CN118666726A - Lipid compounds for delivering therapeutic agents and preparation methods and applications thereof - Google Patents

Abstract

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

Gene therapy technology is a hot topic in modern biomedicine 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 carriers. 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 or a pharmaceutically acceptable form thereof.

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

Another object of the present invention is to provide a pharmaceutical preparation comprising the above compound or a pharmaceutically acceptable form thereof, 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.

<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 or C _1-6 alkoxy; 0 to 3 H in the alkyl or alkoxy is optionally further substituted by halogen, hydroxy, C _1-6 alkyl, C _1-6 alkoxy, halogen-substituted C _1-6 alkyl or hydroxy-substituted 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; 0 to 3 ring Cs in the heterocyclic group are optionally further substituted by N, O or S; 0 to 3 Hs in the heterocyclic group are optionally further substituted by halogen, hydroxyl, C _1-6 alkyl, C _1-6 alkoxy, halogen-substituted C _1-6 alkyl or hydroxyl-substituted C _1-6 alkyl;

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

R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are each independently hydrogen, C _1-12 alkyl, C _1-12 alkenyl, C _1-12 alkynyl, or C _1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C _1-12 alkyl, C _1-12 alkenyl, C _{1-12 alkynyl, C 1-12 alkoxy} , halogen-substituted C _1-12 alkyl, or hydroxy-substituted C _1-12 alkyl;

a and b are each independently selected from integers of 0-10;

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

R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are each independently hydrogen, C _1-12 alkyl, C _1-12 alkenyl, C _1-12 alkynyl, or C _1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C _1-12 alkyl, C _1-12 alkenyl, C _{1-12 alkynyl, C 1-12 alkoxy} , halogen-substituted C _1-12 alkyl, or hydroxy-substituted C _1-12 alkyl;

c and d are each independently selected from integers of 0-10;

L ³ and L ⁴ are each independently selected from alkynylene, heteroatom O, N, S or absent;

R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , and R ¹⁹ are each independently hydrogen, C _1-12 alkyl, C _1-12 alkenyl, C _1-12 alkynyl, or C _1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C _1-12 alkyl, C _1-12 alkenyl, C _1-12 alkynyl, C _1-12 alkoxy, halogen-substituted C _1-12 alkyl, or hydroxy-substituted C _1-12 alkyl;

e, f, and g are each independently selected from integers of 0-10.

According to a specific embodiment 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, propyl or butyl, wherein 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C _1-3 alkyl; or, R ¹ , R ² and the connected N together form a 5-membered or 6-membered heterocyclic group, wherein 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and wherein 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C _1-3 alkyl.

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- or 6-membered heterocyclic group, and the other of R ¹ and R ² is methyl, ethyl, propyl or butyl, and 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C _1-3 alkyl. 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C _1-3 alkyl.

According to a specific embodiment of the present invention, in Formula I, G ¹ , R ¹ , and R ² correspond to the groups represented by any one of Compounds 1 to 24, respectively.

According to a specific embodiment of the present invention, in Formula I,

^L1 is Alkyne, O or absent;

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

a is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

b is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Preferably, at least one of a and b or both of them are not zero.

According to a specific embodiment 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 24, respectively.

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

^L2 is Alkyne, O or absent;

R ⁸ and R ⁹ are each independently hydrogen, and at least two of R ¹⁰ , R ¹¹ , and R ¹² are not hydrogen;

c and d are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, at least one of c and d or both of them are not 0.

According to a specific embodiment 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 24, respectively.

According to a specific embodiment of the present invention, in Formula I,

L ³ and L ⁴ are each independently selected from Alkyne, O or absent;

R ¹³ and R ¹⁴ are each hydrogen; at least two of R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ are not hydrogen; preferably, at least two of R ¹⁶ , R ¹⁸ and R ¹⁹ are not hydrogen;

e, f, g are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

According to a specific embodiment of the present invention, in Formula I,

^L3 is Alkyne, O or absent;

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

e and f are each independently selected from 0, 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 24, respectively.

In the present invention, when the values of a, b, c, d, e, and f are greater than 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 24 shown in Table 1.

Table 1

<Second Aspect>

The present invention provides an intermediate having a structure shown in Formula Ic or Formula Id:

Wherein, each substituent and the values of e, f, and g are as defined in the present invention above.

The intermediates described in the present invention can be used to prepare the compounds described in the present invention or their pharmaceutically acceptable forms.

<Third Aspect>

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

Obtained by alkylation or reductive amination

Thiocarbamates are generated by the action of triphosgene, carbonyldiimidazole or p-nitrophenyl chloroformate

Removing the ketal protecting group and introducing a long fatty chain through a condensation reaction to obtain a compound of formula I or a pharmaceutically acceptable form thereof;

The values of each substituent and e, f, and g are as defined in the present invention.

According to a specific embodiment of the present invention, in the method of preparing the compound or its pharmaceutically acceptable form, It is a key intermediate that can be used to obtain secondary amines containing a long chain or its precursors through alkylation or reductive amination. Then, thiocarbamate is generated by the action of triphosgene, carbonyldiimidazole or p-nitrophenyl chloroformate. Finally, the ketal protecting group is removed, and the second and third fatty long chains are introduced through condensation reaction to obtain a compound of formula I or a salt thereof.

<Fourth Aspect>

The present invention also provides the use of the compound or its pharmaceutically acceptable form in the preparation of liposome nanocarriers.

<Fifth 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 cationic or ionizable lipids.

In some embodiments, the other cationic or ionizable 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 DOT AP, 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 cationic or ionizable lipids 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 of the above compounds or their pharmaceutically acceptable forms such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (3-4):(0-5).

