Synthesis, molecular modelling, and biological evaluation of novel quinoxaline derivatives for treating type II diabetes

Abstract

Quinoxalines are benzopyrazine derivatives with significant therapeutic impact in the pharmaceutical industry. They proved to be useful against inflammation, bacterial, fungal, viral infection, diabetes and other applications. Very recently, in January 2024, the FDA approved new quinoxaline containing drug, erdafitinib for treatment of certain carcinomas. Despite the diverse biological activities exhibited by quinoxaline derivatives and the role of secretory phospholipase A2 (sPLA2) in diabetes-related complications, the potential of sPLA2-targeting quinoxaline-based inhibitors to effectively address these complications remains unexplored. Therefore, we designed novel sPLA2- and α-glucosidase-targeting quinoxaline-based heterocyclic inhibitors to regulate elevated post-prandial blood glucose linked to patients with diabetes-related cardiovascular complications. Compounds 5a–d and 6a–d were synthesised by condensing quinoxaline hydrazides with various aryl sulphonyl chlorides. Biological screening revealed compound 6a as a potent sPLA2 inhibitor (IC₅₀ = 0.0475 µM), whereas compound 6c most effectively inhibited α-glucosidase (IC₅₀ = 0.0953 µM), outperforming the positive control acarbose. Moreover, compound 6a was the best inhibitor for both enzymes. Molecular docking revealed pharmacophoric features, highlighting the importance of a sulfonohydrazide moiety in the structural design of these compounds, leading to the development of potent sPLA2 and α-glucosidase inhibitors. Collectively, our findings helped identify promising candidates for developing novel therapeutic agents for treating diabetes mellitus.

HIGHLIGHTS

• A small, focused library comprising 8 novel compounds was synthesised using a series of substituted quinoxaline sulfonohydrazide derivatives.

• All synthesised compounds were tested against phospholipase A2 (sPLA2) and α-glucosidase enzymes.

• The compounds exhibited activities against α-glucosidase and were potent at nanomolar concentrations against sPLA2 isozymes.

• Structure-based molecular modelling was employed to rationalise the SAR of the compounds.

Introduction

Quinoxaline, characterised by a pyrazine moiety fused with benzene ring, is a constituent of various commercially available drug molecules, including riboflavin. Notably, quinoxaline also accounts for various biological activities; for instance, echinomycin¹ and erdafitinib² containing quinoxaline moiety, the first is an antibacterial and antineoplastic agent with nucleic acid inhibitory activity, the second is approved to treat urothelial carcinoma. Similarly, varenicline is a cholinergic and partial agonist for nicotinic receptor whereas dioxidine and mequindox are antibacterial agents¹. Carbadox controls swine dysentery², panadipion acts as a hepatoprotective agent, and sulfoquinoxaline is a veterinary medicine used for treating coccidiosis in cattle and sheep³. Moreover, quinoxaline analogues with diverse biological activities and synthetic pathways have been patented (Figure 1).

Figure 1.

Example of some quinoxaline drugs.

The primary global public health concerns among patients with diabetes are heart disease, stroke, circulatory failure, and kidney insufficiency⁴. Demir’s studies explore the impact of quinone chemicals, particularly naphthoquinones, benzoquinones, and anthraquinones, on human blood paraoxonase-1 (PON1), an enzyme associated with high-density lipoprotein (HDL). PON1 functions to shield LDL from oxidation and eliminate harmful chemicals through detoxification. The findings emphasise the potential of quinone derivatives as inhibitors and their significance in the treatment of cardiovascular disorders and cancer. Demir concluded that reduced PON1 levels are associated with conditions such as diabetes mellitus, cardiovascular diseases, hyperthyroidism, and chronic renal failure. Demir’s study investigated the interactions between some antihypertension drugs and PON1. Notably, these drugs exhibited potential inhibitor properties for PON1, with IC₅₀ values ranging from 131.40 to 369.40 μm and Ki values from 56.24 ± 6.75 to 286.74 ± 28.28 μm. Midodrine and nadolol exhibited competitive inhibition, while atenolol and pindolol showed non-competitive inhibition. It is essential to consider the potential risks associated with using these drugs, especially for patients with hyperthyroidism and cardiovascular diseases^5,6. Dysregulated lipid metabolism in patients with impaired blood glucose levels results in fat deposits in the arteries, leading to atherosclerosis^7,8, a prominent complication of diabetes. Therefore, a recent consensus has been established regarding the key lipid metabolic markers, such as phospholipase A2 (PLA2), which are associated with coronary heart disease (CHD). PLA2 is a group of enzymes that hydrolyse the sn-2 ester bond of membrane phospholipids to yield fatty acids and lysophospholipids^9,10. Furthermore, the hydrolytic activity of secretory PLA2 (sPLA2s) releases arachidonic acid (AA) to the neighbouring tissues and acts as a precursor of leukotrienes and inflammatory prostaglandin mediators. Dysfunction of sPLA2 in affected tissues is associated with various disorders such as Alzheimer’s disease¹¹, sepsis¹², asthma¹², rheumatoid arthritis^12,13, cancer¹⁴ and many others¹⁵. Chronic elevation of blood glucose levels is associated withs sPLA2s activation, AA generation, inflammatory conditions, CHD, and eicosanoids^16,17.

