Synthesis, Molecular Docking, Molecular Dynamics Studies, and

Article pubs.acs.org/jcim

Synthesis, Molecular Docking, Molecular Dynamics Studies, and Biological Evaluation of 4H‑Chromone-1,2,3,4-tetrahydropyrimidine5-carboxylate Derivatives as Potential Antileukemic Agents Zahra Dolatkhah,† Shahrzad Javanshir,*,† Ahmad Shahir Sadr,‡,§ Jaber Hosseini,† and Soroush Sardari∥

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†

Heterocyclic Chemistry Research Laboratory, Department of Chemistry, Iran University of Science and Technology, Tehran 16846-13114, Iran ‡ Bioinformatics Research Center, Sabzevar University of Medical Sciences, Sabzevar 9613873136, Iran § Bioinformatics Research Center, Cheragh Medical Institute and Hospital, Kabul 1001-1007, Afghanistan ∥ Drug Design and Bioinformatics Unit, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Pasteur Avenue, Tehran 13164, Iran S Supporting Information *

ABSTRACT: A series of 4H-chromone-1,2,3,4-tetrahydropyrimidine-5-carboxylates derivatives were synthesized via a three component one-pot condensation of chromone-3-carbaldehyde, alkyl acetoacetate, and urea or thiourea, using MCM-41SO3H as efficient nanocatalysts and evaluated for their anticancer activity using a combined in silico docking and molecular dynamics protocol to estimate the binding affinity of the title compounds with the Bcr-Abl oncogene. Two programs, AutoDock 4 and AutoDock Vina software were applied to dock the target protein with synthesized compounds and ATP. AutoDock runs resulted in binding energy scores from −7.8 to −10.16 kcal/mol for AutoDock 4 and −6.9 to −8.5 (kcal/mol) for AutoDock Vina. Furthermore, molecular dynamics (MD) simulations are performed using Gromacs for up to 20 ns simulation time investigating the stability of a ligand−protein complex. Finally, a theoretical experiment using MD simulation for 10 ns was performed without defining the initial coordinates, and the affinity binding of ligand to receptors was directly studied, which revealed that the ligand approaches the active sites. The relative free binding energy for the structure 06 (S06), which has the highest binding energy in Autodock 4 and Autodock Vina (−10.10 and −8.5 kcal/mol, respectively), was also evaluated by molecular mechanics (MM) with Poisson− Boltzmann (PB) and a surface area solvation (MM-PBSA) method using g_mmpbsa tools for the last 15 ns MD. On the basis of binding energy scores, a negative binding energy value of 73.6 kcal/mol, S06, was recognized as the dominant potential inhibitors. The cytotoxic properties of S06 was evaluated against three cell lines, acute T cell leukemia (Jurkat), human chronic myelogenous leukemia, (K562) and human foreskin fibroblast (Hu02) using the microculture tetrazolium test MTT assay. Cisplatin was used as the reference agent. The results indicated that S06 has a higher safety index (SI = 0.73, IC50 = 152.64 μg/ mL for Jurkat and IC50 = 110.25 μg/mL for Hu02, P < 0.05 means ± SD for four independent experiments) compared to cisplatin (SI = 0.56, IC50 = 8.86 μg/mL for Jurkat and IC50 = 4.96 μg/mL for Hu02). The in silico results indicated that the proposed structures, which have no toxic effects, are potential tyrosine kinase inhibitors (TKIs) that target Bcr-Abl and thus prevent uncontrolled cell growth (proliferation) but not necessarily cell death (apoptosis) and might potentially constitute an interesting novel class of targeted antileukemic drugs, which deserve further studies.

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in drug discovery (Figure 2).1−4 Moreover, the dihydropyrimidinone-5-carboxylate scaffold is found in various marine natural products including batzelladine alkaloids, which are potent HIV inhibitors.5,6 Another important heterocyclic skeleton is chromone (4H-chromen-4-one or 4H-1-benzopyran-4-one),7−10 which is an important class of natural and synthetic compounds displaying a broad spectrum of pharmacological activities including anticancer, antitubercular,

INTRODUCTION 4H-Cromone-1,2,3,4-tetrahydropyrimidone comprise two bioactive heterocyclic cores, dihydropyrimidinones (DHPMs) and chromones, which contains four pharmacophore features, namely, hydrogen bond acceptor (HBA), hydrogen donor (HBD), hydrophobic (H), and two aromatic ring (AR) (Figure 1). The DHPM substructure evinces a variety of biological and pharmacological activities, such as antiviral, antimitotic, anticarcinogenic, antihypertensive, and most importantly, calcium channel modulators and have been widely exploited © 2017 American Chemical Society

