CN102516351A - Ursolic acid derivative with anti-cancer activity and preparation method thereof - Google Patents
Ursolic acid derivative with anti-cancer activity and preparation method thereof Download PDFInfo
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Abstract
The invention provides an ursolic acid derivative, which is N-[3beta-acetoxy-ursa-12- alkene-28-acyl]-aminoethylenediamine-folic acid. A natural product ursolic acid is structurally transformed to obtain N-[3beta-acetoxy-ursa-12- alkene-28-acyl]-aminoethylenediamine-folic acid (I), N-[3beta-acetoxy-ursa-12-alkene-28-acyl]-aminoethylenediamine (III) and N-[3beta-acetoxy-ursa-12- alkene-28-acyl]-aminoethylenediamine (IV). The in vitro cell proliferation inhibition test shows that the proliferation inhibition effect of the compounds I, III and IV on tested tumor cell lines HepG2, of BGC823, A-375, Hela and the like is superior to that of the ursolic acid, and the toxic effect of the compounds I, III and IV on human embryonic lung fibroblasts (HELF) is lower than that of the ursolic acid. The in vitro anti-tumor effect of the compound III is the best, and the compound I has certain targeting.
Description
Technical field
The present invention relates to a kind of ursolic acid verivate and preparation method thereof, is a kind of ursolic acid verivate with anti-tumor activity and preparation method thereof specifically.
Background technology
Ursolic acid (Ursolic acid UA), has another name called ursonic acid, urson, belongs to pentacyclic triterpenoid, chemical name (3 β)-3-Hydroxyurs-12-en-28-oic acid, and its relative molecular weight is 456.68, molecular formula is C
30H
48O
3, it is distributed widely in occurring in nature with the form of free or glucosides.Pharmaceutical research is found; Ursolic acid has biological effect widely, and ursolic acid has the various biological activity, and is antibacterial etc. like anticancer, anti-HIV, anti-liver injury, antimalarial, anti-inflammatory; Wherein remarkable with antitumour activity especially; Its anticancer spectrum is wide, not only multiple carcinogenic, short cancer thing is had resistant function, and to the kinds of tumor cells body is inside and outside restraining effect is arranged all.Its spinoff is little, and toxicity is low, demonstrates bigger clinical application potentiality.
The chemical structural formula of ursolic acid is:
At present, the research of ursolic acid is reported for work focuses mostly on aspect the separation-extraction technology and pharmaceutical research of ursolic acid both at home and abroad, and less to the structural modification aspect research of ursolic acid, for the research of target property is especially less.Because the remarkable antitumous effect of ursolic acid makes that this research on the one hand is comparatively deep.As Lin Fengping etc. " ursolic acid verivate synthetic, characterize and active " to the inhibition of cancer cells (" applied chemistry ", 2010,27,893-898); " The synthesis of ursolic acid derivatives with cytotoxic activity and the investigation of their preliminary mechanism of action " (Bioorg. Med. Chem. Lett. 2009 of Meng Y. Q. etc.; 17,848 – 854); " the Betulin and ursolic acid synthetic derivatives as inhibitors of Papilloma virus " of Oxana B. etc. (Bioorg. Med. Chem. Lett. 2010,20,4088-4090).Most investigators think that ursolic acid can wait generation, invasion and the transfer that suppresses tumour through chemoprophylaxis, mutation, CDCC, antineoplastic vascular generation and inducing tumor cell differentiation, apoptosis.
