CN1726183A - Cardiolipin molecules and method of synthesis - Google Patents

Cardiolipin molecules and method of synthesis Download PDF

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CN1726183A
CN1726183A CN 200380106116 CN200380106116A CN1726183A CN 1726183 A CN1726183 A CN 1726183A CN 200380106116 CN200380106116 CN 200380106116 CN 200380106116 A CN200380106116 A CN 200380106116A CN 1726183 A CN1726183 A CN 1726183A
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phosphatide
preparation
composition
alkoxyl group
cationic cardiolipin
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M·U·艾哈迈德
K·卡西雷迪
伊姆兰·艾哈迈德
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Neopharm Inc
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Abstract

The invention provides cationic cardiolipin compounds, and methods for synthesizing and using them in liposomal formulation, gene transfection, etc. In particular, the invention provides liposomes comprising cationic cardiolipin analog, pharmaceutical compositions comprising cationic cardiolipin analogs, and methods of using such liposomes and compositions, in delivering active pharmaceutical agents to treat human and animal diseases and/or in diagnostic assays.

Description

Cationic cardiolipin phospholipid analogues and application thereof
Invention field
The composition that the present invention relates to cationic cardiolipin phospholipid molecule, their preparation method and application and contain cationic cardiolipin phosphatide.
Background of invention
Needs to synthetic phospholipid constantly increase, and partly are that it has become the useful carrier (Tyrell waits 1976) of active therapeutic agent, enzyme, microbiotic, antigen and hormone and other compound owing to their application in liposome.Think that cationic-liposome is auxiliary important way (Miller, 1998) to cell delivery anionic species, for example gene or other nucleic acid.Think that cationic-liposome and electronegative nucleotide sequence form mixture by electrostatic interaction, it promotes these reagent to enter in the cell.Thereby when the treatment disease, cation lipid can work in target cell that reagents for anion is delivered into the patient and organ.As a result, the new synthetic method (Bhattacharya etc. 1999) that has needed the clear and definite lipid of development structure.
The present invention relates to cationic cardiolipin phosphatide, it is used for by promoting transmembrane transport or the conveying that improves biologically active agent, particularly polynucleotide, albumen, peptide and drug molecule to the adhesion of biological surface by enhancing.More specifically, relate to the cationic cardiolipin phosphatide that contains ammonium.Some biologically active substance does not need to enter cell and brings into play their biological action, works because they act on cell surface by cell surface receptor.But, manyly can influence the natural biological molecule of cell function and their analogue at ubcellular or molecular level, comprise albumen and polynucleotide or foreign matter, for example medicine preferably enters in the cell, to produce their effect.For these reagent, cytolemma becomes the selectivity barrier of impermeable.
An impressive progress in DNA transfection field is, find the synthetic cation lipid of some liposome or vesicle form, N-[1-(2,3-two oil base oxygen bases) propyl group for example]-N, N, N-trimethyl ammonium muriate (DOTMA), can spontaneously interact with DNA, form lipid-DNA mixture, it can merge mutually with the electronegative lipid of cytolemma, cause absorption and the expression (Felgner waits 1987) of DNA.The LipofectinTM reagent of knowing (Bethesda Research Laboratories, Gaithersburg, MD) be the potent agent that is used for highly anionic polynucleotide are delivered into the living tissue culturing cell, it comprises positively charged polynucleotide to form mixture.To a certain extent, think that cation lipid is that they have the enhanced cell avidity as the reason of the effect of cytofectin, the many zones that have high negative charge on their film surface in the described cell.In addition, comprise the positive charge that exists on the lipid aggregate of cation lipid and make this gathering physical efficiency, particularly nucleic acid in conjunction with polyanion.With the lipid aggregate of this method preparation can be spontaneously with cell surface on negative charge interact, merge with plasma membrane, and the function polynucleotide be delivered in the cell effectively.
Val (being also referred to as diphosphatidylglycerol) has constituted the anionic phosphatide of a class compound, and it is purifying from the cytolemma of the tissue relevant with the height metabolic activity typically, comprises the plastosome (Grunner etc. 1985) of heart and skeletal muscle.But known chromatogram purification technology can not be classified as Val discrete molecular species.As a result, the application of this component in pharmaceutical preparation is restricted, because the preparation that obtains is not uniform.In addition, this compound is anionic, and this has limited its application in cationic-liposome.Electrical charge rejection between Val and the reagents for anion can influence its application in many cases.
The Val of cationic form can attract reagents for anion, and more useful to drug conveying.Such mixture can also be used for stablizing the hydrophobic compound of micella and liposome.Need to be used for the new synthetic method of synthesizing cationic Val.The synthetic method that is used to prepare cationic cardiolipin phosphatide can be used to prepare the even preparation of compound.
Need new synthetic method, it can be used to prepare a large amount of saturated and undersaturated cationic cardiolipin phosphatide kinds with different fatty acid chain lengths.This method can improve the utilizability of more kinds of cationic cardiolipin phosphatide kinds, and can make the lipid variation that can be used to develop the new Liposomal formulation that contains promoting agent, with can obtain at present those compare, it has the composition of more definition.
The invention provides such method and composition.From the description of the invention that provides here, these and other advantage of the present invention and other inventive features will be tangible.
Summary of the invention
The invention provides cationic cardiolipin phosphatide cpd and synthetic and use their method.More specifically, the invention provides the liposome that contains the cationic cardiolipin phospholipid analogues, contain the pharmaceutical composition of cationic cardiolipin phospholipid analogues and use such liposome and method for compositions, for example active medicine is flowed to the patient.
Cationic cardiolipin phosphatide of the present invention can be combined in liposome or other lipid formulations, and described liposome or other lipid formulations can also comprise for example hydrophobic or hydrophilic medicine of promoting agent, and nucleic acid is antisense oligonucleotide or diagnostic reagent for example.Such liposome can be used for the treatment of disease, or is used for diagnosis and/or analytical test.Cationic cardiolipin phosphatide can promote the transportation of biologically active agent in cell.Cationic cardiolipin phosphatide cpd of the present invention can be processed,, like this, cytofectin can be used as to form the lipid aggregate with biologically active agent.
Description of drawings
Fig. 1 has shown the synthetic of the cationic cardiolipin phospholipid analogues that contains the alkyl group side chain that ether connects.
Fig. 2,3 and 4 has shown the synthetic of the spacer cationic cardiolipin phospholipid analogues that contains the alkyl group side chain that ether is connected.
Fig. 5 has shown the synthetic of the spacer cationic cardiolipin phospholipid analogues that contains the alkyl group side chain that ether connects.
Fig. 6 has shown the synthetic of the spacer cationic cardiolipin phosphatide variant that contains the alkyl group side chain that ether connects, cationic cardiolipin phospholipid analogues.
Fig. 7 has shown the synthetic of the cationic cardiolipin phosphatide variant analogue that contains the alkyl group side chain that ether connects.
Fig. 8 has shown the synthetic of the spacer cationic cardiolipin phospholipid analogues that contains the alkyl group side chain that ester connects.
Detailed Description Of The Invention
The invention provides and comprise optically cationic cardiolipin phosphatide pure and/or diastereoisomer Variant and analog and their synthetic method and application. In one embodiment, the present invention Cationic cardiolipin phosphatide variant and analog and their synthetic method with general formula I or X are provided:
Figure A20038010611600191
In formula I and X, Z 1And Z 2Can be-O-C (O)-,-O-,-S-,-NH-C (O)-etc. identical or differently.In addition, in formula I and X, R 1, R 2, R 3And R 4Can be identical or different, and can be H, saturated or unsaturated alkyl, alkenyl or alkynyl (typically, but not necessarily, C independently 1To C 32), it can be randomly hydroxylated, aminating (aminenated), sulfurized, epoxidised, cyclisation, Pegylation, halogenated or replace with their combination.In formula I and X, R 5And R 6Can be identical or different, and can be independently not or contain connector, described connector contains alkyl, the cycloalkyl of alkyl, replacement, the cycloalkyl of replacement (C typically 1To C 32) or alkoxyl group for example contain the ether of the unitary Pegylation of 1 to 500 PEG (polyoxyethylene glycol).
In formula I and X, R 7Can be alkyl, alkoxyl group, the alkoxyl group of replacement, cycloalkyl, the cycloalkyl of replacement, alkenyl, alkynyl, alkyloyl, enoyl-(alkenoyl), the alkynes acyl group (alkynoyl) of hydrogen, alkyl, replacement, it can be randomly hydroxylated, aminating a, sulfurized, epoxidised, cyclisation, Pegylation, halogenated or replace with their combination; Or the alkoxyl group of alkoxyl group or replacement for example contains the ether of the unitary Pegylation of 1 to 500 PEG (polyoxyethylene glycol); Amino acid, peptide, peptide simulation part (peptidomimetic moiety), dipeptides, polypeptide, protein, carbohydrate, sugar or polysaccharide, polyamines, heterogeneous ring compound, nucleosides or polynucleotide or other similar portions.In formula X, two R 7Substituting group can be identical or different, and can contain such part independently.In formula I and X, R 8Group can be identical or different, and can comprise C independently 1To C 25The alkyl of saturated or unsaturated alkyl, alkoxyl group, replacement or the alkoxyl group that replaces." X " among formula I and the X is nontoxic negatively charged ion, for example chlorion, bromide anion, iodide ion etc.
Term " alkyl " comprises saturated or unsaturated straight chain and branched-chain hydrocarbon part.Term " replace alkyl " or " alkoxyl group that replaces " etc. comprise and also carry one or more substituent alkyl or alkoxyl group, and described substituting group is selected from hydroxyl, alkoxyl group (low alkyl group); sulfydryl (low alkyl group), cycloalkyl, the cycloalkyl of replacement; halogen, cyano group, nitro; amino, amide group (amido), imino-; sulfo-,-C (O) H, acyl group; the oxygen acyl group, carboxyl etc.Term " sugar " is meant any naturally occurring or non-natural sugar, for example glucose, seminose, allose, ribose, Fucose, pectinose, semi-lactosi, 2-desoxy sugar, 3-desoxy sugar, 4-desoxy sugar, disaccharides and polysaccharide.Term " amino acid " refers to any naturally occurring or non-natural amino acid.This definition is intended to comprise the a-amino acid and the non-a-amino acid of replacement.A-amino acid is to be defined as such amino acid, and wherein amino is to be connected on the carbon atom that closes on hydroxy-acid group.Term " nucleotide sequence " refers to any one or multiple polynucleotide or polynucleotide passage or construct (for example, DNA or RNA oligomer, mRNA or pDNA).Nucleotide sequence can be linear, cyclic (for example, plasmid) or branch's form and two strands or single stranded form.Nucleotide sequence can comprise conventional phosphodiester bond or unconventional key (for example, amido linkage for example appears in the peptide nucleic acid(PNA) (PNA)).Term " peptide " refers to contain the two or more amino acid whose molecule that connects by peptide bond." peptide bond " or " peptide connection " is to remove the covalent linkage that a water molecules forms between an amino acid whose carboxyl and another the amino acid whose amino, and has chemical structure-C (O)-NR-, and wherein R is H, C 1To C 15Alkyl.Term " polypeptide " refers to 9 polymer of amino acid that surpass by the peptide bond connection.Term " albumen " refers to the high-molecular weight amino acid polypeptide, includes but not limited to hormone, antibody and some antigen (Wheeler, Carl, J. patent No. WO 00/73263A1).
In preferred embodiments, the R among formula I and the X 1, R 2, R 3And R 4Be H or C identical or differently 1To C 32Alkyl, alkenyl, alkynyl, they are randomly hydroxylated, aminating, sulfurized, epoxidised, cyclisation, Pegylation or halogenated.In addition, in preferred embodiments, the R among formula I and the X 5And R 6Be connector, it contains C identical or differently 1To C 32Alkyl or alkoxyl group for example contain the ether of 1 to 500 unitary Pegylation of PEG.In addition, in preferred embodiments, the R in formula I and X 7Substituting group is alkyl, the cycloalkyl of hydrogen, alkyl, replacement, cycloalkyl, peptide, dipeptides, polypeptide, protein, carbohydrate, polysaccharide, heterogeneous ring compound, nucleosides or the polynucleotide of replacement.
In other embodiment preferred, the R among formula I and the X 5, R 6, R 7And R 8In at least one comprise the optional alkyl that replaces or optional alkoxyl group that replaces.For example, the R among formula I and the X 5Or R 6In at least one (or the two) can contain the optional poly-alkoxyl group that replaces, it contains 1 to 500 alkoxyl group, for example 1 to 100 alkoxyl group.In fact, in preferred embodiments, the R among formula I and the X 7And R 8In at least one comprise the optional alkyl that replaces, and R 5And R 6Comprise the optional poly-alkoxyl group that replaces.In an especially preferred embodiment, in formula I and X, at least one R 5And/or R 6(or whole R 5And/or R 6Substituting group) is oxyethyl group (single PEG).
In another preferred embodiment, in formula I and X, R 7Substituting group is amino acid, folic acid, sugar, peptide, polysaccharide, polypeptide, protein, polyamines or peptide simulation part.For example, in formula I and X, preferred R 7Substituting group is histone, spermine, spermidine, or derivatives thereof.In formula I and X, another preferred R 7Substituting group is as O-glycosides or C-glucosides bonded carbohydrate, for example glucose, seminose, semi-lactosi, ribose, pectinose, allose, Fucose, 2-desoxy sugar etc.In other embodiment preferred, in formula I and X, R 7Substituting group can be included in L-or the D-alpha amino acid that has positively charged group on the side chain, for example, and arginine, Histidine, Methionin, ornithine or its analogue.In addition, in preferred embodiments, in formula I and X, R 7Substituting group comprises amino acid, carbohydrate, peptide, polysaccharide, polypeptide, protein, polyamines or the peptide simulation part that has one or more positive charges.About formula X, it has 2 R 7Substituting group, they can be identical or different, and can contain such group independently.
In preferred embodiments, in formula I and X, R 8Group is CH 3In other embodiment preferred, in formula I and X, at least one R 5, R 6, R 7And R 8Comprise the alkyl of replacement or the alkoxyl group of replacement.In the most preferred embodiment, in formula I and X, Z 1And Z 2Be-O-C (O)-or-O-.R 1, R 2, R 3And R 4Be identical, be C 1To C 32Saturated and/or undersaturated alkyl, preferred 10 to 24 carbon atoms.In formula I and X, " X " is most preferably chlorion or bromide anion.
In preferred compound according to general formula I, R 5And R 6Do not exist; R 7Be hydrogen; R 8Be CH 3X is a bromide anion; Z 1And Z 2Be oxygen; It is the cationic cardiolipin phosphatide ether with structure I I:
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Alkyl, alkenyl, alkynyl;
At another preferably in the compound according to general formula I, R 5And R 6Do not exist; R 7Be hydrogen; R 8Be CH 3X is a bromide anion; Z 1And Z 2Be-O-C (O)-; It is the cationic cardiolipin phosphatide ester with structure III:
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 31Alkyl, alkenyl, alkynyl.
At another preferably in the compound according to general formula I, R 5Do not exist; R 6Be OCH 2CH 2R 7Be hydrogen; R 8Be CH 3, X is a bromide anion; Z 1And Z 2Be oxygen; It is the cationic cardiolipin phospholipid ether analogs thing with structure I V:
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Alkyl, alkenyl, alkynyl.
At another preferably in the compound according to general formula I, R 5Do not exist; R 6Be OCH 2CH 2R 7Be hydrogen; R 8Be CH 3, X is a bromide anion; Z 1And Z 2Be-O-C (O)-; It is the cationic cardiolipin phosphatide ester analogs with structure V:
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 31Alkyl, alkenyl, alkynyl.
At another preferably in the compound according to general formula I, R 5Be OCH 2CH 2R 6Do not exist; R 7Be hydrogen; R 8Be CH 3, X is a bromide anion; Z 1And Z 2Be oxygen; It is the cationic cardiolipin phospholipid ether analogs thing with structure VI:
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Alkyl, alkenyl, alkynyl.
At another preferably in the compound according to general formula I, R 5Be OCH 2CH 2R 6Do not exist; R 7Be hydrogen; R 8Be CH 3, X is a bromide anion; Z 1And Z 2Be-O-C (O)-; It is the cationic cardiolipin phosphatide ester analogs with structure VII:
Figure A20038010611600232
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 31Alkyl, alkenyl, alkynyl.
At another preferably in the compound according to general formula I, R 5And R 6Be OCH 2CH 2R 7Be hydrogen; R 8Be CH 3, X is a bromide anion; Z 1And Z 2Be oxygen; It is the cationic cardiolipin phosphatide ether with structure VIII:
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Alkyl, alkenyl, alkynyl.
At another preferably in the compound according to general formula I, R 5And R 6Be OCH 2CH 2R 7Be hydrogen; R 8Be CH 3, X is a bromide anion; Z 1And Z 2Be-O-C (O)-; It is the cationic cardiolipin phosphatide ester with structure I X:
Figure A20038010611600241
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 31Alkyl, alkenyl, alkynyl.
In a preferred compound according to general formula X, R 5And R 6Do not exist; R 7Be hydrogen; X is a bromide anion; R 8Be CH 3, Z 1And Z 2Be oxygen; It is the cationic cardiolipin phosphatide variant ether with structure XI:
Figure A20038010611600242
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Alkyl, alkenyl, alkynyl.
At another preferably in the compound according to general formula X, R 5And R 6Do not exist; R 7Be hydrogen; X is a bromide anion; R 8Be CH 3, Z 1And Z 2Be-O-C (O)-; It is the cationic cardiolipin phosphatide variant ester with structure XII:
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 31Alkyl, alkenyl, alkynyl.
