Design, synthesis and evaluation of imidazo[1,2-a]pyrazin-8(7H)-one derivatives as acetylcholinesterase inhibitors and antioxidants

Design, synthesis and evaluation of imidazo[1,2-a]pyrazin-8(7H)-one derivatives as acetylcholinesterase inhibitors and antioxidants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design, synthesis and evaluation of imidazo[1,2-a]pyrazin-8(7H)-one derivatives as acetylcholinesterase inhibitors and antioxidants Wen-Rong Du, Ben-Ben Wei, Xin-Yuan Guo, Yong Lan, Pan-Pan Shang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4447664/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Aug, 2024 Read the published version in Medicinal Chemistry Research → Version 1 posted 5 You are reading this latest preprint version Abstract A series of 8-(piperazin-1-yl)imidazo[ 1,2-a ]pyrazine derivatives were designed and synthesized as acetylcholinesterase inhibitors (AChEIs) and antioxidants for the treatment of Alzheimer's disease (AD). Moreover, the biological evaluation results demonstrated that these synthesized compounds exhibited moderate inhibitory activities toward acetylcholinesterase (AChE) and radical scavenging activities. Among them, compound 14r was the most potent AChE inhibitor with an IC 50 value of 0.47 µM and moderate inhibitory activity against butyrylcholinesterase (BuChE) (IC 50 = 11.02 µM). Meanwhile compound 14r had the best selectivity of AChE and selectivity index (SI) values was 23.45. Compound 14r has better activity as well as AChE selectivity compared to reference drug galantamine (AChE IC 50 = 5.01 µM, BuChE IC 50 = 18.46 µM, SI = 3.68). Compound 14o had the best antioxidant activity with an IC 50 value of 89.33 µM, which was lower than that of ascorbic acid (IC 50 value = 25.70 µM) as the control drug. Furthermore, the results of molecular docking studies indicated that 14r could simultaneously bind to both catalytic active site and peripheral anionic site of AChE, which was consistent with the mixed inhibition pattern shown by enzyme kinetic studies. The interaction’s stability of 14r-AChE/BuChE were also assessed using a conventional atomistic 100 ns dynamics simulation study, which revealed the conformational stability of representative compound 14r in the cavity of the AChE. In addition, the molecular properties of all compounds were predicted online through the SwissADME, and the best active compound 14r matched the properties of most orally administered drugs. Based on the biological activity and molecular properties, compound 14r as AChEI was valuable for further development. Alzheimer’s disease Acetylcholinesterase inhibitor Antioxidant Molecular docking study Molecular dynamics simulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Alzheimer's disease (AD) is a neurological condition marked by extensive neuronal damage in many brain regions. Currently, AD affects more than 50 million people worldwide, and this number will continue to increase with the aging population growing [ 1 – 3 ]. And by 2050, the number of senior patients with the condition is expected to triple [ 4 – 6 ]. Researchers have made many efforts to treat AD, and various hypotheses have been proposed by researchers, such as the cholinergic hypothesis [ 7 ], the amyloid (Aβ) toxicity hypothesis [ 8 ], the Tau protein abnormality hypothesis [ 9 ], the neuroinflammatory hypothesis [ 10 ], the free radical damage hypothesis [ 11 ] and so on. Due to the complex pathogenesis of AD, a single target drug cannot cure this disease fundamentally [ 12 ]. Dual or multiple target drugs involved in two or more aspects of AD pathogenesis may generate a synergistic effect and ultimately achieve an ideal therapeutic effect [ 13 – 15 ]. Thus, in consideration of the complicated interrelation among multiple pathways, a multitarget strategy possesses a more effective and promising capacity to manage the special disease network of AD with synergistic adjustment toward more pivotal targets, becoming a hot research topic for the potentially curable treatment of AD [ 16 – 19 ]. Among the drugs currently approved by the U.S. Food and Drug Administration for the treatment of AD, most are acetylcholinesterase (AChE) inhibitors [ 20 – 22 ]. Although inadequate for curing AD, inhibition of AChE remains the most successful strategy and has been shown to be transiently capable of improving memory and cognitive function in AD patients [ 23 , 24 ]. Therefore, multitarget-directed ligands (MTDLs) combining AChE inhibitors with other active pharmacophore within a single drug would generate a more significant reduction in AD symptoms [ 25 – 28 ]. Based on this theory, a number of active compounds have been discovered, these compounds not only enhance acetylcholine (ACh) levels in the brain by inhibiting acetylcholinesterase, but also exhibit one or more other anti-AD biological activities such as glycogen synthase kinase 3 beta (GSK-3β) inhibitory activity, monoamine oxidase (MAO) inhibitory activity, and antioxidation, such as compounds 1–3 [ 29 – 31 ]. (Fig. 1 ) Under the guidance of MTDLs theory, dual-target compounds with AChE inhibitory activity and antioxidant activity for the treatment of AD was designed in this paper, and their design philosophy was as follows: compound 4 was found to have strong AChE inhibitory activity. Through structural analysis, it was found that its A region was the key structure with high activity [ 32 ]. Furthermore, compound 5 displayed promising antioxidant activity, and the imidazo[1,2- a ]pyrazine ring on the B region of compound 5 was found to be the important functional group for their antioxidant activity [ 33 ]. In order for region A with antioxidant activity to be connected to region B with AChE inhibitory activity, we converted the imidazo[1,2- a ]pyrazine ring to imidazo[1,2- a ]pyrazin-8(7 H )-one ring, and the two active regions were linked by acetamide. To this end, the target compounds were designed. (Fig. 2 ) Results and discussion Chemical synthesis The synthetic strategy for designed target compounds 14a–14u was depicted in Scheme 1 . Briefly, compound 6a-6k reacted with various Q 1 to provide 7a-7o . Then compounds 7a-7o could be reacted with the chloroacetyl chloride in CH 2 Cl 2 to get compounds 8a-8o under the condition of an ice water bath. In parallel, compound 6a-6k reacted with Q 2 to provide 9a-9b . The removal of Boc group was achieved by dissolving 10a-10b in MeOH solution with HCl gas at room temperature for 2h, then, 10a-10b reacted with the chloroacetyl chloride to yield compounds 11a-11b. Commercially available 2-amino-3-chloropyrazine was condensed with the corresponding α-halocarbonyl derivatives to build 13a-13e . Eventually, the target compounds 14a-14u were synthesized by reaction of compounds 13a-13e with compounds 8a-8o and 11a-11b . The structures of new compounds were confirmed by the 1 H NMR, 13 C NMR, HRMS and IR spectra, and the purity of all the target compounds was determined to be over 95.0% by high-performance liquid chromatography (HPLC) analysis. In vitro AChE and BChE inhibition assay According to Ellman method [ 34 ], the inhibitory activities of target compounds 14a-14u against eeAChE and eqBuChE in vitro were tested with galantamine as the reference compound. EeAChE and eqBuChE were purchased from Sigma-Aldrich. The corresponding IC 50 values were shown in Table 1 . As shown in Table 1 , most of the synthesized target compounds had moderate inhibitory activity against eeAChE and eqBuChE. Compound 14r was the most potent eeAChE inhibitor with an IC 50 value of 0.47 µM and moderate inhibitory activity against eqBuChE (IC 50 = 11.02 µM). Meanwhile compound 14r had the best selectivity of AChE and selectivity index (SI) values was 23.45. Compound 14r has better activity as well as eeAChE selectivity compared to reference drug galantamine (eeAChE IC 50 = 5.01 µM, eqBuChE IC 50 = 18.46 µM, SI = 3.68). For eeAChE, the compounds had better activity at n = 1 compared to n = 2. For example, compound 14i (eeAChE IC 50 = 1.74 µM) had better activity than 14j (eeAChE IC 50 = 2.56 µM). When the substituent on benzene ring of R 2 was an electron donating group, the 14i with two methoxy groups on the benzene ring of R 2 held the highest activity. When the substituent on benzene ring of R 2 was an electron withdrawing group, para substituted fluorine has the best activity than trifluoromethyl, and among compounds with trifluoromethyl as a substituent, meta substituted fluorine has the best activity. When Q was an unsaturated ring, there was a significant increase in activity compared to when Q was a saturated ring. When the substituent of R 1 was meta methoxy, its activity was stronger than that of dimethoxy, para fluorine, and naphthalene ring. In the case of eqBuChE, compound 14j (eqBuChE, IC 50 = 1.49 µM) had the best inhibitory activity to eqBuChE. The compounds with the substituents on R 2 in the ortho position usually exhibited better activity, and the compounds had better activity at n = 2 compared to n = 1. When replaced the R 1 substituent, the structure with a larger spatial resistance at the substituent of R 1 possessed better activity. Kinetic study of AChE inhibition In order to further explore its mechanism of AChE inhibition on 14r with the best inhibitory activity against AChE, compound 14r was selected for enzyme kinetic study. The Lineweaver-Burk plot was constructed for three varied concentration of compound 14r against six different concentrations of substrate (acetylthiocholine iodide, ATCI). As in Fig. 3 (a), the intersecting of double reciprocal curve in the second quadrant showed that compound 14r displayed both competitive and non-competitive inhibitory effects on AChE. Used the slope in the Lineweaver Burk plot to plot the compound concentration (Fig. 5 (b)), the K i value was 0.54 µM, which was consistent with the IC 50 value. The K i value was confirmed the strong affinity of 14r towards AChE. Studies of anti-oxidative activity Stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were used to determine the antioxidant activity of the synthesized compounds 14a - 14u . The compounds for antioxidant activity were evaluated using standard procedures and ascorbic acid was used as a control drug. As shown in Table 2 , among these compounds, compound 14o exhibited excellent antioxidant activity with IC 50 value of 89.33 µM, yet it was lower than that of ascorbic acid. And the remaining compounds had only moderate antioxidant activity. Table 2 Antioxidant activity of 14a-14u . Compd. IC 50 (µM) ± SD a Compd. IC 50 (µM) ± SD a 14a 119.34 ± 16.81 14l 118.88 ± 10.62 14b 186.45 ± 11.48 14m 190.67 ± 12.52 14c 170.85 ± 14.39 14n 97.31 ± 11.11 14d 181.77 ± 8.45 14o 89.33 ± 8.58 14e 162.92 ± 21.08 14p 153.82 ± 13.86 14f 131.78 ± 12.06 14q 168.39 ± 16.76 14g 158.24 ± 9.44 14r 106.81 ± 7.84 14h 203.26 ± 12.30 14s 186.84 ± 16.36 14i 148.19 ± 17.69 14t 161.76 ± 8.72 14j 100.98 ± 15.54 14u 145.76 ± 11.26 14k 92.67 ± 8.39 Ascorbic Acid 25.70 ± 3.82 SD a indicates standard deviation Molecular docking studies To gain insight into the binding mode of compound 14r to AChE (4EY7) and BuChE (5K5E) enzymes, molecular docking studies were performed using AutoDock 4.2, and the docking results were shown in Fig. 4 and Fig. 5 . As shown in Fig. 4 , the 3D model of AChE docked with compound 14r . The aminobenzamide ring could simultaneously produce π-π interactions with Trp-286 and Tyr-341. The amide bond on the pyrazine of 14r could form a hydrogen bonding interaction with Tyr-124. Meanwhile, the meta methoxy substituted benzene ring could simultaneously produce π-π interactions with Trp-86. Additionally, the imidazole ring of 14r could form a π-π stacking with His-447. At the same time, 14r could interact simultaneously with the catalytic active site (CAS) and peripheral anionic site (PAS) after entering the binding pocket of AChE. As shown in Fig. 5 , the 3D model of BuChE docked with compound 14r . The amide bond on the pyrazine of 14r could form a hydrogen bonding interaction with Thr-120. Meanwhile, the aminobenzamide ring could produce π-π interactions with Trp-82. Additionally, the a aminobenzamide ring could form a hydrogen bonding interaction with Tyr-440. According to Figs. 4 and 5 , it was able to find that compound 14r produced more interactions with AChE and relatively few interactions with BuChE. This partly explained the the better activity and selectivity of compound 14r for AChE. Molecular dynamics simulation study The molecular dynamics (MD) simulation is a popular method for determining structural stability and the molecular interaction profiles of proteins and small molecules. Generally, the binding of ligand to protein is a dynamic phenomenon, which occurs in less than one nanosecond. MD simulations over a period of 100 ns were carried out for 14r with targeted enzyme (AChE and BuChE). The MD simulations allowed us to observe the stability and patterns of the established interactions of 14r with the active sites of the enzymes AChE and BuChE. The dynamic behavior of the whole simulated system was examined using a variety of quantitative parameters, including root mean square deviation (RMSD) and root mean square fluctuation (RMSF). The RMSD is an excellent indicator of protein and ligand structural stability, as well as the magnitude of atom position deviation from the starting position. The smaller the deviation, the more stable the conformation, and vice versa. The stability of AChE in the apolipoprotein (APO) form (enzyme without the ligand) was observed from 34 ns (the production phase) of the simulation; as the enzyme complexed with the ligand ( 14r ), its production phase was from 72 ns. The stability of BuChE in the APO form was observed from 23 ns of the simulation; as the enzyme complexed with the ligand ( 14r ), its productive phase was 36 ns (Fig. 6 ). Steady RMSD values indicated well-equilibrated states of the systems during MD simulations. The RMSF analysis indicated the local conformational changes in protein side chains during the whole simulation. The analysis of the RMSF values demonstrated the atomic fluctuations of the APO form of AChE and its complex, as well as the APO form of BuChE and its complex (Fig. 7 ). The mean RMSF values of the APO form and the complexes formed showed a minimal fluctuation for most residues, indicating a high stability and strong interactions between 14r and AChE/BuChE. In silico molecular property analysis Drug-likeness is a complex balance of various molecular properties, such as hydrophobicity, electronic distribution, hydrogen bonding characteristics, molecule size, and flexibility and presence of various pharmacophoric features. Therefore, the properties of the synthesized compounds 14a-14u were predicted online using the SwissADME( http://www.swissadme.ch ). As shown in Table 3 , the synthesized compounds were found to match most of the drug properties. Unfortunately, most of the tested compounds can not pass the blood-brain barrier, so they cannot be used directly as oral drugs and need to be further optimized. Table 3 In silico prediction of molecular properties for target compounds 14a-14u . CODE MW LogP HBA HBD TPSA n violation BBB Rule < 500 < 5 < 10 < 5 < 130Ǻ 2 ≤ 1 - 14a 525.53 3.67 6 2 106.73 1 No 14b 575.54 3.64 8 2 106.73 1 No 14c 575.54 4.17 8 2 106.73 1 No 14d 575.54 4.4 8 2 106.73 1 No 14e 643.54 3.99 11 2 106.73 1 No 14f 537.57 3.11 6 2 115.96 1 No 14g 537.57 3.21 6 2 115.96 1 No 14h 537.57 3.48 6 2 115.96 1 No 14i 567.59 3.45 7 2 125.19 2 No 14j 581.62 4.09 7 2 125.19 2 No 14k 507.54 3.37 5 2 106.73 1 No 14l 539.56 2.97 6 2 106.73 1 No 14m 526.52 2.96 7 2 119.62 1 No 14n 525.53 3.43 6 2 106.73 1 No 14o 532.55 2.94 7 2 147.86 1 No 14p 517.55 3.56 6 1 97.94 1 No 14q 517.55 3.45 6 1 97.94 1 No 14r 525.53 3.34 6 2 106.73 1 No 14s 555.56 3.79 7 2 115.96 1 No 14t 513.49 3.45 