CN103394095B - Two chloro-1,2-cyclohexanediamine close platinum complex and preparation method thereof - Google Patents
Two chloro-1,2-cyclohexanediamine close platinum complex and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of two chloro-1,2-cyclohexanediamine and close platinum complex, close platinum by two chloro-1,2-cyclohexanediamine and obtain with the polymer complex effect with formula (I) structure.Described two chloro-1,2-cyclohexanediamine close platinum and have cyclohexanediamine structure, can form stable inner core, good stability in physiological conditions with after polymer complex owing to piling sum hydrophobic interaction; Described coordination compound can effectively avoid due to intravenous injection enter blood circulation after two chloro-1, the 2-cyclohexanediamine that occur close the problems such as the unexpected release of platinum and non-specific interaction; Described coordination compound can assemble formation micellar structure, by strengthening infiltration and retention effect realizes in the accumulation of tumor locus, thus can realize the object reducing toxic and side effects and raising therapeutic effect.Secondly, institute adopts polymer to have good biocompatibility, can vivo degradation, and therefore prepare two chloro-1,2-cyclohexanediamine conjunction platinum complex dissolubilities are good.？
Description
Technical field
The present invention relates to polymer drug field, particularly two chloro-1,2-cyclohexanediamine close platinum complex and preparation method thereof.
Background technology
Oxaliplatin (oxalate base-1,2-cyclohexanediamine closes platinum) be the cancer therapy drug with cytotoxicity, due to its oncotherapy spectrum wider, with cisplatin without cross resistance, become the representative of third generation platinum medicine, be used for clinical practice in 2002 by the approval of U.S. food Drug Administration.
Oxaliplatin enters after in body to be needed through hydrolysis generation two chloro-1,2-cyclohexanediamine closes platinum or hydration 1,2-cyclohexanediamine closes platinum, and then to be combined with DNA or RNA and to play antitumor action (B.Desoize, etal.CriticalReviewsinOncology/Hematology, 42 (2002) 317-325), therefore two chloro-1,2-cyclohexanediamine close the prodrug that platinum (DACHPt) can be considered as oxaliplatin.As the homologue of oxaliplatin, two chloro-1,2-cyclohexanediamine close platinum and want high a lot of compared to the functioning efficiency of oxaliplatin, but its dissolubility and poor stability, therefore do not find broad application.
Prior art discloses the multiple mode utilizing polymer support to support medicine and develop the method that two chloro-1,2-cyclohexanediamine close platinum preparation.The PEGization liposome that utilizes as I.Han report supports two chloro-1,2-cyclohexanediamine closes platinum derivatives can improve its therapeutic effect, but this loading mode just has the drug release more than 50% in phosphate buffered solution after 2 hours, therefore cannot ensure medicine enter blood after long circulating.The seminar of Kataoka utilizes Polyethylene Glycol-b-polyglutamic acid compound two chloro-1,2-cyclohexanediamine closes DACHPt micelle (H.Cabral prepared by platinum, etal.JournalofControlRelease, 101 (2005) 223-232) can stablize and support platinum medicine, and the tumor locus realizing higher than oxaliplatin 20 times builds up concentration.But its gained micelle forms through crosslinked action between linear polyamino acid side chain, the lyophilized powder of the method gained complex after lyophilizing is difficult to redissolve, and thus limits it and further applies.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of two chloro-1,2-cyclohexanediamine closes platinum complex, there is good dissolubility, stable existence in physiological conditions, and can effectively avoid due to intravenous injection enter blood circulation after the problem such as unexpected release and non-specific interaction that occurs.
The invention discloses a kind of two chloro-1,2-cyclohexanediamine and close platinum complex, close platinum by two chloro-1,2-cyclohexanediamine and formed with the polymer complex with formula (I) structure,
In formula (I), R
1independently be selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10, phenyl or R '-CO-, R ' and be independently selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10 or phenyl;
R
2independently be selected from the alkyl of H atom, the alkyl of C1 ~ C20 or the C1 ~ C20 of replacement;
R
3independently be selected from H atom or cation;
L is independently selected from-CH
2-or-CH
2-CH
2-;
M is the degree of polymerization, 40≤m≤250; X, y, z are the degree of polymerization, 10≤x+y+z≤5000,5%≤y/ (x+y+z)≤80%.
Preferably, R
2independently be selected from the alkyl of the alkyl of C1 ~ C10 or the C1 ~ C10 of replacement, the substituent group of the alkyl of described replacement be selected from ketal, acetal, hydroxyl, aldehyde radical, amino, sulfydryl and saccharide residue one or more;
R
3independently be selected from H atom, metal cation or organic cation.
Preferably, R
3independently be selected from H atom, sodium ion, potassium ion, the subgroup of magnesium, amine ion or aminoacid ion.
Preferably, 30≤x+y+z≤300,5%≤y/ (x+y+z)≤50%.
Preferably, R
1for C6 alkyl, R
2for methyl, R
3for hydrogen atom or sodium ion, L are-CH
2-CH
2-.
