Design, synthesis and biological evaluation of new benzoxazolone/benzothiazolone derivatives as multi-target agents against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2020.113124

In this study, four series of compounds with benzoxazolone and benzothiazolone cores were designed, synthesized and evaluated as multifunctional agents against Alzheimer's disease (AD). Additionally, in order to shed light on the effect of the carbonyl groups of benzoxazolone/benzothiazolone, benzoxazole/benzothiazole-containing analogues were also synthesized and evaluated. Inhibition potency of all final compounds towards cholinesterase enzymes and their antioxidant activity were tested. Subsequently, the anti-inflammatory activity, cytotoxicity, apoptosis, and Aβ aggregation inhibition tests were also performed for selected compounds. The results indicated that compounds 11c, a pentanamide derivative with benzothiazolone core, and 14b, a keton derivative with benzothiazolone core, were considered as promising multi-functional agents for further investigation against AD. The reversibility, kinetic and molecular docking studies were also performed for the compounds with the highest AChE 14b (eeAChE IC₅₀ = 0.34 μM, huAChE IC₅₀ = 0.46 μM) and BChE 11c (eqBChE IC₅₀ = 2.98 μM, huBChE IC₅₀ = 2.56 μM) inhibitory activities.

Full text of DOI:10.1016/j.ejmech.2020.113124

European Journal of Medicinal Chemistry 212 (2021) 113124
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of new benzoxazolone/
benzothiazolone derivatives as multi-target agents against
Alzheimer’s disease
Merve Erdogan a, Burcu Kilic a, Rahsan Ilıkcı Sagkan b, Fatma Aksakal c, Tugba Ercetin d,
,
*
Hayrettin O. Gulcan d, Deniz S. Dogruer a
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara, Turkey
b Department of Medical Biology, Faculty of Medicine, Us¸ak University, Us¸ak, Turkey
c Department of Chemistry, Hacettepe University, Ankara, Turkey
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Eastern Mediterranean University, Gazimagosa, Cyprus
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, four series of compounds with benzoxazolone and benzothiazolone cores were designed,
synthesized and evaluated as multifunctional agents against Alzheimer’s disease (AD). Additionally, in
order to shed light on the effect of the carbonyl groups of benzoxazolone/benzothiazolone, benzoxazole/
benzothiazole-containing analogues were also synthesized and evaluated. Inhibition potency of all final
compounds towards cholinesterase enzymes and their antioxidant activity were tested. Subsequently,
Received 14 October 2020
Received in revised form
27 November 2020
Accepted 18 December 2020
Available online 24 December 2020
the anti-inflammatory activity, cytotoxicity, apoptosis, and A
b aggregation inhibition tests were also
performed for selected compounds. The results indicated that compounds 11c, a pentanamide derivative
with benzothiazolone core, and 14b, a keton derivative with benzothiazolone core, were considered as
promising multi-functional agents for further investigation against AD. The reversibility, kinetic and
molecular docking studies were also performed for the compounds with the highest AChE 14b (eeAChE
Keywords:
Alzheimer’s disease
Benzoxazolone
Benzothiazolone
IC50 ¼ 0.34
m
M, huAChE IC50 ¼ 0.46
m
M) and BChE 11c (eqBChE IC50 ¼ 2.98
m
M, huBChE IC50 ¼ 2.56
mM)
Cholinesterase inhibitory activity
Anti-inflammatory
Molecular docking
inhibitory activities.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
acetylcholine (ACh) deficiency, neuroinflammation, oxidative
stress, and excitotoxicity) have been well determined [3,4]. The
neurotransmitter ACh deficiency in specific regions of the brain
associated with cognitive functions results in progressive memory
and orientation loss and other cognitive impairments combined
with behavioral and neuropsychiatric disturbances. Thus, drugs
including donepezil, rivastigmine, and galantamine, which can in-
crease ACh levels through the inhibition of cholinesterase enzymes
have been developed [5,6]. However, these drugs can only slow
down the progression of cognitive function impairments. There-
fore, the research studies focus on the development of compounds
that may be effective on different pathologies of the disease along
with cholinesterase inhibitory activity [7].
Alzheimer’s disease (AD), accounting for about 70% of all de-
mentia cases, is a progressive neurodegenerative brain disease in
elderly people. The 2019 report of Alzheimer Disease International
has been estimated that there are more than 50 millionpeople living
with dementiaworldwide and theincidence of the diseasewilltriple
by 2050 because of the increase in the average life expectancy.
Accordingly, AD poses a great problem for global health [1,2].
Despite many scientific efforts, the mechanism underlying the
pathogenesis of AD is not exactly explained due to its complex and
multifactorial characteristics. However, many factors involved in
the development of AD (e.g., amyloid-
b
(Ab) plaques and hyper-
phosphorylated neurofibrillary
tangle (NFT) formations,
Recently, many studies have revealed that neuroinflammation
plays a fundamental role in AD. It has been reported that an
incessant immune response in the brain is not only associated with
neurodegeneration but it also facilitates and exacerbates both A
and NFT pathologies [8]. Furthermore, it has been suggested that
chronic neuroinflammation may be a central mechanism driving A
b
* Corresponding author.
E-mail
addresses:
dogruer@gazi.edu.tr,
denizdogruer2002@yahoo.com
b
(D.S. Dogruer).
https://doi.org/10.1016/j.ejmech.2020.113124
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
pathology and progression. In AD, a disruption in the balance of
anti-inflammatory and pro-inflammatory signals results in chronic
neuroinflammation, which is attributed to the activation of
microglia cells and the release of numerous cytokines [8,9].
Microglia, the resident immune cells within the central nervous
system (CNS), are activated once they recognize a threat to the CNS.
Several investigations have demonstrated that activated microglia
ring system, 2-phenylbenzoxazole/benzothiazole derivative-two
compounds were synthesized by replacing the carbonyl group at
the benzoxazolone/benzothiazolone rings with the phenyl ring. As
a result, a total of 39 new final compounds were synthesized,
including the propanamide derivative 28 and the 11 compounds
obtained by structural modifications of the compounds with the
highest AChE (8g, 9g) and BChE inhibitory (9l) activities (Fig. 1).
Subsequently, inhibition potency of all final compounds towards
cholinesterase enzymes and their antioxidant activity were tested.
Additionally, the anti-inflammatory activity, cytotoxicity, apoptosis,
phagocytose A
no longer able to phagocytose A
cated that their capacity to produce pro-inflammatory cytokines is
unaffected. This situation makes overall clearance of A become
b, but after prolonged activation of periods, they are
b. Nevertheless, it has been indi-
b
and Ab aggregation inhibition tests were also performed for
dangerous [10]. Moreover, the continued release of pro-
inflammatory cytokines from microglia worsens the neuro-
inflammation and contribute to neurodegeneration. Recent studies
have demonstrated that reducing the levels of inflammatory
selected compounds. The reversibility, kinetic and molecular
docking studies were also performed for the compounds with the
highest AChE and BChE inhibitory activity.
markers such as interleukin-1
b
(IL-1b
), interleukin-6 (IL-6), tumor
2. Result and discussion
necrosis factor- (TNF- ) and nitric oxide (NO) released from
a
a
stimulated microglia and other immune cells may be effective for
the treatment of AD [11e13].
2.1. Chemistry
Oxidative stress (OS) or nitrative stress (NS) is a condition
caused by increased reactive oxygen species (ROS) and reactive
nitrogen species (RNS) concentrations in cells when the antioxidant
defense system is overwhelmed. The concentrations of ROS and
RNS, steadily produced and eliminated during cellular reactions,
are the key factor of their effect. At very low concentrations, they
are known to play vital role in many biological processes such as
cell growth, cell signaling, smooth muscle relaxation, immune re-
sponses. However, OS/NS, resulting from the increased cellular
concentration of ROS/RNS, leads to the oxidation of cellular mac-
romolecules such as lipids, proteins, and DNA, which is involved in
the development of many pathophysiological conditions. Accord-
ingly, there are several experimental data supporting the idea that
OS is associated with both the initiation and progression of AD.
Thus, compounds with antioxidant activity might be useful for the
treatment of AD [14e16].
The synthetic routes of intermediates 5, 6 and final compounds
8a-n, 9a-n, 10a-c, 11a-c, 14a-c, 21a-b are presented in Schemes
1e6, respectively. Compounds 5 and 6 used as the cardinal in-
termediates in the synthesis of final compounds 8a-n, 9a-n, 10a-c,
and 11a-c were synthesized by the methylation of commercially
available 1,3-benzoxazol-2(3H)one or 1,3-benzothiazol-2(3H)-one
rings with dimethyl sulfate followed by nitration with nitric acid
and then reduction with SnCl2 [31,32]. In order to synthesize
propanamide derivatives bearing benzoxazolone ring, the inter-
mediate 5 was first acylated with 3-chloropropionyl chloride to
obtain the intermediate 7. Then, this compound was reacted with
appropriate secondary amines to get the title compounds (8a-n).
However, the propanamide derivatives carrying the benzothiazo-
lone ring (9a-n) could not be synthesized by the above method,
because of low yield. These compounds were prepared by the re-
action
of
intermediate
6
with
CAS
registered
3-
Benzoxazolone and benzothiazolone rings which are bio-
isosteres of each other have attracted attention because of their
capacity to mimic a phenol or catechol [17]. In addition, these cores
have been used as privileged scaffolds in the design of new com-
pounds due to their wide range of biological properties as well as
anti-inflammatory [18e21] and antioxidant [22] activities. More-
over, various tertiary amine moieties reported as cholinesterase
inhibitory pharmacophores were used to derivatization of the
synthesized compounds in the present study [23,24].
Since a drug with a single-target mechanism of action cannot be
effective in the treatment of complex and multifactorial diseases
such as Alzheimer’s disease, the development of multi-target
compounds that can interact with two or more targets associated
with AD pathogenesis has been adopted as a more effective treat-
ment strategy [7,25,26].
In the light of these facts and as a continuation of our research
for new anti-Alzheimer compounds [27e30], we designed and
synthesized new propanamide derivatives in which the side chain
at the 6th positions of the 3-methyl-benzoxazolone and 3-methyl-
benzothiazolone rings terminate with tertiary amine structures.
After the cholinesterase inhibitory activities of the synthesized
compounds were examined, the compounds with the highest AChE
(acetylcholinesterase) and BChE (butyrylcholinesterase) inhibitory
activities were determined. Subsequently, with the hope of
improving AChE/BChE inhibitory activity, new compounds were
designed by the structural modifications of the compounds. For this
purpose, firstly butanamide and pentanamide derivatives were
prepared to find the optimal length of the linker. Secondly, the
ketone derivatives were synthesized to evaluate the effect of the
functional group. Finally, to get a better insight into the effect of the
substitutedpropanoic acid derivatives prepared by Michael addi-
tion according to our in-house method [30]. To obtain compounds
10a-c and 11a-c in the butanamide and pentanamide series
respectively were used the following steps. First, the reaction of
ethyl 4-bromobutyrate or ethyl 5-bromovalerate with appropriate
amines gave corresponding ester derivatives which were further
hydrolyzed in %5 NaOH solution to give 4-substitutedbutanoic acid
or 5-substitutedpentanoic acid, respectively. Then, compounds
10a-c and 11a-c were prepared by the reaction of intermediate 5 or
6 with 4-substitutedbutanoic acid or 5-substitutedpentanoic acid
in the presence of EDC and DMAP, as shown in Scheme 4. For
preparing compounds 14a-c, Intermediates 12 and 13 were syn-
thesized by the Friedel-Crafts acylation reaction of 1,3-benzoxazol-
2(3H)-one/1,3-benzothiazol-2(3H)-one with 3-chloropropionyl
chloride [33]. Then, compounds 14a-c were obtained by treat-
ment of intermediate 12, 13 with sodium iodide, potassium car-
bonate, and suitable amine derivatives. In the synthesis of final
compounds 21a-b, commercially available 2-amino-5-nitrophenol
and 2-amino-5-nitrobenzenethiol (16) synthesized in our labora-
tory were used as starting materials. The nitration of 1,3-
benzothiazole with nitric acid afforded intermediate 15 [34] that
was subsequently hydrolyzed to 16 (CAS 23451-98-1) with a 20%
solution of potassium hydroxide. Intermediates 17 and 18 were
obtained from the reaction of both starting materials with benzoic
acid in PPA [35], which were further reduced with iron to provide
intermediates 19 and 20 [35]. Then, the reaction of 19 and 20 with
3-(4-benzylpiperazin-1-yl)propanoic acid in the presence of EDC
and DMAP gave 21a and 21b, respectively.
The chemical structures of recently synthesized compounds
were approved by 1H NMR, 13C NMR, HRMS, and elemental analysis.
2

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Fig. 1. The general structure of synthesized compounds.
Scheme 1. Synthesis of the compounds 5e6. Conditions and reagents: (i) Dimethyl sulfate, 1 M NaOH, 30 min, rt. (ii) %65 HNO3, 1 h, 40 ꢀC. (iii) SnCl2, 6 M HCl, EtOH, 30 min, reflux.
Scheme 2. Synthesis of the compounds 8a-n. Conditions and reagents: (i) 3-Chloropropionyl chloride, DMAP, TEA, DMF, 1 h, rt. (ii) NaI, ACN, reflux, 2 h. (iii) Amine derivative,
K2CO3, 3 h, rt.
2.2. Cholinesterase inhibitory activities
Concerning AChE inhibition of propanamide derivatives (8a-n;
9a-n), nonsubstituted benzylpiperazine-bearing compounds 8g
and 9g were more active than other compounds. Between the two
compounds, the stronger inhibitor was compound 9g with ben-
Initially, the percent inhibition of final compounds on AChE
(from electric eel) and BChE (from equine serum) were evaluated
by the modified Ellman’s method using donepezil (10
m
M) as the
zothiazolone core (IC50 ¼ 2.34
m
M) followed by compound 8g with
reference compound. Subsequently, IC50 values of final compounds
showing inhibition above 50% were calculated and the results are
reported in Table 1.
benzoxazolone core (IC50 ¼ 6.94
m
M). When the effect of the sub-
stituents at the para position of the phenyl ring of benzylpiperazine
on the activity was evaluated, it was observed that fluorine at the
3

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Scheme 3. Synthesis of the compounds 9a-n. Conditions and reagents: (i) DCM, overnight, rt. (ii) %5 NaOH, 2 h, 40 ꢀC. (iii) EDC, DMAP, DCM, overnight, rt.
Scheme 4. Synthesis of the compounds 10a-c and 11a-c. Conditions and reagents: (i) ACN, K2CO3, 6 h, reflux. (ii) %5 NaOH, 2 h, 40 ꢀC. (iii) EDC, DMAP, DCM, overnight, rt.
Scheme 5. Synthesis of the compounds 14a-c. Conditions and reagents: (i) 3-Chloropropionyl chloride, AlCl3, DMF, 2 h, 45 ꢀC. (ii) NaI, ACN, reflux, 2 h. (iii) Amine derivative, K2CO3,
3 h, rt.
para position (8h IC50 ¼ 16.10
m
M and 9h IC50 ¼ 7.09
m
M) reduced
at the side chain significantly decreased or abolished AChE inhibi-
tion of the compounds.
As for the BChE inhibition, most of the compounds displayed
negligible BChE inhibitory activity except for 9h, 9l, 9m, and 9n.
Among the active compounds, 4-phenypiperidine-bearing com-
pound 9l with benzothiazolone core displayed the highest inhibi-
tion potential with an IC50 value of 11.14 mM. In addition, none of
the compounds possessing inhibitory activity on BChE are not
benzoxazolone analogues.
inhibitor activity approximately 2.5 times, while the other sub-
stituents abolished the activity compared to their nonsubstituted
benzylpiperazine counterpart. The results showed that sub-
stitutions on the para position of the phenyl ring were not tolerated
except for fluorine substituent. Similarly, as with nonsubstituted
benzylpiperazine derivatives, 4-fluorobenzylpiperazine-carrying
compound 9h with benzothiazolone core was more effective than
the corresponding benzoxazolone derivative (compound 8h).
However, the presence of phenylpiperidine, benzylpiperidine, N-
methylbenzylamine groups, phenylpiperazine, and its derivatives
Overall, these results showed that benzothiazolone ring is not
only important for AChE inhibition but is also critical for BChE
4

