CN117486754A - Lipid compound for delivering therapeutic agent and preparation method and application thereof - Google Patents

Abstract

The invention provides a lipid compound for delivering a therapeutic agent, and a preparation method and application thereof. The invention specifically provides a lipid compound which is a compound with a structural formula I or a pharmaceutically acceptable form thereof. The lipid compounds can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer-bound lipids, to form lipid nanoparticles for delivery of therapeutic agents, such as nucleic acid molecules, for therapeutic or prophylactic purposes, such as vaccination, enriching the class of ionizable lipid compounds.

Description

Lipid compound for delivering therapeutic agent and preparation method and application thereof

Technical Field

The invention belongs to the field of drug delivery, and particularly relates to a lipid compound for delivering a therapeutic agent, a preparation method and application thereof, in particular to a lipid compound for delivering a therapeutic agent (such as a nucleic acid molecule), a lipid carrier containing the lipid compound, a nucleic acid lipid nanoparticle composition and a pharmaceutical preparation, and a preparation method and related application thereof.

Background

The gene therapy technology is a hotspot of research in the field of modern biological medicine, and can prevent and treat cancers, bacterial and viral infections, diseases with genetic etiology and the like by using nucleic acid medicines. Because nucleic acid drugs are easy to degrade and difficult to enter cells, and the like, the nucleic acid drugs are usually required to be encapsulated by a carrier and delivered to target cells, so that the development of safe and efficient delivery carriers becomes a precondition for clinical application of gene therapy.

Lipid nanoparticles (Lipid nanoparticle, LNP) are currently a research hotspot in the field of non-viral gene vectors. In 2018, the FDA approved LNP delivery patisiran (onpattro) for the treatment of hereditary transthyretin amyloidosis, and studies from the use of LNP technology to deliver nucleic acid drugs have been shown to grow in bursts. In particular, at the end of 2020, the FDA approved new coronavirus vaccines against COVID-19 for Moderna and BioNtech & pyroxene, respectively, both of which delivered mRNA drugs using LNP technology, thus achieving prevention of SARS-CoV-2 virus.

LNP is typically composed of four lipid compounds, namely ionizable lipids, neutral lipids, steroids, and polymer-bound lipids, with the choice of ionizable lipids having the greatest impact on LNP. Current nucleic acid therapeutic agents still face challenges including mainly delivery efficiency, low cell permeability, and high sensitivity to degradation by certain nucleic acid molecules, including RNA. Thus, there remains a need to develop new lipid compounds that facilitate in vitro or in vivo delivery of nucleic acid molecules for therapeutic and/or prophylactic purposes.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a lipid compound or a pharmaceutically acceptable form thereof (e.g. salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, etc.), which can be used to prepare lipid nanoparticles with other lipid compounds (e.g. neutral lipids, charged lipids, steroids) for delivering therapeutic agents (e.g. nucleic acid molecules, in particular mRNA) to enhance the efficiency of nucleic acid drug delivery in vivo, and which can be used as lipid carrier for organs enriched in nucleic acid drug needs.

In one embodiment, provided herein is a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof:

in the formula (I) of the present invention,

G ¹ is C _1-10 An alkylene group; g ¹ Preferably isWherein m is selected from integers from 1 to 6;

R ¹ 、R ² each independently is C _1-6 An alkyl group; preferably, R ¹ 、R ² Together with the attached N form a 3-8 membered heterocyclic group, or R ¹ 、R ² Any one of which is with G ¹ Any carbon atom of the two are directly connected to form a 3-8 membered heterocyclic group; further preferably, R ¹ 、R ² Each independently is methyl, ethyl or propyl; alternatively, R ¹ 、R ² Together with the attached N forms a 4-, 5-or 6-membered heterocyclyl;

L ¹ 、L ² 、L ³ 、L ⁴ 、L ⁵ each independently selected from the group consisting of ester groups, amides, carbonates, carbamates, mercapto formates, ureas, phosphates, or none; preferably L ¹ ～L ⁵ Each independently selected from Or none;

R ³ 、R ⁵ 、R ⁷ each independently selected from the group consisting of linear alkanes, branched alkanes, linear heteroatom-containing alkyl, branched heteroatom-containing alkyl, or none; preferably, R ³ 、R ⁵ 、R ⁷ Each independently isWherein X is selected from O, S, se, S-S, se-Se or none; r is R ¹¹ ～R ¹⁴ Is H or C ₁ -C ₈ Linear alkanes of (a); n and o are integers from 1 to 10;

R ⁴ 、R ⁶ 、R ⁸ each independently selected from the group consisting of linear alkyl, linear alkenyl, linear alkynyl, branched alkyl, cycloalkyl, bridged cycloalkyl, or none; preferably, R ⁴ 、R ⁶ 、R ⁸ Each independently isWherein Y is%> Or none; r is R ¹⁵ ～R ¹⁹ Is H or C ₁ -C ₁₀ Linear alkanes, cycloalkanes or bridged cycloalkanes; p and q are integers of 0 to 10.

In one embodiment, provided herein is also a lipid carrier comprising the above lipid compound or a pharmaceutically acceptable form thereof.

In one embodiment, also provided herein are nucleic acid lipid nanoparticle compositions comprising the above lipid compounds or pharmaceutically acceptable forms thereof or the above lipid carriers.

In one embodiment, provided herein is also a pharmaceutical formulation comprising the above lipid compound or a pharmaceutically acceptable form thereof, or the above lipid carrier, or the above nucleic acid lipid nanoparticle composition.

In one embodiment, also provided herein is the use of the above lipid compound or a pharmaceutically acceptable form thereof or the above lipid carrier or the above nucleic acid lipid nanoparticle composition or the above pharmaceutical formulation for the preparation of a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide or a protein drug.

In one embodiment, provided herein is also a method for delivering a nucleic acid drug in vivo, the method comprising administering to a subject in need thereof the nucleic acid lipid nanoparticle composition described above or the pharmaceutical formulation described above.

Solution for solving the problem

< first aspect >

The invention provides a compound shown in a formula (I) or pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof:

wherein in the formula (I),

G ¹ is C _1-10 An alkylene group;

R ¹ 、R ² each independently is C _1-6 An alkyl group;

optionally R ¹ 、R ² Together with the attached N form a 3-8 membered heterocyclic group, or R ¹ 、R ² Any one of which is with G ¹ Any carbon atom of the two are directly connected to form a 3-8 membered heterocyclic group;

L ¹ 、L ² 、L ³ 、L ⁴ 、L ⁵ each independently selected from the group consisting of ester groups, amides, carbonates, carbamates, mercapto formates, ureas, phosphates, or none;

R ³ 、R ⁵ 、R ⁷ each independently selected from the group consisting of linear alkanes, branched alkanes, linear heteroatom-containing alkyl, branched heteroatom-containing alkyl, or none;

R ⁴ 、R ⁶ 、R ⁸ each independently selected from the group consisting of linear alkyl, linear alkenyl, linear alkynyl, branched alkyl, cycloalkyl, bridged cycloalkyl, or none.

In one embodiment, G ¹ Is thatWherein m is selected from integers from 1 to 6.

In one embodiment, R ¹ 、R ² Each independently is methyl, ethyl or propyl; alternatively, R ¹ 、R ² Together with the attached N forms a 4-, 5-or 6-membered heterocyclic group.

In one embodiment, L ¹ ～L ⁵ Each independently selected from Or none.

