CN102924428B - Oligothiophene - Google Patents
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- CN102924428B CN102924428B CN201210405073.XA CN201210405073A CN102924428B CN 102924428 B CN102924428 B CN 102924428B CN 201210405073 A CN201210405073 A CN 201210405073A CN 102924428 B CN102924428 B CN 102924428B
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- thiophenes
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- 150000003839 salts Chemical class 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 81
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 abstract description 16
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- 230000009702 cancer cell proliferation Effects 0.000 abstract 1
- 239000000411 inducer Substances 0.000 abstract 1
- 239000000203 mixture Substances 0.000 description 35
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- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical compound C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 18
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- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 2
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Abstract
The invention discloses oligothiophene as well as a preparation method and application thereof. The structural formula of the oligothiophene is shown as a formula (III) described in the specification. In the formula (III), m is equal to 3, and n is an integer of 1-6. The oligothiophene is applied to five aspects as follows: (1) application for preparing cancer cell proliferation inhibitors; (2) application for preparing cancer cell apoptosis inducers; (3) application for preparing cancer preventing and/or treating anti-cancer drugs; (4) application for preparing mitochondria targeted drugs; and (5) application in cell imaging as mitochondrial dyes. The oligothiophene keeps a framework of a conjugated polymer, so fluorescence properties of the oligothiophene can be used for cell imaging. The oligothiophene shown as the formula (III) is a specific dye for mitochondrion by experiments. Furthermore, the oligothiophene shown as the formula (III) further can be compounded with chlorambucil to form nano-particles. The results show that the compound is featured by the broad-spectrum cancer resistance and capable of increasing the anti-tumor activity.
Description
The application is to be dividing an application of June 15, application number in 2011 are 201110160346.4, denomination of invention is " a kind of oligo-thiophenes and preparation method thereof and application " innovation and creation the applying date.
Technical field
The present invention relates to a kind of oligo-thiophenes and preparation method thereof and application.
Background technology
Cancer has become one of main killer of harm humans health.In the therapeutic process of tumour, resistance is a murderous principal element.Along with the more and more advanced person for the treatment of means, it is more and more general that acquired resistance also becomes.Due to the generation of resistance, many very effective anticarcinogens, as the result for the treatment of of cis-platinum, Chlorambucil, taxol etc. reduces greatly.Therefore find the new lead compound with broad-spectrum anti-tumor activity, find that new compound skeleton is significant.
Conjugated polymers (CPs) is because main chain electron delocalization demonstrates unique physical/chemical, and three ten years in the past have become one of the most noticeable advanced material.Oligo-thiophenes is owing to having retained the skeleton of conjugated polymers, and its photoluminescent property can be used for carrying out cell imaging.Up to the present, there is not yet the report that relevant oligo-thiophenes has multifunctional anticancer activity.
Plastosome is called as the generator of cell, yet due to the singularity of membrane structure in it, allows hardly any molecule to see through.Therefore this character does not allow drug molecule to pass through equally, and to be directed to mitochondrial medicine significant to overcoming resistance in exploitation.
Chlorambucil (chlorambucil) is a kind of medicine that is widely used in treatment chronic lymphocytic leukemia.Reason based on resistance, finding new therapeutic strategy is challenging to overcoming the resistance of chlorambucil.
Summary of the invention
The object of this invention is to provide a kind of oligo-thiophenes with anticancer, cell imaging function and preparation method thereof.
