CN102604083B - Polymer modified lipid material and application thereof - Google Patents
Polymer modified lipid material and application thereof Download PDFInfo
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- CN102604083B CN102604083B CN 201110408374 CN201110408374A CN102604083B CN 102604083 B CN102604083 B CN 102604083B CN 201110408374 CN201110408374 CN 201110408374 CN 201110408374 A CN201110408374 A CN 201110408374A CN 102604083 B CN102604083 B CN 102604083B
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- UKWHYYKOEPRTIC-UHFFFAOYSA-N mercury(ii) oxide Chemical compound [Hg]=O UKWHYYKOEPRTIC-UHFFFAOYSA-N 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000008354 sodium chloride injection Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- SRLOHQKOADWDBV-NRONOFSHSA-M sodium;[(2r)-2,3-di(octadecanoyloxy)propyl] 2-(2-methoxyethoxycarbonylamino)ethyl phosphate Chemical compound [Na+].CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCCNC(=O)OCCOC)OC(=O)CCCCCCCCCCCCCCCCC SRLOHQKOADWDBV-NRONOFSHSA-M 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical group O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000007738 vacuum evaporation Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
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Abstract
The invention relates to a polymer modified lipid material and application thereof. The lipid material is a compound shown in the formula I, or a compound shown in the formula 9 or a physiologically acceptable salt thereof, wherein n is an integer in the range of 5-500, m is an integer in the range of 2-12, and p is an integer in the range of 1-8. The invention also relates to an intermediate of the compound shown in the formula I, an application of the compound shown in the formula I and an administration carrier prepared by the compound shown in the formula I. The administration carrier provided by the invention can be used for increasing the concentration of an anti-cancer medicine on a target part, simultaneously reducing the toxin or side effect on a non-target part and increasing the therapeutic index of the medicine.
Description
Technical field
The present invention relates to a kind of matrix material, particularly a kind of polymer-modified matrix material.The purposes that also relates to this matrix material.Matrix material of the present invention can be used as the solid support material of liposome.In addition, the invention still further relates to the intermediate of synthetic described matrix material.
Background technology
Liposome is a kind of targeted drug carrier, belongs to a kind of novel form of targeting drug delivery system.Because it has targeting, slow-releasing, characteristics such as tissue affinity, hypotoxicity and high stability are subjected to extensive concern in recent years, and many domestic and international investigators are seeking the liposome vectors material that novel targeting is strong and have superperformance.
Folic acid-liposome is a kind ofly by the wetting ability straight chain polymer folate molecule to be connected the receptor type target liposomes that surface of liposome obtains indirectly, folic acid (in this article, can be abbreviated as F) have very high tumor-targeting, so folic acid-liposome provides a kind of comparatively practical antitumor drug targeting vector for medicine is imported at high proportion in the tumour cell.Existing research is at present made the folacin receptor target liposomes with F-PEG-DSPE as fat material parcel antitumor drug, and result of study shows that this liposome has good long Circulation and tumor-targeting.
Poly-(2-ethyl-2-oxazoline) is the polymkeric substance that is obtained by the ring-opening polymerization of 2-ethyl-2-oxazoline (EOz) monomer (PEOz), has characteristics such as good biocompatibility and hypotoxicity.Studies show that the effect of PEOz and PEG is similar, therefore be expected to become the equivalent material of PEG.Discovered in recent years that PEOz had pH susceptibility, its pKa value (4-6) can show different character near the physiological pH value under weak acid environment with under the physiological pH environment.This pH susceptibility is fit to medicine in the release at slightly acidic positions such as tumor tissues and inclusion body very much.
Yet people still can not obtain to gather the compounds that (2-ethyl-2-oxazoline) is combined with phospholipid substance and optional folic acid at present, (for example good water-solubility, biocompatibility and/or hypotoxicity in order to obtain to have desirable properties, with optional tumor-targeting) matrix material, particularly can be used as the solid support material of liposome.
Summary of the invention
The purpose of this invention is to provide a kind of polymer-modified matrix material, it has good water-solubility, biocompatibility and/or hypotoxicity and optional tumor-targeting.The inventor finds, adopt the 2-ethyl-2-oxazoline (in this article, can be abbreviated as EOz) obtain polymer poly (2-ethyl-2-oxazoline) (in this article as monomer through ring-opening polymerization, can be abbreviated as PEOz), this specific functional group of PEOz warp and DSPE are (in this article, can be abbreviated as DSPE) combination, randomly further be combined with folic acid again, can successfully obtain polymer-modified phospholipid substance, these polymer-modified phospholipid substances have the advantageous property of expectation.The present invention is based on above-mentioned discovery and be accomplished.
Put it briefly, the invention provides with the following formula I compound:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.The present invention also provides the intermediate of preparation I compound, the purposes of formula I compound and the drug administration carrier that is made by formula I compound.
First aspect present invention provides with the following formula I compound:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
According to the formula I compound of first aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer, perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer, perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to the formula I compound of first aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to the formula I compound of first aspect present invention, wherein p is 1 to 6 integer, and perhaps p is 1 to 5 integer, and perhaps p is 1 to 4 integer, and perhaps p is 2.
According to the formula I compound of first aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 4 integer.
According to the formula I compound of first aspect present invention, its for following formula Ia compound (be m=6, p=2):
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, perhaps n is 10 to 250 integer, perhaps n is 10 to 200 integer, perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer, perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
Second aspect present invention provides with following formula 9 compounds:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
According to formula 9 compounds of second aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer, perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer, perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to formula 9 compounds of second aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to formula 9 compounds of second aspect present invention, wherein p is 1 to 6 integer, and perhaps p is 1 to 5 integer, and perhaps p is 1 to 4 integer, and perhaps p is 2.
According to formula 9 compounds of second aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 5 integer.
According to formula 9 compounds of second aspect present invention, it is with following formula 9a compound:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, perhaps n is 10 to 250 integer, perhaps n is 10 to 200 integer, perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer, perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
Third aspect present invention provides with following formula 4 compounds:
Or its salt, wherein n is 5 to 500 integer, m is 2 to 12 integer.
According to formula 4 compounds of third aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer, perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer, perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to formula 4 compounds of third aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to formula 4 compounds of third aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer.
According to formula 4 compounds of third aspect present invention, it is with following formula 4a compound:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, perhaps n is 10 to 250 integer, perhaps n is 10 to 200 integer, perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer, perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
Fourth aspect present invention provides the method for preparing described formula 9 compounds of second aspect present invention, and it may further comprise the steps:
(i) in the presence of suitable catalyzer (for example DCC), in suitable organic solvent (for example chloroform), make carboxylic acid and DSPE (DSPE) reaction of the base of Terminal Acetylenes shown in the following formula,
Obtain containing formula 6 compounds (can be described as Terminal Acetylenes base DSPE at this paper) of Terminal Acetylenes base:
(ii) make with the reaction of following formula 4 compound step (i) gained formulas 6 compounds,
Obtain with following formula 9 compounds (can abbreviate PEOz-DSPE as at this paper):
Wherein, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
According to the method for fourth aspect present invention, the reaction of wherein said step (i) is to carry out 5-25 hour (for example 8-20 hour, for example 10-15 hour, for example about 12 hours) under 30-75 ℃ (for example 40-60 ℃, for example about 50 ℃).
According to the method for fourth aspect present invention, the reaction (ii) of wherein said step is carried out in the presence of part (for example PMDETA).
According to the method for fourth aspect present invention, the reaction (ii) of wherein said step is carried out in the presence of reagent (for example CuBr).
According to the method for fourth aspect present invention, the reaction (ii) of wherein said step is to carry out 15-150 hour (for example 30-120 hour, for example 50-100 hour, for example about 72 hours) under 30-75 ℃ (for example 40-60 ℃, for example about 50 ℃).
According to the method for fourth aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer, perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer, perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to the method for fourth aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to the method for fourth aspect present invention, wherein p is 1 to 6 integer, and perhaps p is 1 to 5 integer, and perhaps p is 1 to 4 integer, and perhaps p is 2.
According to the method for fourth aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 5 integer.According to the method for fourth aspect present invention, wherein n is 10 to 100 integer, and m is 4 to 8 integer, and p is 1 to 4 integer.
According to the method for fourth aspect present invention, wherein said formula 4 compounds (can be described as PEOz in this article) are to prepare by the method that may further comprise the steps:
(ia) in the presence of reagent (for example NaI), in organic solvent (for example DMF), make formula
Shown in compound and reaction of sodium azide, obtain formula
Compound;
(ib) in the presence of reagent (for example triethylamine and/or trimethylamine hydrochloride), in organic solvent (for example chloroform), make formula
Compound and Tosyl chloride reaction obtain formula
Compound;
(ic) in organic solvent (for example acetonitrile), make formula
Compound and formula
Monomer reaction, charge into ammonia then with stopped reaction, obtain with following formula 4 compounds:
Wherein, X is halogen (for example fluorine, chlorine, bromine, iodine, preferably chlorine), and n is 5 to 500 integer, and m is 2 to 12 integer.
Fifth aspect present invention provides the method for preparing the described formula I compound of first aspect present invention, and it may further comprise the steps:
(a) in organic solvent (for example triethylamine and/or DMSO), in the presence of catalyzer (for example DCC), make the reaction of folic acid and following formula N-maloyl imines (NHS), obtain the folic acid Acibenzolar (can abbreviate F-NHS as at this paper) with following formula 8:
(b) in the presence of alkali (for example triethylamine), in organic solvent (for example DMSO), make formula 8 compounds and react with following formula 9 compounds,
Obtain formula I compound:
With optional
(c) make gained formula I compound purifying and/or salify,
Wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
According to the method for fifth aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer, perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer, perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to the method for fifth aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to the method for fifth aspect present invention, wherein p is 1 to 6 integer, and perhaps p is 1 to 5 integer, and perhaps p is 1 to 4 integer, and perhaps p is 2.
According to the method for fifth aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 5 integer.
Sixth aspect present invention provides the described formula I compound of first aspect present invention or the purposes of described formula 9 compounds of second aspect present invention in the preparation drug administration carrier.
According to the purposes of sixth aspect present invention, wherein said formula I compound or formula 9 compounds are that one of lipid composition as described drug administration carrier is used.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is pH responsive type drug administration carrier.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is the drug administration carrier with cancer target performance.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is pH responsive type and drug administration carrier with cancer target performance.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is the drug administration carrier that contains or do not contain active constituents of medicine.In one embodiment, described drug administration carrier contains activeconstituents.In one embodiment, described active constituents of medicine is antitumor drug.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is liposome.In one embodiment, described drug administration carrier is initiatively target liposomes of pH responsive type.In one embodiment, described drug administration carrier is folacin receptor mediated pH responsive type active target liposomes.
Seventh aspect present invention provides a kind of drug administration carrier, wherein comprises described formula 9 compounds of the described formula I compound of first aspect present invention or second aspect present invention, and optional active constituents of medicine.
According to the drug administration carrier of seventh aspect present invention, wherein said formula I compound or formula 9 compounds are one of lipid compositions as described drug administration carrier.
According to the drug administration carrier of seventh aspect present invention, it is pH responsive type drug administration carrier.
According to the drug administration carrier of seventh aspect present invention, it is the drug administration carrier with cancer target performance.
According to the drug administration carrier of seventh aspect present invention, it is pH responsive type and drug administration carrier with cancer target performance.
According to the drug administration carrier of seventh aspect present invention, wherein contain or do not contain active constituents of medicine.In one embodiment, described drug administration carrier contains activeconstituents.In one embodiment, described active constituents of medicine is antitumor drug (for example Zorubicin).
According to the drug administration carrier of seventh aspect present invention, it is liposome.In one embodiment, described drug administration carrier is initiatively target liposomes of pH responsive type.In one embodiment, described drug administration carrier is folacin receptor mediated pH responsive type active target liposomes.
Eighth aspect present invention provides the method that treats and/or prevents disease in the experimenter who needs is arranged, it comprises to described experimenter uses each described drug administration carrier of seventh aspect present invention, and wherein said drug administration carrier contains the active constituents of medicine of significant quantity.In one embodiment, described disease is tumour or Cancerous disease.In one embodiment, described active constituents of medicine is antitumor drug (for example Zorubicin).
In either side of the present invention, wherein any one in arbitrary embodiment or a plurality of technical characterictic can be combined in another embodiment of this aspect or be combined in arbitrary embodiment of other side, as long as this combination can be not conflicting; Certainly when making up each other, necessary words can be done suitably to modify to individual features.In addition, for the term that uses in either side of the present invention, its implication is equally applicable to other side.
Be further described with characteristics to various aspects of the present invention below.
All documents that the present invention quotes from, their full content is incorporated this paper by reference into, and if the expressed implication of these documents and the present invention when inconsistent, be as the criterion with statement of the present invention.In addition, various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art, nonetheless, the present invention still wishes at this more detailed description and interpretation to be made in these terms and phrase, the term of mentioning and phrase are as the criterion with the implication that the present invention was explained if any inconsistent with known implication.
As described herein, term " pharmaceutical composition ", it can also refer to composition, is used in to realize treating, prevent, alleviate and/or alleviating disease of the present invention or illness or adverse health situation among the experimenter.Those skilled in the art understand, pharmaceutical composition of the present invention or composition can be the liposomes that is enclosed with medicine (for example Zorubicin), also can be the blank liposome that does not comprise medicine, it mixes the pastille liposome with medicine (for example Zorubicin) before clinical application.
As described herein, term " experimenter " also can refer to " patient ", it typically refers to Mammals, for example people, dog, monkey, ox, horse etc.
As described herein, term " significant quantity " refers to effectively to realize treating, preventing, alleviate, alleviate, eliminate the dosage of relative disease and complication thereof in the experimenter.
As described herein, term " drug administration carrier ", also can be described as " drug delivery system ", one of important component of forming this drug administration carrier is lipid composition, the polymer-modified phosphatide with hydrophilic segment and hydrophobic part that obtains of the present invention for example, for example the present invention obtains has hydrophilic segment and hydrophobic part and comprises target functional group's polymer-modified phosphatide, and for example well known to a person skilled in the art other phospholipid composition.Described drug administration carrier can be lipid microsphere drug delivery system (for example lipoid microsphere), liposome administration system (for example unilamelar liposome, multilamelar liposome, multiphasic liposomes), lipid vesicle drug delivery system etc.This drug administration carrier can be pastille, thus can also be not pastille but face the time spent and medicine mixed with this drug administration carrier medicine is mixed in this drug administration carrier.In addition, drug administration carrier of the present invention also can contain other the acceptable vehicle of pharmacy, for example caffolding agent.For example, in one embodiment, drug administration carrier of the present invention is by being similar to following method preparation: make phospholipid, cholesterol and formula I compound or formula 9 compound formation liposome aqueous suspensions; Add vehicle (for example N.F,USP MANNITOL); Lyophilize obtains the not blank drug administration carrier of pastille; Join in the above-mentioned blank drug administration carrier facing with the preceding solution that will be dissolved with medicine (for example Zorubicin) (for example solution of the aqueous solution, sodium chloride injection preparation), the medicine parcel is entered in liposome, thus can be for clinical use.
In the present invention, when mentioning Zorubicin, it comprises any pharmacologically acceptable salts of Zorubicin itself and Zorubicin, hydrochloric acid for example, and they all can be expressed as DOX in the present invention.Have under the situation of concrete context implication at some, as not statement in addition, mention that Zorubicin all refers to doxorubicin hydrochloride.
In the present invention, mention that PEOz-DSPE refers to the binding substances of PEOz and DSPE, namely as formula 9 compounds of the present invention; Refer to when mentioning F-PEOz-DSPE be connected with folic acid at the binding substances of above-mentioned PEOz and DSPE, namely as formula I compound of the present invention; Refer to the binding substances of PEG and DSPE when mentioning PEG-DSPE, for example PEG2000-DSPE refers to the PEG of molecular weight 2000 and the binding substances of DSPE.
As described herein, term " pH sensitivity ", for example when explanation the present invention " drug administration carrier is pH responsive type drug administration carrier ", refer to that the material that refers to has different character under condition of different pH, for example be included under solutions of weak acidity that activeconstituents in this drug administration carrier compares in easier release under condition of neutral pH.
As described herein, term " cancer target performance ", for example when explanation the present invention " drug administration carrier is the drug administration carrier with cancer target performance ", refer to that the material that refers to has the performance that is directed to the related tissue position, for example after the drug administration carrier administration with the formula I compound of the present invention that comprises the folic acid part, can make this drug administration carrier by the folacin receptor mediated for example tumor locus of relevant histoorgan that initiatively is targeted to because having folic acid functional group.
One of purport of the present invention is folacin receptor mediated pH responsive type Zorubicin initiatively preparation and the application of target liposomes.
In the prior art, the synthetic liposome vectors material that contains the PEOz block of some researchs is confined to its single-ended other functional group that is connected with, and the research that the pH susceptibility of PEOz is applied to liposome seldom, more not about by the particular functional group F-PEOz-DSPE three being coupled together to realize the instruction of the object of the invention.
An object of the present invention is to provide the synthetic method of a kind of liposome vectors material folic acid-poly-(2-ethyl-2-oxazoline)-DSPE (F-PEOz-DSPE).
It is the method for the target liposomes of liposome vectors material preparation pastille (for example Zorubicin) with F-PEOz-DSPE that another object of the present invention provides a kind of.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
One embodiment of the invention provides the novel lipide solid support material F-PEOz-DSPE as shown in the formula structure shown in the I:
Wherein n, m, p are as indicated above.
In one embodiment, the invention provides the compound with following formula Ia:
Wherein n, m, p are as indicated above.
Another embodiment of the invention provides the method for preparation I compound.In a specific embodiment, provide the preparation method with following formula Ia compound:
Wherein n, m, p are as indicated above.
Particularly, be example with formula Ia compound, the synthetic method that the invention provides formula Ia compound can may further comprise the steps:
Step 1: prepare initiator 6-nitrine-own ester of 1-tosic acid (compound 3) by two step substitution reactions, shown in the following reaction formula:
Make 6-chloro-1-hexanol (compound 1) and reaction of sodium azide obtain 6-nitrine-1-hexanol (compound 2), 6-nitrine-1-hexanol obtains 6-nitrine-own ester of 1-tosic acid with the Tosyl chloride reaction again;
Step 2: cause 2-ethyl-2-oxazoline (EOz) cationic ring-opening polymerization with the own ester of 6-nitrine-1-tosic acid, generate poly-(2-ethyl-2-oxazoline) (compound 4a) of an end amino, an endlap nitrogen, shown in the following reaction formula:
The feed ratio of control monomer and initiator can generate the polymkeric substance of different polymerization degree, the present invention exemplarily synthesized the polymerization degree (n) be respectively 18,32 and 64 3 kind poly-(2-ethyl-2-oxazoline) (PEOz);
Step 3: the condensation acylation reaction by carboxylic acid and amine prepares Terminal Acetylenes base DSPE (compound 6), shown in the following reaction formula:
Generate Terminal Acetylenes base DSPE (compound 6) with 4-pentynoic acid and DSPE (DSPE, compound 5) reaction;
Step 4: prepare folic acid Acibenzolar (compound 8) by esterification, shown in the following reaction formula:
Make folic acid (compound 7) and N-maloyl imine reaction generate folic acid Acibenzolar (compound 8);
Step 5: by " clicking (Click) " chemical reaction, an end amino of preparation, the PEOz of an endlap nitrogen are connected with Terminal Acetylenes base DSPE in the performing step two, three, generate PEOz-DSPE (compound 9a), shown in the following reaction formula:
Step 6: by acylation reaction, the folic acid Acibenzolar of preparation is connected with PEOz-DSPE in the performing step four, five, generate F-PEOz-DSPE (compound I a), shown in the following reaction formula:
Moisture in ratio, reaction times, temperature of reaction and the system of the molecular weight of PEOz and initiator and monomer is relevant.The inventor earlier carries out pre-reaction under 70 ℃, the condition of reaction 24h, transformation efficiency is about 60%.Different ratios according to three kinds of initiators and monomer feeds intake accordingly, and the proper extension reaction times, and the moisture content in the hierarchy of control has exemplarily been synthesized molecular weight and has been respectively 1900,3300,6,500 three kinds of polymkeric substance (polymerization degree n is respectively 18,32,64).
The synthetic solid support material that contains the PEOz block of more existing researchs is confined to its single-ended other functional group that is connected with.The present invention successfully is connected with different functional groups with the both-end of polymer P EOz, and this has laid important foundation for realizing the object of the invention.The initiator of the at first synthetic end azido-of the present invention, cause cationic ring-opening polymerization, with ammonia/acetonitrile solution termination reaction, generating an end is that nitrine, an end are amino polymer P EOz, priority reacts with DSPE and the folic acid of Terminal Acetylenes base again, generates target compound.Wherein the reaction of PEOz and DSPE has utilized the principle of " click chemistry ", take place 1 by nitrine and Terminal Acetylenes base, the 3-Dipolar Cycloaddition connects the two, because the molecular weight of PEOz and DSPE is all bigger, reaction between endlap nitrogen and the Terminal Acetylenes base is carried out than the reaction between the small molecules is more difficult, therefore in an example of the present invention temperature of reaction is brought up to 50 ℃, the reaction times extends to 72h, obtains PEOz-DSPE.This reaction has very strong selectivity, to not influence of aminoterminal, has avoided the generation of side reaction.The present invention finds " click chemistry " can be applied in liposome material synthetic pleasantly surprisedly.
In one embodiment, the invention provides the method that the F-PEOz-DSPE that synthesizes with the present invention prepares Evacet.This method for example can be to carry out in the following manner:
At first, the PEOz-DSPE that hydrogenated soy phosphatidyl choline (HSPC), cholesterol (Chol) and the present invention are synthesized (being the compound of formula 9 or formula 9a) or F-PEOz-DSPE (being the compound of formula I or formula Ia) or be used for the PEG of contrast
2000-DSPE is prepared by film dispersion method, can obtain optional pH responsive type lipidosome drug carrier with folic acid targeting; Wherein, in this lipidosome drug carrier, preferred, each component can comprise according to the molar part meter:
Should be appreciated that in this article, more than (1), (2), (3) three can be referred to as " TL " or " lipid mixt "; And above (1), (2), (3) three's mixture or the treated formed lipidosome drug carrier of this three also can be described as " TL " or " lipid mixt ".Certainly, those skilled in the art understand, phrase " the treated formed lipidosome drug carrier of this three " refers to that three kinds of compositions pass through " lipidosome drug carrier " that obtains after the liposome preparation processes, should " lipidosome drug carrier " can also comprise other component in addition, and when mentioning " the treated formed lipidosome drug carrier of this three ", term " TL " refers to above-mentioned (1), (2), (3) three in this liposome.For example phrase " TL amount " refers to above-mentioned in " lipidosome drug carrier " (1), (2), (3) three's amount sum, and other composition in the liposome is not counted in " TL amount ".
Then, to have water miscible Zorubicin and the above-mentioned lipidosome drug carrier that makes (namely) carries out the preparation of drug-loaded liposome by the ammonium sulfate density gradient method, can obtain optional Evacet or Zorubicin contrast liposome with pH responsive type of folic acid targeting.In a preferred embodiment, in the liposome that obtains thus, comprise:
| ?(a) | Antitumor drug (for example Zorubicin) | 1～30 weight part and |
| ?(b) | TL (i.e. above (1), (2), (3) three's sum) | 70～99 weight parts; |
Preferably, comprise:
| ?(a) | Antitumor drug (for example Zorubicin) | 1～20 weight part and |
| ?(b) | TL (i.e. above (1), (2), (3) three's sum) | 80～99 weight parts; |
Preferably, comprise:
| ?(a) | Antitumor drug (for example Zorubicin) | 2～20 weight parts and |
| ?(b) | TL (i.e. above (1), (2), (3) three's sum) | 80～98 weight parts; |
Preferably, comprise:
| (a) | Antitumor drug (for example Zorubicin) | 2～10 weight parts and |
| (b) | TL (i.e. above (1), (2), (3) three's sum) | 90～98 weight parts. |
Therefore, for example, in an embodiment of the drug administration carrier of seventh aspect present invention, this drug administration carrier comprises the TL that following mol ratio is formed:
Thus, for example in an embodiment of the drug administration carrier of seventh aspect present invention, this drug administration carrier comprises again:
(a) active constituents of medicine (for example antitumor drug, for example Zorubicin) and
(b) TL of following mol ratio composition:
And wherein said (a) active constituents of medicine with (b) weight of TL is:
In one embodiment, drug administration carrier (for example for drug-loaded liposome) above except comprising (a) active constituents of medicine and (b) TL, can also comprise other medicines activeconstituents and the acceptable shape agent of other pharmacy, for example water, sodium chloride solution, glucose solution etc.
In one embodiment, the drug administration carrier of seventh aspect present invention is a kind of liposomal pharmaceutical preparation, and wherein each component is formed according to following weight percent:
Wherein in described mixture, each component mol ratio is:
It will be appreciated by those skilled in the art that term " weight part " is a kind of weight unit, it can be microgram, milligram, gram, kilogram etc.Similarly, term " molar part " is a kind of molar weight unit, and it can be micromole, mmole, mole etc.
In one embodiment, the invention provides the preparation method of (initiatively target) liposome of pH responsive type, it can may further comprise the steps substantially:
At first, described each lipid composition (for example phosphatide, cholesterol, formula I of the present invention or formula 9 compounds) is dissolved with chloroform, rotary evaporation is removed solvent, adds (NH
4)
2SO
4Solution is to adipose membrane, and ultra-sonic dispersion gets blank liposome turbid liquor, with high pressure homogenizer suspension is passed through polycarbonate leaching film; Namely get blank liposome;
Then, take by weighing Zorubicin, soluble in water, the aqueous solution is added in the blank liposome, the heating jolting namely gets (initiatively target) liposome of pH responsive type of the present invention.
In one embodiment, the invention provides the preparation method of (initiatively target) liposome of pH responsive type, it can may further comprise the steps particularly:
(1) gets each component by following weight part: Zorubicin 1-30 weight part (preferred 1-20, preferred 2-20, preferred 2-10 weight part), lipid mixt (hydrogenated soy phosphatidyl choline, cholesterol and PEOz-DSPE (or F-PEOz-DSPE or PEG
2000-DSPE)) 70-99 weight part (preferred 80-99, preferred 80-98, preferred 90-98 weight part), in this lipid mixt, the mol ratio of three constituents is: hydrogenated soy phosphatidyl choline 60～70 molar part, cholesterol 20～38 molar part, PEOz-DSPE (or F-PEOz-DSPE or PEG-DSPE) 2～10 molar part;
(2) above-mentioned each component is dissolved rotation vacuum-evaporation film forming (temperature is 10-50 ℃, is preferably 20-40 ℃, more preferably about 35 ℃) with chloroform.With 250mmol/L (NH
4)
2SO
4Solution (pH=4.5-6.5 is preferably pH=5.0-6.0, and preferred pH is about 5.5) adds in the adipose membrane, and (temperature is 40-80 ℃ to ultra-sonic dispersion, be preferably 50-70 ℃, more preferably about 65 ℃, ultra-sonic dispersion 5-60min, preferred 10-30min, more preferably from about 20min), get blank liposome turbid liquor.With high pressure homogenizer with the blank liposome suspension continuously by the aperture the polycarbonate leaching film of 200nm, 100nm, 80nm (be preferably and respectively filter 5 times, 65 ℃ of extruding down), get the blank liposome solution of ammonium sulfate dissolving.Adopt the wet method perfusion to fill chromatography column, pour into sephadex G-25 continuously, (use 5-20 column volume with 10% sucrose solution elution chromatography post, be preferably 10 column volumes), the blank liposome solution that adds the ammonium sulfate dissolving (is preferably flow velocity=1ml/min), collect the milky liquid that contains liposome, with 5% glucose solution elution chromatography post (with 5-20 column volume, being preferably 10 column volumes).It is an amount of that precision takes by weighing Zorubicin in addition, be dissolved in 10% sucrose solution, this solution is added in the collection blank liposome solution, in hot water bath, place (preferred 65 ℃, 30min～60min), jolting frequently, the liposome that has wrapped up medicine is added chromatography column, with 5% glucose solution wash-out, to remove the Zorubicin that is not wrapping to liposome interior, collect effluent liquid, namely get Evacet.
After testing, the prepared liposome of aforesaid method (adopts three kinds of material PEOz-DSPE or F-PEOz-DSPE or PEG respectively
2000All between 90～200nm, entrapment efficiency is all between 85%～98% for-particle diameter DSPE).
Utilize cationic ring-opening polymerization and " click " chemical reaction of success of the present invention have prepared folic acid-poly-(2-ethyl-2-oxazoline)-DSPE (F-PEOz-DSPE) liposome vectors material of three kinds of different molecular weights.Experiment showed, that the liposome that is respectively 18,32,64 PEOz-DSPE and F-PEOz-DSPE preparation with the exemplary synthetic polymerization degree of the present invention has excellent particle size distribution and higher entrapment.In experiment in vitro, the F-PEOz-DSPE liposome shows stronger tumor-targeting than PEOz-DSPE liposome and PEG-DSPE liposome, does not see significant cytotoxicity.
Description of drawings
Fig. 1 is the proton nmr spectra of PEOz
Fig. 2 is the carbon-13 nmr spectra of Terminal Acetylenes base DSPE
Fig. 3 is the high-efficient liquid phase chromatogram of Terminal Acetylenes base DSPE
Fig. 4 is that (Fig. 4 a) and the infared spectrum of PEOz-DSPE (Fig. 4 b) for PEOz
Fig. 5 is the uv-spectrogram of PEOz-DSPE and F-PEOz-DSPE
Fig. 6 is the gel permeation chromatography figure of F-PEOz-DSPE
Fig. 7 is the proton nmr spectra of F-PEOz-DSPE
Fig. 8 has described the cumulative release rate change curve of three kinds of liposomes in the phosphate buffered saline buffer of pH 7.4 and pH 5.0.Among each figure, ordinate zou is cumulative release percentage ratio (%), X-coordinate be time of releasing (hour).Wherein Fig. 8 A is the release profiles of PEG-DSPE liposome, and Fig. 8 B is the release profiles of PEOz-DSPE liposome, and Fig. 8 C is the release profiles of F-PEOz-DSPE liposome.
Fig. 9 has described three kinds of liposomes to the external pharmacodynamics evaluation result of SKOV3 cell (Fig. 9 A) and A549 cell (Fig. 9 B).Among each figure, ordinate zou is inhibiting rate (%), X-coordinate be incubation time (hour).In Fig. 9 A, the post at left side DOX place is represented is that Zorubicin without liposome is to the cell inhibiting rate; 0.25,0.5,1,2,4,12,24 or the time point place of 48h, 3 posts from left to right represent to wrap medicine liposome PEG-DSPE, the cell inhibiting rate of PEOz-DSPE, F-PEOz-DSPE respectively separately; In Fig. 9 B, each post implication is with identical at Fig. 9 A.
Figure 10 A has described the liposome of different film material different concns to the influence of cell absorption.
The influence that the liposome that Figure 10 B has described different film materials is taken in cell under differing temps.
Figure 10 C has described the influence that cell is taken in liposome under different incubation times of different film materials.
Figure 10 D has described the absorption of flow cytometer showed cell to different liposome.
Embodiment
Can further describe the present invention by the following examples, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the test method that use in the test.Though for realizing that the employed many materials of the object of the invention and working method are well known in the art, the present invention still does detailed as far as possible description at this.In the present invention, as not explanation in addition, entrapment efficiency is to measure with classical sephadex column partition method, and the liposome particle diameter uses the particle diameter instrument to measure.
A, embodiment part: preparation F-PEOz-DS
The preparation of embodiment 1:6-nitrine-1-hexanol (compound 2)
As following formula, in the 250mL of drying there-necked flask, add 6-chloro-1-hexanol (compound 1) 10g (73mmol), NaN
314.3g (220mmol), NaI 1.1g (7.3mmol) and the dry 100mLDMF that crosses, stirring reaction 18h under 80 ℃ of oil baths.After reacting end, vacuum rotary steam adds ethyl acetate 200mL dilution, difference water and saturated aqueous common salt washed twice, K to doing
2CO
3Dry organic phase, suction filtration, filtrate decompression is concentrated into dried colourless liquid 10.02g, and yield is 95%.
1H?NMR(300MHz，CDCl
3)δ：1.30-1.71(m，8H)；3.28(t，2H)；3.65(t，2H)。
Embodiment 2: the preparation of initiator 6-nitrine-own ester of 1-tosic acid (compound 3)
As following formula, in the 500mL of drying single port flask, add the dry chloroform of crossing of 6-nitrine-1-hexanol (compound 2) 10g (70mmol), triethylamine 20mL (145mmol), trimethylamine hydrochloride 1.3g (13.5mmol) and 300mL, stirring and dissolving dropwise adds the dry CHCl of 60mL of Tosyl chloride 20g (105mmol) under the ice bath
3Solution, again in room temperature reaction 1h, the reaction solution vacuum rotary steam concentrates behind 0～5 ℃ of reaction 1h, and it is colourless to be washed to organic layer, anhydrous CaCl
2Dry organic phase, suction filtration, filtrate decompression is revolved and is steamed to doing, and gets colourless liquid.(eluent is ethyl acetate: sherwood oil=1: 30), get pure product is colourless liquid 10.39g to crude product, productive rate 50% through the silica gel chromatography column separating purification.
1H?NMR(300MHz，CDCl
3)δ：1.27(m，4H)；1.41-1.61(m，4H)；2.46(s，3H)；3.23(t，2H)；4.02(t，2H)；7.34(d，2H)；7.79(d，2H)。
Proton nmr spectra (not shown) shows that terminal hydroxy group has changed into p-toluenesulfonic esters.In addition, in infared spectrum (not shown), can see at 2097cm
-1There is the charateristic avsorption band of nitrine at the place.The proton nmr spectra endlap nitrogen initiator structure that explanation is synthesized with infared spectrum is identical with expected structure.
Embodiment 3: the preparation of poly-(2-ethyl-2-oxazoline) (compound 4a)
As following formula, add an amount of toluene in the initiator (compound 3), through twice component distillation to remove residual moisture.In three Schlenk bottles that are connected on the vacuum line, add EOz monomer 8.5mL (84mmol) and acetonitrile 25mL respectively, add initiator (compound 3) 0.620mL (2.5mmol) under the nitrogen respectively, 0.310mL (1.3mmol), 0.155mL (0.63mmol) (by three kinds of different molecular weight designs, be denoted as 1,2, No. 3 bottles).The Schlenk bottle is sealed back 70 ℃ of reactions down, and 1, No. 2 bottle respectively reacts 24h, No. 3 bottle reaction 48h.1,2,3 bottle be connected in feed high pure nitrogen on the vacuum line, add NH under the ice bath respectively
3/ acetonitrile solution (ammonia is blasted in the acetonitrile, and molecule number ratio is 1: 16) 23.6mL, 11.8mL, 5.9mL react 24h under the room temperature, are used for termination reaction.Add K afterwards respectively
2CO
325g (181mmol) stirs 24h under the room temperature, is used for removing tosic acid.After reaction finished, suction filtration was removed K
2CO
3, the rotary evaporation desolventizing, minimum of chloroform dissolving dropwise is added in the ether precipitated product 2 times, suction filtration, after the vacuum-drying white solid 6.33g, 5.08g, 4.66g, productive rate is respectively 76%, 61%, 56% (being numbered PEOz1, PEOz2 and PEOz3).
The sign of polymer P EOz and the mensuration of molecular weight
Fig. 1 is PEOz's
1H NMR spectrogram, chemical shift δ=1.13 places are the methyl (CH on the side chain
3) proton peak; δ=2.44 places are by methylene radical (O=C-CH on the side chain
2-) proton causes; Methylene radical (CH on the main chain
2-CH
2-N-) proton peak appears at δ=3.57 places.In addition, in infared spectrum, can see at 2099cm
-1There are the charateristic avsorption band of nitrine, 1639cm in the place
-1Characteristic peak for teritary amide (O=C-N-).More than the synthetic polymer architecture of explanation conforms to design.
For measuring the molecular weight of polymkeric substance, " Click " (" click ") reaction is taken place with compound 1-ethanoyl-2-nitro-4-proyl oxygen base-5-anisole respectively in three kinds of polymkeric substance (PEOz1, PEOz2 and PEOz3), utilize
1The molecular weight that the integration of H on H in the H NMR spectrogram on the phenyl ring and the repeating unit calculates than the molecular weight that calculates polymkeric substance with by turnover ratio is used as shown in Table 1
1The calculation result of H NMR is as the molecular weight of polymkeric substance, and the polymerization degree is respectively 18,32 and 64.
" Click " reaction
The measurement result of table 1 PEOz molecular weight
Embodiment 4: the preparation of Terminal Acetylenes base DSPE (compound 6)
As following formula, take by weighing 4-pentynoic acid 0.8g (8.2mmol) and DCC 1.2g (5.8mmol) and be dissolved in the dry CHCl that crosses of 50mL
3In, activate 4h under the room temperature, add 200 μ L pyridines and DSPE (compound 5) (3g, 50mL CHCl 4mmol)
3Solution, 50 ℃ of reacting by heating 12h, suction filtration is removed by product DCU, organic phase washing 3～5 times, anhydrous Na SO
4Dry 2h, rotary evaporation is to dry, and vacuum-drying 12h gets white solid 2.48g, and productive rate is 75%.
The sign of Terminal Acetylenes base DSPE
ESI-MS:m/z 826.6[M-H]
-, 2300cm in the infared spectrum (not shown)
-1The place is the stretching vibration characteristic peak of Terminal Acetylenes base (H-C ≡ C-).
13C NMR (400MHz, CDCl
3) δ: 173.41 (m); 83.12 (l); 69.75 (k); 69.13 (j); 62.11 (i); 35.12 (h); 34.24 (f); 32.75 (g); 31.90 (e); 29.41 (c); 25.12 (d); 22.84 (b); (a) 14.12 (as Fig. 2).
HPLC:Diamond C
18Chromatographic column (250mm * 4.6mm, 5 μ m); Moving phase is water: methyl alcohol=70: 30, flow velocity 1.0mL/min; Detect wavelength 242nm; 30 ℃ of column temperatures; Sample size 20 μ L.High-efficient liquid phase chromatogram is seen Fig. 3, and the t=10.784min place is the chromatographic peak of Terminal Acetylenes base DSPE, and peak area ratio is 96.94%, and the Terminal Acetylenes base DSPE that visible present embodiment obtains has higher degree.
Embodiment 5: folic acid Acibenzolar (compound 8, preparation F-NHS)
As following formula, take by weighing folic acid (compound 7)) 5g (11.3mmol), N-maloyl imines (NHS) 1.3g (11.3mmol) places the 100mL reaction flask, add triethylamine 25mL, DMSO 40mL, lucifuge is led to nitrogen 10min, treats that folic acid dissolves the back fully and adds DCC 2.5g (12.1mmol), lucifuge room temperature reaction 24h.Suction filtration is removed DCU, and filtrate splashes in the 400mL ethyl acetate, produces yellow mercury oxide, suction filtration, with washing with alcohol filter cake twice, with 30mL DMSO dissolving filter cake, splash into precipitated product in the 300mL ether again, suction filtration, vacuum-drying 12h gets red-brown solid 4.51g, and productive rate is 74%.
Folic acid (F)
1H NMR (300MHz, D
2O/NaOD) δ: 8.01 (1H); 7.20 (2H); 6.17 (2H); 3.98 (2H); 3.87 (1H); 2.04 (2H); 1.751.70 (2H).
F-NHS?
1H?NMR(300MHz，D
2O/NaOD)δ：8.16(1H)；7.26(2H)；6.18(2H)；4.15(2H)；4.05(1H)；2.30(4H)；2.10(2H)；1.851.80(2H)。4 H of δ=2.30 place's correspondences are the H on the succinimide ring that is connected on the folic acid.1726cm in the infared spectrum
-1The place judges to sum up that for the characteristic peak of newly-generated ester (C (=O) O-) folic acid Acibenzolar structure is correct.
The preparation of embodiment 6:PEOz-DSPE (compound 9a)
As following formula, get each 500mg of PEOz1, PEOz2 and PEOz3 (compound 4a) and put into polymerizing pipe, add Terminal Acetylenes base DSPE (compound 6) 400mg (0.48mmol), 250mg (0.30mmol), 140mg (0.17mmol) respectively, and add part PMDETA (ρ=0.829g/mL) 110 μ L, 69 μ L, 38 μ L, CHCl respectively
3Each 2mL.After twice " cooled with liquid nitrogen-vacuumize-thaw " cyclical operation, feed high pure nitrogen, add CuBr 72mg (0.5mmol), 45mg (0.3mmol), 25mg (0.17mmol) respectively, vacuumize, carry out once " cooled with liquid nitrogen-vacuumize " operation again, tube sealing under the vacuum, 50 ℃ are reacted 72h down.After reaction stops, splashing into precipitated product in the ether respectively, filter, the solid water dissolution, it is centrifugal that (3800rpm * 5min), repeat 3 times removes insolubles, merges the aqueous solution, freeze-drying.Obtain light green solid (a small amount of mantoquita removes when dialysing at last) 447mg, 447mg, 477mg, productive rate is respectively 63%, 72% and 85%.
Hydrogen spectrum (not shown) and the INFRARED SPECTRUM of the PEOz-DSPE of the above preparation of test.
1In H NMR (400MHz) collection of illustrative plates, there is faint " Click " fignal center at δ=7.96 places, are the proton peak on the newly-generated triazole ring, and δ=1.17 are the proton peak on the DSPE carbon long-chain.2099cm in its infared spectrum (as Fig. 4 b, Fig. 4 a is the infared spectrum of PEOz)
-1The nitrine characteristic peak at place disappears, and has illustrated " Click " reaction has taken place between nitrine and the alkynyl, namely generates triazole by 1,3-Dipolar Cycloaddition PEOz is connected with DSPE, generates PEOz-DSPE.
Measure through solvability, to be converted to the mole number of DSPE, PEOz1-DSPE, PEOz2-DSPE, the solubleness of PEOz3-DSPE in water (20 ℃) all are more than 10 times of DSPE.
Embodiment 7:F-PEOz-DSPE (compound I preparation a)
As following formula, take by weighing PEOz1-DSPE 300mg (0.11mmol), PEOz2-DSPE 200mg (0.05mmol), PEOz3-DSPE 300mg (0.04mmol) (compound 9a) respectively in three reaction flasks, add F-NHS (compound 8) 180mg (0.33mmol), 80mg (0.15mmol), 66mg (0.12mmol) respectively; Triethylamine 900 μ L, 400 μ L, 240 μ L; Each 1.5mL of DMSO, lucifuge is led to nitrogen 10min, 50 ℃ of lucifuge reaction 72h.After reaction finishes, splash in the ether respectively and be settled out, filter, with chloroform solid is dissolved, filter, get yellow filtrate, the rotary evaporation desolventizing is dissolved with less water, dialysis (MwCO 1000) is two days in distilled water, lyophilize gets faint yellow solid F-PEOz1-DSPE 165mg, F-PEOz2-DSPE 160mg, F-PEOz3-DSPE 184mg respectively, and productive rate is respectively 55%, 80%, 61%.
The sign of F-PEOz-DSPE
Test PEOz-DSPE and the F-PEOz-DSPE uv-absorbing in THF respectively, show that as Fig. 5 the maximum absorption band of PEOz-DSPE is at the 244nm place; The maximum absorption band of F-PEOz-DSPE appears at 256nm and 290nm place, and this also is the charateristic avsorption band of folic acid.The method that detects with differential detection (IR) and dual wavelength is surveyed its gel permeation chromatography figure (chromatographic column: Waters Styragel HR post 1 * 10 respectively
4, 1 * 10
3And
The aperture; Solvent: THF; Flow velocity: 1mL/min; Column temperature: 35 ℃), as shown in Figure 6, the product peak appears at the 22.5min place.
Fig. 7 has shown the proton nmr spectra of F-PEOz-DSPE,
1H NMR (300MHz, DMSO) δ: 8.70 (1H); 7.70 (2H); 6.71 (2H); 4.45 (2H); 4.25 (1H) for being connected the proton peak of the folic acid on the polymkeric substance, δ=8.00 (1H) is the proton peak on the triazole ring, δ=3.35,2.28 and 0.94 are the proton peak of polymer repeat unit, and δ=1.26 are the proton peak of carbon long-chain among the DSPE.More than Ce Shi product structure conforms to the structure of estimating F-PEOz-DSPE.
Measure through solvability, to be converted to the mole number of DSPE, PEOz1-DSPE, PEOz2-DSPE, the solubleness of PEOz3-DSPE in water (20 ℃) all are more than 10 times of DSPE.
Embodiment 8: preparation m is 4 or 8 formula I compound
The method of reference example 1-7, in embodiment 1, be initial substance with 4-chloro-1-butanols and 8-chloro-1-octanol respectively, in embodiment 3, adopt the reaction conditions that obtains n=32, make the formula I compound of n=32, m=4 and p=2 and the formula I compound of n=32, m=8 and p=2.The gained compound characterizes and performance measurement through embodiment 1-7 similar approach, the conforming to of structure structure and design as a result.
Embodiment 9: preparation p is 1 or 4 formula I compound
The method of reference example 1-7, in embodiment 4, be initial substance with 3-butynoic acid and 6-heptynoic acid respectively, in embodiment 3, adopt the reaction conditions that obtains n=18, make the formula I compound of n=18, m=6 and p=1 and the formula I compound of n=18, m=6 and p=4.The gained compound characterizes and performance measurement through embodiment 1-7 similar approach, the conforming to of structure structure and design as a result.
B, preparing carriers example: contain the preparation of the drug administration carrier of active constituents of medicine
Preparation example 1: contain PEG in the film material
2000
The preparation of the Evacet of-DSPE
The consumption of each component: Zorubicin 2-10 weight part; Lipid mixt (hydrogenated soy phosphatidyl choline, cholesterol and PEG
2000The mixture of-DSPE) 90-98 weight part, in this lipid mixt, hydrogenated soy phosphatidyl choline 60-70 molar part, cholesterol 20-38 molar part; And polyoxyethylene glycol-DSPE (PEG
2000-DSPE) 2-10 molar part.
Adopt the preparation of ammonium sulfate density gradient method, get blank liposome with high pressure homogenizer control particle diameter.It is an amount of that other takes by weighing Zorubicin, soluble in water, this solution is added in the collection blank liposome solution, in hot water bath, place (preferred 65 ℃, 30min～60min), jolting frequently, the liposome that has wrapped up medicine is added chromatography column, with 5% glucose solution wash-out, to remove the Zorubicin that is not wrapping to liposome interior, collect effluent liquid, namely get and contain PEG
2000-DSPE Evacet.
With Zorubicin 10 weight parts and lipid mixt (hydrogenated soy phosphatidyl choline 70 molar part, cholesterol 20 molar part and PEG
2000The mixture of-DSPE 10 molar part) 90 weight part feed ratio prepare liposome, test the research of example part with this liposome.With Zorubicin 2 weight parts and lipid mixt (hydrogenated soy phosphatidyl choline 60 molar part, cholesterol 38 molar part and PEG
2000The mixture of-DSPE 2 molar part) 98 weight part feed ratio prepare liposome.With Zorubicin 5 weight parts and lipid mixt (hydrogenated soy phosphatidyl choline 65 molar part, cholesterol 30 molar part and PEG
2000The mixture of-DSPE 5 molar part) 95 weight part feed ratio prepare liposome.These liposomes with the PEG preparation are analogous to business-like PEGization DSPE liposome.
More than the liposome particle diameter that makes of three kinds of feed ratio between 90～200nm, entrapment efficiency is between 85%～98%.
Preparation example 2: contain the preparation of the Evacet of PEOz-DSPE in the film material
The consumption of each component: Zorubicin 2～20 weight parts; ((embodiment 6 makes lipid mixt for hydrogenated soy phosphatidyl choline, cholesterol and PEOz-DSPE, polymerization degree n is respectively 18,32,64) mixture) the 80-98 weight part, in this lipid mixt, hydrogenated soy phosphatidyl choline 60-70 molar part, cholesterol 20-38 molar part; With poly-(2-ethyl-2-oxazoline)-DSPE (PEOz-DSPE, the polymerization degree is respectively 18,32,64) 2-10 molar part.
With reference to the preparation method of preparation example 1, get an amount of hydrogenated soy phosphatidyl choline, cholesterol and PEOz-DSPE (polymerization degree is respectively 18,32,64) with method operation, make and contain PEOz-DSPE Evacet (polymerization degree is respectively 18,32,64).
Prepare liposome with Zorubicin 10 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 70 molar part, cholesterol 20 molar part and PEOz-DSPE (n=18) 10 molar part) 90 weight part feed ratio, test the research of example part with this liposome.Prepare liposome with Zorubicin 2 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 60 molar part, cholesterol 38 molar part and PEOz-DSPE (n=32) 2 molar part) 98 weight part feed ratio.Prepare liposome with Zorubicin 5 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, cholesterol 30 molar part and PEOz-DSPE (n=64) 5 molar part) 95 weight part feed ratio.Prepare liposome with Zorubicin 20 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, cholesterol 30 molar part and PEOz-DSPE (n=64) 6 molar part) 80 weight part feed ratio.
More than the liposome particle diameter that makes of various feed ratio between 90～200nm, entrapment efficiency is between 85%～98%.
Preparation example 3: contain the preparation of the Evacet of F-PEOz-DSPE in the film material
The consumption of each component: Zorubicin 2-20 weight part; ((embodiment 7 makes lipid mixt for hydrogenated soy phosphatidyl choline, cholesterol and F-PEOz-DSPE, polymerization degree n is respectively 18,32,64) mixture) the 80-98 weight part, in this lipid mixt, hydrogenated soy phosphatidyl choline 60-70 molar part, cholesterol 20-38 molar part; And folic acid-poly-(2-ethyl-2-oxazoline)-DSPE (F-PEOz-DSPE, the polymerization degree is respectively 18,32,64) 2-10 molar part.
With reference to the preparation method of preparation example 1, get an amount of hydrogenated soy phosphatidyl choline, cholesterol and F-PEOz-DSPE (polymerization degree is respectively 18,32,64) with method operation, make and contain F-PEOz-DSPE Evacet (polymerization degree is respectively 18,32,64).
Prepare liposome with Zorubicin 10 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 70 molar part, cholesterol 20 molar part and F-PEOz-DSPE (n=18) 10 molar part) 90 weight part feed ratio, test the research of example part with this liposome.Prepare liposome with Zorubicin 2 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 60 molar part, cholesterol 38 molar part and F-PEOz-DSPE (n=64) 2 molar part) 98 weight part feed ratio.Prepare liposome with Zorubicin 5 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, cholesterol 30 molar part and F-PEOz-DSPE (n=32) 5 molar part) 95 weight part feed ratio.Prepare liposome with Zorubicin 20 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, cholesterol 30 molar part and F-PEOz-DSPE (n=32) 6 molar part) 80 weight part feed ratio.Prepare liposome with Zorubicin 30 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, cholesterol 30 molar part and F-PEOz-DSPE (n=32) 5 molar part) 70 weight part feed ratio.
More than the liposome particle diameter that makes of various feed ratio between 90～200nm, entrapment efficiency is between 85%～98%.
C, test routine part: the pharmaceutical composition of liposome or the performance of drug administration carrier are investigated
The measuring method of test example 1, hydrochloric doxorubicin liposome pH susceptibility
1, reagent: first kind of liposome (namely by 10 parts of Zorubicins and 90 parts of liposomes that lipid mixt makes) of making respectively of preparation example 1,2,3 above is designated as PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE respectively; The PBS of pH 7.4 (pH 7.4 for potassium primary phosphate, 0.05M), the PBS of pH 5.0 (pH 5.0 for SODIUM PHOSPHATE, MONOBASIC, 0.05M).
2, test method: three kinds of each 1mL of hydrochloric doxorubicin liposome of accurate absorption pack in the dialysis tubing (MWCO:1000), place pH 7.4 and pH 5.0 phosphate buffered saline buffers of 100mL respectively, six beakers are put into rotary constant temperature oscillator, 37 ℃ of following joltings (40r/min).Regularly (15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 36h 48h) takes out release medium 0.5mL in each beaker, replenishes the fresh release medium of respective amount simultaneously.Measure medicament contg in each time point release medium with the HPLC method, calculate the cumulative release degree, draw release profiles.
3, test-results: three kinds of liposomes are in the phosphate buffered saline buffer of pH 7.4 and pH 5.0, and cumulative release rate change curve is respectively shown in Fig. 8 A, Fig. 8 B, Fig. 8 C in the 48h.The result shows that the release under two kinds of pH conditions of PEG-DSPE liposome does not have notable difference, and the cumulative release rate reaches about 50% (seeing Fig. 8 A) in the 48h.PEOz-DSPE is similar to the release profiles of PEG-DSPE liposome with F-PEOz-DSPE liposome rate of releasing drug in the release medium of pH 7.4, and the cumulative release rate reaches about 50% in the 48h; But they in the release medium of pH 5.0 rate of releasing drug obviously faster than the rate of releasing drug in pH 7.4 release medium, and the cumulative release rate reaches about 100% (seeing Fig. 8 B and Fig. 8 C) in 48h, and visible PEOz-DSPE and two kinds of liposomes of F-PEOz-DSPE demonstrate tangible pH susceptibility.This character is conducive to medicine and discharges under weak acid environments such as tumor tissues and inclusion body, can solve the PEG liposome in the target site deposition and discharge problem slowly, improves curative effect of medication, reduces dosage.
Test example 2, the external evaluating drug effect of liposome
1, reagent: first kind of liposome (namely by 10 parts of Zorubicins and 90 parts of liposomes that lipid mixt makes) of making respectively of preparation example 1,2,3 above is designated as PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE respectively
2, test method:
For investigating folacin receptor (FR) targeting of liposome of the present invention, in this test example, choosing the ovarian cancer cell line SKOV3 cell of FR (+) and the lung adenocarcinoma cell of FR (-) is the cell in vitro experiment that the A549 cell carries out liposome, with targeting and the validity of checking liposome.FR (+) the expression folacin receptor positive wherein, i.e. high expression level folacin receptor, FR (-) expression folacin receptor feminine gender, the i.e. low folacin receptor of expressing.
Because two kinds of cells that use in this test are to the susceptibility difference of medicine, before carrying out liposome toxicity and evaluating drug effect, need to measure Zorubicin (as not indicating in addition, the used reagent Zorubicin of the present invention refers to its hydrochloride, can be expressed as DOX at this paper) to the effective concentration of two kinds of cancer cells.This test will be killed the DOX concentration of 80% cancer cells as active drug concentration, namely will measure DOX to the IC of two kinds of cells
80This result will provide foundation for follow-up toxicity assessment and evaluating drug effect.The doxorubicin hydrochloride of different concns is to SKOV3 and A549 two kinds of cell inhibiting rates, IC as a result after measured
80(SKVO3)=12.5 μ g/mL; IC
80(A549)=100 μ g/mL.In addition after measured, three kinds of blank liposomes (first kind of liposome preparation that makes respectively with reference to preparation example 1,2,3 above, but do not add the medicine Zorubicin) can not influence the growth of two kinds of cancer cells, it there is not toxic action, to the not influence of drug action evaluation of the liposome of parcel DOX.
According to the drug sensitive test result, select two kinds of cells (SKOV3, IC A549)
80As administration concentration, with mtt assay detect three kinds above first kind of liposome (namely by 10 parts of Zorubicins and 90 parts of liposomes that lipid mixt makes, being designated as PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE respectively) of making respectively of preparation example 1,2,3 to the fragmentation effect of different tumour cells.
The same drug sensitive test of cell culture processes; Prepare certain density drug-loaded liposome, make contained drug concentration wherein meet the IC of two kinds of clones
80(SKOV3:12.5 μ g/mL; A549:100 μ g/mL); The three kinds of drug-loaded liposomes (PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE) that prepare are hatched jointly with two kinds of clones respectively, after hatching 15min, 30min, 1h, 2h, 4h, 12h, 24h, 48h, replace the substratum that contains drug-loaded liposome originally with ordinary culture medium and continue culturing cell.Each time period all arranges 5 parallel control holes, arrange simultaneously do not contain liposome under the equal drug level naked medicine group as positive control, the blank liposome under the equal liposome concentration is as negative control and the blank of not doing any processing; More than all experimental group after cultivating 48h, unifiedly detect cytoactive with mtt assay, calculate the inhibiting rate of medicine cell growth, and each group data carried out statistical study.
3, test-results: three kinds of drug-loaded liposomes to the fragmentation effect of SKOV3 and two kinds of cancer cells of A549 shown in Fig. 9 A and Fig. 9 B, the cancer cells inhibiting rate of PEOz-DSPE and two kinds of liposomes of F-PEOz-DSPE is apparently higher than business-like PEG-DSPE liposome, and it is particularly evident in initial 1h, this is consistent with three kinds of liposome extracorporeal releasing test results, PEOz-DSPE and F-PEOz-DSPE liposome have under sour environment that medicine is prominent releases phenomenon, have proved that further the PEOz block can produce pH susceptibility.Two kinds of liposomes of PEOz-DSPE and F-PEOz-DSPE do not have notable difference to the fragmentation effect of the A549 cell of FR (-), and for the SKOV3 cell of FR (+), the F-PEOz-DSPE liposome that contains the folic acid group embodies targeting behind 12h, fragmentation effect is apparently higher than the PEOz-DSPE liposome that does not have the target group.
More than experiments of these test examples show that three kinds of blank liposomes do not disturb the drug effect detected result of drug-loaded liposome to the almost not influence of growth of cell; Two kinds of liposomes of PEOz-DSPE and F-PEOz-DSPE show the cancer cells restraining effect that is better than commercialization PEG-DSPE liposome, as seen the pH susceptibility that embodies of PEOz block is conducive to medicine and plays a role in cancer cells, improve medicine to the restraining effect of cancer cells, thereby improve curative effect of medication; The F-PEOz-DSPE liposome embodies the folic acid targeting, the SKOV3 cell inhibiting effect of FR (+) is better than the PEOz-DSPE liposome of no target, A549 cell to FR (-) does not then show targeting, this folic acid targeting not only can improve medicine to the curative effect of FR (+) tumor tissues, can also reduce medicine to the toxic side effect of other tissue and organ simultaneously.
The biological characteristics of test example 3, F-PEOz--DSPE liposome
1, test method: use the SKOV3 cell, its cell to each folic acid liposome of vitro detection is taken in situation.The folic acid liposome utilizes the 25-NBD-cholesterol to carry out mark, can disperse green fluorescence, observes under fluorescent microscope.Different with 2 used reagents with test example 1, the method for this test example 3 use reference example 1-7 and preparation example 1-3 prepares three kinds of blank liposomes that do not add activeconstituents as reagent:
(1) P2000 liposome (in this test, can be called for short P2000): with reference to the method for preparation example 3, do not add Zorubicin, prepare liposome with the feed ratio of hydrogenated soy phosphatidyl choline 70 molar part, cholesterol 15 molar part, 25-NBD-cholesterol 5 molar part and PEOz1-DSPE (embodiment 6 preparation) 10 molar part.
