Cationic lipids and their applications

Abstract

The present invention discloses a cationic lipid and its application. The cationic lipid can be prepared into lipid particles for introducing nucleic acids or other functional compounds into cells. The nucleic acid introduction agent of the cationic lipid prepared by the present invention is used to introduce functional nucleic acids, and effective gene regulation can be achieved by using the functional nucleic acids delivered to the cytoplasm, and its effect is much higher than that of commercially available products. The cationic lipid of the present invention is shown in formula (I):.

Description

Technical Field

[0001] The invention relates to a lipid compound, in particular to a cationic lipid and lipid particles prepared from the cationic lipid, and to the use of the cationic lipid in delivering therapeutic agents and in drug preparation. Background Art

[0002] Lipid particles are often used to deliver macromolecules (such as DNA, RNA / protein) and small molecules (such as small molecule compounds or medically active small molecules) to cells and tissues in laboratory and clinical research. In particular, those containing cationic lipid components are particularly effective in delivering anionic molecules such as nucleic acids.

[0003] In recent years, drugs based on nucleic acids (especially RNA, including siRNA, miRNA and mRNA, etc.) have attracted more and more attention in regulating protein expression at the gene level. In in vitro and in vivo models, they have demonstrated advantages that traditional small molecule drugs and antibody drugs do not have. Researchers have used lipid-based carrier systems to deliver therapeutic nucleic acids. Although some progress has been made, there is still a problem of low delivery efficiency, resulting in the termination of experiments in many clinical studies due to the efficacy not being as expected. There is still a need in the art for lipid carriers for efficient delivery of nucleic acids. Summary of the invention

[0004] In order to solve the problems existing in the above prior art, the purpose of the present invention is to provide a novel cationic lipid, lipid particle composition and a method for introducing nucleic acid into cells using the composition. These lipid particles protect nucleic acids from degradation and removal, and release nucleic acids in cells, greatly improving the delivery efficiency of nucleic acids and giving full play to the role of RNA drugs in living bodies.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A compound having structural formula (I), which is a cationic lipid or a pharmaceutically acceptable salt thereof:

[0007]

[0008] in:

[0009] R1 is an optionally substituted alkyl, alkenyl, alkynyl or -(CH2) p Q'; when the substituent is -(CH2) p When Q', Q' is OH, alkoxy, -NH2 or alkylamino, and p is 1, 2, 3, 4, 5 or 6;

[0010] R' is absent or substituted hydrogen or alkyl;

[0011] k, l, m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0012] M1 and M2 are each independently absent or substituted with -C(O)O-, -OC(O)-, -SS-, -OC(O)O-, -C(O)NH-, -NH C(O)-, -C(O)S-, -C(S)O-, -NH-C(O)O- or -OC(O)-NH-;

[0013] Q1, Q2 are substituted -O- or -S- or at most one of them is absent;

[0014] R2, R3, and R4 are each independently a substituted C1-C12 alkylene group or a C2-C12 alkenylene group.

[0015] Furthermore, the k, l, m and n also include each independently being 0.

[0016] Furthermore, the R2, R3, and R4 also include C13-C24 substituted alkylene or C13-C24 alkenylene, each of which is independently substituted.

[0017] Preferably, in the compound described in the present invention, R1 is -(CH2)2-OH.

[0018] Preferably, in the compound of the present invention, M1 and M2 are each independently -C(O)NH-, -C(O)O- or -OC(O)-.

[0019] Preferably, the compound of the present invention is selected from II, III, IV, V or their pharmaceutically acceptable salts as shown below,

[0020]

[0021] wherein k, l, m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and R2 is a substituted C1-C12 alkylene or C2-C12 alkenylene.

[0022] Preferably, the compound of the present invention is selected from VI, VII, VIII or their pharmaceutically acceptable salts as shown below,

[0023]

[0024] A compound having structural formula (IA), which is a cationic lipid or a pharmaceutically acceptable salt thereof:

[0025]

[0026] in:

[0027] R1 is an optionally substituted alkyl, alkenyl, alkynyl or -(CH2) p Q'; when the substituent is -(CH2) p When Q', Q' is OH, alkoxy, -NH2 or alkylamino, and p is 1, 2, 3, 4, 5 or 6;

[0028] R' is absent or substituted hydrogen or alkyl;

[0029] k, l, m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0030] M1 and M2 are each independently absent or substituted with -C(O)O-, -OC(O)-, -SS-, -OC(O)O-, -C(O)NH-, -NH C(O)-, -C(O)S-, -C(S)O-, -NH-C(O)O- or -OC(O)-NH-;

[0031] Q1 is substituted -O- or -S-;

[0032] R2, R3, and R4 are each independently a substituted C1-C24 alkylene group or a C2-C24 alkenylene group.

[0033] Preferably, in the compound of the present invention, M1 and M2 are each independently -C(O)NH-, -C(O)O- or -OC(O)-.

[0034] Preferably, R2 in compound IA is independently selected from one of the following structures:

[0035]

[0036] A compound having structural formula (IB), which is a cationic lipid or a pharmaceutically acceptable salt thereof:

[0037]

[0038] in:

[0039] R1 is an optionally substituted alkyl, alkenyl, alkynyl or -(CH2) p Q'; when the substituent is -(CH2) p When Q', Q' is OH, alkoxy, -NH2 or alkylamino, and p is 1, 2, 3, 4, 5 or 6;

[0040] R' is absent or substituted hydrogen or alkyl;

[0041] k, l, k' and l' are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0042] M1 and M2 are each independently absent or substituted with -C(O)O-, -OC(O)-, -SS-, -OC(O)O-, -C(O)NH-, -NH C(O)-, -C(O)S-, -C(S)O-, -NH-C(O)O- or -OC(O)-NH-;

[0043] Q1, Q1' are substituted -O- or -S-;

[0044] R2, R3, and R4 are each independently a substituted C1-C24 alkylene group or a C2-C24 alkenylene group.

[0045] Preferably, in the compound of the present invention, M1 and M2 are each independently -C(O)NH-, -C(O)O- or -OC(O)-.

[0046] Preferably, R2 in the compound formula (IB) is independently selected from one of the following structures:

[0047]

[0048] A lipid particle comprising the above compound.

[0049] Furthermore, the median diameter of the lipid particles of the present invention is 25-300 nm.

[0050] Furthermore, the lipid particles of the present invention also contain non-cationic lipids.

[0051] Furthermore, the non-cationic lipid in the lipid particles of the present invention is a neutral lipid and / or a sterol.

[0052] Preferably, the neutral lipid in the lipid particles described in the present invention is a phospholipid, and the phospholipid comprises one or a combination of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphocholine.

[0053] Preferably, the sterol in the lipid particles of the present invention is cholesterol or a cholesterol derivative.

