CN105294641A - Brefeldin A selenoester derivatives as well as preparation method and application thereof - Google Patents
Brefeldin A selenoester derivatives as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses brefeldin A selenoester derivatives as well as a preparation method and application thereof. The brefeldin A selenoester derivatives have antioxidant activity and antineoplastic activity, wherein the brefeldin A selenoester derivative (I-1), (I-3), (I-4) or (I-5) has a good antioxidant effect, and can be used for removing DPPH (1,1-diphenyl-2-picryl-hydrazyl), ABTS (2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) and superoxide free radical (O<2->); the brefeldin A selenoester derivative as shown in the formula (I-5) has good inhibitory activity for human lung cancer cell A549, and the inhibitory activity is far higher that of BFA (Brefeldin A); obtained results show that the BFA derivatives have broad application prospect in a medical development system, provide a new broader idea for screening and synthesizing the BFA derivatives, and further provide a more effective path for treating relevant diseases.
Description
(1) technical field
The present invention relates to brefeldin A selenium ester class derivative compound and preparation thereof and biologic applications.
(2) background technology
Brefeldin A ((+)-BrefeldinA, be called for short BFA), it is a kind of novel microbiotic, by the mycetogenetic secondary metabolite of a kind of Macrolide, also decumbin or ascochytin can be referred to as, there is various biological activity, comprise antimycotic, antiviral, nematicide, antimitotic, and find that it has high reactivity restraining effect to multiple cancer cells, can also the effect of molecular tool be applied in the research of mammalian signal pathway in addition.But, because there is poorly water-soluble in brefeldin A, transformation period is short, the problems such as the low and bioavailability of targeting is low, it is restricted in all many-sided application, therefore the undesirable element that structural modification exists to overcome itself is carried out to BFA, the scientific research focus enabling it be applied to clinical study as early as possible to have become new.
Selenium is the trace element of needed by human, has irreplaceable effect.The existence form of organoselenium in human body is Sethotope, follows the metabolism of Methionine metabolism approach, synthetic protein, and therefore storage in tissue, absorption are easier to; Can utilize rapidly after absorption of human body, effectively can improve blood in human body in selenium situation.Selenium can directly act on virus, suppresses virus copying in vivo, and can participate in the reparation of cell, prevent multiple virus and disease (as hepatitis B, carditis etc.).When selenium deficiency time, human immunological competence can be made to decline, threaten human health and life, 40 various diseases of the mankind are all too low relevant to human body selenium content in fact, as cancer, cardiovascular diseases, hepatopathy, cataract, pancreatic disease, diabetes, reproductive system disease etc.Research shows, organoselenium has good biological activity, biological active selenium is applied to the structural modification to BFA, can make moderate progress to the pharmacokinetic property of BFA, greatly may improve anti-tumor activity, become the new BFA drug derivative with potential pharmaceutical use.
The present invention is just by the 4-OH to BFA, the chemically modified of 7-OH, obtain BFA selenium ester derivative, have detected its anti-oxidant activity, and detect its rejection ability to human lung cancer cell line (A549), CHL cells (CHL), the anti-tumor activity of research BFA selenium ester derivative and structure activity relationship, for anti-cancer agent screening provides Research foundation.
(3) summary of the invention
The object of the invention is to provide a kind of brefeldin A selenium ester derivative and preparation method thereof, and this analog derivative is preparing the application in anti-oxidant and anti-tumor activity medicine, this analog derivative synthesis technique is simple, there are good anti-oxidant activity and anti-tumor activity, the researching value had.
The technical solution used in the present invention is:
Brefeldin A of the present invention (BFA) structural formula is for shown in (II):
In formula II: 3-, 11-carbon-carbon double bond, 4-OH, 7-OH and 15-CH
3being BFA itself can modification group, in order to synthesize different BFA derivatives, to change its character.The preferred modification group of the present invention is 4-OH, 7-OH, modifies compound used therefor and comprises different aromatic series isoselenocyanates, to synthesize selenium ester compound.
The invention provides the ester derivative of brefeldin A selenium shown in a kind of formula I,
R in formula I
1for H or
r
2for
r
1, R
2middle R
3be phenyl ring, benzyl or 2-methyl-6-ethylphenyl simultaneously.
Further, preferred brefeldin A selenium ester derivative of the present invention is one of following:
Brefeldin A selenium ester derivative of the present invention most preferably is compound shown in formula (I-1), (I-3), (I-4) or (I-5).
