Cationic lipid compound

Abstract

The present invention relates to lipid compounds, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids and / or their analogs, and / or lipid-conjugated polymers to form lipid nanoparticles for delivering therapeutic and / or prophylactic agents. In some instances, the lipid nanoparticles are used to deliver nucleic acids, such as messenger RNA and / or antisense RNA. Methods of using such lipid nanoparticles to treat and / or prevent various diseases are also provided. In one embodiment, a compound having the structure of formula (I) is provided: or a salt or an isomer or an N-oxide thereof, wherein R 1 , R 2 , R 3 , R 4 and R 5 are as defined herein. Pharmaceutical compositions comprising one or more of the compounds of the foregoing structural formula (I) and a therapeutic and / or prophylactic agent are also provided. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids and lipid-conjugated polymers. Such compositions are useful for forming lipid nanoparticles for delivering therapeutic and / or prophylactic agents. In other embodiments, the present 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 of a lipid nanoparticle comprising a compound of structural formula (I) and a therapeutic and / or prophylactic agent, and delivering the composition to the subject.

Description

Technical Field

[0001] The present invention provides cationic lipids which 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 lipids, the lipid nanoparticle composition of the present invention may further comprise 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 Art

[0002] The effective targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids presents a persistent medical challenge. Specifically, delivering nucleic acids to cells is difficult due to the relative instability and low cell permeability of these species. Therefore, 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.

[0003] It has been demonstrated that lipid-based nanoparticle compositions, liposomes, and lipoplexes can be used effectively to transport bioactive substances such as small molecule drugs, proteins, and nucleic acids to cells and / or intracellular compartments. These compositions generally comprise one or more "cationic" lipids, neutral lipids including polyunsaturated lipids (such as phospholipids), structural lipids (such as steroids), and / or lipids containing polyethylene glycol (polymer-conjugated lipids). Cationic lipids include, for example, amine-containing lipids that can be readily protonated.

[0004] However, the use of oligonucleotides in a therapeutic setting currently faces two problems. First, free RNA is readily digested by nucleases in plasma. Second, the ability of free RNA to enter intracellular compartments where the relevant translation machinery is present is limited. Lipid nanoparticles formed from cationic lipids and other lipid components (such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides) have been used to prevent the degradation of RNA in plasma and to promote the cellular uptake of oligonucleotides.

[0005] There is still 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. Additionally, 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 nucleic acid does not result in unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages. Summary of the Invention

[0006] The present invention provides the following compounds and methods involving these compounds:

[0007] In a first aspect, the present invention relates to compounds of the following structural formula (I):

[0008]

[0009] or their N-oxides, or their salts or isomers.

[0010] wherein R in structural formula “I” 1 , R 2 , R 3 , R 4 and R 5 are each independently a combination of two “hydrogen” isotopes (including the isotopes “protium” and “deuterium”), and cannot be “hydrogen” simultaneously;

[0011] R 1 is independent and can be either “H” or “D”, but not “H” simultaneously with R 2 , R 3 , R 4 and R 5 ;

[0012] R 2 and R 3 are each independent and can be “HH”, “HD” or “DD”, but not “H” simultaneously with R 1 , R 4 and R 5 , that is, a combination containing at least one “D”;

[0013] R 4 and R 5 are each independent and can be “HHH”, “HHD”, “HDD” or “DDD”, but not “H” simultaneously with R 1 , R 2 and R 3 , that is, a combination containing at least one “D”;

[0014] R 1 , R 2 , R 3 , R 4 and R 5Each is a combination of two independent "hydrogen" isotopes (including the isotopes "protium" and "deuterium"), and there are 11 specific combination cases, including combinations containing 1 "D", combinations containing 2 "D", combinations containing 3 "D", combinations containing 4 "D", combinations containing 5 "D", combinations containing 6 "D", combinations containing 7 "D", combinations containing 8 "D", combinations containing 9 "D", combinations containing 10 "D", and combinations containing 11 "D".

[0015] In various different embodiments, the compound has one of the structures shown in Table 1 below

[0016] Table 1 Representative Compounds

[0017]

[0018]

[0019]

[0020] In some embodiments, there is provided a composition comprising any one or more of the compounds of structural formula (I) and a therapeutic agent and / or a prophylactic agent.

