CN117045806A - Lipid composition, preparation and use thereof - Google Patents

Lipid composition, preparation and use thereof Download PDF

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CN117045806A
CN117045806A CN202310529268.3A CN202310529268A CN117045806A CN 117045806 A CN117045806 A CN 117045806A CN 202310529268 A CN202310529268 A CN 202310529268A CN 117045806 A CN117045806 A CN 117045806A
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lipid
acid
compound
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Suzhou Huiyi Biomedical Technology Co ltd
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    • 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
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    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
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    • 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
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    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
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    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
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    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
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Abstract

The invention discloses a lipid composition, a preparation method and application thereof, wherein the lipid compound is a compound based on cholic acid or a derivative thereof or a pharmaceutically usable salt thereof, and the lipid compound and the composition thereof can provide more carrier choices for delivery of nucleic acid medicaments, genetic vaccines, polypeptides, proteins, antibodies, small molecule medicaments and the like.

Description

Lipid composition, preparation and use thereof
Technical Field
The invention relates to a lipid composition, a preparation method and application thereof, in particular to application in preparing nucleic acid medicaments, genetic vaccines, polypeptides, proteins, antibodies and small molecule medicaments.
Background
The nucleic acid drug refers to artificially designed DNA or RNA with disease prevention or treatment function, and can radically regulate and control the expression of pathogenic genes by acting on pathogenic target genes or target mRNA, thereby achieving the purpose of disease prevention or treatment. Nucleic acid drugs mainly include antisense nucleic acid (antisense oligonucleotide, ASO), small interfering RNA (small interference RNA, siRNA), micro RNA (miRNA), messenger RNA (mRNA), and the like. By the end of 2021, the FDA has approved more than 10 nucleic acid drugs, and various drug candidates are in clinical or preclinical stages.
mRNA technology demonstrates its unique advantages over traditional biopharmaceutical and vaccine technologies. Therapeutic nucleic acids have the potential to radically alter vaccination, gene therapy, protein replacement therapy, and other therapies for genetic diseases.
The very easily degradable nature of RNA itself dictates the need for a superior delivery system to deliver it into the body. The invention focuses on providing a novel delivery carrier for RNA drugs.
Disclosure of Invention
The invention provides a lipid composition, a preparation method and application thereof, namely lipid based on cholic acid or derivatives thereof, enriches the types of lipid compounds, and provides more choices for delivery of nucleic acid drugs, genetic vaccines, polypeptides, proteins, antibodies, small molecule drugs and the like.
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, non-covalent compounds or prodrugs, and specifically, the "pharmaceutically acceptable salt thereof" refers to an acid addition salt or a base addition salt.
General formula 1:
R 1 =hydrogen atom, alkane, alkene, substituted carbonyl group, alkane containing nitrogen atom, alkene containing nitrogen atom
R 2 =hydrogen atom, alkane, alkene, substituted carbonyl group, alkane containing nitrogen atom, alkene containing nitrogen atom
R 3 =hydroxy, halogen atom, alkane, alkene
R 4 =hydrogen atom, alkane, alkene, alkane containing nitrogen atom, alkene containing nitrogen atom, cyclic compound containing nitrogen atom
X=nh or O
General formula 2:
the acids of the present invention include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, carbonic acid, cinnamic acid, citric acid, cyclic amic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, 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, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid.
The base addition salt according to the present invention refers to a salt 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, potassium, lithium, ammonium, calcium, magnesium, iron, and the like; 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, hydrazinaniline, choline, betaine, benazepine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.
Preferably, the lipid compound mother nucleus is cholic acid or a derivative thereof; wherein the linkage (Linker) comprises one or more of an ester group, an amide group, a urethane group, a carbonate group, or a urea group.
Preferably, the lipid compound wherein the cholic acid or derivative thereof is one or more optionally selected from cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3β -hydroxy-D5-cholanic acid, chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholic acid, 5β -cholic acid, dehydrocholic acid, hyodeoxycholic acid, glycochenodeoxycholic acid, tauroursodeoxycholic acid, taurocholic acid, hyodeoxycholic acid, methyl hyodeoxycholic acid, sodium taurochenoxycholic acid, sodium dehydrocholic acid, sodium cholate, sodium deoxyglycocholate, sodium taurodeoxycholate, sodium taurocholate-3-sulfate disodium salt, sodium tauroursodeoxycholate and sodium taurocholate.
Preferably, the lipid compound is one or more selected from ursodeoxycholic acid derivative lipid compounds or one or more obeticholic acid derivative lipid compounds.
Preferably, the lipid compound is also one or more selected from cholic acid derivative lipid compounds, or one or more of hyodeoxycholic acid derivative lipid compounds, or one or more of chenodeoxycholic acid derivative lipid compounds.
In particular, 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 polymyosine derivative lipid, or a chitosan derivative lipid, or a hyaluronic acid derivative lipid.
Specifically, the lipid compound may also be a conjugate of a lipid compound, the conjugate mainly consisting of three parts of a therapeutic or prophylactic drug, a linker (linker) and a lipid compound. The therapeutic or prophylactic drug comprises one or more of nucleic acid, polypeptide, protein, antibody, saccharide, polyethylene glycol and its derivatives, and small molecule.
In particular, the nucleic acid comprises any form of nucleic acid molecule, e.g., the DNA may be non-coding or coding DNA, and the RNA may be selected from: ASO, mRNA, siRNA, micRNA, etc.
Specifically, the linker (linker) is independently selected from one or more of a non-existent, linear compound or a cyclic compound, and is connected with both ends through an ester group, an amide group, a urethane group, a carbonate group, an ether group, a urea group, or the like.
Specifically, the linker is independently selected from a linear saturated hydrocarbon or an unsaturated hydrocarbon of 2-25C, and two ends of the linker are one or more of carboxyl, hydroxyl, amino, sulfate, sulfonate and phosphate.
Specifically, the linker is independently selected from saturated hydrocarbon or unsaturated hydrocarbon containing 3-8 membered ring, and two ends of the linker are one or more of carboxyl, hydroxyl, amino, sulfate, sulfonate and phosphate.
The present invention also provides a lipid compound which is a lipid based on cholic acid or a derivative thereof, or which is a pharmaceutically acceptable salt, prodrug or stereoisomer of a lipid based on cholic acid or a derivative thereof, the lipid compound having the structure of formula 1 or formula 2:
general formula 1:
r1=hydrogen atom, alkane, alkene, substituted carbonyl group, alkane containing nitrogen atom, alkene containing nitrogen atom
R 2 =hydrogen atom, alkane, alkene, substituted carbonyl group, containing nitrogen Alkanes of atoms, alkenes containing nitrogen atoms
R 3 =hydroxy, halogen atom, alkane, alkene
R 4 =hydrogen atom, alkane, alkene, alkane containing nitrogen atom, alkene containing nitrogen atom, cyclic compound containing nitrogen atom
X=nh or O
General formula 2:
specifically, the lipid compound mother nucleus is cholic acid or a derivative thereof; wherein the linkage (Linker) comprises one or more of an ester group, an amide group, a urethane group, a carbonate group, or a urea group.
Specifically, wherein the cholic acid or derivative thereof is one or more selected from among cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3β -hydroxy-D5-cholanic acid, chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholate, 5β -cholic acid, dehydrocholic acid, hyocholic acid, chenodeoxycholic acid, glycocholic acid, hyodeoxycholic acid, methyl hyodeoxycholic acid, sodium taurochenodeoxycholate, sodium dehydrocholate, sodium deoxyglycocholate, sodium taurodeoxycholate, sodium taurochenodeoxycholate, sodium glycocholate, taurocholate-3-sulfate disodium salt, sodium taurocursodeoxycholate and sodium taurocholate.
Specifically, the lipid compound is one or more of ursodeoxycholic acid derivative lipid compounds, or one or more of obeticholic acid derivative lipid compounds, one or more of cholic acid lipid compounds, one or more of hyodeoxycholic acid derivative lipid compounds, and one or more of chenodeoxycholic acid derivative lipid compounds.
In particular, 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 polymyosine derivative lipid, or a chitosan derivative lipid, or a hyaluronic acid derivative lipid.
Specifically, the lipid compound may also be a conjugate of a lipid compound, the conjugate mainly consisting of three parts of a therapeutic or prophylactic drug, a linker (linker) and a lipid compound. The therapeutic or prophylactic drug comprises one or more of nucleic acid, antibody, polypeptide, protein, saccharide, polyethylene glycol and its derivatives, and small molecule.
In particular, the nucleic acid comprises any form of nucleic acid molecule, e.g., the DNA may be non-coding or coding DNA, and the RNA may be selected from: ASO, mRNA, siRNA, micRNA, etc.
Specifically, the linker (linker) is independently selected from one or more of a non-existent, linear compound or a cyclic compound, and is connected with both ends through an ester group, an amide group, a urethane group, a carbonate group, an ether group, a urea group, or the like.
Specifically, the linker is independently selected from a linear saturated hydrocarbon or an unsaturated hydrocarbon of 2-25C, and two ends of the linker are one or more of carboxyl, hydroxyl, amino, sulfate, sulfonate and phosphate.
Specifically, the linker is independently selected from saturated hydrocarbon or unsaturated hydrocarbon containing 3-8 membered ring, and two ends of the linker are one or more of carboxyl, hydroxyl, amino, sulfate, sulfonate and phosphate.
A composition of lipid compounds comprising a therapeutic or prophylactic agent and a carrier for delivering the therapeutic or prophylactic agent, the carrier comprising one or more of the aforementioned lipid compounds.
Preferably, the composition, the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a polypeptide, a protein, an antibody and a small molecule drug.
Preferably, in the composition, the mass ratio of the carrier to the therapeutic or prophylactic agent is 1:1-100:1.
Preferably, the composition is a nanoparticle preparation, and the average size of the nanoparticle preparation is 20 nm-1000 nm; the polydispersity of the nanoparticle preparation is less than or equal to 0.5.
Preferably, the composition comprises three different lipid components in the carrier, wherein one lipid is a lipid based on cholic acid or a derivative thereof.
Preferably, the composition further comprises a charge-assisted lipid of neutral charge, negative charge or bipolar charge in the carrier.
Preferably, the composition is characterized in that the charge-assisted lipid is one or more of the following: distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof.
Preferably, the composition, the carrier further comprises a structure-modified lipid.
Preferably, the composition, the structure-modified lipid comprises one or more of polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol and dialkylglycerol.
Preferably, the composition, the carrier further includes but is not limited to a lipid of cholic acid or its derivative, a charge-assisted lipid and a structure-modified lipid, wherein the molar ratio of the cholic acid lipid, the charge-assisted lipid and the structure-modified lipid is (30-80): (5-50): (0.5-10).
Preferably, the composition further comprises one or more of a pharmaceutically acceptable excipient or diluent.
Preferably, the carrier further comprises a lipid of cholic acid or a derivative thereof, a charge-assisted lipid, cholesterol or a derivative thereof, and a structure-modified lipid, wherein the molar ratio of the cholic acid lipid, the charge-assisted lipid, the cholesterol or a derivative thereof, and the structure-modified lipid is (30-80): (0.5-10): (5-50): (0.5-2.5).
The lipid compound or the composition can be applied to the preparation of nucleic acid medicaments, gene vaccines, polypeptides, proteins, antibodies and micromolecular medicaments.
The lipid compound or the composition is applied to the preparation of nucleic acid medicaments, gene vaccines, polypeptides, proteins, antibodies and small molecule medicaments, wherein the lipid nano particles have the particle size of 20-1000 nm.
A composition for the preparation of nucleic acid pharmaceuticals, genetic vaccines, polypeptides, proteins, antibodies and small molecule pharmaceuticals comprising a nucleic acid and lipid nanoparticles encapsulating the nucleic acid, wherein each individual lipid nanoparticle comprises a plurality of lipid components, wherein one lipid component is a cholic acid-based lipid compound, including compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent compounds or prodrugs thereof, and wherein the lipid nanoparticle has a nucleic acid encapsulation ratio of at least 70%.
A method of preparing a composition as described herein, wherein the lipid nanoparticle is formed by mixing an mRNA solution and a lipid solution of any of the lipid compounds described herein, 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, isopropanol or dimethyl sulfoxide; wherein the lipid nanoparticle is further purified by dialysis or ultrafiltration.
The composition of the patent further comprises one or more of buffering agent, carbohydrate, mannitol, protein, polypeptide or amino acid, antioxidant, bacteriostat, chelating agent and adjuvant.
The acids of the present invention include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, carbonic acid, cinnamic acid, citric acid, cyclic amic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, 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, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid.
The base addition salt according to the present invention refers to a salt 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, potassium, lithium, ammonium, calcium, magnesium, iron, and the like; the organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholized, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydrazinaniline, choline, betaine, benazepine (bennethamine), ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.
The present invention provides a composition comprising a therapeutic or prophylactic agent and a carrier for delivering the therapeutic or prophylactic agent, the carrier comprising one or more of a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable salt thereof.
In particular, the therapeutic or prophylactic agent is encapsulated within or associated with a carrier.
Specifically, the therapeutic or prophylactic agent comprises one or more of nucleic acid molecules, genetic vaccines, polypeptides, proteins, antibodies and small molecule drugs.
In particular, the nucleic acid includes any form of nucleic acid molecule including, but not limited to, single-stranded DNA, double-stranded DNA, short isoforms, agomir, antagomir, antisense molecules, small interfering RNAs (siRNA), asymmetric interfering RNAs (aiRNA), microRNA (miRNA), dicer-subduct RNAs (dsRNA), small hairpin RNAs (shRNA), transfer RNAs (tRNA), messenger RNAs (mRNA), and other forms of RNA molecules known in the art, or nucleic acid mimics such as Locked Nucleic Acids (LNAs), peptide Nucleic Acids (PNAs), and morpholino oligonucleotides.
According to some specific embodiments, the therapeutic or prophylactic agent comprises at least one mRNA encoding an antigen or protein or peptide, or a fragment or epitope thereof.
More specifically, the mRNA is a monocistronic mRNA or a polycistronic mRNA.
More specifically, the antigen is a pathogenic antigen.
More specifically, the mRNA comprises one or more functional nucleotide analogs including, but not limited to, one or more of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.
In particular, the small molecule compounds include, but are not limited to, active ingredients of therapeutic and/or prophylactic agents that are currently known drugs, such as anti-neoplastic agents, anti-infective agents, local anesthetics, antidepressants, anticonvulsants, antibiotics/antimicrobials, antifungals, antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics, or contrast agents.
Preferably, the lipid comprises three different lipid components, wherein one lipid is a lipid based on cholic acid or a derivative thereof. Preferably, the lipid further comprises a neutral, negatively or amphiphilically charged helper lipid.
More specifically, the lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.
More specifically, the lipids include, but are not limited to, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1
1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dimethylammonium 2- (((2, 3-bis (oleoyloxy) propyl)) phosphate) ethyl hydrogen (DOCP), sphingomyelin (SM), ceramide and derivatives thereof. The lipid may be synthetic or derived from (isolated or modified from) a natural source or compound.
According to some embodiments, the carrier further comprises a structurally modified lipid.
In particular, the structurally modified lipids mainly include disclosed or unpublished lipid compounds, which can improve the stability of the liposome and reduce protein absorption of the liposome, such as one or more of polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, dialkylglycerol.
More specifically, the structurally modified lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG 2000, chol-PEG2000, 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), 4-O- (2 ',3' -di (tetradecyloxy) propyl-1-O- (omega-methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG), pegylated ceramide (PEG-cer), omega-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecyloxy) propyl) carbamate, or 2, 3-di (tetradecyloxy) propyl-N- (omega-methoxy) (polyethoxy) ethyl) carbamate.