[0054] Furthermore, the lipid particles of the present invention further comprise conjugated lipids that inhibit particle aggregation.

[0055] Furthermore, the conjugated lipid for inhibiting particle aggregation of the present invention comprises a PEG-lipid conjugate.

[0056] Preferably, the PEG-lipid conjugate of the present invention comprises one or a mixture of PEG-diacylglycerol (PEG-DAG) conjugate and PEG-dialkoxypropyl (PEG-DAA) conjugate.

[0057] A nucleic acid introduction agent comprises the lipid particles of the present invention and nucleic acid.

[0058] A pharmaceutical composition comprises the lipid particles of the present invention and a therapeutic agent.

[0059] Furthermore, the therapeutic agent of the present invention is a nucleic acid.

[0060] Furthermore, the nucleic acid includes interfering RNA molecules, and also includes single-stranded or double-stranded DNA, RNA or DNA / RNA hybrids, antisense oligonucleotides, ribozymes, plasmids, and immunostimulatory oligonucleotides.

[0061] Furthermore, the interfering RNA molecule includes one or a combination of small interfering RNA, asymmetric interfering RNA, micro RNA, and small hairpin RNA.

[0062] Furthermore, in the pharmaceutical composition of the present invention, the therapeutic agent is completely encapsulated in the lipid particles.

[0063] Furthermore, in the pharmaceutical composition of the present invention, the mass ratio of lipid to therapeutic agent in the lipid particles is (5:1)-(15:1).

[0064] Furthermore, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0065] The pharmaceutical composition of the present invention is used in the preparation of a drug for treating or preventing a disease in a subject, wherein the disease is selected from viral infection diseases, liver diseases or cancer.

[0066] Furthermore, the administration method of the drug is selected from oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous or intradermal.

[0067] A preparation comprising the pharmaceutical composition of the present invention and also comprising a vaccine or an antigen. Thus, the 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 comprises a tumor antigen or an infectious agent antigen.

[0068] Furthermore, in the preparation of the present invention, the antigen is a tumor antigen, and the tumor antigen includes proteins of Ras, p53, Her2, KSA, TRP1, TRP2 or BCR-abl oncogenes.

[0069] Furthermore, in the preparation of the present invention, the antigen is an infectious agent antigen, which includes a viral antigen, a bacterial antigen or a parasite antigen.

[0070] Furthermore, in the preparation of the present invention, the viral antigen includes hepatitis B virus, hepatitis A virus, tuberculosis virus, rubella virus, smallpox virus, influenza virus, measles virus, rotavirus or coronavirus.

[0071] Furthermore, in the preparation described in the present invention, the viral antigen is also derived from Retroviridae, Picornaviridae, Caliciviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Togaviridae, Flaviviridae, Bunyaviridae, Arenaviridae, Dirnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Iridoviridae or an unclassified virus.

[0072] Beneficial effects: The present invention discloses a cationic lipid and lipid particles prepared from the cationic lipid. The lipid particles can be prepared into pharmaceutical compositions or other preparations, and can also be used to prepare nucleic acid introduction agents to introduce functional nucleic acids into cells, effectively exerting the functions of functional nucleic acids. The experimental results show that the introduction of functional nucleic acids using the nucleic acid introduction agent of the cationic lipid prepared by the present invention can achieve effective gene regulation by utilizing the functional nucleic acids delivered to the cytoplasm, and its effect is much higher than that of commercially available products. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 It is the nuclear magnetic resonance image of the compound of formula VII.

[0074] Figure 2 The figure shows the knockout results of mRNA in Jurkat cells by lipid particles prepared containing compounds of formula VI, VII and VIII.

[0075] Figure 3 This is a diagram showing the knockout results of mRNA in Jurkat cells by LNP-4 to LNP-14 lipid particles.

[0076] Figure 4 This is the result of knocking down mRNA in Jurkat cells by LNP-15 to LNP-25 lipid particles.

[0077] Figure 5 The figure shows the knockout results of mRNA in Jurkat cells by lipid particles prepared from compounds of formulas A1 to A12 and B1 to B11.

[0078] Figure 6 The figure shows the knockout results of mRNA in mice by lipid particles prepared from compounds of formula VI, VII and VIII.

[0079] Figure 7The figure shows the knockout results of mRNA in tumor cells in mice using lipid particles prepared from compounds of formula VI, VII and VIII.

[0080] Figure 8 The figure shows the results of mRNA expression in mice mediated by lipid particles prepared containing compounds of formula VI, VII and VIII.

[0081] Fig. 9 The figure shows the results of mRNA expression in mice mediated by lipid particles prepared from compounds of formula A6, A7, A9 and B5. DETAILED DESCRIPTION

[0082] The present invention is further described below in conjunction with specific embodiments, but the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0083] The applicant has developed a cationic lipid prepared into lipid particles. The lipid particles of the present invention can be used to deliver therapeutic agents (such as nucleic acid molecules) or other agents to cells (in vitro or in vivo in a subject), thereby further exerting the function of delivering substances. The present invention provides a cationic lipid having structural formula (I) or a pharmaceutically acceptable salt thereof,

[0084]

[0085] in:

[0086] R1 is an optionally substituted alkyl, alkenyl, alkynyl or -(CH2) p Q'; when the substituent is -(CH2) p When Q', Q' is OH, alkoxy, -NH2 or alkylamino, and p is 1, 2, 3, 4, 5 or 6;

[0087] R' is absent or substituted hydrogen or alkyl;

[0088] k, l, m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0089] M1 and M2 are each independently absent or substituted with -C(O)O-, -OC(O)-, -SS-, -OC(O)O-, -C(O)NH-, -NH C(O)-, -C(O)S-, -C(S)O-, -NH-C(O)O- or -OC(O)-NH-;

[0090] Q1, Q2 are substituted -O- or -S- or at most one of them is absent;

[0091] R2, R3, and R4 are each independently a substituted C1-C12 alkylene group or a C2-C12 alkenylene group.

[0092] In one embodiment, the cationic lipid is selected from Formula II-V or a pharmaceutically acceptable salt thereof,

[0093]

[0094] wherein k, l, m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and R2 is a C1-C12 alkylene group or a C2-C12 alkenylene group.

[0095] In certain embodiments, the cationic lipid is selected from the following formula VI, VII, VIII:

[0096]

[0097] In certain embodiments, the cationic lipid is selected from formulas L1 to L22 in the following table.

