Microwave-Assisted Organic Synthesis, structure–activity relationship, kinetics and molecular docking studies of non-cytotoxic benzamide derivatives as selective butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.07.015

A series of benzamide derivatives 1–12 with various functional groups (–H, –Br, –F, –OCH₃, –OC₂H₅, and –NO₂) was synthesized using an economic, and facile Microwave-Assisted Organic Synthesis, and evaluated for

Full text of DOI:10.1016/j.bmc.2019.07.015

Accepted Manuscript
Microwave-assisted organic synthesis, structure–activity relationship, kinetics
and molecular docking studies of non-cytotoxic benzamide derivatives as se-
lective butyrylcholinesterase inhibitors
Sheeba Wajid, Asma Khatoon, Maria Aqeel Khan, Humaira Zafar, Shama
Kanwal, Atta-ur-Rahman, M. Iqbal Choudhary, Fatima Z. Basha
PII:
S0968-0896(19)30927-7
https://doi.org/10.1016/j.bmc.2019.07.015
BMC 14999
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 June 2019
5 July 2019
9 July 2019
Please cite this article as: Wajid, S., Khatoon, A., Khan, M.A., Zafar, H., Kanwal, S., Atta-ur-Rahman, Iqbal
Choudhary, M., Basha, F.Z., Microwave-assisted organic synthesis, structure–activity relationship, kinetics and
molecular docking studies of non-cytotoxic benzamide derivatives as selective butyrylcholinesterase inhibitors,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.015
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Microwave-assisted organic synthesis, structure–activity relationship, kinetics and
molecular docking studies of non-cytotoxic benzamide derivatives as selective
butyrylcholinesterase inhibitors
Sheeba Wajida, Asma Khatoona, Maria Aqeel Khana, Humaira Zafarb, Shama Kanwala, Atta-ur-
Rahmana, M. Iqbal Choudharya,b*, and Fatima Z. Bashaa*
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biology
Science, University of Karachi-75270, Pakistan.
bDr. Panjwani Center for Molecular Medicine and Drug Research (PCMD), International
Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan
E-mail: bashafz@gmail.com
iqbal.chaudhary@iccbs.com
Abstract
A series of benzamide derivatives 1-12 with various functional groups (-H, -Br, -F, -OCH3, -
OC2H5, and -NO2) were synthesized using an economic, and facile Microwave-Assisted Organic
Synthesis, and evaluated for acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE)
activity in vitro. Structure–activity relationship showed that the substitution of -Br group
influenced the inhibitory activity against BCHE enzyme. Synthesized compounds were found to
be selective inhibitors of BCHE. In addition, all compounds 1-12 were found to be non-
cytotoxic, as compared with standard cycloheximide (IC50 = 0.8 ± 0.2 µM). Among them,
compound 3 revealed the most potent BCHE inhibitory activity (IC50 = 0.8 ± 0.6 µM) when
compared with standard galantamine hydrobromide (IC50 = 40.83 ± 0.37 µM). Enzyme kinetic
studies indicated that compounds 1, 3-4, and 7-8 showed mixed mode of inhibition against
BCHE, while compounds 2, 5-6 and 9 exhibited an uncompetitive pattern of inhibition.
Molecular docking studies further highlighted the interaction of these inhibitors with
catalytically important amino acid residues, such as Glu197, Hip438, Phe329 and many others.
Keywords:
1

Benzamides, Green Chemistry, Microwave-Assisted Organic Synthesis, Acetylcholinesterase,
Butyrylcholinesterase, Enzyme Inhibition, Alzheimer disease.
1. Introduction
Alzheimer’s disease (AD) is a neurological disorder of senior population. This disease was
identified by Dr. Alois Alzheimer in 1906 who also discovered plaques and tangles in the AD
brain [1, 2]. AD may arise with the destruction of nerve cells that also affects the function of
brain. Due to neurodegeneration lifespan of the patients declines that make AD among top ten
causes of death in developed countries [3, 4]. Globally 35 million population has been affected
from AD [5] and it is hypothesized that by the end of 2050, 1 person in 85 people will be
affected. Cholinesterases, comprises of acetylcholinesterse (ACHE) [EC 3.1.1.7], and
butyrylcholinesterase (BCHE) [EC 3.1.1.8], are renowned serine hydrolases, and have
nucleophilic serine residue in the active sites [6]. Acetylcholinesterase (ACHE) is found in
erythrocytes, nerve endings, lungs, spleen and in the all compartments of brain.
Butyrylcholinesterase (BCHE) has capability to hydrolyse various choline-based esters. BCHE is
precisely found in peripheral tissues, brain, serum, neurons, and glial cells [7-9]. BCHE have
ability to hydrolyze many drugs, like cocaine, acetylcholine, and succinylcholine [10-12], and
thus play a vital role in neurodegenerative disorder. In the last stages of Alzheimer`s disease,
there is an increase in the development of amyloid plaque and neurofibrillary tangles with the
increase expression of BCHE enzyme [13]. Researchers also found that level of
acetylcholinesterase (ACHE) is reduced when incubated with Aβ fibrils, while BCHE remains
active [14]. This study highlights the significance of developing selective BCHE inhibitors for
the treatment of AD.
Recently, several natural products or their derivatives were discovered as potential therapy for
the treatment of AD. Galantamine, donepezil, and rivastigmine are well known cholinesterase
inhibitors that have proven to enhance cognitive function with mild to moderate AD patients
[15]. Selective BCHE inhibitors elevate brain acetylcholine, improve cognition and decline β-
amyloid, and thus may serve as modern therapy in the treatment of AD. Therefore, there is a
need to identify BCHE inhibitors. In current study, we focused to study a series of benzamide
derivatives for BCHE enzyme inhibition in vitro.
2

