CN104744549B - Betulic acid derivant and preparation method thereof and the application in antitumor drug is prepared - Google Patents
Betulic acid derivant and preparation method thereof and the application in antitumor drug is prepared Download PDFInfo
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Abstract
The invention discloses a kind of formula (1), betulic acid derivant and preparation method thereof shown in formula (2).Betula platyphylla Suk. fat amine, betulic acid are carried out betulic acid derivant that condensation reaction obtains heterocycle modification respectively with various heterocyclic carboxylic acids.Present invention also offers application of the betulic acid derivant in antitumor drug is prepared.
Description
Technical field
The invention belongs to medicine and its prepare and apply technical field, and in particular to a kind of betulic acid derivant and its
Preparation method and its application in antitumor drug is prepared.
Background technology
Betulic acid (Betulinic acid, BA) is a kind of pentacyclic triterpenoid, is present in silver birch, summer withered
Grass, Fructus Chaenomeliss, Herba Rosmarini Officinalis, in the various plants such as Folium seu Cortex Nerii.Nineteen ninety-five, National Cancer Institute (National Cancer
Institute, NCI) find that betulic acid can optionally kill melanoma cell, and to normal cell without obvious poison
Property.Follow-up research finds betulic acid to tumors such as ovarian cancer, cervical cancer, carcinoma of prostate, breast carcinoma, colon cancer, leukemia
Cell strain is respectively provided with preferable active anticancer.Betulic acid as a kind of natural product because of its novel antitumor mechanism and significantly
Anti-tumor activity, just increasingly showing its it is good independent or with traditional anti-tumor means, the DEVELOPMENT PROSPECT of drug combination.Cause
This, synthesizes its natural product analog by methodology of organic synthesis, carries out structure optimization, and structure yield is higher, anti-tumor activity
More preferable new betulic acid derivant, and the developmental research for antitumor drug is significant.
The content of the invention
The present invention proposes a kind of betulic acid derivant, shown in its structure such as formula (1)：
Wherein,
Different substituent Rs are specifically corresponded to, formula (1) the betulic acid derivant includes：Such as with following formula (B-2),
Formula (B-3), formula (B-4), formula (B-5), formula (B-6), formula (B-14), formula (B-15), formula (B-16), formula (B-17), formula (B-18),
Betulic acid derivant shown in formula (B-19), formula (B-20).
The present invention proposes a kind of betulic acid derivant, shown in its structure such as formula (2)：
Wherein,
Different substituent Rs are specifically corresponded to, formula (2) the betulic acid derivant includes：Such as with following formula (B-23),
Betulic acid derivant shown in formula (B-24).
Present invention also offers the preparation method of betulic acid derivant, wherein, involved Betula platyphylla Suk. fat amine raw material is ginseng
Document Organic Letters are examined,《Organic bulletin》, the method described in 2009,11,461-464 prepares.With Betula platyphylla Suk. fat
Acid is raw material, and C-3 positions hydroxyl Jing IBX aoxidize to obtain betulonic acid, then Jing CH3COONH4And NaCNBH3Reduction amination obtains Betula platyphylla Suk.
Fat amine.The synthetic route of Betula platyphylla Suk. fat amine is as follows：
Present invention also offers the preparation method of formula (1) betulic acid derivant, with Betula platyphylla Suk. fat amine as raw material, with N, N '-
Carbonyl dimidazoles (N, N '-Carbonyldiimidazole, CDI) are condensing agent, DMAP (4-
Dimethylaminopyridine, DMAP) it is condensed with heterocyclic carboxylic acid for catalyst respectively, the heterocyclic carboxylic acid includes：2- pyrazines
Carboxylic acid, 5- methyl -2- pyrazine carboxylic acids .gamma.-pyridinecarboxylic acid, nicotinic acid, 2- chlorine apellagrins, are condensed to yield such as formula (B-2), formula (B-3), formula (B-
4), formula (B-5) and betulic acid derivant shown in formula (B-6)；The reaction scheme of the preparation method is as follows：
Visible above, 2- pyrazine carboxylic acids, 5- methyl -2- pyrazine carboxylic acids, nicotinic acid .gamma.-pyridinecarboxylic acid, 2- chlorine apellagrins are in CDI, DMAP
Under effect, it is condensed to yield as shown in formula (B-2), formula (B-3), formula (B-4), formula (B-5), formula (B-6) with Betula platyphylla Suk. fat amine
Betulic acid derivant.In the preparation method, condensing agent used by condensation reaction is CDI, and catalyst is DMAP, solvent used
For anhydrous methylene chloride, reaction temperature is 25 DEG C of room temperature.
Present invention also offers the preparation method of formula (1) betulic acid derivant, with Betula platyphylla Suk. fat amine as raw material, CDI is contracting
Mixture, DMAP are catalyst, include N-Boc-L- proline, N-Boc-2- piperidine carboxylic acids, N-Boc-3- respectively with heterocyclic carboxylic acid
Piperidine carboxylic acid, N-Boc-4- piperidine carboxylic acids, N-Boc-4- Piperidineacetic acids, N-Boc-4- piperidine propanoic acids, N-Boc- piperidines -4 butyric acid
It is condensed to yield white as shown in formula (B-7), formula (B-8), formula (B-9), formula (B-10), formula (B-11), formula (B-12) and formula (B-13)
Pine gum acid derivative；Then remove Boc protection groups by boron trifluoride diethyl etherate respectively to obtain such as formula (B-14), formula (B-15), formula
(B-16), formula (B-17), formula (B-18), formula (B-19) and betulic acid derivant shown in formula (B-20)；The preparation method
Reaction scheme is as follows：
It is visible above, by N-Boc-L- proline, N-Boc-2- piperidine carboxylic acids, N-Boc-3- piperidine carboxylic acids, N-Boc-4-
Piperidine carboxylic acid, N-Boc-4- Piperidineacetic acids, N-Boc-4- piperidine propanoic acids, N-Boc-4- piperidines butanoic acid CDI, DMAP effect under,
With Betula platyphylla Suk. fat amine be condensed to yield as formula (B-7), formula (B-8), formula (B-9), formula (B-10), formula (B-11), formula (B-12) and
Betulic acid derivant shown in formula (B-13)；Then remove Boc protection groups by boron trifluoride diethyl etherate respectively to obtain such as formula (B-
14), formula (B-15), formula (B-16), formula (B-17), formula (B-18), formula (B-19) and betulic acid derivant shown in formula (B-20).
In the preparation method, condensing agent used by condensation reaction is CDI, and catalyst is DMAP, and solvent used is anhydrous methylene chloride,
Reaction temperature is 25 DEG C of room temperature.The de- Boc protection groups reaction agents useful for same is boron trifluoride diethyl etherate, and solvent used is anhydrous
Ether, reaction temperature are 25 DEG C of room temperature.
