CN115772089B

CN115772089B - Cationic lipid compounds

Info

Publication number: CN115772089B
Application number: CN202111043919.5A
Authority: CN
Inventors: 黄才古; 王帅; 黄铁强
Original assignee: Guangzhou Anovent Pharmaceutical Co Ltd
Current assignee: Guangzhou Anovent Pharmaceutical Co Ltd
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2025-06-10
Anticipated expiration: 2041-09-07
Also published as: WO2023035652A1; CN115772089A

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. Methods of using such lipid nanoparticles for the treatment and/or prevention of various diseases are also provided. In one embodiment, compounds having the structure of formula (I) are provided: Or a salt or isomer or N-oxide thereof, wherein R ₁、R₂、R₃、R₄ and R ₅ are as defined herein. Also provided are pharmaceutical compositions comprising one or more of the compounds of formula (I) above 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 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, the methods 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

Cationic lipid compounds

Technical Field

The present invention provides cationic lipids that can be used in combination with other lipid components (e.g., neutral lipids, steroids, and polymer conjugated lipids) to form a nucleic acid mRNA lipid nanoparticle composition for delivery of one or more therapeutic and/or prophylactic agents to and/or production of polypeptides in mammalian cells or organs. In addition to lipids, the lipid nanoparticle compositions of the present invention may also 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

Efficient targeted delivery of biologically active substances such as small molecule drugs, proteins and nucleic acids presents a long-lasting 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.

It has been 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 transport vehicles. These compositions generally comprise one or more "cationic" lipids, including neutral lipids (e.g., phospholipids) including polyunsaturated lipids, structural lipids (e.g., steroids), and/or polyethylene glycol-containing lipids (polymer conjugated lipids). Cationic lipids include amine-containing lipids such as can be easily protonated.

However, the use of oligonucleotides in therapeutic environments is currently faced with two problems. First, free RNA is readily digested in plasma by nucleases. Second, free RNA has limited ability to enter the intracellular compartment where relevant translation mechanisms exist. Lipid nanoparticles formed from cationic lipids with other lipid components (e.g., neutral lipids, cholesterol, PEG, pegylated lipids, and oligonucleotides) have been used to prevent RNA degradation in plasma and to promote cellular uptake of the oligonucleotides.

There remains a need for improved cationic lipids and lipid nanoparticles for delivery of oligonucleotides. The improved lipid nanoparticle will 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 so that patient treatment at an effective dose of nucleic acid does not create 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 compounds and methods involving these compounds:

In a first aspect, the present invention relates to a compound of the following structural formula (I):

Or an N-oxide thereof, or a salt or isomer thereof.

Wherein R ₁、R₂、R₃、R₄ and R ₅ in structural formula "I" are each independently a combination of 2 "hydrogen" isotopes (including the isotopes "protium" and "deuterium") and cannot be both "hydrogen";

R ₁ has independence, which is represented by either of "H" and "D", but not both R ₂、R₃、R₄ and R ₅ are "H";

R ₂ and R ₃ are independent and may represent "HH", "HD" or "DD", but are not "hydrogen" at the same time as R ₁、R₄ and R ₅, i.e. contain at least one combination of "D";

R ₄ and R ₅ are independent and may represent "HHH", "HHD", "HDD" or "DDD", but are not "H" at the same time as R ₁、R₂ and R ₃, i.e. contain at least one "D" combination;

R ₁、R₂、R₃、R₄ and R ₅ are each independently a combination of 2 "hydrogen" isotopes (including the isotopes "protium" and "deuterium"), and the specific combination is divided into 11 cases, including a combination of 1 "D", a combination of 2 "D", a combination of 3 "D", a combination of 4 "D", a combination of 5 "D", a combination of 6 "D", a combination of 7 "D", a combination of 8 "D", a combination of 9 "D", a combination of 10 "D" and a combination of 11 "D".

In various embodiments, the compounds have one of the structures shown in Table 1 below

Table 1 representative Compounds

In some embodiments, compositions are provided that include any one or more of the compounds of structural formula (I) and a therapeutic and/or prophylactic agent.

