New Benzimidazole-, 1,2,4-Triazole-, and 1,3,5-Triazine-Based Derivatives as Potential EGFR^WT and EGFR^T790M Inhibitors: Microwave-Assisted Synthesis, Anticancer Evaluation, and Molecular Docking Study

Abstract

A new series of benzimidazole, 1,2,4-triazole, and 1,3,5-triazine derivatives were designed and synthesized using a microwave irradiation synthetic approach utilizing 2-phenylacetyl isothiocyanate (1) as a key starting material. All the new analogues were evaluated as anticancer agents against a panel of cancer cell lines utilizing doxorubicin as a standard drug. Most of the tested derivatives exhibited selective cytotoxic activity against MCF-7 and A-549 cancer cell lines. Furthermore, the new target compounds 5, 6, and 7 as the most potent antiproliferative agents have been assessed as in vitro EGFR^WT and EGFR^T790M inhibitors compared to the reference drugs erlotinib and AZD9291. They represented more potent suppression activity against the mutated EGFR^T790M than the wild-type EGFR^WT. Moreover, the compounds 5, 6, and 7 down-regulated the oncogenic parameter p53 ubiquitination. A docking simulation of compound 6b was carried out to correlate its molecular structure with its significant EGFR inhibition potency and its possible binding interactions within the active site of EGFR^WT and the mutant EGFR^T790M.

1. Introduction

Cancer disease is a terrible health epidemic that kills millions of people all over the world in both developed and developing countries.¹ Despite the great progress accomplished in cancer therapy, several limitations still present. Examples are the selectivity for cancer cells, adverse effects, as well as the multiple-drug resistance acquisition by the cancer cells leading them to be unresponsive to conventional therapeutic agents.^2,3 Accordingly, the innovation of new small molecules that are both potent and selective is still a serious challenge in the field of medicinal chemistry. The alteration of different protein expressions and the activity of various receptor tyrosine kinases (RTKs) are considered the main causes of many cancer types since they are responsible for the regulation of different cellular pathways such as proliferation, differentiation, migration, and angiogenesis.^4,5 The epidermal growth factor receptor (EGFR) is a member of tyrosine kinases (TKs).⁶ It is a trans-membrane protein belonging to the erbB/HER-family and plays a pivotal role in governing cellular transduction or communication signaling through the phosphorylation of tyrosine residues in the protein domain.^7,8 EGFR is one of the main tumor markers in many cancer types (such as colon, lung, liver, cervical, ovarian, breast, prostate, and bladder cancers), where its signaling in tumors, as opposed to normal cells, becomes dysregulated, resulting in EGFR overexpression and/or obtaining a gain-of-function mutation.⁹⁻¹³ This act is considered the main cause of tumor cell proliferation, invading the surrounding tissues and resulting in an increased angiogenesis.¹⁴ Accordingly, interrupting EGFR communicating signals is considered to be one of the prime targets to invade tumors caused by its mis-regulation.⁹⁻¹⁴ Targeted drugs inhibiting EGFR can selectively attack the cancer cells rather than normal ones, thus producing a good safety profile and less harm to the body with more patient comfortability.¹⁵ Multiple EGFR suppressors have been developed and classified into different generations. The first generation was gefitinib (Iressa), erlotinib (Tarceva), and icotinib (Conmana).¹⁶⁻²⁰ Studies revealed that acquired drug resistance to the first-generation EGFR-TKIs was revealed due to T790M ″gatekeeper″ and L858R mutations in EGFR about 9–14 months after clinical treatment.²¹⁻²³ The emergence of resistance paved the way toward the development of the second-generation inhibitors (EGFR TKI) (afatinib, dacomitinib, neratinib, and canertinib) that have exhibited a 60–70% objective response rate.¹⁷ The drugs related to this class contain an electrophilic acrylamide side chain that interacts irreversibly with cysteine CYS797 forming covalent complexes, thus overcoming the obstacle of resistance mediated by EGFR^T790M or EGFR^T790M/L858R mutation.²⁴⁻²⁷ On the other hand, due to the high reactivity of the acrylamide moiety, it interacts non-selectively with the cysteine residue in untargeted proteins, leading to toxic side effects such as diarrhea and skin rash that limited their clinical use.²⁸⁻³² Recently, to solve these undesirable side effects, several third-generation inhibitors have been discovered, such as WZ4002,²⁹ osimertinib (AZD9291) (Tagrisso),³³ olmutinib (Olita),³¹ and rociletinib (CO1686).³⁴ These inhibitors do not only produce good anti-tumor activity but also produce good selectivity to EGFR^T790M and EGFR^T790M/L858R kinases.^29,35 Rociletinib and osimertinib were considered as breakthrough therapies in the mutant NSCLC treatment by the US FDA in 2014.³⁶ Studies showed that osimertinib’s efficacy is marred by various side effects such as grade 3 venous thromboembolism and pneumonia. Its toxicity was attributed to AZ5104, which is its main metabolite, lacking selectivity between the mutant and WT EGFR.^29,35−38 Accordingly, much efforts are still needed to discover new EGFR inhibitors of high selectivity to EGFR^T790M and EGFR^T790M/L858R kinase with low side effects (Figure 1).

Figure 1.

Examples of the first, second and third generations of EGFR inhibitors.

Many nitrogen-heterocyclic ring systems have recently been discovered to introduce surprisingly complex biological properties, making them one of the most significant groups in medicinal chemistry. They constitute a basic scaffold in numerous drugs due to their capabilities to imitate and interact with different biological molecules, leading to remarkable pharmacological properties.³⁹⁻⁴² The benzimidazole scaffold participates in various compounds producing a wide range of biological activities such as antimicrobial, antiparasitic, antihistaminic, antiallergic, anticancer, and antioxidant.⁴³⁻⁴⁹ The benzimidazoles could be considered as auxiliary isosters of nucleotides having a potential for chemotherapeutic applications.⁴⁶ In addition, the orally available third-generation EGFR inhibitor nazartinib bears a benzimidazole nucleus.⁵⁰ Moreover, the triazole nucleus plays a vital role in the field of drug discovery. The triazole ring is characterized by significant stability and excellent pharmacological potency due to its electron-rich characteristics and the occurrence of an unsaturated hydrocarbon ring structure. These properties support the triazole structure to interact with various receptors (enzymes) through H-bonding that endows it with significant pharmacological actions.⁵¹⁻⁵³ Currently, triazole derivatives are used to treat a wide variety of diseases specially cancer disease.⁵¹⁻⁵³ Numerous anticancer drugs bearing the 1,2,4-triazole moiety are available in the market such as anastrozole,^54,55 letrozole,⁵⁶ and vorozole.⁵⁷ Furthermore, the s-triazine (1,3,5-triazine) scaffold constitutes a basic template for the design and synthesis of various bioactive compounds with widespread applications in medicinal chemistry.⁵⁸ The s-triazine core has three functionalized branches at positions 2, 4, and 6, a property that leads to easily modulating the physicochemical and biological activities of s-triazine derivatives.⁵⁹ Many studies investigated the notable progress in the design, synthetic approaches, and evaluation of numerous s-triazine candidates with great promising antitumor activity acting via the inhibition of different protein kinases such as CDK2, PI3Kα/mTOR, CA, human topoisomerase IIα, hDHFR, EGFR (EGFR^WT and EGFRT^790M), and tubulin polymerization.⁶⁰⁻⁶⁷ There are various anticancer drugs containing the s-triazine motif that are FDA-approved such as tretamine,⁶⁸ gedatolisib,⁶⁹ HL 010183,⁷⁰ enasidenib,⁷¹ altretamine,⁷² and KY-04031⁷³ (Figure 2).

Figure 2.

Examples of various marketed anticancer drugs bearing 1,2,4-triazole and s-triazine scaffolds.

Microwave-assisted organic synthesis (MAOS) has been widely used in green chemistry in recent years.⁷⁴ Microwave irradiation is an eco-friendly approach without hazardous solvents. It helps in the synthesis of various heterocyclic compounds rapidly and in high yields.^75,76 In addition, MAOS has a great contribution in chemical selectivity, catalyst-free conditions, and the absence of side products during the synthesis processes of various aromatic and heterocyclic compounds.^77,78 Based on the above-mentioned knowledge and in continuation of our previous efforts in the field of design and generation of new bioactive heterocyclic compounds,⁷⁹⁻⁸⁴ this study deals with the design and microwave-assisted organic synthesis of a new set of benzimidazole, 1,2,4-triazole, and s-triazine derivatives targeting the wild-type EGFR-TK (EGFR^WT) and the mutant EGFR-TK (EGFRT^790M).