Research for the ursolic acid structural modification mainly contains both direction, and one is improve ursolic acid water-soluble, and another is the antitumour activity that improves ursolic acid.Yu Zhou etc. are through carrying out chemically modified to 3 and 28 of ursolic acid, and preparation ursolic acid-trolamine verivate has ursolic acid water-soluble and must improve (" medicine biotechnology ", 2010,17 (5): 382-385); It is lower that the ursolic acid A ring open loop structure that Xia Yan etc. propose design is voluntarily modified the route cost, and the reagent low toxicity is for the further investigation of ursolic acid structural modification thing provides basis (" herbal medicine ", 2011,42 (1): 34-37); Meng Yanqiu etc. are lead compound with the natural product ursolic acid; 16 ursolic acid verivates have been synthesized through reactions such as peroxo-, acidylate, esterification, hydrolysis design; Wherein compound 7a etc. has good anticancer activity (" Acta Pharmaceutica Sinica ", 2011,46 (5): 556-560).Jing-Wei Shao etc. modify 3 of ursolic acid and 28, preparation ursolic acid-piperazine and ursolic acid-ethanolamine derivative, its antitumour activity (the Eur. J. Med. Chem. 2011,46 (7): 2652-61) that is significantly improved.Although above-mentioned ursolic acid structural modification thing has shown good prospects for application, also exist target property clear and definite inadequately, promptly lack the limitation of the targeting specific of cancer cells.Therefore for reducing its toxicity and the affinity that increases target organ to healthy tissues, people processed targeted drug with antitumor drug in recent years.Wherein receive extensive concern, become the novel targets of diagnosing tumor and treatment research, be with a wide range of applications with folacin receptor mediated tumor-targeting drug.At present be lead compound with the ursolic acid and being connected of folic acid, the synthetic target Journal of Sex Research that reaches of this compounds is not all seen its report both at home and abroad.
Summary of the invention
The objective of the invention is with the ursolic acid is lead compound, synthetic a kind of ursolic acid verivate with anti-tumor activity and preparation method thereof.
The invention provides a kind of ursolic acid verivate, said ursolic acid verivate is N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid, and structural formula is suc as formula shown in the I:
Its preparation method is following:
(1) ursolic acid and acetic anhydride reaction generates 3-O-ethanoyl ursolic acid; The structural formula of 3-O-ethanoyl ursolic acid is suc as formula shown in the II,
Ⅱ;
(2) 3-O-ethanoyl ursolic acid and oxalyl chloride reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol with reacting ethylenediamine again, and structural formula is suc as formula shown in the III,
(3) N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol and folic acid reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid, and structural formula is suc as formula shown in the I.
Said preparing method's concrete steps are following:
The ursolic acid stirring and dissolving of (1) 1 weight part is added dropwise to the acetic anhydride of 2-3 weight part in the pyridine of 40-50 weight part, add the DMAP of 0.001-0.02 weight part, stirs 16-18 h under the room temperature; After reaction finishes, regulate pH 3-4 with 2 N HCl, steaming desolventizes, suction filtration; The washing filter cake is extremely neutral, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol cools off recrystallization, gets the 3-O-ethanoyl ursolic acid of white powder;
(2) 1 weight part 3-O-ethanoyl ursolic acid are dissolved in 30-35 weight part CH
2Cl
2In, dropwise add the oxalyl chloride of 1-2 weight part, stirring reaction 12-24h under the room temperature steams the gas that desolventizes and react generation, the bullion of 3-O-ethanoyl UA acyl chlorides midbody, directly get into next step reaction; The CH that in the bullion of above-mentioned midbody, adds the 20-30 weight part
2Cl
2, triethylamine regulator solution pH 8-9 adds the quadrol of 1-2 weight part, stirring reaction 12-24h under the room temperature after reaction finishes, adds the water of 5-10 weight part in the reaction solution, regulate pH3-4 with 2N HCl, filter, the washing filter cake to the pH that filtrates for neutral, drying; Appearance on the dried product dry method, column chromatography purification, the absolute ethyl alcohol thermosol, the cooling recrystallization gets white powder N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol;
(3) 1 weight part N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol is dissolved in the anhydrous dimethyl sulphoxide of 20-30 weight part; The folic acid that adds the 1-2 weight part; Stirring at room 30-60min; Add 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride of 1-2 weight part, the N-maloyl imines of 1-2 weight part then, room temperature lucifuge reaction 20-30 h; After reacting end, steaming desolventizes, and adds the water of 5-10 weight part then, regulates pH3-4 with 2N HCl, filters, and the washing filter cake is a neutrality to the pH that filtrates, drying; Appearance on the dried product dry method, the reversed-phase column purifying, the absolute ethyl alcohol thermosol, the cooling recrystallization gets yellow powder shape N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid.
The moving phase that the said column chromatography purification of above-mentioned steps (1) adopts is sherwood oil: ETHYLE ACETATE=3:1; The moving phase that the said column chromatography purification of step (2) adopts is sherwood oil: ETHYLE ACETATE=1:5; The moving phase that the said reversed-phase column purifying of step (3) adopts is methyl alcohol: water=8:1.
