Lipid compound suitable for three-component lipid nanoparticles and application thereof

Abstract

The invention relates to the field of drug delivery, in particular to a lipid compound, a pharmaceutical composition containing the lipid compound and a preparation and application of the pharmaceutical composition. When the lipid compound provided by the invention is applied to a lipid nanoparticle system, traditional four-component lipid nanoparticles can be changed into three-component lipid nanoparticles, so that the preparation efficiency of a lipid nanoparticle drug delivery system is improved, and meanwhile, the lipid compound has relatively good safety.

Description

Technical Field

[0001] The present invention relates to the field of drug delivery, and in particular to a lipid compound, a pharmaceutical composition comprising the lipid compound, and a preparation and application thereof. Background Art

[0002] As an important drug delivery system, lipid nanoparticles (LNPs) have received extensive attention and application in recent years. The lipids of LNPs generally include cholesterol, phospholipids, ionizable / cationic lipids and pegylated lipids. In traditional four-component lipid nanoparticles, cholesterol plays a key role. Cholesterol can enhance the stability of the lipid bilayer, regulate the fluidity of the membrane, and help improve the drug encapsulation rate and cellular uptake efficiency of nanoparticles.

[0003] However, traditional four-component lipid nanoparticles also have some shortcomings. First, the use of cholesterol may increase the complexity and production cost of nanoparticle preparation. The source and quality control of cholesterol may also become a problem, affecting the stability and consistency of nanoparticles.

[0004] Therefore, there is an urgent need for a new type of lipid compound to improve the shortcomings of traditional lipid nanoparticles and enhance the preparation efficiency of lipid nanoparticle drug delivery systems. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of the deficiencies in the prior art, the present invention provides a lipid compound. By applying the above lipid compound to a lipid nanoparticle system, traditional four-component lipid nanoparticles can be converted into three-component lipid nanoparticles, thereby improving the preparation efficiency of the lipid nanoparticle drug delivery system while having better safety.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In one aspect, the present invention provides a lipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein the structural formula of the lipid compound is shown in formula (I):

[0010]

[0011]

[0012] The X 1 , X 2 , X 3 Each independently is O or S;

[0013] The X 4 is selected from O, S, N, substituted or unsubstituted C1-C 10 Alkylene, wherein the alkylene is selected from a straight chain alkylene and a branched alkylene;

[0014] The Z is selected from O or NR 7 , the R 7 is selected from H, substituted or unsubstituted C1-C 10 Alkyl, wherein the alkyl is selected from a linear alkyl, a branched alkyl, a saturated alkyl or an unsaturated alkyl, a substituted or unsubstituted C3-C 12 Cycloalkyl;

[0015] The R 1 , R 2 Each independently selected from steroid derivatives;

[0016] The R 3 , R 4 are each independently selected from H, substituted or unsubstituted C1-C 10 Alkylene, wherein the alkylene is selected from a straight chain alkylene and a branched alkylene;

[0017] The R 5 , R 6 are each independently selected from H, substituted or unsubstituted C1-C 10 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, wherein the alkyl is selected from a linear alkyl, a branched alkyl, a saturated alkyl or an unsaturated alkyl; or R 5 and R 6 Together with the nitrogen atom to which they are attached, they can form a C2-C 12 Heterocyclic group, the C2-C 12 The heterocyclyl group is optionally substituted with one to three C1-C6 alkyl groups.

[0018] In one embodiment, X 1 , X 2 , X 3 , X 4 Both are O.

[0019] In one embodiment, Z is selected from O or NH.

[0020] In one embodiment, the steroid derivative is selected from cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid or α-tocopherol.

[0021] In one embodiment, the R 1 , R 2 All selected

[0022] In one embodiment, the R 1 , R 2 All selected

[0023] In one embodiment, the R 3 Selected from C1-C7 straight chain alkylene.

[0024] In one embodiment, the R 3 Selected from C1-C5 straight chain alkylene.

[0025] In one embodiment, the R 3 Selected from methylene or ethylene.

[0026] In one embodiment, the R 4 Selected from C1-C7 straight chain alkylene.

[0027] In one embodiment, the R 4 Selected from C1-C5 straight chain alkylene.

[0028] In one embodiment, the R 4 Selected from methylene or ethylene.

[0029] In one embodiment, the R 5 , R 6 Each is independently selected from substituted or unsubstituted C1-C5 alkyl.

[0030] In one embodiment, the R 5 , R 6 Each is independently selected from substituted or unsubstituted C1-C3 alkyl.

