Comparative cholinesterase, α-glucosidase inhibitory, antioxidant, molecular docking, and kinetic studies on potent succinimide derivatives

Article abstract of DOI:10.2147/DDDT.S237420

Introduction: The current study was designed to synthesize derivatives of succinimide and compare their biological potency in anticholinesterase, alpha-glucosidase inhibition, and antioxidant assays. Methods: In this research, two succinimide derivatives

Full text of DOI:10.2147/DDDT.S237420

Drug Design, Development and Therapy
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O R I G I N A L R E S E A R C H
Comparative Cholinesterase, α-Glucosidase
Inhibitory, Antioxidant, Molecular Docking, and
Kinetic Studies on Potent Succinimide Derivatives
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
Ashfaq Ahmad,1,2
Introduction: The current study was designed to synthesize derivatives of succinimide and
Farhat Ullah,2 Abdul Sadiq,2
compare their biological potency in anticholinesterase, alpha-glucosidase inhibition, and
2
antioxidant assays.
Muhammad Ayaz,
Muhammad Saeed Jan,
2
Methods: In this research, two succinimide derivatives including (S)-1-(2,5-dioxo-1-phe-
nylpyrrolidin-3-yl) cyclohexanecarbaldehyde (Compound 1) and (R)-2-((S)-2,5-dioxo-1-phe-
nylpyrrolidin-3-yl)-2-phenylpropanal (Compound 2) were synthesized using Michael
addition. Both the compounds, ie, 1 and 2 were evaluated for in-vitro acetylcholinesterase
(AChE), butyrylctcholinesterase (BChE), antioxidant, and α-glucosidase inhibitory poten-
tials. Furthermore, molecular docking was performed using Molecular Operating
Environment (MOE) to explore the binding mode of both the compounds against different
enzymes. Lineweaver–Burk plots of enzyme inhibitions representing the reciprocal of initial
enzyme velocity versus the reciprocal of substrate concentration in the presence of synthe-
sized compounds and standard drugs were constructed using Michaelis–Menten kinetics.
Results: In AChE inhibitory assay, compounds 1 and 2 exhibited IC50 of 343.45 and 422.98
µM, respectively, against AChE enzyme. Similarly, both the compounds showed IC50 of 276.86
and 357.91 µM, respectively, against BChE enzyme. Compounds 1 and 2 displayed IC50 of
157.71 and 471.79 µM against α-glucosidase enzyme, respectively. In a similar pattern, com-
pound 1 exhibited to be more potent as compared to compound 2 in all the three antioxidant
assays. Compound 1 exhibited IC50 values of 297.98, 332.94, and 825.92 µM against DPPH,
ABTS, and H2O2 free radicals, respectively. Molecular docking showed a triple fold in the AChE
and BChE activity for compound 1 compared with compound 2. The compound 1 revealed good
interaction against both the AChE and BChE enzymes which revealed the high potency of this
compound compared to compound 2.
1
Muhammad Shahid,
Abdul Wadood,3
Fawad Mahmood,1
Umer Rashid,4 Riaz Ullah,5
Muhammad Umar Khayam
1
Sahibzada,
Ali S Alqahtani,5
6
Hafiz Majid Mahmood
1Department of Pharmacy, Sarhad University of
Science & Technology, Peshawar, KPK, Pakistan;
2Department of Pharmacy, Faculty of Biological
Sciences, University of Malakand, Chakdara,
18000, KP, Pakistan; 3Department of
Biochemistry, UCS, Shankar Abdul Wali Khan
University, Mardan 23200, Pakistan;
4Department of Chemistry, COMSATS
University Islamabad, Abbottabad Campus,
Abbottabad 22060, Pakistan; 5Department of
Pharmacognosy, Medicinal, Aromatic and
Poisonous Plants Research Center (MAPRC),
College of Pharmacy, King Saud University,
Riyadh, 11451, Saudi Arabia; 6Department of
Pharmacology, College of Pharmacy, King Saud
University, Riyadh 11451, Saudi Arabia
Conclusion: Both succinimide derivatives exhibited considerable inhibitory activities
against cholinesterases and α-glucosidase enzymes. Of these two, compound 1 revealed to
be more potent against all the in-vitro targets which was supported by molecular docking
with the lowest binding energies. Moreover, compound 1 also proved to have antiradical
properties.
