Design, synthesis, in vitro biological assessment and molecular modeling insights for novel 3-(naphthalen-1-yl)-4,5-dihydropyrazoles as anticancer agents with potential EGFR inhibitory activity

Wagdy M Eldehna; Mahmoud A El Hassab; Zainab M Elsayed; Tarfah Al-Warhi; Hazem Elkady; Mahmoud F Abo-Ashour; Mohammed A S Abourehab; Ibrahim H Eissa; Hatem A Abdel-Aziz

doi:10.1038/s41598-022-15050-8

. 2022 Jul 27;12:12821. doi: 10.1038/s41598-022-15050-8

Design, synthesis, in vitro biological assessment and molecular modeling insights for novel 3-(naphthalen-1-yl)-4,5-dihydropyrazoles as anticancer agents with potential EGFR inhibitory activity

Wagdy M Eldehna ^1,^2,^✉, Mahmoud A El Hassab ³, Zainab M Elsayed ⁴, Tarfah Al-Warhi ⁵, Hazem Elkady ⁶, Mahmoud F Abo-Ashour ⁷, Mohammed A S Abourehab ⁸, Ibrahim H Eissa ⁶, Hatem A Abdel-Aziz ⁹

PMCID: PMC9329325 PMID: 35896557

Abstract

Currently, the humanity is in a fierce battle against various health-related challenges especially those associated with human malignancies. This created the urge to develop potent and selective inhibitors for tumor cells through targeting specific oncogenic proteins possessing crucial roles in cancer progression and survive. In this respect, new series of pyrazole-thiazol-4-one hybrids (9a–p) were synthesized as potential anticancer agents. All the synthesized molecules exhibited potent antiproliferative actions against breast cancer (BC) T-47D and MDA-MB-231 cell lines with IC₅₀ ranges 3.14–4.92 and 0.62–58.01, respectively. Moreover, the most potent anti-proliferative counterparts 9g and 9k were assessed against EGFR. They displayed nanomolar inhibitory activity, IC₅₀ 267 ± 12 and 395 ± 17 nM, respectively. Worth noting, both compounds 9g and 9k induced apoptosis in MDA-MB-231 cells, and resulted in a cell cycle arrest at G2/M phase. Furthermore, an in silico analysis including docking and molecular dynamic simulations was performed.

Subject terms: Medicinal chemistry, Medicinal chemistry, Oncology

Introduction

In 2020, 2.3 million women were affected by breast cancer with nearly 685,000 deaths killed by the same disease worldwide. As the year ended, a report of the past 5 years, revealed 7.8 million alive women who diagnosed with breast cancer, giving it rise to be the most prevalent cancer globally¹. More than 90% of BCs are not metastatic upon diagnosis. For breast cancer patients that diagnosed without metastatic disease, the main therapeutic objectives are eradication of the tumor and preventing recurrence. In many cases, patients with nonmetastatic breast cancer can be treated with chemotherapy as a systemic therapy².

RTKs (receptor tyrosine kinases) are found on the cells surface and represent a type of receptor that plays a significant role in the genesis of tumors. RTKs are known to influence cancer stemness, angiogenesis, and metastasis through a variety of downstream signaling pathways³. RTKs are a good target for breast cancer treatment because of their several activities⁴. However, anti-RTK therapy is complicated by the mutations that are created in these biological targets⁵. Different types of RTKs are over-expressed in various human malignancies, including breast cancer, they are namely epidermal growth factor receptors (EGFRs), platelet-derived growth factor receptors (PDGFRs), vascular endothelial growth factor receptors (VEGFRs), fibroblast growth factor receptors (FGFRs) and insulin-like growth factor receptors (IGFRs)^3,6–8. Overexpression of RTKs are associated with increased breast cancer aggressiveness⁸.

EGFR belongs to the ErbB family of RTK. These proteins are stimulated after binding with EGF-family of proteins. Many reports confirmed that the EGFR is involved in the pathogenesis of different cancer types⁹. The EGFR and its agonists are over-expressed in human malignancies producing cell transformation¹⁰. All EGFR types are expressed in breast cancer. EGFR has been reported to be over-expressed in about 14% of human breast tumors¹¹.