1.1. Rational and Design

Computational studies represented that the ATP active pocket of EGFR-TK possesses mainly five regions, as follows: (1) an adenine binding pocket bearing the key amino acid residues that can interact with the adenine ring via hydrogen bond formation, (2) a sugar zone (hydrophilic ribose pocket), (3) hydrophobic zone I (this area is not used by ATP but displays a pivotal role in the inhibitor selectivity), (4) hydrophobic region II (this area is also not used by ATP and can be used to determine the inhibitor specificity), and (5) a phosphate binding area that is important for improving the characteristics of inhibitor pharmacokinetics^85,86 (Figure 3).

Figure 3.

The structure of the ATP-binding site of EGFR-TK.

Additionally, various studies demonstrated that the main pharmacophoric features shared by multiple EGFR-TKIs are four common areas: (i) a flat hetero-aromatic ring system, fitting in the adenine binding pocket and that can participate in H-bonding interactions with different amino acids such as Met793, Thr854, and Thr790 residues; (ii) a terminal hydrophobic head that occupies the hydrophobic zone I; (iii) an imino moiety (NH– spacer) that can participate in generating hydrogen bonds with different amino acid residues present in the linker region; and (iv) a hydrophobic tail that fits in the hydrophobic region II⁸⁷⁻⁸⁹ (Figure 4).

Figure 4.

The designed molecular structures of new benzimidazole, 1,2,4-triazole, and 1,3,5-triazine derivatives.

Since benzimidazole, 1,2,4-triazole, and s-triazine are bio-isosteric, this work deals with the design and MAOS synthesis of new sets bearing one of the previously mentioned heterocyclic nuclei possessing the essential pharmacophoric features of EGFR-TKIs. The first position was the benzimidazole moiety (as compounds 3 and 4), and this scaffold was replaced by 1,2,4-triazole (as compounds 5 and 6) and 1,3,5-triazine nuclei (as compounds 7 and 8) to fit in the adenine binding pocket, where the heterocyclic nitrogen atoms act as hydrogen-bond acceptors leading to excellent EGFR-TK potency.⁹⁰ The second position was the terminal benzyl moiety (hydrophobic head) (as compounds 3 and 4), which might be replaced with aliphatic, heterocyclic, or substituted phenyl structures (as compounds 5–8). The third position was the NH linker, as a site for the creation of different hydrogen bonds. The used linkers may be an imino group as in compounds 5–8 or a carbonothioyl-acetamide linker (as in compound 4). The fourth position was the phenyl group (hydrophobic tail), where a phenyl ring was incorporated at position-2 of the benzimidazole nucleus as in compound 4 or replaced by a benzyl ring at positions-5/3 of the 1,2,4-triazole nucleus or position-6 of the triazine ring to occupy the hydrophobic zone II of the ATP binding site. The fourth position of the benzimidazole 3 was left unsubstituted to find out the impact of the phenyl substitution on the target activity (Figure 4).

All the newly synthesized compounds were screened for their anti-proliferative activities against a panel of human cancer cell lines. Furthermore, the most promising compounds as cytotoxic agents were assessed as EGFR^WT, EGFR^T790M, and p53 ubiquitination inhibitors. Since compound 6b represented the most promising cytotoxic activity as well as EGFR^WT and EGFR^T790M inhibition activity, it was selected as a representative example to emphasize its possible binding patterns in the active pockets of EGFR^WT and EGFR^T790M via a molecular docking study.

2. Results and Discussion

2.1. Chemistry

The new target compounds 3–8 were synthesized in short reaction times and with high yields utilizing a microwave irradiation process (Schemes 1 and 2, Table 1). The chemical structures of the new compounds were elucidated using microanalytical and spectral data (IR, ¹H, ¹³C NMR, MS). 2-Phenylacetyl isothiocyanate (1) was utilized as a key starting material and treated with different aliphatic, heterocyclic, and/or aromatic amines, namely, isopropylamine, 2-aminothiazole, o-phenylenediamine, m-aminophenol, and o-chloro-p-nitroaniline, in acetonitrile at room temperature⁹¹ to afford the corresponding thiourea derivatives 2a–e, respectively. IR spectra of 2a–e represented characteristic absorption bands at the regions 3370–3135, 1690–1660, and 1173–1146 cm^–1 due to NH, C=O, and C=S groups, respectively. ¹H NMR spectra of the new compounds 2a–e revealed singlet signals at the range δ 3.43–3.82 ppm representing the two methylene protons of CH₂-ph. In addition, the expected signals of the aromatic protons appeared at the corresponding region δ 7.20–8.61 ppm, while the two NH protons appeared as D₂O exchangeable signals at the range δ 12.11–13.0 ppm. Compound 2a exhibited a doublet signal at δ 1.06 ppm and another multiplet at δ 3.80–3.84 ppm that is an evidence of the presence of the −CH(CH₃)₂ group. Compounds 2c and 2d exhibited two additional D₂O exchangeable signals at δ 5.01 and 9.66 ppm due to NH₂ and OH groups, respectively.

Scheme 1. Synthesis of New Substituted Benzimidazole Derivatives Utilizing the Microwave Irradiation Synthetic Approach.

Scheme 2. Synthesis of New Substituted 1,2,4-Triazole and 1,3,5-Triazine Derivatives Utilizing the Microwave Irradiation Synthetic Approach.

Table 1. The Reaction Times and the Yields of the Newly Synthesized Compounds Using the Microwave Irradiation Technique.

	microwave irradiation method
compound no.	time (min)	yield (%)
3	2	80
4	3	96
5a	2	98
5b	5	96
5c	7	86
6a	6	95
6b	9	85
6c	10	89
7	5	97
8a	4	96
8b	9	97
8c	7	92
8d	10	98

Microwave irradiation of the thiourea derivative 2c in DMF for 2 min led to its cyclization, forming the corresponding N-(1H-benzo[d]imidazol-2-yl)-2-phenylacetamide benzimidazole (3), while its microwave irradiation with benzoyl chloride afforded^92,93 the corresponding N-(2-phenyl-1H-benzo[d]imidazole-1-carbonothioyl) benzamide (4) (Scheme 1). IR spectra of the later derivatives 3 and 4 exhibited absorption bands at the ranges 3220–3128 and 1670–1699 cm^–1 correlated to NH and C=O groups, respectively. Furthermore, 4 represented an additional band at 1266 cm^–1 due to its C=S moiety. ¹H NMR spectra of compounds 3 and 4 exhibited singlet signals at the region δ 3.70–3.91 due to CH₂-ph and D₂O exchangeable signals at the range δ 11.74–12.78 ppm representing NH groups, while the aromatic-Hs appeared as multiplet signals at their expected up-field region δ 7.36–8.24 ppm. Moreover, ¹³C NMR spectra of both 3 and 4 showed signals at the corresponding regions δ 40.06 and 40.32 ppm referring to CH₂-ph and δ 173.30 and 167.0 due to C=O groups as well as various signals at the range δ 114.05–166.53 ppm representing the aromatic carbons.

On the other hand, treatment of compounds 2a, b, d, and e with hydrazine hydrate or phenyl hydrazine under microwave irradiation afforded the corresponding 1,3,4-triazole derivatives 5a–c and 6a–c, respectively, as the reported methods.⁹¹⁻⁹⁴ IR spectral data of the latter triazole derivatives were devoid of any absorption bands correlated to C=O or C=S groups; instead, they exhibited absorption bands at 3288–3199 cm^–1 contributing to NH groups, 3328 cm^–1 referring to OH of compound 5c, and 1664–1639 cm^–1 due to C=N groups. Furthermore, ¹H NMR data of compounds 5 and 6 showed singlet signals at the region δ 4.23–3.50 ppm representing the presence of the two methylene protons of CH₂-ph and D₂O exchangeable signals at the range δ 7.21–12.31 ppm due to NH and OH protons, in addition to the expected multiplet signals at δ 6.60–8.38 ppm representing the aromatic protons. The −CH(CH₃)₂ residue of 5a was confirmed by the presence of doublet–multiplet signals at δ 1.06 and 3.39 ppm. ¹³C NMR spectra of compounds 5 and 6 represented singlet signals at the range δ 38.83–42.07 ppm ascribed to CH₂-ph and different signals at the region δ 102.52–170.51 ppm referring to the aromatic carbons. Also, the isopropyl residue of 5a appeared as two additional singlets at δ 22.86 and 39.84 ppm.

