CN114409554A - Novel cationic lipid compound, composition and application thereof - Google Patents
Novel cationic lipid compound, composition and application thereof Download PDFInfo
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- 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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- C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
- C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
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- C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
- C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
- C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
- C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
Abstract
The invention discloses a novel cationic lipid for delivering nucleic acid and lipid particles thereof, wherein the cationic lipid is a cationic lipid with the following structural formula I or pharmaceutically acceptable salt, tautomer or stereoisomer thereof,
. The invention also discloses a pharmaceutical composition prepared by using the lipid particle of the invention and application of the pharmaceutical composition as a delivery vector of RNAi, mRNA and gene editing tools.
Description
Technical Field
The invention relates to a lipid compound, in particular to a cationic lipid, lipid particles prepared from the cationic lipid, a delivery therapeutic agent and application of the cationic lipid in disease treatment.
Background
Nucleic acid-based therapeutic techniques have great potential, e.g., siRNA, mRNA, antisense oligonucleotides, ribozymes, dnase, plasmids, antagomir, immunostimulatory nucleic acids, animir, and aptamers. Some nucleic acids may be used to achieve expression of specific cellular products, such as messenger rna (mrna) or plasmids, and may be used for treatment of diseases caused by protein or enzyme deletions, disease prevention, and tumor immunity. Some nucleic acids, such as RNAi and antisense oligonucleotides, can down-regulate the level of a particular mRNA in a cell, thereby down-regulating the synthesis of the corresponding protein.
The Lipid Nanoparticle (LNP) can be used for encapsulating the nucleic acid molecules and has wide application in the fields of siRNA drugs, mRNA vaccines, gene editing tools and the like. LNPs are composed of cationic lipids, which directly determine the encapsulation and delivery efficiency of nucleic acids, and other lipid components (such as neutral lipids, sterols, and PEG lipid conjugates), which are key factors in LNP technology.
Different types, different molecular weights of nucleic acid molecules, and different target tissues have different requirements for the chemical structure of cationic lipids and the LNP component, and therefore methods for developing different structures of cationic lipid chemical structures and compositions thereof are needed to meet the requirements for in vivo delivery of nucleic acids for the treatment and prevention of various diseases.
Disclosure of Invention
The object of the present invention is to provide a novel cationic lipid, a lipid particle composition, and a method for introducing a nucleic acid into a cell using the composition. The cationic lipid compound variety is enriched, more choices are provided for in vivo delivery of nucleic acid drugs, and the method has important significance for development and application of mRNA vaccines/drugs.
In order to achieve the purpose, the invention provides the following technical scheme:
a compound having the structural formula (I), or a pharmaceutically acceptable salt or isomer thereof:
wherein:
R1、R2each independently H, optionally substituted C1-C10Alkyl, optionally substituted C2-C10Alkenyl, optionally substituted C2-C10Alkynyl, optionally substituted C3-C8Cycloalkyl, optionally substituted C3-C8Cycloalkenyl, optionally substituted C3-C8Cycloalkynyl, optionally substituted four-to eight-membered heterocyclyl, optionally substituted C6-C10Aryl or five-to ten-membered heteroaryl,
R1、R2are linked to form a ring or R2、L4Connecting to form a ring;
R3、R3’each independently is optionally substituted C1-C24Alkyl or C2-C24An alkenyl group;
L1、L2、L3、L1’、L2’and L3’Each independently is a bond, optionally substituted C1-C10Alkyl or optionally substituted C2-C10An alkenyl group;
L4is optionally substituted C2-C10Alkyl, optionally substituted C2-C10Alkenyl, optionally substituted C2-C10Alkynyl, optionally substituted C3-C8Cycloalkyl, optionally substituted C3-C8Cycloalkenyl, optionally substituted C3-C8Cycloalkynyl, optionally substituted four-to eight-membered heterocyclyl, optionally substituted C6-C10Aryl or five-to ten-membered heteroaryl, or with R2Connecting to form a ring;
Q1、Q2、Q1’or Q2’One of which is-O-or-S-, and Q1、Q2、Q1’Or Q2’The others of (A) are-O-, -S-or a bond;
one of M or M' is-C (O) O-, -OC (O) -, -S-, -OC (O) O-, -C (O) NH-, -NHC (O) -, -C (O) S-, -C (S) O-, -NH-C (O) O-or-OC (O) -NH-, and the other of M or M' is-C (O) O-, -OC (O) -, -S-, -OC (O) O-, -C (O) NH-, -NHC (O) -, -C (O) S-, -C (S) O-, -NH-C (O) O-, -OC (O) -NH-or a bond.
Further, R is described in the invention1-N(R2)-L4-any one selected from the group consisting of:
wherein m, m', n are each independently any integer from 1 to 10.
Further, R is described in the invention3、R3’Is unsubstituted C1-C24Straight chain alkyl, unsubstituted C3-C24Branched alkyl, unsubstituted C3-C24Straight-chain alkenyl or unsubstituted C4-C24A branched alkenyl group.
Further, R according to the present invention3、R3’Each independently selected from any one of the following:
-CH3；
according to some specific and preferred embodiments, the cationic lipid compound is one or more selected from the group consisting of the compounds represented by the following structures:
further, the invention also discloses a lipid particle comprising the compound.
Further, in the lipid particle of the present invention, the lipid particle further comprises one or more of a neutral lipid, a sterol, or a conjugated lipid that inhibits aggregation of particles.