Correspondence: Abdul Sadiq
Department of Pharmacy,Faculty of
Biological Sciences, University of
Malakand, Chakdara 18000, KP, Pakistan
Tel +92 301-2297-102
Keywords: succinimides, Alzheimer’s disease, cholinesterase, antioxidant, glucosidase,
molecular docking
Email sadiquom@yahoo.com
Introduction
Ashfaq Ahmad
Alzheimer’s disease (AD) is a neurodegenerative disorder of the aging people and
is the common cause of dementia. AD is clinically characterized by progressive
cognitive decline, behavioral turbulence, and imperfection in routine activities.1,2
Department of Pharmacy, Sarhad
University of Information Technology,
Peshawar, Pakistan
Email ashfaqpharma123@gmail.com
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Although the complex pathophysiological aspects of AD impact in stopping various illnesses, such as cancer and
are not quite clear, yet numerous therapeutic agents includ- cardiovascular diseases, and in delaying the growing older
ing cholinesterase (AChE/BChE) inhibitors, BACE1 inhi- populations.20
bitors, metal chelators, amyloid-β-peptide vaccination,
cholesterol-lowering, and anti-inflammatory agents have associated with excessive blood glucose levels within the
been tested as potential AD therapeutics.3–5
body. DM is a persistent ailment of metabolism due to either
Diabetes mellitus (DM) is another metabolic disorder
Acetylcholine is an important neurotransmitter implicated less secretion of insulin or the cells of the body not respond-
in the communication of impulses across the synapse.6–8 This ing properly to the insulin produced and resist the action of
neurotransmitter is cleared by two vital enzymes, acetylcholi- insulin.20,21 It is characterized by excessive blood sugar level
nesterase (AChE) and butyrylcholinesterase (BChE).9,10 In (hyperglycemia) in postprandial and is followed by keto-
Alzheimer’s disease, cholinergic neurons are more effected acidosis and protein-losing.23 α-Glucosidase is a vital biolo-
and there is deficiency of acetylcholine (ACh) at the synaptic gical target/enzyme which catalyzes the degradation of diet
cleft. Consequently, inhibiting enzymes involved in the degra- polysaccharides to monosaccharide. Naturally or syntheti-
dation of ACh at synapse is an important and successful tool to cally prepared glucosidase inhibitors are of therapeutic inter-
restore its level and activity.11,12 Till now, among the five est to put off postprandial hyperglycemia in type 2 diabetes.
clinically approved drugs for AD, four are cholinesterase Amongst those, saccharine derivatives, for example, miglitol
inhibitors.13 The commercially available inhibitors of AChE and acarbose have been approved anti-diabetic drugs.24
such as rivastigmine, donepezil, and galanthamine might also Commercially available α-glucosidase inhibitors, which
help to prevent, for a constrained time, some signs from turn- include voglibose, acarbose, and miglitol are currently used
ing into worse for a few people in the early and middle levels against DM. However, all of these drugs are accompanied by
of the disease.14,15 This signifies the search for more useful adverse effects like renal tumors, hepatic injury, abdominal
and safe cholinesterase inhibitors for the effective manage- pain, diarrhea, and flatulence. Therefore, the scientists are in
ment of AD. In ongoing research, our group synthesized constant search of safe and effective alternatives for the
numerous synthetic inhibitors of AChE and BChE for the management of DM.25
potential management of AD.
Succinimides or 2.5-dioxopyrrolidines, a well-known class
Oxidative stress mediated by free radicals is implicated in of drugs, possess a variety of biological potentials.26,27 The
the progression of various diseases including neurological basic structure can be derivatized by various aryl and/or alkyl
disorders.16,17 Free radicals are generated in the body during groups on nitrogen or carbon positions.28,29 Formerly, succi-
normal metabolic process and are neutralized by immune nimides had been synthesized through various methods.30 The
system antioxidant enzymes like catalase, superoxide dismu- primary nucleus of succinimides can be replaced by employ-
tase, glutathione, and hydroperoxidase. However, due to ing each C and N-position substitutions, leading to diverse
excessive generation of the free radicals or suppression of derivatives having various functionalities. We have previously
immune system leads to improper combating of free radicals, synthesized the keto-esters derivatives (ethyl 3-oxo-2-(2,5-
which leads to oxidative stress. Further, amyloid beta (Aβ) is dioxopyrrolidin-3-yl)butanoate) of succinimide with their
an abnormal protein generated from amyloid precursor pro- positive anticholinesterase and antioxidant potentials.31 The
tein (APP) through the action of beta amyloid cleaving C and N substituted succinimides showed antimicrobial,32
enzyme 1 (BACE1) causing the formation of amyloid antitumor,33 analgesic,34 anti-inflammatory,35 and antispasmo-
plaques.18 Once Aβ is generated, it accumulates around the dic activities.36 Apart from their biological significance, they
neurons, causing their deterioration and disturb electron also find an application in the liquid crystal displays (LCD)
transport chain which can cause mitochondrial dysfunction and the production of water-soluble reactive copolymers and
and inflammation.11 Aβ is considered to be mitochondrial polymers.28
poison causing excessive release of free radicals, which dis-
Basically, succinimide class of drugs is known as antic-
rupt mitochondrial function, electron transport chain, and onvulsant in the market. And, also previously we have reported
function of immune system antioxidant enzymes like cata- the ketoester derivatives of succinimides as anti-Alzheimer’s
lase and hydroperoxides. Consequently, using antioxidants agents.31 So, being a class of drugs for neuropharmacology
and anti-inflammatory agents can potentially sluggish down based on previously evaluated nucleus of pyrrolidine-2,5-
AD development and neurodegeneration.7,11,19 In this regard, dione, this study was a proposed rationale for the designed
supplementation of exogenous antioxidants has a vital compounds as anti-Alzheimer’s agents. Secondly, the
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pyrrolide-2,5-dione moiety has a close structural resemblance 7384 USA), and α-glucosidase from Saccharomyces cerevi-
with thiazolidinedione which is a known class of anti-diabetic siae (CAT No. G 0660) were purchased from Sigma-Aldrich
drugs as shown in Figure 1. This is also obvious from previous USA, while acarbose (CAT No. A 8980) and ascorbic acid
reported studies that asymmetric nitrogenous molecules are were acquired from Sigma-Aldrich France.1H and 13C-NMR
important building blocks of medicinal importance and may spectra were recorded in deuterated solvents on a Bruker
possesses diverse pharmacological potential.37–40
spectrometer at 400 and 100 MHz, respectively, using tetra-
Based on the importance of succinimide scaffold and methyl silane (TMS) as internal reference.