Furthermore, cyclization of the thiazolyl derivative 2b with urea using microwave irradiation gave the corresponding 1,3,5-triazin-2-one derivative 7. On the other hand, microwave irradiation of compounds 2a, b, d, and e with thiourea led to the formation of the corresponding 1,3,5-triazin-2-thione analogues 8a–d, respectively (Scheme 2), following the reported reactions.⁹¹⁻⁹⁴ The IR spectrum of compound 7 showed a characteristic absorption band at 1685 cm^–1 characteristic for the carbonyl group of the triazine ring, in addition to an absorption band at 3206 cm^–1 due to NH moieties. IR spectra of compound 8 exhibited the C=S group as an absorption band at the region1177–1127 cm^–1. Moreover, ¹H NMR data of compound 8 exhibited CH₂-ph protons as a singlet signal at δ 3.51–3.82 ppm, the aromatic protons as multiplet signals in the down field expected region δ 6.65–7.76 ppm, and NH and OH protons as D₂O exchangeable singlets at the region δ 7.96–12.35 ppm. The isopropyl protons of compound 8a appeared as a doublet–multiplet signal at δ 1.05 and 3.36 ppm. In addition, ¹³C NMR spectra showed signals at δ 40.06–42.15 ppm due to CH₂-ph, 22.85 and 40.15 due to −CH(CH₃)₂ of compound 8a, and 113.65–170.75 related to the aromatic carbons. Further support for the suggested structures of the new compounds was gained by their mass spectra, which were in accordance with the proposed structures representing their correct molecular ion peaks beside some other important peaks (cf. Experimental Section).

2.2. Biological Activity

2.2.1. In Vitro Evaluation of Cytotoxic Potentials of the Newly Prepared Derivatives

The newly synthesized compounds 2–8 were investigated for their potential cytotoxic activities against a panel of four different human cancer cell lines—hepatocellular carcinoma (HepG-2), prostate carcinoma (PC-3), breast adenocarcinoma (MCF-7), and non-small cell lung cancer cells (A-549)—and the normal peripheral blood mononuclear cells (PBMCs) using an MTT assay.⁹⁵ Doxorubicin and erlotinib served as reference standards. The concentrations of the tested derivatives that induced 50% inhibition of the cell viability (IC₅₀, μM) were determined and tabulated in Table 2.

Table 2. In Vitro Cytotoxic Potency of the Newly Synthesized Compounds 2–8 against Various Human Cancer Cell Lines and Normal Cells Representing SI of the Most Active Derivatives^a.

	IC50 (mean ± SEM) (μM)
compd. no.	HepG-2	PC-3	MCF-7	A-549	PBMC
2a	57.85 ± 0.07	26.82 ± 2.21	37.73 ± 0.08	34.57 ± 0.06	121.34 ± 11.35
2b	60.85 ± 0.06	44.50 ± 3.51	55.44 ± 0.04	57.48 ± 0.05	133.30 ± 13.67
2c	47.66 ± 0.04	25.44 ± 0.04	21.76 ± 0.08	27.81 ± 0.04	144.56 ± 14.89
2d	34.77 ± 0.03	39.32 ± 0.0	24.74 ± 0.08	20.84 ± 0.04	157.78 ± 16.35
2e	37.76 ± 0.05	40.56 ± 0.05	25.64 ± 0.08	25.96 ± 0.05	168.54 ± 17.36
3	37.73 ± 0.02	17.29 ± 0.03	6.59 ± 0.07	10.42 ± 0.05	179.25 ± 18.75
4	56.65 ± 0.04	25.41 ± 0.05	8.32 ± 0.04	15.61 ± 0.06	186.68 ± 19.36
5a	35.66 ± 0.04	16.49 ± 0.05	5.42 ± 0.05	10.37 ± 0.04	174.90 ± 18.24
	SI = 4.90	SI = 10.60	SI = 32.26	SI = 16.86
5b	38.49 ± 0.02	18.22 ± 0.03	4.18 ± 0.03	8.27 ± 0.03	165.76 ± 17.36
	SI = 4.30	SI = 9.09	SI = 39.65	SI = 20.04
5c	27.59 ± 0.04	18.44 ± 0.04	4.33 ± 0.04	12.30 ± 0.04	146.32 ± 16.15
	SI = 5.23	SI = 7.93	SI = 33.79	SI = 11.89
6a	10.48 ± 0.03	16.30 ± 0.09	4.30 ± 0.04	5.20 ± 0.03	157.45 ± 16.89
	SI = 15.02	SI = 9.65	SI = 36.61	SI = 30.27
6b	5.47 ± 0.02	13.21 ± 0.06	1.29 ± 0.03	3.18 ± 0.03	178.23 ± 17.69
	SI = 32.58	SI = 13.49	SI = 138.16	SI = 56.04
6c	7.73 ± 0.04	26.41 ± 0.05	2.51 ± 0.06	5.80 ± 0.05	187.67 ± 19.76
	SI = 24.27	SI = 7.10	SI = 74.76	SI = 32.35
7	29.75 ± 0.03	17.90 ± 0.03	4.65 ± 0.07	7.43 ± 0.04	165.23 ± 18.05
8a	59.84 ± 0.05	36.55 ± 0.03	13.75 ± 0.08	25.46 ± 0.05	173.45 ± 18.95
	SI = 2.91	SI = 4.74	SI = 12.61	SI = 6.81
8b	60.48 ± 0.05	42.56 ± 0.06	17.95 ± 0.04	37.21 ± 0.03	196.67 ± 20.67
	SI = 3.25	SI = 4.62	SI = 10.95	SI = 5.28
8c	45.49 ± 0.04	32.48 ± 0.04	10.31 ± 0.04	8.29 ± 0.03	188.89 ± 19.86
	SI = 4.15	SI = 5.81	SI = 18.32	SI = 22.78
8d	47.86 ± 0.05	39.55 ± 0.08	14.77 ± 0.05	9.50 ± 0.06	179.09 ± 18.96
	SI = 3.74	SI = 4.52	SI = 12.12	SI = 18.85
DOX	4.51 ± 0.26	8.11 ± 0.05	4.17 ± 0.2	8.20 ± 0.08	250.00 ± 26.56
	SI = 55.43	SI = 30.82	SI = 59.95	SI = 30.48
erlotinib	8.19 ± 0.4	8.89 ± 0.6	4.16 ± 0.2	3.76 ± 0.2	45.75 ± 26.56
	SI = 5.58	SI = 5.14	SI = 10.99	SI = 12.16

^aDOX: doxorubicin; IC₅₀: compound concentration required to inhibit the cell viability by 50%; SEM: standard error mean; each value is the mean of three independent determinations; SI: selectivity index.

Based on the resultant data, the examined compounds showed versatile antiproliferative activities against the tested cell lines. It could be noted that the benzimidazole derivatives 3 and 4, the triazole derivatives 5 and 6, and the triazine derivatives 7 and 8 elicited superior cytotoxicity against MCF-7 and A-549 cell lines. The N-phenyl-1,2,4-triazole compounds 6a–c exhibited the most potent cytotoxic activity against MCF-7 of IC₅₀ values ranging from 1.29 to 4.30 μM that were evidently near those of the reference drugs (doxorubicin and erlotinib) of IC₅₀ 4.17 and 4.16 μM, respectively. Furthermore, the latter derivatives reduced the viability of A-549 cells with 1.3–2.6-folds more potency than doxorubicin and approximately equivalent potency to erlotinib, exhibiting IC₅₀’s ranging from 3.18 to 5.80 μM and IC_{50doxorubicin, erlotinib} of 8.20 and 3.76 μM, respectively. The 3-OH-phenyl derivative 6b was 3.2-folds more potent than doxorubicin and erlotinib against MCF-7 cells and 2.6-folds more potent than doxorubicin against the A-549 cell line. The oxygen atom of the hydroxyl group might produce an additional H-binding interaction with the target protein. In addition, the HepG-2 cell line exhibited promising sensitivity against 6a–c that was slightly higher than its sensitivity against erlotinib but slightly less than that against doxorubicin, exhibiting IC₅₀’s of 5.47–10.48 μM and IC_{50doxorubicin, erlotinib} of 4.51 and 8.19 μM.

