CN111675716B - Fangchinoline derivative with anticancer activity and preparation method and application thereof - Google Patents
Fangchinoline derivative with anticancer activity and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of preparation of fangchinoline derivatives, in particular to fangchinoline with anticancer activityNolinkaline derivative, preparation method and application thereof. The molecular structure of the fangchinoline derivative is as follows:
(ii) a The described
. The invention synthesizes a novel fangchinoline derivative which has the anticancer activity and high activity; the invention improves the bioactivity of fangchinoline by modifying the structure of fangchinoline, and has important practical significance for developing novel anticancer drugs; the fangchinoline derivatives with anticancer activity of the invention use fangchinoline as a raw material to carry out related structural modification, and have the advantages of simple synthesis method, low raw material price and novel structure.
Description
Technical Field
The invention relates to the technical field of preparation of fangchinoline derivatives, in particular to fangchinoline derivatives with anticancer activity, and a preparation method and application thereof.
Background
Cancer is a major public health problem in the world. Cancer is one of the major public health problems in the world, and according to the World Health Organization (WHO) statistical report of 2018, there are about 1810 new cancer cases around the world, 960 ten thousand patients die of cancer, and it is expected that global cancer cases will show a rapid growth trend, increasing to 1900 ten thousand by 2025 and reaching 2400 ten thousand by 2035. The continuous increase in the incidence of cancer worldwide has also driven the rapid growth of the anticancer drug market. However, due to acquired resistance and a variety of molecular cancer types, there are fewer and fewer anticancer drugs currently available. Scientists have studied many new candidates for anti-tumor drugs, but research is still ongoing. Therefore, the search for novel, safe and effective antitumor drugs is urgently needed.
Chemotherapy and radiation therapy are among the most widely used and potential cancer treatment strategies among various interventions. In fact, the use of chemotherapy is largely limited by their adverse effects on normal cells. This has increasingly led to research aimed at identifying new compounds, preferably natural product sources with anti-cancer potential, with minimal collateral damage to normal cells. Relevant molecular mechanism researches show that the fangchinoline and the derivative thereof can induce autophagy in a human liver cancer cell line HepG2 and PLC/PRF/5 at a lower concentration through a P53/sestrin2/AMPK signal. Tetrandrine, with a similar chemical structure, blocks the cell cycle of G1/S and G2/M and stimulates apoptotic cell death and autophagy through PKC- α inhibition and mTOR-dependent mechanisms. However, the natural compound has its own disadvantages such as low bioavailability, which limits the wide clinical application, so the tetrandra derivatives with novel structures try to overcome its own disadvantages by changing the structure-activity relationship through structural modification, and become a hotspot of research in the compound field.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a fangchinoline derivative with anticancer activity.
The invention also provides a preparation method of the fangchinoline derivative with anticancer activity.
The invention also provides an application of the fangchinoline derivative.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a fangchinoline derivative with anticancer activity, which has a molecular structure as follows:
the described
。
The invention also provides a preparation method of the fangchinoline derivative, which comprises the following steps:
(1) dissolving fangchinoline dTTet in dichloromethane, placing the solution in a two-neck flask, magnetically stirring the solution, and adding triethylamine dissolved in dichloromethane to react;
(2) dissolving 2-bromobenzene sulfonyl chloride or p-toluene sulfonyl chloride in dichloromethane, slowly dropwise adding the dichloromethane into a flask under stirring for reaction, and tracking the reaction process by TLC;
(3) after the reaction is finished, concentrating the reaction solution, and purifying by using an eluent through a neutral alumina column to obtain a target product.
Further, in the step (1), the ratio of the fangchinoline to the dichloromethane is 0.06mmol:1 mL; the concentration of triethylamine in dichloromethane was 0.36 mmol/mL.
In the steps (1) and (2) of the invention, the reaction temperature is 0 ℃ and the reaction time is 1 h.
Further, in the step (2), the molar ratio of the 2-bromobenzenesulfonyl chloride or the p-toluenesulfonyl chloride to the dichloromethane is 1: 1.2.
Further, in the step (3), the eluent is dichloromethane: the methanol is in a volume ratio of 120:1, 100:1 and 80: 1.
