Development of Piperazine- and Oxazine-Linked Pyrimidines as p65 Subunit Binders of NF–κB in Human Breast Cancer Cells

Nuclear factor kappa B (NF–κB) is a potential therapeutic target in breast cancer. In the current study, a new class of oxazine– and piperazine–linked pyrimidines was developed as inhibitors of NF–κB, overcoming the complexity of the oxazine structure found in nature and enabling synthesis under laboratory conditions. Among the series of synthesized and tested oxazine–pyrimidine and piperazine–pyrimidine derivatives, compounds 3a and 5b inhibited breast cancer cell (MCF–7) viability with an IC50 value of 9.17 and 6.29 µM, respectively. In silico docking studies showed that the pyrimidine ring of 3a and the 4–methoxybenzyl thiol group of 5b could strongly bind the p65 subunit of NF–κB, with the binding energies −9.32 and −7.32 kcal mol−1. Furthermore, compounds 3a and 5b inhibited NF–κB in MCF–7 breast cancer cells. In conclusion, we herein report newer structures that target NF–κB in BC cells.


Introduction
Breast cancer has become the world's second-leading cause of cancer-related death (lung cancer being first), accounting for about 13.7% of all cancer-related fatalities [1,2].Nuclear factor kappa B (NF-κB) signaling has been extensively studied for over three decades since its discovery by Sen et al. [3].Recent evidence confirms that activation of NF-κB promotes human breast cancer progression.For that reason, NF-κB has emerged as a potential therapeutic target in breast cancer treatment [4,5].The NF-κB family comprises five transcription factors: NF-κB1/p50, NF-κB2/p52, RelA/p65, RelB, and c-Rel [6].These factors can either hetero-or homodimerize to produce NF-κB complexes.The p65 subunit of NF-κB is a critical component in activating and regulating downstream target genes.Most of them are found in the cytoplasm of dormant cells; when they are activated, they move to the nucleus for transcription, which in turn causes hundreds of genes to be activated or repressed directly, indirectly, or both [7].
NF-κB is activated by viral and bacterial antigens, UV radiation, and cytokines such as IL-2 and TNF-α.The nuclear factor supports cell proliferation, apoptosis, and immunological responses to infection and inflammation.However, system disruption is associated with disorders including cancer, immunosuppression, and chronic inflammation [8].NF-κB activation promotes cell survival by inhibiting apoptosis (programmed cell death).It controls the expression of anti-apoptotic proteins that aid cancer cells to avoid cell death and promote their survival, including Bcl-2, Bcl-xL, and inhibitors of apoptosis (IAPs) [9].Additionally, NF-κB signaling promotes cell proliferation by increasing the expression of genes essential for cell cycle progression, including cyclins and cyclin-dependent kinases (CDKs) [10,11].
It has been demonstrated that several drugs, including aspirin, sodium salicylate, and dexamethasone, decrease NF-κB activation by preventing the breakdown of IκB [12][13][14].The current anti-TNF-α antibodies approved by the FDA include infliximab, adalimumab, and golimumab [15].Ongoing research is being conducted to produce innovative small molecules that target the NF-κB pathway.New compounds and therapeutic strategies are continually explored and may emerge as potential candidates for further investigation in breast cancer treatment [16].Piperazines derivatives, among other heterocycles, were discovered as promising anti-cancer agents [17][18][19][20], and many of the FDA-approved drugs include piperazines [21].Novel piperazine compounds could suppress NF-κB translocation to the nucleus [22] and inhibit NF-κB by decreasing TNF-α levels [23].Pyrimidines also play a vital role in anti-cancer drug discovery [24].Ibudilast, spebrutinib, and dasatinib are a few pyrimidine-based drugs (Figure 1) that block the NF-κB pathway [25].Furthermore, many reports have shown that oxazine derivatives might emerge as promising anti-cancer agents [26,27], and that they are potential candidates for NF-κB inhibitors [28][29][30].The oxazine derivative compound 1 decreased the DNA binding ability of NF-κB and NF-κBdependent luciferase expression and IκBα phosphorylation in hepatocellular carcinoma (HCC) and HCT116 cells.Furthermore, treatment of inflammatory bowel disease (IBD)induced mice with compound 1 decreased myeloperoxidase activity in colonic extracts and modulated the colon length and serum levels of cytokines such as TNF-α, IFN-γ, IL-6, IL-1β, and IL-10 [31,32].Similarly, compound 2 inhibited proliferation in HepG2, HCCLM3, and Huh-7 cells in a dose-and time-dependent manner, as well as decreased p65 subunit DNA binding capacity, p65 phosphorylation, and the consequent production of NF-κB-dependent luciferase gene expression in several HCC cell lines [33].From the abovementioned discoveries, Lys28 was observed to be the active site of the p65 subunit in NF-κB.Benzimidazole-clubbed pyrimidines (3) were demonstrated as covalent inhibitors of cysteine in NF-κB inducing kinase [34].Pyrido-pyrimidine (5) inhibited NF-κB activation by suppressing IκBα and LPS-induced phosphorylation levels of p65 and Akt, and by indirectly suppressing the MAPK signaling pathway [35], and pyralopyridine (5)-substituted pyrimidines were discovered as NF-κB transcription inhibitors [36].
Herein, we have synthesized novel oxazine-and piperazine-linked pyrimidine small molecules using thiouracils active in breast cancer cells (MCF-7) that target NF-κB.Alamar Blue assay showed that newly synthesized compounds 3a and 5b produced an IC 50 of 9.17 and 6.29 µM in MCF-7 cells.In silico docking studies showed that compounds 3a and 5b exhibited −9.32 and −7.32 kcal mol −1 binding energy.Lys28 of the p65 subunit of NF-κB and the pyrimidine ring of 3a and 4-methoxy benzyl thiol group of 5b showed strong pi-alkyl interactions.

