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CN116854606B - Cationic lipid compound, composition and application thereof - Google Patents

Cationic lipid compound, composition and application thereof Download PDF

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CN116854606B
CN116854606B CN202311132650.7A CN202311132650A CN116854606B CN 116854606 B CN116854606 B CN 116854606B CN 202311132650 A CN202311132650 A CN 202311132650A CN 116854606 B CN116854606 B CN 116854606B
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dcm
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CN116854606A (en
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刘金良
姜骞
张世超
钱佳乐
荆慧泽
陈宇浩
孟丽苹
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Crystal Pharmaceutical Suzhou Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
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    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • 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
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    • A61K9/5123—Organic compounds, e.g. fats, sugars
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/08—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by a carboxylic acid having the esterifying carboxyl group bound to an acyclic carbon atom of an acyclic unsaturated carbon skeleton

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Abstract

The invention relates to a cationic lipid compound, a composition and application thereof. The compound is shown as the following formula (I). The invention also provides the use of lipid nanoparticles comprising the compounds as key components in nucleic acid delivery, including the components of the delivery vehicle, methods of preparation and methods of use.

Description

Cationic lipid compound, composition and application thereof
Technical Field
The present invention relates to the field of lipid delivery vehicles. In particular, the present invention relates to cationic lipid compounds for delivery of nucleic acids and compositions and uses thereof.
Background
Gene therapy is to introduce exogenous genes into target cells or tissues to replace, compensate, block or correct specific genes, so as to achieve the purpose of treating and preventing diseases. However, the foreign gene has a short circulation time in vivo and is easily degraded, so that it is difficult to enter a target cell or a target tissue. Therefore, searching for a good gene delivery vector, improving the in vivo delivery efficiency of gene drugs, is one of the key directions for improving the effectiveness of gene therapy products.
Currently, the most widely used gene delivery vehicles are lipid nanoparticles, which protect nucleic acids from rapid degradation in vivo, extend circulation time, and enhance targeted delivery. Lipid nanoparticles are composed of different lipid components including cationic lipid compounds, structural lipids, phospholipids, and PEG lipids, among others. Among them, cationic lipid compounds play a key role in nucleic acid entrapment and release, and thus it is important to develop novel, efficient, low-toxic cationic lipid compounds.
To overcome the shortcomings of the prior art, there remains a need for a new class of cationic lipid compounds that meet the delivery needs of gene therapy products for the development of gene-containing therapeutic products. The inventor designs and synthesizes the cationic lipid compound provided by the invention, and the lipid nanoparticle composition formed by the cationic lipid compound has advantages in at least one aspects of encapsulation efficiency, biosafety, transfection efficiency in cells and organisms, protein expression and the like, solves the problems existing in the prior art, and has very important significance for the development of gene therapy products.
Disclosure of Invention
The invention provides a novel cationic lipid compound, and a composition and application thereof.
According to the object of the present invention, there is provided a cationic lipid compound represented by the formula (I) or a pharmaceutically acceptable salt, isomer, stable isotope derivative thereof,
wherein,
L 1 and L 2 Each independently selected from-C (=o) O-, -OC (=o) -, -C (=o) NH-, and-NHC (=o) -;
G 1 and G 2 Each independently selected from the group consisting of substituted or unsubstituted alkylene, alkenylene, and alkynylene, the substitution being by at least one Q 1 Substitution;
R 1 and R is 2 Each independently selected from the group consisting of substituted or unsubstituted alkyl, alkenyl, and alkynyl groups, said substitution being by at least one Q 2 Substitution;
Q 1 each occurrence is independently selected from alkyl, alkenyl, and alkynyl;
Q 2 each occurrence is independently selected from alkyl, alkenyl, and alkynyl.
ADVANTAGEOUS EFFECTS OF INVENTION
The cationic lipid compound provided by the invention has the advantages of simple synthesis process, good stability, high efficiency (high transfection efficiency) and low toxicity; meanwhile, the preparation is stable and uniform and easy to prepare; can be used for transfection of various cell lines. Therefore, the preparation method has excellent transmissibility, and can efficiently deliver active substances (such as siRNA and mRNA in examples) into cells, tissues and organs, so that the efficient regulation and control of the active substances are realized.
Detailed Description
In order to make the technical scheme and the beneficial effects of the invention more obvious and understandable, the following detailed description is given by way of example. Unless defined otherwise, technical and scientific terms used herein have the same meaning as technical and scientific terms in the technical field to which this application belongs.
In one aspect, the present invention provides a cationic lipid compound represented by formula (I) or a pharmaceutically acceptable salt, isomer, stable isotope derivative thereof,
wherein,
L 1 and L 2 Each independently selected from-C (=o) O-, -OC (=o) -, -C (=o) NH-, and-NHC (=o) -;
G 1 and G 2 Each independently selected from the group consisting of substituted or unsubstituted alkylene, alkenylene, and alkynylene, the substitution being by at least one Q 1 Substitution;
R 1 and R is 2 Each independently selected from the group consisting of substituted or unsubstituted alkyl, alkenyl, and alkynyl groups, said substitution being by at least one Q 2 Substitution;
Q 1 each occurrence is independently selected from alkyl, alkenyl, and alkynyl;
Q 2 each occurrence is independently selected from alkyl, alkenyl, and alkynyl.
In certain embodiments, the L 1 Selected from-C (=o) O-and-OC (=o) -.
In certain embodiments, the L 2 Selected from-C (=o) O-and-OC (=o) -.
In certain embodiments, the G 1 Selected from substituted or unsubstituted alkylene groups, said substitution being by at least one Q 1 And (3) substitution.
In certain embodiments, the G 1 Selected from unsubstituted C 2-16 An alkylene group.
In certain embodiments, the G 1 Selected from unsubstituted C 2-8 An alkylene group.
In certain embodiments, the G 1 Selected from unsubstituted C 3 、C 4 、C 5 、C 6 And C 7 An alkylene group.
In certain embodiments, the G 1 Selected from the group consisting of、、、And->。
In certain embodiments, the G 2 Selected from substituted or unsubstituted alkylene groups, said substitution being by at least one Q 1 And (3) substitution.
In certain embodiments, the G 2 Selected from unsubstituted C 2-16 An alkylene group.
In certain embodiments, the G 2 Selected from unsubstituted C 2-8 An alkylene group.
In certain embodiments, the G 2 Selected from unsubstituted C 3 、C 4 、C 5 、C 6 And C 7 An alkylene group.
In certain embodiments, the G 2 Selected from the group consisting of、、、And->。
In certain embodiments, the Q 1 Each occurrence is independently selected from alkyl groups.
In certain embodiments, the Q 1 Each occurrence is independently selected from C 1-6 An alkyl group.
In certain embodiments, the Q 1 Each occurrence is independently selected from C 1-3 An alkyl group.
In certain embodiments, the R 1 Selected from the group consisting of substituted or unsubstituted alkyl and alkenyl groups, said substitution being by at least one Q 2 And (3) substitution.
In certain embodiments, the R 1 Selected from unsubstituted C 6-21 Alkyl and C 6-21 Alkenyl groups.
In certain embodiments, the R 1 Selected from unsubstituted C 8-18 Alkyl and C 6-18 Alkenyl groups.
In certain embodiments, the R 1 Selected from unsubstituted C 8 Alkyl, C 9 Alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 9 Alkenyl, C 10 Alkenyl, C 11 Alkenyl, C 12 Alkenyl, C 13 Alkenyl, C 14 Alkenyl, C 15 Alkenyl, C 16 Alkenyl, C 17 Alkenyl and C 18 Alkenyl groups.
In certain embodiments, the R 1 Selected from unsubstituted C 8 Alkyl, C 9 Alkyl, C 10 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 9 Alkenyl, C 17 Alkenyl and C 18 Alkenyl groups; the alkenyl group contains one or two unsaturated bonds.
