CN105294836A - LPNISKP modified 5-fluorouracil, preparation therefor, nanostructure thereof, activity thereof and application thereof - Google Patents
LPNISKP modified 5-fluorouracil, preparation therefor, nanostructure thereof, activity thereof and application thereof Download PDFInfo
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Abstract
The present invention discloses a compound 5-fluorouracil-1-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro in the following structure, and Leu-Pro-Asn-Ile-Ser-Lys-Pro is normally LPNISKP for short. The present invention discloses a preparation method for LPNISKP modified 5-fluorouracil, a nanostructure of the LPNISKP modified 5-fluorouracil, an antitumor effect of the LPNISKP modified 5-fluorouracil, and effects of LPNISKP modified 5-fluorouracil in preventing adhesion, invasion and migration of antitumor cells, and elucidates applications of the LPNISKP modified 5-fluorouracil in medical science.
Description
Technical field
The present invention relates to 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro.The regular abbreviation of Leu-Pro-Asn-Ile-Ser-Lys-Pro is LPNISKP.Relate to its preparation method, relate to its nanostructure, relate to its inside and outside antitumor action, relate to the effect of its antitumor cell Adhesion, Invasion and migration.Thus the present invention relates to it and preparing antitumor drug, prepare antitumor cell migration and adhere to and attack the application in medicine.The invention belongs to biomedicine field.
Background technology
5 FU 5 fluorouracil is a clinical conventional antitumor drug, and it can be used for the treatment of kinds cancer, cancer of the stomach, liver cancer, colon etc.Although 5 FU 5 fluorouracil has good curative effect for the treatment of tumour, also there are some problems that can not be ignored in it.Such as Half-life in vivo is short, and oral administration biaavailability is low, and dosage is close with toxic dose, and toxic side effect is large.In order to the antitumor action of 5 FU 5 fluorouracil can be played better, improve its tumor killing effect, reduce toxic side effect.People have carried out a large amount of modification work to 5 FU 5 fluorouracil for many years, have synthesized multiple 5 FU 5 fluorouracil derivative, as introduced glucose, porphyrins on 5 FU 5 fluorouracil, metal complexes, amino acid and small peptide etc.Regrettably these effort do not obtain the result expected.In the structural modification of 5 FU 5 fluorouracil, also has the study on the synthesis introduced by RGD tripeptides.Because in the research that some are important, RGD tripeptides has confirmed do not have recognition reaction to integrin receptor, so this modification chemical work that to be a kind of target ambiguous, to 5 FU 5 fluorouracil without any biological significance.
RGD tetrapeptide, namely RGDS, RGDF and RGDV are integrin alphas
vβ
3blocker, there is antithrombotic and Anti cell adhesion active.LAM-111 (laminin-111) participates in the growth of tumour, infiltration, transfer, multiple physiological process such as adhesion and vasculogenesis.Leu-Pro-Asn-Ile-Ser-Lys-Pro sequence is positioned on β 1 chain of laminin-111, and applicant to be once reported in when concentration is 1 μM it and can to adhere to and infiltrate by inhibition tumor cell.Applicant is once them and oestrogenic hormon coupling, and preparation does not have the osteoporosis agent of blood coagulation side effect.Applicant once prepared efficient antithrombotic agent them and the coupling of tetrahydro-beta-carboline-3-carboxylic acid.Applicant is also once with the β-carboline-3-carboxylic acid that amino acid modified tetrahydro-beta-carboline-3-carboxylic acid, β-carboline-3-carboxylic acid and 1-position replace, and the β-carboline-3-carboxylic acid of the tetrahydro-beta-carboline-3-carboxylic acid or the replacement of 1-position that comprise the replacement of 1-position prepares efficient antithrombotic agent or antineoplastic agent.Fig. 1 is the representative of the structure type that contriver creates.Although contriver has paid large quantity research energy, screen hundreds of compound, never there is the antitumor and antitumor compound adhering to infiltration and transporting action simultaneously.
