Molecular hybridization based design and synthesis of new benzo[5,6]chromeno[2,3-b]-quinolin-13(14H)-one analogs as cholinesterase inhibitors

Article abstract of DOI:10.1007/s00044-020-02670-w

A Series of new tacrine analogs were designed, synthesized, characterized by respective spectral data and evaluated for cholinesterase inhibitory activity to be useful in Alzheimer’s disease. Most of the synthesized compounds showed good in vitro inhibitory activities toward acetyl cholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. Among the compounds, 6i, 6o and 6r with increased saturated carboxylic ring size attached to the pyridine moiety and having 3,4-dihydroxy, 3,4,5-trimethoxy substituents on the aromatic ring attached at the stereogenic center have shown equal potency to that of tacrine with IC₅₀values 0.65 ± 0.06, 1.32 ± 0.02 and 0.85 ± 0.05, 1.65 ± 0.12 and 0.92 ± 0.03, 1.91 ± 0.12 μM against AChE and BuChE, respectively. Standard drug tacrine exhibited IC₅₀ values of 0.47 ± 0.02 and 0.65 ± 0.08, while Donepezil showed IC₅₀ 0.71 ± 0.06 and 0.31 ± 0.04 μM against AChE and BuChE, respectively. Docking studies of all the molecules disclosed close hydrogen bond interactions with the binding site.

Full text of DOI:10.1007/s00044-020-02670-w

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:685–701
https://doi.org/10.1007/s00044-020-02670-w
ORIGINAL RESEARCH
Molecular hybridization based design and synthesis of new
benzo[5,6]chromeno[2,3-b]-quinolin-13(14H)-one analogs as
cholinesterase inhibitors
Baswaraju Macha1 Ravindra Kulkarni2 Chandrakant Bagul2 Anil Kumar Garige3 Raghuramrao Akkinepally1
Achaiah Garlapati
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1
Received: 7 August 2020 / Accepted: 16 November 2020 / Published online: 8 January 2021
© Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
A Series of new tacrine analogs were designed, synthesized, characterized by respective spectral data and evaluated for
cholinesterase inhibitory activity to be useful in Alzheimer’s disease. Most of the synthesized compounds showed good
in vitro inhibitory activities toward acetyl cholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. Among the
compounds, 6i, 6o and 6r with increased saturated carboxylic ring size attached to the pyridine moiety and having 3,4-
dihydroxy, 3,4,5-trimethoxy substituents on the aromatic ring attached at the stereogenic center have shown equal potency to
that of tacrine with IC50values 0.65 0.06, 1.32 0.02 and 0.85 0.05, 1.65 0.12 and 0.92 0.03, 1.91 0.12 μM against
AChE and BuChE, respectively. Standard drug tacrine exhibited IC50 values of 0.47 0.02 and 0.65 0.08, while Donepezil
showed IC50 0.71 0.06 and 0.31 0.04 μM against AChE and BuChE, respectively. Docking studies of all the molecules
disclosed close hydrogen bond interactions with the binding site.
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Keywords Alzheimer’s disease Antioxidants Anti-inflammatory Cholinesterase inhibitor Multi targeted ligands
Introduction
loss of cognition which aggrandize to severe with time and
ultimately death. AD is now the fourth leading reason for
human mortality in the western countries following cardi-
ovascular disease, cancer, and cerebrovascular disease [1].
World health organization (WHO) has conjectured that in
the next century, AD will be more widespread than AIDS,
cancer and cardiovascular diseases [2]. In USA, over 17%
of people with age above 75 suffer with AD with an esti-
mated cost of whooping USD 236 billion for the treatment
which may escalate over USD 700 billion by 2050 [3]. The
etiology of AD has not been explicated yet, however quite a
lot of factors including amyloidal-β (Aβ) deposits, tau
protein aggregation, exitotoxity hypothesis, oxidative stress
[4] and declined acetylcholine levels [5] have been impli-
cated in the pathomechanism of AD. It has also been
hypothesized that oxidative damage to the intracellular
structures may also decline mitochondrial metabolism
which act as causative factor of abnormal cellular function
and apparently cell death and initiate inflammatory symp-
toms [6–8]. Currently no complete treatment is available to
cure AD and clinical treatments have only symptomatic
effects. Enumerable drugs have already been employed to
contain the brain Aβ levels either by decreasing its
Alzheimer’s disease (AD) is also discerned as senile
dementia of the Alzheimer’s type (SDAT), a form of brain
disease, affecting a large population of geriatric patients. It
is a progressive neurodegenerative disease involving early
decline in memory associated with the other symptoms such
as disturbances in language, visuospatial abnormalities and
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02670-w) contains supplementary
material, which is available to authorized users.
* Achaiah Garlapati
achaiah_g@yahoo.co.in
1
Medicinal Chemistry Division, University College of
Pharmaceutical Sciences, Kakatiya University,
Warangal, Telangana 506009, India
2
Bharati Vidyapeeth’s Poona College of Pharmacy, Paud Road,
Erandawane, Pune 411038, India
3
Jayamukhi Institute of Pharmaceutical Sciences, Narsampet,
Warangal 506332, India

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Medicinal Chemistry Research (2021) 30:685–701
Fig. 1 Structure of anti-Alzheimer’s drug
production or its clearance, however such approach failed to
enhance the cognitive ability of AD patients in trials. Latest
drug discovery in AD segment targeting BACE (beta-site
amyloid precursor protein cleaving enzyme) have failed to
live up to the expectations due to associated toxicities.
Drugs like tacrine, donepezil, galantamine and rivastigmine
which act by elevating acetylcholine (ACh) levels by inhi-
biting acetylcholinesterase (AChE) and memantine, a
NMDA receptor antagonist (Fig. 1). The first clinical can-
didate to be introduced in the market for treatment of AD
was tacrine and has been discontinued from the therapy due
to its severe hepatotoxicity which is attributed to the free
primary amine group.
Aforementioned AChEIs have demonstrated a great
success in raising ACh levels in brain by decreasing the
activity of AChE in the specific regions of the brain. This
elevated levels of ACh was short lived and brain bio-
chemical analysis of AD patients have revealed that it was
due to uninterrupted BuChE activity despite reduced AChE
level. The drug discovery in AD is gradually drifting from
“one target, one disease” to “multi-target-directed ligands”
(MTDLs) [9], which are capable of simultaneously inhi-
biting both AChE and BuChE [10–12]. Having understood
to some extent the pathogenesis of AD, it is palpable
thought that multi-targeting with single drug could even-
tually control the AD. The multi-targeting could involve
inhibiting ChE and free radicals or AChE and BuChE in
single shot. Replacement or annulating the benzene ring in
tacrine with different heterocyclic rings was considered as
important in design of tacrine like compounds. Eghtedari
et al. [13] reported a series of poly functionalized tacrine
derived compounds, 5-amino-2-phenyl-4H-pyrano [2,3-b]
quinoline-3-carboxylate (I) as potent inhibition of choli-
nesterases. The most potent compound with 4-(3-bromo-
phenyl) moiety exhibited IC50 values of 0.069 and 1.35 μM,
respectively, against AChE and BuChE. A series of tacrine-
based pyrano[2,3-c] pyrazoles (II) were reported as potent
inhibitors of cholinesterases. A few of them showed IC50
values in the nanomolar level [14].
Fig. 2 Design of New 10,11,12,12a-tetrahydro-9H-benzo[5,6]chro-
meno[2,3-b]quinolin-13(14H)-one analogs
Maalej et al. [15] synthesized a number of racemic
tacrine analogs containing7-aryl-9,10,11,12-tetrahydro-7H-
benzo[7,8]chromeno[2,3-b]quinoline-8-amine
structure
(III) as potential candidates for the treatment of Alzhei-
mer’s disease, with potent antioxidant and anticholinesterse
activities. Dgachi et al. [16] designed new benzochrome-
nopyrimidinones (IV). Among the series, compounds with a
piperdine-fused ring (n = 2) and 4-methyl/ methoxy sub-
stituents on phenyl ring were found to be potent AChEIs. In
the present study, we used molecular hybridization to
design benzo[5,6]chromenopyridinones (6a-r) as depicted
in Fig. 2 and report the synthesis and AChE and BuChE
inhibitory activity to be useful as anti-Alzheimer’s agents.
Materials and methods
Chemistry
All chemicals including standard drugs and solvents were
procured from Sigma-Aldrich, HiMedia, Bangalore, India
and others and were used without further purification with
the exception of liquid aldehydes which were purified by
using standard procedures prior to use. Melting point for all
the compounds was recorded in open capillary tubes using
VEEGO VMP-D Digital melting point apparatus. FTIR
spectra were done on JASCO FTIR 4100 series by using
1
KBr pellets and are reported in cm−1. Signals of H NMR
and 13C NMR spectra were measured on a BRUKER-II 400
(400 MHz NMR, 13C NMR 100 MHz) spectrophotometer