In one embodiment, R ³ 、R ⁵ 、R ⁷ Each independently is

Wherein X is O, S, se, S-S, se-Se or none;

R ¹¹ ～R ¹⁴ is H or C ₁ -C ₈ Linear alkanes of (a);

n and o are integers from 1 to 10.

In one embodiment, R ⁴ 、R ⁶ 、R ⁸ Each independently is

Wherein Y is,Or none;

R ¹⁵ ～R ¹⁹ is H or C ₁ -C ₁₀ Linear alkanes, cycloalkanes or bridged cycloalkanes;

p and q are integers from 0 to 10.

In one embodiment, the lipid compound is selected from any one or a combination of at least two of the structural compounds as in table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

TABLE 1

In one embodiment, the pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes, or prodrugs.

< second aspect >

The present invention provides the use of a lipid compound according to the first aspect or a pharmaceutically acceptable form thereof in the preparation of a liposomal nanocarrier.

< third aspect >

The present invention provides a lipid carrier comprising a lipid compound according to the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug.

The lipid carrier has high encapsulation efficiency on nucleic acid drugs, and greatly improves the delivery efficiency of the nucleic acid drugs in vivo.

In one embodiment, the lipid carrier comprises a first lipid compound comprising a lipid compound according to the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, and optionally an ionizable lipid, and a second lipid compound comprising any one or a combination of at least two of an anionic lipid, a neutral lipid, a steroid or a polymer-bound lipid.

In some embodiments, the first lipid compound is a lipid compound of the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug.

In some embodiments, the first lipid compound is a lipid compound of the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, and a combination of an ionizable lipid.

In one embodiment, the ionizable lipid is selected from the group consisting of: 1, 2-diiodoyloxy-N, N-dimethylaminopropane DLinDMA, 1, 2-dioleyloxy-N, N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2-diiodo-4- (2-dimethylaminoethyl) - [1,3] -dioxolane DLin-KC2-DMA, 1, 2-dioleoyl-3-trimethylammonium-propane DOTAP, 1'- (2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) di-dodecane-2-ol C12-200, 3β [ N-N' -dimethylaminoethane) -carbamoyl ] cholesterol, or N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N-trimethylamine chloride DOTMA, or a combination of at least two thereof.

In some embodiments, the anionic lipid is selected from the group consisting of: any one or a combination of at least two of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoyl phosphatidylglycerol (DOPG), 1, 2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), and dimyristoyl phosphatidylglycerol.

In some embodiments, the neutral lipid is selected from the group consisting of: any one of 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dipalmitoyl phosphatidylglycerol (DPPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphate ethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphate ethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE) and 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE) or a lipid modified with an anionic or cationic group. The anionic or cationic modifying group is not limited.

In some embodiments, the steroid is selected from the group consisting of: any one or a combination of at least two of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycoalkali, ursolic acid, alpha-tocopherol, fecal sterol, or corticosteroid.

In some embodiments, the polymer-bound lipid is selected from the group consisting of: 1, 2-dimyristoyl-sn-glycerinomethoxy-polyethylene glycol (PEG-DMG), dimyristoylglycerol-polyethylene glycol (PEG-C-DMG), polyethylene glycol-dimyristoylglycerol (PEG-C14), PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-C-DMA), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), pegylated phosphatidylethanolamine (PEG-PE), PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, tween-20, tween-80, 1, 2-dipalmitoyl-sn-glycero-methoxypolyethylene glycol PEG-DPG, 4-O- (2 ',3' -di (tetradecyloxy) propyl-1-O- (ω -methoxy (polyethoxy) ethyl) succinate (PEG-s-DMG), PEG-dialkoxypropyl (PEG-DAA), one or a combination of two or more of mPEG2000-1, 2-di-O-alkyl-sn 3-carbamoyl glyceride (PEG-c-DOMG) and N-acetylgalactosamine ((R) -2, 3-bis (octadecyloxy) propyl-1- (methoxypoly (ethylene glycol) 2000) propyl carbamate)) (GalNAc-PEG-DSG).

In some embodiments, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid in the lipid carrier is (20-65): 0-20): 5-25): 25-55): 0.3-15; illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid may be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55:15, 65:0:9.7:25:0.3, and the like; wherein, in the first lipid compound, the molar ratio of the lipid compound or a pharmaceutically acceptable form thereof to the ionizable lipid is (1-10): 0-10; illustratively, the molar ratio may be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

In some embodiments, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid in the lipid carrier is (20-55): 0-13): 5-25): 25-51.5): 0.5-15; wherein in the first lipid compound, the molar ratio of the lipid compound or pharmaceutically acceptable form thereof and the ionizable lipid is (3-4): 0-5.

< fourth aspect >

The present invention provides a nucleic acid lipid nanoparticle composition comprising a lipid compound according to the first aspect or a pharmaceutically acceptable form thereof, or a lipid carrier according to the third aspect, and a therapeutic or prophylactic agent.

In one embodiment, the therapeutic or prophylactic agent comprises: any one or a combination of at least two of RNA, DNA, antisense nucleic acids, aptamers, nucleases, immunostimulatory nucleic acids, or peptide nucleic acids.

In one embodiment, the antisense nucleic acid is an antisense oligonucleotide.

In one embodiment, the RNA is any one or a combination of at least two of mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, a microrna inhibitor, a microrna activator, or shRNA.

In one embodiment, the RNA is modified RNA.

In one embodiment, the mRNA includes a sequence encoding an RNA-guided DNA binding agent, more specifically, an mRNA including a Cas protein.

In one embodiment, the DNA comprises a plasmid.

In one embodiment, the nuclease is selected from any one or a combination of at least two of Cas9, cas12, cas13, iscB, tnpB, isrB, and homologs thereof.

In some embodiments, the mass ratio of the therapeutic or prophylactic agent to the compound or pharmaceutically acceptable form thereof is 1 (3-40). Illustratively, the mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, 1:40, and the like.

In some embodiments, the mass ratio of the therapeutic or prophylactic agent to the lipid carrier is 1 (3-40). Illustratively, the mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, 1:40, and the like.

< fifth aspect >

The present invention provides a pharmaceutical composition comprising a lipid compound according to the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or a lipid carrier according to the third aspect, or a nucleic acid lipid nanoparticle composition according to the fourth aspect, and a pharmaceutically acceptable adjuvant.

In some embodiments, the pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizers, tonicity adjusting agents, pH adjusting agents, antioxidants, or buffers.

< sixth aspect >

The present invention provides a pharmaceutical formulation comprising a lipid compound according to the first aspect or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or a lipid carrier according to the third aspect or a nucleic acid lipid nanoparticle composition according to the fourth aspect, and a pharmaceutically acceptable adjuvant.

In one embodiment, the pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizers, tonicity adjusting agents, pH adjusting agents, antioxidants or buffers.

In some embodiments, the dosage form of the pharmaceutical formulation is selected from: any one or a combination of at least two of tablets, capsules, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

In some embodiments, the injection comprises an injection solution, a sterile powder for injection, and a concentrated solution for injection.

In some embodiments, the pharmaceutical formulation has a particle size of 30 to 500nm, and illustratively, the particle size may be 30nm, 50nm, 100nm, 150nm, 250nm, 350nm, 500nm, etc.

In some embodiments, the therapeutic or prophylactic agent has an encapsulation efficiency of greater than 50% in a pharmaceutical formulation. Illustratively, the encapsulation efficiency may be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.