[0021] In some embodiments, there is provided a composition comprising any one or more of the compounds of structure (I) and a therapeutic agent and / or a prophylactic agent. In some embodiments, the composition comprises any one of the compounds of structure (I) and a therapeutic agent and / or a 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.

[0022] 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), and sphingomyelin (SM). In some embodiments, the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0023] In some embodiments, the steroid is selected from one or more of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol. In some embodiments, the preferred steroid is cholesterol.

[0024] In some embodiments, the polyethylene glycolylated lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG).

[0025] In some embodiments, the composition ratios are in the following ranges: about 10 to 60 mol% of the compound, about 0 to 30 mol% of neutral lipid, about 10 to 55 mol% of steroid, and about 0 to 10 mol% of polymer-conjugated lipid.

[0026] In some embodiments of the foregoing compositions, the therapeutic and / or prophylactic agent comprises a nucleic acid. The nucleic acid is RNA, which is selected from the group consisting of siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA, and mixtures thereof. In some embodiments, the RNA is selected from mRNA.

[0027] In other different embodiments, the present invention relates to a method of using a therapeutic and / or prophylactic agent in a subject in need thereof, the method comprising preparing or providing any one of the above compositions and administering the composition to the subject.

[0028] For use purposes, the compounds of the present invention can be used as the active pharmaceutical ingredient, or can be formulated into a pharmaceutical composition (usually in the form of a lipid nanoparticle combined with a therapeutic and / or prophylactic agent). The pharmaceutical composition of the present invention comprises a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structure (I) is effective to form lipid nanoparticles and deliver the therapeutic and / or prophylactic agent. Those skilled in the art can readily determine appropriate concentrations and dosages.

[0029] The use of the compositions of the present invention can be carried out by any acceptable use mode of reagents for similar utilities. The pharmaceutical compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, 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, inhaled, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated to render the active ingredient therein bioavailable in a subject. The form of the composition to be used on an object or patient can be one or more dosage forms, wherein the tablet can be a single-dose unit, and the container of the compound in the form of aerosol of the present invention can contain multiple dose units. Current methods for preparing these dosage forms are known or will be apparent to those skilled in the art. In any case, the composition to be used will contain a therapeutically effective amount of the compound of the present invention or its pharmaceutically acceptable salt so as to treat related diseases or conditions according to the teachings of the present invention.

[0030] The pharmaceutical compositions of the present invention can be in solid or liquid form. On the one hand, the carrier can be granules such that the composition is in the form of a tablet or powder. The carrier can also be a liquid, in which case the composition is an oral syrup or an injectable liquid or an aerosol suitable for inhaled use.

[0031] When to be used for oral use, the pharmaceutical composition is preferably in solid or liquid form, wherein the solid or liquid form herein is considered to include semi-solid, semi-liquid, suspension and gel.

[0032] As a solid composition for oral use, the pharmaceutical composition can be formulated into forms such as powder, granule, tablet, pill, capsule, chewing gum, wafer, etc. Such solid compositions generally will contain one or more inert diluents or edible carriers. Additionally, one or more of the following can be present: 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 mint, etc.; and coloring agents.

[0033] When the pharmaceutical composition is in the form of a capsule, it can contain a liquid carrier in addition to the materials of the above types, such as polyethylene glycol or oil.

[0034] The pharmaceutical composition can be in liquid form, such as syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral use or for injectable delivery. When intended for oral use, the preferred compositions contain one or more of sweeteners, preservatives, coloring agents and flavoring agents in addition to the compounds of the present invention. In compositions for use by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents and isotonic agents can be included.

[0035] The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar forms, can include one or more of the following excipients: sterile diluents such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride; non-volatile oils such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerol, propylene glycol or other solvents that can be used as solvents or suspending media; antibacterial agents such as methylparaben, etc.; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetate, citrate or phosphate; and reagents for adjusting tonicity such as sodium chloride or glucose; reagents used as cryoprotectants such as sucrose or trehalose. Parenteral preparations can be encapsulated in ampoules, disposable syringes or multi-dose bottles made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0036] The pharmaceutical compositions of the present invention can be used for topical use, in which case the carrier can suitably comprise a solution matrix, an emulsion matrix, an ointment matrix or a gel matrix. The matrix can comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifying and stabilizing agents. Thickeners can be present in the pharmaceutical compositions for topical use. If intended for transdermal use, the composition can include a transdermal patch or an iontophoresis device.

