Succinamide derivatives ameliorate neuroinflammation and oxidative stress in scopolamine-induced neurodegeneration

Article abstract of DOI:10.3390/biom10030443

Oxidative stress-mediated neuroinflammatory events are the hallmark of neurodegenerative diseases. The current study aimed to synthesize a series of novel succinamide derivatives and to further investigate the neuroprotective potential of these compounds

Full text of DOI:10.3390/biom10030443

biomolecules
Article
Succinamide Derivatives Ameliorate
Neuroinflammation and Oxidative Stress in
Scopolamine-Induced Neurodegeneration
Sumbal Iqbal 1,2, Fawad Ali Shah 3,† , Komal Naeem 3 , Humaira Nadeem 1,*, Sadia Sarwar 4,
Zaman Ashraf 5, Muhammad Imran 1 , Tariq Khan 6 , Tayyaba Anwar 1 and Shupeng Li 2,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Riphah International
University, Islamabad 747424, Pakistan; sumbalqas@hotmail.com (S.I.);
m.imran.khanzada@gmail.com (M.I.); tayyaba.anwar@riphah.edu.pk (T.A.)
State Key Laboratory of Oncogenomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate
School, Peking University, Shenzhen 518000, China
2
3
4
5
6
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Riphah International University,
Islamabad 747424, Pakistan; fawad.shah@riphah.edu.pk (F.A.S.); komal.naeem@riphah.edu.pk (K.N.)
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Riphah International University,
Islamabad 747424, Pakistan; sadia.sarwar@riphah.edu.pk
Department of Chemistry, Allama Iqbal Open University, Islamabad 747424, Pakistan;
mzchem78@gmail.com
Department of Pharmacy, Capital University of Science and Technology, Islamabad 747424, Pakistan;
tariq.khan@cust.edu.pk
*
†
Correspondence: humaira.nadeem@riphah.edu.pk (H.N.); lips@pkusz.edu.an (S.L.)
Fawad Ali Shah is currently working as Post-Doctorate in Robarts Research Institute, Schulich School of
Medicine & Dentistry, Western University, ON 91761 Canada.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 January 2020; Accepted: 7 March 2020; Published: 13 March 2020
Abstract: Oxidative stress-mediated neuroinflammatory events are the hallmark of neurodegenerative
diseases. The current study aimed to synthesize a series of novel succinamide derivatives and to
further investigate the neuroprotective potential of these compounds against scopolamine-induced
neuronal injury by in silico, morphological, and biochemical approaches. The characterization of
all the succinamide derivatives was carried out spectroscopically via proton NMR (1H-NMR), FTIR
and elemental analysis. Further in vivo experiments showed that scopolamine induced neuronal
injury, characterized by downregulated glutathione (GSH), glutathione S-transferase (GST), catalase,
and upregulated lipid peroxidation (LPO). Moreover, scopolamine increased the expression of
inflammatory mediators such as cyclooxygenase2 (COX2), nuclear factor kappa B (NF-kB), tumor
necrosis factor (TNF-α), further associated with cognitive impairment. On the other hand, treatment
with succinamide derivatives ameliorated the biochemical and immunohistochemical alterations
induced by scopolamine, further supported by the results obtained from molecular docking and
binding affinities.
Keywords: neurodegeneration; succinamide derivatives; neuroprotective; scopolamine; cognitive
impairment; molecular docking
Biomolecules 2020, 10, 443; doi:10.3390/biom10030443
www.mdpi.com/journal/biomolecules

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1. Introduction
Consistent reports have reiterated the involvement of inflammatory cascade and oxidative stress
in the pathophysiology of neurodegenerative disorders including Alzheimer’s disease (AD), ischemic
stroke, and Parkinson’s disease [
health burden and a substantial increase is estimated by 2050 [
significant role in the progression of neuronal pathologies [ ]. Furthermore, astrocytes and microglial
cells are rapidly activated during neuronal insult, followed by brain translocation of granulocytes
and leukocytes from circulating blood [ ]. Moreover, mitochondrial dysfunction and oxidative
phosphorylation decoupling generate numerous reactive oxygen species (ROS), leading to widespread
oxidative stress [ ]. ROS damage lipids, proteins and DNA, and cause intracellular organelle
destruction via plasma and organelle membrane peroxidation [ ]. These could further stimulate the
1
]. Neurodegenerative disorders contribute significantly to the global
2
]. The inflammatory mediators have a
3–5
6,7
8
9
release of biologically active free fatty acids, such as arachidonic acid and DNA fragmentation. Along
with glutamate-induced excitotoxicity and inflammatory factors, the vicious cycles induced by ROS
eventually activate the injury pathways and lead to cell necrosis and apoptosis [10].
Scopolamine is a non-selective muscarinic receptor antagonist, commonly employed to
induce cognitive and memory impairment in experimental models of neurodegenerative disorders.
Scopolamine is associated with elevated acetylcholinesterase activity, altered brain ROS levels, and
increased expression of inflammatory mediators such as TNF-
α and COX2, along with the attenuated
level of an antioxidant such as GSH (glutathione) [11 15]. Previous studies demonstrated the
–
hyperactivity of glial cells coupled to neuroinflammatory cascade in the scopolamine-induced memory
impairment model [16].
Succinamide derivatives are a pharmacologically active class of nitrogen-containing heterocyclic
compounds. Among them, ethosuximide, methsuximide, and phensuximide have shown
anticonvulsant, neuroprotective, and antinociceptive properties and are useful for the treatment
of various neuropsychiatric disorders. The convenient addition of a new functional group into
the succinamide ring, which further modifies the spectroscopic and pharmacological properties of
resultant derivatives, is a characteristic feature of succinamide [17]. Several strategies can be adopted
to synthesize succinamide including multiple coupling agents. A remarkable milestone in this regard
is to employ succinic anhydride, an amide bond precursor, for succinamide synthesis. Succinamide
provided a marked improvement to both pharmacokinetics and pharmacodynamics as demonstrated
previously [18]. A number of succinamide containing agents were reported for anti-inflammatory and
anti-oxidant effects [19,20].
The current study was designed to synthesize a series of novel succinamide derivatives and to
investigate their neuroprotective action in the scopolamine-induced neurodegenerative model. The
results obtained will not only help us to understand the cascading mechanisms leading to cell death
but will also provide a clue for succinamide derivatives to be a potential therapeutic in the future.
2. Materials and Methods
2.1. Drugs and Chemicals
Materials required for initiation were obtained from Sigma Aldrich (St. Louis, MO, USA). Melting
points of all newly synthesized succinamide derivatives were recorded via the Digital Gallen Kamp
apparatus (Sanyo, Osaka, Japan). A Bruker AM-300 (Billerica, Massachusetts, UK) in DMSO-d6 was
employed to determine the proton NMR (1H-NMR) spectra at 300 MHz and TMS was used as an
internal standard. FTIR spectra were recorded by using the FTIR spectrophotometer (ATR eco ZnSe,
Vmax in cm−1). Elemental analysis was performed via LECO 183 CHNS analyzer (Changsha, Hunan,
China). The monitoring of all chemical reactions was carried out by thin-layer chromatography
(TLC). Antibodies (mouse anti-TNF-α, mouse anti-COX2), Avidin-Biotin Complex (ABC) Elite kit,
3,3-diaminobenzidine peroxidase were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Hydrogen peroxide, formalin, 1-chloro-2,4-dinitrobenzene, trichloroacetic acid, glutathione (GSH),

