WO2024152512A1 - Lipid compound for delivery of therapeutic agent, preparation method therefor and use thereof - Google Patents

Abstract

A lipid compound for delivery of a therapeutic agent, a preparation method therefor and a use thereof. The lipid compound is a compound having structural formula (I) or a pharmaceutically acceptable form thereof. The lipid compound may be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer conjugated lipids, to form lipid nanoparticles for delivery of therapeutic agents (e.g., nucleic acid molecules) to achieve therapeutic or prophylactic purposes (e.g., vaccination), enriching the types of ionizable lipid compounds.

Description

Lipid compounds for delivering therapeutic agents and preparation methods and applications thereof Technical Field

The present invention relates to a lipid compound for delivering a therapeutic agent (such as a nucleic acid molecule), a lipid carrier containing the same, a nucleic acid lipid nanoparticle composition and a pharmaceutical preparation, as well as their preparation methods and related applications, and belongs to the technical field of biomedicine and gene therapy.

Background technique

Gene therapy technology is a hot topic in modern biomedical research. Nucleic acid drugs can be used to prevent and treat cancer, bacterial and viral infections, and diseases with genetic causes. Since nucleic acid drugs are easily degraded and difficult to enter cells, they usually need to be encapsulated with a carrier and delivered to the target cells. Therefore, the development of safe and efficient delivery carriers has become a prerequisite for the clinical application of gene therapy.

Lipid nanoparticles (LNP) are currently a research hotspot in the field of non-viral gene vectors. In 2018, the FDA approved the LNP delivery of patisiran (onpattro) to treat hereditary transthyretin amyloidosis. Since then, the research on the delivery of nucleic acid drugs using LNP technology has shown explosive growth. In particular, at the end of 2020, the FDA approved Moderna and BioNtech & Pfizer's new coronavirus vaccines for COVID-19, both of which use LNP technology to deliver mRNA drugs, thereby achieving prevention of the SARS-CoV-2 virus.

LNPs are usually composed of four lipid compounds, namely ionizable lipids, neutral lipids, steroids, and polymer-bound lipids, among which the choice of ionizable lipids has the greatest impact on LNPs.

Currently, nucleic acid therapeutic drugs still face some challenges in their effectiveness, mainly including delivery efficiency, low cell permeability, and high sensitivity to degradation of certain nucleic acid molecules including RNA. Therefore, there is still a need to develop new lipid compounds to promote the in vitro or in vivo delivery of nucleic acid molecules for therapeutic and/or preventive purposes.

Summary of the invention

One object of the present invention is to provide a lipid compound or a pharmaceutically acceptable form thereof (e.g., salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs, etc.), which can be used together with other lipid compounds (e.g., neutral lipids, charged lipids, steroids) to prepare lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acid molecules, specifically including mRNA) to improve the delivery efficiency of nucleic acid drugs in vivo, and lipid compounds with specific structures can be selected as lipid carriers according to the organs where the nucleic acid drugs need to be enriched.

Another object of the present invention is to provide a method for preparing the lipid compound or a pharmaceutically acceptable form thereof.

Another object of the present invention is to provide a lipid carrier comprising the above compound.

Another object of the present invention is to provide a nucleic acid lipid nanoparticle composition comprising the above compound or the above lipid carrier.

Another object of the present invention is to provide a pharmaceutical preparation comprising the above compound, or the above lipid carrier, or the above nucleic acid lipid nanoparticle composition.

Another object of the present invention is to provide the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

Another object of the present invention is to provide a method for delivering a nucleic acid drug in vivo, the method comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.

Another object of the present invention is to provide a method for preventing, improving or treating a disease or condition in a subject in need thereof, the method comprising administering the nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation to the subject.

<First aspect>

The present invention provides a compound of formula I or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, etc.

in:

^G1 is a _C1-6 alkylene group;

R ¹ and R ² are each independently C _1-6 alkyl;

Optionally, R ¹ , R ² and the connected N together form a 3-8 membered heterocyclic group, or, any one of R ¹ and R ² is directly connected to any carbon atom in G ¹ to form a 3-8 membered heterocyclic group;

L ¹ is selected from Heteroatoms O, N, S or absent;

R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are each independently hydrogen, C _1-8 alkyl, or -OC _2-12 alkenyl;

a and b are each independently selected from an integer of 1-10;

L ² is selected from Heteroatoms O, N, S or absent;

R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are each independently hydrogen or C _1-8 alkyl;

c and d are each independently selected from an integer of 1-10;

L ³ selected from Heteroatoms O, N, S or absent;

R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , and R ¹⁷ are each independently hydrogen, C _1-8 alkyl, or -OC _2-12 alkenyl;

e and f are each independently selected from integers of 1-10.

According to some specific embodiments of the present invention, in Formula I, ^G1 is methylene, ethylene, propylene, butylene or pentylene.

According to some specific embodiments of the present invention, in Formula I, R ¹ and R ² are each independently methyl, ethyl or propyl; or, R ¹ , R ² and the connected N together form a 5-membered or 6-membered heterocyclic group. According to other specific embodiments of the present invention, in Formula I, one of R ¹ and R ² is directly connected to any carbon atom in G ¹ to form a 5-membered or 6-membered heterocyclic group, and the other of R ¹ and R ² is methyl, ethyl or propyl.

According to some specific embodiments of the present invention, in Formula I, G ¹ , R ¹ , and R ² correspond to the groups shown in any one of Compounds 1 to 35, respectively.

According to some specific embodiments of the present invention, in Formula I,

^L1 is or does not exist;

At least two of R ³ , R ⁴ and R ⁵ are not hydrogen, and R ⁶ and R ⁷ are hydrogen;

a is an integer selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10);

b is 2, 3, 4, 5, 6, 7, 8 or 9.

According to some specific embodiments of the present invention, in Formula I, L ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , a, and b correspond to the groups or values shown in any one of Compounds 1 to 35, respectively.

According to some specific embodiments of the present invention, in Formula I,

^L2 is or not present; preferably not present;

R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are each independently hydrogen;

c and d are each independently selected from an integer of 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

According to some specific embodiments of the present invention, the sum of c and d is 4, 5, 6, 7, 8, 9, or 10.

According to some specific embodiments of the present invention, L ² is absent; the sum of c and d is 5, 6, 7, 8, 9 or 10.

According to some specific embodiments of the present invention, in Formula I, L ² , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , c, and d correspond to the groups or values shown in any one of Compounds 1 to 35, respectively.

According to some specific embodiments of the present invention, in Formula I,

^L3 is

R ¹³ and R ¹⁴ are each hydrogen; at least two of R ¹⁵ , R ¹⁶ and R ¹⁷ are not hydrogen;

e is 2, 3, 4, 5, 6, 7, 8 or 9;

f is selected from an integer of 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

According to a specific embodiment of the present invention, in Formula I, L ³ , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , e, and f correspond to the groups or values shown in any one of Compounds 1 to 35, respectively.

In the present invention, when the values of a, b, c, d, e, and f are not 1, the corresponding multiple R ^{3 s} , multiple R ^{4 s} , multiple R ^{6 s} , multiple R ^{7 s} , multiple R ⁸ s, multiple R ⁹ s, multiple R ^{10 s} , multiple R ^{11 s} , multiple R ^{13 s} , multiple R ^{14 s} , multiple R ^{15 s} , and multiple R ^{17 s} may be independently the same or different.

According to a specific embodiment of the present invention, the compound is selected from one or more of Compounds 1 to 35 shown in Table 1.

Table 1

<Second Aspect>

The present invention provides a method for preparing the compound of the first aspect or a pharmaceutically acceptable form thereof, the method comprising:

compound of, compound of, Compounds and The compound of formula I or a pharmaceutically acceptable form thereof is synthesized by Ugi reaction;

Wherein, R ¹ -R ¹⁷ , L ¹ -L ³ , G ¹ , a, b, c, d, e, and f are as defined in the present invention above.

<Third Aspect>

The present invention also provides use of the compound described in the first aspect or a pharmaceutically acceptable form thereof in the preparation of liposome nanocarriers.

<Fourth Aspect>

The present invention provides a lipid carrier, which comprises the 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. Such lipid carriers have high encapsulation efficiency for nucleic acid drugs, greatly improving the delivery efficiency of nucleic acid drugs in vivo.

In some embodiments, the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises the 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 other ionizable lipids, and the second lipid compound comprises one or a combination of two or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids.

In some embodiments, the first lipid compound is any one of the compounds described above 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 any one of the compounds described above or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and a combination of other lipid compounds.

In some embodiments, the other lipid compounds include 1,2-dilinoleyloxy-N,N-dimethylaminopropane DLinDMA, 1,2-dioleyloxy-N,N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane DLin-KC2-DMA, 1,2-dioleoyl-3-trimethylammonium-propane DOTAP, 1 ,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)di-dodecan-2-ol C12-200, 3β[N-N’N’-dimethylaminoethane)-carbamoyl]cholesterol DC-Chol and N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylamine chloride DOTMA, or a combination of two or more thereof.

In some embodiments, the anionic lipid includes one or a combination of two or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol DOPG, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine DOPS and dimyristoylphosphatidylglycerol.

In some embodiments, the neutral lipid includes at least one of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine DOPE, 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine DSPC, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine DPPC, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine DOPC, dipalmitoylphosphatidylglycerol DPPG, oleoylphosphatidylcholine POPC, 1-palmitoyl-2-oleoylphosphatidylethanolamine POPE, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine DPPE, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine DMPE, distearoylphosphatidylethanolamine DSPE and 1-stearoyl-2-oleoylphosphatidylethanolamine SOPE, or lipids modified with anionic or cationic modifying groups. Anionic or cationic modifying groups are not limited.

In some embodiments, the steroid includes one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, coproposterol, and corticosteroids.

In some embodiments, the polymer-bound lipids include 1,2-dimyristoyl-sn-glyceromethoxy-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-20, One or more of Wen-80, 1,2-dipalmityl-sn-glycerol-methoxypolyethylene glycol PEG-DPG, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate PEG-s-DMG, PEG-dialkoxypropyl PEG-DAA, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol ester PEG-c-DOMG and N-acetylgalactosamine ((R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propylcarbamate))GalNAc-PEG-DSG.

In some embodiments, in the lipid carrier, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid 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 can be 20:20:5:50:5, 30:5:25:30:10, 20:5:5: 55:15, 65:0:9.7:25:0.3, etc.; wherein, in the first lipid compound, the molar ratio of any one of the above compounds or its pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other lipid compounds is (1-10):(0-10); illustratively, the molar ratio can 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, in the lipid carrier, the molar ratio of the first lipid compound, anionic lipids, neutral lipids, steroids and polymer-bound lipids is (20-55):(0-13):(5-25):(25-51.5):(0.5-15); wherein, in the first lipid compound, the molar ratio of any one of the above compounds or their pharmaceutically acceptable forms such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other lipid compounds is (3-4):(0-5).

<Fifth Aspect>

The present invention provides a nucleic acid lipid nanoparticle composition, which includes the 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 the lipid carrier according to the fourth aspect, and a nucleic acid drug.

In some embodiments, the nucleic acid drug comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA.

In some embodiments, the antisense nucleic acid is an antisense oligonucleic acid.

In some embodiments, the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA.

In some embodiments, the DNA comprises a plasmid.

In some embodiments, the mRNA includes a sequence encoding an RNA-guided DNA binder, more specifically, an mRNA encoding a nuclease or a base editor.

