CN105272980A - (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 - Google Patents
(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 Download PDFInfo
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Abstract
The invention relates to a micromolecule mu-PA inhibitor (3R,12aS)-3-(4-aminobutyl)-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione with the structural formula shown in the specification, relates to a preparation method of the compound and application of the compound to inhibit tumor invasion and migration, resist coagulation and diminish inflammation as the mu-PA inhibitor. The invention belongs to the field of biological medicine.
Description
Technical field
The present invention relates to the small molecules u-PA inhibitor (3S shown in structural formula below, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone, relate to it preparation method and as the application of u-PA inhibitor in Tumor suppression invasion and attack and migration, anti-inflammatory and antithrombotic.The invention belongs to biomedicine field.
Background technology
Plasminogen activating system system is by plasminogen activator (Pas), and Plasminogen activation inhibitor (PAIs) and extracellular plasminogen activator receptor (PAR) form.Plasminogen activating system system relates to cell migration, vasculogenesis, wound healing, fetal development, a series of processes such as tumour cell diffusion and transfer.Tissue-type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA) two kinds of plasminogen activators are mainly contained in mammalian body.
Urokinase type plasminogen activator (u-PA) is a kind of serine protease extracted from people's urine or nephrocyte tissue culture medium, belong to double chain urokinase type Plasminogen Activator (tcu-PA), molecular weight is 55000 or 33000, is the activator of exogenous fibrolysis system.U-PA can arginine (560)-α-amino-isovaleric acid (561) peptide bond of By Direct Pyrolysis Profibrinolysin, makes the strand Profibrinolysin of non-activity change activated double-strand plasmin into.
In tumor tissues, u-PA plasminogen activation is converted into plasmin, and plasmin directly or indirectly causes extracellular matrix degradation, causes the infiltration of tumour cell further, transfer and vasculogenesis, promotes the propagation of tumour.In blood circulation, u-PA participates in by plasminogen activation the balance regulating intravascular coagulation-fibrinolytic, plays an important role to suppression thrombus.
In inflammatory response process, u-PA is by being combined with the u-PAR on inflammatory cell surface, participate in the penetrating power regulating inflammatory cell to blood vessel and tissue, promote that inflammatory cell moves to inflammation part, promote the release of inflammatory reaction and inflammatory factor, and the release of inflammatory factor can induce u-PA to express rising.
Because the enzymic activity of u-PA is applied in the cross of tumour-fibrinolytic-inflammation complexity, so the activity affecting u-PA can affect complicated cross.That is, the u-PA inhibitor inventing outstanding activity is significant at tumour-thrombus-inflammation cross for suppression u-PA.This meaning is embodied in more than 70% cancer patient and dies from metastasis of cancer; There is the cancer patient of about 80% to merge thrombosis, make thrombus become the major reason of cancer patient death; The general concurrent inflammation of cancer patient, inflammation also worsens the prognosis of patient.For this situation, the present invention proposes (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone, its preparation and therapeutic action.
Before this, contriver once reported structure below (3S, 6 ' S)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone reversible tumour cell is to the resistance of Zorubicin.Unfortunately, it for the complication of tumour without any effect.Under contrast, (3S of the present invention, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the beyond thought advantage of 5 '-diketone is, has outstanding inhibition tumor cell invasion and m etastasis, the concurrent inflammatory effect of Tumor suppression, simultaneously inhibition thrombosis.Their structure is shown in Fig. 1, before this, contriver report reversible drug resistance of tumor cell (3S, 6 ' S)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone (left side) and (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] of the present invention [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the structure of 5 '-diketone (right side); The unique difference of their structure is the configuration of the carbon of 6 ' position.