In some embodiments, compositions are provided that include 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 neutral lipids, steroids, and polymer conjugated lipids. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the composition.

In some embodiments, the neutral lipid is selected from one or more of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (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-phosphorylcholine (DSPC).

In some embodiments, the steroid is selected from one or more of cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, alpha-tocopherol. In some embodiments, the preferred steroid is cholesterol.

In some embodiments, the pegylated lipid is 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG)

In some embodiments, the composition ratio ranges from about 10 to 60 mole% of the compound, from about 0 to 30 mole% neutral lipid, from about 10 to 55 mole% steroid, and from about 0 to 10 mole% polymer conjugated lipid.

In some embodiments of the foregoing compositions, 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 various other embodiments, the invention relates to methods of administering a therapeutic and/or prophylactic agent to a subject in need thereof, comprising preparing or providing any of the compositions described above and administering the composition to the subject.

For purposes of use, the compounds of the present invention may be used as bulk drugs, or may be formulated as pharmaceutical compositions (typically in the form of lipid nanoparticles conjugated to therapeutic and/or prophylactic agents). The pharmaceutical compositions of the present invention comprise a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents or excipients. The compounds of structure (I) are effective to form lipid nanoparticles and deliver therapeutic and/or prophylactic agents. The 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 carried out by any acceptable means of use for similarly effective agents. The pharmaceutical compositions of the invention may be formulated as solid, semi-solid, liquid or gaseous forms of preparations such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of using 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 as to render the active ingredient therein bioavailable in the subject. The composition to be administered to a subject or patient may be in the form of one or more dosage forms, wherein the tablet may be a single dosage unit and the container of the compound in aerosol form of the invention 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 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 composition of the present invention may be in solid or liquid form. In one aspect, the carrier may be a granule 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, where solid or liquid forms are considered herein to include semi-solids, semi-liquids, suspensions and gels.

As solid compositions for oral use, pharmaceutical compositions may be formulated in the form of powders, granules, tablets, pills, capsules, chewing gums, wafers, and the like. Such solid compositions will typically contain one or more inert diluents or edible carriers. In addition, there may be one or more of binders such as gelatin, cellulose, etc., excipients such as lactose, etc., disintegrants such as alginic acid, etc., lubricants such as magnesium stearate, etc., glidants such as silica gel, etc., sweeteners such as sucrose or saccharin, flavoring agents such as peppermint, etc., and coloring agents.

When the pharmaceutical composition is in the form of a capsule, it may contain a liquid carrier other than materials of the type described above, 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 a sweetener, preservative, coloring/coloring agent, and flavoring agent in addition to the compounds of the present invention. In the composition for use by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspension 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 sterile diluents such as water for injection, saline solutions, preferably physiological saline, ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono-or diglycerides which may be used as solvents or suspension media, polyethylene glycol, glycerol, propylene glycol or other solvents, antibacterial agents such as methyl paraben and the like, antioxidants such as ascorbic acid or sodium bisulphite, chelating agents such as ethylenediamine tetraacetic acid, buffers such as acetate, citrate or phosphate, and agents for modulating tonicity such as sodium chloride or dextrose, agents for use as cryoprotectants such as sucrose or trehalose. Parenteral formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic. 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 alcohols, and emulsifiers and stabilizers. The thickener may be present in a pharmaceutical composition for topical use. If intended for transdermal use, the composition may include a transdermal patch or 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 coating shell around the active ingredient. The materials forming the coating shell are generally inert and may be sugar, shellac and other enteric coating agents. Or the active ingredient may be encapsulated in a gelatin capsule.

Pharmaceutical compositions of the invention in solid or liquid form may include a compound of the invention in combination with a compound of the invention, which aids in the delivery of the compound. 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 refers to 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 in a single phase, a biphasic system or a triphasic system in order to deliver the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form the drug delivery device. The preferred aerosols can be determined by those skilled in the art without additional experimentation.