Materials and Methods
All chemicals and solvents were purchased from Sigma-Aldrich (Bangalore, In The completion of the reaction was monitored by pre-coated silica gel TLC plates.An ilent mass spectrophotometer was used to record the mass of the synthesized compou 1 H and 13 C NMR (Santa Clara, CA, USA) were recorded on Agilent and Jeol NMR spe photometers (400 MHz).TMS was used as an internal standard, and DMSO was use a solvent.Chemical shifts were expressed as ppm.

Materials and Methods
All chemicals and solvents were purchased from Sigma-Aldrich (Bangalore, India).The completion of the reaction was monitored by pre-coated silica gel TLC plates.An Agilent mass spectrophotometer was used to record the mass of the synthesized compounds. 1H and 13 C NMR (Santa Clara, CA, USA) were recorded on Agilent and Jeol NMR spectrophotometers (400 MHz).TMS was used as an internal standard, and DMSO was used as a solvent.Chemical shifts were expressed as ppm.Substituted thiouracils (1) (1.0 mmol) and various benzyl chlorides (1.2 mmol) were refluxed with KOH (1.2 mmol) in EtOH: H 2 O (1:1) as a solvent for 1 h.After the completion of the reaction, the solid mass was filtered off and washed with aqueous NaHCO 3 solution and water, yielding compound 2.

Synthesis of Compound 3
Compound 2 (1.0 mmol), substituted oxazines (1.0 mmol), and K 2 CO 3 (2 mmol) were refluxed in acetone for 2-3 h.After the completion of the reaction, the crude reaction mass was extracted to ethyl acetate (25 mL × 3).The combined organic layer was distilled under reduced pressure and purified by column chromatography using ethyl acetate and hexane.

Synthesis of Compounds 4/5
Compound 2 (1.0 mmol) was treated with tert-butyl bromoacetate (1.2 mmol) and K 2 CO 3 (1.5 mmol) in refluxing DMF.After the completion of the reaction, reaction mass was extracted with ethyl acetate and the crude product was purified by column chromatography, yielding compound 4. The solution of 4 in trifluoroacetic acid was stirred at room temperature for 1-2 h.After completion of the reaction, it was quenched with sodium bicarbonate and extracted with ethyl acetate.The solid formed was filtered off and dried, yielding compound 5.

Synthesis of Compound 5b/f/k/o
Compound 4 (1.0 mmol) was treated with acetyl piperazines (1.2 mmol) with EDC.HCl and DMAP as catalysts in basic conditions in DCM as solvent under nitrogen atmosphere for 2 h.After completion of the reaction, the crude mass was extracted with ethyl acetate, and the combined organic layer was distilled off and purified through column chromatography.