[0037] The pharmaceutical compositions of the present invention can include various materials that modify the physical form of the solid or liquid dosage forms. The composition can include materials that form a coating shell around the active ingredient. The materials forming the coating shell are usually inert and can be sugars, shellac and other enteric coating reagents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule.

[0038] The pharmaceutical compositions in solid or liquid form of the present invention can include carriers that bind to the compounds of the present invention and facilitate the delivery of the compounds. Such carriers include monoclonal or polyclonal antibodies or proteins.

[0039] The pharmaceutical composition of the present invention may consist of a formulation that can be used as an aerosol. The term "aerosol" refers to a system with colloidal properties and a system composed of a pressurized package. It can be delivered by liquefied gas or compressed gas, or by a suitable pump system for dispersing the active ingredient. The aerosol of the compound of the present invention can be delivered in a single-phase, two-phase system or three-phase system to deliver the active ingredient. The delivery of the aerosol includes necessary containers, activators, valves, sub-containers, etc., which together can form a dosing device. A person skilled in the art can determine the preferred aerosol without additional experiments.

[0040] The pharmaceutical composition of the present invention can be prepared by methods well known in the pharmaceutical field. The pharmaceutical composition for injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a solution. A surfactant can be added to promote the formation of a uniform solution or suspension. The surfactant interacts non-covalently with the compound of the present invention, thereby promoting the dissolution or uniform suspension of the compound in a water-soluble medium.

[0041] The composition of the present invention or its pharmaceutically acceptable salts are used in a therapeutically effective amount, and this amount will vary according to various factors, including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the age, weight, general health status, gender and diet of the subject; the mode and time of use; the excretion rate; drug combinations; the severity of the specific case, etc.

[0042] The composition of the present invention can also be used simultaneously with, before or after using one or more other therapeutic agents. Such therapeutic combinations include preparations using the composition of the present invention alone and combinations using the composition of the present invention and one or more other active ingredients. For example, the composition of the present invention and other active ingredients can be used together with a subject in a single oral dosage formulation (such as a tablet or capsule), or each active ingredient can be used in different oral dosage formulations. When using different dosage formulations, the compound of the present invention and one or more additional active ingredients can be used at the same time, or used sequentially at staggered times; it should be understood that combination therapy includes all these dosing regimens.

[0043] The structural modification and design of the above-mentioned deuterated cationic lipid compounds have achieved more advantageous physical and chemical properties, including more appropriate pKa and better chemical stability, for mRNA nanoliposome compositions, which can achieve more effective binding and delivery of ionic nucleic acid drugs. At the same time, its chemical structure is more stable, facilitating synthesis and being conducive to development as a pharmaceutical excipient.

[0044] The preparation methods of the above-mentioned compounds and compositions are described below and / or are known in the art.

[0045] 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 include trialkylsilyl or diarylalkylsilyl, tetrahydrofuranyl, benzyl, etc. Suitable protecting groups for amino include tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for carboxylic acid include hydroxyl, aryl, or aralkyl esters. The protecting groups can be added or removed according to standard techniques, which are known to those skilled in the art and described herein.

[0046] Those skilled in the art will also recognize that although such protected derivatives of the compounds of the present invention may not thereby have pharmacological activity, they can be administered to mammals and then metabolized in vivo to form the pharmacologically active compounds of the present invention. Such derivatives can therefore be described as "prodrugs". Accordingly, prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0047] In addition, all compounds of the present invention in the form of the free base or free acid 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. The salts of the compounds of the present invention can be converted into their free base or acid form by standard techniques.

[0048] The following examples are provided for purposes of illustration and not limitation.

[0049] In the following examples, unless otherwise indicated, all solvents and reagents used are commercially available and used as received.

[0050] The procedures described below can be used to synthesize Compound I in Table 1.

