Synthesis, Crystal Structures, Molecular Docking, and Urease Inhibitory Activity of Transition Metal Complexes with 2- [[ 4-(4-Fluorophenyl)piperazin-1-yl ]] acetic Acid

Two novel mononuclear complexes, [ Cu( L ) 2 (H 2 O) ] · 2H 2 O ( 1 ) and [ Ni( L ) 2 (H 2 O) 2 ] ( 2 ) ( HL = 2- [ 4-(4-fluorophenyl)pi-perazin-1-yl ] acetic acid) were synthesized and structurally determined by single-crystal X-ray diffraction. Their inhibitory activities were tested in vitro against jack bean urease. Molecular docking was investigated to determine the probable binding mode. The experimental values and docking simulation exhibited that complex 1 had better inhibitory activity than the positive reference acetohydroxamic acid (AHA), showing IC 50 value of 0.15 ± 0.08 μ M, while 2 showed no inhibitory activity.


Introduction
Urease is a nickel-containing metalloenzyme, which can be widely found in various fungus, germs and plants, 1 catalyzing the hydrolysis of urea into ammonia and carbamate. 2,3 Comparing the sequences of jack bean urease and bacteria urease suggests that these two different kinds of urease may have a common evolutionary origin. 4 The catalytic sites in jack bean urease and H. pylori urease display highly conserved amino acid residues, indicating the ureases may have the same catalytic mechanism. In addition, it is a major cause of pathologies induced by Helicobacter pylori (H. pylori) as it allows the bacteria to survive in the extremely acidic environment of stomach during colonization. [5][6][7][8] Meanwhile, the reaction catalyzed by urease will increase the pH value, which is a virulency factor in pathogens responsible for the development of kidney stones, pyelonephritis, peptic ulcers, and other diseases. 9,10 Therefore, the restriction of the activity of urease is an important goal to pursue.
Recently, a compound synthesized by Negar 11 containing 3-methoxybenzylpiperazine pendant demonstrated strong urea enzyme inhibitory activity. We are interested in finding metal complexes which possess potential urease enzyme inhibitory activities. Therefore, 2-[4-(4fluorophenyl)piperazin-1-yl]acetic acid (HL) and two metal complexes bearing L were synthesized. The structures of [Cu(L) 2 (H 2 O)] · 2H 2 O (1) and [Ni(L) 2 (H 2 O) 2 ] (2) were characterized by X-ray diffraction. The inhibitory activity evaluation of the complexes 1 and 2 was performed against jack bean urease in vitro. Docking simulation was investigated from the docking analysis using the AUTODOCK 4.2 program to determine the probable binding mode.

1. Materials and Methods
All chemicals and reagents used in the current study were of analytical grade. Urease (from jack beans, type III, activity 34310 units/mg solid), HEPES buffer, and urea were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cu(NO 3 ) 2 · 3H 2 O, Ni(NO 3 ) 2 · 6H 2 O, 1-(4-fluorophenyl)piperazine, DMSO, and bromoacetic acid were purchased from Aladdin Chemistry Co. Ltd (Shanghai) and used without further purification. All other chemicals and solvents were purchased from Aldrich and used as re-Chen et al.: Synthesis, Crystal Structures, Molecular Docking, ... ceived. Distilled water was used for all procedures. Elemental analyses (C, H, and N) were performed using a Perkin-Elmer 240 elemental analyzer. IR spectra were recorded on a FT-IR Nicolet 5700 Spectrometer from 4000 to 400 cm -1 . The enzyme inhibitory activity was measured on a Bio-Tek Synergy™ HT Microplate reader.

General Synthetic Method for the Preparation of the HL
1-(4-Fluorophenyl)piperazine (1.80 g, 0.010 mol) and bromoacetic acid (1.67 g, 0.012 mol) were added into 50 mL ethanol which containing 0.6 mol/L potassium hydroxide. The resulting mixture was refluxed for 14 h at 75 °C and after cooling the solvent was neutralized with hydrochloric acid to form the precipitate, which was isolated by filtration from ethanol to give pure HL. Yield was 88%.

