Ayoup et al. BMC Chemistry(2024) 18:130
https://doi.org/10.1186/s13065-024-01235-x

BMC Chemistry

RESEARCH

Open AccessNew 1,2,4-oxadiazole derivatives as potential
multifunctional agents for the treatmentof Alzheimer’s disease: design, synthesis,
and biological evaluation
Mohammed Salah Ayoup1,2*, Mariam Ghanem2, Hamida Abdel-Hamid2, Marwa M. Abu-Serie3, Aliaa Masoud4,
Doaa A. Ghareeb4,5,6, Mohammed B. Hawsawi7, Amr Sonousi8,9 and Asmaa E. Kassab8*
Abstract
A series of new 1,2,4-oxadiazole-based derivatives were synthesized and evaluated for their anti-AD potential. The
results revealed that 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). 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). Moreover, oxadiazole derivative 2c (IC50 = 463.85 µM) was more potent antioxidant than quercetin(IC50 = 491.23 µM). Compounds 3b (IC50 = 536.83 µM) and 3c (IC50 = 582.44 µM) exhibited comparable antioxidant
activity to that of quercetin. Oxadiazole derivatives 3b (IC50 = 140.02 µM) and 4c (IC50 = 117.43 µM) showedprominent MAO-B inhibitory potential. They were more potent than biperiden (IC50 = 237.59 µM). Compounds
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). Most of thesynthesized oxadiazole derivatives provided significant protection against induced HRBCs lysis, revealing thenontoxic effect of the synthesized compounds, thus making them safe drug candidates. The results unveiled
oxadiazole derivatives 2b, 2c, 3b, 4a, 4c, and 5a as multitarget anti-AD agents. The high AChE inhibitory potential
can be computationally explained by the synthesized oxadiazole derivatives’ significant interactions with the AChE
active site. Compound 2b showed good physicochemical properties. All these data suggest that 2b could beconsidered as a promising candidate for future development.
Keywords Design, Synthesis, 1,2,4-oxadiazole, Anti-alzheimer, AChE, BuChE, MAO-A, MAO-B, Molecular docking*Correspondence:
Mohammed Salah Ayoup
mayoup@kfu.edu.sa; mohammedsalahayoup@gmail.com
Asmaa E. Kassab
asmaa.kassab@pharma.cu.edu.eg
Full list of author information is available at the end of the article© The Author(s) 2024. 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) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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Ayoup et al. BMC Chemistry(2024) 18:130

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 diminishedintelligence [3, 4]. AD is a very difficult and emotionally
exhausting condition to care for, and few diseases distresspatients 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 heartdisease 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 byseveral 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 thecentral 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 theaxon 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 thehalf-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 levelof 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 treatmentof moderate-to-severe AD [24]. Thus, AChE remains a
potential therapeutic target in the hunt for novel anti-AD
medications.

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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 otherconditions. 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 provedthat following the inhibition of MAO-B, a rise in DA levels and a neuroprotective effect are observed [29–33].
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 [36–39].
Consequently, selective irreversible human MAO-B
inhibitors are efficiently employed in the treatment ofAD, either alone or in conjunction with other medications [40–43]. 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 thatsingle-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 antiAD candidates [45–47].
Due to its distinct bioisosteric characteristics and avery broad range of biological activities, the five-membered 1,2,4-oxadiazole heterocyclic ring has drawn a lotof 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 forthe development of innovative drugs. Throughout thepast fifteen years, there has been a two-fold increase ininterest in the biological applications of 1,2,4-oxadiazoles. The FDA has approved several derivatives basedAyoup et al. BMC Chemistry(2024) 18:130

on 1,2,4-oxadiazoles, which are now being promoted ascommercial 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[50–55].
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 firststrategy involved the incorporation of benzyl, phenyl(electron-rich moieties), or p-trifluoromethyl phenyl(more lipophilic and electron-poor moiety) at position 3of the oxadiazole scaffold. In the second strategy, a phenyl