Present invention also offers the preparation method of formula (2) betulic acid derivant, with betulic acid as raw material, dicyclohexyl
Carbodiimide (Dicyclohexylcarbodiimide, DCC) is condensing agent, and DMAP is catalyst, respectively and heterocyclic carboxylic acid：N-
Boc-2- piperidine carboxylic acids, N-Boc-3- piperidine carboxylic acids, are condensed to yield such as formula (B-21) and betulic acid shown in formula (B-22) is derivative
Thing；Formula (B-21) and formula (B-22) remove Boc protection groups by boron trifluoride diethyl etherate respectively again and obtain such as formula (B-23) and formula (B-
24) betulic acid derivant shown in number；The reaction scheme of the preparation method is as follows：
It is visible above, by betulic acid respectively and heterocyclic carboxylic acid：N-Boc-2- piperidine carboxylic acids, N-Boc-3- piperidine carboxylic acids exist
Such as formula (B-21) and betulic acid derivant shown in formula (B-22) are condensed to yield under DCC, DMAP effect；Then pass through trifluoro respectively
Change borate ether removing Boc protection groups and obtain such as formula (B-23) and betulic acid derivant shown in formula (B-24).The preparation method
In, condensing agent used by condensation reaction is DCC, and catalyst is DMAP, and solvent used is anhydrous methylene chloride, and reaction temperature is room
25 DEG C of temperature.The de- Boc protection groups reaction agents useful for same is boron trifluoride diethyl etherate, and solvent used is absolute ether, reacts temperature
Spend for 25 DEG C of room temperature.
In preparation method of the present invention, the performance level that above reaction is generally reacted come tracking and measuring with thin plate chromatography method, instead
The post-processing approach that should be adopted after finishing includes concentrating, extracts, column chromatography for separation etc., and final product is tested by nuclear magnetic resoance spectrum
Card.
Present invention also offers application of the betulic acid derivant in antitumor drug is prepared.Involved in the present invention is white
Pine gum acid derivative has remarkable result in terms of tumor cell proliferation is suppressed, while having inducing apoptosis of tumour cell and suppressing swollen
The effect of oncocyte migration.
The advantage of betulic acid derivant of the present invention and preparation method thereof includes, by Betula platyphylla Suk. fat amine, betulic acid respectively with
Heterocyclic carboxylic acid is condensed to yield betulic acid derivant.The reaction condition of preparation method of the present invention is gentle, agents useful for same low price,
Environmental friendliness, simple synthetic method, yield are higher.
The anti-tumor activity of most of betulic acid derivant involved in the present invention is remarkably reinforced, as shown in table 1, wherein
Compound B-16, B-17, B-18, B-19, B-20, B-23 and B-24 colon cancer cell line (HCT-116, HT- to non-drug resistance
29), Prostatic cancer cell lines (PC-3, DU-145), breast carcinoma cell strain (MDA-MB-231,4T1, T47D, MCF-7) and multiple medicines
Breast cancer cell line mcf-7/the ADR (adriamycin-resistant ADR and Docetaxel DOC) of drug resistance shows good Inhibit proliferaton and makees
With.Average ICs of the particularly compound B-17 to the tumor cell line of eight plants of non-drug resistances50=1.19 μM, to multidrug resistance breast carcinoma
The IC of cell strain MCF-7/ADR50=0.33 μM, wherein being betulic acid (betulinic to MCF-7/ADR inhibited proliferations
Acid, BA) (IC50=38.5 μM) 117 times.Additionally, compound B-2, B-3, B-4, B-5, B-6 are to testing tumor cell line table
Reveal relatively weak Inhibit proliferaton activity (IC50>25 μM), although not providing concrete test number, in test process substantially
It was observed that each compound has inhibitory activity to each tested tumor cell line.
Experiment shows that betulic acid derivant of the present invention has to tumor cell preferably apoptosis-induced and suppresses migration to live
Property, as shown in Figure 1 and Figure 2.Betulic acid derivant simultaneously involved in the present invention is also tested for normal cell, human desmocyte
The proliferation inhibition activity of cell (HAF), as a result shows these derivants with to the more preferable selectivity of tumor cell.Particularly change
Compound B-17 is up to 13.5 times with the selectivity of HAF cells to multidrug resistance breast cancer cell line mcf-7/ADR, therefore the present invention
Involved derivant shows higher safety, as shown in table 2.Betulic acid derivant of the present invention can be used as potential anti-swollen
Tumor medicine, in particular for the medicine of particular type drug resistance breast carcinoma, with applications well prospect.
Description of the drawings
Fig. 1 represents betulic acid derivant B-17 of the present invention, BA and positive control Docetaxel (DOC) to breast carcinoma
Cell line MDA-MB-231, MCF-7, and the test result of MCF-7/ADR apoptosis inductions；Compound B-17 (5 μM) treatment group
Breast cancer cell MDA-MB-231 apoptosis rates are 31.43%, compare the apoptosis rate of (50 μM) groups 29.52% of BA, show more
Significantly apoptosis-induced effect.Meanwhile, (1 μM) process breast carcinoma of compound BA (80 μM) and positive control Docetaxel DOC
After cell strain MCF-7 and multidrug resistance cell strain MCF-7/ADR, MCF-7/ADR is shown bright to BA and DOC compared with MCF-7
Aobvious drug resistance.Especially, it is after (1 μM) of DOC is processed, far little to the apoptosis-promoting effect of MCF-7/ADR (apoptosis rate 2.96%)
In MCF-7 (apoptosis rate 16.24%), and compound B-17 is to MCF-7/ADR (apoptosis rate 19.16%) and MCF-7 (apoptosis rates
20.32%) almost identical apoptosis-induced effect is shown, illustrates that B-17 can overcome the multidrug resistance of MCF-7/ADR.
Fig. 2 represents the inhibition of metastasis of betulic acid derivant B-17 of the present invention and BA to MDA-MB-231 breast cancer cell lines
Effect；Compare with matched group, compound BA is migrated to MDA-MB-231 breast cancer cells when concentration is respectively 10,25 and 50 μM
Certain inhibitory action is shown.And compound B-17 only just can almost completely inhibit MDA-MB- when concentration is 10 μM
The migration of 231 breast cancer cells.In addition, compound B-17 concentration be 2.5 μM and compound BA when concentration is 50 μM, it is right
The effect of MDA-MB-231 breast cancer cells inhibition of metastasis is suitable, i.e. compound B-17 presses down to the migration of MDA-MB-231 breast cancer cells
Make and use about 20 times of parent compound BA.
Specific embodiment
With reference to specific examples below and accompanying drawing, the present invention is described in further detail, the protection content of the present invention
It is not limited to following examples.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and with appending claims as protection domain.The process of the enforcement present invention,
Condition, reagent, experimental technique etc., in addition to the following content for specially referring to, are the universal knowledege and common knowledge of this area,
The present invention is not particularly limited content.In following embodiments, compound structure is by nmr determination；Reagent is mainly by Shanghai
Traditional Chinese medicines chemical reagents corporation provides；Purifying products mainly by column chromatography, given birth to by Haiyang Chemical Plant, Qingdao by silica gel (200-300)
Produce.
Embodiment 1：The preparation of Betula platyphylla Suk. fat amine
The preparation of betulonic acid：Betulic acid (5g, 10.9mmol) is dissolved in DMSO (50mL) and THF (50mL),
Add IBX (6.16g, 22mmol), room temperature reaction 6 hours.After TLC detection reactions completely, H is added2O (100mL) and acetic acid second
Ester (50mL), sucking filtration remove insoluble matter, and filtrate ethyl acetate (30mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, it is dense
Contracting, obtain betulonic acid white solid (4.5g, it is 90%), not purified to be directly used in next step.
The preparation of Betula platyphylla Suk. fat amine：By betulonic acid (4.5g), ammonium acetate (7.63g, 99mmol), sodium cyanoborohydride
(935mg, 14.9mmol) is dissolved in methanol (100mL), nitrogen protection, room temperature reaction 12 hours.TLC detection reactions are complete
Afterwards, removal of solvent under reduced pressure, adds H2O (100mL), 1M NaOH adjust pH value of solution in alkalescence, and the solid that sucking filtration is separated out is obtained
Crude product, then silica gel column chromatography (DCM：MeOH=10：1) purification, obtain Betula platyphylla Suk. fat amine white solid (3.7g, 82%).
Embodiment 2：The preparation of betulic acid derivant shown in formula (1)
The preparation of B-2：2- pyrazine carboxylic acids (110mg, 0.88mmol), CDI (143mg, 0.88mmol), DMAP (108mg,
0.88mmol) it is dissolved in anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Add in above-mentioned reactant liquor
Betula platyphylla Suk. fat amine (200mg, 0.44mmol) continues reaction 8 hours.After TLC detection reactions completely, removal of solvent under reduced pressure adds H2O
(30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：
MeOH=20：1) purification, obtain white solid (188mg, 76%).1H NMR(400MHz,CDCl3)δ9.27(s,1H),8.75(s,
2H),4.92-4.83(m,1H),4.75(s,1H),4.62(s,1H),3.05-2.98(m,1H),2.30-2.18(m,2H),
2.05-1.94(m,2H),1.87-1.73(m,3H),1.71(s,3H),1.66-1.63(m,1H),1.58-1.48(m,2H),
1.48-1.37(m,6H),1.34(s,1H),1.30-1.19(m,3H),1.13-1.06(m,2H),1.01(s,3H),1.00(s,
3H),0.96(s,3H),0.94(s,3H),0.91(s,3H).
The preparation of B-3：5- methyl -2- pyrazine carboxylic acids (122mg, 0.88mmol), CDI (143mg, 0.88mmol), DMAP
(108mg, 0.88mmol) is dissolved in anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.To above-mentioned reaction
Betula platyphylla Suk. fat amine (200mg, 0.44mmol) is added to continue reaction 8 hours in liquid.After TLC detection reactions completely, removal of solvent under reduced pressure,
Add H2O (30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography
(DCM：MeOH=20：1) purification, obtain white solid (205mg, 81%).1H NMR(400MHz,CDCl3)δ9.13(s,1H),
8.61(s,1H),4.88-4.82(m,1H),4.75(s,1H),4.62(s,1H),3.04-2.98(m,1H),2.66(s,3H),
2.31-2.18 (m, 2H), 2.05-1.95 (m, 2H), 1.85-1.76 (m, 3H), 1.70 (s, 3H), 1.63 (t, J=11.2Hz,
1H),1.54-1.51(m,2H),1.49-1.38(m,7H),1.34(s,1H),1.13-1.04(m,2H),1.00(s,6H),
0.96(s,3H),0.92(s,3H),0.91(s,3H).
The preparation of B-4：.gamma.-pyridinecarboxylic acid (109mg, 1.32mmol), CDI (143mg, 0.88mmol), DMAP (108mg,
0.88mmol) it is dissolved in anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Add in above-mentioned reactant liquor
Betula platyphylla Suk. fat amine (200mg, 0.44mmol) continues reaction 8 hours.After TLC detection reactions completely, removal of solvent under reduced pressure adds H2O
(30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：
MeOH=20：1) purification, obtain white solid (190mg, 77%).1H NMR(400MHz,DMSO-d6)δ12.03(s,1H),8.96
(s, 1H), 8.68 (d, J=4.7Hz, 1H), 8.15 (d, J=7.9Hz, 1H), 8.07 (d, J=9.3Hz, 1H), 7.48 (dd, J
=7.6,5.0Hz, 1H), 4.70 (s, 1H), 4.59 (s, 1H), 3.79-3.73 (m, 1H), 2.98-2.92 (m, 1H), 2.24 (t,
J=10.9Hz, 1H), 2.12 (d, J=8.4Hz, 1H), 1.87-1.74 (m, 3H), 1.66 (s, 3H), 0.97 (s, 3H), 0.89
(s,3H),0.82(s,3H),0.81(s,6H).
The preparation of B-5：Nicotinic acid (109mg, 1.32mmol), CDI (143mg, 0.88mmol), DMAP (108mg,
0.88mmol) it is dissolved in anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Add in above-mentioned reactant liquor
Betula platyphylla Suk. fat amine (200mg, 0.44mmol) continues reaction 8 hours.After TLC detection reactions completely, removal of solvent under reduced pressure adds H2O
(30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：
MeOH=20：1) purification, obtain white solid (192mg, 78%).1H NMR(400MHz,DMSO-d6)δ12.03(s,1H),8.69
(d, J=4.9Hz, 2H), 8.13 (d, J=9.4Hz, 1H), 7.72 (d, J=5.0Hz, 2H), 4.70 (s, 1H), 4.57 (s,
1H), 3.76 (dd, J=15.2,6.3Hz, 1H), 3.17 (s, 1H), 2.98-2.92 (m, 1H), 2.24 (t, J=10.6Hz,
1H),2.13-2.11(m,1H),1.88-1.75(m,3H),1.66(s,3H),1.20-1.08(m,2H),0.97(s,3H),
0.89(s,3H),0.82(s,3H),0.80(s,6H).
