Synthesis, biological evaluation and molecular docking studies of a new series of bis-chalcones

A convenient and efficient reaction for the synthesis of several new bis-hydroxy derivatives of chalcones 4a–h was accomplished via the two-directional Claisen–Schmidt condensation of different ketones 3a–e with premade benzaldehyde 2 under alkaline conditions. Products were obtained with reasonable yields and with excellent purity. The in vitro antioxidant activity of bis-hydroxychalcones was assessed by DPPH radical scavenging activity in comparison with ascorbic acid, and products showed good antioxidant activity. To determine the possible binding mechanisms with the active site of tubulin and bovine xanthine oxidase (BXO), docking analyses were performed. According to the docking results, compounds 4b and 4a showed the best binding affinity of − 9.13 and − 9.11 kcal/mol for tubulin, respectively. Furthermore, as before, the greatest scores for the case of BXO belong to 4b and 4a with the scores of the values − 10.32 and − 9.81 kcal/mol.


Introduction
Chalcones are natural phenolic compounds widely found in nature and considered as the precursors of flavonoids and isoflavonoids in plants. Chemically, they consist of open-chain flavonoids with two aromatic rings connected by carbonyl groups and two α,β-unsaturated carbon atoms [1]. Chalcones form an important class of natural compounds that serve as starting materials for the synthesis of heterocyclic derivatives like pyrazolines [2,3], epoxyketones [4] and imidazoles [5]. In the organic synthesis, chalcones are privileged structures due to their high reactivity and their usefulness in medicinal chemistry and displaying a variety of important pharmacological activities [6][7][8]. Our previous investigation of this type of molecules showed that many natural and synthetic chalcones, bis-chalcones and their derivatives exhibit a wide spectrum of biological activities like anti-malarial [9], antiviral [10], anticancer activities [11,12], anti-inflammatory [13], antibacterial [14], anti-platelet [15,16], antioxidant [17]. The attendance of reactive α,β-unsaturated keto group in chalcones is known responsible for their biological activity. Especially, a large number of chalcones and their derivatives exhibited potent antiproliferative activity due to tubulin inhibition and the disturbance of microtubules which are essential ingredients of the cytoskeleton and affect a series of cellular processes such as keeping of cell shape, mitosis, intracellular transport and regulation of mobility [18][19][20]. Therefore, tubulin inhibitors have been recognized as an attractive target for anticancer agents [21][22][23][24][25].
In the last decade, the use of molecular computational modeling methods for the rational design of drugs accelerates the process of discovering new compounds with pharmaceutical properties and has greatly helped to save costs and reduce time. In addition, molecular docking is a valuable way to better understand receptor-ligand interactions [26,27].
As a part of our search for the synthesis of new compounds and screening for their biological activities, in this study, we report herein the design and synthesis of novel bis-hydroxychalcones. Molecular docking has been carried out to investigate the interaction of inhibitors with the colchicine binding site of tubulin and also interaction of inhibitors with bovine xanthine oxidase (BXO). In addition, the DPPH assay was done for screening the antioxidative activity of new synthetic chalcones by the scavenging of free radicals.

Chemistry
In this study, relying on our prior projects related to the two-directional synthesis and with the aim of developing effective synthesis of new compounds and 1 3 Synthesis, biological evaluation and molecular docking studies… designing novel molecules with pharmacological activities [28,29], the synthesis of bis-hydroxychalcone derivatives (Scheme 1) is reported. Initially, for the synthesis of 4-methoxy/methyl 2,6-diformyl phenol 2 the 4-methoxy/methyl, phenol 1 was participated in the Duff reaction and refluxed for 20 h by using HMTA (hexamethylenetetramine) and TFA (trifluoroacetic acid), after which the medium is acidified and the temperature of the reaction mixture reached 110 °C for additional 30 min reflux. Then, bis-hydroxychalcone derivatives were synthesized from reaction of 2 eq. ketone 3a-e, and 1 eq. of premade benzaldehyde 2 under alkaline conditions. All eight new bis-hydroxychalcone derivatives 4a-h were designed, synthesized, purified and determined.
All of the products described in Scheme 1 were determined by FT-IR, 1 H NMR, and 13 C NMR spectra. The FT-IR spectrum of bis-hydroxychalcones 4a-h indicated the presence of OH stretching vibration band at 3200-3500 cm −1 , the absorption band at the range of 1648-1691 cm −1 corresponding to carbonyl C = O stretching vibration and absorption bands in the 1574-1610 cm −1 and 1456-1465 cm −1 regions related to aromatic C = C stretching peaks.  Also, in the 1 H NMR spectra of bis-hydroxychalcones 4a-h the expected number of various protons appeared within appropriate coupling constants (J) at their anticipated chemical shifts (ppm). Phenol hydrogen signal appeared as a single or broadband in the range of 9.05-9.97 ppm. Aromatic and aliphatic protons were appeared at 6.79-8.28 and 1.25-3.95 ppm, respectively. In the 13 C NMR spectra of bis-hydroxychalcones 4a-h signals attributed to the carbonyl groups appear at 152.77-155.66 ppm. The signals assigned to the aromatic and aliphatic carbons, with the expected number and chemical shifts appeared at 163.64-20.48 ppm. (See the "Supplementary material" file).
The structure of the synthesized compounds is described in Table 1.

