CN115850686A

CN115850686A - Novel deuterated PEG lipid compound, preparation method, composition and application thereof

Info

Publication number: CN115850686A
Application number: CN202111133137.0A
Authority: CN
Inventors: 黄才古; 孙辉; 徐天祥
Original assignee: Guangzhou Anovent Pharmaceutical Co Ltd
Current assignee: Guangzhou Anovent Pharmaceutical Co Ltd
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2023-03-28
Also published as: WO2023045377A1

Abstract

The invention relates to a novel deuterated PEG lipid compound, a preparation method, a composition and application thereof, and particularly discloses a novel deuterated lipid compound shown as a formula A and a formula B, or pharmaceutically acceptable salt or isomer thereof. Also discloses a preparation method, a composition and application of the PEG lipid compound. The compounds are used as functional PEG lipids, pharmaceutical compositions suitable for nucleic acid delivery, can achieve superior stability, water solubility, and intracellular delivery efficiency of the drug, wherein A and formula B are as follows.

Description

Novel deuterated PEG lipid compound, preparation method, composition and application thereof

Technical Field

The present invention provides novel deuterated polymer-conjugated lipids that can be used in combination with other lipid components (e.g., neutral lipids, steroids, and cationic lipids) to form a nucleic acid mRNA lipid nanoparticle composition for delivering one or more therapeutic and/or prophylactic agents to mammalian cells or organs and/or for producing polypeptides in mammalian cells or organs. In addition to the novel deuterated lipids, the lipid nanoparticle compositions of the present invention can include one or more deuterated polymer-conjugated lipids, neutral lipids including polyunsaturated lipids, cationic lipids, steroids, and/or therapeutic and/or prophylactic agents in specific ratios.

Background

Effective targeted delivery of biologically active substances such as small molecule drugs, proteins and nucleic acids presents a long-standing medical challenge. In particular, delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of these species. Accordingly, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.

Studies have shown that bioactive substances such as small molecule drugs, proteins and nucleic acids can be efficiently delivered to cells and/or intracellular compartments using lipid-containing nanoparticle compositions, liposomes and liposome complexes as delivery vehicles. These compositions generally comprise one or more polymer-conjugated lipids, neutral lipids (e.g., phospholipids), structural lipids (e.g., steroids), and/or cation-containing lipids, including polyunsaturated lipids.

However, the use of oligonucleotides in a therapeutic setting is currently facing two problems. First, free RNA is susceptible to nuclease digestion in plasma. Second, the ability of free RNA to enter intracellular compartments where relevant translation mechanisms exist is limited. Lipid nanoparticles formed from pegylated deuterated lipids with other lipid components (such as neutral lipids, cholesterol, cationic lipids, and oligonucleotides) have been used to prevent degradation of RNA in plasma and promote cellular uptake of oligonucleotides, while deuterated polymer-conjugated lipids have not been reported.

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

Disclosure of Invention

The invention provides the following novel polyethylene glycol lipid compounds (as shown in the following formula A and formula B) and preparation methods, compositions and applications of the compounds.

1. The invention relates to a deuterated polyethylene glycol lipid compound (A):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

r1, R2, R3 are each independently a combination of DDD, DDH, DHH, HHH, DD, DH, HH, D, H;

r1, R2, and R3 are all independent, and when R1 and R2 are both "HHH", R3 cannot be "HH", and when R3 is a combination of "HH", one of R1 and R2 cannot be a combination of "HHH";

r1, R2, are each independently a "DDD" combination, R3 is independently a "DD" combination;

the average value of n is 40 to 50, preferably 45.

2. The invention relates to a deuterated polyethylene glycol lipid compound (B):

r1, R2, R3, R4, R5 are each independently a combination of DDD, DDH, DHH, HHH, DD, DH, HH, D, H;

r1, R2, R3, R4 and R5 are all independent, when R1 and R2 are both the combination of 'HHH' and R4 is the combination of 'H', R3 and R5 can not be simultaneously 'HH'; when R3 and R5 are both the combination of 'HH' and R4 is the combination of 'H', R1 and R2 can not be the combination of 'HHH' at the same time; when R1, R2 are both "HHH" combinations, and R3, R5 are both "HH" combinations, R4 cannot be "H" combinations;

r1, R2, R3, R4, R5 are each independently a combination of "DDD", "DD", "D";

the average value of n is 40 to 50, preferably 45.

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

Table 1 representative novel PEG lipid compounds

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In some embodiments, compositions are provided comprising any one or more of the deuterated compounds of structural formulae (a) and (B) and a therapeutic and/or prophylactic agent.

In some embodiments, compositions are provided comprising any one or more of the compounds of structures (a) and (B) and a therapeutic and/or prophylactic agent. In some embodiments, the composition comprises any of the compounds of structures (a) and (B) and a therapeutic and/or prophylactic agent and one or more excipients selected from the group consisting of neutral lipids, steroids, and cationic lipids. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the compositions.

In some embodiments, the neutral lipid is selected from 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dimyristyl-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, it is preferred that the neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

In some embodiments, the steroid is selected from the group consisting of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, it is preferred that the steroid is cholesterol. In some embodiments, the cationic lipid is selected from 4- (N, N-dimethylamino) butanol (6z, 9z,28z, 31z) -heptatriacontan-6, 9,28, 31-tetraen-19-yl ester (DLin-MC 3-DMA), heptadecan-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) caprylate (SM-102), ((4-hydroxybutyl) aza-ethyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), 4- (N-2- (N, N-bis (2-hydroxydodecyl) ethylamino) -1-N (2-ethylamino- (2- (N, N-bis (2-hydroxydodecyl)) -2 hydroxydodecyl) ethylamino piperazine (C12-200), novel lipid compounds of the pyrrolidine class.

In some embodiments, the cationic lipid is preferably selected from the group consisting of ((4-hydroxybutyl) aza-ethyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315) deuterated analogs, deuterated analogs of heptadecan-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate (SM-102).

The cationic lipid in the composition of the invention has the following structure (formula I):

wherein:

l1 is selected from the following structures: -C-, -O (C = O) -, - (C = O) O-, -C (= O) -, -O-, -S (O) X-, -S-, -C (= O) S-, -SC (= O) -, -N-C (= 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-;

r1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl, said hydrocarbyl chains optionally being linked by one or more ester or ether linkages; r3 and R4 are each independently C1-C12 alkyl or C1-C12 alkenyl, or R3 and R4 combine with each other to form a 4-to 10-membered heterocyclic ring, the heteroatoms including one or more of N, O, S, the heterocyclic ring being optionally substituted with 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 cationic lipid compounds in the composition according to the invention are preferably all cationic lipid compounds disclosed in the following patent application nos.: CN2021109304214, CN2021109693918, CN2021109698790, CN2021109700095, CN202110970112X, CN2021109787347, CN2021110306263, CN2021110307694, CN2021110439195, CN2021110310644, CN2021110311986, CN2021111108325, CN2021111057272.

In some embodiments, the composition ratio is in the following range: about 1-10mol% of said compound, about 0-30 mol% of neutral lipids, about 10-55 mol% of steroids and about 30-60mol% of cationic lipids.

In some embodiments of the foregoing composition, the therapeutic and/or prophylactic agent comprises a nucleic acid. Wherein the nucleic acid is RNA selected from the group consisting of: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof. In some embodiments, the RNA is selected from mRNA.

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

For administration purposes, the compounds of the invention (typically in the form of lipid nanoparticles in combination with a therapeutic and/or prophylactic agent) can be administered as a drug substance or can be formulated as a pharmaceutical composition. The pharmaceutical compositions of the present invention comprise compounds of structures (a) and (B) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compounds of structures (a) and (B) 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 acceptable manner of administration of the agents for similar utility. The pharmaceutical composition of the present invention may be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of 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 present invention are formulated so as to allow the active ingredient contained therein to be bioavailable upon 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 may be a single dosage unit, while a container of a compound of the invention in aerosol form may contain a plurality of dosage units. Current methods of preparing these dosage forms are known or will be apparent to those skilled in the art. In any event, the composition to be administered will contain a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in order to treat the relevant disease or condition in accordance with the teachings of the present invention.

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

When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein forms considered herein to be solid or liquid include semi-solid, semi-liquid, suspension, and gel forms.

As solid compositions for oral administration, the pharmaceutical compositions may be formulated into the form of powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, and the like. Such solid compositions will generally contain one or more inert diluents or edible carriers. Additionally, one or more of a binder, such as gelatin, cellulose, and the like; excipients, such as lactose and the like; disintegrating agents such as alginic acid and the like; lubricants, such as magnesium stearate and the like; glidants such as silicone gel and the like; sweetening agents, such as sucrose or saccharin; flavoring agents such as herba Menthae; and a colorant.

