Cationic lipid
阅读说明：本技术 阳离子性脂质 (Cationic lipid ) 是由 松本悟 大森良真 峯野雅博 帆足保孝 于 2018-12-27 设计创作，主要内容包括：本发明提供能够将活性成分、特别是核酸以优良的效率导入到细胞中的技术和该技术中使用的阳离子性脂质等。本发明的化合物或其盐为下述式(I)所表示的化合物或其盐,式中,n表示2～5的整数,R表示直链状C<Sub>1-5</Sub>烷基、直链状C<Sub>7-11</Sub>烯基或直链状C<Sub>11</Sub>链二烯基,波状线各自独立地表示顺式或反式的键。<Image he="229" wi="700" file="DDA0002552703450000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The present invention provides a composition capable of dissolving an active ingredient,In particular, a technique for introducing nucleic acid into cells with excellent efficiency, and a cationic lipid used in the technique. The compound or salt thereof of the present invention is a compound or salt thereof represented by the following formula (I), wherein n represents an integer of 2 to 5, and R represents a linear C 1‑5 Alkyl, straight chain C 7‑11 Alkenyl or straight-chain C 11 Alkadienyl, the wavy lines each independently represent a bond of cis or trans form.)
1. A compound represented by the formula (I) or a salt thereof,
in the formula (I), the compound is shown in the specification,
n represents an integer of 2 to 5,
r represents a straight chain C1-5Alkyl, straight chain C7-11Alkenyl or straight-chain C11An alkadienyl group, a mono-or di-alkenyl group,
the wavy lines each independently represent a cis or trans bond.
(9Z) -tetradec-9-enoic acid 3- ((4- (dimethylamino) butanoyl) oxy) -2, 2-bis (((9Z) -tetradec-9-enoyloxy) methyl) propyl ester or a salt thereof.
3- ((5- (dimethylamino) pentanoyl) oxy) -2, 2-bis (((9Z) -tetradec-9-enoyloxy) methyl) propyl (9Z) -tetradec-9-enoate) or a salt thereof.
(9Z) -tetradec-9-enoic acid 3- ((6- (dimethylamino) hexanoyl) oxy) -2, 2-bis (((9Z) -tetradec-9-enoyloxy) methyl) propyl ester or a salt thereof.
5. A lipid particle comprising the compound of claim 1 or a salt thereof.
6. A composition for nucleic acid introduction, which comprises a lipid particle according to claim 5 and a nucleic acid.
7. The composition of claim 6, wherein the nucleic acid is RNA.
8. The composition of claim 7, wherein the RNA is mRNA or siRNA.
Technical Field
The present invention relates to a cationic lipid capable of introducing a nucleic acid as an active ingredient into various cells, tissues or organs. The present invention also relates to a lipid particle containing the cationic lipid, and a composition containing the lipid particle and a nucleic acid.
Background
In recent years, research and development of nucleic acid drugs containing nucleic acid as an active ingredient have been actively conducted. For example, many studies have been made on nucleic acid drugs having a decomposition action and a function inhibitory action on target mrnas, which contain nucleic acids such as siRNA, miRNA mimic (miRNA mimic), or antisense nucleic acid. In addition, studies have been made on nucleic acid drugs for expressing a target protein in cells, which contain mRNA encoding the target protein, and the like. In connection with these research and development, a technique for introducing nucleic acid into cells, tissues, or organs with high efficiency has been developed as a Drug Delivery System (DDS) technique.
As the DDS technique, a technique has been known in which nucleic acids are mixed with lipids to form complexes, and then the complexes mediate the uptake of the nucleic acids into cells. As the lipid used for forming the complex, a cationic lipid, a hydrophilic polymer lipid, a helper lipid, and the like have been known. As the cationic lipid, for example, compounds described in the following prior art documents are known.
Patent document 1 describes a compound represented by the following formula or a salt thereof.
Provision is made for: in the formula, R1Each independently selected from C which may be substituted8～C24Alkyl and C which may be substituted8～C24Alkenyl groups; r2And R3Each independently selected from hydrogen, C which may be substituted1～C8Alkyl, arylalkyl which may be substituted, etc.; y is1And Y2Each independently selected from hydrogen, C which may be substituted1～C6Alkyl, arylalkyl which may be substituted, etc.; y is3Each independently selected from hydrogen, C which may be substituted, when present1～C8Alkyl, arylalkyl which may be substituted, etc.; m is any integer from 1 to 4, n is any integer from 0 to 3, p is 0 or 1, and the sum of m, n and p is 4; k is any integer of 1-5; q is 0 or 1; and the like.
Patent document 2 describes a compound represented by the following formula or a salt thereof.
Wherein W represents formula-NR1R2Or formula-N+R3R4R5(Z-)，R1And R2Each independently represents C1-4Alkyl or hydrogen atoms, R3、R4And R5Each independently represents C1-4Alkyl radical, Z-Represents an anion, X represents optionally substituted C1-6Alkylene radical, YA、YBAnd YCEach independently represents a methine group which may be substituted, LA、LBAnd LCEach independently represents a methylene group or a bond which may be substituted, RA1、RA2、RB1、RB2、RC1And RC2Each independently represents C which may be substituted4-10An alkyl group.
Disclosure of Invention
Problems to be solved by the invention
Cationic lipids capable of introducing nucleic acids into cells with high efficiency are expected to contribute to the creation of nucleic acid drugs having excellent effects in terms of drug efficacy, safety (low toxicity), and the like, and in terms of therapy. In addition, cationic lipids capable of introducing nucleic acids into various cells are expected to create nucleic acid drugs against various diseases occurring in various tissues. However, at present, no nucleic acid drug has been found that can sufficiently satisfy the above requirements.
The purpose of the present invention is to provide a technique capable of efficiently introducing nucleic acid into cells, a cationic lipid used in the technique, and the like. From another viewpoint, the present invention aims to provide a technique capable of introducing nucleic acids into various cells, a compound used in the technique, and the like.
Means for solving the problems
The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that the above problems can be solved by using a compound represented by the following formula or a salt thereof, and have completed the present invention.
That is, the present invention relates to at least the following inventions.
[1]
Formula (I):
[ in the formula (I),
n represents an integer of 2 to 5,
r represents a straight chain C1-5Alkyl, straight chain C7-11Alkenyl or straight-chain C11An alkadienyl group, a mono-or di-alkenyl group,
the wavy lines each independently represent a cis or trans bond. ]
[2]
(9Z) -tetradec-9-enoic acid 3- ((4- (dimethylamino) butanoyl) oxy) -2, 2-bis (((9Z) -tetradec-9-enoyloxy) methyl) propyl ester or a salt thereof.
[3]
(9Z) -tetradec-9-enoic acid 3- ((5- (dimethylamino) pentanoyl) oxy) -2, 2-bis (((9Z) -tetradec-9-enoyloxy) methyl) propyl ester or a salt thereof.
[4]
(9Z) -tetradec-9-enoic acid 3- ((6- (dimethylamino) hexanoyl) oxy) -2, 2-bis (((9Z) -tetradec-9-enoyloxy) methyl) propyl ester or a salt thereof.
