CN105273055A - RGD tetrapeptide-modified beta-carboline, preparation, activity and application thereof - Google Patents
RGD tetrapeptide-modified beta-carboline, preparation, activity and application thereof Download PDFInfo
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- CN105273055A CN105273055A CN201410261587.1A CN201410261587A CN105273055A CN 105273055 A CN105273055 A CN 105273055A CN 201410261587 A CN201410261587 A CN 201410261587A CN 105273055 A CN105273055 A CN 105273055A
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Abstract
The invention discloses 1-(4-hydroxy-3-methoxycarbonylphenyl)-beta-carboline-3-formyl-RGD tetrapeptide with a structure as shown in the formula. The invention also discloses a preparation method of the compound, a nano-structure of the compound, an anti-tumor effect of the compound, an effect of the compound in inhibiting adhesion, invasion and migration of tumor cells, further discloses anti-inflammatory and antithrombotic effects of the compound and expounds an application of the compound in medical science.
Description
Technical field
The present invention relates to 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val, relate to their preparation method, relate to their nanostructure and application.The invention belongs to biomedicine field.
Technical background
The health of the malignant tumour serious threat mankind.Except self is severe to the prognosis of tumour patient, the inflammation of Complicated by Malignancy, thrombus and transfer worsen the prognosis of patient further.Such as, the malignant tumor patient more than more than 90% is all die from metastases.Metastases depends on 4 factors: 1) tumor cell surface forms microthrombus, escapes macrophage phagocytic, moves to far-end by blood circulation; 2) tumor cell adhesion of far-end is moved to vessel wall; 3) cell adhesion enters healthy tissues to the tumour cell of vessel wall by attacking out blood vessel; 4) inflammation makes the tumour cell entering healthy tissues grow into the nascent tumor of transfer.
Because existing antitumor drug does not possess anti-inflammatory, antithrombotic and anti-metastasis effect, so curative effect is undesirable.It is clinical active demand that invention has antitumor, anti-inflammatory, antithrombotic and inhibiting effect on tumor metastasis medicine simultaneously.
RGD tetrapeptide, namely RGDS, RGDF and RGDV are integrin alphas
vβ
3blocker.Applicant to be once reported in when concentration is 1 μM them and can to adhere to and infiltrate by inhibition tumor cell.Applicant is once them and oestrogenic hormon coupling, and preparation does not have the osteoporosis agent of blood coagulation side effect.Applicant once prepared efficient antithrombotic agent them and the coupling of tetrahydro-beta-carboline-3-carboxylic acid.Applicant is also once with the β-carboline-3-carboxylic acid that amino acid modified tetrahydro-beta-carboline-3-carboxylic acid, β-carboline-3-carboxylic acid and 1-position replace, and the β-carboline-3-carboxylic acid of the tetrahydro-beta-carboline-3-carboxylic acid or the replacement of 1-position that comprise the replacement of 1-position prepares efficient antithrombotic agent or antineoplastic agent.Here is the representative of the structure type that contriver creates.Although contriver has paid large quantity research energy, screen hundreds of compound, never there is the compound of antitumor, anti-inflammatory, antithrombotic and antitumor cell Adhesion, Migration and immersional wetting simultaneously.
Contriver is in the analysis structure of hundreds of compound and the basis of activity change, recognize RGDS, RGDV or RGDF and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid coupling, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser formed, (4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val can have antitumor 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-simultaneously, anti-inflammatory, the compound of antithrombotic and antitumor cell Adhesion, Migration and immersional wetting.Based on this understanding, inventors herein propose the present invention.
Summary of the invention
First content of the present invention is to provide 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser (Phe, Val) of structure below.
Second content of the present invention is to provide the preparation method of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser (Phe, Val), and the method is made up of following steps:
(1) under the vitriol oil exists, 5-formylsalicylic acid is 90 DEG C, microwave reaction 2h in methyl alcohol, generates 5-formylsalicylic acid methyl esters;
(2) under the existence of polyphosphoric acid, L-Trp and phenylcarbinol reaction, generate L-Trp benzyl ester;
(3) under trifluoracetic acid exists, in methylene dichloride, 5-formylsalicylic acid and the ester condensation of L-Trp benzyl are 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-1,2,3,4-tetrahydro-beta-carboline-3-benzyl carboxylates;
(4) 2,3-bis-chloro-5,6-dinitrile-1, under the existence of 4-benzoquinones (DDQ), 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-1,2,3,4-tetrahydro-beta-carboline-3-benzyl carboxylate is oxidized to 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-benzyl carboxylate in tetrahydrofuran (THF) (THF);
(5) at Pd/C and H
2under existence, in methyl alcohol, the reaction of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-benzyl carboxylate generates 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid;
(6) adopt progressively condensation method, under DCC and HOBt exists, react in dry THF, obtain full guard peptide sequence Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
(7) under ice bath hydrogenchloride ethyl acetate solution (4M) in, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl removes Boc respectively and obtain Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
(8) DCC and HOBt exist under, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid in anhydrous THF respectively with Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl generation 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
(9) at Pd/C and H
2under existence, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO in methyl alcohol
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl generates compound 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser respectively, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val.
3rd content of the present invention measures 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, the nanostructure of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val.
4th content of the present invention evaluates 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, the effect of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val inhibition tumor cell propagation.
5th content of the present invention evaluates 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val suppresses the effect of S180 tumor-bearing mice tumor growth.
6th content of the present invention evaluates 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, the effect of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val inhibition tumor cell Adhesion, Invasion and migration.
7th content of the present invention evaluates 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, the anti-inflammatory action of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val.
8th content of the present invention evaluates 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, antithrombotic effect in the In Vitro Anti platelet aggregation of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val and body.
Accompanying drawing explanation
Fig. 1. the structure type representative of the antithrombotic that contriver creates or active compound for anti tumor, in formula, AA is L-amino acid or glycine.
Fig. 2. the synthetic route .i of compound 5a-c) CH
3oH, dense H
2sO
4, 90 DEG C, microwave; Ii) polyphosphoric acid, phenylcarbinol, oil bath 75 DEG C; Iii) CH
2cl
2, TFA; Iv) THF, DDQ; V) CH
3oH, Pd/C, H
2; Vi) DCC, HOBt, NMM, THF; Vii) CH
3oH, 2NNaOH; Viii) in hydrogenchloride/ethyl acetate solution (4N) .4a-c, AA is respectively Ser (Bzl), Phe and Val residue; In 5a-c, AA is respectively Ser, Phe and Val residue.
Fig. 3. compound 5a-c is in pure water solution 1 × 10
-7transmission electron microscope photo under M concentration.
Fig. 4. compound 5a acts on the cell viability figure (n=3) of tumour cell.
Fig. 5. compound 5b acts on the cell viability figure (n=3) of tumour cell.
Fig. 6. compound 5c acts on the cell viability figure (n=3) of tumour cell.