Medicinal Chemistry Research (2021) 30:685–701
687
by taking TMS as internal standard. Pre-coated TLC plates
were used to check the purity of the compounds and spots
were visualized by using iodine vapors and ultra-violet rays.
Elemental analyses were carried out by a CHN-Vario Elico
Micro elemental analyzer. The estimation of biochemical
parameters was carried out using commercially available
test kits (Sigma-Aldrich).
(KBr, cm−1): 3060.21 (C–H, Aromatic), 2968.10 (C–H,
Aliphatic), 1716.96 (C = O), 1516.01 (C = C, Aromatic),
1265.51 (C–O).
14-Phenyl-10, 11, 12, 12a-tetrahydro-9H-benzo [5,6]
chromeno [2,3-b]quinolin-13 (14H)-one (6b)
Compound 6b obtained as yellowish white solid (yield
1
General procedure for synthesis of ethyl
aminobenzochromene-2-carboxylates (4a-f)
30%), m. p. 201–203 °C. H NMR (400 MHz DMSO, δ
ppm): 8.09–8.11 (d, 1H, J = 8.0 Hz, Ar–H), 7.80–7.82 (d,
1H, J = 8.0 Hz, Ar–H), 7.76–7.78 (d, 1H, J = 4.0 Hz,
Ar–H), 7.50–7.53 (t, 1H, J = 4.0 Hz, Ar–H), 7.37–7.39 (t,
1H, J = 4.0 Hz, Ar–H), 7.32–7.36 (m, 4H, Ar–H),
7.23–7.31 (m, 2H, Ar–H), 5.351 (s, 1H), 3.19–3.21 (t, 1H,
J = 4.0 Hz), 2.71–2.76 (m, 2H), 1.90–2.02 (m, 4H),
1.72–1.88 (m, 6H). 13C NMR (100 MHz, DMSO): 191.15,
160.25, 158.15, 154.17, 145.12, 135.18, 130.25, 128.10,
127.38, 127.84 126.24, 125.18, 125.01, 124.25, 116.52,
101.28, 60.21, 57.40, 48.58, 38.04, 29.29. MASS spectrum
m/z: 380.20 [M + H]+ Calc. for C26H21NO2; CHN: C,
82.30; H, 5.58; N, 3.69; O, 8.43; Found: C, 82.36; H, 5.62;
N, 3.65; O, 8.40. IR (KBr, cm−1): 3103.33 (C–H, Aro-
matic), 2922.15 (C–H, Aliphatic), 1718.28 (C = O),
1590.56 (C = C, Aromatic), 1184.54 (C–O).
A mixture of 2-naphthol (1, 10 mmol), aromatic aldehyde
(2a-f, 10 mmol) and ethyl cyanoacetate (3, 10 mmol) were
refluxed in ethanol for 10 h with stirring in the presence of
piperidine (0.2 eq.). The crude precipitate resulted in was
filtered, washed with cold ethanol and dried to produce the
desired compounds (4a-f).
General procedure for the synthesis of benzochromeno[5,6]
pyridinones (6a-r)
Phosphorous oxychloride (0.14 mL, 0.23 g, 1.5 eq.) was
dropped in portion to the solution of the appropriate ethyl
amino benzochromene-2-carboxylates(4a-f) and the appro-
priate cycloalkanones (5a-c, n = 1, 2, 3: 1.5 eq.) in 1,2-
dichloroethane. The mixture was refluxed for 10 h with
stirring and maximum part of solvent was evaporated and
water (10 mL) was added. The solution was basified with
20% aqueous NaOH, and extracted with DCM, the organic
layer washed with water and dried over magnesium sulfate.
The organic volatilities were evaporated, the solid obtained
was washed with ether and filtered to give benzochrome-
nopyridinones (6a-r).
15-Phenyl-9,10,11,12,13,13a-hexahydrobenzo [5,6]
chromeno [2,3-b] cyclohepta [e] pyridin-14 (15H)-one (6c)
Compound 6c obtained as white solid (yield 39%), m. p.
181–191 °C. 1H NMR (400 MHz CDCl3,
δ ppm):
7.91–7.93 (d, 2H, J = 8.0 Hz, Ar–H), 7.71–7.73 (t, 1H, J =
4.0 Hz, Ar–H) 7.71–7.73 (t, 1H, J = 4.0 Hz, Ar–H),
7.47–7.56 (m, 3H, Ar–H), 7.35–7.39 (t, 2H, Ar–H),
7.18–7.20 (d, 2H, Ar–H), 5.53 (s, 1H), 1.69–1.71 (t, 2H, J
= 4.0 Hz), 1.36–1.45 (m, 2H), 1.10–1.33 (m, 2H),
0.92–0.95 (m, 4H). 13C NMR (100 MHz, CDCl3): 193.09,
165.36, 160.20, 156.25, 145.12, 134.10, 131.10, 128.32,
128.04, 127.12, 126.58, 125.10, 125.51 123.24, 113.15,
105.10, 68.15, 58.17, 45.14, 36.14, 20.14; MASS spectrum
m/z: 394.18 [M + H]+ Calc. for C27H23NO2; CHN: C,
82.42; H, 5.89; N, 3.56; O, 8.13; Found: C, 82.40; H, 5.84;
N, 3.52; O, 8.10. IR (KBr, cm−1): 3058.56 (C–H, Aro-
matic), 2976.58 (C–H, Aliphatic), 1724.84 (C = O),
1591.59 (C = C, Aromatic), 1080.34 (C–O).
Characterization of compounds
13-Phenyl-9, 10, 11, 11a-tetrahydrobenzo [5,6]chromeno
[2,3-b]cyclopenta[e] pyridin-12(13H)-one (6a)
Compound 6a obtained as yellowish orange solid (yield
1
27%), m. p. 165–167 °C. H NMR (400 MHz DMSO, δ
ppm): 8.09–8.11 (d, 1H, J = 8.0 Hz, Ar–H), 7.81–7.82 (d,
1H, J = 4.0 Hz, Ar–H) 7.55–7.56 (d, 1H, J = 4.0 Hz,
Ar–H), 7.51–7.53 (t, 1H, J = 4.0 Hz Ar–H), 7.35–7.39 (t,
1H, J = 8.0 Hz Ar–H), 7.23–7.33 (m,7H, Ar–H), 5.351 (s,
1H), 3.18–3.21 (t, 1H, J = 8.0 Hz), 2.71–2.76 (m, 2H),
1.90–2.02 (m, 2H), 1.72–1.88 (m, 6H); 13C NMR
(100 MHz, DMSO): 191.15, 160.25, 158.15, 154.17,
145.12, 135.18, 130.25, 128.10, 127.38, 127.84 126.24,
125.18, 125.01, 124.25, 116.52, 101.28, 60.21, 57.40,
48.58, 38.04, 29.29. MASS spectrum m/z: 366.14 [M + H]
13-(Naphthalen-2-yl)-9,10,11,11a-tetrahydrobenzo[5,6]
chromeno[2,3-b]cyclopenta [e] pyridin-12(13H)-one (6d)
Compound 6d obtained as orange solid (yield 26%), m. p.
158–160 °C. 1H NMR (400 MHz CDCl3,
δ ppm):
8.49–8.51 (d, 1H, J = 8.0 Hz, Ar–H), 7.95–8.00 (t, 1H, J =
10 Hz, Ar–H) 7.89–7.91 (d, 1H, J = 8.0 Hz, Ar–H),
7.77–7.81 (t, 2H, J = 8.0 Hz, Ar–H), 7.63–7.67 (m, 2H,
+
Calc. for C25H19NO2; CHN: C, 82.17; H, 5.24; N, 3.83;
O, 8.76; Found: C, 82.05; H, 5.10; N, 3.78; O, 8.70. IR

