CN105272968B - The diketone of (3S, 6R) 3,6 disubstituted piperazine 2,5, it is prepared and application - Google Patents

The diketone of (3S, 6R) 3,6 disubstituted piperazine 2,5, it is prepared and application Download PDF

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Publication number
CN105272968B
CN105272968B CN201410260126.2A CN201410260126A CN105272968B CN 105272968 B CN105272968 B CN 105272968B CN 201410260126 A CN201410260126 A CN 201410260126A CN 105272968 B CN105272968 B CN 105272968B
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piperazine
indoles
ethyls
diketone
fourths
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CN105272968A (en
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彭师奇
赵明
王玉记
吴建辉
王枫
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Capital Medical University
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Capital Medical University
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Abstract

The invention discloses u PA inhibitor (3S, 6R) 3 (4 fourth amino) 6 (ethyl of indoles 3) piperazine 2,5 diketone, disclose its preparation method, disclose it and suppress tumor cell invasion and migration, further disclose the effect such as its hemostasis and anti-inflammatory.The invention belongs to biomedicine field.

Description

(3S, 6R) -3,6 disubstituted piperazine -2,5- diketone, it is prepared and application
Technical field
The present invention relates to the small molecule u-PA inhibitor shown in following structural formula, it is related to its preparation method and as u- Application of the PA inhibitor in terms of tumor invasion and migration, hemostasis and anti-inflammatory is suppressed.The invention belongs to biomedicine field.
Background technology
Plasminogen activating system is united by plasminogen activator (Pas), Plasminogen activation inhibitor (PAIs) and cell Outer plasminogen activator receptor (PAR) composition.Plasminogen activating system, which is united, is related to cell migration, angiogenesis, wound healing, Embryonic development, a series of processes such as tumour cell diffusion and transfer.Main type plasminogen in a organized way swashs in mammal body Agent (t-PA) living and two kinds of plasminogen activators of urokinase type plasminogen activator (u-PA).
Urokinase type plasminogen activator (u-PA) is a kind of silk extracted from human urine or nephrocyte tissue culture medium Serine protease, category double chain urokinase type Plasminogen Activator (tcu-PA), molecular weight are 55000 or 33000, are exogenous The activator of Fibrinolysis system.U-PA can be made with the arginine (560) of Direct Pyrolysis plasminogen-valine (561) peptide bond Inactive single-stranded plasminogen is changed into active double-strand fibrinolysin.
In tumor tissues, u-PA plasminogen activations are converted into fibrinolysin, and fibrinolysin is caused directly or indirectly extracellular Substrate degradation, the infiltration of tumour cell, transfer and angiogenesis are further resulted in, promote the propagation of tumour.In blood circulation, U-PA participates in the balance of regulation intravascular coagulation-fibrinolytic by plasminogen activation, is played an important role to suppressing thrombus.
During inflammatory response, u-PA participates in regulation inflammatory cell pair by being combined with the u-PAR on inflammatory cell surface The penetrating power of blood vessel and tissue, promote inflammatory cell to be migrated to inflammation part, promote releasing for inflammatory reaction and inflammatory factor Put, and the release of inflammatory factor can induce u-PA expression to rise.
Because u-PA enzymatic activity is applied in the complicated cross of tumour-fibrinolytic-inflammation, so influenceing u-PA work Property can influence complexity cross.That is, the outstanding active u-PA inhibitor of invention is for suppressing u-PA in tumour-blood Bolt-inflammation cross is significant.This meaning is embodied in more than 70% cancer patient and dies from metastasis of cancer；Have about 10% Cancer of late stage patient there is bleeding, such as there is almost unpredictable nose, swallow, lung or massive gastrointestinal bleeding；Extract swollen The intraoperative bleeding of knurl, also deteriorate the prognosis of patient.For this situation, the present invention proposes (3S, 6S) -3- (4- fourth ammonia Base) -6- (indoles -3- ethyls)-piperazine -2,5- diketone, it is prepared and therapeutic action.