[0031] In one embodiment, the R 5 , R 6 are selected from methyl or ethyl.

[0032] In one embodiment, the R 5 , R 6 Each is independently selected from C1-C3 hydroxyalkyl.

[0033] In one embodiment, the R 5 , R 6 They are selected from hydroxymethyl or hydroxyethyl.

[0034] In one embodiment, the lipid compound is selected from any one of the following structural formulas:

[0035]

[0036]

[0037] In one aspect, the present invention provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the lipid compound described in any one of the above embodiments or a pharmaceutically acceptable salt or a stereoisomer thereof.

[0038] In one embodiment, the lipid nanoparticles further include one or more active ingredients, including chemical drugs, biological macromolecules, nucleic acid drugs, etc.

[0039] In one embodiment, the nucleic acid drug is selected from RNA and DNA.

[0040] In one embodiment, the nucleic acid drug is selected from any one of antisense nucleic acid, plasmid, mRNA, interfering nucleic acid, aptamer, antagomir, miRNA, ribozyme and siRNA; preferably any one of DNA, mRNA, miRNA and siRNA.

[0041] In one embodiment, the lipid nanoparticles include phospholipids, PEGylated lipids and the lipid compound described in any one of the above embodiments or a pharmaceutically acceptable salt or a stereoisomer thereof.

[0042] In one embodiment, the phospholipids are selected from one or more of the following compounds: dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DUPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl dimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-divinyl-sn-glycerol-3-phosphocholine, 1,2-diaryl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), 1,2-dihydroxytin-sn-glycerol-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-divinyl alcohol-sn-glycerol-3-phosphoethanolamine, 1,2-divinyl-sn-glycerol-3-phosphoethanolamine, 1,2-diaryl-sn-glycerol-3-phosphoethanolamine, 1,2-dithiohexaenoic acid-sn-glycerol-3-phosphoethanolamine, 1,2-diol-sn-glycerol-3-phosphoethanolamine (DOPG) or sphingomyelin. Preferably, the phospholipid is DOPE or DSPC.

[0043] In one embodiment, the PEGylated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol. Preferably, the PEGylated lipid is DMG-PEG2000.

[0044] In one embodiment, the lipid nanoparticle has a diameter of about 30 nm to about 200 nm.

[0045] In one embodiment, the lipid nanoparticle has a diameter of about 70 nm to about 150 nm.

[0046] In one embodiment, the lipid nanoparticle has a diameter of about 90 nm to about 110 nm.

[0047] In one aspect, the present invention provides a pharmaceutical composition comprising any one of the lipid nanoparticles described above and a pharmaceutically acceptable carrier.

[0048] In one aspect, the present invention provides a method for delivering a drug to mammalian cells, the method comprising administering an effective amount of the above-mentioned pharmaceutical composition or the above-mentioned lipid nanoparticle to a subject.

[0049] In one embodiment, the mammalian cell is in a mammal.

[0050] In one embodiment, the mammal is a human.

[0051] In one embodiment, the pharmaceutical composition or lipid nanoparticle is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0052] In one aspect, the present invention provides use of the above-mentioned pharmaceutical composition or the above-mentioned lipid nanoparticles in the preparation of drugs for preventing and / or inhibiting and / or treating diseases or disorders.

[0053] In one embodiment, the disease or disorder is selected from infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, or metabolic diseases.

[0054] In one aspect, the present invention provides use of the above-mentioned pharmaceutical composition or the above-mentioned lipid nanoparticles in preparing a targeting carrier.

[0055] (III) Beneficial effects

[0056] The present invention provides a lipid compound and a lipid nanoparticle system comprising the lipid compound. Compared with the prior art, the lipid nanoparticle system has the following beneficial effects:

[0057] The lipid compound of the present invention can be used to prepare lipid nanoparticles with phospholipids and PEGylated lipids without the need to add cholesterol, thereby simplifying the composition of the nanoparticles. At the same time, the lipid nanoparticles obtained by using the lipid compound of the present invention have good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0059] Figure 1 Verification Example 2 Effect of LNPs prepared from different lipid compounds on the fluorescence expression intensity of Luciferase in mice;

[0060] Figure 2 Anatomy diagram of the mouse that died in Verification Example 2. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] The technical solution in the embodiment of the present invention is to solve the above technical problems, and the overall idea is as follows:

[0063] The embodiment of the present invention provides a lipid compound, and the lipid compound is applied to the lipid nanoparticle system, so that the traditional four-component lipid nanoparticles can be changed into three-component lipid nanoparticles, without the need to add cholesterol, thereby simplifying the composition of the nanoparticles. At the same time, by optimizing the specific structure of the lipid compound, the lipid nanoparticles obtained by using the lipid compound of the present invention have good safety.