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Medicinal Chemistry Research (2021) 30:685–701
Ar–H), 7.38–7.47 (m, 2H, Ar–H), 7.19–7.23 (t, 1H, J =
8.0 Hz, Ar–H), 6.80–6.82 (d, 1H, J = 9.6 Hz, Ar–H), 6.267
(s, 1H, Ar––H), 5.418 (s, 1H), 1.33–1.44 (m, 4H),
1.10–1.127 (m, 3H), 0.91–0.95 (m, 2H). 13C NMR
(100 MHz, CDCl3): 193.09, 165.36, 160.20, 156.25,
145.12, 134.10, 131.10, 128.32, 128.04, 127.12, 126.58,
125.10, 125.51 123.24, 113.15, 105.10, 68.15, 58.17,
45.14, 36.14, 20.14; Mass spectrum m/z: 416.16 [M + H]+
Calc. for C29H21NO2; CHN: C, 83.83; H, 5.09; N, 3.37; O,
7.70; Found: C, 83.80; H, 5.14; N, 3.32; O, 7.75. IR (KBr,
cm−1): 3071.66 (C–H, Aromatic), 2981.22 (C–H, Alipha-
tic), 1730.51 (C = O), 1591.45 (C = C Aromatic), 1294.70
(C–O).
7.21; Found: C, 83.92; H, 5.72; N, 3.20; O, 7.25. IR (KBr,
cm–1): 3067.12 (C–H, Aromatic), 2938.02 (C–H, Alipha-
tic), 1720.32 (C = O), 1515.12 (C = C, Aromatic), 1124.61
(C–O).
13-(3-Chlorophenyl)-9,10,11,11a-tetrahydrobenzo[5,6]
chromeno [2,3-b] cyclopenta [e]pyridin-12(13H)-one (6 g)
Compound 6 g obtained as yellow solid (yield 48%), m.
p.186–188 °C. 1H NMR (400 MHz CDCl3, δ ppm):
8.09–8.11 (d, 1H, J = 8.0 Hz, Ar–H), 7.80–7.81 (d, 1H, J =
4.0 Hz, Ar–H), 7.76–7.77 (d, 1H, J = 4.0 Hz, Ar–H),
7.50–7.53 (t, 2H, J = 6.0 Hz, Ar–H), 7.17–7.39 (m, 3H,
Ar–H), 7.13–7.14 (d, 1H, J = 4.0 Hz, Ar–H), 7.11–7.12 (d,
1H, J = 4.0 Hz, Ar–H), 5.31 (s, 1H), 3.18–3.21 (t, 1H, J =
4.0 Hz), 2.67–2.74 (m, 2H,), 1.72–1.88 (m, 4H). 13C NMR
(100 MHz, CDCl3):196.10, 160.25, 158.15, 154.17, 145.12,
135.18, 130.25, 128.10, 128.02, 127.38, 127.84 126.24,
126.10, 125.18, 125.01, 124.25, 116.52, 101.28, 60.21,
57.40, 48.58, 38.04, 29.29, 14.09; MASS spectrum m/z:
403.24 [M + H]+ Calc. for C25H18ClNO2: CHN: C, 75.09;
H, 4.54; N, 3.50; O, 8.00; Found: C, 75.15; H, 4.50; N,
3.59; O, 8.04. IR (KBr, cm–1): 3068.23 (C–H, Aromatic),
2980.18 (C–H, Aliphatic), 1720.13 (C = O), 1516.04 (C =
C, Aromatic), 1124.61 (C–O).
14-(Naphthalen-2-yl)-10,11,12,12a-tetrahdro-9H-benzo[5,6]
chromeno[2,3-b]-Quinolin-13(14H)-one (6e)
Compound 6e obtained as orange solid (yield 51%), m. p.
196-198 °C. 1H NMR (400 MHz CDCl3, δ ppm): 8.49–8.51
(d, 1H, J = 8.0 Hz, Ar–H), 7.95–8.00 (t, 2H, J = 8.4 Hz,
Ar–H), 7.89–7.91 (d, 1H, J = 8.0 Hz, Ar–H), 7.77–7.81 (t,
1H, J = 6.0 Hz, Ar–H), 7.63–7.68 (m, 3H, Ar–H),
7.40–7.47 (m, 3H, J = 8.0 Hz, Ar–H), 7.19–7.23 (t, 1H, J =
7.6 Hz, Ar–H), 6.81–6.83 (d, 1H, J = 7.2 Hz, Ar–H), 4.30
(s, 1H), 1.30–1.49 (m, 6H), 1.11–1.15(t, 3H, J = 6.0 Hz).
13C NMR (100 MHz, CDCl3): 196.34, 168.13, 164.53,
153.78, 152.34, 143.63, 142.44, 139.59, 138.74, 136.03,
129.96, 129.40, 127.96, 127.65, 126.73, 125.01, 119.69,
48.15, 38.66, 32.03, 26.21, 22.30. MASS spectrum m/z:
430.52 [M + H]+ Calc. for C26H20N2O4; CHN: C, 73.57; H,
4.75; N, 6.60; O, 15.08; Found: C, 73.53; H, 4.70; N, 6.65;
O, 15.09. IR (KBr, cm–1): 3086.15 (C–H, Aromatic),
2922.18 (C–H, Aliphatic), 1722.81 (C = O), 1549.75 (C =
C, Aromatic), 1210.31 (C–O).
14-(3-Chlorophenyl)-10,11,12,12a-tetrahydro-9H-benzo
[5,6] chromeno [2,3-b] quinolin-13(14H)-one (6 h)
Compound 6 h obtained as cream solid (yield 38%), m.
p.163–165 °C. 1H NMR (400 MHz DMSO, δ ppm):
8.09–8.11(d, 1H, J = 8.0 Hz, Ar–H), 7.80–7.82 (d, 1H, J =
8.0 Hz, Ar–H), 7.76–7.77 (d, 1H, J = 4.0 Hz, Ar–H),
7.50–7.52 (t, 2H, J = 4.2 Hz, Ar–H), 7.29–7.38 (m, 3H,
Ar–H), 7.25–7.26 (d, 1H, J = 4.0 Hz, Ar–H), 5.30 (s, 1H),
3.26–3.28 (t, 1H, J = 4.0 Hz), 3.07–3.11 (m, 2H),
1.82–1.84 (t, 1H, J = 4.2 Hz), 1.70–1.76 (m, 2H). 13C NMR
(100 MHz, DMSO): 196.10, 160.25, 158.15, 154.17,
145.12, 135.18, 130.25, 128.10, 128.02, 127.38, 127.84
126.24, 126.10, 125.18, 125.01, 124.25, 116.52, 101.28,
60.21, 57.40, 48.58, 40.12 38.04, 29.29, 14.09; MASS
spectrum m/z: 415.10 [M + H]+ Calc. for C26H20ClNO2;
CHN: C, 75.45; H, 4.87; N, 3.38; O, 7.73; Found: C, 75.40;
H, 4.89; N, 3.32; O, 7.75. IR (KBr, cm−1): 3089.23 (C–H,
Aromatic), 2965.18 (C–H, Aliphatic), 1715.10 (C = O),
1535.14 (C = C, Aromatic), 1140.12 (C–O).
15-(Naphthalen-2-yl)-9,10,11,12,13,13a-hexahydrobenzo
[5,6] chromeno[2,3-b] cyclohepta [e] pyridin-14(15H)-one
(6 f)
Compound 6 f obtained as orange solid (yield 49%), m.
p.210–212 °C. 1H NMR (400 MHz CDCl3, δ ppm):
8.49–8.51 (d, 1H, J = 8.0 Hz, Ar–H), 7.95–8.00 (t, 2H, J =
8.4 Hz, Ar–H) 7.89–7.91 (d, 1H, J = 8.0 Hz, Ar–H),
7.77–7.81 (t, 2H, J = 6.0 Hz Ar–H), 7.63–7.67 (m, 2H,
Ar–H), 7.28–7.47 (m, 3H, Ar–H), 7.19–7.23 (t, 1H, J =
7.6 Hz, Ar–H), 6.80–6.82 (d, 1H, J = 7.2 Hz, Ar–H), 6.27
(s, 1H, Ar–H), 4.29 (s, 1H), 1.41–1.51 (m, 6H), 1.11–1.14
(t, 5H, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): 196.6,
164.6, 161.3, 152.3, 138.4, 137.2, 135.2, 133.7, 132.9,
131.8, 129.9, 128.0, 127.6, 127.5, 127.4, 127.3, 127.0,
126.5,126.0, 125.1, 120.2, 117.6, 116.1, 109.3, 52.7, 38.9,
38.3, 23.7, 14.9; MASS spectrum m/z: 444.58 [M + H]+
Calc. for C31H25NO2; CHN: C, 83.95; H, 5.68; N, 3.16; O,
15-(3-Chlorophenyl)-9,10,11,12,13,13a-hexahydrobenzo
[5,6] chromeno[2,3-b] cyclohepta [e] pyridin-14 (15H)-one
(6i)
Compound 6i obtained as light yellow solid (yield 45%), m.
p.190–192 °C. 1H NMR (400 MHz DMSO, δ ppm):

Medicinal Chemistry Research (2021) 30:685–701
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8.10–8.12 (d, 1H, J = 8.0 Hz, Ar–H), 7.81–7.82 (d, 1H, J =
4.0 Hz, Ar–H), 7.76–7.78 (d, 1H, J = 8.0 Hz, Ar–H),
7.50–7.54 (t, 2H, J = 8.0 Hz, Ar–H), 7.20–7.34 (m, 3H,
Ar–H), 7.14–7.16 (d, 1H, J = 8.0 Hz, Ar–H), 7.11–7.13 (d,
1H, J = 8.0 Hz, Ar–H), 5.34 (s, 1H), 3.21–3.24 (t, 3H, J =
6.8 Hz), 2.67–2.74 (m, 4H,), 1.72–1.88 (m, 4H). 13C NMR
(100 MHz, DMSO): 196.06, 164.06, 161.03, 151.08,
141.07, 134.80, 133.51, 130.16, 128.28, 128.15, 128.10,
128.04, 126.32, 126.12, 126.04, 123.08, 123.02, 122.10,
118.09, 109.3, 47.05, 35.30, 34.17, 28.21, 27.50, 26.14,
22.30. MASS spectrum m/z: 429.32 [M + H]+ Calc. for
C27H22ClNO2; CHN: C, 75.78; H, 5.18; N, 3.27; O, 7.48;
Found: C, 75.70; H, 5.21; N, 3.29; O, 7.41. IR (KBr, cm–1):
3071.13(C–H, Aromatic), 2957.18 (C–H Aliphatic),
1718.10 (C = O), 1524.12(C = C, Aromatic), 1132.14
(C–O).
123.02, 122.04, 122.01, 119.04, 118.24, 109.03, 52.07,
45.32, 40.51, 38.09, 34.21, 23.25, 14.31. MASS spectrum
m/z: 425.17 [M + H]+ Calc. for C26H20N2O4; CHN: C,
73.57; H, 4.75; N, 6.60; O, 15.08; Found: C, 73.52; H, 4.80;
N, 6.55; O, 15.10. IR (KBr, cm–1): 3068.23 (C–H, Aro-
matic), 2980.18 (C–H, Aliphatic), 1718.13 (C = O),
1525.04 (C = C, Aromatic), 1135.61 (C–O).
15-(3-Nitrophenyl)-9,10,11,12,13,13a-hexahydrobenzo [5,6]
chromeno[2,3-b] cyclohepta [e] pyridine-14 (15H)-one (6 l)
Compound 6 l obtained as white solid (yield 36%), m. p.
1
160–162 °C. H NMR (400 MHz, CDCl3, δ ppm): 8.14 (s,
1H, Ar–H), 8.02–8.04 (d, 1H, J = 8.0 Hz, Ar–H), 7.87–7.88
(d, 1H, J = 4.0 Hz, Ar–H), 7.76–7.78 (d, 1H, J = 8.0 Hz,
Ar–H), 7.46–7.48 (t, 1H, J = 4.0 Hz, Ar–H), 7.34–7.36 (t,
1H, J = 4.2 Hz, Ar–H), 7.30–7.32 (d, 1H, J = 8.0 Hz,
Ar–H), 6.80–6.82 (d, 1H, J = 8.0 Hz, Ar–H), 6.64–6.66 (d,
1H, J = 8.0 Hz, Ar–H), 5.35 (s, 1H), 3.18–3.21 (t, J =
4.2 Hz, 3H), 2.72–2.78 (m, 6H), 1.74–1.96 (m, 8H); 13C
NMR (100 MHz, CDCl3): 196.6, 164.6, 161.03, 152.06,
151.8, 148.04, 133.05, 132.5, 128.8, 128.3, 126.03, 123.08,
123.02, 122.04, 122.01, 119.04, 118.24, 109.03, 74.21,
68.12 52.07, 45.32, 40.51 38.09, 34.21, 23.25, 14.31;
MASS spectrum m/z: 439.28 [M + H]+ Calc. for
C27H22N2O4; CHN: C, 73.96; H, 5.06; N, 6.39; O, 14.60;
Found: C, 73.90; H, 5.08; N, 6.40; O, 14.58. IR (KBr, cm–1):
3071.13 (C–H, Aromatic), 2957.18 (C–H Aliphatic),
1718.10 (C = O), 1524.12 (C = C, Aromatic), 1132.14
(C–O).
13-(3-Nitrophenyl)-9,10,11,11a-tetrahydrobenzo[5,6]
chromeno[2,3-b]cyclopenta [e] pyridin-12(13H)-one (6j)
Compound 6j obtained as light orange solid (yield 21%), m.
p. 186–188 °C. 1H NMR (400 MHz CDCl3, δ ppm): 8.12 (s,
1H, Ar–H), 8.04–8.06 (d, 1H, J = 8.0 Hz, Ar–H), 7.80–7.82
(d, 1H, J = 8.0 Hz, Ar–H), 7.70–7.72 (d, 1H, J = 8.0 Hz,
Ar–H), 7.40–7.43 (t, 1H, J = 4.2 Hz, Ar–H), 7.29–7.31 (t,
1H, J = 6.8 Hz, Ar–H), 7.26–7.28 (d, 1H, J = 8.0 Hz,
Ar–H), 6.70–6.71 (d, 1H, J = 4.0 Hz, Ar–H), 6.62–6.64 (d,
1H, J = 6.8 Hz, Ar–H), 5.35 (s, 1H), 3.18–3.21 (t, 3H, J =
4.0 Hz), 2.72–2.78 (m, 6H), 1.74–1.96 (m, 6H); 13C NMR
(100 MHz, CDCl3): 196.6, 164.6, 161.03, 152.06, 151.8,
148.04, 133.05, 132.5, 128.8, 128.3, 126.03, 123.08,
123.02, 122.04, 122.01, 119.04, 118.24, 109.03, 52.07,
38.09, 34.21, 23.25, 14.31; MASS spectrum m/z: 411.48
[M + H]+ Calc. for C25H18N2O4; CHN: C, 73.16; H, 4.42;
N, 6.83; O, 15.59; Found: C, 73.20; H, 4.45; N, 6.86; O,
15.62. IR (KBr, cm–1): 3071.05(C–H, Aromatic), 2988.18
(C–H, Aliphatic), 1720.14 (C = O), 1545.38 (C = C, Aro-
matic), 1148.20 (C–O).
13-(3,4-Dihydroxyphenyl)-9,10,11,11a-tetrahydrobenzo
[5,6] chromeno [2,3-b] cyclopenta [e]pyridin-12 (13H)-one
(6 m)
Compound 6 m obtained as white solid (yield 48%), m. p.
148–150 °C. 1H NMR (400 MHz DMSO,
δ ppm):
8.09–8.11 (d, 1H, J = 8.0 Hz, Ar–H), 7.80–7.82 (d, 1H, J =
8.0 Hz, Ar–H), 7.75–7.77 (d, 1H, J = 8.0 Hz, Ar–H),
7.49–7.53 (t, 1H, J = 8.2 Hz, Ar–H), 7.35–7.39 (t, 1H, J =
12.0 Hz, Ar–H), 7.25–7.26 (d, 1H, J = 8.0 Hz, Ar–H),
6.75–6.77 (d, 1H, Ar–H), 6.67 (s, 1H, Ar–H), 6.65–6.66 (d,
1H, J = 4.0 Hz, Ar–H), 5.34 (s, 1H), 3.18–3.21 (t, 3H, J =
4.0 Hz), 2.70–2.76 (m, 4H), 1.74–1.96 (m, 4H). 13C NMR
(100 MHz, DMSO): 196.6, 164.6, 161.3, 151.8, 147.6,
133.5, 129.8, 129.4, 128.6, 128.4, 128.2, 126.3, 126.2,
123.8, 123.8, 123.2, 122.1, 118.9, 109.3, 52.7, 38.6, 35.8,
23.7, 14.9; MASS spectrum peak m/z: 366.32[M + H]+
Calc. for C25H19NO4; CHN: C, 75.55; H, 4.82; N, 3.52; O,
16.10; Found: C, 75.58; H, 4.86; N, 3.50; O, 16.08. IR
(KBr, cm–1): 3604.23 (OH), 3067.12 (C–H, Aromatic),
2985.42 (C–H, Aliphatic), 1713.75 (C = O), 1523.20 (C =
C, Aromatic), 1268.31(C–O).
14-(3-Nitrophenyl)-10,11,12,12a-tetrahydro-9H-benzo [5,6]
chromeno [2,3-b] quinolin-13 (14H)-one (6k);
Compound 6k obtained as white solid (yield 40%), m. p.
1
190–192 °C. H NMR (400 MHz DMSO, δ ppm): 8.14 (s,
1H, Ar–H), 8.06–8.07 (d, 1H, J = 4.0 Hz, Ar–H), 7.85–7.87
(d, 1H, J = 8.0 Hz, Ar–H), 7.78–7.79 (d, 1H, J = 8.0 Hz,
Ar–H), 7.41–7.44 (t, 1H, J = 12.0 Hz, Ar–H), 7.31–7.33 (t,
1H, J = 4.0 Hz, Ar–H), 7.28–7.30 (d, 1H, J = 8.0 Hz,
Ar–H), 6.78–6.80 (d, 1H, J = 8.0 Hz, Ar–H), 6.62–6.63 (d,
1H, J = 8.0 Hz, Ar–H), 5.35 (s, 1H), 3.18–3.21 (t, 3H, J =
8.0 Hz), 2.72–2.78 (m, 6H), 1.74–1.96 (m, 8H); 13C NMR
(100 MHz, DMSO): 196.6, 164.6, 161.03, 152.06, 151.8,
148.04, 133.05, 132.5, 128.8, 128.3, 126.03, 123.08,

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14-(3,4-Dihydroxyphenyl)-10,11,12,12a-tetrahydro-9H-
benzo[5,6]chromeno[2,3-b]quinolin-13 (14H)-one (6n)
1H, Ar–H), 7.469–7.506 (t, J = 6.8 Hz, 1H, Ar–H),
7.373–7.395 (d, J = 8.8 Hz, 2H, Ar–H), 6.377 (s, 2H,
Ar–H), 5.351 (s, 1H), 3.797 (s, 3H, OCH3), 3.750 (s, 6H,
OCH3), 1.106–1.378 (m, 4H), 1.024–1.060 (t, 3H, J =
7.2 Hz). 13C NMR (100 MHz, CDCl3): 192.43, 160.15,
159.31, 153.50, 143.20, 138.10, 134.12, 128.19, 128.12,
127.20, 126.10, 124.15, 123.25, 116.32, 105.18, 61.28,
57.10, 49.10, 38.18, 28.65; MASS spectrum m/z: 456.14
[M + H]+ Calc. for C28H25NO5; CHN: C, 73.83; H, 5.53;
N, 3.08; O, 17.56; Found C, 73.80; H, 5.51; N, 3.04; O,
17.54. IR spectrum (KBr, cm−1): 3085.98 (C–H, Aromatic),
2974.62 (C–H, Aliphatic), 1718.40 (C = O), 1585.10 (C =
C, Aromatic, 1289.04 (C–O).
Compound 6n obtained as orange solid (yield 35%), m. p.
157–159 °C. 1H NMR (400 MHz DMSO,
δ ppm):
8.10–8.12 (d, 1H, J = 8.0 Hz, Ar–H), 7.82–7.84 (d, 1H, J =
8.0 Hz, Ar–H), 7.72–7.73 (d, 1H, J = 4.2 Hz, Ar–H),
7.48–7.51 (t, 1H, J = 4.0 Hz, Ar–H), 7.30–7.33 (t, 1H, J =
4.0 Hz, Ar–H), 7.27–7.29 (d, 1H, J = 8.0 Hz, Ar–H),
6.72–6.74 (d, 1H, J = 8.0 Hz, Ar–H), 6.69 (s, 1H, Ar–H),
6.62–6.64 (d, 1H, J = 8.0 Hz, Ar–H), 5.30 (s, 1H),
3.19–3.22 (t, 3H, J = 4.0 Hz,), 2.70–2.76 (m, 4H),
1.74–1.96 (m, 6H). 13C NMR (100 MHz, DMSO): 195.6,
168.6, 163.3, 156.8, 148.2, 132.5, 129.6, 129.3, 128.2,
128.1, 128.0, 126.8, 126.4, 123.8, 123.2, 123.1, 122.8,
118.6, 109.4, 52.1, 38.4, 35.6, 32.8, 30.5, 23.1, 14.9; MASS
spectrum m/z: 412.28 [M + H]+ Calc. for C26H21NO4;
CHN: C, 75.90; H, 5.14; N, 3.40; O, 15.55; Found C, 75.84;
H, 5.18; N, 3.45; O, 15.60. IR (KBr, cm–1): 3082.10 (C–H,
Aromatic), 2975.15 (C–H, Aliphatic), 1720.12(C = O),
1541.20 (C = C, Aromatic), 1150.68 (C–O).
14-(3,4,5-Trimethoxyphenyl)-10,11,12,12a-tetrahydro-9H-
benzo[5,6]chromeno [2,3-b] quinolin-13(14H)-one (6q)
Compound 6q obtained as white solid (yield 34%), m. p.
172–174 °C. 1H NMR (400 MHz CDCl3,
δ ppm):
7.867–7.891(d, J = 9.6 Hz, 2H, Ar–H), 7.526–7.563 (t, J =
8.8 Hz, 1H, Ar–H), 7.462–7.499 (t, J = 14.8 Hz, 1H, Ar–H),
7.373–7.395 (d, J = 8.8 Hz, 2H, Ar–H), 6.378 (s, 2H,
Ar–H), 5.351 (s, 1H), 3.797 (s, 3H, OCH3), 3.750 (s, 6H,
OCH3), 1.106–1.378 (m, 6H), 1.024–1.060 (t, 3H, J =
7.2 Hz). 13C NMR (100 MHz, CDCl3): 93.23, 162.58,
158.38, 152.05,141.65, 135.65, 130.25, 129.85, 129.32,
128.16, 126.32, 125.45, 123.12, 117.12, 102.16, 61.28,
58.32, 48.12, 36.78, 29.54, 25.07. MASS spectrum m/z:
470.10 [M + H]+ Calc. for C29H27NO5; CHN: C, 74.18; H,
5.80; N, 2.98; O, 17.04; Found C, 74.10; H, 5.78; N, 2.95;
O, 17.01. IR (KBr, cm−1): 3086.15 (C–H, Aromatic),
2922.83 (C–H, Aliphatic), 1722.81 (C = O), 1549.75 (C =
C, Aromatic), 1269.79 (C–O).
15-(3,4-Dihydroxyphenyl)-9,10,11,12,13,13a-
hexahydrobenzo [5,6] chromeno [2,3 -b] cyclohepta [e]
pyridin-14 (15H)-one (6o)
Compound 6o obtained as cream white solid (yield 28%),
1
m. p. 165–167 °C. H NMR (400 MHz CDCl3, δ ppm):
8.06–8.08 (d, 1H, J = 8.0 Hz, Ar–H), 7.80–7.82 (d, 1H, J
= 8.0 Hz, Ar–H), 7.70–7.72 (d, 1H, J = 8.0 Hz, Ar–H),
7.40–7.43 (t, 1H, J = 4.0 Hz, Ar–H), 7.29–7.31 (t, 1H, J =
4.0 Hz, Ar–H), 7.26–7.28 (d, 1H, J = 8.0 Hz, Ar–H),
6.70–6.72 (d, 1H, J = 8.0 Hz, Ar–H), 6.68 (s, 1H, Ar–H),
6.62–6.64 (d, 1H, J = 8.0 Hz, Ar–H), 5.35 (s, 1H),
3.18–3.21 (t, 3H, J = 4.0 Hz), 2.72–2.78 (m, 6H),
1.74–1.96 (m, 6H). 13C NMR (100 MHz, CDCl3): 196.2,
170.4, 168.1, 158.2, 145.2, 138.2, 129.5, 129.3, 128.2,
128.1, 128.0, 126.8, 126.4, 123.8, 123.2, 123.1, 122.8,
118.6, 109.4, 58.32, 52.1, 41.23, 38.4, 35.6, 32.8, 30.5,
23.1, 14.9; MASS spectrum m/z: 426.48 [M + H]+ Calc.
for C27H23NO4; CHN: C, 76.22; H, 5.45; N, 3.29; O,
15.04; Found C, 76.20; H, 5.40; N, 3.32; O, 15.10. IR
(KBr, cm–1): 3068.52(C–H, Aromatic), 2980.20 (C–H,
Aliphatic), 1716.34(C = O), 1535.12(C = C, Aromatic),
1135.18(C–O).
15-(3,4,5-Trimethoxyphenyl)-9,10,11,12,13,13a-
hexahydrobenzo[5,6] chromeno [2,3-b] cyclohepta[e]
pyridin-14(15H)-one (6r)
Compound 6r obtained as yellow solid (yield 45%), m.
p.180–182 °C. 1H NMR (400 MHz CDCl3, δ ppm):
7.865–7.919 (m, 5H, Ar–H), 7.473–7.555 (m,2H, Ar–H),
7.316–7.428 (m, 2H, Ar–H), 6.500 (s, 2H, Ar–H), 5.351 (s,
1H), 3.803 (s, 6H, OCH3), 3.757 (s, 3H, OCH3),
1.217–1.431 (m, 6H), 1.035–1.071 (m, 5H); 13C NMR
(100 MHz, CDCl3):192.59, 162.14, 157.81, 152.64, 142.08,
132.54, 131.08, 129.35, 129.09, 128.56, 128.24, 125.58,
125.25, 124.18, 116.04, 101.28, 60.21, 57.40, 48.58, 38.04,
36.24, 29.29, 25.07, MASS spectrum m/z: 484 [M + H]+
Calc. for C30H29NO5; CHN: C, 74.52; H, 6.04; N, 2.90; O,
16.54; Found C, 74.50; H, 6.02; N, 2.95; O, 16.50. IR (KBr,
cm−1): 3051.89 (C–H, Aromatic), 2922.61 (C–H, Alipha-
tic), 1722.32 (C = O), 1588.12 (C = C, Aromatic), 1253.72
(C–O).
13-(3,4,5-Trimethoxyphenyl)-9,10,11,11a-tetrahydrobenzo
[5,6] chromeno [2,3-b]cyclopenta[e] pyridin-12(13H)-one
(6p)
Compound 6p obtained as yellowish orange solid (yield
26%), mp 198–200 °C. 1H NMR (400 MHz CDCl3, δ ppm):
7.864–7.917 (m, 3H, Ar–H), 7.531–7.568 (t, J = 7.6 Hz,

Medicinal Chemistry Research (2021) 30:685–701
691
Pharmacological screening
was taken in a test tube and mixed with occasional shaking.
Solution of appropriate concentration of test compounds
and standards were added and incubated for 20 min fol-
lowed by addition of 30 μL DTNB reagent and 30 μL of
substrate acetyl thiocholine iodide. The intensity of yellow
color thus developed was measured at 412 nm one minute
gap at 37 °C in triplicate. The intensity of yellow color
developed in stoichiometric reaction indicates the amount of
acetyl thiocholine hydrolyzed in the enzymatic reaction.
The relative inhibitions of all the test compounds were
measured with respect to the native esterase activity. The
aforementioned methodology was carried similarly for
BuChE activity using butyrylthiocholine iodide as substrate.
Linear regression plot drawn between % inhibition vs log
concentration of test compounds on MS Excel determines
the IC50 of compounds. The results are presented as mean
standard deviation of the triplicates.
All the tests were organized and conducted in accordance
with the standard guiding principles of the CPCSEA, New
Delhi and protocol was approved by Institutional Animal
Ethical Committee (IAEC/40/UCPSc/KU/2018). 5–6
Weeks male swiss albino mice weighing 20–25 g were used
for behavioral studies and the animals were kept under
standard environmental conditions viz., 20–25 °C tempera-
ture, 12 h lights/dark cycle and given standard pellet diet
and water ad libitum. The animals were allowed to accli-
matize for a week prior to the testing to the laboratory
conditions and randomly divided into five groups of six
animals each as described below:
Group 1: (vehicle control): Animals received PBS
(Phosphate Buffer Solution), p.o
Group 2: (negative control): Mice administered with
β-amyloid peptide by cerebroventricular injection.
Group 3: Mice injected with β-amyloid peptide and
rivastigmine (p.o.) 5 mg/kg.
In vivo estimation of brain cholinesterases
Group 4: Mice injected with β-amyloid peptide and
Tacrine (p.o.) 5 mg/kg
Group 5: Mice injected with β-amyloid peptide and
treated with 200 mg/kg and 400 mg/kg of test
compounds (p.o)
All the groups except 1 were administered with 10 μL
containing 10 μg Aβ (25–35) peptide [17] for the induction
of neurotoxicity at intra-cerebroventicular injection by
identifying bregma point in the skull using stereotaxic
apparatus (INCO, India). On the 14th day of β-amyloid
peptide administration, test compounds and standard drugs
were administered. On the 7th day and 21st day after
β-amyloid peptide treatment, behavioral studies and bio-
chemical parameters are studied [18].
The modified Ellman method was further followed to
estimate brain AChE and BuChE levels in experimental
animals. Rat brain was employed to ascertain the choli-
nesterase inhibitory activity of test compounds in which
animals were sacrificed in order to collect the brain and
homogenate was prepared 0.1 M phosphate buffer (pH
8.0). To a test tube containing 2.6 mL of phosphate buffer
was added 0.4 mL brain homogenate and contents were
mixed thoroughly. 100 μL of DTNB chromophore reagent
was transferred into the above solution and swirled for
proper mixing along with air bubbling. Absorbance of
resultant color developed was measured at 412 nm, when
absorbance reaches a stable value, it was recorded as the
basal reading. Acetyl thiocholine substrate 20 μL was
then transferred into test tube and alternation in absor-
bance was noted for a period of 10 min at intervals of
2 min and later change in the absorbance per minute was
determined.
In vitro AChE and BuChE inhibitory studies [19]
Investigation of AChE and BuChE was carried out by fol-
lowing standard reported procedure and ready to use kits
which provided all reagents were supplied by M/s. Sigma
Aldrich. Test compounds were solubilized in DMSO and
1% bovine serum and finally diluted with phosphate buffer.
Enzyme solutions were prepared to provide a 2.0 unit/ml
and solution of Ellman reagent (5, 5′-dithiobis- 2-
nitrobenzoic acid, DTNB) was prepared by solubilizing
2 mg of DTNB in 200 mL of buffer with occasional vortex.
Determination of IC50 of test compounds in modified Ell-
man method involves formation of thiocholine by the action
of AChE. Thiocholine subsequently reacts with DTNB to
develop yellow color and the intensity of color is propor-
tionate to the enzyme activity in the presence of test com-
pounds is measured at 412 nm spectroscopically. The assay
mixture consisting of 10 μL AChE, 30 μL DTNB solution
In vivo behavior pharmacological activities
Jumping avoidance box (conditioned avoidance test)
[20–22]
Two equal chambers were created by Plexiglas partition in a
box and a gate was provided to have access for the animals
into the adjacent compartment through 14 × 17 cm space.
The test animals are exposed for 30 s to light which is
followed by 10 s sound stimulus and a single low intensity
foot shock (0.5 mA) for three seconds in each trial. Each
mouse from all the groups received 15 such trials in a day
with a gap of 15 s between successive trials repeated for
5 days.

