CN102260315A - Ursolic acid derivatives with anticancer activity and preparation method thereof - Google Patents
Ursolic acid derivatives with anticancer activity and preparation method thereof Download PDFInfo
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Abstract
The invention relates to ursolic acid derivatives with anticancer activity and a preparation method thereof. In the method, a carbonoxyl group at the C-28 position of ursolic acid is connected with natural alpha-amino acid through ethanediamine in a mode of forming an amido bond to synthesize a series of ursolic acid derivatives with anticancer activity. In-vitro pharmacological experiments indicate that the ursolic acid derivatives chemically modified by amino acid have obvious in-vitro inhibition effect on human liver cancer HepG2 cells, human colon cancer HT-29 cells, human gastric cancer AGS and BGC-823cells, and human prostatic cancer PC-3 cells.
Description
Technical field
The present invention is specifically related to a kind of ursolic acid derivative with anticancer activity and preparation method thereof.
Background technology
At present, domestic research report to ursolic acid focuses mostly at the extraction and separation process and the pharmaceutical research of ursolic acid, particularly both at home and abroad antitumous effect research of ursolic acid is appeared in the newspapers.As Wang Tao etc. " ursolic acid antitumor action and Mechanism Study progress thereof " (" medicine biotechnology ", 2008,15[2]); " the pharmacological progress of ursolic acid " of Xiong Bin etc. (" foreign medical science pharmacy fascicle ", 2004,31[[3]); " the ursolic acid antitumor action progress " of Xia Guohao etc. (" foreign medical oncology credit volume ", 2002,29[6]); Or the like.Studies show that ursolic acid has anti-start sudden change, anti-promoting effect, antioxygenation, cytotoxicity, inducing cancer cell differentiation, blood vessel formation against function, cell death inducing.Domestic Bai Yujun etc. has reported 6 kinds of ursolic acid parent nucleus modifiers (West China Journal of Pharmaceutical Sciences, 2003,18[2]); Yang Dingju etc. have reported the polyglycol supported ursolic acid (" China Medicine University's college journal ", 2008,39[1]) of biologically active, and have delivered patent " hydrophilic polyglycol supported ursolic acid medicines and preparation method thereof "; Meng Yanqiu etc. have developed multinomial patents such as series derivates such as ursolic acid modified amino acid, amino alcohol, amine, heterocycle.Yet, when adopting amino acid modified ursolic acid in existing patent or the document, all adopt the mode that the C-28 position carboxyl of ursolic acid is become amido linkage with the alpha-amino group of natural a-amino acid, prepare amino-acid modified ursolic acid derivative, do not relate to the research that natural a-amino acid and ursolic acid is otherwise linked etc. the parties concerned as yet.
Summary of the invention
Based on the problems referred to above, the invention provides a kind of ursolic acid derivative with anticancer activity and preparation method thereof, this derivative is through the ursolic acid derivative through the chemistry of amino acids modification, be connecting arm with the quadrol, a-amino acid and ursolic acid are linked, studies show that this ursolic acid derivative has good antitumour activity.
The present invention implements by following technical solution:
A kind of ursolic acid derivative with anticancer activity, through chemistry of amino acids modification preparation, its structural formula is:
Wherein: R
1Be hydroxyl, acetoxyl group, R
2Be hydroxyl, aminoethyl base, alpha-amino group amido ethylamino-, wherein R
1And R
2Group is not hydroxyl simultaneously.
Described amino acid is natural a-amino acid, and these amino acid are with the C-28 position carboxyl binding by quadrol and ursolic acid of the mode that becomes α-amido linkage;
The preparation method of ursolic acid derivative with anticancer activity is:
(1) ursolic acid and acetic anhydride generate compound 2; Compound 2 generates compound 3 with reacting ethylenediamine after activating through oxalyl chloride; Compound 3 obtains compound 4 through alkaline hydrolysis, acidifying;
(2) compound 3 with the natural a-amino acid reaction of Fmoc protection, removes the Fmoc protecting group with in advance again, has prepared compound 5;
(3) compound 4 with the natural a-amino acid reaction of Fmoc protection, through after taking off the Fmoc protecting group, has prepared compound 6 with in advance again.
The detailed process of described preparation method's step (1) is:
A:5-15 g ursolic acid slowly drips 20-30 mL diacetyl oxide with the dissolving of 40-50 mL pyridine under condition of ice bath, dropwise reaction 6-9 h under the room temperature; Stopped reaction, remove pyridine under reduced pressure, resistates dissolves with methylene dichloride, and be 5% hydrochloric acid, distilled water, saturated nacl aqueous solution washing organic phase with massfraction successively, organic phase is again with the anhydrous sodium sulphate 1-3 h after-filtration that dewaters, filtrate dries to constant weight behind 40-50 ℃ of following concentrating under reduced pressure, in 50-70 ℃ of oven dry down, gets compound 2;
B: get 4-6 g compound 2 and be dissolved in fully in the 30-50 mL methylene dichloride, slow Dropwise 5-7 mL oxalyl chloride under the condition of ice bath continues to keep ice bath and stirs 0.5-2 h after dropwising, and reaction 12-36 h is continued in the back under room temperature; Reaction finishes, and with above-mentioned solution decompression distillation, gets white foam shape solid; Add 40 ~ 60 mL methylene dichloride to dissolving fully and the evaporated under reduced pressure solvent in this solid, products therefrom becomes stock solution with the dissolving of 40-70 mL methylene dichloride;
Get 10-30 mL anhydrous ethylenediamine and be well-dispersed in the 40-60 mL methylene dichloride, ice bath slowly drips above-mentioned stock solution down; Drip and finish, continue ice bath 20-40 min, recession removes ice bath, room temperature reaction 8-16 h; With massfraction is that 5% hydrochloric acid, distilled water, saturated sodium-chloride wash organic layer successively, is neutral until organic layer, and organic layer adds the anhydrous sodium sulphate 1-3 h after-filtration that dewaters again, the filtrate decompression distillation, obtain white solid, the white solid recrystallizing methanol obtains compound 3;
C: get among the mixing solutions 160-170 mL that 4-6 g compound 3 is dissolved in tetrahydrofuran (THF) and methyl alcohol, both volume ratios are (1-2): (1-2); The 3-5 mol/L aqueous sodium hydroxide solution that adds 10-30 mL, stir 8-16 h under the room temperature, remove solvent under reduced pressure, add 200-300 ml redistilled water, fully stir 0.5-2 h under the room temperature, decompress filter, filter cake to neutral, removes solvent with the second distillation water washing under reduced pressure, obtains the white powder solid, the white solid recrystallizing methanol obtains compound 4.