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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 heterocyclicring 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 ringat position 5 of the oxadiazole core via 4- or 6-atom linkers. As donors or acceptors of H bonds, these motifs andlinkers may collaborate with amino acids in the active
site of the AChE enzyme. Such variation in substitutions

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

Ayoup et al. BMC Chemistry(2024) 18:130

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Scheme 1 The reagents and synthesis route used to prepare the target compounds 2–5

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 thisstudy 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 fromthe corresponding esters by refluxing with hydrazine inethanol. 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 3ac, respectively. The structures of the Schiff`s bases wereconfirmed 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 3ac, 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 thehydrazides 1a-c with phthalic anhydride in acetic acidafforded the corresponding N-Phthalimido-protectedAyoup et al. BMC Chemistry(2024) 18:130

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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 appearedat 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 thephthalimido 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 theother hand, the amide 5c was synthesized in a good yieldsuperior to that of the DCC/NO2-HOBt protocol via
a reaction of the acid chloride of quinaldic acid and thehydrazide 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
1
HNMR 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 ofcompounds 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.

AChE and BuChE inhibitory activities
The in vitro AChE and BuChE inhibition potentials of
newly synthesized oxadiazole derivatives were assessedby comparing their half maximum inhibitory concentration (IC50) values to those of anti-AD drugs, donepezil and rivastigmine. The results, which are shownin 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. Theirpotency 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 16.64–70.82µM 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, and17.74 µM, respectively.

Table 1 Biological evaluation results of the synthesized 1,2,4-oxadiazole-based derivatives
Compound
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
Donepezil HCl
Rivastigmine
Quercetin
Biperiden HCl
Diclofenac
Methylene blue

AChE-IC50(µM ± SD) a
N.A.
0.03098 ± 0.0024
N.A.
0.03393 ± 0.0029
0.00098 ± 0.000001
0.01271 ± 0.00102
N.A.
0.02330 ± 0.00069
N.A.
0.03275 ± 0.0011
0.02934 ± 0.0013
0.03769 ± 0.0027
0.04748 ± 0.0028
0.03374 ± 0.00089
0.07920 ± 0.0061
0.12297 ± 0.0.0103

BuChE-IC50(µM ± SD) a
N.A.
N.A.
17.47 ± 2.5
N.A.
35.84 ± 4.5
39.41 ± 5.3
16.64 ± 0.93
17.14 ± 2.3
19.72 ± 0.89
33.07 ± 4.7
N.A.
25.38 ± 1.9
N.A.
21.16 ± 2.970.82 ± 2.3

AChE Selectivity Index b
36,571
3,100
735
1,010
673
627
894

DPPH-IC50(µM ± SD) a
1215.74 ± 51.30
1280.92 ± 42.80
734.85 ± 12.80
1306.54 ± 31.50
991.45 ± 11.40
463.85 ± 31.50
N.A.
536.83 ± 19.30
582.44 ± 42.90
1658.66 ± 62.30
2660.83 ± 26.30
N.A.
885.05 ± 22.90
N.A.
1143.46 ± 68.90

MAO-A-IC50(µM ± SD) a
47.25 ± 8.1
82.54 ± 8.7
146.63 ± 16.9
N.A.
203.91 ± 17.9
N.A.
85.2 ± 5.9
N.A.
114.6 ± 19.5
N.A.
129.7 ± 16.9
143.9 ± 22.4
N.A.
N.A.
N.A.

Hemolysis-IC50(µM ± SD) a
1620.36 ± 57.8
1193.47 ± 78.3
656.56 ± 33.1
1792.14 ± 63.1
4104.89 ± 114.5
4338.69 ± 125.6
622.32 ± 23.4
457.23 ± 19.6
661.47 ± 23.8
522.45 ± 15.9
1788.84 ± 89.3
1040.07 ± 88.9
2016.04 ± 111.3
3559.75 ± 125.3
521.22 ± 11.9

5.88 ± 0.64a

The means of three replicates are used to express all valuesb

AChE Selectivity index = IC50 of BuChE/ IC50 of AChE

491.23 ± 14.8

The IC50 values of compounds that are more powerful than the reference standard are shown in the bold text
N.A. means no activity

MAO-B-IC50(µM ± SD) a
358.61 ± 23.4
534.58 ± 41.5
396.84 ± 19.6
N.A.
346.03 ± 14.3
265.56 ± 13.4
N.A.
140.02 ± 8.7
N.A.
393.68 ± 16.3
713.28 ± 22.3
117.43 ± 9.8
274.43 ± 21.1
N.A.
N.A.

237.59 ± 16.3
1121.94 ± 12.6
143.6 ± 22.1

Ayoup et al. BMC Chemistry(2024) 18:130

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Fig. 2 A bar diagram representing the AChE inhibition of the synthesized compounds

Fig. 3 A bar diagram representing the BuChE inhibition of the synthesized compounds

Fig. 4 A bar diagram representing the antioxidant potential of the synthesized compounds using DPPH assay

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 thanquercetin (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 of1143.46- 1306.54 µM, oxadiazole derivatives 1a, 1b, 2a,and 5c exhibited moderate antioxidant activity.

MAO-B and MAO-A inhibitory activities
The MAO-B and MAO-A inhibitory potentials of all thesynthesized 1,2,4-oxadiazole-based derivatives were evaluated. Biperiden and methylene blue (methylthioninium
chloride) were used as the reference standards. As shownin Table 1; Fig. 5, oxadiazole derivatives 3b (IC50 = 140.02µM) 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 showedsignificant 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

Ayoup et al. BMC Chemistry(2024) 18:130

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Fig. 5 A bar diagram representing the MAO-B and MAO-A inhibition of the synthesized compounds

Fig. 6 A bar diagram representing the anti-hemolytic effect of the synthesized compounds

times higher than that of methylene blue (IC50 = 143.6µM). Compound 4c (IC50 = 143.9 µM) exhibited equipotent MAO-A inhibitory potential to that of methylene
blue.

In vitro determination of the anti-hemolytic effectof synthesized compounds
The inhibition of human red blood cells (HRBCs) membrane lysis under hypnotic conditions was taken as ameasure of the mechanism of anti-hemolytic activity ofthe 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 isrepressed. Eight compounds namely, 1a, 1b, 2a-c, 4b, 5a,and 5b with IC50 values at the range of 1193.47–4338.69µM (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).

Structure-activity relationships (SAR)
The structure-activity relationship studies of the newoxadiazole-based derivatives revealed that the 3,5-disubstituted-1,2,4-oxadiazole pharmacophoric entity is highly
tolerated for AChE inhibition. It was clear that graftinga 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

Ayoup et al. BMC Chemistry(2024) 18:130

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-richphenyl 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 thanlipophilic quinoline counterparts (5a-c) (Fig. 7).
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 mostprominent antioxidant candidates. In hydrazide andhydrazone derivatives, grafting the lipophilic electrondeficient p-trifluoromethylphenyl at position 3 of theoxadiazole 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 byPage 8 of 15

the incorporation of the isoindoline motif (4a-c) than thequinoline 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 andN-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, theprotein crystal structure of AChE (PDB: 7E3H) [59] wasdownloaded 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 theexperimental pose with RMSD of 0.42 Å which is within
the cutoff limit (< 1 Å) (Fig. 8). The active site of AChE

Fig. 7 Structure-activity relationship (SAR) for anti-AD potential of 1,2,4-oxadiazole-based derivatives

Ayoup et al. BMC Chemistry(2024) 18:130

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Fig. 8 A) 2D diagram of donepezil interaction with human acetylcholinesterase; B) 3D of the co-crystallized donepezil inhibitor (magenta) together withthe re-docked donepezil (green) in the gorge of the active site (highlighted in yellow) of human acetylcholinesterase (RMSD = 0.42 Å)

Fig. 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)