In another embodiment, the invention provides cationic cardiolipin phosphatide with general formula X III:
In the compound of formula XIII, R 1, R 2, R 3, R 4, R 5And R 6, X, Z 1And Z 2Can be about as described in formula I and the X as top.And, in formula XIII, R 7, R 9, R 10And R 11Can be about the R among formula I and the X as top 7Described.In formula XIII, R 8Be alkoxyl group, the preferred C of alkenyl, alkoxyl group or replacement of alkyl, alkenyl, the replacement of alkyl, replacement 2-C 32
In a preferred compound according to general formula X III, R 5And R 6There is not R 8It is propyl group; R 7, R 9, R 10And R 11Be hydrogen; Z 1And Z 2Be oxygen; X is a muriate; It is the cationic cardiolipin phosphatide variant ether with structure XIV:
Figure A20038010611600252
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Alkyl, alkenyl, alkynyl.
At another preferably in the compound according to general formula X III, R 5And R 6There is not R 8It is propyl group; R 7, R 9, R 10And R 11Be hydrogen; Z 1And Z 2Be-O-C (O)-; X is a muriate; It is the cationic cardiolipin phosphatide variant ester with structure XV:
Figure A20038010611600261
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 31Alkyl, alkenyl, alkynyl.
Cationic cardiolipin phospholipid molecule according to the present invention can comprise and has different lengths and saturated/undersaturated lipid acid/alkyl chain (for example, at R 1, R 2, R 3And R 4).Usually, the length of fatty acid hydrocarbon chains is about 2 to about 32 carbon atoms; But carbochain more typically is about 10 to about 24 carbon atoms (for example 14 to 20 carbon atoms).Lipid acid is typically classified according to two keys in the hydrocarbon chain and/or triple-linked number (being degree of unsaturation).Saturated fatty acid does not contain any pair of key or triple bond, and is combined with the hydrogen atom of maximum number on each carbon in the chain.The degree of unsaturation of lipid acid depends on two keys or the triple-linked number in the hydrocarbon chain.In this respect, monounsaturated fatty acids contains a two key, and polyunsaturated fatty acid contain 2 or a plurality of pairs of keys (referring to, for example, Oxford Dictionary of Biochemistryand Molecular Biology, revision, A.D.Smith (volume), Oxford UniversityPress (2000) and Molecular Biology of the Cell, the 3rd edition, B.A.Alberts (volume), Garland Publishing, New York (1994)).
That no matter lack or still long, the fatty acid chain of cationic cardiolipin phosphatide of the present invention can also be saturated or unsaturated.The carbon chain length of preferred lipid acid is about C 1To C 32, preferably about C 4To about C 24, and comprise butyric acid (C 4:0), valeric acid (C 5:0), caproic acid (C 6:0), enanthic acid (C 7:0), sad (C S:0), n-nonanoic acid (C 9:0), capric acid (C 10:0), undecanoic acid (C 11:0), dodecylic acid (C 12:0), tridecanoic acid (C 13:0), tetradecanoic acid (tetradecanoic acid) (C 14:0), pentadecylic acid (C 15:0), hexadecanoic acid (palmitinic acid) (C 16:0), margaric acid (C 17:0), octadecanoic acid (stearic acid) (C 18:0), nondecylic acid (C 19:0), arachic acid (eicosanoic acid) (C 20:0), heneicosanoic acid (C 21:0), docosoic acid (docosoic) (C 22:0), tricosanic acid (C 23:0), Lignoceric acid (C 24:0), 10-undecylenic acid (C 11:1), 11-dodecenoic acid (C 12:1), 12-tridecylenic acid (C 13:1), nutmeg olic acid (C 14:1), 10-pentadecylenic acid (C 15:1), Zoomeric acid (C 16:1), oleic acid (C 18:1), linolic acid (C 18:2), linolenic acid (C 18:3), eicosenoic acid (C 20:1), eicosadienoic acid (eicosdienoic acid) (C 20:2), eicosatrienoic acid (C 20:3), arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid) and cis-5,8,11,14, the 17-timnodonic acid, inter alia.About the ether analogs thing, the scope of alkyl chain is C 1To C 32, preferably about C 4To about C 24Other fatty acid chain also can be used as R 1And/or R 2, R 3And/or R 4Substituting group.Such example comprises saturated fatty acid, acetate (or acetate) for example, propionic acid (or propionic acid), butyric acid (or butyric acid), hydroxyhexacosanoic acid (or cerinic acid), octacosanoic acid (or montanic acid), triacontanoic acid (or myricyl acid), n-Dotriacontanoic acid (or lacceroic acid), gheddic acid, three pentadecanoic acids (or ceroplasticacid) etc.; For example trans-2-butene acid of the unsaturated fatty acids of monoene (or Ba Dousuan), cis-2-butene acid (or iso-crotonic acid), 2-hexenoic acid (or isohexenoic acid), 4-capric acid (or obtusilic acid), 9-capric acid (or decylenic acid), 4-dodecenoic acid (or linderic acid), linderic acid (or denticetic acid), 9-dodecenoic acid (or lauroleic acid), 4-tetradecenoic acid (or tsuzuic acid), physeteric acid (or physeteric acid), petroselinic acid (or petroselenic acid), elaidic acid (or elaidic acid), anti-form-1 1-octadecenoic acid (or vaccinic acid), 9-eicosenoic acid (or cis-9-20 carbon acid), 11-eicosenoic acid (or gondoic acid), 11-docosenoic acid (or cetoleic acid), 13-docosenoic acid (or erucic acid), 15-tetracosenoic acid (or Selacholeic acid), 17-hexacosenoic acid (or ximenic acid), 21-lumequeic acid (or lumequeic acid), etc.; The unsaturated fatty acids of diene for example 2,4-pentadienoic acid (or β-vinylacrylic acid), 2,4-Sorbic Acid (or Sorbic Acid), stillingic acid (or stillingic acid), 2, the 4-dodecadienoic acid, 9, the 12-hexadecadienoic acid, cis-9, cis-12-octadecadienoic acid (or α-linolic acid), trans-9, anti-form-1 2-octadecadienoic acid (or linlolelaidic acid), anti-form-1 0, anti-form-1 2-octadecadienoic acid, 11, the 14-eicosadienoic acid, 13,16-two dodecadienoic acids, 17,20-hexacosandienoic acid etc.; The unsaturated fatty acids of triolefin for example 6,10,14-hiragonic acid (or hiragonic acid), 7,10, the 13-hiragonic acid, cis-6, cis-9-cis-12-punicic acid (or gamma-linoleic acid), trans-8, anti-form-1 0-anti-form-1 2-punicic acid (or β-punicic acid), cis-8, anti-form-1 0-cis-12-punicic acid, cis-9, cis-12-cis-15-punicic acid (or alpha-linolenic acid), trans-9, anti-form-1 2-anti-form-1 5-punicic acid (or alpha-linolenic acid), cis-9, anti-form-1 1-anti-form-1 3-punicic acid (or alpha-eleostearic acid), trans-9, anti-form-1 1-anti-form-1 3-punicic acid (or alpha-eleostearic acid), cis-9, anti-form-1 1-cis-13-punicic acid (or punicic acid), 5,8, the 11-eicosatrienoic acid, 8,11,14-eicosatrienoic acid etc.; The unsaturated fatty acids of tetraene for example 4,8,11,14-16 carbon tetraenoic acids, 6,9,12,15-16 carbon tetraenoic acids, 4,8,12,15-therapic acid (or moroctic acid), 6,9,12, the 15-therapic acid, 9,11,13, the 15-therapic acid (or α-or β-therapic acid), 9,12,15, the 18-therapic acid, 4,8,12, the 16-eicosatetraenoic acid, 6,10,14, the 18-eicosatetraenoic acid, 4,7,10, the 13-docosatetratenoic acid, 7,10,13, the 16-docosatetratenoic acid, 8,12,16,19-docosatetratenoic acid etc.; Five-and the unsaturated fatty acids of six-alkene for example 4,8,12,15,18-timnodonic acid (or timnodonic acid), 4,7,10,13,16-clupanodonic acid, 4,8,12,15,19-clupanodonic acid (or clupanodonic acid), 7,10,13,16,19-clupanodonic acid, 4,7,10,13,16,19-docosahexenoic acid, 4,8,12,15,18,21-nisioic acid (or nisinic acid) etc.; The lipid acid of side chain is 3 Methylbutanoic acid (or isovaleric acid) for example, 8-methyl dodecylic acid, 10-methyl undecanoic acid (or different lauric acid), 11-methyl dodecylic acid (or different undecanoic acid), 12-methyl tridecanoic acid (or different tetradecanoic acid), 13-methyl tetradecanoic acid (or different pentadecylic acid), 14-methyl pentadecylic acid (or different palmitinic acid), 15-methyl hexadecanoic acid, the 10-methylheptadecanoic acid, 16-methylheptadecanoic acid (or Unimac 5680), 18-methyl nondecylic acid (or different eicosanoic acid), 20-methyl heneicosanoic acid (Huo docosoic), 22-methyl tricosanic acid (or isotetracosane acid), 24-methyl pentacosoic acid (or different cerinic acid), 26-methyl carboceric acid (or isomonatonic acid), 2,4,6-trimethylammonium octocosoic acid (or mycoceranic or mycoserosic acid), 2-methyl-cis-2-butene acid (angelicic acid), 2-methyl-trans-2-butene acid (or tiglic acid), pyroterebic acid (or pyroterebic acid) etc.
Cationic cardiolipin phospholipid molecule of the present invention can be according to prepared by any suitable process, for example methods known in the art.But typically, they belong to one of 2 kinds of general synthetic methods.In first kind of approach, hydrocarbon chain is connected on the glycerol backbone by ehter bond.The following examples 1-8 (Fig. 1-7) has explained this route of synthesis.According to the 2nd kind of approach of synthetic cationic cardiolipin phosphatide of the present invention, hydrocarbon chain is connected on the glycerol backbone by ester bond.The following examples 9 (Fig. 8) have been explained this route of synthesis.Those skilled in the art can understand, when having described particular compound synthetic in an embodiment, similarly reaction scheme also can be used to make up cationic cardiolipin phospholipid molecule of the present invention, and it is for example at R 1, R 2, R 3, R 4Or other are substituent different in nature.
But after the preparation, cationic cardiolipin phospholipid molecule of the present invention can be included in the composition, Liposomal formulation for example, mixture, emulsion, suspension etc.Such preparation can prepare by any suitable technique according to the type of composition, and this is that those of ordinary skill in the art is known.
Such preparation can comprise the composition except cationic cardiolipin phosphatide of the present invention, for example one or more common lipids or physiologically acceptable carrier.For the purpose that defines, term " altogether lipid " refer to can with any hydrophobic substance of cationic cardiolipin phosphatide combination, it comprises the amphipathic lipid, for example phosphatide and neutral lipid, for example cholesterol and other sterol, and such lipid solubilized form.Altogether lipid can be any natural or synthetic phosphatide or single-, two-or triglycerin.Natural phospholipid is typically from those of animal and plant source, for example phosphatidylcholine, phosphatidylethanolamine, sphingophospholipid, phosphatidylserine or phosphatidylinositols.Other suitable common lipid comprises sterol (cholesterol for example, the derivative of cholesterol, coprostanol, Dihydrocholesterol, cholestane, Cholesteryl hemisuccinate, cholesterol sulfate and its mixture) and tocopherol (for example alpha-tocopherol).Other the suitable lipid altogether that is used for being included in composition of the present invention comprises phosphatidylcholine, for example dimyristoyl phosphatidyl choline, distearoyl phosphatidylcholine, two oil base phosphatidylcholines, two palmitoyl-phosphatidylcholine, two arachidonic phosphatidyl cholines, Yelkin TTS phatidylcholine, fabaceous lecithin phatidylcholine, hydrogenant fabaceous lecithin phatidylcholine and its mixture.In addition, other known cation lipid N-[1-(2,3-two oil base oxygen bases) propyl group for example]-N; N; N-trimethyl ammonium muriate (DOTMA) or 1,2-dioleoyl oxygen base-3-(trimethylammonium ammonia)-propane (DOTAP) can be included in the cationic cardiolipin phospholipid composite of the present invention.When comprising neutral lipid, positively charged ion lipid (comprising cationic cardiolipin phosphatide of the present invention) typically is about 9/1 to about 1/9 with the mol ratio of neutral lipid.
Except cationic cardiolipin phosphatide of the present invention, liposome composition, mixture, emulsion etc. can comprise stablizer, absorption enhancer, oxidation inhibitor, phosphatide, biodegradable polymkeric substance and medicinal actives agent, except other composition.In some embodiments, preferably, composition of the present invention, particularly liposome composition comprise the target agent, carbohydrate or albumen or other part that for example can the binding specificity substrate, for example antibody (or its fragment) or part that can the recognizing cells acceptor.Comprise such reagent (for example carbohydrate or one or more protein, described protein is selected from by antibody, antibody fragment, peptide, peptide hormone, the receptors ligand for example antibody of cell receptor and the group that its mixture is formed), can promote tissue or cell type that the liposome target is predetermined.
Although the invention is intended to comprise cationic cardiolipin phosphatide in the composition of any adequate types, preferred compositions is liposome composition or other composition that contains lipid capsule.That such composition can comprise individual layer or multiwalled capsule, or its mixture.Can produce such Liposomal formulation with any suitable technique.For example, can will can form the composition of lipophilic liposome, for example phosphatidylcholine, cationic cardiolipin phosphatide of the present invention, cholesterol and alpha-tocopherol dissolve or are distributed in the combination of suitable solvent or solvent, and dry.Suitable solvent comprises any nonpolar or slight polar solvent, for example trimethyl carbinol, ethanol, methyl alcohol, chloroform or acetone, and they can evaporate and not stay any pharmaceutically unacceptable resistates.Can carry out drying by any suitable method, for example by freeze-drying.Hydrophilic composition (for example some medicaments, sanitas and other reagent) can be dissolved into polar solvent, comprise in the water that it can be before dry or mixes mutually with lipid during in reconstruct.The exsiccant lipotropic component mixed with hydrophilic mixture can form liposome.Can polar solvent be mixed with the exsiccant lipid film by the method for any homogenised mix consumingly.Eddy current, magnetic agitation and/or supersound process can realize homogenizing.
When containing promoting agent in the liposome, they can dissolve or be dispersed in the suitable solvent, and add to before mixing in the liposome mixture.Typically, hydrophilic promoting agent is directly added in the polar solvent, add hydrophobic promoting agent to be used for dissolving other composition non-polar solvent, but this is unwanted.Promoting agent can be dissolved in the 3rd kind of solvent or the solvent mixture, and adds to before homogenised mix in the mixture of polar solvent and lipid film.
Usually, liposome can be clean neutral, negative charge or positive charge.For example, can from contain phosphatidylcholine, cholesterol, enough stearylamine solution, form cationic-liposome with the clean positive charge that overcomes cationic cardiolipin phosphatide.Now, can go out positively charged liposome from the formulations prepared from solutions that contains phosphatidylcholine, cholesterol and cationic cardiolipin phospholipid analogues of the present invention.
According to preparation their concrete composition and method, liposome of the present invention can be the capsule of multiwalled or individual layer.Liposome can be prepared into has in fact the size in the selected size scope uniformly, and for example about 1 micron or littler, or about 500nm or littler, about 200nm or littler, or about 100nm or littler.A kind of method of effective setting size comprises: the waterborne suspension of liposome is extruded from a series of polycarbonate membranes with selected uniform pore size size; Roughly to extrude the overall dimension of liposome of generation corresponding with passing this film for the pore size of film.
Can give the biodegradable polymkeric substance of liposome for example sucrose, Epicholorohydrin, the hydrophilic sucrose polymer of ramose, polyoxyethylene glycol, polyvinyl alcohol, methoxy poly (ethylene glycol), oxyethyl group polyoxyethylene glycol, polyethylene oxide, polyoxyethylene, polyoxypropylene, rhodia, sodiun alginate, N, the segmented copolymer of N-diethyl amino yl acetate, polyoxyethylene and polyoxypropylene, polyvinylpyrrolidone, polyoxyethylene X-bay ether, wherein X is 9 to 20, and Sorbitan ethoxylate.
Liposome (or other lipid) composition or preparation can be any ideal forms.For example, for pharmaceutical use, composition can be to be ready to be administered to the patient.When such composition contained the lipid capsule of liposome or other type, such preparation is the micro-capsule form in aqueous medium typically.Perhaps, preparation can be exsiccant or freeze dried form, and in this case, composition preferably also comprises cryoprotectant.Suitable cryoprotectant comprises that for example, carbohydrate is trehalose, maltose, lactose, sucrose, glucose and dextran for example, and from performance, most preferred sugar is trehalose and sucrose.Can also use other more complicated sugar, for example, aminoglycoside comprises Streptomycin sulphate and dihydrostreptomycin.
Oxidation inhibitor can be included in the liposome or other lipid formulations that comprises cationic cardiolipin phosphatide of the present invention.Suitable oxidation inhibitor comprises compound for example xitix, tocopherol and deteroximemesylate.Absorption enhancer can be included in the lipid formulations.Suitable absorption enhancer comprises sodium salicylate-SODIUM CHENODIOL, sodium deoxycholate, polyoxyethylene 9-bay ether, CDC-deoxycholate and polyoxyethylene 9-bay ether, Rylo MG 19, ox sulphur-24; 25-dihydro Sodium Fusidate (Na taruo-24,25-dihydrofusidate), Taurodeoxycholate sodium, sweet ammonia SODIUM CHENODIOL (Na glycochenodeoxycholate), oleic acid, linoleic acid plus linolenic acid.Can also comprise the polymeric absorption enhancer, for example Soxylat A 25-7, Sorbitan ethoxylate, polyoxyethylene 10-bay ether, polyoxyethylene 16-bay ether, azone (1-dodecyl-aza-cycloheptane alkane-2-ketone).
