Design, synthesis and assay of 2-(4-phenylpiperazin-1-yl)pyrimidine-5- carboxamide derivatives as acetylcholinesterase inhibitors

A series of 2-(4-phenylpiperazin-1-yl)pyrimidine-5-carboxamide derivatives as acetylcholinesterase inhibitors (AChEIs) were designed and synthesized for the treatment of Alzheimer’s disease (AD). Their bioactivities were evaluated by the Ellman’s method, and the results showed that most of synthesized compounds displayed moderate acetylcholinesterase inhibitory activities in vitro. Among them, compound 6g exhibited the most potent inhibitory activity against AChE with IC50 of 0.90 μM, and poor inhibitory activity against butyrylcholinesterase (BuChE) with IC50 of 7.53 μM, which indicated that compound 6g was a selective AChE inhibitor, and compound 6g as a selective AChE inhibitor was confirmed by the molecular docking studies of compound 6g with AChE and BuChE. Furthermore, the mechanism of inhibition of compound 6g against AChE was analyzed by the kinetic study, and the result indicated that compound 6g was the mixed-type inhibitor of competitive inhibition and non-competitive inhibition. All the above showed that compound 6g could be considered as a lead compound for the development of AD drugs. Graphical abstract Graphical abstract


Introduction
Alzheimer's disease (AD) is a neurodegenerative disease which is clinically characterized as progressive cognitive decline, memory impairment, language dysfunction, and inability to perform daily life tasks as usual [1][2][3][4]. Globally, more than 50 million people are suffering from AD, which is predicted to increase up to 152 million by 2050 [5,6]. Currently, AD treatment is based on reducing the symptomatology, not on stopping or slowing down the progression of the disease. Donepezil, Rivastigmine, Galantamine and Huperzine-A as acetylcholinesterase inhibitors (AChEIs) are the most popular AD treatments, and they can temporarily relieve symptoms and reduce memory impairment. Nevertheless, these drugs offer limited ability to prevent or reverse the progression of the disease [7,8].
The pathogenic mechanism of AD is highly complicated, and the exact etiology remains unknown. The cholinergic deficiency hypothesis, which is the basis of the main therapeutic approach in the treatment of AD, suggests that the low level of acetylcholine (ACh) is the main cause of memory and cognitive impairment in AD patients [9,10]. The increasing studies suggest cholinergic neurotransmitters (ACh, BuCh) play an important role in learning and memory. Acetylcholinesterase (AChE) is selectively responsible for hydrolyzing ACh in the healthy brain, while butyrylcholinesterase (BuChE), possessing wider substrate specificity than AChE, can take over AChE to some extent to modulate acetylcholine, enhancing cognition functions [11][12][13].
Heterocyclic compounds play a wide role in drug discovery processes and possess considerable chemical significance and biological activities. Among them, pyrimidine was widely utilized for the development of AD drugs because of its diverse biological potential [14][15][16] (Fig. 1), and piperazine is also an important scaffold for AD drug design [17][18][19] (Fig. 1).
It is worth mentioning that the 2-(piperazin-1-yl)pyrimidine fragments have been found to possess excellent anti-acetylcholinesterase and have recently been reported for the design of AD drugs. A new series of phenyl sulfonyl-pyrimidine carboxylate derivatives were synthesized and evaluated as the potential multi-target drugs with effective anti-Alzheimer's action [20]. Among them, compound BS-10 ( Fig. 2) exhibited the best AChE inhibitory activity (AChE: IC 50 = 47.33 nM) and could be regarded as a promising lead candidate for AD therapy. Meanwhile, the compound S-1 (Fig. 2) designed by S. Montanari et al. displayed promising AChE inhibition with IC 50 value of 37.4 nM and hence was reported effective therapeutic option for the treatment of AD [21]. Its O-phenyl group and alkyl chain containing N and O atoms caught our attention. Herein a series of 2-(4-phenylpiperazin-1-yl)pyrimidine-5carbo-xamide as AChE inhibitors had been designed and synthesized through inspiring of compound BS-10 and compound S-1. The 2-(piperazin-1-yl)pyrimidine fragments of compound BS-10 was preserved, and O-phenyl group of compound S-1 was linked to C-5 of pyrimidine ring with amide chain to design the target compounds 1 (Fig. 2).

