2,4-Diarylpyrano[3,2-c]chromen-5(4H)-ones as Antiproliferative Agents:

Design, Synthesis and Biological Evaluation

Abstract

In the present study, a series of 2,4-diarylpyrano[3,2-c]chromen-5(4H)-ones were synthesised and evaluated as antiproliferative agents. The compounds were evaluated against a panel of human cancer cell lines. CH-1 exhibited significant cytoxicity against HCT 116 cell lines with an IC₅₀ value of 1.4 and 4.3 µM against “MiaPaCa-2” cell lines. The compound CH-1 was found to induce apoptosis as evidenced by phase contrast microscopy, Hoechst 33258 staining and mitochondrial membrane potential (MMP) loss. The cell phase distribution studies indicated that the apoptotic population increased from 10.22% in the control sample to 57.19% in a sample treated with 20 µM compound CH-1.

Molecular hybridization (MH) is a strategy of rational design of such ligands or prototypes based on the recognition of pharmacophoric sub-units in the molecular structure of two or more known bioactive derivatives which, through the adequate fusion of these sub-units, lead to the design of new hybrid architectures that maintain pre-selected characteristics of the original templates.¹⁾

The strategy is analogous to conventional combination therapy, with the exception that the two drugs are covalently linked and available as a single entity.²⁾ The selection of the two principles in the dual drugs is usually based on their observed (or anticipated) synergistic or additive pharmacological activities to enable the identification of highly active novel chemical entities.^3–5) The technique of drug design has been extensively employed for the design of numerous novel anticancer hybrids.⁵⁾

Chalcones are precursors of flavonoids and possess diverse array of biological activities, such as anti-cancer,^6–8) anti-inflammatory,^9,10) anti-tuberculosis,¹¹⁾ and anti-fungal activities.¹²⁾ Chemically, chalcones (1,3-diaryl-2-propen-1-ones) possess an enone bridge between two aromatic rings.¹³⁾ Chalcones have been one of the most extensively explored class possesing anticancer potential owing to their ease of synthesis and relatively simpler chemical architecture. Chalcone based hybrids have gained enough attention in the recent past as the presence of chalcones in various hybrid scaffolds has been evidenced through number of reports. Figure 1 represents the structure of potent chalcone based hybrids.^14–19)

Fig. 1. Chalcones in Various Hybrid Anticancer Scaffolds

Coumarins form an important class of compounds, which occupy a special role in nature.^20,21) The presence of coumarin architecture in naturally occurring phytoconstituents such as anticancer,^22,23) anti-human immunodeficiency virus (HIV),²⁴⁾ antituberculosis,²⁵⁾ anti-influenza,²⁶⁾ anti-Alzheimer,^27,28) anti-inflammatory,²⁹⁾ antiviral³⁰⁾ and antimicrobial agents²⁰⁾ makes it a privileged structure. Though coumarin posseses diverse array of biological activities but their ability to kill cancer cells through varied mechanisms has been explored in detail.⁵⁾ Coumarins in hybrid scaffolds have also been reported to display promising results.^31–35)

Figure 2 represents the structure of coumarin based hybrids with anticancer potential.

Fig. 2. Coumarins in Various Hybrid Anticancer Scaffolds

Keeping in view of the success of molecular hybridization techniques towards the design of anticancer agents, proved anticancer potential of coumarin and chalcones and the promising results of previously reported coumarin–chalcone hybrids, 2,4-diarylpyrano[3,2-c]chromen-5(4H)-ones as pyran tethered coumarin–chalcone were designed, synthesised and evaluated for antiproliferative effects. The presence of pyran as an structural motiff in various scaffolds possesing anticancer potential justifies its selection as linker.^36,37)

Results

Chemistry

The designed compounds were synthesised by the reaction of 4-hydroxy coumarin with various chalcones in the presence of catalytic amounts of SiO₂–ZnCl₂ (10 mol%).³⁸⁾ The chalcones were synthesised by base catalysed Claisen Schmidt condensation of acetophenones and benzaldehydes and were characterized by the appearance of two doublets with a J value of 15 Hz. The reaction involves the michael addition of 4-hydroxy coumarin to chalcones followed by cyclization leading to 2,4-diarylpyrano[3,2-c]chromen-5(4H)-ones. The products were characterized by the appearance of two doublets at 5.78–5.85 and 4.68–4.73 ppm with a J value of 4.8 Hz for the protons of the pyran (linker) along with the resonance signals for aromatic protons. The carbon resonances for C-4 and C-3 were observed at around 30 and 100 ppm Chart 1 represents the synthesis of target compounds. Figure 3 represents the structures of all the synthesised compounds.

