WO2023029578A1 - 新型阳离子脂质化合物 - Google Patents

新型阳离子脂质化合物 Download PDF

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WO2023029578A1
WO2023029578A1 PCT/CN2022/092599 CN2022092599W WO2023029578A1 WO 2023029578 A1 WO2023029578 A1 WO 2023029578A1 CN 2022092599 W CN2022092599 W CN 2022092599W WO 2023029578 A1 WO2023029578 A1 WO 2023029578A1
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composition
compound
lipid
combination
glycero
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PCT/CN2022/092599
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English (en)
French (fr)
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黄才古
王帅
黄铁强
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广州谷森制药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/12—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/14—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D317/28—Radicals substituted by nitrogen atoms

Definitions

  • the present invention provides novel cationic lipids that can be used in combination with other lipid components (such as neutral lipids, steroids, and polymer-conjugated lipids) to form a nucleic acid mRNA lipid nanoparticle composition combining a A method of delivering one or more therapeutic and/or prophylactic agents to a mammalian cell or organ and/or producing a polypeptide in a mammalian cell or organ.
  • lipid components such as neutral lipids, steroids, and polymer-conjugated lipids
  • the lipid nanoparticle compositions of the invention may also include, in specified proportions, one or more cationic and/or ionizable amino lipids, neutral lipids including polyunsaturated lipids, Lipids, polymer-conjugated lipids, steroids, and/or therapeutic and/or prophylactic agents.
  • nucleic acids Efficient targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids presents a persistent medical problem. Specifically, delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of these species. Accordingly, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.
  • compositions generally comprise one or more "cationic" lipids, neutral lipids including polyunsaturated lipids (such as phospholipids), structured lipids (such as steroids) and/or polyethylene glycol-containing Lipids of alcohols (polymer-conjugated lipids).
  • Cationic lipids include, for example, amine-containing lipids that can be readily protonated.
  • RNA degradation in plasma has been used to prevent RNA degradation in plasma and promote Cellular uptake of oligonucleotides.
  • the improved lipid nanoparticles would provide optimized drug delivery, protect nucleic acids from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids.
  • these preferred lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index such that treatment of a patient at an effective dose of the nucleic acid does not create unacceptable toxicity and/or risk to the patient.
  • the present invention provides these and related advantages.
  • the present invention provides the following novel compounds and methods involving these compounds:
  • the present invention relates to compounds of the following structural formula (I):
  • R 1 , R 2 and R 3 in the structural formula "I" are all independent combinations of two “hydrogen” isotopes (including isotopes “protium” and “deuterium”), and R 1 is specifically expressed as “HH", “HD “ and “DD”combination; combination of R2 and R3 "HHH", “HHD”, “HDD” and “DDD”;
  • R 1 , R 2 and R 3 are all independent, but the isotopes "protium” and “deuterium” in R 1 , R 2 and R 3 cannot be "H” at the same time, that is, a combination containing at least one "D", specifically Combinations are divided into 5 situations, including combinations containing 1 "D", combinations containing 2 "D", combinations containing 3 "D", combinations containing 4 "D” and combinations containing 5 "D” The combination.
  • the compound has one of the structures shown in Table 1 below
  • composition comprising any one or more of the compounds of structural formula (I) and a therapeutic and/or prophylactic agent.
  • compositions comprising any one or more of the compounds of structure (I) and a therapeutic and/or prophylactic agent.
  • the composition comprises any of the compounds of structure (I) and a therapeutic and/or prophylactic agent and one or more compounds selected from the group consisting of neutral lipids, steroids, and polymer conjugates. Lipid excipients. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the compositions.
  • the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Alkaline (DPPC), 1,2-Dimyristyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palm Acyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM) or Various.
  • the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • the steroid is selected from one of cholesterol, steroids, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, ⁇ -tocopherol or Various.
  • the preferred steroid is cholesterol.
  • the pegylated lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)
  • the composition ratio ranges from about 10 to 60 mol% of the compound, about 0 to 30 mol% of neutral lipids, about 10 to 55 mol% of steroids, and about 0 to 10 mol% of polymer-conjugated lipids. quality.
  • the therapeutic and/or prophylactic agent comprises a nucleic acid.
  • the nucleic acid is RNA, which is selected from the following composition: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof.
  • the RNA is selected from mRNA.
  • the present invention relates to methods of administering therapeutic and/or prophylactic agents to a subject in need thereof, the method comprising preparing or providing any of the compositions described above and administering to the subject combination.
  • the compounds of the present invention may be used as drug substances, or may be formulated as pharmaceutical compositions (usually in the form of lipid nanoparticles in combination with therapeutic and/or prophylactic agents).
  • the pharmaceutical composition of the present invention comprises a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • Compounds of structure (I) are effective to form lipid nanoparticles and deliver therapeutic and/or prophylactic agents. Appropriate concentrations and dosages can be readily determined by those skilled in the art.
  • compositions of the present invention may be by any of the acceptable modes of use for agents of similar utility.
  • the pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microgels, etc. Balls and Aerosols.
  • Typical routes of use of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques.
  • the pharmaceutical compositions of the present invention are formulated so that the active ingredients therein are bioavailable in the subject.
  • the composition to be administered to a subject or patient may be in one or more dosage forms where a tablet may be a single dosage unit and a container of the compound of the invention in aerosol form may hold multiple dosage units. Current methods for the preparation of such dosage forms are known, or will be apparent, to those skilled in the art.
  • the composition to be used will contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treating the associated disease or condition in accordance with the teachings of the invention.
  • compositions of the present invention may be in solid or liquid form.
  • the carrier can be particulate, such that the composition is in tablet or powder form.
  • the carrier can also be a liquid in which case the composition is an oral syrup or an injectable liquid or an aerosol suitable for inhalation.
  • the pharmaceutical composition is preferably in solid or liquid form, where solid or liquid forms are considered herein to include semi-solids, semi-liquids, suspensions and gels.
  • the pharmaceutical composition can be formulated in the form of powder, granule, tablet, pill, capsule, chewing gum, flake and the like.
  • Such solid compositions will generally contain one or more inert diluents or edible carriers.
  • binders such as gelatin, cellulose, etc.
