Abstract
Background/Aim: 4H-1-Benzopyran-4-ones (chromones) provide a backbone structure for the chemical synthesis of potent anticancer drugs. In contrast to 2-(N-cyclicamino)chromones, the biological activity of 3-(N-cyclicamino)chromones has not been reported. In this study, cytotoxicity of 15 3-(N-cyclicamino)chromone derivatives was investigated and subjected to quantitative structure–activity relationship (QSAR) analysis. Materials and Methods: Cytotoxicity against four human oral squamous cell carcinoma cell lines and three oral normal mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor-specificity (TS) was evaluated as the ratio of mean 50% cytotoxic concentration (CC50) against normal oral cells to that against human oral squamous cell carcinoma cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by the CC50 against tumor cells. Apoptosis induction was evaluated by morphological observation, western blot analysis and cell-cycle analysis. For QSAR analysis, a total of 3,096 physicochemical, structural and quantum chemical features were calculated from the most stabilized structure optimized using CORINA. Results: 3-(4-phenyl-1-piperazinyl)-4H-1-benzopyran-4-one (3a) had the highest tumor specificity, comparable with that of melphalan, without induction of apoptosis. Compound 3a caused cytostatic growth inhibition and had much lower cytotoxicity against human oral keratinocytes compared to doxorubicin. TS of the 15 3-(N-cyclicamino)chromones was correlated with 3D structure and lipophilicity. Conclusion: Chemical modification of 3a may be a potential choice for designing a new type of anticancer drug.
- 3-(N-Cyclicamino)chromones
- QSAR analysis
- cytotoxicity
- tumor selectivity
- apoptosis induction
- molecular shape
4H-1-Benzopyran-4-one (chromone), found ubiquitously in the plant kingdom (1), provides a backbone structure for synthesis of various derivatives. We reported that 3-styrylchromones (2), 3-styryl-2H-chromenes (3) and 22-azolylchromones (4), had much higher cytotoxicity against human oral squamous cell carcinoma (OSCC) cell lines than against human normal oral mesenchymal cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell), yielding excellent tumor specificity, comparable with that of several anticancer drugs (5).
2-Aminochromone derivatives are reported to have various biological activities including anti-inflammatory activity (6, 7), antimicrobial activity (8), phosphodiesterase inhibition (8, 9), modulation of DNA repair (10), inhibition of DNA-dependent protein kinase and radiosensitization of a human tumor cell line (11), and as potential positron-emission tomographic agents for imaging of DNA-dependent protein kinase in cancer (12). We recently found that among 15 such 2-(N-cyclicamino)chromone derivatives, 7-methoxy-2-(4-morpholinyl)-4H-1-benzopyran-4-one had the highest tumor specificity, comparable with that of doxorubicin, without induction of apoptosis. Tumor specificity of these derivatives was correlated with molecular shape, especially 3D structure (13).
Structure of 15 3-(N-cyclicamino)chromones investigated in this study.
As far as we know, the biological activity of 3-aminochromones, structural isomers of 2-aminochromones, has not been reported. In continuation of discovering new biological activities of chromone derivatives, we investigated a total of 15 3-(N-cyclicamino)chromone derivatives (Figure 1) for their cytotoxicity against four human OSCC cell lines and three human normal oral cell types, and then subjected them to quantitative structure–activity relationship (QSAR) analysis.
Materials and Methods
Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM) from GIBCO BRL (Grand Island, NY, USA); fetal bovine serum, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin, ribonuclease (RNase) A from Sigma-Aldrich Inc. (St. Louis, MO, USA); propidium iodide (PI), dimethyl sulfoxide (DMSO), actinomycin D, and 4% paraformaldehyde phosphate buffer solution from Wako Pure Chem. Ind. (Osaka, Japan); Nonidet-P40 (NP-40) from Nakalai Tesque Inc. (Kyoto, Japan); culture plastic dishes and 96-well plates from Techno Plastic Products AG (Trasadingen, Switzerland).