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Rectangular maze test [23, 24]
Carbon tetrachloride, control and test compounds control
administered orally which is briefed below.
Group-I (normal): rats were administered normal saline
orally for 4 days.
Group-II (tacrine): rats administered tacrine orally
200 mg/kG daily for 4 days.
Group-III: rats received 6n 200 mg/kg orally daily for
4 days.
Group-IV: rats were given 6o 200 mg/kg orally daily for
4 days.
Group-V: rats received 6r for four days 200 mg/kg
orally daily.
Group-VI: rats administered orally CCl4 2 ml/kg of 20%
in olive nut oil single dose.
Blood samples were amassed on the fourth day and were
subjected to centrifugation at 400 rpm for 15 min in order to
separate the serum and refrigerated at −20 °C for further
estimation of biomarkers for hepatotoxicity.
Rectangular Maze test apparatus consist of three inter-
connected chambers A, B and C where in chamber B con-
stituted the maze which is used to explore memory capacity of
test animals. All starved mice were allowed to traverse from
chamber A to C passing through B. Chamber C contained
reward food for animal which traversed from chamber A to C
which is indicated by pilot light. All the test animals were
trained daily to collect the reward food and time duration was
monitored. The animals were considered as trained where in
the time required to complete each maze remains constant for
three consecutive days and time to traverse for completion of
maze was then recorded for each animal before and after
treatment with test compounds and standard drug.
Y-maze test [25, 26]
Special recognition memory of mice can be experimented
using two trial recognition Y-maze test which does not need
learning rule. Working memory of rodents is recorded by the
spontaneous Alternation behavior using Y maze which is
made up of black painted wood. Y maze made up of three
arms, each arm with dimensions 40 cm long, 12 cm high, 3 cm
wide at the bottom and 10 cm wide at the top and converged in
an equilateral triangular central area. In the experiment which
last for 8 min, a mouse is placed in one arm of Y maze and
allowed to move freely throughout maze and arm entries (an
entry is said to completed when mouse hind paw completely
enters the arm) are recorded visually. In Y maze, Alternation
term is defined as successive entries into all the three arms and
% Alternation is calculated by following expression.
Biochemical analysis
Four biochemical markers levels from the serum which
indicate the extent of hepatotoxicity viz. alanine amino
transaminase (ALT), aspartate amino transaminase (AST),
alkaline phosphatase (ALP) and total bilirubin levels were
estimated spectroscopically by employing commercial kit
from BioMed Diagnostics (White City, OR, USA).
Statistical analysis
Biochemical data were passed in to Prism GraphPad 8.0 for
post experimental statistical analysis and results were
expressed as mean SD. Two-way ANOVA followed by
Tukey and Dunnet comparisons test was used to identify
significance among groups. Statistical significance was
accepted for values when P < 0.05.
%alternation ¼ fðNo: ofalternationsÞ=ðTotal arm entries À 2Þg  100
% Alternation hint about the depth of working memory and
higher the % is the greater level of memory of the rodents.
All the mice in groups except group 1 were injected with
β-amyloid protein (10 μg) the day one and all the group
mice were trained over maze. On 7th day maze counts were
recorded and 14th day standard drugs tacrine and
rivastigmine and test compounds administered. On 21st
day, once again the mice were allowed move freely on the
maze and entry counts were recorded.
Docking and molecular dynamics studies
Docking studies were carried out using Schrödinger software
[29] (Version 2019-1, Schrödinger) (Glide module). The
ligands used as inputs for docking were sketched by using
ChemDraw software. Ligands were prepared using OPLS3e
force field in Ligprep (Version 2019-1, Schrödinger). This
minimization helps to assign bond orders and addition of the
hydrogens to the ligands. The generated output file (Best
conformation of the ligands) was used for docking studies.
Protein was prepared by using the protein preparation in
Maestro wizard [30] (Version 2019-1, Schrödinger).
Hydrogen atom was added to the proteins and charges were
assigned andalso generated Het states using epik at pH 7.2.
Water molecules and other heteroatoms were excluded from
the crystal structure as they were not significant for the
Hepatotoxicity [27, 28]
Male Wistar rats with 200–250 g body weight were
employed to estimate the hepatotoxicity and animals are
sorted into six groups with five rats in each groups. The
group animals were provided with amenable environment in
separate polypropylene cages and kept at 25 5 °C under
12 h light/dark cycle. Test animals were provided with
standard pellet diet with free access and water ad libitum.

Medicinal Chemistry Research (2021) 30:685–701
693
function of the protein in docking studies. Finally, the protein
was optimized by using optimized potential liquid simula-
tions (OPLS3) force field. A receptor grid was generated
around the cocrystal ligand (X-ray pose of the ligand in the
protein). The centroid of grid box and Vander Waal radius of
receptor atoms was scaled to 1.00 Å with a partial atomic
charge of 0.25. Glide docking score was used to determine
the best docked structure from the output. Poses of the
generated ligands after docking were analyzed by the help of
XP Visualizer (Version 2019-1, Schrödinger).
Molecular dynamic studies were performed by using
GROMACS 2019 [31] for esterase (PDB ID: 1EVE) in
order to ascertain the stability of complex by applying
CHARMM force field. Ligand topology was also generated
by employing CHARMM General Force Field (CGenFF)
[32]. Protein with 6oligand complex was solvated using
predefined water model (TIP3P) [33] and complex was
neutralized by addition of counter ions and subsequently
minimized by following steepest descent method [34]. Entire
system heated to 300 K for 100 ps timescale and followed by
equilibration which was carried out in two different steps.
First step, equilibration was conducted with constant volume
and temperature and in second step, system was equilibrated
with constant pressure and temperature. Finally, the complex
was subjected to molecular dynamics for 6 ns and in order to
evaluate the stability of structure, various parameters were
evaluated, including RMSD, radius of gyration, RMSF and
structural changes as a function of time.
Scheme 1 Synthesis of 10,11,12,12a-tetrahydro-9H-benzo[5,6]chro-
meno[2,3-b]quinolin-13(14H)-one analogs
illustrated a multiplet at ~0.91–3.28 ppm assignable to satu-
rated carbocyclic protons and aromatic protons resonated as
multiplet in the range of 6.26–7.98 ppm. 3,4 dihydro-2H- pyran
proton was found as a singlet in the range of 4.27–5.56. The
ESI mass spectrometric data of all the compounds showed
peaks at relevant [M + H]+ m/z which complemented the
FTIR and NMR spectra for the confirmation of expected
structures of all the compounds.
Biochemical evaluation
In vitro AChE and BuChE inhibitory activity
All the 18 compounds have been screened against human
AChE and BuChE by following reported methods and
compared the potency with standards Tacrine and Done-
pezil. AChE and BuChE inhibitory activity can be tangibly
correlated with structure of all compounds depending on the
number of carbon atoms in saturated carbocyclic ring
attached to the pyridine ring and substitution in the phenyl
ring. AChE and BuChE inhibitory activities of all the
compounds have been listed in Table 2 and graphically
represented in Figs. 3 and 4.
Unsubstituted compound 6a (n = 1) with cyclopentano
fused pyridine ring showed both AChE and BuChE with IC50
value of 2.1 and 6.16 μM. Most active compound among the
series was found to be 6o (n = 3) with cycloheptane fused
ring system with IC50 of 0.65 and 1.32 μM and compared to
0.47, 0.65 μM of tacrine and 0.71, 0.31 μM of donepezil
against AChE and BuChE, respectively. In general, as the
size of the carbocyclic ring increased the cholinesterase
inhibitory activity was found to increase significantly.
Among the unsubstituted compounds (6p and 6r, Ar =
Phenyl), as the size of carbocyclic ring increased from five to
seven (n = 1 to n = 3), the potency against AChE was
increased by about two times and more than three times
increase in potency was observed against BuChE.
Results and discussions
Chemistry
The synthesis of the target compounds (6a-r) has been
accomplished in two steps as outlined in Scheme 1. A multi-
component reaction involving ethyl cyanoacetate1, aromatic
aldehydes 2a-f, 2-naphthol 3 and catalytic amount of piperidine
in ethanol were refluxed at 80 °C for 6 hours to afford the
respective ethyl 3-amino-1-phenyl-1H-benzo[f]chromene-2-
carboxylates 4a-f in good yield (52–64%). Further, ethyl 3-
amino-1-phenyl-1H-benzo[f]chromene-2-carboxylate 4a-f were
refluxed with the appropriate commercial cycloketones 5a-c in
the presence of phosphorus oxytrichloride for 10 h to yield
compounds13-phenyl-9,10,11,11a-tetrahydrobenzo[5,6]chro-
meno[2,3-b]cycloalkano[e] pyridin-12(13H)-ones 6a-r in con-
siderably low yields (32–45%). The physical characterization
data of 6a-r are listed in Table 1.
All the new compounds exhibited satisfactory spectral data
correlating with their structures, FTIR spectrum of all final
compounds 6a-r showed carbonyl stretching vibration around
1704–1730 cm−1 and aliphatic CH stretching observed at the
2952–3090 cm−1 The 1H NMR data of the compounds
.

