Synthesis, Biological Evaluation and Molecular Modelling Studies of Thiophene Piperazine-Carbamate Derivatives as Multi-Target Agents for Alzheimer’s Disease

Abstract

Alzheimer’s disease is a progressive neurodegenerative condition marked by cognitive deterioration, memory deficits and behavioural changes, underscoring the pressing need for innovative therapeutic strategies. While acetylcholinesterase (AChE) inhibitors remain a cornerstone in managing cholinergic dysfunction in AD, the multifaceted nature of the disease, which also involves oxidative stress, necessitates the development of multi-targeted therapeutic agents. In response to this challenge, a series of novel thiophene piperazine-carbamate hybrids (8a-g) was designed and synthesized to simultaneously inhibit AChE and butyrylcholinesterase (BChE), while also possessing potent antioxidant properties, as evidenced by ABTS radical scavenging activity. In vitro analysis revealed robust inhibition of AChE and BChE across all compounds, with a clear preference for AChE inhibition. Among these hybrids, compound 8e exhibited exceptional potency, achieving AChE inhibition (IC50 = 0.12 ± 0.001 µM), BChE inhibition (IC50 = 12.29 ± 0.02 µM) and antioxidant activity (IC50 = 0.192 ± 0.001 µM). Biophysical kinetic studies confirmed that compound 8e operates via mixed-type inhibition of AChE, with inhibition constants (Ki1 = 0.158 µM, Ki2 = 0.347 µM). Molecular docking studies substantiated that both compounds bind effectively to key residues in the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE, supporting their dual inhibition mechanism. Significantly, compounds 8e, 8d, 8g and 8a stand out as promising candidates for further development due to their dual-target inhibition and antioxidant properties. Structure-activity relationship (SAR) analysis highlighted that shorter, unbranched alkyl chains enhance binding affinity and inhibitory potency, while bulkier or branched groups introduce steric hindrance, reducing efficacy. Collectively, these findings position the thiophene piperazine-carbamate hybrids, particularly compounds 8e and 8d, as potent multi-target agents with significant potential for addressing both cholinergic dysfunction and oxidative stress in Alzheimer’s disease therapy.

Full text

AJ CSIAN OURNAL OF HEMISTRY
AJ CSIAN OURNAL OF HEMISTRY
ASIAN JOURNAL
OF CHEMISTRY
(An International Peer Reviewed Research Journal of Chemistry)
Editor-in-Chief
DR. R.K. AGARWAL
ISSN 0970-7077 (Print)
Asian J. Chem.
ISSN 0975-427X (Online)
Vol.3, No. , pp.7 1 1–236
CODEN: AJCHEW
202January 5
https://doi.org/10.14233/ajchem.2025.33493
Synthesis, Biological Evaluation and Molecular Modelling Studies of Thiophene
Piperazine-Carbamate Derivatives as Multi-Target Agents for Alzheimer’s Disease
BALA Y ESU VALAPARLA1,2, , YELAMANDA RAO KANDRAKONDA3, , SAJITHA KETHINENI1, , VAMSI KATTA4, ,
SURESH BABU DONKA1, , MANJUNADH D. METI5, , UTTAM A. MORE6, , A.G. DAMU3, and SRINIVASULU DODDAGA1,*,
1Department of Chemistry, Sri Venkateswara University, Tirupati-517502, India
2Department of Chemistry, SGK Government Degree College, Vinukonda, Palnadu-522647, India
3Bioorganic Chemistry Research Laboratory, Department of Chemistry, Yogi Vemana University, Kadapa-516005, India
4Indian Institute of Science Education and Research (IISER), Tirupati-517507, India
5Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli-500046, India
6Department of Pharmaceutical Chemistry, Shree Dhanvantary Pharmacy College, Kim, Suram-394110, India
*Corresponding author: E-mail: doddaga_s@yahoo.com
INTRODUCTION
Alzheimer’s disease (AD) is the leading cause of dementia
globally, presenting a significant health, social and economic
burden. The disease typically starts with memory loss and
progresses to cognitive impairments, including difficulties with
visuospatial skills, navigation and executive function. As it
advances, individuals experience greater challenges in daily
activities, severely affecting quality of life. In the later stages,
Received: 10 February 2025; Accepted: 19 March 2025; Published online: 30 April 2025; AJC-21969
Alzheimer’s disease is a progressive neurodegenerative condition marked by cognitive deterioration, memory deficits and behavioural
changes, underscoring the pressing need for innovative therapeutic strategies. While acetylcholinesterase (AChE) inhibitors remain a
cornerstone in managing cholinergic dysfunction in AD, the multifaceted nature of the disease, which also involves oxidative stress,
necessitates the development of multi-targeted therapeutic agents. In response to this challenge, a series of novel thiophene piperazine-
carbamate hybrids (8a-g) was designed and synthesized to simultaneously inhibit AChE and butyrylcholinesterase (BChE), while also
possessing potent antioxidant properties, as evidenced by ABTS radical scavenging activity. In vitro analysis revealed robust inhibition of
AChE and BChE across all compounds, with a clear preference for AChE inhibition. Among these hybrids, compound 8e exhibited
exceptional potency, achieving AChE inhibition (IC50 = 0.12 ± 0.001 µM), BChE inhibition (IC50 = 12.29 ± 0.02 µM) and antioxidant
activity (IC50 = 0.192 ± 0.001 µM). Biophysical kinetic studies confirmed that compound 8e operates via mixed-type inhibition of AChE,
with inhibition constants (Ki1 = 0.158 µM, Ki2 = 0.347 µM). Molecular docking studies substantiated that both compounds bind effectively
to key residues in the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE, supporting their dual inhibition mechanism.
Significantly, compounds 8e, 8d, 8g and 8a stand out as promising candidates for further development due to their dual-target inhibition
and antioxidant properties. Structure-activity relationship (SAR) analysis highlighted that shorter, unbranched alkyl chains enhance
binding affinity and inhibitory potency, while bulkier or branched groups introduce steric hindrance, reducing efficacy. Collectively, these
findings position the thiophene piperazine-carbamate hybrids, particularly compounds 8e and 8d, as potent multi-target agents with
significant potential for addressing both cholinergic dysfunction and oxidative stress in Alzheimer’s disease therapy.
Keywords: Thiophene, Piperazine, Carbamates, Alzheimer, Acetylcholinesterase, Butyrylcholinesterase, Molecular docking.
Asian Journal of Chemistry; Vol. 37, No. 5 (2025), 1049-1059
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behavioural and psychological symptoms such as depression,
anxiety, hallucinations and sleep disturbances become more
prominent. Comorbidities like hypertension, diabetes and cardio-
vascular disease can accelerate cognitive decline. In the most
advanced stages, complications such as mobility impairments,
dysphagia, malnutrition and pneumonia are common, incre-
asing mortality risk and further compromising the patient’s
well-being [1,2]. The global prevalence of AD is staggering,
with over 55.2 million people affected worldwide. Projections

suggest this number could rise to 82 million by 2030 and 139
million by 2050, highlighting the urgent need for more effective
treatments to address this growing public health crisis [3].