< seventh aspect >

The invention provides the use of a lipid compound according to the first aspect or a pharmaceutically acceptable form thereof, a lipid carrier according to the third aspect, a nucleic acid lipid nanoparticle composition according to the fourth aspect, a pharmaceutical composition according to the fifth aspect or a pharmaceutical formulation according to the sixth aspect in the preparation of a nucleic acid drug, a genetic vaccine, a polypeptide or a protein drug.

In one embodiment, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is for use in treating or preventing a disease or disorder in a subject in need thereof.

In one embodiment, the subject is a mammal.

In one embodiment, the mammal is a human.

In one embodiment, the disease or disorder is selected from metabolic disease, genetic disease, cancer, cardiovascular disease, or infectious disease.

In one embodiment, the metabolic disease comprises familial hypercholesterolemia, the genetic disease comprises transthyretin amyloid disease, primary hyperuricemia, or hereditary angioedema, and the infectious disease comprises hepatitis b.

< eighth aspect >

The present invention provides a method of delivering a therapeutic or prophylactic agent to a cell of a subject, the method comprising: administering the nucleic acid lipid nanoparticle composition of the fourth aspect or the pharmaceutical formulation of the sixth aspect to the subject, the administering comprising contacting the subject cells with the lipid nanoparticle composition or pharmaceutical formulation, thereby delivering the therapeutic and/or prophylactic agent to the subject cells.

In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered, for example, via an enteral or parenteral route of administration.

In some embodiments, the nucleic acid lipid nanoparticle composition or pharmaceutical formulation is administered to the subject at a dose of about 0.001mg/kg to about 10 mg/kg.

< ninth aspect >

The present invention provides a method of producing a protein or polypeptide of interest in a cell of a subject, the method comprising: contacting the subject cell with the nucleic acid lipid nanoparticle composition of the fourth aspect, wherein the therapeutic or prophylactic agent is an mRNA, and wherein the mRNA encodes a protein or polypeptide of interest, whereby the mRNA is capable of translating in the cell to produce the protein or polypeptide of interest.

< tenth aspect >

The present invention provides a method for preventing, ameliorating or treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the nucleic acid lipid nanoparticle composition of the fourth aspect or the pharmaceutical formulation of the sixth aspect.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a series of compounds of formula I with novel structure, which can be used as ionizable lipid to prepare lipid carrier together with other lipid compounds, and has controllable particle size, uniform distribution and high encapsulation efficiency; the synthesis method is simple, the yield is high, the synthesis can be fast, and the cost is low. The compound of the invention can be used for delivering nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs, and enriches the variety of ionizable lipid compounds.

Drawings

FIG. 1 is a schematic representation of the delivery strategy for PCSK9 gene editing efficiency detection in mouse liver cells according to the invention.

FIG. 2 shows PCSK9 gene editing efficiency of different lipid compound encapsulated base editors in mouse liver cells.

Detailed Description

For easier understanding of the present invention, certain technical and scientific terms are defined below in detail. Unless otherwise defined explicitly herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, the use of "substantially" means that the standard deviation from the theoretical model or theoretical data is within a range of 5%, preferably 3%, more preferably 1%.

In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

Reference throughout this specification to "some embodiments," "some specific/preferred embodiments," "other specific/preferred embodiments," "embodiments," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[ definition of terms ]

Unless otherwise indicated, the following terms have the following meanings:

the term "pharmaceutically acceptable salt" refers to salts of the compounds of the invention which are substantially non-toxic to the organism. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed from the compounds of the present invention by reaction with pharmaceutically acceptable inorganic/organic acids or inorganic/organic bases, such salts also being referred to as acid addition salts or base addition salts. Common inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like, common organic acids include, but are not limited to, trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like, common inorganic bases include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, and the like, and common organic bases include, but are not limited to, diethylamine, triethylamine, ethambutol, and the like.

The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular plane of asymmetry due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby enabling rotation of plane polarized light. The present invention also includes stereoisomers and mixtures thereof, due to the presence of asymmetric centers and other chemical structures in the compounds of the present invention which may lead to stereoisomers. Since the compounds of the present invention and salts thereof include asymmetric carbon atoms, they can exist as single stereoisomers, racemates, mixtures of enantiomers and diastereomers. Typically, these compounds can be prepared in the form of a racemic mixture. However, if desired, such compounds can be prepared or isolated to give pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in single stereoisomers (purity. Gtoreq.98%, purity. Gtoreq.95%,. Gtoreq.93%,. Gtoreq.90%,. Gtoreq.88%,. Gtoreq.85% or. Gtoreq.80%). The individual stereoisomers of the compounds are prepared synthetically from optically active starting materials containing the desired chiral centers or by preparation of mixtures of enantiomeric products followed by separation or resolution, e.g., conversion to mixtures of diastereomers followed by separation or recrystallization, chromatography, use of chiral resolving agents, or direct separation of the enantiomers on chiral chromatographic columns. Starting compounds having specific stereochemistry are either commercially available or can be prepared according to the methods described herein and resolved by methods well known in the art.

The term "tautomer" (or "tautomeric form") refers to structural isomers having different energies that can be converted to each other by a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or proton transfer tautomers) include, but are not limited to, interconversions by proton transfer, such as keto-enol isomerisation, imine-enamine isomerisation, amide-imine alcohol isomerisation, and the like. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

The term "solvate" refers to a substance formed by the association of a compound of the invention, or a pharmaceutically acceptable salt thereof, with at least one solvent molecule by non-covalent intermolecular forces. Common solvates include, but are not limited to, hydrates, ethanolates, acetonates, and the like.

The term "chelate" is a complex having a cyclic structure, obtained by chelation of two or more ligands with the same metal ion to form a chelate ring.

The term "non-covalent complex" is formed by the interaction of a compound with another molecule, wherein no covalent bond is formed between the compound and the molecule. For example, recombination can occur by van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

The term "prodrug" refers to a derivative compound that is capable of providing a compound of the invention directly or indirectly after administration to a patient. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to a patient, may increase the bioavailability of the compounds of the invention (e.g., are more readily absorbed into the blood) or promote delivery of the parent compound to the site of action (e.g., the lymphatic system). All prodrug forms of the compounds of the invention are within the scope of the invention unless otherwise indicated, and the various prodrug forms are well known in the art.

The term "independently" means that at least two groups (or ring systems) present in the structure that are the same or similar in value range may have the same or different meanings in the particular case. For example, substituent X and substituent Y are each independently hydrogen, halogen, hydroxy, cyano, alkyl or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl; similarly, when the substituent Y is hydrogen, the substituent X may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl.

The terms "comprising" and "including" are used in their open, non-limiting sense.

The term "alkyl" refers to a monovalent, linear or branched alkyl group consisting of only carbon and hydrogen atoms, free of unsaturation, and attached to other moieties by a single bond, including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, and the like. For example, "C _1-30 Alkyl "refers to a saturated monovalent straight or branched hydrocarbon radical containing 1 to 30 carbon atoms.

The term "alkylene" refers to a divalent straight or branched chain alkane group consisting of only carbon and hydrogen atoms, containing no saturation, and linked to other fragments by two single bonds, respectively, including, but not limited to, methylene, 1-ethylene, 1, 2-ethylene, and the like. For example, "C _1-30 Alkylene "refers to a saturated divalent straight or branched chain alkyl group containing from 1 to 30 carbon atoms.

The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting of only carbon and hydrogen atoms. Cycloalkyl groups may include fused, bridged or spiro ring systems. For example, the term "C" as used in the present invention _3-6 Cycloalkyl "refers to cycloalkyl groups having 3 to 6 carbon atoms. For example, cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo [2.2.1 ]Heptyl, and the like.

