CN105218526B - The diketone of (3S, 6S) 3,6 disubstituted piperazine 2,5, it is prepared and therapeutic action - Google Patents
The diketone of (3S, 6S) 3,6 disubstituted piperazine 2,5, it is prepared and therapeutic action Download PDFInfo
- Publication number
- CN105218526B CN105218526B CN201410252598.3A CN201410252598A CN105218526B CN 105218526 B CN105218526 B CN 105218526B CN 201410252598 A CN201410252598 A CN 201410252598A CN 105218526 B CN105218526 B CN 105218526B
- Authority
- CN
- China
- Prior art keywords
- piperazine
- indoles
- ethyls
- diketone
- fourths
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention discloses (the 3S of u PA inhibitor, 6S) 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
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, 6S) -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, 6S) -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, 6S) -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 L- 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 L-Boc-Lys (Z)-L-Trp-OBzl slough Boc generation L-Lys (Z)-
L-Trp-OBzl；
(3) in ethyl acetate and 5%NaHCO3In the presence of L-Lys (Z)-L-Trp-OBzl generation (3S, 6S) -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, 6S) -3- (4- fourths aminobenzyl) -6- (indoles -3- ethyls) -
The reactive ketone of piperazine -2,5- bis- generates (3S, 6S) -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, 6S) -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, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone)
DCC, HOBt, NMM, THF；ii)HCI/EA(4N)；Iii) EA, 5%NaHCO3(3a) or EA, TEA, 80 DEG C (3b, 3c)；iv)
CH3OH, Pd/C, H2。
Fig. 3 (3S, 6S) -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, 6S) -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, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,
The common incubation component of 5- diketone and 5uL UK (400U/mL) and 5uL PLG (5mg/mL)；5 are classified as 10 μ g (3S, 6S) -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；6 be 500 μ g EACA and 5 μ L UK (400U/mL) and 5 μ L PLG (5mg/mL) common incubation component；7 be 250 μ g's
EACA and 5 μ L UK (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 L-Boc-Lys (Z)-L-Trp-OBzl (1)
1.9g (5.0mmol) L-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 3.037g
(92.6%) title compound, it is colorless solid.ESI-MS(m/e)：657[M+H]+。
Embodiment 2 prepares L-Lys (Z)-L-Trp-OBzl (2)
Sterling 2.62g (4mmol) L-Boc-Lys (Z)-L-Trp-OBzl (1) is placed in 50mg eggplant bottles, under ice bath stirring
2.6mL 4N hydrogen chloride-ethyl acetate solution is slowly added dropwise into reaction bulb, adds drying tube, after being reacted 4 hours under ice bath stirring
TLC monitoring raw material points disappear, terminating reaction.Post processing：Ethyl acetate is added to dissolve reaction solution decompressing and extracting with water pump under stirring
Use water pump decompressing and extracting again afterwards, in triplicate；Add absolute ether to be stood after being fully suspended, pour out ether, drain product, weight
Again three times, 2.09g (95%) title compound is obtained, is pale yellow powder.ESI-MS(m/e)：557[M+H]+。
Embodiment 3 prepares (3S, 6S) -3- (4- fourths aminobenzyl) -6- (indoles -3- ethyls)-piperazine -2,5- diketone (3)
2g (3.6mmol) compound L-Lys (Z)-L-Trp-OBzl (2) are dissolved with 150mL ethyl acetate, with 5%
Na2CO3Solution washs three times, anhydrous Na2SO4After drying 30min, filtering, ethyl acetate solution is stirred overnight at room temperature, and next day subtracts
Pressure removes ethyl acetate solution, adds 10mL methanol and disperses, there is a large amount of insoluble matters, filters, and precipitation is colorless solid product
0.725g (45%).ESI-MS(m/e)：449[M+H]+.1H NMR (300MHz, DMSO-d6)：δ/ppm=10.846 (s, 1H),
8.017 (d, J=1.5Hz, 1H), 7.911 (d, J=1.8Hz, 1H), 7.574 (d, J=7.8Hz, 1H), 7.341 (m, 6H),
7.044 (m, 3H), 6.920 (t, J=7.2Hz, 1H), 5.000 (s, 2H), 4.108 (s, 1H), 3.505 (s, 1H), 3.249
(dd, J=4.2Hz, J=14.4Hz, 1H), 3.011 (dd, J=4.8Hz, J=14.4Hz, 1H), 2.719 (q, J=7.2Hz,
2H), 0.981 (m, 3H), 0.513 (m, 3H).
