CN103254279B - The polypeptide conjugates of taxol or Docetaxel - Google Patents
The polypeptide conjugates of taxol or Docetaxel Download PDFInfo
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- CN103254279B CN103254279B CN201310045580.1A CN201310045580A CN103254279B CN 103254279 B CN103254279 B CN 103254279B CN 201310045580 A CN201310045580 A CN 201310045580A CN 103254279 B CN103254279 B CN 103254279B
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Abstract
The invention discloses the polypeptide conjugates of serial taxol or Docetaxel, described taxol or Docetaxel are connected with the amino of polypeptide by different catenation sequence at 2 ' position hydroxyl.The invention also discloses the preparation method of described conjugates and preparing the application in medicine.Conjugates of the present invention possesses excellent anti-cancer activity and cancer cell selectivity, and has good aqueous solubility.
Description
The divisional application of the application's to be the application number submitted on October 10th, 2008 be patent application of 200810216426.5, the denomination of invention of original application is " polypeptide conjugates of taxol or Docetaxel ".
Technical field
The present invention relates to a kind of conjugates of cancer therapy drug, particularly relate to a kind of polypeptide conjugates of taxol or Docetaxel.
Background technology
Taxol (Paclitaxel, PTAX) is the diterpenes natural product of separation and Extraction from the bark of Taxus (Taxus) plant, by recent two decades takes one of the most effective chemical anticarcinogenic drug that institute's research and development go on the market.By being specifically bound to the β position of cellular microtubules tubule, make microtubule polymerization be agglomerate or pencil, taxol effectively can suppress the normal restructuring of microtubule net, makes the division of cell, and breeding and growth are hindered and be stuck in G2 phase and M phase.As the efficient chemical cancer therapy drug of extensively accreditation in the world, taxol is used for clinical by numerous state approval, mammary cancer is comprised, ovarian cancer, colorectal carcinoma with treatment, the esophageal carcinoma, nonsmall-cell lung cancer, bladder cancer, prostate cancer, melanoma (Melanoma), lymphoma, the kinds cancers such as leukocytosis (Leukemias) and cancer of the stomach
1-3.
The complex structure of taxol, is difficult to be prepared by chemical synthesis process at present, because natural resource are limited, makes its price very expensive.Taxol has high efficiency cell toxicity, but lacks cell selective.In vivo except tumor tissues, also can be distributed in healthy tissues, except causing bioavailability and obviously reducing, also make taxol while killing cancer cells, normal cell and tissue are damaged.Taxol cell selective difference result also in clinical application and produces serious toxic side effect, comprise neutrophilic leukocyte to reduce, bone marrow depression, angioedema, ypotension, anaphylaxis, general urticaria, sensation and central neuropathy, paraesthesia, feel to go down, topoanesthesia etc., often make to need the cancer patients of long-term prescription to be difficult to bear.In addition, taxol soluble extreme difference (~ 0.4ug/ml), formulation and administering mode are subject to many limitations.Taxol is added polyoxyethylenated castor oil (CremphorEL) in clinical preparation: in dehydrated alcohol (V:V=1:1), again to pass through intravenous infusion administration after normal saline dilution, except making troubles to patient, polyoxyethylenated castor oil also can cause the anaphylaxis of human body.Need to increase the case that dosage could obtain good therapeutic effect to some, taxol is also difficult to effective use because of its water-soluble extreme difference
2,4-7.
Docetaxel (Docetaxel; DTAX) go acetyl baccatin iii (10-DeacetylbaccatinIII) for raw material to extract from Japanese yew leaf, containing the basic mother nucleus structure of taxol; by its 3, the semi-synthetic paclitaxel analogs obtained after the amino of position carries out tertiary fourth oxygen acidylate
8,9.By the mechanism of action identical with taxol, Docetaxel is also used as effective chemical anticarcinogenic drug clinically.But Docetaxel demonstrates the weakness of cell selective difference in the application equally, while inhibition tumor cell, normal cell and tissue are damaged, cause various with toxic side effect like taxanes
10,11.
Taxol and Docetaxel have following structure:
When for taxol, R
1for C
6h
5-, R
2for CH
3-CO-; When for Docetaxel, R
1for (CH
3)
3cO-, R
2for H-.