The invention also provides application of the fangchinoline derivative in preparation of anti-cancer drugs.
The reaction solution concentration process of the invention adopts a rotary evaporation method to concentrate the reaction solution; the two-neck flask is kept full of nitrogen in the whole reaction process
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention synthesizes a novel fangchinoline derivative which has anticancer activity and high activity; the invention improves the bioactivity of fangchinoline by modifying the structure of fangchinoline, and has important practical significance for developing novel anticancer drugs;
(2) the fangchinoline derivatives with anticancer activity of the invention use fangchinoline as a raw material to carry out related structural modification, and have the advantages of simple synthesis method, low raw material price and novel structure.
Drawings
FIG. 1 is a drawing of the compound 7-O- (m-Br-benzenesulfonyl) fangchinoline prepared in example 1 1 H NMR spectrum;
FIG. 2 is a diagram of the compound 7-O- (m-Br-benzenesulfonyl) fangchinoline prepared in example 1 13 C NMR spectrum;
FIG. 3 is a drawing of the compound 7-O- (p-toluenesulfonyl) fangchinoline prepared in example 2 1 H NMR spectrum;
FIG. 4 is a drawing of the compound 7-O- (p-toluenesulfonyl) fangchinoline prepared in example 2 13 C NMR spectrum;
FIG. 5 is a schematic diagram showing the results of flow cytometry on Compound 1 and Compound 2 according to the present invention;
FIG. 6 is a schematic diagram showing the Western-Blot experiment results of Compound 1 and Compound 2 of the present invention.
FIG. 7 is an image of colony formation by Giemsa staining of A549 cells treated with different concentrations of the compound prepared in example 1 (0, 1, 2, 3. mu.M).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1
A method for preparing fangchinoline derivatives with anticancer activity comprises dissolving 100mg fangchinoline dT et (about 0.18 mmol) in 3 mL dichloromethane, placing in a two-neck flask, repeatedly injecting argon into the flask for three times until the flask is evacuated to fill argon, and controlling temperature to 0 deg.C. Under magnetic stirring, 66.5 mg (about 0.72 mmol) of triethylamine dissolved in2 mL of dichloromethane was added, and after 1 hour of reaction, 50.7 mg (about 0.20 mmol) of 2-bromobenzenesulfonyl chloride was added, and the reaction was followed by TLC. After the reaction is finished, concentrating the reaction solution, purifying the reaction solution by a neutral alumina column, wherein the eluent is dichloromethane: methanol (120: 1, 100:1, 80: 1), separating and purifying to obtain 123.6 mg (about 0.149 mmol) of white powder product, yield is 83%, and the obtained product is 7-O- (m-Br-benzenesulfonyl) fangchinoline through NMR and HRMS analysis, and NMR spectra are shown in figures 1 and 2.
1 H NMR (400 MHz, CDCl3) δ 7.79 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.38 – 7.16 (m, 3H), 7.10 (dd, J = 8.1, 2.5 Hz, 1H), 6.83 – 6.77 (m, 2H), 6.66 (dd, J = 8.3, 2.5 Hz, 1H), 6.38 (d, J = 7.2 Hz, 2H), 6.25 (s, 1H), 6.17 (dd, J = 8.3, 2.0 Hz, 1H), 3.83 (d, J = 9.4 Hz, 3H), 3.65 (dd, J = 11.1, 5.4 Hz, 1H), 3.57 (d, J = 9.9 Hz, 1H), 3.45 (s, 3H), 3.43 – 3.31 (m, 2H), 3.20 (s, 3H), 2.96 – 2.59 (m, 8H), 2.55 (s, 3H), 2.43 (dd, J = 30.0, 9.4 Hz, 2H), 2.20 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 153.77, 150.96, 149.26, 148.70, 147.61, 147.12, 142.29, 139.25, 136.62, 135.06, 134.51, 132.69, 131.39, 130.17, 128.53, 128.32, 127.98, 127.22, 123.98, 122.85, 122.46, 121.77 , 120.61, 116.26, 112.81, 111.47, 106.22, 63.74, 61.38, 56.11 , 55.80, 46.14, 45.02, 43.80, 42.37 , 39.64, 24.33, 22.00;
HRMS(ESI): calcd for C 43 H 43 BrN 2 O 8 S m/z:826.1923, found: 827.1965
The specific reaction scheme is as follows:
example 2
The preparation method of the fangchinoline derivative with anticancer activity comprises the following steps: 100.0 mg of fangchinoline dTTet (about 0.18 mmol) was dissolved in 3 mL of dichloromethane and placed in a two-necked flask filled with argon and the temperature was controlled to 0 ℃. Under magnetic stirring, 66.5 mg (about 0.72 mmol) of triethylamine dissolved in2 mL of dichloromethane was added, and after 1 hour of reaction, 38.0 mg (about 0.20 mmol) of p-toluenesulfonyl chloride was added and the reaction was followed by TLC. After the reaction is finished, concentrating the reaction solution, purifying the reaction solution by using a neutral alumina column, and eluting with dichloromethane: methanol (120: 1, 100:1, 80: 1) is separated and purified to obtain 119.5 mg (about 0.157 mmol) of white powder product with yield of 87%, and the white powder product is analyzed by NMR and HRMS to obtain 7-O- (p-toluenesulfonyl) fangchinoline, and NMR spectra are shown in figures 3 and 4.
1 H NMR (400 MHz, CDCl 3 ) δ7.53 (d, J = 8.1 Hz, 2H), 7.36 – 7.20 (m, 4H), 7.15 (dd, J = 8.0, 1.9 Hz, 1H), 6.87 (s, 2H), 6.75 (dd, J = 8.2, 1.9 Hz, 1H), 6.46 (d, J = 8.7 Hz, 2H), 6.32 – 6.22 (m, 2H), 3.92 (s, 3H), 3.77 – 3.51 (m, 4H), 3.47 (s,3H), 3.28 (s, 3H), 2.97 – 2.69 (m, 8H), 2.64 (s, 3H), 2.54 (d, J = 13.7 Hz, 2H), 2.47 (s, 3H), 2.28 (s, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ153.73, 151.10 , 149.25 , 148.83 , 147.78, 147.10, 144.42, 142.40, 135.23, 134.99 , 134.58,134.37, 132.61 , 132.35, 130.08, 129.13, 128.65, 128.21, 127.64, 123.62 , 122.88, 121.65,121.58 120.98, 116.15, 115.94, 112.74, 111.50 , 106.09 , 64.06, 61.40, 56.11, 55.78,55.72, 45.08, 43.84 , 42.48 , 42.29,41.91, 39.62, 24.32, 22.06, 21.83;
HRMS (ESI) : calcd for C 44 H 46 N 2 O 8 S m/z:726.2975 found:763.3008
The specific synthetic route is as follows:
comparative example 17 Synthesis of O- (m-Naphthalenesulfonyl) fangchinoline
100.0 mg of fangchinoline dTTet (about 0.18 mmol) was dissolved in 3 mL of dichloromethane and placed in a two-necked flask filled with argon and temperature controlled to 0 ℃. Under magnetic stirring, 66.5 mg (about 0.72 mmol) of triethylamine dissolved in2 mL of dichloromethane was added, and after 1 hour of reaction, 45.2 mg (about 0.20 mmol) of 2-naphthalenesulfonyl chloride was added, and the reaction was followed by TLC. After the reaction is finished, concentrating the reaction solution, purifying the reaction solution by a neutral alumina column, wherein the eluent is dichloromethane: methanol (120: 1, 100:1, 80: 1), separating and purifying to obtain 118.0 mg (about 0.148 mmol) of light yellow powder product, with yield of 82%, and analyzing by NMR and HRMS to obtain 7-O- (m-naphthalenesulfonyl) fangchinoline.