[5]
A lipid particle comprising the compound according to claim 1 or a salt thereof.
[6]
A composition for nucleic acid introduction, which comprises a nucleic acid and the lipid particle according to item 5.
[7]
The composition of item 6, wherein the nucleic acid is RNA.
[7a]
The composition of item 6, wherein the nucleic acid is DNA.
[8]
The composition of item 7, wherein the RNA is mRNA or siRNA.
In the present specification, "the compound represented by the formula (I)" may be referred to as "the compound (I)". The "compound represented by the formula (I) or a salt thereof" may be referred to as "the compound of the present invention". The "lipid particle containing the compound represented by the formula (I) or a salt thereof (the compound of the present invention)" is sometimes referred to as "the lipid particle of the present invention". The "composition for introducing nucleic acid containing nucleic acid and lipid particles of the present invention" may be referred to as "the composition of the present invention".
Effects of the invention
According to the present invention, a nucleic acid can be introduced into a cell, a tissue or an organ with excellent efficiency. In addition, according to the present invention, nucleic acids can be introduced into various cells, tissues or organs (e.g., cancer cells). According to the present invention, a drug or a research reagent for introducing a nucleic acid into various cells, tissues or organs can be obtained. In addition, according to the present invention, when a nucleic acid is introduced into a cell, a tissue, or an organ, the expression efficiency of the activity (e.g., drug effect) of the nucleic acid is high.
Detailed Description
The definitions of the substituents used in the present specification are explained in detail below. Unless otherwise specified, each substituent has the following definition.
In the present specification, the term "linear C" means1-5Examples of the alkyl group "include a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group.
In the present specification, the term "linear C" means7-11Alkenyl group "may be exemplified by 1-heptenyl group, 2-heptenyl group, 3-heptenyl group, 4-heptenyl group, 5-heptenyl group, 6-heptenyl group, 1-octenyl group, 2-octenyl group, 3-octenyl group, 4-octenyl group, 5-octenyl group, 6-octenyl group, 7-octenyl group, 1-nonenyl group, 2-nonenyl group, 3-nonenyl group, 4-nonenyl group, 5-nonenyl group, 6-nonenyl group, 7-nonenyl group, 8-nonenyl group, 1-decenyl group, 2-decenyl group, 3-decenyl group, 4-decenyl group, 5-decenyl group, 6-decenyl group, 7-decenyl group, 8-decenyl group, 9-decenyl group, 1-undecenyl group, 2-decenyl group, 3-decenyl group, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl. These linear chains C7-11The alkenyl group contains 1 carbon-carbon double bond, and thus can form cis-and trans-structures, and may be either structure.
In the present specification, the term "linear C" means11Alkadienyl "includes, for example, 1, 3-undecenyl and 1, 4-undecenyl1, 5-undecenyl group, 1, 6-undecenyl group, 1, 7-undecenyl group, 1, 8-undecenyl group, 1, 9-undecenyl group, 1, 10-undecenyl group, 2, 4-undecenyl group, 2, 5-undecenyl group, 2, 6-undecenyl group, 2, 7-undecenyl group, 2, 8-undecenyl group, 2, 9-undecenyl group, 2, 10-undecenyl group, 3, 5-undecenyl group, 3, 6-undecenyl group, 3, 7-undecenyl group, 3, 8-undecenyl group, 3, 9-undecenyl group, 3, 10-undecenyl group, 4, 6-undecenyl group, 4, 7-undecenyl group, 4, 8-undecenyl group, 4, 9-undecenyl group, 4, 10-undecenyl group, 5, 7-undecenyl group, 5, 8-undecenyl group, 5, 9-undecenyl group, 5, 10-undecenyl group, 6, 8-undecenyl group, 6, 9-undecenyl group, 6, 10-undecenyl group, 7, 9-undecenyl group, 7, 10-undecenyl group, 8, 10-undecenyl group. These linear chains C11The alkadienyl group contains 2 carbon-carbon double bonds, and therefore, each may form a cis-form and a trans-form independently of each other, and each may be either structure.
Preferred examples of n and wavy lines in formula (I) are as follows.
n is preferably an integer of 3 to 5, more preferably 3.
The wavy lines are preferably both cis-form bonds.
Preferred specific examples of the compound (I) are as follows.
Compound (A): n is an integer of 3 to 5, R is a cis-form straight chain C7-11Compounds in which both the alkenyl group and the wavy line are cis-form bonds.
Compound (B): n is 4, R is a straight chain C having 2 carbon-carbon double bonds11Compounds in which both the alkadienyl group and the wavy line are cis-form bonds.
Compound (C): n is 2 or 3, R is straight chain C1-5Alkyl groups and wavy lines are compounds having cis-form bonds.
More preferred specific examples of the compound (I) are as follows.
Compound (a 1): n is an integer of 3 to 5, R is a cis-form bond of 5-heptenyl, 7-nonenyl or 9-undecenyl, and wavy lines.
Compound (B1): a compound in which n is 4, R is a bond in which both of 2 carbon-carbon double bonds are cis-form 2, 5-undecabdienyl groups, and both of wavy lines are cis-form.
Compound (C1): a compound wherein n is 2 or 3, R is methyl, propyl or pentyl, and the wavy line is a bond in cis.
The salt of compound (I) is preferably a pharmacologically acceptable salt, and examples thereof include a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, and a salt with a basic or acidic amino acid.
Preferred examples of the salt with an inorganic base include: alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts and ammonium salts. Sodium salt, potassium salt, calcium salt, and magnesium salt are preferable, and sodium salt and potassium salt are more preferable.
Preferred examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [ tris (hydroxymethyl) methylamine ], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, and N, N-dibenzylethylenediamine.
Preferred examples of the salt with an inorganic acid include salts with hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid. Preferably, the salt is a salt with hydrochloric acid or a salt with phosphoric acid.
Preferred examples of the salt with an organic acid include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
Preferred examples of the salt with a basic amino acid include salts with arginine, lysine and ornithine.
Preferred examples of the salt with an acidic amino acid include salts with aspartic acid and glutamic acid.
In the present invention, the compound of the present invention can be used as a cationic lipid. Cationic lipids can form complexes with a variety of molecules in a solvent or dispersion medium. The complex may contain other components in addition to the compound of the present invention. Examples of the other components include other lipid components and nucleic acids.