The term "cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group as defined above, including, but not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and the like. For example, "C _3-30 Cycloalkyl "refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.

The term "branched alkyl" refers to an alkane radical that is attached to the parent molecule and itself forms at least two branched structures.

For example

The term "alkenyl" refers to a monovalent, straight or branched chain, alkyl group consisting of only carbon and hydrogen atoms, containing at least one double bond, and attached to other moieties by a single bond, including, but not limited to, ethenyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups, and the like. For example "C _2-30 Alkenyl "means an alkenyl group containing 2 to 30 carbon atoms and having at least 1 carbon-carbon double bond [ ]>C＝C<) A monovalent straight or branched hydrocarbon group.

The term "alkenylene" refers to a divalent straight or branched chain alkane group consisting of only carbon and hydrogen atoms, containing at least one double bond, and each attached to other fragments by two single bonds, including, but not limited to, vinylidene and the like. For example, "C _2-30 Alkenylene "means an alkylene group containing 2 to 30 carbon atoms and having at least 1 carbon-carbon double bond>C＝C<) A divalent straight or branched hydrocarbon group.

The term "alkynyl" refers to monovalent, straight or branched chain, alkanyl radicals consisting of only carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and attached to other moieties by a single bond, including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. For example "C _2-30 Alkynyl "refers to a monovalent straight or branched hydrocarbon radical containing 2 to 30 carbon atoms and having at least 1 carbon-carbon triple bond.

The term "alkynylene" refers to a divalent straight or branched chain alkane group consisting of only carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and linked to other fragments, respectively, by two single bonds, including, but not limited to, ethynylene and the like. For example, "C _2-30 Alkynylene "refers to a divalent straight or branched chain hydrocarbon radical containing 2 to 30 carbon atoms and having at least 1 carbon-carbon triple bond.

The term "cycloalkenyl" refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting of only carbon and hydrogen atoms. Cycloalkenyl groups may include fused, bridged or spiro ring systems. Such as cyclopropenyl and cyclobutenyl, and the like.

The term "cycloalkenyl" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group as defined above, including, but not limited to, cyclopropenyl, cyclobutenyl, and the like. For example, "C _3-30 The "cycloalkenylene group" means a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group containing 3 to 30 carbon atoms.

The term "branched alkenyl" is an olefinic radical that is attached to the parent molecule and itself forms at least two branched structures. For example

The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g. fused, bridged or spiro) non-aromatic group, the ring atoms of which consist of carbon atoms and at least one heteroatom selected from N, O and S, wherein the S atom is optionally substituted to form S (=o), S (=o) ₂ Or S (=o) (=nr ^x )，R ^x Independently selected from H or C _1-4 An alkyl group. If valence requirements are met, the heterocyclyl may be attached to the remainder of the molecule through any one ring atom. For example, the term "3-8 membered heterocyclyl" as used herein refers to heterocyclyl groups having 3 to 8 ring atoms. For example, the heterocyclic group may be oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl or trithianyl.

The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated pi-electron system. For example, the term "C" as used in the present invention _6-10 Aryl "refers to aryl groups having 6 to 10 carbon atoms. For example, aryl groups canIs phenyl, naphthyl, anthryl, phenanthryl, acenaphthylenyl, azulenyl, fluorenyl, indenyl, pyrenyl, etc.

The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated pi-electron system, the ring atoms of which are made up of carbon atoms and at least one heteroatom selected from N, O and S. If valence requirements are met, the heteroaryl group may be attached to the remainder of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" as used in the present invention refers to heteroaryl groups having 5 to 10 ring atoms. For example, heteroaryl groups can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and its benzo derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, and the like.

The term "bridged cycloalkyl" refers to an all-carbon polycyclic ring system having two carbon atoms not directly attached between the rings, which may contain one or more double bonds within the ring, and having from 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., a 5 to 20 membered bridged cycloalkyl). The bridged cycloalkyl group is preferably a bridged cycloalkyl group having 6 to 14 carbon atoms (i.e., a 6 to 14 membered bridged cycloalkyl group), more preferably a bridged cycloalkyl group having 7 to 10 carbon atoms (i.e., a 7 to 10 membered bridged cycloalkyl group). The bridged cycloalkyl group includes a bicyclic bridged cycloalkyl group and a polycyclic bridged cycloalkyl group (e.g., a tricyclic bridged cycloalkyl group, a tetracyclic bridged cycloalkyl group, etc.), preferably a bicyclic bridged cycloalkyl group or a tricyclic bridged cycloalkyl group. Non-limiting examples include:

the cycloalkyl ring may be fused to an aryl, heteroaryl, or heterocycloalkyl ring, where the ring attached to the parent structure is cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, and the like. Cycloalkyl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate groups.

The term "heteroatom-containing alkyl" refers to an alkyl group having at least one heteroatom, the "heteroatom" being selected from oxygen, nitrogen, sulfur, phosphorus or halogen atoms, having one point of attachment.

The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

The term "hydroxy" refers to-OH.

The term "cyano" refers to-CN.

The term "amino" refers to-NH ₂ 。

The term "nitro" refers to-NO ₂ 。

The term "oxo" refers to (=o).

Unless otherwise indicated, the descriptions provided herein apply to all formulae provided herein (e.g., formula (I), including their sub-formulae) insofar as they apply.

[ preparation method ]

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

In the present invention, "proper amount" means that the amount of the solvent or the amount of the drug to be added is large in adjustable range and less affects the synthesis result, and is not particularly limited.

In the examples described below, both solvents and drugs were used in analytical or chemical purity; redistilling the solvent before use; the anhydrous solvents were treated according to standard methods or literature methods.

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other. All publications and other references mentioned herein are incorporated by reference in their entirety.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Example 1 Compound 1

Synthesis of 3- (diethylamino) propionic acid-7-butyl-17- (10-butyl-3, 9-dioxo-2, 8-dioxahexadecan-1-yl) -8,14,19-trioxo-20-aza-9, 15-dioxaicosazin-18-yl ester

The chemical structure of the compound 1 is shown as a formula (II).

The method specifically comprises the following steps:

Step 1: synthesis of Compounds 1-4

Into a 100 mL round bottom flask was charged 2, 2-dimethyl-1, 3-dioxane-5-carbaldehyde (282.6 mg,1.96mmol,1.0 eq), 3- (diethylamino) propionic acid hydrochloride (356.0 mg,1.96mmol,1.0 eq), 1-isocyanonane (300.0 mg,1.96mmol,1.0 eq), triethylamine (200.0 mg,1.96mmol,1.0 eq) and 10mL dichloromethane. After 16 hours at room temperature, the solvent was removed by concentration under reduced pressure, and the compound 1-isocyanoooctane (350.0 mg, yield 40.4%) was obtained by column chromatography. MS: M/z [ M+H ]] ⁺ ＝443.3。

Step 2: synthesis of Compounds 1-5

Compounds 1-4 (330.0 mg,0.75mmol,1.0 eq) were added sequentially to a 100 mL round bottom flask, p-toluene sulfonic acid (260.0 mg,1.50mmol,2.0 eq) and methanol (10 mL) and reacted at room temperature for 3 hours. To the reaction solution was added 20 ml of saturated sodium bicarbonate solution, and the extracts were each extracted five times with 20 ml of ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to give the compound 3- (diethylamino) propionic acid-4-hydroxy-3- (hydroxymethyl) -1- (nonylamino) -1-oxybutynin-2-yl ester (200 mg, yield 66.6%). MS: M/z [ M+H ]] ⁺ ＝403.3。

Step 3: synthesis of Compound 1

To a 100 ml round bottom flask was added in sequence 5- [ (2-butyl-1-oxyoctylidene) oxy ]Pentanoic acid (448.0 mg,1.49mmol,3.0 eq), 4-lutidine (60.7 mg,0.50mmol,1.0 eq), N, N-diisopropylethylamine (385.2 mg,2.98mmol,6.0 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (285.7 mg,1.49mmol,3.0 eq) and 15 ml dichloromethane were added after stirring for half an hour at room temperature. After 16 hours at room temperature, the solvent was removed by concentration under reduced pressure, 100 ml of water was added to dilute, and extracted three times with 100 ml of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column-chromatographed to give the compound 3- (diethylamino) propionic acid-7-butyl-17- (10-butyl-3, 9-dioxo-2, 8-dioxahexadecan-1-yl) -8,14,19-trioxymethylene-20-aza-9, 15-dioxaicosalan-18-yl ester (200.0 mg, yield 41.6%). MS: M/z [ M+H ]] ⁺ ＝967.8。

¹ H NMR(300MHz,CDCl ₃ )δ6.40-6.20(m,1H),5.31-5.22(m,1H),4.19-4.07(m,7H),3.29-3.19(m,1H),2.82-2.72(m,2H),2.60-2.43(m,7H),2.36-2.27(m,4H),1.76-1.64(m,9H),163-1.53(m,5H),1.51-1.38(m,8H),1.33-1.19(m,35H),1.05-1.01(m,6H),0.89-0.85(m,15H)。

Example 2 Compound 2

Synthesis of 4- (diethylamino) butanoic acid-7-butyl-17- (10-butyl-3, 9-dioxo-2, 8-dioxahexadeca-1-yl) -8,14,19-trioxo-20-aza-9, 15-dioxaicosazin-18-yl ester

The chemical structural formula of the compound 2 is shown in a formula III. The preparation procedure was as described in example 1, except that compound 1-1 was replaced with compound 4- (diethylamino) butanoic acid for synthesis.

/>

The yield was 56.6%. MS: M/z [ M+H ]] ⁺ ＝981.8。

¹ H NMR(400MHz,CDCl ₃ )δ6.19-6.15(m,1H),5.32-5.31(m,1H),4.18-4.15(m,2H),4.11-4.07(m,6H),3.31-3.20(m,2H),2.80-2.74(m,1H),2.54-2.41(m,8H),2.33-2.27(m,6H),1.77-1.64(m,12H),1.61-1.55(m,5H),1.50-1.38(m,7H),1.30-1.20(m,32H),1.02-0.99(m,6H),0.89-0.85(m,15H)。

EXAMPLE 3 Compound 3

Synthesis of (diethylamino) acetic acid-7-butyl-17- (10-butyl-3, 9-dioxo-2, 8-dioxahexadecan-1-yl) -8,14,19-trioxo-20-aza-9, 15-dioxaicosazin-18-yl ester

The chemical structural formula of the compound 3 is shown in the formula IV, the preparation method refers to the example 1, and the preparation method is only different from the example 1 in that the compound 1-1 is replaced by the compound N, N-diethyl glycine for reaction synthesis.

The yield was 47.9%. MS: M/z [ M+H ]] ⁺ ＝953.7。

¹ H NMR(300MHz,CDCl ₃ )δ6.22-6.19(m,1H),6.16-6.13(m,1H),5.32-5.29(m,2H),4.25-4.15(m,3H),4.12-4.07(m,5H),3.31-3.30(m,2H),3.29-3.19(m,2H),2.80-2.76(m,1H),2.66-2.61(m,4H),2.46-2.43(m,2H),2.37-2.27(m,4H),1.84-1.77(m,2H),1.74-1.64(m,8H),1.62-1.53(m,4H),1.50-1.39(m,8H),1.32-1.22(m,31H),1.06-1.03(m,6H),0.88-0.85(m,15H)。

EXAMPLE 4 Compound 4

Synthesis of 3- (diethylamino) propionic acid-7-butyl-17- ({ [ (10Z, 12Z) -1-oxoiden-octadec-9, 12-dienyl ] oxy } methyl) -8,14,19-trioxyiden-20-aza-9, 15-dioxa-icosazin-18-yl ester

The chemical structural formula of the compound 4 is shown in a formula V, and the preparation method is characterized in that the compound is synthesized by substituting linoleic acid for one of the compounds 1-6 according to the preparation method in reference to the example 1.

The yield was 35.1%. MS: M/z [ M+H ]] ⁺ ＝947.8。

¹ H NMR(400MHz,CDCl ₃ )δ6.23-6.18(m,1H),6.18-6.13(m,1H),5.50-5.20(m,6H),4.25-4.07(m,6H),3.38-3.15(m,4H),2.88-2.45(m,6H),2.37-1.74(m,16H),1.68-1.25(m,44H),1.05-0.90(m,18H)。

EXAMPLE 5 Compound 5

Synthesis of 3- (diethylamino) propionic acid-16- ({ [ (10Z, 12Z) -1-oxooctadeca-9, 12-dienyl ] oxy } methyl) -10- (octyloxy) -13, 18-dioxo-19-aza-9, 14-dioxaoctan-17-yl ester

The chemical structural formula of the compound 5 is shown in the formula VI, and the preparation method is described in the example 1, and the difference from the example 1 is that the compounds 1-6 are replaced by linoleic acid and 4, 4-bis (octyloxy) butyric acid for condensation reaction synthesis.

The yield was 37.9%. MS: M/z [ M+H ]] ⁺ ＝991.9。

¹ H NMR(400MHz,CDCl ₃ )δ6.25-6.20(m,1H),6.19-6.15(m,1H),5.86-5.45(m,3H),4.44-4.10(m,8H),3.40-3.27(m,4H),3.27-3.03(m,4H),2.95-2.68(m,4H),2.63-2.48(m,4H),2.40-1.75(m,16H),1.68-1.25(m,47H),1.02-0.89(m,18H)。

EXAMPLE 6 Compound 6

Synthesis of 2- (10-butyl-3, 9-dioxo-2, 8-dioxahexadecan-1-yl) -8-ethyl-3- ({ [ (10Z, 12Z) -octadeca-9, 12-dienyl ] amino } carbonyl) -5-oxo-8-aza-4-oxadec-1-yl 5- [ (2-butyl-1-oxooctyl) oxy ] pentanoate

The chemical structural formula of the compound 6 is shown as a formula VII.

Intermediate 6-5: (6Z, 10Z) -18-isocyanatooctadec-6, 9-diene.

/>

The method specifically comprises the following steps:

step 1: synthesis of Compound 6-2

6-1 (10.00 g,35.66mmol,1.0 eq.) N, N-dimethylformamide (0.26 g,3.57mmol,0.1 eq.) was added to a round bottom flask containing 100 ml of dichloromethane and oxalyl chloride (6.79 g,53.49mmol,1.5 eq.) was slowly added dropwise and reacted for 3 hours at 0deg.C under stirring, and the reaction solution was concentrated under reduced pressure to give crude product. Ammonia water (7.04 g,50.19mmol,1.5 eq), triethylamine (10.16 g,100.38mmol,3.0 eq) and 100 ml dichloromethane were added to a 500 ml round bottom flask, the crude product diluted with dichloromethane was slowly added dropwise at 0℃and reacted at 0℃for 3 hours with stirring, 80 ml water was added to the reaction solution, each extracted 3 times with 100 ml dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography gave the compound (10Z, 12Z) -octadeca-9, 12-dienamide (7.5 g, 80.2% yield). MS: M/z [ M+H ] ] ⁺ ＝280.3。

Step 2: synthesis of Compound 6-3

6-2 (7.50 g,26.84mmol,1.0 eq) was added to a round bottom flask containing 60 mL of tetrahydrofuran, lithium aluminum hydride (4.07 g,107.35mmol,4.0 eq) was slowly added at 0deg.C and stirred for 30 minutes, then the reaction solution was stirred at 65deg.C for 12 hours. The reaction solution was cooled to 0℃and 4.1 ml of water and then 4.1 ml of 15% aqueous sodium hydroxide solution were carefully added dropwiseThen 12.3 ml of water was added dropwise thereto, and the mixture was stirred at room temperature for 1 hour, diluted with some tetrahydrofuran, and dried with a little anhydrous sodium sulfate under stirring for 15 minutes, followed by filtration and concentration to give the compound (10Z, 12Z) -octadeca-9, 12-dien-1-amine (6.00 g, yield 84.3%). MS: M/z [ M+H ]] ⁺ ＝266.3。

Step 3: synthesis of Compound 6-4

Intermediate 6-3 (6.00 g,22.60mmol,1.0 eq) was added to 60 ml ethyl formate, heated to 65℃and refluxed for 12 hours, concentrated under reduced pressure, and column chromatographed to give the compound N- [ (6Z, 10Z) -octadec-6, 9-dien-18-yl]Methane amide (5.50 g, 82.92% yield). MS: M/z [ M+H ]] ⁺ ＝294.3。

Step 4: synthesis of Compound 6-5

To 40 mL of tetrahydrofuran was added compound 6-4 (5.50 g,18.74mmol,1.0 eq), triethylamine (9.48 g,93.70mmol,5.0 eq) and phosphorus oxychloride (4.31 g,28.11mmol,1.5 eq) slowly dropwise at 0deg.C. After the completion of the dropwise addition, the temperature was raised to 25℃and the reaction was carried out for 2 hours. Concentrating under reduced pressure, and column chromatography to give compound (6Z, 10Z) -18-isocyano-octadeca-6, 9-diene (4.30 g, 83.30% yield). MS: M/z [ M+H ] ] ⁺ ＝276.3。

Example 6 was synthesized by substituting compound 1-2 with intermediate 6-5 using the procedure employed in example 1, intermediate 6-5 being (6 z,10 z) -18-isocyano-octadeca-6, 9-diene. The yield was 48.9%. MS: M/z [ M+H ]] ⁺ ＝1089.9。

¹ H NMR(300MHz,CDCl ₃ )δ6.47-6.42(m,1H),6.28-6.23(m,1H),5.39-5.32(m,4H),4.22-4.18(m,3H),4.15-4.07(m,5H),3.28-3.25(m,1H),2.83-2.77(m,5H),2.62-2.56(m,4H),2.52-2.44(m,4H),2.40-2.29(m,4H),2.09-2.04(m,4H),1.76-1.66(m,10H),1.63-1.58(m,4H),1.52-1.42(m,6H),1.36-1.23(m,37H),1.08-1.05(m,6H),0.94-0.86(m,15H)。

EXAMPLE 7 Compound 7

Synthesis of 2-butyloctyl 10- [ 22-methyl-10- (octyloxy) -13, 19-dioxo-18, 22-diaza-9, 14-dioxatetrac-n-18-yl ] -11- (octylamino) -11-oxoundecanoate

The chemical structural formula of the compound 7 is shown in the formula VIII, the preparation procedure is described in reference to example 1, and the difference from example 1 is that the compound 1-2 is replaced by an intermediate 8-isocyanocactanoic acid-2-butyl octyl ester for reaction synthesis.

The yield was 25.5%. MS: M/z [ M+H ]] ⁺ ＝1151.9。

¹ H NMR(300MHz,CDCl ₃ )δ6.54-6.48(m,1H),6.34-6.28(m,1H),5.33-5.24(m,1H),4.21-4.20(m,2H),4.14-4.06(m,5H),3.99-3.97(m,2H),3.31-3.20(m,2H),2.88-2.83(m,1H),2.81-2.73(m,1H),2.66-2.57(m,3H),2.55-2.44(m,3H),2.38-2.29(m,6H),1.82-1.56(m,18H),1.53-1.41(m,6H),1.33-1.21(m,45H),1.09-1.06(m,6H),0.94-0.85(m,18H)。

Example 8 preparation, characterization of lipid nanoparticles and in vivo editing experimental evaluation

1. Design of animal experiment

mRNA and sgRNA delivery experiments targeting the base editor ABE8e of PCSK9

Cholesterol in blood is mainly synthesized by the liver, which is also a main organ for decomposing excessive cholesterol, and a low-density lipoprotein LDL receptor (LDLR) exists on the surface of the liver, which can be combined with cholesterol circulating back to the liver, so that the cholesterol is decomposed into bile acid, and is excreted outside the body through the intestinal tract; PCSK9 is a liver-synthesized protease capable of binding to LDL receptors, promoting their entry into hepatocytes, leading to degradation of LDL receptors by lysosomes, and a reduction in the number of LDL receptors; thus, inhibiting the activity of PSCK9, the amount of LDLR can be increased, and thus the uptake and decomposition ability of cholesterol can be enhanced. Basic and clinical studies show that the PCSK9 gene is an effective target for treating hyperlipidemia and atherosclerosis. FIG. 1 shows the changes in the number of LDL receptors and ultimately the changes in cholesterol metabolism caused before and after editing of a specific site of the PCSK9 gene.

The strategy of PCSK9 gene editing and delivery of the mouse liver cells is shown in figure 1, and the main flow is as follows: the prepared lipid nanoparticle is utilized to target and deliver mRNA and sgRNA encoding ABE8e to mouse liver cells by intravenous injection, mutation is introduced into PCSK9 genes under the action of the ABE8e and the sgRNA, the mutation of bases A to G is realized at a specific site, and the editing efficiency is calculated by sequencing.

The specific experimental design is as follows:

1.1 selecting appropriate mutation sites and editing designs

The single base editor ABE8e realizes accurate base substitution from a to G without a donor template and without DSB, based on which the first exon of the PCSK9 gene is selected as the screening mutation site. mRNA encoding a single-base editing tool ABE8e and sgRNA are delivered into an animal body together through lipid nanoparticles, the mRNA encoding the base editor ABE8e is translated into protein in cytoplasm, the protein forms a complex with the sgRNA and enters a cell nucleus, the base editor ABE8e targets a first exon splice donor site of a PCSK9 gene under the guidance of the sgRNA, adenine (A) on a first exon template strand is deaminated into inosine (I), I can be regarded as G at DNA level for reading and copying, and finally, replacement from A to G is realized, so that the splice donor site is destroyed so that a PCSK9 gene reading frame is terminated in advance.

1.2 preparation of mRNA and sgRNA of base editor ABE8e

The sequence of the first exon and the first intron of the mouse PCSK9 Gene (NCBI Gene ID: 100102) is selected as a targeting region, and a target sequence PCSK9-sgRNA for single-base editing of the PCSK9 Gene is determined.

By analyzing the sequence of the PCSK9 gene across the first exon and the intron, the sgrnas of the targeting region were designed: PCSK9-sgRNA (synthesized by auresli, south kyo) having the sequence:

PCSK9-sgRNA：5’-cccataccttggagcaacgg-3’(SEQ ID NO:1)；

the sgRNA is designed according to the target sequence and oligonucleotide (oligo) is synthesized, and the sequence of the used sgRNA is shown as SEQ ID NO. 1. CACC sequences were added to the 5 'end of the upstream sequence and AAAC sequences to the 5' end of the downstream sequence of each sgRNA. After synthesis, the upstream and downstream sequences were annealed by a preset procedure (95 ℃,5 min; 95 ℃ to 85 ℃ at-2 ℃/S;85 ℃ to 25 ℃ at-0.1 ℃/S; kept at 4 ℃) and the annealed product was ligated to the lenti U6-sgRNA/EF1a-mCherry vector (Addgene, plasmid, # 114199) linearized by BbsI (NEB, R3539S).

Wherein, the system used in the construction of the sgRNA plasmid is as follows:

the linearization system of the lenti U6-sgRNA/EF1a-mCherry vector is as follows: 3 μg of carrier; 6. Mu.L of buffer (NEB: R0539L); bbsI 2. Mu.L; ddH ₂ O was added to 60. Mu.L and digested overnight at 37 ℃.

The linkage system of the sgRNA annealing product and the linearization carrier is as follows: t4 ligase buffer (NEB: M0202L) 1. Mu.L, linearized vector 20ng, annealed oligonucleotide fragment (10. Mu.M) 5. Mu.L, T4 ligase (NEB: M0202L) 0.5. Mu.L, ddH ₂ O was made up to 10. Mu.L and ligated overnight at 16 ℃.

The ligated vector was transformed into E.coli DH5a competent cells (Geotex only, DL 1001). The specific flow is as follows: DH5 alpha competent cells were taken out from-80℃and rapidly inserted into ice for 5 min, after which the pellet was thawed, the ligation product was added and gently mixed by hand-pulling the bottom of the centrifuge tube, and left to stand in ice for 25 min. The mixture was heat-shocked in a 42℃water bath for 45 seconds, quickly returned to ice and allowed to stand for 2 minutes. To the centrifuge tube, 700. Mu.L of sterile LB medium without antibiotics was added, and after mixing, resuscitated at 37℃for 60 minutes at 200 rpm. The bacteria were harvested by centrifugation at 5000rpm for one minute, and about 100. Mu.L of the supernatant was gently swirled to resuspension the pellet and spread on LB medium of Amp antibiotics. The plates were placed in an incubator at 37℃overnight. Single colonies were picked, sequenced and confirmed, positive clones were shaken and plasmids (TIANGEN: DP 120-01) were extracted, and the concentration was determined and stored in a-20deg.C refrigerator for use.

The base editor ABE8E used in this experiment was the high efficiency base editor ABE8E evolved by David R.Liu team (Richter MF, zhao KT, eton E, lapinaite A, newby GA, thuronyi BW, wilson C, koblan LW, zeng J, bauer DE, doudna JA, liu DR.Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nat Biotechnol.2020Jul;38 (7): 883-891.doi:10.1038/s41587-020-0453-z.Epub 2020Mar 16.Erratum in:Nat Biotechnol.2020May 20;PMID:32433547;PMCID:PMC7357821). Plasmid ABE8e (Plasmid # 138489) was purchased from adedge and mRNA of ABE8e was expressed and purified for use by the laboratory.

2. The ionizable lipid or the compound of the invention/DSPC/cholesterol/PEG-lipid was prepared in a molar ratio of 50:10:38.5:1.5.

2.1 Di-oleylmethylene-4-dimethylaminobutyrate (DLin-MC 3-DMA, commonly abbreviated as MC 3) and Compound 1-Compound 7 of the present invention were dissolved in absolute ethanol at the above molar ratios with DSPC, cholesterol, PEG-DMG, respectively.

2.2 ethanol solutions of different lipid carriers were mixed with buffer of mRNA at 1:3 (volume/volume) where the mass ratio of total lipid to mRNA was 40:1,sgRNA:ABE8e mRNA (w/w) was 1:1, and nucleic acid lipid nanoparticles 1-8 were obtained by a microfluidic nano-drug manufacturing system (NanoAssemblr Ignite, canada) at a flow rate of 12 mL/min. The obtained nucleic acid lipid nanoparticles were immediately diluted 40-fold in 1 x DPBS buffer. The diluted nucleic acid lipid nanoparticle solution passes through an overspeed centrifuge tube and is concentrated to reach the target volume. After dilution, the particles were used for DLS particle size measurement and encapsulation efficiency detection.

2.3 the particle size and polydispersity index of lipid nanoparticles were determined by dynamic light scattering using Malvern Zetasizer Nano ZS (Malvern UK, malvern Zetasizer nanoparticle size analyzer) in 173 ° backscatter detection mode. The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions, and the test results for the characterization of the nanolipid particles are shown in table 2.

TABLE 2

Lipid nanoparticles	Ionizable lipid compounds	Particle size (nm)	PDI (polydispersity index)	Encapsulation efficiency (%)
					1	MC3	76.5	0.16	92.3
2	Compound 1	72.5	0.01	89.5
					3	Compound 2	73.5	0.06	97.9
4	Compound 3	74.3	0.06	76.9
					5	Compound 4	70.7	0.01	95.5
6	Compound 5	66.4	0.02	95.3
					7	Compound 6	71.6	0.01	93.5
8	Compound 7	63.9	0.01	94.6

3 in vivo editing experiment evaluation

3.1 lipid nanoparticles comprising a compound of the invention (see Table 2) encapsulating mRNA and sgRNA encoding base editor ABE8e were systemically administered to 6-7 week old C57BL/6 female mice (purchased from Jiangsu Jiujiaka) by tail vein injection at a dose of 0.2 mg/kg. Lipid nanoparticles comprising dioleylmethylene-4-dimethylaminobutyrate (DLin-MC 3-DMA, abbreviated as MC 3) encapsulating mRNA and sgRNA of base editor ABE8e were applied in a similar manner to a comparable group of mice of both week-old and sex-old as positive controls. In addition, PBS buffer was also used as a negative control for mice of comparable age and sex in a similar manner to tail vein injection.

3.2 edit efficiency detection

Editing efficiency detection was performed one week after mice were dosed, liver tissue was taken after mice were sacrificed, genome was extracted after lysis, and efficiency analysis was performed by deep sequencing.

The deep sequencing procedure was as follows:

(1) Primers were designed according to the target gene positions, and the design of the primers for targeting PCSK9 gene is shown in table 3.

TABLE 3 Table 3

Sequence object	Sequence(s)
		PCSK9-F2	5’-accagacggctagatgagca-3’(SEQ ID NO:2)
PCSK9-R2	5’-cccaggacgaggatggagatta-3’(SEQ ID NO:3)

(2) Editing efficiency detection.

The PCR procedure was as follows: 94 ℃ for 2 minutes; 98℃10s,60℃30s,68℃20s,34 cycles; 68℃for 5 minutes. After the PCR is finished, gel electrophoresis is used for verification, a single band with proper size is selected, the amplified product is determined to be correct, and the obtained PCR product is sent to Nanjing gold Style company for sequencing.

(3) And analyzing the reading of the deep sequencing result through the crispress software, analyzing the specific site, and calculating the editing efficiency, wherein the calculated result is shown in table 4, and the editing efficiency corresponding to each lipid nanoparticle can be shown in fig. 2. Table 4 is an in vivo edit efficiency assessment.

TABLE 4 Table 4

As shown in table 4, the ionizable lipid compounds employed in the present invention are capable of effectively delivering nucleic acid molecules, small molecule compounds, and the like; by contrast, the lipid nanoparticle has the advantages of better particle size distribution, high encapsulation efficiency and obviously better delivery effect than the lipid nanoparticle contrast, and can meet the in-vivo delivery requirement.

The description of the exemplary embodiments presented above is merely illustrative of the technical solution of the present invention and is not intended to be exhaustive or to limit the invention to the precise form described. Obviously, many modifications and variations are possible in light of the above teaching to those of ordinary skill in the art. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable others skilled in the art to understand, make and utilize the invention in various exemplary embodiments and with various alternatives and modifications. The scope of the invention is intended to be defined by the claims and equivalents thereof.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims (14)

1. A lipid compound or a pharmaceutically acceptable form thereof, wherein the lipid compound has a chemical structural formula as shown in formula I;

In the formula (I) of the present invention,

G ¹ is C _1-10 An alkylene group;

R ¹ 、R ² each independently is C _1-6 An alkyl group;

L ¹ 、L ² 、L ³ 、L ⁴ 、L ⁵ each independently selected from the group consisting of ester groups, amides, carbonates, carbamates, mercapto formates, ureas, phosphates, or none;

R ³ 、R ⁵ 、R ⁷ each independently selected from the group consisting of linear alkanes, branched alkanes, linear heteroatom-containing alkyl, branched heteroatom-containing alkyl, or none;

R ⁴ 、R ⁶ 、R ⁸ each independently selected from the group consisting of linear alkyl, linear alkenyl, linear alkynyl, branched alkyl, cycloalkyl, bridged cycloalkyl, or none.

2. The compound of claim 1, or a pharmaceutically acceptable form thereof, wherein:

G ¹ is thatWherein m is selected from integers from 1 to 6.

3. The compound or pharmaceutically acceptable form thereof according to claim 1 or 2, wherein:

R ¹ 、R ² together with the attached N form a 3-8 membered heterocyclic group, or R ¹ 、R ² Any one of which is with G ¹ Any carbon atom of the two are directly connected to form a 3-8 membered heterocyclic group;

preferably, R ¹ 、R ² Each independently is methyl, ethyl or propyl; alternatively, R ¹ 、R ² Together with the attached N forms a 4-, 5-or 6-membered heterocyclic group.

4. A compound according to any one of claims 1-3, or a pharmaceutically acceptable form thereof, wherein: l (L) ¹ ～L ⁵ Each independently selected from Or none.

5. The compound or pharmaceutically acceptable form thereof according to any one of claims 1-4, wherein: r is R ³ 、R ⁵ 、R ⁷ Each independently is

Wherein X is selected from O, S, se, S-S, se-Se or none;

R ¹¹ ～R ¹⁴ is H or C ₁ -C ₈ Linear alkanes of (a);

n and o are integers from 1 to 10.

6. The compound or pharmaceutically acceptable form thereof according to any one of claims 1-5, wherein R ⁴ 、R ⁶ 、R ⁸ Each independently is

Wherein Y is,Or none;

R ¹⁵ ～R ¹⁹ is H or C ₁ -C ₁₀ Linear alkanes, cycloalkanes or bridged cycloalkanes;

p and q are integers of 0 to 10.

7. The lipid compound according to any one of claims 1-6, or a pharmaceutically acceptable form thereof, wherein the lipid compound is selected from any one or a combination of at least two of the following structural compounds;

preferably, the pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs.

8. Use of a lipid compound according to any one of claims 1-7, or a pharmaceutically acceptable form thereof, for the preparation of a liposomal nanocarrier.

9. A lipid carrier comprising the lipid compound of any one of claims 1-7 or a pharmaceutically acceptable form thereof;

Preferably, the lipid carrier comprises a first lipid compound comprising the lipid compound of any one of claims 1-7 or a pharmaceutically acceptable form thereof, and optionally an ionizable lipid, and a second lipid compound comprising any one or a combination of at least two of an anionic lipid, a neutral lipid, a steroid, or a polymer-bound lipid;

preferably, the ionizable lipid is selected from: 1, 2-diiodoyloxy-N, N-dimethylaminopropane DLinDMA, 1, 2-dioleyloxy-N, N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2-diiodo-4- (2-dimethylaminoethyl) - [1,3] -dioxolane DLin-KC2-DMA, 1, 2-dioleoyl-3-trimethylammonium-propane DOTAP, 1'- (2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) di-dodecane-2-ol C12-200, 3β [ N-N' -dimethylaminoethane) -carbamoyl ] cholesterol, or N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N-trimethylamine chloride DOTMA, or a combination of at least two thereof.

10. A nucleic acid lipid nanoparticle composition comprising the lipid compound of any one of claims 1-7 or a pharmaceutically acceptable form thereof, or the lipid carrier of claim 9, and a therapeutic or prophylactic agent.

11. The nucleic acid lipid nanoparticle composition of claim 10, wherein the therapeutic or prophylactic agent comprises: any one or a combination of at least two of RNA, DNA, antisense nucleic acids, aptamers, nucleases, immunostimulatory nucleic acids, or peptide nucleic acids;

preferably, the antisense nucleic acid is an antisense oligonucleotide;

preferably, the RNA is any one or a combination of at least two of mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, a microrna inhibitor, a microrna activator or shRNA;

preferably, the RNA is modified RNA;

preferably, the mRNA comprises a sequence encoding an RNA-guided DNA binding agent;

preferably, the DNA comprises a plasmid;

preferably, the nuclease is selected from any one or a combination of at least two of Cas9, cas12, cas13, iscB, tnpB, isrB and homologs thereof.

12. A pharmaceutical composition comprising a lipid compound according to any one of claims 1-7 or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or a lipid carrier according to claim 9 or a nucleic acid lipid nanoparticle composition according to claim 10 or 11, together with a pharmaceutically acceptable adjuvant;

Preferably, the pharmaceutically acceptable auxiliary materials comprise any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifying agents, cosolvents, solubilizers, osmotic pressure regulators, pH regulators, antioxidants or buffers.

13. A pharmaceutical formulation comprising the lipid compound of any one of claims 1-7 or a pharmaceutically acceptable form thereof, or the lipid carrier of claim 9 or the nucleic acid lipid nanoparticle composition of claim 10 or 11, and a pharmaceutically acceptable adjuvant;

preferably, the pharmaceutically acceptable auxiliary materials comprise any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrating agents, emulsifying agents, cosolvents, solubilizers, osmotic pressure regulators, pH regulators, antioxidants or buffers;

preferably, the dosage form of the pharmaceutical formulation is selected from: any one or a combination of at least two of tablets, capsules, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays;

preferably, the injection comprises injection, sterile powder for injection and concentrated solution for injection.

14. Use of the lipid compound of any one of claims 1-7 or a pharmaceutically acceptable form thereof, the lipid carrier of claim 9, the nucleic acid lipid nanoparticle composition of claim 10 or 11, the pharmaceutical composition of claim 12 or the pharmaceutical formulation of claim 13 in the preparation of a nucleic acid drug, a genetic vaccine, a polypeptide or a protein drug;

preferably, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is for use in the treatment or prevention of a disease or disorder in a subject in need thereof;

preferably, the subject is a mammal;

preferably, the mammal is a human;

preferably, the disease or disorder is selected from metabolic, genetic, cancer, cardiovascular or infectious diseases;

preferably, the metabolic disease comprises familial hypercholesterolemia, the genetic disease comprises transthyretin amyloid disease, primary hyperuricemia or hereditary angioedema, and the infectious disease comprises hepatitis b.