Inhibiting sPLA2s holds significant potential in improving diabetes-associated inflammatory and cardiovascular complications. Carbohydrate hydrolases, particularly α-glucosidase¹⁸, play a crucial role in elevating post-prandial blood glucose, as they hydrolyse polysaccharides to monomeric sugars for absorption, contributing to increased postprandial blood glucose levels¹⁹. Scientists isolated aldose reductase (AR) from cow liver and examined the inhibitory effects of several chemicals on AR, α-glycosidase, and α-amylase. All substances exhibited significant inhibition of α-glycosidase and α-amylase, indicating their potential as therapeutic agents for the treatment of diabetes and hyperglycaemia. Pyrazolyl-thiazole derivatives exhibited significant inhibition of both AR and α-glycosidase. Focusing on regulating blood sugar levels after meals may enhance the management of blood sugar and decrease the likelihood of problems. Alpha-glucosidase inhibitors, such as acarbose and miglitol, lower blood sugar levels after meals, enhance lifespan, decrease the risk of cardiovascular disease, stabilise plaques in the carotid arteries, and counteract high blood sugar, oxidative stress, and dysfunction of the inner lining of blood vessels. These studies emphasise the significance of controlling postprandial glucose levels in the treatment of diabetes^20,21. Therefore, inhibiting these enzymes is crucial in regulating postprandial blood glucose, and various inhibitors have been reported and marketed for their antihyperglycemic effect. Demir et al undertook a study to examine how dietary phenolic chemicals can hinder the activity of important enzymes involved in glucose metabolism. More precisely, the researchers directed their attention towards α-amylase, aldose reductase (AR), and α-glycosidase. Aldose Reductase (AR) is an enzyme. AR is the initial enzyme in the polyol pathway, catalysing the conversion of glucose into sorbitol. It has a pivotal function in the development of diabetes complications. The study isolated AR from sheep kidneys and discovered that several quinones efficiently suppressed AR activity. Significantly, anthraquinone demonstrated strong inhibitory effects on AR. The phenolic substances examined in this study shown effective inhibition against both α-amylase and α-glycosidase. These chemicals have the potential to act as specific inhibitors for controlling blood glucose levels. Additional studies were concentrated on bromophenol derivatives. The inhibitory effects of these substances on AR, α-glucosidase, and α-amylase were assessed. Bromophenol derivative had the most potent inhibitory effect against AR. The investigated compounds effectively inhibited α-glucosidase, they also inhibited α-amylase, with IC₅₀ values ranging from 9.63 to 91.47 nM. The bromophenol derivatives show potential as antidiabetic medicines by specifically targeting certain metabolic processes^22,23.

In this study, we synthesised novel heterocyclic sPLA2 inhibitors based on a quinoxalinone scaffold and assessed their activity against sPLA2 IIA. Their ability to inhibit α-glucosidase was evaluated to assess their potential in controlling postprandial blood glucose^9,18,24. The inhibitory results against carbohydrate hydrolases were compared with those of a well-known flavonoid α-glucosidase inhibitor^25–27. Collectively, we believe that our findings would help evaluate the potential of the new compounds in inhibiting proatherogenic sPLA2 targets and controlling hyperglycaemia, thereby offering promising approaches for managing hyperglycaemia and concurrent cardiovascular complications in patients with diabetes.

Materials and methods

General methods

The melting points of the compounds were determined using a Gallenkamp melting point apparatus. The infrared (IR) spectra were recorded in potassium bromide (KBr) discs using a PerkinElmer FT-IR (Spectrum BX) spectrophotometer, with the ν_max measured in cm⁻¹. For the ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectra, a BRUKER Resonance spectrometer (850 MHz) was used, with coupling constants given in Hertz (Hz). Deuterated dimethyl sulfoxide (DMSO)-d6 solvent was procured from Goss Scientific Instruments and stored in silica gel desiccators. The structures of the compounds were confirmed using spectral data, including IR, NMR, and elemental analysis. The purity of all the compounds was established to be over 95% using liquid chromatography-mass spectrometry (LC–MS), and the results were within 0.4% of the calculated values. Any known compounds were identified by comparing their analytical and physicochemical data with previously reported data.