Many EGFRs small molecule inhibitors (EGFRIs) have been developed. In comparison to other ErbB receptors, Gefitinib I (Fig. 1) is a reversible EGFRI with a 200-fold higher affinity for EGFR. It's been approved for the management of individuals with breast tumors and NSCLC who haven't responded to chemotherapies^12,13. Erlotinib II (Fig. 1) is a reversible EGFRI that has been approved by the FDA for the management of NSCLC and metastatic pancreatic malignancy¹⁴. Gefitinib as well as Erlotinib work as ATP analogues through competing with the EGFR receptors' ATP binding spaces¹⁵. Despite the fact that EGFRIs have significant clinical outcomes in treating patients with breast cancer, tumor cells rapidly develop acquired resistance, in which the efficacy of EGFR-targeted therapy is highly limited¹⁶ (Fig. 1).

(A) The reported pharmacophoric features of EGFRIs. (B) Reported FDA EGFR inhibitors (Gefitinib and Erlotinib) and the synthesized compounds in this study (**9a-p**), as well as the binding mode of Erlotinib within EGFR active site.

As depicted by Fig. 1, some pharmacophoric features of EGFR inhibitors are required for maximum affinity against the EGFR ATP binding site. These features include: (1) a central flat aromatic heterocyclic moiety that should be accommodated within the adenine binding pocket to interact with Leu768, Met769, Gln767 and Thr766¹⁷, (2) a terminal hydrophobic head which should occupy the hydrophobic region I to interact with Glu738, (3) a spacer of one atom (NH, spacer) to allow the terminal hydrophobic motif to access the hydrophobic region I¹⁸, (4) a hydrophobic tail that can occupy the hydrophobic region II^19,20. Research about the ribose binding pocket has been limited²¹ (Fig. 1).

Literature investigation revealed that pyrazole moiety is a lead building block in many anticancer molecules through the inhibition of diverse RTKs including EGFR^22,23. In addition, thiazol-4(5H)-one was reported as promising scaffold in the discovery of new antineoplastic agents targeting RTKs especially EGFR^22,24.

Accordingly, the aim of this study is summarized on the design and synthesis of pyrazole- thiazol-4(5H)-one hybrids (9a-p, Fig. 1) having the essential pharmacophoric features of EGFRIs. In such design, the 2-(4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-one moiety was utilized as an aromatic heterocyclic system to be convenient for the adenine binding pocket. In addition, diverse substituted phenyl rings were used as a hydrophobic head to accommodate within the hydrophobic pocket. Furthermore, the naphthyl moiety was used as a hydrophobic tail to occupy a second hydrophobic pocket. In this design, we gave two modifications on the reported EGFRIs. The first one, the NH linker was replaced by –CH= group as a chemical isostere. The second modification was the grafting of different aromatic substituents to explore the effect of such substituents on the biological activities (Fig. 1).

The synthesized molecules were examined as anti-proliferative agents against 2 BC MDA-MB-231 and T-47D cell lines. Then, the most promising members that showed superior cytotoxic activity were further evaluated for their EGFR inhibitory activity. In addition, the effects of these active members against cell cycle and apoptosis induction were examined. Finally, Molecular modelling studies including docking and molecular dynamic simulations were carried out to examine the binding mode of herein reported pyrazole- thiazol-4(5H)-one hybrids against their proposed biological target (EGFR).

Results and discussion

Chemistry

The adopted strategy to prepare the herein proposed pyrazole- thiazol-4(5H)-one hybrids 9a-p was illustrated in Scheme 1. 1-(Naphthalen-1-yl)ethan-1-one 1 was condensed with the appropriate 4-fluorobenzaldehyde 2a or 4-chlorobenzaldehyde 2b in a basic medium through the Claisen-Schmidt condensation reaction to furnish 3-(4-fluorophenyl)-1-(naphthalen-1-yl)propen-1-one 3a and 3-(4-chlorophenyl)-1-(naphthalen-1-yl)propen-1-one 3b, respectively.