Summary of the invention
First content of the present invention is to provide u-PA inhibitor (3S, 6 ' the R)-Tetrahydrocarboline [2,3-b] of structural formula below and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone;
Second content of the present invention is to provide (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the preparation method of 5 '-diketone, the method comprises five reactions steps below:
(1) in the presence of sulphuric acid, L-Trp and formaldehyde reaction generate 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid;
(2) under methyl alcohol and thionyl chloride exist, 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylate methyl ester;
(3) 3S-1 under DCC and HOBt exists, 2,3,4-tetrahydro-beta-carboline-3-carboxylate methyl ester is 3S-1 with D-Boc-Lys (Z) condensation in anhydrous tetrahydro furan, 2,3,4-tetrahydro-beta-carboline-2-(D-Boc-carbobenzoxy-(Cbz) lysyl)-3-carboxylate methyl ester;
(4) (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-benzyloxycarbonyl amino fourth-4-base)-piperazine-2 in hydrogenchloride-ethyl acetate solution ', 5 '-diketone;
(5) at Pd/C and H
2under existence, (3S in methyl alcohol, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-benzyloxycarbonyl amino fourth-4-base)-piperazine-2 ', 5 '-two ketogenesis (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone.
Five reactions steps above can describe by the synthetic route of Fig. 1.
3rd content of the present invention evaluates small molecules u-PA inhibitor (3R, 6S)-3-(4-fourth is amino)-6-(indoles-3-ethyl)-piperazine-2,5-diketone is in inhibition tumor cell Infiltration and metastasis, and hemostasis, the effect of the aspect such as antithrombotic and anti-inflammatory.
Accompanying drawing explanation
Fig. 1 contriver reports the (3S of reversible drug resistance of tumor cell before this, 6 ' S)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone (left side) and (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] of the present invention [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the structure of 5 '-diketone (right side).
Fig. 2 synthesizes (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the route of 5 '-diketone.i)HCHO，H
2SO
4；ii)MeOH，SOCl
2；iii)DCC，HOBt，NMM，THF；iv)HCl/EA(4N)，MeOH，TEA；v)MeOH，Pd/C，H
2。
Fig. 3 (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is in vitro on the impact of UK plasminogen activation vigor.Wherein 1 human plasminogen (PLG) being classified as single component, 2 are classified as hatching altogether of UK and PLG; 3 are classified as 100 μ g (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone and 5 μ LUK (400U/mL) hatch component altogether with 5 μ LPLG's (5mg/mL); 4 are classified as 50 μ g (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone and 5 μ LUK (400U/mL) hatch component altogether with 5 μ LPLG's (5mg/mL); 5 are classified as 10 μ g (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone and 5 μ LUK (400U/mL) hatch component altogether with 5 μ LPLG's (5mg/mL); 6 be the EACA of 500 μ g and 5 μ LUK (400U/mL) with 5 μ LPLG (5mg/mL) hatch component altogether; 7 be the EACA of 250 μ g and 5 μ LUK (400U/mL) with 5 μ LPLG (5mg/mL) hatch component altogether.
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 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (1)
The 0.4mL vitriol oil is added in 800mL distilled water, stirs, add 10gL-Trp, ultrasonic dissolution assisting; Add 20mL formaldehyde solution in reaction solution, stirring at room temperature reacts 10 hours.Aftertreatment: drip strong aqua and adjust pH to 6 in reaction solution, leave standstill, have colorless solid to separate out, filtration is dried, and obtains product crude product 11.32g.ESI-MS (m/e): 215 [M-H]
-.