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thiobarbituric acid, and N-(1-naphthyl) ethylenediamine dihydrochloride were purchased from Sigma
Aldrich (St. Louis, MO, USA). Secondary antibody goat anti-mouse was procured from Abcam
(Cambridge, UK). All solvents, chemicals and reagents used were of 99% HPLC grade.
2.2. General Procedure for One-Pot Synthesis of Succinamide Derivatives(2a-2i)
Succinamide derivatives (2a–2i) were synthesized by dissolving equimolar quantities of succinic
anhydride in dry dichloromethane and cyclohexylamine with continuous stirring at room temperature
for 20 min while the progress of the reaction was monitored by TLC. After reaction completion,
the solid separated was filtered and recrystallized from methanol to get the target compound
4-(cyclohexylamino)-4-oxobutanoic acid (parent compound) [21]. 1 mmol of carboxylic acid was
added to 1 mmol of amine and 3 mmol of triethylamine (Et3N) in dichloromethane, then 1 mmol of
SOCl2 was added at room temperature. The mixture was stirred continuously for 30–60 min at room
temperature. The recovery of the reaction product was performed by evaporating the solvent under
reduced pressure. The resulting residue was extracted in dichloromethane and washed first with 1 N
hydrochloric acid (HCl) and then with 1 N sodium hydroxide (NaOH). The organic phase was dried by
using anhydrous sodium sulphate (Na2SO4), recrystallized with methanol and evaporated to dryness
to afford the corresponding carboxylic amide (Figure 1: general scheme; Figure 2: structures of newly
synthesized succinamide derivatives).
Figure 1. Scheme for the synthesis of new succinamide derivatives. DCM, dichloromethane; rt, room
temperature; SOCl2, thionyl chloride.

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Figure 2. Structures of all newly synthesized succinamide derivatives.
2.3. DPPH Free Radical Scavenging Assay
The anti-oxidant potential of succinamide derivatives was measured by using
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. Stock solution of test compounds
and ascorbic acid (positive control)having concentration of 1 mg/mLwas prepared and further sample
concentrations were prepared by serial dilution method (700, 300, 100, 10, 3, and 1 µg/mL). 1 mmol
DPPH solution was prepared in methanol. Then, 1 mLof the test sample was taken from each dilution
in separate test tubes and 3 mLof already prepared DPPH solution was added in each test tube to
make up the final volume of 4 mL. All test tubes were covered with aluminum foil and kept at room
temperature. In case of the presence of oxidation potential in test compounds, the inherently purple
color of DPPH will change from purple to yellow due to free radical scavenging and absorbance will
be recorded by using UV spectrophotometer at 517 nm. Percent inhibition or percent scavenging will
be calculated by using the formula given as under [22].
Percentage scavenging activity = Absorbance of control − Absorbance of sample × 100
Absorbance of control
2.4. In-Silico Studies
The in-silico studies of newly synthesized succinamide derivatives were carried out to gain a
perception of their potential binding affinities at the active binding sites of the target proteins via
Autodock Vina version 4.2.6 (San Diego, CA, USA). The PDB files of X-ray crystal structures of
selected receptors TNF-α (PDB ID: 2AZ5) and COX2 (PDB ID: 3ln1) were retrieved from the online
protein database http://www.rcsb.org/pdb. Active binding pockets of targets were retrieved from
https://bio.tools/dogsitescorer (DoG Site Scorer) [23]. Preparation of ligand-protein complexes was

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done for molecular docking. Biovia Discovery Studio Visualizer (DSV) was employed to clean the
target proteins by removing water and cocrystallized ligands and saved as PDB format. Mol files of
ligands were generated by drawing their structures in ChemSketch. All files of succinamide derivatives
and ligands were transformed into PDB format via Open Babel [23]. Furthermore, PDBQT files of
targets and ligands were prepared through AutoDock Tools version 1.5.6. Moreover, PyRx, digital
software for molecular docking was used for docking purpose [24]. Biovia DSV was employed to
interpret the best conformational poses and ligand-target molecular interactions. Validation of the
docking procedure was carried out by comparing the best poses of the cocrystallized ligands with the
best poses of re-docked configurations.
2.5. Animals and Experimental Groups
Balb-C mice of either gender (25–30g) were housed at controlled temperature (22–25 ◦C) and
kept on a 12 h: 12 h light–dark cycle, with free access to food and water. Mice were group-housed
throughout the testing period. All the animals were randomly divided into seven groups (n = 10 in
each group): (i) saline-treated control group, containing 2.5% DMSO (10 mL/kg, i.p); (ii) scopolamine
(SCO) treated disease group (1 mg/kg, i.p); (iii)–(vii) compound 2b, 2d, 2e, 2g and 2i (250 ug/kg, i.p) +
SCO. All compounds were initially solubilized in 2.5% dimethyl sulfoxide (DMSO) and volume make
up was done with saline. All experiments were carried out in accordance with the approved protocols
of the research and ethical committee (REC) of Riphah Institute of pharmaceutical science (RIPS),
Riphah International University, Islamabad, Pakistan (REC/RIPS/2018/17, approved on 18-07-2018).
After behavioral studies, animals were sacrificed employing standard protocol using CO2 euthanasia.
For one cohort, brain tissues were extracted and stored at
to collect the supernatant for further analysis (n = 5). For another cohort, brain tissues were fixed in 4%
−
80 ◦C followed by tissues homogenization
formalin, later embedded in paraffin, 4
(n = 5).
µm thin coronal sections were made by a rotary microtome
2.6. Behavioural Studies
2.6.1. Y-Maze Test
Spatial working memory was measured by the Y-maze test. Y-maze is a three-arm horizontal
maze (50 cm long and 10 cm wide) with walls 20 cm high and the arms are symmetrically inclined
at 120◦ to each other. Mice were divided into seven groups (n = 10) and received a single dose per
day for four consecutive days. Group (i) was used as control and received an i.p dose of mixture of
saline and 2.5% DMSO (10 mL/kg), Group (ii) was used as disease group which received an i.p dose of
SCO (1 mg/kg), in Groups (iii)–(vii) (disease + treatment), one hour before the test, mice were treated
with 250 µg/kg dose of test compound i.p and after 30 min SCO (1 mg/kg) was administered. Briefly,
animals were set free for spontaneous movement throughout the Y-maze by placing them at the middle
point of the maze. Animal entries into different arms were recorded by using a digital camera. Each of
the un-interrupted entry was considered to be spontaneous alteration behavior [25]. The percentage of
alteration was determined by the following equation:
% Alteration = ((Number of alterations)/(Total arm entries−2)) × 100
The extent of neurodegeneration was estimated by elevation in percent spontaneous
alteration behavior.
2.6.2. Morris Water Maze Test (MWM)
Mice were distributed into seven groups (n = 10) and received a single dose per day for four
consecutive days. Group (i) was used as control which received an i.p dose of mixture of saline and
2.5% DMSO (10 mL/kg), Group (ii) was used as disease group which received an i.p dose of SCO

