Design and Synthesis of 2-Substitutedphenyl Benzo[D]Thiazole Derivatives and Their β-Amyloid Aggregation and Cholinesterase Inhibitory Activities

Article abstract of DOI:10.1007/s11094-019-02000-4

The occurrence of amyloid-β (Aβ) and reduced cholinergic tranmission are two major hallmarks of Alzheimer’s disease (AD). Therefore, a series of new 2-phenylbenzo[d]thiazoles substituted with azole/piperazine moieties were designed, synthesized, and evaluated as potential dual inhibitors of Aβ aggregation and cholinesterase (ChE) activities. In vitro studies showed that compound 2m containing an imidazole ring strongly inhibited Aβ_1–40 (49.2%) and Aβ_1-42 aggregation (60.6%). All derivatives exhibited weak inhibitory activities against both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Therefore, compound 2m may represent promising therapeutic option for inhibiting Aβ-mediated pathology in AD.

Full text of DOI:10.1007/s11094-019-02000-4

DOI 10.1007/s11094-019-02000-4
Pharmaceutical Chemistry Journal, Vol. 53, No. 4, 19, 2019 (Russian Original Vol. 53, No. 4, April, 2019)
DESIGN AND SYNTHESIS OF 2-SUBSTITUTEDPHENYL
BENZO[D]THIAZOLE DERIVATIVES AND THEIR b-AMYLOID
AGGREGATION AND CHOLINESTERASE INHIBITORY ACTIVITIES
Merve Zengin,1 Oya Unsal-Tan,1 Tuba Tüylü Küçükkýlýnç,2
Beyza Ayazgok,2 and Ayla Balkan1,*
Original article submitted November 5, 2018.
The occurrence of amyloid-b (Ab) and reduced cholinergic tranmission are two major hallmarks of Alzhei-
mer’s disease (AD). Therefore, a series of new 2-phenylbenzo[d]thiazoles substituted with azole/piperazine
moieties were designed, synthesized, and evaluated as potential dual inhibitors of Ab aggregation and
cholinesterase (ChE) activities. In vitro studies showed that compound 2m containing an imidazole ring
strongly inhibited Ab1–40 (49.2%) and Ab1-42 aggregation (60.6%). All derivatives exhibited weak inhibitory
activities against both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Therefore, compound
2m may represent promising therapeutic option for inhibiting Ab-mediated pathology in AD.
Keywords: benzothiazole; Alzheimer’s disease; beta amyloid; cholinesterase.
1. INTRODUCTION
as a result of these studies were approved by the American
Food and Drug Administration (FDA) in 1996, 1996, 2000,
and 2001, respectively, for the treatment of AD [15]. How-
ever, studies also showed that acetylcholinesterase (AChE)
exhibits non-cholinergic functions related to the formation
and storage of amyloid aggregates, thus playing a role in the
early stages of senile plaque development. Therefore, the
cholinergic and amyloid hypotheses cannot be independently
considered [16 – 18]. After AD was demonstrated to be a
multifactorial disease, extensive research has been focused
on adopting a new strategy that involved the design of
multi-target-directed ligands (MTDLs). This approach fea-
tures the development of new molecules that inhibit both am-
yloid aggregation and ChE activity [18 – 20].
Alzheimer’s disease (AD) is a chronic progressive
neurodegenerative disease and is the most common type of
dementia known since ancient times [1 –3]. Distinctive
plaques and neurofibrillary tangles in the brain tissue of de-
mentia patients were described for the first time by Alois
Alzheimer, a German psychiatrist, in 1906. Today, these
findings are accepted as characteristic features of AD [4, 5].
Although many hypotheses regarding the pathophysiology of
AD have been proposed, its pathogenesis cannot be still
completely explained [6]. Reduced cholinergic transmission,
beta amyloid (Ab) protein aggregation, hyperphosphoryl-
ation of tau protein, increased oxidative stress, inflammation,
and exposure to toxic metal ions have been implicated as the
causes or contributors to the progression of AD [7 – 11]. In-
The benzothiazole moiety is an interesting scaffold for
the design of new compounds for non-invasive diagnostics
and treatment of AD [2, 8, 21 – 32]. Hybrid compounds con-
taining a benzothiazole ring and tacrine exhibit significant
creasing cholinergic functions, inhibiting Ab aggregate for-
mation, and reducing tau protein hyperphosphorylation con-
stitute the main treatment approaches for AD [12 – 14].
Research has been primarily focused on cholinesterase
(ChE) inhibitors that can increase cholinergic transmission.
Tacrin, donepezil, rivastigmine, and galantamine developed
AChE and Ab aggregation inhibitory activity [2, 20, 33].
Previous studies have shown that various compounds con-
taining both benzothiazole and piperazine rings exhibit
strong AChE inhibition [22, 23]. In addition, several benzo-
thiazole analogs have been screened as potential amyloid-
binding diagnostic agents for various neurodegenerative dis-
eases [28 – 32]. Based on the above information, a series of
new hybrid compounds bearing benzothiazole and azole/pi-
perazine moieties were designed, synthesized, and evaluated
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacette-
pe University, Ankara, Turkey.
2
Department of Biochemistry, Faculty of Pharmacy, Hacettepe University,
Ankara, Turkey.
