Effect of benzothiazole/piperazine derivatives on intracerebroventricular streptozotocin-induced cognitive deficits

Article abstract of DOI:10.1016/S1734-1140(12)70878-2

Background: In this study, benzothiazole-piperazine compounds were synthesized by condensing the functional groups of donepezil (DNP), FK-960, and sabeluzole, which are known to have therapeutic potential against Alzheimer's disease, with the aim of obtaining new and potent anti-Alzheimer agents. Methods: Initially, acetylcholinesterase/butyrylcholinesterase enzyme inhibition activities of the synthesized test compounds were investigated by Ellman's method. Effects of the compounds on a streptozotocin (STZ) model of Alzheimer's disease (SMAD) were investigated in rats. SMAD was established by intracerebroventricular (icv) injection of STZ (3 mg/kg), bilaterally. The elevated plus maze, Morris water maze, and active avoidance tests were used to examine the effects of test compounds (1, 5, and 10 mg/kg) on learning and memory parameters of icv STZ-injected rats. Effects of the test compounds on spontaneous locomotor activities of rats were examined with the activity cage test. Results: The compounds B2-B5 and DNP exhibited significant selective inhibitory potencies against acetylcholinesterase. Compounds B2 and B3 at 10 mg/kg doses and compounds B4 and B5 at 5 and 10 mg/kg doses, as well as the reference drug DNP (1 and 3 mg/kg), significantly improved the learning and memory parameters of animals in all cognition tests. None of the test compounds changed spontaneous locomotor activities. Conclusion: Results of the present study revealed that compounds B2-B5 repaired the parameters related to the learning and memory deficits of icv STZ-injected rats. Potencies of these test compounds were comparable to the activity of DNP. Copyright

Full text of DOI:10.1016/S1734-1140(12)70878-2

Effect of benzothiazole/piperazine derivatives
on intracerebroventricular streptozotocin-induced
cognitive deficits
1
1
2
1
Ümide Demir Özkay , Özgür Devrim Can , Yusuf Özkay , Yusuf Öztürk
Department of Pharmacology, Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Anadolu University, 26470, Eskiºehir, Turkey
Correspondence: Ümide Demir Özkay, e-mail: udemir@anadolu.edu.tr
Abstract:
Background: In this study, benzothiazole-piperazine compounds were synthesized by condensing the functional groups of donepe-
zil (DNP), FK-960, and sabeluzole, which are known to have therapeutic potential against Alzheimer’s disease, with the aim of ob-
taining new and potent anti-Alzheimer agents.
Methods: Initially, acetylcholinesterase/butyrylcholinesterase enzyme inhibition activities of the synthesized test compounds were
investigated by Ellman’s method. Effects of the compounds on a streptozotocin (STZ) model of Alzheimer’s disease (SMAD) were
investigated in rats. SMAD was established by intracerebroventricular (icv) injection of STZ (3 mg/kg), bilaterally. The elevated
plus maze, Morris water maze, and active avoidance tests were used to examine the effects of test compounds (1, 5, and 10 mg/kg) on
learning and memory parameters of icv STZ-injected rats. Effects of the test compounds on spontaneous locomotor activities of rats
were examined with the activity cage test.
Results: The compounds B2–B5 and DNP exhibited significant selective inhibitory potencies against acetylcholinesterase.
Compounds B2 and B3 at 10 mg/kg doses and compounds B4 and B5 at 5 and 10 mg/kg doses, as well as the reference drug DNP
(1 and 3 mg/kg), significantly improved the learning and memory parameters of animals in all cognition tests. None of the test com-
pounds changed spontaneous locomotor activities.
Conclusion: Results of the present study revealed that compounds B2–B5 repaired the parameters related to the learning and mem-
ory deficits of icv STZ-injected rats. Potencies of these test compounds were comparable to the activity of DNP.
Key words:
Alzheimer’s disease, streptozotocin, donepezil, acetylcholinesterase, elevated plus maze test, Morris water maze test, active
avoidance test, benzothiazole/piperazine
Introduction
Abbreviations: AChE – acetylcholinesterase, AD – Alz-
heimer’s disease, BChE – butyrylcholinesterase, DNP – done-
Alzheimer’s disease (AD) is the most common type
of dementia, comprising 60–70% of all cases [8]. It is
a chronic and irreversible neurodegenerative disorder
characterized by a progressive deterioration of intel-
lectual functions, including memory, language, visio-
spatial skills, problem-solving ability, and abstract
pezil, DTNB – 5,5-dithiobis(2-nitrobenzoic acid), IAL – initial
acquisition latency, ITL – initial transfer latency, icv – in-
tracerebroventricular, 1 RL – first retention latency, 2 RL –
second retention latency, 1 RTL – first retention transfer la-
tency, 2 RTL – second retention transfer latency, SEM – stan-
dard error of the mean, SMAD – streptozotocin model of Alz-
heimer’s disease, STZ – streptozotocin
834