Embodiment
In order to set forth the present invention further, provide a series of embodiment below.These embodiments are illustrative completely, and they are only used for being specifically described the present invention, not should be understood to limitation of the present invention.
Embodiment 1 prepares 5-formylsalicylic acid methyl esters
Take 1.660g (10.0mmol) 5-formylsalicylic acid in microwave reaction tank, add 25mL methyl alcohol and the dense H of 1mL
2sO
490 DEG C of reaction 2h in microwave reactor, TLC monitoring is utilized to disappear to raw material spot, after stopped reaction is down to room temperature, reaction solution is transferred in 100mL eggplant-shape bottle, with strong aqua adjust pH to 7-8, reaction solution is evaporated to after doing, add a large amount of acetic acid ethyl dissolution, ethyl acetate layer uses saturated NaHCO successively
3, saturated NaCl respectively washes three times, then uses anhydrous Na
2sO
4dry 2h, filter, be evaporated to dry, namely having crystal to separate out in left at room temperature over night, obtain 1.635g (90.8%) target compound, is faint yellow needle-like crystal.ESI-MS(m/e)：181[M+H]
+。
Embodiment 2 prepares L-Trp benzyl ester
Take 15.0g (44.4mmol) polyphosphoric acid in 500mL eggplant-shape bottle, add 80mL phenylcarbinol, it is made to dissolve in oil bath 50 DEG C, after solution temperature rises to 75 DEG C, taking 10g (49.0mmol) L-Trp adds wherein, 48h is reacted at 75 DEG C, TLC monitoring is utilized to disappear to raw material spot, after stopped reaction cooling, in reaction flask, 400mL anhydrous diethyl ether is poured under ice bath stirs, now adularescent solid is separated out, stirring spend the night after by it filtration, white solid 200mL ethyl acetate and 10mL aqueous suspension, about adjusting solution ph to 8 with triethylamine, solution becomes clarification shape, leave standstill separatory, the ester layer be separated is used saturated NaHCO successively
3, saturated NaCl respectively washes three times, ethyl acetate layer anhydrous Na
2sO
4dry 2h, filter, be evaporated to dry, obtaining 12.85g (89.2%) target compound, is white solid.ESI-MS(m/e)：295[M+H]
+。
Embodiment 3 prepares 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-1,2,3,4-tetrahydro-beta-carboline-3-benzyl carboxylates (1)
100mLCH is added in 250mL eggplant-shape bottle
2cl
2and 10mLTFA, 11.76g (40.0mmol) L-Trp benzyl ester is taken and 7.92g (44.0mmol) 5-formylsalicylic acid methyl esters adds wherein after stirring, after several minutes, reaction solution becomes blush, after 2 days, reaction solution becomes black, strong aqua is slowly dripped by reaction solution adjust pH to 8 under ice bath stirs, reaction solution is left standstill separatory, by separation of C H
2cl
2layer uses saturated NaHCO successively
3, saturated NaCl respectively washes three times, CH
2cl
2layer anhydrous Na
2sO
4dry 2h, filter, be evaporated to dry, obtaining 14.59g (80%) target compound, is yellow solid.ESI-MS(m/e)：457[M+H]
+。
Embodiment 4 prepares 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-benzyl carboxylate (2)
Take 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-1,2,3,4-tetrahydro-beta-carboline-3-benzyl carboxylate 4.56g (10.0mmol), in 250mL eggplant-shape bottle, dissolves by dry THF, adds DDQ4.54g (20.0mmol), after several minutes, reaction solution becomes muddy, react completely after 4h, filter, leach solid and use saturated NaHCO successively
3, methyl alcohol, ether wash, filter, obtaining 3.75g (82.7%) target compound, is pale solid.ESI-MS(m/e)：453[M+H]
+；Mp：190.4-191.0℃.
1HNMR(300MHz，DMSO)：δ/ppm＝8.87(s，1H)，8.42(m，2H)，8.10(dd，J＝2.1Hz，J＝2.1Hz，1H)，7.69(d，J＝8.1Hz，1H)，7.57(m，3H)，7.37(m，3H)，7.04(d，J＝6.3Hz)，5.46(s，1H)，3.88(s，3H).
13CNMR(75MHz，DMSO)：δ/ppm＝169.28，165.93，142.35，137.07，135.13，131.75，129.30，128.98，128.47，128.41，122.40，121.73，120.62，116.54，114.71，66.39，52.47.Elem.Anal：C
27H
20N
2O
5.C，71.67；H，4.46；N，6.19。
Embodiment 5 prepares 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid (3)
2.26g (5.0mmol) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-benzyl carboxylate is suspended in 120mL methyl alcohol, add 400mgPd/C, reaction solution first takes air away with vacuum pump, then passes into hydrogen, 3 times so repeatedly, react 2 days under room temperature, utilize TLC to monitor to disappear to raw material spot, natural filtration reaction solution, is evaporated to dry by reaction solution, obtaining 1.080g (59.7%) target compound, is yellow solid.ESI-MS(m/e)：361[M-H]
-；Mp：227.1-227.9℃.Elem.Anal：C
20H
14N
2O
5.C，66.30；H，3.89；N，7.73。
Embodiment 6 prepares Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
1) Boc-Arg (NO
2) preparation of-Gly-OBzl
By 1.60g (5.0mmol) Boc-Arg (NO
2) being suspended in 20mL dry THF, in solution, add 0.68g (5.0mmol) HOBt under stirring at room temperature, ice bath adds 1.133g (5.5mmol) DCC under stirring, and obtains reaction solution I, stirs 30 minutes under ice bath.1.69g (5.0mmol) Gly-OBzl is suspended in 20mL dry THF, then adds NMM gradually, regulate pH to 8-9, obtain reaction solution II.Reaction solution II is added in reaction solution I, first under ice bath, stir 1h, then in stirring at room temperature, TLC monitoring disappears to raw material point.Aftertreatment: filtration under diminished pressure removing DCU, filtrate reduced in volume is removed THF, and residue 150mLEA dissolves, and the solution obtained is placed in 250mL separating funnel, uses 5%KHSO successively
4the aqueous solution is washed and is respectively washed 3 times with the saturated NaCl aqueous solution, EA layer anhydrous Na
2sO
4dry 30min, filtration under diminished pressure, filtrate reduced in volume is to dry, and obtaining 2.26g (97%) title compound, is colorless solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1, R
f=0.40.ESI-MS (m/e): 466 [M+H]
+.
2) Boc-Arg (NO
2) preparation of-Gly
By 1.63g (3.5mmol) Boc-Arg (NO
2)-Gly-OBzl is dissolved in 15mL methyl alcohol, solution NaOH (2N) aqueous solution that will obtain under ice bath stirs adjusts pH to 12, and after reaction 2h, TLC monitors raw material point and disappears.Aftertreatment: regulate reaction solution pH to 7 with 2N dilute hydrochloric acid, concentrating under reduced pressure removing methyl alcohol, residue 2N dilute hydrochloric acid regulates pH to 2, extracts 3 times with EA, merges EA layer, is washed till neutrality, anhydrous Na with the saturated NaCl aqueous solution
2sO
4dry 30min, filtration under diminished pressure, filtrate reduced in volume is to dry, and obtaining 1.24g (94%) title compound, is colorless solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1.ESI-MS (m/e): 375 [M-H]
-.
