CN101857630B

CN101857630B - Fluorouracil derivative containing tyrosine-isoleucine-glycine-serine-arginine polypeptide

Info

Publication number: CN101857630B
Application number: CN201010154674A
Authority: CN
Inventors: 艾伟伦
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-04-20
Filing date: 2010-04-20
Publication date: 2012-10-03
Anticipated expiration: 2030-04-20
Also published as: CN101857630A

Abstract

The invention relates to a fluorouracil compound containing a tyrosine-isoleucine-glycine-serine-arginine sequence and a preparation method thereof. The fluorouracil compound a the structural formula of Tyr-Ile-Gly-Ser-Arg-FU, wherein Tyr-Ile-Gly-Ser-Arg expresses a tyrosine-isoleucine-glycine-serine-arginine pentapeptide sequence, and FU expresses the fluorouracil compound. The invention can be combined with a laminin receptor of a cell 67 kDa, selectively transports the fluorouracil compound to a target tissue in vivo to increase the focus local drug concentration, enhances the curative effect, lowers the toxic and side effects and achieves the purpose of targeted therapy.

Description

Contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide

Technical field

The invention belongs to biological and medical technical field.In particular, it relate to contain junket-different bright-the covalently bound and coupling compound that forms and preparation method thereof of Gan-Si-l-arginine polypeptide of sequence and protein and verivate thereof and Fluracil compounds.The fluorouracil derivative of these contain junket-different bright-Gan-Si-arginine polypeptide initiatively target property be oriented to and has the various tumours that the 67kDa laminin receptor is expressed.

Background technology

Malignant tumour is the healthy malignant disease of one type of serious threat human life.Its generation, development and nucleic acid metabolism have very confidential relation.It is generally acknowledged that the nucleic acid anabolism in the tumour cell is apparently higher than normal cell, it is the basic substance of tumour ramp, propagation that intracellular nucleic acid increases.Because required material mainly is purine and pyrimidine in the nucleic acid anabolism, the metabolism that any material that influences purine or pyrimidine metabolic can both RNA, thus stop the propagation and the division of tumour cell.Nineteen fifty-seven to the anabolic characteristics of nucleic acid, designs and synthesizes out 5 FU 5 fluorouracil first, begins to be applied to the clinical treatment malignant tumour.5 FU 5 fluorouracil is that No. 5 position hydrogen of uridylic molecule are replaced the formed verivate in back by fluorine.Because the atomic radius of fluorine is close with the radius of Wasserstoffatoms, the 5 FU 5 fluorouracil that produces with fluorine replacement hydrogen, just closely similar on molecular structure with the molecular structure of uridylic and thymus pyrimidine, thereby become the metabolism that counterfeit substrate has been participated in nucleic acid.Though the molecular structure of 5 FU 5 fluorouracil is similar with pyrimidine, be not the required pyrimidine molecule of the normal anabolism of nucleic acid, like this, just can cause effectively nucleic acid and make the growth of cell and propagation receive obvious suppression.For many years; The fluorouracil drug that with the 5 FU 5 fluorouracil is representative has been brought into play very important antitumor action clinically; Be used to the treatment as: various clinical common malignancy such as cancer of the stomach, liver cancer, carcinoma of the pancreas, the rectum cancer, mammary cancer, ovarian cancer, cervical cancer, bladder cancer have become the core drug in the various clinical chemotherapy regimen.

The same with other antitumor drug, the Fluracil molecular weight is low, diffusion rapidly easily behind the entering body.Can not optionally differentiate tumour cell and normal cell, lack target property, distribute, can not accumulate the drug level higher in the tumour cell above normal tissue cell thereby produce average relatively body tissue to tumour cell.Like this, Fluracil often causes the non-specific injury to healthy tissues when tumour cell is played a role, makes human body generation gastrointestinal reaction, bone marrow depression, cerebellar degeneration cause ataxia, alopecia, dermatitis, nail matrix blackening etc.And the therapeutic dose of fluorouracil drug is often very approaching with toxic dose, makes their application clinically receive strict restriction.

For the antitumor action that increases Fluracil and overcome its toxic side effects; Utilize the acceptor that high expression level more and/or unconventionality expression are arranged in the tumor cell ratio non-tumor cell (normal tissue cell) as target spot; Combination by ligand-receptor has biological characteristicses such as high specific, highly selective and high-affinity, and part is combined to form carrier-Fluracil mixture as carrier with Fluracil.After carrier-Fluracil mixture gets in the body, highly express on the tumor cell film and/or the acceptor of unconventionality expression and combine with it, through the mediation of cell receptor, carrier-Fluracil mixture is by in the cellular uptake entering cell.In cell, because the effect of cellular enzymes, Fluracil molecule and carrier separating also are released.The Fluracil compounds that like this, in tumour cell, can gather high density makes its performance antitumor action.Meanwhile; Because this receptor is not expressed in normal tissue cell or low the expression; Carrier-Fluracil compound can not or seldom with this receptors bind, cell does not just absorb or only absorbs a spot of compound, thereby has changed the distribution of Fluracil; Significantly reduce Fluracil to the infringement that healthy tissues produces, can effectively overcome the toxic side effects that Fluracil produces body.Therefore; Utilize above-mentioned principle; Design can make the Fluracil target be distributed in the drug molecule or the drug delivery system of tumour cell; Increasing the focus local drug concentration, improve curative effect and reduce toxic side effect to reach the purpose of targeted therapy, is one of most active research field in the present antineoplaston.

Utilize ligand-receptor reaction targeted antitumor drug; Need this receptor in target cell (tumour cell), higher expression to be arranged than other non-target cells (normal tissue cell); Could make the medicine of target cell picked-up high density through the ligand-receptor approach, and carry with the orientation that realizes antitumor drug with the drug level formation notable difference that non-target cell is absorbed.Along with deepening continuously to the research of tumour molecular level; Compare with non-tumor cell; In many common clinically many malignant tumours; Find all that like: cell surfaces such as mammary cancer, cervical cancer, colorectal carcinoma, cancer of the stomach, liver cancer, lung cancer, ovarian cancer, pancreas cancer, prostate cancer and thyroid carcinoma 67kDa laminin receptor (67kDa laminin receptor is designated hereinafter simply as " 67LR ") is highly expressed or the data of over-expresses.For example: the expression of on the cytolemma of most of nonsmall-cell lung cancers, finding 67LR presents over-expresses, and finds that expression level and the nodus lymphoideus transferring rate of 67LR are proportionate.In colon cancer patient, the 67LR expression level of tumour cell is higher than cancer beside organism, and with the progress of tumour be proportionate by stages.In the research that patients with gastric cancer 67LR is expressed, find that there is confidential relation in the expression of tumor invasive depth and 67LR.The expression that people use 67LR in the RT-PCR technical Analysis liver cancer all is significantly higher than non-cancer tissue.Secondary liver cancer 67LR no matter protein level still the expression on the mRNA level all be significantly higher than non-metastatic liver cancer, explain that 67LR plays important effect in the transfer of liver cancer.In ovarian cancer, 67LR all raises in the expression of mRNA and protein level, and finds that the relatively poor patient 67LR of ovarian cancer clinical prognosis is over-expresses.In cervical cancer, the expression ratio normal cervical tissues of 67LR is high, in metastatic carcinoma, expresses the highlyest, the higher proliferation of high expression level and the tumour of 67LR be described, highly infiltration and transfer ability are closely related.Because having on many malignant cells, 67LR highly expresses or over-expresses, so 67LR is a kind of very ideal targeting antineoplastic medicine thing specific recognition and bonded target spot.Many experimental results show that 67LR has become the important target of related neoplasms treatment research, has shown the bright prospects to the 67LR targeted therapy.

67LR is the transmembrane glycoprotein that is present on the cytolemma.People such as nineteen eighty-three Terranova find 67LR first in the mankind mastopathy cell.With 67LR bonded part be ln (laminin is designated hereinafter simply as " LN ").The associative list of 67LR and LN reveals biological natures such as high-affinity, highly selective and high specific.In human body, LN is the NCP in the extracellular matrix, is to constitute the very important structural molecule of extracellular matrix, mainly is present in the transparent layer of basement membrane, near the cell based bottom surface.The basic function of LN is to participate in structure, cell adhesion and the expansion of extracellular matrix system.LN can interact with many macromole in the extracellular matrix such as perlecan, nidogen and collagen and form a polymeric matrix network.In matrix, the aminoterminal of nidogen combines with the IV Collagen Type VI, forms the bridge of LN-IV Collagen Type VI, and this is the basis that forms extracellular matrix super large molecular structure.The heterotrimer of the asymmetric cruciform that the LN molecule is made up of a heavy chain (α chain) and two light chains (beta, gamma chain).Contain some specificity structure sequences in the LN molecule; Especially the one section tyrosine-Isoleucine-glycocoll-Serine that is contained with the shaft-like district of β chain galianconism in the LN molecule-l-arginine pentapeptide sequence [Tyr (Y)-Ile (I)-Gly (G)-Ser (S)-Arg (R) is called for short " YIGSR "] receives special concern.This section YIGSR peptide sequence is in the LN molecule 929～932, also is core of the present invention.People such as Graf found that the β chain YIGSR sequence of LN was the recognition site of 67LR in 1987, also were 67LR and LN bonded specificity site.The YIGSR pentapeptide of synthetic can combine 67LR with the LN competition, shifts thereby suppress the intravital lung of mouse melanin tumor cell B16F10.People such as Yudoh report that YIGSR can combine 67LR with the LN competition, thereby suppress adhesion and the migration of oncocyte in LN of tumour cell and LN.Have report YIGSR polypeptide can be competitively with lung carcinoma cell on 67LR combine, make lung carcinoma cell can not combine to cause the transfer ability decline of cancer cells with LN.In addition, have and discover that the YIGSR peptide also has the effect that antineoplastic vascular generates, not only suppress the metastases kitchen range, and suppress the growth of primary tumors.Use the verivate of YIGSR peptide, can avoid YIGSR to receive the destruction of some enzyme in the body, prolong its RT in vivo, bring into play antitumous effect better.

