CN117623978A - Biodegradable amino acid derived ionizable lipid, and preparation method and application thereof - Google Patents

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

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CN117623978A
CN117623978A CN202311373806.0A CN202311373806A CN117623978A CN 117623978 A CN117623978 A CN 117623978A CN 202311373806 A CN202311373806 A CN 202311373806A CN 117623978 A CN117623978 A CN 117623978A
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lipid
amino acid
medicine
ionizable
alkyl
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姜新义
王艳
岳啸
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Shandong University
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    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

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Abstract

The invention belongs to the field of biological medicine, and provides biodegradable amino acid derived ionizable lipid, a preparation method and application thereof, wherein a lipid structure comprises an amino head group, a connecting part with amino acid as a core and a hydrophobic lipid tail chain; the prepared lipid nanoparticle formula comprises the amino acid lipid, phospholipid, PEG lipid, auxiliary lipid and nucleic acid drugs, can safely and efficiently transfect nucleic acid into cells, and has wide application prospects in the gene therapy fields of nano nucleic acid vaccines, nucleic acid drug preparations and the like.

Description

Biodegradable 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 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.
Therapeutic nucleic acids including small interfering RNAs (sirnas), messenger RNAs (mrnas), micrornas (mirnas), antisense oligonucleotides, plasmids, and the like have been developed to correct genetic disorders or acquired diseases caused by abnormal gene expression profiles. Although nucleic acid drugs have high specificity, in the clinical application process, the problems of short half-life, poor stability, easy hydrolysis by endogenous nuclease and the like are faced, and the electronegative phosphate skeleton of the nucleic acid drugs is difficult to interact with electronegative cell membranes to enter cells. Therefore, developing safe and efficient nucleic acid delivery vehicles to improve the stability of nucleic acid drugs and the ability to penetrate cell membranes is a continuing medical challenge.
Non-viral nucleic acid delivery vehicles are primarily Lipid Nanoparticles (LNP) or polymeric nanoparticles that utilize charge interactions between ionizable or cationic lipids and electronegative nucleic acids to deliver nucleic acid drugs into cells for therapeutic purposes. As a novel drug delivery system, lipid nanoparticles (comprising ionizable lipids, auxiliary lipids, phospholipids, polyethylene glycol-lipids, and the like) have been widely used in nucleic acid delivery and clinical research due to their advantages of controllable preparation, large carrier capacity, high transport efficiency, good biocompatibility, no risk of integrating host genome, and the like. The ionizable lipid serves as the core structure of the LNP, and typically contains 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 solve the defects in the prior art, the invention aims to provide biodegradable amino acid derived ionizable lipid, and a preparation method and application thereof. The amino acid derived ionizable lipid provided by the invention has the advantages of mild preparation conditions and easiness in separation and purification, shows better biodegradability and high-efficiency in-vitro and in-vivo transfection efficiency, and can be used as a novel ionizable lipid for delivering nucleic acid drugs in vivo.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect of the invention there is provided a biodegradable amino acid derived ionizable lipid which is a compound of formula (i), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof;
wherein R is 1 、R 2 Are identical or different from each other and are each independently C 6 -C 24 Alkyl, C 6 -C 24 Alkenyl, C 6 -C 24 Alkynyl, substituent group-substituted C 6 -C 24 C substituted by alkyl, substituent groups 6 -C 24 Alkenyl, substituent group-substituted C 6 -C 24 With or without one or more heteroatoms;
R 3 、R 4 are identical or different from each other and are each independently C 6 -C 24 Alkyl, C 6 -C 24 Alkenyl, C 6 -C 24 Alkynyl, substituent group-substituted C 6 -C 24 C substituted by alkyl, substituent groups 6 -C 24 Alkenyl, substituent group-substituted C 6 -C 24 With or without one or more heteroatoms;
R 1 、R 2 and R is R 3 、R 4 The same or different;
R 5 independently selected from hydrogen, C 1 -C 10 C substituted by alkyl, substituent groups 1 -C 10 An alkyl group;
X 1 、X 2 selected from oxygen or nitrogen or sulfur;
a is selected from positive integers from 1 to 3;
b is selected from positive integers from 1 to 3;
c is selected from integers from 0 to 2;
d is selected from integers from 0 to 2.
