Design, synthesis and biological evaluation of benzofuran appended benzothiazepine derivatives as inhibitors of butyrylcholinesterase and antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2018.02.049

A series of bezofuran appended 1,5-benzothiazepine compounds 7a–v was designed, synthesized and evaluated as cholinesterase inhibitors. The biological assay experiments showed that most of the compounds displayed a clearly selective inhibition for butyrylcholinesterase (BChE), while a weak or no effect towards acetylcholinesterase (AChE) was detected. All analogs exhibited varied BChE inhibitory activity with IC₅₀ value ranging between 1.0 ± 0.01 and 72 ± 2.8 μM when compared with the standard donepezil (IC₅₀, 2.63 ± 0.28 μM). Among the synthesized derivatives, compounds 7l, 7m and 7k exhibited the highest BChE inhibition with IC₅₀ values of 1.0, 1.0 and 1.8 μM, respectively. The results from a Lineweaver-Burk plot indicated a mixed-type inhibition for compound 7l with BChE. In addition, docking studies confirmed the results obtained through in vitro experiments and showed that most potent compounds bind to both the catalytic anionic site (CAS) and peripheral anionic site (PAS) of BChE active site. The synthesized compounds were also evaluated for their in vitro antibacterial and antifungal activities. The results indicated that the compounds possessed a broad spectrum of activity against the tested microorganisms and showed high activity against both gram positive and gram negative bacteria and fungi.

Full text of DOI:10.1016/j.bmc.2018.02.049

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Benzofuran appended Benzo-
thiazepine Derivatives as Inhibitors of Butyrylcholinesterase and Antimicrobial
Agents
Manizheh Mostofi, Ghodsi Mohammadi Ziarani, Alireza Foroumadi, Mehdi
Khoobi, Negar Lashgari
PII:
S0968-0896(17)32409-4
https://doi.org/10.1016/j.bmc.2018.02.049
BMC 14235
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
14 December 2017
17 February 2018
27 February 2018
Please cite this article as: Mostofi, M., Mohammadi Ziarani, G., Foroumadi, A., Khoobi, M., Lashgari, N., Design,
Synthesis and Biological Evaluation of Benzofuran appended Benzothiazepine Derivatives as Inhibitors of
Butyrylcholinesterase and Antimicrobial Agents, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
1
0.1016/j.bmc.2018.02.049
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Design, Synthesis and Biological Evaluation of Benzofuran appended
Benzothiazepine Derivatives as Inhibitors of Butyrylcholinesterase and
Antimicrobial Agents
a
a
b
b
Manizheh Mostofi , Ghodsi Mohammadi Ziarani *, Alireza Foroumadi , Mehdi Khoobi and
a
Negar Lashgari
a
Department of Chemistry, Alzahra University, Vanak Square, P.O. Box 1993891176, Tehran, Iran
b
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medicinal Sciences, Tehran, Iran
E-mail: gmziarani@hotmail.com gmohammadi@alzahra.ac.ir
Abstract:
A series of bezofuran appended 1,5-benzothiazepine compounds 7a-v was designed, synthesized
and evaluated as cholinesterase inhibitors. The biological assay experiments showed that most of
the compounds displayed a clearly selective inhibition for butyrylcholinesterase (BChE), while a
weak or no effect towards acetylcholinesterase (AChE) was detected. All analogs exhibited varied
BChE inhibitory activity with IC50 value ranging between 1.0 ± 0.01-72 ± 2.8 µM when compared
with the standard donepezil (IC50, 2.63±0.28 µM). Among the synthesized derivatives, compounds
7
l, 7m and 7k exhibited the highest BChE inhibition with IC50 values of 1.0, 1.0 and 1.8 µM,
respectively. The results from a Lineweaver-Burk plot indicated a mixed-type inhibition for
compound 7l with BChE. In addition, docking studies confirmed the results obtained through in
1