Although the YIGSR polypeptide has multiple biological function, use YIGSR polypeptide and verivate thereof as carrier and the covalently bound formation covalent coupling of Fluracil compounds compound, do not see relevant report so far yet.YIGSR polypeptide and verivate thereof have the following advantages as tumor-targeting fluorouracil drug carrier:

1), neoplasm targeted therapy is divided into organ targeted therapy, cell-targeting treatment and 3 levels of molecular targeted treatment.Molecular targeted treatment is to express or some significant molecule of over-expresses is a target spot with the tumour cell camber; Optionally be directed against this significant molecular target as identifying object; Strengthen the specificity and the selectivity of antineoplaston; Thereby avoiding the non-selectivity toxic side effect and the resistance of chemotherapeutics, is the highest level of specificity in the targeted therapy.In order to realize molecular targeted treatment, confirm that the tumor-marker property molecular target as identifying object is exactly the basis.Just because of finding the 67LR molecule on many malignant cells, there are over-expresses or highly expression than non-tumor cell, make 67LR become and have specific recognition and the significant target spot of bonded in the very molecular targeted treatment of ideal tumour.Though be LN with 67LR bonded part in vivo, the YIGSR polypeptide fragment in the LN molecule is LN and 67LR bonded site, so the YIGSR polypeptide has and excellent specific properties such as 67LR bonded high-affinity, high specific and highly selective.Therefore, the YIGSR polypeptide is the targeting vector of highly effective molecular targeted property antitumor drug.

2), with the compound that contains the YIGSR polypeptide as carrier, and have multiple combination to carry out coupling between the Fluracil compounds, be the covalently bound multiple choices property that provides of YIGSR polypeptide and Fluracil compounds.After YIGSR polypeptide-Fluracil conjugate gets in the body; Through combining with lysosome in the receptor-mediated endocytosis entering cell; Because the significant difference of lysosome and endochylema and born of the same parents' external environment (as: pH value is 4.8 in the lysosome, and endochylema and born of the same parents are outward 7.4), and the effect of plurality of enzymes such as the interior lytic enzyme that enriches of born of the same parents, esterase; Associative key between YIGSR polypeptide-Fluracil conjugate ruptures; The Fluracil compounds can disintegrate down from carrier rapidly, recovers its inherent character, the performance antitumor action.

3), in blood, the small molecules Fluracil gets into that the back arrives body tissue with blood circulation in the body, in passive diffusion way got into each histocyte very soon, Fluracil concentration promptly descended in the blood.And after in YIGSR polypeptide and verivate thereof and the Fluracil compounds coupling formation covalent compound entering body; Can not pass through the simple passive diffusion mode gets in the cell; Need combine with the 67LR on the cytolemma, through the 67LR mediation, cell absorbs the covalent coupling compound with the endocytosis mode.Because it is relevant with the expression level of cell 67LR that cell absorbs coupling compound, there is not 67LR to express in a large number or the normal tissue cell that hangs down expression can not absorb or seldom absorbs the Fluracil compounds, make that the density loss of coupling compound in blood is slow.Therefore, the covalent coupling compound can keep than have link coupled small molecules Fluracil in blood has more lastingly, higher concentration, and such result is highly beneficial to Fluracil performance antitumor action.In healthy tissues, because Fluracil lacks selectivity, diffusion easily causes average relatively body tissue extensively to distribute in vivo, and healthy tissues is produced serious injury.On the contrary, YIGSR polypeptide and verivate thereof and the coupling of Fluracil compounds have changed their distributional patterns in vivo after forming in the covalent compound entering body.As above-mentioned; Because it is relevant with the expression level of cell 67LR that cell absorbs the covalent coupling compound; There are not 67LR expression or low normal tissue cell of expressing to absorb or seldom absorb coupling compound in a large number; Greatly reduced the distribution of medicine in healthy tissues, made medicine drop to minimum the infringement that healthy tissues produces.In tumor tissues, Fluracil is because be distributed to the cause that whole body is organized everywhere relatively fifty-fifty, and their content in tumour cell is very limited, and the antineoplaston that has seriously influenced them is worth.On the other hand; Can selectively discern the tumour cell of 67LR over-expresses after YIGSR polypeptide and verivate thereof and the coupling of Fluracil compounds; Gather high density covalent coupling compound in the tumour cell that 67LR highly expresses, greatly improved the antitumor action of Fluracil.

4), along with the development of modern biotechnology, studying more pharmaceutical carrier at present has liposome, virus, polycomplex and protein (as: monoclonal antibody) etc.Compare with the other medicines carrier, the YIGSR polypeptide is that human body self exists, and is distributed widely in the human body whole body everywhere, participates in the material of human body normal physiological activity.As everyone knows, can be broken down into amino acid behind the polypeptide entering cell is the cell utilization, so the YIGSR polypeptide is nontoxic to human body.In addition; Many passivity targeted systems though can change the distribution of medicine between tumor tissues and nonneoplastic tissue, self also do not exist stability good enough and target property is undesirable; Shortcoming such as engulfed by the scavenger cell of reticuloendothelial system easily, performance target property antitumor action is limited.And YIGSR polypeptide no antigen, non-immunogenicity can not produce anaphylaxis to human body, and simultaneously, YIGSR polypeptide-medicinal composition does not receive immune the influence after getting in the body yet.And it is molecular targeted that YIGSR polypeptide-medicinal composition belongs to initiative, and the targeting specific of tumour is stronger.In a word, YIGSR polypeptide-medicinal composition has more meliority and wide prospect more.

Summary of the invention

The objective of the invention is for overcoming the toxic side effects that the Fluracil compounds produces human body, strengthen the antitumous effect of Fluracil compounds and provide contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide.This verivate can combine with cell 67kDa laminin receptor, optionally carries the Fluracil compound to arrive intravital target tissue, to increase the focus local drug concentration, improves curative effect and reduces toxic side effect, reaches the purpose of targeted therapy.

Technical scheme of the present invention is achieved in that its general formula is following: Tyr-Ile-Gly-Ser-Arg-FU; Wherein, Tyr-Ile-Gly-Ser-Arg representes tyrosine-Isoleucine-glycocoll-Serine-l-arginine pentapeptide sequence; FU representes the Fluracil compounds, and has the chemical structure that describes below:

F representes fluorine atom, X ₁And X ₃Be selected from O, S, C=N and C=NY respectively, wherein Y is selected from hydrogen, C ₁-C ₁₆Alkyl, C ₁-C ₁₆Acyl group and C ₁-C ₁₆Alkylamine, X ₁And X ₃Can be identical or different; X ₂And X ₄Be selected from O, S, SO, SO respectively ₂, NO and NY, wherein Y is selected from hydrogen, C ₁-C ₁₆Acyl group, C ₁-C ₁₆Alkyl, C ₁-C ₁₆Aryl and C ₁-C ₁₆The halogen acyl group; X ₂And X ₄Can be identical or different; R ₁, R ₂And R ₃Be selected from the sugar shown in hydrogen, hydroxyl, saturated or undersaturated heterocyclic group, halogen, nitro, cyanic acid, toluene semi-annular jade pendant acyl group, fluoroform semi-annular jade pendant phenolic ester, trifluoro-acetate or the following formula respectively:

R ₄, R ₅, R ₆And R ₇Be selected from hydrogen, alkyl, hydroxyl, amino, halogen, alkoxyl group, alkyloyl, mercaptan, thioether, acyloxy, monose or oligose respectively.