In some embodiments, the nuclease includes one or more of Cas9 (e.g., GeoCas9, CjCas9, SpCas9), CasX, CasY, Casz, Cpf1, C2c1, C2c2, C2c3, and Argonaute protein (AGO), and homologs thereof (e.g., dCas9 and nCas9).

In some embodiments, the nucleic acid drug may further include a guide RNA, specifically, the guide RNA includes a gRNA nucleic acid.

In some embodiments, the nucleic acid drug includes mRNA and gRNA encoding a nuclease or a base editor.

In some embodiments, the mRNA or gRNA is modified.

In some embodiments, the modification is selected from the group consisting of pseudouracil (ψ), N1-methylpseudouracil (N1Mψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof.

In some embodiments, the mass ratio of the nucleic acid drug to any of the compounds or their pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs is 1:(3-40).

In some embodiments, the mass ratio of the nucleic acid drug to the lipid carrier is 1:(3-40).

Exemplarily, the above mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, etc.

In some embodiments, the ratio (w/w) of the mRNA to the gRNA of the nuclease or base editor is about 10: 1 to about 1: 10. In some embodiments, the ratio (w/w) is about 3: 1 to about 1: 3. In some embodiments, the ratio (w/w) is about 3: 2.

<Sixth Aspect>

The present invention provides a pharmaceutical composition comprising a 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 fourth aspect, or a nucleic acid lipid nanoparticle composition according to the fifth aspect, and a pharmaceutically acceptable excipient, carrier or diluent.

<Seventh Aspect>

The present invention provides a pharmaceutical preparation comprising any one of the above-mentioned compounds or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or the above-mentioned lipid carrier, or the above-mentioned nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable excipient, carrier or diluent.

In some embodiments, the particle size of the pharmaceutical preparation is 30 to 500 nm. For example, the particle size can be 30 nm, 50 nm, 100 nm, 150 nm, 250 nm, 350 nm, 500 nm, etc.

In some embodiments, the encapsulation rate of the nucleic acid drug in the pharmaceutical preparation is greater than 50%. Exemplarily, the encapsulation rate can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.

<Eighth Aspect>

The present invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

The present invention also provides a method for delivering a nucleic acid drug in vivo, the method comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.

The present invention also provides a method for preventing, ameliorating or treating a disease or condition in a subject in need thereof, the method comprising administering the nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation to the subject.

In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation is administered, for example, via an enteral or parenteral administration route. In some embodiments, a dosage of about 0.001 mg/kg to about 10 mg/kg of the nucleic acid lipid nanoparticle composition or pharmaceutical preparation is administered to the subject.

In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is used to treat or prevent a disease or disorder in a subject in need thereof.

In some embodiments, the disease or condition is selected from a metabolic disease, a genetic disease, cancer, a cardiovascular disease, and an infectious disease.

In some embodiments, the metabolic disease includes familial hypercholesterolemia (FH), the hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria (PH1) and hereditary angioedema (HAE), and the infectious disease includes hepatitis B (HEPATITIS B).

In some embodiments, the subject is a mammal or a human.

The present invention provides a series of novel structural compounds of formula I, which can be used as ionizable lipids to prepare lipid carriers together with other lipid compounds, and the particle size is controllable, the distribution is uniform, and the encapsulation rate of negatively charged drugs is very high. The synthesis method of the lipid compound of the present invention is simple, the yield is high, and the synthesis can be rapid and the cost is low. The compounds of the present invention can be used to deliver nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs, enriching the types of ionizable lipid compounds, and are particularly important for the development and application of nucleic acid preventives and therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the delivery strategy for detecting the PCSK9 gene editing efficiency in mouse liver cells in the present invention.

Figure 2 shows the PCSK9 gene editing efficiency of base editors encapsulated by different lipid compounds in mouse liver cells.

Figure 3 shows the PCSK9 gene editing efficiency of base editors encapsulated by different lipid compounds in mouse liver cells.

Detailed ways

For easier understanding of the present invention, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

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

In this specification, the word "may" includes both performing a certain process and not performing a certain process.

In the present specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes cases where the event occurs and cases where it does not occur.

In this specification, the references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

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

[Definition of Terms]

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

The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is substantially non-toxic to an organism. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting the compound of the present invention with a pharmaceutically acceptable inorganic/organic acid or inorganic/organic base, and such salts are also 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, etc., 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, etc., common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, etc., and common organic bases include (but are not limited to) diethylamine, triethylamine, ethambutol, etc.

The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a vertical asymmetric plane due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby being able to rotate plane polarized light. Since there are asymmetric centers and other chemical structures that may cause stereoisomerism in the compounds of the present invention, the present invention also includes these stereoisomers and mixtures thereof. Since the compounds of the present invention and their salts include asymmetric carbon atoms, they can exist in the form of a single stereoisomer, a racemate, an enantiomer, and a mixture of diastereomers. Generally, these compounds can be prepared in the form of a racemic mixture. However, if desired, such compounds can be prepared or separated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or single stereoisomer-enriched (purity ≥98%, purity ≥95%, ≥93%, ≥90%, ≥88%, ≥85% or ≥80%) mixtures. A single stereoisomer of a compound is prepared synthetically from an optically active starting material containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds of a particular stereochemistry are either commercially available or prepared as described herein and resolved by methods well known in the art.

The term "tautomer" (or "tautomeric form") refers to structures of different energies that are interconvertible via a low energy barrier. Isomers. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (or prototropic tautomers) include, but are not limited to, interconversions via proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-imine alcohol isomerization, etc. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

The term "solvate" refers to a substance formed by the combination of a compound of the present invention or a pharmaceutically acceptable salt thereof with at least one solvent molecule through non-covalent intermolecular forces. Common solvates include (but are not limited to) hydrates, ethanolates, acetoneates, etc.

The term "chelate" refers to a complex having a ring structure, which is 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, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

The term "prodrug" refers to a derivative compound that can directly or indirectly provide a compound of the present invention after being applied to a patient. Particularly preferred derivative compounds or prodrugs are compounds that can increase the bioavailability of the compound of the present invention when administered to a patient (e.g., more easily absorbed into the blood), or compounds that promote the delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compounds of the present invention are within the scope of the present invention, and various prodrug forms are well known in the art.

The term "independently" means that at least two groups (or ring systems) with the same or similar value ranges in the structure may have the same or different meanings in specific circumstances. For example, substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl or aryl. When substituent X is hydrogen, substituent Y can be either hydrogen or halogen, hydroxyl, cyano, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X can be either hydrogen or halogen, hydroxyl, cyano, alkyl or aryl.

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

The term "alkyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing no unsaturation, and connected to other fragments by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl and tert-butyl, etc. For example, "C _1-30 alkyl" refers to a saturated monovalent straight or branched hydrocarbon group containing 1 to 30 carbon atoms.

The term "alkylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing no saturation, and connected to other fragments by two single bonds, including (but not limited to) methylene, 1,1-ethylene and 1,2-ethylene, etc. For example, "C _1-30 alkylene" refers to a saturated divalent straight or branched 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 only of carbon atoms and hydrogen atoms. The cycloalkyl group may include a paracyclic, bridged or spirocyclic ring system. For example, the term "C _3-6 cycloalkyl" used in the present invention refers to a cycloalkyl group having 3 to 6 carbon atoms. For example, the cycloalkyl group may be a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo[2.2.1]heptyl group, etc.

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, and cycloheptylene, etc. For example, "C _3-30 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, and cycloheptylene. A divalent group obtained by removing a hydrogen atom from a cycloalkyl group having 30 to 50 carbon atoms.

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

The term "alkenyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by a single bond, including (but not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl and isobutenyl groups. For example, " _C2-30 alkenyl" refers to a monovalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

The term "alkenylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by two single bonds, including (but not limited to) vinylene, etc. For example, " _C2-30 alkenylene" refers to a divalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

The term "alkynyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by a single bond, including (but not limited to) ethynyl, propynyl, butynyl and pentynyl groups. For example, " _C2-30 alkynyl" refers to a monovalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one 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 only of carbon atoms and hydrogen atoms. Cycloalkenyl may include cyclic, bridged or spirocyclic systems. For example, cyclopropenyl and cyclobutenyl, etc.

The term "cycloalkenylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group as defined above, including, but not limited to, cyclopropenylene and cyclobutenylene, etc. For example, " _C3-30 cycloalkenylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group containing 3 to 30 carbon atoms.

The term "branched alkenyl" refers to an alkene radical attached to a parent molecule and forming at least two branched structures. For example

The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as a bicyclic, for example, fused, bridged or spiro) non-aromatic group, whose ring atoms 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)(= ^NRx ), and ^Rx is independently selected from H or _C1-4 alkyl. If the valence bond requirements are met, the heterocyclyl can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "3-8 membered heterocyclyl" used in the present invention refers to a heterocyclyl having 3 to 8 ring atoms. For example, the heterocyclyl group can be oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidonyl, 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 π electron system. For example, the term "C _6-10 aryl" used in the present invention refers to an aryl group having 6 to 10 carbon atoms. For example, the aryl group can be phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthenyl, azulenyl, fluorenyl, indenyl, pyrenyl, etc.

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

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 _-NH2 .

The term "nitro" refers to _-NO2 .

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

The term "subject" includes humans and non-human animals. Non-human animals include vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, cats, horses, cows, chickens, dogs, mice, rats, goats, rabbits and pigs. Preferably, the subject is a human. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

The term "nuclease" refers to an enzyme that catalyzes the cleavage of phosphodiester bonds between nucleotides in a nucleic acid molecule. In some embodiments, the nuclease is selected from a meganuclease, a zinc finger nuclease (ZFNs), a TAL-effector DNA binding domain-nuclease fusion protein (TALEN), and an RNA-guided nuclease or a variant thereof in which the nuclease activity has been reduced or inhibited.

In some embodiments, the RNA-guided nuclease is a naturally occurring CRISPR-Cas protein or an active variant or fragment thereof. CRISPR-Cas systems are classified as class I or class II systems. Class II systems contain single effector nucleases and include types II, V, and VI. Each category is further subdivided into types (types I, II, III, IV, V, VI), some of which are further divided into subtypes (e.g., type II-A, type II-B, type II-C, type V-A, type V-B).

The term "Type II CRISPR-Cas protein", "Type II CRISPR-Cas effector protein" or "Cas9" refers to a CRISPR-Cas effector protein that requires a trans-activating RNA (tracrRNA) and comprises two nuclease domains (RuvC and HNH), each of which is responsible for cutting a single strand of a double-stranded DNA molecule. In other embodiments, the CRISPR-Cas protein is a naturally occurring Type V CRISPR-Cas protein or an active variant or fragment thereof.

As used herein, the term "V-type CRISPR-Cas protein", "V-type CRISPR-Cas effector protein" or "Cas12" refers to a CRISPR-Cas effector protein that cuts dsDNA and comprises a single RuvC nuclease domain or a split RuvC nuclease domain and lacks an HNH domain. In other embodiments, the CRISPR-Cas protein is a naturally occurring VI-type CRISPR-Cas protein or an active variant or fragment thereof. As used herein, the term "VI-type CRISPR-Cas protein", "VI-type CRISPR-Cas effector protein" or "Cas13" refers to a CRISPR-Cas effector protein that does not require tracrRNA and comprises two HEPN domains that cut RNA.

The term "gRNA" refers to a nucleotide sequence that has sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and guide the sequence-specific binding of an associated nuclease to the target nucleotide sequence. For CRISPR-Cas enzymes, the corresponding guide RNA is one or more RNA molecules (usually one or two) that can bind to the Cas enzyme and guide the Cas enzyme to bind to a specific target nucleotide sequence, and in those cases where the Cas enzyme has nickase or nuclease activity, also cut the target nucleotide sequence.