vitro experiments and showed that most potent compounds bind to both the catalytic anionic site
CAS) and peripheral anionic site (PAS) of BChE active site. The synthesized compounds were
(
also evaluated for their in vitro antibacterial and antifungal activities. The results indicated that the
compounds possessed a broad spectrum of activity against the tested microorganisms and showed
high activity against both gram positive and gram negative bacteria and fungi.
Keywords: Alzheimer's disease; Cholinesterase inhibitors; Specific butyrylcholinesterase
inhibitor, Benzofuran; Benzothiazepine, Molecular docking
1
. Introduction
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two important types of
cholinesterase species. They are mainly responsible for the hydrolysis of acetylcholine (ACh) into
choline and acetic acid which is an essential process allowing for the control of the cholinergic
1
2
transmission. Low levels of ACh is a key pathological hallmark of Alzheimer’s disease (AD).
AD is the leading cause of dementia and is characterized by a progressive decline in cognitive
3
,4
function, which typically begins with deterioration in memory. In a healthy brain, ACh is
predominantly (80%) hydrolyzed by AChE, whereas BChE plays a supplementary role. However,
with progression of AD, the AChE activity decreases, whereas the activity of BChE gradually
5
,6
increases. This phenomenon enhances the significance of BChE as an additional therapeutic
7
,8
target for reducing the cholinergic deficiency inherent in AD. Currently, AD therapy is mainly
founded on cholinesterase inhibitors, which are able to increase ACh levels in cholinergic
9
synapses. Recent studies have demonstrated that BChE inhibition results in improved cognitive
2

potential with elevated levels of ACh in brain and hence, it may act as an effective therapeutic
1
0-13
strategy for AD.
,5-Benzothiazeoines are considered privileged scaffolds in drug discovery for cardiovascular and
neurodegenerative diseases. 1,5-Benzothiazepine derivatives have a broad spectrum of therapeutic
1
14
+2
15
16,17
applications as coronary vasodilator,
Ca
channel antagonists,
antidepressant,
1
8
19
20-22
acetylcholinesterase inhibitors, butyrylcholinesterase inhibitors, and antimicrobial agents.
Benzofurans, a groups of naturally occurring substances in many plants, exhibit a wide range of
2
3,24
biological activities.
Benzofuran scaffold has emerged as an important pharmacophore for
2
5,26
27,28
designing antiviral
and antimicrobial agents
and inhibitors of cyclin-dependent kinases
2
9
30,31
(
CDKs) and cholinesterase.
Combination of 1,5-benzothiazepine and benzofuran moiety
exhibited synergistic effect thereby, enhancing their potency.
In the light of above-mentioned findings, and as a continuation of our endeavor to identify new
candidates that might be advantageous in designing new, potent, selective, and less toxic
3
2-35
cholinesterase inhibitors,
we have reported the synthesis of 1,5-benzothiazepine derivatives
containing benzofuran fragments at C-4 position. All synthesized compounds were screened for
their ability to inhibit the enzyme activities of BChE and their in vitro antimicrobial activity. To
better understand the enzyme inhibition mechanisms, in relation to the substituents and their
positions in the presented compounds, molecular modeling studies were also performed.
2
. Results and Discussion
.1. Chemistry
2
The synthesis of 1,5-benzothiazepine derivatives 7a-v was outlined in Scheme 1. First, 2-
acetylbenzofuranes 3a-d were prepared from the ring closure reaction of salicylaldehyde
3

derivatives 1a-d and chloroacetone 2 (Table 1). Then, condensation of 2-acetylbenzofuranes 3a-d
with various benzaldehydes 4a-k under microwave irradiation in the presence of catalytic amount
of piperidine gave α,β-unsaturated carbonyl compounds 5a-v (Table 2). Finally, the thia Michael
addition and further cyclocondensation reaction of compounds 5a-v with 2-aminothiophenol 6 was
accomplished in refluxing ethanol in the presence of catalytic amount of glacial acetic acid to
afford designed compounds 7a-v in good to excellent yields (70-90%). The generality of this
sequence was examined by using different salicylaldehyde derivatives in the first step and (het)aryl
aldehydes in the second step. It was found that substrates containing various functional groups
yielded the corresponding products 7a-v in good to excellent yields.
Scheme 1. General scheme for the synthesis of 2,3-dihydro-1,5-benzothiazepine derivatives 7a-v.
4

Table 1. Synthesis of 2-acetylbenzofuranes 3a-d from chloroacetone and salicylaldehydes.
M.P. [Lit.]
Entry
Salicylaldehyde
Product
Time (h)
Yield (%) M.P. (ºC)
(ºC)
3
6
1
4
72
75
74-76
76
3
6
2
3
4
3
4
5
109-111
110
91
O
O
OCH3
36
78
65
91-93
3
c
36
174-176
175
Table 2. Synthesis of chalcones 5a-v from 2-acetylbenzofuran and benzaldehydes.
2
-Acetyl
Benzaldeh
yde
Time Yield
(min) (%)
M.P
M.P.
Entry
Chalcone
benzofurane
(ºC) [Lit.] (ºC)
5

