De Novo Design of Imidazopyridine-Tethered Pyrazolines That Target Phosphorylation of STAT3 in Human Breast Cancer Cells

In breast cancer (BC), STAT3 is hyperactivated. This study explored the design of imidazopyridine-tethered pyrazolines as a de novo drug strategy for inhibiting STAT3 phosphorylation in human BC cells. This involved the synthesis and characterization of two series of compounds namely, 1-(3-(2,6-dimethylimidazo [1,2-a]pyridin-3-yl)-5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-(substituted)piperazin-1-yl)ethanone and N-substituted-3-(2,6-dimethylimidazo[1,2-a]pyridin-3-yl)-5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazoline-1-carbothioamides. Compound 3f with 2,3-dichlorophenyl substitution was recognized among the tested series as a lead structure that inhibited the viability of MCF-7 cells with an IC50 value of 9.2 μM. A dose- and time-dependent inhibition of STAT3 phosphorylation at Tyr705 and Ser727 was observed in MCF-7 and T47D cells when compound 3f was added in vitro. Calculations using density functional theory showed that the title compounds HOMOs and LUMOs are situated on imidazopyridine-pyrazoline and nitrophenyl rings, respectively. Hence, compound 3f effectively inhibited STAT3 phosphorylation in MCF-7 and T47D cells, indicating that these structures may be an alternative synthon to target STAT3 signaling in BC.