[Preparation]

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

In the present invention, "appropriate amount" means that the amount of the added solvent or the amount of the drug can be adjusted in a wide range and has little effect on the synthesis result, and no specific limitation is made.

In the following examples, all solvents and chemicals used were analytically pure or chemically pure; all solvents were redistilled before use; and all anhydrous solvents were treated according to standard methods or literature methods.

Example

If the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

Example 1. Synthesis of Compound 10-(7-butyl-23-methyl-10,18-dioxy-19,23-diaza-9-oxa-tetracosane-19-yl)-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester

Step 1 Synthesis of Compound 1-2

In a 100 ml round-bottom flask, add n-octylamine (10.00 g, 77.38 mmol, 1.0 eq) and ethyl formate (30 mL). Heat to 60 degrees Celsius, react for 16 hours, and remove the solvent by vacuum distillation to obtain the compound N-octyl methane amide (12.10 g, yield 98.63%). It was used directly in the next reaction without further purification.

Step 2: Synthesis of Compound 1-3

Compound 1-2 (12.00 g, 76.31 mmol, 1.0 eq), triethylamine (63.47 mL, 457.86 mmol, 6.0 eq) and tetrahydrofuran (100 mL) were added to a 250 mL three-necked flask in sequence, and phosphorus oxychloride (18.72 g, 122.10 mmol, 1.6 eq) was slowly added dropwise under an ice bath. After the addition was completed, the reaction was continued at zero degrees Celsius for 3 hours. 100 mL of saturated sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 100 mL of ethyl acetate respectively. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 1-isocyanooctane (8.00 g, yield 75.3%). MS: m/z[M+H] ⁺ =140.1.

Step 3 Synthesis of Compound 1-6

Into a 100 ml round-bottom flask, azelaic acid (10.10 g, 53.65 mmol, 5.0 eq), 4-lutidine (0.66 g, 5.37 mmol, 0.5 eq), N,N-diisopropylethylamine (13.87 g, 107.30 mmol, 10.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.09 g, 16.09 mmol, 1.5 eq) and 50 ml of dichloromethane were added in sequence, and 2-butyloctan-1-ol (2.00 g, 10.73 mmol, 1.0 eq) was added after stirring at room temperature for half an hour. After reacting at room temperature for 16 hours, the solvent was removed by concentration under reduced pressure, and the mixture was diluted with 100 ml of water and extracted three times with 100 ml of ethyl acetate respectively. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to obtain compound 9-[(2-butyloctyl)oxy]-9-oxyylidenenonanoic acid (3.00 g, yield 78.4%).

Step 4 Synthesis of Compound 1-8

In a 100 ml flask, 10-hydroxydecanoic acid (5.00 g, 26.56 mmol, 1.0 eq), dihydropyran (3.35 g, 39.84 mmol, 1.5 eq), p-toluenesulfonic acid (0.23 g, 1.33 mmol, 0.05 eq) and tetrahydrofuran (50 mL) were added. The mixture was reacted at room temperature for 16 hours, concentrated under reduced pressure, and column chromatography was performed to obtain compound 10-(3,4,5,6-tetrahydro-2H-pyran-2-yloxy)decanoic acid (6.30 g, yield 87.1%).

Step 5 Synthesis of Compound 1-9

Compound 1-8 (6.30 g, 23.13 mmol, 1.0 eq), 2-butyloctan-1-ol (6.46 g, 34.70 mmol, 1.5 eq), 4-lutidine (1.41 g, 11.56 mmol, 0.5 eq), N,N-diisopropylethylamine (8.97 g, 69.39 mmol, 3.0 eq) were added to a round-bottom flask containing 60 ml of dichloromethane, and finally 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.65 g, 34.70 mmol, 1.5 eq) was added, and the mixture was reacted at room temperature for 16 hours. After vacuum concentration, the compound 10-(3,4,5,6-tetrahydro-2H-pyran-2-yloxy)decanoic acid-2-butyloctyl ester (5.30 g, yield 52.0%) was obtained by column chromatography.

Step 6 Synthesis of Compound 1-10

In a 100 ml flask, add 20 ml of ethanol, compound 1-9 (5.30 g, 12.03 mmol, 1.0 eq) and p-toluenesulfonic acid (2.07 g, 12.03 mmol, 1.0 eq). Stir at room temperature for 16 hours. Concentrate under reduced pressure to remove the solvent, and obtain compound 10-hydroxydecanoic acid-2-butyloctyl ester (1.60 g, yield 37.3%) by column chromatography.

Step 7 Synthesis of Compound 1-11

Compound 1-10 (0.80 g, 2.24 mmol, 1.0 eq) and sodium bicarbonate (0.47 g, 5.60 mmol, 2.5 eq) were added to a round-bottom flask containing 10 ml of dichloromethane, and Dess-Martin periodinane (1.14 g, 2.69 mmol, 1.2 eq) was added in batches under an ice bath. After the addition was completed, the reaction was continued at 0 degrees Celsius for 2 hours, quenched by adding saturated sodium thiosulfate solution, extracted three times with 30 ml of dichloromethane, and the total The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain compound 9-formylnonanoic acid-2-butyloctyl ester (570.0 mg, yield 71.6%).

Step 8 Synthesis of Compound 1

In a 25 ml round-bottom flask, 5 ml of methanol, compound 1-11 (150.0 mg, 0.42 mmol, 1.0 eq), and 3-(dimethylamino)propan-1-amine (43.0 mg, 0.42 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 1-6 (0.18 g, 0.50 mmol, 1.2 eq) was added, and compound 1-3 (58.0 mg, 0.42 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 10-(7-butyl-23-methyl-10,18-dioxy-19,23-diaza-9-oxa-tetracosane-19-yl)-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester (210.0 mg, yield 53.1%). MS: m/z[M+H] ⁺ =934.8. ¹ H NMR (400MHz, CDCl ₃ ) δ6.68 (s, 1H), 4.72 (s, 1H), 3.97 (d, J = 4.0Hz, 4H), 3.47-3.10 (m, 4H), 2.55-2.10 (m, 14H), 2.07-1.52 (m, 15H), 1.50-1.12 (m, 57H) ),1.05-0.78(m,15H).

Example 2: Synthesis of Compound 9-{7-[(8Z,11Z)-heptadeca-8,11-dienyl]-2-methyl-8-oxyylidene-2,6,9-triazaheptadecan-6-yl}-9-oxyylidenenonanoic acid-2-butyloctyl ester

In a 25 ml round-bottom flask, 5 ml of methanol, compound 2-1 (130.0 mg, 0.49 mmol, 1.0 eq), and 3-(dimethylamino)propan-1-amine (50.0 mg, 0.49 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 1-6 (0.21 g, 0.59 mmol, 1.2 eq) was added, and compound 1-3 (68.0 mg, 0.49 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 9-{7-[(8Z,11Z)-heptadeca-8,11-dienyl]-2-methyl-8-oxygen-2,6,9-triazaheptadecan-6-yl}-9-oxygen-nonanoic acid-2-butyloctyl ester (150.0 mg, yield 36.3%). MS: m/z[M+H] ⁺ =844.8. ¹ H NMR (400MHz, CDCl ₃ ) δ6.69 (s, 1H), 5.50-5.20 (m, 4H), 4.72 (s, 1H), 3.97 (d, J = 4.0Hz, 2H), 3.47-3.10 (m, 4H), 2.85-2.70 (m, 2H), 2.50-1.52 (m, 27H) ,1.50-1.02(m,48H),0.98-0.75(m,12H).

Example 3. Synthesis of Compound 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester

In a 25 ml round-bottom flask, 5 ml of methanol, compound 1-11 (150.0 mg, 0.42 mmol, 1.0 eq), and 3-(diethylamino)propan-1-amine (55.0 mg, 0.42 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hour, compound 1-6 (0.18 g, 0.50 mmol, 1.2 eq) was added. After stirring at room temperature for 0.5 hour, compound 1-3 (58.0 mg, 0.42 mmol, 1.0 eq) was added. The reaction solution was stirred at room temperature for 0.5 hour. The mixture was stirred for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester (130.0 mg, yield 31.9%). MS: m/z[M+H] ⁺ =962.9. ¹ H NMR (400MHz, CDCl ₃ ) δ6.66 (s, 1H), 4.72 (s, 1H), 3.97 (d, J = 4.0Hz, 4H), 3.50-2.80 (m, 6H), 2.78-2.23 (m, 11H), 2.00-1.52 (m, 13H), 1.50-0.98 (m, 64H) ),0.96-0.75(m,15H).

Example 4. Synthesis of Compound 10-[7-butyl-10,18-dioxy-22-(tetrahydro-1H-pyrrol-1-yl)-19-aza-9-oxadocosan-19-yl]-11-(octylamino)-11-oxyylideneundecanoic acid-2-butyloctyl ester

In a 25 ml round-bottom flask, 5 ml of methanol, compound 1-11 (150.0 mg, 0.42 mmol, 1.0 eq), and 3-(tetrahydro-1H-pyrrol-1-yl)propan-1-amine (66.0 mg, 0.42 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 1-6 (0.18 g, 0.50 mmol, 1.2 eq) was added, and compound 1-3 (58.0 mg, 0.42 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 10-[7-butyl-10,18-dioxy-22-(tetrahydro-1H-pyrrol-1-yl)-19-aza-9-oxa-docosapentaenoic acid-19-yl]-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester (70.5 mg, yield 17.2%). MS: m/z[M+H] ⁺ =960.9. ¹ H NMR (400MHz, CDCl ₃ ) δ6.73 (s, 1H), 4.74 (s, 1H), 3.98 (d, J = 4.0Hz, 4H), 3.50-3.05 (m, 5H), 2.90-2.51 (m, 5H), 2.48-2.20 (m, 6H), 2.20-1.55 (m, 17H) ,1.48-1.05(m,59H),0.96-0.75(m,15H).

Example 5. Synthesis of Compound 10-(7-butyl-22-ethyl-10,18-dioxy-19,22-diaza-9-oxa-tetracosane-19-yl)-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester

In a 25 ml round-bottom flask, 5 ml of methanol, compound 1-11 (150.0 mg, 0.42 mmol, 1.0 eq), and 2-(diethylamino)ethan-1-amine (49.0 mg, 0.42 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 1-6 (0.18 g, 0.50 mmol, 1.2 eq) was added, and compound 1-3 (58.0 mg, 0.42 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 10-(7-butyl-22-ethyl-10,18-dioxy-19,22-diaza-9-oxa-tetracosane-19-yl)-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester (121.0 mg, yield 29.9%). MS: m/z[M+H] ⁺ =948.9. ¹ H NMR (400MHz, CDCl ₃ ) δ6.73 (s, 1H), 4.72 (s, 1H), 3.99 (d, J = 4.0Hz, 4H), 3.50-3.05(m,4H),2.75-1.85(m,12H),1.78-1.52(m,14H),1.50-1.01(m,62H),0.98-0.75(m,15H).

