CN115772089A - Novel cationic lipid compounds - Google Patents

Novel cationic lipid compounds Download PDF

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Publication number
CN115772089A
CN115772089A CN202111043919.5A CN202111043919A CN115772089A CN 115772089 A CN115772089 A CN 115772089A CN 202111043919 A CN202111043919 A CN 202111043919A CN 115772089 A CN115772089 A CN 115772089A
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composition
combinations
compound
lipid
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黄才古
王帅
黄铁强
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Guangzhou Anovent Pharmaceutical Co Ltd
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Guangzhou Anovent Pharmaceutical Co Ltd
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Priority to PCT/CN2022/093196 priority patent/WO2023035652A1/en
Publication of CN115772089A publication Critical patent/CN115772089A/en
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    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
    • C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/62—Halogen-containing esters
    • C07C69/63—Halogen-containing esters of saturated acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Abstract

The present invention relates to lipid compounds that can be used alone or in combination with other lipid components, such as neutral lipids, dotted lipids, steroids and/or their analogs, and/or polymer-conjugated lipids, to form lipid nanoparticles for the delivery of therapeutic and/or prophylactic agents. In some examples, lipid nanoparticles are used to deliver nucleic acids, such as messenger RNA and/or anti-RNASense RNA. Also provided are methods of using such lipid nanoparticles for the treatment and/or prevention of various diseases. In one embodiment, compounds having the structure of formula (I) below are provided:
Figure DDA0003250509990000011
or a salt or isomer thereof or an N-oxide thereof, wherein R 1 、R 2 、R 3 、R 4 And R 5 As defined herein. Also provided are pharmaceutical compositions comprising one or more compounds of the foregoing structural formula (I) and a therapeutic and/or prophylactic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from the group consisting of neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions are useful for forming lipid nanoparticles for the delivery of therapeutic and/or prophylactic agents. In other embodiments, the present invention provides methods of administering a therapeutic and/or prophylactic agent to a subject in need thereof, comprising preparing a pharmaceutical combination comprising a lipid nanoparticle of a compound of structural formula (I) and a therapeutic and/or prophylactic agent, and delivering the composition to the subject.

Description

Novel cationic lipid compounds
Technical Field
The present invention provides novel cationic lipids that can be used in combination with other lipid components (such as neutral lipids, steroids, and polymer-conjugated lipids) to form a nucleic acid mRNA lipid nanoparticle composition for delivering one or more therapeutic and/or prophylactic agents to mammalian cells or organs and/or for producing polypeptides in mammalian cells or organs. In addition to the novel lipids, the lipid nanoparticle compositions of the present invention may include one or more cationic and/or ionizable amino lipids, neutral lipids including polyunsaturated lipids, polymer-conjugated lipids, steroids, and/or therapeutic and/or prophylactic agents in specific proportions.
Background
Effective targeted delivery of biologically active substances such as small molecule drugs, proteins and nucleic acids presents a long-standing medical challenge. In particular, delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of these species. Accordingly, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.
Studies have shown that bioactive substances such as small molecule drugs, proteins and nucleic acids can be efficiently delivered to cells and/or intracellular compartments using lipid-containing nanoparticle compositions, liposomes and liposome complexes as delivery vehicles. These compositions generally comprise one or more "cationic" lipids, neutral lipids (e.g., phospholipids), structural lipids (e.g., steroids), and/or polyethylene glycol-containing lipids (polymer-conjugated lipids) including polyunsaturated lipids. Cationic lipids include, for example, amine-containing lipids that can be readily protonated.
However, the use of oligonucleotides in a therapeutic setting is currently facing two problems. First, free RNA is readily digested in plasma by nucleases. Second, the ability of free RNA to enter intracellular compartments where relevant translation mechanisms exist is limited. Lipid nanoparticles formed from cationic lipids with other lipid components (such as neutral lipids, cholesterol, PEG, pegylated lipids, and oligonucleotides) have been used to prevent degradation of RNA in plasma and to promote cellular uptake of oligonucleotides.
There remains a need for improved cationic lipids and lipid nanoparticles for delivery of oligonucleotides. The improved lipid nanoparticles would provide optimized drug delivery, protect nucleic acids from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. In addition, these preferred lipid-nucleic acid particles should be well-tolerated and provide a sufficient therapeutic index such that patient treatment at an effective dose of the nucleic acid does not result in unacceptable toxicity and/or risk to the patient. The present invention provides these and related advantages.
Disclosure of Invention
The present invention provides the following novel compounds and methods involving these compounds:
in a first aspect, the present invention relates to compounds of the following structural formula (I):
Figure BDA0003250509980000011
or an N-oxide thereof, or a salt or isomer thereof.