The present invention also provides a kind of ursolic acid verivate, and said ursolic acid verivate is N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol, and structural formula is suc as formula shown in the IV:
Its preparation method is following:
(1) ursolic acid and acetic anhydride reaction generates 3-O-ethanoyl ursolic acid; The structural formula of 3-O-ethanoyl ursolic acid is suc as formula shown in the II;
(2) 3-O-ethanoyl ursolic acid and oxalyl chloride reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol with reacting ethylenediamine again, and structural formula is suc as formula shown in the III;
(3) N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol and NaOH hydrolysis reaction obtains N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol, and structural formula is suc as formula shown in the IV.
Said preparing method's concrete steps are following:
The ursolic acid stirring and dissolving of (1) 1 weight part is added dropwise to the acetic anhydride of 2-3 weight part in the pyridine of 40-50 weight part, add a spot of DMAP, stirs 16-18 h under the room temperature; After reaction finishes, transfer pH 3-4 with 2 N HCl, steaming desolventizes, suction filtration; The washing filter cake is extremely neutral, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol cools off recrystallization, gets the 3-O-ethanoyl ursolic acid of white powder;
(2) 1 weight part 3-O-ethanoyl ursolic acid are dissolved in 30-35 weight part CH
2Cl
2In, in batches, dropwise add the oxalyl chloride of 1-2 weight part, stirring reaction 12-24 h under the room temperature steams the gas that desolventizes and react generation, the bullion of 3-O-ethanoyl UA acyl chlorides midbody, directly get into next step reaction; The CH that in above-mentioned midbody, adds the 20-30 weight part
2Cl
2, triethylamine regulator solution pH 8-9, the quadrol of adding 1-2 weight part, stirring reaction 12-24 h under the room temperature; After reaction finishes, add the water of 5-10 weight part in the reaction solution, transfer pH3-4, filter with 2N HCl; The washing filter cake is neutrality to the pH that filtrates, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol, the cooling recrystallization gets white powder N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol;
(3) take by weighing N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol of 1-2 weight part, be dissolved in the CH of 20-30 weight part
3Among the OH-THF, said CH
3The volume ratio of OH and THF is 1:1.5-2.5; The 4N NaOH that adds the 4-8 weight part, stirring reaction 3-5h under the room temperature, the water of adding 5-10 weight part in reaction solution, 2 N HCl regulate pH 3-4, remove solvent under reduced pressure, and water washing and precipitating is to neutrality, drying; Appearance on the dried product dry method, column chromatography purification, the absolute ethyl alcohol thermosol, the cooling recrystallization gets N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol.
The moving phase that the said column chromatography purification of above-mentioned steps (1) adopts is sherwood oil: ETHYLE ACETATE=3:1, and the moving phase that step (2), (3) said column chromatography purification adopt is sherwood oil: ETHYLE ACETATE=1:5.
Said quadrol replaces with tn, tetramethylenediamine, pentamethylene diamine or hexanediamine, obtains the ursolic acid verivate of a series of correspondences.
The ursolic acid verivate of above-mentioned preparation has anti-tumor activity, is used for preparing anti-tumor medicine.
Below be the synthetic route of N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid (chemical compounds I), N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol (compound III) and N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol (compound IV):
The invention has the advantages that; The present invention carries out structure of modification to the natural product ursolic acid; Get N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid (I), N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol (III) and N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol (IV); Body outer cell proliferation suppresses experiment and shows; Chemical compounds I, III and IV are superior to ursolic acid to the inhibited proliferation of survey tumor cell line HepG2, BGC823, A-375, Hela etc., and the toxic action of HELF HELF then is lower than ursolic acid.Wherein the extracorporeal anti-tumor effect of compound III is best, and chemical compounds I has certain target property.
