Development of 1-(4-(Substituted)piperazin-1-yl)-2-((2-((4-methoxybenzyl)thio)pyrimidin-4-yl)oxy)ethanones That Target Poly (ADP-Ribose) Polymerase in Human Breast Cancer Cells

A number of uracil amides cleave poly (ADP-ribose) polymerase and therefore novel thiouracil amide compounds were synthesized and screened for the loss of cell viability in a human-estrogen-receptor-positive breast cancer cell line. The synthesized compounds exhibited moderate to significant efficacy against human breast cancer cells, where the compound 5e IC50 value was found to be 18 μM. Thouracil amide compounds 5a and 5e inhibited the catalytical activity of PARP1, enhanced cleavage of PARP1, enhanced phosphorylation of H2AX, and increased CASPASE 3/7 activity. Finally, in silico analysis demonstrated that compound 5e interacted with PARP1. Hence, specific thiouracil amides may serve as new drug-seeds for the development of PARP inhibitors for use in oncology.


Introduction
Poly (ADP-ribose) polymerase (PARP) is an enzyme which catalyzes poly ADPribosylation of specific target proteins. The PARP family contains 17 enzymes, among which PARP1 is the most abundant and widely studied [1][2][3]. It is involved in a number of

Chemical Synthesis of Novel Thiouracil Amides
The synthesis of novel thiouracil derivatives and their characterization is herein reported. In the present investigation, regioisomeric 2-thiopyrimidine acetic acids were utilized as a template to prepare a library of novel amides with various amines, as described in Scheme 1.
The synthetic sequence commenced by reacting 2-thiouracil with substituted benzyl derivative to afford S-benzylated products. The phenolic hydroxyl group in S-benzylated products was treated with tert-butyl acetate to afford tert-butyl esters which were subjected to hydrolysis to afford 2-thiopyrimidine acetic acid. The acid so obtained was coupled with various amines to afford amides 5a-5l. The structures of these newly synthesized amide derivatives were confirmed by 1 H NMR, 13 CNMR, and LC/MS analysis.

Novel Thiouracil Derivatives Produce Loss of Breast Cancer Cell Viability
The synthesized compounds were screened for loss of cell viability using the ER+ breast cancer cell line by Alamar Blue cell viability assays [17][18][19][20][21][22]. The compounds were dissolved in DMSO and cells were treated in a dose-dependent manner for 72 h. Amongst the tested amides, compounds 5a, 5b, 5e, and 5f showed significant loss of cell viability with IC 50 values of 22.68, 25.71, 18.23, and 29.34 µM, respectively, as shown in Figure 1 and Table 1. The other compounds tested from the series exhibited less efficacy. The thiouracil amides possessing tolyl or halophenyl groups displayed significant loss of breast cancer cell viability, according to studies on the structure-activity relationship of this library of compounds. Furthermore, the loss of cell viability produced by the lead compounds (5a, 5e) was compared with Olaparib using immortalized but otherwise normal human mammary epithelial cells (MCF-10A) as an indicator of effect on normal cells. It was observed that these compounds produced loss of cell viability of MCF-10A cells with IC 50 values of 46.9 and 52.3 µM for compounds 5a and 5e, respectively, which were comparable to Olaparib, where IC 50 was observed to be 57.3 µM (Supplementary Materials).  Loss of MCF-7 cell viability produced by compounds 5a, 5b, 5e, and 5f. MCF-7 were exposed to 5a (A), 5b (B), 5e (C), and 5f (D) for 72 h and the viability of cells was analyzed by Alamar Blue assays. The results are presented as mean ± S.E.M. of triplicate determinations.