The structural formula of oligo-thiophenes provided by the present invention is as shown in formula III:
(formula III)
Wherein, m=3; N is the integer in 1-6; R
1be selected from any one in following radicals :-COOH ,-NH
2,-NHCH
3,-N (CH
3)
2,-N
+(CH
3)
3,-N
+(CH
3)
3br
-,-NH (CH
2cH
3) ,-N
+(CH
2cH
3)
3,-O
-n
+(CH
2cH
3)
2,-COOCH
3,-COOCH
2cH
3,-SO
3h ,-SO
3cH
3,-CH
2cOOH ,-CH
2cOOCH
3,-NO
2,-PO
3,-CHO ,-OH ,-N
3,-OCHO ,-CN,
It is three Polythiophenes shown in the single thiophene shown in formula I and formula II that the present invention also protects two intermediates of oligo-thiophenes shown in preparation formula III
(formula I) (formula II)
Wherein, the n in formula I and formula II is the integer in 1-6; R
1be selected from any one in following radicals :-COOH ,-NH
2,-NHCH
3,-N (CH
3)
2,-N
+(CH
3)
3,-N
+(CH
3)
3br
-,-NH (CH
2cH
3) ,-N
+(CH
2cH
3)
3,-O
-n
+(CH
2cH
3)
2,-COOCH
3,-COOCH
2cH
3,-SO
3h-SO
3cH
3,-CH
2cOOH ,-CH
2cOOCH
3,-NO
2,-PO
3,-CHO ,-OH ,-N
3,-OCHO ,-CN ,-Cl ,-I ,-Br,
M=1 in formula II.
The method of preparing oligo-thiophenes shown in right formula III, comprises the steps:
1) 2-(3-thienyl) ethanol is reacted under sodium hydride effect with halogenated alkane shown in formula IV, obtains the compound shown in formula I,
X(CH2)
nR
1 
(formula IV) (formula I)
N in formula IV is the integer in 1-6, and X is Cl, Br or I, R
1with R in formula I
1identical;
2) compound shown in formula I is reacted with N-bromo-succinimide, obtain the compound shown in formula V;
3) compound shown in formula V is reacted under palladium (0) katalysis with single boric acid ester shown in formula VI, obtain the oligo-thiophenes shown in formula II;
(formula V) (formula VI)
R=(CH in formula V, formula VI
2)
nr
1, the integer that n is 1-6, R
1with R in formula I
1identical;
4) compound shown in formula II is reacted with N-bromo-succinimide, obtain the compound shown in formula VII;
(formula VII)
5) compound shown in formula VII is reacted under palladium (0) katalysis with single boric acid ester shown in formula VI, obtain oligo-thiophenes shown in formula III.
Wherein, the palladium (0) in step 3) and step 5) is tetrakis triphenylphosphine palladium.
Another object of the present invention is to provide the application of polymkeric substance shown in formula III.
The application of polymkeric substance shown in of the present invention provided formula III comprises following five aspects:
1) application of polymkeric substance shown in formula III in preparing cancer cell multiplication inhibitor;
2) application of polymkeric substance shown in formula III in preparing cancer cell-apoptosis inductor;
3) shown in formula III, polymkeric substance prevents and/or treats the application in cancer drug in preparation;
4) application of polymkeric substance shown in formula III in the Mitochondrially targeted medicine of preparation;
5) application in cell imaging as plastosome dyestuff.
Wherein, described cancer cells be selected from following at least one: kidney cancer cell, lung carcinoma cell, liver cancer cell, breast cancer cell and colon cancer cell; The preferred human renal carcinoma cell A498 of described kidney cancer cell, the preferred human lung cancer cell A549 of described lung carcinoma cell, the preferred human liver cancer cell HepG2 of described liver cancer cell, the preferred human breast cancer cell MCF-7 of described breast cancer cell, the preferred human colon cancer cell HCT116 of described colon cancer cell;
Described cancer be selected from following at least one: kidney, lung cancer, liver cancer, mammary cancer and colorectal carcinoma.
A further object of the present invention is to provide a kind of mixture with broad spectrum anticancer.
Mixture provided by the present invention is composited by oligo-thiophenes shown in formula III and Chlorambucil salt.
In described mixture, the mol ratio of oligo-thiophenes shown in formula III and Chlorambucil salt is 1:(1-5).The form of described mixture is nano particle, and its particle diameter is 10-100nm.
The preparation method of this mixture, comprises the steps: oligo-thiophenes shown in formula III to be dissolved in water, and dropwise adds wherein the aqueous solution of Chlorambucil salt; Mixture is stirred to 48-96 hour, obtain described mixture.
