CN103030738B - Lawsone copolymer with antineoplastic activity and preparation method thereof - Google Patents

Abstract

The invention provides a hennain copolymer with anti-tumor activity, which is to link 2-hydroxy-1,4-naphthoquinone to N-(2-hydroxypropyl)methacrylamide by polymerization The formed high molecular copolymer with good biocompatibility belongs to the field of high molecular chemistry and application. The copolymer of the present invention superimposes the activity of 2-hydroxyl-1,4-naphthoquinone and N-(2-hydroxypropyl)methacrylamide, which further promotes the inhibitory effect of the polymer on tumors, and reduces the polymerization Toxicity of substances, while showing good biological water solubility. The experiment of inhibiting tumor cell growth in vivo proves that the copolymer of the present invention has a strong tumor inhibiting effect on liver cancer H22 cells, which provides a new idea for preparing antitumor drugs.

Description

A kind of hennain copolymer with antitumor activity and preparation method thereof

technical field

The invention belongs to the field of macromolecular chemistry, and relates to a hennain copolymer with antitumor activity—N-(2-hydroxypropyl)methacrylamide-2-hydroxyl-1,4-naphthoquinone; the invention At the same time, it also relates to a preparation method of the copolymer; the invention also relates to the tumor suppressing effect of the copolymer on liver cancer H22 cells.

Background technique

Cancer has become one of the major threats to human health, and it is on the rise. The main methods of treating cancer today include radiotherapy, chemotherapy, surgical therapy and gene therapy. Among them, radiotherapy and chemotherapy are extremely important non-surgical treatments, but while killing tumor cells, radiotherapy and chemotherapy also cause serious damage to normal cells in the body.

Naphthoquinones are a class of small molecular compounds that widely exist in nature and have various biological activities. According to the structure of the parent, this kind of compound can be divided into naphthoquinones (5,8-dihydroxy-1,4-naphthoquinones), 1,4-naphthoquinones and 1,2-naphthoquinones, etc. Kamei et al. conducted a test on the inhibition of cell growth by quinone compounds, and found that Henna sativa mainly inhibited the S phase of cell growth, and the more the number of phenolic hydroxyl groups in the compound, the greater the cytotoxicity. Hennamol, the chemical name is 2-hydroxy-1,4-naphthoquinone, and its structural formula is as follows:

                                                                            

Due to the poor water solubility of hennain, the selection of co-solvents (absolute ethanol, DMSO, etc.) will cause a variety of toxic and side effects. In addition, after diluting with water during administration, it needs to be filtered through a 0.22 μm microporous membrane.

N-(2-hydroxypropyl)methacrylamide (HPMA) is a polymer drug carrier, its structural formula is as follows:

                             

Because HPMA has good biocompatibility, it can not only reduce the toxic and side effects of drugs, reduce drug resistance, improve the stability of drugs in vivo, but also increase the accumulation of drugs in tumor sites, so that the efficacy of drugs can be better exerted, etc. It has been used clinically as a tumor-targeting drug carrier. Therefore, using N-(2-hydroxypropyl) methacrylamide with good water solubility as a drug carrier, a high molecular weight copolymer with anti-tumor activity is obtained by copolymerization with hennain, which is expected to provide anti-cancer field. more choices.

Contents of the invention

The object of the present invention is to provide a hennacopolymer with antitumor activity.

Another object of the present invention is to provide a preparation method of hennacopolymer with antitumor activity.

(1) Hennacopolymer with antitumor activity

The hennain copolymer with anti-tumor activity of the present invention is a biological phase formed by linking hennain to N-(2-hydroxypropyl)methacrylamide (HPMA) through polymerization. Good polymer copolymer - N-(2-hydroxypropyl)methacrylamide-2-hydroxyl-1,4-naphthoquinone, its structure is as follows:

 

In the formula, n=10~15, m=85~90; number average molecular weight Mn=2.4~3.2×10 ⁴ , Mw/Mn=1.16~1.21.