Wherein said R ₁, R ₂And R ₃Respectively preferably from C ₁-C ₁₆Alkyl, C ₁-C ₁₆Alkoxyl group, C ₃-C ₈Naphthenic base, C ₁-C ₁₆Acyl group, C ₁-C ₁₆Aryl-acyl, can be by C ₁-C ₈Alkyl, C ₃-C ₈Naphthenic base, acyl group, trifluoro acyl group, aryl, C ₁-C ₈Alkenyl, C ₁-C ₈Alkynyl ,-O-C or-O-CY, wherein Y is selected from hydrogen, C ₁-C ₁₆Alkyl, C ₃-C ₈Naphthenic base, alkoxyalkyl, aralkyl, sweet-smelling alkoxy alkyl, aryloxy alkyl and aryl.

Wherein said R ₄, R ₅, R ₆And R ₇Respectively preferably from hydrogen, hydroxyl, amino, halogen, mercaptan, thioether, acyloxy, acidylate amino, trifluoro acidylate amino, morpholino, the substituted morpholino of cyanic acid, one, two, three or the substituted morpholino of four-methoxyl group,, two, three or the substituted morpholino of four-acyloxy, C ₁-C ₁₆Alkoxyl group, C ₃-C ₈Naphthenic base, C ₁-C ₁₆Alkyl, C ₃-C ₈Naphthenic base, acyl group, trifluoro acyl group, aralkyl, aryl, amino acid, random length amino acid polypeptide, protein, non-replacement or commutablely the benzoic ether of nitro is arranged and be present in monose or the oligose in the fluorouracil drug.

Another technical scheme of the present invention is the following compound of general formula: Z ₁-Tyr-Ile-Gly-Ser-Arg-Z ₂-FU, in the formula, Tyr-Ile-Gly-Ser-Arg representes tyrosine-Isoleucine-glycocoll-Serine-l-arginine pentapeptide sequence; FU representes the Fluracil compounds; Z ₁And Z ₂Be selected from hydrogen, hydroxyl, amino, ester group, thioether, mercaptan, carboxyl, carbonyl, halogen, nitro, cyanic acid, aryl, saturated or undersaturated heterocyclic group respectively, Z ₁And Z ₂Can be identical or different.Z ₁And Z ₂Preferred toluene semi-annular jade pendant acyl group, fluoroform semi-annular jade pendant phenolic ester, trifluoro-acetate, one or two henzylates are amino; Acidylate is amino, the trifluoro acyl group, and the trifluoro acidylate is amino, morpholino; The substituted morpholino of cyanic acid, one, two, three or the substituted morpholino of four-methoxyl group, one, two, three or the substituted morpholino of four-acyloxy, C ₁-C ₁₆Acyl group, C ₁-C ₁₆Aryl-acyl, C ₁-C ₁₆Acyloxy, acyloxy, carboxamido-group, C ₁-C ₁₆Alkyl, C ₁-C ₁₆Alkoxyl group, alkoxyalkyl, hydrocarbon alkyl, aralkyl, sweet-smelling alkoxy alkyl, C ₁-C ₁₆Aromatic yl aminocarbonyl or C ₁-C ₁₆The arylamino thiocarbonyl.

Technical scheme of the present invention also is the following compound of general formula: Aaa ₁-Tyr-Ile-Gly-Ser-Arg-Aaa ₂-FU, wherein Tyr-Ile-Gly-Ser-Arg representes tyrosine-Isoleucine-glycocoll-Serine-l-arginine pentapeptide sequence; FU representes the Fluracil compounds; Aaa ₁And Aaa ₂Be selected from an amino acid, random length amino acid polypeptide or protein respectively, Aaa ₁And Aaa ₂Can be identical or different.Better technical scheme is that its general formula is: Z ₁-Aaa ₁-Tyr-Ile-Gly-Ser-Arg-Aaa ₂-Z ₂-FU, structural formula is following:

The compound of P representes to contain junket-different bright-Gan-Si-arginine polypeptide.Another better technical scheme of the present invention is that its general formula is: Z ₁-Aaa ₁-Tyr-Ile-Gly-Ser-Arg-Aaa ₂-Z ₂-FU, structural formula is following:

The compound of P representes to contain junket-different bright-Gan-Si-arginine polypeptide.

Another better concrete technical scheme of the present invention is: described Fluracil compounds is the 5 FU 5 fluorouracil or derivatives thereof; Described Fluracil compounds is the Tegafur or derivatives thereof; Described Fluracil compounds is the Tegadifur or derivatives thereof; Described Fluracil compounds is own aminoacyl Fluracil or derivatives thereof; Described Fluracil compounds is the FUDR or derivatives thereof.

Described contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide, its structure is as follows:

The structural formula of the most preferred technical scheme of the present invention such as Fig. 4, shown in Figure 5, the curative effect of the compound of above-mentioned two kinds of structures is best, and preparation is convenient.

Preparing method of the present invention is to be raw material with 9-fluorenylmethyloxycarbonyl protection amino acid; Adopt the synthetic junket of Wang resin solid-phase polypeptide synthesis method-different bright-Gan-Si-arginine polypeptide; Then with LUTARALDEHYDE as linking agent, with junket-different bright-Gan-Si-arginine polypeptide and fluorouracil compound be cross-linked to form coupling compound.

Another preparation method of the present invention be with junket-different bright-Gan-Si-arginine polypeptide is dissolved in the DMSO 99.8MIN.; Add 1-hydroxy benzo triazole and dicyclohexyl carbon imide subsequently respectively; Again the Fluracil compound is dissolved in adding in the reaction system behind the DMSO 99.8MIN. and reacts, through the preparation HPLC chromatography must contain junket-different bright-the Fluracil coupling compound of Gan-Si-arginine polypeptide.

Another preparation method of the present invention be through dicarboxylicacid with the Fluracil compound with junket-different bright-Gan-Si-arginine polypeptide is coupled at and forms compound.

A preparation method more of the present invention be with penta 4-(N-maleimide ylmethyl) hexanaphthene-1-carboxylic acid succinimide ester as linking agent, will contain junket-different bright-compound of Gan-Si-arginine polypeptide and Fluracil compound be cross-linked to form contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide.

Provided by the invention contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide.It can combine with cell 67kDa laminin receptor, optionally carries the Fluracil compounds to arrive intravital target tissue, to increase the focus local drug concentration, improves curative effect and reduces toxic side effect, reaches the purpose of targeted therapy.

Description of drawings

Figure 1A-Fig. 1 C be respectively junket-different bright-molecular structure of Gan-Si-arginine polypeptide 5 FU 5 fluorouracil compound.

Fig. 2 A-Fig. 2 C be respectively half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil, half Guang-asparagus fern-dried meat-Gan-junket-different is bright-Gan-Si-essence-5 FU 5 fluorouracil, half Guang-asparagus fern-dried meat-Gan-junket-different bright-molecular structure of Gan-Si-essence-5 FU 5 fluorouracil compound.

Fig. 3 A-3C be respectively acetyl-junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil, acetyl-junket-different is bright-Gan-Si-essence-5 FU 5 fluorouracil, acetyl-junket-different bright-molecular structure of Gan-Si-essence-5 FU 5 fluorouracil compound.

Fig. 4 be junket-different bright-molecular structure of Gan-Si-essence-succinyl-2 '-deoxidation-5 FU 5 fluorouracil nucleoside compound.

Fig. 5 be junket-different bright-molecular structure of Gan-Si-essence-glutaryl--oxygen-2 '-deoxidation-5 FU 5 fluorouracil nucleoside compound.

Fig. 6 be junket-different bright-the stable comparison diagram of Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound.

Fig. 7 A-Fig. 7 F be junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil is at the intravital distribution plan of tumor-bearing mice.

Fig. 8 be junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil is to the restraining effect comparison diagram of tumor-bearing mice tumor growth in vivo.

Specific embodiment

At present, there is the clear and definite fluorouracil drug of multiple curative effect to be used to treat various malignant tumours by clinical.Because 5 FU 5 fluorouracil is found first antimetabolic antitumor drug, clinical application range is wide, the time is long.The novel fluorouracil drug of constantly releasing for many years all is to be that the basis is found with the 5 FU 5 fluorouracil.So, below listed examples be to be that typical scenario is described the present invention in detail with the 5 FU 5 fluorouracil.The described method of this specific embodiment and the fact also are applicable to other Fluracil compounds.Equally, for ease of explaining the present invention, with 5 FU 5 fluorouracil covalently bound be with junket-different bright-Gan-Si-arginine polypeptide is a typical scenario.Other can specific recognition with combine cell 67kDa laminin receptor and comprise junket-different bright-Gan-Si-l-arginine polypeptide of sequence and protein and their verivate, also be applicable to the embodiment that describes below.The Fluracil compounds can have multiple mode with contain junket-different bright-the compound coupling of Gan-Si-arginine polypeptide forms covalent conjunct agent, only introduces wherein several kinds of compound methods commonly used here.Therefore, following embodiment is in order to explain rather than limit the present invention.

