Acetic Acid Mediated for One-Pot Synthesis of Novel Pyrazolyl s-Triazine Derivatives for the Targeted Therapy of Triple-Negative Breast Tumor Cells (MDA-MB-231) via EGFR/PI3K/AKT/mTOR Signaling Cascades

Here, we described the synthesis of novel pyrazole-s-triazine derivatives via an easy one-pot procedure for the reaction of β-dicarbonyl compounds (ethylacetoacetate, 5,5-dimethyl-1,3-cyclohexadione or 1,3-cyclohexadionone) with N,N-dimethylformamide dimethylacetal, followed by addition of 2-hydrazinyl-4,6-disubstituted-s-triazine either in ethanol-acetic acid or neat acetic acid to afford a novel pyrazole and pyrazole-fused cycloalkanone systems. The synthetic protocol proved to be efficient, with a shorter reaction time and high chemical yield with broad substrates. The new pyrazolyl-s-triazine derivatives were tested against the following cell lines: MCF-7 (breast cancer); MDA-MB-231 (triple-negative breast cancer); U-87 MG (glioblastoma); A549 (non-small cell lung cancer); PANC-1 (pancreatic cancer); and human dermal fibroblasts (HDFs). The cell viability assay revealed that most of the s-triazine compounds induced cytotoxicity in all the cell lines tested. However, compounds 7d, 7f and 7c, which all have a piperidine or morpholine moiety with one aniline ring or two aniline rings in their structures, were the most effective. Compounds 7f and 7d showed potent EGFR inhibitory activity with IC50 values of 59.24 and 70.3 nM, respectively, compared to Tamoxifen (IC50 value of 69.1 nM). Compound 7c exhibited moderate activity, with IC50 values of 81.6 nM. Interestingly, hybrids 7d and 7f exerted remarkable PI3K/AKT/mTOR inhibitory activity with 0.66/0.82/0.80 and 0.35/0.56/0.66-fold, respectively, by inhibiting their concentrations to 4.39, 37.3, and 69.3 ng/mL in the 7d-treatment, and to 2.39, 25.34 and 57.6 ng/mL in the 7f-treatment compared to the untreated control.

Given the excellent bioactivity and wide range of applications of pyrazole derivatives, many studies have addressed their synthesis and bioactivities [19,20]. Furthermore, the methodology for the synthesis of these derivatives has been summarized in several reviews [7,[21][22][23].
Recently, we described the synthesis of pyrazole-s-triazine derivatives by direct reaction with β-diketone, using triethylamine as a catalyst or using HClO 4 in an aqueous medium [38,39]. In the present work, we describe a one-pot method for the synthesis of pyrazole and fused pyrazole-s-triazine derivatives in the presence of acetic acid via the formation of the enaminedione derivatives of β-diketone.
The search for new compounds with therapeutic efficacy is a major focus in medicinal chemistry. However, the latent progress of resistance or tolerance to these compounds over time, particularly in the context of the treatment of diseases such as cancer, severely limits their medical use. Many representative examples reported for cancer treatment, either approved for human use or in late-stage clinical trials, contain the 1,3,5-triazine (s-triazine) moiety. In 1990, the US FDA approved Hexalen (Altretamine) (Figure 1, compound I), as an example of targeted therapy for ovarian cancer [40]. First authorized in 2017 by the US-FDA, Enasidenib (Idhifa, compound II) is another commercial drug based on the s-triazine scaffold and it is used to treat IDH2-positive acute leukemia [41]. Indeed, Gedatolisib was reported as first-in-class to treat breast cancer via the PI3K/mTOR inhibitor ( Figure 1, compound III) [42]. Several molecules have been reported to be tethered to the s-triazine motif, such as targeted EGFR-TK inhibitors IV [43] and V [44], VI ZSTK474 (as PI3K/MEK dual inhibitors) [45], and Bimiralisib (PQR309) (compound VII) [46][47][48]. Moreover, VIII [49] also shows anti-cancer efficacy as a dual inhibitor of PI3K/mTOR Figure 1). More recently, compound IX possessed EGFR/PI3K/AKT/mTOR signaling cascades inhibitor [50] (Figure 1). In the framework of our ongoing project based on the use of s-triazine as a scaffold for the development of novel agents for cancer treatment [38,39], we tested a new series of pyrazolyl-s-triazine derivatives against MCF-7, MDA-MB-231, U-87 MG, A549, PANC- In the framework of our ongoing project based on the use of s-triazine as a scaffold for the development of novel agents for cancer treatment [38,39], we tested a new series of pyrazolyl-s-triazine derivatives against MCF-7, MDA-MB-231, U-87 MG, A549, PANC-1, and HDF cell lines. To better understand the potential mechanism of action of these compounds in human cancer cells, we also conducted an EGFR enzymatic assay and evaluated the PI3K/AKT/mTOR downstream signaling pathway. Finally, a molecular docking study targeting the EGFR/PI3K/AKT/mTOR cascades was conducted.

