Antibacterial and Antitumor Activities of Biscoumarin and Dihydropyran Derivatives

A novel series of biscoumarin (1–4) and dihydropyran (5–13) derivatives were synthesized via a one-pot multicomponent condensation reaction and evaluated for antibacterial and antitumor activity in vitro. The X-ray crystal structure analysis of four representative compounds, 3, 7, 9 and 11, confirmed the structures of these compounds. Compounds 1–4 showed the most potent antitumor activity among the total 13 derivatives; especially for compounds 1 and 2, they also emerged as promising antibacterial members with better antibacterial activity. In addition, the results of density functional theory (DFT) showed that compared with compounds 3 and 4, biscoumarins 1 and 2 had lower intramolecular hydrogen bonds (HB) energy in their structures.


Introduction
Natural products have a profound impact on both chemical biology and drug discovery, and the great structural diversity of natural products with various interesting biological characteristics has always provided medicinal chemists an important source of inspiration in their search for new molecular entities with pharmacological activity [1,2]. Among them, biscoumarin and dihydropyran derivatives are two important groups of compounds covering a wide range of biological properties, including anti-oxidant, anti-inflammatory [3] and anti-microbial [4] as well as anticancer activities [5,6]. However, many of them are not suitable for therapeutic application due to their relatively lower activity or evident side-effect properties; and for the already marketable antibacterial and antitumor drugs [7,8], resistance has become one of main reasons for their failure in chemotherapy. Hence the search for high-quality novel antibacterial and anticancer agents has always been advisable and emergent [9].  In order to get more effective antibacterial and antitumor agents, it is possible to make modifications on active chemical structures of title compounds. In the present study, a novel series of biscoumarin (1)(2)(3)(4) and dihydropyran (5)(6)(7)(8)(9)(10)(11)(12)(13) derivatives were firstly synthesized (Figure 1), their antibacterial activities were then measured in vitro against Staphylococcus aureus (S. aureus ATCC 29213), methicillin-resistant S. aureus (MRSA XJ 75302), vancomycin-intermediate S. aureus (Mu50 ATCC 700699), and USA 300 (Los Angeles County clone, LAC), and finally their antitumor activities on intestinal epithelial adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell line (4T1) and pancreatic cancer cell line (PANC1) in vitro were then evaluated.

Molecular Structure
The crystal structures of compounds 3, 7, 9 and 11 are given in Figure 2. In the crystal structure of compound 3, two 4-hydroxycoumarin moieties are linked through a methylene bridge, wherein one hydrogen atom has been replaced with a 4-methylthiophenyl group; and two classical intramolecular hydrogen bonds (O3-H3···O4 and O6-H6···O1) between a hydroxyl group of one coumarin fragment and a lacton carbonyl group of another coumarin fragment further stabilize the whole structure. In the crystal structures of compounds 7, 9 and 11, the new formed pyran ring and the adjacent ketone (coumarin) ring are both basically planar, and the two planes are also essentially parallel to each other. However, the aromatic ring makes a torsion angle to the pyran ring in the three compounds.

Hydrogen Bonds Energies in Biscoumarins 1-4
We only used compound 1 as an example to estimate single and total intramolecular hydrogen bonds (HB) energies. The global minimum structure is stabilized by two HBs (1ab); two higher energy structures are stabilized by one HB (1a and 1b, respectively). The corresponding values are listed in Table 1.
Based on our previous calculation results [10], B3LYP/6-31G* exhibited sufficient agreement with experimental data and lower computational cost, so further theoretical study was performed at this level.

Minimal Inhibitory Concentration (MIC) Assay
For compounds 1-13, one drug-sensitive S. aureus (S. aureus ATCC 29213) strain and three MRSA strains (MRSA XJ 75302, Mu50, and USA 300 LAC) were used in the systematic analysis of their antibacterial activities in vitro. Because of the liposolubility of these compounds, they were dissolved into the solution with 1% dimethyl sulfoxide (DMSO) at final concentration. From Table 2, we can see that, among these compounds, compounds 1 and 2 exerted more potent anti-bacterial activity against the tested S. aureus with minimum inhibitory concentration (MIC) values in the range of 2-16 μg/mL. Compared with compounds 1-13, the MIC values of levofloxacin, ceftazidime, ceftriaxone, gentamicin and piperacillin against S. aureus (ATCC 29213) strains were lower (less than 8 μg/mL) but were higher against other three strains at varying degrees.
S means drug susceptibility, R means drug resistance.

In Vitro Antitumor Activity
Intestinal epithelial adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell line (4T1) and pancreatic cancer cell line (PANC1) representing three different tumor types were used in the systematic analysis of the antitumor activities of the newly synthesized compounds 1-13 in vitro. For comparison purpose, the cytotoxicity of carboplatin, a standard antitumor drug, was evaluated under the same condition.
The results showed that all the tested compounds possessed a certain degree of antitumor activities against the three tumor cell lines and their inhibitory action get stronger with the corresponding higher concentration. The related half maximal inhibitory concentration (IC50) and IC90 values (dose of the compound which cause a 50% and 90% reduction of survival values, respectively) are shown in Table 3. As can be seen in Table 3, there is great difference in the antitumor activity between the four different groups of the tested compounds. Biscoumarins 1-4 from the first group showed more potent antitumor activity against the three tested tumor cells (HuTu80, 4T1 and PANC1) with IC50 and IC90 values of 18.78-32.63 μg/mL and 36.05-64.55 μg/mL, respectively, which is much lower than the IC50 and IC90 values (45.85-65.62 μg/mL and 102.14-126.24 μg/mL) of the positive control drug carboplatin. However, the compounds in other three groups demonstrated lower antitumor activity with relatively higher IC50 and IC90 values.
All antibiotics used were purchased from the National Institute for the Control of  (Beijing, China). The cells were cultured in RPMI supplemented with 10% HIFBS and 1% PS. Cells were cultured in a 5% CO2 in a humidified atmosphere at 37 °C.

