5.31mmol)，碳酸钾(1.5g，8.0mmol)和DMF依次加入到反应瓶中，40℃下反应4个小时。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，EA/PE(3:1)洗脱，得淡黄色油状物(1.47g,77.0％)。1H NMR(400MHz,Chloroform-d)δ5.31(d,1H),3.64(t,2H),3.17(q,2H),2.39(m,4H),2.19(s,3H),1.77(s,1H),1.72-1.57(m,4H),1.43(s,9H),0.89(s,9H),0.04(s,6H)。HPLC:96.14％。Synthesis of intermediate 85-2: Add intermediate 85-1 (1.6g, 6.38mmol), diamine (1.0g, 5.31mmol), potassium carbonate (1.5g, 8.0mmol) and DMF into the reaction bottle in sequence, React at 40°C for 4 hours. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with EA/PE (3:1), gave light yellow oil (1.47g, 77.0%). 1 H NMR(400MHz,Chloroform-d)δ5.31(d,1H),3.64(t,2H),3.17(q,2H),2.39(m,4H),2.19(s,3H),1.77(s ,1H),1.72-1.57(m,4H),1.43(s,9H),0.89(s,9H),0.04(s,6H). HPLC: 96.14%.
中间体85-3的合成：将中间体85-2(1.4g，3.88mmol)溶解于DCM中，再加入1mL TFA,室温搅拌5个小时，TLC检测，反应结束后，直接浓缩干， 得到的中间体85-3粗品直接用于下一步。Synthesis of intermediate 85-3: Dissolve intermediate 85-2 (1.4g, 3.88mmol) in DCM, then add 1mL of TFA, stir at room temperature for 5 hours, and detect by TLC. After the reaction is completed, directly concentrate to dryness. The obtained crude intermediate 85-3 was directly used in the next step.
中间体85-4的合成：将中间体85-3(1.5g，3.88mmol)溶解于甲醇中，加入TEA(1.2g，11.64mmol)和中间体59-2(660mg，4.66mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/MeOH(15:1)洗脱，得白色固体中间体85-4(495mg,50.0％)。1H NMR(400MHz,Methanol-d4)δ3.67(t,4H),3.26(s,3H),3.00(q,4H),2.68(s,3H),2.05-1.94(m,2H),1.87(m,5.9Hz,2H)。HPLC:98.26％。ESI-MS m/z Calc.C12H22N3O3[M+H]+256.16,Found 256.3。Synthesis of intermediate 85-4: Dissolve intermediate 85-3 (1.5g, 3.88mmol) in methanol, add TEA (1.2g, 11.64mmol) and intermediate 59-2 (660mg, 4.66mmol), and stir at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/MeOH (15:1), gave white solid intermediate 85-4 (495 mg, 50.0%). 1 H NMR (400MHz, Methanol-d 4 ) δ3.67(t,4H),3.26(s,3H),3.00(q,4H),2.68(s,3H),2.05-1.94(m,2H), 1.87(m,5.9Hz,2H). HPLC: 98.26%. ESI-MS m/z Calc.C 12 H 22 N 3 O 3 [M+H] + 256.16, Found 256.3.
化合物85的合成：将中间体66-3(60mg，0.065mmol)溶解于MeCN中，加入化合物TCFH(27.5mg，0.098mmol)，NMI(16mg，0.20mmol)及中间体85-4(21mg，0.085mmol)。室温搅拌16h。TLC检测，反应结束后，浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色固体状物85(35mg,46.7％)。1H NMR(400MHz,Chloroform-d)δ5.40-5.29(m,9H),4.79(m,1H),4.67(s,1H),4.23(t,2H),3.76(s,2H),3.31(d,3H),2.77(t,4H),2.56(s,2H),2.47(s,2H),2.28-2.17(m,9H),2.05(q,11H),0.97(s,3H),0.94(d,3H),0.91-0.87(m,6H),0.68(s,3H).HPLC：96.01％。ESI-MS m/z Calc.C72H120N3O8[M+H]+1054.9,Found 1055.0。Synthesis of compound 85: Dissolve intermediate 66-3 (60 mg, 0.065 mmol) in MeCN, add compound TCFH (27.5 mg, 0.098 mmol), NMI (16 mg, 0.20 mmol) and intermediate 85-4 (21 mg, 0.085 mmol). Stir at room temperature for 16h. After TLC detection, the reaction was concentrated, followed by silica gel column chromatography and eluted with DCM/CH 3 OH (10:1) to obtain yellow solid 85 (35 mg, 46.7%). 1 H NMR(400MHz,Chloroform-d)δ5.40-5.29(m,9H),4.79(m,1H),4.67(s,1H),4.23(t,2H),3.76(s,2H),3.31 (d,3H),2.77(t,4H),2.56(s,2H),2.47(s,2H),2.28-2.17(m,9H),2.05(q,11H),0.97(s,3H), 0.94 (d, 3H), 0.91-0.87 (m, 6H), 0.68 (s, 3H). HPLC: 96.01%. ESI-MS m/z Calc.C 72 H 120 N 3 O 8 [M+H] + 1054.9, Found 1055.0.
实施例77：熊去氧胆酸衍生物(化合物86)的合成路线如下
Example 77: The synthetic route of ursodeoxycholic acid derivative (compound 86) is as follows
中间体86-1的合成：将中间体66-1(2.00g,4.46mmol),2-己基癸酸(1.71g,6.69mmol),HATU(2.54g,6.69mmol),DIPEA(1.74g,13.38mmol) 溶解于DMF中,室温搅拌20h。TLC检测反应完全，浓缩溶剂得粗品。硅胶柱层析，PE/EA(10:1-1:1)洗脱，得无色油状中间体86-1(1.85g,60.4％).1HNMR(400MHz,CDCl3)δ4.74-4.66(m,1H,C3),3.62-3.55(m,1H,C7),2.29-2.22(m,2H),2.16-2.08(m,1H),2.04-1.98(m,1H),1.44(s,9H,CH3×3),0.96(s,3H,CH3),0.92(d,3H,CH3),0.89-0.86(m,6H),0.67(s,3H,CH3).HPLC:96.40％。Synthesis of intermediate 86-1: Combine intermediate 66-1 (2.00g, 4.46mmol), 2-hexyldecanoic acid (1.71g, 6.69mmol), HATU (2.54g, 6.69mmol), DIPEA (1.74g, 13.38 mmol) Dissolve in DMF and stir at room temperature for 20h. TLC detected that the reaction was complete, and the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (10:1-1:1), gave colorless oily intermediate 86-1 (1.85g, 60.4%). 1 HNMR (400MHz, CDCl 3 ) δ 4.74-4.66 (m,1H,C3),3.62-3.55(m,1H,C7),2.29-2.22(m,2H),2.16-2.08(m,1H),2.04-1.98(m,1H),1.44(s, 9H,CH 3 ×3),0.96(s,3H,CH 3 ),0.92(d,3H,CH 3 ),0.89-0.86(m,6H),0.67(s,3H,CH 3 ).HPLC:96.40 %.
中间体86-2的合成：将亚油酸(2.94g,10.48mmol)溶解于DCM中,加入1滴DMF和草酰氯(2.66g,20.96mmol)，室温搅拌4h。浓缩得酰氯粗品。将中间体86-1(1.80g,2.62mmol)溶解于吡啶中，加入上述酰氯，室温搅拌20h。TLC检测反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE/EA(30:1-1:1)洗脱，得淡黄色油状中间体86-2(1.45g,58.2％)。1HNMR(400MHz,CDCl3)δ5.41-5.29(m,4H),4.82-4.76(m,1H,C7),4.73-4.66(m,1H,C3),2.78-2.75(m,2H),2.28-2.18(m,4H),2.15-2.09(m,1H),2.07-1.98(m,5H),1.44(s,9H,CH3×3),0.97(s,3H,CH3),0.91-0.86(m,12H),0.67(s,3H,CH3).HPLC:99.00％。Synthesis of intermediate 86-2: Dissolve linoleic acid (2.94g, 10.48mmol) in DCM, add 1 drop of DMF and oxalyl chloride (2.66g, 20.96mmol), and stir at room temperature for 4 hours. Concentrate to obtain crude acid chloride. Intermediate 86-1 (1.80g, 2.62mmol) was dissolved in pyridine, the above acid chloride was added, and stirred at room temperature for 20h. After the reaction was detected by TLC, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (30:1-1:1), gave light yellow oily intermediate 86-2 (1.45g, 58.2%). 1 HNMR (400MHz, CDCl 3 ) δ5.41-5.29(m,4H),4.82-4.76(m,1H,C7),4.73-4.66(m,1H,C3),2.78-2.75(m,2H), 2.28-2.18(m,4H),2.15-2.09(m,1H),2.07-1.98(m,5H),1.44(s,9H,CH 3 ×3),0.97(s,3H,CH 3 ),0.91 -0.86(m,12H),0.67(s,3H,CH 3 ). HPLC: 99.00%.
