CN117203186A - Lipid compound containing disulfide bond and composition thereof - Google Patents

Lipid compound containing disulfide bond and composition thereof Download PDF

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Publication number
CN117203186A
CN117203186A CN202280001241.0A CN202280001241A CN117203186A CN 117203186 A CN117203186 A CN 117203186A CN 202280001241 A CN202280001241 A CN 202280001241A CN 117203186 A CN117203186 A CN 117203186A
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lipid
compound
polyethylene glycol
glycol modified
lipid compound
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曾臣
申志高
汪子芊
王鹏
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Southwest University of Science and Technology
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Southwest University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Abstract

A lipid compound and compositions thereof, the lipid compound comprising: lipid compounds represented by general formula (I) and/or (II), or pharmaceutically acceptable salts of the lipid compounds, or solvates of the lipid compounds, or lipid nanoparticles formed by the lipid compounds. The lipid compound is nontoxic, and the formed lipid nanoparticle can efficiently deliver nucleic acid and has high encapsulation efficiency and stability.

Description

Lipid compound containing disulfide bond and composition thereof Technical Field
The application relates to the field of compounds, in particular to a lipid compound containing disulfide bonds and a composition thereof.
Background
mRNA vaccines are bang on the "ten breakthrough technology worldwide" list of 2021 by their great reform in the medical field. The harvesting of new coronal mRNA vaccines has led to a third revolution in biopharmaceuticals following small molecule drugs and protein drugs. The treatment based on mRNA technology can be applied to important fields such as infectious disease prevention, tumor treatment, rare disease treatment, monoclonal antibody and other drug replacement treatment, and the like at a higher speed.
However, nucleic acid therapeutics still face several challenges, mRNA is a negatively charged polymer, is extremely hydrophilic, and is difficult to cross the cell membrane, which is also negatively charged, and to enter the cell itself; mRNA is also very unstable and is easily degraded by ubiquitous nucleases, and the circulation time in blood is very short; mRNA itself is immunogenic and is recognized by the immune system immediately after injection into the human body, thereby eliciting an immune response and being cleared. And mRNA vaccine delivery systems effectively solve this series of challenges. The lipid nanoparticle (Lipid nanoparticles, LNP) technology has high delivery efficiency and good safety, and is the most popular delivery technology at present. LNP is typically composed of cationic or ionizable lipids, helper lipids, structural lipids, and modified polyethylene glycol lipids. Accordingly, there is a need to develop lipid compounds and related methods and compositions capable of delivering nucleic acid drugs to facilitate the delivery of various types of nucleic acid drugs in cells, tissues and bodies.
Disclosure of Invention
According to a first aspect, in an embodiment, there is provided a lipid compound comprising: a lipid compound represented by general formula (I) and/or (II), or a pharmaceutically acceptable salt of said lipid compound, or a solvate of said lipid compound:
wherein:
each L 1 、L 2 And L 3 Independently selected from alkyl groups of 1 to 22 carbon atoms in length, said alkyl groups being linear or containing at least one branched chain, said carbon atoms on said alkyl groups being unsubstituted or at least one carbon atom on said alkyl groups being optionally replaced by at least one selected from the group consisting of: -C (O) -, -NH-, -O-, -S-, C 2 -C 10 Alkenyl, C 2 ～C 10 Alkynyl, C 3 ～C 8 Cycloalkyl and C 6 ～C 10 Arylene groups;
R 1 selected from the following structures: -NH 2 A mono-substituted amino group, a di-substituted amino group, a substituted or unsubstituted nitrogen-containing saturated heterocyclic ring, a substituted or unsubstituted aza aromatic compound;
R 2 and R is 3 Independently selected from the following structures: 1) A hydrogen atom; 2) An alkyl group comprising 1 to 22 carbon atoms, said alkyl group being linear or containing at least one branched chain, said carbon atoms on said alkyl group being unsubstituted or at least one carbon atom on said alkyl group being optionally replaced by at least one selected from the group consisting of: -C (O) -, -NH-, -O-, -S-, C 2 -C 10 Alkenyl, C 2 ～C 10 Alkynyl, C 3 ～C 8 Cycloalkyl and C 6 ～C 10 Arylene groups.
According to a second aspect, in an embodiment, there is provided a nanoparticle formulation comprising the lipid compound of the first aspect.
According to a third aspect, in an embodiment, there is provided a composition comprising the lipid compound of the first aspect, the lipid compound being loaded with an active agent.
According to the lipid compound containing disulfide bonds and the composition thereof, the lipid compound is nontoxic, and the lipid nanoparticle formed by the lipid compound can efficiently deliver active agents (including but not limited to nucleic acid) and has high encapsulation efficiency and stability.
Drawings
FIG. 1.1 is a graph of particle size of Compound 1.
Fig. 1.2 is a particle size diagram of compound 2.
Fig. 1.3 is a particle size diagram of compound 3.
Fig. 1.4 is a particle size diagram of compound 4.
FIG. 2 shows the expression intensity of Fluc mRNA delivered by intramuscular administration.
FIG. 3.1 is a hydrogen spectrum of Compound 1.
FIG. 3.2 is a carbon spectrum of Compound 1.
Fig. 4.1 is a hydrogen spectrum of compound 2.
Fig. 4.2 is a carbon spectrum of compound 2.
Fig. 5.1 is a hydrogen spectrum of compound 3.
Fig. 5.2 is a carbon spectrum of compound 3.
FIG. 6.1 is a hydrogen spectrum of Compound 4.
Fig. 6.2 is a carbon spectrum of compound 4.
FIG. 7.1 is a hydrogen spectrum of Compound 5.
Fig. 7.2 is a carbon spectrum of compound 5.
FIG. 8.1 is a hydrogen spectrum of Compound 6.
Fig. 8.2 is a carbon spectrum of compound 6.
FIG. 9.1 is a hydrogen spectrum of Compound 7.
Fig. 9.2 is a carbon spectrum of compound 7.
FIG. 10.1 is a hydrogen spectrum of Compound 8.
Fig. 10.2 is a carbon spectrum of compound 8.
FIG. 11.1 is a hydrogen spectrum of Compound 9.
Fig. 11.2 is a carbon spectrum of compound 9.
FIG. 12.1 is a hydrogen spectrum of compound 10.
Fig. 12.2 is a carbon spectrum of compound 10.
FIG. 13.1 is a hydrogen spectrum of Compound 11.
FIG. 13.2 is a carbon spectrum of Compound 11.
FIG. 14.1 is a hydrogen spectrum of compound 12.
Fig. 14.2 is a carbon spectrum of compound 12.
FIG. 15.1 is a hydrogen spectrum of Compound 13.
Fig. 15.2 is a carbon spectrum of compound 13.
FIG. 16.1 is a hydrogen spectrum of compound 14.
Fig. 16.2 is a carbon spectrum of compound 14.
FIG. 17.1 is a hydrogen spectrum of compound 15.
FIG. 17.2 is a carbon spectrum of compound 15.
Fig. 18.1 is a hydrogen spectrum of compound 16.
Fig. 18.2 is a carbon spectrum of compound 16.
FIG. 19.1 is a hydrogen spectrum of compound 17.
Fig. 19.2 is a carbon spectrum of compound 17.
FIG. 20.1 is a hydrogen spectrum of compound 18.
Fig. 20.2 is a carbon spectrum of compound 18.
FIG. 21.1 is a hydrogen spectrum of compound 19.
FIG. 21.2 is a carbon spectrum of compound 19.