Contriver is in the analysis structure of hundreds of compound and the basis of activity change, recognize Leu-Pro-Asn-Ile-Ser-Lys-Pro and 5 FU 5 fluorouracil coupling, the 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro of formation can have antitumor adhesion with antitumor cell simultaneously and infiltrate and transporting action.Based on this understanding, inventors herein propose the present invention.
Summary of the invention
First content of the present invention is to provide the 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro of structure below.
Second content of the present invention is to provide the preparation method of 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro, and the method is made up of following steps:
(1) 60 DEG C of 5 FU 5 fluorouracil first reacts 10h in ethyl bromoacetate solution and the 30%NaOH aqueous solution, and then compound of reaction generates 5 FU 5 fluorouracil-1-guanidine-acetic acid in ice-water bath concentrated hydrochloric acid process;
(2) liquid phase synthesis is adopted to prepare Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl;
(3) by Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl and the coupling of 5 FU 5 fluorouracil-1-guanidine-acetic acid, 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl is prepared;
(4) by 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl deprotection, 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro is prepared.
3rd content of the present invention evaluates the activity of 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro antiproliferative effect.
4th content of the present invention evaluates 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro to the restraining effect of mice bearing S180 tumor growth.
5th content of the present invention evaluates 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro antitumor cell Adhesion, Invasion and transporting action.
Accompanying drawing explanation
Fig. 1. the structure type representative of the active compound for anti tumor that contriver creates, in formula, AA is L-amino acid or glycine.
The synthetic route .i of Fig. 2 .5-Fluracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro) DCC, HOBt, NMM, THF; Ii) hydrogenchloride/ethyl acetate solution (4N); Iii) CH
3oH, 2NNaOH; Iv) NaOH (30%), 60 DEG C, ethyl bromoacetate, dense HCl.
Fig. 3 .5-Fluracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro is in pure water solution 1 × 10
-5transmission electron microscope photo under M concentration.
Embodiment
In order to set forth the present invention further, provide a series of embodiment below.These embodiments are illustrative completely, and they are only used for being specifically described the present invention, not should be understood to limitation of the present invention.
Embodiment 1 prepares 5 FU 5 fluorouracil-1-guanidine-acetic acid
1.30g (10mmol) 5 FU 5 fluorouracil is placed in 100mL eggplant bottle, add the aqueous dissolution of 10mLNaOH (30%), reaction is heated to 60 DEG C, treat to dissolve completely, slow dropping 1.75mL ethyl bromoacetate solution, constant temperature 60 DEG C reaction 12h, TLC (ethyl acetate: Glacial acetic acid: water=5: 2: 0.5) show reaction and complete.Reaction solution is cooled to room temperature, under ice bath, slowly drips concentrated hydrochloric acid, regulate pH to 2, stir 0.5h, there is colorless solid to separate out, filter, filter cake is washed three times with frozen water and ether respectively, shady and cool place dries, and obtaining 0.56g (30%) title compound, is colourless powder.ESI-MS(m/e)：189[M+H]
+.
1HNMR(500MHz，DMSO-d
6)：δ/ppm＝13.21(s，1H)，11.89(d，J＝4.5Hz1H)，8.07(d，J＝6.5Hz1H)，4.37(s，2H)。
Embodiment 2 prepares Boc-Leu-Pro-OBzl
11.55g (50mmol) Boc-Leu 100mL anhydrous tetrahydro furan is dissolved in 500mL reaction flask, add 7.43g (55mmol) N-hydroxyl benzotriazole (HOBt) and 12.36g (60mmol) dicyclohexylcarbodiimide (DCC) under ice bath, stir 30min and obtain reactant A.Separately 12.08g (50mmol) HClPro-OBz 150mL anhydrous tetrahydro furan is dissolved in 250mL reaction flask, slowly drips N-methylmorpholine (NMM) under ice bath and regulate pH to 9, obtain reactant B.Under ice bath, reactant B is added in reactant A, slow dropping N-methylmorpholine (NMM) regulates pH to 9, stirred at ambient temperature reaction 12h, thin-layer chromatography TLC (methylene dichloride: methyl alcohol: Glacial acetic acid=20: 1: 0.1) show reaction and complete.Reacting liquid filtering, removal of solvent under reduced pressure, residue column chromatography for separation (methylene chloride-methanol system), obtains 16.35g (78.3%) Boc-Leu-Pro-OBzl.ESI-MS(m/e)：419[M+H]
+。
Embodiment 3 prepares Boc-Leu-Pro
16.35g (39mmol) Boc-Leu-Pro-OBzl dissolve with methanol, ice bath stirs the lower slowly dropping 2NNaOH aqueous solution, under ice bath, thin-layer chromatography TLC (methylene dichloride: methyl alcohol: Glacial acetic acid=20: 1: 0.1) show reaction and complete.Reaction solution 1NHCl is adjusted to neutrality, decompression removing methyl alcohol, continue to add 1NHCl and be acidified to pH=2, extraction into ethyl acetate, merges organic layer, and with saturated sodium-chloride water solution washing to neutral, add anhydrous sodium sulfate drying, filter, filtrate decompression is removed desolventizing and is obtained 13.25g (81%) Boc-Leu-Pro, ESI-MS (m/e): 327 [M-H]
-.
