Synthesis, in vitro biological screening and docking study of benzo[d]oxazole bis Schiff base derivatives as a potent anti-Alzheimer agent

Abstract We have synthesized benzo[d]oxazole derivatives (1–21) through a multistep reaction. Alteration in the structure of derivatives was brought in the last step via using various substituted aromatic aldehydes. In search of an anti-Alzheimer agent, all derivatives were evaluated against acetylcholinesterase and butyrylcholinesterase enzyme under positive control of standard drug donepezil (IC50 = 0.016 ± 0.12 and 4.5 ± 0.11 µM) respectively. In case of acetylcholinesterase enzyme inhibition, derivatives 8, 9 and 18 (IC50 = 0.50 ± 0.01, 0.90 ± 0.05 and 0.3 ± 0.05 µM) showed very promising inhibitory potentials. While in case of butyrylcholinesterase enzyme inhibition, most of the derivatives like 6, 8, 9, 13, 15, 18 and 19 (IC50 = 2.70 ± 0.10, 2.60 ± 0.10, 2.20 ± 0.10, 4.25 ± 0.10, 3.30 ± 0.10, 0.96 ± 0.05 and 3.20 ± 0.10 µM) displayed better inhibitory potential than donepezil. Moreover, derivative 18 is the most potent one among the series in both inhibitions. The binding interaction of derivatives with the active gorge of the enzyme was confirmed via a docking study. Furthermore, the binding interaction between derivatives and the active site of enzymes was correlated through the SAR study. Structures of all derivatives were confirmed through spectroscopic techniques such as 1H-NMR, 13C-NMR and HREI-MS, respectively. Communicated by Ramaswamy H. Sarma


Introduction
Alzheimer's disease (AD) is identified by a progressive, irreversible disorder of the brain, which progressively damages a person's skills like memory and thinking ability (Prince et al., 2016). It was estimated that AD acts as the sixth leading cause of mortality in the US. Moreover, it was figured out that almost 50 million people are suffering globally from this disease (Davies & Maloney, 1976;Perry et al., 1978). In the clinical approach for AD medication, galantamine, donepezil, and rivastigmine are employed, predominantly regulating the cholinergic system in the synaptic cleft by inhibiting cholinesterase. There are two types of cholinesterase in the human brain: acetylcholinesterase (AChE) and butyrylcholinesterase, where it can share 50% of the amino acid sequence with the diameter of 20 A (Chatonnet & Lockridge, 1989). Moreover, different structural features give substrates specificity due to different sequences of amino acids in the active site (Colovic et al., 2013;Lane et al., 2006;Taylor & Radic, 1994).
Furthermore, a detailed study about acetylcholinesterase showed that there are two subunits named catalytic anionic site (CAS) and peripheral anionic site (PAS) in the active site gorge (Dvir et al., 2010;Sussman et al., 1993). The peripheral anionic site (PAS) regulates the consequence of AChE allosterically; this clearly indicated that (PAS) play a role in amyloid-beta (Ab) protein aggregation, respectively (Inestrosa et al., 1996;Selkoe, 1999). Similarly, butyrylcholinesterase (BChE) was found in the blood plasma and a minor amount in the brain. Therefore, it was found that BChE is predominantly involved in cholinergic medication (Huang et al., 2007;Mesulam et al., 2002). Additionally, AD becomes severe as the activity of BChE increase with age compared to AChE (Perry et al., 1978). Therefore, in the beginning, AChE was taken into consideration to cure AD. Still, today many attempts have been found to inhibit AChE and BChE based on promising results about the treatment of AD (Lane et al., 2006). In this regard, many synthetic and medicinal chemists have reported various dual inhibitors for AD treatment (Akincioglu & Gulcin, 2020;Anand & Singh, 2013;Arslan et al., 2020;Cavdar et al., 2019;Kazancioglu & Senturk, 2020;Nuthakki et al., 2019;€ Ozil et al., 2019;Sharma, 2019;Wu et al., 2020;Zilbeyaz et al., 2018).
We have already reported thiazole and benzimidazole derivatives Rahim et al., 2015) as inhibitors for AChE and BChE, respectively. In addition, Benzoxazole derivatives have also been explored for anti-Alzheimer studies (Chigurupati et al., 2016). The basic skeletons of benzimidazole and Benzoxazole are the same, only differ by one atom. These results encourage us to further develop the modified benzoxazole-based moieties in search of a potent anti-Alzheimer agent. Therefore, we designed and synthesized benzo[d]oxazole derivatives (1-21) (see Scheme 1) and screened all derivatives for AChE and BChE inhibitory activity in search of a potent molecule ( Figure 1).

