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Synthesis, in vitro biological evaluation and molecular modelling of new 2-chloro-3-hydrazinopyrazine derivatives as potent acetylcholinesterase inhibitors‏ on PC12 cells

Synthesis, in vitro biological evaluation and molecular modelling of new... Background: The loss of cholinergic neurotransmission in Alzheimer’s disease (AD) patients’ brain is accompanied by a reduced concentration of Acetylcholine (ACh) within synaptic clefts. Thus, the use of acetylcholinesterase inhibitors (AChEIs) to block the cholinergic degradation of ACh is a promising approach for AD treatment. In the present study, a series of 2-chloro-3-hydrazinopyrazine derivatives (CHP1-5) were designed, synthesized, and biologically evaluated as potential multifunctional anti-AD agents. Methods: In addition, the chemical structures and purity of the synthesized compounds were elucidated through 1 13 using IR, H and C NMR, and elemental analyses. Further, the intended compounds were assessed in vitro for their AChE inhibitory and neuroprotective effects. Furthermore, DPPH, FRAP and ABTS assays were utilized to determine their antioxidant activity. The statistical analysis was performed using one-way ANOVA. Results: Based on the results, CHP4 and CHP5 exhibited strong AChE inhibitory effects with the IC values of 3.76 and 4.2 µM compared to the donepezil (0.53 µM), respectively. The study examined the effect and molecular mecha- nism of CHP4 on the Ab1–42-induced cytotoxicity in differentiated PC12 cells. At concentrations of 0–100 μM, CHP4 was non-toxic in PC12. Additionally, Ab1–42 significantly stimulated tau hyperphosphorylation and induced differ - entiated PC12 cell death. Further, CHP4 resulted in diminishing the Ab1–42-induced toxicity in PC12 cell significantly. CHP4 at 30 μM concentration significantly increased the Ab1–42-induced HSP70 expression and decreased tau hyperphosphorylation. Conclusions: According to the results of our studies CHP4 can be considered as safe and efficient AChEI and employed as a potential multifunctional anti-AD agent. Keywords: Acetylcholinesterase inhibitors, Alzheimer’s disease, 2-Chloro-3-hydrazinopyrazine, PC12 cells Background AD is considered as one of the most causes of dementia *Correspondence: h.ghafoori@guilan.ac.ir and neurodegenerative disease among senile individu- Department of Biology, Faculty of Basic Sciences, University of Guilan, als, which may occur in 60% of cases. About 50 mil- Rasht, Iran lion individuals have dementia worldwide, to which Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Taheri et al. BMC Chemistry (2022) 16:7 Page 2 of 12 approximately 10 million new cases are added every year. and S203) in human AChE. Further, the anionic site is The total number of dementia individuals is expected to formed by the side chains of Glu202, Trp86, and Tyr337, reach 82 and 152 million in 2030 and 2050, respectively and considered as responsible for binding the ACh [18]. [1, 2]. AD is characterized by the aggravation of cogni- Some drugs such as tacrine, rivastigmine, donepezil, and tion, function, and behavior, as well as losing memory. galantamine have been consumed for years in most coun- The growth of amyloid-β (Aβ) peptide deposits and tries to diminish AD symptoms, which inhibit AChE. hyperphosphorylated tau protein (Tau-p) in brain tissue However, the number of AD patients is increasing and has been addressed as the most important pathological no definite treatment is currently available in this regard. hallmarks with AD [3]. In addition, heat shock proteins There are many synthetic and natural inhibitors of AChE (HSPs) are an important class of molecular chaper- [19]. Furthermore, novel AChEIs have been designed and ones, which can involve under stress conditions such produced from a series of some 3,5-dimethoxy-N-meth- as hypoxia, oxidative stress, proteotoxic stress, inflam - ylenebenzenamine and 4-(methyleneamino) benzoic mation, and several diseases like cancer, AD, and other acid derivatives comprising of N-methylenebenzenamine neurodegenerative diseases. AD is associated with a type nucleus (imin-metal) [20]. Pyrazine is one of the main disorder of protein accumulation [4–6]. Further, HSP70 types of heterocyclic compounds, synthesized chemically activation stabilizes Tau-p both in neuronal cells and or extracted from natural sources [21]. So far, several syn- brain tissues in the neurodegenerative diseases such as thesized derivatives of pyrazine have been introduced as AD, Parkinson’s disease (PD), and epilepsy. HSP70 pro- important drugs and 2,5-dichloropyrazine is a basic start- teins are up-regulated in response to stress although it’s ing compound for preparing many bioactive pyrazines up-regulation can be damaging because of worsening the [22, 23]. Pyrazine ring has become important in repre- chronic inflammation [7–10]. senting diverse biological activities in interaction with Oxidative stress is detected as one of the causing fac- other scaffolds such as pyrrole, pyrazole, thiophene, oxa - tors in aging, which contributes to the development of zole, pyridine, triazole, tetrazole, imidazole, piperidine, multiple neurodegenerative disease such as AD, and plays and piperazine [24]. The presence of pyrazine ring as a an important role in destroying neuronal cells. Therefore, vital scaffold in different clinically-used drugs exhibits its this process is considered as one of the primary incidents importance in drug design. Considering the above-men- in AD. Accordingly, the effective protection of neuronal tioned reports, the present study sought to design and cells against oxidative stress damage can potentially synthesize several novel 2-chloro-3-hydrazinopyrazine prevent AD [11]. In AD, inflammatory processes gener - derivatives (CHP1-5) based on their pyrazine and formyl ate reactive oxygen species (ROSs) leading to the dys- for assessing their inhibitory effects on the AChE in order function of antioxidant system [12]. AD is related to a to obtain new ligands with efficacy and high inhibitory decrease in ACh level in hippocampus (a part of medial effect. Then, the other biological evaluations such as anti - temporal lobe memory system) and neocortex, especially oxidant activity, enzyme, and cell protective assays were in frontal and temporal lobes by cholinergic system defi - performed. The results suggested CHP4 as an effective ciency such as the death or atrophy of basal forebrain AChEI. Among all  compounds under study,  CHP4 rep- cholinergic neurons [13]. Furthermore, the reduction resented the highest antioxidant properties. Based on the of Ach synaptic availability is caused by basal forebrain results of anti-Alzheimer’s studies and molecular dock- cholinergic loss and memory deficits in AD [14]. Cho - ing ones on this compound, the protective effect of CHP4 linesterases (ChEs) catalyze Ach hydrolysis into choline against the Aβ-induced damage in PC12 cells was caused and acetate, which is an essential process in the choliner- by changing Tau-p expression. In addition, the roles of gic neurotransmission. Two types of ChEs are available, HSP70 in the protective effect of CHP4 were considered AChE (EC3.1.1.7) and Butyrylcholinesterase (BuChE, in the present study. Finally, the results may propose the EC3.1.1.8), the first of which is one of the important fac - usage potential of CHP4 in subsequent research on AD. tors in AD [15, 16]. AChE is involved in the termination of impulse transmission by hydrolyzing ACh in numer- ous cholinergic pathways in the nervous systems. Indeed, Methods the active site of AChE is placed in the bottom of the Chemistry molecule and contains anionic and esteratic subsites, Chemical materials, 2-chloro-3-hydrazinopyrazine; and catalytic machinery and choline-binding pocket are chromene; methoxy benzaldehyde; 3-hydroxybenzalde- related to esteratic and anionic subsites, respectively hyde; 2-hydroxybenzaldehyde; 4-chlorobenzaldehyde; [17]. The catalytic machinery site is placed at the middle glacial acetic acid and TLC-RP 18 (Silica gel 60) were of the gorge and involves the catalytic triad (H447, E334, purchased from Sigma-Aldrich. T aheri et al. BMC Chemistry (2022) 16:7 Page 3 of 12 Synthesis of the pyrazine‑based Schiff‑bases (CHP1‑CHP5) (E)‑3‑((2‑(3‑chloropyrazin‑2‑yl)hydrazineylidene) The 2-chloro-3-hydrazinopyrazine and formyl deriva - methyl)‑6‑methyl‑4H‑chromen‑4‑one (CHP2) tives utilized to synthesize Schiff-bases and other Yellow solid, mp: 265–268 °C, yield: 85%, Mw: 314.76 g/ chemicals were obtained from Sigma-Aldrich and mol, λ (DMF): 365 nm. max −1 Merck. Additionally, double-distilled water was con- FT-IR (KBr, cm ): 719 (C–Cl), 1170 (C–N), 1207 sumed throughout the experiment and n-hexane–ethyl (C–O), 1518 (C=C), 1645 (C=N), 1665 (C=O), 3256 acetate solvent system was used as an eluent. Thin layer (N–H). H NMR (400  MHz, C DCl , δ (ppm)): 2.51 chromatography (TLC Silica gel 60 F254, Merck) was (s, 3H, CH ), 7.46 (d,1H, CH, J = 8.8  Hz),7.56 (d,1H, employed for determining the substrate purity and CH J = 8.8  Hz), 7.88 (s,1H, CH), 8.09 (s,1H, CH), 8.23 monitoring reaction. Further, an electrothermal melt- (s,1H, CH), 8.41 (s,1H, CH), 8.74 (s, 1H, CH), 8.77 ing point device was applied