Design, synthesis and biological evaluation of new 2-benzoxazolinone derivatives as potential cholinesterase inhibitors for therapy of Alzheimer's disease

Article abstract of DOI:10.1691/ph.2011.0815

Currently acetylcholinesterase inhibitor (AChEI) therapy is one of the most frequently used methods in the treatment of Alzheimer's disease; tacrine, donepezil, rivastygmine and galantamine are applied in different stages of AD. In the present study, we p

Full text of DOI:10.1691/ph.2011.0815

ORIGINAL ARTICLES
Department of Pharmaceutical Chemistry and Drug Analysis, Medical University of Lodz, Poland
Design, synthesis and biological evaluation of new 2-benzoxazolinone
derivatives as potential cholinesterase inhibitors for therapy of Alzheimer’s
disease
˙
P. Szyman´ski, A. Janik, E. Zurek, E. Mikiciuk-Olasik
Received November 19, 2010, accepted August 8, 2010
Prof. Dr. Elz˙bieta Mikiciuk-Olasik, Department of Pharmaceutical Chemistry and Drug Analysis, Medical University
of Lodz, ul. Muszynskiego 1, 90-151 Lodz, Poland
elzbieta.mikiciuk-olasik@umed.lodz.pl
Pharmazie 66: 399–403 (2011)
doi: 10.1691/ph.2011.0815
Currently acetylcholinesterase inhibitor (AChEI) therapy is one of the most frequently used methods in
the treatment of Alzheimer’s disease; tacrine, donepezil, rivastygmine and galantamine are applied in
different stages of AD. In the present study, we propose a new series of 2-benzoxazolinone derivatives
as potential cholinesterase inhibitors. These compounds were synthesized by condensation of 6-chloro
acetyl-2-benzoxa zolinone with the corresponding amine and evaluated as acetylcholinesterase inhibitors
using the colorimetric Ellman’s method. Selectivity and the IC50 values were determined for the received
derivatives. All tested compounds exhibited the inhibitory activity towards acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE). Compound 3e showed stronger activity than the standard tacrine, and
compound 3a showed activity similar to that of tacrine for AChE. Compounds 3a, 3b, 3c, and 3e showed
stronger activity than the standard donepezil towards the inhibition of BChE, and the compound 3e showed
stronger activity than donepezil towards AChE.
1. Introduction
basis of the cholinergic hypothesis the first group of drugs –
acetylcholinesterase inhibitors (AChEI) – were introduced for
AD treatment. The first one approved in the United States for
the treatment of Alzheimer’s disease was tacrine, at present there
are four inhibitors registered by the Food and Drug Adminis-
tration (FDA): tacrine, rivastigmine, galantamine and donepezil
(Musial et al. 2007; Shah et al. 2008; Sonkusare et al. 2005;
Calabria et al. 2009).
In the present study, synthesis and biological evaluation of
a series of 2-benzoxazolinone derivatives coupled with the
N-substituted piperazines and primary aryl-aliphatic amines
are described. All obtained compounds were tested for their
selectivity and activity towards the inhibition of acetyl-
cholinesterase and butyrylcholinesterase with the colorimetric
Ellman’s method (Szymanski et al. 2006; Calis et al. 2001).
Alzheimer’s disease (AD) is the most common form of dementia
in the elderly. It is a neurodegenerative disorder characterized
by progressive degeneration of some parts of the brain such
as: hippocampus and associative regions of cerebral cortex.
Irreversible degeneration of cholinergic neurons and synapses
leads to disturbances in memory, cognitive functions, learning,
speech, and emotional behaviour, causing full dependency to
a caregiver and finally death. Alzheimer’s disease affects at
present about 5–10% of the population aged 65 and nearly 50%
over the age of 80 (Magierski et al. 2004; Blennow et al. 2006;
Ellis 2005).