<Sixth 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 fifth 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 including a Cas protein.

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

In some embodiments, the nucleic acid drug includes mRNA and gRNA of the Cas protein.

In some embodiments, the gRNA is modified.

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.

<Seventh 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 fifth aspect, or a nucleic acid lipid nanoparticle composition according to the sixth aspect, and a pharmaceutically acceptable excipient, carrier or diluent.

<Eighth 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.

<Ninth 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.

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.

The present invention provides a series of novel structural formula I compounds, which can be used as ionizable lipids to prepare lipid carriers together with other lipid compounds, and their particle size is controllable, uniformly distributed, and has a very high encapsulation rate. More specifically, the compound of the present invention is an amino liposome, and the liposome compound contains both hydrophobic non-polar long chains and hydrophilic amino groups, and this amphoteric characteristic can be used to form lipid particles. Further, in the compound of the present invention, the group connected to the nitrogen atom in the thiocarbamate is preferably a fatty long chain, and the C number of the fatty long chain is preferably greater than or equal to 3, further preferably greater than or equal to 4, further preferably greater than or equal to 5, and further preferably greater than or equal to 6. With the increase of the fatty long chain, the delivery effect can be significantly improved, and the introduction of the fatty long chain can increase the proportion of the non-polar hydrophobic component in the liposome, thereby increasing membrane fusion to enhance the release of mRNA, promote the synergistic improvement of mRNA delivery, and obtain significantly enhanced delivery efficiency.

The lipid compound synthesis method of the present invention is simple, has a high yield, can be quickly synthesized, and has a low cost. The compound 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 is particularly important for the development and application of nucleic acid preventive 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.

DETAILED DESCRIPTION

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 the present specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 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 structural isomers with different energies that can be interconverted through a low energy barrier. 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 through proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-imino 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, " _C3-30 cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.

The term "branched alkyl" refers to an alkane radical that is attached to 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 "alkynylene" refers to a divalent 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 two single bonds, including (but not limited to) ethynylene, etc. For example, " _C2-30 alkynylene" refers to a divalent 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).

[Preparation method]

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 drugs used were analytically pure or chemically pure; the solvents were redistilled before use; and the 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 conventional conditions or 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 4-[7-butyl-17-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-25-ethyl-8,14,20-trioxy-19,25-diaza-9,15-dioxa-21-thiacosano-19-yl]butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester

Step 1: Synthesis of compound 1-2

In a 1000 ml round-bottom flask, 5-hydroxymethyl-2,2-dimethyl-1,3-dioxane (40.00 g, 273.62 mmol, 1.0 eq), dichloromethane (400 mL), sodium bicarbonate (57.47 g, 684.06 mmol, 2.5 eq), and Dess-Martin periodinane (127.66 g, 300.99 mmol, 1.1 eq) were added respectively. After reacting at 0 degrees Celsius for 2 hours, 1000 ml of saturated sodium thiosulfate solution was slowly added, and the mixture was extracted with dichloromethane (500 mL×2). 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 2,2-dimethyl-1,3-dioxane-5-carboxaldehyde (20.10 g, yield 50.7%).

Step 2: Synthesis of Compound 1-4

In a 300 ml three-necked flask, add 3-chloro-1-diethylaminopropane (21.00 g, 140.32 mmol, 1.0 eq), thiourea (10.89 g, 143.12 mmol, 1.0 eq) and 200 ml of ethanol in sequence. React at 80 degrees Celsius overnight. Concentrate under reduced pressure to obtain a crude compound 3-(diethylamino)propane-1-thiol, which can be directly used in the next step.

Step 3: Synthesis of Compound 1-7

In a 250 ml round-bottom flask, 5-hydroxypentanoic acid benzyl ester (5.00 g, 24.01 mmol, 1.0 eq), 2-butyloctanoic acid (7.21 g, 36.01 mmol, 1.5 eq), 100 ml of dichloromethane and 4-dimethylaminopyridine (2.93 g, 24.01 mmol, 1.0 eq) were added, and finally 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.20 g, 48.02 mmol, 2.0 eq) was added. The mixture was reacted at room temperature for 4 hours, diluted with 100 ml of water, extracted with dichloromethane (100 mL x 2), 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 2-butyloctanoic acid-5-(benzyloxy)-5-oxydipentyl ester (4.50 g, yield 48.0%).

Step 4: Synthesis of Compound 1-8

In a 50 ml round bottom flask, 1-7 (2.00 g, 5.12 mmol, 1.0 eq), methanol (10 mL), tetrahydrofuran (10 mL) and finally Pd/C (545.0 mg, 0.51 mmol, 0.1 eq, 10% purity) were added. The mixture was reacted at room temperature for 16 hours under an atmospheric pressure of hydrogen, and the mixture was filtered and concentrated to obtain compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid (1.54 g, yield 100%).

Step 5: Synthesis of Compound 1-10

In a 2000 ml round-bottom flask, add hex-2-yn-1-ol (65.00 g, 662.28 mmol, 1 eq), dichloromethane (1300 mL), pyridine (52.39 g, 662.28 mmol, 1.0 eq), slowly add phosphorus tribromide (215.13 g, 794.74 mmol, 1.2 equiv.) at 0 degrees Celsius, and warm to room temperature to react for 16 hours after the addition is complete. Add 500 ml of water to quench, extract twice with 500 ml of dichloromethane, combine the organic phases, wash with saturated sodium bicarbonate solution, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound 1-bromohex-2-yn (24.20 g, yield 22.5%) by column chromatography.