What comprise cationic cardiolipin phosphatide of the present invention can be used for multiple use according to composition of the present invention, for example with in peptide, polypeptide, protein, Nucleotide, polynucleotide, small molecules or other reagent body or external human or animal (typically vertebrates) patient, plant or the cultured cells of flowing to.Can use composition with any method, for example, comprise and use in liposome or the lipid utricule ground or the interior transportation of substances of external ground born of the same parents U.S. Patent numbers 5,264,618 such as () Felgner.Composition can also use in the makeup mode, for example, and as the dermatology goods.Therefore, can come compositions formulated according to the target end-use.
In order to be applied to human or animal patient, composition preferably comprises (or pharmaceutically) acceptable medium or carrier on one or more physiology.Can use any such medium, for example be generally used in the lipid formulations those.Therefore, the invention provides pharmaceutical preparation, it contains cationic cardiolipin phosphatide of the present invention (for example preparing as described) and pharmaceutically acceptable carrier or medium.
Believe according to compound of the present invention and can give patient's beneficial effect, and can for example resist the influence of aging, cancer, heart trouble etc.Therefore, can use pharmaceutical preparation of the present invention to treat some individuality valuably, need not to comprise other medicine.But pharmaceutical preparation of the present invention can comprise one or more therapeutical agents of significant quantity on the pharmacology.On this meaning, the significant quantity of therapeutical agent is the amount that is fit to provide result of treatment after using said composition to the human or animal.The significant quantity of particular therapeutic agent depends on this reagent, but the general knowledge of determining to belong to this area of the significant quantity of in composition of the present invention, using.
In one embodiment, pharmaceutical preparation can comprise nucleoside analog or the nucleotide analog as the treatment significant quantity of promoting agent.The suitable nucleoside analog or the example of nucleotide analog comprise nucleosides or the nucleotide analog with antivirus action, the halogenated or azido-derivative and the acyclic nucleosides of for example di-deoxynucleoside acid, two dehydrogenation Nucleotide, nucleosides.For example, have the purine of halogen-replacement or the nucleosides or the nucleotide analog of pyrimidine ring, for example 5-trifluoromethyl-2 '-deoxyuridine or 5 FU 5 fluorouracil; Have halogen-and the nucleosides or the nucleotide analog of the ribose part that replaces of azido-, for example 3 '-azido--3 ' deoxythymidine (AZT), nucleoside analog (carbocyclic nucleoside) with the carbon that replaces the oxygen on the ribose part, or have the nucleotide analog of acyclic pentose, for example acycloguanosine or gancyclovir (gancyclovir) (DHPG) can be included in the composition suitably.It is U.S. Patent numbers 5,223,263 such as () Hosteter known in the art that the liposome of this analogue is carried, and composition of the present invention can be used for such reagent is flowed to the patient that can benefit from such treatment suitably.When they are when passing cell as phospholipid derivative, find that the antiviral efficacy of these analogues increases.These derivatives can be integrated into the liposome structure that is used for being administered to cell, form more stabilized liposomes mixture thus, and it can give target cell with more substantial drug conveying, and has lower toxicity.The effective antiviral lipid derivate of nucleoside analog comprise phosphatidyl 2 ', 3 '-di-deoxynucleoside, 2 ', 3 '-two dehydrogenation nucleosides, 3 '-azido--2 '-deoxynucleoside, 3 '-fluorine deoxynucleoside and 3 '-the fluorine di-deoxynucleoside, 9-β-D-arabinofuranosyladenine (araA), 1-β-D-arbinofuranose cytidine (araC), nucleosides for example has the acycloguanosine and the gancyclovir of acyclic ribose groups, or with the identical nucleoside analog of two glycerine bisphosphate derivatives.The preferred kind that is used to use the lipid derivate of the antiviral or antiretroviral nucleoside analog that cation lipid mediated liposome delivering therapeutic HIV infects is 3 '-azido--2 ', 3 '-the dideoxy pyrimidine 3 '-phospholipid derivative, 3 '-halogenated pyrimidine di-deoxynucleoside or 2 ', 3 '-two dehydrogenations-2 ', 3 '-di-deoxynucleoside, for example phosphatidyl 3 '-azido--3 ' deoxythymidine (pAZT) or phosphatidyl 2-chlorodeoxyadenosine.Use contain acycloguanosine, gancyclovir, 1-(2-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base)-5-iodocytosine (FIAC) or 1-(2 '-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base) nucleoside analog of 5-iodouracil (FIAU), some virus infection be can treat effectively, bleb, cytomegalovirus and hepatitis B infected comprised.Use can be applied to these diseases with the phospholipid derivative of these reagent, preferred phosphatidyl and two glycerine bisphosphate derivatives according to cation lipid liposome delivery system of the present invention.Reported the structure of the lipid derivate of anti-viral nucleoside, synthetic and details that liposome is carried (U.S. Patent number 6 such as Hostetler, 448,392), composition of the present invention can be used for such reagent is flowed to the patient that can benefit from such treatment suitably.
In another embodiment, pharmaceutical preparation can comprise as the reflunomide of the treatment significant quantity of promoting agent or the anti-inflammatory agent of on-steroidal.Other suitable promoting agent comprises the biological activity lipid, for example, and 1-O-octadecyl-2-O-methyl-sn-glyceryl-3-phosphorylcholine.Other suitable promoting agent comprises medicine, anticarcinogen and partial microbiotic clindamycin for example for example, tobramycin, Xin Meisu, gentamicin, tsiklomitsin, erythromycin; Antifungal drug, clotrimazole for example, miconazole, nystain, lactoconazole, econazole and tolnaftate; The vitamin A acid that is used for the treatment of acne; With the reagent that is used for the treatment of herpes simplex with comprise antiviral nucleoside analogs for example acycloguanosine and gancyclovir.These nucleoside analog preparations preferably contain the lipid derivate of antiviral agent, particularly as (Felgner etc., U.S. Patent number 5,459,127) described phosphatidyl glycerol derivative can be incorporated in the liposome or other preparation that contains one or more cationic cardiolipin phospholipid analogues of the present invention like this.The example of the promoting agent that other is suitable comprises antifungal drug, oxygenant, protein, polypeptide and therapeutic polynucleotide.Preferred protein comprises that for example, antibody is monoclonal antibody or its fragment for example.
Be applicable to that important polynucleotide are electronegative new oligonucleotide of different process in the treatment of conveying of cation lipid mediation, comprise being used to eliminate or reduce the antisense polynucleotides sequence (Ts ' o etc. 1987) that gene product generates.Many in these oligonucleotide kinds are rare and synthetic expensive, according to common prior art, can not be encapsulated into effectively in the liposome of electronegative lipid.
The example of therapeutic polynucleotide comprises ribozyme, RNA interfering (RNAi), sense-rna or dna sequence dna, and it can navigate to the target sequence in the cell, for example relevant with morbid state gene (for example, oncogene or virogene).The preferred therapeutic polynucleotide that are used to navigate to target gene are antisense polynucleotides of 10 to 30-mer, the sequence of preferred 15-mer, for example, to the c-raf gene target (referring to, for example United States Patent (USP) 6,126,965, its disclose have sequence 5 '-the anti-c-raf-1 oligonucleotide of 15-mer of GTGCTCCATTGATGC-3 ').When containing oligonucleotide in the composition, they preferably contain one or more thiophosphoric acids and connect, and preferred 2 thiophosphoric acids connect.Most preferably, be included in oligonucleotide in the composition of the present invention and contain 1 thiophosphoric acid at each end and connect, but they may reside in any position from an end of oligonucleotide to another end (for example, endways between).Localized other the preferred gene of therapeutic ribozyme, RNA interfering (RNAi) sense-rna or dna sequence dna comprises virogene, particularly HIV gene, for example rev trans-activator.In other embodiments, the therapeutic polynucleotide can be such, and it does not exist in morbid state or suddenlys change, and maybe can be coded in morbid state is that lack or non-existent gene product.Other polynucleotide therapeutical peptide of can encoding, for example, the synthetic analogues of immunogenic peptide (it can be used as vaccine), natural hormone or natural hormone.
Other suitable promoting agent that can be included in the preparation of the present invention comprises the reagent that can act on following object: peripheral nerve, adrenergic receptor, cholinergic receptor, skeletal muscle, cardiovascular systems, unstriated muscle, vascular system, cynapse site, neural effector engage site, internal secretion and hormone system, immunity system, reproductive system, Skeletal system, digestion and Excretory system, histamine system and central nervous system.Suitable reagent can be selected from, for example protein, enzyme, hormone, Nucleotide (including justice and antisense oligonucleotide), polynucleotide, nucleoprotein, polysaccharide, glycoprotein, lipoprotein, polypeptide, steroid.Promoting agent can be an anodyne, narcotic, anti-dysrhythmia agents, microbiotic, anti-allergic agent, anti-mycotic agent, carcinostatic agent, anti-coagulant, antidepressive, antidiabetic, Anti-epileptics, antiphlogistic reflunomide, be used for the treatment of Alzheimer or parkinsonian medicine, anti ulcer agent, antiprotozoal drug, anxiolytic, thyradin, anti-thyroid preparation, antiviral drug, anorectic medicaments (anoretics), diphosphonate, influence myocardial contraction medicine (cardiac inotropicagents), cardiovascular drug, reflunomide, diuretic(s), the dopaminergic agent, gastrointestinal agent, hemostatic drug, the hypercholesterolemia medicine, depressor (for example dihydropyridine), thymoleptic and cox-2 inhibitor, immunosuppressor, antigout drug, antimalarial drug, steroid, terpinoids, triterpines, retinoids; Antiulcer agent H2-receptor antagonist, hypoglycemic agents, wetting Agent for Printing Inks, makeup, antimigraine, Antimuscarinic agent (antimuscarinic agents), antiphlogiston, for example be used for the treatment of the medicine of rheumatosis, sacroiliitis, psoriasis, inflammatory bowel, regional ileitis; Or be used for the treatment of demyelinating diseases, the medicine that comprises multiple sclerosis, medicament for the eyes, vaccine (for example anti-pneumonia, hepatitis A, hepatitis B, hepatitis C, choleratoxin B subunit, influenza virus, typhoid fever, plasmodium falciparum, diptheria, tetanus, HSV, tuberculosis, HIV, SARS virus, pordetela pertussis, measles, mumps and Rubella Vaccine (MMV), bacterial toxoid, vaccine virus, adenovirus, canary, poliovirus, bacille Calmette-Guerin vaccine (BCG), bacterium friedlanderi pneumonia (klebsiellapneumonia) etc.), histamine receptor antagonists, soporific, protect the kidney agent, transfer the fat agent, muscle relaxant, neural Depressant, neurotrope, opioid agonist and antagonist, cholinomimetic, proteinase inhibitor, prostaglandin(PG) (prostglandins), tranquilizer, sexual hormoue (for example, oestrogenic hormon, male sex hormone), stimulant, sympathomimetic, the synthetic analogues of vasodilator and xantheine and these kinds.Therapeutical agent can be a renal toxicity, for example ciclosporin and amphotericin B, or cardiac toxic, for example amphotericin B and taxol.Exemplary carcinostatic agent comprises melphalan, mustargen, extramustinephosphate, Uramustine, ifosfamide, Mannomustine, trifosfamide, streptozocin, mitobronitol, mitoxantrone (referring to, for example disclosed International Patent Application WO 02/32400), methotrexate, Fluracil, cytosine arabinoside, Tegafur, idoxide, taxanes (for example, safe plain, taxol etc., (referring to, for example disclosed International Patent Application WO 00/01366), daunomycin, daunorubicin, bleomycin, amphotericin, carboplatin, cis-platinum, taxol, BCNU, vincristine(VCR), camptothecine and its derivative (for example SN38 (referring to, for example disclosed International Patent Application WO 02/058622), irinotecan (referring to, for example disclosed International Patent Application WO 03/030864)), little erythromycin (antracyclines), antibody, cytoxines, Dx, etoposide (etopside), cytokine, ribozyme, Interferon, rabbit, the functional deriv of oligonucleotide and aforementioned substances.Other example of the medicine that can carry according to present method comprises ethanedisulphonate prochlorperazine (prochlorperzine edisylate), ferrous sulfate, hexosamine, mecamylamine hydrochloride, procamide, amfetamine sulfate, methamphetamine hydrochloride, benzamphetaminehydrochloride, isoproterenol sulfate, phenmetrazine hydrochloride, urethan of .beta.-methylcholine chloride, Methacholine Chloride, Pilovisc, Tropintran, Scopolamine bromide (scopolamine bromide), isopropamide iodide, tridihexethyl chloride, phenformin hydrochloride, methylphenidate hydrochloride, Zy 15061, cefalexin hydrochloride, diphenidol, meclozine hydrochloride, prochlorperazine maleate, Phenoxybenzamine, Tresten (thiethylperzine maleate), anisindone, the Oragulant Erythrityl Tetranitrate, digoxin, Isoflurophate, acetazolamide, methazolamide, Hydrex, chloropromaide, tolazamide, chlormadinone acetate, phenaglycodol, allopurinol, Rumasal, methotrexate, Acetylsulfisoxazole, erythromycin, hydrocortisone, hydrocorticosterone acetate, cortisone acetate, dexamethasone and its derivative be Betamethasone Valerate for example, triamcinolone, Synrotabs, 17-(S)-estradiol, ethinylestradiol, ethinylestradiol 3-methyl ether, prednisolone, 17-α-hydroxyprogesterone acetate, the 19-norprogesterone, Norethisterone is pregnant, Anazolan, Norethisterone, norethiederone, progesterone, norgesterone, norethynodrel, Asprin, indomethacin, Naproxen Base, fenoprofen, sulindac, indoprofen, nitroglycerin, sorbide nitrate, Proprasylyte, timolol, atenolol USP 23, alprenolol, Cimitidine Type A/AB, clonidine, imipramine, levodopa, chlorpromazine, methyldopa, dopa, theophylline, calglucon, Ketoprofen, Ibuprofen BP/EP, Cephalexin Monohydrate Micro/Compacted, erythromycin, haloperidol, zomepirac, iron lactate, vincamine, vinorelbine (referring to, for example disclosed International Patent Application WO 03/018018), diazepam, Phenoxybenzamine, diltiazem , milrinone, Cefamandole, quanbenz, hydrochlorothiazide, Ranitidine HCL, flurbiprofen, fenufen, R.D. 17345, tolmetin, Warner-Lambert), mefenamic acid, Flufenamic Acid, difuinal, nimodipine, nitrendipine, nisoldipine, nicardipine, felodipine, lidoflazine, tiapamil, Procorum, amlodipine, mioflazine, lisinopril, enalapril, enalaprilat, captopril, Ramipril, famotidine, nizatidine, sucralfate, etintidine, tetratolol, minoxidil, zeisin, diazepam, amitriptyline, and imipramine.Other example is albumen and peptide, and it includes but not limited to bone morphogenetic protein, Regular Insulin, colchicine, hyperglycemic-glycogenolytic factor, thyrotropic hormone, parathyroid gland and pituitrin, peptic hormones, thyrocalcitonin, feritin, prolactin, thyroliberin, thyrotropic hormone, follicle stimulating hormone, chorionic-gonadotropin hormone, gonadotropin releasing hormone, bovine somatotropin, porcine somatotropin, pitocin, beta-hypophamine, GRF, somatostatin, lypressin, Pancreozymin, lutropin, LHRH, LHRH agonist and antagonist, leuproside, Interferon, rabbit (for example total Interferon, rabbit, Intederon Alpha-2a, Interferon Alpha-2b, α-, β-or gamma-interferon), interleukin, tethelin is for example first thiamines (methione)-human growth hormone and des-phenylalanine human growth hormone of human growth hormone and its derivative for example, Trobest and Porcine somatotropin, the fertility inhibitor is prostaglandin(PG) for example, fertility promotor, somatomedin is rhIGF-1 for example, coagulation factors, pancreatic hormone releasing hormone (pancreas hormone releasing factors), pharmaceutical salts or their analogue or the derivative of the analogue of these compounds and derivative and these compounds.Therapeutical agent can be that () mixture for example, two or more reagent, they can use in Liposomal formulation reagent valuably jointly.
Pharmaceutical preparation can be used for therapeutical agent is transported to the intravital different loci of animal by all means, with the therapeutic effect of realizing ideal.By comprising using of body cavity or tissue used or be inserted into to preparation, oral delivery, the suction of aerosol or be blown into, locally apply to skin or mucomembranous surface, or pass through administered parenterally, comprise in intramuscular, intravenous, the tumour, intracutaneous, peritonaeum, subcutaneous using, can realize partly or delivering therapeutic agents capapie with partial.The effect of the cation lipid in these preparations is, by transporting effect and the efficient (Felgner etc., U.S. Patent number 5,459,127) that improves it in the cell that promotes to be contained in therapeutical agent wherein.Therefore, the invention provides by using the method that contains one or more promoting agents of delivery of composition of cationic cardiolipin phosphatide and one or more promoting agents as herein described.This method can be used to therapeutic, and promptly giving needs the human or animal patient (or plant) of treatment to carry one or more promoting agents, and perhaps this method can be used for giving the cell of cultivating with one or more active agent delivery.This method can be used to use all in fact promoting agents.Use about therapeutic, think that for promoting agent stable when having tensio-active agent be conventional.Hydrophilic promoting agent is suitable, and can be included in liposome interior, and the liposome bilayer is set up diffusion barrier like this, prevents that it from spreading all over the health diffusion randomly.Think that hydrophobic promoting agent is specially adapted to the purposes of present method, can show hypotoxic advantage, and they tend to dissolving preferably in the lipid bilayer of liposome because they not only have.