The pathophysiology of AD is multifactorial, involving
genetic, environmental and aging-related factors. Central to
the disease are cholinergic dysfunction, the accumulation of
amyloid-β (Aβ) plaques, neurofibrillary tangles (NFTs), neuro-
inflammation and oxidative stress, which drive cognitive decline
and neuronal damage. Metal ion imbalances and mitochondrial
dysfunction further exacerbate these processes, leading to ther
synaptic failure and neuronal loss. However, the exact causes
of these pathological changes remain unclear, making AD diffi-
cult to treat effectively. The enzymes acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) are responsible for
the breakdown of ACh in the brain. Inhibiting these enzymes
to reduce ACh hydrolysis is a corner-stone of current therap-
eutic strategies for AD. AChE inhibitors such as donepezil,
galantamine and rivastigmine are widely used to increase ACh
levels, thereby improving neurotransmission and alleviating
cognitive symptoms [4]. Recently, BChE has also emerged as
an important target, as it plays a role in regulating ACh levels
and even modulating amyloid-β release. This highlights the
potential benefit of dual inhibition of both AChE and BChE in
AD treatment [5,6].
Another critical area in AD pathogenesis is the oxidative
stress. The overproduction of reactive oxygen species (ROS)
and the decline in cellular antioxidant defenses contribute to
neuronal damage, synaptic dysfunction and cognitive impair-
ment. Oxidative stress exacerbates amyloid-β plaque forma-
tion, tau hyperphosphorylation and neuronal degeneration.
Thus, combining cholinesterase inhibition with antioxidant
activity offers a promising therapeutic approach to mitigate
both cholinergic dysfunction and oxidative damage in AD
[7,8].
AD is a multifactorial disorder, which makes finding a
single, effective treatment particularly challenging. Multi-
target-directed ligands (MTDLs) present a promising strategy,
as they can simultaneously modulate multiple pathological
targets involved in neurodegenerative diseases. MTDLs are
typically synthesized by connecting the pharmacophores of
various ligands, often incorporating small organic molecules
and heterocyclic compounds known for their favourable physi-
cochemical properties and ability to inhibit enzymes [9]. Parti-
cularly, thiophene derivatives are valuable in drug design due
to their broad pharmacological activity, including antipsy-
chotic, antianxiety, antimicrobial, anti-inflammatory and anti-
oxidant effects. Their structural diversity and ability to form
key interactions with biological targets especially via sulphur-
based hydrogen bonding make them potent candidates [10].
Additionally, piperazine, a six-membered nitrogen-containing
heterocycle, has emerged as a critical component in rational
drug design. It is found in a wide range of well-known therap-
eutic agents, such as antipsychotics, antihistamines, antianginals,
antidepressants, anticancer drugs and more [11]. Minor struc-
tural modifications to piperazine can significantly alter the
medicinal potential of resulting molecules, which enhances
its appeal in drug development for diseases like AD [12]. W hen
combined with thiophene, piperazine improves the pharmaco-
logical profile of drugs, offering synergistic effects such as
anti-inflammatory, anticancer, antiviral, antimicrobial and anti-
alzheimer properties [13,14]. The combination of these two
moieties (Fig. 1) allows for an integrated approach to target
multiple AD-related pathways, including cholinergic dysfunc-
tion, oxidative stress and neuroinflammation, as suggested by
recent studies [15].
H
N
O
N
N
F
O
O
S
8f
Aβ aggregates, tau protein aggregates,
neuroinflammation
AChE, BChE, Aβ aggregate s
AChE, BChE
Fig. 1. Core-linker-base architecture of thiophene piperazine-carbamate
hybrids
Furthermore, the carbamate moiety, known for its ability
to enhance chemical stability, metabolic stability and bioavail-
ability, plays a critical role in drug design. Carbamate-based
compounds have demonstrated significant therapeutic efficacy
in a range of disorders, including AD, cancer and infectious
diseases [16]. Notably, rivastigmine, a carbamate-based drug,
is an established inhibitor of acetylcholinesterase and has been
used to treat AD [17,18]. Leveraging these structural principles,
our research focuses on developing novel thiophene pipera-
zine-carbamate derivatives designed to inhibit both AChE and
BChE, which are pivotal enzymes in AD pathophysiology.
These compounds also aim to provide antioxidant and anti-
inflammatory effects, further targeting the complex and multi-
factorial nature of AD. We conducted molecular docking studies
to assess the interaction profiles of these compounds with AChE,
BChE and antioxidant targets, providing valuable insights into
their mechanisms of action and guiding the refinement of these
compounds for improved therapeutic potential [19]. By integ-
rating these multi-target strategies, we aim to develop more
effective agents capable of addressing the diverse pathological
aspects of AD, ultimately improving therapeutic outcomes for
patients, as supported by recent advances in the field.
EXPERIMENTAL
All chemicals were procured from Merck, TCI, Sigma
Aldrich, Avra and used without purification. All the procedures
and biological experiments were carried out by using double
deionized water. The TLC plates with 0.25 mm and an iodine
chamber or UV lamp (254 nm) visualization were utilized for
monitoring all reactions. As per requirement, compoun were
purified using column chromatography with silica gel (60–
120 mesh). On Shimadzu-8400 spectrometer IR spectra were
1050 Valaparla et al. Asian J. Chem.

recorded KBr pellet. 1H NMR and 13C NMR spectra were meas-
ured at 400 MHz and 100 MHz on a Bruker spectrometer with
CDCl3/DMSO-d6 solvents and TMS as an internal standard
for intermediates and target compounds. The mass spectrometer
Xevo TQD Quadrupole (QCA583) was used to measure HR-
MS.
General synthesis of thiophene piperazine carbamate
hybrids (8a-g)
General procedure of synthesis of 3,4-difluoronitro-
benzene (1): o-Difluoro benzene (0.39 mmol) was nitrated by
dropwise addition of a mixture of conc. HNO3 and conc. H2SO4
over 2 h at 10-15 ºC with continuous stirring. After the addition,
the reaction mixture was stirred at 45-50 ºC for 4 h. The reaction
was then quenched by pouring it into ice water. The organic
phase was separated and the aqueous phase was extracted with
CHCl3 (3 × 50 mL). The combined organic extracts were washed
with water (3 × 50 mL) and dried over anhydrous Na2SO4. The
organic layer was concentrated under vacuum and distilled to
obtained residue 1.