3
7
1
2
3
20
20
20
65
80
88
250-252 254
H
O
OCH3
38
39
40
119-122 118-120
4c
170-173
New
H
O
4
20
20
73
72
172-175 178-181
OCH3
4e
5
188-200
New
6
20
25
87
77
165-168 159-160
O
O
O
OCH3
O
OCH3
7
228-231
New
3c
5g
6

O
O
OCH3
8
9
25
15
15
83
70
65
194-197
New
3
c
41
149-151 145-147
10
192-195
New
1
1
1
2
15
15
80
78
287-289
285-288
New
New
4
2
1
3
15
15
69
68
285-287 286
Br
O
O
14
279-281
New
5n
CH3
7

H
O
15
15
75
287-291
New
F
4h
Br
O
O
41
1
1
6
7
15
15
87
80
203-206 202-204
O N
2
5p
223-226
230-233
New
New
H
O
1
8
N
15
87
H
4
k
1
2
2
9
0
1
30
30
30
60
54
72
221-223
230-232
184-187
New
New
New
8

22
30
75
236-238
New
As an initial attempt for optimization of the reaction conditions, the effect of various protic and
aprotic solvents such as acetonitrile, toluene, dichloromethane, methanol, and ethanol for the
cyclocondensation reaction of compound 5i with 2-aminothiophenol 6 was evaluated (Table 3).
The results clearly showed that among the different tested solvents, the best result was obtained
using absolute ethanol under reflux condition which led to the formation of the corresponding
product 7i in high yield (80%).
Table 3. The effect of solvent on the synthesis of 2,3-dihydro-1,5-benzothiazepine 7i.
Entry
Solvent
MeOH
Conditions
r.t.
Time (h)
Yield (%)
1
2
3
4
5
24
8
20
50
30
80
25
MeOH
Reflux
r.t.
Absolute EtOH
Absolute EtOH
24
2
Reflux
60 °C
2
Dry CH Cl
2
24
9

6
7
8
Dry Toluene
Dry CH CN
DMF
Reflux
Reflux
80 °C
6
6
6
45
35
50
3
Then, evaluation of various catalytic systems for the formation of target compound 7i was carried
out. It was observed that in the absence of any catalyst, the reaction did not entirely proceed.
Several catalysts such as conc. HCl, trifluoroacetic acid (TFA), p-toluene sulfonic acid (p-TSA),
ammonium chloride (NH3.HCl) and piperidine were applied in this reaction but the reaction time
and product yields were not appropriate (Table 4, entries 1-5). The most encouraging result was
obtained in the presence of 0.1 equivalent of glacial acetic acid as catalyst for the preparation of
the model compound 7i. Increasing the amount of glacial acetic acid accompanied by the
temperature did not lead to an increase in product yields (Table 4, entries 6-8).
Table 4. The optimization of reaction condition for the synthesis of 2,3-dihydro-1,5-benzothiazepine 7i.
Entry
Catalyst
Conc. HCl
p-TSA
No. Eq.
0.1
Time (h) Yield (%)
1
2
3
4
5
6
7
8
5
6
5
5
6
2
2
2
35
40
55
30
35
80
80
80
0.1
TFA
0.1
3
NH .HCl
0.1
Piperidine
0.1
Glacial AcOH
Glacial AcOH
Glacial AcOH
0.1
0.2
0.3
1
0

Then, in regard to library construction, we extended our study with 22 different substituted
chalcones under optimized reaction condition. All the reactions underwent smoothly and provided
corresponding 1,5-benzothiazepines 7a-v in high yield (Table 5).
Table 5. Synthesis of 1,5-benzothiazepines 7a-v from 2-aminothiophenol and chalcone derivatives.
Time
Yield
(%)
M.P.
(ºC)
Entry
Chalcone
Product
(
h)
1
3
79
72
80
237-239
176-178
138-140
H3CO
O
N
S
3
3
3
2
3
4
7b
O
O
H3CO
5
c
90
176-178
1
1