3) preparation of Boc-Asp (OBzl)-Ser (Bzl)-OBzl
By Boc-Arg (NO
2) preparation method of-Gly-OBzl obtains 2.87g (97%) title compound by 1.62g (5.0mmol) Boc-Asp (OBzl) and 2.29g (5.0mmol) Ser (Bzl)-OBzl, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1, R
f=0.42.ESI-MS (m/e): 592 [M+H]
+.
4) preparation of Asp (OBzl)-Ser (Bzl)-OBzl
2.36g (4.0mmol) Boc-Asp (OBzl)-Ser (Bzl)-OBzl is placed in 50mg eggplant bottle, ice bath stirs the lower slow 15mL4NHCl/EA solution that drips in reaction flask, add drying tube, ice bath stirs lower reaction TLC monitoring raw material point disappearance after 2 hours, termination reaction.Aftertreatment: under stirring with water pump by reaction solution decompressing and extracting, add EA dissolve after again use water pump decompressing and extracting, in triplicate; Leave standstill after adding the abundant suspendible of anhydrous diethyl ether, pour out ether, drain product, in triplicate, obtaining 1.94g (97%) title compound, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1.ESI-MS (m/e): 500 [M+H]
+.
5) Boc-Arg (NO
2) preparation of-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
By Boc-Arg (NO
2) preparation method of-Gly-OBzl is by 1.31g (3.5mmol) Boc-Arg (NO
2)-Gly and 1.87g (3.5mmol) Asp (OBzl)-Ser (Bzl)-OBzl obtains 2.08g (70%) title compound, is colorless solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1, R
f=0.39.ESI-MS (m/e): 849 [M+H]
+.
6) Arg (NO
2) preparation of-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
By the preparation method of Asp (OBzl)-Ser (Bzl)-OBzl by 1.87g (2.2mmol) Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl obtains 1.56g (95%) title compound, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1.ESI-MS (m/e): 747 [M+H]
+.
Embodiment 7 prepares Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
1) preparation of Boo-Asp (OBzl)-Phe-OBzl
By Boc-Arg (NO
2) preparation method of-Gly-OBzl obtains 2.66g (95%) title compound by 1.62g (5.0mmol) Boc-Asp (OBzl) and 2.14g (5.0mmol) Phe-OBzl, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1, R
f=0.41.ESI-MS (m/e): 561 [M+H]
+.
2) preparation of Asp (OBzl)-Phe-OBzl
Obtaining 1.77g (96%) title compound by the preparation method of Asp (OBzl)-Ser (Bzl)-OBzl by 2.24g (4.0mmol) Boc-Asp (OBzl)-Phe-OBzl, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1.ESI-MS (m/e): 461 [M+H]
+.
3) Boc-Arg (NO
2) preparation of-Gly-Asp (OBzl)-Phe-OBzl
By Boc-Arg (NO
2) preparation method of-Gly-OBzl is by 1.31g (3.5mmol) Boc-Arg (NO
2)-Gly and 1.74g (3.5mmol) Asp (OBzl)-Phe-OBzl obtains 2.06g (72%) title compound, is colorless solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1, R
f=0.43.ESI-MS (m/e): 819 [M+H]
+.
4) Arg (NO
2) preparation of-Gly-Asp (OBzl)-Phe-OBzl
By the preparation method of Asp (OBzl)-Ser (Bzl)-OBzl by 1.80g (2.2mmol) Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl obtains 1.50g (95%) title compound, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1.ESI-MS (m/e): 719 [M+H]
+.
Embodiment 8 prepares Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
1) preparation of Boc-Asp (OBzl)-Val-OBzl
By Boc-Arg (NO
2) preparation method of-Gly-OBzl obtains 2.48g (97%) title compound by 1.62g (5.0mmol) Boc-Asp (OBzl) and 1.90g (5.0mmol) Val-OBzl, is colorless oil.TLC condition: CH
2cl
2/ CH
3oH, 20: 1, R
f=0.38.ESI-MS (m/e): 513 [M+H]
+.
2) preparation of Asp (OBzl)-Val-OBzl
Obtaining 1.74g (97%) title compound by the preparation method of Asp (OBzl)-Ser (Bzl)-OBzl by 2.05g (4.0mmol) Boc-Asp (OBzl)-Val-OBzl, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1.ESI-MS (m/e): 413 [M+H]
+.
3) Boc-Arg (NO
2) preparation of-Gly-Asp (OBzl)-Val-OBzl
By Boc-Arg (NO
2) preparation method of-Gly-OBzl is by 1.31g (3.5mmol) Boc-Arg (NO
2)-Gly and 1.57g (3.5mmol) Asp (OBzl)-Val-OBzl obtains 2.10g (78%) title compound, is colorless solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1, R
f=0.41.ESI-MS (m/e): 771 [M+H]
+.
4) Arg (NO
2) preparation of-Gly-Asp (OBzl)-Val-OBzl
By the preparation method of Asp (OBzl)-Ser (Bzl)-OBzl by 1.69g (2.2mmol) Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl obtains 1.51g (97%) title compound, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH, 20: 1.ESI-MS (m/e): 671 [M+H]
+.
Embodiment 9 prepares 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser (5a)
1) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2) preparation of-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (4a)
By Boc-Arg (NO
2) preparation method of-Gly-OBzl is by 0.36g (1.0mmol) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid (3) and 0.78g (1.0mmol) Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl obtains 0.51g (46.2%) title compound, is faint yellow solid.TLC condition: CH
2cl
2/ CH
3oH/HAc, 25: 1: 0.25, R
f=0.30.ESI-MS (m/e): 1094 [M+H]
+.
1hNMR (300MHz, DMSO-d6): δ/ppm=11.25 (s, 1H), 8.81 (m, 2H), 8.44 (m, 6H), 8.19 (dd, J=8.7Hz, J=2.1Hz, 1H), 7.98 (s, 1H), 7.69 (d, J=8.1Hz, 1H), 7.61 (t, J=7.5Hz, 1H), 7.29 (m, 17H), 5.05 (dd, J=15.9Hz, J=3.9Hz, 4H), 4.81 (m, 1H), 4.70 (m, 1H), 4.57 (m, 1H), 4.39 (dd, J=25.5Hz, J=12.3Hz, 2H), 3.94 (s, 3H), 3.83 (d, J=5.4Hz, 1H), 3.71 (m, 2H), 3.61 (dd, J=9.6Hz, J=3.9Hz, 1H), 3.21 (s, 1H), 2.79 (dd, J=16.8Hz, J=5.4Hz, 1H), 2.62 (dd, J=16.5Hz, J=8.7Hz, 1H), 1.87 (s, 2H), 1.78 (m, 1H), 1.59 (m, 2H) .Elem.Anal:C
56h
56n
10o
14.C, 61.53, H, 5.16, N, 12.81.