The present invention also provides a kind of preparation method of described brefeldin A selenium ester derivative, described method for: with aromatic series isoselenocyanates shown in brefeldin A shown in formula II (BFA) and formula III for raw material, with tetrahydrofuran (THF) (THF) for solvent, take sodium hydride as catalyzer, logical nitrogen, react under condition of ice bath, after reaction terminates, reaction solution aftertreatment obtains the selenium of brefeldin A shown in formula I ester derivative;
R in formula III
3for phenyl ring, benzyl or 2-methyl-6-ethylphenyl.
Further, the feed intake ratio of amount of substance of aromatic series isoselenocyanates shown in brefeldin A with formula III shown in described formula II is 1:1; Brefeldin A shown in described formula II is 1:2 with the ratio of sodium hydride-feeding amount of substance; Described tetrahydrofuran (THF) volumetric usage counts 30ml/mmol with the amount of substance of brefeldin A shown in formula II.
Further, reaction solution post-treating method of the present invention is: after reaction terminates, and reaction mixture adds water cancellation, and adds methylene dichloride and extract, organic layer is first washed, afterwards with saturated NaCl solution washing, organic phase collects rear anhydrous magnesium sulfate drying, filters, revolve steaming removing organic solvent and obtain thick product, be separated through thin-layer chromatography, collect the component of Rf=0.5 ~ 0.7, obtain the selenium of brefeldin A shown in formula I ester derivative.
The present invention also provides the application of a kind of described brefeldin A selenium ester derivative in preparation anti-tumor activity medicine, concrete preferred described brefeldin A selenium ester derivative is compound shown in formula (I-5), preferred CHL cells CHL and human lung cancer cell A549.
The invention provides a kind of described brefeldin A selenium ester derivative and prepare the application in anti-oxidation medicine, concrete preferred described brefeldin A selenium ester derivative is compound shown in formula (I-1), (I-3), (I-4) or (I-5), and described brefeldin A selenium ester derivative can remove DPPH, ABTS and superoxide radical (O
2 -).
The reaction formula of preparation BFA selenium ester derivative of the present invention is:
R in formula III
3for phenyl ring, benzyl and 2-methyl-6-ethylphenyl.
R in formula I
1for H or
r
2for
r
1, R
2middle R
3be phenyl ring, benzyl or 2-methyl-6-ethylphenyl simultaneously.
In the preparation method of brefeldin A selenium ester derivative of the present invention, first take purity be 60% sodium hydride be dissolved in anhydrous THF, then phenyl isoselenocyanates (III-1) and BFA (II) is added successively, in a nitrogen atmosphere, ice bath reaction 4 ~ 5h, reaction process with TLC trace detection (ethyl acetate: sherwood oil=1:2, v/v), until (III-1) reacts completely.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: (1) the present invention, by aromatic series isoselenocyanates and brefeldin A generation electrophilic addition reaction, has prepared new brefeldin A selenium ester derivative; (2) the invention provides a kind of new brefeldin A selenium ester derivative, study their structure activity relationship, thus have new research direction and new drug development thinking; (3) such BFA derivative has anti-oxidant activity and anti-tumor activity, wherein the antioxidant effect that had of brefeldin A selenium ester derivative (I-1), (I-3), (I-4) or (I-5), can remove DPPH, ABTS and superoxide radical (O
2 -).Shown in formula (I-5), brefeldin A selenium ester derivative has good inhibit activities to human lung cancer cell A549, far above the inhibit activities of BFA itself; (4) by research of the present invention, the selection in derivative site has vital impact to BFA related activity, brefeldin A selenium ester derivative (I-4) is more weak than the anti-tumor activity of (I-3), illustrates that 4-OH is larger than the impact of 7-OH site on anti-tumor activity.Therefore, the result of gained demonstrates BFA derivative and has broad application prospects in drug development system, for synthesis and screening BFA drug derivative provide new more wide thinking, to providing more effective approach for the treatment of relative disease.