O
5
6
7
8
N
S
3
3
3
3
86
80
80
89
189-191
225-227
227-229
247-249
NH
O
N
S
7
f
OH
H3CO
O
N
S
7h
9
2
80
189-191
1
2

Br
OH
O
1
1
1
0
1
2
2
2
2
70
75
73
253-255
165-167
179-181
N
S
7
j
Br
OCH3
O
13
2
75
181-183
N
S
7m
Br
O
O
14
2
72
175-177
5n
CH3
1
3

Br
F
O
15
2
83
221-223
N
S
7o
Br
O2N
O
16
2
72
175-177
N
S
7
p
1
1
1
7
8
9
2
2
4
85
80
90
197-199
245-247
222-224
Br
NH
O
N
S
7r
O2N
O
O
5s
1
4

O2N
F
O
2
2
2
0
1
2
4
4
4
78
73
85
138-140
223-224
228-230
N
S
7t
O2N
O N
O
2
NH
O
O
N
S
N
5
v
H
7v
A plausible mechanism for the synthesis of compounds 7a-v is proposed as illustrated in Scheme
. The mechanism is most likely to be involved in the intermolecular hydrogen bonding promoted
2
by acetic acid that activates chalcone towards the nucleophilic attack by sulfur in 2-
aminothiophenol to afford the enol intermediate A. Then, 1,3-hydrogen shift occurs to give an
4
3
isomeric keto B that cyclizes to yield the seven-membered ring product.
1
5

R2
HS
R2
CH3
O
H2N
CH COOH
3
R1
6
O C
H
R1
O
O
O
O
5
a-v
R2
O
R2
H C C
3
R2
O
O H
H
R1
O
O
O
R1
S
H N
2
S
O
R1
O H N
2
H N
S
2
A
B
R2
R2
O
H C C O
3
S
-
H O
2
-
H
R1
H
O
S
R1
O
N
O
N
H
7
a-v
Scheme 2. Proposed mechanism for the preparation of benzothiazepine derivatives.
2
.2. Butyrylcholinesterase inhibition and preliminary SAR studies
We designed and synthesized 22 benzofuran-appended benzothiazepine analogues and determined
44
their ability to inhibit BChE using Ellman spectrophotometric method. Structurally, the designed
compounds can be divided into two series: benzofuran-benzothiazepines with substituents on
benzofuran scaffold and benzofuran-benzothiazepines with substituents on aryl ring. According to
the enzyme inhibition data, as depicted in Table 6, most of the target compounds showed potent
BChE inhibition activity with IC50 values within micro molar ranges. As observed, only
compounds 7e, 7f and 7v exerted a weak inhibitory activity towards BChE, while all other
1
6

compounds, except compounds 7p and 7s, inhibited BChE enzymatic activity with a varying
efficiency (Table 6). In particular, compounds 7l, 7m and 7k showed the lowest IC50 values of 1.0,
1
.0, and 1.8 µM, respectively, while IC50 of standard donepezil hydrochloride is 2.63 µM. Thus,
these compounds were chosen for the kinetic studies.
In the compounds with pendant phenyl group at C-2 position of benzothiazepine, the presence of
4
-methoxy group on the phenyl ring was favorable for inhibitory activity. It was also revealed that
cholinesterase inhibition activity of the target compounds depends largely to the steric and
electronic features of the substituents. For example, a twofold reduction in the activity was
observed by the movement of methoxy group from position 2 in compound 7k to position 4 in
compounds 7m. This fact was supported by the docking studies. It can be likely related to the
greater extension of the aromatic system of the phenyl, which could better interact with Trp82
through stacking interaction. Based on docking studies, presence of methoxy group at ortho
position can disrupt the π–π stacking interactions via rotation of the phenyl ring as in compounds
7
k and 7b. Movement of methoxy group to position meta as in compound 7l and 7c have therefore
resulted in better activity due to proper stacking of the pendant phenyl group with Trp82. On the
other hand, the activity would slightly improve when the methoxy group or fluoro-substitution
shift to the para position. It is assumed that the steric hindrance of substituents with amino acids at
the bottom of BChE gorge may be responsible for this effect. Furthermore, the activity of target
compounds is very sensitive to the size of the substituent at para position. The IC50 values of 4-
substituted phenyl analogs 7m, 7n and 7o revealed that the methoxy group on para-position of
phenyl ring is more favorable than methyl or fluoro substituents. Thus, compounds such as 7m and
7
d having bulky groups at para position showed stronger activity. It is worth noting here that in
case of 7b, 7c and 7d, removing 5-bromo substiuent in benzofuran ring, led to a large decrease
1
7