The detailed process of described preparation method's step (2) is:
Getting 2-4 mmol is dissolved in the natural a-amino acid of Fmoc protection and contains 60-70 mL methylene dichloride and 1, in the mixed solvent of 4-dioxane, both volume ratios are (1-2): (1-2), add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride 0.5-0.7 g under the condition of ice bath and keep this thermotonus 20-40 min, the back adds 1-2 g compound 3, room temperature reaction 12-36 h then; Reaction is finished, evaporate solvent, it is methylene dichloride, the triethylamine mixing solutions 50-70mL of 4-6:1 that resistates adds volume ratio, room temperature reaction 48-72 h, and reaction finishes, steam solvent, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: be (9-11) with 1 L volume ratio earlier: 1 ethyl acetate/petroleum ether mixed solvent washes silicagel column, and then is (9-11) with volume ratio: ethyl acetate/ethanol of 1 carries out wash-out, collect required component, obtain compound 5.
The detailed process of described preparation method's step (3) is:
Getting 1-2 g is dissolved in the natural a-amino acid of Fmoc protection and contains 60-70 mL methylene dichloride and 1, in the mixed solvent of 4-dioxane, both volume ratios are (1-2): (1-2), add 0.7-0.8 g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride under the condition of ice bath and keep this thermotonus 20-40 min, the back adds 1-2 g compound 4, room temperature reaction 12-36 h then; Reaction is finished, evaporate solvent, it is methylene dichloride, the triethylamine mixing solutions 100-140 mL of 4-6:1 that resistates adds volume ratio, room temperature reaction 48-72 h, and reaction finishes, steam solvent, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: be (9-11) with 1 L volume ratio earlier: 1 ethyl acetate/petroleum ether mixed solvent washes silicagel column, and then is (4-6) with volume ratio: ethyl acetate/ethanol of 1 carries out wash-out, collect required component, obtain white solid and be compound 6.
The structure of prepared compound 5 is for working as R
1During for acetoxyl group, suc as formula structure shown in (I).
The structure of prepared compound 6 is for working as R
1During for hydroxyl, suc as formula structure shown in (I).
Institute's synthetic has antitumor action suc as formula compound shown in (I), can be applicable to be fit to adopt ursolic acid to carry out the medical treatment situation of antineoplaston.
Preferably: R
2Be alpha-amino group amido ethylamino-, and the alpha-amino group acyl is the residue structure of natural a-amino acid, preferred glycyl residue, methionyl residue, phenylalanyl residue;
Preferably: the preferred glycine of modified amino acid, methionine(Met), phenylalanine.
The invention has the advantages that: the present invention is parent compound with the ursolic acid, and ursolic acid C-28 position carboxyl by quadrol, to form the mode of amido linkage, is linked the ursolic acid derivative of synthetic a series of tool anti-tumor activities with natural a-amino acid.In-vitro pharmacological experiments shows, ursolic acid derivative through the chemistry of amino acids modification in this patent all has the obvious in-vitro suppression activity to human liver cancer cell HepG2, human colon cancer cell HT-29, people's cancer of the stomach ags cell and BGC-823 cell, Human Prostate Cancer Cells PC-3, has simple, the significant advantage of product pharmacologically active of preparation process.
Description of drawings
Fig. 1 has the reacting flow chart through the ursolic acid derivative of chemistry of amino acids modification of antitumour activity for preparation;
Fig. 2 is ursolic acid and structural modification thing thereof the restraining effect (inhibiting rate %) to human colon cancer cell strain HT-29;
Fig. 3 is ursolic acid and structural modification thing thereof the restraining effect (inhibiting rate %) to people's cancer of the stomach BGC-823;
Fig. 4 is ursolic acid and structural modification thing thereof the restraining effect (inhibiting rate %) to people's liver cancer HepG2;
Fig. 5 is ursolic acid and structural modification thing thereof the restraining effect (inhibiting rate %) to people's cancer of the stomach AGS;
Fig. 6 is ursolic acid and structural modification thing thereof the restraining effect (inhibiting rate %) to human prostata cancer PC-3.
Wherein a among Fig. 2-6---compound is the inhibiting rate that records when being 48 h 20 μ mol/L, action time in concentration;
B---the half effective inhibition concentration of the compound when representing to be 48 h action time.