Both MCF-7 and A-549 cell lines displayed an equipotent or a slightly less sensitivity against the 1,2,4-triazole analogues 5a–c than that against the reference drugs, displaying IC₅₀’s ranging from 4.18 to 5.42 and 7.43 to 12.30 μM, respectively. Moreover, the 1,3,5-triazinone 7 was nearly equivalent to doxorubicin against MCF-7 and A-549 cell lines with IC₅₀’s of 4.65 and 7.43 μM, but it showed nearly 2-folds less potency against A-549 compared to erlotinib.

With the exception of compounds 8c and 8d that were as potent as doxorubicin against the A-549 cell line, a detectable drop in the cytotoxic activity was observed by the 1,3,5-triazin-2-thione derivatives 8 against both MCF-7 and A-549 cell lines with IC₅₀ values of 10.31–25.46 and 37.21–25.46 μM, respectively. On the other hand, the tested compounds showed moderate to weak antiproliferative activities against hepatocellular carcinoma (HepG-2) and prostate carcinoma (PC-3) cell lines. On the other hand, all the tested derivatives produced low cytotoxicity against the normal PBMC cell line with IC₅₀ values <100 μM, confirming the safety margin of the newly synthesized derivatives.

2.2.2. In Vitro Inhibition of EGFR^WT and EGFR^T790M Activity

Following the primary screening for cytotoxic potentials, the most active congeners 5a–c, 6a–c, and 7 that revealed the most promising antiproliferative activities were further investigated for their possible mechanism of actions against cancer cells. They were assessed in terms of in vitro kinase inhibitory efficiencies against the wild-type EGFR^WT and the mutant form EGFR^T790M using a homogeneous time resolved fluorescence (HTRF) assay.^96,97 The results are summarized as IC₅₀ values (μM) in Table 3 using erlotinib and AZD9291 as positive controls.

Table 3. Kinase Inhibitory Assay of the Newly Synthesized Derivatives 5–7 in Comparison with Erlotinib and AZD9291 against EGFR^WT and Mutant EGFR^T790Ma.

	IC50 (mean ± SEM) (μM)
compound no.	EGFRWT	EGFRT790M
erlotinib	0.09 ± 0.05	0.55 ± 0.10
AZD9291	0.52 ± 0.03	0.03 ± 0.01
5a	0.25 ± 0.01	0.17 ± 0.05
5b	0.22 ± 0.15	0.13 ± 0.11
5c	0.24 ± 0.30	0.14 ± 0.50
6a	0.18 ± 0.10	0.12 ± 0.18
6b	0.08 ± 0.05	0.09 ± 0.01
6c	0.15 ± 0.02	0.13 ± 0.07
7	0.22 ± 0.05	0.18 ± 0.11

^aIC₅₀: compound concentration required to inhibit the enzymes’ activities by 50%; SEM: standard error mean; each value is the mean of three independent values.

Excellent inhibitory activities were obtained by the examined compounds 5 and 6 against EGFR^WT, which were about 2–6.5 times more potent than AZD9291 representing IC₅₀ values ranging from 0.08 to 0.25 μM and IC_50AZD9291 of 0.52 μM. On the other hand, erlotinib (IC₅₀ of 0.095 μM) represented about 2.7–1.6-folds more potency against the wild form of EGFR compared with 5a–c and 6a and c. Interestingly, compound 6b appeared to be a 1-fold more potent EGFR^WT inhibitor than erlotinib with an IC₅₀ value of 0.08 μM. Additionally, the resultant data investigated that compounds 5a–c, 6a–c, and 7 were 6.1–3.2-folds more active against the mutated form of EGFR^T790M than the reference drug erlotinib, exhibiting IC₅₀’s ranging from 0.09 to 0.18 μM and IC_{50 erlotinib} of 0.55 μM. Reversely, the tested analogues 5, 6, and 7 appeared to be less potent EGFR^T790M suppressors compared to the reference drug AZD9291 with IC₅₀ of 0.03 μM. It is evident that the 3-hydroxyphenyl derivative 6b represented the most promising suppression activity against the wild and the mutant form T790M of EGFR compared with the reference standards erlotinib and AZD9291 (Figure 4). The obtained results were in agreement with the data of cytotoxicity evaluation. The docking study correlated the enhanced activity of 6b to its hydroxyl oxygen that was a site for H-bonding in the active regions of EGFR^WT and EGFR^T790M, while the N-phenyl moiety increased the hydrophobic interaction with the target enzyme.

Moreover, it could be detected that all the compounds exhibited more potent inhibitory activity against the mutant form EGFR^T790M over the wild-type form EGFR^WT, which can overcome the resistance problem to EGFR-TKIs that develops due to the T790M mutation of the EGFR gene.

2.2.3. In Vivo Inhibition of p53 Ubiquitination

The p53 protein plays a crucial role in the regulation of cancer development through its action as a suppressing molecule that binds to E3 ubiquitin ligase, thus inhibiting its role as a transcription activator.^98,99 Therefore, interfering with p53 binding on E3 ligase can interfere with tumor development and progression. Following the reported methodology,⁹⁹ the obtained results exhibited that the compounds 5–7 showed moderate inhibitory actions toward in vivo p53 ubiquitination compared to the reference diphenyl imidazole (DPI). According to Table 4, compounds 5a–c have recorded IC₅₀ values greater than the reference drug of IC₅₀’s of 0.68, 0.62, and 0.60 nM and IC_50DPI of 0.26 ± 0.005 nM. A higher potency was reported by the compounds 6a–c and 7 affording IC₅₀ values ranging from 0.59 to 0.48 nM. Accordingly, these results revealed that the tested analogues can still act as p53 ubiquitination inhibitors and thus can intervene with cancer cell growth and development.

Table 4. IC₅₀ Values Obtained Due to In Vivo p53 Ubiquitination Inhibition of MCF-7 Cells.

	IC50 (mean ± SEM) (nM)
compound no.	p53 ubiquitination
DPI	0.26 ± 0.005
5a	0.68 ± 0.01
5b	0.62 ± 0.05
5c	0.60 ± 0. 03
6a	0.59 ± 0. 01
6b	0.50 ± 0.05
6c	0.48 ± 0.02
7	0.48 0 ± 0.05

2.3. Molecular Modeling Study on EGFR^WT and Mutant EGFR^T790M

In the current docking simulation, the potent kinase inhibitors 5–7 were selected based on the potency and scaffold type to correlate the structure–activity relationship with their behavior and the possible binding interactions within the active sites of EGFR^WT and mutant EGFR^T790M. Thus, the domains of EGFR^WT and mutant EGFR^T790M kinase complexed with erlotinib and AZD9291 (PDB ID: 1M17 and 6JX0)^100,101 were downloaded from the Protein Data Bank. The docking calculations were done using MOE-Dock (Molecular Operating Environment) software version 2014.0901.^102,103 At the beginning, redocking of the native ligands (erlotinib and AZD9291) was achieved within their own binding sites of EGFR^WT and EGFR^T790M, giving energy scores −11.40 and −12.66 kcal/mol with RMDS values (root mean square deviation) of 0.91 and 1.02 Å, respectively. It was noted that the compounds 5–7 approximately displayed similar binding poses with promising energy scores that are depicted in Tables 5 and 6.