The antitumor activity of the copolymer 2-hydroxyl-1,4-naphthoquinone and N-(2-hydroxypropyl)methacrylamide is superimposed, which further promotes the inhibitory effect of the polymer on tumors and greatly prolongs the antitumor activity of the polymer. The residence time of anticancer drugs in the tumor. Moreover, the polymer carrier HPMA also reduces the toxicity of anticancer drugs, and at the same time shows good biocompatibility, thereby reducing the damage to normal tissues, and is an antitumor active substance with application prospects.

The preparation method of the hennain copolymer with antitumor activity of the present invention comprises the following process steps:

(1) Preparation of intermediate: Dissolve 2-hydroxy-1,4-naphthoquinone in acetone, add acid-binding agent, add methacryloyl chloride under nitrogen protection, and react at -5~5°C for 4~5 h time; after the reaction is complete, the reaction solution is concentrated and separated by silica gel column chromatography to obtain an intermediate compound. Its structural formula is:

                          

The molar ratio of the 2-hydroxy-1,4-naphthoquinone to methacryloyl chloride is 1:1-1:1.2.

The acid-binding agent is sodium hydroxide, potassium carbonate, sodium carbonate, triethylamine or trimethylamine, and the molar ratio of methacryloyl chloride to the acid-binding agent is 1:1.1-1:1.

The eluent used in the silica gel column chromatography is: ethyl acetate and petroleum ether in a volume ratio of 1:40 to 1:50.

(2) Preparation of the target compound: Dissolve N-(2-hydroxypropyl)methacrylamide and the intermediate compound in DMSO and acetone, add the initiator azobisisobutyronitrile (AIBN), under nitrogen protection at 50 React at ~60°C for 20~24 hours, precipitate with a mixture of acetone and ether, filter, dissolve the precipitate with anhydrous methanol, and finally centrifuge with an ultrafiltration concentration centrifuge tube with a molecular weight of 3000 to remove small molecules to obtain the target polymer copolymer thing.

The molar ratio of the intermediate compound to N-(2-hydroxypropyl)methacrylamide (HPMA) is 1:4-1:20.

The volume ratio of the DMSO and acetone is 1:1~1:0.5.

The dosage of the initiator azobisisobutyronitrile is 5%-10% of the total mass of the intermediate compound and N-(2-hydroxypropyl)methacrylamide.

Fig. 1 is the H NMR spectrum of the henniferin copolymer with anti-tumor activity prepared by the above method. Through proton nuclear magnetic resonance spectrum analysis, it can be concluded that the high molecular polymer obtained by copolymerization has a chemical shift between 6.5-9ppm, and there is a peak of hydrogen on the aromatic ring in the compound 2-hydroxyl-1,4-naphthoquinone, and the chemical shift is between 6.5 and 9ppm. The characteristic peaks -CH- (connected to -OH) and -CH ₂ (connected to -NH-) on HPMA will appear around 3.70ppm and 2.81ppm, and there is no peak at the chemical shift between 5-6ppm, indicating that the above Methods The product obtained by copolymerization is consistent with the previously designed structure.

