WO2023045366A1 - New type pyrrolidine type lipid compound, preparation method therefor, and composition and use thereof - Google Patents

Abstract

Disclosed is a new type pyrrolidine type compound as represented by formula (A), or an isomer, an N-oxide, or a pharmaceutically acceptable salt and prodrug thereof. Also disclosed are a preparation method therefor, and a composition and the use thereof. The compound is used as a cationic lipid, is suitable for a pharmaceutical composition for nucleic acid delivery, and can achieve a better stability, pKa value and intracellular delivery efficiency of drugs. Formula A is as follows in the description.

Description

Novel lipid compound of pyrrolidine class, preparation method, composition and application thereof technical field

The present invention provides novel cationic lipids that can be used in combination with other lipid components (such as neutral lipids, steroids, and polymer-conjugated lipids) to form a nucleic acid mRNA lipid nanoparticle composition combining a A method of delivering one or more therapeutic and/or prophylactic agents to a mammalian cell or organ and/or producing a polypeptide in a mammalian cell or organ. In addition to the novel lipids, the lipid nanoparticle compositions of the invention may also include, in specified proportions, one or more cationic and/or ionizable amino lipids, neutral lipids including polyunsaturated lipids, Lipids, polymer-conjugated lipids, steroids, and/or therapeutic and/or prophylactic agents.

Background technique

Efficient targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids presents a persistent medical problem. Specifically, delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of these species. Accordingly, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.

Studies have proved that using lipid-containing nanoparticle compositions, liposomes and liposome complexes as transport media can effectively deliver biologically active substances such as small molecule drugs, proteins and nucleic acids to cells and/or intracellularly in the compartment. These compositions generally comprise one or more "cationic" lipids, neutral lipids including polyunsaturated lipids (such as phospholipids), structured lipids (such as steroids) and/or polyethylene glycol-containing Lipids of alcohols (polymer-conjugated lipids). Cationic lipids include, for example, amine-containing lipids that can be readily protonated.

However, the use of oligonucleotides in a therapeutic setting currently faces two problems. First, free RNA is readily digested by nucleases in plasma. Second, cell-free RNA has limited access to intracellular compartments where associated translation machinery exists. Lipid nanoparticles formed from cationic lipids with other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to prevent RNA degradation in plasma and promote Cellular uptake of oligonucleotides.

There remains a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Improved lipid nanoparticles would provide optimized drug delivery, protect nucleic acids from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. In addition, these preferred lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index such that treatment of a patient at an effective dose of the nucleic acid does not create unacceptable toxicity and/or risk to the patient. The present invention provides these and related advantages.

public content

The present invention provides the following novel compounds and methods involving these compounds:

A pyrrolidine compound as shown in formula A, or its isomer, or its N-oxide, or its pharmaceutically acceptable salt, prodrug;

in:

_L is selected from the following structures: -C-, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _X- , - SS-, -C(=O)S-, -SC(=O)-, -NC(=O)-, -C(=O)-N-, _L2 is selected from the following structures: -C-, - (C=O)O-, -C(=O)-, -S(O) _X- , -C(=O)S-, -C(=O)-N-;

R ₁ and R ₂ are each independently C6-C24 alkyl or C6-C24 alkenyl, and the hydrocarbon chains are optionally connected by one or more ester bonds or ether bonds;

R ₃ and R ₄ are each independently C1-C12 alkyl or C1-C12 alkenyl, or R ₃ and R ₄ combine with each other to form a 4- to 10-membered heterocyclic ring, and the heteroatoms include one of N, O, and S One or more heteroatoms, the heterocycle is optionally substituted by 1-6 heteroatoms;

X is C, N, O, S, -S-S-;

M is C1-C12 alkyl or C1-C12 alkenyl;

x is 0, 1 or 2.

In various embodiments, the compound has one of the structures shown in Table 1 below.

Table 1 Compound list

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

In some embodiments, there is provided a composition 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 of the compounds of structure (I) and a therapeutic and/or prophylactic agent and one or more compounds selected from the group consisting of neutral lipids, steroids, and polymer conjugates. Lipid excipients. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the compositions.

In some embodiments, the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Alkaline (DPPC), 1,2-Dimyristyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palm Acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM) and mixtures thereof. In some embodiments, the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

In some embodiments, the steroid is selected from the group consisting of cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof . In some embodiments, the preferred steroid is cholesterol.

In some embodiments, the pegylated lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG).

In some embodiments, the composition ratio ranges from about 10 to 60 mol% of the compound, about 0 to 30 mol% of neutral lipids, about 10 to 55 mol% of steroids, and about 0 to 10 mol% of polymer-conjugated lipids. quality.

In embodiments of some 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 following composition: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof. In some embodiments, the RNA is selected from mRNA.

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

For purposes of administration, the compounds of the invention, typically in the form of lipid nanoparticles in combination with therapeutic and/or prophylactic agents, can be administered as a drug substance, or can be formulated as 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. Compounds of structure (I) are effective to form lipid nanoparticles and deliver therapeutic and/or prophylactic agents. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

Administration of the compositions of the present invention may be by any of the acceptable modes of administration for agents of similar utility. The pharmaceutical composition of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of 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 injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the invention are formulated so as to allow the active ingredient contained therein to be bioavailable upon administration of the composition to a subject. Compositions to be administered to a subject or patient are in the form of one or more dosage units, where a tablet may be a single dosage unit and a container of the compound in aerosol form of the invention may hold multiple dosage units. Current methods for the preparation of such dosage forms are known, or will be apparent, to those skilled in the art. In any event, the composition to be administered will contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treating the associated disease or condition in accordance with the teachings of the invention.

The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier is particulate, such that the composition is in tablet or powder form. The carrier can be a liquid in which case the composition is an oral syrup or an injectable liquid or an aerosol suitable for administration by inhalation.

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.

As solid compositions for oral administration, the pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, flakes and the like. Such solid compositions will generally contain one or more inert diluents or edible carriers. In addition, there may be one or more of the following: 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; and coloring agents.

When the pharmaceutical composition is in capsule form, it may contain liquid carriers other than materials of the above type, such as polyethylene glycol or oil.

Pharmaceutical compositions may be in liquid form, such as syrups, solutions, emulsions or suspensions. Liquids can be used for oral administration or for injectable delivery, as two examples. When intended for oral administration, the compositions of Urim contain, in addition to the compounds of the present invention, one or more of sweetening agents, preservatives, dyeing/coloring agents and darkening agents. In compositions administered by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents may be included.

The liquid pharmaceutical composition of the present invention, whether it is a solution, a suspension or other similar forms, may 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; fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerol, propylene glycol, or other solvents that may be used as a solvent or suspending medium; antibacterial agents , such as methylparaben, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenedipic acid; buffering agents, such as acetate, clematine, or phosphate; Reagents for tonicity, such as sodium chloride or glucose; reagents used as cryoprotectants, such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

The pharmaceutical compositions of the invention may consist of dosage units which can be administered as an aerosol. The term aerosol is used to denote systems ranging from those of a colloidal nature to those consisting of pressurized packs. Delivery may be by liquefied or compressed gas, or by a suitable pump system which dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered as single-phase, bi-phasic or triphasic systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Preferred aerosols can be determined by those skilled in the art without undue experimentation.

The pharmaceutical compositions of the present invention can be prepared by methods well known in the field of pharmacy. Pharmaceutical compositions intended to be administered by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers into a solution. Surfactants can be added to promote the formation of a uniform solution or suspension. Surfactants are compounds that interact non-covalently with the compounds of the invention in order to facilitate dissolution or uniform suspension of the compounds in aqueous delivery systems.

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 number of factors, including the activity of the particular therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; The patient's age, weight, general health, sex, and diet; mode and timing of administration; rate of excretion; drug combination; severity of the specific case, etc.

Compositions of the invention may also be administered concurrently with, before or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the invention and one or more additional active agents, as well as administration of a composition of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, compositions of the invention and other active agents may be administered to a subject together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations. When different dosage formulations are used, the compound of the invention and the one or more additional active agents may be administered at substantially the same time, or sequentially at times staggered from each other; it is understood that combination therapy includes all such dosing regimens.

The structural modification and design of the above-mentioned novel cationic lipid compounds have achieved more advantageous physical and chemical properties, including more suitable pKa and better chemical stability, and are used in mRNA nanoliposome compositions, which can realize anti-ionic nucleic acid The drug is more effectively combined and delivered, and its chemical structure is more stable, which is convenient for synthesis and beneficial to be developed as a pharmaceutical excipient.

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

Those skilled in the art will recognize that in the methods described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl, tetrahydrofuranyl, benzyl, and the like. Suitable protecting groups for amino include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for carboxylic acids include hydrocarbyl, aryl or arylhydrocarbyl esters. Protecting groups can be added or removed according to standard techniques, known to those skilled in the art and described herein.