6 2 97.5 1 No 14u 545.56 3.64 5 2 97.5 1 No MW: 150 < MV < 500, LIPO(Lipophility): -0.7 < LOGP < + 5.0, HBA(H-bond acceptors): 0 < Num. H-bond acceptors < 10, HBD(H-bond donors): 0 < Num. H-bond donors < 5, POLAR(Polarity): 20 Ų < TPSA < 130 Ų, n violation (number violations from Lipinski’s rule): n violation ≤ 1, BBB: The ability of crossing BBB blood brain barrier. Conclusion In summary, a new series of imidazo[1,2- a ]pyrazin-8(7 H )-one derivatives was designed, synthesized, and evaluated for their ChE inhibitory activity and antioxidant activity. Biological assays demonstrated that all synthesized compounds had certain ChE inhibitory activity and antioxidant activity in vitro . Among them, compound 14r showed the strongest inhibitory activity against AChE with an IC 50 value of 0.47 µM, which was superior to galantamine as the control compound. Furthermore, molecular docking studies showed that 14r was able to bind to both the CAS and PAS of AChE, which was consistent with the mixed inhibition pattern shown by enzyme kinetic studies. Moreover, a standard atomistic 100 ns dynamic simulation results of the binding stability of the compound 14r to AChE revealed the conformational stability in the binding cavity. In addition, the synthesized compounds were found to match most of the drug properties. Taken together, compound 14r 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, the reagents were commercially available analytically pure or chemically pure, unless stated otherwise. High Resolution Mass Spectrometry was determined by Thermo QExactive; the nuclear magnetic resonance spectrum was measured by Bruker Avance III 600 MHz nuclear magnetic resonance spectrometer. The purity of the target compounds were 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. Synthesis of Compounds 14a-14u 8a (0.32 g, 1 mmol) was added to a stirred solution of 13a (0.241 g, 1 mmol) and Cs 2 CO 3 (0.326, 1.2 mmol) in CH 3 CN (8 mL). The mixture was stirred at 80°C for 12 h, and then the solvent was evaporated in vacuo. The crude product was dissolved in CH 2 Cl 2 and washed with a saturated solution of sodium carbonate. The organic layer was separated, dried (Na 2 SO 4 ), and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel to afford crude compounds 14a . The crude compounds 14a was recrystallized from ethyl acetate (5 mL) to give pure compounds 14a . The same method produces for compounds 14b-14u . N -(4-fluorobenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)benzamide(14a) : white solid; Yield: 52%; mp: 208.3-208.9°C; Purity: 96.20% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.53 (s, 1H), 9.04 (t, J = 6.0 Hz, 1H), 8.18 (s, 1H), 8.09 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.61–7.53 (m, 2H), 7.42 (t, J = 8.0 Hz, 1H), 7.37–7.30 (m, 2H), 7.19–7.09 (m, 3H), 7.01 (d, J = 8.3 Hz, 2H), 4.79 (s, 2H), 4.44 (d, J = 5.9 Hz, 2H), 3.79 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.10, 165.80, 161.91, 160.31, 159.10, 152.99, 144.09, 138.76, 136.81, 135.79, 135.16, 129.18, 129.13, 128.79, 126.67, 125.67, 121.94, 121.84, 118.50, 114.99, 114.85, 114.20, 112.43, 106.60, 55.14, 50.34, 41.94; HR-ESI + -MS calcd for C 29 H 24 FN 5 O 4 : 526.1890 [M + H] + , found 526.1894 [M + H] + ; IR (KBr), υ (cm-1): 3292, 1679, 1674, 1639. 3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)- N -(4-(trifluoromethyl)benzyl)benzamide(14b) : white solid; Yield: 43%; mp: 186.5-187.2°C; Purity: 98.27% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.48 (s, 1H), 9.12 (t, J = 6.0 Hz, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 5.8 Hz, 1H), 7.53 (d, J = 7.9 Hz, 2H), 7.44 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 5.8 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H), 4.80 (s, 2H), 4.55 (d, J = 6.0 Hz, 2H), 3.80 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.24, 165.82, 159.10, 153.00, 144.52, 144.09, 138.78, 136.81, 135.00, 128.84, 127.83, 127.54, 127.33, 126.67, 125.69, 125.16, 125.14, 125.11, 125.09, 122.00, 121.95, 118.51, 114.21, 112.43, 106.60, 55.14, 50.35, 42.35; HR-ESI + -MS calcd for C 30 H 24 F 3 N 5 O 4 : 576.1858 [M + H] + , found 576.1861 [M + H] + ; IR (KBr), υ (cm-1): 3291, 1676, 1674, 1672. 3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)- N -(3-(trifluoromethyl)benzyl)benzamide(14c) : white solid; Yield: 39%; mp: 231.7-232.6°C; Purity: 98.99% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.49 (s, 1H), 9.12 (t, J = 6.1 Hz, 1H), 8.19 (s, 1H), 8.09 (s, 1H), 7.85 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 5.9 Hz, 1H), 7.66 (s, 1H), 7.64–7.59 (m, 2H), 7.58 (d, J = 4.8 Hz, 2H), 7.56 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 5.7 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H), 4.79 (s, 2H), 4.54 (d, J = 6.0 Hz, 2H), 3.80 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.23, 165.82, 159.10, 152.99, 144.08, 141.14, 138.79, 136.80, 134.97, 131.37, 129.35, 129.08, 128.87, 126.67, 125.69, 123.74, 123.49, 121.95, 118.47, 114.20, 112.43, 106.60, 55.14, 50.34, 42.29; HR-ESI + -MS calcd for C 30 H 24 F 3 N 5 O 4 : 576.1858 [M + H] + , found 576.1865 [M + H] + ; IR (KBr), υ (cm-1): 3259, 1679, 1639, 1631. 3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)- N -(2-(trifluoromethyl)benzyl)benzamide(14d) : white solid; Yield: 52%; mp: 256.5-257.2°C; Purity: 97.55% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.50 (s, 1H), 9.09 (t, J = 5.9 Hz, 1H), 8.19 (s, 1H), 8.11 (s, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 6.0 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 8.4 Hz, 2H), 7.59 (d, J = 5.7 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.46 (q, J = 7.5 Hz, 2H), 7.17 (d, J = 5.8 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 4.80 (s, 2H), 4.65 (d, J = 5.9 Hz, 2H), 3.80 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.41, 165.84, 159.09, 152.99, 144.08, 138.81, 137.56, 136.80, 134.88, 132.62, 128.88, 128.11, 127.24, 126.66, 126.18, 125.73, 125.68, 125.39, 122.07, 122.00, 121.96, 118.51, 114.20, 112.43, 106.60, 55.14, 50.36, 40.07; HR-ESI + -MS calcd for C 30 H 24 F 3 N 5 O 4 : 576.1858 [M + H] + , found 576.1852 [M + H] + ; IR (KBr), υ (cm-1): 3307, 1671, 1655, 1631. N -(3,5-bis(trifluoromethyl)benzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo-[1,2- a ]-pyrazin-7(8 H )-yl)acetamido)benzamide(14e) : yellow solid; Yield: 58%; mp: 223.4-224.1°C; Purity: 98.40% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.49 (s, 1H), 9.19 (t, J = 6.0 Hz, 1H), 8.18 (s, 1H), 8.09 (s, 1H), 8.01 (s, 2H), 7.99 (s, 1H), 7.85 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 6.2 Hz, 2H), 7.45 (t, J = 8.0 Hz, 1H), 7.16 (d, J = 5.8 Hz, 1H), 7.01 (d, J = 9.1 Hz, 2H), 4.79 (s, 2H), 4.63 (d, J = 5.9 Hz, 2H), 3.79 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.42, 165.84, 159.09, 152.99, 143.21, 136.79, 130.25, 130.03, 128.96, 128.13, 126.66, 122.08, 121.94, 118.42, 114.20, 112.43, 106.61, 55.13, 50.34, 42.07; HR-ESI + -MS calcd for C 31 H 23 F 6 N 5 O 4 : 644.1732 [M + H] + , found 644.1726 [M + H] + ; IR (KBr), υ (cm-1): 3295, 1678, 1647, 1612. N -(4-methoxybenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)benzamide(14f) : white solid; Yield: 41%; mp: 208.4–209.0°C; Purity: 99.58% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.47 (s, 1H), 8.94 (t, J = 6.1 Hz, 1H), 8.18 (s, 1H), 8.06 (s, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 5.8 Hz, 1H), 7.56 (d, J = 7.3 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.23 (d, J = 7.1 Hz, 2H), 7.16 (d, J = 5.5 Hz, 1H), 7.01 (d, J = 8.9 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 4.79 (s, 2H), 4.38 (d, J = 6.0 Hz, 2H), 3.79 (s, 3H), 3.71 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 165.96, 165.78, 159.12, 158.16, 144.08, 138.69, 136.80, 135.56, 131.59, 128.77, 128.54, 126.67, 125.56, 121.95, 118.49, 114.21, 113.65, 112.43, 106.61, 55.14, 55.02, 50.33, 42.06; HR-ESI + -MS calcd for C 30 H 27 N 5 O 5 : 538.2090 [M + H] + , found 538.2088 [M + H] + ; IR (KBr), υ (cm-1): 3082, 1675, 1635, 1623. N -(3-methoxybenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)benzamide(14g) : white solid; Yield: 33%; mp: 234.7-235.5°C; Purity: 95.43% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.47 (s, 1H), 8.99 (t, J = 6.1 Hz, 1H), 8.18 (s, 1H), 8.08 (s, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.0 Hz, 1H), 7.62–7.52 (m, 2H), 7.42 (t, J = 7.9 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.17 (d, J = 5.8 Hz, 1H), 7.02 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 6.8 Hz, 2H), 6.80 (d, J = 9.6 Hz, 1H), 4.79 (s, 2H), 4.44 (d, J = 6.0 Hz, 2H), 3.80 (s, 3H), 3.72 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.11, 165.80, 159.28, 159.10, 152.99, 144.09, 141.23, 138.73, 136.81, 135.30, 129.29, 128.80, 126.67, 125.70, 121.96, 121.79, 119.32, 118.52, 114.21, 112.92, 112.43, 112.03, 106.60, 55.14, 54.94, 50.33, 42.56; HR-ESI + -MS calcd for C 30 H 27 N 5 O 5 : 538.2090 [M + H] + , found 538.2097 [M + H] + ; IR (KBr), υ (cm-1): 3271, 1686, 1651, 1620. N -(2-methoxybenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)benzamide(14h) : yellow solid; Yield: 39%; mp: 251.8-252.5°C; Purity: 99.06% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.48 (s, 1H), 8.83 (t, J = 5.9 Hz, 1H), 8.18 (s, 1H), 8.07 (s, 1H), 7.85 (d, J = 8.9 Hz, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.58 (d, J = 5.2 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 6.3 Hz, 2H), 7.01 (d, J = 8.3 Hz, 2H), 6.98 (d, J = 8.2 Hz, 1H), 6.90 (t, J = 7.5 Hz, 1H), 4.79 (s, 2H), 4.43 (d, J = 5.9 Hz, 2H), 3.82 (s, 3H), 3.79 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.24, 165.80, 159.10, 156.53, 153.00, 144.09, 138.72, 136.80, 135.32, 128.79, 127.84, 127.12, 126.83, 126.67, 125.67, 121.95, 121.77, 120.08, 118.53, 114.21, 112.43, 110.43, 106.61, 55.31, 55.14, 50.34, 37.62; HR-ESI + -MS calcd for C 30 H 27 N 5 O 5 : 538.2090 [M + H] + , found 538.2094 [M + H] + ; IR (KBr), υ (cm-1): 3267, 1683, 1627, 1596. N -(3,4-dimethoxybenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyra-zin-7(8 H )-yl)acetamido)benzamide(14i) : white solid; Yield: 42%; mp: 241.9-242.7°C; Purity: 99.83% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.46 (s, 1H), 8.92 (t, J = 6.1 Hz, 1H), 8.18 (s, 1H), 8.07 (s, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 9.4 Hz, 1H), 7.58 (t, J = 6.0 Hz, 2H), 7.42 (t, J = 7.9 Hz, 1H), 7.16 (d, J = 5.8 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 6.94 (s, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 6.3 Hz, 1H), 4.79 (s, 2H), 4.39 (d, J = 6.0 Hz, 2H), 3.80 (s, 3H), 3.72 (d, J = 5.7 Hz, 6H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.01, 165.79, 159.10, 152.99, 148.64, 147.77, 144.09, 138.71, 136.81, 135.42, 132.10, 128.76, 126.67, 125.69, 121.95, 121.72, 119.38, 118.53, 114.20, 112.42, 111.85, 111.55, 106.59, 55.59, 55.44, 55.14, 50.34, 42.41; HR-ESI + -MS calcd for C 31 H 29 N 5 O 6 : 568.2196 [M + H] + , found 568.2203 [M + H] + ; IR (KBr), υ (cm-1): 3298, 1679, 1639, 1616. N -(3,4-dimethoxyphenethyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]p-yrazin-7(8 H )-yl)acetamido)benzamide(14j) : white solid; Yield: 46%; mp: 264.2-264.9°C; Purity: 99.86% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.46 (s, 1H), 8.48 (t, J = 5.5 Hz, 1H), 8.19 (s, 1H), 8.03 (s, 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.1 Hz, 1H), 7.59 (d, J = 5.7 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 5.8 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 6.85 (d, J = 8.1 Hz, 1H), 6.82 (s, 1H), 6.73 (d, J = 8.2 Hz, 1H), 4.79 (s, 2H), 3.80 (s, 3H), 3.70 (d, J = 7.3 Hz, 6H), 3.45 (q, J = 6.8 Hz, 2H), 2.76 (t, J = 7.5 Hz, 2H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.00, 165.77, 159.10, 152.99, 148.59, 147.20, 144.09, 138.68, 136.80, 135.56, 131.97, 128.70, 126.67, 125.67, 121.96, 121.84, 121.62, 120.46, 118.41, 114.21, 112.55, 112.43, 111.96, 106.60, 55.48, 55.33, 55.14, 50.34, 40.99, 34.54; HR-ESI + -MS calcd for C 32 H 31 N 5 O 6 : 582.2352 [M + H] + , found 582.2355 [M + H] + ; IR (KBr), υ (cm-1): 3311, 1671, 1647, 1624. N -benzyl-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)ace-tamido)benzamide(14k) : white solid; Yield: 46%; mp: 247.2-247.8°C; Purity: 99.37% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.48 (s, 1H), 9.02 (t, J = 6.1 Hz, 1H), 8.18 (s, 1H), 8.07 (s, 1H), 7.85 (d, J = 8.9 Hz, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 4.8 Hz, 2H), 7.42 (t, J = 7.9 Hz, 1H), 7.31 (q, J = 5.7, 5.2 Hz, 4H), 7.26–7.20 (m, 1H), 7.16 (d, J = 5.7 Hz, 1H), 7.01 (d, J = 9.0 Hz, 2H), 4.79 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H), 3.79 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.10, 165.79, 159.10, 152.99, 144.08, 139.61, 138.72, 136.80, 135.26, 128.80, 128.23, 127.14, 126.68, 125.67, 121.95, 121.80, 118.50, 114.20, 112.43, 106.61, 55.14, 50.34, 42.60; HR-ESI + -MS calcd for C 29 H 25 N 5 O 4 : 508.1985 [M + H] + , found 508.1996 [M + H] + ; IR (KBr), υ (cm-1): 3291, 1671, 1647, 1624. N -(4-fluorobenzyl)-2-(3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)phenyl)acetamide(14l) : white solid; Yield: 38%; mp: 218.3-218.8°C; Purity: 98.98% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.30 (s, 1H), 8.53 (t, J = 6.0 Hz, 1H), 8.18 (s, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 5.7 Hz, 1H), 7.56 (s, 1H), 7.46 (d, J = 5.9 Hz, 1H), 7.30–7.24 (m, 2H), 7.24 (d, J = 7.8 Hz, 1H), 7.16 (d, J = 5.7 Hz, 1H), 7.10 (t, J = 8.9 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 7.7 Hz, 1H), 4.77 (s, 2H), 4.24 (d, J = 5.9 Hz, 2H), 3.80 (s, 3H), 3.45 (s, 2H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 169.92, 165.52, 161.91, 160.31, 159.10, 152.99, 144.08, 138.57, 136.98, 136.82, 135.58, 135.56, 129.16, 129.11, 128.58, 126.66, 125.71, 124.29, 122.00, 119.79, 117.29, 114.99, 114.85, 114.20, 112.40, 106.53, 55.14, 50.32, 42.35, 41.49; HR-ESI + -MS calcd for C 30 H 26 FN 5 O 4 : 540.2047 [M + H] + , found 540.2041 [M + H] + ; IR (KBr), υ (cm-1): 3251, 1671, 1639, 1631. N -(4-fluorobenzyl)-5-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)nicotinamide(14m) : white solid; Yield: 41%; mp: 178.6-179.5°C; Purity: 98.55% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.71 (s, 1H), 9.31–9.21 (m, 1H), 8.88 (d, J = 2.5 Hz, 1H), 8.77 (d, J = 2.0 Hz, 1H), 8.47 (t, J = 2.3 Hz, 1H), 8.19 (s, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 5.7 Hz, 1H), 7.39–7.33 (m, 2H),, 7.19–7.14 (m, 2H), 7.13 (s, 1H), 7.02 (d, J = 8.8 Hz, 2H), 4.83 (s, 2H), 4.46 (d, J = 6.1 Hz, 2H), 3.80 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 169.18, 166.48, 164.55, 159.12, 144.12, 142.96, 142.85, 136.77, 135.14, 130.04, 129.28, 129.22, 126.68, 125.18, 121.85, 115.06, 114.92, 114.21, 112.48, 106.71, 55.14, 50.32, 41.99; HR-ESI + -MS calcd for C 28 H 23 FN 6 O 4 : 527.1843 [M + H] + , found 527.1844 [M + H] + ; IR (KBr), υ (cm-1): 3302, 1683, 1643, 1620. N -(4-fluorobenzyl)-4-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-y-l)acetamido)benzamide(14n) : white solid; Yield: 53%; mp: 239.3-240.1°C; Purity: 99.63% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.55 (s, 1H), 9.03–8.80 (m, 1H), 8.19 (s, 1H), 8.01–7.87 (m, 2H), 7.86 (s, 2H), 7.74–7.63 (m, 2H), 7.59 (s, 1H), 7.35 (s, 2H), 7.21–7.08 (m, 3H), 7.07–6.94 (m, 2H), 4.80 (s, 2H), 4.44 (s, 2H), 3.80 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 168.17, 167.74, 165.63, 162.87, 160.55, 152.61, 147.63, 144.04, 135.47, 129.21, 129.15, 128.22, 126.67, 121.81, 118.32, 114.99, 114.84, 114.21, 112.29, 106.78, 55.14, 50.29, 41.88; HR-ESI + -MS calcd for C 29 H 24 FN 5 O 4 : 526.1890 [M + H] + , found 526.1892 [M + H] + ; IR (KBr), υ (cm-1): 3267, 1683, 1659, 1631. N -(4-fluorobenzyl)-2-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)thiazole-5-carboxamide(14o) : white solid; Yield: 46%; mp: 216.4–217.0°C; Purity: 99.81% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 12.76 (s, 1H), 9.03 (s, 1H), 8.24–8.06 (m, 2H), 7.85 (d, J = 9.5 Hz, 2H), 7.60 (d, J = 5.8 Hz, 1H), 7.35 (s, 2H), 7.24–7.07 (m, 3H), 7.02 (d, J = 8.5 Hz, 2H), 4.90 (s, 2H), 4.41 (s, 2H), 3.80 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 171.00, 168.34, 164.53, 163.07, 158.97, 154.71, 143.98, 142.43, 140.43, 136.62, 129.33, 129.28, 126.66, 120.08, 115.08, 114.94, 114.20, 112.51, 107.15, 55.13, 46.16, 41.95; HR-ESI + -MS calcd for C 26 H 21 FN 6 O 4 S: 533.1407 [M + H] + , found 533.1416 [M + H] + ; IR (KBr), υ (cm-1): 3326, 1703, 1655, 1619. N -(4-fluorobenzyl)-1-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetyl)piperidine-3-carboxamide(14p) : white solid; Yield: 46%; mp: 188.3-188.9°C; Purity: 98.28% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 8.46 (t, J = 6.1 Hz, 1H), 8.16 (s, 1H), 7.85 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 5.7 Hz, 1H), 7.39–7.29 (m, 1H), 7.28–7.22 (m, 1H), 7.18–7.07 (m, 2H), 7.01 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 5.8 Hz, 1H), 4.91 (d, J = 16.3 Hz, 1H), 4.79 (dd, J = 16.2, 3.2 Hz, 1H), 4.43–4.28 (m, 1H), 4.30–4.07 (m, 2H), 3.91 (d, J = 13.6 Hz, 1H), 3.80 (s, 3H), 3.25–3.05 (m, 1H), 2.72 (q, J = 12.1 Hz, 1H), 2.42–2.23 (m, 1H), 2.03–1.89 (m, 1H), 1.84–1.61 (m, 2H), 1.59–1.29 (m, 1H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 172.49, 172.37, 164.94, 164.84, 159.08, 152.89, 144.00, 136.85, 135.65, 129.13, 128.95, 126.65, 125.72, 115.05, 114.19, 112.33, 106.44, 106.39, 55.14, 48.30, 44.29, 42.10, 41.28, 41.13, 27.52, 23.80; HR-ESI + -MS calcd for C 28 H 28 FN 5 O 4 : 518.2203 [M + H] + , found 518.2208 [M + H] + ; IR (KBr), υ (cm-1): 3315, 1679, 1647, 1620. N -(4-fluorobenzyl)-1-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetyl)piperidine-4-carboxamide(14q) : white solid; Yield: 42%; mp: 205.7-206.4°C; Purity: 97.47% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 8.38 (t, J = 6.0 Hz, 1H), 8.16 (s, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 5.8 Hz, 1H), 7.33–7.20 (m, 2H), 7.14 (t, J = 8.9 Hz, 2H), 7.03 (d, J = 5.8 Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H), 4.84 (d, J = 6.7 Hz, 2H), 4.30 (d, J = 12.8 Hz, 1H), 4.26 (d, J = 6.0 Hz, 2H), 3.95 (d, J = 13.7 Hz, 1H), 3.80 (s, 3H), 3.17–3.04 (m, 1H), 2.69 (t, J = 11.2 Hz, 1H), 2.47–2.42 (m, 1H), 1.81 (d, J = 13.1 Hz, 1H), 1.75 (d, J = 13.3 Hz, 1H), 1.68 (q, J = 11.9, 8.6 Hz, 1H), 1.53–1.39 (m, 1H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 173.74, 164.68, 161.90, 160.29, 159.08, 152.89, 144.00, 136.84, 135.84, 135.82, 128.99, 128.93, 126.65, 125.72, 121.92, 115.01, 114.87, 114.19, 112.31, 106.38, 55.14, 48.27, 43.72, 41.47, 41.20, 40.08, 28.70, 28.06; HR-ESI + -MS calcd for C 28 H 28 FN 5 O 4 : 518.2203 [M + H] + , found 518.2197 [M + H] + ; IR (KBr), υ (cm-1): 3307, 1683, 1643, 1635. N -(4-fluorobenzyl)-3-(2-(2-(3-methoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)benzamide(14r) : white solid; Yield: 58%; mp: 262.1-262.9°C; Purity: 97.56% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.50 (s, 1H), 9.04 (t, J = 6.1 Hz, 1H), 8.33 (s, 1H), 8.09 (s, 1H), 7.77 (d, J = 6.0 Hz, 1H), 7.60 (d, J = 5.7 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 7.7 Hz, 2H), 7.42 (t, J = 7.9 Hz, 1H), 7.35 (q, J = 7.2, 6.3 Hz, 3H), 7.19 (d, J = 5.8 Hz, 1H), 7.14 (t, J = 8.8 Hz, 2H), 6.91 (d, J = 5.5 Hz, 1H), 4.80 (s, 2H), 4.44 (d, J = 5.9 Hz, 2H), 3.83 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.07, 165.76, 161.91, 160.31, 159.66, 153.06, 143.87, 138.73, 136.91, 134.44, 129.87, 129.17, 129.12, 128.80, 122.17, 121.97, 121.82, 118.49, 117.71, 115.00, 114.86, 113.77, 113.70, 110.38, 106.64, 55.09, 50.36, 41.94; HR-ESI + -MS calcd for C 29 H 24 FN 5 O 4 : 526.1890 [M + H] + , found 526.1897 [M + H] + ; IR (KBr), υ (cm-1): 3302, 1690, 1643, 1624. 3-(2-(2-(3,4-dimethoxyphenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamid-o)- N -(4-fluorobenzyl)benzamide(14s) : white solid; Yield: 51%; mp: 247.5-248.3°C; Purity: 99.72% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.48 (s, 1H), 9.03 (t, J = 6.1 Hz, 1H), 8.24 (s, 1H), 8.08 (s, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.58 (d, J = 5.4 Hz, 2H), 7.50 (s, 1H), 7.47 (d, J = 6.2 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.38–7.31 (m, 2H), 7.17 (d, J = 5.7 Hz, 1H), 7.14 (t, J = 8.9 Hz, 2H), 7.03 (d, J = 8.3 Hz, 1H), 4.79 (s, 2H), 4.44 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H), 3.79 (s, 3H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.08, 165.79, 161.92, 160.31, 153.00, 149.01, 148.75, 144.23, 138.73, 136.72, 135.81, 135.79, 135.18, 129.18, 129.12, 128.80, 125.94, 121.98, 121.82, 118.49, 117.82, 115.00, 114.86, 112.73, 112.12, 109.02, 106.60, 55.57, 55.52, 50.33, 41.94; HR-ESI + -MS calcd for C 30 H 26 FN 5 O 5 : 556.1996 [M + H] + , found 556.1995 [M + H] + ; IR (KBr), υ (cm-1): 3283, 1706, 1675, 1635. N -(4-fluorobenzyl)-3-(2-(2-(4-fluorophenyl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)benzamide(14t) : white solid; Yield: 42%; mp: 229.1-229.8°C; Purity: 99.08% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.48 (s, 1H), 9.03 (t, J = 6.0 Hz, 1H), 8.29 (s, 1H), 8.08 (s, 1H), 7.97 (dd, J = 8.6, 5.6 Hz, 2H), 7.77 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 5.8 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.34 (dd, J = 8.5, 5.6 Hz, 2H), 7.28 (t, J = 8.8 Hz, 2H), 7.19 (d, J = 5.8 Hz, 1H), 7.14 (t, J = 8.9 Hz, 2H), 4.80 (s, 2H), 4.44 (d, J = 6.0 Hz, 2H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.07, 165.76, 162.66, 161.91, 161.04, 160.31, 153.01, 143.11, 137.01, 135.17, 129.61, 129.17, 129.12, 128.81, 127.35, 127.29, 122.18, 118.48, 115.73, 115.58, 115.00, 114.86, 113.33, 106.64, 50.37, 41.93; HR-ESI + -MS calcd for C 28 H 21 F 2 N 5 O 3 : 514.1690 [M + H] + , found 514.1693 [M + H] + ; IR (KBr), υ (cm-1): 3295, 1675, 1643, 1627. N -(4-fluorobenzyl)-3-(2-(2-(naphthalen-2-yl)-8-oxoimidazo[1,2- a ]pyrazin-7(8 H )-yl)acetamido)benzamide(14u) : white solid; Yield: 46%; mp: 255.2-255.8°C; Purity: 96.47% (HPLC); 1 H NMR (600 MHz, DMSO- d 6 ) δ 10.50 (s, 1H), 9.04 (t, J = 6.0 Hz, 1H), 8.51 (s, 1H), 8.44 (s, 1H), 8.12–8.05 (m, 2H), 8.00 (t, J = 8.6 Hz, 2H), 7.92 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 5.7 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.55–7.50 (m, 2H), 7.43 (t, J = 7.9 Hz, 1H), 7.37–7.32 (m, 2H), 7.21 (d, J = 5.7 Hz, 1H), 7.14 (t, J = 8.9 Hz, 2H), 4.82 (s, 2H), 4.44 (d, J = 6.1 Hz, 2H); 13 C NMR (151 MHz, DMSO- d 6 ) δ 166.08, 165.78, 161.91, 160.31, 153.07, 143.96, 138.74, 137.20, 135.79, 135.18, 133.21, 132.60, 130.50, 129.18, 129.12, 128.82, 128.30, 128.07, 127.61, 126.47, 126.03, 123.75, 123.72, 118.50, 115.00, 114.86, 113.98, 106.69, 50.37, 41.94; HR-ESI + -MS calcd for C 32 H 24 FN 5 O 3 : 546.1941 [M + H] + , found 546.1944 [M + H] + ; IR (KBr), υ (cm-1): 3271, 1666, 1639, 1618. AChE and BuChE Inhibition Assay The assay was performed according to our previous reports based on the Ellman’s method [ 29 ]. Kinetic analysis of 14r To determine the inhibition type of these compounds against electroporation AChE, a kinetic study was carried out with inhibitor 14r as the representative AChEI. In the process, the used concentrations of inhibitor 0.5×IC 50 , IC 50 , and 2×IC 50 were 0.235, 0.47, and 0.94 µM, respectively. The type of inhibition was established from the analysis of Lineweaver-Burk (LB) reciprocal plots. Secondary plots of slopes of LB versus concentrations of inhibitor (14r) provided inhibition constants (K i ) as intercepts on negative x-axis In vitro antioxidant inhibition test A simple method that has been developed to determine the antioxidant activity of the drug utilizes the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Briefly, 2 mL DPPH solution (0.2 mM, in 95% ethanol) was incubated with 2mL different concentrations of compounds solution. Then, the reaction mixture was shaken and incubated in the dark for 30 min at 35°C. The absorbance was immediately recorded at 517nm against ethanol with a spectrophotometer (Metash, model UV-5200, China). The IC 50 values were given in table. The amount of DPPH radical was calculated following this equation: % inhibition of DPPH= [A 0 -A s ]/A 0 × 100% Where A 0 was the absorbance of control and A s was the absorbance of sample. Standard drug was Ascorbic acid. Docking study For docking analysis, because no significant difference was detected for the amino acid sequences within the active sites of enzymes from human and non-human sources, X-ray structures of AChE (PDB ID: 4EY7) and BuChE (PDB ID: 5K5E) were selected as templates (downloaded from the Protein Data Bank( https://www.rcsb.org/ )). The protein was then prepared by adding hydrogen atoms, removing water and assigning Kollman atomic charges to the protein, moreover, the prepared proteins were converted to pdbqt files using AutoDock, and the ligands were also prepared in the pdbqt files. A grid box spacing of 0.375 Å was constructed over the docking area and docking procedure was carried out. The center of the grid box was placed at the bottom of the active site gorge (AChE [-14.11 -43.83 -27.67]; BuChE [3.36 9.53 14.4]). The dimensions of the active site box were set at 36×36×36 Å. Finally, Pymol visualization software was used to visualize the 3D interaction of docking study ( https://www.pymol.org/pymol.html ). Then, the docking of processed proteins and ligands was performed using AutoDock. Molecular dynamics simulation. MD simulation was performed with the GROMACS 2022.2 package. Complexes of AChE/BuChE and 14r were obtained from the docking poses. The electrostatic potentials (ESP) of each ligand were calculated at the HF/6-31G(d) level by Gaussian 09. Amberff19SB and gaff2 force field were used to create topology parameters of protein and ligands respectively. TIP3P water model was used to create solvent molecules. The time step was set to 2 fs. The protein–ligand systems were energy minimized using 5000 steps steepest descent algorithm. After minimization, the three systems were heated to 300 K during 100 ps in NVT ensemble. The pressure was then equilibrated to 1 atm during a 100 ps NPT simulation. V-rescale was used for temperature coupling and c-rescale was used for pressure coupling. Finally, 100 ns production simulations were performed for each complex. Declarations Supporting Information Summary The supporting information includes experimental data, characterization of the synthesized compounds and their NMR spectra Conflict of interest The authors declare no competing interests Acknowledgements This work was supported by Hebei Natural Science Foundation ( S&T Program of Hebei, NO. B2020201056, H2024201041). References Toro PMJ, Parra PDR, Pacheco MNV (2022) Galarza A G A. Enfermedad de Alzheimer. Recimundo 6(4):68–76 Gustavsson A, Norton N, Fast T, Frölich L, Georges J, Holzapfel D et al (2023) Global estimates on the number of persons across the Alzheimer's disease continuum. Alzh Dement 19(2):658–670 Woźniak K, Gardian-Baj M, Jung M, Hedesz P, Jung M, Żuk-Łapan A et al (2024) Alzheimer's disease-a comprehensive review. J Educ Health Sport 56:195–209 Grossberg G, Urganus A, Schein J, Bungay R, Cloutier M, Gauthier-Loiselle M et al (2023) A real-world assessment of healthcare costs associated with agitation in Alzheimer's dementia. J Med Econ 27(1):99–108 Wilson RS, Segawa E, Boyle PA, Anagnos SE, Hizel LP, Bennett DA (2012) The natural history of cognitive decline in Alzheimer's disease. Psychol Aging 27(4):1008–1017 Woodward MC (2024) Drug treatment of Alzheimer's disease: what has changed in 30 years? J Pharm Pract Res 53(6):314–319 Hampel H, Mesulam MM, Cuello AC, Farlow MR, Giacobini E, Grossberg GT et al (2018) The cholinergic system in the pathophysiology and treatment of Alzheimer's disease. Brain 141(7):1917–1933 Hua KY, Zhao WJ (2022) Current study on diagnosis and treatment of Alzheimer's disease by targeting amyloid b-protein. Folia Neuropathol 61(1):8–15 Iliyasu MO, Musa SA, Oladele SB, Iliya AI (2023) Amyloid-beta aggregation implicates multiple pathways in Alzheimer's disease: Understanding the mechanisms. Front Neurosci 17:1081938 Twarowski B, Herbet M (2023) Inflammatory Processes in Alzheimer's Disease-Pathomechanism, Diagnosis and Treatment: A Review. Int J Mol Sci 24(7):6518 Abdelazim AM, Abomughaid MM (2024) Oxidative stress: an overview of past research and future insights. All Life 17(1):2316092 Tian S, Huang Z, Meng Q, Liu Z (2021) Multi-target drug design of anti-Alzheimer's disease based on tacrine. Mini-Rev Med Chem 21(15):2039–2064 Kou X, Shi X, Pang Z, Yang A, Shen R, Zhao L (2023) A review on the natural components applied as lead compounds for potential multi-target anti-AD theranostic agents. Curr Med Chem 30(40):4586–4604 Siemers E (2015) Drug development in AD: point of view from the industry. JPAD-J Prev Alzheim 2(4):216–218 Maniam S (2024) Screening techniques for drug discovery in Alzheimer's disease. ACS Omega 9(6):6059–6073 Pravin N, Jozwiak K (2022) Effects of linkers and substitutions on multitarget directed ligands for Alzheimer’s diseases: Emerging paradigms and strategies. Int J Mol Sci 23(11):6085 Kasus-Jacobi A, Washburn JL, Laurence RB, Pereira HA (2022) Selecting multitarget peptides for Alzheimer’s disease. Biomolecules 12(10):1386 Carreiras MC, Mendes E, Perry MJ, Francisco AP, Marco-Contelles J (2013) The multifactorial nature of Alzheimer's disease for developing potential therapeutics. Curr Top Med Chem 13(15):1745–1770 Rosini M, Simoni E, Caporaso R, Minarini A (2016) Multitarget strategies in Alzheimer's disease: Benefits and challenges on the road to therapeutics. Future Med Chem 8(6):697–711 Atali S, Dorandish S, Devos J, Williams A, Price D, Taylor J et al (2020) Interaction of amyloid beta with humanin and acetylcholinesterase is modulated by ATP. FEBS Open Bio 10(12):2805–2823 Elsbaey M, Igarashi Y, Ibrahim MAA, Elattar E (2023) Click-designed vanilloid-triazole conjugates as dual inhibitors of AChE and Aβ aggregation. RSC Adv 13(5):2871–2883 Kulshreshtha A, Piplani P (2016) Current pharmacotherapy and putative disease-modifying therapy for Alzheimer's disease. Neurol Sci 37(9):1403–1435 Anand A, Patience AA, Sharma N, Khurana N (2017) The present and future of pharmacotherapy of Alzheimer's disease: A comprehensive review. Eur J Pharmacol 815:364–375 Benek O, Korabecny J, Soukup O (2020) A perspective on multi-target drugs for Alzheimer's disease. Trends Pharmacol Sci 41(7):434–445 Oliveira C, Cagide F, Teixeira J, Amorim R, Sequeira L, Mesiti F et al (2018) Hydroxybenzoic acid derivatives as dual-target ligands: Mitochondriotropic antioxidants and cholinesterase inhibitors. Front Chem 6:126 Zhang C, Wang L, Xu Y, Huang Y, Huang J, Zhu J et al (2022) Discovery of novel dual RAGE/SERT inhibitors for the potential treatment of the comorbidity of Alzheimer's disease and depression. Eur J Med Chem 236:114347 Mao F, Wang H, Ni W, Zheng X, Wang M, Bao K et al (2017) Design, synthesis, and biological evaluation of orally available first-generation dual-target selective inhibitors of acetylcholinesterase (AChE) and phosphodiesterase 5 (PDE5) for the treatment of Alzheimer's disease. ACS Chem Neurosci 9(2):328–345 Dai R, Sun Y, Su R, Gao H (2022) Anti-Alzheimer's disease potential of traditional chinese medicinal herbs as inhibitors of BACE1 and AChE enzymes. Biomed Pharmacother 154:113576 Karaca Ş, Osmaniye D, Sağlık BN, Levent S, Ilgın S, Özkay Y et al (2022) Synthesis of novel benzothiazole derivatives and investigation of their enzyme inhibitory effects against Alzheimer's disease. RSC Adv 12(36):23626–23636 Yao H, Uras G, Zhang P, Xu S, Yin Y, Liu J et al (2021) Discovery of novel tacrine–pyrimidone hybrids as potent dual AChE/GSK-3 inhibitors for the treatment of Alzheimer's disease. J Med Chem 64(11):7483–7506 Soliman AM, Ghorab WM, Lotfy DM, Karam HM, Ghorab MM, Ramadan LA (2023) Novel iodoquinazolinones bearing sulfonamide moiety as potential antioxidants and neuroprotectors. Sci Rep-UK 13(1):15546 Drozdowska D, Maliszewski D, Wróbel A, Ratkiewicz A, Sienkiewicz M (2023) New benzamides as multi-targeted compounds: A study on synthesis, AChE and BACE1 inhibitory activity and molecular docking. Int J Mol Sci 24(19):14901 Myadaraboina S, Alla M, Parlapalli A, Manda S (2018) Novel imidazo[1,2- a ]pyrazine derivatives: Design, synthesis, antioxidant and antimicrobial evaluations. Int J Chem Sci 16:276–288 Ellman GL, Courtney KD, Andres V Jr, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7(2):88–95 Table 1 Table 1 is available in the Supplementary Files section. Scheme 1 Scheme 1 is available in the Supplementary Files section. Supplementary Files Table1.docx scheme1.png Scheme 1. The synthetic route of target compounds 14a-14u. Reagents and conditions: (a) Corresponding carboxylic acid, DIEA, TBTU, CH 2 Cl 2 , rt, 3 h; (b) Chloroacetyl chloride, DIEA, CH 2 Cl 2 , -5 °C, 4 h; (c) HCl/MeOH, rt, 2 h; (d) 2-Amino-3-chloropyrazine, CH 3 CN, H 2 O, 80 °C, 8 h; (e) 8a-8o or 11a-11b, Cs 2 CO 3 , CH 3 CN, 80 °C, 12 h; SupportingInformation.doc Cite Share Download PDF Status: Published Journal Publication published 24 Aug, 2024 Read the published version in Medicinal Chemistry Research → Version 1 posted Editorial decision: Minor Revisions 13 Jul, 2024 Reviewers agreed at journal 13 Jun, 2024 Reviewers invited by journal 09 Jun, 2024 Editor assigned by journal 20 May, 2024 First submitted to journal 20 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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08:21:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4447664/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4447664/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00044-024-03298-w","type":"published","date":"2024-08-24T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58918080,"identity":"7356545d-71e2-4cd5-9f67-7be806f08152","added_by":"auto","created_at":"2024-06-24 06:18:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28747,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of dual-target inhibitors\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/332eec71f9312c12c0b41993.png"},{"id":58919063,"identity":"fa7143e8-7818-47e8-a314-5ff7a877b623","added_by":"auto","created_at":"2024-06-24 06:34:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68855,"visible":true,"origin":"","legend":"\u003cp\u003eDesign strategy for pyrimidinone derivatives.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/b506b7a53e12f902c8fe3e08.png"},{"id":58918597,"identity":"dcfc4962-092c-4151-90de-bce8e1a39f63","added_by":"auto","created_at":"2024-06-24 06:26:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54059,"visible":true,"origin":"","legend":"\u003cp\u003e(a)Lineweaver-Burk plot of inhibition kinetics of compound \u003cstrong\u003e14r\u003c/strong\u003e. (b) Secondary plot for calculation of steady-state inhibition constant (K\u003csub\u003ei\u003c/sub\u003e) of compound \u003cstrong\u003e14r\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/5fe58f3876ac995727463e0c.png"},{"id":58918087,"identity":"d2fdf2b4-5c8c-4bfb-b1d2-30c6154e09b6","added_by":"auto","created_at":"2024-06-24 06:18:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":253233,"visible":true,"origin":"","legend":"\u003cp\u003e3D model of AChE (4EY7) docked with compound \u003cstrong\u003e14r\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/8d325c30d59510029b61dbff.png"},{"id":58918090,"identity":"48367c8b-258a-45e1-98a6-80e4eec3a658","added_by":"auto","created_at":"2024-06-24 06:18:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":230653,"visible":true,"origin":"","legend":"\u003cp\u003e3D model of BuChE (5K5E) docked with compound \u003cstrong\u003e14r\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/59ea817abdd7af265305defd.png"},{"id":58919064,"identity":"f1cfd0f0-550f-472f-b21c-8496ac8de954","added_by":"auto","created_at":"2024-06-24 06:34:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":74833,"visible":true,"origin":"","legend":"\u003cp\u003eTime dependent RMSD plots Apo AChE/BChE protein (red), 14r-AChE/BChE complex (blue), resulted from 100 ns MD\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/a107ca346d1006aba0ecdc0c.png"},{"id":58918085,"identity":"5155453b-829b-4044-b6fc-29e5b88098aa","added_by":"auto","created_at":"2024-06-24 06:18:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":74860,"visible":true,"origin":"","legend":"\u003cp\u003eRMSF plots Apo AChE/BuChE Protein (purple), 14r-AChE/BChE complex (green), resulted from 100 ns MD\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/8bfb070a3617571c164b28c5.png"},{"id":63300264,"identity":"c1df3c9e-cb04-4b1f-ad96-a1a8319fcf32","added_by":"auto","created_at":"2024-08-26 16:13:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2339664,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/e3b7d2a5-152d-4d94-afbf-e7eb5585c8cb.pdf"},{"id":58918596,"identity":"a664c49b-481f-4ec1-b669-55cf9c493c13","added_by":"auto","created_at":"2024-06-24 06:26:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":102106,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/b1110b385e5943ebd1bb3251.docx"},{"id":58918599,"identity":"8dc90bdb-f2e9-43a7-b819-6aa7d495bdcc","added_by":"auto","created_at":"2024-06-24 06:26:30","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":90160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme\u003c/strong\u003e \u003cstrong\u003e1. \u003c/strong\u003eThe synthetic route of target compounds \u003cstrong\u003e14a-14u\u003c/strong\u003e. Reagents and conditions: (a) Corresponding carboxylic acid, DIEA, TBTU, CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e, rt, 3 h; (b) Chloroacetyl chloride, DIEA, CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e, -5 °C, 4 h; (c) HCl/MeOH, rt, 2 h; (d) 2-Amino-3-chloropyrazine, CH\u003csub\u003e3\u003c/sub\u003eCN, H\u003csub\u003e2\u003c/sub\u003eO, 80 °C, 8 h; (e) \u003cstrong\u003e8a-8o \u003c/strong\u003eor\u003cstrong\u003e 11a-11b\u003c/strong\u003e, Cs\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, CH\u003csub\u003e3\u003c/sub\u003eCN, 80 °C, 12 h;\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/b65737c809966aed81fb17ea.png"},{"id":58918601,"identity":"961adf70-7936-4756-a0e4-66ab9fa4f516","added_by":"auto","created_at":"2024-06-24 06:26:30","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3674112,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-4447664/v1/1973429ea272889711e77341.doc"}],"financialInterests":"","formattedTitle":"Design, synthesis and evaluation of imidazo[1,2-a]pyrazin-8(7H)-one derivatives as acetylcholinesterase inhibitors and antioxidants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer's disease (AD) is a neurological condition marked by extensive neuronal damage in many brain regions. Currently, AD affects more than 50\u0026nbsp;million people worldwide, and this number will continue to increase with the aging population growing [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. And by 2050, the number of senior patients with the condition is expected to triple [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearchers have made many efforts to treat AD, and various hypotheses have been proposed by researchers, such as the cholinergic hypothesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], the amyloid (Aβ) toxicity hypothesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the Tau protein abnormality hypothesis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], the neuroinflammatory hypothesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], the free radical damage hypothesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and so on. Due to the complex pathogenesis of AD, a single target drug cannot cure this disease fundamentally [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Dual or multiple target drugs involved in two or more aspects of AD pathogenesis may generate a synergistic effect and ultimately achieve an ideal therapeutic effect [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, in consideration of the complicated interrelation among multiple pathways, a multitarget strategy possesses a more effective and promising capacity to manage the special disease network of AD with synergistic adjustment toward more pivotal targets, becoming a hot research topic for the potentially curable treatment of AD [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the drugs currently approved by the U.S. Food and Drug Administration for the treatment of AD, most are acetylcholinesterase (AChE) inhibitors [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although inadequate for curing AD, inhibition of AChE remains the most successful strategy and has been shown to be transiently capable of improving memory and cognitive function in AD patients [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, multitarget-directed ligands (MTDLs) combining AChE inhibitors with other active pharmacophore within a single drug would generate a more significant reduction in AD symptoms [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Based on this theory, a number of active compounds have been discovered, these compounds not only enhance acetylcholine (ACh) levels in the brain by inhibiting acetylcholinesterase, but also exhibit one or more other anti-AD biological activities such as glycogen synthase kinase 3 beta (GSK-3β) inhibitory activity, monoamine oxidase (MAO) inhibitory activity, and antioxidation, such as compounds \u003cb\u003e1\u0026ndash;3\u003c/b\u003e [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder the guidance of MTDLs theory, dual-target compounds with AChE inhibitory activity and antioxidant activity for the treatment of AD was designed in this paper, and their design philosophy was as follows: compound \u003cb\u003e4\u003c/b\u003e was found to have strong AChE inhibitory activity. Through structural analysis, it was found that its A region was the key structure with high activity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, compound \u003cb\u003e5\u003c/b\u003e displayed promising antioxidant activity, and the imidazo[1,2-\u003cem\u003ea\u003c/em\u003e]pyrazine ring on the B region of compound \u003cb\u003e5\u003c/b\u003e was found to be the important functional group for their antioxidant activity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In order for region A with antioxidant activity to be connected to region B with AChE inhibitory activity, we converted the imidazo[1,2-\u003cem\u003ea\u003c/em\u003e]pyrazine ring to imidazo[1,2-\u003cem\u003ea\u003c/em\u003e]pyrazin-8(7\u003cem\u003eH\u003c/em\u003e)-one ring, and the two active regions were linked by acetamide. To this end, the target compounds were designed. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemical synthesis\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe synthetic strategy for designed target compounds \u003cb\u003e14a\u0026ndash;14u\u003c/b\u003e was depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Briefly, compound \u003cb\u003e6a-6k\u003c/b\u003e reacted with various Q\u003csub\u003e1\u003c/sub\u003e to provide \u003cb\u003e7a-7o\u003c/b\u003e. Then compounds \u003cb\u003e7a-7o\u003c/b\u003e could be reacted with the chloroacetyl chloride in CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e to get compounds \u003cb\u003e8a-8o\u003c/b\u003e under the condition of an ice water bath. In parallel, compound \u003cb\u003e6a-6k\u003c/b\u003e reacted with Q\u003csub\u003e2\u003c/sub\u003e to provide \u003cb\u003e9a-9b\u003c/b\u003e. The removal of Boc group was achieved by dissolving \u003cb\u003e10a-10b\u003c/b\u003e in MeOH solution with HCl gas at room temperature for 2h, then, \u003cb\u003e10a-10b\u003c/b\u003e reacted with the chloroacetyl chloride to yield compounds \u003cb\u003e11a-11b.\u003c/b\u003e Commercially available 2-amino-3-chloropyrazine was condensed with the corresponding α-halocarbonyl derivatives to build \u003cb\u003e13a-13e\u003c/b\u003e. Eventually, the target compounds \u003cb\u003e14a-14u\u003c/b\u003e were synthesized by reaction of compounds \u003cb\u003e13a-13e\u003c/b\u003e with compounds \u003cb\u003e8a-8o\u003c/b\u003e and \u003cb\u003e11a-11b\u003c/b\u003e. The structures of new compounds were confirmed by the \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR, HRMS and IR spectra, and the purity of all the target compounds was determined to be over 95.0% by high-performance liquid chromatography (HPLC) analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eAChE and BChE inhibition assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to Ellman method [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], the inhibitory activities of target compounds \u003cb\u003e14a-14u\u003c/b\u003e against eeAChE and eqBuChE \u003cem\u003ein vitro\u003c/em\u003e were tested with galantamine as the reference compound. EeAChE and eqBuChE were purchased from Sigma-Aldrich. The corresponding IC\u003csub\u003e50\u003c/sub\u003e values were shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, most of the synthesized target compounds had moderate inhibitory activity against eeAChE and eqBuChE. Compound \u003cb\u003e14r\u003c/b\u003e was the most potent eeAChE inhibitor with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.47 \u0026micro;M and moderate inhibitory activity against eqBuChE (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.02 \u0026micro;M). Meanwhile compound \u003cb\u003e14r\u003c/b\u003e had the best selectivity of AChE and selectivity index (SI) values was 23.45. Compound \u003cb\u003e14r\u003c/b\u003e has better activity as well as eeAChE selectivity compared to reference drug galantamine (eeAChE IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.01 \u0026micro;M, eqBuChE IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;18.46 \u0026micro;M, SI\u0026thinsp;=\u0026thinsp;3.68).\u003c/p\u003e \u003cp\u003eFor eeAChE, the compounds had better activity at n\u0026thinsp;=\u0026thinsp;1 compared to n\u0026thinsp;=\u0026thinsp;2. For example, compound \u003cb\u003e14i\u003c/b\u003e (eeAChE IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.74 \u0026micro;M) had better activity than \u003cb\u003e14j\u003c/b\u003e (eeAChE IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.56 \u0026micro;M). When the substituent on benzene ring of R\u003csub\u003e2\u003c/sub\u003e was an electron donating group, the \u003cb\u003e14i\u003c/b\u003e with two methoxy groups on the benzene ring of R\u003csub\u003e2\u003c/sub\u003e held the highest activity. When the substituent on benzene ring of R\u003csub\u003e2\u003c/sub\u003e was an electron withdrawing group, para substituted fluorine has the best activity than trifluoromethyl, and among compounds with trifluoromethyl as a substituent, meta substituted fluorine has the best activity. When Q was an unsaturated ring, there was a significant increase in activity compared to when Q was a saturated ring. When the substituent of R\u003csub\u003e1\u003c/sub\u003e was meta methoxy, its activity was stronger than that of dimethoxy, para fluorine, and naphthalene ring.\u003c/p\u003e \u003cp\u003eIn the case of eqBuChE, compound \u003cb\u003e14j\u003c/b\u003e (eqBuChE, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.49 \u0026micro;M) had the best inhibitory activity to eqBuChE. The compounds with the substituents on R\u003csub\u003e2\u003c/sub\u003e in the ortho position usually exhibited better activity, and the compounds had better activity at n\u0026thinsp;=\u0026thinsp;2 compared to n\u0026thinsp;=\u0026thinsp;1. When replaced the R\u003csub\u003e1\u003c/sub\u003e substituent, the structure with a larger spatial resistance at the substituent of R\u003csub\u003e1\u003c/sub\u003e possessed better activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eKinetic study of AChE inhibition\u003c/h2\u003e \u003cp\u003eIn order to further explore its mechanism of AChE inhibition on \u003cb\u003e14r\u003c/b\u003e with the best inhibitory activity against AChE, compound \u003cb\u003e14r\u003c/b\u003e was selected for enzyme kinetic study. The Lineweaver-Burk plot was constructed for three varied concentration of compound \u003cb\u003e14r\u003c/b\u003e against six different concentrations of substrate (acetylthiocholine iodide, ATCI). As in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (a), the intersecting of double reciprocal curve in the second quadrant showed that compound \u003cb\u003e14r\u003c/b\u003e displayed both competitive and non-competitive inhibitory effects on AChE. Used the slope in the Lineweaver Burk plot to plot the compound concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (b)), the K\u003csub\u003ei\u003c/sub\u003e value was 0.54 \u0026micro;M, which was consistent with the IC\u003csub\u003e50\u003c/sub\u003e value. The K\u003csub\u003ei\u003c/sub\u003e value was confirmed the strong affinity of \u003cb\u003e14r\u003c/b\u003e towards AChE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStudies of anti-oxidative activity\u003c/h2\u003e \u003cp\u003eStable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were used to determine the antioxidant activity of the synthesized compounds \u003cb\u003e14a\u003c/b\u003e-\u003cb\u003e14u\u003c/b\u003e. The compounds for antioxidant activity were evaluated using standard procedures and ascorbic acid was used as a control drug. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, among these compounds, compound \u003cb\u003e14o\u003c/b\u003e exhibited excellent antioxidant activity with IC\u003csub\u003e50\u003c/sub\u003e value of 89.33 \u0026micro;M, yet it was lower than that of ascorbic acid. And the remaining compounds had only moderate antioxidant activity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant activity of \u003cb\u003e14a-14u\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompd.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e(\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCompd.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e(\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e119.34\u0026thinsp;\u0026plusmn;\u0026thinsp;16.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14l\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e118.88\u0026thinsp;\u0026plusmn;\u0026thinsp;10.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e186.45\u0026thinsp;\u0026plusmn;\u0026thinsp;11.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14m\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e190.67\u0026thinsp;\u0026plusmn;\u0026thinsp;12.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e170.85\u0026thinsp;\u0026plusmn;\u0026thinsp;14.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14n\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.31\u0026thinsp;\u0026plusmn;\u0026thinsp;11.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14d\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e181.77\u0026thinsp;\u0026plusmn;\u0026thinsp;8.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14o\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14e\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e162.92\u0026thinsp;\u0026plusmn;\u0026thinsp;21.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14p\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e153.82\u0026thinsp;\u0026plusmn;\u0026thinsp;13.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14f\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e131.78\u0026thinsp;\u0026plusmn;\u0026thinsp;12.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14q\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e168.39\u0026thinsp;\u0026plusmn;\u0026thinsp;16.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14g\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e158.24\u0026thinsp;\u0026plusmn;\u0026thinsp;9.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14r\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e106.81\u0026thinsp;\u0026plusmn;\u0026thinsp;7.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14h\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e203.26\u0026thinsp;\u0026plusmn;\u0026thinsp;12.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14s\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e186.84\u0026thinsp;\u0026plusmn;\u0026thinsp;16.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14i\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e148.19\u0026thinsp;\u0026plusmn;\u0026thinsp;17.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14t\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e161.76\u0026thinsp;\u0026plusmn;\u0026thinsp;8.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14j\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.98\u0026thinsp;\u0026plusmn;\u0026thinsp;15.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14u\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e145.76\u0026thinsp;\u0026plusmn;\u0026thinsp;11.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14k\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.67\u0026thinsp;\u0026plusmn;\u0026thinsp;8.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eAscorbic Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eSD\u003csup\u003ea\u003c/sup\u003e indicates standard deviation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking studies\u003c/h2\u003e \u003cp\u003eTo gain insight into the binding mode of compound \u003cb\u003e14r\u003c/b\u003e to AChE (4EY7) and BuChE (5K5E) enzymes, molecular docking studies were performed using AutoDock 4.2, and the docking results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the 3D model of AChE docked with compound \u003cb\u003e14r\u003c/b\u003e. The aminobenzamide ring could simultaneously produce π-π interactions with Trp-286 and Tyr-341. The amide bond on the pyrazine of \u003cb\u003e14r\u003c/b\u003e could form a hydrogen bonding interaction with Tyr-124. Meanwhile, the meta methoxy substituted benzene ring could simultaneously produce π-π interactions with Trp-86. Additionally, the imidazole ring of \u003cb\u003e14r\u003c/b\u003e could form a π-π stacking with His-447. At the same time, \u003cb\u003e14r\u003c/b\u003e could interact simultaneously with the catalytic active site (CAS) and peripheral anionic site (PAS) after entering the binding pocket of AChE.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the 3D model of BuChE docked with compound \u003cb\u003e14r\u003c/b\u003e. The amide bond on the pyrazine of \u003cb\u003e14r\u003c/b\u003e could form a hydrogen bonding interaction with Thr-120. Meanwhile, the aminobenzamide ring could produce π-π interactions with Trp-82. Additionally, the a aminobenzamide ring could form a hydrogen bonding interaction with Tyr-440.\u003c/p\u003e \u003cp\u003eAccording to Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, it was able to find that compound \u003cb\u003e14r\u003c/b\u003e produced more interactions with AChE and relatively few interactions with BuChE. This partly explained the the better activity and selectivity of compound \u003cb\u003e14r\u003c/b\u003e for AChE.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics simulation study\u003c/h2\u003e \u003cp\u003eThe molecular dynamics (MD) simulation is a popular method for determining structural stability and the molecular interaction profiles of proteins and small molecules. Generally, the binding of ligand to protein is a dynamic phenomenon, which occurs in less than one nanosecond. MD simulations over a period of 100 ns were carried out for \u003cb\u003e14r\u003c/b\u003e with targeted enzyme (AChE and BuChE). The MD simulations allowed us to observe the stability and patterns of the established interactions of \u003cb\u003e14r\u003c/b\u003e with the active sites of the enzymes AChE and BuChE. The dynamic behavior of the whole simulated system was examined using a variety of quantitative parameters, including root mean square deviation (RMSD) and root mean square fluctuation (RMSF).\u003c/p\u003e \u003cp\u003eThe RMSD is an excellent indicator of protein and ligand structural stability, as well as the magnitude of atom position deviation from the starting position. The smaller the deviation, the more stable the conformation, and vice versa. The stability of AChE in the apolipoprotein (APO) form (enzyme without the ligand) was observed from 34 ns (the production phase) of the simulation; as the enzyme complexed with the ligand (\u003cb\u003e14r\u003c/b\u003e), its production phase was from 72 ns. The stability of BuChE in the APO form was observed from 23 ns of the simulation; as the enzyme complexed with the ligand (\u003cb\u003e14r\u003c/b\u003e), its productive phase was 36 ns (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Steady RMSD values indicated well-equilibrated states of the systems during MD simulations.\u003c/p\u003e \u003cp\u003eThe RMSF analysis indicated the local conformational changes in protein side chains during the whole simulation. The analysis of the RMSF values demonstrated the atomic fluctuations of the APO form of AChE and its complex, as well as the APO form of BuChE and its complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The mean RMSF values of the APO form and the complexes formed showed a minimal fluctuation for most residues, indicating a high stability and strong interactions between \u003cb\u003e14r\u003c/b\u003e and AChE/BuChE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn silico molecular property analysis\u003c/h2\u003e \u003cp\u003eDrug-likeness is a complex balance of various molecular properties, such as hydrophobicity, electronic distribution, hydrogen bonding characteristics, molecule size, and flexibility and presence of various pharmacophoric features. Therefore, the properties of the synthesized compounds \u003cb\u003e14a-14u\u003c/b\u003e were predicted online using the SwissADME(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch\u003c/span\u003e\u003cspan address=\"http://www.swissadme.ch\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the synthesized compounds were found to match most of the drug properties. Unfortunately, most of the tested compounds can not pass the blood-brain barrier, so they cannot be used directly as oral drugs and need to be further optimized.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn silico prediction of molecular properties for target compounds \u003cb\u003e14a-14u\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCODE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMW\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLogP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHBA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHBD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTPSA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003en\u003csub\u003eviolation\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBBB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;130Ǻ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e525.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e106.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e575.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e106.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e 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align=\"left\" colname=\"c2\"\u003e \u003cp\u003e537.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e115.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14i\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e567.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e125.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14j\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e581.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e 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align=\"left\" colname=\"c2\"\u003e \u003cp\u003e526.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e119.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14n\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e525.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e106.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14o\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e532.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e147.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14p\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e517.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14q\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e517.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14r\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e525.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e106.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14s\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e555.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e115.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14t\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e513.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14u\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e545.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMW: 150\u0026thinsp;\u0026lt;\u0026thinsp;MV\u0026thinsp;\u0026lt;\u0026thinsp;500, LIPO(Lipophility): -0.7\u0026thinsp;\u0026lt;\u0026thinsp;LOGP\u0026thinsp;\u0026lt;\u0026thinsp;+\u0026thinsp;5.0, HBA(H-bond acceptors): 0\u0026thinsp;\u0026lt;\u0026thinsp;Num. H-bond acceptors\u0026thinsp;\u0026lt;\u0026thinsp;10, HBD(H-bond donors): 0\u0026thinsp;\u0026lt;\u0026thinsp;Num. H-bond donors\u0026thinsp;\u0026lt;\u0026thinsp;5, POLAR(Polarity): 20 \u0026Aring;\u0026sup2; \u0026lt; TPSA\u0026thinsp;\u0026lt;\u0026thinsp;130 \u0026Aring;\u0026sup2;, n\u003csub\u003eviolation\u003c/sub\u003e(number violations from Lipinski\u0026rsquo;s rule): n\u003csub\u003eviolation\u003c/sub\u003e \u0026le; 1, BBB: The ability of crossing BBB blood brain barrier.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, a new series of imidazo[1,2-\u003cem\u003ea\u003c/em\u003e]pyrazin-8(7\u003cem\u003eH\u003c/em\u003e)-one derivatives was designed, synthesized, and evaluated for their ChE inhibitory activity and antioxidant activity. Biological assays demonstrated that all synthesized compounds had certain ChE inhibitory activity and antioxidant activity \u003cem\u003ein vitro\u003c/em\u003e. Among them, compound \u003cb\u003e14r\u003c/b\u003e showed the strongest inhibitory activity against AChE with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.47 \u0026micro;M, which was superior to galantamine as the control compound. Furthermore, molecular docking studies showed that \u003cb\u003e14r\u003c/b\u003e was able to bind to both the CAS and PAS of AChE, which was consistent with the mixed inhibition pattern shown by enzyme kinetic studies. Moreover, a standard atomistic 100 ns dynamic simulation results of the binding stability of the compound \u003cb\u003e14r\u003c/b\u003e to AChE revealed the conformational stability in the binding cavity. In addition, the synthesized compounds were found to match most of the drug properties. Taken together, compound \u003cb\u003e14r\u003c/b\u003e might be a promising lead compound for the development of new anti-AD drugs.\u003c/p\u003e"},{"header":"Experimental Section","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemistry\u003c/h2\u003e \u003cp\u003eAll experiments were carried out under air atmosphere, the reagents were commercially available analytically pure or chemically pure, unless stated otherwise. High Resolution Mass Spectrometry was determined by Thermo QExactive; the nuclear magnetic resonance spectrum was measured by Bruker Avance III 600 MHz nuclear magnetic resonance spectrometer. The purity of the target compounds were 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of Compounds 14a-14u\u003c/h2\u003e \u003cp\u003e \u003cb\u003e8a\u003c/b\u003e (0.32 g, 1 mmol) was added to a stirred solution of \u003cb\u003e13a\u003c/b\u003e (0.241 g, 1 mmol) and Cs\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (0.326, 1.2 mmol) in CH\u003csub\u003e3\u003c/sub\u003eCN (8 mL). The mixture was stirred at 80\u0026deg;C for 12 h, and then the solvent was evaporated in vacuo. The crude product was dissolved in CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e and washed with a saturated solution of sodium carbonate. The organic layer was separated, dried (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel to afford crude compounds \u003cb\u003e14a\u003c/b\u003e. The crude compounds \u003cb\u003e14a\u003c/b\u003e was recrystallized from ethyl acetate (5 mL) to give pure compounds \u003cb\u003e14a\u003c/b\u003e. The same method produces for compounds \u003cb\u003e14b-14u\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14a)\u003c/b\u003e: white solid; Yield: 52%; mp: 208.3-208.9\u0026deg;C; Purity: 96.20% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.53 (s, 1H), 9.04 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 8.18 (s, 1H), 8.09 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 7.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H), 7.61\u0026ndash;7.53 (m, 2H), 7.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.37\u0026ndash;7.30 (m, 2H), 7.19\u0026ndash;7.09 (m, 3H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 4.79 (s, 2H), 4.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 3.79 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.10, 165.80, 161.91, 160.31, 159.10, 152.99, 144.09, 138.76, 136.81, 135.79, 135.16, 129.18, 129.13, 128.79, 126.67, 125.67, 121.94, 121.84, 118.50, 114.99, 114.85, 114.20, 112.43, 106.60, 55.14, 50.34, 41.94; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 526.1890 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 526.1894 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3292, 1679, 1674, 1639.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)-\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-(trifluoromethyl)benzyl)benzamide(14b)\u003c/b\u003e: white solid; Yield: 43%; mp: 186.5-187.2\u0026deg;C; Purity: 98.27% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.48 (s, 1H), 9.12 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 7.78 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 1H), 7.69 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 2H), 7.61 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 2H), 7.44 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.17 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.02 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 4.80 (s, 2H), 4.55 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.80 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.24, 165.82, 159.10, 153.00, 144.52, 144.09, 138.78, 136.81, 135.00, 128.84, 127.83, 127.54, 127.33, 126.67, 125.69, 125.16, 125.14, 125.11, 125.09, 122.00, 121.95, 118.51, 114.21, 112.43, 106.60, 55.14, 50.35, 42.35; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 576.1858 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 576.1861 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3291, 1676, 1674, 1672.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)-\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(3-(trifluoromethyl)benzyl)benzamide(14c)\u003c/b\u003e: white solid; Yield: 39%; mp: 231.7-232.6\u0026deg;C; Purity: 98.99% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.49 (s, 1H), 9.12 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 1H), 8.19 (s, 1H), 8.09 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 7.78 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 1H), 7.66 (s, 1H), 7.64\u0026ndash;7.59 (m, 2H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8 Hz, 2H), 7.56 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 7.44 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.17 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.02 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 4.79 (s, 2H), 4.54 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.80 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.23, 165.82, 159.10, 152.99, 144.08, 141.14, 138.79, 136.80, 134.97, 131.37, 129.35, 129.08, 128.87, 126.67, 125.69, 123.74, 123.49, 121.95, 118.47, 114.20, 112.43, 106.60, 55.14, 50.34, 42.29; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 576.1858 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 576.1865 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3259, 1679, 1639, 1631.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)-\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(2-(trifluoromethyl)benzyl)benzamide(14d)\u003c/b\u003e: white solid; Yield: 52%; mp: 256.5-257.2\u0026deg;C; Purity: 97.55% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.50 (s, 1H), 9.09 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 1H), 8.19 (s, 1H), 8.11 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 7.80 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 7.73 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.65 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 7.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.51 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.46 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 2H), 7.17 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 4.80 (s, 2H), 4.65 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 3.80 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.41, 165.84, 159.09, 152.99, 144.08, 138.81, 137.56, 136.80, 134.88, 132.62, 128.88, 128.11, 127.24, 126.66, 126.18, 125.73, 125.68, 125.39, 122.07, 122.00, 121.96, 118.51, 114.20, 112.43, 106.60, 55.14, 50.36, 40.07; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 576.1858 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 576.1852 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3307, 1671, 1655, 1631.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(3,5-bis(trifluoromethyl)benzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo-[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]-pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14e)\u003c/b\u003e: yellow solid; Yield: 58%; mp: 223.4-224.1\u0026deg;C; Purity: 98.40% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.49 (s, 1H), 9.19 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 8.18 (s, 1H), 8.09 (s, 1H), 8.01 (s, 2H), 7.99 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.0 Hz, 2H), 7.78 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 7.59 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 2H), 7.45 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.1 Hz, 2H), 4.79 (s, 2H), 4.63 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 3.79 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.42, 165.84, 159.09, 152.99, 143.21, 136.79, 130.25, 130.03, 128.96, 128.13, 126.66, 122.08, 121.94, 118.42, 114.20, 112.43, 106.61, 55.13, 50.34, 42.07; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e31\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 644.1732 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 644.1726 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3295, 1678, 1647, 1612.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-methoxybenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14f)\u003c/b\u003e: white solid; Yield: 41%; mp: 208.4\u0026ndash;209.0\u0026deg;C; Purity: 99.58% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.47 (s, 1H), 8.94 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 1H), 8.18 (s, 1H), 8.06 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 7.76 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.56 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 1H), 7.41 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.23 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 2H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.5 Hz, 1H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 6.87 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 4.79 (s, 2H), 4.38 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.79 (s, 3H), 3.71 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 165.96, 165.78, 159.12, 158.16, 144.08, 138.69, 136.80, 135.56, 131.59, 128.77, 128.54, 126.67, 125.56, 121.95, 118.49, 114.21, 113.65, 112.43, 106.61, 55.14, 55.02, 50.33, 42.06; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e: 538.2090 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 538.2088 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3082, 1675, 1635, 1623.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(3-methoxybenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14g)\u003c/b\u003e: white solid; Yield: 33%; mp: 234.7-235.5\u0026deg;C; Purity: 95.43% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.47 (s, 1H), 8.99 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 1H), 8.18 (s, 1H), 8.08 (s, 1H), 7.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 7.78 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.62\u0026ndash;7.52 (m, 2H), 7.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.23 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.17 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.02 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 6.88 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.8 Hz, 2H), 6.80 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.6 Hz, 1H), 4.79 (s, 2H), 4.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.80 (s, 3H), 3.72 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.11, 165.80, 159.28, 159.10, 152.99, 144.09, 141.23, 138.73, 136.81, 135.30, 129.29, 128.80, 126.67, 125.70, 121.96, 121.79, 119.32, 118.52, 114.21, 112.92, 112.43, 112.03, 106.60, 55.14, 54.94, 50.33, 42.56; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e: 538.2090 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 538.2097 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3271, 1686, 1651, 1620.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(2-methoxybenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14h)\u003c/b\u003e: yellow solid; Yield: 39%; mp: 251.8-252.5\u0026deg;C; Purity: 99.06% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.48 (s, 1H), 8.83 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 1H), 8.18 (s, 1H), 8.07 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 7.79 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 7.61 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.2 Hz, 1H), 7.43 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.22 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.3 Hz, 2H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 6.98 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 6.90 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 1H), 4.79 (s, 2H), 4.43 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 3.82 (s, 3H), 3.79 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.24, 165.80, 159.10, 156.53, 153.00, 144.09, 138.72, 136.80, 135.32, 128.79, 127.84, 127.12, 126.83, 126.67, 125.67, 121.95, 121.77, 120.08, 118.53, 114.21, 112.43, 110.43, 106.61, 55.31, 55.14, 50.34, 37.62; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e: 538.2090 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 538.2094 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3267, 1683, 1627, 1596.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(3,4-dimethoxybenzyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyra-zin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14i)\u003c/b\u003e: white solid; Yield: 42%; mp: 241.9-242.7\u0026deg;C; Purity: 99.83% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.46 (s, 1H), 8.92 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 1H), 8.18 (s, 1H), 8.07 (s, 1H), 7.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 7.76 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.4 Hz, 1H), 7.58 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 7.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 6.94 (s, 1H), 6.89 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 6.83 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.3 Hz, 1H), 4.79 (s, 2H), 4.39 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.80 (s, 3H), 3.72 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.01, 165.79, 159.10, 152.99, 148.64, 147.77, 144.09, 138.71, 136.81, 135.42, 132.10, 128.76, 126.67, 125.69, 121.95, 121.72, 119.38, 118.53, 114.20, 112.42, 111.85, 111.55, 106.59, 55.59, 55.44, 55.14, 50.34, 42.41; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e31\u003c/sub\u003eH\u003csub\u003e29\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e: 568.2196 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 568.2203 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3298, 1679, 1639, 1616.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(3,4-dimethoxyphenethyl)-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]p-yrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14j)\u003c/b\u003e: white solid; Yield: 46%; mp: 264.2-264.9\u0026deg;C; Purity: 99.86% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.46 (s, 1H), 8.48 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.5 Hz, 1H), 8.19 (s, 1H), 8.03 (s, 1H), 7.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 7.73 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H), 7.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.50 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.40 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.17 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.02 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 6.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H), 6.82 (s, 1H), 6.73 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 4.79 (s, 2H), 3.80 (s, 3H), 3.70 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 6H), 3.45 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.8 Hz, 2H), 2.76 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.00, 165.77, 159.10, 152.99, 148.59, 147.20, 144.09, 138.68, 136.80, 135.56, 131.97, 128.70, 126.67, 125.67, 121.96, 121.84, 121.62, 120.46, 118.41, 114.21, 112.55, 112.43, 111.96, 106.60, 55.48, 55.33, 55.14, 50.34, 40.99, 34.54; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e: 582.2352 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 582.2355 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3311, 1671, 1647, 1624.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-benzyl-3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)ace-tamido)benzamide(14k)\u003c/b\u003e: white solid; Yield: 46%; mp: 247.2-247.8\u0026deg;C; Purity: 99.37% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.48 (s, 1H), 9.02 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 1H), 8.18 (s, 1H), 8.07 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 7.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H), 7.59 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8 Hz, 2H), 7.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.31 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7, 5.2 Hz, 4H), 7.26\u0026ndash;7.20 (m, 1H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.0 Hz, 2H), 4.79 (s, 2H), 4.46 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.79 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.10, 165.79, 159.10, 152.99, 144.08, 139.61, 138.72, 136.80, 135.26, 128.80, 128.23, 127.14, 126.68, 125.67, 121.95, 121.80, 118.50, 114.20, 112.43, 106.61, 55.14, 50.34, 42.60; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 508.1985 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 508.1996 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3291, 1671, 1647, 1624.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-2-(3-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)phenyl)acetamide(14l)\u003c/b\u003e: white solid; Yield: 38%; mp: 218.3-218.8\u0026deg;C; Purity: 98.98% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.30 (s, 1H), 8.53 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 8.18 (s, 1H), 7.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.56 (s, 1H), 7.46 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 1H), 7.30\u0026ndash;7.24 (m, 2H), 7.24 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.10 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 6.98 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 4.77 (s, 2H), 4.24 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 3.80 (s, 3H), 3.45 (s, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 169.92, 165.52, 161.91, 160.31, 159.10, 152.99, 144.08, 138.57, 136.98, 136.82, 135.58, 135.56, 129.16, 129.11, 128.58, 126.66, 125.71, 124.29, 122.00, 119.79, 117.29, 114.99, 114.85, 114.20, 112.40, 106.53, 55.14, 50.32, 42.35, 41.49; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 540.2047 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 540.2041 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3251, 1671, 1639, 1631.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-5-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)nicotinamide(14m)\u003c/b\u003e: white solid; Yield: 41%; mp: 178.6-179.5\u0026deg;C; Purity: 98.55% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.71 (s, 1H), 9.31\u0026ndash;9.21 (m, 1H), 8.88 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.5 Hz, 1H), 8.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H), 8.47 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.3 Hz, 1H), 8.19 (s, 1H), 7.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 7.60 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.39\u0026ndash;7.33 (m, 2H),, 7.19\u0026ndash;7.14 (m, 2H), 7.13 (s, 1H), 7.02 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 4.83 (s, 2H), 4.46 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 2H), 3.80 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 169.18, 166.48, 164.55, 159.12, 144.12, 142.96, 142.85, 136.77, 135.14, 130.04, 129.28, 129.22, 126.68, 125.18, 121.85, 115.06, 114.92, 114.21, 112.48, 106.71, 55.14, 50.32, 41.99; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eFN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 527.1843 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 527.1844 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3302, 1683, 1643, 1620.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-4-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-y-l)acetamido)benzamide(14n)\u003c/b\u003e: white solid; Yield: 53%; mp: 239.3-240.1\u0026deg;C; Purity: 99.63% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.55 (s, 1H), 9.03\u0026ndash;8.80 (m, 1H), 8.19 (s, 1H), 8.01\u0026ndash;7.87 (m, 2H), 7.86 (s, 2H), 7.74\u0026ndash;7.63 (m, 2H), 7.59 (s, 1H), 7.35 (s, 2H), 7.21\u0026ndash;7.08 (m, 3H), 7.07\u0026ndash;6.94 (m, 2H), 4.80 (s, 2H), 4.44 (s, 2H), 3.80 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 168.17, 167.74, 165.63, 162.87, 160.55, 152.61, 147.63, 144.04, 135.47, 129.21, 129.15, 128.22, 126.67, 121.81, 118.32, 114.99, 114.84, 114.21, 112.29, 106.78, 55.14, 50.29, 41.88; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 526.1890 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 526.1892 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3267, 1683, 1659, 1631.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-2-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)thiazole-5-carboxamide(14o)\u003c/b\u003e: white solid; Yield: 46%; mp: 216.4\u0026ndash;217.0\u0026deg;C; Purity: 99.81% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 12.76 (s, 1H), 9.03 (s, 1H), 8.24\u0026ndash;8.06 (m, 2H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.5 Hz, 2H), 7.60 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.35 (s, 2H), 7.24\u0026ndash;7.07 (m, 3H), 7.02 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 2H), 4.90 (s, 2H), 4.41 (s, 2H), 3.80 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 171.00, 168.34, 164.53, 163.07, 158.97, 154.71, 143.98, 142.43, 140.43, 136.62, 129.33, 129.28, 126.66, 120.08, 115.08, 114.94, 114.20, 112.51, 107.15, 55.13, 46.16, 41.95; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eFN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS: 533.1407 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 533.1416 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3326, 1703, 1655, 1619.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-1-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetyl)piperidine-3-carboxamide(14p)\u003c/b\u003e: white solid; Yield: 46%; mp: 188.3-188.9\u0026deg;C; Purity: 98.28% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 8.46 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 1H), 8.16 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 7.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.39\u0026ndash;7.29 (m, 1H), 7.28\u0026ndash;7.22 (m, 1H), 7.18\u0026ndash;7.07 (m, 2H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 2H), 6.96 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 4.91 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.3 Hz, 1H), 4.79 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.2, 3.2 Hz, 1H), 4.43\u0026ndash;4.28 (m, 1H), 4.30\u0026ndash;4.07 (m, 2H), 3.91 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.6 Hz, 1H), 3.80 (s, 3H), 3.25\u0026ndash;3.05 (m, 1H), 2.72 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.1 Hz, 1H), 2.42\u0026ndash;2.23 (m, 1H), 2.03\u0026ndash;1.89 (m, 1H), 1.84\u0026ndash;1.61 (m, 2H), 1.59\u0026ndash;1.29 (m, 1H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 172.49, 172.37, 164.94, 164.84, 159.08, 152.89, 144.00, 136.85, 135.65, 129.13, 128.95, 126.65, 125.72, 115.05, 114.19, 112.33, 106.44, 106.39, 55.14, 48.30, 44.29, 42.10, 41.28, 41.13, 27.52, 23.80; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 518.2203 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 518.2208 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3315, 1679, 1647, 1620.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-1-(2-(2-(4-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetyl)piperidine-4-carboxamide(14q)\u003c/b\u003e: white solid; Yield: 42%; mp: 205.7-206.4\u0026deg;C; Purity: 97.47% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 8.38 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 8.16 (s, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 2H), 7.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.33\u0026ndash;7.20 (m, 2H), 7.14 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 7.03 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 2H), 4.84 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7 Hz, 2H), 4.30 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.8 Hz, 1H), 4.26 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.95 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.7 Hz, 1H), 3.80 (s, 3H), 3.17\u0026ndash;3.04 (m, 1H), 2.69 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.2 Hz, 1H), 2.47\u0026ndash;2.42 (m, 1H), 1.81 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.1 Hz, 1H), 1.75 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.3 Hz, 1H), 1.68 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.9, 8.6 Hz, 1H), 1.53\u0026ndash;1.39 (m, 1H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 173.74, 164.68, 161.90, 160.29, 159.08, 152.89, 144.00, 136.84, 135.84, 135.82, 128.99, 128.93, 126.65, 125.72, 121.92, 115.01, 114.87, 114.19, 112.31, 106.38, 55.14, 48.27, 43.72, 41.47, 41.20, 40.08, 28.70, 28.06; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 518.2203 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 518.2197 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3307, 1683, 1643, 1635.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-3-(2-(2-(3-methoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14r)\u003c/b\u003e: white solid; Yield: 58%; mp: 262.1-262.9\u0026deg;C; Purity: 97.56% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.50 (s, 1H), 9.04 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 1H), 8.33 (s, 1H), 8.09 (s, 1H), 7.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 7.60 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.50 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 2H), 7.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.35 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2, 6.3 Hz, 3H), 7.19 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.14 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 6.91 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.5 Hz, 1H), 4.80 (s, 2H), 4.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 3.83 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.07, 165.76, 161.91, 160.31, 159.66, 153.06, 143.87, 138.73, 136.91, 134.44, 129.87, 129.17, 129.12, 128.80, 122.17, 121.97, 121.82, 118.49, 117.71, 115.00, 114.86, 113.77, 113.70, 110.38, 106.64, 55.09, 50.36, 41.94; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: 526.1890 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 526.1897 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3302, 1690, 1643, 1624.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3-(2-(2-(3,4-dimethoxyphenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamid-o)-\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)benzamide(14s)\u003c/b\u003e: white solid; Yield: 51%; mp: 247.5-248.3\u0026deg;C; Purity: 99.72% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.48 (s, 1H), 9.03 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 1H), 8.24 (s, 1H), 8.08 (s, 1H), 7.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.4 Hz, 2H), 7.50 (s, 1H), 7.47 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 1H), 7.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.38\u0026ndash;7.31 (m, 2H), 7.17 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.14 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 7.03 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 4.79 (s, 2H), 4.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 3.85 (s, 3H), 3.79 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.08, 165.79, 161.92, 160.31, 153.00, 149.01, 148.75, 144.23, 138.73, 136.72, 135.81, 135.79, 135.18, 129.18, 129.12, 128.80, 125.94, 121.98, 121.82, 118.49, 117.82, 115.00, 114.86, 112.73, 112.12, 109.02, 106.60, 55.57, 55.52, 50.33, 41.94; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e: 556.1996 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 556.1995 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3283, 1706, 1675, 1635.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-3-(2-(2-(4-fluorophenyl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14t)\u003c/b\u003e: white solid; Yield: 42%; mp: 229.1-229.8\u0026deg;C; Purity: 99.08% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.48 (s, 1H), 9.03 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 8.29 (s, 1H), 8.08 (s, 1H), 7.97 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6, 5.6 Hz, 2H), 7.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 7.61 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.34 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 5.6 Hz, 2H), 7.28 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 7.19 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.14 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 4.80 (s, 2H), 4.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.07, 165.76, 162.66, 161.91, 161.04, 160.31, 153.01, 143.11, 137.01, 135.17, 129.61, 129.17, 129.12, 128.81, 127.35, 127.29, 122.18, 118.48, 115.73, 115.58, 115.00, 114.86, 113.33, 106.64, 50.37, 41.93; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e: 514.1690 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 514.1693 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3295, 1675, 1643, 1627.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(4-fluorobenzyl)-3-(2-(2-(naphthalen-2-yl)-8-oxoimidazo[1,2-\u003c/b\u003e \u003cb\u003ea\u003c/b\u003e \u003cb\u003e]pyrazin-7(8\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e)-yl)acetamido)benzamide(14u)\u003c/b\u003e: white solid; Yield: 46%; mp: 255.2-255.8\u0026deg;C; Purity: 96.47% (HPLC); \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 10.50 (s, 1H), 9.04 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H), 8.51 (s, 1H), 8.44 (s, 1H), 8.12\u0026ndash;8.05 (m, 2H), 8.00 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 2H), 7.92 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.78 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 7.65 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.55\u0026ndash;7.50 (m, 2H), 7.43 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.37\u0026ndash;7.32 (m, 2H), 7.21 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.14 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 4.82 (s, 2H), 4.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.08, 165.78, 161.91, 160.31, 153.07, 143.96, 138.74, 137.20, 135.79, 135.18, 133.21, 132.60, 130.50, 129.18, 129.12, 128.82, 128.30, 128.07, 127.61, 126.47, 126.03, 123.75, 123.72, 118.50, 115.00, 114.86, 113.98, 106.69, 50.37, 41.94; HR-ESI\u003csup\u003e+\u003c/sup\u003e-MS calcd for C\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e: 546.1941 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, found 546.1944 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e; IR (KBr), υ (cm-1): 3271, 1666, 1639, 1618.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAChE and BuChE Inhibition Assay\u003c/h2\u003e \u003cp\u003eThe assay was performed according to our previous reports based on the Ellman\u0026rsquo;s method [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eKinetic analysis of 14r\u003c/h2\u003e \u003cp\u003eTo determine the inhibition type of these compounds against electroporation AChE, a kinetic study was carried out with inhibitor \u003cb\u003e14r\u003c/b\u003e as the representative AChEI. In the process, the used concentrations of inhibitor 0.5\u0026times;IC\u003csub\u003e50\u003c/sub\u003e, IC\u003csub\u003e50\u003c/sub\u003e, and 2\u0026times;IC\u003csub\u003e50\u003c/sub\u003e were 0.235, 0.47, and 0.94 \u0026micro;M, respectively. The type of inhibition was established from the analysis of Lineweaver-Burk (LB) reciprocal plots. Secondary plots of slopes of LB versus concentrations of inhibitor (14r) provided inhibition constants (K\u003csub\u003ei\u003c/sub\u003e) as intercepts on negative x-axis\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eantioxidant inhibition test\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA simple method that has been developed to determine the antioxidant activity of the drug utilizes the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Briefly, 2 mL DPPH solution (0.2 mM, in 95% ethanol) was incubated with 2mL different concentrations of compounds solution. Then, the reaction mixture was shaken and incubated in the dark for 30 min at 35\u0026deg;C. The absorbance was immediately recorded at 517nm against ethanol with a spectrophotometer (Metash, model UV-5200, China). The IC\u003csub\u003e50\u003c/sub\u003e values were given in table. The amount of DPPH radical was calculated following this equation:\u003c/p\u003e \u003cp\u003e% inhibition of DPPH= [A\u003csub\u003e0\u003c/sub\u003e-A\u003csub\u003es\u003c/sub\u003e]/A\u003csub\u003e0\u003c/sub\u003e \u0026times; 100%\u003c/p\u003e \u003cp\u003eWhere A\u003csub\u003e0\u003c/sub\u003e was the absorbance of control and A\u003csub\u003es\u003c/sub\u003e was the absorbance of sample. Standard drug was Ascorbic acid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDocking study\u003c/h2\u003e \u003cp\u003eFor docking analysis, because no significant difference was detected for the amino acid sequences within the active sites of enzymes from human and non-human sources, X-ray structures of AChE (PDB ID: 4EY7) and BuChE (PDB ID: 5K5E) were selected as templates (downloaded from the Protein Data Bank(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)). The protein was then prepared by adding hydrogen atoms, removing water and assigning Kollman atomic charges to the protein, moreover, the prepared proteins were converted to pdbqt files using AutoDock, and the ligands were also prepared in the pdbqt files. A grid box spacing of 0.375 \u0026Aring; was constructed over the docking area and docking procedure was carried out. The center of the grid box was placed at the bottom of the active site gorge (AChE [-14.11 -43.83 -27.67]; BuChE [3.36 9.53 14.4]). The dimensions of the active site box were set at 36\u0026times;36\u0026times;36 \u0026Aring;. Finally, Pymol visualization software was used to visualize the 3D interaction of docking study (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pymol.org/pymol.html\u003c/span\u003e\u003cspan address=\"https://www.pymol.org/pymol.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Then, the docking of processed proteins and ligands was performed using AutoDock.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular dynamics simulation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMD simulation was performed with the GROMACS 2022.2 package. Complexes of AChE/BuChE and \u003cb\u003e14r\u003c/b\u003e were obtained from the docking poses. The electrostatic potentials (ESP) of each ligand were calculated at the HF/6-31G(d) level by Gaussian 09. Amberff19SB and gaff2 force field were used to create topology parameters of protein and ligands respectively. TIP3P water model was used to create solvent molecules. The time step was set to 2 fs. The protein\u0026ndash;ligand systems were energy minimized using 5000 steps steepest descent algorithm.\u003c/p\u003e \u003cp\u003eAfter minimization, the three systems were heated to 300 K during 100 ps in NVT ensemble. The pressure was then equilibrated to 1 atm during a 100 ps NPT simulation. V-rescale was used for temperature coupling and c-rescale was used for pressure coupling. Finally, 100 ns production simulations were performed for each complex.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting Information Summary\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supporting information includes experimental data, characterization of the synthesized compounds and their NMR spectra\u003c/p\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests\u003c/p\u003e \u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by Hebei Natural Science Foundation \u003cb\u003e(\u003c/b\u003eS\u0026amp;T Program of Hebei, NO. B2020201056, H2024201041).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eToro PMJ, Parra PDR, Pacheco MNV (2022) Galarza A G A. Enfermedad de Alzheimer. Recimundo 6(4):68\u0026ndash;76\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGustavsson A, Norton N, Fast T, Fr\u0026ouml;lich L, Georges J, Holzapfel D et al (2023) Global estimates on the number of persons across the Alzheimer's disease continuum. Alzh Dement 19(2):658\u0026ndash;670\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoźniak K, Gardian-Baj M, Jung M, Hedesz P, Jung M, Żuk-Łapan A et al (2024) Alzheimer's disease-a comprehensive review. J Educ Health Sport 56:195\u0026ndash;209\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrossberg G, Urganus A, Schein J, Bungay R, Cloutier M, Gauthier-Loiselle M et al (2023) A real-world assessment of healthcare costs associated with agitation in Alzheimer's dementia. J Med Econ 27(1):99\u0026ndash;108\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson RS, Segawa E, Boyle PA, Anagnos SE, Hizel LP, Bennett DA (2012) The natural history of cognitive decline in Alzheimer's disease. Psychol Aging 27(4):1008\u0026ndash;1017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoodward MC (2024) Drug treatment of Alzheimer's disease: what has changed in 30 years? J Pharm Pract Res 53(6):314\u0026ndash;319\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHampel H, Mesulam MM, Cuello AC, Farlow MR, Giacobini E, Grossberg GT et al (2018) The cholinergic system in the pathophysiology and treatment of Alzheimer's disease. Brain 141(7):1917\u0026ndash;1933\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua KY, Zhao WJ (2022) Current study on diagnosis and treatment of Alzheimer's disease by targeting amyloid b-protein. Folia Neuropathol 61(1):8\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIliyasu MO, Musa SA, Oladele SB, Iliya AI (2023) Amyloid-beta aggregation implicates multiple pathways in Alzheimer's disease: Understanding the mechanisms. Front Neurosci 17:1081938\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTwarowski B, Herbet M (2023) Inflammatory Processes in Alzheimer's Disease-Pathomechanism, Diagnosis and Treatment: A Review. Int J Mol Sci 24(7):6518\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdelazim AM, Abomughaid MM (2024) Oxidative stress: an overview of past research and future insights. All Life 17(1):2316092\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian S, Huang Z, Meng Q, Liu Z (2021) Multi-target drug design of anti-Alzheimer's disease based on tacrine. Mini-Rev Med Chem 21(15):2039\u0026ndash;2064\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKou X, Shi X, Pang Z, Yang A, Shen R, Zhao L (2023) A review on the natural components applied as lead compounds for potential multi-target anti-AD theranostic agents. Curr Med Chem 30(40):4586\u0026ndash;4604\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemers E (2015) Drug development in AD: point of view from the industry. JPAD-J Prev Alzheim 2(4):216\u0026ndash;218\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManiam S (2024) Screening techniques for drug discovery in Alzheimer's disease. ACS Omega 9(6):6059\u0026ndash;6073\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePravin N, Jozwiak K (2022) Effects of linkers and substitutions on multitarget directed ligands for Alzheimer\u0026rsquo;s diseases: Emerging paradigms and strategies. Int J Mol Sci 23(11):6085\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasus-Jacobi A, Washburn JL, Laurence RB, Pereira HA (2022) Selecting multitarget peptides for Alzheimer\u0026rsquo;s disease. Biomolecules 12(10):1386\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarreiras MC, Mendes E, Perry MJ, Francisco AP, Marco-Contelles J (2013) The multifactorial nature of Alzheimer's disease for developing potential therapeutics. Curr Top Med Chem 13(15):1745\u0026ndash;1770\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosini M, Simoni E, Caporaso R, Minarini A (2016) Multitarget strategies in Alzheimer's disease: Benefits and challenges on the road to therapeutics. Future Med Chem 8(6):697\u0026ndash;711\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtali S, Dorandish S, Devos J, Williams A, Price D, Taylor J et al (2020) Interaction of amyloid beta with humanin and acetylcholinesterase is modulated by ATP. FEBS Open Bio 10(12):2805\u0026ndash;2823\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElsbaey M, Igarashi Y, Ibrahim MAA, Elattar E (2023) Click-designed vanilloid-triazole conjugates as dual inhibitors of AChE and Aβ aggregation. RSC Adv 13(5):2871\u0026ndash;2883\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulshreshtha A, Piplani P (2016) Current pharmacotherapy and putative disease-modifying therapy for Alzheimer's disease. Neurol Sci 37(9):1403\u0026ndash;1435\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnand A, Patience AA, Sharma N, Khurana N (2017) The present and future of pharmacotherapy of Alzheimer's disease: A comprehensive review. Eur J Pharmacol 815:364\u0026ndash;375\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenek O, Korabecny J, Soukup O (2020) A perspective on multi-target drugs for Alzheimer's disease. Trends Pharmacol Sci 41(7):434\u0026ndash;445\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira C, Cagide F, Teixeira J, Amorim R, Sequeira L, Mesiti F et al (2018) Hydroxybenzoic acid derivatives as dual-target ligands: Mitochondriotropic antioxidants and cholinesterase inhibitors. Front Chem 6:126\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Wang L, Xu Y, Huang Y, Huang J, Zhu J et al (2022) Discovery of novel dual RAGE/SERT inhibitors for the potential treatment of the comorbidity of Alzheimer's disease and depression. Eur J Med Chem 236:114347\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMao F, Wang H, Ni W, Zheng X, Wang M, Bao K et al (2017) Design, synthesis, and biological evaluation of orally available first-generation dual-target selective inhibitors of acetylcholinesterase (AChE) and phosphodiesterase 5 (PDE5) for the treatment of Alzheimer's disease. ACS Chem Neurosci 9(2):328\u0026ndash;345\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai R, Sun Y, Su R, Gao H (2022) Anti-Alzheimer's disease potential of traditional chinese medicinal herbs as inhibitors of BACE1 and AChE enzymes. Biomed Pharmacother 154:113576\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaraca Ş, Osmaniye D, Sağlık BN, Levent S, Ilgın S, \u0026Ouml;zkay Y et al (2022) Synthesis of novel benzothiazole derivatives and investigation of their enzyme inhibitory effects against Alzheimer's disease. RSC Adv 12(36):23626\u0026ndash;23636\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao H, Uras G, Zhang P, Xu S, Yin Y, Liu J et al (2021) Discovery of novel tacrine\u0026ndash;pyrimidone hybrids as potent dual AChE/GSK-3 inhibitors for the treatment of Alzheimer's disease. J Med Chem 64(11):7483\u0026ndash;7506\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoliman AM, Ghorab WM, Lotfy DM, Karam HM, Ghorab MM, Ramadan LA (2023) Novel iodoquinazolinones bearing sulfonamide moiety as potential antioxidants and neuroprotectors. Sci Rep-UK 13(1):15546\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrozdowska D, Maliszewski D, Wr\u0026oacute;bel A, Ratkiewicz A, Sienkiewicz M (2023) New benzamides as multi-targeted compounds: A study on synthesis, AChE and BACE1 inhibitory activity and molecular docking. Int J Mol Sci 24(19):14901\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMyadaraboina S, Alla M, Parlapalli A, Manda S (2018) Novel imidazo[1,2-\u003cem\u003ea\u003c/em\u003e]pyrazine derivatives: Design, synthesis, antioxidant and antimicrobial evaluations. Int J Chem Sci 16:276\u0026ndash;288\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllman GL, Courtney KD, Andres V Jr, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7(2):88\u0026ndash;95\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medicinal-chemistry-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcre","sideBox":"Learn more about [Medicinal Chemistry Research](https://www.springer.com/journal/44)","snPcode":"44","submissionUrl":"https://submission.nature.com/new-submission/44/3","title":"Medicinal Chemistry Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alzheimer’s disease, Acetylcholinesterase inhibitor, Antioxidant, Molecular docking study, Molecular dynamics simulation","lastPublishedDoi":"10.21203/rs.3.rs-4447664/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4447664/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA series of 8-(piperazin-1-yl)imidazo[\u003cem\u003e1,2-a\u003c/em\u003e]pyrazine derivatives were designed and synthesized as acetylcholinesterase inhibitors (AChEIs) and antioxidants for the treatment of Alzheimer's disease (AD). Moreover, the biological evaluation results demonstrated that these synthesized compounds exhibited moderate inhibitory activities toward acetylcholinesterase (AChE) and radical scavenging activities. Among them, compound \u003cstrong\u003e14r\u003c/strong\u003e was the most potent AChE inhibitor with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.47 µM and moderate inhibitory activity against butyrylcholinesterase (BuChE) (IC\u003csub\u003e50\u003c/sub\u003e = 11.02 µM). Meanwhile compound \u003cstrong\u003e14r\u003c/strong\u003e had the best selectivity of AChE and selectivity index (SI) values was 23.45. Compound \u003cstrong\u003e14r\u003c/strong\u003e has better activity as well as AChE selectivity compared to reference drug galantamine (AChE IC\u003csub\u003e50\u003c/sub\u003e = 5.01 µM, BuChE IC\u003csub\u003e50\u003c/sub\u003e = 18.46 µM, SI = 3.68). Compound \u003cstrong\u003e14o\u003c/strong\u003e had the best antioxidant activity with an IC\u003csub\u003e50\u003c/sub\u003e value of 89.33 µM, which was lower than that of ascorbic acid (IC\u003csub\u003e50\u003c/sub\u003e value = 25.70 µM) as the control drug. Furthermore, the results of molecular docking studies indicated that \u003cstrong\u003e14r\u003c/strong\u003e could simultaneously bind to both catalytic active site and peripheral anionic site of AChE, which was consistent with the mixed inhibition pattern shown by enzyme kinetic studies. The interaction’s stability of 14r-AChE/BuChE were also assessed using a conventional atomistic 100 ns dynamics simulation study, which revealed the conformational stability of representative compound \u003cstrong\u003e14r\u003c/strong\u003e in the cavity of the AChE. In addition, the molecular properties of all compounds were predicted online through the SwissADME, and the best active compound \u003cstrong\u003e14r\u003c/strong\u003e matched the properties of most orally administered drugs. Based on the biological activity and molecular properties, compound \u003cstrong\u003e14r\u003c/strong\u003e as AChEI was valuable for further development.\u003c/p\u003e","manuscriptTitle":"Design, synthesis and evaluation of imidazo[1,2-a]pyrazin-8(7H)-one derivatives as acetylcholinesterase inhibitors and antioxidants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 06:18:24","doi":"10.21203/rs.3.rs-4447664/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revisions","date":"2024-07-13T04:35:17+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-13T19:08:03+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-09T04:48:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-21T02:19:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medicinal Chemistry Research","date":"2024-05-20T04:21:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medicinal-chemistry-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcre","sideBox":"Learn more about [Medicinal Chemistry Research](https://www.springer.com/journal/44)","snPcode":"44","submissionUrl":"https://submission.nature.com/new-submission/44/3","title":"Medicinal Chemistry Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a5beb25d-23da-4fe2-aab0-64a1c8806104","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:03:30+00:00","versionOfRecord":{"articleIdentity":"rs-4447664","link":"https://doi.org/10.1007/s00044-024-03298-w","journal":{"identity":"medicinal-chemistry-research","isVorOnly":false,"title":"Medicinal Chemistry Research"},"publishedOn":"2024-08-24 15:57:38","publishedOnDateReadable":"August 24th, 2024"},"versionCreatedAt":"2024-06-24 06:18:24","video":"","vorDoi":"10.1007/s00044-024-03298-w","vorDoiUrl":"https://doi.org/10.1007/s00044-024-03298-w","workflowStages":[]},"version":"v1","identity":"rs-4447664","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4447664","identity":"rs-4447664","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}