Embodiment 4 prepares Boc-Asn-Ile-OBzl
According to the operation of embodiment 2 by 13.66g (58.90mmol) Boc-Asn, 8.75g (64.79mmol) N-hydroxyl benzotriazole (HOBt), 15.77g (76.57mmol) dicyclohexylcarbodiimide (DCC) and 24.21g (58.90mmol) TosIle-OBzl obtain 13.45g (52.5%) Boc-Asn-Ile-OBzl.ESI-MS(m/e)：436[M+H]
+。
Embodiment 5 prepares HClAsn-Ile-OBzl
Under ice-water bath, by the ethyl acetate solution (4N) of 13.45g (30.92mmol) Boc-Asn-Ile-OBzl and 36mL hydrogenchloride, stirring at room temperature reaction 2h, thin-layer chromatography TLC (methylene dichloride: methyl alcohol: Glacial acetic acid=20: 1: 0.1) show reaction and complete.Concentrating under reduced pressure removing ethyl acetate, residue carries out concentrating under reduced pressure with except de-chlorine hydride with a small amount of ether repeatedly, obtains 10.49g (91.28%) HClAsn-Ile-OBzl.ESI-MS(m/e)：334[M-H]
-。
Embodiment 6 prepares Boc-Leu-Pro-Asn-Ile-OBzl
According to the operation of embodiment 2, by 9.56g (28.22mmol) Boc-Leu-Pro, 4.19g (31.04mmol) N-hydroxyl benzotriazole (HOBt), 7.56g (36.69mmol) dicyclohexylcarbodiimide (DCC) and 10.49g (28.22mmol) HClAsn-Ile-OBzl react, obtained 10.37g (56.70%) Boc-Leu-Pro-Asn-Ile-OBzl.ESI-MS(n/e)：646[M+H]
+。
Embodiment 7 prepares Boc-Leu-Pro-Asn-Ile
According to the operation of embodiment 3, obtain 7.95g (89%) Boc-Leu-Pro-Asn-Ile by 10.37g (16.00mmol) Boc-Leu-Pro-Asn-Ile-OBzl.ESI-MS(m/e)：554[M-H]
-。
Embodiment 8 prepares Boc-Lys (Fmoc)-Pro-OBzl
According to the operation of embodiment 2, by 23.4g (50mmol) Boc-Lys (Fmoc), 7.43g (55mmol) N-hydroxyl benzotriazole (HOBt) and 12.36g (60mmol) N, N-dicyclohexylcarbodiimide (DCC), 2.08g (50mmol) HClPro-OBzl1 uses column chromatography (two chloro-alkaline methanol systems), obtains Boc-Lys (Fmoc)-Pro-OBzl20.44g (62.35%).655.8[M+H]
+。
Embodiment 9 prepares HClLys (Fmoc)-Pro-OBzl
According to the operation of embodiment 5, by 20.44g (31.18mmol) Boc-Lys (Fmoc)-Pro-OBzl 16.24g (93.75%) HClLys (Fmoc)-Pro-OBzl obtained with the ethyl acetate solution (4N) of 32mL hydrogenchloride.ESI-MS(m/e)：555.8[M+H]
+。
Embodiment 10 prepares Boc-Ser-Lys (Fmoc)-Pro-OBzl
According to the operation of embodiment 2, by Boc-Ser22.10g (68.30mmol), 4.34g (32.15mmol) N-hydroxyl benzotriazole (HOBt) and 7.83g (40.00mmol) N, N-dicyclohexylcarbodiimide (DCC), 16.24g (29.23mmol) HClLys (Fmoc)-Pro-OBzl uses column chromatography, and (two chloro-alkaline methanol systems) obtains 13.57g (62.50%) Boc-Ser-Lys (Fmoc)-Pro-OBzl.ESI-MS(m/e)：759[M+H]
+。
Embodiment 11 prepares HClSer-Lys (Fmoc)-Pro-OBzl