Benzamide derivatives have been reported for a variety of pharmacological activities, such as
acetylcholinesterase [16], antimicrobial, antioxidant [17], chymotrypsin [18], antiasthmatic [19],
anti-leukotriene [20] and anti-HIV [21] activities, etc. Currently, a number of potent drugs are
available that possess amide functionality as a key structural element, such as aripiprazole
(antipsychotic), phenacetin (analgesic and antipyretic) and atorvastatin (lipid-lowering drug) [22-
24]. Beside other biological properties, this class is also identified as potent cholinesterase
inhibitors. Gao and co-workers have reported benzamide and policnamide analogues as selective
ACHE inhibitors [16]. Darvesh et al. have reported alkyl and aryl derivatives of phenothiazine as
selective BCHE inhibitors [25, 26]. We have designed a series of benzamide derivatives, and
studied their effect on activity and selectivity (BCHE: ACHE) by varying properties of molecule,
such as hydrolytic properties via adding two chloro groups at N-aryl part, and changing
substituents at acyl part, of benzamide moiety (Fig. 1).
H
N
N
N
O
O
Benzamide derivatives
H
N
N
Changing electronics by
substituting dif f erent
Functional groups
O
Changing hydrolytic
properties
O
Policnamide derivatives
Cl
H
N
R
Selective AChE inhibitors
O
Cl
R
O
Designed
R = alkyl or aryl
Benzamide derivatives
N
S
Phenothiazine derivatives
Selective BChE inhibitors
Fig. 1. Designing of benzamide analogues.
Various methods have been reported on amide synthesis, but base-catalyzed reaction of amines
or anilines with benzoyl chlorides is the common reported strategy. Most of the amidation
3

reactions make use of hazardous solvents [dichloromethane (DCM), toluene, and tetrahydrofuran
(THF)] and toxic bases (sodium hydride, and tertiary amines), which add disadvantages to the
method and decreased its utility [27]. Green synthetic methodologies are considered as safe
alternates to the traditional strategies. Their environmentally benign conditions and efficiency
increased the utility of these methods. Microwave-Assisted Organic Synthesis (MAOS) is one of
the considerable green routes by Gedbye and Giguere/Majetich in 1986 [28]. Short reaction time,
simpler and easier workups, high yield, environmentally benign, highly efficient, selective, and
clean reactions are important attributes of this protocol [29-31]. This method has been emerged
as an effective synthetic strategy for many transformations, such as etherification, esterification,
hydrolysis, rearrangement, and addition reactions, etc. [32-37]. It is also an efficient method in
peptide synthesis, material sciences, biochemical processes, polymer chemistry, and
nanotechnology [38-46]. Based on the efficiency of this method, a series of benzamide
derivatives of 2,6-dichloroaniline was prepared using solvent-free and base-free MAOS
methodology with the goal of identifying potential BCHE inhibitors. For this, the inhibition
potential of newly synthesized compounds against ACHE and BCHE enzymes, their Structure-
Activity Relationship, enzyme kinetic parameters and molecular docking studies was
investigated.
2. Experimental
2.1. Material and methods
All chemicals were of analytical grade. Butrylcholinesterase (BCHE) (equine serum) (Sigma
Aldrich), Acetylcholinesterase (ACHE) (electric eel) (Sigma Aldrich), 5,5`-Dithiobis(2-
nitrobenzoic acid) (DTNB) (D218200) (Sigma Aldrich), butrylthiocholine chloride (1866-16-6),
acetylthiocholine iodide (6050-81-3) and sodium dihydrogen phosphate (NaH2PO4) (543840)
were bought from Sigma Aldrich. Galantamine hydrobromide (65789) was isolated from
commercially available tablets, donepezil (Sigma Aldrich), and 2,6-dichloroaniline (820430)
were obtained from Merck, Acros organics, Alfa Aesar, and Sigma Aldrich, respectively. At 300
MHz and 400 MHz, and using appropriate deuterated solvents, NMR spectra were recorded on
spectrometer (Bruker). EI- and ESI-Mass spectra were recorded on a Finningan MAT-321A
(Germany). Infrared (IR) spectra were obtained on Bruker vector 22 spectrometer (United
States).
4