Detailed Description
The application will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to persons skilled in the art from a detailed description of the related operations, which may be presented in the description and general knowledge of one skilled in the art.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, the steps or acts in the method descriptions may be sequentially transposed or modified in a manner apparent to those skilled in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning. The terms "connected" and "coupled," as used herein, unless otherwise indicated, are intended to encompass both direct and indirect connections (couplings).
Disulfide bonds play a great role in folding proteins to form secondary structures, reducing the structural entropy of proteins and increasing the mechanical strength of proteins. For example, animal hair contains many cysteines for disulfide bond formation, and the cysteine content of the membrane protein is higher than that of other proteins. Almost all transmembrane and secreted proteins, after assembly in the golgi apparatus, have been assembled, a signal sequence is added at their ends, which is linked to the protein by disulfide bonds. This indicates that disulfide bonds are stable in vivo and that biocompatibility is excellent. Meanwhile, disulfide bond reaction is a very common bioorthogonal reaction, and some compounds or drugs with specific properties can be connected to some biomacromolecules through disulfide bonds, so that the action mechanism of the biomacromolecules is studied or the performance of antibody drugs is improved. For example, the ADC drugs Mylotarg and Besponsa use disulfide bonds as cleavable linkers (linker) to small molecule toxic drugs.
In one embodiment, the present application introduces disulfide-bonded structures into cationic lipid compounds and produces LNPs. The LNP is stable in blood and once taken into the cell by endocytosis, intracellular reduced Glutathione (GSH) can react with cationic lipids through thiol-disulfide exchange, thereby disrupting the LNP structure and facilitating mRNA release. The disulfide bond-containing lipid compound and the LNP comprising the same can efficiently deliver nucleic acid in vivo, and meanwhile, the LNP has better safety due to biodegradability.
According to a first aspect, in an embodiment, there is provided a lipid compound comprising: a lipid compound represented by general formula (I) and/or (II), or a pharmaceutically acceptable salt of said lipid compound, or a solvate of said lipid compound:
wherein:
each L 1 、L 2 And L 3 Independently selected from alkyl groups of 1 to 22 carbon atoms in length, the carbon atoms on the alkyl groups being unsubstituted or at least one carbon atom on the alkyl groups being optionally replaced by at least one selected from the group consisting of: -C (O) -, -NH-, -O-, -S-, C 2 -C 10 Alkenyl, C 2 ～C 10 Alkynyl, C 3 ～C 8 Cycloalkyl and C 6 ～C 10 Arylene groups;
R 1 selected from the following structures: -NH 2 A mono-substituted amino group, a di-substituted amino group, a substituted or unsubstituted nitrogen-containing saturated heterocyclic ring, a substituted or unsubstituted aza aromatic compound;
R 2 and R is 3 Independently selected from the following structures: 1) A hydrogen atom; 2) An alkyl group comprising 1 to 22 carbon atoms, said alkyl group being linear or containing at least one branched chain, said carbon atoms on said alkyl group being unsubstituted or at least one carbon atom on said alkyl group being optionally replaced by at least one selected from the group consisting of: -C (O) -, -NH-, -O-, -S-, C 2 -C 10 Alkenyl, C 2 ～C 10 Alkynyl, C 3 ～C 8 Cycloalkyl and C 6 ～C 10 Arylene groups.
In one embodiment, the lipid compound comprises at least one of the following structures:
wherein n is an integer of 1 to 9.
In one embodiment, each R 2 And R is 3 Is a hydrogen atom.
In one embodiment, each L 2 And L 3 Independently selected from linear alkylene or alkenyl structures having a length of 8 to 22 carbon atoms.
In one embodiment, each L 2 And L 3 Independently selected from the following structures:
in one embodiment, R 1 May be selected from the following structures:
1) A disubstituted amine group; 2) Substituted or unsubstituted nitrogen-containing saturated four-membered ring, five-membered ring, six-membered ring, seven-membered ring, eight-membered ring; 3) Substituted or unsubstituted nitrogen-containing aromatic compounds.
In one embodiment, the nitrogen-containing aromatic compound includes, but is not limited to, at least one of imidazole, pyrazole, pyridine, pyrrole.
In one embodiment, R 1 Alkane chain- (CH) with ester bond 2 ) n Can be linear and n is an integer from 0 to 6.
In one embodiment, R 1 Selected from any one of the following structures:
in one embodiment, R 1 Alkane chain- (CH) with ester bond 2 ) n -any one selected from the following structures:
1)-(CH 2 ) 1 -；2)-(CH 2 ) 2 -；3)-(CH 2 ) 3 -；4)-(CH 2 ) 4 -；5)-(CH 2 ) 5 -；6)-(CH 2 ) 6 -；7)-(CH 2 )CH(CH 3 )(CH 2 )-。
in one embodiment, the lipid compound is selected from at least one of the following structures:
according to a second aspect, in an embodiment, there is provided a nanoparticle formulation comprising a lipid compound of any one of the first aspects.
In one embodiment, the nanoparticle formulation further comprises at least one of a helper lipid, a structural lipid, and a polyethylene glycol lipid.
In one embodiment, the molar ratio of the lipid compound to the auxiliary lipid, the structural lipid and the polyethylene glycol lipid is (40-60): (5-25): (25-50): (0.1-10).
In an embodiment, the auxiliary lipid is an ionic lipid or a neutral lipid, and may contain cationic functional groups (e.g., amine groups, quaternary ammonium groups) and/or anionic functional groups (e.g., phosphate groups, carboxylic acid groups).
In one embodiment, the nanoparticle formulation has an average particle size of 30nm to 200nm.
In one embodiment, the nanoparticle formulation has a polydispersity index of 0.3 or less.
In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is (3-50): 1.
in one embodiment, the helper lipid includes, but is not limited to, at least one of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, glycolipid, lipid.
In one embodiment, the auxiliary lipids include, but are not limited to, at least one of dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), 2, 3-dioleoyl-propyl) -trimethylamine (DOTAP), and 2, 3-dioleoyl-propyl) -Dimethylamine (DOTAP).
In one embodiment, the structural lipids may be used to stabilize the lipid nanoparticle and aid in fusion with the cell membrane.
In one embodiment, the structural lipid includes, but is not limited to, at least one of cholesterol, vitamin D, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, corticosteroid.
In one embodiment, the hydrophilic polyethylene glycol chains of the polyethylene glycol modified lipid are distributed on the surface of the nanoparticle particles, preventing fusion between the nanoparticles and reducing immunogenicity of the particles from being destroyed by the immune system in vivo, thereby prolonging circulation time in vivo.
In one embodiment, the polyethylene glycol modified lipid includes, but is not limited to, at least one of polyethylene glycol (PEG) modified phosphatidylethanolamine, polyethylene glycol modified phosphatidic acid, polyethylene glycol modified ceramide, polyethylene glycol modified dialkylamine, polyethylene glycol modified diacylglycerol, polyethylene glycol modified dialkylglycerol.
In one embodiment, the polyethylene glycol modified lipids include, but are not limited to, 1- (monomethoxy polyethylene glycol) -2, 3-dimesyl Kou Xianji glycerol (PEG 2000-DMG) (CAS No. 1397695-86-1).
In one embodiment, the lipid nanoparticle is used to deliver an active therapeutic agent.
According to a third aspect, in an embodiment, there is provided a composition comprising the lipid compound of the first aspect, the lipid nanoparticle formed from the lipid compound being loaded with an active agent. The active agent may be bound to the surface of the lipid compound or may be bound to the interior of the lipid compound.
In one embodiment, the composition comprises the nanoparticle formulation of the second aspect loaded with an active agent.