Examples of the other lipid component include structural lipids that can constitute lipid particles. Examples of the structural lipid include those selected from sterols (e.g., cholesterol ester, cholesterol hemisuccinate, etc.), phospholipids (e.g., phosphatidylcholines (e.g., dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, lysophosphatidylcholine, dioleoylphosphatidylcholine, palmitoylphosphatidylcholine, dilinoleoylphosphatidylcholine, MC-1010(NOFCORPORATION), MC-2020(NOF CORPORATION), MC-4040(NOF CORPORATION), phosphatidylserines (e.g., dipalmitoylphosphatidylserine, distearoylphosphatidylserine, palmitoylphosphatidylserine), phosphatidylethanolamines (e.g., dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, palmitoylphosphatidylethanolamine, etc.), phosphatidylethanolamines (e.g., cholesterol hemisuccinate, etc.), lysophosphatidylethanolamine, etc.), phosphatidylinositol, phosphatidic acid, etc.), polyethylene glycol lipid (PEG lipid) (e.g., PEG-DAA, PEG-DAG, PEG-phospholipid conjugate, PEG-Cer, PEG-cholesterol, PEG-C-DOMG, 2KPEG-CMG, GM-020(NOF CORPORATION), GS-050(NOF CORPORATION), etc.). In the present invention, as the structural lipid, it is preferable to use all three of sterols (particularly cholesterol), phospholipids (particularly phosphatidylcholine), and polyethylene glycol lipids.
The ratio of the compound of the present invention to the structural lipid in the mixed lipid component forming the lipid particle of the present invention can be appropriately adjusted depending on the purpose and use. For example, the proportion of the structured lipid is usually 0.008 to 4 mol, preferably 0.4 to 1.5 mol, based on 1 mol of the compound of the present invention. In addition, when another predetermined method is adopted, the ratio of the compound of the present invention to the mixed lipid component is usually 1 to 4 moles, the sterol is usually 0 to 3 moles, the phospholipid is usually 0 to 2 moles, and the polyethylene glycol lipid is usually 0 to1 mole. A more preferable mode for using the compound of the present invention in a mixture with other lipid components is a ratio of 1 to 1.5 moles of the compound of the present invention, 0 to 1.25 moles of sterols, 0 to 0.5 moles of phospholipids, and 0 to 0.125 moles of polyethylene glycol lipids.
The compounds of the present invention may be used to produce the lipid particles of the present invention. The lipid particle of the present invention refers to a complex that does not contain nucleic acid in the complex. The shape of the lipid particle of the present invention is not particularly limited, and examples thereof include a complex in which the compounds of the present invention and the like are aggregated to form a sphere, a complex in which the compounds of the present invention and the like are aggregated without forming a specific shape, a complex in which the compounds of the present invention and the like are dissolved in a solvent, and a complex in which the compounds of the present invention and the like are uniformly or non-uniformly dispersed in a dispersion medium.
The lipid particle of the present invention (for example, a lipid particle composed of the compound of the present invention and a lipid having a structure other than the compound) can be used for producing, for example, the composition of the present invention containing the lipid particle and a nucleic acid (in particular, a nucleic acid which is a substance useful for pharmaceutical use or for research purposes). The composition of the present invention can be used as a drug or an agent. In the composition of the present invention, it is preferable that as much as possible of the nucleic acid is encapsulated by the lipid particle (i.e., encapsulation efficiency is high).
The "nucleic acid" may be any molecule as long as it is a molecule obtained by polymerizing a nucleotide and a molecule having a function equivalent to the nucleotide, and examples thereof include RNA as a polymer of ribonucleotides, DNA as a polymer of deoxyribonucleotides, a polymer obtained by mixing ribonucleotides and deoxyribonucleotides, and a nucleotide polymer containing a nucleotide analog, and further, a nucleotide polymer containing a nucleic acid derivative. In addition, the nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. In addition, a double-stranded nucleic acid also includes a double-stranded nucleic acid in which one strand hybridizes to the other strand under stringent conditions.
The nucleotide analog may be any molecule as long as it is a molecule obtained by modifying a ribonucleotide, a deoxyribonucleotide, RNA or DNA in order to improve or stabilize nuclease resistance as compared with RNA or DNA, to improve affinity with a complementary strand nucleic acid, to improve cell permeability, or to visualize the nucleotide. The nucleotide analog may be a naturally occurring molecule or a non-natural molecule, and examples thereof include a sugar-modified nucleotide analog and a phosphodiester-modified nucleotide analog.
Examples of the sugar-moiety-modified nucleotide analog include nucleotide analogs substituted with 2 '-O-methyl ribose, nucleotide analogs substituted with 2' -O-propyl ribose, nucleotide analogs substituted with 2 '-methoxyethoxy ribose, nucleotide analogs substituted with 2' -O-methoxyethyl ribose, nucleotide analogs substituted with 2'-O- [2- (guanidine) ethyl ] ribose, nucleotide analogs substituted with 2' -O-fluoro ribose, bridge-structure-type artificial Nucleic acids (bridge Nucleic acids) (BNA) having 2 cyclic structures by introducing a bridge structure into the sugar moiety, more specifically, lock-type artificial Nucleic acids (L ocked Nucleic acids) (L NA) and Ethylene-bridge-type artificial Nucleic acids (Ethylene Bridge Nucleic Acids) (BNA), seq id No. [ 32, 23, 32, 123, 32, or similar peptides.
The phosphodiester bond-modified nucleotide analog may be any one as long as it is obtained by adding or substituting an arbitrary chemical substance to or for a part or all of the chemical structure of the phosphodiester bond of a nucleotide, and specific examples thereof include a nucleotide analog substituted with a phosphorothioate bond, a nucleotide analog substituted with an N3 '-P5' phosphoramidite bond, and the like [ cell engineering, 16,1463 1473(1997) ] [ RNAi method and antisense method, lecture (2005) ].
The nucleic acid derivative may be any molecule as long as it is a molecule obtained by adding another chemical substance to the nucleic acid in order to improve nuclease resistance compared to the nucleic acid, stabilize the nucleic acid, improve affinity with a complementary strand nucleic acid, improve cell permeability, or visualize the nucleic acid, and specific examples thereof include a 5' -polyamine addition derivative, a cholesterol addition derivative, a steroid addition derivative, a bile acid addition derivative, a vitamin addition derivative, a Cy5 addition derivative, a Cy3 addition derivative, a 6-FAM addition derivative, and a biotin addition derivative.
The nucleic acid in the present invention is not particularly limited, and may be, for example, a nucleic acid for the purpose of ameliorating a disease, symptom, injury, or disease state, reducing a disease, symptom, injury, or disease state, or preventing the onset of a disease (in the present specification, it may be referred to as "treatment of a disease or the like"), or a nucleic acid for regulating the expression of a desired protein, which is useful for research purposes, although it does not contribute to the treatment of a disease or the like.
Genes or polynucleotides associated with diseases (sometimes referred to as "disease-associated genes" in this specification) can be obtained from, for example, McKumock-Nathans Institute of genetic medicine (McKumock-Nathans Institute of genetic medicine), John Hopkins university (Baltimore, Maryland) and the National Center for Biotechnology Information (National Center for Biotechnology Information), the National library of medicine (Besserda, Maryland), and the like.
Specific examples of the nucleic acid of the present invention include siRNA, shRNA, miRNA mimetic, antisense nucleic acid, ribozyme, mRNA, Decoy nucleic acid, aptamer, plasmid DNA, Cosmid (Cosmid) DNA, and BAC DNA. The nucleic acid is preferably siRNA, mRNA, or the like RNA, or an analogue or derivative thereof obtained by artificially modifying the RNA or the mRNA.