2.2. Synthesis of benzamide derivatives 1-12
General Procedure: 0.5 mmol of benzoyl chlorides and 1.0 mmol of 2,6-dichloroaniline was
flushed with nitrogen gas in microwave (MW) vial. At 130 °C and 200 W, reaction was
irradiated under microwave for 15 mins. The mixture was washed with bicarbonate solution and
5% EtOAc: n-hexanes to obtain benzamide derivatives 1-12.
2.2.1. N-(2,6-Dichlorophenyl) benzamide (1) [47]
Yield: 95%; white solid; IR υ cm-1 (KBr disk): 3239, 1655 (amide), 1570, 1515; 1H-NMR: (400
MHz, DMSO-d6): δ 10.26 (s, 1H, NH), 8.00 (d, 2H, J2',3'/6',5' = 7.6 Hz, H-2', H-6'), 7.52-7.64 (m,
5H, H-3, H-5, H-3', H-5', H-4'), 7.40 (t, 1H, J4,(3,5) = 8.0 Hz, H-4); ESI MS: m/z : 269 [M+H+4]+,
267 [M+H+2]+, 265 [M+H]+.
2.2.2. 2-Bromo-N-(2,6-dichlorophenyl)benzamide (2) CAS # 10282-64-1
Yield: 80%; white solid; IR υ cm-1 (KBr disk): 3184, 1671 (amide), 1575, 1514; 1H-NMR: (400
MHz, DMSO-d6): δ 10.42 (s, 1H, NH), 7.74 (d, 1H, J2',3' = 8.0 Hz, H-5'), 7.57-7.61 (m, 3H, H-3,
H-5, H-3'), 7.52 (t, 1H, J4',(3',5') = 6.8 Hz, H-4'), 7.44 (dd, 1H, J5',4' = 7.6 Hz, J5',2' = 1.6 Hz, H-5'),
7.40 (t, 1H, J4,(3,5) = 8.4 Hz, H-4); ESI MS: m/z : 348 [M+H+4]+, 346 [M+H+2]+, 344 [M+H]+.
2.2.3. 4-Bromo-N-(2,6-dichlorophenyl)benzamide (3) CAS # 282091-65-0
Yield: 95%; white solid; IR υ cm-1 (KBr disk): 3221, 1651 (amide), 1578, 1511; 1H-NMR: (300
MHz, DMSO-d6): δ 10.36 (s, 1H, NH), 7.92 (d, 2H, J2',3'/6',5' = 8.4 Hz, H-2', H-6'), 7.75 (d, 2H,
J3',2'/5',6' = 8.4 Hz, H-3', H-5'), 7.58 (d, 2H, J3,4/5,4 = 8.4 Hz, H-3, H-5), 7.37 (t, 1H, J4,(3,5) = 8.1 Hz,
H-4); ESI MS: m/z: 348 [M+H+4]+, 346 [M+H+2]+, 344 [M+H]+.
2.2.4. N-(2,6-Dichlorophenyl)-4-fluorobenzamide (4) CAS # 330196-46-8
Yield: 69%; white solid; IR υ cm-1 (KBr disk): 3225, 1658 (amide), 1615, 1529; 1H-NMR: (300
MHz, DMSO-d6): δ 10.29 (s, 1H, NH), 8.04 (dd, 2H, J2',3'/6',5' = 8.7 Hz, J2',6'/6',3' = 5.7 Hz, H-2', H-
6'), 7.57 (d, 2H, J3,4/5,4 = 8.4 Hz, H-3, H-5), 7.32-7.39 (m, 3H, H-4, H-3', H-5'); ESI MS: m/z: 288
[M+H+4]+, 286 [M+H+2]+, 284 [M+H]+.
2.2.5. N-(2,6-Dichlorophenyl)-2,4-difluorobenzamide (5) CAS # 948649-43-2
5

Yield: 63%; white solid; IR υ cm-1 (KBr disk): 3225, 1658, 1615 (amide), 1529; 1H-NMR: (400
MHz, DMSO-d6): δ 10.27 (s, 1H, NH), 7.77-7.83 (m, 1H, H-6'), 7.60 (d, 2H, J3,4/5,4 = 8.4 Hz, H-
3, H-5), 7.38-7.46 (m, 2H, H-3', H-4), 7.25 (td, 1H, J5′,(4′,6′) = 8.4 Hz, J5′,(3′,6′) = 1.6 Hz, H-5'); ESI
MS: m/z: 306 [M+H+4]+, 304 [M+H+2]+, 302 [M+H]+.
2.2.6. N-(2,6-Dichlorophenyl)-2,6-difluorobenzamide (6) CAS # 937635-92-2
Yield: 90%; white solid; IR υ cm-1 (KBr disk): 3196, 1662 (amide), 1627, 1526; 1H-NMR: (400
MHz, DMSO-d6): δ 10.69 (s, 1H, NH), 7.55-7.63 (m, 3H, H-3, H-5, H-4'), 7.41 (t, 1H, J4,(3,5)
=
8.0 Hz, H-4), (t, 2H, J3′,(2′,4′)/5′,(4′,6′) = 8.0 Hz, H-3', H-5');ESI MS: m/z: 306 [M+H+4]+, 304
[M+H+2]+, 302 [M+H]+.
2.2.7. N-(2,6-Dichlorophenyl)-3-methoxybenzamide (7) CAS # 199726-36-8
Yield: 72%; white solid; IR υ cm-1 (KBr disk): 3203, 1647 (amide), 1594, 1512; 1H-NMR: (400
MHz, DMSO-d6): δ 10.26 (s, 1H, NH), 7.60 (d, 3H, J3,4/5,4/4',5' = 8.0 Hz, H-3, H-5, H-4'), 7.53 (s,
1H, H-2'), 7.45 (t, 1H, J5',(6',4') = 8.0 Hz, H-5'), 7.40 (t, 1H, J4,(3,5) = 8.4 Hz, H-4), 7.18 (dd, 1H,
J6',5' = 8.0 Hz, J6',2' = 2.0 Hz, H-6'), 3.83 (s, 3H, OCH3-3'); ESI MS: m/z: 300 [M+H+4]+, 298
[M+H+2]+, 296 [M+H]+.
2.2.8. N-(2,6-Dichlorophenyl)-4-methoxybenzamide (8) CAS # 157491-16-2
Yield: 75%; white solid; IR υ cm-1 (KBr disk): 3251, 1650 (amide), 1560; 1H-NMR: (400 MHz,
DMSO-d6): δ 10.10 (s, 1H, NH), 7.99 (d, 2H, J3',2'/5',6' = 8.4 Hz, H-3', H-5'), 7.58 (d, 2H, J3,4/5,4
=
8.0 Hz, H-3, H-5), 7.38 (t, 1H, J4,(3,5) = 8.4 Hz, H-4), 7.08 (d, 2H, J2',3'/6',5' = 8.8 Hz, H-2', H-6'),
3.85 (s, 3H, OCH3-4'); ESI MS: m/z: 300 [M+H+4]+, 298 [M+H+2]+, 296 [M+H]+.
2.2.9. N-(2,6-Dichlorophenyl)-3,4,5-trimethoxybenzamide (9) CAS # 198009-97-1
Yield: 74%; white solid; IR υ cm-1 (KBr disk): 3287, 1670 (amide), 1588, 1490; 1H-NMR: (400
MHz, DMSO-d6): δ 10.20 (s, 1H, NH), 7.60 (d, 2H, J3,4/5,4 = 8.4 Hz, H-3, H-5), 7.42 (t, 1H,
J4,(3,5)= 8.4 Hz, H-4), 7.35 (s, 2H, H-1', H-6'), 3.85 (s, 6H, OCH3-3', OCH3-5'), 3.73 (s, 3H,
OCH3-4');; EI MS m/z (% rel. abund.): 355 (M++2, 16), 355 (M+, 24), 195 (100).
2.2.10. N-(2,6-Dichlorophenyl)-2-ethoxybenzamide (10) CAS # 314055-43-1
6

