Design, Synthesis, Biological Activity, and Molecular Modeling of Novel Spiroquinazoline Derivatives as Acetylcholinesterase Inhibitors for Alzheimer Disease

Abstract The p-toluene sulfonic acid (p-TSA) catalyzed cascade ring closing transformation has been executed for the preparation of novel spiroquinazolinone compounds 4 and 5 by the reaction between anthranilamide and cyclohexanone followed by subsequent acylation. These molecules were then examined against the inhibitory activity of Acetylcholineterase (AchE). The tested compounds revealed moderate anti-AChE activity of IC50 values ranging from 46.675 to 14.256 µM). The described results lead toward the development of compounds 4b and 5c having promising anti-AChE activities with IC50 values at the micromolar level. The docking study suggests that these hybrid spiroquinazolinone scaffold might facilitate the further development of investigated compounds as anti-Alzheimer agents.


Introduction
The quinazoline and quinazolinone derivatives are well regarded as notable pharmacophores for the syntheses of a series of significant and useful pharmaceuticals.][6][7][8][9][10] Due to their precedential and recognized potency as cholinesterase, inhibitor, and antioxidant effects, the phenolic compounds could lead toward the promising structure in the designing of novel AChEIs.In designing our compounds, the phenol pharmacophore was employed in the design strategy to demonstrate the potent spiroquinazolines with possible enhanced AChEI activities.
On the other hand, the acetylcholinesterase (AChE) is a serine protease which plays a decisive role in the hydrolysis of the neurotransmitter, acetylcholine (ACh), into acetate and choline. 11,12ccordingly, AChE has been studied effectively as main target of anti-Alzheimer's disorders.4][15][16][17][18] The reported cholinergic hypothesis suggests the rapid degradation of ACh by AChE and decreases its concentration level in the brain which is responsible for this disease.Therefore, to conserve learning capacity and brain memory functions, the AChE inhibitors require to be exercised in order to regulate the ACh's degradation. 19he drug Donepezil or the brand name Aricept is one of the most effective pharmaceuticals that targets Alzheimer's Disease. 13The computational studies of the inhibition of AChE have revealed a series of effective donepezil analogs embedded with quinolinodonepezil and donepezilpyridyl scaffolds. 20In addition, the quinazoline is an immense pharmacophoric scaffold which is witnessed as AChE inhibitor to treat Alzheimer's Disease. 21Previously, Daoud et al. highlighted the importance of hydrogen bonding interaction of pyrazinamide derivatives with Tyr121 amino acid residues during multi-pronged computational approach on AChE, suggesting that these derivatives are AChE effective inhibitors. 22Figure 1 depicts AChE (top) and (bottom) from the active-site gorge.The peripheral aromatic site is located in the top near the mouth of gorge and plays an obvious role in ligand binding. 23,24Moreover, it interacts with multiple loops and surrounded by several subdomains around the active site at the surface of the protein, including two 'bottleneck', which is the narrowest part of the gorge and mostly contributed by Phe330 and Tyr121 amino acid residues in the middle of the main gorge (Figure 1). 25 The substrates and inhibitors can relocate from the surface to arrive the active site in deeper regions of the gorge, which is known as the 'breathing' of the enzyme, and that is considered as an important motion in order to enlarge the gorge radius to cross over the 'bottleneck'. 24On the other hand, the catalytic active site of AChE includes three amino acid residues, such as 'catalytic triad' which is responsible for the degradation of AChE. 26The catalytic triad is buried at the base of a deep narrow gorge of $20A˚and hence, it is not easy to access. 27Therefore, the search of AChE inhibitors that can concurrently bind to its catalytic and peripheral aromatic binding sites has been witnessed as a desirable research area.Molecular docking is one of the most important methods used to study ligand-protein interactions.It describes the binding position and energy of the macromolecule-ligand complex and gives the best-docked pose with the minimum binding energy. 28Thus enabling a researcher to analyze the complex system's binding pocket and interacting residues. 29gure 1.Backbone of AChE in complex with E2020, Aricept drug, PDB ID: 1EVE.The active-site gorge of AChE is highlighted in grey molecular surface.Two 'bottleneck' residues, Tyr121, and Phe330, are shown as sticks.The important subdomains surrounding the top and the bottom of the active-site gorge are highlighted with yellow (the X-loop, residues 67-94), green (S1, residues 114-150), cyan (S2, residues 225-296), blue (S3, residues 324-400), and magenta (S4, residues 428-450).
Due to the remarkable biological properties of spiro-cyclohexylquinazolinone and in view of the limited number of synthetic available compounds of this class of heterocycles, there is a strong demand for the development of new derivatives.Straightforward approaches using readily available starting materials are preferred.
In this report, the facile syntheses of phenolic derivatives of 1'H-Spiro[cyclohexane-1,2 0 -quinazolin]-4 0 (3'H)-one 4 and 5 have been described, and their inhibition potential of AChE enzyme was investigated along with the detailed in-silico studies.These compounds might show promising anti-AChE activities, since they possess a phenol chromophore on the spiro-cyclohexylquinazolinone scaffold.

Synthesis of 1'H-Spiro[cyclohexane-1,2'-quinazolin]-4'(3'H)-one (1)
The earlier procedures reported by Revathy and Lalitha were adopted for the preparation of compound 1. 30 In which 20 mol% of p-TSA (172.2 g/mol) was added to 1:1 mixture of anthranilamide (1 mmol, 136.15 g/mol) and cyclohexanone (1 mmol, 98.15 g/mol) in 30 mL of absolute ethanol and the reaction mixture was refluxed for 10 min.The reaction mixture was cooled to room temperature.Finally, the precipitate was filtered on a suction pump which was thoroughly washed with ethanol and dried, then recrystallized in ethanol to yield compound 1 as a white powder in 98%.

In vitro inhibition studies on AChE
AChE inhibitory effects of the newly synthesized compounds were examined by Ellman's test. 28n brief, 1700 mL of 50 mM of Tris-HCl buffer of pH around 8.0 and 250 lL of test compounds 4 and 5 at the concentrations ranging from 25 to 400 lg mL À1 , then 20 mL of 10 mM of 5,5 0 -dithiobis-2-nitrobenzoic acid (DTNB) and 10 lL 6.66 mmL À1 AChE were added.The control drug galantamine was used by dissolving in 50 mM Tris-HCl buffer at pH ¼ 8.0.The final mixtures were incubated for 15 min at 37 C, then, 10 lL of acetylthiocholineiodide (200 mM) in buffer solution was added to the mixture.The absorbance of this mixture was measured using the spectrometer at k ¼ 412 nm in every 10 s for 3 min and the blank measurement was performed using the buffer solution.The enzyme inhibition (%) was found from the rate of absorbance changes over time (V¼Abs/Dt) as follows: The measurements were conducted in triplicate.The concentrations of the test sample that inhibit the hydrolysis of the ACh by 50% (IC50) were found by regression analysis between the inhibition percentages against the compounds concentration.

Preparation of ligand and protein structure
The X-ray crystal structure of AChE, PDB ID: 1EVE was retrieved from the Protein Data Bank (www.rcsb.org).The substrate, Aricept, drug bound to AChE was removed from the crystal structure.Prior to docking calculations, hydrogen atoms were added to the proteins.The missing residues of the proteins were fixed.Grid box utility in AutoDock version 4.2.6 was used to select the active site of the co-crystal ligand within the protein structure. 32The 3D structure of the quinazolinone ligands 4 and 5 were built using Gaussview program, 33 and the energy minimization for the ligands were performed using semi-empirical PM3 method available in Gaussian 2003. 34,35lecular docking Docking calculations were performed using a Lamarckian Genetic Algorithm available in Autodock version 4.2.6. 36Docking simulation were applied for the Donepezil bound to the AChE to ensure its accuracy in predicting the binding site, binding conformation, and affinities.The receptor and the ligands were prepared using Autodock Tools program.Then, the Gasteiger partial charges were computed for each molecule and rotatable bonds were defined as free for the ligands and rigid for the receptor.The grid spacing was set at 0.375 Å.In this study, the docking runs were set to terminate after a maximum of 25 Â 106 energy evaluations, a maximum number of 27,000 generations, a mutation rate of 0.02 and the population size was set to use 300 randomly placed individuals.A total of 100 independent docking runs were carried out for docking system.The best possible conformations were then selected and analyzed using Chimera and Discovery Studio 4.0 and programs. 37,38

Chemistry
The plausible mechanism for the formation of spiroquinazolinone (1) has been represented in Figure 3.At first, the protonation of cyclohexanone (A) using p-toluene sulfonic acid may occur, which followed by the addition of anthranilamide (B) leading to the formation of an imine (D).The cyclization reaction occurs through the attack of the amide -NH 2 group on the double bond of imine to form spiroquinazolinone (1) after the H þ shift (Figure 3).
In the second step, the formation of the final amide compound was accomplished by transferring the corresponding carboxylic acid to the acyl derivative and followed by the nucleophilic attack of the spiroquinazolinone (1).
As a spectral evaluation, in 1 H NMR, the amide protons were seen in the range of d¼7.91-8.43ppm as expected.The broad peak at d ¼6.64 ppm was assigned for the amine proton in spiroquinazolinone (1), which disappeared in compounds 4 due to the formation of the amide bond.
The vinylic protons (-HC¼CH-) of compounds 5 exhibited J values in the range of 15.6-15.9Hz, which indicates that all the vinylic derivatives embedded with trans-isomer.The carbonyl peak of the compounds in 13 C NMR was realized in the range of 164.03-167.30ppm.The carbon peak of the spiro-carbon was appeared in the range of 55.37-71.89ppm, while the carbon signals of methylene were found in the range of 20.41-41.23 ppm.
The calculated and measured elemental analyses of the compounds were found compatible.Further, calculated and measured m/z values of the compounds were also found compatible in GCMS analysis.

Enzyme inhibition
The inhibitory activities of the novel spiroquinazolines derivatives against AChE in vitro studies were assessed and expressed as IC 50 values, and these are reported in Table 1.Using Ellman's technique and Galantamine as a reference drug, the potency of compounds 4a-5d to suppress AChE activity was evaluated.

Molecular modeling
Molecular docking of donepezil, E2020, substrates and the synthesized compounds 2a-3d were examined with AChE binding site to understand the ligand-protein interactions and free binding energies at atomic level.As shown in the docking scores (Table 2), compounds 4b and 5d were found most potent derivatives reported in this work with binding free energies of À10.55 and À10.71 kcal mol À1 , respectively.The fused spiro-cyclohexylquinazolinone scaffolds coupled with aryl moieties in derivatives 4 are smaller in comparison with derivatives 5, thereby we expected that its binding poses in the AChE active site gorge would be different and reveals some common pharmacophoric properties with each other, as well as with the AChE-donepezil.
The spiro-quinazolinone ring confers to all derivatives a molecular structure that allows a nice fit of the alicyclic rings within the AChE's gorge.The derivatives 4a-d with the small tails are embedded between the amino acid residues Ser286-Arg289 and interacting with the upper part of the gorge as shown in Figure 4(a).In contrast, the phenyl moiety in derivatives 5a-d are located deeply in the bottom of the gorge and stacks against Trp84 involving p-p interactions.Furthermore, the overlay structures docked pose of the synthesized compounds at the AChE binding site with its original crystal structure of donepezil, E2020, is shown in Figure 4(b).It can be observed that the compounds donepezil located between the two derivatives and have the same binding mode.
In Figure 5, the docking results revealed that all derivatives of compounds 4 and 5 have the same binding mode except 4a and 5b, grey and green, respectively.In addition, near the bottom of the AChE's gorge, the OH group from the phenyl ring in derivatives 5a-d form H-bonds interactions with the Ser124 and Trp84 with distances 2.80 and 2.52 Å, respectively.In contrast, the -OH groups from the phenyl ring in derivatives 4 were located pointing out to the Ser286 and Arg289 and form H-bonds interactions with distances about 1.76 and 1.71 Å, respectively.
Docking simulation of derivatives 4a-d showed that the phenyl and alicyclic moieties interact via hydrophobic interactions with Leu282, Tyr70, Tyr121, Tyr334, Trp279, Ile287, Phe288, Phe290, Phe330, Phe331 as shown in Figure 6(a).While, the hydrophobic regions in derivatives 5a-d interact with many hydrophobic amino acids residues around the bottom of the active site gorge as shown in Figure 6(b).Meanwhile, both derivatives behave the same interactions with the two 'bottleneck' residues, Tyr121 and Phe330.The docking results of the newly synthesized spiro-cyclohexylquinazolinone derivatives complexed with AChE receptor show that the main factor that contributes to the stabilization of the receptor-inhibitor complexes is hydrophobic interactions along with the hydrogen bond formation of the   important bottleneck Tyr121 and Phe330 key residues.In view of all that has been mentioned so far, one may suppose that the spirocyclohexylquinazolinone-attracting scaffold is required for the activity.Figure 7 shows an overview of the relationships between the chemical structures and the target property of studied compounds.However, more research on this topic needs to be undertaken before the association between the spirocyclohexylquinazolinones and AChE active site is more clearly understood.This study suggests that the development of these hybrid spiroquinazolinone scaffold may worth exploring for the preparation of new anti-Alzheimer agents.

Figure 2 .
Figure 2. Synthesis of spiro compound with acyl aromatic compounds.

Figure 3 .
Figure 3. Proposed mechanism for preparation of compound 1.

Figure 4 .
Figure 4. Super positions of the docked poses (a) Derivatives 4 and 5 are embedded in front of Ser124 and Ser286, respectively.(b) Donepezil, yellow color, over lied with derivatives 4 and 5 in the active site of the AChE, PDB ID: 1EVE.

Figure 5 .
Figure 5.The docking poses of derivatives 4a-5d in the AChE binding pocket.The important hydrogen bond and amino acid residues involved in the polar and non-polar interactions are highlighted.

Figure 6 .
Figure 6.Two-dimensional schematic representations of the protein-ligand interactions.(a) 2d-AChE complex; (b) 3d-AChE complex.The important amino acid residues involved in the polar and non-polar interactions are highlighted.

Figure 7 .
Figure 7.The intercorrelations among the spirocyclohexylquinazolinone scaffold and the important amino acid residues in the AChE active site.

Table 2 .
Docking score for 4 and 5 derivatives and E2020 substrate with AChE.