  • excipients such as lactose, etc.
  • disintegrants such as alginic acid, etc.
  • lubricants such as magnesium stearate, etc.
  • Glidants such as silica gel, etc.
  • sweeteners such as sucrose or saccharin
  • flavoring agents such as mint
  • coloring agents such as sucrose or saccharin
  • liquid carriers other than materials of the above type, such as polyethylene glycol or oil.
  • compositions may be in liquid form, such as syrups, solutions, emulsions or suspensions.
  • Liquids may be for oral use or for injectable delivery, as two examples.
  • preferred compositions contain, in addition to the compounds of the present invention, one or more of sweetening agents, preservatives, dyeing/coloring agents and flavor enhancers.
  • one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents may be included.
  • the liquid pharmaceutical composition of the present invention may include one or more of the following auxiliary materials: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as a solvent or suspending medium; antibacterial antioxidants, such as methylparaben, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; Reagents for tonicity, such as sodium chloride or glucose; reagents used as cryoprotectants, such as sucrose or trehalose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or
  • the pharmaceutical composition of the present invention may be intended for topical use, in which case the carrier may suitably comprise a solution base, emulsion base, ointment base or gel base.
  • the base may contain one or more of petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in pharmaceutical compositions for topical use.
  • the composition may include a transdermal patch or iontophoretic device.
  • compositions of the invention can include various materials which modify the physical form of solid or liquid dosage forms.
  • the composition may include materials which form a coating shell around the active ingredient.
  • the materials forming the coating shell are generally inert and can be sugar, shellac and other enteric coating agents.
  • the active ingredient may be enclosed in a gelatin capsule.
  • compositions of the invention in solid or liquid form may include vehicles which facilitate delivery of the compound in combination with the compound of the invention.
  • Such vectors include monoclonal or polyclonal antibodies or proteins.
  • compositions of the invention may consist of formulations available as aerosols.
  • aerosol denotes systems comprising colloidal properties and systems consisting of pressurized packs. Delivery may be by liquefied or compressed gas, or by a suitable pump system which dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered as single-phase, bi-phasic or triphasic systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which together may form a drug delivery device. Preferred aerosols can be determined by those skilled in the art without undue experimentation.
  • the pharmaceutical compositions of the present invention can be prepared by methods well known in the field of pharmacy.
  • the pharmaceutical composition for injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers into a solution.
  • Surfactants can be added to promote the formation of a uniform solution or suspension. Surfactants interact non-covalently with the compounds of the invention thereby facilitating dissolution or homogeneous suspension of said compounds in aqueous media.
  • compositions of the present invention are used in therapeutically effective amounts, which will vary depending on a number of factors, including the activity of the particular therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; The patient's age, weight, general health, sex, and diet; mode and duration of use; rate of excretion; drug combination; severity of the specific case, etc.
  • compositions of the invention may also be administered concurrently with, prior to, or subsequent to administration of one or more other therapeutic agents.
  • Such therapeutic combinations include formulations using the compositions of the invention alone as well as combinations using the compositions of the invention and one or more other active ingredients.
  • the compositions of the present invention and other active ingredients may be administered to a subject together in a single oral dosage formulation such as a tablet or capsule, or each active ingredient may be administered in separate oral dosage formulations.
  • the compound of the invention and one or more additional active ingredients may be administered at the same time, or sequentially at times staggered from each other; it is understood that combination therapy includes all such dosing regimens.
  • novel deuterated cationic lipid compounds have achieved more advantageous physical and chemical properties, including more suitable pKa and better chemical stability, and are used in mRNA nanoliposome compositions, which can realize anti-ion Nucleic acid-like drugs are more effectively combined and delivered, and at the same time, their chemical structure is more stable, which is convenient for synthesis and beneficial for development as pharmaceutical excipients.
  • functional groups of intermediate compounds may need to be protected by suitable protecting groups.
  • suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl, tetrahydrofuranyl, benzyl, and the like.
  • Suitable protecting groups for amino include t-butoxycarbonyl, benzyloxycarbonyl and the like.
  • Suitable protecting groups for carboxylic acids include hydroxy, aryl or aryl esters. Protecting groups can be added or removed according to standard techniques, known to those skilled in the art and described herein.
  • all compounds of the present invention that exist in free base or free acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid according to methods known to those skilled in the art. Salts of compounds of the present invention may be converted to their free base or acid forms by standard techniques.
  • Compound 1 can be synthesized according to the representative route described in Example 1.
  • Compound 2 can be synthesized according to the representative route described in Example 1.
  • Compound 4 can be synthesized according to the representative route described in Example 1.
  • Compound 5 can be synthesized according to the representative route described in Example 1.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • Compound 6 can be synthesized according to the representative route described in Example 1.
  • Embodiment 7 is a diagrammatic representation of Embodiment 7:
  • Compound 7 can be synthesized according to the representative route described in Example 1.
  • Embodiment 8 is a diagrammatic representation of Embodiment 8
  • Compound 8 can be synthesized according to the representative route described in Example 1.
  • Embodiment 9 is a diagrammatic representation of Embodiment 9:
  • Compound 9 can be synthesized according to the representative route described in Example 1.
  • Compound 10 can be synthesized according to the representative route described in Example 1.
  • Compound 11 can be synthesized according to the representative route described in Example 1.
  • Compound 12 can be synthesized according to the representative route described in Example 1.
  • Compound 13 can be synthesized according to the representative route described in Example 1.
  • Compound 14 can be synthesized according to the representative route described in Example 1.
  • Compound 14 can be synthesized according to the representative route described in Example 1.
  • Compound 16 can be synthesized according to the representative route described in Example 1.
  • Compound 17 can be synthesized according to the representative route described in Example 1.
  • Compound 18 can be synthesized according to the representative route described in Example 1.
  • Compound 19 can be synthesized according to the representative route described in Example 1.
  • Compound 20 can be synthesized according to the representative route described in Example 1.
  • Compound 21 can be synthesized according to the representative route described in Example 1.
  • Compound 17 can be synthesized according to the representative route described in Example 1.
  • Compound 23 can be synthesized according to the representative route described in Example 1.
  • Compound 24 can be synthesized according to the representative route described in Example 1.