Embodiment
Compound
2Preparation:
10 g ursolic acid dissolve with 45 mL pyridines, slowly drip 25 mL diacetyl oxides under condition of ice bath, drip reaction 8 h under the complete room temperature.Stopped reaction, remove pyridine under reduced pressure, resistates dissolves with methylene dichloride, and use hydrochloric acid (5%), distilled water, saturated nacl aqueous solution to wash organic phase successively, organic phase is with the anhydrous sodium sulphate 2 h after-filtration that dewater, filtrate dries to constant weight behind 45 ℃ of following concentrating under reduced pressure, and the thick product of gained is 10.50 g.Crude product dehydrated alcohol recrystallization gets white needle-like crystals 8.59 g.Crystallization yields is 83.3%, and overall yield is 80.5%.mp：282~283℃。
1H?NMR(600MHz,?CDCl
3):?5.32?(t,?1H,?
J=3.6?Hz,?H-12),?4.42?(t,?1H,?
J=5.6?Hz,?H-3),?2.22?(d,?1H,?
J=11.2?Hz,?H-18),?2.04?(s,?3H,?CH
3CO),?0.92?(d,?3H,?
J=6.4?Hz,?CH
3),?0.85?(d,?3H,?
J=3.6?Hz,?CH
3),?1.09,?1.00,?0.91,?0.78,?0.74?(s,?15H,?each,?CH
3)。
IR(KBr):?3366,?2972,?2928,?1736,?1700,?1455,?1370,?1246?cm
-1。
The preparation of compound 3
5.0 g compound 2 is dissolved in the 40 mL methylene dichloride fully, slowly splashes into 6 mL oxalyl chlorides under the condition of ice bath, continues to keep ice bath after dropwising and stirs 1 h, reaction 24 h are continued in the back under room temperature.Reaction finishes, and with above-mentioned solution decompression distillation, gets white foam shape solid.Divide respectively to add a small amount of methylene dichloride and evaporated under reduced pressure solvent 2 times, products therefrom is a light yellow solid, with the dissolving of 50 mL methylene dichloride, is stock solution.
20 mL anhydrous ethylenediamines are well-dispersed in the 50 mL methylene dichloride, slowly splash into above-mentioned stock solution under the ice bath.Drip and finish, continue ice bath 30 min, recession removes ice bath, room temperature reaction 12 h.Wash organic layer successively with 5% hydrochloric acid, distilled water, saturated sodium-chloride successively, until organic layer clarification and be neutral, the anhydrous sodium sulphate that organic layer the adds capacity 2 h after-filtration that dewater, the filtrate decompression distillation obtains white solid 5.20 g.The white solid recrystallizing methanol obtains white needle-like crystals 4.09 g, and overall yield is 75.5%.mp：140~142℃。
1H?NMR(600MHz,?CDCl
3):?6.32?(s,?1H,?CO
NHCH
2),?5.33?(s,?1H,?H-12),?4.49?(t,?1H,?
J=7.6?Hz,?H-3),?3.41~3.38?(m,?1H,?CONH
CH 2 CH
2NH
2a),?3.06~3.04?(m,?1H,?CONH
CH 2 CH
2NH
2b),?2.79?(s,?2H,?
CH 2 NH
2),?2.05?(s,?3H,?CH
3CO),?1.09,?0.95,?0.94,?0.86,?0.85,?0.84,?0.79?(s,?21H,?each,?CH
3)。
IR(KBr):?3365,?2950,?2853,?1728,?1631,?1534,?1452,?1388,?1370,?1252,?1026?cm
-1。
The preparation of compound 4
4.32 g compound 3 is dissolved in 80 mL tetrahydrofuran (THF)s/80 mL methyl alcohol fully, adds the 4 mol/L aqueous sodium hydroxide solutions of 20 mL, stirs reaction overnight under the room temperature.Remove solvent under reduced pressure, add 250 mL secondary water, abundant stir about 1 h under the room temperature, decompress filter, filter cake is washed to neutrality with secondary water washing, removes solvent under reduced pressure, obtains white powder solid 3.84 g.The white solid recrystallizing methanol obtains white needle-like crystals 3.45 g, and overall yield is 86.7%.mp：145~147℃。
1H?NMR(600MHz,?DMSO-d
6):?7.13(s,1H,?CO
NHCH
2),?5.20(s,1H,?H-12),?3.5~3.1?(brs,?OH),?3.02~2.98?(m,?2H,?CONH
CH 2 CH
2NH
2),?2.97~2.92?(m,?2H,?CONHCH
2 CH 2 NH
2),?2.15?(d,?1H,?H-18),?0.82?(d,?3H,?
J=5.2?Hz?,CH
3),?1.03,?0.91,?0.89,?0.85,?0.69,?0.67?(s,?18H,?each,?CH
3)。
IR(KBr):?3362,?2925,?2868,?1640,?1535,?1455,?1389,?1043,?999,?663?cm
-1。
The preparation of compound 5
5a is an example with the preparation compound, 0.90 g(closes 3 mmol) Fmoc-Gly is dissolved in fully and contains 20 mL methylene dichloride, 20 mL 1, in the mixed solvent of 4-dioxane, add EDCl HCl 0.65 g(3.3 mmol under the condition of ice bath) and keep this thermotonus 30 min, the back adds 1.08 g(2 mmol) compound 3, room temperature reaction 24 h then; Reaction is finished, and evaporation desolventizes, and resistates adds methylene dichloride/triethylamine (V:V=5:1) mixing solutions, room temperature reaction 48-72 h, TLC(ethyl acetate/petroleum ether=10:1, V/V) detection reaction situation.Reaction finishes, steaming desolventizes, resistates adopts silica gel column chromatography to separate, the column chromatography condition is: (V/V=10:1) mixed solvent washes silicagel column, and then carries out wash-out with ethyl acetate/ethanol (V/V=10:1), collects required component with the 1L ethyl acetate/petroleum ether earlier, obtain white solid 1.06 g, be compound 5a, productive rate is 88.5%, mp:147 ~ 149 ℃.