[0051] The following abbreviations are used herein:

[0052] Detailed Description

[0053] Example 1:

[0054] Representative Route

[0055] Synthesis of Compound 8

[0056]

[0057] 1) Synthesis of Compound 8-02

[0058] Chemical formula: C 41 H 71 D 2 BrO 2

[0059] Molecular weight: 679.95

[0060] To a mixed solution of compound A (5.3 g, 10.0 mmol) and 4,4-d 2 -bromobutyric acid (2.5 g, 15.0 mmol) in DCM, EDC.HCl (2.1 g, 11.0 mmol), DIEA (5.3 ml, 30.0 mmol), and DMAP (0.2 g, 1.5 mmol) were added successively. The reaction was carried out at 45 °C for 2 h. Then the system was diluted with DCM and washed successively with saturated aqueous sodium bicarbonate solution and dilute hydrochloric acid aqueous solution, dried over magnesium sulfate, filtered and concentrated. The obtained residue was purified by silica gel column (0 - 15% ethyl acetate / hexane). Compound 8-02 (5.1 g, 75%) was obtained.

[0061] 2) Synthesis of compound 8

[0062]

[0063] Chemical formula: C 43 H 77 D 2 NO 2

[0064] Molecular weight: 644.12

[0065] 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), K 2 CO 3 (0.27 g, 2.0 mmol) and potassium iodide (0.017 g, 0.1 mmol) were added, and the temperature was raised to 65 °C and stirred for 24 h. After the raw materials reacted completely, the temperature of the system was lowered to room temperature. Ethyl acetate was added to the system, and the organic phase was washed successively with 20 mL of water and 20 mL of saturated saline solution. After drying over anhydrous sodium sulfate, it was concentrated under reduced pressure. The residue was purified by silica gel column (0 - 100% (a mixture of 1% NH 4 OH, 20% MeOH in dichloromethane) dichloromethane) to obtain compound 8 (0.58 g, 90%). C 43 H 77 D 2 NO 2 , Ms m / z: [M + H + 644.6; 1H-NMR (300 MHz): δ 5.4 - 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).

[0066] Example 2:

[0067] Synthesis of Compound 1

[0068]

[0069] Chemical formula: C 43 H 78 DNO 2

[0070] Molecular weight: 643.12

[0071] Compound 1 can be synthesized according to the representative route described in Example 1.

[0072] C 43 H 78 DNO 2 , Ms m / z: [M + H + 643.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: 1 H-NMR (300 MHz): δ 5.4 - 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).

[0073] Example 3:

[0074] Synthesis of Compound 2

[0075]

[0076] Chemical formula: C 43 H 78 DNO 2

[0077] Molecular weight: 643.12

[0078] Compound 2 can be synthesized according to the representative route described in Example 1.

[0079] C 43H 78 DNO 2 ,Ms m / z: [M+H + 643.6; 1 H-NMR(300MHz, CDCl 3 ) δ 5.4 - 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).

[0080] Example 4:

[0081] Synthesis of Compound 3

[0082]

[0083] Chemical formula: C 43 H 78 DNO 2

[0084] Molecular weight: 643.12

[0085] Compound 3 can be synthesized according to the representative route described in Example 1.

[0086] C 43 H 78 DNO 2 ,Ms m / z: [M+H + 643.6; 1 H-NMR(300MHz, CDCl 3 ) δ 5.4 - 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).

[0087] Example 5:

[0088] Synthesis of Compound 4

[0089]

[0090] Chemical formula: C 43 H 78 DNO 2

[0091] Molecular weight: 643.12

[0092] Compound 4 can be synthesized according to the representative route described in Example 1.

[0093] C 43 H 78 DNO 2 , Ms m / z: [M+H + 643.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0094] Example 6:

[0095] Synthesis of Compound 5

[0096]

[0097] Chemical formula: C 43 H 77 D 2 NO 2

[0098] Molecular weight: 644.12

[0099] Compound 5 can be synthesized according to the representative route described in Example 1.

[0100] C 43 H 77 D 2 NO 2 , Ms m / z: [M+H + 644.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0101] Example 7:

[0102] Synthesis of Compound 6

[0103]

[0104] Chemical formula: C 43 H 77 D 2 NO 2

[0105] Molecular weight: 644.12

[0106] Compound 6 can be synthesized according to the representative route described in Example 1.

[0107] C 43 H 77 D 2 NO 2 , Ms m / z: [M+H + 644.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0108] Example 8:

[0109] Synthesis of Compound 7

[0110]

[0111] Chemical formula: C 43 H 77 D 2 NO 2

[0112] Molecular weight: 644.12

[0113] Compound 7 can be synthesized according to the representative route described in Example 1.

[0114] C 43 H 77 D 2 NO 2 , Ms m / z: [M+H + 644.6; 1 H-NMR (300 MHz, CDCl 3)δ 5.4 - 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).