Example 6. Synthesis of the compound (10Z, 12Z)-N-[3-(dimethylamino)propyl]-N-[(10Z, 13Z)-1-(octylamino)-1-oxyylidene nonadeca-10,13-dien-2-yl]octadeca-9,12-dienamide

In a 25 ml round-bottom flask, 5 ml of methanol, compound 2-1 (150.0 mg, 0.57 mmol, 1.0 eq), and compound 1-12 (58.0 mg, 0.57 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 6-1 (0.19 g, 0.68 mmol, 1.2 eq) was added, and compound 1-3 (79.0 mg, 0.57 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound (10Z, 12Z)-N-[3-(dimethylamino)propyl]-N-[(10Z, 13Z)-1-(octylamino)-1-oxyylidene 19-10,13-diene-2-yl] 18-9,12-dieneamide (110.0 mg, yield 25.2%). MS: m/z[M+H] ⁺ =768.7. ¹ H NMR (400MHz, CDCl ₃ ) δ6.75(s,1H),5.50-5.25(m,8H),4.72(s,1H),3.50-2.86(m,10H),2.78-2.52(m,8H),2.50-2.25(m,7H),2.12-1.75(m,12H), 1.47-1.12(m,37H),1.00-0.75(m,9H).

Example 7. Synthesis of the compound 2-butyloctanoate-7-butyl-19-[3-(diethylamino)propyl]-20-[(octylamino)carbonyl]-8,18-dioxyylidene-19-aza-9-oxanonacosan-29-yl ester

Step 1: Synthesis of compound 7-3

2-Butyloctanoic acid (6.00g, 29.95mmol, 1.0eq), 4-dimethylaminopyridine (3.66g, 29.95mmol, 1.0eq), N,N-diisopropylethylamine (7.74g, 59.9mmol, 2.0eq) were added to a round-bottom flask containing 60 ml of DMF, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.46g, 38.94mmol, 1.5eq) was added. After reacting at room temperature for half an hour, decan-1,10-diol (10.44g, 59.9mmol, 2.0eq) was added, and then reacted at room temperature for 2.5 hours. The reaction solution was concentrated under reduced pressure, diluted with 100 ml of water, extracted with 100 ml of ethyl acetate three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography The compound 2-butyloctanoic acid-10-hydroxydecyl ester (4.30 g, yield 40.3%) was obtained.

Step 2: Synthesis of compound 7-4

Compound 7-3 (2.00 g, 5.61 mmol, 1.0 eq) and sodium bicarbonate (1.18 g, 14.03 mmol, 2.5 eq) were added to a round-bottom flask containing 50 ml of dichloromethane, and Dess-Martin periodinane (3.09 g, 7.29 mmol, 1.3 eq) was added in batches under an ice bath. After the addition was completed, the reaction was continued at 0 degrees Celsius for 2 hours, quenched by adding saturated sodium thiosulfate solution, extracted three times with 30 ml of dichloromethane, respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 2-butyloctanoic acid-9-formylnonyl ester (1.99 g, yield 75.43%).

Step 3: Synthesis of compound 7-6

In a round-bottom flask filled with 50 ml of tetrahydrofuran, 9-hydroxynonanoic acid (5.00 g, 28.70 mmol, 1.0 eq), sodium hydroxide (1.15 g, 28.70 mmol, 1.0 eq), and tetrabutylammonium iodide (1.06 g, 2.87 mmol, 0.1 eq) were added in sequence. After the reaction solution was stirred at room temperature for 0.5 hours, benzyl bromide (5.89 g, 34.44 mmol, 1.5 eq) was slowly added dropwise. After the addition was completed, the temperature was raised to 70 degrees Celsius and the reaction was allowed to react for 16 hours. The mixture was concentrated under reduced pressure, diluted with 100 ml of water, extracted three times with 50 ml of ethyl acetate respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain the compound 9-hydroxynonanoic acid benzyl ester (2.30 g, yield 30.3%).

Step 4: Synthesis of compound 7-7

Compound 7-2 (2.07 g, 10.32 mmol, 1.3 eq), 4-dimethylaminopyridine (0.49 g, 3.97 mmol, 0.5 eq), N,N-diisopropylethylamine (2.05 g, 15.88 mmol, 2.0 eq) were added to a round-bottom flask containing 50 ml of dichloromethane, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.83 g, 9.53 mmol, 1.2 eq) was added. After reacting at room temperature for half an hour, compound 7-6 (2.1 g, 7.94 mmol, 1.0 eq) was added and reacted at room temperature for 15.5 hours. The reaction solution was concentrated under reduced pressure, diluted with 100 ml of water, extracted three times with 100 ml of ethyl acetate respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain compound 9-[(2-butyl-1-oxyoctyl)oxy]nonanoic acid benzyl ester (3.55 g, yield 62.0%).

Step 5: Synthesis of Compound 7-8

Compound 7-7 (2.30 g, 5.15 mmol, 1.0 eq) and palladium carbon (1.10 g, 1.03 mmol, 1.0 eq, 10% purity) were added into a round-bottom flask containing 30 ml of ethanol, and a hydrogen balloon was added. The flask was ventilated three times, reacted at room temperature for 16 hours, filtered, and concentrated under reduced pressure to obtain compound 9-[(2-butyl-1-oxyoctyl)oxy]nonanoic acid (1.80 g, yield 98.0%).

Step 6: Synthesis of compound 7

In a 25 ml round-bottom flask, 5 ml of methanol, compound 3-1 (73.0 mg, 0.56 mmol, 1.0 eq), and compound 7-4 (200.0 mg, 0.56 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 7-8 (200.0 mg, 0.56 mmol, 1.0 eq) was added, and compound 1-3 (78.0 mg, 0.56 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 2-butyloctanoic acid-7-butyl-19-[3-(diethylamino)propyl]-20-[(octylamino)carbonyl]-8,18-dioxy-19-aza-9-oxa-nonacosan-29-yl ester (115.0 mg, yield 21.2%). MS: m/z[M+H] ⁺ =962.8. ¹ H NMR (400MHz, CDCl ₃ ) δ6.65(s,1H),4.70(s,1H),4.25-3.98(m,4H),3.25-2.50(m,10H),2.48-2.22(m,5H),2.05-1.75(m,3H),1.73-1.05(m,76H),0. 98-0.75(m,15H).

Example 8 Synthesis of Compound 10-[7-butyl-20-(1-methylpiperidin-3-yl)-10,18-dioxy-19-aza-9-oxaicosane-19-yl]-11-(octylamino)-11-oxyylideneundecanoic acid-2-butyloctyl ester

In a 25 ml round-bottom flask, 5 ml of methanol, compound 1-11 (150.0 mg, 0.42 mmol, 1.0 eq), and compound 8-1 (53.8 mg, 0.42 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 1-6 (0.18 g, 0.50 mmol, 1.2 eq) was added, and compound 1-3 (58.0 mg, 0.42 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 10-[7-butyl-20-(1-methylhexahydropyridin-3-yl)-10,18-dioxy-19-aza-9-oxaicosane-19-yl]-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester (105.0 mg, yield 26.0%). MS: m/z[M+H] ⁺ =960.8. ¹ H NMR (400MHz, CDCl ₃ ) δ6.75 (s, 1H), 4.73 (s, 1H), 3.96 (d, J = 4.0Hz, 4H), 3.61-3.08 (m, 5H), 2.97-2.50 (m, 5H), 2.44-2.20 (m, 6H), 2.20-1.50 (m, 16H) ,1.48-1.05(m,60H),0.98-0.75(m,15H).

Example 9. Synthesis of Compound 10-[7-butyl-21-(1-methyltetrahydro-1H-pyrrol-2-yl)-10,18-dioxy-19-aza-9-oxahenedecane-19-yl]-11-(octylamino)-11-oxyylideneundecanoic acid-2-butyloctyl ester

In a 25 ml round-bottom flask, 5 ml of methanol, compound 1-11 (150.0 mg, 0.42 mmol, 1.0 eq), and compound 9-1 (53.8 mg, 0.42 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 1-6 (0.18 g, 0.50 mmol, 1.2 eq) was added, and compound 1-3 (58.0 mg, 0.42 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 10-[7-butyl-21-(1-methyltetrahydro-1H-pyrrol-2-yl)-10,18-dioxy-19-aza-9-oxahistane-19-yl]-11-(octylamino)-11-oxyundecanoic acid-2-butyloctyl ester (98.0 mg, yield 24.3%). MS: m/z[M+H] ⁺ =960.8. ¹ H NMR (400MHz, CDCl ₃ ) δ6.68 (s, 1H), 4.75 (s, 1H), 3.90 (d, J = 4.0Hz, 4H), 3.55-3.00 (m, 5H), 2.99-2.52 (m, 5H), 2.50-2.25 (m, 6H), 2.18-1.55 (m, 17H) ,1.44-1.03(m,59H),0.98-0.75(m,15H).

Example 10. Synthesis of Compound 10-[7-butyl-20-(1-methyltetrahydro-1H-pyrrol-3-yl)-10,18-dioxy-19-aza-9-oxaicosane-19-yl]-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester

In a 25 ml round-bottom flask, 5 ml of methanol, compound 1-11 (150.0 mg, 0.42 mmol, 1.0 eq), and compound 10-1 (47.9 mg, 0.42 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 1-6 (0.18 g, 0.50 mmol, 1.2 eq) was added, and compound 1-3 (58.0 mg, 0.42 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 10-[7-butyl-20-(1-methyltetrahydro-1H-pyrrol-3-yl)-10,18-dioxy-19-aza-9-oxa-eicosane-19-yl]-11-(octylamino)-11-oxyylidene undecanoic acid-2-butyloctyl ester (210.0 mg, yield 52.1%). MS: m/z[M+H] ⁺ =946.9. ¹ H NMR (400MHz, CDCl ₃ ) δ7.23 (s, 1H), 4.49 (s, 1H), 3.98 (d, J = 4.0Hz, 4H), 3.52-3.00 (m, 6H), 2.99-2.20 (m, 14H), 2.10-1.80 (m, 4H), 1.78-1.05 (m, 66H) ,0.98-0.75(m,15H).

Example 11. Synthesis of Compound 10-({9-[(2-butyloctyl)oxy]-1,9-dioxyylidenonyl}(1-methylpiperidin-4-yl)amino)-11-(octylamino)-11-oxyylideneundecanoic acid-2-butyloctyl ester

In a 25 ml round-bottom flask, 5 ml of methanol, compound 2-1 (150.0 mg, 0.42 mmol, 1.0 eq), and compound 11-1 (47.9 mg, 0.42 mmol, 1.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 1-6 (0.18 g, 0.50 mmol, 1.2 eq) was added, and compound 1-3 (58.0 mg, 0.57 mmol, 1.0 eq) was added after stirring at room temperature for 0.5 hours. The reaction solution was stirred at room temperature for 16 hours, diluted with 50 ml of water, extracted three times with 30 ml of n-hexane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 10-({9-[(2-butyloctyl)oxy]-1,9-dioxyylidenonyl}(1-methylhexahydropyridin-4-yl)amino)-11-(octylamino)-11-oxyylideneundecanoic acid-2-butyloctyl ester (130.0 mg, yield 32.2%). MS: m/z[M+H] ⁺ =946.9. ¹ H NMR (400MHz, CDCl ₃ ) δ7.18 (s, 1H), 4.49 (s, 1H), 3.98 (d, J = 4.0Hz, 4H), 3.60-2.96 (m, 6H), 2.93-2.15 (m, 14H), 2.11-1.91 (m, 4H), 1.78-1.10 (m, 66H) ,0.98-0.75(m,15H).

