Design, synthesis and bioactivity study on oxygen-heterocyclic-based pyran analogues as effective P-glycoprotein-mediated multidrug resistance in MCF-7/ADR cell

Materials and equipment’s

All chemicals were purchased from Sigma-Aldrich Chemical Co. (Sigma-Aldrich Corp., St. Louis, MO, USA). All the melting points were measured with a Stuart Scientific Co. Ltd apparatus, which means they are uncorrected. The IR spectra were recorded on a KBr disc on a Jasco FT/IR 460 plus spectrophotometer. The ¹H/¹³C (500/125 MHz) NMR and ¹³C NMR-APT spectrum (125 MHz) spectra were measured on a BRUKER AV 500 MHz spectrometer in DMSO-d6, using tetramethylsilane (TMS) as an internal standard. The Microwave apparatus utilized is Milestone Sr1, Microsynth. The mass spectra were determined on a Shimadzu GC/MS-QP5050A spectrometer. The elemental analysis was carried out at the Regional Centre for Mycology and Biotechnology (RCMP), Al-Azhar University, Cairo, Egypt, and the results were within ± 0.25%. The reaction courses and product mixtures were routinely monitored by thin layer chromatography (TLC) on silica gel precoated F₂₅₄ Merck plates.

General procedure for synthesis of 1H-benzo[f]chromene derivatives (4a-l)

In an ethanol solution (30 ml), a reaction mixture containing naphthalene-2,7-diol (1) (0.01 mol), malononitrile (2) (0.01 mol), various aromatic aldehydes (3a–l), and piperidine (0.5 ml) was heated for two minutes at 140 °C under Microwave irradiation conditions. Upon the completion of the reaction, the reaction mixture was allowed to cool down to room temperature. The precipitated solid was then removed by filtering, cleaned with methanol, and separated from the ethanol/benzene mixture. The physical and spectral data of compounds 4a–l are as follows:

3-Amino-1-(4-fluorophenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4a)

Yellow needles, yield 94%, m.p. 272–272 ℃ (Literature procedure, reflux condition, yield 92%; m.p. 274–276 °C⁷³).

3-Amino-1-(2-chlorophenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4b)

Buff crystals; yield 86%, m.p. 262–263 ℃ (Literature procedure, reflux condition, yield 75%; m.p. 260–262 °C⁷⁴).

3-Amino-1-(3-chlorophenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4c)

Yellow crystals; yield 88%; m.p. 260–261 ^οC; IR (KBr) υ (cm^−1): 3456, 3358, 3226, 3196 (NH₂ & OH), 2175 (CN); ¹H NMR δ: 10.03 (s, 1H, OH), 7.89–6.97 (m, 11H, Ar and NH₂), 5.13 (s, 1H, H-1); ¹³C NMR δ: 160.28, 157.08, 148.46, 147.86, 133.71, 132.47, 131.21, 130.72 (C-7), 130.03, 127.14, 126.17, 125.75, 120.83, 117.73, 113.68, 113.45, 106.09, 57.58, 37.72. In ¹³C NMR-APT δ: 160.28 ↓, 157.08 ↓, 148.46 ↓, 147.86 ↓, 133.71 ↓, 132.47 ↓, 131.21 ↑, 130.72 ↑, 130.03 ↑, 127.14 ↑, 126.17 ↑, 125.75 ↓, 120.83 ↓, 117.73 ↓, 113.68 ↑, 113.45 ↑, 106.09 ↑, 57.58 ↓, 37.72 ↑; MS m/z (%): 350 (M⁺ + 2, 9.43), 348 (M⁺, 28.56) with a base peak at 238 (100); Anal. Calcd for C₂₀H₁₃ClN₂O₂ (359.33): C, 68.87; H, 3.76; N, 8.03. Found: C, 68.94; H, 3.82; N, 8.10%.

3-Amino-1-(4-chlorophenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4d)

Yellow powder, yield 89%, m.p. 285–286 ℃ (Literature procedure, reflux condition, yield 75%; m.p. 286–288 °C⁷⁵).

3-Amino-1-(4-bromophenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4e)

Yellow powder, yield 89%, m.p. 285–286 ℃ (Literature procedure, reflux condition, yield 75%; m.p. 286–288 °C⁷⁶).

