Novel 2-(5-Aryl-4,5-Dihydropyrazol-1-yl)thiazol-4-One as EGFR Inhibitors: Synthesis, Biological Assessment and Molecular Docking Insights

Introduction Epidermal growth factor receptor (EGFR) regulates several cell functions which include cell growth, survival, multiplication, differentiation, and apoptosis. Currently, EGFR kinase inhibitors are of increasing interest as promising targeted antitumor therapeutic agents. Methods Different thiazolyl-pyrazoline derivatives (7a-o) were synthesized and were first tested for anti-proliferative effect towards the A549 lung cancer cell line and the T-47D breast cancer cell line in MTT assay. Thereafter, thiazolyl-pyrazolines (7b, 7g, 7l, and 7m) were subsequently evaluated for their PK inhibition for EGFR. Moreover, representative promising derivatives (7g and 7m) in cytotoxic and PK inhibition assays were tested to investigate their impact on the apoptosis and cell cycle phases in T-47D cells in order to explore more insights into the antitumor actions of the target thiazolyl-pyrazolines. Furthermore, docking studies were accomplished to evaluate the patterns of binding of thiazolyl-pyrazolines 7b, 7g, 7l, and 7m in the EGFR active pocket (PDB ID: 1M17). Results Testing the thiazolyl pyrazoline compounds 7a-o on A549 and T-47D cell lines showed IC50 arrays between 3.92 and 89.03 µM, and between 0.75 and 77.10 µM, respectively. Also, the tested thiazolyl-pyrazolines (7b, 7g, 7l, and 7m) demonstrated significant sub-micromolar EGFR inhibitory actions with IC50 values 83, 262, 171 and 305 nM, respectively, in comparison to erlotinib (IC50 =57 nM). Discussion Generally, it was observed that the tested thiazolyl pyrazolines showed more potent antiproliferative activity toward breast cancer cells T-47D than toward lung cancer cell lines A549. In particular, thiazolyl pyrazolines 7g and 7m showed the best activity against A549 cells (IC50 = 3.92 and 6.53 µM) and T-47D cells (IC50 = 0.88 and 0.75 µM). Compounds 7g and 7m provoked a sub-G1 phase arrest and cell apoptosis which are in agreement with the expected outcome of EGFR inhibition. Finally, the molecular docking of 7g and 7m in the active site of EGFR revealed a common binding pattern similar to that of erlotinib which involves the accommodation of the 1,3 thiazol-4-one ring and pyrazoline ring of target compounds in the binding region of erlotinib’s quinazoline ring and anilino moiety.