In certain embodiments, the R 1 Selected from the group consisting of、、、、、、、、、、、、、、、、、And->。
In certain embodiments, the R 1 Selected from the group consisting of、、 、、、、、、And->。
In certain embodiments, the R 2 Selected from the group consisting of substituted or unsubstituted alkyl and alkenyl groups, said substitution being by at least one Q 2 And (3) substitution.
In certain embodiments, the R 2 Selected from unsubstituted C 6-21 Alkyl and C 6-21 Alkenyl groups.
In certain embodiments, the R 2 Selected from unsubstituted C 8-18 Alkyl and C 6-18 Alkenyl groups.
In certain embodiments, the R 2 Selected from unsubstituted C 8 Alkyl, C 9 Alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 9 Alkenyl, C 10 Alkenyl, C 11 Alkenyl, C 12 Alkenyl, C 13 Alkenyl, C 14 Alkenyl, C 15 Alkenyl, C 16 Alkenyl, C 17 Alkenyl and C 18 Alkenyl groups.
In certain embodiments, the R 2 Selected from unsubstituted C 8 Alkyl, C 9 Alkyl, C 10 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 9 Alkenyl, C 17 Alkenyl and C 18 Alkenyl groups; the alkenyl group contains one or two unsaturated bonds.
In certain embodiments, the R 2 Selected from the group consisting of、、、、、、、、、、、、、、、、、And->。
In certain embodiments, the R 2 Selected from the group consisting of、、 、、、、、、And->。
In certain embodiments, the Q 2 Each occurrence is independently selected from alkyl, alkenyl.
In certain embodiments, the Q 2 Each occurrence is independently selected from C 1-6 Alkyl, C 2-6 Alkenyl groups.
In certain embodiments, the Q 2 Each occurrence is independently selected from C 1-3 Alkyl, C 2-4 Alkenyl groups.
The invention provides a cationic lipid compound or pharmaceutically acceptable salts, isomers and stable isotope derivatives thereof, which is characterized in that the cationic lipid compound has one of the following structures:
、、、、、、、、、、、、、、、and。
in another aspect, the present invention provides a lipid nanoparticle composition comprising the above cationic lipid compound or a pharmaceutically acceptable salt, isomer, stable isotope derivative thereof.
In certain embodiments, the lipid nanoparticle composition further comprises one or more components of a therapeutic and/or prophylactic agent, a structural lipid, a phospholipid, or a PEG lipid.
In certain embodiments, the therapeutic and/or prophylactic agent is selected from a nucleic acid.
In certain embodiments, the nucleic acid may be exemplified by: siRNA, miRNA, shRNA expression vectors, antisense oligonucleotides, mRNAs, ribozymes, and the like.
In certain embodiments, the nucleic acid may be exemplified by: siRNA, miRNA, mRNA.
In certain embodiments, by phospholipid is meant a lipid that exists in either the form of an uncharged or neutral zwitterionic at physiological pH. As neutral lipids, there may be mentioned: di-oleoyl phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), lecithin (EPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), di-arachidyl phosphatidylcholine (DAPC), di-behenyl phosphatidylcholine (DBPC), di (xylosyl) phosphatidylcholine (DLPC), di-oleoyl phosphatidylcholine (DOPC), sphingomyelin, brain amide, di-oleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), phosphatidylethanolamine (POPE), di-oleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), and the like. The neutral lipid may be used alone or in combination of 2 or more kinds.
In certain embodiments, as the PEG lipid, there may be mentioned: PEG2000-DMG (PEG 2000-dimyristoylglycerol), PEG2000-DPG (PEG 2000-dimyristoylglycerol), PEG2000-DSG (PEG 2000-distearoyl glycerol), PEG5000-DMG (PEG 5000-dimyristoylglycerol), PEG5000-DPG (PEG 5000-dimyristoylglycerol), PEG5000-DSG (PEG 5000-distearoyl glycerol), PEG-cDMA (N- [ (methoxypoly (ethylene glycol) 2000) carbamoyl ] -1, 2-dimyristoxypropyl-3-amine), PEG-C-DOMG (R-3- [ (omega-methoxy-poly (ethylene glycol) 2000) carbamoyl ] -1, 2-dimyristoxypropyl-3-amine), polyethylene glycol (PEG) -Diacylglycerol (DAG), PEG-Dialkyloxypropyl (DAA), PEG-phospholipids, PEG-brain amide (Cer), ALC-59 (2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide), and the like.
In certain embodiments, as PEG-dialkyloxypropyl, there may be mentioned: PEG-dilauryloxypropyl, PEG-dimyristoxypropyl, PEG-dipalmitoxypropyl, PEG-distearyloxy propyl, and the like. The polyethylene glycol modified lipid may be used alone in an amount of 1 or in an amount of 2 or more.
In certain embodiments, as structural lipids, there may be mentioned: cholesterol, dihydrocholesterol, lanosterol, beta-phytosterol, campesterol, stigmasterol, brassicasterol, ergosterol, algae sterol, 3 beta- [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol), and the like. The sterols may be used alone or in combination of 2 or more.
In certain embodiments, the compositions of the present examples contain, for example, 10 to 80 mole%, for example, 20 to 70 mole%, for example, 30 to 60 mole%, of the cationic lipid compound described above, based on the total lipid contained in the composition. The cationic lipid compound may be used alone or in combination of 2 or more.
In certain embodiments, the compositions of the present examples may contain, for example, from 0 to 50 mole%, such as from 0 to 40 mole%, such as from 0 to 30 mole%, such as from 0 to 15 mole%, of phospholipids, based on the total lipid contained in the composition.
In certain embodiments, the compositions of the present examples may contain, for example, from 0 to 10 mole%, such as from 0 to 5 mole%, such as from 0 to 3 mole%, such as from 0 to 2 mole%, such as from 0.5 to 2 mole%, of PEG lipid, based on the total lipid contained in the composition.
In certain embodiments, the compositions of the present examples may contain, for example, from 0 to 70 mole%, for example, from 10 to 60 mole%, for example, from 20 to 50 mole%, of structural lipids, based on the total lipids contained in the composition.
In certain embodiments, the compositions of the present examples contain, for example, 1 to 30 wt%, for example, 2 to 25 wt%, for example, 3 to 20 wt% nucleic acid, for example, 4 to 15 wt% nucleic acid, relative to the total weight of the composition.
In another aspect, the present invention provides the use of a cationic lipid compound as described above, or a pharmaceutically acceptable salt, isomer, stable isotope derivative thereof, or a product of any of the following functions 1) to 4) in a lipid nanoparticle composition as described above:
1) Encapsulating the active;
2) Delivering an active agent to a cell, tissue or organ;
3) Allowing the active substance to exert its activity in cells, tissues or organs;
4) Preventing, diagnosing and/or treating diseases.
In certain embodiments, the present invention provides the use of the above cationic lipid compound or a pharmaceutically acceptable salt, isomer, stable isotope derivative or lipid nanoparticle composition described above in the preparation of a pharmaceutical carrier for delivering a therapeutic and/or prophylactic agent to a cell.
Term interpretation:
unless stated to the contrary, the terms used in the specification and claims have the following meanings.
The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 25 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl 4, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, it may be substituted at any available point of attachment, and the substituents are preferably independently optionally selected from one or more of deuterium, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.
The above alkyl groups are derived from residues derived from the removal of one hydrogen atom from the parent atom, i.e. "alkylene".
The term "alkenyl" refers to an alkyl compound having at least one carbon-carbon double bond in the molecule, wherein alkyl is as defined above. Alkenyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more substituents independently selected from one or more of alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.