3-Amino-1-(4-methylphenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4f)

Colurless crystals; yield 91%; m.p. 258–259 ^οC; IR (KBr) υ (cm^−1): 3448, 3351, 3229, 3199 (NH₂ & OH), 2177 (CN); ¹H NMR δ: 7.82–6.88 (m, 11H, Ar and NH₂), 5.00 (s, 1H, H-1), 2.20 (s, 3H, CH₃); ¹³C NMR δ: 160.11, 156.71, 147.69, 143.00, 136.32, 132.53, 130.60, 129.69, 127.30, 125.73, 121.14, 117.55, 114.31, 113.69, 106.19, 58.83, 37.79, 20.96. In ¹³C NMR-APT δ: 160.11 ↓, 156.71 ↓, 147.69 ↓, 143.00 ↓, 136.32 ↓, 132.53 ↓, 130.60 ↑, 129.69 ↑, 127.30 ↑, 125.73 ↑, 121.14 ↓, 117.55 ↑, 114.31 ↓, 113.96 ↑, 106.19 ↑, 58.83 ↓, 37.79 ↑, 20.96 ↑; MS m/z (%): 328 (M⁺, 100); Anal. Calcd for C₂₁H₁₆N₂O₂ (328.36): C, 76.81; H, 4.91; N, 8.53. Found: C, 76.88; H, 4.97; N, 8.61%.

3-Amino-1-(4-methoxyphenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4g)

Colourless needles, yield 90%, m.p. 229–230 ℃ (Literature procedure, reflux condition, yield 80%; m.p. 228 °C⁷⁷).

3-Amino-1-(4-nitrophenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4h)

Yellow crystals; yield 88%; m.p. 262–263 ^οC; IR (KBr) υ (cm^−1): 3456, 3358, 3226, 3196 (NH₂ & OH), 2175 (CN); ¹H NMR δ: 9.92 (s, 1H, OH), 8.18, 8.17 (dd, 2H, J = 7.5, 7.2 Hz, Ar, H-3,5), 7.83 (d, 1H, J = 8.9 Hz, H-7), 7.77 (d, 1H, J = 8.8 Hz, H-6), 7.43, 7.42 (dd, 2H, J = 7.5, 7.2 Hz, Ar, H-2,6), 7.11 (bs, 2H, NH₂), 7.10 (s, 1H, H-10), 6.98, 6.97 (dd, 1H, J = 8.8, 2.3 Hz, H-8), 6.93 (d, 1H, J = 1.0 Hz, H-5), 5.28 (s, 1H, H-1); ¹³C NMR δ: 160.29, 157.03, 153.34, 147.82, 146.67, 132.43, 130.77, 130.27, 128.69, 125.73, 124.65, 120.66, 117.70, 113.68, 112.91, 106.03, 57.03, 38.49; MS m/z (%): 360 (M⁺ + 1, 100); Anal. Calcd for C₂₀H₁₃N₃O₄ (359.33): C, 66.85; H, 3.65; N, 11.69. Found: C, 66.91; H, 3.72; N, 11.74%.

3-Amino-1-(4-(benzyloxy)phenyl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4i)

Pale yellow crystals; yield 87%; m.p. 269–279 ^οC; IR (KBr) υ (cm^−1): 3457, 3445, 3322, 3226 (NH₂ & OH), 2175 (CN); ¹H NMR δ: 9.87 (s, 1H, OH), 7.77 (d, 1H, J = 8.9 Hz, H-7), 7.74 (d, 1H, J = 8.8 Hz, H-6), 7.43,7.40 (dd, 2H, J = 8.2, 6.8 Hz, Ph, H-2,6), 7.38, 7.37 (2H, dd, J = 8.4, 6.9 Hz, Ph, H-3,5), 7.32 (1H, t, J = 7.1 Hz, Ph, H-4), 7.07, 7.06 (2H, dd, J = 8.8, 6.9 Hz, Ar, H-2,6), 7.05 (1H, s, H-10), 7.00 (1H, d, J = 2.3 Hz, H-5), 6.97 (2H, dd, J = 8.7, 2.3 Hz, Ar, H-3,5), 6.93, 6.92 (1H, d, J = 3.3 Hz, H-8), 6.90 (bs, 2H, NH₂), 5.02 (s, 2H, CH₂), 4.96 (s, 1H, H-1);

¹³C NMR δ: 160.00, 157.52, 156.75, 147.64, 138.38, 137.53, 132.55, 130.60, 129.98, 128.90, 128.45, 128.31, 128.19, 125.71, 121.16, 117.51, 115.29, 114.42, 113.65, 106.21, 69.68, 58.62, 38.12; MS m/z (%): 421 (M⁺ + 1, 100); Anal. Calcd for C₂₇H₂₀N₂O₃ (420.46): C, 77.13; H, 4.79; N, 6.66. Found: C, 77.17; H, 4.84; N, 6.70%.