Chemistry

Synthesis of benzenesulfonohydrazide and bisbenzenesulfonohydrazide derivatives 5a–d and 6a–d

These derivatives were synthesised based on the procedure described by Siddiqa et al. 33 (Scheme 1). Distilled water (15 ml) and tetrahydrofuran (15 ml) were added to 0.005 mol of hydrazide derivatives (3 and 4). The pH was set at 8–10 using sodium carbonate solution, and 0.005 mol of substituted benzene sulphonyl chloride were gradually added (5a–d and 6a–d) The reaction mixture was brought to room temperature and further stirred for 6–10 h, and monitored using thin-layer chromatography (TLC; 8:2 chloroform/methanol). After the completion of the reaction, the separated solid products were filtered, washed, and dried.

Scheme 1.

Synthetic route for the preparation of the target compounds.

N’-(3-chloroquinoxalin-2-yl)-4-methylbenzenesulfonohydrazide 5a

Yield: 50.24%, as a red solid, mp 150–152 °C, IR (KBr, cm⁻¹) ν_max = 3319 (N–H), 3145 (C–H, sp₂), 1563 (C = N), 1160–1335 (S = O), 758 (C–Cl).¹H NMR (850 MHz, DMSO-d₆) δ (ppm): 10.11 (s, 1H, NHSO₂), 9.86 (s,1H, NH), 7.85 (dd, 1H, J = 8.5 Hz, 1.7 Hz, H-5), 7.73 (d, 2H, J = 7.65 Hz, H-2′& H-6′), 7.59 (t, 1H, J = 8.5 Hz, H-6), 7.46 (t, 1H, J = 6.8 Hz, H-7), 7.39 (m, 3H, H-4′ & H-5′), 7.32 (d, 1H, J = 6.8 Hz, H-8).¹³C NMR (850 MHz, DMSO-d₆) δ (ppm): 147.13, 140.33, 139.63, 137.11, 136.01, 133.13, 130.83, 128,93, 128,17, 127.78, 126.82, 126.33. LC–MS (ESI), RT = 4.13–4.31 min, m/z 335.1 [M + H]⁺.

N’-(3-chloroquinoxalin-2-yl)-4-methylbenzenesulfonohydrazide 5b

Yield: 64.36%, as an orange solid, mp 190–193 °C, IR (KBr, cm⁻¹) ν_max = 3312 (N–H), 3147 (C–H, sp²), 1609 (C = N), 1159–1335 (S = O), 757(C–Cl). ¹H NMR (850 MHz, DMSO-d₆) δ (ppm): 9.99 (s, 1H, NHSO₂), 8.30(s, 1H, NH), 7.71 (dd, 1H, J = 10.6 Hz, 5.9 Hz, H-5), 7.65 (t, 1H, J = 9.3 Hz, H-6), 7.48 (d, 2H, J = 7.6 Hz, H-2′ & H- 6′), 7.32 (d, 1H, J = 8.5 Hz, H-8), 7.23(t, 1H, J = 7.6 Hz, H-7), 7.12 (d, 2H, J = 8.5 Hz, H-3′ & H-5′), 2.28 (s, 3H,phenyl-CH3).¹³C NMR (850 MHz, DMSO-d₆) δ (ppm): 151.19, 145.70, 143.59, 138.39, 131.15, 130.73, 129,65, 129.39, 128.62, 125.95, 21.19. LC–MS (ESI), RT = 6.4 min, m/z 349. [M + H]⁺. HR–MS (EI+, m/z) molecular ion requires for C₁₅H₁₃ClN₄O₂S, 348.81; found 349.05.

N’-(3-chloroquinoxalin-2-yl)-4-(trifluoromethyl) benzenesulfonohydrazide (5c)

Yield: 89.37%, as an orange solid, mp 184–186 C˚, IR (KBr, cm¹) ν_max = 3323- (N–H), 3103(C–H,sp²), 1605 (C = N),1167–1345(S = O),766 (C–Cl).¹H NMR (850 MHz, DMSO-d₆) δ (ppm): 9.97(s, 1H, NHSO₂), 8.42(s, 1H, NH), 8.02 (d, 2H, J = 7.6 Hz, H-3 & H-5′), 7.77(d, 2H, J = 8.5 Hz, H-2′&H-6′), 7.72 (d, 1H, J = 8.5 Hz, H-5), 7.54(t, 1H, J = 7.6 Hz, H-7), 7.46(t, 1H, J = 7.6 Hz, H-6), 7.14 (d, 1H, J = 8.5 Hz, 8-H), ¹³C NMR (850 MHz, DMSO-d₆) δ (ppm): 146.69, 144.67, 139.36, 137.08, 135.87,132.74, 132.59, 130.71, 129.07, 127,78, 126.91, 126.21, 126.01. LC–MS (ESI), RT = 4.87 min, m/z 403 [M + H]⁺.