The preparation of B-6：2- chlorine apellagrins (139mg, 1.32mmol), CDI (143mg, 0.88mmol), DMAP (108mg,
0.88mmol) it is dissolved in anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Add in above-mentioned reactant liquor
Betula platyphylla Suk. fat amine (200mg, 0.44mmol) continues reaction 8 hours.After TLC detection reactions completely, removal of solvent under reduced pressure adds H2O
(30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：
MeOH=20：1) purification, obtain white solid (178mg, 68%).1H NMR(400MHz,DMSO-d6)δ12.06(s,1H),8.44
(dd, J=4.8,1.8Hz, 1H), 8.22 (d, J=9.5Hz, 1H), 7.82 (dd, J=7.5,1.8Hz, 1H), 7.51-7.43
(dd, J=7.5,4.8Hz, 1H), 4.69 (s, 1H), 4.57 (s, 1H), 3.69-3.62 (m, 1H), 2.99-2.91 (m, 1H),
2.23 (t, J=10.5Hz, 1H), 2.12 (d, J=7.1Hz, 1H), 1.86-1.78 (m, 2H), 1.66 (s, 3H), 1.63-1.60
(m, 2H), 1.56-1.48 (m, 3H), 1.44-1.33 (m, 11H), 1.24 (s, 1H), 1.09 (t, J=7.0Hz, 3H), 0.97
(s,3H),0.91(s,3H),0.88(s,3H),0.79(s,3H),0.74(s,3H).
Embodiment 3：The preparation of betulic acid derivant shown in formula (1)
The preparation of B-7：N-Boc-L- proline (285mg, 1.32mmol), CDI (322mg, 1.32mmol) are dissolved in nothing
In water dichloromethane (10mL), nitrogen protection, room temperature reaction 2 hours.Betula platyphylla Suk. fat amine is sequentially added in above-mentioned reactant liquor
(300mg, 0.66mmol), DMAP (161mg, 1.32mmol) continue reaction 8 hours.After TLC detection reactions completely, it is removed under reduced pressure
Solvent, adds H2O (30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silicagel column
Chromatography (DCM：MeOH=20：1) purification, obtain white solid (332mg, 77%).1H NMR(400MHz,CDCl3)δ4.73(s,
1H), 4.59 (s, 1H), 4.30 (d, J=5.0Hz, 1H), 3.63 (dd, J=9.6,7.0Hz, 1H), 3.42 (s, 2H), 3.06-
2.94(m,1H),2.32-2.10(m,4H),1.68(s,3H),1.45(s,9H),0.96(s,3H),0.91(s,3H),0.87
(s,3H),0.80(s,3H),0.73(s,3H).
The preparation of B-8：N-Boc-2- piperidine carboxylic acids (303mg, 1.32mmol), CDI (322mg, 1.32mmol) are dissolved in
In anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Betula platyphylla Suk. fat amine is sequentially added in above-mentioned reactant liquor
(300mg, 0.66mmol), DMAP (161mg, 1.32mmol) continue reaction 8 hours.After TLC detection reactions completely, it is removed under reduced pressure
Solvent, adds H2O (30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silicagel column
Chromatography (DCM：MeOH=20：1) purification, obtain white solid (317mg, 72%).1H NMR(400MHz,CDCl3)δ4.73(s,
2H), 4.60 (s, 1H), 4.21-3.89 (m, 1H), 3.81-3.46 (m, 1H), 3.10-2.90 (m, 1H), 2.76 (d, J=
13.4Hz,1H),2.45-2.08(m,4H),1.68(s,3H),1.46(s,9H),0.97(s,3H),0.92(s,3H),0.85
(s,3H),0.79(s,3H),0.70(s,3H).
The preparation of B-9：N-Boc-3- piperidine carboxylic acids (303mg, 1.32mmol), CDI (322mg, 1.32mmol) are dissolved in
In anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Betula platyphylla Suk. fat amine is sequentially added in above-mentioned reactant liquor
(300mg, 0.66mmol), DMAP (161mg, 1.32mmol) continue reaction 8 hours.After TLC detection reactions completely, it is removed under reduced pressure
Solvent, adds H2O (30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silicagel column
Chromatography (DCM：MeOH=20：1) purification, obtain white solid (330mg, 75%).1H NMR(500MHz,CDCl3)δ4.72(s,
1H),4.59(s,1H),4.18-4.97(m,3H),3.65(s,1H),2.99-2.78(m,3H),2.51-2.43(m,1H),
2.30-2.16(m,2H),1.68(s,3H),1.45(s,9H),0.96(s,3H),0.92(s,3H),0.83(s,3H),0.80
(s,3H),0.73(s,3H).
The preparation of B-10：N-Boc-4- piperidine carboxylic acids (303mg, 1.32mmol), CDI (322mg, 1.32mmol) are dissolved in
In anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Betula platyphylla Suk. fat amine is sequentially added in above-mentioned reactant liquor
(300mg, 0.66mmol), DMAP (161mg, 1.32mmol) continue reaction 8 hours.After TLC detection reactions completely, it is removed under reduced pressure
Solvent, adds H2O (30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silicagel column
Chromatography (DCM：MeOH=20：1) purification, obtain white solid (365mg, 83%).1H NMR(400MHz,CDCl3)δ4.73(s,
1H), 4.60 (s, 1H), 4.21-4.06 (m, 3H), 3.65 (s, 1H), 2.99 (d, J=10.2Hz, 1H), 2.76 (d, J=
11.6Hz,2H),2.35-2.14(m,4H),1.68(s,3H),1.44(s,9H),0.97(s,3H),0.92(s,3H),0.83
(s,3H),0.80(s,3H),0.73(s,3H).
The preparation of B-11：N-Boc-4- Piperidineacetic acids (322mg, 1.32mmol), CDI (322mg, 1.32mmol) are dissolved in
In anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Betula platyphylla Suk. fat amine is sequentially added in above-mentioned reactant liquor
(300mg, 0.66mmol), DMAP (161mg, 1.32mmol) continue reaction 8 hours.After TLC detection reactions completely, it is removed under reduced pressure
Solvent, adds H2O (30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silicagel column
Chromatography (DCM：MeOH=20：1) purification, obtain white solid (368mg, 82%).1H NMR(500MHz,CDCl3)δ4.72(s,
1H), 4.60 (s, 1H), 4.06 (d, J=12.2Hz, 2H), 3.71-3.59 (m, 1H), 3.00 (t, J=9.8Hz, 1H), 2.69
(t, J=11.6Hz, 2H), 2.30-2.16 (m, 2H), 1.68 (s, 3H), 1.43 (s, 9H), 0.96 (s, 3H), 0.95 (s, 3H),
0.84(s,3H),0.79(s,3H),0.73(s,3H).