In one embodiment, the active agent includes, but is not limited to, at least one of a prophylactic agent, a therapeutic agent.
In one embodiment, the active agent includes, but is not limited to, a nucleic acid, an immunomodulatory agent, an antigen or fragment thereof, a vaccine, an anti-inflammatory agent, an anti-neoplastic agent, an antibiotic, an agent acting on the central nervous system, a protein, a peptide, a polypeptide, a small molecule, or a mixture thereof.
In one embodiment, the nucleic acid includes, but is not limited to, at least one of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), antisense oligonucleotide (ASO), DNA, and plasmid.
In one embodiment, the mass ratio of the lipid nanoparticle formulation to the active agent is (3-50): 1. the lipid nanoparticle preparation is used as a carrier for loading an active agent and delivering the active agent to a target position of a human or animal body, so as to prevent and/or treat corresponding diseases.
In one embodiment, the application has a novel chemical composition comprising a cationic or ionizable lipid, a helper lipid, a steroid, and polyethylene glycol or modified polyethylene glycol in a molar ratio of 50:10:38.5:1.5. these LNPs will be used to encapsulate nucleic acids, including but not limited to messenger RNA (mRNA), ribosomal RNA (rRNA), micrornas (miRNA), transfer RNAs (tRNA), small interfering RNAs (siRNA), small nuclear RNAs (snRNA), antisense oligonucleotides (ASO), DNA, plasmids, and the like, as active therapeutics that encapsulate the nucleic acid, delivering the nucleic acid into the cell. The active therapeutic agent may also be an immunomodulator, antigen or fragment thereof, vaccine, anti-inflammatory agent, anti-neoplastic agent, antibiotic, agent acting on the central nervous system, protein, peptide, polypeptide and small molecule, or mixtures thereof. The novel LNP can be used to transfect multicellular tissues or organs to provide a novel therapeutic treatment to a patient, which can be any mammal, preferably from humans, mice, rats, pigs, cats, dogs, horses, goats, cows and monkeys, and/or others.
In one embodiment, the present application provides an ionizable cationic lipid compound comprising disulfide bonds, compositions comprising the same, and uses thereof.
In one embodiment, the application also details the preparation of disulfide-bond ionizable lipids and lipid nanoparticles comprising disulfide-bond ionizable lipids, as well as methods of how to introduce nucleic acids for therapeutic purposes.
In one embodiment, a novel disulfide ionizable cationic lipid is provided that forms stable, specific functional Lipid Nanoparticles (LNPs) with phospholipids, sterols, and polyethylene glycol lipids for delivery of a range of drugs (RNA, DNA, proteins, polypeptides, or small molecule drugs). Also discloses a preparation method of the lipid and a formula for encapsulating RNA as a therapeutic drug.
In one embodiment, the application provides a novel disulfide bond-containing cationic lipid useful for the in vivo delivery of mRNA in cells, tissues, and methods of making and using the same. The application enriches the cationic lipid compound types, provides more choices for effective delivery of nucleic acid drugs, gene therapies, small molecule drugs, polypeptides or protein drugs, and has important significance in the development and application of nucleic acid preventive agents and therapeutic agents.
In one embodiment, the present application provides a lipid nanoparticle formulation comprising a novel disulfide-bonded lipid as a cationic lipid, and an auxiliary lipid, cholesterol, and polyethylene glycol ester. The lipid nanoparticle provided by the application is nontoxic, can be used for efficiently delivering nucleic acid, and has high encapsulation efficiency and stability.
Example 1: synthesis of 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-4- (dimethylamino) butyl ester (Compound 1C 4S 18A)
Into a 100mL round bottom flask was added 2,2' -dithiodipyridine (1.8 g,7.2 mmol), methanol (25 mL), followed by slowly dropping dimercaptopropanol (372 mg,3.0 mmol), stirring at room temperature and reacting for 12 hours, and column chromatography of the residue obtained by evaporating the solvent under reduced pressure (dichloromethane/petroleum ether 1/1then dichloromethane) to obtain a pale yellow liquid 2, 3-bis (pyridine disulfide) propanol (323 mg, 70%). To a 50mL round bottom flask was added linoleylthiol (2.3 g,8.0 mmol), methanol (20 mL), 2, 3-bis (pyridine disulfide) propanol (1.23 g,3.6 mmol), and the mixture was stirred at room temperature for 12 hours, followed by column chromatography under reduced pressure and evaporation to dryness to give 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) disulfide) propyl-1-ol (2.02 g, 82%). Dichloromethane (10 mL), 2, 3-bis (((9 z,12 z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-1-ol (554 mg,0.8 mmol), 4-dimethylaminobutyrate (0.96 mmol,161 mg), N-diisopropylethylamine (104 mg,0.8 mmol), 4-dimethylaminopyridine (10 mg,0.08 mmol) were then added to a 25mL round bottom flask and stirred at 0 ℃ for 20 minutes followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (192 mg,1.25 mmol) stirred at room temperature for 12 hours. Solvent column chromatography was removed under reduced pressure to give compound 1 (413 mg, 64%) as a pale yellow oil.
1 H NMR(400MHz,CDCl 3 )δ5.42–5.29(m,8H),4.36(d,J＝5.6Hz,2H),3.28(dq,J＝11.8,5.8Hz,1H),3.05(dd,J＝13.7,6.2Hz,1H),2.92(dt,J＝12.6,3.7Hz,1H),2.77(t,J＝6.4Hz,4H),2.70(t,J＝6.6Hz,4H),2.39(t,J＝7.4Hz,2H),2.34(t,J＝7.4Hz,2H),2.25(s,6H),2.05(dd,J＝13.7,6.8Hz,8H),1.86–1.77(m,2H),1.72–1.62(m,4H),1.42–1.24(m,32H),0.89(t,J＝6.7Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ173.24,130.36,130.21,128.17,128.05,64.17,58.79,49.27,45.37,40.20,39.97,39.00,32.01,31.67,29.80,29.57,29.56,29.49,29.39,29.36,29.36,29.33,29.30,28.65,28.62,27.36,27.35,25.77,22.80,22.72,14.23。
Fig. 3.1 shows a hydrogen spectrum of compound 1, and fig. 3.2 shows a carbon spectrum of compound 1.
HR-MS(ESI)m/z calcd for C 45 H 84 O 2 NS 4 [M+H] + 798.5379,found 798.5364。
Example 2: synthesis of 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-3- (dimethylamino) propyl ester (Compound 2C 3S 18A)
Into a 25mL round bottom flask was added dichloromethane (10 mL), 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-1-ol (554 mg,0.8 mmol), 3-dimethylaminopropionic acid (0.96 mmol,112 mg), N-diisopropylethylamine (104 mg,0.8 mmol), 4-dimethylaminopyridine (10 mg,0.08 mmol), stirred at 0deg.C for 20 min, followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (192 mg,1.25 mmol) and stirred at room temperature for 12 h. Solvent column chromatography was removed under reduced pressure to give compound 2 (489 mg, 78%) as a pale yellow oil.