When the pharmaceutical composition is in the form of a capsule, it may contain a liquid carrier other than the above-mentioned types of materials, such as polyethylene glycol or an oil.

The pharmaceutical composition may be in the form of a liquid, such as a syrup, solution, emulsion or suspension. As two examples, the liquid may be for oral administration or for injection delivery. When intended for oral administration, the compositions of the present invention contain, in addition to the compounds of the present invention, one or more of sweetening agents, preserving agents, coloring/colouring agents and taste enhancers. In compositions for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizing agents and isotonic agents may be included.

The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar form, may include one or more of the following adjuvants, such as sterile diluents, e.g., water for injection, saline solution, preferably normal saline, ringer's solution, isotonic sodium chloride; non-volatile oils such as synthetic monoglycerides or diglycerides which may be used as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as methyl paraben vinegar; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, squarates or phosphates; and agents for regulating tonicity, such as sodium chloride or dextrose; agents used as cryoprotectants, such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

The pharmaceutical compositions of the present invention may be comprised of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from systems of colloidal nature to systems consisting of pressurized packaging. Delivery may be by liquefied or compressed gas, or by a suitable pump system for dispensing the active ingredient. Aerosols of the compounds of the invention may be delivered as a single phase, biphasic system, or triphasic system for delivery of the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. One skilled in the art can determine the preferred aerosol without undue experimentation.

The pharmaceutical compositions of the present invention may be prepared by methods well known in the pharmaceutical arts. Pharmaceutical compositions intended for administration by injection may be prepared by combining the lipid nanoparticles of the present invention in solution with sterile distilled water or other carrier. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the compounds of the present invention in order to facilitate dissolution or uniform suspension of the compounds in an aqueous delivery system.

The compositions of the present invention, or pharmaceutically acceptable salts thereof, are administered in therapeutically effective amounts, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent employed; metabolic stability and length of action of the therapeutic agent; the age, weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; a drug combination; severity of the particular case, etc.

The compositions of the present invention may also be administered simultaneously with, before or after the administration of one or more other therapeutic agents. Such combination therapies include the administration of a single pharmaceutical dosage formulation of a composition of the present invention and one or more additional active agents, as well as the administration of a composition of the present invention and each active agent in its own separate pharmaceutical dosage formulation. For example, the compositions of the invention and other active agents can be administered to a subject together in a single oral dosage composition (e.g., a tablet or capsule), or each agent can be administered in different oral dosage formulations. When different dosage formulations are employed, the compound of the invention and one or more additional active agents may be administered at substantially the same time, or sequentially at staggered times; it is to be understood that combination therapy encompasses all such regimens.

The structure modification and design of the deuterated PEGylated deuterated lipidosome compound realize more advantageous physicochemical properties including more proper pKa and better chemical stability, and the deuterated PEGylated deuterated lipidosome compound is used for an mRNA nanoliposome composition, can realize more effective combination and delivery of ionic nucleic acid drugs, has more stable chemical structure, is convenient to synthesize and is beneficial to being developed into pharmaceutic adjuvant.

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

One skilled in the art will recognize that in the processes described herein, functional groups of starting materials and intermediates include epoxy, hydroxyl, amino, and amide. The method can be used for substitution, reduction, condensation and the like, is suitable for the method for preparing amine by amide reduction, and mainly comprises a metal hydride reduction method, a borane tetrahydrofuran complex reduction method, a catalytic hydrogenation reduction method, a sodium borohydride-Lewis acid system reduction method and the like. The amide production method mainly includes an ammonia ester exchange method, an aliphatic amine and ester exchange method, an acid anhydride-based raw material method, an acid halide-based raw material method, an organic phosphorus-based condensing agent method, an onium salt-based condensing agent method, and the like. The epoxy ring opening is primarily catalyzed by acid and alkaline, and the method can be selected based on the nature of the substrate, and standard techniques are known to those skilled in the art and described herein.

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

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

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

The following examples, unless otherwise indicated, all solvents and reagents used were commercially available and used as received.

The procedure described below can be used for the synthesis of compounds a and B.

The following abbreviations are used herein:

DCM: methylene dichloride

THF: tetrahydrofuran (THF)

MeOH: methanol

HBTU:2- (benzotriazole) -N, N, N, N-tetramethyluronium hexafluorophosphate

DIEA: diisopropylethylamine

DMF: n, N-dimethylformamide

LiAlH ₄ : lithium aluminum tetrahydride

NaOH: sodium hydroxide

PEG: polyethylene glycol

DMSO-d6: deuterated dimethyl sulfoxide

EA: acetic acid ethyl ester

PE: petroleum ether (60-90)

EDCI: 1-Ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride

DMAP: n, N-lutidine.

Detailed Description

Example 1

Representative routes

A compound A: synthesis of N, N-bis (14, 14-D3-tetradecyl) -2, 2-2D-2-methylpolyethylene glycol acetamide

1) Ext> Synthesisext> ofext> 14,14,14ext> -ext> Dext> 3ext> -ext> tetradecanamideext> (ext> dPEGext> -ext> Aext> -ext> 3ext>)ext>

The molecular formula is as follows: c ₁₄ H ₂₆ D ₃ NO

Molecular weight 230.40

Ext> oxalylext> chlorideext> (ext> 38.85mmoext> 1ext>,ext> 4.93gext>)ext> wasext> addedext> toext> aext> solutionext> ofext> 14,14ext>,ext> 14ext> -ext> Dext> 3ext> -ext> tetradecanoicext> acidext> (ext> dPEGext> -ext> Aext> -ext> 1ext>)ext> (ext> 6ext> gext>,ext> 25.9ext> mmoext> 1ext>)ext> inext> tolueneext> (ext> 50ext> mLext>)ext> atext> roomext> temperatureext>.ext> After heating the resulting mixture at 70 ℃ for 2h, the mixture was concentrated. The residue was dissolved in toluene and concentrated again. The residual oil was added to a concentrated ammonia solution (20 m) by syringe at 10 ℃. The reaction mixture was filtered and washed with water. The white solid was dried in vacuo. Ext> theext> desiredext> productext>,ext> 14ext> -ext> Dext> 3ext> -ext> tetradecanamideext> (ext> dPEGext> -ext> Aext> -ext> 3ext>)ext>,ext> wasext> obtainedext> asext> aext> whiteext> solidext> (ext> 3.58gext>,ext> 60ext>%ext>)ext>.ext>

APCI-MS:m/z 231[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 6.8(s,2H),2.28(t,2H,J＝7.2Hz),1.80(m,2H),1.35(m, 2H),1.24(m,18H)。

2) Ext> Synthesisext> ofext> 14,14,14ext> -ext> Dext> 3ext> -ext> tetradecylamineext> (ext> dPEGext> -ext> Aext> -ext> 4ext>)ext>

The molecular formula is as follows: c ₁₄ H ₂₈ D ₃ N

Molecular weight 216.40

To a suspension of 14,14, 14-D3-tetradecanamide (3.58g, 15.54mm01) in THF (70 mL) was added lithium aluminum hydride (1.14g, 30mmol) portionwise at room temperature over a period of 30 min. The mixture was then slowly heated to reflux (65 ℃ oil bath) overnight. The mixture was cooled to 5 ℃ and sodium sulfate hydrate was added. The mixture was stirred for 2h, filtered through a celite layer, and washed with 15% meoh in DCM (200 m). The filtrate and washings were combined and concentrated. The residual solid was dried under vacuum 29197. Ext> 14,14ext>,ext> 14ext> -ext> Dext> 3ext> -ext> tetradecylamineext> (ext> dPEGext> -ext> Aext> -ext> 4ext>)ext> wasext> obtainedext> asext> aext> whiteext> solidext> (ext> 2.86ext>,ext> 85.1ext>%ext>)ext>.ext>

APCI-MS:m/z 217[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.7(s,2H),2.72(m,2H),1.65(m,2H),1.35(m, 2H),1.24(m,20H)。

3) Ext> Synthesisext> ofext> 14,14,14ext> -ext> Dext> 3ext> -ext> tetradecylext> -ext> 14,14,14ext> -ext> Dext> 3ext> -ext> tetradecylamideext> (ext> dPEGext> -ext> Aext> -ext> 5ext>)ext>

The molecular formula is as follows: c ₂₉ H ₅₃ D ₆ NO

Molecular weight 443.82

To a solution of 14,14, 14-D3-myristic acid (1 g,4.3 mmol) in benzene (10 mL) and DMF (1 drop) was added oxalyl chloride (0.82g, 6.45mmol) at room temperature, and the mixture was stirred at room temperature for 3h. After concentration at 60 ℃ to remove toluene, the residual oil was dissolved in 5mL of benzene and cooled to 10 ℃ it was added to a solution of 14, 14-D3-tetradecylamine (0.97g, 4.5 mol) and triethylamine (0.65 g,6.45 mmol) in benzene (5 mL). After the addition was complete, the reaction was stirred at room temperature overnight. After completion of the reaction, water was added, diluted with water, and adjusted to pH5-6 with 1.7M HCl, and a solid was precipitated, stirred at room temperature for 5 hours, filtered, and washed with water. An off-white wet product is obtained, the wet product is heated to 58 ℃ by 5ml of methanol to be dissolved, the temperature is reduced to room temperature, the stirring is carried out for 8 hours, the filtration is carried out, 1ml of methanol is used for leaching, and the obtained solid is dried by air blowing at 55 ℃ for 10 hours, thus obtaining the target compound 14, 14-D3-tetradecyl 14, 14-D3-tetradecylamide as a light white solid (1.77g, 93%).