The preparation of B-12：N-Boc-4- piperidine propanoic acids (340mg, 1.32mmol), CDI (322mg, 1.32mmol) are dissolved in
In anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Betula platyphylla Suk. fat amine is sequentially added in above-mentioned reactant liquor
(300mg, 0.66mmol), DMAP (161mg, 1.32mmol) continue reaction 8 hours.After TLC detection reactions completely, it is removed under reduced pressure
Solvent, adds H2O (30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silicagel column
Chromatography (DCM：MeOH=20：1) purification, obtain white solid (359mg, 78%).1H NMR(500MHz,CDCl3)δ4.73(s,
1H), 4.61 (s, 1H), 4.07 (d, J=12.9Hz, 2H), 3.65 (t, J=8.8Hz, 1H), 3.08-2.92 (m, 1H), 2.65
(t, J=12.3Hz, 2H), 2.32-2.10 (m, 4H), 1.69 (s, 3H), 1.44 (s, 9H), 0.97 (s, 3H), 0.96 (s, 3H),
0.85(s,3H),0.80(s,3H),0.74(s,3H).
The preparation of B-13：N-Boc-4- piperidines butanoic acid (358mg, 1.32mmol), CDI (322mg, 1.32mmol) are dissolved in
In anhydrous methylene chloride (10mL), nitrogen protection, room temperature reaction 2 hours.Betula platyphylla Suk. fat amine is sequentially added in above-mentioned reactant liquor
(300mg, 0.66mmol), DMAP (161mg, 1.32mmol) continue reaction 8 hours.After TLC detection reactions completely, it is removed under reduced pressure
Solvent, adds H2O (30mL), ethyl acetate (20mL × 3) extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silicagel column
Chromatography (DCM：MeOH=20：1) purification, obtain white solid (347mg, 74%).1H NMR(500MHz,CDCl3)δ4.72(s,
1H), 4.61 (s, 1H), 4.05 (d, J=12.2Hz, 2H), 3.65 (t, J=10.0Hz, 1H), 3.00 (t, J=10.2Hz,
1H), 2.65 (t, J=11.9Hz, 2H), 2.33-2.10 (m, 5H), 2.07-1.86 (m, 3H), 1.68 (s, 3H), 1.44 (s,
9H),0.96(s,6H),0.85(s,3H),0.79(s,3H),0.73(s,3H).
The preparation of B-14：Compound B-7 (310mg, 0.47mmol) is dissolved in absolute ether (20mL), is slowly added to three
Fluorination borate ether (5mL), room temperature reaction 10 minutes.After TLC detection reactions completely, Deca saturated sodium bicarbonate solution under ice-water bath
Reaction is quenched, ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：
MeOH=10：1) purification, obtain white solid (197mg, 75%).1H NMR(400MHz,DMSO-d6)δ12.05(s,1H),8.06
(d, J=9.4Hz, 1H), 4.67 (d, J=16.9Hz, 1H), 4.56 (s, 1H), 4.13 (t, J=7.6Hz, 1H), 3.58-3.47
(m, 1H), 3.29-3.16 (m, 2H), 2.98-2.91 (m, 1H), 2.35-2.20 (m, 2H), 2.12 (d, J=9.0Hz, 1H),
(1.94-1.73 m, 5H), 1.65 (s, 3H), 1.17 (t, J=7.1Hz, 3H), 0.95 (s, 3H), 0.88 (s, 3H), 0.79 (s,
3H),0.73(s,3H),0.72(s,3H).
The preparation of B-15：Compound B-8 (300mg, 0.45mmol) is dissolved in absolute ether (20mL), is slowly added to three
Fluorination borate ether (5mL), room temperature reaction 10 minutes.After TLC detection reactions completely, Deca saturated sodium bicarbonate solution under ice-water bath
Reaction is quenched, ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：
MeOH=10：1) purification, obtain white solid (209mg, 82%).1H NMR(400MHz,DMSO-d6) δ 8.01 (d, J=9.5Hz,
1H), 4.69 (s, 1H), 4.56 (s, 1H), 3.71-3.62 (m, 1H), 3.59-3.49 (m, 1H), 3.20 (d, J=12.3Hz,
1H), 2.99-2.86 (m, 2H), 2.22 (dd, J=16.8,7.1Hz, 1H), 2.09 (dd, J=25.0,9.8Hz, 2H), 1.65
(s,3H),0.95(s,3H),0.87(s,3H),0.79(s,3H),0.73(s,3H),0.71(s,3H).
The preparation of B-16：Compound B-9 (310mg, 0.46mmol) is dissolved in absolute ether (20mL), is slowly added to three
Fluorination borate ether (5mL), room temperature reaction 10 minutes.After TLC detection reactions completely, Deca saturated sodium bicarbonate solution under ice-water bath
Reaction is quenched, ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：
MeOH=10：1) purification, obtain white solid (205mg, 78%).1H NMR(400MHz,DMSO-d6)δ7.61-7.50(m,1H),
4.67 (s, 1H), 4.54 (s, 1H), 3.46-3.40 (m, 1H), 3.03-2.94 (m, 1H), 2.86 (dd, J=24.6,11.7Hz,
2H), 2.38-2.24 (m, 2H), 2.12 (d, J=11.6Hz, 1H), 1.64 (s, 3H), 0.93 (s, 3H), 0.86 (s, 3H),
0.77(s,3H),0.71(s,3H),0.69(s,3H).
The preparation of B-17：Compound B-10 (340mg, 0.51mmol) is dissolved in absolute ether (20mL), is slowly added to
Boron trifluoride diethyl etherate (3mL), room temperature reaction 10 minutes.After TLC detection reactions completely, under ice-water bath, Deca saturated sodium bicarbonate is molten
Liquid is quenched reaction, and ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography
(DCM：MeOH=10：1) purification, obtain white solid (239mg, 83%).1H NMR(400MHz,DMSO-d6) δ 7.43 (d, J=
9.4Hz, 1H), 4.65 (s, 1H), 4.53 (s, 1H), 3.49 (s, 1H), 3.23 (d, J=10.7Hz, 1H), 3.08 (d, J=
9.7Hz, 2H), 2.59 (d, J=11.6Hz, 1H), 2.42 (d, J=13.2Hz, 2H), 2.24 (s, 1H), 2.14 (d, J=
10.7Hz,1H),2.09-2.01(m,1H),1.63(s,3H),0.93(s,3H),0.86(s,3H),0.77(s,3H),0.72
(s,3H),0.72(s,3H).
The preparation of B-18：Compound B-11 (340mg, 0.50mmol) is dissolved in absolute ether (20mL), is slowly added to
Boron trifluoride diethyl etherate (3mL), room temperature reaction 10 minutes.After TLC detection reactions completely, under ice-water bath, Deca saturated sodium bicarbonate is molten
Liquid is quenched reaction, and ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography
(DCM：MeOH=10：1) purification, obtain white solid (235mg, 81%).1H NMR(400MHz,DMSO-d6) δ 7.41 (d, J=
9.0Hz, 1H), 4.66 (s, 1H), 4.54 (s, 1H), 3.49-3.44 (m, 1H), 3.03 (d, J=10.3Hz, 3H), 2.14 (d, J
=8.0Hz, 2H), 2.03 (d, J=6.5Hz, 1H), 1.64 (s, 3H), 0.94 (s, 3H), 0.87 (s, 3H), 0.78 (s, 3H),
0.73(s,3H),0.69(s,3H).