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Scheme 6. Synthesis of the compounds 21a-b. Conditions and reagents: (i) H2SO4, HNO3, 45 min, 4e6 ꢀC. (ii) %20 KOH, 5 h, reflux (iii) PPA, 2 h, 140 ꢀC. (iv) Fe, 1.2 N HCl, EtOH, 2 h,
reflux. (v) EDC, DMAP, DCM, overnight, rt.
inhibition.
Furthermore,
the
benzylpiperazine
and
4-
After this incubation period, the reaction was subsequently diluted
100-fold with substrate solution to give final concentrations of
inhibitors 0.1 ꢁ IC50 and 1 ꢁ IC50, respectively. In control studies,
inhibitors were replaced with standard inhibitors and buffer. The
reaction was further incubated at 37 ꢀC for a further 15 min and the
residual enzyme activities were measured, and the results are
displayed in bar graphic in Fig. 2 and Table 2 as well. All mea-
surements were carried out in triplicate and are expressed as
mean SEM.
For reversible enzyme inhibition, the enzyme activities are ex-
pected to be recovered to the levels of approximately 90% and 50%,
respectively, after 100-fold dilution of the preincubations con-
taining inhibitor concentrations of 10xIC50 and 100xIC50 [37]. Thus,
the results obtained pointed out the reversible inhibitor character
of the title molecules under the experimental conditions employed.
phenylpiperidine structures in the propanamide series were
determined to be favorable in terms of AChE and BChE inhibition,
respectively. In this connection, compound 9g with the highest
AChE inhibition (IC50 ¼ 2.34
m
M) and compound 9l with the highest
BChE inhibition (IC50 ¼ 11.14
mM) were selected as the hit com-
pounds in the propanamide series. In addition, some final com-
pounds with benzoxazolone core were also synthesized in order to
make a comparison in terms of AChE inhibition in new series.
From this point of view, butanamide (10a-c) and pentanamide
series (11a-c) were prepared to examine the effect of the carbon
number in the side chain on inhibitory activity. In the butanamide
series, compounds 10a (IC50 ¼ 6.00
mM) and 10b (IC50 ¼ 1.96
mM)
were found to slightly increase AChE inhibition compared to
compounds in the propanamide series. However, compounds in the
pentanamide series reduced AChE inhibition. Regarding BChE in-
hibition of compounds in butanamide and pentanamide series,
2.4. Kinetic studies of enzyme inhibition
Compound 10c from the butanamide series (IC50 ¼ 9.65
more active compared to the propanamide series compound 9l
M) in the
mM) was
To determine the enzyme inhibition types and inhibition con-
stant (Ki) of the two most active molecules, enzyme kinetics studies
were carried out on compound 11c (for eqBChE) and 14b (for
eeAChE). Lineweaver-Burk plots and replots of the slope versus
concentration were utilized to obtain the inhibition constants (Ki),
as shown in Fig. 3. Accordingly, compound 11c was shown to have
mixed-type inhibition and compound 14b was shown to have
noncompetitive type inhibition. The obtained Ki values were
consistent with the measured IC50 values.
(IC50 ¼ 11.14
mM). In addition, compound 11c (IC50 ¼ 2.98
m
pentanamide series exhibited the most potent inhibition for BChE
among all series, indicating that increasing the side chain length
positively affected activity.
In the case of ketone derivatives (14a-c), compound 14b with
the benzothiazolone core possessed the highest AChE inhibitory
activity with an IC50 value of 0.34 mM among all series, indicating
that the ketone function was more suitable for AChE inhibitory
activity instead of the amide function.
Regarding compounds 21a and 21b, removal of carbonyl group
at the second position of benzoxazolone and benzothiazolone rings
abolished the cholinesterase inhibition.
Finally, the IC50 values of compound 14b with the highest
inhibitory activity against electric eel AChE (eeAChE) and compound
11c with the highest inhibitory activity against equine BChE (eqB-
ChE) were also examined against human AChE (huAChE) and human
BChE (huBChE), respectively. New results were parallel with the
results obtained from eeAChE and eqBChE (14b eeAChE
2.5. In vitro antioxidant activity
The antioxidant activities of synthesized compounds were
tested by the ORAC-FL method (Oxygen Radicals Absorbance Ca-
pacity by Fluorescence). The vitamin E analogue Trolox was used as
a standard. According to the data in Table 1, all the compounds
exhibited good ORAC-FL values within the range of 2.61e4.55
Trolox equivalents at 10 mM concentration. The results indicated
that functional group, or atom differences among the synthesized
molecules resulted in little or negligible effect on the antioxidant
effects determined under the ORAC assay conditions.
IC50
IC50 ¼ 2.98
¼
0.34
m
M, huAChE IC50
¼
0.46
M).
mM and 11c eqBChE
mM, huBChE IC50 ¼ 2.56
m
2.3. Reversibility studies of 11c and 14b for AChE/BuChE inhibition
2.6. Inhibitory effects of selected compounds on IL-1
and NO production
b, IL-6, TNF-a
A previously described dilution method was employed to test
the reversibility of enzyme inhibition observed for the most active
compounds in the series (i.e., 14b for huAChE, and 11c for huBChE)
[36]. Briefly, ten times and a hundred times of IC50 concentrations
of the inhibitors were incubated with enzyme at 37 ꢀC for 30 min.
Neuroinflammation plays a vital role in AD and other neuro-
degenerative diseases. So, preventing the production of inflam-
matory factors released from activated microglia has been
considered to be a promising strategy for the treatment of disease.
5

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Table 1
Cholinesterase inhibitory activities and ORAC results of the synthesized compounds.
Compd
X
n
R
eeAChE
eqBChE
ORAC
% inhibition SD (100
32 3.7
m
M)
IC50 SD (
n.t
m
M)
% inhibition SD (100
m
M)
IC50 SD (
n.t
mM)
8a
8b
8c
8d
8e
8f
O
O
O
O
O
O
O
O
2
2
2
2
2
2
2
2
<%10
4.02 0.008
3.85 0.005
3.95 0.006
4.03 0.009
4.28 0.007
3.90 0.004
3.78 0.008
3.92 0.004
35 3.3
30 4.2
33 3.7
29 3.3
35 3.2
81 5.2
66 4.3
n.t
n.t
n.t
n.t
n.t
<%10
n.t
n.t
n.t
n.t
n.t
n.t
n.t
<%10
<%10
<%10
<%10
8g
8h
6.94 1.06
30 2.6
21 1.8
16.10 0.99
8i
8j
O
O
2
2
40 2.1
38 3.2
n.t
n.t
<%10
<%10
n.t
n.t
4.19 0.004
4.20 0.003
8k
O
2
35 3.5
n.t
<%10
n.t
3.96 0.005
8l
O
O
2
2
25 2.7
64 2.3
n.t
<%10
n.t
n.t
4.09 0.005
3.87 0.006
8m
24.60 1.15
47 2.6
8n
O
2
58 1.1
52.40 1.21
42 3.5
n.t
4.30 0.006
9a
9b
9c
9d
9e
9f
S
S
S
S
S
S
S
S
2
2
2
2
2
2
2
2
34 3.0
37 3.2
32 3.4
35 2.8
31 3.0
36 3.1
92 2.4
74 4.1
n.t
<%10
<%10
<%10
<%10
<%10
<%10
<%10
52 0.9
n.t
n.t
n.t
n.t
n.t
n.t
n.t
4.24 0.006
4.03 0.008
4.43 0.007
4.31 0.005
4.55 0.011
4.10 0.006
4.08 0.008
4.10 0.006
n.t
n.t
n.t
n.t
n.t
9g
9h
2.34 0.61
7.09 1.09
37.60 1.84
9i
9j
S
S
2
2
37 3.2
39 3.4
n.t
n.t
<%10
<%10
n.t
n.t
4.01 0.003
4.28 0.008
9k
S
2
35 3.7
n.t
<%10
n.t
4.45 0.006
6

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Table 1 (continued)
Compd
X
n
R
eeAChE
eqBChE
ORAC
% inhibition SD (100
60 4.1
m
M)
IC50 SD (
m
M)
% inhibition SD (100
81 0.8
m
M)
IC50 SD (mM)
9l
S
S
2
2
60.30 2.24
11.14 1.07
4.31 0.007
4.01 0.009
9m
62 4.6
63 1.3
31.90 1.46
60 3.3
70 1.5
75.38 1.99
9n
S
2
49.50 1.54
41.96 1.12
3.90 0.007
10a
10b
10c
11a
11b
O
S
3
3
3
4
4
4
94 0.2
93 0.3
32 2.1
82 1.0
87 1.7
6.00 1.13
1.96 0.42
n.t
<%10
n.t
3.97 0.004
3.91 0.003
2.93 0.04
2.63 0.03
2.67 0.04
<%10
n.t
S
90 1.6
<%10
9.65 1.13
O
S
30.86 1.08
13.96 1.02
n.t
n.t
<%10
11c
14a
14b
14c
21a
S
30 2.3
97 2.3
99 0.6
81 1.2
<%10
n.t
96 0.2
<%10
2.98 1.02
2.56 0.23b
2.61 0.02
3.03 0.04
3.70 0.04
3.10 0.04
2.68 0.04
O
S
0.89 0.14
n.t
e
e
e
e
e
0.34 0.16
0.46 0.04a
<%10
n.t
S
4.32 1.18
79 0.4
<%10
13.97 1.15
n.t
O
n.t
21b
S
<%10
n.t
<%10
n.t
2.64 0.03
n.t
Donepezil
99 0.8
0.062 0.002
0.048 0.004a
88 1.4
3.55 0.07
6.4 0.025b
a
huAChE.
huBChE, n. t: not tested.
b
Fig. 2. Reversibility studies of compound 14b with AChE (a) and compound 11c with BChE (b).
Additionally, inspiring through the anti-inflammatory activity
study conducted with Donepezil by Arikawa et al. we evaluated the
anti-inflammatory activities of the most potent cholinesterase in-
hibitor compounds in the present study [38]. Thus, according to
cholinesterase inhibitory activity results, the most potent 8 com-
pounds (8g, 9g, 10a, 10b, 10c, 11c, 14a, 14b) were selected to
Table 2
AChE and BChE reversibility studies.
AChE
BChE
Donepezil
14b
Donepezil
11c
Control
0.1xIC50
1xIC50
100.0 1.4
89.0 0.9
81.6 0.6
100.0 1.4
72.2 0.7
50.6 0.7
100.0 3.1
95.4 1.2
90.2 1.0
100.0 3.1
98.4 1.5
81.5 0.5
determine their effects on inflammatory markers IL-1
b, IL-6, TNF-a,
and NO. Anti-inflammatory activities of selected compounds were
7

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Fig. 3. Lineweaver-Burk plot of 11c for BChE hydrolysis (a) and slope replot vs 11c concentration (b). Lineweaver-Burk plot of 14b for AChE hydrolysis (c) and slope replot vs 14b
concentration (d).
evaluated according to the reduction of LPS- induced IL-1
TNF- , and NO production in THP 1 cell line. As seen in Fig. 4, the
results revealed that the selected compounds inhibited the release
of IL-1 , IL-6, and TNF- as effectively as donepezil. Significantly,
the most active AChE inhibitor, 14b decreased NO production better
than donepezil. Additionally, the other compounds inhibited NO
production comparable to the effect of donepezil.
b
, IL-6,
(14b) and BChE (11c) inhibitory effects by using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. According to the results presented in Table 5, 11c and 14b
have no significant effect on cell line at their cholinesterase in-
hibitor concentrations.
a
b
a
2.10. Assessment of physicochemical parameters
2.7. Inhibition of 11c and 14b on self-induced Ab1-42 aggregation
The physicochemical properties and BBB penetration scores of
the selected compounds (8g, 9g, 10a, 10b, 10c, 11c, 14a, 14b) in our
study were calculated by employing Molinspiration and admetSAR
online services. Calculated descriptors such as molecular weight,
logP, topological polar surface area (tPSA), volume, number of
hydrogen donors, number of hydrogen acceptors and number of
violations of Lipinski’s rule and blood brain barrier (BBB) perme-
ability were presented in Table 6. According to the calculated data,
the selected compounds have a high rate of drug-likeness because
all of them following the Lipinski’s rule and their predicted BBB
penetration rates over % 90.
With respect to the cholinesterase inhibitory activity results, the
most potent two compounds (11c and 14b) were selected for A
b
aggregation inhibition assay. To investigate the effect of the
selected compounds and references (curcumin and phenol red) on
the aggregation of Ab1ꢂ42, the reported thioflavin T-based fluoro-
metric assay was performed at 50 mM concentration. The effects of
each compound on the Ab1e42 peptide self-aggregation was sum-
marized as the percent (%) inhibition data in Table 3. According to
the results, compounds 11c and 14b were found to be as active as
curcumin.
2.11. Molecular docking
2.8. Effect of 11c and 14b on apoptosis in 3T3 cells
Docking of the synthesized compounds was performed to pre-
dict the binding orientations and non-covalent interactions with
the active sites of the AChE and BChE. In case of docking with AChE,
two active compounds (9g and 14b) and inactive compound (9a)
were selected based on the experimental results on their AChE
inhibitory activities. The most energetically profitable poses of
these compounds in the AChE binding site were given in Fig. 6 with
both three-dimensional representation and two-dimensional
interaction diagram.
Annexin V/PI double staining method was employed to evaluate
the effects of compounds 11c and 14b on apoptosis in 3T3 cells at
10 mM concentration. As shown in Table 4 and Fig. 5, the results
indicated that both 11c and 14b do not have apoptotic effects.
2.9. Cytotoxicity test
Cytotoxicity test was investigated in the 3T3 cell line to deter-
mine the safety profiles of two compounds with the best AChE
Aromatic amino acids Trp84 and Phe330 at the catalytic active
8

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Fig. 4. The effect of donepezil and selected compounds on LPS-induced IL-1
b
secretion (a). The effect of donepezil and selected compounds on LPS-induced IL-6 secretion (b). The
effect of donepezil and selected compounds on LPS-induced TNF-
control).
a secretion (c). The effect of donepezil and selected compounds on LPS-induced NO secretion (d). (P < 0.05 versus
Table 3
Inhibition of A
Trp84 and Trp279 and CeH$$$
characteristics for both active compounds 9g and 14b. For the
inactive compound 9a, $$$ stacking interaction with Trp279 was
p interaction with Phe330 were
b aggregation of selected compounds.
% inhibition Ab42 self-aggregation SEMa
p
p
Compound
conserved, while interactions with other aromatic residues Trp84
and Phe330 were missing. Inactivity of compound 9a can be related
to these missing interactions. Binding free energies obtained with
docking were not significant to explain AChE inhibitory effects of
compound 9a. Other common interactions of all compounds with
AChE were water-mediated contacts, hydrogen bonding interaction
Curcumin
Phenol Red
11c
57.1 3.0
32.2 0.8
55.6 3.7
57.5 5.3
14b
a
Inhibition of Ab42 self-aggregation investigated by the thioflavin-T fluorescence
M inhibitor and 50 M Ab42
assay. Assays were carried out in the presence of 50
m
m
([I] ¼ [Ab42]); Data are the mean of two independent experiments each performed in
between Ser286 and benzothiazole sulphur, and CeH$$$
p in-
triplicate.
teractions with Phe331 and Tyr334 residues, with distances of
2.7 Åe4.4 Å. For the docking of 9g and 14b to AChE, binding affinity
values of ꢂ10.0 and ꢂ9.8 kcal molꢂ1 were obtained, respectively
with a good agreement with the experimental results. Slightly
lower binding free energy value of 14b can be attributed to the
site and Trp279 at the peripheral anionic site of AChE were reported
as important residues for ligand binding affinity and specificity
[39]. As seen in Fig. 6,
p$$$p interactions with the phenyl ring of
Table 4
The effect of 11c and 14b on apoptosis in 3T3 cells. Annexin V-FITC (ꢂ)/Propidium Iodide (þ): Dead cells. Annexin V-FITC (þ)/Propidium Iodide (þ): Late apoptotic cells.
Annexin V-FITC (ꢂ)/Propidium Iodide (ꢂ): Live cells. Annexin V-FITC (þ)/Propidium Iodide (ꢂ): Early apoptotic cells. Data are presented by mean SD in 10 M concentration.
m
Compound
Annexin V-/PIþ
Annexin Vþ/PIþ
Annexin V-/PI-
Annexin Vþ/PI-
% Mean SD
Control
11c
14b
0
0.13 0.06
0.33 0.32
0.27 0.21
98.83 1.44
97.70 1.91
98.33 0.21
0.97 1.42
1.73 1.53
1.03 0.40
0.23 0.15
0.40 0.10
9