In the present invention, the term "siRNA" refers to a double-stranded RNA or an analog thereof having 10 to 30 bases, preferably 15 to 25 bases, and includes a complementary sequence. The siRNA preferably has 1 to 3 bases, more preferably 2 bases, at the 3' end. The complementary sequence portions may be fully complementary or may comprise non-complementary bases, but are preferably fully complementary.
The siRNA in the present invention is not particularly limited, and for example, siRNA for knocking down (knock down) gene expression of a disease-related gene can be used. The disease-associated gene refers to any gene or polynucleotide that produces a transcription or translation product at an abnormal level or in an abnormal form in a cell derived from a diseased tissue compared with a non-disease control tissue or cell. Further, as the siRNA of the present invention, an siRNA for regulating the expression of a desired protein useful for research purposes may be used.
In the present invention, "mRNA" refers to RNA containing a base sequence translatable into protein. The mRNA in the present invention is not particularly limited as long as it is an mRNA that can express a desired protein in a cell. The mRNA is preferably one useful for pharmaceutical applications (for example, for disease treatment) and/or research purposes, and examples of such mRNA include mrnas for expressing a marker protein such as luciferase in cells.
The above-mentioned diseases are not particularly limited, and examples thereof include the diseases described below. Examples of disease-related genes are shown in "()" except for the case where a specific disease example is described. The nucleic acid of the present invention may be a nucleic acid that regulates the expression level of the disease-related genes (or proteins encoded by the genes).
(1) Blood system diseases [ anemia (CDAN1, CDA1, RPS19, DBA, PK L R, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, A L AS2, ANH1, ASB, ABCB7, ABC7, ASAT), naked lymphocyte syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2 82 2TA, C2TA, RFX5), hemorrhagic disease (TBXA2R, FADP 2RX1, P2X1), factor H and factor-like 1 factor deficiency (HF1, CFH, HUS), factor V and factor VIII deficiency (MCFD2), factor VII deficiency (2), factor X deficiency (F2), factor F deficiency (36F 2), factor deficiency (36F), FANCF deficiency), FANCFA deficiency 2), FACFANCFA deficiency (FANCFA 2), FANCF deficiency 2, FACNFF deficiency 2, FACCETF 2, FACNFF, FACPIF deficiency 2, FACFANCH deficiency 2, FACFANCF deficiency 2, FACFANCH deficiency 2, FACFANCF deficiency 2, FACFANCH deficiency 2, FACPIF deficiency 2, FACFANCH deficiency 2, FACFANCF deficiency 36FANCF deficiency 2, FACN deficiency 2, FACFANCF deficiency 36;
(2) inflammatory/immune diseases [ AIDS (KIR3, NKAT, NKB, AMB, KIR3DS, IFNG, CXC 12, SDF), autoimmune lymphoproliferative syndrome (TNFRSF, APT, FAS, CD, A0 PS 1), combined immunodeficiency diseases (I12 RG, SCHDX, IMD), HIV infection (CC 25, SCYA, D17S135, TCP228, I310, CSIF, CMKBR, CCR, DMKBR, CCCKR, CCR), immunodeficiency diseases (CD3, AICDA, AIAID, HIGM, TNFRSF, CD, UNG, HIGM, TNFSF, CD4, IGM, FOXP, IPEX, AIID, XPID, PIDGX, TNFRSF14, TACI), inflammation diseases (I510, I6-1, I7-13, I-17, I-23, CT A), severe immunodeficiency diseases (IDA, IDD, PIDGDX, TNFRSF14, TACI), inflammatory diseases (I, IDS, SLE, SL;
(3) metabolic/hepatic/renal diseases [ amyloidosis neuropathy (TTR, PA B), amyloidosis (APOA, APP, AAA, CVAP, AD, GSN, FGA, YZ, TTR, PA 0B), nonalcoholic steatohepatitis and liver fibrosis (CO 11A), cirrhosis (KRT, CIRH1, NAIC, TEX292, KIAA1988), cystic fibrosis (CFTR, ABCC, CF, MRP), glycogen storage disease (S2C 2A, G3 UT, G6PT, GAA, 4AMP, 5AMPB, AG, GDE, GBE, GYS, PYG, PFKM), hepatocellular adenoma (TCF, HFN1, MODY), hepatic failure (SCKD, SCO), hepatic lipase deficiency (MPRP), UMB granulomatosis (NNB, PDFGR, PDGPR, AXIN, AXTP, JKP, IGF, 2, METK, PKD, PKFH, PPD, PKFCF, PKFCPR, PKFCP, PKFCF, PDPR, PKFCP, PKFCS, PKFCP, PDPR, PKFCP, PKFCPR, PKF, PKPCK, PKFCPR, PKF;
(4) nervous system diseases [ A S (SOD, A S, STEX, FUS, TARDBP, VEGF), Alzheimer 'S disease (APP, AAA, CVAP, AD, APOE, AD, PSEN, AD, STM, APBB, FE 01, NOS, P1 AU, URK, ACE, DCP, ACE, MPO, PACIP, PAXIP 2, PTIP, A2, B MH, BMH, PSEN, AD), autism (BZLAP, MDGA, G O, MECP, RTT, PPMX, MRX, N GN, KIAA1260, AUTSX), fragile X chromosome syndrome (FXR, mG UR), Huntington' S disease (MRIT, SCAIT, PRIP, JPH, JP, SCAHD, TBP, PAR 4A, NURR, DJ, TIR, SNIP, TBP, SCAHD, SNCP, SNK, DRK, DRCP, SALT, SLOT, DRCP, SALT, SLP, SALT;
(5) eye diseases [ age-related macular degeneration (Abcr, Ccl2, CP, Timp3, cathepsin D, Vldlr, Ccr2), cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX 2, BFSP2, CP2, PAX 2, AN2, MGDA, CRYBA 2, CRYB2, CRYGC, CRYG 2, CC 2,2 IM2, MP2, CRYGD, CRYG 2, BSFP2, CP2, CTF 2, CTM, MIP, AQP 2, CRYAB, CRYA2, CTPP2, CRYBB2, CRYGD 2, CRYAYGD, CRYAYG 2, CRYP 2, CROPSCH 2, CR;
(6) neoplastic diseases [ malignant tumor, neovascular glaucoma, infantile hemangioma, multiple myeloma, chronic sarcoma, metastatic melanoma, kaposi's sarcoma, vascular proliferation, cachexia, metastasis of breast cancer, and the like, cancer (e.g., large-intestine cancer (e.g., familial large-intestine cancer, hereditary non-polyposis large-intestine cancer, gastrointestinal stromal tumor, and the like), lung cancer (e.g., non-small cell lung cancer, small-cell lung cancer, malignant mesothelioma, and the like), mesothelioma, pancreatic cancer (e.g., pancreatic ductal cancer, and the like), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, and the like), breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer, and the like), ovarian cancer (e.g., epithelial ovarian cancer, gonadoblastoma, ovarian germ cell tumor, ovarian low grade malignant tumor, and the like), prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, and the like), liver cancer (e.g., primary liver cancer, extrahepatic bile duct cancer, and the like), thyroid cancer (e.g., medullary thyroid cancer, and the like), kidney cancer (e.g., renal cell carcinoma, transitional epithelial carcinoma of the renal pelvis ureter, and the like), uterine cancer, brain cancer (e.g., pineal astrocytoma, hairy cell astrocytoma, diffuse astrocytoma, degenerative astrocytoma, and the like), melanoma, sarcoma, bladder cancer, blood cancer including multiple myeloma, and the like, pituitary adenoma, glioma, acoustic schwannoma, retinal sarcoma, pharyngeal cancer, laryngeal cancer, tongue cancer, thymic tumor, esophageal cancer, duodenal cancer, colon cancer, rectal cancer, hepatocellular carcinoma, pancreatic endocrine tumor, bile duct cancer, gallbladder cancer, penile cancer, ureter cancer, testis tumor, vulva cancer, cervical cancer, and the like), melanoma, and the like, Uterine corpus carcinoma, uterine sarcoma, villous disease, vaginal cancer, skin cancer, mycosis fungoides, basal cell tumor, soft tissue sarcoma, malignant lymphoma, hodgkin's disease, myelodysplastic syndrome, adult T-cell leukemia, chronic myeloproliferative disease, pancreatic endocrine tumor, fibrohistiocytoma, leiomyosarcoma, rhabdomyosarcoma, carcinoma of unknown primary focus, etc.), leukemia (e.g., acute lymphatic leukemia, acute myelogenous leukemia, etc.), chronic leukemia (e.g., chronic lymphatic leukemia, chronic myelogenous leukemia, etc.), myeloproliferative syndrome, etc.), uterine sarcoma (e.g., medulloblastoma, uterine leiomyosarcoma, endometrial tumor, etc.), myelofibrosis, etc. ].