Embodiment 1: junket-different is bright-and Gan-Si-arginine polypeptide synthetic

The present invention is a raw material with 9-fluorenylmethyloxycarbonyl (Fmoc) protection amino acid, adopt the synthetic junket of Wang resin solid-phase polypeptide synthesis method-different bright-Gan-Si-arginine polypeptide.Other polypeptide synthesis method is suitable for junket-different equally bright-Gan-Si-arginine polypeptide synthetic.The peptide that connects of present embodiment polypeptide is l-arginine (Arg) → Serine (Ser) → glycocoll (Gly) → Isoleucine (Ile) → tyrosine (Tyr) in proper order.Activated resin at first.Take by weighing the 10g Wang resin, put it in the reactor drum, add 150mL N, dinethylformamide (DMF) soaks 30 minutes (be used for activated resin, it is fully expanded), uses DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing successively.Fmoc-Arg (Pbf) is fixed on the activatory Wang resin.Add the 1-hydroxy benzo triazole (HOBt) of 1.08g, the NSC 57182 (DCC) of 0.82g, the 4-dimethylamino piperidine (DMAP) of 0.24g and Fmoc-Arg (Pbf)-OH of 5.2g respectively; Reacted 4 hours; Filter; Use methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing reaction resin successively, each washing is no less than 1 minute.With the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the resin that takes a morsel detects to being negative with ninhydrin method (Kaiser method) then.Then, according to meeting peptide be linked in sequence next amino acid, that is: Fmoc-Ser (tBu)-OH.Get HOBt 1.08g, DCC 1.64g and Fmoc-Ser (tBu)-OH3.06g respectively and join in the reaction system reaction 4 hours; Filter; Methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing; With the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the Kaiser method detects to being negative.Connect Fmoc-Gly-OH successively again.Get HOBt 1.08g, DCC 1.64g and Fmoc-Gly-OH 2.65g and join in the reaction system reaction 4 hours; Filter; Methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing, with the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the Kaiser method detects to being negative.Connect reactive polypeptide and connect Fmoc-Ile-OH again.In reaction system, add HOBt 1.08g, DCC 1.64g and Fmoc-Ile-OH 2.82g respectively; Reacted 4 hours, and filtered, methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing; With the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the Kaiser method detects to being negative.The next amino acid that connects the reactive polypeptide access is Fmoc-Tyr (tBu)-OH.Add HOBt 1.08g, DCC 1.64g and Fmoc-Tyr (tBu)-OH 3.68g respectively; Reacted 4 hours, and filtered, methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DM F washing; With the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the Kaiser method detects to being negative.Like this, the peptide bond between the five amino acid all forms.Through aforesaid method synthetic junket-different bright-Gan-Si-arginine polypeptide, need cut down from resin.The method of cutting resin is to add trifluoroacetic acid, m-cresol, 0 ℃ of reaction 1.5h.The G-3 funnel filters, and filtrating concentrates, and removes trifluoroacetic acid, adds anhydrous diethyl ether, is settled out solid, and the G-4 funnel filters, and removes filtrating, and solid is dissolved in distilled water, lyophilize, bullion junket-different bright-Gan-Si-arginine polypeptide.Thick peptide confirms to collect target peak again through RP-HPLC (anti-phase type performance liquid chromatography) separation and purification.Adopt the MALDI-Ms mass spectroscopy to identify products obtained therefrom, relatively come to confirm the synthetic result through molecular weight on the mass spectrum and theoretical value.

Embodiment 2: junket-different is bright-and Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound synthetic

Get the 5 FU 5 fluorouracil of 0.86g and 4-brooethyl-3-nitrobenzoic acid of 1.0g; In the dark join in the 5mL acetonitrile and reacted 4 hours; Add 7mL methyl alcohol reaction 30 minutes under the room temperature, vacuum-drying, it is methyl alcohol that resistates adopts eluent: ETHYLE ACETATE: acetic acid (1: 3: 0.004; V: v: v) column chromatographic isolation and purification, the faint yellow 3-of 197mg (2 '-nitro-4 '-phenylformic acid)-5 FU 5 fluorouracil.

Get the resiniferous junket of 0.5g-different bright-Gan-Si-arginine polypeptide (with reference to 1 described method synthetic of embodiment not with the polypeptide of resin cutting) and 3-(2 '-nitro-4 '-phenylformic acid)-5 FU 5 fluorouracil of 30mg, use 2-(7-azo benzotriazole)-N respectively, N; N ', N '-tetramethyl-urea phosphofluoric acid ester and N, the activation of N-diisopropylethylamine; Filter, N (5 * 4mL) with methylene dichloride (4 * 4mL) wash, form resiniferous junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil; Under the room temperature, add 0.3mL methyl-phenoxide and 26mL trifluoroacetic acid, add the 3.8mL DMSO 99.8MIN. after 1 hour again; 4 ℃ were stirred 1 hour down, removed by filter resin, added 80mL ice ether; Centrifugal collecting precipitate; Ice ether washing (4 * 20mL), through the separation and purification of preparation type HPLC, freeze-drying gets junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound.See Figure 1A through aforesaid method synthetic molecular structure of compounds.

Synthesizing of embodiment 3:1-(N-methyl-N-2-benzyl carbamyl)-5 FU 5 fluorouracil

Get the 2g o-nitro benzyl alcohol and the 2.72mL triethylamine is added in the THF of 40mL.Then, under-20 ℃ of argon gas, slowly drip the methyl semi-annular jade pendant acyl chlorides of 1.06mL.-20 ℃ were stirred down after 2 hours, added 50mL cold water, with t-butyl methyl ether (3 * 30mL) extractions; Organic phase adds 1N Hydrogen chloride (40mL), saturated sodium bicarbonate solution washing, anhydrous magnesium sulfate drying; Vacuum-drying gets the light yellow methylsulfonic acid of 2.61g, adds the THF that 6.72mL contains the 2N methylamine again, and stirring reaction is 8 hours under the room temperature; The MTBE dilution; Organic layer washs through saturated sodium bicarbonate, and anhydrous magnesium sulfate drying, resistates employing eluent are that the quick silicagel column gradient chromatography separation and purification of methyl alcohol-methylene dichloride-1% triethylamine gets 1.52g methyl-(2-nitrobenzyl) amine.

Get 130mg methyl-(2-nitrobenzyl) amine and 20mg gac and join in the 5mL toluene, slowly drip 190 μ L superpalites, stirring reaction is after 24 hours under the room temperature; In reaction system, charge into argon gas 15 minutes removing unnecessary carbonyl chloride, vacuum filtration elimination gac, resistates is added to the anhydrous N of 5mL after the removal of solvent under reduced pressure; In the dinethylformamide; The 5 FU 5 fluorouracil sodium salt that adds 178mg again, stirring reaction is 18 hours under the room temperature, vacuum-drying; It is the silica gel column chromatography separation and purification of acetone-normal hexane that resistates adopts eluent, gets 161mg white 1-(N-methyl-N-2-nitrobenzyl carboxamide)-5 FU 5 fluorouracil solids.

1-(N-methyl-N-2-nitrobenzyl carboxamide)-5 FU 5 fluorouracil of 20mg is joined 5mL contained in the methyl alcohol of 10% palladium-carbon catalyst hydrogenation 40 minutes; Reactant is through filtering, vacuum-drying remove desolvate 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil.

Embodiment 4:1-[N-methyl-N '-[2-(junket-different bright-Gan-Si-essence-) aminobenzyl carboxamide]-5 FU 5 fluorouracil synthetic

With 110mg junket-different bright-Gan-Si-arginine polypeptide joins in the 2mL DMSO 99.8MIN., adds 0.264mL subsequently, 1M hydroxybenzotriazole/SL 1332 and 0.264mL, 1M dicyclohexyl carbon imide/SL 1332.Get 70mg by embodiment 3 method synthetic 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil, join in the above-mentioned reactant after being dissolved in the 2mL DMSO 99.8MIN., with diisopropylethylamine adjustment pH value to 7.0.Stirred 2 hours at 10 ℃.Reactant joins 400mL, in 0.1% bicarbonate of ammonia.Then through the preparation HPLC chromatography get junket-different bright-Gan-Si-essence-1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil compound.Its molecular structure is seen Figure 1B.

Embodiment 5: with LUTARALDEHYDE as the synthetic junket of linking agent-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound

Utilize LUTARALDEHYDE as linking agent, the amino in-Gan-Si-arginine polypeptide bright with junket-different and the amino group in 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil molecule are cross-linked to form coupling compound respectively by 2 symmetric active aldehyde radicals in the LUTARALDEHYDE molecule.The glutaraldehyde cross-linking method has single stage method and two step method.The single stage method concrete grammar is following: get 0.75mg according to method synthetic 1-(N-methyl-N-2-aminobenzyl the carboxamide)-5 FU 5 fluorouracil of embodiment 3 explanation and 1mg junket-different bright-Gan-Si-arginine polypeptide; Join 2mL and contain in the 0.15M sodium chloride solution of 0.1% LUTARALDEHYDE, reaction is 30 minutes under the room temperature.Use then Dowex50W * 8 Zeo-karbs (the H+ type, 5 * 15mm) carry out chromatography, filter through 0.45 μ m strainer again, obtain junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound.Two step method is that 10mg 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil is dissolved among the 0.1M PBS that 2mL contains 1.25% LUTARALDEHYDE; Reaction is 1 hour under the room temperature; Remove unnecessary LUTARALDEHYDE through Sephadex G-25 chromatography; Add then 5mg junket-different bright-Gan-Si-arginine polypeptide, reaction is 2 hours under the room temperature.Use Dowex 50W * 8 Zeo-karb (H then ⁺Type, 5 * 15mm) carry out chromatography, filter through 0.45 μ m strainer, junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound, its molecular structure such as Fig. 1 C.