[0064] Application The lipid nanoparticle delivery system of the present invention can be used to deliver a variety of therapeutic agents, including chemical drugs, macromolecular drugs, nucleic acid drugs, such as mRNA, etc.

[0065] definition:

[0066] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the referenced terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as understood by those skilled in the art.

[0067] References in the specification to "one embodiment" or "some embodiments" or the like indicate that the described embodiment may include a particular aspect, feature, structure, part, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, part, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment mentioned in other parts of the specification. Moreover, when a particular aspect, feature, structure, part, or characteristic is described in conjunction with an embodiment, it is within the knowledge of those skilled in the art to affect or relate such aspect, feature, structure, part, or characteristic with other embodiments, whether or not explicitly described.

[0068] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a compound" includes a plurality of such compounds, such that a compound X includes a plurality of compounds X. Note also that the claims may be drafted to exclude any optional elements. As such, this statement is intended to serve as antecedent basis for use of exclusive terminology, such as "solely," "only," and the like in connection with any element described herein, and / or for use of claim element invocation or "negative" limitations.

[0069] The term "and / or" means any one of the items, any combination of items, or all of the items associated with the term. The phrase "one or more" is readily understood by those skilled in the art, especially when read in the context of its use. For example, one or more substituents on a benzene ring refers to one to five, or one to four substituents (e.g., if the benzene ring is disubstituted).

[0070] The term "about" may refer to a change of ±5%, ±10%, ±20%, or ±25% of a specified value. For example, "about 50%" may have a change of 45 to 55% in some embodiments. For integer ranges, the term "about" may include one or two integers greater than and / or less than the integers quoted at each end of the range. Unless otherwise specified herein, the term "about" is intended to include values, such as weight percentages, that are equivalent to the functional aspects of a single component, composition, or embodiment near the quoted range. The term about may also modify the endpoints of the quoted range as discussed above in this paragraph.

[0071] As will be appreciated by those skilled in the art, all numerical values ​​(including those expressing amounts of ingredients, properties such as molecular weight, reaction conditions, etc.) are approximate and are to be understood as being optionally modified in all instances by the term "about". These values ​​may vary depending on the desired properties sought to be obtained by one skilled in the art utilizing the teachings of this specification. It is also to be understood that such values ​​inherently contain the variations necessarily resulting from the standard deviation present in their respective testing measurements.

[0072] Each operation or step may be described as multiple separate operations in turn in a manner that may be helpful for understanding the embodiments. However, the order of description should not be interpreted as implying that these operations are order-dependent.

[0073] The term "one embodiment" or "some embodiments" may be used in the specification, which may each refer to one or more identical or different embodiments. In addition, the terms "comprising", "including", "having" and the like are synonymous when used in relation to the embodiments.

[0074] As understood by those skilled in the art, for any and all purposes, especially in providing written descriptions, all ranges quoted herein also encompass any and all possible sub-ranges and combinations of their sub-ranges, as well as individual values, particularly integer values, constituting the ranges. The quoted ranges (e.g., weight percentages or carbon groups) include each specific value, integer, decimal, or entity within the range. Any listed range can be easily considered to fully describe and achieve the same range of being decomposed into at least equal two, three, four, five, or ten points. As a non-limiting example, each range discussed herein can be easily decomposed into the lower third, the middle third, and the upper third, etc. As also understood by those skilled in the art, all languages, such as "up to", "at least", "greater than", "less than", "exceed", "or more", etc., include quoted values, and such terms refer to the ranges that can be subsequently decomposed into sub-ranges as discussed above. In the same manner, all ratios quoted herein also include all sub-ratios falling within the wider ratios. Therefore, the specific values ​​quoted for free radicals, substituents, and ranges are only for illustration; they do not exclude other defined values ​​or other values ​​within the defined ranges of free radicals and substituents.

[0075] It is also readily understood by those skilled in the art that when members are grouped together in a common manner, such as in Markush groups, the present invention encompasses not only the complete group listed as a whole, but also each member of the group and all possible subgroups of the main group individually. In addition, for all purposes, the present invention encompasses not only the main group, but also the main group lacking one or more group members. Therefore, the present invention contemplates the explicit exclusion of any one or more members of the cited group. Therefore, this condition can be applied to any of the disclosed classes or embodiments, whereby any one or more of the cited elements, species, or embodiments can be excluded from the class or embodiment, for example, for explicit negative limitations.