Preferably, the mass ratio of the carrier to the therapeutic or prophylactic agent is from 5:1 to 50:1, more preferably from 5:1 to 35:1, and even more preferably from 10:1 to 30:1.
The composition according to the preceding claim, wherein the carrier further comprises, but is not limited to, lipids of cholic acid or derivatives thereof, charge-assisted lipids and structurally modified lipids. The molar ratio of the cholic acid lipid, the charge-assisted lipid, and the structure-modified lipid is (30 to 80): (5-50): (0.5-10).
In some embodiments, the lipid nanoparticle is formed by mixing an mRNA solution and a lipid solution. In some embodiments, the lipid nanoparticle is further purified by tangential flow filtration.
The method according to the preceding claim, wherein the lipid nanoparticle is formed by mixing an mRNA solution and a lipid solution. Wherein the medium of the mRNA solution is HEPES, phosphate, acetate, ammonium sulfate, sodium bicarbonate or citrate. The medium of the lipid solution is ethanol, isopropanol or dimethyl sulfoxide.
Preferably, the pharmaceutical composition is a nanoparticle formulation having an average size of 20nm to 1000nm, preferably 40nm to 150nm, more preferably 50nm to 100nm, more preferably 70nm to 100nm.
Further preferably, the nanoparticle formulation has a polydispersity index of 0.5 or less, further preferably 0.3 or less, more preferably 0.25 or less.
The pharmaceutical compositions of the invention typically further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannitol, sucrose, trehalose, dextrose, or dextran), mannitol, proteins, polypeptides, or amino acids (e.g., glycine and lysine), antioxidants (vitamin E and butylhydroxytoluene), bacteriostats, chelators (e.g., EDTA and glutathione), adjuvants (e.g., aluminum hydroxide), suspending/thickening/preserving agents that make the formulation isotonic with the blood of the recipient, and the like, or the compositions of the invention may be formulated as lyophilized.
In particular, modes of administration of the composition include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, ocular administration, otic administration, nasal administration, oral administration, anal administration, vaginal administration, and the like.
In particular, subjects to which the composition is administered include, but are not limited to, mammals such as cattle, horses, mules, donkeys, camels, pigs, sheep, dogs, foxes, rabbits, and the like, birds such as chickens, ducks, geese, pigeons, and the like, fish, non-human primates, humans.
The invention relates to the field of Lipid Nano Particles (LNP), and the preparation method of the nano particles is simple, has good repeatability, simplifies the production flow and reduces the cost; is favorable for promoting the localization of nucleic acid medicaments. In a specific embodiment, the prepared lipid nanoparticle coated with siRNA has a better cell transfection effect; the LNP nanoparticle has better in-vivo delivery effect no matter the nanoparticle is injected into a mouse body through an vein or injected into the mouse body through a muscle. In addition, in a specific embodiment, the nanoparticle with the mRNA entrapped therein is injected intratumorally, and the tumor site of the mouse is subjected to fluorescent expression after 6 hours of shooting after injection, which indicates that the lipid nanoparticle can be administrated by adopting an intratumoral injection mode. The nanoparticle has good delivery effect and application scene by combining in-vivo and in-vitro evaluation results.
The technical scheme of the invention provides a brand new lipid compound for delivering medicines for treatment or prevention, namely lipid based on cholic acid or derivatives thereof, is different from the prior patent technical scheme of foreign pharmaceutical companies, enriches the types of lipid compounds, provides more choices for delivering nucleic acid medicines, genetic vaccines, polypeptides, proteins, antibodies, small molecular medicines and the like, and can be obviously different from the technical routes of foreign-part-of-the-art and Morgana-type companies.
Drawings
FIG. 1 shows a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 1 (compound 1).
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 2 (compound 2).
FIG. 3 shows the nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 7 (compound 7).
FIG. 4 shows the nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 9 (compound 9).
FIG. 5 shows the nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 10 (compound 10).
FIG. 6 shows a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 13 (compound 13).
FIG. 7 shows a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 16 (compound 16).
FIG. 8 shows a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 17 (compound 17).
Fig. 9 is a nuclear magnetic resonance hydrogen spectrum of obeticholic acid derivative 18 (compound 18).
FIG. 10 shows a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 42 (compound 42).
FIG. 11 shows a nuclear magnetic resonance hydrogen spectrum of lithocholic acid derivative 58 (compound 58).
FIG. 12 is a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 62 (compound 62).
FIG. 13 is a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 63 (compound 63).
FIG. 14 is a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 66 (compound 66).
FIG. 15 shows a nuclear magnetic resonance hydrogen spectrum of chenodeoxycholic acid derivative 69 (compound 69).
FIG. 16 shows a nuclear magnetic resonance hydrogen spectrum of hyodeoxycholic acid derivative 72 (compound 72).
FIG. 17 shows a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 75 (compound 75).
FIG. 18 is a chart showing the nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 85 (compound 85).
FIG. 19 shows a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 87 (compound 87).
FIG. 20 shows a nuclear magnetic resonance hydrogen spectrum of ursodeoxycholic acid derivative 89 (compound 89).
FIG. 21 is a graph showing the results of LNP-entrapped cy3-siRNA transfected cells, wherein a) fluorescent microscopy fields, b) fluorescent microscopy fields, and c) cell flow patterns.
FIG. 22 shows the results of LNP-entrapped EGFP mRNA transfected cells, wherein a) fluorescence microscopy fields, b) fluorescence microscopy fields, c) cell flow charts.
FIG. 23 shows the results of 12h fluorescence imaging of the LNP harboring Luciferase mRNA after intravenous injection in mice.
FIG. 24 shows the results of 12h fluorescence imaging of the LNP harboring Luciferase mRNA after intramuscular injection in mice.
Fig. 25: tumor cell a549 killing effect graph.
Fig. 26: fluorescence imaging results after intramuscular injection of mice.
Fig. 27: fluorescence imaging results of mice after intravenous injection.
Fig. 28: results of fluorescence imaging after intratumoral injection in mice.
Detailed Description
Cholic acid is a naturally occurring steroid molecule in humans and mammals, synthesized in the liver from cholesterol. After eating, cholic acid is actively secreted by liver cells, enters the gall bladder along with bile, enters the intestinal tract to exert the digestion function of the cholic acid, enters the small intestine in the form of sodium salt to help digestion and absorption of lipid, then returns to the liver through portal vein at the terminal of ileum by means of active absorption or passive transportation, is processed and transformed in the liver cells, and is secreted into the small intestine together with newly synthesized bile acid. This EHC (hepatointestinal) process of bile acids circulates 4 to 12 times per day, with about 95% of bile acids being used by aspiration. If the EHC of bile acid is destroyed, not only the digestion and absorption of lipids in the body can be affected, but also cholesterol stones can be formed in the body. Therefore, the compound delivery carrier designed based on cholic acid has the greatest advantage of high liver and intestine circulation efficiency, and participates in the liver and intestine circulation of cholic acid, thereby improving the absorption of the medicine in the liver and gall bladder. Cholic acid has the following structural formula.
The cholic acid molecule has 3 six-membered rings and 1 five-membered ring on the steroid skeleton on the same plane, wherein the ring A and the ring B are reversely connected, so that the molecule forms 1 hole structure. The 3 methyl groups in the molecule are distributed on one side of the plane of the steroid ring to form a hydrophobic part of the molecule, and the 3 hydroxyl groups are distributed on the other side of the plane of the steroid ring to form a hydrophilic part of the molecule together with the carboxyl group at the C24 position.
The special structure of cholic acid molecule determines that it has amphiphilicity, acid-base nature and is easy to carry out chemical modification, so that the cholic acid is used as a building element to prepare high polymer or oligomer, and the functional molecules based on cholic acid have good technical effect in drug delivery.
Cholic acid drugs have been marketed in the chinese or us market for many years, including ursodeoxycholic acid, obeticholic acid, chenodeoxycholic acid, tauroursodeoxycholic acid. Such as ursodeoxycholic acid, for treating cholesterol gallstone and bile reflux gastritis; obeticholic acid for use in the treatment of Primary Biliary Cirrhosis (PBC), in patients who do not respond adequately or who are intolerant to ursodeoxycholic acid. Clinical application for many years shows that the cholic acid compound has better safety. In addition, the cholic acid compound has hydrophilic and oleophilic amphiphilic properties, and the bioavailability of the micromolecular chemical medicine can be improved through a wrapping or coupling mode.
Cholic acid compounds belong to steroid compounds with similar structures and more sites for chemical modification. The cholic acid compound is subjected to structural modification to prepare a novel lipid component, and the novel lipid component can replace ionizable lipid and cholesterol in the original four-component LNP, so that a similar or better mRNA delivery effect can be achieved. This patent study constructs lipid nanoparticle carriers based on cholic acid analogs and explores their use in mRNA drug delivery.
The LNP of the invention can simplify the production flow and reduce the cost; meanwhile, patent blocking of four-component LNP can be avoided, and localization of nucleic acid medicines is facilitated. Because cholic acid or its derivatives are each composed of a rigid steroid ring and an aliphatic side chain, wherein the steroid ring comprises three six-membered rings and a five-membered ring. The side chain structure, steroidal conformation, number of hydroxyl groups, and orientation on the steroidal ring of cholic acid will vary depending on the source alone. The common hydroxyl group of cholic acid or derivative thereof and the carboxyl group of the fatty side chain are good chemical modification sites. Therefore, it is believed that cholic acid or its derivatives may be modified in some common ways to achieve the intended purpose.
Cholic acid or derivatives thereof in this patent are optionally selected from cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3 beta-hydroxy-D5-cholanic acid, chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholic acid, 5 beta-cholic acid, dehydrocholic acid, hyocholic acid, rocholic acid, glycochenodeoxycholic acid, tauroursodeoxycholic acid, taurochenodeoxycholic acid, glycocholic acid, hyodeoxycholic acid, methyl hyodeoxycholic acid, sodium taurochenodeoxycholate, sodium dehydrocholate, sodium deoxyglycocholate, sodium taurodeoxycholate, sodium taurochenodeoxycholate, sodium glycocholate, taurocholate-3-sulfate disodium salt, sodium taurochenodeoxycholate and sodium taurochenocholate, the specific structures are as follows.
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The lipid compound is one or more selected from compounds shown in the following structures; (1) ursodeoxycholic acid derivative lipid:
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(2) obeticholic acid derivative lipid:
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the present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the embodiment can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not interfere with each other.
Example 1: synthesis of ursodeoxycholic acid derivative 1 (Compound 1)
Ursodeoxycholic acid (393 mg,1.0 mmol) was dissolved in DMF (8 mL), HBTU (569 mg,1.5 mmol), DIEA (194 mg,1.5 mmol), N, N-dimethylethylenediamine (132 mg,1.5 mmol) was added sequentially, and the reaction was stirred at room temperature under nitrogen overnight. TLC detection, adding appropriate amount of water after reaction, extracting with ethyl acetate for 3 times, mixing organic layers, drying over anhydrous sodium sulfate, filtering, concentrating solvent to obtain crude productThe product is obtained. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 1 (333 mg, 72%) as a white solid. 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 the nuclear magnetic resonance hydrogen spectrum of the compound 1 is shown in figure 1 in the drawing of the specification.
Example 2: synthesis of ursodeoxycholic acid derivative 2 (Compound 2)
Ursodeoxycholic acid (393 mg,1.0 mmol) was dissolved in DMF (8 mL), HBTU (569 mg,1.5 mmol), DIEA (194 mg,1.5 mmol), 4-pyrrole-1-butylamine (213 mg,1.5 mmol) were added sequentially, and the reaction was stirred at room temperature under nitrogen overnight. TLC detection, after the reaction, adding a proper amount of water, extracting with ethyl acetate for 3 times, combining organic layers, drying with anhydrous sodium sulfate, filtering, concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 2 (403 mg, 78%) as a white solid. 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 nuclear magnetic resonance hydrogen spectrum of the compound 2 is shown in figure 2 in the attached drawing of the specification.
Example 3: synthesis of ursodeoxycholic acid derivative 7 (Compound 7)
Ursodeoxycholic acid (393 mg,1.0 mmol) was dissolved in acetonitrile (10 mL) and K was added sequentially 2 CO 3 (418 mg,3.0 mmol), bnBr (850 mg,5.0 mmol), nitrogen were then stirred at 80℃for 5h. TLC detection, filtering after reaction, concentrating and dissolvingCrude product is obtained. Silica gel column chromatography, PE/EA (1:1) gave compound 6 (390 mg, 81%) as a white solid. 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-Dimethylaminobutyrate (336 mg,2.0 mmol) was dissolved in anhydrous DCM (8 mL), oxalyl chloride (1 mL) was added and stirred at room temperature for 4h. Concentrated to no solvent in vacuo and dissolved with anhydrous DCM (3 mL) for use. Compound 6 (241 mg,0.5 mmol), TEA (202 mg,2.0 mmol) was dissolved in anhydrous DCM (3 mL) and the above stock solution was added dropwise to the reaction solution and reacted overnight at room temperature. An appropriate amount of water was added, extracted 3 times with DCM, the organic layers were combined, dried over anhydrous sodium sulfate, filtered and the solvent concentrated to give crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 7 (213 mg, 60%) as a white solid. 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 nuclear magnetic resonance hydrogen spectrum of the compound 7 is shown in figure 3 in the drawing of the specification.
Example 4: synthesis of ursodeoxycholic acid derivative 9 (Compound 9)
Ursodeoxycholic acid (785 mg,1.0 mmol) was dissolved in DMF (20 mL) and K was added sequentially 2 CO 3 (829mg,6.0mmol)，CH 3 I (850 mg,6.0 mmol), nitrogen, stirring at room temperature overnight. TLC detection, after the reaction, adding a proper amount of water, extracting with ethyl acetate for 3 times, combining organic layers, drying with anhydrous sodium sulfate, filtering, concentrating the solvent to obtain a crude product. Silica gel column chromatography, PE/EA (1:1) eluted, affording compound 8 (700 mg, 86%) as a white solid.
4-Dimethylaminobutyrate (336 mg,2.0 mmol) was dissolvedOxalyl chloride (0.5 mL) was added to anhydrous DCM (8 mL) and stirred at room temperature for 4h. Concentrated to no solvent in vacuo and dissolved with anhydrous DCM (3 mL) for use. Compound 8 (203 mg,0.5 mmol), TEA (202 mg,2.0 mmol) was dissolved in anhydrous DCM (3 mL) and the above stock solution was added dropwise to the reaction solution and reacted overnight at room temperature. An appropriate amount of water was added, extracted 3 times with DCM, the organic layers were combined, dried over anhydrous sodium sulfate, filtered and the solvent concentrated to give crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 9 (221 mg, 70%) as a white solid. 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 nuclear magnetic resonance hydrogen spectrum of the compound 9 is shown in figure 4 of the drawings.
Example 5: synthesis of ursodeoxycholic acid derivative 10 (Compound 10)
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 5h. Concentrated to no solvent in vacuo and dissolved with anhydrous DCM (2 mL) for use. Compound 2 (240 mg,0.34 mmol), TEA (69 mg,0.68 mmol) was dissolved in anhydrous DCM (5 mL) and the above stock solution was added dropwise to the reaction solution and reacted overnight at room temperature. An appropriate amount of water was added, extracted 3 times with DCM, the organic layers were combined, dried over anhydrous sodium sulfate, filtered and the solvent concentrated to give crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 10 (212 mg, 60%) as a pale yellow semi-solid product. 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 nuclear magnetic resonance hydrogen spectrum of the compound 10 is shown in figure 5 of the drawings in the specification.