Synthesis of 1-(2-fluoro-4-nitro-phenyl)piperazine (2):
A mixture of 3,4-difluoronitrobenzene (1, 7.5 mmol) in 50 mL
of acetonitrile was mixed with piperazine (18.8 mmol) and
heated under reflux for 3 h. After cooling to room temperature,
the mixture was concentrated in vacuo. The residue was then
diluted with water and extracted with EtOAc (3 × 50 mL) and
satd. NH4Cl (2 × 50 mL) solution, then dried over anhydrous
Na2SO4. The solvent was removed under vacuum and purified
by column chromatography to obtain compound 2.
Synthesis of tert-butyl 4-(2-fluoro-4-nitrophenyl)piper-
azine-1-carboxylate (3): To a solution of piperazinyl comp-
ound (2, 3.2 mmol) in THF (20 mL), di-tert-butyl dicarbonate
(3.78 mmol), a satd. solution of NaHCO3 (15 mL) and Milli-Q
water (15 mL) were added sequentially. The mixture was stirred
at 25 ºC for 5 h, then extracted twice with CH2Cl2. The com-
bined organic layers were washed with brine, dried over anhyd-
rous Na2SO4. The solvent was removed under vacuum and puri-
fied by using column chromatography led to the formation of
compound 3 as a deep yellow powder.
Synthesis of tert-butyl 4-(4-amino-2-fluorophenyl)
piperazine-1-carboxylate (4): To a solution of compound 3
(6.15 mmol) in THF and CH3OH, cooled to below 5 ºC, a satu-
rated solution of CuSO4 (27 mL) was added. The resulting
mixture was stirred vigorously and NaBH4 (107 mmol) was
added in portions. The mixture was then filtered through celite
and the filtrate was subsequently diluted with 200 mL of Milli-Q
water and 400 mL of CH2Cl2. After the separation of organic
phase which was extracted twice with CH2Cl2. The combined
organic extracts were washed with brine, dried over anhydrous
Na2SO4 and filtered. The solvent was removed under reduced
pressure, yielding a brownish oily residue of the desired amine 4.
Synthesis of tert-butyl 4-(2-fluoro-4-(thiophene-2-
carboxamido)phenyl)piperazine-1-carboxylate (5): To an
ice-cooled solution of compound 4 (10 mmol) in 5 mL of DMF,
thiophene-2-carboxylic acid (12 mmol), hydroxy benzotriazole
(HOBt, 15 mmol), (3-dimethylaminopropyl)ethylcarbodiimide
hydrochloride (EDC·HCl, 20 mmol) and a few drops of N,N-
diisopropylethylamine (DIPEA) were added. The mixture was
stirred at room temperature for 1 h. Subsequently, the reaction
mixture was poured onto crushed ice. The resulting precipitate
was filtered, dried and recrystallized from ethanol to yield com-
pound 5.
Synthesis of N-(3-fluoro-4-(piperazin-1-yl)phenyl)thio-
phene-2-carboxamide (6): Compound 5 (10 mmol) and TFA
(20 mmol) was suspended in 20 mL of DCM at ambient tempe-
rature and stirred for 2 h. After completion of the reaction, the
crude was concentrated under reduced pressure. The compound
was dissolved in 5 mL of water, saturated Na2CO3 solution was
used to adjust the pH 8-9. The aqueous layer was extracted
with DCM (3 × 10 mL) and organic layer was washed with satd.
NaHCO3, brine followed by water. Then dried over with anhy-
drous Na2SO4. The organic layer was concentrated under vacuum
and purified by using column chromatography to yield comp-
ound 6 as a pale yellow solid.
General procedure of synthesis of carbamates of N-(3-
fluoro-4-(piperazin-1-yl)phenyl)thiophen-2-amine hybrids
(8a-g): To a solution of 6, (1.1 mmol), triethylamine (1.3 mmol)
and various substituted chloroformates 7a-g, (2.2 mmol) in 5
mL THF, was added dropwise over a period of 20 min. After
stirring the mixture at 40 ºC for 2 h, it was poured into 10 mL
of water. The solution was neutralized with 1N HCl and the
resulting precipitate was filtered and washed with water
(Scheme-I). The crude product was dried in a vacuum desi-
ccator and further purified by column chromatography to yield
8a-g as solids.
Pentyl-4-(2-fluoro-4-(thiophene-2-carboxamido)-
phenyl)piperazine-1-carboxylate (8a): Pale green solid, yield:
68%; m.p.: 168-172 ºC; FT-IR (KBr, νmax, cm–1): 3325, 2943,
2830, 1738, 1654, 1424, 1114, 1022; 1H NMR (CDCl3, 400
MHz) δ ppm: 9.54 (s, 1H), 7.80 (m, 1H), 7.59-7.55 (m, 1H),
7.48-7.46 (m, 1H), 7.36-7.33 (m, 2H), 7.04 (m, 1H), 6.85-
6.81 (m, 1H), 4.04-4.01 (t, 2H), 3.57 (m, 4H), 2.94 (m, 4H),
1.60-1.57 (m, 2H), 1.33-1.22 (m, 4H), 0.87-0.83 (t, 3H); 13C
NMR (CDCl3, 100 MHz) δ ppm: 160.30, 156.41, 155.43, 154.82,
140.11, 135.87, 134.11, 130.71, 128.64, 119.02, 116.24,
109.34, 65.53, 50.40, 49.68, 28.56, 27.99, 22.22, 13.91; m.f.:
C21H28FN3O3S; calcd. m.w.: 419.52: HRMS: 420.1754 [M+H]+.
tert-Butyl-4-(2-fluoro-4-(thiophene-2-carboxamido)-
phenyl)piperazine-1-carboxylate (8b): Pale yellow solid,
yield: 73%; m.p.: 171-175 ºC; FT-IR (KBr, νmax, cm–1) : 3314,
2943, 2831, 1738, 1654, 1448, 1216, 1022; 1H NMR (CDCl3,
400 MHz) δ ppm: 9.64 (s, 1H), 7.81 (m, 1H), 7.60-7.56 (m, 1H),
7.48 (m, 1H), 7.36-7.33 (m, 1H), 7.04 (m, 1H), 6.86-6.81 (m,
1H), 3.57-3.54 (m, 4H), 2.95 (m, 4H), 0.89-0.87 (d, 9H); 13C
NMR (CDCl3, 100 MHz) δ ppm: 160.29, 153.17, 152.88,
140.17, 135.44, 134.37, 130.72, 128.66, 127.48, 119.07, 116.24,
109.32, 79.34, 50.40, 49.68, 28.39; m.f.: C20H24FN3O3S; calcd.
m.w.: 405.49: HRMS: 406.1597 [M+H]+.