(4) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
The preparation of embodiment 1:7-phenyl brefeldin A list selenium ester (I-1) and the two selenium ester (I-2) of 4 ' 7-phenyl brefeldin A
Reaction formula is as follows:
Take the sodium hydride (2eq that purity is 60%, 1mmol, 44mg) be dissolved in anhydrous THF (15mL), add phenyl isoselenocyanates (III-1) (1eq successively, 0.5mmol, 91mg) with BFA (II) (1eq, 0.5mmol, 140mg), in a nitrogen atmosphere, ice bath reaction 4 ~ 5h, reaction process TLC trace detection (ethyl acetate: sherwood oil=1:2, v/v), until (III-1) reacts completely.Reaction mixture adds 10ml shrend and goes out, and adds 2 × 20ml methylene dichloride and extracts, and after organic layer washing 2 × 20ml, saturated NaCl solution washing 2 × 20ml, collects the anhydrous Mg of organic phase
2sO
4drying, filters, and revolves steaming removing organic solvent and obtains thick product, be separated (ethyl acetate: sherwood oil=1:2 is developping agent, v/v), collect R through thin-layer chromatography
fthe component of=0.5 obtains 7-phenyl brefeldin A list selenium ester (I-1), collects R
fthe component of=0.7 obtains the two selenium ester (I-2) of 4 ' 7-phenyl brefeldin A.
7-phenyl brefeldin A list selenium ester (I-1) (productive rate 50%): MS (ESI) m/z464 (M+H)
+;
1hNMR (400MHz, CDCl
3) δ 7.56 – 7.33 (m, 4H), 7.25 (dd, J=21.2,6.0Hz, 1H), 5.51 (d, J=53.4Hz, 1H), 5.07 (dd, J=15.4,8.4Hz, 1H), 4.86 – 4.59 (m, 1H), 4.36 (s, 1H), 2.72 – 2.45 (m, 2H), 2.42 – 2.16 (m, 1H), 2.03 (s, 1H), 1.94 – 1.59 (m, 4H), 1.47 (dd, J=29.1,12.4Hz, 2H), 1.37 – 1.02 (m, 6H), 0.87 (d, J=9.3Hz, 2H);
13cNMR (126MHz, CDCl
3) δ 188.99,168.73,136.79,134.99,131.62,129.81,129.42,129.24,127.28,121.69,77.35,77.09,76.84,72.016,71.583,65.28,49.93,43.92,41.18,39.00,33.36,30.89,20.66;
IRν
max(cm
-1):3370.36,2932.33,1700.361639.52,1496.57,1455.60,1297.18,1209.30,1168.61，1089.67，972.50,697.72,573.33。
Two selenium ester (I-2) (productive rate 30%): MS (ESI) m/z647 (M+H) of 4 ' 7-phenylbenzene brefeldin A
+; IR ν
max(cm
-1): 2978.39,2351.71,1708.30,1631.36,1594.24,1447.98,1401.77,1362.14,1335.62,1312.58,1230.49,1114.15,1011.41,908.78,695.01,571.62.
The preparation of embodiment 2:7-benzyl brefeldin A list selenium ester (I-3) and the two selenium ester (I-4) of 4 ' 7-benzyl brefeldin A
Reaction formula is as follows:
Take the sodium hydride (2eq that purity is 60%, 1mmol, 44mg) be dissolved in anhydrous THF (15mL), add benzyl isoselenocyanates (III-2) (1eq successively, 0.5mmol, 98mg), BFA (II) (1eq, 0.5mmol, 140mg), in a nitrogen atmosphere, ice bath reaction 3 ~ 4h, reaction process TLC trace detection (ethyl acetate: sherwood oil=1:2, v/v), until (III-1) reacts completely.Reaction mixture adds 10ml shrend and goes out, and adds 2 × 20ml methylene dichloride and extracts, and after organic layer washing 2 × 20ml, saturated NaCl solution washing 2 × 20ml, collects the anhydrous Mg of organic phase
2sO
4drying, filters, and revolves steaming removing organic solvent and obtains thick product, (ethyl acetate: sherwood oil=1:2 is developping agent is separated through thin-layer chromatography, v/v), the component of collecting Rf=0.5 obtains 7-benzyl brefeldin A list selenium ester (I-3), collects R
fthe component of=0.7 obtains the two selenium ester (I-4) of 4 ' 7-benzyl brefeldin A.
7-benzyl brefeldin A list selenium ester (I-3) (productive rate 60%): MS (ESI) m/z478 (M+H)
+;
1hNMR (400MHz, CDCl
3) δ 7.31 (d, J=41.1Hz, 5H), 5.50 (d, J=14.9Hz, 1H), 5.39 (d, J=10.3Hz, 1H), 5.18 – 4.99 (m, 2H), 4.54 (d, J=4.7Hz, 1H), 4.44 – 4.26 (m, 2H), 3.79 (d, J=11.4Hz, 1H), 2.75 (d, J=13.0Hz, 1H), 2.45 – 2.35 (m, 1H), 2.29 (dd, J=22.3,12.8Hz, 2H), 1.67 (d, J=18.1Hz, 5H), 1.62 – 1.42 (m, 3H), 1.40 – 1.23 (m, 3H), 1.16 (d, J=6.6Hz, 3H).