Synthesis of 3-(N-cyclicamino)chromone derivatives. 3-(1-piperidinyl)-4H-1-benzopyran-4-one (1a), 6-methoxy-3-(1-piperidinyl)-4H-1-benzopyran-4-one (1b), 7-methoxy-3-(1-piperidinyl)-4H-1-benzopyran-4-one (1c), 3-(4-phenyl-1-piperidinyl)-4H-1-benzopyran-4-one (2a), 6-methoxy-3-(4-phenyl-1-piperidinyl)-4H-1-benzopyran-4-one (2b), 7-methoxy-3-(4-phenyl-1-piperidinyl)-4H-1-benzopyran-4-one (2c), 3-(4-phenyl-1-piperazinyl)-4H-1-benzopyran-4-one) (3a), 6-methoxy-3-(4-phenyl-1-piperazinyl)-4H-1-benzopyran-4-one (3b), 7-methoxy-3-(4-phenyl-1-piperazinyl)-4H-1-benzopyran-4-one (3c), 3-(4-methyl-1-piperazinyl)-4H-1-benzopyran-4-one (4a), 6-methoxy-3-(4-methyl-1-piperazinyl)-4H-1-benzopyran-4-one (4b), 7-methoxy-3-(4-methyl-1-piperazinyl)-4H-1-benzopyran-4-one (4c), 3-(4-morpholinyl)-4H-1-benzopyran-4-one (5a), 6-methoxy-3-(4-morpholinyl)-4H-1-benzopyran-4-one (5b), 7-methoxy-3-(4-morpholinyl)-4H-1-benzopyran-4-one (5c) were synthesized by condensation reactions of 2,3-epoxychromone derivatives (14) with selected cyclic secondary amines. All compounds were dissolved in DMSO at 40 mM and stored at −20°C before use.
Cell culture. Human normal oral mesenchymal cells (human gingival fibroblast, HGF; human periodontal ligament fibroblast, HPLF; and human pulp cells, HPC) were established from the first premolar tooth extracted from the lower jaw of a 12-year-old girl (15), and cells at 10-18 population-doubling levels were used in this study. Human OSCC cell lines [Ca9-22 (derived from gingival tissue); and HSC-2, HSC-3, HSC-4 (derived from tongue)] were purchased from Riken Cell Bank (Tsukuba, Japan). All of these cells were cultured at 37°C in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml, penicillin G and 100 μg/ml streptomycin sulfate under a humidified 5% CO2 atmosphere. human oral keratinocytes (purchased from Cosmo Bio Co. Ltd., Tokyo, Japan) were cultured in keratinocyte growth supplement (OKGS, Cat, No. 2652) and cells at 7-11 population-doubling levels were used in the present study. Cell morphology was checked periodically under a light microscope (EVOS FL; Thermo Fisher Scientific, Waltham, MA, USA).
Cytotoxic activity of 15 3-(N-cyclicamino)chromones against oral malignant and non-malignant cells. Each value represents the mean of triplicate determinations. Two sets of tumor-specificity index (TS) and potency-selectivity expression (PSE) values were determined using all oral squamous cell carcinoma (OSCC) compared to non-malignant cells, and paired cells derived from the same (gingival) tissue.
Assay for cytotoxic activity. Cells were inoculated at 2×103 cells/0.1 ml in a 96-microwell plate. After 48 h, the medium was replaced with 0.1 ml of fresh medium containing different concentrations of single test compounds. Cells were incubated further for 48 h and the relative viable cell number was then determined by the MTT method (2-5). The relative viable cell number was determined by the absorbance of the cell lysate at 560 nm, using a microplate reader (Infinite F50R; TECAN, Männedorf, Switzerland). Control cells were treated with the same amounts of DMSO and the cell damage induced by DMSO was subtracted from that induced by test agents. The concentration of compound that reduced the viable cell number by 50% (CC50) was determined from the dose–response curve and the mean value of CC50 for each cell type was calculated from triplicate assays.
