Amino acid lipid formed nanodelivery system and its use

Abstract

The application relates to an amino acid lipid formed nano delivery system and application thereof. The ionizable lipid forming the amino acid lipid nano delivery system is an amino acid lipid shown in a general formula (I) or a general formula (II) or a salt thereof, wherein R1 represents an amino acid residue with one hydroxyl group removed from a carboxyl group, the structural formula is NH2-CR-CO-, R is an R group of an amino acid; R2, R3 and R4 are independently linear alkyl groups with 5-40 carbon atoms. The application can enrich the existing nucleic acid drug delivery system, effectively reduce immunogenicity and biological toxicity, and provide more safe and effective nucleic acid drugs for users.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nucleic acid drug delivery system, and particularly relates to an amino acid lipid formed nano delivery system and application thereof. BACKGROUND

[0002] With the development of mRNA vaccines, RNA interference (RNAi), RNAi therapy, RNA drugs, antisense therapy and gene therapy, the demand for introducing RNA into cells has increased. However, RNA is highly unstable. In general, RNA alone is stable in plasma for no more than a few hours. In order to exert a therapeutic effect, an effective delivery system is required for RNA drugs.

[0003] Liposome nanoparticles (LNP) are one of the most commonly used delivery systems for nucleic acid drugs. LNP has high encapsulation efficiency for nucleic acids and can effectively transfect cells, and has strong tissue penetration, which is more conducive to drug delivery. These advantages make LNP an excellent nucleic acid delivery system. The LNP components widely used at present mainly include the following four categories: liposomes, neutral phospholipid auxiliary lipids, cholesterol or its derivatives, and polyethylene glycol lipids. Liposomes are the key component, which are amphiphilic chemical molecules composed of a polar hydrophilic head, a hydrophobic tail, and a connecting group between the two.

[0004] The first generation of liposomes developed earliest is cationic liposomes, that is, the hydrophilic head is positively charged, usually quaternary ammonium salt liposomes. Cationic liposomes can spontaneously self-assemble into stable nanoparticles through electrostatic interaction with negatively charged nucleic acids to deliver nucleic acids into cells, so they are still used in LNP. However, cationic liposomes have relatively high cytotoxicity and are gradually being replaced by ionizable liposomes, that is, the second generation of liposomes.

[0005] Ionizable liposomes are neutral liposomes, that is, the hydrophilic head is usually an alkyl-substituted amine group. In an acidic medium (low pH), the amine group in the ionizable liposome combines with hydrogen ions to become a positively charged compound, which covers the RNA molecule through ionic interaction, forms nanoparticles, and helps the RNA molecule to be released from the cell endosome / lysosome into the cell cytoplasm by interfering with the stability and permeability of the endosome / lysosome membrane. Due to the improvement of effectiveness and toxicity characteristics, LNP formed by ionizable liposomes has become the mainstream nucleic acid delivery system.

[0006] LNP delivery systems that encapsulate RNA drugs stably in serum are also known as SNALP (Serum-stable nucleic acid lipid particles, US8058069). SNALP has helped multiple mRNA vaccines (Pfizer / BioNTech, Moderna) and the first siRNA drug (Patisiran, 2018, Alnylam) to obtain marketing approval. Figure 1 The structure of the liposome delivery system used in Patisiran is shown. The typical liposome used in the market is composed of one tri-substituted amine and two lipids containing linear or branched ester bonds (e.g. SM102, ALC0315).

[0007] One of the most important physicochemical properties of SNALP is the apparent dissociation constant (apparent pKa). The apparent pKa is a property of the nanoparticle determined experimentally. At this pH, the equivalents of dissociated and non-dissociated groups are equal. The most effective SNALP nanoparticles in RNA delivery have an apparent pKa value between 6 and 7 (Cheng et al., Trends in Pharmacological Sciences, 42:448, 2021).

[0008] But SNALP also has disadvantages. The ionizable lipid of SNALP is a synthetic chemical with lower biocompatibility than natural lipids. Side effects such as inflammation, inflammation exacerbation reactions have been reported (Muzykantov et al., Journal of Controlled Release, 344:50, 2022).

[0009] Therefore, how to reduce the toxicity of ionizable lipids while ensuring the encapsulation effect of nucleic acid molecules and the function of in vivo delivery of nucleic acid molecules is one of the key problems in nucleic acid delivery technology. SUMMARY

[0010] The purpose of the present application is to provide a new amino acid lipid or salt thereof, which has a high encapsulation efficiency of nucleic acid molecules, can effectively deliver nucleic acid molecules and successfully express nucleic acid molecules in cells, and can reduce immunogenicity and biological toxicity compared to mainstream delivery systems.

[0011] Another purpose of the present application is to provide a drug delivery system with low immunogenicity, low biological toxicity, which can effectively deliver nucleic acid molecules and ensure the successful expression of nucleic acid molecules.

[0012] Still another purpose of the present application is to provide a nucleic acid drug with low immunogenicity and low biological toxicity.

[0013] To achieve the above object, the technical scheme adopted by the present application is:

[0014] amino acid lipid represented by general formula (I) or general formula (II) or a salt thereof,

[0015]

[0016] wherein,

[0017] R1 represents an amino acid residue with one hydroxyl group removed from a carboxyl group, and its structural formula is NH2-CR-CO-, R is the R group of an amino acid;

[0018] R2, R3, R4 are independently linear alkyl groups with 5-40 carbon atoms.

[0019] In the present application, the amino acid is not limited to L-type or D-type.

[0020] Preferably, the amino acid is glycine, methionine, serine, phenylalanine, alanine, threonine, tyrosine, hydroxyproline, glutamic acid, lysine, cysteine, proline, valine, leucine, isoleucine, tryptophan, glutamine, aspartic acid, asparagine, arginine or histidine.

[0021] Further preferably, the amino acid is glycine, methionine, serine, phenylalanine, alanine, threonine, tyrosine, hydroxyproline, glutamic acid or lysine.

[0022] More preferably, the amino acid is glycine, methionine, serine or phenylalanine.

[0023] Preferably, R2, R3, R4 are independently linear alkyl groups with 5-40 carbon atoms.

[0024] Further preferably, R2, R3, R4 are independently linear alkyl groups with 7-30 carbon atoms.

[0025] More preferably, R2, R3, R4 are independently linear alkyl groups with 7-20 carbon atoms.

[0026] Preferably, R2, R3, R4 are independently linear alkenyl groups with 1-3 double bonds and 5-40 carbon atoms.

[0027] Further preferably, R2, R3, R4 are independently linear alkenyl groups with 1-3 double bonds and 7-30 carbon atoms.

[0028] More preferably, R2, R3, R4 are independently linear alkenyl groups with 1-3 double bonds and 7-20 carbon atoms.

[0029] Preferably, the amino acid lipid or salt thereof is a compound represented by general formula (I).

[0030] In the present application, R2, R3, R4 are the same or different.

[0031] Preferably, R2, R3, R4 are the same.

[0032] and / or, R2, R3, R4 are independently:

[0033]

[0034] According to some embodiments, the amino acid lipid is:

[0035]

[0036]

[0037] The present application also provides a delivery system comprising one or more of the above-mentioned amino acid lipids or salts thereof.

[0038] Preferably, the delivery system further comprises one or more of helper lipids, cholesterol and derivatives thereof, PEGylated lipids.

[0039] Further preferably, the helper lipids are selected from phospholipids and derivatives thereof.