Those skilled in the art will also recognize that while such protected derivatives of compounds of the invention may not thereby be pharmaceutically active, they may be administered to a mammal and then metabolized in vivo to form pharmacologically active compounds of the invention. Such derivatives can thus be described as "prodrugs". Prodrugs of the compounds of the present invention are therefore included within the scope of the present invention.

In addition, all compounds of the present invention that exist in free base or free acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid according to methods known to those skilled in the art. Salts of compounds of the present invention may be converted to their free base or acid forms by standard techniques.

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

In the following examples, unless otherwise indicated, all solvents and reagents used were obtained commercially and used as received.

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

This article uses the following abbreviations:

DIEA: Diisopropylethylamine

HATU: 2-(7-Azabenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate

K ₂ CO ₃ : potassium carbonate

DMF: N,N-Dimethylformamide

NaOH: sodium hydroxide

THF: Tetrahydrofuran

TBAB: Tetrabutylammonium bromide

Detailed ways

Example 1:

representative route

Compound CLPP: N-(diethylamine)ethyl)-1-pentadecacarbonyl-4-pentadecayloxy)pyrrolidone-2-carboxamide

1) Preparation of Intermediate F

At room temperature, raw materials H (1g, 4.32mmol), G (1.51g, 5.18mmol) and DMF (100ml) were added into a 250ml four-necked flask, and stirred to dissolve. K ₂ CO ₃ (0.78g, 5.62mmol) was added, heated to 80°C, stirred for 4h, and the reaction was complete as detected by TLC. Ethyl acetate was added, the organic phase was washed with water, sodium bicarbonate, dilute hydrochloric acid, and water respectively, dried over _Na2SO4 , and concentrated under reduced pressure to obtain a crude intermediate F, which was purified by column chromatography to obtain 1.49 _g of the target compound. Yield 78%.

Molecular formula: C ₂₅ H ₄₇ NO ₅

Molecular weight: 441.64

LC-MS: m/z 442[M+H]

1H NMR(DMSO-d6):δ(ppm)11.02(s,1H)4.25(t,1H,J＝3.5),3.50(m,2H)3.39(m,2H),3.05(m,1H),1.98 (m,2H), 1.40-1.47(m,11H), 1.25-1.33(m,24H), 0.99(m,3H).

2) Preparation of Intermediate D

(DMF)20mL，冰浴冷却，搅拌，≤5℃依次加入0.84g(6.5mmol)二异丙基乙胺(DIEA)、中间体N-取代胺(E,2.34mmol)和0.83g(2.6mmol)O-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU)，室温搅拌3h，TLC检测反应完全。搅拌下，将反应液倒入100g冰水中，继续搅拌0.5h，乙酸乙酯提取3次(30mL×3)，合并乙酸乙酯层，有机相依次水、盐酸、碳酸氢钠、食盐水洗涤，无水MgSO ₄干燥，减压蒸馏后，制得中间体C 粗品，柱层析(石油醚:乙酸乙酯＝5:1洗脱)，得1.07g浅色油状物，收率85.01％。

(DMF) 20mL, cooled in an ice bath, stirred, 0.84g (6.5mmol) diisopropylethylamine (DIEA), intermediate N-substituted amine (E, 2.34mmol) and 0.83g (2.6mmol) were added successively at ≤5°C )O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), stirred at room temperature for 3h, and TLC detected that the reaction was complete. Under stirring, pour the reaction solution into 100 g of ice water, continue stirring for 0.5 h, extract 3 times with ethyl acetate (30 mL×3), combine the ethyl acetate layer, and wash the organic phase with water, hydrochloric acid, sodium bicarbonate, and brine in sequence, After drying over anhydrous MgSO ₄ and distillation under reduced pressure, the crude intermediate C was obtained. Column chromatography (petroleum ether: ethyl acetate = 5:1 elution) gave 1.07 g of light-colored oil with a yield of 85.01%.

Molecular formula: C ₃₁ H ₆₁ N ₃ O ₄

Molecular weight: 539.47

LC-MS: m/z 540[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.27(t,1H,J＝3.5Hz),3.52(m,2H)3.40(m,2H),3.35(m,2H) ,3.02(t,1H,J=3Hz),2.70(m,2H),2.30(m,4H),1.98(m,2H),1.40-1.47(m,11H),1.25-1.33(m,24H) ,1.02-0.99(m,9H).

3) Preparation of Intermediate C

Put (3.13g, 5.8mmol) Intermediate C and 30mL ethyl acetate into a 100mL three-neck flask, stir, cool in an ice-water bath, ≤10°C, add 2.5mL 33% HCl ethanol solution (14.5mmol) dropwise within 0.5h ), stirred at room temperature for 4 hours, TLC detected that the reaction was complete, extracted 3 times with water (30mL×3), combined the aqueous layer, washed the aqueous phase with ethyl acetate, added 30mL dichloromethane to the aqueous layer, stirred, and added Na ₂ CO ₃ to adjust the pH to 8-9, separate layers, extract the aqueous layer twice with dichloromethane (30ml×2), combine the dichloromethane layers, wash with saturated brine three times, dry over anhydrous MgSO ₄ , filter, and concentrate under reduced pressure to obtain The crude intermediate C was purified by column chromatography to obtain 2.45 g of oil, with a yield of 96%.

Molecular formula: C ₂₆ H ₅₃ N ₃ O ₂

Molecular weight: 439.72

LC-MS: m/z 440[M+H]

1H NMR (DMSO-d6): δ (ppm) 8.06 (s, 1H), 4.27 (t, 1H, J = 3.5), 3.52 (m, 2H) 3.40 (m, 2H), 3.35 (m, 2H), 3.02(t,1H,J=3),2.70(m,2H),2.30(m,4H),2.01(s,1H),1.98(m,2H),1.47(m,2H),1.25-1.33( m,24H), 0.99(m,9H).

4) Preparation of Compound 1

To a 100ml reaction flask, add intermediate C (1.03g, 2.34mmol) and 20mL of N,N-dimethylformamide (DMF), cool in an ice bath, stir, and add 0.84g (6.5mmol) di Isopropylethylamine (DIEA), intermediate CLPP-6 (2.34mmol) and 0.83g (2.6mmol) O-(7-azabenzotriazol-1-yl)-N,N,N',N '-Tetramethyluronium hexafluorophosphate (HATU), stirred at room temperature for 3 h, and TLC detected that the reaction was complete. Under stirring, pour the reaction solution into 100 g of ice water, continue stirring for 0.5 h, extract 3 times with ethyl acetate (30 mL×3), combine the ethyl acetate layer, and wash the organic phase with water, hydrochloric acid, sodium bicarbonate, and brine in sequence, After drying over anhydrous MgSO ₄ and distillation under reduced pressure, the crude product of compound 1 was obtained. Column chromatography (petroleum ether: ethyl acetate = 5:1 elution) gave 1.4 g of light yellow oil with a yield of 90%.

Molecular formula: C ₄₁ H ₈₁ N ₃ O ₃

Molecular weight: 664.10

LC-MS: m/z 665[M+H]

1H NMR (DMSO-d6): δ (ppm) 8.06 (s, 1H), 4.27 (t, 1H, J = 3.5), 3.52 (m, 2H) 3.40 (m, 2H), 3.35 (m, 2H), 3.02(t, 1H, J=3), 2.70(m, 2H), 2.36(t, 2H, J=6), 2.30(m, 4H), 2.20(m, 2H), 1.60(m, 2H), 1.47(m,2H),1.25-1.33(m,42H),0.99(m,12H).

The synthesis of example 2 compound 2-hexyl capric acid 6-bromohexyl ester (C1)

In a 500ml reaction flask, add commercially available 2-hexyldecanoic acid (10g, 35.15mmol) and tetrahydrofuran (THF) 200mL, add 8.27g (45.70mmol) 6 bromohexanol, 6.3g potassium carbonate successively, heat up to 68 Stir at ℃ for 8h, and TLC detects that the reaction is complete. Under stirring, ethyl acetate was extracted 3 times (30mL×3), the ethyl acetate layers were combined, the organic phase was washed with water, hydrochloric acid, sodium bicarbonate, and brine in sequence, dried over anhydrous _MgSO4 , and after vacuum distillation, the obtained side Chain 2-octyldecanoic acid 6-bromohexyl ester crude product, column chromatography (petroleum ether: ethyl acetate = 10:1 elution), to obtain 12.56g 2-hexyldecanoic acid 6-bromohexyl ester yellow oil, obtained rate 80%.

Molecular formula: C ₂₂ H ₄₃ BrO ₂

Molecular weight: 419.54

LC-MS: m/z 420[M+H]

1H NMR(DMSO-d6):4.13(m,2H,),3.52(m,2H),2.13(m,1H),1.82(m,2H),1.60(m,6H),1.47(m,2H), 1.29(m,16H),0.90(m,6H).