Wherein R in the structural formula "I 1 、R 2 、R 3 、R 4 And R 5 Are all independent combinations of 2 "hydrogen" isotopes (including isotopes "protium" and "deuterium") and cannot be "hydrogen" at the same time;
R 1 has independence, and is represented by any one of H and D, but not R 2 、R 3 、R 4 And R 5 And is simultaneously "H";
R 2 and R 3 All have independence and can be expressed as HH and HD' or "DD", but not with R 1 、R 4 And R 5 Simultaneously "hydrogen" and "H", i.e., a combination comprising at least one "D";
R 4 and R 5 All have independence and can be expressed as 'HHH', 'HHD', 'HDD' or 'DDD', but not with R 1 、 R 2 And R 3 And is also "H", i.e., a combination containing at least one "D";
R 1 、R 2 、R 3 、R 4 and R 5 Each of which is a separate combination of 2 "hydrogen" isotopes (including the isotopes "protium" and "deuterium"), with particular combinations being divided into 11, including combinations comprising 1 "D", combinations comprising 2 "D", combinations comprising 3 "D", combinations comprising 4 "D", combinations comprising 5 "D", combinations comprising 6 "D", combinations comprising 7 "D", combinations comprising 8 "D", combinations comprising 9 "D", combinations comprising 10 "D" and combinations comprising 11 "D".
In various embodiments, the compound has one of the structures shown in table 1 below
Representative Compounds of Table 1
Figure BDA0003250509980000021
Figure BDA0003250509980000031
Figure BDA0003250509980000041
Figure BDA0003250509980000051
In some embodiments, compositions are provided comprising any one or more of the compounds of structural formula (I) and a therapeutic and/or prophylactic agent.
In some embodiments, compositions are provided comprising any one or more of the compounds of structure (I) and a therapeutic and/or prophylactic agent. In some embodiments, the composition comprises any one of the compounds of structure (I) and a therapeutic and/or prophylactic agent and one or more excipients selected from the group consisting of neutral lipids, steroids, and polymer-conjugated lipids. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the compositions.
In some embodiments, the neutral lipid is selected from one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM). In some embodiments, the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
In some embodiments, the steroid is selected from one or more of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol. In some embodiments, it is preferred that the steroid is cholesterol.
In some embodiments, the pegylated lipid is 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG)
In some embodiments, the composition ratio is in the following range: about 10-60 mol% of the compound, about 0-30 mol% of a neutral lipid, about 10-55 mol% of a steroid, and about 0-10 mol% of a polymer-conjugated lipid.
In some embodiments of the foregoing composition, the therapeutic and/or prophylactic agent comprises a nucleic acid. Wherein the nucleic acid is RNA selected from the group consisting of: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof. In some embodiments, the RNA is selected from mRNA.
In other various embodiments, the invention relates to a method of administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising preparing or providing any of the above compositions and administering the composition to the subject.
For use purposes, the compounds of the present invention may be used as drug substances or may be formulated as pharmaceutical compositions (typically in the form of lipid nanoparticles in combination with a therapeutic and/or prophylactic agent). The pharmaceutical compositions of the present invention comprise a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. A compound of structure (I) effective to form a lipid nanoparticle and deliver a therapeutic and/or prophylactic agent. Appropriate concentrations and dosages can be readily determined by those skilled in the art.
The use of the compositions of the present invention may be through any acceptable use of the agents for similar utility. The pharmaceutical composition of the present invention may be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of use of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated so that the active ingredients therein are bioavailable in the subject. The form of the composition to be administered to a subject or patient may be one or more dosage forms, wherein a tablet may be a single dosage unit, while a container of a compound of the invention in aerosol form may contain a plurality of dosage units. Current methods of preparing such dosage forms are known or will be apparent to those skilled in the art. In any event, the composition to be used will contain a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, in order to treat the relevant disease or condition in accordance with the teachings of the present invention.
The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier may be a particle, such that the composition is in the form of a tablet or powder. The carrier may also be a liquid, in which case the composition is an oral syrup or an injectable liquid or aerosol suitable for inhalation.
When intended for oral use, the pharmaceutical composition is preferably in solid or liquid form, wherein solid or liquid forms are considered herein to include semisolids, semi-liquids, suspensions, and gels.
As a solid composition for oral use, the pharmaceutical composition may be formulated into the form of powder, granules, tablets, pills, capsules, chewing gum, flakes, and the like. Such solid compositions will typically contain one or more inert diluents or edible carriers. Additionally, one or more of a binder, such as gelatin, cellulose, and the like; excipients, such as lactose and the like; disintegrating agents such as alginic acid and the like; lubricants, such as magnesium stearate and the like; glidants such as silicone gel and the like; sweetening agents, such as sucrose or saccharin; flavoring agents, such as peppermint and the like; and a colorant.