APCI-MS:m/z 444[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 8.13(s,1H),3.86(m,2H),2.66(m,2H),1.58(m,4H), 1.35(m,4H),1.24(m,30H)。

4) Ext> Synthesisext> ofext> Next>,ext> Next> -ext> bisext> (ext> 14ext>,ext> 14ext> -ext> Dext> 3ext> -ext> tetradecylext>)ext> amineext> (ext> dPEGext> -ext> Aext> -ext> 6ext>)ext>

The molecular formula is as follows: c ₂₉ H ₅₅ D ₆ N

Molecular weight 429.84

To a suspension of 14,14, 14-D3-tetradecyl-14, 14-D3-tetradecylamide (2g, 4.5mo1) in THF (20 mL) was added lithium aluminum hydride (0.51g, 13.5mmo1) in portions at room temperature. The mixture was heated to 68 ℃ and refluxed overnight. After the reaction was detected to be complete, the mixture was cooled to 0 ℃ and added with aqueous sodium hydroxide solution and stirred for 2h, filtered through a pad of celite and silica gel and washed with ether. The filtrate became cloudy and a precipitate formed, which was stirred for 5min and filtered to give a white solid.

Ext> transferringext> theext> whiteext> solidext> intoext> aext> 20ext> mlext> doubleext> -ext> mouthext> bottleext>,ext> addingext> 5ext> mlext> ofext> methanolext>,ext> heatingext> toext> 60ext> ℃ext> forext> dissolvingext>,ext> coolingext> toext> 25ext> ℃ext> afterext> 30ext> minext>,ext> stirringext> forext> crystallizationext> forext> 10ext> hoursext>,ext> filteringext>,ext> leachingext> withext> 1ext> mlext> ofext> methanolext>,ext> andext> dryingext> theext> solidext> atext> 55ext> ℃ext> forext> 10ext> hoursext> toext> obtainext> Next>,ext> Next> -ext> diext> (ext> 14ext>,ext> 14ext> -ext> Dext> 3ext> -ext> tetradecylext>)ext> amineext> (ext> dPEGext> -ext> Aext> -ext> 6ext>)ext> asext> aext> whiteext> solidext> (ext> 1.45gext>,ext> 75.01ext>%ext>)ext>.ext>

APCI-MS:m/z 430[M+H]+

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.45(s,1H)，2.66(m,4H),1.58(m,4H),1.35(m, 4H),1.24(m,44H)。

5) Ext> Synthesisext> ofext> Next>,ext> Next> -ext> bisext> (ext> 14ext>,ext> 14ext> -ext> Dext> 3ext> -ext> tetradecylext>)ext> -ext> 2ext>,ext> 2ext> -ext> 2ext> Dext> -ext> 2ext> -ext> methylpolyethyleneglycolext> acetamideext> (ext> dPEGext> -ext> Aext>)ext>

The molecular formula is as follows: c ₁₁₇ H ₂₃₁ D ₈ NO ₄₇

Molecular weight: 2485

To a 50mL two-necked flask was added a solution of N, N-bis (14,14,14-D3-tetradecyl) amine (2g, 4.65mmol) and triethylamine (0.71 g, 6.98 mmol) in DCM (40 mL) was added a solution of methyl polyethylene glycol succinimide acetate (mPEG-NHS, 4.65mml,10.1g, PEG MW about 2,000,n = about 45) in DCM (50 m). After stirring at room temperature 3 for 24h, water (30 mL) was added and the aqueous phase was extracted twice with DCM (100 mLx 2). The DCM extracts were combined and washed with 1M HCl (30 mL). After drying the organic phase over sodium sulfate, filtration and concentration of the remaining approximately 30m1 of the organic phase, it was cooled to-20 ℃ and stirred for 8 hours, filtered and rinsed with 3ml of DCM to give a white solid (0.9 g, unreacted starting amine), to which was added triethylamine (18.6 mmol) and acetic anhydride (1.4 mmol, 143mg), and after 10min there was a solid which precipitated and stirred at room temperature for 5 hours, then concentrated to a solid at 40 ℃ and the remaining solid was purified by column chromatography on silica gel (DCM containing 0-8% methanol) to give N, N-bis (14, 14-D3-tetradecyl) -2, 2-2D-2-methylpolyethyleneglycol acetamide as a white solid (5.82 g, yield approximately 50%).

APCI-MS:m/z 2486[M+H] ⁺

¹ H-NMR(300MHz,CDC13)：δppm 3.83-3.465(m,180-200H)，3.39(s,3H),3.28(m,4H)， 1.58(m,4H),1.35(m,4H),1.24(m,44H)。

Example 2

Representative synthetic routes

Compound B3- (2-2-Methylpolyethyleneglycol (n = 45)) -1, 2-diyl-bis (14, 14-3D-tetradecylcarboxy) 1,2, 3-5D-propane

1) Synthesis of 1,2, 3-3D-3-methyl polyethylene glycol-1, 2-propylene oxide

The molecular formula is as follows: c ₉₄ H ₁₈₃ D ₅ O ₄₇

Molecular weight: 2073

A mixed solution of polyethylene glycol monomethyl ether (dEG-B-2, 68.2g, PEG Mw of about 2000, n = about 45, 34.1mmol, 1.2eq) in THF (55 mL) and DMF (10 mL) was cooled at 0-10 deg.C, naH (60% by mass, 1.4g, 34.1mmol, 1.2eq) was added, the mixture was stirred at constant temperature for 0.5hr, a THF solution (10 mL) of 1,2, 3-3D-3-chloro-1, 2-epoxypropane (dEG-B-1, 2.8g,28.4mmol, 1.eq) was slowly added, and the mixture was stirred at room temperature for 18 hours after the addition. TLC showed that the dEG-B-1 was substantially complete, and the reaction was slowly poured into saturated ammonium chloride solution. THF was added and extraction was performed 3 times. The organic phases were combined and washed once with saturated sodium chloride. The organic phase was stirred with silica gel and purified by column chromatography (EA: PE =5, 1, rf = 0.4) to give 39.2g of semi-oil semisolid with a yield of 65%.

APCI-MS:m/z 2074[M+H] ⁺

¹ H-NMR(300MHz,CDC13)：δppm 3.83-3.46(m,180-200H)，3.39(s,3H)。

2) Synthesis of 1,2, 3-3D-3-methyl polyethylene glycol-1, 2-propanediol

The molecular formula is as follows: c ₉₄ H ₁₈₅ D ₅ O ₄₈

Molecular weight: 2091

1,2, 3-3D-3-methyl polyethylene glycol-1, 2-propylene oxide (39.2g, 18.9mmol) is dissolved in 500ml tetrahydrofuran, 2M sodium hydroxide solution (1.13g, 28.35mmol, 14.16ml) is added, the temperature is raised to 50 ℃ and stirred for 6 hours, after the detection of the reaction of the raw materials, the temperature is lowered to room temperature, EA extraction is carried out for 1 time, saturated sodium chloride is used for washing an organic phase silica gel sample for one time, and column chromatography purification (EA) is carried out to obtain 38.7g of white solid with the yield of 97.7%.

APCI-MS:m/z 2092[M+H] ⁺

¹ H-NMR(300MHz,CDC13)：δppm 6.10(s,1H),5.08(s,1H),3.83-3.46(m,180-200H)，3.39(s, 3H)。

3) Synthesis of 3- (2-2-methyl polyethylene glycol (n = 45)) -1, 2-diyl-bis (14, 14-3D-tetradecyloxy) 1,2, 3-5D-propane

The molecular formula is as follows: c ₁₂₂ H ₂₃₁ D ₁₁ O ₅₀

Molecular weight: 2517

38.7g of 1,2, 3-3D-3-methyl polyethylene glycol-based-1, 2-propanediol (dPEG-B-4, 18.50 mmol) was put into a 1000ml four-necked bottle, 400ml of DMF was added, 14-D3-myristic acid (8.99g, 38.85mmol) was added, DIEA (5.62g, 55.5 mmol) and EDCI (10.64g, 55.5 mmol) were added, the temperature was raised to 45 ℃ to react for 5 hours, after completion of the detection reaction, EA was extracted three times, washed once with water, washed once with brine, the organic phase was concentrated at 45 ℃ and purified by column chromatography (EA: PE = 15): 76.46 percent.

APCI-MS:m/z 2518[M+H] ⁺

¹ H-NMR(300MHz,CDC ₁₃ )：δppm 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m, 4H),1.38(m,4H),1.31(m,4H),1.26(m,32H)。

The following compounds can be prepared by employing the techniques described above and the associated procedures well known to those skilled in the art, and using appropriate deuterated reagents.

EXAMPLE 3 Synthesis of Compound 2

The molecular formula is as follows: C117H232D7NO47

Molecular weight: 2484

APCI-MS:m/z 2485[M+H]+

1H-NMR(300MHz,DMSO-d6)：δppm 4.23(s,1H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,4H),1.24(m,44H)。

EXAMPLE 4 Synthesis of Compound 3

The molecular formula is as follows: c ₁₁₇ H ₂₃₂ D ₇ NO ₄₇

Molecular weight: 2484

APCI-MS:m/z 2485[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.465(m,180-200H)，3.39(s,3H),3.28(m,4H), 1.58(m,4H),1.35(m,5H),1.24(m,44H)。

EXAMPLE 5 Synthesis of Compound 4

The molecular formula is as follows: c ₁₁₇ H ₂₃₃ D ₆ NO ₄₇

Molecular weight: 2483

APCI-MS:m/z 2484[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.23(s,2H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,4H),1.24(m,44H)。

The molecular formula is as follows: c ₁₁₇ H ₂₃₃ D ₆ NO ₄₇

Molecular weight: 2483

APCI-MS:m/z 2484[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.23(s,1H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,5H),1.24(m,44H)。

EXAMPLE 7 Synthesis of Compound 6

The molecular formula is as follows: c ₁₁₇ H ₂₃₃ D ₆ NO ₄₇

Molecular weight: 2483

APCI-MS:m/z 2484[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.465(m,180-200H),3.39(s,3H),3.28(m,4H), 1.58(m,4H),1.35(m,6H),1.24(m,44H)。

EXAMPLE 8 Synthesis of Compound 7

The molecular formula is as follows: c ₁₁₇ H ₂₃₄ D ₅ NO ₄₇

Molecular weight: 2482

APCI-MS:m/z 2483[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm3.83-3.465(m,180-200H),3.39(s,3H),3.28(m,4H), 1.58(m,4H),1.35(m,2H),1.24(m,46H),0.88(m,3H)。

EXAMPLE 9 Synthesis of Compound 8

Molecular formula：C ₁₁₇ H ₂₃₄ D ₅ NO ₄₇

Molecular weight: 2482

APCI-MS:m/z 2483[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.56(s,2H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,5H),1.24(m,44H)。

EXAMPLE 10 Synthesis of Compound 9

The molecular formula is as follows: c ₁₁₇ H ₂₃₄ D ₅ NO ₄₇

Molecular weight: 2482

APCI-MS:m/z 2483[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.56(s,1H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,6H),1.24(m,44H)。

EXAMPLE 11 Synthesis of Compound 10

The molecular formula is as follows: c ₁₁₇ H ₂₃₄ D ₅ NO ₄₇

Molecular weight: 2482

APCI-MS:m/z 2483[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.56(s,1H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,2H),1.24(m,44H),0.89(m,3H)。

EXAMPLE 12 Synthesis of Compound 11

The molecular formula is as follows: c ₁₁₇ H ₂₃₅ D ₄ NO ₄₇

Molecular weight: 2482

APCI-MS:m/z 2482[M+H] ⁺

EXAMPLE 13 Synthesis of Compound 12

The molecular formula is as follows: c ₁₁₇ H ₂₃₅ D ₄ NO ₄₇

Molecular weight: 2481

APCI-MS:m/z 2482[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.465(m,180-200H)，3.39(s,3H),3.28(m,4H), 1.58(m,4H),1.35(m,8H),1.24(m,44H)。

The molecular formula is as follows: c ₁₁₇ H ₂₃₅ D ₄ NO ₄₇

Molecular weight: 2481

APCI-MS:m/z 2482[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.465(m,180-200H)，3.39(s,3H),3.28(m,4H), 1.58(m,4H),1.35(m,3H),1.24(m,46H),0.90(m,3H)。

EXAMPLE 15 Synthesis of Compound 14

The molecular formula is as follows: c ₁₁₇ H ₂₃₆ D ₃ NO ₄₇

Molecular weight: 2481

APCI-MS:m/z 2482[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.62(s,2H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,2H),1.24(m,46H),0.90(m,3H)。

EXAMPLE 16 Synthesis of Compound 15

The molecular formula is as follows: c ₁₁₇ H ₂₃₆ D ₃ NO ₄₇

Molecular weight: 2481

APCI-MS:m/z 2482[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.62(s,2H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,7H),1.24(m,44H)。

EXAMPLE 17 Synthesis of Compound 16

The molecular formula is as follows: c ₁₁₇ H ₂₃₆ D ₃ NO ₄₇

Molecular weight: 2481

APCI-MS:m/z 2482[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.62(s,1H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,3H),1.24(m,46H),0.91(m,3H)。

EXAMPLE 18 Synthesis of Compound 17

The molecular formula is as follows: c ₁₁₇ H ₂₃₆ D ₃ NO ₄₇

Molecular weight: 2481

APCI-MS:m/z 2482[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.62(s,1H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,8H),1.24(m,44H)。

EXAMPLE 19 Synthesis of Compound 18

The molecular formula is as follows: c ₁₁₇ H ₂₃₆ D ₃ NO ₄₇

Molecular weight: 2481

APCI-MS:m/z 2482[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.465(m,180-200H)，3.39(s,3H),3.28(m,4H), 1.58(m,4H),1.35(m,4H),1.24(m,46H),0.92(m,3H)。

EXAMPLE 20 Synthesis of Compound 19

The molecular formula is as follows: c ₁₁₇ H ₂₃₇ D ₂ NO ₄₇

Molecular weight: 2480

APCI-MS:m/z 2481[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.465(m,180-200H)，3.39(s,3H),3.28(m,4H), 1.58(m,4H),1.24(m,48H),0.92(m,6H)。

EXAMPLE 21 Synthesis of Compound 20

The molecular formula is as follows: c ₁₁₇ H ₂₃₇ D ₂ NO ₄₇

Molecular weight: 2480

APCI-MS:m/z 2481[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.66(s,1H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.36(m,4H),1.24(m,46H),0.92(m,3H)。

EXAMPLE 22 Synthesis of Compound 21

The molecular formula is as follows: c ₁₁₇ H ₂₃₇ D ₂ NO ₄₇

Molecular weight: 2480

APCI-MS:m/z 2481[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.66(s,2H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.36(m,6H),1.24(m,44H)。

EXAMPLE 23 Synthesis of Compound 22

The molecular formula is as follows: c ₁₁₇ H ₂₃₇ D ₂ NO ₄₇

Molecular weight: 2480

APCI-MS:m/z 2481[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.66(s,2H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.36(m,3H),1.24(m,46H),0.92(m,3H)。

EXAMPLE 24 Synthesis of Compound 23

The molecular formula is as follows: c ₁₁₇ H ₂₃₈ D ₁ NO ₄₇

Molecular weight: 2479

APCI-MS:m/z 2480[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.66(s,1H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.24(m,48H),0.92(m,6H)。

EXAMPLE 25 Synthesis of Compound 24

The molecular formula is as follows: c ₁₁₇ H ₂₃₈ D ₁ NO ₄₇

Molecular weight: 2479

APCI-MS:m/z 2480[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.66(s,2H),3.83-3.465(m,180-200H)，3.39(s,3H), 3.28(m,4H),1.58(m,4H),1.35(m,4H),1.24(m,46H),0.92(m,3H)。

EXAMPLE 26 Synthesis of Compound 26

The molecular formula is as follows: c ₁₂₂ H ₂₃₂ D ₁₀ O ₅₀

Molecular weight: 2516

APCI-MS:m/z 2517[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm3.83-3.46(m,180-200H)，3.39(s,3H),2.35(m,4H), 1.69(m,4H),1.38(m,4H),1.31(m,5H),1.26(m,32H)。

EXAMPLE 27 Synthesis of Compound 27

The molecular formula is as follows: c ₁₂₂ H ₂₃₂ D ₁₀ O ₅₀

Molecular weight: 2516

APCI-MS:m/z 2517[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm3.83-3.46(m,180-200H)，3.39(s,3H),2.35(m,4H), 1.69(m,4H),1.38(m,5H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 28 Synthesis of Compound 28

The molecular formula is as follows: c ₁₂₂ H ₂₃₂ D ₁₀ O ₅₀

Molecular weight: 2516

APCI-MS:m/z 2517[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.6(s,1H),3.83-3.46(m,180-200H)，3.39(s,3H), 2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 29 Synthesis of Compound 29

The molecular formula is as follows: c ₁₂₂ H ₂₃₂ D ₁₀ O ₅₀

Molecular weight: 2516

APCI-MS:m/z 2517[M+H] ⁺

EXAMPLE 30 Synthesis of Compound 30

The molecular formula is as follows: c ₁₂₂ H ₂₃₂ D ₁₀ O ₅₀

Molecular weight: 2516

APCI-MS:m/z 2517[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.65(s,1H),3.83-3.46(m,180-200H)，3.39(s,3H), 2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 31 Synthesis of Compound 31

The molecular formula is as follows: c ₁₂₂ H ₂₃₃ D ₉ O ₅₀

Molecular weight: 2515

APCI-MS:m/z 2516[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.46(m,180-200H)，3.39(s,3H),2.35(m,4H), 1.69(m,4H),1.38(m,6H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 32 Synthesis of Compound 32

The molecular formula is as follows: c ₁₂₂ H ₂₃₃ D ₉ O ₅₀

Molecular weight: 2515

APCI-MS:m/z 2516[M+H] ⁺

EXAMPLE 33 Synthesis of Compound 33

The molecular formula is as follows: c ₁₂₂ H ₂₃₃ D ₉ O ₅₀

Molecular weight: 2515

APCI-MS:m/z 2516[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.6(s,1H),3.83-3.46(m,180-200H)，3.39(s,3H), 2.35(m,4H),1.69(m,4H),1.38(m,5H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 34 Synthesis of Compound 34

The molecular formula is as follows: c ₁₂₂ H ₂₃₃ D ₉ O ₅₀

Molecular weight: 2515

APCI-MS:m/z 2516[M+H] ⁺

EXAMPLE 35 Synthesis of Compound 35

The molecular formula is as follows: c ₁₂₂ H ₂₃₃ D ₉ O ₅₀

Molecular weight: 2515

APCI-MS:m/z 2516[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 4.43(s,1H),3.83-3.46(m,180-200H)，3.39(s,3H), 2.35(m,4H),1.69(m,4H),1.38(m,5H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 36 Synthesis of Compound 36

The molecular formula is as follows: c ₁₂₂ H ₂₃₄ D ₈ O ₅₀

Molecular weight: 2514

APCI-MS:m/z 2515[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.46(m,180-200H)，3.39(s,3H),2.35(m,4H), 1.69(m,4H),1.38(m,4H),1.31(m,2H),1.26(m,34H),0.92(m,3H)。

EXAMPLE 37 Synthesis of Compound 37

The molecular formula is as follows: c ₁₂₂ H ₂₃₄ D ₈ O ₅₀

Molecular weight: 2514

APCI-MS:m/z 2515[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.62(d,1H,J＝5.6Hz),5.58(d,1H,J＝5.6Hz),3.83-3.46 (m,180-200H)，3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,5H),1.26(m,32H)。

EXAMPLE 38 Synthesis of Compound 38

The molecular formula is as follows: c ₁₂₂ H ₂₃₄ D ₈ O ₅₀

Molecular weight: 2514

APCI-MS:m/z 2515[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.62(t,1H,J＝8.3Hz),5.58(d,2H,J＝5.3Hz),3.83-3.46 (m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,5H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 39 Synthesis of Compound 39

The molecular formula is as follows: c ₁₂₂ H ₂₃₄ D ₈ O ₅₀

Molecular weight: 2514

APCI-MS:m/z 2515[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.62(s,2H),5.58(s,1H),3.83-3.46(m,180-200H)， 3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,5H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 40 Synthesis of Compound 40

The molecular formula is as follows: c ₁₂₂ H ₂₃₃ D ₉ O ₅₀

Molecular weight: 2515

APCI-MS:m/z 2516[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.62(s,1H),4.86(s,1H),3.83-3.46(m,180-200H)， 3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,4H),1.26(m,32H)。

EXAMPLE 41 Synthesis of Compound 41

The molecular formula is as follows: c ₁₂₂ H ₂₃₅ D ₇ O ₅₀

Molecular weight: 2513

APCI-MS:m/z 2514[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 3.83-3.46(m,180-200H)，3.39(s,3H),2.35(m,4H), 1.69(m,4H),1.38(m,4H),1.31(m,3H),1.26(m,34H),0.92(s,3H)。

EXAMPLE 42 Synthesis of Compound 42

The molecular formula is as follows: c ₁₂₂ H ₂₃₅ D ₇ O ₅₀

Molecular weight: 2513

APCI-MS:m/z 2514[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.56(s,1H),3.83-3.46(m,180-200H)，3.39(s,3H), 2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,5H),1.26(m,32H)。

EXAMPLE 43 Synthesis of Compound 43

The molecular formula is as follows: c ₁₂₂ H ₂₃₅ D ₇ O ₅₀

Molecular weight: 2513

APCI-MS:m/z 2514[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.56(d,1H,J＝5.5Hz),5.49(d,1H,J＝5.5Hz),3.83-3.46 (m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,6H),1.26(m,32H)。

EXAMPLE 44 Synthesis of Compound 44

The molecular formula is as follows: c ₁₂₂ H ₂₃₅ D ₇ O ₅₀

Molecular weight: 2513

APCI-MS:m/z 2514[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：δppm 5.56(t,1H,J＝8.9Hz),5.49(d,1H,J＝5.5Hz),4.87(d,1H, J＝4.3Hz),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m, 5H),1.26(m,32H)。

EXAMPLE 45 Synthesis of Compound 45

The molecular formula is as follows: c ₁₂₂ H ₂₃₅ D ₇ O ₅₀

Molecular weight: 2513

APCI-MS:m/z 2514[M+H] ⁺

EXAMPLE 46 Synthesis of Compound 46

The molecular formula is as follows: c ₁₂₂ H ₂₃₅ D ₇ O ₅₀

Molecular weight: 2513

APCI-MS:m/z 2514[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m, 4H),1.38(m,4H),1.31(m,3H),1.26(m,34H),0.93(m,3H)。

EXAMPLE 47 Synthesis of Compound 47

The molecular formula is as follows: c ₁₂₂ H ₂₃₆ D ₆ O ₅₀

Molecular weight: 2512

APCI-MS:m/z 2513[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m, 4H),1.38(m,4H),1.31(m,4H),1.26(m,34H),0.93(m,3H)。

EXAMPLE 48 Synthesis of Compound 48

The molecular formula is as follows: c ₁₂₂ H ₂₃₆ D ₆ O ₅₀

Molecular weight: 2512

APCI-MS:m/z 2513[M+H] ⁺

EXAMPLE 49 Synthesis of Compound 49

The molecular formula is as follows: c ₁₂₂ H ₂₃₆ D ₆ O ₅₀

Molecular weight: 2512

APCI-MS:m/z 2513[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：4.65(s,2H),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m, 4H),1.69(m,4H),1.38(m,4H),1.31(m,2H),1.26(m,34H),0.93(m,3H)。

EXAMPLE 50 Synthesis of Compound 50

The molecular formula is as follows: c ₁₂₂ H ₂₃₆ D ₆ O ₅₀

Molecular weight: 2512

APCI-MS:m/z 2513[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(d,1H,J＝6.8Hz),4.65(d,1H,J＝3.2Hz),3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,2H),1.26(m,34H), 0.93(m,3H)。

EXAMPLE 51 Synthesis of Compound 51

The molecular formula is as follows: c ₁₂₂ H ₂₃₆ D ₆ O ₅₀

Molecular weight: 2512

APCI-MS:m/z 2513[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.72(d,1H,J＝8.9Hz),4.65(d,1H,J＝3.2Hz),3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,2H),1.26(m,34H), 0.93(m,3H)。

