Nothing Special   »   [go: up one dir, main page]

CN116675624A - Lipid compound and lipid nanoparticle - Google Patents

Lipid compound and lipid nanoparticle Download PDF

Info

Publication number
CN116675624A
CN116675624A CN202310616858.XA CN202310616858A CN116675624A CN 116675624 A CN116675624 A CN 116675624A CN 202310616858 A CN202310616858 A CN 202310616858A CN 116675624 A CN116675624 A CN 116675624A
Authority
CN
China
Prior art keywords
lipid
independently selected
alkyl
branched alkyl
linear
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310616858.XA
Other languages
Chinese (zh)
Inventor
章波
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Naicheng Biotechnology Co ltd
Original Assignee
Shanghai Naicheng Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Naicheng Biotechnology Co ltd filed Critical Shanghai Naicheng Biotechnology Co ltd
Priority to CN202310616858.XA priority Critical patent/CN116675624A/en
Publication of CN116675624A publication Critical patent/CN116675624A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/64—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups singly-bound to oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—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
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Optics & Photonics (AREA)
  • Nanotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The application relates to a lipid compound and lipid nano-particles, wherein the lipid is a cationic lipid compound with a structure shown as a formula (I) or pharmaceutically acceptable salt, prodrug or stereoisomer thereof,the compound comprises one or more biodegradable groups. The incorporation of the cationic lipid compound into lipid nanoparticles can be used to deliver therapeutic or prophylactic agents, such as nucleic acids, to animal cells or organs for therapeutic and prophylactic effects.

