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

doi:10.21203/rs.3.rs-4447664/v1

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

https://doi.org/10.21203/rs.3.rs-4447664/v1

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published 24 Aug, 2024

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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 IC₅₀ value of 0.47 µM and moderate inhibitory activity against butyrylcholinesterase (BuChE) (IC₅₀ = 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 IC₅₀ = 5.01 µM, BuChE IC₅₀ = 18.46 µM, SI = 3.68). Compound 14o had the best antioxidant activity with an IC₅₀ value of 89.33 µM, which was lower than that of ascorbic acid (IC₅₀ 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

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(7H)-one ring, and the two active regions were linked by acetamide. To this end, the target compounds were designed. (Fig. 2)

Chemical synthesis

The synthetic strategy for designed target compounds 14a–14u was depicted in Scheme 1. Briefly, compound 6a-6k reacted with various Q₁ to provide 7a-7o. Then compounds 7a-7o could be reacted with the chloroacetyl chloride in CH₂Cl₂ to get compounds 8a-8o under the condition of an ice water bath. In parallel, compound 6a-6k reacted with Q₂ 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 ¹H NMR, ¹³C 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 IC₅₀ 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 IC₅₀ value of 0.47 µM and moderate inhibitory activity against eqBuChE (IC₅₀ = 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 IC₅₀ = 5.01 µM, eqBuChE IC₅₀ = 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 IC₅₀ = 1.74 µM) had better activity than 14j (eeAChE IC₅₀ = 2.56 µM). When the substituent on benzene ring of R₂ was an electron donating group, the 14i with two methoxy groups on the benzene ring of R₂ held the highest activity. When the substituent on benzene ring of R₂ 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 R₁ was meta methoxy, its activity was stronger than that of dimethoxy, para fluorine, and naphthalene ring.

In the case of eqBuChE, compound 14j (eqBuChE, IC₅₀ = 1.49 µM) had the best inhibitory activity to eqBuChE. The compounds with the substituents on R₂ in the ortho position usually exhibited better activity, and the compounds had better activity at n = 2 compared to n = 1. When replaced the R₁ substituent, the structure with a larger spatial resistance at the substituent of R₁ 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 IC₅₀ 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 IC₅₀ 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.	IC₅₀(µM) ± SD^a	Compd.	IC₅₀(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	< 130Ǻ²	≤ 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 Å² < TPSA < 130 Å², n_violation(number violations from Lipinski’s rule): n_violation ≤ 1, BBB: The ability of crossing BBB blood brain barrier.

In summary, a new series of imidazo[1,2-a]pyrazin-8(7H)-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 IC₅₀ 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.

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 Cs₂CO₃ (0.326, 1.2 mmol) in CH₃CN (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 CH₂Cl₂ and washed with a saturated solution of sodium carbonate. The organic layer was separated, dried (Na₂SO₄), 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₉H₂₄FN₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₀H₂₄F₃N₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₀H₂₄F₃N₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₀H₂₄F₃N₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₁H₂₃F₆N₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₀H₂₇N₅O₅: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₀H₂₇N₅O₅: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₀H₂₇N₅O₅: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₁H₂₉N₅O₆: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₂H₃₁N₅O₆: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₉H₂₅N₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₀H₂₆FN₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₈H₂₃FN₆O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₉H₂₄FN₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₆H₂₁FN₆O₄S: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₈H₂₈FN₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₈H₂₈FN₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₉H₂₄FN₅O₄: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₀H₂₆FN₅O₅: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₂₈H₂₁F₂N₅O₃: 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); ¹H NMR (600 MHz, DMSO-d₆) 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); ¹³C NMR (151 MHz, DMSO-d₆) 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 C₃₂H₂₄FN₅O₃: 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×IC₅₀, IC₅₀, and 2×IC₅₀ 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 IC₅₀ values were given in table. The amount of DPPH radical was calculated following this equation:

% inhibition of DPPH= [A₀-A_s]/A₀ × 100%

Where A₀ 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.

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).

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Table 1 is available in the Supplementary Files section.

Scheme 1 is available in the Supplementary Files section.

Table1.docx
scheme1.png
Scheme 1. The synthetic route of target compounds 14a-14u. Reagents and conditions: (a) Corresponding carboxylic acid, DIEA, TBTU, CH₂Cl₂, rt, 3 h; (b) Chloroacetyl chloride, DIEA, CH₂Cl₂, -5 °C, 4 h; (c) HCl/MeOH, rt, 2 h; (d) 2-Amino-3-chloropyrazine, CH₃CN, H₂O, 80 °C, 8 h; (e) 8a-8o or 11a-11b, Cs₂CO₃, CH₃CN, 80 °C, 12 h;
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Design, synthesis and evaluation of imidazo[1,2-a]pyrazin-8(7H)-one derivatives as acetylcholinesterase inhibitors and antioxidants

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Abstract

Figures

Introduction

Results and discussion

Chemical synthesis

Kinetic study of AChE inhibition

Studies of anti-oxidative activity

Molecular docking studies

Molecular dynamics simulation study

In silico molecular property analysis

Conclusion

Experimental Section

Chemistry

Synthesis of Compounds 14a-14u

AChE and BuChE Inhibition Assay

Kinetic analysis of 14r

Docking study

Declarations

Conflict of interest

Acknowledgements

References

Table 1

Scheme 1

Supplementary Files

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