2) preparation of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser (5a)
By 0.51g (0.46mmol) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl is dissolved in 15mL methyl alcohol, add 100mgPd/C under stirring at room temperature, use threeway ligation bottle and hydrogen gas bag, reaction solution first takes air away with vacuum pump, then hydrogen is passed into, so 3 times repeatedly, under stirring at room temperature, react 48h, utilize TLC monitoring to disappear to raw material spot, filtration under diminished pressure reaction solution, being evaporated to by reaction solution dry, obtaining 0.188g (52.5%) target compound, is yellow solid.TLC condition: CH
2cl
2/ CH
3oH/HAc/H
2o, 2.5: 0.5: 0.25: 0.1, R
f=0.39.HPLC: moving phase (chromatogram acetonitrile/ultrapure water, 0min, 10: 90, 2min, 15: 85, 5min, 20: 80, 8min, 30: 70, 10min, 50: 50, 12min, 90: 10, 30min, 90: 10), retention time 10.32min, purity 97.8%.ESI-MS (m/e): 778 [M+H]
+dEG C .Mp:169.5-170.4.
(c=0.24, CH
3oH) .IR (KBr): 3325.28,1670.35,1492.90,1448.54,1392.61,1246.02,1068.56em
-1.
1hNMR (500MHz, DMSO-d6): δ/ppm=11.91 (s, 1H), 10.09 (s, 1H), 8.78 (m, 3H), 8.49 (s, 1H), 8.35 (d, J=2.5Hz, 1H), 8.22 (dd, J=8.5Hz, J=2.0Hz, 1H), 7.67 (d, J=8.0Hz, 1H), 7.58 (t, J=7.5Hz, 1H), 7.29 (m, 2H), 7.11 (t, J=5.5Hz, 2H), 6.73 (m, 1H), 4.67 (m, 1H), 4.39 (m, 1H), 3.99 (dd, J=17.0Hz, J=7.0Hz, 1H), 3.94 (s, 3H), 3.58 (m, 2H), 3.51 (m, 1H), 3.21 (m, 1H), 3.02 (m, 1H), 2.58 (dd, J=17.0Hz, J=5.5Hz, 1H), 2.35 (dd, J=17.0Hz, J=5.0Hz, 1H), 2.12 (m, 2H), 1.88 (m, 1H), 1.58 (m, 1H), 1.12 (m, 1H) .Elem.Anal:C
35h
39n
9o
12.C, 54.05, H, 5.05, N, 16.21.
Embodiment 10 prepares 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe (5b)
1) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2) preparation of-Gly-Asp (OBzl)-Phe-OBzl (4b)
By Boc-Arg (NO
2) preparation method of-Gly-OBzl is by 0.36g (1.0mmol) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid (3) and 0.75g (1.0mmol) Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl obtains 0.55g (51.3%) title compound, is pale solid.TLC condition: CH
2cl
2/ CH
3oH/HAc, 25: 1: 0.25, R
f=0.29.HPLC: moving phase (chromatogram acetonitrile/ultrapure water, 0min, 10: 90, 2min, 15: 85, 5min, 20: 80, 8min, 30: 70, 10min, 50: 50, 12min, 90: 10, 30min, 90: 10), retention time 12.77min, purity 97.2%.ESI-MS (m/e): 1063 [M+H]
+.
1hNMR (300MHz, DMSO-d6): δ/ppm=11.90 (s, 1H), 10.77 (s, 1H), 8.83 (s, 1H), 8.77 (d, J=8.1Hz, 1H), 8.52 (t, J=5.1Hz, 2H), 8.37 (m, 4H), 8.18 (dd, J=8.7Hz, J=2.4Hz, 1H), 7.95 (m, 1H), 7.69 (d, J=8.1Hz, 1H), 7.61 (t, J=7.5Hz, 1H), 7.25 (m, 16H), 5.02 (dd, J=9Hz, J=4.5Hz, 4H), 4.73 (m, 2H), 4.46 (dd, J=15Hz, J=7.8Hz, 1H), 3.94 (s, 3H), 3.87 (m, 1H), 3.67 (m, 1H), 3.21 (m, 2H), 2.96 (m, 2H), 2.73 (m, 1H), 2.57 (m, 1H), 1.91 (s, 2H), 1.87 (m, 1H), 1.73 (m, 1H), 1.60 (m, 2H) .Anal:C
55h
54n
10o
13.C, 62.14, H, 5.12, N, 13.18.
2) preparation of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe (5b)
By the preparation method of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser by 0.55g (0.51mmol) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl obtains 0.238g (55.7%) target compound, is pale solid.TLC condition: CH
2cl
2/ CH
3oH/HAc/H
2o, 2.5: 0.5: 0.25: 0.1, R
f=0.37.ESI-MS (m/e): 838 [M+H]
+dEG C .Mp:159.5-160.3.
(c=0.28, CH
3oH) .IR (KBr): 3307.92,1647.21,1624.06,1492.90,1448.54,1288.45,1247.94cm
-1.
1hNMR (300MHz, DMSO-d6): δ/ppm=11.95 (s, 1H), 10.36 (s, 1H), 9.02 (d, J=7.8Hz, 1H), 8.79 (t, J=7.2Hz, 2H), 8.47 (d, J=2.1Hz, 1H), 8.40 (t, J=7.8Hz, 1H), 8.21 (dd, J=8.4Hz, J=2.1Hz, 1H), 7.69 (d, J=8.1Hz, 1H), 7.61 (t, J=7.8Hz, 1H), 7.31 (m, 3H), 7.14 (m, 6H), 4.67 (m, 1H), 4.32 (m, 1H), 4.12 (m, 1H), 3.93 (s, 3H), 3.51 (m, 2H), 3.15 (m, 1H), 2.93 (m, 3H), 2.57 (m, 2H), 2.33 (m, 1H), 2.09 (m, 1H), 1.81 (m, 1H), 1.56 (s, 2H), 1.07 (m, 1H) .Elem.Anal:C
41h
43n
9o
11.C, 58.78, H, 5.17, N, 15.05.
Embodiment 11 prepares 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val (5c)
1) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2) preparation of-Gly-Asp (OBzl)-Val-OBzl (4c)
By Boc-Arg (NO
2) preparation method of-Gly-OBzl is by 0.36g (1.0mmol) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid (3) and 0.71g (1.0mmol) Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl obtains 0.63g (62.1%) title compound, is pale solid.TLC condition: CH
2cl
2/ CH
3oH/HAc, 25: 1: 0.25, R
f=0.31.ESI-MS (m/e): 1015 [M+H]
+.
1hNMR (300MHz, DMSO-d6): δ/ppm=11.91 (s, 1H), 10.75 (s, 1H), 8.83 (s, 1H), 8.77 (d, J=8.1Hz, 1H), 8.45 (t, J=2.7Hz, 2H), 8.37 (m, 3H), 8.18 (m, 2H), 7.95 (m, 1H), 7.69 (d, J=8.4Hz, 1H), 7.59 (t, J=7.8Hz, 1H), 7.35 (m, 12H), 5.05 (dd, J=9.3Hz, J=5.1Hz, 4H), 4.73 (m, 2H), 4.16 (t, J=6.3Hz, 1H), 3.95 (s, 3H), 3.87 (m, 1H), 3.67 (m, 1H), 3.21 (m, 2H), 2.79 (m, 1H), 2.65 (m, 1H), 2.01 (m, 1H), 1.91 (s, 1H), 1.77 (m, 2H), 1.60 (m, 2H), 0.78 (d, J=6.9Hz, 6H) .Elem.Anal:C
51h
54n
10o
13.C, 60.35, H, 5.36, N, 13.80.
2) preparation of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val (5c).
By the preparation method of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser by 0.63g (0.62mmol) 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl obtains 0.268g (53.9%) target compound, is pale solid.TLC condition: CH
2cl
2/ CH
3oH/HAc/H
2o, 2.5: 0.5: 0.25: 0.1, R
f=0.39.HPLC: moving phase (chromatogram acetonitrile/ultrapure water, 0min, 10: 90, 2min, 15: 85, 5min, 20: 80, 8min, 30: 70, 10min, 50: 50, 12min, 90: 10, 30min, 90: 10), retention time 11.68min, purity 95.8%.ESI-MS (m/e): 790 [M+H]
+dEG C .Mp:168.2-169.1.
(c=0.36, CH
3oH) .IR (KBr): 3344.57,2956.87,1670.35,1492.90,1456.26,1386.82,1249.87,756.10cm
-1.
1hNMR (300MHz, DMSO-d6): δ/ppm=11.95 (s, 1H), 10.46 (s, 1H), 9.02 (d, J=6.9Hz, 1H), 8.94 (s, 1H), 8.81 (s, 1H), 8.76 (d, J=7.5Hz, 1H), 8.42 (t, J=8.1Hz, 2H), 8.20 (d, J=7.8Hz, 1H), 7.68 (d, J=8.1Hz, 1H), 7.60 (t, J=7.2Hz, 1H), 7.31 (m, 2H), 7.18 (s, 1H), 6.76 (d, J=13.8Hz, 1H), 4.74 (m, 1H), 4.36 (m, 1H), 4.12 (dd, J=15.6Hz, J=4.8Hz, 1H), 3.95 (s, 3H), 3.56 (m, 2H), 3.21 (m, 2H), 3.00 (m, 1H), 2.64 (m, 1H), 2.26 (m, 2H), 1.62 (s, 2H), 0.80 (s, 6H) .Elem.Anal:C
37h
43n
9o
11.C, 56.27, H, 5.49, N, 15.96.
Experimental example 1 measures the transmission electron microscope photo under compound Plasma Concentration
By 5a-c according to 1 × 10
-7the pure water solution of the concentration configuration compound of M, is layered on uniformly on copper mesh, observes the self-assembly property of compound under transmission electron microscope (TEM, JEM-1230, JEOL).The photo obtained is as Fig. 3.Result shows, 5a-c all can form nano particle in the pure water of pH=7.0, and diameter is between 40-250nm.
Experimental example 2 measures the cytotoxicity of compound 5a-c to tumour cell
1) substratum of compound 5a-c of the present invention containing 0.1%DMSO is mixed with desired concn.
2) tumour cell of experiment is HepG
2(human liver cell cancer cells), HL60 (human promyelocytic leukemia), Bel-7402 (human liver cancer cell), HT-29 (human colon cancer cell), HeLa (human cervical carcinoma cell), A549 (human lung carcinoma cell), S180 (mouse ascites oncocyte) and HCCLM3 (people's height transfer liver cancer cell).
3) experimental technique HL-60, HT-29, Bel-7402, A549 and S180 cell selects RPMI-1640 substratum; HepG
2, HeLa and HCCLM3 cell selects DMEM substratum.In substratum all containing 10% through the foetal calf serum and 1 × 10 of deactivation
5u/L penicillin and 100mg/L Streptomycin sulphate.
Attached cell HepG2, the cultivation of HT-29, Bel-7402, A549, HeLa, HCCLM3 and half attached cell S180: respectively that growth conditions is good, is in the cell of logarithmic phase with 3 × 10
4the density of individual/mL is inoculated in 96 orifice plates, and every hole 100 μ L, is placed in 37 DEG C and 5%CO
2cell incubation case in cultivate 4 hours, then add compound 5a-c through sterilising treatment and the solution be mixed with containing the substratum of 0.1%DMSO by the concentration gradient preset, every hole 25 μ L, control group adds the solvent of isopyknic sample dissolution.Continue cultivation after 48 hours, every hole adds the MTT solution that 25 μ L concentration are 5mg/mL, is placed in 37 DEG C and 5%CO
2cell incubation case in cultivate 4 hours.After careful removing supernatant liquor, every hole adds the DMSO of 100 μ L, and about 10min dissolve purple of vibrating residue (first a ceremonial jade-ladle, used in libation), detects O.D. (absorbancy) value immediately in microplate reader, and wavelength is 570nm.
The cultivation of suspension cell HL60: respectively that growth conditions is good, is in the cell of logarithmic phase with 5 × 10
4the density of individual/mL is inoculated in 96 orifice plates, every hole 100 μ L, then adds by the concentration gradient preset the solution that the compound 5 through sterilising treatment is mixed with the substratum containing 0.1%DMSO, every hole 25 μ L, control group adds the solvent of isopyknic sample dissolution, is placed in 37 DEG C and 5%CO
2cell incubation case in cultivate 48 hours.Every hole adds the MTT solution that 25 μ L concentration are 5mg/mL, and continuing the condition that is placed in is 37 DEG C and 5%CO
2cell incubation case in cultivate 4 hours.The centrifugal 10min of 2500rpm, careful sucking-off supernatant liquor, every hole adds 100 μ LDMSO, and about 10min dissolve purple of vibrating residue (first a ceremonial jade-ladle, used in libation), detects O.D. (absorbancy) value immediately in microplate reader, and wavelength is 570nm.
The activity of compound 5 inhibition tumor cell propagation under each concentration is obtained by following formula:
Cell proliferation (%)=(the average O.D. value of compound 5a-c group average O.D. value/control group) × 100%, experiment repetition 3 times, maps to drug level with cell proliferation, obtains IC by graphing method
50(half effective inhibition concentration) value.
4) the results are shown in Figure 4 to Fig. 6.Result shows, in vitro in cytotoxicity test, have rated compound 5a-c only the high density of 100 and 200 μMs to Bel-7402, HepG
2, HL60, HT-29, HeLa, S180, A549 and HCCLM3 etc. 8 strain tumour cell show weak cytotoxicity.
The anti-tumor in vivo of experimental example 3 assessing compound 5a-c is active
1) physiological saline solution of compound 5a-c tween 80 of the present invention, Zorubicin physiological saline solution is as positive control, and the physiological saline of tween 80 is as negative control;
2) the equal gastric infusion of the physiological saline of compound 5a-c and tween 80, the dosage of compound 5a-c is 10nmol/kg, and the dosage of the physiological saline of tween 80 is 0.2mL/20g, successive administration 7 days, altogether administration 7 times; Zorubicin intraperitoneal administration, dosage is 2 μm of ol/kg, successive administration 7 days, altogether administration 7 times.
3) laboratory animal is ICR male mice (cleaning grade), body weight 20 ± 2g, often organizes 12 mouse.
4) knurl source is mouse S 180 sarcoma, purchased from Department Of Medicine, Peking University's animal experimental center, and maintenance of going down to posterity voluntarily.
5) extract and inoculate eugonic S180 ascitic tumor knurl liquid under animal model and treatment aseptic condition, the liquid of (1: 2) is become fully to mix with normal saline dilution, by freshly prepared 0.2% Trypan Blue of tumor cell suspension, by white blood cell count(WBC) method counting after mixing, contaminate blue person for dead cell, tinter is not viable cell, and is calculated as follows cell concn and cell survival rate.
Viable count/4 × 10 in the block plaid of cell concn=4
4× extension rate=cell count/mL
Cell survival rate=viable count/(viable count+dead cell number) × 100%
Knurl liquid homogenate method survival rate being greater than 90% is prepared into 2.0 × 10
7the cell suspension of individual/mL, in the subcutaneous vaccination of mouse armpit, 0.2mL/ only, manufactures S180 tumor-bearing mice.After tumor inoculation 24h, treatment group mouse oral administration of compound every day 5, dosage is 10nmol/kg.The physiological saline of naive mice oral 0.2mL tween 80 every day.Positive controls mouse abdominal injection every day Zorubicin, dosage is 2 μm of ol/kg.Experiment proceeds to the 8th day, claim Mouse Weight, etherization, de-cervical vertebra puts to death mouse, then fixes the right armpit tumor location of mouse with tweezers, cuts off skin, expose tumour, blunt separation, weighs, and is calculated as follows tumour inhibiting rate: the average knurl of tumour inhibiting rate %=(negative control group average knurl weight-administration group average knurl weight)/negative control group heavy × 100%.Experimental data adopts t inspection and variance analysis, knurl heavy with
represent.The results are shown in Table 1.As can be seen from Table 1, under the oral dosage of 10nmol/kg, the knurl of compound 5a-c treatment group mouse weighs tool significant difference compared with physiological saline group, does not have significant difference compared with Zorubicin group.Visible, the effective dose of compound 5a-c is lower than Zorubicin 200 times.
The anti-tumor in vivo of table 1 compound 5a-c is active
N=12; A) with physiological saline group than p < 0.01, with Zorubicin group than p > 0.05.
The extracorporeal anti-tumor cell adhesion activity of experimental example 4 assessing compound 5a-c
1) the DMEM substratum of compound 5a-c containing 0.1%DMSO is mixed with the solution that concentration is 100nM.
2) cell is HCCLM3 (high-transfer human liver cancer cell).
3) Fn (people's fibronectin).
4) experimental technique
With PBS, Fn is mixed with the solution that concentration is 100 μ g/mL, adds in 96 well culture plates by 100 μ L/ holes, culture plate is placed in 4 DEG C of refrigerator overnight.Next day, absorb and do not wrap by Fn solution, wash 1 time with PBS, every hole adds the PBS solution 30 μ L shrouding containing 2%FBS, at 37 DEG C and 5%CO
2incubator in hatch 3 hours, discard each hole solution.By good for growth conditions, be in the HCCLM3 cell of logarithmic phase with 5 × 10
4the density of individual/mL is inoculated in bag by 96 orifice plates of Fn, and every hole 100 μ L, adds the solution of 25 μ L compound 5a-c simultaneously, make its final concentration be 20nM, at 37 DEG C and 5%CO
2cultivate 2 hours in incubator, wash away the cell do not adhered to PBS, after discarding PBS, every hole adds the MTT solution that 25 μ L concentration are 5mg/mL, is placed in 37 DEG C and 5%CO
2hatch 4 hours in incubator, after careful removing supernatant liquor, every hole adds 100 μ LDMSO, and vibrate about 10min dissolution precipitation, detects O.D. (absorbancy) value immediately under microplate reader 570nm wavelength.The calculation formula of adherence inhibition rate is as follows: adherence inhibition rate (%)=[1-(the OD value of the OD value/blank group cell of compound 5a-c group cell)] × 100%; Experimental data statistics all adopts t inspection and variance analysis, and adherence inhibition rate represents with mean value ± SD.
5) the results are shown in Table 2.As can be seen from Table 2, compound 5a-c obviously suppresses HCCLM3 cell and FN to adhere under 20nM concentration, and adherence inhibition rate is 24.2%.Be 1 μM compared with suppressing SACC-LM cell with the effective concentration of ECM and platelet adhesion reaction with YIGSR disclosed in contriver, the effective concentration of compound 5a-c reduces 50 times.
The extracorporeal anti-tumor cell adhesion activity of table 2 compound 5a-c
n＝3。
The extracorporeal anti-tumor cell-invasive activity of experimental example 5 assessing compound 5a-c
1) compound 3 and the DMEM substratum of 5a-c containing 0.1%DMSO are mixed with the solution that concentration is 100nM.
2) cell is HCCLM3 (high-transfer human liver cancer cell).
3) matrigel is matrigel.
4) experimental technique
The frozen matrigel matrigel4 DEG C in-20 DEG C of refrigerators is spent the night, liquefy; Get 720 μ L plasma-free DMEM medium, add 180 μ LMatrigel, mixing, room on the polycarbonate membrane being added to Transwell cell, 100 μ L/, put into 37 DEG C and 5%CO
25h is hatched in incubator.Absorb residual liquid in cell, every hole adds 50 μ LDMEM substratum, 37 DEG C and 5%CO
230min is hatched in incubator.
After HCCLM3 cell dissociation, wash 3 times with plasma-free DMEM medium, counting, be made into cell suspension, density is 5 × 10
5individual/mL.Every hole adds 100 μ L cell suspensions, adds the solution that 25 μ L add 25 μ L compounds 3 or 5 simultaneously simultaneously, makes its final concentration be 20nM.Blank adds the solution that 25 μ L prepare containing the DMEM substratum of 0.1%DMSO.Lower room adds 600 μ L plasma-free DMEM medium, at 37 DEG C and 5%CO
2cultivate 48 hours in incubator.
Wipe the cell of matrigel and upper indoor with cotton swab after, with the paraformaldehyde fixed cell 30min of 4%.Absorb stationary liquid, wash 3 times with PBS, with the Viola crystallina dye liquor dyeing 30min of 0.1%.Absorb staining fluid, wash 3 times with PBS.
Choose 9 roughly the same visuals field at each cell to observe, take pictures, counting.Experimental data statistics all adopts t inspection and variance analysis, and the cell count of invasion and attack represents with mean value ± SD.
5) the results are shown in Table 3.Can find out, under 20nM concentration, compound 3 is compared with blank group, and the cell count that invasion and attack occur significantly reduces, and illustrates that compound 3 has the effect of obvious anti-HCCLM3 cell invasion under this condition.It can also be seen that, under 20nM concentration, compound 5a-c is compared with compound 3, and the cell count that invasion and attack occur also significantly reduces, and illustrates that the effect of compound 5a-c anti-HCCLM3 cell invasion is obviously than compound the last 3 under this condition.Compare suppressing the effective concentration of SACC-LM cell invasion to be 1 μM with RGDS, RGDV, RGDF disclosed in contriver, the effective concentration of compound 5a-c reduces 50 times.
The extracorporeal anti-tumor cell-invasive activity of table 3 compound 5a-c
N=9; A) with blank group and compound 3 groups than p < 0.01; B) with blank group than p < 0.01.
The extracorporeal anti-tumor cell migration of experimental example 6 assessing compound 5a-c is active
1) compound 3 and the DMEM substratum of 5a-c containing 0.1%DMSO are mixed with the solution that concentration is 100nM.
2) cell is HCCLM3 (high-transfer human liver cancer cell).
3) experimental technique
After HCCLM3 cell dissociation, wash 3 times with plasma-free DMEM medium, counting, be made into cell suspension, density is 2 × 10
6individual/mL.Every hole adds 100 μ L cell suspensions, adds the solution that 25 μ L add 25 μ L compounds 3 or 5a-c simultaneously simultaneously, makes its final concentration be 20nM.Blank adds the solution that 25 μ L prepare containing the DMEM substratum of 0.1%DMSO.Lower room adds 600 μ L plasma-free DMEM medium, at 37 DEG C and 5%CO
2cultivate 6 hours in incubator.
Wipe the cell of matrigel and upper indoor with cotton swab after, with the paraformaldehyde fixed cell 30min of 4%.Absorb stationary liquid, wash 3 times with PBS, with the Viola crystallina dye liquor dyeing 30min of 0.1%.Absorb staining fluid, wash 3 times with PBS.
Choose 9 roughly the same visuals field at each cell to observe, take pictures, counting.Experimental data statistics all adopts t inspection and variance analysis, and the cell count of invasion and attack represents with mean value ± SD.
4) the results are shown in Table 4.Can find out, under 20nM concentration, compound 3 is compared with blank group, and the cell count that invasion and attack occur significantly reduces, and illustrates that compound 3 has the effect of obvious anti-HCCLM3 cell invasion under this condition.It can also be seen that, under 20nM concentration, compound 5a-c is compared with compound 3, and the cell count that invasion and attack occur also significantly reduces, and illustrates that the effect of compound 5a-c anti-HCCLM3 cell invasion is obviously than compound the last 3 under this condition.Compare suppressing the effective concentration of SACC-LM cell invasion to be 1 μM with RGDS, RGDV, RGDF disclosed in contriver, the effective concentration of compound 5a-c reduces 50 times.
The extracorporeal anti-tumor cell migration of table 4 compound 5a-c is active
N=9; A) with blank group and compound 3 groups than p < 0.01.
The interior anti-inflammatory activity of experimental example 7 assessing compound 5a-c
1) experimental technique
18-22gICR male mice is divided into blank group, positive medication group and administration group at random, and tranquillization 1 day before mouse uses, operation room keeps room temp 22 DEG C, often organizes mouse 10.The left ear gabarit of single administration toward small white mouse after 30 minutes is coated with dimethylbenzene (0.03mL), is put to death by small white mouse cervical dislocation after 2 hours.By a left side for mouse, auris dextra is cut, and with the punch tool of diameter 7mm in the same position of two ears, gets circular auricle, weighs respectively, obtains the weight difference of two circle auricles as swelling.Swelling=former of left ear weight-auris dextra former weight.
2) medication and dosage
Gastric infusion.Blank is physiological saline, and dosage is 0.2mL/20g.Positive control is acetylsalicylic acid, and dosage is 1.11mmol/kg.The dosage of compound 5a-c is 10nmol/kg.
3) statistical method
Data statistics all adopts t to check and variance analysis, and swelling represents with mean value ± SDmg.
4) the results are shown in Table 5.Can find out, when dosage is 10nmol/kg, the mouse ear swelling degree of compound 5 treatment group has significant difference compared with NS group, illustrates that compound 5 can suppress mouse to be inflamed.There was no significant difference compared with the mouse ear swelling degree that when dosage is 10nmol/kg, mouse ear swelling degree and the dosage of compound 5 treatment group is the aspirin for treatment group of 1.11mmol/kg, illustrates that the anti-inflammatory activity of compound 5 is stronger than acetylsalicylic acid 111000 times.
The interior anti-inflammatory activity of table 5 compound 5a-c
N=12; A) with physiological saline group than p < 0.01 with acetylsalicylic acid group than p > 0.05.
The In Vitro Anti platelet aggregation activity of experimental example 8 assessing compound 5a-c
1) arachidonic acid (AA) is platelet aggregation
2) SD male rat (cleaning grade), body weight 250 ± 20g, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
3) method
Rat gets blood with after the urethane anesthesia of 20% through carotid artery, adds Sodium Citrate (9: 1, the v/v) anti-freezing of 3.8%, 1mL Trisodium Citrate is added in 9mL whole blood, with the rotating speed of 1000r/min centrifugal 10 minutes, get supernatant, be platelet rich plasma (PRP); By remaining whole blood with the rotating speed of 3000r/min centrifugal 10 minutes, get supernatant, be platelet poor plasma (PPP).
Open platelet aggregation instrument, its system temperature is adjusted to 37 DEG C, adjusting rotary speed is 1000 turns, preheating 30min.Get a glass tubule, add 240 μ LPPP, put into groove instrument being marked with PPP.Get a glass tubule again, put into rotor, add 240 μ LPRP, put into groove instrument being marked with PRP.
Open program, click " runnewpatient ", input sample ID, while with mouse hit OK key, left hand presses the dark buttons of the side on platelet aggregation instrument, can see that the curve in recorder is rising, when curve rises to zero time, left hand unclamps dark buttons, now can see As time goes on, curve is movement steadily near baseline, retouches the hematoblastic accumulation process of meter in real time.
Physiological saline is blank.Treat that baseline stability walks out about 1 minute, use that micro sample adding appliance is disposable adds 5 μ L physiological saline, curve can be observed continue again slowly to move in time near baseline after the rising occurring moment, after curve is steady, use the disposable AA solution adding 5 μ L and prepare of micro sample adding appliance, curve can be observed suddenly decline after the rising occurring moment, illustrate that thrombocyte there occurs gathering under the induction of AA.When curve drops to a certain degree, there is plateau, show that hematoblastic gathering reaches maximum value, after curve is stable, the record MA of thrombocyte within 6 minutes (instrument calculates automatically), replication 6 times, tries to achieve the mean value of platelet aggregation rate under AA effect;
Compound 5 is administration group.Prepare the PRP that a pipe is new, as above method puts into instrument, treat that baseline stability walks out about 1 minute, use the disposable normal saline solution adding the compound 5a-c that 5 μ L prepare of micro sample adding appliance, curve can be observed continue again slowly to move in time near baseline after the rising occurring moment, after curve is steady, use the disposable AA solution adding 5 μ L and prepare of micro sample adding appliance, curve can be observed start to decline after the rising occurring moment, illustrate that thrombocyte there occurs gathering under AA effect, when curve drops to a certain degree, there is plateau, under showing this condition, hematoblastic gathering reaches maximum value, after curve is stable, the record MA of thrombocyte within 6 minutes (instrument calculates automatically), replication 6 times, try to achieve after adding medicine, the mean value of the lower platelet aggregation rate of AA effect,
Computerized compound is to the inhibiting rate of the platelet aggregation that AA induces as follows: inhibiting rate=(physiological saline group aggregation rate-administration group aggregation rate)/physiological saline group aggregation rate × 100%; Platelet aggregation-against rate represents with mean value ± SD.
4) the results are shown in Table 6.Can find out, the platelet aggregation that when concentration is 20nM, compound 5a-c can suppress AA to induce significantly.
Table 6 compound 5a-c In Vitro Anti platelet aggregation
N=3; A) with physiological saline group than p < 0.01.
Antithrombotic acitivity in the body of experimental example 9 assessing compound 5a-c
1) experiment material
SD male rat (cleaning grade), body weight 200 ± 20g, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., is divided into 3 groups at random, often organizes 12 rats.Administering mode is gavage.Blank is physiological saline, and dosage is 0.6mL/200g; Positive control is acetylsalicylic acid, and dosage is 50 μm of ol/kg.The dosage of compound 5 is 10nmol/kg.
Experiment intubate is formed by 3 sections, middle segment length 8.0cm, internal diameter 0.3cm, two ends are identical polyethylene tube, long 10.0cm, internal diameter 0.1cm, external diameter 0.2cm, one end of this pipe pulls into point pipe (for inserting rat carotid artery or jugular vein), all uses the silicon ether silanization of 1% before the inwall use of 3 sections of pipes.The silk thread of the long 6.0cm weighed in advance is put into stage casing polyethylene extra heavy pipe, and the two ends of extra heavy pipe are nested with the non-drawing-down end of two polyethylene tubules respectively (wherein one section silk thread is pushed down 0.5mm fix).For subsequent use by filling heparin-saline solution (50IU/kg) in pipe by sharp pipe end with syringe.
2) model and treatment
Gavage gives the physiological saline that dosage is 0.6mL/200g, or dosage is the acetylsalicylic acid of 50 μm of ol/kg, or dosage is after the compound 5a-c30min of 10nmol/kg, by rat with 20% urethane solution (6mL/kg, i.p.) anaesthetize.Anesthetized rat dorsal position is fixed, isolate the left side external jugular vein of rat, proximal part and distal end penetrate surgical thread respectively, ligation distal end, the left external jugular vein exposed cuts an angle carefully, the non-line ball end point pipe of the intubate prepared is inserted the proximal part of left external jugular vein opening by angle, pushed the heparin-saline (50IU/kg) of correct amount by the sharp pipe of the other end with syringe, now syringe does not withdraw polyethylene tube, be separated right carotid, in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, ligation distal end, right common carotid artery is being cut an angle carefully nearby from bulldog clamp.Extract syringe from the tip of polyethylene tube, the tip of polyethylene tube is inserted the proximal part of artery angle.The two ends of bypass duct all use 4 trumpeter's art suture ligatures to fix.Open bulldog clamp, make blood flow flow to jugular vein by bypass duct from carotid artery.From circulation time timing, take out from bypass duct after 15min and hang with the silk thread of thrombus, accurately weigh, the wet weight of thrombus that is of poor quality before and after silk thread represents with mean value ± SDmg.Data statistics all adopts t to check and variance analysis.
3) the results are shown in Table 7.Can find out, the wet weight of thrombus of compound 5a-c treatment group rat is significantly less than the wet weight of thrombus with saline rats, illustrates that compound 5a-c shows anti-arterial thrombus and generates active while neoplasm growth.
Antithrombotic acitivity in the body of table 7 compound 5a-c
N=10; A) p < 0.01 is compared with NS group.
Claims (7)
1. structure is 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val of following formula.
2. 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser of claim 1, the preparation method of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val, the method is made up of following steps:
(1) under the vitriol oil exists, 5-formylsalicylic acid is 90 DEG C, microwave reaction 2h in methyl alcohol, generates 5-formylsalicylic acid methyl esters;
(2) under the existence of polyphosphoric acid, L-Trp and phenylcarbinol reaction, generate L-Trp benzyl ester;
(3) under trifluoracetic acid exists, in methylene dichloride, 5-formylsalicylic acid and the ester condensation of L-Trp benzyl are 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-1,2,3,4-tetrahydro-beta-carboline-3-benzyl carboxylates;
(4) 2,3-bis-chloro-5,6-dinitrile-1, under the existence of 4-benzoquinones (DDQ), 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-1,2,3,4-tetrahydro-beta-carboline-3-benzyl carboxylate is oxidized to 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-benzyl carboxylate in tetrahydrofuran (THF) (THF);
(5) at Pd/C and H
2under existence, in methyl alcohol, the reaction of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-benzyl carboxylate generates 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid;
(6) adopt progressively condensation method, under DCC and HOBt exists, react in dry THF, obtain full guard peptide sequence Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
(7) under ice bath hydrogenchloride ethyl acetate solution (4M) in, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl removes Boc respectively and obtain Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
(8) DCC and HOBt exist under, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-carboxylic acid in anhydrous THF respectively with Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl generation 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
(9) at Pd/C and H
2under existence, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO in methyl alcohol
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl generates compound 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser respectively, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val.
3. the nanostructure of 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val of claim 1.
4. 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val of claim 1 is preparing the application in antitumor drug.
5. 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser of claim 1,1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val is preparing antitumor cell migration, adheres to and attacks the application in medicine.
6. 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val of claim 1 is preparing the application in anti-inflammatory medicaments.
7. 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Ser, 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Phe and 1-(4-hydroxyl-3-methoxycarbonyl phenyl)-β-carboline-3-formyl-Arg-Gly-Asp-Val of claim 1 is preparing the application in antithrombotic reagent.
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