Cationic lipid compound

Abstract

The present invention relates to lipid compounds of formula (I), 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 lipids conjugated with polymers to form lipid nanoparticles for delivering therapeutic and / or prophylactic agents. In some examples, the 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 to treat and / or prevent various diseases. The structure of formula (I) is as follows: or a salt or isomer or N-oxide thereof, wherein R1 is as defined herein.

Description

Technical Field

[0001] The present invention provides a cationic lipid which can be used in combination with other lipid components (such as neutral lipids, steroids, and lipid-polymer conjugates) to form a nucleic acid mRNA lipid nanoparticle composition for delivering one or more therapeutic and / or prophylactic agents to mammalian cells or organs and / or for producing polypeptides in mammalian cells or organs. In addition to the lipid, the lipid nanoparticle composition of the present invention may further include one or more cationic and / or ionizable amino lipids, neutral lipids including polyunsaturated lipids, lipid-polymer conjugates, 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 poses a persistent medical challenge. Specifically, delivering nucleic acids to cells is difficult due to the relative instability and low cellular 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 as delivery vehicles to effectively transport bioactive substances such as small molecule drugs, proteins, and nucleic acids to cells and / or intracellular compartments. These compositions generally include one or more "cationic" lipids, neutral lipids (such as phospholipids) including polyunsaturated lipids, structural lipids (such as steroids), and / or lipids containing polyethylene glycol (lipid-polymer conjugates). Cationic lipids include 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 susceptible to nuclease digestion 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. Preferably, these lipid nanoparticles would provide an optimized drug:lipid ratio, 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 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 is not associated with 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 , and R 4 are each independently a combination of 2 “hydrogen” isotopes (including the isotopes “protium” and “deuterium”), specifically represented as combinations of “HH”, “HD”, and “DD”;

[0011] R 1 and R 2 are independently selected from any one group consisting of “HH”, “HD”, and “DD”;

[0012] R 3 and R 4 are independently selected from any one group consisting of “HH, HD”, “HD, HH”, “HH, DD”, “DD, HH”, “HD, DD”, and “DD, DD”;

[0013] R 1 , R 2 , R 3 , and R 4 are each independent, but cannot all be “HH” at the same time, that is, at least one “HD” is included;

[0014] In various different embodiments, the compound has one of the structures shown in Table 1 below

[0015] Table 1 Representative Compounds

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028] In some embodiments, there are provided compositions comprising any one or more of the compounds of structural formula (I) and a therapeutic and / or prophylactic agent.

[0029] In some embodiments, there are provided compositions 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 neutral lipids, steroids, and polymer-conjugated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in the various embodiments of the composition.

[0030] In some embodiments, the neutral lipid is selected from 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), and mixtures thereof. In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0031] In some embodiments, the steroid is selected from cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the steroid is cholesterol.

[0032] In some embodiments, the polyethylene glycolated lipid is 1,2-dimyristoyl-sn-glycero methoxypolyethylene glycol (PEG-DMG)

[0033] 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.

[0034] In some embodiments of the foregoing compositions, the therapeutic and / or prophylactic agent comprises a nucleic acid. Wherein 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.

[0035] In other different embodiments, the present 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 one of the above compositions and administering the composition to the subject.

[0036] For the purpose of administration, the compounds of the present invention (usually in the form of lipid nanoparticles combined with a therapeutic and / or prophylactic agent) can be administered as a crude chemical, or can be formulated into a pharmaceutical composition. 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 easily determine the appropriate concentration and dosage.

[0037] The administration of the compositions of the present invention can be carried out by any acceptable mode of administration for reagents of similar utility. 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 administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the active ingredient contained therein to be bioavailable after administration of the composition to a subject. The composition to be administered to a subject or patient is in the form of one or more dosage units, wherein a tablet can be a single dosage unit, and a container of the compound in aerosol form of the present invention can contain multiple dosage units. Current methods of preparing these dosage forms are known or will be apparent to those skilled in the art. In any case, the composition to be administered will contain a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof to treat the relevant disease or condition according to the teachings of the present invention.

[0038] The pharmaceutical composition of the present invention can be in solid or liquid form. On the one hand, the carrier is particulate, such that the composition is in the form of tablets or powders. The carrier can be liquid, in which case the composition is an oral syrup or an injectable liquid or an aerosol, the aerosol being suitable for inhalation administration.

[0039] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where forms considered solid or liquid herein include semi-solid, semi-liquid, suspension and gel forms.

[0040] As a solid composition for oral administration, the pharmaceutical composition can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, etc. Such solid compositions will generally contain one or more inert diluents or edible carriers. Additionally, one or more of the following may 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 peppermint, etc.; and coloring agents.

[0041] 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.

[0042] 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 administration or for injectable delivery. When intended for oral administration, the preferred composition contains, in addition to the compound of the present invention, one or more of sweeteners, preservatives, coloring agents and flavoring agents. In the composition administered by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers and isotonic agents can be included.

[0043] The liquid pharmaceutical composition of the present invention, whether it is in solution, suspension or other similar forms, can include one or more of the following adjuvants: sterile diluents 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, 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 enclosed in ampoules, disposable syringes or multi-dose bottles made of glass or plastic. Physiological saline is the preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0044] The liquid pharmaceutical composition of the present invention intended for parenteral or oral administration should contain an amount of the compound of the present invention that allows for an appropriate dose to be obtained.