  • Compound 25 can be synthesized according to the representative route described in Example 1.
  • Compound 26 can be synthesized according to the representative route described in Example 1.
  • Compound 25 can be synthesized according to the representative route described in Example 1.
  • Compound 29 can be synthesized according to the representative route described in Example 1.
  • Compound 29 can be synthesized according to the representative route described in Example 1.
  • Ethanol was then removed and the external buffer was replaced by PBS by dialysis. Finally, filter the lipid nanoparticles through a sterile filter with a pore size of 0.2 ⁇ m.
  • the particle size of the lipid nanoparticles as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS, is approximately 65-105 nm in diameter, and in some cases, approximately 75-100 nm in diameter.
  • liver tissue approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). 1/4" ceramic balls (MP Biomedicals) were added to each tube, and 500 ⁇ L of Glo Lysis Buffer-GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue.
  • the FastPrep24 instrument MP Biomedicals
  • the Liver tissue was homogenized at 2 ⁇ 6.0 m/s for 15 s. The homogenate was incubated at room temperature for 5 min, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega).
  • FLuc mRNA (L-6107) from Trilink Biotechnologies will express the luciferase protein, which was originally isolated from the firefly (pHotinus pyralis). Fluc is commonly used in mammalian cell culture to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely replaced by 5-methylcytidine and pseudouridine.
  • the pKa of the formulated cationic lipid correlates with the effectiveness of the LNP for delivery of nucleic acids.
  • the preferred pKa range is 5-7.
  • the pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-phthanesulfonic acid (TNS).
  • TNS 2-(p-toluidino)-6-phthanesulfonic acid
  • the vesicles were diluted to contain 24 ⁇ M lipid in 2 mL of a buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM sodium acetate, 130 mM NaCl, wherein the pH value was 2.5-11.0. Aliquots of TNS solution were added to give a final concentration of 1 ⁇ M, and after vortex mixing, fluorescence intensity was measured in a SLM Aminco Series 2 Luminescence Spectrophotometer at room temperature using excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid best-fit analysis was applied to the fluorescence data, and pKa was measured as the pH that yielded half the maximum fluorescence intensity.
  • lipid nanoparticles containing FLuc mRNA were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the ordered mixing method as described in Example 30.
  • Lipid nanoparticles were formulated using the following molar ratios: 50% cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38% cholesterol/2% PEG lipid ("PEG-DMG", i.e., ( 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, the average PEG molecular weight is 2000).
  • PEG-DMG i.e., ( 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, the average PEG molecular weight is 2000.
  • Relative activity was determined by measuring luciferase expression in the liver. The activity was compared at doses of 0.3 and 1.0 mg mRNA/kg and expressed as ng

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Abstract

本申请涉及脂质化合物，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。脂质纳米颗粒可用于递送核酸，例如信使RNA和/或反义RNA。这类脂质纳米颗粒可于制备治疗和/或预防疾病的组合物。

Description

新型阳离子脂质化合物 技术领域
本发明提供新型阳离子脂质，其可用于与其他脂质组分(如中性脂质、类固醇和聚合物缀合的脂质)结合，以便形成一种核酸mRNA脂质纳米粒子组合物将一种或多种治疗剂和/或预防剂递送至哺乳动物细胞或器官和/或在哺乳动物细胞或器官中产生多肽的方法。除新型脂质外，本发明的脂质纳米粒子组合物还可以包括成特定比例的一种或多种阳离子性和/或可离子化氨基脂质、包括多不饱和脂质在内的中性脂质、聚合物缀合的脂质、类固醇、和/或治疗剂和/或预防剂。
