Introduction

The most common progressive neurodegenerative disease in the world and the main leading cause of dementia is Alzheimer’s disease (AD) [1, 2]. The two primary signs of AD are progressive memory loss and diminished intelligence [3, 4]. AD is a very difficult and emotionally exhausting condition to care for, and few diseases distress patients and their loved ones as much and regularly as it does [5]. Based on available data, AD caused an estimated 1.9 million deaths globally in 2015 [6] and an estimated 46.8 million cases of AD worldwide in 2017 [7]. With population growth, it was projected that by 2050, the number of AD patients will triple [8]. After heart disease and cancer, AD is currently the third most common cause of mortality [9]. The etiology of AD remains unclear, despite being recognized as a complex condition with numerous contributing factors explained by several theories such as the cholinergic hypothesis [10], tau hyperphosphorylation [11], amyloid cascade, and oxidative stress [12, 13]. One of the key theories explaining AD pathogenesis is the cholinergic one. Acetylcholine (ACh), a neurotransmitter, is required for the transmission of nerve impulses between muscle and/or nerve cells [14]. ACh works on the nicotinic-sensitive receptor in the central nervous system (CNS) and the muscarinic-sensitive receptor in the peripheral nervous system (PNS), which is also linked to the cardiac and smooth muscles [15]. ACh congregates in a structure known as vesicles at the terminals of neural cells. It diffuses from the vesicles into the synaptic cleft and binds to the nicotinic or muscarinic receptor, which results in the creation of electrical impulses in the cholinergic system when the action potential travels through the nerve cells and reaches the axon terminals [16]. The enzyme acetylcholine esterase (AChE), which is abundant in the synaptic clefts of both the CNS and PNS, hydrolyzes the disseminated ACh into inactive choline and acetate metabolites, shortening the half-life of ACh [17]. Additionally, research has revealed that AChE interacts to amyloid-β (Aβ) in its nonamyloidogenic form via peripheral active site (PAS) and promotes conformational change to its amyloidogenic form [18, 19]. The other cholinesterase (ChE) neurotransmitter, butyrylcholinesterase (BuChE), regulates the level of ACh and maintains regular cholinergic activities [20, 21]. Moreover, both ChEs and Aβ plaque deposition have been related [22, 23]. U.S. Food and Drug Administration (FDA) approved AChE inhibitors (donepezil, rivastigmine, and galantamine) that are used to treat mild-to-moderate AD stages. Simultaneously, the glutamate regulator memantine and a combination of memantine and donepezil were authorized for the treatment of moderate-to-severe AD [24]. Thus, AChE remains a potential therapeutic target in the hunt for novel anti-AD medications.

The connection between neurologic diseases and oxidative stress has garnered more attention recently. Free radicals have been linked to AD, Parkinson’s disease, head trauma, cerebral ischemia-reperfusion, and other conditions. Because the brain uses up a lot of oxygen, has a lot of lipids, and has fewer antioxidant enzymes than other tissues, it is particularly susceptible to damage from free radicals [25]. This implies that therapies for AD that try to get rid of free radicals or stop them from getting created are beneficial [26].

The breakdown of endogenous and exogenous amines is significantly aided by monoamine oxidases (MAOs), which are widely distributed enzymes. The most popular substrates for MAOs are dopamine (DA), norepinephrine (NE), epinephrine, serotonin (5-HT), and 2-phenylethylamine (PEA) [27].

The mammalian family of MAOs comprises two isozymes, MAO-A and MAO-B, which have different selectivities towards substrates and inhibitors. The A isoform preferentially deamines 5-HT and NE, while the MAO-B substrates are PEA and benzylamine [28]. It was proved that following the inhibition of MAO-B, a rise in DA levels and a neuroprotective effect are observed [29,30,31,32,33]. Elevated MAO-B level in aged people [34, 35] induces a rise in reactive oxygen species (ROS) and hydrogen peroxide production, which in turn may cause neuron degeneration [36,37,38,39].

Consequently, selective irreversible human MAO-B inhibitors are efficiently employed in the treatment of AD, either alone or in conjunction with other medications [40,41,42,43]. Until 2021, the FDA had only approved five medications for the treatment of AD since the disease was first identified in 1906. Therefore, there is an urgent need to discover more potent anti-AD drug candidates.

Given the complex nature of AD and recent advancements in systems biology, it was highly indicated that single-target drugs will not be adequate to treat AD or halt its progression [44]. With the ability to target several pathways involved in AD pathogenesis, multi-target-directed ligands (MTDLs) have recently attracted significant interest. The employment of this technique has resulted in the discovery of several promising anti-AD candidates [45,46,47].

Due to its distinct bioisosteric characteristics and a very broad range of biological activities, the five-membered 1,2,4-oxadiazole heterocyclic ring has drawn a lot of interest. It has been discovered and used as a metabolically stable analog of an ester or amide functionality in pharmacologically significant molecules [48]. Consequently, 1,2,4-oxadiazole is an ideal platform for the development of innovative drugs. Throughout the past fifteen years, there has been a two-fold increase in interest in the biological applications of 1,2,4-oxadiazoles. The FDA has approved several derivatives based on 1,2,4-oxadiazoles, which are now being promoted as commercial drugs [49]. The 3,5-disubstituted-1,2,4-oxadiazole structural motif, incorporating aryl and/or heteroaryl scaffolds as compounds I-VI (Fig. 1), is included in several anti-AD and neuroprotective candidates. They exhibited their AD potential through various mechanistic pathways such as cholinesterase inhibition and antioxidant activity. Moreover, some 1,2,4-oxadiazole derivatives demonstrated multifunctional anti-AD potential [50,51,52,53,54,55].