[0115] Example 9:

[0116] Synthesis of Compound 9

[0117]

[0118] Chemical formula: C 43 H 77 D 2 NO 2

[0119] Molecular weight: 644.12

[0120] Compound 9 can be synthesized according to the representative route described in Example 1.

[0121] C 43 H 77 D 2 NO 2 , Ms m / z: [M + H + 644.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0122] Example 10:

[0123] Synthesis of Compound 10

[0124]

[0125] Chemical formula: C 43 H 77 D 2 NO 2

[0126] Molecular weight: 644.12

[0127] Compound 10 can be synthesized according to the representative route described in Example 1.

[0128] C43 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)。

[0129] Example 11:

[0130] Synthesis of Compound 11

[0131]

[0132] Chemical formula: C 43 H 76 D 3 NO 2

[0133] Molecular weight: 645.12

[0134] Compound 11 can be synthesized according to the representative route described in Example 1.

[0135] 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)。

[0136] Example 12:

[0137] Synthesis of Compound 12

[0138]

[0139] Chemical formula: C 43 H 76 D3 NO 2

[0140] Molecular weight: 645.12

[0141] Compound 12 can be synthesized according to the representative route described in Example 1.

[0142] C 43 H 76 D 3 NO 2 , Ms m / z: [M+H + 645.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0143] Example 13:

[0144]

[0145] Chemical formula: C 43 H 76 D 3 NO 2

[0146] Molecular weight: 645.12

[0147] Compound 13 can be synthesized according to the representative route described in Example 1.

[0148] C 43 H 76 D 3 NO 2 , Ms m / z: [M+H + 645.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0149] Example 14:

[0150] Synthesis of Compound 14

[0151]

[0152] Chemical formula: C 43 H 75 D 4 NO 2

[0153] Molecular weight: 646.12

[0154] Compound 14 can be synthesized according to the representative route described in Example 1.

[0155] C 43 H 75 D 4 NO 2 , Ms m / z: [M+H + 646.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0156] Example 15:

[0157] Synthesis of Compound 15

[0158]

[0159] Chemical formula: C 43 H 75 D 4 NO 2

[0160] Molecular weight: 646.12

[0161] Compound 15 can be synthesized according to the representative route described in Example 1.

[0162] C 43 H 75 D 4 NO 2 , Ms m / z: [M+H + 646.6; 1 H-NMR (300 MHz, CDCl 3)δ 5.4 - 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).

[0163] Example 16:

[0164] Synthesis of Compound 16

[0165]

[0166] Chemical formula: C 43 H 75 D 4 NO 2

[0167] Molecular weight: 646.12

[0168] Compound 16 can be synthesized according to the representative route described in Example 1.

[0169] C 43 H 75 D 4 NO 2 , Ms m / z: [M + H + 646.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0170] Example 17:

[0171] Synthesis of Compound 17

[0172]

[0173] Chemical formula: C 43 H 75 D 4 NO 2

[0174] Molecular weight: 646.12

[0175] Compound 17 can be synthesized according to the representative route described in Example 1.

[0176] C 43 H 75 D4 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).

[0177] Example 18:

[0178]

[0179] Synthesis of Compound 18

[0180] Chemical formula: C 43 H 74 D 5 NO 2

[0181] Molecular weight: 647.12

[0182] Compound 18 can be synthesized according to the representative route described in Example 1.

[0183] 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).

[0184] Example 19:

[0185] Synthesis of Compound 19

[0186]

[0187] Chemical formula: C 43 H 74 D 5 NO 2

[0188] Molecular weight: 647.12

[0189] Compound 19 can be synthesized according to the representative route described in Example 1.

[0190] C 43 H 74 D 5 NO 2 , Ms m / z: [M+H + 647.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0191] Example 20:

[0192] Synthesis of Compound 20

[0193]

[0194] Chemical formula: C 43 H 74 D 5 NO 2

[0195] Molecular weight: 647.12

[0196] Compound 20 can be synthesized according to the representative route described in Example 1.

[0197] C 43 H 74 D 5 NO 2 , Ms m / z: [M+H + 647.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0198] Example 21:

[0199] Synthesis of Compound 21

[0200]

[0201] Chemical formula: C 43 H 74 D 5 NO 2

[0202] Molecular weight: 647.12

[0203] Compound 21 can be synthesized according to the representative route described in Example 1.