4-Bromo-N’-(3-chloroquinoxalin-2-yl)benzenesulfonohydrazide 5d

Yield: 50.24%, as a red solid, mp 187–188 °C, IR (KBr, cm⁻¹) ν_max = 3316 (N–H), 3177 (C–H, sp2), 1611 (C = N), 1164–1343 (S = O),760 (C–Cl), 600 (C–Br). ¹H NMR (850 MHz, DMSO-d₆) δ (ppm): 10.25(s, 1H, NHSO₂), 8.40(s, 1H, NH), 7.73 (d, 2H, J = 7.65 Hz, H-3′ & H-5′), 7.59 (m, 1H, 5-H), 7.51 (m, 1H, 8-H), 7.27 (d, 2H, J = 8.5 Hz, H-2′, H-6′), 7.22 (t, 1H, J = 7.65 Hz, H-7), 7.18 (t, 1H, J = 7.65 Hz, H-6). ¹³C NMR (850 MHz, DMSO-d₆) δ (ppm): 147.81, 145.70, 143.48, 139.75, 132.09, 131.15, 130.88, 128.25, 128,22, 127.84, 127.26, 122.24.LC–MS (ESI), RT = 7.2 min, m/z 414.9 [M + H]⁺. HR–MS (EI+, m/z) molecular ion requires for C₁₄H₁₀ BrClN₄O₂S, 413.67; found 414.96.

N’,N’’’-(quinoxaline-2,3-diyl)dibenzenesulfonohydrazide 6a

Yield: 64%, as an orange solid, m.p0.153–155 °C, IR (KBr, cm⁻¹) ν_max = 3316 (N–H), 3168 (C–H, sp²), 1390 (S = O). ¹H NMR (850 MHz, DMSO-d₆) δ (ppm): 10.98 (s, 2H, NHSO₂),8.39 (s, 2H, NH),8.24 (d, 2H, J = 8.5 Hz, H-2′& H-2′’), 8.14 (d, 2H, J = 9.35 Hz, H-6′&H-6′’), 7.95 (m, 2H, H-3′ & H-3′’), 7.89 (m, 2H, H-5′& H-5′’), 7.78 (d, 1H, J = 7.65 Hz, H-8), 7.69 (dd, 1H, J = 7.65 Hz & 1.7 Hz, H-5), 7.64 (t, 1H, J = 7.65 Hz, H-7), 7.57 (t, 1H, J = 7.65 Hz, H-6) ¹³C NMR (850 MHz, DMSO-d₆) δ (ppm): 146.75, 142.79, 141.68, 138.57, 133.12, 129.50,128.03, 127.27. LC–MS (ESI), RT = 3.74–3.76 min, m/z 471.2 [M + H].

N’,N’’’-(quinoxaline-2,3-diyl)bis(4-methylbenzenesulfonohydrazide) 6b

Yield: 76.13%, as a yellow solid, m.p0.140–142 °C, IR (KBr, cm⁻¹) ν_max = 3254 (N–H), 3174 (C–H, sp²), 1630 (C = N), 1165–1394 (S = O). ¹H NMR (850 MHz, DMSO-d₆) δ (ppm): 10.896 (s, 1H, NHSO₂), 8.31 (s, 1H, NH), 8.19 (dd, 4H, J = 8.5 Hz, 1.7 Hz, H-2′ & H-6′ & H-2′’ & H-6′’), 8.04 (t, 1H, J = 8,5 Hz, H-7), 7.98 (t, 1H, J = 7.65 Hz, H-6), 7.68 (dd, 1H, J = 7.22 Hz, 2.55 Hz, H-8), 7.64 (d, 1H, J = 7.65 Hz, H-5), 7.40 (d, 4H, J = 7.65 Hz, H-3′ & H-5′ & H-3′’ & H-5′’), 2.39 (s, 6H, phenyl-CH₃).¹³C NMR (850 MHz, DMSO-d₆) δ (ppm): 146.83, 143.35, 143.08, 135.66, 130.49, 129.93, 129.28, 128.12, 21.49. LC–MS (ESI), RT = 2.71 min, m/z 498.1 [M + H]⁺.

N’,N’’’-(quinoxaline-2,3-diyl)bis(4-trifluoromethyl benzenesulfonohydrazide) 6c

Yield: 46%, as a red solid, mp 220–224 °C, IR (KBr, cm⁻¹) ν_max = 3290 (N–H), 3169 (C–H, sp²), 1632 (C = N), 1168–1322 (S = O). ¹H NMR (850 MHz, DMSO-d₆) δ (ppm): 11.23 (s, 2H, NHSO₂), 8.66 (s, 2H, NH), 8.23 (d, 4H, J = 7.65 Hz, H-3′ & H-5′ & H-3′’ & H-5′’), 8.07 (d, 4H, J = 8.5 Hz, H-2′ & H-6′ & H-2′’ & H-6′’), 7.93 (m, 1H, 7-H), 7.93 (m, 1H, 6-H), 7.76 (d, 2H, J = 8.5 Hz, H-5 & H-8).¹³C NMR (850 MHz, DMSO-d₆) δ (ppm): 146.78, 142.74, 141.68, 137.01, 131.60, 130.25, 130.05, 129.32, 128.08. LC–MS (ESI), RT = 4.19–4.25 min, m/z 607.2 [M + H]⁺.

