CN118388389A - Endogenous amino acid derived ionizable lipid, and preparation method and application thereof - Google Patents

Endogenous amino acid derived ionizable lipid, and preparation method and application thereof Download PDF

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CN118388389A
CN118388389A CN202410477849.1A CN202410477849A CN118388389A CN 118388389 A CN118388389 A CN 118388389A CN 202410477849 A CN202410477849 A CN 202410477849A CN 118388389 A CN118388389 A CN 118388389A
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lipid
amino acid
formula
endogenous amino
derived
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姜新义
郑作林
贾臻
刘颖
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Shandong University
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Shandong University
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Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to endogenous amino acid derived ionizable lipid, and a preparation method and application thereof. The invention provides an endogenous amino acid derivative ionizable lipid compound shown in a formula (I), which is based on endogenous amino acid, so that toxicity is greatly reduced, and safety is remarkably improved. The nucleic acid drug delivery device comprises a hydrophilic amino acid head part and a hydrophobic fatty chain tail part, has obvious amphipathy, can form a liposome structure in an aqueous phase, and can effectively deliver the nucleic acid drug into cells. The structure of the polypeptide contains an amide bond, and the bond can be rapidly hydrolyzed by enzymes in vivo, is easy to metabolize and remove, and has biodegradability. Amino acids produced during degradation play a role in immunopotentiating or enhancing/inhibiting other physiological activities. This property makes this novel lipid compound have great potential in the field of drug delivery and is expected to become an important carrier for future gene therapy and drug delivery.Formula (I).

Description

Endogenous amino acid derived ionizable lipid, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to endogenous amino acid derived ionizable lipid, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Gene therapy (GENE THERAPY) is a therapeutic method for correcting or compensating diseases caused by defective or abnormal genes by introducing exogenous genes into target cells. Nucleic acid vaccine (nucleic ACID VACCINE), also called genetic vaccine (GENETICVACCINE), refers to a nucleic acid sequence (such as DNA, mRNA, etc.) containing encoded immunogenic protein or polypeptide is introduced into a host, expressed by the host cell, and the host cell is induced to generate immune response to the immunogen, so as to achieve the purpose of preventing and treating diseases. Among them, ensuring the smooth introduction of foreign genes is an extremely important part of gene therapy and immunization with genetic vaccines. Among the many methods of gene delivery, methods of developing suitable lipid nanoparticles (i.e., LNP, lipid Nanoparticle) to encapsulate nucleic acids, target them to target cells, and deliver nucleic acids of specific genes into cells are increasingly being used by scientists.
One obvious difference between nucleic acid drugs and common chemical drugs is that nucleic acids carry a large number of phosphates, thus being negatively charged and of large molecular weight. In order to enable better encapsulation by LNP, various lipid compounds such as ionizable lipids have been developed.
Ionizable lipids typically contain one or more ionizable amine groups in the molecular structure, the apparent pKa of which is a key attribute for nucleic acid drug delivery in vivo. The chemical space of the ionizable lipid structure and the great difference of the ionizable lipid structure in cell uptake and endosomal escape exist, so that the development of the ionizable lipid with different chemical spaces, biodegradability and high transfection efficiency has great significance for promoting the deep development and clinical transformation of nucleic acid medicaments.
Disclosure of Invention
In order to overcome the problems, the invention provides an endogenous amino acid derived ionizable lipid, a preparation method and application thereof.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
In a first aspect of the present invention there is provided an endogenous amino acid derived ionizable lipid or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, having the structural formula (i):
Wherein R 1 and R 2 are the same or different from each other and are each independently selected from substituted or unsubstituted linear or branched C 8～C20 alkyl, or substituted or unsubstituted linear or branched C 8～C20 alkenyl, or substituted or unsubstituted linear or branched C 8～C20 alkynyl, wherein R 3、R4 can be H;
R 3 and R 4 are the same or different from each other and are each independently selected from hydrogen, substituted or unsubstituted linear or branched C 8～C20 alkyl, or substituted or unsubstituted linear or branched C 8～C20 alkenyl, or substituted or unsubstituted linear or branched C 8～C20 alkynyl;
x is O, NH or S;
m is a positive integer of 1 to 3;
L 1 is A group.