[0098] Table 1

[0099]

[0100]

[0101] The present invention provides a compound having structural formula (IA), which is a cationic lipid or a pharmaceutically acceptable salt thereof:

[0102]

[0103] in:

[0104] R1 is an optionally substituted alkyl, alkenyl, alkynyl or -(CH2) p Q'; when the substituent is -(CH2) p When Q', Q' is OH, alkoxy, -NH2 or alkylamino, and p is 1, 2, 3, 4, 5 or 6;

[0105] R' is absent or substituted hydrogen or alkyl;

[0106] k, l, m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0107] M1 and M2 are each independently absent or substituted with -C(O)O-, -OC(O)-, -SS-, -OC(O)O-, -C(O)NH-, -NH C(O)-, -C(O)S-, -C(S)O-, -NH-C(O)O- or -OC(O)-NH-;

[0108] Q1 is substituted -O- or -S-;

[0109] R2, R3, and R4 are each independently a substituted C1-C24 alkylene group or a C2-C24 alkenylene group.

[0110] In certain embodiments, M1 and M2 in Formula (IA) are each independently -C(O)NH-, -C(O)O- or -OC(O)-.

[0111] In a specific experimental scheme, R2 in compound IA is independently selected from one of the following structures:

[0112]

[0113] In certain embodiments, the cationic lipid is selected from Formulas A1 to A12 in the following table.

[0114] Table 2

[0115]

[0116]

[0117] A compound having structural formula (IB), which is a cationic lipid or a pharmaceutically acceptable salt thereof:

[0118]

[0119] in:

[0120] R1 is an optionally substituted alkyl, alkenyl, alkynyl or -(CH2) p Q'; when the substituent is -(CH2) p When Q', Q' is OH, alkoxy, -NH2 or alkylamino, and p is 1, 2, 3, 4, 5 or 6;

[0121] R' is absent or substituted hydrogen or alkyl;

[0122] k, l, k' and l' are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0123] M1 and M2 are each independently absent or substituted with -C(O)O-, -OC(O)-, -SS-, -OC(O)O-, -C(O)NH-, -NH C(O)-, -C(O)S-, -C(S)O-, -NH-C(O)O- or -OC(O)-NH-;

[0124] Q1, Q1' are substituted -O- or -S-;

[0125] R2, R3, and R4 are each independently a substituted C1-C24 alkylene group or a C2-C24 alkenylene group.

[0126] In certain embodiments, M1 and M2 in the compound of formula (IB) are each independently -C(O)NH-, -C(O)O- or -OC(O)-.

[0127] In a specific experimental scheme, R2 in the compound of formula (IB) is independently selected from one of the following structures:

[0128]

[0129] In certain embodiments, the cationic lipid is selected from Formulas B1 to B11 in the following table.

[0130] Table 3

[0131]

[0132]

[0133] The compounds of the present invention can be prepared by known organic synthesis techniques, including the methods described in the examples. It should be understood that certain non-critical variables of the embodiments may be changed by reading the description herein, which will be apparent to those skilled in the art.

[0134] The present invention also discloses a lipid particle comprising a cationic lipid of the present invention, wherein the lipid particle is a nanoparticle, and the average diameter of the lipid particle is from about 25 nm to about 300 nm, for example, from about 50 nm to 200 nm or from about 100 nm to about 200 nm. In one embodiment, the lipid particle comprises a compound of formula I of the present invention. Further, in a specific embodiment, the lipid particle comprises a compound of any one of formulas II-V of the present invention or comprises a compound of any one of formulas VI, VII, VIII of the present invention or L1-L22 in Table 1.

[0135] The lipid particles can also be cationic lipids in their pharmaceutically acceptable salts. The lipid particles further include one or more non-cationic lipids, such as neutral lipids. In certain other embodiments, the lipid particles further include one or more conjugated lipids that can reduce or inhibit particle aggregation. The non-cationic lipid component in the lipid particles can include but is not limited to phospholipids, cholesterol (or cholesterol derivatives) or mixtures thereof. Phospholipids include but are not limited to one or more combinations of DOPE, DOPC, DSPC, DPPC. The conjugated lipid components that can reduce or inhibit particle aggregation include but are not limited to polyethylene glycol (PEG)-lipid conjugates, and specific PEG-lipid conjugates include but are not limited to one or more combinations of PEG-DMG, PEG-DAG, PEG-DAA conjugates.

[0136] In a specific embodiment, the lipid granules include cationic lipids, neutral lipids, sterols and PEG-lipid conjugates of the present invention. The lipid granules include cationic lipids of about 25% to about 75% by mole percentage, such as about 30% to about 60% by mole percentage, about 40% to about 70%, about 35%, about 50%, about 70.1% or about 61.5% by mole percentage. The lipid granules include neutral lipids of about 0% to about 20% by mole percentage, such as about 5% to about 10% by mole percentage, about 4% to about 12%, about 15%, about 6.3% or about 0%, in a specific embodiment, the neutral lipids are DSPC or DOPE. The lipid granules include sterols of about 5% to 50% by mole percentage, such as about 10% to 40% by mole percentage, about 15 to 35%, about 6.1%, about 12%, about 31.5% or about 33.7%, in a specific embodiment, the sterol is cholesterol. The lipid particles include about 0.1% to about 20% PEG conjugate by mole, 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. In a specific embodiment, the PEG-lipid conjugate is PEG-DMG.

[0137] In a specific embodiment, the lipid particle comprises, by mole percentage, 25-75% cationic lipid, 0.5-15% neutral lipid, 5-50% sterol and 0.5-20% PEG conjugate.

[0138] Table 4 Exemplary lipid particles according to some non-limiting embodiments (in mol %)

[0139]

[0140]

[0141] Table 5 Exemplary lipid particles according to some non-limiting embodiments (in mol %)

[0142] Lipid particles Compound of formula VII DSPC cholesterol PEG-DAG 1 50.5 10 38.5 1 2 40 15 40 5 3 39.5 6 44 10.5 4 60.7 12.4 20.5 6.4 5 51 9 39.1 0.9

[0143] The present invention also discloses a pharmaceutical composition comprising lipid particles of the present invention and a therapeutic agent or an active agent, wherein the active agent or the therapeutic agent comprises a nucleic acid. In certain embodiments, the nucleic acid comprises an interfering RNA molecule, such as siRNA, miRNA, shRNA or a mixture thereof. In certain embodiments, the nucleic acid comprises a single-stranded or double-stranded DNA, RNA or a DNA / RNA hybrid such as mRNA, antisense oligonucleotides, ribozymes, plasmids, immunostimulatory oligonucleotides or a mixture thereof.

[0144] In some embodiments, the ratio of lipid particle:nucleic acid is between about 0.5:1 and about 12:1 or between about 0.5:1 and about 30:1 by mass ratio.

[0145] The active agent or therapeutic agent is completely encapsulated within the lipid particle, such that the active agent or therapeutic agent in the lipid particle is resistant to enzymatic degradation in aqueous solution, for example, by nucleases or proteases, and the lipid particle is substantially non-toxic to mammals such as humans.