Further, in the lipid particle of the present invention, the neutral lipid is a phospholipid.
Further, in the lipid particle of the present invention, the sterol is cholesterol or a cholesterol derivative.
Further, in the lipid particle of the present invention, the conjugated lipid in which aggregation of the particle is inhibited comprises a PEG-lipid conjugate.
Further, the present invention also discloses a pharmaceutical composition comprising the lipid particle of the present invention and a therapeutic agent; it further comprises a pharmaceutically acceptable carrier.
Further, in the pharmaceutical composition of the present invention, wherein the therapeutic agent is a nucleic acid, wherein the therapeutic agent comprises an interfering RNA molecule, and further comprises a single-or double-stranded DNA, RNA or DNA/RNA hybrid, an antisense oligonucleotide, a ribozyme, a plasmid, or an immunostimulatory oligonucleotide.
Further, the invention also discloses the application of the pharmaceutical composition in preparing a medicament for treating or preventing diseases in a subject.
Has the advantages that: the structure contains a plurality of cationic groups, so that a strong electrostatic effect can be formed between the cationic groups and RNA or DNA, the prepared nanoparticles can stably wrap nucleic acid, the efficient loading of the nucleic acid including mRNA and DNA is realized, and the encapsulation efficiency and the stability are high; the nucleic acid can be rapidly released in cells, and the remarkable delivery efficiency is shown; contains degradable chemical bonds, is easy to be metabolized and excreted in vivo, and has low toxicity. In addition, the preparation method of the cationic lipid compound has the advantages of easily available raw materials, mild reaction conditions, simple operation and the like. The invention provides novel cationic lipid for delivering nucleic acid and lipid particles thereof, enriches the types of cationic lipid compounds, provides more choices for in vivo delivery of nucleic acid drugs, and has important significance for development and application of nucleic acid vaccines/drugs.
Drawings
FIG. 1 is a graph showing the results of the knockdown of mRNA in cells by lipid particles prepared with compounds 1-7.
FIG. 2 is a graph showing the results of the knockdown of lipid particles prepared with compounds 1-7 to ApoB in mice.
FIG. 3 is a graph showing the results of in vivo expression in mice of mRNA-lipid particles comprising compounds 1 to 7, respectively, injected subcutaneously.
Fig. 4 is a graph showing the results of in vivo expression of mRNA-lipid particles comprising MC3, compounds 4 and 6, respectively, injected subcutaneously in mice.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The present invention discloses cationic lipids prepared as lipid particles that can be used to deliver therapeutic agents (e.g., nucleic acid molecules) or other agents to cells (in vitro or in vivo in a subject) to further function in delivering the agent. The present invention provides a cationic lipid having the structural formula (I) or a pharmaceutically acceptable salt thereof,
wherein:
R1、R2each independently H, optionally substituted C1-C10Alkyl, optionally substituted C2-C10Alkenyl, optionally substituted C2-C10Alkynyl, optionally substituted C3-C8Cycloalkyl, optionally substituted C3-C8Cycloalkenyl, optionally substituted C3-C8Cycloalkynyl, optionally substituted four-to eight-membered heterocyclyl, optionally substituted C6-C10Aryl or five-to ten-membered heteroaryl,
R1、R2are linked to form a ring or R2、L4Connecting to form a ring;
R3、R3’each independently is optionally substituted C1-C24Alkyl or C2-C24An alkenyl group;
L1、L2、L3、L1’、L2’and L3’Each independently is a bond, optionally substituted C1-C10Alkyl or optionally substituted C2-C10An alkenyl group;
L4is optionally substituted C2-C10Alkyl, optionally substituted C2-C10Alkenyl, optionally substituted C2-C10Alkynyl, optionally substituted C3-C8Cycloalkyl, optionally substituted C3-C8Cycloalkenyl, optionally substituted C3-C8Cycloalkynyl, optionally substituted four-to eight-membered heterocyclyl, optionally substituted C6-C10Aryl or five-to ten-membered heteroaryl, or with R2Connecting to form a ring;
Q1、Q2、Q1’or Q2’One of which is-O-or-S-, and Q1、Q2、Q1’Or Q2’The others of (A) are-O-, -S-or a bond;
one of M or M' is-C (O) O-, -OC (O) -, -S-, -OC (O) O-, -C (O) NH-, -NHC (O) -, -C (O) S-, -C (S) O-, -NH-C (O) O-or-OC (O) -NH-, and the other of M or M' is-C (O) O-, -OC (O) -, -S-, -OC (O) O-, -C (O) NH-, -NHC (O) -, -C (O) S-, -C (S) O-, -NH-C (O) O-, -OC (O) -NH-or a bond.
In some embodiments, R is as described herein1-N(R2)-L4-any one selected from the group consisting of:
wherein m, m', n are each independently any integer from 1 to 10.
Preferably, R is as defined in the invention3、R3’Is unsubstituted C1-C24Straight chain alkyl, unsubstituted C2-C24Branched alkyl, unsubstituted C3-C24Straight-chain alkenyl or unsubstituted C4-C24A branched alkenyl group.
In some embodiments, R is as described herein3、R3’Selected from any one of the following:
-CH3；
in particular embodiments, the cationic lipid is selected from the following table.
TABLE 1
A lipid particle comprising a compound of formula (I) as described above. Further, in a particular embodiment, the lipid particle comprises a compound as in any one of table 1 of the present invention.