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Fig. 5. The data generated by flow cytometry were plotted in two-dimensional dot plots in which PI is represented versus Annexin V-FICT. Region Q3 (Annexin VePIe) contains the
vital population, region Q4 (Annexin V þ PIe) contains the early apoptotic cells, region Q1 (Annexin V-PIþ) contains the dead cells and region Q2 (Annexin V þ PIþ) contains the
late apoptotic cells.
Table 5
derivative with benzothiazolone core, was the most potent and a
Cytotoxicity assay results of 11c and 14b.
selective inhibitor of AChE, while 11c (IC50 ¼ 2.98
mM), the penta-
namide derivative with benzothiazolone core, was the most potent
and a selective inhibitor of BChE. These results showed that the
benzothiazolone ring was more favorable for both AChE and BChE
inhibition. Moreover, it was observed that the ketone function was
more suitable for AChE inhibitory activity, while the amide group
was more favorable for BChE inhibitory activity. Additionally, all the
tested compounds, including 11c and 14b, displayed good ORAC-FL
Compound
Viability (%) of 3T3 cells
0.1
m
M
1
mM
10 mM
11c
14b
98.9 3.8
97.1 3.4
93.6 0.3
92.6 10.7
101.7 2.9
99.3 12.4
Table 6
Calculated physicochemical parameters of the compounds.
values ranging from 2.61 to 4.55 fold of Trolox at 10 mM concen-
tration. Furthermore, the selected compounds (i.e, 8g, 9g, 10a, 10b,
10c, 11c, 14a, 14b) had a significant effect on inflammatory markers
Compound MWa logPa tPSAa nONa nOHNHa Vola
BBBb
Vioa
(IL-1
Compounds 14b and 11c were not found to be statistically signifi-
cantly different from donepezil in reducing LPS-induced IL-1 , IL-6
and TNF- levels. Notably, compound 14b was found to be better at
reducing LPS-induced NO than the donepezil. Besides, 11c and 14b
exhibited significantly Ab1-42 anti-aggregatory action as much as
curcumin under our experimental condition. Meanwhile, neither
11c nor 14b did not show a cytotoxic or apoptotic effect. Collec-
tively, the above-mentioned multifunctional properties highlighted
11c and 14b as promising potential leads for further studies.
b, IL-6, TNF-a, NO) in LPS stimulated THP1 cells inflammation.
8g
9g
394,48 1,99 70,72
7
6
7
6
5
6
6
5
1
1
1
1
1
1
0
0
365,65 0,9878
374,79 0,9919
382,45 0,9807
391,59 0,9903
378,83 0,9908
408,39 0,9908
353,25 0,9933
362,39 0,9939
0
0
0
0
0
0
0
0
410,54 2,63 57,58
408,50 2,26 70,72
424,57 2,90 57,58
409,56 4,08 54,34
438,60 3,41 57,58
379,46 2,33 58,69
395,53 2,98 45,55
10a
10b
10c
11c
14a
14b
b
a
MW: Molecular weight; logP: log octanol/water partition coefficient; tPSA: Total
Polar Surface Area; nON: number of Hydrogen acceptors; nOHNH: number of
Hydrogen donors; Vol: Molecular volume; Vio: Violation of Lipinski’s rule.
a
Calculated with Molinspiration.
Calculated with admetSAR.
b
4. Experimental
predicted hydrogen bonding interactions with Glu199 and Phe290
residues which were absent in case of docking with 9g.
4.1. Chemistry
In Fig. 7, three-dimensional docking views and interaction dia-
grams were shown for the active compound 11c and inactive
All the chemicals used for the synthesis of the compounds were
purchased from commercial suppliers. Melting points (mp) were
recorded on a Schmelzpunkt SMP-II digital melting point apparatus
and values are uncorrected. Thin-layer chromatography (TLC) was
performed on Merck 60F254 plates. 1H (400 MHz) and 13C
(100 MHz) NMR spectra were recorded with a Varian Mercury
400 MHz FT-NMR spectrometer in DMSO‑d6 using tetramethylsi-
lane as the internal standard (Faculty of Pharmacy, Ankara Uni-
compound 9a bound to the BChE enzyme. Aromatic
with Trp82 and CeH$$$ interaction with Trp231 were conserved
for all these compounds, with distance range of 3.6 Åe4.3 Å. Unlike
9a, additional stabilization by hydrogen bonding and CeH$$$ in-
teractions were obtained between 11c and BChE. It was predicted
two CeH$$$ interactions between phenyl group of 11c and both of
p$$$p stacking
p
p
p
Leu286 and Phe329, and two hydrogen bonding interactions be-
tween benzothiazole sulphur of 11c and Glu197 and His438 resi-
dues of BChE. Higher BChE inhibitory activity of 11c relative to
compound 9a may be attributed to all these non-covalent in-
teractions obtained for this.
versity). All chemical shifts were recorded as d (ppm) and coupling
constants (J) are reported as Hertz. The high-resolution mass
spectra (HRMS) were obtained on a Waters LCT Premier XE Mass
Spectrometer also coupled to an AQUITY Ultra Performance Liquid
Chromatography system at Faculty of Pharmacy, Gazi University,
Ankara, Turkey. Elemental analysis was performed on a Leco 932
CHNS instrument at the Faculty of Pharmacy, Ankara University,
Ankara, Turkey, and the results were within 0.4% of the theoretical
values.
3. Conclusion
Taking into consideration the structure activity relationships of
the synthesized compounds, 14b (IC50 ¼ 0.34
mM), the keton
10

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Fig. 6. 2D and 3D representation of docking poses for the compounds 9g (a), 14b (b) and 9a (c) in the active site of AChE (PDB code: 1EVE).
4.1.1. 3-Methyl-2(3H)-benzoxazolone (1)/3-methyl-2(3H)-
benzothiazolone (2)
4.1.3. 6-Amino-3-methyl-2(3H)-benzoxazolone (5)/6-amino-3-
methyl-2(3H)-benzothiazolone (6)
3-methyl-2(3H)-benzoxazolone (1) and 3-methyl-2(3H)-ben-
zothiazolone (2) were synthesized according to the previously reþ-
ported method. Yield for (1): 80%.; mp: 84 ꢀC. HRMS (ESI) [MþH]
m/z for C8H7NO2 calculated: 150.0555, fþound: 155.0558. Yield for
(2): 90%; mp: 76 ꢀC. HRMS (ESI) [MþH] m/z for C8H7NOS calcu-
lated: 166.0327, found: 166.0319 [31,32].
6-amino-3-methyl-2(3H)-benzoxazolone (5) and 6-amino-3-
methyl-2(3H)-benzothiazolone (6) were synthesized according to
the previously reported method. Yield for (5): 87%.; mp: 154 ꢀC.
HRMS (ESI) [MþH]þ m/z for C8H8N2O2 calculated: 165.0664, found:
165.0665. Yield for (6): 90%; mp: 188.5 ꢀC. HRMS (ESI) [MþH]þ m/z
for C8H8N2OS calculated: 181.0436, found: 181.0440 [31,32].
4.1.4. 3-Chloro-N-(3-methyl-2-oxo-2,3-dihydrobenzoxazol-6-yl)
propanamide (7)
4.1.2. 3-Methyl-6-nitro-2(3H)-benzoxazolone (3)/3-methyl-6-
nitro-2(3H)-benzothiazolone (4)
To the solution of 500 mg (3 mmol) 6-amino-3-methyl-1,3-
benzoxazole-2(3H)-one (5) in 6 ml DMF was added 37 mg
(0.3 mmol) DMAP and 0.32 ml (2.2 mmol) TEA and mixed for
10 min. After reaction mixture cooled in ice bath, 0.38 ml (4 mmol)
3-chloropropionyl chloride was added and then mixed for 1 h at
room temperature. At the end of this period, the reaction mixture
was poured into ice water. The precipitate was filtered in vacuo,
3-methyl-6-nitro-2(3H)-benzoxazolone (3) and 3-methyl-6-
nitro-2(3H)-benzothiazolone (4) were synthesized according to
the previously reported method. Yield for (3): 85%.; mp: 181.5 ꢀC.
HRMS (ESI) [MþH]þ m/z for C8H6N2O4 calculated: 195.0406, found:
195.0410. Yield for (4): 91%; mp: 164 ꢀC. HRMS (ESI) [MþH]þ m/z
for C8H6N2O3S calculated: 211.0177, found: 211.0175 [31,32].
11

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
Fig. 7. 2D and 3D representation of docking pose for the compounds 9a (a) and 11c (b) in the active site of BChE (PDB code: 4BDS).
dried and used for the next step without further purificatþion. Yield:
67%; mp: 204 ꢀC (decomposed). HRMS (ESI) [MþH] m/z for
piperazine), 2.67 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.55 (t, 4H,
J ¼ 4.9 Hz, H2, H6-piperazine), 2.48 (m, 2H, J ¼ 7.0 Hz, -NeCH2eCH2-
C
11H11ClN2O3 calculated: 255.0536, found: 255.0529. (CAS number:
). 13C NMR (DMSO‑d6)
d: 170.0, 154.1, 150.9, 141.7, 134.3, 128.8, 127.1,
1888921-51-4).
118.7, 115.3, 114.4, 108.7, 101.4, 53.6, 52.4, 48.1, 34.1, 27.9. HRMS (ESI)
[MþH]þ m/z for C21H25N4O3 calculated: 381.1927, found: 381.1929.
Anal. Calcd. for C21H24N4O3: C, 66.30; H, 6.36; N, 14.73. Found: C,
65.98; H, 6.39; N, 14.85.
4.1.5. General procedure for the preparation of 3-substituted-N-(3-
methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)propanamide
derivatives (8a-n) (method A)
NaI (3 equivalent) was added to intermediate 7 (1 equivalent) in
ACN (25 ml) and mixture was refluxed for 1.5 h and cooled to room
temperature. Subsequently, anhydrous potassium carbonate (2.5
equivalent) and appropriate amine derivative (2.5 equivalent) were
added. Then, the mixture was refluxed for 5 h. After evaporation
under reduced pressure, the crude product was purified by crys-
tallization from an appropriate solvent.
4.1.5.2. 3-[4-(4-Fluorophenyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzoxazol-6-yl)propanamide (8b). The intermediate 7
(382 mg, 1.5 mmol) was treated with 1-(4-fluorophenyl)piperazine
(685 mg, 3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and
anhydrous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from ACN. Yield: 537 mg, 90%;
mp: 239.3 C. 1H NMR (DMSO‑d6)
d: 10.12 (s, 1H, NeH), 7.72 (d, 1H,
J ¼ 1.7 Hz, H7), 7.26 (dd, 1H, J ¼ 8.3 Hz, J ¼ 1.7 Hz, H5), 7.14 (d, 1H,
J ¼ 8.3 Hz, H4), 7.03e6.99 (m, 2H, H3’, H5’), 6.93e6.90 (m, 2H, H2’,
H6’), 3.28 (s, 3H, NeCH3), 3.05 (t, 4H, J ¼ 4.7 Hz, H3, H5-piperazine),
2.67 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.54 (t, 4H, J ¼ 4.7 Hz, H2, H6-
4.1.5.1. N-(3-methyl-2-oxo-2,3-dihydrobenzoxazol-6-yl)-3-(4-
phenylpiperazin-1-yl)propanamide (8a). The intermediate
7
(382 mg, 1.5 mmol) was treated with phenylpiperazine (0.58 ml,
3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and anhy-
drous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from ACN. Yield: 502 mg, 88%;
piperazine), 2.50e2.47 (m, 2H, -NeCH2eCH2-, with DMSO).13
C
NMR (DMSO‑d6)
d
: 169.9, 155.8 (d, J ¼ 233.9 Hz), 154.0, 147.8, 141.6,
134.3, 127.0, 116.9 (d, J ¼ 7.6 Hz), 115.1 (d, J ¼ 21.4 Hz), 114.3, 108.6,
mp: 215.9 C. 1H NMR (DMSO‑d6)
d: 10.13 (s, 1H, NeH), 7.72 (d, 1H,
101.4, 53.5, 52.3, 48.8, 34.0, 27.9. HRMS (ESI) [MþH]þ m/z for
J ¼ 1.7 Hz, H7), 7.26 (dd, 1H, J ¼ 8.2 Hz, J ¼ 1.7 Hz, H5), 7.19e7.13 (m,
3H, H3’, H5’, H4), 6.90 (d, 2H, J ¼ 8.0 Hz, H2’, H6’), 6.74 (t, 1H,
J ¼ 8.0 Hz, H4’), 3.28 (s, 3H, NeCH3), 3.11 (t, 4H, J ¼ 4.9 Hz, H3, H5-
C
C
21H24FN4O3 calculated: 399.1832, found: 399.1837. Anal. Calcd. for
21H23FN4O3: C, 62.90; H, 5.82; N,14.06. Found: C, 62.54; H, 5.99; N,
14.06.
12