Effect of compounds on icv STZ-induced cognitive deficits
reasoning, as well as behavioral disturbances [12, 62]. rivative, ethoxzolamide, has been reported to suppress
AD is a major health problem and the third largest epileptic seizures [46]. This pharmacophoric group is
cause of death in the world after cardiovascular dis- also important for drug discovery studies in the AD
eases and cancer [53]. Global prevalence of AD was area with several benzothiazole-bearing compounds
reported as 26.6 million in 2006, and it is estimated demonstrating potential therapeutic effects against
that the worldwide prevalence of this disease will AD [6, 11, 30, 38, 41]. Furthermore, another ben-
reach 106.2 million by 2050. Its impact on health care zothiazole-based agent, sabeluzole, has been shown to
costs was predicted at $156 billion per year [51].
Until now, several therapeutic approaches have
delay the clinical progression of AD [13].
Piperidine is another important ring for the devel-
been offered, but only non-competitive N-methyl- opment of new chemical agents, demonstrating thera-
D-aspartate receptor antagonist, memantine, and ace- peutic effects against CNS disorders in particular. Nu-
tylcholinesterase (AChE) inhibitors, tacrine, donepe- merous piperidine-carrying compounds have been
zil (DNP), rivastigmine, and galantamine have been synthesized and investigated for their anti-Alzheimer
approved by the Food and Drug Administration and potential [1, 4, 14, 24, 27, 40, 42, 49]. The chemical
have been applied in the treatment of AD [3, 21, 29]. structure of DNP, an important and widely used drug
However, the effectiveness of these drugs on AD is for the treatment of Alzheimer patients in neurology
limited, and they only provide a palliative therapy for clinics, is also based on a piperidine ring [9, 52, 56,
patients [29, 61]. Besides, treatment with these drugs 60]. Like piperidine, its bioisostere, piperazine has
has several limitations such as short half-life or side- also been subjected to the development of new agents.
effects like hepatotoxicity, nausea, diarrhoea, vomit- FK-960, a new anti-Alzheimer drug candidate, and
ing, anorexia, weight loss, muscle cramps, dizziness, some other piperazine derivative compounds have
hallucinations, confusion, and headache [21, 29]. been demonstrated to possess activities against AD
Therefore, there is still great interest in the develop- [5, 7, 18, 25, 26, 31–36, 53, 58].
ment of more potent and safer agents against AD.
In light of the aforementioned literature and the
Benzothiazole, a heterocyclic ring, is often subjected limitations of drugs currently used for the treatment of
to various drug discovery studies [17]. Some clinically AD, our research group sought to design compounds
available drugs carrying this ring system are used in the carrying benzothiazole and piperazine moieties on the
management of several central nervous system (CNS) same chemical skeleton, with the intention of investi-
diseases. For instance, riluzole, a benzothiazole deriva- gating their potential to treat cognitive defects. Con-
tive, is effective in retarding the progression of amyo- densation studies were carried out according to the mo-
trophic lateral sclerosis [15]. Another benzothiazole de- lecular hybridization strategy, which is an approach
Fig. 1. Designing of the compounds
B1–B5 based on chemical structures
of anti-Alzheimer agents
835