[0040] Still further preferably, the helper lipids are selected from one or more of PC, DPPC, DOPC, DSPC, DOPE, DPPG.

[0041] Further preferably, the PEGylated lipids are selected from one or more of PEG-DMG, PEG-C-DMG, PEG-DSPE.

[0042] Further preferably, the molar ratio of the amino acid lipid or salt thereof, the helper lipids, the cholesterol and derivatives thereof, and the PEGylated lipids is (40-99.5):(0-15):(0-50):(0.5-3).

[0043] The present application also provides use of the delivery system in the preparation of a nucleic acid drug.

[0044] The present application also provides a nucleic acid drug comprising the above-mentioned delivery system and a nucleic acid molecule.

[0045] Preferably, the nucleic acid molecule comprises one or more of a messenger nucleic acid molecule (mRNA), a small interfering nucleic acid molecule (siRNA), a micro nucleic acid molecule (miRNA), a small activating nucleic acid molecule (saRNA), an antisense oligonucleotide molecule (ASO) or an aptamer.

[0046] Preferably, the nucleic acid drug is an amino acid lipid nanoparticle with a particle size of 50-300 nm.

[0047] Further preferably, the nucleic acid drug is an amino acid lipid nanoparticle with a particle size of 50-300 nm, such as 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 nm, 220 nm, 250 nm, 280 nm, 300 nm.

[0048] Preferably, the nucleic acid drug is obtained by mixing and self-assembling the nucleic acid molecule and the amino acid lipid or salt thereof, optionally with one or more of an auxiliary lipid, cholesterol and derivatives thereof, and a PEGylated lipid (PEG-Lipid) through microfluidics.

[0049] Preferably, the nitrogen-to-phosphorus molar ratio (N / P) of the amino acid lipid or salt thereof and the nucleic acid molecule is (1-50):1.

[0050] Further preferably, the nitrogen-to-phosphorus molar ratio of the amino acid lipid or salt thereof and the nucleic acid molecule is (3-15):1, such as 3:1, 5:1, 10:1, 15:1.

[0051] Preferably, the nucleic acid drug further comprises a pharmaceutically acceptable additive.

[0052] Further preferably, the additive comprises an excipient or the like.

[0053] Preferably, the nucleic acid drug is a lyophilized powder or an injection.

[0054] Further preferably, the subject of the nucleic acid drug is a mammal, preferably a human.

[0055] Further preferably, the nucleic acid drug is administered by intramuscular injection, intravenous injection or the like.

[0056] The present application has at least the following beneficial effects:

[0057] The amino acid lipid or salt thereof obtained by adopting the amino acid substitution as the hydrophilic head of the ionizable lipid and combining the hydrophobic carboxylic acid tail has low cytotoxicity, the drug delivery system prepared by using the amino acid lipid or salt thereof can achieve the effect of mainstream nanoliposomes in terms of the encapsulation efficiency of nucleic acid molecules, and can also deliver the nucleic acid molecules into the body and realize the effective expression of the nucleic acid molecules in cells, and compared with mainstream nanoliposomes, the amino acid lipid or salt thereof has lower self-toxicity while ensuring the delivery function.

[0058] Further, the amino acid lipid or salt thereof formed by connecting the amino acid hydrophilic head and the hydrophobic hydrocarbon chain by tris(hydroxymethyl)aminomethane can not only reduce the immunogenicity and biological toxicity of the delivery system, but also has obviously better in-vivo delivery effect without affecting the encapsulation efficiency of nucleic acid molecules.

[0059] The present application can enrich the existing nucleic acid drug delivery system, provide more choices for users, and be beneficial to the development and application of nucleic acid drugs. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 It is a structural schematic diagram of a lipid nanoparticle (LNP);

[0061] Figure 2 It is a cell toxicity test result diagram of the amino acid lipid;

[0062] Figure 3 It is a luciferase antibody detection result diagram;

[0063] Figure 4 It is a gene knockdown effect diagram corresponding to the apparent pKa. DETAILED DESCRIPTION

[0064] In order to solve the problems of the existing nucleic acid delivery system, such as complex composition, high preparation cost, high biological toxicity, strong immunogenicity, and low endosome escape rate, the present application is proposed by the present inventors after long-term research and a large number of practices. The technical solution, implementation process, and principles will be further explained below.

[0065] The present inventors have found that the hydrophilic head amine group in the ionizable liposome can be formed by amino acids, and compared with the non-natural amine group with high toxicity, the amino acid substitution has good biocompatibility and non-immunogenicity, and reduces the cytotoxicity of the ionizable liposome without causing the decrease of the encapsulation efficiency.

[0066] Specifically, the present application provides an amino acid lipid or salt thereof represented by general formula (I) or general formula (II),

[0067]

[0068] wherein R1 represents an amino acid residue with one hydroxyl group missing from the carboxyl group, and has a structure of NH2-CR-CO-, R is the R group of the amino acid; R2, R3, and R4 are independently linear hydrocarbon groups with a carbon number of 5-40.

[0069] The amino acid lipids of the present application can disrupt endosome membranes and safely release nucleic acid molecules into the cytoplasm and achieve expression.

[0070] Based on one of the purposes of the present application, to provide amino acid lipids and nucleic acid drug delivery systems thereof with low immunogenicity and low biological toxicity, in addition to considering the selection of amino acids from natural sources, in the selection of carboxylic acid lipids, not only the efficiency of nucleic acid encapsulation and delivery is considered, but also natural carboxylic acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, oleic acid, and linoleic acid are selected as much as possible, so that the side reactions during delivery can be further reduced, and the immunogenicity and biological toxicity can be reduced.

[0071] In some preferred embodiments, R2, R3, and R4 are independently:

[0072]

[0073] After selecting the hydrophilic amino acid head and the hydrophobic carboxylic acid tail, the inventors selected pentaerythritol (PEL) as the linking group for both. The chemical structure of pentaerythritol is:

[0074]

[0075] Pentaerythritol is a polyhydroxy compound that can be covalently connected to amino acids through an ester bond and can be covalently combined with carboxylic acid lipids through an ester bond. Pentaerythritol has good safety, and because pentaerythritol is covalently combined with amino acids and hydrophobic lipids through an ester bond, it is easily hydrolyzed into small molecules after entering cells and is promptly cleared from the body, effectively reducing immunogenicity and biological toxicity.

[0076] According to this strategy, the inventors synthesized several amino acid pentaerythritol lipids (APL) with pentaerythritol as the linking group, and prepared the corresponding liposome nanoparticles (APLNP). Physicochemical and biological tests found that APLNP can effectively encapsulate mRNA and siRNA.

[0077] The present application further attempts to use Tris instead of pentaerythritol as the linking group in the amino acid liposomes, and the chemical structure of Tris is:

[0078]

[0079] Similar to pentaerythritol, Tris is also a polyhydroxyl multifunctional compound, in which three hydroxyl groups can be covalently connected with carboxylic acid lipids through ester bonds, and the amino group can be covalently combined with amino acids through amide bonds. Compared with pentaerythritol, Tris has similar chemical structure characteristics that can be used as a connecting group for amino acids and hydrophobic chains, and Tris itself is also low immunogenic and low biotoxicity.

[0080] Tris is also called ammonium glycyrrhizinate in medicine, which is a basic buffer with good buffering effect on metabolic acidosis and enzyme activity. It is suitable for metabolic acidosis, respiratory acidosis and other diseases, especially for hyperuricemia, which can effectively promote the dissolution of uric acid. The above application of Tris in the field of biology or medicine proves that Tris is biologically safe.