7-O-(m-Naphthalene-Sulfonyl)dTet 1 H NMR (400 MHz, CDCl 3 ) δ 8.20 (s, 1H), 8.02 – 7.75 (m, 4H), 7.69 (t, J = 7.5 Hz, 1H), 7.56 (dd, J = 15.4, 8.1 Hz, 2H), 6.85 – 6.73 (m, 3H), 6.61 (d, J = 7.4 Hz, 1H), 6.54 (dd, J = 8.2, 2.1 Hz, 1H), 6.30 (d, J = 23.5 Hz, 2H), 6.21 (s, 1H), 6.03 (d, J = 8.2 Hz, 1H), 4.40 (s, 1H), 3.84 (s, 3H), 3.53 (d, J = 10.0 Hz, 1H), 3.45 (d, J = 10.1 Hz, 3H), 3.33 (ddd, J = 21.6, 20.7, 10.6 Hz, 2H), 3.19 (d, J = 14.4 Hz, 3H), 2.94 – 2.68 (m, 6H), 2.53 (s, 3H), 2.51 – 2.31 (m, 4H), 2.20 (d, J = 12.1 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ153.47, 151.48, 149.29, 148.57, 147.57, 147.12, 142.42, 135.28, 134.63, 134.59,134.47, 132.42, 131.87, 129.97, 129.52, 128.73, 128.08, 127.79, 127.59, 123.85, 122.83,121.50, 120.04, 115.89, 112.84, 111.47, 106.42, 63.42 , 61.42, 56.12,56.59,55.77, 44.96, 43.91, 42.41, 42.32,42.23, 39.05, 24.34, 22.05; HRMS (ESI) : calcd for C 47 H 46 N 2 O 8 S m/z:798.2975, found:799.3029 [M+H] + ,Yield: 82%.
Comparative example 27 Synthesis of fangchinoline (O-Br-benzenesulfonyl)
100.0 mg of fangchinoline dTTet (about 0.18 mmol) was dissolved in 3 mL of dichloromethane and placed in a two-necked flask filled with argon and the temperature was controlled to 0 ℃. Under magnetic stirring, 66.5 mg (about 0.72 mmol) of triethylamine dissolved in2 mL of dichloromethane was added, and after 1 hour of reaction, 50.7 mg (about 0.20 mmol) of 3-bromobenzenesulfonyl chloride was added and the reaction was followed by TLC. After the reaction is finished, concentrating the reaction solution, purifying the reaction solution by using a neutral alumina column, and eluting with dichloromethane: methanol (120: 1, 100:1, 80: 1), separating and purifying to obtain 128.0 mg (about 0.155 mmol) of white powder product, wherein the yield is 86%, and the product is analyzed by NMR and HRMS to obtain the 7-O- (O-Br-benzenesulfonyl) fangchinoline.
7-O-(o-Br-benzene -Sulfonyl)dTet 1 H NMR (400 MHz, CDCl 3 ) δ 7.79 (t, J= 1.6 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.38 – 7.16 (m, 3H), 7.10 (dd, J = 8.1, 2.5 Hz, 1H), 6.83 – 6.77 (m, 2H), 6.66 (dd, J = 8.3, 2.5 Hz, 1H), 6.38 (d, J = 7.2 Hz, 2H), 6.25 (s, 1H), 6.17 (dd, J = 8.3, 2.0 Hz, 1H), 3.83 (d, J = 9.4 Hz, 3H), 3.65 (dd, J = 11.1, 5.4 Hz, 1H), 3.57 (d, J = 9.9 Hz, 1H), 3.45 (s, 3H), 3.43 – 3.31 (m, 2H), 3.20 (s, 3H), 2.96 – 2.59 (m, 8H), 2.55 (s, 3H), 2.43 (dd, J = 30.0, 9.4 Hz, 2H), 2.20 (s, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ 153.77, 150.96, 149.26, 148.70, 147.61, 147.12, 142.29, 139.25, 136.62, 135.06, 134.51, 132.69, 131.39, 130.17, 128.53, 128.32, 127.98, 127.22, 123.98, 122.85, 122.46, 121.77 , 120.61, 116.26, 112.81, 111.47, 106.22, 63.74, 61.38, 56.11 , 55.80, 46.14, 45.02, 43.80, 42.37 , 39.64, 24.33, 22.00; HRMS (ESI) : calcd for C 43 H 43 BrN 2 O 8 S m/z:826.1923 found: 827.1844 [M+H] + Yield: 86%.