中间体86-3的合成：将中间体86-2(1.40g,1.44mmol)溶解在DCM中，加入TFA(1mL),室温搅拌20h。TLC检测，反应结束后，反应液直接蒸掉溶剂得粗品。硅胶柱层析，DCM洗脱，得无色油状中间体86-3(0.98g,76.6％)。1HNMR(400MHz,CDCl3)δ5.42-5.30(m,4H),4.83-4.77(m,1H,C7),4.73-4.67(m,1H,C3),2.79-2.75(m,2H),2.43-2.35(m,1H),2.30-2.19(m,4H),2.07-1.99(m,4H),0.97(s,3H,CH3),0.93(d,3H,CH3),0.90-0.86(m,9H),0.68(s,3H,CH3).HPLC:95％。Synthesis of intermediate 86-3: Dissolve intermediate 86-2 (1.40g, 1.44mmol) in DCM, add TFA (1mL), and stir at room temperature for 20h. TLC detection, after the reaction is completed, the solvent is directly evaporated from the reaction solution to obtain the crude product. Silica gel column chromatography, eluting with DCM, gave colorless oily intermediate 86-3 (0.98g, 76.6%). 1 HNMR(400MHz, CDCl 3 )δ5.42-5.30(m,4H),4.83-4.77(m,1H,C7),4.73-4.67(m,1H,C3),2.79-2.75(m,2H), 2.43-2.35(m,1H),2.30-2.19(m,4H),2.07-1.99(m,4H),0.97(s,3H,CH 3 ),0.93(d,3H,CH 3 ),0.90-0.86 (m,9H),0.68(s,3H,CH 3 ). HPLC: 95%.
化合物86的合成：将中间体86-3(200mg,0.22mmol),4-吡咯烷-1基丁-1-醇(48mg,0.34mmol),EDCI(56mg,0.29mmol),DIPEA(87mg,0.67mmol)溶解于DMF中，室温搅拌20h。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(15:1)洗脱，得无色油状化合物86(70mg,30.7％)。1HNMR(400MHz,CDCl3)δ5.39-5.29(m,4H),4.88-4.76(m,1H,C7),4.73-4.67(m,1H,C3),4.08(t,2H,O-CH2),2.85-2.75(m,6H),2.37-2.29(m,1H),2.27-2.16(m,4H),2.07-2.02(m,4H),0.97(s,3H,CH3)，0.92-0.86(m,12H),0.68(s,3H,CH3).HPLC:95.2％。ESI-MS m/z Calc.C66H116NO6[M+H]+1018.87,Found 1018.90。 Synthesis of compound 86: Combine intermediate 86-3 (200mg, 0.22mmol), 4-pyrrolidin-1-ylbutan-1-ol (48mg, 0.34mmol), EDCI (56mg, 0.29mmol), DIPEA (87mg, 0.67 mmol) was dissolved in DMF and stirred at room temperature for 20 h. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (15:1), gave compound 86 (70 mg, 30.7%) as a colorless oil. 1 HNMR (400MHz, CDCl 3 ) δ5.39-5.29(m,4H),4.88-4.76(m,1H,C7),4.73-4.67(m,1H,C3),4.08(t,2H,O-CH 2 ),2.85-2.75(m,6H),2.37-2.29(m,1H),2.27-2.16(m,4H),2.07-2.02(m,4H),0.97(s,3H,CH 3 ),0.92 -0.86(m,12H),0.68(s,3H,CH 3 ). HPLC: 95.2%. ESI-MS m/z Calc.C 66 H 116 NO 6 [M+H] + 1018.87, Found 1018.90.
实施例78：熊去氧胆酸衍生物(化合物87)的合成路线如下
Example 78: The synthetic route of ursodeoxycholic acid derivative (compound 87) is as follows
化合物87的合成：将中间体86-3(300mg，0.34mmol)，中间体74-2(110mg，0.38mmol)，EDCI(85mg，0.44mmol)，DIEA(130mg，1.00mmol)和DMF依次加入反应瓶中。室温搅拌16h。TLC检测，反应结束后，浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色固体化合物87(82.5mg，21.7％)。1H NMR(400MHz,Chloroform-d)δ7.67(s,2H),6.16(s,1H),5.42-5.27(m,4H),4.79(m,1H),4.70(m,1H),3.78(d,2H),3.36(m,2H),3.30(d,3H),2.76(t,2H),2.56(s,2H),2.48(s,2H),2.31-2.17(m,8H),2.14-2.96(m,8H),0.97(s,3H),0.94(d,3H),0.87(m,9H),0.68(s,3H).HPLC：96.1％。ESI-MS m/z Calc.C70H121N4O7[M+H]+1129.92,Found 1130.0。Synthesis of compound 87: Intermediate 86-3 (300 mg, 0.34 mmol), intermediate 74-2 (110 mg, 0.38 mmol), EDCI (85 mg, 0.44 mmol), DIEA (130 mg, 1.00 mmol) and DMF were added to the reaction in sequence in a bottle. Stir at room temperature for 16h. After TLC detection, the reaction was concentrated, followed by silica gel column chromatography and eluted with DCM/CH 3 OH (10:1) to obtain compound 87 (82.5 mg, 21.7%) as a yellow solid. 1 H NMR(400MHz,Chloroform-d)δ7.67(s,2H),6.16(s,1H),5.42-5.27(m,4H),4.79(m,1H),4.70(m,1H),3.78 (d,2H),3.36(m,2H),3.30(d,3H),2.76(t,2H),2.56(s,2H),2.48(s,2H),2.31-2.17(m,8H), 2.14-2.96(m,8H),0.97(s,3H),0.94(d,3H),0.87(m,9H),0.68(s,3H). HPLC: 96.1%. ESI-MS m/z Calc.C 70 H 121 N 4 O 7 [M+H] + 1129.92, Found 1130.0.
实施例79：熊去氧胆酸衍生物(化合物88)的合成路线如下
Example 79: The synthetic route of ursodeoxycholic acid derivative (compound 88) is as follows
化合物88的合成：将中间体86-3(200mg，0.22mmol)，TCFH(94mg，0.34mmol)，NMI(55mg，0.67mmol)，中间体85-4(75mg，0.29mmol)和乙腈依次加入反应瓶中。室温搅拌16h。TLC检测，反应结束后，浓缩，硅胶柱层析，DCM/CH3OH(10:1)洗脱，得黄色固体化合物88(174mg,68.8％)。1H NMR(400MHz,Chloroform-d)δ5.41-5.29(m,4H),4.80(m,1H),4.70(m,1H),4.23(t,2H),3.76(s,2H),3.31(d,3H),2.77(t,2H),2.56(s,2H),2.46(d,2H),2.38(m,1H),2.30-2.19(m,7H),2.09-1.96(m,6H),0.97(s,3H),0.94(d,3H),0.88(m,9H),0.68(s,3H).HPLC：95.90％。ESI-MS m/z Calc.C70H120N3O8[M+H]+1130.75,Found 1130.9。 Synthesis of compound 88: Intermediate 86-3 (200mg, 0.22mmol), TCFH (94mg, 0.34mmol), NMI (55mg, 0.67mmol), intermediate 85-4 (75mg, 0.29mmol) and acetonitrile were added to the reaction in sequence in a bottle. Stir at room temperature for 16h. After TLC detection, the reaction was concentrated, followed by silica gel column chromatography and elution with DCM/CH 3 OH (10:1) to obtain compound 88 (174 mg, 68.8%) as a yellow solid. 1 H NMR(400MHz,Chloroform-d)δ5.41-5.29(m,4H),4.80(m,1H),4.70(m,1H),4.23(t,2H),3.76(s,2H),3.31 (d,3H),2.77(t,2H),2.56(s,2H),2.46(d,2H),2.38(m,1H),2.30-2.19(m,7H),2.09-1.96(m,6H ), 0.97 (s, 3H), 0.94 (d, 3H), 0.88 (m, 9H), 0.68 (s, 3H). HPLC: 95.90%. ESI-MS m/z Calc.C 70 H 120 N 3 O 8 [M+H] + 1130.75, Found 1130.9.
实施例80：熊去氧胆酸与紫杉醇的偶联物(化合物89)的合成路线如下
Example 80: The synthesis route of the conjugate of ursodeoxycholic acid and paclitaxel (compound 89) is as follows
将中间体66-3(250mg,0.272mmol)溶解于DMF中,依次加入紫杉醇(233mg,0.272mmol)，EDCI(68mg,0.354mmol)，DMAP(50.0mg,0.408mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE/EA(10:1)洗脱，得白色固体化合物89(200mg,42％)。1HNMR(400MHz,CDCl3)δ8.15-8.12(m,2H),7.75-7.72(m,2H),7.63-7.58(m,1H),7.54-7.49(m,3H),7.44-7.33(m,8H),6.86(d,1H),6.32-6.22(m,2H),5.95(m,1H),5.68(d,1H),5.50(d,1H),5.40-5.31(m,8H),4.98(d,1H),4.82-4.73(m,1H),4.72-4.61(m,1H),4.50-4.41(m,1H),4.32(d,1H),4.20(d,1H),3.81(d,1H),2.79-2.74(m,4H),0.97(s,3H),0.90-0.85(m,9H),0.64(s,3H).HPLC：96.93％。Dissolve intermediate 66-3 (250 mg, 0.272 mmol) in DMF, add paclitaxel (233 mg, 0.272 mmol), EDCI (68 mg, 0.354 mmol), and DMAP (50.0 mg, 0.408 mmol) in sequence, and stir at room temperature overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, eluting with PE/EA (10:1), gave compound 89 as a white solid (200 mg, 42%). 1 HNMR (400MHz, CDCl 3 ) δ8.15-8.12(m,2H),7.75-7.72(m,2H),7.63-7.58(m,1H),7.54-7.49(m,3H),7.44-7.33( m,8H),6.86(d,1H),6.32-6.22(m,2H),5.95(m,1H),5.68(d,1H),5.50(d,1H),5.40-5.31(m,8H) ,4.98(d,1H),4.82-4.73(m,1H),4.72-4.61(m,1H),4.50-4.41(m,1H),4.32(d,1H),4.20(d,1H),3.81 (d,1H),2.79-2.74(m,4H),0.97(s,3H),0.90-0.85(m,9H),0.64(s,3H). HPLC: 96.93%.