Table 5. Docking Study of Compounds 5–7 within EGFR^WT (PDB Code: 1M17) Using MOE Software Version 2014.0901.

compd. no.	docking score (kcal/mol)	amino acid residues (bond length Å)	atoms of compound	type of bond
erlotinib	–11.40	Met769(2.70)	N1(quinazoline)	H-acc
5a	–10.15	Lys721(2.92)	N-4(1,2,4-triazole)	H-acc
		Met769(2.75)	N(thiazole)	H-acc
5b	–10.32	Lys721(2.65)	N-4(1,2,4-triazole)	H-acc
		Met769(2.95)	N(linker NH)	H-acc
5c	–9.85	Lys702	phenol	arene-cation
		Lys721(2.75)	N-4(1,2,4-triazole)	H-acc
		Met769(2.60)	O(OH)	H-acc
6a	–10.50	Lys721(3.22)	N-2(1,2,4-triazole)	H-acc
		Met769(2.88)	N(thiazole)	H-acc
6b	–10.88	Val702	phenol	arene-cation
		Lys721(3.63)	N-2(1,2,4-triazole)	H-acc
		Met769(3.27)	O(OH)	H-acc
6c	–10.25	Lys721(3.20)	N-2(1,2,4-triazole)	H-acc
		Met769(3.60)	O(NO2)	H-acc
7	–10.36	Lys721(2.70)	N-3(1,3,5-triazine)	H-acc
		Met769(2.80)	N(thiazole)	H-acc

Table 6. Docking Study of Compounds 5–7 within EGFR^T790M (PDB Code: 6JX0) Using MOE Software Version 2014.0901.

compd. no.	docking score (kcal/mol)	amino acid residues (bond length Å)	atoms of compound	type of bond
AZD9291	–12.66	Val726	indole	arene-cation
		Met793(2.88)	N-1(pyrimidine)	H-acc
		Asp800(3.27)	N(N(CH3)2)	H-don
5a	–11.20	Leu718	thiazole	arene-cation
		Met793(2.95)	N-1(1,2,4-triazole)	H-don
		Met793(2.80)	N(linker NH)	H-don
5b	–10.74	Met793(3.15)	N-1(1,2,4-triazole)	H-don
		Met793(3.00)	N(linker NH)	H-don
5c	–10.70	Leu718	phenol	arene-cation
		Met793(2.77)	N-1(1,2,4-triazole)	H-don
		Met793(2.60)	N(linker NH)	H-don
6a	–11.45	Leu718	thiazole	arene-cation
		Met793(3.55)	N-4(1,2,4-triazole)	H-acc
		Met793(3.26)	N(linker NH)	H-don
6b	–11.75	Leu718	phenol	arene-cation
		Met793(3.65)	N-4(1,2,4-triazole)	H-acc
		Met793(3.06)	N(linker NH)	H-don
6c	–11.35	Leu718	2-Cl-4-NO2-C6H3	arene-cation
		Met793(3.22)	N-4(1,2,4-triazole)	H-acc
		Met793(2.90)	N(linker NH)	H-don
7	–11.20	Leu718	thiazole	arene-cation
		Met793(3.15)	N-1(1,3,5-triazine)	H-don
		Met793(2.85)	N(linker NH)	H-don

All the screened derivatives 5–7 afforded H-bonding with the key amino acids Met769 and Met793 within the active sites of EGFR^WT and mutant EGFR^T790M kinases like the original ligands erlotinib and AZD9291, respectively. Furthermore, the existence of 1,2,4-triazoles in compounds 5 and 6, in addition to the 1,3,5-triazine moiety in compound 7, potentiates fixation within the binding pockets of EGFR^WT and EGFR^T790M enzymes through extra H-bonding with Lys721 and Met793, respectively.

By focusing upon compound 6b as the most active inhibitor, it fulfilled the key interactions in the active site of EGFR^WT with energy score −10.88 kcal/mol, where hydrogen bonding was established between N-2 of the 1,2,4-triazole moiety and the side chain of Lys721 (distance: 3.63 Å), as well as the Pi-cation interaction of the phenolic ring with the Val702 residue. The presence of the H-bond acceptor between the hydroxyl oxygen and the backbone of the key amino acid Met769 improved the fitting within the active site of the enzyme (distance: 3.27 Å) (Figure 5).

Figure 5.

2D and 3D schematic binding interactions (A and B) of compound 6b into EGFR^WT (PDB code: 1M17) using the MOE software.

Regarding the docking of 6b within the ATP-binding pocket of EGFR^T790M allowing energy score −13.27 kcal/mol, it was found that N-2 of the 1,2,4-triazole scaffold and the NH linker at position-5 played a vital role in the binding through a bidentate hydrogen-bonded interaction with the backbone of the hinge Met793 (distance: 3.65 and 3.06 Å, respectively). Moreover, the phenolic ring shared fixation through Pi-cation interaction with Leu718 (Figure 6).

Figure 6.

2D and 3D schematic binding interactions (A and B) of compound 6b into EGFR^T790M (PDB code: 6JX0) using the MOE software.

The analysis of the docking results demonstrated that compound 6b with the highest EGFR^WT and EGFR^T790M inhibitory activities adopted good binding mode through its characterized structure of the 1,2,4-triazole core and the phenolic ring linked via the NH group forming hydrophilic and hydrophobic interactions.

3. Conclusions

A new set of benzimidazole, 1,2,4-triazole, and 1,3,5-triazine derivatives was designed and synthesized using microwave irradiation. The cytotoxic activity of all the new analogues was evaluated against a panel of four human cancer cell lines—HepG-2, PC-3, MCF-7, and A-549—in addition to the normal peripheral blood mononuclear cells (PBMCs) using doxorubicin and erlotinib as standard drugs. The gained results represented the significant selective cytotoxicity of some of the examined derivatives against MCF-7 and A-549 cell lines. The most potent cytotoxic activity against MCF-7 cells was revealed by the N-phenyl-1,2,4-triazole analogues 6a–c, exhibiting IC₅₀ values ranging from 1.29 to 4.30 μM that were evidently near those of the reference compounds (doxorubicin and erlotinib) of IC₅₀ of 4.17 and 4.16 μM, respectively. Moreover, A-549 cancer cells represented about 1.3–2.6-folds more sensitivity against the latter derivatives than that against doxorubicin and approximately equal sensitivity to that obtained against erlotinib exhibiting IC₅₀’s ranging from 3.18 to 5.80 μM and IC_{50; doxorubicin, erlotinib} of 8.20 and 3.76 μM, respectively. On the other hand, both MCF-7 and A-549 cell lines displayed an equipotent or a slightly less sensitivity against the 1,2,4-triazole analogues 5a–c and the 1,3,5-triazinone 7 than that against the reference drugs displaying IC₅₀’s ranging from 4.18 to 5.42 and 7.43 to 12.30 μM, respectively. With the exception of compounds 8c and 8d that were as potent as doxorubicin against the A-549 cell line, an observable decrease in the cytotoxic activity was detected in the 1,3,5-triazin-2-thione derivatives 8 against both MCF-7 and A-549 cell lines with IC₅₀ values of 10.31–25.46 and 37.21–25.46 μM, respectively. Moreover, moderate to weak antiproliferative activity against hepatocellular carcinoma (HepG-2) and prostate carcinoma (PC-3) cell lines was detected by the tested compounds. All the tested derivatives represented low cytotoxicity against the normal PBMC cell line with IC₅₀ values <100 μM, confirming the safety margin of the new derivatives.

Furthermore, the new target compounds showing the most promising anticancer activity (5, 6, and 7) were evaluated as in vitro EGFR^WT and EGFR^T790M inhibitors compared to the reference drugs erlotinib and AZD9291. Generally, the target derivatives represented a promising inhibitory effect against EGFR^WT and EGFR^T790M with more potency against the mutant form EGFR^T790M, which is a good property to overcome the EGFR-TKI resistance problem. Also, derivative 6b represented the most potent suppression effect against both EGFR^WT and EGFR^T790M.

Moreover, compounds 5–7 down-regulated the oncogenic parameter p53 ubiquitination, representing approximately an equivalent suppression potency to the reference diphenyl imidazole (DPI). The docking simulation study was performed for the promising inhibitors 5–7, giving energy scores of −11.40 and −12.66 kcal/mol with RMDS values of 0.91 and 1.02 Å, respectively. Compound 6b was chosen as a representative example to find out the binding modes of the compound in the active pocket of EGFR^WT and the mutant EGFR^T790M. It adopted promising binding interactions with the active sites of the tested proteins through its 1,2,4-triazole scaffold and the phenolic ring linked via the NH group forming various hydrophilic and hydrophobic interactions in the active pocket of the wild EGFR^WT and its mutated form EGFR^T790M.

As an overview on the obtained results, it has been investigated that 6b is a new potent antitumor agent exhibiting a safety profile against the normal cells as well as a promising inhibitory impact against EGFR^WT and EGFR^T790M. These advantages together indicated that 6b could be considered as an auspicious lead compound for the future evolution of new more potent anticancer candidates inhibiting EGFR mutations.

