Research Article |
Corresponding author: Hiba Alsaad ( hiba.jassem@uobasrah.edu.iq ) Academic editor: Plamen Peikov
© 2022 Hiba Alsaad, Ammar Kubba, Lubna H. Tahtamouni, Ali H. Hamzah.
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:
Alsaad H, Kubba A, Tahtamouni LH, Hamzah AH (2022) Synthesis, docking study, and structure activity relationship of novel anti-tumor 1, 2, 4 triazole derivatives incorporating 2-(2, 3- dimethyl aminobenzoic acid) moiety. Pharmacia 69(2): 415-428. https://doi.org/10.3897/pharmacia.69.e83158
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A series of 1,2,4 triazole derivatives (H7-12) have been synthesized by reacting an excess of hydrazine hydrate with carbothioamide derivatives (H1-6). The final compounds (HB1-HB6) were synthesized by reacting the triazole derivatives with mefenamic acid using DCC as a coupling agent. The chemical structures were confirmed by FT-IR, 1H, and 13C-NMR spectra, and some physicochemical properties were determined. The cytotoxicity of the different compounds (HB1-HB6) was evaluated by the MTT assay against two human epithelial cancer cell lines, A549 lung carcinoma and Hep G2 hepatocyte carcinoma, and one normal human cell line WI-38 lung fibroblasts. The mode of cell killing (apoptosis versus necrosis), as well as the effect on cell cycle phases were evaluated via flow cytometry. Additionally, EGFR tyrosine kinase inhibition assay was performed. The results presented in the current study indicate that the six tested compounds exhibited cytotoxicity against both cancer cell lines, and the lowest IC50 was achieved with compound HB5 against Hep G2 cancer cells which was found to be highly selective against cancer cells. HB5-treated Hep G2 cells were arrested at the S and G2/M cell cycle phases. Compound HB5 caused cell killing via apoptosis rather than necrosis, and this was achieved by inhibiting EGFR tyrosine kinase activity needed for cell proliferation, and cell cycle progression. In silico pre-ADMET studies confirmed all final compounds don’t cause CNS side effects, with little liver dysfunction effect.
1,2,4 triazole, MTT assay, apoptosis, EGFR tyrosine kinase activity, pre-ADMET
Cancer is one of the major global health burdens representing the second major cause of death worldwide (
Epidermal growth factor has a role in cell growth stimulation and differentiation through binding to its receptor, the epidermal growth factor receptor (EGFR). EGFR is a transmembrane protein belonging to the ErbB family of receptors. The ErbB is a subfamily of tyrosine kinase receptors including EGFR (ErbB-1), HER2/neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). EGFR is often upregulated in several cancers such as breast cancer, non‐small‐cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), and glioblastoma (Sigismund, Avanzato and Lanzetti 2018a;
Heterocyclic compounds are interesting structures for medicinal chemists due to their important chemical and biological properties. Although many heterocyclic compounds have been developed, efforts are still ongoing to produce a new heterocyclic ring system with important biological activity (
The ability of 1,2,4 triazole derivatives to form different types of non-covalent interactions such as hydrogen bonds, van der Waals forces, hydrophobic interactions, and dipole-dipole bonds with various biological targets, is responsible for diverse therapeutics properties including their antibacterial (
Additionally, one of the important medications based on triazole nucleus which possess anti-proliferative activity against different types of cancer are the non-steroidal anti-inflammatory drugs (NSAIDs) (
In this regard, the objective of this study was to design and synthesize some novel 1, 2, 4-triazole derivatives incorporating 2-(2, 3- dimethyl aminobenzoic acid) moiety. Different electron withdrawal and donating groups at the p-position of benzene moieties were chosen to improve the physicochemical properties and cytotoxicity of the new synthetic compounds.
The chemical reagents and solvents were used without further purification from Sigma-Aldrich, Milano, Italy and Merck, Taufirchen, Germany. Mefenamic acid was purchased from Pioneer Company (Erbil, Iraq). Infrared spectra were measured using a Shimadzu model 8400s (Nakagyo, Japan) spectrophotometer on disk of KBr, (v = cm-1). Elemental microanalysis (CHNS) was done using a Euro EA300 elemental analyzer (Carlo Erba, Emmendingen, Germany). The proton (1H) and carbon (13C) NMR spectra were measured using the Inova model Ultra shield at 500, and 125 MHz respectively, δ= ppm was used to express the chemical shift. The solvent used was DMSOd6.
The intermediates and final compounds were synthesized according to Scheme 1.