approximately four fold in the activity of the target compounds (IC50 = 4.0-4.8 µM). To study the
effects of more hydrophilic substituents on benzofuran ring, some compounds bearing methoxy
group on 7 position and nitro group on 5 position of benzofuran ring were synthesized.
Introduction of 5-nitro substituent on the benzofuran ring resulted in inactive compound 7s. In
contrast, 7-methoxy substitution could improve the activity as observed in compound 7g.
Methoxy-substituted benzofurans showed favorable, but lower activity than bromo-substituted
benzofurans whereas nitro-substituted benzofurans showed a significant weak activity. Indeed, the
introduction of a methoxyl group on the benzofuran ring (7g vs 7i) resulted in a four-fold increase
of the activity.
As it could be observed from Table 6, the size of the substituent on the pendant phenyl group,
regardless of its electronic properties, is important in order to show considerable activity. Larger
substituents have therefore exhibited better activities in comparison to those with the smaller size.
The anticholinesterase activity was in the order: OCH > OH> F > H according to the Van der
3
Waals radii, respectively. For R groups with similar volumes other factors should be considered,
such as hydrogen bond acceptor/donor properties, PSA (polar surface area) and the extension of
the aromatic system. This hypothesis was confirmed by docking studies. Astonishing results for
BChE inhibition were obtained after synthesis of compounds 7p and 7s. The data showed that
compounds with more electron-withdrawing substituent on position 3 of pendant phenyl group or
on position 5 of benzofuran moiety exhibited no activity (compounds 7p and 7s).
Structurally, the most designed compounds possess phenyl ring which enables the molecule to
make additional π-π interaction with aromatic or hetero-aromatic amino acid residues of enzyme
binding site. On the other hand, the phenyl moiety was selected as it could establish hydrophobic
and π-π interactions with the aromatic residues of PAS. Furthermore, in order to obtain
1
8

information about the structural requirements for optimally targeting BChE, the phenyl ring was
replaced with other aromatic and hetero-aromatic groups with increasing bulkiness. In particular,
the phenyl ring was replaced by sterically demanding aromatic substituents: 1-naphthyl and 3-
indolyl substitutions (compounds 7e and 7v). Interestingly, inhibition of BChE by the 1-naphthyl
and 3-indolyl substituted compounds 7e and 7v dropped to IC50 of 63 and 72 µM, respectively.
The replacement of the phenyl ring (7b) with the indolyl substitution (7f) reduced the inhibitory
activity of 12.5-fold. Because of these observations, compounds 7q and 7u were prepared carrying
a 1-naphthyl moiety in position C-2 of benzothiazepine as well as compounds 7f and 7r holding
each a 3-indolyl moiety in position C-2 of benzothiazepine. Unfortunately, these compounds gave
only minor inhibition of BChE. The bulkiness of the aromatic group seems to play a main role in
BChE inhibition. On the other hand, the indole and naphthalene moieties were selected as they
could establish hydrophobic and π-π interactions with the aromatic residues of PAS.
R1
R2
O
N
S
7a-v
Table 6. Inhibitory activities of benzofuran linked benzothiazepines 7a-v against BChE.
BChE inhibition,
1
2
R
Entry Compound
R
+
IC50 ± SD (µmol/L)
1
2
3
7a
7b
7c
H
H
H
4-OH
2-OCH
3-OCH
14±0.71
4.8±0.13
4.1±0.14
3
3
1
9

