Green synthesis, molecular docking and anticancer activity of novel 1,4-dihydropyridine-3,5-Dicarbohydrazones under grind-stone chemistry

ABSTRACT The reaction of 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarbohydrazide with variety of carbonyl compounds such as substituted benzaldehydes, cycloalkanones and hetero-cyclic ketones under grinding method under catalyst- and solvent-free conditions at room temperature, in the presence of catalytic drops of acetic acid gave a new series of 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarbohydrazone derivatives in good to excellent yields. The structures of new products were elucidated on the basis of their spectral data and elemental analysis. Most of the newly prepared compounds were evaluated towards HepG2 cell lines and showed good IC50 for some compounds. Additionally, molecular docking of the novel chemical entities using Autodock Vina demonstrated their binding modes within the active site of DYRK1A. GRAPHICAL ABSTRACT


Introduction
Catalyst-and solvent-free synthetic methods are important for both chemical industry and laboratory synthesis because of lower costs, reduced pollution, ease of purification, and mild conditions. Procedures without catalysts and solvents have been advised for cleaner and greener syntheses (1)(2)(3)(4). The grinding technique, as solvent-free reactions, has been widely used in organic synthesis (5)(6)(7).
Although, chemotherapy is useful for liver cancer but the drug resistance development occurs in metastatic cancers (8,9). Resistance to several similar drugs is often observed after sporadic or repeated exposure of tumor cells to only one chemotherapeutic agent (10). The need for effective treatment encouraged the search for design and summarization of new agents as a result of multiple drug resistance in cancer chemotherapy. 1,4-Dihydropyridines are important class of compounds in the field of pharmaceuticals and drugs. Researches on dihydropyridine and its synthetic analogs has revealed that they possess many biological activities such as vasodilates, antihypertensive, antiinflammatory, anti-ischemic agents, antianginal, antithrombotic, anticonvulsant, antimicrobial activities (11)(12)(13)(14)(15)(16)(17)(18). Moreover, many heterocyclic hydrazones represents an interesting class of hetero compounds with a broad spectrum of biological activities, analgesic, anti-inflammatory, antimicrobial, antitubercular, antiplatelet, anticonvulsant, antimalarial and antiviral activities (19)(20)(21)(22)(23). It is quite interesting to note that the 1,4-dihydropyridine and hydrazine derivatives have gained prominence in the recent years for cancer chemotherapy (24)(25)(26)(27)(28)(29)(30). 1,4-Dihydropyridines have been proved to be a new class of multidrug resistance (MDR) reversals in cancer treatment (31).
In order to get a better insight into the mechanism of action of the compounds with promising activities, a brief target prediction was carried out using chemical similarity as the basis for target estimation. Subsequently, Dual specificity tyrosine-phosphorylationregulated kinase 1A (DYRK1A) was selected; owing to the consensus of its prediction for the majority of our active chemical entities. DYRK1A is known to phosphorylate Sirtuin 1 (SIRT1) upon genotoxic stress as part of the negative regulation of apoptosis. Thereafter, Tumor Suppressor p53 (TP53) is deacetylated and deactivated by the active Sirtuin 1 which leads to carcinogenesis due to uncontrolled cellular division ( Figure 1).
Interestingly, despite the fact that Dual specificity tyrosine-phosphorylation-regulated kinases (DYRKs) maintain most of the kinases family conserved characteristics, they still exhibit a few unique features which render them distinguishable from all other kinases. The first notable distinction of DYRKs is the dual kinase activity for both serine and threonine substrates. Moreover, the peculiar HCD motif of the catalytic loop of DYRKs is another remarkable variation from the highly conserved Y/HRD motif in most kinases ( Figure 2). Furthermore, mutation studies unraveled the essential role of the DYRK1A conserved ATP anchor LYS188, through the abolishment of the kinase activity upon altering the aforementioned lysine with an arginine residue. Additionally, the catalytic proton acceptor residue of DYRK1A was identified to be the ASP287, which performs the catalytic transfer of the γ-phosphate

