Rational Design, Synthesis, In Vitro, and In Silico Studies of Dihydropyrimidinone Derivatives as β -Glucuronidase Inhibitors

In the current study, a series of dihydropyrimidinone derivatives were rationally designed as β -glucuronidase inhibitors. These designed compounds were successfully synthesized and characterized through various spectroscopic techniques such as IR, 1 H-NMR, 13 C-NMR, and EI-MS. A structure-activity relationship (SAR) of synthesized derivatives to inhibit β -glucuronidase was also established. In vitro biological evaluations revealed that 4i as the most potent compound in this series has an IC 50 value of 31.52 ± 2.54 μ M compared to the standard D-saccharic acid 1,4-lactone (IC 50 � 41.32 ± 1.82 µ M). Also, molecular docking and dynamics studies of the most potent compound are performed to evaluate interactions between the active compound and binding


Introduction
β-glucuronidase belongs to the glycosidase family with 332 kDa distributed in lysosomes of mammalian tissues and body fluids, while significantly reserved in the endoplasmic reticulum [1]. Human x-ray crystallography of the protein structure reveals three distinct structural domains: (I) jelly roll motif also named lysosomal targeting motif with barrellike structure, (II) immunoglobulin constant domains, and (III) α/β or TIM barrel domain, which possesses the active site of the enzyme comprise of Glu451 as a proton donor, Glu540 known as a catalytic nucleophile, Tyr504 (uncertain catalytic role), and Asp207 (plausible role as Glu540) [2]. e biological function of β-glucuronidase is to cleave the glycosidic bond of glycans (β-linked glucuronides), release glucuronic derivatives, and produced physiological responses. Besides, β-glucuronidase incorporates β-glucuronides into steroids and helps in the recirculation of vitamin D, estrogen, and thyroid hormone [2,3]. Deficiencies in β-glucuronidase result in a metabolic disease known as Sly syndrome or mucopolysaccharidosis type VII [4]. On the other hand, the overexpression of the enzyme is reported in some solid tumors, necrotic areas, and infected tissues [5,6]. β-glucuronidase is also found to be involved in the etiology of hepatic diseases [7], inflammatory joint diseases [8], colon cancer, liver cancer, and AIDS [2,9]. ese reports suggest that the development of specific β-glucuronidase inhibitors has great pharmacological importance. Several molecules from natural origin including flavonoids [10], iminosugars [11], thiosulfinate [12], terpenoids, and steroids [13] have proven to be promising β-glucuronidase inhibitors. Also, some successful synthetic β-glucuronidase inhibitors such as imidazole [14], imidazolopyridine [15], benzimidazole [16], thiazole [17], 1,3,4-thiadiazole [18], and 1,3,4-oxadiazole derived scaffolds were also reported.
Dihydropyrimidinones are heterocyclic compounds synthesized by simple multi-component reactions such as the Biginelli reaction with a variety of methodologies [19,20]. Dihydropyrimidinones have been identified to possess a wide range of applications in pharmacology and medicinal chemistry including anticancer [21], antiinflammatory [22], antimicrobial [23], antidiabetic, and antioxidant agents [24]. is skeleton also showed a broad spectrum of biological activities including calcium channel antagonism/inhibition, Eg5 kinesin, and topoisomerase I inhibitor [25]. Considering β-glucuronidase inhibition, dihydropyrimidone and pyrimidine structures were first reported as a successful example in rational drug design against β-glucuronidase [26,27]. Recently single point modification on pyrimidine resulted in designing thioxotetrahydropyrimidines which showed excellent inhibitory potency in vitro against β-glucuronidase with an IC 50 value of 0.35 μM for the most potent compound. According to their docking and MD research, conformational stability of this compound was seen within the enzyme active site through hydrogen bond interactions with Tyr205, Asp207, and Glu451 residues.
As a result, the dihydropyrimidinone structure was selected due to several reasons. (I) e dihydropyrimidinone structure was chosen due to the significant β-glucuronidase inhibitory potential reported in limited studies. (II) To the best of our understanding, limited data are available concerning SAR of dihydropyrimidinone structure against β-glucuronidase. (III) e unique power of multicomponent reactions to synthesize dihydropyrimidinone structure is recognized as an extremely valuable strategy to reduce time, cost, and energy.
In continuation of our ongoing research on heterocyclic compounds with bioactivity and pharmacological importance of β-glucuronidase inhibitors, herein, we report the design and synthesis of dihydropyrimidinone analogs to evaluate their β-glucuronidase inhibitory potentials.