Chemistry
In the continuation of our efforts for the synthesis of bioactive compounds (Alomari et al., 2019;Seraj et al., 2021;Taha et al., 2020;Wahid et al., 2020). Benzo[d]oxazole-2-thiol as intermediate (I) was synthesized via mixing 2-aminophenol and CS 2 in ethanol and a catalytic quantity of NaOH, and the mixture was refluxed for 3-4 hrs. After completion of the reaction, the solvent was evaporated, and the resulting intermediate (

Acetylcholinesterase inhibitory potential
The screening result about inhibition of derivatives against acetylcholinesterase enzyme showed that practically all derivatives of the series displayed very good potential compared to the standard drug donepezil (See Table 1). The extent of inhibition is 50% inhibitory concentration (IC 50 ), which the derivatives have exhibited ranging between 0.3 to 26.20 mM compared to standard drug donepezil (0.016 ± 0.12 mM). Many of the derivatives like 5, 6, 8, 9, 10, 13, 15, 18, and 19 showed better inhibitory potential; in this regard, derivative 18 (IC 50 ¼ 0.3 ± 0.05 mM) showed very potent inhibitory activity. The varied inhibitory activity showed by derivatives clearly indicates that substituting phenyl B/C ring greatly influences the inhibitory activity because all the derivatives have the same basic skeleton. Still, they are different from each other via substitutions on phenyl ring B/C. The analog 18 is the most active one among the series, and its phenyl ring B bear 2-OMe/5-OMe substitution and phenyl ring C bears 2-OH/3/5-Cl substitution, respectively. It indicated that OMe-groups and Cl-groups activate both phenyl ring B/C for dipole-dipole interactions through electrondonating effects. Moreover, OH-group at two positions on phenyl C provides a decent opportunity for hydrogen binding interaction, therefore exhibited the most potent acetylcholinesterase activity (IC 50 ¼ 0.3 ± 0.05 mM). The compounds 5, 7-10, 13, and 15, having chlorine atom/atoms in ring C showing good activities due to the high electronegative of chlorine and having variation in activity due to changes in ring B. the compounds having nitro 1, 2, 3, 16 and 17 in ring C showed intermediate activities as compared to chlorinated compounds. The compounds 7 and 20 having anthracene as ring C showed weak activities due to the non-polar behavior of the anthracene ring ( Figure 2).

Butyrylcholinesterase inhibitory potential
Almost all derivatives of the series displayed very decent butyrylcholinesterase inhibitory potential compared to standard drug donepezil (See Table 1). Some of the derivatives like 6, 8, 9,10,13,15,18, and 19 emerged with more significant butyrylcholinesterase inhibitory potential than standard drug donepezil (IC 50 ¼ 4.5 ± 0.11 mM). The inhibitory potential of the current work was found much better than our previously reported work. Herein this work, derivatives 18 (IC 50 ¼ 0.96 ± 0.05 mM) displayed the most potent inhibition among the series compared to donepezil.
Regarding the inhibitory activity profile of the derivatives, all derivatives displayed asymmetrical inhibitory potential because they differed from each other by substituting phenyl ring B/C. The detailed study about their potentials is summarized via structure-activity relationship, respectively.
Derivative 18 is the most active one among the series, and its phenyl ring B bear 2-OMe/5-OMe substitution and phenyl ring C bears 2-OH/3/5-Cl substitution, respectively.
Thus, it clearly indicates that OMe-groups and Cl-groups activate both phenyl ring B/C for dipole-dipole interactions through electron-donating effects. Moreover, OH-group at two positions on phenyl C provides a decent opportunity for hydrogen binding interaction, therefore exhibited the most potent acetylcholinesterase and butyrylcholinesterase activity (IC 50 ¼ 0.3 ± 0.05 and 0.96 ± 0.05 mM).    SAR studies showed that the elimination of electrondonating group from phenyl ring directly suppresses the potential of the derivatives. By this consideration, derivative 5 has 4-phenyl substitution on phenyl ring B, and 2-OH/3/5-Cl substitution on phenyl ring C exhibited less inhibitory activity (IC 50 ¼ 1.20 ± 0.10 and 5.20 ± 0.10 mM) as compared to derivative 18. Decreasing the inhibitory activity of derivative 5 directly correlates with the electron-donating group because derivative 5 has no OMe-groups on phenyl ring B like in derivative 18, which have OMe-groups on phenyl ring B. This showed that the prompting extent of derivative 5 toward the active site is fewer than derivative 18 and therefore displayed less inhibitory activity.
Positions of substitution on phenyl ring have their effect on the inhibitory activity of the derivatives. This might be due to the structural alignment of the derivatives with active site residues in binding interaction. Due to this declaration, it was found that derivative 9, in which phenyl ring C comprises of 2/4-Cl substitutions, showed better inhibitory result (IC 50 ¼ 0.90 ± 0.05 and 2.20 ± 0.10 mM) than derivative 10 (IC 50 ¼ 2.70 ± 0.10 and 4.40 ± 0.20 mM) bearing the identical Cl substituents at 3/4 position on phenyl ring C. This clearly illustrates that the position of substituents in isomer plays a vital role in binding interactions. Furthermore, different substituents have different volumes and geometry, and for this reason, derivatives displayed varied inhibitory activity from each other.
It was found that derivative 7 is the least active one among the series compared to other derivatives. This is because there are no substituents on phenyl ring B and C, and by this motive, it only favors pi-pi interaction with the enzyme's active site. It might be due to this reason that derivative 7 displayed the least inhibitory activity (IC 50 ¼ 26.20 ± 0.40 and 37.60 ± 0.50 mM).

Molecular Docking
The experimental acetylcholinesterase inhibition of benzo[d]oxazole bis Schiff base derivatives is displayed in Table 1. From the obtained IC 50 values, it appears that the acetylcholinesterase inhibition of benzo[d]oxazole bis Schiff base derivatives may strongly be affected by the type, number, and positions of the substituted functional groups at the aromatic ring R and R1 of the basic skeleton (Table 1 and Scheme 1). To understand the observed enzymatic inhibition of the synthesized derivatives and the effects of type, number, and positions of the substituted functional groups on acetylcholinesterase inhibition, molecular docking has been performed out to determine the binding modes between the synthesized derivatives from one side and the active site residues of the acetylcholinesterase from another side. Table 2 summarized the calculated binding free energies of the stable complexes' ligand-acetylcholinesterase, the number of established intermolecular hydrogen bonds between the synthesized compounds, and active site residues of acetylcholinesterase, the closest residues to the docked compounds, and their IC 50 values.
All the selected docked compounds fit well into the binding site of acetylcholinesterase, which forms stable complexes with its active site residues with negative bending energies. The negative binding energies may indicate that the acetylcholinesterase inhibition by the selected benzo[d]oxazole bis Schiff base derivatives is a thermodynamically favorable process (Table 2). However, the binding energies of the stable complexes vary moderately, with a maximal variation of 2.7 kcal mol À1 in respect to the most stable complex. Thus, the binding energy of complexes may not be used as a determinant property to distinguish between the observed acetylcholinesterase inhibitions of the tilted compounds. Hence, our focus will be on the effect of the substitute functional groups on the subfamilies (1-3), (5, 9-10 and 12), (8, 13, 15 and 18), and (6 and 19). The compound 1-3 are mono substituted by the nitro functional group at the ortho, meta, and para position of the R1 aromatic ring (Scheme 1). Therefore, the nitro group position has a relatively strong effect on the acetylcholinesterase inhibition. Indeed, at the ortho position, the nitro group shows no interaction with the active site residues into the binding site of acetylcholinesterase.
In contrast, substituted at meta and para positions, the nitro group interacts through hydrogen bonds with amino acids of acetylcholinesterase ( Figure 3). This may explain the  higher acetylcholinesterase inhibition of 2 and 3 compared with 1. One also may relate the higher acetylcholinesterase inhibition of 2 compared to 3 to the number of hydrogen bonds established with the former compared with the latter ( Figure 3). Indeed, the nitro group at meta position formed two strong hydrogen bonds with PHE 295 and ARG 296 of 2.90 and 3.19 Å distances, respectively ( Figure 3).
In the subfamily of compounds 5, 9, 10, and 12, the aromatic ring R1 is substituted by the mono chloro group (12), dichloro groups (9 and 10), and dichloro and hydroxyl groups (5). Their corresponding energies vary slightly with a maximum variation of 0.37 kcal mol À1 . The higher acetylcholinesterase inhibition of 5 compared to 9, 10, and 12 may refer to the number of interactions established between chlorines and hydroxyl functions of the former with the active amino acids of acetylcholinesterase compared with the interactions formed with the latter (Figure 4). Similar behaviors were observed for the subclasses of compounds (8, 13, 15 and 18) and (6 and 19). (Table 2).
The superposition of the best conformations of the docked benzo[d]oxazole bis Schiff base derivatives and standard drug donepezil into the binding site of acetylcholinesterase is shown in Figure 5. The interactions of the reference drug donepezil into the active site of acetylcholinesterase and the superposition between the original ligand donepezil and the docked one are shown in Figure 5. The re-docking of the original ligand donepezil into the active site of acetylcholinesterase is well reproduced. The binding energy of donepezil-acetylcholinesterase is À11 kcal mol-1, which explains the ability of the donepezil to inhibit acetylcholinesterase. The keto group of donepezil showed a strong hydrogen bond interaction with the amino acid Phen B:295 at a distance of 2.81 Ð. Other interactions are carbon-hydrogen, p-r, p-p stacked, and p-Alkyl types ( Figure 5). Experimentally, the donepezil displays higher acetylcholinesterase inhibition compared with the synthesized compounds. As mentioned above, the synthesized compounds and the reference drug donepezil have different basic skeletons. Thus, the binding energy of the stable complexes ligand-receptor may not be used as a strong descriptor to distinguish between their observed acetylcholinesterase inhibition. Indeed, donepezilacetylcholinesterase binding energy is less than those of the selected compounds by 1-2 kcal mol À1 2.6. Molecular dynamics simulation 2.6.1. Inspection of stability of bound C18 with AChE protein The molecular docking study revealed a fit-well pattern of binding by all the compounds in the active site, but solely differences observed in their mode of adopting interactions with active site residues of the AChE protein. Furthermore, the molecular dynamics (MD) simulation study was conducted for potent C18 in the series to illustrate the stability of C18 in complex with AChE protein. Subsequently, it was observed that the AChE þ C18 showed an energetic stabilized behavior in the binding site by adopting intermolecular interactions with active site residues.
The root means square deviation (RMSd) was calculated based on the initial backbone coordinates of the protein-ligand complexes to evaluate the possible deviation in the structure during simulation. The RMSd of AChE bound with C18 relative to the original structures shows that the simulation time of a total of 50 ns is appropriate to reach equilibration at temperature 310 K.
We have observed a gradual increase in the RMSd curve till 10 ns, then oscillated and steady till 50 ns, as shown in Figure 6a. This point deviation pins the point of the potential impact of C18 on the protein structure, which indicates that this compound resides in a good pattern in the active site, resulting in the overall stability of the protein complex.
Moreover, carbon alpha distance analysis was conducted to explore the binding pattern concerning MD simulation time. Subsequently, it was observed that, initially, the distance among C18 and the active site residues remain high till 15 ns, but gradually it decrease with respect to simulation time to 50 ns ( Figure 6b). interestingly, we have noticed that the initial gradual increase is due to the attached R group, which moves in an arbitrary direction, and is finally steady after 15 ns simulation time. Overall, the MD simulation results indicate and witness the stable behavior of C18 in the active site of AChE protein and could be a good candidate against the target.
Placed the hot solution of the reaction mixture in an ice  bath, stirred at 10-14 C for 30 minutes, and then left until crystal formation to achieve pure intermediate (II).

Assay protocol for acetylcholinesterase inhibitory activity
The in vitro AChE inhibitory activity was measured using the methods described earlier (Chigurupati et al., 2016;Zaman et al., 2019). Briefly, stock solutions (1 mg/mL) of test compounds were prepared using DMSO. Working solutions (1-100 lg/mL) were prepared by serial dilutions. The various concentrations of test compounds (10 mL) were pre-incubated with sodium phosphate buffer (0.1 M; pH 8.0; 150 mL), and AChE (Human) solution (0.1 U/mL; 20 mL) for 15 min at 25 C. The reaction was initiated by adding DTNB (10 mM; 10 mL) and ATChI (14 mM; 10 mL). The reaction mixture was mixed using a cyclomixer and incubated for 10 min at room temperature. The absorbance was measured using a microplate reader at 410 nm wavelength against the blank reading containing 10 mL DMSO instead of the test compound. The % inhibition was calculated using the formula described in Eq.
% Inhibition The IC 50 (half maximal inhibitory concentration) was calculated by constructing a non-linear regression graph between % inhibition vs. concentration, using Graph Pad Prism software (version 5.3).

Assay protocol for butyrylcholinesterase inhibitory activity
The in vitro BChE (Human) inhibitory activity was measured using the same methods as described above with slight modification. Herein this assay, BChE solution was used instead of AChE solution (Mansha et al., 2021;. Moreover, for percent inhibition, the same equation was used as given above in acetylcholinesterase assay protocol, respectively.

Molecular docking study
The binding modes between the selected benzo[d]oxazole derivatives and the active site residues of acetylcholinesterase have been investigated through Autodock 4.2.6 software (Morris et al., 2009). The acetylcholinesterase and its original docked donepezil geometries were downloaded from the RCSB data bank website (PDB code 4EY7) (Cheung et al., 2012). The active site is identified based on the co-crystallized receptor-ligand complex structure of acetylcholinesterase. The re-docking of the original ligand donepezil into the active site is well reproduced with an RMSD value of 0.63 Å. Molecular geometries benzo[d]oxazole derivatives were minimized at Merck molecular force field 94 (MMFF94) level44 and saved as PDB files. Docking study is performed using Lamarckian genetic algorithm, with 500 as a total number of runs for binding sites for original ligand the synthesized derivatives. In each respective run, a population of 150 individuals with 27000 generations and 250000 energy evaluations was employed. Operator weights for crossover, mutation, and elitism were set to 0.8, 0.02, and 1, respectively. 2 D and 3 D binding interactions between the docked benzo[d]oxazole derivatives were visualized using Discovery Studio Client (Discovery Studio Client is A Product of Accelrys Inc., San Diego, CA, USA).

Molecular dynamics simulation
Furthermore, compound 18 (C18) from a docked complex with acetylcholinesterase was parameterized using the generalized Amber force field (GAFF) (Wang et al., 2004). GAFF atom types were assigned using the Antechamber module (Wang et al., 2006), and the parameter file was prepared with the tLEaP module implemented in AMBER. Total a system was constructed for molecular dynamic (MD) simulation; AChE protein in the presence of docked C18. All-atom MD simulations and essential dynamics analysis were conducted in AMBER version 2018 (Case et al., 2018). First, the tLEaP module integrated the hydrogen atoms into the crystallographic structure (PDB code 4EY7) (Cheung et al., 2012). Next, the counter ions were added to maintain the overall system neutrality. Then the system was solvated in a truncated octahedral box of the TIP3P water model with a cutoff 8.0 Å buffer. The Particle Mesh Ewald (PME) method was used to treat long-range electrostatic interactions (Darden et al., 1993). The ff14SB forcefield was used for all MD simulations (Maier et al., 2015). The SHAKE algorithm with a tolerance of 10-5Å was applied to constrain all covalent bonds involving hydrogen atoms (Ryckaert et al., 1977). The PMEMD CUDA version was used to accelerate the MD simulation. The steepest descent method was used to minimize the solvated systems for 2000,0 steps, then heating for 400ps, and equilibration in the NVT ensemble for 200ps. According to Langevin's algorithm, the temperature and pressure were coupled with a time constant of 1.0ps, isotropic position scaling, and a relaxation time of 2.0ps (Wu et al., 2016).

Conclusion
We have synthesized benzo [d]oxazole bis Schiff base derivatives (1-21) for anti-Alzheimer activity herein this work. Alteration in the basic skeleton within the structure of the derivatives is brought by using various aromatic aldehydes in the last step of this protocol. With the hope to find a potent anti-Alzheimer agent, in this regard, all derivatives were evaluated for acetylcholinesterase and butyrylcholinesterase with respect to donepezil act as a standard drug. Almost all derivatives displayed very decent potentials, but derivatives 18 emerged as the most potent one among the series. Furthermore, this study showed that both electron-donating/ electron-withdrawing ability of substitutions on phenyl ring B/C proved its potential in activating the phenyl ring. Moreover, OH-group as a substituent on phenyl ring further provides the chance for hydrogen binding interaction as hydrogen donor and hydrogen acceptor toward the enzyme's active site, respectively.