CN102114247A - Conjugate of SSA, PEG and anticancer drugs and preparation thereof - Google Patents
Conjugate of SSA, PEG and anticancer drugs and preparation thereof Download PDFInfo
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- CN102114247A CN102114247A CN2011100439570A CN201110043957A CN102114247A CN 102114247 A CN102114247 A CN 102114247A CN 2011100439570 A CN2011100439570 A CN 2011100439570A CN 201110043957 A CN201110043957 A CN 201110043957A CN 102114247 A CN102114247 A CN 102114247A
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Abstract
The invention provides a conjugate of SSA (somatostatin analog), PEG (polyethylene glycol) and anticancer drugs and the preparation thereof. The macromolecule conjugate is made from the targeting group SSA, the hydrophilic long-chain PEG and the anticancer drugs by adopting linking arms through conjugating. The macromolecule conjugate has tumor-targeting property and long circulation function, can obviously improve the antitumor activity, reduces toxic side effect of the drugs, is more suitable for clinical application, and provides new ideas for research and development of the anticancer drugs.
Description
Technical field
The present invention relates to conjugate of a kind of somatostatin analogue-Polyethylene Glycol-antitumor drug and preparation method thereof.This conjugate is formed by introducing the linking arm coupling by targeting group somatostatin analogue, hydrophilic longer chain polyethylene glycols and antineoplastic agent.The invention still further relates to the preparation method and the application of this conjugate.Compare with antineoplastic agent, this conjugate has better tumor-targeting and long circulatory function.
Background technology
Malignant tumor is one of serious disease of harm humans health, and at present the whole world has malignant tumor patient about 1,700 ten thousand, the annual pathogenesis of cancer number about 2,000,000 to 2,400,000, dead 1,600,000 to 1,800,000 of China.China's malignant tumor number of the infected and death toll constantly increase, and early 1970s, China's pathogenesis of cancer number is about 900,000, dead about 700,000 people; 2005, pathogenesis of cancer number about 1,800,000-2,000,000, dead 1,400,000 to 1,500,000.Whole world cancer also shows a rising trend.1991 to 2000 in the period of, global pathogenesis of cancer and death toll all increase by 22%.2000, global New Development cancer number surpassed 1,000 ten thousand.World Health Organization's prediction, to the year two thousand twenty, annual New Development cancer number will reach 1,500 ten thousand, and cancer has become the new century mankind's first killer, and becomes the maximum public health problem in the whole world.
For a long time, for cancer patient's clinical treatment, adopt the means of chemotherapy and surgical operation and radiation treatment therapeutic alliance mostly.And employed antitumor drug in the chemotherapy, because it easily produces drug resistance, poor selectivity, big to the toxic and side effects of normal structure, from blood, to eliminate rapidly and tumor cell lacked problem such as specificity, the clinical practice of antineoplastic agent meets with bottleneck.In view of above reason, over nearly 20 years, people carry out a large amount of structure of modification to antineoplastic agent, develop a variety of derivants, but the derivant that only a few is only arranged enters the clinical research stage or has gone on the market, and therefore, the exploitation of new antitumoral medicine prodrug seems particularly important.
The prodrug strategy promptly is will bioactive former medicine and certain chemical group be arranged, fragment or molecule form temporary bonding through covalent bond, as adopt esterification, amidatioon, aminomethylation, etherificate, chemical modes such as cyclisation or open loopization are to having-OH,-NH:, the former medicine of-COOH structure is modified, non-activity of the new chemical entities of Xing Chenging own or activity are lower than former medicine like this, but the basic feature that has kept parent drug, simultaneously can improve its transmission performance, the suitable in vivo moment and position, fall temporary transient transhipment group through enzymolysis or hydrolysis cracking, recover to generate former medicine, improve the medicine general effect with this.Prodrug is owing to increase medicine dissolution, avoid first pass effect, increase stability and targeting, reduce advantages such as poisonous side effect of medicine, become in the current drug delivery system research field one of research direction of tool application prospect.For synthetic prodrug, people study the position that antineoplastic agent may be modified, confirmed the position of most possibly carrying out chemical modification on the drug molecules such as some antitumor drug such as taxanes, anthracene nucleus class, pteridine class, camptothecin, ciclosporin class, podophillotoxines, cisplatin class, and it has been modified make prodrug.Linking arm is meant that two ends contain the chemical substance of active group (being mainly amino, carboxyl, sulfydryl), and the one end can be connected to form intermediate by covalent bond with a kind of chemical compound, and its other end can link to each other by covalent bond with another chemical compound.The selection of linking arm depends primarily on the antitumor drug of bonding with it.For example, when antineoplastic agent has amino or hydroxyl, should select to have the linking arm of carboxyl to form ester bond or amido link; When antitumor drug contains carboxyl, then should select to have the linking arm of free amine group to form amido link.The chemical property of linking arm is active, is important pharmaceutical intermediate, can prepare various prodrugs by introducing linking arm, and this brings into play great function in the prodrug design.G.Cavallaro. waiting with paclitaxel (TXL) is parent drug, with the succinic anhydride is linking arm, by the ester bond combination, generate intermediate 2 '-succinyl paclitaxel, and then be connected by ester bond with PHEA, synthesized macromolecular prodrug PHEA-TXL, this method has improved the dissolubility and the stability of former medicine, prolong drug release time, significantly improved tumor-targeting (Cavallaro, G., M.Licciardi, Caliceti, P.et al.Synthesis, physico-chemical and biological characterization of a paclitaxel macromolecular prodrug.[J] Eur JPharm Biopharm 2004,58 (1): 151-159).