Description

Lipid compound and lipid nanoparticle
Technical Field
The present application relates to an ionizable lipid compound, and lipid nanoparticles comprising the same can deliver nucleic acid drugs into animal cells.
Background
Nucleic acid drugs, including mRNA, siRNA, ASO, are delivered to target cells of the human body by certain means, and therapeutic and prophylactic effects are achieved in the human body by translating target proteins, or silencing target mRNAs, and the like. Delivery means include viral vectors and non-viral vectors. Viral vectors have limited their use due to their toxicity and immunogenicity. Non-viral vectors, particularly lipid nanoparticles, encapsulate and deliver nucleic acid drugs to target cells by means of liposome molecular assembly, and have achieved commercial use. The first FDA approved siRNA drug (onettro) from Alnylam corporation and two new mRNA crown vaccines approved in 2020 both employed nanoparticle delivery systems comprising ionizable cationic lipid molecules.
These lipid nanoparticles typically comprise one or more cationic lipids, neutral phospholipids, sterols, polyethylene glycol lipids. Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. Scientists have developed several cationic lipids for nucleic acid drug delivery, but there remains a need for more structurally abundant cationic lipids to achieve functionalized lipid nanoparticles.
Disclosure of Invention
In one aspect, the present application provides a cationic lipid compound having a structure as shown in formula (I):
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein,
L 1 ,L 2 ,L 3 ,L 4 ,L 5 identical to or different from each other and each independently selected from: absence of C 1 -C 24 Straight-chain or branched alkyl, C 2 -C 24 Alkenylene or branched alkenyl;
M 1 and M 2 Each independently selected from-OC (O) -, -C (O) O-, or-nhc=n (CN) NH-;
G 1 and G 2 Each independently selected from H, C 5 -C 10 Aryl or 5-to 10-membered heteroaryl, and C 5 -C 10 The hydrogen atoms on the aryl or 5-to 10-membered heteroaryl groups may each independently optionally be replaced by n-XR 1 Substituted, n is an integer from 1 to 3, X is absent, nitrogen, oxygen, sulfur, selenium, R 1 Is C 4 -C 20 Straight-chain or branched alkyl, C 4 -C 20 Straight or branched alkenyl groups.
In another aspect, the present application provides a lipid nanoparticle comprising a cationic lipid compound according to any one of claims 1 to 9, or an acceptable salt, stereoisomer thereof.
In yet another aspect, the application provides a pharmaceutical composition comprising a lipid compound or nanoparticle composition as described above, and optionally a pharmaceutically acceptable excipient.
In yet another aspect, the application provides a method of producing a polypeptide of interest in a cell of a subject, the method comprising contacting the cell with a nanoparticle composition comprising an mRNA encoding the polypeptide of interest as described above, whereby the mRNA is capable of translation in the cell to produce the polypeptide of interest.
Drawings
FIG. 1 shows luciferase protein expression levels after lipid nanoparticles prepared from different lipid compounds are delivered to B16F10 cells and HSKMC cells.
FIG. 2 shows the fluorescence intensity of lipid nanoparticles prepared from different lipid compounds, injected intramuscularly into mice at the injection site and liver site after 12 hours.
Detailed Description
Definition of terms
The term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising one or more carbon atoms.
The term "alkoxy" refers to an alkyl group as described herein that is attached to the remainder of the molecule through an oxygen atom.
The term "alkylene" refers to a divalent group formed by the corresponding alkyl group losing one hydrogen atom.
The term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms and at least one double bond. Alkenyl groups may include one, two, three, four or more carbon-carbon double bonds.
The term "aryl" refers to an all-carbon monocyclic or fused-polycyclic aromatic ring radical having a conjugated pi-electron system.
The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C and having at least one aromatic ring. Heteroaryl groups may have 5 to 10 ring atoms (5 to 10 membered heteroaryl groups) including 5, 6, 7, 8, 9 or 10 membered, especially 5 or 6 membered heteroaryl.
The term "pharmaceutically acceptable salt" refers to a derivative of the disclosed compounds wherein the parent compound is altered by converting the existing acid or base moiety to its salt form (e.g., by reacting a free basic group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; acidic residues such as alkali metal or organic salts of carboxylic acids, and the like. Representative acid addition salts include, but are not limited to, acetates, adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulphates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptonates, glycerophosphate, hemisulfates, heptanoates, caprates, hydrobromides, hydrochlorides, hydroiodides, 2-hydroxy-ethane sulfonates, lactobionic, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalene sulfonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectates, persulfates, 3-phenylpropionates, phosphates, bitrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates, undecanoates, valerates, and the like. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like; and non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethyl ammonium, tetraethyl ammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