背景技术
生物活性物质如小分子药物、蛋白质和核酸的有效靶向递送提出了一个持久的医学难题。确切地说，将核酸递送至细胞因这些物种的相对不稳定性和低细胞渗透性而变得困难。因此，需要开发有助于将治疗剂和/或预防剂如核酸递送至细胞的方法和组合物。
经研究证明，利用含脂质的纳米颗粒组合物、脂质体和脂质体复合物作运输媒介，可有效地将生物活性物质如小分子药物、蛋白质和核酸运送至细胞和/或细胞内隔室中。这些组合物一般包含一种或多种"阳离子性"脂质，包括多不饱和脂质在内的中性脂质(如磷脂)、结构性脂质(如类固醇)和/或含聚乙二醇的脂质(聚合物缀合的脂质)。阳离子脂质包括如可容易地质子化的含胺脂质。
然而，在治疗环境中使用寡核苷酸目前面临着两个问题。第一，游离的RNA易于在血浆中被核酸酶消化。第二，游离RNA进入存在相关翻译机制的细胞内隔室的能力受限。由阳离子脂质与其他脂质组分(如中性脂质、胆固醇、PEG、PEG化的脂质和寡核苷酸)形成的脂质纳米颗粒已用于阻止RNA在血浆中的降解并促进寡核苷酸的细胞摄取。
仍然有必要改进的用于递送寡核苷酸的阳离子脂质和脂质纳米颗粒。改进后的脂质纳米颗粒会提供优化的药物递送，保护核酸不在血清中被降解和清除，其适于全身或局部递送，并且提供核酸的细胞内递送。另外，这些优选的脂质-核酸颗粒应当是耐受良好的，并且提供足够的治疗指数，使得在有效剂量的核酸下的患者治疗不会对患者产生不可接受的毒性和/或风险。本发明提供这些优点和相关的优点。
发明内容
本发明提供以下新型化合物和涉及这些化合物的方法：
第一方面，本发明涉及以下结构式(I)的化合物：
Figure PCTCN2022092599-appb-000001
或其N-氧化物，或其盐或异构体。
其中结构式“I”中的R 1、R 2和R 3均为独立的2个“氢”同位素(包括同位素“氕”和“氘”)的组合，具体表现R 1为“HH”、“HD”和“DD”的组合；R 2和R 3“HHH”、“HHD”、“HDD”和“DDD”的组合；
R 1、R 2和R 3均有独立性，但R 1、R 2和R 3中的同位素“氕”和“氘”不能同时为“H”，即至少含有一个“D”的组合，具体组合情况分5种情况，包括含1个“D”的组合、含2个“D”的组合、含3个“D”的组合、含4个“D”的组合和含5个“D”的组合。
在各个不同的实施方案中，所述化合物具有以下表1中所示的结构之一
表1代表性化合物
Figure PCTCN2022092599-appb-000002
Figure PCTCN2022092599-appb-000003
Figure PCTCN2022092599-appb-000004
在一些实施方案中，提供了包含结构式(I)的化合物中的任一种或多种和治疗剂和/或预防剂的组合物。
在一些实施方案中，提供了包含结构(I)的化合物中的任一种或多种和治疗剂和/或预防剂的组合物。在一些实施方案中，所述组合物包含结构(I)的化合物中的任一种和治疗剂和/或预防剂以及一种或多种选自中性脂质、类固醇和聚合物缀合的脂质的赋形剂。其他药物可接受的赋形剂和/或载体也包括在组合物的各种实施方案内。
在一些实施方案中，中性脂质选自1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1，2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1，2-二肉豆寇基-sn-甘油-磷酸胆碱(DMPC)、1，2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1，2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、鞘磷脂(SM)中的一种或多种。在一些实施方案中，优选中性脂质为1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)。
在一些实施方案中，类固醇选自胆固醇、类固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚中的一种或多种。在一些实施方案中，优选类固醇为胆固醇。
在一些实施方案中，聚乙二醇化脂质为1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)
在一些实施方案中，所述组合物比例以下范围：约10～60mol％所述化合物、约0～30mol％中性脂质、约10～55mol％类固醇和约0～10mol％聚合物缀合的脂质。
在一些前述组合物的实施方案中，治疗剂和/或预防剂包括核酸。其中核酸为RNA，其选自以下组成：siRNA、aiRNA、miRNA、dsRNA、shRNA、mRNA以及其混合物。在一些实施方案中，所述RNA选自mRNA。
在其他不同的实施方案中，本发明涉及向有需要的受试者使用治疗剂和/或预防剂的方法，该方法包括制备或提供上述组合物中的任一种并向受试者使用该组合物。
出于使用的目的，本发明的化合物可以以原料药使用，或者可以配制为药物组合物(通常是脂质纳米颗粒与治疗剂和/或预防剂结合的形式)。本发明的药物组合物包含结构(I)的化合物和一种或多种药物可接受的载体、稀释剂或赋形剂。结构(I)的化合物以有效形成脂质纳米颗粒并递送治疗剂和/或预防剂。本领域技术人员可以容易地确定适当的浓度和剂量。
本发明组合物的使用可以通过任何用于类似效用的试剂的可接受使用方式来进行。本发明的药物组合物可以配制成固体、半固体、液体或气体形式的制剂，例如片剂、胶囊、粉末、颗粒、软膏、溶液、混悬液、栓剂、注射剂、吸入剂、凝胶、微球和气溶胶。使用这类药物组合物的典型途径包括，但不限于口服、局部、经皮、吸入、胃肠外、舌下、口含、直肠、阴道和鼻内途径。本文使用的术语胃肠外包括皮下注射，静脉内、肌内、皮内、胸骨内注射或输注技术。配制本发明的药物组合物是为了使其中的活性成分在受试者体内生物可利用。待向对象或患者使用的组合物形式可以是一个或多个剂型，其中，片剂可以是单剂量单位，而本发明气溶胶形式的化合物的容器可以容纳多个剂量单位。制备这些剂型的现行的方法是己知的，或者对于本领域技术人员是显而易见的。在任何情况下，待使用的组合物将会含有治疗有效量的本发明化合物或其药物可接受的盐，以便根据本发明的教导治疗相关的疾病或病况。
本发明的药物组合物可以是固体或液体的形式。一方面，载体可以是颗粒，使得组合物是片剂或粉末形式。载体还可以是液体，此时组合物是口服糖浆或可注射液体或气溶胶，所述气溶胶适用于吸入使用。
当要用于口服使用时，药物组合物优选为固体或液体形式，其中本文认为固体或液体的形式包括半固体、半液体、混悬液和凝胶。
作为用于口服使用的固体组合物，药物组合物可以配制成粉末、颗粒、片剂、丸剂、胶囊、咀嚼胶、薄片等形式。这类固体组合物通常将含有一种或多种惰性稀释剂或可食用载体。另外，可以存在以下的一种或多种:粘合剂，如明胶、纤维素等；赋形剂，如乳糖等；崩解剂，如海藻酸等；润滑剂，如硬脂酸镁等；助流剂，如硅胶等；甜味剂，如蔗糖或糖精；调味剂，如薄荷等；以及着色剂。
当药物组合物是胶囊形式时，其可以含有上述类型材料之外的液体载体，如聚乙二醇或油。
药物组合物可以是液体的形式，如糖浆、溶液、乳液或混悬液。作为两种实例，液体可以用于口服使用或用于注射递送。当意图用于口服使用时，优选的组合物含有除本发明化合物之外的甜味剂、防腐剂、染色/着色剂和增味剂中的一种或多种。在通过注射使用的组合物中，可以包括表面活性剂、防腐剂、润湿剂、分散剂、混悬剂、缓冲剂、稳定剂和等渗剂中的一种或多种。
本发明的液体药物组合物，不论其为溶液、混悬液还是其他类似的形式，可以包括以下辅料中的一种或多种:无菌稀释剂，如注射用水、盐水溶液、优选生理盐水、林格氏溶液、等 渗氯化钠；不挥发性油类，如可用作溶剂或混悬介质的合成的单甘酯或双甘酯，聚乙二醇、甘油、丙二醇或其他溶剂；抗菌剂，如尼泊金甲酯等；抗氧化剂，如抗坏血酸或亚硫酸氢钠；螯合剂，如乙二胺四乙酸；缓冲剂，如乙酸盐、柠檬酸盐或磷酸盐；以及用于调节张力的试剂，如氯化钠或葡萄糖；用作冷冻保护剂的试剂，如蔗糖或海藻糖。胃肠外制剂可以封装在玻璃或塑料制作的安瓿、一次性注射器或多剂量瓶中。生理盐水是优选的佐剂。可注射的药物组合物优选为无菌的。