Example 12: Synthesis of 2-butyloctyl 10-(7-butyl-23-methyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-(octylamino)-11-oxyylideneundecanoate

Compound 12-3: 3-[Ethyl(methyl)amino]propan-1-amine

Step 1: Synthesis of compound 12-2

In a 100 ml round-bottom flask, 25 ml acetonitrile, compound 12-1 (4.00 g, 14.92 mmol, 1.0 eq), N-ethylmethylamine hydrochloride (2.14 g, 22.38 mmol, 1.5 eq), potassium carbonate (6.19 g, 44.76 mmol, 3.0 eq) were added in sequence. The reaction solution was heated under reflux and stirred for 16 hours, diluted with 40 ml water, extracted three times with 40 ml ethyl acetate respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 2-(3-ethylmethylamino)propyl-2,3-dihydro-1H-isoindole-1,3-dione (1.80 g, yield 48.8%). MS: m/z[M+H] ⁺ =247.1.

Step 2: Synthesis of compound 12-3

In a 100 ml round-bottom flask, 30 ml of anhydrous ethanol, compound 12-2 (1.80 g, 7.31 mmol, 1.0 eq), and hydrazine hydrate (1.72 g, 29.24 mmol, 4.0 eq, 85% purity) were added in sequence. The reaction solution was heated under reflux and stirred for 16 hours, cooled to 0 degrees Celsius, filtered and concentrated to obtain compound 3-[ethyl(methyl)amino]propan-1-amine (0.50 g, yield 58.9%). MS: m/z[M+H] ⁺ =117.1.

Example 12 was synthesized by Ugi reaction of compound 1-6, compound 1-3, compound 1-11 and compound 12-3 using the method used in Example 1. Yield 41%. MS: m/z [M+H] ⁺ = 948.9. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.66 (s, 1H), 4.69 (s, 1H), 3.95 (d, J = 4.0 Hz, 4H), 3.34-3.15 (m, 4H), 2.38-2.19 (m, 12H), 1.95-1.61 (m, 13H), 1.42-1.27 (m, 62H), 1.10-1.00 (m, 3H), 0.98-0.75 (m, 15H).

Example 13 Synthesis of 2-butyloctyl 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxacosan-19-yl)-11-(octylamino)-11-oxyylideneundecanoate

Compound 13-3: 3-[Ethyl(propyl)amino]propan-1-amine

Step 1: Synthesis of compound 13-2

In a 100 ml round-bottom flask, 25 ml of acetonitrile, compound 13-1 (5.00 g, 18.65 mmol, 1.0 eq), N-ethyl-n-propylamine (2.40 g, 27.97 mmol, 1.5 eq), potassium carbonate (7.73 g, 55.95 mmol, 3.0 eq) were added in sequence, and the reaction solution was heated under reflux. The mixture was stirred for 16 hours, diluted with 40 ml of water, extracted three times with 40 ml of ethyl acetate respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain the compound 2-(3-(ethyl(propyl)amino)propyl)isoindoline-1,3-dione (4.20 g, yield 82.1%). MS: m/z[M+H] ⁺ =275.2.

Step 2: Synthesis of compound 13-3

In a 100 ml round-bottom flask, 30 ml of anhydrous ethanol, compound 12-2 (2.00 g, 7.29 mmol, 1.0 eq), and hydrazine hydrate (1.72 g, 29.16 mmol, 4.0 eq, 85% purity) were added in sequence. The reaction solution was heated under reflux and stirred for 16 hours, cooled to 0 degrees Celsius, filtered and concentrated to obtain compound 3-[ethyl(methyl)amino]propan-1-amine (0.60 g, yield 57.0%). MS: m/z[M+H] ⁺ =145.2.

Example 13 was synthesized by a four-component Ugi reaction of compound 1-6, compound 1-3, compound 1-11 and compound 13-3 using the method used in Example 1. Yield: 37.3%. MS: m/z[M+H] ⁺ =976.9. ¹ H NMR (400 MHz, CDCl ₃ )δ6.65(s,1H),4.69(s,1H),3.96(d,J=8.0Hz,4H),3.23-3.14(m,5H),2.37-2.27(m,12H),1.95-1.43(m,18H),1.32-1.17(m,54H),1.02-0.98(m,3H),0.94-0.81(m,15H).

Example 14 Synthesis of 2-butyloctyl 10-(7-butyl-24-ethyl-10,18-dioxy-19,24-diaza-9-oxacosan-19-yl)-11-(octylamino)-11-oxyylideneundecanoate

Example 14 was synthesized by a four-component Ugi reaction of compound 1-6, compound 1-3, compound 1-11 and 4-(diethylamino)butan-1-amine using the method used in Example 1. Yield: 31.6%. MS: m/z [M+H] ⁺ = 976.9. ¹ H NMR(400MHz,CDCl ₃ )δ6.68(s,1H),4.66(s,1H),3.95(d,J＝5.8Hz,4H),3.38-3.12(m,4H),3.10-2.94(m,4H),2.90–2.77(m,2H),2.42–2.21(m,6H),2.05(d,J＝37.1Hz,2H),1.91(d,J＝8.9Hz,1H),1.76(d,J＝6.9Hz,3H),1.61(s,10H),1.42(d,J＝5.8Hz,3H),1.31(dd,J＝22.7,15.0Hz,59H),0.87(dd,J＝6.3,4.6Hz,17H)。

Example 15 Synthesis of 2-propylheptyl 10-(22-ethyl-9,17-dioxy-6-propyl-18,22-diaza-8-oxacosan-18-yl)-11-(octylamino)-11-oxyylideneundecanoate

Compound 15-1: 2-propylheptyl 9-formylnonanoate

Compound 15-1 was prepared from 2-propylheptan-1-ol using the method for synthesizing compound 1-11.

Compound 15-2: 9-Oxylidene-9-[(2-propylheptyl)oxy]nonanoic acid

Compound 15-2 was prepared from 2-propylheptan-1-ol and azelaic acid using the method for synthesizing compound 1-6.

Example 15 was synthesized by a four-component Ugi reaction of compound 15-2, compound 1-3, compound 15-1 and compound 3-1 using the method used in Example 1. Yield 35.8%. MS: m/z[M+H] ⁺ =906.8. ¹ H NMR (400MHz, CDCl ₃ )δ6.65(s,1H),4.72(s,1H),3.98(d,J＝5.7Hz,4H),3.48–2.98(m,4H),2.50(s,3H),2.39(d,J＝4.5Hz,3H),2.33–2.14(m,4H),1.96(s,1H),1.63(s,11H),1.34(dd,J＝46.2,24.8Hz,52H),1.10–0.57(m,23H).

Example 16 Synthesis of 2-hexyldecyl 10-(25-ethyl-9-hexyl-12,20-dioxy-21,25-diaza-11-oxaheptacosane-21-yl)-11-(octylamino)-11-oxyylideneundecanoate

Compound 16-1: 9-Formylnonanoic acid-2-hexyldecyl ester

Compound 16-1 was prepared from 2-hexyldecan-1-ol using the method for synthesizing compound 1-11.

Compound 16-2: 9-[(2-hexyldecyl)oxy]-9-oxyylidenenonanoic acid

Compound 16-2 was prepared from 2-hexyldecan-1-ol and azelaic acid using the method for synthesizing compound 1-6.

Example 16 was synthesized by a four-component Ugi reaction of compound 16-2, compound 1-3, compound 16-1 and compound 3-1 using the method used in Example 1. Yield: 40.3%. MS: m/z [M+H] ⁺ = 1075.0. ¹ H NMR (400MHz, CDCl ₃ ) δ6.66 (s, 1H), 4.72 (s, 1H), 3.98 (d, J = 5.6Hz, 4H), 3.40-3.06 (m, 4H), 2.50 (s, 3H), 2.38 (s, 3H), 2.33-2.22 (m, 4H), 1.96 (s, 1H), 1.69(d,J＝49.2Hz,11H),1.48-1.10(m,76H),1.07-0.97(m,6H),0.89(t,J＝6.5Hz,17H).

Example 17 Synthesis of 2-butyloctyl 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-(heptylamino)-11-oxyylideneundecanoate

Compound 17-1: 1-Isocyanoheptane

Compound 17-1 was prepared from n-heptylamine using the method for synthesizing compound 1-3.

Example 17 was synthesized by a four-component Ugi reaction of compound 1-6, compound 17-1, compound 1-11 and compound 3-1 using the method used in Example 1. Yield 41.1%. MS: m/z [M+H] ⁺ = 948.9. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.66 (s, 1H), 4.70 (s, 1H), 3.97 (d, J = 4.0 Hz, 4H), 3.39-3.17 (m, 3H), 3.05-2.30 (m, 8H), 1.70-1.55 (m, 17H), 1.40-1.20 (m,63H),0.92-0.85(m,15H).

Example 18 Synthesis of 2-butyloctyl 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-(nonylamino)-11-oxyylideneundecanoate

Compound 18-1: 1-Isocyanononane

Compound 18-1 was prepared from nonan-1-amine using the method for synthesizing compound 1-3.

Example 18 was synthesized by a four-component Ugi reaction of compound 1-6, compound 18-1, compound 1-11 and compound 3-1 using the method used in Example 1. Yield 40.0%. MS: m/z[M+H] ⁺ =976.9. ¹ H NMR (400 MHz, CDCl ₃ )δ6.65(s,1H),4.71(s,1H),3.96(d,J＝4.0Hz,4H),3.23-3.14(m,3H),2.51-2.27(m,10H),1.98-1.33(m,17H),1.30-1.19(m,59H),1.04-0.96(m,6H),0.92-0.82(m,15H).

Example 19: Synthesis of 2-butyloctyl 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-(decylamino)-11-oxyylideneundecanoate

Compound 19-1: 1-isocyanodecane

Compound 19-1 was prepared from decan-1-amine using the method for synthesizing compound 1-3.

Example 19 was synthesized by a four-component Ugi reaction of compound 1-6, compound 19-1, compound 1-11 and compound 3-1 using the method used in Example 1. Yield: 44.2%. MS: m/z [M+H] ⁺ = 990.9. ¹ H NMR (400MHz, CDCl ₃ ) δ6.66 (s, 1H), 4.72 (s, 1H), 3.98 (d, J = 5.7Hz, 4H), 3.44-3.06 (m, 4H), 2.53 (s, 3H), 2.39 (d, J = 4.8Hz, 3H), 2.30 (t, J = 7.4Hz, 4H), 1 .96(s,1H),1.63(s,13H),1.34(dd,J＝47.9,25.1Hz,64H),1.04(s,6H),0.90(d,J＝4.0Hz,15H).

Example 20. Synthesis of 2-butyloctyl 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-[(6-methoxy-6-oxyylidenehexyl)amino]-11-oxyylideneundecanoate

Compound 20-1: Methyl 6-isocyanohexanoate

Compound 20-1 was prepared from methyl 6-aminohexanoate using the method for synthesizing compound 1-3.

Example 20 was synthesized by a four-component Ugi reaction of compound 1-6, compound 20-1, compound 1-11 and compound 3-1 using the method used in Example 1. Yield: 36.9%. MS: m/z [M+H] ⁺ = 978.8. ¹ H NMR (400MHz, CDCl ₃ ) δ6.70 (s, 1H), 4.70 (s, 1H), 3.97 (d, J = 5.3Hz, 4H), 3.67 (d, J = 1.6Hz, 3H), 3.41-3.12 (m, 4H), 2.49 (dd, J = 13.5, 6.5Hz, 4H), 2.43-2.21 (m,9H),1.95(s,1H),1.65(dd,J＝23.2,16.9Hz,14H),1.46(dd,J＝14.8,7.4Hz,3H),1.31(d,J＝23.5Hz,49H),1.01(t,J＝7.0Hz,6H),0.96-0.70(m,12H).