Example 6: synthesis of ursodeoxycholic acid derivative 13 (Compound 13)
Synthesis of intermediate compound 11: compound 6 (3.0 g,6.2 mmol) was dissolved in dry pyridine (20 mL), DMAP (159 mg,1.3 mmol), acetic anhydride (3.2 g,31.0 mmol) was added sequentially, and the reaction was stirred at room temperature overnight under nitrogen. After the reaction was completed by TLC, a proper amount of water was added, extracted 3 times with DCM, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to give crude product. Silica gel column chromatography, PE/EA (2:1) eluted, affording compound 11 (3.0 g, 86%) as a white solid. 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).
Synthesis of intermediate compound 12: compound 11 (1.7 g,3.0 mmol) was dissolved in dry methanol (40 mL) and K was added 2 CO 3 (0.83 g,6.0 mmol) and stirred at room temperature for 2h. TLC detection, after the reaction, filtering, adding proper amount of water, extracting with ethyl acetate for 3 times, combining organic layers, drying with anhydrous sodium sulfate, filtering, concentrating the solvent to obtain crude product. Silica gel column chromatography, PE/EA (2:1) eluted, affording compound 12 (1.1 g, 70%) as a white solid. 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).
Compound 12 (1.0 g,2.2 mmol) was dissolved in DMF (8 mL) and HBTU (1.15 g,3.0 mmol), DIEA (786 mg,6.1 mmol), 4-dimethylaminobutyrate (509 mg,3.0 mmol) was added sequentially and the reaction stirred at room temperature under nitrogen overnight. TLC detection, after the reaction, adding a proper amount of water, extracting with ethyl acetate for 3 times, combining organic layers, drying with anhydrous sodium sulfate, filtering, concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 13 as a white solid (900 mg, 73%). 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 nuclear magnetic resonance hydrogen spectrum of the compound 13 is shown in figure 6 of the drawings in the specification.
Example 7: synthesis of ursodeoxycholic acid derivative 16 (Compound 16)
Compound 6 (965 mg,2.0 mmol) was dissolved in DMF (30 mL), HBTU (1.15 g,3.0 mmol), DIEA (517 mg,4.0 mmol), 4-dimethylaminobutyrate (335 mg,2.0 mmol) under nitrogen were added sequentially and the reaction stirred at room temperature overnight. TLC detection, after the reaction, adding a proper amount of water, extracting with ethyl acetate for 3 times, combining organic layers, drying with anhydrous sodium sulfate, filtering, concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 16 (400 mg, 33%) as a white solid. 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 nuclear magnetic resonance hydrogen spectrum of the compound 16 is shown in figure 7 of the drawings in the specification.
Example 8: synthesis of ursodeoxycholic acid derivative 17 (Compound 17)
Compound 4 (200 mg,0.39 mmol) was dissolved in dry pyridine (4 mL), DMAP (19 mg,0.16 mmol) was added sequentially, acetic anhydride (0.5 mL) was purged with nitrogen, and the reaction was stirred at room temperature overnight. After the reaction was completed by TLC, a proper amount of water was added, extracted 3 times with DCM, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated to give crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 17 (100 mg, 44%) as a white solid. 1 HNMR(400MHz,d 6 DMSO) delta 4.52-4.60 (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=8.0 hz, 3H), 0.63 (s, 3H) the nuclear magnetic resonance hydrogen spectrum of compound 17 is shown in figure 8 of the drawings of the specification.
Example 9: synthesis of Obeticholic acid derivative (Compound 18)
Obeticholic acid (426 mg,1 mmol) was dissolved in DMF (8 mL), HBTU (569 mg,1.5 mmol), DIEA (194 mg,1.5 mmol), N, N-dimethylethylenediamine (132 mg,1.5 mmol) was added sequentially, and the reaction was stirred at room temperature under nitrogen overnight. TLC detection, after the reaction, adding a proper amount of water, extracting with ethyl acetate for 3 times, combining organic layers, drying with anhydrous sodium sulfate, filtering, concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 18 as a white solid (343mg, 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, found492.20, the nuclear magnetic resonance hydrogen spectrum of the compound 18 is shown in figure 9 of the drawings.
Example 10: the synthetic route of ursodeoxycholic acid derivative (compound 19) is as follows
Synthesis of intermediate 19-1: compound 11 (10.2 g,18.0 mmol), palladium on carbon (0.5 g,5 wt%) and methanol were successively added to a reaction flask, and reacted at room temperature for 16 hours under a hydrogen atmosphere. TLC detection, after completion of the reaction, direct filtration and concentration of the organic phase, crude intermediate 19-1 (7.3 g, 85%) was used directly in the next step.
Intermediate 19-synthesis of 2: intermediate 19-1 (7.3 g,15.0 mmol) was dissolved in DMF and HBTU (8.7 g,23 mmol), DIEA (3.0 g,23.0 mmol) and N, N-dimethylpropanediamine (2.4 g,23.0 mmol) were added sequentially and the reaction stirred at room temperature for 16 hours. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 19-2 (6.0 g, 69.5%) as a white solid. 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％。
Synthesis of intermediate 19-3/19-4: intermediate 19-2 (5.4 g,10.0 mmol) was dissolved in THF/MeOH (3:1) and NaOH (2.3 g,58.0 mmol) was dissolved in H 2 O was added to the reaction mixture, and the mixture was stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, crude products are directly obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) afforded intermediate 19-3 (2.7 g, 54.0%) as a white solid and intermediate 19-4 (1.8 g, 41.3%) as a white solid. 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％。
synthesis of Compound 19: linoleic acid (1.4 g,5.0 mmol) was dissolved in DCM, DMF (1 drop) was added dropwise, oxalyl chloride (1.2 g,10.0 mmol) was added dropwise and stirred at room temperature for 4h. The reaction solution was directly concentrated and dried. Intermediate 19-3 (500 mg,1 mmol) was dissolved in DCM and Et was added 3 N (195 mg,2.0 mmol) was added to the acid chloride prepared above, in the chamberThe reaction was stirred at temperature overnight. TLC detection is carried out, and crude products are obtained through direct concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give 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 )。
Example 11: the synthetic route of ursodeoxycholic acid derivative (compound 20) is as follows
Synthesis of intermediate 20-1: compound 17 (1.8 g,3.0 mmol) was dissolved in THF/MeOH (3:1), and an aqueous solution of NaOH (719 mg,18 mmol) was added and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, crude products are directly obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 20-1 (720 mg, 43.1%) as a pale yellow solid. 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。
Synthesis of Compound 20: linoleic acid (1.6 g,6 mmol) was dissolved in DCM, oxalyl chloride (1.5 g,12 mmol) was added and DMF (1 drop) was added and stirred 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 and Et was added 3 N (235 mg,2.3 mmol) was added to the acid chloride prepared above, and the reaction was stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the crude product is directly concentrated. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 20 as a yellow oil (194.4 mg, 22.0%). 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％。
Example 12: the synthetic route of ursodeoxycholic acid derivative (compound 21) is as follows
Linoleic acid (1.5 g,5.3 mmol) was dissolved in DCM, oxalyl chloride (1.3 g,11.0 mmol) was added dropwise and DMF (1 drop) was added and stirred 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 and Et was added 3 N (212 mg,2.1 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, crude products are directly obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 21 as a yellow oil (197mg, 18%). 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％。
Example 13: the synthetic route of ursodeoxycholic acid derivative (compound 22) is as follows
Oleic acid (1.4 g,5.0 mmol) was dissolved in DCM, oxalyl chloride (1.3 g,10.0 mmol) was added and DMF (1 drop) was added and stirred 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 and Et was added 3 N (204 mg,2.0 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and crude products are obtained through direct concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 22 as a yellow oil (98.5 mg, 9.8%). 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 )。
Example 14: the synthetic route of ursodeoxycholic acid derivative (compound 23) is as follows
Stearic acid (1.4 g,5.0 mmol) was dissolved in DCM, oxalyl chloride (1.3 g,10 mmol) was added and DMF (1 drop) was added and stirred 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 and Et was added 3 N (204 mg,2.0 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, crude products are directly obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) afforded 23 as a pale yellow oil (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 )。
Example 15: the synthetic route of ursodeoxycholic acid derivative (compound 24) is as follows
Oleic acid (2.2 g,7.7 mmol) was dissolved in DCM, oxalyl chloride (4.9 g,38.7 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (500 mg,1.0 mmol) was dissolved in DCM and Et was added 3 N (196 mg,2.0 mmol) was added to the acid chloride prepared above, and the reaction was stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 24 as a yellow oil (247.3 mg, 23.6%). 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 )。
Example 16: the synthetic route of ursodeoxycholic acid derivative (compound 25) is as follows
Stearic acid (2.2 g,7.7 mmol) was dissolved in DCM, oxalyl chloride (4.9 g,38.7 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (500 mg,1.0 mmol) was dissolved in DCM and Et was added 3 N (196 mg,2.0 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 25 as a yellow oil (323.8 mg, 30.9%). 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 )。
Example 17: the synthetic route of ursodeoxycholic acid derivative (compound 26) is as follows
Cinnamic acid (1.29 g,8.7 mmol) was dissolved in DCM, oxalyl chloride (11.1 g,87 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (450 mg,0.9 mmol) was dissolved in DCM and Et was added 3 N (180 mg,1.8 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 26 as a yellow oil (116 mg, 16.6%). 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 )。
Example 18: the synthetic route of ursodeoxycholic acid derivative (compound 27) is as follows
2-octyl decanoic acid (0.9 g,3.1 mmol) was dissolved in DCM, oxalyl chloride (2.0 g,16 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (200 mg,0.4 mmol) was dissolved in DCM and Et was added 3 N (78 mg,0.8 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and the reaction liquid is concentrated to obtain crude products. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 27 as a yellow oil (54.1 mg, 12.9%). 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％。
Example 19: the synthetic route of ursodeoxycholic acid derivative (compound 28) is as follows
N-octanoic acid (0.4 g,3.1 mmol) was dissolved in DCM, oxalyl chloride (2.0 g,16 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (200 mg,0.4 mmol) was dissolved in DCM and Et was added 3 N (78 mg,0.8 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 28 as a yellow oil (58.7 mg, 19%). 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％。
Example 20: the synthetic route of ursodeoxycholic acid derivative (compound 29) is as follows
Linoleic acid (1.72 g,6.12 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (0.78 g,6.12 mmol) was added and stirred at room temperature for 5 hours. TLC detection, reaction was concentrated directly to dryness. Intermediate 37-1 (0.50 g,1.02 mmol), triethylamine (3.1 g,30.60 mmol) were dissolved in DCM and the above linoleoyl chloride was added to the reaction solution. Stirring at room temperature for 16 hours. TLC detection, after the reaction is completed, concentrating the solvent to obtain crude product, silica gel column chromatography and DCM/CH 3 OH (20:1) to afford compound 29 as a pale yellow oil (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％。
Example 21: the synthetic route of ursodeoxycholic acid derivative (compound 30) is as follows
Synthesis of intermediate 30-1: lithocholic acid (2.0 g,5.3 mmol) was dissolved in THF, TEA (1.1 g,11 mmol), HBTU (2.4 g,6.4 mmol) and 4-pyrrolidinebutylamine (0.9 g,6.4 mmol) were added sequentially and stirred overnight at room temperature. TLC detection, after the reaction, the reaction solution was concentrated, silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 30-1 (1.9 g, 72%) as a yellow oil. 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 ).
Synthesis of Compound 30: linoleic acid (0.56 g,2.0 mmol) was dissolved in DCM, oxalyl chloride (1.27 g,10 mmol) was added dropwise and DMF (1 drop) was added and stirred 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 and Et was added 3 N (101 mg,1.0 mmol) was slowly added dropwise theretoThe prepared acid chloride in DCM was stirred at room temperature overnight. TLC detection is carried out, and the reaction liquid is concentrated to obtain crude products. Chromatography on silica gel, DCM/CH 3 OH (10:1) to afford compound 30 as a yellow oil (122 mg, 32%). 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％。
Example 22: the synthetic route of ursodeoxycholic acid derivative (compound 31) is as follows
Synthesis of intermediate 31-1: lithocholic acid (2.0 g,5.3 mmol) was dissolved in THF, TEA (1.1 g,11 mmol), HBTU (2.4 g,6.4 mmol) and 3-dimethylaminopropylamine (0.7 g,6.4 mmol) were added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give yellow oily compound 31-1 (1.7 g, 70%). 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 ).
Synthesis of Compound 31: linoleic acid (0.61 g,2.2 mmol) was dissolved in DCM, oxalyl chloride (1.38 g,11 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 5h. The reaction solution was directly concentrated to dryness. Intermediate 31-1 (250 mg,0.5 mmol) was dissolved in DCM and Et was added 3 N (110 mg,1.1 mmol) was added to the acid chloride prepared above, and the reaction was stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give compound 31 as a yellow oil (109 mg, 30%). 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％。
Example 23: the synthetic route of lithocholic acid derivative (compound 32) is as follows
Synthesis of intermediate 32-1: lithocholic acid (1.00 g,2.6 mmol), bromooctadecane (0.97 g,2.9 mmol), potassium carbonate (1.87 g,13.5 mmol) were dissolved in DMSO and stirred at room temperature for 24 hours. TLC detection, diluting the reaction solution with ethyl acetate, washing with water for three times, drying with anhydrous sodium sulfate, filtering, and concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (15:1) to afford compound 32-1 (1.48 g, 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 ).
Synthesis of Compound 32: dimethylaminobutyrate (0.40 g,2.40 mmol) was dissolved in DCM, 1 drop of DMF and oxalyl chloride (0.31 g,2.40 mmol) were added and stirred at room temperature for 4 hours. Directly concentrating to obtain crude acyl chloride. Intermediate 32-1 (0.40 g,0.63 mmol), triethylamine (1.20 g,12.00 mmol) was dissolved in THF, the crude acid chloride was added and stirred at room temperature for 16 hours. TLC detection is carried out, and the reaction liquid is concentrated to obtain crude products. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford 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 ).
Example 24: the synthetic route of ursodeoxycholic acid derivative (compound 33) is as follows
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Palmitic acid (1.0 g,3.8 mmol) was dissolved in DCM, oxalyl chloride (2.5 g,19 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 5h. The reaction solution was directly concentrated to dryness. Compound 2 (250 mg,0.5 mmol) was dissolved in DCM and Et was added 3 N (98 mg,1.0 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. T (T)LC detection, and concentrating the reaction liquid to obtain a crude product after the reaction is finished. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give 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％。
Example 25: the synthesis route of cholesterol derivative (compound 34) is as follows
3- (imidazol-4-yl) propionic acid (0.27 g,1.9 mmol) was dissolved in DCM, oxalyl chloride (0.74 g,5.8 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 5h. The reaction solution was directly concentrated to dryness. Cholesterol (250 mg,0.65 mmol) was dissolved in DCM and Et was added 3 N (131 mg,1.3 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (15:1) eluted, affording 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％。
Example 26: the synthetic route of ursodeoxycholic acid derivative (compound 35) is as follows
Synthesis of intermediate 35-1: lithocholic acid (5.0 g,13 mmol) was dissolved in DMF and K was added sequentially 2 CO 3 (1.8 g,13 mmol) and methyl iodide (1.9 g,13 mmol) were stirred overnight at room temperature. TLC detection, after the reaction was completed, the reaction solution was concentrated, silica gel column chromatography, PE/EA (1:1) eluted, to give 35-1 as a white solid intermediate (3.8 g,75％)。
synthesis of Compound 35: intermediate 35-1 (300 mg,0.77 mmol) was dissolved in DMF and 3- (imidazol-4-yl) propionic acid (129 mg,0.92 mmol), HBTU (433 mg,1.2 mmol) and DIEA (298 mg,2.3 mmol) were added sequentially and stirred at room temperature overnight. TLC was used to check, after the reaction was completed, the reaction solution was concentrated to give crude product, which was chromatographed on silica gel, DCM/MeOH (20:1) eluted to give compound 35 (200 mg, 51%) as a white solid. 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 ).