Cause nearly ten years show great attention to molecular targeted anti-tumor agents R & D Strategy, for the weakness fundamentally overcoming chemical anticarcinogenic drug cell selective difference provides scientific and effective solution.Numerous result of study shows, some tumour cell can express the specific receptor that normal cell does not have to high-density, and some antibody or endogenous polypeptide alternative are incorporated into this receptoroid, the DNA replication dna of tumour cell is suppressed, the growth of tumour, breeding and transfer are interfered and stop.Conduct a research for the specific receptor of tumour cell and corresponding antibody and target polypeptide, just becoming the focus 12-17 in new and effective low toxicity cancer drug development field.Somatostatin receptor (Somatostatinreceptors, STTRs) is the glycoprotein that a class contains 7 TMDs, belongs to g protein coupled receptor family.Be cloned so far from SSTR1 and SSTR2 in 1992, successful clone determine 5 kinds of different STTRs molecular isoforms (STTR1-STTR5), encytosis and the cell biological information of they and cell are transmitted closely related.Most of neuroendocrine tumor, as prostate cancer, mammary cancer, small cell lung cancer, nonsmall-cell lung cancer all have expressed highdensity STTRs.And containing 14 amino acid whose endogenous polypeptide Somatostatins (Somatostatin), with the tumour cell of High Cell Density And High Expression STTRs, there is good affinity and selectivity
15,18.
Sostatin (Octreotide) is on the basis of Somatostatin being carried out to structural modification and sequence adjustment, the efficient target polypeptide medicine researched and developed by chemosynthesis.Sostatin is only containing 8 amino acid but have stronger STTRs affinity and stability, and the internal metabolism transformation period is 50 times of Somatostatin.Its sequence is: D-Phe-c [Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-ol.Except clinically for except the rehabilitation of patient after cancer operation and anticancer transfer, Sostatin also has vital role for research and development for the target anticancer preparation of carcinoid tumor and small cell lung cancer
16-18.By the phenylalanine (Phe of 3 in Sostatin aminoacid sequence
3) obtain Sostatin-Tyr with after tyrosine (Tyr) replacement
3(Tyr
3-Octreotide), except the biological activity keeping Sostatin and targeting, the Tyr in its sequence
3be easy to carry out radioactivity
131i marks, in tumour radiotherapy treatment, have important use
19.
Reference
1.R.M.Straubinger,Biopharmaceuticsofpaclitaxel(taxol):formulation,activityandpharmacokinetics,in:M.Suffness(Ed.),Taxol:ScienceandApplications,CRCPress,NewYork,pp.237–54(1995).
2.C.M.Spencer,D.Faulds,Drugs48:794–847(1994).
3.J.D.Adams,K.Flora,B.R.Goldspiel,J.W.Wilson,R.Finley,J.Natl.CancerInst.Monogr.15:141-7(1993).
4.M.Maroneeral.,Exp.Cel.lRes.270:1-12(2001).
5.H.Xiaoetal.,PNAS.103:10166-73(2006).
6.T.Kiietal,GanToKagakuRyoho.33:621-4(2006)
7.Y.Koderaetal.,Int.J.Clin.Oncol.11:449–53(2006).
8.K.A.Lyseng-Williamson,C.Fenton,Drugs65(17):2513-31(2005).
9.S.J.Clarke,L.P.Rivory，Clin.Pharmacokinet.36(2):99-114(1999).
10.J.P.Snyder,J.H.Nettles,B.Cornett,K.H.Downing,E.Nogales,PNAS.98(9):5312-16(2001).
11.H.P.Rang,M.M.Dale,J.M.Ritter,P.K.Moore.Pharmacology.5thed.London:ChurchillLivingstone;pp.694-8(2003).
12.H.Varmus,Science,312:1162-5(2006).
13.M.C.Garnett,Adv.Drug.Del.Rev.53:171216(2001).
14.J.Baselga,Science,312:1175-8(2006).
15.J.C.Reubl,EndocrineReviews24:389427(2003).
16.O.H.Aina1,T.C.Sroka,M.Chen,K.S.Lam,Biopolymers66:184-99(2002).
17.F.Hudecz,Z.Banoczi,G.Csik,MedicinalResearchReviews.25:679-736(2005).
18.A.V.Schally,A.Nagy,LifeSciences,72:2305-20(2003).
19.M.Ginjetal.,PNAS.103(44):16436-41(2006).
Summary of the invention
The object of the invention is for the deficiencies in the prior art, provide one to possess excellent anti-cancer activity and cancer cell selectivity simultaneously, and there is the taxol of good aqueous solubility or the polypeptide conjugates of Docetaxel.
Another object of the present invention is to the preparation method of the polypeptide conjugates that above-mentioned taxol or Docetaxel are provided.
Another object of the present invention is to provide the polypeptide conjugates of above-mentioned taxol or Docetaxel preparing the application in medicine.
For achieving the above object, present invention employs following technical scheme:
The invention discloses the polypeptide conjugates of a kind of taxol or Docetaxel, described taxol or Docetaxel are connected with the amino of polypeptide by catenation sequence at 2 ' position hydroxyl.