[0146] The present invention also provides a nucleic acid introduction agent, which comprises the lipid particles of the present invention and nucleic acid. The method of introducing nucleic acid into cells is: contacting the cells with the nucleic acid-lipid particles of the present invention. In a specific embodiment, the cells are in a mammal and the mammal is a human.

[0147] In one specific experimental protocol, the cells were grown at a density of about 10 3 to about 10 5 cells / mL, and lipid-nucleic acid particles are added to the cells seeded with 60-80% confluency. In another embodiment, lipid particles can be used to deliver nucleic acids to cells or cell lines, including but not limited to A549, HeLa, MDA-MB-231, K562, Jurkat cells, etc.

[0148] The present invention also discloses the use of the pharmaceutical composition in the preparation of a drug for treating or preventing a disease in a subject, wherein the disease is selected from a viral infection, a liver disease or a cancer; and the drug is administered orally, intranasally, intravenously, intraperitoneally, intramuscularly, intraarticularly, intralesionally, intratracheally, subcutaneously or intradermally. In some specific embodiments, the pharmaceutical composition is administered systemically, for example, via an enteral or parenteral administration route. In some preferred embodiments, the mammal is a human.

[0149] 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 a specific embodiment, the nucleic acid-lipid particle pharmaceutical composition is administered intravenously or intraperitoneally by injection.

[0150] In some embodiments, the nucleic acid-lipid particle pharmaceutical composition can also be directly applied to the tissue to contact the pharmaceutical composition with the target tissue. The application can be local, open or closed surgery. Local refers to the direct application of the pharmaceutical preparation to the tissue exposed to the environment, such as skin, oropharynx, external auditory canal, etc. Open surgery includes cutting the patient's skin and directly observing the operation of the lower tissue of the application of the pharmaceutical preparation with the naked eye. Closed surgery is an invasive surgery, in which the internal target tissue cannot be directly seen, such as lumbar puncture.

[0151] In some embodiments, the pharmaceutical composition may also be inhaled into the respiratory tract, such as the lungs, in the form of an aerosol.

[0152] In a specific embodiment, the pharmaceutical composition comprising lipid-nucleic acid particles further comprises a pharmaceutically acceptable carrier. Typically, physiological saline can be used as a pharmaceutically acceptable carrier. Other suitable carriers include water, 5% glucose, etc., and also include glycoproteins for improving stability, such as albumin, lipoprotein, globulin, etc.

[0153] The present invention also discloses a preparation comprising the pharmaceutical composition of the present invention. In a specific embodiment, the preparation further comprises a vaccine or an antigen, and the vaccine may comprise a lipid particle, and the lipid particle comprises an immunostimulatory oligonucleotide. In a specific embodiment, the antigen is a tumor antigen or is associated with an infectious agent, such as a virus, a bacterium, or a parasite. Tumor antigens, infectious agent antigens, and antigens associated with other diseases are well known in the art. On the one hand, antigens include, but are not limited to, hepatitis B virus, hepatitis A virus, tuberculosis virus, rubella virus, smallpox virus, influenza virus, measles virus, rotavirus, or coronavirus. On the other hand, the antigen is a tumor antigen, including proteins of Ras, p53, Her2, KSA, TRP1, TRP2, or BCR-abl oncogenes.

[0154] In the preparations described in the present invention, the sources of the viral antigens include, but are not limited to, Retroviridae, Picornaviridae, Caliciviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Togaviridae, Flaviviridae, Bunyaviridae, Arenaviridae, Binaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Iridoviridae or unclassified viruses. The following general reaction schemes illustrate methods for preparing the compounds of the present invention, and it should be understood that those skilled in the art can prepare these compounds by similar methods or by other methods known to those skilled in the art. It should also be understood that those skilled in the art can prepare other compounds not explicitly described below in a similar manner as described below by using appropriate starting components and modifying the parameters of the synthesis as needed.

[0155] General reaction scheme

[0156]

[0157] The general reaction schemes provide exemplary methods for preparing compounds of the present invention. R1, R2, R3, R4, k, l, k' and l' in the schemes are as defined herein, and G in the schemes is x , G x’ Each of them is independently taken from G1 to G8. The raw materials used in the general reaction scheme can be purchased or prepared according to methods known in the art.

[0158] Example

[0159] The present invention will be described in more detail below by specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will readily appreciate that certain noncritical parameters can be changed or modified to produce substantially the same result.

[0160] Example 1 Synthesis of Compound VI

[0161]

[0162] Synthesis of compound 3: Diisopropylethylamine (33 mL, 187 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (11 g, 58 mmol) and DMAP (1.14 g, 9 mmol) were added to a solution of compound 1 (10.0 g, 44.2 mmol) and compound 2 (15.0 g, 58 mmol) in dichloromethane (200 mL). The reaction mixture was stirred at room temperature for 14 h. The reaction mixture was then diluted with CH2Cl2 (400 mL) and washed with saturated NaHCO3 aqueous solution (300 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 3 (8.1 g, 38%). 25 H 49 The calculated molecular weight of BrO2(M+H)+ is 461.57 and the measured value is 461.5.

[0163] Synthesis of compound 4: Compound 3 (4 g, 8.6 mmol) and 2-hydroxyethylamine (15 mL, 248 mmol) were dissolved in ethanol (50 mL) and stirred at 62 ° C for 18 hours. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved in ethyl acetate and washed with water. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 4 (3 g, 78%). 27 H 55 The calculated molecular weight of NO3(M+H)+ is 442.68, and the measured value is 442.7. Synthesis of Compound 6: Dissolve 1,2-dibromoethane (20 g, 108.7 mmol) and potassium carbonate (18 g, 130 mmol) in 200 mL DMF, heat to 60°C, then dropwise add the DMF solution of Compound 5 (10 g, 51.5 mmol) and react for 6 hours. After the reaction, remove the organic solvent by rotary evaporation, then dissolve in dichloromethane and wash with water. Dry the organic layer with anhydrous MgSO4, filter and concentrate. Purify the crude product by silica gel column chromatography to obtain Compound 6 (4.3 g, 28%). 13 H 17 The calculated molecular weight of BrO3(M+H)+ is 301.18 and the measured value is 301.2.

[0164] Synthesis of compound 7: Add 30 mL of ethanol to compound 6 (4.0 g, 13.3 mmol) and Pd / C (0.4 g) to dissolve, and react for two hours under a hydrogen environment. After 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 7 (2.1 g, 75%). 11 The calculated molecular weight of BrO3(M+H)+ is 211.06 and the measured value is 211.

