CN115745942A - Novel cationic lipid compounds - Google Patents

Novel cationic lipid compounds Download PDF

Info

Publication number
CN115745942A
CN115745942A CN202111031198.6A CN202111031198A CN115745942A CN 115745942 A CN115745942 A CN 115745942A CN 202111031198 A CN202111031198 A CN 202111031198A CN 115745942 A CN115745942 A CN 115745942A
Authority
CN
China
Prior art keywords
composition
compound
lipid
therapeutic
lipids
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
CN202111031198.6A
Other languages
Chinese (zh)
Inventor
黄才古
谭俊荣
黄铁强
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.)
Guangzhou Anovent Pharmaceutical Co Ltd
Original Assignee
Guangzhou Anovent Pharmaceutical 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 Guangzhou Anovent Pharmaceutical Co Ltd filed Critical Guangzhou Anovent Pharmaceutical Co Ltd
Priority to CN202111031198.6A priority Critical patent/CN115745942A/en
Priority to PCT/CN2022/093716 priority patent/WO2023029599A1/en
Publication of CN115745942A publication Critical patent/CN115745942A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/28—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/46—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and unsaturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/14—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D317/18—Radicals substituted by singly bound oxygen or sulfur atoms
    • C07D317/24—Radicals substituted by singly bound oxygen or sulfur atoms esterified
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/32—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D317/34—Oxygen atoms