The composition of the present invention as a medicament can be manufactured by a method well known in the art of formulation technology using a pharmaceutically acceptable carrier. Examples of the dosage form of the above-mentioned drugs include preparations for parenteral administration (for example, liquid preparations such as injections) containing conventional adjuvants such as buffers and/or stabilizers, and topical preparations such as ointments, creams, liquids, and plasters containing conventional pharmaceutical carriers.
The composition of the present invention can be used for introducing an active ingredient into a plurality of types of cells, tissues or organs. Examples of the cells to which the composition of the present invention can be applied include mesenchymal stem cells, neural stem cells, skin stem cells, spleen cells, nerve cells, glial cells, pancreatic B cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, myoblasts, myosatellite cells, and smooth muscle cells), adipocytes, blood cells (e.g., macrophages, T cells, B cells, natural killer cells, adipocytes, leukocytes, neutrophils, basophils, eosinophils, monocytes, megakaryocytes, and hematopoietic stem cells), synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or mesenchymal cells, ova cells, and the like, Sperm cells or precursor cells that can be induced to differentiate into these cells, stem cells (e.g., including artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells)), primordial germ cells, oocytes, fertilized eggs. Examples of the tissue or organ to which the composition of the present invention can be applied include all tissues or organs in which the above-mentioned cells are present, for example, the brain, various parts of the brain (for example, olfactory bulb, nucleus applanatum, basal ganglia, hippocampus, visual mound, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, corpus callosum, substantia nigra, spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonads, thyroid gland, gall bladder, bone marrow, adrenal gland, skin, lung, digestive tract (for example, large intestine and small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood cells, prostate, placenta, uterus, bone, joints, and muscles (for example, skeletal muscle, smooth muscle, and cardiac muscle). These cells, tissues or organs may be cancer cells, tissues or the like which have become cancerous.
The composition of the present invention is particularly excellent in the efficiency of introducing nucleic acid into cancer cells.
The compounds, lipid particles and compositions of the present invention can be used safely with stability and low toxicity. When the composition of the present invention is used in vivo (in vivo) or as a pharmaceutical, the composition may be administered to a subject (e.g., a human or non-human mammal (preferably a human)) in such a manner that an effective amount of the nucleic acid is delivered to a target cell.
When the composition of the present invention is used in vivo or as a pharmaceutical, it can be safely administered orally or parenterally (for example, topical, rectal, intravenous administration, and the like) by preparing it into pharmaceutical preparations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, and orally disintegrating tablets), powders, granules, capsules (including soft capsules and microcapsules), liquids, troches, syrups, emulsions, suspensions, injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), external preparations (for example, nasal preparations, transdermal preparations, and ointments), suppositories (for example, rectal suppositories and vaginal injections), pills, nasal preparations, pulmonary preparations (inhalants), and drops. These preparations may be controlled-release preparations (e.g., sustained-release microcapsules) such as immediate-release preparations and sustained-release preparations.
The following describes a method for producing the compound of the present invention.
The raw materials and reagents used in the respective steps in the following production methods and the obtained compounds may form salts, respectively. Examples of such salts include the same salts as those of the compounds of the present invention.
When the compound obtained in each step is a free compound, it can be converted into a target salt by a known method. On the other hand, when the compound obtained in each step is a salt, it can be converted into an episome or another salt of the target species by a known method.
The compound obtained in each step may be used in the next reaction as it is or after obtaining a crude product, or the compound obtained in each step may be separated and/or purified from the reaction mixture by separation means such as concentration, crystallization, recrystallization, distillation, solvent extraction, fractionation, chromatography, and the like according to a conventional method.
When the compounds of the raw materials and reagents in each step are commercially available, commercially available products can be used as they are.
The reaction time in the reaction of each step may vary depending on the reagents and solvents used, and is usually 1 minute to 48 hours, preferably 10 minutes to 8 hours, unless otherwise specified.
In the reaction of each step, the reaction temperature may vary depending on the reagents and solvents used, and is usually from-78 ℃ to 300 ℃, preferably from-78 ℃ to 150 ℃, unless otherwise specified.
The pressure in the reaction in each step may vary depending on the reagents and solvents used, and is usually 1 to 20 atmospheres, preferably 1 to 3 atmospheres unless otherwise specified.
For the reaction in each step, a microwave synthesizer such as an Initiator manufactured by Biotage may be used. The reaction temperature may vary depending on the reagents and solvents used, and when not particularly described, is usually from room temperature to 300 ℃, preferably from room temperature to 250 ℃, and more preferably from 50 ℃ to 250 ℃. The reaction time may vary depending on the reagents and solvents used, and is usually 1 minute to 48 hours, preferably 1 minute to 8 hours, unless otherwise specified.
In the reaction of each step, a reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents, based on the substrate, unless otherwise specified. When a reagent is used as the catalyst, the reagent is used in an amount of 0.001 to1 equivalent, preferably 0.01 to 0.2 equivalent, to the substrate. In the case where the reagent doubles as a reaction solvent, a solvent amount of the reagent is used.
