Design, synthesis, docking, ADMET studies, and anticancer evaluation of new 3-methylquinoxaline derivatives as VEGFR-2 inhibitors and apoptosis inducers

Abstract

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a critical role in cancer angiogenesis. Inhibition of VEGFR-2 activity proved effective suppression of tumour propagation. Accordingly, two series of new 3-methylquinoxaline derivatives have been designed and synthesised as VEGFR-2 inhibitors. The synthesised derivatives were evaluated in vitro for their cytotoxic activities against MCF-7and HepG2 cell lines. In addition, the VEGFR-2 inhibitory activities of the target compounds were estimated to indicate the potential mechanism of their cytotoxicity. To a great extent, the results of VEGFR-2 inhibition were highly correlated with that of cytotoxicity. Compound 27a was the most potent VEGFR-2 inhibitor with IC₅₀ of 3.2 nM very close to positive control sorafenib (IC₅₀ = 3.12 nM). Such compound exhibited a strong cytotoxic effect against MCF-7 and HepG2, respectively with IC₅₀ of 7.7 and 4.5 µM in comparison to sorafenib (IC₅₀ = 3.51 and 2.17 µM). In addition, compounds 28, 30f, 30i, and 31b exhibited excellent VEGFR-2 inhibition activities (IC₅₀ range from 4.2 to 6.1 nM) with promising cytotoxic activity. Cell cycle progression and apoptosis induction were investigated for the most active member 27a. Also, the effect of 27a on the level of caspase-3, caspase-9, and BAX/Bcl-2 ratio was determined. Molecular docking studies were implemented to interpret the binding mode of the target compounds with the VEGFR-2 pocket. Furthermore, toxicity and ADMET calculations were performed for the synthesised compounds to study their pharmacokinetic profiles

Keywords:

• Anticancer
• apoptosis
• 3-methylquinoxalin
• molecular docking
• VEGFR-2

1. Introduction

According to WHO reports, cancer is considered the second major cause of death¹. By 2030, the incidence of cancer deaths will reach thirteen million worldwide². Although the high advances in the diagnosis and treatment of cancer diseases, the survival of patients remains poor due to the widespread adverse effects of anticancer agents³. So that, the discovery of new, effective, selective, and less toxic anticancer agents remains one of the most urgent needs⁴.

Angiogenesis process plays an important role in the growth and regeneration of tissues. Such a role is crucial to prevent ischaemic necrosis and facilitate the survival of the damaged tissues⁵. During the normal state, angiogenesis is controlled by some protein kinases (PKs), which comprise VEGFRs, FGFRs, and EGFRs⁶. PKs can be deregulated under pathological conditions, producing a disturbance in angiogenesis process. This leads to an increasing in the rate of cell division, creating tumour disease⁷.

VEGFRs and their specific agonist (VEGF) are overexpressed in many human tumours, especially solid tumours as gliomas and carcinomas⁸. Therefore, VEGFRs are considered as one the most important regulators of angiogenesis and consequently tumour growth⁹. VEGFRs family comprises three subtypes including VEGFR-1, VEGFR-2, and VEGFR-3¹⁰. VEGFR-1 controls embryonic vasculogenesis¹¹. VEGFR-2 regulates both embryonic vasculogenesis and tumour angiogenesis¹². On the other hand, VEGFR-3 is responsible for lymphangiogenesis¹³. So that, VEGFR-2 is now the foremost target for antiangiogenic therapy, and its blocking is a relevant approach for the discovery of new drugs against angiogenesis–dependent malignancies¹⁴. VEGFR-2 inhibitors demonstrated effective suppression of tumour progression. ATP binding site is the main target of most VEGFR-2 inhibitors¹⁵.

The crystal structures of VEGFR-2 revealed the presence of many pockets at the ATP binding site. (i) Hinge region which locates on the front side and comprises two key amino acid residues (Cys919 and Glu917). These residues participate in H-bond interactions with the adenine ring of ATP. (ii) Gatekeeper region which separates between the hinge region and DFG-motif region. (iii) DFG-motif region which locates on the backside and contains two key amino acids (Glu885 and Asp1046). For maximum activity, VEGFR-2 inhibitors have to bind with these two residues via H-bonds. (iv) Allosteric hydrophobic region which consists of much hydrophobic amino acid residues^16–19.

VEGFR-2 inhibitors can be classified into three types. Type I inhibitors (ATP competitive inhibitors), e.g. sunitinib 1 can bind to the adenine binding region of the ATP binding site¹⁵. Type II inhibitors, e.g. sorafenib 2 induce inactive activation of DFG-out confirmation of activation loop. Such type can bind additionally the allosteric hydrophobic pocket of ATP²⁰. Type III inhibitors, e.g. vatalanib 3 can form covalent interaction with cysteine amino acid residue at ATP binding site^21,22.

Many drugs targeting VEGFR-2 have been approved for clinical use in the treatment of different types of cancers (Figure 1). Sorafenib 2 is a potent VEGFR-2 inhibitor²³. It is mainly used in the treatment of advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC)²⁴. Regorafenib 4, a fluoro derivative of sorafenib, has been developed to inhibits VEGFR1-3²⁵. Tivozanib 5, a VEGFR-2 inhibitor, has been approved by the FDA in March 2021 for the treatment of RCC^26–28. Sunitinib 1 is a VEGFR-2 kinase inhibitor that was approved for the treatment of RCC and of gastrointestinal stromal tumours (GIST)²⁹.

Figure 1. Some clinically used VEGFR-2 inhibitors as well as quinoxaline derivatives having VEGFR-2 inhibitory actions.

Binding mode sorafenib, a representative example of VEGFR-2 inhibitors, against VEGFR-2 active site (PDB: 4ASD) was reported in many publications. The heterocyclic (N-methylpicolinamide) moiety is buried in the hinge region forming two H-bonds with Cys919 through the N atom of the pyridine ring and NH group of acetamide moiety. In addition, the urea moiety binds to the receptor at the DFG motif through various H-bonding interactions with Glu885 and Asp1046. The terminal phenyl ring occupies the allosteric site forming hydrophobic interactions with the hydrophobic pocket created by the DFG flipping out^30–32.

Studying the SAR of VEGFR-2 inhibitors and analysing the binding mode of sorafenib revealed that the majority of potent and selective VEGFR-2 inhibitors have four common pharmacophoric features that facilitate their fitting with the active binding pocket^33–35. The first feature is a flat heteroaromatic ring system (ahead) incorporating at least one H-bond acceptor to interact with the crucial amino acid residue Cys919 in the hinge region^30,31. A second feature is a spacer group that gives the inhibitor enough length^36,37, making the third feature (pharmacophore) near at DFG motif to bind with two crucial residues (Glu883 and Asp1044)^37,38. A pharmacophore is a functional group that comprises at least one H-bond acceptor (HBA) and one H-bond donor (HBD) group (typically amide or urea)^31,38. The fourth feature is a terminal hydrophobic moiety that can occupy the created allosteric hydrophobic binding site^16,39.

Apoptosis as a form of cellular suicide is one of the important mechanisms by which anticancer agents can affect cancer cells⁴⁰. Apoptosis is regulated by several mediators⁴¹. Among these are caspases, specifically caspase-3 and caspase-9^42,43. Caspase-3 protease is a major mediator of apoptosis catalysing the cleavage of many vital cellular proteins leading to cell death⁴⁴. Caspase-9 activates other executioner caspases as caspase-3, -6, and -7 initiating apoptosis as they cleave several other cellular targets⁴⁵. In addition, the Bcl-2 family proteins are key regulators of apoptosis. This family comprised pro-apoptotic proteins as BAX that promote cell death and anti-apoptotic proteins as Bcl-2 which suppress cell death^46,47. The balance between pro-apoptotic and anti-apoptotic proteins (BAX/Bcl-2 ratio) regulates cell fate⁴⁸. Current evidence suggested that inhibition of angiogenesis, anti-angiogenic therapies have been shown to increase apoptosis in tumour cells⁴⁹. VEGFR-2 inhibitors were found to induce and accelerate apoptosis in cancer cells which synergistically potentiates their antitumor effect^30,49–51.

Moreover, the literature survey revealed that different scaffolds have been reported as excellent inhibitors of VEGFR-2. These scaffolds comprise quinoline (e.g. lenvatinib 6⁵²), quinazoline⁵³, and indazole⁵⁴. Furthermore, quinoxaline, a bioisostere for the aforementioned scaffolds, is considered an important nucleus for anticancer drugs^3,55–58. Many quinoxaline derivatives were reported to possess significant VEGFR-2 inhibitory activities^39,59–62.

In our previous work, we developed [1,2,4]triazolo[4,3-a]quinoxaline containing derivatives as VEGFR-2 inhibitors. Compounds 8 and 9 were the most potent candidates exhibiting an excellent VEGFR-2 inhibitory activity with a promising cytotoxic efficacy against breast and hepatocellular carcinoma^63,64. In continuation of our work^63–65 aimed at synthesising new anticancer agents targeting VEGFR-2 inhibition, new quinoxaline derivatives were designed and synthesised. The synthesised compounds were evaluated for their anti-proliferative activity. In addition, VEGFR-2 inhibitory activities were estimated for all compounds to hint at the potential mechanism of their cytotoxicity. Furthermore, deep investigations were performed on the most active member to assess its effect on apoptotic (caspase-3, caspase-9, and BAX) and anti-apoptotic (Bcl-2) mediators.