Chalcones 3a-b were then subjected to heterocyclization to produce 3-(naphthalen-1-yl)-4,5-dihydro-pyrazole-1-carbothioamide derivatives 5a-b via refluxing in absolute ethanol with thiosemicarbazide and sodium hydroxide. The previously prepared thioamide compounds 5a-b were heated under reflux in absolute ethanol with ethyl bromoacetate 6 to get the two key intermediates 3-(naphthalen-1-yl)-4,5-dihydropyrazol-1-yl)thiazol-4(5H)-one derivatives 7a-b. Finally, the pyrazole- thiazol-4(5H)-one intermediates 7a-b were reacted with different aldehydes 8a-h via the Knoevenagel condensation by heating in glacial acetic acid and fused sod. acetate to yield the targeted pyrazole- thiazol-4(5H)-one hybrids 9a-p.

Spectral and elemental analyses data supported the proposed structure for target pyrazole- thiazol-4(5H)-one hybrids 9a-p herein reported. ¹H NMR spectra for pyrazolines 9a-p showed three doublet of doublet signals of pyrazoline ring; one for CH and two for CH₂ about δ (5.90) and (3.67 and 4.39) ppm, respectively with three reciprocal values for coupling constant (4.0, 11.2, 18.0) Hz. Moreover, ¹H NMR spectra for compounds 9d-h and 9l-p confirmed their structure through the presence of additional aliphatic singlet signal for OCH₃ around δ 3.80 ppm, whereas, the ¹H NMR spectra of pyrazole- thiazol-4-one hybrids 9c and 9k revealed the presence of another aliphatic signal for (N(CH₃)₂) around δ 3.01 ppm. In turn, the ¹³C NMR spectra for the targeted pyrazole- thiazol-4-one hybrids 9a-p disclosed the presence of aliphatic signals of pyrazoline ring around (46.55 and 62.40) ppm, for CH₂ and CH, respectively, in addition, the signal of (C = O) functional group appeared around δ 179.90 ppm. Lastly, the ¹³C NMR spectra of pyrazolines 9d-h and 9l-p disclosed the presence of the methoxy group around δ 56.0 ppm.

Biological evaluation

In vitro anti-proliferative activity

The cytotoxic effects of herein prepared pyrazole- thiazol-4-one hybrids 9a-p against BC MDA-MB-231 and T-47D cell lines, obtained from American Type Culture Collection (ATCC), were determined using the MTT assay protocol. It is worth to mention that the selected cancer cell lines in this study are EGFR-expressing cell lines^25–28. Doxorubicin and Erolitinib were utilized as positive control (Table 1).

Table 1.

Anti-proliferative activity of pyrazolines 9 BC MDA-MB-231 and T-47D cancer cell lines, as well as towards breast MCF-10A normal cells.


Com	X	R	R₁	R₂	IC₅₀ (µM)^a
Com	X	R	R₁	R₂	MDA-MB-231	T-47D	MCF-10A
9a	F	H	H	H	19.84 ± 1.08	NA^b	ND^c
9b	F	H	H	OH	12.24 ± 0.67	18.61 ± 0.87	ND^c
9c	F	H	H	N(CH₃)₂	2.54 ± 0.138	8.45 ± 0.27	47.03 ± 3.52
9d	F	OMe	H	H	45.02 ± 2.45	NA^b	ND^c
9e	F	H	H	OMe	14.13 ± 0.77	12.37 ± 0.33	ND^c
9f.	F	OH	OMe	H	3.40 ± 0.185	27.1 ± 1.08	42.60 ± 2.81
9g	F	H	OH	OMe	0.62 ± 0.03	3.14 ± 0.11	31.74 ± 3.10
9h	F	H	OMe	OH	5.82 ± 0.317	11.42 ± 0.67	79.45 ± 5.27
9i	Cl	H	H	H	24.68 ± 1.35	NA^b	ND^c
9j	Cl	H	H	OH	6.24 ± 0.34	35.10 ± 2.04	65.37 ± 5.04
9k	Cl	H	H	N(CH₃)₂	1.14 ± 0.06	4.92 ± 0.28	58.51 ± 4.36
9l	Cl	OMe	H	H	58.01 ± 3.16	NA^b	ND^c
9m	Cl	H	H	OMe	18.95 ± 1.03	27.63 ± 1.24	ND^c
9n	Cl	OH	OMe	H	35.15 ± 1.916	67.29 ± 2.51	ND^c
9o	Cl	H	OH	OMe	10.81 ± 0.59	7.36 ± 0.30	ND^c
9p	Cl	H	OMe	OH	6.55 ± 0.357	19.47 ± 0.92	83.62 ± 6.74
Doxorubicin	–	–	–	–	3.67 ± 0.20	2.73 ± 0.09	ND^c
Erolitinib	–	–	–	–	2.88 ± 0.17	3.61 ± 0.24