The term "alkynyl" refers to an alkyl compound having at least one carbon-carbon triple bond in the molecule, wherein alkyl is as defined above. Alkynyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more substituents independently selected from one or more of alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.
The compounds of the present disclosure include isotopic derivatives thereof. The term "isotopically-enriched derivative" refers to a compound that differs in structure only in the presence of one or more isotopically-enriched atoms. For example, with the structures of the present disclosure, replacement of hydrogen with "deuterium" or "tritium", or with 18 F-fluorine labeling [ ] 18 F isotope) instead of fluorine, or with 11 C-， 13 C-, or 14 C-enriched carbon 11 C-， 13 C-, or 14 C-carbon labeling; 11 C-， 13 c-, or 14 C-isotopes) are within the scope of this disclosure. Such compounds may be used, for example, as analytical tools or probes in biological assays, orCan be used as an in vivo diagnostic imaging tracer for diseases or as a tracer for pharmacodynamic, pharmacokinetic or receptor studies. The various deuterated forms of the compounds of the present disclosure mean that each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom. Those skilled in the art are able to refer to the relevant literature for the synthesis of deuterated forms of the compounds. Commercially available deuterated starting materials may be used in preparing the deuterated form of the compound or they may be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated borane, tridentate borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like. Deuterated compounds generally retain activity comparable to non-deuterated compounds and may achieve better metabolic stability when deuterated at certain specific sites, thus achieving certain therapeutic advantages.
"substituted" means that one or more hydrogen atoms, preferably 1 to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. The person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.
"cationic lipid" refers to a lipid that is positively charged at any pH or hydrogen ion activity of its environment, or is capable of being positively charged in response to the pH or hydrogen ion activity of its environment (e.g., the environment in which it is intended to be used). Thus, the term "cation" encompasses both "permanent cations" and "cationizable". In certain embodiments, the positive charge in the cationic lipid results from the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid is positively charged in the environment of its intended use (e.g., at physiological pH). In certain embodiments, the cationic lipid is one or more lipids of formula (I) as described herein.
"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.
The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, and are effective for the intended use.
By "pharmaceutically acceptable salts" is meant salts of the compounds of the present disclosure which are safe and effective when used in a mammal, and which possess the desired biological activity. Salts may be prepared separately during the final isolation and purification of the compounds, or by reacting the appropriate groups with an appropriate base or acid. Bases commonly used to form pharmaceutically acceptable salts include inorganic bases, as well as organic bases. Acids commonly used to form pharmaceutically acceptable salts include inorganic and organic acids.
The term "isomer" means any geometric isomer, tautomer, zwitterionic, stereoisomer, enantiomer or diastereomer of a compound. The compounds may include one or more chiral centers and/or double bonds and thus 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 disclosure encompasses any and all isomers of the compounds described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as enantiomers and stereoisomeric mixtures, e.g., racemates. The enantiomers and mixtures of stereoisomers of a compound and the manner in which it is resolved into its constituent enantiomers or stereoisomers are well known.
As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
The terms involved in the present invention are defined above, and those skilled in the art can understand the above terms in combination with the prior art, and the following is further described based on the contents of the present invention and the definition of the terms.
The "room temperature" is not a specific temperature value, but means a temperature range of 10-30 ℃.
The term "about" when used in reference to a measurable value, such as mass, time, temperature, etc., means that there may be some range of float around a particular value, which may be + -10%, + -5%, + -1%, + -0.5%, or + -0.1%.
The synthetic processes of the present disclosure may tolerate multiple functional groups, and thus various substituted starting materials may be used. These processes generally provide the desired final compound at or near the end of the overall process, although in some cases it may be desirable to further convert the compound to a pharmaceutically acceptable salt thereof.
The compounds of the present disclosure may be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates by employing standard synthetic methods and procedures known to those skilled in the art or apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for preparing organic molecules, functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field.
The following description of synthetic methods is designed to illustrate, but not limit, the general procedure used to prepare the compounds of the present disclosure. The compounds of the invention having the formulae described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature procedures according to the procedures set forth in the representative synthetic methods below. Variables in schemes (e.g. R 1 And R is 2 Etc. as defined herein). It should be noted by those skilled in the art that the order of certain steps may be varied during the reaction procedures and synthetic schemes described herein, such as the introduction and removal of protecting groups. One of ordinary skill in the art will recognize that it may be desirable to protect certain groups from reaction conditions using protecting groupsInfluence.
Preferred protecting groups include, but are not limited to:
for the hydroxyl moiety: TBS, benzyl, THP, ac;
for carboxylic acids: benzyl, methyl, ethyl, allyl esters;
for amines: fmoc, cbz, BOC, DMB, ac, bn, tr, ts, trifluoroacetyl, phthalimide, benzylidene amine;
for diols: ac (×2), TBS (×2) or acetone when taken together;
for thiols: ac;
for benzimidazoles: SEM, benzyl, PMB, DMB;
for aldehydes: dialkyl acetals, such as dimethoxy acetal or diethyl acetyl.
In the reaction schemes described herein, a variety of stereoisomers may be produced. When a particular stereoisomer is not indicated, this is understood to mean all possible stereoisomers that can be produced by the reaction. Those of ordinary skill in the art will recognize that the reaction may be optimized to preferentially yield one isomer, or new schemes may be devised to make a single isomer. If a mixture is produced, the isomers may be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC or preparative SFC.
EXAMPLE 1 Synthesis of Compound T01
Step 1: synthesis of Compound T01-I
To a mixture of 1, 6-hexanediol (27.95 g), DCM (475 mL) was added 2-hexyldecanoic acid (19.51 g), DCC (18.93 g), and DMAP (11.37 g). The reaction system was stirred at room temperature for three days and then filtered. Petroleum ether (500, mL) was added to the filtrate, and the solid was precipitated by stirring. After filtration, the filtrate was washed with dilute hydrochloric acid (1M). The washed organic phase was dried over anhydrous sodium sulfate, filtered and then distilled off in a rotary manner to obtain a crude product of the compound T01-I.
Step 2: synthesis of Compound T01
To compounds T01-I (35.21 g) and NEt at 0deg.C 3 TsCl (25.96 g) was slowly added to a mixture of (13.99 g), DMAP (1.13 g), DCM (200 mL), followed by warming to room temperature and stirring overnight. Saturated sodium bicarbonate aqueous solution was added to the reaction system, and the organic phase was dried over anhydrous magnesium sulfate, filtered and evaporated. The residue is separated and purified by a silica gel column (the eluent is petroleum ether/ethyl acetate) to obtain a compound T01.
1 H NMR (400 MHz, CDCl 3 ) δ 7.78 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.02 (t, J = 6.5 Hz, 4H), 2.45 (s, 3H), 2.34 - 2.25 (m, 1H), 1.68 - 1.16 (32H), 0.87 (t, 6H)。
MS m/z (APCI): [M+H]: 511.43。
EXAMPLE 2 Synthesis of Compound T13
DCC (8.59 g), DMAP (4.25 g) and 1-nonanol (5.11 g) were added sequentially to a solution of 8-bromooctanoic acid (8.50 g) in DCM (50 mL) and stirred at room temperature 19 h. Petroleum ether (50 mL) is added into the reaction system, after shaking, filtration is carried out, and the residue after concentration of the filtrate is separated and purified by a silica gel column (the eluent is petroleum ether/ethyl acetate=50:1), so that the compound T13 is obtained.