In some embodiments, c=d=0, the amino acid is α -glycine;
in some embodiments, c=d=1, the amino acid is β -alanine;
in some embodiments, c=d=2 and the amino acid is gamma-aminobutyric acid.
In some embodiments, R 1 、R 2 、R 3 、R 4 Are identical to or different from each other and are independently selected from C 6 -C 24 An alkyl group; r is R 1 ＝R 2 ，R 3 ＝R 4 ；R 5 Is hydrogen or methyl; x is nitrogen; a=b, c=d.
In some embodiments, R 1 、R 2 、R 3 、R 4 Independently selected from C 8 -C 18 Alkyl, R 5 Methyl, c=d=2.
In some embodiments, the biodegradable 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 the biodegradable amino acid-derived ionizable lipid described above, comprising:
a step of obtaining a compound represented by formula (I) by an esterification reaction or an amidation reaction in an organic solvent in the presence of a catalyst with a compound represented by formula (I1) and with a compound represented by formula (III);
wherein in the formulas (II), (III), R 1 、R 2 、R 5 、X 1 、X 2 A, b and c andthe meaning of the compounds of the formula (I) is the same.
In some embodiments, carboxylic acid (II) and organic amine (III) are commercially available or are prepared according to existing methods.
In some embodiments, when R 5 Is methyl, X is nitrogen, a=b=2 or 3, r 1 、R 2 Independently selected from C 6-24 In the case of alkyl groups, the process for preparing carboxylic acid (II) comprises the steps of: in acetonitrile, under the action of potassium carbonate and potassium iodide, amino acid tert-butyl ester and bromoalkane react to obtain an intermediate a; under the action of trifluoroacetic acid, the methylene dichloride solution of the intermediate a is reacted to obtain the intermediate b, namely the compound shown in the formula (II).
In some preferred embodiments, the molar amount of the tertiary butyl amino acid to the volume ratio of acetonitrile is from 0.01 to 1mol/L; the molar ratio of the potassium carbonate to the tertiary butyl amino acid is 1-3:1; the molar ratio of potassium iodide to tertiary butyl amino acid is 0.1-1:1; the mol ratio of the tertiary butyl amino acid to the bromoalkane is 1:2-2.5; the reaction temperature of the tertiary butyl amino acid and bromoalkane is 60-100 ℃ and the reaction time is 60-80h; the molar ratio of the molar amount of the intermediate 1 to the molar amount of the trifluoroacetic acid is 1-3:1; the reaction temperature of the intermediate 1 is ice bath, and the reaction time is 2-8h.
In some 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, ethylene glycol ether, N-dimethylformamide, or triethanolamine;
in some preferred embodiments, the solvent may be methanol, methylene chloride, acetonitrile, petroleum ether, ethyl acetate, isopropyl alcohol, N-diisopropylethylamine, N-dimethylformamide, or the like; the molar amount of carboxylic acid (II) and the volume ratio of the organic solvent are 0.01-10mol/L.
In some embodiments, the catalyst is selected from one or a combination of two 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), or O-benzotriazol-N, N' -tetramethylurea tetrafluoroborate (TBTU).
In some preferred embodiments, the catalyst may be 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 1-Hydroxybenzotriazole (HOBT), the catalyst and the carboxylic acid (II) in a molar ratio of 2-3:1.
In some embodiments, the molar ratio of carboxylic acid (II) to organic amine (III) is from 2 to 2.1:1 to 1.1.
In some embodiments, the reaction temperature is room temperature and the reaction time is 10 to 30 hours.
In some embodiments, the method for working up the reaction mixture obtained by reacting carboxylic acid (II) with organic amine (III) comprises the steps of: adding saturated sodium chloride solution into the reaction solution, extracting with dichloromethane, drying the organic phase with anhydrous sodium sulfate, filtering, evaporating under reduced pressure, and separating by silica gel column chromatography to obtain lipid; the eluent used for the column chromatography on silica gel is a mixture of dichloromethane and methanol, DCM/meoh=100:0-10:1.
In a third aspect of the invention there is also provided the use of a biodegradable amino acid derived ionizable lipid as described above in a drug delivery vehicle.
In a fourth aspect the present invention provides a lipid nanoparticle or lipid nanoparticle composition comprising a lipid, in particular an ionizable lipid as defined herein. The nanoparticle composition may further comprise a helper lipid, a phospholipid, a PEG lipid, and a drug.