The lipid particles of the present invention have a median diameter of 25-300 nm. For example from about 50nm to 200nm or from about 100nm to about 200 nm.
The lipid particles of the present invention further comprise a non-cationic lipid, which is a neutral lipid and/or a sterol.
In some embodiments, the neutral lipid of the lipid particle of the invention is a phospholipid comprising one or a combination of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, or 1, 2-dioleoyl-sn-glycero-3-phosphocholine.
In some embodiments, the sterol in the lipid particle of the invention is cholesterol or a cholesterol derivative.
In various embodiments, the lipid particles of the present invention further comprise a conjugated lipid that inhibits aggregation of the particles.
In some embodiments, the conjugated lipid that inhibits aggregation of particles of the present invention comprises a PEG-lipid conjugate.
In particular embodiments, the PEG-lipid conjugates of the invention comprise one or a mixture of two of a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkoxypropyl (PEG-DAA) conjugate.
In a particular embodiment, the lipid particle comprises a cationic lipid of the invention, a neutral lipid, a sterol, and a PEG-lipid conjugate. The lipid particle comprises from about 25% to about 75% by mole percent of a cationic lipid, for example from about 30% to about 60%, from about 40% to about 70%, from about 35%, about 50%, about 70.1% or about 61.5% by mole percent. The lipid particle comprises, in mole percent, from about 0% to about 20% of a neutral lipid, for example, from about 5% to about 10%, from about 4% to about 12%, from about 15%, about 6.3% or about 0%, in a particular embodiment, the neutral lipid is DSPC or DOPE. The lipid particle comprises, in mole percent, about 5% to 50% of a sterol, for example about 10% to 40%, about 15% to 35%, about 6.1%, about 12%, about 31.5% or about 33.7% in mole percent, in a particular embodiment the sterol is cholesterol. The lipid particle comprises about 0.1% to about 20% by mole percent of a PEG conjugate, for example about 0.2% to 20%, about 1.5 to 15%, about 10%, about 12%, about 1.5% or about 0.7% by mole percent, in a particular embodiment the PEG-lipid conjugate is PEG-DMG.
In a particular embodiment, the lipid particle comprises, in mole percent, 25-75% cationic lipid, 0.5-15% neutral lipid, 5-50% sterol, and 0.5-20% PEG conjugate.
Table 2 exemplary lipid particles (in mole%) according to some non-limiting embodiments
| Lipid particles | Cationic lipids | DSPC | Cholesterol | PEG- |
| 1 | 50 | 10 | 38.5 | 1.5 |
| 2 | 40 | 10 | 40 | 10 |
| 3 | 39.5 | 9 | 51 | 0.5 |
| 4 | 60.7 | 8.4 | 20.5 | 10.4 |
| 5 | 45 | 15 | 39.1 | 0.9 |
A pharmaceutical composition comprising a lipid particle of the invention and a therapeutic agent.
In some embodiments of the foregoing composition, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from interfering RNA molecules, single/double stranded DNA, RNA or DNA/RNA hybrids, antisense oligonucleotides, ribozymes, plasmids, immunostimulatory oligonucleotides.
In some specific embodiments, the interfering RNA molecules include small interfering RNA, asymmetric interfering RNA, micro RNA, small hairpin RNA or a combination of several.
In some particular embodiments, the single-stranded RNA is selected from antisense RNA and mRNA.
The pharmaceutical composition of the present invention, wherein the therapeutic agent is encapsulated in the lipid particle.
The pharmaceutical composition of the present invention, wherein the mass ratio of the lipid in the lipid particle to the therapeutic agent is (5:1) - (15: 1).
The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.
Use of a pharmaceutical composition according to the invention for the preparation of a medicament for the treatment or prevention of a disease in a subject, wherein the disease is selected from a viral infectious disease, a liver disease or a cancer.
Administration of the compositions of the present invention may be by any acceptable manner of administration of the agents for similar utility. The pharmaceutical composition of the present invention may be formulated into solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, sublingual, buccal subcutaneous or intradermal.
In some embodiments, the nucleic acid-lipid particle pharmaceutical composition is administered intravenously. In other embodiments, the nucleic acid-lipid particle pharmaceutical composition is administered intramuscularly. In particular embodiments, the nucleic acid-lipid particle pharmaceutical composition is administered intravenously or intraperitoneally by injection.
In some embodiments, the nucleic acid-lipid particle pharmaceutical composition can also be applied directly to a tissue to contact the pharmaceutical composition with a target tissue. Application may be topical, open or closed surgery. Topical refers to the application of a pharmaceutical formulation directly to tissue exposed to the environment, such as the skin, oropharynx, external auditory meatus, and the like. Open surgery includes a procedure in which the skin of a patient is incised and the underlying tissue to which the pharmaceutical formulation is applied is directly visualized. Closed surgery is an invasive procedure in which the internal target tissue cannot be directly seen, such as a lumbar puncture.
In some embodiments, the pharmaceutical composition may also be inhaled into the respiratory tract, e.g. the lungs, in the form of an aerosol.
The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar form, may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, ringer's solution, isotonic sodium chloride; non-volatile oils such as synthetic monoglycerides or diglycerides which may be used as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for adjusting tonicity, such as sodium chloride or dextrose; agents used as cryoprotectants, such as sucrose or trehalose.
The pharmaceutical compositions of the present invention may be comprised of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from systems of colloidal nature to systems consisting of pressurized packaging. The delivery may be by liquefied or compressed gas, or by a suitable pump system for dispensing the active ingredient. Aerosols of the compounds of the invention may be delivered as a single phase, biphasic system, or triphasic system for delivery of the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Those skilled in the art can determine the preferred aerosol without undue experimentation.
The compositions of the present invention may also be administered simultaneously with, prior to, or after the administration of one or more other therapeutic agents. Such combination therapies include the administration of a single pharmaceutical dosage formulation of a composition of the present invention and one or more additional active agents, as well as the administration of a composition of the present invention and each active agent in its own separate pharmaceutical dosage formulation. For example, the compositions of the invention and other active agents may be administered to a patient together in a single dosage composition (e.g., a tablet or capsule), or the individual agents may be administered in different dosage formulations. When different dosage formulations are used, the compound of the invention and one or more additional active agents can be administered at substantially the same time (i.e., simultaneously), or at staggered times (i.e., sequentially); it is to be understood that combination therapy encompasses all of these dosing regimens.
A formulation comprising a pharmaceutical composition according to the invention, further comprising a vaccine or antigen. Accordingly, a vaccine may comprise a lipid particle comprising an immunostimulatory oligonucleotide and also associated with an antigen to which an immune response is desired. The antigen includes a tumor antigen or an infectious agent antigen.
In some embodiments, the antigen in the formulations of the invention is a tumor antigen that includes proteins of Ras, p53, Her2, KSA, TRP1, TRP2, or BCR-abl oncogenes.
In some embodiments, the formulation of the invention wherein the antigen is an infectious agent antigen, the infectious agent antigen comprises a viral antigen, a bacterial antigen, or a parasitic antigen.
In some embodiments, in the formulations of the invention, the viral antigen comprises hepatitis b virus, hepatitis a virus, tuberculosis virus, rubella virus, smallpox virus, influenza virus, measles virus, rotavirus, or coronavirus.
In some embodiments, in the formulations of the invention, the viral antigen is further derived from a virus of the family retroviridae, picornaviridae, caliciviridae, coronaviridae, rhabdoviridae, filoviridae, orthomyxoviridae, paramyxoviridae, togaviridae, flaviviridae, bunyaviridae, arenaviridae, binuclear glycoviridae, hepadnaviridae, parvoviridae, papovaviridae, adenoviridae, herpesviridae, iridoviridae or unclassified virus.
The following general reaction schemes 1 and 2 illustrate methods for preparing compounds of the present invention, i.e., compounds of formula I
Or a pharmaceutically acceptable salt, tautomer, or pharmaceutically acceptable salt thereof, wherein R1、R2、R3、R3’、L1、L2、L3、L1’、L2’、L3’、L4、Q1、Q1’、Q2And Q2' is as defined herein. It is understood that one skilled in the art can prepare these compounds by similar methods or by other methods known to those skilled in the art. It will also be appreciated that the skilled person will be able to prepare other compounds of formula I not explicitly specified below in a similar manner to that described below, by using the appropriate starting components and modifying the parameters of the synthesis as required.
The compounds of the present invention may be prepared by known organic synthesis techniques, including the methods described in the examples. It is to be understood that certain non-critical variables of the embodiments may be altered by reading the description herein, as will be apparent to those skilled in the art.
Examples
The present invention will be described in more detail below by way of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. One skilled in the art will readily recognize that certain noncritical parameters may be changed or modified to produce substantially the same results.
Several methods of preparing cationic lipids are shown in examples 1-7 below:
EXAMPLE 1 Synthesis of Compound 1
2-Hexyldecanoic acid (22g, 87.75mmol), DCC (21.3g, 103mmol) and DMAP (1.18g, 8.75mmol) were added to a solution of 6-bromo-1-hexanol (16g, 87.75mmol) in dichloromethane (500mL) and the solution was stirred overnight. The reaction mixture was filtered and the solvent was removed. The residue was dissolved in dichloromethane and washed with dilute hydrochloric acid, and dried over anhydrous magnesium sulfate to give intermediate 1-1(15.5 g).
1-1(3.0g, 7.2mmol) and N, N-dimethylethylenediamine (1.27g, 14.4mmol) were dissolved in ethanol and reacted with stirring at 65 ℃ for 16 hours, then cooled to room temperature and dried under vacuum. The crude product was taken up in ethyl acetate and saturated NaHCO3Extraction, solvent removal by vacuum drying and final purification by silica gel chromatography gave intermediate 1-2(2.9 g).
2-Hexyldecanoic acid (9.8g, 38.2mmol), DCC (9.5g, 45.9mmol) and DMAP (0.52g, 3.8mmol) were added to a solution of 1, 3-propanediol (9.6g, 126mmol) in dichloromethane (200mL) and the solution was stirred overnight. The reaction mixture was filtered and the solvent was removed. The residue was dissolved in dichloromethane and washed with dilute hydrochloric acid, and dried over anhydrous magnesium sulfate to give intermediates 1 to 3(8.5 g).
Intermediate 1-3(4.3g, 12.9mmol) was dissolved in dichloromethane (150mL), treated with sodium salt and 1, 4-dibromobutane (5.6g, 25.8mmol) was added, the reaction stirred overnight and purified by silica gel column to give intermediate 1-4(3.9 g).
Intermediate 1-2(2.9g, 6.8mmol) and intermediate 1-4(2.2g, 6.8mmol) were reacted in a solution of N, N-diisopropylethylamine (0.87g, 6.8mmol) in ethanol at 65 deg.C with stirring for 16 h and then cooled to room temperature before vacuum drying, the crude product was taken up in ethyl acetate and saturated NaHCO3Extraction, removal of the organic phase by vacuum drying and purification of the product by silica gel chromatography gave compound 1(0.67 g).
EXAMPLE 2 Synthesis of Compound 2
1-1(3.0g, 7.2mmol, same synthetic procedure as in example 1) and N, N-diethylethylenediamine (1.67g, 14.4mmol) were dissolved in ethanol and reacted with stirring at 65 ℃ for 16 hours, then cooled to room temperature and dried under vacuum. The crude product was taken up in ethyl acetate and saturated NaHCO3Extraction, solvent removal by vacuum drying and final purification by silica gel column chromatography gave intermediate 2-1(3.1 g). Intermediate 2-1(3.1g, 6.8mmol) and intermediate 1-4(2.2g, 6.8mmol, as in example 1) were reacted in a solution of N, N-diisopropylethylamine (0.87g, 6.8mmol) in ethanol at 65 deg.C with stirring for 16 h and then cooled to room temperature and dried in vacuo, the crude product was extracted with ethyl acetate and saturated NaHCO3Extraction, removal of the organic phase by vacuum drying and purification of the product by silica gel chromatography gave compound 2(0.71 g).
EXAMPLE 3 Synthesis of Compound 3
1-1(3.0g, 7.2mmol, synthetic procedure as in example 1) and 1- (2-aminoethyl) piperidine (1.84g, 14.4mmol) were dissolved in ethanol and reacted with stirring at 65 ℃ for 16 h, then cooled to room temperature and dried in vacuo. The crude product was taken up in ethyl acetate and saturated NaHCO3Extraction, solvent removal by vacuum drying and final purification by silica gel column chromatography gave intermediate 2-1(3.2 g).Intermediate 2-1(3.2g, 6.8mmol) and intermediate 1-4(2.2g, 6.8mmol, as in example 1) were reacted in a solution of N, N-diisopropylethylamine (0.87g, 6.8mmol) in ethanol at 65 deg.C with stirring for 16 h and then cooled to room temperature and dried in vacuo, the crude product was extracted with ethyl acetate and saturated NaHCO3Extraction, removal of the organic phase by vacuum drying and purification of the product by silica gel chromatography gave compound 3(0.68 g).
EXAMPLE 4 Synthesis of Compound 4
To a solution of 8-bromooctanoic acid (10.0g, 44.2mmol) and 9-heptadecanol (15.0g, 58mmol) in dichloromethane (200mL) was added diisopropylethylamine (33mL, 187mmol), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (11g, 58mmol) and DMAP (1.14g, 9 mmol). The reaction mixture was stirred at room temperature for 14 h. Then using CH2Cl2The reaction mixture was diluted (400mL) and saturated NaHCO3Aqueous (300mL) wash. With anhydrous MgSO4The organic layer was dried, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain 4-1(8.1 g).
Compound 4-1(4g, 8.6mmol) and N, N-diethylethylenediamine (14mL, 100mmol) were dissolved in ethanol (50mL) and stirred at 62 ℃ for 18 hours. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved with ethyl acetate and washed with water. With anhydrous MgSO4The organic layer was dried, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 4-2(3.4 g).
1, 2-dibromoethane (20g, 108.7mmol) and potassium carbonate (18g, 130mmol) were dissolved in 200mL of DMF, heated to 60 ℃ and then a solution of benzyl 4-hydroxybutyrate (10g, 51.5mmol) in DMF was added dropwise and reacted for 6 hours. After the reaction was complete, the organic solvent was removed by rotary evaporation, then dissolved in dichloromethane and washed with water. With anhydrous MgSO4The organic layer was dried, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 4-3(4.3 g).
To compound 4-3(4.0g, 13.3mmol) and Pd/C (0.4g) was added 30mL of ethanol to dissolve, and the reaction was carried out under a hydrogen atmosphere for two hours. After the completion of the reaction, the organic solvent was removed by rotary evaporation, and then the crude product was purified by silica gel column chromatography to obtain compound 4-4(2.1 g).
To a solution of compound 4-4(2.0g, 9.5mmol) and N-heptanol (1.1g, 9.5mmol) in dichloromethane (30mL) was added diisopropylethylamine (3.3mL, 19mmol), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (1.8g, 9.5mmol) and DMAP (0.11g, 0.9 mmol). The reaction mixture was stirred at room temperature for 12 h. Then using CH2Cl2The reaction mixture was diluted (100mL) and saturated NaHCO3Aqueous (100mL) wash. With anhydrous MgSO4The organic layer was dried, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 4-5(2.2 g).
To a solution of compound 4-2(1.66g, 3.25mmol) and compound 4-5(1g, 3.25mmol) in dichloromethane (30mL) was added diisopropylethylamine (0.42g, 3.25mmol), and the reaction mixture was stirred at 65 ℃ for 18 h. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved with ethyl acetate and washed with water. With anhydrous MgSO4The organic layer was dried, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 4(0.7 g).
EXAMPLE 5 Synthesis of Compound 5
To a solution of 3-butoxypropylamine (1.0g, 7.6mmol) and 6-bromohexanoic acid (1.48g, 7.6mmol) in dichloromethane (30mL) was added diisopropylethylamine (2.6mL, 15mmol), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (1.4g, 7.6mmol) and DMAP (0.08g, 0.7 mmol). The reaction mixture was stirred at room temperature for 12 h. Then using CH2Cl2The reaction mixture was diluted (100mL) and saturated NaHCO3Aqueous (100mL) wash. With anhydrous MgSO4The organic layer was dried, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 5-1(1.6 g). To a solution of compound 4-2(1.66g, 3.25mmol, synthetic procedure as in example 4) and compound 5-1(1.0g, 3.25mmol) in dichloromethane (30mL) was added diisopropylethylamine (0.42g, 3.25mmol), and the reaction mixture was stirred at 65 ℃ for 18 h. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved with ethyl acetate and washed with water. With anhydrous MgSO4The organic layer was dried, filtered and concentrated. Purifying the crude product by silica gel column chromatographyTo obtain compound 5(0.7 g).
EXAMPLE 6 Synthesis of Compound 6
Undecanoic acid (6.38g, 34.3mmol), DCC (8.5g, 41.2mmol) and DMAP (0.47g, 3.5mmol) were added to a solution of 6-bromo-1-hexanol (6.4g, 35.1mmol) in dichloromethane (200mL) and the solution was stirred overnight. The reaction mixture was filtered and the solvent was removed. The residue was dissolved in dichloromethane and washed with dilute hydrochloric acid, and dried over anhydrous magnesium sulfate to give intermediate 6-1(4.8 g).
6-1(3.8g, 9.1mmol) and N, N-diethylethylenediamine (2.55mL, 18.2mmol) were dissolved in ethanol and reacted at 65 ℃ with stirring for 16 hours, then cooled to room temperature and dried under vacuum. The crude product was taken up in ethyl acetate and saturated NaHCO3Extraction, solvent removal by vacuum drying and final purification by silica gel column chromatography gave intermediate 6-2(3.6 g).
2-Hexyldecanoic acid (9.8g, 38.2mmol), DCC (9.5g, 45.9mmol) and DMAP (0.52g, 3.8mmol) were added to a solution of 1, 3-propanediol (9.6g, 126mmol) in dichloromethane (200mL) and the solution was stirred overnight. The reaction mixture was filtered and the solvent was removed. The residue was dissolved in dichloromethane and washed with dilute hydrochloric acid, and dried over anhydrous magnesium sulfate to give intermediate 6-3(8.5 g).
Intermediate 6-3(4.3g, 12.9mmol) was dissolved in dichloromethane (150mL), treated with sodium salt, 1, 4-dibromobutane (5.6g, 25.8mmol) was added, the reaction stirred overnight, and purified by a silica gel column to give intermediate 6-4(3.9 g).
Intermediate 6-2(3.2g, 8.7mmol) and intermediate 6-4(3.9g, 8.7mmol) were reacted in a solution of N, N-diisopropylethylamine (0.51g, 4mmol) in ethanol at 65 ℃ with stirring for 16 h and then cooled to room temperature before vacuum drying, the crude product was taken up in ethyl acetate and saturated NaHCO3Extraction, removal of the organic phase by vacuum drying and purification of the product by silica gel chromatography gave compound 6(0.76 g).
EXAMPLE 7 Synthesis of Compound 7
1-1(3.0g, 7.2mmol, same synthetic procedure as in example 1) and 3-diethylaminopropylamine (1.87g, 14.4mmol) were dissolved in ethanol and reacted at 65 ℃ with stirring for 16 hours, then cooled to room temperature and dried in vacuo. Using ethyl acetate as crude productEthyl acid and saturated NaHCO3Extraction, solvent removal by vacuum drying and final purification by silica gel chromatography gave intermediate 7-1(3.4 g). Intermediate 7-1(3.2g, 6.8mmol) and intermediate 1-4(2.2g, 6.8mmol, as in example 1) were reacted in a solution of N, N-diisopropylethylamine (0.87g, 6.8mmol) in ethanol at 65 deg.C with stirring for 16 h and then cooled to room temperature and dried in vacuo, the crude product was extracted with ethyl acetate and saturated NaHCO3Extraction, removal of the organic phase by vacuum drying and purification of the product by silica gel chromatography gave compound 7(0.74 g).
Example 8 preparation and characterization of nucleic acid-lipid particles
Cationic lipid, cholesterol, DSPC and PEG-DMG were dissolved in ethanol at a molar ratio of 50/38.5/10/1.5, respectively, and nucleic acid-Lipid Nanoparticles (LNPs) were prepared at a total lipid to nucleic acid (siRNA or mRNA) mass ratio of 10/1. The lipid ethanol solution was mixed with the aqueous nucleic acid solution at a volume ratio of 1/3 using a syringe pump, with a total flow rate of 10 mL/min. The ethanol was then removed by dialysis against PBS buffer pH 7.4. Finally the lipid nanoparticles were filtered through a 0.22 μm sterile filter. Then PBS (155mM NaCl, 3mM Na)2HPO4，1mM KH2PO4pH 7.4) dialysis displacement of the external buffer and final storage at 4 ℃ environment.
The particle size, polydispersity index (PDI) and surface potential of the Lipid Nanoparticles (LNP) prepared above were measured by dynamic light scattering method (Zetasizer Nano; Malvern instruments Ltd.).