1 H NMR(400MHz,CDCl 3 )δ＝5.47–5.26(m,8H),4.45–4.32(m,2H),3.33–3.24(m,1H),3.04(dd,J＝13.2,6.8Hz,1H),2.94(dd,J＝13.6,8.0Hz,1H),2.77(t,J＝6.3Hz,4H),2.70(t,J＝7.3Hz,4H),2.62(t,J＝7.0Hz,2H),2.51(t,J＝7.2Hz,2H),2.24(s,3H),2.04(dd,J＝13.5,6.7Hz,8H),1.73–1.61(m,8H),1.41–1.24(m,32H),0.89(t,J＝6.2Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ172.22,130.36,130.22,128.17,128.05,64.32,54.83,49.29,45.41,40.21,39.97,39.01,32.98,31.68,29.81,29.58,29.50,29.40,29.37,29.35,29.31,28.67,28.63,27.41,27.37,27.36,25.78,22.74,14.25。
HR-MS(ESI)m/z calcd for C 44 H 82 O 2 NS 4 [M+H] + 784.5223,found 784.5222。
Fig. 4.1 shows a hydrogen spectrum of compound 2, and fig. 4.2 shows a carbon spectrum of compound 2.
Example 3: synthesis of 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-3- (pyrrolidin-1-yl) propyl ester (Compound 3C 3 AS18A)
Into a 25mL round bottom flask was added dichloromethane (10 mL), 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-1-ol (554 mg,0.8 mmol), 3- (pyrrolidinyl-1-yl) propionic acid (0.96 mmol,137 mg), N-diisopropylethylamine (104 mg,0.8 mmol), 4-dimethylaminopyridine (10 mg,0.08 mmol), stirred at 0deg.C for 20 min, followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (192 mg,1.25 mmol) and stirred at room temperature for 12 h. The solvent was removed under reduced pressure and column chromatography was performed to give 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-3- (pyrrolidin-1-yl) propyl ester (460 mg, 71%) as a pale yellow oily liquid.
1 H NMR(400MHz,CDCl 3 )δ5.42–5.29(m,8H),4.38(qd,J＝11.6,5.6Hz,2H),3.28(dq,J＝11.9,5.8Hz,1H),3.04(dd,J＝13.8,6.3Hz,1H),2.94(dd,J＝13.8,8.0Hz,1H),2.83–2.75(m,6H),2.70(t,J＝7.3Hz,4H),2.57(t,J＝7.5Hz,2H),2.53(dd,J＝9.2,3.9Hz,4H),2.05(q,J＝6.8Hz,8H),1.81–1.75(m,4H),1.72–1.62(m,4H),1.44–1.22(m,32H),0.89(t,J＝6.9Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ172.22,130.35,130.21,128.16,128.04,64.24,54.14,51.46,49.32,40.16,39.96,39.00,34.23,31.68,29.80,29.57,29.50,29.40,29.37,29.34,29.31,28.66,28.63,27.37,27.35,25.78,23.63,22.73,14.24。
HR-MS(ESI)m/z calcd for C 46 H 84 O 2 NS 4 [M+H] + 810.5379,found 810.5373。
Fig. 5.1 shows a hydrogen spectrum of compound 3, and fig. 5.2 shows a carbon spectrum of compound 3.
Example 4: synthesis of 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-3- (4-methylpiperazin-1-yl) propyl ester (Compound 4C 3 BS18A)
Into a 25mL round bottom flask was added dichloromethane (10 mL), 2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-1-ol (554 mg,0.8 mmol), 3- (4-methylpiperazin-1-yl) propionic acid (0.96 mmol,165 mg), N-diisopropylethylamine (104 mg,0.8 mmol), 4-dimethylaminopyridine (10 mg,0.08 mmol) and stirred at 0deg.C for 20 min followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (192 mg,1.25 mmol) and stirred at room temperature for 12 h. Solvent column chromatography was removed under reduced pressure to give compound 4 (349 mg, 52%) as a pale yellow oil.
1 H NMR(500MHz,CDCl 3 )δ5.41–5.30(m,8H),4.41(dd,J＝11.6,5.7Hz,1H),4.35(dd,J＝11.6,5.4Hz,1H),3.28(dq,J＝7.9,5.7Hz,1H),3.04(dd,J＝13.7,6.3Hz,1H),2.94(dd,J＝13.8,8.1Hz,1H),2.77(t,J＝6.7Hz,4H),2.73–2.65(m,7H),2.57–2.41(m,6H),2.28(s,3H),2.08–2.01(m,8H),1.80–1.71(m,3H),1.66(dt,J＝14.6,7.3Hz,4H),1.41–1.24(m,32H),0.89(t,J＝6.9Hz,6H). 1 3 C NMR(101MHz,CDCl 3 )δ172.19,130.36,130.20,128.16,128.03,64.18,55.13,53.58,52.94,49.29,46.10,40.11,39.95,38.98,32.43,31.67,29.80,29.57,29.49,29.40,29.37,29.36,29.33,29.31,28.66,28.63,27.36,27.35,25.77,22.73,14.24.
HR-MS(ESI)m/z calcd for C 47 H 87 O 2 N 2 S 4 [M+H] + 839.5645,found 839.5651。
Fig. 6.1 shows a hydrogen spectrum of compound 4, and fig. 6.2 shows a carbon spectrum of compound 4.
Example 5: synthesis of 2, 3-di ((hexadecyl-1-yl) dithio) propyl-4- (dimethylamino) butyl ester (Compound 5C 4S 16)
To a 50mL round bottom flask was added linoleylthiol (2.07 g,8.0 mmol), methanol (20 mL), 2, 3-bis (pyridine disulfide) propanol (1.23 g,3.6 mmol), and the mixture was stirred at room temperature for 12 hours, followed by vacuum evaporation to dryness, followed by column chromatography to give 2, 3-bis ((hexadecyl-1-yl) disulfide) propyl-1-ol (1.74, 76%). Dichloromethane (10 mL), 2, 3-bis ((hexadecyl-1-yl) dithio) propyl-1-ol (600 mg,0.8 mmol), 4-dimethylaminobutyrate (0.96 mmol,161 mg), N-diisopropylethylamine (104 mg,0.8 mmol), 4-dimethylaminopyridine (10 mg,0.08 mmol) were then added to a 25mL round bottom flask and stirred at 0 ℃ for 20 min, followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (192 mg,1.25 mmol) and stirred at room temperature for 12 h. Solvent column chromatography was removed under reduced pressure to give compound 5 (402 mg, 67%) as a pale yellow oil.
1 H NMR(400MHz,CDCl 3 )δ4.35(dd,J＝9.2,3.8Hz,2H),3.27(dq,J＝8.0,5.8Hz,1H),3.03(dd,J＝12.4,6.2Hz,1H),2.91(dd,J＝13.7,8.1Hz,1H),2.70(td,J＝7.8,2.1Hz,4H),2.42–2.32(m,4H),2.26(s,6H),1.82–1.77(m,2H),1.71–1.62(m,2H),1.39–1.22(m,52H),0.87(t,J＝6.8Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝173.19,64.16,58.75,49.24,45.32,40.17,39.96,39.03,32.07,31.97,29.84,29.81,29.76,29.68,29.67,29.51,29.39,29.39,29.34,29.31,28.66,28.64,22.84,22.72,14.27。
HR-MS(ESI)m/z calcd for C 41 H 84 O 2 NS 4 [M+H] + 750.5379,found 750.5388。
Fig. 7.1 is a hydrogen spectrum of compound 5, and fig. 7.2 is a carbon spectrum of compound 5.