[0165] Synthesis of compound 8: Diisopropylethylamine (3.3 mL, 19 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.8 g, 9.5 mmol) and DMAP (0.11 g, 0.9 mmol) were added to a solution of compound 7 (2.0 g, 9.5 mmol) and n-hexylamine (0.96 g, 9.5 mmol) in dichloromethane (30 mL). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was then diluted with CH2Cl2 (100 mL) and washed with a saturated NaHCO3 aqueous solution (100 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 8 (2.1 g, 72%). 13 H 12 The calculated molecular weight of BrNO2(M+H)+ is 308.26 and the measured value is 308.2.

[0166] Synthesis of compound 9: Diisopropylethylamine (0.42 g, 3.25 mmol) was added to a solution of compound 4 (1.4 g, 3.25 mmol) and compound 8 (1 g, 3.25 mmol) in dichloromethane (30 mL), and the reaction mixture was stirred at 65 ° C for 18 h. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved in ethyl acetate and washed with water. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 9 (0.6 g, 27%). 41 H 82 The calculated molecular weight of N2O5(M+H)+ is 683.12 and the measured value is 683.2.

[0167] Example 2 Synthesis of Compound VII

[0168]

[0169] Synthesis of compound 3: Diisopropylethylamine (33 mL, 187 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (11 g, 58 mmol) and DMAP (1.14 g, 9 mmol) were added to a solution of compound 1 (10.0 g, 44.2 mmol) and compound 2 (15.0 g, 58 mmol) in dichloromethane (200 mL). The reaction mixture was stirred at room temperature for 14 h. The reaction mixture was then diluted with CH2Cl2 (400 mL) and washed with saturated NaHCO3 aqueous solution (300 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 3 (8.1 g, 38%). 25 H 49The calculated molecular weight of BrO2(M+H)+ is 461.57 and the measured value is 461.5.

[0170] Synthesis of compound 4: Compound 3 (4 g, 8.6 mmol) and 2-hydroxyethylamine (15 mL, 248 mmol) were dissolved in ethanol (50 mL) and stirred at 62 ° C for 18 hours. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved in ethyl acetate and washed with water. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 4 (3 g, 78%). 27 H 55 The molecular weight of NO3(M+H)+ was calculated to be 442.68 and the measured value was 442.7. Synthesis of Compound 7: Diisopropylethylamine (2.6mL, 15mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.4g, 7.6mmol) and DMAP (0.08g, 0.7mmol) were added to a solution of compound 5 (1.0g, 7.6mmol) and compound 6 (1.7g, 7.6mmol) in dichloromethane (30mL). The reaction mixture was stirred at room temperature for 12h. The reaction mixture was then diluted with CH2Cl2 (100mL) and washed with a saturated NaHCO3 aqueous solution (100mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 7 (1.8g, 67%). 16 H 32 The calculated molecular weight of BrNO2(M+H)+ is 350.34 and the measured value is 350.4.

[0171] Synthesis of compound 8: Diisopropylethylamine (0.42 g, 3.25 mmol) was added to a solution of compound 4 (1.4 g, 3.25 mmol) and compound 7 (1.1 g, 3.25 mmol) in dichloromethane (30 mL), and the reaction mixture was stirred at 65 ° C for 18 h. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved in ethyl acetate and washed with water. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 9 (0.72 g, 31%). 43 H 86 The calculated molecular weight of N2O5(M+H)+ is 710.65, and the measured value is 710.6. The NMR result is shown in the figure below: Figure 1 shown.

[0172] Example 3 Synthesis of Compound VIII

[0173]

[0174] Synthesis of compound 3: Diisopropylethylamine (33 mL, 187 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (11 g, 58 mmol) and DMAP (1.14 g, 9 mmol) were added to a solution of compound 1 (10.0 g, 44.2 mmol) and compound 2 (15.0 g, 58 mmol) in dichloromethane (200 mL). The reaction mixture was stirred at room temperature for 14 h. The reaction mixture was then diluted with CH2Cl2 (400 mL) and washed with saturated NaHCO3 aqueous solution (300 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 3 (8.1 g, 38%). 25 H 49 The calculated molecular weight of BrO2(M+H)+ is 461.57 and the measured value is 461.5.

[0175] Synthesis of compound 4: Compound 3 (4 g, 8.6 mmol) and 2-hydroxyethylamine (15 mL, 248 mmol) were dissolved in ethanol (50 mL) and stirred at 62 ° C for 18 hours. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved in ethyl acetate and washed with water. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 4 (3 g, 78%). 27 H 55 The molecular weight of NO3(M+H)+ was calculated to be 442.68 and the measured value was 442.7. Synthesis of Compound 7: Diisopropylethylamine (3.3mL, 19mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.8g, 9.5mmol) and DMAP (1.1g, 0.95mmol) were added to a solution of compound 5 (1.0g, 9.5mmol) and compound 6 (2.2g, 9.5mmol) in dichloromethane (30mL). The reaction mixture was stirred at room temperature for 12h. The reaction mixture was then diluted with CH2Cl2 (100mL) and washed with a saturated NaHCO3 aqueous solution (100mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 7 (2.3g, 74.7%). 16 H 32 The calculated molecular weight of BrNO2(M+H)+ is 324.32 and the measured value is 324.3.

[0176] Synthesis of compound 8: Diisopropylethylamine (0.42 g, 3.25 mmol) was added to a solution of compound 4 (1.4 g, 3.25 mmol) and compound 7 (1.05 g, 3.25 mmol) in dichloromethane (30 mL), and the reaction mixture was stirred at 65 ° C for 18 h. After the reaction was completed, the organic solvent was removed by rotary evaporation, and then dissolved in ethyl acetate and washed with water. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain compound 9 (0.8 g, 36%). 40 H 80 The calculated molecular weight of N2O4S (M+H)+ is 685.15 and the measured value is 685.2.

[0177] Example 4 Synthesis of Compound A6

[0178] 2-Hexyldecanoic acid (8.8 g, 34.3 mmol), DCC (8.5 g, 41.2 mmol) and DMAP (0.47 g, 3.5 mmol) were added to a solution of 6-bromo-1-hexanol (6.4 g, 35.1 mmol) in dichloromethane (200 mL), 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 A6-1 (3.8 g).

[0179] A6-1 (3.8 g, 9.1 mmol) and 4-amino-1-butanol (1.6 g, 18.2 mmol) were dissolved in ethanol and stirred at 65°C for 16 hours, then cooled to room temperature and dried in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain intermediate A6-2 (3.6 g).

[0180] 2-Hexyldecanoic acid (8.7 g, 33.9 mmol), DCC (8.4 g, 40.7 mmol) and DMAP (0.46 g, 3.4 mmol) were added to a solution of 1,3-propylene glycol (10.3 g, 135 mmol) in dichloromethane (200 mL), 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 A6-3 (7.9 g).