1 H NMR (400 MHz, CDCl 3 ) δ 4.05 (t, J = 6.7 Hz, 2H), 3.40 (t, J = 6.9 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.98 - 1.78 (m, 2H), 1.74 - 1.55 (m, 2H), 1.43 (d, J = 4.3 Hz, 2H), 1.38 - 1.19 (m, 17H), 0.87 (td, J = 6.7, 2.6 Hz, 4H)。
EXAMPLE 3 Synthesis of Compound T14
DCC (5.1434 g), DMAP (2.5291 g) were added sequentially to a mixture of 6-bromohexanoic acid (4.4864 g), 2-hexyl-1-decanol (5.0754 g) and DCM (50 mL), and stirred at room temperature for 18 hours, the filter cake was washed once with DCM (20 mL), and the filtrate was evaporated by rotary evaporation. The residue is separated and purified by a silica gel column (the eluent is petroleum ether/ethyl acetate) to obtain a compound T14.
1 H NMR (400 MHz, CDCl 3 ) δ 3.97 (d, J = 5.8 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.88 (dt, J = 14.3, 6.9 Hz, 2H), 1.66 (dt, J = 15.3, 7.4 Hz, 3H), 1.54-1.44 (m, 2H), 1.33-1.21 (m, 24H), 0.88 (t, J = 6.7 Hz, 6H)。
EXAMPLE 4 Synthesis of Compound T15
DCC (6.8020 g) and DMAP (2.3812 g) were added to a mixture of 9-heptadecanol (5.0075 g) and 7-bromoheptanoic acid (4.4718 g), dichloromethane (50 mL), and stirred at room temperature for 24 h. Filtering the reaction system, removing the solvent by rotary evaporation, and separating and purifying the residue by a silica gel column (the eluent is petroleum ether/ethyl acetate) to obtain a compound T15.
1 H NMR (400 MHz, CDCl 3 ) δ 3.96 (d, J = 5.8 Hz, 2H), 3.39 (t, J = 6.8 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 1.85 (p, J = 6.9 Hz, 2H), 1.69 - 1.58 (m, 3H), 1.52 - 1.41 (m, 2H), 1.39 - 1.19 (m, 28H), 0.87 (t, J = 6.6 Hz, 6H)。
EXAMPLE 5 Synthesis of Compound T16
DCC (15.68 g) was added to a mixture of compound 7-bromoheptanoic acid (10.01 g) and 2-hexyl-1-decanol (11.36 g), DMAP (6.65 g), dichloromethane (250 mL) and stirred at room temperature for 16 hours. The reaction system was diluted with dichloromethane (200 mL), filtered and distilled. The residue was purified by silica gel column (petroleum ether/ethyl acetate as eluent) to give compound T16.
1 H NMR (400 MHz, CDCl 3 ) δ 3.97 (d, J = 5.8 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H), 1.86 (dt, J = 14.4, 6.9 Hz, 2H), 1.68 - 1.59 (m, 3H), 1.51 - 1.42 (m, 2H), 1.40 - 1.33 (m, 2H), 1.27 (d, J = 3.7 Hz, 24H), 0.88 (t, J = 6.8 Hz, 6H)。
EXAMPLE 6 Synthesis of Compound T17
DCC (11.75 g) was added to a mixture of compound 8-bromooctanoic acid (8.71 g) and 3-octanol (5.00 g), DMAP (4.72 g), dichloromethane (100 mL) and stirred at room temperature for 16 hours. The reaction system was diluted with dichloromethane (200 mL), filtered and distilled. The residue was purified by separation on a silica gel column (petroleum ether/ethyl acetate as eluent) to give compound T17.
1 H NMR (400 MHz, CDCl 3 ) δ 4.81 (ddd, J = 12.5, 6.8, 5.5 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.95 - 1.69 (m, 3H), 1.65 - 1.49 (m, 6H), 1.46 - 1.41 (m, 2H), 1.30 (ddd, J = 19.2, 7.2, 2.9 Hz, 9H), 0.87 (td, J = 7.2, 2.2 Hz, 6H)。
EXAMPLE 7 Synthesis of Compound T18
DCC (8.75 g), DMAP (5.12 g) and cis-2-nonen-1-ol (5.12 g) were added sequentially to a solution of 8-bromooctanoic acid (7.77 g) in DCM (100 mL) and stirred at room temperature for 18 h. Petroleum ether (100 mL) is added into the reaction system, after shaking, filtration is carried out, and the residue after concentration of the filtrate is separated and purified by a silica gel column (the eluent is petroleum ether/ethyl acetate) to obtain the compound T18.
1 H NMR (400 MHz, CDCl 3 ) δ 5.79 - 5.59 (m, 1H), 5.59 - 5.45 (m, 1H), 4.71 - 4.53 (m, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 2.10 (qd, J = 7.4, 1.4 Hz, 2H), 1.85 (p, J = 6.9 Hz, 2H), 1.69 - 1.59 (m, 2H), 1.49 - 1.22 (m, 14H), 0.93 - 0.83 (m, 3H)。
EXAMPLE 8 Synthesis of Compound T23
DCC (14.51 g), DMAP (7.80 g) and 6-bromohexanol (9.72 g) were added sequentially to a solution of 2-hexyldecanoic acid (15.38 g) in DCM (150 mL), and stirred at room temperature 24. 24 h. Petroleum ether (150, mL) is added into the reaction system, after shaking, filtration is carried out, and the residue after concentration of the filtrate is separated and purified by a silica gel column (the eluent is petroleum ether/ethyl acetate) to obtain the compound T23.
1 H NMR (400 MHz, CDCl 3 ) δ 4.07 (t, J = 6.6 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 2.31 (tt, J = 9.0, 5.3 Hz, 1H), 1.93 - 1.81 (m, 2H), 1.65 (dt, J = 14.0, 6.7 Hz, 2H), 1.52 - 1.36 (m, 6H), 1.36 - 1.20 (m, 20H), 0.88 (t, J = 6.8 Hz, 8H)。
EXAMPLE 9 Synthesis of Compound T27
DCC (0.9288 g), DMAP (0.5352 g) and (9 z,12 z) -octadecane-9, 12-dien-1-ol (1.0486 g) were added sequentially to a solution of 5-bromopentanoic acid (0.7605 g) in DCM (15 mL) and stirred at room temperature for 19 h. Petroleum ether (15, mL) is added into the reaction system, after shaking, filtration is carried out, and the residue after concentration of the filtrate is separated and purified by a silica gel column (the eluent is petroleum ether/ethyl acetate=100:1), so that the compound T27 is obtained.
1 H NMR (400 MHz, CDCl 3 ) δ 5.49 - 5.21 (m, 4H), 4.13 - 4.03 (t, J = 6.7 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H), 2.75 (t, J = 6.5 Hz, 2H), 2.34 (t, J = 7.3 Hz, 2H), 2.05 (q, J = 6.8 Hz, 4H), 1.95 - 1.69 (m, 4H), 1.61 (q, J = 7.0 Hz, 2H), 1.32 (qddd, J = 12.1, 9.4, 6.3, 3.8 Hz, 16H), 0.91 - 0.87 (t, J = 6.8 Hz, 3H)。
EXAMPLE 10 Synthesis of Compound T29
To a mixture of the compounds linoleic acid (1.0169 g), DCC (0.8883 g), DMAP (0.4450 g) and DCM (20 mL) was added 3-bromopropanol (0.5017 g), which was stirred at room temperature for 20 hours. Suction filtration, filter cake washing with DCM (20 mL) and filtrate rotary evaporation. The residue was purified by separation on a silica gel column (petroleum ether/ethyl acetate as eluent) to give compound T29.
1 H NMR (400 MHz, CDCl 3 ) δ 5.58 - 5.15 (m, 4H), 4.21 (t, J = 6.0 Hz, 2H), 3.46 (t, J = 6.6 Hz, 2H), 2.90 - 2.64 (m, 2H), 2.30 (t, J = 7.5 Hz, 2H), 2.18 (q, J = 6.3 Hz, 4H), 2.05 (q, J = 6.8 Hz, 2H), 1.65 - 1.59 (m, 2H), 1.39 - 1.22 (m, 14H), 0.95 - 0.83 (m, 3H)。
EXAMPLE 11 Synthesis of Compound T30
To a mixture of the compounds linoleic acid (0.5960 g), DCC (0.5396 g), DMAP (0.2515 g) and DCM (20 mL) was added 4-bromobutanol (0.4038 g), which was stirred at room temperature for 18 hours. The mixture was filtered under reduced pressure, the filter cake was washed with DCM (20 mL), and the filtrate was evaporated. The residue was purified by column chromatography on silica gel (eluent DCM/MeOH) to give compound T30.