EXAMPLE 52 Synthesis of Compound 52

The molecular formula is as follows: c ₁₂₂ H ₂₃₆ D ₆ O ₅₀

Molecular weight: 2512

APCI-MS:m/z 2513[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.78(m,1H),5.63(d,2H,J＝8.6Hz),4.65(d,2H,J＝3.8Hz), 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,4H),1.26(m, 32H)。

EXAMPLE 53 Synthesis of Compound 53

The molecular formula is as follows: c ₁₂₂ H ₂₃₇ D ₅ O ₅₀

Molecular weight: 2511

APCI-MS:m/z 2512[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m, 4H),1.38(m,4H),1.26(m,36H),0.93(m,6H)。

EXAMPLE 54 Synthesis of Compound 54

The molecular formula is as follows: c ₁₂₂ H ₂₃₇ D ₅ O ₅₀

Molecular weight: 2511

APCI-MS:m/z 2512[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(m,1H),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m, 4H),1.69(m,4H),1.38(m,4H),1.31(m,4H),1.26(m,34H),0.93(m,3H)。

EXAMPLE 55 Synthesis of Compound 55

The molecular formula is as follows: c ₁₂₂ H ₂₃₇ D ₅ O ₅₀

Molecular weight: 2511

APCI-MS:m/z 2512[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(t,1H,J＝7.98Hz),4.68(d,2H,J＝3.59Hz),3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,2H),1.26(m,34H), 0.93(m,3H)。

EXAMPLE 56 Synthesis of Compound 56

The molecular formula is as follows: c ₁₂₂ H ₂₃₇ D ₅ O ₅₀

Molecular weight: 2511

APCI-MS:m/z 2512[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(d,1H,J＝6.5Hz),5.58(d,1H,J＝6.3Hz),4.68(d,2H, J＝3.59Hz),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m, 2H),1.26(m,34H),0.93(m,3H)。

EXAMPLE 57 Synthesis of Compound 57

The molecular formula is as follows: c ₁₂₂ H ₂₃₇ D ₅ O ₅₀

Molecular weight: 2511

APCI-MS:m/z 2512[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(d,1H,J＝6.5Hz),4.68(d,2H,J＝3.59Hz),3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,8H),1.26(m,32H)。

EXAMPLE 58 Synthesis of Compound 58

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(s,1H),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m, 4H),1.69(m,4H),1.38(m,4H),1.26(m,36H),0.93(m,6H)。

EXAMPLE 59 Synthesis of Compound 59

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(d,1H,J＝5.2Hz),5.59(d,1H,J＝6.6Hz)3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,4H)1.26(m,34H),0.93(m, 3H)。

EXAMPLE 60 Synthesis of Compound 60

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(t,1H,J＝8.6Hz),5.59(d,1H,J＝6.6Hz)4.86(d,1H, J＝3.8Hz),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m, 8H),1.26(m,32H)。

EXAMPLE 61 Synthesis of Compound 61

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.62(m,1H),5.59(d,2H,J＝6.6Hz)4.86(d,2H,J＝3.8Hz), 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,6H),1.26(m, 32H)。

EXAMPLE 62 Synthesis of Compound 62

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.59(s,2H),4.86(s,2H),3.83-3.46(m,180-200H),3.39(s, 3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,2H),1.26(m,34H),0.94(m,3H)。

EXAMPLE 63 Synthesis of Compound 63

The molecular formula is as follows: c ₁₂₂ H ₂₃₉ D ₃ O ₅₀

Molecular weight: 2509

APCI-MS:m/z 2510[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.65(d,1H,J＝8.4Hz),4.83(d,1H,J＝3.9Hz),3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.26(m,36H),0.94(m,6H)。

EXAMPLE 64 Synthesis of Compound 64

The molecular formula is as follows: c ₁₂₂ H ₂₃₉ D ₃ O ₅₀

Molecular weight: 2509

APCI-MS:m/z 2510[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.65(m,1H),5.58(d,2H,J＝6.9Hz),4.83(d,2H,J＝3.9Hz), 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,2H),1.26(m, 34H),0.94(m,4H)。

EXAMPLE 65 Synthesis of Compound 65

The molecular formula is as follows: c ₁₂₂ H ₂₃₉ D ₃ O ₅₀

Molecular weight: 2509

APCI-MS:m/z 2510[M+H] ⁺

EXAMPLE 66 Synthesis of Compound 66

The molecular formula is as follows: c ₁₂₂ H ₂₃₉ D ₃ O ₅₀

Molecular weight: 2509

APCI-MS:m/z 2510[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.65(m,1H),5.58(d,2H,J＝6.9Hz),4.83(d,2H,J＝3.9Hz), 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,7H),1.26(m, 32H)。

EXAMPLE 67 Synthesis of Compound 67

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.65(m,1H),5.58(d,2H,J＝6.9Hz),4.83(d,2H,J＝3.9Hz), 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,6H),1.26(m, 32H)。

EXAMPLE 69 Synthesis of Compound 68

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.65(d,1H,J＝6.6Hz),4.83(d,2H,J＝3.9Hz),3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,3H),1.26(m,34H),0.95(m, 3H)。

EXAMPLE 69 Synthesis of Compound 69

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The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.68(t,1H,J＝8.9Hz),5.65(d,1H,J＝6.6Hz),3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,3H),1.26(m,34H),0.95(m, 3H)。

EXAMPLE 70 Synthesis of Compound 70

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.65(s,1H),4.69(s,2H),3.83-3.46(m,180-200H),3.39(s, 3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,4H),1.26(m,34H),0.95(m,3H)。

EXAMPLE 71 Synthesis of Compound 71

The molecular formula is as follows: c ₁₂₂ H ₂₃₈ D ₄ O ₅₀

Molecular weight: 2510

APCI-MS:m/z 2511[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.56(s,2H),4.69(s,2H),3.83-3.46(m,180-200H),3.39(s, 3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.31(m,7H),1.26(m,32H)。

EXAMPLE 72 Synthesis of Compound 72

The molecular formula is as follows: c ₁₂₂ H ₂₄₀ D ₂ O ₅₀

Molecular weight: 2508

APCI-MS:m/z 2509[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.61(t,1H,J＝8.3Hz),4.66(d,2H,J＝7.8Hz),3.83-3.46(m, 180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.26(m,36H),0.96(m,6H)。

EXAMPLE 73 Synthesis of Compound 73

The molecular formula is as follows: c ₁₂₂ H ₂₄₀ D ₂ O ₅₀

Molecular weight: 2508

APCI-MS:m/z 2509[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.79(m,1H),5.66(d,2H,J＝7.6Hz)，4.61(d,2H,J＝7.3Hz), 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.32(m,3H), 1.26(m,34H),0.96(m,3H)。

EXAMPLE 74 Synthesis of Compound 74

The molecular formula is as follows: c ₁₂₂ H ₂₄₀ D ₂ O ₅₀

Molecular weight: 2508

APCI-MS:m/z 2509[M+H] ⁺

EXAMPLE 75 Synthesis of Compound 75

The molecular formula is as follows: c ₁₂₂ H ₂₄₀ D ₂ O ₅₀

Molecular weight: 2508

APCI-MS:m/z 2509[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.79(m,1H),5.66(d,2H,J＝7.6Hz)，4.61(d,2H,J＝7.3Hz), 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.32(m, 8H),1.26(m,32H)。

EXAMPLE 76 Synthesis of Compound 76

The molecular formula is as follows: c ₁₂₂ H ₂₄₀ D ₂ O ₅₀

Molecular weight: 2508

APCI-MS:m/z 2509[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.66(s,2H,)，4.61(s,2H),3.83-3.46(m,180-200H),3.39(s, 3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.32(m,4H),1.26(m,34H),0.93(m,3H)。

EXAMPLE 77 Synthesis of Compound 77

The molecular formula is as follows: c ₁₂₂ H ₂₄₀ D ₂ O ₅₀

Molecular weight: 2508

APCI-MS:m/z 2509[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.70(q,1H,J ₁ ＝9.6Hz,J ₂ ＝3.8Hz),5.66(d,1H,J＝7.3Hz)， 4.61(d,2H,J＝7.3Hz),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m, 4H),1.32(m,4H),1.26(m,34H),0.93(m,3H)。

EXAMPLE 78 Synthesis of Compound 78

The molecular formula is as follows: c ₁₂₂ H ₂₄₁ D ₁ O ₅₀

Molecular weight: 2507

APCI-MS:m/z 2508[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.70(q,1H,J ₁ ＝9.6Hz,J ₂ ＝3.8Hz),5.69(d,1H,J＝7.3Hz), 4.63(d,2H,J＝3.9Hz),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m, 4H),1.26(m,36H),0.93(m,6H)。

EXAMPLE 79 Synthesis of Compound 79

The molecular formula is as follows: c ₁₂₂ H ₂₄₁ D ₁ O ₅₀

Molecular weight: 2507

APCI-MS:m/z 2508[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.69(s,2H)，4.63(s,2H),3.83-3.46(m,180-200H),3.39(s, 3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.26(m,36H),0.93(m,6H)。

EXAMPLE 80 Synthesis of Compound 80

The molecular formula is as follows: c ₁₂₂ H ₂₄₁ D ₁ O ₅₀

Molecular weight: 2507

APCI-MS:m/z 2508[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.70(q,1H,J ₁ ＝9.1Hz,J ₂ ＝42.1Hz),5.69(d,2H,J＝5.9Hz), 4.63(d,1H,J＝3.9Hz),3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m, 4H),1.26(m,36H),0.93(m,6H)。

EXAMPLE 81 Synthesis of Compound 81

The molecular formula is as follows: c ₁₂₂ H ₂₄₁ D ₁ O ₅₀

Molecular weight: 2507

APCI-MS:m/z 2508[M+H] ⁺

¹ H-NMR(300MHz,DMSO-d6)：5.70(m,1H),5.69(d,2H,J＝5.9Hz),4.63(d,1H,J＝3.9Hz), 3.83-3.46(m,180-200H),3.39(s,3H),2.35(m,4H),1.69(m,4H),1.38(m,4H),1.32(m,3H), 1.26(m,34H),0.93(m,6H)。

EXAMPLE 82 Synthesis of Compound 82

The molecular formula is as follows: c ₁₂₂ H ₂₄₁ D ₁ O ₅₀

Molecular weight: 2507

APCI-MS:m/z 2508[M+H] ⁺

Example 83

Luciferase mRNA in vivo evaluation using lipid nanoparticle compositions

Dissolving a cationic lipid, DSPC, cholesterol, and a deuterated PEG-lipid in ethanol at a molar ratio of 50. Lipid Nanoparticles (LNPs) were prepared at a weight ratio of total lipid to mRNA of about 10. Briefly, mRNA was diluted to 0.15mg/mL in 10mL to 50mL citrate buffer (pH = 4). The lipid ethanol solution and the mRNA aqueous solution were mixed using a syringe pump at a ratio of about 1 to 1 (volume/volume) in the range of 10ml/min or more for the total flow rate. The ethanol was then removed and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with a pore size of 0.2 μm. The particle size of the lipid nanoparticles, as determined by quasielastic light scattering using a Malvern Zetasizer Nano ZS, is about 65-105nm in diameter, and in some cases, about 75-100nm in diameter.

R ₁ ，R ₂ ，R ₃ ，R4，R ₅ ，R ₆ ，R ₇ R8 are each independently 2 "hydrogen" isotopes (including hydrogen, deuterium), embodied as a combination of "HH", "HD" and "DD"

R ₁ ，R ₂ ，R ₃ ，R4，R ₅ ，R ₆ ，R ₇ R8 are each independently 2 "hydrogen" isotopes (including hydrogen, deuterium), embodied as a combination of "HH", "HD", and "DD".

The study was performed on 6-8 week old female C57BL/6 mice, 8-10 week old CD-1 mice, according to the guidelines set by the national institute of science and technology. Different doses of mRNA lipid nanoparticles were administered systemically by tail vein injection and animals were euthanized at specific time points (e.g., 5 hours) after administration. Liver and spleen were collected in pre-weighed tubes, weighed, immediately snap frozen in liquid nitrogen, and stored at-80 ℃ until used for analysis.

For the liver, approximately 50mg was cut for analysis in 2mL FastPrep tubes (MP Biomedicals, solon OH). 1/4 "ceramic spheres (MP Biomedicals) were added to each tube, and 500. Mu.L of Glo lysis buffer-GLB (Promega, madison Wis.) equilibrated to room temperature was added to the liver tissue. Liver tissue was homogenized using a FastPrep24 instrument (MP Biomedicals) at 2X 6.0m/s for 15 seconds. The homogenate was incubated at room temperature for 5min, then diluted 1. Specifically, 50. Mu.L of the diluted tissue homogenate was reacted with 50. Mu.L of SteadyGlo substrate, shaken for 10 seconds, followed by incubation for 5 minutes, and then quantified using a SpectraMAX _ L chemiluminescence-type microplate reader (Meigu Motors, inc.). The amount of protein determined was determined by using BCA protein quantification kit (shanghai chromophil medical science and technology ltd). Relative Luminescence Units (RLU) were then normalized to the total μ g of protein assayed. To convert RLU to μ g luciferase, a standard curve was generated with QuantiLum recombinant luciferase (Promega).

Fluciferase protein will be expressed by Fluuc mRNA from Trilink Biotechnologies (L-6107), which was originally isolated from fireflies (Photinus pyralis). Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability. Which emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA was completely replaced by 5-methylcytidine and pseudouridine.

Example 84

Determination of pKa of the prepared lipid

The pKa of the formulated cationic lipid correlates with the effect of the LNP used to deliver the nucleic acid. The preferred pKa range is from 5 to 7. The pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on the fluorescence of 2- (p-toluidinyl) -6-naphthalenesulfonic acid (TNS). Lipid nanoparticles comprising cationic lipids/DSPC/cholesterol/PEG lipids (50/10/38/2 mol%) at a concentration of 0.4mM total lipid in PBS were prepared using an ordered method as described in example 3. TNS was prepared as a 100 μ M stock solution in distilled water. The vesicles were diluted to 24. Mu.M lipid in 2mL of buffer containing 10mM HEPES, 10mM MES, 10mM acetic acid, 130mM NaCl, pH 2.5-11. Aliquots of the TNS solution were added to give a final concentration of l μ M and after vortex mixing the fluorescence intensity was measured in a SLM Aminco Series 2 luminescence spectrophotometer at room temperature using excitation and emission wavelengths of 321nm and 445 nm. Sigmoidal best fit analysis was applied to the fluorescence data and pKa was measured as the pH yielding half the maximum fluorescence intensity.

Example 85

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

For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using an ordered mixing method, as described in example 83. Lipid nanoparticles were formulated using a molar ratio of 50% cationic lipid/10% Distearoylphosphatidylcholine (DSPC)/38% cholesterol/2% deuterated PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 5 hours after administration via tail vein injection as described in example 83. The activities were compared at doses of 0.3 and 1.0mg mRNA/kg and expressed as ng luciferase/g liver measured 5 hours after administration as described in example 83. The results of examples 83 and 84 are shown in Table 2.

Table 2 potency study of mRNA compositions comprising novel PEG lipids

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The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-described embodiments are merely illustrative of several embodiments of the present invention, which are described in more detail and detail, but are not to be construed as limiting the scope of the disclosure. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A deuterated polyethylene glycol lipid compound shown as A has the following structure:

R ₁ 、R ₂ 、R ₃ each independently is a combination of "DDD", "DDH", "DHH", "HHH", "DD", "DH", "HH", "D", "H".

2. The deuterated polyethylene glycol lipid as set forth in claim 1, R ₁ 、R ₂ 、R ₃ All have independence when R is ₁ And R ₂ When both are "HHH", R ₃ Can not be "HH", when R ₃ In the form of a "HH" combination, R ₁ 、R ₂ One of which cannot be a HHH combination.

3. The deuterated polyethylene glycol lipid as recited in claims 1-2, R ₁ 、R ₂ 、R ₃ Independently from each other, DDD, DD.

4. Deuterated polyethylene glycol lipid as recited in claim 1, wherein n has an average value of 40 to 50, preferably 45.