Open in a new tab

^aIC₅₀ value is the mean ± S.D. of 3 separate experiments.

^bNA: Derivatives with IC₅₀ more than 100 mM.

^cND: Not determined.

Generally, the obtained results disclosed that MDA-MB-231 cell line was more susceptible to the influence of the tested members than T-47D. Also, it was noticed that the 4-fluorophenyl derivative 9g (IC₅₀ = 0.62 ± 0.03 against MDA-MB-231 and 3.14 ± 0.11 µM against T-47D) and the 4-chlorophenyl derivative 9k (IC₅₀ = 1.14 ± 0.06 against MDA-MB-231 and 4.92 ± 0.28 µM against T-47D) were the most potent cytotoxic members against the two breast cancer cell lines, comparing to the positive control, doxorubicin (IC₅₀ = 3.67 ± 0.2 and 2.73 ± 0.09 µM against MDA-MB-231 and T-47D, respectively), Table 1.

According to all results, it can be concluded that the 4-fluorophenyl derivatives 9a-h were slightly more advantageous than the 4-chlorophenyl derivatives 9i-p against the explor cell lines.

Regarding the activity toward MDA-MB-231 BC cell line, hybrids 9c, 9f, 9h, 9j and 9p displayed good cytotoxicity with (IC₅₀ = 2.54 ± 0.138, 3.40 ± 0.185, 5.82 ± 0.317, 6.24 ± 0.34 and 6.55 ± 0.357 μM, respectively). In the meantime, compounds 9a, 9b, 9e, 9m, and 9p, with IC₅₀ = 19.84 ± 1.08, 12.24 ± 0.67, 14.13 ± 0.77, 18.95 ± 1.03 and 10.81 ± 0.59 μM, respectively, exhibited moderate cytotoxicity, whereas, pyrazolines 9d, 9i, 9l, and 9n with IC₅₀ ranging from 35.15 ± 1.916 to 58.01 ± 3.16 μM displayed the lowest cytotoxic activity.

Additionally, cytotoxicity evaluation against T-47D breast cancer cell line highlighted that compounds 9c and 9o displayed good anticancer actions with IC₅₀ values of 8.45 ± 0.27 and 7.36 ± 0.30 μM, respectively. In turn, pyrazolines 9b, 9e, 9h, and 9p with (IC₅₀ ranging from 11.42 ± 0.67 to 19.47 ± 0.92 μM) exhibited moderate cytotoxicity. Besides, compound 9f, 9j, 9m, and 9n with IC₅₀ ranging from 27.1 ± 1.08 to 67.29 ± 2.51 μM demonstrated weak cytotoxic activity. Finally, pyrazolines 9a, 9d, 9i, and 9l appeared to be inactive.