1 H NMR (400 MHz, CDCl 3 ) δ 5.83 - 4.99 (m, 4H), 4.10 (t, J = 6.3 Hz, 2H), 3.43 (t, J = 6.6 Hz, 2H), 3.09 - 2.55 (m, 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.05 (q, J = 6.9 Hz, 4H), 1.99 - 1.89 (m, 2H), 1.85 – 1.75 (m, 2H), 1.70 - 1.49 (m, 2H), 1.31 (q, J = 4.4, 2.9 Hz, 14H), 0.94 - 0.84 (m, 3H)。
EXAMPLE 12 Synthesis of Compound T31
DCC (3.1352 g), DMAP (2.0727 g) and 4-decanol (2.0050 g) were added sequentially to a solution of 8-bromooctanoic acid (3.1429 g) in DCM (50 mL) and stirred at room temperature for 16 h. Petroleum ether (50, mL) is added into the reaction system, after shaking, filtration is carried out, and the residue after concentration of the filtrate is separated and purified by a silica gel column (the eluent is petroleum ether/ethyl acetate) to obtain the compound T31.
1 H NMR (400 MHz, CDCl 3 ) δ 4.89 (ddd, J = 12.5, 7.0, 5.5 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.85 (p, J = 7.0 Hz, 2H), 1.70 - 1.57 (m, 2H), 1.48 (ddddd, J = 18.4, 15.2, 9.5, 6.6, 3.3 Hz, 5H), 1.38 - 1.19 (m, 15H), 0.89 (dt, J = 9.9, 7.2 Hz, 6H)。
EXAMPLE 13 Synthesis of Compound T36
To a mixture of compound 8-bromooctanoic acid (3.4143 g), DCC (3.4680 g), DMAP (1.6740 g) and DCM (50 mL) was added 3-nonanol (2.0421 g) and stirred at room temperature for 18 hours. Suction filtration, filter cake washing with DCM (20 mL) and filtrate rotary evaporation. The residue was purified by column chromatography on silica gel (eluent DCM/MeOH) to give compound T36.
1 H NMR (400 MHz, CDCl 3 ) δ 4.81 (p, J = 6.2 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.85 (p, J = 7.0 Hz, 2H), 1.67 - 1.57 (m, 3H), 1.57 - 1.48 (m, 3H), 1.38 - 1.23 (m, 14H), 0.95 - 0.80 (m, 6H)。
EXAMPLE 14 Synthesis of Compound T37
DCC (1.7053 g), DMAP (1.0046 g) and 3-nonanol (1.0241 g) were added sequentially to a solution of 7-bromoheptanoic acid (1.16116 g) in dichloromethane (40 mL) and stirred at room temperature 22 h. Petroleum ether (40, mL) is added into the reaction system, after shaking, filtration is carried out, and the residue after concentration of the filtrate is separated and purified by a silica gel column (the eluent is petroleum ether/ethyl acetate) to obtain the compound T37.
1 H NMR (400 MHz, CDCl 3 ) δ 4.82 (p, J = 6.3 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.86 (p, J = 6.9 Hz, 2H), 1.65 (p, J = 7.5 Hz, 2H), 1.59 - 1.42 (m, 5H), 1.41 - 1.17 (m, 11H), 1.00 - 0.78 (m, 6H)。
EXAMPLE 15 Synthesis of Compound T38
DCC (1.5778 g) was added to a mixture of compound 7-bromoheptanoic acid (1.3154 g) and 4-decanol (0.9976 g), DMAP (0.8145 g), DCM (15 mL), and stirred at room temperature for 16 hours. The reaction was diluted with DCM, filtered off with suction and evaporated. The residue was purified by column chromatography on silica gel (eluent PE/EtOAc) to give compound T38.
1 H NMR (400 MHz, CDCl 3 ) δ 4.88 (ddd, J = 12.5, 7.0, 5.5 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.86 (p, J = 6.9 Hz, 2H), 1.64 (p, J = 7.5 Hz, 2H), 1.54 - 1.41 (m, 6H), 1.40 - 1.25 (m, 12H), 0.89 (dt, J = 9.7, 7.1 Hz, 6H)。
EXAMPLE 16 Synthesis of Compound H05T01
N, N' -dimethylethylenediamine (0.0335 g) and Compound T01 (0.2150 g) were dissolved in 10mL MeCN at room temperature at which point K was added 2 CO 3 (0.1070 g), the reaction was stirred at 70℃for 24. 24 h. The reaction solution was filtered, the filtrate was concentrated by rotary evaporation, and the residue was purified by column chromatography on silica gel (eluent DCM/MeOH, ratio 20:1) to give compound H05T01.
MS m/z (APCI): [M+H]: 427.48。
EXAMPLE 17 Synthesis of Compound H05T13
To a mixture of the compounds N, N' -dimethylethylenediamine (0.5056 g), T13 (0.2639 g) and MeCN (20 mL) was added K 2 CO 3 (0.0943 g) reacting at 75 ℃ with stirring about 18 h; the reaction was filtered and the filter cake was washed with DCM (20 mL), the filtrate was distilled off, and the residue was purified by column chromatography on silica gel (eluent DCM/MeOH) to give Compound H05T13.
MS m/z (APCI): [M+H]: 357.28。
EXAMPLE 18 Synthesis of Compound H05T14
K was added to a mixture of the compound N, N' -dimethylethylenediamine (0.2548 g) with T14 (0.8090 g), meCN (10 mL) 2 CO 3 (0.3535 g), at 75 o C stirred for 16 hours. The reaction was diluted with DCM, filtered under reduced pressure and evaporated. The residue was purified by column chromatography on silica gel (eluent DCM/MeOH) to give compound H05T14.
EXAMPLE 19 Synthesis of Compound H05T23
The compound N, N' -dimethylethylenediamine (0.9001 g) and T23 (2.0013 g) were dissolved in 50mL MeCN at room temperature at which point K was added 2 CO 3 (1.3172 g) the reaction was stirred at 72℃for 22: 22 h. The reaction solution was filtered, the filtrate was concentrated by rotary evaporation, and the residue was purified by column chromatography on silica gel (eluent DCM/MeOH, ratio 20:1) to give compound H05T23.
MS m/z (APCI): [M+H]: 427.54。
EXAMPLE 20 Synthesis of Compound 1
To a mixture of the compound N, N' -dimethylethylenediamine (0.0313 g), the compound T14 (0.3573 g) and acetonitrile (5 mL) was added potassium carbonate (0.1511 g), and the mixture was reacted at 75 ℃ with stirring about 16 h. The reaction system was filtered, the filter cake was washed with dichloromethane (20, mL), the filtrate was distilled off, and the residue was purified by separation on a silica gel column (eluent dichloromethane/methanol) to give compound 1.
1 H NMR (400 MHz, CDCl 3 ) δ 3.96 (d, J = 5.8 Hz, 4H), 2.69 (s, 3H), 2.50 (t, J = 7.8 Hz, 3H), 2.36 (s, 6H), 2.30 (t, J = 7.4 Hz, 4H), 1.73 - 1.50 (m, 10H), 1.41 - 1.16 (m, 54H), 0.87 (t, J = 6.7 Hz, 12H)。
MS m/z (APCI): [M+H]: 765.85。
EXAMPLE 21 Synthesis of Compound 2
K is added to a mixture of the compound H05T01 (0.1038 g) with T15 (0.1282 g), meCN (6 mL) 2 CO 3 (0.09648 g) the reaction was stirred at 75℃for about 18 h. The reaction was filtered and the filter cake was washed with DCM (20, mL) and the filtrate was distilled off and the residue was purified by column chromatography on silica gel (eluent DCM/MeOH) to give compound 2.
