Discovery of new VEGFR-2 inhibitors based on bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxaline derivatives as anticancer agents and apoptosis inducers

Abstract

Herein, a new wave of bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxaline derivatives have been successfully designed and synthesised. The synthesised derivatives were biologically investigated for their cytotoxic activities against HepG2 and MCF-7. Also, the tested compounds were further examined in vitro for their VEGFR-2 inhibitory activity. The most promising derivative 23j was further investigated for its apoptotic behaviour in HepG2 cell lines using flow cytometric and western-plot analyses. Additional in-silico studies were performed to predict how the synthesised compounds can bind to VEGFR-2 and to determine the drug-likeness profiling of these derivatives. The results revealed that compounds 23a, 23i, 23j, 23l, and 23n displayed the highest antiproliferative activities against the two cell lines with IC₅₀ values ranging from 6.4 to 19.4 µM. Furthermore, compounds 23a, 23d, 23h, 23i, 23j, 23l, 23 m, and 23n showed the highest VEGFR-2 inhibitory activities with IC₅₀ values ranging from 3.7 to 11.8 nM, comparing to sorafenib (IC₅₀ = 3.12 nM). Moreover, compound 23j arrested the HepG2 cell growth at the G2/M phase and induced apoptosis by 40.12% compared to the control cells (7.07%). As well, such compound showed a significant increase in the level of caspase-3 (1.36-fold), caspase-9 (2.80-fold), and BAX (1.65-fold), and exhibited a significant decrease in Bcl-2 level (2.63-fold).

1. Introduction

Cancer is a rebound system represented by unrestricted cell growth¹. Cancer originates in the humanoid body from the buildup of genetic and epigenetic variations in the normal cells². In spite of the huge efforts directed towards cancer treatment and prevention, cancer remains one of the foremost public health problems all over the world³. Consequently; increasing interest in the current medicinal chemistry has been dedicated to the design and synthesis of more effective anticancer agents with low side effects⁴.

Angiogenesis is a multi-stage process to produce new vessels from quiescent pre-existing ones^5–7. It is interrelated to several physiological functions and also, it is a fundamental step in numerous diseases including cancer⁸. Furthermore, it has a significant role in tumour progression and development^9,10.

At present, there are main approaches in targeting angiogenesis that has been verified in clinical trials and officially approved in clinical practice¹¹: i) monoclonal antibodies which bind to vascular endothelial growth factor-A (VEGF-A) e.g. bevacizumab¹²; ii) VEGF-trap e.g. aflibercept which binds to VEGF-A, VEGF-B, and placental growth factor (PGF)¹³; iii) monoclonal antibodies targeting VEGF receptors and block the binding of natural VEGFR ligands e.g. ramucirumab, iv) tyrosine kinase inhibitors (TKIs) which are drugs that inhibit the kinase activity of VEGF receptors through binding to the ATP binding site. Hence, TKIs hinder the phosphorylation of the tyrosine residue and subsequent transmission of signalling down the intercellular pathway. Among this class of agents, sorafenib 1 and sunitinib 2 are the prototypes¹⁴. There have been numerous known tyrosine kinase receptors such as vascular endothelial growth factor receptors (VEGFRs) and endothelial growth factor receptors (EGFRs)¹⁵.

It has been stated that the vascular endothelial growth factor (VEGF) can stimulate the production and movement of vascular endothelial cells and regulate the formation of blood vessels^12,16. There are three main vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, and VEGFR-3), which are strategic intermediates in angiogenesis and in the construction of new networks of blood vessels required to hoard oxygen and nourishment for cancer growth¹⁷.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is the most critical regulator of angiogenic factors that plays a significant role in tumour survival, angiogenesis, and migration¹⁸. Binding of VEGFR-2 to VEGF leads to inspiration of downstream signalling pathway and certain endothelial reactions, such as improved permeability of vascular cells and increased endothelial cell multiplying and propagation, consequently, lead to angiogenesis¹⁹. Hence, blockage of VEGF/VEGFR-2 system is considered a favourable approach for anti-angiogenic therapy in retarding cancer growth^20,21. Additionally, VEGFR-2 has been confirmed to motivate apoptosis in cancer cells which synergistically enhances the antitumor effect^22–25.

Apoptosis has two major pathways; the intrinsic pathway which is controlled by the Bcl-2 family (involve BAX and Bcl-2) and the extrinsic pathway which is controlled by a subgroup of tumour necrosis factor receptors superfamily (TNFR)²⁶. Immediately as the apoptosis process is instigated, caspases, a family of protease enzymes, are activated. Caspases are classified into initiator caspases as caspases 8 and 9, effector caspases as caspases 3, 6, and, 7 and inflammatory caspases as caspases 1, 4, 5, 11, and 12²⁶. Caspase 9 is the initiator of intrinsic apoptosis while caspase 3 plays a central role in the execution phase of apoptosis²⁷. Cancer cells can avoid apoptosis by hindering caspase function, decreasing BAX expression level or increasing the gene expression level of anti-apoptotic Bcl-2^27,28.

A literature study revealed that VEGFR-2 inhibitors have main pharmacophoric features^29–31. (i) A flat heteroaromatic ring system that can accommodate the hinge region²⁹. (ii) A central spacer moiety that can occupy the linker region between the hinge region and DFG domain of the enzyme³². (iii) A pharmacophore moiety consists of an H-bond acceptor (HBA) and an H-bond donor (HBD). Such pharmacophore moiety can interact with the two vital amino acid residues (Asp1044 and Glu883) in the DFG motif region³³. (iv) A terminal hydrophobic moiety that occupies the allosteric hydrophobic pocket via numerous hydrophobic interactions³⁴ (Figure 1).

Figure 1.

The essential structural requirements of reported VEGFR-2 inhibitors.

Also, a literature review revealed that many VEGFR-2 inhibitors have been approved as potent anticancer agents such as sorafenib 1³⁵, Sunitinib 2³⁶, lenvatinib 3^37,38, lucitanib 4^37,38, and fruquinitinib 5³⁹. All of them fulfilled the main pharmacophoric features of VEGFR-2 inhibitors (Figure 2).

Figure 2.

Structures of some representative VEGFR-2 inhibitors.

1.1. Rationale of molecular design

Based on facts mentioned above and in the extension of our efforts to discover novel molecules with potential anticancer activity^5,40–48, we design and synthesise a new wave of bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]quinoxaline derivatives having the basic crucial pharmacophoric features of VEGFR-2 inhibitors.

At first, bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]quinoxaline moiety was selected as a heteroaromatic ring system to occupy the hinge region in the ATP binding site. Such moiety was accomplished to attain more rigid structures that could increase binding affinity against the active site. Then, sulfanyl-N-phenylacetamide moiety was utilised as a central spacer to occupy the linker region. Such spacer was expected to increase the flexibility of the designed compounds. Regarding the DFG-motif region, we used two different moieties having the essential HBA/HBD features to play the role of the pharmacophore. The pharmacophores were designed to be amide moiety (compounds 23a–n), or diamide moiety (compounds 24a–c). Finally, the allosteric hydrophobic region can be occupied by different terminal aliphatic moieties (23a–f) or terminal aromatic moieties (23 g–n and 24a–c). These wide varieties of modifications helped us to study the SAR of these compounds as efficient anticancer agents with potential VEGFR-2 inhibitory activities. All modification pathways and molecular design rationale were illustrated and summarised in Figure 3. As shown in Figure 4, the target compounds achieved the essential pharmacophoric requirements of VEGFR-2 inhibitors.

Figure 3.

Schematic representation showing the designing strategy.

Figure 4.

The target compounds fulfilled the pharmacophoric features of VEGFR-2 inhibitors.

To attain our aim of this work, cytotoxicity and VEGFR-2 inhibitory effects of the synthesised compounds were assessed. Besides, flow cytometric analyses were carried out for the most active candidate to estimate its potential against apoptosis induction and cell cycle arrest. Furthermore, such a compound was examined against several apoptotic markers include caspase 3/9 and BAX, and Bcl-2.

2. Results and discussion

2.1. Chemistry

The general synthetic pathways adopted for the synthesis of the designed compounds are illustrated in Schemes 1–3. Scheme 1 depicts the synthesis of potassium bis[1, 2, 4]triazolo[4,3-a]quinoxaline-4-thiolate 14. Initially, o-phenylenediamine 6 was refluxed with oxalic acid 7 in the presence of 4 N HCl to afford 2,3-(1H,4H)-quinoxalinedione 8⁴⁹. Chlorination of compound 8 was done by refluxing with thionyl chloride yielding 2,3- dichloroquinoxaline 9⁴⁹. Subsequent treatment of the latter with hydrazine hydrate in absolute ethanol afforded 2-chloro-3-hydrazinylquinoxaline 10⁵⁰. Heating of compound 10 with triethyl orthoformate gave 4-chloro[1, 2, 4]triazolo[4,3-a]quinoxaline 11⁵⁰. The obtained compound 11 was heated with hydrazine hydrate to afford 4-hydrazinyl-[1, 2, 4] triazolo[4,3-a]quinoxaline 12⁵¹. Moreover, reflux of 12 in an alcoholic mixture of carbon disulphide and potassium hydroxide afforded bis[1, 2, 4]triazolo[4,3-a:3′,4′-c]quinoxaline-3-thiol 13⁴⁴. Heating compound 13 with an alcoholic solution of potassium hydroxide gave the corresponding potassium salt 14⁴⁴. (Scheme 1)

Scheme 1.

Synthetic pathway for compound 14; Reagents and conditions: (i) 4 N conc. HCl/reflux/6 h, (ii) SOCl₂/DCE/reflux/4 h, (iii) NH₂NH₂.H₂O/ethanol/r.t., (iv) triethyl orthoformate/reflux/4 h, (v) NH₂NH₂.H₂O/ethanol/reflux/4 h, (vi) absolute ethanol/KOH/CS₂/reflux/6 h, (vii) absolute ethanol/KOH/heating/10 min.

Scheme 2.

Synthetic pathway for compounds 18a–n and 22a–c; Reagents and conditions: (i) ClCH₂COCl/DMF/NaHCO₃/r.t./1 h, (ii) SOCl₂/DCE/DMF/reflux/4 h, (iii) RNH₂/acetonitrile/TEA/r.t./8 h (iv) methanol/conc.H₂SO₄/reflux/8 h, (v) NH₂NH₂.H₂O/ethanol/reflux/8 h, (vi) acetonitrile/TEA/r.t./8 h.