The targeted drug transmission system (TDDS) claim targeting preparation again, and it can be transported to target tissue, target organ and target cell to greatest extent with medicine, and very little to non-target organ influence, improves the purpose that curative effect reduces toxic and side effects thereby reach.In dissimilar targeting preparations, active target preparation is because it has special affinity interaction to target site, and targeting efficient is high and show one's talent in all targeting preparations, becomes a class targeting preparation of tool application prospect.At present, reported that both at home and abroad the targeting " bullet " that is used for the TDDS design mainly contains: folic acid, transferrins, agglutinin, integration element, galactose, mannose, RGD peptide, NGR peptide, blood vessel intestinal polypeptide, interleukin-2, cell growth factor, epidermal growth factor, low-density lipoprotein Pseudobulbus Bletillae (Rhizoma Bletillae) monoclonal antibody etc.Big quantity research report shows, drug-loading system behind above-mentioned targeting base group modification, in the tumor cell in vitro experimental level, all can show higher targeting, but in animal body owing to the biotic environment complexity, have reasons such as various biological barriers, it reduces greatly in the intravital cancer target effect of tumor animal, even the negative report of appearance (Yokoyama M.Drug targeting with nano-sized carrier systems.[J] .J ArtifOrgans, 2005,8 (2): 77-84.).Therefore, the bottleneck that is selected to the active target preparation exploitation of new and effective targeted molecular.
(somatostatin receptor SSTR) expresses in the most of tumors of human body (human pituitary cancer, gastrointestinal pancreas cancer, hepatocarcinoma, neurocyte cancer and non-small cell carcinoma) cell surface high density somatostatin receptor.Because SSTR is higher than normal structure far away in tumor and metastasis cell surface expression thereof, and big with somatostatin (SST) affinity, can be used as the novel targeted material of the molecular level of diagnosing tumor and treatment.Action range, selectivity are strong because SST exists, the half-life, short (reasons such as 2～3min) limited its application clinically.Therefore, on the basis that keeps SST function amino acid structure, design synthesized somatostatin analogue (somatostatin analog, SSA), as octreotide, vapreotide, Lanreotide, depreotide etc.Wherein octreotide (Octreotide) is the SSA of first synthetic, compare with SST, octreotide improves about 70 times to the SSTR affinity, biological half-life reaches 9h, therefore use the most extensively (Dijkgraaf I clinically, Boerman OC, Oyen WJ, et al.Development and application of peptide-based radiopharmaceuticals.[J] .Anticancer Agents Med Chem, 2007,7 (5): 543-551.), octreotide radioisotope labeling thing is ratified as the clinical tumor diagnostic reagent by FDA.
At present, less for SSA both at home and abroad as the application report of tumor " targeting bullet ", mainly contain the application of following two aspects: (1) is with the phospholipid coupling of octreotide and Pegylation, and make the liposome entrapment medicine and be proved to be tumor tissues targeting and antitumor action (Zhang J preferably, Jin W, Wang X, et al. A novel octreotide modified lipid vesicle improved the anticancer efficacy of doxorubicin in somatos
Polyethylene Glycol (PEG) can delay the medicine clearance rate, the prolong drug half-life, increases antitumor drug accumulating at tumor locus.Polyethylene Glycol can synthesize different macromole conjugates by introducing linking arm miscellaneous with multiple medicine.At present, Shang Weiyou makes macromole conjugate with cancer target and long circulatory function and document and the patent report that is applied to the antitumor drug transmission system with somatostatin analogue, Polyethylene Glycol and antineoplastic agent by introducing the linking arm coupling.
Summary of the invention
The purpose of this invention is to provide a kind of macromole conjugate with cancer target and long circulatory function.This conjugate can be applicable to initiatively targeting drug delivery system, has long circulatory function in tumor-targeting and the body.This conjugate scope of application is extensive, and preparation technology is simple.
Another object of the present invention provides above-mentioned preparation method with macromole conjugate of cancer target and long circulatory function.
A further object of the invention provides the above-mentioned application of macromole conjugate in oncotherapy with cancer target and long circulatory function.
For achieving the above object, the invention provides a kind of macromole conjugate that forms by the coupling of introducing linking arm by tumor-targeting group somatostatin analogue, hydrophilic longer chain polyethylene glycols and antitumor drug.This conjugate can be applicable to targeting drug delivery system, may strengthen antitumous effect, reduces toxic and side effects, improves targeting, is more suitable for clinical practice, for the research and development of antitumor drug and preparation thereof provide new thinking.
Described macromole conjugate with cancer target and long circulatory function is characterized in that the structural representation of this conjugate is as follows:
Wherein, X is a somatostatin analogue, and Spacer is a linking arm.
Described macromole conjugate with cancer target and long circulatory function is characterized in that: provide the somatostatin analogue of targeting group to comprise: the derivant of octreotide, Lanreotide, vapreotide, depreotide and above material.
Described macromole conjugate with cancer target and long circulatory function, wherein, the activation of Polyethylene Glycol two ends, molecular weight is 500～20000.