Before this, it is u-PA inhibitor (structure is shown in Fig. 1) that inventor, which once reported that the diketopiperazine of following structure included CIPPC, There is outstanding styptic activity.Under 1nmol/kg dosage, CIPPC can significantly reduce the mouse rat-tail bleeding time [33,34].Unfortunate It is that CIPPC can promote thrombus to generate under 10nmol/kg dosage.Thrombus is that tumour is important and close disease, is to cause tumour An important factor for I am dead.The tumour patient that act as that CIPPC can promote thrombus to generate brings new threat.Contrast it Under, the unexpected advantage of (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone is, except With having as CIPPC outside outstanding styptic activity, also suppress tumor cell invasion and transfer, suppress the concurrent inflammation of tumour Effect, while thrombosis will not be caused.
The content of the invention
First content of the present invention is to provide u-PA inhibitor (3S, 6R) -3- (4- fourths amino) -6- of following structural formula (indoles -3- ethyls)-piperazine -2,5- diketone；
Second content of the present invention is to provide (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2, The preparation method of 5- diketone, this method include following four reactions steps：
(1) L-Trp-OBzl is D- with L-Boc-Lys (Z) condensations in anhydrous tetrahydro furan in the presence of DCC and HOBt Boc-Lys(Z)-L-Trp-OBzl；
(2) in hydrogen chloride-ethyl acetate solution D-Boc-Lys (Z)-L-Trp-OBzl slough Boc generation D-Lys (Z)- L-Trp-OBzl；
(3) in ethyl acetate and 5%NaHCO3In the presence of D-Lys (Z)-L-Trp-OBzl generation (3S, 6R) -3- (4- fourths Aminobenzyl) -6- (indoles -3- ethyls)-piperazine -2,5- diketone；
(4) in Pd/C and H2In the presence of, in methyl alcohol (3S, 6R) -3- (4- fourths aminobenzyl) -6- (indoles -3- ethyls) - The reactive ketone of piperazine -2,5- bis- generates (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone.
Four reactions steps above can be described with Fig. 2 synthetic route.
The present invention the 3rd content be evaluation small molecule u-PA inhibitor (3S, 6R) -3- (4- fourths amino) -6- (indoles - 3- ethyls)-piperazine -2,5- diketone is in terms of tumor cell invasion and transfer is suppressed, and the effect of hemostasis and anti-inflammatory etc..
Brief description of the drawings
Fig. 1 before this inventor report u-PA inhibitor diketopiperazines CIPPC structure.
Fig. 2 synthesizes the route 1.i of (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone) DCC, HOBt, NMM, THF；ii)HCl/EA(4N)；Iii) EA, 5%NaHCO3(3a) or EA, TEA, 80 DEG C (3b, 3c)；iv) CH3OH, Pd/C, H2。
Fig. 3 (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone activates fine to UK in vitro The influence of lyase original vigor.Wherein 1 is classified as the human plasminogen (PLG) of one pack system, and 2 are classified as UK and PLG common incubation；3 are classified as 100 μ g (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone and 5 μ L UK (400U/mL) and 5 μ L PLG (5mg/mL) common incubation component；4 are classified as 50 μ g (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2, The common incubation component of 5- diketone and 5 μ L UK (400U/mL) and 5 μ L PLG (5mg/mL)；5 are classified as 10 μ g (3S, 6R) -3- (4- fourths Amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone and 5 μ L UK (400U/mL) and 5 μ LPLG (5mg/mL) common incubation Component；6 are classified as 500 μ g EACA and 5 μ LUK (400U/mL) and 5 μ LPLG (5mg/mL) common incubation component；7 are classified as 250 μ g EACA and 5 μ LUK (400U/mL) and 5 μ L PLG (5mg/mL) common incubation component.
Embodiment
In order to which the present invention is expanded on further, a series of embodiments are given below.These embodiments be entirely it is illustrative, it Only be used for the present invention is specifically described, be not construed as limitation of the present invention.