for specifying all melt- (s, 1H, NH). C NMR (100  MHz, C DCl , δ (ppm)): ing points (mp). The visible spectra and FTIR ones in 21.04, 118.30, 125.36, 125.4, 133.8, 134.1, 134.3, 135.50, KBr pellets were respectively measured on a Pharma- 135.54, 136.05, 136.11, 138.51, 141.37, 153.98. cia Biotech spectrophotometer and a Shimadzu 8400 FT-IR spectrophotometer. Furthermore, a FT-NMR (E)‑3‑((2‑(3‑chloropyrazin‑2‑yl)hydrazineylidene)methyl) (400  MHz) Brucker apparatus was used for record- 1 13 phenol (CHP3) ing H and C NMR spectra. Chemical shifts were Yellow solid, mp: 210–215 °C, yield: 89%, Mw: 248.67 g/ expressed in δ ppm using TMS as an internal standard mol, λ (DMF): 365 nm. max and coupling constants (J) were given in Hz. −1 FT-IR (KBr, cm ): 689 (C–Cl), 1170 (C–N), (C–O), The pyrazine-based Schiff-bases were prepared 1666 (C=N), 3291 (O–H), 3420 (N–H). H NMR through the condensation reaction of 2-chloro-3-hy- (400 MHz, CDCl , δ (ppm)): 6.80 (d,1H,CH, J = 8  Hz), drazinopyrazine (0.5 mmol) with various formyl deriva- 7.08 (d, 1H, CH, J = 7.6  Hz), 7.24 (m, 2H, CH), 7.85 tives (0.5  mmol) in ethanol (15  mL) in high yield. As (s,1H,CH), 8.23 (s, 1H, CH), 8.43 (s, 1H, CH), 9.59 a typical procedure, 0.5  mmol 2-chloro-3-hydrazino- (s, 1H, OH), 10.77 (s, 1H, NH). C NMR (100  MHz, pyrazine was added to a mixture of a formyl derivative CDCl , δ (ppm)): 112.76, 117.35, 118.87, 130.32, 132.91, (0.5 mmol), absolute ethanol (15 mL), and glacial acetic 133.79, 136.52, 141.97, 146.31, 148.37, 158.11. acid (organic catalyst) in a 50-mL round-bottom flask and heated to reflux with stirring for 7  h. The reaction progress was monitored through TLC using n-hexane: (E)‑2‑((2‑(3‑chloropyrazin‑2‑yl)hydrazineylidene)methyl) ethyl acetate (6:4) as solvent. After completing the reac- phenol (CHP4) tion, the reaction mixture was cooled to room tem- Brown solid, mp: 140–144  °C, Mw: 248.67  g/mol, λ max perature, and the precipitate was collected by using (DMF): 386 nm. vacuum filtration and washed with deionized water −1 FT-IR (KBr, cm ): 747 (C–Cl), 1053 (C–N), 1168 several times for affording the pyrazine-based Schiff- 1 (C–O), 1666 (C=N), 3287 (O–H), 3420 (N–H). H bases. Then, the products were isolated through recrys - NMR (400  MHz, CDCl , δ (ppm)): 6.93 (d, 2H, CH, tallization from DMF/H O. Finally, the structure of the J = 9.2  Hz), 7.29 (t, 1H, CH, J = 7.6  Hz), 7.46 (d, 1H, obtained Schiff-bases was confirmed by using FT-IR CH, J = 7.6  Hz), 7.89 (s, 1H, CH), 8.27 (dd, 1H, CH, 1 13 and H and C NMR spectroscopy. J = 1.6 Hz), 8.70 (s, 1H, CH), 11.2 (s, 1H, OH), 11.55 (s, 1H, NH). C NMR (100 MHz, CDCl , δ (ppm)): 116.89, 119.32, 119.74, 130.09, 131.20, 132.98, 134.20, 142.08, (E)‑2‑chloro‑3‑(2‑(4‑methoxybenzylidene) hydrazinyl) 147.36, 147.86, 157.69. pyrazine (CHP1) Yellow solid, mp: 196–198  °C, yield: 87%, molecular (E)‑2‑chloro‑3‑(2‑(4‑chlorobenzylidene)hydrazineyl)pyrazine weight (Mw): 262.69 g/mol, λ (DMF): 365 nm. FT-IR max −1 (CHP5) (KBr, cm ): 610 (C–Cl), 1170 (C–N), 1242 (C–O), Brown solid, mp: 201–204 °C, yield: 92%, Mw: 267.11 g/ 1511 (C=C), 1647 (C=N), 3248 (N–H). H NMR mol, λ (DMF): 390 nm. (400 MHz, CDCl , δ (ppm)): 3.86 (s, 3H, OCH ), 6.94 3 3 max −1 FT-IR (KBr, cm ): 707 (C–Cl), 1168 (C–O), 1487 (d, 2H, CH, J = 8.8 Hz),7.73 (d, 2H, CH, J = 8.4  Hz),7.81 (C=C), 1647 (C=N), 3207 (N–H). H NMR (400 MHz, (dd,1H, CH J = 2.4  Hz), 8.07 (s,1H, CH), 8.21(dd,1H, CDCl , δ (ppm)): 7.52 (d, 2H, CH, J = 7.2  Hz), 7.73 (d, CH, J = 2.4  Hz), 8.58 (s,1H, NH). C NMR (100  MHz, 2H, CH, J = 7.2 Hz), 7.87 (dd, 1H, CH, J = 2 Hz), 8.26 (s, CDCl , δ (ppm)): 55.39, 109.94, 114.20, 126.30, 129.04, 1H, CH), 8.50 (s, 1H, CH), 10.92 (S, 1H, NH). C NMR 133.31, 141.52, 146.01, 147.52, 161.33. Taheri et al. BMC Chemistry (2022) 16:7 Page 4 of 12 (100 MHz, CDCl , δ (ppm)): 128.73, 129.37, 133.01, Ab1-42 peptides were dissolved in distilled water and 134.10, 134.23, 134.29, 142.01, 144.70, 148.29. primarily incubated at 37 °C for 7 d before using for con- structing pre-aggregation. Further, PC12 cells were incu- bated with 100 µM aggregated Ab1-42 for 24 h to make a In vitro assessments cellular AD model in vitro. Furthermore, 50 µL of DMSO AChE inhibition assay and 10 µL of 0.5 mg/mL MTT stock solution were added The AChE inhibitory activity of all synthesized novel to each well containing about 1  mL of medium and the derivatives and their controls was evaluated through mixture was incubated for 4 h. Then, the plates were agi - using the Ellman method. In addition, the AChE (Sigma, tated on a plate shaker for 30 min and optical density was EC number 3.1.1.7, CAS number 9000.81.1) from Elec- read at 570  nm by using an ELISA reader (Biotech). A trophorus electricus was utilized in the study [25]. Fur- slightly-modified MTT method of Jamalzadeh et  al. [28] ther, 4 µL of enzyme solution (1 ng commercial enzyme, was employed in the experiments and the cell viability of Tris–HCl, pH 7.4, final concentration of 0.1  mg/mL), the control groups not exposed to CHP4 or Ab1-42 was 60 µL of 15 mM ATCI in 40 µL of water, 500 µL of 5 mM defined as 100%. DTNB in Buffer B (0.1  mg/mL Tris–HCl, pH 7.4, con - taining 100 mM K3PO4 and 1 mM EDTA), and 10 µL of compounds (0.1–10 µM) were poured in a 96-well plate. Western blot analysis Each concentration was assayed in triplicate. Further- To this end, the cells were seeded in the 6-well plate and more, all of the synthesized compounds (CHP1-5) and treated with 20 and 30  µM CHP4 for 24  h. Additionally, their bases were dissolved in DMSO. Finally, absorbance they were washed with cold PBS, followed by lysing with was recorded by using a Pharmacia Biotech Ultrospec lysis buffer (20  mM Tris (pH 7.5), 150  mM NaCl, 1  mM 3000 UV–Vis spectrophotometer at 410 nm. EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50  mM Tris, protease and phosphatase inhibitor cock- Antioxidant activity tails) for 30  min on ice. After centrifuging lysates at The radical scavenging activity of prepared compounds 4 °C in 12,000g for 10 min, the supernatants were trans- was assessed through using ABTS decolorization assay ferred to new tubes. Protein concentration was detected and method reported by Re et al. [26] with few modifica - through using the Bradford assay [29]. Further, the dena- tions. In this regard, ABTS solution was diluted with 80% tured proteins was resolved on SDS-PAGE, transferred ethanol to the absorbance of 0.70 ± 0.05 at 734 nm. After onto PVDF membranes (Millipore, Billerica, MA, USA), adding 100  µL of the obtained compounds in ethanol and blocked with 5% nonfat dry milk in the PBS contain- (1–30  µM) or ascorbic acid, absorbance was measured ing 0.1% Tween-20 (PBST). Then, the membranes were at exactly 20  min in the same wavelength. DPPH assay incubated with the rabbit primary antibodies against was based on the method provided by Ghafouri et  al. Tau-p, HSP70, and β-actin (as a control group) overnight [27] with some modifications. Additionally, the quench - at 4  °C, and exposed with the horseradish peroxidase ing of free radicals by novel synthesized compounds was (HRP)-conjugated second antibody for 2 h at room tem- evaluated spectrophotometrically (UV–Vis Pharmacia perature. Finally, the density of relative protein bands was Biotech Ultrospec 3000) at 517  nm against the absorb- determined through densitometric scanning the blots by ance of DPPH radical. Further, ferric reducing/antioxi- using Image-J program. dant power (FRAP) assay was performed according to the previous study with some modifications [27], the princi - ple of which was based on reducing a ferric 2,4,6-tris(2- In silico studies 3+ pyridyl)-1,3,5-triazine (Fe -TPTZ) to ferrous in the Geometry optimization, MEP and molecular descriptors presence of prepared compounds. Finally, a standard Geometry optimization of all five compounds was curve was drawn by considering the different concentra - performed at DFT level theory with B3LYP hybrid tions (10–1000 µM) of ferrous sulfate. exchange–correlation energy functional method and 6-31G++ (d, p) basis by Gaussian 09 software [30]. From Cell viability the optimized structures, the molecular electrostatic Rattus norvegicus pheochromocytoma PC12 cells (Pastor potential (MEP) of each compound was calculated by the Institute, Iran) were cultured in the Dulbecco’s Modified same software and visualized using GaussView v. 6. The Eagle’s Medium (DMEM) containing 10% fetal bovine capability of molecules to permeate blood–brain bar- serum (FBS) and 1% penicillin/streptomycin (Gibco) at rier (BBB) was predicted by Online BBB Predictor [31] 37 °C with 5% C O . In order to examine the role of CHP4, and other descriptors related to drug likeness property PC12 cells were treated with the different concentra - of each compound was calculated through using Padel tions (10–100 µM) of CHP4 for 24 and 48 h. In addition, descriptor [32]. T aheri et al. BMC Chemistry (2022) 16:7 Page 5 of 12 Docking Results and discussion AutoDock Vina software program was applied for dock- Chemistry ing CHP4 into hAChE protein [33]. In addition, the X-ray Spectroscopic characterization of the pyrazine‑based crystallographic structure of human AChE (PDB ID: Schiff‑bases 6O4W, 2.35  Å resolution) was obtained from the RCSB In the present study, five pyrazine-based Schiff-bases protein data bank (PDB). The protein was prepared were synthesized through condensation reaction, the through removing water, co-factor, and co-crystallized synthetic pathways for which are presented in Fig.  1. ligands by MolSoft ICM [34]. Further, the 2D structure of The chemical structure of compounds was approved 13 1 CHP4 was drawn using MarvinSketch (version 16.8.15) by FT-IR, and C and H NMR spectroscopic data (see by ChemAxon and converted to 3D format by using spectrums in Additional file  1). Additionally the UV–Vis molconverter from Jchem toolkit (http:// www. chema spectra of all compounds were measured between 200 xon. com). All docking calculations were performed by and 800  nm, which their λ was 365–390  nm due to max considering the protein and ligand as inflexible and flex - n–π٭ and/or π–π٭ electronic transitions. ible, respectively. Furthermore, donepezil (DPZ) was docked into 6O4W as a positive control ligand to validate In vitro the docking protocol. Finally, the Vina output file was AChE inhibition assay entered into ADT for analyzing the docking results, and All of obtained imine derivatives (CHP1-5) were evalu- the hydrophobic interactions of AChE-ligand complexes, ated for their inhibitory activities toward AChE through as well as bond lengths were examined. using an in  vitro assay based on the reported proto- col in comparison with donepezil as a standard drug Analysis of binding sites and conserved sequences (IC = 0.53  µM). The preliminary results in Fig.  2 dem- For the purpose of identification of conserved sequences onstrated the highest AChE inhibitory effect in CHP4 of AChE from an evolutionary aspect, ConSurf soft- with a hydroxyl group in the formyl (IC = 3.76  µM). ware was used with HMMER homolog search algorithm, In fact, the activity of the synthesized imine derivatives MAFFT-L-INS-i method for alignments and Bayesian depends on the nature of the substituents attached to for- method for calculations [35]. myl group. Statistical analysis Antioxidant properties Each concentration was assayed in triplicates (n = 3) and The ABTS radical is created by oxidizing ABTS with repeated in three independent experiments. The values potassium persulfate and reduced in the presence of such were expressed as mean ± SD and one-way ANOVA was hydrogen-donating antioxidant compound. The results utilized to determine the significant differences from were obtained at the final concentration (1–30  μM) controls statistically. of compounds and their inhibition was assessed. The Fig. 1 Synthesized pathways for the pyrazine-based compounds (CHP1-CHP5) Taheri et al. BMC Chemistry (2022) 16:7 Page 6 of 12 and least antioxidant effect was respectively obtained in CHP4 and CHP5. Cell viability assays In order to examine the neuroprotective effects of CHP4 against the Ab1-42-induced toxicity, PC12 cells were treated with CHP4 in the presence or absence of 100 µM Ab1-42 for 24  h. Then, cell viability was determined through using MTT assay. As depicted in Fig.  4, the via- bility of the PC12 cells exposed to 100  µM Ab1-42 for 24 h decreased to 49.24% compared to the control group. In addition, CHP4 significantly reduced the Ab1-42-in - Fig. 2 IC50 (µM) values of synthesized compounds against AChE duced cell death in a dose-dependent manner (Fig.  5). activity (IC50 = 0.53 µM). CHP4 > CHP5 > CHP3 > CHP1 > CHP2. The data are expressed in relation to AChE activity with respect to control In the assays, the cell viability of the control group not as mean ± SD (n = 3, P < 0.001). Donepezil is used as a positive control exposed to CHP4 or Ab1-42 was considered as 100%. In the pathological conditions, tau can be more phos- phorylated than the normal phosphorylation, which is compounds were tested to determine by decoloriz- AD hallmarks [36]. Based on the western blot results of ing the ABTS radical and assessed as a quenching of Tau-p and HSP70 in the PC12 cell treated with CHP4 and the absorbance at 734  nm. Based on the results, CHP1 in the presence of Ab1-42, the level of tau phosphoryla- exhibited high scavenging activity (48%) (Fig.  3A). Fig- tion increased significantly in the +Aβ1-42 group com - ure  3B  compares the antiradical effect of  compounds at pared to the control. However, Tau-p expression clearly various concentrations compared to that of ascorbic acid, decreased in response to CHP4 at the final concentration which indicates the lower activity of all compounds. Fur- of 20 and 30 µM. Also donepezil is utilized as a positive ther, the DPPH-reducing abilities of all compounds were control (Fig.  6). Original photos of western blot gel are measured by determining their IC   values. As shown available, in Additional file 1. in Fig.  3B,  the highest scavenging activity on DPPH are respectively observed in CHP2, CHP4, and CHP3, while In silico studies CHP1 and CHP5 represent moderate and low properties, MEP respectively. Furthermore, the antioxidant activity of all First, the optimized structural properties of all com- compounds was estimated by considering their ability to pounds, which can be found in supplementary informa- reduce TPRZ-Fe (III) complex to the TPTZ-Fe (II), the tion, were obtained at DFT level of theory. The colored results of which are displayed in Fig.  3C. The concentra - scheme of MEP visualization is presented in Fig.  7. The 2+ tion values of Fe exhibited significant antioxidant prop - darkest red color indicates the most electron-rich region, erties in all synthesized compounds. Finally, the highest capable of acting as one of the best hydrogen bond Fig. 3 Antioxidant activities of synthesized compounds. Scavenging activity of prepared compounds on A ABTS radicals and B DPPH radicals 2+ (Ascorbic acid is used as a standard), and C concentration values of Fe which indicate significant antioxidant properties in all of the synthesized compounds T aheri et al. BMC Chemistry (2022) 16:7 Page 7 of 12 Fig. 4 PC12 cells exposed to the Ab1-42 peptides in the A absence or B presence of 100 µM Ab1-42 acceptors, whereas the darkest blue color represent the site which is the most sensitive towards nucleophilic attacks [37]. As can be seen in Fig.  7, except for CHP2 that has its most electron-rich region located at its car- bonyl group, the same region is located at N of other compounds. Additionally, while CHP1, CHP2 and CHP4 have their least electron-rich regions at their N–H of hydrazine group, for CHP3 and CHP4 it is related to the phenolic hydrogen. Fig. 5 Protective effects of CHP4 at various concentrations against the Aβ1–42. Aβ1–42 (100 µM)-induced cytotoxicity in PC12 neuronal Drug likeness properties of the compounds cells (Cell viability was assayed through using MTT. The values are First, it was predicted that all compounds are capable of presented as the mean ± SD of three independent experiments. passing from BBB and their diagrams are presented in The cell viability of the control group not exposed to either CHP4 or supplementary information. Lipinski’s rule of five was Ab1-42 is defined as 100%.) employed as the basis of investigating the drug likeness properties of compounds. According to this rule, an ideal drug candidate must not have more than 5 hydrogen bond donors, more than ten hydrogen bond acceptors, a molecular mass less than 500 Da and logP more than 5 [38]. As has been represented in Table  1, apart from the molecular mass, other criteria of the rule apply to the studied compounds. Molecular docking of CHP4 and donepezil Following the end of docking process, the best confor- mation was selected and the scoring functions indi- cated that conformations were the best complement to the AChE. Additionally, AutoDock 4.2 software was applied for all of the docking calculations (see vina results in the Additional file  1). The docking of CHP4 affinity with active site was calculated as − 8.3  kcal/ mol. D72, W84, N85, Y121, S122, F330, Y334 and F331 Fig. 6 Representative western blot analysis of Tau-p and HSP70 in were the residues interacting with CHP4. Moreover, the the PC12 cell treated with CHP4 evolutionary analysis of AChE revealed that out of this Taheri et al. BMC Chemistry (2022) 16:7 Page 8 of 12 Fig. 7 MEP of CHP1 to CHP5. For CHP1 MEP is represented from − 4.636 a.u. to + 4.636 a.u., for CHP2 it is from − 4.646 a.u. to + 4.646 a.u., for CHP3 from − 6.529 a.u. to + 6.529 a.u., for CHP4 from − 7.159 a.u. to + 7.159 a.u. and for CHP5 from − 5.104 a.u. to + 5.104 a.u. The most electron rich region for all compounds, except for CHP2, is located at N and for CHP2 it is O . Furthermore, the least electron rich region for CHP1, CHP2 and 6 19 CHP5 is at H but for CHP3 and CHP4 it is H 11 26 Table 1 Drug-likeness properties of CHP1 to CHP4 LigPlot [40] software (Fig. 8). More figures are available in the additional file. Finally. Descriptor CHP1 CHP2 CHP3 CHP4 CHP5 Hydrogen bond donor 1 1 2 2 1 Discussion Hydrogen bond accep- 5 6 5 5 4 Designing, synthesizing, and producing the molecules valu- tor able as human therapeutic agents are considered one of the AlogP 0.027 0.054 − 0.380 − 0.380 0.82 principal purpose of pharmaceutical and organic chemistry. Number of rotatable 4 5 3 3 3 Pyrazines possess various types of biological activity, which