Example 27: the synthetic route for cholic acid derivative (Compound 36) is as follows
Synthesis of intermediate 36-1: cholic acid (2.00 g,4.89 mmol), methyl iodide (0.73 g,5.13 mmol), potassium carbonate (1.01 g,7.34 mmol) were dissolved in DMF and stirred at room temperature for 16 hours. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) afforded intermediate 36-1 (1.75 g, 84%) as a white solid. 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％。
Synthesis of intermediate 36-2: intermediate 36-1 (1.20 g,2.84 mmol), 4-dimethylaminobutyrate (0.48 g,2.84 mmol), HBTU (1.62 g,4.26 mmol) were dissolved in DMF and DIPEA (1.6 mL,8.52 mmol) was added and stirred at room temperature for 16 h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) afforded intermediate 36-2 (0.82 g, 54%) as a white solid. 1HNMR (400 MHz, 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.
Synthesis of Compound 36: stearic acid (0.77 g,3.36 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (2.13 g,16.80 mmol) was added and stirred at room temperature for 4 hours. And detecting by TLC, and concentrating the reaction liquid after the reaction is finished to remove the solvent to obtain a crude product of the acyl chloride. Intermediate 36-2 (0.30 g,0.56 mmol) was dissolved in DCM and sodium hydride (0.23 g,5.60 mmol) was added and stirred for 20 min. The above stearoyl chloride was added and stirred at room temperature for 16 hours. TLC detection is carried out, after the reaction is finished, a proper amount of water is added for quenching, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (25:1) to afford compound 36 as a pale yellow oil (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).
Example 28: the synthetic route of ursodeoxycholic acid derivative (compound 37) is as follows
Synthesis of intermediate 37-1: ursodeoxycholic acid (2.00 g,5.1 mmol), dimethylaminobutanol (0.72 g,6.1 mmol), EDCI (1.17 g,6.1 mmol), DIPEA (1.98 g,15.3 mmol), DMAP (0.32 g,2.6 mmol) were dissolved in DMF and stirred at room temperature for 16 hours. TLC detects that the raw materials are reacted completely, and the solvent is concentrated to obtain crude products. Silica gel column chromatography, DCM/CH 3 OH (15:1) afforded intermediate 37-1 (1.08 g, 43%) as a white solid. ESI-MS m/z Calc.C 30 H 53 NO 4 [M+H] + 491.76,Found 492.15.
Synthesis of Compound 37 linoleic acid (1.72 g,6.12 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (0.78 g,6.12 mmol) was added and stirred at room temperature for 4 hours. And detecting by TLC, and concentrating the reaction liquid after the reaction is finished to remove the solvent to obtain a crude product of the acyl chloride. Intermediate 37-1 (0.50 g,1.02 mmol) was dissolved in DCM and sodium hydride (0.41 g,10.20 mmol) was added and stirred for 20 min. Then adding the oleoyl chloride and stirring for 16 hours at room temperature. TLC detection, adding proper amount of water for quenching after the reaction is finished, concentrating the solvent to obtain crude productThe product is obtained. Silica gel column chromatography, DCM/CH 3 OH (20:1) to afford compound 37 as a pale yellow oil (160 mg, 18%). 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。
Example 29: the synthetic route of ursodeoxycholic acid derivative (compound 38) is as follows
2-octyl decanoic acid (1.4 g,4.9 mmol) was dissolved in DCM, oxalyl chloride (3.1 g,24 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 4h. The reaction solution was directly concentrated. Intermediate 37-1 (300 mg,0.6 mmol) was dissolved in DCM and Et was added 3 N (124 mg,1.2 mmol) was added to the acid chloride prepared above and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give 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％。
Example 30: the synthetic route of ursodeoxycholic acid derivative (compound 39) is as follows
2-hexyl decanoic acid (0.7 g,2.9 mmol) was dissolved in DCM, DMF (1 drop) was added, oxalyl chloride (1.8 g,15 mmol) was added and stirred at room temperature for 4h. The reaction solution was directly concentrated. Compound 2 (250 mg,0.5 mmol) was dissolved in DCM and Et was added 3 N (98 mg,1.0 mmol) was added to the preparationIs stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give 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％。
Example 31: the synthetic route of ursodeoxycholic acid derivative (compound 40) is as follows
Synthesis of intermediate 40-1: the compound 3-amino-1, 2-propanediol (5.00 g,54.88 mmol) was dissolved in ethanol and Boc anhydride (13.18 g,60.37 mmol) was added and stirred at room temperature for 20h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is directly concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (100:1-19:1) to afford intermediate 40-1 (9.80 g, 93.3%) as a colourless oil. 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)。
Synthesis of intermediate 40-2: intermediate 40-1 (1.60 g,8.37 mmol), stearic acid (5.00 g,17.57 mmol), EDCI (10.00 g,52.16 mmol), DMAP (0.50 g,4.09 mmol) were dissolved in DCM and stirred at room temperature for 16h. After completion of the reaction, TLC was used to concentrate the mixture to give intermediate 40-2 (5.31 g, 87%) as a white, viscous liquid. The crude product is directly put into the next step.
Synthesis of intermediate 40-3: intermediate 40-2 (3.20 g,4.41 mmol) was dissolved in DCM and TFA was added and stirred at room temperature for 7h. The reaction solution was concentrated, and the obtained crude intermediate 40-3 was directly used in one step.
Synthesis of intermediate 40-4: lithocholic acid (500 mg,1.33 mmol), intermediate 40-3 (1.17 g,1.59 mmol), HATU (0.76 g,2.00 mmol), DIPEA (0.74 mL,4.00 mmol) were dissolved in DMF and stirred at room temperature for 20h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 Eluting with OH (50:1) to obtain white solidIntermediate 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 )。
Synthesis of Compound 40: intermediate 40-4 (300 mg,0.31 mmol), 4-N, N-dimethylbutyrate (102 mg,0.61 mmol), py-BOP (470 mg,0.91 mmol), TEA (0.22 mL,1.52 mmol) were dissolved in DMF and stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) to afford compound 40 (90 mg, 26.1%) as an off-white solid. 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 )。
Example 32: the synthetic route of ursodeoxycholic acid derivative (compound 41) is as follows
The compound 2-hexyldecanoic acid (1.22 g,4.79 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (2 mL,23.63 mmol) was added and the reaction was stirred for 3h. TLC detection was performed, and after completion of the reaction, the reaction mixture was directly concentrated. Intermediate 37-1 (400 mg,0.81 mmol) was dissolved in DCM and sodium hydride (320 mg,8.10 mmol) was added and stirred for 30 min. The acid chloride was added and stirred at room temperature for 20 hours. TLC detection is carried out, after the reaction is finished, a proper amount of water is added, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) to give compound 41 (120 mg, 20.5%) as a pale 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％。
Example 33: the synthetic route of ursodeoxycholic acid derivative (compound 42) is as follows
The compound 2-hexyldecanoic acid (1.23 g,4.79 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (2 mL,23.63 mmol) was added and the reaction was stirred for 3h. TLC detection was performed, and after completion of the reaction, the reaction mixture was directly concentrated to dryness. Intermediate 37-1 (400 mg,0.81 mmol), (320 mg,8.10 mmol) was dissolved in DCM, the above acid chloride was added and stirred at room temperature for 20h. TLC detection and concentration of the solvent gave crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) to give compound 42 (40 mg, 5.2%) as a pale 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％。
Example 34: the synthetic route of ursodeoxycholic acid derivative (compound 43) is as follows
Synthesis of intermediate 43-1: ursodeoxycholic acid (786 mg,2.0 mmol) was dissolved in DMF, HBTU (950 mg,2.5 mmol), DIEA (400 mg,3.0 mmol), 4-dimethylaminobutylamine (290 mg,2.5 mmol) were added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 43-1 (730 mg, 75%) as a white solid.
2-octyl decanoic acid (0.9 g,3.1 mmol) was dissolved in DCM, DMF (1 drop) was added dropwise oxalyl chloride (1.9 g,15 mmol) and stirred at room temperature for 4h. The reaction solution was concentrated. Intermediate 43-1 (250 mg,0.5 mmol) was dissolved in DCM and Et was added 3 N (103 mg,1.0 mmol) was added to the solution, and the mixture was stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) elution,yellow oily compound 43 (115 mg, 23%) was obtained. 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 x2),2.33-2.21(m,3H),2.12-2.05(m,2H),1.01(s,3H,CH 3 ),0.98-0.97(d,3H,CH 3 ),0.92-0.88(t,12H,CH 3 x4),0.73(s,3H,CH 3 ).HPLC:93.53％。
Example 35: the synthetic route of ursodeoxycholic acid derivative (compound 44) is as follows
2-hexyl decanoic acid (0.8 g,3.1 mmol) was dissolved in DCM, oxalyl chloride (1.9 g,15 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 4h. The reaction solution was concentrated. Intermediate 43-1 (250 mg,0.5 mmol) was dissolved in DCM and Et was added 3 N (103 mg,1.0 mmol) was added to the solution, and the mixture was stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 44 as a yellow oil (116 mg, 24%). 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％。
Example 36: the synthetic route of ursodeoxycholic acid derivative (compound 45) is as follows
Synthesis of intermediate 45-1: obeticholic acid (840 mg,2.0 mmol) was dissolved in DMF, HBTU (948 mg,2.5 mmol), DIEA (400 mg,3.0 mmol), 4-dimethylaminobutylamine (290 mg,2.5 mmol) was added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 45-1 (746 mg, 72%) as a white solid. 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 )。
Synthesis of Compound 45: 2-octyl decanoic acid (0.7 g,2.9 mmol) was dissolved in DCM, oxalyl chloride (1.8 g,15 mmol) 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 and Et was added 3 N (98 mg,1.0 mmol) was added to the above-prepared acid chloride, and stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 45 as a yellow oil (60 mg, 12%). 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.
Example 37: the synthetic route of ursodeoxycholic acid derivative (compound 46) is as follows
Synthesis of intermediate 46-1: ursodeoxycholic acid (3.92 g,10.0 mmol) was dissolved in DMF, HBTU (4.56 g,12.0 mmol), DIEA (3.87 g,30.0 mmol) and 4-pyrrolidine-1-butanol (1.72 g,12.0 mmol) were added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 46-1 (3.9 g, 75%) as a white solid.
Synthesis of Compound 46: 2-hexyl decanoic acid (0.7 g,2.9 mmol) was dissolved in DCM, oxalyl chloride (1.8 g,14 mmol) was added and DMF (1 drop) was added and stirred at room temperature for 4h. The reaction solution was directly concentrated to dryness. Intermediate 46-1 (250 mg,0.5 mmol) was dissolved in DCM and Et was added 3 N (97 mg,1.0 mmol) and then the acid chloride prepared above were added and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford 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％。
Example 38: the synthetic route of ursodeoxycholic acid derivative (compound 47) is as follows
Synthesis of intermediate 47-1: cholic acid (2.00 g,5.1 mmol) was dissolved in DMF, HATU (2.89 g,7.6 mmol), DIEA (3.8 mL,20.4 mmol) and 4-tetrahydropyrrole butylamine (0.87 g,6.1 mmol) were added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) afforded intermediate 47-1 (1.65 g, 60%) as a yellow solid. 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 ).
Synthesis of Compound 47: intermediate 47-1 (200 mg,0.37 mmol), TEA (0.8 mL,5.62 mmol) was dissolved in DCM, 2-hexyldecanoyl chloride (1031 mg,0.37 mmol) was added to the reaction and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (30:1) to afford compound 47 (150 mg, 39.7%) as a pale 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％。
Example 39: the synthetic route of ursodeoxycholic acid derivative (compound 48) is as follows
Synthesis of intermediate 48-1: intermediate 37-1 (400 mg,0.8 mmol), naH (320 mg,8.0 mmol) was dissolved in DCM, and freshly prepared 2-hexyldecanoyl chloride (1319 mg,4.8 mmol) was added to the reaction solution and stirred overnight at room temperature. TLC detection is carried out, after the reaction is finished, a proper amount of water is added, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) afforded intermediate 48-1 (152 mg, 26%) as a pale yellow oil. 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 )。
Synthesis of Compound 48: intermediate 48-1 (120 mg,0.16 mmol), naH (131 mg,3.28 mmol) was dissolved in DCM, and the now-prepared stearoyl chloride (530 mg,1.75 mmol) was added to the reaction solution and stirred overnight at room temperature. TLC detection is carried out, after the reaction is finished, a proper amount of water is added, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (30:1) to afford 48 (40 mg, 24.5%) as a colourless 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％。
Example 40: the synthetic route of ursodeoxycholic acid derivative (compound 49) is as follows
Synthesis of intermediate 49-1: compound 2 (300 mg,0.58 mmol), TEA (0.8 mL,5.62 mmol) was dissolved in DCM, and freshly prepared 2-hexyldecanoyl chloride (956 mg,3.48 mmol) was added to the reaction solution and stirred at room temperature overnight. TLC detection is carried out, after the reaction is finished, a proper amount of water is added, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (30:1) afforded intermediate 49-1 (155 mg, 35%) as a pale yellow oil. 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 )。
Synthesis of Compound 49: intermediate 49-1 (110 mg,0.14 mmol), naH (117 mg,2.92 mmol) was dissolved in DCM, and freshly prepared lauroyl chloride (319 mg,1.46 mmol) was added to the reaction solution and stirred overnight at room temperature. TLC detection is carried out, after the reaction is finished, a proper amount of water is added, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (30:1) to afford compound 49 (23 mg, 16.8%) as a pale 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％。
Example 41: the synthetic route of ursodeoxycholic acid derivative (compound 50) is as follows
2-octyl decanoic acid (0.82 g,2.89 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (1.83 g,14.4 mmol) was added, the reaction was carried out at room temperature for 4 hours, the reaction solution was concentrated directly, the crude acid chloride was added to ursodeoxycholic acidIn a reaction flask of intermediate 46-1 (250 mg,0.48 mmol), triethylamine (0.96 g,9.64 mmol) and DCM, stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 50 (152 mg, 30.4%) as a pale 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％。
Example 42: the route for the synthesis of the obeticholic acid derivative (compound 51) is as follows
2-octyl decanoic acid (0.82 g,2.89 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (1.83 g,14.4 mmol) was added, the reaction was continued at room temperature for 4 hours, the reaction solution was concentrated 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. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 51 as a pale yellow oil (60 mg, 12%). 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％。
Example 43: the synthetic route of chenodeoxycholic acid derivative (compound 52) is as follows
Synthesis of intermediate 52-1: chenodeoxycholic acid (3.92 g,10.0 mmol) was dissolved in DMF, TEA (3.1 g,30 mmol), HBTU (4.55 g,12.0 mmol) and 4-pyrrolidinebutylamine (1.7 g,12.0 mmol) were added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 52-1 (4.0 g, 78%) as a yellow solid.
Synthesis of compound 52: 2-octyl decanoic acid (0.82 g,2.89 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (1.83 g,14.4 mmol) was added, the reaction was continued at room temperature for 4 hours, the reaction solution was concentrated directly, the crude acid chloride was added to a reaction flask containing intermediate 52-1 (250 mg,0.48 mmol), triethylamine (0.96 g,9.64 mmol) and DCM, and stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 52 (118 mg, 23.6%) as a pale 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％。
Example 44: the synthetic route of ursodeoxycholic acid derivative (compound 53) is as follows
Compound 2 (100 mg,0.19 mmol) was dissolved in THF, naH (46 mg,1.15 mmol) was added, and stirred at room temperature for 20 minutes, and iodooctane (186 mg,0.77 mmol) was added to the above reaction solution. Stir at room temperature overnight. TLC detection is carried out, after the reaction is finished, a proper amount of water is added, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (15:1) to afford compound 53 as a white solid (30 mg, 18.2%). 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％。
Example 45: the route for the synthesis of the obeticholic acid derivative (compound 54) is as follows
2-octyl decanoic acid (0.82 g,2.89 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (1.83 g,14.4 mmol) was added, the reaction was continued at room temperature for 4 hours, the reaction solution was concentrated 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. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (30:1) to afford compound 54 as a pale yellow oil (65 mg, 13%). 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％。
Example 46: the route for the synthesis of the obeticholic acid derivative (compound 55) is as follows
Synthesis of intermediate 55-1: obeticholic acid (1.5 g,3.57 mmol) was dissolved in DMF, EDCI (1.0 g,5.35 mmol), DIEA (1.38 g,10.7 mmol), DMAP (0.22 g, 1.78 mmol), alcohol (0.56 g, 3.9 mmol) was added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) afforded intermediate 55-1 (1.0 g, 52.5%) as a white solid.