Embodiment 1 prepares D-Boc-Lys (Z)-L-Trp-OBzl (1)
1.9g (5.0mmol) D-Boc-Lys (Z) is suspended in 20mL anhydrous tetrahydro furans, is stirred at room temperature lower into solution 0.675g (5.0mmol) HOBt is added, ice bath stirring is lower to add 1.133g (5.5mmol) DCC, obtains reaction solution I, is stirred under ice bath Mix 30 minutes.1.47g (5.0mmol) L-Trp-OBzl is suspended in 20mL anhydrous tetrahydro furans, is then gradually added into NMM, PH to 8-9 is adjusted, obtains reaction solution II.Reaction solution II is added in reaction solution I, 1h is first stirred under ice bath, then stir in room temperature Mix, TLC, which is monitored to raw material point, to disappear.Post processing：It is filtered under diminished pressure and removes DCU, filtrate decompression is concentrated and removes tetrahydrofuran, residual Thing 150mL ethyl acetate is dissolved, and obtained solution is placed in 250mL separatory funnels, uses 5%KHSO successively4The aqueous solution is washed Respectively washed 3 times with the saturation NaCl aqueous solution, ethyl acetate layer anhydrous Na2SO430min is dried, is filtered under diminished pressure, filtrate decompression concentration To doing, obtained yellow foaming material, (CH is purified through silica gel column chromatography2Cl2∶CH3OH=100: 1~20: 1), obtain 2.863g (87.3%) title compound, it is colorless solid.ESI-MS(m/e)：657[M+H]+。
Embodiment 2 prepares D-Lys (Z)-L-Trp-OBzl (2)
Sterling 2.62g (4.0mmol) D-Boc-Lys (Z)-L-Trp-OBzl (1) is placed in 100mL eggplant bottles, ice bath stirs Lower slowly dropwise addition 30mL4N hydrogen chloride-ethyl acetate solution into reaction bulb is mixed, adds drying tube, the lower reaction of ice bath stirring 4 hours TLC monitors raw material point and disappeared afterwards, terminating reaction.Post processing：Add ethyl acetate molten reaction solution decompressing and extracting with water pump under stirring Water pump decompressing and extracting is used after solution again, in triplicate；Add absolute ether to be stood after being fully suspended, pour out ether, drain product, In triplicate, 2g (90.9%) title compound is obtained, is pale yellow powder.ESI-MS(m/e)：557[M+H]+。
Embodiment 3 prepares (3R, 6R) -3- (4- fourths aminobenzyl) -6- (indoles -3- ethyls)-piperazine -2,5- diketone (3)
1.83g (3.29mmol) compound D-Lys (Z)-L-Trp-OBzl (2) are dissolved with 80mL ethyl acetate, with three Ethamine adjusts pH to 9, and 80 DEG C of oil bath is reacted 6 hours, and TLC shows that raw material point disappears substantially.Post processing：Reaction solution is directly used into silica gel Mix sample, silica gel column chromatography purifying (CH2Cl2∶CH3OH=100: 1-20: 1), obtain 1.28g (86.9%) title compound, be nothing Color solid.ESI-MS(m/e)：449[M+H]+.1H NMR (300MHz, DMSO-d6)：δ/ppm=10.89 (s, 1H), 8.03 (s, 1H), 7.85 (s, 1H), 7.56 (d, J=9.0Hz, 1H), 7.35 (m, 6H), 7.19 (t, J=6.0Hz, 1H), 7.05 (m, 2H), 6.95 (t, J=6.0Hz, 1H), 4.99 (s, 2H), 4.07 (s, 1H), 3.25 (dd, J=3.0Hz, J=15.0Hz, 1H), 3.09 (dd, J=3.0Hz, J=15.0Hz, 1H), 2.99 (s, 1H), 2.91 (q, J=6.0Hz, 2H), 1.48 (m, 2H), 1.24 (m, 2H), 1.16 (m, 2H).
