Rational Design, Synthesis and Biological Evaluation of Novel Pyrazoline-Based Antiproliferative Agents in MCF-7 Cancer Cells

Breast cancer is a disease in which cells in the breast divide continuously without control. There are great limitations in cancer chemotherapy. Hence, it is essential to search for new cancer therapeutics. Herein, a novel series of EGFR/HER2 dual inhibitors has been designed based on the hybridization of thiazole and pyrazoline fragments. The synthesized compounds were screened for their anti-proliferative activity against MCF-7 breast cancer cell line and MCF-10 normal breast cell line. Interestingly, synthesized compounds 6e and 6k showed very potent antiproliferative activity towards MCF-7 with IC50 values of 7.21 and 8.02 µM, respectively. Furthermore, enzymatic assay was performed against EGFR and HER2 to prove the dual inhibitory action. Compounds 6e and 6k showed potent inhibitory activity for EGFR with IC50 of 0.009 and 0.051 µM, respectively, and for HER2 with IC50 of 0.013 and 0.027 µM, respectively. Additionally, compounds 6e and 6k significantly stimulated apoptotic breast cancer cell death. Compound 6e was further explored for its anticancer activity in vivo using a Xenograft model. Moreover, computational modeling studies, ADMET studies and toxicity prediction were performed to investigate their potential drug candidates.


Introduction
Cancer is considered one of the most challenging causes of death in the world, as it is estimated to have caused nearly 10 million deaths in 2020 according to WHO [1,2]. Cancer is not a single disease, but is rather a collective term that describes the distress of the fundamental regulatory genes that are responsible for controlling cell proliferation, differentiation, and survival [3]. The traditional treatment strategies were based on the unspecific death induction targeting the DNA synthesis process without discriminating between tumor cells and rapidly dividing normal cells, which lead to many side effects [4][5][6]. Currently, it is an essential demand to develop more safe and selective molecular targeted chemotherapeutic agents [7]. In this regard, one of the powerful approaches is to target transduction-related macromolecules, especially protein kinases [8][9][10].

Rationale of the Work
Molecular hybridization of both thiazole and pyrazoline scaffolds has been used in multi-targeting kinase inhibitors [50], topoisomerases, apoptosis, with significant antitumor activity against MCF-7 and A595 cell lines [27]. Based on the aforementioned protein kinase inhibitory activity of thiazole and pyrazoline nuclei, especially on EGFR and HER2, a strategy of hybridizing these two luminous moieties was designed to synthesize dual EGFR/HER2 hybrid modulators/inhibitors targeting breast cancer [51]. The new scaffold aimed to find novel lead structures with better activity and lower toxicity [52] ( Figure  3).
In this study, a new series of thiazolyl-pyrazoline derivatives was designed, synthesized, and screened for their activity against MCF-7 and MCF-10, and their inhibitory

Rationale of the Work
Molecular hybridization of both thiazole and pyrazoline scaffolds has been used in multi-targeting kinase inhibitors [50], topoisomerases, apoptosis, with significant antitumor activity against MCF-7 and A595 cell lines [27]. Based on the aforementioned protein kinase inhibitory activity of thiazole and pyrazoline nuclei, especially on EGFR and HER2, a strategy of hybridizing these two luminous moieties was designed to synthesize dual EGFR/HER2 hybrid modulators/inhibitors targeting breast cancer [51]. The new scaffold aimed to find novel lead structures with better activity and lower toxicity [52] (Figure 3). activity was further explored as dual-EGFR/HER2 inhibitors. In addition, the promising compounds were investigated for apoptosis activity and in vivo studies. The molecular modelling studies were performed for the designed compounds to confirm their binding modes to EGFR and HER2 at the ATP binding site.