Senile dementia of the Alzheimer type is characterized by
lesionsatthemicro-andmacroscopiclevel. Macroscopiclesions
are manifested by dramatic atrophy of certain parts of the brain
like frontal, parietal and temporal lobes. At the microscopic
level the most characteristic changes are: the presence of neu-
ritic or senile plaques and neurofibrillary tangles. The plaques
are composed of the insoluble form of beta-amyloid which
are deposited around nerves and cells. Neurofibrillary tangles,
deposited within the cells, are mostly made up of hyperfosfory-
lated tau protein (Blennow et al. 2006; Bolognesi et al. 2005;
Musial et al. 2007). The third characteristic lesion is the loss of
number of cholinergic neurons and as a result the decrease in
cholinergic neurotransmission (Bolognesi et al. 2005). On the
basis of many observations two hypotheses have been formu-
lated which try to explain the etiopathogenesis of Alzheimer’s
disease. They are called the amyloid cascade hypothesis and
the cholinergic hypothesis (Magierski et al. 2004; Racchi and
Govoni 2003; Bartus et al. 1982; Sugimoti 2008). On the
2. Investigations and results
Compounds 2a–2c were obtained by the reaction of
6-chloroacethyl-2-benzoxazolinone with the corresponding 4-
substituted aryl-aliphatic amines: 4-bromobenzylamine (2a),
4-bromophenethylamine (2b), and 4-chlorophenethylamine
(2c) in acetonitrile (reflux at 80 ◦C/2 h; Scheme 1). The reaction
was conducted in the presence of argone.
Compounds 2d-2 g were obtained by the reaction of
6-chloroacethyl-2-benzoxazolinone with the corresponding
amines: N-benzylpiperazine (2d), N-phenylpiperazine (2e), N-
methylpiperazine (2f) and N-hydroxyethylpiperazine (2 g) in
acetone (reflux at 60 ◦C, 2 or 4 h; Scheme 2). The reac-
tion was conducted in the presence of argone. Our synthesis
Pharmazie 66 (2011)
399

ORIGINAL ARTICLES
(CH2)n
H
N
H
N
a
+
H2N
Cl
O
O
(CH2)n
NH
X
O
O
O
O
X
2a - 2c
b
n = 1 X = -Br 2a, 3a
n = 2 X = -Br 2b, 3b
H
N
O
(CH2)n
NH
n = 2 X = -Cl
2c, 3c
O
O
X
HCl
3a - 3c
Scheme 1: Scheme of synthesis of 6-chloroacethy-2-benzoxazolinone with 4-substituted aryl-aliphatic amine derivatives (a) acetonitrile/80 ◦C/2 h/reflux; (b) HCl/methanol
H
N
H
N
a
+
O
O
HN
O
O
N
Cl
N
R
O
N
O
R
2d - 2g
b
R= -benzyl
-phenyl
2d,
3d
3e
3f
H
N
2e,
2f,
2g,
O
-methyl
O
N
*
HCl
-hydroxyethyl
3g
O
N
R
3d - 3g
Scheme 2: Scheme of synthesis of 6-chloroacetyl-2-benzoxazolinone with N-substituted piperazine derivatives (a) acetone/60 ◦C/2-4 h/reflux; (b) HCl/methanol
methodology was based on the modification of methods devised
by Calis and Gorkan (2001) and Mentrup et al. (1976). The
crude product was obtained by pouring the warm reaction mix-
ture into the ice water. Monitoring the reaction by TLC showed
that the reaction was usually completed within 1 h (for com-
pounds 2 g-2 h). Purified by the crystallization from methanol,
the compounds were transformed to hydrochlorides (3a–3f) and
examined for their activity towards the inhibition of AChE and
BChE using tacrine and donepezil as standards.
nonlinearregression. StatisticalparametersandvaluesofKm and
vmax for AChE and BChE are shown in Table 1. In accordance
with the standard methodology, we obtained AChE and BChE
inhibition activity and selectivity of the received derivatives
Table 1: Statistical parameters and values of Km and Vmax
for AChE and BChE
Parameters
AChE
BChE
To determine the type of inhibition we used the linear transfor-
mation of the Michaelis-Menten equation: the Lineweaver-Burk
plot (1/v vs. 1/[S]). Michaelis-Menten constans: Km and Vmax
were also obtained from the Lineweaver-Burk plot by the lin-
ear regression of the reaction rate in relation to the substrate
concentration. Values of Ki constans were calculated by using
Km
3.71 × 10−8
M
9.93 × 10−8
M
Vmax
5.79 × 10−8 M/ml/min 8.95 × 10−8 M/ml/min
r2
0.97
0.99
0.00076
Standard error 0.00619
400
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ORIGINAL ARTICLES
Table 2: IC50 values for activities towards acetylcholinesterase and butyrylcholinesterase
a
b
Compd.