Embodiment 4 prepares (3R, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone (4)
By 0.12g (0.26mmol) compound (3R, 6S) -3- (4- fourths aminobenzyl) -6- (indoles -3- ethyls)-piperazine - 2,5- diketone (3) are placed in 50mL reaction bulbs, add the dissolving of 10mL methanol, 30mg Pd/C are added into solution, are passed through H2, room Warm stirring reaction 48 hours, TLC show the basic disappearance of raw material, filter off Pd/C, be concentrated under reduced pressure to obtain 63mg (75%) title compound Thing, it is colourless powder.ESI-MS(m/e)：315[M+H]+.Mp189-190℃.IR(KBr)：3250,2860,2312,1662, 1456,1325,1230,1093,1006,736cm-1.(c=0.32, CH3OH).1HNMR (300MHz, DMSO-d6)：δ/ppm=10.92 (s, 1H), 8.05 (s, 1H), 7.88 (s, 1H), 7.56 (d, J=9.0Hz, 1H), 7.31 (d, J=9.0Hz, 1H), 7.06 (s, 1H), 7.04 (t, J=9Hz, 1H), 6.94 (t, J=9Hz, 1H), 4.07 (s, 1H), 3.26 (dd, J=3Hz, J=12Hz, 1H), 3.02 (dd, J=6.0Hz, J=15Hz, 1H), 2.84 (m, 2H), 2.44 (m, 2H), 1.19 (m, 2H), 1.10 (m, 2H)13C NMR (200MHz, DMSO-d6)：δ/ppm=168.60,168.09,136.37, 128.05,125.02,121.31,119.26,118.85,111.62,108.86,55.89,53.59,41.05,31.94, 31.65,29.39,21.16.Elem.Anal：C17H22N4O2.C, 64.95；H, 7.05；N, 17.82.
Experimental example 1 determines (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone to urokinase (UK) active influence
1) main agents and instrument
Reagent：PAGE gel electrophoresis agents useful for same and aminocaproic acid (EACA) are commercial reagent；
Human plasminogen (PLG) and urokinase (UK) are purchased from Sigma companies.
Instrument：Vertial electrophorestic tank Mini-PROREN Tetra System (BIO-RAD)；
Electrophoresis apparatus Power Pac (BIO-RAD)；
Scanner Scanmaker8700 (MICROTEK).
2) preparation of solution
The preparation of PLG solution：PLG5mg is weighed, is placed in 15mL centrifuge tubes, 1mL physiological saline solutions is added and produces PLG Solution, concentration 5mg/mL；Packing, often pipe 0.1mL, -20 DEG C of preservations are stand-by；
The preparation of UK solution：By whole bottle UK (100,000U) with 10mL physiological saline solutions, mother liquor is obtained；Take 0.1mL mother liquors With normal saline dilution to 2.5mL, UK solution, concentration 400U/mL are obtained；Packing, often pipe 0.1mL, -20 DEG C of preservations are stand-by；
The preparation of EACA solution：50mg compounds are weighed, with 1mL physiological saline solutions, produce mother liquor 1, concentration 50mg/ mL；0.5mL mother liquors 1 are taken, with normal saline dilution to 1mL, obtain solution 2, concentration 25mg/mL；
The preparation of (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone solution：Weigh 5mg (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone, with 1mL physiological saline solutions, concentration is 5mg/mL；- 20 DEG C of preservations are stand-by.
3) preparation of sample
Take 5 μ L UK (400U/mL) solution；Packing, often pipe 0.1mL, is placed in 0.5mL centrifuge tubes, then adds respectively thereto Enter 10 μ L physiological saline or (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone solution, 37 DEG C incubate Educate 15 minutes；5 μ L PLG solution are separately added into each centrifuge tube again, 37 DEG C are incubated 15 minutes；After incubation terminates, then to respectively from 5 μ L5 × SDS electrophoresis sample-loading buffers are separately added into heart pipe, are denatured each centrifuge tube 5 minutes in 100 DEG C of water-baths after mixing, It is separated by electrophoresis after quick ice bath cooling with 12% PAGE gel.