Described catenation sequence is-CO-CH
2-(X)
n-CH
2-CO-;
Wherein, X is O (CH
2cH
2oCH
2cH
2cH
2nHCOCH
2-)
2,-CO-,-O-,-S-,-CH
2-,-CHCH
3-or-C (CH
3)
2-; N is 0 or 1.
Described polypeptide is 3 phenylalanine (Phe of Sostatin or Sostatin
3) obtain Sostatin-Tyr with after tyrosine (Tyr) replacement
3.
Described taxol or Docetaxel at 2 ' position hydroxyl by catenation sequence and Sostatin or Sostatin-Tyr
3n-Amino End Group and/or 5 Methionin (Lys
5) side chain-amino is connected.
The invention also discloses the preparation method of the polypeptide conjugates of above-mentioned taxol or Docetaxel, described method comprises:
Taxol or Docetaxel and linking agent are obtained by reacting on 2 ' position hydroxyl and are connected with catenation sequence-CO-CH
2-(X)
n-CH
2taxol-Linker the intermediate of-CO-OH or Docetaxel-Linker intermediate, wherein X is O (CH
2cH
2oCH
2cH
2cH
2nHCOCH
2-)
2,-CO-,-O-,-S-,-CH
2-,-CHCH
3-or-C (CH
3)
2-, n is 0 or 1;
Taxol-Linker intermediate or Docetaxel-Linker intermediate are connected with the amino of polypeptide by solid phase or liquid-phase synthesis process.
The polypeptide conjugates that the invention also discloses above-mentioned taxol or Docetaxel is for the preparation of the application in the medicine preventing and/or treating tumour.
Owing to have employed above technical scheme, the beneficial effect that the present invention is possessed is:
The synthesized taxol of preparation of the present invention, the polypeptide conjugates of Docetaxel, particularly corresponding Sostatin/Sostatin-Tyr
3conjugates, owing to wherein having the taxol of high efficiency cell toxicity and the target polypeptide Sostatin of Docetaxel and identifiable design cancer-cell surface receptors and Sostatin-Tyr
3maximize favourable factors and minimize unfavourable ones after being interconnected, have complementary advantages, show good cell selective and water-soluble, there is the antitumour activity being better than parent drug taxol and Docetaxel.Conjugates significantly can strengthen the selectivity to cancer cells, avoid or reduce the distribution in normal cell and tissue, while significantly improving bioavailability, by overcoming the weakness of taxol and Docetaxel cell selective difference, effectively reduce the toxic side effect that parent anticarcinogen produces in vivo.In addition, Sostatin and Sostatin-Tyr
3distinctive high-hydrophilic, can make conjugates have good water-soluble, for the selection of formulation preparation and administering mode is provided convenience.And by two taxols-Sostatin conjugates, two Docetaxel-Sostatin conjugates, two taxol-Sostatin-Tyr that a part target polypeptide is connected with two molecule taxols or Docetaxel and prepares
3conjugates and two Docetaxel-Sostatin-Tyr
3conjugates, is conducive to the concentration increasing tumor locus taxol, more effectively improves anti-cancer effectiveness.Conjugates of the present invention has important application prospect in new and effective low toxicity molecular targeted anti-tumor agents research and development field.
Embodiment
The invention discloses series two taxol-Sostatin conjugates, two Docetaxel-Sostatin conjugates, two taxol-Sostatin-Tyr
3conjugates and two Docetaxel-Sostatin-Tyr
3the chemical structure of conjugates and their synthesis preparation method.The chemical structure of involved serial conjugates is as shown in formula I.
Formula I
In formula I, R
1can be C
6h
5-or (CH
3)
3cO-;
R
2can be CH
3-CO-or H-;
R
3can be H-(Phe) or HO-(Tyr);
X can be O (CH
2cH
2oCH
2cH
2cH
2nHCOCH
2-)
2,-CO-,-O-,-S-,-CH
2-,-CHCH
3-or-C (CH
3)
2-; N can be 0 or 1.
Particularly, conjugates of the present invention is the 2` position hydroxyl of taxol and the 2` position hydroxyl of the Docetaxel taxol-Linker intermediate that reacts with dissimilar linking agent respectively and formed and Docetaxel-Linker intermediate (general formula II), taxol-Linker intermediate (R in general formula II
1=C
6h
5-; R
2=CH
3cO-) by cross structure-CO-CH
2-(X)
n-CH
25 Methionin (Lys in the N-Amino End Group of-CO-and Sostatin and aminoacid sequence thereof
5) side chain-amino is connected and obtains two taxol-Sostatin conjugates (R in formula I
3=H-); With Sostatin-Tyr
3n-Amino End Group and aminoacid sequence in 5 Methionin (Lys
5) side chain-amino is connected and obtains two taxol-Sostatin-Tyr
3conjugates (R in formula I
3=HO-); Docetaxel-Linker intermediate (R in general formula II
1=(CH
3)
3cO-; R
2=H-) by cross structure-CO-CH
2-(X)
n-CH
25 Methionin (Lys in the N-Amino End Group of-CO-and Sostatin and aminoacid sequence thereof
5) side chain-amino is connected and obtains two Docetaxel-Sostatin conjugates (R in formula I
3=H-); With Sostatin-Tyr
3n-Amino End Group and aminoacid sequence in 5 Methionin (Lys
5) side chain
-amino is connected and obtains two Docetaxel-Sostatin-Tyr
3conjugates (R in formula I
3=HO-).