*
e-mail: aebalkan@hacettepe.edu.tr
322
0091-150X/19/5304-0322 © 2019 Springer Science+Business Media, LLC

Design and Synthesis of 2-Substitutedphenyl Benzo[d]thiazole Derivatives
323
i
HN
OHC
F
OHC
N
1a-o
4
3'
5'
2'
3a
7a
NH2
N
5
6
ii
2
4'
OHC
N
N
1'
S
SH
6'
7
1a-o
2a-o
Scheme 1. Synthesis of the target compounds. Reagents and conditions: (i) K CO , DMSO, reflux (ii) DMSO, 140°C.
2
3
126.62 – 126.77, 134.44 – 134.51, 153.42 – 153.55, and
165.88 – 166.33 ppm, respectively. In the ESI-MS spectra,
the protonated [M+H]+ and sodiated [M+Na]+ molecular
ions of all compounds were observed. Elemental analysis
agreed well with the theoretical chemical structures of the
synthesized compounds.
in this work as potential dual inhibitors for Ab aggregation
and ChE activity.
2. RESULTS AND DISCUSSION
2.1. Chemistry
In this study, a total of 15 2-(4-substitutedphenyl)benzo-
[d]thiazole derivatives bearing azole or substituted pipera-
zine moieties at the 4 position of the phenyl ring were syn-
thesized, of which two have been previously reported (2a
and 2l) [29, 34, 35]. The target compounds, 2a–o, were ob-
tained using the synthetic route outlined in Scheme 1. The
starting compounds, 4-substituted benzaldehyde derivatives
1a–o, were obtained by aromatic nucleophilic substitution of
4-fluorobenzaldehyde with various azole and piperazine de-
rivatives in the presence of potassium carbonate with yields
of 70 – 81% [36]. The target compounds, 2-(4-substituted-
phenyl) benzo[d]thiazole derivatives, 2a–o were obtained by
treating the substituted benzaldehydes 1a–o with o-amino-
thiophenol at 140°C in moderate yields (55 – 79%). The
structures of the target compounds were elucidated using in-
2.2. Biological Activity
Inhibition of self-mediated Ab
and Ab
aggrega-
1–40
1-42
tion. Inhibitory effects of compounds 2a – 2o on Ab fibril
formation were evaluated using the Thioflavin T method,
and donepezil was used as a reference compound (Table 1).
It was found that compounds 2a – 2o bearing imidazole,
triazole, and benzimidazole rings at the 4 position of the
phenyl ring, respectively, showed good inhibitory activity on
Ab
(49.2 – 74.9%) and Ab
(47.3 – 60.6%) aggrega-
1–40
1-42
tion. Furthermore, compound 2m inhibited Ab
aggrega-
1–40
tion better than donepezil ( p < 0.05).
When the inhibitory activities of piperazine-substituted
compounds (2a – 2k) on Ab fibril formation were examined,
the derivatives bearing small substituents, such as methyl,
ethyl, and acetyl (2a – 2c) groups at the 4-position, showed
1
frared (IR), H- and 13C NMR, electrospray ionization-mass
spectrometry (ESI-MS), and elemental analysis for the com-
pounds not already described in the literature.
good inhibitory effects on Ab
fibril formation (Ab
:
1-42
1-42
63.7 – 82.7%). However, bulky substituents, such as cyclo-
hexyl, phenyl, and benzyl groups, at the 4-position of
piperazine reduced the inhibitory activity (2d – 2k). In par-
ticular, compound 2i exhibited remarkable inhibitory activity
In the IR spectra of the target compounds, the absence of
signals at approximately 2837 – 2812 and 1690 – 1657 cm-1,
which were seen in the spectra of the corresponding alde-
hydes, was evidence for the benzothiazole ring closure. In
on Ab
(61.6%) and Ab
(64.7%) fibril formation.
1
the H-NMR spectra of the target compounds, signals ob-
1-40
1-42
Cholinesterase inhibitory activity. Compounds 2a – 2o
were tested for their inhibitory activity towards AChE and
BChE using the Ellman method and the results are summa-
rized in Table 2. All compounds (except 2e and 2o) showed
no inhibitory activity (7.9 – 48.7%) against either ChE at a
concentration of 100 mM. The IC values of 2e (IC :
served as triplets or triplet of doublets at approximately
7.44 – 7.48 and 7.30 – 7.37 ppm were assigned to H and H ,
5
6
whereas the signals observed as doublets at approximately
7.85 and 8.00 ppm were assigned to H and H of the
4
7
benzothiazole ring, respectively. In addition, the ortho- (2,6-)
and meta- (3,5-) phenyl protons at position 2 of benzothia-
zole were observed at approximately 7.90 and 8.00 ppm, re-
spectively. Furthermore, in the 13C-NMR spectra, C , C , C ,
50
50
78.09 mM for AChE, IC : 107.00 mM for BChE) and 2o
50
(IC : 46.84 mM for AChE and IC : 40.61 mM for BChE)
4
7
6
50
50
C , C , C , and C of the benzothiazole ring appeared at
were evaluated, clearly showing weak inhibitory activities
towards both AChE and BChE.
5
7a
3a
2
122.32 – 122.34,
122.79 – 122.87,
125.49 – 125.59,

324
Merve Zengin et al.