(2) F-P2000 liposome (in this test, can be called for short F-P2000): with reference to the method for preparation example 3, do not add Zorubicin, prepare liposome with the feed ratio of hydrogenated soy phosphatidyl choline 70 molar part, cholesterol 15 molar part, 25-NBD-cholesterol 5 molar part and F-PEOz1-DSPE (embodiment 7 preparation) 10 molar part.
(3) F-P3000 liposome (in this test, can be called for short F-P2000): with reference to the method for preparation example 3, do not add Zorubicin, prepare liposome with the feed ratio of hydrogenated soy phosphatidyl choline 70 molar part, cholesterol 15 molar part, 25-NBD-cholesterol 5 molar part and F-PEOz2-DSPE (embodiment 7 preparation) 10 molar part.
2, the liposome of different concns influence that cell is taken in: SKOV3 and the various liposome that is mixed with different concns were hatched under 37 ℃ 2 hours, and the situation of observation of cell absorption the results are shown in Figure 10A under the fluorescent microscope.As can be seen, the fluorescence intensity of cell (solar flare among the figure) strengthens with the increase of liposome concentration from the graph, and the fluorescence intensity of two groups of F-P2000 and F-P3000 is organized apparently higher than P2000 under same concentrations.The increase that liposome concentration is described can strengthen cell to the absorption of liposome, and the liposome of folic acid target also can significantly improve cell to the absorption of liposome under the condition that concentration equates.Subsequent experimental selects for use the liposome of 200uM concentration to carry out.
3, under the condition of different temperatures cell is taken in the influence of liposome: the SKOV3 cell was hatched respectively 1 hour under 4 ℃ and 37 ℃ of conditions, observe under the fluorescent microscope, the results are shown in Figure 10B, find that cell is difficult to take in liposome under 4 ℃ of conditions, and under 37 ℃ of conditions, can normally take in liposome and make cell send green fluorescence (solar flare among the figure), and fluorescence intensity is obvious higher than P2000 in folic acid target liposomes F-P2000 and F-P3000, illustrates that receptor-mediated folic acid target has increased the absorption of cell to the folic acid liposome.
4, the influence that cell is taken in liposome under the different incubation times: SKOV3 and each liposome are examined under a microscope after hatching different time respectively under 37 ℃, the results are shown in Figure 10C.The fluorescence intensity (solar flare among the figure) of finding cell strengthens gradually to the prolongation of incubation time, and the amount that F-P2000 and F-P3000 were taken in by cell after 20 minutes is obviously high than the P2000 group, has shown that good receptor-mediated target takes in.
5, the flow cytometer showed cell is to the absorption of different liposome: after SKOV3 is hatched different time with various liposomes respectively, with being dispersed into single cell suspension after the trysinization, the green fluorescence intensity that utilizes the flow cytometry cell to disperse, the results are shown in Figure 10D, 4 of three reagent groups post figure among the figure are respectively the incubation time of 5min, 15min, 30min and 50min from left to right.As seen from the figure, fluorescence intensity numerical value strengthens with the prolongation of incubation time, and after hatching 15 minutes, the fluorescence intensity of F-P2000 and F-P3000 group cell is all organized apparently higher than P2000, and along with the prolongation of incubation time significantly strengthens.
Poly-(2-ethyl-2-oxazoline) that the present invention exemplarily synthesizes three kinds of different polymerization degrees (PEOz), and be connected by chemical bond with folic acid with DSPE (DSPE), be intended to synthesize the highly selective liposome vectors material that has tumor-targeting and pH susceptibility simultaneously.PEOz has well water-soluble and biocompatibility, and toxicity is low and have temperature sensitivity and pH susceptibility, and its pH susceptibility can promote the release of medicine in tumor tissues.The great majority research of existing report is that the character of research micella has confirmed its pH susceptibility with the solid support material of PEOz block as micella, and the research that this character is used for liposome at present also seldom.The present invention by special method with the PEOz block as the hydrophilic parts in the liposome vectors material, one end is connected with the target group, one end is connected with hydrophobic part, synthesize novel lipide solid support material F-PEOz-DSPE, it has tumor-targeting and pH susceptibility and can be used as a kind of good liposome material.
Claims (15)
1. with the following formula I compound:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is the integer of l to 8.
2. the compound of claim 1, it is characterized in that following (a) to (d) each or multinomial:
(a) n is 10 to 250 integer.
(b) m is 3 to 10 integer;
(c) p is 1 to 6 integer;
(d) n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 4 integer.
3. the compound of claim 1, it is with following formula Ia compound:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer.
4. with following formula 9 compounds:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
5. the compound of claim 4, it is characterized in that following (a) to (d) each or multinomial:
(a) n is 10 to 250 integer.
(b) m is 3 to 10 integer;
(c) p is 1 to 6 integer;
(d) n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 4 integer.
6. the compound of claim 4, it is with following formula 9a compound:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer.
7. following formula 4 compounds:
Or its salt, wherein n is 5 to 500 integer, m is 2 to 12 integer.
8. the compound of claim 7, it is characterized in that following (a) to (d) each or multinomial:
(a) n is 10 to 250 integer.
(b) m is 3 to 10 integer;
(c) n is 10 to 250 integer, and m is 2 to 8 integer;
(d) it is with following formula 4a compound:
Or acceptable salt on its physiology, wherein n is 5 to 500 integer.
9. prepare the method for each described formula 9 compounds of claim 4-5, it may further comprise the steps:
(i) in the presence of catalyzer DCC, in solvent chloroform, make carboxylic acid and the DSPE reaction of the base of Terminal Acetylenes shown in the following formula,
Obtain containing formula 6 compounds of Terminal Acetylenes base:
(ii) make with following formula 4 compounds and the reaction of step (i) gained formula 6 compounds,
Obtain with following formula 9 compounds:
Wherein, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
10. prepare the method for claim 7 or 8 described formula 4 compounds, it may further comprise the steps:
(ia) in the presence of reagent N aI, in solvent DMF, make formula
Shown in compound and reaction of sodium azide, obtain formula
Compound;
(ib) in the presence of reagent triethylamine and/or trimethylamine hydrochloride, in solvent chloroform, make formula
Compound and Tosyl chloride reaction obtain formula
Compound;
(ic) in solvent acetonitrile, make formula
Compound and formula
Monomer reaction, charge into ammonia then with stopped reaction, obtain with following formula 4 compounds:
Wherein, X is halogen, and n is 5 to 500 integer, and m is 2 to 12 integer.
11. prepare the method for each described formula I compound of claim 1-2, it may further comprise the steps:
(a) in solvent triethylamine and/or DMSO, in the presence of catalyzer DCC, make folic acid and following formula N-maloyl imine reaction, obtain the folic acid Acibenzolar with following formula 8:
(b) in the presence of alkali triethylamine, in solvent DMSO, make formula 8 compounds and react with following formula 9 compounds,
Obtain formula I compound:
With optional
(c) make gained formula I compound purifying and/or salify,
Wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
12. the purposes of each described formula 9 compounds of each described formula I compound of claim 1-3 or claim 4-6 in the preparation drug administration carrier.
13. the purposes of claim 12, it is characterized in that following (a) to (g) each or multinomial:
(a) described formula I compound or formula 9 compounds are that one of lipid composition as described drug administration carrier is used;
(b) described drug administration carrier is pH responsive type drug administration carrier;
(c) described drug administration carrier is the drug administration carrier with cancer target performance;
(d) described drug administration carrier is pH responsive type and drug administration carrier with cancer target performance;
(e) described drug administration carrier is the drug administration carrier that contains or do not contain active constituents of medicine;
(f) described drug administration carrier is liposome;
(g) described drug administration carrier is initiatively target liposomes of pH responsive type.
14. a drug administration carrier wherein comprises each described formula 9 compounds of each described formula I compound of claim 1-3 or claim 4-6, and optional active constituents of medicine.
15. the drug administration carrier of claim 14, it is characterized in that following (a) to (g) each or multinomial:
(a) described formula I compound or formula 9 compounds are that one of lipid composition as described drug administration carrier is used;
(b) described drug administration carrier is pH responsive type drug administration carrier;
(c) described drug administration carrier is the drug administration carrier with cancer target performance;
(d) described drug administration carrier is pH responsive type and drug administration carrier with cancer target performance;
(e) described drug administration carrier is the drug administration carrier that contains or do not contain active constituents of medicine;
(f) described drug administration carrier is liposome;
(g) described drug administration carrier is initiatively target liposomes of pH responsive type.
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