Yield: 57%; white solid; IR υ cm-1 (KBr disk): 3298, 1655 (amide), 1600, 1503; 1H-NMR: (400
MHz, DMSO-d6): δ 9.86 (s, 1H, NH), 7.78 (dd, 1H, J5',4' = 7.6 Hz, J5',2' = 1.6 Hz, H-5'), 7.56 (d,
2H, J3,4/5,4 = 8.4 Hz, H-3, H-5), 7.52 (t, 1H, J4',(3',5') = 8.0 Hz, H-4'), 7.37 (t, 1H, J4,(3,5) = 8.0 Hz,
H-4), 7.21 (d, 1H, J2',3' = 8.0 Hz, H-2'), 7.07 (t, 1H, J3',(5',4') = 8.0 Hz, H-3'), 4.21-4.26 (m, 2H,
CH2-6'), 1.41 (t, 3H, CH3-6'); ESI MS: m/z: 314 [M+H+4]+, 312 [M+H+2]+, 310 [M+H]+.
2.2.11. N-(2,6-Dichlorophenyl)-3-nitrobenzamide (11) CAS # 351155-08-3
Yield: 83%; white solid; IR υ cm-1 (KBr disk): 3225, 1652 (amide), 1571, 1524; 1H-NMR: (300
MHz, DMSO-d6): δ 10.71 (s, 1H, NH), 8.79 (s, 1H, H-2), 8.43-8.47 (m, 1H, H-4'), 8.42 (d, 1H,
J6',5' = 7.8 Hz, H-6'), 7.84 (t, 1H, J5',(4',6') = 8.1 Hz, H-5'), 7.60 (d, 2H, J3,4/5,4 = 8.1 Hz, H-3, H-5),
7.39 (t, 1H, J4,(3,5) = 7.9 Hz, H-4); ESI MS: m/z: 315 [M+H+4]+, 313 [M+H+2]+, 311 [M+H]+.
2.2.12. N-(2,6-Dichlorophenyl)-4-methyl-3-nitrobenzamide (12) CAS # 351492-12-1
Yield: 98%; white solid; IR υ cm-1 (KBr disk): 3229, 1652 (amide), 1570, 1519; 1H-NMR: (400
MHz, DMSO-d6): δ 10.60 (s, 1H, NH), 8.60 (s, 1H, H-2'), 8.23 (dd, 1H, J6′,5′ = 8.0 Hz, J6′,2′ = 1.2
Hz, H-6'), 7.72 (d, 1H, J5′,6′ = 8.4 Hz, H-5'), 7.62 (d, 2H, J3,4/5,4 = 8.0 Hz, H-3, H-5), 7.42 (t, 1H,
J4,(3,5) = 8.4 Hz, H-4), 3.33 (s, 3H, CH3); ESI MS: m/z: 329 [M+H+4]+, 327 [M+H+2]+, 325
[M+H]+.
2.3. BCHE inhibition assay
To determine the mean inhibitory concentrations (IC50), and kinetic mechanisms of the effective
compounds, Ellman’s assay was performed with slight modifications [48]. All reactions were
performed in 96-well microplates, and the reaction volume was kept as 200 µL. BCHE enzyme
(51.732 mU/, 20 µL) was incubated individually with test compounds (0.5 mM) (dissolved in
methanol) in 150 µL of 100 mM sodium phosphate buffer (pH 8.0), and incubated for 15
minutes at 25 °C. The reaction was initiated by adding 10 µL of pre-prepared butyrylthiocholine
chloride (0.5 mM) substrate and in the dark for 15 minutes, and DTNB (0.5 mM), which is a
chemical reagent used to identify thiol groups in the sample was added and plate was read at
412nm in spectrophotometer.
2.4. ACHE inhibition assay
7

ACHE Inhibitory activity was performed with slight modification of Ellman’s assay [48]. All
reactions were performed in 96-well microplates, and the reaction volume was kept as 200 µL.
ACHE enzyme (0.002 mU/, 20 µL) was incubated individually with test compounds (0.5 mM)
(dissolved in methanol) in 150 µL of 100 mM sodium phosphate buffer (pH 8.0), and incubated
for 15 minutes at 25 °C. The reaction was initiated by adding 10 µL of pre-prepared
acetylthiocholine iodide (0.5 mM) substrate and DTNB in the dark for 15 minutes, and plate was
read at 412 nm in spectrophotometer.
2.5. Cytotoxicity assay
MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay was
used to perform cytotoxicity of all compounds in 96 well flat-bottomed micro plates. Assay was
initiated with the addition of 100 μL of media with 3x105 cells. All the cells got attached to the
surface of plate. Media was removed after 24 hours with the addition of fresh media (180 μL)
and 20 μL of test compound (1 to 30 μM). The plate was placed under carbon dioxide incubator
(5%) with 90% humidity at 37 °C for 48 hours. After incubation, MTT dye (200 μL) was added.
The plates were then incubated for 4 hours. Living cells form formazan crystals which were
dissolved by adding 100 μL of DMSO [49]. Absorbance was measured at 540 nm. EZ-Fit
software was used to calculate IC50 values.
2.6. Molecular docking studies
To understand the receptor-ligand interactions of selected butrylcholinesterase inhibitors,
molecular docking studies were performed using Maestro Schrӧdinger2018-1. Crystal structure
human butyrylcholinesterase in complex with the substrate analog butyrylthiocholine (1P0P) was
used for the docking studies. Protein Preparation Wizard using OPLS3 force field was employed
to prepare the receptor and in order to add missing hydrogens. Ligprep module was used to
prepare the ligands in correct protonation and ionization states [50, 51].
Since most of the inhibitors showed mixed type inhibition, they were docked in active site of
butyrylcholinesterase. For the few uncompetitive inhibitors, sitemap was used to generate
possible allosteric sites. Among them, the top ranked site was used for the docking of these
8