The preparation of B-19：Compound B-12 (330mg, 0.47mmol) is dissolved in absolute ether (20mL), is slowly added to
Boron trifluoride diethyl etherate (3mL), room temperature reaction 10 minutes.After TLC detection reactions completely, under ice-water bath, Deca saturated sodium bicarbonate is molten
Liquid is quenched reaction, and ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography
(DCM：MeOH=10：1) purification, obtain white solid (243mg, 86%).1H NMR(400MHz,DMSO-d6) δ 7.42 (d, J=
9.4Hz, 1H), 4.64 (s, 1H), 4.52 (s, 1H), 3.48 (s, 1H), 3.22 (d, J=10.7Hz, 1H), 3.07 (d, J=
9.7Hz, 2H), 2.58 (d, J=11.6Hz, 1H), 2.41 (d, J=13.2Hz, 2H), 2.23 (s, 1H), 2.13 (d, J=
10.7Hz,1H),2.09–2.01(m,1H),1.62(s,3H),0.92(s,3H),0.85(s,3H),0.76(s,3H),0.71
(s,3H),0.71(s,3H).
The preparation of B-20：Compound B-13 (320mg, 0.45mmol) is dissolved in absolute ether (20mL), is slowly added to
Boron trifluoride diethyl etherate (3mL), room temperature reaction 10 minutes.After TLC detection reactions completely, under ice-water bath, Deca saturated sodium bicarbonate is molten
Liquid is quenched reaction, and ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography
(DCM：MeOH=10：1) purification, obtain white solid (217mg, 77%).1H NMR(400MHz,DMSO-d6) δ 7.40 (d, J=
9.0Hz, 1H), 4.65 (s, 1H), 4.53 (s, 1H), 3.43 (d, J=19.6Hz, 1H), 3.02 (d, J=10.3Hz, 3H),
2.20-2.07(m,2H),2.06-1.96(m,1H),1.63(s,3H),0.93(s,3H),0.86(s,3H),0.77(s,3H),
0.71(s,3H),0.68(s,3H).
Embodiment 4：The preparation of betulic acid derivant shown in formula (2)
The preparation of B-21：Betulic acid (300mg, 0.66mmol), N-Boc-3- piperidine carboxylic acids (303mg, 1.32mmol,
DCC (272mg, 1.32mmol) and DMAP (161mg, 1.32mmol) are dissolved in anhydrous methylene chloride (10mL), nitrogen protection,
Room temperature reaction 4 hours.After TLC detection reactions completely, removal of solvent under reduced pressure adds H2O (30mL), ethyl acetate (20mL × 3)
Extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：MeOH=20：1) purification, obtains white solid
(348mg, 79%).1H NMR(400MHz,CDCl3)δ4.74(s,1H),4.61(s,1H),4.51-4.44(m,1H),4.23-
4.14 (m, 1H), 3.93 (d, J=12.8Hz, 1H), 3.02-2.89 (m, 2H), 2.77 (t, J=12.4Hz, 1H), 2.43 (t, J
=10.7Hz, 1H), 2.27 (d, J=12.6Hz, 1H), 2.23-2.16 (m, 1H), 1.97 (dd, J=13.3,5.5Hz, 2H),
1.69 (s, 3H), 1.63-1.57 (m, 4H), 1.45 (s, 9H), 1.30-1.25 (m, 2H), 1.19 (d, J=13.3Hz, 1H),
0.97(s,3H),0.94(s,3H),0.85(s,6H),0.83(s,3H).
The preparation of B-22：Betulic acid (300mg, 0.66mmol), N-Boc-4- piperidine carboxylic acids (303mg, 1.32mmol,
DCC (272mg, 1.32mmol) and DMAP (161mg, 1.32mmol) are dissolved in anhydrous methylene chloride (10mL), nitrogen protection,
Room temperature reaction 4 hours.After TLC detection reactions completely, removal of solvent under reduced pressure adds H2O (30mL), ethyl acetate (20mL × 3)
Extraction, merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography (DCM：MeOH=20：1) purification, obtains white solid
(330mg, 75%).1H NMR(400MHz,CDCl3)δ4.74(s,1H),4.61(s,1H),4.52-4.45(m,1H),3.99
(d, J=13.3Hz, 2H), 3.05-2.96 (m, 1H), 2.85 (td, J=11.3,3.6Hz, 2H), 2.43 (td, J=10.7,
5.4Hz, 1H), 2.31-2.14 (m, 2H), 2.03-1.92 (m, 2H), 1.87 (t, J=10.7Hz, 2H), 1.69 (s, 3H),
1.64 (s, 2H), 1.63-1.56 (m, 4H), 1.45 (s, 9H), 1.38 (s, 3H), 1.30-1.25 (m, 2H), 1.18 (d, J=
13.4Hz,1H),0.97(s,3H),0.94(s,3H),0.85(s,3H),0.83(s,6H).
The preparation of B-23：Compound B-21 (320mg, 0.48mmol) is dissolved in absolute ether (20mL), is slowly added to
Boron trifluoride diethyl etherate (5mL), room temperature reaction 10 minutes.After TLC detection reactions completely, under ice-water bath, Deca saturated sodium bicarbonate is molten
Liquid is quenched reaction, and ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography
(DCM：MeOH=10：1) purification, obtain white solid (213mg, 81%).1H NMR(400MHz,DMSO-d6)δ4.69(s,1H),
4.56 (s, 1H), 4.41 (dd, J=11.0,4.5Hz, 1H), 3.25 (d, J=12.6Hz, 2H), 2.94 (d, J=8.8Hz,
3H), 2.68 (s, 1H), 2.23 (s, 1H), 2.13 (s, 1H), 1.99 (t, J=13.1Hz, 2H), 1.80 (d, J=6.8Hz,
1H),1.65(s,3H),0.95(s,3H),0.88(s,3H),0.81(s,6H),0.79(s,3H).
The preparation of B-24：Compound B-22 (310mg, 0.46mmol) is dissolved in absolute ether (20mL), is slowly added to
Boron trifluoride diethyl etherate (5mL), room temperature reaction 10 minutes.After TLC detection reactions completely, under ice-water bath, Deca saturated sodium bicarbonate is molten
Liquid is quenched reaction, and ethyl acetate (10mL × 3) extraction merges organic faciess, anhydrous Na2SO4It is dried, concentration, silica gel column chromatography
(DCM：MeOH=10：1) purification, obtain white solid (211mg, 80%).1H NMR(400MHz,DMSO-d6)δ4.68(s,1H),
4.55 (s, 1H), 4.40 (dd, J=11.0,4.5Hz, 1H), 3.24 (d, J=12.6Hz, 2H), 2.93 (d, J=8.8Hz,
3H), 2.67 (t, J=11.0Hz, 1H), 1.79 (d, J=6.8Hz, 1H), 1.64 (s, 3H), 0.94 (s, 3H), 0.87 (s,
3H),0.80(s,6H),0.78(s,3H).