Example 6: synthesis of 2, 3-di ((octadecyl-disulfide) propyl) 4- (dimethylamino) butyl ester (Compound 6C 4S 18)
Into a 50mL round bottom flask was charged octadecylmercaptan (1.29 g,4.5 mmol), methanol (20 mL), 2, 3-bis (pyridine disulfide) propanol (0.70 g,2.06 mmol), stirred at room temperature for 12 hours, and then column chromatographed under reduced pressure to give 2, 3-bis (octadecyl-disulfide) propyl-1-ol (1.11 g, 78%). Dichloromethane (10 mL), 2, 3-dioctadecyl-dithio) propyl-1-ol (554 mg,0.8 mmol), 4-dimethylaminobutyrate (0.96 mmol,161 mg), N-diisopropylethylamine (104 mg,0.8 mmol), 4-dimethylaminopyridine (10 mg,0.08 mmol) were then added to a 25mL round bottom flask and stirred at 0 ℃ for 20 min, followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (192 mg,1.25 mmol) and stirred at room temperature for 12 h. Solvent column chromatography was removed under reduced pressure to give compound 6 (268 mg, 57%) as a pale yellow oil.
1 H NMR(400MHz,CDCl 3 )δ＝4.35(dd,J＝5.6,1.2Hz,2H),3.27(dq,J＝8.0,5.8Hz,1H),3.04(dd,J＝13.7,6.2Hz,1H),2.91(dd,J＝13.8,8.1Hz,1H),2.69(td,J＝7.7,2.1Hz,4H),2.36(dt,J＝15.1,7.5Hz,4H),2.24(s,6H),1.85–1.77(m,2H),1.66(dq,J＝14.4,7.1Hz,4H),1.39–1.20(m,60H),0.86(t,J＝6.8Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝173.17,64.14,58.68,49.22,45.25,40.15,39.94,39.00,32.05,31.96,29.82,29.79,29.74,29.65,29.65,29.49,29.37,29.36,29.32,29.29,28.64,28.62,22.81,22.68,14.25。
HR-MS(ESI)m/z calcd for C 45 H 92 O 2 NS 4 [M+H] + 806.6005,found 806.6009。
Fig. 8.1 is a hydrogen spectrum of compound 6, and fig. 8.2 is a carbon spectrum of compound 6.
Example 7:2, 3-bis ((octaalkyl-dithio) propyl) 4- (dimethylamino) butyl ester (Compound 7C 4S 8)
Compound 7 was prepared according to the synthetic method of compound 1.
1 H NMR(500MHz,CDCl 3 )δ＝4.39–4.33(m,2H),3.28(dq,J＝8.0,5.8Hz,1H),3.04(dt,J＝13.7,4.3Hz,1H),2.95–2.88(m,1H),2.70(td,J＝7.6,2.6Hz,4H),2.39(td,J＝7.5,3.3Hz,4H),2.28(s,6H),1.87–1.81(m,2H),1.68(dt,J＝14.6,7.4Hz,4H),1.41–1.35(m,4H),1.32–1.24(m,16H),0.8 8(t,J＝6.8Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝173.02,64.07,58.51,49.10,45.05,40.04,39.85,38.90,31.82,31.80,29.20,29.19,28.53,28.51,22.67,22.41,14.13。
HR-MS(ESI)m/z calcd for C 25 H 52 O 2 NS 4 [M+H] + 526.2875,found 526.2879。
Fig. 9.1 is a hydrogen spectrum of compound 7, and fig. 9.2 is a carbon spectrum of compound 7.
Example 8:2, 3-bis ((decanyl-dithio) propyl) 4- (dimethylamino) butyl ester (Compound 8C 4S 10)
Compound 8 was prepared according to the synthetic method of compound 1.
1 H NMR(500MHz,CDCl 3 )δ＝4.39–4.33(m,2H),3.30–3.25(m,1H),3.05(dd,J＝13.8,6.2Hz,1H),2.92(dd,J＝13.8,8.1Hz,1H),2.72–2.68(m,4H),2.38(t,J＝7.5Hz,2H),2.30(t,J＝7.1Hz,2H),2.22(s,6H),1.84–1.77(m,2H),1.71–1.63(m,4H),1.41–1.35(m,4H),1.32–1.24(m,24H),0.88(t,J＝7.0Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝173.30,64.14,58.86,49.26,45.49,40.19,39.96,39.03,32.06,32.04,29.70,29.67,29.66,29.46,29.38,29.38,29.34,29.31,28.66,28.64,22.94,22.82,14.27。
HR-MS(ESI)m/z calcd for C 29 H 60 O 2 NS 4 [M+H] + 582.3501,found 582.3503。
Fig. 10.1 shows a hydrogen spectrum of compound 8, and fig. 10.2 shows a carbon spectrum of compound 8.
Example 9:2, 3-Di ((dodecyl-dithio) propyl) 4- (dimethylamino) butyl ester (Compound 9C 4S 12)
Compound 9 was prepared according to the synthetic method of compound 1. 1 H NMR(400MHz,CDCl 3 )δ＝4.36(d,J＝5.7Hz,2H),3.28(dq,J＝8.1,5.8Hz,1H),3.08–3.02(m,1H),2.91(dd,J＝13.8,8.2Hz,1H),2.74–2.67(m,4H),2.41(dd,J＝15.5,8.1Hz,4H),2.31(s,6H),1.91–1.82(m,2H),1.71–1.62(m,4H),1.41–1.34(m,4H),1.31–1.24(m,32H),0.87(t,J＝6.8Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝173.07,64.20,58.58,49.22,45.08,40.15,39.98,39.03,32.06,31.86,29.81,29.79,29.76,29.68,29.67,29.50,29.39,29.35,29.32,28.66,28.64,22.84,22.41,14.28。
HR-MS(ESI)m/z calcd for C 33 H 68 O 2 NS 4 [M+H] + 638.4127,found 638.4132。
Fig. 11.1 shows a hydrogen spectrum of compound 9, and fig. 11.2 shows a carbon spectrum of compound 9.
Example 10:2, 3-bis ((tetradecyl-dithio) propyl) 4- (dimethylamino) butyl ester (Compound 10C 4S 14)
Compound 10 was prepared according to the synthetic method of compound 1.
1 H NMR(400MHz,CDCl 3 )δ＝4.39–4.33(m,2H),3.28(dq,J＝8.0,5.8Hz,1H),3.05(dd,J＝13.7,6.2Hz,1H),2.95–2.88(m,1H),2.70(td,J＝7.7,2.3Hz,4H),2.38(t,J＝7.5Hz,2H),2.30(d,J＝7.5Hz,2H),2.23(s,6H),1.85–1.76(m,2H),1.67(dq,J＝14.4,7.1Hz,4H),1.43–1.34(m,4H),1.31–1.22(m,40H),0.88(t,J＝6.8Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝173.30,64.15,58.85,49.26,45.46,40.19,39.97,39.04,32.07,29.85,29.84,29.81,29.77,29.68,29.68,29.52,29.40,29.39,29.35,29.32,28.67,28.65,22.91,22.85,14.28。
HR-MS(ESI)m/z calcd for C 37 H 76 O 2 NS 4 [M+H] + 694.4759,found 694.4756。
Fig. 12.1 is a hydrogen spectrum of compound 10, and fig. 12.2 is a carbon spectrum of compound 10.
Example 11:2, 3-bis ((9Z-octadecyl-9-en-1-yl) dithio) propyl-4- (dimethylamino) butyl ester (Compound 11C 4S 18B)
Compound 11 was prepared according to the synthetic method of compound 1.