ligands. The receptor grid in both the allosteric and active sites has dimensions of 15Ǻ x 15Ǻ x
15Ǻ, and ligands were docked using extra precision mode [52, 53].
3. Result and discussion
3.1. Chemistry
Different substituted benzamides 1-12 were synthesized by reacting acid chlorides with 2,6-
dichloroaniline (Scheme-1).
Cl
MW, 130 oC
15 min
O
NH2
H
N
R
Cl
Cl
Cl
+
R
O
57-98%
Cl
1-12
R = -H, -F, -Br, -OCH3, -OC2H5, -NO2
Scheme-1. Synthesis of benzamide derivatives 1-12.
For optimization, several conditions were used, as shown in Table-1. Initially, reaction was done
using different solvents, such as DCM and THF, with triethylamine (NEt3) as a base. Results
revealed that reaction was completed after overnight stirring and desired benzamide was
obtained in 20-21% yields, with anhydride formation as a major side product (entry 1-2).
Refluxing reaction to 50 °C using NEt3 as base and THF as solvent, decreased reaction time,
however, no appreciable change in amount of desired product was obtained (entry 3). Poor yields
and use of hazardous solvents, switched our attention towards solvent-free reaction protocol. For
this, varying temperatures (80 °C, 100 °C and 130 °C) were used to explore best solvent-free
condition in the presence of NEt3 as a base. No appreciable change in % yield was observed;
however, time of reaction is reduced with increase in temperature (entry 4-6). Switching to
solvent-free as well as base-free conditions slightly vary time and amount of product (entry 7). In
above given conditions, one equivalent of acid halide and 2 equivalents of aniline were used.
Reversing equivalences of two reacting partners and heating at 130 °C in solvent-free and base
(NEt3)-free conditions didn’t show any appreciable change. Besides, anhydride was obtained in
all conditions, which was very difficult to remove from product. Due to less yield, unwanted side
product formation and purification via column chromatography, MW-assisted solvent-free and
9

base-free conditions were explored for the synthesis of benzamide. Interestingly, these
conditions provide benzamide product in 75% yield within 0.25 h (15 mins) at 130 °C (entry 8).
No anhydride formation as side product was observed and precipitates of product (benzamide)
were easily separated by filtration. For additional insights into scope of this protocol, a series of
benzamide derivatives 1-12 was prepared in 57-98% yields (Table-2). All the compounds were
previously reported [47], but present study is the first report of benzamides (1-12) using MW-
assisted organic synthesis.
Table-1: Optimization of benzamide derivative 1.
TEMPERATURE
ENTRY BASE SOLVENT
TIME
% YIELD
(%)
(h)
(°C)
1
2
3
4
5
6
7
8
NEt3
NEt3
NEt3
NEt3
NEt3
NEt3
-
DCM
room temperature
16
16
5
21
20
24
15
17
20
15
75
THF
room temperature
THF
50
80
-
-
-
-
-
4
100
3
130
1.5
2
130
-
130/MW
0.25
3.2. Structure-activity relationship
Benzamide derivatives 1-12, substituted with different functional groups (-H, -Br, -F, -OCH3, -
OC2H5, and -NO2) at various positions, were evaluated for their BCHE inhibitory activity
10

(Table-2). Our parent compound 1 (IC50 = 1.3 ± 0.9 µM) was found to be tenfold potent than the
standard donepezil (IC50 = 8.4 ± 0.34 µM). Therefore, we studied the effect of various electron
donating and accepting substitutions on benzamide moiety. Among the halogen derivatives (2-6),
it was observed that -Br at ortho and para position of compound 2 (IC50 = 1.1 ± 1.7 µM) and 3
(IC50 = 0.8 ± 0.6 µM), respectively, showed excellent BCHE inhibitory activity. However,
activity was decreased when -Br group at para position (compound 3) was replaced with -F
group in compound 4 (IC50 = 16.1 ± 1.2 µM). The substitution of additional ortho -F group at
para position in compound 5 (IC50 = 18.7 ± 1.2 µM), showed a slight decrease in activity, as
compared to compound 4. In contrast, activity was further reduced while switching two -F
groups to two ortho positions in compound 6 (IC50 = 12.6 ± 0.2 µM). Comparison of methoxy
substitution at meta position in compound 7 (IC50 = 5.5 ± 0.5 µM) exhibited good activity than
the standard donepezil (IC50 = 8.4 ± 0.34 µM). However, methoxy substitution at para position
of compound 8 (IC50 = 11.3 ± 1.6 µM) and trimethoxy substitution in compound 9 (IC50 = 20.8 ±
0.6 µM) showed slight decline in the activity as compared to compound 7. In contrast, the ethoxy
substitution in compound 10 (IC50 = 6.0 ± 0.5 µM) showed significant activity as compared with
standard galantamine hydrobromide (IC50 = 40.83 ± 0.37 µM). A marked decline in activity
occurred due to the presence of methoxy and -NO2 groups at para and meta positions,
respectively, of compound 12 (IC50 = 165.0 ± 2.0 µM). However, activity was completely lost
due to the presence of a -NO2 at para position in compound 11.
Benzamide derivatives were also evaluated for their ACHE inhibitory activity. None of the
compounds showed inhibitory activity against ACHE enzyme. Therefore, synthesized
compounds displayed selective inhibition against BCHE enzyme.
Table-2: ChE inhibitory activities of benzamide derivatives 1-12.
Cl
H
N
Ar
O
Cl
IC50 (µM) ± S.E.M.
BCHE ACHE
% YIELD
COMPOUND
Ar
% INHIBITION
11