1 H NMR (400 MHz, CDCl 3 ) δ 4.85 (p, J = 6.3 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H), 2.55 (s, 3H), 2.40 (t, J = 7.6 Hz, 4H), 2.34 - 2.19 (m, 9H), 1.66 - 1.56 (m, 5H), 1.49 (p, J = 5.9 Hz, 8H), 1.37 - 1.16 (m, 56H), 0.87 (t, J = 6.7 Hz, 12H)。
MS m/z (APCI): [M+H]: 793.94。
EXAMPLE 22 Synthesis of Compound 3
K is added to a mixture of the compound H05T01 (0.1065 g) with T17 (0.0976 g), meCN (6 mL) 2 CO 3 (0.0964 g) and stirred at 75℃for about 18 h. The reaction was filtered and the filter cake was washed with DCM (20 mL), the filtrate was distilled off, and the residue was purified by separation on a silica gel column (eluent DCM/MeOH) to give Compound 3.
1 H NMR (400 MHz, CDCl 3 ) δ 4.80 (ddd, J = 12.5, 6.9, 5.5 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H), 3.35 (s, 2H), 2.69 (s, 3H), 2.55 - 2.45 (m, 4H), 2.40 - 2.22 (m, 9H), 1.68 - 1.47 (m, 14H), 1.37 - 1.15 (m, 37H), 0.94 - 0.74 (m, 12H)。
MS m/z (APCI): [M+H]: 681.73。
EXAMPLE 23 Synthesis of Compound 4
K was added to a mixture of the compounds H05T13 (0.1207 g) with T14 (0.1752 g), meCN (6 mL) 2 CO 3 (0.0926 g) and stirred at 75℃for about 18. 18 h. The reaction was filtered and the filter cake was washed with DCM (20 mL), the filtrate was evaporated in vacuo and the residuePurification by column chromatography on silica gel (eluent DCM/MeOH) afforded Compound 4.
1 H NMR (400 MHz, CDCl 3 ) δ 4.05 (t, J = 6.8 Hz, 2H), 3.96 (d, J = 5.8 Hz, 2H), 2.63 (s, 3H), 2.46 (q, J = 8.7, 7.9 Hz, 4H), 2.38 - 2.24 (m, 9H), 1.76 - 1.45 (m, 10H), 1.38 - 1.19 (m, 47H), 0.88 (t, J = 6.7 Hz, 9H)。
MS m/z (APCI): [M+H]: 695.71。
EXAMPLE 24 Synthesis of Compound 5
Compound H05T23 (0.0847 g) and T18 (0.0785 g) were dissolved in 10mL MeCN at room temperature at which point K was added 2 CO 3 (0.0548 g) the reaction was stirred at 75℃for 20: 20 h. The reaction solution was filtered, the filtrate was concentrated by rotary evaporation, and the residue was purified by column chromatography on silica gel (eluent DCM/MeOH, ratio 20:1) to give Compound 5.
1 H NMR (400 MHz, CDCl 3 ) δ 5.70 - 5.58 (m, 1H), 5.58 - 5.48 (m, 1H), 4.62 (dd, J = 7.0, 1.2 Hz, 2H), 4.06 (t, J = 6.6 Hz, 2H), 2.49 (s, 3H), 2.40 - 2.21 (m, 13H), 2.14 - 2.05 (m, 2H), 1.53 - 1.19 (m, 51H), 0.88 (tt, J = 6.2, 3.0 Hz, 9H)。
MS m/z (APCI): [M+H]: 693.76。
EXAMPLE 25 Synthesis of Compound 6
Compounds H05T23 (0.0811 g) and T27 (0.0921 g) were dissolved in 10mL MeCN at room temperature at which point K was added 2 CO 3 (0.0595 g) the reaction was stirred at 75℃for 18: 18 h. Filtering the reaction solution, concentrating the filtrate by rotary evaporation, and separating the residue by silica gel columnPurification by separation (eluent DCM/MeOH, ratio 20:1) afforded Compound 6.
1 H NMR (400 MHz, CDCl 3 ) δ 5.45 - 5.30 (m, 4H), 4.05 (td, J = 6.7, 3.2 Hz,4H), 2.77 (t, J = 6.5 Hz, 2H), 2.53 (s, 3H), 2.39 (d, J = 7.2 Hz, 3H), 2.36 - 2.19 (m, 9H), 2.05 - 1.99 (q, J = 6.6 Hz, 4H), 1.89 (s, 3H), 1.61 - 1.56 (p, J = 6.8 Hz, 6H), 1.43 - 1.15 (m, 47H), 0.88 (q, J = 6.6 Hz, 9H)。
MS m/z (APCI): [M+H]: 775.73。
EXAMPLE 26 Synthesis of Compound 7
K was added to a mixture of Compound H05T14 (0.1024 g) with T15 (0.1631 g), meCN (10 mL) 2 CO 3 (0.0771 g) and stirred at 75℃for 16 hours. The reaction was diluted with DCM, filtered under reduced pressure and evaporated. The residue was purified by column chromatography over silica gel (eluent DCM/MeOH) to give compound 7.
1 H NMR (400 MHz, CDCl 3 ) δ 4.85 (p, J = 6.3 Hz, 1H), 3.96 (d, J = 5.8 Hz, 2H), 2.67 (s, 4H), 2.49 (q, J = 7.8 Hz, 6H), 2.40 - 2.25 (m, 11H), 1.69 - 1.45 (m, 14H), 1.37 - 1.25 (m, 50H), 0.87 (td, J = 6.9, 2.1 Hz, 12H)。
MS m/z (APCI): [M+H]: 793.94。
EXAMPLE 27 Synthesis of Compound 8
K was added to a mixture of the compounds H05T14 (0.1105 g) with T16 (0.1510 g), meCN (10 mL) 2 CO 3 (0.0690 g) and stirred at 75℃for 16 hours. The reaction was diluted with DCM, filtered under reduced pressure and evaporated. The residue was purified by column chromatography over silica gel (eluent DCM/MeOH) to give compound 8.
1 H NMR (400 MHz, CDCl 3 ) δ 3.96 (d, J = 5.8 Hz, 4H), 2.65 (s, 4H), 2.54 - 2.42 (m, 4H), 2.38 - 2.26 (m, 11H), 2.25 - 2.10 (m, 4H), 1.68 - 1.50 (m, 10H), 1.38 - 1.25 (m, 49H), 0.88 (t, J = 6.7 Hz, 12H)。
MS m/z (APCI): [M+H]: 779.83。
EXAMPLE 28 Synthesis of Compound 9
K was added to a mixture of the compounds H05T14 (0.0983 g) with T23 (0.1592 g), meCN (10 mL) 2 CO 3 (0.0784 g) and stirred at 75℃for 16 hours. The reaction was diluted with DCM, filtered under reduced pressure and evaporated. The residue was purified by column chromatography on silica gel (eluent DCM/MeOH) to give compound 9.