[0081] Experiments have found that a reasonable combination of some amino acids, Tris and lipids can achieve good delivery effect, and the delivery effect of fluorescent mRNA can reach or exceed that of mainstream LNP. The knockdown effect of siRNA on target genes after delivery can also reach or exceed that of mainstream LNP. In the control of APLNP related biological experiments, it is found that whether mRNA or siRNA is delivered, ATLNP shows more superior delivery efficiency than APLNP.

[0082] The inventors believe that the amino group in Tris and the amide bond formed by the amino acid play an important role in the nucleic acid delivery of ATLNP. The amino acid lipid or its salt (Amino Acid-Tris-Lipid, ATL) formed by the connection of the hydrophilic head of amino acid and the hydrophobic hydrocarbon chain of Tris has much smaller charge than cationic lipids at physiological pH, and is mainly combined with nucleic acid molecules through hydrogen bonds to form lipid nanoparticles containing nucleic acid molecules. In ATL, which is a key component of ATLNP, the amide bond formed by Tris and amino acid has strong hydrogen bond formation ability, thereby strengthening the binding ability of ATLNP and nucleic acid, which greatly facilitates the delivery of nucleic acid drugs. In APLNP, there is no amide bond, so the hydrogen bond binding ability with nucleic acid is reduced. In the amino acid lipid ATL, which is the main component of ATLNP, the amino acid and Tris are connected by an amide bond, and the amide bond can further enhance the hydrogen bond interaction between the amino acid lipid and the nucleic acid molecule, thereby further improving the delivery efficiency of the nucleic acid molecule.

[0083] Accordingly, the present application provides various amino acid lipids with high encapsulation efficiency, low toxicity, and the ability to successfully deliver nucleic acid molecules into cells and express them, wherein the amino acid lipids represented by general formula (I) are preferred, and the in vivo delivery effect of mRNA and siRNA is better.

[0084] The amino acid lipid nanoparticle delivery system (Amino Acid-Tris-Lipid Nano Particles, ATLNP) prepared using the ionizable liposome-amino acid lipid or salt thereof (Amino Acid-Tris-Lipid, ATL) of the present application can be used for the delivery of nucleic acid molecules, has low immunogenicity and low biological toxicity, effectively delivers nucleic acid molecules, and achieves expression in vivo.

[0085] The technical solutions and technical effects of the present application are further described below in conjunction with specific examples and comparative examples.

[0086] In the following examples, the raw materials and the like used are obtained by market purchase unless otherwise specified.

[0087] In the following examples, room temperature (RT) refers to 20-35°C unless otherwise specified, and the ratio of the eluent referred to in the following examples is a volume ratio.

[0088] In the following examples and comparative examples, the ―C10" and ―C 10 in the name abbreviations are equivalent and all represent chain lipids containing 10 carbon atoms.

[0089] In the following examples, the experimental methods or test methods referred to are conventional methods in the art unless otherwise specified.

[0090] Synthesis Examples

[0091] Example 1. Met-Tris-3MOA

[0092]

[0093] In a 10 mL reaction vial, under N2protection, add myristoleic acid (MOA, 298 mg, 1.31 mmol), N-tert-butoxycarbonyl-tris(hydroxymethyl)aminomethane (Boc-Tris, 88.5 mg, 0.40 mmol), DMAP (50 mg, 0.40 mmol) and DMF (3.5 mL), stir to get a clear solution, add EDCI.HC1 (307 mg, 1.60 mmol). After addition, stir at room temperature for 18 h under N2protection. Dilute with ethyl acetate (EA) (150 mL), wash the organic phase with saturated sodium bicarbonate solution and saturated sodium chloride solution each once, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the crude product on a silica gel column using PE / EA = 30:1 as eluent to give Boc-Tris-3MOA (259 mg, 0.31 mmol, 77% yield) as a colorless oily liquid.

[0094] In a 10 mL reaction vial, under N2protection, add myristoleic acid (MOA, 298 mg, 1.31 mmol), N-tert-butoxycarbonyl-tris(hydroxymethyl)aminomethane (Boc-Tris, 88.5 mg, 0.40 mmol), DMAP (50 mg, 0.40 mmol) and DMF (3.5 mL), stir to get a clear solution, add EDCI.HC1 (307 mg, 1.60 mmol). After addition, stir at room temperature for 18 h under N2protection. Dilute with ethyl acetate (EA) (150 mL), wash the organic phase with saturated sodium bicarbonate solution and saturated sodium chloride solution each once, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the crude product on a silica gel column using PE / EA = 30:1 as eluent to give Boc-Tris-3MOA (259 mg, 0.31 mmol, 77% yield) as a colorless oily liquid.

[0095] In a 10 mL reaction vial, under N2protection, add myristoleic acid (MOA, 298 mg, 1.31 mmol), N-tert-butoxycarbonyl-tris(hydroxymethyl)aminomethane (Boc-Tris, 88.5 mg, 0.40 mmol), DMAP (50 mg, 0.40 mmol) and DMF (3.5 mL), stir to get a clear solution, add EDCI.HC1 (307 mg, 1.60 mmol). After addition, stir at room temperature for 18 h under N2protection. Dilute with ethyl acetate (EA) (150 mL), wash the organic phase with saturated sodium bicarbonate solution and saturated sodium chloride solution each once, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the crude product on a silica gel column using PE / EA = 30:1 as eluent to give Boc-Tris-3MOA (259 mg, 0.31 mmol, 77% yield) as a colorless oily liquid.

[0096] Boc-Met-Tris-3MOA (123 mg, 0.12 mmol) was dissolved in 3 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was stirred at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was transferred to a separatory funnel after being concentrated under reduced pressure, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated. The resulting product was purified by silica gel column chromatography with eluent DCM / CH3OH = 40:1 to obtain Met-Tris-3MOA (97 mg, 0.11 mmol) as a colorless oily liquid with a yield of 92%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0097] 1 H NMR (400 MHz, Chloroform-d) δ 7.65 (s, 1H), 5.32 (s, 6H), 4.40 (s, 6H), 3.46 (d, J = 12.6 Hz, 1H), 2.59 (d, J = 3.9 Hz, 2H), 2.32 (d, J = 15.2 Hz, 6H), 2.10 (s, 3H), 2.01 (s, 12H), 1.77 (d, J = 28.4 Hz, 2H), 1.59 (s, 6H), 1.30 (s, 36H), 0.90 (d, J = 14.0 Hz, 9H).

[0098] Example 2. Gly-Tris-3MOA

[0099]

[0100] Tris-3MOA (168 mg, 0.22 mmol), N-tert-butoxycarbonyl-glycine (Boc-Gly, 77 mg, 0.44 mmol), HBTU (167 mg, 0.44 mmol), HOBt (59 mg, 0.44 mmol), and DMF (3.5 mL) were added in a 10 mL reaction bottle under N2protection, and the mixture was stirred to obtain a clear solution. DIPEA (170 mg, 1.32 mmol) was added. After the addition was completed, the mixture was stirred at room temperature for 18 h under N2protection. The reaction was diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. The organic phase was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography with eluent PE / EA = 10:1 to obtain Boc-Gly-Tris-3MOA (159 mg, 0.18 mmol) as a colorless oily liquid with a yield of 81%.