The invention also discloses the preparation method that a kind of two chloro-1,2-cyclohexanediamine close platinum complex, comprise the following steps:
Two chloro-1,2-cyclohexanediamine close platinum and in an aqueous medium complex reaction occur with the polymer with formula (I) structure, generate two chloro-1,2-cyclohexanediamine and close platinum complex;
In formula (I), R
1independently be selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10, phenyl or R '-CO-, R ' and be independently selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10 or phenyl;
R
2independently be selected from the alkyl of H atom, the alkyl of C1 ~ C20 or the C1 ~ C20 of replacement;
R
3independently be selected from H atom or cation;
L is independently selected from-CH
2-or-CH
2-CH
2-;
M is the degree of polymerization, 40≤m≤250; X, y, z are the degree of polymerization, 10≤x+y+z≤5000,5%≤y/ (x+y+z)≤80%.
Preferably, described two chloro-1,2-cyclohexanediamine conjunction platinum also comprise other derivants with 1,2-cyclohexanediamine part.
Preferably, there is described in the mol ratio that the carboxyl of the polymer of formula (I) structure and two chloro-1,2-cyclohexanediamine close Pt in platinum and be less than 20.
Preferably, there is described in the mol ratio that the carboxyl of the polymer of formula (I) structure and two chloro-1,2-cyclohexanediamine close Pt in platinum and be less than 10.
Preferably, described aqueous medium is water, normal saline, buffer solution, tissue culture medium or body fluid.
Compared with prior art, two chloro-1, the 2-cyclohexanediamine that the present invention utilizes two chloro-1,2-cyclohexanediamine conjunction platinum and the polymer with formula (I) structure to prepare close platinum complex.Described two chloro-1,2-cyclohexanediamine close platinum and have cyclohexanediamine structure, can form stable inner core, good stability in physiological conditions after itself and polymer complex owing to piling sum hydrophobic interaction; Described coordination compound can effectively avoid due to intravenous injection enter blood circulation after two chloro-1, the 2-cyclohexanediamine that occur close the problems such as the unexpected release of platinum and non-specific interaction; Described coordination compound can assemble formation micellar structure, by strengthening infiltration and retention effect (EPR effect) realizes in the accumulation of tumor locus, thus can realize the object reducing toxic and side effects and raising therapeutic effect.Secondly, adopt polymer to have good biocompatibility, can vivo degradation; Described polymer adopts grafting method side chain keyed jointing Polyethylene Glycol, and therefore prepare two chloro-1,2-cyclohexanediamine close platinum complex and have good dissolubility.
Accompanying drawing explanation
Two chloro-1,2-cyclohexanediamine prepared by Fig. 1 embodiment of the present invention 2 close the hydrodynamic radius scattergram of platinum complex;
Fig. 2 is that two chloro-1,2-cyclohexanediamine prepared by the embodiment of the present invention 2 close the releasing curve diagram of platinum complex when pH=7.4 and pH=5.5;
Fig. 3 is the light scattering diagram of two chloro-1,2-cyclohexanediamine conjunction platinum complex micelles prepared by the embodiment of the present invention 2;
In Fig. 4 embodiment of the present invention 2, two of preparation chloro-1,2-cyclohexanediamine conjunction platinum complexes and the naked medicine of oxaliplatin are to the Effect tests result figure of A549 cell.
Detailed description of the invention
In order to understand the present invention further, below in conjunction with embodiment, the preferred embodiment of the invention is described, but should be appreciated that these describe just for further illustrating the features and advantages of the present invention, instead of limiting to the claimed invention.
The embodiment of the invention discloses a kind of two chloro-1,2-cyclohexanediamine and close platinum complex, close platinum by two chloro-1,2-cyclohexanediamine and formed with the polymer complex with formula (I) structure,
In formula (I), R
1independently be selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10, phenyl or R '-CO-, R ' and be independently selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10 or phenyl, R
1be preferably straight chained alkyl, the branched alkyl of C4 ~ C8, the phenyl of C3 ~ C8, be more preferably C6 alkyl;
R
2independently be selected from the alkyl of H atom, the alkyl of C1 ~ C20 or the C1 ~ C20 of replacement, be preferably the alkyl of H atom, the alkyl of C1 ~ C10 or the C1 ~ C10 of replacement, the substituent group of the alkyl of described replacement be selected from ketal, acetal, hydroxyl, aldehyde radical, amino, sulfydryl and saccharide residue one or more;
R
3independently be selected from H atom or cation, be preferably H atom, metal cation or organic cation, be more preferably H atom, sodium ion, potassium ion, the subgroup of magnesium, amine ion or aminoacid ion, most preferably be H atom or sodium ion;
L is independently selected from-CH
2-or-CH
2-CH
2-;
M is the degree of polymerization, 40≤m≤250, is preferably 45≤m≤200; X, y, z are the degree of polymerization, 10≤x+y+z≤5000, are preferably 30≤x+y+z≤300, are more preferably 50≤x+y+z≤250; 5%≤y/ (x+y+z)≤80%, is preferably 5%≤y/ (x+y+z)≤50%.
In the present invention, the polymer with formula (I) structure is preferably R
1for n-hexyl, R
2for methyl, R
3for H atom, L is-CH
2-.Now, polymer has the structure of formula (I-a).
M is the degree of polymerization, 40≤m≤250, is preferably 45≤m≤200; X, y, z are the degree of polymerization, 10≤x+y+z≤5000, are preferably 30≤x+y+z≤300, are more preferably 50≤x+y+z≤250; 5%≤y/ (x+y+z)≤80%, is preferably 5%≤y/ (x+y+z)≤50%.