4
5
6
7
8
9
7d
7e
H
H
4-OCH
3
4.0±0.12
63±2.6
1-Naphthyl
3-Indolyl
H
7f
H
60±2.4
7g
7-OCH
7-OCH
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
3
2.5±0.12
4.1±0.14
8.5±0.43
6.5±0.35
1.8±0.06
1.0±0.01
1.0±0.01
8.5±0.43
13.7±0.6
Inactive
17±0.9
7h
3
4-OH
H
7i
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
7j
4-OH
1
1
1
1
1
1
1
1
1
2
2
2
2
7k
2-OCH
3-OCH
4-OCH
3
3
3
7l
7m
7n
4-CH
3
7o
4-F
7p
3-NO
2
7q
1-Naphthyl
3-Indolyl
H
7r
28±1.38
Inactive
22±1.13
55±2.1
7s
5-NO
5-NO
5-NO
5-NO
2
2
2
2
7t
2-F
7u
1-Naphthyl
3-Indolyl
7v
72±2.8
Donepezil
2.63±0.28
IC50 values are at least from three independent experiments and are expressed as the means ± SD.
2
.3. Kinetic study of BChE inhibition
2
0

To determine the mechanism of enzyme inhibition, the kinetic study was performed using 7l as
representative compound. The relative velocity of the enzyme was determined on three increasing
concentrations (0, 0.5, 1.0 and 2.0 µM) of the substrate BTChI (butyrylthiocholine iodide). The
Lineweaver-Burk plot was then schemed using the reciprocal of velocity (1/v) versus reciprocal of
substrate concentration (1/[S]) (Figure 1, left). Based on the obtained plot, a mixed-type of
inhibition was established for compound 7l. Using the Lineweaver-Burk secondary plot of slope
against concentration of 7l (Figure 1, right), Ki value was determined equal to 0.27 µM.
Figure 1. Left: Lineweavere-Burk plot for the inhibition of BChE by compound 7l at different
concentrations of substrate (BTChI), Right: Secondary plot for calculation of steadystate inhibition constant
(Ki) of compound 7l.
2
.4. Ligand-protein docking study
In order to study the binding mode of the synthesized compounds in active site of BChE, docking
studies were performed using Auto Dock Tools (version 1.5.6). The results of SAR show that most
potent compounds have a methoxy group on 2, 3, or 4-position of the 2-phenyl ring and a bromine
atom on the 5-position of benzofuran. Therefore, the docking studies were performed on the most
2
1

active compounds 7k, 7l, and 7m and their hypothetical analogs with two methoxy groups on 2-
phenyl ring.
In the compound 7k, the benzothiazepine moiety created a hydrophobic interaction with important
4
5,46
amino acid Trp82 in catalytic anionic site (CAS).
Also, this moiety interacted with peripheral
anionic site (PAS) via a π–anion interaction with Asp70. 2-Phenyl ring created a weak
hydrophobic interaction with Pro84. Bromine atom of compound 7k interacted with Trp231,
Leu286, and Val288 via hydrophobic interactions (Figure 2A).
Movement of methoxy substituent into 3 or 4-position (compounds 7l and 7m) caused small
increase of potency in comparison with 2-methoxy inhibitor 7k. In compound 7l, benzothiazepine
and benzofuran moieties interacted with amino acids Trp82 (CAS), Tyr332 (PAS), Ala328,
phe329, Leu286, Val288, and Trp231 (Figure 2B). In addition, 2-phenyl ring of this compound
formed a π-π interaction with Trp82. As can be seen in Figure 2, compound 7m established all the
interactions mentioned above, but instead of interaction of 2-phenyl ring with Trp82 (Figure 2B), a
hydrogen bond formed between 4-methoxy substituent of this ring with NH unit of Gly121(Figure
2
C).
Hypothetically, introduction of another methoxy substituent on 2-phenyl ring in most potent
compounds, with the exception of two cases (2,4-dimethoxy and 3,5-dimethoxy compounds), does
not significantly change the interaction of compounds with the active site (Figure 3). Interaction
mode of 2,3-dimethoxy compound exactly is similar to 3-methoxy compound 7l (Figures 3A and
2
B). 2,4-Dimethoxy shows all the interactions of 4-methoxy derivative 7m, but did not establish π-
π interaction with important amino acid Tyr332 (PAS) (Figures 3B and 2C). Interaction mode of
,5-dimethoxy compound is almost similar to that of 3-methoxy compound 7l (Figures 3C and
2
2
2

2
B). 3,4-Dimethoxy hypothetical derivative shows all interactions that there is between 3 and 4-
methoxy compounds 7l and 7m with active site of BChE (Figures 3D, 2B, and 2C).
Figure 2. Binding mode of the most potent compounds (A) 7k, (B) 7l, and (C) 7m in the enzyme active
site.
2
3