Example 21, 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-[(4-methoxy-4-oxyylidenebutyl)amino]-11-oxyylideneundecanoic acid-2-butyloctyl ester

Compound 21-1: Methyl 4-isocyanobutyrate

Compound 21-1 was prepared from methyl 4-aminobutyrate using the method for synthesizing compound 1-3.

Example 21 was synthesized by a four-component Ugi reaction of compound 1-6, compound 21-1, compound 1-11 and compound 3-1 using the method used in Example 1. Yield: 28.7%. MS: m/z [M+H] ⁺ = 950.8. ¹ H NMR (400MHz, CDCl ₃ ) δ6.80 (s, 1H), 4.69 (s, 1H), 3.97 (d, J = 5.6Hz, 4H), 3.67 (s, 3H), 3.25 (dd, J = 22.0, 15.0Hz, 4H), 2.51 (s, 3H), 2.32 (dd, J = 19.1, 16.1 Hz, 9H), 1.95 (s, 1H), 1.88-1.71 (m, 5H), 1.62 (s, 10H), 1.31 (d, J = 24.2Hz, 50H), 1.03 (s, 5H), 0.90 (d, J = 3.7Hz, 11H).

Example 22: Synthesis of 2-butyloctyl 9-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-10-(octylamino)-10-oxydecanoate

Compound 22-1: 8-Formyloctanoic acid-2-butyloctyl ester

Compound 22-1 was prepared from 9-hydroxynonanoic acid using the method for synthesizing compound 1-11.

Example 22 was synthesized by a four-component Ugi reaction of compound 1-6, compound 1-3, compound 22-1 and compound 3-1 using the method used in Example 1. Yield 47.1%. MS: m/z [M+H] ⁺ = 948.9. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.65 (s, 1H), 4.70 (s, 1H), 3.96 (d, J = 8.0 Hz, 4H), 3.25-3.14 (m, 3H), 2.55-2.26 (m, 8H), 1.97-1.54 (m, 14H), 1.43-1.09 (m, 65H), 0.92-0.82 (m, 15H).

Example 23. Synthesis of 2-butyloctyl 10-(7-butyl-22-ethyl-10,17-dioxy-18,22-diaza-9-oxacosan-18-yl)-11-(octylamino)-11-oxyylideneundecanoate

Compound 23-1: 8-[(2-butyloctyl)oxy]-8-oxyoctanoic acid

Compound 23-1 was prepared from suberic acid using the method for synthesizing compound 1-6.

Example 23 was synthesized by a four-component Ugi reaction of compound 23-1, compound 1-3, compound 1-11 and compound 3-1 using the method used in Example 1. Yield: 32.9%. MS: m/z[M+H] ⁺ =948.9. ¹ H NMR (400 MHz, CDCl ₃ )δ6.63(s,1H),4.70(s,1H),3.96(d,J=8.0Hz,4H),3.77-3.11(m,4H),2.50-2.26(m,12H),1.97-1.56(m,12H),1.44-1.17(m,57H),1.02-0.97(m,6H),0.94-0.84(m,15H).

Example 24: Synthesis of 2-butyloctyl 10-(7-butyl-24-ethyl-10,19-dioxy-20,24-diaza-9-oxacosan-20-yl)-11-(octylamino)-11-oxyylideneundecanoate

Compound 24-1: 10-[(2-butyloctyl)oxy]-10-oxydecanoic acid

Compound 24-1 was prepared from sebacic acid using the method for synthesizing compound 1-6.

Example 24 was synthesized by a four-component Ugi reaction of compound 24-1, compound 1-3, compound 1-11 and compound 3-1 using the method used in Example 1. Yield 35.5%. MS: m/z[M+H] ⁺ =976.9. ¹ H NMR (400 MHz, CDCl ₃ )δ6.64(s,1H),4.70(s,1H),3.96(d,J＝4.0Hz,4H),3.34-3.08(m,2H),2.52-2.01(m,9H),1.75-1.54(m,10H),1.43-1.07(m,70H),1.02-0.97(m,6H),0.94-0.80(m,15H).

Example 25. Synthesis of 2-propylheptyl 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-(nonylamino)-11-oxyylideneundecanoate

Example 25 was synthesized by the four-component Ugi reaction of compound 1-6, compound 18-1, compound 15-1 and compound 3-1 using the method used in Example 1. Yield 52.3%. MS: m/z[M+H] ⁺ =948.9. ¹ H NMR (400 MHz, CDCl ₃ )δ6.64(s,1H),4.70(s,1H),3.96(d,J＝4.0Hz,4H),3.38-3.11(m,4H),2.51-2.21(m,11H),1.72-1.55(m,13H),1.46-1.40(m,2H),1.33-1.17(m,57H),1.02-0.98(m,6H),0.91-0.83(m,15H).

Example 26. Synthesis of 2-hexyldecyl 4-({11-[2-(diethylamino)ethyl]-12-{6-[(2-hexyldecyl)oxy]-6-oxyylidenehexyl}-3,10,13-trioxyylidene-11-aza-2-oxatridecan-13-yl}amino)butyrate

Compound 26-6: 2-Hexyldecyl 4-isocyanobutyrate

Step 1: Synthesis of compound 26-3

Compound 26-1 (6.29 g, 30.93 mmol, 1.5 eq), 2-hexyldec-1-ol (5.00 g, 20.62 mmol, 1.0 eq), 4-lutidine (2.52 g, 20.62 mmol, 1.0 eq), N,N-diisopropylethylamine (7.99 g, 61.86 mmol, 3.0 eq) were added to a round-bottom flask containing 60 ml of dichloromethane, and finally 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.93 g, 30.93 mmol, 1.5 eq) was added, and the mixture was reacted at room temperature for 16 hours. After vacuum concentration, the compound 4-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)butyric acid-2-hexyldecyl ester (7.20 g, yield 81.6%) was obtained by column chromatography. MS: m/z [M+H] ⁺ = 428.4.

Step 2: Synthesis of compound 26-4

Compound 26-3 (7.20 g, 16.84 mmol, 1.0 eq) and dioxane hydrochloride solution (4 M, 40 mL) were added to a 100 mL round-bottom flask, stirred at room temperature for 2 hours, and concentrated under reduced pressure to obtain compound 4-aminobutyric acid-2-hexyldecyl ester hydrochloride (7.2 g, yield 91.4%). MS: m/z [M+H] ⁺ = 328.3.

Step 3: Synthesis of compound 26-5

Compound 26-4 (7.20 g, 15.39 mmol, 1.0 eq) and ethyl formate (50 mL) were added to a 100 mL flask, refluxed overnight, and concentrated under reduced pressure to obtain compound 4-(formylamino)butyric acid-2-hexyldecyl ester (2.63 g, yield 30.8%). MS: m/z [M+H] ⁺ = 356.3.

Step 4: Synthesis of compound 26-6

Compound 26-5 (2.63 g, 7.39 mmol, 1.0 eq), triethylamine (6.15 mL, 44.34 mmol, 6.0 eq) and tetrahydrofuran (20 mL) were added in sequence in a 50 mL three-necked flask, and phosphorus oxychloride (1.71 g, 11.82 mmol, 1.6 eq) was slowly added dropwise under an ice bath. After the addition was completed, the reaction was continued at zero degrees Celsius for 3 hours. 100 mL of saturated sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 30 mL of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 1-isocyanooctane (1.45 g, yield 57.9%).

Compound 26-7: 2-Hexyldecyl 6-formylhexanoate

Compound 26-7 was prepared from 7-hydroxyheptanoic acid and 2-hexyldecan-1-ol using the method for synthesizing compound 1-11.

Example 26 was synthesized by a four-component Ugi reaction of 8-methoxy-8-oxyoctanoic acid, compound 26-6, compound 26-7 and compound 5-1 using the method used in Example 1. Yield: 35%. MS: m/z [M+H] ⁺ = 992.8. ¹ H NMR (400MHz, CDCl ₃ ) δ6.78 (s, 1H), 4.73 (s, 1H), 3.97 (d, J = 4.0Hz, 4H), 3.69 (s, 3H), 3.62-3.07 (m, 3H), 3.05-2.58 (m, 6H), 2.48-2.22 (m, 8H), 2.11-1 .76(m,3H),1.73-1.50(m,10H),1.43-1.02(m,62H),0.98-0.75(m,12H).

Example 27. Synthesis of 2-hexyldecyl 4-({11-[3-(diethylamino)propyl]-12-{6-[(2-hexyldecyl)oxy]-6-oxyylidenehexyl}-3,10,13-trioxyylidene-11-aza-2-oxatridecan-13-yl}amino)butyrate

Example 27 was synthesized by a four-component Ugi reaction of 8-methoxy-8-oxydecanedioic acid, compound 26-6, compound 26-7 and compound 3-1 using the method used in Example 1. Yield: 55%. MS: m/z [M+H] ⁺ = 1006.9. ¹ H NMR (400MHz, CDCl ₃ ) δ6.80 (s, 1H), 4.71 (s, 1H), 3.97 (d, J = 4.0Hz, 4H), 3.67 (s, 3H), 3.50-3.13 (m, 3H), 3.12-2.51 (m, 6H), 2.48-2.20 (m, 8H), 2.10-1 .75(m,3H),1.73-1.50(m,10H),1.48-1.02(m,64H),0.98-0.75(m,12H).

Example 28. Synthesis of 2-hexyldecyl 4-({9-[3-(diethylamino)propyl]-10-{6-[(2-hexyldecyl)oxy]-6-oxyylidenehexyl}-3,8,11-trioxyylidene-9-aza-2-oxaundecane-11-yl}amino)butyrate

Example 28 was synthesized by a four-component Ugi reaction of 6-methoxy-6-oxyylidenehexanoic acid, compound 26-6, compound 26-7 and compound 3-1 using the method used in Example 1. Yield 68.0%. MS: m/z [M+H] ⁺ = 978.8. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.81 (s, 1H), 4.71 (s, 1H), 3.97 (d, J = 4.0 Hz, 4H), 3.67 (s, 3H), 3.55-2.75 (m, 10H), 2.50-2.12 (m, 9H), 2.10-1.50 (m, 15H), 1.48-1.02 (m, 56H), 0.98-0.75 (m, 15H).

Example 29. Synthesis of 2-butyloctyl 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosane-19-yl)-11-({9-[(2-butyloctyl)oxy]-9-oxyylidenonyl}amino)-11-oxyylideneundecanoate

Compound 29-1: 2-Butyloctyl 9-isocyanonanoate

Compound 29-1 was prepared from 9-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)nonanoic acid using the method for synthesizing compound 26-6.

Example 29 was synthesized by a four-component Ugi reaction of compound 1-6, compound 29-1, compound 1-11 and compound 3-1 using the method used in Example 1. Yield: 43.5%. MS: m/z [M+H] ⁺ = 1175.1. ¹ H NMR (400MHz, CDCl ₃ ) δ6.65 (s, 1H), 4.71 (s, 1H), 3.97 (d, J = 4.0Hz, 5H), 3.36-3.15 (m, 4H), 2.49-2.28 (m, 11H), 2.00-1.90 (m, 1H), 1.78-1.56 (m, 16H) ),1.49-1.41(m,2H),1.36-1.18(m,73H),1.03-0.98(m,6H),0.93-0.83(m,18H).

Example 30: Synthesis of 2-hexyldecyl 8-(23-ethyl-9-hexyl-12,18-dioxy-19,23-diaza-11-oxapentacosane-19-yl)-9-(nonylamino)-9-oxyylidenenonanoate

Compound 30-1: 7-Formylheptanoic acid-2-hexyldecyl ester

Compound 30-1 was prepared from 8-hydroxyoctanoic acid and 2-hexyldecan-1-ol using the method for synthesizing compound 1-11.