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
4.1.5.3. 3-[4-(4-Chlorophenyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzoxazol-6-yl)propanamide (8c). The intermediate 7
(382 mg, 1.5 mmol) was treated with 1-(4-chlorophenyl)piperazine
(747 mg, 3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and
anhydrous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from butanol. Yield: 454 mg,
(d, 1H, J ¼ 8.3 Hz, H4), 7.04 (d, 2H, J ¼ 8.8 Hz, H2’, H6’), 3.31 (s, 3H,
NeCH3), 3.25 (t, 4H, J ¼ 4.9 Hz, H3, H5-piperazine), 2.67 (t, 2H,
J ¼ 7.0 Hz, -N-CH2-CH2-), 2.54 (t, 4H, J ¼ 4.9 Hz, H2, H6-piperazine),
2.51e2.47 (m, 2H, -NeCH2eCH2-, with DMSO). 13C NMR (DMSO‑d6)
d
: 169.8,154.0,153.1,141.6,134.2,127.1,125.9 (q, J ¼ 3.9 Hz),124.8 (q,
J ¼ 269.0 Hz), 117.7 (q, J ¼ 31.7), 114.4, 114.0, 108.6, 101.5, 53.5, 52.0,
46.9, 34.0, 27.8. HRMS (ESI) [MþH]þ m/z for C22H24F3N4O3 calcu-
lated: 449.1801, found: 449.1801. Anal. Calcd. for C22H23F3N4O3: C,
58.79; H, 5.17; N, 12.49. Found: C, 58.39; H, 4.95; N, 12.67.
73%; mp: 249.7 C. 1H NMR (DMSO‑d6)
d: 10.11 (s, 1H, NeH), 7.72 (s,
1H, H7), 7.26 (d, 1H, J ¼ 8.2 Hz, H5), 7.19 (d, 2H, J ¼ 9.0 Hz, H3’, H5’),
7.14 (d, 1H, J ¼ 8.2 Hz, H4), 6.91 (d, 2H, J ¼ 9.0 Hz, H2’, H6’), 3.31 (s,
3H, NeCH3), 3.10 (bt, 4H, H3, H5-piperazine), 2.66 (t, 2H, J ¼ 7.0 Hz,
-N-CH2-CH2-), 2.53 (bt, 4H, H2, H6-piperazine), 2.50e2.46 (m, 2H,
4.1.5.7. 3-(4-Benzylpiperazin-1-yl)-N-(3-methyl-2-oxo-2,3-
-NeCH2eCH2-, with DMSO). 13C NMR (DMSO‑d6)
d
: 169.9, 154.0,
dihydrobenzoxazol-6-yl)propanamide (8g). The intermediate
7
149.6, 141.6, 134.2, 128.4, 127.0, 122.1, 116.6, 114.3, 108.6, 101.4, 53.5,
52.1, 47.9, 34.0, 27.9. HRMS (ESI) [MþH]þ m/z for C21H24ClN4O3
calculated: 415.1537, found: 415.1539. Anal. Calcd. for
(382 mg, 1.5 mmol) was treated with 1-benzylpiperazine (670 mg,
3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and anhy-
drous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from ACN. Yield: 414 mg, 70%;
C
21H23ClN4O3: C, 58.68; H, 5.59; N, 13.10. Found: C, 58.28; H, 5.92;
N, 12.97.
mp: 183.5 C. 1H NMR (DMSO‑d6)
d: 10.12 (s, 1H, NeH), 7.69 (d, 1H,
J ¼ 2 0.0 Hz, H7), 7.31e7.19 (m, 6H, H5, phenyl protons), 7.14 (d, 1H,
J ¼ 8.4 Hz, H4), 3.42 (s, 2H, eCH2- C6H5), 3.29 (s, 3H, NeCH3), 2.59 (t,
2H, J ¼ 7.2 Hz, -N-CH2-CH2-), 2.43 (t, 2H, J ¼ 7.2 Hz, -NeCH2eCH2-),
4.1.5.4. 3-[4-(4-Methylphenyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzoxazol-6-yl)propanamide (8d). The intermediate 7
(382 mg, 1.5 mmol) was treated with 1-(4-methylphenyl)pipera-
zine (670 mg, 3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol)
and anhydrous potassium carbonate (525 mg, 3.8 mmol) according
to method A then, it was recrystallized from ethanol. Yield: 473 mg,
2.41e2.35 (m, 8H, piperazine).13C NMR (DMSO‑d6)
d: 169.9, 154.0,
141.7, 138.1, 134.3, 128.7, 128.0, 127.1, 126.7, 114.3, 108.7, 101.4, 61.9,
53.6, 52.5, 52.4, 34.0, 27.9. HRMS (ESI) [MþH]þ m/z for C22H27N4O3
calculated: 395.2083, found: 395.2083. Anal. Calcd. for C22H26N4O3:
C, 66.99; H, 6.64; N, 14.20. Found: C, 66.86; H, 6.64; N, 14.15.
80%; mp: 244 ꢀC. 1H NMR (DMSO‑d6)
d: 10.14 (s, 1H, NeH), 7.74 (d,
1H, J ¼ 2.0 Hz, H7), 7.28 (dd,1H, J ¼ 8.5 Hz, J ¼ 2.0 Hz, H5), 7.16 (d,1H,
J ¼ 8.5 Hz, H4), 7.01 (d, 2H, J ¼ 8.6 Hz, H3’, H5’), 6.82 (d, 2H, J ¼ 8.6 Hz,
H2’, H6’), 3.31 (s, 3H, NeCH3), 3.06 (t, 4H, J ¼ 4.9 Hz, H3, H5-piper-
azine), 2.68 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.56 (t, 4H, J ¼ 4.9 Hz,
H2, H6-piperazine), 2.52e2.49 (m, 2H, -NeCH2eCH2-, with DMSO),
4.1.5.8. 3-[4-(4-Fluorobenzyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzoxazol-6-yl)propanamide (8h). The intermediate 7
(382 mg, 1.5 mmol) was treated with 1-(4-fluorobenzyl)piperazine
(737 mg, 3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and
anhydrous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from butanol. Yield: 457 mg,
2.19 (s, 3H, phenyl-CH3). 13C NMR (DMSO‑d6)
d: 169.9, 154.0, 148.8,
̊
141.6, 134.3, 129.2, 127.4, 127.0, 115.5, 114.3, 108.7, 101.4, 53.6, 52.3,
48.6, 34.0, 27.9, 19.9. HRMS (ESI) [MþH]þ m/z for C22H27N4O3
calculated: 395.2083, found: 395.2083. Anal. Calcd. for
74%; mp: 164.2 C. 1H NMR (DMSO‑d6)
d: 10.12 (s, 1H, NeH), 7.69 (d,
1H, J ¼ 2.0 Hz, H7), 7.31e7.23 (m, 3H, H2’, H6’, H5), 7.15e7.08 (m, 3H,
H3’, H5’, H4), 3.41 (s, 2H, eCH2-C6H5) 3.29 (s, 3H, NeCH3), 2.59 (t, 2H,
J ¼ 7.0 Hz, -N-CH2-CH2-), 2.42 (t, 2H, J ¼ 7.0 Hz, -NeCH2eCH2-),
C
22H26N4O3.0.5C2H5OH: C, 66.16; H, 7.00; N, 13.41. Found: C, 65.76;
H, 7.05; N, 13.42.
2.40e2.30 (m, 8H, piperazine). 13C NMR (DMSO‑d6)
d: 169.9, 161.1
4.1.5.5. 3-[4-(4-Methoxyphenyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzoxazol-6-yl)propanamide (8e). The intermediate 7
(382 mg, 1.5 mmol) was treated with 1-(4-methoxyphenyl)piper-
azine (730 mg, 3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol)
and anhydrous potassium carbonate (525 mg, 3.8 mmol) according
to method A then, it was recrystallized from ethanol. Yield: 498 mg,
(d, J ¼ 240.8 Hz), 154.0, 141.6, 134.3 (d, J ¼ 2.3 Hz), 134.2, 130.5 (d,
J ¼ 8.4 Hz), 127.0, 114.7 (d, J ¼ 20.5 Hz), 114.3, 108.6, 101.4, 60.9, 53.6,
52.4, 52.3, 33.9, 27.9. HRMS (ESI) [MþH]þ m/z for C22H26FN4O3
calculated: 413.1989, found: 413.1986. Anal. Calcd. for C22H25FN4O3:
̊
C, 64.06; H, 6.16; N, 13.43. Found: C, 63.62; H, 6.15; N, 13.50.
81%; mp: 214.7 C. 1H NMR (DMSO‑d6)
d
: 10.12 (s, 1H, NeH), 7.71 (d,
4.1.5.9. 3-[4-(4-Chlorobenzyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzoxazol-6-yl)propanamide (8i). The intermediate 7
(382 mg, 1.5 mmol) was treated with 1-(4-chlorobenzyl)piperazine
(0.7 ml, 3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and
anhydrous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from methanol. Yield: 437 mg,
1H, J ¼ 2.0 Hz, H7), 7.26 (dd,1H, J ¼ 8.4 Hz, J ¼ 2.0 Hz, H5), 7.14 (d,1H,
J ¼ 8.4 Hz, H4), 6.86 (d, 2H, J ¼ 9.0 Hz, H3’, H5’), 6.78 (d, 2H, J ¼ 9.0 Hz,
H2’, H6’), 3.65 (s, 3H, OeCH3), 3.29 (s, 3H, NeCH3), 2.99 (t, 4H,
J ¼ 4.7 Hz, H3,H5-piperazine), 2.66 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-),
2.54 (t, 4H, J ¼ 4.8 Hz, H2,H6-piperazine), 2.49e2.46 (m, 2H,
J ¼ 7.0 Hz, -NeCH2eCH2-, with DMSO).13C NMR (DMSO‑d6)
d
: 169.9,
68%; mp: 170 C. 1H NMR (DMSO‑d6)
d: 10.11 (s, 1H, NeH), 7.71 (d,
154.0, 152.7, 145.3, 141.6, 134.3, 127.0, 117.1, 114.3, 114.1, 108.6, 101.4,
1H, J ¼ 1.6 Hz, H7), 7.36 (d, 2H, J ¼ 8.4 Hz, H2’, H6’), 7.30 (d, 2H,
J ¼ 8.4 Hz, H3’, H5’), 7.26 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.6 Hz, H5), 7.15 (d,
1H, J ¼ 8.4 Hz, H4), 3.44 (s, 2H, eCH2-C6H5), 3.31 (s, 3H, NeCH3),
2.62 (t, 2H, J ¼ 7.2 Hz, -N-CH2-CH2-), 2.44 (t, 2H, J ¼ 7.2 Hz,
-NeCH2eCH2-), 2.42e2.32 (m, 8H, piperazine).13C NMR (DMSO‑d6)
55.0, 53.6, 52.4, 49.5, 34.0, 27.9. HRMS (ESI) [MþH]þ m/z for
C
C
22H27N4O4 calculated: 411.2032, found: 411.2028. Anal. Calcd. for
22H26N4O4$1H2O: C, 61.66; H, 6.58; N, 13.07. Found: C, 61.77; H,
6.68; N, 13.17.
d
: 169.9, 154.0, 141.7, 137.2, 134.3, 131.3, 130.4, 128.0, 127.0, 114.3,
4.1.5.6. 3-[4-(4-Trifluoromethylphenyl)piperazin-1-yl]-N-(3-methyl-
2-oxo-2,3-dihydrobenzoxazol-6-yl)propanamide (8f). The interme-
108.7, 101.4, 61.0, 53.6, 52.5, 52.3, 34.0, 27.9. HRMS (ESI) [MþH]þ m/
̊
z for C22H26ClN4O3 calculated: 429.1693, found: 429.1693. Anal.
Calcd. for C22H25ClN4O3: C, 60.33; H, 5.98; N,12.79. Found: C, 60.29;
H, 6.13; N, 12.79.
diate
7
(382 mg, 1.5 mmol) was treated with 1-(4-
trifluoromethylphenyl)piperazine (875 mg, 3.8 mmol) in the pres-
ence of NaI (675 mg, 4.5 mmol) and anhydrous potassium car-
bonate (525 mg, 3.8 mmol) according to method A then, it was
recrystallized from butanol. Yield: 255 mg, 38%; mp: 257 C. 1H NMR
4.1.5.10. 3-[4-(4-Methoxybenzyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzoxazol-6-yl)propanamide (8j). The intermediate 7
(382 mg, 1.5 mmol) was treated with 1-(4-methoxybenzyl)piper-
azine (784 mg, 3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol)
(DMSO‑d6)
d
: 10.10 (s, 1H, NeH), 7.72 (d, 1H, J ¼ 1.7 Hz, H7), 7.47 (d,
2H, J ¼ 8.8 Hz, H3’, H5’), 7.27 (dd, 1H, J ¼ 8.3 Hz, J ¼ 1.7 Hz, H5), 7.14
13
̊

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
and anhydrous potassium carbonate (525 mg, 3.8 mmol) according
to method A then, it was recrystallized from ethanol. Yield: 216 mg,
42.2, 37.2, 34.1, 31.6, 27.9. HRMS (ESI) [MþH]þ m/z for C23H28N3O3
calculated: 394.2131, found: 394.2130. Anal. Calcd. for C23H27N3O3:
C, 70.21; H, 6.92; N, 10.68. Found: C, 69.91; H, 6.86; N, 10.85.
34%; mp: 168.7 C. 1H NMR (DMSO‑d6)
d: 10.12 (s, 1H, NeH), 7.70 (d,
1H, J ¼ 1.6 Hz, H7), 7.26 (dd,1H, J ¼ 8.4 Hz, J ¼ 1.6 Hz, H5), 7.18 (d, 2H,
J ¼ 8.2 Hz, H2’, H6’), 7.14 (d, 1H, J ¼ 8.4 Hz, H4), 6.86 (d, 2H, J ¼ 8.2 Hz,
H3’, H5’), 3.73 (s, 3H, OeCH3), 3.37 (s, 2H, eCH2-C6H5), 3.31 (s, 3H,
NeCH3), 2.61 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.44 (t, 2H, J ¼ 7.0 Hz,
-NeCH2eCH2-), 2.42e2.32 (m, 8H, piperazine). 13C NMR (DMSO‑d6)
4.1.5.14. 3-[Benzyl(methyl)amino]-N-(3-methyl-2-oxo-2,3-
dihydrobenzoxazol-6-yl)propanamide (8n). The intermediate
7
(382 mg, 1.5 mmol) was treated with N-benzyl(methyl)amine
(0.49 ml, 3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and
anhydrous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from 2-propanol. Yield:
d
: 170.4, 158.6, 154.5, 142.2, 134.8, 130.4, 127.5, 114.8, 113.9, 109.2,
101.9, 61.8, 55.4, 54.1, 52.9, 52.9, 34.5, 28.4. HRMS (ESI) [MþH]þ m/z
for C23H29N4O4 calculated: 425.2189, found: 425.2189. Anal. Calcd.
for C23H28N4O4: C, 64.39; H, 6.69; N, 13.05. Found: C, 64.42; H, 6.47;
N, 13.22.
264 mg, 52%; mp: 165.5 C. 1H NMR (DMSO‑d6)
d: 10.05 (s, 1H, NeH),
7.69 (s, 1H, H7), 7.28e7.20 (m, 6H, H5, phenyl protons), 7.14 (d, 1H,
J ¼ 8.0 Hz, H4), 3.51 (s, 2H, eCH2-C6H5), 3.29 (s, 3H, NeCH3), 2.70 (t,
2H, J ¼ 6.8 Hz, -N-CH2-CH2-), 2.49 (t, 2H, J ¼ 6.8 Hz, -NeCH2eCH2-),
4.1.5.11. 3-[4-(4-Trifluoromethylbenzyl)piperazin-1-yl]-N-(3-methyl-
2-oxo-2,3-dihydrobenzoxazol-6-yl)propanamide (8k). The interme-
2.15 (s, 3H, N2eCH3).13C NMR (DMSO‑d6)
d: 170.0, 154.1, 141.7, 134.3,
128.7, 128.0, 127.1, 126.8, 114.4, 108.7, 101.5, 60.9, 52.8, 41.5, 34.5,
27.9. HRMS (ESI) [MþH]þ m/z for C19H22N3O3 calculated: 340.1661,
found: 340.1662. Anal. Calcd. for C19H21N3O3: C, 67.24; H, 6.24; N,
12.38. Found: C, 66.84; H, 6.47; N, 12.40.
diate
7
(382 mg, 1.5 mmol) was treated with 1-(4-
trifluoromethylbenzyl)piperazine (0.75 ml, 3.8 mmol) in the pres-
ence of NaI (675 mg, 4.5 mmol) and anhydrous potassium car-
bonate (525 mg, 3.8 mmol) according to method A then, it was
recrystallized from 2-propanol. Yield: 451 mg, 65%; mp: 163.8 C. 1H
4.1.6. General procedure for the preparation of 3-
substitutedpropanoic acid intermediates
NMR (DMSO‑d6)
d
: 10.15 (s, 1H, NeH), 7.69 (d, 1H, J ¼ 2.0 Hz, H7),
7.65 (d, 2H, J ¼ 8.2 Hz, H3’, H5’), 7.49 (d, 2H, J ¼ 8.4 Hz, H2’, H6’), 7.24
(dd, 1H, J ¼ 8.7 Hz, J ¼ 2.0 Hz, H5), 7.14 (d, 1H, J ¼ 8.7 Hz, H4), 3.55 (s,
2H, eCH2-C6H5), 3.31 (s, 3H, NeCH3), 2.63 (t, 2H, J ¼ 7.0 Hz, -N-CH2-
CH2-), 2.45 (t, 2H, J ¼ 7 Hz, -NeCH2eCH2-), 2.43e2.38 (m, 8H,
A mixture of appropriate amine derivatives (1 equivalent) and
methyl acrylate (1.2 equivalent) in 15 ml DCM were stirred at room
temperature overnight. After completion of reaction, solvent was
evaporated under reduced pressure and corresponding methyl
ester was produced. To obtain desired propanoic acid, methyl ester
hydrolyzed by heating at 40 ꢀC for 2 h with 5% NaOH solution. End
of this period, reaction mixture was cooled to room temperature
and neutralized with HCl. The resulting precipitate was filtered,
dried and used for the method B without further purification [29].
piperazin). 13C NMR (DMSO‑d6)
d: 169.9, 154.0, 141.6, 134.3, 129.2,
̊
127.4 (q, J ¼ 31.4 Hz), 127.0, 125.6, 124.3 (q, J ¼ 3.6 Hz), 124.2 (q,
J ¼ 269.9 Hz), 114.3, 108.6, 101.4, 61.1, 53.6, 52.4, 52.3, 34.0, 27.9.
HRMS (ESI) [MþH]þ m/z for C23H26F3N4O3 calculated: 463.1957,
found: 463.1957. Anal. Calcd. for C23H25F3N4O3: C, 59.73; H, 5.45; N,
12.11. Found: C, 59.39; H, 5.73; N, 12.08.
4.1.7. General procedure for the preparation of 3-substituted-N-(3-
methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)propanamide
4.1.5.12. 3-(4-Phenylpiperidin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzoxazol-6-yl)propanamide (8l). The intermediate
7
derivatives (9a-n) (method B)
̊
(382 mg, 1.5 mmol) was treated with 4-phenylpiperidine (612 mg,
3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and anhy-
drous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from methanol. Yield: 472 mg,
To a solution of appropriate 3-substitutedpropanoic acid (1.1
equivalent) in DCM (20 ml), EDC (1.2 equivalent), DMAP (0.2
equivalent) and intermediate 6 (1 equivalent) were added respec-
tively in 20 min. The reaction was carried out at room temperature
overnight then, the reaction mixture was evaporated to dryness.
The precipitate was poured into water, filtered, and crystallized
from appropriate solvent.
83%; mp: 171.2 C. 1H NMR (DMSO‑d6)
d: 10.15 (s, 1H, NeH), 7.72 (d,
1H, J ¼ 2.0 Hz, H7), 7.28e7.14 (m, 7H, H4, H5, phenyl protons), 3.29
(s, 3H, NeCH3), 2.98 (d, 2H, J ¼ 11.2 Hz, H2e, H6e-piperidine), 2.65 (t,
2H, J ¼ 7.2 Hz, -N-CH2-CH2-), 2.49e2.43 (m, 3H, H4-piperidine,
-NeCH2eCH2-, with DMSO), 2.05 (td, 2H, J ¼ 11.2 Hz, J ¼ 2.0 Hz, H2a
,
4.1.7.1. N-(3-methyl-2-oxo-2,3-dihydrobenzothiazol-6-yl)-3-(4-
phenylpiperazin-1-yl)propanamide (9a). Compound 9a was ob-
tained by using method B from intermediate 6 (360 mg, 2 mmol), 3-
(4-phenylpiperazin-1-yl)propanoic acid (515 mg, 2.2 mmol), EDC
(460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol). Then it was
recrystallized from methanol. Yield: 476 mg, 60%; mp: 178.6 C. 1H
H6a-piperidine, 1.75e1.59 (m, 4H, H3, H5-piperidine).13C NMR
̊
(DMSO‑d6) d: 170.0, 154.0, 146.1, 141.7, 134.3, 128.2, 127.0, 126.5,
125.8, 114.3, 108.7, 101.4, 53.9, 53.4, 41.7, 34.1, 33.0, 27.9. HRMS (ESI)
[MþH]þ m/z for C22H26N3O3 calculated: 380.1974, found: 380.1973.
Anal. Calcd. for C22H25N3O3: C, 69.64; H, 6.64; N, 11.07. Found: C,
69.51; H, 6.62; N, 11.26.
NMR (DMSO‑d6) d
: 10.14 (s, 1H, NeH), 7.95 (s, 1H, H7), 7.45 (dd, 1H,
J ¼ 8.7 Hz, J ¼ 1.6 Hz, H5), 7.21 (d, 1H, J ¼ 8.7 Hz, H4), 7.18 (t, 2H,
J ¼ 7.6 Hz, H3’, H5’), 6.90 (d, 2H, J ¼ 7.6 Hz, H2’, H6’), 6.74 (t, 1H,
J ¼ 7.6 Hz, H4’), 3.35 (s, 3H, NeCH3), 3.10 (m, 4H, H3, H5-piperazine),
2.67 (t, 2H, J ¼ 6.8 Hz, -N-CH2-CH2-), 2.55 (m, 4H, H2, H6-pipera-
zine), 2.52e2.46 (m, 2H, -NeCH2eCH2-, with DMSO). 13C NMR
4.1.5.13. 3-(4-Benzylpiperidin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzoxazol-6-yl)propanamide (8m). The intermediate
7
(382 mg, 1.5 mmol) was treated with 4-benzylpiperidine (612 mg,
3.8 mmol) in the presence of NaI (675 mg, 4.5 mmol) and anhy-
drous potassium carbonate (525 mg, 3.8 mmol) according to
method A then, it was recrystallized from ethanol. Yield: 312 mg,
(DMSO‑d6) d: 170.0, 168.4, 150.9, 134.8, 133.2, 128.8, 121.5, 118.7,
117.9, 115.3, 113.3, 111.2, 53.7, 52.4, 48.1, 34.0, 28.9. HRMS (ESI)
[MþH]þ m/z for C21H25N4O2S calculated: 397.1698, found: 397.1702.
Anal. Calcd. for C21H24N4O2S: C, 63.61; H, 6.10; N, 14.13; S, 8.09.
Found: C, 63.40; H, 6.04; N, 13.91; S, 7.99.
53%; mp: 164.1 C. 1H NMR (DMSO‑d6)
d: 10.16 (s, 1H, NeH), 7.69 (d,
1H, J ¼ 2 Hz, H7), 7.26e7.12 (m, 7H, H4, H5, phenyl protons), 3.29 (s,
3H, NeCH3), 2.83 (d, 2H, J ¼ 10.8 Hz, H2e, H6e-piperidin), 2.56 (t, 2H,
J ¼ 7 Hz, -N-CH2-CH2-), 2.47 (m, 2H, eCH2-C6H5, with DMSO), 2.41
(t, 2H, J ¼ 7.0 Hz, -NeCH2eCH2-), 1.86 (t, 2H, J ¼ 10.8 Hz, H2a, H6a
-
4.1.7.2. 3-[4-(4-Fluorophenyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzothiazol-6-yl)propanamide (9b). Compound 9b was
obtained by using method B from intermediate 6 (360 mg, 2 mmol),
3-[4-(4-fluorophenyl)piperazin-1-yl]propanoic acid (555 mg,
piperidin), 1.53e1.45 (m, 3H, H3e, H5e, H4-piperidin), 1.18e1.13 (m,
2H, H3a, H5a-piperidin).13C NMR (DMSO‑d6)
d
: 170.0, 154.0, 141.6,
̊
140.2, 134.3, 128.8, 127.9, 127.0, 125.6, 114.3, 108.6, 101.3, 53.9, 52.8,
14