Fig. 2. Synthesis of the compounds
B1–B5
for the logical design of new compounds based on the acetamido)benzothiazole (A), which was then treated
recognition of pharmacophoric sub-unities in 2 or with appropriate piperazine derivatives to obtain final
more bioactive agents that form new hybrid structures products (B1–B5). The synthetic method is given in
whilst preserving pre-selected characteristics of the Figure 2.
original templates [59]. For this purpose, DNP, FK-
960, and sabeluzole, which contain piperidine, pipera-
2-[2-(4-Phenylpiperazin-1-yl)acetylamino]ben-
zothiazole (B1)
zine, and benzothiazole groups, respectively, were se-
lected as model agents (Fig. 1). After a careful litera-
ture survey, we observed that no previous CNS-
related study included the designed compounds.
M.p. 184–186°C. Yield 75%. IR (KBr, cm–1): 3350
(N-H), 1676 (C=O), 1599–1395 (C=C and C=N).
1H-NMR (500 MHz, DMSO-d6, d, ppm): 3.47–3.64
(8H, m, piperazine), 3.92 (2H, s, COCH2), 7.34–7.58
(7H, m, Ar-H), 7.84 (H, d, J = 7.96 Hz, benzothia-
zole), 8.02 (H, d, J = 7.73 Hz, benzothiazole), 10.96
(H, s, NH-CO). MS (Es) m/z: 353 [M + 1].
Materials and Methods
Drugs and chemicals
2-[2-(4-Benzylpiperazin-1-yl)acetylamino]ben-
zothiazole (B2)
Purified AChE (cholinesterase, acetyl C 2888 Type
V-S: from electric eel; 1000–2000 U/mg), purified bu-
tyrylcholinesterase (BChE) (cholinesterase, butyryl
C 4290 from horse serum, highly purified minimum
500 U/mg), streptozotocin (STZ), 1-(4-methylbenzyl)
piperazine, 1-(4-methoxybenzyl)piperazine, and 1-(4-
chlorobenzyl)piperazine were purchased from Sigma-
Aldrich Chemical Co., St. Louis, MO, USA. 5,5-
Dithiobis(2-nitrobenzoic acid) (DTNB), potassium
dihydrogen phosphate, potassium hydroxide, sodium
hydrogen carbonate, acetylthiocholine iodide, and
butyrylthiocholine iodide, used as substrates, were
obtained from Fluka, Buchs, Switzerland. All other
chemicals were purchased from Merck, Darmstadt,
Germany. DNP (Doenza®, Sanovel, Ýstanbul, Turkey),
ketamine (Alfamine®, Woerden, Holland), and xy-
lazine (Alfazyne®, Woerden, Holland) were of com-
mercial grade.
M.p. 118–119°C. Yield 76%. IR (KBr, cm–1): 3350
(N-H), 1676 (C=O), 1599–1395 (C=C and C=N).
1H-NMR (500 MHz, DMSO-d6, d, ppm): 2.14 (2H, s,
C6H5CH2), 2.37–2.66 (8H, m, piperazine), 3.62 (2H,
s, COCH2), 7.24–7.48 (7H, m, Ar-H), 7.83 (H, d, J =
7.94 Hz, benzothiazole), 8.00 (H, d, J = 7.78 Hz, ben-
zothiazole), 11.56 (H, s, NH-CO). MS (Es) m/z: 367
[M + 1].
2-[2-[4-(4-Methoxybenzyl)piperazin-1-yl]ace-
tylamino]benzothiazole (B3)
M.p. 114–115°C. Yield 77%. IR (KBr, cm–1): 3349
(N-H), 1676 (C=O), 1599–1397 (C=C and C=N).
1H-NMR (500 MHz, DMSO-d6, d, ppm): 2.34 (2H, s,
C6H5CH2), 3.26–3.44 (8H, m, piperazine), 3.82 (2H,
s, COCH2), 3.98 (3H, s, OCH3), 6.84–7.38 (6H, m,
Synthesis of compounds
Target compounds were synthesized over 2 steps. Ini- Ar-H), 7.71 (H, d, J = 7.98 Hz, benzothiazole), 7.92
tially, 2-aminobenzothiazole was acetylated with (H, d, J = 7.74 Hz, benzothiazole), 11.54 (H, s,
chloroacetyl chloride in benzene to give 2-(2-chloro- NH-CO). MS (ES) m/z: 397 [M + 1].
836