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(1 mg/kg), in Groups (iii)–(vii) (disease + treatment), one hour before the test, mice were treated
with 250 g/kg dose of test compound i.p and 30 min after SCO (1 mg/kg) was administered. Mice
µ
were subjected to four trials per day for four consecutive days (a minimum of 15 min difference were
maintained between each trial) with the platform in place. Once the mice located the platform, it was
permitted to remain there for 10s. If the mouse was incapable to locate the platform within 60 s, it was
positioned to the platform and stayed for 10s and then removed from the pool. On the fifth day of
the MWM, each mouse was individually subjected to a probe trial session and mice were probed for
time spent in the platform quadrant. Mice were allowed to swim for 60s and escape latency time was
recorded by a video camera [26].
2.7. Hematoxylin Eosin (H&E) Staining
After de-paraffinizing tissue slides using absolute xylene (100%), it was followed by rehydrating
with absolute ethanol, gradient ethanolic concentrations (95% to 70%), and subsequently with distilled
water. Slides were then rinsed with PBS and were kept in hematoxylin for a total of 10 min. Then
slides were placed for 5 min under running tap water in a glass jar. Slides were then probed under the
microscope for nuclear staining, and if staining was not clear, hematoxylin timing was increased. Slides
were then treated with 1% HCl and 1% ammonia water for a short interval and immediately rinsed
with water again. Slides were then immersed in eosin solution for 5–10 min, followed by rinsing with
water and finally air-dried [27]. Slides were then dehydrated using graded ethanol (70%, 95%, and
100%), fixed in xylene and cover-slipped. Images were captured using an Olympus light microscope
(Olympus, Shinjuku, Tokyo, Japan) and analyzed using ImageJ software. Five images per group were
captured and analyzed while focusing on neuronal shape, size, infiltrated cells, and vacuolation.
2.8. Immunohistochemical Analysis
Immunohistochemical analysis was performed as described previously with minor
modifications [28]. After deparaffinization and rehydration procedures, slides were subjected to
an enzymatic method for antigen retrieval step using proteinase K. Slides were then washed using
0.1 M PBS, and endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min.
Again, slides were washed with 0.1 M PBS and incubated with 5% NGS (normal goat serum) containing
0.1% Triton X-100 for a minimum of 1 h in a humidified chamber. After blocking, slides were kept
for overnight incubation at 4 ◦C with primary antibodies as anti-COX-2, and anti-TNF-
α(Dilution 1:
100, Santa Cruz Biotechnology, Dallas, Texas, USA). The next morning, after washing twice with 0.1 M
PBS, they were incubated for 90 min with biotinylated secondary antibodies (dilution factor 1:50) in
a humidified chamber. Slides were again washed and incubated for 1 h with ABC reagents (Santa
Cruz Biotechnology, Dallas, Texas, USA) in a humidified chamber. Slides were then stained in DAB
solution, washed with distilled water, dehydrated in graded ethanol, fixed in xylene and cover-slipped
using mounting medium. Immunohistochemical TIF images of the slides were taken using a light
microscope (Olympus, Shinjuku, Tokyo, Japan) taking three images per slide. Hyper activated TNF-
α
and COX-2 were quantified using Image J software and expressed in terms of the relative integrated
density of the samples comparative to the control.
2.9. Assessment of Antioxidant Enzymes
2.9.1. GSH and GST Analysis
Previously documented assay protocols were employed for measuring glutathione (GSH) and
glutathione-S-transferase (GST) levels. Pre-homogenized samples were treated with PBS followed
by the addition of 2-nitrobenzoic acid solution. Absorbance values were measured at a wavelength
of 412 nm. Similarly, GST levels were also measured by using previously used assay protocol
with slight modification. Briefly, equimolar concentrations of 1-chloro-2,4-dinitrobenzene and
glutathione-S-transferase were added together and after thorough mixing was diluted with 0.1 M PBS.

