Electrochemical Synthesis of New Isoxazoles and Triazoles Tethered with Thiouracil Base as Inhibitors of Histone Deacetylases in Human Breast Cancer Cells

Histone deacetylases (HDACs) are an attractive drug target for the treatment of human breast cancer (BC), and therefore, HDAC inhibitors (HDACis) are being used in preclinical and clinical studies. The need to understand the scope of the mode of action of HDACis, as well as the report of the co-crystal structure of HDAC6/SS-208 at the catalytic site, provoked us to develop an isoxazole-based lead structure called 4-(2-(((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio) pyrimidin-4-yl) morpholine (5h) and 1-(2-(((3-(p-tolyl) isoxazol-5-yl)methyl)thio) pyrimidin-4-yl) piperidin-4-one (6l) that targets HDACs in human BC cells. We found that the compound 5h or 6l could inhibit the proliferation of BC cells with an IC50 value of 8.754 and 11.71 µM, respectively. Our detailed in silico analysis showed that 5h or 6l compounds could target HDAC in MCF-7 cells. In conclusion, we identified a new structure bearing triazole, isoxazole, and thiouracil moiety, which could target HDAC in MCF-7 cells and serve as a base to make new drugs against cancer.


Introduction
Histone deacetylases (HDACs) remove acetyl groups from lysine residues on acetylated histones, leading to changes in chromatin structure and gene expression. In breast cancer (BC), HDACs were found to be overexpressed and regulate the expression of a tumor-suppressor gene called BRCA1, which was involved in DNA repair, cell-cycle control, and cancer-cell progression [1][2][3][4]. In addition, HDACs play an essential role in oncogenic pathways, such as the estrogen-receptor (ER) signaling pathway, which leads to ER-positive BC [5]. Structurally, the catalytic site of HDACs contains four regions of highly conserved amino acids, where the first region is referred to as a zinc-binding domain, which is coordinated by two histidines and one aspartic acid residue [6][7][8]. The second region was known as the catalytic triad (His-Asp-Tyr) domain, which consists of three amino acid Figure 1. Evolution of HDACis based on a hybrid structure bearing triazole or isoxazole and thiouracil-based core heterocycles; Structures 1 and 2 represents 1,2,3-triazoles (pink), 3 and 4 represents thiouracil (red) analogues also 5 and 6 represents isoxazole (blue) derivatives as HDACis inhibitors. The HDAC (cyan-HDAC7) active site, which contains zinc (Zn 2+ , green), was the target of these compounds. Within the active site is a zinc-binding site and two Asp residues (Asp801-Asp707). The walls of the pocket are lined with hydrophobic residues (Pro667, Phe679, Phe737, Phe738, Pro809 and Leu810), while other residues present include His709, His670, ARG547, Gly842 and others. Doted lines (yellow) represents hydrogen bondings.  The HDAC (cyan-HDAC7) active site, which contains zinc (Zn 2+ , green), was the target of these compounds. Within the active site is a zinc-binding site and two Asp residues (Asp801-Asp707). The walls of the pocket are lined with hydrophobic residues (Pro667, Phe679, Phe737, Phe738, Pro809 and Leu810), while other residues present include His709, His670, ARG547, Gly842 and others. Doted lines (yellow) represents hydrogen bondings. The HDAC (cyan-HDAC7) active site, which contains zinc (Zn 2+ , green), was the target of these compounds. Within the active site is a zinc-binding site and two Asp residues (Asp801-Asp707). The walls of the pocket are lined with hydrophobic residues (Pro667, Phe679, Phe737, Phe738, Pro809 and Leu810), while other residues present include His709, His670, ARG547, Gly842 and others. Doted lines (yellow) represents hydrogen bondings.