The mixture providing in the present invention can be used for preparing cancer cell multiplication inhibitor or for the preparation of the application preventing and/or treating in cancer drug.
Wherein, described cancer cells be selected from following at least one: kidney cancer cell, lung carcinoma cell, liver cancer cell, breast cancer cell and colon cancer cell; The preferred human renal carcinoma cell A498 of described kidney cancer cell, the preferred human lung cancer cell A549 of described lung carcinoma cell, the preferred human liver cancer cell HepG2 of described liver cancer cell, the preferred human breast cancer cell MCF-7 of described breast cancer cell, the preferred human colon cancer cell HCT116 of described colon cancer cell;
Described cancer be selected from following at least one: kidney, lung cancer, liver cancer, mammary cancer and colorectal carcinoma.
The invention provides the mitochondrial oligo-thiophenes with multifunctional anticancer activity of a kind of novel target.This oligo-thiophenes is owing to having retained the skeleton of conjugated polymers, and its photoluminescent property can be used for carrying out cell imaging.By test, find, oligo-thiophenes shown in formula III is mitochondrial single-minded dyestuff.In addition, oligo-thiophenes shown in formula III of the present invention also can form with Chlorambucil the mixture with nanostructure by electrostatic interaction.Result shows that this mixture has broad spectrum anticancer, has improved anti-tumor activity.
Accompanying drawing explanation
Fig. 1 is the chemical reaction flow process figure of the synthetic oligo-thiophenes of the present invention.
Fig. 2 is the reacting flow chart of the mixture of the synthetic oligo-thiophenes of the present invention and Chlorambucil salt.
Fig. 3 is the stereoscan photograph of the mixture of the synthetic oligo-thiophenes of the present invention and Chlorambucil salt.
Fig. 4 is cell A498, the HepG2 cell survival rate under single thiophene and oligo-thiophenes effect; Wherein, 1T represents single thiophene 3, and 3T represents oligo- thiophenes 7, and 5T represents oligo-thiophenes 9.
Fig. 5 is cell A498, the MCF-7 cell survival rate under the mixture effect of oligo-thiophenes and Chlorambucil salt.
Fig. 6 is the cell imaging figure of oligo-thiophenes 9.
Fig. 7 is the apoptotic flow cytometer showed figure of oligo-thiophenes 9 induction A498.
Embodiment
Below by specific embodiment, the present invention will be described, but the present invention is not limited thereto.
Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
Prepare single thiophene 2:663 microlitre (6 mmole) 2-(3-thienyl) ethanol joins in the dry DMF solution of sodium hydride (content 70%, 206mg, 6 mmoles) under nitrogen atmosphere.Stir after 30 minutes, it is excessive 1 then to add, 6-dibromo-hexane (4.6 milliliters, 10 mmoles), stirred overnight at room temperature.Add the shrend reaction of going out, methylene dichloride (3 * 100 milliliters) extracts three times, anhydrous magnesium sulfate drying, and after concentrating under reduced pressure, column chromatography (silica gel; Sherwood oil: ethyl acetate=40:1, v/v) separated 0.54 gram of product, the productive rate 31% of obtaining.Single thiophene 2 structural identification data are as follows:
1h NMR (400MHz, CDCl
3) δ 1.61-1.45 (m, 4H), 1.72 (p, 2H; J=6.78), 1.99 (p, 2H, J=6.82); 3.04 (t, 2H, J=6.98), 3.53 (t; 2H, 6.86), 3.57 (t, 2H; J=6.50), 3.76 (t, 2H, J=7.01); 7.15-7.10 (m, 2H), 7.37 (m, 1H);
13c NMR (75MHz, CDCl
3) δ 25.43,28.01,29.57,30.83,32.79,33.82,70.77,71.03,121.04,125.14,128.53,139.46; HREI-MS Calcd.for C
12h
19brOS m/z Acc.Mass290.0340,292.0320; Obs.Mass 290.0342,290.0316.