IRν
max(cm
-1):3423.43,2977.73,2922.45,1710.71,1499.90,1453.62,1389.11,1340.32,1313.00,1267.21,1171.20,1109.10，1072.52,973.89,839.8。
Two selenium ester (I-4) (productive rate 40%): MS (ESI) m/z675 (M+H) of 4 ' 7-dibenzyl brefeldin A
+,
1hNMR (400MHz, CDCl
3) δ 7.38 (s, 1H), 7.32 (t, J=21.2Hz, 8H), 7.16 (d, J=7.1Hz, 1H), 5.75 (d, J=42.1Hz, 1H), 5.56 – 5.45 (m, 1H), 4.78 (d, J=5.2Hz, 1H), 4.51 (d, J=4.6Hz, 1H), 4.35 (dd, J=27.6, 16.6Hz, 2H), 4.10 (d, J=7.1Hz, 1H), 3.81 – 3.66 (m, 1H), 2.74 (d, J=13.0Hz, 1H), 2.45 (d, J=7.1Hz, 1H), 2.29 (d, J=13.2Hz, 1H), 2.02 (s, 1H), 1.62 (s, 2H), 1.58 (s, 2H), 1.51 – 1.43 (m, 2H), 1.37 (s, 1H), 1.24 (s, 4H), 1.20 – 1.13 (m, 4H), 1.04 (d, J=11.4Hz, 1H).IRν
max(cm
-1):3029.14，2926.61,2855.53,1719.95,1520.74,1485.42,1341.54,1243.34,1167.77，1092.55,947.24,909.93,798.85，699.71。
The preparation of embodiment 3:7-2-methyl-6-ethylphenyl brefeldin A list selenium ester (I-5) and the two selenium ester (I-6) of 4 ' 7-2-methyl-6-ethylphenyl brefeldin A
Reaction formula is as follows:
Take the sodium hydride (2eq that purity is 60%, 1mmol, 44mg) be dissolved in anhydrous THF (15mL), add benzyl isoselenocyanates (III-3) (1eq successively, 0.5mmol, 112mg), BFA (II) (1eq, 0.5mmol, 140mg), in a nitrogen atmosphere, ice bath reaction 3 ~ 4h, reaction process TLC trace detection (ethyl acetate: sherwood oil=1:2, v/v), until (III-1) reacts completely.Reaction mixture adds 10ml shrend and goes out, and adds 2 × 20ml methylene dichloride and extracts, and organic layer washing 2 × 20ml, saturated NaCl solution washing 2 × 20ml, collect the anhydrous Mg of organic phase
2sO
4dry, filter, revolve steaming removing organic solvent and obtain thick product, (ethyl acetate: sherwood oil=1:2 is developping agent is separated through thin-layer chromatography, v/v), the component of collecting Rf=0.5 obtains 7-2-methyl-6-ethylphenyl brefeldin A list selenium ester (I-5), and the component of collecting Rf=0.7 obtains 4 ' the two selenium esters (I-6) of 7-2-methyl-6-ethylphenyl brefeldin A.
7-2-methyl-6-ethylphenyl brefeldin A list selenium ester (I-5) (productive rate 60%): MS (ESI) m/z506 (M+H)
+,
1hNMR (400MHz, CDCl
3) δ 7.33 (td, J=7.6, 3.0Hz, 1H), 7.23 (t, J=8.9Hz, 1H), 7.17 (d, J=7.3Hz, 1H), 5.45 (ddd, J=15.1, 9.7, 5.3Hz, 1H), 5.30 (dd, J=15.4, 8.7Hz, 1H), 5.15 – 5.02 (m, 1H), 4.73 (dd, J=6.6, 2.7Hz, 1H), 4.49 (d, J=4.0Hz, 1H), 4.28 (ddd, J=19.3, 11.1, 5.8Hz, 1H), 2.60 – 2.48 (m, 4H), 2.31 (t, J=10.3Hz, 5H), 2.11 – 1.94 (m, 2H), 1.81 (dd, J=16.1, 8.1Hz, 2H), 1.73 – 1.65 (m, 3H), 1.61 – 1.38 (m, 3H), 1.33 (t, J=7.5Hz, 3H), 1.21 – 1.16 (m, 3H),
13cNMR (126MHz, CDCl
3) δ 168.60,142.50,140.90,135.78,135.70,134.97,132.82,130.63,130.53,129.57,127.21,77.03,72.50,71.49,64.87,44.03,41.38,38.61,38.44,33.82,33.59,24.18,23.81,20.47,17.22,14.44.