3. EXPERIMENTAL CHEMICAL PART
(Perkin Elmer) and positions of peaks are reported in cm-1.
1
The H and 13C NMR spectra (DMSO-d /CDCl ) were re-
6
3
3.1. Materials and Methods
corded using Varian Mercury 400 FT NMR spectrophotome-
ter with TMS as the internal standard (chemical shifts re-
ported as d, ppm). The ESI-MS spectra were measured using
Micromass ZQ-4000 single-quadruple mass spectrometer.
Elemental analyses (C, H, N, and S) were performed using
Leco CHNS 932 analyzer.
Melting points of the compounds were determined using
a Stuart SMP20 melting point apparatus and the results are
reported as uncorrected values. ATR-FTIR spectra were ob-
tained using a MIRacle ATR accessory (Pike Technologies)
in conjunction with a Spectrum BX FTIR spectrometer
TABLE 1. Effects of Compounds 2a – 2o on the Inhibition of Self-Mediated Ab
and Ab
Aggregation
1–40
1–42
N
R
S
Comp
R
Ab1–40 aggregation ±SEM (%)
81.3 ± 11.3
Ab1–42 aggregation SEM (%)
82.7 ± 8.1
2a
N
N CH3
N C2H5
2b
2c
68.4 ± 6.5
70.5 ± 7.1
N
O
55.6 ± 5.0*
63.7 ± 7.4*
N
N
C
CH3
2d
2e
³ 100
74.8 ± 8.2
86.8 ± 9.5
N
N
70.1 ± 4.4
N
N
H3CO
2f
³ 100
³ 100
69.7 ± 7.0
³ 100
N
N
OCH3
2g
N
N
Cl
2h
2i
³ 100
73.5 ± 7.2
64.7 ± 7.2
N
N CH2
CH2
61.6 ± 5.2*
N
N
N
CH3
2j
³ 100
³ 100
³ 100
³ 100
N
CH2
CH2
F
2k
N
N
Cl
2l
2m
2n
2o
96.5 ± 10.5
49.2 ± 6.8**
60.9 ± 5.6*
74.9 ± 13.3
³ 100
N
N
60.6 ± 6.8*
47.3 ± 5.5**
60.6 ± 7.3*
N
N
N
N
N
N
N
Controla
100
100
Donepezil
83.1 ± 5.0
61.9 ± 8.7*
a
The control of each assay was non-treated protein; data show mean ± SEM values for at least three independent experiments; * p < 0.05,
** p < 0.001.

Design and Synthesis of 2-Substitutedphenyl Benzo[d]thiazole Derivatives
325
3.2. Synthesis
General procedure for the preparation of 4-substitu-
2.36 (2H, q, -CH -); 2.47 – 2.50 (4H, m, piperazine, over-
2
lapped with DMSO); 3.28 – 3.30 (4H, m, piperazine); 7.04
(2H, d, J = 9.2 Hz, H H ); 7.36 (1H, td, J = 7.6 Hz,
3’,
5’
1
tedbenzaldehydes (1a – 1o). First, 4-fluorobenzaldehyde
and various azole/piperazine derivatives were reacted using
potassium carbonate as catalyst in DMSO as described in the
literature [36].
J = 1.2 Hz, H ); 7.46 (1H, t, J = 8.0 Hz, H ); 7.87 – 7.94
2
6
1
5
(3H, m, H , H , H ); 8.03 (1H, d, J = 8.0 Hz, H ). 13C-NMR
4
2’
6’
7
(100 MHz; DMSO-d ; Me Si; d , ppm): 11.94 (CH -CH );
6
4
C
2
3
47.82 and 52.54 (piperazine); 52.33 (CH -CH ); 114.75;
2
3
General procedure for the preparation of 2-(4-substi-
tuted phenyl)benzo[d]thiazoles (2a – 2o). Initially, 4-sub-
stituted benzaldehydes 1a – 1o (0.02 mol) and 2-aminothio-
phenol (0.02 mol) in 10 mL DMSO were heated under reflux
at 140°C for 3 h. The mixture was then poured into 100 mL
of ice-cold distilled water. The precipitated solid was col-
lected by filtration and purified by crystallization from an ap-
propriate solvent.
121.41; 122.53; 124.03; 124.46; 126.06; 128.76; 134.68;
152.95; 154.33; 168.24. ESI-MS (m/z): 346.31 [M+Na]+
(100%), 324.35 [M+H]+.
2-[4-(4-acetylpiperazin-1-yl)phenyl]benzo[d]thiazole
(2c): yield, 0.48 g (71%); m.p., 292°C (from DMF and H O);
2
Found: C, 67.50; H, 5.79; N, 12.58; S, 9,29. Anal. Calcd. for
C H N OS: C, 67.63; H, 5.68; N, 12.45; S, 9.50%; IR
19 19
3
(n , cm-1): 3053 (C-H), 2994, 2840 (C-H), 1650 (C=O),
max
1600, 1479, 1434, 1424 (C=N, C=C), 1219 (C-N); 1H-NMR
The synthesized compounds were characterized as fol-
lows:
(400 MHz; DMSO-d ; Me Si; d , ppm): 2.04 (3H, s,
6
4
H
2-[4-(4-methylpiperazin-1-yl)phenyl]benzo[d]thiazole
(2a): m.p., 207°C (from methanol) (lit. [35], 211 – 212°C).