Synthesis of Compounds 5d/h/m/q
Compound 4 (1.0 mmol) was treated with N-boc piperazines (1.2 mmol) with EDC.HCl and DMAP were used as catalysts in basic conditions in DCM as solvent under nitrogen atmosphere for 2 h.After completion of the reaction, the crude mass was extracted with ethyl acetate, and the combined organic layer was distilled off and purified through column chromatography, yields 5d, 5h, 5m, or 5q.

Synthesis of Compounds 5c/g/l/p
Compounds 5d, 5h, 5m, or 5q (1.0 mmol) were treated with trifluoroacetic acid.After completion of the reaction, the crude mass was neutralized with K 2 CO 3 and extracted with ethyl acetate.The combined organic layer was distilled off and purified through column chromatography, yielding compounds 5c, 5g, 5l, or 5p, respectively.

Synthesis of Compounds 5e/i/j/n
Compounds 5c, 5g, 5l, or 5p (1 mmol) were treated with 5-bromopyridine-2-carboxylic acid (1.2 mmol) with EDC.HCl and DMAP were used as catalysts in basic conditions in DCM as solvent under nitrogen atmosphere for 2 h.After completion of the reaction, the crude mass was extracted with ethyl acetate, and the combined organic layer was distilled off and purified through column chromatography, yielding compounds 5e, 5i, 5j, or 5n, respectively.

Cell Viability Assay
MCF-7, MDAMB-231, BT549, and SUM159PT cells were purchased from Procell Life Science & Technology Co., Ltd.(Wuhan, China).All carcinoma cell lines were cultured according to ATCC propagation instructions.By following the procedure in Basappa et al. [37], first, 2 × 10 3 MCF-7 cells in 200 µL were grown in MEM enriched with 2% FBS and kept at 37 • C in a humidified 5% CO 2 environment.The compounds (10 mM) were dissolved in DMSO and were stored as a stock solution.The DMSO and the stock solution of compounds were diluted to 0.01, 0.1, 10, 100, and 1000 µM solutions in cell culture medium, keeping a DMSO amount less than 1%.MCF-7 cells (2 × 10 3 ) were incubated for 72 h with exposure to pyrimidines and Alamar Blue reagent was used to evaluate cell viability.

Annexin V Apoptosis and Cell Cycle Analysis Assay
MCF-7 cells were cultured at a density of approximately 1 × 10 5 cells per well on a six cm tissue culture petri dish, and treated with compounds 3a or 5b for 72 h.Following the procedure, attached and floating cells were gathered and rinsed twice with ice-cold phosphate buffer solution.The degree of apoptosis was determined using the Annexin V-AbFluorTM 488/PI Apoptosis Detection Kit (Abbkine, KTA0002, Wuhan, China) following the manufacturer's instructions.A quantity of 1 × 10 5 cells were collected, washed once in ice-cold PBS, and permeabilized with 100 L of 0.5% Triton X-100 to evaluate cell cycle distribution.A quantity of 1 × 10 5 cells werefixed with 75% ethanol at −20 • C overnight and stained with 50 µg/mL PI in 200 µL PBS supplemented with 20 µg/mL (w/v) RNase A (Abbkine, KTA2020, Wuhan, China) for 1 h at 4 • C. Cytofluorometric acquisitions were performed on a BECKMAN COULER CytoFlex at a low flow rate mode.

Western Blot Analysis
MCF-7 cells were treated with compounds 3a or 5b and harvested, and the cell lysates were obtained.SDS-PAGE was used to separate the proteins of interest, and were transferred onto PVDF (Millipore, ISEQ00010, Burlington, MA, USA) membrane.The membrane was sequentially incubated with primary and secondary antibodies and the corresponding proteins were visualized using an ECL kit Clarity™ and Clarity Max™ Western ECL Blotting Substrates (BIO-RAD, Hercules, CA, USA).

Data Analysis and Statistics
The results are presented as mean ± standard deviation.A one-way analysis of variance (one-way ANOVA), with Bonferroni's multiple comparison tests, was used to analyze the statistical change between treatment groups.A 0.05 confidence level was the significant change cutoff.