[0076] The term "C1-C 10 "Alkyl" is a straight or branched chain alkyl group having 1 to 10 carbons. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 1-methylpropyl, n-pentyl, isopentyl, 2-methylbutyl, 1,1-dimethylpropyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl and 2-ethylbutyl, n-heptyl, n-octyl, n-nonyl, n-decyl and various structural isomers, etc.

[0077] The term "C1-C 10 “Alkylene” refers to a straight or branched chain alkylene group containing 1 to 10 carbon atoms. Examples include methylene, ethylene, trimethylene (—CH2CH2CH2—), tetramethylene (—CH2CH2CH2CH2—), pentamethylene (—CH2CH2CH2CH2CH2—), methyl-substituted tetramethylene (—CH(CH3)CH2—), methyl-substituted pentamethylene (—CH(CH3)CH2CH2—), and the like.

[0078] The term "C3-C 12 "Cycloalkyl" means a cycloalkyl group of 3 to 12 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. When two groups are taken together to form a C 3-12 When the cycloalkane ring is cyclopropane-1,1-diyl, cyclobutane-1,1-diyl, cyclopentane-1,1-diyl, cyclohexane-1,1-diyl, etc., the resulting group is divalent.

[0079] When two groups on two carbon atoms combine to form C 3-12 When the carbon ring is a carbon ring, the resulting ring forms a fused ring. Examples include ring structures where two carbon atoms are connected by -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2-. The cycloalkane ring, carbon ring, and cycloalkyl in the cycloalkyl group may be cross-linked rings. 3-12Examples of cross-linked rings in a cycloalkyl group include bicyclo[1.1.0]butane, bicyclo[3.2.1]octane.

[0080] The term "heterocyclic group" in the present invention means a non-aromatic cyclic group containing one or more heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms, and it can be fully saturated or partially unsaturated. The ring can be 3 to 12 members, preferably 3 to 9 members of monocyclic, bicyclic or spirocyclic rings. Examples include oxetane, azetidinyl, piperazinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, tetrahydropyranyl, etc.

[0081] The term "effective amount" in the present invention refers to an amount that is effective in treating a disease, illness and / or condition, or brings about the cited effect. For example, an effective amount can be an amount that is effective in reducing the progression or severity of the condition or symptom being treated. The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, particularly in light of the disclosure provided herein. The term "effective amount" is intended to include an amount of a compound described herein or a combined amount of a compound described herein that is, for example, effective in treating or preventing a disease or condition of a host or treating a symptom of the disease or condition. Therefore, an "effective amount" generally refers to an amount that provides a desired effect.

[0082] The term "treatment" in the present invention includes (i) preventing a disease, pathology or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting a disease, pathology or medical condition, or preventing its development; (iii) alleviating a disease, pathology or medical condition; and / or (iv) reducing symptoms associated with the disease, pathology or medical condition. Thus, the term "treatment" can be extended to prevention, and can include preventing, reducing, stopping or reversing the progression or severity of the condition or symptom being treated. Thus, the term "treatment" can include (as the case may be) medical, therapeutic and / or prophylactic administration.

[0083] The term "inhibit" in the present invention refers to slowing, stopping or reversing the growth or progression of a disease, infection, disorder or cell group. Compared to the growth or progression that occurs in the absence of treatment or contact, the inhibition can be, for example, greater than about 20%, 40%, 60%, 80%, 90%, 95% or 99%.

[0084] The term "RNA" in the present invention refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides or linkers. RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyadenylic acid sequence and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, such as in vivo translation of an mRNA inside a mammalian cell, may produce an encoded polypeptide. RNA may be selected from a non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), cas9mRNA, antisense oligonucleotides and mixtures thereof. Antisense oligonucleotides or small interfering RNA (siRNA) may inhibit the expression of target genes and target proteins in vitro or in vivo.

[0085] The term "delivery" in the present invention refers to providing an entity to a target, for example, delivering a drug and / or a therapeutic agent and / or a preventive agent to a subject, which is a tissue and / or cell of a human and / or other animal.

[0086] The term "pharmaceutically acceptable salt" in the present invention refers to a salt formed when an acidic or basic group in the lipid compound of the present invention reacts with a suitable base or acid.