Embodiment 2 prepares 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylate methyl ester (2)
Add 60mL methyl alcohol in 250mL reaction flask, drip 7mL thionyl chloride under ice bath, activate 30 minutes, in reaction solution, add 6.2g3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (1), stirred overnight at room temperature, TLC shows the basic disappearance of raw material.Aftertreatment: removal of solvent under reduced pressure, resistates 100mL acetic acid ethyl dissolution, triethylamine adjusts PH to 8, purification by silica gel column chromatography (CH
2cl
2: CH
3oH=100: 1-20: 1), obtain 4.625g (70%) 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylate methyl ester is colorless solid.ESI-MS(m/e)：231[M+H]
+。
Embodiment 3 prepares 3S-1,2,3,4-tetrahydro-beta-carboline-2-(D-Boc-carbobenzoxy-(Cbz) lysyl)-3-carboxylate methyl ester (3)
1.9g (5.0mmol) D-Boc-Lys (Z) is suspended in 20mL anhydrous tetrahydro furan, in solution, 0.675g (5.0mmol) HOBt is added under stirring at room temperature, ice bath adds 1.133g (5.5mmol) DCC under stirring, obtain reaction solution I, stir 30 minutes under ice bath; By 1.15g (5.0mmol) 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylate methyl ester (2) is suspended in 20mL anhydrous tetrahydro furan, obtains reaction solution II; Reaction solution II is added in reaction solution I, stir 1h under ice bath, stirred overnight at room temperature.Aftertreatment: filtration under diminished pressure removing DCU, filtrate reduced in volume is removed tetrahydrofuran (THF), and residue 150mL acetic acid ethyl dissolution, is placed in 250mL separating funnel by the solution obtained, uses 5%KHSO successively
4the aqueous solution is washed and is respectively washed 3 times, ethyl acetate layer anhydrous Na with the saturated NaCl aqueous solution
2sO
4dry 30min, filtration under diminished pressure, filtrate reduced in volume, the yellow oily crude product obtained, through purification by silica gel column chromatography (CH
2cl
2: CH
3oH=100: 1-20: 1), obtain 2.785g (94.1%) 3S-1,2,3,4-tetrahydro-beta-carboline-2-(D-Boc-carbobenzoxy-(Cbz) lysyl)-3-carboxylate methyl ester is colorless solid.ESI-MS(m/e)：593[M+H]
+。
(3S, 6 ' R)-Tetrahydrocarboline [2,3-b] prepared by embodiment 4 [3 ', 4 '-b]-6 '-(1-benzyloxycarbonyl amino fourth-4-base)-piperazine-2 ', 5 '-diketone (4)
By 2.368g (4mmol) 3S-1,2,3,4-tetrahydro-beta-carboline-2-(D-Boc-carbobenzoxy-(Cbz) lysyl)-3-carboxylate methyl ester (3) is placed in 100mL eggplant bottle, ice bath stirs the lower slow 30mL4N hydrogenchloride-ethyl acetate solution that adds in reaction flask, add drying tube, ice bath stirs lower reaction TLC monitoring raw material point disappearance after 4 hours, termination reaction.Aftertreatment: stir lower removal of solvent under reduced pressure, add ethyl acetate redissolve after removal of solvent under reduced pressure again, in triplicate; Resistates, with 80mL dissolve with methanol, adjusts pH to 9 with triethylamine, and oil bath 80 DEG C reaction about 6 hours, TLC shows the basic disappearance of raw material.Aftertreatment: removal of solvent under reduced pressure, resistates disperses with 10mL methyl alcohol, filter, collect filter cake and obtain 1.334g (72.5%) (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-benzyloxycarbonyl amino fourth-4-base)-piperazine-2 ', 5 '-diketone is colorless solid.ESI-MS(m/e)：461[M+H]
+。
(3S, 6 ' R)-Tetrahydrocarboline [2,3-b] prepared by embodiment 5 [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone (5)
By 0.200g (0.435mmol) (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-benzyloxycarbonyl amino fourth-4-base)-piperazine-2 ', 5 '-diketone (4) is placed in 50mL reaction flask, add 10mL dissolve with methanol, in solution, add 40mgPd/C, pass into H
2, stirring at room temperature reacts 48 hours, and TLC shows the basic disappearance of raw material; Elimination Pd/C, removal of solvent under reduced pressure obtains 117mg (83%) (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is colourless powder.ESI-MS(m/e)：327[M+H]
+.Mp212-213℃.
(c＝0.30，CH
3OH).IR(KBr)：3261，2933，2856，1672，1456，1332，1242，1141，740cm
-1.
1HNMR(300MHz，DMSO-d6)：δ/ppm＝10.99(s，1H)，8.37(s，1H)，7.44(d，J＝9Hz，1H)，7.32(d，J＝9Hz，1H)，7.06(t，J＝9Hz，1H)，6.98(t，J＝9Hz，1H)，5.41(d，J＝18Hz，1H)，4.41(d，J＝9Hz，1H)，4.21(d，J＝15Hz，1H)，3.97(m，1H)，2.86(m，2H)，2.57(m，2H)，1.76(m，1H)，1.68(m，1H)，1.33(m，3H)，1.08(m，1H).