. Materials and Methods
All reagents and solvents were purchased from commercial suppliers and used without further purification. The reaction was followed up and checks of the purity were done using TLC on silica gel-protected aluminum sheets (Type 60 GF254, Merck, Darmstadt, Germany). Melting points were recorded on a Mel-Temp Apparatus (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in an open capillary and are uncorrected. Fourier transform infrared spectroscopy (FTIR) was conducted on a Shimadzu 8201 PC FTIR spectrophotometer (Shimadzu, Ltd., Kyoto, Japan). 1 H NMR and 13 C NMR spectra were recorded on a JEOL 400 MHz spectrometer (JEOL, Ltd., Tokyo, Japan), and chemical shift (δ) values were expressed in ppm. Elemental analyses were performed on a Perkin-Elmer 2400 elemental analyzer (PerkinElmer, Inc., Waltham, MA, USA). High resolution mass spectrometry (HRMS) was performed using a Bruker ESI-QTOF mass spectrometer (Bruker, Billerica, MA, USA) in positive-ion mode.
General Procedure for the Synthesis of 2-Hydrazino-6-Substituted s-Triazine Derivatives, 3a-l A solution of amine (20 mmol) in acetone (50 mL) was added dropwise over 15 min to a solution of cyanuric chloride 1 (20 mmol) in acetone (50 mL) at 0-5 • C. After complete addition, an aqueous solution of NaHCO 3 , (22 mmol equiv.) in water (50 mL) was added dropwise (10 min) at the same temperature. The reaction mixture was then stirred at 0-5 • C for 2 h. After completion of the reaction and disappearance of the starting materials (TLC, ethyl acetate/hexane 2:8), the second nucleophile (20 mmol) in acetone (50 mL) was added at the same temperature, followed by addition of an aqueous solution of NaHCO 3 (22 mmol equiv.) in water (50 mL). The reaction mixture was stirred at 0 • C for 1 h and then at rt overnight. Excess distilled water was added and the precipitates of products 2a-l (Scheme 1) were collected by filtration, washed with water (2 × 20 mL), and dried at rt to afford the desired products in good yield.
The chloro derivatives 2a-l were reacted with excess hydrazine hydrate (80%) for 6-8 h following the reported method [32,33,50] to afford the desired products 3a-l (Scheme 1) as white solids, which were used directly in the next step.
The spectral data for compounds 2a-l were previously reported by our group [32,33,50] and agreed with the reported data.

Cell Viability Assay (MTT)
To determine the IC 50 of the synthesized compounds 5a-i and 7a-t on the cell lines, an MTT assay was performed [51]. MDA-MB-231, MCF-7, and PANC-1 cells were seeded into 96-well plates at 8 × 10 3 cells/well (Corning, New York, NY, USA), and U-87 and A549 cells and HDFs were seeded at 6.5 × 10 3 cells/well. All cell lines were treated with concentrations of the tested compounds ranging from 0.5 to 500 µg/mL. Cells were then incubated at 37 • C in a 5% CO 2 incubator for 72 h, after which the old media was aspirated and the MTT assay salt (Bioworld, Visalia, CA, USA) in 100 µL of fresh media was added to each well. Next, plates were incubated at 37 • C for 3 h, then 50 µL of solubilization solution (DMSO) was added to each well to determine cell viability. The absorbance of the solution was measured at 560 nm using a Glomax plate reader (Promega, Madison, WI, USA).

EGFR Protein Kinase (PK) Inhibition
The EGFR-TK assay kit (ADP-Glo TM kinase assay, Cat No. V9261, Promega, USA) was used to determine the inhibitory capacity of compounds 7d, 7f and 7c against EGFR.

Apoptosis by Flow Cytometry
To study the growth inhibition of all the cell lines treated with 5a-i and 7a-t, the mechanism of apoptosis was determined by Annexin V/Propidium iodide (PI) stain using flow cytometry. Each cell line was seeded at 1 × 10 5 cells/well in 6-well plates and exposed to the IC 50 concentration of each compound for that specific cell line, as shown in Table 1. After 72 h, the cells were trypsinized using StemPro™ Accutase™ Cell Dissociation Reagent (Gibco™, Inchinnan, UK). The collected cells were then washed with PBS. Next, the Annexin V/PI apoptosis kit (Invitrogen, Waltham, MA, USA) was used to stain the cell pellets, following the manufacturer's instructions. 10,000 events were counted by BD FACS CANTO II and analyzed using BD FACS Diva™ software version 7.0.