[0087] The term "isomer" in the present invention refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. A compound may contain one or more chiral centers and / or double bonds and may therefore exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis-trans isomers). The present application encompasses any and all isomers of the compounds described herein. Enantiomeric and stereoisomeric mixtures of compounds and methods of resolving them into their component enantiomers or stereoisomers are well known.

[0088] The term "pharmaceutically acceptable carrier" in the present invention refers to a diluent, adjuvant, excipient or vehicle administered together with the therapeutic agent, and is suitable for contacting the tissues of humans and / or other animals without excessive toxicity, irritation, allergic reactions or other problems or complications corresponding to a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and glucose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition can also optionally contain a small amount of wetting agent, emulsifier or pH buffer. Oral formulations may contain standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Specifically, for example, excipients include, but are not limited to, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending agents or dispersing agents, sweeteners, and hydration water. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, cross-linked carboxymethylcellulose sodium, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked polyvinyl pyrrolidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

[0089] The pharmaceutical composition of the present invention can act systemically and / or locally. For this purpose, they can be administered by suitable routes, for example, by injection (such as intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermal administration; or by oral, buccal, nasal, transmucosal, local, in the form of ophthalmic preparations or by inhalation administration. For these routes of administration, the pharmaceutical composition of the present invention can be administered in suitable dosage forms. The dosage form includes but is not limited to tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0090] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0091] About the synthesis of compounds:

[0092] Example 1 Synthesis of Compound Cap-2 (Control)

[0093] Compound Cap-2 (control)

[0094]

[0095] Compound Cap-2 (control) synthesis route:

[0096]

[0097] Specifically, compound B (3.5 g, 8.97 mmol) was added to a pyridine (40 ml) solution of compound A (1.0 g, 4.27 mmol), stirred at 75°C for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, saturated sodium bicarbonate solution (100 mL) was added to the reaction solution for quenching, and dichloromethane was used for extraction (100 mL x 3), and the liquid was separated. The organic layer was dried by adding anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1) gave compound C (2.0 g, 51.43%).

[0098] Triethylamine (0.5 g, 4.88 mmol) and DMAP (10 mg, 0.08 mmol) were added to a solution of compound C (1.0 g, 1.22 mmol) in carbon tetrachloride (20 mL), compound D (0.5 g, 4.88 mmol) was dissolved in dichloromethane (10 mL) and added to the above solution, stirred at room temperature for 1 hour, and the reaction was monitored by TLC. After the reaction was complete, saturated saline solution (30 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL x 3), separated, anhydrous sodium sulfate was added to the organic layer to dry, filtered, the filtrate was concentrated, and silica gel column chromatography (dichloromethane: methanol = 30: 1) was performed to obtain compound Cap-2 (control) (200 mg, 16.07%). 1 H NMR(400MHz, CDCl3) δ5.43(d,J=4.0Hz,2H),5.34(s,1H),4.32–4.04(m,4H),3.03-2.84(m,2H),2.65(s,6H),2.47(d,J=8.4Hz,4H),2.30–2.11 (m,3H),2.10–1.82(m,13H),1.60–1.40(m,16H),1.24–1.12(m,11H),1. 05(s,10H), 0.98–0.86(m,25H),0.71(s,6H); MS-ESI(m / z):920.8(M+H) + .

[0099] Example 2 Synthesis of Compound 1

[0100] The structure of compound 1 is shown below:

[0101]

[0102] The synthetic route of compound 1 is as follows:

[0103]

[0104] Specifically, compound B (3.5 g, 8.97 mmol) was added to a pyridine (40 mL) solution of compound A (1.0 g, 4.27 mmol), stirred at 75°C for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, saturated sodium bicarbonate solution (100 mL) was added to the reaction solution, extracted with dichloromethane (100 mL x 3), separated, anhydrous sodium sulfate was added to the organic layer to dry, filtered, the filtrate was concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1) was performed to obtain compound C (2.0 g, 51.43%).

[0105] Triethylamine (366 mg, 3.61 mmol) and DMAP (10 mg, 0.08 mmol) were added to a solution of compound C (740 mg, 0.90 mmol) in carbon tetrachloride (10 mL), compound D (56 mg, 0.90 mmol) was dissolved in dichloromethane (5 mL) and added to the above solution, stirred at room temperature for 16 hours, and the reaction was monitored by TLC. After the reaction was complete, saturated saline solution (20 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL x 3), separated, anhydrous sodium sulfate was added to the organic layer to dry, filtered, the filtrate was concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 3: 1) was performed to obtain compound E (400 mg, 45.57%).