[0101] Boc-Gly-Tris-3MOA (159 mg, 0.18 mmol) was dissolved in 3 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was stirred at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted with dichloromethane twice, the combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting product was purified by silica gel column with eluent DCM / CH3OH = 40:1 to obtain Gly-Tris-3MOA (117 mg, 0.15 mmol) as a colorless oily liquid with a yield of 83%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0102] 1 H NMR (400 MHz, Chloroform-d) δ 7.62 (s, 1H), 5.33 (s, 6H), 4.44 (s, 6H), 3.31 (s, 2H), 2.34 (s, 6H), 2.08-2.00 (m, 12H), 1.60 (d, J = 6.8 Hz, 6H), 1.31 (d, J = 9.4 Hz, 36H), 0.88 (s, 9H).

[0103] Example 3. Gly-Tris-3MA

[0104]

[0105] Myristic acid (MA, 0.75 g, 3.3 mmol), Boc-Tris (0.22 g, 1.0 mmol), DMAP (0.12 g, 1.0 mmol), and DMF (10 mL) were added to a 10 mL reaction bottle under N2protection, and a clear solution was obtained after stirring. EDCI.HCl (0.85 g, 4.4 mmol) was added. After the addition was completed, the reaction was stirred at room temperature for 18 h under N2protection. Ethyl acetate was added (300 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each. The crude product obtained after drying over anhydrous sodium sulfate and concentrating under reduced pressure was purified by silica gel column with eluent PE / EA = 30:1 to obtain Boc-Tris-3MA (0.78 g, 0.9 mmol) as a colorless oily liquid which became a white solid after standing.

[0106] Boc-Tris-3MA (0.78 g, 0.90 mmol) was dissolved in 8 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (2.0 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction solution was concentrated under reduced pressure and then transferred to a separatory funnel, 200 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain white solid Tris-3MA (0.64 g, 0.85 mmol) with a yield of 94%.

[0107] Tris-3MA (0.64 g, 0.85 mmol), Boc-Gly (0.30 g, 1.71 mmol), HBTU (0.65 g, 1.71 mmol), HOBt (0.23 g, 1.70 mmol), and DMF (10 mL) were added to a 10 mL reaction bottle under N2protection, and a clear solution was obtained by stirring. DIPEA 0.81 g, 6.3 mmol) was added. After the addition was completed, the reaction was stirred at room temperature for 18 h under N2protection. Dilution was performed with ethyl acetate (300 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each. After drying with anhydrous sodium sulfate, the crude product obtained after concentration under reduced pressure was purified by silica gel column chromatography with eluent PE / EA = 10:1 to obtain colorless oily liquid Boc-Gly-Tris-3MA (0.66 g, 0.72 mmol, yield 85%).

[0108] Boc-Gly-Tris-3MA (0.66 g, 0.72 mmol) was dissolved in 8.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (2.0 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction solution was concentrated under reduced pressure and then transferred to a separatory funnel, 200 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated. The product obtained was purified by silica gel column chromatography with eluent DCM / CH3OH = 40:1 to obtain white solid Gly-Tris-3MA (0.48 g, 0.59 mmol) with a yield of 82%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0109] 1 H NMR (400 MHz, Chloroform-d) δ 7.62 (s, 1H), 4.44 (s, 6H), 3.32 (s, 2H), 2.32 (d, J = 15.1 Hz, 6H), 1.61 (d, J = 6.8 Hz, 6H), 1.26 (s, 60H), 0.88 (d, J = 13.5 Hz, 9H).

[0110] Example 4. Gly-Tris-3POA

[0111]

[0112] In a 10 mL reaction vial, under N2protection, was placed palmitoleic acid (POA, 251.7 mg, 0.99 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.7 mg, 0.3 mmol) and DMF (3 mL) to give a clear solution. EDCI.HC1 (230.3 mg, 1.2 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. The reaction mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using PE / EA = 30:1 as eluent to give Boc-Tris-3POA (231 mg, 0.25 mmol) as a colorless oily liquid with a yield of 83%.

[0113] In a 10 mL reaction vial, under N2protection, was placed palmitoleic acid (POA, 251.7 mg, 0.99 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.7 mg, 0.3 mmol) and DMF (3 mL) to give a clear solution. EDCI.HC1 (230.3 mg, 1.2 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. The reaction mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using PE / EA = 30:1 as eluent to give Boc-Tris-3POA (231 mg, 0.25 mmol) as a colorless oily liquid with a yield of 83%.

[0114] In a 10 mL reaction vial, under N2protection, was placed palmitoleic acid (POA, 251.7 mg, 0.99 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.7 mg, 0.3 mmol) and DMF (3 mL) to give a clear solution. EDCI.HC1 (230.3 mg, 1.2 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. The reaction mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using PE / EA = 30:1 as eluent to give Boc-Tris-3POA (231 mg, 0.25 mmol) as a colorless oily liquid with a yield of 83%.

[0115] Boc-Gly-Tris-3POA (166 mg, 0.17 mmol) was dissolved in 2.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was transferred to a separatory funnel after being concentrated under reduced pressure, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated bicarbonate solution. The organic layer was separated, the aqueous layer was extracted with dichloromethane twice, the combined organic layer was dried with anhydrous sodium sulfate, filtered, concentrated, and the obtained product was purified by silica gel column with eluent DCM / CH3OH = 40:1 to obtain Gly-Tris-3POA (132 mg, 0.15 mmol) as a colorless oily liquid with a yield of 88%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0116] 1 H NMR (400 MHz, Chloroform-d) δ 7.64 (s, 1H), 5.33 (s, 6H), 4.44 (s, 6H), 3.28 (s, 2H), 2.32 (d, J = 15.2 Hz, 6H), 2.01 (d, J = 18.6 Hz, 12H), 1.60 (d, J = 6.5 Hz, 6H), 1.29 (d, J = 22.7 Hz, 48H), 0.90 (s, 9H).

[0117] Example 5. Gly-Tris-3DOA

[0118]

[0119] In a 10 mL reaction bottle, cis-5-dodecenoic acid (DOA, 196.8 mg, 0.99 mmol), Boc-Tris (66.2 mg, 0.3 mmol), DMAP (36.8 mg, 0.3 mmol) and DMF (3 mL) were added under N2protection, and a clear solution was obtained by stirring. EDCI.HCl (230.2 mg, 1.2 mmol) was added. After the addition was completed, the reaction was stirred at room temperature for 18 h under N2protection. Diluted with ethyl acetate (150 mL), the organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride solution respectively once, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the obtained crude product was purified by silica gel column with eluent PE / EA = 30:1 to obtain Boc-Tris-3DOA (208 mg, 0.27 mmol) as a colorless oily liquid with a yield of 90%.

[0120] Boc-Tris-3DOA (208 mg, 0.27 mmol) was dissolved in 2 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain Tris-3DOA (179 mg, 0.27 mmol) as a colorless oily liquid with a yield of 99%.

[0121] Tris-3DOA (179 mg, 0.27 mmol), Boc-Gly (94.8 mg, 0.54 mmol), HBTU (205 mg, 0.54 mmol), HOBt (73.2 mg, 0.54 mmol), and DMF (3 mL) were added to a 10 mL reaction bottle under N2protection, and a clear solution was obtained by stirring. DIPEA (210 mg, 1.63 mmol) was added. After the addition was completed, the reaction was stirred at room temperature for 18 h under N2protection. Ethyl acetate (150 mL) was added for dilution, and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. The organic phase was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography with PE / EA = 10:1 as the eluent to obtain Boc-Gly-Tris-3DOA (161 mg, 0.20 mmol) as a colorless oily liquid with a yield of 74%.