Introduction
Cancer stands as a baleful fatal challenge, in the current medical era, with regard to morbidity and mortality ranks second only to cardiovascular diseases and is expected to overtake the leading death cause globally in the near future. 1 Accordingly, World Health Organization (WHO) reported that the number of cancer deaths could reach 13 million by the end of 2030. 1,2 It is estimated that one out of every five people under the age of 75 will develop cancer during their lifetime. [1][2][3] Most cancers are characterized by uncontrolled cell division, growth, and impaired proliferation because of the deregulation of the necessary proteins and enzymes that control cell division. 4 Despite the numerous efforts to treat cancer diseases and the significant progress from cancer diagnosis to cancer treatment, drug developers worldwide face a slew of issues stemming from the several undesirable side effects of conventional non-selective chemotherapeutic agents such as systemic toxicity, decreased bioavailability, and drug resistance. 5 The emergence of targeted therapies specifically the signaling-pathways-targeting chemotherapeutic agents construct a new era of selective chemotherapeutic hits that could attack cancer cells and/or the tumor microenvironment responsible for tumor proliferation, resulting in greater efficacy and minor side effects on normal cells. 6,7 One of the most principal signaling pathways was the tyrosine kinase (TK) Epidermal Growth Factor Receptor (EGFR) that regulates several cell functions which include cell growth, survival, multiplication, differentiation, and apoptosis. The EGFR is activated as a result of conformational changes caused by binding interactions of the endogenous EGF ligand within EGFR extracellular binding domain. 8,9 As a result, several tyrosine residues are autophosphorylated resulting in a downstream activation of Mitogen Activated Protein Kinase (MAPK) pathway through the phosphorylation of following proteins in the pathway. Remarkable mutations in multiple proteins of the MAPK pathway, including EGFR, have been linked to various cancer types which include the colorectal carcinoma, hepatocellular carcinoma, pancreatic cancer, breast cancer, as well as the nonsmall cell lung cancer. 10 This explains why EGFR kinase inhibitors are of increasing interest as promising targeted antitumor therapeutic agents.
Both pyrazoline-and thiazole-bearing small molecules have variable forms of biological activities, including antiinflammatory, anti-cholinesterase, antimalarial, anti-carbonic anhydrase, and antimicrobial. [11][12][13][14][15][16][17] In particular, pyrazoloand thiazolo-based small molecules are significant organic compounds that have been extensively studied and reported for their antitumor activity. [18][19][20][21] Surveying literature showed that there has been a remarkable rise in the amount of published studies investigating the antitumor and kinase inhibitory activities of both pyrazoles and thiazoles. 9,21-27 For example, Akhtar et al 28 reported new pyrazoline-bearing EGFR inhibitors (Compound I, Figure 1) with potent cytotoxic effect against several human cancer cell lines which include breast cells MDA-MB231, and lung cells A549. Moreover, in 2021 Kamonpan et al 29 investigated the antitumor effect of a series of thiazole-based chalcone derivatives against lung carcinoma cells A549 overexpressing EGFR. Interestingly, compound II ( Figure 1) showed promising inhibitory activities against both lung carcinoma cells (A549) growth and EGFR TK (IC 50 of 16.30 µM and 33 nM, respectively) upon comparison to the reference drug erlotinib.
In the last few years, many research groups were interested in designing and synthesizing diverse thiazolylpyrazoline hybrids in order to gain the most beneficial antitumor activity of both rings. [30][31][32][33] In 2019, Mohamed et al 34 synthesized novel series of thiazolylpyrazolyl coumarin derivatives as new effective vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors. In particular, the polycyclic lead compound III ( Figure 1) exhibited a remarkably potent VEGFR-2 inhibitory effect (IC 50 = 34 nM), in addition to a significant growth inhibitory effect against different human cancer cell lines especially breast MCF-7 cells (IC 50 = 5.41 μM). Moreover, another new series of dual EGFR/HER2 inhibitors based on a hybrid thiazolyl/pyrazoline scaffold has been reported recently which exhibited promising anti-proliferative activity towards lung A549 and breast MCF-7 cancer cell lines in MTT cytotoxicity assay. 35 Compound IV (Figure 1) inhibited HER2 and EGFR kinases possessing IC 50 of 2.28 μM and 4.34 μM, respectively. Moreover, it exerted moderate anti-proliferative effect against the two examined cell lines with IC 50 of 10.76 μM and 8.05 μM, respectively. 35 By the end of 2019, another study reported two series of dihydropyrazole thiazole hybrids, one of the promising derivatives was Several studies reported 4-fluorophenyl moiety as a key part of several potent EGFR inhibitors with pronounced cytotoxic activity on several cancer cell lines. [38][39][40] The 4-fluorophenyl pyrazole derivative VII ( Figure 1) showed a potent cytotoxic activity on MCF-7 breast cancer cell line with IC 50 of 3.87 μM and a promising EGFR and HER-2 inhibitory activity with IC 50 of 4.87 and 6.23 μM, respectively. 38 The 4-fluorophenyl quinazolines VIII and IX ( Figure 1) exhibited promising cytotoxic activity on MCF-7 and HeLa cell lines with IC 50 of 1.44 μM and 2.70 μM, respectively, on MCF-7 and IC 50 of 4.97 μM and 1.39 μM, respectively, on HeLa cell lines. Moreover, they showed effective EGFR inhibitory activity with IC 50 of 37.66 nM and 59.21 nM, respectively. 39 Finally, the 4-fluorophenyl pyrazolo [3,4-d]pyrimidine derivative X ( Figure 1) proved prominent anticancer activity against OVCAR-4, NCI-H460, and ACHN cell lines with IC 50 of 1.74, 4.44, and 5.53 μM, respectively, more potent than the used reference standard (erlotinib); it inhibited EGFR and HER2 kinases at sub-micromolar level (IC 50 of 0.18 and 0.25 μM, respectively). 40 The pronounced cytotoxic activity of the pyrazoline/thiazole hybrid compounds III-VI and the 4-fluorophenyl derivatives VIII-X which attributed to their remarkable kinase inhibitory activity on EGFR, HER2, and/or VEGFR2 encouraged us to design and synthesize a novel series of 4-fluorophenyl substituted thiazolyl-pyrazoline derivatives (7a-o) ( Figure 2). They were further evaluated for their cytotoxic activity against the lung cancer cell line A549 and the breast cancer cell line T-47D using MTT assay. Derivatives exhibited potent anti-proliferative action were subsequently evaluated for their PK inhibition for EGFR. Representative promising derivatives in cytotoxic and PK inhibition were further tested for their effect on cell apoptosis and cell cycle progression in breast cancer cell line (T-47D). In addition, molecular docking simulations were performed to predict the binding pattern of the target thiazolyl-pyrazoline hybrids in EGFR kinase domain (PDB ID: 1M17).

Chemical Synthesis General
The proton NMR spectra were collected on a Bruker 400 MHz spectrometer for all herein prepared novel intermediates and final target thiazolyl pyrazolines. 13 C NMR spectra were done in deuterated dimethylsulfoxide (DMSO-d6) using 100 MHz frequency. Chemical shifts (δ H ) have been reported relative to the solvent (DMSO-d 6 ) peaks. IR spectra were collected on Shimadzu FT-IR 8400S spectrophotometer. Elemental analyses (EI) were measured at the Regional Center for Microbiology and Biotechnology, Al-Azhar University, Egypt. Compounds 3a-c, 4a-c and 5a-b were reported previously. 41,42 Synthesis of Thiazolidenone Derivatives 5a-c The appropriate pyrazoline thioamide derivative 4a-c (6 mmol) was heated under reflux with equimolar amount of ethyl bromoacetate (1 g, 6 mmol) in absolute EtOH (30 mL), with TLC monitoring. After 4 hours, the starting materials were consumed, and the whole mixture was cooled to lab temperature. The resulting precipitate was collected with filtration, washed with petroleum ether and recrystallized from dioxane to produce the key thiazolidenone intermediates 5a-c in a good yield (87-91%).

Synthesis of the Target Thiazolyl-Pyrazolines 7a-o
To a hot stirring solution of thiazolidenone intermediates 5a-c (1 mmol) in glacial acetic acid (10 mL), the appropriate aldehydes 6a-e (1.1 mmol) and sodium acetate (0.1 g, 1.2 mmol) were mixed. The whole solution was heated for 5 hours then the resulted precipitate was collected by filtration on hot, washed using hexane and crystallized from DMF/ethanol mixture to provide the thiazolyl-pyrazolines 7a-o in a good yield (78-90%).
Full characterisation (physical and spectral data) for the key intermediate (5c) and the target thiazolyl pyrazolines (7a-o) were presented in the Supporting Information.

Biological Evaluation
All experimental procedures utilized in the biological cytotoxicity, 43,44 Annexin V-FITC Apoptosis, 45 cell cycle, 46 and EGFR TK 47 assays were carried out as reported earlier and were shown in the Supporting Information.

Molecular Docking Studies
Molecular docking was carried out using Molecular Operating Environment software (MOE, 2020.0901), using X-ray crystallographic structure of EGFR co-crystallized with erlotinib (PDB code: 1M17). The details of the utilized protocol as well as its validation were provided in the Supporting Information.