AChE inhibition (IC , M)
50
BChE inhibition (IC , M)
50
Selectivity for AChE
Selectivity for BChE
3a
3b
3c
3d
3e
3f
3g
1.82 × 10−11
1.03 × 10−9
2.16 × 10−9
8.04 × 10 −9
5.69 × 10−13
3.11 × 10−8
3.98 × 10−8
1.97 × 10−11
5.76 × 10−11
9.08 × 10−13
3.84 × 10−12
1.64 × 10−12
2.30 × 10 −8
3.18 × 10−12
1.34 × 10 −8
2.53 × 10 −8
5.99 × 10−13
3.08 × 10−10
0.05
3.74 × 10−3
7.57 × 10−3
2.87
20.08
2.67 × 102
1.32 × 103
0.35
5.59
0.04
0.64
0.18
2.32
1.57
Tacrine
Donepezil
0.03
32.93
5.34 × 102
1.87 × 10−3
a
Selectivity for AChE is defined as IC (BChE)/IC (AChE)
50 50
Selectivity for BChE is defined as IC (AChE)/IC (BChE)
50 50
b
towards cholinesterases (Table 2). One can find that 3e is more
active compound towards the inhibition of AChE than tacrine,
while compound 3a has similar activity to tacrine. Compound 3e
has better activity than donepezil. Considering the inhibition of
butyrylcholinesterase we can see that 3a, 3b, 3c and 3e are more
active compounds compared with donepezil. All tested com-
pounds exhibit lower inhibition of butyrylcholinesterase than
tacrine. Out of all described derivatives, compound 3e has the
highest selectivity towards the inhibition of acetylcholinesterase
and derivative 3c has the strongest selectivity towards the inhi-
bition of butyrylcholinesterase. All obtained compounds show
lower selectivity towards the inhibition of acetylcholinesterase
than donepezil and higher (except 3b and 3c) than tacrine. All
compounds are characterized by better selectivity towards the
inhibition of butyrylcholinesterase than donepezil and deriva-
tives 3b and 3c have higher selectivity than tacrine.
4.1.1. 6-{[(4-Bromobenzyl)amino]acetyl}-1,3-benzoxazol-2(3H)-one
(2a)
6-Chloroacetyl-2-benzoxazolinone (0.40 g, 1.89 mmol) was dissolved in
acetonitrile (40 ml, 766 mmol), then 4-bromobenzylamine (0.70 g, 3.78
mmol) was added and the mixture was heated in 80 ◦C under reflux for
75 min. The warm reaction mixture was poured into ice-water. The precipi-
tate was filtered and dried in vacuum. Recrystallization from methanol gave
the product as a white solid. Compound 2a: m.p.: 160–162 ◦C; yield: 53%;
1H NMR (DMSO) (ð ppm): 4.23 (2H, d, J=1.0, CH2), 4.56 (2H, s, CH2),
7.17–7.19 (1H, d, J = 7.6, ArH), 7.45–7.49 (2H, d, J = 8.2, ArH), 7.64–7.66
(2H, d, J = 7.9, ArH); 7.85–7.91 (2H, m, ArH), 9.77 (2H, 2xNH); MS(FAB)
m/z (M+1): 361.1, 170.8; MS-HR calcd: 361.018224, found: 361.018790
C16H13BrN2O3.