Scheme 3.

Synthetic pathway for compounds 23a–n and 24a–c; Reagents and conditions: (i) DMF/KI/reflux/6 h.

Scheme 2 was carried out to prepare the key intermediates (18a–n and 22a–c). At first, acetylation of p-amino benzoic acid 15 with chloroacetyl chloride in DMF in the presence of NaHCO₃ provided the key product 4–(2-chloroacetamido)benzoic acid 16. Compound 16 was acylated using thionyl chloride to produce the benzoyl chloride derivative 17⁵². Furthermore, stirring of compound 17 with commercially available amines namely, ethylamine, n-butylamine, sec-butylamine, tert- butylamine, cyclopentylamine, cyclohexylamine, 4-aminoacetophenone, 3-chloroaniline, 4-chloroaniline, 2,5-dichloroaniline, 4-fluoroaniline, 2-hydroxyaniline, 4-hydroxyaniline, and 2-hydroxy-4-nitroaniline at room temperature in acetonitrile/TEA mixture afforded the corresponding intermediates 18a–n, respectively. On the other hand, to prepare the ester derivatives 20a–c, appropriate acid derivatives 19a–c namely, 2-chlorobenzoic acid, 3-chlorobenzoic, and 2-hydroxybenzoic acid were refluxed in methanol in the presence of sulphuric acid according to the reported procedures^42,53,54. In addition, reflux of 20a–c with hydrazine hydrate afforded the corresponding acid hydrazides 21a–c⁴². In the end, in acetonitrile and TEA mixture, compound 17 was stirred with the acid hydrazides 21a–c to produce the corresponding diamide derivatives 22a–c (Scheme 2).

The structures of compounds 18a–n and 22a–c were confirmed by spectral and elemental data. IR spectra of such compounds showed strong bands around 3370 − 3100 cm⁻¹ corresponding to NHs. Also, IR spectra showed strong C=O absorption bands at a range of 1770 − 1624 cm⁻¹. Moreover, ¹H NMR spectra showed singlet signals around δ 10.50 and 8.35 ppm corresponding to the two amidic NHs. Additionally, CH₂ protons appeared at around δ 4.30 ppm. Matching with such results, ¹³C NMR spectra also confirmed the validity of suggested structures where characteristic peaks were displayed around δ 165.60, 165.05, and 44.00 ppm corresponding to the two C=O and CH₂ groups, respectively.

Scheme 3 demonstrated the synthetic pathway of the final target compounds (23a–n and 24a–c). Compound 14 was heated with the formerly synthesised intermediates (18a–n and 22a–c) in dry DMF using KI to furnish the titled compounds 23a–n and 24a–c, respectively.

The spectral and elemental data supported the structures of the obtained derivatives, where the ¹H NMR spectra of compounds 23a–n and 24a–c displayed characteristic downfield singlet signals around δ 10.75 ppm. The mass spectra were also consistent with the expected structures. Taking compound 23d as a representative example, the IR spectrum demonstrated stretching bands at 2968 and 2929 cm⁻¹ corresponding to aliphatic CH bonds. The ¹H NMR spectrum of this compound showed an up-field singlet signal at δ 1.38 ppm corresponding to tertiary butyl moiety. Furthermore, ¹³C NMR spectrum showed the presence of two peaks at δ 51.16 and 29.11 corresponding to CH and three CH₃ of tert-butyl moiety.

2.2. Biological evaluation

2.2.1. In vitro anti-proliferative activity

All newly prepared compounds were assessed for their in vitro cytotoxic efficiencies via standard MTT method^55–57, against breast cancer (MCF-7) and hepatocellular carcinoma (HepG2) cell lines. Sorafenib was applied as a standard anticancer drug. The growth inhibitory concentration (IC₅₀) values were concluded for each final compound and depicted in Table 1.

Table 1.

In vitro anti-proliferative activities of the tested compounds against MCF-7 and HepG2 cell lines, and in vitro enzymatic inhibitory activities against VEGFR-2.

	IC50 (µM)a		IC50 (nM)a
Comp.	MCF-7	HepG2	VEGFR-2
23a	17.7 ± 1.5	11.3 ± 0.9	7.1 ± 0.4
23b	78.9 ± 6.1	69.1 ± 6.1	71.6 ± 5.2
23c	76.2 ± 6.9	69.8 ± 5.9	47.1 ± 4.2
23d	21.6 ± 1.8	17.5 ± 1.2	11.8 ± 0.8
23e	78.3 ± 6.5	74.8 ± 6.9	39.8 ± 3.1
23f	74.1 ± 6.7	70.4 ± 5.8	39.5 ± 2.9
23g	81.3 ± 7.4	80.7 ± 7.1	62.7 ± 5.7
23h	37.2 ± 3.2	22.3 ± 1.8	11.7 ± 0.9
23i	18.3 ± 1.2	10.8 ± 0.9	9.4 ± 0.7
23j	10.3 ± 0.8	6.4 ± 0.5	3.7 ± 0.2
23k	68.2 ± 5.2	71.8 ± 6.3	49.6 ± 4.1
23l	19.4 ± 1.1	11.3 ± 0.7	5.8 ± 0.3
23m	21.6 ± 1.7	15.2 ± 1.2	9.7 ± 0.7
23n	16.5 ± 1.3	12.7 ± 0.9	7.4 ± 0.5
24a	48.3 ± 3.9	34.5 ± 2.8	23.9 ± 1.9
24b	42.7 ± 3.7	30.3 ± 2.7	22.3 ± 1.8
24c	40.7 ± 3.7	29.8 ± 2.1	20.7 ± 1.6
Sorafenib	3.51 ± 0.22	2.17 ± 0.14	3.12 ± 0.1

^aIC₅₀ values are the mean ± SD of three separate experiments.

Overall, HepG2 cells were more sensitive to the tested compounds than MCF-7 except for compound 23k. Among the series, compound 23j showed the highest anti-proliferative activities against MCF-7 and HepG2 cell lines with IC₅₀ values of 10.3 and 6.4 µM, respectively. In addition, compounds 23a, 23i, 23 l, and 23n exhibited good anti-proliferative activities against the tested cell lines with IC₅₀ values ranging from 7.1 to 19.4 µM, comparing to sorafenib (IC₅₀ = 3.51 and 2.17 µM against MCF-7 and HepG2, respectively). Moreover, compounds 23d, 23h, 23m, 24a, 24b, and 24c displayed moderate anti-proliferative activities against the tested cell lines with IC₅₀ values ranging from 15.2 to 48.3 µM. On the other hand, the rest of the compounds displayed weak anti-proliferative activities against the tested cell lines.

2.2.2. In vitro VEGFR-2 enzyme assay inhibition

All the synthesised compounds were subjected to further assay for their ability to inhibit VEGFR-2 using sorafenib as a positive control. The results were stated as growth inhibitory concentration (IC₅₀) values and illuminated in Table 1.

Compound 23j was the most potent VEGFR-2 inhibitor with an IC₅₀ value of 3.7 nM, nearly equal to that of sorafenib (IC₅₀ = 3.12 nM). Moreover, compounds 23a, 23d, 23h, 23i, 23l, 23m, and 23n showed promising activities with IC₅₀ values ranging from 5.8 to 11.8 nM. On the other hand, compounds 23c, 23e, 23f, 23k, and 24a–c exhibited moderate to weak activity with IC₅₀ values ranging from 20.7 to 49.6 nM. Finally, compounds 23b and 23g exhibited the lowest anti VEGFR-2 activities with IC₅₀ values of 71.6 and 62.7 nM, respectively.

2.2.3. Structure–activity relationship (SAR)

Inspecting the results of different biological analyses (anti-proliferative activity and VEGFR-2 assay); we concluded a valuable SAR.

At First, the effect of pharmacophore moiety on the activity was explored. It was noticed that the amide derivatives 23h (IC₅₀ = 37.2 and 22.3 µM against MCF-7 and HepG2, respectively & 11.7 nM against VEGFR-2) and 23 l (IC₅₀ = 19.4 and 11.3 µM against MCF-7 and HepG2, respectively & 5.8 nM against VEGFR-2) displayed higher activities than the corresponding diamide derivatives 24b (IC₅₀ = 42.7 and 30.3 µM against MCF-7 and HepG2, respectively & 22.3 nM against VEGFR-2) and 24c (IC₅₀ = 40.7 and 29.8 µM against MCF-7 and HepG2, respectively & 20.7 nM against VEGFR-2).

Next, we investigated the effect of the terminal hydrophobic moiety. With respect to the terminal aliphatic hydrophobic moieties, it was found that the VEGFR-2 inhibitory activities decreased in the order of ethyl (23a, IC₅₀ = 7.1 nM) > tert-butyl (23d, IC₅₀ = 11.8 nM) > cyclohexyl (23f, IC₅₀ = 39.5 nM) > cyclopentyl (23e, IC₅₀ = 39.8 nM) > sec-butyl (23c, IC₅₀ = 47.1 nM) > n-butyl (23b, IC₅₀ = 71.6 nM). In addition, the effect of the substitution on the aromatic hydrophobic moieties has been examined. For the amide derivatives, it was found that the VEGFR-2 inhibitory activities decreased in the order of 2,5-dichloro (23j, IC₅₀ = 3.7 nM) >2-hydroxy (23l, IC₅₀ = 5.8 nM) >2-hydroxy −4-nitro (23n, IC₅₀ = 7.4 nM) >4-chloro (23i, IC₅₀ = 9.4 nM) >4-hydroxy (23m, IC₅₀ = 9.7 nM) >4-fluoro (23k, IC₅₀ = 49.6 nM). While the diamide derivatives revealed decrease in VEGFR-2 inhibitory activities in the order of 2- hydroxy (24c, IC₅₀ = 20.7 nM) >3-chloro (24b, IC₅₀ = 22.3 nM) > 2-chloro (24a, IC₅₀ = 23.9 nM).

2.2.4. Correlation of cytotoxicity with VEGFR-2 inhibition

From the aforementioned results, we can conclude that our tested compounds can inhibit VEGFR-2 in the tested cell lines. To prove that inhibition of VEGFR-2 is the major prominent cause of cell mortality, we compared the cytotoxicity results of the synthesised candidates with their corresponding VEGFR-2 inhibitory activities utilising a simple linear regression analysis. The coefficients of determination (R²) were determined. The R² values for MCF-7 and HepG2 were 0.881 (p values >.0001) and 0.800 (p values > .0001), respectively (Figure 5). Such high values of R² indicate the high correlation between the dependent variable (VEGFR-2 inhibition) and the independent one (cytotoxicity).

Figure 5.

Correlation of cytotoxicity with VEGFR2 inhibition on two cell line models MCF-7and HepG2. MCF-7 (p value >.0001) & HepG2 (p value >.0001).

2.2.5. Cellular mechanistic study

Compound 23j which demonstrated remarkable cytotoxic potency and significant inhibitory activity against VEGFR-2 was nominated for further cellular mechanistic study. This involved study of its influence on cell cycle progression and induction of apoptosis in HepG2 cells.

2.2.5.1. Effect on cell cycle progression

In this work, HepG2 cell line was treated with compound 23j at a concentration of 6.4 μM (the IC₅₀ value of compound 23j) and incubated for 24 h. Then, the cells were stained with propidium iodide and analysed for cell distribution during the various phases of the cell cycle against untreated HepG2 cells. Flow cytometry results exhibited that the percent of HepG2 cells decreased at the Sub-G1, G1 and S phases. For Sub-G1 phase, it decreased from 1.46% to 1.21%, for G1 phase it decreased from 57.75 to 37.34% while for S phase it decreased from 28.65% to 25.79%. On the other hand, the percentage of HepG2 cells significantly increased at G2/M phase from 12.13% to 35.64%. Such results indicated that compound 23j inhibited proliferation of HepG2 cells via cessation of the growth of the cell cycle at G2/M phase (Table 2 and Figure 6).

Table 2.

Effect of compound 23j on cell cycle progression in HepG2 cells after 24 h treatment.

Sample	Cell cycle distribution (%)a
	%Sub-G1	%G1	%S	%G2 / M
HepG2	1.46 ± 0.17	57.75 ± 2.34	28.65 ± 2.74	12.13 ± 0.80
Compound 23j/HepG2	1.21 ± 0.15	37.34 ± 2.50**	25.79 ± 1.21	35.64 ± 2.16***

^aValues are given as mean ± SEM of three independent experiments. **p < .01 and ***p < .001.

Figure 6.

Cell cycle analysis of HepG2 cells treated with compound 23j. **p < .01 and ***p < .001.

2.2.5.2. Apoptosis analysis

To confirm the apoptotic ability of compound 23j, a flow cytometry technique was performed. In such technique, HepG2 cells were stained with annexin V/propidium iodide (PI) and incubated for 24 h with compound 23j (6.4 μM). It was revealed that compound 23j triggered more apoptotic cells comparing to untreated control cells. In details, compound 23j induced apoptosis by 40.12% (early apoptosis = 39.97% & late apoptosis = 0.15%), compared to 7.07% in the control cells (early apoptosis = 6.88% & late apoptosis = 0.19%). Such findings revealed that compound 23j could induce apoptosis in HepG2 cells (Table 3 & Figure 7).

Table 3.

apoptotic effect of compound 23j against HepG2 cells.

Sample	Viablea (left bottom)	Apoptosisa		Necrosisa (left top)
		Early (right bottom)	Late (right top)
HepG2	92.80 ± 1.91	6.88 ± 0.90	0.19 ± 0.01	0.14 ± 0.01
Compound 23j/HepG2	59.61 ± 2.88	39.97 ± 2.93***	0.15 ± 0.01	0.24 ± 0.07

^aValues are given as mean ± SEM of three independent experiments. ***p < .001.

Figure 7.

Flow cytometric analysis of apoptosis in HepG2 cells exposed to compound 23j. ***p < .001.

2.2.5.3. Caspase 3/9 assay

To study the effect of compound 23j, the most promising member, on caspase-3 and caspase-9 levels, western blot technique was carried out. HepG2 cells were treated with 23j (6.4 µM) for 24 h. The results revealed that compound 23j produced a significant increase in the cellular levels of caspase-3 (1.36-fold) and caspase-9 (2.80-fold) compared to the control cells (Table 4 and Figure 8).

Table 4.

Effect of compound 23j on levels of BAX, Bcl-2, active caspase-9, and active caspase-3 proteins expression

Sample	Protein expression (normalized to β-actin)a
	BAX	Bcl-2	BAX/Bcl-2 ratio	Caspases-9	Caspases-3
HepG2	1.00 ± 0.13	1.00 ± 0.07	1.00 ± 0.10	1.00 ± 0.08	1.00 ± 0.03
23j/HepG2	1.65 ± 0.06*	0.38 ± 0.03**	4.34 ± 0.22***	2.80 ± 0.25**	1.36 ± 0.10*

^aValues are given as mean ± SEM of three independent experiments. *p < .05, **p < .01 and ***p < .001.

Figure 8.

The immunoblotting of BAX, Bcl-2, Caspase-9, and Caspase-3 (Normalized to β-actin). *p < .05, **p < .01, ***p < .001.

2.2.5.4. Evaluation of BAX and Bcl-2 expressions

BAX and Bcl-2 cellular levels were assessed for compound 23j after 24 h of its application on HepG2 cells using western blot technique. The results showed that compound 23j produced an increase of the pro-apoptotic factor BAX by 1.65-fold while anti-apoptotic protein Bcl-2 demonstrated a concentration decreased by 2.63-fold. Moreover, compound 23j increased BAX/Bcl-2 ratio to be 4.34 which indicates the efficiency of compound 23j on apoptosis cascade (Table 4 and Figure 8).

2.3. Molecular modelling study

2.3.1. Docking study

Molecular docking study was accomplished to get further insight into the binding modes and orientations of the newly synthesised compounds into the ATP binding site of VEGFR-2 enzyme. Sorafenib was used as a reference ligand. Validation of the docking procedure was achieved via re-docking of the co-crystallised ligand against the active pocket of VEGFR-2. The results revealed that the RMSD value between the re-docked pose and the co-crystallised one was 1.02. This value revealed the validity of the docking process (Figure 9). The calculated ΔG (binding free energies) of the synthesised compounds and the reference drug against VEGFR-2 were summarised in Table 5.

Figure 9.

Alignment of the co-crystallised pose (green) and the redocked pose (Orange) of the same ligand inside the protein.

Table 5.

The calculated ΔG of the synthesised candidates and sorafenib (ΔG in kcal/mole).

Comp. No.	ΔG [kcal mol-1]	Compound	ΔG [kcal mol–1]
23a	–22.15	23j	–23.87
23b	–22.86	23k	–22.98
23c	–23.25	23l	–23.26
23d	–21.65	23m	–22.88
23e	–22.97	23n	–22.98
23f	–21.47	24a	–21.30
23g	–23.00	24b	–21.71
23h	–22.37	24c	–22.76
23i	–23.63	Sorafenib	–22.48

Sorafenib interactions with the amino acids of the active site have been studied and displayed in 2 D and 3 D style in Figure 10. The proposed binding mode of sorafenib revealed an affinity value of −22.48 kcal/mol. The N-methylpicolinamide moiety formed five hydrophobic interactions with Val846, Ala864, Phe1045, Leu1033, and Leu838. Likewise, it formed one hydrogen bond with Cys917. Moreover, the phenyl moiety was buried in the linker region forming five hydrophobic interactions with Phe1045, Cys1043, Val846, Val914, and Val897. Furthermore, the pharmacophore moiety (urea group) formed three hydrogen bonds with the two crucial amino acids Glu883 and Asp1044. Finally, the 1-chloro-2-(trifluoromethyl)benzene moiety conquered the allosteric hydrophobic region forming many hydrophobic and electrostatic interactions with Ile886, Leu887 Leu1017, His1024, Ile890, and Asp1044^34,58.

Figure 10.

(A) 3D binding mode of sorafenib into VEGFR-2. (B) 2D binding mode sorafenib into VEGFR-2.

Docking simulation of compound 23i revealed that it has a good fitting into the enzyme active sites with docking score of −23.63 kcal/mol. In DFG region, the amide moiety (pharmacophore) formed two hydrogen bonds with carboxylate moiety of Glu883 (1.50 Å) and NH group of Asp1044 (1.82 Å). Furthermore, the phenyl ring (central spacer) occupied the linker region forming five hydrophobic interactions with Cys1043, Phe1045, Val897, and Val914. Also, the bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]quinoxaline moiety occupied the hinge region forming two hydrophobic and one hydrogen bond interactions with Leu838. Additionally, the terminal hydrophobic (4-chlorophenyl) moiety formed three hydrophobic interactions with Ile886 and Leu887 and one electrostatic interaction with Asp1044 (Figure 11).

Figure 11.

(A) 3D binding mode of compound 23i into VEGFR-2. (B) 2D binding mode of compound 23i into VEGFR-2.

The docking findings of compound 23j revealed affinity value of −23.87 kcal/mol. The amide moiety formed two hydrogen bonds with Glu883 (COO^-, 1.75 Å) and Asp1044 (NH, 2.20 Å). Additionally, in the hinge region, the docked compound formed four hydrophobic interactions via its bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]quinoxaline moiety with Leu838 and Phe916. Also, the central phenyl ring moiety formed three hydrophobic interactions with Val914, Val897, and Cys1043. Finally, the 2,5-dichlorophenyl moiety formed electrostatic and hydrophobic interactions with Asp1044, Ile890, and Leu 887 (Figure 12).

Figure 12.

(A) 3D binding mode of compound 23j into VEGFR-2. (B) 2D binding mode of compound 23j into VEGFR-2.

Compound 23l (affinity value of −23.26 kcal/mol) combined with the receptor protein as follows; In DFG domain, the amide moiety formed two hydrogen bonds with Glu883 (distance: 2.28 Å) and Asp1044 (distance: 1.76 Å). Moreover, in the hinge region, bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]quinoxaline moiety formed three hydrophobic interactions with His814 and Ile886. Also, in the linker region, the central phenyl ring moiety formed two hydrophobic interactions with Asp1044 and Leu887. Additionally, the terminal hydrophobic (2-hydroxyphenyl moiety) formed three hydrophobic interactions with Val897, Val914, and Lys866 (Figure 13).

Figure 13.

(A) 3D binding mode of compound 23 l into VEGFR-2. (B) 2D binding mode of compound 23 l into VEGFR-2.

2.3.2. In silico ADME analysis

To investigate pharmacokinetics properties of the prepared compounds, computer aided ADME studies were accomplished using Accelrys Discovery Studio 4.0 software. Sorafenib was used as a reference molecule. These studies include the estimation of certain parameters. 1) Blood brain barrier penetration which measures the ability of molecule to diffuse through blood brain barrier. 2) Absorption level which determines human intestinal absorption (HIA) of a chemical after its oral administration. A well-absorbed compound is one that is absorbed at least 90% into the blood stream^59,60. iii) Solubility level in which the solubility of a chemical in water was predicted at 25 °C. iv) CYP2D6 binding which analyzes cytochrome P450 2D6 enzyme inhibition^61,62. v) Plasma protein binding which predicts the fraction of drug bound to plasma proteins in the blood⁶³. The results were predicted and listed in Table 6.

Table 6.

ADME screening of the designed compounds

Comp.	BBB levela	Solubility levelb	Absorption levelc	CYP2D6 predictiond	PPB predictione
23a	4	2	0	FALSE	FALSE
23b	4	2	1	FALSE	FALSE
23c	4	2	1	FALSE	FALSE
23d	4	2	1	FALSE	FALSE
23e	4	2	1	FALSE	FALSE
23f	4	1	2	FALSE	FALSE
23g	4	2	2	FALSE	FALSE
23h	4	1	2	FALSE	FALSE
23ai	4	1	2	FALSE	FALSE
23j	4	1	2	FALSE	FALSE
23k	4	1	1	FALSE	FALSE
23l	4	2	2	FALSE	FALSE
23m	4	2	2	FALSE	FALSE
23n	4	2	3	FALSE	FALSE
24a	4	1	2	FALSE	FALSE
24b	4	1	2	FALSE	FALSE
24c	4	2	3	FALSE	FALSE
Sorafenib	4	1	0	FALSE	TRUE

^aBBB: very high (0), high (1), medium (2), low (3), or very low (4).

^bSolubility level: very low (1), low (2), good (3), or optimal (4).

^cAbsorption level: good (0), moderate (1), poor (2), or very poor (3).

^dCYP2D6: inhibitor (TRUE) or non-inhibitor (FALSE).

^ePBB: less than 90% (FALSE) or more than 90% (TRUE).

The results revealed that the estimated compounds have very low BBB penetration levels.

Accordingly, the CNS side effects of all compounds were expected to be low. Regarding absorption levels, compound 23a demonstrated good absorption level, compounds 23b–e and 23k exhibited moderate absorption levels. On the other hand, compounds 23f–j, 23l–n, and 24a–c showed poor and very poor intestinal absorption. With respect to aqueous solubility, all compounds demonstrated low and very low levels of aqueous solubility. Moreover, the effect on cytochrome P450 2D6 was investigated. The results showed that all examined members were non-inhibitors of CYP2D6. Consequently, liver side effect is not expected upon their administration. The plasma protein binding model exhibited that all compounds were anticipated to bind plasma protein less than 90% (Figure 14).

Figure 14.

The ADME plot of the synthesised compounds.

2.3.3. Toxicity studies

Toxicity profile of the prepared candidates was assessed as stated by the validated and constructed models in Discovery studio software version 4.0^64,65. These models involve: 1) FDA rodent carcinogenicity. 2) Carcinogenic potency TD₅₀⁶⁶_. 3) Rat maximum tolerated dose^67,68. 4) Developmental toxicity potential^69,70. 5) Rat oral LD₅₀⁷¹_. 6) Rat chronic LOAEL^72,73.

Regarding FDA rodent carcinogenicity model, all the tested candidates were forecasted to be non-carcinogenic. For carcinogenic potency TD₅₀ rat model, compounds 23a,b, 23 l,m, and 24c showed TD₅₀ values ranging from 33.907 to 44.001 g/kg body weight, which are higher than sorafenib (14.244). With respect to rat maximum tolerated dose model, compounds 23a–c, 23 h–n, and 24a–c demonstrated maximum tolerated dose with a range of 0.099 to 0.391 g/kg body weight which are higher than that of sorafenib (0.089 g/kg body weight). On the other hand, compounds 23d–g showed a maximum tolerated dose lower than that of sorafenib with a range of 0.083 to 0.088 g/kg body weight. Additionally, all compounds were predicted to be non-toxic against developmental toxicity potential model. For rat oral LD₅₀ model, compounds 23a–d, 23g, 23i, 23m, and 24a–c revealed oral LD₅₀ values in a range of 0.864 to 2.477 g/kg body weight which are higher than that of sorafenib (0.823 g/kg body weight). For rat chronic LOAEL model, the tested compounds exhibited LOAEL values ranging from 0.064 to 0.329 g/kg body weight. These values are higher than sorafenib (0.005 g/kg body weight) Table 7.

Table 7.

Toxicity study of the synthesised compounds.

Comp.	FDA rodent carcinogenicity (mouse- female)	Carcinogenic potency TD50 (rat)a	Rat maximum tolerated dose (feed)b	Developmental toxicity potential	Rat oral LD50b	Rat chronic LOAELb
23a	Non-carcinogen	35.802	0.099	Non-toxic	2.209	0.153
23b	Non-carcinogen	33.907	0.145	Non-toxic	1.900	0.329
23c	Non-carcinogen	0.543	0.114	Non-toxic	1.899	0.206
23d	Non-carcinogen	10.185	0.075	Non-toxic	1.978	0.084
23e	Non-carcinogen	0.857	0.088	Non-toxic	0.729	0.221
23f	Non-carcinogen	0.794	0.083	Non-toxic	0.619	0.192
23g	Non-carcinogen	7.641	0.087	Non-toxic	1.071	0.094
23h	Non-carcinogen	4.519	0.133	Non-toxic	0.720	0.072
23i	Non-carcinogen	4.519	0.133	Non-toxic	1.140	0.067
23j	Non-carcinogen	4.056	0.105	Non-toxic	0.546	0.064
23k	Non-carcinogen	4.882	0.144	Non-toxic	0.592	0.073
23l	Non-carcinogen	37.552	0.362	Non-toxic	0.663	0.134
23m	Non-carcinogen	37.552	0.362	Non-toxic	0.864	0.115
23n	Non-carcinogen	13.503	0.262	Non-toxic	0.769	0.094
24a	Non-carcinogen	5.280	0.143	Non-toxic	2.477	0.155
24b	Non-carcinogen	5.080	0.143	Non-toxic	1.754	0.089
24c	Non-carcinogen	44.001	0.391	Non-toxic	1.350	0.211
Sorafenib	Single-carcinogen	14.244	0.089	Toxic	0.823	0.005

^aUnit: mg/kg body weight/day.

^bUnit: g/kg body weight.

3. Conclusion

In the present study, a new series of bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]quinoxaline derivatives were synthesised as potential cytotoxic agents and VEGFR-2 inhibitors. The anticancer efficiency of these derivatives was evaluated against MCF-7 and HepG2 cancer cell lines. Four compounds 23a (IC₅₀ = 17.7 and 11.3 µM), 23i (IC₅₀ = 18.3 and 10.8 µM), 23j (IC₅₀ = 10.3 and 6.4 µM), 23l (IC₅₀ = 19.4 and 11.3 µM), and 23n (IC₅₀ =16.5 and 12.7 µM) were the most active antiproliferative members against MCF-7 and HepG2, respectively. The in vitro VEGFR-2 assay revealed that compounds 23a, 23d, 23h, 23i, 23j, 23l, 23m, and 23n exhibited the highest inhibitory activity versus VEGFR-2 with IC₅₀ values of 7.1, 11.8, 11.7, 9.4, 3.7, 5.8, 9.7, and 7.4 nM, respectively. SAR revealed that the amide derivatives (compounds 23h and 23l) exhibited higher activities than the corresponding diamide derivatives (compounds 24b and 24c). Furthermore, compound 23j, the most potent member, arrested the HepG2 cell growth at G2/M phase and induced apoptosis by 40.12% compared to the control cells (7.07%). Moreover, caspase activation assay was performed for 23j in HepG2 cell lines. The results showed significant increase of caspase 3 (1.36-fold) and caspase 9 (2.80-fold). BAX and Bcl-2 concentration levels were also evaluated and showed a titre increase of the pro-apoptotic protein BAX (1.65-fold) and a decrease of Bcl-2 (2.63-fold). Additionally, compound 23j increased BAX/Bcl-2 ratio to be 4.34. Finally, computational physicochemical assessment of the synthesised compounds showed that they have favourable properties with satisfactory drug-like profiles.

4. Experimental

4.1. Chemistry

All the reagents, chemicals, and apparatus were presented in Supplementary data. Compounds 8, 9, 10, 11, 12, 13, 14, 16, 17, 20a–c, and 21a–c were prepared in accordance with the reported procedures^{41,42,44,49,50,52}.

4.1.1. General procedure for synthesis of compounds 18a–n

A mixture of 17 (2 mmol) and appropriate amines namely, ethylamine, n-butylamine, sec-butylamine, tert-butylamine, cyclopentylamine, cyclohexylamine, 4-aminoacetophenone, 3-chloroaniline, 4-chloroaniline, 2,5-dichloroaniline, 4-flouroaniline, 2-hydroxyaniline, 4-hydroxyaniline, and 2-hydroxy-4-nitroaniline (2 mmol) was stirred in acetonitrile (50 ml) in the presence of trimethylamine (1 ml) for 8 h. The obtained precipitates were filtered and crystallised from ethanol to give the corresponding derivatives, 18a–n, respectively.

4.1.1.1. 4-(2-Chloroacetamido)-N-ethylbenzamide (18a)

Yellow crystal (yield, 80%); m.p. = 190 − 192 °C; FT-IR (ν_max, cm⁻¹): 3376, 3273 (NH), 2971(C–H aliphatic), 1709, 1636 (C=O), 1605 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.49 (s, 1H), 8.36 (s, 1H), 7.80 (d, J = 9.7 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 4.26 (s, 2H), 3.29 − 3.21 (q, 2H), 1.14 − 1.05 (t, 3H).; ¹³C NMR (101 MHz, DMSO-d₆) δ 165.73 (2C), 165.40 (2C), 141.29, 130.24, 128.37, 44.04, 34.42, 15.43, 15.20; Anal. Calcd. for C₁₁H₁₃ClN₂O₂ (240.68): C, 54.89; H, 5.44; N, 11.64. Found: C, 54.72; H, 5.20; N, 11.46%.

4.1.1.2. N-Butyl-4–(2-chloroacetamido)benzamide (18b)

White crystal (yield, 85%); m.p. = 170 − 172 °C; FT-IR (ν_max, cm⁻¹): 3369, 3263 (NH), 3185 (NH), 3099 (C–H aromatic), 2960 (C–H aliphatic), 1706, 1633 (C=O), 1606 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.57 (s, 1H), 8.33 (s, 1H), 7.80 (dd, J = 8.8, 1.8 Hz, 2H), 7.66 − 7.61 (m, 2H), 4.27 (s, 2H), 3.25 − 3.18 (m, 2H), 1.51 − 1.43 (m, 2H), 1.33 − 1.26 (m, 2H), 0.89 − 0.84 (m, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.93, 165.41, 141.30, 130.27, 128.67, 128.31, 119.43, 118.44, 44.44, 44.01, 31.74, 20.12, 14.18; MS (m/z): 269 (M⁺ + 1, 100%). Anal. Calcd. for C₁₃H₁₇ClN₂O₂ (268.74): C, 58.10; H, 6.38; N, 10.42. Found: C, 58.38; H, 6.61; N, 10.32%.

4.1.1.3. N-(Sec-butyl)-4-(2-chloroacetamido)benzamide (18c)

White crystals (yield 78%); m.p. = 180–182 °C; IR (KBr) ν cm⁻¹: 3307, 3113 (NH), 2969, 2933 (CH aliphatic), 1684, 1625 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.54 (s, 1H), 8.06 (d, J = 6.1 Hz, 1H), 7.82 (d, J = 6.3 Hz, 2H), 7.65 (d, J = 7.9 Hz, 2H), 4.28 (s, 2H), 3.89 (dq, J = 11.8, 6.8 Hz, 1H), 1.56 − 1.41 (m, 2H), 1.11 (d, J = 5.1 Hz, 3H), 0.84 (d, J = 4.7 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.46, 165.38, 141.25, 130.45, 128.64, 119.33, 118.74, 118.44, 46.76, 44.05, 29.31, 20.69, 11.29; Anal. Calcd. for C₁₃H₁₇ClN₂O₂ (268.74): C, 58.10; H, 6.38; N, 10.42. Found: C, 58.06; H, 6.65; N, 10.29%.

4.1.1.4. N-(Tert-butyl)-4-(2-chloroacetamido)benzamide (18d)

Yellow crystals (yield 68%); m.p. = 190–192 °C; IR (KBr) ν cm⁻¹: 3314 (NH), 2975 (CH aliphatic), 1666, 1628 (C=O), 1606 (C=N); Anal. Calcd. for C₁₃H₁₇ClN₂O₂ (268.74): C, 58.10; H, 6.38; N, 10.42. Found: C, 58.31; H, 6.55; N, 10.59%.

4.1.1.5. 4-(2-Chloroacetamido)-N-cyclopentylbenzamide (18e)

White crystals (yield 82%); m.p. = 230–232 °C; IR (KBr) ν cm⁻¹: 3290 (NH), 2961 (CH aliphatic), 1680, 1624 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 8.20 (d, J = 7.3 Hz, 1H), 7.81 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 4.31 (s, 2H), 3.03 (qd, J = 7.2, 4.2 Hz, 1H), 1.87 − 1.82 (m, 2H), 1.66 (tt, J = 4.5, 2.5 Hz, 2H), 1.50 (qd, J = 7.7, 6.9, 3.9 Hz, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.75, 165.42, 141.35, 130.26, 128.76, 119.28, 118.81, 118.36, 51.46, 44.00, 32.54, 31.05, 24.05, 8.93; MS (m/z): 281 (M⁺ + 1, 100%); Anal. Calcd. for C₁₄H₁₇ClN₂O₂ (280.75): C, 59.89; H, 6.10; N, 9.98. Found: C, 59.50; H, 6.04; N, 10.25%.

4.1.1.6. 4-(2-Chloroacetamido)-N-cyclohexylbenzamide (18f)

White crystals (yield 78%); m.p. = 220–222 °C; FT-IR ((ν_max, cm⁻¹): 3286 (NH), 3042 (C–H aromatic), 2938, 2854 (C–H aliphatic), 1681, 1624 (C=O), 1610 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.52 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.83 − 7.77 (m, 2H), 7.65 − 7.59 (m, 2H), 4.26 (s, 2H), 3.72 (s, 1H), 1.78 (d, J = 7.1 Hz, 2H), 1.71 (d, J = 8.4 Hz, 2H), 1.58 (d, J = 12.8 Hz, 1H), 1.27 (dd, J = 11.4, 8.6 Hz, 4H), 1.12 (q, J = 10.8, 9.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.38, 165.15, 141.24, 130.41, 129.07, 128.80, 128.47, 119.35, 118.86, 118.41, 48.79, 48.66, 44.42, 44.02, 25.63; Anal. Calcd. for C₁₅H₁₉ClN₂O₂ (294.77): C, 61.12; H, 6.50; N, 9.50. Found: C, 60.88; H, 6.39; N, 9.25%.

4.1.1.7. N-(4-Acetylphenyl)-4-(2-chloroacetamido)benzamide (18g)

White crystals (yield 80%); m.p. = 250–252 °C; FT-IR (ν_max, cm⁻¹): 3326 (NH), 3062 (C–H aromatic), 1674, 1655 (C=O), 1596 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.61 (s, 1H), 10.46 (s, 1H), 7.98 − 7.90 (m, 6H), 7.72 (s, 2H), 4.29 (s, 2H), 2.53 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 197.08, 165.69(2 C), 165.57(2 C), 144.14, 142.15, 132.33, 129.90, 129.85, 129.60, 129.26, 119.97, 119.66, 118.65, 44.05, 27.16; Anal. Calcd. for C₁₇H₁₅ClN₂O₃ (330.76): C, 61.73; H, 4.57; N, 8.47. Found: C, 61.44; H, 4.39; N, 8.29%.

4.1.1.8. 4-(2-Chloroacetamido)-N-(3-chlorophenyl)benzamide (18h)

Yellow crystal (yield, 75%); m.p. = 190 − 192 °C; FT-IR (v max, cm⁻¹): 3291 (NH), 1676, 1644 (C=O), 1593 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (s, 1H), 10.30 (s, 1H), 7.95 − 7.94 (m, 2H), 7.93 (d, J = 1.9 Hz, 1H), 7.74 − 7.68 (m, 3H), 7.36 (t, J = 8.1 Hz, 1H), 7.14 − 7.12 (m, 1H), 4.29 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.56, 165.53, 142.07, 141.20, 141.14, 133.33, 129.86, 129.41, 129.12, 120.24, 119.93, 119.03, 118.66, 118.59, 44.05; Anal. Calcd. for C₁₅H₁₂Cl₂N₂O₂ (323.17): C, 55.75; H, 3.74; N, 8.67. Found: C, 55.64; H, 3.63; N, 8.45%.

4.1.1.9. 4-(2-Chloroacetamido)-N-(4-chlorophenyl)benzamide (18i)

Yellow crystal (yield, 83%); m.p. = 228 − 230 °C; FT-IR (ν_max, cm⁻¹): 3324 (NH), 1666 (C=O amide), 1595 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.59 (s, 1H), 10.27 (s, 1H), 7.93 (d, J = 8.6 Hz, 2H), 7.79 (d, J = 8.9 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 4.29 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.53 (2C), 165.36 (2C), 141.98, 138.67, 130.01, 129.36 (2C), 129.08, 128.58, 128.30, 127.56, 118.63, 44.05; Anal. Calcd. for C₁₅H₁₂Cl₂N₂O₂ (322.17): C, 55.75; H, 3.74; N, 8.67. Found: C, 55.59; H, 3.57; N, 8.40%.

4.1.1.10. 4-(2-Chloroacetamido)-N-(2,5-dichlorophenyl)benzamide (18j)

Yellow crystal (yield, 77%); m.p. = 195 − 197 °C; FT-IR (ν_max, cm⁻¹): 3277, 3104 (NH), 3033 (C–H aromatic), 1770, 1679 (C=O), 1606 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.63 (s, 1H), 10.03 (s, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.73 (dd, J = 5.6, 3.0 Hz, 3H), 7.57 (d, J = 8.6 Hz, 1H), 7.34 (dd, J = 8.6, 2.5 Hz, 1H), 4.29 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.60 (2 C), 165.26, 142.26, 136.86, 131.87, 131.81, 130.80, 129.48 (2C), 129.19, 128.97 (2C), 118.75, 44.02; Anal. Calcd. for C₁₅H₁₁Cl₃N₂O₂ (357.61): C, 50.38; H, 3.10; N, 7.83. Found: C, 50.42; H, 3.18; N, 7.64%.

4.1.1.11. 4-(2-Chloroacetamido)-N-(4-fluorophenyl)benzamide (18k)

White crystal (yield, 70%); m.p. = 238–240 °C; FT-IR (ν_max, cm⁻¹); 3325 (NH), 1666, 1643 (C=O), 1611 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.66 (s, 1H), 10.23 (s, 1H), 7.95 (d, J = 7.0 Hz, 2H), 7.82 − 7.70 (m, 4H), 7.17 (s, 2H), 4.31 (t, J = 2.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.54, 165.21, 141.91, 136.06, 136.02, 130.12, 129.28, 129.04, 122.15, 119.45, 119.02, 118.63, 116.11, 115.88, 44.06; Anal. Calcd. for C₁₅H₁₂ClFN₂O₂ (306.72): C, 58.74; H, 3.94; N, 9.13. Found: C, 58.55; H, 3.89; N, 9.05%.

4.1.1.12. 4-(2-Chloroacetamido)-N-(2-hydroxyphenyl)benzamide (18l)

Brown crystal (yield, 65%); m.p. = 192 − 194 °C; FT-IR (ν_max, cm⁻¹): 3262, 3190 (NH), 3064 (C–H aromatic), 2952 (C–H aliphatic), 1735, 1683 (C=O), 1600 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.70 (s, 1H), 10.00 (s, 1H), 9.49 (s, 1H), 8.11–8.02 (m, 2H), 8.02–7.91 (m, 2H), 7.12 − 6.73 (m, 4H), 4.32 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.77, 165.24, 165.07, 163.82 (2C), 149.77, 144.89, 143.80, 141.98, 131.61, 129.69 (2C), 126.39, 124.39, 44.07; Anal. Calcd. for C₁₅H₁₃ClN₂O₃ (304.72): C, 59.12; H, 4.30; N, 9.19. Found: C, 59.01; H, 4.14; N, 8.92%.

4.1.1.13. 4-(2-Chloroacetamido)-N-(4-hydroxyphenyl)benzamide (18m)

Brown crystal (yield, 85%); m.p. >300 °C; FT-IR (ν_max, cm⁻¹): 3309, 3104 (NH), 3033 (C–H aromatic), 1770, 1671 (C=O), 1607 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.81 (s, 1H), 10.29 (s, 1H), 9.95 (s, 1H), 8.11 (d, J = 9.2 Hz, 2H), 7.85 − 7.80 (m, 2H), 7.50 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8.3 Hz, 2H), 4.30 (d, J = 4.1 Hz, 2H); Anal. Calcd. for C₁₅H₁₃ClN₂O₃ (304.73): C, 59.12; H, 4.30; N, 9.19. Found: C, 59.32; H, 4.15; N, 8.95%.

4.1.1.14. 4-(2-Chloroacetamido)-N-(2-hydroxy-4-nitrophenyl)benzamide (18n)

Greenish yellow crystal (yield, 60%); m.p. = 198–200 °C; FT-IR (ν_max, cm⁻¹): 3596, 3409 (NH), 3074 (C–H aromatic), 1700, 1652 (C=O), 1602 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 11.15 (s, 1H), 10.66 (s, 1H), 9.51 (s, 1H), 8.22 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.85 − 7.67 (m, 4H), 4.30 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.61, 165.05 (2C), 148.61 (2C), 143.65, 142.42, 133.57, 129.12 (2 C), 122.04, 118.83, 115.16, 109.46, 44.07; Anal. Calcd. for C₁₅H₁₂ClN₃O₅ (349.72): C, 51.52; H, 3.46; N, 12.02. Found: C, 51.33; H, 3.57; N, 11.87%.

4.1.2. General procedure for the synthesis of compounds 22a–c

A mixture of 17 (2 mmol) and the acid hydrazides 21a–c namely, 2-chlorobenzohydrazide, 3-chlorobenzohydrazide, and 2-hydroxybenzohyazide (2 mmol) was allowed to stir in acetonitrile (50 ml) in the presence of trimethylamine (1 ml) for 8 h. The precipitated products were filtered, dried, and crystalised from ethanol to give the diamide intermediated, 22a–c, respectively.

4.1.2.1. 2-Chloro-N-(4-(2-(2-chlorobenzoyl)hydrazine-1-carbonyl)phenyl)acetamide (22a)

White crystal (yield, 85%); m.p. = 230 − 232 °C; FT-IR (ν_max, cm⁻¹): 3409, 3249 (NH), 3074 (C–H aromatic), 1673, 1650 (C=O), 1603 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1H), 10.55 (s, 1H), 10.38 (s, 1H), 7.92 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.6 Hz, 2H), 7.56 − 7.46 (m, 4H), 4.33 − 4.28 (m, 2H); Anal. Calcd. for C₁₆H₁₃Cl₂N₃O₃ (366.19): C, 52.48; H, 3.58; N, 11.47. Found: C, 52.72; H, 3.48; N, 11.34%.

4.1.2.2. 2-Chloro-N-(4-(2-(3-chlorobenzoyl)hydrazine-1-carbonyl)phenyl)acetamide (22b)

Yellow crystal (yield, 80%); m.p. = 235 − 237 °C; FT-IR (ν_max, cm⁻¹): 3409, 3283 (NH), 3074 (C–H aromatic), 1676, 1645 (C=O), 1604 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 10.63 (s, 2H), 10.51 (s, 1H), 7.91 (dd, J = 19.0, 9.7 Hz, 4H), 7.74 − 7.66 (m, 3H), 7.58 (d, J = 6.4 Hz, 1H), 4.30 (t, J = 2.4 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.67, 165.58 (2C), 165.00, 142.13, 134.92, 133.83, 129.11, 128.88, 127.83 (2C), 127.59, 119.58, 119.13, 118.75, 44.06; Anal. Calcd. for C₁₆H₁₃Cl₂N₃O₃ (366.19): C, 52.48; H, 3.58; N, 11.47. Found: C, 52.71; H, 3.62; N, 11.60%.

4.1.2.3. 2-Chloro-N-(4–(2-(2-hydroxybenzoyl)hydrazine-1-carbonyl)phenyl)acetamide (22c)

Yellow crystal (yield, 80%); m.p. = 233 − 235 °C; FT-IR (ν_max, cm⁻¹): 3272 (NH), 1687, 1661 (C=O), 1600 (C=N); ¹H NMR (400 MHz, DMSO-d₆) δ 11.97 (s, 1H), 10.69 (d, J = 9.7 Hz, 2H), 10.63 (s, 1H), 7.93 (d, J = 8.0 Hz, 3H), 7.75 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.6 Hz, 1H), 6.96 (q, J = 8.4, 7.4 Hz, 2H), 4.32 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 168.25, 165.62, 165.47, 159.76, 159.72, 142.21, 129.14, 128.87, 128.65, 127.68, 119.58, 119.16, 118.79, 118.12, 114.98, 44.07; Anal. Calcd. for C₁₆H₁₄ClN₃O₄ (347.76): C, 55.26; H, 4.06; N, 12.08. Found: C, 55.45; H, 3.83; N, 11.90%.

4.1.3. General procedure for the synthesis of compounds 23a–n

A mixture of potassium salt of bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]quinoxaline-3-thiol 14 (0.5 g, 0.001 mol) and 4-(2-chloroacetamido)-N-(substituted)benzamide 18a–n (0.001 mol) in DMF (50 ml) was heated on a water bath for 6 h. After cooling to room temperature, the reaction mixture was poured on crushed ice. The obtained precipitates were collected by filtration, dried, and crystalised from ethanol to give the target compounds 23a–n.

4.1.3.1. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)-N-ethylbenzamide (23a)

White crystal (yield, 70%); m.p. = 260–262 °C. FT-IR (ν_max, cm⁻¹): 3267 (NH), 1700, 1655 (C=O), 1596 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.69 (s, 1H), 10.12 (s, 1H), 8.35 (dq, J = 11.5, 3.7, 3.1 Hz, 2H), 7.86 (dd, J = 8.1, 3.1 Hz, 1H), 7.85 − 7.80 (m, 2H), 7.75 − 7.69 (m, 2H), 7.69 − 7.65 (m, 1H), 7.63 (dt, J = 10.1, 5.1 Hz, 1H), 4.41 (d, J = 3.4 Hz, 2H), 3.32 − 3.25 (m, 2H), 1.16 − 1.09 (m, 3H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.67, 165.83, 151.65, 141.85 (2C), 138.29, 135.59, 129.91, 128.73, 128.52 (2C), 128.34, 128.32 (2C), 124.07, 118.76 (2C), 117.13, 34.45 (2C), 15.33; Anal. Calcd. for C₂₁H₁₈N₈O₂S (446.49): C, 56.49; H, 4.06; N, 25.10. Found: C, 56.21; H, 4.15; N, 24.95%.

4.1.3.2. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)-N-butylbenzamide (23b)

Yellowish white crystal (yield, 70%); m.p. = 252–254 °C. FT-IR (ν_max, cm⁻¹): 3299 (NH), 2928 (CH aliphatic), 1676, 1631 (C=O); ¹H NMR (700 MHz, DMSO-d₆) δ 10.68 (s, 1H), 10.02 (s, 1H), 8.65 − 8.58 (m, 1H), 8.50 − 8.45 (m, 1H), 8.33 (q, J = 4.9 Hz, 1H), 7.86 − 7.79 (m, 2H), 7.75 (p, J = 7.6, 7.2 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 4.57 (s, 2H), 3.25 (p, J = 6.6, 5.4 Hz, 2H), 1.50 (q, J = 7.5 Hz, 2H), 1.33 (q, J = 8.0 Hz, 2H), 0.94 − 0.88 (m, 3H); ¹³C NMR (176 MHz, DMSO-d₆) δ 165.93, 165.90, 147.72, 142.11, 141.56, 139.35, 138.78, 130.08, 128.54, 128.37, 128.35, 124.09, 123.20, 118.76, 118.58, 118.07, 39.29, 38.88, 31.78, 20.15, 14.22; MS (m/z): 475 (M⁺ + 1, 50%); Anal. Calcd. for C₂₃H₂₂N₈O₂S (474.54): C, 58.21; H, 4.67; N, 23.61. Found: C, 58.05; H, 4.60; N, 23.55%.

4.1.3.3. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)-N-(sec-butyl)benzamide (23c)

White crystal (yield, 72%); m.p. = 245–247 °C. FT-IR (ν_max, cm⁻¹): 3266, 3127 (NH), 2965, 2910 (CH aliphatic), 1701, 1636 (C=O), 1605 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.68 (s, 1H), 10.01 (s, 1H), 8.58 (d, J = 8.1 Hz, 1H), 8.45 (d, J = 7.9 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.73 (p, J = 7.6 Hz, 2H), 7.65 (d, J = 8.3 Hz, 2H), 4.57 (s, 2H), 3.90 (dd, J = 14.5, 7.5 Hz, 1H), 1.51 (ddq, J = 29.9, 15.4, 8.3, 7.7 Hz, 2H), 1.13 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 7.5 Hz, 3H); ¹³C NMR (176 MHz, DMSO-d₆) δ 165.88, 165.48, 147.74, 142.07, 141.52, 139.32, 138.77, 130.27, 128.63 (2C), 128.34, 128.32, 124.04, 123.15, 118.69 (2C), 118.56, 118.03, 46.82, 38.89, 29.34, 20.77, 11.25; MS (m/z): 475 (M⁺ + 1, 60%); Anal. Calcd. for C₂₃H₂₂ClN₈O₂S (474.54): C, 58.21; H, 4.67; N, 23.61. Found: C, 57.98; H, 4.62; N, 23.57%.

4.1.3.4. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)-N-(tert-butyl)benzamide (23d)

Faint yellow crystal (yield, 75%); m.p. = 248–250 °C. FT-IR (ν_max, cm⁻¹): 3439, 3104 (NH), 3051 (CH aromatic), 2968, 2929 (CH aliphatic), 1651 (C=O), 1600 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.67 (s, 1H), 10.03 (s, 1H), 8.61 (d, J = 8.0 Hz, 1H), 8.50 − 8.47 (m, 1H), 7.79 (t, J = 6.9 Hz, 3H), 7.76 (d, J = 8.1 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 4.57 (s, 2H), 1.38 (s, 9H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.06, 165.85, 147.74, 142.13, 141.39, 139.37, 138.78, 131.16, 128.74 (2C), 128.38, 128.37, 124.12, 123.23, 118.60, 118.57 (2C), 118.08, 51.16, 38.87, 29.11 (3C); MS (m/z): 475 (M⁺ + 1, 100%); Anal. Calcd. for C₂₃H₂₂N₈O₂S (474.54): C, 58.21; H, 4.67; N, 23.61. Found: C, 58.05; H, 4.55; N, 23.44%.

4.1.3.5. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)-N-cyclopentylbenzamide (23e)

Yellow crystal (yield, 77%); m.p. = 244–246 °C. FT-IR (ν_max, cm⁻¹): 3262, 3124 (NH), 2950, 2905 (CH aliphatic), 1699, 1636 (C=O), 1607 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.68 (s, 1H), 10.02 (s, 1H), 8.61 (dd, J = 8.0, 1.6 Hz, 1H), 8.47 (dd, J = 7.8, 1.8 Hz, 1H), 8.17 (d, J = 7.3 Hz, 1H), 7.83 (d, J = 8.6 Hz, 2H), 7.76 (ddd, J = 8.9, 7.6, 1.6 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 4.57 (s, 2H), 4.21 (q, J = 7.1 Hz, 1H), 1.88 (dtd, J = 12.0, 8.8, 7.9, 4.4 Hz, 2H), 1.70 (dt, J = 9.6, 5.3 Hz, 2H), 1.56 − 1.51 (m, 4H); ¹³C NMR (176 MHz, DMSO-d₆) δ 165.88, 165.73, 147.73, 142.12, 141.53, 139.36, 138.78, 130.15, 128.70 (2C), 128.37, 128.35, 124.10, 123.21, 118.66 (2C), 118.59, 118.06, 51.34, 38.87, 32.61 (2C), 24.09 (2C); MS (m/z): 487 (M⁺ + 1, 65%); Anal. Calcd. for C₂₄H₂₂N₈O₂S (486.55): C, 59.25; H, 4.56; N, 23.03. Found: C, 59.08; H, 4.47; N, 22.89%.

4.1.3.6. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)-N-cyclohexylbenzamide (23f)

White crystal (yield, 70%); m.p. = 270–272 °C. FT-IR (ν_max, cm⁻¹): 3269, 3131 (NH), 2930 (CH aliphatic), 1700, 1633 (C=O), 1607 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.68 (s, 1H), 10.02 (s, 1H), 8.61 (dd, J = 7.9, 1.6 Hz, 1H), 8.47 (dd, J = 7.7, 1.8 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.76 (dtd, J = 16.4, 7.5, 1.5 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 4.57 (s, 2H), 3.75 (ddt, J = 15.1, 11.0, 5.3 Hz, 1H), 1.81 (dt, J = 8.6, 4.1 Hz, 2H), 1.74 (dt, J = 11.4, 4.1 Hz, 2H), 1.64 − 1.59 (m, 1H), 1.30 (dd, J = 11.3, 8.6 Hz, 4H), 1.13 (qt, J = 8.4, 4.3 Hz, 1H); ¹³C NMR (176 MHz, DMSO-d₆) δ 165.88, 165.14, 147.73, 142.12, 141.54, 139.36, 138.78, 130.21, 128.69 (2C), 128.37, 128.35, 124.10, 123.22, 118.67 (2C), 118.59, 118.07, 48.73, 38.87, 32.95 (2C), 25.76, 25.45 (2C); MS (m/z): 501 (M⁺ + 1, 100%); Anal. Calcd. for C₂₅H₂₄N₈O₂S (500.58): C, 59.99; H, 4.83; N, 22.39. Found: C, 59.85; H, 4.76; N, 22.11%.

4.1.3.7. N-(4-Acetylphenyl)-4-(2-(bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)benzamide (23g)

Pale white crystal (yield, 80%); m.p. = 275–277 °C. FT-IR (ν_max, cm⁻¹): 3300 (NH), 1655 (C=O), 1593 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.77 (s, 1H), 10.14 (s, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.81 (dd, J = 8.0, 1.5 Hz, 1H), 7.78 − 7.76 (m, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.59 − 7.57 (m, 1H), 7.54 (dd, J = 8.5, 1.3 Hz, 1H), 7.40 − 7.37 (m, 1H), 7.36 − 7.34 (m, 2H), 7.10 (t, J = 7.4 Hz, 1H), 5.19 (s, 2H), 2.49 (s, 3H); ¹³C NMR (176 MHz, DMSO-d₆) δ 165.80, 165.23, 157.97, 154.85, 142.00, 139.71, 133.48, 132.47 (2C), 130.20 (2C), 130.04, 129.29, 129.23 (2C), 129.05 (2C), 123.99, 123.94 (2C), 120.80 (2C), 118.83 (2C), 115.21, 45.80, 21.59; MS (m/z): 537 (M⁺ + 1, 40%), 354 (40%); Anal. Calcd. for C₂₇H₂₀N₈O₃S (536.57): C, 60.44; H, 3.76; N, 20.88. Found: C, 60.10; H, 3.66; N, 20.59%.

4.1.3.8. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)-N-(3-chlorophenyl)benzamide (23h)

Yellowish white crystal (yield, 63%); m.p. = 280–282 °C. FT-IR (ν_max, cm⁻¹): 3267, 3109 (NH), 1701, 1647 (C=O), 1593 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.79 (s, 1H), 10.30 (s, 1H), 10.02 (s, 1H), 8.61 (dd, J = 8.1, 1.5 Hz, 1H), 8.47 (dd, J = 7.9, 1.7 Hz, 1H), 7.98 − 7.95 (m, 3H), 7.78 − 7.73 (m, 4H), 7.70 (ddd, J = 8.3, 2.1, 1.0 Hz, 1H), 7.38 (t, J = 8.1 Hz, 1H), 7.15 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H), 4.59 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.08, 165.52, 147.71, 142.38, 142.12, 141.23, 139.34, 138.78, 133.38, 130.76, 129.63, 129.30 (2C), 128.37, 124.08, 123.65, 123.20, 120.10 (2C), 119.03, 118.86 (2C), 118.59, 118.06, 38.89; Anal. Calcd. for C₂₅H₁₇ClN₈O₂S (528.98): C, 56.77; H, 3.24; N, 21.18. Found: C, 56.37; H, 3.11; N, 20.99%.

4.1.3.9. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)acetamido)-N-(4-chlorophenyl)benzamide (23i)

Yellow crystal (yield, 65%); m.p. = 277–279 °C. FT-IR (ν_max, cm⁻¹): 3382, 3111 (NH), 1674 (C=O), 1595 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.78 (s, 1H), 10.28 (s, 1H), 10.03 (s, 1H), 8.62 (d, J = 8.0 Hz, 1H), 8.53 − 8.44 (m, 1H), 7.96 (d, J = 8.5 Hz, 3H), 7.82 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 9.1 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 4.59 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.07, 165.39, 147.71, 142.29, 142.15, 139.37, 138.79, 138.72, 129.80, 129.26 (2C), 128.98 (2C), 128.38 (2C), 127.57, 124.12, 123.24, 122.27 (2C), 118.86 (2C), 118.61, 118.08, 38.88; Anal. Calcd. for C₂₅H₁₇ClN₈O₂S (528.98): C, 56.77; H, 3.24; N, 21.18. Found: C, 56.44; H, 3.19; N, 20.85%.

4.1.3.10. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]uinoxaline-3-ylthio)acetamido)-N-(2,5-dichlorophenyl)benzamide (23j)

White crystal (yield, 78%); m.p. = 260–262 °C. FT-IR (ν_max, cm⁻¹): 3429 (NH), 1672 (C=O), 1511 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.80 (s, 1H), 10.03 (s, 2H), 8.62 (dd, J = 8.0, 1.5 Hz, 1H), 8.48 (dd, J = 7.9, 1.7 Hz, 1H), 7.99 − 7.97 (m, 2H), 7.77 (dt, J = 5.0, 2.2 Hz, 2H), 7.76 (m, 2H), 7.75 (s, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.38 (dd, J = 8.6, 2.6 Hz, 1H), 4.60 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.11, 165.23, 147.70, 142.59, 142.15, 139.37, 138.79, 136.95, 131.89, 131.35, 129.39 (2C), 128.76, 128.39, 128.37, 128.20, 127.90, 127.39, 124.11, 123.23, 118.94 (2C), 118.60, 118.08, 38.89; MS (m/z): 563 (M⁺, 30%), 420 (65%); Anal. Calcd. For C₂₅H₁₆Cl₂N₈O₂S (563.42): C, 53.30; H, 2.86; N, 19.89. Found: C, 53.01; H, 2.77; N, 19.66%.

4.1.3.11. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]uinoxaline-3-ylthio)acetamido)-N-(4-fluorophenyl)benzamide (23k)

White powder (yield, 80%); m.p. = 250–252 °C. FT-IR (ν_max, cm⁻¹): 3289, 3108 (NH), 1645 (C=O), 1604 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.77 (s, 1H), 10.21 (s, 1H), 10.02 (s, 1H), 8.79 − 8.57 (m, 1H), 8.55 − 8.41 (m, 1H), 7.96 (t, J = 9.5 Hz, 2H), 7.78 (dt, J = 35.0, 12.1 Hz, 5H), 7.26 − 7.14 (m, 3H), 4.59 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.04, 165.20, 147.72, 142.11, 139.34, 138.78, 129.17 (2C), 128.36 (2C), 124.08, 123.19, 122.61 (2C), 122.57 (2C), 118.84 (2C), 118.58, 118.06, 115.68 (2C), 115.55 (2C), 38.90; MS (m/z): 513 (M⁺ + 1, 25%), 372 (100%); Anal. Calcd. For C₂₅H₁₇FN₈O₂S (512.52): C, 58.59; H, 3.34; N, 21.86. Found: C, 58.34; H, 3.22; N, 21.69%.

4.1.3.12. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]uinoxaline-3-ylthio)acetamido)-N-(2-hydroxyphenyl)benzamide (23l)

Off white crystal (yield, 71%); m.p. = 282–284 °C. FT-IR (ν_max, cm⁻¹): 3263 (NH), 1646 (C=O), 1600 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.78 (s, 1H), 10.02 (s, 1H), 9.76 (s, 1H), 9.46 (s, 1H), 8.60 (dd, J = 8.1, 1.4 Hz, 1H), 8.46 (dd, J = 8.0, 1.7 Hz, 1H), 7.97 − 7.96 (m, 2H), 7.75 − 7.73 (m, 3H), 7.69 (dt, J = 8.1, 2.1 Hz, 2H), 7.04 − 7.03 (m, 1H), 6.93 − 6.92 (m, 1H), 6.84 − 6.83 (m, 1H), 4.60 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.05, 165.10, 149.68, 147.72, 142.24, 142.10, 139.33, 138.77, 129.49, 129.08 (2C), 128.36, 128.34, 126.46, 126.03, 124.43, 124.06, 123.17, 119.53 (2C), 118.95, 118.57, 118.06, 116.50, 38.93; MS (m/z): 511 (M⁺ + 1, 70%), 293 (100%); Anal. Calcd. For C₂₅H₁₈N₈O₃S (510.53): C, 58.82; H, 3.55; N, 21.95. Found: C, 58.78; H, 3.50; N, 21.88%.

4.1.3.13. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]uinoxaline-3-ylthio)acetamido)-N-(4-hydroxyphenyl)benzamide (23m)

Pale yellow crystal (yield, 75%); m.p. = 285–287 °C. FT-IR (ν_max, cm⁻¹): 3414 (NH), 1601 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.75 (s, 1H), 10.01 (s, 1H), 9.93 (s, 1H), 9.26 (s, 1H), 8.63 − 8.54 (m, 1H), 8.51 − 8.39 (m, 1H), 7.93 (d, J = 8.7 Hz, 2H), 7.72 (dq, J = 14.7, 7.1, 6.6 Hz, 4H), 7.55 − 7.49 (m, 2H), 6.79 − 6.70 (m, 2H), 4.59 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 165.99, 164.72, 154.09 (2C), 147.74, 142.05, 139.30, 138.77, 131.21, 130.33, 128.98 (2C), 128.33 (2C), 124.01, 123.12, 122.74 (2C), 118.81(2C), 118.55, 118.02, 115.42 (2C), 38.92; MS (m/z): 511(M⁺ + 1, 30%); Anal. Calcd. For C₂₅H₁₈N₈O₃S (510.53): C, 58.82; H, 3.55; N, 21.95. Found: C, 58.63; H, 3.48; N, 21.73%.

4.1.3.14. 4-(2-(Bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]uinoxaline-3-ylthio)acetamido)-N-(2-hydroxy-4-nitrophenyl)benzamide (23n)

Red crystal (yield, 68%); m.p. = 265–267 °C. FT-IR (ν_max, cm⁻¹): 3294, 3101 (NH), 2927 (CH aliphatic), 1653 (C=O), 1603 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 11.14 (s, 1H), 10.82 (s, 1H), 10.02 (s, 1H), 9.49 (s, 1H), 8.61 (dd, J = 8.1, 1.5 Hz, 1H), 8.47 (dd, J = 7.8, 1.8 Hz, 1H), 8.25 (d, J = 8.9 Hz, 1H), 7.98 − 7.96 (m, 2H), 7.79 (dd, J = 8.9, 2.6 Hz, 1H), 7.78 − 7.75 (m, 3H), 7.74 (dd, J = 6.7, 2.1 Hz, 2H), 4.60 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.14, 165.06, 148.55, 147.70, 143.66, 142.72, 142.13, 139.35, 138.78, 133.63, 129.27 (2C), 128.90, 128.38, 128.36, 124.08, 123.20, 121.83, 119.05 (2C), 118.59, 118.07, 115.57, 109.89, 38.91; MS (m/z): 554 (M⁺ − 1, 70%); Anal. Calcd. For C₂₅H₁₇N₉O₅S (555.53): C, 54.05; H, 3.08; N, 22.69. Found: C, 53.85; H, 2.97; N, 22.43%.

4.1.4. General procedure for the synthesis of compounds 24a–c

A mixture of potassium salt of bis([1, 2, 4]triazolo)[4,3-a:3′,4′-c]quinoxaline-3-thiol 14 (0.5 g, 0.001 mol) and 2-chloro-N-(4–(2-(substituted)hydrazine-1-carbonyl)phenyl)acetamide 22a–c (0.001 mol) in DMF (50 ml) was heated on a water bath for 6 h. After cooling to room temperature, the reaction mixture was poured on crushed ice. The obtained precipitates were collected by filtration, dried, and crystalised from ethanol to give the target compounds 24a–c.

4.1.4.1. 2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)-N-(4–(2-(2-chlorobenzoyl)hydrazine-1-carbonyl)phenyl)acetamide (24a)

White crystal (yield, 65%); m.p. = 220–222 °C. FT-IR (ν_max, cm⁻¹): 3185 (NH), 1698 (C=O), 1603 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.77 (s, 1H), 10.54 (s, 1H), 10.37 (s, 1H), 10.03 (s, 1H), 8.63 (dd, J = 8.1, 1.5 Hz, 1H), 8.48 (dd, J = 7.9, 1.7 Hz, 1H), 7.93 (d, J = 8.7 Hz, 2H), 7.77 (dtd, J = 17.1, 7.5, 1.5 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.57 (ddd, J = 8.0, 4.0, 1.5 Hz, 2H), 7.53 (td, J = 7.6, 1.7 Hz, 1H), 7.48 (td, J = 7.4, 1.3 Hz, 1H), 4.60 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.25, 166.08, 165.40, 147.70, 142.36, 142.15, 139.37, 138.79, 135.18, 131.98, 130.93, 130.38, 129.90, 129.06 (2C), 128.37 (2C), 127.64 (2C), 124.12, 123.23, 118.91(2C), 118.60, 118.10, 38.94. MS (m/z): 572 (M⁺, 100%); Anal. Calcd. for C₂₆H₁₈ClN₉O₃S (572): C, 54.60; H, 3.17; N, 22.04. Found: C, 54.39; H, 3.09; N, 21.88%.

4.1.4.2. 2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)-N-(4–(2-(3-chlorobenzoyl)hydrazine-1-carbonyl)phenyl)acetamide (24b)

White crystal (yield, 63%); m.p. = 232–234 °C. FT-IR (ν_max, cm⁻¹): 3260, 3108 (NH), 1639 (C=O), 1603 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 10.78 (s, 1H), 10.62 (s, 1H), 10.49 (s, 1H), 10.02 (s, 1H), 8.62 (dd, J = 8.0, 1.5 Hz, 1H), 8.47 (dd, J = 7.8, 1.7 Hz, 1H), 7.96 (t, J = 1.9 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.91 − 7.88 (m, 1H), 7.78 − 7.76 (m, 1H), 7.75 − 7.71 (m, 3H), 7.70 − 7.69 (m, 1H), 7.59 (t, J = 7.9 Hz, 1H), 4.60 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 166.09, 165.67, 164.99, 147.71, 142.40, 142.13, 139.35, 138.78, 135.00, 133.84, 132.23, 131.11, 129.03 (2C), 128.38, 128.36, 127.72 (2C), 127.66, 126.66, 124.09, 123.21, 118.96, 118.59, 118.07, 38.92; MS (m/z): 572 (M⁺, 100%); Anal. Calcd. for C₂₆H₁₈ClN₉O₃S (572): C, 54.60; H, 3.17; N, 22.04. Found: C, 54.45; H, 3.11; N, 21.95%.

4.1.4.3. 2-(Bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxalin-3-ylthio)-N-(4–(2-(2-hydroxybenzoyl)hydrazine-1-carbonyl)phenyl)acetamide (24c)

Yellow crystal (yield, 60%); m.p. = 270–272 °C. FT-IR (ν_max, cm⁻¹): 3282 (NH), 1648, 1619 (C=O), 1599 (C=N); ¹H NMR (700 MHz, DMSO-d₆) δ 11.96 (s, 1H), 10.78 (s, 1H), 10.67 (s, 1H), 10.59 (s, 1H), 10.02 (s, 1H), 8.62 (dd, J = 8.0, 1.5 Hz, 1H), 8.47 (dd, J = 7.8, 1.7 Hz, 1H), 7.93 (dd, J = 10.4, 7.6 Hz, 3H), 7.77 (ddt, J = 10.8, 8.3, 3.9 Hz, 2H), 7.74 (dd, J = 7.3, 4.8 Hz, 2H), 7.47 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.00 − 6.95 (m, 2H), 4.59 (s, 2H); ¹³C NMR (176 MHz, DMSO-d₆) δ 168.26, 166.09, 165.44, 159.79, 147.71, 142.45, 142.13, 139.35, 138.78, 134.65, 129.07 (2 C), 128.73, 128.38, 128.36, 127.52, 124.09, 123.21, 119.52, 118.96 (2C), 118.59, 118.07, 117.88, 115.01, 38.91; MS (m/z): 554 (M⁺ + 1, 100%); Anal. Calcd. for C₂₆H₁₉N₉O₄S (553.56): C, 56.41; H, 3.46; N, 22.77. Found: C, 56.20; H, 3.39; N, 22.58%.

4.2. Biological testing

4.2.1. In vitro anti-proliferative activity

MTT assay protocol^55–57,74 was applied as described in Supplementary data.

4.2.2. In vitro VEGFR-2 kinase assay

All the synthesised compounds were tested for their inhibitory activity against VEGFR-2 as described in Supplementary data⁷⁵.

4.2.3. Flow cytometry analysis for cell cycle

Cell cycle analysis was performed using propidium iodide (PI) staining and flow cytometry analysis for compound 23j as described in Supplementary data^76,77.

4.2.4. Flow cytometry analysis for apoptosis

Apoptotic effect was assessed for compound 23j as described in Supplementary data^78,79.

4.2.5. Western blot analysis

Western blot technique was performed for compound 23j to determine its effect against caspase3, caspase9, BAX, and Bcl-2 as described in Supplementary data^80–82.

4.3. In silico studies

4.3.1. Docking studies

Docking studies were carried out against VEEGFR-2 (PDB ID: 2OH4) using Discovery Studio 4.0 as described in Supplementary data^{83–85,86,87}.

4.3.2. Admet studies

ADMET studies were carried out as described in Supplementary data^85,87.

4.3.3. Toxicity studies

Toxicity studies were performed as described in Supplementary data.

Funding Statement

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no [RG-1441–364].

Disclosure statement

No potential conflict of interest was reported by the author(s). The authors would like to acknowledge Dr. Mohamed R. Elnagar, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt for his valuable suggestion and technical assistance.