the dire need of the novel drugs, the current study has been
designed in an attempt to synthesize potential derivatives Chemistry
of succinimide and to evaluate them for various biological Both of these compounds were synthesized as per the pre-
activities including the anti-Alzheimer’s and anti-diabetic viously reported procedure.41 Generally, L-isoleucine (0.1
using in-vitro and in-silico simulation approaches.
mol%, 13.12 mg) and potassium hydroxide (0.1 mol%, 5.6
mg) were added into a small reaction vessel containing DCM
(1M) to make a non-covalent catalyst assembly. Afterwards,
excess amount (2.0 equivalent) of the respective aldehydes
(cyclohexanecarboxaldehyde and 2-phenyl-propionaldehyde)
were added into each reaction and stirred for 2 min at ambient
temperature to form enamine. Added one equivalent ratio of
phenyl-maleimide with continuously stirring at room tempera-
ture. The reaction progresses were checked with the help of
TLC (thin layer chromatography). Silica plates 60 (TLC) were
used to perceive the reactions and then observed under UV
(254 nm) to detect reaction spot. Solvent system was n-hexane
and ethyl acetate and in 60:40 ratio, respectively. Once the
reactions completed, it was diluted with distilled water (H2O)
and extracted three times with dichloromethane using separat-
ing funnel. The organic layer was collected and dried in a
rotary evaporator. Then purified with the help of column
chromatography (silica gel 60; 0.063-0.200 mm) using ethyl
acetate and n-hexane as eluents. The Rf values of the synthe-
sized compounds 1 and 2 were 0.46 and 0.42, respectively.
Synthetic schemes of the compounds are presented in Figure 2.
Materials and Methods
Chemicals and Drugs
For synthesis of succinimides, all chemicals, reagents and
solvents, which include N-phenyl-maleimide (CAT No.
A14616), cyclohexane carboxaldehyde (CAT No. 108464),
2-phenyl propionaldehyde (CAT No. 241369), potassium
hydroxide (CAT No. 757551), ethyl acetate (EtOAc), dichlor-
omethane (DCM), dimethyl sulfoxide (DMSO), methanol, n-
hexane, and analytical chromatographic plates (TLC Silica gel
60 F254 ) for reaction progresses observation were purchased
from Sigma-Aldrich USA. For inhibition assays of cholines-
terase, AChE electric eel (CAT No. C 1682) and BChE (lyo-
philized equine serum (CAT No C 1057) were purchased from
authorized dealers of Sigma-Aldrich USA, in Pakistan. While
substrates naming acetylthiocholine iodide (CAT No.A
22300) and butyrylthiocholine iodide (CAT No. B 3253-5G)
along with indicator substance, 5,5-dithio-bis-nitrobenzoic
acid (DTNB) CAT No. D 218200 and a standard drug
galanthamine hydrobromide Lycoris Sp. (CAT No.
Y0001190) were acquired from Sigma-Aldrich, UK.. For
antioxidant assays and α-glucosidase studies, DPPH (CAT
No. D 9132 Sigma-Aldrich USA), ABTS (CAT No.
10102946001 Sigma-Aldrich USA), Gallic acid (CAT No. G
(S)-1-(2,5-Dioxo-1-Phenylpyrrolidin-3-yl)
Cyclohexanecarbaldehyde (Compound 1)
1H NMR (400 MHz, CDCl3) (ppm): 1.33–1.55 (m, 7H),
1.76–1.79 (m, 1H), 1.84–1.88 (m, 2H), 2.58 (dd, J = 18.17,
5.91 Hz, 1H), 2.78 (dd, J = 18.16, 9.48 Hz, 1H), 3.13 (dd,
J = 9.46, 5.89 Hz, 1H), 7.18–7.22 (m, 2H), 7.29–7.32 (m,
1H), 7.36–7.41 (m, 2H), 9.45 (s, 1H). (Figure S1).
13C NMR (100 MHz, CDCl3) (ppm): 21.28, 21.47,
25.18, 28.15, 28.69, 31.63, 42.76, 52.25, 126.72, 128.74,
129.23, 132.02, 174.96, 177.19, 204.70. (Figure S2).
(R)-2-((S)-2,5-Dioxo-1-Phenylpyrrolidin-3-yl)-2-
Phenylpropanal (Compound 2)
1H NMR (400 MHz, CDCl3) (ppm): 1.79 (s, 3H), 2.53 (dd,
J = 18.91, 4.65 Hz, 1H), 2.99 (dd, J = 18.92, 9.45 Hz, 1H),
3.83 (dd, J = 9.45, 4.64 Hz, 1H), 7.05.7.10 (m, 2H), 7.26–
Figure 1 (A) General structure of aldehyde derivatives of succinimides, (B)
Generic structure of thiazolidinedione class of anti-diabetic drugs
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7.30 (m, 2H), 7.36–7.46 (m, 6H), 9.69 (s, 1H).13C NMR added reagent DTNB (5 μl). The resultant three reagents
(100 MHz, CDCl3) (ppm): 16.54, 21.05, 31.37, 33.54,
40.18, 42.51, 51.99, 123.67, 125.99, 127.96, 128.06,
128.60, 128.67, 128.71, 130.97, 133.44, 135.56, 173.64,
176.94, 200.57 (Figures S3 and 4).
mixture was incubated for fifteen min at 30°C using a
water bath. Afterwards, 5 μL of the substrate was added.