4.1.2. 6-({[2-(4-Bromophenyl)ethyl]amino}acetyl)-1,3-benzoxazol-
2(3H)-one (2b)
6-Chloroacetyl-2-benzoxazolinone (0.40 g, 1.89 mmol) was dissolved in
acetonitrile (40 ml, 766 mmol), then 4-chlorophenethylamine (0.59 g,
3.78 mmol) was added and the mixture was heated in 80 ◦C under reflux for
135 min. The warm reaction mixture was poured into ice-water. The pre-
cipitate was filtered and dried in vacuum. Recrystallization from methanol
gave the product as a white solid. Compound 2b: m.p.: 122–125 ◦C, yield:
49%; 1H NMR (DMSO) (ð ppm): 3.07–3.12 (2H, m, CH2CH2), 3.16–3.25
(2H, m, CH2CH2), 4.90 (2H, s, CH2), 7.35–7.42 (3H, m, ArH), 7.56–7.62
(2H, m, ArH), 7.91–7.97 (2H, m, ArH), 11.30 (2H, 2xNH); MS(FAB) m/z
(M+1): 375.3, 184.8; MS-HR calcd: 375.033874; found: 375.034440
3. Discussion
The synthesis and biological evaluation of series of 6-acetyl-
2-benzoxazolinone analogs led to the design of new potential
cholinesterase inhibitors. These compounds were prepared from
6-chloroacetyl-2-benzoxazolinone in the reaction with the cor-
responding amine. Obtained derivatives were evaluated as
cholinesterase inhibitors by using the colorimetric method of
C17H15BrN2O3.
Ellman. Then the IC
values and selectivity for AChE and
50
BChE were calculated and compared with standards (tacrine,
donepezil). We have found four derivatives that show higher
activity towards the inhibition of BChE than donepezil, and one
derivative better than tacrine and donepezil as AChE inhibitor.
The most promising compound is 3e. This derivative possesses
higher inhibitory activity for AChE than tacrine and donepezil,
and is a better butyrylcholinesterase inhibitor than donepezil.
The results suggest that the new derivatives may be potential
drugs for treatment of Alzheimer’s disease. The structure of
6-acetyl-2-benzoxazolinone is an innovation and it opens the
way for the modification of these molecules in searching com-
pounds with better activities and selectivity for the inhibition of
cholinesterases.
4.1.3. 6-({[2-(4-Chlorophenyl)ethyl]amino}acetyl)-1,3-benzoxazol-
2(3H)-one (2c)
6-Chloroacetyl-2-benzoxazolinone (0.40 g, 1.89 mmol) was dissolved in
acetonitrile (40 ml, 766 mmol), then 4-bromophenetylamine (0.59 g, 3.78
mmol) was added and the mixture was heated in 80 ◦C under reflux for
1 h. The warm reaction mixture was poured into ice-water. The precipitate
was filtered and dried in vacuum. Recrystallization from methanol gave the
product as a white solid. Compound 2c: m.p.: 117–120 ◦C; yield 54%;1H
NMR (DMSO) (ð ppm): 3.02–3.08 (2H, m, CH2CH2), 3.08–3.20 (2H, m,
CH2CH2), 4.80 (2H, s, CH2), 7.27–7.32 (3H, m, ArH), 7.40–7.42 (2H, d,
ArH), 7.86–7.91 (2H, m, ArH), 9.37 (2H, 2xNH); MS(FAB) m/z (M+1):
331.1, 138.9, 156.8; MS-HR calcd: 330.084396; found (M+1): 331.084945
C17H15ClN2O3.
4.1.4. 6-[(4-Benzylpiperazin-1-yl)acetyl]-1,3-benzoxazol-2(3H)-one
(2d)
4. Experimental
4.1. Chemistry
6-Chloroacetyl-2-benzoxazolinone (0.20 g, 0.94 mmol) was dissolved in
acetone (20 ml, 272 mmol), then N-benzylpiperazine (0.33 g, 1.89 mmol)
was added and the mixture was heated in 60 ◦C under reflux for 2 h. The
warm reaction mixture was poured into ice-water. The precipitate was fil-
tered and dried in vacuum. Recrystallization from methanol gave the product
as a yellow solid. Compound 2d: m.p. 140–142 ◦C; yield 50%; 1H NMR
(DMSO) (ð ppm): 3.35–3.69 (8H, m, piperaz.), 4.42 (2H, s, ArCH2), 5.02
(2H, s, CH2), 7.31–7.34 (1H, m, ArH), 7.52–7.68 (5H, m, ArH), 7.87–7.96
(2H, m, ArH), 12.11 (1H, NH); Anal. calcd for C: 68.36, N: 11.95, H: 6,02;
found: C: 68.29, N: 11.74, H: 6.30 C20H21N3O3.