Step 6: Synthesis of Compound 1-12

In a 500 ml three-necked flask, 1-bromo-2-hexyne (44.00 g, 273.22 mmol, 1.0 eq), N,N-dimethylformamide (240 mL), propargyl alcohol (19.91 g, 355.19 mmol, 1.3 equiv.), cuprous iodide (67.65 g, 355.19 mmol, 1.3 equiv.), cesium carbonate (115.73 g, 355.19 mmol, 1.3 equiv.), sodium iodide (53.24 g, 355.19 mmol, 1.3 equiv.) were added in sequence, and reacted at room temperature under nitrogen atmosphere for 16 hours. The reaction solution was quenched with 500 ml saturated ammonium chloride solution, extracted with ethyl acetate (500 mL x 2), and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure and subjected to column chromatography to obtain compound nona-2,5-diyn-1-ol (24.00 g, yield 64.5%). MS: m/z [M+H] ⁺ = 137.0.

Step 7: Synthesis of Compound 1-13

In a 500 ml round-bottom flask, nickel acetate tetrahydrate (14.02 g, 79.30 mmol, 0.9 eq) and ethanol (240 mL) were added, sodium borohydride (2.95 g, 70.49 mmol, 0.8 eq) was added at 0 degrees Celsius, and ethylenediamine (21.17 g, 352.44 mmol, 4.0 eq) was added after stirring for 30 minutes, and then 1-12 (12.00 g, 88.11 mmol, 1.0 eq) in ethanol (60 mL) was slowly added dropwise, and hydrogen was replaced three times, and the reaction was carried out at 0 degrees Celsius for 4 hours. The reaction was quenched with saturated ammonium chloride, extracted with ethyl acetate (500 mL x 2), 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 (2Z, 5Z)-nona-2,5-diene-1-ol (8.00 g, yield 64.7%).

Step 8: Synthesis of Compound 1-15

In a 250 ml round-bottom flask, 1-13 (8.00 g, 57.05 mmol, 1.0 eq), 4-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)butyric acid (17.39 g, 85.58 mmol, 1.5 equiv.), dichloromethane (180 mL), 4-dimethylaminopyridine (6.97 g, 57.05 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.87 g, 114.10 mmol, 2.0 eq) were added. The mixture was reacted at room temperature for 16 hours, diluted with 100 ml of water, and then extracted twice with 300 ml of dichloromethane 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 4-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (9.00 g, yield 48.5%), MS: m/z[M+H] ⁺ =326.0.

Step 9: Synthesis of Compound 1-16

In a 250 ml flask, 1-15 (9.00 g, 27.65 mmol, 1.0 eq) and ethyl acetate (150 mL, 4 M) were added. The mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure to obtain the compound 4-aminobutyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (7.24 g, yield 100%). MS: m/z[M+H] ⁺ =226.0.

Step 10: Synthesis of Compound 1-17

In a 250 ml flask, 1-16 (6.00 g, 26.63 mmol, 1.0 eq), tetrahydrofuran (120 mL), 1-2 (3.45 g, 23.96 mmol, 0.9 eq), sodium acetate borohydride (5.06 g, 0.9 eq) were added and reacted at room temperature for 4 hours. 100 ml of water was added to quench the reaction solution, and the reaction solution was extracted twice with 200 ml of dichloromethane, and 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 4-{[(2,2-dimethyl-1,3-dioxan-5-yl)methyl]amino}butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (4.00 g, yield 42.5%). MS: m/z[M+H] ⁺ =354.0.

Step 11: Synthesis of Compound 1-18

In a 100 ml round-bottom flask, 1-17 (4.00 g, 11.31 mmol, 1.0 eq), dichloromethane (80 mL), triethylamine (5.73 g, 56.57 mmol, 5.0 eq), carbonyldiimidazole (7.34 g, 45.26 mmol, 4.0 eq) were added. The mixture was reacted at room temperature for 16 hours, diluted with 100 ml of water, extracted twice with 100 ml of dichloromethane, 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 4-{[(2,2-dimethyl-1,3-dioxane-5-yl)methyl](imidazol-1-ylcarbonyl)amino}butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (2.00 g, yield 39.5%). MS: m/z[M+H] ⁺ =448.0.

Step 12: Synthesis of Compound 1-19

In a 250 ml round-bottom flask, add 1-18 (2.00 g, 4.47 mmol, 1.0 eq) and dichloromethane (100 mL), slowly add methyl trifluoromethanesulfonate (0.62 g, 3.80 mmol, 0.85 eq) at -10 degrees Celsius, stir at -10 degrees Celsius for 1 hour after the addition is complete, slowly add 1-4 (1.65 g, 11.17 mmol, 2.5 eq), and then react at -10 degrees Celsius for 2 hours. 100 ml of water was added to dilute, and the reaction solution was extracted twice with 100 ml of dichloromethane 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-[1-(2,2-dimethyl-1,3-dioxane-5-yl)-8-ethyl-3-oxygen-2,8-diaza-4-thiadecane-2-yl]butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (1.8 g, yield 76.47%). MS: m/z[M+H] ⁺ =527.0.

Step 13: Synthesis of Compound 1-20

In a 50 ml flask, 1-19 (1.80 g, 3.42 mmol, 1.0 eq), methanol (18 mL), p-toluenesulfonic acid (0.29 g, 1.71 mmol, 0.5 eq) were added. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure and column chromatography was performed to obtain compound 4-[10-ethyl-1-hydroxy-2-(hydroxymethyl)-5-oxyde-4,10-diaza-6-thiadodecane-4-yl]butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (0.40 g, yield 24.0%) as a yellow oil. MS: m/z[M+H] ⁺ =487.1.