Result and discussion
Condensation of 1,4-dihydropyridine-3,5-dicarbohydrazide 1 (48) with two moles of each of benzaldehyde derivatives 2a-d, 4, 6 in the presence of one drop of glacial acetic acid by grinding method at room temperature gave in each case a single product proved to be the respective bis(3-hydroxybenzylidene)-2,6dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarbohydrazide derivatives 3a-d, 5 and 7 in an excellent yields (Scheme 1). The structure of the products 3a-d, 5 and 7 was evidenced by their spectral (MS, 1 H NMR and IR) data and elemental analysis. For example, mass spectrum of compound 3a, taken as typical example of the series, showed the molecular ion peak at the expected m/z Reactions of 1 with two equivalents of cycloalkanones 8a-d such as cyclopentanone 8a, cyclohexanone 8b, cycloheptanone 8c, cyclooctanone 8d in presence of one drop of glacial acetic acid under grinding method to give the respective N' 3 ,N' 5 -dicycloalkylidene-2,6-dimethyl-4phenyl-1,4-dihydropyridine-3,5-dicarbohydrazides 9a-d (Scheme 2). The structure of 9a-d was established based on its elemental analysis and spectral data (IR, MS and 1 H NMR). For example, its 1 H NMR spectra showed the expected signals for different protons. Also, mass spectrum showed the molecular ion peak at the expected m/z values.
In a similar mannar, as shown in Scheme 2, compound 1 reacted with two equivalents of tetralone 10 and isatin derivatives 12a-c to give the respective hydrazones 11 and 11a-c as evidenced by their elemental analysis and spectral data (see experimental section).

Antitumor activity
The antitumor activity of the products 3a,c, 5, 9a,d, 11, 13a,c, and 15a,b was investigated against human Hepatocellular carcinoma cell lines using colorimetric MTT assay, in comparison with Harmine standard drug. The   relation between drug concentration and surviving cells is plotted to get the survival curve. The 50% inhibitory concentration (IC 50 ) was obtained and the anti-proliferative activity was expressed as the mean IC 50 of three independent experiments (μM) ± standard deviation from three replicates. The results in Table 1 and Figure 4 showed that: . The in vitro growth inhibitory activities of the tested compounds depend on the electronic environment and structural skeleton of the molecules. . The activity of the tested compounds depends on concentration. . The descending order of inhibitory activity of the tested compounds towards the HepG-2 were as follow: where electron withdrawing group as Cl increases the cytotoxic activity than electron donating group as OH.

Target prediction
After synthesizing the compounds, the chemical structures were submitted to Swiss Target Prediction webserver (49) to predict the best fit target for screening of the novel ligands. DYRK1A was selected based on the significant consensus of its prediction for most of the compounds.

Molecular docking studies
The X-ray crystal structure of the DYRK1A (PDB ID: 3ANR) was selected mainly on the basis of the bound co-crystallized Harmine inhibitor. Harmine is known to be a potent inhibitor of DYRK1A (50), therefore we used it as the reference compound for the biological screening. The molecular docking protocol was validated through re-docking of the co-crystallized ligand and evaluating the RMSD value of its highest ranking docked pose ( Figure 5). The RMSD value for the global docking of the co-crystallized ligand was 0.4495 Å, while local docking resulted in an RMSD value of 0.368 Å.
Initially, the compounds were globally docked to DYR1A to confirm their unbiased affinity towards the ATP binding site. Successfully, the highest affinity poses for all the globally docked ligands were selective for the ATP binding pocket ( Figure 6A). Subsequently, local docking of the compounds into the ATP binding site  was performed, to refine the binding modes of the ligands ( Figure 6B) and obtain a better estimate of their binding affinities to the enzyme (Figure 7).
Finally, compounds 5, 11 and 15b were selected for a more detailed interaction profile analysis. Strikingly, despite having a very good IC 50 yet compound 15b exhibits weak interactions with the ATP binding pocket of DYRK1A. This puzzling finding may be explained by the high drug-likeness of 15b, which renders its pharmacokinetic behavior more suitable to the cellular environment. On the other hand, both compounds 5 and 11 are actually capable of reaching inside the ATP binding site through their lengthy flexible structures, while anchoring themselves to the exposed surface of the enzyme (Figure 8). Furthermore, compound 5 exhibited interactions with both LEU241 and the catalytic ASP307 residues, thus establishing interactions with two of the key residues for DYRK1A activity. Lastly, compound 11 displayed interactions with both the catalytic ASP307 and ATP anchor LYS188, which renders the ATP binding to its pocket unattainable (Figure 9) (44).