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A wavelength of 340 nm was set to take absorbance values of sample homogenate following serial
dilution [28].
2.9.2. LPO Assay
Lipid peroxidation (LPO) assay was carried out by employing previously used protocol with
slight modification [29]. Lipid peroxidation was measured in terms of thiobarbituric acid reactive
substances (TBARS) in tissue sample homogenates of mice.
2.9.3. Catalase Assay
Catalase assay was done in accordance with the specifications as mentioned before with minor
modifications [30]. A quantity of 10
µL of the sample was added to each well followed by the addition
of 290 L of 3% H2O2. Then, the96-well plate was stored at room temperature in darkness for 10 min
µ
and absorbance was measured at a wavelength of 440 nm.
2.10. Enzyme-Linked Immunosorbent Assay (ELISA)
Enzyme-linked immunosorbent assay for COX2, p-NF-κB, and TNF-α was carried out in
accordance with the manufacturer’s instructions (Shanghai Yuchun Biotechnology, Shanghai, China).
Briefly, brain tissues stored at
80◦C were homogenized and the supernatant was collected following
centrifugation (at 13,500 g for 1 h). p-NF- BElisa kit (Cat # SU-B28069), and TNF- Elisa kit (Cat.
−
×
κ
α
# SU-B3098) were purchased from (Shanghai Yuchun Biotechnology, Shanghai, China). Briefly, the
protein samples were reacted with respective antibodies provided in the kit using a 96-well plate and
absorbance values were measured via microplate reader, BioTekEL x808. Results were expressed as a
concentration in picograms per milli Liter (pg/mL). All steps were performed in triplicate.
2.11. Statistical Analysis
Results were expressed as mean
±
SEM and analysis was done by applying one-way analysis
of variance (ANOVA) followed by post hoc Tuckey’s test through Graph Pad Prism 6 (San Diego,
CA, USA). Moreover, the behavioral data was analyzed by two-way grouped analysis. Histological
data were analyzed through ImageJ software. Symbol # denotes significance against the saline group
and *denotes significance against the scopolamine group. Data were considered to show statistical
significance at a value of p < 0.05.
3. Results
3.1. Spectral Analysis
3.1.1. 4-(Cyclohexylamine)-4-oxobutanoic acid (1)
◦
1
White solid: yield 89% m.p (observed):155–157 C; H-NMR (DMSO-d6, 300 MHz,
δ
ppm):
1.08–1.71 (m, 11H, cyclohexyl-H), 2.27(t, J = 6.9 Hz,
amide-NH), 11.99 (s, 1H, −COOH).
−
CH2), 2.50(t, J = 6.5 Hz,
−
CH2), 7.12 (s, 2H,
3.1.2. N-cyclohexyl-4-(morpholin-4-yl)-4-oxobutanamide (2a)
Light brown solid; yield 25%; m.p 180–182◦C; FTIR cm−1: 3292(N-H), 1660(C=O), 2851(C-H);
1H-NMR (DMSO-d6, 300 MHz,
ppm): 1.22–1.72(m, 11H, cyclohexyl-H), 2.49 (t, 2H, J = 7.2 Hz, -CH2),
δ
2.89(t, 2H, J = 7.4 Hz, -CH2), 3.33–3.52 (m, 8H, morpholin-H), 7.62 (s, 2H, amide-NH); elemental
analysis: C14H24N2O3; calculated: C 62.60%, N 10.43%, H 8.94%, found: C 63.17%, N 9.94%, H 9.05%.
3.1.3. N-(4-chlorophenyl)-N0-cyclohexylbutanediamide (2b)
◦
Pale white solid; yield 76%; m.p 210–213 C; FTIR cm−1: 3295(N-H), 1665(C=O), 2854(C-H);
1H-NMR (DMSO-d6, 300 MHz,
δ
ppm): 1.09–1.72(m, 11H, cyclo-H), 2.37 (t, 2H, J = 7.4 Hz, -CH2), 2.53(t,

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2H, J = 7.3 Hz, -CH2), 7.34–7.62 (m, 4H, Aryl-H), 8.31 (s, 1H, amide-NH), 10.09 (s, 1H, amide-NH);
elemental analysis: C16H21N2O2Cl; calculated: C 62.17%, N 9.06%, H 6.80%;found: C 62.50%, N 9.00%,
H 7.13%.
3.1.4. N-cyclohexyl-N0-(pyridin-4-yl)butanediamide) (2c)
Dark maroon solid; yield 25%; m.p 125–127 ◦C; FTIR cm−1:3296(N-H), 1685(C=O), 2927(C-H);
1H-NMR (DMSO-d6, 300 MHz,
δ ppm):1.36–1.74 (m, 11H, cyclo-H), 2.60(t, 2H, J = 7.3 Hz, -CH2), 2.91(t,
2H, J = 7.2 Hz, -CH2), 7.35–8.51(m, 4H, pyridine-H), 9.64(s, 1H, amide-NH), 11.48 (s, 1H, amide-NH);
elemental analysis: C15H21N3O2; calculated: C 65.37%, N 15.25%, H 7.62%; found: C 66.12%, N 15.59%,
H 7.92%.
3.1.5. N-cyclohexyl-N0-(4-methoxyphenyl)butanediamide) (2d)
Off-white solid; yield 55%; m.p 180–182◦C; FTIR cm−1: 3271(N-H), 1661(C=O), 2924(C-H);
1H-NMR (DMSO-d6, 300 MHz,
δ ppm): 1.19–1.14(m, 11H, cyclo-H), 2.59 (t, 2H, J = 7.2 Hz,-CH2), 2.70(t,
2H, J = 7.4 Hz, -CH2), 3.79(s, 3H, -OCH3), 5.77 (d, 1H, J = 6.6 Hz), 7.45–6.83 (m, 4H, Aryl-H), 8.31(s,
1H, amide-NH); elemental analysis: C17H24N2O3; calculated: C 67.02%, N 9.19%, H 7.88%; found: C
67.13%, N 8.98%, H 8.19%.
3.1.6. N-cyclohexyl-N0-(3-methoxyphenyl butanediamide) (2e)
Dark brown solid; yield 56%; m.p 130–133 ◦C; FTIR cm−1: 3296(N-H), 1664(C=O), 2920(C-H);
1H-NMR (DMSO-d6, 300 MHz,
δ ppm): 1.10–1.45 (m, 11H, cyclohexyl-H), 2.68 (t, 2H, J = 7.2 Hz, -CH2),
2.84 (t, 2H, J = 7.3 Hz, -CH2), 3.70 (s, 3H, -OCH3), 6.96–7.62 (m, 4H, Aryl-H), 7.11(s, 1H, amide-NH),
8.89(s, 1H, amide-NH); elemental analysis: C17H24N2O3; calculated: C 67.02%, N 9.19%, H 7.88%;
found: C 66.91%, N 9.20%, H 7.51%.
3.1.7. N-cyclohexyl-N0-(2-methoxyphenyl)butanediamide) (2f)
Light yellow solid; yield 38% 140–142 ◦C FTIR cm−1:3314(N-H), 1669(C=O), 2851(C-H); 1H-NMR
(DMSO-d6, 300 MHz, δ ppm): 1.29–1.87(m, 11H, cyclohexyl-H), 2.38(t, 2H, J = 7.4 Hz, -CH2), 2.94(t,
2H, J = 7.5 Hz, -CH2),3.68(s, 3H,-OCH3),), 7.17-7.33 (m, 4H, Aryl-H),9.86 (s, 1H, amide-NH), 11.42 (s,
1H, amide-NH); elemental analysis: C17H24N2O3; calculated: C 67.02%, N 9.19%, H 7.88%; found: C
67.23%, N 9.31%, H 7.05%.
3.1.8. N-benzyl-N0-cyclohexylbutanediamide (2g)
◦
1
Yellow solid; yield 66% 180–182 C; FTIR cm−1: 3285(N-H), 1662(C=O), 2921(C-H); H-NMR
(DMSO-d6, 300 MHz, ppm 1.41–1.77(m, 11H, cyclohexyl-H), 2.60(t, 2H, J = 7.4 Hz, -CH2), 2.79(t,
δ
2H, J = 7.4 Hz, -CH2), 4.50(s, 2H, -CH2), 7.23–7.86(m, 4H, Aryl-H), 7.19(s, 1H, amide-NH), 7.91(s, 1H,
amide-NH); elemental analysis: C17H24N2O2; calculated: C 70.7%, N 9.70%, H 8.32%; found: C 71.06%,
N 9.60%, H 7.95%.
3.1.9. N-cyclohexyl-N0-(4-hydroxyphenyl)butanediamide (2h)
Off-white solid; yield 30% 155–156◦C; FTIR cm−1: 3309(N-H), 1675(C=O), 2855(C-H); 1H-NMR
(DMSO-d6, 300 MHz, δ ppm): 1.40–1.79(m, 11H, cyclohexyl-H), 2.69(t, 2H, J = 7.3 Hz, -CH2), 2.86(t,
2H, J = 7.4 Hz, -CH2), 6.36(s, 1H, OH), 6.61–7.13(m, 4H, Aryl-H),7.84(s, 1H, amide-NH), 8.47(s, 1H,
amide-NH); elemental analysis: C16H22N2O3; calculated: C 66.12%, N 9.64%, H 7.57%; found: C
66.51%, N 9.35%, H 8.01%.
3.1.10. N,N0-di cyclohexyl butanediamide (2i)
1
White solid; yield 68% 140–142◦C; FTIR cm−1:3284(N-H), 1670 (C=O), 2853(C-H); H-NMR
(DMSO-d6, 300 MHz, δ ppm): 1.56–1.68(m, 11H, cyclohexyl-H), 1.70–1.89(m, 11H, cyclohexyl-H), 2.62(t,