N’,N’’’-(quinoxaline-2,3-diyl)bis(4-bromobenzenesulfonohydrazide) 6d

Yield: 57%, as an orange solid m.p0.150–152 °C, IR (KBr, cm⁻¹) ν_max = 3294 (N–H), 3171 (C–H, sp²), 1631 (C = N), 1171–1393 (S = O). ¹H NMR (850 MHz, DMSO-d₆) δ (ppm): 11.06 (s, 2H, NHSO₂), 8.91 (s, 2H, NH), 8.26 (d, 4H, J = 8.5 Hz, H-3′ & H-5′ & H-3′’ & H-5′’), 8.13 (d, 4H, J = 8.5 Hz, H-2′ & H-6′ & H-2′’ & H-6′’), 7.98 (t, 1H, J = 7.65 Hz, H-7), 7.64 (m, 2H, 5-H, H-8).¹³C NMR (850 MHz, DMSO-d₆) δ (ppm): 146.79, 142.87, 141.80, 136.96, 131.91, 130.34, 129.69, 129.31. LC–MS (ESI), RT = 4.95 min, m/z 627.9 [M + H] ⁺.

Biology

Inhibition of sPLA2 activity

The De Aranjo and Radvany method was employed to assess the inhibitory activity of sPLA2. The substrate comprised 3.5 mM lecithin in a solution containing 3 mM NaTDC (sodium taurodeoxycholate), 100 mM NaCl, 10 mM CaCl₂, and 0.055 mM red phenol, with the volume adjusted to 100 ml with H₂O at a pH of 7.6. Human group IB (pG-IB), IIA (hG-IIA), V (hG-V), X (hG-X), and XII (hG-XII) sPLA2 were dissolved in 10% acetonitrile at a concentration of 0.02 μg/μL. For the assay, 10 μL of each PLA2-containing solution was combined with 10 μL of each compound and incubated for 20 min at room temperature. Subsequently, 1 ml of the PLA2 substrate was added, and the optical density at 558 nm was recorded for 5 min to monitor the hydrolysis kinetics. The inhibition percentage was determined by comparing it with a control experiment lacking any compound.

α-Glucosidase inhibitory activity

The α-glucosidase inhibitory activity was assessed by monitoring the release of 4-nitrophenol, following the method outlined by Andrade-Cetto et al. Briefly, a mixture comprising 20 μL of either the control drug (quercetin), DMSO, or the compound under investigation (ranging between 0.78 and 12.5 μg/mL) was prepared with 180 μL of α-glucosidase enzyme obtained from Saccharomyces cerevisiae (Sigma) and incubated at 37 °C for 2 min. Subsequently, 150 μL of 4-nitrophenyl β-d-glucopyranoside (NPGP) was added, and the samples were further incubated at 37 °C for approximately 20 min. The assay medium contained 10 mM potassium phosphate buffer at pH 6.9, 5 mM 4-NPGP, and 2 U of α-glucosidase. Quercetin was used as the positive control, whereas DMSO served as the negative control, both at the same concentration as the tested compound. Finally, the samples were measured at 405 nm using a microplate reader. The inhibition percentage was determined using the following equation:

$$100-\left(\text{X2 sample}-\text{X1 sample}/\text{X2 control}-\text{X1 control}\right)\times 100,$$

where X1 and X2 represent the absorbance of the initial (T0) and final (T = 15 min) reading, respectively. The IC₅₀ values were determined using nonlinear regression analysis.

Molecular modelling

For this study, the 3D structures of the desired proteins were retrieved from the Protein Data Bank (www.rcsb.org). Specifically, the structures for sPLA2 and α-glucosidase were retrieved from the PDB entries 5G3M and 3W37, respectively, with X-ray resolutions of 1.85 Å and 1.70 Å. The PDB files were curated for missing residues and H-bond additions and were optimised using Biovia Discovery Studio. Subsequently, the refined PDB files were used for molecular docking simulations^28,29.