[0181] Intermediate A6-3 (4.3 g, 12.9 mmol) was dissolved in dichloromethane (150 mL), treated with sodium salt, and then 1,4-dibromobutane (5.6 g, 25.8 mmol) was added. The reaction was stirred overnight and purified by silica gel column to obtain intermediate A6-4 (3.9 g).

[0182] Intermediate A6-2 (3.6 g, 7.1 mmol) and intermediate A6-4 (2.3 g, 7.1 mmol) were stirred in an ethanol solution of N,N-diisopropylethylamine (0.91 g, 7.1 mmol) at 65°C for 16 hours and then cooled to room temperature and dried in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO3, the organic phase was removed by vacuum drying, and the product was purified by silica gel chromatography to obtain compound A6 (0.76 g).

[0183] Example 5 Synthesis of Compound A7

[0184] Intermediate A6-1 (1.18 g, 2.8 mmol) and 3-amino-1-propanol (0.21 g, 2.8 mmol) were dissolved in ethanol and stirred at 65°C for 16 hours, then cooled to room temperature and dried in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain intermediate A7-1 (1.05 g).

[0185] Intermediate A7-1 (1.05 g, 2.1 mmol) and intermediate A6-3 (0.71 g, 2.1 mmol, the synthesis steps are the same as Example 14) were stirred in an ethanol solution of N,N-diisopropylethylamine (0.27 g, 7.1 mmol) at 65°C for 16 hours and then cooled to room temperature and vacuum dried. The crude product was extracted with ethyl acetate and saturated NaHCO3, the organic phase was removed by vacuum drying, and the product was purified by silica gel chromatography to obtain compound A7 (0.39 g).

[0186] Example 6 Synthesis of Compound B3

[0187] Diamyl acetic acid (8.0 g, 39.9 mmol), DCC (9.9 g, 47.9 mmol) and DMAP (0.54 g, 4.0 mmol) were added to a solution of ethylene glycol (8.6 mL, 155 mmol) in dichloromethane (200 mL), 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 obtain intermediate B3-1 in an output of 7.6 g.

[0188] Intermediate B3-1 (3.8 g, 15.5 mmol) was dissolved in dichloromethane (100 mL), treated with sodium salt, and then 1,3-dibromopropane (6.3 g, 31.0 mmol) was added. The reaction was stirred overnight and purified by silica gel column to obtain intermediate B3-2 (3.6 g).

[0189] Intermediate 2 (3.6 g, 9.85 mmol), 4-amino-1-butanol (0.45 g, 4.9 mmol) and N,N-diisopropylethylamine (0.63 g, 4.9 mmol) were dissolved in ethanol and stirred at 65°C for 16 hours, then cooled to room temperature and dried in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain compound B3 (1.6 g).

[0190] Example 7 Synthesis of Compound B4

[0191] Dibutyl acetic acid (8.0 g, 46.4 mmol), DCC (11.5 g, 55.7 mmol) and DMAP (0.62 g, 4.6 mmol) were added to a solution of ethylene glycol (10.3 mL, 186 mmol) in dichloromethane (200 mL), 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 B4-1 (7.8 g).

[0192] Intermediate B4-1 (3.9 g, 16.2 mmol) was dissolved in dichloromethane (150 mL), treated with sodium salt, and then 1,3-dibromopropane (6.6 g, 32.5 mmol) was added. The reaction was stirred overnight and purified by silica gel column to obtain intermediate B4-2 (3.8 g).

[0193] Intermediate B4-2 (3.8 g, 10.5 mmol), 4-amino-1-butanol (0.5 g, 5.2 mmol) and N,N-diisopropylethylamine (0.67 g, 5.2 mmol) were dissolved in ethanol and stirred at 65°C for 16 hours, then cooled to room temperature and dried in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain compound B4 (1.7 g).

[0194] Example 8 Synthesis of Compound B5

[0195] 2-Hexyldecanoic acid (12.5 g, 48.7 mmol), DCC (12.1 g, 58.5 mmol) and DMAP (0.65 g, 4.9 mmol) were added to a solution of ethylene glycol (10.8 mL, 195 mmol) in dichloromethane (200 mL), 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 B5-1 (12.2 g).

[0196] Intermediate B5-1 (6.1 g, 19.1 mmol) was dissolved in dichloromethane (100 mL), treated with sodium salt, and then 1,3-dibromopropane (7.6 g, 38.2 mmol) was added. The reaction was stirred overnight and purified by silica gel column to obtain intermediate B5-2 (5.9 g).

[0197] Intermediate B5-2 (5.9 g, 13.4 mmol), 4-amino-1-butanol (0.6 g, 6.7 mmol) and N,N-diisopropylethylamine (0.86 g, 6.7 mmol) were dissolved in ethanol and stirred at 65°C for 16 hours, then cooled to room temperature and dried in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain compound B5 (2.12 g).

[0198] Example 9 Synthesis of Compound B6

[0199] 2-Butyl octanoic acid (10.2g, 50.9mmol), DCC (12.6g, 61.1mmol) and DMAP (0.68g, 5.1mmol) were added to a solution of ethylene glycol (11.3mL, 204mmol) 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, dried over anhydrous magnesium sulfate, and intermediate B6-1 (10.0g) was obtained.

[0200] Intermediate B6-1 (5.0 g, 18.6 mmol) was dissolved in dichloromethane (100 mL), treated with sodium salt, and then 1,3-dibromopropane (7.5 g, 37.3 mmol) was added. The reaction was stirred overnight and purified by silica gel column to obtain intermediate B6-2.

[0201] Intermediate B6-2 (4.8 g, 12.4 mmol), 4-amino-1-butanol (0.55 g, 6.2 mmol) and N,N-diisopropylethylamine (0.8 g, 6.2 mmol) were dissolved in ethanol and stirred at 65°C for 16 hours, then cooled to room temperature and dried under vacuum. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain compound B6 (1.8 g).

[0202] Example 10 Synthesis of Compound B8

[0203] Intermediate A6-3 (3.8 g, 11.9 mmol) was dissolved in dichloromethane (150 mL), treated with sodium salt, and then 1,2-dibromoethane (4.5 g, 23.9 mmol) was added. The reaction was stirred overnight and purified by silica gel column to obtain intermediate B8-1 (3.6 g).

[0204] Intermediate B8-1 (3.6 g, 8.2 mmol), 4-amino-1-butanol (0.36 g, 4.1 mmol) and N,N-diisopropylethylamine (0.53 g, 4.1 mmol) were dissolved in ethanol and stirred at 65°C for 16 hours, then cooled to room temperature and dried in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain compound B8 (1.2 g).