Embodiment 3 prepares (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone (4)
By 0.448g (1.0mmol) compound (3S, 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, 140mg Pd/C are added into solution, are passed through H2, room
Warm stirring reaction 48 hours, Pd/C is filtered off, be concentrated under reduced pressure to obtain 0.260g (82,8%) title compound, is colourless powder.ESI-
MS(m/e)：315[M+H]+.Mp187-188℃.IR(KBr)：3190.26,3053.32,2920.23,1668.43,
1460.11,1334.74,1099.43,746.45,424.34cm-1·(c=0.31, CH30H).1HNMR
(800MHz, DMSO-d6)：δ/ppm=10.979 (s, 1H), 8.091 (s, 1H), 7.933 (s, 1H), 7.590 (d, J=
8.0Hz, 1H), 7.330 (d, J=8.0Hz, 1H), 7.030 (dd, J=15.2Hz, J=8.0Hz, 2H), 6.935 (t, J=
7.2Hz, 1H), 4.127 (d, J=4.0Hz, 1H), 3.528 (d, J=5.6Hz, 1H), 3.266 (dd, J=4.0Hz, J=
14.8Hz, 1H), 3.009 (dd, J=4.0Hz, J=14.8Hz, 1H), 2.369 (m, 2H), 1.073 (m, 2H), 0.955 (m,
1H), 0.597 (m, 2H), 0.573 (m, 1H)13C NMR (75MHz, DMSO-d6)：δ/ppm=167.49,167.25,
136.35,128.37,125.11,121.11,119.49,118.73,111.63,109.04,55.88,54.19,33.24,
29.22,28.75,20.86. elementary analysis C17H22N4O2：Calculated value C, 64.95；H, 7.05；N, 17.82；Measured value C,
64.80；H, 7.18；N, 17.63.
Experimental example 1 determines (3S, 6S) -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：PLG 5mg are weighed, are 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, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone solution：Weigh 5mg
(3S, 6S) -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, 6S) -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-HCl 2.5mL
12%SDS 0.1mL
10%AP 0.1mL
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-HCl 0.25mL
12%SDS 0.02mL
10%AP 0.02mL
TEMED 2μL
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 albumen
Denaturation, 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, 6S) -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, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone pair
The influence of HCCLM3 cell invasion abilities
1) given the test agent
(3S, 6S) -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；Take 720 μ L serum-frees
DMEM culture mediums, 180 μ L Matrigel are added, mix, add to the polycarbonate membrane upper chamber of Transwell cells, 100 μ L/
It is individual；It is put into 37 DEG C, 5%CO2In incubator, 5h is 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 30min；
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；The 100 μ L cell suspensions of addition per hole, while 25 μ L (3S, 6S) -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 48 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) 1 is the results are shown in Table, (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone can be effective
Ground suppresses HCCLM3 cell invasions.
Table 1. (3S, 6S) -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 ratio, P ＜ 0.05.
Experimental example 3 evaluates (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone pair
The influence of HCCLM3 cell migration abilities
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 NaCl 8.2g, KCl 0.2g, Na in per 1L solution2HPO4·H2O1.56g、KH2PO4
0.2g, 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, 6S) -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, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone can be effective
Ground suppresses HCCLM3 cell migrations.
Influence of the compound 4 of table 2. to HCCLM3 cell migration abilitiesa
A) n=3；B) with physiological saline than P ＜ 0.05.