Synthesis of Compounds
The preparation of pyrazoline derivatives 7a-o was done through the synthetic scheme depicted in Scheme 1. The chalcones intermediate 3a-c were prepared through a condensation reaction of different acetophenones 1a-c with 4-flourobenzaldehyde 2 via the reported procedures. 48 The previously prepared chalcones 3a-c subjected to heterocyclization with thiosemicarbazide in basic condition (using NaOH) to furnish the pyrazoline derivatives 4a-c. The pyrazoline derivatives 4a-c were further cyclized with ethyl bromoacetate in absolute ethyl alcohol to produce the key thiazolidenone intermediates 5a-c. Then, a knoevenagel condensation reaction between thiazolidenones 5a-c and different aldehydes 6a-e, in refluxing acetic acid and sodium acetate, led to the production of thiazolyl-pyrazoline derivatives 7a-o. Scheme 1 illustrates these steps.
All of the prepared thiazolyl-pyrazolines 7a-o showed satisfactory spectroscopic and analytical data that have complete agreement with respective structures. The proton NMR spectra of the targeted thiazolyl-pyrazolines clearly show a couple of doublets of doublet signals attributable to the diastereotopic protons (H A and H B ) of -CH 2 -group at C-4 of the pyrazoline ring around δ 3.50 and 4.0 ppm. Moreover, a third doublets of doublet signal corresponding to the C-5 proton of the pyrazoline motif around δ 5.50 ppm because of the vicinal coupling between the non-equivalent methylene hydrogens at C-4 of the pyrazoline moiety.
In addition, the 1 H NMR spectra of thiazolyl-pyrazolines 7c, 7f-j and 7m confirmed the presence of extra aliphatic signals attributable to the protons of the methoxy group around δ 3.85 ppm, while the spectra of thiazolyl-pyrazolines 7a-e revealed the presence of another aliphatic signals for CH 3 group protons around δ 2.30 ppm. Moreover, the structure of thiazolylpyrazolines 7d, 7i and 7n was confirmed with the existence of a singlet signal of (N(CH 3 ) 2 ) protons at δ 3.0 ppm, whereas, the

Biological Evaluation
Using MTT assay, the novel new prepared thiazolyl-pyrazolines 7a-o were first evaluated for their anti-proliferative effect on the lung cancer cell line A549 and the breast cancer cell line T-47D. Compounds exhibited potent antiproliferative action, were subsequently evaluated for their PK inhibition for EGFR. Representative promising derivatives in cytotoxic and PK inhibition were tested on T-47D cell line apoptosis and cell cycle progression.

In vitro Cytotoxic Activity
It is well-reported that EGFR is frequently overexpressed in diverse human malignancies such as non-small cell lung cancer and breast cancer, as well as, it stands out as a key molecule in the initiation and progression for these tumors. [49][50][51][52] In this study, A549 and T-47D cell lines were selected as representative ones for the NSCLC and breast cancer respectively. Using the MTT assay, all the prepared thiazolyl-pyrazolines 7a-o were tested for their inhibitory effect on two different cell lines including A549 lung cancer and the T-47D breast cancer cell lines, and they were compared to Staurosporine and erlotinib as a standard reference anticancer drugs. 53 IC 50 values of target compounds against both cell lines are shown in Table 1.
Testing the thiazolyl pyrazoline compounds on T-47D cell line exhibited an IC 50 array between 0.75 and 77.10 µM. Compounds 7b, 7g, 7l and 7m showed more potent antiproliferative activity (IC 50 of 0.75, 0.88, 1.15, and 1.66 µM, respectively) than the used reference standard (staurosporine) (IC 50 = 6.83 µM). Moreover, compounds 7e and 7c showed moderate cytotoxic activity with IC 50 of 7.06 and 9.30 µM, respectively, while the remainder compounds showed modest antiproliferative activities with IC 50 ranged between 10 and 100 µM. Generally, the tested pyrazoline derivatives showed more potent antiproliferative activity on the breast cancer cell line (T-47D) than on the lung cancer cell line (A549) ( Table 1). As can be seen in Table 1, the hydrophilic p-hydroxy substituted phenyl ring improves the antiproliferative activity of the tested derivatives against both used cell lines as indicated by the significant potency of thiazolyl pyrazoline 7b, 7g and 7l with IC 50 of 4.41, 3.92 and 8.10 µM, respectively, towards A549 cell line and IC 50 of 1.15, 0.88 and 1.66 µM, respectively, against T-47D cell line compared to staurosporine (IC 50 = 4.29 and 6.83 µM, respectively). Moreover, the p-methoxyphenyl derivatives 7c, 7h and 7m showed potent to moderate antiproliferative activity against both used cell lines with IC 50 of 11.72, 21.73 and 6.53 µM, respectively, against A549 cell line and IC 50 of 9.30, 14.40 and 0.75 µM, respectively, against T-47D cell line. Thiazolyl pyrazoline 7g and 7m showed the most potent antiproliferative activity toward the tested cell lines with IC 50 equal 3.92 and 6.53 µM, respectively, against A549 cell line and IC 50 of 0.88 and 0.75 µM, respectively, toward T-47D cell line.
Upon investigation of impact of the substitution of the benzylidine motif, appended on C-5 of the thiazol-4-one ring, we can see that grafting of different substituents within the para positon such as hydroxyl, methoxy, N,N-dimethylamino and chloro substituents, resulted in an enhanced anti-proliferative activity against lung A549 cell line. This could be evidenced by the lower IC 50  Concerning the SAR about the aryl moiety appended to C-3 of the pyrazoline ring, it is worth mentioning that the bioisosteric replacement of the 4-methyl/methoxy phenyl ring with the 2-thienyl heterocycle led to an enhancement of the antiproliferative activity toward both examined cell lines (A549 and T-47D), except the 4-hydroxyl derivatives 7b and 7g.

EGFR Inhibitory Activity
Compounds with significant antiproliferative impact against the investigated cell lines, viz, 7b, 7g, 7l, and 7m, were biochemically assessed for their EGFR inhibitory activity and erlotinib was used as a reference standard, Table 3 summarizes these results. When compared to the employed reference standard, erlotinib (IC 50 = 57 nM), the tried compounds demonstrated significant nanomolar inhibitory activities with IC 50 values of 83, 262, 171 and 305 nM, respectively. Regarding the correlation of cytotoxicity with EGFR inhibition, it is worth mentioning that the cell growth inhibition profile of thiazolyl-pyrazolines 7b, 7g, 7l and 7m was found to be rather flat, where the measured IC 50 toward lung (A549) and breast (T-47D) cell lines ranged from 3.92 ± 0.18-8.10 ± 0.37 and 0.75 ± 0.05-1.66 ± 0.1 μM, respectively. The same could be observed for the resulting inhibitory activity against EGFR since the obtained IC 50 values span from 83 ± 4 to 305 ± 16 nM, with about 3-fold difference in activity.

Cell Cycle Analysis
Compounds with significant antiproliferative and EGFR inhibitory activities 7g and 7m were next evaluated for the impact on cell cycle using flow cytometry technique utilizing propidium iodide (PI) staining. Cell cycle parameters for T-47D cells were compared after incubation of test compounds for 24 hours and after using DMSO as a negative control. Table 4 and Figure S3 show the results of this experiment.
According to Table 4, a significant increase of cells in the sub-G1 phase is seen, from 2.14% in control to 31.41 and 26.78% in cells incubated with 7g and 7m, respectively, which suggest a sub-G1 phase arrest and cell apoptosis which is the expected outcome of EGFR inhibition. 54,55 The higher efficiency of compound 7g in cell arrest in the sub-G1 phase and in cell apoptosis which is seen in Table 4 compared to its 7m analogue agrees with its more potent antiproliferative effect on T-47D cell line as indicated by its IC 50 of 3.92 μM in comparison to that of 7m (IC 50 of 6.53 μM).

Apoptosis Assay
The potent thiazolyl-pyrazolines 7g and 7m were investigated for action on cells apoptosis using Annexin V-FITC/propidium iodide dual-staining. 56 The apoptotic markers of the tested cell line, T-47D, were analyzed in the presence and absence of compounds 7g and 7m. Programmed cell death is characterized by the phosphatidylserine (PS) phospholipid translocation to the apoptotic cell surface. PS is stained with annexin V fluorescent conjugate then detected with flow cytometry. Additionally, T-47D cancer cells have been stained by the propidium iodide (PI) which is able to penetrate only the cells with broken plasma  membranes; facilitating the differentiation among the early apoptotic cells (positive for PS, but negative for PI), late apoptotic and necrotic cells (positive for both PS and PI).
As can be seen in Table 5 and Figure 3, the total percentage of apoptotic cells in T-47D cell line increases because of compounds 7g and 7m treatment (30.38 and 25.25%, respectively) compared to non-treated cells (0.70%) which is a clear evidence of the apoptotic action of these two target compounds. Moreover, compounds 7g and 7m showed an increase in both early and late apoptotic phases where early phase was increased from 0.52 to 3.22 and 6.28 for 7g and 7m, respectively, while late phase was also increased from 0.18 to 27.16 and 18.97, respectively.
Likewise, The higher apoptotic effect of compound 7g (Table 5) relative to its 7m analogue agrees with its more potent antiproliferative effect on T-47D cell line, as indicated by its IC 50 of 3.92 μM in comparison to that of 7m (IC 50 of 6.53 μM).

Docking Study
The molecular docking was performed to study the binding interactions between thiazolyl pyrazolines 7b, 7g, 7l, and 7m and the EGFR active pocket (PDB ID: 1M17) 57 . The molecular docking procedure was first validated by self-docking of the known active co-crystallized ligand (erlotinib) in the proximity of the active site showing docking score (S) of −10.86 kcal/mol and RMSD of 1.46 Å. The ability of the docking protocol to predict the correct crystal structure pose was demonstrated by the presence of all key interactions between the residues in the active site and docked erlotinib indicating the suitability of it for the intended docking study. The key interactions are represented by the hydrogen bonding with Met 769, a water Hydrogen bond bridge with Thr 766 and cation-pi bond with Lys 721 (for further details, see Supporting Information Figures S1 and S2).
The investigated compounds showed a common binding pattern which involves the accommodation of the 1,3-thiazol-4-one ring in the binding region of erlotinib's quinazoline ring involved in an essential hydrogen bond with the   The pyrazoline ring of the target compounds is accommodated in the binding region of erlotinib's anilino moiety directing the substitutions in position 3 and 5 of the pyrazoline ring towards both sides of the binding site interacting through several interactions with Glu738, Met742, and Asp831 on one side and Arg817 on the other side. The good activity of the tested thiazolyl pyrazoline derivatives was rationalized by their ability to interact with the key amino acids in the binding site as indicated by their docking binding pattern and docking score which ranged between −11.14 and −10.64 kcal/mol that was comparable to that of erlotinib (S = −10.86 kcal/mol) (Figures 4-7, and Table 6).

Conclusions
Novel synthesized thiazolyl-pyrazoline derivatives (7a-o) were investigated in vitro for their potential anticancer effect against the lung cancer cell line A549 and the breast cancer cell line T-47D in MTT assay. Generally, it was observed that the tested thiazolyl pyrazolines showed more potent antiproliferative activity toward breast cancer cells T-47D than toward lung cancer cell lines A549. Four thiazolyl pyrazolines (7b, 7g, 7l, 7m), out of fifteen, appeared to be potent cell growth inhibitors for both the examined cell lines. In particular, thiazolyl pyrazolines 7g and 7m showed the best activity against A549 cells (IC 50 = 3.92 and 6.53 µM, respectively) and T-47D cells (IC 50 = 0.88 and 0.75 µM, respectively). Further in vitro biochemical evaluation was performed for the potent hits (7b, 7g, 7l, 7m) so as to assay their inhibitory effect for the EGFR kinase with respect to the approved EGFR inhibitor erlotinib. Target compounds demonstrated potent nanomolar inhibition activity with IC 50 of 83, 262, 171 and 305 nM, respectively, in comparison to erlotinib (IC 50 =57 nM). Furthermore, a flow-cytometric cell cycle assay was carried out on the breast T-47D cells for the promising thiazolyl pyrazolines 7g and 7m so as to explore their cellular mechanism of action. Compounds 7g and 7m provoked a sub-G1 phase arrest and cell apoptosis which are in agreement with the expected outcome of EGFR inhibition. Finally, the molecular docking of 7g and 7m in the active site of EGFR revealed a common binding pattern similar to that of erlotinib which involves the accommodation of the 1,3 thiazol-4-one ring and pyrazoline ring of target compounds in the binding region of erlotinib's quinazoline ring and anilino moiety. Moreover, the ability of the tested thiazolyl pyrazolines to interact with the key amino acids in the binding site rationalizes their good activity as indicated by their docking binding pattern and docking score which ranged between −11.14 and −10.64 kcal/mol which is comparable to that of erlotinib (S = −10.86 kcal/mol).