In one or more embodiments, L 1 isWhen the amino acid is a tryptophan, the endogenous amino acid is tryptophan.
In one or more embodiments, L 1 isWhen the group is, the endogenous amino acid is kynurenine.
In one or more embodiments, the endogenous amino acid-derived ionizable lipid is selected from one of the following compounds:
in a second aspect of the present invention, there is provided a method for preparing an endogenous amino acid-derived ionizable lipid as described above, comprising:
In an organic solvent, under the action of a catalyst, a compound shown in a formula (II) and a compound shown in a formula (III) are subjected to esterification reaction or amidation reaction to obtain a compound shown in a formula (I);
In the compounds of the formula (II) and the compounds of the formula (III), R 1、R2、R3、R4、X、L1 and m have the same meanings as in the compounds of the formula (I).
In one or more embodiments, when X is nitrogen, m=1, and r 1、R2 is independently selected from C 10～14 alkyl, a method of preparing a compound represented by formula (iii) includes the steps of: in acetonitrile, under the action of potassium carbonate, N-tert-butoxycarbonyl-1, 2-ethylenediamine and bromoalkane react to obtain an intermediate a; reacting a dichloromethane solution of the intermediate a under the action of trifluoroacetic acid to obtain an intermediate b, namely a compound shown in a formula (III);
the specific process is as follows:
Preferably, the volume ratio of the mole amount of the N-t-butoxycarbonyl-1, 2-ethylenediamine to the acetonitrile is 0.01-1 mole/L; the molar ratio of the potassium carbonate to the N-tert-butoxycarbonyl-1, 2-ethylenediamine is 1-3:1; the mol ratio of the N-tert-butyloxycarbonyl-1, 2-ethylenediamine to the bromoalkane is 1:1-1.5; the reaction temperature of the N-tert-butoxycarbonyl-1, 2-ethylenediamine and bromoalkane is 60-100 ℃ and the reaction time is 60-80 h; the molar ratio of the intermediate a to the trifluoroacetic acid is 1-3:1; the reaction temperature of the intermediate a for producing the intermediate b is ice bath, and the reaction time is 2-8 h.
In one or more embodiments, the organic solvent is selected from one or more of methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, methylene chloride, diethyl ether, propylene oxide, acetone, methyl butanone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, dimethyl sulfoxide, ethylene glycol ether, N-dimethylformamide, or triethanolamine;
preferably, when the solvent is methanol, dichloromethane, acetonitrile, ethyl acetate, dimethyl sulfoxide; the volume ratio of the compound shown in the formula (II) to the organic solvent is 0.01-10 mol/L.
In one or more embodiments, the catalyst is selected from one or more of N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT), 2- (7-azobenzotriazole) -N, N '-tetramethylurea Hexafluorophosphate (HATU), O-benzotriazol-tetramethylurea Hexafluorophosphate (HBTU), 4-Dimethylaminopyridine (DMAP), N-Diisopropylethylamine (DIPEA), or O-benzotriazol-N-, N' -tetramethylurea tetrafluoroboric acid (TBTU);
preferably, when the catalyst is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT) and N, N-Diisopropylethylamine (DIPEA), the molar ratio of the catalyst to the compound represented by formula (II) is 1-1.5:1.
In one or more embodiments, the molar ratio of the compound of formula (II) to the compound of formula (III) is from 1:1 to 1.5; preferably 1:1.2.
In one or more embodiments, the reaction temperature is room temperature and the reaction time is 10 to 30 hours.
In one or more embodiments, the method for post-treating a reaction solution obtained by reacting a compound represented by formula (II) with a compound represented by formula (III) comprises the steps of:
adding saturated sodium chloride solution and saturated sodium bicarbonate solution into the reaction solution, extracting with dichloromethane, collecting an organic phase, drying the organic phase by anhydrous sodium sulfate, filtering, evaporating under reduced pressure, and separating by silica gel column chromatography to obtain lipid; the eluent used for the silica gel column chromatography is a mixture of dichloromethane and methanol, DCM/meoh=200:0 to 10:1.
In a third aspect of the invention there is provided the use of an endogenous amino acid derived ionizable lipid of the first aspect or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof as a drug delivery vehicle.