本发明的药物组合物可以用于局部使用，在这种情况下，载体可以适当地包含溶液基质、乳液基质、软膏基质或凝胶基质。所述基质可以包含以下的一种或多种：矿脂、羊毛脂、聚乙二醇、蜂蜡、矿物油、诸如水和醇的稀释剂、以及乳化剂和稳定剂。增稠剂可以存在于用于局部使用的药物组合物中。如果意图用于经皮使用，组合物可以包括经皮贴片或离子电渗装置。
本发明的药物组合物可以包括修饰固体或液体剂型的物理形式的各种材料。组合物可以包括形成活性成分周围的包衣壳的材料。形成包衣壳的材料通常是惰性的，并且可以为糖、虫胶和其他肠溶包衣试剂。或者活性成分可以封装在明胶胶囊中。
本发明固体或液体形式的药物组合物可以包括与本发明化合物结合，有助于所述化合物递送载体。这种载体包括单克隆或多克隆抗体或蛋白质。
本发明的药物组合物可以由可作为气溶胶使用的制剂组成。术语气溶胶表示包含胶体性质的系统和由加压包装组成的系统。可以通过液化气或压缩气来递送，或者通过分散活性成分的适合的泵系统来递送。本发明化合物的气溶胶可以以单相、双相系统或三相系统来递送，以便递送活性成分。气溶胶的递送包括必要的容器、活化剂、阀、子容器等，其在一起可以形成给药装置。本领域技术人员不需额外实验即可确定优选的气溶胶。
本发明的药物组合物可以通过制药领域熟知的方法来制备。通过注射使用的药物组合物可以将本发明的脂质纳米颗粒与无菌的蒸馏水或其他载体结合成溶液来制备。可以加入表面活性剂以促进形成均匀的溶液或混悬液。表面活性剂是通过本发明化合物非共价地相互作用，从而促进所述化合物在水溶性介质中溶解或均匀混悬。
本发明的组合物或其药物可接受的盐以治疗有效量使用，所述量将会根据多种因素变化，包括使用的具体治疗剂的活性；治疗剂的代谢稳定性和作用时长；受试者的年龄、体重、一般健康状况、性别和饮食；使用的方式和时间；排泄速率；药物组合；具体病例的严重性等。
本发明的组合物也可以在使用一种或多种其他治疗剂的同时、之前或之后使用。这类治疗组合包括单独使用本发明组合物的制剂以及使用本发明组合物和其它一种或多种活性成分的组合。例如，本发明组合物和其他活性成分可以以单一口服剂量制剂(如片剂或胶囊)一起向受试者使用，或者各个活性成分以不同的口服剂量制剂使用。当使用不同的剂量制剂时，本发明化合物和一种或多种另外的活性成分可以在同一时间使用，或者在相互交错的时间依次使用；应理解组合治疗包括所有的这些给药方案。
上述新型氘代阳离子脂质化合物的结构修饰和设计，实现了更有优势的理化性质，包括更合适的pKa和更好的化学稳定性，用于mRNA纳米脂质体组合物，可实现对离子类核酸药物更有效结合并递送，同时其化学结构更稳定，便于合成和有利开发为药用辅料。
上述化合物和组合物的制备方法在下文描述，和/或在本领域已知。
本领域的技术人员将会认识到，在本文描述的方法中，中间化合物的官能团可能需要通过适合的保护基保护。这类官能团包括羟基、氨基和羧酸。用于羟基的适合的保护基包括三烷基甲硅烷基或二芳基烷基甲硅烷基、四氢呋喃基、苄基等。用于氨基的适合的保护基包括叔丁氧基羰基、苄氧基羰基等。用于羧酸的适合的保护基包括羟基、芳基或芳烃基酯。保护基可以根据标准技术添加或去除，所述标准技术是本领域技术人员已知的和本文中描述的。
本领域技术人员还将认识到，虽然本发明化合物的这类经保护的衍生物可以不由此具有药物活性，但其可以对哺乳动物施用并且之后在体内代谢形成具有药理活性的本发明化合物。 这类衍生物因此可以被描述为“前药”。所以本发明化合物的前药包括在本发明的范围内。
此外，所有以游离碱或游离酸形式存在的本发明化合物可以根据本领域技术人员已知的方法用适当的无机或有机的碱或酸处理来转化为其药物可接受的盐。本发明化合物的盐可以通过标准技术转化为其游离碱或酸形成。
提供了以下实施例，其目的在于举例展示，并非限定。
以下实施例，除非另外指出，否则使用的所有溶剂和试剂都是商购得到并且以原样使用。
以下描述的程序可用于合成表1中的化合物I。
本文采用了以下缩写:
Figure PCTCN2022092599-appb-000005
具体实施方式
实施例1：
代表性路线
化合物3的合成
Figure PCTCN2022092599-appb-000006
1)化合物B的合成
Figure PCTCN2022092599-appb-000007
化学式:C 37H 66O
分子量:526.93
向化合物A(6.0g，11.4mmol)和PCC(7.39g，34.2mmol)的DCM混合液中加入碳 酸钠(1.0g，5.66mmol)，氮气保护。在室温下反应3h，中控至反应至终点。反应结束垫如硅藻土进行过滤，滤饼用DCM淋洗。合并有机相，涤，硫酸镁干燥后，过滤浓缩，所得残余物通过硅胶柱(0～15％乙酸乙酯/正己烷)纯化。得化合物B(4.2g，收率70％)。
2)化合物3-01的合成
Figure PCTCN2022092599-appb-000008
化学式：C 41H 72D 2O 3
分子量：617.05
将化合物B(4.2g，8.2mmol)、1,2,4-4,4-d 2-丁三醇(3.4g,32.0mmol)和PPTS(200.0mg,0.8mmol)加入到盛有甲苯(60mL)反应瓶中，开启搅拌，在氮气保护下加热回流反应过夜，同时利用分水器进行分水至原料反应完全。将体系降至室温，加入50mL甲苯进行稀释，依次用5％的碳酸钠水溶液和水进行洗涤，经无水硫酸镁干燥后，通过层析柱纯化得3-01(3.0克，59％收率)。
3)化合物3-02的合成
Figure PCTCN2022092599-appb-000009
化学式：C 42H 74D 2O 5S
分子量：695.14
将化合物3-01(3.0g,4.9mmol)和TEA(1.58g,15.6mmol)加入到装有20mL二氯甲烷的反应瓶中。氮气保护后，降至-15℃。将MsCl(1.11g,9.7mmol)的二氯甲烷溶液(约32mL)逐滴加入到上述体系中，约20分钟加料完毕。保温反应2.5小时，中控至原料反应完全。向体系中加入50mL的二氯甲烷进行稀释，依次用饱和碳酸氢钠和饱和食盐水洗涤，有机相经无水硫酸镁干燥，过滤，减压浓缩移除溶剂。粗品通过柱层析进行过柱纯化，得3-02(3.05g，91％收率)
4)化合物3的合成
Figure PCTCN2022092599-appb-000010
化学式：C 43H 77D 2NO 2
分子量：644.12
将化合物3-02(3.0g，4.35mmol)加入到盛有25mL的异丙醇的反应瓶中，开启搅拌并进行氮气保护。将120mL2.0M的二甲胺的甲醇溶液逐渐加入上述体系中，保温反应72小时，至原料反应完全。将体系减压浓缩移除溶剂，所得粗品经通过硅胶柱(0～100％(1％NH 4OH、20％MeOH于二氯甲烷中的混合物)二氯甲烷)纯化，得化合物3(2.49g，收率89％)。
C 43H 77D 2NO 2,Ms m/z:[M+H +]644.6； 1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82～2.78(d,4H),2.26-2.15(m,14H,),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例2：
化合物1的合成
Figure PCTCN2022092599-appb-000011
化学式：C 43H 78DNO 2
分子量：643.12
化合物1可根据实施例1描述的代表性路线合成。
C 43H 78DNO 2,Ms m/z:[M+H +]643.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82～2.78(d,4H),2.43(t,1H),2.26-2.15(m,14H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例3：
化合物2的合成
Figure PCTCN2022092599-appb-000012
分子量：643.12
化合物2可根据实施例1描述的代表性路线合成。
C 43H 78DNO 2,Ms m/z:[M+H +]643.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.43-2.41(t,2H),2.26-2.15(m,13H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例4：