According to the operation of embodiment 5, by 18.28g (35.7mmol) Boc-Ser-Lys (Fmoc)-Pro-OBzl 11.64g (93.80%) HClSer-Lys (Fmoc)-Pro-OBzl obtained with the ethyl acetate solution (4N) of 36mL hydrogenchloride.ESI-MS(m/e)：657[M-H]
-。
Embodiment 12 prepares Boc-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl
According to the operation of embodiment 2, by 10.37g (16.00mmol) Boc-Leu-Pro-Asn-Ile, 2.38g (17.60mmol) N-hydroxyl benzotriazole (HOBt) and 4.28g (20.8mmol) N, N-dicyclohexylcarbodiimide (DCC), 10.87g (16.00mmol) HClSer-Lys (Fmoc)-Pro-OBzl uses column chromatography (two chloro-alkaline methanol systems), obtained 7.33g (38.90%) Boc-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl is colorless solid.Boc-Leu-Pro-Asn-Ile-Ser-Lys(Fmoc)-Pro-OBzl.ESI-MS(m/e)：1180[M+H]+。
Embodiment 13 prepares HClLeu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl
6.46g (93.05%) HClLeu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl is obtained by 7.33g (6.22mmol) Boc-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl and the ethyl acetate solution (4N) of 8mL hydrogenchloride according to the operation of embodiment 5.ESI-MS(n/e)：1078[M-H]
-。
Embodiment 13 prepares 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl (2)
1.21g (5.79mmol) 5 FU 5 fluorouracil-1-guanidine-acetic acid 20mL dry DMF is dissolved in 100mL reaction flask, add 0.859g (6.37mmol) N-hydroxyl benzotriazole (HOBt) and 1.43g (6.95mmol) dicyclohexylcarbodiimide (DCC) under ice bath, stir 30min and obtain reactant A.Separately 6.46g (5.79mmol) HClLeu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl 20mL dry DMF is dissolved in 50mL reaction flask, slowly drip N-methylmorpholine (NMM) under ice bath and regulate pH to 9, obtain reactant B.Under ice bath, reactant B added in reactant A, slowly drip N-methylmorpholine (NMM) and regulate pH to 9, stirred at ambient temperature reaction 12h, TLC (methylene chloride/methanol/Glacial acetic acid=10/1/0.1) display reaction completes.Reacting liquid filtering, filtrate reduced in volume, residue column chromatography purification, obtains 1.44g (20%) 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl.ESI-MS(m/e)：1284.5[M+Cl]
-。
Embodiment 14 prepares 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro (3)
According to the operation of embodiment 3, under ice bath, by obtained 0.193g (18%) 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro (3) ESI-MS (m/e): 936.40 [M-H] of 1.44g (1.15mmol) 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl (2) in NaOH methanol aqueous solution (2N)
-, Mp200-202 DEG C; [α] D25=-21.42 (c=0.4, CH3OH) .IR (KBr): 3844,3331,3323.35,3292,3230,3209,2962,2939,1614,1508,1448,1419,1379,1340,1244,1165,1072,881,821,516cm
-1.