Synthesis of Compound 55: compound 2-octyl decanoic acid (62 5mg,2.2 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (1.4 g,11.0 mmol) was added and the reaction was stirred for 4h. TLC detection, reaction After completion, the oxalyl chloride was removed by concentration. Intermediate 55-1 (200 mg,0.37 mmol) was dissolved in DCM, and the acid chloride prepared above was added and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give compound 55 (130 mg, 33%) as a pale 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％。
Example 47: the route for the synthesis of the obeticholic acid derivative (compound 56) is as follows
Synthesis of intermediate 56-1: obeticholic acid (1.7 g,4.04 mmol) was dissolved in DMF, EDCI (1.17 g,6.06 mmol), DIEA (1.58 g,12.12 mmol), DMAP (0.25 g,2.02 mmol) was added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) afforded intermediate 56-1 (1.5 g, 71.5%) as a white solid. 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 )。
Synthesis of Compound 56: intermediate 56-1 (230 mg,0.44 mmol) was dissolved in DCM and the now prepared acid chloride was added and stirred overnight at room temperature. TLC detection and concentration of the solvent gave crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give compound 56 (130 mg, 28.8%) as a pale 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％。
Example 48: the synthetic route of ursodeoxycholic acid derivative (compound 57) is as follows
Synthesis of intermediate 57-1: ursodeoxycholic acid (1.5 g,3.82 mmol), HBTU (2.17 g,5.73 mmol), DIEA (1.48 g,11.46 mmol), amine (0.66 g,4.2 mmol) and DMF were added sequentially to the reaction flask and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the crude product is obtained by concentration. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 57-1 (1.5 g, 71.5%) as a white solid. 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).
Intermediate 57-1 (250 mg,0.47 mmol) was dissolved in DCM and the now prepared acid chloride was added and stirred overnight at room temperature. TLC detection and concentration of the solvent gave crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 57 (180 mg, 36.0%) as a pale 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.
Example 49: the synthetic route for lithocholic acid derivative (compound 58) is as follows
Synthesis of intermediate 58-1: to the reaction flask was added lithocholic acid (10 g,2.66 mmol), benzyl bromide (5.9 g,3.45 mmol), potassium carbonate (5.5 g,3.98 mmol) and acetonitrile in sequence. The temperature was raised to 40℃and the reaction was allowed to proceed overnight. TLC showed the reaction was complete. The reaction solution was filtered, the filtrate was concentrated and dried, chromatographed on silica gel, DCM/CH 3 OH (20:1) to give 58-1 (10 g, 80%) as an off-white solid. 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％。
Synthesis of intermediate 58-2: intermediate 58-1 (450 mg,0.96 mmol) was dissolved in pyridine, myristoyl chloride (1 mL) was added to the reaction solution, and the reaction was stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE: DCM (3:1-3:2) eluting to give 58-2 (230 mg, 35.4%) as a white solid 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％。
Synthesis of intermediate 58-3: intermediate 58-2 (220 mg,0.33 mmol), pd/C (30 mg) and methanol were added to the flask and stirred under hydrogen at 25℃for 1h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/MeOH (50:1-30:1) eluted to give 58-3 (160 mg, 84.6%) as a white solid. 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％。
Synthesis of Compound 58: intermediate 58-3 (80 mg,0.14 mmol), mPEG2000-NH 2 (349mg, 0.17 mmol), HBTU (70 mg,0.18 mmol), DIPEA (46 mg,0.35 mmol) was dissolved in DMF and stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (15:1) to give 58 as a white waxy solid (210 mg, 27.8%). 1 HNMR(400MHz,CDCl 3 )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-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％。
EXAMPLE 50 the synthetic route for ursodeoxycholic acid derivative (Compound 59) is as follows
Synthesis of intermediate 59-2: methylamine hydrochloride (6755 mg,10 mmol), intermediate 59-1 (4.26 g,30 mmol), TEA (2.0 g,20 mmol) and methanol were added sequentially to the reaction flask and stirred at room temperature for 16h. TLC detection, directly concentrating and drying the reaction solution to obtain a crude product, silica gel column chromatography and DCM/CH 3 OH (30:1) to afford intermediate 59-2 (1.0 g, 71%) as a yellow solid. 1 HNMR(400MHz,CD 3 OD,)δ4.37-4.39(d,3H),3.31-3.25(d,3H)。
Synthesis of intermediate 59-4: intermediate 59-2 (1.0 g,7.08 mmol), intermediate 59-3 (2.4 g,14.1 mmol), TEA (1.4 g,14.1 mmol) and methanol were added sequentially to the reaction flask and stirred at room temperature for 16h. TLC detection. The reaction solution is directly concentrated to obtain crude product, silica gel column chromatography and DCM/CH 3 OH (20:1) to afford intermediate 59-4 (1.05 g, 52.5%) as a white solid. 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)。
Synthesis of intermediate 59-5: intermediate 59-4 (210 mg,0.74 mmol) was dissolved in dichloromethane, then 0.1mL trifluoroacetic acid was added, stirred at room temperature for 16h, TLC detection and reaction solution concentrated to dryness to give crude intermediate 59-5 (150 mg, 110%) which was used directly in the next step.
Synthesis of intermediate 59-6: 2-hexyl decanoic acid (6.4 g,25 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (6.4 g,50 mmol) was added dropwise and stirred at room temperature for 2h. Directly concentrating to obtain crude acyl chloride. Compound 6 (2.4 g,5.0 mmol), TEA (1.5 g,15.0 mmol) was dissolved in DCM, and the above acid chloride was added to the reaction solution and reacted overnight at room temperature. TLC detection and concentration of the solvent gave crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) to afford intermediate 59-6 (2.0 g, 42%) as a pale yellow oil.
Synthesis of intermediate 59-7: a single neck flask was charged with intermediate 59-6 (2.0 g,2.08 mmol), pd/C (100 mg,5 wt%) and methanol in H 2 The reaction was carried out at room temperature for 16h under an atmosphere. TLC showed complete reaction of starting material. The reaction solution was filtered, concentrated and chromatographed on silica gel column. DCM/CH 3 OH (10:1) afforded intermediate 59-7 (1.43 g, 79%) as a colourless oil. 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％。
Synthesis of compound 59: intermediate 59-7 (200 mg,0.223 mmol), HATU (85 mg,0.268 mmol), DIEA (90 mg,0.690 mmol), intermediate 59-5 (49 mg,0.268 mmol) and DMF were added sequentially to the reaction flask and stirred at room temperature overnight. TLC detection and concentration of the solvent gave crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 59 (45.8 mg, 22.1%) as a white solid. 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％。
Example 51: the synthetic route of ursodeoxycholic acid derivative (compound 60) is as follows
Synthesis of intermediate 60-2: intermediate 59-7 (200 mg,0.22 mmol), intermediate 60-1 (134 mg,0.27 mmol), HATU (126 mg,0.33 mmol), TEA (68 mg,0.66 mmol) were dissolved in DMF and stirred at room temperature for 20h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (50:1) afforded intermediate 60-2 (140 mg, 46.6%) as a pale yellow oil. 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％。
Synthesis of Compound 60: intermediate 60-2 (100 mg,0.07 mmol) was dissolved in ethyl acetate solution of hydrogen chloride and stirred at room temperature for 16h. After the TLC detection, the reaction solution is concentrated, added with dichloromethane for dissolution, washed by saturated sodium carbonate aqueous solution, dried by anhydrous sodium sulfate, filtered and concentrated. Purification by reverse phase HPLC to give a white waxy solidCompound 60 (160 mg, 19.3%). 1 HNMR(400MHz,CDCl 3 )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,CH 3 )，0.92(d,3H,CH 3 ),0.89-0.85(m,12H),0.68(s,3H,CH 3 ).HPLC：82.0％。
Example 52: the synthetic route of ursodeoxycholic acid derivative (compound 61) is as follows
Synthesis of intermediate 61-2: intermediate 61-1 (5.0 g,56.2 mmol), TEA (17 g,168.6 mmol) and DCM were added separately to the flask and TBSCl (12.7 g,84.3 mmol) was added and reacted at room temperature for 16 hours. TLC detection, concentration to obtain crude product, silica gel column chromatography, DCM/CH 3 OH (10:1) afforded intermediate 61-2 (9.3 g, 82%) as a yellow oil. 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)。
Synthesis of intermediate 61-3: ursodeoxycholic acid (5.0 g,12.7 mmol) was dissolved in DMF and HBTU (5.8 g,15.2 mmol), DIEA (4.9 g,38.1 mmol) and intermediate 61-2 (3.1 g,15.2 mmol) were added sequentially and stirred overnight at room temperature. TLC detection and concentration of the solvent gave crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 61-3 (5.5 g, 75%) as a white solid. 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)。
Synthesis of intermediate 61-5: intermediate 61-3 (1.15 g,2.0 mmol) was dissolved in DMF, naH (240 mg,6.0 mmol) was added and stirred for half an hour at room temperature, intermediate 61-4 (2.0 g,6.0 mmol) was added and the reaction was stirred at room temperature overnight. TLC detection, adding proper amount of water for quenching, and concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 61-5 (756 mg, 35%) as a white solid. 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)。
Synthesis of intermediate 61-6: intermediate 61-5 (720 mg,0.665 mmol) was dissolved in THF and TBAF-3H was added 2 O (631 mg,2.0 mmol) was stirred at room temperature for 16 hours. TLC detection, adding a proper amount of water, and concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) afforded intermediate 61-6 (142 mg, 72%) as a colourless oil. 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)。
Synthesis of intermediate 61-7: intermediate 61-6 (130 mg,0.134 mmol) was dissolved in DCM and Dess-Martin reagent (85 mg,0.2 mmol) was added and stirred at room temperature for 16 hours. TLC detection, adding a proper amount of water, and concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (30:1) afforded intermediate 61-7 (87 mg, 67%) as a colourless oil. 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)。
Synthesis of Compound 61: intermediate 61-7 (87 mg,0.09 mmol), intermediate 61-8 (13 mg,0.18 mmol) and DCM were added sequentially to a reaction flask, one drop of acetic acid was added, and after stirring at room temperature for 1 hour, naHB (OAc) was added 3 (29 mg,0.145 mmol) and allowed to react overnight at room temperature. TLC detection, adding a proper amount of water, and concentrating the solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (15:1) to afford compound 61 (31 mg, 33%) as a colourless 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％。
Example 53: the synthetic route of ursodeoxycholic acid derivative (compound 62) is as follows
Intermediate 59-7 (100 mg,0.115 mmol) was dissolved in ACN, starting material 62-1 (24 mg,0.138 mmol), NMI (24 mg,0.288 mmol), TCFH (39 mg,0.138 mmol) was added sequentially and the reaction was stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give compound 62 (55 mg, 47%) as a pale 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％。
Example 54: the synthetic route of ursodeoxycholic acid derivative (compound 63) is as follows
Intermediate 59-7 (220 mg, 0.255 mmol) was dissolved in ACN, diol (20 mg,0.115 mmol), NMI (47 mg, 0.514 mmol), TCFH (71 mg, 0.255 mmol) was added sequentially and the reaction stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give compound 63 (135 mg, 63%) as a pale 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)。
Example 55: the synthetic route of ursodeoxycholic acid derivative (compound 64) is as follows
Intermediate 59-7 (100 mg,0.115 mmol), HBTU (52 mg,0.14 m)mol), DMF (3 mL), DIEA (44 mg,0.34 mmol), amine (70 mg,0.6 mmol) and DMF were added sequentially to the reaction flask and stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to give 64 (10 mg) as a pale 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％。
Wherein 59-7 is a carboxylic acid, an intermediate in the preparation of compound 59.
EXAMPLE 56 the synthetic route for ursodeoxycholic acid derivative (Compound 65) is as follows
To a single neck flask was added sequentially 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. Stirring for 16h at room temperature. TLC detection, adding proper amount of water into the reaction solution, concentrating, performing silica gel column chromatography, and DCM/CH 3 OH (10:1) to afford 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)。
EXAMPLE 57 the synthetic route for ursodeoxycholic acid derivative (Compound 66) is as follows
Synthesis of intermediate 66-1: ursodeoxycholic acid (4.00 g,10.00 mmol), TFAA (11.55 g,55.00 mmol) and THF were added sequentially to the flask, tert-butanol (23.25 g,314.00 mmol) was added and stirred at room temperature for 20h. TLC detection reaction, concentration Condensing solvent to obtain crude product. Silica gel column chromatography, DCM/MeOH (20:1) eluting gave intermediate 66-1 (4.30 g, 95.8%) as a white, foamed solid. 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％。
Synthesis of intermediate 66-2: linoleic acid (23.11 g,82.40 mmol) was dissolved in DCM, 1 drop of DMF was added, oxalyl chloride (20.92 g,164.80 mmol) was added and stirred at room temperature for 4h. Directly concentrating to obtain crude acyl chloride. Intermediate 66-1 (3.70 g,8.24 mmol) was dissolved in pyridine, and the acid chloride was added thereto and stirred at room temperature for 20 hours. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (1:1) eluted, afforded intermediate 66-2 (2.51 g, 31.1%) as a colourless oil. 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％。
Synthesis of intermediate 66-3: intermediate 66-2 (2.50 g,2.57 mmol) was dissolved in DCM and TFA (2.5 mL) was added and stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the solvent is directly distilled off from the reaction liquid to obtain a crude product. Silica gel column chromatography, DCM/MeOH (20:1) eluting gave intermediate 66-3 (1.79 g, 75.8%) as a colourless oil. 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％。
Synthesis of compound 66: intermediate 66-3 (250 mg,0.27 mmol), 4-pyrrolidin-1-yl-butan-1-ol (58 mg,0.41 mmol), EDCI (68 mg,0.35 mmol), DIPEA (105 mg,0.82 mmol) were dissolved in DMF and stirred at room temperature for 20h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 66 (170 mg, 59.8%) as a colourless 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。
Example 58: the synthetic route of ursodeoxycholic acid derivative (compound 67) is as follows
Intermediate 66-3 (100 mg,0.11 mmol), 3-dimethylamino-1 propanol (17 mg,0.16 mmol), EDCI (27 mg,0.14 mmol), DIPEA (42 mg,0.33 mmol) were dissolved in DMF and stirred at room temperature for 20h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 67 (73 mg, 67.0%) as a colourless 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。
Example 59: the synthetic route of ursodeoxycholic acid derivative (compound 68) is as follows
Synthesis of Compound 68: intermediate 66-3 (200 mg,0.22 mmol), N, N-dimethylethanolamine (29 mg,0.33 mmol), EDCI (54 mg,0.28 mmol), DMAP (41 mg,0.33 mmol) were dissolved in DMF and stirred at room temperature for 20h. After the TLC detection reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 68 (96 mg, 44.6%) as a colourless 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。
Example 60: the synthetic route of chenodeoxycholic acid derivative (compound 69) is as follows
Synthesis of intermediate 69-1: chenodeoxycholic acid (3.0 g, 7.64) is added into a single-neck flask in sequence
mmol)，K 2 CO 3 (1.6 g 11.46 mmol), KI (130 mg,0.77 mmol), benzyl bromide
(1.5 g,9.17 mmol) and acetonitrile, then the reaction solution was warmed to 40℃and stirred overnight.