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 theAChE active site pocket and the 2,3-dihydroinden-1-one
carbonyl moiety interacts with Phe295 by hydrogenbonding. The benzyl group showed arene interactions
with Trp86 amino acid of the enzyme. The synthesizedmolecules were docked using the same method and thebinding 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 compound2b inhabited a similar space as donepezil piperidine ringand formed Arene-H interaction with Tyr341 (Fig. 9). In
addition, the hydrazide carbonyl forms a hydrogen bondwith 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 withbenzyl 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 doneusing the online software SwissADME. All compounds
showed alignment with Lipinski’s rule of five [60]. Mostof the compounds showed a high probability of gastrointestinal absorption and all of them exhibited a 0.55 oral
bioavailability score (see Supplementary data) and thedocking score and interactions of all target compoundswith the active site of AChE enzyme were collected in(Table 2) with donepezil. Compound 2b showed thehighest in vitro inhibitory activity, high docking binding,and good physicochemical properties as shown in thebioavailability radar map (Fig. 10). All these data suggest

Ayoup et al. BMC Chemistry(2024) 18:130

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

1aDocking score withhuman AChE active
site (PDB: 7E3H)(kcal/mol)-7.6551b-7.8951c-7.8052a-8.8082b-8.8532c
3a-8.391-8.6743b-8.7233c-8.6914a-9.314

4b

9.122

4c

-9.0675a-8.6505b-8.6755c-8.598

Donepezil HCl -9.592

Interactions with human
AChE active site

HB (Asn87)
HB (Tyr124)
HB (Asp74)
HB (Trp86, via water bridge)
Arene-H (Tyr341)
HB (Asn87)
HB (Tyr124)
HB (Ser203, via water bridge)
Arene-arene (Trp286)
Arene-arene (Tyr124)
HB (Ser203, via water bridge)
Arene-H (Tyr341)
Arene-arene (Phe338)
Arene-arene (Trp86)
Arene-arene (Trp86)
HB (Tyr124)
Arene-arene (Trp286)
Arene-H (Trp86)
Arene-H (Tyr341)
Arene-H (Tyr337)
Arene-arene (Trp86)
Arene-H (Ser203, via water
bridge)HB (Phe295)
Arene-H (Trp286)
Arene-H (Ser203, via water
bridge)HB (Trp86, via water bridge)
Arene-arene (Trp86)
Arene-arene (Tyr341)
HB (Trp86, via water bridge)
HB (Thr83, via water bridge)
Arene-arene (Trp86)
Arene-arene (Tyr341)
Arene-H (Tyr337)
Arene-arene (Tyr341)
Arene-arene (Trp86)
Arene-arene (Trp286)
Arene-H (Tyr341)
Arene-H (Gly121)
Arene-arene (Trp86)
Arene-H (Tyr341)
Arene-H (Gly121)
Arene-arene (Trp86)
Arene-H (Ser203, via water
bridge)HB (Asp74)
Arene-arene (Trp86)
Arene-H (Ser203, via water
bridge)HB (Phe295)
Arene-H (Tyr341)
Arene-arene (Trp86)
Arene-arene (Trp286)

Page 10 of 15

that 2b could be considered as a promising candidate for
future development.

Conclusion
New derivatives based on 1,2,4-oxadiazole core were
designed, synthesized and their anti-AD potential wasassessed. 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, thenewly synthesized oxadiazole derivatives exhibited lessselectivity towards BuChE. Furthermore, compared toquercetin (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 thehighest ability to inhibit MAO-B were 3b (IC50 = 140.02µM) 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. Theirpotency 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 astrong 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 wereprominent. 1,2,4-Oxadiazole derivative 2b is a promising anti-AD drug candidate for further research anddevelopment.
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 and3a-c

To a stirred solution of the acid hydrazide 1a-c, (0.318mmol) 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-1,2,4oxadiazol-5-yl)phenoxy)acetohydrazide (2a).