[0045] The pharmaceutical composition of the present invention can be intended for topical administration, 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 composition for topical administration. If intended for transdermal administration, the composition can include a transdermal patch or an iontophoretic device.

[0046] The pharmaceutical composition of the present invention can be intended for rectal administration, in the form of a suppository, which dissolves and releases the drug in the rectum. The composition for rectal administration can contain an oily matrix as a suitable non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0047] The pharmaceutical composition of the present invention can include various materials that modify the physical form of solid or liquid dosage units. The composition can include materials that form a coating shell around the active ingredient. The materials forming the coating shell are generally inert and can be, for example, sugar, shellac, and other enteric coating reagents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule.

[0048] The pharmaceutical composition of the present invention in solid or liquid form can include reagents that bind to the compound of the present invention and thus assist in the delivery of the compound. Suitable reagents that can act in this capacity include monoclonal or polyclonal antibodies or proteins.

[0049] The pharmaceutical composition of the present invention can consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from colloidal systems to systems consisting of pressurized packages. It can be delivered by a liquefied gas or a compressed gas, or by a suitable pump system that disperses the active ingredient. The aerosol of the compound of the present invention can be delivered in a single-phase, two-phase or three-phase system in order to deliver the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together can form a kit. A person skilled in the art can determine the preferred aerosol without undue experimentation.

[0050] The pharmaceutical composition of the present invention can be prepared by methods well known in the pharmaceutical art. The pharmaceutical composition intended for administration by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a solution. Surfactants can be added to facilitate the formation of a uniform solution or suspension. A surfactant is a compound that interacts non-covalently with the compound of the present invention in order to facilitate the dissolution or uniform suspension of the compound in an aqueous delivery system.

[0051] The composition of the present invention or a pharmaceutically acceptable salt thereof is administered in a therapeutically effective amount, which will vary depending on a variety of 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, gender and diet of the subject; the mode and time of administration; the rate of excretion; drug combinations; the severity of the particular case, etc.

[0052] The composition of the present invention can also be administered simultaneously with, before or after the administration of one or more other therapeutic agents. Such combination therapies include single pharmaceutical dosage formulations that administer the composition of the present invention and one or more additional active agents, as well as the administration of the composition of the present invention and the active agents in their own separate pharmaceutical dosage formulations. For example, the composition of the present invention and other active agents can be administered to a subject together in a single oral dosage composition (such as a tablet or capsule), or each agent can be administered in a different oral dosage formulation. When different dosage formulations are used, the compounds of the present invention and one or more additional active agents can be administered at substantially the same time, or sequentially at staggered times; it should be understood that combination therapies include all such dosing regimens.

[0053] The methods for preparing the above-mentioned compounds and compositions are described below and / or are known in the art.

[0054] 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 alkyl, 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.

[0055] Those skilled in the art will also recognize that although such protected derivatives of the compounds of the present invention may not thereby have pharmaceutical activity, they can be administered to mammals and then metabolized in vivo to form the pharmaceutically active compounds of the present invention. Such derivatives can therefore be described as "prodrugs". Therefore, prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0056] In addition, all compounds of the present invention in the form of free base or free acid can be treated with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art to be converted into their pharmaceutically acceptable salts. The salts of the compounds of the present invention can be converted into their free base or acid forms by standard techniques.

[0057] The following examples are provided for illustrative purposes only and are not limiting.

[0058] In the following examples, all solvents and reagents used are commercially available and used as received, unless otherwise indicated.

[0059] The procedures described below can be used to synthesize Compound I in Table 1.

[0060] The following abbreviations are used herein:

[0061] EDC.HCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0062] DCM: Dichloromethane

[0063] DMAP: 4-Dimethylaminopyridine

[0064] DIEA: N,N-Diisopropylethylamine

[0065] EtOH: Ethanol

[0066] K 2 CO 3 : Potassium carbonate

[0067] AcN: Acetonitrile

[0068] KI: Potassium iodide Detailed Description of the Invention

[0069] Example 1:

[0070] Representative Route

[0071] Synthesis of Compound 28

[0072]

[0073] 1) Synthesis of Compound C

[0074]

[0075] Chemical Formula: C 15 H 29 BrO 2

[0076] Molecular Weight: 361.36

[0077] EDC·HCl (2.08 g, 11.0 mmol), DIEA (5.30 mL, 30.0 mmol), and DMAP (0.18 g, 1.5 mmol) were successively added to a DCM mixture of 6-bromohexanoic acid (2.91 g, 15.0 mmol) and n-undecanol (1.72 g, 10.0 mmol). The reaction was carried out at room temperature for 24 h. Then the system was diluted with DCM and washed successively with saturated aqueous sodium bicarbonate and dilute aqueous hydrochloric acid, dried over magnesium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column (0 - 15% ethyl acetate / hexane) to obtain compound C (2.61 g, 75%).

