Gossypol l-arginine schiff base compound with antitumor activities and a method of preparing the same

1.74 g (10 mmol) of L-arginine and 2.07 g (4 mmol) of gossypol were placed in a 100 mL three-necked flask. 50 mL anhydrous ethanol was added to the flask to dissolve the L-arginine and gossypol, and 3 drops of acetic acid was added as a catalyst. The mixture was heated at 60° C. with a water bath under magnetic stir. TLC was used to track the reaction to completion. The hot reaction mixture was filtered and washed with ethanol. The filtrate was cooled, filtered, and washed with ethanol three times to obtain a crude product. The crude product was recrystallized in anhydrous ethanol to give a yellow solid, 1.88 g, a yield of 56.61%.

Yellow crystalline powder. M.P. 182.3° C.; 1 H-NMR (300 MHz, DMSO-d 6 ) δ(ppm): 13.76 (2H, s), 12.32 (2H, s), 10.31 (2H, s), 9.94 (2H, s), 9.58 (2H, s), 9.46 (2H, s), 8.75 (4H, d, J=7.8 Hz), 8.74 (2H, d, J=7.8 Hz), 8.25 (2H, s), 7.81 (2H, s), 3.42 (2H, q, J=8.0 Hz), 3.30 (2H, q, J=8.0 Hz), 2.87 (2H, m, J=7.8 Hz), 2.78 (6H, s), 1.75 (4H, q, J=7.8 Hz), 1.50 (4H, m, J=7.8 Hz), 1.33 (12H, d, J=7.8 Hz); 13 C-NMR (101 MHz, DMSOd 6 ) δ(ppm): 175.7, 163.7, 153.4, 146.1, 153.4, 146.1, 144.1, 132.5, 130.7, 128.7, 119.0, 117.4, 112.2, 109.7, 101.4, 55.5, 37.9, 28.5, 27.1, 24.6, 24.0; MS (ESI) for (M+H)+: 831.4.

1.74 g (10 mmol) of L-arginine and 2.07 g (4 mmol) of gossypol were placed in a 100 mL three-necked flask. 50 mL anhydrous methanol was added to the flask to dissolve the L-arginine and gossypol, and 0.05 g of p-toluenesulfonic acid was added as a catalyst. The mixture was heated at 60° C. with a water bath under magnetic stir. TLC was used to track the reaction to completion. The hot reaction mixture was filtered and washed with methanol. The filtrate was cooled, filtered, and washed with methanol three times to obtain a crude product. The crude product was recrystallized in anhydrous methanol to give a yellow solid, 1.43 g, a yield of 43.06%.

1.74 g (10 mmol) of L-arginine and 2.07 g (4 mmol) of gossypol were placed in a 100 mL three-necked flask. 50 mL anhydrous isopropanol was added to the flask to dissolve the L-arginine and gossypol, and 0.04 g (0.2 mmol) of ZnCl 2 was added as a catalyst. The mixture was heated at 80° C. with a water bath under magnetic stir. TLC was used to track the reaction to completion. The hot reaction mixture was filtered and washed with methanol. The filtrate was cooled, filtered, and washed with ethanol three times to obtain a crude product. The crude product was recrystallized in anhydrous ethanol to give a yellow solid, 0.97 g, a yield of 28.90%.

1.74 g (10 mmol) of L-arginine and 2.07 g (4 mmol) of gossypol were placed in a 100 mL three-necked flask. 50 mL anhydrous ethanol was added to the flask to dissolve the L-arginine and gossypol, and 0.04 g (0.2 mmol) of ZnCl 2 was added as a catalyst. The mixture was heated at 70° C. with a water bath under magnetic stir. TLC was used to track the reaction to completion. The hot reaction mixture was filtered and washed with methanol. The filtrate was cooled, filtered, and washed with ethanol three times to obtain a crude product. The crude product was recrystallized in anhydrous ethanol to give a yellow solid, 0.65 g, a yield of 19.40%.

The Anti-Tumor Activity Test of the Compounds of the Present Invention

The compounds of the present invention were subjected to tumor cell proliferation inhibition test, and conventional MTT method was used.

Cell lines: human hepatoma cells (HepG2), human lung cancer cells (A-549), human gastric cancer cells (SGC-7901). The culture medium was DMEM+15% NBS+double antibody.

Sample solution preparation: after dissolving with DMSO (Merck), PBS (-) was added to obtain 100 μmol/L solution or homogeneous suspension. The solution was diluted with PBS (-) in DMSO to a final concentration of 0.1, 1, 10, 20, 40, 60, 80, 100 μmol/L.

Anti-uterine fibroids drug gossypol was used as control solution, prepared under the same condition.

Cell culture: adherent growth Tumor cells were cultured in 1640 medium containing 10% inactivated neonatal bovine serum and penicillin, streptomycin (1 million U/L), placed in carbon dioxide incubator at 37° C., 5% CO 2 , and saturated humidity. Cells were treated serially passaged 2-3 times. The first culture was washed with PBS 2 times, and digested with trypsin. Fresh culture medium was added evenly, cells were adjusted to a appropriate concentration and transferred into a new culture flask. Cell in an exponential phase were chosen for the tests.

MTT Assay for Cell Viability and IC 50 Determination:

Experimental Principle: Living cells mitochondria in the dehydrogenase can reduce yellow MTT to water-insoluble blue-violet product MT (MTT formazan), deposited in the cells. The amount of production is proportional to the number of living cells. Dead cells do not reduce yellow MTT. DMSO can dissolve blue violet crystals, and the color depth is proportional to the amount contained, so the absorbance measured by the microplate reader can reflect the cell viability.

Methods: The exponential phase cells were digested and counted and seeded in 96-well plates at a density of 2×10 4 /mL at 100 μl per well. After 24 hours of incubation, the cells to be tested were treated with 0.1, 1, 10, 20, 40, 60, 80, 100 μmol/L of the compounds. Each experimental group had 5 wells in each concentration, and the culture medium containing 0.4% DMSO was used as control. After 48 hours, the supernatant was discarded, and 100 μl of MTT ((2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazole hydrobromide) (1 mg/mL) was added to each well. After another 4 hours, the supernatant was discarded, and 100 μl of DMSO was added to each well. After mixing, the absorbance was measured at 570 nm using a microplate reader. An IC 50 calculation software was used to determine the half inhibitory concentration (IC 50 ).

The test results are shown in Table 1. The compounds listed in the table correspond to the compounds described above.

The results show that the gossypol L-arginine Schiff base compound has excellent antitumor activities in the three cell lines tested. Specifically, the compound has better antitumor activities than gossypol against A549 and SCG-7901. The above experimental results indicate that the gossypol L-arginine Schiff base compound of the present invention has good antitumor activities. The compound can be used for the study of antitumor research and drug development.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.