Synthesis, anticancer activity and molecular docking studies of new 4-nitroimidazole derivatives

Imidazoles have occupied a unique position in heterocyclic chemistry, and its derivatives have attracted considerable interests in recent years for their versatile properties in chemistry and pharmacology. Herein, we report the synthesis of 3-(1-benzyl-2-ethyl-4-nitro-1H-imidazol-5-ylsulfanyl)-1-(4-substituted phenyl-piperazin-1-yl)-propan-1-one 5a - p by reaction of 3-(1-benzyl-2-methyl-4-nitro-1H-imidazol-5-ylsulfanyl)-propanoyl chloride ( 3 ) with piperazine nucleophiles. Eighteen compounds were assessed for their antiproliferative inhibition potency against four human cancer cell lines (MCF-7, PC3, MDA MB231 and Du145). Compounds 5f and 5k were the most potent anticancer agents on MCF-7 cell lines cell line with IC 50 value of 1.0 µg/mL, while 5d and 5m exhibited cytotoxic effect on PC3 and DU145 cell lines with IC 50 values of 4.0 and 5.0 µg/mL, respectively. The molecular docking of compounds 5f , 5d and 5m has been studied.


Introduction
Imidazoles have attracted attention since imidazole ring presents in the essential amino acid, histidine which is existed in many proteins and enzymes and plays an important role in the structure and binding function of hemoglobin. Biological studies showed great numbers of substituted imidazoles with wide spectrum of biological activities, such as antitumor, antimicrobial, anti-HIV, antibacterial, antihypertensive, antifungal and anticonvulsant activity. 1-6 Imidazole nucleus and its derivatives are considered as privileged scaffold in medicinal chemistry, they constitute an important class of therapeutic agents and well known as drugs. For example; Dacarbazine (DTIC) (5-(3,3-dimethyl-1-triazeno)imidazol-4-carboxamide was synthesized as an alkylating agent 7 and used in the treatment of metastatic melanoma 8,9 as well as a part of the ABVD chemotherapy regimen to treat Hodgkin's lymphoma 10,11 and in the MAID regimen for sarcoma. 12 Temozolomide (Temodar) is also classified as one of alkylating agents commonly used to treat certain types of brain tumors such as glioblastoma multiforme or anaplastic astrocytoma. 13,14 Furthermore, clotrimazole [1-(2chlorotrityl)-1H-imidazole] is an azole antimycotic agent (antifungal) and used to treat skin infections such as athlete's foot, jock itch, ringworm, and other fungal skin infections (candidiasis). 15,16 Moreover, imidazole ring substituted with nitro group (nitroimidazoles) are also biologically active compounds commonly used as therapeutic agents for treatment of different diseases such as; metronidazole [2-(2-methyl-5-nitroimidazol-1yl)ethanol] (Flagyl) (antibiotic) is used to treat trichomoniasis, amoebiasis, and giardiasis. 17 Misonidazole (1methoxy-3-(2-nitroimidazol-1-yl)propan-2-ol) (radiosensitizer and antineoplastic) is one of the imidazole drugs which used for treatment of hypoxic tumors, 18 meanwhile cimetidine is considered as a potential histamine H2 receptor antagonist that inhibits stomach acid production. 19 In addition, secnidazole (hydroxy-2-propyl)-lmethyl-2-nitro-5-imidazole) and tinidazole (1- [2-(ethylsulfonyl)ethyl]-2-methyl-5-nitroimidazole) has been described for treatment of bacterial vaginosis. 20,21 Some selected structures of biologically active imidazole compounds are shown in Figure 1.
Based on the imidazole pharmacological importance of imidazole derivatives and in contituation of our previous work on imidazole analogues with their antiviral and anticancer activity, [22][23][24][25][26][27][28][29] we report here new derivatives of nitroimidazole-containing piperazine derivatives and evaluation of their anticancer activity as well as the molecular docking study.

Molecular docking study
In silico study using molecular docking was undertaken against targets of imidazol analogues to verify the potential affinity of the most active compounds of the series 5d, 5f and 5m to the target proteins. In docking calculations, compound 5d was docked to the binding pockets of proteins with PDB code 3RUK (chain A), while 5f and 5m were docked with 3U2B (B chain) and 5T8E (C chain), respectively, using Autodock4 32 and the docking results were viewed and analysed by MGLTools. MCF-7, PC-3 and DU145 cell lines, respectively. The binding energy scores of 5d, 5f and 5f were found -9.97, 7.68 and -7.17 kcal mol -1 , respectively, indicating selectivity and potency profiles of these analogs to bind the active site of proteins pockets. Detailed analysis of the binding mode showed that compound 5d is settled down in the protein active site properly. Figure. 2 (A) demonstrated π -π stacking interactions between the aromatic ring (ring B) of 5d and Phe114, together with the same interaction between the pyrrole ring of HEM600 and imidazole scaffold (ring A). Additionally, it showed seven π−H interactions: three interactions between the aromatic ring at N-1 (ring B) and Ala367, Val482 and 371 were observed, while other interactions were indicated between imidazole ring and Ala113 and Ala302. Furthermore, π−H interactions between rings C and D with Ile205 and Arg239, respectively, in addition to the same interaction between OMe group at ring D and Phe300. Figure 2 B showed π−H interactions between imidazole ring and Met7 and Lys4, while ring B revealed the same interaction with Me11 and Arg 5. The phenyl group (ring D) presented a π−H interaction with Met11, in addition to aliphatic hydrophobic action between nitro group and Lys4. Figure 2 (C) demonstrated that compound 5m was able to show π-π interactions between imidazole ring and Trp718 as well as ring D with Phe764. Further π−H and aliphatic hydrophobic interactions with protein receptors-binding residues including His714, Val715, Pro682, Ala748, Met749, Arg752, Met745, Leu707, Leu873 and Met742 were witnessed.