bonds represents a range of their pharmacological activities such Polar surface area 59.4 76.47 70.4 70.4 50.17 as antibacterial, antimycobacterial, antiviral, antifungal, Molecular mass 262.062 314.057 248.046 248.046 266.012 anti-diabetic, anti-cancer, analgesic, hypnotic, diuretic [23], Number of atoms 29 33 26 26 25 and anticholinergic ones [41, 42]. In the present study, sev- eral novel 2-chloro-3-hydrazinopyrazine derivatives were designed, synthesized, and evaluated, which their pyrazine residues, Y121 is highly conserved. In order to validate base was effective (Fig.  1). Pyrazine is an aromatic hetero- the docking results obtained by Vina software, done- cyclic ring, which contains two nitrogen atoms with the pezil was docked in 6O4W as positive control ligand general effect of protonation and hydrogen-bond formation (RMSD: 0.307  Å) with an affinity of − 12.8  kcal/mol [43]. The molecular characteristics of all of the synthesized (Table  2). After docking, the interaction of CHP4 with compounds (Table  1), indicated that these molecules have AChE was evaluated by using UCSF Chimera [39] and T aheri et al. BMC Chemistry (2022) 16:7 Page 9 of 12 Table 2 List of amino acids involved in hydrogen bonding and hydrophobic interactions with CHP4 and donepezil with AChE Compounds UCSF Chimera LigPlot+ H-bonds HI H-bonds HI CHP4 S124b Chain b: Y121b Chain b: W84, Y121, D72, S122, W84, Y121, D72, S122, *F331, *Y334, *F330, N85 *F331, *Y334, *F330, N85 DPZ S228b, S205b Chain b: _ Chain b: F288, A234, W233, *F331, C231, S288,A204, P229, F288, A234, *F331, C231, S288,A204, P229, F290, N230, F290, N230, F120, M208, S200, I287, *F330, N399, F120, M208, *F330, N399, S235, V395, L332, *Y334, V400 S235, V395, L332, *Y334, V400 HI: Hydrophobic Interactions *Common residue Fig. 8 2D (left) and 3D (right) illustration of the complexes of 6O4W with CHP4 (A) and DPZ (B). In the 3D-interaction diagram, the ligand is represented as a solid and protein as a ribbon potential as drug candidates. In the AD therapy, one of and the relationship between AChE inhibitory activity with the aims of treatment is to inhibit AChE [44]. Accordingly, the chemical structures of the compounds was evaluated. some novel compounds have been produced for assessing Based on the preliminary results, CHP4 with a hydroxyl the inhibitory effects on AChE. In this study, the inhibitory group at the ortho position of N-phenyl ring exhibited effects of the novel compounds on AChE were assessed the highest AChE inhibitory effect (IC = 3.76  µM). The 50 Taheri et al. BMC Chemistry (2022) 16:7 Page 10 of 12 effectiveness of CHP4 may be related to the electron with - enhance Aβ clearance by upregulates the expression of drawing property of the phenyl ring [45]. Computational insulin degrading enzyme and transforming growth factor analysis illustrated that CHP4 is capable of having hydro- beta [57]. Some studies have reported the protective role gen interaction with Tyr121 through its nitrogen of pyra- of HSP70 in the CNS, while several others have found the zine ring (Fig. 8), which is the most electron-rich region of pathological and detrimental role of HSP70 in AD. In fact, the compound according to MEP and the same residue in HSP70 acts as a double-sided sword and its role in AD is the protein is a highly conserved sequence from an evolu- still unclear and controversial. According to Miyata et al., tionary aspect. According to Uysal et  al., the formation of HSP70 is considered as an emerging pharmaceutical target mono- and di-substituents to the ortho position of N-phe- for treating neurodegenerative tauopathies [58]. nyl ring improved AChE inhibitory effect slightly [46]. Fur - thermore the hydroxyl group was not involved in hydrogen Conclusion bonding with AChE, in spite the fact that its hydrogen pos- In summary, five compounds (CHP1–CHP5) were sesses the least electron-rich region of the molecule based designed, synthesized, and evaluated. In the following, all on MEP [47]. synthesized compounds were analyzed and identified by In the AD patients, the use of antioxidant therapies for using IR, 1H and 13C NMR. Novel compounds have been the disease is necessary to reduce pathogenic symptoms assayed for assessing the inhibitory effects on AChE, which [48]. The results of the present study demonstrated that CHP4 was effective (IC50 = 3.76  µM). According to the CHP2 represented the most potent antioxidant proper- results of DPPH, ABTS and FRAP assays, CHP4 was cho- ties (IC = 0.026 mM) in the DPPH assay due to its higher sen for subsequent the study. We have shown that CHP4 capacity to donate electron compared to the others (CHP2 has a protective effect on PC12 neuronal cells against the > CHP3 > CHP4 > CHP1 > CHP5). In the ABTS assay, CHP1 toxicity of Ab1-42 peptide. In supplemental studies of west- performed better than the others due to the presence of ern blot analysis, CHP4 reduce the expression of Tau-p as electron-withdrawing and electron-donating group, meth- an important cause to AD On the other hand, a decrease in oxy group, with 48% inhibition (CHP1 > CHP4 > CHP3 > HSP70 expression was also seen, which could be a sign of a CHP5 > CHP2). Furthermore, FRAP assay is based on the diminish in the pathological symptoms of this disease. 3+ 2+ ability of antioxidants to reduce Fe to Fe . The highest The multifunctional properties (an optimal strategy) 3+ ability for F e reduction was observed in CHP4 (3.016 per highlight CHP4 as a promising candidate for further 1  mM) (CHP4 > CHP2 > CHP5 > CHP3 > CHP1). The ortho studies on the development of novel drugs against AD. position of a hydroxyl group on benzoic ring exhibited However, more extensive research should be conducted excellent antioxidant activity, while the meta position led to to assess the exact mechanism of effectiveness, as well as weak effect [49]. Based on previous studies and due to the dose and potential in medical applications. toxicity of iron and its important role in the progression of AD [50–52], CHP4 was selected for following the study. Supplementary Information AD is a common neurodegenerative diseases, the hall- The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13065- 022- 00799-w. mark pathologic characteristics of Aβ plaques, tau hyper- phosphorylation, and neuronal cell death [2]. HSP70 and Additional file 1: Figures S1-S20. 2D Structure image and 1H-NMR, some of the other co-chaperones are involved in regulat- 13C-NMR, FT-IR spectra of compounds CHP1, CHP2, CHP3, CHP4 and ing, phosphorylation, aggregation and degradation of tau, CHP5. Figures S21-S23. Original photos of western bot of CHP4. Tables and potentially implicated in pathogenesis of AD [53– S1-S5. Optimized geometric properties of CHP1 to CHP5. Tables S6-S7. Vina docking results. Figures S24-S28. Diagram of the capability of the 56]. In the present study, the protective effect of CHP4 synthesized compounds in passing from BBB. Figures S29-S30. Protein was examined against Ab1-42 toxicity in PC12 neuronal and ligand interaction, ribbon model of the dimeric structure (S29), and cells, the results of which reflected that CHP4 at thera - binding pocket (S30) of AChE. peutic-relevant concentration protected and rescued neuronal cells from the toxicity of Ab1-42 peptide. Acknowledgements In addition, the effects of CHP4 on the expression The authors thank the Research Council of the Iranian National Science Foun- dation (INSF) and University of Guilan for the financial support to this study. of Tau-p and HSP70 were assessed using western blot analysis, which demonstrates that the exposure of CHP4 Authors’ contributions resulted in decreasing the Ab1-42-induced phosphoryla- All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for tion of tau and increased HSP70 expression. According to all aspects of the work. MT, SA and HN carried out the experiments. MT and Lu et al., HSP700 plays cytoprotective roles in AD, blocks VAM developed the theory and performed the computations. HG, NM & NM Aβ self-assembly, moderates caspase-dependent and cas- verified the analytical methods. AM helped supervise the project, HG super - vised the project. All authors discussed the results and contributed to the final pase-independent apoptotic pathways and reduces it in manuscript. All authors read and approved the final manuscript. neuron cells, it also directly prevents tau aggregation and T aheri et al. BMC Chemistry (2022) 16:7 Page 11 of 12 Funding 12. Butterfield DA, Boyd-Kimball D. Oxidative stress, amyloid-β peptide, and This work is supported by the Iranian National Science Foundation (INSF, Grant altered key molecular pathways in the pathogenesis and progression of No. 99027281) and the University of Guilan. Alzheimer’s disease. J Alzheimers Dis. 2018;62(3):1345–67. 13. Ferreira-Vieira TH, Guimaraes IM, Silva FR, Ribeiro FM. Alzheimer’s Availability of data and materials disease: targeting the cholinergic system. Curr Neuropharmacol. The datasets generated and/or analysed during the current study available 2016;14(1):101–15. from the corresponding author on reasonable request. We have presented all 14. 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Synthesis, in vitro biological evaluation and molecular modelling of new 2-chloro-3-hydrazinopyrazine derivatives as potent acetylcholinesterase inhibitors‏ on PC12 cells