3-Amino-1-(benzo[d][1,3]dioxol-5-yl)-9-hydroxy-1H-benzo[f]chromene-2-carbonitrile (4j)

Colorless crystals; yield 86%; m.p. 260–261 °C; IR (KBr) υ (cm^−1): 3401, 3315, 3207, 3194 (NH₂ & OH), 2187 (CN); ¹H NMR δ: 9.89 (s, 1H, OH), 7.78 (d, 1H, J = 8.9 Hz, H-7), 7.75 (d, 1H, J = 8.8 Hz, H-6), 7.06 (d, 1H, J = 8.9 Hz, H-5), 7.02 (s, 1H, H-10), 6.98 (s,1H, Ar, H-2), 6.93 (bs, 2H, NH₂), 6.81 (s, 1H, J = 8.0, Hz, H-8), 6.64 (s, 1H, Ar, H-5), 6.61 (s, 1H, Ar H-6), 5.96, 5.94 (d, 2H, J = 14 Hz, CH₂), 4.96 (s, 1H, H-1); ¹³C NMR δ: 160.05, 156.80, 147.85, 147.65, 146.32, 140.13, 132.56, 130.61, 129.68, 125.69, 121.07, 120.46, 117.56, 114.21, 113.65, 108.84, 107.77, 106.21, 101.42, 58.48, 38.49; MS m/z (%): 359 (M⁺ + 1, 100); Anal. Calcd for C₂₁H₁₄N₂O₄ (358.35): C, 70.39; H, 3.94; N, 7.82. Found: C, 70.46; H, 3.99; N, 7.88%.

3-Amino-9-hydroxy-1-(pyridin-3-yl)-1H-benzo[f]chromene-2-carbonitrile (4k)

Colorless crystals; yield 80%; m.p. 296–297 ^οC; IR (KBr) υ (cm^−1): 3479, 3309, 3217, 3191 (NH₂ & OH), 2185 (CN); ¹H NMR δ: 9.93 (s, 1H, OH), 8.51 (d, 1H, J = 2.5 Hz, pyridine, H-4), 8.41,8.40 (dd, 1H, J = 4.7, 1.7 Hz, pyridine, H-5), 7.81 (d, 1H, J = 8.9 Hz, H-7), 7.77 (d, 1H, J = 9.4 Hz, pyridine, H-6), 7.44 (d, 1H, J = 8.0 Hz, H-6), 7.30 (d, 1H, J = 8.0 Hz, H-5), 7.09 (d, 1H, J = 8.9 Hz, H-8), 7.07 (bs, 2H, NH₂), 6.98 (s, 1H, pyridine, H-2), 6.97 (d, 1H, H-10), 5.15 (s, 1H, H-1); ¹³C NMR δ: 160.27, 156.99, 148.62, 148.47, 147.84, 141.33, 135.03, 132.30, 130.77, 130.05, 125.72, 124.61, 120.83, 117.66, 113.67, 113.07, 105.92, 57.50, 36.37; MS m/z (%): 315 (M⁺ + 1, 67) with base peak at 237 (100); Anal. Calcd for C₁₉H₁₃N₃O₂ (315.33): C, 72.37; H, 4.16; N, 13.33. Found: C, 72.45; H, 4.21; N, 13.38%.

3-Amino-9-hydroxy-1-(pyridin-4-yl)-1H-benzo[f]chromene-2-carbonitrile (4l)

Colorless crystals; yield 81%; m.p. 294–295 ^οC; IR (KBr) υ (cm^−1): 3428, 3324, 3217, 3198 (NH₂ & OH), 2191 (CN); ¹H NMR δ: 9.92 (s, 1H, OH), 8.48, 8.47 (dd, 2H, J = 8.8,2.4 Hz, pyridine, H-3,5), 7.82 (d, 1H, J = 8.9 Hz, H-7), 7.77 (d, 1H, J = 8.8 Hz, H-6), 7.14 (d, 1H, J = 1.6 Hz, H-5), 7.10 (bs, 2H, NH₂), 7.09 (s, 1H, H-10), 6.99, 6.98 (dd, 2H, J = 8.8, 2.3 Hz, pyridine, H-2,6), 6.92 (d, 1H, J = 2.0 Hz, H-8), 5.12 (s, 1H, H-1); ¹³C NMR δ: 160.40, 157.01, 154.08, 150.55, 147.92, 132.43, 130.75, 130.19, 125.69, 122.68, 120.72, 117.72, 113.64, 112.63, 105.94, 56.79, 38.13; MS m/z (%): 316 (M⁺ + 1, 100); Anal. Calcd for C₁₉H₁₃N₃O₂ (315.33): C, 72.37; H, 4.16; N, 13.33. Found: C, 72.31; H, 4.11; N, 13.27%.