2-[4-(4-ethylpiperazin-1-yl)phenyl]benzo[d]thiazole
(2b): yield, 0.49 g (75%); m.p. 215°C (from methanol);
found: C, 70.29; H, 6.76; N, 12.86; S, 9,69; Anal. Calcd. for
-CO-CH ); 3.24 – 3.38 (4H, m, piperazine); 3.58 – 3.62 (4H,
3
m, piperazine); 7.07 (2H, d, J = 9.2 Hz, H , H ); 7.37 (1H, t,
3’
5’
J = 7.2 Hz, H ); 7.48 (1H, t, J = 7.2 Hz, H ); 7.91–7.95 (3H,
6
5
m, H , H , H ); 8.04 (1H, d, J = 7.6 Hz, H ). ESI-MS (m/z):
4
2’
6’
7
360.25 [M+Na]+ (100%), 338.29 [M+H]+.
C H N S: C, 70.55; H, 6.54; N, 12.99; S, 9.91%; IR (n ,
max
2-[4-(4-cyclohexylpiperazin-1-yl)phenyl]benzo[d]thia-
zole (2d): yield, 0.58 g (77%); m.p., 234°C (from ethyl ace-
tate); Found: C, 73.15; H, 7.10; N, 11.17; S, 8.46; Anal.
Calcd. for C H N S: C, 73.17; H, 7.21; N, 11.13; S, 8.49%;
19 21
3
cm-1): 3051 (C-H), 2979, 2943, 2805, 2767 (C-H), 1602,
1589, 1478, 1436, 1423 (C=N, C=C), 1223 (C-N); 1H-NMR
(400 MHz; DMSO-d ; Me Si; d , ppm): 1.02 (3H, t, -CH );
23 27
3
6
4
H
3
TABLE 2. Anti-Cholinesterase Activities of Compounds 2a -– 2o
AchE IC50a ± SE (mM)
BchE IC50a ± SE (mM)
Comp
AChE inhibition ± SE (%)
BChE inhibition ± SE (%)
2a
–
–
–
–
–
–
–
–
8.2 ± 0.4
12.5 ± 7.2
nd
45.7 ± 2.1
48.7 ± 3.4
25.8 ± 4.9
33.0 ± 3.0
53.9 ± 4.3
nd
2b
2c
2d
nd
2e
56.3 ± 8.4
25.2 ± 3.8
37.6 ± 8.2
10.2 ± 4.4
48.1 ± 6.5
7.9 ± 2.1
22.8 ± 7.3
nd
78.09 ± 1.36
107.00 ± 2.78
2f
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2g
42.3 ± 1.3
24.4 ± 1.2
32.6 ± 2.1
33.9 ± 5.1
30.4 ± 4.5
47.7 ± 7.6
43.3 ± 4.4
34.9 ± 4.8
44.3 ± 6.5
–
2h
2i
2j
2k
2l
2m
20.8 ± 1.2
2n
nd
2o
72.8 ± 10.6
46.84 ± 1.77
6.16 ± 1.03b
40.61 ± 1.59
4.94 ± 1.04
Donepezil
–
a IC values of compounds indicate the concentration that caused 50% enzyme activity loss; b IC (nM) ± SE;
50
50
nd = not determined; data show mean ± SE values for at least three independent experiments; * p < 0.05.

326
Merve Zengin et al.
IR (n , cm-1): 2918, 2848 (C-H), 1605, 1478, 1438, 1427
(C-N); 1H-NMR (400 MHz; CDCl ; Me Si; d , ppm):
max
3
4
H
(C=N, C=C), 1227 (C-N); 1H-NMR (400 MHz; CDCl ;
3.22–3.26 (4H, m, piperazine); 3.50–3.52 (4H, m,
piperazine); 6.99 – 7.10 (4H, m, H , H , H , H ); 7.26
3
Me Si; d , ppm): 1.11 – 1.93 (10H, m, cyclohexyl);
3’
5’
4’’
6’’
4
H
(1H, m, H , overlapped CHCl ); 7.33 (1H, td, J = 8.0 Hz,
2.28 – 2.35 (1H, m, -N-CH-); 2.72 – 2.78 (4H, m,
piperazine); 3.30 – 3.36 (4H, m, piperazine); 6.95 (2H, d,
J = 8.0 Hz, H , H ); 7.31 (1H, td, J = 8.0 Hz, J = 1.2 Hz,
5’’
3
1
J = 1.2 Hz, H ); 7.40 (1H, dd, J = 8.0 Hz, J = 1.6 Hz H );
2
6
1
2
3’’
7.46 (1H, td, J = 7.6 Hz, J = 1.6 Hz, H ); 7.86 (1H, d,
3’
5’
1
2
1
2
5
J = 7.6 Hz, H ); 8.01–8.04 (3H, m, H , H , H ). 13C-NMR
H ); 7.44 (1H, td, J = 8.4 Hz, J = 1.2 Hz, H ); 7.85 (1H, d,
6
1
2
5
4
7
2’
6’
J = 8.0 Hz, H ); 7.95–8.01 (3H, m, H , H , H ). 13C-NMR
(100 MHz; CDCl ; Me Si; d , ppm): 48.32 and 51.04
4
7
2’
6’
3
4
C
(100 MHz; DMSO-d ; Me Si; d , ppm): 25.81; 26.26; 28.92;
(piperazine); 115.04; 120.37; 121.43; 122.53; 124.07;
124.55; 126.13; 127.66; 128.84; 130.74; 134.60; 146.65;
146.89; 153.00; 168.22. ESI-MS (m/z): 428.22 [M+Na]+
(100%), 406.25 [M+H]+.