The term "RNA" refers to ribonucleic acids that may be naturally occurring or non-naturally occurring. The RNA may be selected from: small interfering RNAs (siRNA), asymmetric interfering RNAs (aiRNA), micrornas (miRNA), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shRNA), mRNA, single-stranded guide RNAs (sgRNA), cas9 mRNA and mixtures thereof.
The term "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component may include one or more cationic/ionizable lipids, pegylated lipids, structural lipids, or other lipids, such as phospholipids.
The term "polydispersity index" or "PDI" is a ratio describing the homogeneity of the particle size distribution of a system.
The term "particle size" refers to the average diameter of the nanoparticle composition.
The term "zeta potential" refers to, for example, the surface potential of a lipid in a nanoparticle composition.
The term "encapsulation efficiency" refers to the ratio of the amount of therapeutic or prophylactic agent that becomes part of the nanoparticle composition to the initial total amount of therapeutic or prophylactic agent used in preparing the nanoparticle composition.
The term "delivering" refers to providing an entity to a target. For example, delivering a therapeutic or prophylactic agent to a subject may involve administering a nanoparticle composition comprising the therapeutic or prophylactic agent to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous route). Administration of a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.
The term "target cell" refers to a cell or group of target cells. These cells may be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism may be an animal, preferably a mammal.
The term "expression" refers to translation of mRNA into and/or from a polypeptide or protein.
The term "subject" refers to a target subject intended to be subjected to a treatment, including, but not limited to, humans, other primates, and other mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, or rats. Preferably, the subject may be a mammal, in particular a human.
Lipid compounds
In one aspect, the present application provides a cationic lipid compound having a structure as shown in formula (I):
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein,
L 1 ,L 2 ,L 3 ,L 4 ,L 5 identical to or different from each other and each independently selected from: absence of C 1 -C 24 Straight-chain or branched alkyl, C 2 -C 24 Alkenylene or branched alkenyl;
M 1 and M 2 Each independently selected from-OC (O) -, -C (O) O-, or-nhc=n (CN) NH-;
G 1 and G 2 Each independently selected from H, C 5 -C 10 Aryl or 5-to 10-membered heteroaryl, and C 5 -C 10 The hydrogen atoms on the aryl or 5-to 10-membered heteroaryl groups may each independently optionally be replaced by n-XR 1 Substituted, n is an integer from 1 to 3, X is absent, nitrogen, oxygen, sulfur, selenium, R 1 Is C 4 -C 20 Straight-chain or branched alkyl, C 4 -C 20 Straight or branched alkenyl groups.
In one embodiment, L 1 Selected from C 2 -C 5 Alkyl, L 2 ,L 3 Each independently selected from C 4 -C 9 Straight chain alkyl, L 4 ,L 5 Each independently selected from C 4 -C 20 Linear or branched alkyl.
In one embodiment, G 1 And G 2 Is each independently selected from H, or C 5 -C 10 Aryl groups. Wherein C is 5 -C 10 The hydrogen atoms on the aryl groups may each independently optionally be replaced by n-XR 1 Substituted, n is an integer from 1 to 2, X is nitrogen, oxygen, R 3 Is C 4 -C 20 Linear or branched alkyl;
in certain embodiments, formula (I) comprises the structure of formula (II):
wherein L is 1 ,L 2 ,L 3 ,L 4 ,L 5 ，M 1 ，M 2 As defined herein;
in one embodiment, L 1 Is C 2 -C 3 An alkyl group;
L 2 ,L 3 each independently selected from C 4 -C 9 A linear alkyl group;
L 4 ,L 5 each independently selected from C 4 -C 20 Linear or branched alkyl;
M 1 ，M 2 each independently selected from-OC (O) -, -C (O) O-, -nhc=n (CN) NH-; in certain embodiments, formula (I) comprises the structure of formula (III):
wherein L is 1 ,L 2 ,L 3 ,L 4 ,L 5 ，M 1 ，M 2 As defined herein;
in one embodiment, L 1 Is C 2 -C 3 An alkyl group;
L 2 ,L 3 each independently selected from C 4 -C 9 A linear alkyl group;
L 4 ,L 5 each independently selected from C 4 -C 20 Linear or branched alkyl;
M 1 ，M 2 each independently selected from-OC (O) -, -C (O) O-, -nhc=n (CN) NH-; r is R 2 Is C 4 -C 20 Straight chain alkyl and alkoxy or branched alkyl and alkoxy;
in certain embodiments, formula (I) comprises the structure of formula (IV):
wherein L is 1 ,L 2 ,L 3 ,L 4 ,L 5 ，M 1 ，M 2 As defined herein;
in one embodiment, L 1 Is C 2 -C 3 An alkyl group;
L 2 ,L 3 each independently selected from C 4 -C 9 A linear alkyl group;
L 4 ,L 5 each independently selected from C 4 -C 20 Linear or branched alkyl;
M 1 ，M 2 each independently selected from-OC (O) -, -C (O) O-, -nhc=n (CN) NH-; r is R 3 And R is 4 Is C 4 -C 20 Straight chain alkyl and alkoxy or branched alkyl and alkoxy; in a specific embodiment, the lipid compounds of the present application comprise:
or a pharmaceutically acceptable salt, prodrug, stereoisomer thereof.
The lipid compounds of the present application, including lipid compounds of formula (I), (II), (III), (IV), are ionizable cationic compounds in which the tertiary amine moiety can be protonated at less than physiological pH. The lipid is also a zwitterionic compound. Such zwitterionic forms, whether charged or not, are encompassed within the scope of the present application.
Nanoparticle compositions
The present application relates to a lipid nanoparticle comprising a lipid compound of the application, and may further comprise one or more other lipids.
Cationic lipids
Nanoparticle compositions may comprise one or more cationic and/or ionizable lipids in addition to the lipids of the application (e.g., the lipids of formulas (I), (II), (III), (IV)). Including but not limited to DLinDMA, DODMA, DOTAP, DOTMA, DLin-KC2-DMA, DC-Chol, etc