实施例081：熊去氧胆酸与小核酸的偶联物(化合物90)的合成路线如下
Example 081: The synthesis route of the conjugate of ursodeoxycholic acid and small nucleic acid (compound 90) is as follows
中间体90-1的合成：在室温下，将熊去氧胆酸(1.1g,2.80mmol)溶解于DCM中，依次加入NHS(387mg,3.36mmol)，EDCI(645mg,3.36mmol)，室温搅拌过夜。TLC检测，反应结束后，浓缩溶剂得粗品。硅胶柱层析，PE/ EA(10:1)洗脱，得白色固体90-1(1.08g,79％)。1HNMR(400MHz,CDCl3,)δ3.64-3.53(m,2H),2.83(d,4H),2.60-2.70(m,1H),2.48-2.58(m,1H),2.03-1.97(m,1H),0.97-0.92(m,6H,CH3×2),0.68(s,3H,CH3).HPLC：86.24％。Synthesis of intermediate 90-1: Dissolve ursodeoxycholic acid (1.1g, 2.80mmol) in DCM at room temperature, add NHS (387mg, 3.36mmol) and EDCI (645mg, 3.36mmol) in sequence, and stir at room temperature. overnight. After TLC detection, the solvent was concentrated to obtain crude product. Silica gel column chromatography, PE/ EA (10:1) eluted to obtain white solid 90-1 (1.08g, 79%). 1 HNMR (400MHz, CDCl 3 ,)δ3.64-3.53(m,2H),2.83(d,4H),2.60-2.70(m,1H),2.48-2.58(m,1H),2.03-1.97(m ,1H),0.97-0.92(m,6H,CH 3 ×2),0.68(s,3H,CH 3 ).HPLC: 86.24%.
化合物90的合成：将化合物90-2(330ug,22.2nmol)溶解于去离子水(1.5mL)中，加入中间体90-1(109ug,222nmol)，室温搅拌过夜。制备HPLC纯化，浓缩得粉色油状物(222ug，65％)，HPLC：75.20％。ESI-MS m/z[M+H]+，7410.1/7853.8。Synthesis of compound 90: Dissolve compound 90-2 (330ug, 22.2nmol) in deionized water (1.5mL), add intermediate 90-1 (109ug, 222nmol), and stir at room temperature overnight. Purified by preparative HPLC and concentrated to obtain pink oil (222ug, 65%), HPLC: 75.20%. ESI-MS m/z[M+H] + , 7410.1/7853.8.
实施例082：熊去氧胆酸与多肽的偶联物(化合物91)的合成路线如下Example 082: The synthesis route of the conjugate of ursodeoxycholic acid and polypeptide (compound 91) is as follows
序列：Ursodeoxycholic Acid-WEARLARALARALARHLARALARALRACEASequence: Ursodeoxycholic Acid-WEARLARALARALARHLARARALALRACEA
合成步骤：Synthesis steps:
化合物91为具有30个氨基酸的固相合成多肽，合成工艺采用Fmoc(9-芴基甲氧基羰基)固相合成法。将C-端的第一个丙氨酸(Ala)连接到Wang树脂上，然后依次按照肽序列从C端一次向N端合成，直至最后一个熊去氧胆酸酸(Ursodeoxycholic Acid)完成，得到化合物91肽树脂。化合物91肽树脂经裂解、无水乙醚沉淀、洗涤得到化合物91粗肽。化合物91粗肽经粗滤、纯化、转盐和冻干得到成品具体步骤如下：Compound 91 is a solid-phase synthetic peptide with 30 amino acids, and the synthesis process adopts the Fmoc (9-fluorenylmethoxycarbonyl) solid-phase synthesis method. Connect the first alanine (Ala) at the C-terminus to the Wang resin, and then synthesize it sequentially from the C-terminus to the N-terminus according to the peptide sequence until the last Ursodeoxycholic Acid (Ursodeoxycholic Acid) is completed to obtain the compound 91 peptide resin. The compound 91 peptide resin was cleaved, precipitated with anhydrous ether, and washed to obtain the crude peptide of compound 91. The crude peptide of compound 91 is coarsely filtered, purified, salted and freeze-dried to obtain the finished product. The specific steps are as follows:
1)称取Fmoc-Ala-Wang Resin投入反应柱加入DMF鼓氮气进行溶胀。1) Weigh Fmoc-Ala-Wang Resin and put it into the reaction column, add DMF and bubble nitrogen for swelling.
2)树脂溶胀后DMF洗涤，加入DBLK液脱保护。2) After the resin is swollen, wash it with DMF and add DBLK solution for deprotection.
3)脱完保护后加入DMF洗涤，茚三酮检测显阳性，开始加入氨基酸Fmoc-Glu(OtBu)-OH和缩合剂HBUT，鼓氮气搅拌后加入DIEA进行反应。3) After deprotection, add DMF for washing. If the ninhydrin test is positive, start adding the amino acid Fmoc-Glu(OtBu)-OH and the condensing agent HBUT, stir with nitrogen, then add DIEA for reaction.
4)反应结束后抽掉反应液，DMF洗涤至茚三酮检测显阴性。4) After the reaction, remove the reaction solution and wash with DMF until the ninhydrin test is negative.
5)重复上述2-4步直至完成整个序列的合成，得到化合物91的肽树脂。5) Repeat the above steps 2-4 until the synthesis of the entire sequence is completed, and the peptide resin of compound 91 is obtained.
裂解步骤：Lysis steps:
1)将上述肽树脂加入裂解液进行裂解。1) Add the above peptide resin to the lysis solution for lysis.
2)裂解完成后进行过滤去除滤饼取滤液。2) After the lysis is completed, filter to remove the filter cake and take the filtrate.
3)将滤液加入乙醚中进行沉降。3) Add the filtrate to diethyl ether for sedimentation.
4)沉降完成后离心除去上清液并重复乙醚洗涤三遍。4) After settling, centrifuge to remove the supernatant and repeat washing with ether three times.
5)真空抽干得到化合物91粗肽。 5) Vacuum dryness to obtain the crude peptide of compound 91.
纯化：purification:
1)取上述化合物91粗品用水和乙腈溶解至澄清。1) Take the crude compound 91 and dissolve it with water and acetonitrile until it becomes clear.
2)将溶解液进行过滤得滤液。2) Filter the dissolved solution to obtain the filtrate.
3)滤液上样采用半制备色谱分离纯化。3) The filtrate is loaded and separated and purified using semi-preparative chromatography.
4)纯化后的合格品进行旋蒸浓缩。4) The purified qualified products are concentrated by rotary evaporation.
5)化合物91浓缩液进行冻干，冻干后得到白色粉末成品。5) The concentrated solution of Compound 91 is freeze-dried, and a white powder product is obtained after freeze-drying.
表征：Characterization:
HPLC:87.06％(面积归一化法)；MS：理论分子量：3702.3；实际分子量：3701.94。HPLC: 87.06% (area normalization method); MS: theoretical molecular weight: 3702.3; actual molecular weight: 3701.94.
实施例83：纳米颗粒组合体的制备与测试Example 83: Preparation and testing of nanoparticle assemblies
1)纳米粒子组合物的制备1) Preparation of nanoparticle composition
为了研究用于将治疗剂和/或预防剂递送至细胞的安全且有效的纳米粒子组合物，制备并测试一系列配制物。确切地说，对纳米粒子组合物的脂质组分中的特定成分和其比率进行优化。To investigate safe and effective nanoparticle compositions for delivering therapeutic and/or prophylactic agents to cells, a series of formulations were prepared and tested. Specifically, specific ingredients and their ratios in the lipid component of the nanoparticle composition are optimized.
纳米粒子可以通过混合方法如微流体化和T型接头混合两种流体流制得，所述两种流体流之一含有治疗剂和/或预防剂且另一种具有脂质组分。通过在乙醇中合并根据实施例1-6中合成的脂质、磷脂(如DOPE或DSPC，可从Avanti Polar Lipids(Alabaster,AL)获得)、PEG脂质(如1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇，又称为PEG-DMG，可从Avanti Polar Lipids(Alabaster,AL)获得)和结构性脂质(如胆固醇，可从Sigma-Aldrich(Taufkirchen,Germany)获得；或皮质类固醇(如泼尼松龙、地塞米松、泼尼龙和氢化可的松)；或其组合，制备出浓度为约50mM的脂质组合物。溶液应在例如-20℃下冷藏储存。合并脂质得到所希望的摩尔比并用水和乙醇稀释至在约5.5mM与约25mM之间的最终脂质浓度。Nanoparticles can be produced by mixing two fluid streams, one of which contains therapeutic and/or prophylactic agents and the other of which has a lipid component, by mixing methods such as microfluidization and T-junctions. By combining in ethanol the lipids synthesized according to Examples 1-6, phospholipids (such as DOPE or DSPC, available from Avanti Polar Lipids (Alabaster, AL)), PEG lipids (such as 1,2-dimyristoyl -sn-Glycerylmethoxypolyethylene glycol, also known as PEG-DMG, available from Avanti Polar Lipids (Alabaster, AL)) and structural lipids such as cholesterol, available from Sigma-Aldrich (Taufkirchen, Germany) Obtain; or corticosteroids (such as prednisolone, dexamethasone, prednisolone and hydrocortisone); or a combination thereof, to prepare a lipid composition at a concentration of about 50mM. The solution should be refrigerated, for example, at -20°C Store. Combine the lipids to obtain the desired molar ratio and dilute with water and ethanol to a final lipid concentration between about 5.5mM and about 25mM.