Molecular modeling (docking) studies
Tubulin inhibition To evaluate the possible effects of the synthesized compounds in the treatment of cancer, we proceeded to examine the interaction of compounds 4ah with tubulin (PDB ID: 1SA0). The molecular docking was done by simulation of compounds into the colchicine binding site of tubulin using the Schrodinger program [30]. The 2D ligand-receptor interaction diagrams of the desired compounds with tubulin and the theoretical 3D binding mode of compounds with highest scores to the mentioned receptor are shown in Fig. 1, and their corresponding calculated Glide Score values are collected in Table 2.
In the binding modes of these compounds with tubulin (1SA0), despite the absence of any direct observable interaction, the greatest binding energies have been obtained. Structure such as 4b with terminal naphthalene group as an aromatic moiety fit well in the pocket of D chain surrounded by residues such as Leu-252, Leu-255, Ala-316 and Cys-241 results in stabilizing hydrophobic interaction. On the other hand, the situation of 4a which differs only in substituent at the central benzene ring (methyl instead of methoxy group) with 4b but representing nearly equal score leads to the conclusion that substitution at this position has little effect on the ligand-receptor interaction. Furthermore, obviously the rule of structural extension is playing role in the case of 4b. Lower score of 4c as well as more bended and twisted conformation presumably is the result of induced repulsion in between polar furan moiety and the nearby residues of chain C containing the same charge.

Xanthine oxidase inhibition
To study the effectiveness of synthesized compounds as antioxidant, we have investigated the inhibitory effect of their compounds on bovine xanthine oxidases (PDB ID: 3NRZ) using the Schrodinger software. 2D ligandreceptor interaction diagrams of the compounds 4a-h with BXO and the theoretical 3D binding mode of compounds with highest scores to the mentioned receptor are presented in Fig. 2, while their corresponding calculated binding score values in the interaction with BXO are collected in Table 3.
According to the docking results, the order of three higher scores molecules appears to be 4b > 4a > 4 g, respectively. The most important observed interactions Synthesis, biological evaluation and molecular docking studies… of 4b are hydrogen bonding types, especially because of the presence of two substituents on the central benzene ring. The hydroxyl group behaves as HBD to MET-1038 and as an HBA through the oxygen atom of the methoxy group with ARG-912. These two interactions hold the middle part of the molecule at the entrance of the pocket tightly, thereby decreasing its degrees of freedom of translational motion providing the opportunity to rotation around the single bonds in between vinyl and carbonyl groups adjacent the penetrated naphthalene moiety, as well as around the single bond between carbonyl and naphthalene groups. Decreasing the planarity of these three functional groups increases the fitting ability of the half of the structure in the pocket consisting of residues such as THR-1077, ALA-1078, ALA-1079, SER-1080, SER-1082 and GLN-1040. The results indicate lacking of any pi-pi interaction anywhere, but there is a weak interaction of one hydrogen at the alpha position of terminal benzene ring of naphthalene group with oxygen atom of hydroxyl group of SER-1082; this interaction depends to the degree of approaching of the naphthalene group in the pocket; in other words, to the length of the half part of the molecule, for example in the case of structure 4a this interaction is entirely lost. The absence of methoxy group lowers the degree of establishment at Synthesis, biological evaluation and molecular docking studies…  the place and preference of the other orientation of the naphthalene group. The presence of exo double bond in 4 g restricts the rotation of terminal ring; besides that, the shorten length of 4 g comparing to 4b altogether would lead to lack of appropriate orientation and consequently losing the mentioned interaction.

DPPH assay
In this study, in addition to investigated the inhibition of BXO enzyme through docking study to evaluate the antioxidant properties of the compounds, the DPPH method was employed for screening the antioxidative activity of new synthetic chalcones by the scavenging of free radicals. DPPH (2, 2-diphenylhydroxyl) is a stable free radical with the ability to accept an electron or hydrogen radical and convert it to stable molecules that can use to the assay radical scavenging ability of materials. Interaction of DPPH with new synthetic compounds leads to a decrease in the absorption band at 517 nm, due to the reduction in the amount of DPPH radical. The reduction in absorption band relies on the tested compounds antioxidant capacity. When the antioxidant capacity of the test compound is higher, the decrease in absorption becomes more. The antioxidant activities of compounds were determined in concentrations of 2000, 1000, 500, 250 and 125 μg/mL in MeOH at 517 nm for the DPPH test. Ascorbic acid (vitamin C) was used as a positive standard. Also, their IC 50 values (which are the concentration of tested compounds to scavenge 50% of the DPPH radical concentration) were calculated from the line equation DPPH radical scavenging activity of synthesis compounds ( Table 4). As is shown in Fig. 3 Fig. 4, the stronger antioxidant activity is reflected in a lower IC 50 . Therefore, products 4a-h were determined to exhibit potent antioxidant

3
Synthesis, biological evaluation and molecular docking studies…   activity in a DPPH assay with IC 50 values. The high antioxidant activity of products could be because of the longer conjugated system which can stabilize the free radical resonance via a longer system.