Anionic lipids
The lipid component of the nanoparticle composition may also comprise one or more anionic lipids. Including but not limited to phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, DOPG, DOPS, and the like
PEG lipid
The lipid component of the nanoparticle composition may also comprise one or more PEG or PEG-modified lipids. Including but not limited to PEG lipids can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids.
Structured lipids
The lipid component of the nanoparticle composition may also include one or more structural lipids. Structural lipids may include, but are not limited to: cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassinosteroids, lycorine, lycopersicin, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol.
Phospholipid
The lipid component of the nanoparticle composition may also include one or more phospholipids, which may include, but are not limited to: 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1, 2-dimyristoyl-sn-glycero-phosphatidylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (DUPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-dioleoyl-sn-3-phosphatidylcholine (zides-PC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine (pov), 1-dioleoyl-sn-3-phosphatidylcholine (POPC), 1, 2-dioleoyl-glycero-3-phosphatidylcholine (poyl-2-oleoyl-phosphatidylcholine (POPC), 1-2-dioleoyl-2-oleoyl-glycero-3-phosphatidylcholine (poe-2-oleoyl-2-phosphatidylcholine (18), 1, 2-bis-docosahexaenoic acid-sn-glycerol-3-phosphatidylcholine, 1, 2-bis-phytanic acid-sn-glycerol-3-phosphatidylethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycerol-3-phosphatidylethanolamine, 1, 2-di-linoleoyl-sn-glycerol-3-phosphatidylethanolamine, 1, 2-di-linolenoyl-sn-glycerol-3-phosphatidylethanolamine, 1, 2-di-arachidonoyl-sn-glycerol-3-phosphatidylethanolamine, 1, 2-bis-docosahexaenoic acid-sn-glycerol-3-phosphatidylethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phosphoric acid-rac- (1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl Phosphatidylethanolamine (PE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DMPE), stearoyl phosphatidylethanolamine (PSOtidyl phosphatidylethanolamine, phosphatidylethanolamine (PE), stearoyl phosphatidylethanolamine (PSOtidyl phosphatidylethanolamine), phosphatidylethanolamine (PC), phosphatidylcholine (SOtidyl phosphatidylethanolamine (PSOtidyl), phosphatidylethanolamine (PSOtidyl) and (PSOtidyl), lysophosphatidylethanolamine (LPE) and mixtures thereof. In some embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.
Effects of the application
The application provides a series of cationic lipid compounds with novel structures, which are combined with other lipid compounds to prepare a lipid carrier, wherein the particle size is controllable, the distribution is uniform, and the cationic lipid compounds have excellent encapsulation efficiency and expression efficiency on medicines with negative charges. Furthermore, the compounds of the present application of a specific structure may be selected depending on the organ to which the drug is desired. The effect of the method is obviously better than that of the prior art, and the method has wide and excellent application prospect.
Examples
The following abbreviations are used herein:
THF tetrahydrofuran
MeCN acetonitrile
LAH lithium aluminum hydride
DCM: dichloromethane
DMAP 4-dimethylaminopyridine
LDA lithium diisopropylamide
rt, room temperature
DME 1, 2-Dimethoxyethane
n-BuLi n-butyllithium
CPME cyclopentyl methyl ether
EDCI N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide
DIEA N, N-diisopropylethylamine
PE Petroleum ether
EA ethyl acetate
Example 1: synthesis of Compound CY-1
Synthesis of intermediates 1-3
To a solution of compound 1-1 (5.00 g,9.24mmol,1.00 eq.) in isopropanol (25 mL) under nitrogen protection was added compound 1-2 (4.20 g,12.0mmol,1.30 eq.) and sodium carbonate (2.94 g,27.7mmol,3.00 eq.) and stirred at 80-85℃for 20 hours. The reaction mixture was concentrated under reduced pressure to remove isopropanol, and the residue was diluted with 200mL of water and extracted with ethyl acetate (200 ml×3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2, petroleum ether/ethyl acetate=1/0-0/1) to give 6.11g of compound 1-3 as a yellow oil.
1 H NMR:(400MHz,CDCl 3 )δ5.03-4.99`(m,1H),4.90-4.84`(m,1H),4.06(t,J＝6.4Hz,2H),3.16-3.06(m,2H),2.51-2.40(m,5H),2.29(td,J1＝7.6,J2＝5.2Hz,5H),1.62(t,J＝6.8Hz,6H),1.55-1.48(m,5H),1.45(s,11H),1.31-1.26(m,49H),0.90-0.86(m,9H).
ESI-LCMS:m/z,[M+H] + ＝809.9.
Synthesis of intermediates 1-4
To a solution of compounds 1-3 (5.50 g,6.80mmol,1.00 eq.) in dichloromethane (15 mL) was added hydrochloric acid/ethyl acetate (4.00 m,17.0mL,10.0 eq.). The mixture was stirred at 20-30℃for 0.5 h. LCMS showed complete consumption of compounds 1-3. The reaction mixture was diluted with 40mL of water and adjusted to pH7-8 with saturated sodium bicarbonate. The mixture was extracted with dichloromethane (20 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue, which yielded 4.78g of the compound as a yellow oil.
1 H NMR(400MHz,CDCl 3 )δ4.90-4.83(m,1H),4.06(t,J＝6.8Hz,2H),2.77(t,J＝6.0Hz,2H),2.51(t,J＝6.0Hz,4H),2.42(t,J＝7.6Hz,4H),2.29-2.27`(m,4H),1.62(t,J＝6.8Hz,6H),1.51-1.50(m,4H),1.45 -1.40(m,4H),1.31-1.26(m,48H),0.90-0.86(m,9H).
ESI-LCMS:m/z,[M+H] + ＝709.8.
Synthesis of Compound CY-1