[0078] 2) Synthesis of compound 28-E

[0079]

[0080] Chemical formula: C 19 H 35 D 4 NO 3

[0081] Molecular weight: 333.55

[0082] Compound C (3.48 g, 10.0 mmol) was mixed with 10 mL of ethanol, and 1,1,2,2-d 4 -2-aminoethanol (1.95 g, 30.0 mmol) was added. The reaction was carried out at room temperature for 4 h. The system was concentrated under vacuum. Ethyl acetate and water were added to the residue, and the organic phase was separated and dried over anhydrous sodium sulfate, then concentrated under vacuum. 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 28-E (2.96 g, 89%).

[0083] 3) Synthesis of compound F

[0084]

[0085] Chemical formula: C 25 H 49 BrO 2

[0086] Molecular weight: 461.57

[0087] Compound F can be prepared by referring to the synthesis method of C.

[0088] 4) Synthesis of compound 28

[0089]

[0090] Chemical formula: C42 H 83 D 4 NO 5

[0091] Molecular weight: 714.21

[0092] To a mixture of compound 28-E (0.69 g, 1.0 mmol) and F (0.69 g, 1.5 mmol) in acetonitrile was added K 2 CO 3 (0.27 g, 2.0 mmol) and potassium iodide, and the mixture was heated to 65 °C, stirred for 24 h, cooled, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and the organic phase was washed with saturated brine solution. After drying over anhydrous sodium sulfate, it was concentrated in vacuo. The residue was purified by silica gel column (0 - 100% (mixture of 1% NH 4 OH, 20% MeOH in dichloromethane) dichloromethane) to obtain compound 28 (0.62 g, 90%). C 42 H 83 D 4 NO 5 , Ms m / z: [M + H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.86 (t, 1H), 4.05 (m, 2H), 2.55 - 2.25 (br.m, 5H), 2.28 (m, 4H), 1.88 - 1.41 (m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0093] Example 2:

[0094] Synthesis of Compound 1

[0095]

[0096] Chemical formula: C 44 H 86 DNO 5

[0097] Molecular weight: 711.19

[0098] Compound 1 can be synthesized according to the representative route described in Example 1.

[0099] C 44 H 86 DNO 5 , Ms m / z: [M + H + 711.6; 1 H-NMR (300 MHz, CDCl 3)δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66 - 3.46 (br.m, 1H), 2.76 - 2.36 (br.m, 7H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0100] Example 3:

[0101] Synthesis of Compound 2

[0102]

[0103] Chemical formula: C 44 H 86 DNO 5

[0104] Molecular weight: 711.19

[0105] Compound 2 can be synthesized according to the representative route described in Example 1.

[0106] C 44 H 86 DNO 5 , Ms m / z: [M + H + 711.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.84 (t, 1H), 4.08 (t, 2H), 3.66 - 3.46 (br.m, 2H), 2.74 - 2.34 (br.m, 6H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0107] Example 4:

[0108] Synthesis of Compound 3

[0109]

[0110] Chemical formula: C 44 H 86 DNO 5

[0111] Molecular weight: 711.19

[0112] Compound 3 can be synthesized according to the representative route described in Example 1.

[0113] C 44 H 86 DNO 5 , Ms m / z: [M + H+ 711.6; 1 H-NMR(300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.64~3.44 (br.m, 2H), 2.74~2.34 (br.m, 6H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0114] Example 5:

[0115] Synthesis of Compound 4

[0116]

[0117] Chemical formula: C 44 H 86 DNO 5

[0118] Molecular weight: 711.19

[0119] Compound 4 can be synthesized according to the representative route described in Example 1.

[0120] C 44 H 86 DNO 5 , Ms m / z: [M + H + 711.6; 1 H-NMR(300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.64~3.44 (br.m, 2H), 2.74~2.34 (br.m, 6H), 2.28 (m, 4H), 1.76~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0121] Example 6:

[0122] Synthesis of Compound 5

[0123]

[0124] Chemical formula: C 44 H 85 D 2 NO 5

[0125] Molecular weight: 712.19

[0126] Compound 5 can be synthesized according to the representative route described in Example 1.

[0127] C 44 H 85 D 2 NO 5 ,Ms m / z: [M+H + 712.6; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83(t, 1H), 4.08(t, 2H), 2.76~2.36(br.m, 7H), 2.28(m, 4H), 1.78~1.42(br.m, 12H), 1.38~1.17(m, 50H), 0.87(m, 9H).

[0128] Example 7:

[0129] Synthesis of Compound 6

[0130]

[0131] Chemical formula: C 44 H 85 D 2 NO 5

[0132] Molecular weight: 712.19

[0133] Compound 6 can be synthesized according to the representative route described in Example 1.

[0134] C 44 H 85 D 2 NO 5 ,Ms m / z: [M+H + 712.6; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83(t, 1H), 4.08(t, 2H), 3.64~3.44(br.m, 1H), 2.74~2.34(br.m, 6H), 2.28(m, 4H), 1.78~1.42(br.m, 12H), 1.38~1.17(m, 50H), 0.87(m, 9H).

[0135] Example 8:

[0136] Synthesis of Compound 7

[0137]

[0138] Chemical formula: C 44 H 85 D 2 NO5

[0139] Molecular weight: 712.19

[0140] Compound 7 can be synthesized according to the representative route described in Example 1.

[0141] C 44 H 85 D 2 NO 5 , Ms m / z: [M+H + 712.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.64~3.44 (br.m, 1H), 2.75~2.35 (br.m, 6H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0142] Example 9:

[0143] Synthesis of Compound 8

[0144]

[0145] Chemical formula: C 44 H 85 D 2 NO 5

[0146] Molecular weight: 712.19

[0147] Compound 8 can be synthesized according to the representative route described in Example 1.