Next, we inspected the influence of the substitution on the benzylidine moiety on the cytotoxic activities. Substitution of the benzylidine moiety at C-4 with hydrophilic or lipophilic electron donating groups (like the hydroxyl, methoxy and N,N-dimethylamino groups) resulted in an enhanced cytotoxic activities in comparison to the counterparts bearing unsubstituted benzylidine motif. In details, compounds 9b, 9c and 9e bearing para-substituted benzylidine motifs showed better activity against both MDA-MB-231 (IC₅₀ = 12.24 ± 0.67, 2.54 ± 0.138 and 14.13 ± 0.77 μM, respectively) and T-47D (IC₅₀ = 18.61 ± 0.87, 8.45 ± 0.27 and 3.14 ± 0.11 μM, respectively) cell lines than the unsubstituted analogue 9a (IC₅₀ = 19.84 ± 1.08 and > 100 μM, toward MDA-MB-231 and T-47D cells respectively). Similarly, pyrazolines 9j, 9k and 9m exerted more potent activity towards both MDA-MB-231 (IC₅₀ = 6.24 ± 0.34, 1.14 ± 0.06 and 18.95 ± 1.03 μM, respectively) and T-47D (IC₅₀ = 35.10 ± 2.04, 4.92 ± 0.28 and 27.63 ± 1.24, respectively) cell lines than the corresponding unsubstituted counterpart 9i (IC₅₀ = 24.68 ± 1.35 and > 100 μM, toward MDA-MB-231 and T-47D cells respectively).

It’s noteworthy mentioning that moving the methoxy group from para to ortho position led to a decrease of the cytotoxic influence against both the tested cancer cell lines; hybrids 9d (IC₅₀ = 45.02 ± 2.45 and > 100 μM) and 9l (IC₅₀ = 58.01 ± 3.16 and > 100 μM) vs. compounds 9e (IC₅₀ = 14.13 ± 0.77 and 12.37 ± 0.33 μM) and 9m (IC₅₀ = 18.95 ± 1.03 and 27.63 ± 1.24 μM). Moreover, it was found that the best di-substitution pattern (hydroxyl and methoxy substitution) for the benzylidene moiety is the 3-hydroxy-4-methoxy pattern for the 4-fluorophenyl bearing derivatives 9a-h and the 4-chlorophenyl bearing derivatives 9i-p against both the examined cell lines, except the 4-hydroxy-3-methoxy substitution in compound 9p toward MDA-MB-231 cell line.

Furthermore, the cytotoxic impact for the most potent anti-proliferative thiazolyl pyrazolines reported in this work against MDA-MB-231 cell line (9c, 9f., 9g, 9h, 9j, 9k and 9p; with IC₅₀ value less than 10 µM) was evaluated toward the normal human MCF-10A cells (Table 1). The examined pyrazolines (9c, 9f., 9g, 9h, 9j, 9k and 9p) exerted weak to non-significant activities against MCF-10A cell line (IC₅₀ range: from 31.74 ± 3.10 to 83.62 ± 6.74), which highlights their safety and selectivity toward the cancer cells.

EGFR inhibitory activity

From the aforementioned data presented in Table 1 concerning the cytotoxicity, compounds 9g and 9k were identified as the most promising members among the series and showed superior cytotoxicity against MDA-MB-231 and T-47D cell lines. So, compounds 9g and 9k were further evaluated for their potential EGFR inhibitory action, using erlotinib as a reference EGFR inhibitor.

Table 2 shows the data as IC₅₀ values. The results displayed that the two candidates exhibited EGFR inhibitory activity at the nanomolar level. In comparison to erlotinib (IC₅₀ = 57 ± 3 nM), compounds 9g and 9k exhibited good activity as EGFR inhibitors in nanomolar concentrations (IC₅₀ = 267 ± 12 and 395 ± 17 nM, respectively).

Table 2.

In vitro inhibitory activity of hybrids 9g and 9k towards EGFR.


Compound	IC₅₀ (nM)
Compound	EGFR
9g	267 ± 12
9k	395 ± 17
Erlotinib	57 ± 3

Open in a new tab

Cell cycle analysis

In this work, MDA-MB-231 cells were treated with the most cytotoxic members (compounds 9g and 9k) at a concentration of 0.62 and 1.14 µM, respectively (corresponding to their IC₅₀ values against MDA-MB-23 cells). Investigating the results, it can be noticed that compounds 9g and 9k induced a significant increase in the cell population at Sub-G1 phase (31.05% and 27.86%, respectively), comparing to control cells (2.13%). For the S phase, the percentage of MDA-MB-23 cells was increased from 32.11% in control cells to 35.43% for compound 9g while the % was decreased to 28.45% for compound 9k.