1 H NMR(500MHz,CDCl 3 )δ＝5.37–5.30(m,4H),4.36(dd,J＝5.6,2.0Hz,2H),3.27(dq,J＝7.8,5.8Hz,1H),3.04(dd,J＝13.7,6.2Hz,1H),2.91(dd,J＝13.8,8.1Hz,1H),2.70(td,J＝7.7,2.1Hz,4H),2.36(dd,J＝15.8,7.6Hz,4H),2.26(s,6H),2.03–1.96(m,8H),1.70–1.63(m,2H),1.70–1.62(m,4H),1.38–1.23(m,44H),0.87(t,J＝6.9Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝173.17,130.10,129.88,64.15,58.69,49.21,45.25,40.14,39.93,38.97,32.03,31.94,29.89,29.87,29.83,29.79,29.74,29.65,29.55,29.45,29.37,29.35,29.34,29.31,29.28,28.63,28.61,27.34,27.31,22.82,22.66,14.26.
HR-MS(ESI)m/z calcd for C 45 H 88 O 2 NS 4 [M+H] + 802.5692,found 802.5699。
Fig. 13.1 shows a hydrogen spectrum of compound 11, and fig. 13.2 shows a carbon spectrum of compound 11.
Example 12:2, 3-bis ((tetradecyl-dithio) propyl-4- (dimethylamino) propyl ester (Compound 12C 3S 14)
Compound 12 was prepared according to the synthetic method of compound 1.
1 H NMR(500MHz,CDCl 3 )δ＝4.42–4.34(m,2H),3.28(dq,J＝7.8,5.9Hz,1H),3.04(dd,J＝13.8,6.3Hz,1H),2.93(dd,J＝13.6,8.3Hz,1H),2.73–2.68(m,4H),2.65–2.60(m,2H),2.52(dd,J＝8.8,5.3Hz,2H),2.25(s,6H),1.71–1.63(m,4H),1.37(d,J＝4.8Hz,4H),1.30–1.24(m,40H),0.87(t,J＝6.9Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝172.19,64.31,54.75,49.25,45.34,40.17,39.97,39.03,32.88,32.07,29.84,29.83,29.81,29.76,29.68,29.67,29.51,29.40,29.39,29.35,29.32,28.67,28.64,22.84,14.28。
HR-MS(ESI)m/z calcd for C 36 H 74 O 2 NS 4 [M+H] + 680.4597,found 680.4601。
Fig. 14.1 is a hydrogen spectrum of compound 12, and fig. 14.2 is a carbon spectrum of compound 12.
Example 13:2, 3-bis ((hexadecyl-dithio) propyl) 4- (dimethylamino) propyl ester (Compound 13C 3S 16)
Compound 13 was prepared according to the synthetic method of compound 1.
1 H NMR(500MHz,CDCl 3 )δ＝4.42–4.34(m,2H),3.28(dq,J＝7.8,5.8Hz,1H),3.05(dd,J＝13.7,6.3Hz,1H),2.98–2.90(m,1H),2.71(t,J＝6.8Hz,4H),2.63(dd,J＝10.9,3.9Hz,2H),2.51(dd,J＝8.8,5.3Hz,2H),2.24(s,6H),1.71–1.63(m,4H),1.40–1.35(m,4H),1.30–1.23(m,48H),0.88(t,J＝7.0Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝172.21,64.31,54.79,49.27,45.38,40.18,39.98,39.04,32.94,32.07,29.85,29.81,29.77,29.68,29.52,29.40,29.36,29.32,28.67,28.65,22.84,14.28。
HR-MS(ESI)m/z calcd for C 40 H 82 O 2 NS 4 [M+H] + 736.5223,found 736.5231。
Fig. 15.1 shows a hydrogen spectrum of compound 13, and fig. 15.2 shows a carbon spectrum of compound 13.
Example 14:2, 3-bis ((9Z-octadecyl-9-en-1-yl) dithio) propyl-4- (dimethylamino) propyl ester (Compound 14C 3S 18B)
Compound 14 was prepared according to the synthetic method of compound 1.
1 H NMR(400MHz,CDCl 3 )δ＝5.39–5.30(m,4H),4.42–4.33(m,2H),3.28(dq,J＝12.0,5.9Hz,1H),3.04(dd,J＝13.7,6.3Hz,1H),2.94(dd,J＝13.8,8.0Hz,1H),2.70(t,J＝7.3Hz,4H),2.63(t,J＝6.9Hz,2H),2.51(t,J＝7.0Hz,2H),2.25(s,6H),2.06–1.94(m,8H),1.71–1.64(m,4H),1.40–1.22(m,44H),0.88(t,J＝6.8Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝172.20,130.13,129.91,64.31,54.79,49.26,45.38,40.18,39.96,39.00,32.93,32.06,29.92,29.90,29.86,29.81,29.68,29.57,29.48,29.40,29.38,29.37,29.34,29.31,28.66,28.64,27.37,27.34,22.84,14.29。
HR-MS(ESI)m/z calcd for C 44 H 86 O 2 NS 4 [M+H] + 788.5536found 788.5533。
Fig. 16.1 is a hydrogen spectrum of compound 14, and fig. 16.2 is a carbon spectrum of compound 14.
Example 15:2, 3-bis ((tetradecyl-dithio) propyl) 4- (dimethylamino) pentyl ester (Compound 15C 5S 14)
Compound 15 was prepared according to the synthetic method of compound 1.
1 H NMR(500MHz,CDCl 3 )δ＝4.37(d,J＝5.6Hz,2H),3.28(dq,J＝8.2,5.7Hz,1H),3.05(dd,J＝13.7,6.1Hz,1H),2.90(dd,J＝13.8,8.3Hz,1H),2.71(td,J＝7.5,2.8Hz,4H),2.65(d,J＝7.4Hz,2H),2.53(s,6H),2.40(t,J＝6.9Hz,2H),1.74–1.63(m,6H),1.41–1.35(m,4H),1.31–1.23(m,42H),0.88(t,J＝6.9Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝172.90,64.15,58.45,49.20,44.14,40.11,39.98,39.02,33.61,32.07,29.84,29.83,29.81,29.81,29.76,29.68,29.51,29.40,29.39,29.34,29.32,28.66,28.64,22.84,22.35,14.28。
HR-MS(ESI)m/z calcd for C 38 H 78 O 2 NS 4 [M+H] + 708.4910,found 708.48980。
Fig. 17.1 is a hydrogen spectrum of compound 15, and fig. 17.2 is a carbon spectrum of compound 15.
Example 16:2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) disulfide) propyl-2- (dimethylamino) ethyl ester (Compound 17C 2S 18A)
Compound 16 was prepared according to the synthetic method of compound 1.
1 H NMR(400MHz,CDCl 3 )δ＝5.41–5.30(m,8H),4.45–4.38(m,2H),3.30(dq,J＝7.9,6.0Hz,1H),3.21(s,2H),3.08–3.02(m,1H),2.92(dd,J＝13.7,8.0Hz,1H),2.77(t,J＝6.7Hz,4H),2.70(t,J＝6.7Hz,4H),2.37(s,6H),2.07–2.02(m,8H),1.70–1.63(m,6H),1.39–1.25(m,30H),0.89(t,J＝6.9Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝170.47,130.35,130.20,128.16,128.03,64.32,60.28,49.14,45.41,40.20,39.95,39.00,31.67,29.79,29.57,29.56,29.50,29.39,29.37,29.35,29.33,29.31,28.65,28.62,27.36,27.35,25.77,22.73,14.25。
HR-MS(ESI)m/z calcd for C 43 H 80 O 2 NS 4 [M+H] + 770.5066,found 770.5073。
Fig. 18.1 is a hydrogen spectrum of compound 16, and fig. 18.2 is a carbon spectrum of compound 16.
Example 17:2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-2- (1H imidazol-1-yl) ethyl ester (Compound 18C 2 BS18A)
Compound 17 was prepared according to the synthetic method of compound 1.