Synthesis of Thiouracil Tethered Triazole/Isoxazole Analogues
The present work synthesized novel thiouracil-tethered triazole/isoxazole derivatives and studied their anticancer activity. Triazole analogues (5a-l) were obtained by adding CuI to a stirred solution of substituted azides and intermediates (4a-d) in THF under basic conditions. Additionally, the synthesis of 1,2,3-triazoles was performed via electrochemical oxidation of copper foil cells as the working electrode and platinum as the counter electrode in tertiary butanol:water medium (1:1). The reaction gave a good yield in the presence of an analytical amount of tetrabutylammonium tetrafluoroborate (TBATFB) within 60 min. This method was highly useful for synthesizing 1,2,3-triazoles tethered with a bioactive scaffold (Scheme 1).
adding CuI to a stirred solution of substituted azides and intermediates (4a-d) in THF under basic conditions. Additionally, the synthesis of 1,2,3-triazoles was performed via electrochemical oxidation of copper foil cells as the working electrode and platinum as the counter electrode in tertiary butanol:water medium (1:1). The reaction gave a good yield in the presence of an analytical amount of tetrabutylammonium tetrafluoroborate (TBATFB) within 60 min. This method was highly useful for synthesizing 1,2,3-triazoles tethered with a bioactive scaffold (Scheme 1).
Isoxazole analogues (6a-l) were obtained via a cyclization reaction between N-hydroxy-(substituted) benzimidoyl chloride and intermediates (4a-d) in DMF under basic conditions. Moreover, the synthesis of isoxazole was conducted using the electrolysis method. Under constant current supplying with a density of 5.0 mA/cm −2 into a beakertype undivided cell, N-hydroxy-(substituted) benzimidoyl chlorides and alkynes (4a-d) were reacted by using Pt-plate as the working electrode and an iron rod as the cathode in methanolic medium, and the desired isoxazoles were obtained in good yield when compared to the abovementioned conventional method (Scheme 1).
Furthermore, the N-boc deprotection of compounds (5d-f/6d-f) was accomplished upon treatment with trifluoroacetic acid in DCM to yield (7a-f) (Scheme 2). The ketone group reduction in compounds (5j-l/6j-l) was made on treatment with sodium borohydride in THF to yield (8a-f) (Scheme 3). Isoxazole analogues (6a-l) were obtained via a cyclization reaction between N-hydroxy-(substituted) benzimidoyl chloride and intermediates (4a-d) in DMF under basic conditions. Moreover, the synthesis of isoxazole was conducted using the electrolysis method. Under constant current supplying with a density of 5.0 mA/cm −2 into a beaker-type undivided cell, N-hydroxy-(substituted) benzimidoyl chlorides and alkynes (4a-d) were reacted by using Pt-plate as the working electrode and an iron rod as the cathode in methanolic medium, and the desired isoxazoles were obtained in good yield when compared to the abovementioned conventional method (Scheme 1).
Furthermore, the N-boc deprotection of compounds (5d-f/6d-f) was accomplished upon treatment with trifluoroacetic acid in DCM to yield (7a-f) (Scheme 2). The ketone group reduction in compounds (5j-l/6j-l) was made on treatment with sodium borohydride in THF to yield (8a-f) (Scheme 3).