[0205] Example 11 Synthesis of Compound B9

[0206] Intermediate A6-3 (4.3 g, 12.9 mmol) was dissolved in dichloromethane (150 mL), treated with sodium salt, and then 1,4-dibromobutane (5.6 g, 25.8 mmol) was added. The reaction was stirred overnight and purified by silica gel column to obtain intermediate B9-1 (3.9 g).

[0207] Intermediate B9-1 (3.9 g, 8.3 mmol), 4-amino-1-butanol (0.36 g, 4.1 mmol) and N,N-diisopropylethylamine (0.53 g, 4.1 mmol) were dissolved in ethanol and stirred at 65°C for 16 hours, then cooled to room temperature and dried in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain compound B9 (1.3 g).

[0208] Example 12 Synthesis of Compound B10

[0209] 2-Hexyldecanoic acid (14.5 g, 56.5 mmol), DCC (14.0 g, 67.8 mmol) and DMAP (0.76 g, 5.7 mmol) were added to a solution of 1,5-pentanediol (23.1 g, 222 mmol) in dichloromethane (200 mL), 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 obtain intermediate B10-1 (13.1 g).

[0210] The intermediate B10-1 (6.65 g, 18.4 mmol) was dissolved in dichloromethane (150 mL), treated with sodium salt, and then 1,6-dibromohexane (8.9 g, 36.7 mmol) was added, stirred and reacted overnight, and purified by silica gel column to obtain intermediate B10-2 (6.2 g). The intermediate B10-2 (6.2 g, 11.9 mmol), 4-amino-1-butanol (0.55 g, 5.9 mmol) and N, N-diisopropylethylamine (0.76 g, 5.9 mmol) were dissolved in ethanol and stirred at 65 ° C for 16 hours, then cooled to room temperature and vacuum dried. The crude product was extracted with ethyl acetate and saturated NaHCO3, the solvent was removed by vacuum drying, and finally purified by silica gel chromatography to obtain compound B10 (1.5 g).

[0211] Example 13 Preparation and Characterization of Nucleic Acid-Lipid Particles

[0212] Cationic lipids, DSPC, cholesterol and PEG-lipids were dissolved in ethanol at a molar ratio of 40 / 10 / 40 / 10. The lipid mixture was added to a buffer (50 mM citrate, pH 4) and mixed to a final concentration of ethanol and lipid of 25% (vol / vol) and 5 mg / mL, respectively, and equilibrated at room temperature for 2 min.

[0213] 10% (wt / wt) siRNA (dissolved in 50 mM citrate at pH 4.0, containing 25% ethanol in water) was added to the particles, and the mixture was incubated at 35°C for 30 min to allow the complex to reassemble and encapsulate siRNA. The external buffer was then replaced by dialysis with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5), and finally stored at 4°C.

[0214] The particle size, polydispersity index (PDI) and surface potential of the lipid nanoparticles (LNP) prepared above were measured by dynamic light scattering (ZetasizerNano; Malvern instruments Ltd.).

[0215] The siRNA encapsulation efficiency and siRNA recovery rate were measured using RiboGreen (Invitrogen; Thermo Fischer Scientific). Specifically, the various LNPs prepared above were diluted to 1000 ng / mL with 10mM pH 7.4 HEPES buffer to prepare a sample solution. In addition, the nucleic acid-lipid nanoparticles to be tested were stepwise diluted to 0-2000 ng / mL with 10mM pH7.4 HEPES buffer to prepare a standard curve solution. In addition to these solutions, a measurement solution was prepared in which dextran sulfate, Triton X-100, and Ribogreen were diluted to 0.08 mg / mL, 0.4%, and 5 μL / mL, respectively, with 10mM HEPES buffer. In addition, a solution was prepared in which Triton X-100 was replaced with 10mM HEPES buffer. 50 μL of standard curve solution or sample solution was added to a 96-well plate, and 50 μL of the assay solution containing or not containing Triton X-100 was further added and mixed. After stirring at 700 rpm for 5 minutes, the fluorescence intensity at an excitation wavelength of 500 nm and an observation wavelength of 525 nm was measured. The siRNA recovery rate was calculated by dividing the amount of siRNA measured under conditions containing Triton X-100 by 1000 ng / mL. The siRNA encapsulation rate was calculated by subtracting the amount of siRNA measured under conditions containing Triton X-100 from the amount of siRNA measured under conditions containing Triton X-100, and dividing the amount of siRNA measured under conditions containing Triton X-100 by this value.

[0216] The results are shown in Tables 6 and 7.

[0217] Table 6

[0218]

[0219] Table 7

[0220]

[0221]

[0222] Example 14 In vitro efficacy determination of lipid particles

[0223] To evaluate the efficacy of various nucleic acid-lipid nanoparticle formulations, in vitro assays were performed using siRNA molecules targeting GAPDH. Nucleic acid-lipid nanoparticle formulations were prepared using siRNA against GAPDH as described in Example 13 above.

[0224] Cell culture and siRNA transfection:

[0225] Jurkat cells were plated in 24-well plates in advance to ensure 70-90% confluence on the day of the experiment. 50 μL of nucleic acid-lipid particles (siRNA concentration was 100 nM) were added on the day of the experiment and incubated for 48 h. TM For the control, 2.5 μL of 2 μM siRNA solution was mixed with 46.5 μL Opti-MEM and 1 μL Lipofectamine TM RNAiMAX was mixed. 50 μL of the resulting mixture was added to a single well of a 12-well plate and incubated at room temperature for 20 minutes to form a complex. It was then added to cells and cultured for another 48 hours. The target gene knockout efficiency was determined by qRT-PCR.

[0226] RNA extraction and analysis:

[0227] Wash the cells once with 2 mL of PBS and use the RNeasy Mini Kit TM Total RNA was extracted from the cDNA library using the Transcriptor 1st Strand cDNA Kit (Qiagen) and eluted in a total volume of 30 μL. TM (Roche) and random hexamers were used to reverse transcribe 1 μg of total RNA according to the instructions. One-thirtieth of the resulting cDNA (0.66 μL) was mixed with 5 μL IQ Multiplex Powermix (Bio-Rad), 3.33 μL H2O and 1 μL of a 3 μM mixture of primers and probes specific for the GAPDH target sequence. The CFX96 real-time system of the C1000 thermal cycler (Bio-Rad) was then used for the amplification reaction. The PCR conditions were: 95°C for 3 minutes; then cycled at 95°C for 10 seconds; and 40 cycles at 55°C for 1 minute. Each sample was tested in triplicate. The relative GAPDH mRNA levels were normalized to the target mRNA levels and compared with the mRNA levels obtained in the control samples treated with or without the transfection agent alone.