Molecular Docking
The docking studies were determined by using The Scripps Research Institute's AutoDock4 tools (v.1.5.6) [38].Initially, 3a or 5b structures were obtained from the molecular drawing software tools, and the compounds were converted to the PDBQT format.Later protein preparation was performed by BIOVIA Discovery studio.Before this, the crystal structure of NF-κB (PDB ID: 1IKN) was retrieved from the Protein Data Bank.The protein structure was prepared by removing water molecules and adding hydrogen atoms.Kollman charges were assigned to the protein.Later ligand preparations were performed for both compounds 3a and 5b and further used for docking purposes.Docking simulations were performed using AutoDock4.The Lamarckian Genetic Algorithm was employed for both ligands.The grid box was defined around the active site of the NF-kappa B p65 subunit, and the grid dimensions were 40 Å × 40 Å × 28 Å with a spacing of 0.375 Å.The docking parameters were set to default values, and 10 docking runs were performed for each compound.Later, the resulting docking poses were visualized using BIOVIA Discovery Studio [39], PyMOL [40], and UCSF Chimera1.16[41].

Efficacy of Pyrimidine Derivatives in Breast Cancer Cells
The newly synthesized pyrimidines were examined for inhibition of cell viability of human breast cancer (MCF-7) cells (Tables 1 and 2).Tamoxifen and doxorubicin were used as internal standards and produced a loss of viability of MCF-7 cells, with IC 50 values of 2.96 and 1.84 µM, respectively.Among the oxazine-clubbed pyrimidine compounds, 3a and 3g produced an IC 50 of 9.17 and 13.87 µM, respectively.Among piperazines, clubbed pyrimidine compounds 5a and 5m exhibited IC 50 of 6.29 and 14.58 µM.All other derivative IC 50 values were observed from 17.26 to >100 µM (Figure 3A) (see supplementary file).Among compounds 3(a-g), 4-methoxyphenyl-substituted oxazines (I) were observed to be active compared to 4-chlorophenyl and phenyl-substituted oxazines, whereas in compounds 5(a-p), 4-methoxybenzyl-substituted pyrimidine were more potent than other benzylated derivatives.Also, 5-bromopyridine-substituted piperazines were found to be active, whereas the other piperazines were inactive.Lead molecules 3a and 5b were evaluated against MDA-MB-231, BT-549, and SUM159PT cells (Figure 3B) (Table 3).Among the two oxazine-pyrimidine derivatives, 5b was more potent, with IC 50 of 7.34, 5.98, and 14.81 µM.

Efficacy of Pyrimidine Derivatives in Breast Cancer Cells
The newly synthesized pyrimidines were examined for inhibition of cell viability of human breast cancer (MCF-7) cells (Tables 1 and 2).Tamoxifen and doxorubicin were used as internal standards and produced a loss of viability of MCF-7 cells, with IC50 values of 2.96 and 1.84 µM, respectively.Among the oxazine-clubbed pyrimidine compounds, 3a and 3g produced an IC50 of 9.17 and 13.87 µM, respectively.Among piperazines, clubbed pyrimidine compounds 5a and 5m exhibited IC50 of 6.29 and 14.58 µM.All other derivative IC50 values were observed from 17.26 to >100 µM (Figure 3A)(see supplementary file).We previously described the discovery of 1,2 oxazines as anti-cancer drugs [32], along with their roles in triggering apoptosis, significantly increasing the population of sub-G1 cells and inhibiting the capacity of NF-κB to bind DNA in HCC cells.We therefore used MCF-7 cells to determine the effect of the lead compounds in Figure 4 on apoptosis.Examination of the data showed that the lead compounds stimulated dose-dependent apoptosis of MCF-7 cells (Figure 4).We previously described the discovery of 1,2 oxazines as anti-cancer drugs [32], along with their roles in triggering apoptosis, significantly increasing the population of sub-G1 cells and inhibiting the capacity of NF-κB to bind DNA in HCC cells.We therefore used MCF-7 cells to determine the effect of the lead compounds in Figure 4 on apoptosis.Examination of the data showed that the lead compounds stimulated dose-dependent apoptosis of MCF-7 cells (Figure 4).

Lead Compounds Arrest MCF-7 Cell Cycle at the Sub-G1 Phase
We next investigated whether the lead compounds can hinder specific cell cycle progression.Propidium iodide labeling was used for the flow cytometric study of untreated and treated (lead compounds) MCF-7 cells.Lead compounds increased the proportion of cells in the sub-G1 phase relative to untreated cells [42] (Figure 5).