Isobutyl-4-(2-fluoro-4-(thiophene-2-carboxamido)-
phenyl)piperazine-1-carboxylate (8c): Off-white solid, yield:
74%; m.p.: 171-175 ºC; FT-IR (KBr, νmax, cm–1) : 3321, 2944,
2831, 1738, 1695, 1448, 1216, 1114, 1022; 1H NMR (CDCl3,
400 MHz) δ ppm: 9.61 (s, 1H), 7.81 (m, 1H), 7.60-7.57 (m,
1H), 7.48-7.46 (m, 1H), 7.38-7.34 (m, 2H), 7.05-7.01 (m, 1H),
Vol. 37, No. 5 (2025) Studies of Thiophene Piperazine-Carbamate Derivatives as Multi-Target Agents for Alzheimer’s Disease 1051

F
F F
NO2
F
ab
FO2N
N
N
H
FO2N
N
N
Boc
cd
FH2N
N
N
Boc
e
f
1
234
5
6
8a-g Cl
O
O
R
(7a-g)
N
H
O
N
N
F
O
O
S
H
N
O
N
NH
F
SH
N
O
N
N
F
S
Boc
+
g
8a
8b
8c 8d 8e
8f 8g
R=
Scheme-I: Synthesis of thiophene piperazine-carbamate hybrids (8a-g) [Reagents and conditions: (a) HNO3, H2SO4, 45 ºC; (b) piperazine,
CH3CN, 80 ºC; (c) Boc2O, THF, 25 ºC; (d) CuSO4, THF, NaBH4, CH3OH, 5 ºC; (e) EDC·HCl, HOBt, DIPEA, DMF; (f) TFA,
DCM; g. TEA, THF]
6.85-6.81 (m, 1H), 3.82-3.80 (t, 2H), 3.69-3.56 (m, 4H), 2.99-
2.94 (m, 4H), 0.98-0.96 (d, 6H); 13C NMR (CDCl3, 100 MHz)
δ ppm: 161.22, 156.39, 154.82, 140.10, 134.51, 134.39, 133.72,
129.79, 128.39, 119.76, 119.73, 118.01, 109.76, 79.34, 50.40,
49.68, 28.39, 18.99; m.f.: C20H24FN3O3S; calcd. m.w.: 405.49:
HRMS: 406.1597 [M+H]+.
Ethyl-4-(2-fluoro-4-(thiophene-2-carboxamido)phenyl)-
piperazine-1-carboxylate (8d): Off-white solid, yield: 69%;
m.p.: 171-175 ºC; FT-IR (KBr, νmax, cm–1) : 3325, 2944, 2831,
1701, 1695, 1590, 1446, 1216, 1022; 1H NMR (CDCl3, 400
MHz) δ ppm: 9.62 (s, 1H), 7.81-7.79 (m, 1H), 7.59-7.54 (m,
1H), 7.48-7.45 (m, 1H), 7.40-7.33 (m, 2H), 7.05-7.01 (m, 1H),
6.85-6.79 (m, 1H), 4.09-4.06 (t, 2H), 3.57-3.54 (m, 4H), 2.95-
2.92 (m, 4H), 1.21-1.19 (t, 3H); 13C NMR (CDCl3, 100 MHz)
δ ppm: 160.27, 156.33, 153.89, 140.11, 135.70, 134.23, 130.17,
128.63, 127.47, 118.98, 116.22, 109.29, 71.74, 50.40, 49.68,
13.91; m.f.: C18H20FN3O3S; calcd. m.w.: 377.43; HRMS: 400.1100
[M+Na]+.
Methyl-4-(2-fluoro-4-(thiophene-2-carboxamido)-
phenyl)piperazine-1-carboxylate (8e): Off-white solid, yield:
66%; m.p.: 184-188 ºC; FT-IR (KBr, νmax, cm–1) : 3314, 2943,
2831, 1738, 1695, 1449, 1420, 1216, 1022; 1H NMR (CDCl3,
400 MHz) δ ppm: 9.54 (s, 1H), 7.87-7.79 (m, 1H), 7.59-7.55
(m, 1H), 7.48-7.46 (m, 1H), 7.36-7.33 (m, 2H), 7.05-7.01 (m,
1H), 6.85-6.79 (m, 1H), 4.04-4.01 (t, 2H), 3.57-3.54 (m, 4H),
2.95-2.92 (m, 4H), 1.60-1.57 (m, 2H), 1.33-1.22 (m, 4H), 0.87-
0.83 (t, 3H); 13C NMR (CDCl3, 100 MHz) δ ppm: 160.27, 154.82,
151.90, 140.10, 130.72, 129.46, 158.63, 127.47, 125.19, 121.62,
119.76, 117.95, 109.76, 50.40, 49.68, 49.65; m.f.: C17H18N3FO3S;
calcd. m.w.: 363.41; HRMS: 364.1116 [M+H]+.
(9H-Fluoren-9-yl)methyl-4-(2-fluoro-4-(thiophene-2-
carboxamido)phenyl)piperazine-1-carboxylate (8f): Off-
white solid, yield: 72%; m.p.: 182-186 ºC; FT-IR (KBr, νmax,
cm–1) : 3314, 2943, 2831, 1737, 1695, 1448, 1216, 1022; 1H
NMR (CDCl3, 400 MHz) δ ppm: 9.68 (s, 1H), 7.82-7.79 (m,
1H), 7.68-7.66 (m, 2H), 7.60-7.46 (m, 5H), 7.41-7.20 (m, 6H),
7.02-7.01 (m, 1H), 6.81 (m, 1H), 4.37 (d, 2H), 3.83 (m, 1H),
3.54 (m, 4H), 2.89 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ
ppm: 160.30, 154.91, 144.99, 143.77, 141.09, 140.09, 135.58,
134.29, 130.73, 128.67, 127.61, 127.13, 126.98, 126.73, 125.00,
124.82, 119.85, 119.01, 116.24, 109.29, 67.09 50.42, 49.68,
47.15; m.f.: C30H26FN3O3S; calcd. m.w.: 527.61; HRMS: 528.1920
[M+H]+.
Phenyl-4-(2-fluoro-4-(thiophene-2-carboxamido)-
phenyl)piperazine-1-carboxylate (8g): Off-white solid, yield:
74%; m.p.: 185-189 ºC; FT-IR (KBr, νmax, cm–1) : 3316, 2942,
2831, 1738, 1695, 1448, 1216, 1022; 1H NMR (CDCl3, 400
MHz) δ ppm: 9.54 (s, 1H), 7.87-7.79 (m, 1H), 7.59-7.55 (m,
1H), 7.48-7.46 (m, 1H), 7.36-7.33 (m, 2H), 7.05-7.01 (m, 1H),
6.85-6.79 (m, 1H), 4.04-4.01 (t, 2H), 3.57-3.54 (m, 4H), 2.95-
2.92 (m, 4H), 1.60-1.57 (m, 2H), 1.33-1.22 (m, 4H), 0.87-
0.83 (t, 3H); 13C NMR (CDCl3, 100 MHz) δ ppm: 161.22,
156.39, 154.82, 144.99, 140.10, 134.51, 134.39, 133.72, 133.63,
132.35, 129.79, 128.39, 119.76, 119.73, 118.01, 117.95, 109.76,
50.42, 49.68; m.f.: C22H20FN3O3S; calcd. m.w.: 425.48; HRMS:
448.1100 [M+Na]+.
In vitro biological assays
Cholinesterase inhibitory activity assay: Elmann’s spectro-
photometric method was used to assess cholinesterase (AChE
& BChE) inhibitory activity using EeAChE (Electrophorus
electricus) and EqBChE (from horse serum) with some modifi-
cations [20]. Phosphate buffer solution (pH = 7.7, 200 mM)
was freshly prepared for assay. Test sample solutions (1 mM)
1052 Valaparla et al. Asian J. Chem.

were prepared by dissolving target compounds in appropriate
amount of DMSO and diluted with phosphate buffer solution
(pH = 7.7, 200 mM) to yield final concentration range. In a 96-
well microplate, the following were added in order of 145 µL
phosphate buffer solution, 10 µL test compounds solution, 80 µL
5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) solution (18.5 mg
in 10 mL phosphate buffer solution) and 10 µL AChE or BChE
solution (4 mU/mL enzyme in 10 mL phosphate buffer). To the
assay mixture, added 15 µL of 1 mM acetylthiocholine/butyryl-
thiocholine (ATCh/BTCh) substrate and assayed after 5 min
of pre-incubation at 25 ºC using Elisa microplate reader (Bio-
Rad) at 415 nm absorbance. Galantamine was a reference drug
using the same technique. Control tests were done with the reac-
tion mixture without test sample. The calculation of the inhibition
percentage was performed using the equation (1-Abst/Absc) ×
100, where Abst and Absc denote the absorbance values of AChE
in the presence and absence of the test compounds, respectively.
The IC50 values were determined using interpolation from linear
regression analysis and are reported as the mean ± standard
error of the mean (SEM) [21].
Kinetics study of AChE inhibition: AChE inhibition by
the most potent analogue 8e was kinetically assessed using
Ellman’s method to calculate the inhibition constant Ki and
understand the type of inhibition. Inhibitor, substrate, enzyme
and DTNB stock solutions were prepared in phosphate buffer
solution (pH 7.7) 2, 2.5 and 3.5 µM of the most active inhibitor,
0.1, 0.2, 0.3, 0.4 and 0.5 µM of acetylcholine iodide and a fixed
AChE concentration (4 mU/mL in 10 mL phosphate buffer)
were used in kinetic tests and assessment was resembled enzy-
me inhibition assay. A 96-Well microplate was loaded with 145
µL of 200 mM phosphate buffer solution, 10 µL of test sample
(2, 2.5 and 3.5 µM), 10 µL of ACh enzyme (4 mU/mL) and 80
µL of DTNB and incubated in dark for 5 min at 25 ºC. The
enzymatic reaction was initiated by the addition of 15 µL of
substrate ATCh (0.1-0.5 mM), which was then incubated for 5
min. Kinetic characterization was done spectrometrically using
a Bio-Rad Elisa microplate reader at 415 nm [20,22]. Each time,
a control assay was performed without the test compound.
The Lineweaver-Burk plots were created using GraphPad Prism
8. The slopes and intercepts of the double reciprocal plots were
plotted against the inhibitor concentrations in order to deter-
mine the inhibitor constants (Ki1 and Ki2) associated with the
binding of the inhibitor to the free enzyme and the enzyme-
substrate complex as intercepts on the negative x-axis. Micro-
soft Excel was used for data analysis.
ABTS radical scavenging assay: The antioxidant ability
of the compounds was evaluated using 2,2′-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) (ABTS) radical scavenging
assay. The ABTS radical cation, referred to as ABTS•+, was
generated by mixing equal volumes of 3 mM potassium
persulfate and ABTS stock solution and placing in the dark at
room temperature for 12-18 h. Before beginning the experi-
ment, the work solution of ABTS•+ was freshly prepared by
adding methanol in a ratio of 1:29. After loading the assay
mixture composed of 10 µL test solution and 290 µL ABTS•+
work solution, the 96-well microplate was then incubated for
30 min, plate was then inserted into the Bio-Rad Elisa reader
and the absorbance was determined at 734 nm. The parallel
control assay without test compound was carried out. The stan-
dard utilized was Trolox. A blank was done in each of the assays
using phosphate buffer rather than test compound and Trolox.
The IC50 values were obtained by doing each assay three times,
averaging the results and reporting them as mean ± SEM [23].
Molecular docking studies: Docking studies were cond-
ucted using the Molecular Operating Environment (MOE) soft-
ware package (2015) to explore the interactions of synthesized
thiophene piperazine-carbamate derivatives with Electrophorus
electricus acetylcholinesterase (PDB ID: 1C2O), obtained from
the Protein Data Bank (https://www.rcsb.org/). The compounds
were first sketched using ChemDraw and then imported into
MOE, where they underwent 3D protonation and energy mini-
mization with a 0.01 gradient. The optimized structures were
saved as MDB files for subsequent docking calculations. The
protein structure was also imported into MOE and the structure
preparation wizard was used to correct any issues with the model.
Hydrogen atoms were added in standard geometry, solvent
molecules were removed and the structure was minimized to
achieve the final optimized protein structure, which was saved
for the docking process. In the docking setup, dummy atoms
were designated as the docking site and the triangle matcher
method was chosen for ligand placement. The London dG scor-
ing function was used to evaluate the initial docking poses,
followed by rigid receptor refinement. The final selection of
the best binding poses was based on the GBVI/WSA dG scoring
method. The default values were applied to all scoring methods.
The MDB files for the synthesized thiophene piperazine-
carbamate derivatives were then loaded into the system and
general docking calculations were run automatically. After the
docking simulations were completed, the resulting poses were
thoroughly analyzed to assess the binding interactions between
the ligands and the acetylcholinesterase enzyme [24,25].
RESULTS AND DISCUSSION
The synthesis of thiophene piperazine-carbamate hybrids
(8a-g) follows a multistep synthetic route. It starts with the
nitration of difluorobenzene using a concentrated HNO3 and
H2SO4 mixture, yielding 3,4-difluoronitrobenzene (1). This
intermediate then undergoes nucleophilic substitution with
piperazine yielded 2 which is followed by Boc protection to
form intermediate 3. The nitro group of intermediate 3 is subse-
quently reduced by NaBH4 to give corresponding amine 4. In
the next step, compound 4 undergoes amide bond formation
with thiophene-2-carboxylic acid in the presence of EDC·HCl
and HOBt, yielding amide 5. The Boc group in 5 was removed
using TFA in DCM, resulting an intermediate 6. Finally, inter-
mediate 6 was treated with various substituted chloroformates
(7a-g) under mild conditions, yielding the thiophene piperazine-
carbamate hybrids (8a-g).
Biological activities
AChE and BChE inhibition activity
AChE inhibition activity: All the synthesized thiophene
piperazine-carbamate hybrids (8a-g), along with galantamine
Vol. 37, No. 5 (2025) Studies of Thiophene Piperazine-Carbamate Derivatives as Multi-Target Agents for Alzheimer’s Disease 1053

as a standard, were assessed for their AChE inhibitory activity,
showing notable inhibition with IC50 values ranging from 0.12
± 0.001 µM to 1.90 ± 0.05 µM (Table-1). Among these, comp-
ound 8e (methyl), exhibited the highest potency, with an IC50
of 0.12 ± 0.001 µM, establishing it as the most potent inhibitor
in the series. Compounds 8d (ethyl) and 8g (phenyl) displayed
slightly reduced activity, with IC50 values of 0.16 ± 0.001 µM
and 1.00 ± 0.006 µM, respectively. In contrast, 8c (isobutyl)
and 8f (fluorenylmethyloxy) demonstrated comparatively
lower inhibition, with IC50 values of 1.58 ± 0.04 µM and 1.90
± 0.05 µM respectively, yet all the compounds still exhibited
notable inhibitory activities, confirming their effectiveness,
albeit with a slight reduction in potency relative to the others.
The SAR study revealed that as alkyl chain length increa-
sed, AChE inhibitory activity decreased. Compound 8e (methyl)
exhibited the highest potency, while compounds with longer
alkyl chains, such as 8d (ethyl, IC50 = 0.16 ± 0.001 µM) and 8a
(pentyl, IC50 = 0.28 ± 0.003 µM) showed progressively reduced
inhibition. Furthermore, branching within the alkyl chains also
led to diminished potency, as observed with 8b (tertiary butyl,
IC50 = 0.94 ± 0.02 µM) and 8c (isobutyl, IC50 = 1.58 ± 0.04
µM). These trends suggest that longer and branched hybrids
reduce AChE activity due to steric hindrance, which prevents
optimal binding in the enzyme’s active site and impairs binding
efficiency. Compound 8g (phenyl, IC50 = 1.00 ± 0.006 µM)
showed moderate inhibition, with the phenyl group allowing
adequate binding despite its bulk. In contrast, compound 8f
(fluorenylmethyloxy, IC50 = 1.90 ± 0.05 µM) exhibited the
weakest inhibition, likely due to the steric bulk of the fluorenyl-
methyloxy group, which hindered proper alignment in the
enzyme’s active site. Collectively, the synthesized thiophene
piperazine-carbamate hybrids (8a-g) demonstrated significant
AChE inhibitory activity, with compound 8e showing the
highest potency.
BChE inhibition activity: All the synthesized thiophene
piperazine-carbamate hybrids (8a-g), along with galantamine
as a standard, were evaluated for their BChE inhibitory activity,
demonstrating significant inhibition with IC50 values ranging
from 12.29 ± 0.02 µM to 114.84 ± 0.12 µM. Among these,
compound 8e (methyl) exhibited the highest potency, with an
IC50 of 12.29 ± 0.02 µM, establishing it as the most effective
inhibitor in the series. Compounds 8d (ethyl) and 8g (phenyl)
showed moderate inhibition, with IC50 values of 18.15 ± 0.14
µM and 35.71 ± 0.27 µM, respectively. In contrast, 8a (pentyl),
8b (tertiary butyl), 8c (isobutyl) and 8f (fluorenylmethyloxy)
demonstrated comparatively lower inhibition, with IC50 values
ranging from 42.81 ± 0.24 µM to 114.84 ± 0.12 µM, yet all
compounds still exhibited notable inhibitory activities, confir-
ming their effectiveness, albeit with reduced potency relative
to the most potent derivatives.
The SAR analysis revealed that shorter alkyl chains and
minimal branching enhanced BChE inhibitory activity. Comp-
ound 8e (methyl) showed the highest potency, while compounds
with longer alkyl chains, such as 8d (ethyl, IC50 = 18.15 ±
0.14 µM) and 8a (pentyl, IC50 = 107.45 ± 0.02 µM), exhibited
progressively reduced inhibition. This suggests that while longer
alkyl chains may still provide some inhibition, they introduce
steric hindrance, which reduces binding efficiency. Further-
more, branching in the alkyl chains, as seen with compounds
8b (tertiary butyl, IC50 = 114.84 ± 0.12 µM) and 8c (isobutyl,
IC50 = 106.07 ± 0.04 µM), led to further diminished potency,
likely due to steric bulk hindering optimal binding in the
enzyme’s active site. The presence of aromatic groups also
influenced BChE inhibition. Compound 8g (phenyl, IC50 =
35.71 ± 0.27 µM) showed moderate inhibition, with the phenyl
group facilitating adequate binding despite its bulk. In contrast,
compound 8f (fluorenylmethyloxy, IC50 = 42.81 ± 0.24 µM)
demonstrated the weakest inhibition, likely due to the steric
hindrance of the fluorenylmethyloxy group, which obstructed
proper alignment within the enzyme’s active site. In summary,
the synthesized thiophene piperazine-carbamate hybrids (8a-g)
demonstrated variable but significant BChE inhibitory activity,
with compound 8e showing the highest potency.
Selectivity index: The most potent compounds in both
AChE and BChE assays are compound 8e and compound 8d.
Compound 8e demonstrated the highest potency in AChE
inhibition with an IC50 of 0.12 ± 0.001 µM and BChE inhibition
with an IC50 of 12.29 ± 0.02 µM. Its selectivity index (SI) for
AChE over BChE is 102.41, indicating a strong preference for
inhibiting AChE. Compound 8d also showed potent inhibition
with an AChE IC50 of 0.16 ± 0.001 µM and a BChE IC50 of 18.15
± 0.14 µM, with a SI of 113.43, demonstrating a slightly higher
selectivity for AChE compared to 8e. Both compounds exhibit
high selectivity for AChE inhibition, highlighting their poten-
tial for targeted therapeutic applications in neurodegenerative
diseases such as Alzheimer’s disease.
TABLE-1
AChE AND BChE INHIBITION AND ABTS RADICAL SCAVENGING
ACTIVITIES OF THIOPHENE PIPERAZINE-CARBAMATE HYBRIDS (8a-g)
IC50 (µM) ± S.E.M. IC50 (µM) ± S.E.M.
