Design, synthesis, and apoptotic antiproliferative action of new 1,2,3-triazole/1,2,4-oxadiazole hybrids as dual EGFR/VEGFR-2 inhibitors

Abstract

A novel series of 1,2,3-triazole/1,2,4-oxadiazole hybrids (7a–o) was developed as dual inhibitors of EGFR/VEGFR-2. Compounds 7a–o were evaluated as antiproliferative agents with Erlotinib as the reference drug. Results demonstrated that most of the tested compounds showed significant antiproliferative action with GI₅₀ values ranging from 28 to 104 nM, compared to Erlotinib (GI₅₀ = 33 nM), and compounds 7i–m were the most potent. Compounds 7h, 7i, 7j, 7k, and 7l were evaluated as dual EGFR/VEGFR-2 inhibitors. These in vitro experiments demonstrated that compounds 7j, 7k, and 7l are potent antiproliferative agents that may operate as dual EGFR/VEGFR-2 inhibitors. Compounds 7j, 7k, and 7l were evaluated for their apoptotic potential activity, where findings indicated that compounds 7j, 7k, and 7l promote apoptosis by activating caspase-3, 8, and Bax and down-regulating the anti-apoptotic Bcl-2. Molecular docking simulations show the binding mode of the most active antiproliferative compounds within EGFR and VEGFR-2 active sites.

Keywords:

• Triazole
• oxadiazole
• EGFR
• VEGFR-2
• apoptosis
• antiproliferative

Introduction

Despite enormous efforts over the last two decades to find effective therapies, cancer remains a menace to the globe today¹. While conventional cancer treatments cannot distinguish between healthy and malignant cells, targeted therapy kills cancer cells while preserving healthy cells by disrupting critical metabolic pathways or oncoproteins required for tumour cell growth and survival^1,2.

Protein tyrosine kinases are important in signal transduction pathways that govern cellular processes such as proliferation, differentiation, migration, and angiogenesis^3,4. The epidermal growth factor receptor (EGFR) is a type of membrane receptor tyrosine kinase that is overexpressed in various tumours. Because EGFR tyrosine kinase signal transduction is closely linked to tumour progression, inhibiting receptor activity can effectively inhibit tumours^5–7. Another significant receptor tyrosine kinase that can induce angiogenesis is vascular endothelial growth factor receptor (VEGFR-2)⁸. VEGFR-2, a VEGFR family member, is a major mediator in tumour angiogenesis and is essential for solid tumour formation. Inhibiting VEGFR-2 has been proposed as an effective method for preventing angiogenesis^9,10.

VEGFR-2 and EGFR have been identified as promising therapeutic targets for cancer treatment. They are essential in the signalling pathways that regulate tumour cell proliferation, differentiation, migration, and angiogenesis^11–14. EGFR and VEGFR-2 typically share common downstream signalling pathways as a complex signal network of interconnected circuits. EGFR inhibition can reduce VEGF expression and attenuate angiogenesis while increasing VEGFR-2 expression, eventually leading to EGFR inhibitor resistance^15,16. As a result, inhibiting both EGFR and VEGFR-2 simultaneously has become an effective cancer therapeutic strategy with a synergistic impact^17–19.

The 1,2,4-oxadiazole heterocycle has been widely studied over the last four decades, yielding many analogs with diverse biological effects. Due to the potential for particular interactions (such as hydrogen bonding), the 1,2,4-oxadiazole achieves bioisosteric equivalence with amide and ester moieties^20,21. The 1,2,4-oxadiazole nucleus is the basic core of a number of drugs on the market today, including Oxolamine, Butalamine, Prenoxdiazine, Fasiplon, and Proxazole^22–24. A new series of 1,2,4-oxadiazole-based compounds was developed and tested as anticancer agents that target the EGFR inhibitory pathway. The optimal derivative was compound I (Figure 1), demonstrating equipotent antiproliferative action against a panel of five NSCLC cell lines (IC₅₀ = 0.2–0.6 µM). Cell cycle investigations demonstrated that I's antiproliferative activity is linked to its ability to cause G2/M arrest and, to a lesser extent, apoptosis²⁵.

Figure 1. Structure of anticancer 1,2,4-oxadiazoles I–III.

Saritha et al.²⁶ reported on the design, synthesis, and antiproliferative efficacy of a new series of molecular hybrids containing benzimidazole, thiazolidine-2,4-dione, and 1,2,4-oxadiazole scaffolds against three human cancer cell lines, MCF-7, A-549, and HepG2. Compound (II, Figure 1) demonstrated greater activity against all cell lines, with the highest activity against MCF-7 (IC₅₀ = 1.32 ± 0.05 µM) as compared to the reference Erlotinib (IC₅₀ = 4.15 ± 0.12 µM). Similarly, new isatin-1,2,4-oxadiazole hybrids were produced and tested for cytotoxicity against four human cancer cell lines. Their potential VEGFR-2 inhibition assay was also investigated, with compound (III, Figure 1) having nearly double the inhibitory potency as Sorafenib, with IC₅₀ = 35.64 ± 1.56 nM against VEGFR-2 enzyme assay.

In a recent publication²⁷, we describe the discovery of a new series of 1,2,3-triazole-based antiproliferative agents, compounds 6a–o (Figure 2). The newly synthesised compounds were tested for antiproliferative activity against a panel of four cancer cell lines, with compound 6k (R = 3,4-di-OMe) being the most potent derivative with a GI₅₀ value of 31 nM against the tested four cancer cell lines, outperforming the reference Erlotinib (GI₅₀ = 33 nM). The EGFR and VEGFR-2 inhibitory assay results showed that compound 6k (R = 3,4-di-OMe), the most potent antiproliferative agent, was also the most potent anti-EGFR and anti-VEGFR-2 agent, with IC₅₀ values of 83 ± 05 and 1.80 ± 0.05 nM, respectively, compared to Erlotinib (IC₅₀ = 80 ± 4 nM) and Sorafenib (IC₅₀ = 0.17 ± 0.01 nM).

Figure 2. Structures of previously reported compounds 6a–o and newly synthesised 7a–o.

Motivated by previous findings and in pursuit of a new antiproliferative agent with dual or multi-targeted inhibitory action^27–30, we present here the design, synthesis, and antiproliferative activity of a novel series of 1,2,3-triazole/1,2,4-oxadiazole hybrids (7a–o, Figure 2) as dual EGFR/VEGFR-2 inhibitors. The newly designed 1,2,4-oxadiazoles 7a–o were considered to be sterically fixed counterparts of the previously discovered linear carboximidamide derivatives 6a–o (Figure 2). Such a rigid conformation may result in tight fitting within the EGFR and VEGFR-2 active sites, increasing the efficacy of newly synthesised compounds. The antiproliferative effect of compounds 7a–o against four cancer cell lines was investigated using Erlotinib as the reference drug. Next, the most effective compounds were evaluated for inhibitory action against EGFR and VEGFR-2, which were identified as possible targets for their action. In addition, the apoptotic potential of the most active compounds was assessed. Finally, docking analysis and ADME studies were performed for the most potent derivatives.