4) PAGE gel electrophoresis
Reagent prepares：
30% deposit sol solution：Acrylamide (Acr) 29.0g, methylene bisacrylamide (Bis) 1.0g are after mixing plus super Pure water (up-H2O), 37 DEG C of dissolvings, are settled to 100mL, brown bottle is stored in room temperature；
1.5M Tris-HCl(pH8.0)：Tris18.17g adds up-H2O dissolves, and concentrated hydrochloric acid adjusts pH to 8.0, is settled to 100mL：
1M Tris-HCl(pH6.8)：Tris12.11g adds up-H2O dissolves, and concentrated hydrochloric acid adjusts pH to 6.8, is settled to 100mL；
12%SDS：Electrophoresis level SDS12.0g adds up-H2O is adjusted to pH7.2, is settled to 100mL in 68 DEG C of hydrotropies, concentrated hydrochloric acid；
10% ammonium persulfate (AP)：100mg AP add up-H2O 1mL；
Coomassie brilliant blue dyeing liquor：Methanol-water-acetic acid=45mL+45mL+10mL, add 100mg Coomassie brilliant blues and consolidate Body, it is configured to dyeing liquor；
Destainer：Methanol-water-acetic acid=10mL+90mL+10mL, is configured to destainer.
Operating procedure：
Using rectilinear electrophoresis slot device
(1) preparation of polyacrylamide gel
1. the preparation of separation gel (12%)：
Ultra-pure water 4.0mL
30% deposit sol solution 3.3mL
1.5M Tris-HCl2.5mL
12%SDS0.1mL
10%AP0.1mL
The above-mentioned mixed liquors of 1mL are taken, add TEMED (N, N, N ', N '-tetramethylethylenediamine) 10 μ L back covers, remaining plus TEMED4 μ L, Poured into after mixing between glass plate, with water seal top, pay attention to putting down liquid level, gel polymerize completely need to about 60min.
2. concentrate the preparation of glue (4%)：
Ultra-pure water 1.4mL
30% deposit sol solution 0.33mL
1M Tris-HCl0.25mL
12%SDS0.02mL
10%AP0.02mL
TEMED2μL
Water on separation gel is gone, adds above-mentioned mixed liquor, immediately by between comb insertion glass plate, polymerization completely needs big About 30min.
(2) sample treatment：Sample is added to 5 × SDS sample-loading buffers of respective amount, 100 DEG C of heating 3-5min make egg Leucismus, take out, fast cooling.
(3) loading：Sample after processing is added in sample cell, and adds 20 μ L Protein Marker product and compares.
(4) electrophoresis：1 × electrophoretic buffer is added in electrophoresis tank, connects power supply, negative pole is upper, and positive pole is under, electrophoresis When, concentrate glue voltage 80V, 30min, separation gel voltage 100V to electrophoresis to bromjophenol blue row to electrophoresis tank lower end stop (about needing 1.5h)。
(5) dye：Glue is taken out from glass plate, Coomassie brilliant blue dyeing liquor dyeing, in shaking table (60RPM) room temperature 10min。
(6) decolourize：Glue is taken out from dyeing liquor, is put into destainer, is decolourized overnight in shaking table (60RPM) room temperature.
(7) by the glue scanner scanning after decolouring, result is observed.
5) result is shown in Fig. 3, (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone concentration according to Suppress the activity of UK hydrolysis human plasminogens badly.It is UK inhibitor to illustrate it.
Experimental example 2 determines (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone to HCCLM3 The influence of cell invasion ability
1) given the test agent
(3S, 6R) -3- (4- fourths amino) -6- (indoles -3- the ethyls)-DMEM of piperazine -2,5- diketone containing 0.1%DMSO Culture medium is configured to 50 μM of concentration；
RGDS is configured to 100 μM of concentration with the DMEM culture mediums containing 0.1%DMSO.
2) cell line
HCCLM3 (high-transfer human liver cancer cell), purchased from ATCC.
3) key instrument and consumptive material
Super-clean bench：VS-1300-U clean benches, SuZhou Antai Air Tech Co., Ltd.；
Cell incubation case：INC153, memmer company；
Microscope：Zeiss companies；
Transwell cells, 12 porocyte culture plates and the 25cm in 8.0 μm of apertures2Blake bottle：Coming Costar are public Department.
4) main agents
DMEM culture medium dry powders：Gibco companies；
PBS：Contain NaCl8.2g, KCl0.2g, Na in per 1L solution2HPO4·H2O1.56g、KH2PO40.2g, PH value 7.4；
Hyclone：Hyclone companies；
0.25% trypsin solution：Hyclone companies；
Penicillin, streptomysin：Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., Shiyao Group；
DMSO (dimethyl sulfoxide (DMSO))：Hyclone companies；
Matrigel (matrigel)：BD companies；
Crystal violet dye liquor：Green skies company.