In the reactions of the respective steps, unless otherwise specified, these reactions are carried out under a solvent-free condition or after being dissolved or suspended in an appropriate solvent. Specific examples of the solvent include those described in examples and those listed below.
Alcohols: methanol, ethanol, isopropanol, isobutanol, tert-butanol, 2-methoxyethanol, and the like;
ethers: diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, 1, 2-dimethoxyethane, etc.;
aromatic hydrocarbons: chlorobenzene, toluene, xylene, and the like;
saturated hydrocarbons: cyclohexane, hexane, heptane, etc.;
amides: n, N-dimethylformamide, N-methylpyrrolidone, and the like;
halogenated hydrocarbons: methylene chloride, carbon tetrachloride, and the like;
nitriles: acetonitrile and the like;
sulfoxides: dimethyl sulfoxide and the like;
aromatic organic bases: pyridine, etc.;
acid anhydrides: acetic anhydride, etc.;
organic acids: formic acid, acetic acid, trifluoroacetic acid, and the like;
inorganic acids: hydrochloric acid, sulfuric acid, and the like;
esters: ethyl acetate, isopropyl acetate, and the like;
ketones: acetone, methyl ethyl ketone, etc.;
and (3) water.
Two or more of the above solvents may be mixed in an appropriate ratio and used.
When a base is used in the reaction of each step, for example, the following bases or bases described in examples can be used.
Inorganic bases: sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.;
alkaline salts: sodium carbonate, calcium carbonate, sodium bicarbonate, and the like;
organic bases: triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, N-dimethylaniline, 1, 4-diazabicyclo [2.2.2] octane, 1, 8-diazabicyclo [5.4.0] -7-undecene, imidazole, piperidine and the like;
metal alkoxides: sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and the like;
alkali metal hydrides: sodium hydride and the like;
metal amides: sodium amide, lithium diisopropylamide, lithium hexamethyldisilazide, and the like;
organic lithium species: n-butyllithium, sec-butyllithium, and the like.
When an acid or an acid catalyst is used in the reaction in each step, for example, the following acids or acid catalysts, or the acids or acid catalysts described in examples can be used.
Inorganic acids: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, and the like;
organic acids: acetic acid, trifluoroacetic acid, citric acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, etc.;
lewis acid: boron trifluoride diethyl etherate, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous ferric chloride, etc.
Unless otherwise stated, the reactions of the respective steps are carried out according to known methods, for example, fifth edition of Experimental Chemistry lecture, Vol.13-19 (edited by Japan chemical society), New Experimental Chemistry lecture, Vol.14-15 (edited by Japan chemical society), second edition of precise ORGANIC Chemistry revision (L. F. Tietz, Th. Eicher, south Jiangtang), revised ORGANIC famous reaction structure and points (Dongxiang Xiong, lecture Co., Ltd.), ORGANIC Synthesis Association (ORGASYNTHESE collectiviue) Vol.I-VII (John Wiley & SonsInc.), laboratory Modern ORGANIC Synthesis Standard Experimental step Association (model ORGANIC Synthesis in the L inorganic Collection A of Standard Experimental Procedures) (Jie Jace L I, Oxford publishing, synthetic Heterocyclic Chemistry (Hetrehenysical publication) of Experimental laboratory A collectives of Experimental research, Vol.14, Vol.82, Vol.7, Vol.54, published by York Highua, published by Yokogaku university, Japan chemical society, and the methods of synthetic Chemistry, Vol.7, Vol.82, Vol.7, and Vol.
In each step, the protection or deprotection reaction of the functional group is carried out according to a known method, for example, Wiley-Interscience corporation, 2007 "Protective Groups in organic Synthesis, fourth edition" (Theodora W.Greene, Peter G.M.Wuts); the method described in "Protecting Groups (Protecting Groups) third edition" (p.j. kocienski) published by Thieme corporation 2004, or the method described in examples.
Examples of the protecting group for a hydroxyl group and a phenolic hydroxyl group of an alcohol include: ether-type protecting groups such as methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, and tetrahydropyranyl ether; carboxylate type protecting groups such as acetate; sulfonate-type protecting groups such as methanesulfonate; and carbonate-type protecting groups such as t-butyl carbonate.
Examples of the protective group for the carbonyl group of an aldehyde include: acetal type protecting groups such as dimethyl acetal; and a cyclic acetal-type protecting group such as cyclic 1, 3-dioxane.
Examples of the protecting group of the carbonyl group of the ketone include: ketal-type protecting groups such as dimethyl ketal; cyclic ketal type protecting groups such as cyclic 1, 3-dioxane; oxime type protecting groups such as O-methyloxime; hydrazone-type protecting groups such as N, N-dimethylhydrazone.
Examples of the protecting group for a carboxyl group include: ester-type protecting groups such as methyl ester; amide-type protecting groups such as N, N-dimethylamide.
Examples of thiol protecting groups include: ether-type protecting groups such as benzyl sulfide; ester-type protecting groups such as thioacetate, thiocarbonate, and thiocarbamate.
Examples of the protecting group of an aromatic heterocycle such as an amino group, imidazole, pyrrole or indole include: carbamate-type protecting groups such as benzyl carbamate; amide-type protecting groups such as acetamide; alkylamine-type protecting groups such as N-triphenylmethylamine, and sulfonamide-type protecting groups such as methanesulfonamide.
The removal of the protecting group can be carried out by a known method, for example, a method using an acid, a base, ultraviolet rays, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylhalosilane (for example, trimethyliodosilane, trimethylbromosilane), a reduction method, or the like.
When the reduction reaction is carried out in each step, examples of the reducing agent to be used include metal hydrides such as lithium aluminum hydride, sodium triacetoxyborohydride, sodium cyanoborohydride, diisobutylaluminum hydride (DIBA L-H), sodium borohydride, tetramethyltriacetoxyborohydride, borane-tetrahydrofuran complex, borane-like compounds, raney nickel, raney cobalt, hydrogen gas, formic acid, and the like.
In the case where the oxidation reaction is carried out in each step, examples of the oxidizing agent to be used include: peracids such as m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, t-butyl hydroperoxide and the like; perchlorates such as tetrabutylammonium perchlorate; chlorates such as sodium chlorate; hypochlorites such as sodium hypochlorite; sodium periodate and other high iodine acids; iodine reagents with high valency such as iodosobenzene; manganese-containing reagents such as manganese dioxide and potassium permanganate; lead such as lead tetraacetate; chromium-containing reagents such as pyridinium chlorochromate (PCC), Pyridinium Dichromate (PDC), jones reagent, and the like; halogen compounds such as N-bromosuccinimide (NBS); oxygen gas; ozone; a sulfur trioxide-pyridine complex; osmium tetroxide; selenium dioxide; 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ), and the like.
In the case where the radical cyclization reaction is carried out in each step, examples of the radical initiator to be used include: azo compounds such as Azobisisobutyronitrile (AIBN); water-soluble radical initiators such as 4-4' -azobis-4-cyanovaleric acid (ACPA); triethylboron in the presence of air or oxygen; benzoyl peroxide, and the like. Examples of the radical reaction reagent to be used include tributylstannane, tris (trimethylsilyl) silane, 1,2, 2-tetraphenyldisilane, diphenylsilane, and samarium iodide.