Moreover, compounds 9g and 9k exhibited insignificant decrease in the cell population (31.0217% and 40.61%, respectively) at G0-G1 phase, comparing to the control cells (53.45%). Additionally, a marked decrease in the cell population for compounds 9g (2%) and 9k (3.08%) was observed at the G2/M phase, comparing to control cells (12.31%). These findings verify that the cytotoxicity of compounds 9g and 9k against MDA-MB-231cells was due to arresting the cell growth at G2/M phase (Fig. 2, Table 3).

Effect of 9g and 9k on the phases of cell cycle of MDA-MB-231 cells.

Table 3.

Influence of pyrazole-thiazol-4-one hybrids 9g and 9k on the cell cycle phases of MDA-MB-231 cells.

Com	%G0-G1	%S	%G2/M	%Sub-G1
9g	31.0217	35.43	2	31.05
9k	40.61	28.45	3.08	27.86
Control	53.45	32.11	12.31	2.13

Open in a new tab

Apoptosis assay

The most potent anticancer agents in the current work 9g and 9k were chosen for the assessment of apoptosis in MDA-MB-231cell line using Annexin V/propidium iodide (PI) double staining assay method. In this method, MDA-MB-231 cells were incubated with compounds 9g and 9k at the IC₅₀ concentrations (0.62 and 1.14 µM, respectively) for 48 h.

The results revealed that compounds 9g and 9k triggered more apoptotic cells in comparison to control cells. In particular, pyrazoline 9g induced apoptosis by 32.32% (early apoptosis = 8.39% & late apoptosis = 23.93%) while pyrazoline 9k induced apoptosis by 34.22% (early apoptosis = 14.22% & late apoptosis = 20%) compared to 0.94% in the control cells (0.73% and 0.21% for early and late apoptosis, respectively). From these findings, we concluded that compounds 9g and 9k could induce apoptosis in MDA-MB-231 cells (Fig. 3 and Table 4).

Effect of pyrazolines 9g and 9k on the percentage of annexin V-FITC-positive staining in MDA-MB-231 cells.

Table 4.

Distribution of apoptotic cells within MDA-MB-231 cell line upon treatment with 9g and 9k, using AnnexinV-FITC/PI assay.

Com	Early apoptosis (lower right %)	Late apoptosis (upper right %)	Total (L.R % + U.R %)
9g	8.39	23.93	32.32
9k	14.22	20	34.22
Control	0.73	0.21	0.94

Open in a new tab

Modeling studies

Docking study

Elucidating the potential binding mode between new compounds and their target became inevitable in lead discovery studies to provide futuristic insights for further optimization. Accordingly, both compounds 9k and 9g were docked into the predetermined active site of EGFR using MOE 2019.02.

Exploring the binding mode of the crystal reference erlotinib with the EGFR has revealed the essential hydrogen bond interactions with the key residues Met 769 and Leu 768. Besides, the phenyl and quinazoline rings of erlotinib participated in carbon hydrogen bond interactions with Glu 738 and Gln 767, respectively. The importance of solvent molecules in EGFR active site was unequivocal, acting as a bridge for two interactions between the nitrogen of quinazoline ring with Cys 751 and Thr 766, Fig. 4.

The binding mode of the crystal reference erlotinib with the EGFR receptor.

Interestingly, the docking of both compounds 9k and 9g induced a favorable binding upon interacting with EGFR through a binding pattern perfectly aligned with that of erlotinib. The synthesized compounds 9g and 9k achieved docking scores of −11.8 and −11.4 kcal/mol respectively, that nearly match the docking score of erlotinib (− 12.0 kcal/mol). As depicted from Fig. 5, the carbonyl group of the thiazol-4(5H)-one ring in compound 9g acted as a hydrogen bond acceptor for 2 H-bonds with the key residues Met 769 and Leu 768, in addition the para methoxy group of compound 9g was engaged in a carbon hydrogen bond interaction with Glu738. Once again, the water molecules played a crucial role in linking compound 9g with Thr766 and Gln767.

The 2D interaction diagram of EGFR receptor with 9g (left) 9k (right).