Compounds AChE BChE Selectivity for AChE ABTS
8a 0.28 ± 0.003 107.45 ± 0.02 383.75 1.089 ± 0.022
8b 0.94 ± 0.02 114.84 ± 0.12 122.17 1.157 ± 0.012
8c 1.58 ± 0.04 106.07 ± 0.04 67.13 1.619 ± 0.34
8d 0.16 ± 0.001 18.15 ± 0.14 113.43 0.434 ± 0.02
8e 0.12 ± 0.001 12.29 ± 0.02 102.41 0.192 ± 0.001
8f 1.90 ± 0.05 42.81 ± 0.24 22.53 2.487 ± 0.013
8g 1.00 ± 0.006 35.71 ± 0.27 35.71 1.491 ± 0.015
Galantamine 0.01 ± 0.003 2.06 ± 0.03 206 –
Trolox – – – 0.112 ± 0.002
1054 Valaparla et al. Asian J. Chem.

ABTS radical scavenging activity: All the synthesized
hybrid compounds 8a-g, alongside Trolox as a standard, were
evaluated for their ABTS radical scavenging abilities, showing
notable antioxidant activity, with IC50 values ranging from 0.19
± 0.001 µM to 2.49 ± 0.013 µM. Among these, compound 8e
(methyl) exhibited the highest potency, with an IC50 of 0.19 ±
0.001 µM, establishing it as the most effective radical scavenger
in the series. Compound 8d (ethyl) showed slightly reduced
activity, with an IC50 of 0.43 ± 0.02 µM, but still demonstrated
substantial radical scavenging potential. In contrast, compound
8f (fluorenylmethyloxy) and compound 8c (isobutyl) exhibited
comparatively lower activity, with IC50 values of 2.49 ± 0.013
µM and 1.62 ± 0.34 µM, respectively, yet both maintained
notable antioxidant activity, confirming their effectiveness
despite a slight reduction in potency compared to the others.
The SAR analysis revealed that shorter alkyl chains signifi-
cantly enhance ABTS radical scavenging activity. Compound
8e (methyl) emerged as the most potent scavenger, with an IC50
of 0.19 ± 0.001 µM. As alkyl chain length increased, the scaven-
ging activity progressively diminished, as seen in 8d (ethyl,
IC50 = 0.43 ± 0.02 µM) and 8a (pentyl, IC50 = 1.09 ± 0.022
µM), suggesting that shorter alkyl chains provide more efficient
interactions with the ABTS radicals, while longer chains reduce
radical scavenging efficiency. Moreover, branching within the
alkyl chains further decreased antioxidant activity, as observed
with compounds 8b (tertiary butyl, IC50 = 1.16 ± 0.012 µM)
and 8c (isobutyl, IC50 = 1.62 ± 0.34 µM). The incorporation of
aromatic groups also influenced radical scavenging potency.
Compound 8g (phenyl, IC50 = 1.49 ± 0.015 µM) demonstrated
moderate activity, as phenyl group, though bulkier than alkyl
chains, still allowed sufficient interaction with the ABTS radicals.
On the other hand, compound 8f (fluorenylmethyloxy, IC50 =
2.49 ± 0.013 µM) exhibited the weakest scavenging activity,
likely due to the steric hindrance imposed by the larger fluor-
enylmethyloxy group, which obstructed optimal interaction
with the radicals. Overall, the synthesized hybrid compounds
(8a-g) displayed significant ABTS radical scavenging activity,
with compound 8e demonstrating the highest potency.
Kinetic study on AChE inhibition: Compound 8e demon-
strates superior potency in inhibiting AChE, BChE and exhib-
iting antioxidant activity, making it an attractive candidate for
further study. Given its outstanding performance in all three
assays, we extended our investigation to explore the binding
mechanism and inhibition strategy of the compound. Inhibition
kinetics of 8e against AChE was analyzed by assessing enzyme
activity at various substrate (ATCh) concentrations (0.1, 0.2,
0.3, 0.4 and 0.5 mM) and inhibitor concentrations (IC50 = 2.0,
2.5 and 3.5 µM). A Lineweaver-Burk plot was constructed using
the reciprocals of initial velocity and substrate concentration
(1/v vs. 1/S), revealing a mixed-type inhibition pattern charact-
erized by increasing slopes (decreased Vmax) and intercepts
(higher Km) as the concentration of 8e increased (Fig. 2). This
indicates that 8e interacts with both the catalytic active site (CAS)
and the peripheral anionic site (PAS) of AChE. The inhibitor
constants Ki1 and Ki2 were determined to be 0.158 µM and 0.347
µM, respectively, through secondary plots of concentration
versus slope and intercept. These kinetic findings not only
confirm the potent inhibition of AChE by 8e but also suggest
that the compound binds effectively to both key sites (CAS and
PAS) within the enzyme. This dual interaction further supports
its strong inhibitory activity, providing valuable insights for the
design of more effective therapeutics targeting cholinesterases.
Molecular docking analysis: All the synthesized hybrid
compounds 8a-g exhibited strong activity in AChE, BChE and
ABTS assays, with compound 8e demonstrating the highest
potency across these tests (Table-2). To investigate the mecha-
nisms behind their biological activities, molecular docking
studies focused on key regions of the AChE enzyme, including
the catalytic active site (CAS), peripheral anionic site (PAS),
catalytic radius and mid-gorge regions, which are crucial for
enzyme-substrate and enzyme-inhibitor interactions.
Compound 8e, with a binding energy of -7.50 kcal/mol
and an RMSD of 3.31 Å, showed the most efficient binding
by interacting with SER203 and HIS447 in the CAS, with mini-
mal steric hindrance from its methyl group, contributing to its
high potency. Compound 8b (binding energy -8.29 kcal/mol,
RMSD 1.54 Å) exhibited strong binding, but the bulky tertiary
butyl group induced steric hindrance, reducing its inhibitory
activity. Similarly, compound 8a (binding energy -8.11 kcal/
mol, RMSD 1.89 Å), despite stable binding, suffered from steric
clashes due to its pentyl group, limiting its fit and reducing
potency. Compound 8c (binding energy -7.27 kcal/mol, RMSD
1.31 Å) showed good binding at SER203 and TRP86, but the
isobutyl group caused steric clashes, reducing its effectiveness.
Compound 8d (binding energy -6.91 kcal/mol, RMSD 1.25
Å) had a slightly lower binding energy but interacted effectively
with GLU202 and TYR337 in the CAS and mid-gorge region.
However, the longer ethyl group caused some steric hindrance,
limiting its overall potency. Compounds 8f (binding energy
-8.42 kcal/mol, RMSD 2.12 Å) and 8g (binding energy -7.50
kcal/mol, RMSD 1.74 Å) exhibited strong binding but faced
steric interference from the larger functional groups (fluorenyl-
methyloxy in 8f and bulky groups in 8g), which diminished
their inhibitory potency. In conclusion, while several comp-
ounds exhibited high binding energies and favourable docking
parameters, 8e outperformed due to its optimal binding fit,
which minimized steric hindrance and enabled more effective
enzyme inhibition (Fig. 3).