1.1. Rational of design

Considering compounds 7 and 8 as leading compounds^63,64. The design included the replacement of [1,2,4]triazolo[4,3-a]quinoxaline in compound 7 and/or bis([1,2,4]triazolo)[4,3-a:3′,4′-c]quinoxaline of compound 8 by 3-methylquinoxaline scaffold. 3-Methylquinoxaline is considered as bioisostere for N-methylpicolinamide moiety of sorafenib⁶¹. Two quinoxaline moieties (3-methylquinoxalin-2(1H)-one and 3-methylquinoxaline-2-thiol) were used as a biological isostere. As in the lead compounds, N-phenylacetamide moiety was utilised as a linker. The pharmacophore (HBD/HBA) was designed to be an amide, diamide, and/or hydrazide groups. Amide pharmacophore served as hydrogen bond donor and acceptor in many reported VEGFR-2 inhibitors^61,63,64,66. Finally, different, aliphatic, and un/substituted aromatic derivatives were selected to be the terminal hydrophobic moieties to occupy the allosteric hydrophobic pocket (Figure 2).

Figure 2. Rational design of new VEGFR-2 inhibitors.

2. Results and discussion

2.1. Chemistry

To reach the designed compounds, four Schemes 1–4 were adopted. The synthesis was initiated by the reaction of o- phenylenediamine 9 with sodium pyruvate 10 in glacial acetic acid according to the reported procedure to furnish 3-methylquinoxalin-2(1H)-one 11⁶⁷. This starting material 11 was subjected to subsequent treatment with potassium hydroxide to produce the corresponding potassium salt 12⁶⁷. Chlorination of compound 11 was achieved using phosphorous oxychloride to afford 2-chloro-3-methylquinoxaline 13⁶⁸. Refluxing the latter with thiourea in absolute ethanol resulted in 3-methylquinoxaline-2-thiol 14^69,70 which was underwent heating with alcoholic potassium hydroxide to provide the corresponding potassium salt 15 (Scheme 1).

Scheme 1. General procedure for preparation of the key potassium salts 12 and 15.

According to the literature^71–73, stirring of commercially available p-amino benzoic acid 16 with chloroacetyl chloride in dry DMF in cold conditions yielded 4–(2-chloroacetamido)benzoic acid 17. Treatment of 17 with thionyl chloride in 1,2 dichloroethane and a catalytic amount of DMF gave the key intermediate 4–(2-chloroacetamido)benzoyl chloride 18^63,74.

On the other hand, methyl esters of appropriate acid derivatives, namely benzoic acid 21a and 4-nitrobenzoic acid 21b were prepared by refluxing carboxylic acids in methanol with the presence of sulphuric acid⁷⁵. The ester derivatives 22a,b were treated with hydrazine hydrate to get the corresponding acid hydrazides 23a,b^76,77. The produced acid hydrazides 23a,b were then allowed to stir with 4-(2-chloroacetamido)benzoyl chloride 18 in acetonitrile and TEA to furnish the intermediates 24a,b, respectively. Likewise, stirring of phenyl hydrazine 20 with 4-(2-chloroacetamido)benzoyl chloride 18 yielded the corresponding intermediate 2-chloro-N-(4–(2-phenylhydrazine-1-carbonyl)phenyl) acetamide 25. Compound 18 was stirred at room temperature in acetonitrile in the presence of a catalytic amount of TEA with appropriate amines 19a–j namely, tertiary butyl amine, cyclohexyl amine, benzyl amine, phenethyl amine, aniline, 2-methyl aniline, 2,6 dimethoxy aniline, 2,6 dimethyl aniline, 4-nitro-aniline, and 3-chloropyridine to give the corresponding chloroacetamide intermediates 26a–j, respectively (Scheme 2).

Scheme 2. General procedure for preparation of the intermediates 24a,b, 25, and 26a–j.

Synthesis of the final target compounds was illustrated in Schemes 3, 4. The 3-methylquinoxalin-2(1H)-one derivative (compounds 27a–f, 28, and 29) were obtained by heating of potassium salt 12 in dry DMF and a catalytic amount of KI with the previously prepared intermediates 26a,d,g–j, 24 b, and 25, respectively (Scheme 3).

Scheme 3. General procedure for preparation of the target compounds 27a–f, 28, and 29.

Furthermore, heating of potassium salt 15 with the intermediates 26a–g,i,j, 24a,b, and 25 in dry DMF and a catalytic amount of KI afforded the final target compounds 30a–i, 31a,b, and 32 (methylquinoxaline-2-thiol derivatives) (Scheme 4).

Scheme 4. General procedure for preparation of the target compounds 30a–i, 31a,b, and 32.

IR spectra of compounds 27a–f and 30a–i exhibited the presence of characteristic stretching bands at 3428–3215 cm⁻¹ corresponding to NH groups. Furthermore, the NMR spectra of compounds 27a–f and 30a–i supported their assigned structures. ¹H NMR charts of these compounds revealed the appearance of two singlet signals around δ 10.5 ppm attributed to the introduced two amidic NH protons. It also demonstrated increased integration of the aromatic protons corresponding to the additional phenyl ring. A characteristic up-field singlet signal equivalent to CH₂ of acetamide moiety was detected at about δ 4.30 ppm. Additionally, a singlet signal of CH₃ group of 3-methylquinoxalin-2(1H)-one moiety appeared around δ 2.49 ppm. ¹³C NMR spectra displayed the presence of two peaks at the aliphatic region attributed to the CH₂ and CH₃ groups around δ 40.0 and 22.5 ppm, respectively.

¹H NMR spectra of compounds 28, 29, 31a,b, and 32 demonstrated the presence of the amidic protons of hydrazide moiety at a range of δ 10.02–12.71 ppm. Additionally, ¹³C NMR spectrum of compounds 31b as an example of these compounds revealed the appearance of two peaks at δ 35.45 and 22.18 ppm corresponding to CH₂ and CH₃ groups, respectively. Mass spectroscopic analysis displayed a base peak at 517.0 (m/z) corresponding to the exact mass of the compound 31b.

2.2. Biological testing

2.2.1. In vitro anti-proliferative activity

In vitro cytotoxic activities of the synthesised compounds were evaluated against two different cancer cell lines; breast cancer (MCF-7) and hepatocellular carcinoma (HepG2) using standard MTT colorimetric assay as described by Mosmann^78,79. Sorafenib was used as a reference cytotoxic drug. The results of cytotoxicity were expressed as growth inhibitory concentration (IC₅₀) values and summarised in Table 1.

Table 1. In vitro cytotoxicity of the synthesised compounds against MCF-7 and HepG2 cell lines, their VEGFR-2 inhibitory activities on cancer HepG2 cell line, and cytotoxicity for compounds 27a and 30f against normal HepG2cell line.

Compounds	Cytotoxicity on cancer cells IC50 (µM)^a		VEGFR-2 IC50 (nM)^a	Cytotoxicity on normal hepatocytes IC50 (µM)^a
	MCF-7	HepG2
27a	7.7	4.5	3.2	19.94
27b	62.3	41.5	22.5	NT
27c	57.2	42.6	11.6	NT
27d	30.7	24.1	21.8	NT
27e	42.3	27.5	21.7	NT
27f	22.7	19.7	10.7	NT
28	17.2	11.7	4.2	NT
29	23.5	17.5	9.8	NT
30a	61.9	43.7	29.8	NT
30b	43.8	34.2	8.7	NT
30c	41.9	34.1	13.8	NT
30d	42.7	32.4	15.7	NT
30e	31.3	24.3	27.4	NT
30f	18.1	10.7	4.9	15.73
30g	61.7	40.8	38.9	NT
30h	21.3	17.7	11.8	NT
30i	17.2	12.7	6.1	NT
31a	23.1	18.7	10.7	NT
31b	19.2	13.7	5.1	NT
32	59.5	41.3	23.8	NT
Sorafenib	3.51	2.17	3.12	17.31
Compound 7	7.2	4.1	3.4	NT
Compound 8	4.4	3.3	3.2	NT

NT: not tested.

^aAll IC₅₀ values are calculated as the mean of at least three different experiments.

General investigation of the cytotoxicity results clarified that the examined compounds had greater anti-proliferative activities against HepG2 than MCF-7 cells. In particular, compound 27a was found to be the most potent derivative. Such compound showed strong anti-proliferative activities against MCF-7 and HepG2 cancer cell lines with IC₅₀ values of 7.7 and 4.5 µM, respectively. These values were close to that of the sorafenib (IC₅₀ = 3.51 and 2.17 µM, respectively).

Additionally, compounds 28 (IC₅₀ = 17.2 and 11.7 µM), 30f (IC₅₀ = 18.1 and 10.7 µM), 30i (IC₅₀ = 17.2 and 12.7 µM), and 31b (IC₅₀ = 19.2 and 13.7 µM) demonstrated promising cytotoxicity against MCF-7 and HepG2, respectively. Compounds 27f, 29, 30h, and 31a showed moderate anti-proliferative activities against the two tested cell lines with IC₅₀ values ranging from 17.5 to 23.5 µM. While compounds 27d, 27e, and 30e showed moderate activities against only HepG2 cells with IC₅₀ values ranging from 24.1 to 27.5 µM.

