CN115724806B - Cationic lipid compounds - Google Patents
Cationic lipid compounds Download PDFInfo
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Abstract
本发明涉及脂质化合物，其可以单独使用，或者与其他脂质组分例如中性脂质、带点脂质、类固醇和/或它们的类似物、和/或聚合物缀合的脂质联合使用，以形成用于递送治疗剂和/或预防剂的脂质纳米颗粒。在一些实例中，将脂质纳米颗粒用于递送核酸，例如信使RNA和/或反义RNA。还提供了使用这类脂质纳米颗粒来治疗和/或预防各种疾病的方法。在一个实施方案中，提供了具有下式(I)结构的化合物：或其盐或其异构体或其N‑氧化物，其中R1如文中所定义还提供了包括一种或多种前述结构式(I)的化合物和治疗剂和/或预防剂的药物组合物。在一些实施方案中，药物组合物还包括选自中性脂质、带电脂质、类固醇和聚合物缀合的脂质的一种或多种组分。这类组合物对于形成用于递送治疗剂和/或预防剂的脂质纳米颗粒是有用的。在其他实施方案中，本发明提供了向有需要的受试者施用治疗剂和/或预防剂的方法，所述方法包括制备包含结构式(I)的化合物的脂质纳米颗粒物和治疗剂和/或预防剂的药物组合，并向受试者递送组合物。The present invention relates to lipid compounds, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids and/or their analogs, and/or polymer-conjugated lipids to form lipid nanoparticles for delivering therapeutic and/or preventive agents. In some examples, lipid nanoparticles are used to deliver nucleic acids, such as messenger RNA and/or antisense RNA. Methods for treating and/or preventing various diseases using such lipid nanoparticles are also provided. In one embodiment, a compound having the structure of the following formula (I) is provided: Or its salt or its isomer or its N-oxide, wherein R 1 is as defined in the text Also provided are pharmaceutical compositions comprising one or more compounds of the aforementioned structural formula (I) and therapeutic and/or preventive agents. In some embodiments, the pharmaceutical composition also includes one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions are useful for forming lipid nanoparticles for delivering therapeutic and/or preventive agents. In other embodiments, the present invention provides a method for administering a therapeutic and/or preventive agent to a subject in need thereof, the method comprising preparing a lipid nanoparticle comprising a compound of structural formula (I) and a pharmaceutical combination of a therapeutic and/or preventive agent, and delivering the composition to the subject.
Description
Technical Field
The present invention provides cationic lipids that can be used in combination with other lipid components (e.g., neutral lipids, steroids, and polymer conjugated lipids) to form a nucleic acid mRNA lipid nanoparticle composition for delivery of one or more therapeutic and/or prophylactic agents to and/or production of polypeptides in mammalian cells or organs. In addition to lipids, the lipid nanoparticle compositions of the present invention may also include one or more cationic and/or ionizable amino lipids, neutral lipids including polyunsaturated lipids, polymer-conjugated lipids, steroids, and/or therapeutic and/or prophylactic agents in specific proportions.
Background
Efficient targeted delivery of biologically active substances such as small molecule drugs, proteins and nucleic acids presents a long-lasting 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.
It has been demonstrated 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 transport vehicles. These compositions generally comprise one or more "cationic" lipids, including neutral lipids (e.g., phospholipids) including polyunsaturated lipids, structural lipids (e.g., steroids), and/or polyethylene glycol-containing lipids (polymer conjugated lipids). Cationic lipids include amine-containing lipids such as can be easily protonated.
However, the use of oligonucleotides in therapeutic environments is currently faced with two problems. First, free RNA is readily digested by nucleases in plasma. Second, free RNA has limited ability to enter the intracellular compartment where relevant translation mechanisms exist. Lipid nanoparticles formed from cationic lipids with other lipid components (e.g., neutral lipids, cholesterol, PEG, pegylated lipids, and oligonucleotides) have been used to prevent RNA degradation in plasma and to promote cellular uptake of the oligonucleotides.
There remains a need for improved cationic lipids and lipid nanoparticles for delivery of oligonucleotides. The improved lipid nanoparticle will 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 so that patient treatment at an effective dose of nucleic acid does not create unacceptable toxicity and/or risk to the patient. The present invention provides these and related advantages.
Disclosure of Invention
The present invention provides the following compounds and methods involving these compounds:
in a first aspect, the present invention relates to a compound of the following structural formula (I):
Or an N-oxide thereof, or a salt or isomer thereof.