Received: March 8, 2016 Published: May 19, 2017 1246

DOI: 10.1021/acs.jcim.6b00138 J. Chem. Inf. Model. 2017, 57, 1246−1257

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Journal of Chemical Information and Modeling

Figure 1. Pharmacophoric features of 4H-chromone-1,2,3,4-tetrahydropyrimidone generated by LigandScout 3.12 colored as follows: HBD (green), HBA (red), H (mustard), and AR (blue).

theless, there was only one investigation report using this precursor for the preparation of chromone derivatives and investigation of their biological activities.45 The authors in this research have used p-TsOH as catalyst under reflux conditions in ethanol, but they did not state the time of reactions. Given the apparent biological importance of these compounds and in continuation of our interest in the synthesis of biologically active heterocyclic compounds,46−50 the synthesis of substituted 4H-chromone-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives was performed via a three-component one-pot condensation of chromone-3-carbaldehyde, alkyl acetoacetate, and urea/thiourea in the presence of MCM-41-SO3H nanoparticles as catalyst (Scheme 1).

Figure 2. Representative bioactive structures that incorporate the dihydropyrimidinone template.

anti-inflammatory, antimicrobial, antihistaminic, antihypertensive, and anti-HIV activity.11−19 Therefore, the fusion of more than one pharmacophore in the same framework leading to a hybrid molecule has considerable interest in the purpose of engendering a chemical unit that is undoubtedly medically more effective than its separate constituents. The classical multistep preparation of a complex molecule commonly comprises a large number of synthetic operations, including extraction and purification processes in each separate step, which leads to a decrease in efficiency and yield beside generating large quantities of waste.20 Organic synthesis by means of one-pot, tandem, domino, or cascade reactions21,22 have become a noteworthy field of research in organic chemistry. Multicomponent reactions (MCRs) are a valuable tool for building chemical libraries of biologically active compounds with high levels of molecular complexity and variety, consequently accelerating drug discovery programs.23−28 Additionally, the improvement of already known RCMs is also of significant interest for the present organic process. The Biginelli reaction29−33 is one of the classical and valuable multicomponent reactions for the production of 3,4-dihydropyrimidines, and it provides quick access to miscellaneous pharmacologically active groups of pyrimidine derivatives from commercially accessible precursors. Chromone-3-carbaldehyde is a valuable precursor for the synthesis of many biologically active compounds.34−44 Never-

Scheme 1. Synthesis of Substituted 4H-Chromone-1,2,3,4tetrahydropyrimidine-5-carboxylate Derivatives

The diverse biological activities of 4H-chromone-1,2,3,4tetrahydropyrimidones inspired us to screen biologically the considered synthesized compounds. These synthesized compounds were evaluated for their potential anticancer activities centered on chronic myeloid leukemia (CML). In this study, potential binding interactions between synthesized compounds and Abl kinase were explored by in silico molecular docking studies. Docking calculations and molecular dynamics (MD) simulations were combined to dock the synthesized compounds into protein receptors. Molecular docking was carried out by using two different docking programs AutoDock 451 and AutoDockVina.52 The computational approach used in this study, that is, molecular docking followed by the MD 1247


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no ligand structure is known. (2) No protein structure but active ligand structures are known. (3) Protein and ligand structures are known. (4) Only the protein structure is known (Figure 3). To identify the mutual interactions, a structure for one ligand may be sufficient, but it is advantageous to have 3D information for multiple ligands.

simulations, is reliable and accurate for forecasting protein− ligand binding structures and binding free energies.

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RESULTS AND DISCUSSION At the onset of this methodology, various reaction parameters were optimized for the three-component Biginelli condensation of chromone-3-carbaldehyde 1, ethyl acetoacetate 2, and urea 3 as a model reaction for the synthesis of 4H-chromone-1,2,3,4tetrahydropyrimidine-5-carboxylates in the presence of a heterogeneous acidic MCM-41-SO3H nanocatalyst since the Biginelli reaction works well under acidic conditions (Scheme 2). Scheme 2. Model Reaction Using 3-Formyl Chromone 1, Ethyl Acetoacetate 2, and Urea 3