6: half Guang-asparagus fern-dried meat-Gan-junket of embodiment-different is bright-Gan-Si-arginine polypeptide synthetic

On the basis of tyrosine-Isoleucine-glycocoll-Serine-l-arginine pentapeptide, increase one or more amino acid; As increase half Guang-asparagus fern-dried meat-Gan-junket of forming behind halfcystine, aspartic acid, proline(Pro) and the glycocoll-different bright-Gan-Si-arginine polypeptide, also have specific recognition and the characteristic that combines cell 67kDa laminin receptor.Utilize half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide is as carrier; The coupling compound that forms with Fluracil compounds covalent attachment also can be positioned fluorouracil compound target tropism to have in the various tumour cells of 67kDa laminin receptor height or over-expresses.Bright with the junket of embodiment 1 explanation-different-Gan-Si-arginine polypeptide compound method is the same, any known polypeptide synthesis method also all be applicable to half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide carrier synthetic.As space is limited, the present invention also is to be raw material with 9-fluorenylmethyloxycarbonyl (Fmoc) protection amino acid, adopt synthetic half Guang of Wang resin solid-phase polypeptide synthesis method-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide.Other polypeptide synthesis method is suitable for equally half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide synthetic.

Present embodiment synthetic polypeptide, be adopt l-arginine (Arg) → Serine (Ser) → glycocoll (Gly) → Isoleucine (Ile) → tyrosine (Tyr) → glycocoll (Gly) → proline(Pro) (Pro) → aspartic acid (Asp) → halfcystine (Cyc) connect peptide in sequence.Activated resin and make Fmoc-Arg (Pbf) be fixed to identical that method and embodiment 1 on the activatory Wang resin describes.And, meet method that reactive polypeptide inserts Fmoc-Ser (tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH and Fmoc-Tyr (tBu)-OH successively and embodiment 1 and describe also identically, insert the 6th amino acid up to connecing the reactive polypeptide peptide, promptly insert Fmoc-Gly-OH.Get HOBt 1.08g, DCC 1.64g and Fmoc-Gly-OH 2.65g and join in the reaction system reaction 4 hours; Filter; Methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing, with the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the Kaiser method detects to being negative.The seven amino acid that connects the access of reactive polypeptide peptide is Fmoc-Pro-OH.Add HOBt 1.08g, DCC1.64g and Fmoc-Pro-OH 2.82g respectively; Reacted 4 hours, and filtered, methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing; With the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the Kaiser method detects to being negative.The 8th amino acid that connects the access of reactive polypeptide peptide is Fmoc-Asp (tBu)-OH.Add HOBt 1.08g, DCC 1.64g and Fmoc-Asp (tBu)-OH3.26g respectively; Reacted 4 hours, and filtered, methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing; With the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the Kaiser method detects to being negative.The 9th amino acid that connects the access of reactive polypeptide peptide is Fmoc-Cyc (Acm)-OH.Add HOBt1.08g, DCC 1.64g and Fmoc-Cyc (Acm)-OH 3.75g respectively; Reacted 4 hours, and filtered, methylene dichloride/DMF/ methyl alcohol/DMF/ methyl alcohol/DMF washing; With the Fmoc group on the DMF solution deresinate that contains 20% piperidines, the Kaiser method detects to being negative.Like this, the peptide bond between nine amino acid all forms.Through aforesaid method synthetic polypeptide, need cut down from resin.Cutting resin German side method is to add trifluoroacetic acid, m-cresol, 0 ℃ of reaction 1.5h.The G-3 funnel filters, and filtrating concentrates, and removes trifluoroacetic acid, adds anhydrous diethyl ether, is settled out solid, and the G-4 funnel filters, and removes filtrating, and solid is dissolved in distilled water, lyophilize, remove resin get bullion half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide.Thick peptide confirms to collect target peak again through RP-HPLC (anti-phase type performance liquid chromatography) separation and purification.Adopt the MALDI-Ms mass spectroscopy to identify products obtained therefrom, relatively come to confirm the synthetic result through molecular weight on the mass spectrum and theoretical value.

7: half Guang-asparagus fern-dried meat-Gan-junket of embodiment-different is bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound synthetic

The method of present embodiment synthesizes half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-3-oil of mirbane formyl-5 FU 5 fluorouracil compound is with reference to embodiment 2.Get the 5 FU 5 fluorouracil of 0.86g and 4-brooethyl-3-nitrobenzoic acid of 1.0g; In the dark join in the 5mL acetonitrile and reacted 4 hours; Add 7mL methyl alcohol reaction 30 minutes under the room temperature, vacuum-drying, it is methyl alcohol that resistates adopts eluent: ETHYLE ACETATE: acetic acid (1: 3: 0.004; V: v: column chromatography separation and purification v) gets the faint yellow 3-of 197mg (2 '-nitro-4 '-phenylformic acid)-5 FU 5 fluorouracil product.

Get resiniferous half Guang of 1.0g-asparagus fern-dried meat-Gan-junket-different bright-3-(2 '-nitro-4 '-phenylformic acid)-5 FU 5 fluorouracil of Gan-Si-arginine polypeptide (polypeptide and resin do not cut) (polypeptide synthesis method of describing with reference to embodiment 6) and 30mg, use 2-(7-azo benzotriazole)-N respectively, N; N ', N '-tetramethyl-urea phosphofluoric acid ester and N, the activation of N-diisopropylethylamine; Filter, N, dinethylformamide (5 * 4mL) with methylene dichloride (4 * 4mL) wash; Form resiniferous half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil, under the room temperature, add 0.3mL methyl-phenoxide and 26mL trifluoroacetic acid; Add the 3.8mL DMSO 99.8MIN. after 1 hour again; 4 ℃ were stirred 1 hour down, removed by filter resin, added 80mL ice ether; Centrifugal collecting precipitate; Ice ether washing (4 * 20mL), through the separation and purification of preparation type HPLC, freeze-drying gets half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound (molecular structure is seen Fig. 2 A).

8: half Guang-asparagus fern-dried meat-Gan-junket of embodiment-different is bright-Gan-Si-essence-1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil synthetic

Present embodiment is with reference to the method for embodiment 4; With half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide; (N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil is covalently bound with pressing embodiment 3 method synthetic 1-, form half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil compound.With 150mg half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide joins in the 2mL DMSO 99.8MIN., adds 0.264mL subsequently, 1M hydroxybenzotriazole/SL 1332 and 0.264mL, 1M dicyclohexyl carbon imide/SL 1332.Get 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil of 70mg, join in the above-mentioned reactant after being dissolved in the 2mL DMSO 99.8MIN., with diisopropylethylamine adjustment pH value to 7.0.Stirred 2 hours at 10 ℃.Reactant joins 400mL, in 0.1% bicarbonate of ammonia.Then through the preparation HPLC chromatography get half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil compound.Its molecular structure is seen Fig. 2 B.

Embodiment 9: with LUTARALDEHYDE as synthetic half Guang of linking agent-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound

Present embodiment is with reference to the method for embodiment 5; Utilize LUTARALDEHYDE as linking agent, the amino in-Gan-Si-arginine polypeptide bright with Guang-asparagus fern-dried meat-Gan-junket-different and the amino group in 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil molecule are cross-linked to form coupling compound respectively by 2 symmetric active aldehyde radicals in the LUTARALDEHYDE molecule.The glutaraldehyde cross-linking method has single stage method and two step method.The single stage method concrete grammar is following: get 0.75mg according to method synthetic 1-(N-methyl-N-2-aminobenzyl the carboxamide)-5 FU 5 fluorouracil of embodiment 3 explanation and 1.5mg half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide; Join 2mL and contain in the 0.15M sodium chloride solution of 0.1% LUTARALDEHYDE, reaction is 30 minutes under the room temperature.Use Dowex 50W * 8 Zeo-karb (H then ⁺Type, 5 * 15mm) carry out chromatography, filter through 0.45 μ m strainer again, obtain half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound.Two step method is that 10mg 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil is dissolved among the 0.1M PBS that 2mL contains 1.25% LUTARALDEHYDE; Reaction is 1 hour under the room temperature; Remove unnecessary LUTARALDEHYDE through Sephadex G-25 chromatography; Add then 8mg half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine polypeptide, reaction is 2 hours under the room temperature.Use Dowex50W * 8 Zeo-karb (H then ⁺Type, 5 * 15mm) carry out chromatography, filter through 0.45 μ m strainer, half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound, its molecular structure such as Fig. 2 C.