13CNMR(200MHz，DMSO-d
6)：δ/ppm＝166.88，165.61，135.82，129.91，126.24，120.93，118.60，117.51，111.00，105.31，55.71，53.41，41.08，32.44，32.27，26.15，20.61.Elem.Anal：C
18H
22N
4O
2.C，66.24；H，6.79；N，17.17。
Experimental example 1 measures (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is on the active impact of urokinase (UK)
1) main agents and instrument
Reagent: SDS-PAGE gel electrophoresis agents useful for same and hexosamine (EACA) are commercial reagent;
Human plasminogen (PLG) and urokinase (UK) available from Sigma.
Instrument: Vertial electrophorestic tank Mini-PRORENTetraSystem (BIO-RAD);
Electrophoresis apparatus PowerPac (BIO-RAD);
Scanner Scanmaker8700 (MICROTEK).
2) preparation of solution
The preparation of PLG solution: take PLG5mg, is placed in 15mL centrifuge tube, adds 1mL physiological saline solution and namely obtains PLG solution, and concentration is 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 solution, obtain mother liquor; Get 0.1mL mother liquor with normal saline dilution to 2.5mL, obtain UK solution, concentration is 400U/mL; Packing, often pipe 0.1mL ,-20 DEG C of preservations are stand-by;
The preparation of EACA solution: take 50mg compound, with 1mL physiological saline solution, obtains mother liquor 1, and concentration is 50mg/mL; Get 0.5mL mother liquor 1, with normal saline dilution to 1mL, obtain solution 2, concentration is 25mg/mL;
(3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the preparation of 5 '-diketone solution: take 5mg (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone, with 1mL physiological saline solution, concentration is 5mg/mL;-20 DEG C of preservations are stand-by.
3) preparation of sample
Get 5 μ LUK (400U/mL) solution; Packing, often pipe 0.1mL, be placed in 0.5mL centrifuge tube, add 10 μ L physiological saline or (3S wherein respectively again, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone solution, hatches 15 minutes for 37 DEG C; In each centrifuge tube, add 5 μ LPLG solution respectively again, hatch 15 minutes for 37 DEG C; After hatching end, then in each centrifuge tube, add 5 μ L5 × SDS electrophoresis sample-loading buffers respectively, by the sex change 5 minutes in 100 DEG C of water-baths of each centrifuge tube after mixing, the SDS-PAGE gel electrophoresis with 12% after quick ice bath cooling is separated.
4) SDS-PAGE gel electrophoresis
Reagent prepares:
30% deposit sol solution: acrylamide (Acr) 29.0g, methylene bisacrylamide (Bis) 1.0g, adds ultrapure water (up-H after mixing
2o), 37 DEG C of dissolvings, be settled to 100mL, brown bottle is stored in room temperature;
1.5MTris-HCl (pH8.0): Tris18.17g adds up-H
2o dissolves, and concentrated hydrochloric acid adjusts pH to 8.0, is settled to 100mL;
1MTris-HCl (pH6.8): Tris12.11g adds up-H
2o dissolves, and concentrated hydrochloric acid adjusts pH to 6.8, is settled to 100mL;
12%SDS: electrophoresis level SDS12.0g adds up-H
2o is in 68 DEG C of hydrotropies, and concentrated hydrochloric acid is adjusted to pH7.2, is settled to 100mL;
10% ammonium persulphate (AP): 100mgAP adds up-H
2o1mL;
Coomassie brilliant blue staining fluid: methanol-water-acetic acid=45mL+45mL+10mL, adds 100mg Xylene Brilliant Cyanine G solid, is mixed with staining fluid;
Destainer: methanol-water-acetic acid=10mL+90mL+10mL, is mixed with destainer.