化合物4的合成
Figure PCTCN2022092599-appb-000013
分子量：644.14
化合物4可根据实施例1描述的代表性路线合成。
C 43H 77D 2NO 2,Ms m/z:[M+H +]644.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.43-2.41(t,2H),2.26-2.15(m,12H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例5：
化合物5的合成
Figure PCTCN2022092599-appb-000014
化合物5可根据实施例1描述的代表性路线合成。
C 43H 77D 2NO 2,Ms m/z:[M+H +]644.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.42(t,1H),2.26-2.15(m,13H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例6：
化合物6的合成
Figure PCTCN2022092599-appb-000015
分子量：644.14
化合物6可根据实施例1描述的代表性路线合成。
C 43H 77D 2NO 2,Ms m/z:[M+H +]644.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.43-2.41(t,2H),2.26-2.15(m,12H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例7：
化合物7的合成
Figure PCTCN2022092599-appb-000016
分子量：645.14
化合物7可根据实施例1描述的代表性路线合成。
C 43H 76D 3NO 2,Ms m/z:[M+H +]645.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.26-2.14(m,13H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例8：
化合物8的合成
Figure PCTCN2022092599-appb-000017
化合物8可根据实施例1描述的代表性路线合成。
C 43H 76D 3NO 2,Ms m/z:[M+H +]645.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.42(t,1H),2.26-2.15(m,12H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例9：
化合物9的合成
Figure PCTCN2022092599-appb-000018
化合物9可根据实施例1描述的代表性路线合成。
C 43H 76D 3NO 2,Ms m/z:[M+H +]645.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82 (m,3H),2.82-2.78(d,4H),2.44-2.43(t,1H),2.26-2.15(m,12H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例10：
化合物10的合成
Figure PCTCN2022092599-appb-000019
分子量：645.14
化合物10可根据实施例1描述的代表性路线合成。
C 43H 76D 3NO 2,Ms m/z:[M+H +]645.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.41(t,2H),2.26-2.15(m,11H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例11：
化合物11的合成
Figure PCTCN2022092599-appb-000020
分子量：645.14
化合物11可根据实施例1描述的代表性路线合成。
C 43H 76D 3NO 2,Ms m/z:[M+H +]645.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.41(t,2H),2.26-2.15(m,11H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例12：
化合物12的合成
Figure PCTCN2022092599-appb-000021
化合物12可根据实施例1描述的代表性路线合成。
C 43H 75D 4NO 2,Ms m/z:[M+H +]646.7；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.26-2.15(m,12H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例13：
化合物13的合成
Figure PCTCN2022092599-appb-000022
化合物13可根据实施例1描述的代表性路线合成。
C 43H 75D 4NO 2,Ms m/z:[M+H +]646.7；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.26-2.15(m,12H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例14：
化合物14的合成
Figure PCTCN2022092599-appb-000023
化学式：C 43H 75D 4NO 2
分子量：646.15
化合物14可根据实施例1描述的代表性路线合成。
C 43H 75D 4NO 2,Ms m/z:[M+H +]646.7；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.41(t,2H),2.26-2.14(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例15：
化合物15的合成
Figure PCTCN2022092599-appb-000024
化合物14可根据实施例1描述的代表性路线合成。
C 43H 75D 4NO 2,Ms m/z:[M+H +]646.7；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.41(t,2H),2.26-2.15(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例16：
化合物16的合成
Figure PCTCN2022092599-appb-000025
化合物16可根据实施例1描述的代表性路线合成。
C 43H 75D 4NO 2,Ms m/z:[M+H +]646.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.43(t,1H),2.26-2.14(m,11H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例17：
化合物17的合成
Figure PCTCN2022092599-appb-000026
化学式：C 43H 74D 5NO 2
分子量：647.14
化合物17可根据实施例1描述的代表性路线合成。
C 43H 74D 5NO 2,Ms m/z:[M+H +]647.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.41(t,2H),2.26-2.14(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例18：
化合物18的合成
Figure PCTCN2022092599-appb-000027
分子量：647.14
化合物18可根据实施例1描述的代表性路线合成。
C 43H 74D 5NO 2,Ms m/z:[M+H +]647.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.43(t,1H),2.26-2.14(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例19：