Results and discussion

Chemistry

Scheme 1 outlines the synthetic steps for the target compounds 7a–o. Compound 1 was hydrolysed in aqueous NaOH (20%) and refluxed for two hours to produce compound 2³¹. On the other hand, compounds 4a–o were prepared according to a reported procedure in THF via an iodine-catalyzed transformation of the formyl group in 3a–o to a cyano group utilising ammonia as a nitrogen source³². Amidoximes 5a–o were prepared in THF by nucleophilic attack of hydroxylamine at the cyanide carbon of 4a–o³³. Aryl carboximidamides 6a–o were prepared by reacting carboxylic acid 2 with the appropriate amidoximes 5a–o in dry acetonitrile using the activating agent N,N'-carbonyl diimidazole (CDI)²⁷.

Scheme 1. Synthetic pathway of compounds 7a–o

Reagents and reaction conditions: (a) 20% aq. NaOH, reflux, 2 h, 80%; (b) 33% aq. NH₃, THF, I₂, r.t 3–5 h, 40–85%; (c) NH₂OH·HCl, Na₂CO₃, methanol, reflux, 6–8 h; (d) CDI, dry CH₃CN, r.t 10 h; (e) CH₃CN, reflux, overnight, 46–90%.

The target compounds 7a–o were synthesised by refluxing the appropriate carboximidamides 6a–o in dry acetonitrile. After the reaction was completed (as monitored by TLC), the excess solvent evaporated under reduced pressure. The precipitates were washed with cold water and filtered, crystalised from acetonitrile to yield 1,2,4-oxadiazoles 7a–o in good yields.

The structures of new compounds 7a–o were elucidated using NMR, IR, and elemental microanalysis. FTIR spectra of target compounds 7a–o confirmed the disappearance of (NH₂) and (C = O) peaks of the precursor aryl carboximidamides 6a–o. The presence of (CH) peaks at 3144, 3001, 2965 cm⁻¹, (C = N and C = C) peaks at 1602, 1579, 1468 cm⁻¹, and (Ar–CH bending) at 856 cm⁻¹ in the FTIR spectrum of compound 7g as a representative example verified the prior observations.

Also,¹H NMR spectra proved the cyclisation reaction by revealing the absence of amino (NH₂) wide signals in compounds 6a–e and the appearance of (N–CH₂) signals at (δ = 6.37–6.14 ppm) compared with compounds’ 6a–o (N–CH₂) signals that appeared in the range of (δ = 6.66–5.61 ppm). ¹H NMR spectrum of compound 7g confirmed the elucidated structure by showing a singlet triazole C–H signal at δ = 8.82 ppm, and nine aromatic protons appeared as five distinct signals in the aromatic region. Moreover, ¹³C NMR spectra confirmed the cyclisation reaction by the disappearance of the C = O signal of their precursors 6a–o at δ = 166 ppm and the appearance of (N–CH₂) signal at (δ = 45.6–45 ppm). ¹³C NMR spectrum of compound 7g confirmed the elucidated structure by the appearance of 12 aromatic signals at δ = 175.1–122.9, CF₃ quartette signal at δ = 124 with J = 272.7 Hz, and N–CH₂ signal as δ = 45.3 ppm.

Biology

Cell viability assay

A cell viability experiment was performed on the MCF-10A (normal human mammary gland epithelial) cell line to assess the effect of compounds 7a–o on normal cell lines^34,35. Before testing cell viability, 50 µM of the examined compound is used in this investigation for four days. Compounds 7a–o have no cytotoxic impact and are greater than 87% cell viability, as shown in Table 1.

Table 1. Antiproliferative activity of compounds 7a–o and Erlotinib.

Comp.	Cell viability% (50 µM)	R	Antiproliferative activity IC50 ± SEM (nM)
			A-549	MCF-7	Panc-1	HT-29	Average (GI50)
7a	90	H	94 ± 9	98 ± 9	96 ± 9	96 ± 9	96
7b	91	4-F	102 ± 10	106 ± 10	104 ± 10	104 ± 10	104
7c	89	4-Cl	87 ± 8	94 ± 9	90 ± 9	92 ± 9	90
7d	88	4-Br	80 ± 8	84 ± 8	80 ± 8	82 ± 8	82
7e	91	4-CH3	98 ± 9	102 ± 9	100 ± 10	102 ± 10	101
7f	90	4-OMe	48 ± 4	51 ± 5	48 ± 4	46 ± 4	48
7g	87	4-CF3	66 ± 6	68 ± 6	68 ± 6	66 ± 6	67
7h	90	3-Cl	40 ± 4	44 ± 4	40 ± 4	42 ± 4	42
7i	89	3-Br	36 ± 3	39 ± 3	37 ± 3	37 ± 3	37
7j	91	2,4-Di-Cl	33 ± 3	36 ± 3	34 ± 3	36 ± 3	35
7k	89	3,4-Di-OMe	30 ± 3	35 ± 3	32 ± 3	32 ± 3	32
7l	88	3,4,5-Tri-OMe	26 ± 2	30 ± 3	28 ± 2	28 ± 2	28
7m	87	2,3-C4H4	42 ± 4	48 ± 4	46 ± 4	44 ± 4	45
7n	89	3,4-C4H4	68 ± 6	74 ± 7	70 ± 7	72 ± 7	71
7o	91	1,3-Dioxole	74 ± 7	80 ± 8	78 ± 7	76 ± 7	77
Erlotinib	–	–	30 ± 3	40 ± 3	30 ± 3	30 ± 3	33

Antiproliferative assay

The antiproliferative activity of 1,2,4-oxadiazoles 7a–o was evaluated against four different human cancer cell lines, namely, colon cancer (HT-29) cell line, lung cancer (A-549) cell line, pancreatic cancer (Panc-1) cell line, and breast cancer (MCF-7) cell line using Erlotinib as the reference drug^36,37. The median inhibitory concentration (IC₅₀) is shown in Table 1. The results showed that the tested compounds 7a–o had promising antiproliferative action with GI₅₀ values ranging from 28 to 104 nM. The six most active derivatives were 7h–m, with GI₅₀ values ranging from 28 to 45 nM, compared to Erlotinib’s 33 nM.

Compound 7l (R = 3,4,5-tri-OMe) was the most potent derivative, with a GI₅₀ value of 28 nM against the four cancer cell lines examined, outperforming the reference Erlotinib (GI₅₀ = 33 nM). Compound 7l was shown to be more potent than Erlotinib against all four human cancer cell lines examined. Compound 7k (R = 3,4-dimethoxy) scored second in activity against the four cancer cell lines with a GI₅₀ value of 32 nM, equipping to the reference Erlotinib (GI₅₀ = 33 nM). Compound 7k was more potent than Erlotinib against the breast cancer (MCF-7) cell line, having an IC₅₀ value of 35 ± 3 nM versus 40 ± 3 nM for Erlotinib. According to the results of compounds 7f (R = 4-OMe) and 7k (R = 3,4-di-OMe), the number of methoxy groups substantially impacts the activity of these compounds. Compounds 7f (R = 4-OMe) and 7k (R = 3,4-di-OMe) were less potent than compound 7l (R = 3,4,5-tri-OMe), indicating that the trimethoxy groups were best suited for antiproliferative action, Figure 3.

Figure 3. GI₅₀ of compounds 7f, 7k, and 7l compared to Erlotinib.