Wherein R 1、R2、R3 and R 4 in structural formula "I" are each independently a combination of 2 "hydrogen" isotopes (including the isotopes "protium" and "deuterium"), and are specifically represented by a combination of "HH", "HD" and "DD";
R 1 and R 2 are independently selected from any one of the group consisting of "HH", "HD" and "DD";
R 3 and R 4 are independently selected from any one of the group consisting of "HH, HD", "HD, HH", "HH, DD", "DD, HH", "HD, DD" and "DD, DD";
R 1、R2、R3, and R 4 are independent, but cannot be both "HH", i.e., contain at least one "HD";
In various embodiments, the compounds have one of the structures shown in Table 1 below
Table 1 representative Compounds
In some embodiments, compositions are provided that include any one or more of the compounds of structural formula (I) and a therapeutic and/or prophylactic agent.
In some embodiments, compositions are provided that include any one or more of the compounds of structure (I) and a therapeutic and/or prophylactic agent. In some embodiments, the composition comprises any one of the compounds of structure (I) and a therapeutic and/or prophylactic agent and one or more excipients selected from neutral lipids, steroids, and polymer conjugated lipids. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the composition.
In some embodiments, the neutral lipid is selected from the group consisting of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (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-phosphorylcholine (DSPC).
In some embodiments, the steroid is selected from cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the preferred steroid is cholesterol.
In some embodiments, the pegylated lipid is 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG)
In some embodiments, the composition ratio ranges from about 10 to 60 mole% of the compound, from about 0 to 30 mole% neutral lipid, from about 10 to 55 mole% steroid, and from about 0 to 10 mole% polymer conjugated lipid.
In some embodiments of the foregoing compositions, 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 various other embodiments, the invention relates to methods of administering a therapeutic and/or prophylactic agent to a subject in need thereof, comprising preparing or providing any of the compositions described above and administering the composition to the subject.
For administration purposes, the compounds of the present invention (typically in the form of lipid nanoparticles conjugated to therapeutic and/or prophylactic agents) may be administered as bulk drugs or may be formulated as pharmaceutical compositions. The pharmaceutical compositions of the present invention comprise a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents or excipients. The compounds of structure (I) are effective to form lipid nanoparticles and deliver therapeutic and/or prophylactic agents. The 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 mode of administration for agents of similar utility. The pharmaceutical compositions of the invention may be formulated as solid, semi-solid, liquid or gaseous forms of preparations 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 composition of the present invention is 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 the tablet may be a single dosage unit and the container of the compound in aerosol form of the invention 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 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 composition 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 suitable for administration by inhalation.
When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein solid or liquid forms are considered herein to include semi-solid, semi-liquid, suspension and gel forms.
As solid compositions for oral administration, pharmaceutical compositions may be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, and the like. Such solid compositions will typically contain one or more inert diluents or edible carriers. In addition, one or more of binders such as gelatin, cellulose, etc., excipients such as lactose, etc., disintegrants such as alginic acid, etc., lubricants such as magnesium stearate, etc., glidants such as silica gel, etc., sweeteners such as sucrose or saccharin, flavoring agents such as peppermint, etc., and coloring agents may be present.
When the pharmaceutical composition is in the form of a capsule, it may contain a liquid carrier other than materials of the type described above, such as polyethylene glycol or 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 flash comprise, in addition to the compounds of the present invention, one or more of a sweetener, preservative, coloring/coloring agent and an odorant. In the composition to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers 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 sterile diluents such as water for injection, saline solutions, preferably physiological saline, ringer's solution, isotonic sodium chloride, non-volatile oils such as synthetic mono-or diglycerides which may be used as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol or other solvents, antimicrobial agents such as nipagin Jin Jiacu and the like, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylene tetracetic acid, buffers such as acetate, twisted or phosphate salts, and agents for modulating tonicity such as sodium chloride or dextrose, agents for use as cryoprotectants such as sucrose or trehalose. Parenteral formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic. Saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.
The pharmaceutical compositions of the invention may be intended for topical administration, in which case the carrier may suitably comprise a solution base, an emulsion base, an ointment base or a suspoemulsion base. The matrix may comprise one or more of petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohols, and emulsifiers and stabilizers. The thickener may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoretic device.
The pharmaceutical compositions of the present invention may include a variety of materials that modify the physical form of the solid or liquid dosage unit. The composition may include a material that forms a coating shell around the active ingredient. The materials forming the coating shell are generally inert and may be sugar, shellac, and other enteric coating agents. Or the active ingredient may be encapsulated in a gelatin capsule.