Described macromole conjugate with cancer target and long circulatory function, wherein, Spacer 1 is the carboxylic acid of dicarboxylic acids, dicarboxylic anhydride, terminal tool primary amino radical or the carboxylic acid of terminal tool sulfydryl, linking arm alkylidene number is 1～8; Spacer 2 is the carboxylic acid of diamidogen, dicarboxylic acids, dicarboxylic anhydride, terminal tool primary amino radical or the carboxylic acid of terminal tool sulfydryl, and linking arm alkylidene number is 1～8.
Described macromole conjugate with cancer target and long circulatory function, wherein, the selection of linking arm is as follows: the selection of Spacer 1 depends primarily on the somatostatin analogue with its bonding, somatostatin analogue contains the N-terminal amino group, should select the carboxylic linking arm of at least one end, when the other end of Polyethylene Glycol contains carboxyl, can be directly and the somatostatin analogue covalent bond; The selection of Spacer 2 depends primarily on the antineoplastic agent with its bonding, when antineoplastic agent has amino or hydroxyl, the linking arm that should select to have carboxyl when antitumor drug contains carboxyl, then should select to have the linking arm of N-terminal amino group to form amido link to form ester bond or amido link.
Described macromole conjugate with cancer target and long circulatory function, wherein, antineoplastic agent is mainly arbitrary material or derivatives thereof of taxanes, anthracene nucleus class, pteridine class, ciclosporin class, camptothecin, podophillotoxines, cisplatin class antineoplastic agent, preferred paclitaxel, amycin and methotrexate.
Described preparation method with macromole conjugate of cancer target and long circulatory function comprises:
(1) carboxylic antineoplastic agent is dissolved in the solvent, the employing diamidogen is a linking arm, adds catalyst and dehydrant, obtains medicine-linking arm intermediate; The carboxylic Polyethylene Glycol of one end is dissolved in the reaction dissolvent, with the intermediate that obtains in the presence of catalyst and dehydrant, carry out amide reaction, obtain medicine-linking arm-Polyethylene Glycol complex; This complex is carried out condensation reaction by introducing suitable linking arm and somatostatin analogue in the presence of catalyst and dehydrant, also can with this complex directly and somatostatin analogue in the presence of catalyst and dehydrant, carry out condensation reaction, obtain somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates.
(2) antineoplastic agent that will contain amino or hydroxyl is dissolved in the solvent, and the employing dicarboxylic acids is a linking arm, adds catalyst and dehydrant, obtains medicine-linking arm intermediate; One end is contained amino Polyethylene Glycol is dissolved in the reaction dissolvent, with the intermediate that obtains in the presence of catalyst and dehydrant, carry out the amide reaction, obtain medicine-linking arm-Polyethylene Glycol complex; This complex is carried out condensation reaction by introducing suitable linking arm and somatostatin analogue in the presence of catalyst and dehydrant, also can with this complex directly and somatostatin analogue in the presence of catalyst and dehydrant, carry out condensation reaction, obtain somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates.
(3) antineoplastic agent that will contain amino or hydroxyl is dissolved in the solvent, and the employing dicarboxylic anhydride is a linking arm, adds catalyst and dehydrant, obtains medicine-linking arm intermediate; One end is contained amino Polyethylene Glycol is dissolved in the reaction dissolvent, with the intermediate that obtains in the presence of catalyst and dehydrant, carry out the amide reaction, obtain medicine-linking arm-Polyethylene Glycol complex; This complex is carried out condensation reaction by introducing suitable linking arm and somatostatin analogue in the presence of catalyst and dehydrant, also can with this complex directly and somatostatin analogue in the presence of catalyst and dehydrant, carry out condensation reaction, obtain somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates.
(4) carboxylic antineoplastic agent is dissolved in the solvent, adopting the carboxylic acid of terminal tool sulfydryl or the carboxylic acid of terminal tool primary amino radical is linking arm, adds catalyst and dehydrant, obtains medicine-linking arm intermediate; One end is contained amino Polyethylene Glycol is dissolved in the reaction dissolvent, with the intermediate that obtains in the presence of catalyst and dehydrant, carry out the amide reaction, obtain medicine-linking arm-Polyethylene Glycol complex; This complex is carried out condensation reaction by introducing suitable linking arm and somatostatin analogue in the presence of catalyst and dehydrant, also can with this complex directly and somatostatin analogue in the presence of catalyst and dehydrant, carry out condensation reaction, obtain somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates.
Above-mentioned reaction sequence can be put the cart before the horse, promptly earlier with somatostatin analogue and PEG direct covalent bond or pass through the linking arm coupling in the presence of catalyst, reaction makes somatostatin analogue-(linking arm)-PEG intermediate, then in the presence of catalyst and dehydrant, this intermediate links to each other with antitumor drug by introducing linking arm, promptly gets the macromole conjugate with cancer target and long circulatory function.
Described macromole conjugate with cancer target and long circulatory function can be made into arbitrary acceptable forms clinically, comprises injection, oral formulations.