1h-NMR (500MHz, D
2o): δ/ppm=7.79-7.75 (m, 1H), 4.53-4.31 (m, 1H), 4.21-4.20 (m, 1H), 3.82-3.71 (m, 3H), 3.62-3.61 (m, 2H), 3.00-2.94 (m, 2H), 2.85-2.68 (m, 2H), 2.28-2.01 (m, 2H), 2.04-1.77 (m, 8H), 1.67-1.53 (m, 7H), 1.45-1.37 (m, 4H), 1.19-1.12 (m, 1H), 0.93-0.79 (m, 13H). purity: 99.05% moving phase: CH
3oH: H
2o: Glacial acetic acid=95: 5: 0.1, retention time: 8.94min.
Experimental example 1 measures the transmission electron microscope photo of compound 3
By compound 3 according to 1 × 10
-5the pure water solution of the concentration configuration compound of M, is layered on uniformly on copper mesh, observes the self-assembly property of compound under transmission electron microscope (TEM, JEM-1230, JEOL).The photo obtained is as Fig. 3.Result shows, compound 3 all can form nano particle in water, and diameter is at 40-400nm.
Experimental example 2 measures the cytotoxicity of compound 3 pairs of cells
1) substratum of compound 3 of the present invention containing 0.1%DMSO is mixed with desired concn.
2) tumour cell of experiment is HepG
2(human liver cell cancer cells), HL60 (human promyelocytic leukemia), BEL-7402 (human liver cancer cell), MCF-7 (human breast cancer cell), HeLa (human cervical carcinoma cell), U-2OS (human osteosarcoma cell), S180 (mouse ascites oncocyte), L02 (Human normal hepatocyte) and Haca-T (strain of people's epidermis normal cell).
3) experimental technique HL-60, BEL-7402, MCF-7 and S180 cell selects RPMI-1640 substratum; HeLa, HepG
2, U-2OS, L02 and Haca-T cell selects DMEM substratum.In substratum all containing 10% through the foetal calf serum and 1 × 10 of deactivation
5u/L penicillin and 100mg/L Streptomycin sulphate.
Attached cell HepG
2, the cultivation of MCF-7, BEL-7402, HeLa, Haca-T, U-2OS and half attached cell S180: respectively that growth conditions is good, is in the cell of logarithmic phase with 3 × 10
4the density of individual/mL is inoculated in 96 orifice plates, and every hole 100 μ L, is placed in 37 DEG C and 5%CO
2cell incubation case in cultivate 4 hours, then add by the concentration gradient preset the solution that the compound 3 through sterilising treatment is mixed with the substratum containing 0.1%DMSO, every hole 25 μ L, control group adds the solvent of isopyknic sample dissolution.Continue cultivation after 48 hours, every hole adds the MTT solution that 25 μ L concentration are 5mg/mL, is placed in 37 DEG C and 5%CO
2cell incubation case in cultivate 4 hours.After careful removing supernatant liquor, every hole adds the DMSO of 100 μ L, and about 10min dissolve purple of vibrating residue (first a ceremonial jade-ladle, used in libation), detects O.D. (absorbancy) value immediately in microplate reader, and wavelength is 570nm.
The cultivation of suspension cell HL60: respectively that growth conditions is good, is in the cell of logarithmic phase with 5 × 10
4the density of individual/mL is inoculated in 96 orifice plates, every hole 100 μ L, then adds by the concentration gradient preset the solution that the compound 3 through sterilising treatment is mixed with the substratum containing 0.1%DMSO, every hole 25 μ L, control group adds the solvent of isopyknic sample dissolution, is placed in 37 DEG C and 5%CO
2cell incubation case in cultivate 48 hours.Every hole adds the MTT solution that 25 μ L concentration are 5mg/mL, and continuing the condition that is placed in is 37 DEG C and 5%CO
2cell incubation case in cultivate 4 hours.The centrifugal 10min of 2500rpm, careful sucking-off supernatant liquor, every hole adds 100 μ LDMSO, and about 10min dissolve purple of vibrating residue (first a ceremonial jade-ladle, used in libation), detects O.D. (absorbancy) value immediately in microplate reader, and wavelength is 570nm.