Absorbance was recorded via double beam UV spectro-
photometer (Model UV-1800, Japan) at 412 nm. For
negative control, cuvette contained all components except
test samples. Whereas in the case of positive control,
galantamine (10 µg/mL) was used as standard. UV absor-
bance values, for 4 min at 30°C, beside the reaction time
were recorded.43 All experiments were performed in tri-
plicate and inhibitory potential values were obtained as
Anti-Cholinesterase Studies
The synthesized compounds were tested against cholines-
terase inhibitory potentials following Ellman’s assay.42
The assays are based on acetylthiocholine iodide or butyr-
ylthiocholine iodide hydrolysis by particular enzymes
AChE and BChE upon the addition of 5-thio-2-nitrobenzo-
ate anion which give yellow colour complex. The resultant
V
Percent Enzyme activity ¼
ꢀ 100
yellow
color
compound
is
detected
with
Vmax
spectrophotometer.
where, V is the rate of reaction of the tested sample which
is A2-A1 and Vmax is the enzyme activity in the absence
of inhibitor or tested drug.
Preparation of Solutions
Our tested samples were dissolved in methanol, ran-
ging from 15.62 to 1000 µg/mL. The dilutions of
AChE (518 U/mg) and BChE (7–16 U/mg) were
freshly prepared in phosphate buffer with pH 8 until
the concentrations of 0.03 and 0.01 U/mL, respectively,
were achieved. Along with that, solutions of DTNB
(0.2273 mM), ATChI and BTChI (0.5 mM), each in
distilled water were prepared and were preserved in
air-tight Eppendorf tubes in the refrigerator at the tem-
perature of 8°C for 15 min as a final solution. A
calculated positive control drug (galantamine) was
solubilized in methanol having the same concentrations
as mentioned before.
Antioxidant Studies
DPPH Radicals Scavenging Assay
The synthesized compounds were screened for antiradical
potentials against DPPH inhibitory analysis following our
previously reported method.44,45 Different dilutions of
tested compounds were prepared (15.62–1000 µg/mL) in
methanol, 0.1 mL of each was mixed with 3000 μL of the
previously prepared DPPH solution. The tested samples
were incubated for 30 min at 25°C and the absorbance
values were noted at 517 nm using a spectrophotometer.
Ascorbic acid was used as a standard drug in this assay. In
this assay, each concentration was taken in triplicate and
the data obtained were presented as mean SEM. The %
scavenging activity was calculated using the following
standard formula:
Spectroscopic Analysis
Using UV spectroscopic analysis, previously prepared
solutions of an enzyme (5 μl) was taken in cleaned
cuvette, then our tested samples (205 μl), and finally
Figure 2 Synthetic schemes of the compounds 1 and 2.
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% Scavenging activity of sample
10 μL of the test sample were added to them in a test tube
and the sample mixture was kept back for 15 min at 37°C.
After 15 min of incubation, the substrate solution having a
volume of 20 μL was added and once again incubated for the
same time at the same temperature. After incubation, 0.2 M
sodium carbonate solution (80 μL) was added to terminate
the reaction. Finally, the optical densities of the samples were
measured via double beam spectrophotometer at 405 nm.
Acarbose served as a positive control. The calculation of
percent enzyme inhibition was carried out using the follow-
ing equation;
absorbance of control ꢁ absorbance of plant extract
¼
ꢀ 100
absorbance of control
ABTS Free Radicals Scavenging Assay
Further confirming antioxidant potentials of synthesized
succinimides, free radicals scavenging of 2, 2-azinobis [3
ethylbenzthiazoline]- 6-sulfonic acid (ABTS).46,47 The
mixture of ABTS 7 mM and potassium persulfate 2.45
mM was prepared. The solution was kept in a dark cabinet
for the production of free radicals. The absorbance of
ABTS solution was adjusted to 0.7 at 745 nm using
methanol (50%) as diluent. To execute the assay, ABTS
solution (3 ml) was mixed with samples (300 μl) and
incubated at 25°C for 15 minutes. The absorbance of the
incubated mixture was recorded at 745 nm. Ascorbic acid
Percent enzyme inhibition
Control absorption ꢁ Sample absorption
¼
ꢀ 100
Control absorption
was used as a positive control. The procedure was impli- Molecular Docking Studies
cated in triplicate and ABTS free radicals scavenging Molecular docking was performed using Dock Module
activity was obtained in percentage using the formula;
implemented in Molecular Operating Environment
(MOE) software49,50 to explore the binding mode of the
tested compounds against acetylcholinesterase (AChE),
butyrylcholinesterase (BChE), and alpha-glucosidase
enzymes. First, the 3D structures for both the compounds
were generated using MOE-builder module implemented
in MOE. Next, the compounds were protonated, and
energy minimized using the default parameters of the
MOE (gradient: 0.05, Force Field: MMFF94X). The struc-
tural coordinates for AChE and BChE were retrieved from
protein databank (PDB code; 1acl and 1p0p). Due to the
unavailability of the crystallographic structure of the α-
glucosidase enzyme, we have used the homology model
described by Carreiro et al for docking purpose. All the
structure was subjected to MOE for preparation.
% scavenging effect
control absorbance ꢁ sample absorbance
¼
ꢀ 100
control absorbance
Hydrogen Peroxide Free Radicals Scavenging Assay
The hydrogen peroxide scavenging activity of the samples
was conducted following the procedure of Ruch et al.20
Hydrogen peroxide solution (2 mM) was prepared in 50
mM phosphate buffer having pH of 7.4. Various samples
having volume of 0.1 mL were taken in test tubes and their
volumes were made 0.4 mL by addition of 50 mM phos-
phate buffer. Hydrogen peroxide solution (0.6 mL) was
added to it and vortexed. After 10 min, the absorbance of
each sample was measured at 230 nm against the blank.