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
2.2 mmol), EDC (460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol).
Then it was recrystallized from butanol. Yield: 588 mg, 71%; mp:
53.7, 52.5, 49.5, 34.0, 28.9. HRMS (ESI) [MþH]þ m/z for C22H27N4O3S
calculated: 427.1804, found: 427.1802. Anal. Calcd. for C22H26N4O3S:
C, 61.95; H, 6.14; N, 13.14; S, 7.52. Found: C, 62.17; H, 6.20; N, 13.09;
S, 7.52.
176.4 C. 1H NMR (DMSO‑d6)
d: 10.13 (s, 1H, NeH), 7.94 (d, 1H,
J ¼ 1.7 Hz, H7), 7.45 (dd, 1H, J ¼ 8.7 Hz, J ¼ 1.7 Hz, H5), 7.21 (d, 1H,
J ¼ 8.7 Hz, H4), 7.03e6.99 (m, 2H, H3’, H5’), 6.93e6.89 (m, 2H, H2’,
H6’), 3.35 (s, 3H, NeCH3), 3.05 (t, 4H, J ¼ 4.8 Hz, H3, H5-piperazine),
2.67 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.55 (t, 4H, J ¼ 4.8 Hz, H2, H6-
piperazine), 2.51e2.47 (m, 2H, -NeCH2eCH2-, with DMSO). 13C
4.1.7.6. 3-[4-(4-Trifluoromethylphenyl)piperazin-1-yl]-N-(3-methyl-
2-oxo-2,3-dihydrobenzothiazol-6-yl)propanamide
(9f).
Compound 9f was obtained by using method B from intermediate 6
(360 mg, 2 mmol), 3-[4-(4-trifluoromethylphenyl)piperazin-1-yl]
propanoic acid (665 mg, 2.2 mmol), EDC (460 mg, 2.4 mmol) and
DMAP (49 mg, 0.4 mmol). Then it was recrystallized from ethanol.
NMR (DMSO‑d6)
d
: 170.0, 168.4, 155.9 (d, J ¼ 234.7 Hz), 147.8 (d, J ¼
2.3 Hz), 134.9, 133.2, 121.5, 117.9, 117.0 (d, J ¼ 7.6 Hz), 115.1 (d, J ¼
22.1 Hz), 113.3, 111.2, 53.6, 52.4, 48.9, 34.0, 28.9. HRMS (ESI)
[MþH]þ m/z for C21H24FN4O2S calculated: 415.1604, found:
415.1608. Anal. Calcd. for C21H23FN4O2S: C, 60.85; H, 5.59; N, 13.52;
S, 7.74. Found: C, 60.46; H, 5.90; N, 13.17; S, 7.60.
Yield: 743 mg, 80%; mp: 213.6 C. 1H NMR (DMSO‑d6)
d: 10.11 (s, 1H,
NeH), 7.95 (d, 1H, J ¼ 1.6 Hz, H7), 7.50e7.47 (m, 3H, H3’, H5’, H5), 7.23
(d, 1H, J ¼ 8.4 Hz, H4), 7.05 (d, 2H, J ¼ 8.4 Hz, H2’, H6’), 3.37 (s, 3H,
NeCH3), 3.28 (t, 4H, J ¼ 4.5 Hz, H3, H5-piperazine), 2.70 (t, 2H,
J ¼ 6.8 Hz, -N-CH2-CH2-), 2.57 (t, 4H, J ¼ 4.5 Hz, H2, H6-piperazine),
2.54e2.49 (m, 2H, -NeCH2eCH2-, with DMSO). 13C NMR (DMSO‑d6)
4.1.7.3. 3-[4-(4-Chlorophenyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzothiazol-6-yl)propanamide (9c). Compound 9c was
obtained by using method B from intermediate 6 (360 mg, 2 mmol),
3-[4-(4-chlorophenyl)piperazin-1-yl]propanoic acid (591 mg,
2.2 mmol), EDC (460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol).
Then it was recrystallized from butanol. Yield: 620 mg, 72%; mp:
d
: 169.9, 168.3, 153.1, 134.9, 133.2, 126.0 (q, J ¼ 3.8 Hz), 124.9 (q, J ¼
269.0 Hz), 121.4, 117.9, 117.7 (q, J ¼ 35.5 Hz), 114.0, 113.3, 111.1, 53.5,
52.0, 46.9, 34.0, 28.8. HRMS (ESI) [MþH]þ m/z for C22H24F3N4O2S
calculated: 465.1572, found: 465.1574. Anal. Calcd. for
200.3 C. 1H NMR (DMSO‑d6)
d
: 10.11 (s, 1H, NeH), 7.93 (d, 1H,
C22H23F3N4O2S: C, 56.89; H, 4.99; N, 12.06; S, 6.90. Found: C, 56.97;
H, 4.86; N, 12.11; S, 6.91.
J ¼ 2.0 Hz, H7), 7.48 (dd, 1H, J ¼ 8.7 Hz, J ¼ 2 0.0 Hz, H5), 7.23 (d, 1H,
J ¼ 8.7 Hz, H4), 7.21 (d, 2H, J ¼ 8.4 Hz, H3’, H5’), 6.94 (d, 2H, J ¼ 8.4 Hz,
H2’, H6’), 3.37 (s, 3H, NeCH3), 3.22e3.10 (m, 6H, H3, H5-piperazine,
-N-CH2-CH2-), 2.66e2.50 (m, 6H, H2,H6-piperazine, -NeCH2eCH2-).
4.1.7.7. 3-(4-Benzylpiperazin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzothiazol-6-yl)propanamide (9g). Compound 9g was
obtained by using method B from intermediate 6 (360 mg, 2 mmol),
3-(4-benzylpiperazin-1-yl)propanoic acid (546 mg, 2.2 mmol), EDC
(460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol). Then it was
recrystallized from 2-propanol. Yield: 575 mg, 70%; mp: 183.3 C. 1H
13C NMR (DMSO‑d6)
d: 169.5, 168.3, 149.4, 134.9, 133.2, 128.5, 122.5,
̊
121.4, 118.0, 116.8, 113.3, 111.2, 53.1, 51.9, 47.4, 33.3, 28.9. HRMS (ESI)
[MþH]þ m/z for C21H24ClN4O2S calculated: 431.1309, found:
431.1317. Anal. Calcd. for C21H23ClN4O2S$1H2O: C, 56.18; H, 5.61; N,
12.48; S, 7.14. Found: C, 56.14; H, 5.75; N, 12.38; S, 7.09.
NMR (DMSO‑d6)
d
: 10.15 (s, 1H, NeH), 7.94 (d, 1H, J ¼ 2.0 Hz, H7),
7.44 (dd, 1H, J ¼ 8.6 Hz, J ¼ 2.0 Hz, H5), 7.33e7.22 (m, 6H, H4, phenyl
protons), 3.44 (s, 2H, eCH2- C6H5), 3.37 (s, 3H, NeCH3), 2.61 (t, 2H,
J ¼ 6.9 Hz, -N-CH2-CH2-), 2.45 (t, 2H, J ¼ 6.9 Hz, -NeCH2eCH2-),
4.1.7.4. 3-[4-(4-Methylphenyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzothiazol-6-yl)propanamide (9d). Compound 9d was
obtained by using method B from intermediate 6 (360 mg, 2 mmol),
3-[4-(4-methylphenyl)piperazin-1-yl]propanoic acid (546 mg,
2.2 mmol), EDC (460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol).
Then it was recrystallized from butanol. Yield: 738 mg, 90%; mp:
2.40e2.32 (m, 8H, piperazine). 13C NMR (DMSO‑d6)
d: 170.0, 168.4,
138.1, 134.9, 133.2, 128.7, 128.1, 126.8, 121.4, 117.8, 113.3, 111.3, 62.0,
̊
53.7, 52.6, 52.4, 34.0, 28.9. HRMS (ESI) [MþH]þ m/z for C22H27N4O2S
calculated: 411.1855, found: 411.1847. Anal. Calcd. for C22H26N4O2S:
C, 64.36; H, 6.38; N, 13.65; S, 7.81. Found: C, 64.39; H, 6.37; N, 13.58;
S, 7.79.
216.8 C. 1H NMR (DMSO‑d6)
d: 10.15 (s, 1H, NeH), 7.95 (d, 1H,
J ¼ 2.0 Hz, H7), 7.46 (dd, 1H, J ¼ 8.8 Hz, J ¼ 2 Hz, H5), 7.22 (d, 1H,
J ¼ 8.8 Hz, H4), 6.99 (d, 2H, J ¼ 8.4 Hz, H3’, H5’), 6.80 (d, 2H, J ¼ 8.4 Hz,
H2’, H6’), 3.35 (s, 3H, NeCH3), 3.05 (t, 4H, J ¼ 4.7 Hz, H3, H5-piper-
azine), 2.67 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.55 (t, 4H, J ¼ 4.7 Hz,
H2, H6 piperazine), 2.52e2.47 (m, 2H, -NeCH2eCH2-, with DMSO),
4.1.7.8. 3-[4-(4-Fluorobenzyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzothiazol-6-yl)propanamide (9h). Compound 9h was
obtained by using method B from intermediate 6 (360 mg, 2 mmol),
3-[4-(4-fluorobenzyl)piperazin-1-yl]propanoic acid (585 mg,
2.2 mmol), EDC (460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol).
Then it was recrystallized from 2-propanol. Yield: 608 mg, 71%;
2.18 (s, 3H, phenyl-CH3). 13C NMR (DMSO‑d6)
d ppm: 170.0, 168.4,
148.9, 135.0, 133.2, 129.3, 127.5, 121.5, 117.9, 115.6, 113.3, 111.3, 53.7,
̊
52.4, 48.6, 34.0, 28.9, 20.0. HRMS (ESI) [MþH]þ m/z for
C
C
22H27N4O2S calculated: 411.1855, found: 411.1861. Anal. Calcd. for
22H26N4O2S: C, 64.36; H, 6.38; N, 13.65; S, 7.81. Found: C, 64.27; H,
mp: 176.7 C. 1H NMR (DMSO‑d6)
d: 10.15 (s, 1H, NeH), 7.94 (d, 1H,
J ¼ 2.0 Hz, H7), 7.44 (dd, 1H, J ¼ 8.8 Hz, J ¼ 2.0 Hz, H5), 7.32e7.28 (m,
2H, H2’, H6’), 7.23 (d, 1H, J ¼ 8.8 Hz, H4), 7.14e7.10 (m, 2H, H3’, H5’),
3.42 (s, 2H, eCH2-C6H5), 3.37 (s, 3H, NeCH3), 2.61 (t, 2H, J ¼ 7.0 Hz,
-N-CH2-CH2-), 2.45 (t, 2H, J ¼ 7.0 Hz, -NeCH2eCH2-), 2.40e2.30 (m,
6.44; N, 13.55; S, 7.76.
4.1.7.5. 3-[4-(4-Methoxyphenyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzothiazol-6-yl)propanamide (9e). Compound 9e was
obtained by using method B from intermediate 6 (360 mg, 2 mmol),
3-[4-(4-methoxyphenyl)piperazin-1-yl]propanoic acid (582 mg,
2.2 mmol), EDC (460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol).
Then it was recrystallized from butanol. Yield: 734 mg, 86%; mp:
8H, piperazine). 13C NMR (DMSO‑d6)
d
: 170.0, 168.4, 161.1 (d, J ¼
240.8 Hz), 134.3, 134.3, 133.2, 130.5 (d, J ¼ 7.6 Hz), 121.4, 117.8, 114.8
̊
(d, J ¼ 21.3 Hz), 113.3, 111.2, 61.0, 53.5, 52.5, 52.4, 34.0, 28.9. HRMS
(ESI) [MþH]þ m/z for C22H26FN4O2S calculated: 429.1761, found:
429.1754. Anal. Calcd. for C22H25FN4O2S: C, 61.66; H, 5.88; N, 13.07;
S, 7.48. Found: C, 61.27; H, 5.81; N, 12.93; S, 7.48.
192.6 C. 1H NMR (DMSO‑d6)
d: 10.17 (s, 1H, NeH), 7.96 (d, 1H,
J ¼ 2.0 Hz, H7), 7.46 (dd, 1H, J ¼ 8.9 Hz, J ¼ 2 Hz, H5), 7.23 (d, 1H,
J ¼ 8.9 Hz, H4), 6.87 (d, 2H, J ¼ 8.8 Hz, H3’, H5’), 6.79 (d, 2H, J ¼ 8.8 Hz,
H2’, H6’), 3.67 (s, 3H, OeCH3), 3.36 (s, 3H, NeCH3), 3.00 (t, 4H,
J ¼ 4.8 Hz, H3,H5-piperazine), 2.68 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-),
4.1.7.9. 3-[4-(4-Chlorobenzyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzothiazol-6-yl)propanamide (9i). Compound 9i was
obtained by using method B from intermediate 6 (360 mg, 2 mmol),
3-[4-(4-chlorobenzyl)piperazin-1-yl]propanoic acid (622 mg,
2.2 mmol), EDC (460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol).
Then it was recrystallized from ethanol. Yield: 783 mg, 88%; mp:
2.56 (t, 4H, J ¼ 4.8 Hz, H2,H6-piperazine), 2.51e2.48 (m, 2H,
̊
-NeCH2eCH2-, with DMSO). 13C NMR (DMSO‑d6)
d: 170.0, 168.4,
152.8, 145.3, 135.0, 133.2, 121.5, 117.9, 117.2, 114.2, 113.3, 111.3, 55.1,
15