Experimental
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus. Elemental analyses were measured by using a German made  Elementar vario LIII CHNS analyzer. IR spectra were recorded in KBr discs on Pye Unicam SP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer at 70 eV. The 1 H and 13 C NMR spectra were recorded on a Varian 6 Mercury VX-300 NMR spectrometer (Karlsruhe, Germany). 1 H NMR (300 MHz) and 13 C NMR (75 MHz) were run in DMSO-d 6 and chemical shifts are expressed in ppm units using TMS as an internal reference. Antitumor activity was evaluated at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt.

Cytotoxicactivity
The cytotoxic activity of the synthesized compounds was evaluated against liver Carcinoma (HepG2) cell line at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt according to the reported methods (51,52). For more details, see supporting information file.

Molecular modeling
The molecular docking study was performed using Auto-Dock Vina (53). Additionally, small molecules file conversions were achieved through the use of Open Babel 2.4.0 (54). Finally, the molecular visualization was done using Discovery Studio Visualizer version 16.0.0.400. The target preparation, ligand preparation and molecular docking procedure that were performed are discussed in this section.

Target preparation
The crystal structure of the target was acquired through the RCSB PDB website (PDB ID: 3anr). Next, Auto Dock Tools target preparation scripts were used to prepare the target molecule for molecular docking (55,56).

Ligand preparation
The SMILES file of the ligands was converted into PDBQT format, with the generation of 3D coordinates option enabled using Open Babel 2.4.0.

Docking:
The global docking of the ligands was initially performed first using AutoDock Vina, then followed by the local docking to the ATP binding pocket of DYRK1A.

Conclusion
In conclusion, a new series of 2,6-dimethyl-4-phenyl-1,4dihydropyridine-3,5-dicarbohydrazone derivatives 4a-j were synthesized using grinding technique under solvent-and catalyst-free conditions at ordinary temperature, in the presence of catalytic drops of glacial acetic acid gave a new series of good to excellent yields. Most of the new compounds assayed for their in vitro anticancer activity against HepG2 cell lines and showed good IC 50 . The molecular docking of the novel chemical entities using Autodock Vina demonstrated their binding modes within the active site of DYRK1A.

Author contributions
All authors contributed toward data analysis, drafting and revising the paper and agree to be accountable for all aspects of the work.

Disclosure statement
No potential conflict of interest was reported by the authors. Dr. Zeinab A. Muhammad was born in Cairo, Egypt. she graduated with B.Sc. degree in special chemistry department from El-Azhar University, Cairo, after getting his M.Sc. degree in organic chemistry in 2012, Cairo University, she had a permanent position at the National Organization for Drug Control and Research (NODCAR) in Cairo. She earned a Ph.D. degree in organic chemistry from Cairo University in 2015. She joined the scientific school of Prof. A. S. Shawali in 2009, His research interests include heterocyclic and medicinal chemistry with emphasis on the design and synthesis of novel bioactive heterocycles and the study of their biomedical applications with the aim of developing new therapeutic agents and she has published scientific papers and reviews all in international journals in the fields of physical organic chemistry, chemistry of hydrazonoyl halides and bioactive heterocyclic chemistry.

Notes on contributors
Associate Prof. Hassan M. Abdel-aziz was born in 1971 in Beni-Suef, Egypt. He graduated from Cairo University, Egypt in 1993. In 2017 he promoted to a full Associate Prof of Organic Chemistry at Beni-Suef University, faculty of science, Chemistry department, Egypt. He worked for more than 20 year on synthesized biological active Heterocyclic compounds and treatment of some diseases such as cancer. He published more than 20 paper in high impact internarial journals. He worked for more than 10 years on molecular biology, reclassification of some Falconiformes and Owls birds, immunology, parasitology, physiology, the biological use of newly synthesized organic compounds and anticancer drugs. He published 8 papers in high impact international journals.