[0106] Compound F (29 mg, 0.25 mmol), DCC (56 mg, 0.27 mmol) and DMAP (10 mg, 0.08 mmol) were added to a solution of compound E (200 mg, 0.23 mmol) in dichloromethane (10 mL), and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After the reaction was complete, saturated sodium bicarbonate solution (20 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (50 mL x 3). The organic layer was separated and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and subjected to silica gel column chromatography (dichloromethane: methanol = 20: 1) to obtain compound 1 (50 mg, 22.73%). 1 H NMR(400MHz, CDCl3)δ5.46–5.38(m,2H),4.96(s,1H),4.44–4.34(m,2H),4.2 8–4.19(m,4H), 3.34(s,2H), 3.07(s,2H), 2.80(s,6H), 2.47(d,J=7.6Hz,4H) ,2.08–1.81(m,14H),1.71–1.45(m,22H),1.33–1.26(m,6H),1.05(s,10H),0 .98–0.93(m,9H),0.92–0.86(m,14H),0.71(s,6H); MS-ESI(m / z):978.9(M+H) + .

[0107] Example 3 Synthesis of Compound 2

[0108] The structural formula of compound 2 is as follows

[0109]

[0110] The preparation method is the same as compound 1, and compound 2 can be prepared by using N,N-dimethylglycine instead of compound F as the raw material. 1HNMR(400MHz, CDCl3)δ5.46–5.38(m,2H),4.96(s,1H),4.44–4.34(m,2H),4. 28–4.19(m,2H), 3.34(s,2H), 3.07(s,2H), 2.80(s,6H), 2.47(d,J=7.6Hz,4H) ,2.08–1.81(m,14H),1.71–1.45(m,22H),1.33–1.26(m,6H),1.05(s,10H),0 .98–0.93(m,9H),0.92–0.86(m,14H),0.71(s,6H); MS-ESI(m / z):964.8(M+H) + .

[0111] Example 4 Synthesis of Compound 3

[0112] The structural formula of compound 3 is as follows

[0113]

[0114] The synthetic route of compound 3 is shown below

[0115]

[0116] Specifically, compound B (3.5 g, 8.97 mmol) was added to a pyridine (40 ml) solution of compound A (1.0 g, 4.27 mmol), stirred at 75°C for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, saturated sodium bicarbonate solution (100 ml) was added to the reaction solution to quench, 100 mL of dichloromethane was added to extract three times, the liquids were separated, anhydrous sodium sulfate was added to the organic layer to dry, filtered, the filtrate was concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1) was performed to obtain compound C (2.0 g, 51.43%).

[0117] Triethylamine (366 mg, 3.61 mmol) and DMAP (10 mg, 0.08 mmol) were added to a solution of compound C (740 mg, 0.90 mmol) in carbon tetrachloride (10 ml), compound D (56 mg, 0.90 mmol) was dissolved in dichloromethane (5 mL) and added to the above solution, stirred at room temperature for 16 hours, and the reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated saline solution was added to the reaction solution for dilution, 50 mL of dichloromethane was added for extraction three times, the liquids were separated, anhydrous sodium sulfate was added to the organic layer for drying, filtration was performed, the filtrate was concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 3: 1) was performed to obtain compound E (400 mg, 45.57%).

[0118] To a solution of compound E (333 mg, 0.38 mmol) in dichloromethane (10 ml), TEA (115 mg, 0.57 mmol) and compound F (99 mg, 0.57 mmol) were added, stirred at room temperature for 16 hours, and the reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated sodium bicarbonate solution was added to the reaction solution for dilution, 50 mL of dichloromethane was added for extraction three times, the liquids were separated, anhydrous sodium sulfate was added to the organic layer for drying, filtered, the filtrate was concentrated, and silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) was performed to obtain compound G (303 mg, 83.57%).

[0119] Ammonia water (2 ml) was added to a tetrahydrofuran (10 ml) solution of compound G (120 mg, 0.13 mmol), and the mixture was heated and stirred at 70°C in a closed system for 16 hours. The reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated saline was added to the reaction solution for dilution, and 50 mL of ethyl acetate was added for three extractions. The organic layer was dried by adding anhydrous sodium sulfate, filtered, and the filtrate was concentrated and subjected to silica gel column chromatography (dichloromethane: methanol = 20: 1) to obtain compound H (64 mg, 58.13%).