TLC showed complete reaction of starting material. The reaction solution was concentrated, chromatographed on silica gel, DCM/CH 3 OH (10:1) to afford intermediate 69-1 (1.9 g, 52.8%) as a white solid. 1H NMR (400 MHz, 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 69-3: 2-octyl decanoic acid (4.25 g,16.6 mmol) was dissolved in DCM, DMF (1 drop) and oxalyl chloride (10.5 g 82.9 mmol) were added and stirred at room temperature for 4h. The reaction solution is directly concentrated to remove the solvent to obtain the crude product of the acyl chloride. The acid chloride was added to a reaction flask containing intermediate 69-1 (1.0 m,2.07 mmol) and pyridine and stirred at room temperature for 16h. TLC showed complete reaction of starting material. Concentrating, and performing silica gel column chromatography. DCM/CH 3 OH (20:1) afforded intermediate 69-3 (530 mg, 27.5%) as a colourless oil. 1H NMR (400 MHz, chloroform-d) delta 7.37-7.30 (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 69-4: to a single neck flask was added sequentially intermediate 69-3 (530 mg,0.552 mmol), meOH, pd/C (27 mg,5 wt%). In H 2 The reaction was carried out at room temperature for 16h under an atmosphere. TLC showed complete reaction of starting material. The reaction solution was filtered, concentrated and chromatographed on silica gel column. DCM/CH 3 OH (20:1) to afford intermediate 69-4 (330 mg, 69%) as a colourless oil. 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％。
Synthesis of Compound 69: to a single neck flask was added successively 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), DIEA (59.5 mg,0.460 mmol) and DMF. The reaction was stirred at room temperature for 16h. TLC showed complete reaction of starting material. The reaction solution was concentrated and chromatographed on silica gel column. DCM/CH 3 OH (10:1) to afford compound 69 (111.8 mg, 61.1%) as a colourless 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％。
Example 61: the synthetic route of chenodeoxycholic acid derivative (compound 70) is as follows
To a single neck flask was added sequentially intermediate 69-4 (160 mg,0.184 mmol), dimethylbutanol (28 mg,0.221 mmol), EDCI (45.9 mg, 0.235 mmol), DIEA (59.5 mg,0.460 mmol) and DMF. The reaction was stirred at room temperature for 16h. TLC showed complete reaction of starting material. Concentrating, and performing silica gel column chromatography. DCM/CH 3 OH (10:1) to afford compound 70 (160.9 mg, 89.9%) as a colourless 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] + 968.86Found967.86。HPLC：95.68％。
Example 62: the synthetic route of chenodeoxycholic acid derivative (compound 71) is as follows
Synthesis of intermediate 71-1: obeticholic acid (2.0 g,4.75 mmol) was dissolved in ACN, and benzyl bromide (1.63 g,9.51 mmol), K was added 2 CO 3 (2.63 g,19.02 mmol) and stirred overnight at 40 ℃. TLC detection is carried out, after the reaction is finished, filtering is carried out, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (3:1) eluted, affording 71-1 (1.7 g, 70%) as a white solid. 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％。
Synthesis of intermediate 71-3: intermediate 71-1 (650 mg,1.27 mmol) was dissolved in pyridine, and then freshly prepared acid chloride 71-2 (2.80 g,10.18 mmol) was added thereto and stirred at room temperature for 3h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (5:1) eluted, gave 71-3 (495mg, 52%) as a colorless oil. 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％。
Synthesis of intermediate 71-4: intermediate 71-3 (399mg, 0.227 mmol) was dissolved in MeOH and 10% Pd/C (59 mg,15% wt) was added and the reaction stirred under hydrogen atmosphere for 16h. TLC detection is carried out, after the reaction is finished, filtering is carried out, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/MeOH (30:1) eluting gave 71-4 as a colorless oil (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％。
Synthesis of Compound 71: intermediate 71-4 (140 mg,0.212 mmol) was dissolved in DMF and 4-pyrrolidin-1-yl-butan-1-ol (46 mg,0.319 mmol), EDCI (89 mg,0.319 mmol) and DMAP (59 mg,0.48 mmol) were added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/MeOH (25:1) gave 71 as a colorless oil (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。
Example 63: the synthetic route of hyodeoxycholic acid derivative (compound 72) is as follows
Synthesis of intermediate 72-1: hyodeoxycholic acid (8.0 g,20.4 g) is sequentially added into a single-neck flask
mmol)，KHCO 3 (4.1 g,40.8 mmol), benzyl bromide (4.2 g,24.5 mmol) and acetonitrile.
The reaction was then warmed to 40 ℃ and stirred overnight. TLC showed complete reaction of starting material. Concentrating the mixture to obtain a concentrated solution,
silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 72-1 (9.1 g, 92%) as a white solid.
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％。
Synthesis of intermediate 72-2: 2-hexyl decanoic acid (11.10 g,43.28 mmol) was dissolved in DCM, 1 drop of DMF and oxalyl chloride (10.99 g,86.57 mmol) were added and stirred at room temperature for 4h. Directly concentrating to obtain crude acyl chloride. Intermediate 72-1 (2.10 g,4.35 mmol) was dissolved in pyridine, the acid chloride was added, and the reaction was continued at room temperatureStirring for 20h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (20:1-5:1) afforded intermediate 72-2 (2.85 g, 68.3%) as a colourless oil. 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％。
Synthesis of intermediate 72-3: intermediate 72-2 (1.20 g,1.25 mmol) Pd/C (100 mg) was dissolved in methanol and stirred under hydrogen atmosphere at 25℃for 16h. TLC detection is carried out, after the reaction is finished, filtering is carried out, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/MeOH (15:1) eluting gave intermediate 72-3 (1.02 g, 93.6%) as a colourless oil. 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％。
Synthesis of compound 72: intermediate 72-3 (300 mg,0.34 mmol), 4-pyrrolidin-1-butyl-1-ol (74 mg,0.52 mmol), EDCI (86 mg,0.45 mmol), DIPEA (134 mg,1.04 mmol) were dissolved in DMF and stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) afforded compound 72 (94 mg, 27.4%) as a colourless 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％。
Example 64: the synthetic route of ursodeoxycholic acid derivative (compound 73) is as follows
Intermediate 72-3 (300 mg,0.34mmol), 4-dimethylamino-1-butanol (61 mg,0.52 mmol), EDCI (86 mg,0.45 mmol), DIPEA (134 mg,1.04 mmol) were dissolved in DMF and stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) to afford compound 73 (175 mg, 52.4%) as a colourless 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％。
Example 65: the synthetic route of ursodeoxycholic acid derivative (compound 74) is as follows
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Synthesis of intermediate 74-1: intermediate 59-2 (1.0 g,7.09 mmol), amine (1.9 g,8.5 mmol), TEA (860 mg,8.50 mmol) and MeOH were added sequentially to the reaction flask. Stirring for 16h at room temperature. TLC detection, direct concentration to remove solvent after reaction, silica gel column chromatography, DCM/CH 3 OH (10:1) to afford intermediate 74-1 (2.5 g, 40%) as an off-white solid. HPLC:99.42%.
Synthesis of intermediate 74-2: intermediate 74-1 (500 mg,1.41 mmol) was dissolved in DCM and the compound HCl-EA (9.5 mL,7.05 mmol) was added. The reaction was carried out at room temperature for 4 hours. After completion of the TLC detection reaction, the reaction was directly concentrated to give off-white solid intermediate 74-2 (400 mg, 97.5%).
Synthesis of Compound 74: intermediate 59-7 (200 mg,0.23 mmol), intermediate 74-2 (82.0 mg,0.28 mmol), EDCI (85 mg,0.44 mmol), DIEA (130 mg,1.00 mmol) and DMF were added sequentially to the reaction flask. The reaction was carried out at room temperature for 16h. TLC detection, after the reaction, the reaction solution is concentrated and subjected to silica gel column chromatography. DCM/CH 3 OH (10:1) to afford 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％。
Example 66: the synthetic route of ursodeoxycholic acid derivative (compound 75) is as follows
Synthesis of intermediate 75-1: linoleic acid (2.90 g,10.36 mmol) was dissolved in DCM, 1 drop of DMF and oxalyl chloride (2.63 g,20.72 mmol) were added and the reaction stirred at room temperature for 4h. TLC detection was performed, and after completion of the reaction, oxalyl chloride was removed by concentration. Compound 6 (0.50 g,1.03 mmol) was dissolved in pyridine, and the acid chloride was added thereto and stirred at room temperature overnight. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (30:1-1:1) afforded intermediate 75-1 (180 mg, 17.3%) as a colourless oil. 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％。
Synthesis of intermediate 75-2: intermediate 75-1 (180 mg,0.12 mmol) Pd/C (20 mg) was dissolved in methanol and stirred under a hydrogen atmosphere at 25℃for 16h. TLC detection is carried out, after the reaction is finished, filtering is carried out, and the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/MeOH (20:1) eluting gave intermediate 75-2 (110 mg, 67.4%) as a colourless oil. 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％。
Synthesis of compound 75: intermediate 75-2 (105 mg,0.11 mmol), 4- (N, N-dimethyl) aminobutanol (16 mg,0.14 mmol), HATU (56 mg,0.15 mmol), DIPEA (37 mg,0.29 mmol) were dissolved in DMF and stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (10:1) washesDe-molding gave 75 (40 mg, 34.4%) as a pale yellow oil. 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％。
Example 67: the route for the synthesis of the obeticholic acid derivative (compound 76) is as follows
To a one-necked flask was successively added obeticholic acid (2.0 g,4.75 mmol), diamine (279 mg,2.38 mmol), HBTU (2.35 g,6.18 mmol), DIEA (1.85 g,14.26 mmol) and THF, followed by stirring overnight at room temperature. TLC showed complete reaction of starting material. The reaction solution was concentrated, chromatographed on silica gel, DCM/CH 3 OH (10:1) to afford compound 76 (800 m g, 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％。
Example 68: the route for the synthesis of the obeticholic acid derivative (compound 77) is as follows
To the reaction flask was added compound 79 (300 mg,0.37 mmol), DMAP (4.5 mg,0.04 mmol), pyridine and acetic anhydride (560 mg,5.49 mmol) in sequence, and stirred overnight at room temperature. TLC showed complete reaction of starting material. The reaction solution was concentrated and chromatographed on silica gel column. DCM/CH 3 OH (20:1) to afford compound 77 (115 mg, 49.1%) as a white solid. 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％。
Example 69: the route for the synthesis of the obeticholic acid derivative (compound 78) is as follows
To the reaction flask was added compound 80 (300 mg,0.37 mmol), DMAP (4.5 mg,0.04 mmol), pyridine and acetic anhydride (560 mg,5.49 mmol) in sequence, and stirred overnight at room temperature. TLC showed complete reaction of starting material. The reaction solution was concentrated, chromatographed on silica gel, DCM/CH 3 OH (20:1) to afford compound 78 as a white solid (115 mg, 49.1%). 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] + 1034.73Found 1034.80.HPLC：96.86％。
Example 70: the route for the synthesis of the obeticholic acid derivative (compound 79) is as follows
Lithocholic acid (2.0 g,5.3 mmol), amine (610 mg,2.66 mmol), HBTU (2.42 g,6.37 mmol), DIEA (1.08 g,10.62 mmol) and THF were added sequentially to the reaction flask and stirred overnight at room temperature. TLC showed complete reaction of starting material. The reaction solution was concentrated, chromatographed on silica gel, DCM/CH 3 OH (10:1) to afford 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] + 834.70Found 834.80.HPLC：98.13％。
Example 71: the synthetic route of ursodeoxycholic acid derivative (compound 80) is as follows
Ursodeoxycholic acid (4.0 g,10.2 mmol), amine (597 mg,5.1 mmol), HBTU (4.63 g,12.21 mmol), DIEA (2.06 g,15.9 mmol) and THF were added sequentially to the reaction flask. Stir at room temperature overnight. TLC showed complete reaction of starting material. The reaction solution was concentrated, chromatographed on silica gel, DCM/CH 3 OH (10:1) to afford compound 80 as a white solid (1.8 g, 40%). 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] + 866.69Found 866.80.HPLC：95.68％。
Example 72: the synthetic route of ursodeoxycholic acid derivative (compound 81) is as follows
Intermediate 59-7 (85 mg,0.1 mmol), 4- (N, N-dimethyl) aminopropanol (16 mg,0.15 mmol), HATU (56 mg,0.15 mmol), DIPEA (37 mg,0.29 mmol) were dissolved in DMF and stirred at room temperature for 16h. TLC detection is carried out, and after the reaction is finished, the reaction liquid is concentrated into a solvent to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) to afford compound 81 (50 mg, 52%) as a pale 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％。
Example 73: the synthetic route of ursodeoxycholic acid derivative (compound 82) is as follows
Synthesis of compound 82: intermediate 66-3 (200 mg,0.22 mmol), 4-pyrrolidine-1-propanol (42 mg,0.33 mmol), EDCI (54 mg,0.28 mmol), DIPEA (85 mg,0.65 mmol) were dissolved in DMF and stirred at room temperature for 20h. After the TLC detection reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) to afford 82 (60 mg, 26.8%) as a colorless oily compound. 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。
Example 74: the synthetic route of ursodeoxycholic acid derivative (compound 83) is as follows
Synthesis of compound 83: intermediate 66-3 (200 mg,0.22 mmol), 4-pyrrolidin-1-ethanol (38 mg,0.33 mmol), EDCI (54 mg,0.28 mmol), DIPEA (85 mg,0.65 mmol) were dissolved in DMF and stirred at room temperature for 20h. After the TLC detection reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) to afford compound 83 (55 mg, 24.9%) as a colourless 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,Found1014.90。
Example 75: the synthetic route of ursodeoxycholic acid derivative (compound 84) is as follows
Compound 66-3 (160 mg,0.17 mmol), 74-2 (76 mg,0.26 mmol), EDCI (43 mg,0.23 mmol), DIPEA (112 mg,0.87 mmol) were dissolved in DMF and stirred at room temperature for 20h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (20:1) afforded 84 as a colorless oil (100 mg, 50.0%). 1 HNMR(400MHz,CDCl 3 )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-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。
Example 76: the synthetic route of ursodeoxycholic acid derivative (compound 85) is as follows
Synthesis of intermediate 85-1: compound 3-bromo-1 propanol (5.0 g,35.72 mmol) and TEA (10.8 g,107.16 mmol) were dissolved in DCM, TBSCl (8.1 g,53.58 mmol) was added and stirred at room temperature for 16 hours. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (10:1) eluted, gave a colorless oil (7.3 g, 81.1%).
Synthesis of intermediate 85-2: intermediate 85-1 (1.6 g,6.38 mmol), diamine (1.0 g,5.31 mmol), potassium carbonate (1.5 g,8.0 mmol) and DMF were added sequentially to the reaction flask and reacted at 40℃for 4 hours. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, EA/PE (3:1) gave a pale yellow oil (1.47 g, 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％。
Synthesis of intermediate 85-3: intermediate 85-2 (1.4 g,3.88 mmol) was dissolved in DCM, 1mL of TFA was added, stirred at room temperature for 5 hours, TLC detection was performed, after the reaction was complete, directly concentrated to dryness, and the crude intermediate 85-3 obtained was directly used in the next step.