[0122] Boc-Gly-Tris-3DOA (154 mg, 0.19 mmol) was dissolved in 2.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated. The obtained product was purified by silica gel column chromatography with DCM / CH3OH = 40:1 as the eluent to obtain Gly-Tris-3DOA (97 mg, 0.13 mmol) as a colorless oily liquid with a yield of 68%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0123] 1H NMR (400 MHz, Chloroform-d) δ 7.65 (s, 1H), 5.31 (s, 6H), 4.45 (s, 6H), 3.29 (s, 2H), 2.31 (s, 6H), 2.09 - 1.99 (m, 12H), 1.66 (d, J = 14.9 Hz, 6H), 1.29 (s, 24H), 0.89 (d, J = 6.6 Hz, 9H).

[0124] Example 6. Gly-Tris-3 OA

[0125]

[0126] In a 10 mL reaction vial, under N2protection, oleic acid (OA, 282.6 mg, 1.0 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.9 mg, 0.3 mmol) and DMF (3 mL) were added, stirred to get a clear solution, EDCI.HC1 (230.5 mg, 1.2 mmol) was added. After addition, the reaction was stirred at room temperature for 18 h under N2protection. Diluted with ethyl acetate (150 mL), the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively once, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the obtained crude was purified by silica gel column, eluent PE / EA = 30: 1, to get colorless oily liquid Boc-Tris-3 OA (272 mg, 0.27 mmol), yield 90%.

[0127] Under N2protection, Boc-Tris-3 OA (272 mg, 0.27 mmol) was dissolved in 3 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and LC-MS was used to track the reaction. The reaction was stopped after 2 h. The reaction was concentrated under reduced pressure, transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted with dichloromethane twice, the combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to get colorless oily liquid Tris-3 OA (240 mg, 0.26 mmol), yield 96%.

[0128] In a 10 mL reaction vial, Tris-3OA (240 mg, 0.26 mmol), Boc-Gly (93 mg, 0.53 mmol), HBTU (201 mg, 0.53 mmol), HOBt (72 mg, 0.53 mmol) and DMF (3 mL) were added under N2protection, the mixture was stirred to get a clear solution, DIPEA (205 mg, 1.58 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. The mixture was diluted with ethyl acetate (150 mL), the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively once, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the obtained crude product was purified by silica gel column, eluent PE / EA = 10:1, to obtain Boc-Gly-Tris-3OA (189 mg, 0.18 mmol) as a colorless oily liquid, the yield was 69%.

[0129] Boc-Gly-Tris-3OA (189 mg, 0.18 mmol) was dissolved in 3.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS, and stopped after 2 h. The reaction was concentrated under reduced pressure, transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted with dichloromethane twice, the combined organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the obtained product was purified by silica gel column, eluent DCM / CH3OH = 40:1, to obtain Gly-Tris-3OA (139 mg, 0.14 mmol) as a colorless oily liquid, the yield was 78%, and the results of nuclear magnetic resonance hydrogen spectrum were as follows:

[0130] 1 H NMR (400 MHz, Chloroform-d) δ 7.64 (s, 1H), 5.34 (s, 6H), 4.44 (s, 6H), 3.34 (s, 2H), 2.32 (t, J = 7.5 Hz, 6H), 2.02 (d, J = 12.1 Hz, 12H), 1.56 (s, 6H), 1.28 (d, J = 12.7 Hz, 60H), 0.86 (s, 9H).

[0131] Example 7. Gly-Tris-3Lin

[0132]

[0133] In a 10 mL reaction vial, under N2protection, linoleic acid (Lin, 280.6 mg, 1.0 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.9 mg, 0.3 mmol) and DMF (3 mL) were added to give a clear solution, and EDCI.HC1 (230.5 mg, 1.2 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. The reaction mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using PE / EA = 30:1 as the eluent to give Boc-Tris-3Lin (241 mg, 0.24 mmol) as a colorless oily liquid, with a yield of 80%.

[0134] In a 10 mL reaction vial, under N2protection, linoleic acid (Lin, 280.6 mg, 1.0 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.9 mg, 0.3 mmol) and DMF (3 mL) were added to give a clear solution, and EDCI.HC1 (230.5 mg, 1.2 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. The reaction mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using PE / EA = 30:1 as the eluent to give Boc-Tris-3Lin (241 mg, 0.24 mmol) as a colorless oily liquid, with a yield of 80%.

[0135] In a 10 mL reaction vial, under N2protection, linoleic acid (Lin, 280.6 mg, 1.0 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.9 mg, 0.3 mmol) and DMF (3 mL) were added to give a clear solution, and EDCI.HC1 (230.5 mg, 1.2 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. The reaction mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using PE / EA = 30:1 as the eluent to give Boc-Tris-3Lin (241 mg, 0.24 mmol) as a colorless oily liquid, with a yield of 80%.

[0136] Boc-Gly-Tris-3Lin (136 mg, 0.13 mmol) was dissolved in 3.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was stirred at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was transferred to a separatory funnel after being concentrated under reduced pressure, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting product was purified by a silica gel column with eluent DCM / CH3OH = 40:1 to obtain Gly-Tris-3Lin (101 mg, 0.11 mmol) as a colorless oily liquid with a yield of 85%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0137] 1 H NMR (400 MHz, Chloroform-d) δ 7.65 (s, 1H), 5.39-5.30 (m, 12H), 4.44 (s, 6H), 3.28 (s, 2H), 2.77 (d, J = 13.0 Hz, 6H), 2.33 (d, J = 7.5 Hz, 6H), 2.06 (s, 12H), 1.62 (d, J = 7.0 Hz, 6H), 1.31 (tdd, J = 9.8, 4.5, 2.2 Hz, 42H), 0.89 (d, J = 13.7 Hz, 9H).

[0138] Example 8. Gly-Tris-3PA

[0139]

[0140] In a 10 mL reaction bottle, palmitic acid (PA, 260 mg, 1.01 mmol), Boc-Tris (68.2 mg, 0.31 mmol), DMAP (40.5 mg, 0.33 mmol), and DMF (3.5 mL) were added under N2protection, and the resulting clear solution was stirred. EDCI.HCl (235 mg, 1.22 mmol) was added. After the addition was completed, the reaction was stirred at room temperature for 18 h under N2protection. Ethyl acetate was added (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting crude product was purified by a silica gel column with eluent PE / EA = 30:1 to obtain Boc-Tris-3PA (235 mg, 0.25 mmol) as a colorless oily liquid with a yield of 81%.

[0141] Boc-Tris-3PA (235 mg, 0.25 mmol) was dissolved in 3 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction solution was concentrated under reduced pressure and then transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain white solid Tris-3PA (212 mg, 0.25 mmol) with a yield of 98%.

[0142] Tris-3PA (212 mg, 0.25 mmol), Boc-Gly (90 mg, 0.51 mmol), HBTU (193 mg, 0.51 mmol), HOBt (69 mg, 0.51 mmol), and DMF (3 mL) were added to a 10 mL reaction bottle under N2protection, and a clear solution was obtained by stirring. DIPEA (195 mg, 1.51 mmol) was added. After the addition was completed, the reaction was stirred at room temperature for 18 h under N2protection. Dilution was performed with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each. Anhydrous sodium sulfate was used for drying, and the obtained crude product was purified by silica gel column chromatography with eluent PE / EA = 10:1 to obtain colorless oily liquid Boc-Gly-Tris-3PA (193 mg, 0.19 mmol) with a yield of 76%.