On the other hand, compounds 30b, 30c, and 30d displayed weak cytotoxic activities against the two cell lines with IC₅₀ values ranging from 32.4 to 43.8 µM. While compounds 27d, 27e, and 30e showed weak activities against only MCF-7 with IC₅₀ values ranging from 30.7 to 42.3 µM. Compounds 27b_, 27c, 30a, 30g, and 32 showed weak activities against only HepG2 with IC₅₀ values ranging from 40.8 to 43.7 µM. In the contrast, these compounds were inactive against the MCF-7 cell line.

Comparing to the lead compounds 7 (IC₅₀ = 7.2 and 4.1 µM) and 8 (IC₅₀ = 4.4 and 3.3 µM), it was found that the most active compounds 27a (IC₅₀ = 7.2 and 4.1 µM) was slightly less active than these compounds against MCF-7 and HepG2, respectively.

2.2.2. In vitro kinase inhibition assay

The newly prepared compounds have been further assayed for their inhibitory activity towards sorafenib's crucial target (VEGFR-2). The results were reported as 50% inhibition concentration values (IC₅₀, expressed as nM) in comparison to sorafenib as a reference drug (Table 1).

To a great extent, the reported results were in good agreement with that of cytotoxicity. This may clarify the possible mechanism of cytotoxic action for the designed compounds. Most of the tested compounds exhibited excellent, moderate, to weak VEGFR-2 inhibitory activities with IC₅₀ values ranging from 3.2 to 38.9 nM, compared to positive control sorafenib (IC₅₀ = 3.12 nM).

Among them, compound 27a was the most potent VEGFR-2 inhibitor with an IC₅₀ value of 3.2 nM. Besides, compounds 28, 29, 30b, 30f, 30i, and 31b showed strong VEGFR-2 inhibitory activities with IC₅₀ values of 4.2, 9.8, 8.7, 4.9, 6.1, and 5.1 nM respectively. Furthermore, compounds 27c, 27f, 30c, 30d, 30h, and 31a displayed moderate activities with IC₅₀ values ranging from 10.7 to 15.7 nM. Finally, compounds 27b, 27d, 27e, 30a, 30e, 30g, and 32 presented weak activities with an IC₅₀ value range of 21.7–38.9 nM.

Comparing to the lead compounds 7 (IC₅₀ = 3.4 nM) and 8 (IC₅₀ = 3.2 nM), it was found that the most active compounds 27a (IC₅₀ = 3.2 nM) had comparable VEGFR-2 inhibitory activity with these lead compounds.

2.2.3. Cytotoxicity against primary rat hepatocytes (normal hepatic cells)

It is of high importance for anticancer agents to have minimum side effects on normal cells. To assess the selectivity of the synthesised compounds against cancer cells over normal ones, the cytotoxicity of the most active anti-proliferative compounds 27a and 30f was evaluated in vitro against primary rat hepatocytes using sorafenib as reference⁸⁰. The two compounds 27a and 30f showed cytotoxic activity against cancer HepG2 cell line (4.5-fold and 1.5-fold, respectively) more than cytotoxic activity against normal hepatic cells in comparison to sorafenib (8-fold) (Table 1).

2.2.4. Structure-activity relationship

It was noticed that the synthesised compounds were more effective against HepG2 than the MCF-7 cell line (Figure 3). The obtained data from VEGFR-2 inhibition was highly matched with that of cytotoxicity. So that, the SAR can be built on the results of cytotoxicity and/or VEGFR-2 inhibition. SAR of the newly synthesised compounds was studied along with the pharmacophoric features outlined in the rationale of molecular design.

Figure 3. Correlation of cytotoxicity of the synthesised compounds on the two tested cell lines; MCF-7 and HepG2 showing higher sensitivity against HepG2 than MCF-7 cell line.

Firstly, the effect of the flat heteroaromatic ring on biological activity was examined. Comparing the cytotoxicity and VEGFR-2 inhibitory activities of the synthesised compounds incorporating 3-methylquinoxalin-2(1H)one moiety (compounds 27a–f, 28, and 29) with that incorporating 3-methylquinoxaline-2-thiol moiety (compounds 30a–i, 31a,b, and 32), it was found that 3-methylquinoxalin-2(1H)one moiety was more valuable than methylquinoxaline-2-thiol nucleus.

In addition, the impact of pharmacophore (HBD/HBA) moiety was explored. Regarding 3-methylquinoxalin-2(1H)one derivatives, it was found that compounds comprising the amide group (compounds 27a–f) were more active than that containing diamide group (compound 28), which in turn was more active than that with hydrazide moiety (compound 29). Concerning for 3-methylquinoxaline-2-thiol series, despite maintaining the same order of activity, it was found that compounds bearing amide (compounds 30a–i), diamide (compounds 31a,b), and hydrazide (compound 32) groups showed decreased activity than that incorporating 3-methylquinoxalin-2(1H)one nucleus.

Furthermore, the structure of the terminal hydrophobic tail gave wide varieties of biological activity. In cornering methylquinoxalin-2(1H)one derivatives, it was noticed that hydrophobic aliphatic moiety was critical for activity as found in the most potent member 27a containing tertiary butyl tail.

For the hydrophobic aromatic tail, the heterocyclic ring (compound 27f) displayed high activity than non-heteroaromatic ones (compounds 27a–d). Then, the effect of the substitution on the aromatic rings was examined. it was observed that the cytotoxicity and VEGFR-2 inhibitory activities were highly fluctuated by substitution with electron-withdrawing (EWG) and electron-donating (EDG)groups. The unsubstituted phenyl ring (compound 27b) markedly decreased the biological activity. Substitution with EDG as a 2,6-dimethoxy group (compound 27c) increased the cytotoxicity. Changing the substitutions to be 2,6-dimethyl groups (compound 27d) markedly decreased the activity. Additionally, it was found that the cytotoxicity decreased upon substitution with the electron-withdrawing group as in compound 27e.

For 3-methylquinoxaline-2-thiol derivatives, it was found that aromatic tails (compound 30c-i) were more effective than aliphatic ones (compounds 30a and 30b). For aliphatic derivatives, the bulky tertiary butyl tail (compound 30a) showed cytotoxic activity higher than the alicyclic one (compound 30b). About aromatic tail, comparing the IC₅₀ of compounds 30f (2-tolyl derivative), 30h (4-nitrophenyl derivative), and 30e (phenyl derivative), indicated that substitution with EDG group was more advantageous than substitution with EWG, which was more favourable than the unsubstituted one. Shifting the 2-tolyl into 5-chloro-2-pyridinyl (hetero-aromatic) moiety (compound 30i) produced a mild decrease in activity. While changing the 2-tolyl into 2,6-dimethoxy phenyl moiety (compound 30g) produced a dramatic decrease in activity.

Investigating the activity of compounds 30c, 30d, and 30e demonstrated that, insertion of carbon bridge between the phenyl moiety (hydrophobic tail) and the pharmacophore moiety produced an increase in activity with higher priority for a one-carbon bridge (compound 30c) over than two-carbon one (compound 30d).

2.2.5. Cell cycle analysis

Tissue homeostasis is tightly controlled by the balance between cell proliferation and cell death⁸¹. This creates a great link between the cell cycle and apoptosis⁸². Therefore manipulation of the cell cycle efficiently affected apoptotic response⁸³.

Flow cytometry analysis as described by Wand et al.^84,85 was used to investigate the effect of the most active compound 27a on the cell cycle progression and apoptosis induction utilising HepG2 cell line. HepG2 cells were treated with 4.5 µM (IC₅₀ of compound 27a) for 24 h, then analysed for its effect on cell cycle distribution. The cell cycle parameters of the incubated cells were compared with untreated control cells (Table 2 and Figure 4).

Figure 4. Flow cytometric analysis of cell cycle phases after treatment with compound 27a. (A) histograms showing the cell cycle distribution of control and treated cells. (B) Column graphs showing the percentage of cells in each phase of the cell cycle.

Table 2. Effect of compound 27a on cell cycle progression in HepG2 cells.

Sample	Cell cycle distribution (%)^a
	%Sub-G1	%G1	%S	%G2/M
HepG2	1.13 ± 0.08	59.93 ± 1.16	29.32 ± 1.08	9.62 ± 0.98
Compound 27a/HepG2	1.24 ± 0.17	37.66 ± 2.16***	26.96 ± 1.55	34.14 ± 1.22****

^aValues are given as mean ± SEM of three independent experiments.

***p < 0.001 and ****p < 0.0001 indicate statistically significant differences from the corresponding control (HepG2) group in unpaired t-tests.

The data of flow cytometric analysis revealed the presence of massive accumulation of the treated HepG2 cells at the G2/M (34.14%, 3.5-fold) compared to control cells (9.62%). In addition, a slight difference was observed at the S phase in the percentages of the treated cells (26.96%) and control ones (29.32%). On other hand, the percentage of HepG2 cells increased in the sub G1 phase from 1.13% (control cell) to 1.24% (treated cell). Also, it decreased at the G1 phase from 59.93% (control cell) to 37.66% (treated cell). Such outcomes indicated that compound 27a had a high ability to hinder cell cycle progression of HepG2 cells at the G2/M phase.

2.2.6. Detection of apoptosis

As compound 27a produced a high accumulation of HepG2 cells at the G2/M phase, such compound was further investigated for its apoptotic effect using Annexin V and PI double staining assay⁸⁶. In such a procedure, HepG2 cells were treated with compound 27a at a concentration of 4.5 µM and incubated for 24 h.