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2H, J = 7.1 Hz, -CH2), 2.76(t, 2H, J = 7.3 Hz, -CH2), 7.14(s, 1H, amide-NH), 8.46 (s, 1H, amide-NH);
elemental analysis: C16H28N2O2; calculated: C 68.47%, N 9.98%, H 9.98%, found: C 69.05%, N 8.95%,
H 10.13%.
3.2. Effect of Succinamide Derivatives on DPPH Free Radical Scavenging Assay
As illustrated in Figure 3 compounds 2g
potential when compared to commercially available reference standard ascorbic acid. Although
compounds 2a 2c, and 2h also demonstrated antioxidant activity to some extent, it was not comparable
, 2i, 2d, 2b, and 2e exhibited significant antioxidant
,
with ascorbic acid or other succinamide derivatives. The order of antioxidant ability of succinamide
derivatives was found to be ascorbic acid > compound 2e > compound 2i > compound 2b > compound
2g > compound 2d > compound 2c > compound 2f > compound 2a > compound 2h (Figure 3).
Five newly synthesized succinamide derivatives (2b, 2d, 2e, 2g, and 2i) relatively exhibited a higher
antioxidant potential and were selected for further in vivo studies.
Figure 3. Illustrates percent free radical scavenging of novel succinamide derivatives (2a–2i) and
ascorbic acid against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. Values are expressed as mean
± SEM.
3.3. Evaluation of In-silico studies
Molecular docking of five newly synthesized succinamide derivatives (2a
–2i) and cocrystallized
ligands were carried out against active binding sites of TNF-α and COX2. The results obtained are
illustrated in Table 1.
Table 1. Binding energy values of docking. TNF, tumor necrosis factor; COX, cyclooxygenase.
Binding Energies (kcal/mol)
Compounds
COX2
TNFα
2b
2d
2e
2g
2i
−6.8
−7.6
−7.8
−7.9
−6.4
−5.5
−6.1
−6.0
−5.7
−5.9
Cocrystallized ligands were re-docked against the same target protein in order to validate the
procedure being employed for molecular docking. A comparison of the best binding poses generated

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before and after re-docking of cocrystallized molecules against TNF-
α
and COX2 are illustrated in
Figure 4A (A,B). The 2D and 3D images of the interaction of succinamide derivative 2b with COX2 are
illustrated in Figure 4B (A,B). Succinamide derivative 2b possesses an amide bond which established a
hydrogen bond with Gly B:225 residue with a bond distance of 2.29 Å, while chloro benzyl group made
hydrophobic contacts with Leu A:145. Figure 4B (C,D) illustrates the binding pose of compound 2d
with COX2. The carbonyl group was stabilized by hydrogen bond interactions with ARG A:376 with a
bond distance of 2.85 Å and π–alkyl and π–π T-shaped contacts were observed with o–anisidine ring
involving Val A:538 and Phe A:142 residues respectively. Another π–alkyl interaction was observed
between the cyclohexyl group and Phe A:142 residue. The binding modes and molecular interactions
with the lowest energy of succinamide derivative 2e with COX2 are represented in Figure 4B (E,F).
The N-H and carbonyl groups in amide were stabilized by forming a hydrogen bond with Asn
A:375 and Arg A:376 with a bond distance of 2.60 Å and 3.68 Å respectively. The cyclohexylamine
scaffold exhibited a
π–alkyl bond with PheA:142 and m-anisidine moiety interacted with Phe A:142
by T-shaped interaction. Ligand–receptor interaction of succinamide derivative 2g with COX2 is
π–π
demonstrated in Figure 4B (G,H). In this case, 3 hydrogen bonds were depicted with NH of the amide
bond involving binding residues of Gln B:374, Gly B:225 and ASN B:375 with a bond distance of 2.35,
2.33, and 2.99 Å respectively. Another hydrogen bond of Arg B:376 was established to stabilize the
carbonyl group of amide (3.65 Å). Binding mode and interaction of succinamide derivative 2i with
COX2 are shown in Figure 4B (I,J). The binding interaction of succinamide derivative 2i against COX2
demonstrated four H bonds, three between the Gln A:47, Ala A:134 and Trp A:28, and carbonyl group
(2.37, 3.06, and 2.90 Å) and the fourth between the NH group and Ala A:134 (2.62 Å). The 2D and
3D images of the interaction of succinamide derivative 2b with TNF-α are shown in Figure 4C (A,B).
Succinamide derivative 2b possesses an amide bond which formed a hydrogen bond with Gly B:225
(2.29 Å), while chloro benzylamine group made hydrophobic contacts with Leu A:145. Figure 4C (C,D)
illustrates the binding pose of compound 2d with TNF-
stabilized by very strong hydrogen bond interactions with Leu A:26 and Asn A:46 (1.77 and 2.4 Å).
The binding interaction of succinamide derivative 2e against TNF- is illustrated in Figure 4C (E,F).
α. Two carbonyl groups of the amide were
α
N–H and Carbonyl groups in amide were stabilized by establishing hydrogen bond with Asn A:46,
Leu A:26, and Glu A:135 (5.08, 1.76, and 2.89 Å) respectively. The m-anisidine scaffold exhibited a
hydrogen bond with Asn A:46 (2.86 Å). Ligand-target binding of succinamide derivative 2g against
TNF-
amide bond involving binding residues of Glu A:135 with a bond distance of 2.61 Å. The molecular
interaction of succinamide derivative 2i with TNF is represented in Figure 4C (I,J). The binding mode
of 2i with TNF- demonstrated four H bonds, three between the carbonyl group and Gln A:47, Ala
α is represented in Figure 4C (G,H). In this case, 1 hydrogen bond was established with NH of the
α
α
A:134, and Trp A:28 (2.37, 3.06, and 2.90 Å)and the fourth between the NH group and Ala A:134
(2.62 Å). The cyclohexylamine moiety was further stabilized by hydrophobic interactions.