Step 14: Synthesis of Compound 1

In a 50 ml flask were added 1-20 (275.0 mg, 0.56 mmol, 1.0 eq), 1-8 (424.4 mg, 1.41 mmol, 2.5 equiv.), 4-dimethylaminopyridine (69.0 mg, 0.56 mmol, 1.0 eq), acetonitrile (5 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (324.9 mg, 1.69 mmol, 3.0 eq). The mixture was reacted at room temperature for 16 hours, diluted with 50 ml of water, extracted twice with 100 ml of ethyl acetate, 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-[7-butyl-17-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-25-ethyl-8,14,20-trioxy-19,25-diaza-9,15-dioxa-21-thiacosadecan-19-yl]butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (220.5 mg, 37.1%). MS: m/z[M+H] ⁺ =1052.2. ¹ HNMR:(300MHz,CDCl ₃ )δ5.732-5.531(m,2H),5.500-5.246(m,2H),4.694(d,J＝6.6Hz,2H),4.083(d,J＝5.1Hz,8H),3.68-3.314(m,4H),2.932-2.884(m,4H),2.516-2.458 (m,7H),2.394-2.299(m,8H ),2.046-2.022(m,2H),1.731-1.717(m,2H),1.695-1.684(m,8H),1.673-1.626(m,6H),1.597-1.552(m,6H),1.467-1.307(m,24H),1.032-0.932 (m,6H),0.978-0.854(m,15H).

Example 2: Synthesis of 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-hexyl-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester

Step 1: Synthesis of compound 2-3

In a 100 ml round-bottom flask, methanesulfonic acid-(2,2-dimethyl-1,3-dioxan-5-yl)methyl ester (2.00 g, 8.92 mmol, 1.0 eq), n-hexylamine (4.51 g, 44.60 mmol, 5.0 eq), potassium carbonate (1.85 g, 13.38 mmol, 1.5 eq), potassium iodide (1.63 g, 9.81 mmol, 1.1 eq), acetonitrile (30 mL) were added in sequence, reacted at 80 degrees Celsius for 16 hours, concentrated under reduced pressure, and column chromatography was performed to obtain the compound [(2,2-dimethyl-1,3-dioxan-5-yl)methyl](hexyl)amine (1.80 g, yield 88.0%). MS: m/z[M+H] ⁺ =230.2.

Step 2: Synthesis of compound 2-4

Compound 2-3 (1.80 g, 7.85 mmol, 1.0 eq), dichloromethane (20 mL) and triethylamine (3.97 g, 39.25 mmol, 5 eq) were added to a 100 mL round-bottom flask in sequence, and then carbonyldiimidazole (3.82 g, 23.55 mmol, 3.0 eq) was added in batches. The reaction solution was stirred at room temperature for 3 hours, concentrated under reduced pressure, diluted with 50 mL of water, extracted three times with 50 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 N-[(2,2-dimethyl-1,3-dioxan-5-yl)methyl]-N-hexylimidazole-1-carboxamide (2.30 g, yield 90.6%). MS: m/z[M+H] ⁺ =324.2.

Step 3: Synthesis of Compound 2-5

Compound 2-4 (1.00 g, 3.09 mmol, 1.0 eq) and dichloromethane (15 mL) were added to a 50 mL round-bottom flask, and methyl trifluoromethanesulfonate (0.48 g, 2.94 mmol, 0.95 eq) was added under ice bath. The reaction solution was stirred under ice bath for 0.5 hours, and then compound 1-4 (0.68 g, 4.65 mmol, 1.5 eq) and tetramethylethylenediamine (1.08 g, 9.30 mmol, 3.0 eq) were added, and then reacted under ice bath for 2.5 hours. The mixture was extracted three times with 50 mL of dichloromethane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound {[(2,2-dimethyl-1,3-dioxan-5-yl)methyl](hexyl)amino}methanethioic acid-S-[3-(diethylamino)propyl]ester (0.49 g, yield 39.1%). MS: m/z [M+H] ⁺ = 403.3.

Step 4: Synthesis of Compound 2-6

Compound 2-5 (0.49 g, 1.21 mmol, 1.0 eq) and 10 ml methanol were added to a 50 ml round-bottom flask, followed by p-toluenesulfonic acid (0.42 g, 2.42 mmol, 2.0 eq). The reaction solution was stirred at room temperature for 2 hours and concentrated to obtain compound {hexyl [3-hydroxy-2- (hydroxymethyl) propyl] amino} methanethioic acid-S- [3- (diethylamino) propyl] ester (0.44 g, yield 100%, crude product), which was used directly in the next step without further purification. MS: m/z [M+H] ⁺ = 363.3.

Step 5: Synthesis of Compound 2

In a 25 ml round-bottom flask, compound 1-8 (1.09 g, 3.63 mmol, 3.0 eq), N,N-diisopropylethylamine (0.78 g, 6.05 mmol, 5.0 eq), 4-dimethylaminopyridine (0.15 g, 1.21 mmol, 1.0 eq), dichloromethane (10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.70 g, 3.63 mmol, 3.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 2-6 (0.44 g, 1.21 mmol, 5.0 eq) was added. mol, 1.0eq), the reaction solution was continued to react at room temperature for 15.5 hours, concentrated under reduced pressure, diluted with 30 ml of water, extracted three times with 20 ml of petroleum ether respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain the compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-hexyl-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester (430.0 mg, yield 38.2%). MS: m/z[M+H] ⁺ =927.7. ¹ H NMR (300MHz, CDCl ₃ ) δ4.20-4.00(m,8H),3.49-3.10(m,4H),2.90-2.79(m,2H),2.60-2.23(m,12H),1.80-1.52(m,17H),1.50-1.43(m,4H),1.30-1 .11(m,30H),1.08-0.98(m,6H),0.97-0.80(m,15H).