1 H NMR(500MHz,CDCl 3 )δ＝7.51(s,1H),7.10(s,1H),6.96(s,1H),5.42–5.29(m,8H),4.74(s,2H),4.49(qd,J＝11.6,5.6Hz,2H),3.29(dq,J＝8.6,5.7Hz,1H),3.02(dd,J＝13.8,5.8Hz,1H),2.83–2.75(m,5H),2.70(td,J＝7.5,2.6Hz,4H),2.05(q,J＝6.9Hz,8H),1.72–1.62(m,4H),1.40–1.25(m,32H),0.89(t,J＝6.9Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝167.18,138.07,130.36,130.18,129.97,128.17,128.02,120.11,65.50,48.74,48.09,40.00,39.78,38.92,31.67,29.78,29.56,29.55,29.49,29.38,29.35,29.34,29.29,28.62,28.59,27.34,25.77,22.72,14.24。
HR-MS(ESI)m/z calcd for C 44 H 77 O 2 N 2 S 4 [M+H] + 793.4862,found 793.48682。
Fig. 19.1 is a hydrogen spectrum of compound 17, and fig. 19.2 is a carbon spectrum of compound 17.
Example 18:2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-3-morpholin-propyl ester (Compound 19C 3CS 18A)
Compound 18 was prepared according to the synthetic method of compound 1.
1 H NMR(400MHz,CDCl 3 )δ＝5.44–5.27(m,8H),4.39(qd,J＝11.6,5.5Hz,2H),3.71–3.68(m,4H),3.28(dq,J＝8.1,5.7Hz,1H),3.05(dd,J＝13.7,6.1Hz,1H),2.93(dd,J＝13.7,8.2Hz,1H),2.77(t,J＝6.5Hz,4H),2.70(dd,J＝14.8,7.3Hz,6H),2.53(t,J＝7.1Hz,2H),2.49–2.46(m,2H),2.05(dd,J＝13.7,6.8Hz,8H),1.67(ddd,J＝12.9,10.6,5.6Hz,6H),1.32(td,J＝13.3,8.5Hz,32H),0.89(t,J＝6.8Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝171.96,130.23,130.07,128.04,127.91,66.93,64.05,53.92,53.40,49.16,39.99,39.84,38.88,32.12,31.55,29.67,29.44,29.37,29.27,29.23,29.21,29.19,28.53,28.50,27.22,25.65,22.60,14.11。
HR-MS(ESI)m/z calcd for C 46 H 84 O 3 NS 4 [M+H] + 826.5329,found 826.5330。
Fig. 20.1 shows a hydrogen spectrum of compound 18, and fig. 20.2 shows a carbon spectrum of compound 18.
Example 19:2, 3-bis (((9Z, 12Z) -octadecyl-9, 12-dien-1-yl) dithio) propyl-3- (1H imidazol-1-yl) propyl ester (Compound 20C 3 DS18A)
Compound 19 was prepared according to the synthetic method of compound 1.
1 H NMR(400MHz,CDCl 3 )δ＝7.52(s,1H),7.05(s,1H),6.94(s,1H),5.43–5.28(m,8H),4.40(d,J＝5.6Hz,2H),4.27(t,J＝6.6Hz,2H),3.26(dq,J＝8.3,5.7Hz,1H),3.02(dd,J＝13.8,6.0Hz,1H),2.85–2.75(m,6H),2.69(td,J＝7.5,2.8Hz,3H),2.09–1.99(m,8H),1.65(dt,J＝14.6,7.3Hz,4H),1.41–1.24(m,34H),0.89(t,J＝6.8Hz,6H). 13 C NMR(101MHz,CDCl 3 )δ＝170.29,137.39,130.36,130.19,129.92,128.18,128.03,118.93,64.81,48.97,42.28,40.00,38.97,36.00,31.67,29.79,29.56,29.49,29.39,29.35,29.34,29.29,28.64,28.60,27.35,25.77,22.72,14.24。
HR-MS(ESI)m/z calcd for C 45 H 79 O 2 N 2 S 4 [M+H] + 807.5019,found 807.5018。
Fig. 21.1 is a hydrogen spectrum of compound 19, and fig. 21.2 is a carbon spectrum of compound 19.
Preparation and characterization of nanolipid particles (LNP formulations)
Method of preparing LNP formulation: the disulfide cationic lipids prepared in each example were combined with DSPC, cholesterol, PEG2000-DMG at 50:10:38.5:1.5 in absolute ethanol at a total lipid concentration of 10mM. Luciferase mRNA (Fluc mRNA) was dissolved in aqueous sodium acetate (10 mm, ph=4.6) and mRNA concentration was 0.1mg/mL. Using a microfluidic instrument to control the flow rate ratio of the ethanol solution to the sodium acetate aqueous solution to be 1: and 3, preparing a solution of lipid nano particles in a microfluidic chip, diluting and centrifuging with PBS at 4 ℃ to remove ethanol, diluting and centrifuging again for three times, and diluting the obtained concentrated solution with PBS for later use. The size and polydispersity index (PDI) of the lipid nanoparticles were determined by dynamic light scattering using Malvern Zetasizer Nano ZS in 173 ° backscatter detection mode. The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific). The final lipid nanoparticle particle size, PDI and encapsulation efficiency are shown in table 1. FIGS. 1.1, 1.2, 1.3, and 1.4 are particle size diagrams of compounds 1, 2,3, and 4, respectively.
TABLE 1
LNP formulation Cationic lipids (Code) Size(nm) PDI Encapsulation efficiency
1 Compound 1 C4S18A 111.0 0.115 94.1％
2 Compound 2 C3S18A 109.0 0.143 93.0％
3 Compound 3 C3AS18A 108.6 0.150 95.3％
4 Compound 4 C3BS18A 104.3 0.135 96.5％
5 Compound 5 C4S16 105.9 0.123 96.6％
6 Compound 6 C4S18 108.1 0.098 94.8％
7 Compound 7 C4S8 82.5 0.325 91.8％
8 Compound 8 C4S10 100.0 0.266 90.5％
9 Compound 9 C4S12 52.0 0.251 89.2％
10 Compound 10 C4S14 102.0 0.139 95.6％
11 Compound 11 C4S18B 62.1 0.338 91.8％
12 Compound 12 C3S14 89.6 0.297 90.1％
13 Compound 13 C3S16 110.3 0.383 92.3％
14 Compound 14 C3S18B 78.1 0.235 94.2％
15 Compound 15 C5S14 66.9 0.358 91.4％
16 Compound 16 C2S18A 119.6 0.264 91.2％
17 Compound 17 C2BS18A 63.2 0.323 93.6％
18 Compound 18 C3CS18A 82.8 0.325 92.7％
19 Compound 19 C3DS18A 64.7 0.146 86.5％
As can be seen from Table 1, LNPs prepared from the cationic lipid compounds prepared in examples 1 to 19 are capable of encapsulating nucleic acid molecules, and the obtained mRNA-LNP has good uniformity and high encapsulation efficiency.
Animal experiment
Female BALB/c mice of 6 weeks old are selected, the weight is about 20g, the feeding environment is SPF-grade feeding room, and animal experiments are strictly performed by referring to national institutes of health guidelines and animal ethics requirements. 3 mice were randomly selected and injected intramuscularly with lipid nanoparticles at a dose of 0.5 mg/kg. After 12 hours, 200. Mu.L of 10mg/mL of D-potassium fluorescein salt was intramuscularly injected into each mouse, and after 10 minutes, the mice were placed under a living imaging system (PerkinElmer IVIS Spectrum), and the total fluorescence intensity of each mouse was observed and recorded by photographing. And then imaged at 12 hours. Intramuscular administration mode the expression intensity of the delivery Fluc mRNA is shown in fig. 2 and table 2. In FIG. 2, the expression intensities of Fluc mRNA of mice in the treatment groups of compounds 3, 1, 2, and 4 are shown in order from left to right.