Synthesis of Example 3 Compound 6-(heptadecyl-9 oxy)-6-oxoylhexanoic acid (C2)

Into a 500ml reaction flask, add commercially available 9-heptadecanol (10g, 38.99mmol) and N,N-dimethylformamide (DMF) 100mL, add 5.7g (38.99mmol) adipic acid, hydrogen Sodium oxide (2.34g, 58.49mmol) was heated up to 80°C and stirred for 6h, and the reaction was complete as detected by TLC. Under stirring, ethyl acetate was extracted 3 times (30mL×3), the ethyl acetate layers were combined, the organic phase was washed with water, sodium bicarbonate, hydrochloric acid, and brine in sequence, dried over anhydrous _MgSO4 , and after vacuum distillation, the obtained side The crude product of chain 6-(heptadecyl-9oxyl)-6-carbonylhexanoic acid was subjected to column chromatography (petroleum ether:ethyl acetate=1:1 elution) to obtain 9g of 6-(heptadecyl-9oxyl )-6-oxoylhexanoic acid yellow oil, yield 60.03%.

Molecular formula: C ₂₃ H ₄₄ O ₄

Molecular weight: 384.60

LC-MS: m/z 385[M+H]

1H NMR(DMSO-d6):11.87(s,1H),4.47(m,1H),2.32(m,2H),2.21(m,2H),1.64(m,2H),1.52(m,2H), 1.49(m,4H),1.29(m,4H),1.26(m,20H),0.90(m,6H).

The synthesis of example 4 compound 6-bromohexyl-9-heptadecane ether (C3)

To a 500ml reaction flask, add commercially available 9-heptadecanol (10g, 38.99mmol) and 50mL of N,N-dimethylformamide (DMF), and then add 5.7g (38.99mmol) of 6-bromohexanol in sequence , sodium hydroxide (2.34g, 58.49mmol) and 2.34g of water, add 0.3 equivalent of TBAB, raise the temperature to 60°C and stir for 12h, TLC detects that the reaction is complete. Under stirring, ethyl acetate was extracted 3 times (30mL×3), the ethyl acetate layers were combined, the organic phase was washed with water, sodium bicarbonate, hydrochloric acid, and brine in sequence, dried over anhydrous _MgSO4 , and after vacuum distillation, the obtained side Chain 6-bromohexyl-9-heptadecane ether crude product, column chromatography (petroleum ether: ethyl acetate = 20:1 elution), obtained 11..4g 6-(heptadecyl-9-oxyl)-6- Carbonyl hexanoic acid yellow oil, yield 70%.

Molecular formula: C ₂₃ H ₄₇ BrO

Molecular weight: 419.53

LC-MS: m/z 420[M+H]

1H NMR(DMSO-d6):3.52(m,2H),3.35(m,2H),3.10(m,1H),1.82(m,2H),1.52(m,2H),1.49(m,2H), 1.38(m,4H),1.29(m,6H),1.26(m,20H),0.90(m,6H).

Compounds of Examples 2 to 60 were prepared by a synthetic method similar to that of Examples 1, 2, 3, and 4.

Synthesis of Example 5 Compound 2

Prepare light yellow compound 2 by a synthetic method similar to Example 1

Molecular formula: C ₄₂ H ₈₃ N ₃ O ₃

Molecular weight: 678.13

LC-MS: m/z 679[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.27(t,1H,J＝3.5Hz),3.52(m,2H),3.40(m,2H),3.35(m,2H ),3.02(t,1H,J=3Hz),2.70(m,2H),2.36(m,6H),2.30(m,4H),2.20(m,2H),1.60(m,2H),1.47( m, 2H), 1.25-1.33 (m, 46H), 0.99 (m, 12H).

Synthesis of Example 6 Compound 3

Prepare light yellow compound 3 by a synthetic method similar to that of Example 1

Molecular formula: C ₄₄ H ₈₇ N ₃ O ₃

Molecular weight: 706.18

LC-MS: m/z 707[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.27(t,1H,J＝3.5),3.52(m,2H),3.40(m,2H),3.35(m,2H) ,3.02(t,1H,J=3),2.70(m,2H),2.36(m,6H),2.30(m,6H),2.20(m,2H),1.60(m,2H),1.47(m ,2H),1.25-1.33(m,50H),0.99(m,12H).

Synthesis of Example 7 Compound 4

Prepare light yellow compound 4 by a synthetic method similar to that of Example 1

Molecular formula: C ₄₄ H ₈₃ N ₃ O ₃

Molecular weight: 702.15

LC-MS: m/z 703[M+H]

1H NMR (DMSO-d6): δ (ppm) 8.06 (s, 1H), 5.60 (d, 2H, J = 2.6Hz), 5.44 (m, 2H), 5 4.27 (t, 1H, J = 3.5), 3.52(m,2H),3.40(m,2H),3.35(m,2H),3.02(t,1H,J=3),2.70(m,2H),2.65(m,2H),2.36(t, 2H, J＝6), 2.30(m, 4H), 2.20(m, 2H), 1.60(m, 2H), 1.47(m, 2H), 1.25-1.33(m, 36H), 0.99(m, 12H) .

Synthesis of Example 8 Compound 5

Prepare light yellow compound 5 by a synthetic method similar to that of Example 1

Molecular formula: C ₄₄ H ₈₉ N ₃ O ₂

Molecular weight: 691.70

LC-MS: m/z 692[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),3.37(m,2H),3.30(m,2H)3.23(m,1H),2.63(m,2H),2.44(m, 4H), 2.36(m, 4H), 1.47(m, 2H), 1.40(m, 2H), 1.25-1.33(m, 48H), 0.99(m, 12H).

Synthesis of Example 9 Compound 6

Prepare light yellow compound 6 by a synthetic method similar to that of Example 1

Molecular formula: C ₄₁ H ₈₃ N ₃ O ₂

Molecular weight: 650.12

LC-MS: m/z 651[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),3.37(m,2H),3.30(m,2H)3.23(m,1H),2.63(m,2H),2.44(m, 4H), 2.36(m, 4H), 1.47(m, 2H), 1.40(m, 2H), 1.25-1.33(m, 42H), 0.99(m, 12H).

Synthesis of Example 10 Compound 7

Prepare light yellow compound 7 by a synthetic method similar to that of Example 1

Molecular formula: C ₄₄ H ₈₇ N ₃ O ₃

Molecular weight: 706.18

LC-MS: m/z 707[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.46(t,1H,J＝5),3.62(m,2H),3.40(m,2H),3.35(m,2H) ,3.02(t,1H,J=3),2.70(m,2H),2.45(m,1H),2.36(t,2H),2.30(m,4H),2.20(m,2H),1.5-1.25 (m,54H),0.99(m,12H).

Synthesis of Example 11 Compound 8

Prepare light yellow compound 8 by a synthetic method similar to that of Example 1

Molecular formula: C ₃₂ H ₆₃ N ₃ O ₃

Molecular weight: 537.93

LC-MS: m/z 540[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.46(t,1H,J＝5),3.62(m,2H),3.40(m,2H),3.35(m,2H) ,3.02(t,1H,J=3),2.70(m,2H),2.45(m,1H),2.36(t,2H),2.30(m,4H),2.20(m,2H),1.5-1.25 (m,32H),0.99(m,9H).

Synthesis of Example 12 Compound 9

Prepare light yellow compound 9 by a synthetic method similar to embodiment 1 and embodiment 2

Molecular formula: C ₄₈ H ₉₅ N ₃ O ₄

Molecular weight: 778.34

LC-MS: m/z 779[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.46(t,1H,J＝5),3.80(m,2H),3.62(m,2H),3.40(m,2H) ,3.35(m,2H),3.02(t,1H,J=3),2.70(m,2H),2.45(m,1H),2.36(t,2H,J=6),2.30(m,4H) ,2.20(m,2H),1.5-1.25(m,50H),0.90(m,15H).

Synthesis of Example 13 Compound 10

Prepare yellow compound 10 by a synthetic method similar to embodiment 1 and embodiment 3

Molecular formula: C ₄₇ H ₉₁ N ₃ O ₅

Molecular weight: 778.32

LC-MS: m/z 779[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.46(t,1H,J＝5),3.80(m,2H),3.62(m,2H),3.40(m,2H) ,3.35(m,2H),3.02(t,1H,J＝3),2.70(m,2H),2.45(m,1H),2.36(t,2H,J＝6),2.30(m,4H) ,2.20(m,2H),1.5-1.25(m,50H),0.99(m,15H).

Synthesis of Example 14 Compound 11

Prepare light yellow compound 11 by a synthetic method similar to embodiment 1 and embodiment 4

Molecular formula: C ₄₉ H ₉₅ N ₃ O ₅

Molecular weight: 806.32

LC-MS: m/z 807[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),3.40(t,1H,J＝4.2),3.35(m,4H),3.30(m,3H),3.10(m,1H) ,2.65(m,2H),2.45(m,8H),1.52(m,2H),1.44(m,6H),1.38(m,6H),1.29(m,6H),1.26(m,40H), 0.90(m,15H).