通过将脂质溶液与包括治疗剂和/或预防剂以在约5:1与约50:1之间的脂质组分比治疗剂和/或预防剂wt:wt比率组合，制备出包含治疗剂和/或预防剂和脂质组分的纳米粒子组合物。使用迈安纳-S微流体纳米粒子制备系统，将脂质溶液以在约10mL/min～约18mL/min之间的流动速率迅速地注入治疗剂和/或预防剂溶液中，制造出分散液，其中水比乙醇比率在约2:1与约5:1之间。 A formulation containing a therapeutic agent is prepared by combining a lipid solution with a therapeutic agent and/or prophylactic agent in a lipid component to therapeutic agent and/or prophylactic agent wt:wt ratio of between about 5:1 and about 50:1. Nanoparticle compositions of agents and/or prophylactic agents and lipid components. Using the Maianna-S microfluidic nanoparticle preparation system, the lipid solution is rapidly injected into the therapeutic agent and/or preventive agent solution at a flow rate between about 10 mL/min and about 18 mL/min to create a dispersion. , wherein the water to ethanol ratio is between about 2:1 and about 5:1.
2)纳米颗粒组合体的表征2) Characterization of nanoparticle assemblies
纳米颗粒理化性状的测定：可以使用Zetasizer NanoZS(Malvern Instruments Ltd,Malvern,Worcestershire,UK)测定纳米粒子组合物的粒度、多分散指数(PDI)和ζ电势，粒度是在1×PBS中测定且ζ电势是在15mM的PBS中测定的。Determination of the physical and chemical properties of nanoparticles: Zetasizer NanoZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, polydispersity index (PDI) and ζ potential of the nanoparticle composition. The particle size is measured in 1×PBS and ζ Potentials were measured in 15mM PBS.
纳米颗粒包封率的测定：对于包含RNA的纳米粒子组合物，可以使用QUANT-ITTM RNA(Invitrogen Corporation Carlsbad,CA)测定评价纳米粒子组合物对RNA的包封情况。在TE缓冲溶液(10mM Tris-HCl、1mM EDTA，pH 7.5)中将样品稀释至约5μg/mL的浓度。将50μL稀释过的样品转移至聚苯乙烯96孔板上并向各孔中添加50μL TE缓冲液或50μL 2％Triton X-100溶液。在37℃温度下孵育板15分钟。将试剂以1:100稀释于TE缓冲液中，并向各孔中添加100μL该溶液。可以使用荧光板读取器(NivoTM Multimode Plate Readers,PerkinElmer，GER)在例如约480nm激发波长和例如约520nm发射波长下测量荧光强度。从各样品的荧光度值中减去试剂空白的荧光度值并通过用完全样品(未添加Triton X-100)的荧光强度除以被破坏的样品(由添加Triton X-100引起)的荧光度值来测定游离RNA的百分比。具体数据见表1。Determination of nanoparticle encapsulation efficiency: For nanoparticle compositions containing RNA, the QUANT-ITTM RNA (Invitrogen Corporation Carlsbad, CA) assay can be used to evaluate the encapsulation of RNA by the nanoparticle composition. Samples were diluted in TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 7.5) to a concentration of approximately 5 μg/mL. Transfer 50 μL of the diluted sample to a polystyrene 96-well plate and add 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution to each well. Incubate the plate at 37°C for 15 minutes. Dilute the reagent 1:100 in TE buffer and add 100 μL of this solution to each well. A fluorescent plate reader can be used ( Nivo ™ Multimode Plate Readers, PerkinElmer, GER) measure fluorescence intensity at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample and determined by dividing the fluorescence intensity of the intact sample (without the addition of Triton X-100) by the fluorescence of the disrupted sample (caused by the addition of Triton X-100). value to determine the percentage of free RNA. See Table 1 for specific data.
表1：不同化合物制备的纳米颗粒组合体的特征
Table 1: Characteristics of nanoparticle assemblies prepared from different compounds
3)纳米颗粒组合体的体外性能研究 3) Study on the in vitro properties of nanoparticle assemblies
对于包含RNA的纳米颗粒组合体，可以采用ONE-GlO+TOX Luciferase Reporter and Cell Viability Assay(Promega Corporation，US)评价其转染效果和细胞毒性。根据包封率测定中测得的RNA浓度计算所需纳米颗粒组合体的体积，将纳米颗粒组合体稀释到20ng/μL，按照聚苯乙烯96孔板2×105细胞/孔的细胞密度向每孔中添加5μL稀释液进行转染。可以使用荧光板读取器(NivoTM Multimode Plate Readers,PerkinElmer，GER)测量化学发光强度。具体数据见表2。For nanoparticle combinations containing RNA, ONE-GlO+TOX Luciferase Reporter and Cell Viability Assay (Promega Corporation, US) can be used to evaluate its transfection effect and cytotoxicity. Calculate the volume of the required nanoparticle assembly based on the RNA concentration measured in the encapsulation efficiency determination, dilute the nanoparticle assembly to 20ng/μL, and add it according to the cell density of 2×10 5 cells/well in a polystyrene 96-well plate. Add 5 μL of diluent to each well for transfection. A fluorescent plate reader can be used ( Nivo ™ Multimode Plate Readers, PerkinElmer, GER) were used to measure chemiluminescence intensity. See Table 2 for specific data.
表2：不同化合物制备的纳米组合体的体外性能研究
Table 2: In vitro performance study of nanoassemblies prepared with different compounds
转染效果：可以提供MC3的倍数。Transfection effect: can provide multiples of MC3.
实施例84：脂质纳米颗粒的制备和检测(验证体外递送siRNA能力)Example 84: Preparation and detection of lipid nanoparticles (to verify the ability to deliver siRNA in vitro)
1)将实施例1合成的化合物-1，与DOPE(1、2-二油酰-SN-甘油-3-磷酰乙醇胺)和DMG-PEG2000(1、2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇)以65:30:5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将cy3标记的siCont(Sense 5’to 3’:CUUACGCUGAGUACUUCGAdTdT-Cy3；Antisense 5’to 3’:UCGAAGUACUCAGCGUAAGdTdT)在50mM柠檬酸盐缓冲液(pH＝4)中稀释得到siRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和siRNA溶液，以总脂质与siRNA的重量比为约20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液(磷酸缓冲盐溶液)代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封cy3-siCont的制剂。1) Compound-1 synthesized in Example 1, and DOPE (1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine) and DMG-PEG2000 (1, 2-dimyristoyl-rac-glycerol -3-methoxypolyethylene glycol) was dissolved in absolute ethanol at a molar ratio of 65:30:5 to prepare a lipid ethanol solution, and cy3-labeled siCont (Sense 5'to 3':CUUACGCUGAGUACUUCGAdTdT-Cy3; Antisense 5'to 3':UCGAAGUACUCAGCGUAAGdTdT) was diluted in 50mM citrate buffer (pH=4) to obtain a siRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the siRNA solution in a volume of 1:3. Prepare liposomes at a weight ratio of about 20:1 to siRNA. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution (phosphate buffered saline). The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain a formulation encapsulating cy3-siCont.
通过动态光散射，使用纳米粒度及电位分析仪(NS-90Z)测定脂质纳米颗粒大小。使用Quant-it Ribogreen RNA定量分析试剂盒测定脂质纳米颗粒的包封效率。测得脂质纳米颗粒的粒径为82nm，PDI(多分散指数)是0.13，包封率 是96.5％。Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the lipid nanoparticles was measured to be 82nm, the PDI (polydispersity index) was 0.13, and the encapsulation rate It's 96.5%.
2)将上述制备的包载siRNA的脂质纳米颗粒转染293T细胞。将293T细胞以1*105个细胞/孔的密度铺到24孔板上，加入含有10％小牛血清的DMEM培养液中培养24小时至细胞密度长到70％后，移除24孔板中的培养液。将制备的LNP/cy3-siRNA复合物用DMEM培养基稀释至1ml，加入24孔板中。37℃，5％CO2条件下培养箱继续孵育24h。2) Transfect 293T cells with the siRNA-encapsulated lipid nanoparticles prepared above. Plate 293T cells on a 24-well plate at a density of 1* 105 cells/well, add DMEM culture medium containing 10% calf serum, and culture for 24 hours until the cell density reaches 70%, then remove the 24-well plate. culture medium in. Dilute the prepared LNP/cy3-siRNA complex with DMEM medium to 1 ml and add it to a 24-well plate. Continue incubation for 24h in the incubator at 37°C and 5% CO2 .
然后采用荧光显微镜拍照记录cy3-siRNA的转染情况。细胞转染效果见说明书附图中的图21。荧光显微镜结果表明，制备的包载siRNA的脂质纳米颗粒具有较好的细胞转染效果。流式细胞仪测试结果表明68.90％的细胞被转染。Then a fluorescence microscope was used to take pictures to record the transfection status of cy3-siRNA. The cell transfection effect is shown in Figure 21 in the accompanying drawings of the instructions. Fluorescence microscopy results showed that the prepared lipid nanoparticles encapsulating siRNA had good cell transfection effect. Flow cytometry test results showed that 68.90% of cells were transfected.