Fig. 1
figure 1

Structure of 1,2,4-oxadiazole derivatives I-VI [50,51,52,53,54,55] with anti-AD potential and design strategy for the synthesized 3,5-disubstituted-1,2,4-oxadiazole derivatives

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Our goal was to design and synthesize novel compounds based on the 1,2,4-oxadiazole scaffold, considering the previously mentioned findings (Fig. 1). The first strategy involved the incorporation of benzyl, phenyl (electron-rich moieties), or p-trifluoromethyl phenyl (more lipophilic and electron-poor moiety) at position 3 of the oxadiazole scaffold. In the second strategy, a phenyl moiety has been grafted to position 5 of the synthesized oxadiazole derivatives. The ortho position of this phenyl ring was decorated with different biologically active pharmacophoric entities, such as hydrazide or N-acylhydrazone. The N-acylhydrazone scaffold either incorporates phenyl triazole (more lipophilic and electron-rich moiety) or o-nitrophenyl (polar and electron-poor moiety). The bicyclic polar isoindoline motif or lipophilic quinoline motif featuring five- or six-membered heterocyclic ring fused to benzene, respectively was also introduced at the ortho position of the phenyl ring. Linker properties were studied in the third strategy, by bridging the aryl or heteroaryl motif at the ortho position of the phenyl ring at position 5 of the oxadiazole core via 4- or 6-atom linkers. As donors or acceptors of H bonds, these motifs and linkers may collaborate with amino acids in the active site of the AChE enzyme. Such variation in substitutions ensured different electrical, steric, and lipophilic environments, which could affect the activity of the target molecules. Several in vitro assays, such as AChE inhibition, BuChE inhibition, antioxidant activity, MAO-B inhibition, and MAO-A inhibition were performed to evaluate the synthetic oxadiazole derivatives’ potential to treat AD.

Results and discussion

Chemistry

In continuation of our previous work [56, 57], herein this study focuses on the functionalization of the 1,2,4-oxadiazole scaffold by constructing novel 3,5-diaryl derivatives containing phenyl, benzyl or 4-trifluoromethylphenyl at C3 position and a pharmacophoric group at C5 of the 1,2,4-oxadiazole scaffold. The phenyl moiety at the C5 position incorporates substituents at the ortho position with hydrogen bond donor/acceptor characters. The acetic acid hydrazide derivatives 1a-c were prepared from the corresponding esters by refluxing with hydrazine in ethanol. Condensation of the hydrazide with o-nitrobenzaldehyde or 2-phenyl-2 H-1,2,3-triazole-4-carbaldehyde afforded the corresponding Schiff`s bases 2a-c and 3a-c, respectively. The structures of the Schiff`s bases were confirmed by IR and NMR spectra, where; the IR spectra showed a strong band at a wave number of 3231 to 3323 cm− 1, corresponding to NH stretching, also a strong band appeared at 1705 to 1716 cm− 1 for 2a-c and 3a-c, revealing the presence of the C = O group. The NMR spectra of 2a-c and 3a-c confirmed their existence as a mixture of E/Z isomers. Where 2a and 2c exist in a ratio (5:2) and 2b, 3a, and 3c exist in a ratio (2:1), while 3b exists in a ratio (3:2) for E/Z, respectively. The 1HNMR spectra of 2a-c and 3a-c showed the proton of NH resonating downfield at δH: 11.94 to 11.68 ppm for the major and minor isomers. Furthermore, the 13CNMR spectra showed a characteristic signal for ArOCH2 resonating at δC: range 65.9 to 66.1 ppm. Furthermore, refluxing of the hydrazides 1a-c with phthalic anhydride in acetic acid afforded the corresponding N-Phthalimido-protected hydrazide 4a-c. The structure of 4a-c was confirmed by IR spectra, where the NH group appeared at wave number 3247 to 3182 cm− 1, corresponding to NH stretching. A strong band appeared at the range 1739 –1737 cm− 1, corresponding to the C = O group. The 1HNMR spectral analyses confirmed the structure of 4a-c, where the NH proton appeared at range δH: 11.08–11.01 ppm, also the aliphatic protons of ArOCH2 for 4a-c appeared at δH: 5.14, 5.06, and 5.13 ppm, respectively. Moreover, 13CNMR spectra of 4a-c showed the characteristic signals corresponding to the carbonyl carbon of the phthalimido group at δC: 165.38, 165.1, and 165.31 ppm, respectively. Coupling of the hydrazide 1a, b with quinaldic acid using DCC/6-NO2-HOBt protocol afforded the amide products 5a, b in excellent yields. On the other hand, the amide 5c was synthesized in a good yield superior to that of the DCC/NO2-HOBt protocol via a reaction of the acid chloride of quinaldic acid and the hydrazide 1c. The structures of 5a-c were confirmed by IR spectra, where the NH group appeared at wave number 3435 to 3255 cm− 1, corresponding to NH stretching. A strong band appeared at the range 1719 –1666 cm− 1, corresponding to the C = O groups of the amides. The 1HNMR spectral analyses confirmed the structure of 5a-c, where the NH proton appeared at range δH: 10.96–10.15 ppm, also the aliphatic protons of ArOCH2 of compounds 5a-c appeared at δH: 4.88, 4.82, and 5.0 ppm, respectively. Moreover, 13CNMR spectra of 5a-c showed the characteristic signals corresponding to the carbonyl carbon of amide at δC: 167.1 to 169.9 ppm. The characteristic carbon of CF3 of 5c appeared at δC: 126.4 ppm.