When the pharmaceutical composition is in the form of a capsule, it may contain a liquid carrier other than the above-mentioned types of materials, such as polyethylene glycol or oil.
The pharmaceutical composition may be in the form of a liquid, such as a syrup, solution, emulsion or suspension. As two examples, the liquid may be for oral use or for injection delivery. When intended for oral use, preferred compositions contain one or more of sweetening agents, preserving agents, coloring/colouring agents and taste-enhancing agents in addition to the compounds of the invention. In the composition for use by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.
The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar form, may include one or more of the following adjuvants, a sterile diluent such as water for injection, saline solution, preferably physiological saline, ringer's solution, isotonic sodium chloride; non-volatile oils such as synthetic monoglycerides or diglycerides which may be used as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as methyl paraben and the like; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for regulating tonicity, such as sodium chloride or dextrose; agents used as cryoprotectants, such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.
The pharmaceutical compositions of the invention may be for topical use, in which case the carrier may suitably comprise a solution base, an emulsion base, an ointment base or a gel base. The matrix may comprise one or more of: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. The thickening agent may be present in a pharmaceutical composition for topical use. If intended for transdermal use, the composition may comprise a transdermal patch or an iontophoretic device.
The pharmaceutical compositions of the present invention may include various materials that modify the physical form of the solid or liquid dosage form. The composition may include a material that forms a shell around the active ingredient. The material forming the coating is generally inert and may be sugar, shellac and other enteric coating agents. Or the active ingredient may be encapsulated in a gelatin capsule.
The pharmaceutical compositions of the present invention in solid or liquid form may include a delivery vehicle for the compound in association with the compound of the present invention. Such vectors include monoclonal or polyclonal antibodies or proteins.
The pharmaceutical composition of the present invention may consist of a formulation that can be used as an aerosol. The term aerosol denotes both systems comprising colloidal properties and systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system for dispersing the active ingredient. Aerosols of the compounds of the invention may be delivered as a single phase, biphasic system, or triphasic system for delivery of the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form a drug delivery device. One skilled in the art can determine the preferred aerosol without additional experimentation.
The pharmaceutical compositions of the present invention may be prepared by methods well known in the pharmaceutical art. The lipid nanoparticles of the present invention may be prepared by combining the lipid nanoparticles with sterile distilled water or other carriers into a solution for injection. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are non-covalently interacted by the compounds of the present invention to facilitate dissolution or uniform suspension of the compounds in an aqueous medium.
The compositions of the present invention, or pharmaceutically acceptable salts thereof, are used in therapeutically effective amounts, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent being used; metabolic stability and length of action of the therapeutic agent; the age, weight, general health, sex, and diet of the subject; the manner and time of use; the rate of excretion; a pharmaceutical composition; severity of the particular case, etc.
The compositions of the present invention may also be used simultaneously with, before or after the use of one or more other therapeutic agents. Such therapeutic combinations include formulations using the compositions of the present invention alone as well as combinations using the compositions of the present invention and one or more other active ingredients. For example, the compositions of the present invention and the other active ingredients may be administered to a subject together in a single oral dosage formulation (e.g., a tablet or capsule), or each active ingredient may be administered in a different oral dosage formulation. When different dosage formulations are employed, the compound of the invention and one or more additional active ingredients may be administered at the same time, or sequentially at staggered times; it is to be understood that combination therapy encompasses all of these dosing regimens.
The structure modification and design of the novel deuterated cationic lipid compound realize more advantageous physicochemical properties including more appropriate pKa and better chemical stability, and the novel deuterated cationic lipid compound is used for mRNA nanoliposome compositions, can realize more effective combination and delivery of ionic nucleic acid drugs, has more stable chemical structure, is convenient to synthesize and is beneficial to development as a pharmaceutical adjuvant.
Methods for preparing the above compounds and compositions are described below, and/or are known in the art.
One skilled in the art will recognize that in the methods described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, and carboxylic acid. Suitable protecting groups for hydroxyl include trialkylsilyl or diarylalkylsilyl groups, tetrahydrofuranyl, benzyl groups, and the like. Suitable protecting groups for amino groups include tert-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for carboxylic acids include hydroxy, aryl or arylalkyl esters. Protecting groups may be added or removed according to standard techniques known to those skilled in the art and described herein.