Effect of compounds on icv STZ-induced cognitive deficits
2-[2-[4-(4-Methylbenzyl)piperazin-1-yl]ace-
tylamino]benzothiazole (B4)
1%) and put into a 100-ml volumetric flask. The vol-
ume was made up with water to 5 units/ml and further
diluted to 2.5 units/ml. All solutions were freshly pre-
pared before enzymatic assays and used rapidly. Stock
solutions of the synthesized compounds were pre-
pared in 2% DMSO. Enzyme activities were deter-
mined in the presence of 6 different concentrations of
test compound. Each concentration was assayed in
triplicate. The samples were investigated immediately
after preparation. Enzyme solution (100 μl) and test
compound solution (100 μl) were added to a cuvette
containing the phosphate buffer (3.0 ml, 0.1 M; pH =
8.0). After 5-min incubation, the required aliquots of
DTNB solution (100 μl) and acetylthiocholine iodide
(20 μl) or butyrylthiocholine iodide (20 μl) were
added. After rapid and immediate mixing, the absorp-
tion was measured at 412 nm. As a reference, an iden-
tical solution of the enzyme without the inhibitor was
processed following the same protocol. DNP was
used as a positive control [20].
M.p. 140–142°C. Yield 74%. IR (KBr, cm–1): 3348
(N-H), 1676 (C=O), 1599–1395 (C=C and C=N).
1H-NMR (500 MHz, DMSO-d6, d, ppm): 1.18 (3H, s,
CH3), 2.25 (2H, s, C6H5CH2), 2.41–2.64 (8H, m,
piperazine), 3.56 (2H, s, COCH2), 7.04–7.46 (6H, m,
Ar-H), 7.78 (H, d, J = 7.93 Hz, benzothiazole), 7.98
(H, d, J = 7.71 Hz, benzothiazole), 12.04 (H, s,
NH-CO). MS (ES) m/z: 381 [M + 1].
2-[2-[4-(4-Chlorobenzyl)piperazin-1-yl]ace-
tylamino]benzothiazole (B5)
M.p. 131–133°C. Yield 71%. IR (KBr, cm–1): 3237
(N-H), 1694 (C=O), 1605–1398 (C=C and C=N).
1H-NMR (500 MHz, DMSO-d6, d, ppm): 2.11 (2H, s,
C6H5CH2), 2.36–2.68 (8H, m, piperazine), 3.65 (2H,
s, COCH2), 7.25–7.44 (6H, m, Ar-H), 7.84 (H, d, J =
7.93 Hz, benzothiazole), 7.98 (H, d, J = 7.77 Hz, ben-
zothiazole), 12.10 (H, s, NH-CO). MS (ES) m/z: 401
[M + 1].
Animals
All experiments were carried out using adult Wistar
rats weighing 200–250 g. The animals were housed in
Conversion of DNP HCl to DNP form
a temperature controlled (24
1°C) room under
Ninety Doenza® tablets, including 900 mg DNP HCl,
were granulated and dissolved in 250 ml of distilled
water. The obtained suspension was stirred at room
temperature for 30 min and filtered. The filtrate was
treated with 10% NaOH (10 ml) and sequentially ex-
tracted with ethyl acetate (3 × 10 ml). Ethyl acetate
extracts were pooled and dried over anhydrous
Na2SO4; the solvent was removed under reduced
pressure to give 780 mg DNP.
a 12-h light/12-h dark cycle and were acclimatized to
the laboratory environment at least 48 h before the ex-
perimental session. Rats were maintained in groups
no greater than 4 per cage prior to and 1 per cage after
intracerebroventricular (icv) STZ injections. During
experiments, food and tap water were allowed ad libi-
tum. The experimental protocols were approved by
the Local Ethical Committee on Animal Experimenta-
tion of the Eskiºehir Osmangazi University, Turkey.
AChE/BChE activity assay
Icv administration of STZ
The in vitro AChE/BChE activities of test compounds
were evaluated prior to animal studies. Enzyme activ- Prior to surgery, rats were anesthetized immediately
ity was investigated using a slightly modified color- with a combination of ketamine (100 mg/kg, ip) and
imetric method of Ellman et al. [10]. The evaluation xylazine (5 mg/kg, ip). The animal’s head was fixed
of enzyme activity was performed using a specific in the stereotaxic frame (Harvard Apparatus, 51650,
chromogenic reagent, DTNB. DTNB solution Massachusetts, USA). The scalp was cleaned with io-
(100 ml) was prepared in water containing DTNB dine solution, and a sagittal incision was made to the
(0.396 g) and sodium hydrogen carbonate (0.15 g). midline of the scalp. Two holes were drilled (Harvard
Solutions of acetylthiocholine iodide and butyrylthio- Apparatus, BS4 72-4951, Massachusetts, USA) in the
choline iodide were prepared in 10 ml of water by ad- skull bilaterally over the lateral ventricles. According
justing concentrations to 0.075 M. AChE or BChE to Paxinos and Watson’s atlas [45], the following co-
(500 units) was dissolved in gelatin solution (1 ml, ordinates were used for icv injection: 0.8 mm poste-
837

rior to the bregma, 1.5 mm lateral to the sagittal su-
Activity cage tests were performed just before (on
ture, and 3.6 mm ventral from the surface of the brain. day 14) and after ‘DNP’ and ‘test compounds’ treat-
STZ was dissolved in citrate buffer (pH 4.4) shortly ments (on day 21) to check the presence of possible
before application. Bilateral icv injection of STZ alterations in spontaneous locomotor activities of rats
(3 mg/kg, 10 μl/injection site) was applied gradually in caused by these compounds [44, 48, 57].
2 divided doses, on days 1 and 3. The skin was su-
tured after injections. The same surgical procedures
2. Elevated plus maze measurements
were also performed in the control group, although cit-
rate buffer (10 μl/injection site) was injected instead of
STZ, on the first and third day. After surgery, rats were
housed individually, and special care was undertaken
until spontaneous feeding resumed [2, 37, 44, 57].
The acquisition and retention of memory processes
were evaluated using the elevated plus maze. The ap-
paratus was made of wood and comprised 2 opposite
open arms (50 × 10 cm), crossed with 2 closed arms
(50 × 10 cm) with 40 cm high walls. The arms were
connected by a central square (10 × 10 cm), and the
maze was elevated 50 cm above the floor. The rats
were placed individually at one end of an open arm,
facing away from the central square. The latency time
to enter into the closed arms was recorded as initial
transfer latency (ITL) and then, rats were allowed to
explore the maze for 30 s. The cut-off time was fixed
at 300 s and rats that could not find the closed arms
within this period were eliminated from the experi-
ments.
Administration of drugs and test compounds
The animals were randomly divided into the follow-
ing groups: the control group, which received icv
citrate buffer; the STZ model of AD (SMAD) group,
which received icv STZ; the DNP-injected SMAD
groups; and the test compound-injected SMAD
groups.
Two weeks after injection of STZ, DNP (1 and
3 mg/kg) was applied to the ‘DNP-injected SMAD
groups’, and 5 test compounds (1, 5, and 10 mg/kg)
were applied to the respective ‘test compound-
injected SMAD groups’ via intraperitoneal injection
over 1 week. Control groups were treated with sun-
flower oil, which is the solvent of the reference drug
DNP and test compounds.
Elevated plus maze tests were performed on days
13, 14, and 21 after icv STZ injection. Acquisition of
memory was assessed on day 13. On days 14 and 21,
latency times to enter the closed arms were measured
again and recorded as first retention transfer latency
(1 RTL) and second retention transfer latency (2
RTL), respectively [22, 57].
Behavioral tests
3. Morris water maze measurements
Activity cage, elevated plus maze, and Morris water
maze tests were performed in the same experimental
groups. In order to avoid potential interactions be-
tween the tests, different experimental groups were
used for active avoidance measurements.
Morris water maze tests were performed to evaluate
spatial learning and memory of animals [39]. The
water maze apparatus consisted of a circular water
tank (150 cm diameter × 60 cm high) which was filled
up to 40 cm with water at 25 1°C. The pool was
divided virtually into 4 equal quadrants. A round plat-
form was placed 2 cm below the surface of water and
remained in the same quadrant for the entire experi-
1. Activity cage measurements
The horizontal and vertical locomotor activities of ment [16, 28, 54].
rats were recorded by the activity cage apparatus,
Before the training session, rats were allowed to
which contains 2 pairs of 16 photocells positioned swim freely in the pool for 60 s without a platform. On
3 cm and 12 cm above the floor (UgoBasile, 7420, day 13, animals were subjected to 4 trials. The rats
Varese, Italy). Interruptions of light beams to the pho- were left in the water, facing the wall of the maze from
tocells during horizontal and vertical movements of the starting points, which were in the middle of each
the animals were automatically recorded for 10 min. quadrant. The latency time to find the hidden platform
The apparatus was placed in a sound-attenuated and was recorded as initial acquisition latency (IAL) [22,
well-ventilated room [44, 48, 57].
23]. Trials were given to rats for a maximum time of 60
838