Comparative example 37 Synthesis of O- (p-Cl-benzenesulfonyl) fangchinoline
100.0 mg of fangchinoline dTTet (about 0.18 mmol) was dissolved in 3 mL of dichloromethane and placed in a two-necked flask filled with argon and the temperature was controlled to 0 ℃. Under magnetic stirring, 66.5 mg (about 0.72 mmol) of triethylamine dissolved in2 mL of dichloromethane was added, and after 1 hour of reaction, 42.0 mg (about 0.20 mmol) of 4-chlorobenzenesulfonyl chloride was added, and the reaction was followed by TLC. After the reaction is finished, concentrating the reaction solution, purifying the reaction solution by a neutral alumina column, wherein the eluent is dichloromethane: methanol (120: 1, 100:1, 80: 1), separating and purifying to obtain 114.2 mg (about 0.146 mmol) of light yellow powder product, wherein the yield is 81%, and the product is analyzed by NMR and HRMS to obtain the 7-O- (p-Cl-benzenesulfonyl) fangchinoline.
7-O-(p-Cl-benzene -Sulfonyl)dTet 1 H NMR (400 MHz, CDCl 3 ) δ 7.52 (d, J= 8.6 Hz, 2H), 7.47 – 7.27 (m, 4H), 7.11 (dd, J = 8.1, 2.4 Hz, 1H), 6.79 (s, 2H), 6.68 (dd, J = 8.2, 2.4 Hz, 1H), 6.37 (d, J = 12.4 Hz, 2H), 6.26 – 6.18 (m, 2H), 3.86 (s, 3H), 3.62 – 3.54 (m, 2H), 3.45 (s, 3H), 3.33 (ddd, J = 23.1, 12.3, 8.9 Hz, 2H), 3.21 (s, 3H), 2.92 – 2.62 (m, 8H), 2.57 (s, 3H), 2.49 – 2.37 (m, 2H), 2.20 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 153.64, 150.93, 149.42, 148.61, 147.68, 147.10, 142.23, 140.15, 135.93, 135.10, 134.40, 132.60, 130.30, 130.05, 129.34, 128.92, 128.50,128.37, 128.21,128.02,123.80, 122.77, 121.87, 120.41, 115.80, 112.79, 111.49, 106.13, 63.96, 61.37, 56.07, 55.79, 53.54, 45.08, 43.84 , 42.54, 42.34, 42.04, 39.15, 24.58, 22.12 ; HRMS (ESI) : calcd for C 43 H 43 ClN 2 O 8 S m/z:782.2829 found:783.2862 [M+H] + Yield: 81%.
Comparative example 47 Synthesis of O- (O-pyridine-sulfonyl) fangchinoline
100.0 mg of fangchinoline dTTet (about 0.18 mmol) was dissolved in 3 mL of dichloromethane and placed in a two-necked flask filled with argon and the temperature was controlled to 0 ℃. Under magnetic stirring, 66.5 mg (about 0.72 mmol) of triethylamine dissolved in2 mL of dichloromethane was added, and after 1 hour of reaction, 35.4 mg (about 0.20 mmol) of 3-pyridinesulfonyl chloride was added and the reaction was followed by TLC. After the reaction is finished, concentrating the reaction solution, purifying the reaction solution by a neutral alumina column, wherein the eluent is dichloromethane: methanol (120: 1, 100:1, 80: 1), separating and purifying to obtain 108.0 mg (about 0.144 mmol) of white powder product, the yield is 80%, and the product is analyzed by NMR and HRMS to obtain 7-O- (O-pyridine-sulfonyl) fangchinoline.
