Background

Cancer is the predominant cause of mortality and a remarkable obstacle to elevating life anticipation in every nation on Globe [1]. By 2025, hepatic cancer will have an effect on over 1 million people per year [2]. In USA, hepatocellular carcinoma (HCC) has been the rapidly growing cause of cancer-related death since the early twentieth century as stated by the Surveillance Epidemiology End Results (SEER). It is expected to become the third biggest cause of cancer-related death by 2030, if developments continue [3]. It is becoming a significant issue that has an impact on people’s health and quality of life everywhere. Hepatitis B surface antigen (HBsAg) positivity is seen in about 80% of primary HCC patients in China, where the virus is a significant risk factor for primary HCC [4]. Threats include the diabetes or obesity-related NASH, stubborn alcohol intake, and HBV or HCV infection [5].

The majority of HCC patients about 80% are now detected in advanced stages of the illness and are not candidates for tumour removal surgery. Systemic chemotherapy with cytotoxic drugs (5-fluorouracil, doxorubicin, cisplatin) and targeted therapy with the tyrosine kinase inhibitor sorafenib are the primary therapies for these patients; nonetheless, chemotherapy resistance continues to be a major therapeutic problem [6].

HCC, a hyper-vascular tumour, develops and progresses due to angiogenesis, a key feature of malignancy. It exhibits high neoangiogenic activity because of the need to generate new blood vessels for tumour growth. During liver carcinogenesis, the tumour is primarily supplied by arterial input, while the surrounding parenchyma receives most of its blood supply through the portal system [7]. Angiogenesis is primarily regulated by VEGF and its receptors (VEGFR’s) [8]. High levels of circulating VEGF in HCC have been linked with quicker disease development and a worse prognosis [7].

Three subtypes of VEGFR exist: VEGFR-1, VEGFR-2 and VEGFR-3. Of them, VEGFR-2 is essential for the angiogenesis of tumours. After being activated by VEGF, VEGFR-2 dimerizes and then auto-phosphorylates the tyrosine receptor to start downstream signal transduction. Tumour angiogenesis is a result of these signalling pathways [9]. Preventing autophosphorylation and dimerization of the receptor is the aim of the development of new VEGFR-2 inhibitors [10].

Fibroblast growth factor receptor-4 (FGFR-4) is a tyrosine kinase receptor that preferentially binds to FGF19 to promote autophosphorylation in trans. Through downstream MAP kinase and AKT signalling pathways, it mediates cellular effects. The FGF19-FGFR4 complex has been confirmed to be an oncogenic driver of HCC through aberrant signalling [11].

Resistance is the major concern in treating the cancer patients (e.g. sorafenib). Individual targeting of VEGFR-2/FGFR-4 may cause resistance. The concept of dual inhibition signifies that VEGFR-2 and FGFR-4 pathways are interrelated, involve common downstream signalling pathways responsible for angiogenesis and are the major determinant for the development of the resistance; they can be dual targeted for effective inhibition [12, 13].

Quinoxaline is a promising scaffold with a diverse spectrum of actions, including the anti-inflammatory, anti-bacterial, anti-mycobacterial, anti-viral, anticancer, etc. The FDA-approved VEGFR-2 inhibitors like pazopanib and vandetanib possess quinazoline as a basic nucleus. Quinoxaline is a bioisostere of the quinazoline nucleus. Also, FGFR-4 inhibitors like erdafitinib possess quinoxaline as the basic moiety [9, 14,15,16,17,18,19,20].

The present work aims to design, synthesize, characterize and perform the in vitro cell cytotoxicity evaluation against the HepG2 cells for the developed quinoxaline derivatives with the objective of overcoming the resistance.

Materials and methods

Pharmacophore identification

The Pharmit server (http://pharmit.csb.pitt.edu/) was used for pharmacophore modelling, making it easier to generate pharmacophores and check them against a variety of large chemical compound repositories, including PubChem, CHEMBL, Zinc database, etc. The pharmacophore identification is done by providing the RCSB PDB ID: 6GQO and 6YI8 as inputs in the webserver [21]. Consideration of the pharmacophore is done while designing the ligands.

Designing of ligands

A virtual library consisting of 50 newly designed ligands is constructed by employing the principles of pharmacophore modelling and molecular hybridization. Our designing methodology aimed to develop the selective inhibitors for the hydrophobic (allosteric pocket) and the gatekeeper region of the target proteins. The ligands are drawn using the Chemsketch software (ACD labs, version 2023.1.0) and saved in the. mol format for further computational work [22]. The novelty of the designed ligands is confirmed through a search of chemical databases such as PubChem and the Zinc 20 database [23, 24].

Docking

Protein selection, retrieval and preparation

The target proteins selected for the study were the signalling pathways involved in the cancer cell growth, proliferation and angiogenesis such as VEGFR-2 (PDB ID:6GQO) and FGFR-4 (PDB ID:6YI8). The X-ray crystallographic structures with the most suitable resolution were chosen from RCSB protein data bank and validated by the Ramachandran plot. The chosen protein is entered into the protein repair and analysis server (PRAS; https://www.protein-science.com/) to check and correct any missing hydrogen atoms, residues or heavy atoms [25,26,27].

Active site prediction

The CB-DOCK2 web server, which clusters the solvent-accessible surface, is used to predict cavities for the targets' active sites. It is a structure-based approach to cavity prediction.