Example 3: Synthesis of 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-(3-hydroxypropyl)-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester

Step 1: Synthesis of compound 3-2

3-aminopropan-1-ol (5.00 g, 66.57 mmol, 1.0 eq) and imidazole (11.33 g, 166.42 mmol, 2.5 eq) were added to 100 ml of dichloromethane in sequence, and then tert-butyldiphenylsilyl chloride (27.45 g, 99.85 mmol, 1.5 eq) was added in batches. The reaction solution was stirred at room temperature for 16 hours, concentrated under reduced pressure, and column chromatography was performed to obtain compound 3-{[(2-methylpropan-2-yl)diphenylsilyl]oxy}propan-1-amine (19.00 g, yield 91.0%). MS: m/z[M+H] ⁺ =314.2.

Step 2: Synthesis of compound 3-3

2-1 (3.00 g, 13.38 mmol, 1.0 eq), 3-2 (8.39 g, 26.76 mmol, 2.0 eq), potassium carbonate (3.70 g, 26.76 mmol, 2.0 eq), potassium iodide (2.22 g, 13.38 mmol, 1.0 eq) were added to 50 ml of acetonitrile in sequence, and the reaction solution was reacted at 80 degrees Celsius 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, and column chromatography was performed to obtain compound 9-(2,2-dimethyl-1,3-dioxacyclohexane-5-yl)-2,2-dimethyl-3,3-diphenyl-8-aza-4-oxa-3-silanonane (4.70 g, yield 79.5%). MS: m/z[M+H] ⁺ =442.3.

Step 3: Synthesis of compound 3-4

3-3 (4.70 g, 10.64 mmol, 1.0 eq) and triethylamine (5.38 g, 53.20 mmol, 5.0 eq) were added to 50 ml of dichloromethane in sequence, and then carbonyldiimidazole (5.18 g, 31.92 mmol, 3.0 eq) was added in batches. After the reaction solution was reacted at 25 degrees Celsius for 3 hours, it was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain the compound N-[(2,2-dimethyl-1,3-dioxane-5-yl)methyl]-N-(2,2-dimethyl-3,3-diphenyl-4-oxa-3-silanylheptan-7-yl)imidazole-1-carboxamide (4.40 g, yield 77.2%). MS: m/z[M+H] ⁺ =536.3.

Step 4: Synthesis of Compound 3-5

3-4 (2.0 g, 3.73 mmol, 1.0 eq) was added to 20 ml of dichloromethane, and methyl trifluoromethanesulfonate (0.61 g, 3.73 mmol, 1.0 eq) was added under ice bath. After stirring for 1 hour, tetramethylethylenediamine (1.30 g, 11.19 mmol, 3.0 eq) and 1-4 (0.82 g, 5.59 mmol, 1.5 eq) were added. The reaction solution was continued to react under ice bath for 2 hours, and then concentrated under reduced pressure. Column chromatography was performed to obtain the compound [9-(2,2-dimethyl-1,3-dioxane-5-yl)-2,2-dimethyl-3,3-diphenyl-8-aza-4-oxa-3-silanonan-8-yl]methanethioic acid-S-[3-(diethylamino)propyl] ester (1.8 g, yield 78.5%). MS: m/z [M+H] ⁺ = 615.4.

Step 5: Synthesis of Compound 3-6

In 10 ml methanol, 3-5 (0.90 g, 1.46 mmol, 1.0 eq) and p-toluenesulfonic acid (0.50 g, 2.92 mmol, 2.0 eq) were added. After the reaction solution was stirred at room temperature for 2 hours, it was concentrated under reduced pressure to obtain a crude compound [11-hydroxy-10-(hydroxymethyl)-2,2-dimethyl-3,3-diphenyl-8-aza-4-oxa-3-silanhexadecane-8-yl]methanethioic acid-S-[3-(diethylamino)propyl] ester (0.84 g, theoretical yield 100%, crude product). It was used directly in the next step without purification. MS: m/z[M+H] ⁺ =575.3.

Step 6: Synthesis of Compound 3-7

1-8 (1.32 g, 4.38 mmol, 3.0 eq), N,N-diisopropylethylamine (0.57 g, 4.38 mmol, 3.0 eq), 4-dimethylaminopyridine (0.36 g, 2.92 mmol, 2.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.84 g, 4.38 mmol, 3.0 eq) were added to 20 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 3-6 (0.84 g, 1.46 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-4-(2,2-dimethyl-3,3-diphenyl-4-oxa-3-silanahept-7-yl)-10-ethyl-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester (1.40 g, yield 84.1%). MS: m/z[M+H] ⁺ =1139.8.

Step 7: Synthesis of compound 3

3-7 (300.0 mg, 0.26 mmol, 1.0 eq) and tetrabutylammonium fluoride tetrahydrofuran solution (0.78 mL, 0.78 mmol, 1.0 M, 3.0 eq) were added to 15 mL of tetrahydrofuran. The reaction solution was stirred at room temperature for 1 hour, concentrated under reduced pressure, diluted with 50 mL of water, extracted three times with 30 mL of petroleum ether, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain the compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-(3-hydroxypropyl)-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester (110.0 mg, yield 46.4%). MS: m/z[M+H] ⁺ =901.6. ¹ H NMR (300MHz, CDCl ₃ ) δ4.25-4.00(m,8H),3.75-3.30(m,6H),2.98-2.78(m,2H),2.73-2.25(m,12H),1.80-1.50(m,16H),1.50-1.12(m,30H),1.10- 0.98(m,6H),0.97-0.98(m,6H),0.97-0.75(m,12H).