IRν
max(cm
-1):3850.15,3730.10，2973.67,2929.67,2855.26，2081.30,1972.27,1721.45,1632.50,1453.17，1360.42,1232.54，1208.27,1179.93,1090.70,977.43,914.10,844.18，696.91。
Two selenium ester (I-6) (productive rate 40%): MS (ESI) m/z731 (M+H) of 4 ' 7-2-methyl-6-ethylphenyl brefeldin A
+;
1hNMR (400MHz, CDCl
3) δ 7.31 (s, 1H), 7.26 – 6.94 (m, 5H), 5.52 (s, 1H), 5.25 (s, 1H), 5.07 (s, 1H), 4.69 (s, 1H), 4.24 (d, J=87.7Hz, 1H), 2.31 – 2.20 (m, 4H), 2.04 (s, 1H), 1.59 (s, 9H), 1.27 (dd, J=37.2,15.5Hz, 18H), 0.87 (s, 3H).
The mensuration of embodiment 4 each sample anti-oxidant activity
(1) sample preparation
Weigh respectively sample BFA, I-1, I-3, I-5, I-4, VE (vitamin-E) each 3mg, be dissolved in respectively in anhydrous methanol, preparation sample concentration is 300 μ g/mL.Carry out mensuration, the superoxide radical (O of the mensuration of removing DPPH (1,1-phenylbenzene-2-trinitrophenyl-hydrazine) free radical ability, total reducing power ability respectively
2 -) clearance rate mensuration and remove the mensuration of ABTS free radical ability.Each experimental group carries out three groups of parallel laboratory tests, averages.
(2) mensuration of scavenging ability of DPPH free radical
Experimental group, control group, blank zeroing group is divided in mensuration process.The DPPH methanol solution 200 μ L getting 1mmol/L adds in test tube, then the anhydrous methanol adding 800 μ L dilutes, the DPPH methanol solution of preparation 0.2mmol/L.(OD value is designated as A to experimental group
i) every test tube adds the DPPH methanol solution of 2mL sample liquid and 2mL, 0.2mmol/L; (OD value is designated as A to control group
j) every test tube adds 2mL sample liquid and 2mL anhydrous methanol; (OD value is designated as A to blank zeroing group
0) every test tube adds DPPH methanol solution and the 2mL anhydrous methanol of 2mL, 0.2mmol/L.Lucifuge reaction 60min at 25 DEG C, the absorbancy of working sample when ultraviolet wavelength is 517nm.
Sample to the calculation formula of DPPH radical scavenging activity is:
R％＝[1-((A
i-A
j)/A
0)]×100％
(3) mensuration of total reducing power ability
Mensuration process only needs experimental group.In every test tube, after the Tripotassium iron hexacyanide damping fluid of the PBS damping fluid of the pH=6.6 of the sample liquid of 2mL and the 0.2mol/L of 2mL and the mass concentration 1% of 2mL mixes, in 50 DEG C of water-baths, react 30min, then the mass concentration adding 2mL is respectively the TCA aqueous solution of 10%, the centrifugal 10min of 3000rpm.Get the anhydrous methanol of supernatant liquor 2mL and 2mL and the FeCl of 0.4mL mass concentration 0.1%
3the aqueous solution mixes.Lucifuge reaction 10min, the absorbancy of working sample when ultraviolet wavelength is 700nm.OD
700be worth larger, illustrate its reducing power and resistance of oxidation stronger.