Effect of compounds on icv STZ-induced cognitive deficits
Tab. 1. AChE IC (μ/M) and BChE IC (μ/M) of synthesized com-
pounds. Results are presented as the mean SEM
s (cut-off time) to find the hidden platform, and rats
were allowed to stay on it for 30 s [16, 19].
On days 14 and 21, latency times to attain the hid-
den platform were recorded as first retention latency
(1 RL) and second retention latency (2 RL), respec-
tively [22, 23].
Comp.
R
AChE
IC50 (μ/M)
BChE IC50 Selectivity*
(μ/M)
22.6
3.07
104.8
9.15
4.64
55.6
B1
B2
B3
B4
B5
DNP
4. Active avoidance measurements
0.52
28.9
4.63
0.072
Learning-related behaviors of rats were assessed by
two-way active avoidance apparatus (UgoBasile,
7530, Varese, Italy). An auditory (70 dB, 670 Hz) and
light stimulus (10 W), administered for a maximum of
11 s, served as the conditioned stimulus. One second
later, a 0.4 mA electric shock was administered for a
maximum time of 10 s, which served as the uncondi-
tioned stimulus. The total time for each trial was 22 s;
the time between 2 consecutive trials was 4 s. Fifty
trials were given every 3 consecutive days. The first
session was carried out 10 min after the accommoda-
tion period. The total number of avoidances (number
of crossings during unconditioned stimulus) and la-
tency periods (as time before the first crossing) of ani-
mals were recorded on days 11, 14, and 21 [55].
0.13
16.6
1.27
127.7
143.3
107.8
181.7
O-CH3
0.019
0.061
8.74
0.36
CH
3
0.0044
0.086
9.27
0.41
0.0029
0.023
4.18
0.34
0.0015
* BChE IC (μ/M)/AChE IC (μ/M)
was comparable with the reference drug DNP. Moder-
ate inhibitory activity was observed with compounds
B2 and B3 (Tab. 1).
Statistical analyses
The data used in statistical analyses were obtained
from 7 animals for each group. Experimental data of
all tests were analyzed by repeated measures ANOVA
followed by Tukey’s HSD multiple comparison test
using GraphPad Prism 4.03 (GraphPad Software, San
Diego, CA, USA). The results were expressed as the
mean standard error of the mean (SEM). Differ-
ences between data sets were considered as signifi-
cant when p value was less than 0.05.
Confirmation of the cognitive deficits with
behavioral tests
Before administration of DNP and test compounds,
elevated plus maze, Morris water maze, and active
avoidance tests were performed on control and icv
STZ-injected animals in order to confirm the estab-
lishment of an AD model in icv STZ-injected rats.
In the elevated plus maze test, there was a signifi-
cant difference between the ITL (on day 13) and 1
RTL (on day 14) values of the icv citrate buffer-
injected control group, reflecting the unimpaired cog-
nitive ability and memory behavior of control ani-
mals. However, the difference between these parame-
ters was not significant in the icv STZ-injected
Results
Cholinesterase activity
The inhibitory potencies of the test compounds against groups, indicating the establishment of SMAD in these
AChE and BChE enzyme activities were evaluated by groups, as expected (Fig. 3). Similar results were ob-
Ellman’s test. The IC50 values for the inhibition of served for the IAL (on day 13) and 1 RL (on day 14)
cholinesterase activity are shown in Table 1. Phenyl- parameters in Morris water maze (Fig. 4), and for la-
substituted compound B1 exhibited a poor inhibitory tency times (Fig. 5) and number of avoidance (Fig. 6)
activity on both AChE and BChE. However, com- parameters in active avoidance tests. Spontaneous lo-
pounds B4 and B5 exhibited notable activity, which comotor activity did not differ significantly between
839