7-O-(o-pyridine -Sulfonyl)dTet 1 H NMR (400 MHz, CDCl 3 ) δ 8.77 (dd, J = 10.1, 3.3 Hz, 2H), 7.84 (d, J = 8.1 Hz, 1H), 7.31 (dd, J = 8.0, 3.5 Hz, 2H), 7.19 (s, 1H), 7.10 (dd, J = 8.1, 2.3 Hz, 1H), 6.79 (s, 2H), 6.69 (dd, J = 8.2, 2.3 Hz, 1H), 6.41 (d, J = 16.1 Hz, 2H), 6.27 – 6.17 (m, 2H), 3.86 (s, 3H), 3.75 – 3.60 (m, 2H), 3.40 (s, 3H), 3.34 (s, 3H), 2.96 – 2.76 (m, 6H), 2.68 (d, J = 12.2 Hz, 2H), 2.59 (s, 3H), 2.49 – 2.35 (m, 2H), 2.21 (s, 3H);13 C NMR (101 MHz, CDCl 3 ) δ 153.77, 150.36, 149.28, 148.84, 147.83, 147.11 , 142.15, 136.06, 135.09, 134.42, 134.03, 132.95, 132.60, 130.37, 128.62, 127.93, 127.71, 123.84, 123.28, 122.81, 121.87, 121.08, 115.94, 112.68, 111.47, 105.92, 63.80, 61.33, 56.12 , 55.81, 55.56, 53.49, 50.82, 43.68 , 42.30, 41.87, 39.89 , 24.21, 22.09; HRMS (ESI) : calcd for C 42 H 43 N 3 O 8 S m/z:749.2804 found: 750.2824[M+H] + Yield: 80%.
The compound has anticancer activity on A549 cells:
(1) flow cytometry
Taking out the A549 cells, placing the A549 cells in a super clean bench, and digesting and blowing the A549 cells into a single cell suspension by pancreatin. The cells were seeded at a density of 5000 cells per well in 6-well plates and placed in 5% CO 2 ，37℃And incubating for 24 hours in an incubator. Preparing culture media containing 4m of different concentrations: weighing 1 mg of each of the compounds 1 and 2, and adding 403.5 muL of DMSO respectively to dissolve the compounds into 3 mM mother liquor; a volume of the mother liquor was removed and diluted with 1640 medium to a concentration gradient (0, 1, 2, 3. mu.M) of medium. Discarding the old culture medium, adding 1640 culture medium containing compounds 1 and 2 with concentration gradient of 0, 1, 2, and 3 μ M, respectively, and placing the culture dish in 5% CO 2 And incubating for 48 h in an incubator at 37 ℃.
And (3) detecting apoptosis: trypsinized, blown into single cell suspension, moved to a centrifuge tube, centrifuged at 1000r for 5min, the supernatant discarded, and PBS re-suspended cells counted. 8 ten thousand cells were taken from each concentration of compound 1 and 2 treated cells, centrifuged at 1000r for 5min, the supernatant was discarded, 195. mu.l Annexin V-FITC binding solution, 5. mu.l Annexin V-FITC, and 10. mu.l propidium iodide staining solution were added in sequence, and mixed gently. Incubate at 25 ℃ for 21min in the dark, followed by ice-bath in the dark. Detection was performed using a flow cytometer.
And (3) periodic detection: and (4) digesting and blowing the cell suspension into a single cell suspension by using pancreatin, transferring the cell suspension into a centrifuge tube, centrifuging the centrifuge tube at 1000r for 5min, discarding supernatant, and resuspending the cells by using PBS and counting the cells. 12 ten thousand cells were taken and added to 1mL ice-cooled 70% ethanol, and the mixture was gently and evenly blown and fixed overnight at 4 ℃. Centrifuging for 5min at about 1000r, removing supernatant, resuspending cells with PBS, centrifuging for 5min at 1000r again, removing supernatant, preparing staining solution: 0.5 mL staining buffer, 25. mu.L propidium iodide staining solution, and 5. mu.L LRNase A. 0.5 mL of prepared staining solution was added to each sample, and the mixture was incubated at 37 ℃ for 30 min in the dark. And (4) detecting by using a flow cytometer.