Synthesis of intermediate 85-4: intermediate 85-3 (1.5 g,3.88 mmol) was dissolved in methanol, TEA (1.2 g,11.64 mmol) and intermediate 59-2 (660 mg,4.66 mmol) were added and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/MeOH (15:1) eluting gave intermediate 85-4 (495mg, 50.0%) as a white solid. 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。
Synthesis of Compound 85: intermediate 66-3 (60 mg,0.065 mmol) was dissolved in MeCN and the compound TCFH (27.5 mg,0.098 mmol), NMI (16 mg,0.20 mmol) and intermediate 85-4 (21 mg,0.085 mmol) were added. Stirring for 16h at room temperature. TLC detection, concentration after reaction, silica gel column chromatography, DCM/CH 3 OH (10:1) to give 85 as a yellow solid (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。
Example 77: the synthetic route of ursodeoxycholic acid derivative (compound 86) is as follows
Synthesis of intermediate 86-1: intermediate 66-1 (2.00 g,4.46 mmol), 2-hexyldecanoic acid (1.71 g,6.69 mmol), HATU (2.54 g,6.69 mmol)) DIPEA (1.74 g,13.38 mmol) was dissolved in DMF and stirred at room temperature for 20h. TLC detection reaction is complete, and the solvent is concentrated to obtain crude products. Silica gel column chromatography, PE/EA (10:1-1:1) afforded intermediate 86-1 (1.85 g, 60.4%) as a colourless oil. 1 HNMR(400MHz,CDCl 3 )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,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％。
Synthesis of intermediate 86-2: linoleic acid (2.94 g,10.48 mmol) was dissolved in DCM, 1 drop of DMF and oxalyl chloride (2.66 g,20.96 mmol) were added and stirred at room temperature for 4h. Concentrating to obtain crude acyl chloride. Intermediate 86-1 (1.80 g,2.62 mmol) was dissolved in pyridine, and the acid chloride was added thereto and stirred at room temperature for 20 hours. After the TLC detection reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (30:1-1:1) afforded intermediate 86-2 (1.45 g, 58.2%) as a pale yellow oil. 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％。
Synthesis of intermediate 86-3: intermediate 86-2 (1.40 g,1.44 mmol) was dissolved in DCM and TFA (1 mL) was added and stirred at room temperature for 20h. TLC detection is carried out, and after the reaction is finished, the solvent is directly distilled off from the reaction liquid to obtain a crude product. Silica gel column chromatography, DCM elution gave intermediate 86-3 (0.98 g, 76.6%) as a colorless oil. 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％。
Synthesis of Compound 86: intermediate 86-3 (200 mg,0.22 mmol), 4-pyrrolidin-1-yl-butan-1-ol (48 mg,0.34 mmol), EDCI (56 mg,0.29 mmol), DIPEA (87 mg,0.67 mmol) were dissolved in DMF and stirred at room temperature for 20h. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, DCM/CH 3 OH (15:1) elution to give a colourless oilCompound 86 (70 mg, 30.7%). 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,Found1018.90。
Example 78: the synthetic route of ursodeoxycholic acid derivative (compound 87) is as follows
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 sequentially to the reaction flask. Stirring for 16h at room temperature. TLC detection, concentration after reaction, silica gel column chromatography, DCM/CH 3 OH (10:1) to afford 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。
Example 79: the synthetic route of ursodeoxycholic acid derivative (compound 88) is as follows
Synthesis of compound 88: intermediate 86-3 (200 mg,0.22 mmol), TCFH (94 mg,0.34 mmol), NMI (55 mg,0.67 mmol), intermediate 85-4 (75 mg,0.29 mmol) and acetonitrile were added sequentially to the reaction flask. Stirring for 16h at room temperature. TLC detection, concentration after reaction, silica gel column chromatography, DCM/CH 3 Eluting with OH (10:1) to obtain yellow solid compound88(174mg,68.8％)。 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。
Example 80: the synthetic route for the conjugate of ursodeoxycholic acid and paclitaxel (compound 89) is as follows
Intermediate 66-3 (250 mg,0.272 mmol) was dissolved in DMF and paclitaxel (233 mg,0.272 mmol), EDCI (68 mg,0.354 mmol) and DMAP (50.0 mg,0.408 mmol) were added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (10:1) eluted, affording compound 89 (200 mg, 42%) as a white solid. 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％。
Example 081: the synthetic route for the conjugate of ursodeoxycholic acid and small nucleic acid (compound 90) is as follows
Synthesis of intermediate 90-1: ursodeoxycholic acid (1.1 g,2.80 mmol) was dissolved in DCM at room temperature, NHS (387 mg,3.36 mmol), EDCI (640 mg,3.36 mmol) was added sequentially and stirred overnight at room temperature. TLC detection is carried out, and after the reaction is finished, the solvent is concentrated to obtain a crude product. Silica gel column chromatography, PE/EA (10:1) eluted, yielding 90-1 (1.08 g, 79%) as a white solid. 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％。
Synthesis of Compound 90: compound 90-2 (330 ug,22.2 nmol) was dissolved in deionized water (1.5 mL), intermediate 90-1 (109 ug,222 nmol) was added, and stirred overnight at room temperature. Purification by preparative HPLC, concentration gave a pink oil (222 ug, 65%), HPLC:75.20%. ESI-MS M/z [ M+H ]] + ，7410.1/7853.8。
Example 082: the synthetic route for the conjugate of ursodeoxycholic acid and polypeptide (compound 91) is as follows: ursodeoxycholic Acid-WEARLARALARALARHLARALARALRACEA synthesis steps:
compound 91 is a solid phase synthetic polypeptide having 30 amino acids, and the synthetic process employs Fmoc (9-fluorenylmethoxycarbonyl) solid phase synthesis. The first alanine (Ala) at the C-terminus was attached to Wang resin and then sequentially synthesized from the C-terminus to the N-terminus once according to the peptide sequence until the last ursodeoxycholic acid (Ursodeoxycholic Acid) was completed to give compound 91 peptide resin. The compound 91 peptide resin is subjected to cracking, anhydrous diethyl ether precipitation and washing to obtain compound 91 crude peptide. The specific steps of rough filtering, purifying, salt transferring and freeze drying the compound 91 crude peptide to obtain a finished product are as follows:
1) Fmoc-Ala-Wang Resin was weighed into the reaction column and swollen with DMF nitrogen.
2) After the resin swells, DMF is washed, and DBLK solution is added for deprotection.
3) After deprotection, DMF was added for washing, ninhydrin detection was positive, and the amino acid Fmoc-Glu (OtBu) -OH and condensing agent HBUT were added, followed by stirring with nitrogen and DIEA reaction.
4) After the reaction is finished, the reaction solution is pumped out, and the DMF is washed until ninhydrin detection shows negative.
5) Repeating the steps 2-4 until the synthesis of the whole sequence is completed, and obtaining the peptide resin of the compound 91.
And (3) a cracking step:
1) And adding the peptide resin into the lysate for cleavage.
2) And filtering to remove filter cakes after the completion of the pyrolysis, and taking filtrate.
3) The filtrate was added to diethyl ether for sedimentation.
4) After completion of sedimentation, the supernatant was removed by centrifugation and ether washing was repeated three times.
5) Vacuum drying to obtain compound 91 crude peptide.
Purifying:
1) The crude compound 91 was taken and dissolved with water and acetonitrile until clear.
2) Filtering the solution to obtain filtrate.
3) And separating and purifying filtrate by semi-preparative chromatography.
4) And (5) carrying out rotary evaporation concentration on the purified qualified product.
5) And freeze-drying the concentrated solution of the compound 91 to obtain a white powder finished product.
Characterization:
HPLC:87.06% (area normalization method); MS: theoretical molecular weight: 3702.3; actual molecular weight: 3701.94.
Example 83: preparation and testing of nanoparticle assemblies
1) Preparation of nanoparticle compositions
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, the specific ingredients and their ratios in the lipid component of the nanoparticle composition are optimized.
Nanoparticles can be made by mixing two fluid streams, one of which contains a therapeutic and/or prophylactic agent and the other has a lipid component, by mixing methods such as microfluidization and T-junctions. Lipid compositions having a concentration of about 50mM were prepared by combining in ethanol a lipid synthesized according to examples 1-9, a phospholipid (such as DOPE or DSPC, available from Avanti Polar Lipids (Alabaster, AL)), a PEG lipid (such as 1, 2-dimyristoyl-sn-glycerogxypolyethylene glycol, also known as PEG-DMG, available from Avanti Polar Lipids (Alabaster, AL)), and a structural lipid (such as cholesterol, available from Sigma-Aldrich (Taufkirchen, germany)), or a corticosteroid (such as prednisolone, dexamethasone, prednisolone and hydrocortisone), or a combination thereof.
Nanoparticle compositions comprising therapeutic and/or prophylactic agents and lipid components are prepared by combining a lipid solution with a composition comprising therapeutic and/or prophylactic agents in a lipid component ratio therapeutic and/or prophylactic agent wt:wt ratio of between about 5:1 and about 50:1. Using a micana-S microfluidic nanoparticle preparation system, a lipid solution is rapidly injected into a therapeutic and/or prophylactic agent solution at a flow rate between about 10mL/min and about 18mL/min, producing a dispersion, wherein the water to ethanol ratio is between about 2:1 and about 5:1.
2) Characterization of nanoparticle assemblies
Determination of physical and chemical properties of nanoparticles: the particle size, polydispersity index (PDI) and zeta potential of the nanoparticle composition can be determined using a Zetasizer NanoZS (Malvern Instruments Ltd, malvern, worcestershire, UK), the particle size being determined in 1 x PBS and the zeta potential being determined in 15mM PBS.
Determination of nanoparticle encapsulation efficiency: for nanoparticle compositions comprising RNA, the encapsulation of RNA by the nanoparticle composition can be evaluated using a QUANT-ITTM RNA (Invitrogen Corporation Carlsbad, CA) assay. The samples were diluted to a concentration of about 5. Mu.g/mL in TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 7.5). mu.L of diluted samples were transferred to polystyrene 96-well plates and 50. Mu.L of TE buffer or 50. Mu.L of 2% Triton X-100 solution was added to each well. Plates were incubated at 37℃for 15 min. Reagents were diluted 1:100 in TE buffer and 100. Mu.L of the solution was added to each well. Can use fluorescent plate reader Nivo TM Multimode Plate Readers, perkinElmer, GER) at an excitation wavelength of, for example, about 480nm 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 obtained by using the fluorescence intensity of the complete sample (without Triton X-100 added)The percentage of free RNA was determined by dividing by the fluorescence value of the destroyed sample (caused by addition of Triton X-100). The specific data are shown in Table 1.
Table 1: characteristics of nanoparticle combinations prepared from different compounds
3) In vitro performance studies of nanoparticle combinations
For nanoparticle combinations containing RNA, ONE-GlO + TOX Luciferase Reporter and Cell Viability Assay (Promega Corporation, US) can be used to evaluate transfection effect and cytotoxicity. The volume of the desired nanoparticle assembly was calculated from the concentration of RNA measured in the encapsulation efficiency assay, the nanoparticle assembly was diluted to 20 ng/. Mu.L and 2X 10 in a polystyrene 96-well plate 5 Cell/well cell density 5 μl of dilution was added to each well for transfection. Can use fluorescent plate readerNivo TM Multimode Plate Readers, perkinElmer, GER) measure the chemiluminescent intensity. The specific data are shown in Table 2.
Table 2: in vitro performance studies of nanocomposites prepared with different compounds
Transfection effect: can provide multiples of MC3
Example 84: preparation and detection of lipid nanoparticles (verifying in vitro siRNA delivery ability)
1) The compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 65:30:5 with DOPE (1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine) and DMG-PEG2000 (1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol) to prepare a lipid ethanol solution, and Cy3 labeled SiCont (Sense 5'to 3': CUUACGCUGAGUACUUUCGADGTdT-Cy 3; the anti-sense 5'to 3' was diluted in 50mM citrate buffer (pH=4) to give siRNA solution, the ethanol lipid solution and siRNA solution were mixed in a 1:3 volume using a microfluidic device, liposomes were prepared at a total lipid to siRNA weight ratio of about 20:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution (phosphate buffered saline). The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give an encapsulated cy3-sicon formulation.
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 nanoparticle was measured to be 82nm, the pdi (polydispersity index) was 0.13, and the encapsulation efficiency was 96.5%.
2) The siRNA-entrapped lipid nanoparticle prepared above was transfected into 293T cells. 293T cells were grown at 1X 10 5 The density of individual cells/well was spread on a 24-well plate, and after culturing in DMEM medium containing 10% calf serum for 24 hours until the cell density became 70%, the medium in the 24-well plate was removed. The LNP/cy3-siRNA complex prepared was diluted to 1ml with DMEM medium and added to a 24-well plate. 37 ℃,5% CO 2 The incubator was incubated under the conditions for an additional 24h.
Transfection of cy3-siRNA was then recorded using fluorescence microscopy. The effect of cell transfection is shown in figure 21 of the accompanying drawings. The fluorescence microscope result shows that the prepared lipid nanoparticle coated with siRNA has better cell transfection effect. Flow cytometry test results indicated 68.90% of cells were transfected.
Example 85: preparation and detection of lipid nanoparticles (verifying in vitro mRNA delivery ability)
1) Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 65:30:5 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and EGFP mRNA (encoding green fluorescent protein mRNA) was diluted in 20-100mM citrate buffer (ph=4) to obtain an mRNA solution, the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a total lipid to mRNA weight ratio of about 20:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation encapsulating EGFP mRNA.
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 LNP formulation particle size was 95nm, PDI 0.19, and encapsulation efficiency was 93.1%.
2) The mRNA-entrapped lipid nanoparticle prepared above was transfected into 293T cells. 293T cells were grown at 1X 10 5 The density of individual cells/well was spread on a 24-well plate, and after culturing in DMEM medium containing 10% calf serum for 24 hours until the cell density became 70%, the medium in the 24-well plate was removed. The LNP/EGFP complex prepared was diluted to 1ml with DMEM medium and added to a 24-well plate. 37 ℃,5% CO 2 Incubators were incubated for 24h under the conditions. The transfection of EGFP was then recorded using fluorescence microscopy. The effect of cell transfection is shown in FIG. 22 of the drawings. The fluorescent microscope result shows that the prepared lipid nanoparticle for encapsulating mRNA has better cell transfection effect. Flow cytometry results indicated 77.79% of cells were transfected.
Example 86: preparation and detection of lipid nanoparticles (verification of in vivo delivery effect of mRNA)
1) Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 65:30:5 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 50mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a total lipid to mRNA weight ratio of 20:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
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 nanoparticle was measured to be 98nm, the pdi was 0.22, and the encapsulation efficiency was 88.9%.
2) LNPs prepared as described above were injected into 6-week-old SPF-grade BALB/c mice via tail vein or muscle, respectively, at a dose of 100ul (containing 10. Mu.g mRNA) per mouse, and after 12 hours, the fluorescence intensity in the mice was measured with a biopsy imager, and the exposure time of the biopsy imager was set at 30s. The results of in vivo imaging of lipid nanoparticles entrapped with Luciferase mRNA in mice are shown in figures 23 and 24 of the accompanying drawings. The results indicate that when the prepared LNP is injected into mice by intravenous injection, the delivery site of LNP in the body is mainly the abdomen. Whereas when LNP is injected into mice via muscle, the site of delivery of LNP in the body is mainly the abdomen and liver. All the results show that LNP has better in vivo delivery effect.
Example 87
The synthesized lipid compound-1 was dissolved in absolute ethanol at a molar ratio of 89.9:10:0.1 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 50mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, and the ethanol lipid solution and the mRNA solution were mixed in a volume of 1:3 using a microfluidic device, liposomes were prepared at a weight ratio of total lipid to mRNA of 20:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
The particle size was determined to be 1249nm, the PDI was determined to be 0.67, and the encapsulation efficiency was determined to be 31%.