2. Antitumor activity experiment

1. In vitro tumor cell growth inhibition experiment

Use the tetrazolium salt reduction method (MTT) to test the H22 cell line: take the liver cancer H22 cells in the logarithmic phase of growth, adjust the cell concentration to 2×10 ⁴ cells/ml, add 90 μl /well, edge wells were filled with sterile PBS. Incubate at 5% CO ₂ at 37°C, place in the incubator until adhered to the wall, and then add drugs. For the high molecular weight copolymer of the present invention and hennafosin (2-hydroxy-1,4-naphthoquinone), 5 gradients with concentrations of 0.2, 1, 5, 25, and 125 μg/ml were respectively set. Four replicate wells were set up in both the experimental group and the control group. After adding the drug, the cells were cultured in a carbon dioxide incubator at a temperature of 37 °C for 24, 48, and 72 h, and then they were taken out and centrifuged first, and then the supernatant in the 96-well plate was discarded. After carefully washing with PBS for 2-3 times, add 20 μl of MTT (tetrazolium, 5 mg/ml, ie 0.5% MTT) solution to each well, and place in a carbon dioxide incubator at 37°C for 4 hours. To terminate the culture, carefully aspirate the culture medium in the well. Add 150 μl of DMSO to each well, shake on a shaker at low speed for 10 min, and fully dissolve the crystals. The absorbance OD value of each well was measured on a microplate reader at 570 nm. The cell growth inhibition rate was calculated according to the following formula:

Inhibition rate = [(1-average OD value of experimental group)/average OD value of control group]×100%

The test results are shown in Table 1 and Figure 2.

Table 1 Anticancer activity data of high molecular weight copolymer and hennain in vitro for 72h

Among them, the IC ₅₀ of the copolymer =7.7μg/ml, and the IC ₅₀ of 2-hydroxy-1,4-naphthoquinone =25.9μg/ml.

2. In vivo inhibition of tumor cell growth experiments

The H22 solid tumor inoculated in Kunming mice was tested by intravenous injection and tumor direct injection: the inoculated Kunming decimals were randomly divided into groups, 6 mice in each group. Tumor-bearing mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (7 mg/kg), and 0.1 mL of 100 μCi (3.7 MBq) of normal saline was injected into the tail vein. , polymer copolymer, administered once a day for 18 consecutive days, a total of 18 times. During the administration period, the longest diameter (L) and shortest diameter (W) of the transplanted tumor were measured with a vernier caliper every 2 days, and the volume of the transplanted tumor was calculated V = (L×W ² )/2, and the tumor weight inhibition rate (%) IR = (control Average tumor volume in the treatment group - average tumor volume in the treatment group)/Tumor volume in the control group × 100. The test results are shown in Table 2 and Figure 3.

Table 2 Inhibition rate of hennain and hennain-like polymers on liver cancer H22 cells (%)

 

In summary, the hennain-based high molecular polymer prepared by the present invention exhibits good biological water solubility, reduces the toxicity of the polymer, and has a good inhibitory effect on liver cancer H22 tumor cells. Anticancer drugs provide a new way of thinking.

Description of drawings

Fig. 1 is the proton nuclear magnetic resonance spectrum of the hennain copolymer with antitumor activity of the present invention;

Fig. 2 is the 72h anticancer activity curve of the polymer copolymer of the present invention and hennain in vitro;

Fig. 3 is the in vivo anticancer activity curve of the polymer copolymer of the present invention and henifrin.

Detailed ways

The synthesis and structural characterization of the polymer copolymer of the present invention will be further described below through specific examples.

Example 1

(1) Preparation of intermediate compound (a): Weigh 0.5279 g (3 mmol) of 2-hydroxy-1,4-naphthoquinone in a flask, dissolve it in 30 ml of acetone, add 0.4146 g (3 mmol) of Water K ₂ CO ₃ was used as an acid-binding agent. Under the protection of nitrogen, 0.3763 g (3.6 mmol) of methacryloyl chloride was slowly dropped into an ice-water bath with constant stirring, and reacted at room temperature. During the reaction, TCL was used to monitor the progress of the reaction. Suction filtration after the reaction was completed, the filtrate was concentrated and the crude product was separated with a silica gel column (ethyl acetate/petroleum ether = 1/40 (v/v)), the product components were collected, and the solvent was evaporated to obtain 0.7432 g of a yellow solid. The rate is 88%.