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Figure 4. Best binding pose for 6,7-dimethyl-3-[(methyl{2-[methyl({1-[3(trifluoromethyl)phenyl]-1h-indol
-3-yl} methyl)amino]ethyl}amino)methyl]-4h-chromen-4-one and celecoxib in the binding pockets of and
tumor necrotic factor (TNF-
ligand, cyan = re-docked ligand) (
StudioVisualizer illustrating both 2D and 3Dposes. Interactions between 2b and COX2 (
and COX2 ( ), 2e and COX2 ( ), 2g and COX2 ( ), and 2i and COX2 ( ). ( )Post docking
analysis visualized by Discovery Studio Visualizer in both 2D and 3D styles. Interactions between 2b
and TNF- ), 2d and TNF- ), 2e and TNF- ), 2g and TNF- ), and 2i and TNF
(I,J) respectively.
α
) and cyclooxygenase-2 (COX-2) respectively (dark-blue = co-crystallized
).( ) Post dock analysis performed through Biovia Discovery
), 2d
A,B B
A,B
C
,D
E,
F
G,
H
I
,J
C
α
(
A
,B
α
(C
,D
α
(E
,F
α
(
G,H
α
3.4. Effect of Succinamide Derivatives on Alteration Behaviour
The Y-maze task was performed to analyze the spatial working memory using spontaneous
alteration behavior (%). The entire series of selected synthesized compounds showed increased
spontaneous alteration behavior (%) in the Y-maze test. Alteration behavior (%) of SCO (1 mg/Kg)
treated group showed significant difference against saline control in all trials carried out on 4 consecutive
days (### p < 0.001 vs. saline group). Alteration behavior (%) of SCO+2g (250
µg/Kg) group were not

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significantly reduced on day 1 and 2 but exhibited a significant decrease of * p < 0.05 vs. SCO group on
day 3 and ** p < 0.01 vs. SCO group on day 4, respectively (Figure 5A). Alteration behavior (%) of
SCO+2i (250
group but revealed a significant reduction of * p < 0.05 vs. SCO group on day 3 and * p < 0.05 vs.
SCO group on day 4, respectively (Figure 5A). Alteration behavior (%) of SCO+2b (250 g/Kg) group
µg/Kg) group were insignificant on day 1 and 2 in comparison to scopolamine treated
µ
did not show marked difference from disease group on day 1 and 2, but demonstrated a significant
difference of ** p < 0.01 vs. SCO group on day 3 and ** p < 0.01 vs. SCO group on day 4, respectively
(Figure 5A). Alteration behavior (%) of the SCO+2e (250
on day 1 and 2 but showed marked decrease of ** p < 0.01 vs. SCO group on day 3 and *** p < 0.001 vs.
SCO group on day 4, respectively (Figure 5A). Alteration behavior (%) of the SCO+2d (250 g/Kg)
µg/Kg) group were not significantly decreased
µ
group were again insignificant on day 1 and 2 but exhibited decreased alteration behavior (%) of
* p < 0.05 vs. SCO group on day 3 and * p < 0.05 vs. SCO group on day 4, respectively (Figure 5A).
Hence, significantly decreased % alteration behavior in succinamide derivatives treated mice indicates
a reversal of scopolamine-induced learning and behavioral deficit.
Figure 5.
a significant difference against the saline group; *, **, *** show significant difference against the
scopolamine (SCO) group. p < 0.05 is considered to be significant. ( ) Average escape latency time.
Mean SEM for the mice (n = 10). # denotes a significant difference against the saline group; *, **, ***
(A
) % alteration behavior. Data were analyzed by mean
±SEM with (n = 10). # denotes
B
±
show significant difference against the scopolamine (SCO) group. p < 0.05 is considered to be significant.
3.5. Effect of Succinamide Derivatives on Escape Latency Time
In the MWM test, escape latency time for the newly synthesized compounds was measured in
four trials per day for four consecutive days. Latency time of SCO (1 mg/Kg) treated group was
insignificant on day 1 as compared to the saline group but showed noticeably increased latency time
##
###
###
with a significance of p < 0.01 vs. saline group,
vs. saline group on day 2, 3, and 4 respectively (Figure 5B). SCO+2g (250
significant decrease of latency time on day 1 and 2, however, a markedly reduced escape latency time
p < 0.001 vs. saline group and
p < 0.001
µ
g/Kg) group exhibited no