The activity of compound 3 inhibition tumor cell propagation under each concentration is obtained by following formula:
Cell proliferation (%)=(the average O.D. value of compound 3 groups average O.D. value/control group) × 100%, experiment repetition 3 times, maps to drug level with cell proliferation, obtains IC by graphing method
50(half effective inhibition concentration) value.
4) the results are shown in Table 1 and table 2.Result shows, in vitro in cytotoxicity test, compound 3 only the high density of 100 and 200 μMs to MCF-7, Bel-7402, HepG
2, HL60, U-2OS, HeLa, S180, L02 and Haca-T etc. 9 strain tumour cell show weak cytotoxicity.
Cytotoxicity (the IC of table 1 compound 3 pairs of tumour cells
50, mean value ± SD μM)
Cytotoxicity (the IC of table 2 compound 3 pairs of tumour cells
50, mean value ± SD μM)
The anti-tumor in vivo of experimental example 3 assessing compound 3 is active
1) compound 3 physiological saline solution of the present invention, 5 FU 5 fluorouracil physiological saline solution as positive control, with physiological saline as negative control;
2) compound 3 and the equal gastric infusion of physiological saline, the dosage of compound 3 is 10 μm of ol/kg, and the dosage of physiological saline is 0.2mL/20g, and the dosage of 5 FU 5 fluorouracil is 10 μm of ol/kg and 150 μm ol/kg, successive administration 7 days, altogether administration 7 times.
3) laboratory animal is ICR male mice (cleaning grade), body weight 20 ± 2g, often organizes 12 mouse.
4) knurl source is mouse S 180 sarcoma, purchased from Department Of Medicine, Peking University's animal experimental center, and maintenance of going down to posterity voluntarily.
5) extract and inoculate eugonic S180 ascitic tumor knurl liquid under animal model and treatment aseptic condition, the liquid of (1: 2) is become fully to mix with normal saline dilution, by freshly prepared 0.2% Trypan Blue of tumor cell suspension, by white blood cell count(WBC) method counting after mixing, contaminate blue person for dead cell, tinter is not viable cell, and is calculated as follows cell concn and cell survival rate.
Viable count/4 × 10 in the block plaid of cell concn=4
4× extension rate=cell count/mL
Cell survival rate=viable count/(viable count+dead cell number) × 100%
Knurl liquid homogenate method survival rate being greater than 90% is prepared into 2.0 × 10
7the cell suspension of individual/mL, in the subcutaneous vaccination of mouse armpit, 0.2mL/ only, manufactures S180 tumor-bearing mice.After tumor inoculation 24h, treatment group mouse oral administration of compound every day 3,5 FU 5 fluorouracil or physiological saline, dosage is same as above, successive administration 7 days, altogether administration 7 times.8th day, claim Mouse Weight, etherization, de-cervical vertebra puts to death mouse, then fixes the right armpit tumor location of mouse with tweezers, cuts off skin, expose tumour, blunt separation, weighs, and is calculated as follows tumour inhibiting rate: the average knurl of tumour inhibiting rate %=(the average knurl of negative control group heavy-the average knurl weight of compound 3 groups)/negative control group heavy × 100%.Experimental data adopts t inspection and variance analysis, and knurl is heavy to be represented with mean value ± SDg.The results are shown in Table 3.Can find out, under 10 μm of ol/kg oral dosages, the heavy not only there was no significant difference heavy with the knurl of saline-treated mice of knurl of 5 FU 5 fluorouracil treatment mouse, and be obviously greater than the knurl weight that compound 3 treats mouse, illustrate that oral 10 μm of ol/kg5-Fluracils are invalid, and oral 10 μm of ol/kg compounds 3 are effective.In addition, the knurl that 10 μm of ol/kg compounds 3 treat mouse is heavily significantly less than the knurl weight of 1501mol/kg5-fluorouracil in treatment mouse, illustrates that the effective dose of compound 3 is lower than 5 FU 5 fluorouracil more than 15 times.
Table 3 compound 3 is on the impact of S180 tumor weight
N=12; A) with physiological saline group and 5 FU 5 fluorouracil (10 μm of ol/kg) group than P < 0.01, with 5 FU 5 fluorouracil (150 μm of ol/kg) group than P < 0.05.