Synthesis of Example 15 Compound 12

Prepare light yellow compound 12 by a synthetic method similar to embodiment 1 and embodiment 4

Molecular formula: C ₅₇ H ₁₁₅ N ₃ O ₄

Molecular weight: 906.56

LC-MS: m/z 907[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),3.40(t,1H,J＝4.2),3.35(m,6H),3.30(m,3H),3.10(m,2H) ,2.65(m,2H),2.45(m,8H),1.52(m,4H),1.44(m,8H),1.38(m,10H),1.29(m,10H),1.26(m,40H), 0.90(m,18H).

Synthesis of Example 16 Compound 13

Yellow compound 13 was prepared by a synthetic method similar to that of Example 2 and 4

Molecular formula: C ₅₅ H ₁₀₇ N ₃ O ₆

Molecular weight: 906.53

LC-MS: m/z 907[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.76(t,1H,J＝6.8Hz),4.47(m,1H),3.35(m,2H),3.30(m,3H ),3.10(m,1H),2.72(t,2H,J=2.9Hz),2.45(m,8H),2.30(m,4H),2.20(d,2H,J=1.3Hz),1.64(m ,4H), 1.50(m,8H), 1.38(m,4H), 1.29(m,10H), 1.26(m,36H)0.90(m,18H).

Synthesis of Example 17 Compound 14

Yellow compound 14 was prepared by a synthetic method similar to that of Example 1, 2, and 4

Molecular formula: C ₅₆ H ₁₁₁ N ₃ O ₅

Molecular weight: 906.57

LC-MS: m/z 907[M+H]

1H NMR (DMSO-d6): δ (ppm) 8.06 (s, 1H), 4.13 (t, 2H, J = 5.2Hz), 3.39 (t, 1H, J = 3.7Hz), 3.35 (m, 4H), 3.30(m,3H),3.10(m,1H),2.60(t,2H,J＝1.3Hz),2.45(m,8H),2.20(m,3H),1.60(m,6H),1.50(m ,2H), 1.43(m,8H), 1.38(m,6H), 1.29(m,10H), 1.26(m,36H), 0.90(m,18H).

Synthesis of Example 18 Compound 15

Yellow compound 15 was prepared by a synthetic method similar to that of Example 1 and 4

Molecular formula: C50H101N3O4

Molecular weight: 806.41

LC-MS: m/z 807[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.76(t,1H,J＝7.9Hz),3.35(m,2H),3.30(m,3H),3.10(m,1H ),2.70(t,2H,J＝3.1Hz),2.45(m,8H),2.20(m,3H),1.60(m,4H),1.50(m,2H),1.43(m,2H),1.38 (m,6H), 1.29(m,10H), 1.26(m,36H), 0.90(m,18H).

Synthesis of Example 19 Compound 16

Prepare yellow compound 16 by a synthetic method similar to Example 1, 4

Molecular formula: C50H101N3O3

Molecular weight: 792.38

LC-MS: m/z 800[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),3.39(t,1H,J＝4.3Hz),3.35(m,4H),3.30(m,3H),3.10(m,1H ),2.70(t,2H,J=3.1Hz),2.45(m,8H),2.20(d,2H,J=2.3Hz),1.50(m,2H),1.43(m,6H),1.38(m ,6H), 1.29(m,8H), 1.26(m,42H), 0.90(m,15H).

Synthesis of Example 20 Compound 17

Prepare yellow compound 17 by a synthetic method similar to Example 1, 4

Molecular formula: C49H97N3O4

Molecular weight: 792.33

LC-MS: m/z 793[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.76(t,1H,J＝6.8Hz),3.35(m,2H),3.30(m,3H),3.10(m,1H ),2.70(t,2H,J=3.1Hz),2.45(m,8H),2.35(m,2H),2.20(d,2H,J=2.3Hz),1.66(m,2H),1.50(m ,2H), 1.43(m,2H), 1.38(m,8H), 1.29(m,6H), 1.26(m,40H), 0.90(m,15H).

Synthesis of Example 21 Compound 18

Prepare yellow compound 18 by a synthetic method similar to Example 1, 4

Molecular formula: C ₅₂ H ₁₀₅ N ₃ O ₃

Molecular weight: 820.43

LC-MS: m/z 821[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),3.39(t,1H,J＝4.3Hz),3.35(m,4H),3.30(m,3H),3.10(m,1H ),2.70(t,2H,J=3.1Hz),2.45(m,8H),2.20(d,2H,J=2.3Hz),1.50(m,2H),1.43(m,6H),1.38(m ,6H), 1.29(m,8H), 1.26(m,48H), 0.90(m,15H).

Synthesis of Example 22 Compound 19

Prepare yellow compound 19 by a synthetic method similar to Example 1, 4

Molecular formula: C52H105N3O3

Molecular weight: 820.43

LC-MS: m/z 821[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.76(t,1H,J＝6.8Hz),3.35(m,2H),3.30(m,3H),3.10(m,1H ),2.70(t,2H,J=3.1Hz),2.45(m,8H),2.35(m,2H),2.20(d,2H,J=2.3Hz),1.66(m,2H),1.50(m ,2H), 1.43(m,2H), 1.38(m,8H), 1.29(m,6H), 1.26(m,46H), 0.90(m,15H).

Synthesis of Example 23 Compound 20

Prepare yellow compound 20 by a synthetic method similar to Example 1, 4

Molecular formula: C50H98N2O4

Molecular weight: 791.34

LC-MS: m/z 792[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.18 (t, 2H, J = 9.2Hz), 3.40 (t, 1H, J = 4.2), 3.35 (m, 4H), 3.21 (t, 1H, J = 6.2Hz), 3.10(m, 1H), 2.93(m, 2H), 2.65(d, 2H, J=2.5HZ), 2.45(m, 6H), 1.52(m, 6H), 1.44(m, 6H) ,1.38(m,8H),1.29(m,8H),1.26(m,40H),0.90(m,9H).

Synthesis of Example 24 Compound 21

Prepare yellow compound 21 by a synthetic method similar to Example 1, 4

Molecular formula: C49H99N3O5

Molecular weight: 810.35

LC-MS: m/z 811[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.18(s,2H),3.40(m,5H,),3.35(m,4H),3.30(m,3H),3.10( m,1H),2.65(d,2H,J=2.5HZ),2.59(m,4H),2.45(m,4H),2.10(d,1H,J=3.3Hz),1.85(d,1H,J =3.8Hz) 1.52 (m, 2H), 1.44 (m, 6H), 1.38 (m, 6H), 1.29 (m, 8H), 1.26 (m, 44H), 0.90 (m, 9H).

Synthesis of Example 25 Compound 22

Prepare yellow compound 22 by a synthetic method similar to Example 1, 4

Molecular formula: C50H99N3O4

Molecular weight: 806.36

LC-MS: m/z 807[M+H]

1H NMR(DMSO-d6):δ(ppm)4.18(s,2H),3.40(t,1H,J＝4.1Hz),3.35(m,4H),3.30(m,1H),3.18(t,1H ,J=3.2Hz),3.10(m,1H),2.98(t,2H,J=3.35Hz),2.65(d,1H,J=2.5HZ),2.36(d,1H,J=2.83Hz), 2.44(m,2H),2.30(m,8H),2.14(s,3H),1.52(m,2H),1.44(m,6H),1.38(m,6H),1.29(m,8H),1.26( m,40H), 0.90(m,9H).

Synthesis of Example 26 Compound 23

Yellow compound 23 was prepared by a synthetic method similar to Example 1 and 4

Molecular formula: C49H96N2O5

Molecular weight: 793.32

LC-MS: m/z 794[M+H]

1H NMR(DMSO-d6):δ(ppm)4.18(s,2H),3.58(m,4H),3.40(t,1H,J＝4.1Hz),3.35(m,4H),3.30(m,1H ),3.18(t,1H,J=3.2Hz),3.10(m,1H),2.98(t,2H,J=3.35Hz),2.65(d,1H,J=2.5HZ),2.52(m,4H ),2.45(m,2H)2.36(d,1H,J=2.83Hz),2.17(d,1H,J=1.82Hz),2.03(d,1H,J=1.56Hz),1.52(m,2H) ,1.44(m,6H),1.38(m,6H),1.29(m,8H),1.26(m,40H),0.90(m,9H).

Synthesis of Example 27 Compound 24

Prepare yellow compound 24 by a synthetic method similar to Example 1, 4

Molecular formula: C49H96N2O4

Molecular weight: 777.32

LC-MS: m/z 778[M+H]

1H NMR(DMSO-d6):δ(ppm)4.23(s,2H),3.40(t,1H,J＝4.1Hz),3.35(m,4H),3.30(m,1H),3.18(t,1H ,J=3.2Hz),3.10(m,1H),2.98(t,2H,J=3.35Hz),2.65(d,1H,J=2.5HZ),2.52(m,4H),2.36(d,1H ,J=2.83Hz),2.17(d,1H,J=1.87Hz),2.00(d,1H,J=1.72Hz),1.70(m,4H),1.52(m,2H),1.44(m,6H ), 1.38(m,6H), 1.29(m,8H), 1.26(m,40H), 0.90(m,9H).