Cytotoxicity of Thiouracil Tethered Triazole/Isoxazole Analogues on MCF-7 Cells
Initially, the novel thiouracil tethered triazole/isoxazole analogues were subjected to cell viability studies against human breast cancer (BC) (MCF-7) cells (Table 1), using the Alamar Blue assay [32][33][34]. For over four decades, numerous research groups have extensively utilized MCF-7, a BC cell line exhibiting a well-characterized molecular profile and
The isoxazole derivative (6l) exhibited a binding energy of −6.60 kcal/mol, indicating a strong affinity for the HDAC7 binding site. Similarly, the triazole derivative (5h) demonstrated a slightly higher binding energy of −6.42 kcal/mol, suggesting an even more robust interaction with the target protein. Both compounds displayed favourable binding energies, indicating their potential as HDAC7 inhibitors, and cartoon representations of docked compounds are depicted in Figure 4A,B. In comparison, the co-crystal ligand (TMP269) exhibited a binding energy of −6.19 kcal/mol, which falls within the range of the isoxazole and triazole compounds. This suggests that the co-crystal ligand also possesses a significant binding affinity for HDAC7. We initially retrieved HDAC7 co-crystal structure from RCBS PDB (PDB ID: 3ZNR) and used it for docking purposes [37]. The Scripps Research Institute's Auto-Dock4 tools (ADT)v1.5.6 was used for docking. The protein structure HDAC7, compounds 6l and 5h, and co-crystal ligand (TMP269) were prepared using discovery studio BIOVIA Discovery Studio 2021 client and Avogadro, and later it was docked using AutoDock tools (ADT).
The isoxazole derivative (6l) exhibited a binding energy of −6.60 kcal/mol, indicating a strong affinity for the HDAC7 binding site. Similarly, the triazole derivative (5h) demonstrated a slightly higher binding energy of −6.42 kcal/mol, suggesting an even more robust interaction with the target protein. Both compounds displayed favourable binding energies, indicating their potential as HDAC7 inhibitors, and cartoon representations of docked compounds are depicted in Figure 4A,B. In comparison, the co-crystal ligand (TMP269) exhibited a binding energy of −6.19 kcal/mol, which falls within the range of the isoxazole and triazole compounds. This suggests that the co-crystal ligand also possesses a significant binding affinity for HDAC7. An analysis of the binding pocket revealed critical interactions for the isoxazole, triazole, and co-crystal ligands. The isoxazole compound (6l) formed hydrogen bonds with residue PHE-738 with a bond distance of 1.83 Å, stabilizing its binding within the pocket ( Figure 5A). Moreover, 6l had π-sigma bond formation with the residue LEU-810 with a bond distance of 3.65 Å, and π-π T-shaped bonds formed with residue PHE-738 with a bond distance of 5.63 Å, and alkyl bond formed with residues LEU-810 and PRO-542, respectively. The triazole compound (5h) displayed hydrogen bond interactions with residue HIS-709 having a bond distance of 2.69 Å, and π-alkyl bonds were formed with residues PRO-242, HIS-670, PHE-679, PHE-737, PRO-809, and LEU-810, contributing to its strong binding ( Figure 5B). The co-crystal ligand (TMP269) demonstrated hydrogen bond interactions with residues PHE-738 with a bond distance of 2.36 Å, and a π-alkyl bond formed with residues HIS-709 and PHE-738 with bond distances of 5.43 Å and 4.73 Å, respectively. Furthermore, TMP269 had a π-sigma bond and salt bridge interactions with residues LEU-810 and ASP-626, respectively ( Figure 5C). These interactions play a crucial role in maintaining the stability and specificity of the ligand-protein complex.
The results of the molecular docking study highlight the good binding affinity of both the isoxazole (6l) and triazole (5h) compounds towards the HDAC7 binding site, with binding energies of −6.60 and −6.42 kcal/mol, respectively. These findings suggest that both compounds have the potential to be effective inhibitors of HDAC7. Furthermore, the co-crystal ligand (TMP269) exhibited a favourable binding energy of −6.19 kcal/mol, indicating its potency as an HDAC7 inhibitor. The critical interactions observed within the binding pocket emphasize the role of specific residues in facilitating ligand-protein interactions for all three compounds. An analysis of the binding pocket revealed critical interactions for the isoxazole, triazole, and co-crystal ligands. The isoxazole compound (6l) formed hydrogen bonds with residue PHE-738 with a bond distance of 1.83 Å, stabilizing its binding within the pocket ( Figure 5A). Moreover, 6l had πsigma bond formation with the residue LEU-810 with a bond distance of 3.65 Å, and π-π T-shaped bonds formed with residue PHE-738 with a bond distance of 5.63 Å, and alkyl bond formed with residues LEU-810 and PRO-542, respectively. The triazole compound (5h) displayed hydrogen bond interactions with residue HIS-709 having a bond distance of 2.69 Å, and π-alkyl bonds were formed with residues PRO-242, HIS-670, PHE-679, PHE-737, PRO-809, and LEU-810, contributing to its strong binding ( Figure 5B). The co-crystal ligand (TMP269) demonstrated hydrogen bond interactions with residues PHE-738 with a bond distance of 2.36 Å, and a π-alkyl bond formed with residues HIS-709 and PHE-738 with bond distances of 5.43 Å and 4.73 Å, respectively. Furthermore, TMP269 had a π-sigma bond and salt bridge interactions with residues LEU-810 and ASP-626, respectively ( Figure 5C). These interactions play a crucial role in maintaining the stability and specificity of the ligand-protein complex.
The results of the molecular docking study highlight the good binding affinity of both the isoxazole (6l) and triazole (5h) compounds towards the HDAC7 binding site, with binding energies of −6.60 and −6.42 kcal/mol, respectively. These findings suggest that both compounds have the potential to be effective inhibitors of HDAC7. Furthermore, the co-crystal ligand (TMP269) exhibited a favourable binding energy of −6.19 kcal/mol, indicating its potency as an HDAC7 inhibitor. The critical interactions observed within the binding pocket emphasize the role of specific residues in facilitating ligand-protein interactions for all three compounds. These results provide valuable insights into the potential therapeutic utility of the isoxazole (6l) and triazole (5h) compounds as HDAC7 inhibitors-also, the 3D surface view of 6l and 5h is represented in Figure 6.  These results provide valuable insights into the potential therapeutic utility of the isoxazole (6l) and triazole (5h) compounds as HDAC7 inhibitors-also, the 3D surface view of 6l and 5h is represented in Figure 6.