化合物19的合成
Figure PCTCN2022092599-appb-000028
分子量：647.14
化合物19可根据实施例1描述的代表性路线合成。
C 43H 74D 5NO 2,Ms m/z:[M+H +]647.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.41(t,2H),2.26-2.15(m,9H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例20：
化合物20的合成
Figure PCTCN2022092599-appb-000029
分子量：647.14
化合物20可根据实施例1描述的代表性路线合成。
C 43H 74D 5NO 2,Ms m/z:[M+H +]647.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.43(t,1H),2.26-2.14(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例21：
化合物21的合成
Figure PCTCN2022092599-appb-000030
化学式：C 43H 74D 5NO 2
分子量：647.14
化合物21可根据实施例1描述的代表性路线合成。
C 43H 74D 5NO 2,Ms m/z:[M+H +]647.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.43(t,1H),2.26-2.14(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例22：
化合物21的合成
Figure PCTCN2022092599-appb-000031
化合物17可根据实施例1描述的代表性路线合成。
C 43H 74D 5NO 2,Ms m/z:[M+H +]647.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.43(t,1H),2.26-2.14(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例23：
化合物23的合成
Figure PCTCN2022092599-appb-000032
化合物23可根据实施例1描述的代表性路线合成。
C 43H 73D 6NO 2,Ms m/z:[M+H +]648.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.26-2.14(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例24：
化合物24的合成
Figure PCTCN2022092599-appb-000033
化合物24可根据实施例1描述的代表性路线合成。
C 43H 73D 6NO 2,Ms m/z:[M+H +]648.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.26-2.14(m,10H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例25：
化合物25的合成
Figure PCTCN2022092599-appb-000034
化学式：C 43H 73D 6NO 2
分子量：648.14
化合物25可根据实施例1描述的代表性路线合成。
C 43H 73D 6NO 2,Ms m/z:[M+H +]648.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.43(t,1H),2.26-2.14(m,9H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例26：
化合物26的合成
Figure PCTCN2022092599-appb-000035
分子量：648.14
化合物26可根据实施例1描述的代表性路线合成。
C 43H 73D 6NO 2,Ms m/z:[M+H +]648.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.44-2.42(t,2H),2.26-2.15(m,8H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例27：
化合物27的合成
Figure PCTCN2022092599-appb-000036
分子量：649.14
化合物25可根据实施例1描述的代表性路线合成。
C 43H 72D 7NO 2,Ms m/z:[M+H +]649.7；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82-2.78(d,4H),2.26-2.14(m,9H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H)。
实施例28：
化合物28的合成
Figure PCTCN2022092599-appb-000037
分子量：649.15
化合物29可根据实施例1描述的代表性路线合成。
C 43H 72D 7NO 2,Ms m/z:[M+H +]649.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82～2.78(d,4H),2.43(s,1H),2.26-2.15(m,8H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例29：
化合物29的合成
Figure PCTCN2022092599-appb-000038
分子量：650.16
化合物29可根据实施例1描述的代表性路线合成。
C 43H 71D 8NO 2,Ms m/z:[M+H +]650.6；1H-NMR(300MHz):δ5.4-5.27(m,8H),3.89-3.82(m,3H),2.82～2.80(d,4H),2.26(m,8H),1.51-1.50(m,6H),1.33-1.26(m,36H),0.88(t,6H).
实施例30
利用脂质纳米颗粒组合物的荧光素酶mRNA体内评价
将阳离子脂质、DSPC、胆固醇和PEG-脂质以50:10:38:2或48:10:40:2的摩尔比溶解在乙醇中。以约10:1至30:1的总脂质与mRNA的重量比制备脂质纳米颗粒(LNP)。简而言之，将mRNA在10mL至50mL柠檬酸盐缓冲液(pH＝4.0)稀释至0.15mg/mL。使用注射器泵，将脂质的乙醇溶液与mRNA水溶液以约1:5至1:3(体积/体积)的比例混合，总流速为10mL/min以上。然后去除乙醇，并通过透析用PBS替代外部的缓冲液。最后，将脂质纳米颗粒通过0.2μm孔径的无菌过滤器过滤。使用Malvern Zetasizer Nano ZS通过准弹性光散射测定的脂质纳米颗粒的粒径为直径大约65-105nm，并且在一些情况下，直径大约75-100nm。
根据国家科学技术委员会制定的指南，在6-8周龄的雌性C57BL/6小鼠，8-10周龄CD-1小鼠上进行研究。通过尾静脉注射全身性给予不同剂量的mRNA脂质纳米颗粒，并在给药后的特定时间点(例如5小时)使动物安乐死。将肝脏和脾脏收集在预先称重的管中，确定重量，立即在液氮中快速冷冻，并且在-80℃下储存，直至用于分析。
对于肝脏，切割约50mg以便在2mL FastPrep管(MP Biomedicals，Solon OH)中进行分析。向各个管中加入1/4"陶瓷球(MP Biomedicals)，并将平衡至室温的500μL的Glo裂解缓冲液-GLB(Promega，Madison WI)加入到肝脏组织中。使用FastPrep24仪器(MP Biomedicals)将肝脏组织在2×6.0m/s下均匀化15秒。将匀浆在室温下孵育5分钟，然后在GLB中进行1:4稀释，并使用SteadyGlo荧光素酶测定系统(Promega)进行评估。具体地，将50μL的稀释的组织匀浆与50μL的SteadyGlo底物反应，摇振10秒，接着孵育5分钟，然后使用SpectraMAX_L化学发光型酶标仪(美谷分子仪器(上海)有限公司)定量。通过使用BCA蛋白质定量试剂盒(上海易色医疗科技有限公司)来确定测定的蛋白质的量。然后将相对发光度单位(RLU)归一化成所测定蛋白质的总μg。为了将RLU转化成μg荧光素酶，用QuantiL μM重组荧光素酶(Promega)生成了标准曲线。