Lead Compounds Increased PARP1 Cleavage, Phospho-H2AX Levels, and Caspase-3/7 Activation in MCF-7 Cells
The effects of compounds 5a, 5e, and Olaparib on the cleavage of PARP1 in MCF-7 cells were performed using the previously reported protocol [24][25][26][27]. MCF-7 cells were incubated with 5a, 5e, or Olaparib at the indicated concentrations. As observed in Figure 3A, treatment of breast cancer cells with either compound 5a or 5e produce PARP cleavage comparable to that observed with Olaparib. Figure 3. Compounds 5a, 5e, and Olaparib cleave PARP1 (A) and increase phosphorylation of H2A-X (B) and CASPASE-3/7 activity (C) in MCF-7 cells. MCF-7 cells were treated with or without compounds 5a, 5e, or Olaparib at the indicated concentrations. The respective protein bands were detected by Western blot analysis. GAPDH or β-ACTIN were used as input controls. Data are expressed as mean ± SEM. ** p ≤ 0.01; *** p ≤ 0.001. Phosphorylation of Ser139 in H2AX is an indicator of cellular DNA damage which occurs upon PARP inhibition [28]. For example, Olaparib has been reported to enhance H2AX phosphorylation in various cancer cell lines [29]. Therefore, the effect of compounds 5a or 5e or Olaparib on the level of p-H2AX in MCF-7 cells was determined ( Figure 3B). It was observed that compounds 5a and 5e increased phosphorylation of H2AX in MCF-7 cells comparable to that observed with Olaparib.
The effect of compounds 5a or 5e and Olaparib on CASPASE 3/7 activity was also examined as an indicator of apoptosis [30][31][32]. Hence, CASPASE 3/7 activity was determined after treatment of breast cancer cells with compounds 5a or 5e or Olaparib. Both compounds 5a and 5e increased CASPASE 3/7 activity in a dose-dependent manner and were roughly comparable to that observed with Olaparib ( Figure 3C).

Density Functional Theory (DFT) Calculations
DFT calculations were performed to understand the stability of compound 5e in the catalytical grove of PARP1 [33][34][35]. According to the frontier molecular orbital theory, the highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) are the most effective factors in bioactivity. HOMO has the priority to provide electrons, while LUMO can accept electrons. Thus, studies on the frontier orbital energy may provide useful information of the biological mechanism [36]. The B3LYP level of theory was used to calculate the energy of HOMO, LUMO, and the E gap value, which were used to study the chemical stability, molecular polarizability, and chemical softness or hardness of compound 5e. The distribution and energy levels of the HOMO and LUMO for 5e are shown in Figure 4. The positive phase of the molecule is represented in red color, whereas the negative phase is in green color. It is clear from the plots that the HOMO is localized on the 1-(4-methylphenyl)piperazine ring. In contrast, LUMO is delocalized on the methoxypyrimidine ring and sulfur atom. The HOMO and LUMO values of conformer 5e are −5.740 eV and −1.124 eV, respectively, and the energy separation between HOMO and LUMO is 4.615 eV. The stabilization and interactions of the compounds with the catalytic site of the enzyme may be explained by global reactivity descriptors and are represented in Table 2. The ionization energy can be expressed as I = −E HOMO = 5.740 eV, and the electron affinity can be expressed as A = −E LUMO = 1.124 eV. Hardness represents the resistance of a chemical system against the deformation of the electron cloud under the slight interference encountered in the chemical process and can be expressed as η = (E LUMO − E HOMO )/2. The hardness and softness values of the 5e molecule are 2.307 eV and 0.216 e −1 V −1 , respectively. With the smaller value of softness, the molecule is chemically stable.  MEPs are fundamental measures of the interaction strength of the nearby charges, nuclei, and electrons at a particular position, thus enabling visualization of the charge distribution and charge-related characteristics of molecules. To facilitate interpretation of the electrostatic potential data, a visual representation with a chromatogram is used. The B3LYP/6-31+G (d,p) level of theory was used to study the molecular electrostatic potential in the intermolecular interaction region of conformer 5e. The resulting MEP map with a range of −6.351 × 10 −2 -6.351 × 10 −2 depicts its potential distribution (see Figure 5). The O atom of the C=O bond of molecule 5e is surrounded by a positive charge, indicating a possible nucleophilic attack site. Furthermore, a negatively charged region is located on the hydrogen atoms of the piperazine ring, indicating a probable electrophilic attack site. To determine the conformational stability of compounds at the PARP1 binding site, MD simulations are employed to assess structural and dynamic changes throughout the protein-ligand interactions [37]. The complex structures are solved using orthorhombic simple point charge (SPC) water models. On periodic boundary conditions, the system was determined to have a cut-off for Lennard-Jones interactions of 10 Å. In the integration steps, a time step of 2.0 fs was used. The system temperature and pressure were set to 300 K and 1.01325 bar, respectively, using the Nose-Hoover thermostat and Martyna-Tobias-Klein protocols. Before the production run, the system was minimized, equilibrated, and 100 ns MD simulations were then performed [38].