The extracorporeal anti-tumor cell adhesion activity of experimental example 4 assessing compound 3
1) 5 FU 5 fluorouracil and compound 3 are mixed with concentration with the DMEM substratum containing 0.1%DMSO is 100 μMs, solution.
2) cell is HepG
2.
3) Fn (people's fibronectin).
4) experimental technique
With PBS, Fn is mixed with the solution that concentration is 100 μ g/mL, adds in 96 well culture plates by 100 μ L/ holes, culture plate is placed in 4 DEG C of refrigerator overnight.Next day, absorb and do not wrap by Fn solution, wash 1 time with PBS, every hole adds the PBS solution 30 μ L shrouding containing 2%FBS, at 37 DEG C and 5%CO
2incubator in hatch 3 hours, discard each hole solution.By good for growth conditions, be in the HCCLM3 cell of logarithmic phase with 5 × 10
4the density of individual/mL is inoculated in bag by 96 orifice plates of Fn, and every hole 100 μ L, adds the solution of 25 μ L compounds 3 simultaneously, makes its final concentration be 20nM, at 37 DEG C and 5%CO
2cultivate 2 hours in incubator, wash away the cell do not adhered to PBS, after discarding PBS, every hole adds the MTT solution that 25 μ L concentration are 5mg/mL, is placed in 37 DEG C and 5%CO
2hatch 4 hours in incubator, after careful removing supernatant liquor, every hole adds 100 μ LDMSO, and vibrate about 10min dissolution precipitation, detects O.D. (absorbancy) value immediately under microplate reader 570nm wavelength.The calculation formula of adherence inhibition rate is as follows: adherence inhibition rate (%)=[1-(the OD value of the OD value/blank group cell of compound 3 groups of cells)] × 100%; Experimental data statistics all adopts t inspection and variance analysis, and adherence inhibition rate represents with mean value ± SD.
5) the results are shown in Table 4.As can be seen from result, compound 3 when 20nM concentration, obvious anti-HepG
2cell and FN adhere to.Suppress SACC-LM cell to be compared with in the of 1 μM with the effective concentration of ECM and platelet adhesion reaction with Leu-Pro-Asn-Ile-Ser-Lys-Pro disclosed in contriver, the effective concentration of compound 3 reduces 50 times.
The extracorporeal anti-tumor cell adhesion activity of table 4 compound 3
n＝3.
The extracorporeal anti-tumor cell-invasive activity of experimental example 5 assessing compound 3
1) 5 FU 5 fluorouracil and compound 3 are mixed with containing DMEM substratum O.1%DMSO the solution that concentration is 100 μMs.
2) cell is HepG
2.
3) matrigel is matrigel.
4) experimental technique
The frozen matrigel matrigel4 DEG C in-20 DEG C of refrigerators is spent the night, liquefy; Get 720 μ L plasma-free DMEM medium, add 180 μ LMatrigel, mixing, room on the polycarbonate membrane being added to Transwell cell, 100 μ L/, put into 37 DEG C and 5%CO
25h is hatched in incubator.Absorb residual liquid in cell, every hole adds 50 μ LDMEM substratum, 37 DEG C and 5%CO
230min is hatched in incubator.
HepG
2after cell dissociation, wash 3 times with plasma-free DMEM medium, counting, be made into cell suspension, density is 5 × 10
5individual/mL.Every hole adds 100 μ L cell suspensions, adds the solution that 25 μ L add 25 μ L5-Fluracils and compound 3 simultaneously simultaneously, makes its final concentration be 20nM.Blank adds the solution that 25 μ L prepare containing the DMEM substratum of 0.1%DMSO.Lower room adds 600 μ L plasma-free DMEM medium, at 37 DEG C and 5%CO
2cultivate 48 hours in incubator.
Wipe the cell of matrigel and upper indoor with cotton swab after, with the paraformaldehyde fixed cell 30min of 4%.Absorb stationary liquid, wash 3 times with PBS, with the Viola crystallina dye liquor dyeing 30min of 0.1%.Absorb staining fluid, wash 3 times with PBS.