Gene Expression Analysis Using RT-qPCR
To further examine the apoptotic pathway, we assessed the gene expression of P53, Bax, Caspases-3, -8, and -9 as proapoptotic genes, Bcl-2 as the anti-apoptotic gene, and the downstream pathway of PI3K/AKT/mTOR. MDA-MB-231 cells were treated with compound 7f at its IC 50 value for 48 h. After completing the treatment, cells were collected, and total RNA was extracted using the RNeasy ® Mini Kit (Qiagen, Hilden, Germany). cDNA was then synthesized using 500 ng of RNA (i-Script cDNA synthesis kit, BioRad, Hercules, CA, USA). Finally, each RT-qPCR reaction was performed following routine work [53]. The Ct values were then collected to calculate the relative gene expression in all samples by normalization to the β-actin housekeeping gene [54,55].

CDOCKER Docking
CDOCKER is a CHARMm-based simulated annealing/molecular dynamics method that uses rigid receptors for docking [56]. The CDOCKER protocol includes the following steps: (i) A set of ligand conformations is generated using high-temperature molecular dynamics starting with different random seeds. (ii) Random orientations of the conformations are produced by translating the center of the ligand to a specified location within the receptor active site and performing a series of random rotations. (iii) A softened energy is calculated, and the orientation is kept if the energy is less than a specified threshold. This process continues until either the desired number of low-energy orientations is found, or the maximum number of poor orientations has been attempted. (iv) Each orientation is subjected to simulated annealing molecular dynamics. The temperature is heated to a high preset temperature and then cooled to the target temperature. (v) A final minimization of the ligand in the rigid receptor using non-softened potential is performed. For each final pose, the CHARMm energy (interaction energy plus ligand strain) and the interaction energy alone is calculated. The poses are sorted by CHARMm energy and the top-scoring (most negative, thus favorable to binding) poses are retained. To enhance performance and shorten calculation times, a non-bond energy grid is used to calculate interaction energy rather than the full potential energy terms commonly used by CHARMm.
The following CDOCKER parameters were implemented in this study. A binding site sphere of 10.14 Å radius surrounding the copied co-crystallized ligand from the EGFR structure (PDB code: 6v6o) was implemented. The conformers of the starting ligands were energy-minimized and then heated to 1000 K over 1000 molecular dynamics steps to generate 50 starting random conformations for each ligand. Each random conformer was rotated 50 times within the binding pocket for subsequent energy refinement. The van der Waals energies of the resulting conformers/poses were examined and those of ≥300 kcal/mol were discarded. Surviving conformers/poses were subjected to a cycle of simulated annealing over 2000 heating steps to the targeted temperature of 700 K, followed by 5000 cooling steps to the targeted temperature of 300 K. The docked poses were energy-minimized to a gradient tolerance of zero kcal/mol/Å. A total of 599 poses were saved for subsequent scoring.
LigScore1 and LigScore2 scores were calculated using the CFF force field (version 1.02) and grid-based energies with a grid extension of 7.5 Å across the binding site. PMF scores were calculated using cutoff distances of 12.0 Å for carbon-carbon interactions and other atomic interactions, while PMF04 scores were calculated employing cutoff values of 6.0 and 9.0 Å for carbon-carbon interactions and other atomic interactions, respectively. -CDOCKER Energy and -CDOCKER Interaction Energy were calculated using the Momany-Rone ligand partial charge method. Docked conformers/poses were selected based on consensus among the 9 scoring functions [62,63]. The consensus function assigned a value of 1 for any molecular pose ranked within the highest 20% by the particular scoring function; otherwise, it was assigned a zero value (i.e., fit was within the lowest 80%). Subsequently, the consensus function summed up the scores for each molecular pose/conformer and ranked the molecular orientation. Docked poses of a particular ligand that achieved consensus among at least 8 scoring functions were selected and saved.

Chemistry
Hydrazino s-triazine derivatives 3a-l required for this study were prepared following our reported method [32,33,50] (Scheme 1), where cyanuric chloride 1 was reacted with the first nucleophile at 0 • C in the presence of NaHCO 3 as a base and acetone-water as a solvent for 2 h. The second nucleophile was added at the same temperature in the presence of 1 equiv. of NaHCO 3 and the reaction was stirred at room temperature (rt) for 24 h. The products 2-chloro-4,6-disubstituted s-triazine derivatives 2a-l were treated with hydrazine hydrate in ethanol under reflux for 6-8 h to afford the hydrazino derivatives 3a-l [32,33,50], which were used directly in the next step without further purification. To optimize the reaction conditions for the incorporation of the pyrazole ring into the s-triazine scaffold, we started with the study of 4-hydrazinyl-6-morpholino-N-phenyl-1,3,5-triazin-2-amine 3d as the model substrate (Table 1). Initially, ethylacetocacetate 4 (1 equiv.) was reacted with neat DMF-DMA (1.2 equiv.) at rt for 5-10 min to generate the enaminedinone intermediate I (Scheme 2), followed by the addition of 4-hydrazinyl-6morpholino-N-phenyl-1,3,5-triazin-2-amine 3d (1 equiv.) in 5% acetic acid in ethanol. The reaction mixture was refluxed for 4 h and monitored by TLC (n-hexane-ethylacetate, 1:1), which showed two products. The reaction did not promote at all with longer time (8 h) ( Table 1, entries 1 and 2). The 1 H-NMR spectrum (Supporting information, Figure S1) showed the two products 5d and 5d' in a 1:1 ratio. Given that the yield of these products (5d and 5d', Scheme 2) was low, we hypothesized that this parameter could be affected by reaction time or acid loading in the medium. To optimize the reaction conditions for the incorporation of the pyrazole ring into the s-triazine scaffold, we started with the study of 4-hydrazinyl-6-morpholino-N-phenyl-1,3,5-triazin-2-amine 3d as the model substrate (Table 1). Initially, ethylacetocacetate 4 (1 equiv.) was reacted with neat DMF-DMA (1.2 equiv.) at rt for 5-10 min to generate the enaminedinone intermediate I (Scheme 2), followed by the addition of 4-hydrazinyl-6-morpholino-N-phenyl-1,3,5-triazin-2-amine 3d (1 equiv.) in 5% acetic acid in ethanol. The reaction mixture was refluxed for 4 h and monitored by TLC (n-hexane-ethylacetate, 1:1), which showed two products. The reaction did not promote at all with longer time (8 h) ( Table 1, entries 1 and 2). The 1 H-NMR spectrum (Supporting information, Figure S1) showed the two products 5d and 5d' in a 1:1 ratio. Given that the yield of these products (5d and 5d', Scheme 2) was low, we hypothesized that this parameter could be affected by reaction time or acid loading in the medium. To optimize the reaction conditions for the incorporation of the pyrazole ring into the s-triazine scaffold, we started with the study of 4-hydrazinyl-6-morpholino-N-phenyl-1,3,5-triazin-2-amine 3d as the model substrate (Table 1). Initially, ethylacetocacetate 4 (1 equiv.) was reacted with neat DMF-DMA (1.2 equiv.) at rt for 5-10 min to generate the enaminedinone intermediate I (Scheme 2), followed by the addition of 4-hydrazinyl-6morpholino-N-phenyl-1,3,5-triazin-2-amine 3d (1 equiv.) in 5% acetic acid in ethanol. The reaction mixture was refluxed for 4 h and monitored by TLC (n-hexane-ethylacetate, 1:1), which showed two products. The reaction did not promote at all with longer time (8 h) ( Table 1, entries 1 and 2). The 1 H-NMR spectrum (Supporting information, Figure S1) showed the two products 5d and 5d' in a 1:1 ratio. Given that the yield of these products (5d and 5d', Scheme 2) was low, we hypothesized that this parameter could be affected by reaction time or acid loading in the medium. Encouraged by this result, we focused on the solvent effect ( Table 1). The reaction was repeated by altering the AcOH ratio (10%, 20% and 33.3%). Screening of AcOH loading revealed that the increasing percentage of AcOH was crucial, as the chemical yield of the cyclized product rather than the open form was enhanced. The optimal reaction condition was 33.3% AcOH, affording the final cyclized compound 5d in 90% yield in 6 h (Entry 5), which increased to 95% after 8 h (Entry 6). The tentative synthetic reaction pathway is shown in Scheme 2. It comprises the initial formation of the enamine intermediate I in situ, followed by nucleophilic attack by the hydrazine derivative in the presence of AcOH to afford the final cyclized product. The hydrazine derivative 3d could possible attack the enamine intermediate I via two pathways (A or B). The enamine intermediate I is typically more reactive in bath A (5d) via initial addition-elimination amine-exchange of the dimethylamino group by the hydrazine derivative to afford the product 5d through the open analogue 5d' after removal of water molecule form the intermediate II [64,65]. This analogue of pyrazole derivative 5d is more favor than its analogue in path B which first formation of the Schiff base (intermediate III) then Nu-attack to the enamine to afford the cyclized analogue 5d" after removal of NEMe 2 (Scheme 3) [64,65]. Encouraged by this result, we focused on the solvent effect ( Table 1). The reaction was repeated by altering the AcOH ratio (10%, 20% and 33.3%). Screening of AcOH loading revealed that the increasing percentage of AcOH was crucial, as the chemical yield of the cyclized product rather than the open form was enhanced. The optimal reaction condition was 33.3% AcOH, affording the final cyclized compound 5d in 90% yield in 6 h (Entry 5), which increased to 95% after 8 h (Entry 6). AcOH-EtOH (1:2) 8 traces ~95 The tentative synthetic reaction pathway is shown in Scheme 2. It comprises the initial formation of the enamine intermediate I in situ, followed by nucleophilic attack by the hydrazine derivative in the presence of AcOH to afford the final cyclized product. The hydrazine derivative 3d could possible attack the enamine intermediate I via two pathways (A or B). The enamine intermediate I is typically more reactive in bath A (5d) via initial addition-elimination amine-exchange of the dimethylamino group by the hydrazine derivative to afford the product 5d through the open analogue 5d' after removal of water molecule form the intermediate II [64,65]. This analogue of pyrazole derivative 5d is more favor than its analogue in path B which first formation of the Schiff base (intermediate III) then Nu-attack to the enamine to afford the cyclized analogue 5d'' after removal of NEMe2 (Scheme 3) [64,65].  Under the optimized conditions and configured the synthetic pathway, various hydrazine derivatives 3a-i, including various heterocycles (morpholine and piperidine) and, substituted aryls employed in this reaction and furnished the products 5a-i, as indicated in Scheme 4 in excellent yield. The spectral data for the synthesized compounds 5a-i are provided in the supporting information, see Figures S2-S9). Under the optimized conditions and configured the synthetic pathway, various hydrazine derivatives 3a-i, including various heterocycles (morpholine and piperidine) and substituted aryls employed in this reaction and furnished the products 5a-i, as indicated in Scheme 4 in excellent yield. The spectral data for the synthesized compounds 5a-i are provided in the supporting information, see Figures S2-S9).