De novo drug design retains the potential for the discovery of new and potent lead molecules for oncology [20]. Pharmacology has heavily utilized imidazopyridine scaffolds in drug development [21]. Ten approved drugs contain imidazopyridine, and another 12 are in active clinical development [22,23]. A synthetic imidazopyridine compound 16, was synthesized, tested, and determined to reduce the level of phospho-STAT3 and downstream signaling cascades in hepatocellular carcinoma cells, which was attributed to an increase in SHP-1A [24]. Furthermore, the design and synthesis of an imidazopyridine scaffoldbearing compound (P3971) has been identified as a potent STAT3 inhibitor with an IC50 value of 350 nM and demonstrated significant antiproliferative activity against a variety of cancerous cell lines including HCT116 and H460 [25]. Additionally, pyrazoles may provide better pharmacological effects, and have also generated a number of drugs such as pyrazofurin, celecoxib, ramifenazone, lonazolac, and rimonabant [26][27][28][29][30]. We reported the synthesis of pyridine-fused pyrazoles as a STAT3 inhibitor and an inhibitor of cancer cell growth [31]. Additionally, the pyrazole-based compound MNS1-Leu inhibited IL-6-induced STAT3 phosphorylation in patient-derived HGG cells without adversely affecting Akt, STAT1, JAK2, or ERK1/2 phosphorylation [32]. Compound C6 was also discovered to be a STAT3-specific inhibitor that had the strongest anti-proliferation activities against breast cancer cells with an IC 50 value of 160 nM [33]. In light of this, we developed a conventional de novo design and identified the essential output structures comprising imidazopyridine, pyrazole, pyrrole, and proposed a series of imidazopyridine-tethered pyrazolines (ITP) that could target STAT3 in breast cancer cells based on synthetic accessibility and chemical stability ( Figure 1). ous studies, various drugs such as tamoxifen, vinflunine, anastrazole, 5-fluorouracil, Doxorubicin, paclitaxel/docetaxel, ribociclib, olaparib, and other drugs have been shown to possess therapeutic potential for women with BC depending on the stage and the specific molecular subtype [5][6][7][8][9][10][11][12]. Genes that encode transcription factors are directly involved in breast cancer progression, proliferation, apoptosis, metastasis, and chemotherapy resistance [13,14]. STAT3 is one such transcription factor that harbors six functional domains, including the terminal-NH2 domain, the coiled-coil domain, the DNA-binding domain, the SRC homology 2 domain, and the transactivation domain [15,16]. STAT3 is activated by both tyrosine and serine phosphorylation and translocates into the nucleus to regulate transcription [17][18][19].
De novo drug design retains the potential for the discovery of new and potent lead molecules for oncology [20]. Pharmacology has heavily utilized imidazopyridine scaffolds in drug development [21]. Ten approved drugs contain imidazopyridine, and another 12 are in active clinical development [22,23]. A synthetic imidazopyridine compound 16, was synthesized, tested, and determined to reduce the level of phospho-STAT3 and downstream signaling cascades in hepatocellular carcinoma cells, which was attributed to an increase in SHP-1A [24]. Furthermore, the design and synthesis of an imidazopyridine scaffold-bearing compound (P3971) has been identified as a potent STAT3 inhibitor with an IC50 value of 350 nM and demonstrated significant antiproliferative activity against a variety of cancerous cell lines including HCT116 and H460 [25]. Additionally, pyrazoles may provide better pharmacological effects, and have also generated a number of drugs such as pyrazofurin, celecoxib, ramifenazone, lonazolac, and rimonabant [26][27][28][29][30]. We reported the synthesis of pyridine-fused pyrazoles as a STAT3 inhibitor and an inhibitor of cancer cell growth [31]. Additionally, the pyrazole-based compound MNS1-Leu inhibited IL-6-induced STAT3 phosphorylation in patient-derived HGG cells without adversely affecting Akt, STAT1, JAK2, or ERK1/2 phosphorylation [32]. Compound C6 was also discovered to be a STAT3-specific inhibitor that had the strongest anti-proliferation activities against breast cancer cells with an IC50 value of 160 nM [33]. In light of this, we developed a conventional de novo design and identified the essential output structures comprising imidazopyridine, pyrazole, pyrrole, and proposed a series of imidazopyridine-tethered pyrazolines (ITP) that could target STAT3 in breast cancer cells based on synthetic accessibility and chemical stability ( Figure 1).
In order to understand the lead structure specificity in the bioactivity of the ITP compounds, DFT calculations were performed. From the frontier molecular orbital (FMOs) theory, HOMO and LUMO are the most influential factors in bioactivity. HOMO has the priority to provide electrons, while LUMO can accept electrons. Moreover, the difference in energy between these two FMOs can be used to predict the strength and stability of molecular complexes [41]. Figure 4 shows the molecular orbital of compounds, while Table 2 lists the calculated global chemical reactivity descriptor parameters of compounds.
HOMO-LUMO levels indicate the interactions between the compound and the protein target. Usually, the HOMO of the compound interacts with the LUMO of the target for binding, and vice versa. A higher HOMO and lower LUMO energies of the molecule imply greater target stability and binding. The lower HOMO-LUMO gap indicates that the lead has lower kinetic stability or higher chemical reactivity and polarizability. Compounds 4g and 3f have the most considerable ionization potential among the compounds, 6.905 eV and 6.764 eV, respectively. All of the synthesized molecules are stable within the permitted limits. Molecules with high polarizability are chemically more soft or reactive, related to chemical hardness. Figure 4 shows contour plots of the HOMO and LUMO for compounds 3f and 4g. The green and red contours surrounding the atoms represent the negative and positive lobes of wave functions, respectively. The green and red contours encircling the atoms depict the wave function's negative and positive lobes. It is clear from the plots that the HOMO is localized on the dimethylimidazole-pyridin, pyrazoline sites, and O and S atoms of all the molecules. In contrast, LUMO is localized on the nitrophenyl ring in all the molecules.