Chart 1. Synthesis of 2,4-Diarylpyrano[3,2-c]chromen-5(4H)-ones

Fig. 3. Structures of the Synthesized Compounds

Biological Evaluation

All the synthetics were assayed for in vitro cytotoxicity against four human cancer cell lines³⁹⁾ viz. HCT 116 (colon cancer), MCF-7 (breast cancer), MiaPaCa-2 (pancreatic cancer) HL-60 (leukemia cancer).¹³⁾ The results of the cytotoxicity studies are presented in Table 1. All the synthetics were screened against the cell lines at 50 µM. The molecules displaying percentage inhibition (Table 1) of greater than 50% against the cell lines employed were only evaluated at different concentrations and IC₅₀ values (Table 1) were calculated. It was observed that HL-60 and MCF-7 cell lines were almost resistant to the exposure of test compounds whereas HCT 116 and MiaPaCa-2 cell lines were the most sensitive. So these cell lines were selected for further studies at different concentrations. Among the series, CH-1 with an unsubstituted phenyl rings at both 2nd and 4th position was found to be the most potent against HCT 116 cell lines (percentage inhibition=95% and IC₅₀=1.4 µM) and MiaPaCa-2 cell line (percentage inhibition=84.11% and IC₅₀ value=4.3 µM). CH-5, 13, 16 possessing a nitro substituted phenyl ring either as Ring A or Ring B also displayed significant cytotoxicity with percentage inhibition ranging from 75–82% and IC₅₀ value of 7.44–11.27 µM against HCT 116 cell lines and percentage inhibition ranging from 62–73% and IC₅₀ value of 11.13–20.40 µM against MiaPaCa-2 cell lines. Tables 1 and 2 also reveal some interesting structure cytotoxicity relationship of cytotoxic acitivity against HCT 116 and MiaPaCa-2 cell lines. Any substitution on the phenyl rings (Ring A and Ring B) irrespective of their electronic nature resulted in decreased cytotoxic effect (Compare CH-1 with CH-2, 3, 4, 5, 6, 7, 9, 13, 14, 15, 16, 17, 18, 19, 20). However, the decline in the activity was less with nitro substituted phenyl rings (either as Ring A or Ring B) as compared to halogens, methyl and methoxy substituted phenyl rings (Compare CH-5, 13, 16 with CH-2, 3, 4, 6, 7, 9, 14, 15, 17, 18, 19, 20). Replacement of phenyl ring (Ring B) with a hetero aromatic ring and bicyclic aromatic ring also resulted in diminished activity profile. (Compare CH-1 with CH-8, 11 and 12.) To get some mechanistic insight into the mode of action of the most potent compound CH-1 in HCT 116 cell lines, a few more experiments such as phase contrast microscopy, Hoechst 33258 staining for nuclear morphology, mitochondrial membrane potential loss, cell cycle phase distribution were carried out.

Table 1. Percentage Inhibition of the Designed Hybrids

Compound code	Percentage inhibition at 50 µM
	MCF-7	HCT-116	MiaPaCa-2	HL-60
CH-1	17.25	95.07	84.11	NA
CH-2	NA	69.18	62.27	10.18
CH-3	NA	59.09	52.84	NA
CH-4	NA	57.82	56.87	NA
CH-5	NA	75.97	73.19	NA
CH-6	13.97	51.03	53.31	NA
CH-7	NA	59.91	57.93	NA
CH-8	NA	43.20	40.37	10.01
CH-9	12.64	41.19	46.21	NA
CH-10	NA	46.79	43	NA
CH-11	NA	45.27	40.40	NA
CH-12	NA	34.48	44.44	NA
CH-13	NA	75.44	62.22	NA
CH-14	NA	47.44	41.13	NA
CH-15	11.48	42.66	37.54	NA
CH-16	NA	82.29	64.22	NA
CH-17	NA	13.23	17.77	10.11
CH-18	NA	19.05	22.44	NA
CH-19	NA	21.31	19.21	NA
CH-20	NA	15.63	NA	NA
	NA	24.09	16.45	NA
	NA	18.43	8.56	NA
Adriamycin (1 µM)	—	—	73	—
Mitomycin (1 µM)	83	—	—	—
5-Fluorouracil (20 µM)	—	69	—	—

Table 2. IC₅₀ Values of Selected Analogues

Compound code	IC50 (µM)
	HCT 116	MiaPaCa-2
CH-1	1.4	4.3
CH-2	19.18	27.27
CH-3	20.09	32.84
CH-4	24.82	33.12
CH-5	9.32	15.43
CH-6	18.03	21.31
CH-7	19.91	27.93
CH-13	11.27	20.40

To confirm the apoptosis induction results obtained from cell cycle analysis, nuclear morphological changes of cells by fluorescence microscopy was studied. After the treatment at 5, 10, and 20 µM of compound CH-1, characteristic changes of apoptosis such as nuclear condensation, membrane blebbing and formation of apoptotic bodies were observed in the morphology of treated cells in a concentration-dependent manner, whereas the nuclei of untreated cells was found to be of normal intact morphology. The number of apoptotic bodies increased with increased concentration of CH-1. Therefore, the mode of HCT 116 cell death induced by CH-1 was apoptosis.