Example 4: Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester

Step 1: Synthesis of compound 4-1

At room temperature, 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid (12.46 g, 41.47 mmol, 2.1 eq), 2-hydroxymethylpropane-1,3-diol (2.00 g, 18.85 mmol, 1.0 eq), 4-dimethylaminopyridine (2.3 g, 18.85 mmol, 1 eq), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (7.23 g, 37.70 mmol, 2.0 eq) and N,N-diisopropylethylamine (7.31 g, 56.55 mmol, 3.0 eq) were added to a round-bottom flask containing 100 ml of dichloromethane and stirred at room temperature for 4 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 give 2-butyloctanoic acid-18-butyl-8-(hydroxymethyl)-5,11,17-trioxydeca-6,10,16-trioxa-tetracosane-1-yl ester (4.50 g, yield 35.6%).

Step 2: Synthesis of compound 4-2

At room temperature, compound 4-1 (2.00 g, 2.98 mmol, 1.0 eq) and triethylamine (0.90 g, 8.94 mmol, 3.0 eq) were added to 30 ml of dichloromethane, and methylsulfonic anhydride (0.78 g, 4.47 mmol, 1.5 eq) was slowly added at zero degrees Celsius, and the temperature was slowly restored to room temperature and reacted for 4 hours. The reaction solution was diluted with water, extracted with dichloromethane 3 times, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain methanesulfonic acid-12-butyl-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-5,11-dioxy-4,10-dioxahexadecan-1-yl ester (2.00 g, yield 89.6%).

Step 3: Synthesis of compound 4-3

At room temperature, compound 4-2 (1.00 g, 1.34 mmol, 1.0 eq), benzylamine (287.2 mg, 2.68 mmol, 2.0 eq), potassium carbonate (555.6 mg, 4.02 mmol, 3.0 eq), potassium iodide (111.2 mg, 0.68 mmol, 0.5 eq) were added to 20 ml of acetonitrile, nitrogen was protected, heated to 90 degrees Celsius, and reacted overnight. The reaction solution was concentrated, diluted with water, extracted with ethyl acetate three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 2-butyloctanoic acid-4-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-7-oxy-1-phenyl-2-aza-6-oxaundecane-11-yl ester (400.0 mg, yield 39.4%). MS: m/z [M+H] ⁺ = 760.6

Step 4: Synthesis of compound 4-4

4-3 (400.0 mg, 0.52 mmol, 1.0 eq) was put into a reaction flask, followed by addition of 10 ml of methanol and palladium carbon (55.3 mg, 0.52 mmol, 0.1 eq, 10% purity), and stirred overnight at room temperature under a hydrogen atmosphere. The reaction solution was filtered and concentrated, and purified by column chromatography to obtain 2-butyloctanoic acid-8-(aminomethyl)-18-butyl-5,11,17-trioxy-6,10,16-trioxa-tetracosane-1-yl ester (246.0 mg, yield 70.0%). MS: m/z[M+H] ⁺ =670.5

Step 5: Synthesis of compound 4-5

At room temperature, compound 4-4 (246.0 mg, 0.37 mmol, 1.0 eq), 4-nitrophenyl chloroformate (148.0 mg, 0.73 mmol, 2.0 eq) and pyridine (174.0 mg, 2.20 mmol, 3.0 eq) were added to a reaction flask, followed by dichloromethane (10 mL), and stirred overnight at room temperature under nitrogen protection. The reaction solution was washed with water, washed with saturated brine, dried, concentrated, and purified by column chromatography to obtain 2-butyloctanoic acid-4-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-1-[(4-nitrophenyl)oxy]-1,7-dioxy-2-aza-6-oxaundecane-11-yl ester (182.0 mg, 59.4%). MS: m/z[M+H] ⁺ =835.5.

Step 6: Synthesis of compound 4

At room temperature, compound 4-5 (182.0 mg, 0.22 mmol, 1.0 eq), 3-(diethylamino)propane-1-thiol (48.0 mg, 1.33 mmol, 1.5 eq), 4-dimethylaminopyridine (27.0 mg, 0.22 mmol, 1.0 eq), pyridine (35.0 mg, 0.44 mmol, 2.0 eq) were added to 10 ml of acetonitrile and stirred at room temperature for 4 hours. The reaction solution was washed with ethyl acetate, saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester (70.0 mg, yield 38.0%). MS: m/z[M+H] ⁺ =843.6. ¹ H NMR (300MHz, CDCl ₃ ) δ6.44(s,1H),4.40-4.20(s,1H),4.15-4.01(m,6H),3.45-3.20(m,1H),3.15-2.78(m,7H),2.45-2.03(m,8H),1.80-1.50(m,1 2H),1.48-1.05(m,38H),0.90-0.75(m,12H).

Example 5: Synthesis of 2-butyloctanoate-22-butyl-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-(6-ethyl-1-oxy-6-aza-2-thiooctan-1-yl)-5,15,21-trioxy-10-aza-6,14,20-trioxaoctacosane-1-yl ester

Step 1: Synthesis of compound 5

1-8 (0.17 g, 0.56 mmol, 2.0 eq), N,N-diisopropylethylamine (0.11 g, 0.84 mmol, 3.0 eq), 4-dimethylaminopyridine (34.0 mg, 0.28 mmol, 1.0 eq), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.11 g, 0.56 mmol, 2.0 eq) were added to 20 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 3 (0.25 g, 0.28 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound 2-butyloctanoic acid-22-butyl-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-(6-ethyl-1-oxy-6-aza-2-thiaoctane-1-yl)-5,15,21-trioxy-10-aza-6,14,20-trioxaoctacosane-1-yl ester (40.0 mg, yield 12.2%). MS: m/z[M+H] ⁺ =1184.0. ¹ H NMR (300MHz, CDCl ₃ ) δ4.25-4.03(m,12H),3.56-3.11(m,4H),2.92-2.73(m,2H),2.46-2.22(m,16H),1.89-1.50(m,20H),1.56-1.43(m,4H),1.39- 1.21(m,40H),1.07-0.97(m,6H),0.99-0.80(m,18H).