Scheme 4. Synthesis of pyrazole-s-triazine derivatives 5a-i.
To explore a cyclized 1,3-dicarbonyl compounds instead of ethylacetoacetate, for example, 5,5-dimethyl-1,3-cyclohexandione 6a or 1,3-cyclohexadionone 6b were employed in the reaction under the optimized reaction condition used for the synthesis of 5a-i but did not afford the final compound 7. In contrast, when the reaction was run in a neat AcOH, complete reaction occurred after 8-12 h, as shown by TLC (n-hexane-ethylacetate 1:1). After completion of the reaction, the acidic solution was poured into ice-cold water and extracted with AcOEt or CHCl3. Next, the organic phase was washed with 10% Na2CO3 solution and NaCl solution and then dried over anhydrous MgSO4. The desired products 7a-t (Scheme 5) were obtained after evaporation of the solvent.
The structures of all the products obtained were established by IR, NMR ( 1 H and 13 C) elemental analysis, and HRMS-ESI (Figures S10-S29 for the NMR spectrum and Figures  S30-S38 for the HRMS). In addition, compound 7t (CCDC No.: 2177427) was assigned based on single crystal X-ray diffraction analysis [66][67][68][69] (see Supporting Information). To explore a cyclized 1,3-dicarbonyl compounds instead of ethylacetoacetate, for example, 5,5-dimethyl-1,3-cyclohexandione 6a or 1,3-cyclohexadionone 6b were employed in the reaction under the optimized reaction condition used for the synthesis of 5a-i but did not afford the final compound 7. In contrast, when the reaction was run in a neat AcOH, complete reaction occurred after 8-12 h, as shown by TLC (n-hexane-ethylacetate, 1:1). After completion of the reaction, the acidic solution was poured into ice-cold water and extracted with AcOEt or CHCl 3 . Next, the organic phase was washed with 10% Na 2 CO 3 solution and NaCl solution and then dried over anhydrous MgSO 4 . The desired products 7a-t (Scheme 5) were obtained after evaporation of the solvent.
The structures of all the products obtained were established by IR, NMR ( 1 H and 13 C), elemental analysis, and HRMS-ESI (Figures S10-S29 for the NMR spectrum and Figures  S30-S38 for the HRMS). In addition, compound 7t (CCDC No.: 2177427) was assigned based on single crystal X-ray diffraction analysis [66][67][68][69]

Cytotoxicity
The anticancer activity of the pyrazolyl-s-triazine derivatives (5a-i and 7a-t; Table 2) was studied in the following cell lines: human breast cancer (MCF-7 and MDA-MB-231); glioblastoma (U-87 MG); non-small cell lung cancer (A549); pancreatic cancer (PANC-1); and human dermal fibroblasts (HDFs). Most of the derivatives affected the viability of the six cancer cell lines, as determined by the MTT cell viability assay [51] (Table 2 and Figure  2). The most effective compounds were 7d, 7f, and 7c. Of note, the substitution of the triazine ring had a marked impact on anticancer activity ( Table 2). In most cases, compounds 5a-i (carboxylate derivatives) showed lower activity than 7a-t (5,5-dimethyl-1,3cyclohexadione and 1,3-cyclohexadionone derivatives). Moreover, 5,5-dimethyl-1,3-cyclohexadione s-triazine derivatives 7a-i showed higher activity against most cancer cells compared with 1,3-cyclohexadionone derivatives 7j-t. In general, compounds with the aniline moiety exerted greater activity compared to those with a morpholine or piperidine ring. Interestingly, piperidine and morpholine can be considered to belong to the same family in some aspects of their synthetic chemistry. The presence of the piperidine moiety was detrimental for anticancer activity, as observed in compounds with both the piperidine and aniline ring attached to the triazine ring, compared to morpholine analogs. Compounds with two aniline moieties showed the highest activity (Table 2)