¹ H NMR (400 MHz, CCl ₃ , δ, ppm): 2.111 (s, 3H, -CH ₃ ), 5.915 (s, 1H, -C=CH ₂ ), 6.455 (s, 1H, -C=CH ₂ ), 6.862 (s, 1H, naphthyl ring), 7.809 (d, 2H, naphthyl ring), 8.138 (t, 2H, naphthyl ring). ¹³ C NMR (100 MHz, CCl ₃ , δ, ppm): 18.148, 125.966 , 126.885, 129.330, 130.986, 131.858, 133.949, 134.345, 154.571, 164.175, 178.583, 184.492.

(2) Preparation of copolymer: Weigh 0.2720 g (95%, 1.9 mmol) of HPMA into a Shleck bottle, heat and dissolve with 0.5 ml of DMSO, then add 0.5 ml of acetone; weigh 0.0242 g (5%, 0.1 mol) intermediate compound into the Shleck bottle, stir until dissolved, add 0.0149 g (5%, wt) azoisobutyronitrile (AIBN) when cooled to room temperature, vacuumize and nitrogen cycle 3~5 times, seal Finally, keep the temperature at about 55°C and react for 24 hours. Precipitate with a mixture of acetone and ether (volume ratio 7:3), filter and dissolve the precipitate with 1ml of anhydrous methanol, centrifuge with an ultrafiltration concentration centrifuge tube with a molecular weight of 3000, remove small molecules, and obtain 80mg of polymer. The yield is about 27%.

M _n =3.2×10 ⁴ , M _w /M _n =1.21. ¹ H NMR (400 MHz, D ₂ O, δ, ppm): 0.712 (-CH ₃ ), 0.975 (-CH ₃ ), 1.375-1.871 ( -CH ₂ -), 2.880-2.998 (-CH ₂ - connected with -OH), 3.714 (-CH- connected with -NH-), 6.894 (naphthyl ring), 7.892 (naphthyl ring) ,8.614 (naphthyl ring). ¹³ C NMR (400 MHz, DMSO-d6, δ, ppm): 22.59, 31.77, 45.64, 48.63, 65.91, 122.37, 129.58, 129.94, 131.57, 133.41, 156.99, 163.74, 1734.15.

Example 2

(1) Preparation of intermediate compound: Same as Example 1.

(2) Preparation of copolymer: Weigh 0.1589 g (92%, 1.15 mmol) of HPMA into a Shleck bottle, heat and dissolve with 0.5 ml of DMSO, then add 0.5 ml of acetone; weigh 0.0242 g (8%, 0.1 mol) intermediate compound into the Shleck bottle, stir until dissolved, add 0.0145 g (8%, wt) azoisobutyronitrile (AIBN) when cooled to room temperature, vacuumize and nitrogen cycle 3~5 times, seal Finally, keep the temperature at about 55°C and react for 24 hours. Precipitate with a mixture of acetone and ether (volume ratio 7:3), filter and dissolve the precipitate with 1ml of anhydrous methanol, centrifuge with an ultrafiltration concentration centrifuge tube with a molecular weight of 3000, remove small molecules, and obtain 56mg of polymer. The yield is about 38.2%.

M _n =2.4×10 ⁴ , M _w /M _n =1.16. ¹ H NMR (400 MHz, D ₂ O, δ, ppm): 0.785(-CH ₃ ), 0.942 (-CH ₃ ), 1.521-1.639 ( -CH ₂ -), 2.854-2.998 (-CH ₂ - connected with -OH), 3.541(-CH- connected with -NH-), 7.012 (naphthyl ring), 7.924 (naphthyl ring) , 8.607 (naphthyl ring). ¹³ C NMR (400 MHz, DMSO-d6, δ, ppm): 23.89, 31.41, 46.74, 49.85, 67.90, 124.26, 130.14, 131.27, 133.46, 136.255, 158.30, 165.07, 716.81, 2

Example 3

(1) Preparation of intermediate compound: Same as Example 1.