[0204] C 43 H 74 D 5 NO 2 , Ms m / z: [M+H + 647.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0205] Example 22:

[0206] Synthesis of Compound 22

[0207]

[0208] Chemical formula: C 43 H 73 D 6 NO 2

[0209] Molecular weight: 648.12

[0210] Compound 22 can be synthesized according to the representative route described in Example 1.

[0211] C 43 H 73 D 6 NO 2 , Ms m / z: [M+H + 648.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0212] Example 23:

[0213] Synthesis of Compound 23

[0214]

[0215] Chemical formula: C 43 H 72 D 7 NO 2

[0216] Molecular weight: 649.12

[0217] Compound 23 can be synthesized according to the representative route described in Example 1.

[0218] C 43 H 72 D 7 NO 2 , Ms m / z: [M+H + 649.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0219] Example 24:

[0220] Synthesis of Compound 24

[0221]

[0222] Chemical formula: C 43 H 71 D 8 NO 2

[0223] Molecular weight: 650.13

[0224] Compound 24 can be synthesized according to the representative route described in Example 1.

[0225] C 43 H 71 D 8 NO 2 , Ms m / z: [M+H + 650.7; 1 H-NMR (300 MHz, CDCl 3)δ 5.4 - 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).

[0226] Example 25:

[0227] Synthesis of Compound 25

[0228]

[0229] Chemical formula: C 43 H 70 D 9 NO 2

[0230] Molecular weight: 651.13

[0231] Compound 25 can be synthesized according to the representative route described in Example 1.

[0232] C 43 H 70 D 9 NO 2 , Ms m / z: [M + H + 651.6; 1 H-NMR (300 MHz, CDCl 3 ) δ 5.4 - 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).

[0233] Example 26:

[0234] Synthesis of Compound 26

[0235]

[0236] Chemical formula: C 43 H 69 D 10 NO 2

[0237] Molecular weight: 652.13

[0238] Compound 26 can be synthesized according to the representative route described in Example 1.

[0239] C 43 H 69 D 10 NO2 , Ms m / z: [M+H + 652.6; 1 H-NMR(300MHz, CDCl 3 ) δ 5.4 - 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).

[0240] Example 27:

[0241] Synthesis of Compound 27

[0242]

[0243] Chemical formula: C 43 H 69 D 10 NO 2

[0244] Molecular weight: 652.13

[0245] Compound 27 can be synthesized according to the representative route described in Example 1.

[0246] C 43 H 69 D 10 NO 2 , Ms m / z: [M+H + 652.6; 1 H-NMR(300MHz, CDCl 3 ) δ 5.4 - 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).

[0247] Example 28:

[0248] Synthesis of Compound 28

[0249]

[0250] Chemical formula: C 43 H 69 D 10 NO 2

[0251] Molecular weight: 652.13

[0252] Compound 28 can be synthesized according to the representative route described in Example 1.

[0253] 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).

[0254] Example 29:

[0255] Synthesis of Compound 29

[0256]

[0257] Chemical formula: C 43 H 68 D 11 NO 2

[0258] Molecular weight: 653.13

[0259] Compound 29 can be synthesized according to the representative route described in Example 1.

[0260] 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).

[0261] Example 30

[0262] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0263] Dissolve cationic lipid, DSPC, cholesterol, and PEG-lipid in ethanol at a molar ratio of 50:10:38:2 or 48:10:40:2. Prepare lipid nanoparticles (LNPs) at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, dilute mRNA to 0.15 mg / mL in 10 mL to 50 mL of citrate buffer (pH = 4.0). Using a syringe pump, mix the ethanol solution of the lipid with the aqueous mRNA solution at a ratio of approximately 1:5 to 1:3 (volume / volume) with a total flow rate of more than 10 mL / min. Then remove the ethanol and replace the external buffer with PBS by dialysis. Finally, filter the lipid nanoparticles through a sterile filter with a pore size of 0.2 μm. The particle size of the lipid nanoparticles determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS is approximately 65 - 105 nm in diameter, and in some cases, approximately 75 - 100 nm in diameter.