Few drops of conc. H2SO4 were added to 10 mmol of benzoic acid (1) or p-nitro benzoic acid (2) in 30 mL MeOH, the mixture was then refluxed for 3h. The product was formed after neutralization of the reaction mixture with Na2CO3. Compound (3) was obtained by extracting the aqueous layer with chloroform, and dried using anhyd. MgSO4. While compound (4) was filtrated after precipitation and recrystallized with EtOH. The physical properties are mentioned in Table
Compound | Appearance | Yield % | M.p.\ b.p οC | * Rf |
---|---|---|---|---|
3 | Oily liquid | 90 | 199 | 0.8 |
4 | Pale- yellow | 92 | 94-95 | 0.83 |
5 | White crystal | 85 | 120-121 | 0.49 |
6 | Yellow crystal | 88 | 142-143 | 0.41 |
H1 | White powder | 78 | 148-149 | 0.35 |
H2 | White powder | 79 | 166-168 | 0.36 |
H3 | White powder | 84 | 160-161 | 0.33 |
H4 | Yellow powder | 72 | 168-169 | 0.33 |
H5 | Yellow powder | 80 | 180-181 | 0.32 |
H6 | Yellow powder | 75 | 175-177 | 0.34 |
H7 | Red powder | 62 | 160-161 | 0.54 |
H8 | Brown powder | 50 | 178-179 | 0.56 |
H9 | Orange powder | 70 | 185-186 | 0.53 |
H10 | Brown powder | 63 | 190-191 | 0.6 |
H11 | Brown powder | 64 | 202-203 | 0.62 |
H12 | Orange powder | 67 | 193-194 | 0.61 |
HB1 | Dark-orange powder | 47 | 206-207 | 0.83 |
HB2 | Dark-brown powder | 52 | 211-212 | 0.85 |
HB3 | Brown powder | 48 | 218-219 | 0.83 |
HB4 | Brown powder | 56 | 220-221 | 0.92 |
HB5 | Dark-red powder | 50 | 230-231 | 0.95 |
HB6 | Dark-brown powder | 43 | 225-226 | 0.94 |
Methyl benzoate (3): FT-IR(v = cm-1): 1724 (C=O) of ester.
Methyl 4-nitrobenzoate (4): FT-IR: 1731(v = cm-1): (C=O) of ester.
To 10 mmol of compound (3) or (4) in 15mL EtOH, 30 mmol of hydrazine hydrate was added. The mixture was refluxed for 4h, the solvent was then reduced using a rotary evaporator, and the residual was added to ice water. The formed precipitate was filtered, and recrystallized from EtOH (
N-(aminomethyl) benzamide (5): FT-IR(v=cm-1) :3501.11 and 3456.81 (-NH) str. of NH2; 3344.47 (-NH) str. of (CONH); 1660 (C=O) str. of amide.
N-(aminomethyl)-4-nitrobenzamide (6): FT-IR(v=cm-1): 3380.66 and 3290.27 (-NH) str. ofNH2; 3130.69 (-NH) str. of (CONH); 1683.2 (C=O) str. of amide.
To 5 mmol of compound (5) or (6) in 10 mL MeOH, 5 mmol of different aryl isothiocyanate derivatives in 5 mL MeOH was added. The reaction was stirred under 50 οC for 1h. The precipitate was filtered and washed with hot MeOH (
2-benzoyl-N-phenylhydrazine-1-carbothioamide (H1): FT-IR(v=cm-1): 3233.16 br. (-NH) str. of (CONHNHCSNH); 3171.65 str. of (Ar-CH).; 1690.11 str. of (C=O) amide; 1566.82, 1507.22 and 1450.69 str of (Ar-C=C); 1189.5 str. of (C=S)
2-benzoyl-N-(4-chlorophenyl)hydrazine-1-carbothioamide (H2): FT-IR(v=cm-1): 3262.91 br. (-NH) str. of (CONHNHCSNH); 1683.56 str. of (C=O) amide; 1181.14 str. of (C=S); 1088.46 str. of (C-Cl).
2-benzoyl-N-(4-methoxyphenyl)hydrazine-1-carbothioamide (H3): FT-IR(v=cm-1): 3263.81 br. str. (-NH) of (CONHNHCSNH); 3144.91 str. of (Ar -CH); 1679.98 (C=O) str. of amide; 1542.25, 1522.5 and 1452.11 str. of (Ar-C=C); 1244 asym str. of (C-O); 1182.55 str. of (C=S); 1027 sym str. of (C-O).
2-(4-nitrobenzoyl)-N-phenylhydrazine-1-carbothioamide (H4): FT-IR((v=cm-1): 3237.2 br. str. (-NH) of (CONHNHCSNH); 1686.31(C=O) str. of amide; 1546.22, 1511.24 and 1453.61 str. of (Ar-C=C); 1182.3 str. of (C=S).
N-(4-chlorophenyl)-2-(4-nitrobenzoyl)hydrazine-1-carbothioamide(H5): FT-IR(v=cm-1): 3265.33 br. str. (-NH) of (CONHNHCSNH); 1690.76 (C=O) str. of amide; 1177.42 str. of (C=S); 1087.56 str. of (C-Cl).
N-(4-methoxyphenyl)-2-(4-nitrobenzoyl)hydrazine-1-carbothioamide(H6):FT-IR (v=cm-1): 3271.55 br. str. (-NH) of (CONHNHCSNH); 1684.58 (C=O) str. of amide; 1566.35; 1511.64 and 1455.17 Ar(C=C) str.; 1245.22 asym str. of (C-O); 1185.55 str. of (C=S); 1029 sym str. of (C-O).
2 mmol of compounds (H1-6) was suspended in 10 mL of MeOH, and 10 mmol of hydrazine hydrate was added. A red mixture was formed and refluxed for 20h. The solvent was reduced with a rotary evaporator. 5 mL of Et2O were added to the residual and kept for 24h at a cold place. The precipitate was washed repeatedly with diethyl ether, and dried overnight at 25 οC (
N3,5- diphenyl-4H-1,2,4- triazole-3,4- diamine (H7): FT-IR(v=cm-1): 3390.11 and 3340.01 (NH) str. of 1ο amine; 3329.61 str. of (-NH) of sec. amine; 1680.58 (C=N) str. of imine.
N3- (4-chlorophenyl)-5-phenyl-4H-1,2,4- triazole-3,4- diamine (H8) : FT-IR(v=cm-1): 3385.78 and 3347.13 str. (NH) of 1ο amine; 3325.62 str. (-NH) of sec. amine; 1685.35 (C=N) str.of imine.
N3- (4-methoxyphenyl)-5-phenyl-4H-1,2,4- triazole-3,4- diamine (H9): FT-IR(v=cm-1): 3400 and 3359.2 str. (NH) of 1ο amine; 3333.53 str. (NH) of sec. amine; 1688.34 (C=N) str. of imine.
5- (4-nitrophenyl)-N3-phenyl-4H-1,2,4- triazole-3,4-diamine (H10): FT-IR(v=cm-1): 3393.45 and 3352.9 str. (NH) of 1ο amine; 3311.71 str. (NH) of sec. amine; 1672.71 (C=N) str. of imine; 1542.16 (N-O) asym str. of NO2, 1376 (N-O) sym str. of (NO2).
N3- (4-chlorophenyl)-5-(4-nitrophenyl)-4H-1,2,4- triazole-3,4-diamine (H11) : FT-IR(v=cm-1): 3420.23 and 3372 str. (NH) of 1ο amine; 3304.15 str. (NH) of sec. amine; 1690.21 (C=N) str. of imine; 1542.61 (N-O) asym str. of NO2, 1379 (N-O) sym str. of (NO2).
N3- (4-methoxyphenyl)-5-(4-nitrophenyl)-4H-1,2,4- triazole-3,4-diamine (H12): FT-IR(v=cm-1): 3411 and 3362 str. (NH) of 1ο amine; 3332.65 str. (NH) of sec. amine; 1683.71 str. (C=N) of imine; 1543.11 (N-O) asym str. of NO2, 1380 (N-O) sym str. of (NO2).
10 mmol of mefenamic acid was dissolved in (5mL dioxan and 10 mL THF) on ice bath, to this solution, 10 mmol of DMAP, and 10 mmol of the corresponding compounds (H7-12) were added, respectively. 10 mmol of N, N-- dicyclohexylcarbodiimide (DCC) in 5 mL of dichloromethane CH2Cl2 was added. The mixture was stirred at 0 οC for 3 days. The product was filtered to remove DCU, and the solvent was reduced using a rotary evaporator. EtOAc (5mL) was added to the residual, and the mixture was washed with 10% HCl, 5%NaHCO3, and H2O, respectively. The organic layer was dried with anhyd. MgSO4, and left to dry for 24h (
2-((2,3-dimethylphenyl)amino)-N-(3-phenyl-5-(phenylamino)-4H-1,2,4-triazol-4-yl)benzamide (HB1): FT-IR(v=cm-1): 3303.3 (NH) str; 1680.4 (C=O) str, 1665 (C=N). 1 HNMR(500MHz,DMSOd6,δ=ppm) : 2.1 (s, 3H, CH3), 2.3 (s, 3H, CH3), 3.7(s, 2H, aliph. NH), 6.0–8.2 (m, 17H, Ar.CH), 8.5 (s, 1H, NH-C=O). 13C-NMR(125MHz,DMSOd6,δ=ppm); 18.9 and 20.2 C(methyl); 115.4, 118.2, 118.8, 121.3, 125.5, 126.1, 126.9, 127.3, 127.7, 128.5, 129.0, 129.4, 130.0, 131.4, 132.6, 133.5, 138.2, 138.8, 139.3, 145.2,C(Aromatic), 155.03 and 157 C(Traiazole), 180 C(carbonyl). CHN analysis: Calcd, for (C29H26N6O), MW: 473.22, C: 73.40; H: 5.52; N: 17.71. Observed C: 73.37; H: 5.53; N: 17.70.
N-(3-((4-chlorophenyl)amino)-5-phenyl-4H-1,2,4-triazol-4-yl)-2-((2,3-dimethylphenyl)amino)benzamide (HB2): FT-IR(v=cm-1): 3320.81 (N-H) str; 1681.61 (C=O) str; 1660.23 (C=N) str. 1HNMR(500MHz,DMSOd6,δ=ppm): (2.4 (s, 3H, CH3), 2.7 (s, 3H, CH3), 4.2(s, 2H, aliph. NH), 6.4–7.9(m, 16H, Ar. CH), 8.8 (s, 1H, NH-C=O). 13C-NMR(125MHz,DMSOd6,δ=ppm): 19.2 and 20.3 C(methyl), 115.4, 118.1, 118.8, 121.3, 125.5, 126.3, 126.8, 127.3, 127.7, 128.1, 128.7, 129.0, 130.0, 131.3, 132.5, 133.5, 138.2, 138.8, 139.3, 145.2 C(Aromatic), 155.2 and 156.9 C(Traiazole), 181.2 C(carbonyl). CHN analysis: Calcd. for (C29H25ClN6O): MW: 509.01, C: 68.43; H: 4.95; Cl: 6.96; N: 16.51. Observed: C: 68.44; H: 4.93; CI: 6.98; N: 16.52.
2-((2,3-dimethylphenyl)amino)-N-(3-((4-methoxyphenyl)amino)-5-phenyl-4H-1,2,4-triazol-4-yl)benzamide (HB3): FT-IR(v=cm-1): 3326.62 (NH), 2874.99, and 2825.51 (C-H) aliph. str , 1688.22 (C=O); 1654.65 (C=N). 1 HNMR 500MHz, DMSOd6, δ=ppm): 2.3 (s, 3H, CH3), 2.7 (s, 3H, CH3), 3.5(s, 3H, -OCH3), 4.1(s, 2H, aliph. NH), 6.5–8.2(m, 16H, Ar. CH), 8.4 (s, 1H, NH-C=O). 13CNMR(125MHz,DMSOd6,δ=ppm): 19.2 and 20.3 C(methyl); 62.9 C(methoxy) 115.1, 115.8, 118.1, 118.7, 120.3, 121.3, 126.1, 126.9, 127.3, 128.5, 129.0, 129.6, 130.1, 131.4, 132.6, 133.7, 138.1, 138.8, 139.3, 145.2 C(Aromatic);152.5 and 157.1 C(Traiazole),181.2 C(carbonyl). Elemental analysis: Calcd.for (C30H28N6O2): MW: 504.59, C: 71.41; H: 5.59; N: 16.66. Observed C: 71.4; H: 5.58; N: 16.68.
2-((2,3-dimethylphenyl)amino)-N-(3-(4-nitrophenyl)-5-(phenylamino)-4H-1,2,4-triazol-4-yl)benzamide(HB4): FT-IR(v=cm-1): 3301.54 (NH); 2860.05, and 2843.12 (C-H) aliph. str, 1669.88 (C=O), 1645.76 (C=N). 1HNMR(500MHz,DMSOd6,δ=ppm): 2.0 (s, 3H, CH3), 2.2 (s, 3H, CH3), 4.1(s, 2H, aliph. NH), 6.9–8.5(m, 16H, Ar. CH), 8.3 (s, 1H, NH-C=O). 13CNMR(125MHz,DMSOd6,δ=ppm): 19.3 and 20.4 C(methyl); 114.2, 118.2, 118.7, 121.9, 124.8, 126.1, 126.7, 127.4, 127.6, 128.5, 129.0, 129.7, 131.4, 132.5, 133.6, 138.2, 138.8, 139.5, 145.7, 149.3C(Aromatic); 152.0 and 157.0 C(Traiazole), 182.0 C(carbonyl). Elemental analysis: Calcd. for (C29H25N7O3): MW: 519.57, C: 67.04; H: 4.85; N: 18.87. Observed: C: 67.01; H: 4.85; N:18.89.
N-(3-((4-chlorophenyl)amino)-5-(4-nitrophenyl)-4H-1,2,4-triazol-4-yl)-2-((2,3-dimethylphenyl)amino)benzamide (HB5): FT-IR(v=cm-1): 3301.99 (NH); 2889.27, and 2853.45 (C-H) aliph. str 1686.2 (C=O) str, 1666.3 (C=N). 1HNMR: 2.0 (s, 3H,CH3), 2.5 (s, 3H, CH3), 3.5(s, 2H, aliph. NH), 6.6–8.8(m, 15H, Ar-CH), 8.7 (s, 1H, NH-C=O). 13CNMR(125MHz,DMSOd6,δ=ppm): 19.2 and 20.21 C(methyl), 114.3, 118.2, 118.9, 121.6, 125.8, 126.1, 126.8, 127.4, 127.9, 128.4, 129.3, 129.9, 131.3, 132.4, 133.5, 133.9, 138.8, 139.7, 146.3, 149.5 C(Aromatic); 152.0 and 155.0 C(Traiazole), 183.0 C(carbonyl). Elemental analysis: calcd. for (C29H24ClN7O3): MW: 554.01, C: 62.87; H: 4.37; Cl: 6.40; N: 17.70. Observed: C: 62.87; H: 4.34; CI: 6.42; N: 17.68.
2-((2,3-dimethylphenyl)amino)-N-(3-((4-methoxyphenyl)amino)-5-(4-nitrophenyl)-4H-1,2,4-triazol-4-yl)benzamide (HB6): FT-IR(v=cm-1): 3300.72 (NH) str, 1683.22 (C=O) str, 1660.1 (C=N) str. 1HNMR(500MHz,DMSOd6,δ=ppm): 2.3 (s, 3H, CH3), 2.4 (s, 3H, CH3), 3.5 (s, 3H, OCH3), 4.2(s, 2H, aliph. NH), 6.2–8.9(m, 15H, Ar-CH), 8.5 (s, 1H, NH-C=O). 13CNMR; 18.4 and 20.5 C(methyl); 61.4 C (methoxy); 115.3, 115.9, 118.1, 119.0, 120.2, 121.6,125.5, 126.1, 126.9, 127.7, 128.3, 129.1, 129.8, 131.3, 132.5, 133.6, 138.3, 139.8, 145.8, 149.5 C(Aromatic), 152 and 157 C(Traiazole), 181.9 C(carbony). Elemental analysis: calcd. for (C30H27N7O4): MW: 549.59, C: 65.56; H: 4.95; N: 17.84. Observed C: 65.55; H: 4.99; N: 17.84.
The chemical structure of the designed compounds was compared with crystal ligands to choose the molecular targets. A protein data bank (PDB) (https://www.rcsb.org/) was used to select the target site, additionally, the main requirements for binding with essential amino acids in the target site were determined.
Molecular Operating Environment 19.0901 software was used to predict the binding modes of the designed compounds inside target sites of EGFR tyrosine kinase. The ligand-binding sites were created from a co-crystallized structure within PDB (ID: 1M17) (https://www.rcsb.org).
Initially, water molecules were reduced from the complex. The clean protein options and protein report and utility were used to correct unfilled valence atoms and crystallographic disorders. The protein energy was reduced using MMFF94 force fields, while fixed atom constraint was applied to obtain the protein rigid binding site. 2D structures were generated using ChemBioDraw Ultra16.0 and saved in MDL-SD form. The 3D structures were protonated after opening in MOE, 0.05 RMSD kcal/mol was applied to minimize the energy, and the docking protocol was used for docking the minimized structures. CDOCKER protocol was used to accomplish the process of molecular docking. The ligands were permitted to be flexible while the receptor was held rigid. Thirty conformation poses were used in the placement process, scored by London DG, and the best 10 docking scores (DG) of fitted poses with the active site at EGFR tyrosine kinase (scored by GBVI/WSA) were used and 3D perspective was done by Discovery Studio 2019 Client software.
These methods were also used to anticipate the binding profile, ideal orientation of each docking pose, affinity, and binding free energy (∆G) of the prepared compounds with EGFR tyrosine kinase.
In-silico study ADMET along with drug-likeness prediction assist the drug discovery and development process. Here, Pre-ADME online software was utilized for estimating absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling of the tested compounds. These descriptors involve human gastrointestinal absorption, bioavailability, penetration to the blood-brain barrier, binding to plasma protein, inhibition of liver enzymes, and carcinogenesis. The drug-likeness of the derivatives were predicted following Lipinski’s Rule. The measured parameters were number of H-bond donors and acceptors, clog p, and Topological Polar Surface Area (TPSA). The study was done using Chem. Informatics on the web (http://www.molinspiration.com).
Human A549 lung cancer cell line was grown in Ham’s F-12K medium (Thermo Scientific, USA) supplemented with fetal bovine serum (10%) (FBS; Gibco, USA), human Hep G2 hepatocyte carcinoma cell line and human WI-38 cell line (lung fibroblasts) were grown in Eagle’s minimum essential medium (LONZA, Switzerland), to which 10% FBS was added. Trypsin-EDTA (Millipore-Merck, USA) was used during subcultures. Growth of cells was established at 37 °C in 5% CO2 and 95% air.
The MTT assay was used to evaluate the proliferation of control and treated cells (Mosmann 1983). 96-well plates were used throughout the experiment. 30,000 cells were added to each well containing the appropriate media and grown for 24 h. The stock solutions of each of the tested compounds were prepared in DMSO. Eight concentrations (100, 30, 10, 3, 1, 0.3, 0.1, and 0.03 µM) were prepared in the growth media for each compound and added to the cells for 48 h. MTT salt (Freshly prepared; 3-(4,5-dimethylthiazol-2yl)-2,5- diphenyltetrazolium bromide) (5 mg/ml; Sigma) was then added to each well to obtain 0.5 μg/μL as a final concentration. 200 μL of DMSO and isopropanol mixture 1:1 was added to each well and incubated for 30–45 min. Cell proliferation was detected by measuring the absorbance of each well at 590 nm using Multiskan EX (Thermo Scientific, USA) MicroPlate Reader. The experiment was performed three times in triplicates.
Hep G2 cells were treated with the IC50 concentration of drug HB5 (2.87 µM) for 48 h (see Table
Hep G2 cells were treated with the IC50 concentration of drug HB5 (2.87 µM) for 48 h. Apoptosis (early and late)/necrosis induction was detected in control and treated cells by phosphatidylserine translocation to the cell surface using annexin V-FITC apoptosis detection kit (Elabscience Biotechnology, USA).
The quantity of BAX, Bcl-2, p53, and caspase 3 mRNA in control and HB5 (at the IC50 concentration)-treated Hep G2 cells were assessed by qRT-PCR. Total RNA from vehicle-treated control (0.01% DMSO) and HB5-treated Hep G2 cells were extracted according to the manufacturer’s instructions (RNeasy mini kit, Qiagen, Germany). After RNA extraction, cDNA was prepared using the Revert Aid First Strand cDNA Synthesis kit (Thermo Scientific, USA). Amplification of target cDNA for apoptosis markers and GAPDH [as a normalization (housekeeping) gene] was done using one-step RT-PCR SYBR Green kit Master Mix (Bio‐Rad Laboratories, USA) on Rotor-Gene Q real-time PCR thermal cycler instrument. cDNA (2 μL aliquots) was mixed with forward primer (1 μL), reverse primer (1 μL) (Table
The sequence of qRT-PCR primers, forward (F) and reverse (R), used in the current study.
Gene | Primer sequence | Reference |
---|---|---|
p53 | F: 5’- GCCCAACAACACCAGCTCCT -3’ | ( |
R: 5’-CCTGGGCATCCTTGAGTTCC -3’ | ||
BAX | F: 5’- CCCGAGAGGTCTTTTTCCGAG -3’ | ( |
R: 5’- CCAGCCCATGATGGTTCTGAT -3’ | ||
Bcl-2 | F: 5’- TTGTGGCCTTCTTTGAGTTCGGTG -3’ R: 5’- GGTGCCGGTTCAGGTACTCAGTCA -3’ | ( |
Caspase-3 | F: 5’- ACATGGAAGCGAATCAATGGACTC -3’ R: 5’- AAGGACTCAAATTCTGTTGCCACC -3’ | (Peluffo, Young and Stouffer 2005) |
GAPDH | F: 5’- GACCCCTTCAT GACCTCAAC -3’ | |
R: 5’- CTTCTCCATGGTGGT GAAGA -3’ |
The in-vitro inhibitory activity of compound HB5 and erlotinib [standard receptor-tyrosine kinase inhibitor (TKI)] against EGFR tyrosine kinase was done using EGFR Kinase Assay Kit (BPS Bioscience, USA). Briefly, EGFR and its substrate were incubated either with HB5 or erlotinib (1000, 300, 100, 30, 10, 3, 1, and 0.3 nM) in the enzymatic buffer at 30 °C for 40 min to initiate the enzymatic reaction. Detection reagent (Kinase-Glo MAX; Promega, USA) was added to terminate the reaction, followed by incubation for 15 min at 25 °C. The remaining activity of EGFR tyrosine kinase was observed by measuring chemiluminescence using BioTek Synergy2 Microplate Reader (BioTek, USA). The concentration-percent remaining EGFR tyrosine kinase activity curve was used to calculate the concentration that caused 50% kinase activity inhibition (the effective concentration that inhibits 50% of EGFR kinase activity; EC50). All samples and controls were verified in triplicate.
Results are recorded as mean ± SEM. GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. The student’s t-test was used to determine significance between means, p-value < 0.05 was considered significant.
The binding form of the reference (Erlotinib) displayed an energy binding of -6.55 kcal/ mol against EGFR tyrosine kinase. The quinazoline ring formed pi-Alkyl interactions with amino acids Val702, Leu694, Leu820, Ala719, and one H-bonding with Met769 with a distance of 2.66 °A. The 3-ethynyl phenyl moiety interacted with Lys721 and Leu764 by pi-alkyl interactions as shown in Fig.
The energy of binding (∆G) of the tested compounds (HB1-6), the number of H-bonds, and pi interactions are displayed in Table
The energy of binding (∆G) kcal/mol of the candidate compounds (HB1-HB6) against EGFR tyrosine kinase target site PDB ID: 1M17.
Ligand | RMSD value (Å) | Docking score(∆G) (kcal/mol) | Interactions | |
---|---|---|---|---|
H-bond | pi | |||
Compound HB1 | 1.85 | -7.08 | – | 7 |
Compound HB2 | 1.90 | -7.47 | 1 | 8 |
Compound HB3 | 1.98 | -7.50 | – | 6 |
Compound HB4 | 1.45 | -7.82 | 1 | 6 |
Compound HB5 | 1.74 | -8.32 | 1 | 7 |
Compound HB6 | 1.99 | -7.15 | 1 | 9 |
Erlotinib | 1.90 | -6.55 | 1 | 6 |
The 2D docking of compounds (HB1-6) against EGFR tyrosine kinase focusing on the interacting amino acids. H-bonds are shown in green dashed lines, and the pi interactions are shown in purple, orange, and pink dashed lines. Superimposition of each compound with erlotinib inside EGFR tyrosine kinase target site is shown.
The 4H-1,2,4-triazole ring of compound HB1 generated pi interactions with the essential amino acids Leu820, Ala719, and Val702 of EGFR tyrosine kinase, while the phenyl moiety formed other pi interactions with Ala719, Lys721, and Val702, and the phenylamino moiety formed pi interaction with leu694. The binding features of compound HB2 to EGFR tyrosine kinase was through 4H-1,2,4-triazole ring which showed pi interactions with the essential amino acids Leu820, Ala719, and Val702 of EGFR tyrosine kinase, while the phenyl moiety formed another sulfur-pi interaction with Met742, and pi-alkyl interaction with Lys721. The 4-chloro phenylamino moiety formed pi interaction with Leu694, and the (2,3-dimethyl phenyl) amino moiety formed one H-bond with Asp831 (2.45 °A), and pi-pi interaction with Phe699, while the benzamide moiety interacted with Cys773 by sulfur-pi interaction.
The 4H-1, 2, 4-triazole ring of HB3 formed pi interactions with Val702, while the phenyl moiety formed another sulfur-pi interaction with Cys773, and the 4-methoxy phenylamino moiety formed pi interactions with Lys721, Ala719, and Val702. In addition, the benzamide moiety interacted with Leu694 by pi-alkyl interaction.
Compound HB4 interacted with different essential amino acids of EGFR tyrosine kinase: the 4H-1,2,4-triazole ring created pi interactions with Leu820, Ala719, and Val702, while the phenylamino moiety formed pi interaction with Lys721. The 4-nitrophenyl group interacted with Leu694 by pi interactions, and the (2, 3-dimethyl phenyl) amino moiety formed one H -bond with Asp831 (2.28 °A), while the benzamide moiety interacted with Cys773 by sulfur-pi interaction.
The 4H-1, 2, 4-triazole ring of compound HB5 created pi interaction with EGFR tyrosine kinase amino acid Leu694, while the (2,3-dimethyl phenyl) amino moiety formed pi interactions with Cys773 and Arg817, and the 4-nitrophenyl group interacted with Leu820, Ala719, Val702, and Lys721 by pi interactions, and one H- bond with Thr766 (2.33 °A).
Finally, the binding features of compound HB6 to essential amino acids of EGFR tyrosine kinase were mediated by 4H-1,2,4-triazole ring which demonstrated pi interactions with Leu820, Ala719, Leu694, and one H-bond with Met769 (2.44 °A), while the 4-methoxy phenylamino moiety formed pi-alkyl and pi-sulfur interactions with Ala719 and Cys751, respectively. The 4-nitrophenyl group formed pi-sigma interaction with Leu694, the (2, 3-dimethyl phenyl) amino moiety interacted with Cys773 by sulfur-pi interaction, and the benzamide moiety interacted with Asp831and Val702 by pi-anion and pi-alkyl interactions, respectively.
The ADMET properties of drugs are usually predicted by intestinal absorption, activity against colon cancer cell line (Caco-2), P-glycoprotein binding, and skin permeability levels (
Properties | HB1 | HB2 | HB3 | HB4 | HB5 | HB6 | Erlotinib |
---|---|---|---|---|---|---|---|
Human gastrointestinal absorption | High | Low | Low | Low | Low | Low | High |
Caco2 | 66.8 | 36.76 | 41.8 | 45.8 | 56.8 | 30.8 | 45.8 |
Bioavailability score | 0.55 | 0.17 | 0.17 | 0.55 | 0.17 | 0.17 | 0.55 |
BBB penetration | NO | NO | NO | NO | NO | NO | Yes |
PPB | 95.35 | 80.46 | 81.30 | 91.69 | 84.36 | 89.39 | 75.69 |
CYP1A2 inhibitor | NO | NO | NO | NO | NO | NO | NO |
CYP2C19 inhibitor | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
CYP2C9 inhibitor | NO | NO | NO | NO | NO | NO | NO |
CYP2D6 inhibitor | NO | NO | NO | NO | NO | NO | NO |
CYP3A4 inhibitor | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
Carcinogenicity | NO | NO | NO | NO | NO | mutant | NO |
Ames Test | NO | NO | NO | NO | NO | NO | mutant |
The drug-likeness study indicated that all of the synthetic compounds (HB1-6) have most of Lipinski’s Rule criteria for oral administration. In addition, it is known that for good bioavailability after oral administration the value of TPSA must not exceed 140 A°(
Properties | HB1 | HB2 | HB3 | HB4 | HB5 | HB6 | Erlotinib |
---|---|---|---|---|---|---|---|
clog P | 3.43 | 3.58 | 4.07 | 1.95 | 2.03 | 2.60 | 4.06 |
clog S | -10.83 | -10.93 | -11.41 | -10.16 | -10.25 | -10.07 | -7.26 |
H-bond acceptor | 3 | 4 | 3 | 5 | 6 | 5 | 6 |
H-bond donor | 3 | 3 | 3 | 3 | 3 | 3 | 1 |
Skin permeation (log Kp) cm/s | -4.19 | -4.39 | -3.95 | -4.85 | -4.79 | -4.35 | -6.35 |
TPSA (A°) | 83.87 | 93.10 | 83.87 | 129.35 | 138.92 | 129.69 | 129.69 |
The cytotoxic effects of the 6 compounds (HB1-HB6) were evaluated against Hep G2 and A549 human cancer cell lines using MTT assay (Suppl. material
In-vitro anti-proliferative activities of the tested compounds against Hep G2 and A549 cell lines. Data are presented as the mean of the IC50 values (µM) from three different experiments.
Compound | HepG2/IC50 | A549/IC50 |
---|---|---|
HB1 | 34.81 | 12.40 |
HB2 | 12.96 | 18.62 |
HB3 | 16.36 | 10.82 |
HB4 | 8.29 | 9.94 |
HB5 | 2.87 | 9.69 |
HB6 | 17.87 | 18.74 |
To study the selectivity of compound HB5 against cancer cells, the cytotoxicity of this compound was evaluated against the normal lung fibroblast cell line WI-38. The IC50 concentration of compound HB5 against WI-38 cells was 23.67 µM (Suppl. material
The effect of 48 h treatment of compound HB5 on Hep G2 cell cycle progression was evaluated by staining cells with PI followed by flow cytometry (Fig.