Choose 9 roughly the same visuals field at each cell to observe, take pictures, counting.Experimental data statistics all adopts t inspection and variance analysis, and the cell count of invasion and attack represents with mean value ± SD.
5) the results are shown in Table 5.Can find out, compound 3 is under the concentration of 20nM compared with blank group, and the cell count that invasion and attack occur has significant difference, and illustrate under this condition, compound 3 has obvious anti-HepG
2cell invasion effect.Suppress the effective concentration of SACC-LM cell invasion to be compared with in the of 1 μM with Leu-Pro-Asn-Ile-Ser-Lys-Pro disclosed in contriver, the effective concentration of compound 3 reduces 50 times.
The extracorporeal anti-tumor cell-invasive activity of table 5 compound 3
N=9; A) with blank group and 5 FU 5 fluorouracil group than p < 0.01.
The extracorporeal anti-tumor cell migration of experimental example 6 assessing compound 3 is active
1) 5 FU 5 fluorouracil and 3 is mixed with the DMEM substratum containing 0.1%DMSO the solution that concentration is 20 μMs.
2) cell is HepG
2.
3) experimental technique
HepG
2after cell dissociation, wash 3 times with plasma-free DMEM medium, counting, be made into cell suspension, density is 2 × 10
6individual/mL.Every hole adds 100 μ L cell suspensions, adds the solution that 25 μ L add 25 μ L5-Fluracils and compound 3 simultaneously simultaneously, makes its final concentration be 20nM.Blank adds the solution that 25 μ L prepare containing the DMEM substratum of 0.1%DMSO.Lower room adds 600 μ L plasma-free DMEM medium, at 37 DEG C and 5%CO
2cultivate 6 hours in incubator.
Wipe the cell of upper indoor with cotton swab after, with the paraformaldehyde fixed cell 30min of 4%.Absorb stationary liquid, wash 3 times with PBS, with the Viola crystallina dye liquor dyeing 30min of 0.1%.Absorb staining fluid, wash 3 times with PBS.
Choose 9 roughly the same visuals field at each cell to observe, take pictures, counting.Experimental data statistics all adopts t inspection and variance analysis, and the cell count of migration represents with mean value ± SD.
4) the results are shown in Table 6.Can find out, under 20nM concentration, compound 3 is compared with blank group, and the cell count that migration occurs significantly reduces, and illustrates that compound 3 has obvious anti-HepG under this condition
2cell migration effect.Suppress the effective concentration of SACC-LM cell migration to be compared with in the of 1 μM with Leu-Pro-Asn-Ile-Ser-Lys-Pro disclosed in contriver, the effective concentration of compound 3 reduces 50 times.
The extracorporeal anti-tumor cell migration of table 6 compound 3 is active
N=9; A) with blank group and 5 FU 5 fluorouracil group than p < 0.01.
Claims (5)
1. the compound 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro of structure below.
2. the preparation method of the 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro of claim 1, the method is made up of following steps:
(1) 60 DEG C of 5 FU 5 fluorouracil first reacts 10h in ethyl bromoacetate solution and the 30%NaOH aqueous solution, and then compound of reaction generates 5 FU 5 fluorouracil-1-guanidine-acetic acid in ice-water bath concentrated hydrochloric acid process;
(2) liquid phase synthesis is adopted to prepare Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl;
(3) by Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl and the coupling of 5 FU 5 fluorouracil-1-guanidine-acetic acid, 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl is prepared;
(4) by 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Fmoc)-Pro-OBzl deprotection, 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro is prepared.
3. the nanostructure of the 5 FU 5 fluorouracil-1-base acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro of claim 1.
4. 5 FU 5 fluorouracil-1-base acetyl-the Leu-Pro-Asn-Ile-Ser-Lys-Pro of claim 1 is preparing the application in antitumor drug.
5. 5 FU 5 fluorouracil-1-base acetyl-the Leu-Pro-Asn-Ile-Ser-Lys-Pro of claim 1 is preparing the application in antitumor cell Adhesion, Migration and invasion and attack medicine.
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