Phase contrast microscopy was performed to observe significant changes in the morphology of cells.³⁹⁾ The changes increased with increasing dose of CH-1. Treatment with 5 µM CH-1 showed blebbing and shrinkage of cells (Fig. 4); the amount of apoptosis (blebbing and shrinkage) increased significantly in cells treated with 20 µM CH-1.

Fig. 4. Compound CH-1 Induced Cell Death in HCT 116 Cell Line

Cells were treated with the indicated concentrations of compound CH-1 and observed for morphological changes under a microscope (1×81, Olympus).

Hoechst staining method was employed to observe the changes in nuclear morphology. Pronounced changes in nuclear morphology and formation of apoptotic bodies were observed in the morphology of treated cells in a concentration-dependent manner, whereas the nuclei of untreated cells was found to be of normal intact morphology. The results suggested that compound CH-1 was able to induce apoptotic cell morphology in HCT 116 cells⁴⁰⁾ (Fig. 5).

Fig. 5. Alteration in Nuclear Morphology by Treatment with Compound CH-1

HCT 116 cells were incubated with different concentrations of compound CH-1, collected after centrifugation at 400×g, washed once with PBS, and then stained with Hoechst 33258 for 30 min.

Mitochondrial integrity is required for cells to be functional, and mitochondrial potential loss is considered as one of the major causes of cell death³⁹⁾ Compound CH-1 induced mitochondrial membrane potential (MMP) loss, which increased with increasing doses. The loss was significant at 5 µM treatment and further increased to 50.8% when the cells were treated with 20 µM CH-1 (Fig. 6). This demonstrates the apoptotic potential of the compound.

Fig. 6. Compound CH-1-Induced Time-Dependent MMP Loss in HCT 116

HCT 116 cells (0.5×10⁶) were treated for 6 h with the indicated concentrations of compound CH-1, washed once with PBS, and stained with RH 123.

Cell cycle phase distribution studies were also carried out.⁴⁰⁾ HCT 116 cell treated with compound CH-1 for 6 h showed considerable increase in apoptotic population (sub G0/G1): the apoptotic population increased from 10.22% in control sample to 57.19% in sample treated with 20 µM compound CH-1 (Fig. 7).

Fig. 7. Induction of G0/G1 Population

HCT 116 (1×10⁶) were seeded in 12-well plates and treated with different concentrations of compound CH-1 (5, 10, 20 µM) for 6 h. After completion, cells were collected at 400 g, washed once with PBS, and fixed in 70% ethanol overnight. Cells were then washed once with PBS and stained with 100 µg of PI for 30 min. Modfit software was used to differentiate between phases and determine the amount of apoptotic population.

2,4-Diarylpyrano[3,2-c]chromen-5(4H)-ones as coumarin–chalcone hybrids were synthesized and evaluated against a panel of cancer cell lines. CH-1 displayed remarkable cytotoxicity against HCT 116 cells with an IC₅₀ value of 1.4 µM. The compound was further investigated in detail to explore the mechanism of cell death induced by compound CH-1 in HCT 116 cells. Apoptotic potential of the compound was observed through phase contrast microscopy and Hoechst staining. Mitochondrial membrane potential loss has been considered as a marker of cell death. CH-1 also induced mitochondrial membrane potential loss significantly, and flow cytometric analysis showed that CH-1 significantly increased the apoptotic subG1 population at 5, 10 and 20 µM.

Experimental

The reagents were purchased from Sigma-Aldrich, Merck, CDH, Loba Chem., Spectro Chem., India and used without further purification. All yields refer to isolated products after purification. Products were characterized by spectral data. ¹H-NMR and ¹³C-NMR spectra were recorded on Bruker Advance II 400 NMR Spectrometer and JEOL AL 300 NMR Spectrometer. The spectra were measured in CDCl₃ relative to tetramethylsilane (TMS) (0.00 ppm). Melting points were determined in open capillaries and were uncorrected.

Procedure for the Synthesis of 2,4-Diaryl Pyrano[3,2-c]coumarins

The mixture of 4-hydroxy coumarin (1 mmol), differently substituted chalcones (1 mmol) and SiO₂ (200–400 mesh)-ZnCl₂ (10 mol%) was heated on an oil bath at 100°C for 4 h.³⁸⁾ The reaction mixture was extracted with water and ethyl acetate. The ethyl acetate fraction was concentrated and subjected to column chromatography. The product was eluted with increasing percentage of ethyl acetate in hexane. The remaining reactions were carried out following these general procedures. In each occasion, the spectral data (¹H-NMR, ¹³C-NMR and MASS) of known compounds such as 2,4-diphenylpyrano[3,2-c]chromen-5(4H)-one (CH-1),⁴¹⁾ 4-(3-nitrophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-16),⁴¹⁾ 2-phenyl-4-p-tolylpyrano[3,2-c]chromen-5(4H)-one (CH-17).⁴¹⁾ 2-(4-Chlorocyclohexa-2,4-dienyl)-4-(4-methoxyphenyl)pyrano[3,2-c]chromen-5(4H)-one (CH-18),⁴¹⁾ 4-(4-chlorophenyl)-2-(4-methylcyclohexa-2,4-dienyl)pyrano[3,2-c]chromen-5(4H)-one (CH-19),⁴¹⁾ 2-(4-methylcyclohexa-2,4-dienyl)-4-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-20).⁴¹⁾

The physical data of compounds are provided below.