Molecular docking was performed using the NRGSuite software, which is freely available and integrated as a plugin for PyMOL (www.pymol.org). NRGSuite offers a user-friendly interface tailored for molecular docking to explore protein–ligand interactions. It facilitates the identification of surface cavities within a protein and utilises them as target binding sites for docking simulations, employing the FlexAID algorithm. The software utilises a genetic algorithm for conformational exploration of ligand poses, simulating ligand and side-chain flexibility, and enabling covalent docking simulations. In this study, a flexible-rigid docking protocol was implemented with the following default parameters to optimise the performance of NRGSuite:

Binding sites input method: spherical shape (diameter: 18 Å), spacing of the 3-dimensional grid: 0.375 Å, side-chain flexibility: disabled, ligand flexibility: enabled, ligand pose as reference: disabled, constraints: none, hetero groups: includes water molecules, van der Waals permeability: 0.1, solvent types: none specified, number of chromosomes: 1000, number of generations: 1000, fitness model: share, reproduction model: population boom, number of top complexes: 5.

Results and discussion

Chemistry

Quinoxaline derivatives have numerous chemical, biochemical, and pharmaceutical properties, and their activity on diabetes remains a research focus^30,31. Furthermore, to develop compounds capable of simultaneously inhibiting multiple enzymes, such as sPLA2 and carbohydrate hydrolases, we regarded the quinoxalinone nucleus as an important pharmacophore in enzyme inhibition^32,33.

A novel series of quinoxaline sulfonohydrazide (5a–d) and (6a–d) derivatives were synthesised as potential inhibitors of sPLA2 and α-glucosidase (Scheme 1). Building upon our previous research, we initiated the synthesis with 1,4-dihydro-quinoxaline-2,3-dione 1, obtained through the reaction between o-phenylene diamine and oxalic acid^33,34. Subsequently, compound 1 was used to synthesise 2,3-dichloroquinoxaline 2 via reaction with POCl₃, as previously described³⁵. Compound 2 was subjected to nucleophilic substitution reaction with hydrazine hydrate to yield 2-chloro-3-hydrazineylquinoxaline 3 and 2,3-dihydrazineylquinoxaline 433^,36,37.

In this study, we synthesised the desired quinoxaline sulfonohydrazide derivatives (5a–d) and (6a–d) through direct nucleophilic substitution reactions between hydrazide (3 and 4) and aryl sulphonyl chloride derivatives in aqueous media under dynamic pH control. The assigned structures for compounds N’-(3-chloroquinoxalin-2-yl)-4-(substituted-phenyl) sulfonohydrazide (5a–d) and N’,N’’’-(quinoxaline-2,3-diyl)bis(4- substituted-phenyl) sulfonohydrazide (6a–d) were confirmed based on their IR, ¹H NMR, ¹³CNMR, HR-MS, and LC–MS spectral data. The infrared spectrum of compounds 5a–d revealed the lack of a peak corresponding to the NH₂ group at a frequency of 3440 cm⁻¹. The absorption bands observed at v_max 3312–3322 cm⁻¹ and 757–765 cm⁻¹ are attributed to the NH groups and chloride groups, respectively. The sulphonyl groups were seen at ν_max 1160–1167 and 1335–1345 cm⁻¹. The ¹H NMR spectrum verified the structure of compound 5b and exhibited a peak corresponding to methyl groups located at position 4′. The presence of methyl groups causes an increase in electron density around the aromatic protons due to electronic processes. Consequently, the protons are protected from the magnetic field outside and are observed at a lower frequency. This causes the aromatic proton peaks in the ¹H NMR spectrum to shift towards lower frequencies relative to the unsubstituted sulfonohydrazide derivative 5a. In addition, the NH groups of (NH) and (NHSO₂) were observed as a wide single peak at chemical shifts of 8.30 and 9.99 ppm, respectively. The ¹³C NMR spectrum provided further confirmation of the reported structure of these compounds. The signals in the range of δc = 122 to 151 ppm correspond to the carbon atoms in the quinoxaline ring. The chemical 5b showed the appearance of a spectral line at δc = 21.19 ppm, which can be attributed to the presence of carbon atoms in the methyl group. Furthermore, there are six additional spectral lines of toluene seen at δc = 125.95, 128.62, 129.39, and 143.59 ppm. These lines correspond to the (4′-C), (3′-C, 5′-C), (2′-C, 6′-C), and (1′-C) positions, respectively. The structures of these compounds (5a–d) were verified using liquid chromatography-electrospray ionisation mass spectrometry (LC/ESI-MS) in positive ionisation mode [M + H]. All compounds exhibited a retention time (RT). The retention time (RT) and liquid chromatography–mass spectrometry (LC–MS) analysis using electrospray ionisation (ESI) of chemical 5a show a range of 4.13–4.31 min and a mass-to-charge ratio (m/z) of 335.1 [M + H]⁺. The HR-MS (EI+, m/z) spectrum of compound 5b exhibited the molecular ion (m/z) [M + H]⁺ calculated for C₁₅H₁₃ClN₄O₂S as 348.81, while the observed value was 349.