[0148] C 44 H 85 D 2 NO 5 , Ms m / z: [M+H + 712.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.64~3.44 (br.m, 1H), 2.75~2.35 (br.m, 6H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0149] Example 10:

[0150] Synthesis of Compound 9

[0151]

[0152] Chemical formula: C 44 H 85 D 2 NO 5

[0153] Molecular weight: 712.19

[0154] Compound 9 can be synthesized according to the representative route described in Example 1.

[0155] C 44 H 85 D 2 NO 5 , Ms m / z: [M+H + 712.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66~3.46 (br.m, 2H), 2.76~2.36 (br.m, 5H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0156] Example 11:

[0157] Synthesis of Compound 10

[0158]

[0159] Chemical formula: C 44 H 85 D 2 NO 5

[0160] Molecular weight: 712.19

[0161] Compound 10 can be synthesized according to the representative route described in Example 1.

[0162] C 44 H 85 D 2 NO 5 , Ms m / z: [M+H + 712.6; 1 H-NMR (300 MHz, CDCl 3)δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66 - 3.46 (br.m, 2H), 2.76 - 2.36 (br.m, 6H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0163] Example 12:

[0164] Synthesis of Compound 11

[0165]

[0166] Chemical formula: C 44 H 85 D 2 NO 5

[0167] Molecular weight: 712.19

[0168] Compound 11 can be synthesized according to the representative route described in Example 1.

[0169] C 44 H 85 D 2 NO 5 , Ms m / z: [M + H + 712.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.65 - 3.45 (br.m, 2H), 2.73 - 2.33 (br.m, 5H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0170] Example 13:

[0171] Synthesis of Compound 12

[0172]

[0173] Chemical formula: C 44 H 85 D 2 NO 5

[0174] Molecular weight: 712.19

[0175] Compound 12 can be synthesized according to the representative route described in Example 1.

[0176] C44 H 85 D 2 NO 5 , Ms m / z: [M+H + 712.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66~3.46 (br.m, 2H), 2.85~2.45 (br.m, 5H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0177] Example 14:

[0178] Synthesis of Compound 13

[0179]

[0180] Chemical formula: C 44 H 84 D 3 NO 5

[0181] Molecular weight: 713.20

[0182] Compound 13 can be synthesized according to the representative route described in Example 1.

[0183] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.66~2.36 (br.m, 6H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0184] Example 15:

[0185] Synthesis of Compound 14

[0186]

[0187] Chemical formula: C 44 H 84 D 3 NO 5 ,

[0188] Molecular weight: 713.20

[0189] Compound 14 can be synthesized according to the representative route described in Example 1.

[0190] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.73~2.33 (br.m, 6H), 2.30 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0191] Example 16:

[0192] Synthesis of Compound 15

[0193]

[0194] Chemical formula: C 44 H 84 D 3 NO 5

[0195] Molecular weight: 713.20

[0196] Compound 15 can be synthesized according to the representative route described in Example 1.

[0197] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.66~2.36 (br.m, 6H), 2.29 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0198] Example 17:

[0199] Synthesis of Compound 16

[0200]

[0201] Chemical formula: C 44 H 84 D 3 NO 5

[0202] Molecular weight: 713.20

[0203] Compound 16 can be synthesized according to the representative route described in Example 1.

[0204] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66~3.46 (br.m, 1H), 2.76~2.36 (br.m, 5H), 2.28 (m, 4H), 1.78~1.42 (br.m, 14H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0205] Example 18:

[0206] Synthesis of Compound 17

[0207]

[0208] Chemical formula: C 44 H 84 D 3 NO 5

[0209] Molecular weight: 713.20

[0210] Compound 17 can be synthesized according to the representative route described in Example 1.

[0211] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3)δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66 - 3.46 (br.m, 2H), 2.76 - 2.36 (br.m, 4H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0212] Example 19:

[0213] Synthesis of Compound 18

[0214]

[0215] Chemical formula: C 44 H 84 D 3 NO 5

[0216] Molecular weight: 713.20

[0217] Compound 18 can be synthesized according to the representative route described in Example 1.

[0218] C 44 H 84 D 3 NO 5 , Ms m / z: [M + H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66 - 3.46 (br.m, 2H), 2.76 - 2.36 (br.m, 4H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0219] Example 20:

[0220] Synthesis of Compound 19

[0221]

[0222] Chemical formula: C 44 H 84 D 3 NO 5

[0223] Molecular weight: 713.20

[0224] Compound 19 can be synthesized according to the representative route described in Example 1.

[0225] C44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83(t, 1H), 4.08(t, 2H), 3.66~3.46(br.m, 1H), 2.86~2.46(br.m, 5H), 2.28(m, 4H), 1.74~1.44(br.m, 12H), 1.38~1.17(m, 50H), 0.87(m, 9H).

[0226] Example 21:

[0227] Synthesis of Compound 20

[0228]

[0229] Chemical formula: C 44 H 84 D 3 NO 5

[0230] Molecular weight: 713.20

[0231] Compound 20 can be synthesized according to the representative route described in Example 1.

