Bis (Diamines) Cu and Zn Complexes of Flurbiprofen as Potential Cholinesterase Inhibitors: In Vitro Studies and Docking Simulations

: Alzheimer’s disease (AD) causes dementia and continuous damage to brain cells. Cholinesterase inhibitors can alleviate the condition by increasing communication between the nerve cells and reducing the risk of dementia. In an effort to treat Alzheimer’s disease, we synthesized ﬂurbiprofen-based diamines (1,2 diaminoethane and 1,3 diaminopropane) Zn(II), Cu(II) metal complexes and characterized them by single-crystal X-ray analysis, NMR, (FT)-IR, UV-Vis, magnetic susceptibility, elemental analysis and conductivities measurements. Synthesized diamine metal complexes appeared in ionic forms and have distorted octahedral geometry based on conductivity studies, magnetic susceptibility and electronic studies. Single crystal X-ray diffraction analysis conﬁrmed (2b) Cu(H 2 O) 2 (L1) 2 (L2) 2 complex formation. Moreover, we tested all synthesized metal complexes against the cholinesterase enzyme that showed higher inhibition potential. In general, copper metal complexes showed higher inhibitory activities than simple metal complexes with ﬂurbiprofen. These synthesized metal complexes may derive more effective and safe inhibitors for


Introduction
Around the globe, 2.5 to 4.0 million elderly people suffer from Alzheimer's disease (AD), manifested by long-term neurodegeneration, loss of neural functions and cognitive abilities that ultimately lead to death [1]. Studies show that numerous disorders, including AD, correlate with high cholinesterase activity. Cholinesterase belongs to the serine hydrolases family, consisting of acetylcholinesterase (AChE) and butyryl-cholinesterase (BChE) [2,3]. Many physiological processes have been controlled by cholinesterase in either a direct or indirect way. Cholesterol overexpression may lead to numerous disorders like ataxia, myasthenia gravis and Parkinson's disease [4,5]. To combat and treat these disorders, researchers have been looking for synthetic and natural inhibitors of cholinesterase. Physostigmine existed as the first cholinesterase inhibitor (ChEI) explored for the cure of AD [6]. Tacrine was the first approved drug for treating AD. Donepezil was permitted in 1996 for the treatment of mild-to-moderate AD. All these compounds, rivastigmine, galantamine, metrifonate, phenserine, physostigmine and tacrine, showed effective inhibition of human AChE and BuChE [7][8][9][10][11][12][13]. To target neuroinflammation and vesicant-induced inflammation, studies have focused on NSAID-AChEI complexes [14][15][16][17]. Current "secondgeneration" AChEIs also demonstrate some side effects such as diarrhoea, nausea, anorexia and vomiting [18]. So far, no proper treatment exists for these neurodegenerative disorders like Alzheimer's disease; hence, researchers have been avidly seeking a proper cure for these neurodegenerative problems.
Currently, several potent inhibitors for cholinesterase exist in the market as first-line treatment for these neurodegenerative problems. However, effective treatment requires more effective inhibitors with safe and extraordinary potential for cholinesterase control, which demands further exploration of anticholinesterase compounds. Recently, organic metal complexes have shown positive effects on these neurodegenerative disorders [19]. For example, Pt(II) complexes with 1,10-phenanthroline ligands can inhibit cholinesterase and reduce Aβ aggregation and Aβ-induced synaptotoxicity [20]. Similarly, previous studies have prepared and analyzed metal complexes of bis (thiosemicarbazone) for cholinesterase inhibition along with a reduction in levels of Aβ aggregations [21].
Some studies showed that Cu or Zn complexes with 8-hydroxyquinoline ligands could induce metal-dependent metalloprotease activity, which degrades Aβ aggregations and ultimately reduces the risk of AD [22][23][24]. Mono-, bis-and tris-diamine conjugated ligands have been found more water-soluble with an enhanced H-bonding network. By using this property, various ligand-metal complexes have been designed with anti-cancer properties [25][26][27][28]. Flurbiprofen-tacrine conjugates and tris-diamine conjugates of flurbiprofen have been reported as cholinesterase inhibitors, where flurbiprofen backbone was found to augment the inhibitory activity [17,29]. Here, we present synthesis and characterization of a relatively new class of flurbiprofen (a propionic acid carboxylate) and its transition metal complexes with diamines (1,2-diaminoethane and 1,3-diaminopropane) as effective cholinesterase inhibitors.