In a similar manner, compound 9k engaged in two hydrogen bonds via its carbonyl group with the essential residues Met 769 and Leu 768. The N,N-dimethyl aniline ring of compound 9k involved in one hydrogen bond with Lys 721 and three carbon hydrogen bonds with Glu738, Asp831 and a water molecule. Unlike compound 9g, the water effect was less significant in case of compound 9k, which explains the superiority of compound 9g in docking score and biological activity. Noteworthy, the difference in the kinase activity of compounds 9g and 9k could be attributed to the grafting of different substituents (para-fluoro in 9g and para-chloro in 9k) to 4,5-dihydro-1H-pyrazolyl ring. The large size of the chlorine atom hinders the ability of compound 9k to get closer enough with the solvent molecules unlike the smaller fluorine atom that allows compound 9g to get closer enough to the water molecule attached to the Thr766 and Gln767, Fig. 5–6.

The 3D interaction diagram of EGFR receptor with 9g (left) 9k (right).

Molecular dynamics

RMSD and RMSF analysis

In the current study, further in silico investigations were achieved through molecular dynamic simulations. Molecular dynamics (MD) simulation provides many valuable information and parameters to study the dynamicity of biological systems. Amongst these information, MD could provide insights into precise estimation of the binding strength of a docked complex of a ligand and a target. Accordingly, the predicted binding co-ordinates retrieved from the docking of EGFR with 9g, 9k and erlotinib were moved forward to MD simulation. To provide a comparative mean for the effect of each ligand on the stability of the EGFR enzyme, the later was subjected to MDS using the Apo form.

As demonstrated by Fig. 7, all the three inhibitors were able to stabilize the EGFR enzyme as indicated by their lower RMSD values comparing to the RMSD value of Apo EGFR. The EGFR-9g and EGFR-9k complexes had RMSD values of 1.8 and 2.1 Å, respectively, that are very comparable to that of EGFR-erlotinib (1.75 Å), while the RMSD of the Apo EGFR reached 4.5 Å. In cancer cells, EGFR serves as the switch-on for the intracellular signaling pathways to trigger cell division and uncontrolled growth. In this respect, the high dynamicity seen in the Apo EGFR as discerned from the high RMSD values is perfectly aligned with its intended oncogenic function. The capability of compounds 9g and 9k to restrict the dynamic nature of the EGFR via the formation of stable complexes as indicated by the lower RMSD values is a valid indicator for their inhibitory impact on EGFR (Fig. 7).

To further endorse the outputs of the RMSD calculations, the RMSF values for all the residues in the four systems have been computed. As expected the RMSF values lead to the same conclusion retrieved from the RMSD calculations in which the average RMSF of the Apo EGFR residues reached 4.2 Å, while the average RMSF values for the EGFR residues in complex with 9g, 9k and erlotinib reached an average 1.8, 2.1, 1.7 Å respectively (Fig. 8). To this end, both RMSD and RMSF values validated the proposed binding mode between 9g and 9k with the EGFR active site and attributed the inhibitory activity of both molecules to their ability to form a stable complex with the EGFR.

The RMSF analysis for the MD simulations.

Binding free energy calculations using MM-PBSA approach

Attempting to further evaluate the strength of binding between the EGFR enzyme and the newly prepared compound 9g and 9k, the g_mmpbsa package generated by Kumari et al. was brought in action to calculate the binding free energies between the EGFR enzyme and the proposed molecules 9g and 9k. The generated trajectories from the production stage were used to calculate all the forms of binding free energy. These energy types include Electrostatic energy, van der Waal energy, Polar solvation energy and SASA energy. All the previous types of energy were calculated to the three complexes containing EGFR bound to either 9g, 9k or erlotinib (Table 5).

Table 5.

The binding free energies of 9g, 9k and erlotinib in complex with EGFR.