The pharmaceutical compositions of the present invention may be prepared by methods well known in the pharmaceutical arts. The lipid nanoparticle of the present invention may be prepared by combining the lipid nanoparticle with sterile distilled water or other carrier into a solution by injection of the pharmaceutical composition. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants are non-covalent interactions through 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, the metabolic stability and length of action of that therapeutic agent, the age, body weight, general health, sex, and diet of the subject, the manner and time of use, the rate of excretion, drug combination, the severity of the particular case, and the like.

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 and combinations using the compositions of the present invention and one or more other active ingredients. For example, the compositions of the invention and other active ingredients may be used together in a single oral dosage formulation (e.g., tablet or capsule) to a subject, or the individual active ingredients may be used in different oral dosage formulations. When different dosage formulations are used, the compounds of the invention and one or more additional active ingredients may be used at the same time or sequentially at staggered times, it being understood that combination therapy includes all of these dosage regimens.

The structural modification and design of the deuterated cationic lipid compound realize more advantageous physicochemical properties including more proper pKa and better chemical stability, can be used for the mRNA nano liposome composition, can realize more effective combination and delivery of ionic nucleic acid medicaments, and has more stable chemical structure, thereby being convenient for synthesis and favorable development as pharmaceutical excipients.

Methods for preparing the above compounds and compositions are described below and/or are known in the art.

Those skilled in the art will recognize that in the methods described herein, the functional groups of the 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 groups include trialkylsilyl or diarylalkylsilyl groups, tetrahydrofuranyl groups, benzyl groups, and the like. Suitable protecting groups for the amino group include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for carboxylic acids include hydroxy, aryl or arene esters. Protecting groups may be added or removed according to standard techniques known to those skilled in the art and described herein.

Those skilled in the art will also recognize that while such protected derivatives of the compounds of the present invention may not be pharmaceutically active therefrom, 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 thus be described as "prodrugs". Prodrugs of the compounds of the present invention are therefore included within the scope of the present invention.

Furthermore, all compounds of the invention which are present in the free base or free acid form can be converted into their pharmaceutically acceptable salts by treatment with suitable inorganic or organic bases or acids according to methods known to the person skilled in the art. Salts of the compounds of the invention may be formed by conversion to their free base or acid by standard techniques.

The following examples are provided for purposes of illustration and not limitation.

All solvents and reagents used in the following examples are commercially available and used as such unless otherwise indicated.

The procedure described below can be used to synthesize compound I in table 1.

The following abbreviations are used herein:

Detailed Description

Example 1:

Representative route

Synthesis of Compound 8

1) Synthesis of Compound 8-02

Chemical formula C ₄₁H₇₁D₂BrO₂

Molecular weight 679.95

EDC.HCl (2.1 g,11.0 mmol), DIEA (5.3 ml,30.0 mmol), DMAP (0.2 g,1.5 mmol) were added sequentially to a mixture of Compound A (5.3 g,10.0 mmol) and 4,4-d ₂ -bromobutyric acid (2.5 g,15.0 mmol) in DCM. After reaction for 2h at 45 ℃, the system was diluted with DCM, washed with saturated aqueous sodium bicarbonate, then with dilute aqueous hydrochloric acid, dried over magnesium sulfate, filtered and concentrated, and the residue was purified by a silica gel column (0-15% ethyl acetate/n-hexane). Compound 8-02 (5.1 g, 75%) was obtained.

2) Synthesis of Compound 8

Chemical formula C ₄₃H₇₇D₂NO₂

Molecular weight 644.12

To a mixture of Compound 8-02 (0.68 g,1.0 mmol) and dimethylamine (0.09 g,2.0 mmol) in DMF (5 mL) was added K ₂CO₃ (0.27 g,2.0 mmol) and potassium iodide (0.017 g,0.1 mmol) in this order, the mixture was warmed to 65℃and stirred for 24 hours, and after the reaction of the starting materials was completed, the system temperature was lowered to room temperature. 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, concentration was performed under reduced pressure in vacuo. Purifying the residue by a silica gel column (0-100% (1% NH ₄ OH, 20% MeOH in dichloromethane) to obtain the compound 8(0.58g,90％).C₄₃H₇₇D₂NO₂,Ms m/z:[M+H⁺]644.6;¹H-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

Chemical formula C ₄₃H₇₈DNO₂

Molecular weight 643.12

Compound 1 can be synthesized according to the representative route described in example 1.