General.
Melting points of synthesized compounds were determined on a Kofler hot stage apparatus. 1 H and 13 C NMR spectra were determined on a Bruker 500 MHz NMR. e letters s, d, t, q, and m are used to indicate singlet, doublet, triplet, quadruplet, and multiplet. Coupling constants (J values) are quoted in hertz (Hz). e mass spectrometric analysis was conducted by using ESI mode on the Agilent 7000-triple quad instrument at 70 eV (Santa Clara, CA, USA). IR spectra were recorded on an FTIR-470 Shimadzu spectrophotometer.

General Procedure for the Synthesis of Dihydropyrimidinone
Compounds (4a-i). Dihydropyrimidinone analogs were synthesized by refluxing N,N-diethyl-3-oxobutanamide (1, 1 mmol), urea (2, 1.2 mmol), and different aldehyde (3, 1 mmol) in the presence of FeCl 3 .6H 2 O (15% mmol, 0.0405 mg) in 20 mL acetonitrile for 24 hours. After completing the reaction (monitored by TLC, n-hexane/ethyl acetate, 3 : 7 v/v), the reaction mixture was cooled to room temperature and the reaction mixture was diluted with water, poured into crushed ice, and allowed to stir for 1 h to obtain maximum precipitation. e precipitated product was filtered off and washed with cold water. All products were recrystallized from hot ethanol to give pure products.

Molecular Docking Details.
Molecular docking was carried out to study the interactions between the most potent compound and the active site of the enzyme (1BHG, apo human β-glucuronidase) using AutoDock tools. e twodimensional structure of 4i was sketched and optimized using HyperChem and was saved in PDB format. Before docking, the PDB file was converted into pdbqt format using AutoDock Tools while adding Gasteiger charges, polar hydrogen atoms, and merged nonpolar hydrogen atoms. e compound was docked into the active site of β-glucuronidase using default parameters 100 runs for each ligand, 27000 as the maximum number of generations. e grid box was set with 30, 30, and 30 points with the center of 84.55, 77.88, and 91.26 in the x, y, and z directions, respectively. All other options were set as default.

Molecular Dynamics Simulation.
e molecular dynamics simulation of compound 4i in complex with human β-glucuronidase, PID: 1BHG, was carried out using the AMBER 18 molecular dynamics package on a Centos Linux server equipped with GPUs. Amber99sb force field was applied to define the atom types and dynamics simulation parameters. An octahedral box was defined around the solute and was filled with the TIP3P water molecules and the appropriate number of Na+ ions replaced water molecules to neutralize the system. en, the system was targeted to energy minimization in explicit solvent by the steepest descent algorithm once with restraints and once without restraints. en, NVT heating with putting restraints on the solute was carried out during a 500 ps time so that solvent molecules and counter-ions immersed the macromolecule. During NVT step, the temperature was controlled near to 300 K. e periodic boundary simulations based on the particle mesh Ewald (PME) method were applied and the SHAKE algorithm was used to constrain the covalent bond lengths. en, under the 500 ps NPT ensemble equilibration phase, the pressure of the system was regulated with an average pressure of 1 atm in such a way that the density of water could relax. Isotropic position scaling was applied to preserve the pressure periodic boundary constant. e production MD run was performed for 100 ns simulation time on the well-equilibrated system at 300 K and pressure of 1 atm. After termination, the trajectory was corrected for periodic boundary conditions and was downsampled to maintain 1000 frames for any postanalysis. To define the equilibrium time range, the root-mean-square deviation (RMSD)Å for the protein backbone atoms of each snapshot was then calculated during the simulation run time against the first frame. All interaction analysis and image preparations were performed by Discovery Studio Visualizer v20.1.0.19295.