The distribution of electrostatic potential (EP) over atomic sites is represented by the molecular electrostatic potential (MEP) profile, which can be connected to the partial charge distribution, the electronegativity of atoms in lead molecules, and their interactions. The MEP plots of compounds 3f and 4g are shown in Figure 5, where the EP varies from the negative (red) to the positive value (blue) in the sequence given by the color spectrum: red (negative) < orange < yellow < green < blue (positive). The negative EPs are located on the O atomic sites of C=O and N-O2, which indicate that these sites are electron-rich. The positive EPs are seen on H atoms, particularly for H attached to the N, indicating this is an electron-deficient site. Depending on the nature of EPs, these sites would prefer to bind to sites having the opposite potential in the binding pocket or hydrogen bonding interaction (Table 3). For example, the electron-rich C=O should combine with positively charged protons of amino acid residues present in the binding pocket.  In order to understand the lead structure specificity in the bioactivity of the ITP compounds, DFT calculations were performed. From the frontier molecular orbital (FMOs) theory, HOMO and LUMO are the most influential factors in bioactivity. HOMO has the priority to provide electrons, while LUMO can accept electrons. Moreover, the difference in energy between these two FMOs can be used to predict the strength and stability of molecular complexes [41]. Figure 4 shows the molecular orbital of compounds, while Ta-  HOMO-LUMO levels indicate the interactions between the compound and the protein target. Usually, the HOMO of the compound interacts with the LUMO of the target for binding, and vice versa. A higher HOMO and lower LUMO energies of the molecule imply greater target stability and binding. The lower HOMO-LUMO gap indicates that the lead has lower kinetic stability or higher chemical reactivity and polarizability. Compounds 4g and 3f have the most considerable ionization potential among the compounds, 6.905 eV and 6.764 eV, respectively. All of the synthesized molecules are stable within the permitted limits. Molecules with high polarizability are chemically more soft or reactive,  spectrum: red (negative) < orange < yellow < green < blue (positive). The negative EPs are located on the O atomic sites of C = O and N-O2, which indicate that these sites are electron-rich. The positive EPs are seen on H atoms, particularly for H attached to the N, indicating this is an electron-deficient site. Depending on the nature of EPs, these sites would prefer to bind to sites having the opposite potential in the binding pocket or hydrogen bonding interaction (Table 3). For example, the electron-rich C = O should combine with positively charged protons of amino acid residues present in the binding pocket.  The rigid docking method analyzed the synthesized compounds 3f and 4g [42]. Au-toDock4.2 was used to determine the orientation of inhibitors bound to STAT3 (PDB ID:  The rigid docking method analyzed the synthesized compounds 3f and 4g [42]. AutoDock4.2 was used to determine the orientation of inhibitors bound to STAT3 (PDB ID: 1BG1) and the conformation with the highest binding energy value for each molecule. The binding modes of STAT3 inhibitors were analyzed using the PyMOL software to identify new STAT3 inhibitors. The binding site at the SH2 domain of STAT3 was described by Becker et al. [43]. It was used to elucidate the interactions that contributed to the compounds' binding affinity to STAT3.
The promising binding modes of 3f and 4g at the SH2 domain of the STAT3 protein were analyzed. Figures 6 and 7 show the ligand and receptor complex poses with the highest binding energy. The binding energies of 3f and 4g to the SH2 domain of STAT3 were observed to be −9.27 kcal/mol and −6.95 kcal/mol, respectively, indicating that the molecule has a high affinity for the target. The binding patterns of the lead molecules 3f and 4g were studied. Both molecules bound to the same site on the receptor molecule (STAT3) and exhibited similar interactions with the vital amino acids of the SH2 domain of STAT3. The docking results showed that the ketone group of 3f forms a hydrogen bond with Leu706 of the SH2 domain, and the nitro group of the same molecule interacts with ARG688 via a salt bridge. One of the oxygens in the nitro group of 4g forms a hydrogen bond and the other oxygen participated in the interaction through a salt bridge with the same ARG688 (Figure 8). To summarize, the presence of nitro groups (Figures 8 and 9) in the molecules and structural flexibility facilitates its interaction with the SH2 domain of STAT3. of STAT3. The docking results showed that the ketone group of 3f forms a hydrogen bond with Leu706 of the SH2 domain, and the nitro group of the same molecule interacts with ARG688 via a salt bridge. One of the oxygens in the nitro group of 4g forms a hydrogen bond and the other oxygen participated in the interaction through a salt bridge with the same ARG688 (Figure 8). To summarize, the presence of nitro groups (Figures 8 and 9) in the molecules and structural flexibility facilitates its interaction with the SH2 domain of STAT3.        Finally, we attempted to understand the effect of compound 3f on the blocking of pSTAT3 into nuclear functionally since pSTAT3 dimers could enter the nucleus to give transcription. For this purpose, we conducted an immunocytochemistry assay using MCF-7 and T47D cells. We observed that compound 3f inhibited the nuclear translocation of STAT3 in MCF-7 and T47D cells. We also analyzed the distribution of phospho-STAT3 in the nucleus and cytoplasm using fluorescent-labeled antibodies. Compound 3f could block the nuclear translocation of pSTAT3 in MCF-7 and T47D cells, as shown in Figure 10A,B, respectively. Finally, we attempted to understand the effect of compound 3f on the blocking of pSTAT3 into nuclear functionally since pSTAT3 dimers could enter the nucleus to give transcription. For this purpose, we conducted an immunocytochemistry assay using MCF-7 and T47D cells. We observed that compound 3f inhibited the nuclear translocation of STAT3 in MCF-7 and T47D cells. We also analyzed the distribution of phospho-STAT3 in the nucleus and cytoplasm using fluorescent-labeled antibodies. Compound 3f could block the nuclear translocation of pSTAT3 in MCF-7 and T47D cells, as shown in Figure  10A and Figure 10B, respectively.