Compound 7j (R = 2,4-di-Cl) was the third most active against the four cancer cell lines, with a GI₅₀ value of 35 nM. Compound 7j was likewise more potent than Erlotinib against the breast cancer (MCF-7) cell line, with an IC₅₀ value of 36 ± 3 nM versus Erlotinib’s IC₅₀ of 40 ± 3 nM. Another key point drawn from the findings in Table 1 is the effect of halogen atom type, position, and number on antiproliferative activity. Compounds 7b (R = 4-F), 7c (R = 4-Cl), 7d (R = 4-Br), 7i (R = 3-Br), and 7h (R = 3-Cl) demonstrated good antiproliferative action with GI₅₀ values ranging from 37 to 104 nM, being less potent than compound 7j (R = 2,4-di-Cl) indicating that the number of halogen atoms had a significant impact on the antiproliferative action of these compounds and that dihalo derivatives are more potent than mono halo substituted derivatives.

Compounds 7i (R = 3-Br) and 7h (R = 3-Cl) demonstrated significant antiproliferative activity, with GI₅₀ values of 37 and 42 nM, respectively, demonstrating that the bromine atom is better tolerated for antiproliferative action than the chlorine atom. Furthermore, compounds 7c (R = 4-Cl) and 7d (R = 4-Br) had weak antiproliferative activity, with GI₅₀ values of 90 nM and 82 nM, respectively, approximately twice as weak as the 3-halosubstituted derivatives 7i and 7h. Finally, with a GI₅₀ value of 104 nM, the 4-fluoro derivative 7b (R = 4-F) was the least potent of all synthesised derivatives, 2.5-fold less active than the 3-halo-substituted derivatives 7i and 7h. These findings indicated that the type, position, and number of halogen atoms had a significant effect on antiproliferative activity, with activity increasing in the order 2,4-di-Cl > 3-Br > 3-Cl > 4-Br > 4-Cl > 4-F, Figure 4.

Figure 4. GI₅₀ of compounds 7b, 7c, 7d, 7h, 7i, 7j and Erlotinib.

The antiproliferative impact of the 1-naphthyl derivative 7m (R = 2,3-C₄H₄) was promising, with a GI₅₀ value of 45 nM, which was 1.4-fold less potent than the reference Erlotinib (GI₅₀ = 33 nM). On the other hand, the 2-naphthyl derivative 7n (R = 3,4-C₄H₄) demonstrated weak antiproliferative activity with a GI₅₀ value of 77 nM, being 2.4-fold and 1.7-fold less potent than Erlotinib and the 1-naphthyl derivative 7m, respectively.

The unsubstituted derivative, compound 7a (R = H), and the 4-methyl derivative 7e (R = 4-CH₃) were 3-fold less potent than the reference Erlotinib, with GI₅₀ values of 96 and 101 nM, respectively, demonstrating the effect of the substitution pattern on the phenyl ring of the 1,2,4-oxadiazole moiety.

EGFR inhibitory assay

The inhibitory effects of the most potent antiproliferative derivatives 7h–l on EGFR, as a possible target for their antiproliferative action, were investigated using Erlotinib as the reference drug^28,38. Table 2 shows the results as IC₅₀ values.

Table 2. IC₅₀ values of compounds 7h, 7i, 7j, 7k, 7l, Erlotinib and Sorafenib against EGFR and VEGFR-2.

Compd. no.	EGFR inhibition IC50 ± SEM (nM)	VEGFR-2 inhibition IC50 ± SEM (nM)
7h	105 ± 10	6.90 ± 0.07
7i	97 ± 09	5.50 ± 0.05
7j	89 ± 08	4.70 ± 0.04
7k	82 ± 07	3.80 ± 0.03
7 l	76 ± 06	2.40 ± 0.02
Erlotinib	80 ± 05	–
Sorafenib	–	0.17 ± 0.01

–, not determined.

Generally, compounds 7h–l displayed potential anti-EGFR action, with IC₅₀ values ranging from 76 to 105 nM. The results of this cell-based experiment are consistent with those of the antiproliferative assay, in which the most potent antiproliferative agent, 7l (R = 3,4,5-tri-OMe), was also the most potent anti-EGFR, with an IC₅₀ value of 76 ± 06 nM, exceeding the reference Erlotinib, which had an IC₅₀ value of 80 ± 05 nM. Compounds 7k (R = 3,4-dimethoxy) and 7j (R = 2,4-di-Cl) were placed second and third in EGFR inhibitory activity, with IC₅₀ values of 82 and 89 nM, respectively, and were comparable to the reference Erlotinib.

These findings highlighted the significance of the methoxy group and the number of chlorine atoms in antiproliferative and anti-EGFR activities. Compounds 7h (R = 3-Cl) and 7i (R = 3-Br) displayed moderate anti-EGFR activity, with IC₅₀ values of 105 ± 10 and 97 ± 9 nM, respectively, and were found to be less potent than the reference Erlotinib, which had an IC₅₀ value of 80 ± 05. These findings indicated that derivatives 7j, 7k, and 7l, which require additional structural modifications to develop more potent derivatives, may be capable of inhibiting cancer cell proliferation by targeting the EGFR.

VEGFR inhibitory assay

The inhibitory efficacy of compounds 7h–l against VEGFR-2 was determined by applying kinase-glo-luminescent kinase assays with Sorafenib as the control medication³⁹. Table 2 displays the results as IC₅₀ values.

The results showed that the investigated compounds inhibited VEGFR-2 significantly, with IC₅₀ values ranging from 2.40 to 6.90 nM, compared to Sorafenib, which had an IC₅₀ value of 0.17 nM. The most potent derivatives were 7j, 7k, and 7l, with IC₅₀ values of 2.40, 3.80, and 4.70 nM, respectively. Again, compound 7l was the most potent derivative as a VEGFR-2 inhibitor, with an IC₅₀ value of 2.40 ± 0.02 nM. These findings demonstrate that compounds 7j, 7k, and 7l are potent antiproliferative agents that may act as dual EGFR/VEGFR-2 inhibitors.

Apoptosis assay

Cancer can be treated by regulating or stopping the uncontrolled multiplication of cancer cells. Using the cell’s natural dying process is a highly effective method. Apoptosis evasion is a trait of cancer and is not specific to the aetiology or kind of cancer; hence, targeting apoptosis is useful for many types of cancer. Many anticancer drugs target various stages of both the intrinsic and extrinsic pathways ^40–42. Compounds 7j, 7k, and 7 l, the most potent derivatives in all in vitro studies, were tested for their capacity to initiate the apoptosis cascade and reveal their proapoptotic potential.

Caspase-3 activation assay

Caspases are vital for the induction and maintenance of apoptosis. Caspase-3 is an essential caspase that cleaves many cell proteins, causing apoptosis ^43,44. Compounds 7j, 7k, and 7 l were investigated as caspase-3 activators against the human epithelial (A-594) cancer cell line ⁴⁵, and the findings are shown in Table 3.

Table 3. Caspase-3, caspase-8, Bax, and Bcl-2 levels of compounds 7j, 7k, and 7l.