(2) Preparation of copolymer: Weigh 0.2488 g (90%, 1.8 mmol) of HPMA into a Shleck bottle, heat and dissolve with 0.2 ml of DMSO, then add 0.2 ml of acetone; weigh 0.0484 g (10%, 0.2 mol) intermediate compound into the Shleck bottle, stir until dissolved, add 0.0238 g (8%, wt) azoisobutyronitrile (AIBN) when cooled to room temperature, vacuumize and nitrogen cycle 3~5 times, seal Finally, keep the temperature at about 55°C and react for 24 hours. Precipitate with a mixture of acetone and ether (volume ratio 7:3), filter and dissolve the precipitate with 1ml of anhydrous methanol, centrifuge with an ultrafiltration concentration centrifuge tube with a molecular weight of 3000, remove small molecules, and obtain 85mg of polymer. The yield is about 28.6%.

M _n =2.4×10 ⁴ , M _w /M _n =1.16. ¹ H NMR (400 MHz, D ₂ O, δ, ppm): 0.808(-CH ₃ ), 1.019 (-CH ₃ ), 1.579-1.724 ( -CH ₂ -), 2.914-3.042 (-CH ₂ - connected with -OH), 3.688(-CH- connected with -NH-), 7.504-7.554 (naphthyl ring), 7.853-7.869 (naphthyl ring). ¹³ C NMR (400 MHz, DMSO-d6, δ, ppm): 23.64, 30.59, 46.17, 49.50, 67.15, 123.61, 129.86, 130.67, 132.71, 135.17, 157.46, 164.57, 176.28, 17.17

Example 4

(1) Preparation of intermediate compound: Same as Example 1.

(2) Preparation of copolymer: Weigh 0.2032 g (88%, 1.47 mmol) HPMA into a Shleck bottle, heat and dissolve with 0.5 ml of DMSO, then add 0.5 ml of acetone; weigh 0.0484 g (12%, 0.2 mol) intermediate compound into the Shleck bottle, stir until dissolved, add 0.0252 g (10%, wt) azoisobutyronitrile (AIBN) when cooled to room temperature, vacuumize and nitrogen cycle 3~5 times, seal Finally, keep the temperature at about 55°C and react for 24 hours. Precipitate with a mixture of acetone and ether (volume ratio 7:3), filter and dissolve the precipitate with 1ml of anhydrous methanol, centrifuge with an ultrafiltration concentration centrifuge tube with a molecular weight of 3000, remove small molecules, and obtain 79mg of polymer. The yield is about 31.2%.

M _n =2.4×10 ⁴ , M _w /M _n =1.16. ¹ H NMR (400 MHz, D ₂ O, δ, ppm): 0.796(-CH ₃ ), 0.981 (-CH ₃ ), 1.484-1.651 ( -CH ₂ -), 2.875-2.980 (-CH ₂ - connected with -OH), 3.617(-CH- connected with -NH-), 6.968 (naphthyl ring), 7.871 (naphthyl ring) , 8.640 (naphthyl ring). ¹³ C NMR (400 MHz, DMSO-d6, δ, ppm): 22.81, 30.27, 44.59, 48.73, 67.09, 122.48, 128.61, 130.09, 131.47, 135.85, 156.90, 164.72, 177.14, 3.

Example 5

(1) Preparation of intermediate compound: Same as Example 1.

(2) Preparation of copolymer: Weigh 0.3179 g (85%, 2.3 mmol) of HPMA into a Shleck bottle, heat and dissolve with 0.2 ml of DMSO, then add 0.2 ml of acetone; weigh 0.0968 g (15% , 0.4 mol) intermediate compound into the Shleck bottle, stir until dissolved, add 0.0415 g (10%, wt) azoisobutyronitrile (AIBN) when cooled to room temperature, vacuumize and nitrogen cycle 3~5 times , keep the temperature at about 55°C after sealing, and react for 24 hours. Precipitate with a mixture of acetone and ether (volume ratio 7:3), filter and dissolve the precipitate with 1ml of anhydrous methanol, centrifuge with an ultrafiltration concentration centrifuge tube with a molecular weight of 3000, remove small molecules, and obtain 93mg of polymer. The yield is about 22.4%.