The identification of the structures of compounds 6a and 6b was determined using spectroscopic data. The compounds’ IR spectrum revealed absorption bands around ν_max 3316–3254 cm⁻¹, indicating the existence of NH groups. Additionally, the sulphonyl groups were seen at roughly ν_max 1394–1165 cm⁻¹. The structures of these compounds were verified using ¹H NMR and ¹³C NMR spectroscopy. The 1H NMR spectra of compounds 6a–d showed that the NH₂ group of 2,3-dihydrazineylquinoxaline 4 disappeared, and two NH protons at the (NH) and (NHSO₂) groups appeared as broad singlets at specific chemical shift values. The configuration of these compounds was verified using ¹H NMR spectroscopy. The compounds exhibited distinct singlets at 8.31–8.91 and 10.89–11.23 ppm, indicating the presence of NH groups at (NH) and (NHSO₂), respectively. In the ¹H NMR spectra of compound 6b, an additional singlet at about 2.39 ppm was detected, corresponding to the (CH₃) group. Consequently, these protons are protected from the magnetic field outside and are observed at a lower frequency, while the protons in the phenyl ring are observed in the aromatic part of the spectrum for all these compounds. The LC–MS (ESI) analysis of compound 6a revealed a retention time (RT) range of 3.74–3.76 min and a mass-to-charge ratio (m/z) of 471. The ion observed was [M + H]⁺.

Inhibition of sPLA2 and α-glucosidase

To assess the anti-inflammatory activity of the eight compounds under investigation, we used human sPLA2 pG-IIA. Data reported in Table 1 highlights that compounds 6a and 6b demonstrated the most promising results in inhibiting the catalytic activity of the proinflammatory hG-IIA sPLA2. Almost all tested compounds exhibited low micromolar IC₅₀ values against sPLA2 (IC₅₀ <3 μM), whereas compound 6d showed high micromolar IC₅₀ values against sPLA2. These results suggest the potential for future optimisation of the compounds to achieve absolute selective activity against sPLA2. Sub micromolar IC₅₀ values against proinflammatory sPLA2 were observed for some of the compounds. For example, compounds 6a and 6b exhibited potent activities against sPLA2 hG-IIA (IC₅₀ 0.0475–0.0765 μM).

Table 1.

Inhibitory activity of the title compounds against sPLA2(IIA)and α-glucosidase expressed as IC₅₀(µM) ± standard deviation.

IC50 (µM)
Compound	sPLA2(IIA)	α-Glucosidase
5a	2.041 ± 0.0459	1.472 ± 0.1255
5b	1.525 ± 0.2117	2.792 ± 0.4999
5c	2.589 ± 0.2924	29.39 ± 0.5315
5d	2.122 ± 0.1322	13.30 ± 0.2627
6a	0.0475 ± 0.0008	3.151 ± 0.0320
6b	0.0765 ± 0.0017	0.9601 ± 0.1120
6c	2.237 ± 1.046	0.0953 ± 0.0054
6d	4.931 ± 0.2410	8.294 ± 0.3669
Acarbose	–	283.3 ± 161.5
Oleanolic acid	3.88 ± 0.1839	–

The compounds exhibited moderate inhibitory activity against the pancreatic α-glucosidase enzyme, as indicated by IC₅₀ values <15 μM (Table 1). The synthetic quinoxaline sulfonohydrazide derivatives 5–6 (a–d) were evaluated for their α-glucosidase inhibitory activity and demonstrated significantly improved activity than that of the standard drug acarbose (IC₅₀ = 283.3 µM). Various structural features of molecules, including the –Br group, –CF₃ group, and different substitutions “R” at the benzene sulphonyl hydrazide ring, appeared to contribute to their inhibitory activity. Molecular docking results further supported these findings, indicating that the –CF₃ group is involved in lipophilic interaction.

IC₅₀ values of compounds 5a, 5b, 6a, 6b, and 6c indicate significantly higher activity against α-glucosidase compared with that of positive controls. The inhibitory activity was more prominent in 6a–6d compared with that in 5a–5d. Moreover, among compounds 6a–6d, 6c exhibited the most potent inhibitory activity (IC₅₀ = 0.0953 µM), which can be attributed to the electron-withdrawing substituent at the para-position of the bisbenzenesulfonohydrazide moiety.

Based on the IC₅₀ values, compound 6a demonstrated the most effective inhibition for both enzymes (Figure 2). Comparing these results with that of docking studies indicated that compound 6a was successfully docked into the active sites of sPLA2 and α-glucosidase. Furthermore, the docking results revealed that the best-scored conformation was observed for compounds containing the benzenebis–sulfonylhydrazide moiety. Molecular docking studies further proved the strong inhibitory activity of 6a–6d relative to other sulfonohydrazide derivatives. A comprehensive understanding of the interactions between the ligands and enzyme active sites would help design novel potent inhibitors.

Figure 2.

inhibition of sPLA2(IIA)and α-glucosidase by 6a points are average inhibition % at each concentration.