The characterization data for the synthesized compounds is given below:

4-(4-Chlorophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-2)

Yield 80%; mp: 87–88°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.01 (1H, d, J=7.8 Hz), 7.73 (2H, d, J=7.8 Hz), 7.60 (1H, m), 7.25–7.48 (9H, m), 5.79 (1H, d, J=4.8 Hz), 4.68 (1H, d, J=4.8 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 31.172(CH), 102.923 (=CH–), 103.480 (=C–), 114.560 (Ar-C), 116.862 (Ar-CH–), 122.761 (Ar-C), 124.237 (Ar-C), 124.905 (Ar-C), 125.532 (Ar-C), 127.028 (Ar-C), 128.736 (Ar-C), 129.465 (=C–O), 132.148 (=C–O), 140.657 (Ar-C–Cl), 147.646, (=C–O), 161.482 (O–C=O). Anal. Calcd for C₂₄H₁₅ClO₃: C, 74.52; H, 3.91. Found: C, 74.68; H, 4.07.

4-(4-Bromophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-3)

Yield 78%; mp: 168–169°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.02 (1H, d, J=6.6 Hz), 7.73 (2H, d, J=6.3 Hz), 7.57 (1H, m), 7.39–7.45 (5H, m), 7.26–7.36 (4H, m), 5.80 (1H, d, J=4.8 Hz), 4.68 (1H, d, J=4.8 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 36.207 (CH), 103.114 (=CH–), 103.220 (=C–), 114.428 (Ar-C), 116.905 (Ar-C), 121.207 (Ar-CH), 122.718 (Ar-C), 124.274 (Ar-C), 124.723 (Ar-C), 128.744 (Ar-C), 129.443 (Ar-C), 130.242 (Ar-CH), 131.720 (Ar-CH), 132.204 (=C–O), 132.449 (=C–O), 142.564 (Ar-C-Br), 147.255 (=C–O), 161.428 (O–C=O). Anal. Calcd for C₂₄H₁₅BrO₃: C, 66.84; H, 3.51. Found: C, 67.02; H, 3.76.

4-(4-Fluorophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-4)

Yield 89%; mp: 142–143°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.03 (1H, d, J=7.5 Hz), 7.75 (2H, d, J=7.8 Hz), 7.59 (1H, m), 7.34–7.49 (7H, m), 7.00 (2H, m), 5.83 (1H, d, J=4.8 Hz), 4.72 (1H, d, J=4.8 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 36.523 (CH), 102.146 (=CH), 102.425 (=C–), 114.316 (Ar-C=), 116.756 (Ar-CH), 122.605 (Ar-CH), 125.182 (Ar-CH), 125.750 (Ar-CH), 125.345 (Ar-CH), 126.326 (Ar-CH), 126.130 (Ar-CH), 128.308 (Ar-CH), 128.546 (Ar-CH), 128.726 (Ar-CH), 128.819 (Ar-CH), 130.343 (Ar-CH), 130.416 (Ar-CH), 131.456 (Ar-CH), 147.101 (Ar-C), 147.834 (=C–O), 150.435 (=C–O), 156.315 (=C–O), 158.613 (Ar-C-F), 161.289 (O–C=O). Anal. Calcd for C₂₄H₁₅FO₃: C, 77.83; H, 4.08. Found: C, 78.11; H, 3.95.

4-(4-Nitrophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-5)