In a fourth aspect of the invention there is provided a lipid nanoparticle composition comprising an endogenous amino acid-derived ionizable lipid of the first aspect, a helper lipid, cholesterol, a PEG lipid, and a entrapped drug.
In one or more embodiments, the auxiliary lipid is selected from a variety of sources including distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine-maleimide (DOPE-MAL), dipalmitoyl phosphatidylethanolamine (DPPE), dipyristoyl phosphatidylcholine (DMPE), distearoyl phosphatidylethanolamine (DSPE), and 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), preferably dioleoyl phosphatidylethanolamine (DOPE).
In one or more embodiments, the cholesterol is selected from one or both of cholesterol or 10α -hydroxycholesterol; preferably, the cholesterol is cholesterol or a mixture of cholesterol and 10 alpha-hydroxycholesterol (mass ratio 1:1).
In one or more embodiments, the PEG lipid is selected from polyethylene glycol-dimyristoyl glycerol (PEG-DMG), polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE), polyethylene glycol-dimethacrylate (PEG-DMA); polyethylene glycol-dimyristoylglycerol (PEG-DMG) is preferred.
In one or more embodiments, the encapsulated drug comprises one or more of a biological drug or a chemical drug; the biological medicine comprises one or more of nucleic acid medicine, protein medicine, polypeptide medicine or polysaccharide medicine; preferably, the nucleic acid drug comprises one or more of messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA (microRNA), circular RNA (circRNA), long non-coding RNA (lncRNA), plasmid DNA (plasmid DNA) and small circle DNA (mini circle DNA); the chemical medicine comprises one or more of small molecule medicine, fluorescein or developer; further preferably, the nucleic acid agent is mRNA.
In one or more embodiments, the lipid nanoparticle composition has a diameter of 1nm to 1000nm; preferably 100 to 200nm.
In one or more embodiments, the endogenous amino acid derived ionizable lipid, helper lipid, cholesterol, PEG lipid of the first aspect is in a molar ratio of 15-45:20-35:10-40:0.5-5;
The mass ratio of the endogenous amino acid derived ionizable lipid to the encapsulated drug of the first aspect is 1-100:1.
In one or more embodiments, the targeting molecule can be modified on the lipid nanoparticle composition to provide targeting functions to target a particular cell, tissue or organ. The targeting molecule may be in the whole lipid nanoparticle composition or may be located only on the surface of the lipid nanoparticle composition. The targeting molecule may be a protein, peptide, glycoprotein, lipid, small molecule, nucleic acid, etc., examples of which include, but are not limited to, antibodies, antibody fragments, low Density Lipoproteins (LDL), transferrin (transferrin), asialoglycoprotein (asialycoprotein), receptor ligands, sialic acids, aptamers, etc.
In one or more embodiments, the lipid nanoparticle composition can be prepared using a variety of methods known in the art, including, but not limited to, thin film hydration, extrusion, nano-precipitation, and microfluidic methods, among others. Preferred methods of preparation include, but are not limited to, the following: film hydration method: the lipid is dissolved in an organic solvent to form a thin film, which is then hydrated by the addition of water or a buffer solution to form nanoparticles. Nano precipitation method: the lipid and nucleic acid drug are dissolved in an organic solvent and an aqueous phase, respectively, and then the two phases are mixed to form nanoparticles by precipitation. Microfluidic method: the lipid and the nucleic acid solution are mixed in a microfluidic manner by utilizing a microfluidic technology, and the nano-particles are prepared through the control of a microchannel. Further preferred, the lipid nanoparticle composition is prepared by microfluidic methods.
In a fifth aspect of the present invention, there is provided the use of the lipid nanoparticle composition of the fourth aspect for the preparation of a genetic medicament comprising an active ingredient and a delivery vehicle, the active ingredient being a nucleic acid medicament, the delivery vehicle being the lipid nanoparticle composition of the fourth aspect.