General formula II
In the inventive method, polytype linking agent can be selected.The condition that selected linking agent need meet for: can with taxol and Docetaxel 2` position hydroxyl reaction generate product represented by general formula II.Linking agent used in the present invention is as Glyoxylic acid hydrate acid anhydride, Succinic anhydried, Pyroglutaric acid, 3-carbonyl Pyroglutaric acid, 3-methylglutaric acid acid anhydride, 3,3 '-dimethylated pentanedioic acid acid anhydride etc.When taxol or Docetaxel and linking agent react, for different linking agents, the condition of ligation is substantially identical, and those skilled in the art can suitably adjust reaction conditions according to the difference of linking agent and obtain required product.
Above formula I also can be expressed as:
Two taxol-Sostatin conjugates:
Two taxol-Sostatin-Tyr
3conjugates:
Two Docetaxel-Sostatin conjugates:
Two Docetaxel-Sostatin-Tyr
3conjugates:
Invention creates solid phase and chemical synthesis in liquid phase technology to combine and carry out the method that substep is connected; with 2 of dissimilar linking agent and taxol or Docetaxel; position hydroxyl is connected and after obtaining taxol-Linker intermediate and Docetaxel-Linker intermediate, then Sostatin free with being connected to solid phase synthesis resin, side chain protected but N-Amino End Group respectively or Sostatin-Tyr
3n-Amino End Group by solid phase synthesis process be connected, obtain taxol-Sostatin conjugates, taxol-Sostatin-Tyr
3conjugates, Docetaxel-Sostatin conjugates or Docetaxel-Sostatin-Tyr
3conjugates; ; Again with liquid-phase synthesis process by taxol-Linker or Docetaxel-Linker and taxol-Sostatin conjugates, the taxol-Sostatin-Tyr to cut from solid phase synthesis resin
3conjugates, Docetaxel-Sostatin conjugates or Docetaxel-Sostatin-Tyr
35 Methionin (Lys in the aminoacid sequence of conjugates
5) side chain-amino is connected and synthesize preparation series couple taxol-Sostatin conjugates, two taxol-Sostatin-Tyr
3conjugates, two Docetaxel-Sostatin conjugates and two Docetaxel-Sostatin-Tyr
3conjugates.Concrete steps are:
(1) taxol-Linker intermediate general formula I I represented and Docetaxel-Linker intermediate respectively with the Sostatin derivative be connected on solid phase synthesis resin and Sostatin-Tyr
3the N-Amino End Group of derivative is connected by solid phase synthesis process, thus obtains the taxol-Sostatin conjugates derivative, Docetaxel-Sostatin conjugates derivative, the taxol-Sostatin-Tyr that are connected with resin respectively
3conjugates derivative and Docetaxel-Sostatin-Tyr
3conjugates derivative;
(2) said derivative is cut from resin respectively, after purified, obtain taxol-Sostatin conjugates, Docetaxel-Sostatin conjugates, taxol-Sostatin-Tyr that general formula III represents
3conjugates and Docetaxel-Sostatin-Tyr
3conjugates;
(3) taxol-Sostatin conjugates that the taxol-Linker intermediate represented by general formula II, Docetaxel-Linker intermediate represent with general formula III respectively under liquid phase synthesis condition, Docetaxel-Sostatin conjugates, taxol-Sostatin-Tyr
3conjugates and Docetaxel-Sostatin-Tyr
35 Methionin (Lys in polypeptid acid sequence in conjugates
5) side chain-amino is connected, thus obtains two taxols-Sostatin conjugates, two Docetaxel-Sostatin conjugates, the two taxol-Sostatin-Tyr represented by general formula I
3conjugates and two Docetaxel-Sostatin-Tyr
3conjugates.
General formula III
In general formula II and general formula III, R
1can be C
6h
5-or (CH
3)
3c-O-; R
2can be CH
3-CO-or H-; X can be O (CH
2cH
2oCH
2cH
2cH
2nHCOCH
2-)
2,-CO-,-O-,-S-,-CH
2-,-CHCH
3-or-C (CH
3)
2-; N can be 0 or 1.