Complex	ΔE_binding (kJ/mol)	ΔE_{Electrostatic} (kJ/mol)	ΔE_{polar solvation} (kJ/mol)	ΔE_{Vander Waal} (kJ/mol)	SASA (kJ/mol)
9g	− 288.1 ± 4.9	− 123.3 ± 5.2	87.1 ± 1.2	− 224.3 ± 7.5	− 27.6 ± 0.2
9k	− 251.9 ± 3.8	− 105.7 ± 3.1	72.1 ± 1.7	− 196.1 ± 6.1	− 22.2 ± 0.2
Erlotinib	− 286.8 ± 4.4	− 118.3 ± 3.8	88.5 ± 2.4	− 230.9 ± 6.4	− 26.1 ± 0.4

Open in a new tab

Interestingly, the calculated binding free energy for the three small molecules were comparable to each other in which compound 9g and 9k achieved binding free energies of − 288.1 ± 4.9 and − 251.9 ± 3.8 (kJ/mol) respectively, whereas the crystal reference erlotinib achieved − 286.8 ± 4.4 (kJ/mol). These results augmented all the in silico calculations giving credit to the predicted binding mode of both 9g and 9k within EGFR. Moreover, all the MD and energy calculations favored compound 9g over compound 9k as consistent with the early results of enzyme assay.

In terms of binding energy the energy contribution for each residue was calculated to provide deep insights into the effect of compounds 9g and 9k upon engaging in the binding pocket of EGFR. This contribution was calculated through decomposing the total binding free energy of each complex into per residue contribution energy. As demonstrated by Fig. 9, the binding of compound 9g to the EGFR active site resulted in favor energy contribution in all the Key residues especially those of close contact to compound 9g such as Met769 and Leu768 Glu738 Thr766 and Gln767. In a similar fashion, the binding of compound 9k contributed favorably to the free energy of the surrounding residues significantly to Met769, Leu768 and Glu738 and in lesser extent to Thr766 and Gln767. Consistent with the results of docking, MD and MMPBSA analyses, the per residue decomposition results supported the predicted binding mode of compounds 9g and 9k with EGFR kinase.

The per residue contribution for the binding free energy of 9g and 9k with EGFR.

Conclusions

In the current work, new series of pyrazole-thiazol-4(5H)-one hybrids (9a-p) were synthesized as potential anti-breast cancer molecules. The anti-proliferative activities for the synthesized molecules (9a-p) were evaluated against MDA-MB-231 and T-47D BC cell lines. The obtained results showed that MDA-MB-231 cell line was more sensitive to the influence of the tested members than T-47D. Also, it was noticed that the 4-fluorophenyl derivative 9g (IC₅₀ = 0.62 ± 0.03 against MDA-MB-231 and 3.14 ± 0.11 µM against T-47D) and the 4-chlorophenyl derivative 9k (IC₅₀ = 1.14 ± 0.06 against MDA-MB-231 and 4.92 ± 0.28 µM against T-47D) were the most potent cytotoxic members against the two BC cell lines, comparing to the positive control, doxorubicin (IC₅₀ = 3.67 ± 0.2 and 2.73 ± 0.09 µM against MDA-MB-231 and T-47D, respectively). Accordingly, both compounds 9g and 9k were tested for their activity against EGFR, where their achieved nanomolar inhibitory activity; IC₅₀ = 267 ± 12 and 395 ± 17 nM for 9g and 9k, respectively. Further investigations unveiled their ability to arrest cell cycle at G2/M phase in addition to apoptosis induction in the MDA-MB-231 cell line as evidenced by Annexin V analysis. Interestingly, the docking of both compounds 9g and 9k induced a favorable binding upon interacting with EGFR through a binding pattern perfectly aligned with the crystal reference erlotinib. Compound 9g achieved docking score (− 11.8 kcal/mol) favorable than 9k (− 11.4 kcal/mol) and both scores are close to the docking score of erlotinib (− 12.0 kcal/mol). The docking results were further endorsed by 200 ns of molecular dynamic simulations alongside with MM-PBSA calculations and both revealed that compound 9g had more stable complex with EGFR than 9k with RMSD of 1.8, Å and binding free energy of − 288.1 ± 4.9 kJ/mol. The results of per residue decomposition supported predicted binding mode of compounds 9g and 9k with EGFR enzyme and showed favorable energy contribution of the key residues in the active site especially upon the binding compound 9g. The overall molecular modeling results attributed the superiority of compound 9g to the smaller size of the fluorine substituent that enables perfect fitting of compound 9g in EGFR active pocket.