Biological screening

Cell culture

The tumor cell lines (PC-3, SKOV-3, and HeLa), resistant cell strains (MCF-7/ADR) and the normal cell lines, (HFL-1, WI-38) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Cytotoxicity evaluation using viability assay

The tumor cell lines were suspended in medium at concentration 5 × 104 cells well^−1 in Corning® 96-well tissue culture plates and then incubated for 24 h. The tested compounds with concentrations ranging from 0 to 100 μM were then added into 96-well plates (six replicates) to achieve six different concentrations for each compound. Six vehicle controls with media or 0.5% DMSO were run for each 96 well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by the MTT test⁵³. Briefly, the media was removed from the 96 well plates and replaced with 100 μl of fresh culture RPMI 1640 medium without phenol red then 10 μl of the 12 mM MTT stock solution (5 mg of MTT in 1 ml of PBS) to each well including the untreated controls. The 96-well plates were then incubated at 37 °C and 5% CO₂ for 4 h. An 85-μl aliquot of the media was removed from the wells, and 50 μl of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37 °C for 10 min. Then, the optical density was measured at 590 nm with the microplate reader (Sunrise, TECAN, Inc, USA) to determine the number of viable cells and the percentage of viability was calculated as [1−(ODt/ODc)] × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC₅₀), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each concentration.

In vitro analysis of P-gp content

The content of P-gp in the MCF-7/ADR cell lysates after incubation with varying conc. (12.5–100 µM) of tested compounds 4a–c following exposure for 48 h. was determined using commercial human P-gp (Permeability Glycoprotein) ELISA Kit (MBS2506188, MyBioSource Inc., San Diego, CA, USA). Absorption was recorded at 450 nm with a Spectramax Gemini fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA)⁷⁸.

Rhodamine 123 accumulation assay

P-gp activity was determined by measuring intracellular accumulation of rhodamine 123 in MCF-7/ADR cells in the absence or presence of compounds 4a–c according to commercial Rhodamine Competitive ELISA Kit (AKR-5142, Cell Biolabs Inc., San Diego, CA, USA) which provides a convenient method for the detection of total rhodamine in extracts from cells⁷⁹. Absorbance at 450 nm of each well was measured using Spectramax Gemini fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA). The total content of Rhodamine in each sample was determined by comparison with a Rhodamine standard curve.

Cell cycle assay

Cell cycle arrest and distribution were done using Propidium Iodide Flow Cytometry Kit (ab139418, Abcam) as previously described⁸⁰. Cells were cultured in 60-mm dishes, after 24 h cells were cultured for an additional 24 h in the absence (control) or presence of the different newly synthesized derivatives (IC₅₀ value). The cells were then harvested and fixed in a 100% ice cold ethanol at + 4 °C for at least 2 h. After rewashing with PBS, the cells were incubated with a 200 μl 1× Propidium Iodide (PI) + RNase Staining Solution for 30 min at room temperature in the dark. The DNA content in each cell nucleus was determined by a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Finally, Cell cycle phase distribution was analyzed using Cell Quest Pro software (BD Biosciences) showing collected propidium iodide fluorescence intensity on FL2.

Annexin V-FITC apoptosis assay

Apoptosis assay was performed with an Annexin V-FITC/PI double staining apoptosis detection kit (K101, Biovison) using a flow cytometer⁸¹. Cells were cultured in 60-mm dishes, after 24 h cells were cultured for an additional 24 h in the absence (control) or presence of the different newly synthesized derivatives (IC₅₀ value). Cells were harvested by the trypsinization, washed twice with 4 °C PBS, and re-suspended in the binding buffer. Subsequently, the Annexin V-FITC and Propidium iodide (PI) solutions were added to stain the cells before the analysis by the flow cytometry, where a minimum of 10,000 cells per sample were acquired. The Annexin V-FITC binding (FL1) and PI (FL2) were analyzed, using the Cell Quest Pro software (BD Biosciences).

Molecular docking

Jaguar was used for generating all possible tautomeric and stereo-isomeric stats for the structures⁸². Crystal structures of P-glycoprotein protein was taken from the protein data bank bonded with 5-florouracil as reference drug. All ligands were imported into Ligprep module and redocked into appropriate binding sites using Glide’s module. The Glide-tool was applied to perform the molecular docking, then a grid for protein charged using the default aspects of force field. The (SP) scoring function produced for study the binding affinity, and then charged with Charm force field. The low-root-square-devotion RMSD score utilized to get the other poses. Schrodinger builder were applied to draw.

Statistics

Statistical analysis and figures were performed by GraphPad Prism 5.01 (Graph Pad software, San Diego, CA. USA).