6
4
C
63.55 (cyclohexyl); 48.27 and 48.77 (piperazine); 114.69;
121.40; 122.52; 123.93; 124.44; 126.05; 128.76; 134.67;
153.05; 154.34; 168.30. ESI-MS (m/z): 400.39 [M+Na]+,
378.41 [M+H]+ (100%).
2-[4-(4-benzylpiperazin-1-yl)phenyl]benzo[d]thiazole
(2h): yield, 0.55 g (71%); m.p., 221°C (from acetone);
Found: C, 75.03; H, 6.22; N, 10.95; S, 8.35; Anal. Calcd. for
2-[4-(4-(2-methoxyphenyl)piperazin-1-yl)phenyl]benzo-
[d]thiazole (2e): yield, 0.64 g (79%); m.p., 218°C (from
ethyl acetate); Found: C, 71.65; H, 5.84; N, 10.40; S, 8.09;
Anal. Calcd. for C H N OS: C, 71.79; H, 5.77; N, 10.47; S,
C H N S: C, 74.77; H, 6.01; N, 10.90; S, 8.32%; IR (n ,
max
24 23
3
24 23
3
cm-1): 3058, 3023 (C-H), 2886, 2830, 2786 (C-H), 1606,
1477, 1436, 1425 (C=N, C=C), 1251 (C-N); 1H-NMR
(400 MHz; CDCl ; Me Si; d , ppm): 2.63 (4H, bs,
7.99%; IR (n , cm-1): 3060 (C-H), 2951, 2829 (C-H), 1602,
max
1498, 1479, 1439, 1421 (C=N, C=C), 1223 (C-N), 1029
1
(C-O); H-NMR (400 MHz; DMSO-d ; Me Si; d , ppm):
3
4
H
piperazine); 3.35 (4H, bs, piperazine); 3.59 (2H, s, -CH -);
6
4
H
2
3.08–3.13 (4H, m, piperazine); 3.42–3.46 (4H, m,
6.94 (2H, d, J = 9.2 Hz, H , H ); 7.26–7.36 (6H, m, H , H ,
3’
5’
6
2’’
piperazine); 3.79 (3H, s, -OCH ); 6.86 – 6.99 (4H, m,
3
H , H , H , H ); 7.44 (1H, t, J = 7.2 Hz, H ); 7.85 (1H, d,
3’’ 4’’ 5’’ 6’’ 5
J = 7.2 Hz, H ); 7.95–8.01 (3H, m, H , H , H ). 13C-NMR
H -H ); 7.11 (2H, d, J = 9.2 Hz, H , H ); 7.36 (1H, td,
3’’
6’’
3’
5’
4
7
2’
6’
J = 7.6 Hz, J = 0.8 Hz, H ); 7.47 (1H, td, J = 7.6 Hz,
1
2
6
1
(100 MHz; CDCl ; Me Si; d , ppm): 47.85 and 52.77
3
4
C
J = 1.2 Hz, H ); 7.91 – 7.95 (3H, m, H , H , H ); 8.04 (1H,
2
5
4
2’
6’
(piperazine); 62.99 (-CH -); 114.76; 121.41; 122.54; 123.99;
d, J = 8.0 Hz, H ). 13C-NMR (100 MHz; CDCl ; Me Si; d ,
2
7
3
4
C
124.46; 126.06; 127.25; 128.33; 128.77; 129.19; 134.68;
153.01; 154.34; 168.27. ESI-MS (m/z): 408.25 [M+Na]+,
386.29 [M+H]+ (100%).
ppm): 48.21 and 50.53 (piperazine); 55.45 (-O-CH ); 111.38;
3
114.91; 118.32; 121.08; 121.43; 122.55; 123.38; 124.18;
124.49; 126.08; 128.81; 134.69; 151.34; 152.31; 153.07;
154.31; 168.25; 154.33; 168.24. ESI-MS (m/z): 424.29
[M+Na]+ (100%), 402.33 [M+H]+.
2-[4-(4-(3-methylbenzyl)piperazin-1-yl)phenyl]benzo-
[d]thiazole (2i): yield, 0.58 g (72%); m.p., 186°C (from ace-
tone); Found: C, 75.04; H, 6.24; N, 10.53; S, 8.13; Anal.