5. The method for preparing deuterated methyl polyethylene glycol compounds as recited in claims 1-2, wherein the method comprises the following steps:

1) Ext>ext> underext>ext> theext>ext> actionext>ext> ofext>ext> anext>ext> acylext>ext> chlorideext>ext> reagentext>ext>,ext>ext> aext>ext> deuteratedext>ext> generalext>ext> formulaext>ext> compoundext>ext> dPEGext>ext> -ext>ext> Aext>ext> -ext>ext> 1ext>ext> firstlyext>ext> generatesext>ext> acylext>ext> chlorideext>ext>,ext>ext> andext>ext> thenext>ext> performsext>ext> amidationext>ext> reactionext>ext> underext>ext> theext>ext> actionext>ext> ofext>ext> ammoniaext>ext> waterext>ext> toext>ext> obtainext>ext> aext>ext> deuteratedext>ext> generalext>ext> formulaext>ext> intermediateext>ext> dPEGext>ext> -ext>ext> Aext>ext> -ext>ext> 3ext>ext>;ext>ext>

2) Ext>ext> underext>ext> theext>ext> actionext>ext> ofext>ext> aext>ext> reducingext>ext> agentext>ext>,ext>ext> carryingext>ext> outext>ext> reductionext>ext> reactionext>ext> onext>ext> aext>ext> deuteratedext>ext> generalext>ext> -ext>ext> formulaext>ext> intermediateext>ext> dPEGext>ext> -ext>ext> Aext>ext> -ext>ext> 3ext>ext> toext>ext> obtainext>ext> aext>ext> deuteratedext>ext> generalext>ext> -ext>ext> formulaext>ext> intermediateext>ext> dPEGext>ext> -ext>ext> Aext>ext> -ext>ext> 4ext>ext>;ext>ext>

3) Ext>ext>ext> performingext>ext>ext> amidationext>ext>ext> reactionext>ext>ext> onext>ext>ext> theext>ext>ext> deuteratedext>ext>ext> generalext>ext>ext> formulaext>ext>ext> intermediateext>ext>ext> dPEGext>ext>ext> -ext>ext>ext> Aext>ext>ext> -ext>ext>ext> 4ext>ext>ext> andext>ext>ext> theext>ext>ext> generalext>ext>ext> formulaext>ext>ext> intermediateext>ext>ext> dPEGext>ext>ext> -ext>ext>ext> Aext>ext>ext> -ext>ext>ext> 1ext>ext>ext> accordingext>ext>ext> toext>ext>ext> theext>ext>ext> methodext>ext>ext> inext>ext>ext> theext>ext>ext> stepext>ext>ext> 1ext>ext>ext>)ext>ext>ext> toext>ext>ext> obtainext>ext>ext> aext>ext>ext> deuteratedext>ext>ext> generalext>ext>ext> formulaext>ext>ext> intermediateext>ext>ext> dPEGext>ext>ext> -ext>ext>ext> Aext>ext>ext> -ext>ext>ext> 5ext>ext>ext>;ext>ext>ext>

4) Ext>ext> accordingext>ext> toext>ext> theext>ext> methodext>ext> ofext>ext> theext>ext> stepext>ext> 2ext>ext>)ext>ext>,ext>ext> carryingext>ext> outext>ext> reductionext>ext> reactionext>ext> onext>ext> theext>ext> intermediateext>ext> dPEGext>ext> -ext>ext> Aext>ext> -ext>ext> 5ext>ext> withext>ext> theext>ext> deuteratedext>ext> generalext>ext> formulaext>ext> toext>ext> obtainext>ext> anext>ext> intermediateext>ext> dPEGext>ext> -ext>ext> Aext>ext> -ext>ext> 6ext>ext> withext>ext> theext>ext> deuteratedext>ext> generalext>ext> formulaext>ext>;ext>ext>

5) Ext>ext> inext>ext> anext>ext> organicext>ext> solventext>ext>,ext>ext> carryingext>ext> outext>ext> amidationext>ext> reactionext>ext> onext>ext> aext>ext> deuteratedext>ext> generalext>ext> formulaext>ext> intermediateext>ext> dPEGext>ext> -ext>ext> Aext>ext> -ext>ext> 6ext>ext> andext>ext> aext>ext> deuteratedext>ext> generalext>ext> formulaext>ext> compoundext>ext> dPEGext>ext> -ext>ext> Aext>ext> -ext>ext> 7ext>ext> underext>ext> theext>ext> actionext>ext> ofext>ext> baseext>ext> catalysisext>ext> toext>ext> obtainext>ext> aext>ext> deuteratedext>ext> polyethyleneext>ext> glycolext>ext> lipidext>ext> compoundext>ext> withext>ext> aext>ext> generalext>ext> formulaext>ext> Aext>ext>;ext>ext>

Synthetic route of general formula A.

6. A deuterated polyethylene glycol lipid compound shown as B has the following structure:

R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ each independently is a combination of DDD, DDH, DHH, HHH, DD, DH, HH, D, H.

7. The deuterated polyethylene glycol lipid as recited in claim 6, R ₁ 、R ₂ 、R ₃ R4 and R5 are independent, when R is ₁ 、R ₂ Are all "HHH" combinations, R ₄ In the case of the "H" combination, R ₃ 、R ₅ Cannot be simultaneously "HH"; when R is ₃ 、R ₅ Are all "HH" combinations, R ₄ In the case of the "H" combination, R ₁ 、R ₂ Cannot be simultaneously combined as "HHH"; when R is ₁ 、R ₂ Are all "HHH" combinations, R ₃ 、R ₅ All in combination of "HH", R ₄ It cannot be an "H" combination.

8. The deuterated polyethylene glycol lipid of claim 6, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Each independently a combination of "DDD", "DD", "D".

9. The deuterated polyethylene glycol lipid as recited in claim 6, wherein n has an average value of 40 to 50, preferably 45.

10. The method of claim 6, comprising the steps of:

1) In an organic solvent, carrying out substitution reaction on a deuterated general formula compound dPEG-B-1 and deuterated methyl polyethylene glycol dPEG-B-2 to obtain a deuterated general formula intermediate dPEG-B-3;

2) Under the alkaline catalysis, carrying out epoxy hydrolysis reaction on the deuterated general-formula intermediate dPEG-B-3 to obtain a deuterated general-formula intermediate dPEG-B-4;

3) Under the action of a condensing agent, carrying out esterification reaction on a deuterated general formula intermediate dPEG-B-4 and a deuterated general formula compound dPEG-B-5 to obtain a deuterated general formula compound dPEG-B;

synthetic route of general formula B.

11. A combination of a compound according to any one of claims 1 to 10 and a therapeutic and/or prophylactic agent.

12. The composition of claim 11, further comprising one or more excipients selected from the group consisting of neutral lipids, steroids, and cationic lipids.

13. A composition as claimed in claim 12, wherein the neutral lipids of the component are selected from one or more of the following in admixture: 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dimyristyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and Sphingomyelin (SM).

14. The composition of claim 12, wherein the neutral lipid is DSPC.

15. A composition as claimed in any one of claims 11 to 12, wherein the steroid in the component is selected from a mixture of one or more of: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol.

16. The composition of claim 15, wherein the steroid is cholesterol.

17. A composition as claimed in any one of claims 11 to 16 wherein the cationic lipid in the component is selected from a mixture of one or more of: 4- (N, N-dimethylamino) butanol (6Z, 9Z,28Z, 31Z) -heptatriacontan-6, 9,28, 31-tetraen-19-yl ester (DLin-MC 3-DMA), heptadecan-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate (SM-102), ((4-hydroxybutyl) aza-ethyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), 4- (N-2- (N, N-bis (2-hydroxydodecyl) ethylamino) -1-N (2-N (2-ethylamino- (2- (N, N-bis (2-hydroxydodecyl)) -2 hydroxydodecyl) ethylamino piperazine (C12-200).

18. The composition of claim 17, wherein the cationic lipid is ((4-hydroxybutyl) aza-ethyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), heptadecan-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate (SM-102).

19. The composition of claim 12, wherein the cationic lipid is ((4-hydroxybutyl) aza-ethyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315) deuterated analog, deuterated analog of heptadecan-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate (SM-102), as shown in structural formula C, D;

R ₁ ，R ₂ ，R ₃ ，R4，R ₅ ，R ₆ ，R ₇ r8 are each independently 2 "hydrogen" isotopes: including hydrogen, deuterium, embodied as a combination of "HH", "HD" and "DD";

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R ₁ ，R ₂ ，R ₃ and R4, each independently are 2 "hydrogen" isotopes: including hydrogen, deuterium, as embodied by a combination of "HH", "HD" and "DD".

20. A composition as claimed in claim 12, wherein the cationic lipid has the following structure:

21. the composition of any one of the preceding claims, wherein the therapeutic and/or prophylactic agent is a vaccine or compound capable of eliciting an immune response, including nucleic acids.

22. The composition of claim 11, wherein the nucleic acid is RNA selected from the group consisting of: siRNA, airRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof.

23. The composition of claim 22, wherein the RNA is mRNA.

24. A method of administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising preparing or providing a composition according to any one of the preceding claims, and administering the composition to the subject.

25. The subject of any one of the preceding claims is a mammal or a human.