Synthesis of Example 28 Compound 25

Prepare yellow compound 25 by a synthetic method similar to Example 1, 4

Molecular formula: C48H96N2O4

Molecular weight: 765.31

LC-MS: m/z 766M+H]

1H NMR(DMSO-d6):δ(ppm)4.10(s,2H),3.40(t,1H,J＝4.1Hz),3.35(m,4H),3.30(m,1H),3.18(t,1H ,J=3.2Hz),3.10(m,1H),2.65(d,1H,J=2.5HZ),2.36(d,1H,J=2.83Hz),2.32(m,2H),2.17(m,6H ),,1.70(m,2H),1.52(m,2H),1.44(m,6H),1.38(m,6H),1.29(m,8H),1.26(m,40H)0.90(m,9H) .

Synthesis of Example 29 Compound 26

Prepare yellow compound 26 by a synthetic method similar to Example 1, 4

Molecular formula: C57H115N3O6

Molecular weight: 938.56

LC-MS: m/z 939[M+H]

1H NMR(DMSO-d6):δ(ppm)8.06(s,1H),4.52(s,2H),3.45(m,4H),3.40(t,1H,J＝4.1Hz),3.35(m,6H ),3.30(m,3H),3.10(m,2H),2.65(d,1H,J＝2.5HZ),2.58(m,4H),2.46(m,4H),2.36(d,1H,J＝ 2.83Hz), 2.32(m, 2H), 2.10(d, 1H, J=3.8Hz), 1.95(d, 1H, J=3.01Hz), 1.52(m, 4H), 1.44(m, 8H), 1.38 (m,8H), 1.29(m,10H), 1.26(m,40H), 0.90(m,12H).

Synthesis of Example 30 Compound 27

Prepare yellow compound 27 by a synthetic method similar to Example 1, 4

Molecular formula: C56H112N2O5

Molecular weight: 893.52

LC-MS: m/z 894[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.18 (t, 2H, J = 2.3Hz), 3.45 (t, 1H, J = 3.3Hz), 3.35 (m, 6H), 3.30 (m, 3H), 3.10(m,2H),2.65(d,1H,J=2.5HZ),2.38(m,2H),2.36(d,1H,J=2.83Hz),2.15(s,6H), 2.10(d,1H ,J=3.8Hz),1.95(d,1H,J=3.01Hz),1.68(m,2H),1.52(m,4H),1.44(m,8H),1.38(m,8H),1.29(m ,10H), 1.26(m,40H)0.90(m,12H).

Synthesis of Example 31 Compound 28

Prepare yellow compound 28 by a synthetic method similar to Example 1, 4

Molecular formula: C58H114N2O5

Molecular weight: 919.56

LC-MS: m/z 920[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.18 (t, 2H, J = 2.3Hz), 3.45 (t, 1H, J = 3.3Hz), 3.35 (m, 6H), 3.30 (m, 3H), 3.10(m,2H),2.97(m,2H),2.65(d,1H,J=2.5HZ),2.46(m,4H),2.38(m,2H),2.36(d,1H,J=2.83Hz ),2.10(d,1H,J=3.8Hz),1.95(d,1H,J=3.01Hz),1.52(m,4H),1.47(m,4H),1.44(m,8H),1.38(m ,10H), 1.29(m,10H), 1.26(m,40H)0.90(m,12H).

Synthesis of Example 32 Compound 29

Prepare yellow compound 29 by a synthetic method similar to Example 1, 4

Molecular formula: C58H114N2O6

Molecular weight: 935.56

LC-MS: m/z 936[M+H]

1H NMR(DMSO-d6):δ(ppm)5.52(s,1H),4.18(t,2H,J＝2.3Hz),4.01(m,1H),3.45(t,1H,J＝3.3Hz), 3.35(m,6H),3.20(t,1H,J=6.7Hz),3.10(m,2H),2.97(m,2H),2.65(d,1H,J=2.5HZ),2.45-2.53(m ,6H),2.36(d,1H,J=2.83Hz),2.10(d,1H,J=3.8Hz),1.99(d,1H,J=3.6Hz),1.82(m,4H),1.52(m ,4H), 1.44(m,8H), 1.38(m,10H), 1.29(m,10H), 1.26(m,40H)0.90(m,12H).

Synthesis of Example 33 Compound 30

Prepare yellow compound 30 by a synthetic method similar to Example 1, 4

Molecular formula: C57H112N2O5

Molecular weight: 905.53

LC-MS: m/z 906[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.18 (t, 2H, J = 2.3Hz), 3.45 (t, 1H, J = 3.3Hz), 3.35 (m, 6H), 3.20 (t, 1H, J =6.7Hz),3.10(m,2H),2.97(m,2H),2.65(d,1H,J=2.5HZ),2.53(m,4H),2.45(m,2H),2.36(d,1H ,J=2.83Hz),2.10(d,1H,J=3.8Hz),1.99(d,1H,J=3.6Hz),1.69(m,4H),1.52(m,4H),1.44(m,8H ), 1.38(m,10H), 1.29(m,10H), 1.26(m,40H)0.90(m,12H).

Synthesis of Example 34 Compound 31

Yellow compound 31 was prepared by a synthetic method similar to Example 1 and 4

Molecular formula: C58H115N3O5

Molecular weight: 934.57

LC-MS: m/z 935[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.18 (t, 2H, J = 2.3Hz), 3.45 (t, 1H, J = 3.3Hz), 3.35 (m, 6H), 3.20 (t, 1H, J =6.7Hz),3.10(m,2H),2.97(m,2H),2.65(d,1H,J=2.5HZ),2.53(m,4H),2.45(m,2H),2.36(d,1H ,J=2.83Hz),2.30(m,8H),2.20(d,1H,J=3.8Hz),2.15(s,3H),1.99(d,1H,J=3.6Hz),1.52(m,4H ), 1.44(m,8H), 1.38(m,10H), 1.29(m,10H), 1.26(m,40H), 0.90(m,12H).

Synthesis of Example 35 Compound 32

Yellow compound 32 was prepared by a synthetic method similar to that of Example 1 and 4

Molecular formula: C50H99N3O6

Molecular weight: 838.41

LC-MS: m/z 839[M+H]

1H NMR (DMSO-d6): δ (ppm) 8.06 (s, 1H), 4.76 (t, 1H, J = 7.9Hz), 4.52 (s, 2H), 3.48 (m, 4H), 3.35 (m, 2H ),3.30(m,3H),3.10(m,1H),2.70(d,2H,J＝3.1Hz),2.62(m,4H),2.45(m,4H),2.20(m,3H),1.60 (m,4H), 1.50(m,2H), 1.43(m,2H), 1.38(m,6H), 1.29(m,10H), 1.26(m,36H), 0.90(m,12H).

Synthesis of Example 36 Compound 33

Yellow compound 33 was prepared by a synthetic method similar to Example 1 and 4

Molecular formula: C49H96N2O5

Molecular weight: 793.37

LC-MS: m/z 794[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.76 (t, 1H, J = 7.9Hz), 4.12 (m, 2H), 3.35 (m, 2H), 3.20 (m, 1H), 3.10 (m, 1H ),2.70(d,2H,J＝3.1Hz),2.45(m,4H),2.20(m,7H),2.31(m,2H),1.70(m,6H),1.50(m,6H),1.43 (m,2H), 1.38(m,8H), 1.29(m,10H), 1.26(m,36H), 0.90(m,12H).

Synthesis of Example 37 Compound 34

Prepare yellow compound 34 by a synthetic method similar to Example 1, 4

Molecular formula: C48H94N2O5

Molecular weight: 793.37

LC-MS: m/z 794[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.76 (t, 1H, J = 7.9Hz), 4.12 (m, 2H), 3.35 (m, 2H), 3.20 (m, 1H), 3.10 (m, 1H ),2.70(d,2H,J＝3.1Hz),2.45(m,4H),2.20(m,7H),2.31(m,2H),1.70(m,6H),1.50(m,6H),1.43 (m,2H),1.38(m,8H),1.29(m,8H),1.26(m,36H),0.90(m,12H).

Synthesis of Example 38 Compound 35

Prepare yellow compound 35 by a synthetic method similar to Example 1, 4

Molecular formula: C44H86N2O5

Molecular weight: 723.18

LC-MS: m/z 724[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.76 (t, 1H, J = 7.9Hz), 4.12 (m, 2H), 3.35 (m, 2H), 3.20 (m, 1H), 3.10 (m, 1H ),2.70(d,2H,J＝3.1Hz),2.45(m,4H),2.20(m,7H),2.31(m,2H),1.70(m,6H),1.50(m,6H),1.43 (m,2H), 1.38(m,8H), 1.29(m,10H), 1.26(m,34H), 0.90(m,12H).