The encapsulation efficiency and recovery rate of nucleic acids were measured by RiboGreen (Invitrogen; Thermo Fischer Scientific). Specifically, the method comprises the following steps: each LNP prepared above was diluted to 1000ng/mL with 10mM HEPES buffer pH 7.4 and made into a sample solution. In addition, the nucleic acid-lipid nanoparticles to be detected are stepwise diluted to 0-2000 ng/mL with 10mM HEPES buffer solution with pH 7.4, and then prepared into a standard curve solution. In addition to these solutions, measurement solutions were prepared by diluting dextran sulfate, Triton X-100, and Ribogreen to 0.08mg/mL, 0.4%, and 5. mu.L/mL, respectively, with 10mM HEPES buffer. In addition, a solution in which Triton X-100 was replaced with 10mM HEPES buffer was prepared. After adding 50. mu.L of the standard curve solution or the sample solution to a 96-well plate and further adding 50. mu.L of the measurement solution with or without Triton X-100, respectively, and mixing them, stirring them at 700rpm for 5 minutes, the fluorescence intensities at an excitation wavelength of 500nm and an observation wavelength of 525nm were measured. The RNA recovery rate was calculated by dividing the amount of RNA measured under the condition containing Triton X-100 by 1000 ng/mL. The RNA amount measured under the condition not containing Triton X-100 was subtracted from the RNA amount measured under the condition containing Triton X-100, and the RNA encapsulation efficiency was calculated by dividing the RNA amount measured under the condition containing Triton X-100 by the value.
The results of mRNA encapsulation by LNP prepared from the cationic lipids of compounds 1-7 are shown in table 3. The result shows that the nanoparticles prepared from the cationic lipid 1-7 have a good wrapping effect on mRNA, and the nanoparticles are still well distributed after being prepared for 14 days.
TABLE 3
The cationic lipid/cholesterol/DSPC/PEG-DMG ratios were all 50/38.5/10/1.5.
Example 9 in vitro assay of lipid nanoparticles
To evaluate the efficacy of various nucleic acid-lipid nanoparticle formulations, in vitro assays were performed using sirnas targeting the apob (apolipoprotein b) gene, siApoB (sense: 5 '-gucaucacugaauccauaudtdt-3'; antisense: 5 '-augguuuguugugaugaggacdtdt-3'). Nucleic acid-lipid nanoparticle formulations were prepared for siApoB using the methods described in example 8 above.
Cell culture and siRNA transfection:
HepG2 cells were plated in 24-well plates in advance to ensure 70-90% confluence on the day of the experiment. 50 μ L of nucleic acid-lipid particles (siRNA concentration 50nM) were added on the day of the experiment and incubation continued for 24 h.
RNA extraction and analysis:
cells were washed once with 2mL PBS and RNeasy Mini Kit was usedTM(Qiagen) Total RNA was extracted and added at 30. mu.LTotal volume elution. Using Transcriptor 1st Strand cDNA KitTM(Roche) and random hexamers 1. mu.g total RNA was reverse transcribed according to the instructions. One-thirtieth (0.66. mu.L) of the cDNA thus obtained was mixed with 5. mu.L of IQ Multiplex Powermix (Bio-Rad) and 3.33. mu. L H2O and 1. mu.L of a 3. mu.M mixture containing primers and probes specific for the ApoB target sequence were mixed together. The CFX96 real-time system of the C1000 thermal cycler (Bio-Rad) was then used for the amplification reaction. The PCR conditions were: 95 ℃ for 3 minutes; then circulating at 95 ℃ for 10 seconds; and 40 cycles were performed at 55 ℃ for 1 minute. Three replicates per sample were tested. The relative ApoB mRNA levels were normalized to target mRNA levels and compared to mRNA levels obtained in control samples treated or untreated with transfection agent alone.
Figure 1 provides in vitro gene knockout results for lipid nanoparticles prepared from the cationic lipids of compounds 1-7 of table 1. Overall, these 7 lipid nanoparticles were effective in inhibiting target mRNA levels when HepG2 cells were administered. In particular, the lipid nanoparticles prepared from compounds 2, 4 and 6 can achieve a target mRNA knockout efficiency of 95% or more.
Example 10: in vivo Performance of siRNA-lipid nanoparticles
To further evaluate LNP performance, in vivo experiments were performed using formulations with siRNA against ApoB. The nanoformulations were prepared from the compounds shown in table 1 as shown in example 8. A single dose (1mg/kg, 0.5mg/kg or 0.1mg/kg) of the lipid particle formulation was administered to approximately 6-week-old female mice by intravenous administration via the tail vein in an administration volume of 10. mu.L/g body weight. Mice were sacrificed 24 hours later and mouse livers were dissected. The amount of ApoB mRNA in the sample was quantified by qRT-PCR, and the amount of ApoB mRNA expression in each administration group was shown as a relative value with the amount of ApoB mRNA expression in the untreated group (NT) being 1. Figure 2 shows the data with error bars, n-mean ± SD of 5 animals/group. The results show that the nano-preparations prepared from the compounds 1 to 7 respectively can remarkably reduce the level of ApoB mRNA, wherein the nano-preparation prepared from the compound 4 has the inhibition efficiency of 90 percent under the dosage of 0.1 mg/kg.