Prepare single thiophene 3: in the tetrahydrofuran solution of single thiophene 2 (291 milligrams, 1 mmole), add the aqueous solution (3 milliliters) of excessive Trimethylamine 99, stirring at room 24 hours, pressure reducing and steaming Trimethylamine 99, obtains white precipitate, is single thiophene 3.Structural identification data are as follows:
1h NMR (400MHz, CDCl
3) δ 7.27 (m, 1H), 7.02-6.98 (m, 2H), 3.65-3.56 (m, 4H), 3.47 (m, 11H), 2.91 (t, 2H, 6.78), 1.75 (br, 2H), 1.58 (br, 2H), 1.42 (br, 4H);
13c NMR (75MHz, CDCl
3) δ 23.02,25.76,25.81,29.25,30.61,53.27,66.64,70.37,70.79,120.96,125.10,128.48,139.34; ESI-MS m/z:270.2 (M); Anal.Calcd.for C
15h
28brNOS:C51.42, H 8.06, and N 4.00; Found C 51.11, H 7.95, N 4.12.
Prepare single thiophene 4(R=(CH
2)
6br): in being dissolved with the tetrahydrofuran (THF) of 1.00 grams of single thiophene 2 and the mixing solutions of acetic acid (12 milliliters, 1:1, v/v) disposablely add 1.22 grams of N-bromo-succinimides, mixture stirring at room 1 hour, then pour in 30 ml waters and extract three times with Skellysolve A (3 * 15 milliliters), anhydrous magnesium sulfate drying, after concentrating under reduced pressure, column chromatography (silica gel; Sherwood oil: ethyl acetate=40:1, v/v) separated 1.38 grams of products, the productive rate 90% of obtaining.Single thiophene 4 structural identification data are as follows:
1h NMR (400MHz, CDCl
3) δ=7.26 (s, 1H), 6.87 (s, 1H), 3.56 (t, J=6.5,2H), 3.42 (dd, J=11.6,5.7,4H), 2.79 (t, J=6.5,2H), 1.95-1.79 (m, 2H), 1.65-1.51 (m, 2H), 1.51-1.30 (m, 4H).
13c NMR (100MHz, CDCl
3) δ=139.75,131.53,110.43,109.03,87.18,70.83,69.49,34.03,32.82,30.13,29.55,28.03,25.46.EI-MS m/z:448; Anal.Calcd.for C
12h
17br
3oS:C 32.10, and H 3.82; Found C 32.05, H 4.01.
Prepare single boric acid ester 5(R=(CH
2)
6br): at the DMF(108 of 2.65 grams of single thiophene 2 milliliter) in solution, splash into 54 milliliters of the DMF solution of the N-bromo-succinimide of 1.62 grams.Mixture stirring at room 24 hours, then pours in 30 ml waters and extracts three times with Skellysolve A (3 * 15 milliliters), anhydrous magnesium sulfate drying, and after concentrating under reduced pressure, column chromatography (silica gel; Sherwood oil: ethyl acetate=40:1, v/v) 2.80 grams of 2 single bromination products of separated single thiophene 2, productive rate 90%.In 20 milliliters of tetrahydrofuran solutions of-78 ℃ of single bromination products that contain 1.00 grams, by syringe, add 1.04 milliliters of n-Butyl Lithiums (hexane solution of 2.72M).Mixture continues to stir 2 hours at-78 ℃, then adds 0.7 milliliter of 2-isopropoxy-4,4,5,5-tetramethyl--1,3,2-dioxy boron pentane, mixture continues to stir 1 hour at-78 ℃, is heated to room temperature, continues to stir 12 hours, then pour in 30 ml waters and extract three times with Skellysolve A (3 * 15 milliliters), anhydrous magnesium sulfate drying, after concentrating under reduced pressure, column chromatography (silica gel; Sherwood oil: ethyl acetate=40:1, v/v) separated 0.57 gram of product, the productive rate 51% of obtaining.Single boric acid ester 5 structural identification data are as follows: 1H NMR (400MHz, CDCl3) δ=7.50 (d, J=3.8, 1H), 7.07 (d, J=3.8, 1H), 3.61 (t, J=6.9, 2H), 3.43 (dt, J=13.5, 6.2, 4H), 3.18 (t, J=6.8, 2H), 1.93-1.80 (m, 2H), 1.64-1.53 (m, 2H), 1.51-1.36 (m, 4H), 1.33 (s, 12H) .13C NMR (100MHz, CDCl3) δ=150.34, 131.49, 130.82, 83.71, 71.81, 70.54, 33.95, 32.82, 30.75, 29.63, 28.05, 25.46, 24.86.EI-MS m/z:416.Anal.Calcd.for C18H30BBrO3S:C 51.82, H7.25, Found C 51.85, H 7.13.