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Abstract

Background: The loss of cholinergic neurotransmission in Alzheimer’s disease (AD) patients’ brain is accompanied by a reduced concentration of Acetylcholine (ACh) within synaptic clefts. Thus, the use of acetylcholinesterase inhibitors (AChEIs) to block the cholinergic degradation of ACh is a promising approach for AD treatment. In the present study, a series of 2-chloro-3-hydrazinopyrazine derivatives (CHP1-5) were designed, synthesized, and biologically evaluated as potential multifunctional anti-AD agents. Methods: In addition, the chemical structures and purity of the synthesized compounds were elucidated through 1 13 using IR, H and C NMR, and elemental analyses. Further, the intended compounds were assessed in vitro for their AChE inhibitory and neuroprotective effects. Furthermore, DPPH, FRAP and ABTS assays were utilized to determine their antioxidant activity. The statistical analysis was performed using one-way ANOVA. Results: Based on the results, CHP4 and CHP5 exhibited strong AChE inhibitory effects with the IC values of 3.76 and 4.2 µM compared to the donepezil (0.53 µM), respectively. The study examined the effect and molecular mecha- nism of CHP4 on the Ab1–42-induced cytotoxicity in differentiated PC12 cells. At concentrations of 0–100 μM, CHP4 was non-toxic in PC12. Additionally, Ab1–42 significantly stimulated tau hyperphosphorylation and induced differ - entiated PC12 cell death. Further, CHP4 resulted in diminishing the Ab1–42-induced toxicity in PC12 cell significantly. CHP4 at 30 μM concentration significantly increased the Ab1–42-induced HSP70 expression and decreased tau hyperphosphorylation. Conclusions: According to the results of our studies CHP4 can be considered as safe and efficient AChEI and employed as a potential multifunctional anti-AD agent. Keywords: Acetylcholinesterase inhibitors, Alzheimer’s disease, 2-Chloro-3-hydrazinopyrazine, PC12 cells Background AD is considered as one of the most causes of dementia *Correspondence: h.ghafoori@guilan.ac.ir and neurodegenerative disease among senile individu- Department of Biology, Faculty of Basic Sciences, University of Guilan, als, which may occur in 60% of cases. About 50 mil- Rasht, Iran lion individuals have dementia worldwide, to which Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Taheri et al. BMC Chemistry (2022) 16:7 Page 2 of 12 approximately 10 million new cases are added every year. and S203) in human AChE. Further, the anionic site is The total number of dementia individuals is expected to formed by the side chains of Glu202, Trp86, and Tyr337, reach 82 and 152 million in 2030 and 2050, respectively and considered as responsible for binding the ACh [18]. [1, 2]. AD is characterized by the aggravation of cogni- Some drugs such as tacrine, rivastigmine, donepezil, and tion, function, and behavior, as well as losing memory. galantamine have been consumed for years in most coun- The growth of amyloid-β (Aβ) peptide deposits and tries to diminish AD symptoms, which inhibit AChE. hyperphosphorylated tau protein (Tau-p) in brain tissue However, the number of AD patients is increasing and has been addressed as the most important pathological no definite treatment is currently available in this regard. hallmarks with AD [3]. In addition, heat shock proteins There are many synthetic and natural inhibitors of AChE (HSPs) are an important class of molecular chaper- [19]. Furthermore, novel AChEIs have been designed and ones, which can involve under stress conditions such produced from a series of some 3,5-dimethoxy-N-meth- as hypoxia, oxidative stress, proteotoxic stress, inflam - ylenebenzenamine and 4-(methyleneamino) benzoic mation, and several diseases like cancer, AD, and other acid derivatives comprising of N-methylenebenzenamine neurodegenerative diseases. AD is associated with a type nucleus (imin-metal) [20]. Pyrazine is one of the main disorder of protein accumulation [4–6]. Further, HSP70 types of heterocyclic compounds, synthesized chemically activation stabilizes Tau-p both in neuronal cells and or extracted from natural sources [21]. So far, several syn- brain tissues in the neurodegenerative diseases such as thesized derivatives of pyrazine have been introduced as AD, Parkinson’s disease (PD), and epilepsy. HSP70 pro- important drugs and 2,5-dichloropyrazine is a basic start- teins are up-regulated in response to stress although it’s ing compound for preparing many bioactive pyrazines up-regulation can be damaging because of worsening the [22, 23]. Pyrazine ring has become important in repre- chronic inflammation [7–10]. senting diverse biological activities in interaction with Oxidative stress is detected as one of the causing fac- other scaffolds such as pyrrole, pyrazole, thiophene, oxa - tors in aging, which contributes to the development of zole, pyridine, triazole, tetrazole, imidazole, piperidine, multiple neurodegenerative disease such as AD, and plays and piperazine [24]. The presence of pyrazine ring as a an important role in destroying neuronal cells. Therefore, vital scaffold in different clinically-used drugs exhibits its this process is considered as one of the primary incidents importance in drug design. Considering the above-men- in AD. Accordingly, the effective protection of neuronal tioned reports, the present study sought to design and cells against oxidative stress damage can potentially synthesize several novel 2-chloro-3-hydrazinopyrazine prevent AD [11]. In AD, inflammatory processes gener - derivatives (CHP1-5) based on their pyrazine and formyl ate reactive oxygen species (ROSs) leading to the dys- for assessing their inhibitory effects on the AChE in order function of antioxidant system [12]. AD is related to a to obtain new ligands with efficacy and high inhibitory decrease in ACh level in hippocampus (a part of medial effect. Then, the other biological evaluations such as anti - temporal lobe memory system) and neocortex, especially oxidant activity, enzyme, and cell protective assays were in frontal and temporal lobes by cholinergic system defi - performed. The results suggested CHP4 as an effective ciency such as the death or atrophy of basal forebrain AChEI. Among all  compounds under study,  CHP4 rep- cholinergic neurons [13]. Furthermore, the reduction resented the highest antioxidant properties. Based on the of Ach synaptic availability is caused by basal forebrain results of anti-Alzheimer’s studies and molecular dock- cholinergic loss and memory deficits in AD [14]. Cho - ing ones on this compound, the protective effect of CHP4 linesterases (ChEs) catalyze Ach hydrolysis into choline against the Aβ-induced damage in PC12 cells was caused and acetate, which is an essential process in the choliner- by changing Tau-p expression. In addition, the roles of gic neurotransmission. Two types of ChEs are available, HSP70 in the protective effect of CHP4 were considered AChE (EC3.1.1.7) and Butyrylcholinesterase (BuChE, in the present study. Finally, the results may propose the EC3.1.1.8), the first of which is one of the important fac - usage potential of CHP4 in subsequent research on AD. tors in AD [15, 16]. AChE is involved in the termination of impulse transmission by hydrolyzing ACh in numer- ous cholinergic pathways in the nervous systems. Indeed, Methods the active site of AChE is placed in the bottom of the Chemistry molecule and contains anionic and esteratic subsites, Chemical materials, 2-chloro-3-hydrazinopyrazine; and catalytic machinery and choline-binding pocket are chromene; methoxy benzaldehyde; 3-hydroxybenzalde- related to esteratic and anionic subsites, respectively hyde; 2-hydroxybenzaldehyde; 4-chlorobenzaldehyde; [17]. The catalytic machinery site is placed at the middle glacial acetic acid and TLC-RP 18 (Silica gel 60) were of the gorge and involves the catalytic triad (H447, E334, purchased from Sigma-Aldrich. T aheri et al. BMC Chemistry (2022) 16:7 Page 3 of 12 Synthesis of the pyrazine‑based Schiff‑bases (CHP1‑CHP5) (E)‑3‑((2‑(3‑chloropyrazin‑2‑yl)hydrazineylidene) The 2-chloro-3-hydrazinopyrazine and formyl deriva - methyl)‑6‑methyl‑4H‑chromen‑4‑one (CHP2) tives utilized to synthesize Schiff-bases and other Yellow solid, mp: 265–268 °C, yield: 85%, Mw: 314.76 g/ chemicals were obtained from Sigma-Aldrich and mol, λ (DMF): 365 nm. max −1 Merck. Additionally, double-distilled water was con- FT-IR (KBr, cm ): 719 (C–Cl), 1170 (C–N), 1207 sumed throughout the experiment and n-hexane–ethyl (C–O), 1518 (C=C), 1645 (C=N), 1665 (C=O), 3256 acetate solvent system was used as an eluent. Thin layer (N–H). H NMR (400  MHz, C DCl , δ (ppm)): 2.51 chromatography (TLC Silica gel 60 F254, Merck) was (s, 3H, CH ), 7.46 (d,1H, CH, J = 8.8  Hz),7.56 (d,1H, employed for determining the substrate purity and CH J = 8.8  Hz), 7.88 (s,1H, CH), 8.09 (s,1H, CH), 8.23 monitoring reaction. Further, an electrothermal melt- (s,1H, CH), 8.41 (s,1H, CH), 8.74 (s, 1H, CH), 8.77 ing point device was applied for specifying all melt- (s, 1H, NH). C NMR (100  MHz, C DCl , δ (ppm)): ing points (mp). The visible spectra and FTIR ones in 21.04, 118.30, 