1H?NMR(600MHz,?CDCl
3):?7.62(s,?1H,?CO
NHCH
2),?6.31(s,?1H,?CO
NHCH
2),?5.29?(s,1H,?H-12),?4.42?(d,?1H,?
J=7.4?Hz,?H-3),?3.54~3.52?(m,?1H,?HN
CHCON),?3.10~3.08?(m,?1H,?HN
CHCON),?3.36,?3.30?(s,?1H,?each,?COHN
CH 2 CH
2NHCO),?3.27?(s,?1H,?COHNCH
2 CH 2 NHCO),?3.22?(d,?1H,?
J=13.4?Hz,?COHNCH
2 CH 2 NHCO),?2.58?(t,?3H,?
J=11.1?Hz,?1H,?NH
2),?1.97?(s,?3H,?CH
3COO),?1.01,?0.86,?0.86,?0.80,?0.78,?0.78,?0.69?(s,?21H,?each,?CH
3)。
IR(KBr):?3397,?2925,?2854,?1736,?1681,?1640,?1530,?1457,?1370,?1246,?1027,?805?cm
-1。
Closing 3 mmol with method with 1.11 g() Fmoc-Met replaces Fmoc-Gly to react, reaction finishes, steaming desolventizes, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is that ethyl acetate/petroleum ether (V/V=3:1) is carried out wash-out, collects required component, make 1.20 g compound 5b, be white solid, productive rate is 89.2%, mp:96 ~ 98 ℃.
1H?NMR(600MHz,?CDCl
3):?7.67?(s,?1H,?CO
NHCH
2a),?6.34?(s,?1H,?CO
NHCH
2b),?5.28?(s,?1H,?H-12),?4.42?(d,?1H,?
J=7.4?Hz,?H-3),?3.43,3.39?(s,?2H,?each,?COHN
CH 2 CH
2NHCO),?3.31,?3.27?(s,?2H,?each,?COHN
CH 2 CH
2NHCO),?3.11~3.10?(m,?1H,?HN
CHCON),?2.54?(m,?2H,?CH
3S
CH 2 ),?2.04?(s,?3H,?CH
3CO),?1.97?(s,?3H,?SCH
3),?1.65?(t,?2H,?
J=12.4?Hz,?
CH 2 CH
2SCH
3),?1.01,?0.88,?0.87,?0.80,?0.79,?0.78,?0.69?(s,?21H,?each,?CH
3)。
IR(KBr):?3355,?2925,?2871,?1736,?1650,?1526,?1455,?1370,?1246,?1027,?804?cm
-1。
Closing 3 mmol with method with 1.16 g() Fmoc-Phe replaces Fmoc-Gly to react, reaction finishes, steaming desolventizes, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is that ethyl acetate/petroleum ether (V/V=2:1) is carried out wash-out, collects required component, make 1.33 g compound 5c, be white solid, productive rate is 96.8%, mp:82 ~ 84 ℃.
1H?NMR?(600MHz,?CDCl
3):?8.63?(s,?1H,?CO
NHCH
2),?6.86?(s,?1H,?CO
NHCH
2),?5.34?(s,?1H,?H-12),?4.49?(s,?1H,?H-3),?3.88?(s,?1H,?HN
CHCON),?3.40?(s,?1H,?Ar-
CH 2 ),?3.30?(s,?2H,?NH
2),?3.16?(s,?1H,?Ar-
CH 2 ),?3.12?(t,?2H,?COHN
CH 2 CH
2NHCO),?3.10?(t,?2H,?COHN
CH 2 CH
2NHCO),?2.05?(s,?3H,?CH
3COO),?1.07,?0.94,?0.94,?0.86,?0.86,?0.86,?0.74?(s,?21H,?each,?CH
3)。
IR(KBr):?3363,?2924,?2853,?1735,?1648,?1525,?1455,?1370,?1246,?1095,?1027,?803,?746,?701,?665cm
-1。
The preparation of compound 6
6a is an example with the preparation compound, 1.08 g(closes 3.6 mmol) Fmoc-Gly is dissolved in fully and contains 30 mL methylene dichloride, 30 mL1, in the mixed solvent of 4-dioxane, add 0.72 g EDCl HCl(3.6 mmol under the condition of ice bath) and keep this thermotonus 30 min, the back adds 1.49 g(3 mmol) compound 4, room temperature reaction 24 h then; Reaction is finished, and evaporation desolventizes, and resistates adds methylene dichloride/triethylamine (V:V=5:1) mixing solutions, room temperature reaction 48-72 h, TLC(ethyl acetate/ethanol=10:1, V/V) detection reaction situation.Reaction finishes, steaming desolventizes, resistates adopts silica gel column chromatography to separate, the column chromatography condition is: (V/V=10:1) mixed solvent washes silicagel column, uses ethyl acetate/ethanol (V/V=5:1) to carry out wash-out then, collects required component with 1 L ethyl acetate/petroleum ether earlier, obtain white solid 1.45 g, be compound 6a, productive rate is 87.1%, mp:205 ~ 208 ℃.