The hydrogen peroxide scavenging activity was measured
using the following standard formula:
Further, the protonation was done using default para-
meters of structure preparation module of MOE. Next, all
the structure was subjected to energy minimization to get
minimal energy conformation. Finally, refined structures
were used for docking study using the default parameters
of MOE; Placement: Triangle Matcher, rescoring 1:
London dG, Refinement: Forcefield, Rescoring 2: GBVI/
WSA. Before running the docking protocol, we have
selected a total of ten conformations for the ligand. The
top-ranked conformations based on docking score were
selected for protein–ligand interaction (PLI) analysis.
Hydrogen peroxide scavenging activity
1 ꢁ absorbance of synthetic compound
¼
ꢀ 100
absorbance of controldrug
α-Glucosidase Inhibition Assay
α-Glucosidase inhibitory activity was carried out with chro-
mogenic assay.48 The solution of α-glucosidase was prepared
to have concentration of 0.5 unit/mL. The freshly prepared
solution (20 μL) was mixed thoroughly with phosphate buf-
fer (pH 6.9) having a volume of 120 μL. Likewise, p-nitro-
phenyl- α-D-glucopyranoside substrate solution (5 mM) was Statistical Analysis
prepared in the same buffer. Then, the test samples with All experiments were performed in triplicate and data was
concentration ranges of 31.25–1000 μL having volume presented as mean SEM. One-way ANOVA followed by
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multiple comparison DUNNET test was used for the sta- both compounds have considerable inhibitory activities
against AChE and BChE and could be possibly effective
against Alzheimer’s disease.
tistical comparison of test compounds with standard drugs.
P-value < 0.05 was considered as statistically significant.
Numerous researchers are trying to explore novel com-
pounds which could surpass the efficacy of galanthamine
(anti-Alzheimer’s disease), acarbose (anti-diabetic), and
many more.51,52 Various drugs are still under trials to get
approved by the FDA. The exploration should go onwards
to get efficacious, feasible, and economic drugs with eter-
nal and easy availability. So, the current research work is
an attempt to sort out some novel potential compounds
which could be a panacea soon.
Results and Discussion
Cholinesterase Inhibition Studies
In the current study, two potent cholinesterase inhibitors
were selected from the synthesized compounds
for detailed study. The anticholinesterase potentials of
both compounds (S)-1-(2,5-dioxo-1-phenylpyrrolidin-3-
yl) cyclohexanecarbaldehyde and (R)-2-((S)-2,5-dioxo-1-
phenylpyrrolidin-3-yl)-2-phenylpropanal
have
been
To cure a specific disease determines the target in the
etiology of that specific disease. As this is known from
decades that AD is effectively managed by those drugs
which stop the breakdown of neurotransmitter acetylcho-
line (ACh) and thus increasing the concentration in the
body. By effectively blocking the enzyme responsible for
the breakdown of acetylcholine, ie, acetylcholinesterase/
butyrylcholinesterase, the amount of acetylcholine could
be an increase in the synaptic cleft and hence the symp-
recorded to be significant in comparison with the standard
drug galanthamine. Among the two compounds, (S)-1-
(2,5-dioxo-1-phenylpyrrolidin-3-yl) cyclohexanecarbalde-
hyde has shown higher enzyme inhibition with IC50 values
of 343.45 and 276.86 μM against AChE and BChE,
respectively (Table 1). Similarly, (R)-2-((S)-2,5-dioxo-1-
phenylpyrrolidin-3-yl)-2-phenylpropanal showed IC50
values of 422.98 and 357.91 μM against AChE and
BChE, respectively. Briefly, it may be concluded that toms of AD are avoided.53
Table 1 Results of Cholinesterase Inhibitory Potentials of Synthesized Compounds
Sample Conc.
Conc.
(%) Inhibition
AChE
IC50
(%) Inhibition
BChE
IC50
(μg/mL)
(µM)
(µM)
(S)-1-(2,5-dioxo-1-phenylpyrrolidin-3-yl)
cyclohexanecarbaldehyde
1000
500
78.42 0.43**
72.76 0.71***
68.56 1.06***
57.03 0.35***
41.08 0.47***
23.91 0.88***
12.80 1.50***
343.45
422.98
76.56
81.49 0.60**
75.76 0.61***
69.65 0.91***
59.83 1.21***
44.58 0.63***
26.08 1.04***
15.45 0.90***
276.86
357.91
59.16
250
125
62.5
31.25
15.62
(R)-2-((S)-2,5-dioxo-1-phenylpyrrolidin-3-yl)-2-
phenylpropanal
1000
500
77.23 0.22***
70.45 0.90***
59.90 0.60***
48.00 0.30***
39.12 0.54***
27.20 0.47***
21.32 0.87***
79.05 1.03***
72.54 0.60***
62.12 0.54***
51.90 0.45***
42.01 0.97***
31.20 0.47***
23.40 0.82***
250
125
62.5
31.25
15.62
Galanthamine (Std.)
1000
500
91.93 1.01
84.65 0.98
78.98 0.72
71.16 0.86
66.83 1.21
57.93 0.67
47.08 0.47
93.90 0.96
86.90 0.48
80.74 0.68
73.23 0.40
69.03 0.23
59.33 0.49
49.74 0.68
250
125
62.5
31.25
15.62
Notes: The data is represented as mean SEM (n=3). Values were significantly different as compared to the positive control, **P < 0.01, ***P < 0.001.