实施例85：脂质纳米颗粒的制备和检测(验证体外递送mRNA能力)Example 85: Preparation and detection of lipid nanoparticles (to verify the ability to deliver mRNA in vitro)
1)将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以65:30:5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将EGFP mRNA(编码绿色荧光蛋白mRNA)在20-100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为约20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒同0.22μm无菌过滤器过滤，得到包封EGFP mRNA的制剂。1) Dissolve the lipid compound-1 synthesized in Example 1 with DOPE and DMG-PEG2000 in absolute ethanol at a molar ratio of 65:30:5 to prepare a lipid ethanol solution, and add EGFP mRNA (encoding green fluorescent protein mRNA ) was diluted in 20-100mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, with a weight ratio of total lipid to mRNA. To prepare liposomes at approximately 20:1, use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles are then filtered with a 0.22μm sterile filter to obtain a preparation encapsulating EGFP mRNA.
通过动态光散射，使用纳米粒度及电位分析仪(NS-90Z)测定脂质纳米颗粒大小。使用Quant-it Ribogreen RNA定量分析试剂盒测定脂质纳米颗粒的包封效率。测得LNP制剂粒径为95nm，PDI 0.19，包封率是93.1％。Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the LNP preparation was measured to be 95nm, PDI 0.19, and the encapsulation rate was 93.1%.
2)将上述制备的的包载mRNA的脂质纳米颗粒转染293T细胞。将293T细胞以1*105个细胞/孔的密度铺到24孔板上，加入含有10％小牛血清的DMEM培养液中培养24小时至细胞密度长到70％后，移除24孔板中的培养液。将制备的LNP/EGFP复合物用DMEM培养基稀释至1ml，加入24孔板中。37℃，5％CO2条件下培养箱孵育24h。然后采用荧光显微镜拍照记录EGFP的转染情况。细胞转染效果见说明书附图中的图22。荧光显微镜结果表明，制备的包载mRNA的脂质纳米颗粒具有较好的细胞转染效果。流式细胞仪结果表明77.79％的细胞被转染。 2) Transfect 293T cells with the mRNA-encapsulated lipid nanoparticles prepared above. Plate 293T cells on a 24-well plate at a density of 1* 105 cells/well, add DMEM culture medium containing 10% calf serum, and culture for 24 hours until the cell density reaches 70%, then remove the 24-well plate. culture medium in. Dilute the prepared LNP/EGFP complex with DMEM medium to 1 ml and add it to a 24-well plate. Incubate for 24h in an incubator at 37°C and 5% CO2 . Then a fluorescence microscope was used to take pictures to record the transfection status of EGFP. The cell transfection effect is shown in Figure 22 in the accompanying drawings of the instructions. Fluorescence microscopy results showed that the prepared lipid nanoparticles encapsulating mRNA had good cell transfection effect. Flow cytometry results showed that 77.79% of cells were transfected.
实施例86：脂质纳米颗粒的制备和检测(验证mRNA体内递送效果)Example 86: Preparation and detection of lipid nanoparticles (to verify the in vivo delivery effect of mRNA)
1)将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以65:30:5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在50mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒同0.22μm无菌过滤器过滤，得到包封mRNA的制剂。1) Dissolve the lipid compound-1 synthesized in Example 1 with DOPE and DMG-PEG2000 in absolute ethanol at a molar ratio of 65:30:5 to prepare a lipid ethanol solution, and add Luciferase mRNA (encoding luciferase mRNA ) were diluted in 50mM citrate buffer (pH=4) to obtain an mRNA solution. Use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3. The weight ratio of total lipid to mRNA is Prepare liposomes at 20:1, remove ethanol using dialysis or tangential flow filtration, and replace with PBS solution. The lipid nanoparticles are then filtered with a 0.22 μm sterile filter to obtain a preparation that encapsulates mRNA.
通过动态光散射，使用纳米粒度及电位分析仪(NS-90Z)测定脂质纳米颗粒大小。使用Quant-it Ribogreen RNA定量分析试剂盒测定脂质纳米颗粒的包封效率。测得脂质纳米颗粒的粒径为98nm，PDI为0.22，包封率为88.9％。Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the lipid nanoparticles was measured to be 98 nm, the PDI was 0.22, and the encapsulation rate was 88.9%.
2)将上述制备的LNP分别通过尾静脉或肌肉注入6周龄SPF级别BALB/c小鼠体内，剂量为每只小鼠100ul(含10μg mRNA)，12h小时候后用活体成像仪测试小鼠体内的荧光强度，活体成像仪的曝光时间设置为30s。包载Luciferase mRNA的脂质纳米粒，在小鼠活体成像结果见说明书附图中的图23和图24。结果表明，当制备的LNP通过静脉注射进小鼠体内时，LNP在体内的递送部位主要是腹部。而当LNP通过肌肉注射进小鼠体内时，LNP在体内的递送部位主要是腹部和肝脏。上述结果均表明，LNP具有较好的体内递送效果。2) Inject the LNPs prepared above into 6-week-old SPF grade BALB/c mice through the tail vein or muscle respectively. The dose is 100ul per mouse (containing 10μg mRNA). After 12 hours, the mice are tested with an in vivo imager. of fluorescence intensity, and the exposure time of the in vivo imager was set to 30 s. The results of in vivo mouse imaging of lipid nanoparticles encapsulating Luciferase mRNA are shown in Figure 23 and Figure 24 in the appendix of the instruction manual. The results showed that when the prepared LNP was injected intravenously into mice, the delivery site of LNP in the body was mainly the abdomen. When LNP was injected intramuscularly into mice, the main delivery sites of LNP in the body were the abdomen and liver. The above results all show that LNP has good in vivo delivery effect.
实施例87Example 87
将合成的脂质化合物-1，与DOPE和DMG-PEG2000以89.9:10:0.1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在50mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。The synthesized lipid compound-1, DOPE and DMG-PEG2000 were dissolved in absolute ethanol at a molar ratio of 89.9:10:0.1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was dissolved in 50mM lemon respectively. Dilute the ethanol lipid solution and the mRNA solution in acid salt buffer (pH=4) using a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3 to prepare the lipid with a weight ratio of total lipid to mRNA of 20:1. For plastids, use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为1249nm，PDI为0.67，包封率为31％。 The detected particle size was 1249nm, the PDI was 0.67, and the encapsulation rate was 31%.
实施例88Example 88
将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以89.5:10:0.5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在50mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 89.5:10:0.5 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was Dilute the mRNA solution in 50mM citrate buffer (pH=4), use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 20: 1. Prepare liposomes. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为467nm，PDI为0.40，包封率为78％。The detected particle size was 467 nm, the PDI was 0.40, and the encapsulation rate was 78%.
实施例89Example 89
将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以89:10:1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 89:10:1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was Dilute the mRNA solution in 100mM citrate buffer (pH=4). Use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 20: 1. Prepare liposomes. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为347nm，PDI为0.41，包封率为81％。The detected particle size was 347nm, the PDI was 0.41, and the encapsulation rate was 81%.
实施例90Example 90
将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以85:10:5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 85:10:5 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was Dilute the mRNA solution in 100mM citrate buffer (pH=4). Use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 20: 1. Prepare liposomes. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为94nm，PDI为0.50，包封率为85％。 The detected particle size was 94nm, the PDI was 0.50, and the encapsulation rate was 85%.
实施例91Example 91
将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以64:35:1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 64:35:1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was Dilute the mRNA solution in 100mM citrate buffer (pH=4). Use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 20: 1. Prepare liposomes. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为82nm，PDI为0.11，包封率为96％。The detected particle size was 82nm, the PDI was 0.11, and the encapsulation rate was 96%.
实施例92Example 92
将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以39:60:1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为40:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 39:60:1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was Dilute the mRNA solution in 100mM citrate buffer (pH=4), use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipids to mRNA is 40: 1. Prepare liposomes. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为145nm，PDI为0.23，包封率为76％。The detected particle size was 145nm, the PDI was 0.23, and the encapsulation rate was 76%.
实施例93Example 93
将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以39:60:1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在20mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为40:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 39:60:1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was Dilute the mRNA solution in 20mM citrate buffer (pH=4), use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 40: 1. Prepare liposomes. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为302nm，PDI为0.38，包封率为73％。 The detected particle size was 302nm, the PDI was 0.38, and the encapsulation rate was 73%.
实施例94Example 94
将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以60:39.2:0.8的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在20mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为40:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。The lipid compound-1 synthesized in Example 1, DOPE and DMG-PEG2000 were dissolved in absolute ethanol at a molar ratio of 60:39.2:0.8 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding luciferase mRNA) was Dilute the mRNA solution in 20mM citrate buffer (pH=4), use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 40: 1. Prepare liposomes. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为302nm，PDI为0.38，包封率为73％。The detected particle size was 302nm, the PDI was 0.38, and the encapsulation rate was 73%.
实施例95：脂质纳米颗粒的制备Example 95: Preparation of lipid nanoparticles
如表1所示，将化合物19至化合物88分别与DSPC:胆固醇:DMG-PEG2000以50:10:38.5:1.5摩尔比的比例溶解于乙醇中。按照实施例083中的纳米粒子组合物的制备方法进行制备。As shown in Table 1, compounds 19 to 88 were dissolved in ethanol with DSPC:cholesterol:DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5. Prepare according to the preparation method of the nanoparticle composition in Example 083.