[0143] Boc-Gly-Tris-3PA (193 mg, 0.19 mmol) was dissolved in 3.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction solution was concentrated under reduced pressure and then transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained product was purified by silica gel column chromatography with eluent DCM / CH3OH = 40:1 to obtain white solid Gly-Tris-3PA (151 mg, 0.17 mmol) with a yield of 89%. The results of nuclear magnetic resonance hydrogen spectrum are as follows:

[0144] 1 H NMR (400 MHz, Chloroform-d) δ 7.65 (s, 1H), 4.44 (s, 6H), 3.28 (s, 2H), 2.32 (d, J = 15.2 Hz, 6H), 1.61 (d, J = 14.2 Hz, 6H), 1.25 (s, 72H), 0.88 (d, J = 13.7 Hz, 9H).

[0145] Example 9. Met-Tris-3MA

[0146]

[0147] In a 10 mL reaction vial, Tris-3MA (212 mg, 0.28 mmol), Boc-Met (140 mg, 0.56 mmol), HBTU (215 mg, 0.56 mmol), HOBt (76 mg, 0.56 mmol) and DMF (3.5 mL) were added under N2protection, stirred to get a clear solution, DIPEA (230 mg, 1.78 mmol) was added. After addition, the reaction was stirred at room temperature for 18 h under N2protection. Diluted with ethyl acetate (150 mL), the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively once, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the obtained crude product was purified by silica gel column, eluent PE / EA = 10:1, to obtain Boc-Met-Tris-3MA (215 mg, 0.22 mmol, yield 78%) as a colorless oily liquid.

[0148] Under N2protection, Boc-Met-Tris-3MA (215 mg, 0.22 mmol) was dissolved in 3.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS, and stopped after 2 h. The reaction was concentrated under reduced pressure, transferred to a separatory funnel, 200 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted with dichloromethane twice, the combined organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the obtained product was purified by silica gel column, eluent dichloromethane / CH3OH = 40:1, to obtain Met-Tris-3MA (179 mg, 0.20 mmol) as a white solid, with a yield of 91%, and the results of nuclear magnetic resonance hydrogen spectrum were as follows:

[0149] 1 H NMR (400 MHz, Chloroform-d) δ 7.67 (s, 1H), 4.43 (s, 6H), 3.45 (d, J = 12.6 Hz, 1H), 2.58 (s, 2H), 2.32 (d, J = 15.2 Hz, 6H), 2.11 (s, 3H), 1.72 (s, 1H), 1.61 (d, J = 14.2 Hz, 7H), 1.26 (s, 60H), 0.88 (d, J = 13.7 Hz, 9H).

[0150] Example 10. Met-Tris-3LA

[0151]

[0152] Under N2protection, add lauric acid (LA, 220 mg, 1.1 mmol), Boc-Tris (70 mg, 0.31 mmol), DMAP (60 mg, 0.49 mmol) and DMF (3.5 mL) into a 10 mL reaction flask, stir to get a clear solution, add EDCI.HC1 (260 mg, 1.35 mmol). After addition, stir at room temperature for 18 h under N2protection. Dilute with ethyl acetate (150 mL), wash the organic phase with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively once, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product on a silica gel column with PE / EA = 30:1 as eluent to obtain Boc-Tris-3LA (239 mg, 0.31 mmol) as a colorless oily liquid with a yield of 99%.

[0153] Under N2protection, dissolve Boc-Tris-3LA (239 mg, 0.31 mmol) in 3 mL dichloromethane, then add trifluoroacetic acid (0.75 mL). React at room temperature, track the reaction by LC-MS, stop the reaction after 2 h. Concentrate the reaction under reduced pressure, transfer to a separatory funnel, add 120 mL dichloromethane, and adjust the organic layer to pH = 7-8 with saturated sodium bicarbonate solution, separate the organic layer, extract the aqueous layer with dichloromethane twice, combine the organic layers and dry over anhydrous sodium sulfate, filter, and concentrate to obtain Tris-3LA (197 mg, 0.29 mmol) as a colorless oily liquid with a yield of 93%.

[0154] Under N2protection, add Tris-3LA (197 mg, 0.29 mmol), Boc-Met (159 mg, 0.60 mmol), HBTU (230 mg, 0.60 mmol), HOBt (819 mg, 0.60 mmol) and DMF (3.5 mL) into a 10 mL reaction flask, stir to get a clear solution, add DIPEA (238 mg, 1.83 mmol). After addition, stir at room temperature for 18 h under N2protection. Dilute with ethyl acetate (150 mL), wash the organic phase with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively once, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the obtained crude product on a silica gel column with PE / EA = 10:1 as eluent to obtain Boc-Met-Tris-3LA (210 mg, 0.23 mmol) as a colorless oily liquid with a yield of 79%.

[0155] Boc-Met-Tris-3LA (210 mg, 0.23 mmol) was dissolved in 3.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was stirred at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted with dichloromethane twice, the combined organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the obtained product was purified by silica gel column with eluent DCM / CH3OH = 40:1 to give Met-Tris-3LA (157 mg, 0.20 mmol) as a white solid with a yield of 87%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0156] 1 H NMR (400 MHz, Chloroform-d) δ 7.66 (s, 1H), 4.46 (s, 6H), 3.45 (d, J = 12.6 Hz, 1H), 2.60 (d, J = 11.2 Hz, 2H), 2.34 - 2.29 (m, 6H), 2.11 (s, 3H), 1.76 (d, J = 29.5 Hz, 1H), 1.63 (s, 7H), 1.26 (s, 48H), 0.88 (d, J = 13.7 Hz, 9H).

[0157] Example 11. Met-Tris-3C10

[0158]

[0159] Boc-Tris (89 mg, 0.40 mmol), DMAP (49 mg, 0.40 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle under N2protection, and the mixture was stirred to obtain a clear solution. EDCI.HCl (307 mg, 1.60 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively once. The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the obtained crude product was purified by silica gel column with eluent PE / EA = 30:1 to give Boc-Tris-3C10 (156 mg, 0.23 mmol) as a colorless oily liquid with a yield of 58%.

[0160] Boc-Tris-3C10 (156 mg, 023 mmol) was dissolved in 3 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain Tris-3C10 (138 mg, 0.23 mmol) as a colorless oily liquid with a yield of 99%.

[0161] Tris-3C10 (138 mg, 0.24 mmol), Boc-Met (125 mg, 0.50 mmol), HBTU (190 mg, 0.50 mmol), HOBt (68 mg, 050 mmol), and DMF (3.5 mL) were added to a 10 mL reaction bottle under N2protection, and a clear solution was obtained by stirring. DIPEA (195 mg, 1.51 mmol) was added. After the addition was completed, the reaction was stirred at room temperature for 18 h under N2protection. Ethyl acetate (150 mL) was added for dilution, and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each. The crude product obtained after drying with anhydrous sodium sulfate and concentration under reduced pressure was purified by silica gel column chromatography with PE / EA = 10:1 as the eluent to obtain Boc-Met-Tris-3C10 (165 mg, 0.20 mmol) as a colorless oily liquid with a yield of 79%.