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 antisense RNA. Also provided are methods of using such lipid nanoparticles for the treatment and/or prevention of various diseases. At one isIn an embodiment, compounds having the following structures (I) and (II) are provided:
Figure DDA0003245350640000011
or a salt or isomer thereof or an N-oxide thereof, wherein R 1 、R 2 And R 3 Also provided are pharmaceutical compositions as defined herein comprising one or more compounds of the foregoing structural formulae 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 invention provides a method of administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising preparing a pharmaceutical combination comprising a lipid nanoparticle of a compound of the foregoing structural formula 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 and/or producing a polypeptide in a mammalian cell or organ. 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 demonstrated 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 easily protonated.
However, the use of oligonucleotides in a therapeutic setting currently faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. 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 delivering 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 BDA0003245350630000021
or a salt or isomer thereof or an N-oxide thereof, wherein:
R 1 、R 2 is alkyl or hydrogen, but not simultaneously hydrogen.
In another aspect, the present invention relates to compounds of the following structural formula (II):
Figure BDA0003245350630000022
or a salt or isomer thereof or an N-oxide thereof, wherein:
R 3 is methyl or hydrogen.
In various embodiments, the compound has one of the structures shown in table 1 below
Representative Compounds of Table 1
Figure BDA0003245350630000023
Figure BDA0003245350630000031
In some embodiments, compositions comprising any one or more of the compounds of the formulae and a therapeutic and/or prophylactic agent are provided.
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-dimyristoyl-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, it is preferred that the 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, airRNA, 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 for 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 these 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 semi-solids, 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, tablets, and the like. Such solid compositions will generally 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 silica 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 an 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. Injectable pharmaceutical compositions are 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 an envelope 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 arts. 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 and at staggered times; it is 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, tetrahydrofuranyl, benzyl, 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 aralkyl 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 present invention may be formed by conversion to their free base or acid 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 procedures described below can be used to synthesize related compounds.
The following abbreviations are used herein:
edc.hcl: 1-Ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride
DCM: methylene dichloride
DMAP: 4-dimethylaminopyridine
DIEA N, N-diisopropylethylamine
THF: tetrahydrofuran (THF)
PCC: pyridinium chlorochromate salts
Detailed Description
Example 1:
represents line 1: synthesis of Compound 1
Figure BDA0003245350630000081
1) Compound A
Figure BDA0003245350630000082
Chemical formula C 9 H 19 NO 2
Molecular weight 173.26
4-bromomethyl-2, 5-trimethyl-1, 3-dioxolane (10.5 g, 0.05mol) and a tetrahydrofuran solution of dimethylamine (2M, 1250mL) were sequentially added to a reaction flask, and after stirring at room temperature for 2 days, the mixture was concentrated in vacuo to obtain a crude compound A (9.2 g).
2) Compound B
Figure BDA0003245350630000083
The chemical formula is as follows: c 6 H 15 NO 2
Molecular weight: 133.19
The crude compound a (9.2 g), and water (10 mL) were added to the reaction flask, the temperature was controlled at 25 ℃, the system pH was adjusted to 4 with 5M hydrochloric acid, stirred for 3 hours, concentrated in vacuo, the residue was dissolved with DCM, sodium bicarbonate and anhydrous sodium sulfate were added in sequence, filtered and concentrated in vacuo, and the residue was purified by column to give compound B (4.7 g, 0.03 mol).
3) Compound 1
Figure BDA0003245350630000091
The chemical formula is as follows: c 42 H 79 NO 2
Molecular weight: 630.10
A reaction flask was charged with Compound B (1.3g, 0.01mol), linoleol methanesulfonate (6.9g, 0.02mol), tetrabutylammonium hydrogensulfate (1.7g, 5mmol) and toluene (50 ml) in this order. 50ml of 40% aqueous sodium hydroxide solution was added dropwise at 25 ℃. Stirring at room temperature for 3 days, adding water to dilute the system, and extracting with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and the residue was purified by column chromatography to give compound 1 (2.5g, 4mmol).
C 42 H 79 NO 2 ，Ms m/z:[M+H + ]631； 1 H-NMR(300MHz,CDCl 3 )δ:ppm 5.40-5.30 (4H，m)，5.30-5.00(4H,m)，3.60-3.30(6H,m)，2.76(4H，m)，2.53(2H， m)，2.31-2.07(14H，m),1.49-1.26(36H，m)1.10(3H，s)，0.88(6H， m)。
Example 2:
compound 2
Figure BDA0003245350630000092
The chemical formula is as follows: c 42 H 79 NO 2
Molecular weight: 630.10
Compound 2 can be synthesized according to representative route 1 described in example 1.
C 42 H 79 NO 2 ，Ms m/z:[M+H + ]631； 1 H-NMR(300MHz,CDCl 3 )δ:ppm 5.50-5.35 (4H，m)，5.30-5.10(4H,m)，3.60-3.30(6H,m)，2.76(4H，m)，2.38-2.05 (16H，m),1.49-1.16(39H，m)，0.88(6H，m)。
Example 3:
compound 3
Figure BDA0003245350630000101
The chemical formula is as follows: c 43 H 81 NO 2
Molecular weight: 644.13
Compound 3 can be synthesized according to representative route 1 described in example 1.
C 43 H 81 NO 2 ，Ms m/z:[M+H + ]645； 1 H-NMR(300MHz,CDCl 3 )δ:ppm 5.50-5.35 (4H，m)，5.30-5.10(4H,m)，3.50-3.20(6H,m)，2.80(4H，m)，2.50-2.20 (8H，m)，2.18-2.01(8H，m),1.49-1.16(38H，m)，0.88(9H，m)。
Example 4:
representative line 2: synthesis of Compound 4
Figure BDA0003245350630000102
1) Compound C
Figure BDA0003245350630000111
The chemical formula is as follows: c 37 H 68 O
Molecular weight: 528.95
Compound 1 can be synthesized according to the representative route described in example 1.
Under the protection of nitrogen, magnesium chips (1.1g, 46mmol), THF (6 mL) and a plurality of iodine simple substances are added into a reaction bottle, tetrahydrofuran (15 mL) solution of bromolinolene (14g, 42.5 mmol) is dripped into a system in batches, the temperature of the system is kept at 40 ℃ for reaction for 5 hours, then the temperature is reduced to 0 ℃, then THF solution of ethyl formate (3.3g, 44.6 mmol) is dripped into the system for reaction for 18 hours at room temperature. Cooling to-10 deg.C, and adding 2N hydrochloric acid dropwise for acidification. Extracted with n-hexane, washed with saturated brine, and concentrated. The residue was dissolved in ethanol (25 mL), potassium hydroxide (1.7 g) in water (5 mL) was added, the mixture was stirred for 3 hours, vacuum concentrated to remove ethanol, neutralized with 2N hydrochloric acid, extracted with N-hexane, concentrated in vacuo, and the residue was subjected to column chromatography to give Compound C (8.7 g,16.4 mmol).
2) Compound D
Figure BDA0003245350630000112
The chemical formula is as follows: c 37 H 66 O
Molecular weight: 526.93
To a solution of Compound C (8.0 g,15.1 mmol) in dichloromethane (100 mL) was added PCC (8.0 g,37.1 mmol) and sodium carbonate (0.8 g) in that order under nitrogen. After stirring at room temperature for 3 hours, the mixture was filtered, concentrated, and the residue was subjected to column chromatography to obtain Compound D (6.4 g, 12.1mmol).
3) Compound E
Figure BDA0003245350630000113
Molecular weight: 643.01
Compound D (6.0g, 11.4 mmol) and boron trifluoride diethyl ether (1.1g, 7.7 mmol) were added in portions to a tetrahydrofuran (15 ml) solution of malic acid (762mg, 5.7 mmol) at a temperature of 0 ℃, stirred at room temperature for 2 days, concentrated in vacuo, the residue was dissolved with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography to give Compound E (1.8g, 2.8mmol).
4) Compound F
Figure BDA0003245350630000121
The chemical formula is as follows: c 41 H 72 O 4
Molecular weight: 629.02
Adding a compound E (1.5g, 2.3mmol) and THF (10 mL) into a reaction bottle in sequence, adding sodium borohydride (105mg, 2.8mmol) in batches, cooling to 0 ℃, injecting boron trifluoride diethyl etherate (392mg, 2.8mmol), heating to 50 ℃, preserving heat for reaction for 5 hours, adding a sodium bicarbonate aqueous solution for quenching, extracting by ethyl acetate, drying by anhydrous sodium sulfate, concentrating in vacuum, and purifying the residue by a column to obtain a compound F (1.2g, 1.9mmol).
5) Compound G
Figure BDA0003245350630000122
The chemical formula is as follows: c 42 H 74 O 6 S
Molecular weight: 707.11
Compound F (1.0g, 1.6 mmol), DCM (10 mL) and triethylamine (240 mg, 2.4 mmol) are sequentially added into a reaction flask, cooled to 0 ℃, a dichloromethane (5 mL) solution of methanesulfonyl chloride (230mg, 2.0 mmol) is dropwise added, the temperature is returned to room temperature, the reaction is carried out for 8 hours, a DCM dilution system is sequentially washed by water, a sodium bicarbonate aqueous solution and saturated saline solution, an organic phase is dried by anhydrous sodium sulfate and then concentrated, and a residue is subjected to column purification to obtain compound G (1.0g, 1.4mmol).
6) Compound 4
Figure BDA0003245350630000131
The chemical formula is as follows: c 43 H 77 NO 3
Molecular weight: 656.09
Under nitrogen protection, compound G (1.0G, 1.4 mmol) and dimethylamine in THF (2M, 35mL) were added sequentially to the reaction flask, stirred at room temperature for 5 days, concentrated in vacuo, and the residue was passed through the column. To give compound 4 (0.6 g, 0.9 mmol)
C 43 H 77 NO 3 ,Ms m/z:[M+H + ]657； 1 H-NMR(300MHz,CDCl 3 )δ:ppm 5.50-5.10(8H，m)，4.00(1H,t)，2.80(4H，m)，2.60-2.50(2H，m)，2.40 (6H，m)2.20-2.00(8H，m),1.83(2H，m)，1.63(4H，m)，1.49-1.16(36H， m)，0.88(6H，m)。
Example 5:
compound 5:
Figure BDA0003245350630000132
the chemical formula is as follows: c 44 H 79 NO 3
Molecular weight: 670.12
Compound 5 can be synthesized according to representative scheme 2 described in example 4.
C 44 H 79 NO 3 ,Ms m/z:[M+H + ]671； 1 H-NMR(300MHz,CDCl 3 )δ:ppm 5.50-5.10(8H，m)，2.80(4H，m)，2.60-2.50(2H，m)，2.40-2.10(14H，m),1.83 (2H，m)，1.63(4H，m)，1.49-1.16(39H，m)，0.88(6H，m)。
Example 6
Luciferase mRNA in vivo evaluation using lipid nanoparticle compositions
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 mRNA aqueous solution are mixed using a syringe pump at a ratio of about 1 to 1 (volume/volume) with a total flow rate of 10mL/min or more. 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 75-100 nm in diameter.
The study was carried out 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. Different 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-dose. Liver and spleen were collected in pre-weighed tubes, weighed, immediately snap frozen in liquid nitrogen, and stored at-80 ℃ until used for analysis.
For the 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. 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).
Fluuc mRNA from Trilink Biotechnologies (L-6107) will express luciferase protein, 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 7
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 solution 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 8
Determination of potency 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 6. 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 6, relative activity was determined by measuring luciferase expression in liver 5 hours after administration via tail vein injection.the activities were compared at doses of 0.3 and 1.0mg mRNA/kg and expressed as ng luciferase/g liver measured 5 hours after administration as described in example 6. Examples 6 and 7 results are shown in Table 2.
Table 2 comparison of lipids exhibiting Activity with mRNA
Figure BDA0003245350630000161
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 more specific and detailed, but not construed as limiting the scope of the patent disclosure. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims (17)