The pharmaceutical compositions of the present invention in solid or liquid form may include agents that bind to the compounds of the present invention and thereby facilitate delivery of the compounds. Suitable agents that may act with this capability include monoclonal or polyclonal antibodies or proteins.
The pharmaceutical composition of the present invention may be composed of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems, from systems of colloidal nature to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system for dispersing the active ingredient. Aerosols of the compounds of the invention may be delivered in a single phase, a biphasic system or a triphasic system in order to deliver the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. The preferred aerosols can be determined by one skilled in the art 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 lipid nanoparticles of the present invention in solution with sterile distilled water or other carrier. Surfactants may be added to promote the formation of a uniform 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 being used, the metabolic stability and length of action of that therapeutic agent, the age, body weight, general health, sex, and diet of the subject, the mode and time of administration, the rate of excretion, drug combination, the severity of the particular case, and the like.
The compositions of the present invention may also be administered simultaneously, before or after the administration of one or more other therapeutic agents. Such combination therapies include administration of a single pharmaceutical dosage formulation of the present composition and one or more additional active agents, as well as administration of the present composition and each of the active agents in its own separate pharmaceutical dosage formulation. For example, the compositions of the invention and the other active agent may be administered together to a subject in a single oral dosage composition (e.g., a tablet or capsule), or the individual agents may be administered in different oral dosage formulations. When different dosage formulations are used, 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 relative to each other, with the understanding that combination therapy includes all of these dosage regimens.
The structural modification and design of the deuterated cationic lipid compound realize more advantageous physicochemical properties including more proper pKa and better chemical stability, can be used for the mRNA nano liposome composition, can realize more effective combination and delivery of ionic nucleic acid medicaments, and has more stable chemical structure, thereby being convenient for synthesis and favorable development as pharmaceutical excipients.
Methods for preparing the above compounds and compositions are described below and/or are known in the art.
Those skilled in the art will recognize that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino and carboxylic acid. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl groups, tetrahydrofuranyl groups, benzyl groups, and the like. Suitable protecting groups for the amino group include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for carboxylic acids include hydrocarbyl, aryl or arene esters. Protecting groups may be added or removed according to standard techniques known to those skilled in the art and described herein.
Those skilled in the art will also recognize that while such protected derivatives of the compounds of the present invention may not be pharmaceutically active therefrom, 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 thus be described as "prodrugs". Prodrugs of the compounds of the present invention are therefore included within the scope of the present invention.
Furthermore, all compounds of the invention which are present in the free base or free acid form can be converted into their pharmaceutically acceptable salts by treatment with suitable inorganic or organic bases or acids according to methods known to the person skilled in the art. Salts of the compounds of the invention may be formed by conversion to their free base or acid by standard techniques.
The following examples are provided for purposes of illustration and not limitation.
All solvents and reagents used in the following examples are commercially available and used as such unless otherwise indicated.
The procedure described below can be used to synthesize compound I in table 1.
The following abbreviations are used herein:
DCM: dichloromethane
EtOH-ethanol
K 2CO3 sodium carbonate
EA ethyl acetate
DMSO-dimethyl sulfoxide
Detailed Description
Example 1:
Representative route
Synthesis of Compound 8D
1) Synthesis of Compound C
Into a 25ml double-necked flask, NH3 (1 g,.7M in MeOH) and THF were added, bromoethanol (4 eq.) was added, and the mixture was reacted at 65℃for 6 hours, and filtered to obtain 1.5g of the objective compound as an off-white solid, the yield was 75%
Chemical formula C6H15NO3
Molecular weight 149.19
LC-MS:m/z 150[M+H]
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)7.31(s,1H)4.16(3H,s),3.99(t,6H,J=6)3.48(t,6H,J=8).
2) Synthesis of Compound 8D-4
To a 25ml double flask was added 1.5g of 8D-3 (10.05 mmol), 15ml of chloroform, SOCl2 (13.07 mmol,1.6 g) was added, stirred at room temperature for 3 hours, pH=8-9 was adjusted with sodium bicarbonate, extracted 1 time with 30ml of DCM, washed once with 6ml of saline, the organic phase was concentrated to give a yellow liquid, 10ml of THF was added, 1.3 equivalents of hydrochloric acid was added, salted out, stirred at room temperature for 5 hours, filtered to give 1.7g of a white solid, yield 70%.