Create in invention simultaneously and adopt chemical synthesis in liquid phase to carry out the synchronous method be connected, by taxol (Docetaxel)-Linker intermediate under liquid phase synthesis condition, with Sostatin and Sostatin-Tyr
3n-Amino End Group and their aminoacid sequences in 5 lysine side-chains-amino connects simultaneously, thus two taxol (the Docetaxel)-Sostatin conjugates of synthesis preparation series and two taxol (Docetaxel)-Sostatin-Tyr
3conjugates.Concrete steps are:
Taxol-the Linker that general formula I I is represented or Docetaxel-Linker intermediate respectively with Sostatin and Sostatin-Tyr
3react under liquid phase synthesis condition, make the 2`-hydroxyl of taxol/Docetaxel by cross structure-CO-CH
2-(X)
n-CH
2-CO-and Sostatin and Sostatin-Tyr
3n-Amino End Group and their aminoacid sequence in 5 Methionin (Lys
5) side chain-amino is connected simultaneously, and product, after high performance liquid phase instrument (HPLC) separation and purification, obtains the two taxols-Sostatin conjugates represented by general formula I and two taxol-Sostatin-Tyr
3conjugates or two Docetaxel-Sostatin conjugates and two Docetaxel-Sostatin-Tyr
3conjugates.
Serial conjugates of the present invention is by two molecule taxols or Docetaxel and Sostatin and Sostatin-Tyr
3in N-Amino End Group and their aminoacid sequences in 5 Methionin (Lys
5) side chain-amino is interconnected and synthesizes series two taxol-Sostatin conjugates, two Docetaxel-Sostatin conjugates, the two taxol-Sostatin-Tyr of preparation
3conjugates and two Docetaxel-Sostatin-Tyr
3conjugates, can make to have the taxol of high efficiency cell toxicity and Docetaxel and identifiable design cancer-cell surface receptors, have good aqueous solubility Sostatin and Sostatin-Tyr
3mutually maximize favourable factors and minimize unfavourable ones, have complementary advantages.While maintenance taxol and Docetaxel antitumour activity, by Sostatin and Tyr
3the target function of-Sostatin, conjugates significantly can strengthen the selectivity to cancer cells, avoid or reduce the distribution in normal cell and tissue, while significantly improving bioavailability, by overcoming the weakness of taxol and Docetaxel cell selective difference, effectively reduce the toxic side effect that parent anticarcinogen produces in vivo.In addition, Sostatin and Sostatin-Tyr
3distinctive high-hydrophilic, can make conjugates have good water-soluble, for the selection of formulation preparation and administering mode is provided convenience.And by two taxols-Sostatin conjugates, two Docetaxel-Sostatin conjugates, two taxol-Sostatin-Tyr that a part target polypeptide is connected with two molecule taxols or Docetaxel and prepares
3conjugates and two Docetaxel-Sostatin-Tyr
3conjugates, is conducive to the concentration increasing tumor locus taxol or Docetaxel, more effectively improves anti-cancer effectiveness.The serial conjugates that the present invention relates to resists molecular targeted cancer drug field of researching and developing to have important application prospect at new and effective low toxicity.
The present invention is described by the synthesis preparation method of following examples to each intermediate and conjugates.Embodiment does not limit the present invention, and protection scope of the present invention is defined by the claims.
Embodiment 1: taxol-2 ' synthesis of-glyoxylic esters
Taxol (0.854g, 1.00mmol) be dissolved in anhydrous tetrahydro furan (10ml), add anhydrous arsenic pyridine (0.5ml), Glyoxylic acid hydrate acid anhydride (Diglycolicanhydride, 0.139g, 1.20mmol), after 0 ° of C stirs 24 hours, add ethyl acetate (100ml), and be adjusted to pH3-4 with hydrochloric acid (1.0M).Acetic acid ethyl fluid uses water (30mlx3) successively, and saturated aqueous common salt (20mlx2) extracts, anhydrous sodium sulfate drying, reclaim under reduced pressure ethyl acetate after filtering.Gained resistates acetone-diethyl ether recrystallization, obtains taxol-2 '-glyoxylic esters sterling 0.801g, yield 82.6%.ESIMS:971.3 [M+H]
+(molecular weight: 969.99).
Embodiment 2: Docetaxel-2 ' synthesis of-succinate
Docetaxel (0.808g, 1.00mmol) be dissolved in anhydrous tetrahydro furan (8ml), add anhydrous arsenic pyridine (0.5ml), Succinic anhydried (Succinicanhydride, 0.121g, 1.20mmol), after 0 ° of C stirs 16 hours, add ethyl acetate (80ml), and be adjusted to pH3-4 with hydrochloric acid (1.0M).Acetic acid ethyl fluid uses water (30mlx3) successively, and saturated aqueous common salt (20mlx2) extracts, anhydrous sodium sulfate drying, reclaim under reduced pressure ethyl acetate after filtering.Gained resistates acetone-diethyl ether recrystallization, obtains Docetaxel-2 '-succinate sterling 0.728g, yield 80.1%.ESI – MS:909.2 [M+H]
+(molecular weight: 907.96).