When the Wittig (Wittig) reaction is carried out in each step, the Wittig reagent used may be an alkylene phosphine or the like. The alkylene phosphanes may be produced by a known method, for example, by reactingSalts are prepared by reaction with strong bases.
When the Horner-Emmons reaction is carried out in each step, examples of the reagent to be used include phosphoryl acetates such as methyl dimethylphosphorylacetate and ethyl diethylphosphorylacetate; alkali such as alkali metal hydrides and organic lithium.
When Friedel-Crafts reaction is carried out in each step, the reagent used may be a Lewis acid, an acid chloride or an alkylating agent (for example, haloalkanes, alcohols, olefins, etc.). Alternatively, an organic acid or an inorganic acid may be used instead of the lewis acid, and an acid anhydride such as acetic anhydride may be used instead of the acid chloride.
When the aromatic nucleophilic substitution reaction is carried out in each step, a nucleophilic agent (e.g., amines, imidazole, etc.) and a base (e.g., basic salts, organic bases, etc.) can be used as a reagent.
When a nucleophilic addition reaction using a carbanion, a nucleophilic 1, 4-addition reaction using a carbanion (Michael addition reaction), or a nucleophilic substitution reaction using a carbanion is performed in each step, examples of the base for generating a carbanion include organolithium, metal alkoxide, inorganic base, and organic base.
When the Grignard (Grignard) reaction is carried out in each step, examples of the Grignard reagent include aryl magnesium halides such as phenylmagnesium bromide; alkyl magnesium halides such as methyl magnesium bromide and isopropyl magnesium bromide. The Grignard reagent can be prepared by a known method, for example, by reacting a haloalkane or a haloarene with magnesium metal using an ether or tetrahydrofuran as a solvent.
In the case of conducting Knoevenagel condensation reaction in each step, an active methylene compound (e.g., malonic acid, diethyl malonate, malononitrile, etc.) and a base (e.g., an organic base, a metal alkoxide, an inorganic base) sandwiched between two electron-withdrawing groups can be used as a reagent.
In the case where Vilsmeier-Haack (Vilsmeier-Haack) reaction is carried out in each step, sulfonyl chloride and amide derivatives (e.g., N-dimethylformamide and the like) may be used as a reagent.
When the azide reaction of an alcohol, a haloalkane or a sulfonic acid ester is carried out in each step, examples of the azide used include diphenyl phosphorazidate (DPPA), trimethylsilylazide and sodium azide. For example, when an alcohol is azidated, there are a method using diphenyl phosphorazidate and 1, 8-diazabicyclo [5,4,0] undec-7-ene (DBU), a method using trimethylsilylazide and a Lewis acid, and the like.
In the case of the amination reaction in which reduction is performed in each step, examples of the reducing agent used include sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen gas, formic acid, and the like. When the substrate is an amine compound, examples of the carbonyl compound used include aldehydes such as acetaldehyde and ketones such as cyclohexanone, in addition to paraformaldehyde. When the substrate is a carbonyl compound, examples of the amine used include primary amines such as ammonia and methylamine; secondary amines such as dimethylamine, etc.
In the case where the mitsunobu reaction is carried out in each step, as the reagent, azodicarboxylic acid esters (e.g., diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), etc.) and triphenylphosphine may be used.
When the esterification reaction, the amidation reaction, or the urethanization reaction is performed in each step, examples of the reagent used include acid halides such as acid chloride and acid bromide; activated carboxylic acids such as acid anhydride, active ester and sulfate. As the activator of carboxylic acid, there may be mentioned: carbodiimide-based condensing agents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSCD); triazine-based condensing agents such as 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine hydrochloride-n-hydrate (DMT-MM); carbonate-based condensing agents such as 1, 1-Carbonyldiimidazole (CDI); diphenylphosphoryl azide (DPPA); benzotriazol-1-yloxy-tris (dimethylamino)Salt (BOP reagent); iodinated 2-chloro-1-methyl-pyridinium salt (yamamoto reagent); thionyl chloride; lower alkyl haloformates such as ethyl chloroformate; o- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium Hexafluorophosphate (HATU); sulfuric acid; or combinations thereof, and the like. When a carbodiimide-based condensing agent is used, an additive such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), Dimethylaminopyridine (DMAP), or the like may be further added during the reaction.
In the case where the coupling reaction is carried out in each step, examples of the metal catalyst to be used include: palladium compounds such as palladium (II) acetate, tetrakis (triphenylphosphine) palladium (0), dichlorobis (triphenylphosphine) palladium (II), dichlorobis (triethylphosphine) palladium (II), tris (dibenzylideneacetone) dipalladium (0), 1' -bis (diphenylphosphino) ferrocene palladium (II) chloride, palladium (II) acetate and the like; nickel compounds such as tetrakis (triphenylphosphine) nickel (0); rhodium compounds such as tris (triphenylphosphine) rhodium (III) chloride; a cobalt compound; copper compounds such as copper oxide and copper (I) iodide; platinum compounds, and the like. A base may be further added to the reaction, and examples of such a base include inorganic bases and basic salts.
When the thiocarbonylation reaction is carried out in each step, phosphorus pentasulfide can be typically used as the thiocarbonylating agent, but a reagent having a1, 3,2, 4-dithiadiphosphetane-2, 4-disulfide structure such as2, 4-bis (4-methoxyphenyl) -1,3,2, 4-dithiadiphosphetane-2, 4-disulfide (lawson (L owesson) reagent) may be used in addition to phosphorus pentasulfide.
When the Wohl-Ziegler (Wohl-Ziegler) reaction is carried out in each step, examples of the halogenating agent used include N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide (NCS), bromine, sulfuryl chloride, and the like. Further, the reaction can be accelerated by adding a radical initiator such as heat, light, benzoyl peroxide, azobisisobutyronitrile, or the like to the reaction.
In the case where the halogenation reaction of the hydroxyl group is carried out in each step, examples of the halogenating agent used include acid halides of hydrohalic acid and inorganic acid, specifically, hydrochloric acid, thionyl chloride, phosphorus oxychloride and the like are mentioned in the case of chlorination, and 48% hydrobromic acid and the like are mentioned in the case of bromination. Further, a method of obtaining a halogenated alkane from an alcohol by the action of triphenylphosphine with carbon tetrachloride, carbon tetrabromide, or the like can also be used. Alternatively, a method of synthesizing a haloalkane compound by a two-stage reaction of converting an alcohol into a sulfonate and then reacting the sulfonate with lithium bromide, lithium chloride, or sodium iodide may be used.
When the albuzov (Arbuzov) reaction is carried out in each step, the reagents used include: halogenated alkanes such as ethyl bromoacetate; and phosphites such as triethyl phosphite and tris (isopropyl) phosphite.