In some embodiments, the helper lipid is selected from the group consisting of a steroid, cholesterol hemisuccinate, cholesterol, and alkyl resorcinol. Preferably cholesterol.
In some embodiments, the phospholipid is selected from one or a combination of two or more of distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), diethyl pyrocarbonate (DEPC), dilauroyl phosphatidylcholine (DLPC), phosphatidylcholine (POPC), egg yolk lecithin (EPC), hydrogenated Soybean Phosphatidylcholine (HSPC), sphingomyelin (SM), or dimyristoyl phosphatidylcholine (DMPC); preferably dioleoyl phosphatidylethanolamine (DOPE) and dipalmitoyl phosphatidylcholine (DPPC).
In some embodiments, the PEG lipid is selected from one or a combination of two or more of DSPE-PEG, DMG-PEG, DPPE-PEG, or DMA-PEG; preferably, the PEG lipid is DSPE-PEG.
In some embodiments, the molar ratio of amino acid derived ionizable lipid, helper lipid, phospholipid, and PEG lipid is 20-50:20-60:10-40:0.5-10; the mass ratio of the amino acid derived ionizable lipid to the drug is 1-100:1.
In some embodiments, the lipid nanoparticle has a diameter in the range of 1nm to 1000 nm. For example, the particle diameter is in the range of 20nm to 800nm, or in the range of 50nm to 500nm, or in the range of 50nm to 200nm, or in the range of 1nm to 100 nm. When the diameter of the lipid nanoparticle is in the range of 1nm to 1000nm, it is a nanoparticle generally described in the art.
According to the present invention, the lipid nanoparticle may be prepared using any method known in the art. These methods include, but are not limited to, liposome extrusion, thin film differentiation, nano-precipitation, microfluidic and impingement jet mixing, and other methods known to those of ordinary skill in the art. Preferably, the preparation method of the lipid nanoparticle comprises the steps of: dissolving ionizable lipid, auxiliary lipid, phospholipid and PEG lipid in ethanol to obtain lipid mixed ethanol phase; dispersing the drug in citric acid buffer solution with pH=4 to obtain drug water phase; rapidly mixing the lipid mixed ethanol phase and the drug water phase by utilizing micro-flow control to prepare a solution containing lipid nanoparticles; then the lipid nanoparticle is prepared through the steps of dialysis, ultrafiltration and the like.
In some embodiments, the drug comprises one or a combination of two or more of a biopharmaceutical or a chemical drug; the biological medicine comprises one or more than two of nucleic acid medicine, protein medicine, polypeptide medicine or polysaccharide medicine; further preferred, the nucleic acid drug comprises one or more than two of small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), circular mRNA, long non-coding RNA (lncRNA), plasmid DNA, mini circle DNA (mcDNA), antisense oligonucleotides (ASOs), small activating RNA (saRNA) or Aptamer (Aptamer); the chemical medicine comprises one or more than two of small molecule medicine, fluorescein or developer. Most preferably, the drug is an mRNA, including linear mRNA and circular mRNA.
In some embodiments, the targeting molecule may be modified on the lipid nanoparticle to provide targeting functions to target a particular cell, tissue or organ. The targeting molecule may be in the whole lipid nanoparticle or may be located only on the surface of the lipid nanoparticle. 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 (asialoglycoprotein), receptor ligands, sialic acids, aptamers, etc.
In a fifth aspect, the present invention provides the use of an amino acid derived ionizable lipid according to the first aspect of the invention and a lipid nanoparticle according to the fourth aspect for the preparation of a genetic medicament comprising an active ingredient and a delivery vehicle, said active ingredient being a nucleic acid medicament, said delivery vehicle being a composition as described above.
The lipid nanoparticle of the present invention can be used for the prevention and treatment of various diseases of human and/or animals by oral, rectal, intravenous, intramuscular injection, intravaginal, intranasal, subcutaneous, intraperitoneal, buccal, or oral, injection or inhalation.
Further, the nucleic acid drug is useful for preventing and/or treating cancer, inflammation, fibrosis disease, autoimmune disease, infection, psychotic disorder, hematopathy, chromosomal disease, genetic disease, connective tissue disease, digestive disease, otorhinolaryngologic disease, endocrine disease, ocular disease, reproductive disease, heart disease, kidney disease, lung disease, metabolic disorder, oral disease, musculoskeletal disease, neonatal screening, nutritional disease, parasitic disease, skin disease, and the like.