AChE and BuChE assay
To evaluate the AChE and BuChE inhibitory activities of target compounds, the Ellman's method [22] was performed with Huperzine-A as reference compound. The results were shown in Tables 1 and 2. Visibly, most of the synthesized target compounds exhibited moderate inhibitory activities against AChE with IC 50 values ranging from 0.90 to 42.2 μM. Among them, compound 6k and 12d displayed good AChE inhibitory activities, and their IC 50 values for inhibiting AChE both reached 1.00 μM. Compound 6g was found to be the most potent AChE inhibitors, with IC 50 value of 0.90 μM. Meanwhile, the synthesized compounds exhibited a certain ability to inhibit BuChE, and several compounds inhibited BuChE with IC 50 value less than 5 μM, for example, compounds 6e, 6h and 6n exhibited the better BuChE inhibitory activities with the IC 50 values of 3.42 μM, 3.87 μM and 3.96 μM, respectively. Notably, compound 6g inhibited AChE with IC 50 value of 0.90 μM, while it inhibited BuChE with IC 50 value of 7.53 μM, which revealed 8.3 times selectivity for AChE over BuChE. Through the analysis and comparison of structure and activity data (Table 1), the following structure-activity relationships could be found: (1) The inhibitory activity of AChE from compounds with Q group as piperazine ring was significantly better than that of compounds with Q group as ethylenediamine and propanediamine, for example, Scheme 2 The synthesis of target compounds 12a-12d and 13a-13d compound 6g (piperazine ring, IC 50 = 0.90 μM) was 9.5 fold more active than 6m (ethylenediamine chain, IC 50 = 8.52 μM) and 6.1 fold more active than 6n (propylenediamine chain, IC 50 = 5.50 μM). However, the inhibition of BuChE by compounds whose Q group was a piperazine ring or a diaminoalkyl chain was similar.
(2) The R 1 substitution as morpholine resulted in the best AChE inhibitory activity (6g: IC 50 = 0.90 μM), whereas the AChE inhibitory activity was worst when the R 1 substituent was N, O-dimethylhydroxylamine (6a: IC 50 = 42.2 μM), and the AChE inhibitory activity of 6g was 46.9 fold more active than that of 6a. (3) After connecting the piperazine ring and the phenyl ring A by amide bond, and removing the nitro group on the phenyl ring A, the AChE inhibitory activity would decline significantly, for example, the IC 50 value of 6g for inhibiting AChE was 0.90 μM, which was 5.1 times more active than that of compound 7 (IC 50 = 4.61 μM). (4) The AChE inhibitory activity decreased when the nitro group on the phenyl ring B was replaced by other groups, but the least decrease in activity occurred when all hydrogens on the phenyl ring B were replaced by fluorine atoms. Furthermore, the position of the substituent also had some influence on AChE inhibitory activity with para-substituted compounds having better AChE inhibitory activity than that of metasubstituted compounds, such as 12b (IC 50 = 5.36 μM) and 12a (IC 50 = 25.0 μM). (5) The inhibitory activity was not significantly improved by connecting pyrimidine ring to the aromatic ring B through two amide bonds, but it was found that extending the length of one carbon chain resulted in the decrease of the inhibitory activity against AChE, such as 13d (IC 50 = 10.0 μM, n = 2) and 13b (IC 50 = 6.85 μM, n = 1). Moreover, when R 3 was substituted with different groups, the order of AChE inhibitory activity was 13b (4-NO 2 ) > 13a (4-CF 3 ) > 13c (4-CN), and compound 13b (4-NO 2 ) with IC 50 value of 6.85 μM is more favorable than others.

Kinetic analysis
In order to know the mechanism of AChE inhibition, compound 6g was conducted for kinetic analysis of AChE inhibition. The Lineweaver-Burk plot was constructed for three varied concentration of compound 6g against six different concentrations of substrate (acetylthiocholine iodide, ATCI). According to the characteristics of enzymatic kinetics, the double-reciprocal curve of the competitive inhibition type is on the Y-axis and the double-reciprocal curve of the non-competitive inhibition type is on the negative semi-axis of the X-axis. the double-reciprocal curve of the mixed inhibition type is assigned to the second quadrant. As shown in Fig. 3, the straight lines of different concentrations of 6g intersected in the second quadrant (Fig. 3). As the concentration of 6g increases, the slope and vertical axis intercept increased, which indicated that compound 6g displayed both competitive and non-competitive inhibitory effects on AChE.