(4) superoxide radical (O
2 -) mensuration of clearance rate
Experimental group, control group, blank zeroing group is divided in mensuration process.Get PBS damping fluid (adjusting pH to 8.0 with NaOH (the 1M)) 1mL of 0.2mol/L, add the anhydrous methanol of 99mL, constant volume dilution is 2mmol/L, pH=8.35, get this damping fluid 4.5mL again and add every test tube, preheating 20min under 25 DEG C i.e. room temperature.(OD value is designated as A to experimental group
x) every test tube 25mmol/L pyrogallol aqueous solution of adding 0.1mL sample liquid and 0.4mL mixes; (OD value is designated as A to control group
i) every test tube anhydrous methanol of adding 0.1mL sample liquid and 0.4mL mixes; (OD value is designated as A to blank zeroing group
0) every test tube 25mmol/L pyrogallol aqueous solution of adding 0.1mL anhydrous methanol and 0.4mL mixes; React 5min at 25 DEG C, every test tube drips concentrated hydrochloric acid (material concentration: 12mol/L) with termination reaction.The absorbancy of working sample when ultraviolet wavelength is 325nm.
Superoxide radical (O
2 -) calculation formula of clearance rate is:
R％＝[(A
0-(A
x-A
i))/A
0]×100％
(5) mensuration of ABTS free radical ability is removed
Experimental group, control group is divided in mensuration process.5mLABTS (2,2-joins nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts) after reagent and Potassium Persulphate 88 μ L lucifuge react 12-16h, use PBS damping fluid (10mmol/L, pH=7.4) reaction solution being diluted to its absorbancy when ultraviolet wavelength is 734nm is 0.70 ± 0.02, obtain ABTS free radical storing solution, for subsequent use.(OD value is designated as A to experimental group
x) every test tube adds 1mL sample solution and 3mLABTS free radical storing solution; (OD value is designated as A to control group
0) every test tube adds 1mL anhydrous methanol and 3mLABTS free radical storing solution.Lucifuge reaction 5min at 25 DEG C, the absorbancy of working sample when ultraviolet wavelength is 734nm.
Sample to the calculation formula of ABTS radical scavenging activity is:
R％＝[(A
0-A
x)/A
0]×100％
Each experimental result is in table 1 and table 2.
Table 1 each sample scavenging ability of DPPH free radical measures and measures with total reducing power
Note: 1) * represents that sample is too small to DPPH radical scavenging activity, cannot calculate;
2) A in scavenging ability of DPPH free radical determination experiment
0value is 0.303.
Table 2 each sample removes superoxide radical (O
2 -) ability measures and ABTS free radical ability measures
Note: remove superoxide radical (O
2 -) A in ability determination experiment
0value is 0.344.
As can be seen from Table 1, chemical compounds I-3, I-4 pair of DPPH free radical have good Scavenging activity, and wherein the Scavenging activity of chemical compounds I-3 is comparatively strong, and higher than VE Scavenging activity; Chemical compounds I-1, I-3, total reducing power of I-5 and VE be more or less the same, and improve compared with BFA itself.
As can be seen from Table 2, chemical compounds I-1, I-3, I-5, I-4 is to removing superoxide radical (O
2 -) ability is comparatively strong, the Scavenging activity of I-3, I-5, higher than VE, still declines to some extent compared with BFA itself, but impact is little.Chemical compounds I-1, I-3, I-5, the removing ABTS free radical ability overall capacity of I-4 is strong, wherein the Scavenging activity of I-1, I-3, I-5, I-4 is stronger, the raising in certain limit is had, far above the Scavenging activity of VE compared with the Scavenging activity of BFA itself.
The mensuration of embodiment 5 each sample cell growth rejection ability
(1) people's lung cancer (A549) cell cultures
Culture condition: RPMI1640 nutrient solution+10%/bovine serum, 37 DEG C, 5%CO
2incubator.
Conditions of cryopreservation: RPMI1640 nutrient solution+10%/foetal calf serum+10%DMSO.
Propagating method: cell grows into 80-90% and still goes down to posterity for during individual layer in culturing bottle.Discard old liquid, in culturing bottle, add 2mLPBS damping fluid (pH value is 7.2 ± 0.1), clean, discard, repeat twice.Add Digestive system (0.25% trypsinase+0.03%EDTA) 1mL to digest, basis of microscopic observation cell departs from completely after a bottle wall is separated into 3-5 cell of uniting and adds 2mL (RPMI1640 nutrient solution+10%/bovine serum) nutrient solution termination digestion, in unicellular under piping and druming cell to microscope, cell suspension moves in centrifuge tube, the centrifugal 5min of 1000r/min, abandon supernatant, RPMI1640 nutrient solution re-suspended cell repeats twice.Be dispensed in T25 culturing bottle after the mixing of nutrient solution re-suspended cell, add nutrient solution to 4-5mL, 37 DEG C, 5%CO
2cultivate in incubator.