Conclusion
The synthesized thiophene piperazine-carbamate hybrids
(8a-g) exhibited promising multi-target activity, effectively
inhibiting AChE and BChE while also demonstrating strong
antioxidant properties. Among these, compound 8e, featuring
a methyl group, emerged as the most potent, showing superior
AChE and BChE inhibition as well as excellent antioxidant
activity. The kinetic studies revealed that 8e exhibited mixed-
type inhibition against AChE, interacting with both the CAS
and PAS, which was substantiated by the molecular docking
studies. The results showed that 8e formed optimal interactions
with key residues in both sites, with a well-fitted structure
explaining its superior potency. In contrast, compounds like
8d (ethyl) and 8g (phenyl), although potent, exhibited slightly
reduced activity, likely due to steric hindrance from their longer
Vol. 37, No. 5 (2025) Studies of Thiophene Piperazine-Carbamate Derivatives as Multi-Target Agents for Alzheimer’s Disease 1055

2.0
1.8
1.6
1.4
1.2
[v] (min/A)
–1 –1
∆
-12 -9 -6 -3 0 3 6 9 12
[ATCh] mm
–1 –1
0 M
0.1 M
0.2 M
0.5 M
(a)
0.12
0.10
0.08
0.06
0.04
0.02
0
Slope
-0.2 0 0.2 0.4 0.6
Concentration (µM)
y = 0.1509x + 0.0239
R = 0.9899
2
(b)
-0.4 -0.2 0 0.2 0.4 0.6
Concentration (µM)
y = 0.8342x + 0.2895
R = 0.9999
2
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
Intercept
(c)
Fig. 2. Kinetics of AChE inhibition for compound 8e; (a) Lineweaver Burk plot for 8e (b) Secondary plots of slope and various concentrations
of 8e (c) Intercept and various concentrations of 8e
TABLE-2
THE BINDING SCORES, RMSD VALUES, LIGAND, RECEPTOR, INTERACTIONS, BINDING SITES
DISTANCES AND ENERGY FOR THE THIOPHENE PIPERAZINE-CARBAMATE HYBRIDS (8a-g)
Compound Score (Kcal/mol) RMSD Receptor Interactions Binding site
8a -8.11 1.89 SER 293 (B) π-H 3.84
8b -8.29 1.54 SER 203 (B) H-acceptor 3.10
PHE 295 (B) H-acceptor 2.88
HIS 447 (B) π-H 3.95
8c -7.27 1.31 SER 203 (B) H-acceptor 2.80
TRP 86 (B) H-π 3.54
TRP 286 (B) π-π 3.93
8d -6.91 1.25 GLU 202 (B) H-donor 3.21
TYR 337 (B) H-π 4.46
8e -7.50 3.31 SER 203 (B) H-acceptor 3.05
HIS 447 (B) π-H 3.85
TRP 286 (B) H-acceptor 2.88
8f -8.42 2.12 SER 203 (B) H-acceptor 3.03
HIS 447 (B) π-H 3.85
8g -7.50 1.74 GLN 291 (B) H-donor 3.99
TRP 286 (B) H-π 3.58
TRP 86 (B) H-π 3.81
Galantamine -5.82 1.62 TRP 86 (B) H-π 3.78
or bulkier groups, as confirmed by docking data. The SAR
studies further emphasized that shorter alkyl chain such as the
one in 8e, facilitated more efficient binding and dual inhibition,
while longer and branched chains, as seen in compounds 8a
(pentyl) and 8b (tertiary butyl) respectively, caused steric clashes
that reduced potency. These findings, drawn from biological
assays, kinetics and docking studies, highlight the potential
of compound 8e as a potent multi-target agent, addressing both
cholinergic dysfunction and oxidative stress in Alzheimer’s
disease therapy, making it the most promising candidate for
further development.
ACKNOWLEDGEMENTS
The authors express their gratitude to the Department of
Bioorganic Chemistry, Yogi Vemana University, Kadapa, India
for supporting in vitro studies. The authors also express their
gratitude to Department of Chemistry, University of Hyderabad,
for providing the molecular docking study.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
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Polar
Acidic
Basic
Greasy
Polar
Acidic
Basic
Greasy
Sidechain acceptor
Sidechain donor
Backbone acceptor
Backbone donor
Sidechain acceptor
Sidechain donor
Backbone acceptor
Backbone donor
Ligand
exposure
Ligand
exposure
Solvent residue
Metal complex
Solvent contact
Metal/ion contact
Solvent residue
Metal complex
Solvent contact
Metal/ion contact
Arene-arene
Arene-H
Arene-cation
Arene-arene
Arene-H
Arene-cation
Receptor
exposure
Receptor
exposure
Proximity
contour
Proximity
contour
8a
8b
8c
Vol. 37, No. 5 (2025) Studies of Thiophene Piperazine-Carbamate Derivatives as Multi-Target Agents for Alzheimer’s Disease 1057

Polar
Acidic
Basic
Greasy
Polar
Acidic
Basic
Greasy
Polar
Acidic
Basic
Greasy
Sidechain acceptor
Sidechain donor
Backbone acceptor
Backbone donor
Sidechain acceptor
Sidechain donor
Backbone acceptor
Backbone donor
Sidechain acceptor
Sidechain donor
Backbone acceptor
Backbone donor
Ligand
exposure
Ligand
exposure
Ligand
exposure
Solvent residue
Metal complex
Solvent contact
Metal/ion contact
Solvent residue
Metal complex
Solvent contact
Metal/ion contact
Solvent residue
Metal complex
Solvent contact
Metal/ion contact
Arene-arene
Arene-H
Arene-cation
Arene-arene
Arene-H
Arene-cation
Arene-arene
Arene-H
Arene-cation
Receptor
exposure
Receptor
exposure
Receptor
exposure
Proximity
contour
Proximity
contour
Proximity
contour
8d
8e
8f
1058 Valaparla et al. Asian J. Chem.

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Polar
Acidic
Basic
Greasy
Sidechain acceptor
Sidechain donor
Backbone acceptor
Backbone donor
Ligand
exposure
Solvent residue
Metal complex
Solvent contact
Metal/ion contact
Arene-arene
Arene-H
Arene-cation
Receptor
exposure
Proximity
contour
8g
Fig. 3. The 2D & 3D binding modes of 8a, 8b, 8c, 8d, 8e, 8f, 8g in the active site of AChE
Vol. 37, No. 5 (2025) Studies of Thiophene Piperazine-Carbamate Derivatives as Multi-Target Agents for Alzheimer’s Disease 1059