In the present invention, the polymer with formula (I) structure is preferably R
1for n-hexyl, R
2for methyl, R
3for H atom, L is-CH
2-CH
2-.Now, polymer has the structure of formula (I-b).
M is the degree of polymerization, 40≤m≤250, is preferably 45≤m≤200; X, y, z are the degree of polymerization, 10≤x+y+z≤5000, are preferably 30≤x+y+z≤300, are more preferably 50≤x+y+z≤250; 5%≤y/ (x+y+z)≤80%, is preferably 5%≤y/ (x+y+z)≤50%.
In the present invention, the polymer with formula (I) structure is preferably R
1for n-hexyl, R
2for H atom, R
3for H atom, L is-CH
2-.Now, polymer has the structure of formula (I-c).
M is the degree of polymerization, 40≤m≤250, is preferably 45≤m≤200; X, y, z are the degree of polymerization, 10≤x+y+z≤5000, are preferably 30≤x+y+z≤300, are more preferably 50≤x+y+z≤250; 5%≤y/ (x+y+z)≤80%, is preferably 5%≤y/ (x+y+z)≤50%.
In the present invention, the polymer with formula (I) structure is preferably R
1for n-hexyl, R
2for H atom, R
3for H atom, L is-CH
2-CH
2-.Now, polymer has the structure of formula (I-d).
M is the degree of polymerization, 40≤m≤250, is preferably 45≤m≤200; X, y, z are the degree of polymerization, 10≤x+y+z≤5000, are preferably 30≤x+y+z≤300, are more preferably 50≤x+y+z≤250; 5%≤y/ (x+y+z)≤80%, is preferably 5%≤y/ (x+y+z)≤50%.
The embodiment of the invention also discloses a kind of two chloro-1,2-cyclohexanediamine closes the preparation method of platinum complex, comprise the following steps: two chloro-1,2-cyclohexanediamine closes platinum and in an aqueous medium complex reaction occurs with the polymer with formula (I) structure, generate two chloro-1,2-cyclohexanediamine and close platinum complex;
In formula (I), R
1independently be selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10, phenyl or R '-CO-, R ' and be independently selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10 or phenyl, R
1be preferably straight chained alkyl, the branched alkyl of C4 ~ C8, the phenyl of C3 ~ C8, be more preferably C6 alkyl;
R
2independently be selected from the alkyl of H atom, the alkyl of C1 ~ C20 or the C1 ~ C20 of replacement; be preferably the alkyl of H atom, the alkyl of C1 ~ C10 or the C1 ~ C10 of replacement, the substituent group of the alkyl of described replacement be selected from ketal, acetal, hydroxyl, aldehyde radical, amino, sulfydryl and saccharide residue one or more;
R
3independently be selected from H atom or cation, be preferably H atom, metal cation or organic cation, be more preferably H atom, sodium ion, potassium ion, the subgroup of magnesium, amine ion or aminoacid ion, most preferably be H atom or sodium ion;
L is independently selected from-CH
2-or-CH
2-CH
2-;
M is the degree of polymerization, 40≤m≤250, is preferably 45≤m≤200; X, y, z are the degree of polymerization, 10≤x+y+z≤5000, are preferably 30≤x+y+z≤300, are more preferably 50≤x+y+z≤250; 5%≤y/ (x+y+z)≤80%, is preferably 5%≤y/ (x+y+z)≤50%.
In the present invention, the described polymer with formula (I) structure is for supporting two chloro-1,2-cyclohexanediamine closes the carrier of platinum, two chloro-1,2-cyclohexanediamine close platinum at least one chloride ion as leaving group substitute by the carboxyl in polymer formula (I) Suo Shi, remaining chloride ion is likely substituted by hydrone or remains unchanged, and another ligand 1,2-cyclohexanediamine remains unchanged.If be two carboxyl generation mating reactions that two chloro-1,2-cyclohexanediamine close platinum and described polymer, it can be coordinate in molecule, and can be also intermolecular cooperation, to this, the present invention there is no particular restriction.
Described two chloro-1,2-cyclohexanediamine close platinum and have cyclohexanediamine structure, can form stable inner core, good stability in physiological conditions after itself and polymer complex owing to piling sum hydrophobic interaction; And can effectively avoid due to intravenous injection enter blood circulation after the problem such as unexpected release and non-specific interaction that occurs.
In the present invention, described two chloro-1,2-cyclohexanediamine close platinum and preferably include various stereomeric homologue (as JournalofMedicinalChemistry, the report in 21 (1978) 1315-1318), and any platinum medicine with 1,2-cyclohexanediamine part.
In the present invention, described in there is the mol ratio (COO-/Pt) that the carboxyl of formula (I) structural polymer and two chloro-1,2-cyclohexanediamine close platinum be preferably less than 20, be more preferably and be less than 10.
