Corresponding author: Ali H. Abbas ( alih.phchm@tu.edu.iq ) Academic editor: Georgi Momekov
© 2021 Ali H. Abbas, Ammar A. Razzak Mahmood, Lubna H. Tahtamouni, Zainab A. Al-Mazaydeh, Majdoleen S. Rammaha, Fatima Alsoubani, Rheda I. Al-bayati.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Abbas AH, Mahmood AAR, Tahtamouni LH, Al-Mazaydeh ZA, Rammaha MS, Alsoubani F, Al-bayati RI (2021) A novel derivative of picolinic acid induces endoplasmic reticulum stress-mediated apoptosis in human non-small cell lung cancer cells: synthesis, docking study, and anticancer activity. Pharmacia 68(3): 679-692. https://doi.org/10.3897/pharmacia.68.e70654
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Thirteen new derivatives of picolinic acid (4–7) were designed and synthesized from the starting parent molecule, picolinic acid. The new compounds were characterized by ATR-FTIR, 1HNMR, and CHNS analysis. A molecular docking study was performed to evaluate the binding affinity of the synthesized compounds toward EGFR kinase domain that indicated occupation of the critical site of EGFR kinase pocket and excellent positioning of the compounds in the pocket. The cytotoxic activity of the compounds against two human cancer cell lines (A549 and MCF-7), the non-tumorigenic MCF10A cell line, and white blood cells (WBC) was evaluated using the MTT assay. Compound 5 showed anticancer activity against A549 lung cancer cells (IC50 = 99.93 µM) but not against MCF-7 breast cancer cells or normal cells. Compound 5 mediated cytotoxicity in A549 lung cancer cells by inducing apoptotic cell death, as suggested by fragmented nuclei after DAPI staining, and agarose gel electrophoresis. Moreover, compound 5 triggered the activation of caspases 3, 4 and 9. However, compound 5 treatment did not affect the release of cytochrome c from the mitochondria to the cytosol, as compared to the vehicle-treated control cells. Nevertheless, compound 5-treated cells reported greater release of smac/DIABLO to the cytosol. In the same context, both compound 5 and thapsigargin (specific inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)) enhanced eIF2 phosphorylation, reflecting the activation of the atypical ER stress pathway and the potential applicability of compound 5 in lung cancer treatment.
Picolinic acid, ER stress pathway, Docking, A549 lung cancer cells
The Global Cancer Observatory 2020 clearly reported that there were 19.3 million new cancer cases and nearly 10 million cancer deaths in 2020. Malignant diseases are a major cause of mortality and morbidity in every region of the world irrespective of the human development index (
Lung cancer is the leading cause of cancer-related deaths globally, with an overall five-year survival rate of 15% (
Endoplasmic reticulum (ER) is a major organelle with several cellular functions and is a vital site for the maintenance of homeostasis. When ER-related pathways of protein folding regulation, post-translational changes, cellular metabolism and/or calcium signaling are disturbed, the accumulation of ER-related proteins might ultimately lead to ER stress (ERS). Under overwhelming ERS, the cells initiate autophagy, followed by large-scale degradation and apoptosis (
The epidermal growth factor receptor (EGFR)/Her1/ErbB1 is a cell-surface receptor that belongs to the ErbB family of tyrosine kinases. EGFR has earned a great attention as a molecular target in cancer therapy, owing to its abnormal expression (upregulation) in many epithelial tumors, and its influence on growth and survival in malignant states (
Picolinic acid (PA) has already been identified as an activator of macrophage pro-inflammatory functions, which provided the first indications of the independent bioactive properties of picolinic acid even in the absence of a co-stimulatory agent (
Picolinic acid was purchased from Sigma-Aldrich. Reactions were monitored using thin-layer chromatography (TLC) on silica gel(60).F254 (Merck, Germany) and exposed to UV254 nm light. The infrared spectra were recorded using Shimadzu Specac GS 10800-R IRAffnity-1Spectrometer (ύ, cm-1).
CHNS microanalysis was carried out using Euro EA3000 elemental analyzer.1HNMR of the synthesized compounds were measured on Inova show Ultra shield 500MHz. using tetramethylsilane (TMS) as an internal standard, the chemical shifts were expressed as (δ, ppm), and DMSO-d6 was used as a solvent.
An accurately weighed amount of picolinic acid (1) (10 g, 0.0812 mol) was added to 70 mL of abs. EtOH in a 250 mL round bottom flask, and the mixture was stirred until a clear solution was obtained. The solution was cooled down to -10 °C and 3 mL of conc. H2SO4 was added drop wise with continuous stirring, which was accompanied by the appearance of an increasing amount of a white precipitate during the addition process. After the acid addition was completed, the white precipitate ceased to form, and the mixture was then refluxed with stirring at 80 °C for 48 h. As the temperature of the mixture increased during the refluxing process, the precipitate dissolved again, and a clear solution reappeared. At the end of the reflux period, the solution was concentrated, and the residue was dissolved in 25 mL of D.W, basified with 5% NaHCO3, extracted thrice with 25 mL of CH2Cl2, filtered over anhyd. MgSO4, and concentrated to yield a clear, colorless oil that was used in the next step without further purification.