Calculation of tumor-specificity index (TS). TS was calculated using the following equation: TS=mean CC50 against three normal oral cell types/mean CC50 against OSCC cell lines. Since both Ca9-22 and HGF cells were derived from the gingival tissue (16), the relative sensitivity of these cells was also compared (as: mean CC50 against HGF/mean CC50 against Ca9-22).
Calculation of potency-selectivity expression (PSE). PSE was calculated by the following equation: PSE=mean CC50 against normal oral cell types/(CC50 against OSCC cell lines)2 ×100 (HGF, HPLF vs. Ca9-22, HSC-2); and as mean CC50 against HGF/(CC50 against Ca9-22)2 ×100 using the pair of cell types from the same tissue (gingiva) (see Table I).
Western blot analysis. Cells were washed with phosphate-buffered saline (PBS) and re-suspended in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholic acid, 1% NP-40 and protease inhibitors (RIPA buffer). After ultrasonication using Bioruptor (UCD-250; Cosmo Bio) for 12.5 min (10 s on, 20 s off) at 250 W at 4°C, the soluble cellular extracts were recovered after centrifugation for 10 min at 16,000 × g. The protein concentration of each sample was determined using BCA Protein Assay Reagent Kit (Thermo Fisher Scientific) and cell extracts were subjected to western blot analysis. The blots were probed with antibodies to poly (ADP-ribose) polymerase (PARP), caspase 3 (both from Cell Signaling Technology Inc., Beverly, MD, USA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA), followed by a horseradish peroxidase-conjugated anti-α-rabbit IgG secondary antibody (DAKO, Glostrup, Denmark). The immune complexes were visualized using Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific). Western blot results were documented and quantified using ImageQuant LAS 500 (GE Healthcare, Tokyo, Japan) (17).
Cell-cycle analysis. Treated and untreated cells (approximately 106 cells) were harvested, fixed with 1% paraformaldehyde in PBS without calcium and magnesium ions [PBS(−)]. Fixed cells were then washed twice with PBS(−) and treated for 30 min with 400 μl of 0.2 mg/ml RNase A (preheated for 10 min at 100°C to inactivate DNase) to degrade RNA. Cells were then washed twice with PBS(−) and stained for 15 min with 0.01% propidium iodide (PI) in the presence of 0.01% NP-40 in PBS(−) – to prevent cell aggregation. After filtering through Falcon® cell strainers (40 μM) (Corning, NY, USA) to remove aggregated cells, PI-stained cells were subjected to cell sorting (SH800 Series; SONY Imaging Products and Solutions Inc., Kanagawa, Japan). Cell-cycle analysis was performed with Cell Sorter Software version 2.1.2. (SONY Imaging Products and Solution Inc.).
Cytotoxicity of compounds 2a and 3a against four human oral squamous cell carcinoma cell lines Ca9-22, HSC-2, HSC-3 and HSC-4, and human normal oral cells human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF) and human pulp cells (HPC). Cells were incubated for 48 h without (control) or with the indicated concentrations of 2a (left) or 3a (right), and cell viability was determined by the MTT method, and expressed as a percentage that of the control. Each value represents the mean±S.D. of triplicate assays.
Toxicity of compound 3a against human oral keratinocytes (HOK) and human oral squamous cell carcinoma (OSCC) cell lines compared to doxorubicin.
Estimation of CC50 values. Since the CC50 values had a distribution pattern close to a logarithmically normal distribution, we used the negative log CC50 (pCC50) values for the comparison of cytotoxicity between compounds. The mean pCC50 values for normal cells and tumor cell lines were defined as N and T, respectively (3).
Calculation of chemical descriptors. The 3D structure of each chemical structure (Marvin Sketch ver 16; ChemAxon, Budapest, Hungary, http://www.chemaxon.com) was optimized by CORINA Classic (Molecular Networks GmbH, Nürnberg, Germany) with forcefield calculations (amber-10: EHT) in Molecular Operating Environment (MOE) version 2018.0101 (Chemical Computing Group Inc., Quebec, Canada). The number of structural descriptors calculated from MOE (18) and Dragon 7.0 (19) (Kode srl., Pisa, Italy) was 344 and 5,255, respectively. Among them, the number of descriptors used for analysis was 302 and 2,794 (total 3,096), respectively.
Number of descriptors showing significant correlation with mean negative log of the concentration of compound that reduced the viable cell number by 50% (pCC50) for tumor cells (T), normal cells (N) and normal cells versus tumor cells (T–N).
Effect of 3a on cell morphology (A), apoptosis induction (B) and cell-cycle analysis (C) in oral squamous cell carcinoma cell line HSC-2. Cells were incubated for 24 or 48 h with the indicated concentrations of 3a or 1 μM actinomycin D (Act D) as positive control and then assessed for morphology under light microscopy (EVOS FL; Thermo Fisher Scientific), cell-cycle distribution by cell sorting and apoptosis induction by western blot. Bar=100 μm. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; PARP: poly (ADP-ribose) polymerase.
Statistical treatment. The CC50 values were expressed as mean±S.D. of triplicate assays. The relation among cytotoxicity, TS and chemical descriptors was investigated using simple regression analyses by JMP Pro version 13.2.0 (SAS Institute Inc., Cary, NC, USA). The significance level was set at p<0.05.
Results
Cytotoxicity. A total of 15 3-(N-cyclicamino)chromone derivatives were synthesized, without (A series) or with the introduction of methoxy group at the C-6 position (B series) or the C-7 position (C series) of the benzopyran-4-one (chromone) ring attached by 1-piperidinyl (1a, 1b, 1c), 4-phenyl-1-piperidinyl (2a, 2b, 2c), 4-phenyl-1-piperazinyl (3a, 3b, 3c), 4-methyl-1-piperazinyl (4a, 4b, 4c) or 4-morpholinyl (5a, 5b, 5c) group at the C-3 position (Figure 1).
We first investigated the effect of introduction of substituent groups at the C-3 position on the induction of cytotoxicity of chromone. The compound with a 4-phenyl-1-piperazinyl group (3a) had the highest cytotoxicity against four human OSCC cell lines (mean CC50=32 μM), followed by compounds with 4-phenyl-1-piperidinyl (2a), 1-piperidinyl (1a), and then those with 4-methyl-1-piperazinyl group (4a) and 4-morpholinyl (5a) (>400 μM). These compounds showed very weak cytotoxicity against human normal oral cells (HGF, HPLF, HPC) (CC50 >386 μM) (Table I).
Introduction of a methoxy group at the C-6 position of 2a and 3a did not further increase, but rather reduced their cytotoxicity against OSCC cells (compare with 2b and 3b, Table I). Introduction of a methoxy group to the C-7 position of 2a and 3a also failed to increase cytotoxicity against OSCC cells (compare with 2c and 3c, Table I).
Determination of correlation coefficient between chemical descriptors and cytotoxicity of 15 3-(N-cyclicamino)chromones against tumor cells. The mean negative log of the concentration of compound that reduced the viable cell number by 50% (CC50) values (T) against tumor cells were plotted. The following chemical descriptors were used: RDF075v, E3m: shape and size; RDF075p, RDF090p: shape and polarizability; Mor06s: 3D shape and electric state; SpMAD_AEA(dm): shape and dipole moment.
Determination of correlation coefficient between chemical descriptors and cytotoxicity of 15 3-(N-cyclicamino)chromones against normal cells. The mean negative log of the concentration of compound that reduced the viable cell number by 50% (CC50) values (N) against normal cells were plotted. The following chemical descriptors were used: Mor28s: 3D shape and electric state; CATS2D_02_AL, CATS3D_02_AL: hydrogen-bond acceptor and lipophilicity, Inflammat-80, Depressant-80: drug-likeness; TDB05i: 3D shape and ionization potential.
Determination of correlation coefficient between chemical descriptors and tumor specificity of 15 3-(N-cyclicamino)chromones [defined as: cytotoxicity against tumor cells–cytotoxicity against normal cells (T–N)]. The following chemical descriptors were used: CATS3D_11_LL, CATS3D_12_LL: lipophilicity; VE3sign_G, Chi_G/D: 3D shape; J_D/Dt: shape; FCASA-: shape and negative charge.