Embodiment 5：Suppress tumor cell proliferation test
Using srb assay in breast carcinoma cell strain MDA-MB-231, T47D, 4T1, MCF7 and MCF-7/ADR, carcinoma of prostate is thin
Born of the same parents strain DU-145 and PC-3, colon cancer cell line HCT-116 and HT-29, in human fibroblasts strain HAF, detection compound is to phase
The suppression ratio for answering cell to breed.Calculation of half inhibitory concentration (IC50), and be compared with positive control (amycin).
1st, test philosophy：
Srb assay：Sulforhodamine B (SRB) is a kind of pink anionic dye, soluble in water, in acid condition may be used
Specifically combined with the basic amino acid of intracellular constitutive protein matter, which produces absworption peak at 515nm wavelength.Necessarily reading
In the range of value, light absorption value and the linear positive correlation of cell concentration, therefore can be used as the detection by quantitative of cell number.
2nd, sample test：
(1) each cell is inoculated on 96 orifice plates with appropriate density, and after culture in 24 hours, experimental group gives different dense
The compound treatment of degree 96 hours, matched group use normal incubation medium culture.(2), after medicine is acted on 96 hours, culture plate is taken out,
25 μ L50%TCA solution are added to fix cell per hole, 4 DEG C are placed more than 1 hour.(3) culture plate after fixing is taken out, is washed with water
Wash 5 times, dry naturally or after hair-dryer is dried up, 50 μ L 0.4%SRB solution are added per hole, is dyeed 10 minutes, is discarded dyeing liquor,
1% glacial acetic acid is washed 5 times and is dried naturally or hair-dryer is dried up.(4) with 100 μ L Tris-base alkali liquor (10mM) dissolve with it is thin
The protein bound dyestuff of born of the same parents, using measure absorbance value at microplate reader 515nm.(5) test in triplicate, IC50Use GraphPad
Prism computed in software is obtained.
3rd, conclusion：
Each test compound is to individual tumor cell line half-inhibition concentration (IC50) data are shown in Table 1.Betula platyphylla Suk. as shown in table 1
Inhibit proliferaton activity of the fat acid derivant to tumor cell line, compound B-16, B-17, B-18, B-19, B-20, B-23 and B-
The antiproliferative activity of 24 pairs of surveyed tumor cell lines is much better than parent compound betulic acid (BA).Compound B-16, B-17, B-
18th, B-19, B-20, B-23 and B-24 compared to parent compound (betulic acid BA) to eight kinds of drug susceptibility-types tumor cell lines
(HCT-116, HT-29, PC-3, DU-145, MDA-MB-231,4T1, T47D, MCF-7) and multidrug resistance breast cancer tumor cells
Strain MCF-7/ADR shows good Inhibit proliferaton effect.Particularly compound B-17 is to eight kinds of drug susceptibility-types cancerous cell
Average IC in strain50=1.19 μM, to IC in multidrug resistance breast cancer cell line mcf-7/ADR50=0.33 μM, wherein, it is right
MCF-7/ADR inhibited proliferations are parent compound (betulic acid, BA) (IC50=38.5 μM) 117 times.Compound B-2,
B-3, B-4, B-5, B-6 show relatively weak Inhibit proliferaton activity (IC to testing tumor cell line50>25 μM), although not having
Providing specific numerical value, but each compound is substantially observed in test process there is suppression to live each tested tumor cell line
Property.
Betulic acid and its derivant are to human fibroblasts proliferation inhibition activity and Correlation selection sex index (SI) data
It is shown in Table 2.As shown in table 2, betulic acid derivant of the present invention has preferable selectivity to the Proliferation Ability of tumor cell line；Phase
Than in tumor cell, most of test compounds show relatively low cytotoxicity to Normal human fibroblast (HAF).Chemical combination
Thing B-16 and B-17 shows the selection of 1.0 to 8.2 and 1.8 to 13.5 times respectively relative to human fibroblasts to tumor cell
Property.Particularly compound B-17 shows preferable selectivity to multidrug resistance breast cancer cell line mcf-7/ADR, itself and HAF
Up to 13.5 times of the selectivity index (SI) of cell.Positive control ADR shows obvious cell toxicant to human fibroblasts
Property (IC50=0.016 μM), it is only 1.8 which suppresses cell propagation selectivity index (SI).
Embodiment 6：B-17, BA and positive control Docetaxel DOC to breast cancer cell line MDA-MB-231, MCF-7,
The test of MCF-7/ADR apoptosis inductions
1st, experimental principle：
In normal cell, Phosphatidylserine (PS) is only distributed in the inner side of cell membrane lipid bilayer, and withers in cell
Die early stage, the Phosphatidylserine (PS) in cell membrane is by turning on one's side laterally in adipose membrane.Annexin V are had with Phosphatidylserine
High affinity, therefore can be combined with the after birth of apoptosis early stage cell by exposed Phosphatidylserine on the outside of cell.Therefore
Annexin V are by one of sensitive indexes as detection early apoptosis of cells.By Annexin V carry out fluorescein (EGFP,
FITC) labelling is using the Annexin V that marked as fluorescent probe, detectable thin using fluorescence microscope or flow cytometer
The generation of born of the same parents' apoptosis.Propidium iodide (Propidium Iodide, PI) is a kind of nucleic acid dye, and it can not pass through complete cell
Film, but the cell and dead cell to apoptosis middle and advanced stage, PI can pass through cell membrane and incarnadine nucleus.Therefore by Annexin
V matches use with PI, it is possible to which the cell differentiation in different apoptosis periods comes.
2nd, sample test：
(1) MDA-MB-231, MCF-7, MCF-7/ADR cell is inoculated in 6 orifice plates with proper density, cultivates through 24 hours
Afterwards, B-17, BA or DOC of variable concentrations gradient is added to process cell.After (2) 24 hours, cell is collected, setting does not contaminate group, list
Double dye groups of dye PI groups, single dye Annexin V groups and variable concentrations.(3) Jing after PBS washings, often pipe is added the cell collected
100 μ L 1 × combination buffer, do not contaminate group and be not added with PI and Annexin V, single dye PI groups add 5 μ L PI, single dye
Annexin V groups add 5 μ L Annexin V, the double dye groups of Annexin-V-PI to add each 5 μ L of PI and Annexin V, mix.
(4) room temperature lucifuge is incubated 15 minutes, cell is transferred in streaming pipe, upper machine testing.