The results revealed that compound 27a induced an increase of HepG2 cells at early (40.47%) and late (0.35%) stages of apoptosis by about five times more than the untreated cells (8.52 and 0.14%, respectively) (Table 3 and Figure 5).

Figure 5. Flow cytometric analysis of apoptosis in HepG2 cells exposed to compound 27a.

Table 3. Stages of the cell death process in HepG2 cells after treatment with compound 27a.

Sample	Viable^a (left bottom)	Apoptosis^a		Necrosis^a (left top)
		Early (right bottom)	Late (right top)
HepG2	91.20 ± 1.16	8.52 ± 1.16	0.14 ± 0.01	0.14 ± 0.011
Compound 27a/HepG2	58.98 ± 2.64	40.47 ± 2.55***	0.35 ± 0.06	0.20 ± 0.04

^aValues are given as mean ± SEM of three independent experiments.

***p < 0.001 indicates statistically significant difference from the corresponding control (HepG2) group in unpaired t-test.

2.2.7. Effects on the levels of active caspase-3 and caspase-9

To analyse the effect of the most active compound 27a on protein expression levels of caspase-3 and caspase-9, Western blot analysis was utilised⁸⁷. In this test, HepG2 cells were treated with compound 27a at its cytotoxic concentration (4.5 µM) for 24 h. The results displayed a marked increase in the level of caspase-3 (2.5-fold) and caspase-9 (3.43-fold) compared to the control cells (Table 4 and Figure 6). Such findings are consistent with previous reports declared that VEGFR-2 inhibitors can up-regulate both caspase-3 and caspase-9 to induce apoptosis^88,89.

Figure 6. The immunoblotting of caspase-3, caspase-9, BAX, and Bcl-2 (normalised to β-actin). (A) Representative Western blot images show the effect of compound 27a (at its IC₅₀ concentration) on the expression levels of BAX, Bcl-2, active caspases-9, and active caspases-3 proteins in HepG2 cells.

Table 4. Effect of compound 27a on the levels of active caspases-3, active caspases-9, BAX, and Bcl-2 proteins in HepG2 cells treated for 24 h.

Sample	Protein expression (normalised to β-actin)^a
	Caspases-3	Caspases-9	BAX	Bcl-2	BAX/Bcl-2 ratio
HepG2	1.00 ± 0.10	1.00 ± 0.10	1.00 ± 0.13	1.00 ± 0.11	1.00 ± 0.09
27a/HepG2	2.51 ± 0.27**	3.43 ± 0.49**	2.60 ± 0.20**	0.52 ± 0.02*	5.03 ± 0.36***

^aValues are given as mean ± SEM of three independent experiments.

*p < 0.05, **p < 0.01, ***p < 0.001 indicate statistically significant differences from the corresponding control (HepG2) group in unpaired t-tests.

2.2.8. Effects on the levels of Bcl-2 family and BAX/Bcl-2 ratio

In this study, BAX and Bcl-2 expression levels in the HepG2 cell line after the treatment with compound 27a were determined by quantitative Western blotting. The results revealed that compound 27a significantly affected the apoptosis pathway in HepG2 cells as it increased the BAX level by 2.6-fold compared to the control cell. Also, it decreased the Bcl-2 level by 2-fold less than the control cells. In addition, the BAX/Bcl-2 ratio was increased 5-fold in the treated cells compared to the control cell (Table 4 and Figure 6). Meanwhile, compound 27a increased BAX/Bcl-2 ratio, it could trigger apoptosis in the experienced cells^90,91.

2.3. In silico studies

2.3.1. Molecular docking studies

Computational docking studies were carried out against two VEGFR-2 crystal structures (PDB ID; 2OH4 and 4ASD)⁹². Docking studies were performed to explore the binding mode of the synthesised compounds against the ATP binding pocket of VEGFR-2. The docking experiments were carried out using MOE.14 software. Sorafenib was used as a reference molecule. The binding free energies (ΔG) of the tested ligands and sorafenib against each protein were presented in Table 5.

Table 5. The calculated ΔG (binding free energies) of the synthesised compounds and reference drug against VEGFR-2 (PDB ID; 2OH4 and 4ASD) (ΔG in Kcal/mole).

Comp.	VEGFR-2 (PDB ID: 2OH4) ΔG [Kcal/mole]	VEGFR-2 (PDB ID: 4ASD) ΔG [Kcal/mole]
27a	−25.71	−28.56
27b	−25.45	−25.51
27c	−10.82	−11.23
27d	−14.22	−20.33
27e	−23.61	−22.41
27f	−20.71	−21.06
28	−26.41	−29.46
29	−23.48	−25.22
30a	−16.47	−22.47
30b	−17.08	−23.62
30c	−21.59	−28.38
30d	−27.45	−24.17
30e	−20.74	−19.11
30f	−23.22	−16.83
30g	−18.23	−25.34
30h	−24.05	−20.07
30i	−20.62	−27.93
31a	−26.77	−29.15
31b	−25.01	−23.61
32	−21.66	−23.49
Sorafenib	−30.07	−36.05

At first, the co-crystallised ligands of each protein were re-docked into the active pockets of VEGFR-2. The resulted RMSD values between the original co-crystallised ligands and the re-docked ones were 1.04 and 1.15 Å. Such values approved the validation of docking processes (Figures 7 and 8).

Figure 7. Alignment of the co-crystallised pose and the re-docked pose of the same ligand (PDB ID; 2OH4).

Figure 8. Alignment of the co-crystallised pose and the re-docked pose of the same ligand (PDB ID; 4ASD).

Docking of sorafenib as a reference compound was performed to compare its binding mode with those of the target compounds. The proposed binding mode of the docked sorafenib was the same in the two pockets. The urea moiety bound the receptor through three hydrogen bonding interactions with the crucial amino acids; Glu883 (Glu885) and Asp1044 (Asp1046). Moreover, the N-methylpicolinamide moiety occupied the hinge region, where the pyridine moiety formed one hydrogen bond with Cys917 (Cys919) (Figures 9 and 10).

Figure 9. 2D binding mode of sorafenib into VEGFR-2 (PDB ID; 2OH4).

Figure 10. 2D binding mode sorafenib into VEGFR-2 (PDB ID; 4ASD).

The results of docking studies against VEGFR-2 (PDB ID; 2OH4) showed that the tested ligands have binding modes similar to that of sorafenib against the VEGFR-2 active pocket. From each series, the most cytotoxic compound was selected to analyse its biding mode against the active pocket. Compound 27a as a representative example of 3-methylquinoxalin-2(1H)one series revealed an affinity value of −25.71 kcal/mol. The pharmacophore (amide) moiety bound to the key amino acids Asp1046 and Glu388, where the NH group formed a hydrogen bond with Glu388 while the C=O group formed another hydrogen bond with Asp1046. Four hydrophobic interactions took place between the phenyl ring (linker) and the amino acid residues Val897, Cys1043, Val914, and Lys866 in the linker region. Furthermore, the 3-methylquinoxalin-2(1H)one moiety occupied the hinge region and was involved in five hydrophobic interactions with Leu838, Leu1033, Phe1045, and Phe916. The tert-butyl moiety occupied the allosteric pocket (Figure 11).

Figure 11. 2D binding mode compound 27a into VEGFR-2 (PDB ID; 2OH4).

The proposed binding mode of 30f as an example of 3-methylquinoxaline-2-thiol series against VEGFR-2 (PDB ID; 2OH4) was like that of sorafenib. The docking score of such a compound was −23.22 kcal/mol. Compound 30f interacted with the key amino acid Asp1046 via its hydrogen bond acceptor (C=O) group of amide moiety, while the hydrogen bond donor (NH) group interacted with the carboxylate moiety of Glu388. Three hydrophobic interactions were observed between the spacer phenyl ring and amino acid residues Lys866, Val897, and Val914. In addition, 3-methylquinoxaline-2-thiol moiety formed seven hydrophobic interactions with Phe1045, Val846, Leu838, Phe916, and Leu1033. Finally, the terminal 2-tolyl moiety occupied the allosteric biding site forming two hydrophobic interactions with Ile868 (Figure 12).

Figure 12. 2D binding mode compound 30f into VEGFR-2 (PDB ID; 2OH4).

The proposed binding mode of 28 against VEGFR-2 (PDB ID; 4ASD) was like that of sorafenib. The docking score of such a compound was −29.46 kcal/mol. Compound 28 interacted with Cys1045 via its hydrogen bond acceptor (C=O) group of amide moiety, while the hydrogen bond donor (NH) group interacted with the carboxylate moiety of Glu885. Two hydrophobic interactions were observed between the spacer phenyl ring and amino acid residues Lys868, Phe1047. In addition, 3-methylquinoxaline moiety formed one hydrogen bond with the key amino acid Cys919 and one hydrophobic interaction with Leu840. Finally, the terminal p-nitrophenyl moiety occupied the allosteric biding site forming an extra hydrogen bond with Cys1024 (Figure 13).

Figure 13. 2D binding mode of compound 28 into VEGFR-2. PDB ID; 4ASD.

2.3.2. In silico ADMET study

To predict the pharmacokinetics properties of the newly synthesised compounds, computer-aided ADME studies were performed using Discovery Studio 4.0 software. Sorafenib was used as a reference drug. These studies include the assessment of certain parameters as blood-brain barrier (BBB) penetration, absorption level, aqueous solubility, CYP2D6 binding, and plasma protein binding. Predictions of ADME properties for the studied compounds were listed in Supplementary Data).

The results showed that compounds 30a–f have medium BBB diffusion levels while the rest of the compounds showed low to very low levels. Consequently, the CNS side effects were anticipated to be minimal for the majority of the synthesised compounds. Regarding, aqueous solubility, compounds 27a–c, 27e, 28, and 29 exhibited good levels, while compounds 27d, 27f, 30a–i, 31a,b, and 32 showed poor levels. With respect to absorption parameter, all compounds demonstrated good absorption levels except compounds 27e showed moderate absorption level and compounds 28, 30h, and 31b which showed poor to very poor intestinal absorption levels. Moreover, the effect on cytochrome P450 2D6 was investigated. The results showed that all the tested compounds were non-inhibitors of CYP2D6. Consequently, hepatotoxicity is not expected upon their administration. The plasma protein binding model displayed that compounds 27a, 28, 30a–c, 30g,h, 31a,b, and 32 were anticipated to bind plasma protein <90%. On the other hand, compounds 27b–f, 29, 30d–f, and 30i were expected to bind plasma protein more than 90% (Figure 14).

Figure 14. The expected ADMET study for the synthesised compounds.

2.3.3. In silico toxicity study

The toxicity profile of the synthesised compounds was determined based on the validated and constructed models in Discovery studio 4.0 software^93,94. This includes the prediction of certain parameters as FDA rodent carcinogenicity, carcinogenic potency TD₅₀, rat maximum tolerated dose, developmental toxicity potential, rat oral LD₅₀, rat chronic lowest observed adverse effect level (LOAEL), ocular irritancy, and skin irritancy.

Regarding the FDA rodent carcinogenicity model compounds 27a–f, 28, 30b, 30g, and 30i were forecasted to be non-carcinogenic. For carcinogenic potency, TD₅₀ mouse model compounds 27a,b, 29, 30a, 30c–f, 30h, i, 31a,b, and 32 showed TD₅₀ values ranging from 15,600 to 225,175 mg/kg body weight, which is higher than sorafenib (14,244 mg/kg body weight), while compounds 27c–f, 28, 30b, and 30g showed carcinogenic potency TD₅₀ lower than that of sorafenib. With respect to the rat maximum tolerated dose model (MTD), compounds 29, 30e,f, 30i, 31a, and 32 demonstrated maximum tolerated dose with a range of 0.096 to 0.194 g/kg body weight, which is higher than that of sorafenib (0.089 g/kg body weight). In Addition, all compounds were predicted to be non-toxic against the developmental toxicity potential model except compounds 27f, 30g, and 30i. For the rat oral LD₅₀ model, all compounds revealed oral LD₅₀ values in a range of 1.523 to 19.408 g/kg body weight which is higher than that of sorafenib (0.823 g/kg body weight). For the rat chronic LOAEL model, the examined members displayed LOAEL values ranging from 0.036 to 0.376 g/kg body weight. These values are higher than sorafenib (0.005 g/kg body weight). Additionally, all the tested compounds were predicted to be mild irritants against the ocular irritancy model and non-irritant against the skin irritancy model. The values of toxicity parameters were depicted in the Supplementary Data.

3. Conclusion

New twenty quinoxaline derivatives were designed and synthesised as anticancer agents with VEGFR-2 inhibitory activity. The synthesised compounds were evaluated in vitro for their anti-proliferative activities against breast cancer (MCF-7) and hepatocellular carcinoma (HepG2). Compound 27a was the most potent derivative revealing strong anti-proliferative activity against MCF-7 and HepG2 cell lines with IC₅₀ values of 7.7 and 4.5 µM, respectively, comparing to sorafenib (IC₅₀ = 3.51 and 2.17 µM, respectively). In addition, compounds 28 (IC₅₀ = 17.2 and 11.7 µM), 30f (IC₅₀ = 18.1 and 10.7 µM), 30i (IC₅₀ = 17.2 and 12.7 µM), and 31b (IC₅₀ = 19.2 and 13.7 µM) demonstrated promising cytotoxicity against MCF-7 and HepG2, respectively. The results of the VEGFR-2 enzyme assay were highly correlated with that of cytotoxicity, where the most potent antiproliferative derivatives exhibited good VEGFR-2 inhibitory effects. Compounds 27a was the most potent VEGFR-2 inhibitor with an IC₅₀ value of 3.2 nM in comparison to sorafenib (IC₅₀ = 3.12 nM). SAR revealed that 3-methylquinoxalin-2(1H)one moiety was more efficient than 3-methylquinoxaline-2-thiol moiety as a heterocyclic ring. The amide moiety was more advantageous as a pharmacophore than the corresponding diamide and hydrazide moieties. In addition, the tert-butyl moiety was the most effective hydrophobic tail. Cell cycle analysis of compounds 27a revealed that such compound can arrest HepG2 cell growth at G2/M phase by 3.5-fold greater than control cell. Moreover, compound 27a induced apoptosis in HepG2 cells by five times more than the control cells. Furthermore, it increased the level of caspase-3 and caspase-9 by 2.5-folds and 3.43-fold, respectively. Also, compound 27a showed an increase in the BAX level (2.6-fold), decrease in the Bcl-2 level (2-fold), and elevation of BAX/Bcl-2 ratio (5-fold) for the control cell. The results of docking studies revealed that the proposed binding modes of the designed compounds were similar to that of sorafenib. Finally, this work presents compound 27a as a lead candidate that can be further optimised for the synthesis of a promising VEGFR 2 inhibitor.

4. Experimental

4.1. Chemistry and material

All the reagents, chemicals, and apparatus were presented in Supplementary Data. Compounds 11⁶⁷, 12⁶⁷, 13⁶⁸, 14^69,70, 15^69,70, and the intermediates (24a,b, 25, 26a–j)^63,64 were prepared according to the reported procedures.

4.1.1. General procedure for the synthesis of target compounds 27a–f, 28, and 29

A mixture of potassium salt 12 (0.5 g, 2 mmol) and appropriate intermediates 26a,d,g–j, 24b, and 25 (1 mmol) in dry DMF (15 ml) with the presence of potassium iodide (0.2 g, 1.2 mmol), was heated over a water bath for 3 h. The reaction mixture was then cooled, poured into ice-cooled water (150 ml) with continuous stirring. The solid separated was filtered, washed with water several times, then dried and crystallised from the proper solvent to afford the final target compounds 27a-f, 28, and 29, respectively.

4.1.1.1. N-(tert-butyl)-4-(2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetamido)benzamide (27a)

The product was crystallised from EtOH/DCM mixture (50:50) as dark green crystal (0.3 g, 80%); m. p. = 230–232 °C; FT-IR (ν max, cm⁻¹): 3291 (NH), 3077 (CH aromatic), 2967 (CH aliphatic), 1648 (C=O), 1601 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm):10.66 (s, 1H), 7.81–7.79 (m, 3H), 7.64–7.61 (m, 3H), 7.58–7.56 (m, 1H), 7.53 (dd, J = 8.5, 1.3 Hz, 1H), 7.38 (s, 1H), 5.16 (s, 2H), 2.49 (s, 3H), 1.38 (s, 9H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 166.00, 165.63, 157.96, 154.85, 141.25, 133.48, 132.46, 131.07, 130.18(2 C), 129.27(2 C), 128.76, 123.92, 118.55(2 C), 115.20, 51.16, 45.76, 29.11(3 C), 21.59; MS (m/z): 393.19 (M⁺ +1, base beak, 100%); Anal. Calcd. for C₂₂H₂₄N₄O₃ (392.46): C, 67.33; H, 6.16; N, 14.28; Found C, 67.74; H, 6.32; N, 14.39%.

4.1.1.2. 4-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)acetamido)-N-phenethylbenzamide (27b)

The product was crystallised from EtOH/DCM mixture (50:50) as white crystal (0.34 g, 85%); m. p. = 283–285 °C; FT-IR (ν max, cm-1): 3274 (NH), 3038 (CH aromatic), 2921 (CH aliphatic), 1676, 1656, 1630 (C=O), 1604 (C=N); ¹H NMR (700 MHz, DMSO d6) δ (ppm):10.69 (s, 1H), 10.39 (s, 1H), 8.48 (t, J = 5.6 Hz, 1H), 7.82–7.81 (m, 1H), 7.80 (q, J = 1.7 Hz, 1H), 7.67–7.62 (m, 2H), 7.57 (ddd, J = 8.5, 7.1, 1.5 Hz, 1H), 7.52 (dd, J = 8.5, 1.3 Hz, 1H), 7.38 (ddd, J = 8.1, 7.1, 1.2 Hz, 1H), 7.30 (tt, J = 7.7, 1.7 Hz, 2H), 7.26–7.23 (m, 2H), 7.22–7.19 (m, 1H), 5.16 (s, 2H), 3.47 (ddd, J = 8.6, 7.5, 5.9 Hz, 2H), 2.86–2.82 (m, 2H), 2.49 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 165.97, 165.69, 157.97, 154.84, 141.52, 140.03, 133.46, 132.46, 130.18(2 C), 129.28(2 C), 129.11(2 C), 128.81(2 C), 128.56, 126.55, 123.92, 118.79(2 C), 115.19, 45.76, 41.32, 35.64, 21.59; MS (m/z): 441.2 (M⁺ +1, 28.68%); Anal. Calcd. for C₂₆H₂₄N₄O₃ (440.50): C, 70.89; H, 5.49; N, 12.72; Found C, 70.99; H, 5.13; N, 12.64%.

4.1.1.3. N-(2,6-Dimethoxyphenyl)-4-(2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetamido)-benzamide (27c)

The product was crystallised from ethanol/gl.acetic acid mixture (80:20) as yellow crystal (0.31 g, 80%); m. p. < 300 °C; FT-IR (ν max, cm⁻¹): 3422, 3293 (NH), 3058 (CH aromatic), 2929 (CH aliphatic), 1661 (C=O), 1602 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm):10.76 (s, 1H), 10.52 (s,1H), 7.97 (d, J = 6.5 Hz, 1H), 7.94–7.93 (m, 2H), 7.83 (d, J = 6.7 Hz, 1H), 7.77 (d, J = 8.7 Hz, 2H), 7.73–7.71 (m, 1H), 7.68 (t, J = 1.4 Hz, 1H), 7.57 (m, 2H), 7.48 (d, J = 1.3 Hz, 1H), 4.33 (s, 2H), 3.32 (s, 6H), 2.68 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm):167.11, 166.26, 165.46, 155.45, 151.97, 142.71, 140.82, 139.36, 135.19, 131.97, 130.93, 130.38, 130.05, 129.89, 129.05(2 C), 128.91, 128.68, 127.64, 127.43, 127.38, 118.85, 35.46(3 C), 22.19; Anal. Calcd. for C₂₆H₂₄N₄O₅ (472.50): C, 66.09; H, 5.12; N, 11.86; Found C, 66.01; H, 5.15; N, 11.64%.

4.1.1.4. N-(2,6-Dimethylphenyl)-4-(2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetamido)-benzamide (27d)

The product was crystallised from EtOH/DCM mixture (50:50) as yellowish white crystal (0.35 g, 85%); m. p. = 240–242 °C; FT-IR (ν max, cm⁻¹): 3429 (NH), 3067 (CH aromatic), 2934 (CH aliphatic), 1656, (C=O), 1603 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 10.76 (s, 1H), 9.67 (s, 1H), 7.99 (d, J = 8.5 Hz, 2H), 7.81 (dd, J = 8.0, 1.5 Hz, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.58 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.54 (dd, J = 8.6, 1.3 Hz, 1H), 7.42–7.35 (m, 1H), 7.12 (s, 3H), 5.19 (s, 2H), 2.50 (s, 3H), 2.18 (s, 6H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 165.77, 164.83, 157.98, 154.86, 141.88, 136.12, 135.89, 133.48, 132.48, 130.19(2 C), 129.64(2 C), 129.29(2 C), 129.02(2 C), 128.16, 127.07, 123.94, 118.95, 115.21, 45.79, 21.60, 18.56(2 C); MS (m/z): 440.18 (M+, 27.13%); Anal. Calcd. for C₂₆H₂₄N₄O₃ (440.50): C, 70.89; H, 5.49; N, 12.72; Found C, 70.54; H, 5.39; N, 12.52%.

4.1.1.5. 4-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)acetamido)-N-(4-nitrophenyl)benzamide (27e)

The product was crystallised from ethanol/gl.acetic acid mixture (80:20) as brown crystal (0.29 g, 78%); m. p. > 300 °C; FT-IR (ν max, cm⁻¹): 3428 (NH), 3060 (CH aromatic), 2955 (CH aliphatic), 1653, (C=O), 1599 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 10.83 (s, 1H), 10.72 (s, 1H), 8.29–8.26 (m, 2H), 8.08–8.06 (m, 2H), 8.02–8.00 (m, 2H), 7.81 (dt, J = 8.0, 1.8 Hz, 1H), 7.77–7.74 (m, 2H), 7.59–7.53 (m, 2H), 7.39 (ddd, J = 8.2, 7.0, 1.4 Hz, 1H), 5.19 (s, 2H), 2.49 (s, 3H); ¹³C NMR (176 MHz, DMSO-d₆) δ (ppm): 165.92, 157.96, 154.85, 146.10, 142.82, 142.58, 133.46, 130.20(2 C), 129.62(2 C), 129.29(2 C), 129.18(2 C), 125.27(2 C), 123.95, 120.24(2 C), 118.90, 115.21, 45.82, 21.59. Anal. Calcd. for C₂₄H₁₉N₅O₅ (457.45): C, 63.02; H, 4.19; N, 15.31; Found C, 63.17; H, 4.13; N, 15.64%.

4.1.1.6. N-(5-Chloropyridin-2-yl)-4-(2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetamido)-benzamide (27f)

The product was crystallised from ethanol/gl.acetic acid mixture (80:20) as dark brown crystal (0.36 g, 90%); m. p. < 300 °C; FT-IR (ν max, cm⁻¹): 3414 (NH), 3030 (CH aromatic), 2962 (CH aliphatic), 1660 (C=O), 1600 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 10.78 (s, 1H), 10.21 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.81–7.79 (m, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.58 (td, J = 7.6, 6.9, 1.5 Hz, 1H), 7.56–7.53 (m, 1H), 7.40–7.36 (s, 1H), 7.19 (t, J = 8.9 Hz, 2H), 5.19 (s, 2H), 2.49 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 165.82, 165.15, 158.00, 157.96, 154.85, 142.05, 136.06, 133.46, 132.48, 130.18, 129.86(2 C), 129.21(2 C), 123.93, 122.62, 122.58, 118.86, 115.68, 115.55, 115.19, 45.80, 21.59; MS (m/z): 448.1 (M⁺ +1, base beak, 100%); Anal. Calcd. for C₂₃H₁₈ClN₅O₃ (447.88): C, 61.68; H, 4.05; N, 15.64; Found C, 61.17; H, 4.13; N, 15.36%.

4.1.1.7. 2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)-N-(4-(2-(4-nitrobenzoyl)hydrazine-1-carbonyl) phenyl)acetamide (28)

The product was crystallised from EtOH/DCM mixture (50:50) as white crystal (0.28 g, 74%); m. p. = 257–259 °C; FT-IR (ν max, cm⁻¹): 3271 (NH), 3055 (CH aromatic), 2944 (CH aliphatic), 1656 (C=O), 1601(C=N); ¹H NMR (700 MHz, DMSO-d6) δ 10.82 (s, 1H), 10.56 (s, 2H), 8.01 (m, 2H), 7.94 (m, 2H), 7.81 (d, J = 7.9 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H), 7.56 (dd, J = 22.6, 8.2 Hz, 3H), 7.39 (d, J = 7.6 Hz, 1H), 5.19 (s, 2H), 2.49 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ 165.86, 164.81 (2C), 157.97, 154.85, 149.86, 142.31, 138.75, 133.45, 132.47(2 C), 130.20 (2C), 129.46(2C), 129.06(3 C), 124.25(2C), 119.01(2C), 115.20, 45.81, 21.59. Anal. Calcd. for C₂₅H₂₀N₆O₆ (500.47): C, 60.00; H, 4.03; N, 16.79; Found C, 60.78; H, 4.54; N, 16.56%.

4.1.1.8. 2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)-N-(4-(2-phenylhydrazine-1-carbonyl) phenyl) acetamide (29)

The product was crystallised from ethanol/gl.acetic acid mixture (80:20) as greenish yellow crystal (0.3 g, 75%); m. p. < 300 °C; FT-IR (ν max, cm⁻¹): 3291 (NH), 3041 (CH aromatic), 2967 (CH aliphatic), 1648 (C=O), 1601 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm): 10.70 (s, 1H), 10.31 (s, 1H), 10.02 (s, 1H), 8.33 (dd, J = 8.1, 1.4 Hz, 1H), 8.26–7.75 (m, 4H), 7.75–7.72 (m, 2H), 7.72–7.69 (m, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.59–7.56 (m, 1H), 7.49–7.46 (m, 1H), 7.38 (t, J = 8.1 Hz, 1H), 7.17–7.14 (m, 1H), 5.22 (s, 2H), 2.74 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 157.97, 155.40, 154.86, 133.47, 132.47(2C), 130.20(3 C), 129.78, 129.29(2C), 129.05(3 C), 128.34(2C), 123.95, 123.50(2C), 115.69, 115.20, 45.79, 21.59; Anal. Calcd. for C₂₄H₂₁N₅O₃ (427.46): C, 67.44; H, 4.95; N, 16.38; Found C, 67.73; H, 4.76; N, 16.54%.

4.1.2. General procedure for the synthesis of target compounds 30a–i, 31a,b, and 32

Equimolar amounts of potassium salt 15 (0.3 g, 1 mmol) and appropriate intermediates 26a–g,i,j, 24a,b, and 25 (1 mmol) in dry DMF (15 ml) with the presence of potassium iodide (0.2 g, 1.2 mmol) was heated on a water-bath for 3 h. After cooling, the reaction mixture was poured into an ice-water (150 ml) with continuous stirring. The resulted precipitate was filtered and crystallised from the proper solvent to give the final target compounds 30a–i, 31a,b, and 32, respectively.

4.1.2.1. N-(tert-Butyl)-4-(2-((3-methylquinoxalin-2-yl)thio)acetamido)benzamide (30a)

The product was crystallised from ethanol as brown crystal (0.15 g, 70%); m. p. = 231–233 °C; FT-IR (ν max, cm⁻¹): 3315 (NH), 3048 (CH aromatic), 2969 (CH aliphatic), 1673, 1631 (C=O), 1599 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 10.64 (s, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.73–7.71 (m, 1H), 7.70–7.68 (m, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.62 (s, 1H), 4.30 (s, 2H), 2.67 (s, 3H), 1.37 (s, 9H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 166.88, 166.11, 155.47, 151.98, 141.71, 140.81, 139.35, 130.97, 130.03(2C), 128.91(2C), 128.72, 128.68(2C), 127.36, 118.51, 51.14, 35.39, 29.11(3 C), 22.19; MS (m/z): 409.1 (M⁺ +1, base beak, 100%); Anal. Calcd. for C₂₂H₂₄N₄O₂S (408.52): C, 64.68; H, 5.92; N, 13.71; Found C, 64.56; H, 5.89; N, 13.32%.

4.1.2.2. N-Cyclohexyl-4-(2-((3-methylquinoxalin-2-yl)thio)acetamido)benzamide (30b)

The product was crystallised from ethanol as dark brown crystal (0.18 g, 85%); m. p. = 238–240 °C; FT-IR (ν max, cm⁻¹): 3289 (NH), 3055 (CH aromatic), 2928, 2851 (CH aliphatic), 1671, 1628 (C=O), 1607 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm): 10.66 (s, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.96 (s, 1H), 7.84–7.81 (m, 3H), 7.71 (ddd, J = 8.3, 6.9, 1.6 Hz, 1H), 7.70–7.67 (m, 3H), 4.30 (s, 2H), 3.77–3.72 (m, 1H), 2.67 (s, 3H), 1.83–1.72 (m, 4H), 1.63–1.59 (m, 1H), 1.33–1.27 (m, 4H), 1.12 (s, 1H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 166.92, 165.21, 155.47, 151.98, 141.88, 140.82, 139.36, 130.04(2C), 128.91(2C), 128.68(2C), 127.37, 118.62(2C), 48.71, 35.38, 32.95(2C), 25.75, 25.44(2C), 22.18; MS (m/z): 435.1 (M⁺ +1, base beak, 100%); Anal. Calcd. for C₂₄H₂₆N₄O₂S (434.56): C, 66.33; H, 6.03; N, 12.89; Found C, 66.39; H, 6.09; N, 12.98%.

4.1.2.3. N-benzyl-4-(2-((3-methylquinoxalin-2-yl)thio)acetamido)benzamide (30c)

The product was crystallised from ethanol as yellow crystal (0.16 g, 75%); m. p. = 235–237 °C; FT-IR (ν max, cm⁻¹): 3287 (NH), 3071 (CH aromatic), 2933 (CH aliphatic), 1674, 1633, (C=O), 1607 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm): 10.69 (s, 1H), 8.94 (s, 1H), 7.96 (dd, J = 8.0, 1.5 Hz, 1H), 7.88 (d, J = 8.5 Hz, 2H), 7.82 (dd, J = 8.3, 1.6 Hz, 1H), 7.73–7.70 (m, 3H), 7.68 (td, J = 7.6, 1.5 Hz, 1H), 7.32 (d, J = 6.3 Hz, 4H), 7.24 (tt, J = 5.9, 2.4 Hz, 1H), 4.47 (d, J = 5.9 Hz, 2H), 4.31 (s, 2H), 2.67 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 166.97, 166.10, 155.45, 151.97, 142.13, 140.81, 140.27, 139.35, 130.04(2C), 129.49(2C), 128.90(2C), 128.73(2C), 128.69, 128.67, 127.67, 127.38, 127.16, 118.77, 43.02, 35.41, 22.18; MS (m/z): 443.1 (M⁺ +1, 79.98%); Anal. Calcd. for C₂₅H₂₂N₄O₂S (442.54): C, 67.85; H, 5.01; N, 12.66; Found C, 67.73; H, 5.09; N, 12.78%.

4.1.2.4. 4-(2-((3-Methylquinoxalin-2-yl)thio)acetamido)-N-phenethylbenzamide (30d)

The product was crystallised from ethanol as brown crystal (0.18 g, 85%); m. p. = 220–222 °C; FT-IR (ν max, cm⁻¹): 3299 (NH), 3026 (CH aromatic), 2925 (CH aliphatic), 1664, 1630 (C=O), 1607 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 10.67 (s, 1H), 8.47 (s, 1H), 7.98–7.94 (m, 1H), 7.85–7.79 (m, 3H), 7.72 (ddd, J = 8.4, 7.0, 1.7 Hz, 1H), 7.71–7.66 (m, 3H), 7.32–7.27 (m, 2H), 7.26–7.23 (m, 2H), 7.23–7.18 (m, 1H), 4.31 (s, 2H), 3.47 (ddd, J = 8.7, 7.5, 5.9 Hz, 2H), 2.84 (t, J = 7.5 Hz, 2H), 2.67 (s, 3H); ¹³C NMR (176 MHz, DMSO d6) δ (ppm): 166.95, 166.05, 155.45, 151.97, 141.98, 140.81, 140.04, 139.35, 130.03(2C), 129.76 (2C), 129.11(2C), 128.90, 128.81, 128.68(2C), 128.53, 127.38, 126.54, 118.75, 41.32, 35.66, 35.40, 22.18; MS (m/z): 457.2 (M⁺ +1, base beak, 100%); Anal. Calcd. for C₂₆H₂₄N₄O₂S (456.56): C, 68.40; H, 5.30; N, 12.27; Found C, 68.39; H, 5.09; N, 12.32%.

4.1.2.5. 4-(2-((3-Methylquinoxalin-2-yl)thio)acetamido)-N-phenylbenzamide (30e)

The product was crystallised from ethanol as brown crystal (0.18 g, 85%); m. p. = 218–220 °C; FT-IR (ν max, cm⁻¹): 3268 (NH), 3039 (CH aromatic), 2970, 2908 (CH aliphatic), 1666, 1640 (C=O), 1597 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm): 10.75 (s, 1H), 10.14 (s, 1H), 7.98–7.95 (m, 3H), 7.83 (dd, J = 8.0, 1.6 Hz, 1H), 7.80–7.76 (m, 4H), 7.72 (ddd, J = 8.3, 6.9, 1.6 Hz, 1H), 7.68 (ddd, J = 8.4, 7.0, 1.6 Hz, 1H), 7.37–7.33 (m, 2H), 7.09 (tt, J = 7.4, 1.2 Hz, 1H), 4.33 (s, 2H), 2.67 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 167.07, 165.34, 155.44, 151.97, 142.46, 140.82, 139.74, 139.36, 130.02(2C), 129.93, 129.20(2C), 129.04(2C), 128.89, 128.68(2C), 127.37, 123.97, 120.79, 118.79, 35.44, 22.18; MS (m/z): 429.1 (M⁺ +1, base beak, 100%); Anal. Calcd. for C₂₄H₂₀N₄O₂S (428.51): C, 67.27; H, 4.70; N, 13.08; Found C, 67.04; H, 4.89; N, 13.42%.

4.1.2.6. 4-(2-((3-Methylquinoxalin-2-yl)thio)acetamido)-N-(o-tolyl)benzamide (30f)

The product was crystallised from EtOH/DCM mixture (50:50) as white crystal (0.2 g, 90%); m. p. = 247–249 °C; FT-IR (ν max, cm⁻¹): 3262 (NH), 3056 (CH aromatic), 2911 (CH aliphatic), 1659, 1641 (C=O), 1607 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm): 10.75 (s, 1H), 9.77 (s, 1H), 7.97 (dd, J = 8.4, 4.1 Hz, 3H), 7.84 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.73 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 7.5 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 4.33 (s, 2H), 2.68 (s, 3H), 2.24 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 167.05, 165.12, 155.46, 151.98, 142.42, 140.82, 139.36, 136.99, 134.13, 130.75, 130.05, 129.57(2C), 129.14(2C), 128.91, 128.69, 127.38, 127.03, 126.44, 126.33, 118.82, 35.44, 22.19, 18.40; MS (m/z): 443.1 (M⁺ +1, base beak, 100%); Anal. Calcd. for C₂₅H₂₂N₄O₂S (442.54): C, 67.85; H, 5.01; N, 12.66; Found C, 67.73; H, 5.09; N, 12.78%.

4.1.2.7. N-(2,6-Dimethoxyphenyl)-4-(2-((3-methylquinoxalin-2-yl)thio)acetamido) benzamide (30g)

The product was crystallised from EtOH/DCM mixture (50:50) as light brown crystal (0.17 g, 80%); m. p. = 258–260 °C; FT-IR (ν max, cm⁻¹): 3298 (NH), 3033 (CH aromatic), 2931 (CH aliphatic), 1665 (C=O), 1607 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 10.77 (s, 1H), 9.37 (s, 1H), 7.98 (t, J = 8.3 Hz, 4H), 7.93 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.75–7.72 (m, 1H), 7.71–7.67 (m, 1H), 7.35–7.33 (m, 1H), 4.34 (s, 2H), 3.99–3.97 (s, 3H), 2.69 (s, 6H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 167.09, 164.78, 155.46, 151.99, 151.84, 142.57, 140.83, 139.37, 137.68, 130.06, 129.45(2C), 129.05, 129.00, 128.92, 128.69, 127.39, 126.88, 124.35, 119.00, 118.90, 110.11, 56.46(2C), 35.46, 22.20; Anal. Calcd. for C₂₆H₂₄N₄O₄S (488.56): C, 63.92; H, 4.95; N, 11.47; Found C, 63.56; H, 4.45; N, 11.56%.

4.1.2.8. 4-(2-((3-Methylquinoxalin-2-yl)thio)acetamido)-N-(4-nitrophenyl)benzamide (30h)

The product was crystallised from ethanol as pale yellow crystal (0.12 g, 60%); m. p. = 200–202 °C; FT-IR (ν max, cm⁻¹): 3316 (NH), 3028 (CH aromatic), 2921 (CH aliphatic), 1682, 1650 (C=O), 1596 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 10.76 (s, 1H), 10.26 (s, 1H), 7.96 (dd, J = 9.2, 2.3 Hz, 3H), 7.82 (td, J = 7.6, 7.0, 1.8 Hz, 3H), 7.82–7.76 (m, 2H), 7.71 (ddd, J = 8.3, 6.9, 1.6 Hz, 1H), 7.68 (ddd, J = 8.3, 6.9, 1.6 Hz, 1H), 7.43–7.38 (m, 2H), 4.33 (s, 2H), 2.67 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 167.10, 165.42, 155.43, 151.96, 142.61, 140.81, 139.36, 138.73, 130.02, 129.61(3 C), 129.25(2C), 128.96(2C), 128.89, 128.68, 127.55, 127.36, 122.24, 118.80, 35.43, 22.18; Anal. Calcd. for C₂₄H₁₉N₅O₄S (473.51): C, 60.88; H, 4.04; N, 14.79; Found C, 60.39; H, 4.58; N, 14.66%.

4.1.2.9. N-(5-Chloropyridin-2-yl)-4-(2-((3-methylquinoxalin-2-yl)thio)acetamido)benzamide (30i)

The product was crystallised from ethanol as brown crystal (0.19 g, 90%); m. p. = 190–192 °C; FT-IR (ν max, cm⁻¹): 3274 (NH), 3041 (CH aromatic), 2945 (CH aliphatic), 1679 (C=O), 1599 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 10.86 (s, 1H), 10.78 (s, 1H), 8.44 (t, J = 2.6 Hz, 1H), 8.24–8.23 (m, 1H), 8.04–8.02 (m, 2H), 7.96 (d, J = 3.3 Hz, 2H), 7.91 (d, J = 4.8 Hz, 2H), 7.82 (d, J = 6.6 Hz, 1H), 7.76 (s, 1H), 7.72–7.71 (m, 1H), 4.32 (s, 2H), 2.67 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 167.35, 167.16, 155.42, 151.96, 151.48, 146.72, 140.80, 139.35, 138.27, 130.95, 130.04(2C), 129.69(2C), 128.91, 128.67(2C), 127.38, 118.86, 118.71, 116.18, 35.45, 22.18; MS (m/z): 464.1 (M⁺ +1, 68.32%),; Anal. Calcd. for C₂₃H₁₈ClN₅O₂S (463.94): C, 59.54; H, 3.91; N, 15.10; Found C, 59.56; H, 3.59; N, 15.35%.

4.1.2.10. N-(4-(2-benzoylhydrazine-1-carbonyl)phenyl)-2-((3-methylquinoxalin-2-yl)thio) acetamide (31a)

The product was crystallised from ethanol as brown crystal (0.13 g, 65%); m. p. = 195–197 °C; FT-IR (ν max, cm⁻¹): 3262 (NH), 3038 (CH aromatic), 2921 (CH aliphatic), 1657 (C=O), 1601 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm): 10.76 (s, 1H), 10.48 (s, 1H), 10.42–10.41 (s, 1H), 7.94 (d, J = 6.8 Hz, 4H), 7.83 (d, J = 6.8 Hz, 1H), 7.77 (d, J = 4.6 Hz, 1H), 7.72 (d, J = 1.6 Hz, 1H), 7.69–7.67 (m, 1H), 7.60 (d, J = 6.1 Hz, 2H), 7.53 (m, 3H), 4.33 (s, 2H), 2.68 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 167.11, 166.36, 165.77(2C), 155.45, 151.97, 142.68, 140.82, 139.36, 133.09, 132.32, 130.05(2C), 129.00(3 C), 128.98(2C), 128.68(2C), 127.93, 127.38, 118.90, 35.45, 22.19; MS (m/z): 472.1 (M⁺ +1, 53.18%); Anal. Calcd. for C₂₅H₂₁N₅O₃S (471.54): C, 63.68; H, 4.49; N, 14.85; Found C, 63.88; H, 4.68; N, 14.99%.

4.1.2.11. 2-((3-Methylquinoxalin-2-yl)thio)-N-(4-(2-(4-nitrobenzoyl)hydrazine-1-carbonyl) phenyl) acetamide (31b)

The product was crystallised from ethanol as brownish red crystal (0.15 g, 70%); m. p. = 196–198 °C; FT-IR (ν max, cm⁻¹): 3261 (NH), 3044 (CH aromatic), 2911 (CH aliphatic), 1664 (C=O), 1601 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm): 10.81 (s, 2H), 10.55 (s, 1H), 8.39 (t, J = 8.7 Hz, 4H), 8.17 (s, 2H), 7.96 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 3.8 Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 4.33 (s, 2H), 2.67 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm): 165.70, 164.87, 164.85, 164.75, 155.44, 149.98, 149.89, 139.36, 138.67, 130.08(2C), 129.52(2C), 129.47(3 C), 127.38(3 C), 124.32(2C), 119.10, 118.93, 35.45, 22.18; MS (m/z): 517.0 (M⁺ +1, base beak, 100%); Anal. Calcd. for C₂₅H₂₀N₆O₅S (516.53): C, 58.13; H, 3.90; N, 16.27; Found C, 58.21; H, 3.98; N, 16.48%.

4.1.2.12. 2-((3-Methylquinoxalin-2-yl)thio)-N-(4-(2-phenylhydrazine-1-carbonyl)phenyl) acetamide (32)

The product was crystallised from ethanol as white crystal (0.18 g, 85%); m. p. = 185–187 °C; FT-IR (ν max, cm⁻¹): 3272 (NH), 3083 (CH aromatic), 2931 (CH aliphatic), 1676 (C=O), 1598 (C=N); ¹H NMR (700 MHz, DMSO-d6) δ (ppm): 12.71 (s, 1H), 10.76 (s, 1H), 10.20 (s, 1H), 7.92–7.88 (m, 2H), 7.84–7.79 (m, 2H), 7.76–7.73 (m, 2H), 7.69–7.65 (m, 2H), 7.38–7.31 (m, 2H), 7.15 (t, J = 7.7 Hz, 1H), 6.78 (d, J = 12.6, 8.0, 7.3 Hz, 2H), 4.32 (s, 2H), 2.69 (s, 3H); ¹³C NMR (176 MHz, DMSO-d6) δ (ppm):155.42, 151.96, 143.54, 140.81, 139.35, 131.08, 130.96, 130.03(2C), 129.79(3 C), 129.19(3 C), 128.9(2C)0, 119.77, 118.91, 118.87, 112.78, 112.01, 35.45, 22.18; Anal. Calcd. for C₂₄H₂₁N₅O₂S (443.53): C, 64.99; H, 4.77; N, 15.79; Found C, 64.54; H, 4.79; N, 15.88%.

4.2. Biological testing

4.2.1. In vitro anti-proliferative activity

The in vitro antiproliferative activities were assessed using the MTT assay protocol^78,79,95. As shown in Supplementary Data.

4.2.2. In vitro VEGFR-2 enzyme inhibition assay

The synthesised compounds were estimated for their in vitro inhibition on human VEGFR-2 in HepG2 cell line; using ELISA kit⁹⁶ as described in Supplementary Data.

4.2.3. Flow cytometry analysis for cell cycle

Cell cycle analysis for the most potent candidate 27a has been carried out through Flow cytometric analysis as described in Supplementary Data^84,85.

4.2.4. Flow cytometry analysis for apoptosis

Apoptosis analysis for compound 27a in HepG2 cells was carried out using Annexin V-FITC as shown in Supplementary Data^30,86.

4.2.5. Determination of active caspase-3 and caspase-9 levels

Quantitative assay of caspase-3 and caspase-9 activation was performed using Caspase- Invitrogen Caspase-3 ELISA Kit (KHO1091) and Invitrogen Caspase 9 Human ELISA Kit (BMS2025)^87,97–99 as shown in Supplementary Data.

4.3. In silico studies

4.3.1. Docking studies

The docking studies were performed utilising MOE.14 software as described in Supplementary Data^57,100,101. The final figures were visualised using Discovery studio 4.0¹⁰².

4.3.2. ADMET study

ADMET descriptors were determined using Discovery studio 4.0. as described in Supplementary Data^101,103.

4.3.3. Toxicity study

The toxicity parameters were calculated using Discovery studio 4.0. as described in Supplementary Data¹⁰².

Acknowledgement

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no (RG-1441–333).

Disclosure statement

The authors declare that no conflict of interest exists. The authors give valuable appreciation to Dr. Mohamed R. Elnagar, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt for his valuable technical assistance.