β-Glucuronidase
Bioassay. β-glucuronidase inhibitory activity was determined by monitoring the absorbance of p-nitrophenol formed from the substrate at 405 nm by the spectrophotometric method according to the literature report. Briefly, to a 96-well microplate, 185 μl of 0.1 M acetate buffer (pH � 7.0) and 5 μl of test compound (3X concentration) were added and gently mixed. en, 10 μl of 2.5 unit/mL enzyme was added to start the reaction. After incubation for 30 minutes at room temperature, 50 μL of 0.4 mM para nitrophenyl-b-d-glucuronide as substrate was added and the absorbance was measured. DMSO was used to dissolve the test compound (3X concentration). e IC 50 values were determined graphically from inhibition curves (log inhibitor concentration vs. percent of inhibition) [28,29].
In 2016, Ali et al. introduced dihydropyrimidones derivatives as a novel class of β-glucuronidase inhibitors via a "one-pot" three-component reaction. In vitro, SAR evaluation confirmed that polar groups with hydrogen bonding interactions have better inhibitory activity compared to the rest of synthesized compounds. e most potent compounds containing para-trifluoromethyl group (Figure 1, compound G) showed good inhibitory potential (IC 50 � 9.38 ± 0.15 μM) compared to the standard drug d-saccharic acid 1,4-lactone (IC 50 � 48.4 ± 1.25 μM).
Previously, our research group reported thioxo-tetrahydropyrimidine as potent β-glucuronidase inhibitors with IC 50 values ranging from 0.35 to 42.05 μM. e most potent compound in this series was compound H (Figure 1) bearing the thiophene motif. It was also concluded that meta substitutions on phenyl ring are more favorable to anti-β glucuronidase activity.
e SARs indicated that the thiophene ring is important for inhibiting the β-glucuronidase. Molecular dynamics suggested that hydrogen bonds interaction with Tyr205, Asp207, and Glu451 with ß-glucuronidase results in conformational stability which justifies the high potency of the mentioned compound [28].
e literature review has shown that the introduction/ modification of specific groups on heterocyclic moiety may change their biological activities. Some structural modifications were planned based on single-point replacement on compound H (Figure 1) via replacing the thio atom with oxo while keeping diethyl acetamide linker in the structure with the hope to enhance their β-glucuronidase inhibitory potential and H-bound interactions. Additionally, the structure-activity relationship (SAR) and molecular docking study against β-glucuronidase were discussed.

Chemistry.
Dihydropyrimidinone analogs 4a-i were synthesized by treating commercially available β-ketoamide with different aldehydes in acetonitrile (Scheme 1). In a typical reaction, to a stirring mixture of N,N-diethyl-3oxobutanamide (1) and thiourea (2) e introduction of the 4-methoxy group at the R position slightly increased the activity (4g, IC 50 = 73.12 ± 2.69) compared to an unsubstantiated one (4a). Position and bioisosteric replacement of 4-methoxy with 3-hydroxy (4h) substitution on phenyl ring contributed to a better β-glucuronidase inhibitory activity with an IC 50 value of 46.33 ± 4.13 μM. In most cases, the para-substitution on the phenyl ring was found to be weakly active, while switching from para to meta, better inhibitory potential was observed. 4i containing thiophene moiety with IC 50 = 31.52 μM emerged as the most active inhibitor of the series, producing a substantial improvement of β-glucuronidase inhibitorypotential.