Described macromole conjugate with cancer target and long circulatory function is mainly used in treatments such as breast carcinoma, nonsmall-cell lung cancer, hepatocarcinoma, ovarian cancer, glioma, incidence cancer.This macromole conjugate may strengthen antitumous effect, reduces poisonous side effect of medicine, and prolong drug improves targeting in the holdup time of tumor tissues, is more suitable for clinical practice, for the research and development of antitumor drug and preparation thereof provide new thinking.
The specific embodiment
To the present invention's further instruction in addition, but following embodiment does not limit this patent scope below by embodiment.
Embodiment 1
The preparation of octreotide-Polyethylene Glycol-paclitaxel conjugate (OCT-PEG-PTX) (is linking arm with the succinic anhydride)
(1) 35mg paclitaxel (PTX) is dissolved in the pyridine, adds the 4mg succinic anhydride, stirring at room reaction 4h.Rotary evaporation is removed pyridine, vacuum drying.Add an amount of distilled water, stir 30min, filter collecting precipitation, precipitation is dissolved in proper amount of acetone, slowly add distilled water, collect crystallization, vacuum drying gets 2 '-succinyl paclitaxel.Through nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
(2) the 2 '-succinyl paclitaxel that obtains in (1) is dissolved in the acetonitrile, adds 200mg H
2N-PEG-COOH (PEG
5000), add 16mg 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCHCl) and 10mgN-N-Hydroxysuccinimide (NHS), stirring at room 12h.Get above-mentioned reactant liquor, add the absolute ether precipitation, the reduced vacuum drying gets PEG-2 '-succinyl paclitaxel.
(3) the PEG-2 '-succinyl paclitaxel that obtains in (2) is dissolved in N, in the dinethylformamide (DMF), add the 40mg octreotide, add 10mg 1-hydroxyl-BTA (HOBt) and 16mg 1-ethyl-3-(3-dimethylamino-propyl) carbon imidodicarbonic diamide hydrochlorate (EDCHCl) then, add 8 μ l N again, N-diisopropylethylamine (DIPEA), stirring at room reaction 4h.Above-mentioned reactant liquor dilutes with big water gaging, dialysis, and lyophilizing promptly gets OCT-PEG-PTX.Through dissolubility, nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
Embodiment 2
The preparation of octreotide-Polyethylene Glycol-paclitaxel conjugate (OCT-PEG-PTX) (is linking arm with the succinic acid)
(1) 35mg paclitaxel (PTX) is dissolved in the pyridine, adds the 4mg succinic acid, stirring at room reaction 4h.Rotary evaporation is removed pyridine, vacuum drying.Add an amount of distilled water, stir 30min, filter collecting precipitation, precipitation is dissolved in proper amount of acetone, slowly add distilled water, collect crystallization, vacuum drying gets 2 '-succinyl paclitaxel.Through nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
(2) the 2 '-succinyl paclitaxel that obtains in (1) is dissolved in the acetonitrile, adds 200mg H
2N-PEG-COOH (PEG
5000), add 16mg 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCHCl) and 10mg N-hydroxy-succinamide (NHS), stirring at room 12h.Get above-mentioned reactant liquor, add the absolute ether precipitation, the reduced vacuum drying gets PEG-2 '-succinyl paclitaxel.
(3) PEG-2 ' that (2) are obtained-succinyl paclitaxel is dissolved in N, in the dinethylformamide (DMF), add the 40mg octreotide, add 10mg 1-hydroxyl-BTA (HOBt) and 16mg 1-ethyl-3-(3-dimethylamino-propyl) carbon imidodicarbonic diamide hydrochlorate (EDCHCl) then, add 8 μ l N again, N-diisopropylethylamine (DIPEA), stirring at room reaction 4h.Above-mentioned reactant liquor dilutes with big water gaging, dialysis, and lyophilizing promptly gets OCT-PEG-PTX.Through dissolubility, nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
Embodiment 3
The preparation of vapreotide-Polyethylene Glycol-paclitaxel conjugate (VAP-PEG-PTX) (is linking arm with the succinic anhydride)
(1) 35mg paclitaxel (PTX) is dissolved in the pyridine, adds the 4mg succinic anhydride, stirring at room reaction 4h.Rotary evaporation is removed pyridine, vacuum drying.Add an amount of distilled water, stir 30min, filter collecting precipitation, precipitation is dissolved in proper amount of acetone, slowly add distilled water, collect crystallization, vacuum drying gets 2 '-succinyl paclitaxel.Through nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
(2) with 200mg (Boc) HN-PEG-CONHS (PEG
2000) be dissolved in an amount of acetonitrile, the 40mg vapreotide is dissolved in an amount of N, in the dinethylformamide (DMF), the PEG drips of solution is added in the vapreotide solution, adds 5mg dimethylamino naphthyridine (DMAP) then, 4 ℃ of airtight stirring 12h.Add trifluoroacetic acid (TFA) in the above-mentioned reactant liquor, stirring at room reaction 4h, reactant liquor dilutes with big water gaging, dialysis, lyophilizing promptly gets VAP-PEG-NH
2, through nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
(3) VAP-PEG-NH that (2) are made