[0162] Boc-Met-Tris-3C10 (165 mg, 0.20 mmol) was dissolved in 3.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the combined organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated. The product obtained was purified by silica gel column chromatography with DCM / CH3OH = 40:1 as the eluent to obtain Met-Tris-3C10 (131 mg, 0.18 mmol) as a white solid with a yield of 90%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0163] 1H NMR (400 MHz, Chloroform-d) δ 7.67 (s, 1H), 4.43 (d, J = 24.6 Hz, 6H), 3.45 (d, J = 11.5 Hz, 1H), 2.58 (d, J = 31.9 Hz, 2H), 2.32 (d, J = 15.2 Hz, 6H), 2.10 (s, 3H), 1.76 (d, J = 27.6 Hz, 1H), 1.63 (s, 7H), 1.27 (d, J = 7.3 Hz, 36H), 0.89 (d, J = 6.6 Hz, 9H).

[0164] Example 12. Met-PEL-3MOA

[0165]

[0166] Under N2protection, a 500 mL reaction flask was charged with pentaerythritol (PEL, 5.0 g, 36.6 mmol), imidazole (2.67 g, 38.6 mmol) and anhydrous DMF (235 mL), a solution of tert-butyldimethylsilyl chloride (TBDMS-Cl, 2.94 g, 19.6 mmol) in 15 mL of anhydrous DMF was added dropwise with stirring. After addition, the mixture was stirred at room temperature for 24 h under N2protection. Concentrated under reduced pressure, diluted with ethyl acetate (300 mL), the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively once, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the crude obtained was purified by silica gel column, eluent PE / EA = 1:1, to give PEL(TBS) (2.26 g, 9.02 mmol) as colorless oily liquid (solidified after standing at room temperature) in 46% yield.

[0167] Under N2protection, a 10 mL reaction flask was charged with PEL(TBS) (65.8 mg, 0.26 mmol), myristoleic acid (MOA, 198 mg, 0.87 mmol), DMAP (32.5 mg, 0.26 mmol) and DMF (3 mL), a clear solution was obtained with stirring, EDCI.HC1 (203.2 mg, 0.26 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2protection. Diluted with ethyl acetate (150 mL), the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively once, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the crude obtained was purified by silica gel column, eluent PE / EA = 50:1, to give PEL(TBS)-3MOA (210 mg, 0.24 mmol) as colorless oily liquid in 92% yield.

[0168] PEL(TBS)-3MOA (210 mg, 0.24 mmol) was dissolved in 5 mL of tetrahydrofuran, followed by the addition of a solution of tetrabutylammonium fluoride in tetrahydrofuran (TBAF, 1 M, 1.5 mL) and acetic acid (AA, 0.5 mL) under N2protection. After 3 days of reaction at room temperature, it was concentrated under reduced pressure, diluted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, once each, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using PE / EA = 10:1 as the eluent to obtain PEL-3MOA (144 mg, 0.19 mmol) as a colorless oily liquid, with a yield of 79%.

[0169] Boc-Met-PEL-3MOA (149 mg, 0.15 mmol) was dissolved in 3.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was concentrated under reduced pressure, transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted with dichloromethane twice, the combined organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the resulting product was purified by silica gel column chromatography using DCM / CH3OH = 40:1 as the eluent to obtain Met-PEL-3MOA (105 mg, 0.12 mmol) as a colorless oily liquid, with a yield of 80%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0170] Boc-Met-PEL-3MOA (149 mg, 0.15 mmol) was dissolved in 3.0 mL of dichloromethane under N2protection, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. After 2 h, the reaction was stopped. The reaction was concentrated under reduced pressure, transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with saturated sodium bicarbonate solution. The organic layer was separated, the aqueous layer was extracted with dichloromethane twice, the combined organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the resulting product was purified by silica gel column chromatography using DCM / CH3OH = 40:1 as the eluent to obtain Met-PEL-3MOA (105 mg, 0.12 mmol) as a colorless oily liquid, with a yield of 80%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows:

[0171] 1H NMR (400 MHz, Chloroform-d) δ 5.34 (d, J = 11.8 Hz, 6H), 4.14 (d, J = 15.3 Hz, 8H), 3.62 (d, J = 15.5 Hz, 1H), 2.62 (d, J = 14.4 Hz, 2H), 2.31 (d, J = 15.1 Hz, 6H), 2.10 (s, 3H), 2.02 (s, 12H), 1.78 (d, J = 26.5 Hz, 2H), 1.59 (d, J = 6.8 Hz, 6H), 1.32 (d, J = 17.4 Hz, 36H), 0.90 (d, J = 14.0 Hz, 9H).

[0172] Cytotoxicity test of amino acid lipids

[0173] The cytotoxicity of the compounds obtained in Examples 1-7 was investigated using the CTG method. 2.5 x 104cells / mL of 293T cells were placed in a 24-well plate, then a certain concentration of the tested compound was added for incubation for 24 h, and then transferred to a 96-well plate for further incubation for 24 h, then 100 μL of CTG and complete culture medium were added. After incubation at room temperature for 10 min, the absorbance value at 540 nm was detected using an enzyme marker to calculate the cell survival rate, and the results are shown in Table 1. 5 Figure 2 The results show that all the tested amino acid lipids have low cytotoxicity (among them, MC3 is the ionizable lipid DLin-MC3-DMA used in the Onpattro preparation).

[0174] Preparation of lipid nanoparticles

[0175] ​Take the amino acid lipids obtained in Examples 1-7, 10-12 above and the ionizable lipid SM102 used in the Moderna Covid-19 Vaccine (Spikevax) and mix them with DSPC, cholesterol, PEG-DMG in a molar ratio of 50 / 10 / 38.5 / 1.5 in anhydrous ethanol to obtain a lipid solution (a). Dissolve Fluc mRNA in citric acid buffer according to N / P = 5:1 of amino acid lipid and mRNA to obtain Fluc mRNA solution (b). Mix the lipid solution (a) and the mRNA solution (b) at a flow rate ratio of 1:3 using microfluidics to obtain product (c). After dialysis of product (c) in PBS buffer at pH = 7.2-7.4 for 24 h, concentrate at 4°C to obtain Fluc mRNA-loaded amino acid lipid nanoparticles ATLNP 1-7, 10, 11, APLNP 1 and SM102-LNP (mRNA). The encapsulation efficiency of all LNP samples is greater than 90%. The Fluc mRNA used in this example is provided by Trilink. The particle size and zeta potential of the Fluc mRNA-loaded ATLNP and APLNP are shown in Table 1.