When the sulfone esterification reaction is carried out in each step, examples of the sulfonating agent to be used include methanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonic anhydride, p-toluenesulfonic anhydride, trifluoromethanesulfonic anhydride and the like.
In the case where the hydrolysis reaction is carried out in each step, an acid or a base may be used as a reagent. In addition, when the acid hydrolysis reaction of the tert-butyl ester is performed, formic acid, triethylsilane, or the like may be added to reductively trap the tert-butyl cation formed as a by-product.
When the dehydration reaction is carried out in each step, examples of the dehydrating agent used include sulfuric acid, phosphorus pentoxide, phosphorus oxychloride, N' -dicyclohexylcarbodiimide, alumina, polyphosphoric acid, and the like.
The compound (I) can be produced, for example, by the following production method. In the compound (I), both of the compound having a cis bond and the compound having a trans bond can be produced by the same production method as that shown below. In the present invention, particularly in the case of esterification, a compound (I) having a desired structure can be synthesized by using an appropriate raw material corresponding to the structure of the objective compound (I). In addition, the salt of compound (I) can be obtained by appropriately mixing with an inorganic base, an organic acid, a basic or acidic amino acid.
Hereinafter, a method for producing lipid particles containing the compound of the present invention and a composition for nucleic acid introduction containing the lipid particles and nucleic acid will be described.
The lipid particle of the present invention can be produced by a known method for preparing a lipid particle from a lipid component by mixing the compound of the present invention (cationic lipid) with another lipid component as needed. For example, the lipid component (mixture) can be prepared as a lipid particle dispersion by dissolving the above-mentioned lipid component in an organic solvent, and mixing the obtained organic solvent solution with water or a buffer (for example, emulsification method). The mixing may be performed using a micro fluid mixing system, for example, a NanoAssemblr device (Precision NanoSystems). The resulting lipid particles can be desalted or dialyzed and sterile filtered. Further, pH adjustment and osmotic pressure adjustment may be performed as necessary.
The compound (I) can obtain various structures by a combination of n, R and the definition of wavy line of formula (I). In the production of lipid particles, one compound having a specific structure may be used alone or a mixture of a plurality of compounds having different structures may be used as the compound (I).
Examples of the "other lipid component" include the structural lipids described above, for example, sterols, phospholipids, and polyethylene glycol lipids. The "other lipid component" is used, for example, in an amount of 0.008 to 4 mol based on 1 mol of the compound of the present invention. The compounds of the invention are preferably used in admixture with other lipid components, in particular cholesterol, phosphatidylcholine and polyethylene glycol lipids. A preferable mode for mixing and using the compound of the present invention and other lipid components is a mixture of 1 to 4 moles of the compound of the present invention, 0 to 3 moles of sterols, 0 to 2 moles of phospholipids, and 0 to1 mole of polyethylene glycol lipids. A more preferred embodiment when the compound of the present invention is used in admixture with other lipid components is a mixture of 1 to 1.5 moles of the compound of the present invention, 0 to 1.25 moles of sterols, 0 to 0.5 moles of phospholipids, and 0 to 0.125 moles of polyethylene glycol lipids.
The concentration of the compound of the present invention or the mixture of the compound of the present invention and other lipid components in the organic solvent solution is preferably 0.5 to 100mg/m L.
Examples of the organic solvent include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, t-butanol, acetone, acetonitrile, N-dimethylformamide, dimethyl sulfoxide and a mixture thereof. The organic solvent may contain 0 to 20% of water or a buffer solution.
Examples of the buffer include acidic buffers (e.g., acetate buffer, citrate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, and phosphate buffer), neutral buffers (e.g., 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer, Tris (hydroxymethyl) aminomethane (Tris) buffer, phosphate buffer, and Phosphate Buffered Saline (PBS)).
When the mixing is performed using a microfluid mixing system, it is preferable to mix 1 to 5 parts by volume of water or a buffer solution to1 part by volume of an organic solvent solution, and in this system, the flow rate of a mixed solution (mixed solution of an organic solvent solution and water or a buffer solution) is preferably 0.1 to 10m L/min, and the temperature is preferably 15 to 45 ℃.
The composition of the present invention can be produced by adding a nucleic acid as an active ingredient to water or a buffer solution in advance at the time of producing lipid particles or a lipid particle dispersion solution to produce a lipid particle dispersion solution containing an active ingredient, and the active ingredient is preferably added so that the concentration of the active ingredient in water or the buffer solution is 0.05 to 2.0mg/m L.
The composition of the present invention may be produced by mixing lipid particles or a lipid particle dispersion with an active ingredient or an aqueous solution thereof by a known method to obtain a lipid particle dispersion containing the active ingredient. The lipid particle dispersion can be prepared by dispersing the lipid particles in an appropriate dispersion medium. Alternatively, an aqueous solution of the active ingredient may be prepared by dissolving the active ingredient in a suitable solvent.
The content of the compound of the present invention in the composition of the present invention excluding the dispersion medium and the solvent is preferably 40 to 70% by weight.
The content of the active ingredient in the composition of the present invention excluding the dispersion medium and the solvent is preferably 1 to 20% by weight.
The dispersion medium of the lipid particle dispersion or the dispersion containing the composition can be replaced with water or a buffer by dialysis. In the dialysis, an ultrafiltration membrane with the cut-off molecular weight of 10-20K is used and is implemented at the temperature of 4-room temperature. Dialysis can be repeated. Displacement of the dispersion medium may use Tangential Flow Filtration (TFF). After the replacement of the dispersion medium, pH adjustment and osmotic pressure adjustment may be performed as necessary.
Hereinafter, a method for analyzing lipid particles containing the compound of the present invention and a composition containing the lipid particles and a nucleic acid as an active ingredient will be described.
The particle diameter of the lipid particle (in the composition) can be measured by a known means. For example, it can be calculated as the Z-average particle size by cumulant analysis of the autocorrelation function using a particle size measuring apparatus Zetasizer Nano ZS (Malvern Instruments) based on the NIBS (non-contact back scattering) technique. The particle diameter (average particle diameter) of the lipid particles (in the composition) is preferably 10 to 200 nm.
The concentration and encapsulation efficiency of the nucleic acid (e.g., siRNA, mRNA) as an active ingredient in the composition of the present invention can be measured by a known means. For example, using Quant-iTTMThe concentration and the encapsulation efficiency can be determined by fluorescently labeling nucleic acids with Ribo Green (registered trademark) (Invitrogen) and measuring the fluorescence intensity. The concentration of nucleic acid in the composition can be calculated using a standard curve prepared from an aqueous solution of nucleic acid having a known concentration, and the encapsulation efficiency can be calculated based on the difference in fluorescence intensity between the presence and absence of addition of Triton-X100 (surfactant for disintegrating lipid particles). The concentration of nucleic acid in the composition refers to the total concentration of nucleic acid encapsulated by lipid particles and nucleic acid not encapsulated, and the encapsulation efficiency refers to the proportion of nucleic acid encapsulated by lipid particles in the total nucleic acid in the composition.