Example 6: Synthesis of 2-butyloctanoic acid-(24Z)-8-(10-butyl-3,9-dioxyylidene-2,8-dioxahexadecan-1-yl)-10-(6-ethyl-1-oxyylidene-6-aza-2-thiaoctan-1-yl)-18-{[(5Z)-oct-5-enyl]oxy}-5,15-dioxyylidene-10-aza-6,14,19-trioxacosaccharide-24-en-1-yl ester

Step 1: Synthesis of compound 6-2

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 6-3

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 (6 mL), and water (6 mL) were added to a jar, 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 (1.5 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).

Step 3: Synthesis of compound 6

6-3 (0.14 g, 0.42 mmol, 1.5 eq), N,N-diisopropylethylamine (72.0 mg, 0.56 mmol, 2.0 eq), 4-dimethylaminopyridine (51.0 mg, 0.42 mmol, 1.5 eq), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (81.0 mg, 0.42 mmol, 1.5 eq) were added to 20 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 3 (0.25 g, 0.28 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to obtain 2-butyloctanoic acid-(24Z)-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-(6-ethyl-1-oxy-6-aza-2-thiaoctane-1-yl)-18-{[(5Z)-oct-5-enyl]oxy}-5,15-dioxy-10-aza-6,14,19-trioxacositol-24-en-1-yl ester (130.0 mg, yield 38.3%). MS: m/z[M+H] ⁺ =1123.9. ¹ H NMR (300MHz, CDCl ₃ ) δ5.47-5.25(m,4H),4.55-4.48(m,1H),4.20-4.01(m,8H),3.71-3.52(m,2H),3.51-3.28(m,5H),2.98-2.81(m,2H),2.65-2.26 (m,14H),2.12-1.80(m,10H),1.81-1.50(m,20H),1.48-1.13(m,38H),1.11-0.78(m,22H).

Example 7. 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-compound 6 of the present invention are dissolved in anhydrous ethanol with DSPC, cholesterol, PEG-DMG according to the above-mentioned molar ratio. Luciferase mRNA (L-6107, TriLink BioTechnologies, Inc.) is dissolved in 100mM pH 4 enzyme-free citric acid buffer (mRNA concentration is 0.2mg/mL). The ethanol solution of different lipid carriers is mixed with the buffer of mRNA with 1:3 (volume/volume) (wherein the mass ratio of total lipid and mRNA is 40:1), and nucleic acid lipid nanoparticles LNP A-F are obtained by microfluidic nano drug manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12ml/min. The nucleic acid lipid nanoparticles obtained are immediately diluted into 1×DPBS buffer with 40 times the volume. The diluted nucleic acid lipid nanoparticle solution is concentrated to reach the target volume through an ultracentrifuge tube. After dilution, it was used for DLS particle size measurement and encapsulation efficiency detection.

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

The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions. The test results are shown in Table 2.

Table 2

Lipid Nanoparticles Ionizable lipid compounds Particle size (nm) PDI (Polydispersity Index) Encapsulation rate (%) LNP A MC3 76.5 0.16 92.3 LNP B Compound 1 116.4 0.07 94.8 LNP C Compound 2 117.1 0.02 75.7 LNP D Compound 3 78.7 0.15 94.5 LNP E Compound 4 113.0 0.11 86.1 LNP F Compound 5 62.1 0.02 97.3 LNP G Compound 6 70.6 0.01 96.5

Example 8: Animal Experiment

mRNA and sgRNA delivery experiment of base editor ABE8e targeting PCSK9

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). The LDL receptor can bind to cholesterol that circulates back to the liver, and then break down cholesterol into bile acid, which is excreted out of the body through the intestines. PCSK9 is a protease synthesized by the liver that can bind to the LDL receptor and promote the entry of the LDL receptor into liver cells, causing the LDL receptor to be degraded by the lysosomes and the number of LDL receptors to decrease. Therefore, inhibiting the activity of the enzyme PSCK9 can increase the number of LDLRs, thereby enhancing the ability to absorb and break down 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 via 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. The CACC sequence was added to the 5' end of the upstream sequence of each sgRNA, and the AAAC sequence was added to the 5' end of the downstream sequence. After synthesis, the upstream and downstream sequences were annealed by a preset program (95°C, 5min; 95°C-85°C at -2°C/s; 85°C-25°C at -0.1°C/s; maintained at 4°C), 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 ligation system of sgRNA annealing product and linearized vector was as follows: 1 μL of T4 ligase buffer (NEB: M0202L), 20 ng of linearized vector, 5 μL of annealed oligo fragment (10 μM), 0.5 μL of T4 ligase (NEB: M0202L), ddH ₂ O was added to make up to 10 μL, and ligated at 16°C overnight.

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 an 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 anadenine base editor 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 in the laboratory for future use.

3. Animal research

Lipid nanoparticles containing the compounds of the present invention (see Table 2) encapsulating mRNA and sgRNA encoding the base editor ABE8e 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 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

The editing efficiency was tested one week after the mice were given the drug. The mice were killed and their liver tissues were taken out. The genome was extracted after lysis and the efficiency was analyzed by deep sequencing.