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
184.9 C. 1H NMR (DMSO‑d6)
d
: 10.11 (s, 1H, NeH), 7.93 (d, 1H,
found: 396.1739. Anal. Calcd. for C22H25N3O2S: C, 66.81; H, 6.37; N,
10.62; S, 8.11. Found: C, 66.83; H, 6.43; N, 10.65; S, 8.12.
J ¼ 2.1 Hz, H7), 7.44 (dd, 1H, J ¼ 8.5 Hz, J ¼ 2.1 Hz, H5), 7.35 (d, 2H,
J ¼ 8.2 Hz, H2’, H6’), 7.29 (d, 2H, J ¼ 8.2 Hz, H3’, H5’),7.22 (d, 1H,
J ¼ 8.5 Hz, H4), 3.43 (s, 2H, eCH2-C6H5), 3.36 (s, 3H, NeCH3), 2.62 (t,
2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.46 (t, 2H, J ¼ 7.0 Hz, -NeCH2eCH2-),
4.1.7.13. 3-(4-Benzylpiperidin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzoxazol-6-yl)propanamide (9m). Compound 9m was
obtained following method B using intermediate 6 (360 mg,
2.42e2.32 (m, 8H, piperazine). 13C NMR (DMSO‑d6)
d: 169.6, 168.3,
137.2, 134.9, 133.1, 131.3, 130.4, 128.0, 121.4, 117.8, 113.2, 111.1, 60.9,
2
mmol), 3-(4-benzylpiperidin-1-yl)propanoic acid (544 mg,
53.6, 52.4, 52.3, 33.9, 28.8. HRMS (ESI) [MþH]þ m/z for
2.2 mmol), EDC (460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol).
Then it was recrystallized from 2-propanol. Yield: 606 mg, 74%; mp:
C
22H26ClN4O2S calculated: 445.1465, found: 445.1465. Anal. Calcd.
156.4 C. 1H NMR (DMSO‑d6)
d: 10.19 (s, 1H, NeH), 7.96 (d, 1H,
for C22H25ClN4O2S: C, 59.38; H, 5.66; N, 12.59; S, 7.21. Found: C,
59.43; H, 5.87; N, 12.69; S, 7.31.
J ¼ 2.3 Hz, H7), 7.43 (dd, 1H, J ¼ 9.0 Hz, J ¼ 2.3 Hz, H5), 7.28e7.13 (m,
6H, H4, phenyl protons), 3.37 (s, 3H, NeCH3), 2.85 (d, 2H, J ¼ 10.9 Hz,
H2e, H6e-piperidin), 2.58 (t, 2H, J ¼ 6.9 Hz, -N-CH2-CH2-), 2.52e2.46
4.1.7.10. 3-[4-(4-Methoxybenzyl)piperazin-1-yl]-N-(3-methyl-2-oxo-
2,3-dihydrobenzothiazol-6-yl)propanamide (9j). Compound 9j was
obtained by using method B from intermediate 6 (360 mg, 2 mmol),
3-[4-(4-methoxybenzyl)piperazin-1-yl]propanoic acid (612 mg,
2.2 mmol), EDC (460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol).
Then it was recrystallized from 2-propanol. Yield: 608 mg, 69%;
(m, 2H, eCH2-C6H5, with DMSO), 2.43 (t, 2H,
J
¼
6.9 Hz,
-NeCH2eCH2-), 1.87 (t, 2H, J ¼ 10.9 Hz, H2a, H6a-piperidine),
1.55e1.42 (m, 3H, H3e, H5e, H4-piperidine), 1.21e1.10 (m, 2H, H3a
,
H5a-piperidine). 13C NMR (DMSO‑d6)
d: 170.1, 168.4, 140.3, 135.0,
133.2, 128.9, 128.0, 125.7, 121.5, 117.8, 113.2, 111.3, 54.0, 52.9, 42.3,
37.3, 34.1, 31.7, 28.9. HRMS (ESI) [MþH]þ m/z for C23H28N3O2S
calculated: 410.1902, found: 410.1902. Anal. Calcd. for C23H27N3O2S:
C, 67.45; H, 6.65; N, 10.26; S, 7.83. Found: C, 67.83; H, 6.68; N, 10.31;
S, 7.83.
mp: 175.2 C. 1H NMR (DMSO‑d6)
d: 10.12 (s, 1H, NeH), 7.93 (d, 1H,
J ¼ 2.0 Hz, H7), 7.43 (dd, 1H, J ¼ 8.4 Hz, J ¼ 2.0 Hz, H5), 7.22 (d, 1H,
J ¼ 8.4 Hz, H4), 7.17 (d, 2H, J ¼ 8.2 Hz, H2’, H6’), 6.86 (d, 2H, J ¼ 8.2 Hz,
H3’, H5’), 3.72 (s, 3H, OeCH3), 3.36 (s, 5H, eCH2-C6H5, NeCH3), 2.61
(t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.44 (t, 2H, J ¼ 7.0. Hz,
-NeCH2eCH2-), 2.38e2.30 (m, 8H, piperazine). 13C NMR (DMSO‑d6)
4.1.7.14. 3-[Benzyl(methyl)amino]-N-(3-methyl-2-oxo-2,3-
dihydrobenzoxazol-6-yl)propanamide (9n). Compound 9n was ob-
tained following method B using intermediate 6 (360 mg, 2 mmol),
3-[benzyl(methyl)amino]propanoic acid (425 mg, 2.2 mmol), EDC
(460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol). Then it was
recrystallized from ethanol. Yield: 270 mg, 38%; mp: 119.9 C. 1H
d
: 169.9, 168.3, 158.1, 134.9, 133.1, 129.9, 129.8, 121.4, 117.8, 113.4,
̊
113.2, 111.1, 61.3, 54.8, 53.6, 52.4, 52.3, 33.9, 28.8. HRMS (ESI)
[MþH]þ m/z for C23H29N4O3S calculated: 441.1960, found:
441.1958. Anal. Calcd. for C23H28N4O3S: C, 62.70; H, 6.41; N,12.72; S,
7.28. Found: C, 62.59; H, 6.59; N, 12.69; S, 7.32.
NMR (DMSO‑d6)
d
: 10.07 (s, 1H, NeH), 7.94 (d, 1H, J ¼ 2.0 Hz, H7),
7.46 (dd, 1H, J ¼ 8.7 Hz, J ¼ 2.0 Hz, H5), 7.30e7.25 (m, 5H, phenyl
protons), 7.23 (d,1H, J ¼ 8.7 Hz, H4), 3.50 (s, 2H, eCH2-C6H5), 3.37 (s,
3H, NeCH3), 2.69 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.54e2.48 (m, 2H,
4.1.7.11. 3-[4-(4-Trifluoromethylbenzyl)piperazin-1-yl]-N-(3-methyl-
2-oxo-2,3-dihydrobenzothiazol-6-yl)propanamide
(9k).
Compound 9k was obtained by using method B from intermediate
6 (360 mg, 2 mmol), 3-[4-(4-trifluoromethylbenzyl)piperazin-1-yl]
propanoic acid (696 mg, 2.2 mmol), EDC (460 mg, 2.4 mmol) and
DMAP (49 mg, 0.4 mmol). Then it was recrystallized from butanol.
-NeCH2eCH2-, with DMSO), 2.15 (s, 3H, N2eCH3). 13C NMR
̊
(DMSO‑d6) d: 170.0, 168.3, 138.8, 134.9, 133.1, 128.5, 127.9, 126.7,
121.4, 117.9, 113.3, 111.1, 61.0, 52.9, 41.5, 34.5, 28.8. HRMS (ESI)
[MþH]þ m/z for C19H22N3O2S calculated: 356.1433, found:
356.1435. Anal. Calcd. for C19H21N3O2S: C, 64.20; H, 5.95; N,11.82; S,
9.02. Found: C, 63.92; H, 6.14; N, 11.75; S, 8.98.
Yield: 574 mg, 60%; mp: 190.8 C. 1H NMR (DMSO‑d6)
d: 10.11 (s, 1H,
NeH), 7.93 (d, 1H, J ¼ 2.0 Hz, H7), 7.66 (d, 2H, J ¼ 8.2 Hz, H3’, H5’),
7.51 (d, 2H, J ¼ 8.2 Hz, H2’, H6’), 7.44 (dd, 1H, J ¼ 8.5 Hz, J ¼ 2.0 Hz,
H5), 7.22 (d, 1H, J ¼ 8.5 Hz, H4), 3.54 (s, 2H, eCH2-C6H5), 3.36 (s, 3H,
NeCH3), 2.62 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.47 (t, 2H, J ¼ 7.0 Hz,
-NeCH2eCH2-), 2.42e2.37 (m, 8H, piperazine). 13C NMR (DMSO‑d6)
4.1.8. General procedure for the preparation of 4-
substitutedbutanoic acid intermediates
d
: 169.9, 168.3, 143.3, 134.9, 133.1, 129.2, 127.5 (q, J ¼ 31.5 Hz), 124.9
The appropriate amine derivative (1 equivalent), ethyl 4-
bromobutyrate (1 equivalent) and potassium carbonate (1.5
equivalent) were dissolved in 20 ml ACN and stirred under reflux
condition for 6 h. The end of this period, potassium carbonate was
filtered and ACN was evaporated to dryness. The remaining residue
(corresponding ester derivatives) was hydrolyzed with 5% NaOH
solution by heating at 40 C for 2 h and obtained the sodium salt of
the corresponding butanoic acid derivatives. Then, the reaction
mixture was cooled down and neutralized with HCl solution. To
obtain the carboxylic acid derivatives, water was evaporated under
̊
(q, J ¼ 3.8 Hz), 124.2 (q, J ¼ 269.7 Hz), 121.4, 117.8, 113.2, 111.2, 61.2,
53.6, 52.5, 52.3, 34.0, 28.8. HRMS (ESI) [MþH]þ m/z for
C
23H26F3N4O2S calculated: 479.1729, found: 479.1727. Anal. Calcd.
for C23H25F3N4O2S: C, 57.73; H, 5.27; N, 11.71; S, 6.70. Found: C,
57.78; H, 5.48; N, 11.62; S, 6.74.
4.1.7.12. 3-(4-Phenylpiperidin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzoxazol-6-yl)propanamide (9l). Compound 9l was ob-
tained following method B using intermediate 6 (360 mg, 2 mmol),
3-(4-phenylpiperidin-1-yl)propanoic acid (513 mg, 2.2 mmol), EDC
(460 mg, 2.4 mmol) and DMAP (49 mg, 0.4 mmol). Then it was
recrystallized from ethanol. Yield: 577 mg, 73%; mp: 185.1 C. 1H
reduced pressure and the mixture was separated from sodium
̊
chloride with the aid of acetone. The resulting oil gained by evap-
orating acetone was solidified with ether and used in the synthesis
of the butanamide derivatives.
NMR (DMSO‑d6)
d
: 10.19 (s, 1H, NeH), 7.98 (d, 1H, J ¼ 2.0 Hz, H7),
7.47 (dd, 1H, J ¼ 8.8 Hz, J ¼ 2.0 Hz, H5), 7.30e7.15 (m, 6H, H4, phenyl
protons), 3.37 (s, 3H, NeCH3), 3.00 (d, 2H, J ¼ 11.6 Hz, H2e, H6e
-
4.1.9. General procedure for the preparation of 4-
substitutedbutanamide derivatives bearing benzoxazolone or
benzothiazolone (10a-c)
4-Substituted-N-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-
6-yl)butanamide derivative (10a) and 4-substituted-N-(3-methyl-
2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)butanamide derivatives
(10b and 10c) were synthesized by following method B as
piperidine), 2.67 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.51e2.44 (m, 3H,
H4-piperidine, -NeCH2eCH2-, with DMSO), 2.06 (td, 2H, J ¼ 11.6 Hz,
J ¼ 2.0. Hz, H2a, H6a-piperidine), 1.76e1.59 (m, 4H, H3, H5-piperi-
̊
dine). 13C NMR (DMSO‑d6)
d: 170.2, 168.4, 146.1, 135.0, 135.2, 128.2,
126.6,125.9,121.5, 117.9, 113.3, 111.3, 54.0, 53.4, 41.7, 34.2, 33.0, 28.9.
HRMS (ESI) [MþH]þ m/z for C22H26N3O2S calculated: 396.1746,
16