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Table 2 DPPH Free Radicals Scavenging Activity Results
Table 4 Hydrogen per Oxide (H2O2) Free Radicals Scavenging
Activity Result
Sample Conc.
Conc.
(%) Inhibition
IC50
μg/mL
(μM)
Compound Name
Conc.
(%) Inhibition
IC50
μg/mL
μM
(S)-1-(2,5-dioxo-1-
1000
500
79.53 1.92***
72.83 1.89***
68.40 1.97***
58.50 1.61***
44.66 0.88***
34.05 1.40***
28.23 1.71***
297.89
406.72
96.53
phenylpyrrolidin-3-yl)
cyclohexanecarbaldehyde
(S)-1-(2,5-dioxo-1-
1000
500
79.58 1.12
45
250
72.90 0.96
phenylpyrrolidin-3-yl)
cyclohexanecarbaldehyde
125
250
68.77 1.24
62.5
31.25
15.62
125
58.76 0.71
62.5
31.25
15.62
44.90 1.55***
34.42 0.43***
28.03 0.35***
(R)-2-((S)-2,5-dioxo-1-
phenylpyrrolidin-3-yl)-2-
phenylpropanal
1000
500
77.04 1.61***
70.34 1.10***
61.31 1.97***
50.07 0.97***
36.36 0.91***
23.55 1.85***
13.70 1.60***
(R)-2-((S)-2,5-dioxo-1-
phenylpyrrolidin-3-yl)-2-
phenylpropanal
1000
500
71.77 1.24***
65.08 0.47***
55.76 0.71***
47.80 1.50***
41.90 0.96***
34.03 0.35***
28.80 1.50***
145
250
125
250
62.5
31.25
15.62
125
62.5
31.25
15.62
Ascorbic acid (Std.)
1000
500
89.00 0.57
83.96 1.91
77.00 1.73
69.66 1.36
61.40 1.85
55.23 1.21
48.70 0.90
Acarbose (Std)
1000
500
83.62 1.67
75.08 1.04
71.95 2.01
63.87 1.27
57.67 0.88
49.90 0.48
41.80 0.37
26
250
125
250
62.5
31.25
15.62
125
62.5
31.25
15.62
Notes: Values represent % radical scavenging (mean SEM) of three replicates.
Values significantly different as compared to positive control. ***P < 0.001.
Notes: The data is represented as mean SEM, (n=3). Values were significantly
different as compared to the positive control, ***P<0.001 (highly significant)
Table 5 Alpha-Glucosidase Inhibitory Potentials of Succinimide
Table 3 ABTS Free Radicals Scavenging Activity Results
Derivatives
Compound Name
Conc.
(%) Inhibition
IC50
Compound Name
Conc.
(%) Inhibition
IC50
μg/mL
(μM)
μg/mL
(μM)
(S)-1-(2,5-dioxo-1-
1000
500
79.87 1.27***
67.31 0.58***
61.22 1.28***
54.49 0.60***
40.51 0.54***
26.87 0.85***
16.60 1.63***
332.94
497.82
85.17
(S)-1-(2,5-dioxo-1-
1000
500
79.58 1.12ns
72.90 0.96ns
68.77 1.24ns
58.76 0.71*
44.90 1.55***
34.42 0.43***
28.03 0.35***
157.71
471.79
40.27
phenylpyrrolidin-3-yl)
cyclohexanecarbaldehyde
phenylpyrrolidin-3-yl)
cyclohexanecarbaldehyde
250
250
125
125
62.5
31.25
15.62
62.5
31.25
15.62
(R)-2-((S)-2,5-dioxo-1-
phenylpyrrolidin-3-yl)-2-
phenylpropanal
1000
500
75.76 0.61***
68.51 0.54***
59.87 0.85***
49.31 0.58***
38.10 0.90***
22.83 1.21***
12.33 1.67***
(R)-2-((S)-2,5-dioxo-1-
phenylpyrrolidin-3-yl)-2-
phenylpropanal
1000
500
71.77 1.24***
65.08 0.47***
55.76 0.71***
47.80 1.50***
41.90 0.96***
34.03 0.35***
28.80 1.50***
250
250
125
125
62.5
31.25
15.62
62.5
31.25
15.62
Ascorbic acid (Std.)
1000
500
87.73 0.78
81.70 1.60
75.03 0.23
67.00 0.00
58.00 1.15
52.67 0.89
46.00 0.58
Acarbose (Std)
1000
500
83.62 1.67
75.08 1.04
71.95 2.01
63.87 1.27
57.67 0.88
49.90 0.48
41.80 0.37
250
250
125
125
62.5
31.25
15.62
62.5
31.25
15.62
Notes: Values represent % radical scavenging (mean SEM) of three replicates.
Values significantly different as compare to positive control, ***P < 0.001.
Notes: The data is represented as mean SEM, (n=3). Values were significantly different as
compared to the positive control, *P<0.1 (less significant), **P<0.001 (highly significant).
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Figure 3 The PL interaction profiles for Compound 1 and Compound 2 against AChE, BChE, and α-glucosidase enzyme. (A–C) indicates the surface of the corresponding
enzyme. (A1–C1) represent the PL interaction profile for compound 1 against AChE, BChE, and α-glucosidase enzyme. (A2–C2) Represent the PL interaction profile for
compound 2 against AChE, BChE, and α-glucosidase enzyme. Compound 1 and Compound 2 were colored into Magenta, while residues into white. Hydrogen bonding is
shown in black color dotted lines, and the both-sided arrows indicate the pi-stacking interaction.
be depicted by the IC50 values (IC50 of compound 1 =
Antioxidant Studies
297.89 μM, IC50 of Ascorbic acid = 96.53 μM). The
DPPH assay shows that the range of concentrations from
15.62 to 1000 μg/mL is active against the free radicals.