C₄₃H₇₈DNO₂,Ms m/z:[M+H⁺]643.6;¹H-NMR(300MHz,CDCl₃)δ:¹H-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

Chemical formula C ₄₃H₇₈DNO₂

Molecular weight 643.12

Compound 2 can be synthesized according to the representative route described in example 1.

C₄₃H₇₈DNO₂,Ms m/z:[M+H⁺]643.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₈DNO₂

Molecular weight 643.12

Compound 3 can be synthesized according to the representative route described in example 1.

C₄₃H₇₈DNO₂,Ms m/z:[M+H⁺]643.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₈DNO₂

Molecular weight 643.12

Compound 4 can be synthesized according to the representative route described in example 1.

C₄₃H₇₈DNO₂,Ms m/z:[M+H⁺]643.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₇D₂NO₂

Molecular weight 644.12

Compound 5 can be synthesized according to the representative route described in example 1.

C₄₃H₇₇D₂NO₂,Ms m/z:[M+H⁺]644.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₇D₂NO₂

Molecular weight 644.12

Compound 6 can be synthesized according to the representative route described in example 1.

C₄₃H₇₇D₂NO₂,Ms m/z:[M+H⁺]644.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₇D₂NO₂

Molecular weight 644.12

Compound 7 can be synthesized according to the representative route described in example 1.

C₄₃H₇₇D₂NO₂,Ms m/z:[M+H⁺]644.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₇D₂NO₂

Molecular weight 644.12

Compound 9 can be synthesized according to the representative route described in example 1.

C₄₃H₇₇D₂NO₂,Ms m/z:[M+H⁺]644.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₇D₂NO₂

Molecular weight 644.12

Compound 10 can be synthesized according to the representative route described in example 1.

C₄₃H₇₇D₂NO₂,Ms m/z:[M+H⁺]644.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₆D₃NO₂

Molecular weight 645.12

Compound 11 can be synthesized according to the representative route described in example 1.

C₄₃H₇₆D₃NO₂,Ms m/z:[M+H⁺]645.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₆D₃NO₂

Molecular weight 645.12

Compound 12 can be synthesized according to the representative route described in example 1.

C₄₃H₇₆D₃NO₂,Ms m/z:[M+H⁺]645.6;¹H-NMR(300MHz,CDCl₃)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:

Chemical formula C ₄₃H₇₆D₃NO₂

Molecular weight 645.12

Compound 13 can be synthesized according to the representative route described in example 1.

C₄₃H₇₆D₃NO₂,Ms m/z:[M+H⁺]645.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₅D₄NO₂

Molecular weight 646.12

Compound 14 can be synthesized according to the representative route described in example 1.

C₄₃H₇₅D₄NO₂,Ms m/z:[M+H⁺]646.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₅D₄NO₂

Molecular weight 646.12

Compound 15 can be synthesized according to the representative route described in example 1.

C₄₃H₇₅D₄NO₂,Ms m/z:[M+H⁺]646.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₅D₄NO₂

Molecular weight 646.12

Compound 16 can be synthesized according to the representative route described in example 1.

C₄₃H₇₅D₄NO₂,Ms m/z:[M+H⁺]646.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₅D₄NO₂

Molecular weight 646.12

Compound 17 can be synthesized according to the representative route described in example 1.

C₄₃H₇₅D₄NO₂,Ms m/z:[M+H⁺]646.6;¹H-NMR(300MHz,CDCl₃)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:

synthesis of Compound 18

Chemical formula C ₄₃H₇₄D₅NO₂

Molecular weight 647.12

Compound 18 can be synthesized according to the representative route described in example 1.