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
mentioned in section 4.1.7 from intermediate 5 or 6 (1 equivalent),
the corresponding butanoic acid (1.1 equivalent), EDC (1.2 equiva-
lent) and DMAP (0.2 equivalent).
4.1.11. General procedure for the preparation of 5-
substitutedpentanamide derivatives bearing benzoxazolone or
benzothiazolone (11a-c)
5-Substituted-N-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-
6-yl)pentanmide derivative (11a) and 5-substituted-N-(3-methyl-
2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)pentanmide derivatives
(11b and 11c) were synthesized by following method B as
mentioned in section 4.1.7 from intermediate 5 or 6 (1 equivalent),
the corresponding pentanoic acid (1.1 equivalent), EDC (1.2 equiv-
alent) and DMAP (0.2 equivalent).
4.1.9.1. 4-(4-Benzylpiperazin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzoxazol-6-yl)butanamide (10a). Compound 10a was ob-
tained by using method B from intermediate 5 (350 mg, 2 mmol), 4-
(4-benzylpiperazin-1-yl)butanoic acid (616 mg, 2.2 mmol), EDC
(491 mg, 2.4 mmol) and DMAP (52 mg, 0.4 mmol). Then it was
recrystallized from ACN. Yield: 153 mg, 18%; mp: 190.3 C. 1H NMR
(DMSO‑d6)
d
: 9.94 (s,1H, NeH), 7.73 (d,1H, J ¼ 1.6 Hz, H7), 7.30e7.20
4.1.11.1. 5-(4-Benzylpiperazin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzoxazol-6-yl)pentanamide (11a). Compound 11a was
obtained by using method B from intermediate 5 (600 mg,
3.6 mmol), 5-(4-benzylpiperazin-1-yl)pentanoic acid (1110 mg,
4 mmol), EDC (841 mg, 4.4 mmol) and DMAP (89 mg, 0.7 mmol).
Then it was recrystallized from acetone-hexane. Yield: 347 mg,
(m, 6H, phenyl protons, H5), 7.15 (d, 1H, J ¼ 1.6 Hz, H4), 3.41 (s, 2H,
eCH2-C6H5), 3.30 (s, 3H, NeCH3), 2.40e2.24 (m, 12H, H-piperazine,
-CH2-CH2-CH2-), 1.72 (p, 2H, J ¼ 7.2 Hz, -CH2-CH2-CH2-). 13C NMR
(DMSO‑d6) d: 171.0, 154.1, 141.7, 138.2, 134.5, 128.7, 128.0, 127.0,
126.8, 114.4, 108.7, 101.4, 62.0, 57.2, 52.7, 52.6, 34.3, 28.0, 22.1. HRMS
(ESI) [MþH]þ m/z for C23H29N4O3 calculated: 409.2240, found:
409.2246. Anal. Calcd. for C23H28N4O3: C, 67.63; H, 6.91; N, 13.72.
Found: C, 67.29; H, 7.29; N, 11.75; S, 13.64.
22%; mp: 138.4 C. 1H NMR (DMSO‑d6)
d: 9.92 (s, 1H, NeH), 7.70 (d,
1H, J ¼ 1.6 Hz, H7), 7.30e7.17 (m, 6H, phenyl protons, H5), 7.12 (d,1H,
J ¼ 8.8 Hz, H4), 3.40 (s, 2H, eCH2-C6H5), 3.28 (s, 3H, NeCH3),
2.36e2.21 (m, 12H, H-piperazine, NeCH2eCH2eCH2eCH2-), 1.56 (p,
2H, J ¼ 7.5 Hz, NeCH2eCH2eCH2eCH2-), 1.41 (p, 2H, J ¼ 7.5 Hz,
4.1.9.2. 4-(4-Benzylpiperazin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzothiazol-6-yl)butanamide (10b). Compound 10b was
obtained by using method B from intermediate 6 (400 mg,
2.2 mmol), 4-(4-benzylpiperazin-1-yl)butanoic acid (641 mg,
2.42 mmol), EDC (512 mg, 2.64 mmol) and DMAP (54 mg,
0.44 mmol). Then it was recrystallized from butanol. Yield: 150 mg,
NeCH2eCH2eCH2eCH2-). 13C NMR (DMSO‑d6)
d: 171.0, 154.1, 141.7,
138.2, 134.5, 128.7, 128.0, 127.0, 126.8, 114.4, 108.7, 101.4, 62.1, 57.5,
52.7, 52.6, 36.1, 27.9, 25.9, 23.0. HRMS (ESI) [MþH]þ m/z for
C
C
24H31N4O3 calculated: 423.2396, found: 423.2387. Anal. Calcd. for
24H30N4O3: C, 68.22; H, 7.16; N, 13.26. Found: C, 67.97; H, 7.45; N,
9.88.
16%; mp: 87.3 C. 1H NMR (DMSO‑d6)
d: 9.92 (s, 1H, NeH), 7.94 (d,1H,
J ¼ 2.0 Hz, H7), 7.41 (dd, 1H, J ¼ 8.8 Hz, J ¼ 2.0 Hz, H5), 7.30e7.18 (m,
6H, phenyl protons, H4), 3.39 (s, 2H, eCH2-C6H5), 3.31 (s, 3H,
NeCH3), 2.38e2.24 (m, 12H, H-piperazine, -CH2-CH2-CH2-), 1.69 (p,
4.1.11.2. 5-(4-Benzylpiperazin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzothiazol-6-yl)pentanamide (11b). Compound 11b was
obtained by using method B from intermediate 6 (600 mg,
3.3 mmol), 5-(4-benzylpiperazin-1-yl)pentanoic acid (1012 mg,
3.6 mmol), EDC (767 mg, 4 mmol) and DMAP (81 mg, 0.7 mmol).
Then it was recrystallized from acetone-hexane. Yield: 420 mg,
2H, J ¼ 7.2 Hz, eCH2-CH2-CH2-). 13C NMR (DMSO‑d6)
d: 170.1, 168.2,
138.1, 135.0, 133.0, 128.6, 128.0, 126.7, 121.3, 117.8, 113.2, 111.1, 62.0,
57.1, 52.6, 52.5, 34.2, 28.8, 22.1. HRMS (ESI) [MþH]þ m/z for
̊
C
C
23H29N4O2S calculated: 425.2011, found: 425.2015. Anal. Calcd. for
23H28N4O2S$3H2O: C, 57.72; H, 7.16; N, 11.71; S, 6.81. Found: C,
41%; mp: 120.8 C. 1H NMR (DMSO‑d6)
d: 9.96 (s, 1H, NeH), 7.97 (d,
1H, J ¼ 2.0 Hz, H7), 7.47 (dd, 1H, J ¼ 9.0 Hz, J ¼ 2.0 Hz, H5), 7.31e7.21
(m, 6H, phenyl protons, H4), 3.43 (s, 2H, eCH2-C6H5), 3.36 (s, 3H,
NeCH3), 2.40e2.24 (m,12H, H-piperazine, NeCH2eCH2eCH2eCH2-
), 1.59 (p, 2H, J ¼ 7.4 Hz, NeCH2eCH2eCH2eCH2-), 1.44 (p, 2H,
57.49; H, 7.00; N, 11.80; S, 6.70.
4.1.9.3. 4-(4-Phenylpiperidin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzothiazol-6-yl)butanamide (10c). Compound 10c was
obtained by using method B from intermediate 6 (500 mg,
2.8 mmol), 4-(4-phenylpiperidin-1-yl)butanoic acid (756 mg,
3.1 mmol), EDC (639 mg, 3.4 mmol) and DMAP (68 mg, 0.6 mmol).
Then it was recrystallized from 2-propanol. Yield: 600 mg, 53%;
J ¼ 7.4 Hz, NeCH2eCH2eCH2eCH2-). 13C NMR (DMSO‑d6)
d: 171.0,
168.3, 138.2, 135.1, 133.1, 128.7, 128.0, 126.8, 121.4, 117.9, 113.3, 111.2,
̊
62.0, 57.5, 52.7, 52.6, 36.1, 28.9, 25.8, 23.0. HRMS (ESI) [MþH]þ m/z
for C24H31N4O2S calculated: 439.2168, found: 439.2188. Anal. Calcd.
for C24H30N4O2S.2,5H2O: C, 59.60; H, 7.29; N, 11.58; S,6.63. Found:
C, 59.50; H, 7.18; N, 11.97; S, 6.49.
mp: 104.6 C. 1H NMR (DMSO‑d6)
d: 9.95 (s, 1H, NeH), 7.99 (d, 1H,
J ¼ 2.0 Hz, H7), 7.48 (dd, 1H, J ¼ 8.6 Hz, J ¼ 2.0 Hz, H5), 7.23e7.07 (m,
6H, phenyl protons, H4), 3.35 (s, 3H, NeCH3), 2.91 (d, 2H, J ¼ 11.4 Hz,
H2e, H6e-piperidine), 2.44e2.28 (m, 5H, H4-piperidine, -N-CH2-
CH2-CH2-), 1.91 (td, 2H, J ¼ 11.4 Hz, J ¼ 1.8 Hz, H2a, H6a-piperidin),
1.76 (p, 2H, J ¼ 6.8 Hz, -NeCH2eCH2-CH2-) 1.68e1.48 (m, 4H, H3,
4.1.11.3. 5-(4-Phenylpiperidin-1-yl)-N-(3-methyl-2-oxo-2,3-
dihydrobenzothiazol-6-yl)pentanamide (11c). Compound 11c was
obtained by using method B from intermediate 6 (600 mg,
3.3 mmol), 5-(4-phenylpiperidin-1-yl)pentanoic acid (967 mg,
3.6 mmol), EDC (767 mg, 4 mmol) and DMAP (81 mg, 0.7 mmol).
Then it was recrystallized from 2-propanol. Yield: 340 mg, 24%;
H5-piperidine). 13C NMR (DMSO‑d6)
d: 171.2, 168.4, 146.3, 135.4,
̊
133.0, 128.1, 126.6, 125.9, 121.4, 117.8, 113.1, 111.2, 57.7, 53.7, 41.9,
34.7, 33.1, 28.9, 22.4. HRMS (ESI) [MþH]þ m/z for C23H28N3O2S
calculated: 410.1902, found: 410.1899. Anal. Calcd. for
mp: 169.2 C. 1H NMR (DMSO‑d6)
d: 9.96 (s, 1H, NeH), 7.95 (d, 1H,
J ¼ 2.4 Hz, H7), 7.46 (dd, 1H, J ¼ 8.8 Hz, J ¼ 2.4 Hz, H5), 7.26e7.12 (m,
6H, phenyl protons, H4), 3.37 (s, 3H, NeCH3), 2.92 (d, 2H, J ¼ 11.5 Hz,
H2e, H6e-piperidine), 2.49e2.26 (m, 5H, H4-piperidine, -N-CH2-
C
23H27N4O2S$3H2O: C, 63.94; H, 6.88; N, 9.73; S, 7.42. Found: C,
63.96; H, 7.07; N, 9.88; S, 7.51.
CH2-CH2-CH2-), 1.92 (td, 2H, J ¼ 11.5 Hz, J ¼ 2.4 Hz, H2a, H6a
-
piperidine), 1.70e1.54 (m, 6H, H3, H5-piperidine, -NeCH2eCH2-
4.1.10. General procedure for the preparation of 5-
substitutedpentanoic acid intermediates
CH2-CH2-) 1.46 (p, 2H, J ¼ 7.5 Hz, -NeCH2eCH2-CH2-). 13C NMR
̊
(DMSO‑d6) d: 171.1, 168.3, 146.3, 135.1, 133.1, 128.2, 126.6, 125.9,
5-Substitutedpentanoic acid intermediates were produced by
following the procedure in section 4.1.8 from appropriate amin
derivatives (1 equivalent), ethyl 5-bromovalerate (1 equivalent)
and potassium carbonate (1.5 equivalent).
121.4, 117.9, 113.3, 111.2, 57.9, 53.8, 42.0, 33.1, 28.9, 26.1, 23.1. HRMS
(ESI) [MþH]þ m/z for C24H30N3O2S calculated: 424.2059, found:
424.2048. Anal. Calcd. for C24H29N3O2S: C, 68.05; H, 6.90; N, 9.92; S,
7.55. Found: C, 68.10; H, 7.20; N, 9.90; S, 7.56.
17

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
4.1.12. 6-(3-Chloropropanoyl)-3-methylbenzoxazol-2(3H)-one (12)
and 6-(3-chloropropanoyl)-3-methylbenzothiazol-2(3H)-one (13)
2H, J ¼ 7.2 Hz, -CH2-CH2-N-), 2.51e2.42 (m, 1H, H4-piperidine), 2.06
(td, 2H, J ¼ 11.6 Hz, J ¼ 2.1 Hz, H2a, H6a-piperidine), 1.76e1.50 (m,
6-(3-Chloropropanoyl)-3-methylbenzoxazol-2(3H)-one
(12)
4H, H3, H5-piperidine). 13C NMR (DMSO‑d6)
d: 197.7, 169.3, 146.2,
and 6-(3-chloropropanoyl)-3-methylbenzothiazol-2(3H)-one (13)
were synthesized according to the previously reportedþmethod
[33]. Yield for (12): 72%.; mp: 184 ꢀC. HRMS (ESI) [MþH] m/z for
141.2, 131.9, 128.2, 126.9, 126.6, 125.9, 123.1, 121.6, 111.0, 53.7, 53.3,
41.7, 35.8, 33.0, 29.2. HRMS (ESI) [MþH]þ m/z for C22H25N2O2S
calculated: 381.1637, found: 381.1625. Anal. Calcd. for C22H24N2O2S:
C, 69.44; H, 6.37; N, 7.36; S, 8.43. Found: C, 69.29; H, 6.77; N, 7.45; S,
8.73.
C
11H10ClNO3 calculated: 240.0427, found: 240.0423. Yield for (13):
85%; mp: 132 ꢀC. HRMS (ESI) [MþH]þ m/z for C11H10ClNO2S
calculated: 256.0199, found: 256.0192.
4.1.14. 6-Nitrobenzothiazole (15)
4.1.13. General procedure for the preparation of 6-(3-
substitutedpropanoyl)-3-methylbenzoxazol-2(3H)-one (14a-c) and
6-(3-substitutedpropanoyl)-3-methylbenzothiazol-2(3H)-one (15a-
c) derivatives
6-(3-Substitutedpropanoyl)-3-methylbenzoxazol-2(3H)-one
(14a) and 6-(3-substitutedpropanoyl)-3-methylbenzothiazol-
2(3H)-one (14b and 14c) derivatives were synthesized by following
method A as mentioned in section 4.1.5 from intermediate 12 or 13
(1 equivalent), the corresponding amine derivative (1.5 equivalent),
NaI (3 equivalent) and K2CO3 (2.5 equivalent).
Commercially available benzothiazole (3 g, 22 mmol) was taken
to the flask and 6 ml 98% sulfuric acid was added in ice bath. After
stirring for a while 65% nitric acid was added in portions. The
experiment was completed by keeping the reaction mixture be-
tween 4 and 6 ꢀC. The mixture was poured into ice water and the
precipitated was filtered and recrystallized from ethanol. Yield:
33%, mp: 173e174 ꢀC [34].
4.1.15. 2-Amino-5-nitrobenzenethiol (16) (CAS 23451-98-1)
15 (500 mg, 2.8 mmol) was refluxed with 30 ml 20% potassium
hydroxide solution for 5 h. After the completion of the reaction, the
mixture was cooled and neutralized with 37% HCl solution. The
precipitate was collected by filtration and used in the next step
without further purification. Yield: 59%, mp: 87e89 ꢀC.
4.1.13.1. 3-Methyl-6-(3-(4-benzylpiperazin-1-yl)propanoyl)benzox-
azol-2(3H)-one (14a). The intermediate 12 (300 mg, 1.3 mmol) was
treated with 1-benzylpiperazine (343 mg, 2 mmol) in the presence
of NaI (563 mg, 3.9 mmol) and anhydrous potassium carbonate
(456 mg, 3.3 mmol) according to method A then, it was recrystal-
lized from ethanol. Yield: 202 mg, 43%; mp: 129.6 C. 1H NMR
4.1.16. 6-Nitro-2-phenylbenzoxazole (17) and 6-nitro-2-
phenylbenzothiazole (18)
(DMSO‑d6)
d
: 7.93 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.6 Hz, H5), 7.86 (d, 1H,
2-Amino-5-nitrophenol (commercially available) (1 g,
6.5 mmol) or 2-amino-5-nitrobenzenethiol (16) (1.1 g, 6.5 mmol)
and (0.79 g, 6.5 mmol) benzoic acid were taken in a round bottom
flask and covered with polyphosphoric acid. The reaction mixture
was stirred in oil bath at 140 ꢀC for 2 h. After this period, the
mixture was cooled, poured into ice water, and then neutralized
with sodium hydroxide solution. The precipitate was filtered, dried,
and used in the next step. For (17) Yield: 90%, mp: 180 ꢀC. For (18)
Yield: 82%, mp: 188 ꢀC [35].
J ¼ 1.6 Hz, H7), 7.34 (d, 1H, J ¼ 8.4 Hz, H4), 7.33e7.21 (m, 5H, phenyl
protons), 3.43 (s, 2H, eCH2-C6H5), 3.37 (s, 3H, NeCH3), 3.16 (t, 2H,
J ¼ 7.2 Hz, eCH2-CH2-N), 2.65 (t, 2H, J ¼ 7.2 Hz, -CH2-CH2-N-),
2.42e2.34 (m, 8H, H-piperazine). 13C NMR (DMSO‑d6)
d: 197.6,
154.1,141.8,138.2,135.9,131.2,128.7,128.0,126.8,125.0,108.7,108.6,
62.0, 53.0, 52.6, 52.5, 35.6, 28.2. HRMS (ESI) [MþH]þ m/z for
C
C
22H26N3O3 calculated: 380.1974, found: 380.1989. Anal. Calcd. for
22H25N3O3: C, 69.64; H, 6.64; N, 11.07. Found: C, 69.24; H, 6.84; N,
11.05.
4.1.17. 6-Amino-2-phenylbenzoxazole (19) and 6-amino-2-
phenylbenzothiazole (20)
4.1.13.2. 3-Methyl-6-[3-(4-benzylpiperazin-1-yl)propanoyl]benzo-
thiazol-2(3H)-one (14b). The intermediate 13 (300 mg, 1.2 mmol)
was treated with 1-benzylpiperazine (317 mg, 1.8 mmol) in the
presence of NaI (540 mg, 3.6 mmol) and anhydrous potassium
carbonate (415 mg, 3 mmol) according to method A then, it was
recrystallized from ethanol. Yield: 100 mg, 22%; mp: 99.9 C. 1H
The solution 6-nitro-2-phenylbenzoxazole (17) (0.9 g, 3.8 mmol)
or 6-nitro-2-phenylbenzothiazole (18) (0.96 g, 3.8 mmol) in 40 ml
ethanol was refluxed with 7 ml 1.2 N HCl. After starting reflux for a
while, iron (0.6 g, 11 mmol) was added and stirred under reflux
condition till the completion of reaction. Then the reaction mixture
was filtered through Celite® to remove iron. The filtrate was taken
and made alkaline with sodium hydroxide solution. The precipitate
was filtered and used without further purification. Yield for (19):
48%, mp: 173 ꢀC. Yield for (20): 40%, mp: 206 ꢀC [35].
NMR (DMSO‑d6)
d
: 8.31 (d, 1H, J ¼ 2.0 Hz, H7), 7.97 (dd, 1H,
J ¼ 8.4 Hz, J ¼ 2.0 Hz, H5), 7.37 (d, 1H, J ¼ 8.4 Hz, H4), 7.30e7.18 (m,
5H, phenyl protons), 3.41 (s, 2H, eCH2-C6H5), 3.40 (s, 3H, NeCH3),
3.14 (t, 2H, J ¼ 7.2 Hz, eCH2-CH2-N), 2.63 (t, 2H, J ¼ 7.2 Hz, -CH2-
CH2-N-), 2.40e2.32 (m, 8H, H-piperazine). 13C NMR (DMSO‑d6)
d:
197.6, 169.3,141.2, 138.2,131.8,128.7, 128.1,126.9,126.8,123.2,121.6,
4.1.18. General procedure for the preparation of compounds 21a
and 21b
111.0, 62.0, 53.0, 52.7, 52.6, 35.6, 29.2. HRMS (ESI) [MþH]þ m/z for
̊
C
C
22H26N3O2S calculated: 396.1746, found: 396.1734. Anal. Calcd. for
22H25N3O2S: C, 66.81; H, 6.37; N, 10.62; S, 8.11. Found: C, 66.44; H,
3-(4-Benzylpiperazin-1-yl)-N-(2-phenylbenzoxazol-6-yl)prop-
anamide
(21a)
and
3-(4-benzylpiperazin-1-yl)-N-(2-
6.54; N, 10.57; S, 7.95.
phenylbenzothiazol-6-yl)propanamide (21b) were synthesized by
method B as mentioned in section 4.1.7 from intermediate 19 or 20
(1 equivalent), 3-(4-benzylpiperazine-1-yl)propanoic acid (1.1
equivalent), EDC (1.2 equivalent) and DMAP (0.2 equivalent).
4.1.13.3. 3-Methyl-6-[3-(4-phenylpiperidin-1-yl)propanoyl]benzo-
thiazol-2(3H)-one (14c). The intermediate 13 (500 mg, 2 mmol)
was treated with 1-phenylpiperidine (476 mg, 3 mmol) in the
presence of NaI (900 mg, 6 mmol) and anhydrous potassium car-
bonate (691 mg, 5 mmol) according to method A then, it was
recrystallized from acetone-hexane. Yield: 53 mg, 7%; mp: 103.7 C.
4.1.18.1. 3-(4-Benzylpiperazin-1-yl)-N-(2-phenylbenzoxazol-6-yl)
propanamide (21a). Compound 21a was obtained by using method
B from intermediate 19 (450 mg, 2.1 mmol), 3-(4-benzylpiperazin-
1-yl)propanoic acid (585 mg, 2.4 mmol), EDC (493 mg, 2.6 mmol)
and DMAP (52 mg, 0.4 mmol). Then it was recrystallized from
acetone-hexane. Yield: 155 mg, 16%; mp: 139.5 C. 1H NMR
1H NMR (DMSO‑d6)
d
: 8.33 (d, 1H, J ¼ 1.3 Hz, H7), 8.30 (dd, 1H,
J ¼ 8.5 Hz, J ¼ 1.3 Hz, H5), 7.41 (d, 1H, J ¼ 8.5 Hz, H4), 7.30e7.15 (m,
5H, phenyl protons), 3.45 (s, 3H, NeCH3), 3.22 (t, 2H, J ¼ 7.2 Hz,
eCH2-CH2-N-), 3.01 (d, 2H, J ¼ 11.6 Hz, H2e, H6e-piperidine), 2.71 (t,
(DMSO‑d6) d
: 10.37 (s, 1H, NeH), 8.25 (s, 1H, H7), 8.18e8.15 (m, 2H,
18
̊