In the present invention two chloro-1,2-cyclohexanediamine closes in the preparation method of platinum complex, two chloro-1,2-cyclohexanediamine closes platinum and the polymer with formula (I) structure coordinating in an aqueous medium can by the method for direct hybrid reaction, also can by first using silver nitrate pretreatment two chloro-1,2-cyclohexanediamine conjunction platinum obtains two hydration-1,2-cyclohexanediamine conjunction platinum and obtains with the method for the polymer reaction with formula (I) structure further.Preferably under lucifuge bar, the polymer with formula (I) structure is dissolved in aqueous medium, adjust ph, adds 1,2-cyclohexanediamine and close platinum, carry out mating reaction, after dialysis or ultrafiltration, obtain two chloro-1,2-cyclohexanediamine conjunction platinum complex micelles.Described aqueous medium is water, normal saline, buffer solution, tissue culture medium or body fluid, and mixing add not affecting into the organic solvent in micelle limit of power as ethanol, acetonitrile, DMF etc., be preferably deionized water.The pH value of described deionized water is preferably 6.5 ~ 8.5, is more preferably 7.0 ~ 8.0.The carboxyl concentration in an aqueous medium of described polymer is preferably 0.1mM ~ 100mM, is more preferably 1mM ~ 60mM, most preferably is 2mM ~ 20mM.After polymer is dissolved in aqueous medium, preferably its pH value is adjusted to 6.0 ~ 10.0, is more preferably adjusted to 7.0 ~ 9.0; Then add two chloro-1,2-cyclohexanediamine and close platinum generation mating reaction, the time of described mating reaction is preferably 24 ~ 72 hours, is more preferably 48 ~ 72 hours; The temperature of described mating reaction is preferably 20 DEG C ~ 40 DEG C.
According to the present invention two chloro-1,2-cyclohexanediamine closes the preparation method of platinum complex, and described two chloro-1, the 2-cyclohexanediamine obtained close platinum complex and are present in aqueous medium with the form of micelle, the hydrodynamic radius of described micelle is preferably 10nm ~ 2000nm, is more preferably 10nm ~ 300nm.
The coordination compound utilizing the method to obtain can through sterilizing, concentrated, add the process such as adjuvant after directly preserve in liquid form or for intravenous injection, make lyophilized powder after also can being carried out post processing and preserve or use.A small amount of freeze drying protectant can be added, as one or more in micromolecule aminoacid, maltose, sucrose, lactose, glucose, mannitol in described freeze-drying process.
The present invention is also not particularly limited the described source with the polymer of formula (I) structure, preferably prepares in accordance with the following methods:
There is the polymer of formula (II) structure, under dehydrant and catalyst combined effect, with the polymer generation graft reaction with formula (III) structure, obtain the polymer with formula (I) structure;
In formula (II), R
1independently be selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10, phenyl or R '-CO-, R ' and be independently selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10 or phenyl, R
1be preferably straight chained alkyl, the branched alkyl of C4 ~ C8, the phenyl of C3 ~ C8, be more preferably C6 alkyl;
R
3independently be selected from H or cation, be preferably H atom, metal cation or organic cation, be more preferably H atom, sodium ion, potassium ion, the subgroup of magnesium, amine ion or aminoacid ion, most preferably be H atom or sodium ion;
L is independently selected from-CH
2-or-CH
2-CH
2-;
N, z are the degree of polymerization, 10≤n+z≤5000, are preferably 30≤n+z≤300, are more preferably 50≤n+z≤250; 5%≤z/ (n+z)≤80%, is preferably 5%≤z/ (n+z)≤50%;
In formula (III), R
2independently be selected from alkyl or substituted alkyl; be preferably the alkyl of H atom, the alkyl of C1 ~ C10 or the C1 ~ C10 of replacement, the substituent group of the alkyl of described replacement be selected from ketal, acetal, hydroxyl, aldehyde radical, amino, sulfydryl and saccharide residue one or more;
M is the degree of polymerization, 40≤m≤250, is preferably 45≤m≤200.
The present invention's preparation has the reaction of the polymer of formula (I) structure preferably under the condition of inert gas shielding; after the polymer with formula (II) structure and the polymer dissolution with solvents with formula (III) structure under the effect of dehydrant and catalyst, there is graft reaction and obtain the polymer with formula (I) structure.Described reaction temperature is preferably room temperature; The described response time is preferably 1 ~ 5 day; Described noble gas is preferably nitrogen, and the logical nitrogen time is preferably 0.5 ~ 1 hour; Described solvent is preferably dimethyl sulfoxide, and DMF or dioxane, be more preferably dimethyl sulfoxide; Described dehydrant is preferably one or more of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride or N, N-DIC or N, N'-dicyclohexylcarbodiimide, is more preferably N, N-DIC; Described catalyst is preferably 4-lutidines.Preferably dialysis 72 hours after having the polymer of formula (I) structure described in obtaining, change water 12 times, lyophilization obtains the lyophilized powder of the polymer with formula (I) structure.
The present invention when preparation has the polymer of formula (I) structure, with have formula (II) structure polymer and there is the polymer of formula (III) structure for raw material; The source of the present invention to the block copolymer with formula (II) or formula (III) structure is not particularly limited.Wherein, formula (II) is preferably prepared in accordance with the following methods:
γ-benzyl-Pidolidone or γ-benzyl-L-aspartate are reacted under the effect of triphosgene, generates γ-benzyl-Pidolidone-N-carboxylic acid anhydride monomer or γ-benzyl-L-aspartate-N-carboxylic acid anhydride monomer; The present invention preferably under inert gas conditions, γ-benzyl-Pidolidone or γ-benzyl-L-aspartate are added in oxolane, add triphosgene reaction, sedimentation in solvent again after reaction, after obtaining solid, recrystallization, drying obtain γ-benzyl-Pidolidone-N-carboxylic acid anhydride monomer or γ-benzyl-L-aspartate-N-carboxylic acid anhydride monomer, and described solvent is preferably petroleum ether or normal hexane; Described noble gas is preferably nitrogen; The present invention is also unrestricted to described sedimentation, recrystallization and drying mode, sedimentation well known to those skilled in the art, recrystallization or drying mode.
After obtaining γ-benzyl-Pidolidone-N-carboxylic acid anhydride monomer or γ-benzyl-L-aspartate-N-carboxylic acid anhydride monomer, under the effect of initiator with formula (IV) structure, carried out ring-opening polymerization, after removing benzyl, obtained formula (II) amino acid copolymer; Or under the effect of initiator with formula (IV) structure, γ-benzyl-Pidolidone-N-carboxylic acid anhydride monomer or γ-benzyl-L-aspartate-N-carboxylic acid anhydride monomer are carried out ring-opening polymerization, after removing benzyl, be mixed to get formula (II) amino acid copolymer with sodium ion, potassium ion or magnesium ion;
R
1-NH
2(IV)；
In formula (IV), R
1independently be selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10, phenyl or R '-CO-, R ' and be independently selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10 or phenyl, R
1be preferably straight chained alkyl, the branched alkyl of C4 ~ C8, the phenyl of C3 ~ C8, be more preferably C6 alkyl.The present invention preferably under inert gas conditions; the dimethyl formamide solution of the initiator with formula (IV) structure is joined in the dimethyl formamide solution of γ-benzyl-Pidolidone-N-carboxylic acid anhydride monomer or γ-benzyl-L-aspartate-N-carboxylic acid anhydride monomer; sedimentation in a solvent after reaction; filtration obtains solid, obtains formula (II) amino acid copolymer after dry after debenzylation protecting group.Described solvent is preferably ether; Described noble gas is preferably nitrogen; The present invention is also unrestricted for described sedimentation, filtration and drying mode, sedimentation well known to those skilled in the art, filtration and drying mode.
In order to understand the present invention further, close platinum complex and preparation method thereof below in conjunction with embodiment to two chloro-1,2-cyclohexanediamine provided by the invention and be described, protection scope of the present invention is not limited by the following examples.
Embodiment 1
By the γ-benzyl-Pidolidone of 10g, under the condition of dry inert gas, add in 100ml anhydrous tetrahydro furan, fully react under the effect of 6.5g triphosgene, then petroleum ether sedimentation is added, obtain solid, after recrystallization, drying, finally obtain 7.85g γ-benzyl-Pidolidone-N-carboxylic acid anhydrides (BLG-NCA) monomer; Under the condition of noble gas, dimethyl formamide (DMF) solution of n-hexylamine is joined in the DMF solution of BLG-NCA, the amount of substance of n-hexylamine and γ-benzyl-Pidolidone-N-carboxylic acid anhydrides (BLG-NCA) compares 1:140, ether sedimentation is added after reaction, filtration obtains solid, obtains the glutamic that the degree of polymerization is the band benzyl protecting group of 140 after drying; Obtaining formula (II) topology convergence degree after Deprotection is the glutamic of 140, is designated as PLG
140.
The 1.010gPLG of above-mentioned preparation is added in the reaction bulb of drying
140, 2.020g number-average molecular weight is the poly glycol monomethyl ether of 5000, is designated as mPEG
5k, to stir, logical nitrogen 0.5 hour with the dmso solution of 20mL drying, under room temperature, nitrogen protection condition, add 24.7mg4-lutidines and 0.31mlN, N-DIC, room temperature, stirring reaction 3 days, obtain product; Product is dialysed 72 hours, change water 12 times, the polymer lyophilized powder of formula (I-b) structure after lyophilization, must be had.
Carry out nuclear magnetic resonance, NMR test to described polymer, calculating percent grafting is 5.00%, and reaction conversion ratio is 93%.The described polymer y=7 with formula (I-b) structure, is designated as P (Glu)
140-g-(mPEG
5k)
7.
Embodiment 2
P (Glu) prepared by 54.0mg embodiment 1
140-g-(mPEG
5k)
7be dissolved in 25mL distilled water, adjust ph is about 8.0, adds chloro-1, the 2-cyclohexanediamine of 24.6mg bis-and closes platinum, 37 DEG C of lucifuges reaction 72h, pure water dialysis 24h, change water 6 times to remove free two chloro-1,2-cyclohexanediamine closes platinum, obtains the micelle that two chloro-1,2-cyclohexanediamine close platinum complex; Described two chloro-1,2-cyclohexanediamine are closed the micelle aseptically quick freeze of platinum complex, lyophilization obtains two chloro-1,2-cyclohexanediamine and closes platinum complex lyophilized powder, and its conversion ratio is 85%.
Measure the hydrodynamic radius of micelle, the results are shown in Figure 1, Fig. 1 be the embodiment of the present invention 2 prepare two chloro-1,2-cyclohexanediamine closes the hydrodynamic radius scattergram of platinum complex, result shows, the hydrodynamic radius that two chloro-1,2-cyclohexanediamine close platinum complex micelle is 41nm.
Obtain two chloro-1,2-cyclohexanediamine are closed platinum complex lyophilized powder redissolve, its Pt content utilizes inductivity coupled plasma mass spectrometry to measure, according to following formulae discovery encapsulation efficiency (DLE) and retention volume (DLC);
The encapsulation efficiency that prepare two chloro-1,2-cyclohexanediamine close platinum complex is 87.2%, and retention volume is 0.91mmolPt/g.
These embodiments that embodiment 3 is added are different drug loading
P (Glu) prepared by 54.0mg embodiment 1
140-g-(mPEG
5k)
7be dissolved in 25mL distilled water, adjust ph is about 8.0, adds chloro-1, the 2-cyclohexanediamine of 12.3mg bis-and closes platinum, 37 DEG C of lucifuges reaction 72h, pure water dialysis 24h, change water 6 times to remove free two chloro-1,2-cyclohexanediamine closes platinum, obtains the micelle that two chloro-1,2-cyclohexanediamine close platinum complex; Described two chloro-1,2-cyclohexanediamine are closed the micelle aseptically quick freeze of platinum complex, lyophilization obtains two chloro-1,2-cyclohexanediamine and closes platinum complex lyophilized powder, and its conversion ratio is 93%.
Embodiment 4
P (Glu) prepared by 54.0mg embodiment 1
140-g-(mPEG
5k)
7be dissolved in 25mL distilled water, adjust ph is about 8.0, adds chloro-1, the 2-cyclohexanediamine of 6.2mg bis-and closes platinum, 37 DEG C of lucifuges reaction 72h, pure water dialysis 24h, change water 6 times to remove free two chloro-1,2-cyclohexanediamine closes platinum, obtains the micelle that two chloro-1,2-cyclohexanediamine close platinum complex; Described two chloro-1,2-cyclohexanediamine are closed the micelle aseptically quick freeze of platinum complex, lyophilization obtains two chloro-1,2-cyclohexanediamine and closes platinum complex lyophilized powder, and its conversion ratio is 98%.
Embodiment 5
P (Glu) prepared by 54.0mg embodiment 1
140-g-(mPEG
5k)
7be dissolved in 25mL distilled water, adjust ph is about 8.0, adds chloro-1, the 2-cyclohexanediamine of 3.1mg bis-and closes platinum, 37 DEG C of lucifuges reaction 72h, pure water dialysis 24h, change water 6 times to remove free two chloro-1,2-cyclohexanediamine closes platinum, obtains the micelle that two chloro-1,2-cyclohexanediamine close platinum complex; Described two chloro-1,2-cyclohexanediamine are closed the micelle aseptically quick freeze of platinum complex, lyophilization obtains two chloro-1,2-cyclohexanediamine and closes platinum complex lyophilized powder, and its conversion ratio is 98%.
Embodiment 6
Under the condition of 37 DEG C, take two chloro-1 of 5mg embodiment 2 preparation respectively, 2-cyclohexanediamine closes platinum complex, be dissolved in 5mL0.01MpH value be 7.4 phosphate and pH value be in the acetate buffer solution of 5.5, then be transferred to bag filter, the molecular cut off of bag filter is 3500, dialyses with the buffer of the corresponding pH value of 40mL, sample 3mL respectively at 12h, 24h, 48h, 72h and 120h, and add the buffer of respective amount; Utilize inductivity coupled plasma mass spectrometry to carry out quantitative analysis, obtain the variation relation that cumulative percentage release increased along with the time, release result as shown in Figure 2.Fig. 2 is that two chloro-1,2-cyclohexanediamine prepared by the embodiment of the present invention 2 close the releasing curve diagram of platinum complex when pH=7.4 and pH=5.5, and as shown in Figure 2, two chloro-1,2-cyclohexanediamine close the feature accelerating under platinum complex has slow-release capability and acid pH to discharge.
Embodiment 7
Under the condition of 37 DEG C, take two chloro-1,2-cyclohexanediamine conjunction platinum complexes prepared by 5mg embodiment 2, being dissolved in 5mL0.01MpH value is in the phosphate buffered solution of 7.4, utilizes dynamic light scattering technique to test scattering strength and the particle diameter of different time points.The results are shown in Figure shown in 3.Fig. 3 is the light scattering diagram of two chloro-1,2-cyclohexanediamine conjunction platinum complex micelles prepared by embodiment 2; A () is the scattering strength change within the testing time, (b) is the particle size results in the testing time.Result shows, within the time of 240h, scattering strength declines limited, and particle diameter does not have significant change substantially.Illustrate that two chloro-1,2-cyclohexanediamine close the stability that platinum complex micelle has overlength thus.
Comparative example 1
According to document N.Nishiyama, etal.CancerResearch, the record of 63 (2003) 9877-8983, the stability of Platinum complexes wherein can only maintain 30 hours.
Comparative example 2
P (Glu) prepared by 54.0mg embodiment 1
140-g-(mPEG
5k)
7be dissolved in 25mL distilled water, adjust ph is about 8.0, adds 19.5mg cisplatin, 37 DEG C of lucifuges reaction 72h, pure water dialysis 24h, changes water 6 times to remove cisplatin, obtains the micelle of Platinum complexes; By the micelle of described Platinum complexes aseptically quick freeze, lyophilization obtains Platinum complexes lyophilized powder, and its conversion ratio is 85%.