2(1) the 2 '-succinyl paclitaxel that makes is dissolved in N, in the dinethylformamide (DMF), add 10mg 1-hydroxyl-BTA (HOBt) and 16mg 1-ethyl-3-(3-dimethylamino-propyl) carbon imidodicarbonic diamide hydrochlorate (EDCHCl), add 8 μ l N again, N-diisopropylethylamine (DIPEA), stirring at room reaction 4h.Above-mentioned reactant liquor dilutes with big water gaging, dialysis, and lyophilizing promptly gets VAP-PEG-DOX.Through dissolubility, nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
Embodiment 4
The preparation of octreotide-Polyethylene Glycol-amycin conjugate (OCT-PEG-DOX) (is linking arm with the glutaric anhydride)
(1) 20mg amycin (DOX) is dissolved in N, in the dinethylformamide (DMF), adds 5 μ l triethylamines (TEA), add the 4mg glutaric anhydride again, stirring at room 12h.Get above-mentioned reactant liquor, remove by filter precipitation, filtrate distribution is in the sodium bicarbonate aqueous solution of chloroform and 5ml 5%.Repeat aforesaid operations three times, discard chloroform layer.Water layer is transferred pH3.0 with hydrochloric acid, reuse ethyl acetate extraction three times.After the washing of ethyl acetate layer reuse sodium-chloride water solution, vacuum drying promptly gets 3 '-glutaryl amycin, through nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
(2) with 200mg (Boc) HN-PEG-CONHS (PEG
5000) be dissolved in an amount of acetonitrile, the 40mg octreotide is dissolved in an amount of N, in the dinethylformamide (DMF), the PEG drips of solution is added in the octreotide solution, adds 5mg dimethylamino naphthyridine (DMAP) then, 4 ℃ of airtight stirring 12h.Add trifluoroacetic acid (TFA) in the above-mentioned reactant liquor, stirring at room reaction 4h, reactant liquor dilutes with big water gaging, dialysis, lyophilizing promptly gets OCT-PEG-NH
2, through nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
(3) OCT-PEG-NH that (2) are made
2(1) the 3 '-glutaryl amycin that makes is dissolved in N, in the dinethylformamide (DMF), add 10mg 1-hydroxyl-BTA (HOBt) and 16mg 1-ethyl-3-(3-dimethylamino-propyl) carbon imidodicarbonic diamide hydrochlorate (EDCHCl), add 8 μ l N again, N-diisopropylethylamine (DIPEA), stirring at room reaction 4h.Above-mentioned reactant liquor dilutes with big water gaging, dialysis, and lyophilizing promptly gets OCT-PEG-DOX.Through dissolubility, nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
Embodiment 5
The preparation of vapreotide-Polyethylene Glycol-amycin conjugate (VAP-PEG-DOX) (is linking arm with the succinic acid)
(1) 20mg amycin (DOX) is dissolved in N, in the dinethylformamide (DMF), adds 5 μ l triethylamines (TEA), add the 4mg succinic acid again, stirring at room 12h.Get above-mentioned reactant liquor, remove by filter precipitation, filtrate distribution is in the sodium bicarbonate aqueous solution of chloroform and 5ml5%.Repeat aforesaid operations three times, discard chloroform layer.Water layer is transferred pH3.0 with hydrochloric acid, reuse ethyl acetate extraction three times.After the washing of ethyl acetate layer reuse sodium-chloride water solution, vacuum drying promptly gets 3 '-succinyl amycin, through nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
(2) with 200mg (Boc) HN-PEG-CONHS (PEG
5000) be dissolved in an amount of acetonitrile, the 40mg vapreotide is dissolved in an amount of N, in the dinethylformamide (DMF), the PEG drips of solution is added in the vapreotide solution, adds 5mg dimethylamino naphthyridine (DMAP) then, 4 ℃ of airtight stirring 12h.Add trifluoroacetic acid (TFA) in the above-mentioned reactant liquor, stirring at room reaction 4h, reactant liquor dilutes with big water gaging, dialysis, lyophilizing promptly gets VAP-PEG-NH
2, through nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
(3) VAP-PEG-NH that (2) are made
2(1) the 3 '-succinyl amycin that makes is dissolved in N, in the dinethylformamide (DMF), add 10mg 1-hydroxyl-BTA (HOBt) and 16mg 1-ethyl-3-(3-dimethylamino-propyl) carbon imidodicarbonic diamide hydrochlorate (EDCHCl), add 8 μ l N again, N-diisopropylethylamine (DIPEA), stirring at room reaction 4h.Above-mentioned reactant liquor dilutes with big water gaging, dialysis, and lyophilizing promptly gets VAP-PEG-DOX.Through dissolubility, nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
Embodiment 6
The preparation of octreotide-Polyethylene Glycol-methotrexate conjugate (OCT-PEG-MTX) (with 1, the 3-propane diamine is a linking arm)
(1) 20mg methotrexate (MTX) is dissolved in the dichloromethane (DCM), adds 5mg dimethylamino naphthyridine (DMAP) and 16mg dicyclohexyl carbon imidodicarbonic diamide (DCC), add 10 μ l 1 again, 3-propane diamine, stirring at room 12h.Get above-mentioned reactant liquor, remove by filter precipitation, filtrate distribution is in the sodium bicarbonate aqueous solution of chloroform and 5ml 5%.Repeat aforesaid operations three times, discard chloroform layer.Water layer is transferred pH3.0 with hydrochloric acid, reuse ethyl acetate extraction three times.After the washing of ethyl acetate layer reuse sodium-chloride water solution, vacuum drying gets the aminopropyl methotrexate.