The deep sequencing steps are as follows:

See Table 3 for primer design:

Table 3

Sequence Objects sequence PCSK9-F2 5’-ACCAGACGGCTAGATGAGCA-3’(SEQ ID NO:2) PCSK9-R2 5’-CCCAGGACGAGGATGGAGATTA-3’(SEQ ID NO: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, 5 min.

After PCR, gel electrophoresis was performed 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 Figure 2.

Table 4

Lipid Nanoparticles Ionizable lipid compounds Editing efficiency (%) LNP A MC3 20 LNP B Compound 1 55 LNP C Compound 2 30 LNP D Compound 3 10 LNP E Compound 4 11 LNP F Compound 5 44 LNP G Compound 6 63

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 (15)

1. 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 or C _1-6 alkoxy; 0 to 3 H in the alkyl or alkoxy is optionally further substituted by halogen, hydroxy, C _1-6 alkyl, C _1-6 alkoxy, halogen-substituted C _1-6 alkyl or hydroxy-substituted 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; 0 to 3 ring Cs in the heterocyclic group are optionally further substituted by N, O or S; 0 to 3 Hs in the heterocyclic group are optionally further substituted by halogen, hydroxyl, C _1-6 alkyl, C _1-6 alkoxy, halogen-substituted C _1-6 alkyl or hydroxyl-substituted C _1-6 alkyl; L ¹ is selected from alkynylene, heteroatom O, N, S or absent; R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are each independently hydrogen, C _1-12 alkyl, C _1-12 alkenyl, C _1-12 alkynyl, or C _1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C _1-12 alkyl, C _1-12 alkenyl, C _{1-12 alkynyl, C 1-12 alkoxy} , halogen-substituted C _1-12 alkyl, or hydroxy-substituted C _1-12 alkyl; a and b are each independently selected from integers of 0-10; L ² is selected from alkynylene, heteroatom O, N, S or absent; R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are each independently hydrogen, C _1-12 alkyl, C _1-12 alkenyl, C _1-12 alkynyl, or C _1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C _1-12 alkyl, C _1-12 alkenyl, C _{1-12 alkynyl, C 1-12 alkoxy} , halogen-substituted C _1-12 alkyl, or hydroxy-substituted C _1-12 alkyl; c and d are each independently selected from integers of 0-10; L ³ and L ⁴ are each independently selected from alkynylene, heteroatom O, N, S or absent; R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , and R ¹⁹ are each independently hydrogen, C _1-12 alkyl, C _1-12 alkenyl, C _1-12 alkynyl, or C _1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C _1-12 alkyl, C _1-12 alkenyl, C _1-12 alkynyl, C _1-12 alkoxy, halogen-substituted C _1-12 alkyl, or hydroxy-substituted C _1-12 alkyl; e, f, and g are each independently selected from integers of 0-10. 2. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: ^G1 is methylene, ethylene, propylene, butylene or pentylene. 3. A compound according to claim 1 or 2, or a pharmaceutically acceptable form thereof, wherein: R ¹ and R ² are each independently methyl, ethyl, propyl or butyl, wherein 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C _1-3 alkyl; or, R ¹ , R ² and the connected N together form a 5-membered or 6-membered heterocyclic group, wherein 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and wherein 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C _1-3 alkyl; Alternatively, one of R ¹ and R ² is directly connected to any carbon atom in G ¹ to form a 5- or 6-membered heterocyclic group, and the other of R ¹ and R ² is methyl, ethyl, propyl or butyl, and 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C _1-3 alkyl; 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C _1-3 alkyl. 4. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable form thereof, wherein: ^L1 is Alkyne, O or absent; At least two of R ³ , R ⁴ and R ⁵ are not hydrogen, and R ⁶ and R ⁷ are hydrogen; a is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; b is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, at least one of a and b or both of them are not zero. 5. A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable form thereof, wherein: ^L2 is Alkyne, O or absent; R ⁸ and R ⁹ are each independently hydrogen, and at least two of R ¹⁰ , R ¹¹ , and R ¹² are not hydrogen; c and d are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, at least one of c and d or both of them are not 0. 6. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable form thereof, wherein: L ³ and L ⁴ are each independently selected from Alkyne, O or absent; R ¹³ and R ¹⁴ are each hydrogen; at least two of R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ are not hydrogen; e, f, g are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 7. The compound or pharmaceutically acceptable form thereof according to any one of claims 1 to 6, wherein the compound is selected from one or more of Compound 1 to Compound 24; Preferably, the pharmaceutically acceptable form is selected from a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug. 8. An intermediate having a structure shown in Formula Ic or Formula Id: Wherein, each substituent and the values of e, f, and g are as defined in any one of claims 1-7. 9. A method for preparing a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, the method comprising: Obtained by alkylation or reductive amination Thiocarbamates are generated by the action of triphosgene, carbonyldiimidazole or p-nitrophenyl chloroformate Removing the ketal protecting group and introducing a long fatty chain through a condensation reaction to obtain a compound of formula I or a pharmaceutically acceptable form thereof; Wherein, each substituent and the values of e, f, and g are as defined in any one of claims 1-7. 10. 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. 11. A lipid carrier comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof; Preferably, the lipid carrier comprises 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 lipid compounds, and the second lipid compound comprises one or a combination of two or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids. 12. 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 11, and a nucleic acid drug. 13. The nucleic acid lipid nanoparticle composition according to claim 12, wherein 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. 14. 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 11, or a nucleic acid lipid nanoparticle composition according to claim 12 or 13, and a pharmaceutically acceptable excipient, carrier or diluent. 15. Use of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 11, or a nucleic acid lipid nanoparticle composition according to claim 12 or 13, or a pharmaceutical preparation according to claim 14 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.