来自Trilink Biotechnologies的FLuc mRNA(L-6107)将表达荧光素酶蛋白，其最初从萤火虫(pHotinus pyralis)中分离出来。Fluc通常用于哺乳动物细胞培养物中以测量基因表达和细胞活力。其在底物萤光素存在下发射出生物性光。这种加帽并且聚腺昔酸化的mRNA被5-甲基胞苷和假尿苷完全取代。
实施例31
所配制得脂质的pKa的测定
所配制的阳离子脂质的pKa与用于递送核酸的LNP的效果相关。优选的pKa范围是5～7。使用基于2-(对甲苯胺基)-6-荼磺酸(TNS)的荧光的分析，在脂质纳米颗粒中测定各阳离子脂质的pKa。如实施例27中所述，使用有序的方法来制备在PBS中的浓度为0.4mM总脂质的包含阳离子脂质/DSPC/胆固醇/PEG脂质(50/10/38/2mol％)的脂质纳米颗粒。将TNS在蒸馏水中制备成100μM储备溶液。将囊泡稀释成在2mL缓冲溶液中含24μM脂质，所述缓冲溶液含有10mM HEPES、10mM MES、10mM乙酸按、130mM NaCl，其中pH值为2.5～11.0。加入等份的TNS溶液以产生lμM的终浓度，并且在涡旋混合之后，在室温下使用321nm和445nm的激发波长和发射波长在SLM Aminco Series 2发光分光光度计中测量荧光强度。对荧光数据应用S形最佳拟合分析，并将pKa测量为产生半数最大荧光强度的pH。
实施例32
使用体内荧光素酶mRNA表达的啮齿动物模型测定含有各种阳离子脂质的脂质纳米颗粒制剂的效能
为了比较的目的，如实施例30所述，使用有序混合方法，将这些脂质也用于配制含有FLuc mRNA(L-6107)的脂质纳米颗粒。使用以下摩尔比来配制脂质纳米颗粒:50％阳离子脂 质/10％二硬脂酰磷脂酰胆碱(DSPC)/38％胆固醇/2％PEG脂质("PEG-DMG"，即，(1-(单甲氧基一聚乙二醇)-2，3一二肉豆蔻酰基甘油，平均PEG分子量为2000)。如实施例30所述，在经由尾静脉注射施用之后的5小时，通过测量肝脏中的荧光素酶表达来确定相对活性。在0.3和1.0mg mRNA/kg的剂量下比较所述活性，并表达成在如实施例30所述的施用之后5小时测量的ng荧光素酶/g肝脏。实施例31及32结果如表2所示。
表2与mRNA表现出活性的比较脂质
Figure PCTCN2022092599-appb-000039
Figure PCTCN2022092599-appb-000040
Figure PCTCN2022092599-appb-000041
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对公开专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (15)

  1. 一种式“I”化合物：
    Figure PCTCN2022092599-appb-100001
    或其N-氧化物，或其盐或异构体。
    其中结构式“I”中的R 1、R 2和R 3均为独立的2个“氢”同位素(包括同位素“氕”和“氘”)的组合，具体表现R 1为“HH”、“HD”和“DD”的组合；R 2和R 3“HHH”、“HHD”、“HDD”和“DDD”的组合；
    R 1、R 2和R 3均为独立的2个“氢”同位素(包括同位素“氕”和“氘”)的组合，具体组合情况分8种情况，包括含1个“D”的组合、含2个“D”的组合、含3个“D”的组合、含4个“D”的组合、含5个“D”的组合、含6个“D”的组合、含7个“D”的组合和含8个“D”的组合。
  2. 选自表1中的化合物的化合物。
  3. 包含权利要求1～2中任一项所述的化合物和治疗剂和/或预防剂的组合物。
  4. 如权利要求3所述的组合物，其还包含选自中性脂质、类固醇以及聚合物缀合的脂质中的一种或多种赋形剂。
  5. 如前述权利要求4所述的组合物，其中所述组分中的中性脂质选自以下的一种或多种物质混合：1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1，2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1，2-二肉豆寇基-sn-甘油-磷酸胆碱(DMPC)、1，2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1，2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)和鞘磷脂(SM)。
  6. 如前述权利要求5所述的组合物，其中所述中性脂质为DSPC。
  7. 如前述权利要求4～6所述的组合物，其中所述组分中的类固醇选自以下一种或多种物质混合：胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚。
  8. 如前述权利要求7所述的组合物，其中所述类固醇为胆固醇。
  9. 如前述权利要求4～8所述的组合物，其中所述组分中的聚合物缀合的脂质为聚乙二醇化脂质。
  10. 如前述权利要求中9所述的组合物，其中所述聚乙二醇化脂质为1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)
  11. 如前述权利要求中任一项所述的组合物，其中所述治疗剂和/或预防剂是能够引起免疫响应的疫苗或化合物，其中包括核酸。
  12. 如前述权利要求11的组合物，其中所述核酸为RNA，其选自以下组成：siRNA、aiRNA、miRNA、dsRNA、shRNA、mRNA以及其混合物。
  13. 如前述权利要求12的组合物，其中所述RNA为mRNA。
  14. 向有需要的受试者施用治疗剂和/或预防剂的方法，所述方法包括制备或提供上述权利要求中任一项所述的组合物，并向所述对象施用所述组合物。
  15. 如前述权利要求中任一项所述的受试者为哺乳动物或人。
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JUSTIN B. LEE, KAIXIN ZHANG, YUEN YI C. TAM, YING K. TAM, NATHAN M. BELLIVEAU, VANESSA Y.C. SUNG, PAULO J.C. LIN, ERIC LEBLANC, MA: "Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo", INTERNATIONAL JOURNAL OF CANCER, UNION INTERNATIONALE CENTRE LE CANCER., vol. 131, no. 5, 1 September 2012 (2012-09-01), pages E781 - E790, XP055083267, ISSN: 00207136, DOI: 10.1002/ijc.27361 *
PATEL SIDDHARTH; RYALS RENEE C.; WELLER KYLE K.; PENNESI MARK E.; SAHAY GAURAV: "Lipid nanoparticles for delivery of messenger RNA to the back of the eye", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 303, 1 January 1900 (1900-01-01), AMSTERDAM, NL , pages 91 - 100, XP085712832, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2019.04.015 *
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