N, N-carbonyldiimidazole (1.42 g,8.76mmol,1.30 eq) was added dropwise to a solution of compounds 1 to 4 (4.78 g,6.74mmol,1 eq) in tetrahydrofuran (20 mL) at 20-30 ℃. After the addition, the mixture was stirred at 20-30 ℃ for 1 hour, then N, N-diisopropylethylamine (1.74 g,13.48mmol,2.35ml,2.00 eq.) and hydroxylamine hydrochloride (562.06 mg,8.09mmol,1.20 eq.) were added dropwise. The resulting mixture was stirred at 20-30℃for 20 hours. LCMS showed complete consumption of compounds 1-4. The reaction mixture was diluted with 30mL of water and extracted with ethyl acetate (20 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2, petroleum ether/ethyl acetate=1/0-0/1) to give 2.25g of compound CY-1 as a yellow oil.
1 H NMR(400MHz,CDCl 3 )δ6.65(s,1H),6.51(s,1H),4.90-4.84`(m,1H),4.06(t,J＝6.8Hz,2H),3.36-3.32`(m,2H),2.57(t,J＝5.6Hz,2H),2.43(t,J＝6.0Hz,4H),2.42(t,J＝7.2Hz,4H),2.32-2.27`(m,4H),1.62-1.61(m,6H),1.51-1.50(m,4H),1.45 -1.43(m,4H),1.31-1.26(m,48H),0.90-0.86(m,9H).
ESI-LCMS:m/z,[M+H] + ＝768.8.
Example 2: synthesis of lipid Compound CY-2
Synthesis of intermediate 2-2
A mixture containing the compound 2-1 (18.0 g,89.5mmol,12.5mL,1.00 eq.) n-hexyne (22.1 g,268.58mmol,30.2mL,3.00 eq.), cuprous iodide (3.41 g,17.9mmol,0.20 eq.), dichlorobis (triphenylphosphine palladium) (12.6 g,17.9mmol,0.20 eq.) and triethylamine (400 mL) was protected with nitrogen, and the mixture was stirred at 80-90℃for 32 hours. TLC showed complete consumption of compound 2-1. The reaction mixture was filtered and concentrated under reduced pressure to remove triethylamine. The residue was diluted with 300mL of water and extracted with ethyl acetate (100 mL. Times.3). The combined organic layers were washed with water (50 ml×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2, petroleum ether/ethyl acetate=1/0-0/1) to give 11.2g of compound 2-2 as a yellow oil.
1 H NMR(400MHz,CDCl 3 )δ7.35(d,J＝7.6Hz,2H),7.15(d,J＝7.2Hz,2H),3.82(t,J＝6.4Hz,2H),2.84(t,J＝6.4Hz,2H),2.41(t,J＝6.4Hz,2H),1.62-1.46(m,5H),0.96(t,J＝6.8Hz,3H).
Synthesis of intermediate 2-3
To a solution of compound 2-2 (11.2 g,55.4mmol,1.00 eq.) in methanol (50 mL) under protection of N2 was added palladium on carbon catalyst (5.60 g,10% purity). The suspension was degassed and purged three times with hydrogen. The mixture was stirred under hydrogen (50 Psi) at 60-70℃for 24 hours. TLC showed complete consumption of reactant 2-2. The mixture was filtered and concentrated under reduced pressure to give a residue, 10.4g of compound 2-3 were obtained as a yellow oil.
1 H NMR(400MHz,CDCl 3 )δ7.14(s,4H),3.86(t,J＝6.4Hz,2H),2.85(t,J＝6.8Hz,2H),2.63-2.57(m,2H),1.63-1.57(m,2H),1.36-1.31(m,6H),0.90(t,J＝6.8Hz,3H).
Synthesis of intermediate 2-4
To a solution of compound 2-3 (10.4 g,50.2mmol,1.00 eq.) and 8-bromooctanoic acid (12.3 g,55.2mmol,1.10 eq.) in dichloromethane (50 mL.) were added 4-dimethylaminopyridine (313 mg,5.02mmol,0.10 eq.) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (10.6 g,55.2mmol,1.10 eq.). The mixture was stirred at 20-30℃for 16 hours. TLC showed complete consumption of compounds 2-3 and concentration of the mixture under reduced pressure gave a residue. The residue was purified by column chromatography (SiO 2, petroleum ether/ethyl acetate=1/0-0/1) to give 15.6g of compound 2-4 as a yellow oil.
1 H NMR(400MHz,CDCl 3 )δ7.13(s,4H),4.29(t,J＝7.2Hz,2H),3.41(t,J＝6.8Hz,2H),2.92(t,J＝6.8Hz,2H),2.59(t,J＝7.6Hz,2H),2.30(t,J＝7.2Hz,2H),1.88-1.84(m,2H),1.63-1.61(m,4H),1.48-1.40(m,2H),1.38-1.32(m,10H),0.90(t,J＝6.4Hz,3H).
Synthesis of intermediate 2-5
To a solution of compound 2-4 (15.6 g,37.9mmol,1.00 eq.) in isopropanol (75 mL) was added 1-1 (22.6 g,41.7mmol,1.10 eq.) sodium carbonate (12.1 g,0.114mol,3.00 eq.) and the mixture was stirred under nitrogen for 12 hours at 80-85 ℃. LCMS showed that compounds 2-4 remained and the reaction mixture was concentrated under reduced pressure to remove isopropanol. The residue was diluted with 200mL of water and extracted with ethyl acetate (200 ml×3), and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to column chromatography (SiO 2, petroleum ether/ethyl acetate=1/0-/1) to obtain 18.1g of compound 2-5 as a yellow oil.
1 H NMR(400MHz,CDCl 3 )δ7.12(s,4H),4.98(s,1H),4.90-4.84(m,1H),4.27(t,J＝7.2Hz,2H),3.15-3.14(m,2H),2.90(t,J＝7.2Hz,2H),2.58(t,J＝7.6Hz,2H),2.49(t,J＝5.6Hz,2H),2.38(t,J＝6.8Hz,4H),2.30-2.29(m,4H),1.61-1.60(m,7H),1.63-1.61(m,4H),1.52-1.50(m,5H),1.45(s,9H),1.39-1.38(m,5H),1.30-1.26(m,41H),0.88(t,J＝6.4Hz,9H).
ESI-LCMS:m/z,[M+H] + ＝871.9
Synthesis of intermediate 2-6
To a solution of compound 2-5 (18.1 g,20.8mmol,1.00 eq.) in dichloromethane (60 mL) was added hydrochloric acid/ethyl acetate (4.00 m,30mL,5.77 eq.). The mixture was stirred at 20-30℃for 1 hour. LCMS showed complete consumption of compounds 2-6. The reaction mixture was diluted with 20mL of water and adjusted to pH7-8 with saturated sodium bicarbonate. The mixture was extracted with dichloromethane (20 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. 15.5g of compound 2-6 are obtained as a yellow oil.
1H NMR:EW43818-10-P1B(400MHz,CDCl3)δ7.12(s,4H),4.89-4.82(m,1H),4.26(t,J＝7.2Hz,2H),3.47(s,4H),2.96-2.88(m,6H),2.57(t,J＝7.6Hz,2H),2.31-2.26(m,4H),1.72(s,4H),1.61-1.60(m,6H),1.51-1.50(m,4H),1.35-1.31(m,13H),1.31-1.30(m,7H),1.30-1.26(m,22H),0.88(t,J＝6.4Hz,9H).
ESI-LCMS:m/z,[M+H]+＝771.7.
Synthesis of Compound CY-2
N, N-carbonyldiimidazole (2.73 g,16.9mmol,1.30 eq.) was added dropwise to a solution of compounds 2-6 (10.0 g,12.9mmol,1.00 eq.) in tetrahydrofuran (50 mL) at 20-30 ℃. After the addition, the mixture was stirred at 20-30 ℃ for 1 hour, then N, N-diisopropylethylamine (3.35 g,25.9mmol,4.52ml,2.00 eq.) and hydroxylamine hydrochloride (1.08 g,15.6mmol,1.20 eq.) were added. The resulting mixture was stirred at 20-30℃for 12 hours. LCMS showed complete consumption of compounds 2-6. The reaction mixture was diluted with 50mL of water and extracted with ethyl acetate (50 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2, petroleum ether/ethyl acetate=1/0-0/1) to give 6.37g of compound CY-3 as a yellow oil.