4. Experimental Section

4.1. Chemistry

The instruments used for measuring the melting points, spectral data (IR, mass, ¹H NMR and ¹³C NMR, X-ray) and elemental analysis are provided in detail in the Supplementary Information.

4.1.1. Synthesis of the Thiourea Derivatives 2a–e

A mixture of 2-phenylacetyl isothiocyanate (1) (0.01 mol) and different amine derivatives, namely, isopropylamine, 2-aminothiazole, o-phenylenediamine, m-aminophenol, and o-chloro-p-nitroaniline (0.01 mol) in dry acetonitrile (20 mL), was stirred at room temperature for 3 h. The solid product obtained was filtered and recrystallized from ethanol to give the corresponding thiourea derivatives 2a–e, respectively.

4.1.1.1. N-(Isopropylcarbamothioyl)-2-phenylacetamide (2a)

Pale yellow crystals; yield 98%, m.p. 70–72 °C. IR (KBr) (υ, cm^–1): 3287, 3192 (NH), 3064 (CH_arom), 2974, 2930, 2875 (CH_aliph), 1660 (C=O), 1639 (C=N), 1173 (C=S); ¹H NMR (DMSO-d₆) δ: 1.06 (d, 6H, CH₃), 3.43 (s, 2H, CH₂-ph), 3.80–3.84 (m, 1H, CH(CH₃)₂), 7.20–7.33 (m, 5H, Ar-H, J = 7.27 Hz), 7.96 (br.s, 1H, NH, D₂O exchangeable), 11.35 (br.s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 22.85, 42.89, 46.86, 126.70, 128.63, 129.33, 137.11, 169.56, 179.12; MS (70 eV) m/z (%): 236 (M⁺, 19). Anal. calcd for C₁₂H₁₆N₂Os (236.33): C, 60.99; H, 6.82; N, 11.85. Found: C, 60.86; H, 7.12; N, 11.67.

4.1.1.2. 2-Phenyl-N-(thiazol-2-ylcarbamothioyl) Acetamide (2b)

Pale brown crystals; yield 90%, m.p. 242–244 °C; IR (KBr) (υ, cm^–1): 3267, 3171 (NH), 3080 (CH_arom), 2947, 2893 (CH_aliph), 1685 (C=O), 1567 (C=N), 1166 (C=S); ¹H NMR (DMSO-d₆) δ: 3.78 (s, 2H, CH₂-ph), 7.20–7.26 (d, 2H, thiazole-H₄, H₅), 7.34–7.63 (m, 5H, Ar-H, J = 7.67 Hz), 8.01 (br.s, 1H, NH, D₂O exchangeable), 12.37 (br.s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 42.12, 113.96, 130.36, 138.06, 127.27, 128.88, 129.70, 135.47, 158.46, 169.62; MS (70 eV) m/z (%): 277 (M⁺, 18). Anal. calcd for C₁₂H₁₁N₃OS₂ (277.36): C, 51.97; H, 4.00; N, 15.15. Found: C, 51.62; H, 3.87; N, 14.86.

4.1.1.3. N-((2-Aminophenyl)carbamothioyl)-2-phenylacetamide (2c)

Yellow crystals; yield 94%; m.p. 188–190 °C; IR (KBr) (υ, cm^–1): 3370, 3327, 3135 (NH, NH₂), 3030 (CH_arom), 2950, 2830 (CH_aliph), 1690 (C=O), 1670 (C=N), 1167 (C=S); ¹H NMR (DMSO-d₆) δ: 3.82 (s, 2H, CH₂-ph), 5.01 (br.s, 2H, NH₂, D₂O exchangeable), 6.57–7.80 (m, 9H, Ar-H, J = 7.10 Hz), 11.63 (br.s, 1H, NH, D₂O exchangeable), 12.13 (br.s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 42.80, 109.96, 116.32, 127.40, 127.67, 128.87, 129.90, 132.80, 134.45, 143.70, 173.30, 180.67; MS (70 eV) m/z (%): 285 (M⁺, 14). Anal. calcd for C₁₅H₁₅N₃Os (285.37): C, 63.13; H, 5.30; N, 14.73. Found: C, 63.09; H, 5.27; N, 14.69.

4.1.1.4. N-((3-Hydroxyphenyl)carbamothioyl)-2-phenylacetamide (2d)

Beige powder; yield 98%, m.p. 260–262^o C; IR (KBr) (υ, cm^–1): 3304 (OH), 3246, 3199 (NH), 3063 (CH_arom), 2906, 2820 (CH_aliph), 1684 (C=O), 1609 (C=N), 1146 (C=S); ¹H NMR (DMSO-d₆) δ: 3.83 (s, 2H, CH₂-ph), 6.67–7.36 (m, 9H, Ar-H, J = 7.05 Hz), 9.66 (br.s, 1H, OH, D₂O exchangeable), 11.67 (br.s, 1H, NH, D₂O exchangeable), 12.41 (br.s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 39.94, 111.28, 113.86, 114.90, 127.48, 128.93, 129.95, 134.74, 139.13, 157.98, 173.75, 178.85; MS (70 eV) m/z (%): 286 (M⁺, 39). Anal. calcd for C₁₅H₁₄N₂O₂S (286.35): C, 62.92; H, 4.93; N, 9.78. Found: C, 61.21; H, 5.21; N, 9.67.

4.1.1.5. N-((2-Chloro-4-nitrophenyl)carbamothioyl)-2-phenylacetamide (2e)

Yellow powder; yield 97%, m.p. 105–107^o C; IR (KBr) (υ, cm^–1): 3275, 3198 (NH), 3091, 3009 (CH_arom), 2921, 2815 (CH_aliph), 1684 (C=O), 1625 (C=N), 1147 (C=S); ¹H NMR (DMSO-d₆) δ: 3.86 (s, 2H, CH₂-ph), 6.84–7.35 (m, 5H, Ar-H, J = 7.69 Hz), 7.94–8.63 (m, 3H, Ar-H, J = 7.31, 7.63 Hz), 12.11 (br.s, 1H, NH, D₂O exchangeable), 12.85 (br.s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 42.77, 120.43, 122.98, 125.13, 126.92, 130.02, 134.44, 136.36, 145.35, 151.83, 173.98, 180.07; MS (70 eV) m/z (%): 349 (M⁺, 29). Anal. calcd for C₁₅H₁₂ClN₃O₃S (349.79): C, 51.51; H, 3.46; N, 12.01. Found: C, 51.13; H, 3.67; N, 11.69.

4.1.2. Synthesis of N-(1H-Benzo[d]imidazol-2-yl)-2-phenylacetamide (3)

A solution of compound 2c (2.85 g; 0.01 mol) in ethyl alcohol (5 mL) was irradiated under MW for 2 min at 80 °C. After cooling at room temperature, the precipitate was filtered and recrystallized from ethanol to give the corresponding compound 3.

White crystals; yield (80%); m.p. 206–208 °C; IR (KBr) (υ, cm^–1): 3200, 3128 (NH), 3067, 3028 (CH_arom), 2910, 2820 (CH_alkyl), 1670 (C=O), 1512 (C=N); ¹H NMR (DMSO-d₆) δ: 3.76 (s, 2H, CH₂-ph), 6.57–7.80 (m, 9H, Ar-H, J = 7.28, 7.68 Hz), 11.74 (br. s, 1H, NH, D₂O exchangeable), 12.11 (br.s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 40.06, 116.32, 116.50, 127.43, 128.87, 128.90, 129.88, 130.01, 134,70, 143.66, 173.30; MS (70 eV) m/z (%): 251 (M⁺, 5). Anal. calcd for C₁₅H₁₃N₃O (251.29): C, 71.70; H, 5.21; N, 16.72. Found: C, 71.53; H, 5.03; N, 16.48.