[0120] Compound I (8 mg, 0.08 mmol), EDCI (22 mg, 0.12 mmol) and HOBt (12 mg, 0.09 mmol) were added to a solution of compound H (64 mg, 0.07 mmol) in dichloromethane (10 ml), and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated sodium bicarbonate solution was added to the reaction solution for dilution, and 50 mL of dichloromethane was added for extraction three times. The organic layer was separated by adding anhydrous sodium sulfate to dry, filtered, and the filtrate was concentrated and subjected to silica gel column chromatography (dichloromethane: methanol = 20: 1) to obtain compound 3 (70 mg, 99.71%). 1H NMR (400MHz, CDCl3) δ7.57(s,1H),5.46–5.39(m,2H), 4.33–4.17(m,2H), 4.16–4.07(m,2H), 3.71–3.52(m,2H), 3.01(s,2H), 2.50–2.4 4(m,4H),2.33(s,6H),2.09–1.02(m,50H),0.98–0.93(d,J=6.4Hz,10H),0.92–0.88(m,16H),0.71(s,6H); MS-ESI(m / z):963.8(M+H)+.

[0121] Example 5 Synthesis of Compound 4

[0122] The structural formula of compound 4 is as follows

[0123]

[0124] The preparation method is the same as compound 3, and N,N-dimethylalanine is used as the raw material instead of compound I to obtain compound 4. MS-ESI (m / z): 977.8 (M+H)+.

[0125] Verification of compound effects:

[0126] Verification Example 1 Lipid Nanoparticle (LNP) Encapsulation

[0127] The basic preparation method of LNP is as follows:

[0128] The mRNA stock solution was dispersed in a 20mM acetic acid solution (pH = 5.3) to a final concentration of 200μg / mL (aqueous phase). The corresponding compounds prepared in Example 1-5 were mixed in a molar ratio of DOPE:DMG-PEG2000 = 60:30:0.75 to form a mixed lipid (oil phase). The flow rates of the aqueous phase and the oil phase were controlled to mix the mRNA with the lipid mixture by T mixing to obtain the mRNA encapsulated in LNP. The encapsulated LNP was diluted with a buffer, then concentrated by ultrafiltration, and the buffer was replaced, and the LNP was finally concentrated to 100μg / mL of mRNA, and the pH of the LNP was adjusted to about 7-8. Finally, the total content and free content of mRNA in the LNP were detected using a Ribogreen kit and 10% OTG as a demulsifier, and the encapsulation rate of the LNP was calculated. The final LNP product was diluted with diluent, added to the particle size pool (1 ml), and placed on the Malvern ZetaSizer instrument to detect the particle size of the LNP. The characterization data of the prepared LNP are shown in Table 1.

[0129] Since compounds 3 and 4 had obvious precipitation during the LNP preparation process and had poor drugability, no further studies were conducted.

[0130] Table 1 Characterization data of LNPs of corresponding compounds of Examples 1-3

[0131] serial number Particle size (nm) PDI Encapsulation efficiency Cap-2 (control) 115.6 0.05031 99.87 Compound 1 196.6 0.2136 99.19 Compound 2 138.4 0.036 97.60

[0132] Verification Example 2: Verification of LNP delivery effect

[0133] 1. Encapsulate the mRNA expressing Luciferase into the LNP formulations of compound Cap-2 (control), compound 1, and compound 2.

[0134] 2. The loaded LNP formulation was injected intravenously into Balb / c mice at a dose of 1 mg / kg, with 5 mice in each group.

[0135] 3. Detect the Luciferase fluorescence expression intensity of each mouse at 6 hours.

[0136] 4. Detection of fluorescence expression intensity: D-luciferin sodium salt (dose: 150 mg / kg) was injected intraperitoneally into each mouse 10 minutes before the test. The mice were then anesthetized with isoflurane and placed in an IVIS instrument, and bioluminescence was selected for detection.

[0137] 5. Determine the delivery effect of the compound based on the fluorescence intensity results.

[0138] The results are as follows Figure 1 As shown, the expression effects of compound 1 group and Cap-2 group were basically equivalent, and there was no significant difference in the expression levels between the two groups, while the expression level of compound 2 group was significantly lower than that of Cap-2 group.