Operation steps:
Adopt rectilinear electrophoresis chamber device
(1) preparation of polyacrylamide gel
1. the preparation of separation gel (12%):
Ultrapure water 4.0mL
30% deposit sol solution 3.3mL
1.5MTris-HCl2.5mL
12％SDS0.1mL
10％AP0.1mL
Get the above-mentioned mixed solution of 1mL, add TEMED (N, N, N ', N '-Tetramethyl Ethylene Diamine) 10 μ L back covers, more than add TEMED4 μ L, pour into after mixing between sheet glass, with water seal top, note liquid level is put down, gel is polymerized completely needs about 60min.
2. the preparation of concentrated glue (4%):
Ultrapure water 1.4mL
30% deposit sol solution 0.33mL
1MTris-HCl0.25mL
12％SDS0.02mL
10％AP0.02mL
TEMED2μL
Gone by water on separation gel, add above-mentioned mixed solution, insert between sheet glass immediately by comb, polymerization needs about 30min completely.
(2) sample preparation: 5 × SDS sample-loading buffer sample being added respective amount, 100 DEG C of heating 3-5min make protein denaturation, take out, fast cooling.
(3) loading: the sample after process is added in sample pool, and adds 20 μ L Protein Marker product and compare.
(4) electrophoresis: add 1 × electrophoretic buffer in electrophoresis chamber, connect power supply, negative pole is upper, positive pole under, during electrophoresis, concentrated glue voltage 80V, 30min, separation gel voltage 100V walk to electrophoresis chamber lower end to electrophoresis to bromjophenol blue and stop (about needing 1.5h).
(5) dye: taken out from sheet glass by glue, Coomassie brilliant blue staining fluid dyes, in shaking table (60RPM) room temperature 10min.
(6) decolour: glue is taken out from staining fluid, puts into destainer, spend the night in the decolouring of shaking table (60RPM) room temperature.
(7) by the glue scanner scanning after decolouring, observations.
5) the results are shown in Figure 2, (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone concentration dependant ground suppresses UK to be hydrolyzed the activity of human plasminogen.Illustrate that it is the inhibitor of UK.
Experimental example 2 measures (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is on the impact of HCCLM3 cell invasion ability
1) given the test agent
(3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the DMEM substratum of 5 '-diketone containing 0.1%DMSO is mixed with 50 μMs of concentration;
The DMEM substratum of RGDS containing 0.1%DMSO is mixed with 100 μMs of concentration.
2) cell strain
HCCLM3 (high-transfer human liver cancer cell), purchased from ATCC.
3) key instrument and consumptive material
Super clean bench: VS-1300-U clean bench, SuZhou Antai Air Tech Co., Ltd.;
Cell incubation case: INC153, memmer company;
Microscope: Zeiss company;
The Transwell cell in 8.0 μm of apertures, 12 porocyte culture plate and 25cm
2culturing bottle: CorningCostar company.
4) main agents
DMEM culture medium dry powder: Gibco company;
PBS damping fluid: containing NaCl8.2g, KCl0.2g, Na in every 1L solution
2hPO
4h
2o1.56g, KH
2pO
40.2g, pH value 7.4;
Foetal calf serum: Hyclone company;
0.25% trypsin solution: Hyclone company;
Penicillin, Streptomycin sulphate: Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., Shiyao Group;
DMSO (dimethyl sulfoxide (DMSO)): Hyclone company;
Matrigel (matrigel): BD company;
Viola crystallina dye liquor: green skies company.
5) evaluation method
Bag is by matrigel: spent the night by the frozen matrigel4 in-20 DEG C of refrigerators DEG C, 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, 5%CO
2in incubator, hatch 5h;
Aquation basilar membrane; Absorb residual liquid in cell, every hole adds the DMEM substratum of 50 μ L, 37 DEG C, 5%CO
230min is hatched in incubator;
Inoculating cell: digestion HCCLM3 cell, plasma-free DMEM medium washes 3 times, and counting, be made into cell suspension, density is 5 × 10
5individual/mL; Every hole adds 100 μ L cell suspensions, adds 25 μ L (3S, 6 ' R)-Tetrahydrocarboline [2 simultaneously, 3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone makes final concentration be 10 μMs, and RGDS final concentration is 20 μMs; Lower room adds the DMEM substratum containing 10%FBS of 600 μ L, at 37 DEG C, 5%CO
2cultivate 48 hours in incubator;
Violet staining: the cell wiping matrigel and upper indoor with cotton swab; 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;
Counting: choose 9 roughly the same visuals field at each cell and observe, take pictures, counting; Experimental data statistics all adopt t inspection and variance analysis, cell count with
represent.