Figure 3. Binding mode of the hypothetical compounds (A) 2,3-dimethoxy , (B) 2,4-dimethoxy, and (C)
,5-dimethoxy (D) 3,4-dimetoxy and (E) 3,5-dimethoxy analogs of compounds7k, 7l, and 7m in the
enzyme active site.
2
2
.5. Antibacterial activity
2
4

All target compounds 7a-v were screened in vitro for their antibacterial activity against three
gram-positive bacterial strains [Bacillus subtilis (ATCC 6633), Staphylococcus epidermidis (ATCC
1
2228) and Staphylococcus aureus (ATCC 29737)] and six gram-negative bacterial strains
Salmonella paratyphi-A serotype (ATCC 5702), Pseudomonas aeruginosa (ATCC 27853),
Klebsiella pneumoniae (ATCC 10031), Shigella dysenteriae (PTCC 1188), Proteus vulgaris
PTCC 1182) and Escherichia coli (ATCC 10536)] using disk diffusion method. Rifampicin and
[
(
Gentamicin were used as reference drugs.
Tables 7 and 8 represent the zone of inhibition (mm) and minimum inhibitory concentrations
-1
(
MICs, µg mL ) obtained for some target compounds. In general, the higher susceptibilities (lower
MICs) were observed with gram-positive bacteria and poorer susceptibilities with gram-negative
bacteria. The results of survey indicated that compounds 7e, 7o, 7p, 7q, 7t and 7u were active
against gram-positive and gram-negative bacterial strains and among them, compound 7u (IZ 30
mm) was the most active derivative against S. epidermidis. This increased potency may be due to
the presence of naphthalene moiety on 2 position of benzodiazepine ring and introduction of NO
group into the benzofuran ring in this compound. Analysis of the Structure-Activity Relationship
SAR) also revealed that replacement of H atom by halogen or nitro groups on aryl ring at 2
2
(
position of benzothiazepine enhanced antimicrobial activity. With respect to gram negative
bacteria, compounds 7p (IZ 23 mm), and 7t (IZ 23 mm) with nitro or fluoro substituents on phenyl
ring at 2 position of benzothiazepine caused a remarkable increase in antimicrobial activity against
S. paratyphi and P. vulgaris, respectively.
Table 7. Antibacterial activity data of tested compounds against Gram positive bacteria.
Test microorganism
2
5

S. aureus (ATCC 29737)
B. subtilis (ATCC 6633) S. epidermidis (ATCC 12228)
Sample
IZ
MIC
IZ
MIC
IZ
MIC
7
e
10
10
-
5.0
5.0
-
-
-
-
9
5.0
5.0
-
7o
7p
7q
-
9
-
19
12
-
12.5
5.0
10
17
20
10
21
5.0
2.5
0.25
0.25
0.50
-
-
7
t
-
10
30
40
35
5.0
0.25
0.25
0.50
7
u
15
13
21
0.125
0.125
0.50
Rifampicin
Gentamicin
Notes: A dash (–) indicates no antimicrobial activity. NA
-1
MIC - minimum inhibitory concentrations (µg mL )
IZ - Inhibition zone (mm)
Table 8. Antibacterial activity data of tested compounds against Gram negative bacteria.
Test microorganism
S.
E. coli
P. aeruginosa
K. Pneumoniae S. dysenteriae P. vulgaris
Sample
paratyphi
IZ MIC
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC IZ MIC
7
7
7
o
p
q
11
50
-
-
-
-
15
-
50
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
23 0.50
-
-
-
-
-
-
-
-
14
-
50
-
19 12.5
23 0.125
7
t
12
50
-
-
2
6