Scheme 1
scheme 1

The reagents and synthesis route used to prepare the target compounds 2–5

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AChE and BuChE inhibitory activities

The in vitro AChE and BuChE inhibition potentials of newly synthesized oxadiazole derivatives were assessed by comparing their half maximum inhibitory concentration (IC50) values to those of anti-AD drugs, donepezil and rivastigmine. The results, which are shown in Table 1, are graphically represented in Figs. 2 and 3. Eleven compounds (1b, 2a-c, 3b, 4a-c, and 5a-c) exhibited excellent inhibitory potential against AChE, with IC50 values ranging from 0.00098 to 0.07920 µM. Their potency was 1.55 to 125.47 times higher than that of donepezil (IC50 = 0.12297 µM). Oxadiazole derivatives 1a, 1c, 3a, and 3c exhibited nonexistent efficacy towards AChE. In contrast, the newly synthesized oxadiazole derivatives with IC50 values in the range of 16.64–70.82 µM exhibited less selectivity towards BuChE when compared to rivastigmine (IC50 = 5.88 µM). Compounds 3a, 3b and 1c showed the most prominent inhibitory potential against BuChE with IC50 values of 16.64, 17.14, and 17.74 µM, respectively.

Table 1 Biological evaluation results of the synthesized 1,2,4-oxadiazole-based derivatives
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Fig. 2
figure 2

A bar diagram representing the AChE inhibition of the synthesized compounds

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Fig. 3
figure 3

A bar diagram representing the BuChE inhibition of the synthesized compounds

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1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity

The antioxidant activity of the synthesized oxadiazole derivatives was evaluated using the DPPH assay. Quercetin, a common antioxidant, was used for comparison of the IC50 values. As can be shown in Table 1; Fig. 4, among tested oxadiazole derivatives, N-acylhydrazone derivative 2c (IC50 = 463.85 µM) was a more potent antioxidant than quercetin (IC50 = 491.23 µM). N-Acylhydrazone derivatives 3b (IC50 = 536.83 µM) and 3c (IC50 = 582.44 µM) exhibited comparable antioxidant activity to that of quercetin. Compounds 1c, 2b, and 5a (IC50 values of 734.85, 991.45, and 885.05 µM, respectively) showed significant antioxidant potential. With IC50 values in the range of 1143.46- 1306.54 µM, oxadiazole derivatives 1a, 1b, 2a, and 5c exhibited moderate antioxidant activity.

Fig. 4
figure 4

A bar diagram representing the antioxidant potential of the synthesized compounds using DPPH assay

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MAO-B and MAO-A inhibitory activities

The MAO-B and MAO-A inhibitory potentials of all the synthesized 1,2,4-oxadiazole-based derivatives were evaluated. Biperiden and methylene blue (methylthioninium chloride) were used as the reference standards. As shown in Table 1; Fig. 5, oxadiazole derivatives 3b (IC50 = 140.02 µM) and 4c (IC50 = 117.43 µM) showed excellent MAO-B inhibitory potentials. They were more potent than biperiden (IC50 = 237.59µM). Compounds 2c and 5a with IC50 values of 265.56 and 274.43 µM, respectively, exhibited equipotent MAO-B inhibitory activity to that of biperiden. Oxadiazole derivatives 1a, 1c, 2b, and 4a with IC50 values in the range of 346.03–396.84 µM showed significant MAO-B inhibitory activity. Five oxadiazole derivatives (1a, 1b, 3a, 3c, and 4b) exhibited remarkable MAO-A inhibitory potential, with IC50 values ranging from 47.25 to 129.7 µM. Their potency was 1.1 to 3.03 times higher than that of methylene blue (IC50 = 143.6 µM). Compound 4c (IC50 = 143.9 µM) exhibited equipotent MAO-A inhibitory potential to that of methylene blue.

Fig. 5
figure 5

A bar diagram representing the MAO-B and MAO-A inhibition of the synthesized compounds

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In vitro determination of the anti-hemolytic effect of synthesized compounds

The inhibition of human red blood cells (HRBCs) membrane lysis under hypnotic conditions was taken as a measure of the mechanism of anti-hemolytic activity of the synthesized 1,2,4-oxadiazole-based derivatives. Inhibition of hemolysis was expressed as IC50 value, which is the inhibitory concentration at which 50% of hemolysis is repressed. Eight compounds namely, 1a, 1b, 2a-c, 4b, 5a, and 5b with IC50 values at the range of 1193.47–4338.69 µM (Table 1), provided higher protection against induced lyses than diclofenac (IC50 = 1121.94 µM). Compound 4c (IC50 = 1040.07 µM), exhibited protection against induced lyses comparable to that of diclofenac. The results revealed the nontoxic effect of the synthesized 1,2,4-oxadiazole derivatives, thus making them safe drug candidates (Fig. 6).

Fig. 6
figure 6

A bar diagram representing the anti-hemolytic effect of the synthesized compounds

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Structure-activity relationships (SAR)

The structure-activity relationship studies of the new oxadiazole-based derivatives revealed that the 3,5-disubstituted-1,2,4-oxadiazole pharmacophoric entity is highly tolerated for AChE inhibition. It was clear that grafting a benzyl moiety at position 3 of the oxadiazole ring (1b, 2b, 3b, 4b, and 5b) exhibited higher AChE inhibitory activity than phenyl and p-trifluoromethylphenyl moieties. It was intriguing to observe that among oxadiazole derivatives featuring the hydrazide scaffold at the ortho position of the phenyl ring of the 5th position of the oxadiazole ring, only compound 1b incorporating the benzyl moiety showed potent AChE inhibitory activity. Shifting the hydrazide moiety into the N-acylhydrazone scaffold improved the AChE inhibitory potential. Further analysis of hydrazone derivatives revealed that the polar and electron-poor o-nitrophenyl motif (2a-c) was more tolerated for AChE inhibition than the lipophilic and electron-rich phenyl triazole motif (3a-c). The incorporation of isoindoline or quinoline scaffold into the hydrazide moiety demonstrated a good impact on the AChE inhibition. An interesting phenomenon is that polar isoindoline derivatives (4a-c) scored higher AChE inhibitory potential than lipophilic quinoline counterparts (5a-c) (Fig. 7).

Fig. 7
figure 7

Structure-activity relationship (SAR) for anti-AD potential of 1,2,4-oxadiazole-based derivatives

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Regarding the antioxidant potential, it was concluded that the 1,2,4-oxadiazole derivatives incorporating the N-acylhydrazone scaffold (2 and 3) emerged as the most prominent antioxidant candidates. In hydrazide and hydrazone derivatives, grafting the lipophilic electron-deficient p-trifluoromethylphenyl at position 3 of the oxadiazole ring (1c, 2c, and 3c) exhibited significant antioxidant potential. The lipophilic quinoline motif displayed more efficient antioxidant activity than the polar isoindoline motif (Fig. 7).

The 1,2,4-oxadiazole hydrazide derivatives (1a-c) showed mild MAO-B inhibition. The incorporation of N-acylhydrazone scaffold (2b, 2c, and 3b) showed a significant impact on the MAO-B inhibition. It was noteworthy that MAO-B inhibition was more enhanced by the incorporation of the isoindoline motif (4a-c) than the quinoline motif (5a-c). It is worth noting that inserting the lipophilic electron-deficient p-trifluoromethylphenyl at position 3 of the oxadiazole ring (1c, 2c, and 4c) highly improved MAO-B inhibitory potential (Fig. 7).

Regarding MAO-A inhibition, both hydrazide and N-acylhydrazone scaffolds were tolerated for activity. The incorporation of an electron-rich phenyl triazole motif in the N-acylhydrazone scaffold (3a and 3c) exhibited a better impact on MAO-A inhibition than the electron-poor o-nitrophenyl motif (2a-c). An interesting phenomenon was that MAO-A inhibition was enhanced by the incorporation of the isoindoline motif (4b and 4c), in contrast, adding the quinoline moiety in compounds 5a-c completely abolished MAO-A inhibitory potential.

Molecular docking study

The purpose of the docking study is to predict the binding interactions between the synthesized drug candidates and the active site of AChE enzyme as well as to rationalize the biological activities of the target molecules. MOE (2020.09) software [58] was used in this study. First, the protein crystal structure of AChE (PDB: 7E3H) [59] was downloaded from the protein data bank and the docking procedure was validated step before docking the synthesized molecules. In the validation step, the co-crystallized molecule (donepezil) was docked in the active site of AChE enzyme to get a pose that overlapped the experimental pose with RMSD of 0.42 Å which is within the cutoff limit (< 1 Å) (Fig. 8). The active site of AChE consists of a narrow groove that is 20 Å deep and contains the catalytic site deep at the bottom of the groove. The donepezil structure is deeply embedded inside the AChE active site pocket and the 2,3-dihydroinden-1-one carbonyl moiety interacts with Phe295 by hydrogen bonding. The benzyl group showed arene interactions with Trp86 amino acid of the enzyme. The synthesized molecules were docked using the same method and the binding modes with the human AChE binding site were evaluated. The compounds generally displayed analogous interactions and occupied the same space with the binding site as donepezil.

The 3-benzyl-1,2,4-oxadiazole moiety of compound 2b inhabited a similar space as donepezil piperidine ring and formed Arene-H interaction with Tyr341 (Fig. 9). In addition, the hydrazide carbonyl forms a hydrogen bond with Ser203 via a water bridge which improved the stability of the molecule in the active site. The 5-phenyl moiety formed Arene-H interaction with Phe338. Finally, the nitro phenyl group showed Arene-H interaction with Trp86 same as donepezil. Most compounds with benzyl substitution showed better interactions and higher binding energies than their phenyl and trifluoro phenyl congeners which rationalize their higher inhibition activity towards AChE enzyme. In addition, all compounds with benzyl substitution showed Arene-H interaction with Tyr341 which may highlight the importance of this interaction with binding stability.

In addition, the prediction of several properties such as ADME and other physicochemical properties was done using the online software SwissADME. All compounds showed alignment with Lipinski’s rule of five [60]. Most of the compounds showed a high probability of gastrointestinal absorption and all of them exhibited a 0.55 oral bioavailability score (see Supplementary data) and the docking score and interactions of all target compounds with the active site of AChE enzyme were collected in (Table 2) with donepezil. Compound 2b showed the highest in vitro inhibitory activity, high docking binding, and good physicochemical properties as shown in the bioavailability radar map (Fig. 10). All these data suggest that 2b could be considered as a promising candidate for future development.