Reactions were monitored by TLC using 25 DC-Alufolien Kieselgel 60F254
plates (Merck). Melting points were measured on the Electrothermal appara-
tusinopencapillariesandwereuncorrected. IRspectrawererecordedinKBr
using a Mattson Infinity Series FT-IR spectrophotometer. 1H NMR spectra
were recorded with a Varian Mercury 300 MHz spectrometer, using tetram-
ethylsilane as an internal standard. Mass spectra were performed by the
Centre of Molecular and Macromolecular Studies in Lodz (Polish Academy
of Sciences) on an apparatus Finnigan Mat 95. All the results of elemental
analyses were in an acceptable range.
Pharmazie 66 (2011)
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ORIGINAL ARTICLES
4.1.5. 6-[(4-Phenylpiperazin-1-yl)acetyl]-1,3-benzoxazol-2(3H)-one
4.1.11. 6-[(4-Benzylpiperazin-1-yl)acetyl]-1,3-benzoxazol-2(3H)-one
(2e)
hydrochloride (3d)
6-Chloroacetyl-2-benzoxazolinone (0.20 g, 0.94 mmol) was dissolved in
acetone (20 ml, 272 mmol), then N-phenylpiperazine (0.31 g, 1.86 mmol)
was added and the mixture was heated in 60 ◦C under reflux for 2 h. The
warm reaction mixture was poured into ice-water. The precipitate was fil-
tered and dried in vacuum. Recrystallization from methanol gave the product
as a white solid. Compound 2e: m.p. 182–185 ◦C; yield 17%; 1H NMR
(DMSO) (ð ppm): 2.49–2.68 (4H, m, piperaz.), 3.10–3.37 (4H, m, piperaz.),
3,88 (2H, s, CH2), 6.74–6.79 (1H, m, ArH), 6.91–6.94 (2H, d, J = 7.7,
ArH), 7.17–7.23 (3H, m, ArH), 7.89–7.93 (2H, m, ArH), 12.57 (1H, NH);
MS(FAB) m/z (M+1): 338.2, 175.0; MS-HR calcd: 338.149918; found:
(M+1): 338.150466 C19H19N3O3.
Compound 2d (0.20 g, 0.57 mmol) was dissolved in methanol (3 ml),
HCl/methanol was added dropwise. The reaction mixture was stirred in
crushed ice. The precipitate was isolated and next recrystallisated from
methanol, isolated and dried in vacuum. Compound 3d: m.p.: 232–236 ◦C;
yield: 55%; IR(KBr) v(cm−1): 1630.5, 1692.2, 1775.4, 3016.6, 3367.0;
1H NMR (DMSO) (ð ppm): 3.30–3.60 (8H, m, piperaz.), 4.39 (2H, s,
ArCH2), 5.00 (2H, s, CH2), 7.26–7.29 (1H, m, ArH), 7.45–7.65 (5H, m,
ArH), 7.85–7.90 (2H, m, ArH), 12.39 (1H, NH); MS(FAB) m/z (M+1):
352.1, 189.0; MS-HR: calcd: 352.165568; found: (M+1): 352.166116.
C20H22ClN3O3.