1
2
3
100.0
88.0
84.0
1.3 ± 0.9*
1.1 ± 1.7*
0.8 ± 0.6*
inactive
inactive
inactive
75
80
95
Br
Br
4
5
86.5
80.7
16.1 ± 1.2
18.7 ± 1.2
inactive
inactive
69
63
F
F
F
6
7
70.6
95.2
12.6 ± 0.2
5.5 ± 0.5*
inactive
inactive
90
72
F
F
OCH3
8
90.7
11.3 ± 1.6
inactive
75
OCH3
12

9
69.16
20.8 ± 0.6
inactive
74
H3CO
OCH3
OCH3
10
11
112.2
6.0 ± 0.5*
inactive
inactive
inactive
57
83
OC2H5
-
NO2
NO2
12
69.1
165 ± 2.0
inactive
98
CH3
Donepezil
8.4 ± 0.37
2.8 ± 0.7
Galantamine
hydrochloride
40.83 ± 0.37
3.25 ± 0.3
* More potent as compared to standard (Donepezil): IC50 = 8.4 ± 0.37 µM.
3.3. Cytotoxicity
Cytotoxicity evaluation of benzamide derivatives 1-12 against 3T3 cell lines and cycloheximide
as standard, showed them as non-cytotoxic compounds (Fig. 2).
13

CYTOTOXICITY
95%
95%
95%
95%
95%
95%
95%
95%
95%
95%
95%
95%
49%
48%
41%
41%
40%
34%
34%
33%
32%
29%
15%
14.80%
7
1
2
3
4
5
6
8
9
10
11
12
COMPOUND (percent inhibition)
CYCLOHEXAMIDE(percent inhibition)
*Standard (Cycloheximide) = IC50 = 0.8 ± 0.2 µM
Fig. 2. Cytotoxicity evaluation of benzamide derivatives 1-12.
3.4. Kinetic parameters of inhibition by benzamide derivatives
Kinetics studies of most active compounds were performed to study the mechanism of inhibition
(Table-3). Inhibitor’s effect was studied by plotting reciprocal of the reaction rate against the
reciprocal of substrate concentrations as Lineweaver-Burk plot. Secondary replots of
Lineweaver-Burk plot (slope vs concentration of inhibitor) and Dixon plots (1/Vmax vs inhibitor
concentration) were used to confirm the modes of inhibition. Various kinetic parameters were
analyzed such as Km (substrate concentration at which enzyme velocity is half of the maximum),
and Vmax (maximum velocity of the enzyme). Compounds 7 and 9 are shown below as
representative examples (Fig. 3 and 4). Compound 1, 3-4, and 7-8 showed a mixed-type of
inhibition, as apparent Km increased while apparent Vmax decreased. This mode is obtained
when inhibitor can bind to the active site as well as to enzyme-substrate complex. On contrary,
compounds 2, 5, 6, and 9 showed uncompetitive inhibitor as their apparent Vmax and Km
decreased.
14

Table-3: Kinetic studies of benzamide derivatives.
Compound IC50 (Mean ± S.E.M µM) Ki (µM) Type of Inhibition
1
2
3
4
5
6
7
8
9
1.3 ± 0.9*
1.1 ± 1.7*
0.8 ± 0.6*
16.1 ± 1.2*
18.7 ± 1.2*
12.6 ± 0.2*
5.5 ± 0.5*
11.3 ± 1.6*
20.8 ± 0.6*
4.4 ± 0.3
2.4 ± 0.8
2.6 ± 0.7
9.9 ± 0.1
27.6 ± 0.4
5.6 ± 0.8
13.0 ± 0.2
18.0 ± 0.4
23.5 ± 0.2
Mixed
Uncompetitive
Mixed
Mixed
Uncompetitive
Uncompetitive
Mixed
Mixed
Uncompetitive
Galantamine
hydrochloride
(Standard)
40.83 ± 0.37
21.5 ± 0.7
Mixed
15

OCH3
Cl
H
N
O
Cl
N-(2,6-Dichlorophenyl)-3-methoxybenzamide (7)
Figure-3: Graphical representation of kinetic parameters and inhibition of
butrylcholinesterase activity by compound 7.
Graph (A) shows Line-weaver-Burk plot, Inhibitor concentrations = 36, 32,
28, and 24 µM.
▼= control
Graph (B) shows secondary replot of Lineweaver-Burk plot
Graph (C) shows Dixon plot
16

Cl
O
O
O
N
H
CH3
CH3
Cl
O-CH3
N-(2,6-Dichlorophenyl)-3,4,5-trimethoxybenzamide (9)
Figure-4: Graphical representation of kinetic parameters and inhibition of
butrylcholinesterase activity by compound 9.
Graph (A) shows Line-weaver-Burk plot, Inhibitor concentrations = 36, 32,
28, and 24 µM.
▼= control
Graph (B) shows secondary replot of Lineweaver-Burk plot
Graph (C) shows Dixon plot
17