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was observed on day 3 (* p < 0.05 vs. SCO group) and 4 (** p < 0.01 vs. SCO group), respectively
(Figure 5B). The latency time of the SCO+2i (250 g/Kg) group was found to be significantly decreased
on day 3 (* p < 0.05 vs. SCO group) and day 4 (* p < 0.05 vs. SCO group) respectively (Figure 5B). The
SCO+2d (250 g/Kg) group demonstrated no significant reduction of latency time on day 1 and 2, but
decreased escape latency was found on day 3 (* p < 0.05 vs. SCO group) and day 4 (* p < 0.05 vs. SCO
group) respectively (Figure 5B). The latency time of the SCO+2b (250 g/Kg) group was decreased on
day 3(** p < 0.01 vs. SCO group) and day 4 (** p < 0.01 vs. SCO group) respectively. A significant
reduction of latency time was seen in the SCO+2e (250 g/Kg) group on day 3 (** p < 0.01 vs. SCO
µ
µ
µ
µ
group) and day 4 (*** p < 0.001 vs. SCO group) respectively (Figure 5B). Therefore, we may perceive
that this significant decrease in escape latency time in succinamide derivatives treated groups indicate
a reversal of scopolamine-induced cognitive deficit.
3.6. Effect of Succinamide Derivatives on Scopolamine-Induced Neurodegeneration
Neuroprotective potential of succinamide derivatives was further elucidated by hematoxylin
and eosin staining. The scopolamine treated group demonstrated vigorous histological changes inthe
cortex and hippocampus of the brain of treated mice;as compared to the saline control group (Figure 5,
p < 0.01). As illustrated, scopolamine administration elicited abnormal histological features including
altered scalloped morphology of neurons associated with pyknosis, cytoplasmic eosinophilia, and
nuclear basophilia. Succinamide derivatives administration demonstrated marked mitigation of
scopolamine-induced neurodegeneration. Hence, a greater degree of cellular intactness and integrity
was observed in succinamide derivatives treated groups (p < 0.05, Figure 6).
Figure 6. H&E staining showing the extent of surviving neurons in the cortex and hippocampus
(dentate gyrus, dg). Bar 50
cytoplasmic swelling, vacuoles, scalloped neurons with intense cytoplasmic eosinophilia, and nuclear
basophilia. These changes resulted from neuronal necrosis. Some cells had a shrunken appearance,
µ
m, magnification 40 , n = 5 group. Surviving neurons were marked by
×
along with pyknotic nuclei (A). # shows significant difference relative to saline; * shows significant
difference relative to scopolamine. Data presented as means ± SEM. ** p < 0.01, * p < 0.05 (B).
3.7. Succinamide Derivatives Mediated Downregulation of Neuroinflammation
Marked elevation in the expression of TNF-α reactive cells has been observed in the scopolamine
treated hippocampus and cortex comparative to saline control (Figure 7B, p < 0.001), followed by

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further validation through ELISA (Figure 7A, p < 0.05). Significant attenuation of these detrimental
effects was carried out by the administration of succinamide derivatives (Figure 7, p < 0.05).
Figure 7.
gyrus, dg). Bar 50
of TNF- in the scopolamine group. ***, ** and * show p < 0.001, p < 0.01 and p < 0.05, respectively, and
illustrate significant variation relative to scopolamine, while ### p < 0.001 shows significant variation
relative to saline control group. Results are expressed as mean SEM. ( ) The protein expression of
TNF- quantified through ELISA. The data is presented as mean
SEM. # p < 0.05 relative to saline
(
A
,B) Immunohistochemistry results for TNF-α in the cortex and hippocampus (dentate
µ
m, magnification 40 (n = 5). Histograms show a comparatively higher expression
×
α
±
C
α
±
control group, while * p < 0.05 and ** p < 0.01 relative to scopolamine (n = 5).
3.8. Succinamide Derivatives Attenuated the Scopolamine-Induced Inflammatory Mediators
The interaction of inflammatory mediators with its designated targets results in sequential
induction of downstream signaling pathways including JNK (c-Jun N-terminal kinase), SEK1
(extracellular signal-regulated kinase 1/stress-activated protein) and ASK1 (apoptosis signal-regulating
kinase 1). The overall outcome is the proteosome detachment of IkB and NF-kB translocation into the
nucleus to activate the inflammation triggering transcription machinery such as COX2 [5]. ELISA results
confirmed the overexpression of COX2 and p-NFkB in the scopolamine treated brain (Figure 8A,B,
p < 0.01). Succinamide derivatives demonstrated a noticeable decrease in COX2 overexpression
(p < 0.05). Further validation was performed through immunohistochemical analysis and a similar
pattern of COX2 expression was found in the scopolamine treated brain whereas succinamide
derivatives significantly downregulated the COX2 levels (Figure 8C, p < 0.05).

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Figure 8.
(A
,
B) Immunohistochemistry results for COX2 in the cortex and hippocampus (dentate gyrus,
dg). Bar 50
µ
m, magnification 40 (n = 5). Histograms show a comparatively higher expression of
×
COX2 in the scopolamine group. ***, ** and * show p < 0.001, p < 0.01, and p < 0.05, respectively, and
illustrate significant variation relative to scopolamine, while ### p < 0.001 shows significant variation
relative to saline control group. Results are expressed as mean
COX2 quantified through ELISA. The data is presented as mean SEM. * p < 0.05 relative to scopolamine,
while p < 0.05 and p < 0.01 relative to saline control group (n = 5). ( )The protein expression
of p-NFkB quantified through ELISA. The data is presented as mean SEM. * p < 0.05 relative to
±
SEM. (C) The protein expression of
±
#
##
D
±
scopolamine, while # p < 0.05 and ## p < 0.01 relative to saline control group (n = 5).
3.9. Effect of Succinamide Derivatives on Antioxidant Enzymes
Table 2 illustrates the data obtained for scopolamine-induced antioxidant enzymes variation and
its attenuation carried out by succinamide derivatives administration. Scopolamine treatment caused
the accumulation of ROS and reduced the levels of antioxidant enzymes including catalase, GSH,
and GST in the brain (p < 0.001). Administration of succinamide derivatives produced significant
mitigation of downregulated antioxidant enzymes as illustrated in Table 2.
Table 2. Effect of succinamide derivatives on antioxidant enzymes and lipid peroxidation.
GSH
Catalase
(µmol
H2O2/min/mg of
Protein)
GST
(µmol/mg of
Protein)
LPO
(nmol/TBARS/mg
of Protein)
(µmol CDNB
Conjugate/min/mg
of Protein)
Groups
Saline
Scopolamine
88.25 ± 0.15
27.8 ± 2.89 ###
45.1 ± 3.8 *
67.90 ± 0.56
26.78 ± 2.78 ###
33.90 ± 1.23 *
32.78 ± 0.90 *
33.33 ± 0.67 *
35.33 ± 0.67 **
37.23 ± 0.78 ***
165.6 ± 1.650
88.80 ± 3.890 ###
125.1±3.230 **
120.5 ± 8.890 *
127.4 ± 4.900 **
130.9 ± 6.890 **
135.9 ± 8.890 ***
85.15 ± 0.91
123.70 ± 3.71###
87.45±8.90 **
96.89 ± 4.56 *
92.78 ± 2.78 *
85.12 ± 4.89 **
78.89 ± 6.89 ***
2g
2i
2d
2b
2e
43.78 ± 3.7 *
44.10 ± 3.78 *
50.13 ± 3.89 **
55.23 ± 2.89 ***
Symbols *** or ### show significant difference at p < 0.001, while * and ** show significant difference at p < 0.05 and
p < 0.01, respectively. The symbol * shows a significant difference relative to scopolamine and # shows significant
difference relative to saline. Data are shown as mean
GSH, glutathione; TBARS, thiobarbituric acid reactive substances; LPO, lipid peroxidation; CAT, catalase.
±
SEM (n=5). Abbreviations: GST, glutathione S-transferase;