4.1.12. 6-[(4-Phenylpiperazin-1-yl)acetyl]-1,3-benzoxazol-2(3H)-one
hydrochloride (3e)
4.1.6. 6-[(4-Methylpiperazin-1-yl)acetyl]-1,3-benzoxazol-2(3H)-one
(2f)
Compound 2e (0.10 g, 0.30 mmol) was dissolved in methanol (3 ml),
HCl/methanol was added dropwise. The reaction mixture was stirred in
crushed ice. The precipitate was isolated and next recrystallisated from
methanol, isolated and dried in vacuum. Compound 3e: m.p.: 260–264 ◦C;
yield: 50%; IR(KBr) v(cm−1): 1618.7, 1690.4, 1769.6, 2923.9, 3338.9; 1H
NMR (DMSO) (ð ppm): 3.25–3.83 (8H, m, piperaz.), 5.17 (2H, s, CH2),
6.85–6.90 (1H, m, ArH), 7.01–7.10 (2H, d, J = 7.9, ArH), 7.25–7.33 (3H,
m, ArH), 7.86–7.90 (2H, m, ArH), 12.52 (1H, NH). C19H20ClN3O3.
6-Chloroacetyl-2-benzoxazolinone (0.20 g, 0.94 mmol) was dissolved in
acetone (20 ml, 272 mmol), then N-methylpiperazine (0.19 g, 1.86 mmol)
was added and the mixture was heated in 60 ◦C under reflux for 2 h. The
warm reaction mixture was poured into ice-water. The precipitate was fil-
tered and dried in vacuum. Recrystallization from methanol gave the product
as a white solid. Compound 2f: m.p. 130–134 ◦C; yield: 23%; 1H NMR
(DMSO) (ð ppm): 2.80 (3H, s, CH3), 2.82–3.45 (8H, m, piperaz.), 4.91
(2H, s, CH2), 7.26–7.29 (1H, m, ArH), 7.85–7.89 (2H, m, ArH), 12.36 (1H,
s, NH); MS(FAB) m/z (M+1): 276.1, MS-HR calcd: 276.134268; found:
(M+1): 276.134816. C14H17N3O3.
4.1.13. 6-[(4-Methylpiperazin-1-yl)acetyl]-1,3-benzoxazol-2(3H)-one
hydrochloride (3f)
Compound 2f (0.10 g, 0.36 mmol) was dissolved in methanol (3 ml),
HCl/methanol was added dropwise. The reaction mixture was stirred in
crushed ice. The precipitate was isolated and next recrystallisated from
methanol, isolated and dried in vacuum. Compound 3f: m.p.: 274–278 ◦C;
yield: 18%; IR(KBr) v(cm−1): 1622.5, 1683.6, 1782.7, 2820.8, 2962.9,
3456.4; 1H NMR (DMSO) (ð ppm): 2.73 (3H, s, CH3), 2.82–3.46 (8H,
m, piperaz.), 4.86 (2H, s, CH2), 7.26–7.29 (1H, m, ArH), 7.85–7.90 (2H, m,
ArH), 12.33 (1H, s, NH). C14H18ClN3O3.
4.1.7. 6-{[4-(2-Hydroxyethyl)piperazin-1-yl]acetyl}-1,3-benzoxazol-
2(3H)-one (2 g)
6-Chloroacetyl-2-benzoxazolinone (0.30 g, 1.42 mmol) was dissolved in
acetone (20 ml, 272 mmol), then N-hydroxyethylpiperazine (0.37 g, 1.86
mmol) was added and the mixture was heated in 60 ◦C under reflux for
4 h. The warm reaction mixture was poured into ice-water. The precipitate
was filtered and dried in vacuum. Recrystallization from methanol gave the
product as a white solid. Compound 2 g: m.p.: 182–184 ◦C; yield: 26%; 1H
NMR (DMSO) (ð ppm): 2.36–2.49 (4H, m, CH2CH2), 3.30–3.46 (8H, m,
piperaz.), 5.11 (2H, s, CH2), 7.18–7.22 (1H, m, ArH), 7.87–7.93 (2H, m,
ArH). C15H19N3O4.