Molecular docking study
The potential binding mode of compound 6g with AChE (PDB: 4EY7) and BuChE (PDB: 5K5E) were investigated by the molecular docking study, which was performed by using Autodock 4.2 with structure images created by Pymol 1.5. The protein structure of AChE (PDB code: 4EY7) and BuChE (PDB code: 5K5E) was downloaded from the Protein Data Bank (https://www.rcsb.org/). Then the downloaded protein was prepared by adding hydrogen atoms, removing water and assigning the Kollman atomic charges to the protein. Auto-Dock was used to switch the prepared protein into pdbqt file and prepare the ligand in pdbqt file. A grid box spacing of 0.375 Å was constructed over the docking area and docking procedure was carried out. Finally, Pymol (https://www.pymol. org/pymol.html) and BIOVIA Discovery Studio (Free Dow nload: BIOVIA Discovery Studio Visualizer -Dassault Systèmes (3ds.com)) software was used to visualize the 3D and 2D interaction of docking study. As shown in Fig. 4, The nitro group on the phenyl ring B of 6g can participate in hydrogen bonds with Asp-74 and Trp-86 simultaneously. The oxygen atom of the amide bond at C-5 of the pyrimidine ring involved in a hydrogen bond with Tyr-124. Meanwhile, hydrogen bonds were observed between the N atom of the pyrimidine ring and Phe-295, and the nitro group on the phenyl Huperzine-A ---5.28 ± 1.20 >10000.00 SD indicates standard deviation ring A involved in two hydrogen bonds with Tyr-341 and Ser-293, respectively. Additionally, the pyrimidine ring formed π-π interaction with Trp-286, and the phenyl ring B of 6g interacted with Trp-86 through π-π stacking effect. Therefore, 6g was strongly bound with the optimal conformation of AChE and stabilized in the cavity. Moreover, as shown in Fig. 4, the phenyl ring A of 6g interacted with Gly-116 through π-Amide. The oxygen atom of the amide bond at C-5 of the pyrimidine ring involved in a hydrogen bond with Asn-289 Fig. 5. As shown in Fig. 6, through the conformational overlap of compounds 6g, BS-10, and S-1, it was found that the docking model of the lead compound BS-10, S-1 has an obvious fold relative to compound 6g. This may be the reason why the inhibitory activity of compound 6g is lower than that of BS-10, S-1. Adding several rotatable carbon bonds between the piperazine ring and the benzene ring may give the compound better activity.

Conclusion
In summary, a series of 2-(4-phenylpiperazin-1-yl)pyrimidine-5-carboxamide derivatives were synthesized and evaluated as potential acetylcholinesterase inhibitors (AChEIs) against AD. Among them, compound 6g emerged as the most potent AChE inhibitor (IC 50 = 0.90 μM) with mixed type of inhibitory pattern, and 6g also exhibited 8.3 times AChE selectivity over BuChE (IC 50 = 7.53 μM), which indicated that compound 6g was a good selective AChE inhibitor. The molecular docking studies displayed that compound 6g was bound up with AChE and BuChE through hydrogen bond and π-π interaction. Taken together, the 2-(4-phenylpiperazin-1-yl)pyrimidine-5-carboxamide derivatives provided a useful template, and compound 6g might be a promising lead compound for the development of new anti-AD drugs.

Experimental Section
Chemistry All experiments were carried out under air atmosphere, 4chloro-3-nitrobenzamide derivatives, N-methyl-3-phenoxypropan-1-amine derivatives, N-(2-aminoethyl)benzamide derivatives, 4-(morpholine-4-carbonyl)benz-oic acid and other reagent materials were commercially available analytically pure or chemically pure, unless stated otherwise. Reaction progress was observed by thin layer chromatography on the glass-backed silica gelsheets (Silica Gel 60 GF254) and visualized under UV light (254 nm). High Resolution Mass Spectrometry was determined by TSQ Quantum Ultra Mass Spectrometer, and the nuclear magnetic resonance spectrum was measured by Bruker Avance III 600 MHz nuclear magnetic resonance spectrometer. The infrared (IR) spectra were run as KBr disk on FTIR-850 spectrophotometer (Tianjin Gangdong Sci. & Tech Co., Ltd.). The purity of the target compounds was determined by LC-3000 HPLC system (Beijing Chuangxin tongheng Technology Co., Ltd.). The melting point was determined by SGW X-4 micro melting point apparatus (Shanghai  Precision Scientific Instrument Co., Ltd.). The 96 plate was read by 1420 Victor Microplate Reader. Acetylcholinesterase and Butyrylcholinesterase were purchased from Sigma, Huperzine-A was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

General procedure for the synthesis of target compounds 6a-6n
To a solution of compounds 5a-5c (1.00 mmol) and 4chloro-3-nitrobenzamide derivatives (2.00 mmol) in DMF (5 mL) were added DIEA (195 mg, 3.0 mmol). The reaction mixture was stirred at 120°C overnight with stirring. After the consumption of the starting material (monitored by TLC), the H 2 O (15 mL) was added and the aqueous layer was extracted twice with ethyl acetate (15 mL). Then, the organics was washed with brine and dried over Na 2 SO 4 , filtered, and concentrated in vacuo to give crude compounds 6a-6n. The crude compounds 6a-6n were purified by column chromatography on silica gel to afford compounds 6a-6n.