Fig. 8
figure 8

A) 2D diagram of donepezil interaction with human acetylcholinesterase; B) 3D of the co-crystallized donepezil inhibitor (magenta) together with the re-docked donepezil (green) in the gorge of the active site (highlighted in yellow) of human acetylcholinesterase (RMSD = 0.42 Å)

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Fig. 9
figure 9

A) 2D diagram and B) 3D diagram of the interactions of compound 2b (green) with the AChE active site (highlighted in yellow) (PDB: 7E3H)

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Fig. 10
figure 10

Bioavailability radar map of donepezil and 2b. The inner pink area shows the ideal range for each of the following characteristics: solubility: log S should not exceed 6, saturation: the fraction of carbons in the sp3 hybridization should not be less than 0.25, flexibility: no more than 9 rotatable bonds, polarity: TPSA between 20 and 130 Å2, size: molecular weight between 150 and 500 g/mol, and lipophilicity: XLOGP3 between − 0.7 and + 5.0

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Table 2 The docking score and interactions of all target compounds
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Conclusion

New derivatives based on 1,2,4-oxadiazole core were designed, synthesized and their anti-AD potential was assessed. Eleven compounds with IC50 values ranging from 0.00098 to 0.07920 µM demonstrated excellent inhibitory potential against AChE. Their efficacy was 1.55 to 125.47 times greater than donepezil’s. In contrast, the newly synthesized oxadiazole derivatives exhibited less selectivity towards BuChE. Furthermore, compared to quercetin (IC50 = 491.23 µM), oxadiazole derivative 2c (IC50 = 463.85 µM) exhibited greater antioxidant capacity. Comparable antioxidant activity to that of quercetin was shown by compounds 3b (IC50 = 536.83 µM) and 3c (IC50 = 582.44 µM). The oxadiazole compounds with the highest ability to inhibit MAO-B were 3b (IC50 = 140.02 µM) and 4c (IC50 = 117.43 µM). Compared to biperiden (IC50 = 237.59 µM), they exhibited more potent MAO-B inhibition. Oxadiazole derivatives 1a, 1b, 3a, 3c, and 4b exhibited excellent MAO-A inhibitory potential. Their potency was 1.1 to 3.03 times higher than that of methylene blue. Compound 4c exhibited equipotent MAO-A inhibitory potential to that of methylene blue. The majority of synthesized oxadiazole derivatives demonstrated a strong protective effect against induced lysis of human red blood cells, indicating their safety as potential therapeutic options. Oxadiazole derivatives 2b, 2c, 3b, 4a, 4c, and 5a were shown as multitarget anti-AD agents. A computational explanation for the high AChE inhibitory potential is the strong interactions between the synthesized oxadiazole derivatives and the AChE active site. The physicochemical properties of compound 2b were prominent. 1,2,4-Oxadiazole derivative 2b is a promising anti-AD drug candidate for further research and development.

Experimental

Chemistry

Materials and equipment

The materials and equipment were reported in the Supporting Information section.

General method for the synthesis of Schiff`s base 2a-c and 3a-c

To a stirred solution of the acid hydrazide 1a-c, (0.318 mmol) in ethanol (20 mL) o-nitrobenzaldehyde or 2-phenyl-2 H-1,2,3-triazole-4-carbaldehyde (0.35 mmol) was added and the reaction mixture was heated under reflux for 9 h, then left to cool to room temperature. The solid formed was filtered off to give the desired product.

(E/Z)-N’-(2-Nitrobenzylidene)-2-(2-(3-phenyl-1,2,4-oxadiazol-5-yl)phenoxy)acetohydrazide (2a).

Off-white powder; (yield 65.0%); m.p = 190–191 ˚C; Rf = 0.1 (n-hexane: EtOAc, 2:1); IR (KBr, cm− 1): 3317 (NH), 1714 (CO), 1526, 1306 (NO2); NMR for the major product (2a-E isomer): 1H NMR (500 MHz, DMSO-d6) δH: 11.94 (s, 0.67 H, NH), 8.35 (s, 0.67 H, CH = N), 8.11–7.139 (m, 13H, ArH), 5.383 (s, 1.37 H, ArOCH2); 13C NMR (125 MHz, DMSO-d6) δC: 175.7, 169., 168.1, 157.8, 148.4, 144.1, 140, 135.1, 134.1, 132.1, 131.1, 129.8, 129.1, 128.6, 127.6, 126.9, 125.1, 121.7, 114.5, 113.1, 65.9; Anal. calcd for C23H17N5O5 (443.12): C, 62.30; H, 3.86; N, 15.79; Found: C, 62.66; H, 3.91; N, 15.59.

(E/Z)-2-(2-(3-Benzyl-1,2,4-oxadiazol-5-yl)phenoxy)-N’-(2-nitrobenzylidene)acetohydrazide (2b)

White powder; (yield 72.0%); m.p = 184–186 ˚C; Rf = 0.1 ( n-hexane: EtOAc, 2:1); IR (KBr, cm− 1): 3231 (NH), 1705 (CO), 1517 and 1287 (NO2); NMR for the major product (2b, E-isomer): 1HNMR (500 MHz, DMSO-d6) δH: 11.92 (s, 0.66 H, NH), 8.34 (s, 0.67 H, CH = N), 8.11–7.139 (m, 13H, ArH), 5.34 (s, 1.1 H, ArOCH2), 4.13 (d, J = 8.5 Hz, 2 H, ArCH2); 13C NMR (125 MHz, DMSO-d6) δC: 175.3, 169.7, 169.4, 157.6, 148.5, 144.1, 140.0, 136.5, 134.9, 134.1, 131.7, 131.1, 129.5, 129.1, 128.6, 127.4, 125.1, 121.6, 114.4, 113.1, 65.9, 31.9; Anal. calcd for C24H19N5O5 (457.14), C, 63.02; H, 4.19; N, 15.31; Found: C, 62.91; H, 4.32; N, 15.24.