(2) Western-Blot experiment
Preparing a solution:
preparation of 10% SDS: 10 g SDS powder + 100 mL deionized water. Preparation of 10% APS: 1mL DDH2O + 10% PAGE gel coagulant, mix well. Preparation of 1X electrophoresis liquid: 200 mL of 5 XTris-glycine electrophoresis solution + 800 mL of ultrapure water was stirred until completely dissolved. 1, preparing a 1X film transfer solution: Tris-Base 5.81 g + Glycine 2.91 g + 200 mL, mixed well and dissolved, added with 800 mL ultrapure water, mixed well. Preparing a TBST solution: 100 mL of 10 × TBS + 900 mL of ultrapure water + 2 mL of Tween-20, and mixing. Preparation of 5% milk sealing liquid: 100 mL TBST solution + 5 g skim milk powder, dissolved. Preparing a 4X Loading Buffer solution: 1.2 mL of 1M Tris-HCl + 0.4g SDS +1.4 mL deionized water + 2 mL glycerol + 0.01% bromophenol blue, dissolved, mixed well and added with beta-mercaptoethanol (10%) before use. Preparation of 10% separation gel: 3.3 mL of 30% acrylamide, 4 mL of deionized water, and 1.5 mL of Tris-Base (pH 8.8) + 100 μ L10% SDS, 50 μ L10% APS, and 15 μ L TEMED. Preparation of 5% concentrated gum: 670 μ L30% acrylamide + 3.6 mL deionized water + 625 μ L1.0 mL Tris-Base (pH 6.8) + 50 μ L10% SDS + 25 μ L10% APS + 15 μ L TEMED.
Extraction of cell protein sample:
a549 cells which are incubated for two days with compounds 1 and 2 (0, 1, 2 and 3 mu M) with different concentrations are taken out from an incubator, the culture medium is discarded, 2 mL of precooled PBS is added for washing twice, 1mL of PBS is added, adherent cells are scraped by a curette, blown into cell suspension, and centrifuged at 12200r and 4 ℃ for 3 min. And (4) removing the supernatant, adding the protein lysate, mixing uniformly, and performing ice bath for 30 min. 12200r, centrifuging at 4 deg.C for 16 min, and collecting supernatant protein solution. Storing at-80 deg.C.
Determination of protein content:
preparation of 25 mg/mL protein standard: 1mL of the protein standard preparation was added to a tube of protein standard (25 mg BSA). ② 0.5 mg/mL protein standard preparation: the 25 mg/mL protein standard was diluted to 0.5 mg/mL protein standard. Preparing a BCA working solution: preparing BCA working solution from the BCA reagent A and the BCA reagent B according to the ratio of 50:1, and mixing uniformly. Detecting the protein concentration: a. 0.5 mg/mL of the protein standard was diluted to 0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.5 mg/mL and added to a 96-well plate at 21. mu.L per well. b. Different protein sample solutions were added to each well at 20 μ L. c. 210 muL of BCA working solution is added into each air, and the mixture is placed at 37 ℃ for 25 min. d. A562 was detected with a microplate reader and a standard curve was made. e. The protein content of the sample was calculated from the standard curve.
Preparing glue:
taking a clean and dry glue-making glass plate, clamping the clean and dry glue-making glass plate on a mold, injecting prepared 10% separation glue, sealing the separation glue by using ultrapure water, and standing the separation glue for 30 min at room temperature. Pouring out ultrapure water, injecting prepared 5% concentrated glue, inserting comb, and standing at room temperature for 30 min.
Electrophoresis
Inserting the prepared gel into an electrophoresis tank, filling the prepared 1X electrophoresis solution, and vertically pulling out the comb. And adding 5 mu L protein Marker into one hole, adding 60 mu g sample protein into the experimental hole, and adding a proper amount of Loading Buffer into the rest holes for balancing. Connecting a power supply, keeping the voltage at 90V, setting time according to requirements, and performing electrophoresis.
Rotary film
After electrophoresis is finished, the PAGE gel is taken out, and excess gel is cut off. And (3) cutting a PVDF membrane with a proper size, soaking the PVDF membrane in methanol for 2 min for activation, and placing the PVDF membrane in 1X membrane transferring liquid for balancing for 10 min. Sponge-two layers of filter paper-PVDF membrane-PAGE gel-two layers of filter paper-sponge are stacked and fixed in a membrane rotating plate according to the sequence from the anode to the cathode, the membrane rotating plate is placed in an electrophoresis tank and connected with a power supply, the constant current is 300 mA, and the time is set according to requirements.
Ponceau dyeing
And (3) putting the converted PVDF membrane into a ponceau solution for dyeing, and rinsing in ultrapure water. Placing in a preservative film, and cutting the strips according to the requirement.
