Synthesis and evaluation of coumarin derivatives against human lung cancer cell lines

Series of novel coumarin derivatives [I (a–d) and II (a–d)] were successfully synthesized and their structures were determined based on infrared 1H-nuclear magnetic resonance (NMR), HRMS, and single crystal X-ray crystallography. Additionally, the new synthesized compounds were evaluated to identify the molecular characteristics that contribute to their cytotoxicity, which was tested against SK-LU-1, SPC-A-1 and 95D human lung cancer cell lines, using the MTT assay. The results of this study showed that compounds Ic, Id, IIc, and IId exhibited an efficient percentage of inhibition of cell proliferation.


Introduction
Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries (1)(2)(3). Currently, the traditional therapies of surgery, chemotherapy and radiation play an important role in the systemic treatment of cancer (4)(5)(6). However, the treatment outcome is generally poor. Thus, it is very important to find an effective alternative treatment for cancer (7)(8)(9).
Coumarins are an important class of compounds of both natural and synthetic origin. Many compounds that contain the coumarin moiety exhibit useful and diverse pharmaceutical and biological activities, and there has been a growing interest in their synthesis (10)(11)(12). Some of these coumarin derivatives have been found useful in photochemotherapy, antitumor and anti-HIV therapy, as CNS-stimulants, antibacterial, anticoagulant, antifungal, and antioxidant agents, and as dyes (13)(14)(15). Natural, semi-synthetic and synthetic coumarins are useful substances in drug research. Coumarins can be used not only to treat cancer, but also to treat the side effects caused by radiotherapy (16)(17)(18). In this paper, series of coumarin derivatives [I (a-d) and II (a-d)] ( Figure 1) have been synthesized and their potential antitumor activity was investigated.

Apparatus and materials
To obtain infrared (IR) spectra (400-4000 cm -1 ), we used a Brucker Equinox-55 spectrophotometer (Bruker, Germany), to obtain 1H-nuclear magnetic resonance (NMR) spectra we used a Varian Inova-400 spectrometer (Virian USA; at 400 MHz), and for the Mass spectra, we used a micrOTOF-Q II mass spectrometer (Bruker, Germany). The melting points were taken on a XT-4 micro melting apparatus (Ledon, China), and the thermometer was uncorrected.

Synthesis and characterization of compounds I (a-d) and II (a-d)
Compounds I (a-d) were synthesized according to the methods of a previous report (19). A mixture of aromatic aldehydes (10 mmol) and 4-hydroxycoumarin (20 mmol) was dissolved in 100 mL of acetic anhydride (Ac2O). A few drops of piperidine were added, and the mixture was stirred for 3 h at room temperature. After reaction completion, determined by thin-layer chromatography, water was added until precipitation occurred. After filtering the precipitates, they were sequentially washed with icecooled water and ethanol and then dried in a vacuum. The synthesis method for compounds II (a-d) was similar to that of compounds I (a-d), in which Ac2O solvent was replaced by ethanol.

Crystal structure determination
According to the evaporation of chloroform solution, suitable single crystals of compound I b became available. The diffraction data were acquired on a Bruker Smart Apex CCD area detector using a graphite monochromated Mo Ka radiation (l=0.71073 Å) at room temperature. The structure was solved by using the program SHELXL-97 (20) and Fourier difference techniques, and refined by full-matrix least-squares method on F2. Hydrogen atoms attached to carbon were placed in geometrically idealized positions and refined using a riding model. Crystallographic data for compound I b are reported in Table 1.

Cytotoxicity assay
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide) assay was performed as described previously (21). Cells were seeded onto a 96-well plate at a concentration of 10 4 cells/well and allowed to adhere overnight. Five replicates were prepared for each treatment and cultured for 48 or 72 h. After 20 mL of MTT (5 mg/mL) was added to each well, the cells were cultured for another 4 h. The supernatant was discarded. After 150 mL of DMSO was added to each well, the samples were incubated at 37°C for 30 min and then swirled for 10 min. The absorbance at 570 nm was measured using a microplate reader. Experiments were repeated three times.

Molecular structure
In order to confirm the configuration of the product, single crystals of compound I b were cultured for X-ray diffraction analysis. The crystal structure of compound I b is given in Figure 2. One can see a chloroform solvent molecule incorporated in the asymmetric unit. In general, the molecular dimensions of the title compound agree with the corresponding values of other similar compounds (9). In the title crystal structure, two 4-hydroxycoumarin moieties are linked through a methylene bridge, wherein  one hydrogen atom has been replaced with a 3,4dimethylphenyl residue. Two hydroxyl groups form an ether bond by releasing a water molecule. This results in the formation of a big, essential planar heterocyclic ring system with 4.349°and 5.217°dihedral angle between the newly formed ether ring and the two original coumarin rings, respectively. The dihedral angle between the mean planes of the heterocyclic ring system and the 3,4dimethylphenyl ring is 87.219°. The crystal packing structure along the a axis is shown in Figure 3. Its crystal structure represents a threedimensional supra-molecular network. Within the threedimensional structure, there is one type of C-HyO; a hydrogen bonding is observed among these organic ligands. Detailed information of C-HyO hydrogen bonding is reported in Table 2. In other words, the threedimensional supra-molecular framework was generated by means of the C-HyO hydrogen bonding interactions.

Pharmacology
The synthesized compounds I (a-d) and II (a-d) were screened in vitro for their antitumor activities against SK-LU-1, SPC-A-1 and 95D human lung cancer cell lines using the standard MTT method, with the antitumor drug docetaxel used as a positive control. The in vitro cytotoxicity screening assays were conducted at different  Table 3.
Among all of the investigated compounds, I c, I d, II c and II d exhibited the most potent growth inhibition in the three lung cancer cells, with an IC50 value of 20-25 mM, indicating that they are more potent than docetaxel, which exhibited an IC50 value of 25-30 mM. In contrast, other compounds exhibited significantly weaker activity, because the cytotoxicity was lower than that of docetaxel in the three cell lines, with an IC50 value of 4100 mM.
In conclusion, among the coumarin compounds that we successfully synthesized, compounds I c, I d, II c and II d had a strong electron-withdrawing group (-CH2Cl, -Cl, -CN and -CF3) in the phenyl ring with the highest cytotoxic activity against the cancer cell lines tested. Therefore, the four compounds are potential anticancer drugs, yet further tests are required to determine their in vivo antitumor activity.