M _n =2.9×10 ⁴ , M _w /M _n =1.19. ¹ H NMR (400 MHz, D ₂ O, δ, ppm): 0.789 (-CH ₃ ), 0.986 (-CH ₃ ), 1.259-1.261 ( -CH ₂ -), 2.889-2.908 (-CH ₂ - connected with -OH), 3.684 (-CH- connected with -NH-), 7.017 (naphthyl ring), 7.894 (naphthyl ring) ,8.627(naphthyl ring). ¹³ C NMR (400 MHz, DMSO-d6, δ, ppm): 20.17, 30.64, 43.57, 47.19, 63.74, 121.40, 128.09, 129.16, 132.71, 133.89, 154.72, 162.64, 173.05, 8.9

Claims (9)

1. A hennain copolymer with antitumor activity is formed by linking 2-hydroxyl-1,4-naphthoquinone to N-(2-hydroxypropyl)methacrylamide by polymerization A biocompatible high molecular copolymer, its structure is as follows:    In the formula, n=10~15, m=85~90. 2. The preparation method of the hennacopolymer with antitumor activity as claimed in claim 1, comprising the following steps: (1) Preparation of intermediate compounds: Dissolve 2-hydroxy-1,4-naphthoquinone in acetone, add acid-binding agent, add methacryloyl chloride under nitrogen protection, and react at -5~5°C for 4~ 5 h; after the reaction was complete, the reaction solution was concentrated and separated by silica gel column chromatography to obtain the intermediate compound; (2) Preparation of the target compound: Dissolve N-(2-hydroxypropyl)methacrylamide and the intermediate compound in DMSO and acetone, add the initiator azobisisobutyronitrile, and store at 50~60°C under nitrogen protection React for 20-24 hours, precipitate with a mixture of acetone and ether, filter, dissolve the precipitate with anhydrous methanol, and finally centrifuge with an ultrafiltration concentration centrifuge tube with a molecular weight of 3000 to remove small molecules to obtain the target polymer copolymer. 3. The preparation method of the hennain copolymer with antitumor activity as claimed in claim 2, characterized in that: in step (1), the 2-hydroxyl-1,4-naphthoquinone and methacryloyl chloride The molar ratio is 1:1~1:1.2. 4. The preparation method of the hennamonin copolymer having antitumor activity as claimed in claim 2, characterized in that: in step (1), the acid-binding agent is sodium hydroxide, potassium carbonate, sodium carbonate, three ethylamine or trimethylamine. 5. The preparation method of the hennain copolymer with antitumor activity as claimed in claim 2, characterized in that: in step (1), the molar ratio of the methacryloyl chloride to the acid-binding agent is 1:1.1 ~1:1. 6. The preparation method of the hennamonin copolymer with antitumor activity as claimed in claim 2, characterized in that: in step (1), the eluent used in the silica gel column chromatography is: ethyl acetate and petroleum Ether at a volume ratio of 1:40~1:50. 7. The preparation method of the hennamonin copolymer with antitumor activity as claimed in claim 2, characterized in that: in step (2), the intermediate compound and N-(2-hydroxypropyl)methyl The molar ratio of acrylamide is 1:4~1:20. 8. The preparation method of the hennamonin copolymer with anti-tumor activity as claimed in claim 2, characterized in that: in step (2), the amount of the initiator azobisisobutyronitrile is the intermediate compound and 5%~10% of the total mass of N-(2-hydroxypropyl)methacrylamide. 9. The preparation method of the hennain copolymer with antitumor activity as claimed in claim 2, characterized in that: in the mixed solution of acetone and ether described in step (2), the volume ratio of acetone and ether is 7: 3~7:1.