Effect of compound HB5 on cell cycle progression in Hep G2 cells after 48 h treatment. Values are given as mean ± SEM of three independent experiments.
Cell cycle distribution (%) | ||||
---|---|---|---|---|
Sub-G1 | G1 | S | G2/M | |
Control Hep G2 | 0.63 ± 0.18 | 57.77 ± 2.68 | 25.35 ± 1.69 | 16.25 ± 1.34 |
HB5-treated Hep G2 | 1.16 ± 0.17 | 35.56 ± 5.08* | 37.87 ± 3.87* | 25.41 ± 2.18* |
Flow cytometric analysis of Hep G2 cell cycle phases 48h-post compound HB5 treatment. Representative histograms showing the distribution of cell cycle phases of control and HB5-treated cells. Hep G2 hepatocyte carcinoma cells were treated with 2.87 µM (IC50 value) of compound HB5 for 48 h.
To investigate the mode of cell death (apoptosis versus necrosis) caused by 48h of compound HB5 treatment, Hep G2 cells were treated, stained with PI and annexin V-FITC, and analyzed by flow cytometry (Fig.
Effect of compound HB5 on the mode of cell death in Hep G2 cells after 48h treatment. Values are given as mean ± SEM of three independent experiments.
% Viable | % Apoptosis | % Necrosis | ||
---|---|---|---|---|
Early | Late | |||
Control Hep G2 | 93.55 ± 0.29 | 5.69 ± 0.30 | 0.62 ± 0.05 | 0.14 ± 0.03 |
HB5-treated Hep G2 | 28.21 ± 1.80**** | 70.44 ± 1.80**** | 1.21 ± 0.20 | 0.14 ± 0.01 |
Treating Hep G2 hepatocyte carcinoma cells with compound HB5 induces apoptosis. A Representative flow cytometric charts for control and HB5-treated Hep G2 hepatocyte carcinoma cells showing the distribution of viable, apoptotic, and necrotic cells. Hep G2 cells were treated with compound HB5 (2.87 µM) for 48 h, fixed and stained with PI and fluorescent annexin V. Viable cells (bottom left; annexin V negative/PI negative), early apoptotic cells (bottom right; annexin V positive, PI negative), necrotic cells (top left; annexin V positive, PI positive), and late apoptotic cells (top right; annexin V positive, PI negative); B Total RNA was extracted from control and HB5-treated cells, reverse transcribed, and assayed for p53, BAX, Bcl-2, and caspase-3 gene expression by qRT-PCR. Data are presented as mean ± SEM of three independent experiments of the fold change in the ratio of relative mRNA levels of target gene/GAPDH (housekeeping gene). Fold change in control cells was set at 1 arbitrary unit. *p < 0.05, **p < 0.01 as compared to control cells.
Additionally, the level (fold change) of mRNA of apoptosis markers such as, p53, BAX (pro-apoptosis), Bcl-2 (anti-apoptosis), and caspase-3 was measured by qRT-PCR. The results shown in Fig.
EGFR is one of the most frequently mutated genes in solid epithelial cancers, either through overexpression of the EGFR protein or a kinase-activating mutation (
All tested compounds have the same pattern of pharmacophoric queries of EGFR tyrosine kinase inhibitors, however, compound HB5 has the highest fitting with these queries. Compound HB5 showed the lowest ∆G score (-8.32 Kcal/mol; Table
The SAR of the compounds (HB1-6) revealed several common findings (Fig.
A series of 1,2,4 triazole derivatives (HB1-6) have been successfully synthesized, and their chemical structures were confirmed using FT-IR, 1H, and 13CNMR spectra and elemental microanalysis. After performing molecular docking studies and MTT assay for the tested compounds, the results showed a high correlation between the expected results from the molecular modeling and the wet-lab biological evaluations, which indicated high selectivity against the EGFR tyrosine kinase target site. Additionally, in silico pre-ADMET study showed that all tested compounds have no CNS side effects, and carcinogenicity score close to zero. Finally, compound HB5 showed the strongest cytotoxicity and highest selectivity against cancer cells through its stable interaction with EGFR tyrosine kinase leading to inhibition of its activating, and thus inducing cancer cell apoptosis.
The authors declare that there is no conflict of interest.
The authors are grateful to Department of Pharmaceutical Chemistry, College of Pharmacy, University of Basrah, Iraq, for supporting the current work.
Figure S1
Data type: JPG file
Explanation note: HB5-treated Hep G2 hepatocyte carcinoma and A549 lung cancer cells show reduced cell proliferation. Representative line chart of the MTT assay results. Cells were treated for 48h and the growth inhibition percentage (Inhibition %) relative to the vehicle-treated control cells was measured. Percent inhibition was calculated according to the following formula: optical density (OD) treated/OD vehicle-treated control×100%. The experiment was performed three times in triplicates.
Figure S2
Data type: JPG file
Explanation note: The EGFR tyrosine kinase inhibitory activity of compound HB5 was comparable to that of erlotinib. The inhibitory activities of compound HB5 and erlotinib (a standard TKI) against EGFR kinase were evaluated by chemiluminescence and the EC50 values were determined in nM.