(E/Z)-N’-(2-Nitrobenzylidene)-2-(2-(3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)phenoxy) acetohydrazide (2c)

White powder; (yield, 61.0%) m.p = 222–224 ˚C; Rf = 0.1 ( n-hexane: EtOAc, 2:1); IR (KBr, cm− 1): 3315 (NH), 1716 (CO), 1531 and 1322 (NO2); NMR for the major product ( E-isomer) product: 1H NMR (500 MHz, DMSO-d6) δH: 11.94 (s, 0.61 H, NH), 8.36 (s, 0.63 H, CH = N), 8.28–7.15 (m, 12 H, ArH), 5.395 (s, 1.32 H, ArOCH2); 13C NMR (125 MHz, DMSO-d6) δC: 176.1, 169.3, 167.2, 164.7, 157.8, 148.5, 144.1, 140.0, 135.3, 134.1, 131.9, 131.1, 130.7, 129.1, 128.7, 128.5, 126.8, 125.1, 121.8, 114.6, 112.9, 66.1; Anal. calcd for C24H16F3N5O5 (511.11), C, 56.37; H, 3.15; N, 13.69; Found: C, 56.51; H, 3.35; N, 13.43.

(E/Z)-2-(2-(3-Phenyl-1,2,4-oxadiazol-5-yl)phenoxy)-N’-((2-phenyl-2 H-1,2,3-triazol-4-yl)methylene) acetohydrazide (3a)

White powder (yield 73.0%); m.p = 224–228 ˚C; Rf = 0.33 ( n-hexane: EtOAc, 2:1); IR (KBr, cm− 1): 3319 (NH), 1715 (CO); NMR for major product (3a, E-isomer) product: 1H NMR (500 MHz, DMSO-d6) δH: 11.89 (s, 0.61 H, NH), 8.46–7.14 (m, 16 H, ArH, HC = N, triazolo -H), 5.39 (s, 1.14 H, ArOCH2); 13C NMR (125 MHz, DMSO-d6) δC: 175.7, 169.2, 168.1, 157.8, 145.8, 19.3, 135.4, 135.1, 134.8, 132.1, 131.7, 130.4, 129.8, 128.7, 127.6, 126.9, 121.8, 118.9, 114.6, 113.2, 66.1; Anal. calcd for C25H19N7O3 (465.15), C, 64.51; H, 4.11; N, 21.06; Found: C, 64.28; H, 4.32; N, 21.29.

(E/Z)-2-(2-(3-Benzyl-1,2,4-oxadiazol-5-yl)phenoxy)-N’-((2-phenyl-2 H-1,2,3-triazol-4-yl)methylene) acetohydrazide (3b)

White powder (yield 75.0%); m.p = 206–207 ˚C; Rf = 0.33 ( n-hexane: EtOAc, 2:1); IR (KBr, cm− 1): 3310 (NH), 1709 (CO); NMR for major product (3b, E-isomer): 1H NMR (500 MHz, DMSO-d6) δH: 11.88 (s, 0.58 H, NH), 8.48–7.07 (m, 16 H, ArH, HC = N, and triazolo -H), 5.35 (s, 1.2 H, ArOCH2), 4.13 (s, 1.18 H, ArCH2); 13C NMR (125 MHz, DMSO-d6) δC: 175.3, 169.7, 169.2, 157.6, 145.8, 139.3, 136.5, 135.1, 134.9, 131.7, 130.4, 129.4, 129.1, 128.6, 127.4, 121.7, 119.0, 114.4, 113.1, 65.9, 31.9; Anal. calcd for C26H21N7O3 (479.17), C, 65.13; H, 4.41; N, 20.45; Found: C, 64.90; H, 4.52; N, 20.61.

(E/Z)-N’-((2-Phenyl-2 H-1,2,3-triazol-4-yl)methylene)-2-(2-(3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)phenoxy)acetohydrazide (3c)

White powder (yield 72.0), m.p = 210–214 ˚C; Rf = 0.27 ( n-hexane: EtOAc, 2:1); IR (KBr, cm− 1): 3323 (NH), 1707 (CO); NMR for the major product (3c, E-isomer): 1H NMR (500 MHz, DMSO-d6) δH: 11.905 (s, 0.61 H, NH), 8.46 (s, 0.55 H, triazolo-H), 8.32–7.16 (m, 15 H, ArH, and ArCH = N), 5.403 (s, 1.2 H, ArOCH2); 13CNMR (125 MHz, DMSO-d6) δC: 1761, 169.1, 167.2, 157.9, 145.8, 139.6, 135.3, 135.1, 134.8, 131.9, 131.7, 131.4, 130.7, 130.4, 128.7, 128.5, 126.8, 121.8, 118.9, 114.9, 112.9, 66.1; Anal. calcd for C26H18F3N7O3 (533.14), C, 58.54; H, 3.40; N, 18.38; Found: C, 58.21; H, 3.32; N, 18.51.