Molecular modelling and docking

Molecular docking, a pivotal aspect of the structure-based drug design, enables the identification of crucial structural features responsible for interacting with residues within the active site of a target enzyme. It provides valuable insights into the pharmacophoric patterns and is a valuable tool in the drug discovery pipeline. In this study, we performed molecular docking using NRGSuite, with all compounds docked in the active site of sPLA2 and α-glucosidase (PDB 5G3M and 3W37, respectively). The active site of sPLA2 comprises residues including ILE2, LEU5, ALA6, VAL9, PRO17, ILE18, TYR20, MET21, CYS27, GLY28, CYS43, HIS46, ASP47, LYS61, ILE94, and LEU98 (Figure 3).

Figure 3.

The active site of sPLA2 (pdb 5G3M) using molecular surface area (green: hydrophobic, red: oxygen, blue: nitrogen).

Similarly, the active site of α-glucosidase comprises residues including ASP232, ILE233, ALA234, PHE236, ASN237, TRP329, ASP357, ILE358, ILE396, TRP432, TRP467, ASP469, MET470, SER474, PHE476, ARG552, TRP565, ASP568, ASP597, PHE601, ALA602, ARG624, and HIS626 (Figure 4).

Figure 4.

The active site of α-glucosidase (pdb 3W37) using molecular surface area (green: hydrophobic, red: oxygen, blue: nitrogen).

The results of molecular docking for the most active compounds 6a and 6c against sPLA2 and α-glucosidase, respectively, are depicted in Figure 5.

Figure 5.

3D-representation of molecular docking poses for 6a in sPLA2 and 6c in α-glucosidase (white and green: hydrophobic, red: oxygen, blue: nitrogen).

The compound 6a occupies a substantial portion of the active site of sPLA2 through a combination of lipophilic, mild polar, and H-bond interactions with different residues (Figure 5a). Owing to its symmetrical nature, it adopts a fork-like shape with two tines, each represented by –NH–NH–SO₂–Ph moiety, which occupies the adjacent sides of the active site. The oxygen atoms of –SO₂– groups are responsible for H-bonding with Gly28 (distance 4.88 Å) and His46 (distance 2.83 Å). The third H-bond is between Lys61 (distance 3.14 Å) and the nitrogen atom of the pyrazine ring. The two benzene rings attached to –SO₂– groups engage in hydrophobic interactions with Ile2, Leu5, Ala6, Val9, Met21, and Asp47. Notably, these benzene rings are oriented perpendicular to each other, optimising available space and facilitating hydrophobic interactions.

Similarly, compound 6c exhibits a unique conformation within the active site of $a$-glucosidase, facilitating lipophilic, mildly polar, and H-bond interactions with different residues (Figure 5b). One of the –NH–NH–SO₂–PhCF₃ moieties occupy the inner cavity of the active site, whereas the second NH–NH–SO₂–PhCF₃ moiety bends slightly, engaging in hydrophobic interactions. The oxygen atom of the second NH–NH–SO₂–PhCF₃ moiety forms a H-bonding with Ala234 (distance 2.25 Å). In contrast, the second H-bond occurs between Asp568 (distance 2.46 Å) and the nitrogen atom of the first NH–NH–SO₂–PhCF₃ moiety. The two Ph–CF₃ groups attached to the SO₂ groups contribute to hydrophobic interactions.

Therefore, molecular docking revealed that the –SO₂, pyrazine ring, and para-substituted benzene rings attached to –SO₂–groups are critical pharmacophoric features. The –NH–NH– moiety serves as a linker, enhancing the flexibility of the molecule and facilitating the adoption of a bioactive conformation.

Conclusions

In conclusion, we synthesised a novel small library of quinoxaline-based scaffold, evaluated its enzyme inhibitory activity against sPLA2 and α-glucosidase, and conducted molecular docking analysis. Seven of the selected compounds, were synthesised based on the sulfonohydrazide scaffold. Their potential as inhibitors of both sPLA2 and α-glucosidase was evaluated for the first time. According to docking studies, five compounds, namely 5c, 6a, 6b, 6c, and 6d exhibited the highest docking scores, fitting well at the active site and being stabilised by ionic bonds, hydrogen bonds, and hydrophobic interactions. Furthermore, we proposed an optimal method for synthesising our target compounds to ensure high purity and yield. Our findings suggest that compounds 6a–d, particularly 6a (IC₅₀ = 0.0471 µM), may serve as promising candidate and lead compound for further development of an inhibitors against both sPLA2 and α-glucosidase.

Funding Statement

This work did not receive funding from any sources.

Disclosure statement

The authors declare report there are no competing interests to declare.

Disclaimer

The views expressed in this paper are those of the author(s) and do not necessarily reflect those of the SFDA or its stakeholders. Guaranteeing the accuracy and validity of the data is the sole responsibility of the research team.