3.5. Molecular docking studies of benzamide derivatives
Molecular docking studies were carried out to understand the interaction of active ligands with
the amino acid residues of butrylcholinesterase. Crystal structure of human butyrylcholinesterase
in complex with the substrate analog butrylthiocholine (1P0P) was used. The structure of BCHE
is well characterized using X-ray studies. It consists of an acyl pocket (Val288, and Leu286), a
catalytic triad (Glu325, Ser198, and Hip438), an oxyanion hole (Gly116, Gly117, and Ala199),
an anion site (Trp82, Tyr128, and Phe329), and a peripheral anionic site (Asp70, and Tyr332)
(Fig. 5) [54].
Acyl Phosphate
O
xy
an
io
Pe
ri
n
H
ph
er
al
A
ni
on
Pe
ri
ph
er
al
A
ni
on
ic
Si
te
A
ni
on
ic
Si
Fig. 5. Active site of human butrylcholinesterase (1P0P) indicating the catalytically important
amino acid residues.
18

The standard inhibitor galantamine hydrobromide was docked into the active site of the enzyme.
The standard inhibitor was able to form hydrogen bonds with Hip438 and Asp70. Both the
interacting amino acid residues are catalytically important as Hip438 is part of the catalytic triad,
while Asp70 belongs to the peripheral anionic site.
Fig. 6. 3D Representation of docked pose of galantamine hydrobromide (IC50 = 40.83 ± 0.8
µM). Two hydrogen bonds are shown with Asp70 and Hip438.
Compounds 1, 3-4, and 7-8 were also found to be mixed type inhibitors in our kinetic studies, so
they were docked into the active site. The parent compound 1 with no substitution on benzamide
moiety showed hydrogen bonding with Glu197 (Fig. 7). Compound 3 with Br group at para
position showed H-bond with Pro285, while the aromatic ring was involved in π-π stacking
interactions with Phe329 in the anionic site (Fig. 8). The replacement of Br with flouro group in
compound 4 slightly changed the docked pose of this compound and it formed H-bond with
HIP438 in the peripheral anionic site (Fig. 9).
19

Compound 7 with OCH3 group at meta position also showed H-bond with HIP438 in the
peripheral anionic site (Fig. 10). Although the change in position of OCH3 group from meta to
para in compound 8 showed H-bond with Pro285. The π-π stacking interaction was also
observed with Phe239 (Fig. 11).
Fig. 7. 3D Representation of docked pose of compound 1 (IC50 = 1.3 ± 0.9 µM). Two
hydrogen bonds are shown with Glu197.
20

Fig. 8. 3D Representation of docked pose of compound 3 (IC50 = 0.8 ± 0.6 µM). A hydrogen
bond is shown with Pro285 (black dotted lines) and π-π stacking interaction with Phe329
(blue dotted line).
21

Fig. 9. 3D Representation of docked pose of compound 4 (IC50 = 16.1 ± 1.2 µM). A
hydrogen bond is shown with Hip438.
22

Fig. 10. 3D Representation of docked pose of compound 7 (IC50 = 5.5 ± 0.5 µM). Compound
7 interacted with Hip438 via H-bonding.
23

Fig. 11. 3D Representation of docked pose of compound 8 (IC50 = 11.3 ± 1.6 µM).
Compound 8 interacted with Pro285 and Phe329 via H-bonding (black dotted lines) , and π-
π stacking interactions (blue dotted lines), respectively.
The uncompetitive inhibitors (2, 5, 6, and 9) interacted with the amino acid residues of the
allosteric sites (identified via sitemap analysis). For instance, compound 2 with Br group at ortho
position showed H- bonds with Asn228 and Asp304 (Fig. 12). Compound 5 with diflouro
substitution at ortho and para positions showed H-bonds with Asn228 and Thr-523 (Fig. 13).
The difluro substitutions at two ortho positions in compound 6, decreased the three hydrogen
bonds (in compound 5) to only one with Asn228 (Fig. 14). While the H-bond with Thr523 was
retained. Compound 9 with tri-methoxy substitutions at ortho, meta, and para positions showed
H-bond with Asn228 and Tyr396 (Fig. 15).
Fig. 12. 3D Representation of docked pose of compound 2 (IC50 = 1.1 ± 1.7 µM). The ligand
showed H-bonds with Asn228 .
24

Fig. 13. 3D Representation of docked pose of compound 5 (IC50 = 18.7 ± 1.2 µM). Three
hydrogen bonds are shown with Asn228 and one with Thr523.
25

Fig. 14. 3D Representation of docked pose of compound 6 (IC50 = 12.6 ± 0.2 µM). Hydrogen
bonds are shown with Asn228 and Thr523.
26

Fig. 15. 3D Representation of docked pose of compound 9 (IC50 = 20.8 ± 0.6 µM). Hydrogen
bonds are shown with Asn228 and Tyr396.
4. Conclusions
A series of benzamide derivatives 1-12 was designed, synthesized using solvent-free and base-
free MAOS methodology and evaluated for their BCHE inhibition. SAR study showed that
compounds with halogen substitution had more potent inhibition activity as compared with
methoxy, ethoxy and nitro substituted ones. Kinetics and molecular docking studies identified
the binding sites of selected inhibitors. The significant inhibitors showed interactions with
catalytically important amino acid residues of BCHE such as Glu197, Phe329, Hip438 and many
others. Current treatment of Alzheimer`s disease is based on cholinesterase enzyme inhibition
(palliative treatment). Although this is not the ultimate cure and other aspect of
pathophysiological mechanism need to be considered, but currently anti-cholinesterase’s
medicines seem to be the only viable option of alleviating the symptoms of AD.
Acknowledgements
Authors are thankful to the Higher Education Commission, Pakistan for providing financial
assistance. The authors would like to thank Dr. Rasheeda Swaleh for her constructive criticism in
the manuscript.
27