[0228] Figure 2 , Figure 3 and Figure 4 Provides in vitro gene knockout results using lipid nanoparticles LNP-1 to LNP-25, Figure 5The results of in vitro gene knockout using lipid nanoparticles A1 to A12 and B1 to B11 are provided. In general, when administered to Jurkat cells, LNP-1 to LNP-25 can effectively inhibit the target mRNA level. In particular, LNP-1, LNP-2 and LNP-3 can achieve a knockout efficiency of more than 80% for the target mRNA, and A6, A7, A9 and B5 can achieve a knockout efficiency of more than 90% for the target mRNA, which is significantly higher than Lipofectamine TM RNAiMAX control group.

[0229] Example 15: In vivo performance of nucleic acid-lipid nanoparticles

[0230] In order to further evaluate the performance of LNP, in vivo experiments were performed using preparations with siRNA for Factor VII. Nanoformulations were prepared by the method shown in Example 13. Single doses (1 mg / kg, 0.5 mg / kg or 0.1 mg / kg) of lipid particle preparations were administered to approximately 4-week-old female mice via intravenous administration via the tail vein, with a dosing volume of 10 μL / g body weight. Blood was collected after 24 hours, and the blood sample was centrifuged at 1000 g, 10 minutes, 4 ° C, and the supernatant was recovered to obtain plasma.

[0231] Factor VII (FVII) in plasma was quantified using BIOPHEN FVII CHROMOGENIC ASSAY (Sysmex BioMed), and the FVII expression level in each administration group was shown as a relative value (relative FVII amount in plasma), with the FVII expression level in the untreated group (NT) being taken as 1. Figure 6 Data with error bars are shown, mean ± SD of n=5 animals / group. The result is shown in that when the dosage of lipid formulation is 0.1 mg / kg, three administration groups (LNP-VI, LNP-VII and LNP-VIII) containing compounds VI, VII and VIII reach 80% knockout efficiency of FVII. Especially, LNP-VII shows the highest knockout efficiency.

[0232] Example 16: Performance of Nucleic Acid-Lipid Nanoparticle Formulations for Gene Knockout in Tumors in Vivo

[0233] To further evaluate the delivery efficiency of cationic lipids containing Formula VI, VII and VIII in tumors, we conducted a subcutaneous tumor model. siRNA targeting HPRT1 mRNA was used to prepare nanoformulations according to the method shown in Example 13. 50 μL 3×10 6Cells were established Hep3B tumors. 10-17 days after tumor inoculation, mice were randomly divided into treatment groups. Nucleic acid-lipid preparations were administered by tail vein injection, based on the mg siRNA / kg body weight basis calculated according to individual animal body weight. mRNA expression data were expressed as the contrast between the treatment group and the untreated group (NT). Figure 7 Data with error bars are shown, mean ± SD of n = 5 animals / group. The results show that when the dose of lipid formulation is 1 mg / kg, the three administration groups (LNP-VI, LNP-VII and LNP-VIII) containing compounds VI, VII and VIII have a knockout efficiency of 70% on HPRT1 mRNA. In particular, LNP-VII shows the highest knockout efficiency (> 85%).

[0234] Example 17: In vivo mRNA expression assay

[0235] Nanoformulations (LNP-VI, LNP-VII, and LNP-VIII) were prepared according to the method shown in Example 13, and diluted with PBS to make the mRNA 10 μg / mL, and administered subcutaneously to the neck of female mice aged 6 weeks. After 24 hours, the pre-prepared fluorescein (VivoGlo TM Luciferin (In Vivo Grade, Promega) aqueous solution was intraperitoneally administered to each mouse, and 30 minutes later, the mouse was monitored using IVIS. TM LuminaII (Caliper Life Sciences) was used to observe and quantify the luminescence at the administration site.

[0236] The results are shown in Figure 8 Luminescence was confirmed in LNP-VI, LNP-VII, and LNP-VIII, indicating mRNA expression activity. Among them, LNP-VI showed the strongest luminescence, indicating high mRNA expression activity.

[0237] Example 18 In vivo evaluation of subcutaneous injection of mRNA-lipid nanoparticle compositions

[0238] The in vivo effect of mRNA lipid nanoparticles was studied on 6-week-old Kunming mice, and in vivo experiments were performed using preparations of mRNA expressing fluorescent protein. mRNA-lipid nanoparticles were prepared using compounds A6, A7, A9 and B5 according to the method shown in Example 13, and diluted with PBS to make the mRNA concentration of 10 μg / mL, and administered subcutaneously to the neck of female mice (dosage of 0.5 mg / kg). At 12 hours after administration, the pre-prepared fluorescein (VivoGlo TMLuciferin (In Vivo Grade, Promega) aqueous solution was intraperitoneally administered to each mouse, and 30 minutes later, the mouse was monitored using IVIS. TM LuminaII (Caliper Life Sciences) was used to observe and quantify the luminescence at the administration site.

[0239] The results are shown in Fig. 9 Luminescence was confirmed in all experimental groups, indicating mRNA expression activity. Among them, the experimental group containing compound B5 showed the strongest luminescence, indicating high mRNA expression activity.

Claims

1. A compound having the following structural formula, which is a cationic lipid or a pharmaceutically acceptable salt thereof, wherein the cationic lipid is selected from the following formula VI, VII, VIII or formula L1 to L22, wherein the general structural formula of L1 to L22 is as shown in formula I-I, 2. A lipid particle comprising the compound of claim 1.

3. lipid granule according to claim 2, wherein said lipid granule also comprises non-cationic lipid.

4. lipid granule according to claim 3, wherein said non-cationic lipid is neutral lipid and / or sterol.

5. lipid granules according to claim 4, wherein the neutral lipid is a phospholipid, and the phospholipid comprises one or more combinations of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphocholine.

6. lipid granule according to claim 4, wherein the sterol is cholesterol or a cholesterol derivative.

7. lipid granule according to claim 3, wherein said lipid granule also comprises the lipid of the putative combination that suppresses particle aggregation.

8. lipid granules according to claim 7, the lipid of the conjugation of wherein said inhibition particle aggregation comprises a PEG-lipid conjugate.

9. lipid granule according to claim 8, wherein said PEG-lipid conjugate comprises one or two mixings of PEG-diacylglycerol conjugate, PEG-dialkoxypropyl conjugate.

10. A pharmaceutical composition comprising the lipid particles of claim 2 and a therapeutic agent, wherein the therapeutic agent is a nucleic acid.

11. The pharmaceutical composition according to claim 10, wherein the nucleic acid comprises an interfering RNA molecule, and further comprises single-stranded or double-stranded DNA, RNA or DNA / RNA hybrid, antisense oligonucleotides, ribozymes, plasmids, immunostimulatory oligonucleotides. The pharmaceutical composition according to claim 11 , further comprising a pharmaceutically acceptable carrier.

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