One skilled in the art will also recognize that while such protected derivatives of the compounds of the present invention may not be pharmaceutically active thereby, they may be administered to a mammal and thereafter metabolized in vivo to form the compounds of the present invention which are pharmacologically active. Such derivatives may therefore be described as "prodrugs". Prodrugs of the compounds of the invention are therefore included within the scope of the invention.
Furthermore, all compounds of the invention in free base or free acid form can be converted into their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid according to methods known to those skilled in the art. Salts of the compounds of the invention may be formed by conversion to the free base or acid thereof by standard techniques.
The following examples are provided for the purpose of illustration and not limitation.
The following examples, unless otherwise indicated, all solvents and reagents used were commercially available and used as received.
The procedure described below can be used to synthesize compound I in table 1.
The following abbreviations are used herein:
Figure BDA0003250509980000071
Detailed Description
Example 1:
representative routes
Synthesis of Compound 8
Figure BDA0003250509980000081
1) Synthesis of Compound 8-02
Chemical formula C 41 H 71 D 2 BrO 2
Molecular weight 679.95
To compound A (5.3g, 10.0mmol) and 4,4-d in that order 2 EDC.HCl (2.1g, 11.0 mmol), DIEA (5.3ml, 30.0 mmol) and DMAP (0.2g, 1.5 mmol) were added to a DCM mixture of bromobutyric acid (2.5g, 15.0 mmol). After 2 hours of reaction at 45 ℃, the system was diluted with DCM, washed with saturated aqueous sodium bicarbonate solution and dilute aqueous hydrochloric acid solution in this order, dried over magnesium sulfate, filtered and concentrated, and the residue was purified by silica gel column (0 to 15% ethyl acetate/n-hexane). Compound 8-02 (5.1g, 75%) was obtained.
2) Synthesis of Compound 8
Figure BDA0003250509980000082
The chemical formula is as follows: c 43 H 77 D 2 NO 2
Molecular weight: 644.12
To a mixture of compounds 8-02 (0.68g, 1.0 mmol) and dimethylamine (0.09g, 2.0 mmol) in DMF (5 mL) in that order was added K 2 CO 3 (0.27g, 2.0mmol) and potassium iodide (0.017g, 0.1mmol) are heated to 65 ℃, stirred for 24 hours, and the temperature of the system is reduced to room temperature after the raw materials are completely reacted. Ethyl acetate was added to the system, and the organic phase was washed with 20mL of water and 20mL of a saturated saline solution in this order. After drying over anhydrous sodium sulfate, it was concentrated under reduced pressure. The residue was passed through a silica gel column (0-100% (1% 4 OH, 20% MeOH in dichloromethane) to yield Compound 8 (0.58g, 90%). C 43 H 77 D 2 NO 2 ,Ms m/z:[M+H + ]644.6； 1 H-NMR(300MHz):δ5.4-5.27(m,8H), 4.47(m,1H),2.82～2.78(d,4H),2.32(t,2),2.26(s,6),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 2:
synthesis of Compound 1
Figure BDA0003250509980000091
The chemical formula is as follows: c 43 H 78 DNO 2
Molecular weight: 643.12
Compound 1 can be synthesized according to the representative route described in example 1.
C 43 H 78 DNO 2 ,Ms m/z:[M+H + ]643.6； 1 H-NMR(300MHz,CDCl 3 )δ: 1 H-NMR(300MHz): δ5.4-5.27(m,8H),3.04(t,2H),2.82～2.78(d,4H),2.47(t,2H),2.26(s,6H),2.16(m,8H),1.80(m, 2H),1.49(m,4H),1.33-1.26(m,36H),0.88(t,6H)。
Example 3:
synthesis of Compound 2
The chemical formula is as follows: c 43 H 78 DNO 2
Molecular weight: 643.12
Figure BDA0003250509980000092
Compound 2 can be synthesized according to the representative route described in example 1.
C 43 H 78 DNO 2 ,Ms m/z:[M+H + ]643.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 4.47(m,1H),3.04(t,2H),2.82～2.78(d,4H),2.47(t,1H),2.26(s,6H),2.16(m,8H),1.80(m,2H), 1.49(m,4H),1.33-1.26(m,36H),0.88(t,6H)。
Example 4:
synthesis of Compound 3
Figure BDA0003250509980000093
Compound 3 can be synthesized according to the representative route described in example 1.