Fig. 3. Elevated plus maze response latencies of the animals in the experimental groups before the DNP and test compounds administrations.
(A) SMAD groups which would be treated with DNP and compound B2. (B) SMAD groups which would be treated with DNP and compound B3.
(C) SMAD groups which would be treated with DNP and compound B4. (D) SMAD groups which would be treated with DNP and compound B5.
Significance against control values, *** p < 0.001. Values are given as the mean SEM
Behavioral tests
the control and icv STZ-injected groups on day 14
(data not shown).
STZ-injected animals (icv) were administrated 1, 5,
and 10 mg/kg doses of test compounds B2, B3, B4,
and B5 and 1 and 3 mg/kg doses of DNP over 1 week.
After this application period, on day 21, behavioral
experiments were repeated to observe the effect of
treatments on animals with AD.
DNP and test compounds (B2, B3, B4, and B5) were
administrated to animals just after impairments in learn-
ing and memory parameters of icv STZ-injected ani-
mals, which were confirmed by the above-mentioned
behavioral tests. Due to the poor potency against AChE,
compound B1 was not assessed in the in vivo tests.
Fig. 4. IAL and 1 RL values of experimental groups in Morris water maze test before the DNP and test compounds administrations. (A) SMAD
groups which would be treated with DNP and compound B2. (B) SMAD groups which would be treated with DNP and compound B3. (C) SMAD
groups which would be treated with DNP and compound B4. (D) SMAD groups which would be treated with DNP and compound B5. Signifi-
cance against control values, *** p < 0.001. Values are given as the mean SEM
840

Effect of compounds on icv STZ-induced cognitive deficits
Fig. 5. Latency times of experimental groups in active avoidance test before the DNP and test compounds administrations. (A) SMAD groups
which would be treated with DNP and compound B2. SMAD groups which would be treated with DNP and compound B3. (C) SMAD groups
which would be treated with DNP and compound B4. (D) SMAD groups which would be treated with DNP and compound B5. Significance
against control values, ** p < 0.01. Values are given as the mean SEM
Fig. 6. Number of avoidances of experimental groups in active avoidance test before the DNP and test compounds administrations. (A) SMAD
groups which would be treated with DNP and compound B2. (B) SMAD groups which would be treated with DNP and compound B3. (C) SMAD
groups which would be treated with DNP and compound B4. (D) SMAD groups which would be treated with DNP and compound B5. Signifi-
cance against control values, **p < 0.01. Values are given as the mean SEM
In elevated plus maze tests, compounds B4 and B5 creased the 2 RTL times, as expected. The effects of
at 5 and 10 mg/kg doses and compounds B2 and B3 at B2, B3, B4, and B5 were comparable to that of DNP,
10 mg/kg doses significantly decreased the 2 RTL as shown in Figure 7.
times of animals. B4 and B5 were significantly more
In Morris water maze tests, similarly to the results
effective than B2 and B3 (at 10 mg/kg doses) in terms of elevated plus maze tests, B4 and B5 at 5 and
of the 2 RTL time-shortening effects (Fig. 7). At 10 mg/kg and B2 and B3 at 10 mg/kg significantly re-
1 and 3 mg/kg doses, the reference drug DNP de- duced the 2 RL times of animals. At 10 mg/kg
841