5) evaluation method
It is coated with matrigel：4 DEG C of the matrigel frozen in -20 DEG C of refrigerators is stayed overnight, becomes liquid；720 μ L are taken without blood Clear DMEM culture mediums, add 180 μ L Matrigel, mix, and add to the polycarbonate membrane upper chamber of Transwell cells, 100 μ L/；It is put into 37 DEG C, 5%CO2In incubator, 5 h are incubated；
Aquation basilar memebrane：Residual liquid in cell is absorbed, 50 μ L DMEM culture mediums, 37 DEG C, 5%CO are added per hole2Training Support in case and be incubated 30 min；
Inoculating cell：HCCLM3 cells are digested, plasma-free DMEM medium is washed 3 times, is counted, is made into cell suspension, density For 5 × 105Individual/mL；100 μ L cell suspensions are added per hole, while add 25 μ L (3S, 6R) -3- (4- fourths amino) -6- (Yin Diindyl -3- ethyls)-piperazine -2,5- diketone makes final concentration of 10 μM, final concentration of 20 μM of RGDS；Lower room adds containing for 600 μ L 10%FBS DMEM culture mediums, in 37 DEG C, 5%CO2Cultivated 48 hours in incubator；
Violet staining：Matrigel and upper indoor cell are wiped with cotton swab；Cell 30 is fixed with 4% paraformaldehyde Min, fixer is absorbed, is washed 3 times with PBS；30 min are dyed with 0.1% crystal violet dye liquor, dyeing liquor is absorbed, 3 is washed with PBS It is secondary；
Count：9 roughly the same visual field observations are chosen in each cell, takes pictures, counts；Experimental data statistics uses T examine and variance analysis, cell number with (It is individual) represent.
6) 1 is the results are shown in Table, (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone can be effective Ground suppresses HCCLM3 cell invasions.
Table 1. (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone is invaded HCCLM3 cells Attack the influence of abilitya
A) n=3；B) with physiological saline than P ＜ 0.05.
Experimental example 3 evaluates (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone to HCCLM3 The influence of cell migration ability
1) given the test agent
Compound 4 is configured to 50 μM of concentration with the DMEM culture mediums containing 0.1%DMSO；
RGDS is configured to 100 μM of concentration with the DMEM culture mediums containing 0.1%DMSO.
2) cell line
HCCLM3 (high-transfer human liver cancer cell).
3) key instrument and consumptive material
Super-clean bench：VS-1300-U clean benches, SuZhou Antai Air Tech Co., Ltd.；
Cell incubation case：INC153, memmer company；
Microscope：Zeiss companies；
Transwell cells, 12 porocyte culture plates and the 25cm in 8.0 μm of apertures2Blake bottle：Coming Costar are public Department.
4) main agents
DMEM culture medium dry powders：Gibco companies；
PBS：Contain NaCl8.2g, KCl0.2g, Na in per 1L solution2HPO4·H2O1.56g、KH2PO40.2g, PH value 7.4；
Hyclone：Hyclone companies；
0.25% trypsin solution：Hyclone companies；
Penicillin, streptomysin：Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., Shiyao Group；
DMSO (dimethyl sulfoxide (DMSO))：Hyclone companies；
Crystal violet dye liquor：Green skies company.
5) evaluation method
Inoculating cell：HCCLM3 cells are digested, plasma-free DMEM medium is washed 3 times, is counted, is made into cell suspension, density For 2 × 106Individual/mL；The 100 μ L cell suspensions of addition per hole, while 25 μ L (3S, 6R) -3- (4- fourths amino) -6- of addition (indoles - 3- ethyls)-piperazine -2,5- diketone makes final concentration of 10 μM, final concentration of 20 μM of RGDS；600 μ L's of lower room addition contains 10%FBS DMEM culture mediums, in 37 DEG C, 5%CO2Cultivated 6 hours in incubator；
Violet staining：Matrigel and upper indoor cell are wiped with cotton swab；Cell is fixed with 4% paraformaldehyde 30min, fixer is absorbed, is washed 3 times with PBS；30min is dyed with 0.1% crystal violet dye liquor, dyeing liquor is absorbed, 3 is washed with PBS It is secondary；
Count：9 roughly the same visual field observations are chosen in each cell, takes pictures, counts；Experimental data statistics uses T examine and variance analysis, cell number with (It is individual) represent.