Yield 84%; mp: 190–191°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.18 (2H, d, J=9.00 Hz), 8.04 (1H, d, J=7.5 Hz), 7.73–7.81 (2H, m), 7.58–7.61 (3H, m), 7.35–7.48 (5H, m,), 5.78 (1H, d, J=4.8 Hz), 4.85 (1H, d, J=4.8 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 36.721 (CH), 102.141 (=CH), 102.425 (=C–), 114.200 (Ar-C=), 116.986 (Ar-CH), 117.586 (Ar-CH), 122.807 (Ar-CH), 123.013 (Ar-CH), 123.251 (Ar-CH), 123.489 (Ar-CH), 123.929 (Ar-CH), 124.070 (Ar-CH), 124.220 (Ar-CH), 124.466 (Ar-CH), 124.795 (Ar-CH), 125.041 (Ar-CH), 128.026 (Ar-CH), 128.308 (Ar-CH), 128.556 (Ar-CH), 128.826 (Ar-CH), 128.939 (Ar-CH), 129.427 (Ar-CH), 129.740 (Ar-CH), 130.283 (Ar-CH), 130.536 (Ar-CH), 131.566 (Ar-CH), 132.119 (Ar-CH), 132.560 (Ar-CH), 133.077 (Ar-CH), 147.101 (Ar-C), 147.834 (=C–O), 150.613 (Ar-C-NO₂), 152.867 (=C–O), 156.315 (=C–O), 161.289 (O–C=O) . Anal. Calcd for C₂₄H₁₅NO₅: C, 72.54; H, 3.80; N, 3.52. Found: C, 72.48; H, 4.02; N, 3.76.

4-(3,4-Dimethoxyphenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-6)

Yield 76%; mp: 85–86°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.02 (1H, d, J=7.8 Hz), 7.74 (2H, d, J=6.3 Hz), 7.57 (1H, m), 7.36–7.46 (5H, m), 6.99 (1H, s), 6.93 (1H, d, J=8.4 Hz), 6.80 (1H, d, J=8.1 Hz), 5.85 (1H, d, J=5.1 Hz), 4.66 (1H, d, J=4.8 Hz), 3.86 (6H, s). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 36.753 (CH), 55.335 (–OCH₃), 102.958 (CH=), 102.595 (=C–), 114.617 (Ar-C), 116.850 (Ar-CH), 122.445 (Ar-CH), 122.234 (Ar-CH), 125.345 (Ar-CH), 126.125 (Ar-CH), 126.349 (Ar-CH), 126.874 (Ar-CH), 128.675 (Ar-CH), 128.786 (Ar-CH), 128.760 (Ar-CH), 129.458 (Ar-CH), 130.954 (Ar-CH), 131.964 (Ar-CH), 135.752 (Ar-C), 146.770 (=C–O), 150.715 (=C–O), 155.533 (=C–O), 157.534 (=C–O), 161.538 (O–C=O). Anal. Calcd for C₂₆H₂₁O₅: C, 75.72; H, 4.89. Found: C, 75.84; H, 5.05.

4-(4-Methoxyphenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-7)

Yield 81%; mp: 132–133°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.00 (1H, d, J=7.5 Hz), 7.72 (2H, d, J=7.8 Hz), 7.42 (1H, m), 7.41 (2H, d, J=7.5 Hz), 7.15–7.35 (5H, m), 6.84 (2H, d, J=8.4 Hz), 5.81 (1H, d, J=5.1 Hz), 4.66 (1H, d, J=5.1 Hz), 3.84 (3H, s). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 35.745 (CH), 55.299 (–OCH₃), 103.873 (=CH), 114.005 (=C–), 114.613 (Ar-CH), 116.816 (Ar-CH), 122.685 (Ar-CH), 124.182 (Ar-CH), 124.650 (Ar-CH), 126.130 (Ar-CH), 128.475 (Ar-CH), 128.697 (Ar-CH), 129.230 (Ar-CH), 129.587 (Ar-CH), 131.964 (Ar-CH), 132.685 (Ar-CH), 135.832 (Ar-C), 146.770 (=C–O), 152.715 (=C–O), 155.510 (=C–O), 158.754 (=C–O), 161.568 (O–C=O). Anal. Calcd for C₂₅H₁₈O₄: C, 78.52; H, 4.74. Found: C, 78.69; H, 4.99.

4-(Naphthalen-2-yl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-8)

Yield 90%; mp: 178–179°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 7.74–8.14 (4H, m), 7.43–7.58 (5H, m), 7.28–7.36 (7H, m), 5.90 (1H, d, J=3.9 Hz), 4.89 (1H, d, J=3.9 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 36.823 (CH), 103.706 (Ar-C), 116.882 (Ar-CH), 122.743 (Ar-CH), 124.220 (Ar-CH), 124.735 (Ar-CH), 125.810 (Ar-CH), 126.103 (Ar-CH), 126.511 (Ar-CH), 127.199 (Ar-CH), 127.616 (Ar-CH), 127.940 (Ar-CH), 128.428 (Ar-CH), 128.706 (Ar-C), 129.304 (Ar-C), 132.068 (Ar-C), 132.712 (Ar-C), 152.713 (=C–O), 155.507 (=C–O), 158.752 (=C–O), 161.565 (O–C=O). Anal. Calcd for C₂₈H₁₈O₃: C, 83.57; H, 4.51. Found: C, 83.33; H, 4.77.