4.1.14. 6-{[4-(2-Hydroxyethyl)piperazin-1-yl]acetyl}-1,3-
benzoxazol-2(3H)-one hydrochloride (3 g)
Compound 2 g (0.10 g, 0.33 mmol) was dissolved in methanol (3 ml),
HCl/methanol was added dropwise. The reaction mixture was stirred in
crushed ice. The precipitate was isolated and next recrystallisated from
methanol, isolated and dried in vacuum. Compound 3 g: m.p.: 268–272 ◦C;
yield: 32%; IR(KBr) v(cm−1): 1609.4, 1662.9, 1760.0, 2924.5, 3419.1; 1H
NMR (DMSO) (ð ppm): 2.41–2.51 (4H, m, CH2CH2), 3.42–3.50 (8H, m,
piperaz.), 5.14 (2H, s, CH2), 7.14–7.17 (1H, m, ArH), 7.84–7.87 (2H, m,
ArH); MS(FAB) m/z (M+1): 306.2, 143.0; MS-HR: calcd: 306,144833;
found: (M+1) 306,145381
4.1.8. 6-{[(4-Bromobenzyl)amino]acetyl}-1,3-benzoxazol-2(3H)-one
hydrochloride (3a)
Compound 2a (0.20 g, 0.55 mmol) was dissolved in methanol (3 ml),
HCl/methanol was added dropwise. The reaction mixture was stirred in
crushed ice. The precipitate was isolated and next recrystallisated from
methanol, isolated and dried in vacuum. Compound 3a: m.p.: 262–266 ◦C;
yield: 51%; IR(KBr) v(cm−1): 1561.6, 1619.5, 1679.5, 1755.2, 2928.5,
3123.6; 1H NMR (DMSO) (ð ppm): 4.17 (2H, d, J = 0.4, CH2), 4.73 (2H,
s, CH2), 7.25–7.28 (1H, d, J = 7.9, ArH), 7.52–7.55 (2H, d, J = 8.7, ArH),
7.64–7.66 (2H, d, J = 8.3, ArH), 7.83–7.89 (2H, m, ArH), 9.70 (2H, 2×NH).
4.2. Enzymatic assay
Cholinesterases and their potential inhibitor activities were determined
by the spectrophotometric method of Ellman with some modifications.
Hydrolysis rates (v) were measured at 8 various substrate concentrations
[S] – acetylthiocholine iodide in 3 ml of the sample volume contained
also phosphate-buffered solution (0.1 M, pH 8.0), a solution of 5.50-
dithiobisnitrobenzoic acid (DTNB, 0.05 ml, 0.5 M), enzyme (AChE or
BChE, 0.05 ml, 5 U/ml) and the appropriate inhibitor. The hydrolysis was
monitored by the formation of the thiolate dianion of DTNB and spec-
trophotometric assay at 412 nm after 1 min. Determination samples without
inhibitor gave 100% of AChE or BChE activity. The IC50 values for
inhibitor concentration was calculated by using linear transformation of
the Michaelis-Menten equation: the Lineweaver-Burk plot.
C
16H14BrClN2O3.
4.1.9. 6-({[2-(4-Bromophenyl)ethyl]amino}acetyl)-1,3-benzoxazol-
2(3H)-one hydrochloride (3b)
Compound 2b (0.20 g, 0.53 mmol) was dissolved in methanol (3 ml),
HCl/methanol was added dropwise. The reaction mixture was stirred in
crushed ice. The precipitate was isolated and the next recrystallisated from
methanol, isolated and dried in vacuum. Compound 3b: m.p.: 270–271 ◦C;
yield 56%; IR(KBr) v(cm−1): 1491.2, 1620.2, 1679.0, 1757.1, 2930.7,
3130.3; 1H NMR (DMSO) (ð ppm): 3.02–3.07 (2H, m, CH2CH2), 3.18-
1.23 (2H, m, CH2CH2), 4.80 (2H, s, CH2), 7.23–7.30 (3H, m, ArH),
7.53–7.57 (2H, m, ArH), 7.86–7.92 (2H, m, ArH), 11.26 (2H, 2×NH).
Acknowledgement: This work was supported by the grant from Polish Min-
istry of Science and Higher Education (No NN 405 302 636).
C17H16BrClN2O3.
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