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Medicinal Chemistry Research (2021) 30:685–701
Table 1 Physical data of
benzochromopyridones 6a-r
Comp.
n
Ar
M. Form
M. Wt
Rfa
% Yield
6a
6b
6c
6d
6e
6 f
6 g
6 h
6i
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Phenyl
C25H19NO2
C26H21NO2
C27H23NO2
C29H21NO2
C30H23NO2
C31H25NO2
C25H18ClNO2
C26H20ClNO2
C27H22ClNO2
C25H18N2O4
C26H20N2O4
C27H22N2O4
C25H19NO4
C26H21NO4
C27H23NO4
C28H25NO5
C29H27NO5
C30H29NO5
365
379
393
415
429
443
399
413
427
410
424
438
397
411
425
455
469
483
0.6
0.5
0.6
0.4
0.5
0.8
0.5
0.7
0.5
0.7
0.5
0.6
0.6
0.5
0.6
0.5
0.7
0.4
27
30
39
26
51
49
48
38
45
21
40
36
48
35
28
26
34
45
Phenyl
Phenyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Nitro phenyl
3-Nitro phenyl
3-Nitro phenyl
3,4-Dihydroxyphenyl
3,4-Dihydroxyphenyl
3,4-Dihydroxyphenyl
3,4,5 Trimethoxy phenyl
3,4,5 Trimethoxy phenyl
3,4,5 Trimethoxy phenyl
6j
6k
6 l
6 m
6n
6o
6p
6q
6r
aMobile phase: Hexane: ethyl acetate
Effect of the nature of aryl group and substituents on AChE
and BuChE inhibitory activity was studied to understand the
structure activity relationship. The simple phenyl group in 6a-c
(Ar = Ph) when replaced with naphthyl (6d-f, Ar = 2-naph-
thyl) resulted in marginal decrease in both cholinesterase
inhibitory activities. This apparently hinted that large bulky
substituent (Ar) could be detrimental for cholinesterase inhi-
bitory activity. Substitution with 3-chloro group as in 6g-i (Ar
= 3-chlorophenyl) enhanced cholinesterase inhibitory activity
significantly and compound 6i with seven membered carbo-
cyclic ring showed potent activity with IC50 value of 0.92 and
1.91 μM against AChE and BuChE, respectively.
with 3,4,5-trimethoxyphenyl groups as in 6p-r have also
shown enhanced activity over unsubstituted compounds, 6a-c,
in both in vitro cholinesterase inhibitory activity.
In vitro brain AChE and BuChE inhibitory activity
Six potent compounds, 6 h, 6i, 6n, 6o, 6q and 6r, were
further tested for in vivo brain AChE and BuChE inhibitory
activity along with tacrine and donepezil as standards by
following the reported methodology. The in vivo brain
AChE and BuChE inhibitory activities of tested compounds
have been listed in Table 3 and depicted in Fig. 5.
Insertion of electron withdrawing group (2-nitro) as in 6j-l
resulted in decreased cholinesterase inhibitory activity when
compared with their corresponding unsustituted compounds,
6a-c, which indicated that electron withdrawing groups hinder
the cholinesterase inhibitory activity. To explore further the
SAR, compounds with two OH groups have been synthesized
(6m-o) and to our expected observations, all the compounds
were superior over 6a-c and compound 6o (Ar = 3,5-dihy-
droxyphenyl; n = 3) was found to be most potent among all the
synthesized compounds with IC50 value of 0.65 and 1.32 μM
against AChE and BuChE, respectively. Three compounds
Two chloro substituted compounds, 6h (n = 2) and 6i (n =
3), demonstrated potent brain AChE inhibitory activity with
IC50 value of 44.14 and 30.35 μM, respectively, while dihy-
droxy compounds 6n (n = 2) and 6o (n = 3) showed potent
AChE inhibitory activity with IC50 of 28.16 and 24.34 μM,
respectively. Similarly, two trimethoxy substituted compounds
6q and 6r showed IC50 of 38.21 and 28.12 μM. In similar
fashion to in vitro AChE and BuChE inhibitory activity as
mentioned in the previous section, increase in the size of the
enhanced the brain AChE inhibitory activity of synthesized
compounds and compound 6o proved to be the most potent in

Medicinal Chemistry Research (2021) 30:685–701
695
Table 2 In vitro inhibitory activity of AChE/ BuChE of compounds (6a-r)
Comp.
n
Ar
AChEa
BuChEa
SIb
6a
6b
6c
6d
6e
6 f
6 g
6 h
6i
1
2
3
1
2
3
1
2
3
1
2
3
1
Phenyl
2.10 0.02 6.17 0.02 2.84
1.52 0.04 5.02 0.04 2.19
1.12 0.02 2.78 0.04 2.77
2.35 0.06 6.41 0.03 1.67
1.80 0.02 5.35 0.03 3.0
1.36 0.03 4.26 0.03 2.50
1.35 0.02 3.45 0.03 2.46
1.08 0.03 2.38 0.03 2.11
0.92 0.03 1.91 0.02 2.07
2.90 0.02 7.64 0.02 1.76
2.02 0.04 5.59 0.02 2.66
1.65 0.03 3.24 0.03 1.98
1.91 0.02 5.86 0.03 1.84
Phenyl
Phenyl
Fig. 3 AChE inhibitory activity (IC50 in μM) of compounds 6a-r,
tacrine and donepezil. The values represent the mean SD from three
independent measurements
2-Naphthyl
2-Naphthyl
2-Naphthyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Nitro phenyl
3-Nitro phenyl
3-Nitro phenyl
6j
6k
6 l
6 m
3,4-
Dihydroxyphenyl
6n
6o
6p
6q
6r
2
3
1
2
3
3,4-
1.58 0.03 4.15 0.04 2.38
0.65 0.06 1.32 0.06 2.03
1.84 0.05 5.14 0.04 1.79
1.26 0.02 3.25 0.02 2.42
0.85 0.06 1.65 0.06 1.94
Dihydroxyphenyl
3,4-
Dihydroxyphenyl
Fig. 4 BuChE inhibitory activity (IC50 in μM) of compounds 6a-r,
tacrine and donepezil. The values represent the mean SD (μM) from
three independent measurements
3,4,5
Trimethoxy phenyl
3,4,5
Trimethoxy phenyl
3,4,5
Trimethoxy phenyl
Table 3 In vivo inhibitory activity of brain AChE/ BuChE of
compounds 6 h, 6i, 6n, 6o, 6q and 6r
Tacrine
−
−
0.47 0.02 0.65 0.08 1.38
0.71 0.06 0.31 0.04 1.34
Donepezil
aIC50 Data are expressed as (mean SD) in μM, analyzed by one-way
ANOVA followed by Tukey’s multiple comparison test with *p <
0.05, **p < 0.01 as significant n = 3
bSelectivity index = IC50 (BuChE)/ IC50 (AChE)
Group
AChEa
BuChEa
6 h
44.14 0.18
30.35 0.15
28.16 0.41
24.34 0.24
38.21 0.20
28.12 0.20
18.10 0.19
20.20 0.14
50.25 0.52
35.10 0.18
37.14 0.24
31.35 0.17
30.23 0.18
40.35 0.34
34.23 0.51
24.62 0.32
26.25 0.25
55.20 0.15
inhibiting brain AChE and BuChE. However, in BuChE
inhibitory activity, compound 6 h (n = 2) was slightly better
active than its seven membered analog 6i (n = 3) and com-
pound 6o was also the most potent against BuChE with IC50 of
30.23 μM which was quite close to both tacrine and donepezil.
6i
6n
6o
6q
6r
Tacrine
Donepezil
Control
Behavioral studies
The statistical analysis was carried out by using Graphpad
prism software version 5.0 and results were compared by one-
way ANOVA followed by Tukey’s Multiple Comparison Test.
A P ≤ 0.001 was considered as statistically significant.
aIC50 Data are expressed as (mean SD) in μM, analyzed by one-way
ANOVA followed by Tukey’s multiple comparison test with *p <
0.05, **p < 0.01 as significant n = 3

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Y maze model demonstrates a sensitive measure of spatial
tive control group. Similarly, groups treated with compounds
6i, 6 h and 6r afforded a significant reduction in transfer
latency (P ≤ 0.001) compared with the negative control group.
With the aim to assess avoidance behaviors, mice were
subjected to conditioning tests in jumping box as this is reli-
able test for measuring the integrity of the learning and
memory processes. Active avoidance behavioral responses,
calculated as a cumulative number of shock avoidance during
15 trials in a day. In avoidance test, groups of animals
administered with test compounds 6i and 6r showed sig-
nificantly increased number of conditioned stimulus responses
(avoidances) on the last day of study (P ≤ 0.001) as compared
with the negative control group animals. Animals treated with
Tacrine and Donepezil showed significantly increased the
number of conditioned stimulus responses (avoidances) on the
last day of study (P ≤ 0.001) as compared with the negative
control group animals. While non-significant difference was
observed between test group 6i and 6r as compared with these
standard drugs signifies increase in the memory.
recognition memory in rodents. The result of the Y maze
experiment is reported as % Alternations for both before and
after the administration of test compounds (Table 4). All the
test compounds and standards tacrine and donepezil have been
administered with 400 mg/kg dose (p.o). The negative control
group showed significant decrease in the Alternation when
compared with the normal control. The results of the standards
treated groups showed significant increase in the % Alterna-
tions with respect 6i and 6r groups which are comparable with
standard drug donepezil.
The learning scores (Transfer latency) obtained by each
group in rectangular maze were suggestive of the fact that
mice took lesser time on last day of study. Negative control
group owed an increase in transfer latency score due to the
memory deficit induced by β-amyloid peptide when compared
to vehicle group. The transfer latency scores observed for the
standard Tacrine treated animals are indicative of a significant
(P ≤ 0.001) memory enhancing potential compared to Nega-
Hepatotoxicity studies
Three compounds 6n, 6o and 6r were subjected for assessing
the serum ALP, ALT, AST and total bilirubin levels, which
indicate the extent of hepatotoxicity caused by the test com-
pounds and are compared with tacrine as standard in carbon
tetrachloride induced hepatotoxicity test protocol. The serum
biomarkers’ data of test compounds, standard and control
groups has been listed in Table 5. Tacrine has been included in
the study as standard due its high hepatotoxicity due to which
tacrine has been discontinued from the market. Single dose of
CCl4 has resulted in drastic 3 to 4 fold increase in serum
biomarkers’ levels when compared with normal saline admi-
nistered control group. The tacrine treated group also
Fig. 5 Brain AChE and BuChE inhibitory activity (IC50 in μM) of
compounds 6a-r, tacrine and donepezil. The values represent the mean
SD (μM) from three independent measurements
Table 4 Effect on biochemical
parameters of liver function tests
Groups
Bilirubin
ALP*
ALT*
ASTa
6n
24.96 1.68
21.63 1.42
26.76 1.19
64.56 3.95
21.03 1.64
117.5 7.25
82.43 2.25
75.0 3.26
51.43 0.86
45.33 1.75
63.73 1.43
82.33 1.10
41.06 1.47
133.06 3.66
133.6 3.14
124.83 1.59
150.63 7.90
198.2 7.51
6o
6r
94.53 1.75
117.86 4.80
85.93 1.82
261.16 7.13
Tacrine
Control
CCl4 group
123.53 2.75
395.16 10.30
aData expressed in U/L, as (mean SD) n = 6 animals in each group analyzed by one-way ANOVA
followed by Tukey’s multiple comparison test with ***p < 0.001 as significant