1H?NMR(600MHz,?DMSO-d
6):?8.56?(s,?1H,?CO
NHCH
2a),?7.39?(s,?1H,?CO
NHCH
2b),?5.21?(s,?1H,?H-12),?4.31?(s,?1H,?HO-3),?4.03~4.02?(d,?1H,?
J=6.3?Hz,?H
2N
CH 2 CON),?3.46?(s,?1H,?H
2N
CH 2 CON),?3.35?(brs,?2H,?NH
2),?3.10?(s,?1H,?CONH
CH 2 CH
2NHCO),?3.03~2.99?(m,?1H,?CONH
CH 2 CH
2NHCO),?2.18?(d,?2H,?
J=10.1?Hz,?H-18),?1.02,?0.91,?0.88,?0.84,?0.82,?0.67,?0.67?(s,?21H,?each,?CH
3)。
IR(KBr):?3412,?3087,?2926,?1683,?1641,?1535,?1452,?1377,?1251,?1044,?997?cm
-1。
Closing 3.6 mmol with method with 1.34 g() Fmoc-Met replaces Fmoc-Gly to react, reaction finishes, steaming desolventizes, and resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: (V/V=10:1) mixed solvent washes silicagel column with 1 L ethyl acetate/petroleum ether earlier, carry out wash-out with ethyl acetate then, collect required component, make compound 6b, be white solid, productive rate is 89.2%, mp:99 ~ 101 ℃.
1H?NMR(600MHz,?DMSO-d
6):?8.01?(s,?1H,?CO
NHCH
2),?7.28?(s,?1H,?CO
NHCH
2),?5.28?(s,?1H,?H-12),?4.34?(s,?1H,?HO-3),?3.38?(brs,?2H,?NH
2),?3.25?(s,?1H,?HN
CHCON),3.15(s,?1H,?CONH
CH 2 CH
2NHCO),?3.04?(s,1H,?CONH
CH 2 CH
2NHCO),?2.17?(d,?2H,?
J=10.1?Hz,?H-18),?2.08?(s,?3H,?SCH
3),?1.63?(t,?2H,?
J=12.4?Hz,?
CH 2 CH
2SCH
3),?1.08,?0.96,?0.94,?0.90,?0.87,?0.72,?0.72?(s,?21H,?each,?CH
3)。
IR(KBr):?3378,?2924,?2854,?1641,?1527,?1455,?1260,?1093,?1028,?802?cm
-1。
Closing 3.6 mmol with method with 1.39 g() Fmoc-Phe replaces Fmoc-Gly to react, reaction finishes, steaming desolventizes, and resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: (V/V=10:1) mixed solvent washes silicagel column with the 1L ethyl acetate/petroleum ether earlier, carry out wash-out with ethyl acetate then, collect required component, make compound 6c, be white solid, productive rate is 96.8%, mp:154 ~ 157 ℃.
1H?NMR(600MHz，?DMSO-d
6):?8.49?(s,?1H,?CO
NHCH
2),?7.35?(s,?1H,?CO
NHCH
2),?7.30~7.24?(5H,?Ar-H),?5.20?(s,?1H,?H-12),?4.31?(s,?1H,?HO-3),?3.79?(s,?1H,?HN
CHCON),?3.38?(s,?1H,?Ar-
CH 2 ),?3.06?(s,?1H,?Ar-
CH 2 ),?3.34?(brs,?2H,?NH
2),?3.03~3.00?(m,?1H,?CONH
CH 2 CH
2NHCO),?2.91~2.88?(m,?1H,?CONHCH
2 CH 2 NHCO),?2.16?(d,?1H,?
J=10.2?Hz,?H-18),?1.02,?0.90,?0.89,?0.85,?0.81,?0.67,?0.67?(s,?21H,?each,?CH
3)。
IR(KBr):?3355,?2926,?2869,?1673,?1638,?1538,?1455,?1251,?1042,?746,?700,?663?cm
-1。
Performance test
Antitumour activity through the ursolic acid derivative of chemistry of amino acids modification
37 ℃, 5%CO
2Conventional BGC-823, HepG2, HT-29, AGS and the PC-3 of cultivating under the condition.Cancer cells is inoculated in 96 orifice plates, and cell concn is 10
4Individual/hole, medicine to be measured DMSO hydrotropy, the DMSO final concentration is no more than 0.5%, adopts the antitumour activity of mtt assay test ursolic acid modifier acidic amino acid.The results are shown in Table 2 ~ 6.