[0264] Conduct studies on female C57BL / 6 mice at 6 - 8 weeks of age and CD-1 mice at 8 - 10 weeks of age according to the guidelines established by the National Science and Technology Council. Systemically administer different doses of mRNA lipid nanoparticles by tail vein injection and euthanize the animals at specific time points after administration (e.g., 5 hours). Collect the liver and spleen in pre-weighed tubes, determine the weight, immediately snap-freeze in liquid nitrogen, and store at -80 °C until used for analysis.

[0265] For the liver, approximately 50 mg was cut for analysis in a 2 mL FastPrep tube (MP Biomedicals, Solon OH). A 1 / 4" ceramic bead (MP Biomedicals) was added to each tube, and 500 μL of Glo Lysis Buffer - GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue. The liver tissue was homogenized at 2 × 6.0 m / s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). The homogenate was incubated for 5 minutes at room temperature, then diluted 1:4 in GLB and evaluated using the SteadyGlo Luciferase Assay System (Promega). Specifically, 50 μL of the diluted tissue homogenate was reacted with 50 μL of the SteadyGlo substrate, shaken for 10 seconds, then incubated for 5 minutes, and then quantified using a SpectraMAX_L chemiluminescent microplate reader (Molecular Devices (Shanghai) Co., Ltd.). The amount of protein assayed was determined by using a BCA Protein Assay Kit (Shanghai Yise Medical Technology Co., Ltd.). The relative light units (RLU) were then normalized to the total μg of protein assayed. To convert RLU to μg luciferase, a standard curve was generated using QuantiL μM recombinant luciferase (Promega).

[0266] FLuc mRNA (L - 6107) from Trilink Biotechnologies will express the luciferase protein, which was originally isolated from the firefly (Photinus pyralis). Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely substituted with 5 - methylcytidine and pseudouridine.

[0267] Example 31

[0268] Determination of the pKa of the formulated lipid

[0269] The pKa of the formulated cationic lipid is related to the efficacy of the LNP used for nucleic acid delivery. The preferred pKa range is 5 - 7. The pKa of each cationic lipid is determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). As described in Example 27, an ordered method is used to prepare lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG lipid (50 / 10 / 38 / 2 mol%) at a total lipid concentration of 0.4 mM in PBS. TNS is prepared as a 100 μM stock solution in distilled water. The vesicles are diluted to contain 24 μM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, where the pH ranges from 2.5 to 11. Equal aliquots of the TNS solution are added to give a final concentration of 1 μM, and after vortex mixing, the fluorescence intensity is measured in an SLM Aminco Series 2 luminescence spectrophotometer at excitation and emission wavelengths of 321 nm and 445 nm at room temperature. A sigmoidal best-fit analysis is applied to the fluorescence data, and the pKa is measured as the pH that gives half-maximal fluorescence intensity.

[0270] Example 32

[0271] The efficacy of lipid nanoparticle formulations containing various cationic lipids was determined using a rodent model of in vivo luciferase mRNA expression.

[0272] 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 the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38% cholesterol / 2% PEG lipid ("PEG-DMG", i.e., (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, average PEG molecular weight of 2000). As described in Example 30, the relative activity was determined by measuring luciferase expression in the liver 5 hours after administration via tail vein injection. The activity was compared at doses of 0.3 and 1.0 mg mRNA / kg and expressed as ng luciferase / g liver measured 5 hours after administration as described in Example 30. The results of Examples 31 and 32 are shown in Table 2.

[0273] Table 2 Comparison of lipids showing activity with mRNA

[0274]

[0275]

[0276]

[0277]

[0278] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0279] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the disclosed patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A cationic lipid compound, which is a compound having one of the following structures: Compound 24: Compound 26: Compound 27: Compound 28: Compound 29:

2. A composition comprising the compound according to claim 1 and a therapeutic agent and / or a prophylactic agent.

3. The composition according to claim 2, which further comprises one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

4. The composition according to claim 3, wherein the neutral lipid is selected from one or more substances mixed from the following: 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin.

5. The composition according to claim 4, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine.

6. The composition according to claim 3, wherein the steroid is selected from one or more substances mixed from the following: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol.

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

8. The composition according to claim 3, wherein the polymer-conjugated lipid is a polyethylene glycolated lipid.

9. The composition according to claim 8, wherein the polyethylene glycolated lipid is 1,2-dimyristoyl-sn-glycero-methoxypolyethylene glycol.

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

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

12. The composition according to claim 11, wherein the nucleic acid is selected from one or more substances mixed from the following: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA.

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

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