Chemistry
The synthetic routes of the thiazolyl-pyrazoline derivatives 3a-c, 6a-l and 9a-f are considered in Charts 1-3. First, the 3-(4-aryl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-ones 2a-c were obtained through the Claisen-Schmidt reaction [53] by direct condensation of aromatic aldehydes 1a-c with dimethoxy acetophenone in the presence of sodium hydroxide [54][55][56]. This step was followed by the reaction of compounds 2a-c with thiosemicarbazide under a basic condition to give 3-(3,4-dimethoxyphenyl)-5-(4-aryl)-4,5-dihydro-1Hpyrazole-1-carbothioamides 3a-c, respectively (Chart 1) [57,58]. This reaction proceeded through a condensation reaction followed by subsequent cyclization via Michael addition reaction. The IR spectra for compounds 3a-c showed the characteristic NH2 band in the range 3422-3260 cm −1 , in addition to a band at 1265-1248 cm −1 referring to C = S group. Additionally, 1 H NMR of compounds 3a-c displayed three doublets of doublet (dd) of pyrazoline ring due to ABX pattern. These three (dd) signals of HA, HB, and HX appeared at δ 3.12-3.14, 3.8 and 5.8-5.9 ppm, respectively. Moreover, NH2 exchangeable proton signal of compounds 3a-c appeared around δ 8.0 ppm. 13 C NMR of compounds 3a-c revealed the signals for CHAHB and CHX carbons of pyrazoline ring around δ 42.9 and 62.8 ppm, respectively, as well as C = S signals at δ 176.2 ppm. In this study, a new series of thiazolyl-pyrazoline derivatives was designed, synthesized, and screened for their activity against MCF-7 and MCF-10, and their inhibitory activity was further explored as dual-EGFR/HER2 inhibitors. In addition, the promising compounds were investigated for apoptosis activity and in vivo studies. The molecular modelling studies were performed for the designed compounds to confirm their binding modes to EGFR and HER2 at the ATP binding site.
Next, the reaction of carbothioamide compounds 3a-c with an equimolar amount of phenacyl bromide derivatives 5a-d [59] in absolute ethanol yielded the corresponding thiazolyl-pyrazoline derivatives 6a-l in good yields (Chart 2) [60]. Compounds 5a-d were prepared by bromination of corresponding acetophenone using bromine and acetic acid in diethyl ether as solvent. The IR spectra of compounds 6a-l showed the disappearance of the NH 2 characteristic band in the region 3422-3260 cm −1 . 1 H NMR spectra also revealed the absence of NH 2 proton around δ 8.0 ppm. 1 H NMR spectra of compounds 6a-l showed the signals of aromatic protons at δ 7.8-6.9 ppm to be assignable to thiazolyl and phenyl protons. In addition, an extra signal at δ 2.3 ppm appeared due to the presence of CH 3 in compounds 6b, 6f, and 6j. The ABX pattern was confirmed by COSY spectra for compound 6k to prove the formation of the pyrazoline ring of two non-equivalent protons at C4 (H A and H B ) and one proton at C5 (H X ). 13 C NMR spectra of compounds 6a-l showed the signals of the aromatic carbons at 103.5-165 ppm. 13 C NMR spectra of compounds 6g, 6c, and 6k (4-FC 6 H 4 ) showed the splitting of the three carbon atoms at δ 115.9-115.7, 128-127.8, 131.7-131.54 ppm due to their coupling with the fluorine atom. Also, 13 C NMR spectra revealed the appearance of singlet signal at δ 21.2-21.3 ppm which belongs to CH 3 (tolyl carbon) of compounds 6b, 6f, and 6j. Mass spectra of compounds 6a-l showed the molecular ions peak in accord with their molecular weight.
IR spectra of compounds 9d-f displayed a band at 1708-1695 cm −1 representing C=O. 1 H NMR spectrum also showed a typical quartet-triplet pattern of the ethyl protons at δ 4.2 and 1.25 ppm, respectively. However, signals of thiazole CH 3 appeared at δ 2.36-2.35 ppm.
Furthermore, 13 C NMR spectrum revealed the signals of ethyl carbons at δ 14.8 and 60.6 ppm, and signal at 165.1-165.2 ppm attributed C=O of ester.

Cytotoxicity
The MTT assay was used to test the activity and selectivity of all synthesized compounds for cytotoxic activity against MCF-7 cancer cells and MCF-10A normal breast cells. As seen in Table 1, compounds 3c, 6b and 6f were found to be more cytotoxic against MCF-7, with IC 50 ranges of 11.09, 13.36 and 11.05 µM, respectively. Interestingly, compounds 6e, 9f and 6k exhibited remarkable cytotoxic activities, with IC 50 values of 7.21, 8.35 and 8.02 µM, respectively, compared to Lapatinib (IC 50 = 7.45 µM) with non-cytotoxic activity against the MCF-10A. Other compounds exhibited relatively moderate-to-weak cytotoxic activities. Hence, three compounds; 6e, 9f, and 6k, were chosen to be studied for their potential molecular targets and ability to induce apoptosis in MCF-7 cells (Figure 4).