Materials and Methods
All chemicals and solvents for chemistry were purchased from Sigma-Aldrich and TCI chemicals, INDIA. The completion of the reaction was monitored by pre-coated silica gel TLC plates. 1 H and 13 C NMR were recorded on an Agilent NMR spectrophotometer (400 MHz); TMS was used as an internal standard and CDCl3 was used as a solvent, chemical shifts are expressed as ppm.

Materials and Methods
All chemicals and solvents for chemistry were purchased from Sigma-Aldrich and TCI chemicals, INDIA. The completion of the reaction was monitored by pre-coated silica gel TLC plates. 1 H and 13 C NMR were recorded on an Agilent NMR spectrophotometer (400 MHz); TMS was used as an internal standard and CDCl 3 was used as a solvent, chemical shifts are expressed as ppm.

Synthesis of 2-Pyrazoline Derivatives (3a-i) from 2,6-Dimethyl-3-(5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl)imidazo[1,2-a]pyridine
One mmol of (1) was dissolved in toluene and kept in an ice bath. After 15 min triethylamine (1.5 mmol) was added in portions followed chloroacetyl chloride (1.5 mmol) and the reaction was monitored by TLC. After the completion of the reaction, toluene was distilled off under reduced pressure and the residue was extracted with chloroform. The crude solid was used directly in the next reaction without further purification. The crude solid (1 mmol) and substituted piperazines (1 mmol) were dissolved in acetone and refluxed overnight with triethylamine (1 mmol). TLC was monitored and acetone was distilled off, the crude obtained was extracted with chloroform and recrystallized using DCM-Hexane to afford substituted pyrazoline derivatives(3a-i).

Synthesis of 2-Pyrazoline Derivatives
Pyrazoline derivative (1) (1 mmol) and substituted isothiocyanates (1.2 mmol) were dissolved in toluene and refluxed overnight with triethylamine (1 mmol). The reaction was monitored by TLC and reaction mass was filtered, washed with hexane to remove unreacted isothiocyanate, and dried in hot air oven to yield pure substituted thiourea derivatives (4a-h).

