Novel deuterated polyethylene glycol lipid compounds

Abstract

This invention relates to a novel deuterated PEG lipid compound, its preparation method, composition, and application. Specifically, it discloses a novel deuterated lipid compound as shown in Formula A and Formula B, or a pharmaceutically acceptable salt thereof, or an isomer thereof. The invention also discloses the preparation method, composition, and application of the above-mentioned PEG lipid compound. These compounds, as functional PEG lipids, are suitable for drug compositions for nucleic acid delivery, achieving superior stability, water solubility, and intracellular drug delivery efficiency, as shown in Formulas A and B below.

Description

Technical Field

[0001] This invention provides novel deuterated polymer-conjugated lipids that can be combined with other lipid components (such as neutral lipids, steroids, and cationic lipids) to form a nucleic acid mRNA lipid nanoparticle composition for delivering one or more therapeutic and / or preventative agents to mammalian cells or organs and / or for generating peptides in mammalian cells or organs. In addition to the novel deuterated lipids, the lipid nanoparticle compositions of this invention may also include one or more deuterated polymer-conjugated lipids, neutral lipids including polyunsaturated lipids, cationic lipids, steroids, and / or therapeutic and / or preventative agents in specific proportions. Background Technology

[0002] The efficient targeted delivery of bioactive substances such as small molecule drugs, proteins, and nucleic acids presents a persistent medical challenge. Specifically, the delivery of nucleic acids to cells is difficult due to the relative instability and low cell permeability of these species. Therefore, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, to cells.

[0003] Studies have demonstrated that lipid-containing nanoparticle compositions, liposomes, and liposome complexes can be used as transport media to effectively deliver bioactive substances such as small molecule drugs, proteins, and nucleic acids into cells and / or intracellular compartments. These compositions generally contain one or more polymer-conjugated lipids, including neutral lipids (such as phospholipids), including polyunsaturated lipids, structural lipids (such as steroids), and / or cationic lipids.

[0004] However, the use of oligonucleotides in therapeutic settings currently faces two challenges. First, free RNA is readily digested by nucleases in plasma. Second, the ability of free RNA to enter intracellular compartments containing relevant translation mechanisms is limited. Lipid nanoparticles formed from PEGylated deuterated lipids and other lipid components (such as neutral lipids, cholesterol, cationic lipids, and oligonucleotides) have been used to prevent RNA degradation in plasma and promote cellular uptake of oligonucleotides, while deuterated polymer-conjugated lipids have not been reported.

[0005] Therefore, there remains a need to improve deuterated polyethylene glycol-modified lipids and lipid nanoparticles for delivering oligonucleotides. Improved lipid compounds and lipid nanoparticles enhance physicochemical properties, provide optimized drug delivery, protect nucleic acids from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. Furthermore, these preferred lipid-nucleic acid particles should be well-tolerated and provide a sufficient therapeutic index so that treatment of patients with effective doses of nucleic acid does not pose unacceptable toxicity and / or risks. This invention provides these and related advantages. Summary of the Invention

[0006] This invention provides the following novel polyethylene glycol lipid compounds (such as Formula A and Formula B below), methods for preparing these compounds, compositions thereof, and applications thereof. The functional PEGylated deuterated lipids provided by this invention can achieve better physicochemical properties—water solubility and stability—and better intracellular nucleic acid delivery efficiency.

[0007] 1. This invention relates to deuterated polyethylene glycol lipid compounds (A):

[0008] (A) n≈45

[0009] Or a drug-acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0010] R1, R2, and R3 are each an independent combination of DDD, DDH, DHH, HHH, DD, DH, HH, D, and H;

[0011] R1, R2, and R3 are all independent. When R1 and R2 are both "HHH", R3 cannot be "HH". When R3 is a "HH" combination, neither R1 nor R2 can be a "HHH" combination.

[0012] R1 and R2 are each independently a "DDD" combination, and R3 is independently a "DD" combination;

[0013] The average value of n is 40-50, with 45 being more optimal.

[0014] 2. This invention relates to deuterated polyethylene glycol lipid compounds (B):

[0015] (B) n≈45

[0016] Or a drug-acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0017] R1, R2, R3, R4, and R5 are each independently a combination of DDD, DDH, DHH, HHH, DD, DH, HH, D, and H;

[0018] R1, R2, R3, R4, and R5 are all independent. When R1 and R2 are both "HHH" combinations and R4 is a "H" combination, R3 and R5 cannot both be "HH". When R3 and R5 are both "HH" combinations and R4 is a "H" combination, R1 and R2 cannot both be "HHH" combinations. When R1 and R2 are both "HHH" combinations and R3 and R5 are both "HH" combinations, R4 cannot be a "H" combination.

[0019] R1, R2, R3, R4, and R5 are each an independent combination of "DDD", "DD", and "D";

[0020] The average value of n is 40-50, with 45 being more optimal.

[0021] In various embodiments, the compound has one of the structures shown in Table 1 below.

[0022] Table 1 Representative novel PEG lipid compounds

[0023]

[0024] In some embodiments, a composition is provided comprising one or more of deuterated compounds of structural formulas (A) and (B) and a therapeutic and / or preventive agent.

[0025] In some embodiments, a composition is provided comprising one or more compounds of structures (A) and (B) and a therapeutic and / or preventative agent. In some embodiments, the composition comprises one or more excipients selected from neutral lipids, steroids, and cationic lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0026] In some embodiments, the neutral lipid is selected from 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), sphingomyelin (SM), and mixtures thereof. In some embodiments, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC) is preferred as the neutral lipid.

[0027] In some embodiments, the steroid is selected from cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, cholesterol is preferred as the steroid.

[0028] In some embodiments, the cationic lipid is selected from 4-(N,N-dimethylamino)butanol (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decoxy)hexyl)amino)octanoate (SM-102), ((4-hydroxybutyl)aza-ethyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 4-(N-2-(N,N-di(2-hydroxydodecyl)ethylamino)-1-N(2-N(2-ethylamino-(2-(N,N-di(2-hydroxydodecyl))-2-hydroxydodecyl)ethylaminopiperazine (C12-200), and novel lipid compounds of the pyrrolidine class.

[0029] In some embodiments, the cationic lipid is preferably a deuterated analog of ((4-hydroxybutyl)aza-ethyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) or a deuterated analog of heptadecano-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decoxy)hexyl)amino)octanoate (SM-102).

[0030] The cationic lipids in the composition of the present invention have the following structure (Formula I):

[0031]

[0032] in:

[0033] L1 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-;

[0034] R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl, and the hydrocarbon chain is optionally linked by one or more ester or ether bonds; R3 and R4 are each independently C1-C12 alkyl or C1-C12 alkenyl, or R3 and R4 are combined with each other to form a 4 to 10-membered heterocycle, wherein the heteroatom includes one or more heteroatoms selected from N, O, and S, and the heterocycle is optionally substituted by 1 to 6 heteroatoms;

[0035] X is C, N, O, S, -SS-; M is C1-C12 alkyl or C1-C12 alkenyl; x is 0, 1 or 2.

[0036] The cationic lipid compounds in the compositions of the present invention are preferably all of the cationic lipid compounds disclosed in the following patent application numbers: CN2021109304214, CN2021109693918, CN2021109698790, CN2021109700095, CN202110970112X, CN2021109787347, CN2021110306263, CN2021110307694, CN2021110439195, CN2021110310644, CN2021110311986, CN2021111108325, CN2021111057272.

[0037] In some embodiments, the composition comprises the following proportions: about 1-10 mol% of the compound, about 0-30 mol% of neutral lipids, about 10-55 mol% of steroids, and about 30-60 mol% of cationic lipids.

[0038] In some embodiments of the aforementioned compositions, the therapeutic and / or preventative agents comprise nucleic acids. The nucleic acid is RNA, selected from the following: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA, and mixtures thereof. In some embodiments, the RNA is selected from mRNA.

[0039] In other different embodiments, the present invention relates to a method of administering a therapeutic and / or preventive agent to a subject in need, the method comprising preparing or providing any of the above-described compositions and administering the composition to the subject.

[0040] For application purposes, the compounds of the present invention (typically in the form of lipid nanoparticles combined with therapeutic and / or preventative agents) can be administered as active pharmaceutical ingredients or formulated as pharmaceutical compositions. 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 used to effectively form lipid nanoparticles and deliver therapeutic and / or preventative agents. Those skilled in the art can readily determine appropriate concentrations and dosages.

[0041] The compositions of the present invention can be administered by any acceptable method of administration of an agent with similar efficacy. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the active ingredient contained therein to be bioavailable after administration to a subject. The composition to be administered to a subject or patient is in the form of one or more dose units, wherein tablets may be single dose units, while containers for the compounds of the present invention in aerosol form may contain multiple dose units. Current methods for preparing these dosage forms are known or will be apparent to those skilled in the art. In any case, the composition to be administered will contain a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, in order to treat the relevant disease or condition according to the teachings of the present invention.

[0042] The pharmaceutical compositions of the present invention can be in solid or liquid form. On one hand, the carrier is a microparticle, making the composition a tablet or powder. The carrier can also be a liquid, in which case the composition is an oral syrup, an injectable liquid, or an aerosol suitable for inhalation.

[0043] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein the solid or liquid form is considered herein to include semi-solid, semi-liquid, suspension and gel forms.

[0044] As solid compositions intended for oral administration, pharmaceutical compositions can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, sheets, etc. These solid compositions typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders, such as gelatin, cellulose, etc.; excipients, such as lactose, etc.; disintegrants, such as alginate, etc.; lubricants, such as magnesium stearate, etc.; glidants, such as silica gel, etc.; sweeteners, such as sucrose or saccharin; flavoring agents, such as menthol, etc.; and coloring agents.

[0045] When the pharmaceutical composition is in capsule form, it may contain a liquid carrier other than the materials described above, such as polyethylene glycol or oil.

[0046] The pharmaceutical composition may be in liquid form, such as 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 composition contains one or more of the following, in addition to the compounds of the present invention: sweeteners, preservatives, coloring / staining agents, and flavor enhancers. In compositions intended for injection, one or more of the following may be included: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0047] The liquid pharmaceutical compositions of the present invention, whether in the form of a solution, suspension, or other similar form, 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; non-volatile oils, such as synthetic mono- or diglycerides that can be used as solvents or suspension media, polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, thiocyanates, or phosphates; and agents for regulating tension, such as sodium chloride or glucose; and agents used as cryoprotectants, such as sucrose or trehalose. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0048] The pharmaceutical compositions of the present invention can consist of dosage units that can be administered as aerosols. The term aerosol is used to refer to a variety of systems, ranging from colloidal systems to systems consisting of pressurized packaging. Delivery can be made by liquefied or compressed gas, or by a suitable pump system for dispersing the active ingredient. Aerosols of the compounds of the present invention can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes necessary containers, activators, valves, sub-containers, etc., which together can form a kit. Preferred aerosols can be determined by those skilled in the art without extensive experimentation.

[0049] The pharmaceutical compositions of the present invention can be prepared by methods well known in the pharmaceutical industry. Pharmaceutical compositions intended for injection can be prepared by conjugating the lipid nanoparticles of the present invention into a solution with sterile distilled water or other carriers. Surfactants can be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that interact nonvalently with the compounds of the present invention to promote the dissolution or homogeneous suspension of said compounds in an aqueous delivery system.

[0050] The compositions of the present invention or pharmaceutically acceptable salts thereof are administered in therapeutically effective amounts, said amounts which will vary depending on a variety of factors, including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the age, weight, general health status, sex, and diet of the subject; the manner and time of administration; the rate of excretion; the combination of drugs; the severity of the specific case, etc.

[0051] The compositions of the present invention can also be administered concurrently with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include single-dose formulations of the composition of the present invention and one or more additional active agents, as well as formulations of the composition of the present invention and each active agent in its own individual pharmaceutical dosage form. For example, the composition of the present invention and other active agents can be administered to a subject together as a single oral dosage composition (such as tablets or capsules), or the individual agents can be administered in different oral dosage forms. When using different dosage forms, the compounds of the present invention and one or more additional active agents can be administered substantially simultaneously, or sequentially at alternating times; it should be understood that combination therapies include all of these administration regimens.

[0052] The structural modification and design of the above-mentioned deuterated PEGylated deuterated lipid compounds have achieved more advantageous physicochemical properties, including a more suitable pKa and better chemical stability. When used in mRNA nanoliposome compositions, they can achieve more effective binding and delivery of ionic nucleic acid drugs. At the same time, their chemical structure is more stable, which facilitates synthesis and is beneficial for development into pharmaceutical excipients.

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

[0054] Those skilled in the art will recognize that the functional groups of the raw materials and intermediates described herein include epoxy, hydroxyl, amino, and amide groups. Substitution, reduction, and condensation methods are employed. Suitable methods for the reduction of amides to amines include metal hydride reduction, borane-tetrahydrofuran complex reduction, catalytic hydrogenation reduction, and sodium borohydride-Lewis acid system reduction. Methods for preparing amides mainly include amine transesterification, aliphatic amine transesterification, anhydride-based methods, acyl halide-based methods, organophosphorus condensing agent methods, and onium salt condensing agent methods. Epoxy ring-opening mainly involves acidic and basic catalysis; the method can be selected based on the substrate properties. The standard techniques described herein are known to those skilled in the art.

[0055] Those skilled in the art will also recognize that while such reduced and condensed derivatives of the compounds of the present invention may not thereby possess pharmaceutical activity, they can be administered to mammals and subsequently metabolized in vivo to form the pharmacologically active compounds of the present invention. Such derivatives can therefore be described as "prodrugs." Thus, prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0056] Furthermore, all compounds of the present invention existing in the form of free bases or free acids can be converted into pharmaceutically acceptable salts by treatment with suitable inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of the compounds of the present invention can be converted into their free base or acid forms using standard techniques.

[0057] The following embodiments are provided for illustrative purposes only and are not intended to be limiting.

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

[0059] The procedure described below can be used to synthesize compounds A and B.

[0060] The following abbreviations are used in this article:

[0061] DCM: Dichloromethane

[0062] THF: Tetrahydrofuran

[0063] MeOH: Methanol

[0064] HBTU: 2-(benzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate

[0065] DIEA: Diisopropylethylamine

[0066] DMF: N,N-dimethylformamide

[0067] LiAlH4: Lithium aluminum hydride

[0068] NaOH: Sodium hydroxide

[0069] PEG: Polyethylene Glycol

[0070] DMSO-d6: Deuterated dimethyl sulfoxide

[0071] EA: Ethyl acetate

[0072] PE: Petroleum ether (60-90)

[0073] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0074] DMAP: N,N-dimethylpyridine. Detailed Implementation

[0075] Example 1

[0076] Representative routes

[0077] Synthesis of Compound A: N,N-bis(14,14,14-D3-tetradecyl)-2,2-2D-2-methylpolyethylene glycol acetamide

[0078]

[0079] 1) Synthesis of 14,14,14-D3-tetradecanoamide (dPEG-A-3)

[0080]

[0081] Molecular formula: C 14 H 26 D3NO

[0082] Molecular weight: 230.40

[0083] Oxaloyl chloride (38.85 mmol, 4.93 g) was added to a toluene (50 mL) solution of 14,14,14-D3-tetradecanoic acid (dPEG-A-1) (6 g, 25.9 mmol) at room temperature. The resulting mixture was heated at 70 °C for 2 h and then concentrated. The residue was dissolved in toluene and concentrated again. The remaining oily substance was added to a concentrated ammonia solution (20 mL) via syringe at 10 °C. The reaction mixture was filtered and washed with water. The white solid was dried under vacuum. The desired product, 14,14,14-D3-tetradecanoamide (dPEG-A-3), was obtained as a white solid (3.58 g, 60%).

[0084] APCI-MS: m / z 231 [M+H] +

[0085] 1 H-NMR (300MHz, DMSO-d6): δ ppm 6.8(s, 2H), 2.28(t, 2H, J=7.2Hz), 1.80(m, 2H), 1.35(m, 2H), 1.24(m, 18H).

[0086] 2) Synthesis of 14,14,14-D3-tetradecadeamine (dPEG-A-4)

[0087]

[0088] Molecular formula: C 14 H 28 D3N

[0089] Molecular weight: 216.40

[0090] At room temperature, lithium aluminum hydride (1.14 g, 30 mmol) was added in portions to a suspension of 14,14,14-D3-tetradecanoamide (3.58 g, 15.54 mmol) in THF (70 mL) over a period of 30 min. The mixture was then slowly heated to reflux (65 °C oil bath) overnight. The mixture was cooled to 5 °C and sodium sulfate hydrate was added. The mixture was stirred for 2 h, filtered through a diatomaceous earth layer, and washed with DCM (200 mL) containing 15% MeOH. The filtrate and washings were combined and concentrated. The remaining solid was dried under vacuum. 14,14,14-D3-tetradecanoamide (dPEG-A-4) was obtained as a white solid (2.86, 85.1%).

[0091] APCI-MS: m / z 217 [M+H] +

[0092] 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).

[0093] 3) Synthesis of 14,14,14-D3-tetradecyl-14,14,14-D3-tetradecanoamide (dPEG-A-5)

[0094]

[0095] Molecular formula: C 28 H 51 D6NO

[0096] Molecular weight: 429.81

[0097] At room temperature, oxaloyl chloride (0.82 g, 6.45 mmol) was added to a solution of 14,14,14-D3-myristic acid (1 g, 4.3 mmol) in benzene (10 mL) and DMF (1 drop). The mixture was stirred at room temperature for 3 h. Toluene was removed by concentration at 60 °C. The remaining oily substance was dissolved in 5 mL of benzene, cooled to 10 °C, and then added to a solution of 14,14,14-D3-tetradecylamine (0.97 g, 4.5 mol) and triethylamine (0.65 g, 6.45 mmol) in benzene (5 mL). After the addition was complete, the reaction mixture was stirred at room temperature overnight. After confirming the reaction was complete, water was added, diluted with water, and adjusted to pH 5-6 with 1.7 M HCl. A solid precipitated. The mixture was stirred at room temperature for 5 h, filtered, and washed with water. A grayish-white wet product was obtained. The wet product was dissolved in 5 ml of methanol at 58 °C, cooled to room temperature and stirred for 8 hours, filtered, and washed with 1 ml of methanol. The resulting solid was dried in a forced-air oven at 55 °C for 10 hours to obtain the target compound 14,14,14-D3-tetradecyl 14,14,14-D3-tetradecylamide, which was a pale white solid (1.77 g, 93%).

[0098] APCI-MS: m / z 430 [M+H] +

[0099] 1 H-NMR (300MHz, DMSO-d6): δppm 8.13(s, 1H), 3.86(m, 2H), 2.66(m, 2H), 1.65(m, 2H), 1.24(m, 44H).

[0100] 4) Synthesis of N,N-bis(14,14,14-D3-tetradecyl)amine (dPEG-A-6)

[0101]

[0102] Molecular formula: C 28 H 53 D6N

[0103] Molecular weight: 415.82

[0104] At room temperature, lithium aluminum hydride (0.51 g, 13.5 mmol) was added in portions to a suspension of 14,14,14-D3-tetradecyl-14,14,14-D3-tetradecylamide (2 g, 4.5 mol) in 20 mL of THF. The mixture was heated to 68 °C and refluxed overnight. After the reaction was complete, the mixture was cooled to 0 °C, and an aqueous sodium hydroxide solution was added and stirred for 2 h. The mixture was then filtered through a diatomaceous earth and silica gel mat and washed with ether. The filtrate became turbid and a precipitate formed. After stirring for 5 min, the precipitate was filtered to obtain a white solid.

[0105] The white solid was transferred to a 20 ml double-necked flask, 5 ml of methanol was added, and the mixture was heated to 60 °C to dissolve completely. After 30 min, the temperature was lowered to 25 °C, and the mixture was stirred to crystallize for 10 hours. The crystals were then filtered, washed with 1 ml of methanol, and dried at 55 °C for 10 hours to obtain N,N-di(14,14,14-D3-tetradecyl)amine (dPEG-A-6), a white solid (1.45 g, 75.01%).

[0106] APCI-MS: m / z 416[M+H]+

[0107] 1 H-NMR (300MHz, DMSO-d6): δppm 3.45(s, 1H), 2.66(m, 4H), 1.35(m, 4H), 1.24(m, 44H).

[0108] 5) Synthesis of N,N-bis(14,14,14-D3-tetradecyl)-2,2-2D-2-methylpolyethylene glycol acetamide (dPEG-A)

[0109]

[0110] Molecular formula: C 117 H 231 D8NO 47

[0111] Molecular weight: 2485

[0112] Add a solution of N,N-di(14,14,14-D3-tetradecyl)amine (2 g, 4.65 mmol) and triethylamine (0.71 g, 6.98 mmol) in DCM (40 mL) to a 50 mL double-necked flask, then add a solution of methyl polyethylene glycol succinimide acetate (mPEG-NHS, 4.65 mL, 10.1 g, PEG MW approx. 2,000, n = approx. 45) in DCM (50 mL). Stir at room temperature for 24 h, then add water (30 mL) and extract the aqueous phase twice with DCM (100 mL x 2). Combine the DCM extracts and wash with 1 M HCl (30 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated to approximately 30 ml. The mixture was then cooled to -20°C and stirred for 8 hours, filtered, and washed with 3 ml of DCM to obtain a white solid (0.9 g, unreacted initial amine). Triethylamine (18.6 mmol) and acetic anhydride (1.4 mmol, 143 mg) were added to the filtrate. After 10 min, a solid precipitated. The mixture was stirred at room temperature for 5 h, and then concentrated to a solid state at 40°C. The remaining solid was purified by column chromatography on silica gel (containing 0-8% methanol in DCM) to obtain N,N-bis(14,14,14-D3-tetradecyl)-2,2-2D-2-methylpolyethylene glycol acetamide as a white solid (5.82 g, yield approximately 50%).

[0113] APCI-MS: m / z 2486 [M+H] +

[0114] 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).

[0115] Example 2

[0116] Representative synthetic routes

[0117] Compound B 3-(2-2-methylpolyethylene glycol (n=45))-1,2-diyl-bis(14,14,14-3D-tetradecanoyl)1,1,2,3,3-5D-propane

[0118] 1) Synthesis of 1,1,2,3,3-3D-3-methylpolyethylene glycol-1,2-epoxypropane

[0119]

[0120] Molecular formula: C 94H 183 D5O 47

[0121] Molecular weight: 2073

[0122] A mixture of THF (55 mL) and DMF (10 mL) of polyethylene glycol monomethyl ether (dEG-B-2, 68.2 g, PEG Mw approx. 2000, n = approx. 45, 34.1 mmol, 1.2 eq) was heated in an ice bath at 0-10 °C. NaH (60% mass fraction, 1.4 g, 34.1 mmol, 1.2 eq) was added, and the mixture was stirred for 0.5 h. Then, a THF solution (10 mL) of 1,1,2,3,3-3D-3-chloro-1,2-epoxypropane (dEG-B-1, 2.8 g, 28.4 mmol, 1.0 eq) was slowly added. After the addition was complete, the mixture was allowed to heat naturally and stirred for 18 h. After TLC showed that dEG-B-1 had essentially reacted completely, the reaction mixture was slowly poured into a saturated ammonium chloride solution. The mixture was extracted three times with THF. The organic phases were combined and washed once with saturated sodium chloride. The sample was mixed with organic silica gel and purified by column chromatography (EA:PE=5:1, Rf=0.4) to obtain 39.2 g of semi-oil and semi-solid, with a yield of 65%.

[0123] APCI-MS: m / z 2074 [M+H] +

[0124] 1 H-NMR (300MHz, CDC13): δppm 3.83-3.46 (m, 180-200H), 3.39 (s, 3H).

[0125] 2) Synthesis of 1,1,2,3,3-3D-3-methylpolyethylene glycol-1,2-propanediol

[0126]

[0127] Molecular formula: C 94 H 185 D5O 48

[0128] Molecular weight: 2091

[0129] 1,1,2,3,3-3D-3-methylpolyethylene glycol-1,2-epoxypropane (39.2 g, 18.9 mmol) was dissolved in 500 mL of tetrahydrofuran, and 2M sodium hydroxide solution (1.13 g, 28.35 mmol, 14.16 mL) was added. The mixture was heated to 50 °C and stirred for 6 hours. After the reaction was complete, the mixture was cooled to room temperature, extracted once with EA, washed once with saturated sodium chloride, and the organic phase was mixed with silica gel. The solution was purified by column chromatography (EA) to obtain 38.7 g of a white solid, with a yield of 97.7%.

[0130] APCI-MS: m / z 2092 [M+H] +

[0131] 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).

[0132] 3) Synthesis of 3-(2-2-methylpolyethylene glycol (n=45))-1,2-diyl-bis(14,14,14-3D-tetradecanoyl)1,1,2,3,3-5D-propane

[0133]

[0134] Molecular formula: C 122 H 231 D 11 O 50

[0135] Molecular weight: 2517

[0136] 38.7 g of 1,1,2,3,3-3D-3-methylpolyethylene glycol-1,2-propanediol (dPEG-B-4, 18.50 mmol) was added to a 1000 ml four-necked flask, along with 400 ml of DMF, 8.99 g of 14,14,14-D3-myristic acid (38.85 mmol), 5.62 g of DIEA (55.5 mmol), and 10.64 g of EDCI (55.5 mmol). The mixture was heated to 45 °C and reacted for 5 hours. After the reaction was complete, the mixture was extracted three times with EA, washed once with water, and washed once with brine. The organic phase was concentrated at 45 °C and purified by column chromatography (EA:PE = 15:1) to obtain 35.6 g of white solid (dPEG-B), yield: 76.46%.

[0137] APCI-MS: m / z 2518 [M+H] +

[0138] 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).

[0139] The following compounds can be prepared by employing the above-described techniques and relevant steps known to those skilled in the art, and by using suitable deuterated reagents.

[0140] Example 3: Synthesis of Compound 2

[0141]

[0142] 2 (n≈45)

[0143] Molecular formula: C 117 H 232 D7NO 47

[0144] Molecular weight: 2484

[0145] APCI-MS: m / z 2485 [M+H] +

[0146] 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, 4H), 1.24(m, 44H).

[0147] Example 4: Synthesis of Compound 3

[0148]

[0149] Molecular formula: C 117 H 232 D7NO 47

[0150] Molecular weight: 2484

[0151] APCI-MS: m / z 2485 [M+H] +

[0152] 1H-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),

[0153] Example 5: Synthesis of Compound 4

[0154]

[0155] Molecular formula: C 117 H 233 D6NO 47

[0156] Molecular weight: 2483

[0157] APCI-MS: m / z 2484 [M+H] +

[0158] 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).

[0159] Example 6: Synthesis of Compound 5

[0160]

[0161] Molecular formula: C 117 H 233 D6NO 47

[0162] Molecular weight: 2483

[0163] APCI-MS: m / z 2484 [M+H] +

[0164] 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).

[0165] Example 7: Synthesis of Compound 6

[0166]

[0167] 6 (n≈45)

[0168] Molecular formula: C 117 H 233 D6NO 47

[0169] Molecular weight: 2483

[0170] APCI-MS: m / z 2484 [M+H] +

[0171] 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).

[0172] Example 8: Synthesis of Compound 7

[0173]

[0174] 7 (n≈45)

[0175] Molecular formula: C 117 H 234 D5NO 47

[0176] Molecular weight: 2482

[0177] APCI-MS: m / z 2483 [M+H] +

[0178] 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).

[0179] Example 9: Synthesis of Compound 8

[0180]

[0181] 8 (n≈45)

[0182] Molecular formula: C 117 H 234D5NO 47

[0183] Molecular weight: 2482

[0184] APCI-MS: m / z 2483 [M+H] +

[0185] 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).

[0186] Example 10: Synthesis of Compound 9

[0187]

[0188] 9 (n≈45)

[0189] Molecular formula: C 117 H 234 D5NO 47

[0190] Molecular weight: 2482

[0191] APCI-MS: m / z 2483 [M+H] +

[0192] 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).

[0193] Example 11: Synthesis of Compound 10

[0194]

[0195] 10 (n≈45)

[0196] Molecular formula: C 117 H 234 D5NO 47

[0197] Molecular weight: 2482

[0198] APCI-MS: m / z 2483 [M+H]+

[0199] 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).

[0200] Example 12: Synthesis of Compound 11

[0201]

[0202] 11 (n≈45)

[0203] Molecular formula: C 117 H 235 D4NO 47

[0204] Molecular weight: 2482

[0205] APCI-MS: m / z 2482 [M+H] +

[0206] 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).

[0207] Example 13: Synthesis of Compound 12

[0208]

[0209] 12 (n≈45)

[0210] Molecular formula: C 117 H 235 D4NO 47

[0211] Molecular weight: 2481

[0212] APCI-MS: m / z 2482 [M+H] +

[0213] 1H-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).

[0214] Example 14: Synthesis of Compound 13

[0215]

[0216] 13 (n≈45)

[0217] Molecular formula: C 117 H 235 D4NO 47

[0218] Molecular weight: 2481

[0219] APCI-MS: m / z 2482 [M+H] +

[0220] 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).

[0221] Example 15: Synthesis of Compound 14

[0222]

[0223] 14 (n≈45)

[0224] Molecular formula: C 117 H 236 D3NO 47

[0225] Molecular weight: 2481

[0226] APCI-MS: m / z 2482 [M+H] +

[0227] 1H-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).

[0228] Example 16: Synthesis of Compound 15

[0229]

[0230] 15 (n≈45)

[0231] Molecular formula: C 117 H 236 D3NO 47

[0232] Molecular weight: 2481

[0233] APCI-MS: m / z 2482 [M+H] +

[0234] 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).

[0235] Example 17: Synthesis of Compound 16

[0236]

[0237] 16 (n≈45)

[0238] Molecular formula: C 117 H 236 D3NO 47

[0239] Molecular weight: 2481

[0240] APCI-MS: m / z 2482 [M+H] +

[0241] 1H-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).

[0242] Example 18: Synthesis of Compound 17

[0243]

[0244] 17 (n≈45)

[0245] Molecular formula: C 117 H 236 D3NO 47

[0246] Molecular weight: 2481

[0247] APCI-MS: m / z 2482 [M+H] +

[0248] 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).

[0249] Example 19: Synthesis of Compound 18

[0250]

[0251] 18 (n≈45)

[0252] Molecular formula: C 117 H 236 D3NO 47

[0253] Molecular weight: 2481

[0254] APCI-MS: m / z 2482 [M+H] +

[0255] 1H-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).

[0256] Example 20: Synthesis of Compound 19

[0257]

[0258] 19 (n≈45)

[0259] Molecular formula: C 117 H 237 D2NO 47

[0260] Molecular weight: 2480

[0261] APCI-MS: m / z 2481 [M+H] +

[0262] 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).

[0263] Example 21: Synthesis of Compound 20

[0264]

[0265] 20 (n≈45)

[0266] Molecular formula: C 117 H 237 D2NO 47

[0267] Molecular weight: 2480

[0268] APCI-MS: m / z 2481 [M+H] +

[0269] 1H-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).

[0270] Example 22: Synthesis of Compound 21

[0271]

[0272] 21 (n≈45)

[0273] Molecular formula: C 117 H 237 D2NO 47

[0274] Molecular weight: 2480

[0275] APCI-MS: m / z 2481 [M+H] +

[0276] 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).

[0277] Example 23: Synthesis of Compound 22

[0278]

[0279] 22 (n≈45)

[0280] Molecular formula: C 117 H 237 D2NO 47

[0281] Molecular weight: 2480

[0282] APCI-MS: m / z 2481 [M+H] +

[0283] 1H-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).

[0284] Example 24: Synthesis of Compound 23

[0285]

[0286] 23 (n≈45)

[0287] Molecular formula: C 117 H 238 D1NO 47

[0288] Molecular weight: 2479

[0289] APCI-MS: m / z 2480 [M+H] +

[0290] 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).

[0291] Example 25: Synthesis of Compound 24

[0292]

[0293] 24 (n≈45)

[0294] Molecular formula: C 117 H 238 D1NO 47

[0295] Molecular weight: 2479

[0296] APCI-MS: m / z 2480 [M+H] +

[0297] 1H-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).

[0298] Example 26: Synthesis of Compound 26

[0299]

[0300] 26 (n≈45)

[0301] Molecular formula: C 122 H 232 D 10 O 50

[0302] Molecular weight: 2516

[0303] APCI-MS: m / z 2517 [M+H] +

[0304] 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).

[0305] Example 27: Synthesis of Compound 27

[0306]

[0307] 27 (n≈45)

[0308] Molecular formula: C 122 H 232 D 10 O 50

[0309] Molecular weight: 2516

[0310] APCI-MS: m / z 2517 [M+H] +

[0311] 1H-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).

[0312] Example 28: Synthesis of Compound 28

[0313]

[0314] 28 (n≈45)

[0315] Molecular formula: C 122 H 232 D 10 O 50

[0316] Molecular weight: 2516

[0317] APCI-MS: m / z 2517 [M+H] +

[0318] 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).

[0319] Example 29: Synthesis of Compound 29

[0320]

[0321] 29 (n≈45)

[0322] Molecular formula: C 122 H 232 D 10 O 50

[0323] Molecular weight: 2516

[0324] APCI-MS: m / z 2517 [M+H] +

[0325] 1H-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).

[0326] Example 30: Synthesis of Compound 30

[0327]

[0328] 30 (n≈45)

[0329] Molecular formula: C 122 H 232 D 10 O 50

[0330] Molecular weight: 2516

[0331] APCI-MS: m / z 2517 [M+H] +

[0332] 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).

[0333] Example 31: Synthesis of Compound 31

[0334]

[0335] 31 (n≈45)

[0336] Molecular formula: C 122 H 233 D9O 50

[0337] Molecular weight: 2515

[0338] APCI-MS: m / z 2516 [M+H] +

[0339] 1H-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).

[0340] Example 32: Synthesis of Compound 32

[0341]

[0342] 32 (n≈45)

[0343] Molecular formula: C 122 H 233 D9O 50

[0344] Molecular weight: 2515

[0345] APCI-MS: m / z 2516 [M+H] +

[0346] 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).

[0347] Example 33: Synthesis of Compound 33

[0348]

[0349] 33 (n≈45)

[0350] Molecular formula: C 122 H 233 D9O 50

[0351] Molecular weight: 2515

[0352] APCI-MS: m / z 2516 [M+H] +

[0353] 1H-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).

[0354] Example 34: Synthesis of Compound 34

[0355]

[0356] 34 (n≈45)

[0357] Molecular formula: C 122 H 233 D9O 50

[0358] Molecular weight: 2515

[0359] APCI-MS: m / z 2516 [M+H] +

[0360] 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).

[0361] Example 35: Synthesis of Compound 35

[0362]

[0363] 35 (n≈45)

[0364] Molecular formula: C 122 H 233 D9O 50

[0365] Molecular weight: 2515

[0366] APCI-MS: m / z 2516 [M+H] +

[0367] 1H-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).

[0368] Example 36: Synthesis of Compound 36

[0369]

[0370] 36 (n≈45)

[0371] Molecular formula: C 122 H 234 D8O 50

[0372] Molecular weight: 2514

[0373] APCI-MS: m / z 2515 [M+H] +

[0374] 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).

[0375] Example 37: Synthesis of Compound 37

[0376]

[0377] 37 (n≈45)

[0378] Molecular formula: C 122 H 234 D8O 50

[0379] Molecular weight: 2514

[0380] APCI-MS: m / z 2515 [M+H] +

[0381] 1H-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).

[0382] Example 38: Synthesis of Compound 38

[0383]

[0384] 38 (n≈45)

[0385] Molecular formula: C 122 H 234 D8O 50

[0386] Molecular weight: 2514

[0387] APCI-MS: m / z 2515 [M+H] +

[0388] 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).

[0389] Example 39: Synthesis of Compound 39

[0390]

[0391] 39 (n≈45)

[0392] Molecular formula: C 122 H 234 D8O 50

[0393] Molecular weight: 2514

[0394] APCI-MS: m / z 2515 [M+H] +

[0395] 1H-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).

[0396] Example 40: Synthesis of Compound 40

[0397]

[0398] 40 (n≈45)

[0399] Molecular formula: C 122 H 233 D9O 50

[0400] Molecular weight: 2515

[0401] APCI-MS: m / z 2516 [M+H] +

[0402] 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).

[0403] Example 41: Synthesis of Compound 41

[0404]

[0405] 41 (n≈45)

[0406] Molecular formula: C 122 H 235 D7O 50

[0407] Molecular weight: 2513

[0408] APCI-MS: m / z 2514 [M+H] +

[0409] 1H-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).

[0410] Example 42: Synthesis of Compound 42

[0411]

[0412] 42 (n≈45)

[0413] Molecular formula: C 122 H 235 D7O 50

[0414] Molecular weight: 2513

[0415] APCI-MS: m / z 2514 [M+H] +

[0416] 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).

[0417] Example 43: Synthesis of Compound 43

[0418]

[0419] 43 (n≈45)

[0420] Molecular formula: C 122 H 235 D7O 50

[0421] Molecular weight: 2513

[0422] APCI-MS: m / z 2514 [M+H] +

[0423] 1H-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).

[0424] Example 44: Synthesis of Compound 44

[0425]

[0426] 44 (n≈45)

[0427] Molecular formula: C 122 H 235 D7O 50

[0428] Molecular weight: 2513

[0429] APCI-MS: m / z 2514 [M+H] +

[0430] 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).

[0431] Example 45: Synthesis of Compound 45

[0432]

[0433] 45 (n≈45)

[0434] Molecular formula: C 122 H 235 D7O 50

[0435] Molecular weight: 2513

[0436] APCI-MS: m / z 2514 [M+H] +

[0437] 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).

[0438] Example 46: Synthesis of Compound 46

[0439]

[0440] 46 (n≈45)

[0441] Molecular formula: C 122 H 235 D7O 50

[0442] Molecular weight: 2513

[0443] APCI-MS: m / z 2514 [M+H] +

[0444] 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).

[0445] Example 47: Synthesis of Compound 47

[0446]

[0447] 47 (n≈45)

[0448] Molecular formula: C 122 H 236 D6O 50

[0449] Molecular weight: 2512

[0450] APCI-MS: m / z 2513 [M+H] +

[0451] 1H-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).

[0452] Example 48: Synthesis of Compound 48

[0453]

[0454] 48 (n≈45)

[0455] Molecular formula: C 122 H 236 D6O 50

[0456] Molecular weight: 2512

[0457] APCI-MS: m / z 2513 [M+H] +

[0458] 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).

[0459] Example 49: Synthesis of Compound 49

[0460]

[0461] 49 (n≈45)

[0462] Molecular formula: C 122 H 236 D6O 50

[0463] Molecular weight: 2512

[0464] APCI-MS: m / z 2513 [M+H] +

[0465] 1H-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).

[0466] Example 50: Synthesis of Compound 50

[0467]

[0468] 50 (n≈45)

[0469] Molecular formula: C 122 H 236 D6O 50

[0470] Molecular weight: 2512

[0471] APCI-MS: m / z 2513 [M+H] +

[0472] 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).

[0473] Example 51: Synthesis of Compound 51

[0474]

[0475] 51 (n≈45)

[0476] Molecular formula: C 122 H 236 D6O 50

[0477] Molecular weight: 2512

[0478] APCI-MS: m / z 2513 [M+H] +

[0479] 1H-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).

[0480] Example 52: Synthesis of Compound 52

[0481]

[0482] 52 (n≈45)

[0483] Molecular formula: C 122 H 236 D6O 50

[0484] Molecular weight: 2512

[0485] APCI-MS: m / z 2513 [M+H] +

[0486] 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).

[0487] Example 53: Synthesis of Compound 53

[0488]

[0489] 53 (n≈45)

[0490] Molecular formula: C 122 H 237 D5O 50

[0491] Molecular weight: 2511

[0492] APCI-MS: m / z 2512 [M+H] +

[0493] 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).

[0494] Example 54: Synthesis of Compound 54

[0495]

[0496] 54 (n≈45)

[0497] Molecular formula: C 122 H 237 D5O 50

[0498] Molecular weight: 2511

[0499] APCI-MS: m / z 2512 [M+H] +

[0500] 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).

[0501] Example 55: Synthesis of Compound 55

[0502]

[0503] 55 (n≈45)

[0504] Molecular formula: C 122 H 237 D5O 50

[0505] Molecular weight: 2511

[0506] APCI-MS: m / z 2512 [M+H] +

[0507] 1H-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).

[0508] Example 56: Synthesis of Compound 56

[0509]

[0510] 56 (n≈45)

[0511] Molecular formula: C 122 H 237 D5O 50

[0512] Molecular weight: 2511

[0513] APCI-MS: m / z 2512 [M+H] +

[0514] 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).

[0515] Example 57: Synthesis of Compound 57

[0516]

[0517] 57 (n≈45)

[0518] Molecular formula: C 122 H 237 D5O 50

[0519] Molecular weight: 2511

[0520] APCI-MS: m / z 2512 [M+H] +

[0521] 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).

[0522] Example 58: Synthesis of Compound 58

[0523]

[0524] 58 (n≈45)

[0525] Molecular formula: C 122 H 238 D4O 50

[0526] Molecular weight: 2510

[0527] APCI-MS: m / z 2511 [M+H] +

[0528] 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).

[0529] Example 59: Synthesis of Compound 59

[0530]

[0531] 59 (n≈45)

[0532] Molecular formula: C 122 H 238 D4O 50

[0533] Molecular weight: 2510

[0534] APCI-MS: m / z 2511 [M+H] +

[0535] 1H-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).

[0536] Example 60: Synthesis of Compound 60

[0537]

[0538] 60 (n≈45)

[0539] Molecular formula: C 122 H 238 D4O 50

[0540] Molecular weight: 2510

[0541] APCI-MS: m / z 2511 [M+H] +

[0542] 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).

[0543] Example 61: Synthesis of Compound 61

[0544]

[0545] 61 (n≈45)

[0546] Molecular formula: C 122 H 238 D4O 50

[0547] Molecular weight: 2510

[0548] APCI-MS: m / z 2511 [M+H] +

[0549] 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).

[0550] Example 62: Synthesis of Compound 62

[0551]

[0552] 62 (n≈45)

[0553] Molecular formula: C 122 H 238 D4O 50

[0554] Molecular weight: 2510

[0555] APCI-MS: m / z 2511 [M+H] +

[0556] 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).

[0557] Example 63: Synthesis of Compound 63

[0558]

[0559] 63 (n≈45)

[0560] Molecular formula: C 122 H 239 D3O 50

[0561] Molecular weight: 2509

[0562] APCI-MS: m / z 2510 [M+H] +

[0563] 1H-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).

[0564] Example 64: Synthesis of Compound 64

[0565]

[0566] 64 (n≈45)

[0567] Molecular formula: C 122 H 239 D3O 50

[0568] Molecular weight: 2509

[0569] APCI-MS: m / z 2510 [M+H] +

[0570] 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).

[0571] Example 65: Synthesis of Compound 65

[0572]

[0573] 65 (n≈45)

[0574] Molecular formula: C 122 H 239 D3O 50

[0575] Molecular weight: 2509

[0576] APCI-MS: m / z 2510 [M+H] +

[0577] 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).

[0578] Example 66: Synthesis of Compound 66

[0579]

[0580] 66 (n≈45)

[0581] Molecular formula: C 122 H 239 D3O 50

[0582] Molecular weight: 2509

[0583] APCI-MS: m / z 2510 [M+H] +

[0584] 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).

[0585] Example 67: Synthesis of Compound 67

[0586]

[0587] 67 (n≈45)

[0588] Molecular formula: C 122 H 238 D4O 50

[0589] Molecular weight: 2510

[0590] APCI-MS: m / z 2511 [M+H] +

[0591] 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).

[0592] Example 69: Synthesis of Compound 68

[0593]

[0594] 68 (n≈45)

[0595] Molecular formula: C 122 H 238 D4O 50

[0596] Molecular weight: 2510

[0597] APCI-MS: m / z 2511 [M+H] +

[0598] 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).

[0599] Example 69: Synthesis of Compound 69

[0600]

[0601] 69 (n≈45)

[0602] Molecular formula: C 122 H 238 D4O 50

[0603] Molecular weight: 2510

[0604] APCI-MS: m / z 2511 [M+H] +

[0605] 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).

[0606] Example 70: Synthesis of Compound 70

[0607]

[0608] 70 (n≈45)

[0609] Molecular formula: C 122 H 238 D4O 50

[0610] Molecular weight: 2510

[0611] APCI-MS: m / z 2511 [M+H] +

[0612] 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).

[0613] Example 71: Synthesis of Compound 71

[0614]

[0615] 71 (n≈45)

[0616] Molecular formula: C 122 H 238 D4O 50

[0617] Molecular weight: 2510

[0618] APCI-MS: m / z 2511 [M+H] +

[0619] 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).

[0620] Example 72: Synthesis of Compound 72

[0621]

[0622] 72 (n≈45)

[0623] Molecular formula: C 122 H 240 D2O 50

[0624] Molecular weight: 2508

[0625] APCI-MS: m / z 2509 [M+H] +

[0626] 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).

[0627] Example 73: Synthesis of Compound 73

[0628]

[0629] 73 (n≈45)

[0630] Molecular formula: C 122 H 240 D2O 50

[0631] Molecular weight: 2508

[0632] APCI-MS: m / z 2509 [M+H] +

[0633] 1H-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).

[0634] Example 74: Synthesis of Compound 74

[0635]

[0636] 74 (n≈45)

[0637] Molecular formula: C 122 H 240 D2O 50

[0638] Molecular weight: 2508

[0639] APCI-MS: m / z 2509 [M+H] +

[0640] 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).

[0641] Example 75: Synthesis of Compound 75

[0642]

[0643] 75 (n≈45)

[0644] Molecular formula: C 122 H 240 D2O 50

[0645] Molecular weight: 2508

[0646] APCI-MS: m / z 2509 [M+H] +

[0647] 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).

[0648] Example 76: Synthesis of Compound 76

[0649]

[0650] 76 (n≈45)

[0651] Molecular formula: C 122 H 240 D2O 50

[0652] Molecular weight: 2508

[0653] APCI-MS: m / z 2509 [M+H] +

[0654] 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).

[0655] Example 77: Synthesis of Compound 77

[0656]

[0657] 77 (n≈45)

[0658] Molecular formula: C 122 H 240 D2O 50

[0659] Molecular weight: 2508

[0660] APCI-MS: m / z 2509 [M+H] +

[0661] 1 H-NMR (300MHz, DMSO-d6): 5.70(q, 1H, J1=9.6Hz, J2=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).

[0662] Example 78: Synthesis of Compound 78

[0663]

[0664] 78 (n≈45)

[0665] Molecular formula: C 122 H 241 D1O 50

[0666] Molecular weight: 2507

[0667] APCI-MS: m / z 2508 [M+H] +

[0668] 1 H-NMR (300MHz, DMSO-d6): 5.70(q, 1H, J1=9.6Hz, J2=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).

[0669] Example 79: Synthesis of Compound 79

[0670]

[0671] 79 (n≈45)

[0672] Molecular formula: C 122 H 241 D1O 50

[0673] Molecular weight: 2507

[0674] APCI-MS: m / z 2508 [M+H] +

[0675] 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).

[0676] Example 80: Synthesis of Compound 80

[0677]

[0678] 80 (n≈45)

[0679] Molecular formula: C 122 H 241 D1O 50

[0680] Molecular weight: 2507

[0681] APCI-MS: m / z 2508 [M+H] +

[0682] 1 H-NMR(300MHz, DMSO-d6): 5.70(q, 1H, J1=9.1Hz, J2=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).

[0683] Example 81: Synthesis of Compound 81

[0684]

[0685] 81 (n≈45)

[0686] Molecular formula: C 122 H 241 D1O 50

[0687] Molecular weight: 2507

[0688] APCI-MS: m / z 2508 [M+H] +

[0689] 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).

[0690] Example 82: Synthesis of Compound 82

[0691]

[0692] 82 (n≈45)

[0693] Molecular formula: C 122 H 241 D1O 50

[0694] Molecular weight: 2507

[0695] APCI-MS: m / z 2508 [M+H] +

[0696] 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).

[0697] Example 83

[0698] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0699] Cationic lipids, as shown in structural formulas E and F, DSPC, cholesterol, and deuterated 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 total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, mRNA was diluted to 0.15 mg / mL in 10 to 50 mL of citrate buffer (pH=4). Using a syringe pump, the ethanol solution of lipids was mixed with the aqueous mRNA solution at a ratio of approximately 1:5 to 1:3 (volume / volume) at a total flow rate of at least 10 mL / min. The ethanol was then removed, and the external buffer was replaced with PBS for 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 quasi-elastic light scattering using a Malvern Zetasizer Nano ZS, was approximately 65–105 nm in diameter, and in some cases, approximately 75–100 nm.

[0700]

[0701] Structural E

[0702] R1, R2, R3, R4, R5, R6, R7, and R8 are each two independent hydrogen isotopes (including hydrogen and deuterium), specifically represented by combinations of "HH", "HD", and "DD".

[0703]

[0704] Structural F

[0705] R1, R2, R3, R4, R5, R6, R7, and R8 are each two independent hydrogen isotopes (including hydrogen and deuterium), specifically represented by combinations of "HH", "HD", and "DD".

[0706] The study was conducted in 6-8 week old female C57BL / 6 mice and 8-10 week old CD-1 mice, following guidelines established by the State Science and Technology Commission. Different doses of mRNA lipid nanoparticles were administered systemically via tail vein injection, and the animals were euthanized at specific time points (e.g., 5 hours) after administration. Liver and spleen were collected in pre-weighed tubes, their weight determined, and immediately rapidly frozen in liquid nitrogen and stored at -80°C until analysis.

[0707] For liver, approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). Add 1 / 4" ceramic beads (MP Biomedicals) to each tube and add 500 μL of Glo lysis buffer-GLB (Promega, Madison WI) equilibrated to room temperature to the liver tissue. Homogenize the liver tissue at 2 × 6.0 m / s for 15 seconds using a FastPrep24 instrument (MPBiomedicals). Incubate the homogenate at room temperature for 5 minutes, then dilute 1:4 in GLB and evaluate using a SteadyGlo luciferase assay system (Promega). Specifically, react 50 μL of diluted tissue homogenate with 50 μL of SteadyGlo substrate, shake for 10 seconds, incubate for 5 minutes, and then quantify using a SpectraMAX_L chemiluminescence microplate reader (Meigu Molecular Instruments (Shanghai) Co., Ltd.). The amount of protein measured was determined using a BCA protein quantification kit (Shanghai Esee Medical Technology Co., Ltd.). The relative luminescence units (RLU) were then normalized to the total μg of the measured protein. To convert RLU to μg... A standard curve was generated using QuantiLum recombinant luciferase (Promega).