The beneficial effects of the invention are that
1. The invention provides an amino acid derived ionizable lipid, which has the characteristics of biodegradability and high transfection efficiency, and the amino acid component of the ionizable lipid is derived from naturally occurring amino acids (such as glycine, beta-alanine, gamma-aminobutyric acid and the like). The ionizable lipid synthesis raw materials are cheap and easy to obtain, the design is reasonable, the operation is convenient, and the lipid nano delivery system composed of the ionizable lipid synthesis raw materials can be widely used for delivering nucleic acid medicines.
2. The amino acid-derived ionizable lipids provided by the present invention are uncharged under physiological conditions (ph=7.4), but positively charged under acidic conditions, binding negatively charged nucleic acid macromolecules in the form of electrostatic interactions. Protonation and positively charging occur under the acidic condition of the endosome, and the protonation and the negatively charged lipid interact to easily form an unstable inverted hexagonal phase, so that the fusion of the LNP and the endosome membrane is promoted, or the endosome escape of the LNP is realized through a proton sponge effect.
3. In the lipid nanoparticle formula provided by the invention, the structure and the proportion of the ionizable lipid influence the stability of the lipid nanoparticle and the nucleic acid transfection efficiency; in order to realize efficient nucleic acid transfection, the invention selects amino acid derived ionizable lipid, auxiliary lipid, phospholipid and PEG lipid with the molar ratio of 20-50:20-60:10-40:0.5-10, and optimizes the prescription, and the optimized proportion can realize efficient transfection of nucleic acid drugs; in some embodiments, to further achieve efficient pulmonary delivery of nucleic acid agents, DPPC is added to the phospholipid fraction at a molar ratio of DOPE to DPPC of 0.2-5. The mass ratio of the amino acid derived ionizable lipid to the drug is 1-100:1.
4. The amino acid derivative ionizable lipid provided by the invention can effectively deliver nucleic acid drugs, can realize efficient transfection of nucleic acid in vivo and in vitro, has a transfection effect equivalent to that of a marketed product (composed of DLin-MC 3), has good biocompatibility, and has wide application prospects in delivery of nucleic acid drugs such as mRNA and the like.
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 is a synthetic route pattern of lipid nanoparticles of the present invention.
FIG. 2 shows particle diameters and PDI before and after atomization of lipid nanoparticles according to Experimental example 1 of the present invention.
FIG. 3 is a Zeta potential characterization of lipid nanoparticles before and after atomization in Experimental example 1 of the present invention.
FIG. 4 is a transmission electron microscope image before and after atomizing lipid nanoparticles in experimental example 1 of the present invention.
FIG. 5 shows the results of examining the encapsulation efficiency of lipid nanoparticles in experimental example 1 of the present invention.
FIG. 6 is a result of examining transfection efficiency of lipid nanoparticles in Experimental example 2 of 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.
Term interpretation:
the term "alkyl" by itself or as part of another substituent means a compound of formula C x H 2x+1 Wherein x is a number greater than or equal to 1. Typically, the alkyl groups of the present invention contain from 1 to 24 carbon atoms. The alkyl group may be linear or branched and may be substituted with one or more groups selected from halogen, hydroxy, amino, oxo, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
The term "alkenyl" or "alkene" as used herein refers to a straight, cyclic or branched hydrocarbon group containing at least one carbon-carbon double bond, unless otherwise specified. In some embodiments, the alkenyl group has 6 to 24 carbons, also known as C 6-24 Alkenyl groups. Alkenyl group inclusion exampleSuch as ethenyl, propenyl, n-butenyl, isobutenyl, and the like. The alkenyl group may be alkenyl that is unsubstituted or substituted with one or more substituents (e.g. 1, 2, 3 or 4) selected from those defined above for substituted alkyl groups.
The term "alkynyl" or "alkyne" as used herein, unless otherwise specified, refers to a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond. In some embodiments, alkynyl groups have 6 to 24 carbons, also known as C 6-24 Alkynyl groups. Alkynyl groups include, for example, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. Alkynyl groups may be alkynyl groups which are unsubstituted or substituted with one or more substituents (e.g. 1, 2, 3 or 4) selected from those defined above for substituted alkyl groups.
"substituted" means that one or more hydrogen atoms in the group are replaced, independently of one another, by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) the possible substitutions without undue effort.