1 H NMR(400MHz,CDCl 3 )δ7.12(s,4H),6.65(s,1H),6.52(s,1H),4.90-4.84(m,1H),4.27(t,J＝7.2Hz,2H),3.36(d,J＝4.8Hz,2H),2.90(t,J＝7.2Hz,2H),2.60-2.58(m,4H),2.46(t,J＝7.2Hz,4H),2.32-2.28(m,4H),1.61-1.58(m,6H),1.52-1.50(m,4H),1.50-1.45(m,4H),1.30-1.26(m,42H),0.88(t,J＝6.4Hz,9H).
ESI-LCMS:m/z,[M+H] + ＝830.7.
Example 3: synthesis of Compounds CY-3 to CY-6
The synthesis process of CY-3 to CY-6 is consistent with CY-1/2, and only relevant intermediates need to be replaced.
Example 4: preparation of lipid nanoparticles
Preparation of lipid solution: the molar ratio is 50:10: 38.5:1.5 ionizable lipids: DSPC: cholesterol: mPEG2000-DMG in ethanol solution;
mRNA solution preparation: dissolving a certain mass of luciferase mRNA in 20mM citrate buffer solution at ph=4.0;
preparation of lipid nanoparticles: respectively sucking 1mL of luciferase mRNA and 3mL of lipid solution by using a syringe, inserting the luciferase mRNA and the 3mL of lipid solution into a microfluidic chip, and setting parameters as follows: volume:4.0mL; flowrate ratio 3:1,Total flow rate: mixing at 18mL/min to obtain lipid nanoparticle solution;
solution replacement: adding the lipid nanoparticle solution into an ultrafiltration tube for centrifugal ultrafiltration, and replacing the phosphate buffer solution for multiple times to obtain a finished product.
Example 5: characterization of lipid nanoparticles
The particle size, polydispersity index (PDI) and zeta potential of the nanoparticle composition can be determined using Zetasizer Nano ZS (Malvern Instruments Ltd, malvern, worcestershire, UK), the particle size being determined in 1 x PBS and the zeta potential being determined in 15mM PBS. For nanoparticle compositions containing RNA, QUANT-IT can be used TM RNA assay (Invitrogen Corporation Carlsbad, CA) the encapsulation of RNA by nanoparticle compositions was evaluated. The samples were diluted to a concentration of about 5. Mu.g/mL in TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 7.5). mu.L of diluted samples were transferred to polystyrene 96-well plates and 50. Mu.L of TE buffer or 50. Mu.L of 2% Triton X-100 solution was added to each well. The plates were incubated at 37℃for 15 minutes. The RIBOGREENR reagent is diluted 1:100 in TE buffer and 100. Mu.L of the solution is added to each well. Fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilabel Counter;Perkin Elmer,Waltham,MA) at an excitation wavelength of, for example, about 480nm and an emission wavelength of, for example, about 520 nm. The fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample and the percentage of free RNA was determined by dividing the fluorescence intensity of the complete sample (without Triton X-100 addition) by the fluorescence value of the destroyed sample (caused by Triton X-100 addition).
TABLE 1 physicochemical characterization of lipid nanoparticles prepared from lipid compounds synthesized in the examples of the present application
Sequence number Cationic lipids Size (nm) PDI Potential (mV) Encapsulation efficiency (%)
1 MC3 91±1.1 0.13±0.02 -0.71±0.13 89.2
2 CY-1 109±0.1 0.05±0.01 21.1±0.91 92.2
3 CY-2 84±0.9 0.17±0.01 21.5±1.32 95.0
4 CY-3 83±0.3 0.14±0.01 20.0±1.13 97.2
5 CY-4 87±0.2 0.15±0.01 17.8±0.42 90.2
6 CY-5 73±1. 0.19±0.03 13.1±0.71 93.2
7 CY-6 87±0.06 0.13±0.02 13.8±0.42 95.5
Example 6: in vitro cell expression level detection
In vitro cell expression evaluation method: B16F10 cells and HSKMC cells were cultured and plated in 96-well plates with 100000 cells per well. 100ng of LNP sample was taken and mixed with cell incubation for 24 hours (n=3), and protein expression was measured 24 hours after administration using a luciferase quantification kit. The positive control of the application is commercial cationic lipid MC3, and the negative control is PBS. As can be seen in fig. 1, the lipid nanoparticles prepared from each compound had a certain degree of in vitro cellular protein expression.
Example 7: in vivo protein expression studies in mice
After the lipid nanoparticles are introduced into the mice by intravenous injection, intramuscular injection, etc., the time course of evaluating protein expression can be measured by enzyme-linked immunosorbent assay (ELISA), bioluminescence imaging, or other methods. The delivery performance of the lipid molecules can be evaluated by comparing the amount of translated protein with a positive control. Specifically, a sample of each lipid nanoparticle containing 3. Mu.g of luciferase mRNA was taken, and Balb/C mice were intramuscular injected, and at 6 hours after the administration, 0.15mg/kg of D-luciferin substrate was intraperitoneally injected, and within 15 minutes after the substrate injection, the samples were placed under a small animal living body imager to perform fluorescence intensity detection at the liver site and the injection site. As shown in fig. 2, all lipid nanoparticles were able to observe a distinct fluorescent signal, with many compounds being stronger than the commercially available MC3 lipid fluorescent signal.
The embodiments are described above in order to facilitate the understanding and application of the present application by those of ordinary skill in the art. It will be apparent to those skilled in the art that various modifications can be made to these embodiments and that the general principles described herein may be applied to other embodiments without the use of inventive faculty. Accordingly, the present application is not limited to the embodiments herein, and those skilled in the art, based on the present disclosure, make improvements and modifications within the scope and spirit of the application.