Materials and Methods
All chemicals and solvents were purchased from Sigma-Aldrich (Bangalore, India). Pre-coated silica gel TLC plates monitored the completion of the reaction. An Agilent mass spectrophotometer was used to record the mass of the synthesized compounds. 1 H and 13 C NMR were recorded on Bruker (400 MHz) and Jeol NMR spectrophotometers (500 MHz). TMS was used as an internal standard, and deuterated DMSO and chloroform were used as solvents. Chemical shifts are expressed as ppm.  [38]. Reactant (2) was refluxed in POCl3 for 20 min after completion of the reaction [TLC: (EtOAc:Hexane) (1:9)]; the reaction mixture was quenched in ice-cold water and neutralized by potassium carbonate [39]. Then, the reaction mass was extracted to the ethyl acetate layer and concentrated using a rotary evaporator to afford 4-chloro-2-(prop-2-yn-1-ylthio)pyrimidine (3). For Compound (3) (1 mmol), substituted piperazines/morpholine/4-piperidinone (1 mmol) were dissolved in acetone and refluxed in the presence of base triethylamine (Et3N) (2 mmol) for 8-10 h [40]. TLC was monitored for the completion of the reaction, and the crude intermediates (4ad) were purified by column chromatography (EtOAc:Hexane) (2:8) (Scheme 1).
B. Electrochemical method: In the presence of copper (Cu) foil and platinum (Pt) electrodes, the azides and alkynes dissolved in tert-butyl alcohol and water (1:1) medium were subjected to a 0.3 voltage of current in the catalytic amount of TBATFB (0.1 mmol) for 1 h. The reaction was monitored by TLC [(EtOAc:Hexane) (4:6)], and soon after the completion

Materials and Methods
All chemicals and solvents were purchased from Sigma-Aldrich (Bangalore, India). Pre-coated silica gel TLC plates monitored the completion of the reaction. An Agilent mass spectrophotometer was used to record the mass of the synthesized compounds. 1 H and 13 C NMR were recorded on Bruker (400 MHz) and Jeol NMR spectrophotometers (500 MHz). TMS was used as an internal standard, and deuterated DMSO and chloroform were used as solvents. Chemical shifts are expressed as ppm.
B. Electrochemical method: In the presence of copper (Cu) foil and platinum (Pt) electrodes, the azides and alkynes dissolved in tert-butyl alcohol and water (1:1) medium were subjected to a 0.3 voltage of current in the catalytic amount of TBATFB (0.1 mmol) for 1 h. The reaction was monitored by TLC [(EtOAc:Hexane) (4:6)], and soon after the completion of the reaction, the 1,2,3-triazoles were extracted in ethyl acetate and purified using column chromatography (Scheme 1).
B. Electrochemical method: Beaker-type undivided cells containing platinum (Pt) and iron (Fe) rod as electrodes and methanol as media were kept at room temperature. N-hydroxy-(substituted) benzimidoyl chlorides and alkynes were added to the solution, and the current 5.0 mA/cm −2 was passed to the reaction mixture. The reaction was stirred for 60 min under stirring, and the reaction was monitored by TLC [(EtOAc:Hexane) (3:7)] and was terminated when the substrate was consumed. The products were isolated as mentioned in the abovementioned procedure (Scheme 1).

General Procedure for the Synthesis of Compounds (7a-f)
To a stirred solution of compounds (5d-f/6d-f) in dichloromethane (DCM), 0.5 mL of trifluoroacetic acid was added slowly at 0-4 • C, and the reaction mixture was stirred at room temperature for 1 h [43]. After completion of the N-boc deprotection [TLC: (MeOH: EtOAc) (1:9)], the solvent was distilled off using a rotary evaporator. The residues were neutralized by a 20% K 2 CO 3 solution, extracted with an ethyl acetate layer, and dried over Na 2 SO 4 . Then, ethyl acetate was concentrated in a rotary evaporator and recrystallized with the appropriate solvent to afford the target compounds (7a-f) (Scheme 2).