Colorless oil, yield (70%), Rf = 0.4, ATR-FTIR (ύ, cm-1): 3059 Ar(CH) str, 2981, 2935, 2904 and 2873 (CH) str. of aliph. (CH2) & (CH3), 1716 str. of (C=O) (conj ester), 1585 (C=N) str, 1465,1438 Ar(C=C) str, 1392,1369 (CH2) and (CH3) bend, 1172 (C-O) str of ester, 748 &705 (CH) bend. of heterocyclic.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 8.71 (dd, 1H, J=1.1, 3.8 Hz, pyr-H), 8.04 (d, 1H, J=7.8 Hz, pyr-H), 7.98 (t, 1H, J=7.8 Hz, pyr-H), 7.63 (ddd, 1H, J=1.1, 4.7, 7.4 Hz, pyr-H), 4.33 (q, 2H, J=7.1 Hz, CH2), 1.31 (t, 3H, J=7.1 Hz, CH3).
Ethyl picolinate compound (2) (5.6 g, 0.037 mol), and hydrazine hydrate 99.5% (an excess amount (9.0 mL ,0.185 mol), were added to 40 mL of abs. EtOH in a 250 mL round bottom flask, and the mixture was first stirred overnight at room temperature (RT), after that it was refluxed at 80 °C for 12 h. It was noticed that the colorless solution changed to pale pink with time. At the end of the reflux time, half of the solvent was removed under reduced pressure and the residue was poured into ice. The precipitate was filtered and washed with ice-cold EtOH to give a product, which was recrystallized from 70% EtOH to yield compound (3).
Off-white crystals, yield (80%), m.p (98–100 °C), Rf = 0.85, ATR-FTIR (ύ, cm-1): 3363 (NH) str. of sec. amide, 3290,3209 (NH). str of prim. amine, 3016 Ar(CH) str, 1670 str of (C=O) of amide (amide I band), 1647 (NH) bend, 1593 (C=N) str, 1566 (NH) bend. of (amide II band), 1516,1469,1431 Ar(C=C) str, 1246 (C-N) str, 752,702 (CH) bend of heterocyclic.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 9.89 (br s, 1H, NH), 8.60 (d, 1H, J=3.8 Hz, pyr-H), 7.96–8.0 (m, 2H, pyr-H), 7.56 (t, 1H, J=5.5 Hz, pyr-H), 4.61 (br s, 2H, NH2).
To a solution of compound (3) (0.5 g, 0.00364 mol) in 25 mL of abs. EtOH was added separately (A): 1-isothiocyanato-4-nitrobenzene(0.657 g, 0.00364 mol), (B): 1-bromo-4-isothiocyanatobenzene (0.78 g, 0.00364 mol), (C): 1-fluoro-4-isothiocyanatobenzene (0.558 g, 0.00364 mol), (D): 1-isothiocyanato-4-methylbenzene (0.544 g, 0.00364 mol), the reaction mixture was stirred at 40–50 °C for 4 h, then kept stirring overnight. Half of the solvent was removed under reduced pressure, and the residue was poured into ice. The precipitate was filtered, and washed with ice-cold EtOH to give a product, and recrystallized from 70% EtOH to yield the corresponding final target compounds (4–7).
The following target compounds were synthesized by the above-mentioned general procedure.
Yellow crystals, yield (95%), m.p(210–212 °C), Rf = 0.14, ATR-FTIR (ύ, cm-1): 3313 (NH) str. of sec. amide, 3228,3151(NH) str. of thioamide, 3078 Ar(CH) str, 1681 (C=O) str. of amide (amide I band), 1627 (C=N) str, 1581,1496,1462 Ar(C=C) str, 1556(NH) bend. of (amide II band), 1531,1300 asym/sym. str. of NO2 group, respectively, 1261 (C-N) str, 1230 (C=S) str, 813 (Ar- p-substitution), 736,698 (CH) bend. of heterocyclic.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.87 (br s, 1H, C(=S) NH), 10.16 (br s, 1H, NH(C=S)), 10.06 (br s, 1H, C(=O) NH), 8.72 (d, 1H, J=4.4 Hz, pyr-H), 8.22–8.21 (d, 2H, J=8.8 Hz, Ar-H), 8.09–8.08 (m, 2H, pyr-H), 7.94 (d, 2H, J=8.8 Hz, Ar-H), 7.69–7.64 (m, 1H, pyr-H).
CHNS analysis: Calcd. For (C13H11N5O3S): C,49.21; H,3.49; N,22.07; S,10.10. Observed: C,49.31; H,3.66; N,22.17; S,10.19.
White crystals, yield (92%), m.p (190–193 °C), Rf = 0.26, ATR-FTIR (ύ, cm-1): 3232 (NH) str of sec. amide, 3201,3124 (NH) str. of thioamide, 3082 Ar(CH) str, 1654 (C=O) str of amide (amide I band), 1604 (C=N) str, 1585,1543,1481 Ar(C=C) str, 1570 (NH) bend. of (amide II band), 1265 (C-N) str, 1222 (C=S) str, 825 (Ar- p-substitution), 736,690 (CH) bend. of heterocyclic, 720 (C-Br) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.77 (s, 1H, C(=S)NH), 9.86 (br s, 2H, C(=O)NH-NH), 8.70 (d, 1H, J=3.8 Hz, pyr-H), 8.09–8.01 (d, 2H, Ar-H), 7.66 (dd, 1H, J=5.5, 6.6 Hz, pyr-H), 7.52–7.51 (d, 2H, Ar-H), 7.48 (m, 2H, pyr-H).
CHNS analysis: Calcd. For (C13H11BrN4OS): C,44.46; H, 3.16; N,15.95; S,9.13. Observed: C,44.38; H, 3.00; N,16.08; S,9.32.
White crystals, yield (90%), m.p (188–191 °C), Rf = 0.25, ATR-FTIR (ύ, cm-1): 3290 (NH) str. of sec. amide, 3251,3151 (NH) str. of thioamide, 3093 Ar(CH) str, 1651 (C=O) str. of amide (amide I band), 1616 (C=N) str, 1558(NH) bend. of (amide II band), 1508,1481,1458 Ar(C=C) str, 1265 (C-N) str, 1215 (C=S) str, 829(Ar-p-substitution), 762,725 (CH) bend. of heterocyclic, 698 (C-F) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.75 (s, 1H, C(=S) NH), 9.75 (br s, 2H, C(=O) NH-NH), 8.71 (d, 1H, J=4.4 Hz, pyr-H), 8.09–8.02 (m, 2H, pyr-H), 7.66 (dd, 1H, J=5.5, 6.6 Hz, pyr-H), 7.46 (d, 2H, Ar-H), 7.17 (d, 2H, J=8.8 Hz, Ar-H).
CHNS analysis: Calcd. For (C13H11FN4OS): C,53.78; H,3.82; N,19.30; S,11.04. Observed: C,53.74; H,3.69; N, 19.52; S,11.02.
White crystals, yield (80%), m.p (190–192 °C), Rf = 0.28, ATR-FTIR (ύ, cm-1): 3290 (NH) str. of sec. amide, 3244,3201(NH) str. of thioamide, 3093,3024 Ar(CH) str, 2912,2854 (CH) str. of aliph.(CH3), 1651 (C=O) str. of amide (amide I band), 1608 (C=N) str, 1589,1512,1481 Ar(C=C) str, 1550(NH) bend. of (amide II band), 1454, 1354(CH) bend. of (CH3), 1265 (C-N) str, 1222 (C=S) str, 817 (Ar- p-substitution), 786,744 (CH) bend. of heterocyclic.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.72 (s, 1H, C (=S) NH), 9.67 (br s, 2H, (C=O) NH-NH), 8.70 (d, 1H, J=4.9 Hz, pyr-H), 8.09–8.04 (m, 2H, pyr-H), 7.66 (t, 1H, J=6.2 Hz, Pyr-H), 7.33 (d, 2H, Ar-H), 7.13 (d, 2H, J=8.2 Hz, Ar-H), 2.29 (s, 3H, CH3).
CHNS analysis: Calcd. For (C14H14N4OS): C,58.72; H,4.93; N,19.57; S,11.20. Observed: C,58.95; H,4.99; N,19.68; S,11.01.
Hydrazinecarbothioamide compounds (5, 6, and 7) (0.001 mol) were separately added to 2N NaOH (6.6 mL), stirring at RT for about (15 min), a clear yellow solution appeared which was then refluxed for 3 h. The reaction mixture was cooled to RT, and then acidified with 2N HCl to pH = 3. The resulting solid was filtered and recrystallized from 70% EtOH to afford the corresponding final compounds (5A, 6A, and 7A).
The following target compounds were synthesized by the above-mentioned general procedure.
White crystals, yield (77%), m.p (260–262 °C), Rf = 0.7, ATR-FTIR (ύ, cm-1): 3105 (NH) str, 3074, 3032 Ar(CH) str, 2762 (SH) str.(w), 1581,1554 (C=N) str, 1489,1458,1442 Ar(C=C) str, 1276 (C-N) str, 1234 (C=S) str, 817 (Ar-p-substitution), 790,763 (CH)bend. of heterocyclic, 744(C-Br) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 14.30 (s, 1H, NH), 8.37 (d, 1H, pyr-H), 7.93 (dd, 1H, J=1.6, 7.7 Hz, pyr-H), 7.90 (t, 1H, pyr-H), 7.67–7.65 (m, 2H, Ar-H), 7.43 (ddd, 1H, J=1.1, 4.8, 7.3 Hz, pyr-H), 7.31–7.29 (m, 2H, Ar-H).
CHNS analysis: Calcd. For (C13H9BrN4S): C,46.86; H,2.72; N,16.81; S,9.62. Observed: C,46.87; H,2.96; N,16.85; S,9.48.
White crystals, yield (72%), m.p (229–231 °C), Rf = 0.68, ATR-FTIR (ύ, cm-1): 3240 (NH) str, 3093, 3070 Ar(CH) str, 2765 (SH) str.(w), 1600,1570 (C=N) str, 1539, 1508,1485 Ar(C=C) str, 1288 (C-N) str, 1222 (C=S) str, 837 (Ar-p-substitution), 786,750 (CH)bend. of heterocyclic, 705 (C-F) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 14.27 (s, 1H, NH), 8.36 (dd, 1H, J=1.4, 5.2 Hz, pyr-H), 7.93 (dd, 1H, J=1.1, 7.7 Hz, pyr-H), 7.88 (t, 1H, pyr-H), 7.42 (t, 1H, J=6.7 Hz, pyr-H), 7.38 (m, 2H, Ar-H), 7.29–7.28 (m, 2H, Ar-H).
CHNS analysis: Calcd. For (C13H9FN4S): C,57.34; H,3.33; N,20.58; S,11.78. Observed: C,57.06; H,3.35; H,20.40; S,11.93.
White crystals, yield (77%), m.p (236–239 °C), Rf = 0.49, ATR-FTIR (ύ, cm-1): 3086, 3028 Ar(CH) str, 2958,2924 (CH) str. of aliph. (CH3), 2762 (SH) str.(w), 1585,1554 (C=N) str, 1512,1492,1460 Ar(C=C) str, 1442,1362 (CH). bend. of (-CH3), 1234 (C-N) str, 1211 (C=S) str, 813 (Ar-p-substitution), 790,740 (CH)bend. of heterocyclic.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 14.22 (s, 1H, NH), 8.39 (m, 1H, pyr-H), 7.91 (d, 1H, pyr-H), 7.80 (d, 1H, J=7.7 Hz, pyr-H), 7.42 (dd, 1H, J=5.5, 6.6 Hz, pyr-H), 7.24 (d, 2H, J=7.7 Hz, Ar -H), 7.17 (d, 2H, J=8.2 Hz, Ar-H), 2.35 (s, 3H, CH3).
CHNS analysis: Calcd. For (C14H12N4S): C,62.66; H,4.51; N,20.88; S,11.95. Observed: C,62.62; H,4.61; N,20.66; S,12.07.
Hydrazinecarbothioamide compounds (5, 6, and 7) (0.001 mol) were separately added to concentrated H2SO4 (5 mL) at 0 °C and stirred for 3 h at RT. The reaction mixture was left to stir for 3 h at RT and it was noticed that a clear yellow solution appeared. The reaction mixture was neutralized with 2N NaOH and filtered, then washed with a plenty amount of water. The compound was recrystallized with 70% EtOH to give the final compounds (5B, 6B, and 7B).
The following target compounds were synthesized by the above-mentioned general procedure.
Faint pink crystals, yield (85%), m.p (244–247 °C), Rf = 0.81, ATR-FTIR (ύ, cm-1): 3174 (NH) str. of sec. amine, 3043 Ar(CH) str, 1612 (NH) bend, 1585,1562 (C=N) str, 1504,1489, 1431 Ar(C=C) str, 1273 (C-N) str, 825 (Ar-p-substitution), 779,740 (CH) bend. of heterocyclic, 713 (C-Br) str, 609 (C-S-C) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.76 (s, 1H NH), 8.65 (d, 1H, J=3.8 Hz, pyr-H), 8.15 (d, 1H, J=8.2 Hz, pyr-H), 7.99 (t, 1H, J=7.7 Hz, pyr-H), 7.68 (d, 2H, J=8.2 Hz, Ar-H), 7.55 (d, 2H, J=8.8 Hz, Ar-H), 7.52 (t, 1H, pyr-H)
CHNS analysis: Calcd. For (C13H9BrN4S): C,46.86; H,2.72; N,16.81; S,9.62. Observed: C,46.71; H,2.71; N,16.85; S,9.83.
Light brown crystalline powder, yield (80%), m.p (223–225 °C), Rf = 0.75, ATR-FTIR (ύ, cm-1): 3217 (NH) str. of sec. amine, 3055 Ar(CH) str, 1627 (NH) bend, 1581,1562 (C=N) str, 1504,1458,1431 Ar(C=C) str, 1280 (C-N) str, 829 (Ar-p-substitution), 779,736 (CH) bend. of heterocyclic, 713 (C-F) str, 613 (C-S-C) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.64 (br s, 1H, NH), 8.65 (d, 1H, pyr-H), 8.16 (d, 1H, J=7.7 Hz, pyr-H), 7.99 (t, 1H, J=6.9 Hz, pyr-H), 7.72 (d, 2H, Ar-H), 7.50 (t, 1H, pyr-H), 7.23 (d, 2H, J=8.2 Hz, Ar-H).
CHNS analysis: Calcd. For (C13H9FN4S): C,57.34; H,3.33; N,20.58; S,11.78. Observed: C,57.26; H,3.27; N,20.41; S,11.58.
Mustard like color crystalline powder, yield (90%), m.p (221–222 °C), Rf = 0.7, ATR-FTIR (ύ, cm-1): 3248 (NH) str. of sec. amine, 3012 Ar(CH) str, 2935,2885 (CH) str. of aliph.(CH3), 1612 (NH) bend, 1585,1562 (C=N) str, 1516,1504 Ar(C=C) str, 1437,1381 (CH). bend. of (CH3), 1280 (C-N) str, 837 (Ar-p-substitution), 740,713 (CH) bend. of heterocyclic, 617 (C-S-C) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.53 (s, 1H, NH), 8.65 (d, 1H, J=4.4 Hz, pyr-H), 8.16 (d, 1H, J=7.7 Hz, pyr-H), 7.98 (t, 1H, J=7.7 Hz, pyr H), 7.57–7.55 (m, 2H, Ar-H), 7.49 (m, 1H, pyr-H), 7.20–7.19 (m, 2H, Ar-H), 2.29 (s, 3H, CH3).
CHNS analysis: Calcd. For (C14H12N4S): C,62.66; H,4.51; N,20.88; S,11.95. Observed: C,62.87; H,4.44; N,20.90; S,12.07.
To a suspension of hydrazinecarbothioamide compounds (5,6, and 7) (0.001 mol) in 25 mL of EtOH, a few drops of 5N NaOH were added, with cooling and stirring at RT resulting in the formation of a clear solution. To this, iodine in potassium iodide solution (5%) was added drop wise with stirring till the color of iodine persisted at RT, and a light brown precipitate appeared. The reaction mixture was then refluxed for 6 h on a water bath, then kept stirring at RT overnight. It was then concentrated, cooled, and the solid separated out was filtered, dried, and recrystallized from 70% EtOH to the corresponding final compounds (5C, 6C and 7C).
The following target compounds were synthesized by the above-mentioned general procedure.
Light brown crystals, yield (75%), m.p (282–285 °C), Rf = 0.73, ATR-FTIR (ύ, cm-1): 3248 (NH) str. of sec. amine, 3070 Ar(CH) str, 1612 (NH) bend, 1581,1546 (C=N) str, 1489,1458,1438 Ar(C=C) str, 1292 (C-O-C) str. of oxadiazole, 1234 (C-N) str, 817 (Ar-p-substitution), 786,736 (CH) bend. of heterocyclic, 721 (C-Br) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 11.04 (s, 1H, NH), 8.74 (d, 1H, J=3.8 Hz, pyr-H), 8.11 (d, 1H, J=7.7 Hz, pyr H), 8.09 (t, 1H, pyr-H), 7.56–7.62 (m, 5H, Ar and pyr-H).
CHNS analysis: Calcd. For (C13H9BrN4O): C, 49.23; H,2.86; N,17.67. Observed: C,49.20; H,2.76; N,17.75.
Light brown crystalline powder, yield (80%), m.p (242–245 °C), Rf = 0.66, ATR-FTIR (ύ, cm-1): 3259 (NH) str. of sec. amine, 3078 Ar(CH) str, 1620 (NH) bend, 1589,1558 (C=N) str, 1504,1458,1438 Ar(C=C) str, 1292 (C-O-C) str. of oxadiazole, 1249 (C-N) str, 829 (Ar-p-substitution), 786,736 (CH) bend. of heterocyclic, 717 (C-F) str.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.89 (s, 1H, NH), 8.73 (d, 1H, J=4.9 Hz, pyr-H), 8.10 (d, 1H, J=8.2 Hz, pyr-H), 8.02 (t, 1H, pyr-H), 7.66–7.63 (d, 2H, Ar-H), 7.57 (dd, 1H, J=5.5, 6.6 Hz, pyr-H), 7.24 (t, 2H, J=8.8 Hz, Ar-H).
CHNS analysis: Calcd. For (C13H9FN4O): C,60.94; H,3.54; N,21.87. Observed: C, 60.99; H, 3.37; N, 21.64.
Faint pink crystalline powder, yield (85%), m.p (219–222 °C), Rf = 0.46, ATR-FTIR (ύ, cm-1): 3263 (N-H) str. of sec.amine, 3062 Ar(CH) str, 2916,2858 (CH) str. of aliph.(CH3), 1608 (NH) bend, 1581,1558 (C=N) str, 1543, 1516,1458 Ar(C=C) str, 1438,1345 (CH). bend. of (CH3), 1292(C-O-C) str. of oxadiazole, 1249 (C-N) str, 817 (Ar-p-substitution), 767,721 (CH) bend. of heterocyclic.
1H NMR (500 MHz, DMSO-d6, δ=ppm): 10.72 (br s, 1H, NH), 8.72 (d, 1H, J=4.4 Hz, pyr-H), 8.09 (d, 1H, J=8.2 Hz, pyr-H), 8.00 (t, 1H, J=7.7 Hz, pyr-H), 7.56–7.51 (m, 3H, Ar and pyr-H), 7.18 (d, 2H, J=8.2 Hz, Ar-H), 2.27 (s, 3H, CH3).
CHNS analysis: Calcd. For (C14H12N4O): C,66.65; H,4.79; N,22.21. Observed: C,66.53; H,5.02; N,22.25.
The synthesis of the target compounds (4–7) and their intermediates is depicted in (Fig.
Human A549 lung cancer cell line was cultured in Ham’s F-12K (Kaighn’s) medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Capricorn Scientific, Germany), human MCF-7 breast cancer cells were cultured in RPMI-1640 medium (Euroclone, Italy) supplemented with 10% FBS, the mammary non-tumorigenic MCF10A cell line was cultured in DMEM/F12 (Gibco, USA) supplemented with 5% horse serum (Invitrogen, USA), EGF (20 ng/mL, Sigma), hydrocortisone (0.5 mg/mL, Sigma), cholera toxin (100 ng/mL, Sigma), and insulin (10 µg/mL, Sigma), and human normal white blood cells (WBC) were isolated from peripheral blood after lysis of erythrocytes with ammonium chloride (
Cytotoxicity of the different picolinic acid derivatives (4–7) against different cell lines (A549, MCF-7 and MCF10A), and WBC was assessed using the MTT (Tetrazolium Salt Reduction) test (
Apoptosis was recorded by visual analysis of apoptotic nuclei after staining with DAPI. Cells were plated on sterile glass cover slips for 24 h. Thereafter, the cells were treated with the various compound(s) at the GI50 concentration. Cells were fixed with 4% paraformaldehyde (Sigma, USA) in phosphate buffered saline (PBS) for 45 min. Cells were then washed 3 times 3 min each with PBS. Cells were then mounted with Prolong Gold antifade containing DAPI (Invitrogen). Images were captured using 100× NA 1.3 objective on Nikon Eclipse Ti-E microscope with a CCD camera and operated by NIS-Elements software. Furthermore, DNA fragmentation was analyzed by agarose gel electrophoresis. Briefly, DNA from control and treated cells was extracted as per the manufacturer instructions (Wizard Genomic DNA Purification, Promega). The DNA samples were electrophoresed on a 1.5% agarose gel containing 5 μl/100 mL RedSafe nucleic acid staining solution (iNtRON Biotechnology, South Korea). The gel was examined and photographed using ultraviolet gel documentation system (FluorChem R System, Oxford, UK).
The quantity of caspase 3, caspase 4, caspase 8, caspase 9 mRNA was determined by qRT-PCR (
Control and GI50-treated cells were washed 3 times with cold PBS, and then lysed with protein lysis buffer [10% sodium dodecyl sulfate (SDS), 1 M Tris buffer pH 7.5, 1 M sodium floride (NaF), 1M dithiothreitol (DTT), 0.1 M ethylene glycol tetra-acetic acid (EGTA) and distilled water] on ice. The cell lysate was collected and boiled for 5 min and sonicated. Aliquots of lysates were diluted in 4× SDS-PAGE sample buffer (0.5 M Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 20% 2-mercaptoethanol and 0.16% bromophenol blue) and proteins were resolved by electrophoresis on 10% or 12.5% SDS-polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes and were blocked using 2% (w/v) BSA in Tris-buffered saline (TBS), and incubated overnight at 4 °C with the different primary antibodies: mouse monoclonal anti-caspase 3 (1:500; Invitrogen, USA), mouse monoclonal anti-caspase 9 (1:1000; Invitrogen, USA), rabbit polyclonal anti-caspase 4 (1:1000; Invitrogen, USA), mouse monoclonal anti-caspase 8 (1:1000; Invitrogen, USA), rabbit polyclonal anti-smac/DIABLO (1:1000; R&D Systems, Germany), mouse GAPDH (1:6000; CHEMICON, USA), rabbit monoclonal phospho-eukaryotic initiation factor-2 (p-EIF-2) (1:2000; Cell Signaling Technology, USA) rabbit polyclonal anti-tubulin (1:3000; abcam, USA), diluted in 1% BSA in TBS containing 0.05% Tween 20. After washing and incubation with appropriate secondary antibodies conjugated to IRDye 680 or 800 nm fluorescent dyes, the membranes were washed, and the bands were visualized on FluorChem R system (Oxford, UK). Signals were quantified using AlphaView software (ProteinSimple, USA).
5×107 control and GI50-treated cells were collected by centrifugation and washed with ice cold PBS at 2,900 rpm for 5 min and re-suspended with 1X cytosolic extraction buffer mix containing dithiothreitol (DTT) and protease inhibitors. The cells were incubated on ice for 10 min, homogenized and centrifuged at 3,100 rpm for 10 min. The supernatant was centrifuged again at 12,000 rpm for 30 min to obtain the cytosolic fraction, while the pellet was resuspended in mitochondrial extraction buffer to get mitochondrial fraction (Cytochrome c Release Apoptosis Assay Kit, Abcam, USA).
The EGFR(T790M/L858R) Kinase Assay Kit (BPS Bioscience, USA) was used to assess the inhibitory activity of the cytotoxic compound(s) against EGFR kinase. The reaction process was halted by addition of detection reagent (Kinase-Glo Max reagent, Promega). The remaining activity of EGFR kinase was detected by measuring chemiluminescence. The effective concentration that inhibits 50% of EGFR kinase activity (EC50) was calculated from the compounds’ concentration versus EGFR kinase remaining activity curve. Each experiment was performed in duplicates.
Results are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). The student’s t-test was used to test for significant differences between means. p value≤0.05 was considered significant.
In order to identify the molecular targets, the newly synthesized compounds (4–7) would eventually work on, and in order to compare them with several other ligands, and to ascertain the pharmacophoric functionality that would allow binding with critical amino acid(s) in a target site, target site selection was done by (https://www.rcsb.org/) protein data bank. The tested compounds were practically examined against a large number of binding sites, and the results were used to identify relevant proteins for docking studies. After selecting a protein for the target location, several processes were put forth to gain an understanding of the molecular binding modes of the tested compounds within the pocket of the epidermal growth factor receptor tyrosine kinase (ATP binding site of EGFR kinase) using the MOE 2015 Software. The binding sites were generated by co-crystallizing the ligand within the crystal protein (PDB code: 1M17) (
The cytotoxicity of 13 different picolinic acid derivatives (4, 5, 5A, 5B, 5C, 6, 6A, 6B, 6C, 7, 7A, 7B, and 7C) against two human cancer cell lines, A549 and MCF-7 was explored. According to the findings, compound 5 was the only one demonstrating cytotoxicity against A549 lung cancer cells (GI50 = 35.1 µg/mL (99.93 µM) after 48 h of treatment). None of the remaining compounds were effective against the A549 lung carcinoma cells, most of them showed an average of 25% maximum growth inhibition after 72 h of treatment with the highest concentration (50 µg/mL), while compounds 4, 5C, and 6 showed an average of 43% growth inhibition. However, none of the different compounds exhibited cytotoxic activities against MCF7 breast cancer cells, with the majority of the compounds showing an average of 25% maximum growth inhibition after 72 h of treatment with the highest concentration (50 µg/mL), while compounds 4 and 5 showed an average of 42% growth inhibition.
The cytotoxicity of compound 5 was tested against two non-cancer cell types, the non-tumorigenic mammary MCF10A cells, and WBC (Fig.
The results presented in Fig.
Compound 5 induces apoptosis in A549 lung cancer cells. A) Fluorescence images of A549 lung cancer cells showing fragmented nuclei after DAPI staining. Scale bar: 10 µm, B) Detection of DNA fragmentation by agarose gel electrophoresis. Cells were treated and genomic DNA was extracted and electrophoresed on 1.5% agarose gels. M: DNA molecular weight marker; C: vehicle-treated control cells; 1%: 1% DMSO-treated cells; 5%: 5% DMSO-treated cells; C.5: Compound 5-treated cells. Arrows indicate DNA fragments. Three experiments were performed with similar results.
To investigate the role of caspase activation in inducing the apoptotic cell death by compound 5, the mRNA levels of caspase 3, caspase 4, caspase 8 and caspase 9 were evaluated by qRT-PCR (Fig.
Compound 5 causes caspase activation. A) qRT-PCR analysis of caspase 3, 4, 8 and 9 mRNA in compound 5-treated cells (GI50) as compared to vehicle-treated control cells [set as 1 arbitrary unit (a.u.)]. Values were normalized to β-actin. Scale bars: mean ± SEM of three independent experiments performed in triplicates. *p < 0.05, **p < 0.01 compared to vehicle-treated control cells, B) Representative Western blots showing cleavage “activation’ of procaspase 3 to the active form p17, procaspase 4 to the active form p20, and procaspase 9 to the active forms p35/p37. C: vehicle-treated control cells; 0.5%: 0.5% DMSO-treated cells; 1%: 1% DMSO-treated cells; 5%: 5% DMSO-treated cells; C.5: Compound 5-treated cells. The experiment was repeated three times.
Since compound 5 activated caspases 3, 4 and 9 (Fig.
Compound 5 induces the ER-mediated apoptosis. A) Representative Western blot showing the enhanced release of Smac/DIABLO but not cytochrome c from the mitochondria (MF) into the cytosol (CF) of compound 5-treated A549 lung cancer cells (GI50). Equal protein loading was controlled by staining membranes with Ponceau S (a representative section of the stained membrane is shown). The experiment was repeated three times and the corresponding quantification is shown in (B), B) Quantification of smac/DIABLO and cytochrome C levels in vehicle-treated control A549 cells and cells treated with GI50 amount of compound 5 or 5% DMSO as a negative control. M: mitochondria fraction; C: cytosol fraction. Scale bars: mean ± SEM of three independent experiments. ** p < 0.01 compared to vehicle-treated control cells, C) Representative Western blot showing the induction of phosphorylation of eukaryotic initiation factor-2 (eIF-2) in A549 cells treated with GI50 amounts of compound 5. For comparison purposes, thapsigargin (TG), an ER stress-causing drug was used (3 μM, 2 h) as a positive control. The experiment was repeated three times.
The CDOCKER protocol was used for the molecular docking procedure. CDOCKER is a grid-based molecular docking method that docks ligands into receptor binding sites that used a CHARMM-based molecular dynamics (MD) scheme. Mostly during refinement, the receptor was kept rigid, while the ligands were allowed to be flexible. Each molecule was given the opportunity to take seven distinct interactions with the protein. The docking scores (-CDOCKER interaction energy) of the best-fitting poses with the active site, the EGFR kinase’s ATP binding site, was recorded (Table
Docking scores (∆G, kcal/mol) of the tested compounds against (EGFR Kinase) target site PDB ID: 1M17.
Comp. | Bonds NO. | Score (∆G) kcal/mol | RMSD value/ ⁰A | E-place | |
---|---|---|---|---|---|
H.B | pi | ||||
4 | 1 | – | -5.49 | 2.18 | -63.43 |
5 | 1 | – | -5.90 | 1.69 | -70.81 |
5A | 1 | 2 | -5.52 | 2.14 | -51.75 |
5B | 1 | 2 | -5.40 | 1.74 | -62.23 |
5C | 2 | 1 | -5.74 | 2.58 | -64.46 |
6 | 1 | 1 | -5.45 | 3.08 | -70.28 |
6A | 1 | 1 | -5.89 | 2.24 | -69.22 |
6B | 1 | – | -6.06 | 1.44 | -61.70 |
6C | 2 | – | -5.96 | 1.23 | -57.89 |
7 | 1 | 1 | -5.06 | 3.34 | -59.31 |
7A | 1 | 1 | -6.05 | 2.04 | -64.00 |
7B | 1 | 2 | -5.07 | 3.13 | -58.98 |
7C | – | 1 | -5.68 | 1.74 | -52.84 |
Erlotinib | 1 | 1 | -5.90 | 2.36 | -80.69 |
For EGFR tyrosine kinase, the key binding site consists of the amino acids Asp776, Thr766, leu694, Cys773, Gly772, and Val702 (
The higher the score of ΔG the greater the affinity of the tested compound for EGFR binding site, which results in higher toxicity towards cancer cells (including the low EGFR-expressing MCF-7 cells) compared to healthy cells which express low levels of EGFR (
Although molecular docking studies have several shortcomings such as choosing an inaccurate binding site of the target protein, screening using unsuitable database, or inconsistency between docking results and in vitro experimental results (the current work is a good example), it helps researchers in solving the problem of choosing samples for in vitro protein-ligand experiments (
The physical characters of drugs play a key role in their molecular activity. One of these parameters is the calculated partition coefficient (Clog p) which predicts drug movement patterns in the human body. All of the target compounds have (Clog p) less than<5 and are in clear violation of Lipinski rule of five. The topological polar surface area (TPSA) of a compound indicates the surface belonging to polar atoms in the compound. An increased TPSA is associated with lower membrane permeability, and compounds with higher TPSA seemed to be better p-glycoprotein substrates (responsible for drug efflux from cell). Once comparing the compounds, lower TPSA reported to be much more desirable for drug-like property (Table
Comp. | Clog p | n. H-bond acceptors | n. H-bond donors | n. rotatable bonds | n. violation | TPSA |
---|---|---|---|---|---|---|
4 | 0.92 | 4 | 3 | 7 | 0 | 143.96 |
5 | 2.25 | 2 | 3 | 6 | 0 | 98.14 |
5A | 3.25 | 2 | 1 | 2 | 0 | 78.59 |
5B | 3.38 | 3 | 1 | 3 | 0 | 78.94 |
5C | 2.85 | 4 | 1 | 3 | 0 | 63.84 |
6 | 1.95 | 2 | 3 | 6 | 0 | 98.14 |
6A | 2.93 | 3 | 1 | 2 | 0 | 78.59 |
6B | 3.06 | 4 | 1 | 3 | 0 | 78.94 |
6C | 2.54 | 5 | 1 | 3 | 0 | 63.84 |
7 | 1.96 | 2 | 3 | 6 | 0 | 98.14 |
7A | 2.94 | 2 | 1 | 2 | 0 | 78.95 |
7B | 3.09 | 3 | 1 | 3 | 0 | 78.94 |
7C | 2.56 | 4 | 1 | 3 | 0 | 63.84 |
Erlotinib | 3.23 | 6 | 1 | 10 | 0 | 74.73 |
The online admetSAR chemformatics software was also used to investigate the ADMET features, and the profile of the synthesized compounds, in order to identify the potential and safer drug candidates, and to filter compounds that are the most likely to fail in subsequent stages of drug development due to unfavorable ADMET properties. The anticipated ADMET analysis of the target compounds is shown in (Table
Comp. | BBB | HIA | CYTP450 3A4 | CYP2D6 | CYP2C9 | PPB |
---|---|---|---|---|---|---|
4 | No | 40.5 | Non-inhibitor | Non-inhibitor | Non-inhibitor | 100% |
5 | No | 93.07 | Inhibitor | Non-inhibitor | Inhibitor | 100% |
5A | No | 96.7 | Non-inhibitor | Non-inhibitor | Inhibitor | 91.8% |
5B | No | 96.3 | Inhibitor | Inhibitor | Inhibitor | 90.3% |
5C | Yes | 95.92 | Non-inhibitor | Non-inhibitor | Non-inhibitor | 91.8% |
6 | No | 91.63 | Non-inhibitor | Non-inhibitor | Non-inhibitor | 100% |
6A | No | 96.8 | Non-inhibitor | Non-inhibitor | Non-inhibitor | 87.8% |
6B | No | 96.6 | Inhibitor | Inhibitor | Non-inhibitor | 86.3% |
6C | Yes | 95.7 | Non-inhibitor | Non-inhibitor | Non-inhibitor | 80.4% |
7 | No | 91.9 | Non-inhibitor | Non-inhibitor | Non-inhibitor | 99.0% |
7A | No | 96.80 | Non-inhibitor | Non-inhibitor | Inhibitor | 92.5% |
7B | No | 96.6 | Inhibitor | Inhibitor | Inhibitor | 91.1% |
7C | Yes | 95.8 | Inhibitor | Non-inhibitor | Non-inhibitor | 96.5% |
Erlotinib | Yes | 96.04 | Inhibitor | Inhibitor | Inhibitor | 93.1% |
The target compounds were reported to have better human intestinal absorption, except for compound 4 that possessed HIA value of 40.5 (
Comp. | Ames test | Carcinogen on mouse | Carcinogen on rat | HERG inhibitor | Carcinogenicity | TA100-NA |
---|---|---|---|---|---|---|
4 | Mutagen | Negative | Positive | Medium risk | 0 | Positive |
5 | Mutagen | Negative | Positive | Medium risk | 0 | positive |
5A | Mutagen | Positive | Positive | Medium risk | 0 | positive |
5B | Mutagen | Negative | positive | Medium risk | 1* | Negative |
5C | Mutagen | Positive | positive | Medium risk | 0 | Negative |
6 | Mutagen | Negative | positive | Medium risk | 0 | positive |
6A | Mutagen | Positive | positive | Medium risk | 0 | positive |
6B | Mutagen | Positive | positive | Medium risk | 1* | Negative |
6C | Mutagen | Positive | positive | Medium risk | 0 | Negative |
7 | Mutagen | Positive | Negative | Medium risk | 0 | Positive |
7A | Mutagen | Negative | Negative | Medium risk | 1* | Negative |
7B | Mutagen | Negative | Negative | Medium risk | 0 | Negative |
7C | Mutagen | Positive | Positive | Medium risk | 0 | Positive |
Erlotinib | Mutagen | Negative | Negative | Medium risk | 0 | Negative |
Novel picolinic derivatives (4–7) were synthesized and confirmed by spectroscopic analysis, including ATR-FTIR, 1HNMR, and CHNS. The MTT assay was used to assess their cytotoxic effects against two human cancer cell lines (A549 and MCF-7) as well as, non-tumorigenic MCF10A and WBC. Compound 5 exhibited anticancer activity against A549 lung cancer cells (IC50 = 99.93 µM), but still had no effect on MCF-7 breast cancer cells or normal cells. Compound 5 exerted cytotoxicity in A549 lung cancer cells by inducing apoptosis, as made evident by fragmented nuclei after DAPI staining and agarose gel electrophoresis. Furthermore, compound 5 activated caspases 3, 4, and 9. Moreover, when compared to vehicle-treated control cells, compound 5 treatment did not result in the release of cytochrome c from the mitochondria to the cytosol, however, it showed increased cytosolic Smac/DIABLO release. Finally, both compound 5 and thapsigargin increased eIF2 phosphorylation, proving activation of the ER stress pathway. A major problem in cancer treatment is chemoresistance, thus the search for new bioactive compounds that induce cancer cell killing in new and atypical mechanisms that avoid chemoresistance is of great interest. The current work demonstrated that the new derivative of picolinic acid, compound 5, modulates ER stress to induce cancer cell death demonstrating its applicability in cancer treatment.
The authors sincerely acknowledge the support of Department of Pharmaceutical Chemistry, College of Pharmacy-University of Baghdad.
Conflicts of interest: The authors declare no conflicts of interest.