Several test reactions were carried out, and the effects of solvent, temperature, catalyst, and catalyst loading were studied. The results revealed that the reaction was faster in DMSO (Supporting Information (SI), Figure S1, series 5). To test the feasibility of this catalyst, initially the model reaction was carried out at room temperature. To our delight, the corresponding 4H-chromone-1,2,3,4-tetrahydropyrimidine-5carboxylate was obtained in good yield. However, it was found that temperature does not play a considerable role in improving the reaction yield (SI, Figure S1, series 5−7). The best result was achieved with a molar ratio of 1:1:1.2 for chromone-3-carbaldehyde 1, ethyl acetoacetate 2, and urea 3, respectively. In order to verify the efficiency of the catalyst, the model reaction was carried out in the presence of various catalysts such as chitosan, L-proline, Fe3O4, and silica gel at room temperature. Experimental results showed that the reaction proceeded at room temperature in a short time and with high yield using catatlytic quantities of MCM-41-SO3H in DMSO. (SI, Figure S2). In order to examine the scope and generality of this procedure, a series of Biginelli-like products were synthesized under optimized conditions (Supporting Information, Table S1). Reactions were performed easily, and the corresponding products are obtained in high yields in a short reaction time. Obviously, recycling and reusability are valued features of heterogeneous catalysts from an economic and environmental point of view. MCM-41-SO3H was used in the model reaction at optimal conditions. The recovered catalyst was washed with EtOH and EtOAc, respectively, dried and then used in the next reaction. The reduction in catalytic activity observed after four runs could be due to catalyst losses during filtration. A conceivable mechanism is also suggested for the formation of tetrahydropyrimidone (SI, Scheme S1).

LIGAND PREPARATION First, the structure of all the synthesized compounds were drawn using gaussview, and then fully optimized geometries and properties of the electronic and structural properties of the all synthesized compounds were derived by means of the density functional theory (DFT) method54 with the B3LYP functional.55 For all systems, a geometry optimization calculation was performed using the STO-3G56 basis set. The calculations were carried out using the Gaussian 03 package.57 The freely available program Open Babel58 was used to generate SMILES strings from the optimized structures representation, using them for a similarity study by PubChem Structure Search (Table 1). All virtual screening hits were also checked for Pan Assay Interference (PAINS) compounds using the FAF Drugs 3.0 program and were approved.

COMPUTATIONAL ANALYSIS In a computer-assisted drug design method, pharmacophorebased molecular docking is one of the well-known methods used to discover the exactness of binding orientation (poses) of the ligands into the protein active site.53 Four different situations for the pharmacophore exploration may be met when starting a virtual screening: (1) No protein structure and

TARGET PROTEIN PREPARATION Tyrosine kinase (TK) activation plays a significant role in the development of several carcinogenesis. Protein TKs are the enzymes that ensure the phosphorylation of proteins. Phosphorylation governs most aspects of cell life. It is therefore not surprising that abnormal phosphorylation of a protein is a cause or consequence of many diseases. The notion underlying

Figure 3. Four different circumstances for the pharmacophore exploration.

In our case, with the structural information being present for both ligands and the receptor protein, the LigandFit module was used to catch the proper orientation of the molecules in the active site of inactive conformation of the Abl (Abelson leukemia virus) kinase domain. It is the catalytic domain of the Bcr-Abl oncoprotein. The molecular docking involves three steps: (i) ligand preparation, (ii) target protein preparation, (iii) and molecular docking.

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Journal of Chemical Information and Modeling Table 1. SMILES Code of Synthesized Compounds

Table 2. Estimated Inhibition Constants, Free Energy of Binding, H-Bond Interactions, and Hydrophobic Interactions between Synthesized Compounds and Abl Kinase Domain Structure (2HYY) Free Binding Energy (kcal/mol) Entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 a

Ligand

AutoDock Vina

AutoDock 4

ATP S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15

−6.6 −7.1 −7.0 −7.8 −7.4 −6.9 −8.5 −6.9 −7.3 −7.2 −7.2 −7.6 −7.5 −7.0 −7.6 −7.7

−8.51 −8.86 −7.31 −9.15 −10.06 −8.35 −10.10 −8.51 −10.16 −8.04 −9.78 −9.48 −7.41 −7.78 −8.02 −8.11

Hydrogen bonds E286,V299,L301,T315,E316 A269,K271,I313,T315,E316 E286, T315, D381 K271,A268,T315,D381 A269,K271,I313,T315,E316,D381 E286,T315,D381 A286,K271,I314,T315,E316,D381 T315 A269,K271,I313,T315,E316,M318,D381 E286,V299,T315,D381 E286,T315,D381 E286,A380,D381 H361,D381 D381 H361,D381 D381