Customizing grid boxes involves tailoring them according to cavity characteristics such as centre, size and volume predictions. This customization optimizes molecular docking simulations, improving the accuracy of predicting interactions between ligands and receptors [28]. The active site prediction details of the proteins, VEGFR-2 and FGFR-4 are available in the Table 1.

Table 1 Active site dimensions of the target proteins
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Virtual screening and rescoring

The molecular docking is done to understand the binding affinity of the designed ligands against the VEGFR-2 and FGFR-4 using the Dockthor-VS software. It is available to assist drug development teams in conducting precise and effective docking-based virtual screening evaluations. For rigid-receptor and flexible-ligand docking, a grid-based method is used. To determine the posture, a multiple-solution genetic algorithm and the molecular force field scoring function (MMFF94S) are employed [29].

ADMET profiling

The qualitative concept of Drug-likeness is used in drug discovery phases of drug design to describe how “drug-like” an element is related to aspects like bioavailability. An established method for estimating drug similarity is to confirm that Lipinski's rule of five is followed [30].

SwissADME enables the ADME parameter prediction in addition to the computation of physiochemical descriptors. Many techniques exist for identifying log p, including topological approaches, fragment approaches, p-glycoprotein substrate, CYP450 inhibitors for pharmacokinetic predictions and calculation of the Lipinski’s rule [31].

Osiris property explorer is an online tool to check the toxicity profile of the molecule. It checks for undesired effects like tumourigenicity, mutagenicity, irritant effect and reproductive toxicity [32].

Visualization of interactions

Visualization of the interactions between the receptor and ligand is done using Biovia discovery studio (Dassault Systems, V21.1.0.20298) and the UCSF Chimera version 1.17.3 software. The nature of interactions and the interacting residues, and their distance are studied [33, 34].

Selection of ligands for the synthesis

The ligands with best docking score, suitable pharmacokinetic properties, i.e. S42, S31, S26, S6, S21, are selected for the synthesis. The rescoring procedure is carried out using the Autodock version 4.2.6 software to confirm the effectiveness of the ligands against the targets [35]. The ligands selected above are designated by the sample code SA-1, SA-2, SA-3, SA-4 and SA-5, respectively. The synthetic schemes of the ligands are portrayed in the Schemes 1, 2, 3, 4, 5 and 6.

Scheme 1
scheme 1

Synthesis of 3-methyl quinoxaline–2-one. Reagents and conditions: 1). stirring, Δ, 3 h

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Scheme 2
scheme 2

Synthesis of the compound SA-1. Reagents and conditions: 1). Pocl3, 0–5 °C. 2). Chloroacetyl chloride, TEA, DMF, reflux, Δ, 8 h. 3). Anhydrous K2CO3, DMF, reflux, 8 h, Δ

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Scheme 3
scheme 3

Synthesis of the compound SA-2. Reagents and conditions: 1). Δ, reflux, 10 h. 2). Chloroacetyl chloride, anhydrous K2CO3, chloroform, reflux, Δ, 10 h. 3). Anhydrous K2CO3, DMF, reflux, 8 h, Δ

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Scheme 4
scheme 4

Synthesis of the compound SA-3. Reagents and conditions: 1). Chloroacetyl chloride, anhydrous K2CO3, chloroform, reflux, Δ, 10 h. 2). Anhydrous K2CO3, DMF, reflux, 10 h, Δ

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Scheme 5
scheme 5

Synthesis of the compound SA-4. Reagents and conditions: 1). Con. H2SO4, 0–5 °C. 2). Chloroacetyl chloride, anhydrous K2CO3, chloroform, reflux, Δ, 10 h. 3). Anhydrous K2CO3, DMF, reflux, 10 h, Δ

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Scheme 6
scheme 6

Synthesis of the compound SA-5. Reagents and conditions: 1). chloroacetylchloride, TEA, stirring. 2). Anhydrous K2CO3, DMF, reflux, 8 h, Δ

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Synthesis

Synthesis of 3-methyl-1H- quinoxaline-2-one:

%yield = 68%, m.p: 210–213 °C, Rf = 0.47 (hexane: ethyl acetate-6:4)

The reported approach is followed in the synthesis of 3-methyl-1H-quinoxaline-2-one. 25 ml of aqueous acetic acid 20% was mixed with 1.85 mmol of sodium pyruvate and 1.85 mmol of O-phenylenediamine in a round-bottom flask equipped with a stirring bar. 3 h was spent stirring the reaction at room temperature. After then, the precipitate that had developed was gathered by filtering, and pure product was obtained by recrystallizing it from ethanol–water 4:1 v/v [36].

Synthesis of SA-1

Step 1: According to the common procedure for synthesis of 3-methyl quinoxalin-2-one

Step 2: Synthesis of 5-(4-methylphenyl)-1,3,4-thiadiazol-2-amine: %yield = 56%. m.p: 201–203 °C, Rf = 0.42 (ethanol: ethyl acetate-8:2)

Dropwise addition of POCl3 (0.3 mol) is done to a combination of p-toluic acid (0.1 mol) and thiosemicarbazide (0.1 mol) at a 0–5 °C, and the mixture was kept there for half an hour. For 6 h, the reflux condition is set for the reaction mixture, and with stirring, about 50 ml of H20 was added to the reaction mixture, once it had cooled. The mixture’s pH was brought to a range of 8–9 using a 50% NaOH solution. The crude product filtered, water washed, dried and recrystallized from ethanol [37].