4.1.3. Synthesis of 2-Phenyl-N-(2-phenyl-1H-enzo[d]imidazole-1-carbonothioyl)acetamide (4)

A mixture of compound 2c (2.85 g; 0.01 mol) and benzoyl chloride (0.01 mol) in ethyl alcohol (5 mL) was irradiated under MW radiation for 3 min at 80 °C, and then it was treated with cold water. The formed solid was filtered, washed with water, dried, and recrystallized from ethanol to give compound 4. Yellow crystals; yield 96%, m.p. 260–280 °C; IR (KBr) (υ, cm^–1): 3219 (NH), 3063, 3026 (CH_arom), 2960, 2820 (CH_aliph), 1699 (C=O), 1625 (C=N), 1266 (C=S); ¹H NMR (DMSO-d₆) δ: 3.91 (s, 2H, CH₂-ph),7.36–8.24 (m, 14H, Ar-H, J = 7.48, 7.94 Hz), 12.78 (br.s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 40.32, 114.05, 125.02, 128.92, 129.09, 129.37, 129.60, 129.87, 129.98, 131.99, 134.18, 144.71, 166.53, 167.0; MS (70 eV) m/z (%): 371 (M⁺, 68). Anal. calcd for C₂₂H₁₇N₃OS (371.46): C, 71.14; H, 4.61; N, 11.31. Found: C, 71.11; H, 4.58; N, 11.28.

4.1.4. Synthesis of 1,2,4-Triazole Derivatives 5a–c and 6a–c

A mixture of 2a, b, and d (0.01 mol) and hydrazine hydrate or phenyl hydrazine (0.01 mol) was heated in MW for 2–10 min at 120–150 °C in the presence of DMF as a solvent. After cooling, the reaction mixture was poured into ice water. The obtained precipitate was filtered and recrystallized from ethanol to give corresponding products 5a–c and 6a–c, respectively.

4.1.4.1. 5-Benzyl-N-isopropyl-4H-1,2,4-triazol-3-amine (5a)

White needles, yield 98%, m.p. 84–86 °C; IR (KBr) (υ, cm^–1): 3288 (NH), 3065, 3029 (CH_arom), 2975, 2930, 2874 (CH_aliph), 1639 (C=N); ¹H NMR (DMSO-d₆) δ: 1.06 (d, 6H, CH₃), 3.39 (m, 1H, CH), 3.82 (s, 2H, CH₂-ph), 7.21 (br. s, 1H, NH, D₂O exchangeable), 7.22–7.31 (m, 5H, Ar-H, J = 7.26, 7.30 Hz), 7.95 (br. s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 22.86, 39.84, 42.90, 126.69, 128.62, 129.34, 137.13, 155.40, 169.51; MS (70 eV) m/z (%): 216 (M⁺, 69). Anal. calcd for C₁₂H₁₆N₄ (216.28): C, 66.64; H, 7.46; N, 25.90. Found: C, 66.60; H, 7.40; N, 25.88.

4.1.4.2. N-(5-Benzyl-4H-1,2,4-triazol-3-yl)thiazol-2-amine (5b)

White powder; yield 96%, m.p. 220–222 °C; IR (KBr) (υ, cm^–1): 3199 (NH), 3056, 3020 (CH_arom), 2922, 2850 (CH_aliph), 1663 (C=N); ¹H NMR (DMSO-d₆) δ: 3.84 (s, 2H, CH₂-ph), 7.16, 7.25 (2d, 2H, thiazole-H₄, H₅), 7.30–7.32 (m, 5H, Ar-H, J = 7.17, 7.23, 7.30 Hz), 7.45 (br. s, 1H, NH, D₂O exchangeable), 12.31 (br. s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 42.07, 114.09, 127.39, 128.95, 129.64, 135.26, 138.04, 158.37, 159.04, 169.86; MS (70 eV) m/z (%): 257 (M⁺, 18). Anal. calcd for C₁₂H₁₁N₅S (257.31): C, 56.01; H, 4.31; N, 27.22. Found: C, 55.97; H, 4.29; N, 26.98.

4.1.4.3. 3-((5-Benzyl-4H-1,2,4-triazol-3-yl)amino)phenol (5c)

Pale brown crystals; yield 86%, m.p. 110–112 °C; IR (KBr) (υ, cm^–1): 3328 (OH), 3270, 3145 (NH), 3086, 3027 (CH_arom), 2964, 2840 (CH_aliph), 1662 (C=N). ¹H NMR (DMSO-d₆) δ: 3.50 (s, 2H, CH₂-ph), 6.62–7.31 (m, 9H, Ar-H, J = 7.37, 7.74 Hz), 8.10 (br. s, 1H, NH, D₂O exchangeable), 9.30 (br. s, 1H, NH, D₂O exchangeable), 9.60 (br. s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 38.83, 102.52, 110.41, 115.98, 125.06, 128.07, 130.15, 136.49, 143.80, 156.12, 159.54, 160.08, 165.13; MS (70 eV) m/z (%): 266 (M⁺, 81). Anal. calcd for C₁₅H₁₄N₄O (266.30): C, 67.65.17; H, 5.30; N, 21.04. Found: C, 67.40; H, 5.18; N, 20.98.

4.1.4.4. N-(3-Benzyl-1-phenyl-1H-1,2,4-triazol-5-yl)thiazol-2-amine (6a)

Brown powder, yield 95%, m.p. 170–172 °C; IR (KBr) (υ, cm^–1): 3285 (NH), 3084, 3026 (CH_arom), 2919 (CH_aliph), 1664 (C=N). ¹H NMR (DMSO-d₆) δ: 3.50 (s, 2H, CH₂-ph), 6.67, 7.10 (2d, 2H, thiazole-H₄, H₅), 7.26–7.74 (m, 10H, Ar-H, J = 7.10, 7.37 Hz), 10.23 (br. s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 40.08, 112.49, 126.99, 128.53, 128.71, 128.75, 129.12, 129.48, 129.83, 136.38, 149.75, 170.43; MS (70 eV) m/z (%): 333 (M⁺, 10). Anal. calcd for C₁₈H₁₅N₅S (333.41): C, 64.84; H, 4.53; N, 21.01. Found: C, 64.81; H, 4.50; N, 20.97.

4.1.4.5. 3-((3-Benzyl-1-phenyl-1H-1,2,4-triazol-5-yl)amino)phenol (6b)

White powder, yield 85%, m.p. 158–160 °C; IR (KBr) (υ, cm^–1): 3285 (OH), 3206 (NH), 3084, 3027 (CH_arom), 2915 (CH_aliph), 1664 (C=N); ¹H NMR (DMSO-d₆) δ: 3.51 (s, 2H, CH₂-ph), 6.66–7.73 (m, 14H, Ar-H, J = 6.68, 7.16 Hz), 9.09 (br. s, 1H, NH, D₂O exchangeable), 9.90 (br. s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 40.94, 112.52, 118.98, 127.02, 128.77, 129.15, 129.49, 136.36, 149.73, 170.51; MS (70 eV) m/z (%): 342 (M⁺, 25). Anal. calcd for C₂₁H₁₈N₄O (342.39): C, 73.67; H, 5.30; N, 16.36. Found: C, 73.96; H, 5.15; N, 15.96.

4.1.4.6. 3-Benzyl-N-(2-chloro-4-nitrophenyl)-1-phenyl-1H-1,2,4-triazol-5-amine (6c)

White powder, yield 89%, m.p. 180–182 °C; IR (KBr) (υ, cm^–1): 3284 (NH), 3084, 3026 (CH_arom), 2915, 2840 (CH_aliph), 1664 (C=N); ¹H NMR (DMSO-d₆) δ: 4.23 (s, 2H, CH₂-ph), 6.67–7.60 (m, 10H, Ar-H, J = 8.18 Hz), 8.22–8.38 (d, 2H, Ar-H, J = 7.59 Hz), 9.22 (s, 1H, Ar-H), 9.90 (br. s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 40.39, 118.43, 122.26, 124.83, 126.12, 128.04, 136.14, 138.36, 139.25, 151.83, 158.32, 162.15; MS (70 eV) m/z (%): 406 (M⁺, 49). Anal. calcd for C₂₁H₁₆ClN₅O₂ (405.84): C, 62.15; H, 3.97; N, 17.26. Found: C, 62.11; H, 3.95; N, 17.24.

4.1.5. Synthesis of 4-Benzyl-6-(thiazol-2-ylamino)-1,3,5-triazin-2(5H)-one (7)

A mixture of 2b (2.77 g; 0.01 mol) and urea (0.60 g; 0.01 mol) in DMF (5 mL) was heated under MW irradiation for 5 min at 130 °C. After cooling to room temperature, the reaction mixture was poured onto ice. The obtained precipitate was filtered and recrystallized from ethanol to give the corresponding compound 7.