C 43 H 78 DNO 2 ,Ms m/z:[M+H + ]643.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 4.47(m,1H),3.04(t,1H),2.82～2.78(d,4H),2.47(t,2H),2.26(s,6H),2.16(m,8H),1.80(m,2H), 1.49(m,4H),1.33-1.26(m,36H),0.88(t,6H)。
Example 5:
synthesis of Compound 4
Figure BDA0003250509980000094
C 43 H 78 DNO 2 ,Ms m/z:[M+H + ]643.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 4.47(m,1H),3.04(t,2H),2.82～2.78(d,4H),2.47(t,2H),2.25(s,5H),2.16(m,8H),1.80(m,2H), 1.49(m,4H),1.33-1.26(m,36H),0.88(t,6H)。
Example 6:
synthesis of Compound 5
The chemical formula is as follows: c 43 H 77 D 2 NO 2
Figure BDA0003250509980000101
Molecular weight: 644.12
Compound 5 can be synthesized according to the representative route described in example 1.
C 43 H 77 D 2 NO 2 ,Ms m/z:[M+H + ]644.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,2H),2.82～2.78(d,4H),2.47(t,1H),2.26(s,6H),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 7:
synthesis of Compound 6
The chemical formula is as follows: c 43 H 77 D 2 NO 2
Figure BDA0003250509980000102
Molecular weight: 644.12
Compound 6 can be synthesized according to the representative route described in example 1.
C 43 H 77 D 2 NO 2 ,Ms m/z:[M+H + ]644.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,1H),2.82～2.78(d,4H),2.47(t,2H),2.26(s,6H),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 8:
synthesis of Compound 7
The chemical formula is as follows: c 43 H 77 D 2 NO 2
Molecular weight: 644.12
Figure BDA0003250509980000103
Compound 7 can be synthesized according to the representative route described in example 1.
C 43 H 77 D 2 NO 2 ,Ms m/z:[M+H + ]644.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,2H),2.82～2.78(d,4H),2.47(t,2H),2.26(s,5H),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 9:
synthesis of Compound 9
Figure BDA0003250509980000111
The chemical formula is as follows: c 43 H 77 D 2 NO 2
Molecular weight: 644.12
Compound 9 can be synthesized according to the representative route described in example 1.
C 43 H 77 D 2 NO 2 ,Ms m/z:[M+H + ]644.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 4.47(m,1H),3.04(t,2H),2.82～2.78(d,4H),2.47(t,1H),2.26(s,5H),2.16(m,8H),1.80(m,2H), 1.49(m,4H),1.33-1.26(m,36H),0.88(t,6H)。
Example 10:
synthesis of Compound 10
Figure BDA0003250509980000112
The chemical formula is as follows: c 43 H 77 D 2 NO 2
Molecular weight: 644.12
Compound 10 can be synthesized according to the representative route described in example 1.
C 43 H 77 D 2 NO 2 ,Ms m/z:[M+H + ]644.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 4.47(m,1H),3.04(t,1H),2.82～2.78(d,4H),2.47(t,2H),2.26(s,5H),2.16(m,8H),1.80(m,2H), 1.49(m,4H),1.33-1.26(m,36H),0.88(t,6H)。
Example 11:
synthesis of Compound 11
The chemical formula is as follows: c 43 H 76 D 3 NO 2
Figure BDA0003250509980000113
Molecular weight: 645.12
Compound 11 can be synthesized according to the representative route described in example 1.
C 43 H 76 D 3 NO 2 ,Ms m/z:[M+H + ]645.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,1H),2.82～2.78(d,4H),2.47(t,2H),2.26(s,5H),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 12:
synthesis of Compound 12
Figure BDA0003250509980000114
The chemical formula is as follows: c 43 H 76 D 3 NO 2
Molecular weight: 645.12
Compound 12 can be synthesized according to the representative route described in example 1.
C 43 H 76 D 3 NO 2 ,Ms m/z:[M+H + ]645.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,2H),2.82～2.78(d,4H),2.47(t,2H),2.26(s,4H),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 13:
the chemical formula is as follows: c 43 H 76 D 3 NO 2
Figure BDA0003250509980000121
Molecular weight: 645.12
Compound 13 can be synthesized according to the representative route described in example 1.
C 43 H 76 D 3 NO 2 ,Ms m/z:[M+H + ]645.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 4.47(m,1H),3.04(t,1H),2.82～2.78(d,4H),2.26(s,6H),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 14:
synthesis of Compound 14
Figure BDA0003250509980000122
The chemical formula is as follows: c 43 H 75 D 4 NO 2
Molecular weight: 646.12
Compound 14 can be synthesized according to the representative route described in example 1.
C 43 H 75 D 4 NO 2 ,Ms m/z:[M+H + ]646.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,1H),2.82～2.78(d,4H),2.26(s,6H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m, 36H),0.88(t,6H)。
Example 15:
synthesis of Compound 15
Figure BDA0003250509980000123
The chemical formula is as follows: c 43 H 75 D 4 NO 2
Molecular weight: 646.12
Compound 15 can be synthesized according to the representative route described in example 1.