Compound 30-2: 7-[(2-hexyldecyl)oxy]-7-oxyylideneheptanoic acid

Compound 30-2 was prepared from pimelic acid and 2-hexyldecan-1-ol using the method for synthesizing compound 1-6.

Example 30 was synthesized by a four-component Ugi reaction of compound 30-2, compound 18-1, compound 30-1 and compound 3-1 using the method used in Example 1. Yield 45.3%. MS: m/z[M+H] ⁺ =1032.9. ¹ H NMR (400 MHz, CDCl ₃ )δ6.63(s,1H),4.73(s,1H),4.05-3.88(m,4H),3.31-3.15(m,3H),2.44-2.30(m,10H),1.98-1.33(m,20H),1.30-1.19(m,68H),1.04-0.96(m,6H),0.92-0.82(m,15H).

Example 31. Synthesis of 2-butyloctyl 10-(7-butyl-23-ethyl-10,18-dioxy-19,23-diaza-9-oxapentacosan-19-yl)-11-({8-[(2-butyloctyl)oxy]-8-oxyoctyl}amino)-11-oxyundecanoate

Compound 31-1: 7-Formylheptanoic acid-2-hexyldecyl ester

Compound 31-1 was prepared from 8-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)octanoic acid using the method for synthesizing compound 26-6.

Example 31 was synthesized by a four-component Ugi reaction of compound 1-6, compound 31-1, compound 1-11 and compound 3-1 using the method used in Example 1. Yield: 39.8%. MS: m/z [M+H] ⁺ = 1161.0. ¹ H NMR (400MHz, CDCl ₃ ) δ6.65 (s, 1H), 4.69 (s, 1H), 3.96 (d, J = 8.0Hz, 5H), 3.38-3.12 (m, 4H), 2.53-2.26 (m, 11H), 1.99-1.89 (m, 1H), 1.77-1.54 (m, 16H) ),1.47-1.39(m,3H),1.33-1.17(m,71H),1.02-0.98(m,6H),0.93-0.84(m,18H).

Example 32: Synthesis of 2-butyloctyl 10-[(3Z)-18-ethyl-10-{[(5Z)-oct-5-enyl]oxy}-13-oxydeoxy-14,18-diaza-9-oxa-eicos-3-en-14-yl]-11-(nonylamino)-11-oxydeoxyundecanoate

Compound 32-3: 4,4-bis{[(5Z)-oct-5-enyl]oxy}butanoic acid

Step 1: Synthesis of compound 32-2

At room temperature, 4,4-dimethoxybutyronitrile (25.00 g, 193.56 mmol, 1.0 eq), cis-5-octen-1-ol (49.63 g, 387.12 mmol, 2.0 eq) and 4-methylbenzenesulfonate pyridine (2.43 g, 9.68 mmol, 2.0 eq) were placed in a sealed pot, heated and stirred to 110 degrees Celsius, and reacted for 72 hours. The reaction solution was extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 12 grams of the title compound, as well as a monosubstituted intermediate and raw material. The intermediate and raw material were placed in a sealed pot again and stirred at 110 degrees Celsius for 72 hours. The above operation was repeated and combined to obtain 4,4-bis[(5Z)-octadecyl-5-en-1-yloxy]butyronitrile (26.52 g, 41.8%). MS: m/z[M+H] ⁺ =357.3. ¹ HNMR (400MHz-CDCl ₃ ) δ5.42-5.28(m,4H),4.57(t,J＝5.3Hz,1H),3.61(dt,J＝9.3,6.6Hz,2H),3.44(dt,J＝9.3,6.6Hz,2H),2.42(t,J＝7.3Hz,2H),2.12-1 .90(m,9H),1.66-1.54(m,5H),0.97(t,J＝7.5Hz,6H).

Step 2: Synthesis of compound 32-3

At room temperature, compound 6-2 (2.00 g, 6.22 mmol, 1.0 eq), potassium hydroxide (1.05 g, 18.66 mmol, 3.0 eq), ethanol (6mL), water (6mL) were added to a stuffy pot, heated to 110 degrees Celsius, and reacted overnight. The reaction solution was concentrated to dryness, diluted with water, neutralized with 1N HCl aqueous solution to pH = 5, extracted with ethyl acetate three times, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4,4-bis[(5Z)-octadecyl-5-en-1-yloxy]butyric acid (1.50 g, yield 70.8%). ¹ H NMR (400MHz, CDCl ₃ ) δ5.41-5.27(m,4H),4.45(t,J＝5.6Hz,1H),3.51(dt,J＝9.0,6.7Hz,2H),3.39(dt,J＝9.0,6.7Hz,2H),2.43(t,J＝7.2Hz,2H),2.12-1 .98(m,8H),1.81(q,J＝7.3Hz,2H)1.65-1.49(m,4H),1.44-1.32(m,4H),0.94(t,J＝7.5Hz,6H).

Example 32 was synthesized by a four-component Ugi reaction of compound 32-3, compound 18-1, compound 1-11 and compound 3-1 using the method used in Example 1. Yield: 41.1%. MS: m/z [M+H] ⁺ = 960.8. ² .05-1.93(m,2H),1.83-1.52(m,15H),1.49-1.41 ₍ m,2H),1.36-1.03(m,52H),1.01-0.95(m,15H).

Example 33. Synthesis of 7-butyl-19-[3-(diethylamino)propyl]-20-[(octylamino)carbonyl]-10,18-dioxyylidene-19-aza-9-oxadocosan-22-yl 4,4-bis{[(5Z)-oct-5-enyl]oxy}butyrate

Compound 33-1 was synthesized by the method employed in Example 1 via a four-component Ugi reaction of compound 1-6, compound 1-3, 3-hydroxypropanal, and compound 3-1.

Compound 33-1 (500.0 mg, 0.733 mmol, 1.0 eq), compound 32-3 (374.4 mg, 1.10 mmol, 1.5 eq), 4-dimethylaminopyridine (89.6 mg, 0.733 mmol, 1.0 eq), N,N-diisopropylethylamine (284.3 mg, 2.20 mmol, 3.0 eq) were added to a round-bottom flask containing 10 ml of dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (210.9 mg, 1.10 mmol, 1.5 eq) was added, and the reaction was carried out at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, diluted with 100 ml of water, extracted three times with 30 ml of ethyl acetate respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain the compound 4,4-bis{[(5Z)-oct-5-enyl]oxy}butyric acid-7-butyl-19-[3-(diethylamino)propyl]-20-[(octylamino)carbonyl]-10,18-dioxy-19-aza-9-oxadocosan-22-yl ester (270.5 mg, yield 36.7%). MS: m/z[M+H] ⁺ =1004.9. ¹ H NMR (400MHz, CDCl ₃ ) δ6.54(s,1H),5.38-5.24(m,4H),4.69(s,1H),4.51-4.47(m,1H),3.89-3.21(m,10H),2.77-2.34(m,12H),1.95-1.51(m,18H), 1.36-1.03(m,51H),1.01-0.95(m,15H).

Example 34. Synthesis of 7-butyl-19-[3-(diethylamino)propyl]-20-[(octylamino)carbonyl]-10,18-dioxyylidene-19-aza-9-oxatricosan-23-yl 4,4-bis{[(5Z)-oct-5-enyl]oxy}butyrate

Example 34 is prepared by the method used in Example 33, wherein compound 1-6, compound 1-3, 4-hydroxybutyraldehyde and compound 3-1 are reacted to obtain the product of Ugi reaction, and then subjected to condensation reaction with compound 32-3. Yield 48.8%. MS: m/z[M+H] ⁺ =1018.9. ¹ H NMR (400MHz, CDCl ₃ )δ6.54(s,1H),5.45-5.20(m,4H),4.71(s,1H),4.54-4.46(m,1H),3.95-3.22(m,10H),2.80-2.42(m,12H),2.12-1.47(m,23H),1.33-1.02(m,48H),0.99-0.83(m,15H).

Example 35. Synthesis of 7-butyl-20-[3-(diethylamino)propyl]-19-[(nonylamino)carbonyl]-10,21-dioxyylidene-20-aza-9-oxapentacosane-25-yl 4,4-bis{[(5Z)-oct-5-enyl]oxy}butyrate

Compound 35-1 was synthesized by the method employed in Example 1 via a four-component Ugi reaction of 5-hydroxyvaleric acid, compound 1-3, compound 1-11, and compound 3-1.

Compound 35-1 (330.0 mg, 0.45 mmol, 1.0 eq), compound 32-3 (229.8 mg, 0.67 mmol, 1.5 eq), 4-dimethylaminopyridine (55.0 mg, 0.45 mmol, 1.0 eq), N,N-diisopropylethylamine (174.5 mg, 1.35 mmol, 3.0 eq) were added to a round-bottom flask containing 10 ml of dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (128.4 mg, 0.67 mmol, 1.5 eq) was added, and the reaction was carried out at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, diluted with 100 ml of water, extracted three times with 30 ml of ethyl acetate respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain the compound 4,4-bis{[(5Z)-oct-5-enyl]oxy}butyric acid-7-butyl-20-[3-(diethylamino)propyl]-19-[(nonylamino)carbonyl]-10,21-dioxyylidene-20-aza-9-oxapentacosane-25-yl ester (190.1 mg, yield 39.8%). MS: m/z[M+H] ⁺ =1060.9. ¹ H NMR (400MHz, CDCl ₃ ) δ6.63(s,1H),5.45-5.22(m,4H),4.72(s,1H),4.61-4.48(m,1H),4.05-3.88(m,4H),3.33-3.10(m,4H),2.44-2.25(m,10H),1 .85-1.32(m,17H),1.30-0.96(m,64H),0.98-0.86(m,15H).

Example 36. Preparation and characterization of lipid nanoparticles

1. Lipid nanoparticles were prepared by mixing ionizable lipid or the compound of the present invention/DSPC/cholesterol/PEG-lipid at a molar ratio of 50:10:38.5:1.5.

Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated as MC3) and compound 1 to compound 35 of the present invention were dissolved in anhydrous ethanol with DSPC, cholesterol, and PEG-DMG according to the above molar ratios. Luciferase mRNA (L-6107, TriLink BioTechnologies, Inc.) was dissolved in 100mM enzyme-free citric acid buffer at pH 4 (mRNA concentration was 0.2mg/mL). The ethanol solutions of different lipid carriers were mixed with the mRNA buffer at a ratio of 1:3 (volume/volume) (wherein the mass ratio of total lipids to mRNA was 40:1), and nucleic acid lipid nanoparticles were obtained by passing through a microfluidic nanodrug manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12ml/min. The obtained nucleic acid lipid nanoparticles were immediately diluted into 1×DPBS buffer at a volume of 40 times. The diluted nucleic acid lipid nanoparticle solution was passed through an ultracentrifuge tube and concentrated to the target volume. After dilution, it was used for DLS particle size measurement and encapsulation efficiency detection.

2. Determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscattering detection mode Particle size and polydispersity index of lipid nanoparticles.

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. The test results are shown in Table 2.