Cytotoxicity
The anticancer activity of the pyrazolyl-s-triazine derivatives (5a-i and 7a-t; Table 2) was studied in the following cell lines: human breast cancer (MCF-7 and MDA-MB-231); glioblastoma (U-87 MG); non-small cell lung cancer (A549); pancreatic cancer (PANC-1); and human dermal fibroblasts (HDFs). Most of the derivatives affected the viability of the six cancer cell lines, as determined by the MTT cell viability assay [51] (Table 2 and Figure 2). The most effective compounds were 7d, 7f, and 7c. Of note, the substitution of the triazine ring had a marked impact on anticancer activity ( Table 2). In most cases, compounds 5a-i (carboxylate derivatives) showed lower activity than 7a-t (5,5-dimethyl-1,3-cyclohexadione and 1,3-cyclohexadionone derivatives). Moreover, 5,5-dimethyl-1,3cyclohexadione s-triazine derivatives 7a-i showed higher activity against most cancer cells compared with 1,3-cyclohexadionone derivatives 7j-t. In general, compounds with the aniline moiety exerted greater activity compared to those with a morpholine or piperidine ring. Interestingly, piperidine and morpholine can be considered to belong to the same family in some aspects of their synthetic chemistry. The presence of the piperidine moiety was detrimental for anticancer activity, as observed in compounds with both the piperidine and aniline ring attached to the triazine ring, compared to morpholine analogs. Compounds with two aniline moieties showed the highest activity (Table 2). Thus, 5d, which contains morpholine and aniline, showed IC 50 values of 39.4 ± 1.9, 42.2 ± 3.4, 73.8 ± 21.0, 26.4 ± 2.7, 10.5 ± 2.4, and 33.4 ± 3.7 (µM) against MDA-MB-231, U-87 MG, PANC-1, A549, MCF-7 and HDFs, respectively. In contrast, compounds with two morpholine 5a, two piperidine 5b, one morpholine and one piperidine 5c and one piperidine and aniline 5e showed lower activity, with higher IC 50 values. In addition, compounds with substituted aniline (p-bromo-, p-chloro-, or p-methoxyanilne) exerted lower activity than unsubstituted aniline in this series of compounds. The same behavior was observed with series of compounds 7a-t (Table 2). tuted aniline in this series of compounds. The same behavior was observed with series of compounds 7a-t (Table 2). In general, the IC50 of 7d, 7f, and 7c revealed a reduction in the viability of the cancer cell lines tested compared to the normal fibroblasts. Interestingly, there were some variations in the IC50 values of the compounds in the distinct cancer cell lines. For instance, 7c showed similar IC50 values against MCF-7, U-87 MG, and PANC-1 cell lines (15.7-17.1 µM) while the values against the MDA-MB-231 and A549 cells were 28.2 and 49.8 µ M, respectively. Compound 7d was found to be more powerful against MCF-7, U-87 MG, and A549 cells, with IC50 values of 8.3, 10.9, and 12.4 µ M, respectively. On the other hand, the MDA-MB-231 cancer cell line showed resistance to 7d compared to the HDFs. Compound 7f was the least effective against the cancer cell lines. However, it showed selectivity against the MDA-MB-231 cell line compared to the other compounds and the IC50 of other cell lines (Table 2, Figure 2). tuted aniline in this series of compounds. The same behavior was observed with series of compounds 7a-t (Table 2). In general, the IC50 of 7d, 7f, and 7c revealed a reduction in the viability of the cancer cell lines tested compared to the normal fibroblasts. Interestingly, there were some variations in the IC50 values of the compounds in the distinct cancer cell lines. For instance, 7c showed similar IC50 values against MCF-7, U-87 MG, and PANC-1 cell lines (15.7-17.1 µM) while the values against the MDA-MB-231 and A549 cells were 28.2 and 49.8 µ M, respectively. Compound 7d was found to be more powerful against MCF-7, U-87 MG, and A549 cells, with IC50 values of 8.3, 10.9, and 12.4 µ M, respectively. On the other hand, the MDA-MB-231 cancer cell line showed resistance to 7d compared to the HDFs. Compound 7f was the least effective against the cancer cell lines. However, it showed selectivity against the MDA-MB-231 cell line compared to the other compounds and the IC50 of other cell lines (Table 2, Figure 2). tuted aniline in this series of compounds. The same behavior was observed with series of compounds 7a-t (Table 2). In general, the IC50 of 7d, 7f, and 7c revealed a reduction in the viability of the cancer cell lines tested compared to the normal fibroblasts. Interestingly, there were some variations in the IC50 values of the compounds in the distinct cancer cell lines. For instance, 7c showed similar IC50 values against MCF-7, U-87 MG, and PANC-1 cell lines (15.7-17.1 µM) while the values against the MDA-MB-231 and A549 cells were 28.2 and 49.8 µ M, respectively. Compound 7d was found to be more powerful against MCF-7, U-87 MG, and A549 cells, with IC50 values of 8.3, 10.9, and 12.4 µ M, respectively. On the other hand, the MDA-MB-231 cancer cell line showed resistance to 7d compared to the HDFs. Compound 7f was the least effective against the cancer cell lines. However, it showed selectivity against the MDA-MB-231 cell line compared to the other compounds and the IC50 of other cell lines (Table 2, Figure 2).