4-(3-Chlorophenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-9)

Yield 72%; mp: 132–133°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.02 (1H, d, J=8.1 Hz), 7.72 (2H, d, J=7.8 Hz), 7.59 (1H, m), 7.19–7.31 (5H, m), 7.31–7.46 (4H, m), 5.80 (1H, d, J=3.0 Hz), 4.69 (1H, d, J=3.00 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 36.422 (CH), 102.858 (CH=), 102.995 (=C–), 114.317 (Ar-C), 116.800 (Ar-CH), 122.745 (Ar-CH), 124.321 (Ar-CH), 124.676 (Ar-CH), 126.716 (Ar-CH), 127.397 (Ar-CH), 128.451 (Ar-CH), 128.705 (Ar-CH), 129.434 (Ar-C), 129.841 (Ar-C), 132.272 (Ar-C), 134.431 (Ar-C), 145.574 (Ar-C-Cl), 147.152 (=C–O), 152.726 (=C–O), 156.025 (=C–O), 161.308 (O–C=O). Anal. Calcd for C₂₄H₁₅ClO₃: C, 74.52; H, 3.91. Found: C, 74.68; H, 3.82.

2-(4-Methoxyphenyl)-4-(thiophen-2-yl)pyrano[3,2-c]chromen-5 (4H)-one (CH-10)

Yield 70%; mp: 134–135°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.04 (1H, d, J=7.8 Hz), 7.92 (2H, d, J=8.7 Hz), 7.67 (1H, d, J=9.00 Hz), 7.54 (1H, m), 7.32–7.38 (3H, m), 6.91–6.99 (2H, m), 6.93 (2H, d, J=9.00 Hz), 5.81 (1H, d, J=5.1 Hz), 5.02 (1H, d, J=5.1 Hz), 3.80 (3H, s). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 31.118 (CH), 55.435 (–OCH₃), 102.864 (CH=), 103.523 (CH=), 113.695 (Ar-C), 114.109 (Ar-CH), 114.607 (Ar-CH), 116.724 (Ar-CH), 116.853 (Ar-CH), 122.751 (Ar-CH), 124.031 (Ar-CH), 124.215 (Ar-CH), 124.839 (Ar-CH), 125.081 (Ar-CH), 125.442 (Ar-CH), 126.360 (Ar-CH), 127.026 (Ar-CH), 130.329 (Ar-C), 130.622 (Ar-C), 132.107 (Ar-C), 132.606 (Ar-C), 147.445 (Ar-C–OCH₃), 147.981 (=C–O), 152.326 (=C–O), 155.551 (=C–O), 161.595 (O–C=O). Anal. Calcd for C₂₃H₁₆SO₄: C, 71.12; H, 4.15; S, 8.25. Found: C, 70.92; H, 3.96; S, 7.99.

2-Phenyl-4-(thiophen-2-yl)pyrano[3,2-c]chromen-5 (4H)-one (CH-11)

Yield 86%; mp: 168–169°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.00 (1H, d, J=7.5 Hz), 7.76 (2H, d, J=6.3 Hz), 7.58 (1H, m), 7.34–7.48 (5H, m), 7.19 (1H, d, J=4.5 Hz), 7.12 (1H, d, J=3.6 Hz), 6.95 (1H, dd, J=3.6 Hz and 5.1 Hz), 5.95 (1H, d, J=5.1 Hz), 5.06 (1H, d, J=5.1 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 31.161 (CH), 102.907 (CH=), 103.452 (CH=), 114.539 (Ar-C), 116.846 (Ar-CH), 122.762 (Ar-CH), 124.254 (Ar-CH), 124.894 (Ar-CH), 125.522 (Ar-CH), 127.028 (Ar-CH), 128.736 (Ar-CH), 129.468 (Ar-C), 132.162 (Ar-C), 132.498 (Ar-C), 147.643 (=C–O), 152.700 (=C–O), 155.539 (=C–O), 161.491 (O–C=O). Anal. Calcd for C₂₂H₁₄SO₃: C, 73.72; H, 3.94; S, 8.95. Found: C, 73.93; H, 4.12; S, 9.11.

4-(Naphthalen-1-yl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-12)

Yield 75%; mp: 192–193°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.39 (1H, d, J=8.7 Hz), 8.08 (1H, d, J=7.8 Hz), 7.89 (1H, d, J=7.8 Hz), 7.74 (1H, d, J=7.2 Hz), 7.50–7.68 (5H, m), 7.32–7.45 (7H, m), 5.96 (1H, d, J=4.5 Hz), 5.57 (1H, d, J=4.5 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 32.100 (CH), 103.029 (CH=), 103.775 (CH=), 114.538 (Ar-C), 116.964 (Ar-CH), 122.702 (Ar-CH), 122.939 (Ar-CH), 124.270 (Ar-CH), 124.640 (Ar-CH), 125.799 (Ar-CH), 125.931 (Ar-CH), 126.584 (Ar-CH), 127.736 (Ar-CH), 128.613 (Ar-CH), 129.022 (Ar-C), 129.181 (Ar-C), 130.839 (Ar-C), 132.153 (Ar-C), 132.665 (Ar-CH), 134.084 (Ar-CH), 140.024 (Ar-CH), 146.361 (=C–O), 152.892 (=C–O), 156.972 (=C–O), 161.420 (O–C=O). Anal. Calcd for C₂₈H₁₈O₃: C, 83.57; H, 4.51. Found: C, 83.49; H, 4.66;

2-(4-Nitrophenyl)-4-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-13)

Yield 79%; mp: 120–124°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.31 (1H, d, J=9.00 Hz), 7.92 (2H, d, J=8.7 Hz), 7.25–7.61 (10H, m), 6.06 (1H, d, J=5.4 Hz), 4.76 (1H, d, J=4.5 Hz). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 34.753 (CH), 102.458 (CH=), 102.735 (=C–), 114.217 (Ar-C), 116.430 (Ar-CH), 121.825 (Ar-CH), 121.435 (Ar-CH), 122.468 (Ar-CH), 125.647 (Ar-CH), 126.368 (Ar-CH), 127.348 (Ar-CH), 127.987 (Ar-CH), 128.376 (Ar-CH), 128.736 (Ar-CH), 128.973 (Ar-CH), 128.969 (Ar-CH), 129.678 (Ar-CH), 129.468 (Ar-C), 136.458 (Ar-C), 144.401 (=C–O), 151.872 (=C–O), 155.802 (=C–O), 157.383 (Ar-C-NO₂), 161.614 (O–C=O). Anal. Calcd for C₂₄H₁₅NO₅: C, 72.54; H, 3.80; N, 3.52. Found: C, 72.44; H, 4.04; N, 3.78.

4-(2-Methoxyphenyl)-2-phenylpyrano[3,2-c]chromen-5(4H)-one (CH-14)

Yield 82%; mp: 168–169°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 8.03 (1H, d, J=7.5 Hz), 7.70 (2H, d, J=7.5 Hz), 7.36–7.49 (5H, m), 7.17–7.25 (3H, m), 6.87–6.98 (2H, m), 5.86 (1H, d, J=5.4 Hz), 5.12 (1H, d, J=4.5 Hz), 3.89 (3H, s). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 32.969 (CH), 55.785 (–OCH₃), 102.480 (CH=), 103.468 (CH=), 114.646 (Ar-C), 116.371 (Ar-CH), 122.134 (Ar-CH), 122.611 (Ar-CH), 122.793 (Ar-CH), 124.118 (Ar-CH), 124.545 (Ar-CH), 125.225 (Ar-CH), 125.686 (Ar-CH), 128.177 (Ar-CH), 128.929 (Ar-CH), 129.430 (Ar-CH), 131.658 (Ar-C), 131.909 (Ar-C), 132.012 (Ar-C), 132.968 (Ar-C), 146.401 (=C–O), 152.872 (=C–O), 156.802 (=C–O), 161.414 (O–C=O). Anal. Calcd for C₂₅H₁₈O₄: C, 78.52; H, 4.74. Found: C, 78.69; H, 4.56.

4-(4-Hydroxy-3-methoxyphenyl)-2-(3,4-dimethoxyphenyl)pyrano[3,2-c]chromen-5(4H)-one (CH-15)

Yield 79%; mp: 122–123°C; ¹H-NMR (CDCl₃, 300 MHz, δ, TMS=0): 7.98 (1H, br s), 7.36–7.56 (6H, m), 6.75–7.00 (4H, m), 6.06 (1H, d, J=4.5 Hz), 4.76 (1H, d, J=4.5 Hz), 3.95 (3H, s), 3.93 (3H, s), 3.87 (3H, s). ¹³C-NMR (CDCl₃, 75 MHz, δ, TMS=0): 36.469 (CH), 55.628 (–OCH₃), 102.129 (CH=), 103.775 (CH=), 114.538 (Ar-C), 115.264 (Ar-CH), 116.964 (Ar-CH). 119.483 (Ar-CH), 122.702 (Ar-CH), 122.939 (Ar-CH), 123.365 (Ar-C), 125.563 (Ar-CH), 126.549 (Ar-CH), 128.458 (Ar-CH), 128.929 (Ar-CH), 129.430 (Ar-CH), 131.658 (Ar-C), 135.589 (Ar-C), 141.247 (=C–OH), 140.387 (=C–O), 149.367 (=C–O), 150.538 (=C–O), 151.369 (=C–O), 159.983 (=C–O), 161.283 (O–C=O). Anal. Calcd for C₂₇H₂₂O₇: C, 70.73; H, 4.84. Found: C, 71.01; H, 5.06

Biological Evaluation

Cell Culture, Growth Conditions, and Treatment

Human colon carcinoma cell lines HCT 116 were obtained from National Cancer Institute (NCI), Bethesda, MD, U.S.A. The cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 µg/mL), L-glutamine (0.3 mg/mL), pyruvic acid (0.11 mg/mL), and 0.37% NaHCO₃. Cells were grown in a CO₂ incubator (Thermocon Electron Corporation, Waltham, MA, U.S.A.) at 37°C in an atmosphere of 95% air and 5% CO₂ with 98% humidity. Different derivatives of a coumarin–chalcone hybrid series (1–20) were dissolved in 0.1% dimethyl sulfoxide (DMSO) and delivered to cell culture in complete medium.