Calcd. for C H N S: C, 75.15; H, 6.31; N, 10.52; S, 8.03%;
2-[4-(4-(3-methoxyphenyl)piperazin-1-yl)phenyl]benzo-
[d]thiazole (2f): yield, 0.58 g (72%); m.p., 248°C (from
chloroform and methanol); Found: C, 71.39; H, 5.95; N,
10.28; S, 7.78; Anal. Calcd. for C H N OS: C, 71.79; H,
25 25
3
IR (n , cm-1): 2918, 2845 (C-H), 1606, 1478, 1437, 1426
max
1
(C=N, C=C), 1249 (C-N); H-NMR (400 MHz; DMSO-d ;
6
24 23
3
Me Si; d , ppm): 2.29 (3H, s, -CH ); 2.47 – 2.51 (4H, m,
5.77; N, 10.47; S, 7.99%; IR (n , cm-1): 2975, 2880, 2837
4
H
3
max
piperazine, overlapped DMSO); 3.28–3.32 (4H, m,
piperazine); 3.47 (2H, s, -CH -); 7.01–7.13 (5H, m, H , H ,
(C-H), 1602, 1479, 1434, 1425 (C=N, C=C), 1224 (C-N),
1
1036 (C-O); H-NMR (400 MHz; CDCl ; Me Si; d , ppm):
2
3’
5’
3
4
H
H , H , H ); 7.20 (1H, t, J = 7.2 Hz, H ); 7.35 (1H, td,
2’’
4’’
6’’
5’’
3.36 (4H, bs, piperazine); 3.49 (4H, bs, piperazine); 3.81
(3H, s, -OCH ); 6.46–6.64 (3H, m, H , H , H ); 7.01 (2H,
J = 8.4 Hz, J = 1.2 Hz, H ); 7.46 (1H, td, J = 8 Hz,
1
2
6
1
3
2’’
4’’
6’’
J = 1.2 Hz, H ); 7.88 (2H, d, J = 8.8 Hz, H , H ); 7.94 (1H,
2
5
2’
6’
d, J = 8.4 Hz, H , H ); 7.21 (1H, t, J = 8.4 Hz, H ); 7.33
3’
5’
5’’
d, J = 7.6 Hz, H ); 8.02 (1H, d, J = 7.6 Hz, H ;). 13C-NMR
4
7
(1H, td, J = 7.8 Hz, J = 1.2 Hz, H ); 7.45 (1H, td,
1
2
6
(100 MHz; CDCl ; Me Si; d , ppm): 21.40 (Ar-CH ); 47.81
J = 7.8 Hz, J = 1.2 Hz, H ); 7.86 (1H, d, J = 8.0 Hz, H );
3
4
C
3
1
2
5
4
7.99 – 8.04 (3H, m, H , H , H ). 13C-NMR (100 MHz;
and 52.81 (piperazine); 63.02 (-CH -); 114.74; 121.40;
2
7
2’
6’
122.53; 123.96; 124.45; 126.05; 126.30; 127.99; 128.19;
128.76; 129.94; 134.68; 137.95; 153.01; 154.33; 168.27.
ESI-MS (m/z): 422.26 [M+Na]+, 400.30 [M+H]+ (100%).
2-[4-(4-(4-fluorobenzyl)piperazin-1-yl)phenyl]benzo-
[d]thiazole (2j): yield, 0.55 g (68%); m.p., 216°C (from ace-
tone); Found: C, 71.73; H, 5.58; N, 10.56; S, 8.00; Anal.
Calcd. for C H FN S: C, 71.44; H, 5.50; N, 10.41; S,
CDCl ; Me Si; d , ppm): 47.95 and 49.12 (piperazine);
3
4
C
55.24 (-O-CH ); 102.97; 109.16; 115.05; 121.43; 122.57;
3
124.55; 126.12; 128.84; 129.95; 134.66; 152.75; 160.66;
168.14. ESI-MS (m/z): 424.29 [M+Na]+ (100%), 402.33
[M+H]+.
2-[4-(4-(2-chlorophenyl)piperazin-1-yl)phenyl]benzo-
[d]thiazole (2g): yield: 0.45 g (55%); m.p., 238°C (from
chloroform); Found: C, 68.23; H, 4.83; N, 10.51; S, 7.91;
Anal. Calcd. for C H ClN S: C, 68.05; H, 4.97; N, 10.35;
24 22
3
7.95%; IR (n , cm-1): 2841 (C-H), 1602, 1507, 1477, 1436,
max
1424 (C=N, C=C), 1249 (C-N); 1H-NMR (400 MHz; CDCl ;
3
23 20
3
S, 7.90%; IR (n , cm-1): 3069 (C-H), 2975, 2883, 2819
Me Si; d , ppm): 2.60 (4H, bs, piperazine); 3.34 (4H, bs,
4
H
max
(C-H), 1603, 1588, 1477, 1438, 1420 (C=N, C=C), 1224
piperazine); 3.54 (2H, s, -CH -); 6.93–7.04 (4H, m, H , H ,
2 3’ 5’

Design and Synthesis of 2-Substitutedphenyl Benzo[d]thiazole Derivatives
327
H , H ); 7.30 – 7.36 (3H, m, H , H , H ); 7.44 (1H, td,
131.83; 134.48; 138.46; 142.53; 152.59; 153.42; 165.88.
ESI-MS (m/z): 279.24 [M+H]+, 301.20 [M+Na]+ (100%).