In the context of the present invention, the alkyl, alkene and alkyne moieties as defined herein may further comprise one or more heteroatoms, for example carbon in the alkyl, alkene or alkyne chain is replaced by a heteroatom such as one selected from nitrogen, oxygen or sulfur.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
Example 1
Synthesis of intermediate tert-butyl 4-N, N' -didecylaminobutylate (GA 10 a):
to a solution of tert-butyl 4-aminobutyrate hydrochloride (978.45 mg,5 mmol) in acetonitrile (20 mL) was added 1-bromodecane (2.4 g,11 mmol), potassium carbonate (1.3 g,10 mmol) and potassium iodide (166 mg,1 mmol). The reaction mixture was stirred at 85℃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, the oil residue was purified by column chromatography (200-300 mesh silica gel, eluent: petroleum ether/ethyl acetate=50:1-20:1) to give tert-butyl 4-N, N' -didecylaminobutyrate (1.32 g, 60.03% yield).
Synthesis of intermediate 4-N, N' -didecylaminobutyric acid (GA 10 b):
trifluoroacetic acid (2.5 mL) was added to a solution of GA10a (439.77 mg,1 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 (3X 10 mL) and saturated sodium chloride solution (3X 10 mL), and the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator to give intermediate GA10b.
Example 2
The synthesis of the intermediates 4- [ di (dodecyl) amino ] butanoic acid-2-methylpropan-2-yl ester (GA 12 a) and 4- [ di (dodecyl) amino ] butanoic acid (GA 12 b) was as described in example 1, except that 1-bromodecane was replaced with 1-bromododecane (11 mol); other steps and conditions were consistent with example 1. The single step yield of intermediate GA12a was 38.9%.
Example 3
Synthesis of intermediate (GA 14 a) and 4- [ ditetradecyl ] amino ] butanoic acid (GA 14 b) as described in example 1, except that 1-bromodecane was replaced with 1-bromotetradecane (11 mol); other steps and conditions were consistent with example 1. The single step yield of intermediate GA14a was 64.73%.
Example 4
Synthesis of N- (11-decyl-3-methyl-7-oxo-3,6,11-triazadi-undec-1-yl) -4- (didecylamino) butanamide (GAE 10)
GA10b (314.6 mg,0.84 mmol) was weighed out and dissolved in 4mL of methylene chloride. N-methyl-diaminoethylamine (46.9 mg,0.4 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (184.03 mg,0.96 mmol), 1-Hydroxybenzotriazole (HOBT) (129.72 mg,0.96 mmol), N-Diisopropylethylamine (DIPEA) (115.12. Mu.l, 1.2 mmol) were added to the solution, and stirred at room temperature for 24h. The reaction was monitored by thin layer chromatography. The reaction mixture was washed with saturated brine (3X 10 mL), and the organic phase was dried over anhydrous sodium sulfate. The residue after concentration by rotary evaporator was purified by column chromatography (column: 200-300 mesh silica gel; eluent: DCM/meoh=100:0-10:1) to give GAE10 (111.3 mg, 43.76% yield). 1 H NMR(400MHz,CDCl 3 )δ8.37(s,2H)，3.33(s,4H)，3.07(s,4H)，3.00(s,8H)，2.58(d,J＝17.71Hz,8H)，2.26(s,3H)，2.04(s,4H)，1.69(s,8H)，1.22(d,J＝27.68,56H)，0.80(t,J＝5.12Hz,12H).
Example 5
Synthesis of 4- [ di (dodecyl) amino ] -N- (3-methyl-7-oxo-11-dodecyl-3,6,11-triazaditridec-1-yl) butanamide (GAE 12)
GAE12 was prepared as described in example 4, except that GA10b (0.84 mmol) was replaced with GA12b (0.63 mmol); other steps and conditions and ratios of reagents were consistent with example 4. GAE12 (55.3 mg, 19.2%) was obtained. 1 H NMR(400MHz,CDCl 3 )δ8.03(s,2H),3.32(t,4H),3.05(s,4H),2.95(s,8H),2.62(t,J＝5.82Hz,4H),2.52(d,J＝4.95Hz,4H),2.21(s,3H),2.08(m,4H),1.74(s,8H),1.29(m,72H),0.88(t,J＝6.78Hz,12H).