Compd. no.	Caspase-3		Caspase-8		Bax		Bcl-2
	Conc (Pg/mL)	Fold change	Conc (ng/mL)	Fold change	Conc (Pg/mL)	Fold change	Conc (ng/mL)	Fold reduction
7j	460 ± 4	7.0	2.00	22	291	32	0.95	5
7k	530 ± 5	8.0	2.30	25	310	34	0.80	6
7l	587 ± 5	9.0	2.55	28	362	40	0.60	8
Staurosporine	465 ± 4	7.0	1.85	21	288	32	1.00	5
Control	65	1.0	0.09	1	9	1	5.00	1

Compounds 7k and 7l showed a promising increase in caspase-3 protein levels of up to 530 ± 5 and 587 ± 5 pg/mL, respectively. They increased caspase 3 protein levels in the A-594 cancer cell line by 8- and 9-fold compared to untreated control cells. Compounds 7k and 7l were more active than the Staurosporine control, which exhibited 465 ± 4 pg/mL caspase-3 overexpression. Compound 7j was the least active derivative, with a caspase-3 overexpression of 460 ± 4 pg/mL and was comparable to the reference Staurosporine as a caspase-3 level inducer. According to these results, the examined compounds 7j, 7k, and 7l may have apoptotic potential activity, which may account for their antiproliferative effects.

Caspase-8, Bax, and Bcl-2 levels assays

Using Staurosporine as a control, the effects of compounds 7j, 7k, and 7l on the levels of caspase-8, Bax, and the anti-apoptotic protein Bcl-2 against the A-594 cancel cell line were further examined. Table 3 presents the results.

Table 3 demonstrated that compound 7l (2.55 ng/mL) had the highest levels of caspase-8 overexpression, followed by compound 7k (2.30 ng/mL) and finally compound 7j (2.00 ng/mL) when compared to the reference Staurosporine (1.85 ng/mL). The investigated compounds 7j, 7k, and 7l elevated caspase-8 levels by 22, 25, and 28 times, respectively, compared to the untreated control cell.

Furthermore, compounds 7k and 7l boosted Bax induction 34- and 40-fold (310 and 362 pg/mL, respectively) over untreated A-594 cancer cells, outperforming Staurosporine (288 pg/mL, a 32-fold induction), while compound 7j, on the other hand, induces Bax at a level comparable to Staurosporine. They were finally compared to Staurosporine, derivatives 7j, 7k, and 7l significantly reduced anti-apoptotic Bcl-2 protein levels in the A-594 cell line (5-, 6-, and 8-folds, respectively). These data suggest that compounds 7j, 7k, and 7l promote apoptosis by activating caspase-3, 8, and Bax and down-regulating the anti-apoptotic Bcl-2.

Docking study

EGFR active site

In silico docking simulations were performed for the most potent antiproliferative compounds, 7h, 7i, 7j, 7k, and 7l, to study their molecular interaction with the epidermal growth factor receptor tyrosine kinase EGFR. Molecular operating environment (MOE) software⁴⁶ was used, as well as the crystal structure of the EGFR in complex with Erlotinib (PDB: 1M17)⁷.

MOE minimizations were performed with the force field (OPLS-AA) using Born solvation. Before simulations, the protein–ligand complex was protonated and corrected. The simulation results of the compounds were compared with Erlotinib, and the data are shown in Table 4. The docking protocol was validated by redocking the co-crystallised ligand into the EGFR binding site, where the docking S score obtained for the docked ligand was −10.70 kcal/mol with an RMSD value of 1.48 Å.

Table 4. Ligand–protein complex interactions of the tested compounds 7h, 7i, 7j, 7k, and 7l within the active site of EGFR.

Compd.	MOE score (kcal/mol)	Hydrogen bond interactions	Hydrophobic interactions	Pi–H interactions
Erlotinib	−10.70	Met769	Leu694, Leu820, Val702, Gly722, Thr766, Thr830	Leu694
7h	−8.18	–	Lys721, Asp831, Val702, Leu820, Leu694, Phe699, Gly772	Gly772, Cys773
7i	−8.45	Leu764	Lys721, Asp831, Leu820, Leu694, Phe699, Gly772	Leu820
7j	−8.77	–	Gly772 Lys721, Asp831, Val702, Leu820, Leu694, Phe699	Gly772, Leu820
7k	−9.52	–	Phe699, Gly772 Lys721, Asp831, Val702, Leu820, Leu694	Gly772
7l	−9.57	Lys721	Leu694, Phe699, Gly772 Lys721, Asp831, Val702, Leu820	Leu694

Concerning docking score analysis, compounds 7k and 7l exhibited the highest negative values (−9.52 and −9.57 kcal/mol, respectively), compatible with their in vitro EGFR inhibition effects (Table 4). Inspection of the ligand–protein complexes exposed that the ligand–protein interactions were mainly hydrophobic. The new 1,2,4-oxadiazole compounds are sterically fixed analogs of the open carboximidamide derivatives of previous work²⁷. Such a rigid conformation resulted in tight fitting within the EGFR active site with derivatives (R = H, 4-OMe, 3-Cl, 3-Br, 2,4-di-Cl, and 3,4,5-tri-OMe) as the cyclized structure better fits within the bioactive conformation. Compounds 7j, 7k, and 7l adopted an orientation within the large binding site where the phenyl triazole scaffold was inserted deeply into the hydrophobic pocket, aligned with Erlotinib phenyl acetylene moiety. The latter scaffold formed stacking with Phe699 and pi–H interaction with Val702. At the same time, the ligand 2,4-dichloro, 3,4-di-methoxy, or 3,4,5-trimethoxyphenyl moiety directed past the Erlotinib ether linkages at the gate of the binding site. This substituted phenyl moiety formed stacking with Pro770 and pi–H interaction with Leu694 and other hydrophobic interactions with nearby residues Lys721, Asp831, Val702, Leu694, and Gly772.

The trimethoxy derivative 7l showed the best binding mode and hydrophobic interactions with the surrounding amino acid residues owing to its increased van der Waals volume within the active site relative to the other derivatives (Figure 5C, D). However, the ligand loses H-bond interaction with the key amino acid residue Met769 compared with the H-bond formed by the quinazoline nitrogen of Erlotinib (3.56 Å) (Figure 5E, F). Alternatively, the ligand stabilises its complex by accepting a strong H-bond from Lys721 in the case of compound 7l with (3.50 Å) and a weak one from Thr830 in the case of compound 7k at the hydrophobic hinge (Figure 5A, B).

Figure 5. Docking representation models of compound 7k, 7l, and Erlotinib within the binding site of EGFR (H-bond: blue dashed lines, Pi–H; green dashed lines). (A) 3D-docked model of compound 7k (cyan) showing the protein surface (grey); (B) 2D-docked model of compound 7k; (C) 3D-docked model of compound 7l (cyan) showing the protein surface (grey); (D) 2D-docked model of compound 7l; (E) 3D-docked model of compound Erlotinib (pink) showing the protein surface (grey); (F) 2D-docked model of compound Erlotinib.

On the other hand, compounds 7h and 7i exhibited another orientation where m-chloro and m-bromophenyl moiety were better accommodated within the active site than 4-chloro and 4-bromo substituents. The m-halophenyl moiety stacked with Phe699 deeply inside the hydrophobic cleft of the enzyme and formed a halogen bond interaction with Leu764 in the case of compound 7i with (R = m-Br). However, the phenyl triazole scaffold formed stacking with Pro770 and pi–H interactions with Cys773 and Gly772 at the gate of the binding site. This indicated that the active site tolerated the m-halophenyl rather than the p-halophenyl moiety with a bulkier halogen atom (Figure 6).

Figure 6. Docking representation model of compound 7h (cyan) aligned with compound 7i (pink) showing the protein surface (grey) of the site of EGFR (H-bond or halogen bond: blue dashed lines, Pi–H; green dashed lines).

VEGFR active site

Moreover, the most potent VEGFR-2 inhibitors, 7h, 7i, 7j, 7k, and 7l, were docked against vascular endothelial growth factor VEGFR-2. The crystal structure of VEGFR-2 in complex with Sorafenib (PDB: 4ASD)⁴⁷ was used in the present study. The docking protocol was validated by redocking the co-crystallised ligand with an S score of −10.73 kcal/mol with an RMSD value of 0.46 Å. Again, the trimethoxy compound 7l showed the highest negative score (−9.10 kcal/mol) among the tested compounds (Table 5). Inspection of the docked complexes revealed that the compounds bind tightly within the binding pocket. Compounds 7l and 7k exhibited good fitting (Figure 7A–C), where the phenyl triazole scaffold bi-stacked between Phe918 and Phe92 forming pi–H interaction with Leu840. However, there were missing H-bond interactions with Cys919 at the gate of the binding site. This indicated the significance of introducing an H-bond forming substituent at the phenyl ring to enhance the fitting. On the opposite end of the active site, the dimethoxy or trimethoxy phenyl moiety formed stacking with His1026 and pi–H interaction with Glu885. Interestingly, the oxadiazole moiety accepted a H-bond from Asp1046 and formed pi–H interactions with Phe1047 (7k) or Lys868 (7l).

Figure 7. Docking representation model of compounds 7k and 7l within the binding site of VEGFR-2 (H-bond: blue dashed lines, Pi–H; green dashed lines). (A) 3D-docked model of compound 7k (yellow) aligned with compound 7l (cyan) showing the lipophilicity surface of active site (purple; hydrophilic, white; neutral; green; lipophilic); (B) 2D-docked model of compound 7k; (C) 2D-docked model of compound 7l.

Table 5. Ligand–protein complex interactions of the tested compounds 7h, 7i, 7j, 7k, and 7l within the active site of VEGFR-2.

Compd.	MOE score (kcal/mol)	Hydrogen bond interactions	Hydrophobic interactions	Pi–H interactions
Sorafenib	−10.73	Cys919, Glu885	Val916, Leu889, Leu840, Asp1046, Cys1045 and Phe1047	Phe1047
7h	−8.24	Asp1046	Leu889, Leu840, Asp1046, Cys1045 and Phe1047	Lys868
7i	−8.11	Asp1046	Val916, Leu889, Leu840, Asp1046, Cys1045 and Phe1047	Leu840 Leu889, Lys868, Asp1046
7j	−7.92	Asp1046	Val916, Leu889, Leu840, Asp1046, Cys1045	…………………
7k	−8.71	Asp1046	Val916, Leu889, Leu840, Asp1046, Cys1045	Lys868, Leu840
7l	−9.10	Asp1046	Phe1047, Val916, Leu889, Leu840, Asp1046, Cys1045	Lys868, Leu840

Other derivatives showed comparable binding modes within the active site; however, compound 7l showed the best fitting due to its enhanced van der Waals volume. Results of the docking simulations attributed to investigate the impact of introducing fixed geometry to the open carboximidamide derivatives on the binding modes within active sites and confirm dual EGFR/VEGFR-2 kinase inhibitory effects of compounds 7h, 7i, 7j, 7k, and 7l.

Moreover, the most potent compound 7l adopts DFG-in conformation of active form EGFR as erlotinib forming H-bond with Lys721 at the ATP binding site. However, the compound missed interaction with Asp831 at the DFG sequence. On the other hand, the same compound probes DFG-out conformation of inactive VEGFR-2 forming H-bond with Asp1046 that is equivalent to Asp831 at EGFR. We assumed that this new ligand could act as dual type I/II kinase inhibitor as stabilising DFG-in conformation at EGFR as well as DFG-out conformation at VEGFR-2 and thus could also inhibit phosphorylation, Figure 8.

Figure 8. (A) Binding mode of compound 7l (cyan) to active site of EGFR showing DFG motif in purple. (B) Binding mode of compound 7l (cyan) to active site of VEGFR-2 showing DFG motif in purple.

In silico ADME/pharmacokinetics studies

The most potent antiproliferative 1,2,4-oxadiazole hybrids 7h, 7i, 7j, 7k, and 7l were examined for their ADME/Pharmacokinetics properties using the web tool Swiss ADME⁴⁸ by entering a list of the compounds’ SMILES (Simplified Molecule Input Line Entry Specification) provided by ChemDraw software.

The in silico pharmacokinetic data (Table 6) showed that all 1,2,4-oxadiazole hybrids are orally active as they obey Lipinski’s rules of five with zero violation. Also, those hybrids exhibit high intestinal absorbance and are non-substrate for P-gp. None of those hybrids are likely to cross BBB except 7h, 7i, and 7j. Based on Lipinski’s rules, log P should be ≤5, 1,2,4-oxadiazole tested hybrids exhibited good permeability as indicated by log P values in the range of 2.77–3.85. Amongst compounds, the most potent antiproliferative compound, 7l, exhibited the lowest log P values and the highest TPSA, indicating the relevance of the latter physicochemical parameters to its highest activity. All tested compounds will likely be metabolised by CYP1A2, CYP2C19, and CYP2C9 but will be CYP2D3 inhibitors. However, only compounds 7k and 7l are considered substrates for CYP3A4. The predicted ADME properties are shown in (Table 7).

Table 6. Physicochemical and pharmacokinetic properties (Lipinski parameters) of compounds 7h, 7i, 7j, 7k, and 7l.

Compd.	MW	^nROTB	HBA	HBD	Violations	MR	TPSA	log P
7h	338	4	5	0	0	89.5	70	3.33
7i	382	4	5	0	0	92.2	70	3.43
7j	372	4	5	0	0	94.5	70	3.85
7k	363	6	7	0	0	97.5	88	2.77
7l	393	7	8	0	0	103.9	97	2.76

Table 7. ADME properties of compounds 7h, 7i, 7j, 7k, and 7l.

Compd.	GI abs.	BBB	P-gp substrate	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor
7h	High	Yes	No	Yes	Yes	Yes	No	No
7i	High	Yes	No	Yes	Yes	Yes	No	No
7j	High	Yes	No	Yes	Yes	Yes	No	No
7k	High	No	No	Yes	Yes	Yes	No	Yes
7l	High	No	No	Yes	Yes	Yes	No	Yes

Structure activity relationship (SAR) analysis

1. In general, cyclisation of amidoximes 6a–o to 1,2,4-oxadiazole derivatives 7a–o increased the activity of the newly synthesised compounds. The new 1,2,4-oxadiazole compounds are sterically fixed analogues of the open carboximidamide derivatives 6a–o. Such a rigid conformation may result in tight fitting within the receptor active site.

2. The substitution on the phenyl group of the 1,2,4-oxadiazole moiety significantly increased the activity (except for methyl group) of 7a–o, and substitution with methoxy groups increased the activity more than halogen atoms. Similar reasoning applies to the amidoxime derivatives 6a–o, where substitution of the amidoxime moiety’s phenyl group with electron donating or electron withdrawing groups’ increases activity.

3. The number of methoxy groups substantially impacts the activity of 7a–o compounds where the trimethoxy derivative > dimethoxy > mono-methoxy one. In 6a–o, the dimethoxy derivative was the most tolerated for activity.

4. The type, position, and number of halogen atoms had a significant effect on antiproliferative activity of 7a–o, with activity increasing in the order 2,4-di-Cl > 3-Br > 3-Cl > 4-Br > 4-Cl > 4-F. The dihalo derivative was also more active than the mono-halo derivative in 6a–o, but the 4-position is better tolerated than the 3-position.

5. When the phenyl group in 6a–o and 7a–o is replaced with a naphthalene moiety, the activity increases, and 1-naphthyl is tolerated better than 2-naphthyl.

Conclusion

Fifteen novel 1,2,3-triazole/1,2,4-oxadiazole hybrids (7a–o) were synthesised and tested as antiproliferative agents with dual EGFR/VEGFR-2 inhibitory activity. The newly synthesised compounds showed promising antiproliferative activity, with the hybrid 7h–l being the most potent class. In vitro investigations revealed that compounds 7j, 7k, and 7l were effective antiproliferative agents that could operate as dual EGFR/VEGFR-2 inhibitors. Moreover, apoptotic-inducing activity experiments indicate that compounds 7j, 7k, and 7l enhance apoptosis by activating caspase-3, 8, and Bax and down-regulating the anti-apoptotic Bcl-2. Docking simulations highlighted the output of including 1,2,4-oxadiazole scaffold to improve the binding of the compounds within the active sites of EGFR and VEGFR-2. The cyclized structures fit well within the bioactive conformations. In addition, the phenyl triazole scaffold bound considerably within the hydrophobic pocket of binding sites.

Moreover, there is a future concern about introducing an H-bond forming substituent at the phenyl ring to tolerate binding with Cys919 within the VEGFR-2 active site. Also, docking results revealed that 7l and 7k exhibited the best binding mode within both active sites. In silico ADME and pharmacokinetic study, the compounds were predicted to have acceptable bioavailability and pharmacokinetic profiles.

Materials and methods

Chemistry

General details: see Appendix A

Compounds 1, 2, 5a–o, and 6a–o were prepared according to previously reported literature^27,31–33.

General procedures for the synthesis of compounds 7a–o

A stirred solution of the appropriate N'-(((4-phenyl-1H-1,2,3-triazol-1-yl)acetyl)oxy)benzene-carboximidamide (6a–o) (0.78 mmole) in dry acetonitrile (20 mL) was refluxed overnight. After the reaction was completed (monitored with TLC), the excess solvent evaporated under reduced pressure. The obtained precipitate was washed with cold water, filtered, and crystallised from acetonitrile.

3-Phenyl-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7a)⁴⁹

Yield: 0.19 g (78%), white solid, mp: 146–148 °C, R_f: 0.72 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3126, 3100, 2964 (CH), 1596, 1580, 1467 (C = N and C = C), 766 and 689 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.81 (s, 1H, triazole-CH), 7.99 (d, J = 7.9 Hz, 2H, Ar–H), 7.90 (d, J = 8.5 Hz, 2H, Ar–H), 7.62–7.56 (m, 3H, Ar–H), 7.48 (t, J = 7.6 Hz, 2H, Ar–H), 7.37 (t, J = 7.4 Hz, 1H, Ar–H), 6.29 (s, 2H, N–CH₂). Anal. Calc. (%) for C₁₇H₁₃N₅O: C, 67.32; H, 4.32; N, 23.09. Found: C, 67.49; H, 4.60; N, 23.37.

3-(4-Fluorophenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7b)

Yield: 0.12 g (48%), white solid, mp: 137–139 °C, R_f: 0.74 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3125, 3099, 2979, 2952 (CH), 1608, 1578, 1469 (C = N and C = C), 848, 770, 700 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.81 (s, 1H, triazole-CH), 8.05 (q, J = 4.8 Hz, 2H, Ar–H), 7.90 (d, J = 7.4 Hz, 2H, Ar–H), 7.48 (t, J = 7.4 Hz, 2H, Ar–H), 7.39 (q, J = 8.6 Hz, 3H, Ar–H), 6.30 (s, 2H, N–CH₂). ¹³C NMR (100 MHz, δ ppm DMSO-d₆): 175.1, 167.7, 166.2, 162.9, 147.3, 130.7, 130.2 (d, J = 9.2 Hz), 129.5, 128.6, 125.7, 123.1, 117.0 (d, J = 22.4 Hz), 45.5. Anal. Calc. (%) for C₁₇H₁₂FN₅O: C, 63.55; H, 3.76; N, 21.80. Found: C, 63.38; H, 3.91; N, 22.03.

3-(4-Chlorophenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7c)

Yield: 0.16 g (61%), white solid, mp: 150–152 °C, R_f: 0.71 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3137, 3086, 2977, 2957 (CH), 1598, 1569, 1467 (C = N and C = C), 844, 766, 697 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.80 (s, 1H, triazole-CH), 8.00 (d, J = 8.5 Hz, 2H, Ar–H), 7.89 (d, J = 7.4 Hz, 2H, Ar–H), 7.63 (d, J = 8.5 Hz, 2H, Ar–H), 7.47 (t, J = 7.6 Hz, 2H, Ar–H), 7.36 (t, J = 7.3 Hz, 1H, Ar–H), 6.29 (s, 2H, N–CH₂). Anal. Calc. (%) for C₁₇H₁₂ClN₅O: C, 60.45; H, 3.58; N, 20.73. Found: C, 60.71; H, 3.69; N, 20.95.

3-(4-Bromophenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7d)

Yield: 0.22 g (74%), white solid, mp: 158–160 °C, R_f: 0.73 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3122, 3093 (CH), 1592, 1564, 1459 (C = N and C = C), 838, 766, 697 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.80 (s, 1H, triazole-CH), 7.92 (d, J = 8.6 Hz, 2H, Ar–H), 7.90 (d, J = 7.1 Hz, 2H, Ar–H), 7.78 (d, J = 8.6 Hz, 2H, Ar–H), 7.48 (t, J = 7.7 Hz, 2H, Ar–H), 7.37 (t, J = 7.5 Hz, 1H, Ar–H), 6.29 (s, 2H, N–CH₂). Anal. Calc. (%) for C₁₇H₁₂BrN₅O: C, 53.42; H, 3.16; N, 18.32. Found: C, 53.66; H, 3.40; N, 18.59.

3-(4-Methylphenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7e)

Yield: 0.20 g (83%), white solid, mp: 140–142 °C, R_f: 0.69 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3121, 3089, 2966, 2935 (CH), 1604, 1574, 1466 (C = N and C = C), 834, 769, 693 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.81 (s, 1H, triazole-CH), 7.92–7.87 (m, 4H, Ar–H), 7.48 (d, J = 7.5 Hz, 2H, Ar–H), 7.40–7.35 (m, 3H, Ar–H), 6.28 (s, 2H, N–CH₂), 2.38 (s, 3H, Ar–CH₃). ¹³C NMR (100 MHz, δ ppm DMSO-d₆): 174.7, 168.4, 147.3, 142.4, 130.7, 130.4, 129.5, 128.7, 127.5, 125.8, 123.3, 123.1, 45.6, 21.6. Anal. Calc. (%) for C₁₈H₁₅N₅O: C, 68.13; H, 4.76; N, 22.07. Found: C, 68.35; H, 4.88; N, 21.89.