实施例96：脂质纳米颗粒的表征Example 96: Characterization of lipid nanoparticles
使用Malvern Zetasizer NanoZS(马尔文纳米粒度测试仪器)测定，通过动态光散射法测定颗粒的粒径(Size)，多分散指数(PDI)和电动电位(Zeta P)。使用QUANT-ITTM RNA(Invitrogen Corporation Carlsbad,CA)测定脂质纳米颗粒的包封效率。按照实施例083中纳米颗粒组合体的表征方法，如表3所示：脂质纳米颗粒制剂大小大部分在100nm左右，电位在±20mv之间，多分散指数在0.1-0.3左右变化，包封率部分在80％-99％左右。The particle size (Size), polydispersity index (PDI) and electrokinetic potential (Zeta P) of the particles were determined by dynamic light scattering using the Malvern Zetasizer NanoZS (Malvern Nanoparticle Size Testing Instrument). The encapsulation efficiency of lipid nanoparticles was determined using QUANT-ITTM RNA (Invitrogen Corporation Carlsbad, CA). According to the characterization method of the nanoparticle assembly in Example 083, as shown in Table 3: the size of the lipid nanoparticle preparation is mostly around 100 nm, the potential is between ±20mv, the polydispersity index varies around 0.1-0.3, and the encapsulation The rate part is around 80%-99%.
表3：脂质纳米颗粒的表征


Table 3: Characterization of lipid nanoparticles


实施例96：脂质纳米颗粒的体外细胞转染实验Example 96: In vitro cell transfection experiment of lipid nanoparticles
将293T细胞以3X10^4个/孔的密度种植在96孔细胞培养板中，长至24h至贴壁。按照实施例83中制备方法制备，将表4中的化合物按照实施例83中的方法制备成脂质纳米颗粒制剂，加入细胞中。培养24h后，加入显色液，用酶标仪检测各孔发光值。表4展示了脂质纳米制剂可以携带mRNA转染进293T细胞中并表达目的荧光蛋白，A、B、C、D分别代表化合物与商业化的MC3-LNP发光通量进行倍比后归一化的结果，如表4所示，有31个化合物的发光通量高于MC3-LNP组，说明脂质纳米颗粒制剂可以有效的携带核酸进入细胞。293T cells were seeded in a 96-well cell culture plate at a density of 3X10^4 cells/well and grown for 24 hours to adhere. Prepare according to the preparation method in Example 83. Prepare the compounds in Table 4 into lipid nanoparticle preparations according to the method in Example 83 and add them to cells. After culturing for 24 hours, add chromogenic solution and detect the luminescence value of each well with a microplate reader. Table 4 shows that the lipid nanoformulation can carry mRNA and be transfected into 293T cells and express the target fluorescent protein. A, B, C, and D respectively represent the compound and the commercial MC3-LNP luminous flux after normalization. As a result, as shown in Table 4, the luminescence flux of 31 compounds was higher than that of the MC3-LNP group, indicating that the lipid nanoparticle preparation can effectively carry nucleic acids into cells.
说明：A、B、C、D分别代表化合物与MC3-LNP的发光通量进行倍比后归一化的结果，倍率范围如下：Note: A, B, C, and D respectively represent the normalized results after multiplying the luminous flux of the compound and MC3-LNP. The magnification range is as follows:
A：≥1A：≥1
B：≥0.5且＜1 B: ≥0.5 and <1
C：≥0.1且＜0.5C: ≥0.1 and <0.5
D：＜0.1D：＜0.1
表4：脂质纳米颗粒体外细胞转染评价结果

Table 4: Evaluation results of lipid nanoparticles for in vitro cell transfection

实施例97：化合物46包裹紫杉醇杀伤细胞Example 97: Compound 46 encapsulates paclitaxel to kill cells
将化合物46与DSPC、胆固醇、PEG2000按照实施例83中的方法制备除醇，然后加入0.2、1、5、10、20mg/ml的紫杉醇溶液，评价载紫杉醇LNP对肿瘤细胞A549杀伤效果，由如图25的结果可知，载紫杉醇的脂质纳米颗粒在高浓度时相比游离紫杉醇有较好的肿瘤细胞杀伤效果。Compound 46, DSPC, cholesterol, and PEG2000 were prepared according to the method in Example 83 to remove alcohol, and then 0.2, 1, 5, 10, and 20 mg/ml paclitaxel solutions were added to evaluate the killing effect of paclitaxel-loaded LNP on tumor cells A549, as follows: The results in Figure 25 show that paclitaxel-loaded lipid nanoparticles have a better tumor cell killing effect than free paclitaxel at high concentrations.
实施例98：挑选化合物27进行LNP处方筛选Example 98: Select compound 27 for LNP formulation screening
以化合物27进行处方筛选，按照以下表格中的处方组成进行配比，实施例83中描述的方法制备，结果如表5所示，脂质纳米颗粒制剂大小在70nm与170nm之间，电位在±20mv之间，多分散指数在0.1-0.3之间变化，包封率部分在80％-90％左右，细胞评价结果的发光通量值大部分高于MC3-LNP组，说明脂质纳米颗粒制剂可以改变处方组成，携带核酸进入细胞。Compound 27 was used for prescription screening, and the formula was proportioned according to the prescription composition in the following table. It was prepared by the method described in Example 83. The results are shown in Table 5. The size of the lipid nanoparticle preparation was between 70nm and 170nm, and the potential was within ± Between 20mv, the polydispersity index varies between 0.1-0.3, the encapsulation rate is around 80%-90%, and the luminous flux values of the cell evaluation results are mostly higher than those of the MC3-LNP group, indicating that lipid nanoparticle preparations can Change the composition of the prescription to carry nucleic acids into cells.
表5：脂质纳米颗粒的表征与细胞评价

Table 5: Characterization and cellular evaluation of lipid nanoparticles

实施例99：脂质纳米颗粒制剂在小鼠体内的荧光蛋白表达实验Example 99: Fluorescent protein expression experiment of lipid nanoparticle preparation in mice
按照实施例83纳米颗粒组合体的制备方法制备样品，以MC3-LNP为阳性参照。Samples were prepared according to the preparation method of the nanoparticle assembly in Example 83, using MC3-LNP as a positive reference.
采用6-8w的雌性SPF级BALB/c小鼠(n＝3)，饲养于SPF级动物房，温度20-26℃，湿度40-70％，饲料、饮水充足。分别以100μL(0.05μg/μL)剂量通过肌肉注射方式，采用实施例83中制剂的制备方法制备，对照组采用MC3-LNP进行体内效果对比。给药6h后，腹腔注射200ul浓度为15mg/ml的D-荧光素钾盐，D-荧光素钾盐注射5min后进行活体成像。全部组活体成像都结束后再次注射D-荧光素钾盐，5分钟后进行器官成像。A、B、C、D分别代表化合物与商业化的MC3-LNP荧光表达值进行倍比后归一化的结果，如表6所示，有化合物27、37、39、42、43、46、50、66、69、74、82、86、87在局部给药均达到了MC3-LNP水平，其中化合物42、43、50、69超过了MC3-LNP水平，说明脂质纳米颗粒制剂可以有效的递送mRNA，并在局部高效表达目的基因蛋白。表7和图26是化合物与商业化的MC3-LNP在腹部表达的对比结果，如表7所示：其中有化合物52、67、75、81、82、83在腹部荧光水平超过MC3-LNP水平，说明胆酸系列脂质化合物可以高效表达目的基因蛋白。Female SPF grade BALB/c mice (n=3) aged 6-8w were used and kept in an SPF-grade animal room with a temperature of 20-26°C, a humidity of 40-70%, and sufficient feed and drinking water. The preparations were prepared by intramuscular injection at a dose of 100 μL (0.05 μg/μL), using the preparation method of the preparation in Example 83, and MC3-LNP was used in the control group for comparison of in vivo effects. 6 hours after administration, 200ul of D-luciferin potassium salt with a concentration of 15 mg/ml was injected intraperitoneally, and in vivo imaging was performed 5 minutes after the injection of D-luciferin potassium salt. After in vivo imaging in all groups was completed, D-fluorescein potassium salt was injected again, and organ imaging was performed 5 minutes later. A, B, C, and D respectively represent the normalized results of compound and commercial MC3-LNP fluorescence expression values after fold ratio. As shown in Table 6, there are compounds 27, 37, 39, 42, 43, 46, 50, 66, 69, 74, 82, 86, and 87 all reached the MC3-LNP level when administered locally, and compounds 42, 43, 50, and 69 exceeded the MC3-LNP level, indicating that lipid nanoparticle formulations can be effective Delivers mRNA and efficiently expresses the target gene protein locally. Table 7 and Figure 26 are the comparison results of the abdominal expression of compounds and commercial MC3-LNP, as shown in Table 7: among them, the fluorescence level of compounds 52, 67, 75, 81, 82, and 83 in the abdomen exceeds the level of MC3-LNP. , indicating that cholic acid series lipid compounds can efficiently express the target gene protein.
说明：A、B分别代表化合物与MC3-LNP的发光值进行倍比后归一化的结 果，倍率范围如下：Note: A and B respectively represent the normalized results after multiplying the luminescence values of the compound and MC3-LNP. As a result, the magnification range is as follows:
A：≥0.5A：≥0.5
B：≥0.1且＜0.5B: ≥0.1 and <0.5
表6：脂质纳米颗粒制剂在小鼠体内肌肉局部荧光表达比值及评分
Table 6: Local fluorescence expression ratio and score of lipid nanoparticle preparation in muscle of mice
表7：脂质纳米颗粒制剂在小鼠体内腹部荧光表达比值及评分

Table 7: Abdominal fluorescence expression ratio and score of lipid nanoparticle preparations in mice

实施例100：脂质纳米颗粒制剂静脉注射的荧光蛋白表达Example 100: Fluorescent protein expression by intravenous injection of lipid nanoparticle formulations
将化合物52、化合物67、化合物75采用实施例83中制剂的制备方法制备，按照实施例099进行静脉注射给药，对照组采用MC3-LNP进行体内效果对比，如图27所示，化合物52、化合物67、化合物75弱于MC3-LNP效果，但均有荧光表达。Compound 52, compound 67, and compound 75 were prepared using the preparation method of the preparation in Example 83, and administered intravenously according to Example 099. The control group used MC3-LNP for in vivo effect comparison. As shown in Figure 27, compound 52, Compound 67 and compound 75 are weaker than MC3-LNP, but both have fluorescent expression.