Synthesis of Example 39 Compound 36

Yellow compound 36 was prepared by a synthetic method similar to that of Example 1 and 4

Molecular formula: C51H88N2O5

Molecular weight: 819.41

LC-MS: m/z 820[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.76 (t, 1H, J = 7.9Hz), 4.12 (m, 2H), 3.35 (m, 2H), 3.20 (m, 1H), 3.10 (m, 1H ),2.97(m,2H),2.70(d,2H,J＝3.1Hz),2.45(m,4H),2.20(m,1H),2.31(m,2H),1.50(m,2H),1.43 (m,2H), 1.38(m,8H), 1.29(m,10H), 1.26(m,36H), 0.90(m,12H).

Synthesis of Example 40 Compound 37

Prepare yellow compound 37 by a synthetic method similar to Example 1, 4

Molecular formula: C50H96N2O6

Molecular weight: 821.38

LC-MS: m/z 822[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.76 (t, 1H, J = 7.9Hz), 4.12 (m, 2H), 3.59 (m, 4H), 3.35 (m, 2H), 3.20 (m, 1H ),3.10(m,1H),2.97(m,2H),2.70(d,2H,J＝3.1Hz),2.45(m,4H),2.20(m,1H),2.31(m,2H),1.50 (m,2H),1.43(m,2H),1.38(m,6H),1.29(m,10H),1.26(m,36H),0.90(m,12H).

Synthesis of Example 41 Compound 38

Yellow compound 38 was prepared by a synthetic method similar to that of Example 1 and 4

Molecular formula: C49H94N2O6

Molecular weight: 835.41

LC-MS: m/z 836[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.76 (t, 1H, J = 7.9Hz), 4.49 (s, 1H), 4.20 (m, 2H), 3.59 (m, 1H), 3.35 (m, 2H ),3.20(m,1H),3.10(m,1H),2.97(m,2H),2.70(d,2H,J=3.1Hz),2.45(m,6H),2.20(m,1H),2.31 (m,2H),1.77(m,4H),1.50(m,2H),1.43(m,2H),1.38(m,6H),1.29(m,10H),1.26(m,36H),0.90( m,12H).

Synthesis of Example 42 Compound 39

Prepare yellow compound 39 by a synthetic method similar to Example 1, 4

Molecular formula: C50H96N2O5

Molecular weight: 805.38

LC-MS: m/z 806[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.76 (t, 1H, J = 7.9Hz), 4.20 (m, 2H), 3.35 (m, 2H), 3.20 (m, 1H), 3.10 (m, 1H ),2.97(m,2H),2.70(d,2H,J＝3.1Hz),2.45(m,6H),2.20(m,1H),2.31(m,2H),1.71(m,4H),1.50 (m,2H),1.43(m,2H),1.38(m,6H),1.29(m,10H),1.26(m,36H),0.90(m,12H).

Synthesis of Example 43 Compound 40

Prepare yellow compound 40 by a synthetic method similar to Example 1, 4

Molecular formula: C51H99N3O5

Molecular weight: 834.42

LC-MS: m/z 835[M+H]

1H NMR (DMSO-d6): δ (ppm) 4.76 (t, 1H, J = 7.9Hz), 4.20 (m, 2H), 3.35 (m, 2H), 3.20 (m, 1H), 3.10 (m, 1H ),2.97(m,2H),2.70(d,2H,J＝3.1Hz),2.45(m,2H),2.31(m,2H),2.29(m,8H),2.18(m,1H),2.14 (s,3H), 1.50(m,2H), 1.43(m,2H), 1.38(m,6H), 1.29(m,10H), 1.26(m,36H), 0.90(m,12H).

Synthesis of Example 44 Compound 41

Yellow compound 41 was prepared by a synthetic method similar to that of Examples 1, 2, and 4

Molecular formula: C56H111N3O7

Molecular weight: 938.57

LC-MS: m/z 938[M+H]

1H NMR(DMSO-d6):δ(ppm)4.20(s,2H),4.18(m,2H),4.15(m,2H),3.46(t,1H,J＝3.2Hz),3.43(m,4H ),3.35(m,4H),3.20(m,3H),3.10(m,1H),2.62(d,2H,J＝2.1Hz),2.57(m,4H),2.45(m,4H),2.18 (m,1H),2.14(d,2H,J＝3.5Hz),1.62(m,6H),1.50(m,2H),1.43(m,8H),1.38(m,6H),1.29(m, 10H), 1.26(m,36H), 0.90(m,12H).

Synthesis of Example 45 Compound 42

Yellow compound 42 was prepared by a synthetic method similar to that of Examples 1, 2, and 4

Molecular formula: C57H110N2O7

Molecular weight: 935.51

LC-MS: m/z 936[M+H]

1H NMR(DMSO-d6):δ(ppm)4.72(s,1H),4.18(m,2H),4.15(m,2H),3.46(t,1H,J＝3.2Hz),3.72(m,1H ),3.35(m,4H),3.20(m,3H),3.10(m,1H),2.62(d,2H,J=2.1Hz),2.57(m,4H),2.45(m,6H),2.18 (m,1H),2.14(d,2H,J＝3.5Hz),1.82(m,4H),1.62(m,6H),1.50(m,2H),1.43(m,8H),1.38(m, 6H), 1.29(m,10H), 1.26(m,36H)0.90(m,12H).

Synthesis of Example 46 Compound 43

Yellow compound 43 was prepared by a synthetic method similar to Example 1, 2, and 4

Molecular formula: C57H111N3O6

Molecular weight: 934.58

LC-MS: m/z 963[M+H]

1H NMR(DMSO-d6):δ(ppm)4.33(m,2H),4.15(m,2H),3.46(t,1H,J＝3.2Hz),3.35(m,4H),3.20(m,3H ),3.10(m,1H),2.97(m,2H),2.62(d,2H,J=2.1Hz),2.45(m,2H),2.29(m,8H),2.18(m,4H),2.14 (d,2H,J=3.5Hz),1.62(m,6H),1.50(m,2H),1.43(m,8H),1.38(m,6H),1.29(m,10H),1.26(m, 36H), 0.90(m, 12H).

Synthesis of Example 47 Compound 44

Yellow compound 44 was prepared by a synthetic method similar to Example 1, 2, and 4

Molecular formula: C57H110N2O6

Molecular weight: 919.57

LC-MS: m/z 920[M+H]

1H NMR(DMSO-d6):δ(ppm)4.33(m,2H),4.15(m,2H),3.46(t,1H,J＝3.2Hz),3.35(m,4H),3.20(m,3H ),3.10(m,1H),2.97(m,2H),2.62(d,2H,J＝2.1Hz),2.45(m,6H),2.18(m,4H),2.14(d,2H,J＝ 3.5Hz), 1.62(m, 6H), 1.50(m, 6H), 1.43(m, 8H), 1.38(m, 8H), 1.29(m, 10H), 1.26(m, 36H), 0.90(m, 12H).

Synthesis of Example 48 Compound 45

Yellow compound 45 was prepared by a synthetic method similar to Example 1, 2, and 4

Molecular formula: C56H108N2O6

Molecular weight: 905.54

LC-MS: m/z 906[M+H]

1H NMR(DMSO-d6):δ(ppm)4.33(m,2H),4.15(m,2H),3.46(t,1H,J＝3.2Hz),3.35(m,4H),3.20(m,3H ),3.10(m,1H),2.97(m,2H),2.62(d,2H,J=2.1Hz),2.45(m,6H),2.14(d,2H,J=3.5Hz),1.62(m ,10H), 1.50(m,2H), 1.43(m,8H), 1.38(m,6H), 1.29(m,10H), 1.26(m,36H), 0.90(m,12H).

Synthesis of Example 49 Compound 46

Yellow compound 46 was prepared by a synthetic method similar to that of Example 1 and 3

Molecular formula: C57H104N2O10

Molecular weight: 977.46

LC-MS: m/z 978[M+H]

1H NMR (DMSO-d6): δ (ppm) 5.14 (t, 1H, J = 9.55Hz), 4.47 (m, 2H), 4.29 (t, 1H, J = 8.96Hz), 4.18 (m, 2H), 3.83(d,2H,J=5.67Hz),3.57(m,4H),2.97(m,2H),2.69(d,2H,J=4.23Hz),2.50(m,4H),2.35(m,6H ), 2.05(m,2H), 1.64(m,6H), 1.26-1.50(m,58H), 0.90(m,12H).

Synthesis of Example 50 Compound 47

Yellow compound 47 was prepared by a synthetic method similar to that of Example 1 and 3

Molecular formula: C58H107N3O9

Molecular weight: 990.51

LC-MS: m/z 991[M+H]

1H NMR (DMSO-d6): δ (ppm) 5.14 (t, 1H, J = 9.55Hz), 4.47 (m, 2H), 4.29 (t, 1H, J = 8.96Hz), 4.18 (m, 2H), 3.83(d,2H,J=5.67Hz),2.97(m,2H),2.69(d,2H,J=4.23Hz),2.35(m,6H),2.29(m,8H),2.14(s,3H ), 2.05(m,2H), 1.64(m,6H), 1.26-1.50(m,58H), 0.90(m,12H).