3. General Synthetic Method for
the Complexes HL (0.2 mmol) in methanol solution (5 mL) was added to aqueous solution (5 mL) of the corresponding metal acetate (0.2 mmol). The resulting solution was stirred for 15 min at room temperature and then filtered. The filtrate was left to stand at room temperature for a few days to give the corresponding block crystals suitable for X-ray diffraction analysis. The crystals were isolated, washed three times with methanol and dried in a vacuum desiccator. The elemental analyses and characteristic IR data for the complexes were as follows: (1) [Cu(L) 2

4. Crystal Structure Determination
Single crystals of complexes 1 and 2 were mounted on a thin glass fiber at room temperature. The reflection data were collected on a Bruker D8 VENTURE PHO-TON diffractometer with graphite monochromatic Mo-K α radiation (λ = 0.71073 Å) using the generic omega scan technique. The structures were solved by direct methods and refined on F 2 by full matrix least-squares with SHELXS-97 program. 13,14 All of the non-hydrogen atoms were refined anisotropically. The water H atoms were located in a difference Fourier map and refined freely. The remaining H atoms were placed in idealized positions and constrained to ride on their parent atoms. Owing to too much water molecules in the asymmetric unit, many numbers of refined parameters may have been limited deliberately leading to the R Flagged nonhydrogen atoms. The crystallographic data are summarized in Table 1.

5. Measurement of Jack Bean Urease Inhibitory Activity
The measurement of urease inhibitory activity was carried out according to the literature reported before. 15 The assay mixture consisted of 25 μL of jack bean urease (40 kU/L) (dissolved in distilled water) and 25 μL of the acquired complexes of different concentrations (dissolved in DMSO/H 2 O mixture (1:1 v/v)) was pre-incubated for 1 h at 37 °C in a 96-well plates. Following, the addition of 200 μL of 100 mM HEPES buffer pH 6.8 containing 500 mM urea and 0.002% phenol red to each well and then incubated at 37 °C. 16 Finally, the reaction was measured at 570 nm by a Microplate reader (Bio-Tek Synergy™ HT, Instruments, Inc. USA), which was required to produce enough ammonium carbonate to raise the pH of a HEPES buffer from 6.8 to 7.7. 17 The results were analyzed using SPSS 19.0 (International Business Machines Corporation, Armonk, NY, USA) program and showed in Table 4.

6. Molecular Docking
Molecular docking of complexes 1 and 2 with the active site of jack bean urease (3LA4) was performed by the AUTODOCK 4.2 program suite. The structures of complexes in docking protocol were used as crystal struc-tures. The graphical user interface AutoDockTools was intended to install the enzymes: all hydrogens were added at every inhibitor enzyme interaction, Gasteiger charges were calculated and non-polar hydrogens were merged to carbon atoms. The Ni initial parameters are set as r = 1.170 Å, q = +2.0, and Vander Waals well depth of 0.100 kcal/mol. The 3D structures of ligand molecules were saved in Mol2 format with the assistance of the program MERCURY. The partial charges of Mol2 file were further modified by using the ADT package so that the charges of the non-polar hydrogen atoms would be assigned to the atom to which the hydrogen is attached. 18 The resulting file was saved as pdbqt file.
The docking input files were generated by Au-toDockTools program. A grid box size of 60 × 60 × 60 pointing in x, y and z directions were built. The maps were centered on the Ni842 atom in the catalytic site of the protein in all docking. The binding mode of potential urease inhibitor complex 1 with 3LA4 was displayed in Figure 7 and

1. Synthesis and Spectroscopic Studies
HL was prepared by electrophilic substitution reaction between 1-(4-fluorophenyl)piperazine and bromoacetic acid according to the method of Sadashiva with suitable modification (Scheme 1). 12 The yield of HL was 88%. The ligand was stable and could dissolve in the polar solvent such as methanol and N,N-dimethylformamide (DMF). Generally, treatment of the ligand with salts Cu(NO 3 ) 2 · 3H 2 O and Ni(NO 3 ) 2 ·6H 2 O with 2 : 1 molar ratio at ambient temperature led to the formation of the complexes. Crystal of complexes suitable for X-ray diffraction were isolated after slow evaporation of the solvent over several days.
The IR spectra of these complexes were similar. They all show broad band ranging from 3450 cm -1 to 3200 cm -1 indicating the O-H stretching of the water molecules. The separation value Δν [ν as (COO -) -ν s (COO -)] of the carboxylic based complex can be used to distin-  19,20 The C-N stretching vibration was noticed in the region of 1330-1300 cm -1 . 21

2. X-ray Structure Analysis
The related bond distances and bond angles data was listed in Table 2. The data of hydrogen-bonds was exhibited in Table 3. The ORTEP plots of the compounds were presented in Figure 1 and Figure 2.