Embodiment 3: taxol-2 '-dihydroxy acetyl-Sostatin-Tyr
3the synthesis of conjugates
To be connected with solid phase synthesis resin, form intramolecular disulfide bond, the Sostatin-Tyr that side chain protected but N-Amino End Group are free
3-resin is (containing Sostatin-Tyr
30.10mmol) be placed in solid phase synthesis device, pump solvent after embathing with anhydrous methylene chloride and logical people's drying nitrogen.By taxol-2 '-glyoxylic esters (0.243g, 0.25mmol) be dissolved in anhydrous methylene chloride (10ml), add PyBop(0.156g, 0.30mmol), N, N-diisopropylethylamine (0.039g, 0.30mmol) after stirring 10 minutes, stirs with drying nitrogen in instillation above-mentioned solid phase synthesis reactor.Drained by solvent after 1 hour, use dimethyl formamide (x6) successively, methylene dichloride (x5) and ether (x3) washing resin are also drained.Added by the resin of drying in trifluoroacetic acid/dichloromethane (V:V=1:5,20ml) solution, stir after 1 hour and filter, resin methylene dichloride (5mlx3) washs and filters.Decompressing and extracting after filtrate merges, residue with diethyl ether washing (x3), the solid that collected by suction ether is insoluble, obtains taxol-2 after drying '-dihydroxy acetyl-Sostatin-Tyr
3conjugates crude product 0.192g, yield 96.6%.ESI – MS:1988.6 [M+H]
+(molecular weight: 1987.22).
Embodiment 4: Docetaxel-2 ' synthesis of-succinyl-Sostatin conjugates:
To be connected with solid phase synthesis resin, and form intramolecular disulfide bond, the free Sostatin resin (containing Sostatin 0.10mmol) of side chain protected but N-Amino End Group is placed in solid phase synthesis device, pumps solvent and logical people's drying nitrogen with anhydrous methylene chloride after embathing.By Docetaxel-2 '-succinate (0.227g, 0.25mmol) be dissolved in anhydrous methylene chloride (10ml), add PyBop(0.156g, 0.30mmol), N, N-diisopropylethylamine (0.039g, 0.30mmol) after stirring 10 minutes, stirs with drying nitrogen in instillation above-mentioned solid phase synthesis reactor.Drained by solvent after 1 hour, use dimethyl formamide (x6) successively, methylene dichloride (x5) and ether (x3) washing resin are also drained.Added by the resin of drying in trifluoroacetic acid/dichloromethane (V:V=1:5,20ml) solution, stir after 1 hour and filter, resin methylene dichloride (5mlx3) washs and filters.Decompressing and extracting after filtrate merges, residue with diethyl ether washing (x3), the solid that collected by suction ether is insoluble, obtains Docetaxel-2 after drying ' and-succinyl-Sostatin conjugates crude product 0.182g, yield 95.3%.ESIMS:1910.6 [M+H]
+(molecular weight: 1909.19).
Embodiment 5: two (taxol-2 '-dihydroxy acetyl)-Sostatin-Tyr
3the synthesis of conjugates
Taxol-2 '-dihydroxy acetyl-Sostatin-Tyr
3crude product 0.159g(0.08mmol) add anhydrous dimethyl formamide (5ml).By taxol-2 '-glyoxylic esters (0.088g, 0.09mmol) is dissolved in anhydrous methylene chloride (6ml), adds PyBop(0.052g, 0.10mmol), DIPEA (0.013g, 0.10mmol) instills above-mentioned solution after stirring 10 minutes.After reaction solution stirs 1 hour, decompression pumps methylene dichloride, and in resistates instillation frozen water (60ml), the solid of separating out in collected by suction water also uses distilled water wash (x3).Gained solids with methanol-re-crystallizing in ethyl acetate, obtains two (taxol-2 '-dihydroxy acetyl)-Sostatin-Tyr
3conjugates sterling 0.218g, yield 92.6%.ESI – MS:2940.6 [M+H]
+(molecular weight: MW.2939.20).