Embodiment 2
The concrete preparation method of this ursolic acid derivative is:
A:5 g ursolic acid dissolves with 40 mL pyridines, slowly drips 20 mL diacetyl oxides under condition of ice bath, dropwises reaction 6 h under the room temperature; Stopped reaction, remove pyridine under reduced pressure, resistates dissolves with methylene dichloride, and be 5% hydrochloric acid, distilled water, saturated nacl aqueous solution washing organic phase with massfraction successively, organic phase is again with the anhydrous sodium sulphate 1 h after-filtration that dewaters, filtrate dries to constant weight behind 40 ℃ of following concentrating under reduced pressure, and bake out temperature is 60 ℃, gets compound 2;
B: get 4 g compounds 2 and be dissolved in fully in the 30 mL methylene dichloride, slow Dropwise 5 mL oxalyl chloride continues to keep ice bath and stirs 0.5 h under the condition of ice bath after dropwising, and reaction 12 h are continued in the back under room temperature; Reaction finishes, and with above-mentioned solution decompression distillation, gets white foam shape solid; Add 40 mL methylene dichloride and evaporated under reduced pressure solvent in this solid, products therefrom dissolves with 40 mL methylene dichloride, becomes stock solution;
Get 10 mL anhydrous ethylenediamines and be well-dispersed in the 40 mL methylene dichloride, ice bath slowly drips above-mentioned stock solution down; Drip and finish, continue ice bath 20 min, recession removes ice bath, room temperature reaction 8 h; With massfraction is that 5% hydrochloric acid, distilled water, saturated sodium-chloride wash organic layer successively, and until organic layer clarification and be neutral, organic layer adds the anhydrous sodium sulphate 1 h after-filtration that dewaters, the filtrate decompression distillation, obtain white solid, the white solid recrystallizing methanol obtains compound 3;
C: get 160 mL in the mixing solutions that 4 g compounds 3 are dissolved in tetrahydrofuran (THF) and methyl alcohol, both volume ratios are 1:2; The 3 mol/L aqueous sodium hydroxide solutions that add 10 ml, stir 8 h under the room temperature, remove solvent under reduced pressure, add 200 ml redistilled waters, fully stir 0.5 h under the room temperature, decompress filter, filter cake to neutral, removes solvent with the second distillation water washing under reduced pressure, obtains the white powder solid, the white solid recrystallizing methanol obtains compound 4.
The detailed process of described preparation method's step (2) is:
Getting 2 mmol is dissolved in Fmoc-Leu and contains 60 mL methylene dichloride and 1, in the mixed solvent of 4-dioxane, both volume ratios are 2:1, add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride 0.5 g under the condition of ice bath and keep this thermotonus 20 min, the back adds 1 g compound 3, room temperature reaction 12 h then; Reaction is finished, evaporate solvent, it is methylene dichloride, triethylamine mixing solutions 50 mL of 4:1 that resistates adds volume ratio, room temperature reaction 48 h, and reaction finishes, steam solvent, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: is earlier the ethyl acetate/petroleum ether mixed solvent flushing silicagel column of 9:1 with 1 L volume ratio, and then is that ethyl acetate/ethanol of 11:1 carries out wash-out with volume ratio, collect required component, obtain compound 5.
The detailed process of described preparation method's step (3) is: get 1 g and be dissolved in Fmoc-Leu and contain 60 mL methylene dichloride and 1, in the mixed solvent of 4-dioxane, both volume ratios are 1:2, add 0.8 g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride under the condition of ice bath and keep this thermotonus 20 min, the back adds 1 g compound 4, room temperature reaction 12 h then; Reaction is finished, evaporate solvent, it is methylene dichloride, triethylamine mixing solutions 100 mL of 4:1 that resistates adds volume ratio, room temperature reaction 48 h, and reaction finishes, steam solvent, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: is earlier the ethyl acetate/petroleum ether mixed solvent flushing silicagel column of 9:1 with 1 L volume ratio, and then is that ethyl acetate/ethanol of 4:1 carries out wash-out with volume ratio, collect required component, obtain white solid and be compound 6.
The concrete preparation method of this ursolic acid derivative is:
A:15 g ursolic acid dissolves with 50 mL pyridines, slowly drips 30 mL diacetyl oxides under condition of ice bath, dropwises reaction 9 h under the room temperature; Stopped reaction, remove pyridine under reduced pressure, resistates dissolves with methylene dichloride, and be 5% hydrochloric acid, distilled water, saturated nacl aqueous solution washing organic phase with massfraction successively, organic phase is again with the anhydrous sodium sulphate 3 h after-filtration that dewater, filtrate dries to constant weight behind 50 ℃ of following concentrating under reduced pressure, and bake out temperature is 70 ℃, gets compound 2;
B: get 6 g compounds 2 and be dissolved in fully in the 50 mL methylene dichloride, slowly drip 7 mL oxalyl chlorides under the condition of ice bath, continue to keep ice bath after dropwising and stir 2 h, reaction 36 h are continued in the back under room temperature; Reaction finishes, and with above-mentioned solution decompression distillation, gets white foam shape solid; Add 60 mL methylene dichloride and evaporated under reduced pressure solvent in this solid, products therefrom dissolves with 70 mL methylene dichloride, becomes stock solution;
Get 30 mL anhydrous ethylenediamines and be well-dispersed in the 60 mL methylene dichloride, ice bath slowly drips above-mentioned stock solution down; Drip and finish, continue ice bath 40 min, recession removes ice bath, room temperature reaction 16 h; With massfraction is that 5% hydrochloric acid, distilled water, saturated sodium-chloride wash organic layer successively, and until organic layer clarification and be neutral, organic layer adds the anhydrous sodium sulphate 3 h after-filtration that dewater, the filtrate decompression distillation, obtain white solid, the white solid recrystallizing methanol obtains compound 3;
C: get among mixing solutions 170 mL that 6 g compounds 3 are dissolved in tetrahydrofuran (THF) and methyl alcohol, both volume ratios are 2:1; The 5 mol/L aqueous sodium hydroxide solutions that add 30 ml, stir 16 h under the room temperature, remove solvent under reduced pressure, add 300 mL redistilled waters, fully stir 2 h under the room temperature, decompress filter, filter cake to neutral, removes solvent with the second distillation water washing under reduced pressure, obtains the white powder solid, the white solid recrystallizing methanol obtains compound 4.