Lead Compounds Arrest MCF-7 Cell Cycle at the Sub-G1 Phase
We next investigated whether the lead compounds can hinder specific cell cycle progression.Propidium iodide labeling was used for the flow cytometric study of untreated and treated (lead compounds) MCF-7 cells.Lead compounds increased the proportion of cells in the sub-G1 phase relative to untreated cells [42] (Figure 5).

Lead Compounds Inhibited the Phosphorylation of Human p65 Protein (Serine-536 Amino Acid) of NF-κB Subunit in MCF-7 Cells
NF-κB activation is regulated by the enzyme inhibitor of κB (IκB) and kinase (IKK), which phosphorylates subunit p65 at serine 536, and which inhibits the NF-κB signaling pathway.Western blot analysis was used to examine if the lead compounds impacted the expression of the p65 protein or the levels of phospho-p65 in MCF-7 cells.Lead compounds 5b and 3a, as shown in Figure 6A,B, produce a concentration-dependent decrease of phospho-p65 levels relative to p65 protein expression in MCF-7 cells 24 h after treatment.In this study, we performed in silico analysis to evaluate the binding energies and critical interactions of two novel compounds, 3a or 5b, targeting p65, the active site of NF-κB.Initially, the NF-κB structure was retrieved from the Protein Data Bank (PDB ID: 1IKN) and further used for molecular docking simulations using AutoDock4 tools.Molecular docking simulation revealed that novel compound 3a demonstrated a binding energy of −9.32 kcal/mol, indicating a strong binding affinity for the active site of the NF-κB p65 subunit, while 5b exhibited a binding energy of −7.32 kcal/mol, indicating a relatively weaker binding affinity.Further key interactions revealed that compound 3a formed a hydrogen bond with the residue GLN-29.The hydrogen bond plays a crucial role in stabilizing the binding of 3a to the active site.One π-anion bond and one π-lone pair bond formed with the residues GLU-225 and GLU-222, respectively.Additionally, hydrophobic interactions (π-alkyl) were observed between 3a and specific residues like LYS-28, ARG-30, ARG-50, and HIS-181 in the binding pocket, contributing to its overall binding affinity.In comparison, 5b engaged in hydrogen bonding interactions with residues GLN-29 and ILE-224 within the active site.These hydrogen bonds contribute to the binding stability of 5b.One π-sigma bond formed with the residue ARG-50.Furthermore, hydrophobic interactions were observed (LYS-28 and PRO-275), further enhancing the binding of 5b to the target protein (Figure 7B).The results of the docking study revealed that both 3a and 5b have potential as inhibitors of the NF-kappa B p65 subunit.

Discussion
Pyrimidines have been demonstrated to be effective inhibitors of NF-κB, and many of the pyrimidine-based drugs such as ibudilast, spebrutinib, and dasatinib are also reported as inhibitors of the NF-κB pathway.In the present work, we have designed and synthesized a new series of oxazine-and piperazine-clubbed pyrimidine derivatives as novel inhibitors of NF-κB.Loss of cell viability in MCF-7 cells revealed 3a and 5b to be the most potent among the series.Further efficacy of the lead compounds was studied by apoptosis and cell cycle assays, and Western blot analysis.The lead compounds increased the proportion of cells in the sub-G1 phase relative to untreated cells and induced apoptosis in MCF-7 cells.Lead molecules 5b and 3a produced a concentration-dependent decrease of phospho-p65 levels in MCF-7 cells.Additionally, an in silico docking study of lead compounds also supported the above data by prediction of strong binding to the p65 subunit of NF-κB.

Conclusions
In this study, novel compounds consisting of oxazines and piperazines linked to pyrimidines were synthesized and evaluated in MCF-7 breast cancer cells.Compounds 3a and 5b exhibited IC 50 s of 9.17 and 6.29 µM, respectively.Through in silico investigation, it was determined that compounds 3a and 5b potentially bind to the active site of NF-κB.Subsequent biological assays confirmed that lead compounds 3a and 5b effectively inhibited NF-κBp65 phosphorylation in MCF-7 cells, presenting a promising chemical entity targeting NF-κB in breast cancer cells.