Ayoup et al. BMC Chemistry(2024) 18:130

Page 11 of 15

Fig. 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

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, DMSOd6) 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);
13
C 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-B enz yl-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, DMSOd6) 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, DMSOd6) 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-2H-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, DMSOd6) 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-2H-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-2H-1,2,3-triazol-4-yl)
methylene)-2-(2-(3-(4-(trifluoromethyl)phenyl)-1,2,4oxadiazol-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,

Ayoup et al. BMC Chemistry(2024) 18:130

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 (0.318mmol) 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 tocool to room temperature. The solid formed was filtered
off to afford the desired product.
N-(1,3-Dioxoisoindolin-2-yl)-2-(2-(3-phenyl-1,2,4oxadiazol-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-(1,3dioxoisoindolin-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,

Page 12 of 15

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, 0.645mmol) 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 (0.3225mmol) 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, thecrude product was refluxed in ethanol (10 mL) for 1 h
and filtered while hot, the precipitate was washed withhot ethanol (10 mL), collected, and dried to get thedesired 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);1
H 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);1
H 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

Ayoup et al. BMC Chemistry(2024) 18:130

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 and3321 (NH), 1718 and 1683 (CO); 1H NMR (500 MHz,DMSO-d6) δH: 10.96 (s, 1H, OCN-H), 10.45 (s, 1H, OCNH), 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 areavailable 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 theProtonate 3D algorithm where the protonation statesof 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 ligandreceptor interactions.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s13065-024-01235-x.

Page 13 of 15

Supplementary Material 1
Acknowledgements
The authors acknowledge the Deanship of Scientific Research, Vice Presidency
for Graduate Studies and Scientific Research at King Faisal University, Saudi
Arabia, for financial support under the annual funding track [GrantA440]”.
Author contributions
MSA: design of the work, writing the chemistry part MG: synthesis of thetarget compounds and interpretation of spectral data HA: writing thechemistry part MMA: revise the manuscript AM: Biological evaluation DAG:
Biological evaluation MBH: revise and editing the manuscript AS: Design
the rational, Molecular docking and interpretation the biological result AEK:
Design the rational, writing and interpretation biological part.
Funding
This work was supported by “the Deanship of Scientific Research, Vice
Presidency for Graduate Studies and Scientific Research, King Faisal University,
Saudi Arabia [Project No. GrantA440]”.
Data availability
This published article and its additional file includes some data generated
or analyzed during this study. Other datasets used and analyzed during thecurrent study are available from the corresponding author on reasonable
request.

Declarations
Ethics approval and consent to participate
We obtained informed consent from all of the participants in the study,
they are three individual participants from our research teamwork. Also,
we obtained ethics approval from Faculty of science, Alexandria University.
Medical Research Ethics Committee (Approval number AU04 608 24 01 2024).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
We obtained informed consent from all of the participants in the study,
they are three individual participants from our research teamwork. Also,
we obtained ethics approval from Faculty of science, Alexandria University.
Medical Research Ethics Committee (Approval number AU04 608 24 01 2024).
Not applicable.
Author details
1
Department of Chemistry, College of Science, King Faisal University,
Al-Ahsa 31982, Saudi Arabia
2
Chemistry Department, Faculty of Science, Alexandria University, P.O. Box426, Alexandria 21321, Egypt
3
Medical Biotechnology Department, Genetic Engineering andBiotechnology Research Institute, City of Scientific Research andTechnological Applications (SRTA-City), Alexandria, Egypt
4
Bio-screening and Preclinical Trial Lab, Biochemistry Department, Facultyof Science, Alexandria University, Alexandria 21511, Egypt
5Center of Excellence for Drug Preclinical Studies (CE-DPS),
Pharmaceutical and Fermentation Industry Development Center, City of
Scientific Research & Technological Applications (SRTA-city), New Borg El
Arab, Alexandria, Egypt
6
Research Projects Unit, Pharos University in Alexandria, Alexandria, Egypt
7
Department of Chemistry, Faculty of Science, Umm Al-Qura University,
Makkah 21955, Saudi Arabia
8
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Cairo University, Kasr El-Aini Street, Cairo, P.O. Box 11562, Cairo, Egypt
9
University of Hertfordshire hosted by Global Academic Foundation, New
Administrative Capital, Cairo, Egypt
Received: 29 February 2024 / Accepted: 25 June 2024

Ayoup et al. BMC Chemistry(2024) 18:130

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