Conclusions
Several new series of bis-hydroxychalcones were synthesized through a reliable procedure for great yields and high purity. Products demonstrated high antioxidant activity in the presence of DPPH and products possessed the higher antioxidant activity than ascorbic acid. The high antioxidant activity of compounds could be because of the longer conjugated system which can fixate the free radical by resonance via a longer system. The docking study of the synthesized molecules at the active sites of enzymes tubulin (1SA0) and BXO (3NRZ) has been explored. According to the calculated ligand-receptor interactions of the compounds, ligands 4a and 4b gained the highest scores toward both of protein.

Materials and methods
All chemical materials and solvents were prepared from Merck, Aldrich, and Fluka and were used without further purification. The purity definition of the synthesized compounds and reaction monitoring was carried out by thin layer chromatography (TLC) on silica-gel polygram SILG/UV 254 plates. The FT-IR spectra were recorded in KBr disks on a Shimadzu IR-470 spectrometer. 1 H NMR and 13 C NMR spectra were recorded on 400,500,100 and 125 MHz Bruker spectrometers using DMSO-d6 and CDCl 3 as solvents. Spectra were internally referenced to tetramethylsilane (TMS). All chemical shifts were reported as (ppm) values. Melting points were determined on a Büchi B-545 apparatus in open capillary tubes and are uncorrected.

General procedure for the synthesis of 4-methoxy/methyl 2,6-diformyl phenol 2
Firstly, 4-methoxy/methyl 2,6-diformyl phenol 2 was synthesized through the Duff reaction; in this way HMTA (hexamethylenetetramine) (110 mmol) was added to a solution of 4-methoxy/methyl phenol 1 (50 mmol) in CF3CO2H (TFA) (55 ml), and the mixture was stirred well at 118-125 °C for 20 h in an oil bath. Then, HCl 3 N (75 ml) was added to the reaction mixture and again stirred for 30 min. After the completion of the reaction, the resulting mixture was poured into a 200 ml flask. The needle-shaped precipitate was filtered and washed with H2O: EtOH (1:1). Then, resulting solid was recrystallized from EtOH to give the pure product.

General procedure for the synthesis of bis-hydroxychalcones (4a-h)
Aromatic aldehydes 2 (1 mmol), ketones 3a-f (2 mmol) and EtOH (5 ml) were added in a pyrex flask (50 ml) placed in an ice bath. To this mixture was added a solution of KOH (8 ml, 60%) dropwise with continuous stirring for 30 min, and the mixture was allowed to stir for another 12 h at r. t. Then, the crude mixture was neutralized with dilute HCl and the resulting precipitate was filtered and washed with H 2 O:EtOH (60:40), recrystallized from EtOH to give the pure bis-chalcones 4a-h. Products as powdered solids with % yields are shown in Table 1.

Molecular docking
The tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: It has been shown that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly. Molecular modeling was performed using the Schrodinger program, providing information to understand their binding mode with the colchicine binding site of tubulin (PDB ID: 1SA0) emphasizing the presence of the active site in between chains C and D, and that of bovine xanthine oxidase (BXO) with (PDB ID: 3NRZ) in chain C. The 3D-crystal structure of the desired proteins with a resolution of 3.58 and 1.8 Å for 1SA0 and 3NRZ, respectively, was obtained in PDB format from protein data bank [31]. The preparation of proteins for docking by protein preparation wizard involves removing all hetero atoms, eliminating water molecules, addition of hydrogen to the heavy atoms and assigning charges and proper bond order. Tautomeric states were generated at pH of 7.0 ± 2 employing Epik [32], and the energy minimization of protein structure was performed using OPLS3 force filed [33,34]. Also, the ligands were prepared using ligprep module in Schrodinger suite 2017-1 with an OPLS3 force field. The molecular docking simulation was done using glide docking [35] implemented in Schrodinger suite 2018-1 with the prepared ligands and a standard drug, docked into the active site of the receptor.

Antioxidant activity by DPPH assay
The DPPH radical scavenging activity of products was estimated according to the literature [31]. An appropriate amount of DPPH was dissolved in MeOH to give a concentration of 6.25 × 10-5 M. Then, 3.5 mL of fresh DPPH solution was added to 0.5 mL of sample solutions in different concentrations (2000, 1000, 500, 250, 125, 62.5 mg/mL in MeOH) and the samples were shaken vigorously and were kept in dark for 30 min at 37 °C; then, the reduction in the absorbance of the resulting solution was measured at 517 nm. MeOH was used as blank, and a sample of 3.5 mL DPPH solution containing 0.5 mL of MeOH instead of sample was used as control.
Inhibition of free radical DPPH in percent was evaluated as follows: Radical scavenging activity% = (A blank − A sample ∕ A blank ) × 100 where A blank is the absorbance of control reaction (DPPH + MeOH) and A sample is the absorbance of the test.
compounds and all the reagents. IC 50 values of the test compounds were calculated by plotting inhibition percentage against sample concentration.