Hydrophobic interactions ALL

a

Kib (μM) 0.57 0.32 4.40 0.20 0.04 0.75 0.40 0.58 0.04 1.28 0.07 0.11 3.72 1.97 1.32 1.13

All residues are involved in hydrophobic interactions. bInhibitory constant (Ki = exp(ΔG/RT)) was estimated by AutoDock 4.

therapy.64 Imatinib resistance is caused by reactivation of BcrAbl kinase as a result of overproduction or mutation.65 The conception and optimization of inhibitors active against a resistant mutants residue is the current main focus in research on CML. Different approaches to overcome resistance to imatinib have been described which has led to different agents such as agents targeting the pathways activated by Bcr-Abl, agents affecting the stability of Bcr-Abl, and an additional inhibitor of Abl kinase.66 Since imatinib binds to an inactive conformation,67 we used the inactive conformation of the protein kinase to study the binding affinity of selected synthesized compounds to design a possible inhibitor. The crystal structure (2.40 Å resolution) of inactive conformation of the Abl kinase domain (2HYY) was retrieved from the Protein Data Bank (PDB) and used as a target for any of synthesized compounds in this study.68 Theoretical models of the protein target are generated using UCSF Chimera,69 PyMol,70 and Swiss-PDBViewer 4.0.4. Chain A of the crystal structure of the Abl kinase domain (PDB code ID: 2hyy) was extracted using the UCSF Chimera, and since some amino acids side chain atoms were missing during the X-ray crystallography, a reconstruction of the whole

much of anti-TK drugs exploration is to identify small molecules that unswervingly inhibit the catalytic activity of the kinase by interfering with the ATP binding and so impairing the abnormal signal transduction resulting in uncontrolled cell proliferation.59 Chronic myeloid leukemia (CML) represents approximately 15%−20% of all leukemia in adulthood, with an annual incidence of 1−2 cases per 100,000 individuals. The main feature of CML is the Bcr-Abl fusion gene resulting from a mutual translocation of chromosome 9 and 22,t (9; 22), in a hematopoietic stem cell.60 This oncogene, encoding a chimeric Bcr-Abl protein, stimulates the abnormal activity of Abl tyrosine kinase. Imatinib mesylate (STI571) marketed under the mark Gleevec, crowned by success in the field of targeted therapy, targets the tyrosine kinase activity of Bcr-Abl.61 The discovery of imatinib is an example illustrating the success of targeted treatment designed for specific cancer.62 Crystallographic studies have revealed the detailed binding of STI571 to the inactive conformation of Abl kinase.63 The imatinib therapy has been efficient in the vast majority of patients with chronic phase CML. Nevertheless, drug resistance has been observed in some patients after some years of 1249


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Journal of Chemical Information and Modeling side chain was endeavored using Swiss PDB Viewer 4.0.4. The three-dimensional structure images were prepared using PyMol.

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MOLECULAR DOCKING STUDIES In this study, the LigandFit module was used to find the suitable orientation of the molecules in the active site of the inactive conformation of the Abl kinase domain. It is the catalytic domain of the Bcr-Abl oncoprotein. AutoDockTools 1.5.4 (ADT) were used to prepare input PDBQT files and to calculate a grid box. A grid map consisted of 54 Å × 50 Å × 50 Å points around the active site, and a grid spacing of 0.375 Å was used. The center of the grid was set to the coordinates of the STI571 ligand (imatinib)71 in the Abl kinase domain (PDB code ID: 2HYY). A Lamarckian genetic algorithm (LGA) was used for the conformational search. Every Lamarckian job involved 50 runs. The initial population was 150 structures, and the maximum number of energy evaluations and generations was 2.5 × 10. The default values were selected for all other parameters. The final structures were grouped and classified according to the most favorable binding energy. This procedure was applied to all synthesized compounds in the similar manner. AutoDock Vina was also used to dock and predict the binding affinity (kcal/mol) of all synthesized compounds between chains A of the PDB structure 2HYY of the Abl kinase domain. The results are shown in Table 2 and Figure 4. A more negative score indicates which of these compounds (ligand) are more likely to dock with an Abl kinase domain structure (receptor) and achieve more favorable interactions. The docking model of Abl kinase in a complex with ATP (entry 1, Table 2) as the main substrate of kinases was generated by AutoDock 4 and AutoDock Vina. The reliability of the applied docking protocol was assessed by redocking imatinib (STI) into the active site of the Abl kinase domain (Figure 5).