(2) the aminopropyl methotrexate that obtains in (1) is dissolved in N, in the dinethylformamide (DMF), adds 200mgHOOC-PEG-COOH (PEG
2000), add 16mg 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCHCl) and 10mg N-hydroxy-succinamide (NHS), stirring at room 12h.Get above-mentioned reactant liquor, add the absolute ether precipitation, the reduced vacuum drying gets PEG-aminopropyl methotrexate.
(3) the PEG-aminopropyl methotrexate that obtains in (2) is dissolved in N, in the dinethylformamide (DMF), add the 40mg octreotide, add 10mg 1-hydroxyl-BTA (HOBt) and 16mg 1-ethyl-3-(3-dimethylamino-propyl) carbon imidodicarbonic diamide hydrochlorate (EDCHCl) then, add 8 μ l N again, N-diisopropylethylamine (DIPEA), stirring at room reaction 4h.Above-mentioned reactant liquor dilutes with big water gaging, dialysis, and lyophilizing promptly gets OCT-PEG-MTX.Through dissolubility, nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
Embodiment 7
The preparation of Lanreotide-Polyethylene Glycol-methotrexate conjugate (LAN-PEG-MTX) (is linking arm with 3 '-mercaptopropionic acid) (1) is dissolved in 20mg methotrexate (MTX) in the dichloromethane (DCM), add 5mg dimethylamino naphthyridine (DMAP) and 16mg dicyclohexyl carbon imidodicarbonic diamide (DCC), add 10 μ l, 3 '-mercaptopropionic acid again, stirring at room 12h.Get above-mentioned reactant liquor, remove by filter precipitation, filtrate distribution is in the sodium bicarbonate aqueous solution of chloroform and 5ml 5%.Repeat aforesaid operations three times, discard chloroform layer.Water layer is transferred pH3.0 with hydrochloric acid, reuse ethyl acetate extraction three times.After the washing of ethyl acetate layer reuse sodium-chloride water solution, vacuum drying gets the methotrexate thioesters.
(2) the methotrexate thioesters that obtains in (1) is dissolved in N, in the dinethylformamide (DMF), adds 200mgNH
2-PEG-COOH (PEG
5000), add 16mg 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCHCl) and 10mg N-hydroxy-succinamide (NHS), stirring at room 12h.Get above-mentioned reactant liquor, add the absolute ether precipitation, the reduced vacuum drying gets PEG-methotrexate thioesters.
(3) the PEG-methotrexate thioesters that obtains in (2) is dissolved in N, in the dinethylformamide (DMF), add the 40mg Lanreotide, add 10mg 1-hydroxyl-BTA (HOBt) and 16mg 1-ethyl-3-(3-dimethylamino-propyl) carbon imidodicarbonic diamide hydrochlorate (EDCHCl) then, add 8 μ l N again, N-diisopropylethylamine (DIPEA), stirring at room reaction 4h.Above-mentioned reactant liquor dilutes with big water gaging, dialysis, and lyophilizing promptly gets LAN-PEG-MTX.Through dissolubility, nuclear magnetic resonance, NMR
1HNMR and mass spectrum MS measure and determine its structure.
Embodiment 8
OCT-PEG-PTX 30g
Water for injection is settled to desired volume
Get recipe quantity OCT-PEG-PTX, add water for injection 4L dissolving, hydro-oxidation sodium or hydrochloric acid adjust pH to 3～7, adding 0.05% medicinal carbon stirred 30 minutes, remove by filter active carbon then, it is 5L that filtrate is surveyed the qualified back adding of pH value water for injection to liquor capacity, through the extremely clarification of 0.45 μ m, 0.22 μ m filtering with microporous membrane, fill is prepared into 1000 bottles of aqueous injection;
Or:
Get recipe quantity OCT-PEG-PTX, add water for injection 4L dissolving, hydro-oxidation sodium or hydrochloric acid adjust pH to 3～7, add 0.05% medicinal carbon and stirred 30 minutes, remove by filter active carbon then, it is 5L that filtrate is surveyed the qualified back adding of pH value water for injection to liquor capacity, through the extremely clarification of 0.45 μ m, 0.22 μ m filtering with microporous membrane, fill, lyophilization is prepared into 1000 of lyophilized injectable powders.
Embodiment 9
OCT-PEG-DOX 10g
Water for injection is settled to desired volume
Get recipe quantity OCT-PEG-DOX, add water for injection 800ml dissolving, hydro-oxidation sodium or hydrochloric acid adjust pH to 3～7, adding 0.05% medicinal carbon stirred 30 minutes, remove by filter active carbon then, it is 1000ml that filtrate is surveyed the qualified back adding of pH value water for injection to liquor capacity, through the extremely clarification of 0.45 μ m, 0.22 μ m filtering with microporous membrane, fill is prepared into 1000 bottles of aqueous injection.
Or:
Get recipe quantity OCT-PEG-DOX, add water for injection 800ml dissolving, hydro-oxidation sodium or hydrochloric acid adjust pH to 3～7, add 0.05% medicinal carbon and stirred 30 minutes, remove by filter active carbon then, it is 1000ml that filtrate is surveyed the qualified back adding of pH value water for injection to liquor capacity, through the extremely clarification of 0.45 μ m, 0.22 μ m filtering with microporous membrane, fill, lyophilization is prepared into 1000 of lyophilized injectable powders.