Chemical formula C 6H12NCl3. HCl
Molecular weight 241.02
LC-MS:m/z 205[M+H]
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)7.25(s,1H),3.82(t,6H,J=4)3.54(t,6H,J=7).
3) Synthesis of Compound 8D-6
Into a 25ml two-necked flask, 1.7g of 8D-4 (6.45 mmol) was added, 20ml of toluene was added, 8D-5 (14.84 mmol,0.95 g) was added, KOH (45.16 mmol,2.9 g) was added, the mixture was stirred at 115℃for 8 hours, after completion of the reaction, the mixture was cooled to room temperature, extracted 3 times with 25X 3ml of EA, the organic phase was combined and washed once with 10ml of brine, the organic phase was concentrated to give a yellow liquid, 20ml of THF was added, 3.5 equivalents of hydrochloric acid was added, cheng Yanxi crystals were stirred at room temperature for 8 hours, 1.29g of a white solid was obtained by filtration, and the yield was 60%
Chemical formula C 10H17D8N5
Molecular weight 223.39
LC-MS:m/z 224[M+H]
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)8.44(s,6H),3.32(s,2H,)2.53(m,2H),2.39(m,2H)2.22(m,6H).
4) Synthesis of Compound 8D
To a 25ml two-port flask was added 0.9g of 8D-6base (4.03 mmol), 8D-6 (22.16 mmol,4.08 g) was added and warmed to 80-90℃and stirred for 5 days, after completion of the reaction, cooled to room temperature, and the desired product was isolated by silica gel chromatography (gradient elution DCM to 175:22:3DCM/MeOH/NH4OH (aq)) and concentrated to give 1.8g of yellow oil in 40% yield
Chemical formula C 70H137D8N5O5
Molecular weight 1145.01
LC-MS:m/z 1146[M+H]
H NMR(300MHz,DMSO-d6):5.4(s,5H),3.49(m,5H)2.22-2.71(m,20H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 2:
Synthesis of Compound 1
Chemical formula C 70H144DN5O5
Molecular weight 1138.01
Compound 1 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,27H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 3:
synthesis of Compound 2
Chemical formula C 70H144DN5O5
Molecular weight 1138.01
Compound 2 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,27H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 4:
Synthesis of Compound 3
Chemical formula C 70H144DN5O5
Molecular weight 1138.01
Compound 3 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,27H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 5:
Synthesis of Compound 4
Chemical formula C 70H144DN5O5
Molecular weight 1138.01
Compound 4 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,27H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 6:
Synthesis of Compound 5
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 5 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,26H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 7:
synthesis of Compound 6
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 6 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,26H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 8:
Synthesis of Compound 7
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 7 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,26H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 9:
synthesis of Compound 8
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 8 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,26H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 10:
synthesis of Compound 9
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 9 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,26H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 11:
synthesis of Compound 10
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 10 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,26H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 12:
Synthesis of Compound 11
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 11 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,26H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 13:
synthesis of Compound 12
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 12 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,)2.22-2.71(m,26H),1.47(m,10H)1.28(m,80H),0.90(m,10H).
Example 14:
synthesis of Compound 13
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 13 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,26H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 15:
Synthesis of Compound 14
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 14 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,26H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 16:
synthesis of Compound 15
Chemical formula C 70H143D2N5O5
Molecular weight 1139.01
Compound 15 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,26H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 17:
Synthesis of Compound 16
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 16 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 18:
Synthesis of Compound 17
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 17 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 19:
Synthesis of Compound 18
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 18 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 20:
synthesis of Compound 19
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 19 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 21:
Synthesis of Compound 20
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 20 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 22:
Synthesis of Compound 21
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 21 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 23:
synthesis of Compound 22
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 22 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 24:
Synthesis of Compound 23
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 23 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 25:
Synthesis of Compound 24
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 24 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 26:
Synthesis of Compound 25
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 25 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 27:
synthesis of Compound 26
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 26 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 28:
synthesis of Compound 27
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 27 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 29:
Synthesis of Compound 28
Chemical formula C 70H142D3N5O5
Molecular weight 1140.01
Compound 28 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 30:
synthesis of Compound 29
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 29 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 31:
synthesis of Compound 30
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 30 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 32:
Synthesis of Compound 31
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 31 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 33:
Synthesis of Compound 32
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 32 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 34:
synthesis of Compound 33
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 33 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 35:
Synthesis of Compound 34
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 34 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 36:
Synthesis of Compound 35
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 35 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,25H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 37:
Synthesis of Compound 36
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 36 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 38:
synthesis of Compound 37