Bioinformatic Analysis of the Binding of Compound 5e towards the Catalytical Grove of PARP1
Compound 5e was studied using molecular docking modeling to assess the binding of compound 5e towards the catalytical grove of PARP1 [39]. Figure 6A depicts the binding pose of compound 5e with PARP1. Docking simulations revealed the prominent interactions of compound 5e with PARP1 active site residues ( Figure 6B). The docking score was assigned to compound 5e that binds to the active site with −7.33 kcal/mol energy and forms potential hydrogen bonds. TYR49 is bonded with the nitrogen of the pyrimidine ring and ARG217 with the oxygen of methoxybenzene with the bond distances 2.50 and 3.56 Å (Figure 7), respectively. Furthermore, pi-pi stacking interactions exist with ILE218 and TYR235. In addition to the latter, hydrophobic interactions have been discovered in the active site [39]. 100 ns MD simulations were run for the molecule in the PARP1 binding pocket, and the associated coordinates throughout the MD simulation were gathered separately in the trajectory file. Figure 8 depicts the root-mean-square deviations (RMSDs) of Cα atoms in the investigated systems. After 30 ns of MD simulations, all of the studied systems have relaxed and show no significant fluctuations in RMSD values. The evaluated system exhibited an average RMSD of less than 3.0 Å. The LigFitProt RMSD plot displays the RMSD of ligands when the protein-ligand complex is initially aligned as a reference state with the backbone atoms of the protein.  Root-mean-square fluctuation (RMSF) values were also calculated during simulations to investigate the effect of defined hits on the mobility of the target protein's backbone atoms (Figure 9). Figure 10A depicts associations that appear in the chosen path for more than 6% of the simulation time (0.00-100.00 ns). Hydrogen bonds with TYR49 and π-π stacking interactions with TYR228 produce significant interactions in the molecule. ASP105, ARG217, ALA219, GLY233, and TYR235 have stable non-bonded chemical contacts throughout the simulations, as shown by the protein-ligand interactions graph Figure 10B. The stability between compound 5e and PARP1 is furthermore enhanced by the interaction with the amino acid residues ARG217, GLY233, ALA219, and TYR49 of PARP1 via water bridges [40].

Materials and Methods
All organic chemicals used were purchased from Sigma-Aldrich. 1 H and 13 C NMR spectra were recorded on a Bruker WH-200 (400 MHz) and JEOLJSM-ECS (400 MHz) spectrometer in CDCl 3 as solvent, using TMS as an internal standard, and chemical shifts are expressed as ppm. High-resolution mass spectra were determined on a Bruker Daltonics instrument. Mass spectra were determined on an Agilent LC-MS and the elemental analyses were carried out using an Elemental Vario Cube CHNS rapid analyzer. The progress of the reaction was monitored by TLC precoated silica gel G plates [41].

Synthesis of Compound 2
To a stirred solution of 2-thiouracil, p-methoxy benzyl bromide in DMF was added potassium carbonate, and the reaction mixture was heated to 80 • C. The reaction was monitored by thin-layer chromatography, quenched with water, and extracted with ethyl acetate. The organic layer was washed with brine and dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography with silica-60 to yield the alcohol 2 in 81% yield.

Synthesis of Compound 3
To a stirred solution of alcohol 2, tert-butyl bromoacetate in DMF was added potassium carbonate (1.2 eq), and the reaction mixture was heated to 80 • C. The reaction was monitored by thin-layer chromatography. After completion of the reaction, it was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine and dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography with silica-60 to afford the ester 3 in 85% yield.

Synthesis of Compound 4
To a stirred solution of ester 3 in dichloromethane, was added trifluoroacetic acid and stirred overnight. After completion of the reaction as monitored by thin-layer chromatography, it was quenched with sodium bicarbonate and extracted with ethyl acetate. The organic layer was washed with brine and dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography with silica-60 to afford acid in 65% yield.