Embodiment 10: with 4-(N-maleimide ylmethyl) hexanaphthene-1-carboxylic acid succinimide ester be the synthetic 1-(N-methyl-N-2-aminobenzyl carboxamide) of linking agent-5 FU 5 fluorouracil-half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine compounds

4-(N-maleimide ylmethyl) hexanaphthene-1-carboxylic acid succinimide ester (SMCC) is a kind of special-shaped difunctional polypeptide commonly used or proteinic linking agent.Amino on the active ester of SMCC molecule one end and 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil molecule forms the amine key of covalency; The maleimide groups of the SMCC molecule the other end and half Guang-asparagus fern-dried meat-Gan-junket-different is bright-and sulfydryl on Gan-Si-arginine polypeptide (CDPGYIGSR) forms stable thioether bond, thereby 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil and CDPGYIGSR is coupled at together.Specific as follows: 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil of getting 6mg is dissolved in 0.5mL DMSO 99.8MIN. (DMSO) and 8mL, in the phosphoric acid buffer of pH 8.0 (PBS), adds the SMCC of 20 μ L triethylamines and 2mg again; Stirred 2 hours under the room temperature, transfer pH to 5.5, get 4.5mg CDPGYIGSR and add in the reactor drum; Stirred 2 hours under the room temperature; Add the acetonitrile/water that 2mL contains 0.1% trifluoroacetic acid (70/30, v/v) solution, last appearance is to half preparation HPLC.Obtain 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil-hexanaphthene acid amides-maleimide ylmethyl-half Guang-asparagus fern-dried meat-Gan-junket-different bright-Gan-Si-arginine compounds.

Embodiment 11: acetyl-junket-different is bright-and Gan-Si-arginine polypeptide synthetic

Acetyl-junket-different is bright-Gan-Si-arginine polypeptide compound be junket-different bright-verivate of Gan-Si-arginine polypeptide, have specific recognition and the characteristic that combines cell 67kDa laminin receptor.Because acetyl-junket-different bright-Gan-Si-arginine polypeptide compound synthetic, with junket-different bright-Gan-Si-arginine polypeptide acetylize and forming, so junket-different is bright-compound method of Gan-Si-arginine polypeptide is identical with the method that embodiment 1 explains.According to the method for embodiment 1 explanation accomplish resiniferous junket-different bright-Gan-Si-arginine polypeptide after, polypeptide is cut down from resin.Add trifluoroacetic acid, m-cresol, 0 ℃ of reaction 1.5h.The G-3 funnel filters, and filtrating concentrates, and removes trifluoroacetic acid; Add anhydrous diethyl ether, be settled out solid, the G-4 funnel filters; Remove filtrating, solid is dissolved in distilled water, resin is removed in lyophilize; Add 5% acetate, through RP-HPLC (anti-phase type performance liquid chromatography) separation and purification, obtain acetyl junket-different bright-Gan-Si-arginine polypeptide.

Embodiment 12: acetyl-junket-different is bright-and Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound synthetic

Present embodiment synthesis of acetyl-junket-different is bright-and the method for Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound is with reference to embodiment 2.Get the 5 FU 5 fluorouracil of 0.86g and 4-brooethyl-3-nitrobenzoic acid of 1.0g; In the dark join in the 5mL acetonitrile and reacted 4 hours; Add 7mL methyl alcohol reaction 30 minutes under the room temperature, vacuum-drying, it is methyl alcohol that resistates adopts eluent: ETHYLE ACETATE: acetic acid (1: 3: 0.004; V: v: column chromatography separation and purification v) gets the faint yellow 3-of 197mg (2 ' nitro-4 '-phenylformic acid)-5 FU 5 fluorouracil powder.

Get acetyl-junket that 0.65g comprises resin-different bright-Gan-Si-arginine polypeptide (the method institute synthetic of describing with reference to embodiment 11 not acetyl-the junket of cutting resin-different bright-Gan-Si-arginine polypeptide) and 3-(2 '-nitro-4 '-phenylformic acid)-5 FU 5 fluorouracil of 30mg, use 2-(7-azo benzotriazole)-N respectively, N; N ', N '-tetramethyl-urea phosphofluoric acid ester and N, the activation of N-diisopropylethylamine; Filter, N, dinethylformamide (5 * 4mL) with methylene dichloride (4 * 4mL) wash; Form resiniferous acetyl-junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil, under the room temperature, add 0.3mL methyl-phenoxide and 26mL trifluoroacetic acid; Add the 3.8mL DMSO 99.8MIN. after 1 hour again, 4 ℃ were stirred 1 hour down, remove by filter resin; Add 80mL ice ether, centrifugal collecting precipitate, ice ether washing (4 * 20mL); Through the separation and purification of preparation type HPLC; Freeze-drying, obtain acetyl-junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound, its molecular structure is seen Fig. 3 A.

Embodiment 13: acetyl-junket-different is bright-and Gan-Si-essence-1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil synthetic

Present embodiment is with reference to the method for embodiment 4; With acetyl-junket-different bright-Gan-Si-arginine polypeptide; (N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil is covalently bound with pressing embodiment 3 method synthetic 1-, form acetyl-junket-different bright-Gan-Si-essence-1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil compound.With 150mg acetyl-junket-different bright-Gan-Si-arginine polypeptide joins in the 2mL DMSO 99.8MIN., adds 0.264mL subsequently, 1M hydroxybenzotriazole/SL 1332 and 0.264mL, 1M dicyclohexyl carbon imide/SL 1332.Get 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil of 70mg, join in the above-mentioned reactant after being dissolved in the 2mL DMSO 99.8MIN., with diisopropylethylamine adjustment pH value to 7.0.Stirred 2 hours at 10 ℃.Reactant joins 400mL, in 0.1% bicarbonate of ammonia.Then through the preparation HPLC chromatography get acetyl-junket-different bright-Gan-Si-essence-1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil compound.Its molecular structure is seen Fig. 3 B.

Embodiment 14: with LUTARALDEHYDE as linking agent synthesis of acetyl-junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound

Present embodiment is with reference to the method for embodiment 5; Utilize LUTARALDEHYDE as linking agent, the amino in-Gan-Si-arginine polypeptide bright with acetyl-junket-different and the amino group in 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil molecule are cross-linked to form coupling compound respectively by 2 symmetric active aldehyde radicals in the LUTARALDEHYDE molecule.The glutaraldehyde cross-linking method has single stage method and two step method.The single stage method concrete grammar is following: get 0.75mg according to method synthetic 1-(N-methyl-N-2-aminobenzyl the carboxamide)-5 FU 5 fluorouracil of embodiment 3 explanation and 1.5mg acetyl-junket-different bright-Gan-Si-arginine polypeptide; Join 2mL and contain in the 0.15M sodium chloride solution of 0.1% LUTARALDEHYDE, reaction is 30 minutes under the room temperature.Use Dowex 50W * 8 Zeo-karb (H then ⁺Type, 5 * 15mm) carry out chromatography, filter through 0.45 μ m strainer again, obtain acetyl-junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound.Two step method is that 10mg 1-(N-methyl-N-2-aminobenzyl carboxamide)-5 FU 5 fluorouracil is dissolved among the 0.1M PBS that 2mL contains 1.25% LUTARALDEHYDE; Reaction is 1 hour under the room temperature; Remove unnecessary LUTARALDEHYDE through the SephadexG-25 chromatography; Add then 8mg acetyl-junket-different bright-Gan-Si-arginine polypeptide, reaction is 2 hours under the room temperature.Use then Dowex 50W * 8 Zeo-karbs (the H+ type, 5 * 15mm) carry out chromatography, filter through 0.45 μ m strainer, acetyl-junket-different bright-Gan-Si-essence-5 FU 5 fluorouracil compound, its molecular structure such as Fig. 3 C.

Embodiment 15: junket-different is bright-Gan-Si-essence-succinyl-oxygen-2 '-deoxidation-5 FU 5 fluorouracil nucleoside compound synthetic

2 '-deoxidation-5 FU 5 fluorouracil nucleosides (5-FdUrd) is the verivate of 5 FU 5 fluorouracil; Through using ester bond of a carboxyl and the oh group formation of 3 of 5-FdUrd ' position of dicarboxylicacid transcribed spacer such as Succinic Acid; Another carboxyl of Succinic Acid and junket-different is bright-free amine group in Gan-Si-arginine polypeptide molecule forms a carboxylic acid amides key, thereby by the dicarboxylicacid transcribed spacer with 5-FdUrd with junket-different bright-Gan-Si-arginine polypeptide be coupled at form junket-different bright-Gan-Si-essence-succinyl-oxygen-2 '-deoxidation-5 FU 5 fluorouracil nucleoside compound.The 5-FdUrd that gets 147.5mg dissolves in the 2mL pyridine, adds 304.5mg dimethoxytrityl methyl chloride, stirs 16 hours under the room temperature, adds 10mL, 5% ice sodium hydrogencarbonate, dichloromethane extraction, anhydrous Na ₂SO ₄Drying, resistates adopt eluent be the silica gel column chromatography separating purification of methyl alcohol-methylene dichloride get 2 of 223.6mg '-deoxidation-5 '-oxygen-[two (4-p-methoxy-phenyl) phenyl methyl]-5 FU 5 fluorouracil nucleosides product.