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3.10. Effect of Succinamide Derivatives on Scopolamine-Induced Lipid Peroxidation (LPO)
Multiple studies reported malondialdehyde (MDA) as a critical marker for the estimation of
oxidative burden. Thiobarbituric acid reactive substances (TBARS) is frequently employed assay
for assessment of malondialdehyde (MDA), an important final product of LPO. TBARS assay was
conducted accordingly and data obtained are shown in Table 2. Succinamide derivatives (2g
, 2i, 2d,
2b, and 2e) significantly down regulated LPO level.
4. Discussion
Previous studies revealed that several substituted succinamic acids showed anti-inflammatory
activity and antitumor properties [17]. In the present study, a series of novel succinamide derivatives
were designed and synthesized. Molecular docking results against COX2 and TNF-α were obtained
to rank and reveal their binding and atom interacting details. In vitro, free radical scavenging assay
showed that all these compounds exhibited antioxidant ability in a dose-dependent way. Further in vivo
results showed that new derivatives markedly reduced scopolamine-induced neuroinflammation,
oxidative stress, and neuronal degeneration. Moreover, they have prominently ameliorated the
cognitive deficits induced by scopolamine.
The last two decades witnessed the identification of multiple oxidative species and inflammatory
mediators involved in the pathophysiology of neurodegenerative disorders [31], where cognition
related hippocampus and cortex are adversely affected [32,33]. Accordingly, the administration of
anti-inflammatory substances including COX2 inhibitors attenuates the inflammatory cascade and
improves behavioral deficits [34]. Similarly, previous studies demonstrated the anti-amnesic activity of
meloxicam and selegiline through the augmentation of endogenous antioxidant enzymes [35]. The
succinamide moiety constitutes a vital part of several drugs and drug candidates [36]. A number of
succinamide derivatives have been demonstrated as potential antioxidants, anti-inflammatory and
neuroprotective agents [19
derivatives was carried out via in-vitro DPPH free radical scavenging assay. Five newly synthesized
succinamide derivatives (2b 2d 2e 2g, and 2i) relatively exhibited a higher antioxidant potential in
,37,38]. Preliminary screening for the antioxidant potential of succinamide
,
,
,
preliminary screening and were selected for further in vivo studies. The antioxidant capacities of these
derivatives could be attributed to their possible proton donor ability to neutralize the free radicals.
Neuroprotection by succinamide derivatives as demonstrated by the reduced neuronal loss was
possibly mediated through their antioxidant and anti-inflammatory effects. Succinamide moiety relies
on a basic scaffold “pyrrolidine-2,5-dione”, comprising of 5 membered rings containing a nitrogen
atom with two carbonyl groups. Interestingly, frequently used natural/synthetic anti-Alzheimer’s
drugs also possess an aromatic ring with a nitrogen atom and carbonyl group in their structure [19].
Whether these shared functional groups are responsible for the observed in vitro and in vivo effects
warranted further structural and functional analysis.
For learning and memory deficit, Y maze test was used in which the percent alteration behavior
indicates short term memory while the total number of arm entries represents general locomotor
activity [39]. Results of % alteration behavior showed significant improvement of short term memory
deficit in succinamide treated groups (Figure 5A). However, general locomotor activity (number of arm
entries) did not show any improvement as no significant difference was observed in succinamide treated
groups as compared to the scopolamine group (data not shown). These results were further corroborated
by the Morris water maze test assessing hippocampus-dependent spatial learning aptitude [40,41],
where succinamide derivatives caused considerable shortening of scopolamine-prolonged escape
latency time (Figure 5B), which indicates an improvement in scopolamine-induced spatial memory
impairment [40,42].
ROS induced oxidative stress is closely linked with neuroinflammation, which further exacerbates
neurodegenerative disorders. Various inflammatory mediators and proinflammatory cytokines are
involved in neuroinflammation such as TNF-
oxide synthase, and nuclear factor kappa B [43
α
, COX2, interleukin 6, interleukin 10, inducible nitric
44]. Besides their free radical scavenging effects,
,

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neuroprotection by succinamide derivatives could also result from their augmentation on endogenous
antioxidant proteins (GST, GSH, and catalase), as well as from amelioration of LPO and proinflammatory
mediators. Taken together, our data supported the hypothesis that these drugs can attenuate the
scopolamine-induced neuronal toxicity by modulating cytokines expression, inflammatory cascade,
and antioxidant enzymes.
5. Conclusions
In conclusion, scopolamine-induced neurodegeneration activates various inflammatory mediators
such as NFkB/COX2/TNF-α along with ROS mediated oxidative stress. Our newly synthesized
succinamide derivatives ameliorated scopolamine-induced oxidative stress and inflammatory cascade,
perhaps by regulating the ROS/TNF-α/COX2/NFkB pathway, ultimately providing neuroprotection
against neuronal inflammation.
Author Contributions: Conceptualization, H.N. and F.A.S.; data curation, S.I., K.N., M.I., and T.K.; formal analysis,
M.I., T.K., T.A., and S.S.; funding acquisition, H.N., F.A.S., K.N., T.A., S.S., M.I., T.K., and S.L.; investigation, S.I. and
K.N.; methodology, H.N., F.A.S., S.I., and K.N.; project administration, H.N., F.A.S., and S.S.; resources, H.N. and,
F.A.S.; software, S.I., K.N., and T.A.; supervision, H.N. and S.L.; writing—original draft, K.N.; writing—review
and editing, H.N., F.A.S., S.I., S.S., M.I., T.A., T.K., Z.A. and S.L. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions,
Shenzhen, 518055, China. Grant NO: 2019SHIBS0004.
Conflicts of Interest: The authors have no conflict of interest.
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