125.36, 125.4, 133.8, 134.1, 134.3, 135.50, KBr pellets were respectively measured on a Pharma- 135.54, 136.05, 136.11, 138.51, 141.37, 153.98. cia Biotech spectrophotometer and a Shimadzu 8400 FT-IR spectrophotometer. Furthermore, a FT-NMR (E)‑3‑((2‑(3‑chloropyrazin‑2‑yl)hydrazineylidene)methyl) (400  MHz) Brucker apparatus was used for record- 1 13 phenol (CHP3) ing H and C NMR spectra. Chemical shifts were Yellow solid, mp: 210–215 °C, yield: 89%, Mw: 248.67 g/ expressed in δ ppm using TMS as an internal standard mol, λ (DMF): 365 nm. max and coupling constants (J) were given in Hz. −1 FT-IR (KBr, cm ): 689 (C–Cl), 1170 (C–N), (C–O), The pyrazine-based Schiff-bases were prepared 1666 (C=N), 3291 (O–H), 3420 (N–H). H NMR through the condensation reaction of 2-chloro-3-hy- (400 MHz, CDCl , δ (ppm)): 6.80 (d,1H,CH, J = 8  Hz), drazinopyrazine (0.5 mmol) with various formyl deriva- 7.08 (d, 1H, CH, J = 7.6  Hz), 7.24 (m, 2H, CH), 7.85 tives (0.5  mmol) in ethanol (15  mL) in high yield. As (s,1H,CH), 8.23 (s, 1H, CH), 8.43 (s, 1H, CH), 9.59 a typical procedure, 0.5  mmol 2-chloro-3-hydrazino- (s, 1H, OH), 10.77 (s, 1H, NH). C NMR (100  MHz, pyrazine was added to a mixture of a formyl derivative CDCl , δ (ppm)): 112.76, 117.35, 118.87, 130.32, 132.91, (0.5 mmol), absolute ethanol (15 mL), and glacial acetic 133.79, 136.52, 141.97, 146.31, 148.37, 158.11. acid (organic catalyst) in a 50-mL round-bottom flask and heated to reflux with stirring for 7  h. The reaction progress was monitored through TLC using n-hexane: (E)‑2‑((2‑(3‑chloropyrazin‑2‑yl)hydrazineylidene)methyl) ethyl acetate (6:4) as solvent. After completing the reac- phenol (CHP4) tion, the reaction mixture was cooled to room tem- Brown solid, mp: 140–144  °C, Mw: 248.67  g/mol, λ max perature, and the precipitate was collected by using (DMF): 386 nm. vacuum filtration and washed with deionized water −1 FT-IR (KBr, cm ): 747 (C–Cl), 1053 (C–N), 1168 several times for affording the pyrazine-based Schiff- 1 (C–O), 1666 (C=N), 3287 (O–H), 3420 (N–H). H bases. Then, the products were isolated through recrys - NMR (400  MHz, CDCl , δ (ppm)): 6.93 (d, 2H, CH, tallization from DMF/H O. Finally, the structure of the J = 9.2  Hz), 7.29 (t, 1H, CH, J = 7.6  Hz), 7.46 (d, 1H, obtained Schiff-bases was confirmed by using FT-IR CH, J = 7.6  Hz), 7.89 (s, 1H, CH), 8.27 (dd, 1H, CH, 1 13 and H and C NMR spectroscopy. J = 1.6 Hz), 8.70 (s, 1H, CH), 11.2 (s, 1H, OH), 11.55 (s, 1H, NH). C NMR (100 MHz, CDCl , δ (ppm)): 116.89, 119.32, 119.74, 130.09, 131.20, 132.98, 134.20, 142.08, (E)‑2‑chloro‑3‑(2‑(4‑methoxybenzylidene) hydrazinyl) 147.36, 147.86, 157.69. pyrazine (CHP1) Yellow solid, mp: 196–198  °C, yield: 87%, molecular (E)‑2‑chloro‑3‑(2‑(4‑chlorobenzylidene)hydrazineyl)pyrazine weight (Mw): 262.69 g/mol, λ (DMF): 365 nm. FT-IR max −1 (CHP5) (KBr, cm ): 610 (C–Cl), 1170 (C–N), 1242 (C–O), Brown solid, mp: 201–204 °C, yield: 92%, Mw: 267.11 g/ 1511 (C=C), 1647 (C=N), 3248 (N–H). H NMR mol, λ (DMF): 390 nm. (400 MHz, CDCl , δ (ppm)): 3.86 (s, 3H, OCH ), 6.94 3 3 max −1 FT-IR (KBr, cm ): 707 (C–Cl), 1168 (C–O), 1487 (d, 2H, CH, J = 8.8 Hz),7.73 (d, 2H, CH, J = 8.4  Hz),7.81 (C=C), 1647 (C=N), 3207 (N–H). H NMR (400 MHz, (dd,1H, CH J = 2.4  Hz), 8.07 (s,1H, CH), 8.21(dd,1H, CDCl , δ (ppm)): 7.52 (d, 2H, CH, J = 7.2  Hz), 7.73 (d, CH, J = 2.4  Hz), 8.58 (s,1H, NH). C NMR (100  MHz, 2H, CH, J = 7.2 Hz), 7.87 (dd, 1H, CH, J = 2 Hz), 8.26 (s, CDCl , δ (ppm)): 55.39, 109.94, 114.20, 126.30, 129.04, 1H, CH), 8.50 (s, 1H, CH), 10.92 (S, 1H, NH). C NMR 133.31, 141.52, 146.01, 147.52, 161.33. Taheri et al. BMC Chemistry (2022) 16:7 Page 4 of 12 (100 MHz, CDCl , δ (ppm)): 128.73, 129.37, 133.01, Ab1-42 peptides were dissolved in distilled water and 134.10, 134.23, 134.29, 142.01, 144.70, 148.29. primarily incubated at 37 °C for 7 d before using for con- structing pre-aggregation. Further, PC12 cells were incu- bated with 100 µM aggregated Ab1-42 for 24 h to make a In vitro assessments cellular AD model in vitro. Furthermore, 50 µL of DMSO AChE inhibition assay and 10 µL of 0.5 mg/mL MTT stock solution were added The AChE inhibitory activity of all synthesized novel to each well containing about 1  mL of medium and the derivatives and their controls was evaluated through mixture was incubated for 4 h. Then, the plates were agi - using the Ellman method. In addition, the AChE (Sigma, tated on a plate shaker for 30 min and optical density was EC number 3.1.1.7, CAS number 9000.81.1) from Elec- read at 570  nm by using an ELISA reader (Biotech). A trophorus electricus was utilized in the study [25]. Fur- slightly-modified MTT method of Jamalzadeh et  al. [28] ther, 4 µL of enzyme solution (1 ng commercial enzyme, was employed in the experiments and the cell viability of Tris–HCl, pH 7.4, final concentration of 0.1  mg/mL), the control groups not exposed to CHP4 or Ab1-42 was 60 µL of 15 mM ATCI in 40 µL of water, 500 µL of 5 mM defined as 100%. DTNB in Buffer B (0.1  mg/mL Tris–HCl, pH 7.4, con - taining 100 mM K3PO4 and 1 mM EDTA), and 10 µL of compounds (0.1–10 µM) were poured in a 96-well plate. Western blot analysis Each concentration was assayed in triplicate. Further- To this end, the cells were seeded in the 6-well plate and more, all of the synthesized compounds (CHP1-5) and treated with 20 and 30  µM CHP4 for 24  h. Additionally, their bases were dissolved in DMSO. Finally, absorbance they were washed with cold PBS, followed by lysing with was recorded by using a Pharmacia Biotech Ultrospec lysis buffer (20  mM Tris (pH 7.5), 150  mM NaCl, 1  mM 3000 UV–Vis spectrophotometer at 410 nm. EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50  mM Tris, protease and phosphatase inhibitor cock- Antioxidant activity tails) for 30  min on ice. After centrifuging lysates at The radical scavenging activity of prepared compounds 4 °C in 12,000g for 10 min, the supernatants were trans- was assessed through using ABTS decolorization assay ferred to new tubes. Protein concentration was detected and method reported by Re et al. [26] with few modifica - through using the Bradford assay [29]. Further, the dena- tions. In this regard, ABTS solution was diluted with 80% tured proteins was resolved on SDS-PAGE, transferred ethanol to the absorbance of 0.70 ± 0.05 at 734 nm. After onto PVDF membranes (Millipore, Billerica, MA, USA), adding 100  µL of the obtained compounds in ethanol and blocked with 5% nonfat dry milk in the PBS contain- (1–30  µM) or ascorbic acid, absorbance was measured ing 0.1% Tween-20 (PBST). Then, the membranes were at exactly 20  min in the same wavelength. DPPH assay incubated with the rabbit primary antibodies against was based on the method provided by Ghafouri et  al. Tau-p, HSP70, and β-actin (as a control group) overnight [27] with some modifications. Additionally, the quench - at 4  °C, and exposed with the horseradish peroxidase ing of free radicals by novel synthesized compounds was (HRP)-conjugated second antibody for 2 h at room tem- evaluated spectrophotometrically (UV–Vis Pharmacia perature. Finally, the density of relative protein bands was Biotech Ultrospec 3000) at 517  nm against the absorb- determined through densitometric scanning the blots by ance of DPPH radical. Further, ferric reducing/antioxi- using Image-J program. dant power (FRAP) assay was performed according to the previous study with some modifications [27], the princi - ple of which was based on reducing a ferric 2,4,6-tris(2- In silico studies 3+ pyridyl)-1,3,5-triazine (Fe -TPTZ) to ferrous in the Geometry optimization, MEP and molecular descriptors presence of prepared compounds. Finally, a standard Geometry optimization of all five compounds was curve was drawn by considering the different concentra - performed at DFT level theory with B3LYP hybrid tions (10–1000 µM) of ferrous sulfate. exchange–correlation energy functional method and 6-31G++ (d, p) basis by Gaussian 09 software [30]. From Cell viability the optimized structures, the molecular electrostatic Rattus norvegicus pheochromocytoma PC12 cells (Pastor potential (MEP) of each compound was calculated by the Institute, Iran) were cultured in the Dulbecco’s Modified same software and visualized using GaussView v. 6. The Eagle’s Medium (DMEM) containing 10% fetal bovine capability of molecules to permeate blood–brain bar- serum (FBS) and 1% penicillin/streptomycin (Gibco) at rier (BBB) was predicted by Online BBB Predictor [31] 37 °C with 5% C O . In order to examine the role of CHP4, and other descriptors related to drug likeness property PC12 cells were treated with the different concentra - of each compound was calculated through using Padel tions (10–100 µM) of CHP4 for 24 and 48 h. In addition, descriptor [32]. T aheri et al. BMC Chemistry (2022) 16:7 Page 5 of 12 Docking Results and discussion AutoDock Vina software program was applied for dock- Chemistry ing CHP4 into hAChE protein [33]. In addition, the X-ray Spectroscopic characterization of the pyrazine‑based crystallographic structure of human AChE (PDB ID: Schiff‑bases 6O4W, 2.35  Å resolution) was obtained from the RCSB In the present study, five pyrazine-based Schiff-bases protein data bank (PDB). The protein was prepared were synthesized through condensation reaction, the through removing water, co-factor, and co-crystallized synthetic pathways for which are presented in Fig.  1. ligands by MolSoft ICM [34]. Further, the 2D structure of The chemical structure of compounds was approved 13 1 CHP4 was drawn using MarvinSketch (version 16.8.15) by FT-IR, and C and H NMR spectroscopic data (see by ChemAxon and converted to 3D format by using spectrums in Additional file  1). Additionally the UV–Vis molconverter from Jchem toolkit (http:// www. chema spectra of all compounds were measured between 200 xon. com). All docking calculations were performed by and 800  nm, which their λ was 365–390  nm due to max considering the protein and ligand as inflexible and flex - n–π٭ and/or π–π٭ electronic transitions. ible, respectively. Furthermore, donepezil (DPZ) was docked into 6O4W as a positive control ligand to validate In vitro the docking protocol. Finally, the Vina output file was AChE inhibition assay entered into ADT for analyzing the docking results, and All of obtained imine derivatives (CHP1-5) were evalu- the hydrophobic interactions of AChE-ligand complexes, ated for their inhibitory activities toward AChE through as well as bond lengths were examined. using an in  vitro assay based on the reported proto- col in comparison with donepezil as a standard drug Analysis of binding sites and conserved sequences (IC = 0.53  µM). The preliminary results in Fig.  2 dem- For the purpose of identification of conserved sequences onstrated the highest AChE inhibitory effect in CHP4 of AChE from an evolutionary aspect, ConSurf soft- with a hydroxyl group in the formyl (IC = 3.76  µM). ware was used with HMMER homolog search algorithm, In fact, the activity of the synthesized imine derivatives MAFFT-L-INS-i method for alignments and Bayesian depends on the nature of the substituents attached to for- method for calculations [35]. myl group. Statistical analysis Antioxidant properties Each concentration was assayed in triplicates (n = 3) and The ABTS radical is created by oxidizing ABTS with repeated in three independent experiments. The values potassium persulfate and reduced in the presence of such were expressed as mean ± SD and one-way ANOVA was hydrogen-donating antioxidant compound. The results utilized to determine the significant differences from were obtained at the final concentration (1–30  μM) controls statistically. of compounds and their inhibition was assessed. The Fig. 1 Synthesized pathways for the pyrazine-based compounds (CHP1-CHP5) Taheri et al. BMC Chemistry (2022) 16:7 Page 6 of 12 and least antioxidant effect was respectively obtained in CHP4 and CHP5. Cell viability assays In order to examine the neuroprotective effects of CHP4 against the Ab1-42-induced toxicity, PC12 cells were treated with CHP4 in the presence or absence of 100 µM Ab1-42 for 24  h. Then, cell viability was determined through using MTT assay. As depicted in Fig.  4, the via- bility of the PC12 cells exposed to 100  µM Ab1-42 for 24 h decreased to 49.24% compared to the control group. In addition, CHP4 significantly reduced the Ab1-42-in - Fig. 2 IC50 (µM) values of synthesized compounds against AChE duced cell death in a dose-dependent manner (Fig.  5). activity (IC50 = 0.53 µM). CHP4 > CHP5 > CHP3 > CHP1 > CHP2. The data are expressed in relation to AChE activity with respect to control In the assays, the cell viability of the control group not as mean ± SD (n = 3, P < 0.001). Donepezil is used as a positive control exposed to CHP4 or Ab1-42 was considered as 100%. In the pathological conditions, tau can be more phos- phorylated than the normal phosphorylation, which is compounds were tested to determine by decoloriz- AD hallmarks [36]. Based on the western blot results of ing the ABTS radical and assessed as a quenching of Tau-p and HSP70 in the PC12 cell treated with CHP4 and the absorbance at 734  nm. Based on the results, CHP1 in the presence of Ab1-42, the level of tau phosphoryla- exhibited high scavenging activity (48%) (Fig.  3A). Fig- tion increased significantly in the +Aβ1-42 group com - ure  3B  compares the antiradical effect of  compounds at pared to the control. However, Tau-p expression clearly various concentrations compared to that of ascorbic acid, decreased in response to CHP4 at the final concentration which indicates the lower activity of all compounds. Fur- of 20 and 30 µM. Also donepezil is utilized as a positive ther, the DPPH-reducing abilities of all compounds were control (Fig.  6). Original photos of western blot gel are measured by determining their IC   values. As shown available, in Additional file 1. in Fig.  3B,  the highest scavenging activity on DPPH are respectively observed in CHP2, CHP4, and CHP3, while In silico studies CHP1 and CHP5 represent moderate and low properties, MEP respectively. Furthermore, the antioxidant activity of all First, the optimized structural properties of all com- compounds was estimated by considering their ability to pounds, which can be found in supplementary informa- reduce TPRZ-Fe (III) complex to the TPTZ-Fe (II), the tion, were obtained at DFT level of theory. The colored results of which are displayed in Fig.  3C. The concentra - scheme of MEP visualization is presented in Fig.  7. The 2+ tion values of Fe exhibited significant antioxidant prop - darkest red color indicates the most electron-rich region, erties in all synthesized compounds. Finally, the highest capable of acting as one of the best hydrogen bond Fig. 3 Antioxidant activities of synthesized compounds. Scavenging activity of prepared compounds on A ABTS radicals and B DPPH radicals 2+ (Ascorbic acid is used as a standard), and C concentration values of Fe which indicate significant antioxidant properties in all of the synthesized compounds T aheri et al. BMC Chemistry (2022) 16:7 Page 7 of 12 Fig. 4 PC12 cells exposed to the Ab1-42 peptides in the A absence or B presence of 100 µM Ab1-42 acceptors, whereas the darkest blue color represent the site which is the most sensitive towards nucleophilic attacks [37]. As can be seen in Fig.  7, except for CHP2 that has its most electron-rich region located at its car- bonyl group, the same region is located at N of other compounds. Additionally, while CHP1, CHP2 and CHP4 have their least electron-rich regions at their N–H of hydrazine group, for CHP3 and CHP4 it is related to the phenolic hydrogen. Fig. 5 Protective effects of CHP4 at various concentrations against the Aβ1–42. Aβ1–42 (100 µM)-induced cytotoxicity in PC12 neuronal Drug likeness properties of the compounds cells (Cell viability was assayed through using MTT. The values are First, it was predicted that all compounds are capable of presented as the mean ± SD of three independent experiments. passing from BBB and their diagrams are presented in The cell viability of the control group not exposed to either CHP4 or supplementary information. Lipinski’s rule of five was Ab1-42 is defined as 100%.) employed as the basis of investigating the drug likeness properties of compounds. According to this rule, an ideal drug candidate must not have more than 5 hydrogen bond donors, more than ten hydrogen bond acceptors, a molecular mass less than 500 Da and logP more than 5 [38]. As has been represented in Table  1, apart from the molecular mass, other criteria of the rule apply to the studied compounds. Molecular docking of CHP4 and donepezil Following the end of docking process, the best confor- mation was selected and the scoring functions indi- cated that conformations were the best complement to the AChE. Additionally, AutoDock 4.2 software was applied for all of the docking calculations (see vina results in the Additional file  1). The docking of CHP4 affinity with active site was calculated as − 8.3  kcal/ mol. D72, W84, N85, Y121, S122, F330, Y334 and F331 Fig. 6 Representative western blot analysis of Tau-p and HSP70 in were the residues interacting with CHP4. Moreover, the the PC12 cell treated with CHP4 evolutionary analysis of AChE revealed that out of this Taheri et al. BMC Chemistry (2022) 16:7 Page 8 of 12 Fig. 7 MEP of CHP1 to CHP5. For CHP1 MEP is represented from − 4.636 a.u. to + 4.636 a.u., for CHP2 it is from − 4.646 a.u. to + 4.646 a.u., for CHP3 from − 6.529 a.u. to + 6.529 a.u., for CHP4 from − 7.159 a.u. to + 7.159 a.u. and for CHP5 from − 5.104 a.u. to + 5.104 a.u. The most electron rich region for all compounds, except for CHP2, is located at N and for CHP2 it is O . Furthermore, the least electron rich region for CHP1, CHP2 and 6 19 CHP5 is at H but for CHP3 and CHP4 it is H 11 26 Table 1 Drug-likeness properties of CHP1 to CHP4 LigPlot [40] software (Fig. 8). More figures are available in the additional file. Finally. Descriptor CHP1 CHP2 CHP3 CHP4 CHP5 Hydrogen bond donor 1 1 2 2 1 Discussion Hydrogen bond accep- 5 6 5 5 4 Designing, synthesizing, and producing the molecules valu- tor able as human therapeutic agents are considered one of the AlogP 0.027 0.054 − 0.380 − 0.380 0.82 principal purpose of pharmaceutical and organic chemistry. Number of rotatable 4 5 3 3 3 Pyrazines possess various types of biological activity, which bonds represents a range of their pharmacological activities such Polar surface area 59.4 76.47 70.4 70.4 50.17 as