Getting 4mmol is dissolved in Fmoc-Lys and contains 70 mL methylene dichloride and 1, in the mixed solvent of 4-dioxane, both volume ratios are 1:2, add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride 0.7 g under the condition of ice bath and keep this thermotonus 40 min, the back adds 2 g compounds 3, room temperature reaction 36 h then; Reaction is finished, evaporate solvent, it is methylene dichloride, triethylamine mixing solutions 70 mL of 6:1 that resistates adds volume ratio, room temperature reaction 72 h, and reaction finishes, steam solvent, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: earlier with the ethyl acetate/petroleum ether mixed solvent flushing silicagel column of 1 L volume ratio 9-11:1, and then be that ethyl acetate/ethanol of 9:1 carries out wash-out with volume ratio, collect required component, obtain compound 5.
Getting 2 g is dissolved in Fmoc-Lys and contains 70 mL methylene dichloride and 1, in the mixed solvent of 4-dioxane, both volume ratios are 2:1, add 0.7 g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride under the condition of ice bath and keep this thermotonus 40 min, the back adds 2 g compounds 4, room temperature reaction 36 h then; Reaction is finished, evaporate solvent, it is methylene dichloride, the triethylamine mixing solutions 140mL of 6:1 that resistates adds volume ratio, room temperature reaction 48h, and reaction finishes, steam solvent, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: is earlier the ethyl acetate/petroleum ether mixed solvent flushing silicagel column of 11:1 with 1 L volume ratio, and then is that ethyl acetate/ethanol of 6:1 carries out wash-out with volume ratio, collect required component, obtain white solid and be compound 6.
The above only is preferred embodiment of the present invention, and all equalizations of being done according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (9)
1. ursolic acid derivative with anticancer activity is characterized in that: described ursolic acid derivative is through chemistry of amino acids modification preparation, and its structural formula is:
(I),
Wherein: R
1Be hydroxyl, acetoxyl group, R
2Be hydroxyl, aminoethyl base, alpha-amino group amido ethylamino-, wherein R
1And R
2Group is not hydroxyl simultaneously.
2. ursolic acid derivative with anticancer activity according to claim 1 is characterized in that: described amino acid is natural a-amino acid, and these amino acid link by quadrol and ursolic acid parent nucleus C-28 position carboxyl in the mode that becomes α-amido linkage.
3. the preparation method of a ursolic acid derivative with anticancer activity as claimed in claim 1, it is characterized in that: described preparation method is:
(1) ursolic acid and acetic anhydride generate compound 2; Compound 2 generates compound 3 with reacting ethylenediamine after activating through oxalyl chloride; Compound 3 obtains compound 4 through alkaline hydrolysis, acidifying;
(2) compound 3 with the natural a-amino acid reaction of Fmoc protection, removes the Fmoc protecting group with in advance again, has prepared compound 5;
(3) compound 4 with the natural a-amino acid reaction of Fmoc protection, through after taking off the Fmoc protecting group, has prepared compound 6 with in advance again.
4. the preparation method of ursolic acid derivative with anticancer activity according to claim 3 is characterized in that: the detailed process of described preparation method's step (1) is:
A:5-15 g ursolic acid slowly drips 20-30 mL diacetyl oxide with the dissolving of 40-50 mL pyridine under condition of ice bath, dropwise reaction 6-9 h under the room temperature; Stopped reaction, remove pyridine under reduced pressure, resistates dissolves with methylene dichloride, and be 5% hydrochloric acid, distilled water, saturated nacl aqueous solution washing organic phase with massfraction successively, organic phase is again with the anhydrous sodium sulphate 1-3 h after-filtration that dewaters, filtrate dries to constant weight behind 40-50 ℃ of following concentrating under reduced pressure, in 50-70 ℃ of oven dry down, gets compound 2;
B: get 4-6 g compound 2 and be dissolved in fully in the 30-50 mL methylene dichloride, slow Dropwise 5-7 mL oxalyl chloride under the condition of ice bath continues to keep ice bath and stirs 0.5-2 h after dropwising, and reaction 12-36 h is continued in the back under room temperature; Reaction finishes, and with above-mentioned solution decompression distillation, gets white foam shape solid; Add 40 ~ 60 mL methylene dichloride to dissolving fully and the evaporated under reduced pressure solvent in this solid, products therefrom becomes stock solution with the dissolving of 40-70 mL methylene dichloride;
Get 10-30 mL anhydrous ethylenediamine and be well-dispersed in the 40-60 mL methylene dichloride, ice bath slowly drips above-mentioned stock solution down; Drip and finish, continue ice bath 20-40 min, recession removes ice bath, room temperature reaction 8-16 h; With massfraction is that 5% hydrochloric acid, distilled water, saturated sodium-chloride wash organic layer successively, is neutral until organic layer, and organic layer adds the anhydrous sodium sulphate 1-3 h after-filtration that dewaters again, the filtrate decompression distillation, obtain white solid, the white solid recrystallizing methanol obtains compound 3;
C: get among the mixing solutions 160-170 mL that 4-6 g compound 3 is dissolved in tetrahydrofuran (THF) and methyl alcohol, both volume ratios are 1-2:1-2; The 3-5 mol/L aqueous sodium hydroxide solution that adds 10-30 mL, stir 8-16 h under the room temperature, remove solvent under reduced pressure, add 200-300 mL redistilled water, fully stir 0.5-2 h under the room temperature, decompress filter, filter cake to neutral, removes solvent with the second distillation water washing under reduced pressure, obtains the white powder solid, the white solid recrystallizing methanol obtains compound 4.