The ABTS free radicals scavenging assay has been tabu-
lated as Table 3. Compound 1 has shown significant antiox-
idant potential by inhibiting 79.87 1.27***, 67.31 0.58***,
61.22 1.28***, 54.49 0.60***, 40.51 0.54***, 26.87
0.85***, 16.60 1.63*** percent ABTS free radicals at
Oxidative stress is implicated in the progression of various
diseases including AD and DM. The etiological factors of
the majority of diseases have been sorted out to be the
oxidative stress, which is developed due to increased con-
centration of free radicals inside our body including the
brain. The free radicals could be able to initiate neuronal
anomalies which lead to AD.54 The free radicals have also
been reported to be one of the etiological factors of dia-
betes mellitus.55 So in the current study, it has also been
evidenced that the synthesized compounds can effectively
Table 6 The Docking Score for Both the Compounds Against
scavenge the free radicals, ie, the DPPH, ABTS, and AChE, BChE, and α-Glucosidase Enzymes
H2O2. Therefore, in the current research project, the suc-
Enzyme
Compound
Docking Score (S)
cinimide derivatives have proved to be effective against
free radicals, AD, and diabetes mellitus.
AChE
Compound 1
Compound 2
−6.50632524
−4.43696213
The synthesized compounds showed considerable
DPPH radicals scavenging as shown in Table 2. The anti-
oxidant potential of compound 1 has been recorded to be
comparatively higher than 2. In comparison with ascorbic
acid, the results of compound 1 are encouraging which can
BChE
Compound 1
Compound 2
−6.67689438
−5.60227823
α-Glucosidase
Compound 1
Compound 2
−6.79298449
−5.32736778
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the concentrations of 1000 to 15.62 μg/mL, respectively. The recorded as 157.71 and 40.27 μM, respectively. The percent
IC50 calculated for compound 1 and standard sample (ascor-
bic acid) were 332.94 and 85.17 μM, respectively. In the
same way, the antioxidant activity of compound 2 was also
recorded to be noteworthy as shown in Table 3.
enzyme inhibition of compound 1 was calculated as 79.58
1.12, 72.90 0.96, 68.77 1.24, 58.76 0.71, 44.90 1.55***,
34.42 0.43***, and 28.03 0.35*** at the concentrations of
1000, 500, 250, 125, 62.5, 31.25, and 15.62 μg/mL, respec-
tively. Similarly, compound 2 also demonstrated notable
enzyme inhibition.
The results of hydrogen peroxide free radicals scavenging
have been summarized in Table 4. It is obvious from the table,
that compound 1 was observed to be more potent than com-
pound 2 in this assay. The observed IC50 values for compounds
1 and 2 were 45 and 145 µM, respectively. In comparison, the
standard drug ascorbic acid exhibited an IC50 of 26 µM.
Diabetes mellitus (DM) is indicated by the increased
blood glucose level in the body due to a metabolic disorder
of carbohydrates, fats and proteins. Two main reasons are
there for increased concentration of glucose, ie, decreased
secretion of insulin and cellular resistance to the action of
insulin.56 The postprandial concentration of glucose could be
effectively decreased by the acarbose, which is the α-gluco-
sidase inhibitor drug.57 The α-glucosidase is responsible for
the conversion of large units of carbohydrates into glucose
α-Glucosidase Inhibitory Assay
Table 5 represents the summarized results of the a-glucosi-
dase assay. The percent a-glucosidase inhibition by com-
pound 1 goes parallel with the results of standard drug, ie,
acarbose. The IC50 value of compound 1 and acarbose was units which is effectively absorbed from the intestine. As far
Figure 4 Represent the 2D interaction for both compounds. (A1–C1) represent the 2D interaction for compound 1 against AChE, BChE, and α-glucosidase enzyme. (A2–C2)
for compound 2 against AChE, BChE, and α-glucosidase enzyme.
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as the α-glucosidase inhibition is concerned, the succinimide compound 1 involved in an interaction with the side chain
and backbone donor of residue Gly118, Ser122. Also,
residue Trp84 shared his pi-electron with the benzene
ring via π-π interaction. Compound 2 only shared the π-
electron with the Trp84 via π-π interaction. In the case of
the docking results for BChE activity revealed that both
the compounds shared a typical interaction with Trp82.
Also, the compound 1 is involved in some additional
interactions with essential residues in the active site;
Gly116 and Trp430 (Figure 3B), while the compound 2,
unfortunately, lack additional interaction, which indicate
the decreased potency of compound 2 as compared with
high potent compound 1 against both the enzymes.
derivatives in our current study have shown considerable
inhibitions of this enzyme which goes parallel with the
activity of standard drug acarbose.
Molecular Docking Studies
Acetylcholinesterase and Butyrylcholinesterase
Molecular docking was performed to explore the binding
mode of both compounds against the target enzyme (AChE
and BChE). Molecular docking results showed good agree-
ment with experimental results. We have noticed a triple fold
in the AChE and BChE activity for compound 1 compared
with compound 2.
Similarly, molecular docking results for compound 1
revealed good interaction against both the AChE and
BChE enzymes (Figure 3A), which evident the high
potency of this compound compared with compound 2.