[0139] In addition, 6 days after the above delivery effect test, 2 animals in the Cap-2 control group died and were subsequently dissected. From the appearance of the dead mice: the whole body was black, especially the extremities, and the abdomen was swollen. The dissection results showed that the gastrointestinal tract was swollen, other tissues and organs were compatible and indistinguishable, and the spleen was almost invisible and became elongated.

[0140] There was no animal death in compound 1 group and compound 2 group.

[0141] The above results show that the lipid compound 1 prepared by the present invention can not only be prepared with phospholipids and PEGylated lipids to obtain lipid nanoparticles, but also does not require the addition of cholesterol, thereby simplifying the composition of the nanoparticles. At the same time, the lipid nanoparticles obtained by using the lipid compound 1 of the present invention have better safety than the control group.

[0142] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that: The structural formula of the lipid compound is shown in formula (I): The X 1 , X 2 , X 3 Each independently is O or S; The X 4 is selected from O, S, N, substituted or unsubstituted C1-C 10 Alkylene, wherein the alkylene is selected from a straight chain alkylene and a branched alkylene; The Z is selected from O or NR 7 , the R 7 is selected from H, substituted or unsubstituted C1-C 10 Alkyl, wherein the alkyl is selected from a linear alkyl, a branched alkyl, a saturated alkyl or an unsaturated alkyl, a substituted or unsubstituted C3-C 12 Cycloalkyl; The R 1 , R 2 Each independently selected from steroid derivatives; The R 3 , R 4 are each independently selected from H, substituted or unsubstituted C1-C 10 Alkylene, wherein the alkylene is selected from a straight chain alkylene and a branched alkylene; The R 5 , R 6 are each independently selected from H, substituted or unsubstituted C1-C 10 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, wherein the alkyl is selected from a linear alkyl, a branched alkyl, a saturated alkyl or an unsaturated alkyl; or R 5 and R 6 Together with the nitrogen atom to which they are attached, they can form a C2-C 12 Heterocyclic group, the C2-C 12 The heterocyclyl group is optionally substituted with one to three C1-C6 alkyl groups.

2. The lipid compound or pharmaceutically acceptable salt thereof or stereoisomer thereof according to claim 1, characterized in that: The X 1 , X 2 , X 3 , X 4 All are O.

3. The lipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The Z is selected from O or NH.

4. The lipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The steroid derivative is selected from cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid or α-tocopherol; preferably, the R 1 , R 2 All selected More preferably, the R 1 , R 2 All selected 5. The lipid compound or pharmaceutically acceptable salt thereof or stereoisomer thereof according to claim 1, characterized in that: The R 3 is selected from C1-C7 straight chain alkylene; preferably, the R 3 is selected from C1-C5 straight chain alkylene; more preferably, the R 3 Selected from methylene or ethylene.

6. The lipid compound or pharmaceutically acceptable salt thereof or stereoisomer thereof according to claim 1, characterized in that: The R 4 is selected from substituted or unsubstituted C1-C7 straight chain alkylene; preferably, the R 4 is selected from C1-C5 straight chain alkylene; more preferably, the R 4 Selected from methylene or ethylene.

7. The lipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The R 5 , R 6 are each independently selected from substituted or unsubstituted C1-C5 alkyl; preferably, the R 5 , R 6 are selected from hydroxymethyl or hydroxyethyl; More preferably, the R 5 , R 6 are selected from methyl or ethyl.

8. The lipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The lipid compound is selected from any one of the following structural formulas:

9. A lipid nanoparticle, comprising the lipid compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

10. The lipid nanoparticle according to claim 9, characterized in that The lipid nanoparticles also include one or more active ingredients, which include chemical drugs, biological macromolecules, and nucleic acid drugs; preferably, the nucleic acid drugs are selected from RNA and DNA; preferably, the nucleic acid drugs are selected from any one of antisense nucleic acid, plasmid, mRNA, interfering nucleic acid, aptamer, antagomir, miRNA, ribozyme and siRNA; preferably, any one of DNA, mRNA, miRNA and siRNA.

11. The lipid nanoparticle according to claim 9, characterized in that The lipid nanoparticles also include phospholipids and PEGylated lipids.

12. The lipid nanoparticle according to any one of claims 9 to 11, characterized in that The diameter of the lipid nanoparticle is about 30 nm to about 200 nm; preferably, the diameter of the lipid nanoparticle is about 70 nm to about 150 nm; preferably, the diameter of the lipid nanoparticle is about 90 nm to about 110 nm.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the lipid nanoparticles according to any one of claims 9 to 12 and a pharmaceutically acceptable carrier.

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