Synthesis of Example 51 Compound 48

Prepare yellow compound 48 by a synthetic method similar to Example 1, 3

Molecular formula: C57H106N2O11

Molecular weight: 995.48

LC-MS: m/z 996[M+H]

1H NMR (DMSO-d6): δ (ppm) 5.14 (t, 1H, J = 9.55Hz), 4.47 (m, 2H), 4.29 (t, 1H, J = 8.96Hz), 4.18 (m, 2H), 4.16(s,2H),3.83(d,2H,J=5.67Hz),3.42(m,4H),2.97(m,2H),2.69(d,2H,J=4.23Hz),2.57(m,4H ),2.35(m,6H),2.05(m,2H),1.64(m,6H),1.26-1.50(m,58H),0.90(m,12H).

Synthesis of Example 52 Compound 49

Yellow compound 49 was prepared by a synthetic method similar to that of Example 1 and 3

Molecular formula: C58H106N2O10

Molecular weight: 991.49

LC-MS: m/z 936[M+H]

1H NMR (DMSO-d6): δ (ppm) 5.14 (t, 1H, J = 9.55Hz), 4.47 (m, 2H), 4.29 (t, 1H, J = 8.96Hz), 4.18 (m, 2H), 4.49(s,2H),3.83(d,2H,J=5.67Hz),3.60(m,1H),2.97(m,2H),2.69(d,2H,J=4.23Hz),2.57(m,4H ),2.35(m,6H),2.05(m,2H),1.77(m,4H),1.64(m,6H),1.26-1.50(m,58H),0.90(m,12H).

Synthesis of Example 53 Compound 50

Prepare yellow compound 50 by a synthetic method similar to Example 1 and 3

Molecular formula: C44H83N3O5

Molecular weight: 734.27

LC-MS: m/z 735[M+H]

1H NMR (DMSO-d6): δ (ppm) 5.14 (t, 1H, J = 9.55Hz), 4.29 (t, 1H, J = 8.96Hz), 4.18 (m, 2H), 3.83 (d, 2H, J =5.67Hz),2.97(m,2H),2.69(d,2H,J=4.23Hz),2.29(m,8H),2.27(m,1H),2.14(m,3H),2.13(m,1H ), 1.60 (m, 4H), 1.26-1.50 (m, 48H), 0.90 (m, 12H).

Synthesis of Example 54 Compound 51

Prepare yellow compound 51 by a synthetic method similar to Example 1

Molecular formula: C43H82N2O7

Molecular weight: 739.24

LC-MS: m/z 7940[M+H]

1H NMR (DMSO-d6): δ (ppm) 5.14 (t, 1H, J = 9.55Hz), 4.29 (t, 1H, J = 8.96Hz), 4.18 (m, 2H), 4.16 (s, 2H), 3.83(d,2H,J=5.67Hz),3.42(m,2H),2.97(m,2H),2.69(d,2H,J=4.23Hz),2.57(m,4H),,2.27(m, 1H), 2.13(m, 1H), 1.60(m, 4H), 1.26-1.50(m, 48H), 0.90(m, 12H).

Synthesis of Example 55 Compound 52

Prepare yellow compound 52 by a synthetic method similar to Example 1

Molecular formula: C44H82N2O6

Molecular weight: 735.26

LC-MS: m/z 736[M+H]

1H NMR (DMSO-d6): δ (ppm) 5.14 (t, 1H, J = 9.55Hz), 4.49 (s, 1H), 4.29 (t, 1H, J = 8.96Hz), 4.18 (m, 2H), 3.83(d,2H,J=5.67Hz),3.60(m,1H),2.97(m,2H),2.69(d,2H,J=4.23Hz),2.51(m,4H),,2.27(m, 1H), 2.13(m, 1H), 1.77(m, 4H), 1.60(m, 4H), 1.26-1.50(m, 48H), 0.90(m, 12H).

Synthesis of Example 56 Compound 53

Yellow compound 53 was prepared by a synthetic method similar to that of Example 1 and 2

Molecular formula: C55H107N3O8

Molecular weight: 938.47

LC-MS: m/z 995[M+H]

1H NMR(DMSO-d6):δ(ppm)8.01(s,1H),4.16(s,2H),4.13(m,4H),3.42(m,2H),3.39(t,1H,J＝3.2Hz ),3.35(m,2H),3.30(m,3H),2.69(d,2H,J＝4.23Hz),2.57(m,4H),2.43(m,4H),2.13(m,2H),1.60 (m,12H),1.26-1.50(m,52H),0.90(m,12H).

Synthesis of Example 57 Compound 54

Yellow compound 54 was prepared by a synthetic method similar to that of Example 1 and 2

Molecular formula: C55H104N2O8

Molecular weight: 921.44

LC-MS: m/z 922[M+H]

1H NMR(DMSO-d6):δ(ppm)4.18(m,2H),4.13(m,4H),3.57(m,4H),3.39(t,1H,J＝3.2Hz),3.35(m,2H ),3.18(t,1H,J=3.51Hz),2.69(d,2H,J=4.23Hz),2.50(m,4H),2.43(m,2H),2.13(m,2H),1.60(m ,12H),1.26-1.50(m,52H),0.90(m,12H).

Synthesis of Example 58 Compound 55

Yellow compound 55 was prepared by a synthetic method similar to that of Example 1 and 2

Molecular formula: C56H107N3O7

Molecular weight: 934.49

LC-MS: m/z 935[M+H]

1H NMR(DMSO-d6):δ(ppm)4.18(m,2H),4.13(m,4H),3.57(m,4H),3.39(t,1H,J＝3.2Hz),3.35(m,2H ),3.18(t,1H,J=3.51Hz),2.97(m,2H),2.69(d,2H,J=4.23Hz),2.29(m,8H),2.14(s,3H),2.13(m ,2H), 1.60(m,12H), 1.26-1.50(m,52H), 0.90(m,12H).

Synthesis of Example 59 Compound 56

Yellow compound 56 was prepared by a synthetic method similar to that of Example 1 and 2

Molecular formula: C56H106N2O8

Molecular weight: 935.47

LC-MS: m/z 936[M+H]

1H NMR(DMSO-d6):δ(ppm)4.49(s,1H)4.18(m,2H),4.13(m,4H),,3.60(m,1H),3.39(t,1H,J＝3.2Hz ),3.35(m,2H),3.18(t,1H,J＝3.51Hz),2.97(m,2H),2.69(d,2H,J＝4.23Hz),2.52(m,4H),2.13(m ,2H), 1.77(m,4H), 1.60(m,12H), 1.26-1.50(m,52H), 0.90(m,12H).

Synthesis of Example 60 Compound 57

Prepare yellow compound 57 by a synthetic method similar to Example 1,2

Molecular formula: C57H106N2O9

Molecular weight: 963.48

LC-MS: m/z 964[M+H]

1H NMR (DMSO-d6): δ (ppm), 4.76 (d, 1H, J = 8.22Hz), 4.49 (s, 1H), 4.47 (m, 1H), 4.18 (m, 2H), 4.13 (m, 2H), 3.60(m, 1H), 3.39(t, 1H, J=3.2Hz), 3.35(m, 2H), 3.18(t, 1H, J=3.51Hz), 2.97(m, 2H), 2.71( d,2H,J＝4.23Hz),2.52(m,4H),2.32(m,4H),2.13(m,1H),1.77(m,4H),1.64(m,4H),1.60(m,6H ), 1.26-1.50 (m, 50H), 0.90 (m, 12H).

Synthesis of Example 61 Compound 58

Yellow compound 58 was prepared by a synthetic method similar to that of Example 1 and 2

Molecular formula: C57H106N3O8

Molecular weight: 963.48

LC-MS: m/z 936[M+H]

1H NMR(DMSO-d6):δ(ppm),4.76(d,1H,J＝8.22Hz),,4.47(m,1H),4.18(m,2H),4.13(m,2H),3.39(t ,1H,J=3.2Hz),3.35(m,2H),3.18(t,1H,J=3.51Hz),2.97(m,2H),2.71(d,2H,J=4.23Hz),2.32(m ,4H),2.29(m,8H),2.14(s,3H),2.13(m,1H),1.64(m,4H),1.60(m,6H),1.26-1.50(m,50H),0.90( m,12H).

Synthesis of Example 62 Compound 59

Prepare yellow compound 59 by a synthetic method similar to Example 1,2

Molecular formula: C56H104N2O9

Molecular weight: 949.45

LC-MS: m/z 950[M+H]

1H NMR(DMSO-d6):δ(ppm),4.76(d,1H,J=8.22Hz),,4.47(m,1H),4.18(m,2H),4.13(m,2H),3.57(m ,4H),3.39(t,1H,J=3.2Hz),3.35(m,2H),3.18(t,1H,J=3.51Hz),2.97(m,2H),2.71(d,2H,J= 4.23Hz), 2.50(m, 4H), 2.32(m, 4H), 2.14(s, 3H), 2.13(m, 1H), 1.64(m, 4H), 1.60(m, 6H), 1.26-1.50( m,50H), 0.90(m,12H).