2-[4-(1H-benzimidazole-1-yl)phenyl]benzo[d]thiazole
(2o): yield, 0.46 g (70%); m.p., 202°C (from acetonitrile);
Found: C, 73.46; H, 4.17; N, 12.95; S, 9.71; Anal. Calcd. for
3’’
5’’
6
2’’
6’’
J = 7.6 Hz, J = 1.2 Hz, H ); 7.85 (1H, d, J = 7.6 Hz, H );
1
2
5
4
7.94 – 8.20 (3H, m, H , H , H ). 13C-NMR (100 MHz;
7
2’
6’
CDCl ; Me Si; d , ppm): 47.81 and 52.66 (piperazine);
3
4
C
62.10 (-CH -); 114.81; 115.15; 121.41; 122.54; 124.09;
2
124.48; 126.07; 128.77; 130.66; 134.67; 152.93; 154.32;
160.92; 163.35; 168.22. ESI-MS (m/z): 426.23 [M+Na]+,
404.27 [M+H]+ (100%).
C H N S: C, 73.37; H, 4.00; N, 12.83; S, 9.79%; IR (n ,
max
cm-1): 3058 (C-H), 1604, 1524, 1487, 1453 (C=N, C=C),
20 13
3
1225 (C-N); 1H-NMR (400 MHz; DMSO-d ; Me Si; d ,
6
4
H
2-[4-(4-(3-chlorobenzyl)piperazin-1-yl)phenyl]benzo-
[d]thiazole (2k): yield, 0.59 g (70%); m.p., 199°C (from ac-
etone); Found: C, 68.23; H, 5.25; N, 10.12; S, 7.63; Anal.
Calcd. for C H ClN S: C, 68.64; H, 5.28; N, 10.01; S,
ppm): 7.31–7.39 (2H, m, benzimidazole H , H ); 7.48 (1H,
5
6
td, J = 8 Hz, J = 1.2 Hz, H ); 7.56 (1H, td, J = 7.2 Hz,
1
2
6
1
J = 1.2, H ); 7.75 (1H, d, J = 8 Hz, benzimidazole H ); 7.80
2
5
4
24 22
3
7.64%; IR (n , cm-1): 3055 (C-H), 2882, 2842 (C-H), 1606,
(1H, d, J = 6.8 Hz, benzimidazole H ); 7.90 (2H, d,
7
max
1477, 1435, 1425 (C=N, C=C), 1249 (C-N); 1H-NMR
J = 9.2 Hz, H , H ); 8.09 (1H, d, J = 7.6 Hz, H ); 8.17 (1H,
3’
5’
4
(400 MHz; CDCl ; Me Si; d , ppm): 2.60–2.62 (4H, m,
d, J = 7.6 Hz, H ); 8.31 (2H, d, J = 8.4 Hz, H , H ); 8.68
7 2’ 6’
(1H, s, benzimidazole H ). 13C-NMR (100 MHz; DMSO-d ;
3
4
H
piperazine); 3.33 – 3.38 (4H, m, piperazine); 3.55 (2H, s,
-CH -); 7.06 (2H, d, J = 8, H , H ); 7.24–7.30 (3H, m,
2
6
Me Si; d , ppm): 110.87; 120.09; 122.42; 122.76; 122.97;
2
3’
5’
4
C
H -H ); 7.32 (1H, td, J = 8, J = 1.2, H ); 7.38 (1H; s;
4’’
6’’
1
2
6
123.72; 124.02; 125.69; 126.77; 128.83; 131.70; 132.64;
134.60; 138.23; 143.14; 143.97; 153.56; 166.10. ESI-MS
(m/z): 328.26 [M+H]+, 350.22 [M+Na]+ (100%).
H ); 7.44 (1H, td, J = 8, J = 1.2, H ); 7.85 (1H, d, J = 8,
2’’
1
2
5
H ); 7.96 – 8.02 (3H, m, H , H , H ). 13C-NMR (100 MHz;
4
7
2’
6’
CDCl ; Me Si; d , ppm): 47.84 and 52.75 (piperazine);
3
4
C
62.30 (-CH -); 114.82; 121.41; 122.54; 124.48; 126.07;
2
EXPERIMENTAL BIOLOGICAL PART
127.18; 127.43; 128.77; 129.06; 129.59; 134.27; 134.67;
152.93; 154.32; 168.22. ESI-MS (m/z): 420 [M+H]+ (100%),
422 [M+H+2]+, 442 [M+Na]+, 444 [M+Na+2]+.
Inhibition of self-mediated Ab
and Ab
aggre-
1–40
1 – 42
gation. Inhibition of Ab
and Ab
aggregation was me-
1–42
2-[4-(1H-pyrazol-1-yl)phenyl]benzo[d]thiazole
[34]: m.p., 178°C.
(2l)
1–40
asured using the ThT method [37]. The inhibitor (100 mM)
and Ab (5 mM) were incubated in the assay medium
2-[4-(1H-imidazole-1-yl)phenyl]benzo[d]thiazole (2m):
yield, 0.59 g (70%); m.p., 199°C (from acetonitrile); Found:
C, 69.73; H, 3.71; N, 15.25; S, 11.36; Anal. Calcd. for
1–40/1–42
containing 0.01 M NaCl in 0.05 M potassium phosphate
buffer (pH 7.4) at 37°C for 48 h. The 100 mM Ab
±
1–40/1–42
C H N S: C, 69.29; H, 4.00; N, 15.15; S, 11.56%; IR (n ,
max
16 11
3
cm-1): 3130, 3056 (C-H), 1606, 1529, 1486, 1440 (C=N,
inhibitor mixture was added to thioflavin T (ThT; 200 mM) in
50 mM glycine-NaOH buffer, pH 8.0 and the reduction in the
fluorescence intensity at Exc: 448 nm Em: 490 nm was mea-
sured using an RF 5301 PC spectrofluorophotometer.