EGFR Enzymatic Assay
The capacity of 7d, 7f, and 7c to inhibit EGFR was tested (

EGFR Enzymatic Assay
The capacity of 7d, 7f, and 7c to inhibit EGFR was tested (Table 3). Compounds 7d and 7f exhibited potent EGFR inhibitory activity, with IC50 values of 59.24 and 70.3 nM, In general, the IC 50 of 7d, 7f, and 7c revealed a reduction in the viability of the cancer cell lines tested compared to the normal fibroblasts. Interestingly, there were some variations in the IC 50 Figure 2).

EGFR Enzymatic Assay
The capacity of 7d, 7f, and 7c to inhibit EGFR was tested (

PI3K/AKT/mTOR Downstream Signaling Pathway
To study the molecular target for the promising cytotoxicity of 7d, 7f, and 7c, which showed the highest cytotoxic activity and promising EGFR inhibitory capacity, these compounds were tested against the PI3K, AKT, and mTOR downstream inhibition pathway. The PI3K/AKT/mTOR signaling cascade is important in many cellular processes, including growth and proliferation, apoptosis, survival, and metabolism, all of which contribute to tumor progression [70,71].
These compounds showed promising capacity to inhibit PI3K/AKT/mTOR (Table 4). In this regard, 7d and 7f exhibited remarkable PI3K/AKT/mTOR inhibitory activity by 0.66/0.82/0.8 and 0.35/0.56/0.66-fold, respectively, by inhibiting their concentrations to 4.39, 37.3, and 69.3 ng/mL in the 7d-treatment, and to 2.39, 25.34, and 57.6 ng/mL in the 7f-treatment compared to the untreated control; while compound 7c did not show inhibitory activity compared to the control. Values are expressed as Mean ± SD of three independent replicates.

Apoptosis by Flow Cytometry
The results of the apoptosis assay showed significant induction of apoptosis by 7d, 7f, and 7c in all the cancer cell lines compared to the normal cell line. In particular, the greatest induction of apoptosis by these three compounds occurred in the MDA-MB-231 and PANC1 cancer cell lines. In this regard, they induced total apoptosis in MDA-MB-231 cells by 26.1%, 31.54%, and 17.2%, respectively, compared to 1.43% in the untreated control (Figures 3 and 4). Additionally, they induced total apoptosis in PANC1 cells by 31.7%, 30.4%, and 40.3%, respectively, compared to 11% in the untreated control (Figures 3 and S39). Furthermore, 7f showed more specific activation of apoptosis in the A549 cancer cell line (27.7% compared to 0.11% in control) compared to the other two compounds (Figures 3 and S39), and it induced total apoptosis in MCF-7 cells (21.53% compared to 0.67% in control). Histograms for Annexin V/PI stainting for the tested compounds in cancer cells were supported in the Supporting Information Figure S39. Results elucidated that cytotoxic activities in cancer cells were due to apoptosis rather than necrosis.  To confirm the apoptosis-inducing activity of the compounds in MDA-MB-231 cells, we conducted gene expression analysis using RT-qPCR in both untreated and treated cells. As seen in Figure 5, treatment with compound 7f increased the expression of the following pro-apoptotic genes: a 3.8-fold increase in P53, a 2.8-fold increase in Bax, and a   To confirm the apoptosis-inducing activity of the compounds in MDA-MB-231 cells, we conducted gene expression analysis using RT-qPCR in both untreated and treated cells. As seen in Figure 5, treatment with compound 7f increased the expression of the