Embodiment 10
LAN-PEG-MTX 5g
Water for injection is settled to desired volume
Get recipe quantity LAN-PEG-MTX, add water for injection 800ml dissolving, hydro-oxidation sodium or hydrochloric acid adjust pH to 3～7, adding 0.05% medicinal carbon stirred 30 minutes, remove by filter active carbon then, it is 1000ml that filtrate is surveyed the qualified back adding of pH value water for injection to liquor capacity, through the extremely clarification of 0.45 μ m, 0.22 μ m filtering with microporous membrane, fill is prepared into 1000 bottles of aqueous injection.
Or:
Get recipe quantity LAN-PEG-MTX, add water for injection 800ml dissolving, hydro-oxidation sodium or hydrochloric acid adjust pH to 3～7, add 0.05% medicinal carbon and stirred 30 minutes, remove by filter active carbon then, it is 1000ml that filtrate is surveyed the qualified back adding of pH value water for injection to liquor capacity, through the extremely clarification of 0.45 μ m, 0.22 μ m filtering with microporous membrane, fill, lyophilization is prepared into 1000 of lyophilized injectable powders.
Claims (10)
1. one kind has cancer target and the macromole conjugate of growing circulatory function, it is characterized in that this conjugate is on hydroxyl, carboxyl or the amino at antineoplastic agent, by introducing the end reaction of linking arm and Polyethylene Glycol, form medicine-linking arm-Polyethylene Glycol intermediate, the other end of Polyethylene Glycol passes through to introduce the amino of linking arm or direct and somatostatin analogue with covalent bonds then, form somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates, its structural representation is as follows:
Wherein, X is a somatostatin analogue, and Spacer is a linking arm.
2. the macromole conjugate with cancer target and long circulatory function according to claim 1 is characterized in that described somatostatin analogue comprises the derivant of octreotide, Lanreotide, vapreotide, depreotide or above material.
3. the macromolecular prodrug chemical compound with cancer target and long circulatory function according to claim 1 is characterized in that, the activation of described Polyethylene Glycol (PEG) two ends, and molecular weight is 500～20000.
4. the macromole conjugate with cancer target and long circulatory function according to claim 1, it is characterized in that, described linking arm: Spacer 1 is the carboxylic acid of dicarboxylic acids, dicarboxylic anhydride, terminal tool primary amino radical or the carboxylic acid of terminal tool sulfydryl, and linking arm alkylidene number is 1～8; Spacer 2 is the carboxylic acid of diamidogen, dicarboxylic acids, dicarboxylic anhydride, terminal tool primary amino radical or the carboxylic acid of terminal tool sulfydryl, and linking arm alkylidene number is 1～8.
5. the macromole conjugate with cancer target and long circulatory function according to claim 1, it is characterized in that, the selection of linking arm is as follows: the selection of Spacer 1 depends primarily on the somatostatin analogue with its bonding, somatostatin analogue contains the N-terminal amino group, should select the carboxylic linking arm of at least one end, when the other end of Polyethylene Glycol contains carboxyl, can be directly and the somatostatin analogue covalent bond; The selection of Spacer 2 depends primarily on the antineoplastic agent with its bonding, when antineoplastic agent has amino or hydroxyl, the linking arm that should select to have carboxyl when antitumor drug contains carboxyl, then should select to have the linking arm of N-terminal amino group to form amido link to form ester bond or amido link.
6. the macromole conjugate with cancer target and long circulatory function according to claim 1, it is characterized in that, described antineoplastic agent is arbitrary material or derivatives thereof of taxanes, anthracene nucleus class, pteridine class, ciclosporin class, camptothecin, podophillotoxines, cisplatin class antineoplastic agent, preferred paclitaxel, amycin and methotrexate.
7. the described preparation method with macromole conjugate of cancer target and long circulatory function of claim 1 is characterized in that comprising the following steps:
(1) carboxylic antineoplastic agent is dissolved in the solvent, the employing diamidogen is a linking arm, adds catalyst and dehydrant, obtains medicine-linking arm intermediate; The carboxylic Polyethylene Glycol of one end is dissolved in the reaction dissolvent, with the intermediate that obtains in the presence of catalyst and dehydrant, carry out amide reaction, obtain medicine-linking arm-Polyethylene Glycol complex; This complex is carried out condensation reaction by introducing suitable linking arm and somatostatin analogue in the presence of catalyst and dehydrant, also can with this complex directly and somatostatin analogue in the presence of catalyst and dehydrant, carry out condensation reaction, obtain somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates.
(2) antineoplastic agent that will contain amino or hydroxyl is dissolved in the solvent, and the employing dicarboxylic acids is a linking arm, adds catalyst and dehydrant, obtains medicine-linking arm intermediate; One end is contained amino Polyethylene Glycol is dissolved in the reaction dissolvent, with the intermediate that obtains in the presence of catalyst and dehydrant, carry out the amide reaction, obtain medicine-linking arm-Polyethylene Glycol complex; This complex is carried out condensation reaction by introducing suitable linking arm and somatostatin analogue in the presence of catalyst and dehydrant, also can with this complex directly and somatostatin analogue in the presence of catalyst and dehydrant, carry out condensation reaction, obtain somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates.