Fig. 7. 2 RTL values of control, SMAD, DNP-treated (1 and 3 mg/kg) SMAD and compound-treated (1, 5 and 10 mg/kg B2–B5) SMAD groups
in elevated plus maze test on day 21. Significance against control values, *** p < 0.001; significance against SMAD group values, p < 0.01,
p < 0.001; significance against 1 mg/kg DNP-treated group values, p < 0.01, p < 0.001; significance against 3 mg/kg DNP-treated group
values, p < 0.05, p < 0.01, p < 0.001; significance against 1 mg/kg compound-treated group values, p < 0.05, p < 0.01, p < 0.001; signifi-
cance against 5 mg/kg compound-treated group values, p < 0.05, p < 0.01; significance against 10 mg/kg compound B2-treated group
values, p < 0.05, p < 0.01; significance against 10 mg/kg compound B3-treated group values, p < 0.01, p < 0.001. Values are given as the
mean SEM
Fig. 8. 2 RL values of control, SMAD, DNP-treated (1 and 3 mg/kg) SMAD and compound-treated (1, 5 and 10 mg/kg B2–B5) SMAD groups
in Morris water maze test on day 21. Significance against control values, ***p < 0.001; significance against SMAD group values, p < 0.05,
p
< 0.01, p < 0.001; significance against 1 mg/kg DNP-treated group values, p < 0.05, p < 0.01; significance against 3 mg/kg DNP-treated
group values, p < 0.05, p < 0.01, p < 0.001; significance against 1 mg/kg compound-treated group values, p < 0.05, p < 0.01, p < 0.001;
significance against 5 mg/kg compound-treated group values, p < 0.05, p < 0.01; significance against 10 mg/kg compound B2-treated
group values, p < 0.05, p < 0.01, significance against 10 mg/kg compound B3-treated group values, p < 0.01, p < 0.001. Values are given
as the mean SEM
842

Effect of compounds on icv STZ-induced cognitive deficits
Fig. 9. Latency time values of control, SMAD, DNP-treated (1 and 3 mg/kg) SMAD and compound-treated (1, 5 and 10 mg/kg B2–B5) SMAD
groups in avoidance test on day 21. Significance against control values, *** p < 0.001; significance against SMAD group values, p < 0.05,
p < 0.01, p < 0.001; significance against 1 mg/kg DNP-treated group values, p < 0.05, p < 0.01, p < 0.001; significance against 3 mg/kg
DNP-treated group values, p < 0.05, p < 0.001; significance against 1 mg/kg compound-treated group values, p < 0.05, p < 0.001; signifi-
cance against 5 mg/kg compound-treated group values, p < 0.05; significance against 10 mg/kg compound B2-treated group values, p <
0.05, p < 0.01; significance against 10 mg/kg compound B3-treated group values, p < 0.01, p < 0.001. Values are given as the mean SEM
Fig. 10. Number of avoidances of control, SMAD, DNP-treated (1 and 3 mg/kg) SMAD and compound-treated (1, 5 and 10 mg/kg B2–B5)
SMAD groups in avoidance test on day 21. Significance against control values, *** p < 0.001; significance against SMAD group values,
p < 0.05, p < 0.01, p < 0.001; significance against 1 mg/kg DNP-treated group values, p < 0.05, p < 0.01; significance against 3 mg/ kg
DNP-treated group values, p < 0.05, p < 0.01, p < 0.001; significance against 1 mg/kg compound-treated group values, p < 0.05,
p < 0.01, p < 0.001; significance against 5 mg/kg compound-treated group values, p < 0.05; significance against 10 mg/kg compound
B2-treated group values, p < 0.05, p < 0.01; significance against 10 mg/kg compound B3-treated group values, p < 0.05, p < 0.01. Values
are given as the mean SEM
843