6) the results are shown in Table 1, (3R, 6S)-3-(4-fourth is amino)-6-(indoles-3-ethyl)-piperazine-2,5-diketone can suppress HCCLM3 cell invasion effectively.
Table 1. (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is on the impact of HCCLM3 cell invasion ability
a
A) n=3; B) with physiological saline ratio, P < 0.05.
Experimental example 3 is evaluated (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is on the impact of HCCLM3 cell migration ability
1) given the test agent
(3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the DMEM substratum of 5 '-diketone containing 0.1%DMSO is mixed with 50 μMs of concentration;
The DMEM substratum of RGDS containing 0.1%DMSO is mixed with 100 μMs of concentration.
2) cell strain
HCCLM3 (high-transfer human liver cancer cell).
3) key instrument and consumptive material
Super clean bench: VS-1300-U clean bench, SuZhou Antai Air Tech Co., Ltd.;
Cell incubation case: INC153, memmer company;
Microscope: Zeiss company;
The Transwell cell in 8.0 μm of apertures, 12 porocyte culture plate and 25cm
2culturing bottle: ComingCostar company.
4) main agents
DMEM culture medium dry powder: Gibco company;
PBS damping fluid: containing NaCl8.2g, KCl0.2g, Na in every 1L solution
2hPO
4h
2o1.56g, KH
2pO
40.2g, pH value 7.4;
Foetal calf serum: Hyclone company;
0.25% trypsin solution: Hyclone company;
Penicillin, Streptomycin sulphate: Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., Shiyao Group;
DMSO (dimethyl sulfoxide (DMSO)): Hyclone company;
Viola crystallina dye liquor: green skies company.
5) evaluation method
Inoculating cell: digestion HCCLM3 cell, plasma-free DMEM medium washes 3 times, and counting, be made into cell suspension, density is 2 × 10
6individual/mL; Every hole adds 100 μ L cell suspensions, adds 25 μ L (3S, 6 ' R)-Tetrahydrocarboline [2 simultaneously, 3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone makes final concentration be 10 μMs, and RGDS final concentration is 20 μMs; Lower room adds the DMEM substratum containing 10%FBS of 600 μ L, at 37 DEG C, 5%CO
2cultivate 6 hours in incubator;
Violet staining: the cell wiping matrigel and upper indoor with cotton swab; 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;
Counting: choose 9 roughly the same visuals field at each cell and observe, take pictures, counting; Experimental data statistics all adopt t inspection and variance analysis, cell count with
represent.
6) the results are shown in Table 2, (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone can suppress HCCLM3 cell migration effectively.
Table 2. (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is on the impact of HCCLM3 cell migration ability
a
A) n=3; B) with physiological saline than P < 0.05.
Experimental example 4 is evaluated (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the impact that 5 '-diketone generates rat carotid artery thrombus
1) experiment material
Test-compound: (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone;
Positive control is acetylsalicylic acid (Aspirin); Negative control is physiological saline (NS);
Laboratory animal: SD male rat (cleaning grade), body weight 180-220g, is provided by Beijing Vital River Experimental Animals Technology Co., Ltd.; Every 10 rats one group, blank and each one group of positive control;
Solvent: physiological saline.
2) Pharmaceutical formulations
Take (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] according to quantity and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone, adds physiological saline successively to desired concn; Acetylsalicylic acid is normal saline solution.
3) dosage and administering mode
Dosage: acetylsalicylic acid is 50 μm of ol/kg; (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is 5 μm of ol/kg; According to rat body weight, every 100g is to 0.3mL liquid or physiological saline; Gastric infusion.
4) foundation of animal model
SD male rat 70, body weight 180-220g, is divided into compound group, acetylsalicylic acid group and blank group at random, often organizes 10.Each group of according to dosage gastric infusion.Administration is after 30 minutes, and each group rat, with 10% chloral hydrate anesthesia, is isolated arteria carotis communis and the total vein of neck, causes rat artery thrombus model with rat carotid artery-venous cannula bypass circuit tuft method; Circulate 15 minutes, take out the strap bolt silk thread in bypass tube, wipe the floating blood in surface away, claim strap bolt silk thread weight in wet base, after deducting silk thread weight, obtain thrombosed weight in wet base, record data.
5) statistical method
This experimental data statistics all adopt t inspection and variance analysis, wet weight of thrombus with
represent.
6) the results are shown in Table 3, (3S, 6 ' R)-Tetrahydrocarboline [2 under 5 μm of ol/kg dosage, 3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone suppresses rat to form thrombus effectively, namely has anti thrombotic action.
Table 3. (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is on the impact of rat carotid artery thrombus growing amount
a
A) n=10; B) with physiological saline than P > 0.05; C) with physiological saline than P < 0.01.
Experimental example 6 evaluates the impact of (3R, 6S)-3-(4-fourth is amino)-6-(indoles-3-ethyl)-piperazine-2,5-diketone p-Xylol inducing mouse ear swelling
1) experiment material
Test-compound: (3R, 6S)-3-(4-fourth is amino)-6-(indoles-3-ethyl)-piperazine-2,5-diketone;
Positive control is acetylsalicylic acid (Aspirin); Negative control is physiological saline (NS);
Laboratory animal: ICR male mice (cleaning grade), body weight 18-22g, ties up the experiment of tonneau China by Beijing
Thing Technology Co., Ltd. provides.Every 10 mouse one group, blank and each one group of positive control.
2) Pharmaceutical formulations
Take (3R, 6S)-3-(4-fourth is amino)-6-(indoles-3-ethyl)-piperazine-2,5-diketone according to quantity, add physiological saline successively to desired concn; Acetylsalicylic acid is normal saline solution.
3) dosage and administering mode
Dosage: acetylsalicylic acid is 1.11mmol/kg; (3R, 6S)-3-(4-fourth is amino)-6-(indoles-3-ethyl)-piperazine-2,5-diketone is 0.05 μm of ol/kg; According to Mouse Weight, every 10g is to 0.1mL liquid or physiological saline; Gastric infusion.
4) foundation of animal model
ICR male mice 70, body weight 18-22g, is divided into (3R, 6S)-3-(4-fourth is amino)-6-(indoles-3-ethyl)-piperazine-2,5-diketone group, acetylsalicylic acid group and blank group at random, often organizes 10.Each group of according to dosage gastric infusion.Administration is after 30 minutes, uniform application 30 μ L dimethylbenzene inside the left ear auricle of mouse, after 2 hours, cervical dislocation puts to death mouse, respectively outer for left and right two ears auricle is cut and overlapped and be stacked together, to beat at same position with the punch tool of diameter 7mm and get circular auricle, weigh, record and calculate two ear weight differences; Mouse ear swelling degree=left auricle weight-auris dextra sheet weight.
5) statistical method
This experimental data statistics all adopt t inspection and variance analysis, mouse ear swelling degree with
represent.
6) the results are shown in Table 5, (3S under 0.05 μm of ol/kg dosage, 6S)-3-(4-fourth amino)-6-(indoles-3-ethyl)-piperazine-2, the 5-diketone mice ear that effectively suppresses dimethylbenzene to cause, i.e. inflammation-inhibiting effectively.
Table 5. (3R, 6S)-3-(4-fourth is amino)-6-(indoles-3-ethyl)-piperazine-2,5-diketone is on the impact of ICR mice ear degree
a
A) n=10; B) with physiological saline than P < 0.05; C) with physiological saline than P < 0.01.
Claims (5)
1. the below u-PA inhibitor shown in structural formula, (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone.