实施例101：脂质纳米颗粒制剂在瘤内给药表达情况Example 101: Expression of lipid nanoparticle preparation after intratumoral administration
以化合物27进行处方进行瘤内给药，按照实施例83中描述的方法制备。采用C57小鼠，建立的EG7-OVA模型，肿瘤生长至500mm3进行给药，瘤内注射，在注射后6h拍照，观察表达和分布情况。如图28所示，小鼠瘤内部位有荧光表达，说明脂质纳米颗粒可以采用瘤内注射方式给药。Compound 27 was formulated for intratumoral administration and prepared according to the method described in Example 83. The EG7-OVA model was established using C57 mice. The tumor was grown to 500mm3 before drug administration and intratumoral injection. Photographs were taken 6 hours after injection to observe the expression and distribution. As shown in Figure 28, there is fluorescence expression in the intratumoral site of mice, indicating that lipid nanoparticles can be administered via intratumoral injection.
实施例102：挑选化合物42和化合物58进行LNP处方筛选Example 102: Selecting compound 42 and compound 58 for LNP formulation screening
以化合物42进行处方筛选，按照以下表格中的处方组成进行配比，实施例83中描述的方法制备，结果如表8所示，脂质纳米颗粒制剂大小在50nm与200nm之间，电位在±5mv之间，多分散指数在0.1附近，包封率大部分在90％以上，细胞评价结果的发光通量值大部分高于MC3-LNP组，说明脂质纳米颗粒制剂可以改变处方组成，携带核酸进入细胞。 Compound 42 was used for prescription screening, and the formula was proportioned according to the prescription composition in the following table. It was prepared by the method described in Example 83. The results are shown in Table 8. The size of the lipid nanoparticle preparation was between 50 nm and 200 nm, and the potential was within ± 5 mv, the polydispersity index is around 0.1, the encapsulation rate is mostly above 90%, and the luminous flux values of the cell evaluation results are mostly higher than the MC3-LNP group, indicating that lipid nanoparticle preparations can change the composition of the prescription and carry nucleic acids Enter the cell.
表8：脂质纳米颗粒的表征与细胞评价
Table 8: Characterization and cellular evaluation of lipid nanoparticles
实施例103：脂质纳米颗粒制剂在小鼠体内的荧光蛋白表达实验Example 103: Fluorescent protein expression experiment of lipid nanoparticle preparation in mice
将实施例102中得到的纳米颗粒组合体。采用6-8w的雌性SPF级BALB/c小鼠(n＝3)，饲养于SPF级动物房，温度20-26℃，湿度40-70％，饲料、饮水充足。分别以100μL(0.05μg/μL)剂量通过肌肉注射方式，采用实施例83中制剂的制备方法制备，对照组采用MC3-LNP进行体内效果对比。给药6h后，腹腔注射200ul浓度为15mg/ml的D-荧光素钾盐，D-荧光素钾盐注射5min后进行活体成像。全部组活体成像都结束后再次注射D-荧光素钾盐，5分钟后进行器官成像。A、B分别代表各组配方与经典配方编号13荧光表达值进行倍比后归一化的结果，如表9所示，有7个配方在肌肉局部给药均达到了经典配方水平，说明脂质纳米颗粒制剂可以通过配方改变有效的递送mRNA，并在肌肉局部高效表达目的基因蛋白。其中配方1、8、9在脾脏荧光表达水平远远超过经典配方水平，说明胆酸系列脂质化合物可以通过改变配方的比例在脾脏中高效表达基因蛋白。另外配方3为去掉DSPC的三组分配方，结果显示在肌肉局部荧光表达值高于经典配方，说明三组分配方是可以形成纳米颗粒并且在体内有效表达荧光蛋白。The nanoparticle assembly obtained in Example 102 was used. Female SPF grade BALB/c mice (n=3) aged 6-8w were used and kept in an SPF-grade animal room with a temperature of 20-26°C, a humidity of 40-70%, and sufficient feed and drinking water. The preparations were prepared by intramuscular injection at a dose of 100 μL (0.05 μg/μL), using the preparation method of the preparation in Example 83, and MC3-LNP was used in the control group for comparison of in vivo effects. 6 hours after administration, 200ul of D-luciferin potassium salt with a concentration of 15 mg/ml was injected intraperitoneally, and in vivo imaging was performed 5 minutes after the injection of D-luciferin potassium salt. After in vivo imaging in all groups was completed, D-fluorescein potassium salt was injected again, and organ imaging was performed 5 minutes later. A and B respectively represent the normalized results of the fluorescence expression values of each group of formulas and the classic formula No. 13. As shown in Table 9, there are 7 formulas that have reached the level of the classic formula when administered locally in the muscle, indicating that lipid Plasma nanoparticle preparations can effectively deliver mRNA through formula changes and efficiently express the target gene protein locally in the muscle. Among them, the fluorescence expression levels of formulas 1, 8, and 9 in the spleen far exceeded the level of classic formulas, indicating that bile acid series lipid compounds can efficiently express gene proteins in the spleen by changing the proportion of the formula. In addition, Formula 3 is a three-component formula without DSPC. The results show that the local fluorescence expression value in the muscle is higher than the classic formula, indicating that the three-component formula can form nanoparticles and effectively express fluorescent proteins in the body.
说明：A、B分别代表各组配方与经典配方编号13的发光值进行倍比后归 一化的结果，倍率范围如下：Note: A and B respectively represent the luminescence values of each group of formulas and the classic formula No. 13 after being doubled. The result of normalization, the magnification range is as follows:
A：≥0.5A：≥0.5
B：≥0.1且＜0.5B: ≥0.1 and <0.5
表9：脂质纳米颗粒制剂在小鼠体内肌肉局部、脾脏荧光表达比值及评分
Table 9: Fluorescence expression ratio and score of lipid nanoparticle preparation in local muscles and spleen of mice
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。 The above are only the preferred embodiments of the present invention. It should be pointed out that those of ordinary skill in the art can also make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (25)

  1. 一种脂质化合物，所述脂质化合物为基于胆酸或其衍生物的脂质，或所述脂质化合物为基于胆酸或其衍生物的脂质的药物可用的盐、前药或立体异构体，所述脂质化合物具有如下通式1或通式2的结构：A lipid compound, which is a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable salt, prodrug or steric compound of a lipid based on cholic acid or a derivative thereof. Isomers, the lipid compound has the structure of the following general formula 1 or general formula 2:
    通式1：
    General formula 1:
    通式2：
    General formula 2:
  2. 根据权利要求1所述的脂质化合物，所述脂质化合物母核为胆酸或其衍生物；其中所述连接键(Linker)包含酯基、酰胺基、氨基甲酸酯基、碳酸酯基或脲基的一种或多种。The lipid compound according to claim 1, wherein the core of the lipid compound is cholic acid or a derivative thereof; wherein the linker includes an ester group, an amide group, a carbamate group, and a carbonate group. Or one or more urea groups.
  3. 根据权利要求1所述的脂质化合物，其中胆酸或其衍生物是任选自胆酸、奥贝胆酸、熊去氧胆酸、熊果胆酸、3β-羟基-D5-胆烯酸、鹅去氧胆酸、石胆酸、脱氧胆酸、牛磺胆酸、5β-胆酸、去氢胆酸、猪胆酸、络胆酸、甘氨鹅脱氧胆酸、牛磺熊去氧胆酸、牛磺鹅去氧胆酸、甘氨胆酸、猪脱氧胆酸、猪去氧胆酸甲酯、牛磺猪去氧胆酸钠、去氢胆酸钠、胆酸钠、脱氧甘胆酸钠、牛磺脱氧胆酸钠、牛磺胆酸钠、牛磺鹅去氧胆酸钠、甘氨胆酸钠盐、牛磺胆酸-3-硫酸酯二钠盐、牛磺熊去氧胆酸钠和牛磺石胆酸钠的一种或多种。The lipid compound according to claim 1, wherein cholic acid or its derivative is optionally selected from the group consisting of cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, and 3β-hydroxy-D5-cholenoic acid. , chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholic acid, 5β-cholic acid, dehydrocholic acid, hyocholic acid, cholic acid, glycinechenodeoxycholic acid, tauroursodeoxy Cholic acid, taurochenodeoxycholic acid, glycocholic acid, hyodeoxycholic acid, hyodeoxycholic acid methyl ester, sodium taurohodeoxycholate, sodium dehydrocholic acid, sodium cholate, deoxyglycholic acid Sodium, sodium taurodeoxycholate, sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate disodium salt, tauroursodeoxycholate One or more of sodium bisulfate and sodium taurocholate.
  4. 根据权利要求1所述的脂质化合物，所述脂质化合物为选自熊去氧胆酸衍生物脂质化合物的一种或多种，或奥贝胆酸衍生物脂质化合物的一种或多种。The lipid compound according to claim 1, which is one or more selected from the group consisting of ursodeoxycholic acid derivative lipid compounds, or one or more obeticholic acid derivative lipid compounds. Various.