Example 6
Synthesis of 4- [ di (tetradecyl) amino ] -N- (3-methyl-7-oxo-11-tetradecyl-3,6,11-triazacyclopentadec-1-yl) butanamide (GAE 14)
GAE14 was prepared as described in example 4, except that GAE10b (0.84 mmol) was replaced with GAE14b (0.84 mmol); other steps and conditions were consistent with example 4. GAE14 (85.47 mg, 21.63% yield) was obtained. 1 H NMR(400MHz,CDCl 3 )δ8.37(s,2H),3.44(s,4H),3.13(s,4H),3.00(d,J＝8.00,8H),2.84(d,J＝8.19Hz,4H),2.62(s,4H),2.44(s,3H),2.11(s,4H),1.75(s,12H),1.28(m,88H),0.87(t,J＝6.50Hz,12H).
Example 7
Synthesis of 4- [ di (dodecyl) amino ] -N- (13-dodecyl-4-methyl-9-oxo-4,8,13-triazacyclopentadec-1-yl) butanamide (GAP 12)
GAP12 was prepared as described in example 4, except that GA10b (0.84 mmol) was replaced with GA12b (0.84 mmol), N-methyl-diaminoethylamine was replaced with N, N-bis (3-aminopropyl) methylamine (0.4 mmol); other steps and conditions and implementationsExample 4 was consistent. GAP12 (58.3 mg, 14.75%) was obtained. 1 H NMR(400MHz,CDCl3)δ7.43(s,2H),3.29(dd,J1＝6.15Hz,J2＝12.13Hz,4H),2.63(d,J＝25.78Hz,12H),2.39(t,J＝6.33Hz,4H),2.31(t,J＝6.83Hz,4H),2.18(s,3H),1.87(m,4H),1.66(m,4H),1.53(s,8H),1.25(s,72H),0.87(t,J＝6.64Hz,12H).
Example 8
N- (13-decyl-4-methyl-9-oxo-4,8,13-triazaditridec-1-yl) -4- (didecylamino) butanamide (GAP 10) synthesis.
GAP10 was prepared as described in example 4, except that N-methyl-diaminoethylamine (0.4 mmol) was replaced with N, N-bis (3-aminopropyl) methylamine (0.4 mmol); other steps and conditions and reagent molar ratios were consistent with example 4. GAP10 (56.5 mg, 16.11% yield) was obtained. 1 H NMR(400MHz,CDCl 3 )δ7.15(s,2H),3.29(dd,J1＝6.24Hz,J2＝6.25Hz,4H),2.44(m,8H),2.37(s,J＝6.46Hz,4H),2.24(d,J＝7.06Hz,8H),2.17(s,3H),1.78(m,4H),1.64(m,4H),1.44(s,8H),1.24(s,56H),0.87(t,J＝6.65Hz,12H).
Example 9
Lipid GAE14 was selected as a representative compound to prepare lipid nanoparticles. The ionizable lipid (GAE 14), cholesterol, DOPE, DSPE-PEG2k used to prepare the lipid nanoparticles were first dissolved in ethanol at a molar ratio of 35:25:30:0.5 to prepare an ethanol phase solution. The EGFP (or Luciferase) mRNA was then added to 10-50mM citrate buffer (ph=4) to obtain an aqueous mRNA solution, and the mRNA lipid nanocomposite was prepared by rapidly and uniformly mixing the ethanol phase solution with the aqueous solution. Wherein the ionizable lipid: the weight ratio of mRNA was 10:1. The lipid nanoparticle for encapsulating mRNA can be obtained through dialysis, ultrafiltration and other operations.
Further prescription optimization is carried out to achieve the purposes of good atomization stability and deep lung delivery of the lipid nanoparticles, a natural lung surfactant component DPPC is added into a single phospholipid component, and the optimized prescription is further obtained through screening. Including but not limited to ionizable lipids, cholesterol, DOPE, DPPC, DSPE-PEG2k molar ratio 35:35:6:4:1.5. Lipid nanoparticles, labeled NGAE14, were prepared and further characterized according to the procedure and prescription ratios described above.