1
C=C), 1252 (C-N); H-NMR (400 MHz; DMSO-d ; Me Si;
6
4
d , ppm): 7.16 (1H, s, imidazole H ); 7.46 (1H, td,
H
4
J = 7.6 Hz, J = 0.8 Hz, H ); 7.54 (1H, t, J = 8 Hz, H );
1
2
6
1
5
7.84 – 7.89 (3H, m, H , H , H ); 8.06 (1H, d, J = 8.4 Hz,
Donepezil (100 mM) was used as the positive controls.
Cholinesterase inhibitory activity. AChE (electric eel),
BChE (equine serum), acetylthiocholine iodide, and S-buty-
rylthiocholine iodide were obtained from Sigma-Aldrich and
5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) was obtained
from Calbiochem (Los Angeles, CA, USA). The cholines-
terase inhibitory activity of the target compounds were deter-
mined using Ellman’s assay [38]. Reactions were initiated by
addition of an enzyme into media containing the substrate
(0.05 – 0.4 mM) and 0.125 mM DTNB in 100 mM 3(N-mor-
pholino)propanesulfonic acid buffer, pH 8.0, at 25°C and
monitored spectrophotometrically at 412 nm using UV-Vis
Model 1700 Shimadzu PC spectrophotometer.
4
3’
5’
H ); 8.14 (1H, d, J = 7.2 Hz, imidazole H ); 8.19 (2H, d,
7
5
J = 8.8 Hz, H , H ); 8.41 (1H, s, imidazole H ). 13C-NMR
2’
6’
2
(100 MHz; DMSO-d ; Me Si; d , ppm): 117.75; 120.64;
6
4
C
122.34; 122.87; 125.59; 126.71; 128.68; 130.28; 131.00;
134.51; 135.60; 138.88; 153.52; 166.12. ESI-MS (m/z):
278.13 (100%) [M+H]+, 300.12 [M+Na]+.
2-[4-(1H-1, 2, 4-triazole-1-yl)phenyl]benzo[d]thiazole
(2n): yield, 0.43 g (77%); m.p., 222°C (from acetone);
Found: C, 67.87; H, 3.72; N, 20.08; S, 11.51; Anal. Calcd.
for C H N S: C, 64.73; H, 3.62; N, 20.1; S, 11.52%; IR
15 10
4
(n , cm-1): 3089 (C-H), 1606, 1529, 1503, 1487, 1439
max
1
(C=N, C=C), 1222 (C-N); H-NMR (400 MHz; DMSO-d ;
6
Me Si; d , ppm): 7.46 (1H, td, J = 7.6 Hz, J = 1.2 Hz, H );
4
H
1
2
6
Statistical analysis. Statistical analysis was performed
using GraphPad Prism software using one-way ANOVA
analysis (Bonferroni’s post-hoc test) in comparison to con-
trol, with p-values less than 0.05 considered statistically sig-
nificant.
7.54 (1H, td, J = 7.6 Hz, J = 1.2 Hz, H ); 8.05–8.08 (3H,
1
2
5
m, H , H , H ); 8.15 (1H, d, J = 8 Hz, H ); 8.25 (2H, d,
4
3’
5’
7
J = 9.2 Hz, H , H ); 8.29 (1H; s; triazole H ); 9.42 (1H, s,
2’
6’
3
triazole H ). 13C-NMR (100 MHz; DMSO-d ; Me Si; d ,
5
6
4
C
ppm): 119.80; 122.24; 122.82; 125.55; 126.62; 128.54;

328
Merve Zengin et al.
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A series of new 2-phenylbenzo[d]thiazoles substituted
with azole/piperazine moieties were designed and synthe-
sized. All compounds were evaluated for their Ab aggrega-
tion and ChE inhibitory activity, and three azole-substituted
compounds 2m – 2o (with imidazole, triazole, and benzimi-
dazole rings, respectively) showed stronger inhibitory effects
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on Ab
and Ab
aggregation. Furthermore, pipera-
1 – 42
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103, 8628 – 8633 (2006).
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methyl, ethyl, and acetyl (2a – 2c), were more promising
than bulky moieties such as cyclohexyl, phenyl, and benzyl
(2d – 2k), for Ab aggregation inhibition (except for 2i).
However, no one compound exhibited good inhibitory activ-
ity against both AChE and BChE. Therefore, imidazole 2m
may be promising therapeutic option for inhibiting Ab-medi-
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ACKNOWLEDGEMENTS
23. U. A. Mohsen, Z. A. Kaplancikli, Y. Özkay, and L. Yurttaº,
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The authors gratefully acknowledge financial support
from the Hacettepe University Scientific Research Fund
(Project no: THD-2018 – 16569).
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CONFLICTS OF INTEREST
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There are no conflicts of interest to declare.
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