X-ray Crystallography
For X-ray diffraction experiments, single crystals of compounds 3, 7, 9 and 11 were grown from methanol. The X-ray diffraction data were collected on a Bruker SMART APEX II CCD diffractometer (Bruker Optics, Ettlingen, Germany) equipped with a graphite monochromated Mo Kα radiation (λ = 0.71073 Å) by using the ω-2θ scan technique at room temperature. The structure was solved by direct methods using SHELXS-97 (Sheldrick 1997, University of Gottingen, Germany) and refined using the full-matrix least squares method on F 2 with anisotropic thermal parameters for all non-hydrogen atoms by using SHELXL-97 [15]. Hydrogen atoms were generated geometrically. The crystal data and details concerning data collection and structure refinement are given in Table 4

Quantum Chemical Calculations
All calculations were carried out using the Gaussian 09 package [16]. Density functional theory (DFT), Becke's three-parameter hybrid function (B3LYP), and LYP correlation function were used to fully optimize all the geometries on the energy surface without constraints. To obtain precise results that are in conjunction with experimental results, three basis sets, namely 6-31G*, 6-31 + G**, and 6-311G*, were tested. Frequency calculations at the B3LYP (with basis sets 6-31G*) level of theory were carried out to confirm stationary points as minima and to obtain the zero-point energies and the thermal correlation data at 1 atm and 298 K.

Minimal Inhibitory Concentration (MIC) Assay
Based on the CLSI broth microdilution method [17], the determination of minimum inhibitory concentrations (MICs) via microdilution assay was performed in sterilized 96-well polypropylene microtiter plates (Sigma-Aldrich) in a final volume of 200 μL. Bacteria were grown overnight in nutrient broth. Mueller-Hinton (MH) broth (100 μL) containing bacteria (5 × 10 5 CFU/mL) was added to 100 μL of the culture medium containing the test compound (0.12 μg/mL-256 μg/mL in serial 2-fold dilutions). The plates were incubated at 37 °C for 20 h in an incubator. About 50 µL of 0.2% triphenyl tetrazolium chloride (TTC), a colorimetric indicator, was added to each well of microtiter plates and incubated at 35 °C for 1.5 h. The TTC-based MIC was determined as the lowest concentration of oxacillin that showed no red color change indicating complete growth inhibition.

Cell Viability Assay
Viability of intestinal epithelial adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell line (4T1) and pancreatic cancer cell line (PANC1) was determined by using the MTT assay as described by Mosmann [18]. Cells reaching 70%-80% confluency were treated with various concentrations of the synthesized compounds with 1% dimethyl sulfoxide (DMSO) as a negative control. After 48 h incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added and incubated for an additional 4 h. Subsequently, the medium was aspirated carefully, and 150 μL of DMSO was added. After incubation for 15 min, the optical density was measured at 490 nm using FlexStation 3 benchtop multi-mode microplate reader (Molecular Devices). Data were recorded and analyzed for the assessment of the effects of the test substances on cell viability and growth inhibition. The IC50 and IC90 values were calculated using regression equation as explained before. The results are presented as the average percentage viability to the negative control (1% DMSO). The percentage of cell viability was calculated using the following formula: % cell viability = (absorbance of treated/absorbance of untreated) × 100. The percentage of inhibition was plotted against the concentration in Microsoft excel and the IC50 was calculated using the regression equation.

Conclusions
In this work, we synthesized two new series of biscoumarin and dihydropyran derivatives; evaluated their antibacterial activities in vitro against one-drug-sensitive S. aureus (S. aureus ATCC 29213) strain and three MRSA strains (MRSA XJ 75302, Mu50, and USA 300 LAC); and then measured their antitumor activities on intestinal epithelial adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell line (4T1) and pancreatic cancer cell line (PANC1) in vitro.
In addition, X-ray structural analysis showed that biscoumarins 1-4 had two classical intramolecular O-H···O hydrogen bonds in their structures. Their corresponding intramolecular hydrogen bonds energy was calculated to be −116.20463, −115.7031595, −118.0346035 and −121.970228 kJ/mol, respectively.
Among the synthesized compounds, compounds 1-4 had more potent antitumor activity against the tested three cancer cell lines with the IC50 and IC90 values of 18.78-32.63 μg/mL and 36.05-64.55 μg/mL, respectively, which is much lower than that of the positive control drug carboplatin; compounds 1 and 2, with lower intramolecular hydrogen bonds energy, also showed the most potent antibacterial effect on four S. aureus bacterial strains with the MIC values of 2-16 μg/mL. The reason may be that intramolecular HB strength is related to the stability of chemical structure, which further affects the binding affinity between molecules and target protein.