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Figure 4. Docking of structures (1−15) [product 4(a−o)] into the active site of Abl kinase (green, PDB: 2HYY) with D381 and A269 as the most common ligand−protein interactions in all ligand binding poses.

MOLECULAR DYNAMICS SIMULATION Less computationally intensive docking methods such as combination of docking and molecular dynamics simulation (MDS)72 have also been developed recently for various applications. Taking into consideration the dynamics of protein before or during the docking stage has become essential, especially for entities that are subjected to flexibility. To provide more reliable protein−ligand complexes, a synergistic combination of rapid and inexpensive docking protocols and precise but more expensive MDS can be used. By this methodology, we are thus able to measure the affinity of the most promising molecules on various conformations of the receptor and thus to take into account some movements of the protein in a reduced calculation time. Therefore, the combination of these techniques are used in this study. The MD simulations are carried out considering the predefined binding site after docking and without any prior information and prepositioning of coordinates of the binding site for the ligand.

The solvated system (chain A of kinase domain, S06 and water) was neutralized by adding seven Na+ counterions. To equilibrate the system, the solutes (chain A of kinase domain, counterions, and S06) were subjected to the position-restrained dynamics simulation (NVT and NPT) at 299.177 K for 100 ps. Finally, the full system was subjected to an MD production run for 20 ns at 300 K temperature and 1 bar pressure. The simulation results were analyzed using the backbone RMSD values of Abl1, the H-bonding between Abl1, and the ligands, as well as some energies, such as the Lennard-Jones−Short Range (LJ−SR), the Lennard-Jones−Long Range (LJ−LR), and the Coulombic potential−Short Range (Coul−SR) energies from 0 to 20000 ps being analyzed (Figure 6a-6d). The protein−ligand complexes remained distinguished throughout the simulation resulting in ligand RMSD from ∼0.0005 to 01152 nm (Figure 6a) and backbone RMSD from ∼0.07 to 0.23 nm (Figure 6b). This magnitude of deviation, together with a small difference in the average RMSD value, leads to the conclusion that the simulation produced a stable trajectory. Analysis of RMSD of the ligand−protein complex suggested stability of ligands in the binding site during MD simulation. For S06 (entry 7, Table 3) that obtained the best docking score, MD simulations were then applied to explore conformations of the protein−ligand complex (S06 and chain

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MD SIMULATION CONSIDERING PREDEFINED BINDING SITE AFTER DOCKING MD simulations of the protein−ligand complexes were carried out with the GROMACS 4.5.4 package using the GROMOS96 43a1 force field.73,74 The conformations with lowest binding energies were taken as the initial conformation for MD simulations. The topology parameters of protein were created by using the Gromacs program. The complex was immersed in a cubic box of simple point charge (SPC) water molecules.75 1250


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Figure 5. Redocked STI structure to domain A of Abl as verification of performance of docking software.

Figure 6. RMSD evolution of (a) ligand and (b) backbone. Time evolution of (c) temperature and (d) potential energies during 20 ns MD.

Table 3. Free Binding Energies Calculated Using MM-PBSA Methoda energy component for S06 σM b a

R-PB

L-PB

LR-PB

ΔGpolar

ΔGnp (SASA)

ΔGsolv

LRVdW

LRElec

ΔEMM

ΔGbind

Docking ΔG

−9889 3.08

−142 0.07

−9754 3.08

277 4.40

−131 1.6

146 4.68

−198 0.14

−282 0.35

−480 0.32

−308 0.50

−42 −

All values are given in kj mol−1. bStandard error of the mean.

A of kinase domain), followed by optimization of the final structures of the complex in 20 ns and calculation of accurate energies. An additional run of 20 ns molecular dynamics simulation on the Abl−S06 complex has revealed that, except for amino acid residues LYS 271, VAL 299, THR 315, ALA 380, ASP 381, and PHE 382, the rest of the residues in the ATP binding site determined by docking were altered, and some new residues such as GLN 252, MET 290, PHE 317, and HIS 361 were positioned in proximity of the ligands and could participate in the interaction. The outcomes also indicated that at the end of the MD simulations that while the critical hydrogen bonds between THR 315 and the carbonyl group in chromone ring

and ALA 380 and the carbonyl group in ester remained (blue lines) a new hydrogen bond was formed between the carbonyl group in the pyrimidone ring as the H-bond acceptor (HBA) and NH group of LYS 271 as the H-bond donor (Figure 7).