3rd, conclusion：
Compound B-17, BA and positive control docetaxel DOC inducing mammary cancerous cell MDA-MB-231, MCF-7, with
And MCF-7/ADR apoptosis, as shown in Figure 1.Wherein, compound B-17 (5 μM) treatment group breast cancer cell MDA-MB-231
Apoptosis rate is 31.43%, compares the apoptosis rate of (50 μM) groups 29.52% of BA, shows the apoptosis-induced effect for becoming apparent from.Together
When, compound BA (80 μM) and (1 μM) of positive control docetaxel DOC processes breast cancer cell line mcf-7 and multidrug resistance is thin
After born of the same parents strain MCF-7/ADR, MCF-7/ADR shows drug resistance obvious to BA and DOC compared with MCF-7.Especially, DOC
After (1 μM) is processed, it is 5.5 times of MCF-7/ADR (2.96%) to MCF-7 apoptosis-promoting effects (16.24%), and compound B-17
Almost identical apoptosis-induced effect is shown to MCF-7 (20.32%) and MCF-7/ADR (19.16%), B-17 energy gram is illustrated
Take the multidrug resistance of MCF-7/ADR.
Embodiment 7：B-17, BA suppress the test of MDA-MB-231 breast cancer cells migration
1st, experimental principle：
In Transwell cells, cell is separated into upper and lower two parts by filter membrane., above, chemotactic factor is under for target cell
Face, chemotactic factor form gradient by filter membrane, and cell then passes through fenestra along gradient, is attached to filter membrane lower surface, dyes and count
The cell number of filter membrane lower surface can measure the chemotactic ability of chemotactic factor, be mainly used in various cytokines to malignant tumor
Cell invasion and the impact of transfer.Transwell cells are put in culture plate, upper indoor splendid attire serum-free medium, lower room
Interior splendid attire serum-containing medium, levels culture fluid are separated by with polycarbonate membrane.Cell is seeded in into upper interior, due to poly- carbonic acid
Ester film has permeability, and the serum composition in lower floor's culture fluid can promote the downward room migration of the cell of upper interior, cell Jing medicines
The impact of medicine cell migration can be studied after process.
2nd, sample test：
(1) Transwell cells are placed in 24 orifice plates, in upper room, add 200 μ L to contain 8 × 104MDA-MB-231
The serum-free medium of cell, the complete medium for adding 700 μ L to contain 10% serum in lower room.(2) by variable concentrations gradient
BA and B-17 compounds add the upper and lower cells of Transwell.(3) 24 orifice plates are steadily put into into cell culture incubator, cell is moved
After moving 12 hours, after absorbing culture medium, 30 minutes are fixed with 4% paraformaldehyde.(4) upper chamber surface is carefully wiped with cotton swab, remove
The cell not migrated in upper surface.(5) to moving to 0.1% violet staining of the cell of lower surface 5 minutes.(6) it is being inverted
On microscope, each cell randomly selects 5 visuals field and carries out cell counting and count.
3rd, conclusion：
The anti-migration effect test of compound B-17 and BA, as shown in Figure 2.Compare with matched group, compound BA is in concentration
Certain inhibitory action is shown to the migration of MDA-MB-231 breast cancer cells when respectively 10,25 and 50 μM.And compound
B-17 just can almost completely inhibit the migration of MDA-MB-231 breast cancer cells only when concentration is 10 μM.In addition, compound
B-17 concentration be 2.5 μM and compound BA when concentration is 50 μM, MDA-MB-231 breast cancer cells inhibition of metastasis is acted on
Quite, i.e. compound B-17 is about 20 times of parent compound BA to the effect of MDA-MB-231 breast cancer cells inhibition of metastasis.
Table 1, betulic acid derivant to tumor cell line HCT-116, HT-29, PC-3, DU145, MDA-MB-231,
The external antiproliferative activity of 4T1, T47D, MCF-7, MCF-7/ADR.
Srb assay detection cell propagation (96 hours).
Table 2, betulic acid and its derivant are to human fibroblasts proliferation inhibition activity.
Srb assay detection cell propagation (96 hours)；Selectivity index SI：IC50(tumor cell line) is divided by IC50(into fiber
Cell strain).
Claims (9)
1. a kind of betulic acid derivant, it is characterised in that shown in its structure such as formula (1)：
Wherein,
2. a kind of betulic acid derivant, it is characterised in that shown in its structure such as formula (2)：
Wherein,
3. a kind of preparation method of betulic acid derivant, it is characterised in that with the Betula platyphylla Suk. fat amine shown in formula (3) as raw material, with
N, N '-carbonyl dimidazoles be condensing agent, with DMAP as catalyst, respectively with 2- pyrazine carboxylic acids, 5- methyl -2- pyrroles
Piperazine carboxylic acid .gamma.-pyridinecarboxylic acid, nicotinic acid, the condensation of 2- chlorine apellagrins, obtain such as formula (B-2), formula (B-3), formula (B-4), formula (B-5) and formula (B-
6) betulic acid derivant shown in；The reaction scheme of the preparation method is as follows：
4. a kind of preparation method of betulic acid derivant, it is characterised in that with the Betula platyphylla Suk. fat amine shown in formula (3) as raw material, with
CDI is condensing agent, with DMAP as catalyst, respectively with N-Boc-L- proline, N-Boc-2- piperidine carboxylic acids, N-Boc-3- piperidines
Formic acid, N-Boc-4- piperidine carboxylic acids, N-Boc-4- Piperidineacetic acids, N-Boc-4- piperidine propanoic acids, the condensation of N-Boc-4- piperidines butanoic acid,
Obtain the chemical combination as shown in formula (B-7), formula (B-8), formula (B-9), formula (B-10), formula (B-11), formula (B-12) and formula (B-13)
Thing；The aforementioned compound for obtaining obtains corresponding such as formula (B-14), formula with boron trifluoride diethyl etherate removing Boc protection groups again respectively
(B-15), formula (B-16), formula (B-17), formula (B-18), formula (B-19) and betulic acid derivant shown in formula (B-20)；The system
The reaction scheme of Preparation Method is as follows：
5. the method as described in any one of claim 3-4, it is characterised in that the solvent of the condensation reaction is anhydrous dichloro
Methane, reaction temperature are 25 DEG C of room temperature.
6. method as claimed in claim 3, it is characterised in that solvent used is absolute ether, and reaction temperature is room temperature 25
℃。
7. method as claimed in claim 4, it is characterised in that the removing Boc protection groups reaction agents useful for same is borontrifluoride
Borate ether, solvent used are absolute ether, and reaction temperature is 25 DEG C of room temperature.
8. application of the betulic acid derivant as described in any one of claim 1～2 in antitumor drug is prepared.
9. it is as claimed in claim 8 to apply, it is characterised in that the apoptosis of the derivative induced tumor cell of the betulic acid,
Suppress tumor cell propagation, suppress tumor cell migration.
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