Step 3: Synthesis of 2-chloro-N-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl] acetamide: %yield = 76%. 196–197 °C, Rf = 0.67 (toluene: ethyl acetate-7:3)

A solution of 1,3,4-thiadiazole derivative (0.001 mol) and three drops of triethylamine in DMF (10 ml) was stirred and dropwise added chloroacetyl chloride (0.004 mol) over the course of two hours at 0–5 °C. The mixture was refluxed for six hours at 50 °C, and then, icy water was poured over the reaction combination. The resultant solid was filtered out, water washed and recrystallized from the 1:1 combination of ethanol and chloroform [38].

Step 4: Synthesis of 2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)-N-[5-(4-methylphenyl) -1,3,4-thiadiazol-2-yl] acetamide: %yield = 66%. Rf = 0.74 (hexane: ethyl acetate-6:4).

3-methyl quinoxaline-2-one (1.29 mmol) in DMF (4 ml) was added to anhydrous K2CO3 (1.45 mmol), and agitation of the mixture is done for 1 h at room temperature. The intermediate from step 3 (1.29 mmol) was then gradually introduced to the mixture in 3 ml of DMF, heated to 60–70 °C and stirred until the reaction was finished. Ice water was added after bringing the mixture to the room temperature. The solid residue is dried and recrystallized from ethanol [39].

Synthesis of SA-2

Step 1: According to the common procedure for synthesis of 3-methyl quinoxalin-2-one.

Step 2: Synthesis of 4-[(1E)-N-(1,3-benzothiazol-2-yl) ethanimidoyl] aniline: %yield = 63%. m.p: 171–172 °C, Rf = 0.58 (hexane: ethyl acetate-7:3).

A round-bottom flask containing 20 ml of alcohol was used to make a p-aminoacetophenone (0.01 M) solution. 15 ml of alcohol is used to dissolve 2-aminobenzothiazole (0.01 M) before adding it. The combination refluxed for ten to twelve hours. By distilling alcohol at lower pressure, the volume was cut in half. On top of crushed ice, the resultant solution was poured. After being separated, the dried precipitate was recrystallized from the ethanol [40].

Step 3: Synthesis of N-{4-[(1E)-N-(1,3-benzothiazol-2-yl) ethanimidoyl] phenyl}-2-chloroacetamide: %yield = 72%. m.p: 156–158 °C, Rf = 0.70 (hexane: ethyl acetate-7:3).

For about 10 h, equimolar volumes of the step 2 product (0.1 mol) and chloroacetyl chloride (0.1 mol) in chloroform (30 ml) were refluxed in the presence of K2CO3 (0.1 mol). The leftover solvent was agitated with 50 ml of water after the excess solvent was eliminated in a vacuum. After that, 30 ml of 5% NaHCO3 and 30 ml of water were used to wash the residue. The ethanol-derived crude product was dried and crystallized [41].

Step 4: Synthesis of (E)-N-(4-(1-(benzo[d]thiazol-2-ylimino) ethyl) phenyl)-2-(3-methyl-2-oxoquinoxalin-1(2H)-yl) acetamide: %yield = 66%. Rf = 0.53 (hexane: ethyl acetate-6:4)

3-methyl quinoxaline-2-one (1.29 mmol) in DMF (4 ml) was mixed with anhydrous K2CO3 (0.2 g, 1.45 mmol) and allowed to stand at room temperature for one hour. The intermediate from step 3 (1.29 mmol) was then gradually added to the mixture in 3 ml of DMF, heated to 50 °C and stirred until the reaction was finished. After the mixture had reached room temperature, it was placed in an ice water bath. After that, the solid residue is dried and recrystallized from ethanol [39].

Synthesis of SA-3

Step 1: According to the common procedure for synthesis of 3-methyl quinoxalin-2-one.

Step 2: Synthesis of N-(1,3-benzothiazol-2-yl)-2-chloroacetamide: %yield = 78%. m.p:189–190 °C, Rf = 0.54 (hexane: ethyl acetate-6:4)

Equimolar volumes of 2-aminobenzothiazole (0.1 mol) and chloroacetyl chloride (0.1 mol) in 30 ml of chloroform were refluxed along with K2CO3 (0.1 mol) for about 10–12 h. The leftover solvent was agitated with 50 ml of water after the excess solvent was eliminated in a vacuum. After that, 30 ml of 5% NaHCO3 and 30 ml of water were used to wash the residue. The crude product was dried and recrystallized with ethanol [41].

Step 3: Synthesis of N-(1,3-benzothiazol-2-yl)-2-(3-methyl-2-oxoquinoxalin-1(2H)-yl) acetamide: %yield = 82%. Rf = 0.49 (hexane: ethyl acetate-6:4)

3-methyl quinoxaline-2-one (1.29 mmol) in DMF (4 ml) was added to anhydrous K2CO3 (0.2 g, 1.45 mmol), and agitation of the mixture was done for one hour at room temperature. The intermediate from step 2 (1.29 mmol) was then gradually added to the mixture in 3 ml of DMF, heated to 50 °C and stirred until the reaction was finished. After the combination had reached room temperature, it was placed in an ice water bath. After drying and for recrystallizing the solid residue, a 1:1 combination of ethanol and chloroform is used [39].

Synthesis of SA-4

Step 1: According to the common procedure for synthesis of 3-methyl quinoxalin-2-one.