Synthesis of Example 63 Compound 60

Prepare yellow compound 60 by a synthetic method similar to Example 1,2

Molecular formula: C52H107N3O9

Molecular weight: 966.54

LC-MS: m/z 967[M+H]

1H NMR(DMSO-d6):δ(ppm),4.76(d,1H,J＝8.22Hz),,4.47(m,1H),4.16(s,2H),4.13(m,2H),3.42(m ,2H),3.39(t,1H,J＝3.2Hz),3.35(m,2H),3.30(m,2H),3.18(t,1H,J＝3.51Hz),2.71(d,2H,J＝ 4.23Hz), 2.46(m, 2H), 2.32(m, 4H), 2.14(s, 3H), 2.13(m, 1H), 1.64(m, 4H), 1.60(m, 6H), 1.26-1.50( m,50H), 0.90(m,12H).

Example 64

In vivo evaluation of luciferase mRNA using a lipid nanoparticle composition

Cationic lipids, DSPC, cholesterol and PEG-lipids were dissolved in ethanol at a molar ratio of 50:10:38:2 or 48:10:40:2. Lipid nanoparticles (LNPs) were prepared at a weight ratio of total lipid to mRNA of about 10:1 to 30:1. Briefly, mRNA was diluted to 0.15 mg/mL in 10 mL to 50 mL citrate buffer (pH=4). Using a syringe pump, mix the lipid in ethanol solution with the mRNA in water solution at a ratio of about 1:5 to 1:3 (vol/vol) at a total flow rate of 10 mL/min or more. Ethanol was then removed and the external buffer was replaced by 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, as 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.

The study was performed on 6-8 week old female C57BL/6 mice, 8-10 week old CD-1 mice according to the guidelines established by the National Science and Technology Commission. Different doses of mRNA lipid nanoparticles were administered systemically via tail vein injection, and animals were euthanized at specific time points (eg, 5 hours) after administration. Livers and spleens were collected in pre-weighed tubes, weight determined, immediately snap frozen in liquid nitrogen, and stored at -80°C until analysis.

For liver, approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). 1/4" ceramic balls (MP Biomedicals) were 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. Using the FastPrep24 instrument (MP Biomedicals), the Liver tissue was homogenized at 2x6.0m/s for 15 s. The homogenate was incubated at room temperature for 5 min, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically , 50 μL of diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, then incubated for 5 minutes, and then quantified using a SpectraMAX_L chemiluminescent microplate reader (Megu Molecular Instruments (Shanghai) Co., Ltd.). Use the BCA protein quantification kit (Shanghai Yise Medical Technology Co., Ltd.) to determine the amount of assayed protein. The relative luminescence units (RLU) are then normalized to the total μg of assayed protein. In order to convert RLU into μg fluorescein Enzymes, standard curves were generated using QuantiLum recombinant luciferase (Promega).

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 culture to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely replaced by 5-methylcytidine and pseudouridine.

Example 65

Determination of the pKa of the prepared lipid

The pKa of the formulated cationic lipid correlates with the effectiveness of the LNP for delivery of nucleic acids. The preferred pKa range is 5-7. The pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-phthanesulfonic acid (TNS). As described in Example 64, a sequenced method was used to prepare cationic lipid/DSPC/cholesterol/PEG lipid (50/10/38/2 mol %) containing cationic lipid/DSPC/cholesterol/PEG lipid (50/10/38/2 mol %) at a concentration of 0.4 mM total lipid in PBS. Lipid nanoparticles. TNS was prepared as a 100 μM stock solution in distilled water. The vesicles were diluted to contain 24 μM lipid in 2 mL of a buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM sodium acetate, 130 mM NaCl, wherein the pH value was 2.5-11. Aliquots of TNS solution were added to give a final concentration of 1 μΜ, and after vortex mixing, fluorescence intensity was measured in a SLM Aminco Series 2 Luminescence Spectrophotometer at room temperature using excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid best-fit analysis was applied to the fluorescence data, and pKa was measured as the pH that yielded half the maximum fluorescence intensity.

Example 66

Determination of the Potency of Lipid Nanoparticle Formulations Containing Various Cationic Lipids Using a Rodent Model of In Vivo Luciferase mRNA Expression

For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the ordered mixing method as described in Example 64. 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, the average PEG molecular weight is 2000). As described in Example 64, 5 hours after administration via tail vein injection, by Relative activity was determined by measuring luciferase expression in the liver. The activity was compared at doses of 0.3 and 1.0 mg mRNA/kg and expressed as ng luciferase measured 5 hours after administration as described in Example 64 /g liver.The results of embodiment 64 and 65 are shown in table 2.

Table 2. Comparative lipids exhibiting activity with mRNA

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the scope of the disclosed patents. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (19)

A pyrrolidine compound as shown in formula A, or its isomer, or its N-oxide, or its pharmaceutically acceptable salt, prodrug;

in: _L is selected from the following structures: -C-, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _X- , - SS-, -C(=O)S-, -SC(=O)-, -NC(=O)-, -C(=O)-N-, _L2 is selected from the following structures: -C-, - (C=O)O-, -C(=O)-, -S(O) _X- , -C(=O)S-, -C(=O)-N-; R ₁ and R ₂ are each independently C6-C24 alkyl or C6-C24 alkenyl, and the hydrocarbon chains are optionally connected by one or more ester bonds or ether bonds; R ₃ and R ₄ are each independently C1-C12 alkyl or C1-C12 alkenyl, or R ₃ and R ₄ combine with each other to form a 4- to 10-membered heterocyclic ring, and the heteroatoms include one of N, O, and S One or more heteroatoms, the heterocycle is optionally substituted by 1-6 heteroatoms; X is C, N, O, S, -S-S-; M is C1-C12 alkyl or C1-C12 alkenyl; x is 0, 1 or 2. The compound of claim 1, wherein L ₁ is one of -O(C=O)- or -O-, and L ₂ is one of -C- or -C(=O)-. The compound of claim ₁ , wherein R and _R or both have one of the following structures:

The compound as claimed in claim 1, wherein X is N or O, and M is C1-C6 alkyl. It is characterized in that: wherein MN(R ₃ )(R ₄ ) has one of the following structures:

A preparation method of the pyrrolidine compound A described in any one of claims 1-5, which comprises the following steps: (1) In an organic solvent, under the action of an acid-binding agent or a condensing agent, the compound H and the compound G of the general formula undergo a substitution/condensation reaction to obtain the intermediate F of the general formula; (2) In an organic solvent, under the action of a condensing agent, the intermediate F of the general formula is condensed with the compound E of the general formula to obtain the intermediate D of the general formula; (3) In an organic solvent, under acid catalysis, the intermediate D of the general formula is subjected to a deprotection reaction to obtain the intermediate C of the general formula; (2) In an organic solvent, under the action of an acid-binding agent and a condensing agent, the intermediate C of the general formula is substituted/condensed with the compound B of the general formula to obtain the pyrrolidine compound of the general formula A;

A composition comprising a compound of any one of the preceding claims and a therapeutic agent. The composition according to claim 7, which further comprises one or more components selected from neutral lipids, steroids and polymer lipids. The composition according to claim 8, wherein the neutral lipid comprises one or more components of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM, preferably DSPC. The composition of any one of claims 7-9, wherein the molar ratio of the compound to the neutral lipid is about 2:1 to 8:1. The composition according to any one of claims 7-10, wherein the steroid comprises one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol One or more, preferably cholesterol. The composition of claim 11, wherein the molar ratio of the compound to the steroid is about 1:1 to 5:1. The composition according to any one of claims 7-12, wherein the polymer lipid is a pegylated lipid comprising PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or PEG-dialkoxypropyl carbamate. The composition as claimed in claim 13, wherein said PEGylated lipid has the following structure (I):

Or its isomer, pharmaceutically acceptable salt, prodrug, wherein: R ₇ and R ₈ are each independently a linear or branched, saturated or unsaturated hydrocarbyl chain containing 10 to 30 carbon atoms, wherein the hydrocarbyl chains are optionally connected by one or more ester bonds; w is an average value of 30 to 60. The composition as claimed in claim 14, wherein R ₇ and R ₈ are each independently a linear, saturated or unsaturated hydrocarbyl chain containing 12 to 22 carbon atoms. The composition of any one of claims 7-15, wherein the therapeutic agent is a nucleic acid. The composition of any one of claim 16, wherein the nucleic acid is selected from antisense nucleic acid, small interfering nucleic acid (siRNA), micronucleic acid (miRNA) and messenger nucleic acid (mRNA). Use of the composition according to any one of claims 7-17 in the preparation of medicines or vaccines for the treatment of the following diseases, which include infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases , neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases. The composition according to any one of claims 7-18, the route of administration to the patient comprises: intravenous, intramuscular, subcutaneous, intradermal, intranasal or inhalation administration.