6) 2 are the results are shown in Table, (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone can be effective Ground suppresses HCCLM3 cell migrations.
Table 2. (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone moves to HCCLM3 cells The influence of shifting abilitya
A) n=3；B) with physiological saline than P ＜ 0.05.
It is small to ICR that experimental example 4 evaluates (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone The influence in mouse rat-tail bleeding time
1) experiment material
Test-compound：Compound 4；
Positive control is 6-aminocaprolc acid (EACA)；Negative control is physiological saline (NS)；
Experimental animal：ICR male mices (cleaning grade), body weight 18-22g, tieing up tonneau China experimental animal technology by Beijing has Limit company provides；Every one group of 10 mouse, blank and each one group of positive control；
Solvent：Physiological saline.
2) medicine is prepared
Weigh Compound 4 according to quantity, physiological saline is added to required concentration；6-aminocaprolc acid (EACA) is physiological saline Solution.
3) dosage and administering mode
Dosage：EACA is 1.96mmol/kg；(3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine - 2,5- diketone are 0.05 μm of ol/kg；According to mouse weight, per 10g to 0.1mL decoctions or physiological saline；Gastric infusion.
4) foundation of animal model
ICR male mices 70, body weight 18-22g, are randomly divided into compound group, EACA groups and blank control group, every group 10 Only.Each group according to dosage gastric infusion.After administration 30 minutes, mouse is loaded in fixing device, makes its tail outside, with milli Meter ruler accurate measurement, made marks at away from rat-tail end 3mm, cut with profit and go out quick shearing in rat-tail end mark, treat blood energy Start timing during enough voluntarily spillings, every 30 seconds, gently wipe incision position drop of blood away with filter paper, until blood stops naturally, that is, filter Paper inhale when without blood untill.
5) statistical method
This experimental data statistics using t examine and variance analysis, the bleeding time withRepresent.
6) 3 are the results are shown in Table, (3S, 6R) -3- (4- fourths the amino) -6- at 3920 times lower than positive control EACA dosage （Indoles -3- ethyls)-piperazine -2,5- diketone still effectively shorten the mouse bleeding time, illustrate that it can effectively stop blooding.
Table 3. (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone goes out to ICR mouse rat-tails The influence of blood timea
A) n=10；B) with physiological saline than P ＜ 0.01；C) with physiological saline than P ＞ 0.05.
Experimental example 5 determines (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5-2 ketone to rat neck The influence of arterial thrombus generation
1) experiment material
Test-compound：(3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone；
Positive control is aspirin (Aspirin)；Negative control is physiological saline (NS)；
Experimental animal：SD male rats (cleaning grade), body weight 180-220g, tieing up tonneau China experimental animal technology by Beijing has Limit company provides；Every one group of 10 rats, blank and each one group of positive control；
Solvent：Physiological saline.
2) medicine is prepared
(3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone is weighed according to quantity, sequentially adds life Salt solution is managed to required concentration；Aspirin is normal saline solution.
3) dosage and administering mode
Dosage：Aspirin is 50 μm of ol/kg；(3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine Piperazine -2,5- diketone is 0.05 μm of ol/kg；According to rat body weight, per 100g to 0.3mL decoctions or physiological saline；Gastric infusion.
4) foundation of animal model
SD male rats 70, body weight 180-220g, are randomly divided into compound group, aspirin group and blank control group, Every group 10.Each group according to dosage gastric infusion.After administration 30 minutes, each group rat isolates neck with 10% chloral hydrate anesthesia Total artery and the total vein of neck, rat artery thrombus model is caused with rat carotid artery-venous cannula bypass circuit tuft method；Follow Ring 15 minutes, the band bolt silk thread in shunt valve is taken out, wipe surface away and float blood, claim band bolt silk thread weight in wet base, after subtracting silk thread weight, i.e., Obtain thrombosed weight in wet base, record data.