Embodiment
Below for practical implementation example of the present invention and comparative example can further be well understood to the present invention, but they are not to qualification of the present invention:
Embodiment 1
The preparation of 3-O ethanoyl ursolic acid (compound ii):
Take by weighing 0.7062 g ursolic acid, add 30 mL pyridines, be added dropwise to 1.8 mL acetic anhydride after the stirring and dissolving, add a spot of DMAP; Stir 16 h under the room temperature, after reaction finishes, transfer pH 3-4 with 2 N HCl, steaming desolventizes; Suction filtration, the washing filter cake is extremely neutral, 100 ℃ of dryings of normal pressure, appearance on the dried product dry method; Column chromatography purification (sherwood oil (60-90 ℃): ETHYLE ACETATE=3:1), and the absolute ethyl alcohol thermosol, the cooling recrystallization gets 3-O ethanoyl ursolic acid.
Proterties: white powder; Productive rate: 86.17%.
IRData (KBr, cm
-1) and ownership: υ: 3293 (O-H stretching vibrations), 2927 (C-H stretching vibrations), 1736 (s, C=O stretching vibrations), 1370 (UA A district characteristic absorbance), 1245 (s, C-O-C asymmetrical stretching vibration) cm
-1
?ESI-MS:m/z?497.5?[M-H]
+,521.5[M+Na]
?+。
1H?NMR(400MHz,CDCl
3)δ:5.23(t,
J=2.8Hz,1H,H-12),4.49(t,
J=7.2Hz,?1H,H-3),2.17(d,
J=10.8Hz,?1H,H-18),2.04(s,3?H,CH
3COO),1.06(s,3?H,CH
3),0.97(s,3H,?CH
3),0.94(s,3?H,CH
3),0.92(s,3H,CH
3),0.82(d,?
J=6.4Hz,?3?H,CH
3),0.78(s,3?H,CH
3),0.76(s,3?H,CH
3)。
Embodiment 2
The preparation of N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol (compound III):
Take by weighing 0.1954 g 3-O ethanoyl ursolic acid and be dissolved in 20 mL CH
2Cl
2In, in batches, dropwise adding oxalyl chloride 0.19 mL, stirring reaction 24 h under the room temperature steam the gas that desolventizes and react generation, get the bullion of 3-O-ethanoyl UA acyl chlorides midbody, directly get into next step reaction.In above-mentioned midbody, add 20 mL CH
2Cl
2, triethylamine regulator solution pH 8-9 adds quadrol 0.19 mL, and stirring reaction 3 h under the room temperature after the reaction end, add 10 mL zero(ppm) water in the reaction solution, transfer pH3-4 with 2N HCl, filter, and the washing filter cake is neutral to the pH that filtrates, drying.Column chromatography purification (chloroform: acetone (v:v)=1:5), the absolute ethyl alcohol thermosol, the cooling recrystallization gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol.
Proterties: white powder; Productive rate: 78.2%.
IRData (KBr, cm
-1) and ownership: υ: 3395 (NH
2Stretching vibration), 2927 (C-H stretching vibrations), 1735 (s, C=O stretching vibrations); 1640 (s, C=O stretching vibration, acid amides I peaks), 1526 (N-H flexural vibration; Acid amides II peak), 1370 (UA A district characteristic absorbance), 1246 (C-O-C stretching vibrations).
?ESI-MS:m/z?541.5?[M-H]
+。
?1H?NMR(400MHz,?CDCl
3)δ:?5.34(t,?J=2.8?Hz,?1?H,?H-12),?4.49?(t,?J?=?6.0?Hz,?1?H,?H-3),?3.47?(m,?J=5.6?Hz,?2H,NHC
H 2),?3.16?(m,?J=5.2?Hz,?2H,?C
H 2NH
2),?2.91?(t,?J=5.6?Hz,?2H,?N
H 2),?2.17?(d,?1?H,?J=3.6?Hz,?H-18),?2.05?(s,?3H,?CH
3COO),?1.25?(s,?3?H,?CH
3),?1.09?(s,?3?H,?CH
3),?0.97-0.93?(m,?6H,?2×CH
3),?0.89-0.85?(m,?9H,?3×CH
3),?0.78?(s,?1?H?CH
3).