Chemotherapeutics also is applicable to this method preferably.The liposome or other lipid formulations that contain chemotherapeutics can be injected directly in the tumor tissues, so that chemotherapeutics is delivered directly in the cancer cells.In some cases, particularly behind tumor resection, Liposomal formulation directly can be implanted in the cavity of generation, maybe can be applied in the remnant tissue as dressing.Use after surgery under the situation of Liposomal formulation, can use to have about 1 micron large diameter liposome, because they need not to pass vascular system.
Particularly preferred embodiment comprises and is used for the treatment of or prevents Herpes Simplex and the application in treatment or prevention herpes infection of such preparation.Pointed as this paper, except cationic cardiolipin phosphatide of the present invention, such preparation comprise ideally effective concentration on the pharmacology acycloguanosine, gancyclovir, 1-(2-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base)-5-iodocytosine (FIAC) or 1 (2 '-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base) 5-iodouracil (FIAU).For example, composition can be administered to by topical application (being administered to mucosal tissue ideally) and infect herpes simplex or be in individuality in the risk that infects herpes simplex.
The invention provides the method for administration of pharmaceutical preparations, described pharmaceutical preparation also comprises vehicle nontoxic, that inert is pharmaceutically suitable except the Liposomal formulation of promoting agent.Pharmaceutically suitable vehicle comprises solid, semisolid or liquid diluent, filler and various pharmaceutical adjunct.The present invention also comprises pharmaceutical preparations in dosage units.This means that preparation is the form of single part, for example bottle, syringe, capsule, pill, suppository or peace are cutd open, and wherein the content of promoting agent in Liposomal formulation is corresponding with the mark or the multiple of single-dose dosage.Dose unit can contain for example 1,2,3 or 4 single-dose dosage, or 1/2,1/3 or 1/4 single-dose dosage.Single-dose dosage preferably contains the active dose of applied once, its usually with every day dosage whole, 1/2,1/3 or 1/4 corresponding.
Tablet, drageeing, capsule, pill, granula, suppository, solution, suspension and emulsion, paste, ointment, gel, creme, lotion, powder and sprays can be suitable pharmaceutical preparations.Except liposomal active agent, suppository can contain suitable water miscible or water-insoluble vehicle.Suitable vehicle is such, and liposomal active agent of the present invention is sufficiently stable therein, to be used for the treatment of purposes, and the mixture of polyoxyethylene glycol, some fat and ester or these materials for example.Ointment, paste, creme and gel can also contain suitable vehicle, and liposomal active agent is stable therein.
Promoting agent or its pharmaceutical preparation can be administered to patient or intravital cell, intravenously ground, hypodermically, partly (for example for example, give skin or dermal tissue, or give mucosal tissue), per os ground, stomach other places, intraperitoneal ground and/or rectum ground, or by method known or exploitation, direct injection advances in the site that tumour maybe needs to treat.And, in some embodiments, give patient's cell paste other places administration of pharmaceutical preparations ideally, it comprises cationic cardiolipin phosphatide of the present invention and optional promoting agent, after this cell is turned back to the patient.Such treatment can be effectively, and for example, the genetic expression in the cell that imports again can be resisted disease of patient effectively, for example treats under the situation of cancer.
Cationic cardiolipin phosphatide of the present invention and above-mentioned preparation can promote the method for the disease of treatment vertebrates (for example people or inhuman animal), it comprises the step of using pharmaceutical preparation as described herein to the patient, and said preparation typically comprises treatment of diseases is had specific therapeutical agent.The method according to this invention is administered to the vertebrates that needs are treated with preparation as herein described (containing promoting agent ideally), and its amount and administration position are enough to treat vertebrate disease.In some embodiments, therapeutical agent is integrated at least one cell of patient, it brings into play the effect (for example, when therapeutical agent was DNA or RNA, it was expressed later) of treatment disease therein in the cell that is ingested.In other embodiments, reagent plays a role resist the disease in patient's inner cell other places.Pharmaceutical preparation be with the mode that is suitable for preparation type be administered to the patient (for example, be administered to skin or mucomembranous surface skin, parenteral injection be injected into intracoelomic cavity or the tissue in, dosage forms for oral administration etc.).Will be appreciated that the method according to this invention is treated disease effectively, eliminate a disease ideally or its symptom in, need not thoroughly to eradicate the influence of disease.In fact, the reduction of the seriousness by disease, infection or the reduction of disease of patient tempo can detect the successful treatment of the method according to this invention.
In one embodiment, the method disease is a cancer, and in this case, pharmaceutical preparation can contain suitable carcinostatic agent, chemotherapeutics for example as herein described (for example, cancer therapy drug) or be selected from following polynucleotide: ribozyme, RNA interfering (RNAi) and sense-rna or dna sequence dna.Preferred anticancer polynucleotide are c-raf antisense oligonucleotides.In another embodiment, disease is a virus infection, for example herpes simplex or HIV.In order to treat virus infection, pharmaceutical preparation typically comprises antiviral drug, and is for example as herein described.For example, in order to treat HSV, preparation can comprise acycloguanosine, gancyclovir, 1-(2-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base)-5-iodocytosine (FIAC) or 1 (2 '-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base) 5-iodouracil (FIAU).In order to treat HIV, composition can comprise antiviral nucleosides, for example 3 '-azido--3 '-deoxythymidine (AZT).
The present invention also provides promoting agent has been imported method in one or more cells.The method according to this invention has prepared the composition that contains cationic cardiolipin phosphatide as herein described, and it also comprises promoting agent.Cell is contacted with reagent, make cell take in promoting agent, perhaps, can prepare the composition that does not contain promoting agent, exist under the situation of promoting agent, cell is contacted with composition, make cell take in promoting agent.
One or more cells can be vitro cell culture.When one or more cells when being external, by it is mixed mutually with substratum, can give cell with the delivery of composition that contains cationic cardiolipin phosphatide and/or promoting agent, perhaps, one or more cells can be that plant or animal (for example people) host are intravital.In this case, can preparation as described herein and delivering composition.
When promoting agent was polynucleotide, method of the present invention can be used to use the one or more cells of this polynucleotide transfection.This method can be used for external ground transfectional cell (for example at culture), perhaps gives delivering therapeutic or the diagnostic polynucleotide in ground in the cell paste.For example, according to the gene therapy scheme, this method can be used for gene is flowed to cells in culture or patient.For therapeutic is used, the present invention thereby gene therapy method is provided, it comprises that the patient to the needs treatment uses the pharmaceutical composition that comprises one or more nucleic acid, wherein said composition comprises cationic cardiolipin phosphatide.In this respect, polynucleotide can be can encoding gene expression construct, this gene after the method according to this invention transfection at cell inner expression.In order to express, such gene construct comprises that ideally suitable regulatory element promotes to express.Therefore, except encoding sequence, expression construct typically comprises the promotor that is operably connected on the encoding sequence.In addition, encoding sequence can comprise other regulatory element, for example ribosome entry site(RES), enhanser etc.The structure of expression construct is the common sense of this area.
The application of expection comprises the method for and use amphiphilic lipids known now with those and uses conventional cation lipid technology and the corresponding transfection method of method that described amphiphilic lipids comprises commercial cation lipid preparation, for example Lipofectin TMTherefore, lipid composition disclosed herein can be used to promote the to encode DNA of polypeptide of therapeutic activity or the iuntercellular of mRNA sequence carried (Felgner etc., U.S. Patent number 5,459,127).They can be used to the liposome of gene product, polypeptide or the oneself protein of expressing similarly and carry.Therefore, the DNA of cation lipid mediation and mRN polynucleotide or proteic conveying can provide the treatment of genetic diseases, it by shortage is provided or non-existent gene product treat any genetic diseases and carry out, dcc gene and/or its product have wherein been identified, for example duchenne's dystrophy (Kunkel etc. 1989).
By using transfection method recited above and test kit with being injected in cation lipid and DNA, RNA or the direct body of albumen in the cell of animal.But verified recently, cation lipid promotes the cells in vitro transfection especially effectively.Therefore, by using the cation lipid carrying method, some cells of external ground transfection animal, and cell imported in the animal again, can carry out top treatment alternatively.Thereby the ability of the transfectional cell expeditiously of cation lipid provides immune alternative method.Conveying by the cation lipid mediation can import antigenic gene from the cell that animal is taken out.The transfectional cell of present energy antigen expressed is injected in the animal again, and immunity system wherein can be reacted to endogenic antigen now.By being total to the gene of injection adjuvant or lymphokine or such lymphokine of encoding, further stimulate lymphocyte, can strengthen this process (Felgner etc., U.S. Patent number 5,459,127).
The present invention also provides the test kit that the polynucleotide transfection is advanced cell.This test kit comprises cationic cardiolipin phosphatide as herein described, and it can also comprise the ideal polynucleotide that are used for transfection.When existing, polynucleotide can be to pack dividually or packaging together with cationic cardiolipin phosphatide, for example in preparation as herein described.This test kit can also comprise about using this test kit to carry out the specification sheets of transfection.Specification sheets can comprise, for example, and about polynucleotide and cationic cardiolipin phosphatide being mixed with the explanation of the preparation that can be used for transfectional cell.This test kit can also include the reagent that is beneficial to transfection, for example buffer reagent, substratum etc.In addition, this test kit can comprise the container that is used to store cationic cardiolipin phosphatide, is used to store the container of reagent, is used for the container of the preparation of storage bag cation Val and polynucleotide, or is used to prepare the container of preparation.In addition, this test kit can include the material that is beneficial to transfection, for example suction pipe or Straw cap, culture dish or bottle or other suitable material.
The invention still further relates to method to the cell delivery promoting agent.Comprise promoting agent and the liposome by aforesaid method synthetic cationic cardiolipin phosphatide variant/analogue by preparation, can realize this method.Then liposome is flowed to cell.This also can finish by liposome is added in the cell culture medium.
The following examples have further been explained the present invention, but certainly, not should be understood to limit by any way its scope.
Embodiment
Synthetic [Fig. 1] of embodiment 1. cationic cardiolipin phospholipid analogues (4)
(R, S)-1,2-two-(tetradecyl oxygen base)-3-dimethylaminopropanecompounds (2)
Sodium hydride (60% in three neck round-bottomed flasks, in oil) (6.7g, 167.92mmol) and the suspension of DMF (100mL) in, dropwise added 1-dimethylamino-2 through 1.5 hours at 0 ℃, ammediol (1) (5g, 41.98mmol) DMF (25mL) solution, make it reach room temperature then, and stirred 1 hour.In reaction mixture, dropwise added through 90 minutes four decyl bromides (46.5g, 167.27mmol).Make it reach room temperature then, and stirred 1 hour.Make the temperature of reaction mixture rise to 82 ℃ gradually, and stirred 24 hours in this temperature.Reaction mixture is cooled to 0 ℃, adds several icy water very lentamente, water (750mL) dilutes this mixture then.With hexane extraction waterbearing stratum several (6 * 150mL).Organic layer is through dried over sodium sulfate.Use the hexane solution of 20% ethyl acetate,, obtain 1 of colorless oil, 2-two-(tetradecyl oxygen base)-3-dimethylaminopropanecompounds (2) (13g, 62%) (Wheeler etc. 1987) through silicon gel (70-230 order) column chromatography purification crude compound.TLC (SiO 2) methyl alcohol/chloroform (1: 9) Rf～0.51 1H NMR (CDCl 3, 500MHz): δ 0.88 (t, J=6.8Hz, 6H), 1.25 (s, 44H), 1.54-1.56 (m, 4H), 2.25 (s, 6H, N-CH 3), 2.26-2.41 (m, 2H, CH 2N), and 3.43-3.52 (m, 7H).
(R, S)-1,3-two-(1,2-tetracosyl oxygen base propyl group-3-N, N, N-Dimethyl Ammonium bromide) propane-2- Alcohol (4)
With 1, (4.3g, 84.0mmol) with 1, (0.73g, 3.36mmol) solution in dehydrated alcohol (100mL) (Bhattacharya etc. 1999) refluxed 5 days 3-glycerin dibromohydrin (3) 2-two-(tetradecyl oxygen base)-3-dimethylaminopropanecompounds (2).With the reaction mixture cooling, evaporating solvent obtains waxy stone shape solid.Crude compound is dissolved in the hexane (200mL), and, remains on 0 ℃ and spend the night stirring at room 6 hours.The solid of filtering separation, and with hexane wash (8 * 10mL), to remove raw material 1,2-two-(tetradecyl oxygen base)-3-dimethylaminopropanecompounds.Crude compound is carried out column chromatography (silica gel, 70-230 order), with the dichloromethane solution wash-out of 1-6% methyl alcohol, to obtain the cationic cardiolipin phospholipid analogues (4) (3.2g, 77%) of white solid.TLC (SiO 2) methyl alcohol/chloroform (1: 9) Rf～0.11. 1HNMR (CDCl 3, 500MHz): δ 0.88 (t, J=6.8Hz, 12H), 1.24-1.31 (m, 88H), 1.56-1.60 (m, 8H), and 3.42-3.68 (m, 25H), 3.79-3.85 (m, 4H), 4.39 (t, J=6.8Hz, 2H), and 4.53-4.60 (m, 2H), 5.18-5.24 (m, 1H), 6.50-6.61 (m, 1H). 13C?NMR(CDCl 3，125MHz)：δ14.03，22.60，29.28，29.35，29.37，29.39，29.52，29.55，29.57，29.58，29.61，29.62，29.98，30.0，31.84，52.74，52.89，53.16，54.85，54.89，55.01，62.28，62.57，65.02，68.53，68.63，68.88，69.29，69.39，69.80，70.02，72.02，72.82，72.87.IR(cm -1)：3407(br)，3223(br)，2956(s)H，2920(s)，1633，1467，1377，1116，971，893，720.ESI-MS?1162.5[M+1-Br]，1080.6[M+1-2Br]，540.7[M+1-2Br/2]。Molecular formula C 69H 144Br 2N 2O 5Ultimate analysis; Calculated value C:66.74, H:11.69, N:2.26, Br:12.87; Measured value C:65.85, H:11.52, N:2.31, Br:13.21.
Synthetic [Fig. 2] of embodiment 2. cationic cardiolipin phospholipid analogues (11)
1,3 two [(2-oxyethyl group tetrahydrochysene-2H-pyrans)]-2-benzyloxy-glycerine (7)
Under argon atmospher, at 0 ℃, to the sodium hydride (59.3g, the 1.48mol that stir, 60%, in oil) in the suspension in dry DMF (300mL), added 2-benzyloxy 1 through 2 hours, ammediol (5) (90g, DMF 0.49mol) (700mL) solution, with temperature maintenance below 15 ℃.In stirring at room after 2 hours, 0 ℃, added through 3 hours 2-(2-bromine oxethyl) tetrahydrochysene-2H-pyrans (6) (310g, 1.48mol), with temperature maintenance below 10 ℃.With reaction mixture stirring at room 12 hours.Reaction mixture is cooled to 0 ℃, adds frozen water very lentamente, with the unnecessary sodium hydride of cancellation.Concentrated reaction mixture under reduced pressure, to remove the DMF of maximum, water (1L) dilutes thick solution, with ethyl acetate (2 * 500mL) extractions.With aqueous saturated sodium-chloride (500mL) washing organic layer, through dried over sodium sulfate.Concentrated solvent under reduced pressure.Use the hexane solution of 10-30% ethyl acetate, by silica gel (230-400 order) column chromatography purification crude compound, obtain 1 of colorless oil, 3-two-[(2-oxyethyl group tetrahydrochysene-2H-pyrans)]-2-benzyloxy-glycerine (7) (154g, 71%).TLC (SiO 2) hexane/ethyl acetate (3: 2) Rf～0.40. 1H?NMR(CDCl 3，300MHz)：δ1.41-1.82(m，12H)，3.41-3.98(m，17H)，4.61(brs，2H)，4.78(s，2H，OCH 2Ph)，7.24-7.45(m，5H，Ph-H)。
3,7-two oxa-s-5-benzyloxy-1,9-nonanediol (8)
To 1,3-two [(2-oxyethyl group tetrahydrochysene-2H-pyrans)]-2-benzyloxy-glycerine (7) (50g in methyl alcohol 0.11mol) (500mL) solution, adds ether (5mL) solution of 1N HCl, and stirring at room 2 hours.With sodium bicarbonate neutralization reaction mixture, become neutrality up to solution.Filter reaction mixture under reduced pressure concentrates.Crude compound is dissolved in the ethyl acetate (1L), water (100mL) washing, and through dried over sodium sulfate.Under reduced pressure concentrate organic layer,, use eluent ethyl acetate through silicon gel (70-230 order) column chromatography purification crude compound, use the ethyl acetate solution wash-out of 5% methyl alcohol subsequently, obtain 3 of colorless oil, 7-two oxa-s-5-benzyloxy-1,9-nonanediol (8) (27g, 88%).TLC (SiO 2) ethyl acetate Rf～0.10. 1H NMR (CDCl 3, 300MHz): δ 3.01 (brs, 2H, OH), 3.50-3.81 (m, 13H), 4.68 (s, 2H, OCH 2Ph), and 7.21-7.42 (m, 5H, Ph-H).
1,9-two bromo-3,7-two oxa-s-5-benzyloxy-nonane (9)
Under argon atmospher, at 0 ℃, to 3,7-two oxa-s-5-benzyloxy-1,9-nonanediol (8) (27g, in anhydrous methylene chloride 0.1mol) (400mL) solution, add triphenyl phosphine (65.5g, 0.25mol), add then carbon tetrabromide (79.4g, 0.24mol).Reaction mixture was stirred 2 hours at 0 ℃.Water (300mL) diluted reaction mixture separates organic layer, through dried over sodium sulfate.Under reduced pressure concentrate organic layer, the hexane solution that uses 20% ethyl acetate obtains 1 of colorless oil through silicon gel (70-230 order) column chromatography purification crude compound, 9-two bromo-3,7-two oxa-s-5-benzyloxy-nonane (9) (36g, 91%).TLC (SiO 2) hexane/ethyl acetate (3: 2) R f～0.60. 1H?NMR(CDCl 3，300MHz)：δ3.45(t，J＝5.5Hz，4H，CH 2Br)，3.60-3.67(m，4H，OCH 2)，3.72-3.82(m，5H)，4.70(s，2H，OCH 2Ph)，7.28-7.38(m，5H，Ph-H)。
1,3-two-(2-bromine oxethyl) propane-2-alcohol (10)
Figure A20038010611600441
With 1,9 two bromo-3, (36g 90.90mmol) is dissolved in the ethanol (110mL) 7-two oxa-s-5-benzyloxy-nonane (9), under the pressure of 50psi, carries palladium (3.6g) hydrogenation 2 hours with 10% carbon.After leaching catalyzer, vapourisation under reduced pressure solution.Thick material is carried out silica gel column chromatography (70-230 order), and the hexane solution wash-out with 60% ethyl acetate obtains 1 of colorless oil, 3-two-(2-bromine oxethyl) propane-2-alcohol (10) (26g, 94%).TLC (SiO 2) hexane/ethyl acetate (1: 1) R f～0.30. 1HNMR(CDCl 3，300MHz)：δ2.56(d，J＝4.5Hz，1H，OH)，3.45(t，J＝6.0Hz，4HCH 2Br)，3.54-3.64(m，4H，OCH 2)，3.82(t，J＝5.7Hz，4H，OCH 2)，3.96-4.02(m，1H)。
(R, S)-1,3-two (1,2-tetracosyl oxygen base propyl group-3-N, N-dimethyl-3-oxyethyl group ammonium bromide) propane-2-alcohol (11)
Figure A20038010611600442
At 78-80 ℃, with 1, (50.2g, 98.36mmol) with 1, (10g, dehydrated alcohol 32.78mmol) (600mL) solution refluxed 5 days 3-two-(2-bromine oxethyl) propane-2-alcohol (10) 2-two-tetradecyl oxygen base-3-dimethylaminopropanecompounds (2).With the reaction mixture cooling, evaporating solvent obtains waxy stone shape solid.Add hexane (400mL), and stirred 1 hour.Leach solid, with hexane (6 * 100mL) washings.By recrystallization [ratio of compound/methanol/acetone is (1: 3: 50)] purifying compounds, remain on-20 ℃ and spend the night.The solid of filtering separation washs with cold acetone.Repeated recrystallization 2 times obtains analytically pure sample.Dry compound is 24 hours under the vacuum, then through P 2O 536 hours, obtain the cationic cardiolipin phospholipid analogues (11) (30g, 69%) of white solid.TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.11. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，12H)，1.25(s，88H)，1.52-1.71(m，8H)，3.41-3.68(m，29H)，3.95-4.19(m，14H)，4.66(brs，1H)。 13C?NMR(CDCl 3，125MHz)：δ13.92，22.50，25.89，26.03，29.12，29.27，29.32，29.42，29.48，29.50，29.54，29.87，31.74，52.95，53.02，53.50，65.02，65.07，66.57，68.63，68.84，69.15，71.80，72.65，73.29。IR(cm -1)：3323(br)，2918(s)，2873(s)，1468(s)，1123(brs)。ESI-MS?1248.6[M+1-Br]，584.3[M+1-2Br/2]。Molecular formula C 73H 152Br 2N 2O 7Ultimate analysis; Calculated value C:65.93, H:11.52, N:2.11, Br:12.02; Measured value C:65.93, H:11.40, N:2.11, Br:11.99.
Synthetic [Fig. 3] of embodiment 3. cationic cardiolipin phospholipid analogues (19)
(R)-and 4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane (13).
Figure A20038010611600451
Under argon atmospher, at 0 ℃, to the sodium hydride (30.3g that stirs, 0.75mol, 60%, in oil) the suspension of anhydrous tetrahydro furan (500mL) in, added R (-)-2 through 1 hour, 2-dimethyl-1, (50g 0.37mol), keeps internal temperature and is lower than 20 ℃ 3-dioxolane-4-methyl alcohol (12).In stirring at room after 1 hour, 0 ℃, added through 1 hour bromotoluene (97.1g, 0.56mol).Add finish after, with reaction mixture stirring at room 14 hours.Reaction mixture is cooled to 0 ℃, adds cold water very lentamente, (500mL) dilutes this mixture with aqueous saturated ammonium chloride.With ethyl acetate (500mL) extraction waterbearing stratum, water (300mL) washing.Under reduced pressure concentrate organic layer, the crude product (121g) that obtains pulpous state (R)-4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane (13).TLC (SiO 2) hexane/ethyl acetate (1: 9) R fNot～0.26 (not purified, that crude product is carried out next step operation) 1H NMR (CDCl 3, 300MHz): δ 1.39 (s, 3H, CH 3), 1.40 (s, 3H, CH 3), 3.41-3.55 (m, 2H), 3.68-3.74 (m, 1H), 4.01-4.05 (m, 1H), 4.20-4.31 (m, 1H), 4.55 (brs, 2H, OCH 2Ph), and 7.25-7.35 (m, 5H, Ph-H).
(S)-(-)-and 3-benzyloxy-1,2-propylene glycol (14)
To (R)-4-benzyloxymethyl-2, in 2-dimethyl-1,3-dioxolane (13) methyl alcohol (700mL) solution (120g), add dense HCl (20mL), and stirring at room 10 hours.With sodium bicarbonate neutralization reaction mixture, become neutrality up to solution.Filter reaction mixture, and under reduced pressure concentrate.Crude compound is dissolved in methylene dichloride (600mL), separates organic layer, and through dried over sodium sulfate.Decompression is concentrated organic layer down.The hexane solution that uses the 10-20% ethyl acetate is as eluent, use the ethyl acetate solution of 10% methyl alcohol subsequently, through silicon gel (230-400 order) column chromatography purification crude compound, obtain (S)-(-)-3-benzyloxy-1 of pulpous state, 2-propylene glycol (14) (64g, 93%).TLC (SiO 2) ethyl acetate R f～0.44. 1H?NMR(CDCl 3，300MHz)：δ3.42-3.61(m，4H)，3.79-3.83(m，3H)，4.47(s，2H，OCH 2Ph)，7.23-7.32(m，5H，Ph-H)。
(S)-1,2-two-tetradecyl oxygen base-3-O-benzyl propane (15).
Under argon atmospher, at 0 ℃, to the sodium hydride (54.5g that stirs, 1.36mol, 60%, in oil) dry DMF (220mL) suspension in, added (S)-(-)-3-benzyloxy-1 through 1 hour, 2-propylene glycol (14) (62g, 0.34mol) DMF (400mL) solution, keep internal temperature and be lower than 20 ℃.In stirring at room after 2 hours, 0 ℃ added through 2 hours the tetradecyl bromine (377.4g, 1.36mol).Add finish after, stirring at room reaction mixture 2 hours, make temperature be increased to 70 ℃ gradually, stirred then 5 hours.Reaction mixture is cooled to 0 ℃, adds cold water very lentamente, and dilute with aqueous saturated ammonium chloride (500mL).With ethyl acetate (1L) extraction waterbearing stratum, and water (3 * 1L) washings.Under reduced pressure concentrate organic layer, through silicon gel (70-230 order) column chromatography purification crude product, use the hexane solution wash-out of 2-10% ethyl acetate, to obtain (S)-1 of colorless oil, 2-two-tetradecyl oxygen base-3-O-benzyl propane (15) (146g, 75%).TLC (SiO 2) hexane/ethyl acetate (1: 9) R f～0.53. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，6H)，1.25(brs，44H)，1.49-1.58(m，4H)，3.39-3.60(m，9H)，4.54(m，2H，OCH 2Ph)，7.23-7.32(m，5H，Ph-H)。
(R)-1,2-two-tetradecyl oxygen base-propane-3-alcohol (16)
Figure A20038010611600471
With (S)-1, (70g, 0.12mmol) solution is dissolved in the ethyl acetate (280mL) 2-two-tetradecyl oxygen base-3-O-benzyl propane (15), under the pressure of 50psi, with 10% palladium (3g) hydrogenation 12 hours.After leaching catalyzer, vapourisation under reduced pressure solution.Resistates is dissolved in the hot ethanol (500mL), remains on-20 ℃ and spend the night.The solid of filtering separation, and vacuum-drying are to obtain (R)-1 of white solid, 2-two-tetradecyl oxygen base-propane-3-alcohol (16) (54g, 92%) .TLC (SiO 2) hexane/ethyl acetate (1: 9) R f～0.17. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，6H)，1.25(brs，48H)，1.59-1.57(m，4H)，2.2(t，J＝5.7Hz，1H，OH)，3.37-3.73(m，9H)。
(S)-1,2-two-tetradecyl oxygen base-3-N-PROPYLE BROMIDE (17)
Under argon atmospher, at 0 ℃, to (R)-1,2-two-tetradecyl oxygen base-propane-3-alcohol (16) (52g, in anhydrous methylene chloride 0.1mol) (280mL) solution, the adding triphenyl phosphine (35.1g, 0.13mol).(46.2g, methylene dichloride 0.13mol) (240mL) solution further stirred 3 hours at 0 ℃ dropwise to add carbon tetrabromide through 1 hour in this reaction mixture.Water (500mL) diluted reaction mixture separates organic layer, through dried over sodium sulfate.Under reduced pressure concentrate organic layer, use the hexane solution of 1-5% ethyl acetate, through silicon gel (230-400 order) column chromatography purification crude compound, to obtain (S)-1 of colorless oil, 2-two-tetradecyl oxygen base-3-N-PROPYLE BROMIDE (17) (53g, 90%).TLC (SiO 2) hexane/ethyl acetate (1: 9) R f～0.72. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，6H)，1.25(brs，48H)，1.51-1.61(m，4H)，3.39-3.61(m，9H)。
(R) 1,2-two-tetradecyl oxygen base-3-dimethylaminopropanecompounds (18)
In the screw-cap pressure bottle, with (S)-1, (50g 0.09mol) is dissolved in the methanol solution (400mL) of 2M dimethylamine 2-two-tetradecyl oxygen base-3-N-PROPYLE BROMIDE (17).The sealing load bottle is in the oil bath of stirring, 88-90 ℃ of heating 60 hours.Cooling pressure bottle, and concentrated solution under reduced pressure.Thick resistates is dissolved in the ethyl acetate (500mL), and water (500mL) washing.Under reduced pressure concentrate organic layer, the hexane solution that uses the 5-20% ethyl acetate through silicon gel (230-400 order) column chromatography purification, obtains light buttery (R)-1 as eluent, 2-two-tetradecyl oxygen base-3-dimethylaminopropanecompounds (18) (41g, 88%).TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.51. 1HNMR(CDCl 3，300MHz)：δ0.88(t，J＝6.8Hz，6H)，1.25(s，44H)，1.51-1.58(m，4H)，2.25(s，6H，N-CH 3)，2.37(t，J＝4.6Hz，2H，N-CH 2)，3.41-3.62(m，7H)。
(R) 1,3-two-(1,2-tetracosyl oxygen base propyl group-3-N, N-dimethyl-3-oxyethyl group ammonium bromide) propane-2-alcohol (19)
Figure A20038010611600482
With (R)-1, (35.6g, 69.8mmol) with 1, (7.1g, dehydrated alcohol 23.2mmol) (430mL) solution refluxed 5 days at 78-80 ℃ 3-two-(2-bromine oxethyl) propane-2-alcohol (10) 2-two-tetradecyl oxygen base-3-dimethylaminopropanecompounds (18).Thermal reaction mixture is transferred in the Erlenmeyer flask, and dropwise added acetone (4.3L) through 2 hours.Mixture being remained on-20 ℃ spends the night.Leach solid,, obtain pure white (colorless white) solid (28g) with cold acetone (500mL) washing.By at warm methyl alcohol (140mL): recrystallization comes the thick solid of purifying in the mixture of acetone (1.4L), is stored in-20 ℃ then and spends the night.Separate, cross filter solid, with cold acetone (300mL) washing.Repeated recrystallization 2 times obtains analytically pure sample.Dry compound 24 hours, then under vacuum through P 2O 536 hours, obtain (R)-cationic cardiolipin phospholipid analogues (19) (24g, 78%) of white solid.TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.13. 1H?NMR(CDCl 3，500MHz)：δ0.88(t，J＝6.7Hz，12H)，1.25(s，88H)，1.52-1.71(m，8H)，3.41-3.68(m，30H)，3.95-4.19(m，13H)，4.63(brs，1H，OH)。 13C?NMR(CDCl 3，125MHz)：δ13.94，22.52，25.90，26.04，29.20，29.28，29.33，29.44，29.50，29.52，29.55，29.88，31.76，52.99，53.07，53.49，65.01，65.07，66.56，68.66，68.84，69.17，71.82，72.62，72.64，73.30，73.28.IR(cm -1)：3409(br，OH)，2918(s)，2873(s)，1468(s)，1124(br?s)。ESI-MS 1248.5[M+1-Br], 584.2[M+1-2Br/2] molecular formula C 73H 152Br 2N 2O 7Ultimate analysis; Calculated value C:65.93, H:11.52, N:2.11, Br:12.02; Measured value C:65.65, H:11.49, N:2.13, Br:12.17.
Synthetic [Fig. 4] of embodiment 4. cationic cardiolipin phospholipid analogues (27)
(S)-and 4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane (21)
Figure A20038010611600491
Under argon atmospher, at 0 ℃, to the sodium hydride (22.7g that stirs, 0.57mol, 60%, in oil) the suspension of anhydrous tetrahydro furan (350mL) in, added S (-)-2 through 1 hour, 2-dimethyl-1, (50g 0.37mol), keeps internal temperature and is lower than 20 ℃ 3-dioxolane-4-methyl alcohol (20).In stirring at room after 1 hour, 0 ℃ added through 1 hour the benzyl bromide (97.1g, 0.56mol).Add finish after, stirring at room reaction mixture 18 hours.Reaction mixture is cooled to 0 ℃, adds several frozen water (20mL) very lentamente, (500mL) dilutes this mixture with aqueous saturated ammonium chloride.With ethyl acetate (750mL) extraction waterbearing stratum.Under reduced pressure concentrate organic layer, obtain (S)-4-benzyloxymethyl-2 of pulpous state, 2-dimethyl-1,3-dioxolane (21) crude product (83g).TLC (SiO 2) hexane/ethyl acetate (1: 9) R f～0.26.
(R)-(-)-and 3-benzyloxy-1,2-propylene glycol (22)
Figure A20038010611600501
To (S)-4-(benzyloxymethyl)-2, in the solution of 2-dimethyl-1,3-dioxolane (21) methyl alcohol (800mL) (83g), add dense HCl (20mL), and stirring at room 15 hours.With sodium bicarbonate neutralization reaction mixture, become neutrality up to solution.Filter reaction mixture, and under reduced pressure concentrate.Crude compound is dissolved in the methylene dichloride (200mL), separates organic layer,, under reduced pressure concentrate through dried over sodium sulfate.The hexane solution that uses the 10-20% ethyl acetate is as eluent, use the ethyl acetate solution of 10% methyl alcohol subsequently,, obtain (R)-(-)-3-benzyloxy-1 of pulpous state through silicon gel (230-400 order) column chromatography purification crude compound, 2-propylene glycol (22) (65g, 94%).TLC (SiO 2) ethyl acetate R f～0.44. 1H?NMR(CDCl 3，300MHz)：δ3.42-3.61(m，4H)，3.75-3.99(m，3H)，4.47(s，2H，OCH 2Ph)，7.22-7.32(m，5H，Ph-H)。
(R)-1,2-two-tetradecyl oxygen base-3-O-benzyl propane (23)
Under argon atmospher, at 0 ℃, to the sodium hydride (38.4g that stirs, 0.96mol, 60%, in oil) the suspension of dry DMF (200mL) in, added (R)-(-)-3-benzyloxy-1,2-propylene glycol (22) (35g through 1 hour, 0.19mol) DMF (150mL) solution, keep internal temperature and be lower than 20 ℃.In stirring at room after 2 hours, 0 ℃ added through 2 hours the tetradecyl bromide (266g, 0.97mol).Add finish after, stirring at room 2 hours, make temperature be increased to 60 ℃ gradually reaction mixture, stirred then 20 hours.Reaction mixture is cooled to 0 ℃, adds several frozen water very lentamente, and dilute with aqueous saturated ammonium chloride (500mL).With hexane (3 * 500mL) extraction waterbearing stratums, and water (500mL) and salt solution (500mL) washing.Under reduced pressure concentrate organic layer,, use the hexane solution wash-out of 2-10% ethyl acetate, obtain (R)-1 of colorless oil, 2-two-tetradecyl oxygen base-3-O-benzyl propane (23) (88g, 80%) through silicon gel (70-230 order) column chromatography purification crude product.TLC (SiO 2) hexane/ethyl acetate (1: 9) R f～0.53. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.6Hz，6H)，1.25(brs，44H)，1.49-1.58(m，4H)，3.39-3.60(m，9H)，4.54(m，2H，OCH 2Ph)，7.23-7.32(m，5H，Ph-H)。
(S) ,-1,2-two-tetradecyl oxygen base-propane-3-alcohol (24)
With (R)-1, (83.8g 0.14mol) is dissolved in the ethyl acetate (450mL) 2-two-tetradecyl oxygen base-3-O-benzyl propane (23), under the pressure of 50psi, carries palladium (3.1g) hydrogenation 12 hours with 10% carbon.After leaching catalyzer, vapourisation under reduced pressure solution.Resistates is dissolved in the hot hexane (200mL), remains on-20 ℃ and spend the night.The solid of filtering separation, drying obtains (S)-1 of white solid, 2-two-tetradecyl oxygen base-propane-3-alcohol (24) (64.8g, 92%).TLC (SiO 2) hexane/ethyl acetate (1: 9) R f～0.17. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，6H)，1.25(brs，44H)，1.51-1.57(m，4H)，2.22(t，J＝5.2Hz，1H，OH)，3.40-3.73(m，9H)。
(R)-1,2-two-tetradecyl oxygen base-3-N-PROPYLE BROMIDE (25)
Figure A20038010611600512
Under argon atmospher, at 0 ℃, to (S)-1,2-two-tetradecyl oxygen base-propane-3-alcohol (24) (36.8g, in the solution of anhydrous methylene chloride 59.0mmol) (375mL), the adding triphenyl phosphine (27.9g, 106.0mmol).In 1 hour time, dropwise add carbon tetrabromide (36.2g, methylene dichloride 109.0mmol) (60mL) solution to reaction mixture.0 ℃ of further stirred reaction mixture 2 hours.Water (3 * 300mL) dilutions then.Separate organic layer,, under reduced pressure concentrate through dried over sodium sulfate.Use the hexane solution of 1-5% ethyl acetate, through silicon gel (230-400 order) column chromatography purification crude compound, obtain (R)-1 of colorless oil, 2-two-tetradecyl oxygen base-3-N-PROPYLE BROMIDE (25) (37.8g, 87%).TLC (SiO 2) hexane/ethyl acetate (1: 9) R f～0.72. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，6H)，1.25(brs，44H)，1.50-1.60(m，4H)，3.40-3.68(m，9H)。
(S)-1,2-two-tetradecyl oxygen base-3-dimethylaminopropanecompounds (26)
Figure A20038010611600521
In the screw-cap pressure bottle, with (R)-1, (36.7g 0.07mol) is dissolved in the methanol solution (450mL) of 2M dimethylamine 2-two-tetradecyl oxygen base-3-N-PROPYLE BROMIDE (25).The sealing load bottle, at 92 ℃, heating is 75 hours in the oil bath of stirring.Cooling pressure bottle, and concentrated solution under reduced pressure.Thick resistates is dissolved in the ethyl acetate (500mL), and washes (500mL) with water.Under reduced pressure concentrate organic layer, the hexane solution that uses the 5-20% ethyl acetate through silicon gel (230-400 order) column chromatography purification, obtains light buttery (S)-1 as eluent, 2-two-tetradecyl oxygen base-3-dimethylaminopropanecompounds (26) (28.7g, 80%).TLC (SiO 2) methyl alcohol/chloroform (2: 8) R f～0.66. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，6H)，1.25(s，44H)，1.51-1.57(m，4H)，2.15，2.25(s，6H，N-CH 3)，2.34-2.36(m，2H，N-CH 2)，3.38-3.61(m，7H)。
(S)-1,3-two-(1,2-tetracosyl oxygen base propyl group-3-N, N-dimethyl-3-oxyethyl group ammonium bromide) propane-2-alcohol (27)
Figure A20038010611600522
With (S)-1, (22.5g, 43.9mmol) with 1, (4.48g, dehydrated alcohol 14.6mmol) (220mL) solution refluxed 5 days at 78-80 ℃ 3-two-(2-bromine oxethyl) propane-2-alcohol (10) 2-two-tetradecyl oxygen base-3-dimethylaminopropanecompounds (26).Hot solution is transferred in the Erlenmeyer flask, dropwise added acetone (2.5L), simultaneously stirred solution.Bottle is stored 15 hours in refrigerator (25 ℃).Leach white solid, with cold acetone (100mL) washing.Product is dissolved in the chloroform (100mL), adds acetone (1L).Bottle is stored 15 hours at-25 ℃.The white solid of filtering separation is with cold acetone washing (100mL).Repeated recrystallization process 3 times.Product with hexane (1L) grind into powder, is filtered, and under high vacuum through P 2O 5Dry 24 hours, obtain (S)-cationic cardiolipin phospholipid analogues (27) (15.5g, 80%) of white solid.TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.13. 1HNMR(CDCl 3，300MHz)：δ0.88(t，J＝6.6Hz，12H)，1.25(s，88H)，1.52-1.71(m，8H)，3.40-3.71(m，36H)，3.92-4.11(m，7H)，4.74(d，J＝5.7Hz，1H，OH)。 13CNMR(CDCl 3，125MHz)：δ13.91，22.48，25.86，26.0，29.16，29.24，29.30，29.40，29.46，29.48，29.51，29.84，31.72，52.93，53.01，53.46，64.98，66.51，86.63，68.8，69.12，71.77，72.61，73.26.IR(cm -1)：3397(br，OH)，2917(s)，1467(s)，1122(br?s)。ESI-MS?1248.5[M+1-Br]，584.4[M+1-2Br/2]。
Synthetic [Fig. 5] of embodiment 5. cationic cardiolipin phospholipid analogues (32)
2-[2-(2,3-two-tetradecyl oxygen base-propoxy-)-oxyethyl group]-tetrahydrochysene-pyrans (28)
Under argon atmospher, at 0 ℃, to the sodium hydride (3.3g that stirs, 82.6mmol, 60%, in oil) the suspension of anhydrous tetrahydro furan (50mL) in, in 2 hours, add 1,2-two (tetradecyl oxygen base) propane-3-alcohol (16) (20g, 41.3mmol) THF (150mL) solution, holding temperature is lower than 15 ℃.After 2 hours, tetrahydrochysene-(25.9g, 123mmol), holding temperature is lower than 10 ℃ to 2H-pyrans (6) to add 2-(2-bromine oxethyl) at 0 ℃ through 3 hours in stirring at room.With reaction mixture stirring at room 48 hours.Reaction mixture is cooled to 0 ℃, adds frozen water very lentamente, with the unnecessary sodium hydride of cancellation, and with aqueous saturated ammonium chloride (300mL) dilution, with ethyl acetate (2 * 150ml) extractions.Water (100mL) washing organic layer through dried over sodium sulfate, under reduced pressure concentrates.Use the hexane solution of 1-8% ethyl acetate,, obtain 2-[2-(2, the 3-two-tetradecyl oxygen base propyl group) oxyethyl group of colorless oil through silicon gel (230-400 order) column chromatography purification crude compound] tetrahydrochysene-pyrans (28) (16.3g, 66%).TLC (SiO 2) hexane: ethyl acetate (1: 4) RF～0.30. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，12H)，1.25(s，44H)，1.53-1.87(m，10H)，3.40-4.04(m，15H)，4.62-4.67(m，1H)。
2-(2,3-two-tetradecyl oxygen base-propoxy-)-ethanol (29)
Figure A20038010611600541
To 2-[2-(2,3-two-tetradecyl oxygen base propoxy-) oxyethyl group] tetrahydropyrans (28) (16g in methyl alcohol 26.75mmol) (500mL) solution, adds ether (5mL) solution of 1N HCl, and stirring at room 2 hours.With sodium bicarbonate neutralization reaction mixture, become neutrality up to solution.Filter reaction mixture, and under reduced pressure concentrate.Resistates is dissolved in the ethyl acetate (500mL), and water (200mL) washing, through dried over sodium sulfate.Under reduced pressure concentrate organic layer,, use the hexane solution wash-out of 10-15% ethyl acetate, obtain 2-(2, the 3-two-tetradecyl oxygen base propoxy-) ethanol (29) (10g, 71%) of colorless oil through silicon gel (230-400 order) column chromatography purification crude compound.TLC (SiO 2) hexane/ethyl acetate (1: 4) R f～0.18. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，12H)，1.25(s，44H)，1.51-161(m，4H)，2.58(t，J＝6Hz，OH)，3.41-3.71(m，13H)。
1-[1-(2-bromo-ethoxyl methyl)-2-tetradecyl oxygen base oxethyl] tetradecane (30)
Under argon atmospher, at 0 ℃, to 2-(2,3-two-tetradecyl oxygen base propoxy-) ethanol (29) (20g, 37.8mmol) the solution of anhydrous methylene chloride (200mL) in, add triphenyl phosphine (14.8g, 56.8mmol), add subsequently carbon tetrabromide (17.5g, 53.0mmol).Reaction mixture was stirred 2 hours at 0 ℃.Water (100mL) diluted reaction mixture separates organic layer, through dried over sodium sulfate.Under reduced pressure concentrate organic layer, the hexane solution of the ethyl acetate of use 5%, through silicon gel (230-400 order) column chromatography purification crude compound, obtain 1-[1-(2-bromine oxethyl the methyl)-2-tetradecyl oxygen base oxethyl of colorless oil] tetradecane (30) (17g, 76%).TLC (SiO 2) hexane/ethyl acetate (1: 5) R f～0.65. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，12H)，1.25(s，44H)，1.51-157(m，4H)，3.40-3.61(m，11H)，3.79(t，J＝6Hz，2H)。
1,3-two-(1,2-tetracosyl oxygen base-4-oxa--hexyl-6-N, N-Dimethyl Ammonium bromide) propane-2-alcohol (32)
Figure A20038010611600551
With 1-[1-(2-bromine oxethyl methyl)-2-tetradecyl oxygen base oxethyl]-tetradecane (30) (6g, 10.15mmol) and 1, (0.48g, dehydrated alcohol 3.38mmol) (65mL) solution refluxed 5 days at 78-80 ℃ 3-two (dimethylamino)-2-propyl alcohol (31).Hot solution is transferred in the Erlenmeyer flask, dropwise added acetone (650mL), simultaneously stirred solution.With bottle store overnight in refrigerator (25 ℃).Leach white solid, with cold acetone (100mL) washing.Solid is dissolved in the methylene dichloride, adds acetone (ratio 1: 10).With bottle-25 ℃ of store overnight.Filter white solid, with cold acetone washing (20mL).Repeated recrystallization process 2 times.Desciccate under the vacuum obtains the cationic cardiolipin phospholipid analogues (32) (0.82g, 18%) of white solid.TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.12. 1HNMRδ(CDCl 3，300MHz)：δ0.88(t，J＝6.8Hz，6H)，1.25(s，88H)，1.49-1.60(m，8H)，3.39-3.82(m，31H)，3.81-4.01(m，8H)，4.52(d，J＝12.6Hz，2H)，5.21-5.35(m，1H)。ESI-MS：1248.7[M+1-Br]，584.6[M+1-2Br -/2]。Molecular formula C 73H 152Br 2N 2O 7Ultimate analysis; Calculated value C:65.93, H:11.52, N:2.11, Br:12.02; Measured value C:64.46, H:11.34, N:2.21, Br:11.80.
Synthetic [Fig. 6] of embodiment 6. cationic cardiolipin phospholipid analogues (34)
[2-(2,3-two-tetradecyl oxygen base propoxy-) ethyl]-dimethylamine (33)
Figure A20038010611600561
In the screw-cap pressure bottle, with 1-[1-(2-bromine oxethyl methyl)-2-tetradecyl oxygen base oxethyl] tetradecane (30) (10g, 16.92mmol) methanol solution (100mL) of adding 2M dimethylamine.The sealing load bottle heated 60 hours at 88-90 ℃ in the oil bath of stirring.Cooling pressure bottle, and concentrated solution under reduced pressure.Crude compound is dissolved in the ethyl acetate (300mL), and water (100mL) washing.Under reduced pressure concentrate organic layer, the hexane solution that uses the 5-20% ethyl acetate is as eluent, and through silicon gel (230-400 order) column chromatography purification, [2-(2 to obtain light buttery, 3-two-tetradecyl oxygen base propoxy-) ethyl] dimethylamine (33) (8g, 85%).TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.46. 1HNMR(CDCl 3，300MHz)：δ0.88(t，J＝6.7Hz，12H)，1.25(s，44H)，1.52-1.56(m，4H)，2.17(s，6H，N-CH 3)，2.78(t，J＝5.8Hz，2H)，3.39-3.46(m，4H)，3.49-3.56(m，5H)，3.71(t，J＝5.4Hz，2H)。
1,3-two-(1,2-two-tetradecyl oxygen base-4,10-two oxa-s-decyl-7-N, N-Dimethyl Ammonium bromide) propane-2-alcohol (34)
With (R)-[2-(2,3-two-tetradecyl oxygen base propoxy-) ethyl] dimethylamine (33) (5g, 8.99mmol) and 1, (0.91g, dehydrated alcohol 2.99mmol) (60mL) solution refluxed 5 days at 78-80 ℃ 3-two-(2-bromine oxethyl) propane-2-alcohol (10).Thermal reaction mixture is transferred in the Erlenmeyer flask, in 2 hours, dropwise added acetone (600mL), remain on-20 ℃ and spend the night.Leach solid,, obtain pure white solid (5g) with cold acetone (100mL) washing.At warm methyl alcohol: the thick solid of recrystallization purifying in the acetone (1: 10 ratio) is stored in-20 ℃ then and spends the night.Separate, cross filter solid, with cold acetone washing (50mL).Repeated recrystallization 2 times obtains pure compound.Dry compound under vacuum obtains the cationic cardiolipin phospholipid analogues (34) (1.82g, 43%) of white solid.TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.13. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.9Hz，12H)，1.25(s，88H)，1.48-1.59(m，8H)，3.38-3.65(m，35H)，3.89-4.13(m，16H)，4.77(brs，1H，OH)。ESI-MS：1336.4[M+1-Br]，628.4[M+1-2Br -/2]。Molecular formula C 77H 160Br 2N 2O 9Ultimate analysis; Calculated value C:65.22, H:11.37, N:1.98, Br:11.27; Measured value C:63.94, H:11.28, N:2.03, Br:9.94.
Synthetic [Fig. 6] of embodiment 7. cationic cardiolipin phosphatide variant analogues (36)
(R, S)-1,3-two-(1,2-two-tetradecyl oxygen base propyl group-3-N, N-Dimethyl Ammonium bromide) butane-2,3-glycol (36)
With 1, and 2-two-(tetradecyl oxygen base)-3-dimethylaminopropanecompounds (2) (6g, 11.72mmol) with 1,4-two bromo-2, (1.16g, the solution of dehydrated alcohol 4.68mmol) (72mL) refluxed 7 days 3-butyleneglycol (35).With the reaction mixture cooling, evaporating solvent obtains waxy stone shape solid.Crude compound is dissolved in the hexane (200mL), and, maintains 0 ℃ and spend the night stirring at room 6 hours.The solid of filtering separation, and with hexane (8 * 10mL) washing, to remove raw material 1,2-two-(tetradecyl oxygen base)-3-dimethylaminopropanecompounds.Thick material is carried out column chromatography (silica gel, 70-230 order), use the dichloromethane solution wash-out of 1-10% methyl alcohol, obtain the cationic cardiolipin phosphatide variant analogue (36) (1.9g, 32%) of white solid.TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.07. 1H?NMR(CDCl 3，500MHz)：δ0.88(t，J＝6.8Hz，12H)，1.24-1.31(m，88H)，1.51-1.62(m，8H)，2.13(brs，2H，OH)，3.33-3.65(m，22H)，3.75-3.82(m，6H)，4.05-4.25(m，6H)，4.68(brs，2H)，4.39(d，J＝13Hz，2H)。 13C?NMR(CDCl 3，125MHz)：δ14.05，22.63，25.98，26.14，29.30，29.42，29.63，31.87，52.58，53.28，54.83，67.27，68.87，69.40，72.04，73.17。ESI-MS：1190.3[M+1-Br -]，555.3[M+1-2Br -/2]。Molecular formula C 71H 148Br 2N 2O 6Ultimate analysis; Calculated value C:66.32, H:11.60, N:2.18, Br:12.43; Measured value C:65.20, H:11.46, N:2.19, Br:12.24.
Synthetic [Fig. 7] of embodiment 8. cationic cardiolipin phosphatide variant analogues (38)
According to the 2-benzyl-1 of the described method preparation of U.S. Patent application PCT/US03/27806 such as () Ahmad, 3-two [(1,2-two myristoyl-sn-glyceryl-3)-phosphoryl] glycerine dicyano ethyl ester compound (37)
Under argon atmospher, to 1,2-myristyl-sn-glycerine (24) (9.2g, 19.01mmol) and N, N-diisopropyl ethyl amine (5.46g, 42.26mmol) the mixture of anhydrous ether (150mL) in, add 2-cyano ethyl di-isopropyl torak acid amides (2-cyanoethyldiisopropylchlorophosphoramidite) (5g, 21.13mmol).Mixture stirring at room 3 hours, is filtered diisopropylamine hydrochloride, concentrated filtrate under reduced pressure, the dry raw material is 1 hour under vacuum, obtains phosphoramidite (phosphoramidite) intermediate (13g), is used for next step phosphorylation like this.