(2) CHL cells (CHL) is cultivated
Culture condition: RPMI1640 nutrient solution+10%/bovine serum, 37 DEG C, 5%CO
2incubator.
Conditions of cryopreservation: RPMI1640 nutrient solution+10%/foetal calf serum+10%DMSO.
Propagating method: cell grows into 80-90% and still goes down to posterity for during individual layer in culturing bottle.Discard old liquid, in culturing bottle, add 2mLPBS damping fluid (pH value is 7.2 ± 0.1), clean, discard, repeat twice.Add Digestive system (0.25% trypsinase+0.03%EDTA) 1mL to digest, basis of microscopic observation cell departs from completely after a bottle wall is separated into 3-5 cell of uniting and adds 2mL (RPMI1640 nutrient solution+10%/bovine serum) nutrient solution termination digestion, in unicellular under piping and druming cell to microscope, cell suspension moves in centrifuge tube, the centrifugal 5min of 1000r/min, abandon supernatant, RPMI1640 nutrient solution re-suspended cell repeats twice.Be dispensed in T25 culturing bottle after the mixing of nutrient solution re-suspended cell, add nutrient solution to 4-5mL, 37 DEG C, 5%CO
2cultivate in incubator.
(3) anti-tumor activity experiment (mtt assay)
A549 cell attachment to T25 bottle about 90% time, through digestion make cell suspending liquid, be evenly laid on 96 orifice plates, concentration is 1.6-2 × 10
4individual/hole.Experiment establishes 6 concentration gradient dosing groups, 1 control group, 1 zeroing group, and often group 3 is parallel, and control group is with solubility promoter alternatives to medication, and zeroing group adds same volume substratum, and 96 orifice plate rims add PBS damping fluid.Cell in 37 DEG C, 5%CO
224h cultivated by incubator, to cell attachment merisis to after accounting for bottle wall 70-80%, it is 2.5 μMs that dosing group adds final concentration respectively, 1.875 μM, 1.25 μM, 0.625 μM, 0.3125 μM, the drug solution 200 μ L (described drug solution is that the RPMI1640 nutrient solution of compound respectively containing volumetric concentration 8% solubility promoter DMSO embodiment I-4 prepared is formulated) of 0.15625 μM, control group adds the RPMI1640 nutrient solution 200 μ L containing volumetric concentration 8% solubility promoter DMSO, zeroing group then adds RPMI1640 nutrient solution 200 μ L, in 37 DEG C, 5%CO
224h cultivated by incubator.Dosing group, control group and zeroing group add 20 μ LMTT (5mg/mL, PBS now joined film and use) effect 4h respectively, careful sucking-off nutrient solution, then add 200 μ LDMSO respectively, and oscillator plate shakes up about 10min and fully dissolves to precipitation; Use microplate reader to measure absorbancy in 570nm place, calculate respective inhibiting rate, utilize SPSS20.0 computed in software IC50 value.
(4) anti-tumor activity control experiment (mtt assay)
CHL cell attachment to T25 bottle about 90% time, through digestion make cell suspending liquid, be evenly laid on 96 orifice plates, concentration is 1.6-2 × 10
4individual/hole.Experiment establishes 6 concentration gradient dosing groups, 1 control group, 1 zeroing group, and often group 3 is parallel, and control group is with solubility promoter alternatives to medication, and zeroing group adds same volume substratum, and 96 orifice plate rims add PBS damping fluid.Cell in 37 DEG C, 5%CO
224h cultivated by incubator, to cell attachment merisis to after accounting for bottle wall 70-80%, it is 2.5 μMs that dosing group adds final concentration respectively, 1.875 μM, 1.25 μM, 0.625 μM, 0.3125 μM, the drug solution 200 μ L (described drug solution is that the compound prepared by embodiment 1-4 is formulated with the RPMI1640 nutrient solution containing volumetric concentration 8% solubility promoter DMSO respectively) of 0.15625 μM, control group adds the RPMI1640 nutrient solution 200 μ L containing volumetric concentration 8% solubility promoter DMSO, and zeroing group then adds RPMI1640 nutrient solution 200 μ L.In 37 DEG C, 5%CO
224h cultivated by incubator.Dosing group, control group and zeroing group add 20 μ LMTT (5mg/mL, PBS now joined film and use) effect 4h respectively, careful sucking-off nutrient solution, and then add 200 μ LDMSO respectively, and oscillator plate shakes up about 10min and fully dissolves to precipitation; Use microplate reader to measure absorbancy in 570nm place, calculate respective inhibiting rate, utilize SPSS20.0 computed in software IC50 value.
Sample experiments the results are shown in Table 3.
Table 3BFA selenium ester class derivative compound to A549 and CHL cell inhibitory activity
Note: 1), * represents does not have inhibit activities or inhibit activities to be far longer than 500 μMs.
2), SI=IC50 (A549)/IC50 (CHL), SI is less, and the selectivity of drug on tumor cell is higher.
As shown in table 3, BFA selenium ester derivative goes out anti-tumor activity to A549 cells show, and wherein monoester compound I-5 is active best, increases substantially compared to the activity of BFA own; Monoester compound I-1, I-3 is better active, decreases compared to the activity of BFA own; The own activity decrease of specific activity BFA of diester compound I-4 about 13 times, but the inhibition of BFA selenium ester derivative to A549 cell to be far longer than CHL cell inhibiting, this selectivity contrast all improves with BFA itself.
Claims (10)
1. the selenium of brefeldin A shown in a formula I ester derivative,
R in formula I
1for H or
r
1, R
2middle R
3be phenyl ring, benzyl or 2-methyl-6-ethylphenyl simultaneously.
2. brefeldin A selenium ester derivative as claimed in claim 1, is characterized in that described brefeldin A selenium ester derivative is one of following:
3. brefeldin A selenium ester derivative as claimed in claim 2, is characterized in that described brefeldin A selenium ester derivative is compound shown in formula (I-1), (I-3), (I-4) or (I-5).
4. the preparation method of brefeldin A selenium ester derivative described in a claim 1, it is characterized in that described method for: with aromatic series isoselenocyanates shown in brefeldin A and formula III shown in formula II for raw material, take tetrahydrofuran (THF) as solvent, take sodium hydride as catalyzer, logical nitrogen, react under condition of ice bath, after reaction terminates, reaction solution aftertreatment obtains the selenium of brefeldin A shown in formula I ester derivative;
R in formula III
3for phenyl ring, benzyl or 2-methyl-6-ethylphenyl.
5. preparation method as claimed in claim 4, is characterized in that the feed intake ratio of amount of substance of aromatic series isoselenocyanates shown in brefeldin A with formula III shown in described formula II is 1:1; Brefeldin A shown in described formula II is 1:2 with the ratio of sodium hydride-feeding amount of substance; Described tetrahydrofuran (THF) volumetric usage counts 30ml/mmol with the amount of substance of brefeldin A shown in formula II.
6. preparation method as claimed in claim 4, it is characterized in that described reaction solution post-treating method is: after reaction terminates, reaction mixture adds water cancellation, and adds methylene dichloride and extract, and organic layer is first washed, afterwards with saturated NaCl solution washing, organic phase collects rear anhydrous magnesium sulfate drying, filters, and revolves steaming removing organic solvent and obtains thick product, be separated through thin-layer chromatography, obtain the selenium of brefeldin A shown in formula I ester derivative.
7. described in a claim 1, brefeldin A selenium ester derivative is preparing the application in anti-oxidation medicine.
8. apply as claimed in claim 7, it is characterized in that described brefeldin A selenium ester derivative is compound shown in formula (I-1), (I-3), (I-4) or (I-5).
9. the application of brefeldin A selenium ester derivative described in a claim 1 in preparation anti-tumor activity medicine.
10. apply as claimed in claim 9, it is characterized in that described brefeldin A selenium ester derivative is compound shown in formula (I-5).
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106928209A (en) * | 2017-03-10 | 2017-07-07 | 沈阳药科大学 | Brefeldin A derivative and its production and use |
| CN106928213A (en) * | 2017-03-10 | 2017-07-07 | 沈阳药科大学 | Double furazan NO donor substitutive derivatives of 4,7 of brefeldin A and its production and use |
| CN106928210A (en) * | 2017-03-10 | 2017-07-07 | 沈阳药科大学 | 4 Preparation method and uses of furazan NO donor type derivants of brefeldin A |
| CN106928210B (en) * | 2017-03-10 | 2019-04-02 | 沈阳药科大学 | The Preparation method and use of the position the 4- furazan NO donor type derivant of brefeldin A |
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| CN106928213B (en) * | 2017-03-10 | 2019-08-30 | 沈阳药科大学 | Double furazan NO donor substitutive derivatives in the position 4,7- of brefeldin A and its preparation method and application |
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