1. A compound having the following structure (I):
Figure FDA0003245350620000011
or a salt or isomer thereof or an N-oxide thereof, wherein:
R 1 、R 2 are independent short chain alkyl groups containing at least 1 carbon, i.e. not both hydrogen.
2. The compound of claim 1, wherein the short chain alkyl is a linear alkane.
3. The compound of claim 2, wherein the lower alkyl is methyl, ethyl, n-propyl.
4. A compound having the following structure (II):
Figure FDA0003245350620000012
or a salt or isomer thereof, or an N-oxide thereof, wherein:
R 3 is hydrogen or methyl.
5. A composition comprising a compound according to any one of claims 1 to 4 and a therapeutic and/or prophylactic agent.
6. The composition of claim 5, further comprising one or more excipients selected from the group consisting of neutral lipids, steroids, and polymer-conjugated lipids.
7. A composition as claimed in claim 6, 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-dimyristoyl-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).
8. The composition of claim 7, wherein the neutral lipid is DSPC.
9. A composition as claimed in any one of claims 5 to 8, 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.
10. The composition of claim 9, wherein the steroid is cholesterol.
11. The composition of claims 5-10, wherein the polymer-conjugated lipid in the component is a pegylated lipid.
12. The composition of claim 11, wherein the pegylated lipid is 1, 2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG).
13. 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 a nucleic acid.
14. The composition of claim 13, wherein the nucleic acid is RNA selected from the group consisting of: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof.
15. The composition of claim 14, wherein the RNA is mRNA.
16. A method of administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising preparing or providing a composition according to any one of the preceding claims, and administering the composition to the subject.
17. The subject of any one of the preceding claims is a mammal or a human.
CN202111031198.6A 2021-09-03 2021-09-03 Novel cationic lipid compounds Pending CN115745942A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202111031198.6A CN115745942A (en) 2021-09-03 2021-09-03 Novel cationic lipid compounds
PCT/CN2022/093716 WO2023029599A1 (en) 2021-09-03 2022-05-19 Novel cationic lipid compound