Embodiment 3
The preparation of N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol (compound IV):
Take by weighing N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol 0.5859g, be dissolved in 20 ml CH
3Among the OH-THF (1:1.5 v:v), add 2.0ml 4N NaOH, stirring reaction 3.5 h under the room temperature; React completely, in mixture, add an amount of water, 2 N HCl transfer pH 3-4; Remove solvent under reduced pressure, water washing and precipitating is extremely neutral, and column chromatography purification (chloroform: acetone=1:5); The absolute ethyl alcohol thermosol, the cooling recrystallization gets N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol.
Proterties: white powder; Productive rate: 85.7%.
IRData (KBr, cm-1) and the ownership: υ: 3384 (O-H stretching vibrations), 2926 (C-H stretching vibrations), 1636 (s, C=O stretching vibration, acid amides I peaks), 1528 (N-H flexural vibration, acid amides II peaks), 1378 (UA A district characteristic absorbance).
?ESI-MS:m/z?499.1?[M-H]
+。
?1H?NMR(400MHz,?CDCl
3)δ:?5.31(t,?J=2.8?Hz,?1?H,?H-12),?4.32?(m,?J?=?5.6?Hz,?1?H,?H-3),?3.49?(m,?J=6.0?Hz,?2H,NHC
H 2),?3.17?(m,?J=5.2?Hz,?2H,?C
H 2NH
2),?2.92?(t,?J=5.2?Hz,?2H,?N
H 2),?2.17?(d,?1?H,?J=4.8?Hz,?H-18),?1.24?(s,?3?H,?CH
3),?1.10?(s,?3?H,?CH
3),?0.97-0.93?(m,?6H,?2×CH
3),?0.89-0.85?(m,?9H,?3×CH
3),?0.78?(s,?1?H?CH
3).
Embodiment 4
The preparation of N-[3 β-acetyl oxygen-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid (chemical compounds I):
Taking by weighing N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol 0.203g is dissolved among the 20ml DMSO; Add 0.219g folic acid; Stirring at room 30 min; Add 0.09 g EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), 0.082g NHS (N-maloyl imines) then, the room temperature lucifuge is reacted 20 h.After the reaction end, desolventize with the oil pump steaming, add 20ml water then, regulate pH3-4 with 2N HCl, filter, the washing filter cake is a neutrality to the pH that filtrates, drying; Appearance on the dried product dry method, and the reversed-phase column purifying (methyl alcohol: water=8:1), and the absolute ethyl alcohol thermosol, the cooling recrystallization makes N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid.
Proterties: orange-yellow powder; Productive rate: 49.5%.
IRData (KBr, cm-1) and the ownership: υ: 3328 (NH
2Stretching vibration), 2928 (C-H stretching vibrations), 1733 (s, C=O stretching vibrations); 1640 (s, C=O stretching vibration, acid amides I peaks), 1516 (N-H flexural vibration; Acid amides II peak), 1371 (UA A district characteristic absorbance), 1245 (C-O-C stretching vibrations).
?ESI-MS:m/z?963.3?[M-H]
+。
1 H?NMR(400MHz,?CDCl
3)δ:7.73-7.70?(m,?J=3.6?Hz,?2?H,?2×Ar-H?),?7.55-7.52?(m,J=3.2?Hz,?2?H,2×Ar-H),5.30?(t,?J=3.2?Hz,?1?H,?H-12),?4.49?(m,?J?=?7.2?Hz,?1?H,?C
HCOOH),?4.31?(d,J=6.0?Hz,?2?H,Ar-NH-C
H 2),?4.22?(t,?J?=?5.6?Hz,?1?H,?H-3),?3.48?(m,?J=7.2?Hz,?2?H,C
H 2NH),?2.89?(t,?J=2.0?Hz,?2?H,?C
H 2CHCOOH),?2.62?(m,?9?H,?3×CH
3),?2.17?(d,?1?H,?J=4.0?Hz,?H-18),?2.05?(s,?3?H,?CH
3COO),?1.25?(s,?3?H,?CH
3),?1.09?(s,?3?H,?CH
3),?0.98-0.94?(m,?6H,?2×CH
3),?0.89-0.85?(m,?9H,?3×CH
3),?0.76?(s,?1?H?CH
3).
Embodiment 5
The external antitumour activity of ursolic acid (UA) and verivate thereof (chemical compounds I, II, III, IV)--the inhibited proliferation of mtt assay detection of drugs pair cell
Get and be in one bottle in logarithmic phase cell in good condition, tryptic digestion processes 5 * 10
4The cell suspension of individual/mL.