General Synthetic Method for the Preparation of 5a-5l
To a stirred solution of the acid (100 mg, 0.33 mol) in dry dichloromethane (4 mL) under nitrogen atmosphere, was added EDC. HCl (93.7 mg, 0.49 mol). Then, DMAP (4 mg, 10 mol%), amine derivative (130.8 mg, 0.49 mol) was added to the reaction mixture and stirred for five minutes. Triethylamine (0.15 mL, 0.99 mol) was then added slowly, dropwise, to the reaction mixture and it was stirred for 2-3 h. After completion of the reaction, as indicated by TLC, it was quenched with water (5 mL) and extracted with ethyl acetate (2 × 10 mL). The combined organic layers were washed with brine (5 mL) and dried over anhydrous sodium sulfate. Evaporation of the solvent gave the crude residue which was purified by silica gel column chromatography using petroleum ether/EtOAc (3:7) as eluent to furnish amides 5a-5l.

Assay of PARP1 Catalytic Activity
The catalytic activity of PARP1 was determined using the PARP/Apoptosis Universal Colorimetric Assay Kit (4677-096-K, R&D Systems Ltd., Abingdon, UK) [46]. Compounds 5a or 5e or Olaparib were added to recombinant human PARP enzyme prior to evaluation as per manufacturer's instructions.

Western Blot Analysis
Western blot analysis was performed to determine the levels of cleaved PARP1, and the phosphorylation ofH2AX in MCF-7 cells using previously reported methods [29]. Cells at 50-60% confluence were treated with compounds and incubated for 60 h in 2% FBS. MCF-7 cells were also treated with Olaparib, 5a, or 5e, and the levels of cleaved PARP1 and the phosphorylation of p-H2AX were determined using the respective antibodies, as previous [29].

CASPASE-3/7 Activity Assay
CASPASE-3/7 activity was determined by the Caspase-Glo ® 3/7 assay kit (Promega corporation, Madison, WI, USA) according to the manufacturer's instructions [30]. MCF-7 cells were seeded in 96-well plates and incubated overnight. Compounds or vehicle were added to cells after 12 h. After a 24 h incubation period, Caspase-Glo ® 3/7 reagent and media (1:1) were added into each well and processed according to the manufacturer's instructions.

DFT Calculations for the Compound 5e
Computational DFT studies for the 5e molecule were performed using Gaussian09 [47]. The molecule was built and visualized by Gaussview 5 program package. The molecular geometry of the 5e molecule was optimized at B3LYP (Becke's three parameter (B3)) level with a 6-311+G (d,p) basis set [48].

Bioinformatics Studies for the Compound 5e
The Scripps Research Institute's AutoDock Tools (ADT) (v1.5.7) [49,50] was used to generate grid and docking parameter files.The crystal structure of PARP1 with the ligand was used to retrieve the crystal structure of PARP1 with a ligand (PDB ID: 4HHY) and use it as afor the docking purpose (PDB ID: 4HHY). With the centroid rendered from inhibitor in the crystallographic structure, the grid box size of 65 × 65 × 65 Å with 0.481 Å spacing is defined. The empirical free-energy function and the Lamarckian genetic algorithm were used to perform molecular docking with the macromolecule, with an initial population of 150 randomly placed individuals, a maximum number of 2,500,000 energy evaluations, a mutation rate of 0.02, a crossover rate of 0.80, and 10 docking runs. Visualization plots were examined using BIOVIA Discovery Studio Visualizer (v21.10.20298) [51] and PyMOL (v2.5.2) [52,53].

Statistical Analysis
The data were analyzed by Student's t-test and p < 0.05 was considered statistically significant (GraphPad Prism 5.0; GraphPad Software, La Jolla, CA, USA).

Conclusions
A series of thiouracil derivatives containing amide linkages were synthesized and screened for loss of viability of breast cancer cells. The compounds exhibited moderate to significant activity in MCF-7 cells. Compounds 5a and 5e inhibited PARP1 activity, produced PARP cleavage, and enhanced phosphorylation of H2AX. Computational methodologies demonstrated conclusively that compound 5e interacts with PARP.