  5. 根据权利要求1所述的脂质化合物，所述脂质化合物还为选自胆酸脂质化合物的一种或多种、猪去氧胆酸衍生物脂质化合物的一种或多种、鹅去氧胆酸衍生物脂质化合物的一种或多种。 The lipid compound according to claim 1, the lipid compound is also selected from one or more cholic acid lipid compounds, one or more hyodeoxycholic acid derivative lipid compounds, goose deoxycholic acid derivative lipid compounds, One or more oxycholic acid derivatives lipid compounds.
  6. 根据权利要求1所述的脂质化合物，其特征在于所述脂质化合物可以为可离子化脂质、或阳离子脂质、或阴离子脂质、或中性脂质、或聚乙二醇衍生物脂质、或聚肌氨酸衍生物脂质、或壳聚糖衍生物脂质、或透明质酸衍生物脂质。The lipid compound according to claim 1, characterized in that the lipid compound can be an ionizable lipid, a cationic lipid, an anionic lipid, a neutral lipid, or a polyethylene glycol derivative. Lipid, or polysarcosine derivative lipid, or chitosan derivative lipid, or hyaluronic acid derivative lipid.
  7. 一种脂质化合物的组合物，其特征在于所述组合物包括治疗或预防剂和用于递送所述治疗或预防剂的载体，所述载体包括前述权利要求1-6中所述的脂质化合物的一种或多种。A composition of lipid compounds, characterized in that the composition includes a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising the lipid described in the preceding claims 1-6 One or more compounds.
  8. 根据权利要求7所述的组合物，其特征在于，所述治疗或预防剂包括核酸分子、多肽、蛋白、抗体及小分子药物中的一种或多种。The composition according to claim 7, wherein the therapeutic or preventive agent includes one or more of nucleic acid molecules, polypeptides, proteins, antibodies and small molecule drugs.
  9. 根据权利要求7所述的组合物，其特征在于所述载体与所述治疗或预防剂的质量比为1:1～100:1。The composition according to claim 7, characterized in that the mass ratio of the carrier to the therapeutic or preventive agent is 1:1 to 100:1.
  10. 根据权利要求7所述的组合物，其特征在于，所述组合物为纳米颗粒制剂，所述纳米颗粒制剂的平均尺寸为20nm～1000nm；所述纳米颗粒制剂的多分散系数≤0.5。The composition according to claim 7, characterized in that the composition is a nanoparticle preparation, the average size of the nanoparticle preparation is 20 nm to 1000 nm; the polydispersity coefficient of the nanoparticle preparation is ≤ 0.5.
  11. 根据权利要求7所述的组合物，其特征在于，所述载体中包含三种不同的脂质组分，其中一种脂质是基于胆酸或其衍生物的脂质。The composition of claim 7, wherein the carrier contains three different lipid components, one of which is a lipid based on cholic acid or a derivative thereof.
  12. 根据权利要求7所述的组合物，其特征在于，所述载体中还包括中性电荷、阴性电荷或双性电荷的电荷辅助脂质。The composition according to claim 7, wherein the carrier further includes a charge-assisted lipid with neutral charge, negative charge or amphiphilic charge.
  13. 根据权利要求12所述的组合物，其特征在于，所述电荷辅助脂质为如下的一种或多种：二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基磷脂酰胆碱(DOPC)、二棕榈酰基磷脂酰胆碱(DPPC)、二油酰基磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二油酰基磷脂酰乙醇胺(DOPE)、棕榈酰油酰基磷脂酰胆碱(POPC)、棕榈酰油酰基-磷脂酰乙醇胺(POPE)、二油酰基-磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-甲酸酯(DOPE-mal)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷脂酰乙醇胺(DMPE)、二硬脂酰基-磷脂酰基-乙醇胺(DSPE)、16-O-单甲基PE,16-O-二甲基PE,18-1-反式PE,1-硬脂酰基-2-油酰基-磷脂酰乙醇胺(SOPE)或其混合物。The composition according to claim 12, wherein the charge-auxiliary lipid is one or more of the following: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) ), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine Base (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE) -mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O -DimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or mixtures thereof.
  14. 根据权利要求7所述的组合物，其特征在于，所述载体中还包括结构修饰脂质。 The composition according to claim 7, wherein the carrier further includes a structurally modified lipid.
  15. 根据权利要求14所述的组合物，其特征在于，所述结构修饰脂质包括聚乙二醇、葡聚糖、聚乳酸或氨基酸修饰的磷脂酰乙醇胺、磷脂酸、神经酰胺、二烷基胺、二酰基甘油、二烷基甘油中的一种或多种。The composition according to claim 14, wherein the structurally modified lipid includes polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine , one or more of diacylglycerol and dialkylglycerol.
  16. 根据权利要求7所述的组合物，其特征在于，所述载体还包括但不限于胆酸或其衍生物的脂质、电荷辅助脂质以及结构修饰脂质，所述胆酸脂质、所述电荷辅助脂质以及所述结构修饰脂质的摩尔比为(30～80)：(5-50)：(0.5～10)。The composition according to claim 7, wherein the carrier further includes, but is not limited to, lipids of cholic acid or its derivatives, charge-assisted lipids and structurally modified lipids, and the cholic acid lipid, the The molar ratio of the charge-assisted lipid and the structurally modified lipid is (30-80): (5-50): (0.5-10).
  17. 根据权利要求7所述的组合物，其特征在于，所述载体还包括胆酸或其衍生物的脂质、电荷辅助脂质、胆固醇或其衍生物以及结构修饰脂质，所述胆酸脂质、所述电荷辅助脂质、所述胆固醇或其衍生物、所述结构修饰脂质的摩尔比为(30～80)：(0.5～10)：(5-50)：(0.5～2.5)。The composition according to claim 7, wherein the carrier further includes lipids of cholic acid or its derivatives, charge-assisted lipids, cholesterol or its derivatives, and structurally modified lipids, and the cholic acid lipid The molar ratio of the mass, the charge-assisted lipid, the cholesterol or its derivatives, and the structurally modified lipid is (30~80): (0.5~10): (5-50): (0.5~2.5) .
  18. 根据权利要求7所述的组合物，其特征在于，所述组合物还包括药物可用的赋形剂或稀释剂中的一种或多种。The composition of claim 7, further comprising one or more pharmaceutically acceptable excipients or diluents.
  19. 根据权利要求1-18中任一项脂质化合物或组合物用于在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用。The lipid compound or composition according to any one of claims 1 to 18 is used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
  20. 根据权利要求1-18中任一项脂质化合物或组合物用于在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用中，其中所述脂质纳米颗粒具有20～1000nm粒径。The lipid compound or composition according to any one of claims 1 to 18 is used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, wherein the lipid nanoparticles have a particle size of 20~ 1000nm particle size.
  21. 用于制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物的组合物，其包含核酸和包载所述核酸的脂质纳米颗粒，其中每个单独的脂质纳米颗粒包含多种脂质组分，其中一种脂质组分是基于胆酸的脂质化合物，包括其化合物或其药学上可用的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价化合物或前体药物，并且其中所述脂质纳米颗粒具有至少70％的核酸包封比例。Compositions for preparing nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, which include nucleic acids and lipid nanoparticles encapsulating the nucleic acids, wherein each individual lipid nanoparticle contains a variety of lipids A lipid component, wherein one of the lipid components is a cholic acid-based lipid compound, including compounds thereof or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non- A covalent compound or prodrug, and wherein the lipid nanoparticle has a nucleic acid encapsulation ratio of at least 70%.
  22. 制备权利要求21所述组合物的方法，其中所述脂质纳米颗粒通过将mRNA溶液和包括权利要求1-6中任一种脂质化合物的脂质溶液混合而形成，其中mRNA溶液的介质为HEPES、磷酸钠、乙酸钠、硫酸铵、碳酸氢钠或柠檬酸钠；脂质溶液的介质为乙醇、异丙醇或二甲亚砜；其中所述脂质纳米颗粒通过透析或超滤进一步纯化。 A method for preparing the composition of claim 21, wherein the lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution including any one of the lipid compounds of claims 1-6, wherein the medium of the mRNA solution is HEPES, sodium phosphate, sodium acetate, ammonium sulfate, sodium bicarbonate or sodium citrate; the medium of the lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide; wherein the lipid nanoparticles are further purified by dialysis or ultrafiltration .
  23. 根据权利要求21所述的组合物，还包含缓冲剂、碳水化合物、甘露醇、蛋白质、多肽或者氨基酸、抗氧化剂、抑菌剂、螯合剂、佐剂中的一种或多种。The composition according to claim 21, further comprising one or more of a buffer, carbohydrate, mannitol, protein, polypeptide or amino acid, antioxidant, bacteriostatic agent, chelating agent, and adjuvant.
  24. 根据权利要求21所述的组合物,其给药方式包括静脉注射、肌肉注射、皮下注射、皮内注射、瘤内注射、眼部给药、耳部给药、经鼻给药、经口腔给药、经肛门给药、经阴道给药。The composition according to claim 21, its administration mode includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, eye administration, ear administration, nasal administration, or oral administration. medicine, transanal administration, transvaginal administration.
  25. 根据权利要求21所述的组合物，其给药对象包括哺乳动物如牛、马、骡、驴、骆驼、猪、羊、犬、狐或兔，禽类如鸡、鸭、鹅或鸽子，鱼类，非人灵长类，人。 The composition according to claim 21, its administration targets include mammals such as cattle, horses, mules, donkeys, camels, pigs, sheep, dogs, foxes or rabbits, poultry such as chickens, ducks, geese or pigeons, fish , non-human primates, humans.
PCT/CN2023/093558 2022-05-13 2023-05-11 Lipid compound, and composition, preparation and use thereof WO2023217237A1 (en)

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