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MD SIMULATION WITHOUT PRESETTING LIGAND COORDINATES Receptor flexibility, especially backbone flexibility, is still a major problem in docking studies. In this step, the MD simulation of the ligand and receptor was carried out without presetting the coordinates for the ligand. The ligand and the receptor have been simulated in their original coordinates after being placed in the water box. During the 10 ns simulation, the 1251


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denotes to the entropic contribution to the free energy in vacuum, where T and S represent the temperature and entropy, respectively. Gx = EMM + Gsolv − TSMM

(2)

The solvation free energy is the sum of polar and nonpolar free energy. Gsolv = Gpolar + Gnon‐polar

Here, EMM contains the bonded and nonbonded interactions energy which comprise both electrostatic (Eelec) and van der Waals (Evdw) interactions.

Figure 7. Interacting residues of chain A of kinase domain complex with structure S06: (a) before additional run of 20 ns simulation and (b) after additional run of 20 ns simulation.

EMM = Enon‐bonded + E bonded + (E vdw + Eelec)

affinity of the ligand moved toward the active site (Figure 8). The average distance between the ligand and the active sites of the receptor decrease from 25.74 to12.18 Å after 10 ns of simulation.

(4)

We have estimated the MM/PBSA free binding energy using g_mmpbsa tool.77,78 The free binding energy was calculated on snapshots collected from a 15 ns MD simulation. MD snapshots were saved every 2 ps, giving 7500 structures per trajectory. The g_mmpbsa tool does not calculate the entropic term (S). Consequently, the term TS in eq 2 was excluded in this study, and the relative binding energy calculation is carried out on static structures of the ligand−protein complexes, which have been previously minimized (Figure 9a−e). Previous reports showed the greatest variation in the estimation of entropy among the five distinct energy terms in MMPBSA calculation. This larger fluctuation of entropy rather than enthalpy during MD trajectories could affect the stable prediction of the MMPBSA protocol, so that the estimation of enthalpy alone is better than free energy compared to experimental affinities.79 The results obtained are summarized in Table 3. The binding energy of S06 predicted by docking was also reported for comparison. The correlation difference between the two methods can be elucidated taking into account that in the MMPBSA technique the effects of the solvent are contemplated with an implicit solvent model. We have already observed that within the complexes the conformation of the ligand remains stable throughout the simulation with RMSDs relative to the initial structures. This stability is also reflected in the mean values and the fluctuations of the angles of folding and torsion between the ligand and receptor, which remain close to the values observed in the monomeric simulations (Figure 9e). These results show that the junctions within the complex are rigid and unaffected by the existence or the type of the ligand interface.

Figure 8. (a) Average distance between the ligand and the active sites of the receptor before 10 ns of MD simulation and (b) after 10 ns of MD simulation.

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REDOCKING OF LIGAND−RECEPTOR AFTER 20 NS OF SIMULATION In order to investigate the fitness of the ligand and the receptor, the redocking of the ligand−receptor was performed after a 20 ns of MD simulation. The results indicated that the binding energy of the ligand−receptor obtained after 20 ns of MD simulation remained constant out of 100 runs (−9.75 kcal/ mol).

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EXPERIMENTAL SECTION Biological Activities Testing. S06 was tested for its cytotoxicity on Jurkat (T cell leukemia) and K562 (myelogenous leukemia) cell lines, and the number of viable cells was estimated by MTT assay (Figure 10). The compounds were dissolved in DMSO (1 mL) and tested on cells at concentrations of 5, 10, 30, 50, 75, 100, 130, 170, 200 μg/ mL for 48 h. The results showed that S06 has no cytotoxicity effect on K562 (myelogenous leukemia) apoptosis in dose and time-dependent manners as assessed by cell viability (Figure 10a). The sensitivity of the Jurkat cells (inhibitory activity of 60% at concentration of 200 μg/mL, Figure 10b) was more than K562 cells (inhibitory activity of 7% at concentration of 200 μg/mL; Figure 10a), which may be related to the higher sensitivity of lymphocytic cells compared with myeloid cells. An IC50 value of 152.64 μg/mL was obtained following 24 h incubation for four independent experiments. The results were