Cell Viability Assay
MCF-7 cells were procured from Procell Life Science and Technology Co. LTD, Wuhan, China. Human breast cancer T47D, BT474, and SK-BR-3 cells were obtained from American Type Culture Collection (Washington, DC, NW, USA). Cells (2000) were cultured in MEM or Leibovitz's L-15 medium enriched with 2% FBS and maintained in a humidified atmosphere of 5% CO 2 at 37 • C. A stock solution of DMSO-dissolved compounds was prepared, and this solution was diluted with culture medium as required. MCF-7 Cells (4 × 10 3 ) were cultured in 96-well plates for 12 h and then treated for 72 h with compounds at concentrations of 0, 0.01, 0.1, 10, 100, and 1000 mM. Using Alamar Blue, the compounds were evaluated for their inhibitory effects [44,45]

Western Blot Analysis
Equal protein concentrations of cell lysate were resolved on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, followed by transfer to nitrocellulose membranes as reported previously [47]. Incubation was carried out overnight at 4 • C with antibodies after treatment with 5% skim milk. Afterward, the membranes were washed, probed with HRPconjugated secondary antibodies for 2 h, and then visualized using chemiluminescence.

In Silico DFT Calculations
The theoretical calculations were performed using Gaussian 09 [48] and Gaussview 5 program. The polarized and diffused basis set 6-311+G(d, p) provides accurate values for all theoretical calculations. The computational studies utilized the most useful and precise hybrid method of B3LYP [49]. The structures of the compounds were fully optimized with no constraint. The global chemical reactivity descriptors (GCRD) were evaluated to understand the chemical properties of a molecule, such as ionization potential, electron affinity, chemical hardness (η), softness (S), potential (µ), electronegativity (χ), and electrophilic index (ψ). The global hardness [η = (E LUMO − E HOMO )/2], softness (S = 1/2η), chemical potential [µ = (E HOMO + E LUMO )/2], electronegativity [χ = (I+A)/2], and electrophilic index (ψ = µ 2 /2η) were calculated by taking the energies of HOMO as ionization potential (I) and LUMO as electron affinity (A). Chemical hardness, softness, and potential were used to understand the chemical reactivity of the molecular system [50].

Docking Simulation
AUTODOCK4.0 [51] software was employed for molecular docking studies. The docking receptor STAT3 (PDB ID: 1BG1) was retrieved from the RCSB Protein Data Bank. The graphical user interface AUTODOCK TOOLS was utilized to build up the protein molecule. The water molecules were removed from the protein crystal and only polar hydrogens were applied. The predicted gasteigers charge was found to be −25.9962. For both dockings, the grid box size was 127 × 127 × 85 with a grid spacing of 0.55Å. The receptor and the complex were saved in the pdbqt file format. Using Lamarckian genetic algorithm searches, twenty runs were performed. The default parameters were employed, with a maximum of 2.5 × 10 6 energy assessments and an initial population of 50 randomly placed individuals [52]. The autogrid4.exe and autodock4.exe functions were executed at the end of the docking process to generate glg and dlg files. Maestro (v2020.4) [53] and PyMOL (v2.5.2) [54] were used to generate the interaction pictures and visualization plots.

Immunocytochemistry Assay
As described earlier, STAT3 phosphorylation in cells was quantified [55]. After compound 3f treatment (10 µM for 4 h), cells were fixed for 20 min with paraformaldehyde (4%). Thereafter, cells were treated with 0.2% Triton X-100 in phosphate-buffered saline for permeabilization, followed by blocking with 5% bovine serum albumin for 1 h. Then, the preparation was incubated overnight at 4 • C with a rabbit polyclonal anti-human STAT3 antibody (dilution, 1:100). The next day, slides were subjected to washing and incubation with Alexa Fluor 594 (dilution, 1:1000) anti-Rabbit IgG1 for 1 h at room temperature in the dark. In the next step, DAPI (5 µg/mL) was used for counterstaining the nuclei. The slides were mounted and analyzed under an Olympus FluoView FV1000 confocal microscope (Tokyo, Japan).

Conclusions
A series of imidazopyridine-tethered-purazoles were synthesized and screened for loss of viability of breast cancer cells. The lead compound 3f inhibited STAT3 phosphorylation in MCF-7 and T47D cells. The DFT calculations and molecular docking experiments showed a theoretical bioactivity correlation for compound 3f towards STAT3. In conclusion, compound 3f effectively inhibited the phosphorylation of STAT3 in MCF-7 and T47D cells, indicating that ITPs may be an alternative method to target STAT3 in BC.