Experimental example 1
Characterization of ionizable lipid nanoparticles:
the lipid nanoparticle prepared in example 9 was further characterized. The lipid nanoparticles were characterized by morphology by transmission electron microscopy, the dynamic light scattering laser particle sizer (Malvern Zetasizer Nano ZS) was characterized for nanoscale, polydisperse coefficient PDI, and Zeta potential, and the Quant-iT RiboGreen RNAAssay Kit RNA quantitative detection kit was subjected to encapsulation efficiency measurement, as shown in FIGS. 2-5 (only the lipid nanoparticles GAE14L prepared by the prescription: ionizable lipid, cholesterol, DOPE, DPPC, DSPE-PEG2k molar ratio 35:35:6:4:1.5 were shown). The lipid nanoparticle is spherical, has uniform particle diameter (PDI < 0.3), has potential close to neutral, good encapsulation efficiency, and stable performance before and after atomization.
Experimental example 2
In vitro transfection efficiency investigation of ionizable lipid nanoparticles:
taking MLE12 cells in logarithmic growth phase, inoculating the MLE12 cells into a 96-well plate filled with DMEM/F12 culture medium, and plating the MLE12 cells at a density of 1.times.10 per well 5 Cells were prepared for transfection (37 ℃ C., about 12 hours) after cell attachment. Lipid nanoparticles containing 0.2 μg of mRNA were added per well, with 3 multiplex wells per group. After 24 hours of transfection, the proportion of fluorescence of each group of cells was measured using a fluorescence microscope or flow cytometer.
Overall experimental results show that different prescription LNP particle sizes prepared with different ionizable lipids remain substantially within 200nm, but that there is a large difference in delivery efficiency. Further, the lipid nanoparticle with high transfection efficiency is prepared by optimizing a prescription, so that the requirements of treatment of different diseases can be met, and the risk brought by multiple drug administration can be reduced. The results of a partial flow assay are shown in figure 6 (only GAE14L lipid nanoparticles are shown) where the positive control is a lipid nanoparticle prepared by commercial DLin-MC 3. The LNP transfection efficiencies of the remaining lipids GAE12, GAE10, GAP12 with the formulations were 84.7%, 73.8%, 54.3%, 67.5%, respectively.
The above description is only of some embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations of the present invention will be apparent to 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. Biodegradable amino acid derived ionizable lipid, characterized in that it is a compound of formula (i), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof;
wherein R is 1 、R 2 Are identical or different from each other and are each independently C 6 -C 24 Alkyl, C 6 -C 24 Alkenyl, C 6 -C 24 Alkynyl, substituent group-substituted C 6 -C 24 C substituted by alkyl, substituent groups 6 -C 24 Alkenyl, substituent group-substituted C 6 -C 24 With or without one or more heteroatoms;
R 3 、R 4 are identical or different from each other and are each independently C 6 -C 24 Alkyl, C 6 -C 24 Alkenyl, C 6 -C 24 Alkynyl, substituent group-substituted C 6 -C 24 C substituted by alkyl, substituent groups 6 -C 24 Alkenyl, substituent group-substituted C 6 -C 24 With or without one or more heteroatoms;
R 1 、R 2 and R is R 3 、R 4 The same or different;
R 5 independently selected from hydrogen, C 1 -C 10 C substituted by alkyl, substituent groups 1 -C 10 An alkyl group;
X 1 、X 2 selected from oxygen or nitrogen or sulfur;
a is selected from positive integers from 1 to 3;
b is selected from positive integers from 1 to 3;
c is selected from integers from 0 to 2;
d is selected from integers from 0 to 2.
2. The biodegradable amino acid derived ionizable lipid of claim 1, wherein c = d = 0, said amino acid is α -aminoacetic acid;
or, c=d=1, the amino acid is β -alanine;
or, when c=d=2, the amino acid is gamma-aminobutyric acid;
or, R 1 、R 2 、R 3 、R 4 Are identical to or different from each other and are independently selected from C 6 -C 24 An alkyl group; r is R 1 ＝R 2 ，R 3 ＝R 4 ；R 5 Is hydrogen or methyl; x is nitrogen; a=b, c=d;
or, R 1 、R 2 、R 3 、R 4 Independently selected from C 8 -C 18 Alkyl, R 5 Methyl, c=d=2.
3. The biodegradable amino acid derived ionizable lipid of claim 1, wherein said biodegradable amino acid derived ionizable lipid is selected from one of the following compounds:
4. a method of preparing a biodegradable amino acid derived ionizable lipid according to any one of claims 1-3, comprising:
a step of obtaining a compound represented by formula (1) by an esterification reaction or an amidation reaction with a compound represented by formula (I1) and a compound represented by formula (III);
wherein in the formulas (II), (III), R 1 、R 2 、R 5 、X 1 、X 2 A, b and c have the same meaning as in the compounds of the formula (I).