[0232] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83(t, 1H), 4.08(t, 2H), 3.66~3.46(br.m, 2H), 2.76~2.36(br.m, 4H), 2.28(m, 4H), 1.78~1.42(br.m, 12H), 1.38~1.17(m, 50H), 0.87(m, 9H).

[0233] Example 22:

[0234] Synthesis of Compound 21

[0235]

[0236] Chemical formula: C 44 H 84 D 3 NO5

[0237] Molecular weight: 713.20

[0238] Compound 21 can be synthesized according to the representative route described in Example 1.

[0239] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66~3.46 (br.m, 2H), 2.66~2.36 (br.m, 4H), 2.32 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0240] Example 23:

[0241] Synthesis of Compound 22

[0242]

[0243] Chemical formula: C 44 H 84 D 3 NO 5

[0244] Molecular weight: 713.20

[0245] Compound 22 can be synthesized according to the representative route described in Example 1.

[0246] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66~3.46 (br.m, 1H), 2.76~2.36 (br.m, 5H), 2.28 (m, 4H), 1.78~1.42 (br.m, 14H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0247] Example 24:

[0248] Synthesis of Compound 23

[0249]

[0250] Chemical formula: C 44 H 84 D 3 NO 5

[0251] Molecular weight: 713.20

[0252] Compound 23 can be synthesized according to the representative route described in Example 1.

[0253] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.56~3.36 (br.m, 2H), 2.76~2.46 (br.m, 4H), 2.32 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0254] Example 25:

[0255] Synthesis of Compound 24

[0256]

[0257] Chemical formula: C 44 H 84 D 3 NO 5

[0258] Molecular weight: 713.20

[0259] Compound 24 can be synthesized according to the representative route described in Example 1.

[0260] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300 MHz, CDCl 3)δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66 - 3.46 (br.m, 2H), 2.72 - 2.32 (br.m, 4H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0261] Example 26:

[0262] Synthesis of Compound 25

[0263]

[0264] Chemical formula: C 44 H 84 D 3 NO 5

[0265] Molecular weight: 713.20

[0266] Compound 25 can be synthesized according to the representative route described in Example 1.

[0267] C 44 H 84 D 3 NO 5 , Ms m / z: [M + H + 713.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.86 - 3.56 (br.m, 1H), 2.75 - 2.35 (br.m, 5H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0268] Example 27:

[0269] Synthesis of Compound 26

[0270]

[0271] Chemical formula: C 44 H 84 D 3 NO 5 ,

[0272] Molecular weight: 713.20

[0273] Compound 26 can be synthesized according to the representative route described in Example 1.

[0274] C44 H 84 D 3 NO 5 ,Ms m / z: [M+H + 713.6; 1 H-NMR (300MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.67~3.47 (br.m, 1H), 2.66~2.36 (br.m, 5H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0275] Example 28:

[0276] Synthesis of Compound 27

[0277]

[0278] Chemical formula: C 44 H 84 D 3 NO 5

[0279] Molecular weight: 713.20

[0280] Compound 27 can be synthesized according to the representative route described in Example 1.

[0281] C 44 H 84 D 3 NO 5 , Ms m / z: [M+H + 713.6; 1 H-NMR (300MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.76~3.46 (br.m, 2H), 2.56~2.36 (br.m, 4H), 2.33 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0282] Example 29:

[0283] Synthesis of Compound 29

[0284]

[0285] Chemical formula: C 44 H 83 D 4 NO5

[0286] Molecular weight: 714.21

[0287] Compound 1 can be synthesized according to the representative route described in Example 1.

[0288] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.86~2.36 (br.m, 5H), 2.48 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0289] Example 30:

[0290] Synthesis of Compound 30

[0291]

[0292] Chemical formula: C 44 H 83 D 4 NO 5

[0293] Molecular weight: 714.21

[0294] Compound 30 can be synthesized according to the representative route described in Example 1.

[0295] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.76~2.36 (br.m, 5H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0296] Example 31:

[0297] Synthesis of Compound 31

[0298]

[0299] Chemical formula: C 44 H 83 D 4 NO 5

[0300] Molecular weight: 714.21

[0301] Compound 31 can be synthesized according to the representative route described in Example 1.

[0302] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.86~3.66 (br.m, 2H), 2.76~2.36 (br.m, 3H), 2.32 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0303] Example 32:

[0304] Synthesis of Compound 32

[0305]

[0306] Chemical formula: C 44 H 83 D 4 NO 5

[0307] Molecular weight: 714.21

[0308] Compound 32 can be synthesized according to the representative route described in Example 1.

[0309] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.86 - 3.66 (br.m, 2H), 2.76 - 2.36 (br.m, 3H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0310] Example 33:

[0311] Synthesis of Compound 33

[0312]

[0313] Chemical formula: C 44 H 83 D 4 NO 5

[0314] Molecular weight: 714.21

[0315] Compound 33 can be synthesized according to the representative route described in Example 1.

[0316] C 44 H 83 D 4 NO 5 , Ms m / z: [M + H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.86 - 2.36 (br.m, 5H), 2.32 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0317] Example 34:

[0318] Synthesis of Compound 34

[0319]

[0320] Chemical formula: C 44 H 83 D 4 NO 5

[0321] Molecular weight: 714.21

[0322] Compound 34 can be synthesized according to the representative route described in Example 1.

[0323] C 44 H 83 D4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83(t, 1H), 4.08(t, 2H), 2.86~2.36(br.m, 5H), 2.33(m, 4H), 1.78~1.42(br.m, 12H), 1.38~1.17(m, 50H), 0.87(m, 9H).

[0324] Example 35:

[0325] Synthesis of Compound 35

[0326]

[0327] Chemical formula: C 44 H 83 D 4 NO 5

[0328] Molecular weight: 714.21

[0329] Compound 35 can be synthesized according to the representative route described in Example 1.

[0330] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83(t, 1H), 4.08(t, 2H), 3.66~3.46(br.m, 1H), 2.76~2.36(br.m, 4H), 2.28(m, 4H), 1.78~1.42(br.m, 12H), 1.38~1.17(m, 50H), 0.87(m, 9H).

[0331] Example 36:

[0332] Synthesis of Compound 36

[0333]

[0334] Chemical formula: C 44 H 83 D 4 NO 5

[0335] Molecular weight: 714.21

[0336] Compound 36 can be synthesized according to the representative route described in Example 1.

[0337] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66~3.46 (br.m, 1H), 2.76~2.36 (br.m, 4H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0338] Example 37:

[0339] Synthesis of Compound 37

[0340]

[0341] Chemical formula: C 44 H 83 D 4 NO 5

[0342] Molecular weight: 714.21

[0343] Compound 37 can be synthesized according to the representative route described in Example 1.

[0344] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.96~2.56 (br.m, 5H), 2.38 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0345] Example 38:

[0346] Synthesis of Compound 38

[0347]

[0348] Chemical formula: C 44 H 83 D 4 NO 5

[0349] Molecular weight: 714.21

[0350] Compound 38 can be synthesized according to the representative route described in Example 1.

[0351] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.94~3.54 (br.m, 2H), 2.86~2.46 (br.m, 3H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0352] Example 39:

[0353] Synthesis of Compound 39

[0354]

[0355] Chemical formula: C 44 H 83 D 4 NO 5

[0356] Molecular weight: 714.21

[0357] Compound 39 can be synthesized according to the representative route described in Example 1.

[0358] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3)δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.68 - 3.48 (br.m, 1H), 2.87 - 2.47 (br.m, 4H), 2.33 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0359] Example 40:

[0360] Synthesis of Compound 40

[0361]

[0362] Chemical formula: C 44 H 83 D 4 NO 5

[0363] Molecular weight: 714.21

[0364] Compound 40 can be synthesized according to the representative route described in Example 1.

[0365] C 44 H 83 D 4 NO 5 , Ms m / z: [M + H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.76 - 3.56 (br.m, 1H), 2.78 - 2.38 (br.m, 4H), 2.28 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0366] Example 41:

[0367] Synthesis of Compound 41

[0368] Chemical formula: C 44 H 83 D 4 NO 5

[0369] Molecular weight: 714.21

[0370] Compound 41 can be synthesized according to the representative route described in Example 1.

[0371] C 44 H 83 D 4 NO5 , Ms m / z: [M+H + 714.6; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83(t, 1H), 4.08(t, 2H), 3.66~3.46(br.m, 1H), 2.82~2.62(br.m, 4H), 2.28(m, 4H), 1.78~1.42(br.m, 12H), 1.38~1.17(m, 50H), 0.87(m, 9H).

[0372] Example 42:

[0373] Synthesis of Compound 42

[0374]

[0375] Chemical formula: C 44 H 83 D 4 NO 5

[0376] Molecular weight: 714.21

[0377] Compound 42 can be synthesized according to the representative route described in Example 1.

[0378] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83(t, 1H), 4.08(t, 2H), 3.76~3.56(br.m, 1H), 2.76~2.36(br.m, 4H), 2.28(m, 4H), 1.78~1.42(br.m, 12H), 1.38~1.17(m, 50H), 0.87(m, 9H).

[0379] Example 43:

[0380] Synthesis of Compound 43

[0381]

[0382] Chemical formula: C 44 H 83 D 4 NO 5

[0383] Molecular weight: 714.21

[0384] Compound 43 can be synthesized according to the representative route described in Example 1.

[0385] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 714.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.66~3.46 (br.m, 1H), 2.79~2.49 (br.m, 4H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0386] Example 44:

[0387] Synthesis of Compound 44

[0388]

[0389] Chemical formula: C 44 H 82 D 5 NO 5

[0390] Molecular weight: 715.21

[0391] Compound 44 can be synthesized according to the representative route described in Example 1.

[0392] C 44 H 83 D 4 NO 5 , Ms m / z: [M+H + 715.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.01~2.81 (br.m, 4H), 2.34 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0393] Example 45:

[0394] Synthesis of Compound 45

[0395]

[0396] Chemical formula: C 44 H 82 D 5 NO 5

[0397] Molecular weight: 715.21

[0398] Compound 45 can be synthesized according to the representative route described in Example 1.

[0399] C 44 H 82 D 5 NO 5 , Ms m / z: [M+H + 715.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.76~2.46 (br.m, 4H), 2.35 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0400] Example 46:

[0401] Synthesis of Compound 46

[0402]

[0403] Chemical formula: C 44 H 82 D 5 NO 5

[0404] Molecular weight: 715.21

[0405] Compound 46 can be synthesized according to the representative route described in Example 1.