Conclusions
In conclusion, we have reported the synthesis of new 4-nitroimidazole derived substituted arylpiperazine at C-5. The structures of the new synthesized 4-nitroimidazole derivatives were confirmed by the spectral and mass data. The synthesized compounds were evaluated for their activity against breast cancer (MCF-7 and MDA MB231) and prostate cancer (PC3 and DU145) cell lines. Two derivatives, 5f and 5h exhibited significant cytotoxic activity on MCF cell lines (IC50 1.0 µg/mL), while compounds 5d and 5m showed cytotoxic effect on PC3 and DU145 cell lines with IC50 values of 4.0 and 5.0 µg/mL, respectively. These studies revealed that such molecules have high scope and potential for further investigations. Molecular docking studies were in agreement with the anticancer activity data. Studies on extensive diversification, mechanistic analysis and application of pharmacognosy principles, especially compounds 5f and 5h, are currently under process to come up with better leads.

Experimental Section
General. Melting points were measured on a Mettler FP1 melting point apparatus and are uncorrected. Reaction progress was monitored by thin layer chromatography (TLC) on Alugram SIL G UV254 (Macherey-Nagel). All new compounds were analyzed for C, H, and N using a 2400 CHN Elemental Analyzer by Perkin Elmer. The observed results agreed with the calculated percentages to within ±0.4%. 1 H and 13 C-NMR spectra were recorded on a Bruker DRX-300 instrument. Chemical shifts are given in parts per million (ppm), and tetramethylsilane (TMS) was used as internal standard for spectra obtained in CDCl3. Mass spectra were measured on LC-MS 8050 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) equipped with a binary solvent delivery system (LC-30AD), a controller (CBM 20A), an autosampler (SIL-30A), column thermostat (CTO-20AC).

Synthesis of 3-(1-benzyl-2-ethyl-4-nitro-1H-imidazol-5-ylsulfanyl)propionyl chloride (4).
A solution of 3 (0.5 mmol) and thionyl chloride (5 mL) was heated under reflux at 75-80 o C for 3-4 h. Excess of thionyl chloride was removed under vacuum to afford compound 4. The crude product 4 was used directly for the next step without further purification. General procedure for the preparation of 4-nitroimidazole analogues (5a-p). Compound 4 (0.5 mmol) was dissolved in CHCl3 (15 mL), piperazine derivatives (0.7 mmol) and three drops of pyridine were added, the reaction mixture was stirred at room temperature for 18 h. After cooling, the mixture was evaporated to dryness. The residue was partitioned between CHCl3 (50 mL) and water (50 mL) and the combined organic extracts were dried over anhydrous sodium sulfate (Na2SO4), filtered and evaporated to dryness. The residue was purified by thin-layer chromatography (TLC) and eluted with (CHCl3-MeOH; 9.5:0.5) to give 5a-p.   ). 13  the following densities: MCF-7, MDA MB231, PC-3, and DU145 cells (3 x 10 3 cells/well). Cells were allowed to adhere for 24 h. Then, the medium was replaced with fresh medium alone or with the tested compounds at increasing concentrations from 0 to 250 uM for cancerous cell lines and to 500 uM for the normal dermal fibroblast cells. The reference drugs cisplatin (0-100 uM) and doxorubicin (0-10 uM) were included as positive controls for growth inhibition. After 72 h, cell viability was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay [31]. All experimental conditions were tested in triplicate and the experiment was performed three times. Half maximal inhibitory concentrations (IC50, the concentration required for 50% in vitro inhibition of growth) were calculated for each experiment using Graphpad prism software (Version 8, San Diego, CA, USA). IC50 values were reported as mean + SD Dock and virtual screening Preparations of ligands and proteins. The structures of ligands 5d, 5f and 5m were prepared by Avogadro (v. 1.0.1) 33 software and saved as PDB file formate. Then, the two ligands were prepared selecting torsions and the structures were converted from PDB formate to PDBQT. The PDBQT files for the proteins and the ligands, united atom Kollman charges, fragmental volumes, and solvation parameters were performed by the MGLTools software. Ligand structures were energy minimized with the MMFF94 force field. The native ligands and crystallographic water molecules were removed from the PDB structures and the polar hydrogens were added before docking. Grid map calculations. AutoDock grid maps were calculated for each compound using AutoGrid4, based on the active site coordinates of each protein crystal structure. The size of all grid boxes 60 x 60 x 60 xyz points with a grid spacing of 0.375 Å. The grid center dimensions were 85.44, 52.99, and 46.41for x, y and z respectively. Maps were calculated for each atom type in each ligand along with an electrostatic and desolavation map using dielectric value of -0.1465. Molecular docking simulations. Molecular docking simulations were undertaken using the Autodock program. 32 Protein structures were prepared using UCSF Chimera 1.15. 34 In Autodock program, the Lamarkian Genetic Algorithm (LGA) was used for pose sampling and the number of energy simulations was set to 2500000. The default scoring function was used for calculating the docking scores. Autogrid was used to prepare the maps. The results of molecular docking were visualized in Biovia Discovery Studio 2020 software 35 and then analyzing the docking results. All docking simulations performed to validate the method, using the ligands present in crystal structures, were able to reproduce the ligand-protein interaction geometries. The image of the native ligands for 3RUK, 3U2B and 5T8E against the redocked native ligand with AutoDock is shown in Figure 2, meanwhile the root-mean-square deviation of atomic positions (RMSD) was 0.405 Å.