5. The method of preparing a biodegradable amino acid derived ionizable lipid of claim 4, comprising one or more of the following conditions:
i. 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, dichloromethane, diethyl ether, propylene oxide, acetone, methyl butanone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, N-dimethylformamide or triethanolamine;
ii. The catalyst is selected from one or more than two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, N-hydroxysuccinimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, O-benzotriazol-tetramethylurea hexafluorophosphate, 4-dimethylaminopyridine or O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid;
or the catalyst is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole;
the mol ratio of the compound shown in the formula (I1) to the compound shown in the formula (III) is 1:1-1.1;
iv, the reaction temperature is room temperature, and the reaction time is 10-30h.
6. Use of a biodegradable amino acid derived ionizable lipid according to any of claims 1-3 for the preparation of a drug delivery vehicle.
7. A lipid nanoparticle comprising the biodegradable amino acid-derived ionizable lipid of any one of claims 1-4, a helper lipid, a phospholipid, and a PEG-lipid.
8. The lipid nanoparticle of claim 7, wherein the helper lipid is selected from the group consisting of a steroid, a cholesterol hemisuccinate, a cholesterol, and an alkyl resorcinol;
or, the phospholipid is selected from one or more than two of distearoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, diethyl pyrocarbonate, dilauroyl phosphatidylcholine, egg yolk lecithin, hydrogenated soybean phosphatidylcholine, sphingomyelin or dimyristoyl phosphatidylcholine;
or, the PEG lipid is selected from one or more than two of DSPE-PEG, DMG-PEG, DPPE-PEG or DMA-PEG;
or, the molar ratio of the biodegradable amino acid derived ionizable lipid, the auxiliary lipid, the phospholipid and the PEG lipid is 20-50:20-60:10-40:0.5-10; the mass ratio of the ionizable lipid to the medicine is 1-100:1;
the medicine comprises one or more than two of biological medicine or chemical medicine; the biological medicine comprises one or more than two of nucleic acid medicine, protein medicine, polypeptide medicine or polysaccharide medicine;
or, the nucleic acid drug comprises one or more than two of small interfering RNA, messenger RNA, microRNA, circular mRNA, long-chain non-coding RNA, plasmid DNA, mini circle DNA, antisense oligonucleotide, small activating RNA or nucleic acid aptamer;
or, the chemical medicine comprises one or more than two of small molecule medicine, fluorescein or developer;
alternatively, the drug is an mRNA, including linear mRNA and circular mRNA.
9. A method of preparing lipid nanoparticles according to claim 7 or 8, comprising: liposome extrusion, thin film differentiation, nano precipitation, microfluidic and impact jet mixing;
or, the preparation method of the lipid nanoparticle comprises the following steps: dissolving biodegradable amino acid derived ionizable lipid, auxiliary lipid, phospholipid and PEG lipid in ethanol to obtain lipid mixed ethanol phase; dispersing the medicine in citric acid buffer solution with pH=4-4.5 to obtain medicine water phase; mixing the lipid mixed ethanol phase and the drug water phase by utilizing micro-flow control to prepare a solution containing lipid nanoparticles; then the lipid nanoparticle is prepared through dialysis and ultrafiltration.
10. Use of a biodegradable amino acid derived ionizable lipid according to any of claims 1-3, and a lipid nanoparticle according to claim 7 or 8, for the preparation of a genetic medicament, characterized in that said genetic medicament comprises: an active ingredient and a delivery carrier, wherein the active ingredient is a nucleic acid drug.
CN202311373806.0A 2023-10-23 2023-10-23 Biodegradable amino acid derived ionizable lipid, and preparation method and application thereof Pending CN117623978A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117964577A (en) * 2024-03-29 2024-05-03 天津全和诚生物技术有限公司 Cationic lipid compound, preparation method thereof, composition containing cationic lipid compound and application of cationic lipid compound

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117964577A (en) * 2024-03-29 2024-05-03 天津全和诚生物技术有限公司 Cationic lipid compound, preparation method thereof, composition containing cationic lipid compound and application of cationic lipid compound

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