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BINDING FREE ENERGIES CALCULATED BY MM/PBSA The MM-PBSA method allows us to estimate the free binding energy of complexation from molecular dynamics trajectories.76 This method can be used in applications of virtual screens or docking to refine the classification of poses. In this technique, ΔGbind is assessed from the free energies of the ligand (L)− receptor (R) system in eq 1 ΔG bind = G RL − (G R + G L)

(3)

(1)

The free energy for each individual entity is given by eq 2 where, x symbolizes the receptor or ligand or receptor−ligand complex, EMM is the molecular mechanics potential energy in vacuum, and Gsolv is the free energy of solvation. Here, TS 1252


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Figure 9. Binding free-energy components and RMS fluctuation calculated by MM-PBSA.

presented as mean ± SD, considered statistically significant (P < 0.05). %Viability = 100 (O.D Test Item/O.D of Control) were O.D. is optical density. %Activity = 100 − %Viability. The cytotoxicity assay on normal cells (Hu02) was performed, and the results indicated that unlike cancer cells, in all tested concentrations, Hu02 cells were unaffected by S06, so the proposed compound was not cytotoxic toward normal cell lines (Figure 10c). Cisplatin, [cisdiamminedichloroplatinum(II)], was used as reference agent. The result obtained showed that the cytotoxic activity of (S06) was lower than cisplatin with a higher safety index (SI = 0.73) compared to that of cisplatin (SI = 0.56). SI was obtained using the following equation: SI =

and were used without further purification. Melting points were determined using an electrothermal 9100 instrument. Infrared (IR) spectra were acquired on a Shimadzu FT-IR-8400S spectrometer. 1H NMR (300 MHz) and 13C NMR (60 MHz) spectra were recorded on a Bruker DRX-300 AVANCE. All chemical shifts are given relative to TMS. Jurkat, Hu02, and K562 cell lines were obtained from Institute Pasteur (TehranIran). Characterization of Nanocatalyst MCM-41-SO3H. MCM-41 was sulfonated by covalently bonded sulfonic acid on the inside surface of channels to provide the silica-supported nanomaterial with Brønsted acid properties. MCM-41 was synthesized according to the described method in the literature.80,81 To synthesize MCM-41-SO3H, a 100 mL suction flask equipped with a constant pressure dropping funnel containing chlorosulfonic acid (0.7 mol) and a gas inlet tube for conducting HCl gas over an adsorbing solution was charged with 60.0 g of MCM-41. Then, chlorosulfonic acid was added

IC50 of compound in normal cell line IC50 of compound in cancer cell line

Instruments and Characterization. All chemicals were purchased from Merck, Fluka, and Sigma-Aldrich companies 1253


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dropwise over a period of 30 min at room temperature. HCl gas evolved from the reaction vessel immediately. After completion of addition, the mixture was shaken for 30 min and the white solid (MCM-41-SO3H) obtained was then characterized by scanning electron microscopy (SEM), FT-IR spectroscopy, and acid−base titration. In FT-IR spectroscopy (Figure 11a), the broad band in the region of 3000−3400 cm−1 is assigned to the O−H stretching vibration of hydroxyl groups. The bands at 1324 and 1284 cm−1 are due to the symmetric and asymmetric stretching vibrations of SO of the sulfonic acid group. Moreover, strong bands at 1176 and 1070 cm−1 are assigned to the hydroxyl chain and terminal bonds Si−O asymmetric stretching vibrations, and bands at 887 and 850 cm−1 are related to their symmetric stretching vibrations. In the SEM micrograph, as shown in Figure 11b, the powder is aggregated due to strong hydrogen bonding between the SO3H groups, and some pores are with widths less than 100 nm. So, these confirm that the pores are in the nanoscale. Cytotoxicity Determination of Compounds against Normal Cells. Hu02 (human foreskin fibroblast) IBRC C10309 was obtained from the Iranian Biological Resource Center (Tehran) and cultured in RPMI 1640 medium supplemented with 10% heat inactivated FBS, 2 mM glutamine (Sigma, USA), penicillin (Sigma, USA) (100 IU/ml), and streptomycin (Sigma, USA) (100 μg/mL) at 37 °C in an incubator (5% CO2). The cells were harvested with trypsin (Sigma, USA) (0.25%) and counted using a Neubauer slide, and then, 104 cells were seeded into each well of the 96-well plates. Various concentrations of test compounds (500, 50, 5, and 0.5 μg/mL) were prepared by serial dilution of the stock solution with DMSO. The cells were then incubated (100 μL of the indicated concentrations of compounds/well) at 37 °C. The solvent control wells were loaded with 100 μL of DMSO (concentration ranges of 1 to 0.12%, serial dilution was achieved by RPMI w/10% FBS). Various concentrations of cisplatin (Mylan, Greece) (10, 1, 0.1, and 0.01 μg/mL) were prepared, and 100 μL of each concentration was added to the wells as a positive control. A 12-well cell-culture plate was used as a negative control without any treatments. Three wells were seeded for each concentration, and for each cell line, triple plates were used. Thereafter, the cells were incubated for 24 h. Each single dose and combination was done in triplicate in each test. In order to perform the MTT test, for each concentration studied, 20 μL of a solution of tetrazolium salt MTT (Sigma) (5 mg/mL, in PBS buffer) is added to each well and incubated