5) statistical method
This experimental data statistics using t examine and variance analysis, wet weight of thrombus withRepresent.
6) 4 be the results are shown in Table, under 0.05 μm of ol/kg dosage (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls) - Piperazine -2,5- diketone does not promote mouse thrombosis to act on, that is, does not have thrombosis risk.
Table 4. (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone is to SD rat carotid arteries The influence of thrombus growing amounta
A) n=10；B) with physiological saline than P ＞ 0.05；C) with physiological saline than P ＜ 0.01.
Experimental example 6 evaluates (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone paraxylene The influence of inducing mouse ear swelling
1) experiment material
Test-compound：(3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone；
Positive control is aspirin (Aspirin)；Negative control is physiological saline (NS)；
Experimental animal：ICR male mices (cleaning grade), body weight 18-22g, it is limited that tonneau China experiment thing technology is tieed up by Beijing Company provides.Every one group of 10 mouse, blank and each one group of positive control.
2) medicine is prepared
(3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone is weighed according to quantity, sequentially adds life Salt solution is managed to required concentration；Aspirin is normal saline solution.
3) dosage and administering mode
Dosage：Aspirin is 1.11mmol/kg；(3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls) - Piperazine -2,5- diketone is 0.05 μm of ol/kg；According to mouse weight, per 10g to 0.1mL decoctions or physiological saline；Gavage is given Medicine.
4) foundation of animal model
ICR male mices 70, body weight 18-22g, it is randomly divided into (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- second Base)-piperazine -2,5- diketone group, aspirin group and blank control group, every group 10.Each group according to dosage gastric infusion.Administration 30 After minute, 30 μ L dimethylbenzene are uniformly smeared on the inside of the left ear auricle of mouse, cervical dislocation puts to death mouse after 2 hours, respectively will The outer auricle of left and right two ear, which is cut and overlapped, to be stacked together, and is beaten with diameter 7mm card punch in same position and takes circular auricle, Weigh, record and calculate two ear weight differences；Mouse ear swelling degree=left auricle weight-auris dextra piece weight.
5) statistical method
This experimental data statistics using t examine and variance analysis, mouse ear swelling degree withRepresent.
6) 5 be the results are shown in Table, under 0.05 μm of ol/kg dosage (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls) - Piperazine -2,5- diketone effectively suppresses mice ear caused by dimethylbenzene, i.e., effectively suppresses inflammation.
Table 5. (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone is to ICR mice ears The influence of degreea
A) n=10；B) with physiological saline than P ＜ 0.05；C) with physiological saline than P ＜ 0.01.

Claims (5)

1. a kind of u-PA inhibitor, (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone, structure Formula is as follows：
2. prepare the side of (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine-2,5-dione of claim 1 Method, this method are made up of following steps：
(1) L-Trp-OBzl is D-Boc- with D-Boc-Lys (Z) condensations in anhydrous tetrahydro furan in the presence of DCC and HOBt Lys(Z)-L-Trp-OBzl；
(2) D-Boc-Lys (Z)-L-Trp-OBzl sloughs Boc generations D-Lys (Z)-L- in hydrogen chloride-ethyl acetate solution Trp-OBzl；
(3) in ethyl acetate and 5%NaHCO3In the presence of D-Lys (Z)-L-Trp-OBzl cyclizations generation (3S, 6R) -3- (4- fourth ammonia Base benzyl) -6- (indoles -3- ethyls)-piperazine-2,5-dione；
(4) in Pd/C and H2In the presence of, in methyl alcohol (3S, 6R) -3- (4- fourths aminobenzyl) -6- (indoles -3- ethyls)-piperazine - 2,5- diketone sloughs benzyl generation (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine-2,5-dione.
3. (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- the ethyls)-piperazine-2,5-diones of claim 1 are preparing tumour The application shifted and invade in profit suppression disease medicament.
4. (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- the ethyls)-piperazine-2,5-diones of claim 1 are preparing hemostasis The application of agent.
5. (3S, 6R) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine-2,5-dione of claim 1 is preparing anti-inflammatory The application of agent.
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