[0406] C 44 H 82 D 5 NO 5 , Ms m / z: [M+H + 715.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.76~2.36 (br.m, 4H), 2.33 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0407] Example 47:

[0408] Synthesis of Compound 47

[0409]

[0410] Chemical formula: C 44 H 82 D 5 NO 5

[0411] Molecular weight: 715.21

[0412] Compound 47 can be synthesized according to the representative route described in Example 1.

[0413] C 44 H 82 D 5 NO 5 , Ms m / z: [M+H + 715.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.72~2.42 (br.m, 4H), 2.33 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0414] Example 48:

[0415] Synthesis of Compound 48

[0416]

[0417] Chemical formula: C 44 H 82 D 5 NO 5

[0418] Molecular weight: 715.21

[0419] Compound 48 can be synthesized according to the representative route described in Example 1.

[0420] C 44 H 82 D 5 NO 5 , Ms m / z: [M+H + 715.6; 1 H-NMR (300 MHz, CDCl 3)δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.76 - 2.36 (br.m, 4H), 2.32 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0421] Example 49:

[0422] Synthesis of Compound 49

[0423]

[0424] Chemical formula: C 44 H 82 D 5 NO 5

[0425] Molecular weight: 715.21

[0426] Compound 49 can be synthesized according to the representative route described in Example 1.

[0427] C 44 H 82 D 5 NO 5 , Ms m / z: [M + H + 715.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.86 - 2.56 (br.m, 4H), 2.34 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0428] Example 50:

[0429] Synthesis of Compound 50

[0430]

[0431] Chemical formula: C 44 H 82 D 5 NO 5

[0432] Molecular weight: 715.21

[0433] Compound 50 can be synthesized according to the representative route described in Example 1.

[0434] C 44 H 82 D 5 NO5 , Ms m / z: [M+H + 715.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.84~3.54 (br.m, 2H), 2.79~2.39 (br.m, 2H), 2.28 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0435] Example 51:

[0436] Synthesis of Compound 51

[0437] Chemical formula: C 44 H 82 D 5 NO 5

[0438] Molecular weight: 715.21

[0439] Compound 51 can be synthesized according to the representative route described in Example 1.

[0440] C 44 H 82 D 5 NO 5 , Ms m / z: [M+H + 715.6; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.95~3.55 (br.m, 2H), 2.76~2.36 (br.m, 2H), 2.30 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0441] Example 52:

[0442] Synthesis of Compound 52

[0443] Chemical formula: C 44 H 81 D 6 NO 5

[0444] Molecular weight: 716.22

[0445] Compound 52 can be synthesized according to the representative route described in Example 1.

[0446] C44 H 81 D 6 NO 5 ,Ms m / z: [M + H + 716.7; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.88~2.58 (br.m, 3H), 2.35 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0447] Example 53:

[0448] Synthesis of Compound 53

[0449] Chemical formula: C 44 H 81 D 6 NO 5

[0450] Molecular weight: 716.22

[0451] Compound 53 can be synthesized according to the representative route described in Example 1.

[0452] C 44 H 81 D 6 NO 5 ,Ms m / z: [M + H + 716.7; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.92~2.62 (br.m, 3H), 2.41 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0453] Example 54:

[0454] Synthesis of Compound 54

[0455] Chemical formula: C 44 H 81 D 6 NO 5

[0456] Molecular weight: 716.22

[0457] Compound 54 can be synthesized according to the representative route described in Example 1.

[0458] C 44 H 81 D 6 NO 5 ,Ms m / z: [M+H + 716.7; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.01~2.81 (br.m, 3H), 2.42 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0459] Example 55:

[0460] Synthesis of Compound 55

[0461]

[0462] Chemical formula: C 44 H 81 D 6 NO 5

[0463] Molecular weight: 716.22

[0464] Compound 55 can be synthesized according to the representative route described in Example 1.

[0465] C 44 H 81 D 6 NO 5 , Ms m / z: [M+H + 716.7; 1 H-NMR(300MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.92~2.52 (br.m, 3H), 2.35 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0466] Example 56:

[0467] Synthesis of Compound 56

[0468]

[0469] Chemical formula: C 44 H 81 D 6 NO 5

[0470] Molecular weight: 716.22

[0471] Compound 56 can be synthesized according to the representative route described in Example 1.

[0472] C 44 H 81 D 6 NO 5 , Ms m / z: [M+H + 716.7; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 2.86~2.56 (br.m, 3H), 2.45 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0473] Example 57:

[0474] Synthesis of Compound 57

[0475]

[0476] Chemical formula: C 44 H 81 D 6 NO 5

[0477] Molecular weight: 716.22

[0478] Compound 57 can be synthesized according to the representative route described in Example 1.

[0479] C 44 H 81 D 6 NO 5 , Ms m / z: [M+H + 716.7; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.98~3.58 (br.m, 1H), 2.85~2.45 (br.m, 2H), 2.45 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0480] Example 58:

[0481] Synthesis of Compound 58

[0482]

[0483] Chemical formula: C 44 H 81 D 6 NO 5

[0484] Molecular weight: 716.22

[0485] Compound 58 can be synthesized according to the representative route described in Example 1.