C₄₃H₇₄D₅NO₂,Ms m/z:[M+H⁺]647.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₄D₅NO₂

Molecular weight 647.12

Compound 19 can be synthesized according to the representative route described in example 1.

C₄₃H₇₄D₅NO₂,Ms m/z:[M+H⁺]647.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₄D₅NO₂

Molecular weight 647.12

Compound 20 can be synthesized according to the representative route described in example 1.

C₄₃H₇₄D₅NO₂,Ms m/z:[M+H⁺]647.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₄D₅NO₂

Molecular weight 647.12

Compound 21 can be synthesized according to the representative route described in example 1.

C₄₃H₇₄D₅NO₂,Ms m/z:[M+H⁺]647.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₃D₆NO₂

Molecular weight 648.12

Compound 22 can be synthesized according to the representative route described in example 1.

C₄₃H₇₃D₆NO₂,Ms m/z:[M+H⁺]648.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₂D₇NO₂

Molecular weight 649.12

Compound 23 can be synthesized according to the representative route described in example 1.

C₄₃H₇₂D₇NO₂,Ms m/z:[M+H⁺]649.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₁D₈NO₂

Molecular weight 650.13

Compound 24 can be synthesized according to the representative route described in example 1.

C₄₃H₇₁D₈NO₂,Ms m/z:[M+H⁺]650.7;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₇₀D₉NO₂

Molecular weight 651.13

Compound 25 can be synthesized according to the representative route described in example 1.

C₄₃H₇₀D₉NO₂,Ms m/z:[M+H⁺]651.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₆₉D₁₀NO₂

Molecular weight 652.13

Compound 26 can be synthesized according to the representative route described in example 1.

C₄₃H₆₉D₁₀NO₂,Ms m/z:[M+H⁺]652.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₆₉D₁₀NO₂

Molecular weight 652.13

Compound 27 can be synthesized according to the representative route described in example 1.

C₄₃H₆₉D₁₀NO₂,Ms m/z:[M+H⁺]652.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₆₉D₁₀NO₂

Molecular weight 652.13

Compound 28 can be synthesized according to the representative route described in example 1.

C₄₃H₆₉D₁₀NO₂,Ms m/z:[M+H⁺]652.6;¹H-NMR(300MHz,CDCl₃)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

Chemical formula C ₄₃H₆₈D₁₁NO₂

Molecular weight 653.13

Compound 29 can be synthesized according to the representative route described in example 1.

C₄₃H₆₈D₁₁NO₂,Ms m/z:[M+H⁺]653.6;¹H-NMR(300MHz,CDCl₃)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 compositions

The cationic lipid, DSPC, cholesterol and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38:2 or 48:10:40:2. Lipid Nanoparticles (LNP) were prepared at a weight ratio of total lipid to mRNA of about 10:1 to 30:1. Briefly, mRNA was diluted to 0.15mg/mL in 10mL to 50mL citrate buffer (ph=4.0). The ethanol solution of the lipid and the aqueous solution of mRNA are mixed in a ratio of about 1:5 to 1:3 (volume/volume) using a syringe pump, and the total flow rate is 10mL/min or more. The ethanol was then removed and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with a pore size of 0.2 μm. The lipid nanoparticles have a particle size of about 65-105nm in diameter, and in some cases, about 75-100nm in diameter, as determined by quasi-elastic light scattering using Malvern Zetasizer Nano ZS.

According to guidelines established by the national science and technology Committee, studies were performed on female C57BL/6 mice of 6-8 weeks of age and CD-1 mice of 8-10 weeks of age. 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) after administration. The livers and spleens were collected in pre-weighed tubes, weighed, immediately flash frozen in liquid nitrogen, and stored at-80 ℃ until used for analysis.

For the liver, about 50mg was cut for analysis in 2mL FastPrep tubes (MP Biomedicals, solon OH). 1/4 "ceramic balls (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 liver tissue. Liver tissue was homogenized at 2X6.0 m/s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). The homogenates were incubated for 5min at room temperature, then 1:4 diluted in GLB, and evaluated using 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 chemiluminescent microplate reader (America molecular instruments (Shanghai)). The amount of protein assayed was determined by using BCA protein quantification kit (shanghai easy color medical science co.ltd). The Relative Luminosity Units (RLU) were then normalized to the total μg of protein assayed. To convert RLU to μg luciferase, a standard curve was generated with QuantiL μm recombinant luciferase (Promega).