General method for the synthesis of 4a-c

To a stirred solution of the acid hydrazide 1a-c (0.318 mmol) in acetic acid (20 mL) phthalic anhydride (81.9 mg, 0.472 mmol) was added and the reaction mixture was heated near the boiling point for 8 h, then left to cool to room temperature. The solid formed was filtered off to afford the desired product.

N-(1,3-Dioxoisoindolin-2-yl)-2-(2-(3-phenyl-1,2,4-oxadiazol-5-yl)phenoxy)acetamide (4a)

White solid; (76% yield); m.p = 256–259 °C; Rf = 0.66 (n-hexane: EtOAc, 1:1); IR (KBr, cm− 1): 3198 (NH), 1739 (CO); 1H NMR (500 MHz, DMSO-d6) δH: 11.08 (s, 1H, N-H ), 8.12–8.10 (m, 1H, Ar-H), 7.96–7.93 (m, 6 H, Ar-H), 7.73–7.70 ( m, 1H, Ar-H), 7.44–7.23 (m, 5 H, Ar-H), 5.14 (s, 2 H, ArOCH2); 13C NMR (125 MHz, DMSO-d6) δC: 174.7, 168.3, 167.8, 165.4, 156.5, 136.1, 135.5, 132.1, 131.4, 129.7, 129.5, 127.5, 126.7, 124.5, 122.7, 115.0, 113.1, 67.3; Anal. calcd for C24H16N4O5 (440.11), C, 65.45; H, 3.66; N, 12.72; Found: C, 65.41; H, 3.42; N, 12.81.

2-(2-(3-Benzyl-1,2,4-oxadiazol-5-yl)phenoxy)-N-(1,3-dioxoisoindolin-2-yl)acetamide (4b)

White solid (78% yield); m.p = 206–209 °C; Rf = 0.32 ( n-hexane: EtOAc, 2:1); IR (KBr, cm− 1): 3182 (NH), 1737 (CO); 1H NMR (500 MHz, DMSO-d6) δH: 11.06 (s, 1H, N-H ), 8.04–79.24 (m, 5 H, Ar-H), 7.69–7.66 (m, 1H, Ar-H), 7.27–7.19 ( m, 2 H, Ar-H), 7.075–7.026 (m, 5 H, Ar-H), 5.06 (s, 2 H, ArOCH2), 4.07 (s, 2 H, ArCH2); 13C NMR (125 MHz, DMSO-d6) δC:174.3, 169.5, 167.7, 165.2, 156.3, 135.96, 135.92, 15.5, 131.1, 129.9, 128.9, 128.7, 127.22, 124.4, 122.7, 115.0, 112.9, 67.4, 31.7; Anal. calcd for C25H18N4O5 (454.13), C, 66.08; H, 3.99; N, 12.33; Found: C, 66.25; H, 4.17; N, 12.14.

N-(1,3-Dioxoisoindolin-2-yl)-2-(2-(3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)phenoxy)acetamide (4c)

White solid; (70% yield); m.p = 210–214 °C; Rf = 0.33 ( n-hexane: EtOAc, 2:1); IR (KBr, cm− 1): 3247 (NH), 1738 (CO); 1H NMR (500 MHz, DMSO-d6) δH:: 11.01 (s, 1H, N-H ), 8.18 (d, J = 8.5 Hz, 2 H, Ar-H), 8.13 (dd, J = 7.5 Hz,1.5 Hz, 1H, Ar-H), 7.97–7.89 ( m, 4 H, Ar-H), 7.78 (d, J = 8.0 Hz, 2 H, Ar-H), 7.74–7.66 (m, 1H, Ar-H), 7.32 (d, J = 8.5 Hz, 2 H, Ar-H), 7.30–7.20 (m, 1H, Ar-H), 5.13 (s, 2 H, ArOCH2); 13C NMR (125 MHz, DMSO-d6) δC: 175.3, 167.7, 165.3, 156.7, 136.0, 135.7, 131.6, 130.7, 129.7, 128.5, 128.2, 126.6, 126.6, 125.3, 124.4, 122.7, 115.1, 113.1, 67.3; Anal. calcd for C25H15F3N4O5 (508.10), C, 59.06; H, 2.97; N, 11.02; Found: C, 60.11; H, 2.71; N, 11.19.

General method for the synthesis of the amides 5a, b

DCC (90 mg, 0.387 mmol) and Et3N (65.15 mg, 0.645 mmol) were added to a solution of quinaldic acid (56.43 mg, 0.3225 mmol) and 6-nitro HOBt (69.73 mg, 0.387 mmol) in DMF (2mL) and the solution was stirred for 5 min at 0 °C. The acid hydrazide 1a or 1b (0.3225 mmol) was added and the solution was stirred at room temperature for 16 h. The reaction mixture was poured into cold water and the formed solid was filtered. To purify the crude product from the side products, the crude product was refluxed in ethanol (10 mL) for 1 h and filtered while hot, the precipitate was washed with hot ethanol (10 mL), collected, and dried to get the desired peptide.