Under the condition of 37 DEG C, take the Platinum complexes of the above-mentioned preparation of 5mg, being dissolved in 5mL0.01MpH value is in the phosphate buffered solution of 7.4, utilizes dynamic light scattering technique to test scattering strength and the particle diameter of different time points.Experimental result shows, within the time of 60h, scattering strength drops to 0.2 by 1.0, and micelle particle diameter obviously diminishes, and micelle decomposes gradually.Illustrate that Platinum complexes micelle stability 1,2-cyclohexanediamine chloro-not as good as two closes platinum complex micelle thus.
Embodiment 8
Collect logarithmic (log) phase A549 cell, adjustment cell concentration, inoculates in 96 orifice plates, containing 100 μ L(about 10 in every hole
4individual) cell, abandon culture fluid after 37 DEG C of cultivation 24h;
By culture medium prepared by embodiment 2 two chloro-1, it is the sample of 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, a 1.25 μ g/mL6 concentration that 2-cyclohexanediamine conjunction platinum complex closes Pt concentration dilution in platinum according to two chloro-1,2-cyclohexanediamine respectively; Same being diluted respectively by naked for oxaliplatin medicine by culture medium is the sample that Pt concentration is respectively 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, a 1.25 μ g/mL6 concentration;
Each sample is added 96 orifice plates, and every hole adds 200 μ L, often kind of concentration 6 multiple holes, at 37 DEG C, and saturated humidity, 5%CO
248h and 72h is cultivated in cell culture incubator, every hole adds 20 μ L concentration is afterwards the tetrazolium bromide of 5mg/mL, stop cultivating after continuing to cultivate 4h, suck culture fluid in hole, every hole adds 150 μ L dimethyl sulfoxide, low-speed oscillation 10min, the absorption value of each hole at 492nm place is detected by microplate reader, convert the survival rate obtaining using the oxaliplatin of each concentration naked medicine platinum and two chloro-1,2-cyclohexanediamine to close cell after platinum complexes, the results are shown in Figure 4.Fig. 4 is that two chloro-1,2-cyclohexanediamine prepared by the embodiment of the present invention 2 close platinum complexes and the naked medicine of oxaliplatin to the Effect tests result figure of A549 cell.In Fig. 4,
for using the survival rate of the naked medicine of oxaliplatin 48 hours inner cells of variable concentrations;
for the survival rate using two of variable concentrations chloro-1,2-cyclohexanediamine to close platinum complex 48 hours inner cells;
for using the survival rate of the naked medicine of oxaliplatin 72 hours inner cells of variable concentrations;
for the survival rate using two of variable concentrations chloro-1,2-cyclohexanediamine to close platinum complex 48 hours inner cells.As shown in Figure 4, two chloro-1,2-cyclohexanediamine conjunction platinum complexes have the drug effect and dosage-time-dependent relation that naked medicine is suitable with oxaliplatin.
Embodiment 9
Be divided at random by 6 Wistar rats 2 groups (n=3, average weight is 240g), tail vein injection oxaliplatin (Oxaliplatin) and two chloro-1,2-cyclohexanediamine close platinum complex (calculating dosage for 3mg/kg with Pt) respectively.Predetermined time point (5min, 0.5,1,4,10,24h), by eye socket collect blood sample to containing heparin sodium centrifuge tube in, centrifugalize obtains serum.Get 200 μ L blood serum samples, after nitric acid digestion, standardize solution tests platinum content to 4mL by inductivity coupled plasma mass spectrometry (ICP-MS), investigates the blood substance dynamic metabolism of two kinds of medicines.Result shows, and two chloro-1, the 2-cyclohexanediamine conjunction metabolism times of platinum complex in Wistar rat blood obviously extended, plasma drug level half-life (t
1/2) reach 2h.
Embodiment 10
Inject 100 μ L in C57BL/6 mice (6 weeks, 15g, purchased from Jilin University's Experimental Animal Center) right front oxter and suspend 1.0 × 10
6the PBS solution of individual B16F1 cell (Mus source K-1735).After 5 days, gross tumor volume rises to 30-70mm
3in time, starts to treat for the first time, is labeled as the 0th day.By mouse weights and measurement tumor volume, and be divided into 3 groups at random, often organize 6: Saline group (i.e. matched group), oxaliplatin group (Oxaliplatin, Pt dosage: 3.0mg/kg), two chloro-1,2-cyclohexanediamine closes platinum complex group (DACHPtMicelles, Pt dosage 3.0mg/kg), implemented tail vein injection at the 0th, 2,4,6,8 day respectively.Measure major diameter (a) and the minor axis (b) of tumor, calculate gross tumor volume (V=a × b × b/2).Put to death whole mice in the 14th day, solution cuts tumor tissues and weighs, and calculates tumour inhibiting rate (TI).TI(%)=(Saline group Mean tumor mass-administration group Mean tumor mass)/Saline group Mean tumor mass × 100%.Oxaliplatin group is 30.2% to the melanomatous tumour inhibiting rate in Mus source, and the tumour inhibiting rate that two chloro-1,2-cyclohexanediamine close platinum complex group is 40.1%, demonstrates good tumor inhibitory effect.