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 37 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 39:
Synthesis of Compound 38
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 38 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 40:
Synthesis of Compound 39
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 39 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 41:
synthesis of Compound 40
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 40 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 42:
Synthesis of Compound 41
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 41 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 43:
Synthesis of Compound 42
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 42 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 44:
synthesis of Compound 43
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 43 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 45:
Synthesis of Compound 44
Chemical formula C 70H141D4N5O5
Molecular weight 1141.01
Compound 44 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 46:
synthesis of Compound 45
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 45 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 47:
Synthesis of Compound 46
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 46 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 48:
Synthesis of Compound 47
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 47 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 49:
Synthesis of Compound 48
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 48 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 50:
Synthesis of Compound 49
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 49 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 51:
Synthesis of Compound 50
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 50 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 52:
Synthesis of Compound 51
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 51 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 53:
Synthesis of Compound 52
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 53 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,24H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 54:
Synthesis of Compound 53
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 53 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 55:
synthesis of Compound 54
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 54 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 56:
Synthesis of Compound 55
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 55 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 57:
Synthesis of Compound 56
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 56 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 58:
Synthesis of Compound 57
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 57 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 59:
Synthesis of Compound 58
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 58 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 60:
Synthesis of Compound 59
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 59 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 61:
Synthesis of Compound 60
Chemical formula C 70H140D5N5O5
Molecular weight 1142.01
Compound 60 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,23H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 62:
Synthesis of Compound 61
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 61 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 63:
Synthesis of Compound 62
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 62 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 64:
Synthesis of Compound 63
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 63 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 65:
synthesis of Compound 64
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 64 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 66:
synthesis of Compound 65
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 65 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 67:
Synthesis of Compound 66
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 66 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 68:
Synthesis of Compound 67
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Substance 67 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 69:
Synthesis of Compound 68
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 68 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 70:
Synthesis of Compound 69
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 69 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 71:
synthesis of Compound 70
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 70 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 72:
Synthesis of Compound 71
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 71 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 73:
Synthesis of Compound 72
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 72 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 74:
Synthesis of Compound 73
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 73 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 75:
Synthesis of Compound 74
Chemical formula C 70H139D6N5O5
Molecular weight 1143.01
Compound 74 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,22H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 76:
Synthesis of Compound 75
Chemical formula C 70H138D7N5O5
Molecular weight 1144.01
Compound 75 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,21H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 77:
synthesis of Compound 76
Chemical formula C 70H138D7N5O5
Molecular weight 1144.01
Compound 76 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,21H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 78:
Synthesis of Compound 77
Chemical formula C 70H138D7N5O5
Molecular weight 1144.01
Compound 77 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,21H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 79:
Synthesis of Compound 78
Chemical formula C 70H138D7N5O5
Molecular weight 1144.01
Compound 78 can be synthesized according to the representative route described in example 1.
H NMR(300MHz,DMSO-d6):1H NMR(DMSO-d6):δ(ppm)5.4(s,5H),3.49(m,5H,),2.22-2.71(m,21H),1.47(m,10H),1.28(m,80H),0.90(m,10H).
Example 80
Luciferase mRNA in vivo evaluation using lipid nanoparticle compositions
The cationic lipid, DSPC, cholesterol and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38:2 or 48:10:40:2. Lipid Nanoparticles (LNP) were prepared at a weight ratio of total lipid to mRNA of about 10:1 to 30:1. Briefly, mRNA was diluted to 0.15mg/m L in 10ml to 50ml citrate buffer (ph=4). The ethanol solution of the lipid and the aqueous solution of mRNA are mixed in a ratio of about 1:5 to 1:3 (volume/volume) using a syringe pump, and the total flow rate is 10ml/min or more. 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 0.2um pore size. The lipid nanoparticles have a particle size of about 65-105nm in diameter, and in some cases, about 75-100nm in diameter, as determined by quasi-elastic light scattering using Malvern Zetasizer Nano ZS.
According to guidelines established by the national science and technology Committee, studies were performed on female C57BL/6 mice of 6-8 weeks of age and CD-1 mice of 8-10 weeks of age. 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. The livers and spleens were collected in pre-weighed tubes, weighed, immediately flash frozen in liquid nitrogen, and stored at-80 ℃ until used for analysis.