General Procedure for the Synthesis of Compounds (8a-f)
To a stirred solution of compounds (5j-l/6j-l) (1 mmol) in THF, sodium borohydride (1.2 mmol) was added, and the reaction mass was stirred at room temperature for 1.5 h. After completion of ketone group reduction [TLC: (EtOAc: Hexane) (7:3)], the solvent was distilled off using a rotary evaporator. The residues were neutralized by the slow addition of water, extracted with an ethyl acetate layer, and dried over Na 2 SO 4 . Then, ethyl acetate was distilled off, and the crude was purified through column chromatography or recrystallized with the appropriate solvents to afford the final compounds (8a-f) (Scheme 3).

Cell Viability Assay
First, 2 × 10 3 MCF-7 cells 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 [44]. DMSOdissolved compounds were kept as a stock solution and diluted with a cell culture medium to the desired concentration. Cancer cells (4 × 10 3 ) were grown overnight in 96-well plates, cultured, and treated with triazoles/isoxazoles at 0, 0.01, 0.1, 10, 100, and 1000 µM concentrations for 72 h. The inhibitory effect of the compounds was assessed using the Alamar Blue reagent.

Molecular Docking Studies
The Scripps Research Institute's AutoDock4 tools (ADT) v1.5.6 [45] were used to determine the docking studies. The co-crystal structure of HDAC7 (PDB ID: 3ZNR) was obtained from the Protein Data Bank (PDB), and the structure was downloaded in PDB format for further analysis. Then, ligand structures, including the novel isoxazole (7d), triazole (5h) analogues, and the co-crystal ligand, were prepared. The ligand structures were built and optimized using molecular modeling software (Discovery Studio & Avogadro) [46] to ensure proper geometry and energy minimization [47][48][49][50][51][52][53]. The ligand structures were saved in the appropriate file format (PDB) for AutoDock4 Tools. Later, the HDAC7 crystal structure (PDB ID: 3ZNR) was loaded into AutoDock4 Tools, and water molecules, cofactors, and other non-essential entities were removed to isolate the protein of interest. The protein structure was prepared by adding polar hydrogen atoms and assigning Kollman charges. A grid box was defined around the HDAC7 to guide the docking simulation with dimensions 60Å × 60 Å × 70 Å with a spacing of 1Å. Then, docking was initiated by selecting macromolecule (HDAC7) and ligand (7d) with a genetic algorithm as the search parameter was set, and the output file was the Lamarckian default parameter file. Initially, 150 were randomly placed individually with a maximum number of 2.5 × 10 6 energy evaluations having a mutation rate of 0.02 and crossover rate of 0.80, and 10 docking runs were performed for compound 7d. Similarly, all steps were performed for the compound 5h and co-crystal ligand (TMP269). Visualization of docking results was examined using Discovery Studio [54], pymol [55], and UCSF chimera 1.16 [56].

Conclusions
In conclusion, the novel thiouracil-tethered triazole-based compounds were synthesized by both conventional and electrolytic methods. The synthesis of 1,2,3-triazoles via electrochemical oxidation provided a good yield and was found to be highly useful for synthesizing bioactive molecules based on triazoles. Moreover, the thiouracil-tethered isoxazole-based compounds were synthesized and presented a better procedure to prepare at a high yield. Additionally, we offered a unique green protocol for synthesizing isoxazole-and triazole-based compounds in the presence of the thiouracil group. Herein, in a target-based study, we further examined the discovered compound's mode of action in silico by considering the reference co-crystal structure of HDAC and its inhibitors and showed that the newly synthesized compounds could mimic HDACis in BC cells. The lead compound, such as 6l or 5h, could effectively inhibit BC cell proliferation with a lower IC 50 value. Furthermore, we discovered through an in silico investigation that these lead compounds could bind to the catalytic areas of HDAC. In conclusion, we provided a new chemical entity bearing triazole, isoxazole, and thiouracil moiety, which might target HDAC in BC cells.