1 H NMR (400 MHz, CDCl 3 ) δ 4.05 (t, J = 6.6 Hz, 2H), 3.96 (d, J = 5.8 Hz, 2H), 2.66 (s, 4H), 2.49 (d, J = 7.8 Hz, 4H), 2.38 - 2.27 (m, 10H), 2.24 - 2.09 (m, 5H), 1.69 - 1.50 (m, 12H), 1.46 - 1.25 (m, 45H), 0.87 (dt, J = 7.0, 3.3 Hz, 12H)。
MS m/z (APCI): [M+H]: 765.78。
EXAMPLE 29 Synthesis of Compound 10
K is added to a mixture of the compound H05T01 (0.1002 g) with T30 (0.1127 g), meCN (6 mL) 2 CO 3 (0.0956 g) stirring at 75 ℃Reaction was about 18 h. The reaction was filtered and the filter cake was washed with DCM (20, mL) and the filtrate was distilled off, and the residue was purified by separation on a silica gel column (eluent DCM/MeOH) to give compound 10.
1 H NMR (400 MHz, CDCl 3 ) δ 5.28 (tddd, J = 12.7, 8.7, 4.5, 1.7 Hz, 4H), 4.00 (q, J = 6.6 Hz, 4H), 2.70 (t, J = 6.5 Hz, 2H), 2.60 (s, 3H), 2.53 - 2.37 (m, 4H), 2.32 (s, 3H), 2.29 - 2.18 (m, 6H), 1.98 (q, J = 6.7 Hz, 4H), 1.66 - 1.45 (m, 10H), 1.33 - 1.11 (m, 43H), 0.81 (q, J = 6.7 Hz, 9H)。
MS m/z (APCI): [M+H]: 761.81。
EXAMPLE 30 Synthesis of Compound 11
To a mixture of compound H05T01 (0.1084 g) with T29 (0.1163 g), acetonitrile (6 mL) K was added 2 CO 3 (0.0936 g) and stirred at 75℃for about 18 h. The reaction was filtered and the filter cake was washed once with DCM (20 mL), the filtrate was distilled off, and the residue was purified by separation on a silica gel column (eluent DCM/MeOH) to give Compound 11.
1 H NMR (400 MHz, CDCl 3 ) δ 5.48 - 5.16 (m,4H), 4.08 (dt, J = 17.6, 6.6 Hz, 4H), 2.76 (t, J = 6.4 Hz, 2H), 2.52 (s, 3H), 2.42 (dt, J = 13.9, 7.5 Hz, 4H), 2.33 - 2.21 (m, 9H), 2.04 (q, J = 6.9 Hz, 4H), 1.87 - 1.74 (m, 2H), 1.68 - 1.53 (m, 6H), 1.41 - 1.17 (m, 43H), 0.95 - 0.80 (m, 9H)。
MS m/z (APCI): [M+H]: 747.82。
EXAMPLE 31 Synthesis of Compound 12
To a mixture of the compounds H05T14 (0.1241 g) and T17 (0.1131 g), meCN (6 mL) K was added 2 CO 3 (0.0757 g) the reaction was stirred at 75℃for about 18 h. The reaction was filtered and the filter cake was washed once with DCM (20 mL), the filtrate was distilled off, and the residue was purified by separation on a silica gel column (eluent DCM/MeOH) to give Compound 12.
1 H NMR (400 MHz, CDCl 3 ) δ 4.81 (ddd, J = 12.4, 6.8, 5.5 Hz, 1H), 3.96 (d, J = 5.8 Hz, 2H), 2.64 (s, 3H), 2.47 (dt, J = 15.1, 7.6 Hz, 4H), 2.41 - 2.19 (m, 9H), 1.71 - 1.44 (m, 10H), 1.38 - 1.19 (m, 43H), 0.96 - 0.80 (m, 12H)。
MS m/z (APCI): [M+H]: 681.85。
EXAMPLE 32 Synthesis of Compound 13
K was added to a mixture of the compounds H05T14 (0.1256 g) with T31 (0.1237 g), meCN (6 mL) 2 CO 3 (0.0801 g) the reaction was stirred at 75℃for about 18 h. The reaction was filtered and the filter cake was washed once with DCM (20 mL), the filtrate was distilled off, and the residue was purified by separation on a silica gel column (eluent DCM/MeOH) to give Compound 13.
1 H NMR (400 MHz, CDCl 3 ) δ 4.88 (ddd, J = 12.4, 7.0, 5.4 Hz, 1H), 3.96 (d, J = 5.8 Hz, 2H), 2.52 (s, 3H), 2.38 (s, 4H), 2.34 - 2.18 (m, 9H), 1.63 (dt, J = 10.0, 7.2 Hz, 5H), 1.56 - 1.44 (m, 9H), 1.38 - 1.14 (m, 43H), 0.98 - 0.78 (m, 12H)。
MS m/z (APCI): [M+H]: 709.89。
EXAMPLE 33 Synthesis of Compound 14
K was added to a mixture of the compounds H05T14 (0.1249 g) with T36 (0.1151 g), meCN (6 mL) 2 CO 3 (0.0831, g) and stirred at 75℃for about 18 h. The reaction was filtered and the filter cake was washed once with DCM (20, mL) and the filtrate was distilled off, and the residue was purified by separation on a silica gel column (eluent DCM/MeOH) to give compound 14.
1 H NMR (400 MHz, CDCl 3 ) δ 4.80 (p, J = 6.2 Hz, 1H), 3.96 (d, J = 5.8 Hz, 2H), 2.68 (s, 3H), 2.48 (d, J = 8.4 Hz, 4H), 2.41 - 2.22 (m, 9H), 1.73 - 1.46 (m, 12H), 1.38 - 1.20 (m, 43H), 0.87 (dp, J = 7.5, 2.7 Hz, 12H)。
MS m/z (APCI): [M+H]: 695.84。
EXAMPLE 34 Synthesis of Compound 15
K is added to a mixture of the compound H05T01 (0.1522 g) with T38 (0.1486 g), meCN (5 mL) 2 CO 3 (0.1107. 0.1107 g) and stirred at 75℃for 16 hours. The reaction was diluted with DCM, filtered off with suction and evaporated. The residue was purified by column chromatography over silica gel (eluent DCM/MeOH) to give compound 15.
1 H NMR (400 MHz, CDCl 3 ) δ 4.88 (ddd, J = 12.5, 7.0, 5.5 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H), 2.75 (d, J = 46.8 Hz, 7H), 2.50 (t, J = 6.0 Hz, 4H), 2.36 (d, J = 4.5 Hz, 6H), 2.32 - 2.25 (m, 3H), 1.68 - 1.47 (m, 15H), 1.41 - 1.24 (m, 36H), 0.88 (dt, J = 10.0, 7.1 Hz, 12H)。
MS m/z (APCI): [M+H]: 695.90。
EXAMPLE 35 Synthesis of Compound 16
K is added to a mixture of the compound H05T01 (0.1035 g) with T36 (0.1052 g), meCN (5 mL) 2 CO 3 (0.0757 g) and stirred at 75℃for 16 hours. The reaction was diluted with DCM, filtered off with suction and evaporated. The residue was purified by column chromatography over silica gel (eluent DCM/MeOH) to give compound 16.
1 H NMR (400 MHz, CDCl 3 ) δ 4.81 (ddd, J = 12.4, 6.8, 5.5 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H), 2.60 (s, 4H), 2.43 (q, J = 7.6, 6.7 Hz, 6H), 2.35 - 2.25 (m, 9H), 1.68 - 1.47 (m, 14H), 1.37 - 1.24 (m, 38H), 0.90 - 0.84 (m, 12H)。
MS m/z (APCI): [M+H]: 695.97。
EXAMPLE 36 Synthesis of Compound 17
Compound H05T14 (0.1060 g) and T37 (0.0869 g) were dissolved in 10mL MeCN at room temperature at which point K was added 2 CO 3 (0.0657 g) the reaction was stirred at 75℃for 18 h. The reaction solution was filtered, the filtrate was concentrated by rotary evaporation, and the residue was purified by column chromatography on silica gel (eluent DCM/MeOH) to give compound 17.