Synthesis of Flurbiprofen Metal Complexes (1a-b)
The potassium salt of flurbiprofen (FLP-K) was prepared by adding 20 mL of 0.01 mol flurbiprofen acid (2.44 g) solution in de-ionized water to 0.01 mol KOH (0.56 g). A dropwise addition of 0.005 mol metal acetate solution (10 mL) to the FLP-K solution with continuous stirring for 20 min in a round bottom flask at room temperature resulted in the formation of the metal-flurbiprofen complex (1a-b) with the characteristic colored precipitates. The precipitates were filtered, followed by subsequent washings with de-ionized water and ethanol. After washing, precipitates were dried at the room temperature.
A white solid product, C 36 H 48 F 2 N 4 O 6 Zn (3a), with a yield of 80%, was synthesized using the method mentioned in Section 2.3. The melting point was observed as 218-220 • C. Analytical calculations show (%): C, 58.73; H, 6.57; N, 7.61, found: C, 57.64; H, 6.55; N, 7.96. 1 The general method given in Section 2.3 was used to obtain the compound C 36

Molecular Docking Simulations
The Three-dimensional structures of human AChE and BChE were obtained from the Protein Data Bank (PDB) with PDB IDs of 4EY4 (X-ray structure with 2.15 Å resolution) and 6ESY (X-ray structure with 2.80 Å resolution), respectively, and attached ligands/water molecules were removed. The Cu-bisdiamine-flurbiprofen complex, 2b (C34H44F2N4O6Cu), was docked against the human AChE and BChE enzymes to map the protein-ligand interactions and binding energies. Cu-bisdiamines and flurbiprofen were also docked individually to calculate the efficacy of 2b. The crystal structure of 2b was used in the docking where the AMBER03 force field and a modified AutoDock-LGA algorithm module were used in YASARA software [31] to perform molecular docking simulations. Binding energies and dissociation constants were obtained by running the 100 simulations for each ligand, where the seed value was set to 1000 [32]. Proteinligand interactions were extracted and mapped using PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. New York, NY, USA) and LigPlus http://www.ebi.ac.uk/thornton-srv/software/LIGPLOT/).

Determination of AChE and BChE Inhibitory Activity
A modified Ellman's method [33] was used to determine the AChE and BChE inhibitory activity of metal complexes [34]. Phosphate buffer (0.1 M KH 2 PO 4 /K 2 HPO 4 , pH 8.0) was used to prepare metal complex samples, enzymes and standard solutions. 0.03 U/mL of AChE and BChE were added to a 10 µL metal complex solution (0-50 µM), and the mixture was incubated for 10 min at room temperature. Then, 25 µL of 1 mM of either ATChI or BTChI was added, and the sample mixture was incubated for the next 15 min followed by the addition of 25 µL of 3 mM DTNB before taking absorbance at Crystals 2021, 11, 208 5 of 14 412 nm using a µQuant microplate spectrophotometer. Microwells with DTNB were used as blank. All reactions were made in triplicate, and the IC50 values were calculated for each sample. Galantamine and donepezil are well-known cholinesterase inhibitors and were used as reference drugs.

Synthesis
In this study, we synthesized transition metal complexes of flurbiprofen with propanediamine (1,3-diaminopropane) and ethylene-diamine (1,2-diaminoethane). Next, we evaluated these metal complexes using magnetic susceptibility, elemental analysis and FT-IR, UV-VIS spectroscopy, conductivity measurements and X-ray analysis, 1 H-NMR, 13 C-NMR. These metal complexes are ionic based on conductivity rearmaments and have distorted octahedral geometry based on electronic studies and magnetic susceptibility studies. Synthesis of these metal complexes caused little to no pollution (green synthesis) because we used harmless solvents like water and ethanol. These metal complexes can only be obtained in water, but for precise crystals, ethanol was also used. Figure 1 illustrates the scheme of synthesis for these metal complexes.
Crystals 2021, 11, x 5 of 14 mixture was incubated for 10 min at room temperature. Then, 25 µL of 1 mM of either ATChI or BTChI was added, and the sample mixture was incubated for the next 15 min followed by the addition of 25 μL of 3 mM DTNB before taking absorbance at 412 nm using a μQuant microplate spectrophotometer. Microwells with DTNB were used as blank. All reactions were made in triplicate, and the IC50 values were calculated for each sample. Galantamine and donepezil are well-known cholinesterase inhibitors and were used as reference drugs.

Synthesis
In this study, we synthesized transition metal complexes of flurbiprofen with propane-diamine (1,3-diaminopropane) and ethylene-diamine (1,2-diaminoethane). Next, we evaluated these metal complexes using magnetic susceptibility, elemental analysis and FT-IR, UV-VIS spectroscopy, conductivity measurements and X-ray analysis, 1 H-NMR, 13 C-NMR. These metal complexes are ionic based on conductivity rearmaments and have distorted octahedral geometry based on electronic studies and magnetic susceptibility studies. Synthesis of these metal complexes caused little to no pollution (green synthesis) because we used harmless solvents like water and ethanol. These metal complexes can only be obtained in water, but for precise crystals, ethanol was also used. Figure 1 illustrates the scheme of synthesis for these metal complexes.

X-ray Crystallography
Among all the simple and bis (1,2-diaminoethan and 1,3-diaminoprpane)-derived metal complexes of flurbiprofen, only Cu(H2O)2(L1)2 (L)2 (2b) yielded blue crystals which were suitable for X-ray analysis. The coordination sphere appears octahedral with a basal plane A (N1/N2/N1 i /N2 i i = 1 − x, − y, 1 − z) around the copper cation in (2b) with two apical O-atoms from two water and four nitrogen atoms from two 1,2-diaminoethane. In the equatorial plane, the copper atom appears in the centre. The Cu-N bonds fall within

X-ray Crystallography
Among all the simple and bis (1,2-diaminoethan and 1,3-diaminoprpane)-derived metal complexes of flurbiprofen, only Cu(H 2 O) 2 (L1) 2 (L) 2 (2b) yielded blue crystals which were suitable for X-ray analysis. The coordination sphere appears octahedral with a basal plane A (N1/N2/N1 i /N2 i i = 1 − x, − y, 1 − z) around the copper cation in (2b) with two apical O-atoms from two water and four nitrogen atoms from two 1,2-diaminoethane. In the equatorial plane, the copper atom appears in the centre.  The crystal contains an infinite polymeric network due to hydrogen bonding of N-H···O, O-H···O and C-H···F with two-dimensional crystallographic base vectors [100], [1] in the plane (0 1 0). The ORTEP diagram of 2b with a 50% probability level and the two-dimensional polymeric network are displayed in Figure 2.

FT-IR Analysis
We also performed an FT-IR analysis of metal complexes for the structural confirmation of metal complexes. IR peaks near 1419-1476 cm −1 suggest that these peaks belong to (aromatic-CH) functional groups. The peaks appearing near the 461-479 cm −1 range reflect (M←O) metal-oxygen bonds formation and peaks near 523-572 cm −1 show the formation of (M←N) metal-nitrogen bonds. IR analysis of the complexes does not show the (M-N) metal-nitrogen peaks for (1a-b) due to synthesis from transition metals and flurbiprofen acid. However, if the addition of (1,3-diaminopropane) and (1,2-diaminoethane) to simple flurbiprofen complexes (1a-b) leads to the formation of (2a-b) and (3a-b), the (M-N) metal peaks appear near 514-580 cm −1 , suggesting the formation of a metal-ligand chelating bond between the nitrogen of 1,2-diaminoethane and 1,3-diaminopropane and metal.

UV/vis and Magnetic Susceptibility
In addition to other spectroscopic techniques, UV-visible analysis was performed to support the complex formation and confirm the symmetry of metal complexes. The symmetry of transition metal complexes was deduced from several peaks observed. For each metal complex, the electronic spectra of 3d-transition metals were recorded in 10

Anticholinesterase Activity
Anti-cholinesterase activities of transition metal complexes of diamines (1,2-diaminoethane, 1,3-diaminopropane) with flurbiprofen (1a-b, 2a-b and 3a-b) are shown in Table 1. We observed that metal complexes of flurbiprofen derived from 1,2-diaminoethane (2a-b) show most potency as a cholinesterase inhibitor, with the lowest IC50 values compare to other series (1a-b and 3a-b). In general, the trend in cholinesterase inhibition follows the order 2a-b >3a-b >1a-b. This sequence indicates that nitrogen-containing transition metal complexes possess more cholinesterase inhibitory potency as compared to simple transition metal complexes. Table 2 shows the inhibitory activities of diamine-based metal in vitro.

Molecular Docking Studies
Molecular docking results are in agreement with in vitro choline esterase activities for the metal complex Cu(H 2 O) 2 (C 2 H 8 N 2 ) 2 (C 15 H 12 FO 2 ) 2 . FLP docking with hAChE shows π-π stacking with Trp 86 and Tyr 124 , while Tyr 337 forms a hydrogen bond (Figure 3a). These interactions look similar to donepezil binding to the PAS (Peripheral Anionic Site), but interestingly Trp 286 does not seem to be involved in FLP binding. Tyr337 is considered an entry point for the CAS (Catalytic Binding Site) [35,36] and it seems here FLP-Tyr 337 interactions hinder the FLP entry deep into the CAS. The bis-diamine metal complex was found to interact with Glu 81 , Trp 86 and Asp 131 . Bound water molecules were found to interact with Glu 81 and Trp 86 . Met 85 interacts with metal ion, while one of the amines from each bis-diamine complex forms H-bonds with Asp 131 (Figure 3b).  The FLP-bis-diamine complex reached the entrance at the gorge of CAS while interacting with Tyr 341 . Non-polar interactions to Tyr 337 , Phe 338 and Phe 295 in the CAS gorge stabilize the complex interactions within the active site (Figure 3c). The complex cannot enter entirely into the CAS gorge; it also loses the interactions with Trp 286 . The bis-diamine metal complex interacts with Glu 392 viz bound water molecule. The interactions within the CAS gorge account for high binding energy and stabilized ligand binding. The Phe 295 interactions, seen here, are also responsible for donepezil inhibition towards hAChE [35,37]. Zephycandidine A and galanthamine have been shown to interact with AChE via Tyr 337 and Trp 286 .
In the case of hBChE, the bis-diamine metal complex shows backbone interactions with Gln 119 , Ser 287 , Leu 286 and Val 288 (Figure 4b). The metal complex is stabilized by Gly 116 , Gly 117 , Gln 119 , Phe 329 and Tyr 332 . FLP interacts with hBChE via π-π stacking to Trp 82 and Phe 329 , while Asp 70 , Ser 79 , Gly 115 , Gly 116 , Ser 198 , Ala 328 , Tyr 332 , Trp 430 and His 438 stabilize the ligand binding (Figure 4a). Here it seems that a highly electronegative fluorine atom in FLP was the reason for its strong interactions with Gly 115 and Gly 116 . Tarcine, an inhibitor of hBChE, has also been found to share the same binding site [38] and its binding is stabilized by π-π stacking with Trp 82 .

Conclusions
Since no proper cure for AD exists, cholinesterase inhibitors such as Donepezil have delayed the progression of AD therapeutically. Second-generation cholinesterase inhibitors, such as rivastigmine and galantamine, have entered the treatment of AD. This study aims to develop novel inhibitors to further advance AD treatment by utilizing the fascinating properties of transition metal complexes [39][40][41]. We have synthesized and characterized Zn(II) and Cu(II) transition metal complexes with flurbiprofen and diamines. All the metal complexes demonstrated distorted octahedral geometry on the basis of electronic spectra and B.M values. Metal complexes, along with 1,2-diaminoethane, 1,3-diaminopropane, and flurbiprofen, show an electrolytic nature, while simple metal flurbiprofen complexes are non-electrolytic. Among all synthesized metal complexes, most of the synthesized metal complexes exhibited elevated cholinesterase inhibitory activity. Copper complexes exhibit the highest activity against the cholinesterase enzyme as compared to zinc metal complexes. Furthermore, bis (1,2-diaminoethane) metal flurbiprofen complexes and bis (1,3-diaminopropane) metal flurbiprofen complexes show better cho- The FLP-bis-diamine complex was found to perfectly occupy the position in the hBChE PAS and CAS gorge. The metal ion was found to coordinate with Glu 238 . Here, interactions with hBChE show that FLP-bis-diamine complex enters the gorge fully, where Phe 357 , Phe 358 and Tyr 396 are involved in π-π stacking. While FLP loses the π-π stacking to Trp 82 , its binding is stabilized by Leu 286 , Val 293 and Asn 396 (Figure 4c). The results suggest that the FLP-bis-diamine complex may be an ideal hAChE and hBuChE inhibitor due to the presence of extensive π-π stacking and hydrogen and hydrophobic interactions. The metal-bound FLP-bis-diamine complex shows stable interactions in comparison to individual the FLP or bis-diamine metal complex.

Conclusions
Since no proper cure for AD exists, cholinesterase inhibitors such as Donepezil have delayed the progression of AD therapeutically. Second-generation cholinesterase inhibitors, such as rivastigmine and galantamine, have entered the treatment of AD. This study aims to develop novel inhibitors to further advance AD treatment by utilizing the fascinating properties of transition metal complexes [39][40][41]. We have synthesized and characterized Zn(II) and Cu(II) transition metal complexes with flurbiprofen and diamines. All the metal complexes demonstrated distorted octahedral geometry on the basis of electronic spectra and B.M values. Metal complexes, along with 1,2-diaminoethane, 1,3-diaminopropane, and flurbiprofen, show an electrolytic nature, while simple metal flurbiprofen complexes are non-electrolytic. Among all synthesized metal complexes, most of the synthesized metal complexes exhibited elevated cholinesterase inhibitory activity. Copper complexes exhibit the highest activity against the cholinesterase enzyme as compared to zinc metal complexes. Furthermore, bis (1,2-diaminoethane) metal flurbiprofen complexes and bis (1,3diaminopropane) metal flurbiprofen complexes show better cholinesterase inhibition as compared to simple metal flurbiprofen complexes. In conclusion, the diamines synthesized in this study show potential in AD treatment as novel cholinesterase inhibitors.