It is small to ICR that experimental example 4 evaluates (3S, 6S) -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：(3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone；
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, 6S) -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, 6S) -3- (4- fourths the amino) -6- at 3920 times lower than positive control EACA dosage
(indoles -3- ethyls)-piperazine -2,5- diketone still effectively shortens the mouse bleeding time, illustrates that it can effectively stop blooding.
Table 3. (3S, 6S) -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 ＜ 0.05；C) with physiological saline than P ＜ 0.01；D) with physiological saline than P ＞
0.05.
Experimental example 5 determines (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone to rat
The influence of carotid thrombosis 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, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- diketone to diformazan
The influence of benzene inducing mouse ear swelling
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：ICR male mices (cleaning grade), body weight 18-22g, the experiment of tonneau China is tieed up by Beijing
Thing Technology Co., Ltd. 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, 6S) -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 (4)
1. u-PA inhibitor (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine -2,5- shown in formula
The preparation method of diketone,
The preparation method is made up of following steps：
(1) L-Trp-OBzl is L-Boc- with L-Boc-Lys (Z) condensations in anhydrous tetrahydro furan in the presence of DCC and HOBt
Lys(Z)-L-Trp-OBzl；
(2) L-Boc-Lys (Z)-L-Trp-OBzl sloughs Boc generations L-Lys (Z)-L- in hydrogen chloride-ethyl acetate solution
Trp-OBzl；
(3) in ethyl acetate and 5%NaHCO3In the presence of L-Lys (Z)-L-Trp-OBzl cyclizations generation (3S, 6S) -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, 6S) -3- (4- fourths aminobenzyl) -6- (indoles -3- ethyls)-piperazine -
2,5- diketone sloughs benzyl generation (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine-2,5-dione.
2. u-PA inhibitor (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine-2,5-dione of formula
Prepare metastases and invade the application moistened in suppressing disease medicament,
3. u-PA inhibitor (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine-2,5-dione of formula
Application in styptic is prepared,
4. u-PA inhibitor (3S, 6S) -3- (4- fourths amino) -6- (indoles -3- ethyls)-piperazine-2,5-dione of formula
Application in antiinflammatory is prepared,
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410252598.3A CN105218526B (en) | 2014-06-10 | 2014-06-10 | The diketone of (3S, 6S) 3,6 disubstituted piperazine 2,5, it is prepared and therapeutic action |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410252598.3A CN105218526B (en) | 2014-06-10 | 2014-06-10 | The diketone of (3S, 6S) 3,6 disubstituted piperazine 2,5, it is prepared and therapeutic action |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105218526A CN105218526A (en) | 2016-01-06 |
| CN105218526B true CN105218526B (en) | 2018-02-06 |
Family
ID=54987859
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410252598.3A Expired - Fee Related CN105218526B (en) | 2014-06-10 | 2014-06-10 | The diketone of (3S, 6S) 3,6 disubstituted piperazine 2,5, it is prepared and therapeutic action |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105218526B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108948137B (en) * | 2017-05-18 | 2021-07-02 | 首都医科大学 | 3S-indolylethyl-6S-polar amino acid modified piperazine-2, 5-diketone and synthesis, activity and application thereof |
| CN108929320B (en) * | 2017-05-22 | 2020-10-16 | 首都医科大学 | 3R-indolylmethyl-6R-oxazolidinone modified piperazine-2, 5-dione, synthesis, activity and application thereof |
| CN108976204B (en) * | 2017-05-31 | 2020-09-01 | 首都医科大学 | 3S-indolylmethyl-6R-aspartic acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof |
| CN108976210B (en) * | 2017-05-31 | 2020-07-28 | 首都医科大学 | 3S-indolylmethyl-6R-aromatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof |
| CN108976211B (en) * | 2017-06-01 | 2020-07-28 | 首都医科大学 | 3S-indolylmethyl-6R-aliphatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof |
| CN108976207B (en) * | 2017-06-01 | 2020-07-28 | 首都医科大学 | 3S-indolylmethyl-6R-fatty side chain amino acid modified piperazine-2, 5-diketone, synthesis, activity and application |
| CN108976208B (en) * | 2017-06-01 | 2020-07-28 | 首都医科大学 | 3S-indolylmethyl-6R-Met modified piperazine-2, 5-dione, and synthesis, activity and application thereof |
| CN110551120B (en) * | 2018-06-04 | 2020-10-20 | 首都医科大学 | 6-amino amido n-hexanoyl carboline benzyl carboxylate, preparation, activity and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK181687A (en) * | 1986-04-23 | 1987-10-24 | Fujisawa Pharmaceutical Co | PIPERAZINE DERIVATIVES AND PROCEDURES FOR PREPARING THEREOF |
| CA2774959C (en) * | 2000-08-04 | 2016-05-31 | Dmi Biosciences, Inc. | Method of using diketopiperazines and composition containing them |
| GB2372740A (en) * | 2001-01-17 | 2002-09-04 | Xenova Ltd | Diketopiperazines |
| CN102234278A (en) * | 2010-04-26 | 2011-11-09 | 首都医科大学 | (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives, and synthesis method and application thereof |
-
2014
- 2014-06-10 CN CN201410252598.3A patent/CN105218526B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN105218526A (en) | 2016-01-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105294660B (en) | The diketone of (3R, 6S) 3,6 disubstituted piperazine 2,5, it is prepared and application | |
| CN105218526B (en) | The diketone of (3S, 6S) 3,6 disubstituted piperazine 2,5, it is prepared and therapeutic action | |
| CN105503833B (en) | Indoles piperazine-diketone, preparation and therapeutic effect | |
| CN105272968B (en) | The diketone of (3S, 6R) 3,6 disubstituted piperazine 2,5, it is prepared and application | |
| Xie et al. | Three-dimensional bio-printing of primary human hepatocellular carcinoma for personalized medicine | |
| Liu et al. | Hypoxia-inducible factor-1α is essential for hypoxia-induced mesenchymal stem cell mobilization into the peripheral blood | |
| US20210010034A1 (en) | Transient cellular reprogramming for reversal of cell aging | |
| CN105272980A (en) | (3R,12aS)-3-(4-aminobutyl)-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione, and preparation and application thereof | |
| CN107106613A (en) | For the composition for including source of human stem cell excretion body into fat induction, adipose tissue regeneration, skin-whitening or improvement wrinkle | |
| CN102250203B (en) | Beta-carboline aminoacid benzyl ester, preparation method and application thereof | |
| CN109641042A (en) | For treating the Hete rocyclic derivatives of RSV | |
| Abbas et al. | Vertebrate cell culture as an experimental approach–limitations and solutions | |
| CN109678987A (en) | A kind of hydrogen sulfide releasing agent HA-ADT, preparation method and application | |
| CN115260083A (en) | Preparation method and application of mitochondrion-targeted viscosity response fluorescent probe | |
| CN103656630A (en) | Method for purifying plasmin in animal medicine and preparing traditional Chinese medicine composition | |
| CN111759748A (en) | Anti-aging peptide composition and application thereof | |
| CN101469322B (en) | Cell preparation for liver injury and hepatic cirrhosis and preparation thereof | |
| CN104873498A (en) | Application of melatonin in preparation of medicine for preventing premature senility of mesenchymal stem cells | |
| CN105396136A (en) | Application of CCN1(Cyr61) to treatment of diseases related to skin injuries and atrophoderma | |
| CN108159045A (en) | The application of red pineapple flower alkali A and its derivative in prevention or treatment central nervous system disease drug is prepared | |
| CN114276388A (en) | Ferrocene piperazine amide compound and preparation method and application thereof | |
| CN108752241A (en) | Acyl group guanidine for treating arthropathy | |
| CN107513071B (en) | A kind of multi-functional podophyllotoxin derivative and its preparation method and application | |
| CN106432424A (en) | Polypeptide capable of inhibiting tumor metastasis, and application of polypeptide | |
| CN109550056A (en) | Matrix metalloproteinase inhibitory polypeptide gold nano grain composition and its application, preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180206 Termination date: 20210610 |