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
H2’, H6’), 7.70 (d, 1H, J ¼ 8.4 Hz, H4), 7.61e7.58 (m, 3H, H3’, H4’, H5’),
7.37 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.6 Hz, H5), 7.32e7.21 (m, 5H, phenyl
protons), 3.44 (s, 2H, eCH2-C6H5), 2.65 (t, 2H, J ¼ 6.4 Hz, -N-CH2-
CH2-), 2.50e2.47 (m, 2H, -NeCH2eCH2-, with DMSO), 2.45e2.38
using modified Ellman’s method for the measurement of residual
enzyme activities of dilutions [36]. The enzyme AChE/BChE (0.5 U/
mL), ATI/BTI (0.01 M) and DTNB (0.01 M) were diluted in tris-HCl
buffer (pH-8). Briefly, 10 ꢁ IC50 and 100 ꢁ IC50 concentrations of
(m, 8H, piperazine). 13C NMR (DMSO‑d6)
d: 170.3, 161.8, 150.3, 138.1,
these inhibitors were incubated with 40
mL of diluted enzyme at
137.1, 136.8, 131.5, 129.1, 128.7, 128.0, 126.9, 126.7, 126.4, 119.5, 116.4,
37 ꢀC for 30 min, then 20
mL of DTNB solution was added. After this
101.1, 61.9, 53.5, 52.5, 52.3, 34.1. HRMS (ESI) [MþH]þ m/z for
incubation period, the reaction was subsequently diluted 100-fold
with substrate solution to give final concentrations of inhibitors
0.1 ꢁ IC50 and 1 ꢁ IC50, respectively. In control studies, inhibitors
were replaced with donepezil or buffer. The reaction was further
incubated at 37 ꢀC for a further 15 min and the residual enzyme
activities were measured, and the bar graphs were constructed. All
measurements were carried out in triplicate and are expressed as
mean SEM.
C
C
27H29N4O2 calculated: 441.2291, found: 441.2275. Anal. Calcd. for
27H28N4O2: C, 73.61; H, 6.41; N, 12.72. Found: C, 73.42; H, 6.75; N,
12.73.
4.1.18.2. 3-(4-Benzylpiperazin-1-yl)-N-(2-phenylbenzothiazol-6-yl)
propanamide (21b). Compound 21b was obtained by using method
B from intermediate 20 (400 mg, 1.7 mmol), 3-(4-benzylpiperazin-
1-yl)propanoic acid (483 mg, 1.9 mmol), EDC (407 mg, 2.1 mmol)
and DMAP (43 mg, 0.3 mmol). Then it was recrystallized from
acetone-hexane. Yield: 200 mg, 25%; mp: 162.9 C. 1H NMR
4.2.3. Kinetic characterization of cholinesterase inhibition
Enzyme kinetic studies were performed similar as enzyme in-
hibition assay. The increase of the absorbances was measured with
different inhibitor concentrations and without inhibitor for pro-
posed substrate concentrations. To determine the inhibition model,
reciprocal plots of 1/V versus 1/[S] were constructed by using re-
ported method with minor modifications. Slopes of the reciprocal
plots were plotted against the concentration of inhibitor, for esti-
mation of Ki. All processes were assayed in triplicate and data
analysis was performed with GraphPad Prism software (Version
7.0). The results were displayed as mean SD [29,30].
(DMSO‑d6)
d
: 10.36 (s, 1H, NeH), 8.51 (d, 1H, J ¼ 1.2 Hz, H7),
8.04e8.01 (m, 2H, H2’, H6’), 7.95 (d, 1H, J ¼ 8.8 Hz, H4), 7.54e7.50 (m,
4H, H5, H3’, H4’,H5’), 7.31e7.19 (m, 5H, phenyl protons), 3.42 (s, 2H,
eCH2-C6H5), 2.63 (t, 2H, J ¼ 7.0 Hz, -N-CH2-CH2-), 2.50e2.47 (m, 2H,
-NeCH2eCH2-, with DMSO), 2.42e2.36 (m, 8H, piperazine). 13C
NMR (DMSO‑d6) d: 170.3, 165.5, 149.3, 138.1, 136.9, 135.1, 132.8,
131.0, 129.2, 128.7, 128.0, 126.8, 126.7, 122.8, 118.8, 111.1, 61.9, 53.6,
52.5, 52.3, 34.0. HRMS (ESI) [MþH]þ m/z for C27H29N4OS calculated:
457.2062, found: 457.2078. Anal. Calcd. for C27H28N4OS: C, 71.02; H,
6.18; N, 12.27; S, 7.02. Found: C, 70.62; H, 6.48; N, 12.26; S, 7.06.
4.2.4. Oxygen radical absorbance capacity (ORAC-Fluorescein)
assay
4.2. Biological studies
The antioxidant activity was determined by the oxygen radical
absorbance capacity fluorescein (ORAC-FL) method [40,41]. Each
assay was conducted with 75 mM phosphate buffer (pH 7.4), and
4.2.1. Cholinesterase inhibition assay
AChE and BChE % inhibitory activities of the 42 test compounds
at 100
mM concentration were determined by the modified Ellman’s
the final reaction mixture was 200
mL. Test samples (20 mL) and
method and compounds displaying more than 50% inhibition were
tested for IC50 determination. The formation of the yellow color was
measured at 412 nm using molecular devices, Versamax microplate
reader. Electric eel AChE (type-VI-S, EC 3.1.1.7), recombinant human
AChE, equine serum BChE (EC 3.1.1.8), recombinant human BChE,
5,50-dithiobis-2-nitrobenzoic acid (DTNB), acetylthiocholine iodide
(ATI), butyrylthiocholine iodide (BTI) and Tris HCl were purchased
fluorescein (120 L, 150 nM final concentration) were placed in the
m
wells of a black 96-well plate. The mixture was pre-incubated for
15 min at 37 ꢀC, and then AAPH (i.e., 2,20- azobis(2-
methylpropionamidine) dihydrochloride (AAPH) solution (60
mL,
12 mM final concentration) was added rapidly using an autosam-
pler. The plate was immediately placed in a Varioskan Flash
Multimode Reader (Thermo Scientific) and the fluorescence
recorded every minute for 90 min with excitation at 485 nm and
emission at 535 nm. The plate was automatically shaken prior to
from Sigma Aldrich. Donepezil and Galantamine were used as the
̊
reference drug at 10 mM concentration. Sample compounds were
dissolved in DMSO (DMSO was 1% and has no inhibitory effect at
this amount). For AChE inhibitory activity tested, in each well of the
each reading. Trolox was used as standard (0.5e8 mM, final con-
centration). A blank (FL þ AAPH) using phosphate buffer instead of
plate were contained 168
10 L of 6.8 mM DTNB solution, 10
and 2
L of each sample solution and incubated for 10 min at 25 ꢀC.
Then the reactions were initiated by the addition of 10 L ATI so-
mL of 50 mM of Tris HCl buffer (pH 8.0),
antioxidant and trolox calibration were carried out in each assay.
m
mL of AChE solution (0.4 U/mL),
The samples were measured at 10 mM concentration. All the reac-
m
tion mixture was prepared in duplicate, and three independent
assays were performed for each sample. Antioxidant curves (fluo-
rescence versus time) were normalized to the curve of the blank in
the same assay, and then the area under the fluorescence decay
curve (AUC) was calculated. The net AUC of a sample was obtained
by subtracting the AUC of the blank. ORACFL values were expressed
as Trolox equivalents by using the standard curve calculated for
each sample, where the ORAC-FL value of Trolox was taken as 1,
indicating the antioxidant potency of the tested compounds.
m
lution (10 mM). Changes in absorbance was recorded for 10 min at
412 nm. As a control, an identical solution of the reaction mixture
without the sample solution was processed following the same
protocol. The blank reading contained 178
mL buffer, 10 mL DTNB,
2
m
L DMSO and 10 L ATI solution. BChE inhibitory activities of the
m
test compounds were measured through the same methodology
described, except employing 0.8 U/mL BChE solution as the enzyme
source and the BTI solution (30 mM) as the substrate of the reac-
tion. Each experiment was performed in triplicate; absorbance
values were corrected by subtracting the absorbance of blank and
the inhibition rate (%) was calculated. To determine the IC50 values,
graphic from log [inhibitor] vs. % inhibition was analyzed with the
use of GraphPad Prism software (Version 7.0). The results were
displayed as mean SD [29,30].
4.2.5. Inhibiting inflammatory factors production of LPS-stimulated
THP1 cells
4.2.5.1. Cell culture and LPS stimulation of THP1 cells. The THP1 cell
line was obtained from Sigma by Atlas Biotechnology, and cultured
in complete medium (RPMI 1640 medium supplemented with 10%
FCS, 100 U/mL penicillin, and 100 m
g/ml streptomycin) at 37 ꢀC in a
humidified incubator containing 5% CO2. After overnight culture in
4.2.2. Reversibility studies for AChE/BuChE inhibition
24-well plate (4 ꢁ 104 cells/well), the cells were treated with or
Reversibility studies were carried with the dilution method
without 10 mM concentration of compound that were added 1 h
19

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
prior to with 100 ng/ml LPS from Escherichia coli serotype O111:B4
(Sigma Aldrich, USA) for 24 h. Untreated cells were used as a
negative control. Cells treated with LPS were used as an inflam-
mation positive group. After 24 h, to determine the effect of
selected compounds on cytokine levels from experimental test
group. All experiments were at least in triplicate [42,43].
at 37 ꢀC for 4 h. After the incubation, the supernatant was removed,
the formazan crystals were dissolved in 0.1 ml of SDS and the plates
were incubated for 24 h. Then, the optical densities were read at
570 nm in multi-well spectrophotometer (Thermo Fisher Scientific,
Finland). The relative cell viability of the test group was calculated
using the following formula: relative cell viability of the test group
(%) ¼ (optical density of the test group/optical density of the un-
treated group) ꢁ 100. All controls and test groups were performed
at least in triplicates [29,30].
4.2.5.2. Nitric oxide (NO) production and cytokine measurement.
After overnight culture in a 24-well plate (4 ꢁ 104 cells/well, 500
mL
medium/well), the cells were treated with or without various
concentrations of madde isimleri that were added 1 h prior to with
100 ng/ml LPS from Escherichia coli serotype O111:B4 (Sigma
Aldrich, USA) for 24 h. After incubation period, Cell-free superna-
tants were collected and stored at ꢂ20 ꢀC until assayed for NO
levels. The concentrations of NO in the supernatants of THP1 cell
cultures were determined using an ELISA kit, according to the
manufacturer’s instructions (Bt-Laboratory). Total protein extrac-
tion from the cell pellets according to the GeneALL ProtinExTM
Animal cell/tissue Protein Isolation Kit (GeneALL Biotechnology).
Human Interleukin 1 Beta, Human Interleukin 6, and Human Tumor
Necrosis Factor Alpha (Bt-Laboratory) levels of protein samples
were measured by enzyme immunoassay (EIA). Absorbance read-
ings were carried out 450 nm using Microplate Reader (Thermo
Fisher Scientific, Finland). Concentrations of unknown samples
were determined from a standard curve obtained with the stan-
dards. According to the kit protocol, the sensitivities were 10,07 pg/
4.2.9. Statistical analysis
According to the experimental design, at least 3 independent
experiments were performed, and the data were presented as
means standard deviation (means SD) values. One-way ANOVA
with post hoc analysis by using Tukey comparison test was per-
formed for multiple comparisons between groups. P value < 0.05
was considered as statistically significant. Statistics were evaluated
using SPSS 17.0.
4.3. Assessment of physicochemical parameters
Calculated descriptors (molecular weight, log P, topological
polar surface area (tPSA), volume, number of hydrogen donors,
number of hydrogen acceptors and number of violations of Lip-
inski’s rule) of the compounds were computed employing the
Molinspiration online service [47]. Blood brain barrier (BBB)
permeability values were calculated by admetSAR online service
[48].
ml, 1,03 ng/l, 1,52 ng/l, and 1,12 mmol/l for Human Interleukin 1
Beta, Human Interleukin 6, Human Tumor Necrosis Factor Alpha,
and Human Nitric Oxide respectively [43].
4.2.6. Ab aggregation inhibition assay/thioflavin T (ThT) assay
All the chemicals used for the assay purchased from Sigma-
Aldrich and measurements carried out at Thermo Biolite 24 well
multidish plate by using SpectraMax i3x Multi-Mode microplate
reader. To investigate the effect of the selected compounds and
references (Donepezil and phenol red) on the aggregation of Ab1ꢂ42
at 50 mM concentration, a reported thioflavin T-based fluorometric
assay was performed. Samples were dissolved in DMSO and
analyzed in triplicate [44].
4.4. Molecular modeling studies
Before the molecular docking, the geometry of the initial
structures of ligands considered was optimized by using the
Gaussian 09 software [49] with the semi-empirical parametric
method 6 (PM6) [50]. The crystal structures of the AChE and BChE
enzymes were retrieved from the RCSB Protein Data Bank (http://
www.rcsb.org/pdb/), under the accession codes 1EVE [39] and
4BDS [51], respectively. Molecular Operating Environment (MOE)
v2014.0901 [52] was used for molecular docking studies. The co-
crystallized bound compounds and water molecules farther than
4.5 Å from ligand or receptor were deleted from the crystal struc-
ture. Water molecules closer than 4.5 Å were conserved since
water-mediated contacts were reported as crucial for binding and
specificity [39]. Enzyme-ligand complexes were energy minimized
to a gradient of 0.01 kcal/(mol Å), and protonated by means of the
force field AMBER99. Charges on the enzyme and ligands were
assigned using the force fields AMBER99 and MMF94X, corre-
spondingly. Possible ligand-binding sites were identified by the
“Site Finder” module of the MOE. For each ligand, Triangle Matcher
Algorithm and scoring function of London dG was used for ranking
1000 docked poses. Then a force field refinement at default pa-
rameters was carried out for the 30 poses followed by a rescoring of
GBVI/WSA dG. All poses generated with docking were analyzed and
the best-scored pose with the lowest binding free energy for each
compound was selected for further investigation of interactions
with the corresponding enzyme.
4.2.7. Annexin V-FITC/PI apoptosis measurement
Annexin V/PI binding assay based on the cell membrane exter-
nalization of phosphatidylserine, apoptosis detection procedure
[45,46]. 3T3 fibroblast cells were treated with 10 mM concentrations
of compounds for 24 h. After incubation period, control and test
groups were trypsinized, washed PBS and cells were stained with
Annexin V conjugated with FITC and PI using the Annexin V-FITC/PI
Apoptosis Detection Kit (BD Pharmingen, USA), according to the
manufacturer’s protocols. Then, viable and apoptotic cells were
analyzed with the BD Accuri C6 Flow Cytometer (BD Bioscience,
USA). Untreated cells served as a negative control.
4.2.8. Cell culture and cell proliferation test (MTT assay)
3T3 fibroblast cell line (ATCC CCL92) was kindly provided by Sap
Institute Cell Bank. The cells were cultured as adherent monolayers
in T25 tissue culture plates in DMEM supplemented with 10%
inactivated fetal bovine serum (Sigma-Aldrich, USA), 2 mM l-
glutamine, 100 U/mL penicillin, and 100 mg/ml streptomycin at
37 ꢀC in a humidified incubator containing 5% CO2. The cells incu-
bated with selected compounds at concentrations 0.1e10
m
M. At
The docking procedure was validated by re-docking the co-
crystallized ligands of AChE and BChE. The most energetically sta-
ble ligand poses obtained by re-docking were largely similar with
the ligands in the crystal structure of enzymes with the root mean
square deviation (RMSD) values of 0.196 Å for AChE and 0.149 Å for
BChE.
the end of 24 h, the cell viability was determined MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) Cell Pro-
liferation Kit (Roche Diagnostics, Germany) according to the man-
ufacturer’s protocol. 10
ml of MTT solution was added to wells and
plates were incubated in a humidified atmosphere of 5% CO2 in air
20

M. Erdogan, B. Kilic, R.I. Sagkan et al.
European Journal of Medicinal Chemistry 212 (2021) 113124
molecules24081583.
Declaration of competing interest
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free radicals in oxidative stress-related human diseases, Trends Pharmacol.
Sci. 38 (2017) 592e607, https://doi.org/10.1016/j.tips.2017.04.005.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
The authors declare no conflict of interest.
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