Claims (9)

1. A lipid compound, characterized in that the lipid compound is a cationic lipid compound having a structure as shown in formula (I):
wherein,,
L 1 ,L 2 ,L 3 ,L 4 ,L 5 identical to or different from each other and each independently selected from: absence of C 1 -C 24 Straight-chain or branched alkyl, C 2 -C 24 Alkenylene or branched alkenyl;
M 1 and M 2 Each independently selected from-OC (O) -, -C (O) O-, or-nhc=n (CN) NH-;
G 1 and G 2 Each independently selected from H, C 5 -C 10 Aryl or 5-to 10-membered heteroaryl, and C 5 -C 10 The hydrogen atoms on the aryl or 5-to 10-membered heteroaryl groups may each independently optionally be replaced by n-XR 1 Substituted, n is an integer from 1 to 3, X is absent, nitrogen, oxygen, sulfur, selenium, R 1 Is C 4 -C 20 Straight-chain or branched alkyl, C 4 -C 20 Straight or branched alkenyl groups.
2. The lipid compound according to claim 1, wherein L 1 Is C 2 -C 5 Alkyl, L 2 ,L 3 Each independently selected from C 4 -C 9 Straight chain alkyl, L 4 ,L 5 Each independently selected from C 4 -C 20 Linear or branched alkyl.
3. The lipid compound according to claim 1 or 2, wherein G 1 And G 2 Is each independently selected from H, or C 5 -C 10 Aryl groups. Wherein C is 5 -C 10 The hydrogen atoms on the aryl groups may each independently optionally be replaced by n-XR 1 Substituted, n is an integer from 1 to 2, X is nitrogen, oxygen, R 1 Is C 4 -C 20 Linear or branched alkyl.
4. A lipid compound according to any one of claims 1 to 3, characterized in that it has the structure according to formula (II):
L 1 is C 2 -C 3 An alkyl group;
L 2 ,L 3 each independently selected from C 4 -C 9 A linear alkyl group;
L 4 ,L 5 each independently selected from C 4 -C 20 Linear or branched alkyl;
M 1 ，M 2 each independently selected from the group consisting of-OC (O) -, -C (O)O-，-NHC＝N(CN)NH-。
5. A lipid compound according to any one of claims 1 to 3, characterized in that it has the structure according to formula (III):
L 1 is C 2 -C 3 An alkyl group;
L 2 ,L 3 each independently selected from C 4 -C 9 A linear alkyl group;
L 4 ,L 5 each independently selected from C 4 -C 20 Linear or branched alkyl;
M 1 ，M 2 each independently selected from-OC (O) -, -C (O) O-, -nhc=n (CN) NH-;
R 2 is C 4 -C 20 Straight chain alkyl and alkoxy or branched alkyl and alkoxy.
6. A lipid compound according to any one of claims 1 to 3, characterized in that it has the structure according to formula (IV):
L 1 is C 2 -C 3 An alkyl group;
L 2 ,L 3 each independently selected from C 4 -C 9 A linear alkyl group;
L 4 ,L 5 each independently selected from C 4 -C 20 Linear or branched alkyl;
M 1 ，M 2 each independently selected from-OC (O) -, -C (O) O-, -nhc=n (CN) NH-;
R 3 is C 4 -C 20 Straight chain alkyl and alkoxy or branched alkyl and alkoxy.
R 4 Is C 4 -C 20 Straight chain alkyl and alkoxy or branched alkyl and alkoxy.
7. The lipid compound according to claim 1, characterized in that it has the structure as follows:
8. a lipid nanoparticle comprising a lipid compound according to any one of claims 1-7.
9. The lipid nanoparticle of claim 8, wherein the lipid nanoparticle comprises a neutral lipid, a polyethylene glycol lipid, a sterol lipid, and one or more bioactive agents; preferably, the neutral lipid is one of DSPC, DOPE, DPPC or POPC, the polyethylene glycol lipid is PEG2000-DMG, the sterol lipid is cholesterol, and the bioactive agent is RNA, mRNA, siRNA, ASO, DNA, tRNA, rRNA, miRNA, plasmid or snRNA.
CN202310616858.XA 2023-05-29 2023-05-29 Lipid compound and lipid nanoparticle Pending CN116675624A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310616858.XA CN116675624A (en) 2023-05-29 2023-05-29 Lipid compound and lipid nanoparticle