Example 11 in vivo evaluation of subcutaneously injected mRNA-lipid nanoparticle compositions
In vivo effects of mRNA lipid nanoparticles in kunming mice 6 weeks old were studied and in vivo experiments were performed using preparations of mRNA expressing fluorescent protein. mRNA-lipid nanoparticles were prepared from compounds 1 to 7, respectively, as shown in example 8, diluted with PBS to an mRNA concentration of 10. mu.g/mL, and subcutaneously administered in the neck of 6-week-old female mice (dose of 0.5 mg/kg). At 12 hours after administration, the previously prepared fluorescein (VivoGlo)TMLuciferin, In Vivo Grade, Promega) In water, was administered intraperitoneally to each mouse 30 minutes later using IVISTMLuminaII (caliper Life sciences) was observed for luminescence at the site of administration and quantified.
The results are shown in FIG. 3. Luminescence was confirmed in each experimental group, and mRNA expression activity was shown. Among them, the group containing compound 6 showed a strong luminescence amount and a high mRNA expression activity.
Example 12 in vivo evaluation of subcutaneously injected mRNA-lipid nanoparticle compositions
In vivo effects of mRNA lipid nanoparticles in kunming mice 6 weeks old were studied and in vivo experiments were performed using preparations of mRNA expressing fluorescent protein. mRNA-lipid nanoparticles were prepared from compounds 4, 6 and MC3 as shown in example 8, respectively, and diluted with PBS to give an mRNA concentration of 10. mu.g/mL, which were administered subcutaneously at the necks of 6-week-old female mice (at doses of 0.1mg/kg, 0.5mg/kg and 1.0mg/kg, respectively). At 12 hours after administration, the previously prepared fluorescein (VivoGlo)TMLuciferin, In Vivo Grade, Promega) In water, was administered intraperitoneally to each mouse 30 minutes later using IVISTMLuminaII (caliper Life sciences) was observed for luminescence at the site of administration and quantified.
The results are shown in FIG. 4. Luminescence was confirmed for each experimental group, with the nanoparticle composition prepared from compound 6 showing the strongest luminescence intensity, indicating higher mRNA expression activity.
Claims (10)
1. A compound having the structural formula (I), or a pharmaceutically acceptable salt or isomer thereof:
wherein:
R1、R2each independently H, optionally substituted C1-C10Alkyl, optionally substituted C2-C10Alkenyl, optionally substituted C2-C10Alkynyl, optionally substituted C3-C8Cycloalkyl, optionally substituted C3-C8Cycloalkenyl, optionally substituted C3-C8Cycloalkynyl, optionally substituted four-to eight-membered heterocyclyl, optionally substituted C6-C10Aryl or five-to ten-membered heteroaryl, R1、R2Are linked to form a ring or R2、L4Connecting to form a ring;
R3、R3’each independently is optionally substituted C1-C24Alkyl or C2-C24An alkenyl group;
L1、L2、L3、L1’、L2’and L3’Each independently is a bond, optionally substituted C1-C10Alkyl or optionally substituted C2-C10An alkenyl group;
L4is optionally substituted C2-C10Alkyl, optionally substituted C2-C10Alkenyl, optionally substituted C2-C10Alkynyl, optionally substituted C3-C8Cycloalkyl, optionally substituted C3-C8Cycloalkenyl, optionally substituted C3-C8Cycloalkynyl, optionally substituted four-to eight-membered heterocyclyl, optionally substituted C6-C10Aryl or five-to ten-membered heteroaryl, or with R2Connecting to form a ring;
Q1、Q2、Q1’or Q2’One of which is-O-or-S-, and Q1、Q2、Q1’Or Q2’The others of (A) are-O-, -S-or a bond;
one of M or M' is-C (O) O-, -OC (O) -, -S-, -OC (O) O-, -C (O) NH-, -NHC (O) -, -C (O) S-, -C (S) O-, -NH-C (O) O-or-OC (O) -NH-, and the other of M or M' is-C (O) O-, -OC (O) -, -S-, -OC (O) O-, -C (O) NH-, -NHC (O) -, -C (O) S-, -C (S) O-, -NH-C (O) O-, -OC (O) -NH-or a bond.
2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: the R is1-N(R2)-L4-any one selected from the group consisting of:
wherein m, m', n are each independently any integer from 1 to 10.
The R is3、R3’Is unsubstituted C1-C24Straight chain alkyl, unsubstituted C3-C24Branched alkyl, unsubstituted C3-C24Straight-chain alkenyl or unsubstituted C4-C24A branched alkenyl group.
3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: the R is3、R3’Each independently selected from any one of the following:
-CH3；
4. the compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is one or more selected from the group consisting of compounds represented by the following structures:
5. a lipid particle comprising one or more of the compounds of any one of claims 1-4 or pharmaceutically acceptable salts thereof.
6. The lipid particle of claim 5, wherein the lipid particle further comprises one or a combination of neutral lipids, sterols, or conjugated lipids that inhibit aggregation of the particles.
7. The lipid particle of claim 6, wherein the neutral lipid is a phospholipid; wherein the sterol is cholesterol or a cholesterol derivative; wherein the conjugated lipid that inhibits aggregation of particles comprises a PEG-lipid conjugate.
8. A pharmaceutical composition comprising the lipid particle of claim 5 and a therapeutic agent; it further comprises a pharmaceutically acceptable carrier.
9. The pharmaceutical composition of claim 8, wherein the therapeutic agent is a nucleic acid, wherein the therapeutic agent comprises an interfering RNA molecule, further comprising a single or double stranded DNA, RNA or DNA/RNA hybrid, an antisense oligonucleotide, a ribozyme, a plasmid, or an immunostimulatory oligonucleotide.
10. Use of the pharmaceutical composition of claim 9 in the manufacture of a medicament for treating or preventing a disease in a subject.
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