For the liver, about 50mg was cut for analysis in 2mL FastPrep tubes (MP Biomedicals, solon OH). 1/4 "ceramic balls (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 liver tissue. Liver tissue was homogenized at 2x6.0 m/s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). The homogenates were incubated for 5min at room temperature, then 1:4 diluted in GLB, and evaluated using SteadyGlo luciferase assay system (Promega). Specifically, 50uL of diluted tissue homogenate was reacted with 50uL of SteadyGlo substrate, shaken for 10 seconds, followed by incubation for 5 minutes, and then quantified using a spectromax_l chemiluminescent microplate reader (molecular instruments (Shanghai) inc.). The amount of protein assayed was determined by using BCA protein quantification kit (shanghai easy color medical science co.ltd). The Relative Luminosity Units (RLU) were then normalized to the total ug of protein assayed. To convert RLU to μg luciferase, a standard curve was generated with QuantiLum recombinant luciferase (Promega).
The FLuc mRNA from Trilink Biotechnologies (L-6107) will express a luciferase protein, which was originally isolated from firefly (Photinus pyralis). Fluc is commonly used in mammalian cell culture to measure gene expression and cell viability. Which emits bioluminescence in the presence of a substrate luciferin. This capped and polyadenylation mRNA is completely replaced by 5-methylcytidine and pseudouridine.
Example 65
Determination of pKa of formulated lipid
The pKa of the formulated cationic lipid is related to the effect of the LNP used to deliver the nucleic acid. The preferred pKa range is 5 to 7. The pKa of each cationic lipid was determined in lipid nanoparticles using an analysis based on fluorescence of 2- (p-toluidinyl) -6-naphthalene sulfonic acid (TNS). Lipid nanoparticles comprising cationic lipid/DSPC/cholesterol/PEG lipid (50/10/38/2 mol%) at a concentration of 0.4mM total lipid in PBS were prepared using an ordered procedure as described in example 64. TNS was prepared as a 100uM stock solution in distilled water. The vesicles are diluted to contain 24uM lipid in 2mL of buffer solution, wherein the buffer solution contains 10mM HEPES, 10mM MES, 10mM acetic acid and 130mM NaCl, and the pH value is 2.5-11. Aliquots of TNS solution were added to give final concentrations of l uM and after vortexing, fluorescence intensities were measured in a SLM Aminco Series 2 luminescence spectrophotometer at room temperature using excitation and emission wavelengths of 321nm and 445 nm. An sigmoid best fit analysis was applied to the fluorescence data and pKa was measured as pH yielding half maximum fluorescence intensity.
Example 66
Determination of potency of lipid nanoparticle formulations containing various cationic lipids using rodent models of in vivo luciferase mRNA expression
For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the ordered mixing method as described in example 64. Lipid nanoparticles were formulated using a molar ratio of 50% cationic lipid/10% distearoyl phosphatidylcholine (DSPC)/38% cholesterol/2% PEG lipid ("PEG-DMG", i.e., (1- (monomethoxy-polyethylene glycol) -2,3 dimyristoyl glycerol, average PEG molecular weight 2000). The relative activity was determined by measuring luciferase expression in the liver 5 hours after administration via tail vein injection as described in example 64.
Table 2 comparative lipids showing Activity with mRNA
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the disclosure. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (13)
1. A cationic lipid compound which is a compound of one of the following structures:
;
;
;
;
;
。
2. a composition comprising a compound of claim 1 and a therapeutic and/or prophylactic agent.
3. The composition of claim 2, further comprising one or more excipients selected from the group consisting of neutral lipids, steroids, and polymer conjugated lipids.
4. A composition according to claim 3, wherein the neutral lipid is selected from the group consisting of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine and sphingomyelin.
5. The composition of claim 4, wherein the neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine.
6. A composition according to claim 3, wherein the steroid is selected from the group consisting of cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycopersine, ursolic acid, alpha-tocopherol.
7. The composition of claim 6, wherein the steroid is cholesterol.
8. The composition of claim 3, wherein the polymer-conjugated lipid is a pegylated lipid.
9. The composition of claim 8, wherein the pegylated lipid is 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol.
10. The composition of any one of claims 2-9, wherein the therapeutic and/or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
11. The composition of claim 10, wherein the therapeutic and/or prophylactic agent is a nucleic acid.
12. The composition of claim 11, wherein the nucleic acid is a mixture of one or more selected from the group consisting of siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA.
13. The composition of claim 12, wherein the nucleic acid is mRNA.
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| CN105903022A (en) * | 2009-05-05 | 2016-08-31 | 阿尔尼拉姆医药品有限公司 | Lipid compositions |
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