Embodiment 6: the synthesis of two (Docetaxel-2 '-succinyl)-Sostatin conjugates
Docetaxel-2 '-succinyl-Sostatin crude product 0.153g(0.08mmol) add anhydrous dimethyl formamide (5ml).By Docetaxel-2 '-succinate (0.082g, 0.09mmol) is dissolved in anhydrous methylene chloride (6ml), adds PyBop(0.052g, 0.10mmol), DIPEA (0.013g, 0.10mmol) instills above-mentioned solution after stirring 10 minutes.After reaction solution stirs 1 hour, decompression pumps methylene dichloride, and in resistates instillation frozen water (60ml), the solid of separating out in collected by suction water also uses distilled water wash (x3).Gained solids with methanol-re-crystallizing in ethyl acetate, obtains two (Docetaxel-2 '-succinyl)-Sostatin conjugates sterling 0.204g, yield 91.1%.ESI – MS:2800.6 [M+H]
+(molecular weight: MW.2799.14).
Embodiment 7: liquid phase method synthesis two (taxol-2 '-dihydroxy acetyl)-Sostatin-Tyr
3conjugates
Taxol-2 '-glyoxylic esters (0.214g, 0.22mmol) be dissolved in anhydrous dimethyl formamide or anhydrous methylene chloride (8ml), add PyBop(0.156g, 0.30mmol), N, N-diisopropylethylamine (0.039g, 0.3mmol), stirs and after 10 minutes, reaction solution is added the Sostatin-Tyr being chilled to 0 ° of C
3in anhydrous dimethyl formamide (10ml) solution of (0.104g, 0.10mmol).After 0 ° of C stirs 3 hours, instilled by reaction solution in frozen water (100ml), the solid of separating out in collected by suction water also uses distilled water wash (x3).Gained solid with after high performance liquid phase instrument (HPLC) separation and purification two (taxol-2 '-dihydroxy acetyl)-Sostatin-Tyr
3conjugates sterling 0.196g, yield 66.6%.ESI-MS:2940.6 [M+H]
+(molecular weight: MW.2939.20).
Embodiment 8: liquid phase method synthesis two (Docetaxel-2 '-succinyl)-Sostatin conjugates
Docetaxel-2 '-succinate (0.200g, 0.22mmol) be dissolved in anhydrous dimethyl formamide anhydrous methylene chloride (8ml), add PyBop(0.156g, 0.30mmol), DIPEA (0.039g, 0.3mmol), stir and reaction solution is added in anhydrous dimethyl formamide (10ml) solution of the Sostatin (0.102g, 0.10mmol) being chilled to 0 ° of C after 10 minutes.After 0 ° of C stirs 3 hours, instilled by reaction solution in frozen water (100ml), the solid of separating out in collected by suction water also uses distilled water wash (x3).Gained solid with after high performance liquid phase instrument (HPLC) separation and purification two (Docetaxel-2 '-succinyl)-Sostatin conjugates sterling 0.186g, yield 66.4%.ESI – MS:2800.6 [M+H]
+(molecular weight: MW.2799.14).
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
Claims (4)
1. a preparation method for the polypeptide conjugates of taxol or Docetaxel, described method comprises:
Taxol or Docetaxel and linking agent are obtained by reacting on 2 ' position hydroxyl and are connected with catenation sequence-CO-CH
2-(X)
n-CH
2taxol-Linker the intermediate of-CO-OH or Docetaxel-Linker intermediate, wherein X is O (CH
2cH
2oCH
2cH
2cH
2nHCOCH
2-)
2,-CO-,-O-,-S-,-CH
2-,-CHCH
3-or-C (CH
3)
2-, n is 0 or 1;
By taxol-Linker intermediate or Docetaxel-Linker intermediate be connected to solid phase synthesis resin, 3 phenylalanine (Phe of Sostatin that side chain protected but N-Amino End Group dissociate or Sostatin
3) replace with tyrosine (Tyr) after the Sostatin-Tyr that obtains
3carry out ligation by solid phase synthesis process, prepare taxol-Linker and be connected to Sostatin or Sostatin-Tyr
3the taxol-Sostatin conjugates of peptide chain N-Amino End Group or taxol-Sostatin-Tyr
3conjugates, or Docetaxel-Linker is connected to Sostatin or Sostatin-Tyr
3the Docetaxel-Sostatin conjugates of peptide chain N-Amino End Group or Docetaxel-Sostatin-Tyr
3conjugates;
Described solid phase synthesis specifically refers to,
By Sostatin-resin free to the intramolecular disulfide bond that is connected with solid phase synthesis resin, formed, side chain protected but N-Amino End Group or Sostatin-Tyr
3-resin is placed in solid phase synthesis device;
Taxol-Linker intermediate or Docetaxel-Linker intermediate are dissolved in anhydrous methylene chloride, and instill in solid phase synthesis device after adding PyBop and DIPEA respectively by the mol ratio of 1:1 ~ 1.5, and stir with drying nitrogen;
Washing resin also collects product.