The invention has the beneficial effects that:
(1) The invention provides an endogenous amino acid derived ionizable lipid compound, which is based on endogenous amino acid, greatly reduces toxicity and remarkably improves safety. The compound comprises a hydrophilic amino acid head part and a hydrophobic fatty chain tail part, has obvious amphipathy, can form a liposome structure in a water phase, and can effectively deliver nucleic acid medicines into cells. In addition, the secondary compound has an amide bond in the structure, and the bond can be rapidly hydrolyzed by enzymes in vivo, is easy to metabolize and remove, and has biodegradability. Amino acids produced by this degradation process play a role in immunopotentiating or enhancing/inhibiting other physiological activities. This property makes this novel lipid compound have great potential in the field of drug delivery and is expected to become an important carrier for future gene therapy and drug delivery.
(2) The preparation method of the endogenous amino acid ionizable lipid compound provided by the invention is based on the simple reaction of common carboxylic acid functional groups and organic amine compounds, and has the advantages of easily obtained raw materials, scientific synthetic route, simple operation steps, mild reaction conditions and high product separation efficiency.
(3) In the lipid nanoparticle composition, the structure and proportion of ionizable lipids have a significant impact on the lysosomal escape capacity of the nucleic acid; the ratio of helper lipid to ionizable lipid can also have an effect; the choice of different phospholipids and the proportion of ionizable lipids also affect the transfection efficiency of nucleic acids. In order to achieve efficient transfection of nucleic acids, the invention defines that the molar ratio of endogenous amino acid ionizable lipid, auxiliary lipid, cholesterol and PEG lipid is 15-45:20-35:10-40:0.5-5; the mass ratio of the endogenous amino acid ionizable lipid to the drug is 1-100:1.
(4) The endogenous amino acid derived ionizable lipid provided by the invention can effectively deliver nucleic acid drugs, can realize efficient transfection of nucleic acids in vivo and in vitro, and has the advantages of greatly reducing toxicity and remarkably improving safety based on endogenous amino acids, so that the endogenous amino acid derived ionizable lipid has wide application prospects in delivery of nucleic acid drugs.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 a shows the synthetic routes of the compounds 1 to 3 in examples 1 to 3 according to the invention, b shows the synthetic route in example 4, c shows the synthetic route of the compound 5;
FIG. 2 is a graph showing particle diameters (a) and Zeta potentials (b) of lipid nanoparticles LNP-1 to 5;
FIG. 3 shows the encapsulation efficiency of lipid nanoparticle LNP-3;
FIG. 4 is a transmission electron microscope image of lipid nanoparticle LNP-3
FIG. 5 shows the results of evaluation of transfection efficiency of lipid nanoparticles in the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
In FIG. 1, a is the synthetic route for the compounds 1 to 3 in examples 1 to 3, b is the synthetic route in example 4, and c is the synthesis of the compound 5 in example 5.
EXAMPLE 1 Synthesis of Compound 1
(1) Synthesis of intermediate N1, N1-didecyl-1, 2-ethylenediamine (intermediate b (Z11 b)):
Into a 250mL round bottom flask equipped with a magneton, t-butoxycarbonyl-1, 2-ethylenediamine (10 mmol), 1-bromodecane (22 mmol), potassium carbonate (20 mmol) and acetonitrile (60 mL) were added and heated under reflux at 80℃for 72h. After the reaction is finished, the reaction solution is filtered by suction, the solid is removed, the solvent is removed by rotary evaporation under reduced pressure, and the product is separated and purified by column chromatography (200-300 meshes of silica gel, eluent: methanol/dichloromethane volume ratio=1:20), thus obtaining intermediate a (Z11 a), and the yield is 75%.
Trifluoroacetic acid (2.5 mL) was added to a solution of intermediate a (Z11 a) (300.65 mg,7 mmol) in glacial dichloromethane (6 mL) and stirred for 6 hours. To the reaction mixture was added saturated sodium bicarbonate solution (5 mL). The organic layer was separated, washed with saturated sodium bicarbonate (3×10 mL) and saturated sodium chloride solution (3×10 mL), and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator to give intermediate b (Z11 b) in 97% yield.
(2) Intermediate b (Z11 b) (691 mg,2.27 mmol) was weighed and dissolved in 20mL of dichloromethane. N-Boc-L-tryptophan (268 mg,2.72 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (803 mg,2.73 mmol), 1-Hydroxybenzotriazole (HOBT) (268 mg,2.72 mmol), N-Diisopropylethylamine (DIPEA) (351 mg,2.72 mmol) were added to the solution and stirred at room temperature for 17h. The reaction was monitored by thin layer chromatography. The reaction mixture was washed with saturated brine (3×10 mL), and the organic phase was dried over anhydrous sodium sulfate. After concentration on a rotary evaporator, purification by column chromatography (column: 200-300 mesh silica gel; eluent: DCM/meoh=100:0-10:1) afforded intermediate c (936 mg, 65.73% yield).
Trifluoroacetic acid (2.5 mL) was added to a solution of intermediate c (936 mg,1.49 mmol) in ice dichloromethane (6 mL) and stirred for 6 hours. To the reaction mixture was added saturated sodium bicarbonate solution (5 mL). The organic layer was separated, washed with saturated sodium bicarbonate (3×10 mL) and saturated sodium chloride solution (3×10 mL), and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator to give compound 1 in 95% yield.
1H NMR(400MHz,Chloroform-d)δ8.15(s,1H),7.61(dd,J＝61.4,6.2Hz,2H),7.38-7.06(m,4H),3.70(dd,J＝9.0,4.2Hz,1H),3.49-3.23(m,3H),2.90(dd,J＝14.4,9.0Hz,1H),2.41(dt,J＝42.7,6.8Hz,6H),1.25(d,J＝3.5Hz,32H),0.88(t,J＝6.7Hz,6H).
EXAMPLE 2 Synthesis of Compound 2
(1) N1, N1, -Didodecyl ethane-1, 2-diamine (intermediate b (Z12 b)) and intermediate a (Z12 a) and were synthesized as described in example 1, except that 1-bromododecane was replaced with 1-bromodecane (22 mol); other steps and conditions were consistent with example 1. The single step yield of intermediate a (Z12 a) was 78%.
(2) The intermediate b (Z11 b) in example 1 was replaced with Z12b in this example, and the other conditions were exactly the same as in example 1, to synthesize compound 2, and finally yield 896mg of compound 2 was obtained in 61%.
1H NMR(400MHz,Chloroform-d)δ8.15(s,1H),7.61(dd,J＝61.4,6.2Hz,2H),7.38-7.06(m,4H),3.70(dd,J＝9.0,4.2Hz,1H),3.49-3.23(m,3H),2.90(dd,J＝14.4,9.0Hz,1H),2.41(dt,J＝42.7,6.8Hz,6H),1.25(d,J＝3.5Hz,42H),0.88(t,J＝6.7Hz,6H).
Example 3
N1, N1, -Bitetradecylethane-1, 2-diamine (intermediate b (Z13 b)) and intermediate a (Z13 a) were synthesized as described in example 1, except that 1-bromotetradecane was replaced by 1-bromodecane (22 mol); other steps and conditions were consistent with example 1. The single step yield of intermediate a (Z13 a) was 79%.
(2) The intermediate b (Z11 b) in example 1 was replaced with Z13b in this example, and the other conditions were exactly the same as those in example 1, to thereby synthesize compound 3, and finally yield 931mg of compound 3 was obtained in 59%.
1H NMR(400MHz,Chloroform-d)δ8.15(s,1H),7.61(dd,J＝61.4,6.2Hz,2H),7.38-7.06(m,4H),3.70(dd,J＝9.0,4.2Hz,1H),3.49-3.23(m,3H),2.90(dd,J＝14.4,9.0Hz,1H),2.41(dt,J＝42.7,6.8Hz,6H),1.25(d,J＝3.5Hz,50H),0.88(t,J＝6.7Hz,6H).
Example 4
Synthesis of intermediate N, N, -didodecyl-ethanolamine (intermediate d)
To an acetonitrile solution (18 mL) of ethanolamine (3.6 mmol) were added 1-bromodecane (8 mmol), potassium carbonate (16 mmol) and potassium iodide (0.4 mmol). The reaction mixture was stirred at 80℃under reflux for 48 hours. The reaction was monitored by thin layer chromatography. The reaction mixture was cooled to room temperature, filtered to remove potassium carbonate and potassium iodide, and concentrated under reduced pressure. After concentrating to dryness, purification by column chromatography (200-300 mesh silica gel, eluent: dichloromethane/methanol=50:1-20:1) afforded N, -didodecyl-ethanolamine, intermediate d (1.14 g, 92% yield).
Intermediate d (410 mg,1.2 mmol) was weighed and dissolved in 5mL DMSO. Kynurenine (213 mg,1 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (230 mg,1.2 mmol), 1-Hydroxybenzotriazole (HOBT) (162 mg,1.2 mmol) 9, triethylamine (607 mg,6 mmol) were added to the solution and stirred at room temperature for 17h. The reaction was monitored by thin layer chromatography. The reaction mixture was washed with saturated brine (3×10 mL), and the organic phase was dried over anhydrous sodium sulfate. After concentration by rotary evaporator, the mixture was purified by column chromatography (column: 200-300 mesh silica gel; eluent: PE/ea=100:0-3:1) to give compound 4 (339 mg, yield 64%).
1H NMR(400MHz,Chloroform-d)δ7.52-7.61(m,1H),7.45(dd,J＝7.9,1.4Hz,1H),7.22(td,J＝7.6,1.2Hz,1H),6.86(dd,J＝8.2,1.1Hz,1H),4.35(t,J＝6.9Hz,2H),3.99(t,J＝7.5Hz,1H),2.87(t,J＝6.9Hz,2H),2.7-2.8(m,4H),2.73(s,2H),1.37(p,J＝2.7Hz,4H),1.22(ddt,J＝11.0,8.9,6.4Hz,16H),-3.77(s,18H),0.78-0.86(m,7H).
Example 5
Compound 3 (10 mmol), 1-bromooctane (22 mmol), potassium carbonate (20 mmol) and acetonitrile (60 ml) were weighed and heated under reflux at 80℃for 48h. After the reaction, the reaction solution is filtered by suction, the solid is removed, the solvent is removed by rotary evaporation under reduced pressure, and the product is separated and purified by column chromatography (200-300 meshes of silica gel, eluent: methanol/dichloromethane volume ratio=1:20), thus obtaining the compound 5 with the yield of 75%.
1H NMR(400MHz,Chloroform-d)δ8.15(s,1H),7.61(dd,J＝61.4,6.2Hz,2H),7.38-7.06(m,3H),3.70(dd,J＝9.0,4.2Hz,1H),3.49-3.23(m,3H),3.15(dd,J＝10.1,9.0Hz,1H),,2.90(dd,J＝14.4,9.0Hz,1H),2.45(m,6.8Hz,10H),1.27(d,J＝3.6Hz,72H),0.88(m,12H).
Experimental example 1
Lipid nanoparticle in vitro delivery mRNA performance test
Preparation of lipid nanoparticles: the prepared amino acid ionizable lipid (compounds 1-5), cholesterol, DMG-PEG and DOPE are dissolved in ethanol according to the molar ratio of 15:25:1:20 to prepare a lipid ethanol solution (wherein the molar concentration of the amino acid ionizable lipid is 10 mg/mL). mRNA of Green Fluorescent Protein (GFP) was dissolved in potassium hydrogen phthalate-sodium hydroxide buffer at pH=4 to give mRNA solution (10 ng/. Mu.L). Lipid ethanol solution and mRNA solution were prepared into amino acid ionizable lipids using NanoAssemblr microfluidic device (Precision NanoSystems company): rapidly mixing the mRNA in a weight ratio of 10:1 to prepare a solution containing lipid nanoparticles; after ethanol was removed by dialysis of the solution containing lipid nanoparticles, lipid nanoparticles LNP-1 to 5 (LNP-1 (Compound 1), LNP-2 (Compound 2), LNP-3 (Compound 3), LNP-4 (Compound 4), and LNP-5 (Compound 5), respectively) were obtained.
Characterization of the lipid nanoparticles LNP-1 to 5 obtained was performed, and FIG. 2 is a graph showing particle diameters (a) and Zeta potentials (b) of the lipid nanoparticles LNP-1 to 5. FIG. 3 shows the encapsulation efficiency of the lipid nanoparticle LNP-3, and FIG. 4 shows a transmission electron microscope image of the lipid nanoparticle LNP-3.
As can be seen from FIG. 2, the particle size of the lipid nanoparticles LNP-1 to 5 is within 200nm, and the potential is slightly negatively charged. From FIG. 3, it can be seen that the encapsulation efficiency of the lipid nanoparticle LNP-3 is good (90.+ -. 1.06%). From FIG. 4 it can be seen that the lipid nanoparticle LNP-3 is spheroidized.
Evaluation of transfection efficiency of lipid nanoparticles LNP-1 to 5 in Hep3B cell line:
Hep3B cells in logarithmic growth phase were inoculated into 24-well plates containing DMEM medium at a density of 1×10 4/well, cultured overnight at 37 ℃, fresh medium containing lipid nanoparticle LNP-1-5 (containing 300ng mRNA) was added to each well, the Control group was added with the same volume of PBS, and after incubation for 12 hours at 37 ℃, the transfection efficiency was measured by flow cytometry and was high, and the result is shown in FIG. 5.
As can be seen from fig. 5, although the difference in particle diameters of the lipid nanoparticles LNP-1 to 5 is small, there is a large difference in delivery efficiency. The highest transfection efficiency of LNP-3 is 89+ -2.1%, and the transfection efficiencies of LNP-1, LNP-2, LNP-4, LNP-5 are 83.9+ -1.6%, 79.6+ -1.35%, 59.1+ -3.1%, 71.7+ -2.3%, respectively.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. An endogenous amino acid-derived ionizable lipid or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, characterized in that the endogenous amino acid-derived ionizable lipid has a structural formula represented by formula (i):
Wherein R 1 and R 2 are the same or different from each other and are each independently selected from substituted or unsubstituted linear or branched C 8～C20 alkyl, or substituted or unsubstituted linear or branched C 8～C20 alkenyl, or substituted or unsubstituted linear or branched C 8～C20 alkynyl, wherein R 3、R4 can be H;
R 3 and R 4 are the same or different from each other and are each independently selected from hydrogen, substituted or unsubstituted linear or branched C 8～C20 alkyl, or substituted or unsubstituted linear or branched C 8～C20 alkenyl, or substituted or unsubstituted linear or branched C 8～C20 alkynyl;
x is O, NH or S;
m is a positive integer of 1 to 3;
L 1 is A group.
2. An endogenous amino acid-derived ionizable lipid or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein when L 1 isWhen the amino acid is a group, the endogenous amino acid is tryptophan;
when L 1 is When the group is, the endogenous amino acid is kynurenine.
3. The endogenous amino acid-derived ionizable lipid or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein the endogenous amino acid-derived ionizable lipid is selected from one of the following compounds:
4. a method of preparing an endogenous amino acid-derived ionizable lipid according to claim 1, comprising:
In an organic solvent, under the action of a catalyst, a compound shown in a formula (II) and a compound shown in a formula (III) are subjected to esterification reaction or amidation reaction to obtain a compound shown in a formula (I);
In the compounds of the formula (II) and the compounds of the formula (III), R 1、R2、R3、R4、X、L1 and m have the same meanings as in the compounds of the formula (I).
5. The process according to claim 4, wherein when X is nitrogen and m=1 and r 1、R2 is independently selected from C 10～14 alkyl, the process for preparing the compound of formula (iii) comprises the steps of: in acetonitrile, under the action of potassium carbonate, N-tert-butoxycarbonyl-1, 2-ethylenediamine and bromoalkane react to obtain an intermediate a; reacting a dichloromethane solution of the intermediate a under the action of trifluoroacetic acid to obtain an intermediate b, namely a compound shown in a formula (III);
the specific process is as follows:
Preferably, the volume ratio of the mole amount of the N-t-butoxycarbonyl-1, 2-ethylenediamine to the acetonitrile is 0.01-1 mole/L; the molar ratio of the potassium carbonate to the N-tert-butoxycarbonyl-1, 2-ethylenediamine is 1-3:1; the mol ratio of the N-tert-butyloxycarbonyl-1, 2-ethylenediamine to the bromoalkane is 1:1-1.5; the reaction temperature of the N-tert-butoxycarbonyl-1, 2-ethylenediamine and bromoalkane is 60-100 ℃ and the reaction time is 60-80 h; the molar ratio of the intermediate a to the trifluoroacetic acid is 1-3:1; the reaction temperature of the intermediate a for producing the intermediate b is ice bath, and the reaction time is 2-8 h.
6. The process according to claim 4, wherein the organic solvent is one or more selected from the group consisting of methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, methylene chloride, diethyl ether, propylene oxide, acetone, methyl butanone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, dimethyl sulfoxide, N-dimethylformamide and triethanolamine; preferably, when the solvent is methanol, dichloromethane, acetonitrile, ethyl acetate, dimethyl sulfoxide; the volume ratio of the compound shown in the formula (II) to the organic solvent is 0.01-10 mol/L;
Or, the catalyst is selected from one or more of N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), O-benzotriazol-tetramethylurea Hexafluorophosphate (HBTU), 4-Dimethylaminopyridine (DMAP), N, N-Diisopropylethylamine (DIPEA) or O-benzotriazol-N-, N, N ', N' -tetramethylurea tetrafluoroboric acid (TBTU); preferably, when the catalyst is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT) and N, N-Diisopropylethylamine (DIPEA), the molar ratio of the catalyst to the compound represented by formula (II) is 1-1.5:1;
or the mol ratio of the compound shown in the formula (II) to the compound shown in the formula (III) is 1:1-1.5; preferably 1:1.2.
Or the reaction temperature is room temperature, and the reaction time is 10-30 hours;
or, the post-treatment method of the reaction liquid obtained by reacting the compound shown in the formula (II) and the compound shown in the formula (III) comprises the following steps:
adding saturated sodium chloride solution and saturated sodium bicarbonate solution into the reaction solution, extracting with dichloromethane, collecting an organic phase, drying the organic phase by anhydrous sodium sulfate, filtering, evaporating under reduced pressure, and separating by silica gel column chromatography to obtain lipid; the eluent used for the silica gel column chromatography is a mixture of dichloromethane and methanol, DCM/meoh=200:0 to 10:1.
7. Use of an endogenous amino acid-derived ionizable lipid of claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, as a drug delivery vehicle.
8. A lipid nanoparticle composition comprising the endogenous amino acid-derived ionizable lipid of claim 1, a helper lipid, cholesterol, a PEG lipid, and a entrapped drug.
9. The lipid nanoparticle composition of claim 8, wherein the helper lipid is selected from a variety of sources, including distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine-maleimide (DOPE-MAL), dipalmitoyl phosphatidylethanolamine (DPPE), dipyrimidinylphosphatidylcholine (DMPE), distearoyl phosphatidylethanolamine (DSPE), and 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), preferably dioleoyl phosphatidylethanolamine (DOPE);
or, the cholesterol is selected from one or two of cholesterol or 10 alpha-hydroxy cholesterol; preferably, the cholesterol is cholesterol or a mixture of cholesterol and 10 alpha-hydroxy cholesterol (mass ratio is 1:1);
or, the PEG lipid is selected from polyethylene glycol-dimyristoyl glycerol (PEG-DMG), polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE), polyethylene glycol-dimethacrylate (PEG-DMA); preferably polyethylene glycol-dimyristoyl glycerol (PEG-DMG);
Or, the encapsulated drug comprises one or more of biological drugs or chemical drugs; the biological medicine comprises one or more of nucleic acid medicine, protein medicine, polypeptide medicine or polysaccharide medicine; preferably, the nucleic acid drug comprises one or more of messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA (microRNA), circular RNA (circRNA), long non-coding RNA (lncRNA), plasmid DNA (plasmid DNA) and small circle DNA (mini circle DNA); the chemical medicine comprises one or more of small molecule medicine, fluorescein or developer; further preferred, the nucleic acid agent is mRNA;
Or, the lipid nanoparticle composition has a diameter of 1nm to 1000nm; preferably 100 to 200nm;
or, the molar ratio of the endogenous amino acid derived ionizable lipid, the auxiliary lipid, the cholesterol and the PEG lipid is 15-45:20-35:10-40:0.5-5;
the endogenous amino acid-derived ionizable lipid of claim 1 in a mass ratio of 1-100:1 to the encapsulated drug.
10. Use of the lipid nanoparticle composition of claim 9 in the preparation of a genetic medicament comprising an active ingredient which is a nucleic acid medicament and a delivery vehicle which is the lipid nanoparticle composition of claim 9.
CN202410477849.1A 2024-04-19 2024-04-19 Endogenous amino acid derived ionizable lipid, and preparation method and application thereof Pending CN118388389A (en)

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