[0708] FLuc mRNA (L-6107) from Trilink Biotechnologies expresses the luciferase protein, originally isolated from the firefly (Photinus pyralis). Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability. It emits bioluminescence in the presence of its substrate, luciferin. This capped and polyadenylated mRNA is completely replaced by 5-methylcytidine and pseudouridine.

[0709] Example 84

[0710] Determination of pKa of the prepared lipids

[0711] The pKa of the formulated cationic lipids is correlated with the effectiveness of LNPs for nucleic acid delivery. The preferred pKa range is 5–7. The pKa of each cationic lipid in the lipid nanoparticles was determined using fluorescence analysis based on 2-(p-toluidine)-6-carboxylic acid (TNS). As described in Example 3, lipid nanoparticles containing cationic lipids / DSPC / cholesterol / PEG lipids (50 / 10 / 38 / 2 mol%) at a concentration of 0.4 mM total lipids in PBS were prepared using an ordered method. A 100 μM stock solution of TNS was prepared in distilled water. The vesicles were diluted to a 2 mL buffer solution containing 24 μM lipids, which contained 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH of 2.5–11. Equal portions of TNS solution were added to produce a final concentration of 1 μM, and after vortex mixing, fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 fluorescence spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. S-shaped best-fit analysis was applied to the fluorescence data, and pKa was measured as the pH at which the half-maximum fluorescence intensity was produced.

[0712] Example 85

[0713] The efficacy of lipid nanoparticle formulations containing various cationic lipids was determined using a rodent model with in vivo luciferase mRNA expression.

[0714] For comparative purposes, as described in Example 83, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using an ordered mixing method. The lipid nanoparticles were formulated using the following molar ratio: 50% cationic lipid / 10% distearate phosphatidylcholine (DSPC) / 38% cholesterol / 2% deuterated PEG lipid. As described in Example 83, relative activity was determined by measuring luciferase expression in the liver 5 hours after administration via tail vein injection. The activity was compared at doses of 0.3 and 1.0 mg mRNA / kg, expressed as ng luciferase / g liver as measured 5 hours after administration as described in Example 83. The results of Examples 83 and 84 are shown in Table 2.

[0715] Table 2. Efficacy studies of mRNA compositions containing novel PEG lipids.

[0716]

[0717]

[0718]

[0719] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0720] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the disclosed patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A deuterated polyethylene glycol lipid compound, which is a compound of one of the following structures: ###0001### ###0002### wherein n = 45. Compound 1: ; Compound 3: ; Compound 4: ; Compound 5: ; Compound 7: ; 2. A method of preparing the deuterated polyethylene glycol lipid compound of claim 1, comprising the following steps: 1) under the action of an acyl chloride reagent, a deuterated general formula compound dPEG-A-1 first generates an acyl chloride, then under the action of ammonia, an amidation reaction is carried out to obtain a deuterated general formula intermediate dPEG-A-3; 2) under the action of a reducing agent, the deuterated general formula intermediate dPEG-A-3 is subjected to a reduction reaction to obtain a deuterated general formula intermediate dPEG-A-4; 3) according to the method of step 1), the deuterated general formula intermediate dPEG-A-4 is subjected to an amidation reaction with a general formula intermediate dPEG-A-1 to obtain a deuterated general formula intermediate dPEG-A-5; 4) according to the method of step 2), the deuterated general formula intermediate dPEG-A-5 is subjected to a reduction reaction to obtain a deuterated general formula intermediate dPEG-A-6.

3. A deuterated polyethylene glycol lipid compound, which is a compound of one of the following structures: ###0003### ###0004### wherein n = 45.

4. A method of preparing the deuterated methyl polyethylene glycol compound of claim 3, comprising the following steps: 1) in an organic solvent, a deuterated general formula compound dPEG-B-1 is subjected to a substitution reaction with a deuterated methyl polyethylene glycol dPEG-B-2 to obtain a deuterated general formula intermediate dPEG-B-3; 2) under basic catalysis, the deuterated general formula intermediate dPEG-B-3 is subjected to an epoxy hydrolysis reaction to obtain a deuterated general formula intermediate dPEG-B-4; 3) under the action of a condensing agent, the deuterated general formula intermediate dPEG-B-4 and a deuterated general formula compound dPEG-B-5 are subjected to an esterification reaction to obtain a deuterated general formula compound dPEG-B.

5. A composition of a compound as claimed in claim 1 or 3 and a therapeutic and / or prophylactic agent.

6. A composition as claimed in claim 5, further comprising one or more excipients selected from neutral lipids, steroids and cationic lipids.

7. A composition as claimed in claim 6, wherein the neutral lipids in the component are selected from one or more of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM). 5) amidation of the deuterated intermediate dPEG-A-6 with the deuterated compound dPEG-A-7 in an organic solvent in the presence of a base to yield the deuterated polyethylene glycol lipid compound of general formula; .

8. A composition as claimed in claim 7, wherein the neutral lipid is DSPC. Compound 27: ; Compound 30: ; Compound 32: ; Compound 34: ; Compound 37: ; 9. A composition as claimed in claim 6, wherein the steroids in the component are selected from one or more of the following: cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol. ​ ​ ​ ​ 。 ​ ​ ​ ​ ​ 10. The composition of claim 9, wherein the steroid is cholesterol.

11. The composition of claim 6, wherein the cationic lipid in the component is selected from one or more of the following: 4-(N,N-dimethylamino)butan-1-ol (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-((decyloxy)hexyl)amino)octanoate (SM-102), ((4-hydroxybutyl)azan-ethyl)bis(hexan-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 4-(N-2-(N,N-di(2-hydroxydodecyl)ethylamino)-1-N(2-N(2-ethylamino-(2-(N,N-di(2-hydroxydodecyl))-2-hydroxydodecyl)ethylamino)piperazine (C12-200).

12. The composition of claim 11, wherein the cationic lipid is ((4-hydroxybutyl)azan-ethyl)bis(hexan-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-((decyloxy)hexyl)amino)octanoate (SM-102).

13. The composition of claim 6, wherein the cationic lipid is ((4-hydroxybutyl)azan-ethyl)bis(hexan-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) deuterated analog, heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-((decyloxy)hexyl)amino)octanoate (SM-102) deuterated analog as shown in the following structures C, D; Structure Formula C Structure Formula C R1, R2, R3, R4, R5, R6, R7, R8 are each independently two "hydrogen" isotopes, specifically a combination of "HH", "HD", and "DD". Structural D R1, R2, R3, R4, are each independently two "hydrogen" isotopes, specifically a combination of "HH", "HD", and "DD".

14. The composition of any one of claims 6-13, wherein the therapeutic and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

15. The composition of claim 14, wherein the therapeutic and / or prophylactic agent is a nucleic acid.

16. The composition of claim 15, wherein the nucleic acid is an RNA.

17. The composition of claim 16, wherein the nucleic acid is selected from one or more of the group consisting of: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA.

18. The composition of claim 17, wherein the RNA is an mRNA.

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