antibacterial, antimycobacterial, antiviral, antifungal, Molecular mass 262.062 314.057 248.046 248.046 266.012 anti-diabetic, anti-cancer, analgesic, hypnotic, diuretic [23], Number of atoms 29 33 26 26 25 and anticholinergic ones [41, 42]. In the present study, sev- eral novel 2-chloro-3-hydrazinopyrazine derivatives were designed, synthesized, and evaluated, which their pyrazine residues, Y121 is highly conserved. In order to validate base was effective (Fig.  1). Pyrazine is an aromatic hetero- the docking results obtained by Vina software, done- cyclic ring, which contains two nitrogen atoms with the pezil was docked in 6O4W as positive control ligand general effect of protonation and hydrogen-bond formation (RMSD: 0.307  Å) with an affinity of − 12.8  kcal/mol [43]. The molecular characteristics of all of the synthesized (Table  2). After docking, the interaction of CHP4 with compounds (Table  1), indicated that these molecules have AChE was evaluated by using UCSF Chimera [39] and T aheri et al. BMC Chemistry (2022) 16:7 Page 9 of 12 Table 2 List of amino acids involved in hydrogen bonding and hydrophobic interactions with CHP4 and donepezil with AChE Compounds UCSF Chimera LigPlot+ H-bonds HI H-bonds HI CHP4 S124b Chain b: Y121b Chain b: W84, Y121, D72, S122, W84, Y121, D72, S122, *F331, *Y334, *F330, N85 *F331, *Y334, *F330, N85 DPZ S228b, S205b Chain b: _ Chain b: F288, A234, W233, *F331, C231, S288,A204, P229, F288, A234, *F331, C231, S288,A204, P229, F290, N230, F290, N230, F120, M208, S200, I287, *F330, N399, F120, M208, *F330, N399, S235, V395, L332, *Y334, V400 S235, V395, L332, *Y334, V400 HI: Hydrophobic Interactions *Common residue Fig. 8 2D (left) and 3D (right) illustration of the complexes of 6O4W with CHP4 (A) and DPZ (B). In the 3D-interaction diagram, the ligand is represented as a solid and protein as a ribbon potential as drug candidates. In the AD therapy, one of and the relationship between AChE inhibitory activity with the aims of treatment is to inhibit AChE [44]. Accordingly, the chemical structures of the compounds was evaluated. some novel compounds have been produced for assessing Based on the preliminary results, CHP4 with a hydroxyl the inhibitory effects on AChE. In this study, the inhibitory group at the ortho position of N-phenyl ring exhibited effects of the novel compounds on AChE were assessed the highest AChE inhibitory effect (IC = 3.76  µM). The 50 Taheri et al. BMC Chemistry (2022) 16:7 Page 10 of 12 effectiveness of CHP4 may be related to the electron with - enhance Aβ clearance by upregulates the expression of drawing property of the phenyl ring [45]. Computational insulin degrading enzyme and transforming growth factor analysis illustrated that CHP4 is capable of having hydro- beta [57]. Some studies have reported the protective role gen interaction with Tyr121 through its nitrogen of pyra- of HSP70 in the CNS, while several others have found the zine ring (Fig. 8), which is the most electron-rich region of pathological and detrimental role of HSP70 in AD. In fact, the compound according to MEP and the same residue in HSP70 acts as a double-sided sword and its role in AD is the protein is a highly conserved sequence from an evolu- still unclear and controversial. According to Miyata et al., tionary aspect. According to Uysal et  al., the formation of HSP70 is considered as an emerging pharmaceutical target mono- and di-substituents to the ortho position of N-phe- for treating neurodegenerative tauopathies [58]. nyl ring improved AChE inhibitory effect slightly [46]. Fur - thermore the hydroxyl group was not involved in hydrogen Conclusion bonding with AChE, in spite the fact that its hydrogen pos- In summary, five compounds (CHP1–CHP5) were sesses the least electron-rich region of the molecule based designed, synthesized, and evaluated. In the following, all on MEP [47]. synthesized compounds were analyzed and identified by In the AD patients, the use of antioxidant therapies for using IR, 1H and 13C NMR. Novel compounds have been the disease is necessary to reduce pathogenic symptoms assayed for assessing the inhibitory effects on AChE, which [48]. The results of the present study demonstrated that CHP4 was effective (IC50 = 3.76  µM). According to the CHP2 represented the most potent antioxidant proper- results of DPPH, ABTS and FRAP assays, CHP4 was cho- ties (IC = 0.026 mM) in the DPPH assay due to its higher sen for subsequent the study. We have shown that CHP4 capacity to donate electron compared to the others (CHP2 has a protective effect on PC12 neuronal cells against the > CHP3 > CHP4 > CHP1 > CHP5). In the ABTS assay, CHP1 toxicity of Ab1-42 peptide. In supplemental studies of west- performed better than the others due to the presence of ern blot analysis, CHP4 reduce the expression of Tau-p as electron-withdrawing and electron-donating group, meth- an important cause to AD On the other hand, a decrease in oxy group, with 48% inhibition (CHP1 > CHP4 > CHP3 > HSP70 expression was also seen, which could be a sign of a CHP5 > CHP2). Furthermore, FRAP assay is based on the diminish in the pathological symptoms of this disease. 3+ 2+ ability of antioxidants to reduce Fe to Fe . The highest The multifunctional properties (an optimal strategy) 3+ ability for F e reduction was observed in CHP4 (3.016 per highlight CHP4 as a promising candidate for further 1  mM) (CHP4 > CHP2 > CHP5 > CHP3 > CHP1). The ortho studies on the development of novel drugs against AD. position of a hydroxyl group on benzoic ring exhibited However, more extensive research should be conducted excellent antioxidant activity, while the meta position led to to assess the exact mechanism of effectiveness, as well as weak effect [49]. Based on previous studies and due to the dose and potential in medical applications. toxicity of iron and its important role in the progression of AD [50–52], CHP4 was selected for following the study. Supplementary Information AD is a common neurodegenerative diseases, the hall- The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13065- 022- 00799-w. mark pathologic characteristics of Aβ plaques, tau hyper- phosphorylation, and neuronal cell death [2]. HSP70 and Additional file 1: Figures S1-S20. 2D Structure image and 1H-NMR, some of the other co-chaperones are involved in regulat- 13C-NMR, FT-IR spectra of compounds CHP1, CHP2, CHP3, CHP4 and ing, phosphorylation, aggregation and degradation of tau, CHP5. Figures S21-S23. Original photos of western bot of CHP4. Tables and potentially implicated in pathogenesis of AD [53– S1-S5. Optimized geometric properties of CHP1 to CHP5. Tables S6-S7. Vina docking results. Figures S24-S28. Diagram of the capability of the 56]. In the present study, the protective effect of CHP4 synthesized compounds in passing from BBB. Figures S29-S30. Protein was examined against Ab1-42 toxicity in PC12 neuronal and ligand interaction, ribbon model of the dimeric structure (S29), and cells, the results of which reflected that CHP4 at thera - binding pocket (S30) of AChE. peutic-relevant concentration protected and rescued neuronal cells from the toxicity of Ab1-42 peptide. Acknowledgements In addition, the effects of CHP4 on the expression The authors thank the Research Council of the Iranian National Science Foun- dation (INSF) and University of Guilan for the financial support to this study. of Tau-p and HSP70 were assessed using western blot analysis, which demonstrates that the exposure of CHP4 Authors’ contributions resulted in decreasing the Ab1-42-induced phosphoryla- All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for tion of tau and increased HSP70 expression. According to all aspects of the work. MT, SA and HN carried out the experiments. MT and Lu et al., HSP700 plays cytoprotective roles in AD, blocks VAM developed the theory and performed the computations. HG, NM & NM Aβ self-assembly, moderates caspase-dependent and cas- verified the analytical methods. AM helped supervise the project, HG super - vised the project. All authors discussed the results and contributed to the final pase-independent apoptotic pathways and reduces it in manuscript. All authors read and approved the final manuscript. neuron cells, it also directly prevents tau aggregation and T aheri et al. BMC Chemistry (2022) 16:7 Page 11 of 12 Funding 12. Butterfield DA, Boyd-Kimball D. Oxidative stress, amyloid-β peptide, and This work is supported by the Iranian National Science Foundation (INSF, Grant altered key molecular pathways in the pathogenesis and progression of No. 99027281) and the University of Guilan. Alzheimer’s disease. J Alzheimers Dis. 2018;62(3):1345–67. 13. Ferreira-Vieira TH, Guimaraes IM, Silva FR, Ribeiro FM. Alzheimer’s Availability of data and materials disease: targeting the cholinergic system. Curr Neuropharmacol. The datasets generated and/or analysed during the current study available 2016;14(1):101–15. from the corresponding author on reasonable request. We have presented all 14. 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Journal

BMC ChemistrySpringer Journals

Published: Feb 22, 2022

Keywords: Acetylcholinesterase inhibitors; Alzheimer's disease; 2-Chloro-3-hydrazinopyrazine; PC12 cells

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