2. 2. Crystal Structure of Complex [ [Ni(L) 2 (H 2 O) 2 ] ] (2)
Complex 2 crystallized in the triclinic space group Pî. In the crystal structure of 2, the asymmetric unit contains one molecule. As shown in Figure 4, the central nickel ion is six-coordinate, which lies on the inversion center. It adopts a pseudo-octahedral coordination environ-ment, 25 which defined by two nitrogen donors and two oxygen donors from two ligand molecules in the equatorial planet. The sum of the equatorial angles O2-Ni1-N2, N2-Ni1-O2A, O2A-Ni1-N2A and N2A-Ni1-O2 for complex 2 (= 360.00°) is equal to the ideal value (360.00°), which ensures the planarity of equatorial plane. Unlike complex 1, these two newly formed five-membered rings were coplanar, the dihedral angle is 0.00(63)°. The axial positions are occupied by O-atom donors (O1W and O1WA) from two coordinated water molecules. The axial Ni-O average distance (2.064 Å) is shorter than the equatorial Ni-N average distance (2.260 Å), showing the squashed octahedron surrounding the Ni(II) center. The Ni-O and Ni-N distance were all similar to those reported values of Ni(II) complexes. 26 Like complex 1, the adjacent molecules are forming infinite one-dimensional catenulate structure through O1W-H1W1···O1 vi and O1WA-H1W1A···O1A vi (symmetry code: (vi) -x + 2, -y, -z + 1) intermolecular hydrogen bonds ( Figure 5). These contiguous chains stack in a faceto-face fashion in ab plane, the hydrogen bonds exist between the carboxyl group of ligand L and the oxygen atom of coordinated water molecule to form intermolecular O1W-H1W2···O1 vii and O1WA-H1W2A···O1A vii (symmetry code: (vii) x, y + 1, z) hydrogen bonding interaction, leading to the construction of 2D supramolecular sheet in the ab plane ( Figure 6).

Inhibitory Activity Against Jack Bean Urease
The inhibiting urease abilities of the HL and complexes 1 and 2 were studied based on the literature repor-  ted phenol red method against jack bean urease. The results are summarized in Table 4. It was found that comparing to the ase reference acetohydroxamic acid (AHA, IC 50 = 26.99 ± 1.43 μM), copper ion and nickel ion as salt showed IC 50 value of 1.71 ± 0.56 μM and 8.01 ± 1.21μM, while HL and complex 2 exhibited no urease inhibitory activities. Interestingly, compared with recently reported urease inhibition study by others in our group, we found that the urease inhibitory activity of HL was weaker than that of organic compounds synthesized by Sheng and coworkers. 27 However, after coordinated with copper ion, the inhibitory activity improved distinctly. In addition, compared with other antiurease research by coordinated Cu(II) ion (0.14 μM by Sheng, 28 0.46 μM by Wu 29 and 22.40 ± 0.08 μM by You 30 ), complex 1 showed similar or even better activities against urease, which resulted in the improved inhibitory activity. The result indicated that inhibitory activities of metal complexes were influenced by ligand substituents, electronic configurations, and by the nature of metal center. acid (Arg439 and Arg639). The results of the molecular docking indicated that the complex 1 could be well fitted in the active pocket of jack bean urease.

4. Molecular Docking
To find out feasible urea enzyme inhibitors, molecular docking of complexes 1 and 2 with 3LA4 was simulated with the AUTODOCK 4.2 program. Additional interactions have been established in a variety of conformations because of the flexibilities of the amino acid residues of jack bean urease. The results indicate that 1 may have interaction with 3LA4 as reflected by the binding energy of the amino acid residues with the corresponding complex 1 showed -2.22 kcal/mol, while complex 2 showed +46.71 kcal/mol. The optimized cluster (50 occurrences) was ranked by energy level in the best conformation of the inhibitor-urease modeled structures, where the lowest intermolecular energy showed -3.15 kcal/mol.
The binding model of complex 1 with urease (3LA4) is presented in Figure 7 and 8. All the amino acid residues which had interacted with complex 1 were showed. In the binding model, the O atom of 1 as acceptor received one strong hydrogen bonding interaction with Gln635. The hydrogen-bonding distance of Gln635 N-H]O8 was 2.618 Å. In addition, Polar interaction exists between the benzene ring of complex 1 and the amino

Conclusion
This paper reports the synthesis, crystal structures, urease inhibitory activities and molecular docking of two transition metal complexes with 1-(4-fluorophenyl) piperazine acetic acid ligand. The molecular docking and the urease inhibitory activity studies of the complexes against jack bean urease valuably lead to the development of new urease inhibitors. The inhibitory activity tested in vitro against jack bean urease exhibits that complex 1 displays the best inhibitory activity of IC 50 0.15 ± 0.08 μM. Importantly, we only focused on finding more effective and potent urease inhibitors for structure-activity relationship research of the complexes 1 and 2, detailed researches are continuing to explore the toxicity of these complexes of urease inhibitory activity for the environment and humans.