2. prepare (3S, 6 ' the R)-Tetrahydrocarboline [2,3-b] of claim 1 and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', the method for 5 '-diketone, the method is made up of following steps:
(1) in the presence of sulphuric acid, L-Trp and formaldehyde reaction generate 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid;
(2) under methyl alcohol and thionyl chloride exist, 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylate methyl ester;
(3) 3S-1 under DCC and HOBt exists, 2,3,4-tetrahydro-beta-carboline-3-carboxylate methyl ester is 3S-1 with D-Boc-Lys (Z) condensation in anhydrous tetrahydro furan, 2,3,4-tetrahydro-beta-carboline-2-(D-Boc-carbobenzoxy-(Cbz) lysyl)-3-carboxylate methyl ester;
(4) (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] [3 ', 4 '-b]-6 '-(1-benzyloxycarbonyl amino fourth-4-base)-piperazine-2 in hydrogenchloride-ethyl acetate solution ', 5 '-diketone;
(5) at Pd/C and H
2under existence, (3S in methyl alcohol, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-benzyloxycarbonyl amino fourth-4-base)-piperazine-2 ', 5 '-two ketogenesis (3S, 6 ' R)-Tetrahydrocarboline [2,3-b] and [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone.
3. (3S, 6 ' the R)-Tetrahydrocarboline [2,3-b] of claim 1 [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is as metastases and the application of invading profit inhibitor.
4. (3S, 6 ' the R)-Tetrahydrocarboline [2,3-b] of claim 1 [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is as the application of anti-inflammatory agent.
5. (3S, 6 ' the R)-Tetrahydrocarboline [2,3-b] of claim 1 [3 ', 4 '-b]-6 '-(1-amino fourth-4-base)-piperazine-2 ', 5 '-diketone is as the application of antithrombotic agent.
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| CN108947975A (en) * | 2017-05-18 | 2018-12-07 | 首都医科大学 | Piperazine -2,5- diketone of 3S- indolylethyl -6S- fatty acid/amino acid modification, synthesis, activity and application |
| CN108947976A (en) * | 2017-05-18 | 2018-12-07 | 首都医科大学 | Piperazine -2,5- diketone of 3S- indolylethyl -6S- ArAA modification, synthesis, activity and application |
| CN108947981A (en) * | 2017-05-27 | 2018-12-07 | 首都医科大学 | Piperazine -2,5- diketone of 3R- indole methyl -6S- fatty acid/amino acid modification, synthesis, activity and application |
| CN110577517A (en) * | 2018-06-11 | 2019-12-17 | 首都医科大学 | Methyl indole and aromatic amino acid modified 2, 5-diketopiperazine, synthesis, activity and application thereof |
| CN110577518A (en) * | 2018-06-11 | 2019-12-17 | 首都医科大学 | methyl indole and amide side chain amino acid modified diketopiperazine, synthesis and application thereof |
| CN110577516A (en) * | 2018-06-08 | 2019-12-17 | 首都医科大学 | Amino acid and tranexamic acid modified diketopiperazines, their preparation and use |
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| CN108947976A (en) * | 2017-05-18 | 2018-12-07 | 首都医科大学 | Piperazine -2,5- diketone of 3S- indolylethyl -6S- ArAA modification, synthesis, activity and application |
| CN108947976B (en) * | 2017-05-18 | 2020-06-19 | 首都医科大学 | 3S-indolylmethyl-6S-aromatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof |
| CN108947975B (en) * | 2017-05-18 | 2020-11-27 | 首都医科大学 | 3S-indolylethyl-6S-fatty amino acid modified piperazine-2, 5-diketone and synthesis, activity and application thereof |
| CN108947981A (en) * | 2017-05-27 | 2018-12-07 | 首都医科大学 | Piperazine -2,5- diketone of 3R- indole methyl -6S- fatty acid/amino acid modification, synthesis, activity and application |
| CN108947981B (en) * | 2017-05-27 | 2020-06-19 | 首都医科大学 | 3R-indolylmethyl-6S-aliphatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof |
| CN110577516A (en) * | 2018-06-08 | 2019-12-17 | 首都医科大学 | Amino acid and tranexamic acid modified diketopiperazines, their preparation and use |
| CN110577517A (en) * | 2018-06-11 | 2019-12-17 | 首都医科大学 | Methyl indole and aromatic amino acid modified 2, 5-diketopiperazine, synthesis, activity and application thereof |
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