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310616858.XA CN116675624A (en) 2023-05-29 2023-05-29 Lipid compound and lipid nanoparticle

Publications (1)

Publication Number Publication Date
CN116675624A true CN116675624A (en) 2023-09-01

Family

ID=87782963

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310616858.XA Pending CN116675624A (en) 2023-05-29 2023-05-29 Lipid compound and lipid nanoparticle

Country Status (1)

Country Link
CN (1) CN116675624A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110520409A (en) * 2017-03-15 2019-11-29 摩登纳特斯有限公司 Compound and composition for Intracellular delivery therapeutic agent
CN114746398A (en) * 2019-09-19 2022-07-12 摩登纳特斯有限公司 Headgroup lipid compounds and compositions for intracellular delivery of therapeutic agents
CN115557875A (en) * 2022-08-19 2023-01-03 上海耐澄生物科技有限公司 Lipid compound and lipid nanoparticles
US20230112857A1 (en) * 2020-01-10 2023-04-13 Modernatx, Inc. Methods of making tolerogenic dendritic cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110520409A (en) * 2017-03-15 2019-11-29 摩登纳特斯有限公司 Compound and composition for Intracellular delivery therapeutic agent
CN114746398A (en) * 2019-09-19 2022-07-12 摩登纳特斯有限公司 Headgroup lipid compounds and compositions for intracellular delivery of therapeutic agents
US20230112857A1 (en) * 2020-01-10 2023-04-13 Modernatx, Inc. Methods of making tolerogenic dendritic cells
CN115557875A (en) * 2022-08-19 2023-01-03 上海耐澄生物科技有限公司 Lipid compound and lipid nanoparticles

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU ZHAOXING ET AL: "Syntheses and biological evaluation of novel hydroxamic acid derivatives containing purine moiety as histone deacetylase inhibitors", 《CHEMICAL & PHARMACEUTICAL BULLETIN》, vol. 66, no. 4, 31 December 2018 (2018-12-31), pages 439 - 451 *

Similar Documents

Publication Publication Date Title
AU2020200576B2 (en) Biodegradable lipids for delivery of nucleic acids
EP3397614B1 (en) Ionizable cationic lipid
CN110741087B (en) Targeting composition
CA2980721C (en) Nucleic acid-containing lipid nanoparticles
JP6182457B2 (en) Lipid nanoparticles for drug delivery systems containing cationic lipids
JP5902616B2 (en) Cationic lipid
EP4282855A1 (en) Ionizable lipid molecule, preparation method therefor, and application thereof in preparation of lipid nanoparticle
US20240360072A1 (en) Cationic Lipid Compound and Composition for Delivery of Nucleic Acids and Use Thereof
CA2969664C (en) Cationic lipid
JP5902617B2 (en) Cationic lipid
WO2013089152A1 (en) Lipid nanoparticles containing combinations of cationic lipids
WO2013065825A1 (en) Cationic lipid
ES2948971T3 (en) Cationic lipid
EP3988537A1 (en) Biodegradable lipids for the delivery of active agents
TW201619137A (en) Cationic lipid
EP3153172A1 (en) Ckap5-gene-silencing rnai pharmaceutical composition
CN116675624A (en) Lipid compound and lipid nanoparticle
JP6495408B2 (en) Cationic lipid
CN117919199A (en) Application of blank lipid nanoparticles in preparation of in-vivo delivery products
CN118515706A (en) Phosphorylcholine structural compound and preparation method thereof
US20140294978A1 (en) Cationic lipid
CN117925729A (en) Transfection reagent based on blank lipid nano-particles, preparation method and application thereof
CN117024323A (en) Degradable and ionizable cationic lipid material and application thereof
CN117942406A (en) Use of preformed vectors for the preparation of products for in vitro gene delivery of immune cells and stem cells
CN118724740A (en) Amino lipids, lipid nanoparticles and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination