New benzoxazole derivatives as potential VEGFR-2 inhibitors and apoptosis inducers: design, synthesis, anti-proliferative evaluation, flowcytometric analysis, and in silico studies

Abstract

A new series of benzoxazole derivatives were designed and synthesised to have the main essential pharmacophoric features of VEGFR-2 inhibitors. Cytotoxic activities were evaluated for all derivatives against two human cancer cell lines, MCF-7 and HepG2. Also, the effect of the most cytotoxic derivatives on VEGFR-2 protein concentration was assessed by ELISA. Compounds 14o, 14l, and 14b showed the highest activities with VEGFR-2 protein concentrations of 586.3, 636.2, and 705.7 pg/ml, respectively. Additionally, the anti-angiogenic property of compound 14b against human umbilical vascular endothelial cell (HUVEC) was performed using a wound healing migration assay. Compound 14b reduced proliferation and migratory potential of HUVEC cells. Furthermore, compound 14b was subjected to further biological investigations including cell cycle and apoptosis analyses. Compound 14b arrested the HepG2 cell growth at the Pre-G1 phase and induced apoptosis by 16.52%, compared to 0.67% in the control (HepG2) cells. The effect of apoptosis was buttressed by a 4.8-fold increase in caspase-3 level compared to the control cells. Besides, different in silico docking studies were also performed to get better insights into the possible binding mode of the target compounds with VEGFR-2 active sites.

Keywords:

• Anti-proliferative
• apoptosis
• benzoxazole
• VEGFR-2 inhibitors

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1. Introduction

Angiogenesis, a complex process of new blood vessel creation, is crucial for cell development and reproduction^1,2. Considering the similar function in cancerous cells, uncontrolled or abnormal angiogenesis has been linked to tumour progression and metastasis³. Therefore, finding efficient anti-angiogenesis agents could be considered as a hopeful approach for cancer treatment⁴.

Growth factors, including vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), control angiogenesis^5–7. Three main vascular endothelial growth factor receptor subtypes are well-defined namely, VEGFR-1, VEGFR-2, and VEGFR-3⁸. These receptors are the key players' intermediates in controlling tumour angiogenesis and in the development of new blood vessel networks essential to supply nutrition and oxygen for tumour growth⁹. Among the three VEGFRs subtypes, VEGFR-2 plays the most critical role in promoting tumour angiogenesis¹⁰. Following its activation by VEGF, VEGFR-2 initiates downstream signal transduction via dimerisation and then autophosphorylation of tyrosine receptor. These signalling pathways result in tumour angiogenesis¹¹. Thus, hindering the VEGF/VEGFR-2 signalling pathway or reducing its response by tyrosine kinases inhibitors (TKIs) is a supreme significant target in anti-angiogenesis therapy against cancer¹². Over the last decades, several small molecules have been approved for obstructing this critical pathway in angiogenesis^13,14. Development of tumour resistance to the effect of the current clinically used small-molecule TKIs opens the door for the investigation of the effectiveness of new chemotypes.

Through our trip in finding novel anticancer agents^9,15–21, our research team has introduced several small molecules serving as ATP competitive inhibitors of VEGFR-2 depending on different scaffolds including, quinoxaline 1^22,23, bis([1,2,4]triazolo)[4,3-a:3′,4′-c]quinoxaline 2²⁴, quinazolin-4(3H)-one 3²⁵, thieno[2,3-d]pyrimidine 4^26,27, 4-phenylphthalazine 5²⁸ and 5-benzylidenethiazolidine-2,4-dione 6²⁹ derivatives. The potency of some derivatives against the VEGFR-2 enzyme exceeded the already marketed VEGFR-2 inhibitors. All these derivatives achieved the basic pharmacophoric requirements needed to fit with the VEGFR-2 active binding site including (a) a “hinge-binding” head segment which is a flat heteroaromatic ring system that occupies the hinge region of ATP binding site³⁰, (b) a central aromatic linker to cross the kinase gatekeeper residues linking the hinge-binding segment with the hydrogen-bonding moiety³¹, (c) a “hydrogen-bonding moiety” which interacts via hydrogen bonding with Glu883 and/or Asp1044 residues in DFG motif of the enzyme, and (d) a terminal lipophilic tail that occupied the allosteric lipophilic pocket through numerous hydrophobic interactions³² (Figure 1).

Figure 1. Some reported VEGFR-2 inhibitors with the essential pharmacophoric features of VEGFR-2 inhibitor agents.

1.1. Rationale of molecular design

In view of the above-mentioned pharmacophoric requirements and depending on our ongoing project to develop novel cytotoxic small molecules based on various chemotypes, it is considered of interest to begin a research work directed towards the design of a new series of anti-angiogenic VEGFR-2 inhibitors. A cross-hybridisation approach between different pharmacophoric elements of the well-known TKIs was the idea of the current study. The approach applied for designing the new target compounds is demonstrated in Figure 2.

Figure 2. Summary of the proposed VEGFR-2 inhibitors modifications.

Herein, and while conserving the carboxamide moiety of sunitinib to serve as a hydrogen-bonding donor/acceptor moiety, a molecular replacement of the indolinylidene core of sunitinib by a benzoxazole core was performed in the hope of testing the effect of the introduction of another heteroatom to the aromatic scaffold in binding with the hinge region of ATP binding site.

Additionally, the fluorine atom in sunitinib was also replaced either by hydrogen, methyl, or chlorine bioisosteres. On the other side, the terminal phenyl ring of sorafenib was kept playing its key role of occupying the allosteric lipophilic pocket. Contrariwise, the 4-chlorine atom and the 3-trifluoro methyl group of sorafenib were replaced by different substituents as represented in the target compounds. Regarding the central aromatic linker moiety, we found that our previously reported linker of compound 3²⁵ could give the opportunity to the designed compounds to be oriented into the DFG motif and allosteric binding site as well (Figure 2).

The wide diversity of modifications enabled us to study the SAR of the designed candidates as potent anti-proliferative agents with potential VEGFR-2 inhibitory effects. To confirm such a design, in silico molecular docking studies of the designed compounds were performed against the prospective biological target (VEGFR-2).

2. Results and discussion

2.1. Chemistry

The target benzoxazole derivatives 14a–o were synthesised following the general methodologies outlined in Schemes 1–3. The starting compounds, 2-mercapto-benzoxazoles 8a–c were synthesised by refluxing the appropriate 2-aminophenol derivatives 7a–c, carbon disulphide, and potassium hydroxide in methanol following the reported procedure³³. Then, compounds 8a–c were treated with alcoholic KOH to afford the corresponding potassium salts, 9a–c (Scheme 1). On the other hand, 4-aminobenzoic acid 10 was reacted with chloroacetyl chloride in DMF to afford the chloroacetamide intermediate 11. Acylation of compound 11 was performed using thionyl chloride to yield 4–(2-chloroacetamido)benzoyl chloride 12 as described in the reported procedures^14,34. Treating of 12 with commercially available amines namely, 2-methoxyaniline, 2,6-dimethoxyaniline, 2,6-dimethylaniline, 2,4-dichloroaniline, and 4-hydroxyaniline, in acetonitrile containing triethylamine (TEA), afforded the target key intermediates 13a–e (Scheme 2).

Scheme 1. General synthetic route of target salts 9a–c.

Scheme 2. General synthetic route of target intermediates 13a–e.

The potassium salts 9a–c were heated in dry DMF with the formerly prepared derivatives 13a–e to afford the final target compounds 14a–o, (Scheme 3).

Scheme 3. General synthetic route of target final compounds 14a–o.

2.2. Biological testing

2.2.1. Breast cancer and hepatocellular carcinoma, in vitro anti-proliferative activities

The anti-proliferative activities of the newly synthesised compounds were assessed in vitro against two human cancer cell lines namely, breast cancer (MCF-7) and hepatocellular carcinoma (HepG2) cell lines, using the standard MTT method³⁵. The tested cell lines were chosen carefully depending on their VEGF overexpression. Sorafenib, the potent VEGFR-2 inhibitor drug, was co-assayed as a positive control. The cytotoxicity results were demonstrated in Table 1. A general observation of the obtained results revealed that all the newly synthesised members had high inhibitory activities towards the two cancer cell lines with IC₅₀ values ranging from 4.054 ± 0.17 to 32.53 ± 1.97 µM for MCF-7 and from 3.22 ± 0.13 to 32.11 ± 2.09 µM for HepG2.

Table 1. In vitro cytotoxic activities of the assessed compounds against MCF-7 and HepG2 cell lines.

Comp. ID	In vitro cytotoxicity IC50 (µM)
	MCF-7	HepG2
14a	4.054 ± 0.17	3.95 ± 0.18
14b	4.754 ± 0.21	4.61 ± 0.34
14c	19.36 ± 1.29	20.19 ± 1.82
14d	15.8 ± 1.04	18.47 ± 1.26
14e	16.52 ± 1.18	25 ± 1.47
14f	21.06 ± 1.5	22.05 ± 1.79
14g	5.8 ± 0.22	10.73 ± 0.83
14h	9.44 ± 0.59	19.29 ± 1.07
14i	6.94 ± 0.22	3.22 ± 0.13
14j	14.76 ± 0.99	11.86 ± 0.79
14k	7.75 ± 0.24	11.42 ± 0.93
14l	6.87 ± 0.23	6.70 ± 0.47
14m	32.53 ± 1.97	32.11 ± 2.09
14n	7.098 ± 0.5	9.93 ± 0.85
14o	18.15 ± 1.42	7.01 ± 0.52
Sorafenib	7.28 ± 0.58	3.38 ± 0.21

With reference to their cytotoxic activity, it was noticed that counterparts incorporating 5-chlorobenzo[d]oxazole moiety were slightly more advantageous than the unsubstituted benzo[d]oxazole analogs. However, the 5-methylbenzo[d]oxazole-containing derivatives displayed less potent inhibitory activity against the tested cell lines.

With respect to the 5-chlorobenzo[d]oxazole-based members, the best cytotoxic activities against both MCF-7 and HepG2 cell lines appeared with the parent derivative bearing a terminal 2-methoxy phenyl moiety 14b with IC₅₀ values of 4.75 ± 0.21 and 4.61 ± 0.34 µM, respectively. Meanwhile, on 5-chlorobenzo[d]oxazole-based derivatives, the 2,5-dichloro phenyl and the 4-hydroxy phenyl containing compounds, 14k and 14n, exhibited almost equipotent cytotoxic activity against the tested cell lines (IC₅₀ = 7.75 ± 0.24, 11.42 ± 0.93 µM for 14k, 7.098 ± 0.5 and 9.93 ± 0.85 µM for 14n). However, the rest of the substituent provided moderate IC₅₀ values against the tested cell lines.

As for unsubstituted benzo[d]oxazole derivatives, results of the in vitro anti-proliferative screening revealed that hybridisation of the nucleus with terminal 2-methoxy phenyl moiety 14a improved the inhibitory activity against HepG2 (IC₅₀ = 3.95 ± 0.18 µM) and MCF-7 (IC₅₀ = 4.054 ± 0.17 µM) as well. In addition, derivative 14g, bearing 2,6-dimethyl phenyl moiety, possessed noticeable inhibitory activity against MCF-7 cell line with IC₅₀ of 5.8 ± 0.22 µM with a moderate effect regarding HepG2 cell (IC₅₀ = 10.73 ± 0.83 µM). Cytotoxic activities were slightly decreased regarding derivatives bearing terminal 2,5-dichloro phenyl 14j or 2,6-dimethoxy phenyl 14d moieties with IC₅₀ values ranging from 11.86 ± 0.79 to 18.47 ± 1.26 µM. Substitution of the terminal phenyl ring with 4-hydroxy group 14m decreased the potency compared to other derivatives.

Lastly in this regard, concerning 5-methylbenzo[d]oxazole containing derivatives, it was found that compound 14i displayed the strongest anti-proliferative effect against HepG2 cell line (IC₅₀ = 3.22 ± 0.13 µM) compared to the reference drug, sorafenib. Compound 14i, moreover, showed a strong effect with respect to MCF-7 cells with IC₅₀ of 6.94 ± 0.22 µM. It is also noteworthy that member 14l, 2,5-dichloro phenyl, strongly inhibited the MCF-7 and HepG2 proliferation with IC₅₀ values of 6.87 ± 0.23 and 6.70 ± 0.47 µM, respectively. Other modifications of the terminal phenyl ring did not increase the cytotoxic activity with increasing the IC₅₀ range (7.01 ± 0.52 to 22.05 ± 1.79 µM) comparing the other derivatives (Figure 3).

Figure 3. In vitro cytotoxic activities of different chemical compounds. *Significant from Sorafenib group at p < 0.001.

2.2.2. Assessment of VEGFR-2 protein concentration

The effect of the most cytotoxic compounds 14b, 14n, 14l, 14i, 14o, and 14a was investigated on VEGFR-2 in HepG2 cells compared to sorafenib as a reference drug. HepG2 cells were treated with sorafenib (3.38 µM), 14b (4.61 µM), 14n (9.93 µM), 14l (6.70 µM), 14i (3.22 µM), 14o (7.01 µM), and 14a (3.95 µM). The inhibitory effects of the tested compounds on VEGFR-2 protein concentrations were summarised in Table 2 and Figure 4.

Figure 4. The effect of the most cytotoxic compounds 14b, 14n, 14l, 14i, 14o, and 14a were investigated on VEGFR-2 in HepG2 cells compared to sorafenib as a reference drug. HepG2 cells were treated with sorafenib (3.38 µM), 14b (4.61 µM), 14n (9.93 µM), 14l (6.70 µM), 14i (3.22 µM), 14o (7.01 µM), and 14a (3.95 µM). Data are represented as mean ± SEM of three different experiments. *Significant from the control group at p-value <0.001.

Table 2. The inhibitory effects of the assessed compounds on VEGFR-2 protein concentration in HepG2 cells compared to Sorafenib.

Comp. ID	VEGFR-2 protein concentration (pg/ml)
	HepG2
Control	1573 ± 36.9
Sorafenib	547.8 ± 13.5
14a	852.9 ± 16.3
14b	705.7 ± 20.3
14i	974.7 ± 25.4
14l	636.2 ± 22.4
14n	893.3 ± 6.34
14o	586.3 ± 16.1

Data are represented as mean ± SEM of three different experiments.

Compound 14o exhibited the most potent VEGFR-2 inhibitory effect (VEGFR-2 protein concentration = 586.3 ± 16.1 pg/ml) which was comparable to that of sorafenib (547.8 pg/ml). Additionally, compounds 14b and 14l showed promising effects with VEGFR-2 protein concentrations of 705.7 ± 20.3 and 636.2 ± 22.4 pg/ml, respectively. On the other hand, compounds 14n, 14i, and 14a showed moderate to weak effects with VEGFR-2 protein concentrations of 893.3 ± 6.34, 974.7 ± 25.4, and 852.9 ± 16.3 pg/ml, respectively.

2.2.3. Wound healing assay

The compound 14b reduced human umbilical vascular endothelial cell (HUVEC) proliferation and migratory potential.

One of the hallmarks of angiogenesis is cell migration, which happens in the earlier stages of the angiogenic cascade. A wound-healing assay was performed to investigate the migratory effect of compound 14b. Compound 14b and sorafenib extremely reduced the HUVECs migration potential exhibiting deeply reduced wound healing patterns after 72 h. Wound closure (%) was significantly lower in the compound 14b group (47.2 ± 2.88) and sorafenib group (39.8 ± 1.9) when compared to the control group (95.86 ± 4.51) (Figure 5).

Figure 5. Effects of compound 14b on endothelial cell migration in HUVEC cells compared to sorafenib. (A) Control/HUVECs, (B) HUVECs were treated with compound 14b for 72 h. (C) HUVECs were treated with sorafenib for 72 h. (D) Represents the graphical illustration for % of wound closure in control, sorafenib and 14b treated cells. Data are represented as mean ± SEM of three different experiments. *Significant from the control group at p < 0.001.

2.2.4. Cell cycle analysis

Compound 14b which demonstrated remarkable cytotoxic potency and significant inhibitory effect against VEGFR-2 was nominated for further cellular mechanistic study. This involved study of its impact on cell cycle progression and induction of apoptosis in HepG2 cells.

The cell cycle process was analysed after exposure of HepG2 cells to 14b with a concentration of 4.61 µM for 48 h. Flow cytometry data³⁶ revealed that the percentage of cells arrested at the Pre-G1 phase increased from 1.49% (in control cells) to 24.59% (in 14b) treated cells. In addition, the percentage of HepG2 cells mild increased at the S phase from 35.21 to 37.26%. Such findings revealed that compound 14b arrested the HepG2 cell growth mostly at the Pre-G1 phase (Table 3 and Figure 6).

Figure 6. Flow cytometry analysis for cell cycle distribution of HepG2 cells. (A) Control (HepG2 cells), (B) The representative histogram shows the cell cycle distribution of cells treated with 14b, and (C) Represents the graphical illustration for cell cycle distribution analysis among different treated cells. *Significant from the control group at p < 0.001.

Table 3. Flow cytometry analysis for cell cycle distribution of HepG2 cells.

Comp. ID	%G0/G1	%S	%G2/M	%Pre-G1
Control	46.55 ± 3.82	35.21 ± 3.13	18.24 ± 1.30	1.49 ± 0.12
14b	39.91 ± 3.52	37.26 ± 3.22	22.83 ± 2.41	24.59 ± 1.99

Data are represented as mean ± SEM of three different experiments.

2.2.5. Apoptosis analysis

To quantify the apoptosis triggered by 14b, Annexin-V/propidium iodide (PI) staining assay was conducted³⁷. In such a procedure, compound 14b at a concentration of 4.61 µM was applied on HepG2 cells for 48 h. As shown in Table 4 and Figure 7, the apoptotic effect of 14b in HepG2 cells was about twenty-four times more than observed in control cells. In detail, compound 14b induced apoptosis by 16.52%, compared to 0.67% in the control cells.

Figure 7. Compound 14b induced apoptosis in HepG2 cells. (A) Control (HepG2 cells), (B) 14b, and (C) Represent the graphical illustration for % of apoptotic and necrotic cells among cells among control (HepG2) cells and compound 14b treated cells. *Significant from the control group at p < 0.001.

Table 4. Compound 14b induced apoptosis in HepG2 cells.

Comp. ID	Apoptosis	Necrosis
	Total	Total
Control	0.67 ± 0.02	0.82 ± 0.074
14b	16.52 ± 1.35	8.07 ± 0.82

Percentages are reported as mean ± SEM of three different experiments. *P < 0.001 indicates a statistically significant difference from the corresponding control (HepG2) group.

2.2.6. Caspase-3 determination

To investigate the effect of the synthesised compounds on caspase-3 level, the most promising member 14b was applied on the most sensitive cells (HepG2) at a concentration of 4.61 µM for 48 h. The results revealed that compound 14b produced a significant increase in the level of caspase-3 (4.8-fold) compared to the control (HepG2) cells (Figure 8).

Figure 8. Effects of compound 14b on Caspase 3 level in HepG2 cells. Values are reported as mean ± SEM of three different experiments. *p < 0.001 indicates statistically significant differences from the control (HepG2) group.

2.3. Docking study

A docking study was carried out in the hope of getting an insight into the mode of interaction of the synthesised compounds to their biomolecular targets^38–40. Thus, VEGFR-2 kinase domain crystal structure PDB ID: 2OH4 in complex with a benzimidazole-urea inhibitor was adopted for the current study. After protonation and preparation of the protein, the validity of the used docking protocol was checked by redocking of the bound benzimidazole-urea inhibitor. The redocking validation step successfully regenerated the experimental binding pattern of the co-crystallized ligand with high efficiency. The docking pose reproduced the key interactions accomplished by the co-crystallized ligand in the active site via binding with Cys917 in the hinge region, Asp1044 of the DFG motif, and Glu883 in the α-C helix. The reproduced binding mode in addition to the small RMSD (0.71 Å) between the docked pose and the co-crystallized ligand proved the effectiveness of the adopted protocol for the planned docking study (Figure 9).

Figure 9. Superimposition of the co-crystallized molecule (mint green) and the docking pose (red) of the same molecule inside the VEGFR-2 kinase active site.

Sorafenib, a potent VEGFR-2 inhibitor used in the experimental in vitro assays, was used as a reference in the docking study as well. Sorafenib interacted by its urea NH groups with the carboxylate side chain of Glu883 in the α-C helix through H-bond interactions. While the urea carbonyl group was involved in an H-bond interaction with the NH group of Asp1044 of the DFG motif. On the hinge region, sorafenib was found to interact by an H-bond with Cys917. Sorafenib interacted, furthermore, via several hydrophobic interactions with the hydrophobic pocked formed by Lys886, Val897, Ile886, Phe1045, and Cys917 (Figure 10).

Figure 10. 3D representation of sorafenib with VEGFR-2 active site.

Investigation of the docking results revealed that the synthesised compounds were able to identify the VEGFR-2 kinase ATP binding site and interact with key amino acids thereof in a manner like that of sorafenib. The studied compounds all occupied the same orientation of sorafenib in the VEGFR-2 kinase active pocket. Thus, as displayed in Figures 11–13, the benzoxazole moieties of the designed compounds 14b, 14n, and 14l were oriented towards the hinge region of the active site forming an H-bond between their nitrogen and Cys917 residue. On the other side, the benzamide scaffolds of the titled compounds were accommodated in the pocket central area, the gate area, interacting via one H-bond with the carboxylate side chain of Glu883 and another one with the NH moiety of Asp1044 of the conserved DFG motif in VEGFR-2. However, the orientation of the later moieties allowed the compounds’ hydrophobic substituents to fit in the hydrophobic allosteric pocket in the active site permitting these hydrophobic substituents to interact with hydrophobic side chains of Ile886, Leu887, Ile890, Val896, Val897, Leu1017, and Ile1042 residues lining the back pocket of VEGFR-2.

Figure 11. The predicted binding pattern of 14b with the active site of VEGFR-2.

Figure 12. The predicted binding pattern of 14n with the active site of VEGFR-2.

Figure 13. The predicted binding pattern of 14l with the active site of VEGFR-2.

2.4. In silico ADME analysis

Results of ADME analysis were illustrated in the Supplementary Data.

2.5. Toxicity studies

The toxicity profiles of all the tested compounds were examined. This involves using seven constructed toxicity models (illustrated in Table 5) utilising Discovery studio 4.0 software (Supplementary Data).

3. Conclusion

A new series of benzoxazole derivatives was designed hoping to discover novel VEGFR-2 inhibitor agents. Fifteen compounds were synthesised and tested in vitro for their anti-proliferative activities against two human cancer cell lines, MCF-7 and HepG2. The tested members exhibited a promising cytotoxic effect with IC₅₀ values ranging from 3.22 ± 0.13 to 32.53 ± 1.97 µM. Amongst, six compounds were further investigated for their in vitro effect against VEGFR-2 enzyme level. Compounds 14o, 14l, 14b and showed the highest effect with a VEGFR-2 protein concentration of 586.3, 636.2, and 705.7 pg/ml, respectively. Also, compound 14b reduced HUVEC cells proliferation and migratory potential. Moreover, Caspase-3 activation assay was performed for compound 14b on HepG2 cells. It produced a significant increase in the level of caspase-3 (4.8-fold) compared to the control HepG2 cells. Furthermore, Compound 14b arrested the cell cycle in the Pre-G1 phase with induction of apoptosis in HepG2 cells. Besides, different in silico studies including docking, ADMET, and toxicity were performed. However, the in silico studies supported the previous results via prediction of the possible binding interactions of the designed compounds with the VEGFR-2 active site.

4. Materials and methods

4.1. Chemistry

4.1.1. General

All the reagents, chemicals, apparatus were described in Supplementary Data. Compounds 8a–c, 9a–c, 11, 12, and 13a–e were obtained according to the reported procedures^14,33,34.

4.1.2. General procedure for preparation of the target compounds 14a-o

A mixture of potassium salts 9a–c (0.001 mol) and the appropriate 4–(2-chloroacetamido)-N-(substituted) phenyl benzamide 13a–e (0.001 mol), and KI (0.001 mol) in DMF (10 ml) was heated on a water bath for 6 h. After completion of the reaction, the mixture was poured on crushed ice. The precipitates were filtered, dried, and crystallised from methanol to afford the corresponding target compounds 14a–o.

4.1.2.1. 4-(2-(Benzo[d]oxazol-2-ylthio)acetamido)-N-(2-methoxyphenyl)benzamide 14a

Yellow powder (yield, 70%); m. p. = 267–269 °C; HPLC purity 97.50%; IR (KBr, cm⁻¹): 3273, 3183 (NH), 3096, 3049 (CH aromatic) 2960, 2845 (CH aliphatic), 1674, 1702 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 9.35 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 8.3 Hz, 2H), 7.69 − 7.63 (m, 2H), 7.37 − 7.32 (m, 2H), 7.18 (t, J = 7.9 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 4.47 (s, 2H), 3.85 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.96, 164.76, 164.33, 151.81 (d, J = 2.7 Hz), 142.16, 141.68, 129.72, 129.06, 127.38, 126.02, 125.18, 124.86, 124.59, 120.68, 118.97, 118.74, 111.78, 110.73, 56.17, 37.31; MS (m/z) for C₂₃H₁₉N₃O₄S (433.48): 433 (base peak, 100%).

4.1.2.2. 4-(2-((5-Chlorobenzo[d]oxazol-2-yl)thio)acetamido)-N-(2-methoxyphenyl)benzamide 14b

Yellow powder (yield, 72%); m. p. = 262–264 °C; HPLC purity 97.55%; IR (KBr, cm⁻¹): 3272 (NH), 3099, 3043 (CH aromatic) 2946, 2865 (CH aliphatic), 1675 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 9.35 (s, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.82 − 7.79 (m, 1H), 7.76 (d, J = 3.0 Hz, 2H), 7.74 − 7.69 (m, 2H), 7.38 (dd, J = 8.7, 2.1 Hz, 1H), 7.20 − 7.16 (m, 1H), 7.10 (d, J = 8.2 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 4.48 (s, 2H), 3.85 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.42, 165.75, 164.74, 151.81, 150.62, 142.96, 142.10, 129.74, 129.47, 129.05, 127.37, 126.02, 124.79, 124.60, 120.67, 118.97, 118.52, 112.02, 111.79, 56.18, 37.42.

4.1.2.3. N-(2-Methoxyphenyl)-4–(2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamido)benzamide 14c

Yellowish white crystal (yield, 74%); m. p. = 258–260 °C; HPLC purity 100.00%; IR (KBr, cm⁻¹): 3273, 3188 (NH), 3054, 3049 (CH aromatic) 2948, 2849 (CH aliphatic), 1703, 1658 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 9.35 (s, 1H), 7.99 − 7.97 (m, 2H), 7.81 (dd, J = 7.9, 1.7 Hz, 1H), 7.77 − 7.74 (m, 2H), 7.53 (d, J = 8.3 Hz, 1H), 7.45 − 7.43 (m, 1H), 7.20 − 7.14 (m, 2H), 7.13 − 7.10 (m, 1H), 7.00 − 6.96 (m, 1H), 4.45 (s, 2H), 3.85 (s, 3H), 2.40 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.99, 164.75, 164.18, 151.79, 150.09, 142.16, 141.88, 134.58, 129.72, 129.04, 127.39, 126.00, 125.65, 124.55, 120.68, 118.96, 118.67, 111.78, 110.10, 56.18, 37.29, 21.40.

4.1.2.4. 4-(2-(Benzo[d]oxazol-2-ylthio)acetamido)-N-(2,6-dimethoxyphenyl)benzamide 14d

White crystal (yield, 65%); m. p. = 250–252 °C; IR (KBr, cm⁻¹): 3276, 3193 (NH), 3054 (CH aromatic) 2937 (CH aliphatic), 1653 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.79 (s, 1H), 9.41 (s, 1H), 8.00 (s, 1H), 7.76 (s, 2H), 7.67 (s, 2H), 7.40 (s, 1H), 7.36 (s, 1H), 7.35 (s, 2H), 7.23 − 7.18 (m, 1H), 6.96 − 6.84 (m, 1H), 4.47 (s, 2H), 4.06 (s, 3H), 3.99 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.98, 164.78, 164.33, 151.90, 151.83, 142.21, 141.68, 129.12, 126.84, 125.20, 124.87, 124.46, 119.00, 118.75, 110.75, 110.11, 56.46, 37.31.

4.1.2.5. 4-(2-((5-Chlorobenzo[d]oxazol-2-yl)thio)acetamido)-N-(2,6-dimethoxyphenyl)-benzamide 14e

Yellowish powder (yield, 68%); m. p. = 248–250 °C; HPLC purity 95.44%; IR (KBr, cm⁻¹): 3285 (NH), 2936, 2854 (CH aliphatic), 1769, 1664 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.79 (s, 1H), 9.41 (s, 1H), 8.02 (s, 2H), 7.93 (d, J = 8.2 Hz, 2H), 7.78 − 7.77 (m, 2H), 7.71 (s, 1H), 7.40 (s, 2H), 7.38 (d, J = 2.1 Hz, 1H), 4.48 (s, 2H), 4.00 (s, 3H), 3.99 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.42, 165.78, 164.77, 151.91, 150.63, 142.96, 142.15, 137.75, 129.70, 129.48, 129.11, 124.81, 124.48, 118.99, 118.53, 112.04, 110.10, 56.45, 37.40.

4.1.2.6. N-(2,6-Dimethoxyphenyl)-4–(2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamido)-benzamide 14f

Greenish white crystal (yield, 72%); m. p. = 270–272 °C; HPLC purity 97.33%; IR (KBr, cm⁻¹): 3268 (NH), 3069 (CH aromatic) 2930, 2855 (CH aliphatic), 1654 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 9.40 (s, 1H), 7.95 (s, 2H), 7.76 (s, 2H), 7.52 (s, 2H), 7.34 (s, 2H), 7.13 (s, 1H), 6.67 (d, J = 65.9 Hz, 1H), 4.45 (s, 2H), 3.98 (s, 6H), 2.40 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.01, 164.77, 151.88, 150.09, 142.21, 141.88, 137.73, 134.59, 129.11, 126.84, 125.65, 124.43, 118.99, 118.68, 110.10, 56.45, 37.30, 21.41.

4.1.2.7. 4-(2-(Benzo[d]oxazol-2-ylthio)acetamido)-N-(2,6-dimethylphenyl)benzamide 14g

White crystal (yield, 75%); m. p. = 266–268 °C; HPLC purity 96.25%; IR (KBr, cm⁻¹): 3262 (NH), 3061 (CH aromatic) 2928, 2856 (CH aliphatic), 1651 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 9.70 (s, 1H), 8.07 (dd, J = 9.3, 2.4 Hz, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.55 (t, J = 6.8 Hz, 1H), 7.35 (dd, J = 6.3, 2.7 Hz, 2H), 7.13 (s, 2H), 4.46 (s, 2H), 2.19 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.92, 164.84, 164.33, 151.82, 142.01, 141.68, 136.14, 135.89, 129.67, 129.05, 128.18, 127.09, 125.20, 124.87, 118.96, 118.75, 110.75, 37.27, 18.57; MS (m/z) for C₂₄H₂₁N₃O₃S (431.51): 431 (base peak, 100%).

4.1.2.8. 4-(2-((5-Chlorobenzo[d]oxazol-2-yl)thio)acetamido)-N-(2,6-dimethylphenyl)benzamide 14h

White powder (yield, 72%); m. p. = 262–264 °C; HPLC purity 95.37%; IR (KBr, cm⁻¹): 3278, 3225 (NH), 3022 (CH aromatic) 2979, 2918, 2859 (CH aliphatic), 1656 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.78 (s, 1H), 9.71 (s, 1H), 8.03 (s, 2H), 7.76 (s, 3H), 7.38 (s, 2H), 7.13 (s, 3H), 4.49 (s, 2H), 2.20 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.43, 165.73, 164.86, 150.62, 142.97, 141.98, 136.15, 135.89, 129.71, 129.48, 129.07, 128.18, 127.09, 124.77, 118.99, 118.51, 111.99, 37.43, 18.58.

4.1.2.9. N-(2,6-Dimethylphenyl)-4–(2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamido)benzamide 14i

Yellowish green crystal (yield, 68%); m. p. = 263–265 °C; HPLC purity 96.77%; IR (KBr, cm⁻¹): 3256, 3106 (NH), 2923, 2861 (CH aliphatic), 1761, 1641 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.76 (s, 1H), 9.70 (s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.3 Hz, 1H), 7.44 (s, 1H), 7.17 − 7.14 (m, 2H), 7.13 (s, 2H), 4.44 (s, 2H), 2.41 (s, 3H), 2.19 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.95, 164.85, 164.19, 150.08, 142.01, 141.88, 136.14, 135.88, 134.59, 129.66, 129.05, 128.18, 127.09, 125.66, 118.95, 118.67, 110.11, 37.25, 21.41, 18.57.

4.1.2.10. 4-(2-(Benzo[d]oxazol-2-ylthio)acetamido)-N-(2,5-dichlorophenyl)benzamide 14j

White powder (yield, 74%); m. p. = 265–267 °C; HPLC purity 97.14%; IR (KBr, cm⁻¹): 3269, 3186 (NH), 3058 (CH aromatic) 2992, 2936 (CH aliphatic), 1659 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.80 (s, 1H), 10.06 (s, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.78 (s, 2H), 7.76 (s, 1H), 7.68 − 7.64 (m, 2H), 7.61 (d, J = 8.6 Hz, 1H), 7.40 − 7.38 (m, 1H), 7.36 − 7.32 (m, 2H), 4.47 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.03, 165.24, 164.32, 151.82, 142.55, 141.67, 136.95, 131.90, 131.36, 129.43, 128.79, 128.23, 127.95, 127.42, 125.19, 124.87, 118.96, 118.75, 110.75, 37.29; MS (m/z) for C₂₂H₁₅Cl₂N₃O₃S (472.34): 471 (base peak, 100%), 473 (M⁺ + 1, 70%).

4.1.2.11. 4-(2-((5-Chlorobenzo[d]oxazol-2-yl)thio)acetamido)-N-(2,5-dichlorophenyl)-benzamide 14k

Off white powder (yield, 70%); m. p. = 259–261 °C; HPLC purity 99.13%; IR (KBr, cm⁻¹): 3271, 3187 (NH), 3059 (CH aromatic) 2977, 2939 (CH aliphatic), 1664 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.81 (s, 1H), 10.05 (s, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.77 (s, 2H), 7.75 (s, 2H), 7.71 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.37 (dd, J = 8.7, 4.0 Hz, 2H), 4.47 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.40, 165.83, 165.23, 150.62, 142.96, 142.51, 136.93, 131.90, 131.35, 129.48, 129.43, 128.81, 128.22, 127.93, 127.41, 124.79, 118.97, 118.52, 112.02, 37.41; MS (m/z) for C₂₂H₁₄Cl₃N₃O₃S (506.78): 506 (M^+, 36%), 345 (base peak, 100%).

4.1.2.12. N-(2,5-Dichlorophenyl)-4–(2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamido)-benzamide 14l

White powder (yield, 76%); m. p. = 255–257 °C; HPLC purity 100.00%; IR (KBr, cm⁻¹): 3274, 3182 (NH), 3052 (CH aromatic), 2953 (CH aliphatic), 1699, 1660 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 9.36 (s, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.80 (s, 1H), 7.64 (d, J = 7.9 Hz, 2H), 7.38 − 7.31 (m, 2H), 7.17 (d, J = 7.8 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 6.98 (t, J = 7.7 Hz, 1H), 4.48 (s, 2H), 3.85 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.98, 164.78, 164.33, 151.82, 151.80, 142.22, 141.69, 129.69, 129.03, 127.38, 126.01, 125.17, 124.84, 124.58, 120.67, 118.98, 118.74, 111.77, 110.72, 56.17, 37.30.

4.1.2.13. 4-(2-(Benzo[d]oxazol-2-ylthio)acetamido)-N-(4-hydroxyphenyl)benzamide 14m

Brownish powder (yield, 60%); m. p. = 280–282 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.74 (s, 1H), 9.95 (s, 1H), 9.27 (s, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.9 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.37 − 7.31 (m, 2H), 6.75 (d, J = 8.3 Hz, 2H), 4.46 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.91, 164.74, 164.33, 154.11, 151.82, 141.85, 141.67, 131.21, 130.38, 129.05, 125.18, 124.85, 122.76, 118.83, 118.74, 115.43, 110.73, 37.29.

4.1.2.14. 4-(2-((5-Chlorobenzo[d]oxazol-2-yl)thio)acetamido)-N-(4-hydroxyphenyl)benzamide 14n

Brownish white powder (yield, 62%); m. p. = 272–274 °C; HPLC purity 94.00%; IR (KBr, cm⁻¹): 3267 (NH), 3067 (CH aromatic), 2930 (CH aliphatic), 1649 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.75 (s, 1H), 9.96 (s, 1H), 9.28 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.75 (s, 1H), 7.73 (s, 2H), 7.67 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.7 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 4.48 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.41, 165.71, 164.75, 154.12, 150.60, 142.95, 141.81, 131.22, 130.41, 129.47, 129.05, 124.73, 122.79, 118.86, 118.49, 115.45, 111.93, 37.46.

4.1.2.15. N-(4-Hydroxyphenyl)-4–(2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamido)benzamide 14o

Yellowish crystal (yield, 65%); m. p. = 269–271 °C; HPLC purity 95.16%; IR (KBr, cm⁻¹): 3264 (NH), 3043 (CH aromatic), 2926 (CH aliphatic), 1634 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 10.75 (s, 1H), 9.96 (s, 1H), 9.28 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.3 Hz, 1H), 7.42 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.77 (d, J = 8.3 Hz, 2H), 4.44 (s, 2H), 2.39 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.41, 165.71, 164.75, 154.12, 150.60, 142.95, 141.81, 131.22, 130.41, 129.47, 129.05, 124.73, 122.79, 118.86, 118.49, 115.45, 111.93, 37.46.

4.2. Biological evaluation

4.2.1. In vitro anti-proliferative activity

The anti-proliferative activity of all tested compounds was performed on MCF-7 and HepG2 cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay^35,41–44. The MTT assay is based on the capability of living cells to reduce the yellow product MTT to a blue product, formazan, by a reduction reaction occurring in the mitochondria. Briefly, in MTT assay, 5000 cells/well were plated in a 96-well plate and allowed to grow 24 h, then treated with Roswell Park Memorial Institute (RPMI) 1640 media that contain increased concentrations (0, 0.1, 1, 10, 100, and 1000 µM) of tested compounds. Each experiment was carried out in triplicate. Then media were removed and 100 µL of MTT was added to each well and incubated for 4 h. The formed formazan crystals were solubilised by adding 100 µL of dimethyl sulfoxide (DMSO) solution and absorbance was measured at 570 nm using ELISA microplate reader (Epoc-2 C micro-plate reader, Bio Tek, VT, USA). The IC₅₀ values [the concentration required for 50% inhibition of cell viability] were calculated and the results are expressed as the relative percentage of the control cells (100% of cell viability).

4.2.2. In vitro VEGFR-2 protein concentration assay

The in vitro assessment of VEGFR-2 protein concentration after exposure of HepG-2 cells to the most cytotoxic candidates was carried out using Enzyme-Linked Immunosorbent Assay (ELISA) kit (Cat. NO. EK0544) (AVIVA System Biology, USA) according to manufacturer instructions⁴⁵.

4.2.3. Wound healing assay

Allow 10 min for the 24-well plate with CytoSelect™ Wound Healing Inserts to warm up at room temperature before applying 500 ml of HUVECs cell suspension (1.0 × 10⁶) in media containing 10% foetal bovine serum (FBS) to each well. In a cell culture incubator, incubate the cells until they form a monolayer. Remove the implant from the well gently to begin the wound healing assay. Aspirate the media from the wells slowly and discard it.

To eliminate dead cells and debris, wash wells with the medium. Finally, fill wells with medium to keep cells hydrated, and examine them under a light microscope. The wells were subsequently filled with media containing the indicated concentrations of compound 14b or sorafenib for 72 h. A light microscope is used to monitor the wound closure. Calculate the percentage of cells that have closed into the wound field⁴⁶.

4.2.4. Analysis of the cell cycle distribution phases

The propidium iodide staining followed by flow cytometric analysis was conducted according to the cell cycle kit (PN C03551) and previously published works^43,47 to investigate the effect of compound 14b on the cell cycle phases. In Brief, HepG2 cells were allowed to grow in 25 cm³ flask until reach 70–80% confluence, then treated with compound 14b for 48 h. Then the cells were harvested and fixed. The cells were centrifuged at 2000 rpm for 5 min then, the supernatant was aspirated. The pellet of fixed cells was resuspended in a 0.5 ml cell cycle kit, vortexed, and incubated at 25 °C for 15 min. Finally, DNA was stained with 50 µg/ml propidium iodide for 30 min. Flow cytometric analysis of cell cycle performed on a COULTER^® EPICS^® XL™ Flow Cytometer (USA).

4.2.5. Annexin V-FITC apoptosis assay

For the detection of apoptosis in treated cells, Annexin V—FITC—apoptosis detection kit (PN IM3546) was used, followed by flow cytometric analysis according to manufacturer protocol. In this assay, HepG-2 cells were allowed to grow in a 25 cm³ flask until 70–80% confluence. Then HepG-2 cells were treated with compound 14b for 48 h followed by a wash in PBS and suspended in 1× binding buffer. To 100 µL of the cell suspensions, 1 µL of annexin V-FITC solution and 5 µL of dissolved PI were added and incubated for 15 min in the dark. Then 400 µL of ice-cold 1× binding buffer was added and mixed gently. The flow cytometric analysis for the percentage of apoptotic cells was performed on a COULTER^® EPICS^® XL™ Flow Cytometer (USA)^42,48.

4.2.6. Caspase-3 determination

The effect of compound 14b on Caspase-3 level was assessed using ELISA kit (Catalog # KHO1091) according to manufacturer instructions.

4.3. Molecular docking studies

Molecular docking studies of synthesised compounds were carried out against VEGFR-2 (PDB ID: 2OH4, resolution: 2.05 Å) using MOE.14 software^27,49–54 as shown in Supplementary Data.

Acknowledgements

The authors extend their appreciation to the Research Center at AlMaarefa University for funding this work under TUMA project number “TUMA-2021-4”.

Disclosure statement

No potential conflict of interest was reported by the authors.

Correction Statement

This article was originally published with errors, which have now been corrected in the online version. Please see Correction (10.1080/14756366.2022.2024999).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.