Potential EGFR/HER2 Kinase Inhibitory Assay
To further elucidate the mechanisms behind the cytotoxic effects on MCF-7 cells, compounds 3c, 6e, 6k, 6f and 9f were tested for their ability to inhibit EGFR and HER2.

Potential EGFR/HER2 Kinase Inhibitory Assay
To further elucidate the mechanisms behind the cytotoxic effects on MCF-7 cells, compounds 3c, 6e, 6k, 6f and 9f were tested for their ability to inhibit EGFR and HER2. As seen in Table 2, the tested compounds exhibited promising dual EGFR/HER2 inhibition activities. Remarkably, compounds 6k and 6e had IC 50 values of 0.014 and 0.009 µM against EGFR and IC 50 values of 0.027 and 0.013 µM against HER2, respectively, in a comparable way to Lapatinib. Due to their potent cytotoxic and EGFR/HER2 inhibitory effects, compounds 6k and 6e merited investigation for their ability to induce apoptosis.

Apoptotic Investigation Annexin V/PI Staining with Cell Cycle Analysis
Flow cytometric analysis of Annexin V/PI staining was used to examine apoptotic cell death in untreated and treated MCF-7 cells to determine the apoptotic activity of the two promising compounds, 6k and 6e (IC 50 = 8.02, 7.21 µM, 48 h). As seen in Figure 5A, compounds 6k, and 6e significantly stimulated apoptotic breast cancer cell death with 22.44% (14.38% for late apoptosis, 8.06% for early apoptosis) and 27.99% (18.52% for late apoptosis, 9.47% early apoptosis), respectively, compared to 0.68% in the control.
Cytotoxic substances were tested using DNA flow cytometry to determine what percentage of cells were in each cell cycle following treatment. Figure 5B, the percentage of cells in the S phase increased from 34.18 to 44.52 % after treatment with compound 6e, indicating that compound 6e induced cell cycle arrest in the S phase, while the percentage of cells in the G2/M phase was reduced by 2.01% after treatment with compound 6k, compared to the control (9.7%). This result indicates that compound 6k induced cell cycle arrest in the G2/M-phase. By comparison, other phases were not significantly changed in either treatment.

RT-PCR
Further validation of the apoptosis-inducing activities of both tested compounds 6k and 6e in MCF-7 cells, the gene expression levels of apoptosis-related genes in both untreated and treated MCF-7 cells through the RT-PCR. As seen in Figure 6, compounds 6k and 6e treatments increased P53 level by 3.91-and 4.8-fold, Bax level by 5.24-and 6.39-fold, caspase 3 level by 5.79-and 7.12-fold, caspase 8 level by 3.91-and 1.71-fold, caspase 9 level by 6.07-and 8.19-fold, while both compound treatments decreased Bcl-2 level "as the anti-apoptotic gene" by 0.41-and 0.32-fold compared to untreated control. cells in the S phase increased from 34.18 to 44.52 % after treatment with compound 6e, indicating that compound 6e induced cell cycle arrest in the S phase, while the percentage of cells in the G2/M phase was reduced by 2.01% after treatment with compound 6k, compared to the control (9.7%). This result indicates that compound 6k induced cell cycle arrest in the G2/M-phase. By comparison, other phases were not significantly changed in either treatment. The data demonstrated is the mean of 3independent experimental runs (mean ± SD). ** p < 0.001 and * p < 0.05 compared to control using an unpaired t test by GraphPad prism.

RT-PCR
Further validation of the apoptosis-inducing activities of both tested compounds 6k and 6e in MCF-7 cells, the gene expression levels of apoptosis-related genes in both untreated and treated MCF-7 cells through the RT-PCR. As seen in Figure 6, compounds 6k and 6e treatments increased P53 level by 3.91-and 4.8-fold, Bax level by 5.24-and 6.39fold, caspase 3 level by 5.79-and 7.12-fold, caspase 8 level by 3.91-and 1.71-fold, caspase 9 level by 6.07-and 8.19-fold, while both compound treatments decreased Bcl-2 level "as the anti-apoptotic gene" by 0.41-and 0.32-fold compared to untreated control.
P53 is a tumor suppressor gene that is essential for apoptosis. The activation of caspases 3 and 9 and the decreased expression of the Bcl-2 gene may cause p53 apoptosis. The results showed the intrinsic apoptotic pathway through activation of P53, Bax, and caspases 3 and 9 levels. Further, the treatment-induced upregulation of caspase 8 gene promotes an extrinsic apoptotic pathway in MCF-7 cells. As a result, our RT-PCR findings corroborated those of previous studies, showing that the apoptotic pathway is highlighted by the upregulation of proapoptotic genes and the downregulation of anti-apoptotic genes (Table 3). The data demonstrated is the mean of 3-independent experimental runs (mean ± SD). ** p < 0.001 and * p < 0.05 compared to control using an unpaired t test by GraphPad prism.
P53 is a tumor suppressor gene that is essential for apoptosis. The activation of caspases 3 and 9 and the decreased expression of the Bcl-2 gene may cause p53 apoptosis. The results showed the intrinsic apoptotic pathway through activation of P53, Bax, and caspases 3 and 9 levels. Further, the treatment-induced upregulation of caspase 8 gene promotes an extrinsic apoptotic pathway in MCF-7 cells. As a result, our RT-PCR findings corroborated those of previous studies, showing that the apoptotic pathway is highlighted by the upregulation of proapoptotic genes and the downregulation of anti-apoptotic genes ( Table 3). Values are expressed as mean ± SD of three independent replicates. Data were normalized using β-actin as housekeeping gene.  Values are expressed as mean ± SD of three independent replicates. Data were normalized using βactin as housekeeping gene.

In Vivo Studies
For evaluation of the anticancer activity of compound 6e, mice, MCF-7 cells (Xenograft model) were treated with compounds 6e and lapatinib at a dose (10 mg/ kg BW, IP). As seen in Figure 7A, an increase in the mice weight of the MCF-7 control mice reached 32.28 g, compared to 24.5 g in normal mice, while treatment with compound 6e and lapatinib reduced the mice weight to 28.23 g and 27.35 g, respectively. The reduction in the mice's weight was consequently related to a decrease in the tumor mass; tumor volume decreased from 89.8 mg to 28.4 mm 3 , respectively, upon treatment with compound 6e. Hence, compound 6e treatment enhanced tumor inhibition ratio by 52.46% compared to 50.53% in Lapatinib treatment Figure 7B.

In Vivo Studies
For evaluation of the anticancer activity of compound 6e, mice, MCF-7 cells (Xenograft model) were treated with compounds 6e and lapatinib at a dose (10 mg/ kg BW, IP). As seen in Figure 7A, an increase in the mice weight of the MCF-7 control mice reached 32.28 g, compared to 24.5 g in normal mice, while treatment with compound 6e and lapatinib reduced the mice weight to 28.23 g and 27.35 g, respectively. The reduction in the mice's weight was consequently related to a decrease in the tumor mass; tumor volume decreased from 89.8 mg to 28.4 mm 3 , respectively, upon treatment with compound 6e. Hence, compound 6e treatment enhanced tumor inhibition ratio by 52.46% compared to 50.53% in Lapatinib treatment Figure 7B.
As seen in Figure 7D, liver enzymes of ALT, AST were significantly elevated to 70.9, 70.65 (U/L), respectively in MCF-7 control mice compared to normal mice at 38.9 and 48.9 (U/L), respectively, caused by tumor inoculation-related hepatocellular damage, while liver enzymes were markedly mitigated by treatment with compound 6e. Liver enzymes were reduced to 41.8, 59.9 U/L, respectively, and this significantly reduced the toxicity to the liver caused by the cancer. While Lapatinib treatment decreased ALT to 40.8 U/L, AST fell to 57.7 U/L compared to the MCF-7 group As seen in Figure 7C, the hemoglobin and red blood cell concentrations dropped dropped dramatically to 5.7 (g/dL) and 2.9 (10 6 /L), respectively. Conversely, white blood cell count was elevated to 7.0 (10 3 /L) from normal control levels. Routine consequences of tumor proliferation are reduced levels of RBC, hemoglobin, and increased WBC counts [63,64]. CBC was nearly maintained at normal levels following treatment with compound 6e, where Hb (7.0 g/dL) and RBC's count (4.8 10 6 /µL) were raised, and WBCs count was lowered (4.12 10 3 /µL). While Lapatinib-treatment increased Hb to 7.43 g/dL, RBC's count to 4.99 10 6 /µL and decreased WBC's count to 4.21 10 3 /µL compared to MCF-7 group.
As seen in Figure 7D, liver enzymes of ALT, AST were significantly elevated to 70.9, 70.65 (U/L), respectively in MCF-7 control mice compared to normal mice at 38.9 and 48.9 (U/L), respectively, caused by tumor inoculation-related hepatocellular damage, while liver enzymes were markedly mitigated by treatment with compound 6e. Liver enzymes were reduced to 41.8, 59.9 U/L, respectively, and this significantly reduced the toxicity to the liver caused by the cancer. While Lapatinib treatment decreased ALT to 40.8 U/L, AST fell to 57.7 U/L compared to the MCF-7 group In agreement with the amelioration of biochemical parameters, histopathological examinations Figure 7E in liver sections in 6e-treated mice showed normal nuclei morphology in most of the cells with slight hydrophobic degeneration compared to MCF-7 control In agreement with the amelioration of biochemical parameters, histopathological examinations Figure 7E in liver sections in 6e-treated mice showed normal nuclei morphology in most of the cells with slight hydrophobic degeneration compared to MCF-7 control mice, which showed chronic inflammation (red arrow), and hepatocytes showing hydropic degeneration (arrowheads).
Taken together, amelioration in hematological, and biochemical results of compound 6e treatment agreed with the in vitro ones of cytotoxicity, EGFR/HER2 targeting, and apoptosis induction.

Molecular Docking
To discover the most appropriate drug target for the proposed compounds, SwissTar-getPrediction [65] was used. The studied compounds revealed that kinase proteins were the first class to be targeted, with a probability of 40% (Figure 8).

Molecular Docking
To discover the most appropriate drug target for the proposed compounds, Swis-sTargetPrediction [65] was used. The studied compounds revealed that kinase proteins were the first class to be targeted, with a probability of 40% (Figure 8). Molecular docking was carried out to estimate the interaction of the designed hybrid compounds with EGFR/HER2 kinase domains to rationalize their biological activity, and to reveal their probable binding pattern. Crystal structures were downloaded (PDB codes: 1XKK [66] and 3RCD [67]) for EGFR and HER2, respectively. Initially, autodocking of the co-crystallized ligands in EGFR and HER2 active sites was performed to validate the docking method. In the EGFR active site, the docking poses of lapatinib formed the key interactions with the active site through H-bond formation with amino acid residues Met793, Asp800, and Leu788 at distance of 3.17, 3.3, and 3.19 Å, respectively. In addition, arene H interactions with Leu844 and Leu718 were formed as illustrated in Figure 9A. As for HER2, the docking pose of TAK-285 produced the key interactions with the active site; it interacts through an H bond with amino acid residues Asp863, Gly727, and 2 H bonds with Met801 at distance of 3.22, 3.18, 3.02, 3.82 Å, respectively. Besides, π-H interactions with Phe864, Lys753, Leu852, and Leu800 were formed as seen in Figure 9B. The method validation was demonstrated by the resulted small RMSD values between the docked and the co-crystallized ligand poses in EGFR (0.917 Å, Figure S1) and HER2 (0.625 Å, Figure  S2). Molecular docking was carried out to estimate the interaction of the designed hybrid compounds with EGFR/HER2 kinase domains to rationalize their biological activity, and to reveal their probable binding pattern. Crystal structures were downloaded (PDB codes: 1XKK [66] and 3RCD [67]) for EGFR and HER2, respectively. Initially, autodocking of the co-crystallized ligands in EGFR and HER2 active sites was performed to validate the docking method. In the EGFR active site, the docking poses of lapatinib formed the key interactions with the active site through H-bond formation with amino acid residues Met793, Asp800, and Leu788 at distance of 3.17, 3.3, and 3.19 Å, respectively. In addition, arene H interactions with Leu844 and Leu718 were formed as illustrated in Figure 9A. As for HER2, the docking pose of TAK-285 produced the key interactions with the active site; it interacts through an H bond with amino acid residues Asp863, Gly727, and 2 H bonds with Met801 at distance of 3.22, 3.18, 3.02, 3.82 Å, respectively. Besides, π-H interactions with Phe864, Lys753, Leu852, and Leu800 were formed as seen in Figure 9B. The method validation was demonstrated by the resulted small RMSD values between the docked and the co-crystallized ligand poses in EGFR (0.917 Å, Figure S1) and HER2 (0.625 Å, Figure S2). The newly synthesized thiazole-pyrazoline hybrids showed comparable bin patterns in both tyrosine kinases. Binding features of compounds 6e, 6k, 3c, and 9f w studied in more detail and compared to the docked co-crystallized inhibitors of e EGFR ( Figure 10) and HER2 (Figure 11). From binding modes analyses, we can conc that: The newly synthesized thiazole-pyrazoline hybrids showed comparable binding patterns in both tyrosine kinases. Binding features of compounds 6e, 6k, 3c, and 9f were studied in more detail and compared to the docked co-crystallized inhibitors of either EGFR ( Figure 10) and HER2 ( Figure 11). From binding modes analyses, we can conclude that:

In Silico Physicochemical Descriptors, Pharmacokinetic Properties and Bioactivity Prediction
Different parameters were measured to explore drug-likeness properties (Table S1) of the target thiazolyl-pyrazoline compounds, all of which obey Veber's rule and Lipinski's rule and may meet the criteria for orally active drugs, except for 6l which has 2 violations of the Lipinski rule. All compounds were predicted to have a clogP value in the range of 2.00-6.00, TPSA ≤ 110.72 Å 2 , rotatable bonds (RB) ≤ 9, H-bond acceptor moieties (HBA) ≤ 7, H-bond donor moieties (HBD) ≤ 1 and molar refractivity (MR) ≤ 147.86. Compound solubility was in the range of poorly soluble to moderately soluble, except for compounds 3a and 3b which were soluble. Bioavailability radar (Figures S3 and S4) indicated that molecules 3a-c and 9a-e are expected to be orally bioavailable.
In silico ADME prediction of pharmacokinetic properties was investigated for the target compounds (Table S2). All compounds showed very high HIA values, ranging from 97.5-99.6%, indicating very high GI absorption. All compounds exerted a strong bound effect on plasma protein (binding values of 87.45-93.09%). All compounds are not blood-brain barrier permeant and have skin p values from −2.35 to −3.43. On inspecting the boiled-egg model ( Figure S5), all compounds were found to be in the white region indicating their proper GI absorption.
An in silico phase I metabolism study indicated that all compounds could inhibit CYP2C9, and that all compounds have CYP3A4-inhibiting activity except 6l. None of the thiazolyl-pyrazoline hybrids could inhibit CYP1A2.

In Silico Physicochemical Descriptors, Pharmacokinetic Properties and Bioactivity Prediction
Different parameters were measured to explore drug-likeness properties (Table S1) of the target thiazolyl-pyrazoline compounds, all of which obey Veber's rule and Lipinski's rule and may meet the criteria for orally active drugs, except for 6l which has 2 violations of the Lipinski rule. All compounds were predicted to have a clogP value in the Toxic properties such as irritant, mutagenic, and reproductive effects using were predicted Osiris server (Table S3). All compounds have no mutagenic, irritant, or reproductive fragments except for 3a, 3b, and 3c, which have reproductive effects due to the 1-ethyl hydrazine-1-carbothioamide fragment.

Chemistry
NMR spectra were recorded by a Bruker spectrometer. 1 H NMR spectra were run at 400 MHz, and 13 C NMR spectra were run at 100 MHz in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts (δH and δC) were reported relative to the solvent (DMSO-d6). Infrared spectra were recorded on a Shimadzu FT-IR 8400S spectrophotometer as KBr disks at the faculty of science, Sohag University. Elemental analysis was performed using FLASH 2000 CHNS/O analyzer, at the Regional Center for Microbiology and Biotechnol-

Chemistry
NMR spectra were recorded by a Bruker spectrometer. 1 H NMR spectra were run at 400 MHz, and 13 C NMR spectra were run at 100 MHz in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shifts (δ H and δ C ) were reported relative to the solvent (DMSO-d 6 ). Infrared spectra were recorded on a Shimadzu FT-IR 8400S spectrophotometer as KBr disks at the faculty of science, Sohag University. Elemental analysis was performed using FLASH 2000 CHNS/O analyzer, at the Regional Center for Microbiology and Biotechnology, Al-Azhar University. The reaction progress was monitored using thin-layer chromatography (TLC)-precoated silica gel G plates, and the formation of products was visualized by irradiation with UV light (254 nm). Melting points were determined using Stuart Scientific apparatus and were uncorrected. All the chemicals were purchased from Alpha Aesar and Sigma-Aldrich (Figures S6-S79).

Synthesis of 3-(4-Aryl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (2a-c)
A solution of dimethoxy acetophenone (1.08 g, 6.0 mmol) in absolute ethanol (30 mL) was stirred with an equimolar weight of the appropriate aromatic aldehyde 1a-c (6.0 mmol), NaOH (2 g, 50 mmol) in water (15 mL) was added within 30 min. The reaction mixture was stirred for 5 h at room temperature. The reaction progress was monitored using TLC and methylene chloride/methanol (9:1) system until the completion of the reaction. Then, the resulting precipitate was filtered and washed with ethanol, and then recrystallized from absolute ethanol to give a yield of 95-98%. The appropriate amount of phenacyl bromide 5a-d (0.6 mmol) was added to a solution of pyrazoline derivatives 3a-c (0.6 mmol) in absolute ethanol (15 mL), before it was refluxed at 90 • C for 5 h. After cooling, the obtained solid was filtered off, washed with ethanol, and recrystallized from EtOH/DMF to give compounds 6a-l.

Cytotoxicity
Both breast cancer (MCF-7) and normal breast (MCF-10A) cells were purchased from National Research Institute, Egypt, and maintained in complete media of RPMI-1640 medium L-Glutamine (Lonza Verviers SPRL, Belgium, cat#12-604F). Cells were incubated following standard tissue culture work. Cell viability was measured 48 h later, utilizing MTT colorimetric assay (Promega, Madison, WI, USA) [63]. Absorbance was consequently assessed (at 570 nm) using an ELISA microplate reader (BIO-RAD, model iMark, Tokyo, Japan). Using GraphPad Prism 7, the IC 50 values were determined, and the viability at each concentration was determined in comparison to the control [68,69].

EGFR/HER2 Kinase Inhibitory Assay
The EGFR Kinase Assay Kit (BPS Bioscience kit, Cat#40321) and the HER2 Kinase Assay Kit (BPS Bioscience kit, Cat#40721) were used to evaluate the potential inhibitory activities against EGFR and HER2, respectively. The inhibitory potency of compounds 3c, 6b, 6e, 6k, and 9f against EGFR and HER2 kinases was determined using kinase inhibitory assays. Compounds' autophosphorylation percentage inhibition was computed according to the following equation: 100 − [( A control A treated − Control)], then IC 50 values were calculated using GraphPad prism software [70].

Investigation of Apoptosis
Annexin V/PI Staining and Cell Cycle Analysis MCF-7 breast cancer cells were seeded into 6-well culture plates (3-5 × 10 5 cells/well) and incubated for 24 h. Subsequently, compounds 6k and 6e (at their IC 50 values) were used to treat the seeded cells for 48 h. After that, the collection of the media supernatants and the cells was performed before undergoing rinsing with ice-cold PBS. The cells were suspended in 100 µL of annexin-binding buffer solution "25 mM CaCl 2 , 1.4 M NaCl, and 0.1 M Hepes/NaOH, pH 7.4" and incubated with "Annexin V-FITC solution (1:100) and propidium iodide (PI)" at a concentration equal 10 µg/mL in the dark for 30 min. Stained cells were then obtained by a Cytoflex FACS machine, and data analysis was performed using cytExpert software [71][72][73].

Real-Time Polymerase Chain Reaction for the Selected Genes
Furthermore, to explore the apoptotic pathway, gene expression of proapoptotic genes (P53, Bax, Caspapses-3, 8,9) and antiapoptotic gene (Bcl-2) was evaluated using routine RT-PCR in the untreated and treated MCF-7 cells. Ct values were calculated to determine the relative genes expression in all samples by normalization to β-actin (housekeeping gene) [71,74].

In Vivo Study Ethics Statement
The experimental protocol was approved by the Research Ethics Committee at Suez Canal University (Approval number REC-10-2021; October 2021), Chemistry Department, Faculty of Science, Suez Canal University.

Animal, Tumor Inoculation Experiment Design
In vivo experiments for lethal dose (LD 50 ), experimental design, tumor mass, blood parameters, histopathological examinations were accomplished as previously described [71]. After inoculating the tumor cells, masses of the solid tumor started to appear ten days later. Seven administrations of 6e and Lapatinib were completed, each at a dose of 10 mg/Kg BW/IP. Solid tumor masses' weight and volume were measured at the end of the experiment. Animals from each group were sacrificed, and their blood was drawn to measure hemoglobin (Hb), red blood cell (RBC), white blood cell (WBC), and liver enzymes (ALT, AST, albumin, and total protein) levels.

Molecular Docking
Two PDB codes for the molecular targets in the proposed antiproliferative mechanism were selected. Both 1XKK and 3RCD were chosen as crystal structures for EGFR and HER2 receptors, respectively. Docking program used was AutoDock Vina [75,76], following multiple ligands' docking protocol regarding water molecule deletion, as well as both ligand and receptor preparation [77]. Grid box size was prepared based on the active residues of the kinase domains of the receptors. After docking simulation, poses with the highest negative binding free energy (kcal/mol) were selected as the best poses for corresponding ligand binding. Visualization of the 3D receptor binding site, disposition of the original (co-crystallized) ligand, and the main ligand receptor interaction in terms of hydrogen bonding with the key amino acid residues was performed using Chimera software [78].

In Silico Physicochemical Descriptors, Pharmacokinetic Properties, and Bioactivity Prediction
In silico estimation of physicochemical properties, pharmacokinetics and drug-likeness was performed using SwissADME web tool [65] and PreADMET server [79]. An osiris server [80] was used for toxicity prediction by measuring irritant, mutagenic, tumorigenic, and reproductive effects.

Conclusions
Herein, a novel series of pyrazoline derivatives 3a-c, and thiazolyl-pyrazolines 6a-l and 9a-f were designed and synthesized as dual EGFR/HER2 inhibitors. All compounds were assessed for their anti-antiproliferative activities towards MCF-7 and MCF-10 (cancer and normal breast cell line). The results revealed that compounds 3c, 6b, 6e, 6f, 6k, and 9f displayed potent and selective anticancer activity against MCF-7 cell lines compared to lapatinib and were safe for the normal breast cell. Moreover, the highly potent compounds were investigated for their inhibitory properties against EGFR and HER2 compared to lapatinib. Interestingly, compound 6e showed potent EGFR and HER2 inhibitory effects (IC 50 = 0.009 and 0.013 µM, respectively), superior to the reference drug lapatinib (IC 50 = 0.006 and 0.017 µM, respectively). Moreover, compounds 6e and 6k also significantly induced apoptosis in the targeted breast cell line with 27.99% and 22.44%, respectively, compared to 0.68% for the control. Furthermore, the in vivo study for compound 6e showed a tumor inhibition ratio by 52.46% compared to 50.53% in lapatinib treatment. Molecular docking results revealed that compounds 6e and 6k had good binding affinity to the ATP binding site of EGFR with H bonds formed with the crucial amino acid residue Met793. Besides, compound 6e also anchored well in the HER2 binding site and participating in the crucial interaction with Thr862. According to the in vitro, in vivo, and in silico studies, compound 6e stands out as a potential drug candidate for the development of future anticancer agents, having a dual inhibitory activity against EGFR and HER2 receptors.