3-(4-Methoxyphenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7f)

Yield: 0.19 g (75%), white solid, mp: 148–150 °C, R_f: 0.70 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3133, 2977, 2843 (CH), 1617, 1576, 1469 (C = N and C = C), 843, 769, 698 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.80 (s, 1H, triazole-CH), 7.93 (d, J = 8.9 Hz, 2H, Ar–H), 7.90 (d, J = 7 Hz, 2H, Ar–H), 7.48 (t, J = 7.5 Hz, 2H, Ar–H), 7.37 (t, J = 7.4 Hz, 1H, Ar–H), 7.11 (d, J = 8.9 Hz, 2H, Ar–H), 6.26 (s, 2H, N–CH₂), 3.83 (s, 3H, Ar–OCH₃). Anal. Calc. (%) for C₁₈H₁₅N₅O₂: C, 64.86; H, 4.54; N, 21.01. Found: C, 65.08; H, 4.67; N, 21.28.

3-(4-Trifluoromethylphenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7g)

Yield: 0.13 g (46%), white solid, mp: 166–168 °C, R_f: 0.7 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3144, 3001, 2965 (CH), 1602, 1579, 1468 (C = N and C = C), 856, 767, 694 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.82 (s, 1H, triazole-CH), 8.21 (d, J = 8.1 Hz, 2H, Ar–H), 7.94 (d, J = 8.4 Hz, 2H, Ar–H), 7.90 (d, J = 7.5 Hz, 2H, Ar–H), 7.48 (t, J = 7.5 Hz, 2H, Ar–H), 7.37 (t, J = 7.3 Hz, 1H, Ar–H), 6.33 (s, 2H, N–CH₂). ¹³C NMR (100 MHz, δ ppm DMSO-d₆): 175.1, 167.2, 147.1, 131.8 (q, J = 32.2 Hz), 130.3, 129.6, 129.2, 128.4, 128.2, 126.5 (q, J = 3.5 Hz), 125.5, 124.0 (q, J = 272.7 Hz), 122.9, 45.3. Anal. Calc. (%) for C₁₈H₁₂F₃N₅O: C, 58.22; H, 3.26; N, 18.86. Found: C, 58.43; H, 3.39; N, 19.07.

3-(3-Chlorophenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7h)

Yield: 0.17 g (67%), white solid, mp: 128–130 °C, R_f: 0.71 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3121, 3095, 2966 (CH), 1606, 1574, 1465 (C = N and C = C), 892, 796, 767, 736, 693 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.80 (s, 1H, triazole-CH), 7.97–7.94 (m, 2H, Ar–H), 7.89 (d, J = 7.3 Hz, 2H, Ar–H), 7.69 (d, J = 8.7 Hz, 1H, Ar–H), 7.60 (t, J = 8.1 Hz, 1H, Ar–H), 7.47 (t, J = 7.6 Hz, 2H, Ar–H), 7.37 (t, J = 7.3 Hz, 1H, Ar–H), 6.30 (s, 2H, N–CH₂). ¹³C NMR (100 MHz, δ ppm DMSO-d₆): 174.8, 167.0, 146.9, 134.0, 131.8, 131.5, 130.2, 129.0, 128.2, 127.5, 126.5, 125.8, 125.3, 122.7, 45.1. Anal. Calc. (%) for C₁₇H₁₂ClN₅O: C, 60.45; H, 3.58; N, 20.73. Found: C, 60.72; H, 3.79; N, 21.01.

3-(3-Bromophenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7i)

Yield: 0.23 g (77%), white solid, mp: 139–141 °C, R_f: 0.72 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3143, 3067, 3002 (CH), 1595, 1564, 1471 (C = N and C = C), 903, 800, 767, 737, 694 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.80 (s, 1H, triazole-CH), 8.10 (t, J = 1.7 Hz, 1H, Ar–H), 7.99 (two t, J = 8, 2.5 Hz, 1H, Ar–H), 7.89 (d, J = 7.1 Hz, 2H, Ar–H), 7.84–7.80 (m, 1H, Ar–H), 7.54 (t, J = 7.9 Hz, 1H, Ar–H), 7.48 (t, J = 7.5 Hz, 2H, Ar–H), 7.37 (t, J = 7.4 Hz, 1H, Ar–H), 6.30 (s, 2H, N–CH₂). Anal. Calc. (%) C₁₇H₁₂BrN₅O: C, 53.42; H, 3.16; N, 18.32. Found: C, 53.68; H, 3.29; N, 18.57.

3-(2,4-Dichlorophenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7j)

Yield: 0.16 g (54%), white solid, mp: 137–139 °C, R_f: 0.73 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3146, 3080, 2992 (CH), 1602, 1588, 1469 (C = N and C = C), 881, 762, 690 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.80 (s, 1H, triazole-CH), 7.93–7.88 (m, 4H, Ar–H), 7.64 (two d, J = 8.4, 2.1 Hz, 1H, Ar–H), 7.48 (t, J = 8.4 Hz, 2H, Ar–H), 7.37 (t, J = 7.4 Hz, 1H, Ar–H), 6.32 (s, 2H, N–CH₂). ¹³C NMR (100 MHz, δ ppm DMSO-d₆): 174.1, 166.0, 146.9, 136.8, 133.2, 133.0, 130.6, 130.2, 129.0, 128.2, 128.1, 125.3, 123.8, 122.7, 45.0 Anal. Calc. (%) C₁₇H₁₁Cl₂N₅O: C, 54.86; H, 2.98; N, 18.82. Found: C, 54.98; H, 3.12; N, 19.09.

3-(3,4-Dimethoxyphenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7k)

Yield: 0.25 g (89%), white solid, mp: 174–176 °C, R_f: 0.68 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3125, 3096, 2943, 2838 (CH), 1601, 1583, 1499 (C = N and C = C), 864, 765, 687 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.80 (s, 1H, triazole-CH), 7.89 (d, J = 7.3 Hz, 2H, Ar–H), 7.57 (two d, J = 8.4, 1.8 Hz, 1H, Ar–H), 7.49–7.45 (m, 3H, Ar–H), 7.36 (t, J = 7.3 Hz, 1H, Ar–H), 7.11 (d, J = 8.5 Hz, 1H, Ar–H), 6.26 (s, 2H, N–CH₂), 3.82 (s, 6H, Ar–(OCH₃)₂). ¹³C NMR (100 MHz, δ ppm DMSO-d₆): 174.0, 167.8, 151.7, 149.1, 146.9, 130.3, 129.0, 128.2, 125.3, 122.7, 120.7, 117.8, 112.0, 109.5, 55.7, 55.6, 45.1. Anal. Calc. (%) C₁₉H₁₇N₅O₃: C, 62.80; H, 4.72; N, 19.27. Found: C, 62.67; H, 4.89; N, 19.51.

3-(3,4,5-Trimethoxyphenyl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7l)

Yield: 0.21 g (69%), white solid, mp: 154–156 °C, R_f: 0.74 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3143, 3000, 2947, 2847 (CH), 1604, 1581, 1467 (C = N and C = C), 762, 689 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.81 (s, 1H, triazole-CH), 7.89 (d, J = 7 Hz, 2H, Ar–H), 7.48 (t, J = 7.5 Hz, 2H, Ar–H), 7.37 (t, J = 7.4 Hz, 1H, Ar–H), 7.26 (s, 2H, Ar–H), 6.27 (s, 2H, N–CH₂), 3.85 (s, 6H, Ar–(OCH₃)₂), 3.74 (s, 3H, Ar–OCH₃). Anal. Calc. (%) C₂₀H₁₉N₅O₄: C, 61.06; H, 4.87; N, 17.80. Found: C, 61.28; H, 4.95; N, 18.04.