Step 2: Synthesis of 5-(3-nitrophenyl)-5H-[1, 3] thiazolo[4,3-b][1,3,4]thiadiazol-2-amine: %yield = 82%, m.p: 183–184 °C, Rf = 0.68 (hexane: ethyl acetate-7:3)

Thioglycolic acid (0.02 M) and aromatic aldehyde (0.02 M) were combined, and after 10–15 min, thiosemicarbazide (0.022 M) was added. After cooling, parts of 10 ml of conc.H2SO4 were introduced. After homogenizing the mixture, it was kept refrigerated for 18–24 h. After applying 30–50 g of ice to the reaction mass, the precipitated solid was decanted, water was added, and then, the suspension was neutralized with a 40% NaOH solution. Then, ethanol is used for the recrystallization [42].

Step 3: Synthesis of 2-chloro-N-[5-(3-nitrophenyl) 5H [1, 3] thiazolo [4,3b] [1, 3, 4] thiadiazol-2-yl] acetamide: %yield = 83%, Rf = 0.82 (hexane: ethyl acetate-7:3)

Equimolar volumes of the step 2 product (0.1 mol) and chloroacetyl chloride (0.1 mol) in 30 ml of chloroform were refluxed for about 10–12 h while K2CO3 (0.1 mol) was present. After the excess solvent was eliminated in a vacuum, 50 ml of water was introduced to the residue and mixed. 30 ml of 5% NaHCO3 and 30 ml of water were used to wash the residue. After being dried and crystallized from ethanol, the crude product yielded a light-yellow solid [41].

Step 4: Synthesis of 2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)-N-(5-(3-nitrophenyl)-5H-thiazolo[4,3b] [1, 3, 4] thiadiazol-2-yl) acetamide: %yield = 63%. Rf = 0.67 (hexane: ethyl acetate-6:4)

3-methyl quinoxaline-2-one (1.29 mmol) in DMF (4 ml) was added to anhydrous K2CO3 (0.2 g, 1.45 mmol), and the mixture was agitated for one hour at room temperature. The intermediate from step 3 (1.29 mmol) was then gradually added to the mixture in 3 ml of DMF, heated to 50 °C and stirred until the reaction was finished. After the combination had reached room temperature, it was placed in an ice water bath. After drying, residue is recrystallized using a 1:1 combination of ethanol and chloroform [39].

Synthesis of SA-5

Step 1: According to the common procedure for synthesis of 3-methyl quinoxalin-2-one.

Step 2: Synthesis of 4-(2-chloroacetamido) benzoic acid: %yield = 74%. m.p: 243–244 °C, Rf = 0.63 (ethanol: ethyl acetate-7:3)

A transparent solution was obtained by thoroughly stirring 0.01 mol of triethylamine and 0.01 mol of p-aminobenzoic acid in ethanol and cooling it for 30 min. Following an hour of stirring and the addition of 0.01 mol of chloroacetyl chloride to the solution, the precipitate was filtered, dried and then recrystallized using ethanol [43].

Step 3: Synthesis of 4-[2-(3-methyl-2-oxoquinoxalin-1(2H)-yl) acetamido] benzoic acid: %yield = 68%. Rf = 0.55 (hexane: ethyl acetate-6:4)

3-methyl quinoxaline-2-one (1.29 mmol) in DMF (4 ml) was added to anhydrous K2CO3 (1.45 mmol), and the mixture was agitated for one hour at room temperature. The intermediate from step 2 (1.29 mmol) was then gently added to the mixture in 3 ml of DMF, heated to 50 °C and stirred for 8 h. After the combination had reached room temperature, it was placed in an ice water bath. The resultant solid residue is dried and recrystallized with ethanol [39].

Molecular dynamics (MD) simulation and MM-GBSA calculation

The MD simulation is carried out for the receptor–ligand complex using the Schrodinger's desmond software (Maestro version 13.1.137) to understand the stability of protein–ligand complex and the changes occurring in the conformation upon the simulation.

The complex was centred in an orthorhombic cubic box, and TIP3P water molecules were added along with buffers at a distance of 10 Å from the protein atom to the box edge. The boundary condition box volume has also been decided based on complex type, in addition to counterions such as Na+ and Cl− that have been put to randomly neutralize the system. The VEGFR2_SA4 complex comprises 45,594 atoms with 13,490 waters, compared to 41,873 atoms with 12,480 waters in the FGFR4_SA4 complex for MD simulations.

By using the desmond protocol, the solvated built system was minimized. Relaxation is carried out following the application of the OPLS 3e forcefield parameters. The Berendsen NVT ensemble was used to model the system while maintaining a temperature of 10 K to limit heavy atoms on the solute.

The setup includes the isothermal isobaric ensemble at 300 K, 1 atm of pressure and 200 ps of thermostat relaxation time (NPT). The Nose–Hoover thermostat approach and the Martyne–Tobias–Klein barostat approach are used to keep the temperature and pressure scales at 1 atm and 300 K, respectively. Following the production simulation procedure lasting 100 ns, the NPT ensemble was launched [44].

FastDRH, which is an open source software is employed to examine protein–ligand interaction structures, structure-truncated MM-GBSA free energy calculation processes and various poses based per-residue energy decomposition analysis was well integrated into a user-friendly and versatile web platform in this server. It includes the docking protocol based on user-defined selection of AutoDock Vina and AutoDock-GPU docking engines (http://cadd.zju.edu.cn/fastdrh/overview). The free energy calculation indicates the stability of the protein ligand complex [45].

Characterization

Synthetic Chemicals, solvents and others were obtained from Commercial grade suppliers. With the aid of TLC (GF254, Merck, Germany), we examined the completeness of the chemical reaction (254 nm) with visualization either by iodine chamber method or UV chamber method. The appropriate solvent system is employed, based on the nature of the compound. The melting temperatures of synthetic compounds in open capillary tubes were determined using the Guna™ melting point apparatus.

Characterization of the synthesized compounds is done by using the UV, IR, NMR and HRMS. The infrared spectra were recorded using the ABB MB 3000 FTIR spectrophotometer (KBr pellet method). The Bruker AVANCE 400 MHz spectrophotometer was utilized to record the 1H NMR spectra. Tetramethylsilane (TMS) was used as an internal standard, and DMSO-d6 was used as the sample solvent. The chemical shift values were reported in delta scale as ppm values. The 13C NMR is recorded using the Bruker 100 MHz spectrophotometer with DMSO as sample solvent. The mass spectrum was recorded using the Waters-Xevo G2-XS-QToF High-Resolution Mass Spectrometer (ESI Positive mode) with methanol as the sample solvent.

MTT-based cytotoxicity studies

From NCCS, the HepG2 cell line was procured and stock cell was cultured in DMEM (Dulbecco's Modified Eagle Medium) medium supplemented with 10% inactivated Foetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 μg/ml) in a humidified atmosphere of 5% CO2 at 37 °C until confluent.

Using the appropriate medium containing 10% FBS, the monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 × 105 cells/ml. In each well of the 96-well microtiter plate, 100 μl of the diluted cell suspension (1 × 104 cells/well) was added.

After 24 h, when a partial monolayer had formed, 100 μl of different test sample concentrations was added to the partial monolayer on microtiter plates, the supernatant was disposed of, and the monolayer was once again washed with media. The plate was then incubated for 24 h at 37 °C in an atmosphere with 5% CO2. After the incubation time, 20 μl of MTT (2 mg/1 ml of MTT) was added to each well.

Following the incubation period, each well received 20 μl of MTT (2 mg/1 ml of MTT in PBS), with the test solutions in the wells being removed. For 4 h, the plate was incubated at 37 °C with 5% CO2. Once the supernatant was removed, 100 μl of DMSO was added, and the formazan that had formed was gently dissolved by gently shaking the plate. A microplate reader operating at 570 nm was used to calculate the absorbance [46].

Results

Virtual library of ligands

The pharmacophoric features for the VEGFR-2 include the hydrophobic group, hydrogen bond acceptor, hydrogen bond donor and aromatic group. The pharmacophoric features for the FGFR-4 include the hydrogen bond acceptor and hydrophobic group. The structures of the ligands are depicted in Supplementary Table 1.

Docking

The Ramachandran plot as represented in Fig. 1 denotes that above 90% of the amino acid residues of the VEGFR-2 and FGFR-4 are situated in the most favourable region. The results of active site prediction of the selected proteins are depicted in the Table 1. The designed ligands were docked against the targets VEGFR-2 and the FGFR-4, and the results are tabulated in Table 2. The sorafenib and the erdafitinib are selected as standard drugs for comparison of the docking scores against the targets VEGFR-2 and FGFR-4, respectively. Validation of the docking protocol is done by observing the RMSD values of the co-crystallized ligand, and it is seen below 2 angstroms.

Fig. 1
figure 1

Ramachandran plots of the A VEGFR-2, B FGFR-4. Ramachandran plot of receptor structure (the red, dark yellow, light-yellow and white area describes the most favoured, allowed, generously allowed and disallowed regions, respectively)

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Table 2 Docking scores from Dockthor-VS webserver
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Most of the compounds are found to exhibit comparable docking scores with the standard drugs. The docking scores lie from the range of − 7.511 to − 10.467 kcals/mol and in the range of − 6.917 to − 8.929 kcals/mol against the VEGFR-2 and FGFR-4, respectively.

Prioritized ligands for synthesis

The ligands S6, S21, S26, S31, S42 are selected for the synthesis, and rescoring is done with results tabulated in Table 3.

Table 3 Rescoring results by Autodock software version 4.2.6
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ADMET results

We found that the ligands prioritized for the synthesis obey the Lipinski’s rule of five. The molecular weight of them lies in the range of 337.33–480.52 daltons and the log p value lies around the range of 2.05–3.82, which are similar to the reference, sorafenib and erdafitinib. Most of the designed ligands in the virtual library were found to exhibit the desirable drug-like properties such as good gastrointestinal absorption and notable membrane permeability. We believe that the optimum properties of our designed ligands will help the molecules in achieving the optimum bioactivity. The prioritized ligands showed absence of toxicity (data are available in the supplementary file).

Visualization of interactions

The binding interactions of the ligands synthesized are depicted in Table 4. The designed ligands exhibit the crucial interactions like the hydrogen bonding, van der Waals and hydrophobic connections with the amino acids ASP 1046 (DFG domain), GLU 885, LEU 889, VAL 916 (allosteric pocket, gate keeper amino acid) of the VEGFR-2 protein and ALA 554, GLU 551, CYS 552 (cysteine residues in the hinge region), ALA 553, ARG 483 of the FGFR-4 protein. The 2D binding modes of the selected ligands were comparable to the standards, sorafenib and the erdafitinib, giving assurance of the suitable interactions with key residues.

Table 4 Binding interactions of the VEGFR-2 and the FGFR-4
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Synthesis

SA-1:

Orangish brown colour powder

IUPAC name:

2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)-N-[5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl] acetamide

m.p:

196–198 °C; UV spectrum, λmax (nm): 227.0; 1H NMR (DMSO-d6, 400 MHz, ppm): δ, 2.40 (s,3H,CH3 of tolyl), 2.51(s,3H, CH3 of 3-methyl quinoxaline-2-one), 3.36(s,2H, –CH2– of acetamide), 7.24 (ddd,3H,aromatic), 7.47 (ddd,1H,aromatic), 7.86 (ddd, 3H,aromatic), 8.06 (ddd,1H,aromatic), 12.334(s,1H, NH of acetamide); 13C NMR (100 MHz, DMSO): 166.52 (s,1C), 162.71 (s,1C), 159.63 (s,1C), 158.25 (s,1C), 157.93 (s,1C), 155.35 (s,1C), 154.84 (s,1C), 142.61 (s,1C),141.12 (s,1C), 140.99 (s,1C), 133.21 (s,1C), 132.07 (s,1C), 130.26 (s,1C), 129.73 (s,1C), 128.91 (s,2C), 128.25 (s,2C), 21.40 (s,1C), 20.97 (s,1C); FTIR (KBr, cm−1): 3479 (NH stretching), 3062 (aromatic C–H), 2962 (aliphatic C–H), 2923 (aliphatic C–H, methyl group), 1658 (carbonyl group of the amide), 1612 (C=C stretching,), 1566 (C=N stretching), 694 (C–S–C linkage), 1303 (C–N stretching); HRMS (ESI–MS, positive mode, m/z): molecular weight: calculated: 391.449, [M + H]+: 392.1182, [M + Na]+: 414.0999 (adduct formation)

SA-2:

Brown colour powder

IUPAC name:

(E)-N-(4-(1-(benzo[d]thiazol-2-ylimino) ethyl) phenyl)-2-(2-methyl-3-oxoquinoxalin-4(3H)-yl) acetamide

m.p:

192–194 °C; UV spectrum, λmax (nm): 279.5; 1H NMR (DMSO-d6, 400 MHz, ppm): δ, 2.40 (s,3H, CH3 of methyl), 2.478 (s,3H, CH3 of 3-methyl quinoxaline-2-one), 3.38 (s,2H, CH2 of acetamide), 7.28 (ddd,5H,aromatic),7.50 (ddd,3H,aromatic),7.70 (ddd,3H,aromatic), 7.76 (ddd,1H,aromatic), 11.42 (s,1H, NH of acetamide); 13C NMR (100 MHz, DMSO): 166.01 (s,1C), 159.64 (s,1C), 158.13 (s,1C), 157.95 (s,1C), 155.46 (s,1C), 154.82 (s,1C), 153.28 (s,1C), 133.37 (s,2C), 132.48 (s,1C), 132.43 (s,2C), 132.13 (s,1C), 131.03 (s,1C), 130.19 (s,1C), 129.98 (s,1C), 125.86 (s,1C), 123.44 (s,1C), 123.32 (s,1C), 121.24 (s,2C), 121.17 (s,1C), 118.18 (s,1C), 45.74 (s,1C), 21.58 (s,1C), 21.74 (s,1C); FTIR (KBr, cm−1): 3438.46 (NH stretching–secondary amide), 3060 (aromatic C–H), 2954 (aliphatic C–H), 2919 (aliphatic C–H), 1660 (carbonyl group of the amide), 1596 (C=C stretching), 1540 (C=N stretching), 653 (C–S–C linkage), 1267 (C–N stretching); HRMS (ESI–MS, positive mode, m/z): molecular weight: calculated:467.547, [M + H]+: 468.1494

SA-3:

Orange colour powder

IUPAC name:

N-(benzo[d]thiazol-2-yl)-2-(3-methyl-2-oxoquinoxalin-1(2H)-yl) acetamide

m.p:

187–189 °C; UV spectrum, λmax (nm): 227.0; 1H NMR (DMSO-d6, 400 MHz, ppm): δ, 2.50 (s,3H, CH3 of 3-methyl quinoxaline-2-one), 3.35 (s,2H, CH2 of acetamide), 7.24 (ddd,4H,aromatic), 7.52 (ddd,2H,aromatic),7.68 (ddd,1H,aromatic), 7.98 (ddd,1H,aromatic), 12.31(s,1H, NH of acetamide); 13C NMR (100 MHz, DMSO): 159.66 (s,1C), 155.37 (s,1C), 154.86 (s,1C), 137.13 (s,1C), 132.40 (s,1C), 132.11 (s,1C), 129.73 (s,1C), 128.31 (s,1C), 126.72 (s,1C), 124.27 (s,1C), 123.46 (s,2C), 122.29 (s,1C), 121.17 (s,2C), 115.70 (s,1C), 43.02 (s,1C), 20.99 (s,1C); FTIR (KBr, cm−1): 3442 (NH stretching–secondary amide), 3060 (aromatic C–H), 2960, 2917, 2848 (aliphatic C–H), 1664 (carbonyl group of the amide), 1602 (C=C stretching), 1560 (C=N stretching), 690 (C–S–C linkage), 1268 (C–N stretching); HRMS (ESI–MS, positive mode, m/z): molecular weight: calculated: 350.396, [M + H]+: 351.0916, [M + Na]+: 373.0731 (adduct formation).

SA-4:

Dark brown colour

IUPAC name:

2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)-N-(5-(3-nitrophenyl)-5H-thiazolo[4,3b] [1, 3, 4] thiadiazol-2-yl) acetamide

m.p:

196–198 °C; UV spectrum, λmax (nm): 202.0; 1H NMR (DMSO-d6, 400 MHz, ppm): δ, 2.50 (s, 1H, CH3 of 3-methyl quinoxaline-2-one), 3.39 (s, 1H, CH2 of acetamide), 7.246 (ddd,4H,aromatic), 7.246 (ddd,4H,aromatic),8.10 (ddd,1H,aromatic),8.17 (ddd,1H,aromatic), 8.35 (s,1H, CH of thiazole), 8.492 (s, 1H, CH of thiazole), 12.35 (s, 1H, NH of acetamide); 13C NMR (100 MHz, DMSO): 159.65 (s,1C), 155.40 (s,1C), 154.02 (s,1C), 150.62 (s,1C), 148.33 (s,1C), 132.40 (s,1C), 130.72 (s,1C), 129.72 (s,1C), 129.32 (s,1C), 128.29 (s,1C), 125.05 (s,1C), 123.96 (s,1C), 122.91 (s,2C), 111.74 (s,1C), 115.73 (s,2C), 119.81 (s,1C), 39.33 (s,1C), 20.98 (s,1C), 18.78 (s,1C), FTIR (KBr, cm−1): 3440 (NH stretching–secondary amide), 3008 (aromatic C–H), 2962, 2908, 2846 (aliphatic C–H), 1666 (carbonyl group of the amide), 1604 (C=C stretching), 1566 (C=N stretching), 1527 (NO2 stretching), 1350 (C–N stretching); HRMS (ESI–MS, positive mode, m/z): molecular weight: calculated: 480.517, [M + H]+: 481.1707

SA-5:

Buff yellow colour

IUPAC name:

4-(2-(3-methyl-2-oxoquinoxalin-1(2H)-yl) acetamido) benzoic acid.

m.p:

183–185 °C; UV spectrum, λmax (nm): 202.5; 1H NMR (DMSO-d6, 400 MHz, ppm): δ, 2.50 (s,3H), 3.40 (s,2H), 7.27 (ddd,4H,aromatic), 7.43 (ddd,1H,aromatic), 7.53 (ddd,2H,aromatic), 8.01 (ddd,1H,aromatic), 8.56 (s,1H, NH of acetamide), 9.9 (s,1H, Carboxyl); 13C NMR (100 MHz, DMSO): 171.92 (s,1C), 166.65 (s,1C), 165.25 (s,1C), 150.5 (s,1C), 136.12 (s,1C), 135.89 (s,1C), 133.39 (s,2C), 132.41 (s,1C), 129.54 (s,1C), 128.18 (s,1C), 123.84 (s,1C), 118.47 (s,2C), 116.31 (s,2C), 45.56 (s,1C), 21.62 (s,1C); FTIR (KBr, cm−1): 3426 (NH stretching–secondary amide), 3386 (carboxylic OH), 3012 (aromatic C–H), 2964, 2917, 2848 (aliphatic C–H), 1664 (carbonyl group of the amide), 1604 (C=C stretching), 1533 (C=N stretching), 1360 (C–O stretching), 1180 (C–N stretching); HRMS (ESI–MS, positive mode, m/z): molecular weight: calculated: 337.335, [M + H]+: 338.1141

Chemistry

Based on the results from in silico studies, synthetic feasibility and the chemicals available in our laboratory, we prioritized the ligands for our synthesis.

The first step involves the condensation reaction between the O-phenylene diamine and the sodium pyruvate in the acidic environment. Few amines are readily available; the others were prepared according to the reported procedures. Various amines selected were subjected to the chloro-acetylation using the chloroacetylchloride in the presence of either TEA or K2CO3 resulting in the intermediates. Then, final step involves the condensation of the 3-methyl quinoxaline-2-one derivative with the chloroacetamide derivatives.

The FTIR spectrum of the compounds displayed the presence of characteristic absorption frequencies in the region of 3000–3100 corresponding to the aromatic CH, 3300–3500 amide group and the carbonyl stretching around 1600–1700 cm−1.

The 13C NMR spectrum revealed the presence of carbonyl peaks in the range of 160 ppm and aliphatic peaks around 20–30 ppm.

The methyl protons of the 3-methyl quinoxaline-2-one are observed around 2.50 ppm, methylene protons of the chloroacetamide linkage around the 3.3–3.5 ppm and the amide peaks as singlet around the 8.5–12 ppm. The ESI + mode mass spectrum of the compounds was found to be close with the molecular weight corresponding to the M + 1 peak and M + Na (adduct formation) peak.

Cytotoxicity test

The synthesized compounds exhibited the potent anticancer activity on the HepG2 cell culture. The varying concentrations of 1–512 µg/ml and the cell viability in the cells were observed against the control. The highly active hit compound is found to be SA-4 with IC5O value of 54.73 µM bearing the thiazolo-thiadiazole nucleus. The results obtained from the in vitro cytotoxicity results correlate with the results of the in silico studies. Table 5 represents the IC50 values of the synthesized compounds.

Table 5 IC50 values of the synthesized compounds
Full size table

Molecular dynamics simulation

MD simulation studies were conducted using the Schrodinger’s Desmond (academic version) on the compound exhibiting the highest activity in the MTT assay. The objective was to gain insights into the real-time conformational stability of the complex and to optimize it for generating improved leads in future research endeavours.