The anhydrous CH of phosphoramidite upward and 1H-tetrazolium (0.45M solution is in acetonitrile for 1.59g, 22.77mmol) 2Cl 2In the mixture (80mL), add 2-benzyloxy 1, ammediol (5) (1.55g, CH 8.54mmol) 2Cl 2(20mL) solution.Reaction mixture stirring at room 3 hours, is cooled to-40 ℃, and by part ground add a tert-butyl hydroperoxide (6.8g, 75.91mmol).After 30 minutes, reaction mixture is heated to room temperature-40 ℃ of stirrings, uses CH 2Cl 2(200mL) dilution is with 5% aqueous NaHCO 3(50mL), salt solution (50mL) washing, through dried over sodium sulfate.Decompression is concentrated organic layer down.Resistates is gone up purifying at silica gel (230-400 order), uses the hexane solution wash-out of 50-75% ethyl acetate, obtains (37) (7.1g, 61%) of colourless pulpous state.TLC (SiO 2) ethyl acetate/hexane (3: 1) R f～0.29. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝7.0Hz，12H)，1.22-1.39(m，88H)，1.51-1.57(m，8H)，2.58-2.79(m，4H)，3.39-3.87(m，13H)，4.04-4.32(m，10H)，4.59-4.68(m，2H)，7.27-7.36(m，5H)。
1,3-two [(1,2-two-tetradecyl oxygen base-sn-glyceryl-3)-phosphoryl] glycerine two ammoniums muriate-n-propane ester (38)
Figure A20038010611600591
Under 50psi, with 2-benzyl-1,3-two-(0.74g, ethanol 0.53mmol) (25mL) solution is through Pd (OH) for [(1,2-myristyl-sn-glyceryl-3)-phosphoryl] glycerine dicyano ethyl ester (37) 2(210mg) hydrogenation is 24 hours.Through the bed of diatomaceous earth filtering catalyst, use washing with alcohol.Under reduced pressure concentrate alcohol layer, vacuum-drying is spent the night, and obtains cationic cardiolipin phosphatide variant analogue (38) 0.6g of pulpous state. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.8Hz)，1.24(brs，88H)，1.51-1.61(m，8H)，2.80-2.88(m，1H)，3.01-3.3.18(m，3H)，3.40-4.21(m，28H)。By in ether, adding 1N HCl solution, aminocompound is transformed into hydrochloride salt.
Synthetic [Fig. 8] of embodiment 9. cationic cardiolipin phosphatide ester analogs (44)
Toluene-4-sulfonic acid 2,2-dimethyl-[1,3] dioxolane-4-ylmethyl ester (39).
Figure A20038010611600592
To R (-)-2,2-dimethyl-1,3-dioxolane-4-methyl alcohol (12) (40g; 0.30mol) and the mixture of pyridine (250mL) in, 0 ℃, add through 1 hour portioning ground the tosyl group muriate (63.3g, 0.33mol); make it rise to room temperature then, and stirred 12 hours.Concentrated reaction mixture under reduced pressure, water (1.5L) dilution crude compound is with ethyl acetate (500mL) extraction.Organic layer under reduced pressure concentrates through dried over sodium sulfate, obtains toluene-4-sulfonic acid 2-[2-(2-methoxy ethoxy) oxyethyl group of light solid state] ethyl ester (39) is (87g).TLC (SiO 2) ethyl acetate/hexane (1: 4) R f～0.22. 1H?NMR(CDCl 3，300MHz)：δ1.29(s，3H)，1.32(s，3H)，2.44(s，3H，Ar-CH 3)，2.73(dd，J＝10.6Hz，1H)，3.97-4.0(m，3H)，4.22-4.30(m，1H)，7.35(dd，J＝8.4Hz，2H，Ar-H)，7.77(dd，J＝8.4Hz，2H，Ar-H)。
2,2-dimethyl-[1,3] dioxolane-4-ylmethyl dimethylamine (41).
Method 1: under argon atmospher, to (R)-(+)-2,2-dimethyl-1, (31g 0.23mol) and in anhydrous methanol (100mL) mixture of the methanol solution (70mL) of 2M dimethylamine, adds anhydrous sodium sulphate (50g) to 3-dioxolane-4-formaldehyde (40).In stirring at room mixture 2 hours.Reaction is cooled to 0 ℃, and portioning adding sodium borohydride (7.1g, 0.18mol), in stirred overnight at room temperature.Concentrated reaction mixture under reduced pressure.Crude compound is dissolved in the ethyl acetate (100mL), and water (100mL) washing.Under reduced pressure concentrate organic layer, the hexane solution that uses the 20-40% ethyl acetate is as eluent, through silicon gel (230-400 order) column chromatography purification, obtain light buttery 2,2-dimethyl-[1,3] dioxolane-4-ylmethyl dimethylamine (41) (7g, 19%).TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.45. 1H?NMR(CDCl 3，500MHz)：δ1.35(s，3H)，1.41(s，3H)，2.28(s，6H，N-CH 3)，2.35(dd，J＝12.5Hz，N-CH 2)，2.49(dd，J＝12.5Hz，N-CH 2)，3.58(dd，J＝8Hz，1H)，4.07(dd，J＝8Hz，1H)，4.20-4.25(m，1H)。
Method 2: in the screw-cap pressure bottle, with toluene-4-sulfonic acid 2-[2-(2-methoxy ethoxy) oxyethyl group] (50g 0.17mol) is dissolved in the methanol solution (400mL) of 2M dimethylamine ethyl ester (39).Pressure bottle was heated 48 hours at 88-90 ℃ in the oil bath of stirring.Cooling pressure bottle, and concentrated solution under reduced pressure.Crude compound is dissolved in the ethyl acetate (100mL), and water (100mL) washing.Decompression is concentrated organic layer down, obtains light buttery 2,2-dimethyl [1,3] dioxolane-4-base-methyl dimethoxy amine (41) (11g, 40%).
3-dimethylaminopropanecompounds-1,2-glycol (42)
To 2,2-dimethyl-[1,3] dioxolane-4-base-methyl dimethoxy amine (41) (5g in the solution of methyl alcohol 31.44mmol) (100mL), adds the ethereal solution (10mL) of 1M HCl, and stirring at room 7 hours.With sodium bicarbonate neutralization reaction mixture, become neutrality up to solution.Filter reaction mixture, and under reduced pressure concentrate.With warm ethyl acetate (5 * 100mL) extraction crude compounds.Decompression is concentrated organic layer down, obtains the 3-dimethylaminopropanecompounds-1 of light pulpous state, 2-glycol (42) (1.4g, 37%).TLC (SiO 2) methyl alcohol: chloroform (1: 4) R f～0.13. 1H?NMR(CDCl 3，300MHz)：δ2.23(dd，J＝5.1Hz，1H，N-CH 2)，2.38(s，6H，N-CH 3)，2.47-2.56(m，1H，N-CH 2)，3.49(dd，J＝11.2Hz，1H)，3.68-3.81(m，4H)。
1,2-two myristoyl-3-dimethylaminopropanecompounds (43)
Figure A20038010611600612
To ice-cooled (R)-(+)-3-dimethylaminopropanecompounds-1,2-glycol (42) (3g, 25.2mmol) and in the solution of pyridine (100mL), dropwise added through 30 minutes mnyristoyl chlorination thing (19.8g, 75.6mmol).Reaction mixture stirring at room 4 days, is under reduced pressure concentrated.Use CH 2Cl 2(200mL) dilution crude compound, water (100mL) and salt solution (100mL) washing are through dried over sodium sulfate.Under reduced pressure concentrate organic layer, use the hexane solution of 5%EtOAc, go up the purifying resistates, obtain (43) (2.9g, 21%) of light pulpous state at silicon gel (230-400 order).TLC (SiO 2) ethyl acetate R f～0.54. 1H?NMR(CDCl 3，300MHz)：δ0.88(t，J＝6.9Hz，6H)，1.25(brs，40H，-)，1.60(t，J＝6.9Hz，4H)，2.13(brs，2H，OH)，3.33-3.65(m，22H)，3.75-3.82(m，6H)，2.25(s，6H，N-CH 3)，2.30-2.33(m，1H，N-CH 2)，2.44(t，J＝6.3Hz，1H，N-CH 2)，4.08(dd，J＝11.8Hz，1H)，4.36(dd，J＝11.8Hz，1H)，5.15-5.23(m，1H)。
(R)-1,3-two (1,2-two myristoyl propyl group-3-N, N-dimethyl-3-oxyethyl group ammonium bromide) propane-2-alcohol (44)
Figure A20038010611600621
With 1, (4g, 7.42mmol) with 1, (0.75g, dehydrated alcohol 2.47mmol) (45mL) solution refluxed 5 days 3-two-(2-bromine oxethyl) propane-2-alcohol (10) 2-two-(tetradecyl oxygen base)-3-dimethylamine (43).With the reaction mixture cooling, evaporating solvent obtains waxy stone shape solid.Compound is carried out column chromatography (silicon gel, 70-230 order), use the dichloromethane solution wash-out of 0-10% methyl alcohol, obtain cationic cardiolipin phosphatide ester analogs (44) (67mg, 2%).TLC (SiO 2) methyl alcohol/chloroform (1: 9) R f～0.10 1H NMR (CDCl 3, 300MHz): δ 0.88 (t, J=7.2Hz), 1.25 (brs, 80H), 1.60 (brs, 8H), 2.37 (t, J=7.5Hz, 8H), 3.48-3.83 (m, 24H), 3.99-4.20 (m, 8H), 4.65-4.75 (m, 2H), 5.14-5.25 (m, 2H).
All reference that this paper quotes, comprise publication, patent application and patent, include but not limited to list below, be hereby expressly incorporated by reference, its degree, and is here set forth with its form in full respectively and especially in conjunction with as a reference as every piece of document.
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The term " a kind of " that uses in the context (particularly in the context of claim below) of describing invention and " being somebody's turn to do " and similar object are construed as and have covered odd number and plural number, unless explanation or clearly inconsistent in context is arranged in this article in addition.Term " comprises ", " having ", " comprising " and " containing " be construed as open term (promptly referring to " including, but are not limited to "), except as otherwise noted.Only enumerating as simple method of numerical range herein, all in this scope, unless this paper has explanation in addition, each single value all is combined in the specification sheets the single value of each that mention, is to quote separately in this article as it individually.Can realize all methods as herein described with any suitable order, unless explanation or clearly inconsistent in context is arranged in this article in addition.(for example, " for example ") use only is intended to the present invention is described better, and does not limit the scope of the invention, except as otherwise noted for any and all embodiment provided herein or exemplary language.It is essential to finishing the present invention that any language in the specification sheets all can not be understood as any unstated key element of hint.
This paper has described the preferred embodiments of the invention, comprises realization known for inventor preferred forms of the present invention.After the explanation of reading the front, the variation of these embodiment preferred is conspicuous for those of ordinary skill in the art.The contriver expects that it is suitable that the technician adopts such variation, and the contriver thinks that the method beyond can specifying by this paper realizes the present invention.Therefore, the present invention includes all improvement and the equivalent of the theme of quoting in the appended claim of applicable law permission.And any combination that key element recited above is carried out with its all possible variation is included in the present invention, unless explanation or clearly inconsistent in context is arranged in this article in addition.

Claims (134)

1. cationic cardiolipin phospholipid analogues with general formula I structure:
Figure A2003801061160002C1
Wherein, Z 1And Z 2Be in the same manner or differently-O-C (O)-,-O-,-S-or-NH-C (O)-;
R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Saturated or unsaturated alkyl, alkenyl or alkynyl, it is randomly hydroxylated, aminating a, sulfurized, epoxidised, cyclisation, Pegylation, halogenated or replace with their combination;
R 5And R 6Be identical or different, can be for lacking or contain connector, described connector contains C 1To C 32The alkoxyl group of the cycloalkyl of the alkyl of alkyl, replacement, cycloalkyl, replacement or alkoxyl group or replacement for example contains the ether of the unitary Pegylation of 1 to 500 PEG (polyoxyethylene glycol);
R 7Be alkyl, alkoxyl group, the alkoxyl group of replacement, cycloalkyl, the cycloalkyl of replacement, alkenyl, alkynyl, alkyloyl, enoyl-, the alkynes acyl group of hydrogen, alkyl, replacement, randomly hydroxylated, aminating, sulfurized, epoxidised, cyclisation, Pegylation, halogenated or replace with their combination; Or the alkoxyl group of alkoxyl group or replacement for example contains the ether of the unitary Pegylation of 1 to 500 PEG (polyoxyethylene glycol); Amino acid, peptide, peptide simulation part, dipeptides, polypeptide, protein, carbohydrate, sugar or polysaccharide, polyamines, heterogeneous ring compound, nucleosides or polynucleotide;
R 8Group is C identical or differently 1To C 25The alkyl of saturated or unsaturated alkyl, alkoxyl group, replacement or the alkoxyl group that replaces;
X is nontoxic negatively charged ion.
2. the analogue of claim 1, wherein said compound is the cationic cardiolipin phosphatide ether with formula II structure:
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 32Alkyl, alkenyl or alkynyl.
3. the composition of claim 1, wherein said compound is the cationic cardiolipin phosphatide ester with formula III structure:
Figure A2003801061160003C1
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 31Alkyl, alkenyl or alkynyl.
4. the composition of claim 1, wherein said compound is the cationic cardiolipin phosphatide ether with formula IV structure:
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 32Alkyl, alkenyl or alkynyl.
5. the composition of claim 1, wherein said compound is the cationic cardiolipin phosphatide ester with formula V structure:
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 31Alkyl, alkenyl or alkynyl.
6. the composition of claim 1, wherein said compound is the cationic cardiolipin phosphatide ether with formula VI structure:
Figure A2003801061160004C1
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 32Alkyl, alkenyl or alkynyl.
7. the composition of claim 1, wherein said compound is the cationic cardiolipin phosphatide ester with formula VII structure:
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 31Alkyl, alkenyl or alkynyl.
8. the composition of claim 1, wherein said compound is the cationic cardiolipin phosphatide ether with formula VIII structure:
Figure A2003801061160004C3
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 32Alkyl, alkenyl or alkynyl.
9. the composition of claim 1, wherein said compound is the cationic cardiolipin phosphatide ester with formula IX structure:
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 31Alkyl, alkenyl or alkynyl.
10. cationic cardiolipin phosphatide variant with general formula X structure:
Figure A2003801061160005C2
Wherein, Z 1And Z 2Be in the same manner or differently-O-C (O)-,-O-,-S-or-NH-C (O)-;
R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Saturated or unsaturated alkyl, alkenyl or alkynyl, randomly hydroxylated, aminating, sulfurized, epoxidised, cyclisation, Pegylation, halogenated or replace with their combination;
R 5And R 6Identical or different, can lack or contain connector, described connector contains C 1To C 32The alkoxyl group of the cycloalkyl of the alkyl of alkyl, replacement, cycloalkyl, replacement or alkoxyl group or replacement for example contains the ether of the unitary Pegylation of 1 to 500 PEG (polyoxyethylene glycol);
R 7Group can be alkyl, alkoxyl group, the alkoxyl group of replacement, cycloalkyl, the cycloalkyl of replacement, alkenyl, alkynyl, alkyloyl, enoyl-, the alkynes acyl group of hydrogen, alkyl, replacement identical or differently, randomly hydroxylated, aminating, sulfurized, epoxidised, cyclisation, Pegylation, halogenated or replace with their combination; Or the alkoxyl group of alkoxyl group or replacement for example contains the ether of the unitary Pegylation of 1 to 500 PEG (polyoxyethylene glycol); Amino acid, peptide, peptide simulation part, dipeptides, polypeptide, protein, carbohydrate, sugar or polysaccharide, polyamines, heterogeneous ring compound, nucleosides or polynucleotide;
R 8Group is C identical or differently 1To C 25The alkyl of saturated or unsaturated alkyl, alkoxyl group, replacement or the alkoxyl group that replaces;
X is nontoxic negatively charged ion.
11. the composition of claim 10, wherein said compound are the cationic cardiolipin phosphatide ether with formula XI structure:
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 32Alkyl, alkenyl or alkynyl.
12. the composition of claim 10, wherein said compound are the cationic cardiolipin phosphatide esters with formula XII structure:
Figure A2003801061160006C2
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 31Alkyl, alkenyl or alkynyl.
13. the cationic cardiolipin phosphatide of claim 1 or 10, wherein, R 5, R 6, R 7And R 8In at least one comprise the optional alkyl that replaces or optional alkoxyl group that replaces.
14. the cationic cardiolipin phosphatide of claim 13, wherein, at least one R 6Comprise the optional poly-alkoxyl group that replaces, it contains 1 to 500 alkoxyl group.
15. the cationic cardiolipin phosphatide of claim 14, wherein, at least one R 6Comprise the optional poly-alkoxyl group that replaces, it contains 1 to 100 alkoxyl group.
16. the cationic cardiolipin phosphatide of claim 13, wherein, at least one R 5Comprise the optional poly-alkoxyl group that replaces, it contains 1 to 500 alkoxyl group.
17. the cationic cardiolipin phosphatide of claim 16, wherein, at least one R 5Comprise the optional poly-alkoxyl group that replaces, it contains 1 to 100 alkoxyl group.
18. the cationic cardiolipin phosphatide of claim 13, wherein, R 7And R 8In at least one contain the optional alkyl that replaces, and R 5And R 6It is the optional poly-alkoxyl group that replaces.
19. the cationic cardiolipin phosphatide of claim 18, wherein, R 5And R 6In at least one contain the optional poly-alkoxyl group that replaces, it contains 1 to 100 alkoxyl group.
20. the cationic cardiolipin phosphatide of claim 1 or 10, wherein, R 5Or R 6In at least one be oxyethyl group (single PEG).
21. the cationic cardiolipin phosphatide of claim 1 or 10, wherein, at least one R 7Be amino acid, folic acid, sugar, peptide, polysaccharide, polypeptide, protein, polyamines or peptide simulation part.
22. the cationic cardiolipin phosphatide of claim 21, wherein, at least one R 7Be histone, spermine, spermidine, or derivatives thereof.
23. the cationic cardiolipin phosphatide of claim 1 or 10, wherein, at least one R 7Be as O-glycosides or C-glucosides bonded sugar.
24. the cationic cardiolipin phosphatide of claim 23, wherein, described sugar is the sugar of glucose, seminose, semi-lactosi, ribose, pectinose, allose, Fucose or 2-deoxidation.
25. the cationic cardiolipin phosphatide of claim 21, wherein, at least one R 7Be L-or the D-alpha amino acid that on side chain, has positively charged group.
26. the cationic cardiolipin phosphatide of claim 25, wherein, described amino acid is arginine, Histidine, Methionin, ornithine or its analogue.
27. the cationic cardiolipin phosphatide of claim 21, wherein, at least one R 7Be amino acid, sugar, peptide, polysaccharide, polypeptide, protein, polyamines or the peptide simulation part that has one or more positive charges.
28. cationic cardiolipin phosphatide with general formula X III structure:
Figure A2003801061160007C1
Wherein, Z 1And Z 2Be in the same manner or differently-O-C (O)-,-O-,-S-or-NH-C (O)-;
R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Saturated or unsaturated alkyl, alkenyl or alkynyl, randomly hydroxylated, aminating, sulfurized, epoxidised, cyclisation, Pegylation, halogenated or replace with their combination;
R 5And R 6Be identical or different ground, can lack or contain connector, described connector contains C 1To C 32The alkoxyl group of the cycloalkyl of the alkyl of alkyl, replacement, cycloalkyl, replacement or alkoxyl group or replacement for example contains the ether of the unitary Pegylation of 1 to 500 PEG (polyoxyethylene glycol);
R 7, R 9, R 10And R 11Be alkyl, alkoxyl group, the alkoxyl group of replacement, cycloalkyl, the cycloalkyl of replacement, alkenyl, alkynyl, alkyloyl, enoyl-, the alkynes acyl group of hydrogen, alkyl, replacement identical or differently, randomly hydroxylated, aminating, sulfurized, epoxidised, cyclisation, Pegylation, halogenated or replace with their combination; Or the alkoxyl group of alkoxyl group or replacement for example contains the ether of the unitary Pegylation of 1 to 500 PEG (polyoxyethylene glycol); Amino acid, peptide, peptide simulation part, dipeptides, polypeptide, protein, carbohydrate, sugar or polysaccharide, polyamines, heterogeneous ring compound, nucleosides or polynucleotide;
R 8Be C 2To C 32The alkoxyl group of alkane thiazolinyl, alkoxyl group or the replacement of the alkyl of alkyl, replacement, alkane thiazolinyl, replacement;
X is nontoxic negatively charged ion.
29. cationic cardiolipin phosphatide with claim 28 of general formula X IV structure:
Figure A2003801061160008C1
Wherein, R 1, R 2, R 3And R 4Be H, C identical or differently 1To C 32Alkyl, alkenyl or alkynyl.
30. cationic cardiolipin phosphatide with claim 28 of general formula X V structure:
Figure A2003801061160008C2
Wherein, R 1, R 2, R 3And R 4Be H or C identical or differently 1To C 31Alkyl, alkenyl or alkynyl.
31. the cationic cardiolipin phosphatide of claim 28, wherein, R 5, R 6, R 7, R 8, R 9, R 10Or R 11In at least one optional alkyl that replaces or optional alkoxyl group that replaces.
32. the cationic cardiolipin phosphatide of claim 31, wherein, at least one R 6Contain the optional poly-alkoxyl group that replaces, it contains 1 to 500 alkoxyl group.
33. the cationic cardiolipin phosphatide of claim 32, wherein, at least one R 6Contain the optional poly-alkoxyl group that replaces, it contains 1 to 100 alkoxyl group.
34. the cationic cardiolipin phosphatide of claim 31, wherein, at least one R 5Contain the optional poly-alkoxyl group that replaces, it contains 1 to 500 alkoxyl group.
35. the cationic cardiolipin phosphatide of claim 34, wherein, at least one R 5Contain the optional poly-alkoxyl group that replaces, it contains 1 to 100 alkoxyl group.
36. the cationic cardiolipin phosphatide of claim 28, wherein, R 7, R 9, R 10Or R 11In at least one contain the optional alkyl that replaces, and R 5And R 6It is the optional poly-alkoxyl group that replaces.
37. the cationic cardiolipin phosphatide of claim 36, wherein, R 5And R 6In at least one contain the optional poly-alkoxyl group that replaces, it contains 1 to 100 alkoxyl group.
38. the cationic cardiolipin phosphatide of claim 28, wherein, R 5Or R 6In at least one be oxyethyl group (single PEG).
39. the cationic cardiolipin phosphatide of claim 28, wherein, R 7, R 9, R 10Or R 11In at least one be amino acid, folic acid, sugar, peptide, polysaccharide, polypeptide, protein, polyamines or peptide simulation part.
40. the cationic cardiolipin phosphatide of claim 39, wherein, R 7, R 9, R 10Or R 11In at least one be histone, spermine, spermidine, or derivatives thereof.
41. the cationic cardiolipin phosphatide of claim 39, wherein, R 7, R 9, R 10Or R 11In at least one be as O-glycosides or C-glucosides bonded sugar.
42. the cationic cardiolipin phosphatide of claim 41, wherein, described sugar is the sugar of glucose, seminose, semi-lactosi, ribose, pectinose, allose, Fucose or 2-deoxidation.
43. the cationic cardiolipin phosphatide of claim 39, wherein, R 7, R 9, R 10Or R 11In at least one be L-or the D-alpha amino acid that on side chain, has positively charged group.
44. the cationic cardiolipin phosphatide of claim 43, wherein, described amino acid is arginine, Histidine, Methionin, ornithine or its analogue.
45. the cationic cardiolipin phosphatide of claim 39, wherein, R 7, R 9, R 10Or R 11In at least one be the amino acid that has one or more positive charges, sugar, peptide, polysaccharide, polypeptide, protein, polyamines or peptide simulation part.
46. a composition, it contains each the cationic cardiolipin phosphatide among the with good grounds claim 1-45.
47. claim 46 composition, it also contains one or more common fat.
48. the composition of claim 46 or 47, it contains one or more liposomes.
49. the composition of each among the claim 46-48, that it contains individual layer or multiwalled vesicle or its mixture.
50. the composition of each among the claim 46-49, wherein, described liposome has about 1 micron diameter to 500nm.
51. the composition of claim 48, it is the vesicle form in the aqueous medium.
52. the composition of claim 48, it is a lyophilized form.
53. the composition of claim 52, it also comprises cryoprotectant.
54. the composition of each among the claim 46-53, it also comprises the neutral lipid kind.
55. the composition of claim 54, wherein, the mol ratio of cation lipid kind and neutral lipid kind is about 9/1 to about 1/9.
56. the composition of each among the claim 46-55, it also comprises the lipid that is selected from by the following group of forming: phosphatidylethanolamine, phosphatidylcholine, sphingophospholipid, cholesterol, sterol and tocopherol.
57. the composition of claim 56, it also comprises the phosphatidylcholine that is selected from by the following group of forming: dimyristoyl phosphatidyl choline, distearoyl phosphatidylcholine, two oil base phosphatidylcholines, two palmitoyl phosphatidylcholines, two arachidonic phosphatidyl cholines, Yelkin TTS phatidylcholine, fabaceous lecithin phatidylcholine, hydrogenant fabaceous lecithin phatidylcholine and its mixture.
58. the composition of claim 56, it comprises the sterol that is selected from by the following group of forming: cholesterol, the derivative of cholesterol, coprostanol, Dihydrocholesterol, cholestane, Cholesteryl hemisuccinate, cholesterol sulfate and its mixture.
59. a preparation, it comprises each composition and the physiologically acceptable carrier among the claim 46-58.
60. the preparation of claim 59, it also comprises a kind of promoting agent.
61. the preparation of claim 60, wherein, described promoting agent is the nucleoside analog or the nucleotide analog of treatment significant quantity.
62. the preparation of claim 61, wherein, described analogue is halogenation or the azido-derivative or the acyclic nucleosides of di-deoxynucleoside, two dehydrogenation nucleosides, nucleosides.
63. the preparation of claim 61, wherein, described nucleoside analog is the anti-viral nucleoside that is selected from by the following group of forming: acycloguanosine, gancyclovir, 1-(2-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base)-5-iodocytosine (FIAC) or 1-(2 '-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base) 5-iodouracil (FIAU).
64. the preparation of claim 61, wherein, described analogue is the anti-viral nucleoside that is selected from by the following group of forming: 3 '-azido--2 ', 3 '-dideoxy pyrimidine, 3 '-halogenated pyrimidine di-deoxynucleoside or 2 ', 3 '-two dehydrogenations-2 ', 3 '-di-deoxynucleoside.
65. the preparation of claim 61, wherein, described analogue is an anti-viral nucleoside, it is 3 '-azido--3 ' deoxythymidine (AZT).
66. the preparation of claim 61, wherein, described therapeutical agent is the phosphatidyl derivant or bisphosphate two glyceride derivative of described analogue.
67. the preparation of claim 66, wherein, described promoting agent is the anti-inflammatory agent of corticosteroid or on-steroidal.
68. the preparation of claim 60, wherein, described promoting agent is anti-inflammatory agent, microbiotic, anti-mycotic agent, oxygenant or the anti-viral nucleoside of corticosteroid, on-steroidal.
69. the preparation of claim 60, wherein, described promoting agent is protein, polypeptide or polynucleotide.
70. the preparation of claim 69, wherein, described promoting agent is protein or peptide.
71. the preparation of claim 69, wherein, described promoting agent is polynucleotide.
72. the preparation of claim 71, wherein, described polynucleotide are ribozyme, RNA interfering (RNAi) or sense-rna or dna sequence dna.
73. the preparation of claim 72, wherein, described polynucleotide are antisense polynucleotides of 10 to 30-mer.
74. the preparation of claim 73, wherein, described antisense polynucleotides is the sequence of 15-mer.
75. the preparation of each in claim 73 or 74, wherein, described antisense oligonucleotide contains one or more thiophosphatephosphorothioates and connects.
76. the preparation of claim 75, wherein, described antisense oligonucleotide contains 2 thiophosphatephosphorothioates and connects.
77. the preparation of claim 76, wherein, described thiophosphatephosphorothioate connects each end that is present in antisense oligonucleotide.
78. the preparation of claim 76, wherein, described thiophosphatephosphorothioate connects and is present between the end of antisense oligonucleotide.
79. the preparation of each among the claim 73-78, wherein, described oligonucleotide is the c-raf antisense oligonucleotide.
80. the preparation of each among the claim 72-78, wherein, described polynucleotide are at HIV.
81. the preparation of claim 80, wherein, described polynucleotide are at the rev trans-activator.
82. the preparation of claim 71, wherein, described polynucleotide encoding is that lack or non-existent gene product at morbid state.
83. the preparation of claim 71, wherein, the synthetic analogues of described polynucleotide encoding immunogenic peptide, natural hormone or natural hormone.
84. the preparation of claim 71, wherein, described polynucleotide are that lack or non-existent at morbid state.
85. the preparation of claim 71, wherein, described polynucleotide encoding therapeutical peptide.
86. the preparation of claim 85, wherein, described polypeptide is that lack or non-existent at morbid state.
87. the preparation of claim 85, wherein, described polypeptide is natural hormone or its synthetic analogues.
88. the preparation of claim 85, wherein, described polypeptide is an immunogen.
89. the preparation of claim 60, wherein, described promoting agent is a medicine.
90. the preparation of claim 89, wherein, described promoting agent is an anticarcinogen.
91. the preparation of claim 69, wherein, described promoting agent is monoclonal antibody or its fragment.
92. the preparation of claim 60, wherein, described promoting agent is the biological activity lipid.
93. the preparation of each among the claim 59-92, it is the pharmaceutical preparation that contains one or more pharmaceutical carriers.
94. the pharmaceutical preparation of claim 93, it is formulated into and is used for topical application.
95. the pharmaceutical preparation of claim 94, it is formulated into and is applied to skin or mucomembranous surface.
96. the pharmaceutical preparation of claim 93, it is formulated into and is used for parenteral administration.
97. the pharmaceutical preparation of claim 93, its be formulated into be used for Orally administered.
98. the pharmaceutical preparation of each among the claim 93-97, it contains multiple actives.
99. the method for the vertebrate disease of treatment, it comprises to the vertebrates of needs treatments uses each the step of preparation among the claim 59-98, and amount of using and position are enough to this vertebrate disease of treatment.
100. the method for claim 99, it comprises described preparation is administered to described vertebrate cell externally, then this cell is returned described vertebrates.
101. the method for claim 99, it comprises and will be administered to described vertebrate cell in the described preparation body.
102. right is wanted 101 method, it comprises described preparation is administered to skin or mucomembranous surface partly.
103. the method for claim 101, it comprise be injected into described vertebrate body cavity or the tissue in.
104. the method for claim 101, it comprises Orally administered described preparation.
105. the method for each among the claim 99-104, wherein, described disease is a cancer.
106. the method for claim 105, wherein, described composition contains anticarcinogen.
107. the method for claim 105 or 106, wherein, described composition comprises the polynucleotide that are selected from by the following group of forming: ribozyme, RNA interfering (RNAi) and sense-rna or dna sequence dna.
108. the method for claim 107, wherein, described polynucleotide are c-raf antisense oligonucleotides.
109. the method for each among the claim 99-104, wherein, described disease is a virus infection.
110. the method for claim 109, wherein, described composition comprises antiviral agent.
111. the method for claim 109 or 110, wherein, described virus infection is a herpes simplex.
112. the method for claim 111, wherein, described composition comprises: acycloguanosine, gancyclovir, 1-(2-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base)-5-iodocytosine (FIAC) or 1-(2 '-deoxidation-2 '-fluoro-1-β-D-arbinofuranose base) 5-iodouracil (FIAU).
113. the method for claim 109 or 110, wherein, described virus infection is HIV.
114. the method for claim 113, wherein, described composition comprises anti-viral nucleoside.
115. the method for claim 114, wherein, described anti-viral nucleoside is 3 '-azido--3 '-deoxythymidine (AZT).
116. the method for each among the claim 99-115, wherein, described vertebrates is the people.
117. one kind with the method in the promoting agent transfered cell, it comprises: (a) preparation contain among the claim 1-45 each cationic cardiolipin phosphatide and the composition of described promoting agent, (b) described cell is contacted with described composition, make described cell take in described promoting agent thus.
118. one kind with the method in the promoting agent transfered cell, it comprises: (a) preparation contains each the composition of cationic cardiolipin phosphatide among the claim 1-45, (b) exist under the situation of described promoting agent, described cell is contacted with described composition, make described cell take in described promoting agent thus.
119. the method for claim 117 or 118, wherein, described contact procedure is to carry out external.
120. the method for each among the claim 117-119, wherein, described promoting agent is polynucleotide.
121. the method for claim 120, wherein, described polynucleotide are ribozyme, RNA interfering (RNAi) and sense-rna or dna sequence dna.
122. method with the polynucleotide transfectional cell, it comprises: (a) preparation contain among the claim 1-45 each cationic cardiolipin phosphatide and the composition of described polynucleotide, (b) described cell is contacted with described composition, make described cell take in described polynucleotide thus.
123. method with the polynucleotide transfectional cell, it comprises: (a) preparation contains each the composition of cationic cardiolipin phosphatide among the claim 1-45, (b) exist under the situation of described polynucleotide, described cell is contacted with described composition, make described cell take in described polynucleotide thus.
124. the method for claim 122 or 123, wherein, described cell is external.
125. the method for claim 122 or 123, wherein, described cell is intravital.
126. the method for each among the claim 122-125, wherein, described polynucleotide are ribozyme, RNA interfering (RNAi) or sense-rna or dna sequence dna.
127. the method for claim 126, wherein, described polynucleotide are expression construct of encoding gene, and described gene is being expressed in described cell after the transfection.
128. test kit that is used for transfectional cell, described test kit comprise among the claim 1-45 each cationic cardiolipin phosphatide and one or more be selected from key element by the following group of forming: polynucleotide, these polynucleotide and cationic cardiolipin phosphatide are mixed with the specification sheets of preparation, use the specification sheets of cationic cardiolipin phosphatide transfectional cell, be used to promote the reagent of transfection, be used to store the container of cationic cardiolipin phosphatide, be used to store the container of polynucleotide, be used to store the container of reagent, the container that is used for the preparation of storage bag cation Val and polynucleotide, or the material that is used to prepare the container of preparation and promotes transfection.
129. a composition, it contains cationic cardiolipin phosphatide and nucleic acid.
130. the composition of claim 129, it also comprises one or more pharmaceutical carriers.
131. the composition of claim 129, it is a liposome composition.
132. a gene therapy method, it comprises that the patient to the needs treatment uses the pharmaceutical composition that comprises one or more nucleic acid, and wherein said composition comprises cationic cardiolipin phosphatide.
133. the method for claim 132, wherein, said composition also comprises one or more pharmaceutical carriers.
134. the method for claim 132, wherein, said composition is a liposome composition.
CN 200380106116 2002-10-16 2003-10-16 Cardiolipin molecules and method of synthesis Pending CN1726183A (en)

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USPCT/US03/13917 2003-05-04
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN102366409A (en) * 2011-09-14 2012-03-07 海南灵康制药有限公司 Nizatidine liposome solid preparation

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US7824708B2 (en) * 2006-12-11 2010-11-02 Access Business Group International Llc Liposome containing cardiolipin for improvement of mitochondrial function
CN106124783B (en) * 2016-06-30 2018-10-02 深圳市亚辉龙生物科技股份有限公司 Coated nanometer magnetic bead of modified cuorin and preparation method thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102366409A (en) * 2011-09-14 2012-03-07 海南灵康制药有限公司 Nizatidine liposome solid preparation

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