In-Vitro Cytotoxicity Assay

In-vitro cytotoxicity against four human cancer cell lines was determined using 96-well tissue culture plate. The cells were allowed to grow in carbon dioxide incubator (37°C) for 24 h. Test materials in complete growth medium (100 µL) were added after 24 h of incubation to the wells containing cell suspension. The plates were further incubated for 48 h in a carbon dioxide incubator. The cell growth was stopped by gentle layering trichloroacetic acid (50%, 50 µL) on top of the medium in all the wells. The plates were incubated at 4°C for 1 h to fix the cells attached to the bottom of the wells. The liquid of all the wells was gently pipetted out and discarded. The plates were washed five times with distilled water to remove trichloroacetic acid, growth medium low-molecular weight metabolites, serum on a mechanical stirrer. The optical density (OD) was recorded on ELISA reader at 540 nm. The cell growth was determined by subtracting the mean OD value of respective blank from the mean OD value of the experimental set. Percent growth in the presence of test material was calculated considering the growth in the absence of any test material as 100%, and in turn, percent growth inhibition in the presence of test material was calculated proteins, etc. and air-dried. The plates were stained with sulforhodamine B dye (0.4% in 1% acetic acid, 100 µL) for 30 min. The plates were washed five times with 1% trichloroacetic acid and then air-dried. The adsorbed dye was dissolved in Tris–HCl buffer (100 µL, 0.01 M, pH 10.4), and the plates were gently stirred for 10 min.

Phase Contrast Microscopy

Phase contrast microscopy was performed to assess the morphological changes on cells after treatment with compound CH-1. Cells were incubated in six-well plates and treated with different concentrations of compound CH-1 for 6 h. After completion of that time, cells were subjected to photography. Apoptosis was assessed by using a microscope (1×70, Olympus), and photographs were taken by using DP-12 camera.

Hoechst 33258 Staining of Cells for Nuclear Morphology

HCT 116 cells were treated with the indicated concentrations of compound CH-1 for 6 h. Cells were centrifuged at 400×g for 5 min and washed twice with PBS. Cells were then stained with 1 mL of staining solution (Hoechst 33258, 10 µg/mL of 0.01 M citric acid, and 0.45 M disodium phosphate containing 0.05% Tween 20) and stained for 30 min under subdued light at room temperature. After staining, the cells were resuspended in 50 µL of mounting fluid (PBS–glycerol, 1 : 1), and 10 µL of mounting solution containing the cells was spread on clean glass slides and covered with coverslips. The slides were then observed for any nuclear morphological alterations and apoptotic bodies under an inverted fluorescence microscope (Olympus 1×70, magnification 30×) using UV excitation.

Measurment of Mitochondrial Membrane Potential

For mitochondrial membrane potential, 1×10⁶ HCT 116 cells were seeded in a 12-well plate and incubated with compound CH-1 (5, 10, 20 µM) for 6 h. RH123 (200 nM/mL) was added 30 min before termination of the experiment. Cells were collected at 400 g and washed once with PBS, and the mitochondrial membrane potential was measured in the FL-1 channel of the flow cytometer.

Cell Cycle Analysis

The effect of compound CH-1 on different phases of the cell cycle was assessed by propidium iodide fluorescence. HCT 116 cells (1×10⁶ per well) were incubated with different concentrations of compound CH-1 (5, 10, 20 µM) for 6 h. The cells were then washed twice with ice-cold PBS, harvested, fixed with ice-cold PBS in 70% ethanol, and stored at −20°C overnight. After fixation, these cells were incubated with RNase A (0.1 mg/mL) at 37°C for 90 min, stained with propidium iodide (100 µg/mL) for 30 min on ice in the dark, and then measured for DNA content using a BD FACS flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Data were collected in list mode, and 10000 events were analyzed for FL2-A vs. FL2-W. Modfit software was used to distinguish different phases of the cell cycle.

Acknowledgments

One of the author, Mr. Dinesh Kumar is thankful to UGC, New Delhi for providing research fellowship under UPE (Focus Area-Health Care, Drug Development and Sports Medicines) Scheme (Sanction No. 25994/Estt./A-11). The authors are grateful to UGC, New Delhi for providing the status to university, UPE (University with potential for excellence) and Grant for the establishment centre of emerging life sciences.

Conflict of Interest

The authors declare no conflict of interest.

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