5. the preparation method of ursolic acid derivative with anticancer activity according to claim 3, it is characterized in that: the detailed process of described preparation method's step (2) is: get 2-4 mmol and be dissolved in the natural a-amino acid of Fmoc protection and contain 60-70 mL methylene dichloride and 1, in the mixed solvent of 4-dioxane, both volume ratios are 1-2:1-2, add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride 0.5-0.7 g under the condition of ice bath and keep this thermotonus 20-40 min, the back adds 1-2g compound 3, room temperature reaction 12-36 h then; Reaction is finished, evaporate solvent, it is methylene dichloride, the triethylamine mixing solutions 50-70 mL of 4-6:1 that resistates adds volume ratio, room temperature reaction 48-72 h, and reaction finishes, steam solvent, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: is earlier the ethyl acetate/petroleum ether mixed solvent flushing silicagel column of 9-11:1 with 1 L volume ratio, and then is that ethyl acetate/ethanol of 9-11:1 carries out wash-out with volume ratio, collect required component, obtain compound 5.
6. the preparation method of ursolic acid derivative with anticancer activity according to claim 3, it is characterized in that: the detailed process of described preparation method's step (3) is: get 1-2 g and be dissolved in the natural a-amino acid of Fmoc protection and contain 60-70 mL methylene dichloride and 1, in the mixed solvent of 4-dioxane, both volume ratios are 1-2:1-2, add 0.7-0.8 g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride under the condition of ice bath and keep this thermotonus 20-40 min, the back adds 1-2 g compound 4, room temperature reaction 12-36 h then; Reaction is finished, evaporate solvent, it is methylene dichloride, the triethylamine mixing solutions 100-140 mL of 4-6:1 that resistates adds volume ratio, room temperature reaction 48-72 h, and reaction finishes, steam solvent, resistates adopts silica gel column chromatography to separate, and the column chromatography condition is: is earlier the ethyl acetate/petroleum ether mixed solvent flushing silicagel column of 9-11:1 with 1 L volume ratio, and then is that ethyl acetate/ethanol of 4-6:1 carries out wash-out with volume ratio, collect required component, obtain white solid and be compound 6.
7. the preparation method of ursolic acid derivative with anticancer activity according to claim 5 is characterized in that: the structure of prepared compound 5 is for working as R
1During for acetoxyl group, suc as formula structure shown in (I).
8. the preparation method of ursolic acid derivative with anticancer activity according to claim 6 is characterized in that: the structure of prepared compound 6 is for working as R
1During for hydroxyl, suc as formula structure shown in (I).
9. ursolic acid derivative with anticancer activity according to claim 1 is characterized in that: institute's synthetic has antitumor action suc as formula compound shown in (I), can be used for treating tumor disease.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102516351A (en) * | 2011-11-22 | 2012-06-27 | 福州大学 | Ursolic acid derivative with anti-cancer activity and preparation method thereof |
| CN102532246A (en) * | 2012-02-27 | 2012-07-04 | 浙江大学 | Oleanolic acid derivative as well as preparation method and application thereof |
| CN106749486A (en) * | 2016-11-30 | 2017-05-31 | 沈阳化工大学 | A kind of oleanolic acid derivate and its application with ethylenediamine as linking arm |
| CN113004368A (en) * | 2021-01-29 | 2021-06-22 | 南京林业大学 | Ursolic acid pyrimidine amide derivatives, and preparation method and application thereof |
| CN113150059A (en) * | 2021-01-29 | 2021-07-23 | 南京林业大学 | Ursolic acid pyrimidine methyl ester derivative and preparation method and application thereof |
| CN113429451A (en) * | 2021-05-31 | 2021-09-24 | 湖南华诚生物资源股份有限公司 | Momordica grosvenori acid acylation derivative and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101157715A (en) * | 2007-11-20 | 2008-04-09 | 沈阳化工学院 | Ursolic acid chemical modified compound amino alcohol having antitumor activity |
| CN101928321A (en) * | 2010-03-02 | 2010-12-29 | 福州大学 | Acidic amino acid chemically-modified ursolic acid derivatives with anti-cancer activities |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101157715A (en) * | 2007-11-20 | 2008-04-09 | 沈阳化工学院 | Ursolic acid chemical modified compound amino alcohol having antitumor activity |
| CN101928321A (en) * | 2010-03-02 | 2010-12-29 | 福州大学 | Acidic amino acid chemically-modified ursolic acid derivatives with anti-cancer activities |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102516351A (en) * | 2011-11-22 | 2012-06-27 | 福州大学 | Ursolic acid derivative with anti-cancer activity and preparation method thereof |
| CN102532246A (en) * | 2012-02-27 | 2012-07-04 | 浙江大学 | Oleanolic acid derivative as well as preparation method and application thereof |
| CN106749486A (en) * | 2016-11-30 | 2017-05-31 | 沈阳化工大学 | A kind of oleanolic acid derivate and its application with ethylenediamine as linking arm |
| CN113004368A (en) * | 2021-01-29 | 2021-06-22 | 南京林业大学 | Ursolic acid pyrimidine amide derivatives, and preparation method and application thereof |
| CN113150059A (en) * | 2021-01-29 | 2021-07-23 | 南京林业大学 | Ursolic acid pyrimidine methyl ester derivative and preparation method and application thereof |
| CN113004368B (en) * | 2021-01-29 | 2021-11-19 | 南京林业大学 | Ursolic acid pyrimidine amide derivatives, and preparation method and application thereof |
| CN113429451A (en) * | 2021-05-31 | 2021-09-24 | 湖南华诚生物资源股份有限公司 | Momordica grosvenori acid acylation derivative and preparation method thereof |
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