doses, B4 and B5 were significantly more effective ported that DNP preferentially inhibits AChE rather
than B2 and B3 (Fig. 8). DNP was effective in de-
creasing 2 RL times, as expected (Fig. 8).
than BChE [43]. This finding was in accordance with
the results of our enzymatic study. The results from in
vitro AChE enzyme inhibition assays encouraged us
to investigate the potential anti-Alzheimer effects of
compounds B2, B3, B4, and B5 in vivo, whereas com-
pound B1 was not evaluated in vivo due to its poor po-
tency against AChE.
In active avoidance tests, latency times were signifi-
cantly decreased (Fig. 9), and avoidance numbers were
significantly increased (Fig. 10) by B4 and B5 (5 and
10 mg/kg) and B2 and B3 (10 mg/kg) treatment. B4
and B5 were significantly more effective than B2 and
B3, at 10 mg/kg doses (Fig. 9 and 10). Once again,
DNP improved the measured learning and memory
parameters in this test (Fig. 9 and 10).
Icv injection of STZ to rats at 3 mg/kg has been ac-
cepted as an appropriate animal model for sporadic
AD. The glucosamine-nitrosourea compound STZ
when applied into the brain bilaterally induces signifi-
cant cognitive impairment in animals without affect-
ing peripheral blood glucose levels. SMAD is charac-
terized by progressive deterioration of memory, cere-
bral glucose, and energy metabolism. Desensitization
of neuronal insulin receptors has been suggested as
the cause of impaired cerebral glucose and energy
metabolism in this animal model. Icv injection of STZ
causes a cholinergic deficiency by inhibiting the syn-
thesis of adenosine triphosphate and acetyl-CoA.
Cholinergic deficiency was accompanied by reduced
choline acetyltransferase activity in the hippocampus
and increased AChE activity in rat brains [37, 50]. Bi-
lateral injection of STZ causes more widespread dam-
age and marked impairment in memory compared to
unilateral damage [50, 57].
Differences in the numbers of horizontal and verti-
cal movements between the groups were not statisti-
cally significant on day 21 (data not shown).
Discussion
In the present study, benzothiazole-piperazine com-
pounds were synthesized by condensing the func-
tional groups of DNP, FK-960, and sabeluzole, which
are known to have therapeutic potential against AD,
with the aim of obtaining new and potent anti-
Alzheimer agents.
The first step of this study was to evaluate the pos-
sible inhibitory activity of synthesized compounds on
AChE and BChE enzymes by Ellman’s method. Fol-
lowing this in vitro method, based on the relationship
between inhibition of cholinesterase activities and
treatment of AD, we planned to continue with in vivo
tests to assess the learning and memory parameters of
the experimental animals.
The occurrence of SMAD in rats was confirmed
with behavioral tests. The elevated plus maze, Morris
water maze, and active avoidance tests were applied to
the icv citrate buffer-injected control groups and indi-
cated that learning and memory functions of these con-
trol groups were intact (Figs. 3–6). In contrast, learning
and memory parameters of icv STZ-injected animals
were significantly impaired when compared to control
groups (Figs. 3–6), as expected [2, 37, 44, 57].
Results from Ellman’s method revealed that some
of the tested benzothiazole-piperazine derivatives ex-
hibited a remarkable AChE inhibitory potential. Com-
pounds B2, B3, B4, and B5 possessed significant in-
hibitory potencies against AChE, whereas BChE ac-
tivity was not affected by the test compounds. DNP
also selectively inhibited AChE.
Just after these behavioral tests, DNP and test com-
pounds B2, B3, B4, and B5 were administrated to ‘icv
STZ-injected animals’ for 1 week. The rats were taken
through elevated plus maze, Morris water maze, and
active avoidance tests after all administrations were
completed. In all these cognitive tests, compounds
B2, B3, B4, and B5 improved the impaired learning
and memory parameters of icv STZ-injected animals.
B4 and B5 were found to be more effective than B2
and B3, in all performed tests. STZ-injected rats
treated with reference drug DNP demonstrated sig-
nificant improvement in the measured learning pa-
It is well established that acetylcholine, a neuro-
transmitter degraded by AChE in nerve synapses, is
required for the proper function of cholinergic trans-
mission in the regulation of learning and memory pro-
cesses [47]. The enhancement of cholinergic activity
by the inhibition of AChE is the mainstay of sympto-
matic treatment of AD [47]. For example, the cen-
trally acting cholinesterase inhibitor DNP is used for
the symptomatic treatment of AD. It has been re- rameters, as expected (Figs. 7–10).
844

Effect of compounds on icv STZ-induced cognitive deficits
The locomotor activity values of reference and test proposed anti-Alzheimer activities were related, at
compound-treated groups were similar to those of the least partially, to the AChE enzyme inhibitory activi-
control animals. Therefore, it may be suggested that ties of the test compounds. However, probable addi-
these compounds did not change the spontaneous lo- tional mechanisms need to be investigated in further,
comotor activities of animals or affect the results of detailed studies.
cognitive tests.
Chemical structure-pharmacological activity rela-
Acknowledgments:
This work was financially supported by Anadolu University
Research Foundation (Eskiºehir, Turkey). Project No. 070321.
tionships revealed that substituents on the fourth posi-
tion of the piperazine ring have a substantial effect on
pharmacological activity. Compounds B2–B5, which
bear 4-substituted benzyl piperazines, indicated sig-
nificant pharmacological activity in vitro, compared
with the phenyl-substituted piperazine compound B1.
This result suggests that the benzyl moiety on the
piperazine ring substantially increases the AChE in-
hibitory activity. This may be explained by chemical
differences between compounds B1 and B2–B5. In
compound B1, N-phenyl substitution decreases the
electron density of the piperazine ring. On the other
hand, a methylene group between aromatic and
piperazine rings enhances the electron density of
piperazine in compounds B2–B5. It is well known
that electron density plays an important role in biomo-
lecular interactions, which is a pre-condition of phar-
macological activity. Thus, it may be suggested that
varying electron density caused differences in the ac-
tivities of B1 and B2–B5 in vitro. Lipophilicity is an-
other important structural feature affecting pharma-
cological activity. Due to the different substitution
patterns of compounds B2–B5, their lipophilicity var-
ies and can be ordered as B5 > B4 > B3 > B2. They
are similarly ordered in terms of their efficacies. The
compounds B4 and B5 possess greater pharmacologi-
cal activity than compounds B2 and B3 both in in vi-
tro and in vivo tests. This interesting finding suggests
that compounds B4 and B5 may pass through the
CNS more readily than B2 and B3 due to their more
lipophilic nature, which accounts for their improved
pharmacological activity.
The authors would like to thank Dr. Erolº ener from Anadolu
University, Faculty of Pharmacy, Department of Analytical
Chemistry, for his help in stereotaxic applications.
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Received: September 26, 2011; in the revised form: March 5, 2012;
accepted: March 26, 2012
847