Example 88
Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 89.5:10:0.5 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 50mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, and the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a weight ratio of total lipid to mRNA of 20:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
The particle size was found to be 467nm, the PDI was found to be 0.40 and the encapsulation efficiency was found to be 78%.
Example 89
Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 89:10:1 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 100mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a total lipid to mRNA weight ratio of 20:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
The particle size was detected as being 347nm, the PDI was 0.41, and the encapsulation efficiency was 81%.
Example 90
Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 85:10:5 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 100mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a total lipid to mRNA weight ratio of 20:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
The particle size was 94nm, the PDI was 0.50, and the encapsulation efficiency was 85%.
Example 91
Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 64:35:1 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 100mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a weight ratio of total lipid to mRNA of 20:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
The particle size was 82nm, PDI was 0.11, and the encapsulation efficiency was 96%.
Example 92
Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 39:60:1 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 100mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a weight ratio of total lipid to mRNA of 40:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
The particle size was 145nm, PDI was 0.23, and the encapsulation efficiency was 76%.
Example 93
Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 39:60:1 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 20mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a weight ratio of total lipid to mRNA of 40:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
The particle size was measured to be 302nm, the PDI was 0.38, and the encapsulation efficiency was 73%.
Example 94
Lipid compound-1 synthesized in example 1 was dissolved in absolute ethanol at a molar ratio of 60:39.2:0.8 with DOPE and DMG-PEG2000 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding Luciferase mRNA) was diluted in 20mM citrate buffer (ph=4) to obtain mRNA solutions, respectively, and the ethanol lipid solution and the mRNA solution were mixed at a volume of 1:3 using a microfluidic device, liposomes were prepared at a weight ratio of total lipid to mRNA of 40:1, ethanol was removed by dialysis or tangential flow filtration, and replaced with PBS solution. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter to give a formulation of encapsulated mRNA.
The particle size was measured to be 302nm, the PDI was 0.38, and the encapsulation efficiency was 73%.
Example 95: preparation of lipid nanoparticles
As shown in table 1, compounds 19 to 88 were dissolved in ethanol in a ratio of 50:10:38.5:1.5 molar ratio to DSPC: cholesterol: DMG-PEG2000, respectively. The nanoparticle composition of example 083 was prepared according to the method of preparation.
Example 96: characterization of lipid nanoparticles
The particle Size (Size), polydispersity index (PDI) and Zeta potential (Zeta P) of the particles were determined by dynamic light scattering using Malvern Zetasizer NanoZS (malvern nanoparticle Size tester). The encapsulation efficiency of the lipid nanoparticles was determined using QUANT-ITTM RNA (Invitrogen Corporation Carlsbad, CA). Following the characterization method of nanoparticle assemblies in example 083, as shown in table 3: the lipid nanoparticle preparation has a size of about 100nm, a potential of + -20 mv, a polydispersity index of about 0.1-0.3, and an encapsulation efficiency of about 80% -99%.
Table 3: characterization of lipid nanoparticles
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Example 96: in vitro cell transfection experiments of lipid nanoparticles
293T cells were seeded at a density of 3X10≡4 cells/well in 96 well cell culture plates for 24h to adhere. The compounds of table 4 were prepared as lipid nanoparticle formulations and added to cells as in example 83, prepared as in example 83. After culturing for 24 hours, adding a color developing solution, and detecting the luminescence value of each hole by using an enzyme-labeled instrument. Table 4 shows that lipid nanoformulations can carry mRNA transfected into 293T cells and express fluorescent protein of interest, A, B, C, D represents the results of normalization of compounds after doubling the luminous flux of commercial MC3-LNP, respectively, as shown in Table 4, with 31 compounds having higher luminous flux than MC3-LNP group, indicating that lipid nanoparticle formulations can effectively carry nucleic acid into cells.
Description: A. b, C, D represents the normalized results of the compound and MC3-LNP luminous flux, respectively, after doubling, the range of multiplying power is as follows:
A：≥1
b: more than or equal to 0.5 and less than 1
C: more than or equal to 0.1 and less than 0.5
D：＜0.1
Table 4: in vitro cell transfection evaluation result of lipid nanoparticle
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Example 97: compound 46 encapsulates paclitaxel killer cells
The compound 46, DSPC, cholesterol and PEG2000 are subjected to alcohol removal according to the method in the embodiment 83, and then 0.2, 1, 5, 10 and 20mg/ml of paclitaxel solution is added to evaluate the killing effect of paclitaxel-loaded LNP on tumor cells A549, and as shown in the result of FIG. 25, the paclitaxel-loaded lipid nanoparticle has better killing effect on tumor cells than free paclitaxel at high concentration.
Example 98: selection of Compound 27 for LNP prescription screening
The compound 27 was screened for prescription, and the formulation was prepared according to the formulation shown in the following table, and the method described in example 83, and the results are shown in table 5, wherein the lipid nanoparticle preparation size was between 70nm and 170nm, the potential was between ±20mv, the polydispersity index was varied between 0.1 and 0.3, the encapsulation efficiency was partially around 80% -90%, and the luminous flux value of the cell evaluation result was mostly higher than that of the MC3-LNP group, indicating that the lipid nanoparticle preparation can change the formulation, carrying nucleic acid into cells.
Table 5: characterization of lipid nanoparticles and cellular evaluation
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Example 99: fluorescent protein expression experiments of lipid nanoparticle formulations in mice
Samples were prepared according to the method for preparing nanoparticle assemblies of example 83, with MC3-LNP as a positive reference.
Female SPF-class BALB/c mice (n=3) with the weight of 6-8w are adopted, and are bred in SPF-class animal houses, the temperature is 20-26 ℃, the humidity is 40-70%, and the feed and the drinking water are sufficient. The preparation of the formulation of example 83 was carried out by intramuscular injection at a dose of 100. Mu.L (0.05. Mu.g/. Mu.L), respectively, and the control was subjected to in vivo efficacy comparison using MC 3-LNP. After 6 hours of administration, 200ul of D-potassium fluorescein salt at a concentration of 15mg/ml was intraperitoneally injected, and 5 minutes of injection of D-potassium fluorescein salt was followed by in vivo imaging. After all groups were imaged, D-potassium fluorescein was injected again and 5 minutes later for organ imaging. A. B, C, D represent the results of compound doubling and normalization to commercial MC3-LNP fluorescent expression values, respectively, as shown in Table 6, with compounds 27, 37, 39, 42, 43, 46, 50, 66, 69, 74, 82, 86, 87 reaching MC3-LNP levels upon local administration, wherein compounds 42, 43, 50, 69 exceeded MC3-LNP levels, indicating that lipid nanoparticle formulations can efficiently deliver mRNA and express target gene proteins locally. Table 7 and FIG. 26 are results of comparison of compounds with commercially available MC3-LNP expressed in the abdomen, as shown in Table 7: wherein, the fluorescence level of the compounds 52, 67, 75, 81, 82 and 83 exceeds the MC3-LNP level in the abdomen, which indicates that the cholic acid series lipid compounds can efficiently express the target gene protein.
Description: A. b represents the normalized result after the compound and the luminescence value of MC3-LNP are multiplied, the multiplying power range is as follows:
A：≥0.5
b: more than or equal to 0.1 and less than 0.5
Table 6: lipid nanoparticle formulation in vivo muscle local fluorescence expression ratio and score of mice
Sample numbering Ratio of Scoring of
Compound 23 0.1 B
Compound 25 0.1 B
Compound 27 0.7 A
Compound 29 0.1 B
Compound 32 0.1 B
Compound 37 0.7 A
Compound 38 0.3 B
Compound 39 0.7 A
Compound 42 1.6 A
Compound 43 1.5 A
Compound 44 0.3 B
Compound 46 0.9 A
Compound 47 0.4 B
Compound 49 0.1 B
Compound 50 2.1 A
Compound 52 0.3 B
Compound 55 0.3 B
Compound 66 0.5 A
Compound 68 0.35 B
Compound 69 1.1 A
Compound 73 0.3 B
Compound 74 0.5 A
Compound 75 0.2 B
Compound 81 0.3 B
Compound 82 0.9 A
Compound 83 0.33 B
Compound 84 0.49 B
Compound 85 0.37 B
Compound 86 0.7 A
Compound 87 0.51 A
Compound 88 0.47 A
Table 7: lipid nanoparticle formulations in vivo in mice for abdominal fluorescence expression ratio and scoring
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Example 100: intravenous fluorescent protein expression of lipid nanoparticle formulations
Compound 52, compound 67, and compound 75 were prepared using the preparation method of example 83, and intravenous administration was performed according to example 099, and the control group was compared with the MC3-LNP for in vivo effects, as shown in fig. 27, and compound 52, compound 67, and compound 75 were weaker than the MC3-LNP, but all had fluorescent expression.
Example 101: lipid nanoparticle formulations for intratumoral administration
Intratumoral administration was prescribed with compound 27 and prepared as described in example 83. The EG7-OVA model established by adopting a C57 mouse is adopted, and the tumor grows to 500mm 3 Administration was performed, intratumoral injection, photographing was performed 6 hours after injection, and expression and distribution were observed. As shown in fig. 28, the intratumoral site of the mouse has fluorescent expression, which indicates that the lipid nanoparticle can be administered by intratumoral injection.
Example 102: compound 42 and compound 58 were selected for LNP prescription screening
Compound 42 was screened for prescription, and the formulation was prepared according to the formulation in the following table, and the method described in example 83, and the results are shown in table 8, wherein the lipid nanoparticle formulation size was between 50nm and 200nm, the potential was between ±5mv, the polydispersity index was around 0.1, the encapsulation efficiency was mostly above 90%, and the luminous flux value of the cell evaluation result was mostly higher than that of the MC3-LNP group, indicating that the lipid nanoparticle formulation could change the formulation, carrying nucleic acid into cells.
Table 8: characterization of lipid nanoparticles and cellular evaluation
Example 103: fluorescent protein expression experiments of lipid nanoparticle formulations in mice
The nanoparticle assembly obtained in example 102 was assembled. Female SPF-class BALB/c mice (n=3) with the weight of 6-8w are adopted, and are bred in SPF-class animal houses, the temperature is 20-26 ℃, the humidity is 40-70%, and the feed and the drinking water are sufficient. The preparation of the formulation of example 83 was carried out by intramuscular injection at a dose of 100. Mu.L (0.05. Mu.g/. Mu.L), respectively, and the control was subjected to in vivo efficacy comparison using MC 3-LNP. After 6 hours of administration, 200ul of D-potassium fluorescein salt at a concentration of 15mg/ml was intraperitoneally injected, and 5 minutes of injection of D-potassium fluorescein salt was followed by in vivo imaging. After all groups were imaged, D-potassium fluorescein was injected again and 5 minutes later for organ imaging. A. B represents the normalized result of the double ratio of each formula and the fluorescence expression value of classical formula number 13, and as shown in Table 9, 7 formulas reach classical formula level when being locally administered into muscle, which indicates that the lipid nanoparticle preparation can effectively deliver mRNA through formula change and efficiently express target gene protein in muscle. Wherein the fluorescence expression level of the formulas 1, 8 and 9 in spleen far exceeds the classical formula level, which indicates that the cholic acid series lipid compounds can efficiently express the gene protein in spleen by changing the proportion of the formulas. In addition, formulation 3 was a three-component formulation with DSPC removed, and the results showed higher local fluorescence expression values in muscle than the classical formulation, indicating that the three-component formulation can form nanoparticles and efficiently express fluorescent proteins in vivo.
Description: A. b represents the normalized result of the light emitting value of each group of formulas and classical formula number 13 after the multiplying power is calculated as follows:
A：≥0.5
b: more than or equal to 0.1 and less than 0.5
Table 9: lipid nanoparticle formulations in mice muscle local, spleen fluorescence expression ratio and scoring
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (15)

1. A composition comprising a lipid compound, characterized in that the composition comprises a therapeutic or prophylactic agent and a carrier for delivering the therapeutic or prophylactic agent, the carrier comprising one or more of the lipid compounds; the carrier comprises three different lipid components, wherein one lipid is a lipid based on cholic acid or a derivative thereof; the lipid compound is a lipid based on cholic acid or a derivative thereof, or the lipid compound is a pharmaceutically acceptable salt, prodrug or stereoisomer of a lipid based on cholic acid or a derivative thereof, the lipid compound having the structure of formula 1 or formula 2:
General formula 1:
R 1 =hydrogen atom, alkane, alkene, substituted carbonyl group, alkane containing nitrogen atom, alkene containing nitrogen atom
R 2 =hydrogen atom, alkane, alkene, substituted carbonyl group, alkane containing nitrogen atom, alkene containing nitrogen atom
R 3 =hydroxy, halogen atom, alkane, alkene
R 4 =hydrogen atom, alkane, alkene, alkane containing nitrogen atom, alkene containing nitrogen atom, cyclic compound containing nitrogen atom
X=nh or O
linker=alkane, S-S,
general formula 2:
2. the composition of claim 1, wherein the carrier further comprises a charge-assisted lipid of neutral charge, negative charge, or bipolar charge.
3. The composition of claim 2, wherein the charge-assisted lipid is one or more of the following: distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof.
4. The composition of claim 1, wherein the carrier further comprises a structurally modified lipid.
5. The composition of claim 4, wherein the structurally modified lipid comprises one or more of polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, dialkylglycerol.
6. The composition of claim 1, wherein the carrier further comprises, but is not limited to, lipids of cholic acid or derivatives thereof, charge-assisted lipids, and structure-modified lipids, the molar ratio of said cholic acid lipids, said charge-assisted lipids, and said structure-modified lipids being (30-80): 5-50): 0.5-10.
7. The composition of claim 1, wherein the carrier further comprises a lipid of cholic acid or a derivative thereof, a charge-assisted lipid, cholesterol or a derivative thereof, and a structure-modified lipid, wherein the molar ratio of the cholic acid lipid, the charge-assisted lipid, the cholesterol or a derivative thereof, and the structure-modified lipid is (30-80): 0.5-10): (5-50): 0.5-2.5.
8. The composition of claim 1, further comprising one or more of a pharmaceutically acceptable excipient or diluent.
9. Use of a composition according to any one of claims 1-8 for the preparation of nucleic acid pharmaceuticals, genetic vaccines, polypeptides, proteins, antibodies and small molecule pharmaceuticals.
10. Use of a composition according to any one of claims 1-8 for the preparation of nucleic acid drugs, genetic vaccines, polypeptides, proteins, antibodies and small molecule drugs, wherein the lipid nanoparticle has a particle size of 20-1000 nm.
11. A composition for the preparation of nucleic acid pharmaceuticals, genetic vaccines, polypeptides, proteins, antibodies and small molecule pharmaceuticals comprising a nucleic acid and lipid nanoparticles encapsulating the nucleic acid, wherein each individual lipid nanoparticle comprises a plurality of lipid components, wherein one lipid component is a cholic acid-based lipid compound, including compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent compounds or prodrugs thereof, and wherein the lipid nanoparticle has a nucleic acid encapsulation ratio of at least 70%.
12. A method of preparing the composition of claim 11, wherein the lipid nanoparticle is formed by mixing an mRNA solution and a lipid solution comprising a lipid compound, 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, isopropanol or dimethyl sulfoxide; wherein the lipid nanoparticle is further purified by dialysis or ultrafiltration.
13. The composition of claim 11, further comprising one or more of a buffer, a carbohydrate, mannitol, a protein, a polypeptide or amino acid, an antioxidant, a bacteriostatic agent, a chelating agent, an adjuvant.
14. The composition of claim 11, wherein the administration comprises intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, ocular administration, otic administration, nasal administration, oral administration, anal administration, vaginal administration.
15. The composition of claim 11, which is administered to a subject comprising a mammal, bovine, equine, mule, donkey, camel, porcine, ovine, canine, fox or rabbit, avian such as chicken, duck, goose or pigeon, fish, non-human primate, human.
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