Molecular modelling studies and synthesis of novel quinoxaline derivatives with potential inhibitory effect on GSK-3β

Purpose: To synthesize quinoxaline derivatives and investigate their inhibitory effects on glycogen synthase kinase (GSK)-3 β in vitro. Methods: Quinoxaline derivatives were synthesized via reaction between synthon 1 and DL- 2-amino succinic acid, and subsequent lactamization reaction. The new compounds were tested against GSK-3 β in vitro to select the most potent compound which was then used for molecular modelling. Results: Novel quinoxaline derivatives with quinolone nucleus were successfully synthesized via simple chemical reactions. The compounds markedly inhibited GSK-3 β , with compound 45 [3-(carboxymethyl)- 5-fluoro-10-(4-fluorophenyl)-2,7-dioxo-1,2,3,4,7,10-hexahydropyrido [2,3-f] quinoxaline-8-carboxylic acid] achieving the best effect (IC 50 = 0.18 μ M). The half maximal inhibitory concentrations (IC 50 ) of the compounds were in micromolar range. Molecular modelling revealed several interactions between compound 45 and the binding site of GSK-3 β . Conclusion: These results indicate that 3-(carboxymethyl)-5-fluoro-10-(4-fluorophenyl)-2,7-dioxo- 1,2,3,4,7,10-hexahydropyrido [2,3-f] quinoxaline-8-carboxylic acid is a potent inhibitor of GSK-3 β and is thus a promising scaffold for the development of novel drugs that can effectively inhibit GSK-3 β signaling pathway.

Molecular modelling is used to unravel binding interactions between newly synthesized compounds and potential target enzymes/proteins [9,10]. The technique has been successfully employed for the elucidation of the crystal lattice structure of GSK-3β.
Heterocyclic compounds are a class of substances, which play critical roles in drug discovery through their incorporation into the structures of a large variety of drugs used for the treatment of diverse diseases. Quinoxaline is an important heterocyclic nucleus with a wide spectrum of biological activity. Quinoxaline scaffold possesses promising therapeutic properties such as anticancer, antimalarial, antiinflammatory, antimicrobial and anti-HIV effects [11]. It has been used as scaffold in drugs that function as protein kinase inhibitors [11]. Quinoline pharmacophore possesses antibacterial, anticancer and kinase inhibitory activities [12,13]. The aim of this study was to synthesize quinoxaline derivatives and investigate their inhibitory effects on GSK-3β in vitro.

EXPERIMENTAL Chemicals and reagents
All reagents and chemicals used in this study were of analytical grade, and they were products of Sigma-Aldrich (USA). Glycogen synthase kinase (GSK)-3β assay kit was obtained from Thermo Fisher Scientific Co. Ltd (USA).

Technique
Melting point (mp) was measured with Stuart scientific electrothermal heating apparatus (EA3000 A). Infra-red (IR) spectrum was recorded using Shimadzu FT-IR spectrophotometer (8400F). Proton and carbon nuclear magnetic resonance (NMR) spectra were analyzed with Bruker Avance spectrometer (DPX-300), while molecular mass was measured with high resolution mass spectrophotometer (Bruker APEX-4).

Synthesis of synthon I derivatives
Synthon I was produced as a product of the reaction between the synthons (a, b and c) and DL-2-amino succinic acid (Scheme 1) [ 13].

Synthesis of synthon II
Exactly 6.0 g of sodium dithionite powder (43.5 mmol) was dissolved in 0.33 L of distilled water and then gradually added to 1.0 g of synthon I for 45 min at room temperature (Scheme 2). Reduction of nitro group on position 8 in synthon I to amino group was done via addition of aqueous sodium dithionite in potassium carbonate. Spontaneous lactamization led to the formation of synthon II. The reductive cyclization process was fast and direct, lasting 60 -120 min. [17,18].
Spectrum analysis was carried out to confirm that the synthesized compound was synthon II.

Preparation of synthon II derivatives for in vitro assay
Exactly 10 mg of each synthon II derivative (compounds 6, 23 and 45) was dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions of required concentrations, which were sent to Thermo Fisher Scientific Co. Ltd. (USA) where activity of GSK-3β was assayed.

Hits profiling against GSK-3β
To determine IC50 values of synthon II derivatives, inhibition of GSK-3β using Z'-LYTE GSK-3β assay was performed with varied concentrations of each compound (Z'-LYTETM Screening Protocol and Assay Conditions, 2016). The inhibition and concentration data were used to determine IC50 for each compound, and the most active compound was then selected. Solution of 10 mM concentration of each synthetic compound (6, 23 and 45) was prepared in DMSO and sent for analysis at Thermo Fisher Scientific Co. Ltd. (USA) [16].

Docking settings
LibDock is a site-feature docking algorithm that docks ligands into active sites under guidance by binding hotspots. The most potent of synthesized quinoxaline derivatives (based on IC50 values) were docked into the binding pocket of GSK-3β (PDB code: 3Q3B; resolution of 2.7 Ǻ) using LibDock (Discovery Studio version 4.5). Apart from ensuring the fitting of hypothesized molecule into the binding pocket, the procedure also aided the visualization of bonds formed between the compound and amino acids in the binding pocket of GSK-3β. Molecular modelling provided clues to binding forces and inhibitory activity of the compound. The LibDock tool consisted of two major parts: allocation of binding site in the receptor, and running of docking procedure. Docking was performed via allocation of conformations of the ligand to polar receptor interaction sites and apolar ones (hotspots). A catalyst was added to ensure that conformations formed on the fly. CHARMM-based algorithm was used to analyze the interaction between the complexes formed.

Properties of synthesized compounds
Novel quinoxaline derivatives with quinolone nucleus were successfully synthesized via simple chemical reactions. The properties of the synthesized synthons are shown below:

Inhibition of GSK-3β activity in vitro
In vitro inhibitory activities of synthon II derivatives (compounds 6, 23 and 45) were tested against human recombinant GSK-3β. Each compound was screened at an initial concentration of 10 nM. The results showed that inhibitory effect of compound 45 was better than those of compounds 6 and 23 (Table 1; Figure  4).

Molecular modelling results
The binding interactions of compound 45 with GSK-3β were determined using LibDock tool. The procedure revealed many interactions between compound 45 and the binding site of the enzyme (Figures 5 and 6).

DISCUSSION
The need to find novel GSK-3β inhibitors is of great importance, since the enzyme is involved in many biological processes. In this study, many compounds were synthesized and tested against GSK-3β. The results of in vitro activity assay showed that compound 45 exhibited potent inhibitory activity against GSK-3β. Molecular modelling revealed that compound 45 assumed different conformations inside the binding pocket of GSK-3β. The quinoline nucleus exhibited remarkable interactions. It interacted with Leu 188 through sigma bond formation, and with Leu 132, Val 70 and Ala 83 via pi-alkyl interaction.
In other poses, the quinoline nucleus of compound 45 formed sigma bond with Val 70, and pi-alkyl interaction with Ala 83 and Leu 188.