TABLE 2
LNP formulation Cationic lipids Total luminous intensity (p/sec/cm < 2 >/sr)
1 Compound 1 1.98E+08
2 Compound 2 3.41E+08
3 Compound 3 1.41E+08
4 Compound 4 2.51E+08
As can be seen from fig. 2 and table 2, LNP vectors prepared from a plurality of cationic lipid compounds of the present application are capable of delivering Fluc mRNA to cells and expressing luciferase at high levels and emitting light.
The experimental data prove that LNP prepared by the cationic lipid can successfully encapsulate nucleic acid molecules, has good uniformity and encapsulation efficiency, and can transport nucleic acid into animal bodies for expression translation.
The foregoing description of the application has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the application pertains, based on the idea of the application.

Claims (10)

  1. A lipid compound comprising: a lipid compound represented by general formula (I) and/or (II), or a pharmaceutically acceptable salt of said lipid compound, or a solvate of said lipid compound:
    wherein:
    each L 1 、L 2 And L 3 Independently selected from alkyl groups of 1 to 22 carbon atoms in length, said alkyl groups being linear or containing at least one branched chain, said carbon atoms on said alkyl groups being unsubstituted or at least one carbon atom on said alkyl groups being optionally replaced by at least one selected from the group consisting of: -C (O) -, -NH-, -O-, -S-, C 2 -C 10 Alkenyl, C 2 ～C 10 Alkynyl, C 3 ～C 8 Cycloalkyl and C 6 ～C 10 Arylene groups;
    R 1 selected from the following structures: -NH 2 Mono-, di-, substituted or unsubstituted nitrogen-containing saturated heterocycles, substituted or unsubstituted amino groupsAn unsubstituted aza aromatic compound;
    R 2 and R is 3 Independently selected from the following structures: 1) A hydrogen atom; 2) An alkyl group comprising 1 to 22 carbon atoms, said alkyl group being linear or containing at least one branched chain, said carbon atoms on said alkyl group being unsubstituted or at least one carbon atom on said alkyl group being optionally replaced by at least one selected from the group consisting of: -C (O) -, -NH-, -O-, -S-, C 2 -C 10 Alkenyl, C 2 ～C 10 Alkynyl, C 3 ～C 8 Cycloalkyl and C 6 ～C 10 Arylene groups.
  2. The lipid compound of claim 1, wherein the lipid compound comprises at least one of the following structures:
    wherein n is an integer of 1 to 9, R 1 、R 2 、R 3 、L 2 And L 3 As defined in claim 1.
  3. The lipid compound according to claim 2, wherein each R 2 And R is 3 Is a hydrogen atom;
    L 2 and L 3 Independently selected from linear alkylene or alkenyl structures having a length of 8 to 22 carbon atoms;
    preferably, each L 2 And L 3 Independently selected from the following structures:
  4. the lipid compound according to claim 1, wherein R 1 Selected from the following structures:
    1) A disubstituted amine group; 2) Substituted or unsubstituted nitrogen-containing saturated four-membered ring, five-membered ring, six-membered ring, seven-membered ring, eight-membered ring; 3) Substituted or unsubstituted nitrogen-containing aromatic compounds;
    preferably, the nitrogen-containing aromatic compound comprises at least one of imidazole, pyrazole, pyridine, pyrrole.
  5. The lipid compound according to claim 2, wherein R 1 Alkane chain- (CH) with ester bond 2 ) n -is a linear chain, n is an integer from 0 to 6;
    preferably, R 1 Selected from any one of the following structures:
    preferably, R 1 Alkane chain- (CH) with ester bond 2 ) n -any one selected from the following structures:
    1)-(CH 2 ) 1 -；2)-(CH 2 ) 2 -；3)-(CH 2 ) 3 -；4)-(CH 2 ) 4 -；5)-(CH 2 ) 5 -；6)-(CH 2 ) 6 -；7)-(CH 2 )CH(CH 3 )(CH 2 )-。
  6. the lipid compound according to any one of claims 1 to 5, wherein the lipid compound is selected from at least one of the compounds represented by the following structures;
  7. a nanoparticle formulation comprising a lipid compound according to any one of claims 1 to 6.
  8. The nanoparticle formulation of claim 7, further comprising at least one of a helper lipid, a structural lipid, and a polyethylene glycol modified lipid;
    preferably, the molar ratio of the lipid compound to the auxiliary lipid, the structural lipid and the polyethylene glycol modified lipid is (40-60): (5-25): (25-50): (0.1-10);
    preferably, the helper lipid is an ionic lipid or a neutral lipid;
    preferably, the helper lipid contains cationic and/or anionic functionalities;
    preferably, the cationic functional group comprises an amine group, a quaternary ammonium group;
    preferably, the anionic functional groups include phosphate groups, carboxylic acid groups;
    preferably, the nanoparticle formulation has an average particle size of 30nm to 200nm;
    preferably, the nanoparticle formulation has a polydispersity index of 0.3 or less;
    preferably, the auxiliary lipid comprises at least one of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, glycolipid, and lipid;
    preferably, the auxiliary lipid comprises at least one of dioleoyl phosphatidylethanolamine, distearoyl phosphatidylcholine, (2, 3-dioleoyl-propyl) -trimethylamine, (2, 3-dioleoyl-propyl) -dimethylamine;
    preferably, the structural lipid comprises at least one of cholesterol, vitamin D, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, corticosteroids;
    preferably, the hydrophilic polyethylene glycol chains of the polyethylene glycol modified lipid are distributed on the surface of the nanoparticle preparation;
    preferably, the polyethylene glycol modified lipid comprises at least one of polyethylene glycol modified phosphatidylethanolamine, polyethylene glycol modified phosphatidic acid, polyethylene glycol modified ceramide, polyethylene glycol modified dialkylamine, polyethylene glycol modified diacylglycerol, polyethylene glycol modified dialkylglycerol;
    preferably, the polyethylene glycol modified lipid comprises 1- (monomethoxy polyethylene glycol) -2, 3-two-meat bean Kou Xianji glycerol.
  9. A composition comprising a lipid compound according to any one of claims 1 to 6, wherein the lipid compound forms lipid nanoparticles loaded with an active agent.
  10. The composition of claim 9, comprising a nanoparticle formulation according to any one of claims 7 to 8, said nanoparticle formulation being loaded with an active agent;
    preferably, the active agent comprises at least one of a prophylactic agent, a therapeutic agent;
    preferably, the active agent comprises a nucleic acid, an immunomodulator, an antigen or fragment thereof, a vaccine, an anti-inflammatory agent, an anti-tumour agent, an antibiotic, a medicament acting on the central nervous system, a protein, a peptide, a polypeptide, a small molecule drug, or a mixture thereof;
    preferably, the nucleic acid comprises at least one of messenger RNA, ribosomal RNA, microrna, transfer RNA, small interfering RNA, small nuclear RNA, antisense oligonucleotide, DNA, plasmid;
    preferably, the mass ratio of the lipid nanoparticle preparation to the active agent is (3-50): 1.
CN202280001241.0A 2022-05-13 2022-05-13 Lipid compound containing disulfide bond and composition thereof Pending CN117203186A (en)

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