Figure 10. Cytotoxic activity of (a) S06 on K562 (IC50= 0.20 μM), (b) S06 on Jurkat (IC50 = 0.44 μM) and Hu02 (IC50 = 0. 32 μM), and (c) cisplatine on Jurkat (IC50 = 0.03 μM) and Hu02 (IC50 = 0.02 μM).

Figure 11. (a) FT-IR spectroscopy. (b) SEM micrograph of MCM-41-SO3H. 1254



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at 37 °C for 3 h. To calculate the percentage of cytotoxicity of lead compounds, the purple precipitated formazan was dissolved in DMSO (100 μL), and the optical density was measured at 545 nm by an ELISA reader (Organon Tekninka, The Netherlands).

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.6b00138. Optimization of reaction condition, general procedure, and mechanism. (PDF)

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CONCLUSION

In summary, the present procedure provides an efficient and improved modification of the Biginelli-type reaction which provides 4H-chromone-1,2,3,4-tetrahydropyrimidone of potential synthetic and pharmaceutical importance. MCM-41-SO3H was identified as an effective, nanobased, and reusable catalyst, and the reaction proceeded at room temperature. In addition, the products were isolated without the need for purification by chromatography. Due to mentioned advantages, the present method is preferred over the previously reported methods in order to produce a 4H-chromone-1,2,3,4-tetrahydropyrimidone-based library of structurally diverse small molecules useful for medicinal chemistry and drug discovery. The in silico inhibition studies of synthesized compounds binding to the Abl kinase domain showed that all the compounds, and particularly S06 (4f), exhibited high Abl binding affinities (−10.10 kcal/ mol by AutoDock 4 and −8.50 by AutoDock Vina). These results are in accordance with the fact that S06 configuration can assume in the Abl binding site a favorable orientation in analogy with the proposed binding mode of Imatinib in the ATP site of Abl. For a more detailed study of binding modes of S06 and to elucidate the effects of ligand binding on Abl conformation, a molecular dynamics simulation was performed on a ligand−protein complex. A modification in the position and orientation of S06 in the ATP site was observed in terms of the MD simulation, indicating the usefulness of MD simulation after docking of S06. The molecular dynamics simulations revealed the stability of the ligand−protein complex. The results further suggested that THR 315, ASP 381, and LYS 271 could play an important role for the further optimization of selective inhibitors. We performed a theoretical experiment using MD simulation for 10 ns without defining the initial coordinates, and we directly studied the binding affinity of the ligands with the target molecule, which revealed that the ligand approached the active sites. On the other hand, the obtained results from redocking suggest that the active site is fully compliant with the ligand and may be considered as a competitive inhibitor of ATP. We also evaluated the relative free binding energies by MM/PBSA using g_mmpbsa tool for the last 15 ns MD. The result showed a negative binding energy value of −73.6 kcal/mol which is higher than docking result (−10.10 kcal/mol). The cytotoxic properties of S06 evaluated by a cytotoxicity assay using the MTT method on specified human cell lines (Jurkat, K562, and Hu02) indicated that S06 is not cytotoxic compared to cisplatin. The in silico results indicated that the proposed structure, which has no toxic effects, is a potential tyrosine kinase inhibitor (TKI) that targets Bcr-Abl and thus prevents cell proliferation, but not necessarily apoptosis, and might potentially constitute an interesting novel class of targeted antileukemic agents, which deserve further studies. However, the enzyme inhibitory activity of the compound must be approved by a special in vitro test such as the Abl (Y253F) kinase assay, which is in our future plans.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] ORCID

Shahrzad Javanshir: 0000-0002-3161-0456 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the support of the Research Council of the Iran University of Science and Technology, Tehran, Iran.

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