Get 2 of 74.5mg '-deoxidation-5 '-the 4-Dimethylamino pyridine of oxygen-[two (4-p-methoxy-phenyl) phenyl methyl]-5 FU 5 fluorouracil nucleosides and 10.2mg Succinic anhydried (coupling agent) and 82.2mg; Add the 16mL methylene dichloride; Stirred 16 hours under the room temperature, add 10mL, 5% ice sodium hydrogencarbonate; Dichloromethane extraction, anhydrous Na ₂SO ₄Drying, resistates adopt eluent be the silica gel column chromatography separating purification of methyl alcohol-methylene dichloride get 2 '-deoxidation-3 '-oxygen-half succinate-5 '-oxygen-[two (4-p-methoxy-phenyl) phenyl methyl]-5 FU 5 fluorouracil nucleosides.

Get the resiniferous junket of 185mg-different bright-Gan-Si-arginine polypeptide (the method institute synthetic of describing with reference to embodiment 1 is not sloughed the polypeptide of resin) and 42mg 2 '-deoxidation-3 '-oxygen-half succinate-5 '-oxygen-[two (4-p-methoxy-phenyl) phenyl methyl]-5 FU 5 fluorouracil nucleosides, use 2-(7-azo benzotriazole)-N respectively, N; N ', N '-tetramethyl-urea phosphofluoric acid ester and N, the activation of N-diisopropylethylamine; Filter, N (5 * 4mL) with methylene dichloride (in the solution that 4 * 4mL) washings, adding are made up of the methyl-phenoxide of the trimethylchlorosilane of the trifluoroacetic acid of 86mL, 0.72mL and 0.99mL; Effect is 1 hour under the room temperature; In reactor drum, add the 12.5mL DMSO 99.8MIN., 4 ℃ were stirred 1 hour down, filter and remove resin; Add the 600mL dry diethyl ether; Centrifugal, dry diethyl ether washing (4 * 20mL), then through the preparation HPLC chromatography get junket-different bright-Gan-Si-essence-succinyl-oxygen-2 '-deoxidation-5 FU 5 fluorouracil nucleoside compound.Its molecular structure is seen Fig. 4.

Embodiment 16: junket-different is bright-Gan-Si-essence-glutaryl--oxygen-2 '-deoxidation-5 FU 5 fluorouracil nucleoside compound synthetic

2 '-deoxidation-5 fluorouracil nucleoside (5-FdUrd) is the verivate of 5 FU 5 fluorouracil; Through using ester bond of a carboxyl and the oh group formation of 3 of 5-FdUrd ' position of dicarboxylicacid transcribed spacer such as pentanedioic acid; Another carboxyl of pentanedioic acid and junket-different is bright-free amine group in Gan-Si-arginine polypeptide molecule forms a carboxylic acid amides key, thereby by the dicarboxylicacid transcribed spacer with 5-FdUrd with junket-different bright-Gan-Si-arginine polypeptide be coupled at form junket-different bright-Gan-Si-essence-glutaryl--oxygen-2 '-deoxidation-5 fluorouracil nucleoside compound.The 5-FdUrd that gets 147.5mg dissolves in the 2mL pyridine, adds 4,4 of 304.5mg '-dimethoxytrityl methyl chloride, stirs 16 hours under the room temperature, adds 10mL, 5% ice sodium hydrogencarbonate, dichloromethane extraction, anhydrous Na ₂SO ₄Drying, resistates adopt eluent be the silica gel column chromatography separating purification of methyl alcohol-methylene dichloride get 2 of 223.6mg '-deoxidation-5 '-oxygen-[two (4-p-methoxy-phenyl) phenyl methyl]-5 FU 5 fluorouracil nucleosides product.

Get 2 of 74.5mg '-deoxidation-5 '-the 4-Dimethylamino pyridine of oxygen-[two (4-p-methoxy-phenyl) phenyl methyl]-5 FU 5 fluorouracil nucleosides and 12mg Pyroglutaric acid (coupling agent) and 82.2mg; Add the 16mL methylene dichloride; Stirred 16 hours under the room temperature, add 10mL, 5% ice sodium hydrogencarbonate; Dichloromethane extraction, anhydrous Na ₂SO ₄Drying, resistates adopt eluent be the silica gel column chromatography separating purification of methyl alcohol-methylene dichloride get 2 '-deoxidation-3 '-oxygen-half glutarate-5 '-oxygen-[two (4-p-methoxy-phenyl) phenyl methyl]-5 FU 5 fluorouracil nucleosides.

Get the resiniferous junket of 185mg-different bright-Gan-Si-arginine polypeptide (the method institute synthetic of describing with reference to embodiment 1 is not sloughed the polypeptide of resin) and 42mg 2 '-deoxidation-3 '-oxygen-half glutarate-5 '-oxygen-[two (4-p-methoxy-phenyl) phenyl methyl]-5 FU 5 fluorouracil nucleosides, use 2-(7-azo benzotriazole)-N respectively, N; N ', N '-tetramethyl-urea phosphofluoric acid ester and N, the activation of N-diisopropylethylamine; Filter, N, dinethylformamide (5 * 4mL) with methylene dichloride (4 * 4mL) wash; In the solution that adding is made up of the methyl-phenoxide of the trimethylchlorosilane of the trifluoroacetic acid of 86mL, 0.72mL and 0.99mL, effect is 1 hour under the room temperature, in reactor drum, adds the 12.5mL DMSO 99.8MIN.; 4 ℃ were stirred 1 hour down; Filter and remove resin, add the 600mL dry diethyl ether, centrifugal; Dry diethyl ether washing (4 * 20mL), then through the preparation HPLC chromatography get junket-different bright-Gan-Si-essence-glutaryl--oxygen-2 '-deoxidation-5 FU 5 fluorouracil nucleoside compound.Its molecular structure is seen Fig. 5.

Embodiment 17: junket-different is bright-and the stability of Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound

With junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound (by embodiment 2 synthetic compounds) joins respectively in blood plasma-phosphoric acid buffer of phosphoric acid buffer and 80% (v/v); 5 FU 5 fluorouracil content on different time point in the test sample also compares the result, assess contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide is at external release characteristics.Concrete grammar is following: press junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound in the content of 5 FU 5 fluorouracil; The 50mg compound is joined in the mixed solution of 1mL, 0.1mM phosphoric acid buffer (pH 7.4) and 0.8mL human plasma and 0.2mL, 0.1mM phosphoric acid buffer 37 ℃ of following mixings respectively.On different time points (0,2,4,8,24,48 and 72 hour), get the ZnCl of 0.1mL sample and 0.1mL, 0.1M respectively ₂The solution mixing is that vibration made protein precipitation in 5 minutes on 37 ℃ the constant temperature oscillator in temperature, 14, and under the 000rpm centrifugal 10 minutes, get the 0.1mL supernatant and add mixing in 1mL, 0.05M, pH 4.1 acetate buffer solutions.0.45 making HPLC, μ m membrane filtration, 10 μ L samples detect.Detected result is seen Fig. 6, and in 80% blood plasma, junket in 24 hours-different is bright-and the degradation rate of Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound is 82%.And in pH was 7.4 phosphoric acid buffer, junket in 24 hours-different was bright-degradation rate of Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound is 46%.The result shows, contain junket-different bright-degraded of Fluracil compounds in blood of Gan-Si-arginine polypeptide is very fast.