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111031198.6A CN115745942A (en) 2021-09-03 2021-09-03 Novel cationic lipid compounds

Publications (1)

Publication Number Publication Date
CN115745942A true CN115745942A (en) 2023-03-07

Family

ID=85332429

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111031198.6A Pending CN115745942A (en) 2021-09-03 2021-09-03 Novel cationic lipid compounds

Country Status (2)

Country Link
CN (1) CN115745942A (en)
WO (1) WO2023029599A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117257965A (en) * 2023-11-21 2023-12-22 深圳瑞吉生物科技有限公司 Nucleic acid delivery carrier composition and application thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1981044B (en) * 2004-06-07 2010-05-05 普洛体维生物治疗公司 Lipid encapsulated interfering RNA
CN104119242B (en) * 2008-10-09 2017-07-07 泰米拉制药公司 The amino lipids of improvement and the method for delivering nucleic acid
EP2516010A2 (en) * 2009-12-23 2012-10-31 Novartis AG Lipids, lipid compositions, and methods of using them
CN112979483B (en) * 2021-05-14 2021-08-06 苏州艾博生物科技有限公司 Cationic lipid compound, composition containing same and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117257965A (en) * 2023-11-21 2023-12-22 深圳瑞吉生物科技有限公司 Nucleic acid delivery carrier composition and application thereof
CN117257965B (en) * 2023-11-21 2024-02-23 深圳瑞吉生物科技有限公司 Nucleic acid delivery carrier composition and application thereof

Also Published As

Publication number Publication date
WO2023029599A1 (en) 2023-03-09

Similar Documents

Publication Publication Date Title
CN103857654B (en) The compound of siRNA activity is conveyed and is strengthened for targeted drug
JP2022549253A (en) Carbonate-containing lipid compounds and compositions for intracellular delivery of therapeutic agents
CN113185421B (en) Lipid compounds and compositions thereof
US7585519B2 (en) Esters of L-carnitine or alkanoyl L-carnitines useful as cationic lipids for the intracellular delivery of pharmacologically active compounds
WO2023109881A1 (en) High-efficiency and low-toxicity dna and rna lipid delivery carrier
WO2023186149A1 (en) Lipid compound, composition containing same, and use thereof
WO2023024513A1 (en) Novel cationic lipid compound
WO2023178795A1 (en) Cationic lipid compounds used for nucleic acid delivery, composition and use
WO2023015904A1 (en) Novel cationic lipid compound
CN115745942A (en) Novel cationic lipid compounds
CN113398276B (en) Preparation and application of brain glioma targeted berberine and folic acid modified lipid material
CN115745819A (en) Novel cationic lipid compounds
CN113214171B (en) Amphiphilic dendrimers, synthesis and use thereof as drug delivery systems
CN115772089A (en) Novel cationic lipid compounds
CN115710193A (en) Novel cationic lipid compounds
CN115745815A (en) Novel cationic lipid compounds
CN115710191A (en) Novel cationic lipid compounds
CN115703714A (en) Novel cationic lipid compounds
CN115745941A (en) Novel cationic lipid compounds
CN114409554A (en) Novel cationic lipid compound, composition and application thereof
CN115925604A (en) Pyrrolidine novel lipid compound, preparation method, composition and application thereof
CN115710196A (en) Novel cationic lipid compounds
CN115894281A (en) Novel cationic lipid compounds, preparation method, composition and application thereof
CN115724806A (en) Novel cationic lipid compound (II)
CN115850686A (en) Novel deuterated PEG lipid compound, preparation method, composition and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication