Design , synthesis , and biological evaluation of 7 H-thiazolo [ 3 , 2-b ]-1 , 2 , 4-triazin-7-one derivatives as novel acetylcholinesterase inhibitors

The docking study on a novel series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives with acetylcholinesterase from Torpedo californica has demonstrated that the ligands bind to the dualsite of the enzyme. The synthesis and characterization of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives was described. The crystal structure of 6-benzyl-3-{4-[2-(1-piperidinyl)-2oxoethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one has been characterized by X-ray diffraction. All target compounds have been screened for their efficacy as acetylcholinesterase inhibitor. The study of AChE inhibitory activity was carried out using the Ellman colorimetric assay with huperzine-A as a reference against targets. Most of the target compounds exhibit more than 50% inhibition at 10 μM. Some derivatives showed good inhibition against AChE. The preliminary structure-activity relationships were discussed.


Introduction
Alzheimer's disease (AD), a neurodegenerative disease affecting the elderly population throughout the world, is clinically characterized by an impairment of the cognitive function.2][3] However, the clinical usefulness of marketed AChE inhibitors often cause some adverse effects, for example, donepezil can lead to diarrhea, and rivastigmine can cause vomiting, therefore, it is necessary and urgent to find more effective AChE inhibitors to treat AD.
In order to gain insight into the recognition between the AChE and the ligands, docking simulations were done on the three-dimensional structures of AChE.The three-dimensional structure of AChE from Torpedo californica has been determined by x-ray analysis in 1991. 4ChE is an α/β serine hydrolase consisting of 537 residues with a 12-stranded mixed β sheet surrounded by 14 α helices.The active site is striking in that it is a deep, narrow tunnel approximately 20 Å long.Fourteen highly conserved aromatic residues line a substantial portion of the surface of the gorge.This tunnel penetrates halfway into the enzyme and widens out at the end.At the bottom of the gorge, there is the catalytic active site (CAS), which is assigned to the Ser-His-Glu catalytic triad (Ser200-His440-Glu327).In the middle of the gorge, there is the anionic binding locus, in which the quaternary ammonium functionality of many ligands interacts with the side chains of Trp84, Glu199, and Phe330, and Tyr130.] Since three-dimensional structure information of the complex structure of ligands and AChE is available, a docking model built with AutoDock 4.0 software was generated.In previous research in this field, 6 a series of 5H-thiazolo[3,2-a]pyrimidine derivatives have been designed and synthesized as acetylcholinesterase inhibitors, but these derivatives bind difficultly to the catalytic sites of AChE due to big stereospecific blockade.In order to find some novel AChE inhibitors which could simultaneously bind to catalytic and peripheral sites, active substructures of 5H-thiazolo[3,2-a]pyrimidine derivatives and some clinical drugs were integrated, using the principles of bioisosterism, hybridization, and structural optimization.A novel series of 7Hthiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were thus designed on the basis of docking results.
Comparison of the three-dimensional structures of human AChE (1B41) and TcAChE (1EVE) has revealed a high degree of similarity, especially with regard to the active sites.The biggest difference between human AChE and TcAChE in the binding site was that Phe330 of TcAChE was replaced by Tyr337 of human AChE, therefore Phe330 was mutated to Tyr337, which might reflect a real difference between human AChE and TcAChE.Another difference is the sequence number of residues (Figure 1).Structure comparison was prepared by the structure editing tools DockPrep in UCSF Chimera (UCSF Chimera, Version 1, USA).Until now, the structure of the complex of human AChE and inhibitors is not available, so donepezil was docked into human AChE.The proposed binding model of donepezil with the key residues in the gorge site is shown in Figure 2. The binding model suggests that donepezil is bound near the bottom of the gorge, and it shows parallel π-π stacking against the six-membered ring of Trp86 indole.The piperidinic nitrogen of the ligand, which contains a positive charge,   Based on the docking, all the complexes between the enzymes and the target molecules suggest this kind of interactions.The inhibitors are depicted as sticks in Figure 3 b]-1,2,4-triazin-7one 6f with the Tyr124, Tyr133 and Tyr337 of AChE.However, the π-π interaction plays an important role, giving the ligand-AChE complexes high stability and at the same time improving the recognition process between this enzyme and the target molecules.The π-π interaction is formed between the aromatic ring of the target molecules and the aromatic ring from the Trp86 of AChE.

Chemistry
The target compounds 5a-5e were obtained in satisfactory yields, and the synthetic pathways are described in Schemes 1.A typical synthesis was developed for our target compounds described in Scheme 1, the aromatic aldehydes were easily converted to 4-arylmethylideneoxazol-5(4H)-ones 1 by cyclization in good yield.Subsequently, the hydrolysis of 1 in the acetone aqueous solution resulted in α-(acetylamino)cinnamic acids 2, in practice, it was found that addition of a small amount of sodium acetate exhibited a remarkable enhancement in hydrolyzing 1 to 2, and the yields of hydrolysis ranged from 85 to 92%. 2 were then converted into the corresponding aryl pyruvic acids (3) by treatment with 1 mol.L -1 hydrochloric acid aqueous solution.The reaction of 3 with thiosemicarbazone gave the cyclized products 3-thioxo-1,2,4-triazin-5(2H)-ones 4. 6-Arylmethyl-3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7 -one derivatives 5 were prepared by reaction of 4 with substituted phenacyl chlorides in the presence of acetic acid.The target compounds 6 could be obtained with ordinary Williamson reaction in Scheme 2. [13][14][15] The chemical structures of all novel compounds synthesized herein were fully characterized by mass analysis, infrared spectra, and proton NMR spectroscopic data reported in the experimental section.

Crystallography
The final X-ray measurement has been carried out by using an BRUKER SMART 1000CCD diffractometer with graphite monochromated MoKa (λ=0.71073Å)radiation.Intensity data were collected in the range of 1.77-25.01°using a moving crystal, moving detector (ω/2θ) scan technique.A total of 3858 independent 'observed' reflections were used in structural analysis.The atomic scattering factors were taken from the International Tables for X-ray Crystallography.All non-hydrogen atoms were refined with anisotropy thermal parameters and the hydrogen atoms were subjected to isotropic refinement.The structure was solved by direct methods using SHELXL-97 refined anisotropically.A summary of the crystal data and structure refinement parameters are collected in Table 1.The structure of 6d is shown in Figure 6.
In the stable crystal structures, the intermolecular interaction is very important.An X-ray diffraction study demonstrated that crystalline 6d was packed as dimers, where two molecules are linked to each other by O-H and N-H hydrogen bonds through a crystallographic center of symmetry (Figure 7).
Complete crystallographic data for the structure of target compound 6d reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as a CIF file (CCDC 732102).Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336-033, e-mail: deposit@ccdc.cam.ac.uk).

Inhibition of AChE
Table 2 illustrates the biological activity of the target compounds against human AChE, in comparison to the huperzine-A.Human AChE (Sigma C-1682) 0.5unit was used.Each performed in double.The incubation time was 20 min, with gentle shake.
The compounds 5f-5i showed moderate activity, this was probably due to the presence of a hydroxy group, which would be able to form a hydrogen bond with the AChE receptor.While the compounds 6a, 6e and 6g with 2-(4-morpholinyl)-2-oxoethoxy group led to a huge decrease in activity than that bearing other groups on the phenyl ring at the C3 position of the parent nucleus (the compounds 6d and 6f), indicating that the binding of some compounds to the active site in the enzyme could be limited by hydrophobic interactions.
The compounds 6d and 6f are more potent than 6b and 6c, the reason is that probably compounds 6b and 6c both have big substituents at the ortho position on the phenyl ring at C3 position of the parent nucleus, resulting in a larger steric hindrance which limits the access of the target molecules to the enzyme-binding site.

Conclusions
In conclusion, 6-arylmethyl-3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives represent a class with a novel scaffold for highly active and selective AChE inhibitors.Further efforts aiming at developing potent AChE inhibitors based on modification of the compounds 5 and 6 will be continued in our laboratory.

Docking studies Preparation of the enzyme
The coordinates for the enzyme were those deposited in the Protein Data Bank for the human acetylcholinesterase (1B41) after eliminating the inhibitor (Fasciculin-2) and water molecules.The missing residues were built and the polar hydrogen atoms to amino acid residues were added.This work was completed by SPDB Viewer.(GSK SPDB Viewer, Version3.7,Swiss).Kollman charges were assigned to all atoms of the enzyme in AutoDock.

Molecular docking
For docking studies, the latest version of AutoDock (4.0) was chosen because its algorithm allows full flexibility of small ligands.It has been shown that it successfully reproduces many crystal structure complexes and includes an empirical binding free energy evaluation.Docking to AChE was carried out using the hybrid Lamarckian Genetic Algorithm, with an initial population of 100 randomly placed individuals and a maximum number of 1.0×10 7 energy evaluations.The dimension of the active site box was set at 60 Å ×60 Å ×60 Å, which ensured an appropriate size of the accessible space.Active site box centre was set at the centre of active gorge of AChE.Resulting docked orientations within a root-mean square deviation of 1.0 Å were clustered together.The lowest energy cluster returned by AutoDock for each compound was used for further analysis.All other parameters were maintained at their default settings. 16

Chemistry
Melting points were determined on Koffler hot-plate apparatus and are uncorrected. 1H-NMR spectra were obtained in CDCl 3 or DMSO-d 6 on a Bruker spectrometers instrument operating at 300 MHz or 600 MHz.The mass spectra (MS) were obtained by electronic impact (EI) at 70 eV in an Agilent spectrometer (with direct insertion probe) or by electrospray (ESI) in a Waters spectrometer.The IR spectra were obtained using a Bruker AFS55 spectrometer.The C, H and N analyses were performed on a Perkin Elmer 240C elemental analyzer.

Synthesis of aryl pyruvic acids (3)
The compound 2 (10 mmol) was added to 60 mL 1mol/L HCl aqueous solution.The solution was refluxed for 7h.The solution was cooled and extract with EtOAc (3×20 mL).The organic layer was dried and evaporated to give aryl pyruvic acid as a solid.

Crystal growth
The solubility of the compound is determined by adding the solvent to a known amount of compound till it is completely dissolved.It is found that the target compound 6d is sparingly in water, and moderately soluble in ethanol and methanol.It is easily soluble in dichloromethane, chloroform, N,N-dimethylformamide.Crystals were grown by the slow evaporation technique at room temperature by using ethanol as a solvent.An aqueous solution of MNC was prepared in a vessel covered with perforated sheet, and kept in a dust free atmosphere.At the period of super saturation, tiny crystals were nucleated [24] .They were allowed to grow to a maximum dimension and then harvested.Thus, grown light yellowish transparent crystals of dimension 13×2×5 mm.

Biology. Inhibition of AChE
The inhibitory potency against AChE was evaluated by means of an Ellman's test [25] .AChE stock solution was prepared by dissolving human AChE 0.5 unit in 100 mM PBS buffer (pH 7.4).The tested target compounds (10 µM) were prepared in DMSO.The assay solution consisted of 100mM PBS buffer (pH 7.4), with the addition of 10 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent), AChE (5 µl), drug (10 µl), and 12.5 mM acetylthiocholine iodide water solution.The final assay volume was 900 µl.Incubate the reaction at 37°C for 15 min with continuous gentle shaking.Add 50 ml acetylthiocholine iodide and 50 ml DTNB.Incubate at 37°C for about 20 min with continuous gentle shake, wait until the yellow color developed.Measure at 412 nm.Calculate the specific inhibition rates.

Figure 2 .
Figure 2. Docking model of donepezil in the active site of human AChE.

Figure 3 .
Figure 3. Docking model of 6d in the active site of human AChE.

Figure 4 .
Figure 4. Docking model of 6e in the active site of human AChE.

Figure 5 .
Figure 5. Docking model of 6f in the active site of human AChE.

Figure 7 .
Figure 7.The molecular packing of 6d, the molecular packing viewed along the a axis.Intermolecular C-H...O and C-H...N interactions are shown as dashed lines.

Table 2 .
Inhibition of AChE activities by the targets at 10 µM (n=2)