Oligo-thiophenes 6(R=(CH
2)
6br): at 0.51 mmole list thiophene 4,4.09 mmole salt of wormwood, the Pd (PPh of 0.09 mmole
3)
4in the toluene and water mixed solvent of (tetrakis triphenylphosphine palladium (0)) (toluene/water=3/1, v/v), the disposable single boric acid ester 5(R=(CH that adds 0.60 mmole
2)
6br) tetrahydrofuran solution.Mixture stirs after 20 minutes and continues to reflux 20 hours under nitrogen.Cool to room temperature, adds 15 ml waters.Mixture dichloromethane extraction, anhydrous magnesium sulfate drying, concentrated rear column chromatography (silica gel; Sherwood oil: ethyl acetate=500:35, v/v) purifying obtains product.The structural identification data of oligo-thiophenes 6 are as follows:
1h NMR (400MHz, CDCl
3) δ=7.31 (s, 1H), 7.18 (s, 1H), 7.07 (s, 1H), 7.03 (s, 1H), 6.99 (d, J=2.9,1H), 3.65 (s, 2H), 3.55 (s, 4H), 3.44 (s, 2H), 3.39 (s, 10H), 3.06 (s, 2H), 2.83 (s, 2H), 2.77 (s, 2H), 1.84 (s, 6H), 1.55 (s, 6H), 1.43 (s, 6H), 1.36 (s, 6H).
13c NMR (100MHz, CDCl
3) δ=139.27,139.04,135.89,135.71,131.74,130.34,129.62,129.15,129.09,128.02,125.87,123.90,70.86,70.80,70.72,33.94,32.79,29.59,29.44,28.04,25.45.MS (MALDI-TOF): 870.3 (M), 790.3 (M-Br) .Anal.Calcd.for C
36h
53br
3o
3s:C 49.72, and H 6.14; Found C 49.76, H 6.23.
Oligo-thiophenes 7(R '=(CH
2)
6n
+(CH
3)
3br): reactions steps is synthetic referring to single thiophene 3.The aqueous solution that adds excessive Trimethylamine 99 in the tetrahydrofuran solution of oligo-thiophenes 6, stirring at room 24 hours, pressure reducing and steaming Trimethylamine 99, obtains oligo-thiophenes 7.Structural identification data are as follows:
1h NMR (400MHz, MeOD) δ=7.54 (d, J=3.9,1H), 7.40 (d, J=3.8,1H), 7.24 (s, 1H), 7.17 (d, J=3.9,1H), 7.12 (d, J=3.9,1H), 3.80-3.69 (m, 2H), 3.65 (s, 4H), 3.58-3.44 (m, 6H), 3.40 (s, 6H), 3.19 (s, 27H), 3.11 (s, 2H), 2.87 (s, 2H), 2.82 (s, 2H), 1.81 (s, 6H), 1.63 (s, 6H), 1.45 (s, 12H).
13c NMR (100MHz, MeOD) δ=139.64,139.20,136.08,135.73,131.22,130.29,129.48,128.97,128.63,128.03,125.93,124.03,70.41,70.35,70.30,70.25,66.45,59.26,52.28,48.52,48.31,48.09,47.88,47.67,47.45,47.24,47.03,29.38,29.11,25.76,25.54,25.48,22.58.ESI-MS m/z:269.1 (M-3Br), 443.7 (M-2Br).