Embodiment 11
Inject 100 μ L in Balb/C nude mice (6 weeks, 15g) right front oxter and suspend 1.0 × 10
6the PBS solution of individual A549 lung adenocarcinoma cell.After 20 days, gross tumor volume rises to 200mm
3in time, starts to treat for the first time, is labeled as the 0th day.By mouse weights and measurement tumor volume, and be divided into 3 groups at random, often organize 6: Saline group (i.e. matched group), oxaliplatin group (Oxaliplatin, Pt dosage: 3.0mg/kg), two chloro-1,2-cyclohexanediamine closes platinum complex group (DACHPtMicelles, Pt dosage 3.0mg/kg), implemented tail vein injection at the 0th, 4,8 day respectively.Measure major diameter (a) and the minor axis (b) of tumor, calculate gross tumor volume (V=a × b × b/2).Put to death whole mice in the 14th day, solution cuts tumor tissues and weighs, and calculates tumour inhibiting rate (TI).TI(%)=(Saline group Mean tumor mass-administration group Mean tumor mass)/Saline group Mean tumor mass × 100%.Oxaliplatin group is 40.2% to the melanomatous tumour inhibiting rate in Mus source, and the tumour inhibiting rate that two chloro-1,2-cyclohexanediamine close platinum complex group is 49.7%, demonstrates good tumor inhibitory effect.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
To the above-mentioned explanation of the disclosed embodiments, professional and technical personnel in the field are realized or uses the present invention.To be apparent for those skilled in the art to the multiple amendment of these embodiments, General Principle as defined herein can without departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention can not be restricted to these embodiments shown in this article, but will meet the widest scope consistent with principle disclosed herein and features of novelty.
Claims (9)
1. chloro-1, a 2-cyclohexanediamine closes platinum complex, closes platinum formed with the polymer complex with formula (I) structure by two chloro-1,2-cyclohexanediamine,
In formula (I), R
1independently be selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10, phenyl or R '-CO-, R ' and be independently selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10 or phenyl;
R
2independently be selected from the alkyl of H atom, the alkyl of C1 ~ C20 or the C1 ~ C20 of replacement; The substituent group of the alkyl of the C1 ~ C20 of described replacement be selected from ketal, acetal, hydroxyl, aldehyde radical, amino, sulfydryl and saccharide residue one or more;
R
3independently be selected from H atom or cation;
L is independently selected from-CH
2-or-CH
2-CH
2-;
M is the degree of polymerization, 40≤m≤250; X, y, z are the degree of polymerization, 10≤x+y+z≤5000,5%≤y/ (x+y+z)≤80%.
2. two chloro-1,2-cyclohexanediamine according to claim 1 close platinum complex, it is characterized in that, R
2independently be selected from the alkyl of the alkyl of C1 ~ C10 or the C1 ~ C10 of replacement;
R
3independently be selected from H atom, metal cation or organic cation.
3. two chloro-1,2-cyclohexanediamine according to claim 2 close platinum complex, it is characterized in that, R
3independently be selected from H atom, sodium ion, potassium ion, the subgroup of magnesium, amine ion or aminoacid ion.
4. two chloro-1,2-cyclohexanediamine according to claim 1 close platinum complex, it is characterized in that, 30≤x+y+z≤300,5%≤y/ (x+y+z)≤50%.
5. two chloro-1,2-cyclohexanediamine according to claim 1 close platinum complex, it is characterized in that, R
1for C6 alkyl, R
2for methyl, R
3for hydrogen atom or sodium ion, L are-CH
2-CH
2-.
6. the preparation method of chloro-1, a 2-cyclohexanediamine conjunction platinum complex, comprises the following steps:
Two chloro-1,2-cyclohexanediamine close platinum and in an aqueous medium complex reaction occur with the polymer with formula (I) structure, generate two chloro-1,2-cyclohexanediamine and close platinum complex;
In formula (I), R
1independently be selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10, phenyl or R '-CO-, R ' and be independently selected from the straight chained alkyl of C2 ~ C10, the branched alkyl of C3 ~ C10 or phenyl;
R
2independently be selected from the alkyl of H atom, the alkyl of C1 ~ C20 or the C1 ~ C20 of replacement;
R
3independently be selected from H atom or cation;
L is independently selected from-CH
2-or-CH
2-CH
2-;
M is the degree of polymerization, 40≤m≤250; X, y, z are the degree of polymerization, 10≤x+y+z≤5000,5%≤y/ (x+y+z)≤80%.
7. preparation method according to claim 6, is characterized in that, described in there is the mol ratio that the carboxyl of the polymer of formula (I) structure and two chloro-1,2-cyclohexanediamine close Pt in platinum be less than 20.
8. preparation method according to claim 6, is characterized in that, described in there is the mol ratio that the carboxyl of the polymer of formula (I) structure and two chloro-1,2-cyclohexanediamine close Pt in platinum be less than 10.
9. preparation method according to claim 6, is characterized in that, described aqueous medium is water, normal saline, buffer solution, tissue culture medium or body fluid.
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| CN102863627A (en) * | 2012-10-10 | 2013-01-09 | 中国科学院长春应用化学研究所 | Cisplatin complex and preparation method thereof |
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