Milk closure
Placing the cut target strip in 5% milk sealing solution, and sealing with shaking table for 1 h.
Combined with an antibiotic
The blocked target band is placed in an antibody incubation box, the corresponding primary antibody is added, and the incubation is carried out overnight at 4 ℃.
Bound secondary antibody
Wash 3 times with TBST (5 min each), add the corresponding secondary antibody, incubate for 2 h at room temperature.
Development
After binding the secondary antibody, wash 3 times with TBST for 5min once. ECL luminophore (1: 1) was dropped onto the target strip and the strip was scanned across the membrane to take a picture in a chemiluminescent imaging system.
FIG. 5 shows the results of the flow cytometry experiments of the compounds 1 and 2, and the ability of the compounds 1 and 2 to induce apoptosis is checked by flow cytometry, and the results show that the compounds 1 and 2 trigger the apoptosis of A549 cells, and the percentage of apoptotic cells is obviously increased along with the increase of concentration. Compounds 1, 2 (1, 2 and 3) compared to the control groupμ M) significantly increased apoptosis of A549 cells in a dose-dependent manner; FIG. 6 shows the results of Western-Blot experiments on Compounds 1 and 2, wherein the protein levels of cyclin B1 and CDK1 were significantly reduced and the protein level expression of P21 was significantly increased in a concentration-dependent manner after treatment with Compounds 1 and 2. These results indicate that compounds 1, 2 may induce a549 cell G by down-regulating cyclin B1 and CDK1 and up-regulating P21 2 Retardation in the/M phase. .
(II) the cytotoxicity of the compounds synthesized in examples and comparative examples against human cancer cell line A549 was tested, and IC was calculated by Graphpad software 50 The value is obtained. Specific results are shown in table 1.
TABLE 1
The compound 7-O- (m-Br-benzenesulfonyl) fangchinoline (IC) is found by experiments 50 0.59 μ M), the activity of which is 14.34 times higher than that of tetrandrine, 12.15 times higher than that of tetrandrine, and 2.12 times higher than that of 10-hydroxycamptothecin. And the activity of the compounds is obviously different from that of the compounds with different substituents.
(III) to further evaluate the anti-proliferative effect of the compounds, a colony formation assay was performed using the compound 7-O- (m-Br-benzenesulfonyl) fangchinoline as an example. As shown in FIG. 7, studies on 7-O- (m-Br-benzenesulfonyl) fangchinoline base-treated A549 cells showed that: the compound significantly inhibited colony formation of A549 cells and decreased in a dose-dependent manner.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A fangchinoline derivative with anticancer activity is characterized in that the molecular structure of the fangchinoline derivative is as follows:
。
2. a method for preparing the fangchinoline derivative according to claim 1, comprising the steps of:
(1) dissolving fangchinoline in dichloromethane, placing in a two-neck flask, magnetically stirring, and adding triethylamine dissolved in dichloromethane for reaction;
(2) dissolving 2-bromobenzene sulfonyl chloride in dichloromethane, slowly dropwise adding the dichloromethane into a flask under stirring for reaction, and tracking the reaction process by TLC;
(3) after the reaction is finished, concentrating the reaction solution, and purifying by using an eluent through a neutral alumina column to obtain the 7-O- (m-Br-benzenesulfonyl) fangchinoline.
3. The preparation method according to claim 2, wherein in the step (1), the ratio of fangchinoline to dichloromethane is 0.06mmol:1 mL; the concentration of triethylamine in dichloromethane was 0.36 mmol/mL.
4. The process according to claim 2 or 3, wherein the reaction temperature in steps (1) and (2) is 0 ℃ and the reaction time is 1 hour.
5. The method according to claim 2, wherein in step (2), the molar ratio of 2-bromobenzenesulfonyl chloride to dichloromethane is 1: 1.2.
6. The method according to claim 2, wherein in the step (3), the eluent is dichloromethane: the methanol is in a volume ratio of 120:1, 100:1 and 80: 1.
7. Use of the fangchinoline derivative of claim 1 in the preparation of an anticancer agent.
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