Oligo-thiophenes 8(R=(CH
2)
6br): at-20 ℃, (44 milligrams of N-bromo-succinimides (NBS), 0.247 mmole) be dissolved in 10 milliliters of DMF(N, dinethylformamide) in, dropwise join (107 milligrams of oligo-thiophenes 6,0.123 mmole), in 5 milliliters of DMF solution, whole process added in 5 minutes.Mixture continues to stir 2 hours, is heated to room temperature, reaction overnight.Reaction soln is joined in 100 ml waters to Skellysolve A extraction three times, anhydrous magnesium sulfate drying, concentrated rear column chromatography (silica gel; Sherwood oil: ethyl acetate=500:35, v/v) purifying obtains 6,110 milligrams of product two bromo oligo-thiophenes, productive rate 93%.Two bromo oligo-thiophenes 6 react to obtain product oligo-thiophenes 8 with single boric acid ester 5 shown in embodiment 2, and concrete reactions steps is synthetic referring to oligo-thiophenes 6.The structural identification data of oligo-thiophenes 8 are as follows:
1h NMR (400MHz, CDCl
3) δ=7.26 (s, 2H), 7.19 (d, J=4.3,2H), 7.10 (d, J=3.9,2H), 7.05 (s, 1H), 7.00 (d, J=4.4,2H), 3.67 (t, J=7.0,6H), 3.59 (t, J=6.4,4H), 3.52-3.29 (m, 20H), 3.07 (s, 6H), 2.83 (t, J=6.2,4H), 1.84 (dd, J=12.9,6.3,10H), 1.58 (d, J=6.2,10H), 1.40 (dd, J=10.5,6.2,20H).
13c NMR (100MHz, CDCl
3) δ=139.64,136.28,136.25,136.03,135.95,135.81,134.18,131.76,131.67,130.48,129.51,129.40,129.15,128.21,128.09,124.16,71.07,71.00,70.86,70.83,70.68,34.06,32.91,30.08,29.95,29.75,29.71,28.18,27.18,25.62,25.56, MS (MALDI-TOF): 1448.4 (M), 1366.4 (M-Br) .Anal.Calcd.for C
60h
87br
5o
3s:C 49.76, and H 6.06; Found C 49.67, H 6.06.
Oligo-thiophenes 9(R '=(CH
2)
6n
+(CH
3)
3br): reactions steps is synthetic referring to single thiophene 3.The aqueous solution that adds excessive Trimethylamine 99 in the tetrahydrofuran solution of oligo-thiophenes 8, stirring at room 24 hours, pressure reducing and steaming Trimethylamine 99, obtains oligo-thiophenes 9.Structural identification data are as follows:
1h NMR (400MHz, MeOD) δ=7.46 (s, 2H), 7.34 (s, 1H), 7.31 (s, 1H), 7.27 (s, 1H), 7.16 (d, J=2.5,2H), 3.79 (s, 6H), 3.71 (s, 4H), 3.62-3.49 (m, 10H), 3.42 (s, 10H), 3.20-3.23 (m, 45H), 3.14 (s, 6H), 2.91 (s, 4H), 1.83 (s, 10H), 1.68 (s, 10H), 1.48 (d, J=5.2,20H).
13c NMR (100MHz, MeOD) δ=140.12,136.92,136.39,136.31,136.16,135.43,134.09,131.08,130.97,130.49,130.46,129.35,129.01,128.58,128.16,128.11,124.27,70.48,70.40,70.31,70.26,70.13,66.42,58.84,52.35,29.60,29.52,29.48,29.22,29.18,25.79,25.60,25.50,22.66,22.63.ESI-MS m/z:268.9 (M-5Br), 356.3 (M-4Br), 501.2 (M-3Br).
By the oligo-thiophenes 9(2.41 milligram of embodiment 3 preparations, 1.38 micromoles) be dissolved in 5 ml water solution, dropwise add the aqueous solution of 5 milliliters of Chlorambucil sodium salts (0.450 milligram, 1.38 micromoles).Mixture vigorous stirring 48 hours, lyophilize, obtains mixture 1 (oligomer: Chlorambucil=1:1, mol/mol).