Embodiment 18: and junket-different is bright-and Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound tests at vitro cytotoxicity

We adopt blue (MTT) method of tetramethyl-paddy nitrogen azoles, assess contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide is to the growth-inhibiting effect of cultured cell in vitro.The junket that present embodiment uses-different is bright-and Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound is the method synthetic that provides by embodiment 2.The cell behaviour rectum cancer cell SW480 that is adopted, gastric carcinoma cells SGC-7901, normal liver cell LH-02 and mouse melanotic tumor cell B16.These cells are all available from Wuhan University China typical culture collection center.Cell culture fluid is to contain 10% calf serum RPMI-1640 substratum.The concrete operations of mtt assay are following: get and be in logarithmic phase SW480, SGC-7901, LH-02 and B16 cell, 500g is centrifugal 5 minutes behind the tryptic digestion.In 96 well culture plates, add 5 * 10 respectively by every hole ³Cell cultures 24 hours.Every hole add 100 μ L contain the junket of different concns gradient-different bright-the serum-free RPMI-1640 nutrient solution of Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound (YIGSR-5-FU) or 5 FU 5 fluorouracil (5-FU), the concentration of YIGSR-5-FU is in contained 5-FU wherein.Add 100 μ L serum-free RPMI-1640 nutrient solutions in the blank group cell.Cultivate down after 5,48 hours for 37 ℃, the MTT/PBS solution 50 μ L that every hole adds 1mg/mL continue to cultivate 4 hours.Liquid in the careful absorption hole, every hole adds DMSO 99.8MIN. 200 μ L, shakes after 10 minutes, with the absorbance (OD value) in automatic ELIASA each hole of mensuration under the 492nm wavelength, the OD value (I of record 5-FU or YIGSR-5-FU _Sample), and the OD value (I of record PBS blank control group _Control).Calculate cell survival rate (Cell Viability) through formula: CellViability (%)=100 * (I _Sample/ I _Control).Calculate the half-inhibition concentration (IC of medicine again according to cell survival rate ₅₀), and the result listed in table 1.

Table 1:MTT method detection of drugs is to the growth-inhibiting effect of each cell

Embodiment 19: and junket-different is bright-and Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil compound is in the intravital distribution of tumor animal and to the target property of tumour

For understand contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide is in the intravital distribution of tumor animal; And compare with there not being the former medicine 5 FU 5 fluorouracil of link coupled; At first set up the tumor animal model, 106 of C57BL/6 mouse, mouse 6～8 weeks of age; Body weight 18～22 grams, male and female are not limit.Get mouse melanosarcoma B16 cell strain, the animal inoculation pvaccination tumour is after two weeks, and the inoculation part forms about 90～120mm ³The size lump.Laboratory animal is divided into 24 groups at random, 8 groups of tumor animals (5～6 every group) is wherein arranged through its tail vein injection 5 FU 5 fluorouracil (5-FU), and dosage is the 24mg/kg body weight.Other 8 treated animals (5～6 every group) via the tail vein inject the method synthetic junket that provides by embodiment 2-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil (YIGSR-5-FU), giving with dosage is to count the 24mg/kg body weight by the amount of its contained 5-FU.Also having 8 treated animals (1～2 every group) to inject saline water via the tail vein is the blank group, and its blood plasma and tissue sample are used for the production standard curve.Tumor animal 15,30 minutes, 1,2,4,8,12 and 24 hour after injection, it is centrifugal immediately to get its blood, keeps somewhere blood plasma and frozen under-70 ℃, is convenient to the sample centralized detecting.Put to death animal subsequently and get its liver, heart, lung, kidney and tumor tissues and weigh, with being cut into small pieces behind the structural bloodstain of SPSS flush away, again with homogenizer with tissue homogenate.Sample after with label frozen in-70 ℃ up to detection.Adopt HPLC to detect the medicament contg in animal plasma and the tissue sample, the result is presented at Fig. 7.

5-FU and the YIGSR-5-FU distribution situation in blood is seen Fig. 7 A.After YIGSR-5-FU gets in the body, on each time point, be high all than the 5-FU experimental group.Such data declaration, 5-FU arrive body tissue with blood circulation after getting in the body, get into very soon in each histocyte, and blood Chinese traditional medicine concentration is descended rapidly.And after in the coupling compound entering body, can not pass through the simple passive diffusion mode and get into histocyte, need combine with the 67LR on the cytolemma, could get in the cell through the 67LR mediation.Because it is relevant with the expression level of cell 67LR that cell absorbs coupling compound, the normal tissue cell that does not have the 67LR expression in a large number or hang down the 67LR expression can not absorb or ingestion of drugs seldom, makes that the density loss of coupling compound in blood is slow.Therefore, coupling compound can keep than have link coupled small molecules Fluracil compounds in blood has more lastingly, higher concentration, and such result is highly beneficial to its performance antitumor action.Can know that from Fig. 7 B behind the administration 5-FU 30 minutes, 5-FU concentration was 39.1 μ g/g tissues in the liver, 5-FU concentration begins to descend after 4 hours.Different with 5-FU, on each time point (0.25,0.5,1,2,4,8,12 and 24 hour), the 5-FU concentration in the compound experimental group animal liver is low than the 5-FU experimental group all.In the lung of animal and nephridial tissue, also found similar drug distribution situation (Fig. 3 C and Fig. 3 D).Fig. 7 F has shown 5-FU or the YIGSR-5-FU distribution in tumor tissues.5-FU is because be distributed to the cause that whole body is organized everywhere relatively fifty-fifty; Medicament contg in the tumour cell is very limited; In the 5-FU experimental group, administration after 0.5 hour tumor tissues Chinese traditional medicine concentration be 21.17 μ g/g tissue, density loss is to 1.2 μ g/g after 24 hours.On the other hand, in YIGSR-5-FU laboratory animal group, administration is after 2 hours, and the drug level that detects in the tumor tissues arrives the climax, peak concentration average out to 61.72 μ g/g tissue.Explain that YIGSR-5-FU has the effect that cancer target is carried medicine, tumor by local gathers high density YIGSR-5-FU, helps improving the antitumor action of the 5-FU that discharges in the YIGSR-5-FU molecule.

Embodiment 20: junket-different is bright-and Gan-Si-essence-3-oil of mirbane formyl-5 FU 5 fluorouracil compound antitumor action in animal body

For judge contain junket-different bright-antitumous effect of the fluorouracil derivative of Gan-Si-arginine polypeptide, the C57BL/6 mouse (body weight 18～22 grams) of selecting mouse 6～8 weeks of age totally 70 be laboratory animal, male and female are not limit.Get mouse melanosarcoma B16 cell strain and set up the plantation property tumor model of animal.All laboratory animal are divided into 3 groups at random, and the 1st day behind tumor inoculation, the 8th day and the 15th day are through the tail intravenously administrable respectively.The I treated animal is the blank group through tail vein injection 0.5mL saline water.The II treated animal injects the 5 FU 5 fluorouracil (5-FU) of 6mg/kg through the tail vein by its body weight; The III treated animal through the tail vein give with press embodiment 2 method synthetic junket-different bright-Gan-Si-essence-3-(2 '-nitro-4 '-benzoyl)-5 FU 5 fluorouracil (YIGSR-5-FU), to dosage be to count the 6mg/kg body weight by the amount of contained 5 FU 5 fluorouracil wherein.Every day is with vernier caliper measurement and write down the major diameter (a) and the minor axis (b) of knurl body, calculates the volume (V) of animal knurl body according to following formula: V (mm ³)=(a * b ²)/2.Through observing the existing state of knurl body size variation and animal, and compare, estimate the antitumor action of YIGSR-5-FU with 5-FU.

Shown in Fig. 8 A, accept the animal of 5-FU treatment, the tumor growth of its inoculation is slow, treated for three weeks after, the tumour of knurl body volume ratio blank treated animal has dwindled 36%, be between the two significant difference ( ^*P＜0.05).Correspondingly, accept of the prolongation (see Fig. 8 B) of the animals survived time of 5-FU treatment than the blank group.The animal of accepting YIGSR-5-FU treatment is after three weeks, and knurl body size is nearlyer 70% than having dwindled of blank treated animal, and the antitumor action of YIGSR-5-FU is very obvious, both compare the difference highly significant ( ^*P＜0.01) (sees Fig. 8 A).And, compare with the animal of accepting 5-FU treatment, its knurl body volume also reduced 36% ( ^*P＜0.05).The animals survived time is longer, and the antitumous effect of YIGSR-5-FU is more obvious than 5-FU.Explain that YIGSR-5-FU has the targeted effect to tumor tissues, alleviated the toxic side effects of medicine, strengthened antitumor action tumor-bearing mice.

Claims

One kind contain junket-different bright-fluorouracil derivative of Gan-Si-arginine polypeptide; Its general structure is following: Tyr-Ile-Gly-Ser-Arg-FU; Wherein, Tyr-Ile-Gly-Ser-Arg representes tyrosine-Isoleucine-glycocoll-Serine-l-arginine pentapeptide sequence; FU representes 3-(2 '-nitro-4 '-phenylformic acid)-5 FU 5 fluorouracil.
One kind contain junket-different bright-preparation method of the fluorouracil derivative of Gan-Si-arginine polypeptide; It is with junket-different bright-Gan-Si-arginine polypeptide is dissolved in the DMSO 99.8MIN.; Add 1-hydroxy benzo triazole and dicyclohexyl carbon imide subsequently respectively; Again 3-(2 '-nitro-4 '-phenylformic acid)-5 FU 5 fluorouracil is dissolved in adding in the reaction system behind the DMSO 99.8MIN. and reacts, through the preparation HPLC chromatography must contain junket-different bright-the Fluracil coupling compound of Gan-Si-arginine polypeptide.