[0486] C 44 H 81 D 6 NO 5 , Ms m / z: [M+H + 716.7; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.89~3.69 (br.m, 1H), 2.86~2.46 (m, 2H), 2.55 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0487] Example 59:

[0488] Synthesis of Compound 59

[0489]

[0490] Chemical formula: C 44 H 81 D 6 NO 5

[0491] Molecular weight: 716.22

[0492] Compound 59 can be synthesized according to the representative route described in Example 1.

[0493] C 44 H 81 D 6 NO 5 , Ms m / z: [M+H + 716.7; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.02~2.82 (m, 2H), 2.45 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0494] Example 60:

[0495] Synthesis of Compound 60

[0496]

[0497] Chemical formula: C 44 H 80 D 7 NO 5

[0498] Molecular weight: 717.22

[0499] Compound 60 can be synthesized according to the representative route described in Example 1.

[0500] C 44 H 80 D 7 NO 5 , Ms m / z: [M+H + 717.7; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.03~2.83 (br.m, 2H), 2.45 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0501] Example 61:

[0502] Synthesis of Compound 61

[0503]

[0504] Chemical formula: C 44 H 80 D 7 NO 5

[0505] Molecular weight: 717.22

[0506] Compound 61 can be synthesized according to the representative route described in Example 1.

[0507] C 44 H 80 D 7 NO 5 , Ms m / z: [M+H + 717.7; 1 H-NMR (300 MHz, CDCl 3)δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.89 - 3.69 (s, 1H), 2.96 - 2.76 (s, 1H), 2.45 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0508] Example 62:

[0509] Synthesis of Compound 62

[0510]

[0511] Chemical formula: C 44 H 80 D 7 NO 5

[0512] Molecular weight: 717.22

[0513] Compound 62 can be synthesized according to the representative route described in Example 1.

[0514] C 44 H 80 D 7 NO 5 , Ms m / z: [M + H + 717.7; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.20 - 3.10 (br.m, 2H), 2.45 (m, 4H), 1.78 - 1.42 (br.m, 12H), 1.38 - 1.17 (m, 50H), 0.87 (m, 9H).

[0515] Example 63:

[0516] Synthesis of Compound 63

[0517]

[0518] Chemical formula: C 44 H 79 D 8 NO 5

[0519] Molecular weight: 718.23

[0520] Compound 63 can be synthesized according to the representative route described in Example 1.

[0521] C 44 H 79 D8 NO 5 , Ms m / z: [M+H + 718.7; 1 H-NMR (300 MHz, CDCl 3 ) δ: ppm 4.83 (t, 1H), 4.08 (t, 2H), 3.15 (s, 1H), 2.45 (m, 4H), 1.78~1.42 (br.m, 12H), 1.38~1.17 (m, 50H), 0.87 (m, 9H).

[0522] Example 64

[0523] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0524] The cationic lipid, DSPC, cholesterol, and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:37:2 or 48:10:42:2. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, the mRNA was diluted to 0.15 mg / mL in 10 mL to 50 mL of citrate buffer (pH = 4). Using a syringe pump, the ethanol solution of the lipid was mixed 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 15 mL / min. Then, the ethanol was 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 um. The particle size of the lipid nanoparticles determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS was approximately 65 - 105 nm in diameter, and in some cases, approximately 75 - 100 nm in diameter.

[0525] Studies were conducted on female mice at 6 - 8 weeks of age and male mice at 8 - 10 weeks of age according to the guidelines established by the National Science and Technology Council. Different doses of mRNA lipid nanoparticles were administered systemically via tail vein injection, and the animals were euthanized at specific time points after administration (e.g., 5 hours). The liver and spleen were collected in pre-weighed tubes, weighed, immediately snap-frozen in liquid nitrogen, and stored at -80 °C until used for analysis.

[0526] For the liver, approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (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 x 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 Centro XS3LB 960 photometer (Berthold Technologies, Germany). The amount of protein assayed was determined by using a BCA protein assay kit (Pierce, Rockford IL). The relative light units (RLU) were then normalized to the total μg of protein assayed. To convert RLU to ng of luciferase, a standard curve was generated using QuantiLum recombinant luciferase (Promega).

[0527] 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.

[0528] Example 65

[0529] Determination of the pKa of the formulated lipid

[0530] The pKa of the formulated cationic lipid is related to the efficacy of the LNP 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-naphthalene sulfonic acid (TNS). As described in Example 3, an ordered method is used to prepare lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG lipid (50 / 10 / 40 / 1.5 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, with a pH ranging 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.

[0531] Example 66

[0532] The efficacy of lipid nanoparticle formulations containing various cationic lipids was determined using a rodent model of in vivo luciferase mRNA expression.

[0533] The cationic lipids shown in Table 2 were previously tested with nucleic acids. For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the in-line mixing method as described in Example 3. Lipid nanoparticles were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 40% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, average PEG molecular weight of 2000). As described in Example 64, the relative activity was determined by measuring luciferase expression in the liver 5 hours after administration via tail vein injection. The activities were 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 64.

[0534] Table 2 Lipids Compared for Activity with mRNA

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543] 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.

[0544] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed. However, 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 this 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 6: Compound 12: Compound 40: Compound 45: Compound 53: Compound 56:

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.

Images