Molecular Docking Interactions.
Interactions of the most potent compound 4i within the active site of the target protein are shown in Figures 2(a) and 2(b). e Asn484 and Lys606 of β-glucuronidase showed hydrogen bond interactions via oxygen in amide and nitrogen in pyrimidine of 4i with a distance of 2.95 and 2.55Å, respectively. Compound 4i also made two other hydrogen bonds with Asp207 and Tyr504. Asp207 was involved in pi-sigma hydrophobic interaction with the thiophene ring (3.72Å). e other in pisigma hydrophobic interaction with a distance of 3.66Å was observed between diethylamide and Tyr508. It seems that in vitro inhibition potential of 4i against β-glucuronidase was supported by performing molecular docking studies. Docking scores and the predicted interactions of top-ranked confirmation of all derivatives are reported in Table 2 which showed the consistency of computational and biological results.

Molecular Dynamics Simulation.
e trajectory stability was examined via RMSD analysis of the protein backbone atoms against the primary conformation calculated for the complex; see Figure 3(a). e RMSD value mounted rapidly from zero for the initial frame, and afterward formed a regular profile after nearly 10 ns. e number of formed hydrogen bonds during the simulation time between ligand 4i and protein residues was also monitored; see Figure 3(b). e maximum number of hydrogen bonds made was 3 and in 37.5% of simulation time there was at least one hydrogen bond between the ligand and protein residues. e interaction pattern of compound 4i is shown in Figure 4. ere were three hydrogen bonds with residues Glu451, Asn502, and Glu540. Two NHs in tetrahydropyrimidine-2-one group donated two hydrogen bonds to Glu451 and Glu540 while carbonyl of this group accepted one hydrogen bond from Asn502. Sulfur in thiophene participated in a pi-sulfur interaction with Trp587. Residue His385 took part in a pi-pi stacked interaction with thiophene which along with hydrogen bond interactions enhanced the affinity of compound 4i in the β-glucuronidase active site. One of the ethanamine groups and the methyl attached to the tetrahydropyrimidine-2-one group were subjected to the solvent and did not show any interaction with protein. As a comparison to our previous study on thioxo-tetrahydropyrimidine derivatives [28], thiourea with   3.6. In Silico ADME Evaluation. In silico ADME/Tox studies of the synthesized compounds were performed using http:// biosig.unimelb.edu.au/pkcsm/prediction server. Based on results reported in Table 3, all compounds pass the Lipinski's rule of 5 (MW < 500, cLogP < 5, HB donor ≤ 5, HB acceptor ≤ 10).

Conclusion
Here, a series of dihydropyrimidinones derivatives as β-glucuronidase inhibitors were designed and synthesized. Newly synthesized compounds depicted various degrees of β-glucuronidase inhibitory potential. Amongst, the key structural feature that enabled better β-glucuronidase inhibition was presented in compound 4i possessing thiophene ring at R position with an IC 50 value of 31.52 μM. Also, plausible SAR suggested that the introduction of a meta group on the phenyl ring in comparison with para is more beneficial for β-glucuronidase inhibition. SAR assessment for thioxo-tetrahydropyrimidine derivatives of our previous work with IC 50 value ranging from 0.35 to 42.05 µM [28] and the present study (4a-i dihydropyrimidinones analogs) with IC 50 of 31.52 to >100 confirmed the favorable role of thioxo-tetrahydropyrimidine scaffold compared to dihydropyrimidinone ring. Interestingly, in both sets of compounds, analogs bearing thiophene substituent demonstrated superior β-glucuronidase inhibitory potency compared to the rest of the derivatives. ese results were also supported by molecular docking and dynamics studies demonstrating efficient binding interaction between the compound 4i and β-glucuronidase.
Data Availability e datasets used and analyzed during the current study are available from the corresponding author on reasonable request. All data are in the form of tables and figures.

Disclosure
is study was part of the Pharm.D thesis of Yasaman Moghdani.

Conflicts of Interest
All authors state that there are no conflicts of interest.