By change Chlorambucil sodium salt add-on (0.9 milligram, 2.76 micromoles; 1.35 milligrams, 4.14 micromoles; 1.78 milligrams, 5.52 micromoles; 2.25 milligram, 6.90 micromoles) with similarity method synthesising complex 2 (oligomer: Chlorambucil sodium salt=1:2, mol/mol), mixture 3 (oligomer: Chlorambucil sodium salt=1:3, mol/mol), mixture 4 (oligomer: Chlorambucil sodium salt=1:4, mol/mol), mixture 5 (oligomer: Chlorambucil sodium salt=1:5, mol/mol).Its stereoscan photograph as shown in Figure 3.
Utilize above-mentioned synthetic oligo-thiophenes and mixture thereof to adopt mtt assay to carry out anti tumor activity in vitro mensuration.Human lung cancer cell A549's cell strain, human liver cancer cell HepG2, human renal carcinoma cell A498, human breast cancer cell MCF-7, human colon cancer cell HCT116 and normal human embryonic lung diploid fibroblast HPF used in test are all purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre.
Concrete steps are as follows: human lung cancer cell A549's cell strain, after trysinization suspends, with the DMEM cell culture fluid containing 10% serum, adjust cell concn to 4-7 * 10
4individual/mL, joins 96 porocyte culture plates, and 100 μ L/ holes, establish parallel control hole, put into 5%CO
2in incubator, cultivate 24h for 37 ℃ and make cell attachment.The oligo-thiophenes that adds respectively different concns, the medicinal composition of embodiment 4 preparations, arranges zeroing hole, control wells, continues to cultivate 48h.Take out cell, outwell nutrient solution, add PBS (pH 7.4) solution of 1mg/mL MTT, 100 μ L/ holes, 37 ℃ are continued to cultivate 4h.After 4h, outwell liquid, add DMSO 150 μ L/ holes, micro-oscillator concussion 5min, fully dissolves blue coloured particles formazan wherein.Culture plate is put into microplate reader, and 520nm measures OD value.Calculate as follows survival rate: survival rate (%)=administration group cell mean light absorbency value/cellular control unit mean light absorbency value * 100%.Finally use SPSS (Ver.13.0) software to process experimental data, calculate cytoactive.
Carry out revision test 3 times, data and result are mean value.With human lung cancer cell A549 and the liver cancer cell HepG2 of vitro culture, human renal carcinoma cell A498, human breast cancer cell MCF-7, human colon cancer cell HCT116, and normally human embryonic lung diploid fibroblast HPF is model, MTT experimental procedure is the same.
Result is as follows:
1) various cells and oligo-thiophenes 9 act on respectively 48 hours, the IC of 9 pairs of cells of oligo-thiophenes
50the results are shown in Table 1.
Table 1
| A498 | HCT116 | HepG2 | A549 | HPF | MCF-7 | |
| IC 50(μM) | 6.96 | 12.03 | 12.74 | 8.81 | 16.74 | 18.07 |
As shown in Table 1, oligo-thiophenes 9 has stronger selective inhibitory to human renal carcinoma cell strain A498 in vitro.
2) various cells and mixture 1,2,3,4 or 5 act on respectively 48 hours, mixture 1,2,3,4,5 and oligo-thiophenes 9, the IC of Chlorambucil to cell
50the results are shown in Table 2.
Table 2
| IC 50(μM) | A498 | A549 | MCF-7 | HepG2 | | HCT116 |
| Mixture | ||||||
| 1 | 2.60 | 5.86 | 2.68 | NA a | 7.05 | 5.66 |
| |
2.54 | 5.28 | 2.29 | NA a | 7.01 | 5.95 |
| |
2.61 | 4.94 | 2.27 | NA a | 7.02 | 5.11 |
| |
2.11 | 3.37 | 1.97 | NA a | 6.90 | 4.91 |
| |
2.45 | 4.57 | 2.43 | NA a | 7.79 | 6.04 |
| Oligo- |
6.96 | 8.81 | 18.07 | 12.74 | 16.74 | 12.03 |
| Chlorambucil | NA a | NA a | NA a | NA a | NA a | NA a |
aNot Available
Utilize above-mentioned synthetic oligo-thiophenes to carry out cell imaging, adopt Confocal laser scanning microscope (FV1000-IX81, Olympus, Japan) to characterize.