Alpha-Glucosidase Inhibition
The molecular docking results for both the compounds
against the α-glucosidase enzyme revealed that com-
pound 1 showed best fit-well binding mode in the active
In the case of AChE, 1-methylpyrrolidin-2-one moiety of site of the corresponding enzyme and adopted various
Figure 5 Lineweaver–Burk plots of acetylcholinesterase inhibition representing the reciprocal of initial enzyme velocity versus the reciprocal of substrate concentration in
the presence of compound 1 and galantamine.
Figure 6 Lineweaver–Burk plots of acetylcholinesterase inhibition representing the reciprocal of initial enzyme velocity versus the reciprocal of substrate concentration in
the presence of Compound 2 and galantamine.
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Ahmad et al
interactions; Arg439 and Arg212 and some additional compound 2, which showed less PL profile. Most
hydrophobic interaction, ie, Phe300 (Figure 3C). While importantly, the compound 1 shared a common moiety
compound 2 only showed interaction with Phe300, (1-methylpyrrolidin-2-one) with various interaction with
which evident the less potency of this compound com- different residues, hence separate the high potent com-
pared with compound 1 against the corresponding pound 1 from less potent compound 2.. The details for
enzyme. Overall, the molecular docking results deli- docking score for compound 1 and compound 2 are
neated that the result for compound 1 agrees with the enlisted in Table 6. The 2D interaction for both com-
experimental results as compared with the results for pounds embedded in Figure 4.
Figure 7 Lineweaver–Burk plots of butyrylcholinesterase inhibition representing the reciprocal of initial enzyme velocity versus the reciprocal of substrate concentration in
the presence of Compound 1 and the standard galantamine.
Figure 8 Lineweaver–Burk plots of butyrylcholinesterase inhibition representing the reciprocal of initial enzyme velocity versus the reciprocal of substrate concentration in
the presence of Compound 2 and the standard galantamine.
Figure 9 Lineweaver–Burk plots representing the reciprocal of initial α-glucosidase velocity versus the reciprocal of substrate concentration in the presence of different
concentrations of compound 1 and the standard acarbose.
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Figure 10 Lineweaver–Burk plots representing the reciprocal of initial α-glucosidase velocity versus the reciprocal of substrate concentration in the presence of different
concentrations of compound 2 and the standard acarbose.
thus showing potent inhibitory proclivity against α-
Michaelis–Menten Kinetics
glucosidase.
The synthesized compounds showed strong inhibitory poten-
tials against acetylcholinesterase, butyrylcholinesterase, and
glucosidase as revealed from the corresponding Vmax and Km
values which were determined using Michaelis–Menten
kinetics and confirmed from the Lineweaver–urk plots for
the respective enzymes (Figures 5–10). For acetylcholines-
terase inhibition, the Vmax and Km values were determined as
84.93µg/min and 69.70 µg/mL for Compound 1, and 78.65
µg/min and 63.17µg/mL for Compound 2, respectively. The
standard galantamine showed a robust inhibition of acetyl-
cholinesterase having a Vmax and Km values of 1.875 µg/min
and 2.566 µg/mL and 1.488 µg/min and 2.633 µg/mL,
respectively.
Conclusions
In conclusion, both succinimide derivatives, ie, (S)-1-
(2,5-dioxo-1-phenylpyrrolidin-3-yl) cyclohexanecarbalde-
hyde (1) and (R)-2-((S)-2,5-dioxo-1-phenylpyrrolidin-3-
yl)-2-phenylpropanal (2) exhibited considerable inhibitory
activities against cholinesterases and α-glucosidase
enzymes. Among both, compound 1 revealed more potent
activity against all enzymes which was supported by
molecular docking with the lowest binding energies
against all enzymes. Likewise, compounds showed con-
siderable anti-radical potentials and thus warranted
Figure 5 Lineweaver–Burk plots of acetylcholinester- further in-vivo studies for potential application in AD
ase inhibition representing the reciprocal of initial enzyme and diabetes.
velocity versus the reciprocal of substrate concentration in
the presence of compound 1 and galantamine.
Acknowledgments
Similarly, the Vmax and Km values for butyrylcholines-
The authors extend their appreciation to the Deanship of
terase inhibition also revealed a potent inhibitory potential
Scientific Research at King Saud University, Riyadh,
of Compound 1 (1.553 µg/min and 4.250 µg/mL) and
Saudi Arabia for funding this work through research
Compound 2 (2.867 µg/min and 7.438 µg/mL). A high-
group No. 1441-341. AS is also grateful to the Higher
grade inhibitory activity was observed with the standard
Education Commission of Pakistan for NRPU project
galantamine (2.566 and 2.641 µg/min and 2.633 and
No. 10562/KPK/NRPU/R&D/HEC/2017.
2.574 µg/mL, respectively).
The Lineweaver–Burk plots for the inhibition of α-
Author Contributions
glucosidase by compounds 1 and 2 and acarbose are
All authors contributed towards data analysis, drafting and
shown in Figures 7 and 8. The dissociation constant
critically revising the paper, gave final approval of the
(Km) and Vmax values of compound 1 for α-glucosidase
version to be published, and agreed to be accountable for
were 5.017 µg/mL and 2.360 µg/min, respectively.
all aspects of the work.
Similarly, for compound 2, the Km and Vmax were
observed as 7.565 µg/mL and 3.542 µg/min, respectively.
The effect was comparable to that of standard, acarbose
Disclosure
(2.328 and 3.176 µg/mL and 2.639 and 2.616 µg/min), The authors report no conflicts of interest in this work.
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