Compound 7f Upregulated Pro-Apoptotic Genes and Downregulated Anti-Apoptotic Ones
To confirm the apoptosis-inducing activity of the compounds in MDA-MB-231 cells, we conducted gene expression analysis using RT-qPCR in both untreated and treated cells. As seen in Figure 5, treatment with compound 7f increased the expression of the following pro-apoptotic genes: a 3.8-fold increase in P53, a 2.8-fold increase in Bax, and a 6.7-, 3.06-, and 7-fold increase in caspases 3, 8, and 9, respectively. In contrast, this treatment caused a 0.17-fold decrease in the expression of the anti-apoptotic gene Bcl-2. In addition, this treatment induced a 0.61-, 0.32-, and 0.18-fold decrease in the PI3K/AKT/mTOR downstream pathway. These results regarding behavior of upregulating the proapoptotic genes and down-regulating the antiapoptotic gene agreed with previous literatures [50,72,73] on proving apoptosis induction in cancer cells. Apoptosis activity upon treatment with compound 7f, a derivative of pyrazolyl s-triazine moieties, was elucidated via EGFR inhibition and its downstreaming pathway of PI3K/AKT/mTOR.
We selected docked conformers/poses based on consensus among the 9 scoring functions [60,61]. The consensus function assigned a value of 1 for any molecular pose ranked within the highest 20% by the particular scoring function; otherwise, a zero value was assigned (i.e., the fit was within the lowest 80%). Docked poses of a particular ligand that achieved consensus among at least 8 scoring functions were selected.
The best-docked poses of 7d, 7f, and 7c interacted with several amino acids in the active site ( Figure 6). Interactions included hydrogen bonding, and hydrophobic and electrostatic interactions. The three ligands showed slightly different binding modes in the active site (Figure 6), particularly 7d and 7f.
The central triazine ring of the three compounds is involved in hydrogen bonding with Lys745 (K745) and hydrophobic interactions with Val726 (V726). One terminal of the three compounds also participates in hydrophobic interactions and either hydrogen or electrostatic bonding with Arg841 (R841). The other terminals of the three compounds are involved in several hydrophobic interactions with Leu718 (L718), Met790 (M790), Ala743 (A743), and Leu788 (L788). Of note, the binding site of the three compounds is enriched in basic amino acids (K745 and R841) and hydrophobic amino acids (V726, L718, M790,
We selected docked conformers/poses based on consensus among the 9 scoring functions [60,61]. The consensus function assigned a value of 1 for any molecular pose ranked within the highest 20% by the particular scoring function; otherwise, a zero value was assigned (i.e., the fit was within the lowest 80%). Docked poses of a particular ligand that achieved consensus among at least 8 scoring functions were selected.
The best-docked poses of 7d, 7f, and 7c interacted with several amino acids in the active site ( Figure 6). Interactions included hydrogen bonding, and hydrophobic and electrostatic interactions. The three ligands showed slightly different binding modes in the active site (Figure 6), particularly 7d and 7f.
The central triazine ring of the three compounds is involved in hydrogen bonding with Lys745 (K745) and hydrophobic interactions with Val726 (V726). One terminal of the three compounds also participates in hydrophobic interactions and either hydrogen or electrostatic bonding with Arg841 (R841). The other terminals of the three compounds are involved in several hydrophobic interactions with Leu718 (L718), Met790 (M790), Ala743 (A743), and Leu788 (L788). Of note, the binding site of the three compounds is enriched in basic amino acids (K745 and R841) and hydrophobic amino acids (V726, L718, M790, A743, and L788). All the binding poses in Figure 6 show a comparable number and type of interactions (i.e., consensus score ≥ 8), which are believed to play a significant role in the high affinity of these compounds. of interactions (i.e., consensus score ≥ 8), which are believed to play a significant role in the high affinity of these compounds.

Conclusions
Here, we reported an easy one-pot procedure for the synthesis of pyrazole-s-triazine derivatives via the reaction of β-dicarbonyl compounds in the presence of DMF-DMA with 4,6-disubstituted 2-hydrazinyl-s-triazine in the presence of acetic acid. This method achieved a novel pyrazole and pyrazole-fused cycloalkanones in 80-95% yield and could find applications for the preparation of a variety of pyrazolo-s-triazine derivatives with biological activity of interest. Most of the tested compounds showed promising cytotoxicity against a panel of cancer cells and a safe profile against normal cells. Interestingly, compounds 7c, 7d, and 7f induced apoptosis in MDA-MB-231 cells through the EGFR/PI3K/AKT/mTOR signaling pathway. Hence, these compounds emerge as potential target-oriented chemotherapeutic agents against breast cancer.

Conclusions
Here, we reported an easy one-pot procedure for the synthesis of pyrazole-s-triazine derivatives via the reaction of β-dicarbonyl compounds in the presence of DMF-DMA with 4,6-disubstituted 2-hydrazinyl-s-triazine in the presence of acetic acid. This method achieved a novel pyrazole and pyrazole-fused cycloalkanones in 80-95% yield and could find applications for the preparation of a variety of pyrazolo-s-triazine derivatives with biological activity of interest. Most of the tested compounds showed promising cytotoxicity against a panel of cancer cells and a safe profile against normal cells. Interestingly, compounds 7c, 7d, and 7f induced apoptosis in MDA-MB-231 cells through the EGFR/PI3K/AKT/mTOR signaling pathway. Hence, these compounds emerge as potential target-oriented chemotherapeutic agents against breast cancer.