Concrete steps are: human renal carcinoma cell A498 is with cultivating in having the culture dish of glass bottom 24 hours containing the DMEM cell culture fluid of 10% serum, when cell count reaches while approaching 60, add 10 micromolar oligo-thiophenes 9 to continue to cultivate 12 hours, and contrast ware is set simultaneously.Remove plastosome fluorescence dye (M7512, Invitrogen) 100 nmoles that nutrient solution adds respectively 37 ° of C preheatings, dye 15 minutes.Dyeing finishes the rear nutrient solution of carefully removing, and with PBS washing 3 times, adds the PBS solution of mass concentration 4% paraformaldehyde, fixes 15 minutes.After fixedly completing, with PBS washing three times, then use Confocal laser scanning microscope (FV1000-IX81, Olympus, Japan) to observe, see Fig. 6.Oligo-thiophenes is used to 405nm laser, and plastosome dyestuff is used 559nm laser.Result shows that oligo-thiophenes 9 can realize the imaging to cell, and plastosome dyestuff relatively finds that this oligo-thiophenes can selectivity target plastosome.Therefore under lower concentration, oligo-thiophenes of the present invention is a kind of new plastosome dyestuff.
Apoptosis can be measured with Annexin V-FITC and the two dyeing of PI, and it is quantitative that flow cytometer (FACSCalibur, Becton, Dickinson and Company, USA) is used for carrying out apoptosis.Phosphatidylserine (Phosphatidylserine, PS) is normally at the inner side of cytolemma, but apoptotic early stage, PS can be turned to the surface of cytolemma from the inner side of cytolemma, be exposed in extracellular environment.Annexin-V is that a kind of molecular weight is the Ca of 35 ~ 36KD
2+dependency phospholipids incorporate albumen, can with PS high-affinity specific binding.Annexin-V is carried out to fluorescein (FITC, PE) or biotin mark, using the Annexin-V of mark as fluorescent probe, utilize flow cytometer or fluorescent microscope can detect apoptotic generation.Propidium iodide (propidine iodide, PI) is a kind of nucleic acid dye, and it can not see through complete cytolemma, but at cell and the dead cell of apoptosis middle and advanced stage, and PI can permeate through cell membranes and made that nucleus is red to be dyed.Therefore Annexin-V is mated to use with PI, just cell and the dead cell in apoptosis late period morning can be made a distinction.
Concrete steps: A498 cell is cultivated 24 hours in 35 * 35mm Tissue Culture Dish, with 1 * PBS washing once, then add 1 milliliter of DMEM nutrient solution.The oligo-thiophenes 9 that adds respectively different concns, with normal cultured cells in contrast, continues to cultivate 24 hours.To normally cultivate and the A498 cell of apoptosis-induced adherent culture is first used 0.25% trysinization, after centrifugal, with PBS, wash 2 times, the Annexin-V(20 mcg/ml that adds 100 microlitre Binding Buffer and FITC mark) 10 microlitres, room temperature lucifuge 30 minutes, add again PI(50 mcg/ml) 5 microlitres, lucifuge reaction is after 5 minutes, add 400 microlitre Binding Buffer, with FACScan, carry out flow cytometry detection by quantitative (being generally no more than 1h) immediately, negative control is set simultaneously.
In experimentation, use 488nm laser, with Channel FL-1 and FL-2, collect the signal from FITC and PI respectively.Collecting cell adds up to 3 * 10
4individual.The results are shown in Figure 7.Show oligo-thiophenes 9 cell death inducing significantly.
Claims (1)
1. the oligo-thiophenes shown in formula II or its salt:
Wherein, m=1; N is the integer in 1-6;
R
1be selected from any one in following radicals :-N
+(CH
3)
3br
-,-N (CH
3)
2,-NH (CH
2cH
3) ,-NHCH
3with-NH
2.
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