1 H NMR (400 MHz, CDCl 3 ) δ 4.81 (p, J = 6.3 Hz, 1H), 3.96 (d, J = 5.8 Hz, 2H), 2.52 (s, 3H), 2.38 (s, 3H), 2.34 - 2.20 (m, 9H), 1.70 (s, 12H), 1.67 - 1.43 (m, 12H), 1.29 (dd, J = 17.3, 5.6 Hz, 33H), 0.87 (ddt, J = 7.4, 4.7, 2.9 Hz, 9H)。
MS m/z (APCI): [M+H]: 681.98。
EXAMPLE 37 preparation of nanolipid particles (LNP)
The cationic lipid compound described in the present invention was dissolved in ethanol with distearyl phosphatidylcholine, cholesterol and 2- [ (polyethylene glycol) -2000] -N, N-tetracosanamide in a molar ratio of 50:10:38.5:1.5 to prepare a mixed lipid ethanol solution. The Fluc-mRNA was dissolved in 10 mM citrate buffer (ph=4.4).
A mixed solution of the nanolipid particle composition was prepared by mixing a lipid ethanol solution with an mRNA citrate buffer (10 mm, ph=4.4) solution at a volume ratio of 1:3 (mass ratio of cationic lipid to mRNA of 15:1) using two microinjection pumps. Diluting the mixed solution to 30 times of the original volume by using PBS buffer solution, performing ultrafiltration concentration by using a 100kDa ultrafiltration tube, and then fixing the volume to a certain volume by using the PBS buffer solution to obtain the nano lipid particle composition solution.
EXAMPLE 38 determination of encapsulation efficiency, particle size and polydispersity index (PDI) of nanolipid particles
Encapsulation efficiency of lipid nanoparticle compositions (prepared according to example 37) was determined using a Quant-iT Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK) according to the instructions of the kit.
60. Mu.L of the nanolipid particle composition solution was added to a micro-sample cell, and the particle size and Polydispersity (PDI) were measured using a Zetasizer nano instrument (90 ℃ scattering angle, 25 ℃) from Markov, and the measurement was repeated 2 times for each sample, and the average of the two results was taken. The results are shown in Table 1.
TABLE 1
Cationic lipids Particle size (nm) PDI Encapsulation efficiency
Compound 1 75.44 0.12 79%
Compound 2 79.21 0.13 87%
Compound 3 70.13 0.15 87%
Compound 4 81.68 0.10 86%
Compound 5 77.61 0.14 90%
Compound 6 87.23 0.10 62%
Compound 7 79.84 0.16 83%
Compound 8 81.10 0.09 83%
Compound 9 70.81 0.12 91%
Compound 10 84.78 0.10 75%
Compound 11 85.04 0.08 78%
Compound 12 79.74 0.10 83%
Compound 13 75.63 0.11 95%
Compound 14 78.11 0.12 91%
Compound 15 70.65 0.12 89%
Compound 16 74.31 0.16 91%
Compound 17 77.37 0.08 91%
As can be seen from Table 1, the polydisperse coefficient of the nano-lipid particles prepared by the cationic lipid of the invention is 0.05-0.2, and the particle size is 70-90 nm, which indicates that the nano-lipid particles are uniform in size and good in dispersibility, and can be used for delivering mRNA; furthermore, the nano lipid particles prepared by adopting the cationic lipid disclosed by the invention have high encapsulation efficiency. The encapsulation efficiency is a key quality attribute of the liposome, and can reflect the encapsulation degree of the drug in the liposome. The nano lipid particles have high encapsulation efficiency, are favorable for efficiently encapsulating active substances (such as siRNA and mRNA) and improve drug loading capacity.
Example 39 cytotoxicity assay
HeLa cells (human cervical cancer cells) were passaged into 96-well plates for culture 24 h (37 ℃,5% CO) 2 ) mu.L of DMEM medium was added to each well. LNP solutions containing 2. Mu.g/mL Fluc-mRNA were incubated 24 with cells h. Mu. L Cell Counting Kit-8 (CCK-8) reagent was added to each well and incubated at 1.5 h. According to the kit instructions, the absorbance of each well at 450 nm was measured using an enzyme-labeled instrument, the stronger the absorption, i.e., the more viable cells, the lower the drug cytotoxicity. Relative cell viability was calculated according to the following formula:
A s for the absorbance of the sample, A b For 96 well plate background absorbance, A c Absorbance was measured for the blank (blank is normal culture HeLa cells). The results are shown in Table 2.
TABLE 2
Cationic lipids Relative cell viability
Compound 3 100%
Compound 4 93%
Compound 12 97%
Compound 17 96%
As can be seen from table 2, the lipid nanoparticle compositions prepared from the cationic lipids of the present invention do not exhibit significant cytotoxicity and can safely act on drug delivery and cell transfection.
Example 40 in vivo validation of lipid nanoparticle compositions delivery mRNA performance (tail vein injection)
3 female ICR mice with the age of 6-8 weeks and the weight of 20-25 g are randomly selected from each group, and the lipid nanoparticle composition solution is injected into the tail vein according to the dosage of 100 mu g/kg body weight. After 6 hours, 200. Mu.L of 15/mg/mL potassium D-fluorescein was intraperitoneally injected into each mouse, respectively. After 10 minutes, the mice were placed under a IVIS Lumina XRMS series III in vivo imaging system, and the total fluorescence intensity of each mouse was observed and recorded by photographing. The results are shown in Table 3.
TABLE 3 Table 3
Cationic lipids Tail vein injection transfection intensity (p/s)
Compound 1 4.2E+10
Compound 2 3.7E+10
Compound 3 8.8E+10
Compound 4 4.5E+10
Compound 5 4.7E+10
Compound 6 1.8E+10
Compound 7 3.0E+10
Compound 8 4.2E+10
Compound 9 3.2E+10
Compound 10 2.4E+10
Compound 11 1.9E+10
Compound 12 9.6E+10
Compound 13 4.6E+10
Compound 14 7.2E+10
Compound 15 4.3E+10
Compound 16 6.4E+10
Compound 17 1.0E+11
As can be seen from table 3, the lipid nanoparticle composition prepared from the cationic lipid according to the present invention has good mRNA delivery performance, and can make mRNA be expressed in mice with high efficiency.
Example 41 in vivo validation of lipid nanoparticle compositions delivery mRNA properties (intramuscular injection)
3 female ICR mice with the age of 6-8 weeks and the weight of 20-25 g are randomly selected from each group, and lipid nanoparticle composition solution is injected into leg muscles according to the dosage of 100 mu g/kg body weight. After 6 hours, 200. Mu.L of 15/mg/mL potassium D-fluorescein solution was intraperitoneally injected into each mouse, respectively. After 10 minutes, the mice were anesthetized, the luminous intensity of each mouse was observed using a IVIS Lumina XRMS series III in vivo imaging system, and photographed for recording. The results are shown in Table 4.
TABLE 4 Table 4
Cationic lipids Intramuscular injection transfection intensity (p/s)
Compound 3 9.4E+09
Compound 12 1.4E+10
Compound 17 2.5E+10
As can be seen from table 4, the lipid nanoparticle composition prepared from the cationic lipid according to the present invention has good mRNA delivery performance, and can make mRNA expressed in mice with high efficiency.
It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the disclosure. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims (3)

1. A cationic lipid compound or a pharmaceutically acceptable salt thereof, wherein the cationic lipid compound has one of the structures shown below:
、and。
2. a lipid nanoparticle composition comprising the cationic lipid compound of claim 1 or a pharmaceutically acceptable salt thereof.
3. Use of the cationic lipid compound of claim 1 or a pharmaceutically acceptable salt thereof or the lipid nanoparticle composition of claim 2 in the manufacture of a pharmaceutical carrier for delivering a therapeutic and/or prophylactic agent to a cell.
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