Medicinal Chemistry Research (2021) 30:685–701
697
Table 5 Behavioral effects in Y-maze, rectangular maze and jumping box tests
Comp
Y-maze test (% Alternations)
Rectangular maze (sec)
Jumping box (sec)
Before treatment
Before treatment
After treatment
Before treatment
After treatment
After treatment
6a
22.15 1.25
20.15 1.56
21.15 1.38
20.32 1.03
25.62 1.30
21.30 1.30
22.02 1.38
20.10 2.25
23.42 1.03
20.12 1.25
22.20 1.00
21.27 1.02
20.35 1.12
21.22 1.29
22.14 1.32
20.23 1.23
21.05 1.35
20.12 1.03
20.60 1.18
21.28 2.42
23.19 2.18
47.61 3.15
32.13 1.08
33.84 1.65
35.12 1.46
31.20 2.14
32.32 1.87
34.24 3.14
42.13 1.87
46.14 2.65
50.61 1.87
30.20 2.10
31.15 1.50
32.65 1.46
37.61 3.15
39.12 1.46
42.12 2.58
36.15 1.23
38.35 1.35
48.25 1.46
54.32 2.06
48.32 2.58
142.87 2.53
47.83 2.53
149.63 2.20
148.26 1.23
151.16 1.86
150.18 1.24
149.28 1.35
148.28 1.40
151.12 3.40
152.72 2.37
150.15 1.85
149.21 1.98
150.14 2.48
150.76 1.37
151.12 2.40
148.28 1.87
149.24 1.35
150.28 2.07
151.23 1.65
150.18 1.42
150.42 2.21
151.42 1.21
34.12 1.09
12.5 2.18
134.45 2.35
130.01 2.40
124.32 1.48
138.12 1.24
136.20 2.35
130.18 2.40
104.15 1.40
98.75 2.37
90.15 1.82
132.07 2.35
129.07 1.32
126.75 1.37
120.15 1.40
116.18 1.25
110.07 2.35
125.30 1.07
120.10 1.25
102.24 2.65
78.61 2.21
92.61 2.21
30.10 2.45
31.23 2.54
30.15 2.10
31.6 3.56
26.15 2.45
28.18 2.54
30.23 2.54
29.05 1.23
32.6 1.62
26.20 2.15
34.10 3.18
29.5 0.22
30.23 1.54
28.18 2.54
30.20 2.45
30.16 1.65
31.40 1.65
30.18 1.20
28.32 0.71
28.32 0.71
21.01 2.45
19.43 2.54
17.56 2.10
22.15 2.56
18.08 1.45
16.12 1.54
14.12 2.54
12.17 1.23
10.24 1.62
20.43 2.15
18.42 3.18
16.17 0.22
16.12 1.20
14.16 1.54
12.45 2.45
17.23 1.25
14.13 2.35
12.33 1.20
8.30 0.71
6b
6c
6d
6e
6 f
6 g
6 h
6i
6j
6k
6 l
6 m
6n
6o
6p
6q
6r
Tacrine
Donepezil
-ve
9.30 0.71
Vehicle control
demonstrated trend of increase in the levels of biomarkers;
however, levels were not high as of CCl4 treated group. All the
three test compounds have shown bilirubin level on par with
control group and compound 6r exhibited slightly elevated
ALP level where as 6n and 6o treated groups have ALP level
slightly lesser than control group. ALT and AST levels of 6r
treated group were found to be higher than control group while
compounds 6n and 6o treated groups showed similar profile as
of control group. The aforementioned data on levels of four
biomarkers and bilirubin revealed that synthesized compounds
are devoid of hepatotoxicity unlike the standard tacrine Fig. 6.
Fig. 6 Effect of test compounds, tacrine, on CCl4 induced serum levels
of Bilirubin, ALP, ALT and AST. The values represent the mean SD
from three independent measurements
Docking and molecular dynamics studies
In vitro studies of synthesized compounds showed the
potential cholinesterase inhibitory activity and among all, the
compound 6r showed promising cholinesterase inhibitory
activity. These result encouraged us to perform docking
studies to get the insight in to the binding mode of synthesized
compounds within binding pocket of AChE and BChE. All
structures of ligands were built using maestro and further
prepared using LigPrep form Schrodinger package. Protein

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Table 6 Docking energies of 6a-r in AChE and BuChE
structures were obtained from the Protein Data Bank (PDB ID:
acetyl cholinesterase-1EVE and butylcholinesterase-4BDS)
and necessary correction to the protein structure were done
using Protein Preparation Wizard in Schrodinger package.
Docking studies were performed using Glide docking software
and docking protocol was validated by docking the cocrystal
ligand which resulted with RMSD of docked conformation
and cocrystal ligand pose was found to be 0.6. The binding
interactions of compounds with AChE and BChE have been
listed in Tables 6 and 7.
Docking study was performed on the both R and S enan-
tiomers of all compounds, among both conformations, R
enantiomers were better in demonstrating docking interactions.
Binding poses of synthesized compounds with AChE have
shown that these molecules bind well within binding pocket of
enzyme. Among the all synthesized molecules, 6r with potent
cholinesterase inhibitory activity, has shown the highest
binding score. In superimposed pose of 6r with cocrystal
ligand, pentacycline ring was coinciding with skeleton of
cocrystal ligand as depicted in Fig. 7. However, 3,4,5-trii-
methoxyphenyl ring has occupied the empty additional space
available in binding pocket. In binding pocket, 6rwas involved
in π-π stacking interactions with Phe331 and His440. 3-
methoxy group formed hydrogen bond with side chain of
Ser122 while 4-methoxy group was involved in hydrogen
Comp.
Docking score AChE
Docking score BChE
Cocrystal Ligand
6r-R
−8.633
−11.85
−10.666
−10.579
−10.529
−9.607
−9.5
−6.015
−8.803
−8.213
−8.185
−7.78
6p-R
6m-R
6q-R
6o-R
6f-R
−7.408
−7.388
−7.311
−7.046
−6.981
−6.914
−6.894
−6.799
−6.68
6d-R
6n-R
6l-R
−8.88
−8.584
−8.381
−8.36
6e-R
6g-R
6k-S
−8.278
−8.049
−7.759
−6.262
−6.195
−6.194
−6.046
−5.595
6j-S
6c-S
−6.517
−6.483
−6.098
−5.775
−5.417
6b-S
6a-S
6i-S
6h-R
Table 7 Analysis of Amino-acid
PDB 4BDS
H-bond Interactions
Aromatic bond Interactions
pi–pi Interactions
residues participated in the
interactions with the proteins
(4BDS, 1EVE)
6q
6n
6 h
SER198
HIS438, ALA328
PHE329
–
–
–
ALA328
ASP70, TRP82
–
ASN83, PHE329
Crystal ligand
HIS438
HIS438
TRP82
PDB 1EVE
H-bond Interactions
Aromatic bond Interactions
pi-pi Interactions
TRP-279
6q
6n
–
–
SER122
ASP72, PHE331 GLU199
TYR121, TYR334
HIS440, PHE331
6 h
–
–
ASP72, GLU199
TYR130, PHE331
TYR121, PHE331 HIS440
Crystal ligand
–
Fig. 7 Superimposed pose of 6r (yellow) with co-crystal ligand (green)
Fig. 8 Docking pose of 6r (yellow) with AChE (green) where
hydrogen bonds are shown in red dotted line

Medicinal Chemistry Research (2021) 30:685–701
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bonding with side chain carbonyl carbon of Asn80 (Fig. 8).
Cyclopentane ring was found to involve in hydrophobic
contacts with Tyr334 and Phe331 and Naphthalene ring was in
close proximity with Trp84, Glu199, His440 and Gly441.
Moreover, docking studies on the BuChE showed the similar
results as acetylcholinesterase. Compound 6r has the highest
binding score and 3,4,5-trimethoxyphenyl was involved in
hydrogen bonding interactions with enzyme; 3-methoxy group
with His148, whereas 4-methoxy was forming with Gly78 and
Trp82 (Fig. 9).
Molecular dynamics study was performed on the protein
complex with ligand 6o to explore the stability of complex
based on data garnered on RMSD, Rg, RMSF, pre and post
molecular dynamics structure analysis as a function of time.
RMSD of backbone atoms was found around 0.2 nm which
supports the stability of the structure (Fig. 10a) and RMSD
for ligand heavy atoms settled in the range of 0.06 nm (Fig.
10b) and also the integrity was maintained throughout the
simulation. Flexible and rigid regions were analyzed using
RMSF which revealed the stability of the complex (Fig.
10c), except for the terminal residues. Radius of gyration
was observed around 2.3 nm which indicates the tight
compactness of the protein ligand complex (Fig. 10d).
Moreover, the comparison of the pre and post molecular
dynamics structure shown that there are no substantial
changes in the structure (Fig. 11) with RMSD of 1.282
which also hinted that the complex was stable.
Fig. 9 Docking pose of 6r (cyan) with BChE (green) where hydrogen
bonds are shown in red dotted line
Fig. 10 Molecular dynamics analysis for esterase. a RMSD for protein backbone, b RMSD for the ligand, c RMSF for protein and ligand and d
radius of gyration

700
Medicinal Chemistry Research (2021) 30:685–701
study. Authors thank the Principal, University College of Pharma-
ceutical Sciences, Kakatiya University, Warangal for providing
necessary facilities. Authors RK and CB express gratitude Mr. Raghu
Rangaswami and Dr. Prajwal Nandekar, Schrodinger, Bangalore for
granting free one-monthlicense of Schrodinger Suite 2019 for docking
studies. Both RK and CB appreciate the Principal and Management of
Bharati Vidyapeeth’s Poona College of Pharmacy, Pune for providing
all computational facilities.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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Fig. 11 Pre (green) and post (cyan) molecular dynamics structure of
esterase
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