Pale brown crystals, yield 97%, m.p. 242–244 °C IR (KBr) (υ, cm^–1): 3206 (NH), 3081 (CH_arom), 2950, 2885 (CH_aliph), 1686 (C=O), 1626 (C=N); ¹H NMR (DMSO-d₆) δ: 3.77 (s, 2H, CH₂-ph), 6.93–7.47 (m, 7H, Ar-H, J = 7.32 Hz), 11.19 (br. s, 1H, NH, D₂O exchangeable), 12.35 (br. s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 40.13, 113.96, 127.27, 128.88, 129.69, 135.47, 138.10150.41, 158.44, 169.63; MS (70 eV) m/z (%): 285 (M⁺, 32). Anal. calcd for C₁₃H₁₁N₅OS (285.32): C, 54.72; H, 3.89; N, 24.55. Found: C, 54.70; H, 3.86; N, 24.51.

4.1.6. Synthesis 1,3,5-Triazin-2-thione Derivatives 8a–d

An equimolar ratio of 2a, b, d, and e (0.01 mol) and thiourea (0.74 g; 0.01 mol) in DMF (5 mL) was heated in MW for 2–10 min at 130–150 °C. After cooling to room temperature, the reaction mixture was poured onto ice. The obtained precipitate was filtered and recrystallized from ethanol to give corresponding products 8a–d, respectively.

4.1.6.1. 6-Benzyl-4-(isopropylimino)-3,4-dihydro-1,3,5-triazine-2(1H)-thione (8a)

Pale brown crystals, yield 96%, m.p. 260–262 °C; IR (KBr) (υ, cm^–1): 3289, 3110 (NH), 3065, 3030 (CH_arom), 2975, 2930, 2875 (CH_aliph), 1639 (C=N), 1175 (C=S); ¹H NMR (DMSO-d₆) δ: 1.05 (d, 6H, CH₃), 3.36 (m, 1H, CH), 3.82 (s, 2H, ph-CH₂), 7.21–7.30 (m, 5H, Ar-H, J = 7.12 Hz), 7.96 (br. s, 1H, NH, D₂O exchangeable), 11.35 (br. s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 22.85, 40.15, 42.88, 126.71, 128.60, 128.64, 129.27, 129.33, 132.66, 137.11, 169.56; MS (70 eV) m/z (%): 260 (M⁺, 29). Anal. calcd for C₁₃H₁₆N₄S (260.36): C, 59.97; H, 6.19; N, 21.52. Found: C, 59.93; H, 6.15; N, 21.49.

4.1.6.2. 6-Benzyl-4-(thiazol-2-ylimino)-3,4-dihydro-1,3,5-triazine-2(1H)-thione (8b)

Brown crystals, yield 97%, m.p. 151–153 °C; IR (KBr) (υ, cm^–1): 3220 (NH), 3072, 3028 (CH_arom), 2918, 2862 (CH_aliph), 1620 (C=N), 1164 (C=S); ¹H NMR (DMSO-d₆) δ: 3.77 (s, 2H, CH₂-ph), 7.20–7.28 (m, 5H, Ar-H, J = 7.34 Hz), 7.35–7.48 (2d, 2H, thiazole-H₄, H₅, J = 7.20, 7.27 Hz), 8.33 (br. s, 1H, NH, D₂O exchangeable), 12.35 (br. s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 40.06, 113.97, 127.28, 128.88, 129.69, 135.47, 138.11, 158.42, 169.62; MS (70 eV) m/z (%): 301 (M⁺, 42). Anal. calcd for C₁₃H₁₁N₅S₂ (301.39): C, 51.81; H, 3.68; N, 23.24. Found: C, 51.78; H, 3.79; N, 23.22.

4.1.6.3. 6-Benzyl-4-((3-hydroxyphenyl)imino)-3,4-dihydro-1,3,5-triazine-2(1H)-thione (8c)

Brown crystals, yield 92%, m.p. 147–149 °C. IR (KBr) (υ, cm^–1): 3357 (OH), 3216 (NH), 3050, 3022 (CH_arom), 2925, 2815 (CH_aliph), 1603(C=N), 1177 (C=S); ¹H NMR (DMSO-d₆) δ: 3.51 (s, 2H, ph-CH₂), 6.65–7.76 (m, 9H, Ar-H, J = 6.68, 7.28 Hz), 8.73 (br. s, 1H, NH, D₂O exchangeable), 9.09 (br. s, 1H, NH, D₂O exchangeable), 9.90 (br. s, 1H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 41.90, 112.20, 113.65, 114.65, 127.40, 128.95, 129.90, 134.74, 139.13, 150.42, 156.90, 170.75; MS (70 eV) m/z (%): 310 (M⁺, 32). Anal. calcd for C₁₆H₁₄N₄OS (310.37): C, 61.92; H, 4.55; N, 18.05. Found: C, 61.89; H, 4.52; N, 18.03.

4.1.6.4. 6-Benzyl-4-((2-chloro-4-nitrophenyl)imino)-3,4-dihydro-1,3,5-triazine-2(1H)-thione (8d)

Yellow powder, yield 98%, m.p. 70–72 °C. IR (KBr) (υ, cm^–1): 3198 (NH), 3098, 3066 (CH_arom), 2923, 2850 (CH_aliph), 1625 (C=N), 1127 (C=S); ¹H NMR (DMSO-d₆) δ: 3.60 (s, 2H, CH₂-ph), 6.66–7.74 (m, 8H, Ar-H, J = 7.01 Hz), 8.73 (br. s, 1H, NH, D₂O exchangeable), 9.09 (br. s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ: 42.15, 112.28, 113.80, 114.78, 127.80, 128.90, 129.70, 135.04, 138.83, 147.60, 156.75, 169.95 ; MS (70 eV) m/z (%): 374 (M⁺, 19). Anal. calcd for C₁₆H₁₂ClN5O₂S (373.82): C, 51.41; H, 3.24; N, 18.74. Found: C, 51.39; H, 3.21; N, 18.70.

4.2. Biological Activity

4.2.1. In Vitro Cytotoxic Assay (MTT)

The potential cytotoxic properties of the prepared compounds 2–8 were evaluated against a panel of four human cancer cell lines, including hepatocellular carcinoma (HepG-2), prostate carcinoma (PC-3), breast adenocarcinoma (MCF-7), and non-small cell lung cancer cells (A-549), and the normal peripheral blood mononuclear cells (PBMCs) using the MTT assay⁹⁵ depending on the development of purple formazan crystals by mitochondrial dehydrogenases. More details were provided in Supporting Information.

4.2.2. In Vitro Inhibition Assay of EGFR^WT and Mutant EGFR^T790M Activities

The compounds that exhibited the most potent cytotoxic activity were further examined for their inhibitory activities against both EGFR^WT and EGFR^T790M. A homogeneous time resolved fluorescence (HTRF) assay^96,97 was applied in this test with EGFR^WT and EGFR^T790M (Sigma). More details were provided in Supplementary Information.

4.2.3. In Vivo Determination of p53 Ubiquitination

The potential of different prepared derivatives as potent p53 ubiquitination inhibitors was evaluated using the standard procedure and protocol previously applied.^98,99 Briefly, cells were allowed to grow for 24 h to reach 50% confluency. Thereafter, of 1 μg p53, 4 μg MDM2 and 1 μg HIS-ubiquitin were transfected with the Gene Juice reagent, and then cells were grown for another 20 h. More details were provided in Supplementary Information.

4.3. Molecular Modeling Study on EGFR^WT and Mutant EGFR^T790M

The domains of EGFR^WT and mutant EGFR^T790M kinase complexed with erlotinib and AZD9291 (PDB ID: 1M17 and 6JX0)^100,101 were downloaded from the Protein Data Bank. The docking calculations were done using MOE-Dock (Molecular Operating Environment) software version 2014.0901.^88,89 More details were provided in the Supplementary Information.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c06836.

• Experimental section; chemistry, in vitro cytotoxic assay (MTT), in vitro inhibition assay of EGFR^WT and mutant EGFR^T790M activities, in vivo determination of p53 ubiquitination, molecular modeling study on EGFR^WT and mutant EGFRT790M, ¹H-NMR spectra of the new compounds, ¹³C-NMR spectra of the new compounds, IR spectra of the new compounds, and mass spectra of the new compounds (PDF)

The authors declare no competing financial interest.