N’-(2-(2-(3-Phenyl-1,2,4-oxadiazol-5-yl)phenoxy)acetyl)quinoline-2-carbohydrazide (5a)

White solid (76% yield); m.p = 241–243 °C; Rf = 0.3 (n-hexane: EtOAc, 1:1); IR (KBr, cm− 1): 3341and 3270 (NH), 1719 and 1666 (CO); 1H NMR (500 MHz, DMSO-d6) δH: 10.87 (s, 1H, CON-H ), 10.15 (s, 1H, CON-H ), 8.10 (m, 1H, Ar-H), 7.98 (s, 2 H, Ar-H), 7.66 ( s, 1H, Ar-H), 7.50–7.44 (d, 5 H, Ar-H), 7.29 (s, 2 H, ArH ), 7.20 (s, 2 H, ArH ), 7.02 (s, 1H, ArH ), 6.90 (s, 1H, ArH ), 4.88 (s, 2 H, ArOCH2); 13C NMR (125 MHz, DMSO-d6) δC: 174.6, 170.5, 168.4, 167.1, 156.5, 136.5, 135.6, 135.4, 132.1, 131.3, 129.8, 127.7, 127.5, 126.6, 124.4, 12.4, 121.5, 119.3, 118.9, 14.7, 112.7, 111.8, 108.4, 67.4. Anal. calcd for C26H19N5O4 (465.14), C, 67.09; H, 4.11; N, 15.05; Found: C, 67.18; H, 4.25; N, 14.91.

N’-(2-(2-(3-Benzyl-1,2,4-oxadiazol-5-yl)phenoxy)acetyl)quinoline-2-carbohydrazide (5b)

White solid (74% yield); m.p = 206–209 °C; Rf = 0.03 (n-hexane: EtOAc, 1:1); IR (KBr, cm− 1): 3355 and 3255 (NH), 1718 and 1667 (CO); 1H NMR (500 MHz, DMSO-d6) δH: 10.85 (s, 1H, OCN-H ), 10.27 (s, 1H, OCN-H ), 7.97 (s, 1H, Ar-H), 7.57 (s, 2 H, Ar-H), 7.21–6.90 ( m, 12 H, Ar-H), 4.82 (s, 2 H, ArOCH2), 3.95 (s, 2 H, ArCH2); 13C NMR (125 MHz, DMSO-d6) δC:174.2, 169.9, 169.91, 166.6, 156.5, 136.6, 136.2, 135.4, 131.1, 129.5, 129.3, 129.1, 127.7, 127.4, 124.4, 122.3, 121.5, 119.3, 118.8, 114.7, 112.8, 111.8, 108.5, 67.4, 31.1. Anal. calcd for C27H21N5O4 (479.16), C, 67.63; H, 4.41; N, 14.61; Found: C, 67.51; H, 4.52; N, 14.43.

N’-(2-(2-(3-(4-(Trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)phenoxy)acetyl)quinoline-2-carbohydrazide (5c)

A mixture of quinaldic acid (22.88 mg, 0.1322 mmol) and SOCl2 (5.0 ml) was refluxed for 2 h, then the excess thionyl chloride was evaporated under reduced pressure. Dry THF (5.0 mL), Et3N (2.0 mL), and the acid hydrazide 1c (50 mg, 0.1322 mmol) were added, and the mixture was stirred at room temperature for 16 h. The reaction mixture was poured into cold water and the formed precipitate was filtered. The crude product was recrystallized from ethanol, filtered, and dried to get the desired peptide 5c (92% yield) as off-white powder, m.p = 202–203 °C; Rf = 0.7 (EtOAc : n-hexane, 1:1); IR (KBr, cm− 1): 3435 and 3321 (NH), 1718 and 1683 (CO); 1H NMR (500 MHz, DMSO-d6) δH: 10.96 (s, 1H, OCN-H), 10.45 (s, 1H, OCN-H), 8.55 (d, J = 8.5 Hz, 1H, Ar-H), 8.19–8.13 (m, 4 H, Ar-H), 8.09–8.03 (m, 2 H, Ar-H), 7.88 (t, J = 7.0 Hz, 1H, Ar-H), 7.729 (t, J = 7.0 Hz, 2 H, ArH ), 7.61 (d, J = 8.5 Hz, 2 H, ArH ), 7.35 (d, J = 8.5 Hz, 1H, ArH), 7.25 (t, J = 8.0 Hz, 1H, ArH ), 5.00 (s, 2 H, ArOCH2); 13C NMR (125 MHz, DMSO-d6) δC: 174.9, 167.5, 167.2, 163.8, 156.4, 149.4, 146.7, 138.6, 135.9, 131.2, 131.2, 130.5, 129.7, 129.5, 128.9, 128.7, 128.5, 126.5, 126.47, 126.44, 122.6, 119.2, 114.9, 112.4, 67.7. Anal. calcd for C27H18F3N5O4 (533.13), C, 60.79; H, 3.40; N, 13.13; Found: C, 60.56; H, 3.53; N, 13.24.

Biological evaluation

The biological experiments were conducted in compliance with the previously documented protocols and are available in the Supplementary Materials; AChE and BuChE inhibitory assays [61], DPPH radical scavenging activity [62], MAO-B and MAO-A inhibitory assays [63], and the anti-hemolytic effect of synthesized compounds [64].

Molecular modeling studies

The x-ray structure of the of AChE (PDB: 7E3H) was downloaded from the protein databank (PDB) website, (https://www.rcsb.org/) at a resolution of 1.90 Å and 2.00 Å respectively. All the molecular modeling and docking studies were carried out using MOE 2020.09 (Chemical Computing Group, Canada) as the computational software. First, all the hydrogen atoms were added using the Protonate 3D algorithm where the protonation states of the amino acid residues were assigned, and the partial charges of atoms were added. In addition, the compounds were drawn using the builder tool and energy was minimized using the MMFF94x force field. MOE induced-fit Dock tool used to dock the synthesized compounds into the active site. The selection of the final docked ligand–enzyme poses was according to the criteria of binding energy score and combined with ligand-receptor interactions.