2. preparation method according to claim 1, is characterized in that: described method comprises step further:
To prepare and under liquid phase synthesis condition, carry out ligation with taxol-Linker intermediate or Docetaxel-Linker intermediate further from the conjugates that solid phase synthesis resin is separated, preparing Sostatin or Sostatin-Tyr
3n-Amino End Group and 5 Methionin (Lys
5) epsilon-amino of side chain is connected to two taxols-Sostatin conjugates or the two taxol-Sostatin-Tyr of taxol-Linker
3conjugates; Or Sostatin or Sostatin-Tyr
3n-Amino End Group and 5 Methionin (Lys
5) epsilon-amino of side chain is connected to two Docetaxels-Sostatin conjugates or the two Docetaxel-Sostatin-Tyr of Docetaxel-Linker
3conjugates.
3. preparation method according to claim 2, is characterized in that: describedly under liquid phase synthesis condition, carry out reaction refer to,
To prepare and be dissolved in anhydrous dimethyl formamide from the conjugates that solid phase synthesis resin is separated;
Taxol-Linker intermediate or Docetaxel-Linker intermediate are dissolved in anhydrous methylene chloride, and the described conjugates be separated from solid phase synthesis resin of instillation is dissolved in the solution of anhydrous dimethyl formamide after adding PyBop and DIPEA respectively by the mol ratio of 1:1 ~ 1.5;
Product is collected after abundant reaction.
4. a preparation method for the polypeptide conjugates of taxol or Docetaxel, described method comprises:
Taxol or Docetaxel and linking agent are obtained by reacting on 2 ' position hydroxyl and are connected with catenation sequence-CO-CH
2-(X)
n-CH
2taxol-Linker the intermediate of-CO-OH or Docetaxel-Linker intermediate, wherein X is O (CH
2cH
2oCH
2cH
2cH
2nHCOCH
2-)
2,-CO-,-O-,-S-,-CH
2-,-CHCH
3-or-C (CH
3)
2-, n is 0 or 1;
By 3 phenylalanine (Phe of taxol-Linker intermediate or Docetaxel-Linker intermediate and Sostatin or Sostatin
3) replace with tyrosine (Tyr) after the Sostatin-Tyr that obtains
3react under liquid phase synthesis condition, prepare Sostatin or Sostatin-Tyr
3n-Amino End Group and 5 Methionin (Lys
5) epsilon-amino of side chain is connected to two taxol-Sostatin or the Sostatin-Tyr of taxol-Linker or Docetaxel-Linker
3conjugates or two Docetaxel-Sostatin or Sostatin-Tyr
3conjugates;
Described reaction under liquid phase synthesis condition refers to,
Taxol-Linker intermediate or Docetaxel-Linker intermediate be dissolved in anhydrous dimethyl formamide or anhydrous methylene chloride and instill Sostatin or Sostatin-Tyr after adding PyBop and DIPEA respectively by the mol ratio of 1:1 ~ 1.5
3anhydrous dimethyl formamide solution in, reaction after separation and purification products therefrom.
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| WO2011124031A1 (en) * | 2010-04-08 | 2011-10-13 | 江苏天一时制药有限公司 | Polypeptide conjugate of paclitaxel or docetaxel |
| CN103421085B (en) * | 2012-05-18 | 2015-07-29 | 中国科学院上海药物研究所 | For increasing the oligopeptides of the solubleness of taxol or the similar medicine based on paclitaxel structure |
| CN105418732A (en) * | 2015-12-02 | 2016-03-23 | 烟台海安药物研发有限公司 | Docetaxel Weirui peptide conjugate and preparation method thereof |
| CN106188273A (en) * | 2016-07-13 | 2016-12-07 | 烟台海安药物研发有限公司 | Paclitaxel vapreotide conjugates and preparation method thereof |
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| EP1118336A2 (en) * | 2000-01-13 | 2001-07-25 | Academia Sinica | Somatostatin analogs to specific delivery of anti-tumor drugs into tumor cells |
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Non-Patent Citations (3)
| Title |
|---|
| Direct Solid-Phase Synthesis of Octreotide Conjugates: Precursors for Use as Tumor-Targeted Radiopharmaceuticals;Hsing-Pang Hsieh et al.;《Bioorganic & Medicinal Chemistry》;19991231;第7卷;1797-1803 * |
| Targeting delivery of paclitaxel into tumor cells via somatostatin receptor endocytosis;Chun-Ming Huang et al.;《Chemistry & Biology》;20000609;第7卷(第7期);453-461 * |
| 紫杉醇2奥曲肽耦连药靶向治疗非小细胞肺癌;孙美丽 等;《中国肿瘤生物治疗杂志》;20070831;第14卷(第4期);368-372 * |
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| CN103254279A (en) | 2013-08-21 |
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