[0176] Table 1

[0177] Sample Amino Acid Lipid (ATL / APL) Size (nm) PDI Zeta Potential (mV) ATLNP 1 Met-Tris-3MOA (Example 1) 88.3 0.18 -5.3 ATLNP 2 Gly-Tris-3MOA (Example 2) 66.2 0.09 -0.7 ATLNP 3 Gly-Tris-3MA (Example 3) 85.0 0.18 2.4 ATLNP 4 Gly-Tris-3POA (Example 4) 71.1 0.11 -0.3 ATLNP 5 Gly-Tris-3DOA (Example 5) 62.3 0.16 -0.7 ATLNP 6 Gly-Tris-3OA (Example 6) 81.6 0.07 -0.2 ATLNP 7 Gly-Tris-3Lin (Example 7) 84.6 0.13 0.2 ATLNP 10 Met-Tris-3LA (Example 10) 66.8 0.12 -4.2 ATLNP 11 Met-Tris-3C10 (Example 11) 57.3 0.10 -3.0 APLNP 1 Met-PEL-3MOA (Example 12) 79.5 0.22 -3.6 SM102-LNP (mRNA) — 117 0.12 -1.3

[0178] Take the amino acid lipids synthesized in Examples 1-7, 9, 12 and the ionizable lipid SM102 used in the Moderna Covid-19 Vaccine (Spikevax) formulation and mix them with DSPC, cholesterol, PEG-C-DMG in a molar ratio of 50 / 10 / 38.5 / 1.5 in anhydrous ethanol to obtain a lipid solution (a). Dissolve siRNA in citric acid buffer according to N / P = 5:1 of amino acid lipid and siRNA to obtain siRNA solution (b). Mix the lipid solution (a) and the siRNA solution (b) at a flow rate ratio of 1:3 using microfluidics to obtain product (c). After dialysis of product (c) in PBS buffer at pH = 7.2-7.4 for 24 h, concentrate at 4°C to obtain siRNA-loaded amino acid lipid nanoparticles ATLNP 12-18, 20), APLNP 2 and SM102-LNP (siRNA), and the sample encapsulation efficiency is greater than 90%. The siRNA sequence used in this example is hmTF-25-2 (US20210324384A1). The particle size and zeta potential of the siRNA-loaded ATLNP and APLNP are shown in Table 2.

[0179] Table 2

[0180]

[0181]

[0182] Cell and animal experiments

[0183] Take nanoparticles ATLNP 1-7, ATLNP 10, ATLNP 11, APLNP 1, 20 μg, intramuscular injection of the inner thigh of BABL / C mice, parallel three experiments. Observe the mouse live imaging at two time points of 24h and 72h, measure the fluorescence intensity, the results are shown in Table 3. As can be seen from Table 3, all mice injected with ATLNP 1-7, ATLNP 10, ATLNP 11 samples in the muscle, fluorescence appeared after 24h, indicating that the mRNA encapsulated ATLNP can effectively deliver Fluc mRNA into cells, and express fluorescent protein in mice in vivo. The mice injected with APLNP 1 sample in the muscle, the fluorescence intensity appeared after 24h was significantly lower than that of the corresponding ATLNP (ATLNP 1 vs APLNP 1), indicating that the APLNP sample with pentaerythritol as the connecting group has lower efficiency than the corresponding ATLNP sample with Tris as the connecting group in delivering Fluc mRNA into cells.

[0184] Table 3

[0185]

[0186] Take ATLNP 1-7 and SM102-LNP(mRNA), intramuscular injection of BABL / C mice with 20 μg Fluc mRNA containing ATLNP sample, parallel three experiments. Two weeks after the first administration, the same dose of intramuscular injection of booster administration once, and then one week after the second administration, 100-200 μL of blood sample was collected from the orbital venous plexus of the mouse. The blood sample was placed in a 4°C refrigerator overnight, then the serum was separated, and the luciferase was coated on the enzyme-labeled plate and incubated at room temperature overnight. After dilution, the mouse serum was added to the enzyme-labeled plate (using luciferase antibody as positive control, using a mixture of phosphate and Tween 20 as negative control), and after full reaction, the plate was washed, then IgG-HRP antibody was added, and after full reaction, the plate was washed, then TMB reaction solution was added, and after full reaction, stop solution was added to stop the reaction. The absorbance value was read at 450nm wavelength on the enzyme-labeled instrument, and the luciferase antibody content was detected, and the results are shown in Table 4. Figure 3 The results show that the immunogenicity of ATLNP 1-7 is lower than or comparable to that of SM102-LNP, and the antibody level of ATLNP 1, 2, and 3 is significantly lower than that of SM102-LNP(mRNA), indicating that the immunogenicity of ATLNP 1, 2, and 3 is lower.

[0187] The siRNA-encapsulated samples ATLNP 12-20 and APLNP 2 were used to transfect 293T cells, and SM102-LNP(siRNA) formulation was used as a comparison. Total RNA was collected 24 h after transfection and reverse transcription was performed. The mRNA expression level of the target gene was detected by QPCR. The knockdown effect of TGF-β1 siRNA on the mRNA expression level of the target gene TGF-β1 is shown in Table 4. The results show that the samples ATLNP 13-18 and 20 have good gene silencing efficiency on TGF-β1 at different concentrations. The knockdown effect of ATLNP 12 and APLNP 2 on the mRNA expression of TGF-β1 is found to be that: with the same amino acid head (Met) and carboxylic acid lipid tail (MOA), APLNP 21 (Met-PEL-3MOA) with pentaerythritol as the connecting group has a significantly lower gene silencing efficiency at different concentrations than ATLNP 12 (Met-Tris-3MOA) with Tris as the connecting group.

[0188] Table 4

[0189]

[0190] The apparent pKa (determination method see Hope et al., Angew. Chem. Int. Ed., 51:1, 2012) and the corresponding gene knockdown effect (KD) of siRNA-encapsulated ATLNP 13, ATLNP 15-18 and SM102-LNP(siRNA) were determined, and the results are shown in Table 4. Figure 4 The pKa value of the ATLNP nanoparticles is between 3 and 5, which indicates that the ATLNP nanoparticles have very low cationic charge at physiological pH (pH 7). There is no obvious correlation between the delivery performance of the ATLNP and the apparent pKa value, indicating that the siRNA delivery mechanism of the ATLNP is different from that of the SNALP. In combination with the characteristics of the ATL chemical structure, it is inferred that hydrogen bonds are the main and most important interaction for RNA delivery in the ATLNP system.

[0191] Although the present application describes certain embodiments of the amino acid lipid, the delivery system containing the same and the preparation method, and a lot of details have been described for the purpose of illustration, the embodiments of the present application are not limited by the above-mentioned embodiments, and any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be included in the protection scope of the present application.

Claims

1. A delivery system characterized by, which comprises any one of the following amino acid lipids: 、 、 or .

2. The delivery system of claim 1, wherein, The delivery system further comprises one or more of auxiliary lipids, cholesterol and derivatives thereof, PEGylated lipids.

3. The delivery system of claim 2, wherein, The auxiliary lipids are selected from phospholipids and derivatives thereof; and / or, the PEGylated lipids are selected from one or more of PEG-DMG, PEG-C-DMG, PEG-DSPE; and / or, the molar ratio of the amino acid lipids, the auxiliary lipids, the cholesterol and derivatives thereof, and the PEGylated lipids is (40-99.5):(0-15):(0-50):(0.5-3).

4. Use of the delivery system according to any one of claims 1 to 3 in the preparation of a nucleic acid drug.

5. A nucleic acid drug, characterized by, The nucleic acid drug comprises the delivery system according to any one of claims 1 to 3 and a nucleic acid molecule, which is mRNA or siRNA, when the nucleic acid molecule is mRNA, the amino acid lipid in the delivery system is or ; when the nucleic acid molecule is siRNA, the amino acid lipid in the delivery system is or .

6. The nucleic acid drug of claim 5, wherein The nucleic acid drug is an amino acid lipid nanoparticle with a particle size of 50-300 nm; and / or, the nucleic acid drug is obtained by mixing the nucleic acid molecule with the amino acid lipid, optionally with one or more of the auxiliary lipids, the cholesterol and derivatives thereof, and the PEGylated lipids, and self-assembling by microfluidics; and / or, the molar ratio of the amino acid lipid and the nucleic acid molecule is (1-50):1; and / or, the nucleic acid drug further comprises a pharmaceutically acceptable additive; and / or, the nucleic acid drug is a lyophilized powder or an injection.

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