References
1.
Holtzman DM, Morris JC, Goate AM. Alzheimer’s disease: the challenge of the second
century. Science translational medicine. 2011;3(77):77sr1-sr1.
2.
Khan MA, Fazal-ur-Rehman S, Hameed A, Kousar S, Dalvandi K, Yousuf S, Choudhary
MI, Basha FZ. Regioselective synthesis of novel 2, 3, 4, 4 a-tetrahydro-1 H-carbazoles and their
cholinesterase inhibitory activities. RSC Advances. 2015;5(73):59240-50.
3.
of gerontology and geriatrics. 2009;49:237-43.
4. Aranda-Abreu GE, Hernández-Aguilar ME, Manzo Denes J, García Hernández LI,
Zanetti O, Solerte S, Cantoni F. Life expectancy in Alzheimer's disease (AD). Archives
Herrera Rivero M. Rehabilitating a brain with Alzheimer's: a proposal. Clin Interv Aging.
2011;6:53-9. Epub 02/22. doi: 10.2147/CIA.S14008. PubMed PMID: 21472092.
5.
burden of Alzheimer’s disease. Alzheimer's & dementia. 2007;3(3):186-91.
6. Asojo OA, Ngamelue M, Homma K, Lockridge O. Cocrystallization studies of full-
Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM. Forecasting the global
length recombinant butyrylcholinesterase (BChE) with cocaine. Acta Crystallographica Section
F: Structural Biology and Crystallization Communications. 2011;67(4):434-7.
7.
amygdala and hippocampal formation. Journal of Comparative Neurology. 1998;393(3):374-90.
8. Darvesh S, Hopkins DA, Geula C. Neurobiology of butyrylcholinesterase. Nature
Reviews Neuroscience. 2003;4(2):131.
9. Liston DR, Nielsen JA, Villalobos A, Chapin D, Jones SB, Hubbard ST, Shalaby IA,
Darvesh S, Grantham D, Hopkins D. Distribution of butyrylcholinesterase in the human
Ramirez A, Nason D, White WF. Pharmacology of selective acetylcholinesterase inhibitors:
implications for use in Alzheimer's disease. European journal of pharmacology. 2004;486(1):9-
17.
10.
Xue L, Ko M-C, Tong M, Yang W, Hou S, Fang L, Liu J, Zheng F, Woods JH, Tai H-H.
Design, preparation and characterization of high-activity mutants of human butyrylcholinesterase
specific for detoxification of cocaine. Molecular pharmacology. 2010:mol. 110.068494.
11.
Masson P, Froment M-T, Fortier P-L, Visicchio J-E, Bartels CF, Lockridge O.
Butyrylcholinesterase-catalysed hydrolysis of aspirin, a negatively charged ester, and aspirin-
related neutral esters. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular
Enzymology. 1998;1387(1-2):41-52.
28

12.
Mesulam M-M, Guillozet A, Shaw P, Levey A, Duysen E, Lockridge O.
Acetylcholinesterase knockouts establish central cholinergic pathways and can use
butyrylcholinesterase to hydrolyze acetylcholine. neuroscience. 2002;110(4):627-39.
13.
Perry EK, Perry RH, Blessed G, Tomlinson BE. Changes in brain cholinesterases in
senile dementia of Alzheimer type. Neuropathology and applied neurobiology. 1978;4(4):273-7.
Epub 1978/07/01. PubMed PMID: 703927.
14.
Darreh-Shori T, Modiri N, Nordberg A. ApoE and amyloid beta deflate the cholinergic
neurotransmission by boosting the activity and stability of cholinesterases in the brain.
Alzheimer's & Dementia: The Journal of the Alzheimer's Association. 2009;5(4):P305. doi:
10.1016/j.jalz.2009.04.463.
15.
Birks J. Cholinesterase inhibitors for Alzheimer’s disease. Cochrane Database Syst Rev.
2006;1(1):CD005593.
16.
Gao X-h, Liu L-b, Liu H-r, Tang J-j, Kang L, Wu H, Cui P, Yan J. Structure–activity
relationship investigation of benzamide and picolinamide derivatives containing dimethylamine
side chain as acetylcholinesterase inhibitors. Journal of enzyme inhibition and medicinal
chemistry. 2018;33(1):110-4.
17.
Yang X, Peng T, Yang Y, Li W, Xiong J, Zhao L, Ding Z. Antimicrobial and antioxidant
activities of a new benzamide from endophytic Streptomyces sp. YIM 67086. Natural product
research. 2015;29(4):331-5.
18.
Lum RT, Nelson MG, Joly A, Horsma AG, Lee G, Meyer SM, Wick MM, Schow SR.
Selective inhibition of the chymotrypsin-like activity of the 20S proteasome by 5-methoxy-1-
indanone dipeptide benzamides. Bioorganic & medicinal chemistry letters. 1998;8(3):209-14.
19.
Ashton MJ, Cook DC, Fenton G, Karlsson J-A, Palfreyman MN, Raeburn D, Ratcliffe
AJ, Souness JE, Thurairatnam S, Vicker N. Selective Type IV Phosphodiesterase Inhibitors as
Antiasthmatic Agents. The Syntheses and Biological Activities of 3-(Cyclopentyloxy)-4-
methoxybenzamides and Analogs. Journal of Medicinal Chemistry. 1994;37(11):1696-703. doi:
10.1021/jm00037a021.
20.
Goto S, Guo Z, Futatsuishi Y, Hori H, Taira Z, Terada H. Quantitative structure-activity
relationships of benzamide derivatives for anti-leukotriene activities. Journal of medicinal
chemistry. 1992;35(13):2440-5.
29