The FLuc mRNA (L-6107) from Trilink Biotechnologies will express a luciferase protein, which was originally isolated from firefly (pHotinus pyralis). Fluc is commonly used in mammalian cell culture to measure gene expression and cell viability. Which emits bioluminescence in the presence of a substrate luciferin. This capped and polyadenylation mRNA is completely replaced by 5-methylcytidine and pseudouridine.

Example 31

Determination of pKa of formulated lipid

The pKa of the formulated cationic lipid is related to the effect of the LNP used to deliver the nucleic acid. The preferred pKa range is 5 to 7. The pKa of each cationic lipid was determined in lipid nanoparticles using an analysis based on fluorescence of 2- (p-toluidinyl) -6-naphthalene sulfonic acid (TNS). Lipid nanoparticles comprising cationic lipid/DSPC/cholesterol/PEG lipid (50/10/38/2 mol%) at a concentration of 0.4mM total lipid in PBS were prepared using an ordered procedure as described in example 27. TNS was prepared as a 100. Mu.M stock solution in distilled water. The vesicles are diluted to contain 24 mu M of lipid in 2mL of buffer solution, wherein the buffer solution contains 10mM HEPES, 10mM MES, 10mM acetic acid and 130mM NaCl, and the pH value is 2.5-11. Aliquots of TNS solution were added to give final concentrations of l. Mu.M, and after vortexing, fluorescence intensities were measured in a SLM Aminco Series-luminescence spectrophotometer at room temperature using excitation and emission wavelengths of 321nm and 445 nm. An sigmoid best fit analysis was applied to the fluorescence data and pKa was measured as pH yielding half maximum fluorescence intensity.

Example 32

Determination of potency of lipid nanoparticle formulations containing various cationic lipids using rodent models of in vivo luciferase mRNA expression

For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the ordered mixing method as described in example 30. Lipid nanoparticles were formulated using a molar ratio of 50% cationic lipid/10% distearoyl phosphatidylcholine (DSPC)/38% cholesterol/2% PEG lipid ("PEG-DMG", i.e., (1- (monomethoxy-polyethylene glycol) -2,3 dimyristoyl glycerol, average PEG molecular weight 2000). The relative activity was determined by measuring luciferase expression in the liver 5 hours after administration via tail vein injection as described in example 30.

Table 2 comparative lipids showing Activity with mRNA

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the disclosure. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A cationic lipid compound which is a compound of one of the following structures:

Compound 24:

Compound 26:

compound 27:

compound 28:

Compound 29:

2. a composition comprising a compound of claim 1 and a therapeutic and/or prophylactic agent.

3. The composition of claim 2, further comprising one or more excipients selected from the group consisting of neutral lipids, steroids, and polymer conjugated lipids.

4. A composition according to claim 3, wherein the neutral lipid is selected from the group consisting of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine and sphingomyelin.

5. The composition of claim 4, wherein the neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine.

6. A composition according to claim 3, wherein the steroid is selected from the group consisting of cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycopersine, ursolic acid, alpha-tocopherol.

7. The composition of claim 6, wherein the steroid is cholesterol.

8. The composition of claim 3, wherein the polymer-conjugated lipid is a pegylated lipid.

9. The composition of claim 8, wherein the pegylated lipid is 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol.

10. The composition of any one of claims 2-9, wherein the therapeutic and/or prophylactic agent is a vaccine or compound capable of eliciting an immune response.

11. The composition of claim 10, wherein the therapeutic and/or prophylactic agent is a nucleic acid.

12. The composition of claim 11, wherein the nucleic acid is a mixture of one or more selected from the group consisting of siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA.

13. The composition of claim 12, wherein the nucleic acid is mRNA.