C 43 H 75 D 4 NO 2 ,Ms m/z:[M+H + ]646.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,2H),2.82～2.78(d,4H),2.26(s,5H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m, 36H),0.88(t,6H)。
Example 16:
synthesis of Compound 16
The chemical formula is as follows: c 43 H 75 D 4 NO 2
Figure BDA0003250509980000131
Molecular weight: 646.12
Compound 16 can be synthesized according to the representative route described in example 1.
C 43 H 75 D 4 NO 2 ,Ms m/z:[M+H + ]646.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.47(t,1H),2.26(s,6H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m, 36H),0.88(t,6H)。
Example 17:
synthesis of Compound 17
Figure BDA0003250509980000132
The chemical formula is as follows: c 43 H 75 D 4 NO 2
Molecular weight: 646.12
Compound 17 can be synthesized according to the representative route described in example 1.
C 43 H 75 D 4 NO 2 ,Ms m/z:[M+H + ]646.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,1H),2.82～2.78(d,4H),2.47(t,1H),2.26(s,5H),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 18:
Figure BDA0003250509980000133
synthesis of Compound 18
The chemical formula is as follows: c 43 H 74 D 5 NO 2
Molecular weight: 647.12
Compound 18 can be synthesized according to the representative route described in example 1.
C 43 H 74 D 5 NO 2 ,Ms m/z:[M+H + ]647.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.26(s,6H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88 (t,6H)。
Example 19:
synthesis of Compound 19
Figure BDA0003250509980000134
The chemical formula is as follows: c 43 H 74 D 5 NO 2
Molecular weight: 647.12
Compound 19 can be synthesized according to the representative route described in example 1.
C 43 H 74 D 5 NO 2 ,Ms m/z:[M+H + ]647.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,1H),2.82～2.78(d,4H),2.26(s,5H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m, 36H),0.88(t,6H)。
Example 20:
synthesis of Compound 20
Figure BDA0003250509980000141
The chemical formula is as follows: c 43 H 74 D 5 NO 2
Molecular weight: 647.12
Compound 20 can be synthesized according to the representative route described in example 1.
C 43 H 74 D 5 NO 2 ,Ms m/z:[M+H + ]647.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.47(t,1H),2.26(s,5H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m, 36H),0.88(t,6H)。
Example 21:
synthesis of Compound 21
Figure BDA0003250509980000142
The chemical formula is as follows: c 43 H 74 D 5 NO 2
Molecular weight: 647.12
Compound 21 can be synthesized according to the representative route described in example 1.
C 43 H 74 D 5 NO 2 ,Ms m/z:[M+H + ]647.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 3.04(t,2H),2.82～2.78(d,4H),2.47(t,1H),2.26(s,3H),2.16(m,8H),1.80(m,2H),1.49(m,4H), 1.33-1.26(m,36H),0.88(t,6H)。
Example 22:
synthesis of Compound 22
Figure BDA0003250509980000143
The chemical formula is as follows: c 43 H 73 D 6 NO 2
Molecular weight: 648.12
Compound 22 can be synthesized according to the representative route described in example 1.
C 43 H 73 D 6 NO 2 ,Ms m/z:[M+H + ]648.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.26(s,5H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88 (t,6H)。
Example 23:
synthesis of Compound 23
The chemical formula is as follows: c 43 H 72 D 7 NO 2
Figure BDA0003250509980000151
Molecular weight: 649.12
Compound 23 can be synthesized according to the representative route described in example 1.
C 43 H 72 D 7 NO 2 ,Ms m/z:[M+H + ]649.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.26(s,4H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88 (t,6H)。
Example 24:
synthesis of Compound 24
The chemical formula is as follows: c 43 H 71 D 8 NO 2
Figure BDA0003250509980000152
Molecular weight: 650.13
Compound 24 can be synthesized according to the representative route described in example 1.
C 43 H 71 D 8 NO 2 ,Ms m/z:[M+H + ]650.7； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.26(s,3H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88 (t,6H)。
Example 25:
synthesis of Compound 25
Figure BDA0003250509980000153
The chemical formula is as follows: c 43 H 70 D 9 NO 2
Molecular weight: 651.13
Compound 25 can be synthesized according to the representative route described in example 1.
C 43 H 70 D 9 NO 2 ,Ms m/z:[M+H + ]651.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.26(s,2H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88 (t,6H)。
Example 26:
synthesis of Compound 26
Figure BDA0003250509980000154
The chemical formula is as follows: c 43 H 69 D 10 NO 2
Molecular weight: 652.13
Compound 26 can be synthesized according to the representative route described in example 1.
C 43 H 69 D 10 NO 2 ,Ms m/z:[M+H + ]652.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.26(s,1H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88 (t,6H)。
Example 27:
synthesis of Compound 27
Figure BDA0003250509980000161
The chemical formula is as follows: c 43 H 69 D 10 NO 2
Molecular weight: 652.13
Compound 27 can be synthesized according to the representative route described in example 1.
C 43 H 69 D 10 NO 2 ,Ms m/z:[M+H + ]652.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 4.47(m,1H),2.82～2.78(d,4H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88 (t,6H)。
Example 28:
synthesis of Compound 28
Figure BDA0003250509980000162
The chemical formula is as follows: c 43 H 69 D 10 NO 2
Molecular weight: 652.13
Compound 28 can be synthesized according to the representative route described in example 1.
C 43 H 69 D 10 NO 2 ,Ms m/z:[M+H + ]652.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.46(t,1H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88(t, 6H)。
Example 29:
synthesis of Compound 29
Figure BDA0003250509980000163
The chemical formula is as follows: c 43 H 68 D 11 NO 2
Molecular weight: 653.13
Compound 29 can be synthesized according to the representative route described in example 1.
C 43 H 68 D 11 NO 2 ,Ms m/z:[M+H + ]653.6； 1 H-NMR(300MHz,CDCl 3 )δ5.4-5.27(m,8H), 2.82～2.78(d,4H),2.16(m,8H),1.80(m,2H),1.49(m,4H),1.33-1.26(m,36H),0.88(t,6H)。
Example 30
Luciferase mRNA in vivo evaluation using lipid nanoparticle composition
The cationic lipid, DSPC, cholesterol and PEG-lipid were dissolved in ethanol at a molar ratio of 50. Lipid Nanoparticles (LNPs) were prepared at a weight ratio of total lipid to mRNA of about 10. Briefly, mRNA was diluted to 0.15mg/mL in 10mL to 50mL citrate buffer (pH = 4.0). The lipid ethanol solution and the aqueous mRNA solution were mixed using a syringe pump at a ratio of about 1:5 to 1:3 (volume/volume) with a total flow rate of 10 mL/min or greater. The ethanol was then removed and the external buffer was replaced by PBS by dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with a pore size of 0.2 μm. The particle size of the lipid nanoparticles as determined by quasielastic light scattering using a Malvern Zetasizer Nano ZS was approximately 65-105nm in diameter, and in some cases approximately 75-100nm in diameter.
The study was performed on 6-8 week old female C57BL/6 mice, 8-10 week old CD-1 mice, according to the guidelines set by the national institute of science and technology. Various doses of mRNA lipid nanoparticles were administered systemically by tail vein injection and animals were euthanized at specific time points (e.g., 5 hours) post-administration. Liver and spleen were collected in pre-weighed tubes, weighed, snap frozen immediately in liquid nitrogen, and stored at-80 ℃ until used for analysis.
For liver, approximately 50mg was cut for analysis in 2mL FastPrep tubes (MP Biomedicals, solon OH). 1/4 "ceramic spheres (MP Biomedicals) were added to each tube, and 500. Mu.L of Glo lysis buffer-GLB (Promega, madison Wis.) equilibrated to room temperature was added to the liver tissue. Liver tissue was homogenized using a FastPrep24 instrument (MP Biomedicals) at 2X 6.0m/s for 15 seconds. The homogenate was incubated at room temperature for 5 minutes, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically, 50. Mu.L of the diluted tissue homogenate was reacted with 50. Mu.L of SteadyGlo substrate, shaken for 10 seconds, followed by incubation for 5 minutes, and then quantified using a SpectraMAX _ L chemiluminescence-type microplate reader (Meigu Motors, inc.). The amount of the protein determined was determined by using BCA protein quantification kit (shanghai chromophil medical science and technology ltd). The Relative Luminescence Units (RLU) were then normalized to the total μ g of protein assayed. To convert RLU to μ g luciferase, a standard curve was generated with QuantiL μ M recombinant luciferase (Promega).
Fluciferase protein will be expressed by Fluuc mRNA from Trilink Biotechnologies (L-6107), which was originally isolated from fireflies (Photinus pyralis). Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability. Which emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA was completely replaced by 5-methylcytidine and pseudouridine.