VEGFR-2

According to the RMSD plot, the VEGFR-2 apoprotein experiences conformational alterations up to the 20 ns mark, followed by a stabilization phase from 25 to 50 ns, maintaining consistent levels with minor fluctuations thereafter until 100 ns. Throughout the simulation, the protein's RMSD values remained below 5 Å.

Observations reveal that the SA4_VEGFR2 complex maintains RMSD values below 2.5 Å from 0 to 20 ns, followed by a notable increase in fluctuations between 20 and 40 ns, which persist until the conclusion of the simulation with minor variations, suggesting moderate stability of the complex throughout the simulation period (as mentioned in Fig. 2). Notably, the overall RMSD values remain below 3.0 Å. However, the analysis of the ligand RMSD indicates relatively minor fluctuations, suggesting the need for optimization to achieve a more stable complex. The RMSF plot also indicates the fluctuations in some regions (Figs. 3, 4).

Fig. 2
figure 2

RMSD plot of SA4_VEGFR2 complex. Picture depicting the RMSD plot of the SA-4_VEGFR2 complex with x axis showing the time in nanoseconds and the y axis showing the protein RMSD in angstroms

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Fig. 3
figure 3

RMSF plot of SA4_VEGFR2 complex. Picture depicting the RMSF plot of the SA-4_VEGFR2 complex with x axis showing the residue index and the y axis showing the protein RMSF in angstroms

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Fig. 4
figure 4

Amino acid interaction histogram of SA4_VEGFR2 complex. Picture depicting the amino acid interaction histogram of SA4_VEGFR2 complex with residue notation in the x axis and the y axis containing the interaction fraction

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The ligand–protein contact map (as denoted in Fig. 5) reveals that the charged (negative) interactions are observed with the amino acid A: GLU 917 and amino acid of the gatekeeper region A: ASP 1046 in the DFG domain for a period of 89 and 99% throughout the simulation period. Hydrophobic interaction is observed with A: PHE 918 for 54% of the simulation period. The Fig. 6 shows the contact map analysis of the SA4_VEGFR2 complex, indicating a mixed pattern of broken and established contacts with aminoacids of protein.

Fig. 5
figure 5

SA4-VEGFR2 complex contact map. Diagram representing the protein ligand contact map giving information on interaction type and time of contact

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Fig. 6
figure 6

SA4-VEGFR2 complex contact analysis of MD trajectory. Diagram depicting the MD trajectory of the SA4-VEGFR2 complex; orange regions denote the maintained contacts and white regions denote the broken contacts

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FGFR-4

The RMSD plot of the FGFR-4 apoprotein undergoes fluctuations up to 40 ns and then reaches the plateau phase of equilibrium till the end of the 100 ns simulation period. The maximum RMSD values around 5 Å reveal that the protein contains flexible residues with frequent movements in it.

The RMSD plot for the SA4_FGFR4 complex illustrates that for the initial 20 ns, the RMSD values remain consistently below 2.5 Å, indicating relative stability. Subsequently, there is a plateau phase lasting until around 80 ns, characterized by fluctuations with higher RMSD. Beyond 80 ns, there is a drastic increase in RMSD values, suggesting inferior stability of the complex, possibly due to the flexibility of certain protein residues. Furthermore, the higher RMSD value of the ligand indicates more pronounced conformational fluctuations within the binding site, highlighting the necessity for structural optimization in the ligand design (as represented in the Fig. 7). In the Fig. 8, the RMSF plot reveals the more fluctuating regions of the protein. The ligand-protein contact map (as denoted in Fig. 9) reveals that the hydrogen bonding and water bridges are observed with the amino acid B:GLU 551.

Fig. 7
figure 7

RMSD plot of SA4_ FGFR4 complex. Picture depicting the RMSD plot of the SA-4_FGFR4 complex with x axis showing the time in nanoseconds and the y axis showing the protein RMSD in angstroms

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Fig. 8
figure 8

RMSF plot of SA4_ FGFR4 complex. Picture depicting the RMSF plot of the SA-4_FGFR4 complex with x axis showing the residue index and the y axis showing the protein RMSF in angstroms

Full size image
Fig. 9
figure 9

Amino acid interaction histogram of SA4_FGFR4 complex. Picture depicting the amino acid interaction histogram of SA4_FGFR4 complex with residue notation in the x axis and the y axis containing the interaction fraction

Full size image

The MM-GBSA analysis showed that the sorafenib and erdafitinib showed binding energy value of − 38.52 and − 50.66 kcal/mol. The protein–ligand complex of the best compound SA-4 showed a binding energy value of − 50.65 and − 42.42 kcal/mol against VEGFR-2 and FGFR-4, which show that our complex is stable and robust in the cavity of proteins.

Conclusion

In this study, a library of 50 ligands is designed and they were docked against the targets VEGFR-2 and FGFR-4. Then, it is subjected to the drug-likeness and pharmacokinetic profiling using the in silico tools. The synthesized compounds were characterized and evaluated for anticancer efficacy against the HepG2 cell line. It is found that the compound SA-4 exhibited the highest activity in the MTT assay (IC50 =54.732 µM). MD simulation study and MM-GBSA reveal the stability of the SA-4 complex and additional requirement of hit optimization. We conclude that further hit to lead optimization of the compound SA-4 will bring more active, efficacious molecules in the area of cancer research.