Figure 4 .
Figure 4. Evaluation of apoptosis in MCF-7 cells induced by lead compounds (5b, A and 3a, B) at 5 and 10 µM for 24 h.Control MCF-7 cells or MCF-7 cells treated with lead compounds (5b, A and 3a, B) were stained with Annexin V-AbFluor™ 488/PI Apoptosis Detection (Abbkine, KTA0002, Wuhan, China) followed by flow cytometry analysis.The percentage of apoptotic cells is indicated.Significant changes from the control group are shown by (* p < 0.05, *** p < 0.01) of three independent assays.

Figure 4 .
Figure 4. Evaluation of apoptosis in MCF-7 cells induced by lead compounds (5b, A and 3a, B) at 5 and 10 µM for 24 h.Control MCF-7 cells or MCF-7 cells treated with lead compounds (5b, A and 3a, B) were stained with Annexin V-AbFluor™ 488/PI Apoptosis Detection (Abbkine, KTA0002, Wuhan, China) followed by flow cytometry analysis.The percentage of apoptotic cells is indicated.Significant changes from the control group are shown by (* p < 0.05, *** p < 0.01) of three independent assays.

2023, 11 , 2716 15 of 20 Figure 5 .
Figure 5. Flow cytometry analyzed MCF-7 cell cycle proportions.Representative cell cycle distribution histograms showing apoptosis in MCF-7 cells treated for 72 h with lead compounds such as A (5b) and B (3a) at concentrations of 5 and 10 mM, and analysis of the number of cells at each cell cycle stage with all phases.

Figure 5 .
Figure 5. Flow cytometry analyzed MCF-7 cell cycle proportions.Representative cell cycle distribution histograms showing apoptosis in MCF-7 cells treated for 72 h with lead compounds such as A (5b) and B (3a) at concentrations of 5 and 10 mM, and analysis of the number of cells at each cell cycle stage with all phases.

3. 5 .
Lead Compounds Inhibited the Phosphorylation of Human p65 Protein (Serine-536 Amino Acid) of NF-κB Subunit in MCF-7 Cells NF-κB activation is regulated by the enzyme inhibitor of κB (IκB) and kinase (IKK), which phosphorylates subunit p65 at serine 536, and which inhibits the NF-κB signaling pathway.Western blot analysis was used to examine if the lead compounds impacted the expression of the p65 protein or the levels of phospho-p65 in MCF-7 cells.Lead compounds 5b and 3a, as shown in Figure6A,B, produce a concentration-dependent decrease of phospho-p65 levels relative to p65 protein expression in MCF-7 cells 24 h after treatment.

Figure 5 .
Figure 5. Flow cytometry analyzed MCF-7 cell cycle proportions.Representative cell cycle distribution histograms showing apoptosis in MCF-7 cells treated for 72 h with lead compounds such as A (5b) and B (3a) at concentrations of 5 and 10 mM, and analysis of the number of cells at each cell cycle stage with all phases.

3. 6 .
In Silico Analysis of Novel Compounds 3a and 5b Targeting the NF-Kappa B p65 Subunit

Figure 7 .
Figure 7. (A) Cartoon representation of both docked compounds 3a (Black) and 5b (Yellow) inside the binding pocket of the p65 subunit of NF-κB; (B) 2D interactions of compound 3a and 5b with the walls of the binding pocket, respectively andshowing bond distance (Å) between respective aminoacid and ligands.Pyrimidine ring of 3a and 4-methoxybenzylthiol group of 5b showed pi-alkyl interaction with Lys28 of p65 subunit of NF-κB; (C) Representation of three-dimensional surface

Figure 7 .
Figure 7. (A) Cartoon representation of both docked compounds 3a (Black) and 5b (Yellow) inside the binding pocket of the p65 subunit of NF-κB; (B) 2D interactions of compound 3a and 5b with the walls of the binding pocket, respectively andshowing bond distance (Å) between respective aminoacid and ligands.Pyrimidine ring of 3a and 4-methoxybenzylthiol group of 5b showed pialkyl interaction with Lys28 of p65 subunit of NF-κB; (C) Representation of three-dimensional surface view of docked compounds (3a: Black, 5b: Yellow) inside the grove of NF-κB and its enlarged view for better understandings.
ND = Not Determined.