(3) antineoplastic agent that will contain amino or hydroxyl is dissolved in the solvent, and the employing dicarboxylic anhydride is a linking arm, adds catalyst and dehydrant, obtains medicine-linking arm intermediate; One end is contained amino Polyethylene Glycol is dissolved in the reaction dissolvent, with the intermediate that obtains in the presence of catalyst and dehydrant, carry out the amide reaction, obtain medicine-linking arm-Polyethylene Glycol complex; This complex is carried out condensation reaction by introducing suitable linking arm and somatostatin analogue in the presence of catalyst and dehydrant, also can with this complex directly and somatostatin analogue in the presence of catalyst and dehydrant, carry out condensation reaction, obtain somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates.
(4) carboxylic antineoplastic agent is dissolved in the solvent, adopting the carboxylic acid of terminal tool sulfydryl or the carboxylic acid of terminal tool primary amino radical is linking arm, adds catalyst and dehydrant, obtains medicine-linking arm intermediate; One end is contained amino Polyethylene Glycol is dissolved in the reaction dissolvent, with the intermediate that obtains in the presence of catalyst and dehydrant, carry out the amide reaction, obtain medicine-linking arm-Polyethylene Glycol complex; This complex is carried out condensation reaction by introducing suitable linking arm and somatostatin analogue in the presence of catalyst and dehydrant, also can with this complex directly and somatostatin analogue in the presence of catalyst and dehydrant, carry out condensation reaction, obtain somatostatin analogue-(linking arm)-Polyethylene Glycol-linking arm-drug conjugates.
Above-mentioned reaction sequence can be put the cart before the horse, promptly earlier with somatostatin analogue and PEG direct covalent bond or pass through the linking arm coupling in the presence of catalyst, reaction makes somatostatin analogue-(linking arm)-PEG intermediate, then in the presence of catalyst and dehydrant, this intermediate links to each other with antitumor drug by introducing linking arm, promptly gets the macromole conjugate with cancer target and long circulatory function.
8. the solvent described in the claim 7 is meant dichloromethane, chloroform, pyridine, N, a kind of or its mixed solution of dinethylformamide or acetonitrile, catalyst is a dimethylamino naphthyridine, the 1-hydroxy benzo triazole, N-hydroxy-succinamide, 2-(7-azo BTA)-N, N, N ', a kind of or its mixture in N '-tetramethylurea hexafluorophosphoric acid ester, dehydrant is a dicyclohexyl carbon imidodicarbonic diamide, 1-ethyl-3-(3-dimethyl aminopropyl) carbon imidodicarbonic diamide hydrochlorate, N, N-diisopropylethylamine or N, a kind of or its mixture of N-DIC, response time is 2～24h, the molar ratio of antitumor drug and Polyethylene Glycol is 1: 1～10, the mol ratio of somatostatin analogue and Polyethylene Glycol is 1: 1～10, the mol ratio of catalyst and Polyethylene Glycol is 1: 1～10, the mol ratio of catalyst and dehydrant is 1: 1～10, preferred 1: 1.
9. the described macromole conjugate with cancer target and long circulatory function of claim 1 can be made into arbitrary acceptable forms clinically, comprises injection, oral formulations.
10. the described application with macromole conjugate of cancer target and long circulatory function of claim 1 is characterized in that being mainly used in treatments such as breast carcinoma, nonsmall-cell lung cancer, hepatocarcinoma, ovarian cancer, glioma, incidence cancer.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988999A (en) * | 2012-05-09 | 2013-03-27 | 中国药科大学 | Curcumin-polysaccharide conjugate as well as preparation method and application thereof |
| CN104551003A (en) * | 2014-12-30 | 2015-04-29 | 燕山大学 | Method for preparing nano-platinum screw rod by taking lanreotide acetate as template |
| CN106619571A (en) * | 2017-01-03 | 2017-05-10 | 西南交通大学 | Polymer nanocarrier capable of promoting endocytosis and cell nucleus targeting and preparation method of polymer nanocarrier |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6552007B2 (en) * | 2000-01-13 | 2003-04-22 | Academia Sinica | Use of somatostatin analogs for the delivery of anti-tumor drugs to tumor cells |
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2011
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6552007B2 (en) * | 2000-01-13 | 2003-04-22 | Academia Sinica | Use of somatostatin analogs for the delivery of anti-tumor drugs to tumor cells |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988999A (en) * | 2012-05-09 | 2013-03-27 | 中国药科大学 | Curcumin-polysaccharide conjugate as well as preparation method and application thereof |
| CN102988999B (en) * | 2012-05-09 | 2015-04-08 | 中国药科大学 | Curcumin-polysaccharide conjugate as well as preparation method and application thereof |
| CN104551003A (en) * | 2014-12-30 | 2015-04-29 | 燕山大学 | Method for preparing nano-platinum screw rod by taking lanreotide acetate as template |
| CN104551003B (en) * | 2014-12-30 | 2016-08-24 | 燕山大学 | A kind of method preparing Platinum Nanoparticles screw rod for template with lanreotide acetate |
| CN106619571A (en) * | 2017-01-03 | 2017-05-10 | 西南交通大学 | Polymer nanocarrier capable of promoting endocytosis and cell nucleus targeting and preparation method of polymer nanocarrier |
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Application publication date: 20110706 |