Cell suspension moves into 96 orifice plates, and every hole 100 μ L make a circle and fill with PBS in week, put 37 ℃, 5% CO
2Cultivate 24 h in the incubator.
removes old substratum; Adding is tried verivate, and (will be tried verivate storage liquid with substratum dilutes; Set different effects concentration: 5,10,15,20,25,30 μ mol/L); Every hole 100 μ L; Other establishes blank group, UA control group (UA 60 μ mol/L or 20 μ mol/L) and taxol control group (60 μ mol/L or 20 μ mol/L), establishes 6 multiple holes for every group.Behind drug effect 24 h, inhale and abandon the pastille substratum, in every hole, add serum-free, no phenol red 1640 substratum, 100 μ L, add MTT solution 10 μ L again, continue to hatch 4 h, stop cultivating.
The careful suction abandoned supernatant in 96 orifice bores; Every hole adds 150 μ L DMSO; 10 min vibrate; On ELIASA, measure each hole absorbance value (OD value) in 490 nm wavelength, calculate the proliferation inhibition rate of cell: inhibiting rate (%)=(the average OD value of the average OD value of 1-medication group ÷ blank group) * 100%, use the half-inhibition concentration (IC that SPSS16.0 software carries out data processing and calculates cancer cell multiplication
50), the result sees table 1.
Shown in table 1 result, institute's synthetic UA verivate all has certain inhibited proliferation to different tumour cells, and wherein the restraining effect of chemical compounds I, III and IV is better than UA, and they also are lower than UA to Normocellular toxic side effect.Wherein, the antitumour activity data of chemical compounds I can find out, to the IC of the Hela cell of folacin receptor high expression level
50Be starkly lower than other cancer cells (HepG2, BGC, A-375), explain that this compound has certain target property; Compare with other compound, chemical compounds I is to the IC of normal cell (HELF cell)
50Be worth very highly, explain that chemical compounds I has hypotoxicity.In a word, chemical compounds I, III and IV have the active potentiality of good anticancer.
Table 1 ursolic acid verivate is to the inhibited proliferation of different tumour cells
mean(±SD?fold,?n≥6),compared?with?UA?,*
P<0.05;**
P<0.01
Claims (8)
1. ursolic acid verivate, it is characterized in that: said ursolic acid verivate is N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid, and structural formula is suc as formula shown in the I:
Ⅰ。
2. the preparation method of a ursolic acid verivate as claimed in claim 1 is characterized in that:
(1) ursolic acid and acetic anhydride reaction generates 3-O-ethanoyl ursolic acid; The structural formula of 3-O-ethanoyl ursolic acid is suc as formula shown in the II,
Ⅱ;
(2) 3-O-ethanoyl ursolic acid and oxalyl chloride reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol with reacting ethylenediamine again, and structural formula is suc as formula shown in the III,
(3) N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol and folic acid reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid, and structural formula is suc as formula shown in the I.
3. the preparation method of ursolic acid verivate according to claim 2 is characterized in that: said preparing method's concrete steps are following:
The ursolic acid stirring and dissolving of (1) 1 weight part is added dropwise to the acetic anhydride of 2-3 weight part in the pyridine of 40-50 weight part, add the DMAP of 0.001-0.02 weight part, stirs 16-18 h under the room temperature; After reaction finishes, regulate pH 3-4 with 2 N HCl, steaming desolventizes, suction filtration; The washing filter cake is extremely neutral, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol cools off recrystallization, gets the 3-O-ethanoyl ursolic acid of white powder;
(2) 1 weight part 3-O-ethanoyl ursolic acid are dissolved in 30-35 weight part CH
2Cl
2In, dropwise add the oxalyl chloride of 1-2 weight part, stirring reaction 12-24h under the room temperature steams the gas that desolventizes and react generation, the bullion of 3-O-ethanoyl UA acyl chlorides midbody, directly get into next step reaction; The CH that in the bullion of above-mentioned midbody, adds the 20-30 weight part
2Cl
2, triethylamine regulator solution pH 8-9 adds the quadrol of 1-2 weight part, stirring reaction 12-24h under the room temperature after reaction finishes, adds the water of 5-10 weight part in the reaction solution, regulate pH3-4 with 2N HCl, filter, the washing filter cake to the pH that filtrates for neutral, drying; Appearance on the dried product dry method, column chromatography purification, the absolute ethyl alcohol thermosol, the cooling recrystallization gets white powder N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol;
(3) 1 weight part N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol is dissolved in the anhydrous dimethyl sulphoxide of 20-30 weight part; The folic acid that adds the 1-2 weight part; Stirring at room 30-60min; Add 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride of 1-2 weight part, the N-maloyl imines of 1-2 weight part then, room temperature lucifuge reaction 20-30 h; After reacting end, steaming desolventizes, and adds the water of 5-10 weight part then, regulates pH3-4 with 2N HCl, filters, and the washing filter cake is a neutrality to the pH that filtrates, drying; Appearance on the dried product dry method, the reversed-phase column purifying, the absolute ethyl alcohol thermosol, the cooling recrystallization gets yellow powder shape N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid.
5. the preparation method of a ursolic acid verivate as claimed in claim 4 is characterized in that:
(1) ursolic acid and acetic anhydride reaction generates 3-O-ethanoyl ursolic acid; The structural formula of 3-O-ethanoyl ursolic acid is suc as formula shown in the II;
(2) 3-O-ethanoyl ursolic acid and oxalyl chloride reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol with reacting ethylenediamine again, and structural formula is suc as formula shown in the III;
(3) N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol and NaOH hydrolysis reaction obtains N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol, and structural formula is suc as formula shown in the IV.
6. the preparation method of ursolic acid verivate according to claim 5 is characterized in that: said preparing method's concrete steps are following:
The ursolic acid stirring and dissolving of (1) 1 weight part is added dropwise to the acetic anhydride of 2-3 weight part in the pyridine of 40-50 weight part, add a spot of DMAP, stirs 16-18 h under the room temperature; After reaction finishes, transfer pH 3-4 with 2 N HCl, steaming desolventizes, suction filtration; The washing filter cake is extremely neutral, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol cools off recrystallization, gets the 3-O-ethanoyl ursolic acid of white powder;
(2) 1 weight part 3-O-ethanoyl ursolic acid are dissolved in 30-35 weight part CH
2Cl
2In, in batches, dropwise add the oxalyl chloride of 1-2 weight part, stirring reaction 12-24 h under the room temperature steams the gas that desolventizes and react generation, the bullion of 3-O-ethanoyl UA acyl chlorides midbody, directly get into next step reaction; The CH that in above-mentioned midbody, adds the 20-30 weight part
2Cl
2, triethylamine regulator solution pH 8-9, the quadrol of adding 1-2 weight part, stirring reaction 12-24 h under the room temperature; After reaction finishes, add the water of 5-10 weight part in the reaction solution, transfer pH3-4, filter with 2N HCl; The washing filter cake is neutrality to the pH that filtrates, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol, the cooling recrystallization gets white powder N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol;
(3) take by weighing N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol of 1-2 weight part, be dissolved in the CH of 20-30 weight part
3Among the OH-THF, said CH
3The volume ratio of OH and THF is 1:1.5-2.5; The 4N NaOH that adds the 4-8 weight part, stirring reaction 3-5h under the room temperature, the water of adding 5-10 weight part in reaction solution, 2 N HCl regulate pH 3-4, remove solvent under reduced pressure, and water washing and precipitating is to neutrality, drying; Appearance on the dried product dry method, column chromatography purification, the absolute ethyl alcohol thermosol, the cooling recrystallization gets N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol.
7. one kind like claim 2 or 5 described ursolic acid verivates, it is characterized in that: said quadrol replaces with tn, tetramethylenediamine, pentamethylene diamine or hexanediamine, obtains the ursolic acid verivate of a series of correspondences.
8. one kind like claim 1 or 4 described ursolic acid verivates or by the application of ursolic acid verivate in the preparation antitumor drug of claim 2,3,5 or 6 said preparing methods' preparations.
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CN107312057A (en) * | 2017-06-13 | 2017-11-03 | 佛山科学技术学院 | A kind of conjugate containing cancer cell targeted molecular and its production and use |
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