Example 31
Determination of pKa of the prepared lipid
The pKa of the formulated cationic lipid correlates with the effect of the LNP used to deliver the nucleic acid. The preferred pKa range is from 5 to 7. The pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on the fluorescence of 2- (p-toluidinyl) -6-naphthalenesulfonic acid (TNS). Lipid nanoparticles comprising cationic lipids/DSPC/cholesterol/PEG lipids (50/10/38/2 mol%) at a concentration of 0.4mM total lipid in PBS were prepared using an ordered method as described in example 27. TNS was prepared as a 100 μ M stock solution in distilled water. The vesicles were diluted to 24. Mu.M lipid in 2mL of buffer containing 10mM HEPES, 10mM MES, 10mM acetic acid, 130mM NaCl, pH 2.5-11. Aliquots of the TNS solution were added to give a final concentration of l μ M and after vortex mixing the fluorescence intensity was measured in a SLM Aminco Series 2 luminescence spectrophotometer at room temperature using excitation and emission wavelengths of 321nm and 445 nm. Sigmoidal best fit analysis was applied to the fluorescence data and pKa was measured as the pH yielding half the maximum fluorescence intensity.
Example 32
Determination of the efficacy of lipid nanoparticle formulations containing various cationic lipids using rodent models of luciferase mRNA expression in vivo
For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using an ordered mixing method, as described in example 30. Lipid nanoparticles were formulated using a molar ratio of 50% cationic lipid/10% Distearoylphosphatidylcholine (DSPC)/38% cholesterol/2% PEG lipid ("PEG-DMG", i.e., (1- (monomethoxy-polyethylene glycol) -2,3 dimyristoyl glycerol, average PEG molecular weight 2000.) As described in example 30, relative activity was determined by measuring luciferase expression in liver 5 hours after administration via tail vein injection, comparing the activity at doses of 0.3 and 1.0mg mRNA/kg, and expressing ng luciferase/g liver measured 5 hours after administration as described in example 30. Examples 31 and 32 results are shown in Table 2.
Table 2 comparison of lipids exhibiting Activity with mRNA
Figure BDA0003250509980000181
Figure BDA0003250509980000191
Figure BDA0003250509980000201
Figure BDA0003250509980000211
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the patent disclosure. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (15)

1. A compound of formula "I":
Figure FDA0003250509970000011
or an N-oxide thereof, or a salt or isomer thereof.
Wherein R in the structural formula "I 1 、R 2 、R 3 、R 4 And R 5 Are all independent combinations of 2 "hydrogen" isotopes (including isotopes "protium" and "deuterium") and cannot be "hydrogen" at the same time;
R 1 has independence, and is expressed as any one of 'H' and 'D', but not asR 2 、R 3 、R 4 And R 5 And is simultaneously 'H';
R 2 and R 3 All have independence, and can be represented by HH, HD or DD but not R 1 、R 4 And R 5 A combination of both "hydrogen" and "H", i.e., at least one "D";
R 4 and R 5 All have independence and can be expressed as 'HHH', 'HHD', 'HDD' or 'DDD', but not with R 1 、R 2 And R 3 And is also "H", i.e., a combination containing at least one "D";
R 1 、R 2 、R 3 、R 4 and R 5 Each independently a combination of 2 "hydrogen" isotopes (including the isotopes "protium" and "deuterium"), particular combinations being split into 11 cases, including combinations comprising 1 "D", combinations comprising 2 "D", combinations comprising 3 "D", combinations comprising 4 "D", combinations comprising 5 "D", combinations comprising 6 "D", combinations comprising 7 "D", combinations comprising 8 "D", combinations comprising 9 "D", combinations comprising 10 "D" and combinations comprising 11 "D".
2. A compound selected from the compounds in table 1.
3. A composition comprising a compound according to any one of claims 1 to 2 and a therapeutic and/or prophylactic agent.
4. The composition of claim 3, further comprising one or more excipients selected from the group consisting of neutral lipids, steroids, and polymer-conjugated lipids.
5. A composition as claimed in claim 4, wherein the neutral lipids of the component are selected from a mixture of one or more of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and Sphingomyelin (SM).
6. The composition of claim 5, wherein the neutral lipid is DSPC.
7. A composition as claimed in any one of claims 4 to 6, wherein the steroid in the component is selected from a mixture of one or more of: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol.
8. The composition of claim 7, wherein the steroid is cholesterol.
9. The composition of claims 4-8, wherein the polymer-conjugated lipid in the component is a pegylated lipid.
10. The composition of claim 9, wherein the pegylated lipid is 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG).
11. The composition of any one of the preceding claims, wherein the therapeutic and/or prophylactic agent is a vaccine or compound capable of eliciting an immune response, including nucleic acids.
12. The composition of claim 11, wherein the nucleic acid is RNA selected from the group consisting of: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof.
13. The composition of claim 12, wherein the RNA is mRNA.
14. A method of administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising preparing or providing the composition of any one of the preceding claims, and administering the composition to the subject.
15. The subject of any one of the preceding claims is a mammal or a human.
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