3-(Naphthalen-1-yl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7m)

Yield: 0.21 g (78%), white solid, mp: 144–146 °C, R_f: 0.69 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3120, 3088, 2975, 2943 (CH), 1607, 1592, 1464 (C = N and C = C), 767, 747, 694 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.86 (s, 1H, triazole-CH), 8.76 (d, J = 8.3 Hz, 1H, Ar–H), 8.19 (d, J = 7.7 Hz, 2H, Ar–H), 8.08 (d, J = 7.9 Hz, 1H, Ar–H), 7.91 (d, J = 7.2 Hz, 2H, Ar–H), 7.72–7.63 (m, 3H, Ar–H), 7.48 (t, J = 7.5 Hz, 2H, Ar–H), 7.37 (t, J = 7.3 Hz, 1H, Ar–H), 6.37 (s, 2H, N–CH₂). ¹³C NMR (100 MHz, δ ppm DMSO-d₆): 173.6, 168.3, 146.9, 133.5, 132.3, 130.3, 129.7, 129.5, 129.0, 128.9, 128.2, 127.9, 126.7, 125.5, 125.4, 125.3, 122.7, 122.5, 45.2. Anal. Calc. (%) C₂₁H₁₅N₅O: C, 71.38; H, 4.28; N, 19.82. Found: C, 71.09; H, 4.41; N, 20.04.

3-(Naphthalen-2-yl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7n)

Yield: 0.23 g (84%), white solid, mp: 180–182 °C, R_f: 0.66 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3117, 3088, 2971, 2939 (CH), 1600, 1462 (C = N and C = C), 764, 756, 689 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.81 (s, 1H, triazole-CH), 8.61 (s, 1H, Ar–H), 8.12 (d, J = 7.8 Hz, 1H, Ar–H), 8.06 (t, J = 7.5 Hz, 2H, Ar–H), 8.00 (d, J = 7.8 Hz, 1H, Ar–H), 7.91 (d, J = 7.9 Hz, 2H, Ar–H), 7.66–7.59 (m, 2H, Ar–H), 7.48 (t, J = 7.6 Hz, 2H, Ar–H), 7.37 (t, J = 7.4 Hz, 1H, Ar–H), 6.31 (s, 2H, N–CH₂). Anal. Calc. (%) C₂₁H₁₅N₅O: C, 71.38; H, 4.28; N, 19.82. Found: C, 71.21; H, 4.52; N, 20.06.

3-(2H-1,3-Benzodioxol-5-yl)-5-[(4-phenyl-1H-1,2,3-triazol-1-yl)methyl]-1,2,4-oxadiazole (7o)

Yield: 0.18 g (67%), white solid, mp: 149–151 °C, R_f: 0.67 (hexane:ethyl acetate, 2:1, v/v). IR (FTIR): ν_max (cm⁻¹) 3141, 2982, 2914 (CH), 1583, 1452 (C = N and C = C), 761, 692 (Ar–CH bending). ¹H NMR (400 MHz, δ ppm DMSO-d₆): 8.80 (s, 1H, triazole-CH), 7.89 (d, J = 7.1 Hz, 2H, Ar–H), 7.55 (two d, J = 8.13, 1.7 Hz, 1H, Ar–H), 7.48 (d, J = 7.6 Hz, 2H, Ar–H), 7.43 (d, J = 1.6 Hz, 1H, Ar–H), 7.37 (t, J = 7.4 Hz, 1H, Ar–H), 7.08 (d, J = 8.13 Hz, 1H, Ar–H), 6.25 (s, 2H, –O–CH₂–O–), 6.14 (s, 2H, N–CH₂). ¹³C NMR (100 MHz, δ ppm DMSO-d₆): 174.1, 167.6, 150.3, 148.1, 146.9, 130.3, 129.0, 128.2, 125.3, 122.7, 122.3, 119.2, 109.0, 106.5, 102.0, 45.1. Anal. Calc. (%) C₁₈H₁₃N₅O₃: C, 62.24; H, 3.77; N, 20.16. Found: C, 62.45; H, 3.85; N, 20.42.

Biology

Assay of cell viability effect

To investigate the effect of compounds 7a–o on normal cell lines, a cell viability experiment was performed on the MCF-10A (human mammary gland epithelial) cell line. This experiment uses 50 µM of the investigated compound for four days^34,35. Refer to Appendix A (Supp. File) for more details.

Assay of antiproliferative effect

The antiproliferative activity of 1,2,4-oxadiazoles 7a–o against four human cancer cell lines was assessed using Erlotinib as the reference medication^36,37. See Appendix A for more details.

Assay of EGFR inhibitory activity

The inhibitory effects of the most potent antiproliferative compounds 7h–l on EGFR, as a potential target for their antiproliferative action, were studied using Erlotinib as a control medication³⁸. For more information, see Appendix A.

Assay of VEGFR-2 inhibitory activity

Compounds 7h–l’s inhibitory activity against VEGFR-2 was assessed using kinase-glo-luminescent kinase assays with Sorafenib as the control drug³⁹. For more information, see Appendix A.

Apoptosis-inducing activity assay

Compounds 7j, 7k, and 7l, the most potent derivatives in all in vitro studies, were tested for their capacity to initiate the apoptosis cascade and reveal their proapoptotic potential⁴⁵. For more information, see Appendix A.

Docking

All molecular modelling calculations and docking simulation studies were performed on a Processor Intel (R) Pentium (R) CPU N3510@ 1.99 GHz and 4 GB memory with Microsoft Windows 8.1 pro (64 Bit) operating system using Molecular Operating Environment (MOE 2019.0102, 2020; Chemical Computing Group, Canada) as the computational software. MOE minimizations were performed until an RMSD gradient of 0.01 kcal/mol/Å with the force field (OPLS-AA) to calculate the partial charges automatically using Born solvation. Before simulations, the protein was corrected, hydrogens were added, and ionisation states were assigned; the system was optimised via protonation, distant water molecules were deleted, and the receptor was minimised using the QuickPrep function. Then, both ligand and pocket were isolated in 3D, and molecular surface was drawn around the binding site to visualise the space available for docked ligands. The compounds’ database was created after the compounds’ structures were prepared, partially charged, and minimised. Initially, self-docking was performed, and ligand conformations were generated with the bond rotation method. These are then placed on the site with the Triangle Matcher method and ranked with the London dG scoring function. The retain option specifies the number of poses (30) to pass to the refinement for energy minimisation in the pocket before restoring with the GBVI/WSA dG scoring function. Docking simulation was performed using a compound database mdb file. A triangle matching with London dG scoring was chosen for initial placement, and then the top 30 poses were refined using force field (OPLS-AA) and GBVI/WSA dG scoring. The output database dock file was created with different poses for each ligand and arranged according to the final score function (S), which is the score of the last stage that was not set to zero.

Acknowledgements

This work was funded by the Researchers Supporting Project Number [RSP2023R273] at King Saud University, Riyadh, Saudi Arabia. The authors also acknowledge support from the KIT-Publication Fund of the Karlsruhe Institute of Technology.

Disclosure statement

The author reported no potential conflicts of interests.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This work was funded by the Researchers Supporting Project Number [RSP2023R273] at King Saud University, Riyadh, Saudi Arabia. The authors also acknowledge support from the KIT-Publication Fund of the Karlsruhe Institute of Technology.