Table 2

Example 37: Animal Experiment

mRNA and sgRNA delivery experiment of base editor ABE8e targeting PCSK9

The cholesterol in the blood is mainly synthesized by the liver, which is also the main organ for breaking down excess cholesterol. On the surface of the liver, there is a low-density lipoprotein LDL receptor (LDLR), which can bind to the cholesterol circulating back to the liver and convert the cholesterol into It is decomposed into bile acids and excreted from the body through the intestines; PCSK9 is a protease synthesized by the liver that can bind to LDL receptors, promote LDL receptors to enter liver cells, and cause LDL receptors to be degraded by lysosomes, resulting in a decrease in the number of LDL receptors; therefore, inhibiting the activity of the enzyme PSCK9 can increase the number of LDLRs, thereby enhancing the ability to absorb and decompose cholesterol. Basic and clinical studies have shown that the PCSK9 gene is an effective target for the treatment of hyperlipidemia and atherosclerosis. Figure 1 shows the changes in the number of LDL receptors before and after editing of specific sites in the PCSK9 gene and the resulting changes in cholesterol metabolism.

The strategy for gene editing and delivery of PCSK9 in mouse liver cells is shown in Figure 1. The main process is as follows: mRNA and sgRNA encoding ABE8e are delivered to mouse liver cells through intravenous injection using the prepared lipid nanoparticles. Under the action of ABE8e and sgRNA, mutations are introduced into the PCSK9 gene, and mutations from base A to G are achieved at specific sites. The editing efficiency is calculated by sequencing.

The specific experimental design is as follows:

1. Select appropriate mutation sites and editing design

The single-base editor ABE8e achieves precise base replacement from A to G without the need for a donor template and without causing DSB. Based on this, the first exon of the PCSK9 gene was selected as the mutation site for screening. The mRNA encoding the single-base editing tool ABE8e and sgRNA were co-delivered into the animal body through lipid nanoparticles. The mRNA encoding the base editor ABE8e was translated into protein in the cytoplasm, and entered the nucleus after forming a complex with the sgRNA. Under the guidance of the sgRNA, the base editor ABE8e targeted the splicing donor site of the first exon of the PCSK9 gene, deaminated adenine (A) on the first exon template chain into inosine (I), and I was read and replicated as G at the DNA level, ultimately achieving the replacement of A to G, thereby destroying the splicing donor site and causing the PCSK9 gene reading frame to terminate prematurely.

2. Preparation of mRNA and sgRNA for base editor ABE8e

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

By analyzing the sequence across the first exon and intron of the PCSK9 gene, an sgRNA targeting the region was designed: PCSK9-sgRNA (synthesized by Nanjing GenScript Co., Ltd.), and the sequence of the PCSK9-sgRNA is:

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

According to the target sequence, sgRNA was designed and oligonucleotides were synthesized. The sgRNA sequence used is shown in SEQ ID NO: 1. CACC sequence was added to the 5' end of the upstream sequence of each sgRNA, and AAAC sequence was added to the 5' end of the downstream sequence. If the first base at the 5' end of the upstream sequence is not G, CACCG was added, and C was added to the 3' end of the corresponding downstream sequence. After synthesis, the upstream and downstream sequences were annealed by a preset program (95℃, 5min; 95℃-85℃ at -2℃/s; 85℃-25℃ at -0.1℃/s; maintained at 4℃), and the annealed products were connected to the lenti U6-sgRNA/EF1a-mCherry vector (Addgene, Plasmid#114199) linearized with BbsI (NEB, R3539S).

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

The linearization system of lenti U6-sgRNA/EF1a-mCherry vector is as follows: 3 μg of vector; 6 μL of buffer (NEB: R0539L); 2 μL of BbsI; ddH ₂ O to 60 μL, and enzyme digestion at 37°C overnight.

The sgRNA annealing product and the linearized vector ligation system are as follows: T4 ligase buffer (NEB: M0202L) 1 μL, linearized vector 20 ng, annealed oligo fragment (10 μM) 5 μL, T4 ligase (NEB: M0202L) 0.5 μL, ddH ₂ O to make up to 10 μL, Ligation was allowed to proceed overnight at 16°C.

The ligated vector was transformed into E. coli DH5a competent cells (Weidi Biotechnology, DL1001). The specific process is as follows: DH5α competent cells were taken out from -80℃ and quickly inserted into ice. After 5 minutes, the bacterial block was melted, and the ligation product was added and gently mixed by hand at the bottom of the centrifuge tube, and then allowed to stand in ice for 25 minutes. Heat shock in a 42℃ water bath for 45 seconds, quickly put back into ice and let stand for 2 minutes. Add 700μl of sterile LB medium without antibiotics to the centrifuge tube, mix well, and recover at 37℃, 200rpm for 60 minutes. Centrifuge at 5000rpm for one minute to collect the bacteria, and take about 100μl of supernatant to gently blow and resuspend the bacterial block and spread it on the LB medium with Amp antibiotics. The plate was inverted and placed in a 37℃ incubator for overnight culture. Pick a single colony, shake the positive clone after sequencing confirmation, extract the plasmid (TIANGEN: DP120-01), and then measure the concentration, and store it in a -20℃ refrigerator for later use.

The base editor ABE8e used in this experiment is a highly efficient base editor ABE8e evolved by David R. Liu's 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 or with improved Cas domain compatibility and activity. Nat Biotechnol. 2020 Jul；38(7):883-891. doi:10.1038/s41587-020-0453-z. Epub 2020 Mar 16. Erratum in: Nat Biotechnol. 2020 May 20；PMID:32433547；PMCID:PMC7357821). Plasmid ABE8e (Plasmid#138489) was purchased from Addgene, and the mRNA of ABE8e was expressed and purified by the laboratory for future use.

3. Animal research

Lipid nanoparticles (mass ratio of total lipids to mRNA is 40:1) containing the compound of the present invention (see Table 2) encapsulating mRNA and sgRNA encoding the base editor ABE8e (ABE8e mRNA:sgRNA ratio (w/w) is 3:2) were systemically administered to 6-7 week old C57BL/6 female mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) via tail vein injection at a dose of 0.2 mg/kg.

Lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, abbreviated as MC3) encapsulating the mRNA and sgRNA of the base editor ABE8e were administered to mice of the same age and gender in a similar manner as a positive control. In addition, PBS buffer was also injected into the tail vein of mice of the same age and gender in a similar manner as a negative control.

4. Editing efficiency detection

One week after administration, the editing efficiency of the mice was tested. The mice were killed and liver tissue was taken. The genome was extracted after lysis and the efficiency was analyzed by deep sequencing. The deep sequencing steps are as follows. The designed primers are shown in Table 3.

table 3

The PCR program was as follows: 94°C, 2 min; 98°C for 10 s, 60°C for 30 s, 68°C for 20 s, 34 cycles; 68°C for 5 min.

After PCR, gel electrophoresis was used to verify that the amplified product was correct by selecting a single band of appropriate size, and the obtained PCR product was sent to Nanjing GenScript for sequencing. The deep sequencing results were analyzed by Crispresso software for reading, specific site analysis, and editing efficiency calculation. The calculation results are shown in Table 4, and the editing efficiency corresponding to each lipid nanoparticle can be seen in Figures 2 and 3.

Table 4

As shown in Table 4, the ionizable lipid compound used in the present invention can effectively deliver drugs such as nucleic acid molecules and small molecule compounds; and by comparison, the lipid nanoparticles of the present invention have a better particle size distribution, a high encapsulation rate, and a delivery effect that is significantly better than that of the comparative lipid nanoparticles, and can meet the needs of in vivo delivery.

The description presented in the above exemplary embodiments is only used to illustrate the technical solution of the present invention, and is not intended to be exhaustive, nor is it intended to limit the present invention to the precise form described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described to explain the specific principles of the present invention and its practical application, so that other technicians in the field can easily understand, implement and use the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be limited by the scope of protection requested and its equivalent form.

Claims (19)

A compound of formula I or a pharmaceutically acceptable form thereof,
in: ^G1 is a _C1-6 alkylene group; R ¹ and R ² are each independently C _1-6 alkyl; Optionally, R ¹ , R ² and the connected N together form a 3-8 membered heterocyclic group, or, any one of R ¹ and R ² is directly connected to any carbon atom in G ¹ to form a 3-8 membered heterocyclic group; L ¹ is selected from Heteroatoms O, N, S or absent; R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are each independently hydrogen, C _1-8 alkyl, or -OC _2-12 alkenyl; a and b are each independently selected from an integer of 1-10; L ² is selected from Heteroatoms O, N, S or absent; R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are each independently hydrogen or C _1-8 alkyl; c and d are each independently selected from an integer of 1-10; ^L3 is Heteroatom O, N or S; R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , and R ¹⁷ are each independently hydrogen, C _1-8 alkyl, or -OC _2-12 alkenyl; e and f are each independently selected from integers of 1-10. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: ^G1 is methylene, ethylene, propylene, butylene or pentylene. The compound according to claim 1 or 2, or a pharmaceutically acceptable form thereof, wherein: R ¹ and R ² are each independently methyl, ethyl or propyl; or, R ¹ , R ² and the connected N together form a 5-membered or 6-membered heterocyclic group. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable form thereof, wherein: ^L1 is or does not exist; At least two of R ³ , R ⁴ and R ⁵ are not hydrogen, and R ⁶ and R ⁷ are hydrogen; a is an integer selected from 1 to 10; b is 2, 3, 4, 5, 6, 7, 8 or 9. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable form thereof, wherein: ^L3 is R ¹³ and R ¹⁴ are each hydrogen; at least two of R ¹⁵ , R ¹⁶ and R ¹⁷ are not hydrogen; e is 2, 3, 4, 5, 6, 7, 8 or 9; f is selected from integers of 1-10. The compound or pharmaceutically acceptable form thereof according to any one of claims 1 to 5, wherein the compound is selected from one or more of Compound 1 to Compound 35. A compound or a pharmaceutically acceptable form thereof according to any one of claims 1 to 6, wherein the pharmaceutically acceptable form is selected from a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug. A method for preparing a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, the method comprising: compound of, compound of, Compounds and The compound of formula I or a pharmaceutically acceptable form thereof is synthesized by Ugi reaction; Wherein, R ¹ -R ¹⁷ , L ¹ -L ³ , G ¹ , a, b, c, d, e, and f are as defined in any one of claims 1-5. Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof in the preparation of liposome nanocarriers. A lipid carrier comprising the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof. The lipid carrier according to claim 10, comprising a first lipid compound and a second lipid compound, wherein the first lipid compound comprises a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof and optionally other ionizable lipids, and the second lipid compound comprises one or a combination of two or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids. A nucleic acid lipid nanoparticle composition comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof or a lipid carrier according to claim 10 or 11, and a nucleic acid drug. According to the nucleic acid lipid nanoparticle composition of claim 12, the nucleic acid drug comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA; preferably, the antisense nucleic acid is an antisense oligonucleic acid; preferably, the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator or shRNA; preferably, the DNA comprises a plasmid. A pharmaceutical preparation comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 10 or 11, or a nucleic acid lipid nanoparticle composition according to claim 12 or 13, and a pharmaceutically acceptable excipient, carrier or diluent. Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or the lipid carrier according to claim 10 or 11, or the nucleic acid lipid nanoparticle composition according to claim 12 or 13, or the pharmaceutical preparation according to claim 14 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. According to the use of claim 15, the nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation is used to treat or prevent a disease or condition in a subject in need thereof. The use according to claim 16, wherein the subject is a mammal or a human. According to the use according to claim 16 or 17, the disease or condition is selected from metabolic diseases, hereditary diseases, cancer, cardiovascular diseases and infectious diseases; preferably, the metabolic diseases include familial hypercholesterolemia (FH), the hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria (PH1) and hereditary angioedema (HAE), and the infectious diseases include hepatitis B (HEPATITIS B). 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 claim 12 or 13, or the pharmaceutical preparation of claim 14.