7
u
11
-
50
-
-
-
-
-
-
-
-
-
11
7
50
-
8
-
-
-
Rifampicin
11
20
0.50
0.50
0.25
0.25
0.25 10 0.125
0.50 23 0.50
Gentamicin 21 0.50
22
18
Notes: A dash (–) indicates no antimicrobial activity. NA
-1
MIC - minimum inhibitory concentrations (µg mL )
IZ - Inhibition zone (mm)
2
.6. Antifungal activity
The antifungal activity of some seven-membered ring products against yeasts [Candida albicans
ATCC 10231)] and filamentous fungi [Aspergillus niger (ATCC 16404) and Aspergillus
(
brasiliensis (PTCC 5011] were evaluated in vitro. Antifungal activity was expressed in terms of
MIC values and the zone of inhibition (in mm) in comparison with standard drug Nystatin (Table
9
). Among the tested compounds, compounds 7e, 7o, 7p, 7q, 7t and 7u showed good biological
activities against fungi C. albicans, A. niger and A. brasiliensis. The results showed that
compounds 7p and 7t were the most potent anti-fungal agents.
By comparing the zone of inhibition values of compounds it could be concluded that the halogen
and nitro substitutions had positive effect in some cases depends on its position, significantly
improve antifungal activity. Compounds 7o with para-substituent and 7t with ortho-substituent
fluoro on phenyl rings were activate against C. albicans. Furthermore, in compound 7p the
introduction of m-NO group to the aryl ring caused a significant increase in antifungal activity
2
against A. niger (IZ = 20 mm) and A. brasiliensis (IZ = 23 mm). In compounds 7e, 7q and 7u, the
presence of naphthalene moiety on 2 position of benzodiazepine ring resulted in better activity
against C. albicans (IZ = 13-14 mm).
2
7

Table 9. Antifungal activity data of tested compounds.
Test microorganism
C. albicans
A. niger
A. brasiliensis
IZ MIC
Sample
IZ
MIC
IZ
MIC
7
e
14
13
-
25
25
-
-
-
-
-
-
7o
7p
7q
-
-
-
20
0.25
23
-
0.50
14
19
13
33
25
-
-
-
-
7
t
0.125
2.5
-
-
-
-
-
7
u
-
-
Nystatin
0.125
27
0.25
23
0.50
Notes: A dash (–) indicates no antimicrobial activity. NA
-1
MIC - minimum inhibitory concentrations (µg mL )
IZ - Inhibition zone (mm)
3
. Conclusion
In summary, twenty-two substituted 1,5-benzothiazepine derivatives bearing benzofuran moiety
have been synthesized and fully characterized. The synthetic pathway was quick and effective. All
compounds were evaluated in vitro for their ability to inhibit AChE and BChE and the resulting
products showed good levels of inhibition against BChE. In particular, compounds 7l, 7m, and 7k
expressed the highest BChE-inhibiting activities. Kinetic analysis studies revealed that compound
7
l features a mixed-type inhibition activity on BChE. Furthermore, molecular modeling studies
indicate that compounds 7l, 7m, and 7k are able to bind to both CAS and PAS in BChE. In
addition, the antibacterial activities of compounds have been evaluated on a panel of pathogenic
Gram+ and Gram- bacteria and the results showed that most of compounds exhibited moderate to
2
8

good activity against the tested microorganisms. Considering these overall results, our findings
could be extended to design and develop new potentially selective BChE inhibitors containing
benzofuran moieties as potential agents for the treatment of AD.
4
. Experimental section
.1. General information
4
All commercially available reagents were purchased from Merck AG, Aldrich or Acros Organics
and used without further purification. Column chromatography was carried out on silica gel (70–
2
30 mesh). TLC was conducted on silica gel 250 micron, F254 plates. For the synthesis of
compounds 7 the experiments were performed using a microwave oven (ETHOS 1600, Milestone)
with a power of 300 W specially designed for an organic synthesis and modified with a condenser
and mechanical stirrer. Melting points were measured on a Kofler hot stage apparatus and are
uncorrected. The IR spectra were taken using Nicolet FT-IR Magna550 spectrographs (KBr disks).
Mass spectra of the products were obtained with an HP (Agilent technologies) 5937 Mass
1
13
Selective Detector. H NMR and C NMR spectra were recorded on a Bruker Avance 300 and 500
MHz NMR instruments. The chemical shifts (δ) and coupling constants (J) are expressed in parts
per million and hertz, respectively. Elemental analyses were carried out by a CHN-Rapid Heraeus
elemental analyzer. The results of elemental analyses (C, H, N) were within ±0.4% of the
calculated values.
4
.2. General procedure for the preparation of 2-acetyl benzofuran derivatives 3a-d
A mixture of freshly distilled salicylaldehyde 1a-d (0.1 mol), chloroacetone 2 (0.1 mol) and
anhydrous potassium carbonate (0.1 mol) in ethyl methyl ketone (25 mL) was refluxed for 3-5 h.
Progress of the reaction was monitored by TLC. After completion of the reaction, the potassium
2
9