Tumor specificity. Among 15 compounds, 3a had the highest TS (TS>12.3), followed by 2a (TS>7.7) and then 3b (TS=4.7). The TS values of other compounds was below 3 (Table I). The TS value of 3a was much lower than that for doxorubicin (TS>85.6), but very close to that of melphalan (TS=16.3). Compound 3a caused cytostatic growth inhibition of OSCC cells (Figure 2). Furthermore, 3a was much less cytotoxic against human oral keratinocytes as compared with doxorubicin (Table II).
Considering that HGF is the normal cell type corresponding to Ca9-22 OSCC cell line (since both derive from gingival tissues), TS values were also generated by dividing the average CC50 value towards HGF cells by the CC50 value towards Ca9-22 cells (C/A, Table I). The TS values derived in this way for 3a (TS=8.5) and 2a (TS=8.4) were slightly higher than that of melphalan (TS=8.0) (Table I).
PSE value. In order to identify the most promising compounds in terms of both good potency and selectively cytotoxicity, PSE values were calculated. PSE values of 2a and 3a (>15.5 and >38.1) against malignant cells were 9.5% and 23.4% that of melphalan (PSE=162.8), respectively. For gingival tissue, PSE values of 2a and 3a (PSE=19.6 and 18.4) were 48.5% and 45.5% that of melphalan (PSE=40.4), respectively (Table I).
Type of cell death induced by 3a. When HSC-2 cells were incubated for 24 h with increasing concentrations (20, 40, 80 or 160 μM) of 3a, cells became gradually enlarged (upper column in Figure 3A). In contrast, actinomycin D treatment induced cell shrinkage, characteristic of apoptosis (Figure 3A).
Cell-cycle analysis demonstrated that actinomycin D, but not 2a nor 3a, produced significantly (p<0.001) higher proportions of sub-G1 cells (50.9±1.3%) vs. control (4.0±0.01%), a characteristic feature of apoptotic cells (Figure 3B). These data indicate apoptosis induction by 2a and 3a is unlikely.
Western blot analysis demonstrated that 3a did not lead to caspase-3 activation, as evidenced by lack of cleavage PARP and caspase-3, in contrast to actinomycin D treatment (Figure 3C).
Computational analysis. We next performed the QSAR analysis of the 15 3-(N-cyclicamino)chromones in regards to their cytotoxicity against tumor cells and normal cells. Since so many chemical descriptors were significantly correlated with cytotoxicity and TS (p<0.05 and p<0.005, respectively) (Table III), the top six chemical descriptors were chosen for QSAR analysis (Figures 4, 5 and 6, and Table IV).
The cytotoxicity of 15 3-(N-cyclicamino)chromones against human OSCC cell lines was correlated positively with RDF075v (shape and size) (r2=0.898, p<0.0001), RDF075p (shape and polarizability) (r2=0.871, p<0.0001), RDF090p (shape and polarizability) (r2=0.867, p<0.0001) and E3m (shape and size) (r2=0.856, p<0.0001), while negatively with Mor06s (3D shape and electric state) (r2=0.871, p<0.0001) and SpMAD_AEA(dm) (shape and dipole moment) (r2=0.869, p<0.0001) (Figure 4).
Properties of descriptors that significantly affected cytotoxicity against tumor cells (T) and normal cells (N), and tumor specificity (T–N).
The cytotoxicity of 15 3-(N-cyclicamino)chromones against human normal oral mesenchymal cells was correlated positively with CATS3D_02_AL (hydrogen-bond acceptor and lipophilicity) (r2=0.548, p=0.0016), CATS2D_02_AL (hydrogen-bond acceptor and lipophilicity) (r2=0.535, p=0.0020), Inflammat-80 (drug-likeness) (r2=0.519, p=0.0025) and Depressant-80 (drug-likeness) (r2=0.519, p=0.0025), while negatively with Mor28s (3D shape and electoric state) (r2=0.664, p=0.0002) and TDB05i (3D shape and ionization potential) (r2=0.508, p=0.0029) (Figure 5).
The TS of 15 3-(N-cyclicamino)chromones was correlated positively with CATS3D_12_LL (lipophilicity) (r2=0.912, p<0.0001), FCASA-(shape and negative charge) (r2=0.847, p<0.0001), CATS3D_11_LL (lipophilicity) (r2=0.845, p<0.0001) and Chi_G/D (3D shape) (r2=0.842, p<0.0001), while negatively with VE3sign_G (3D shape) (r2=0.888, p<0.0001) and J_D/Dt (shape) (r2=0.861, p<0.0001) (Figure 6).
Discussion
The present study demonstrated for the first time that among 15 3-(N-cyclicamino)chromones, (2a and 3a had the highest tumor specificity, comparable with that of melphalan (Table I). Furthermore, 3a was much less cytotoxic against human normal oral keratinocytes compared with doxorubicin. We found that these compounds led to cytostatic growth inhibition, only reducing the viable cell number by 60% after 48 h (Figure 2). Compound 3a did not produce an increased G1 cell population nor induced caspase-3 activation, suggesting that 3a does not induce apoptotic cell death.
We recently reported that 7-methoxy-2-(4-morpholinyl)-4H-1-benzopyran-4-one [5c which had the highest tumor specificity among 15 2-(N-cyclicamino)chromone derivatives (13)], led to cytotoxic growth inhibition in OSCC cell lines without induction of apoptosis. These results suggest that these structural isomers, 2-(N-cyclicamino)chromones and 3-(N-cyclicamino)chromones, have no apoptosis-inducing activity, despite exhibiting different modes of growth inhibition (either cytotoxic or cytostatic). There are a variety of types of cell death reported (20). Further study is needed to determine which type of cell death 3a induces in human OSCC cell lines.
QSAR analysis demonstrated that cytotoxicity of 15 3-(N-cyclicamino)chromones against tumor cell lines was highly significantly correlated with descriptors of shape, size, polarizability, electric state, and dipole moment (p-values from 8.03×10−7 to 8.42×10−8) (Figure 4). Their tumor specificity was also significantly positively correlated lipophilicity, 3D shape, and negative charge (p-values from 1.45×10−6 to 3.18×10−8) (Figure 6). Taken together, these data suggest that tumor specificity is positively related with chemical descriptors that reflect 3D structure and lipophilicity.
When we compared the A series of compounds of 3-(N-cyclicamino)chromones (this study) with their structural isomers, 2-(N-cyclicamino)chromones (13), there were common properties: 4-phenyl-1-piperazinyl derivatives (3a) of both groups had the highest cytotoxicity against four human OSCC cell lines, followed by 4-phenyl-1-piperidinyl derivatives (2a) in both cases [Table I and (13)]. Furthermore, TS of both groups of compounds were correlated with 3D structure (mass or shape) reflected by Mor17v, Mor17m and Mor22m for 2-(N-cyclicamino)chromones, and FCASA-, Chi_G/D, VE3sign_G, J_D/Dt for 3-(N-cyclicamino) chromones. In addition, we found that both 3a (this study) and 7-methoxy-2-(4-morpholinyl)-4H-1-benzopyran-4-one [5c, described as the most tumor-specific compound among 15 2-(N-cyclicamino)chromones (13)] were much less toxic towards human oral keratinocytes compared with doxorubicin (Table II), further substantiating them as new promising anticancer drug candidates.
Chemical modification using 3a as a lead compound may be a potential choice for designing a new type of anticancer drug.
Acknowledgements
This work was partially supported by KAKENHI from the Japan Society for the Promotion of Science (JSPS) (15K08111, 16K11519).
Footnotes
↵* These Authors contributed equally to this work.
This article is freely accessible online.
Conflicts of Interest
The Authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
- Received June 8, 2018.
- Revision received July 1, 2018.
- Accepted July 4, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved