NO-donating tacrine hybrid compounds improve scopolamine-induced cognition impairment and show less hepatotoxicity

Article abstract of DOI:10.1021/jm801131a

A series of tacrine - NO donor hybrid compounds are synthesized and evaluated for cholinesterase inhibitory activity, cognition improving activity, and hepatotoxicity. The pharmacological results indicate that hybrid compounds 1, 2, and 3a potently inhibit cholinesterase in vitro and significantly improve the scopolamine-induced cognition impairment, whereas an analogue (3h) of 2 without the NO donor moiety does not. Compared to tacrine, 1 and 2 show much less hepatotoxicity. Molecular modeling studies suggest that 2 may interact with the catalytic and the peripheral anionic site of acetylcholinesterase.

Full text of DOI:10.1021/jm801131a

7666
J. Med. Chem. 2008, 51, 7666–7669
NO-Donating Tacrine Hybrid Compounds
Improve Scopolamine-Induced Cognition
Impairment and Show Less Hepatotoxicity
Lei Fang,†,‡ Dorothea Appenroth,§ Michael Decker,b
Michael Kiehntopf,∞ Amelie Lupp,§ Sixun Peng,‡
Christian Fleck,§ Yihua Zhang,*,‡ and Jochen Lehmann*,†
Figure 1. Structures of tacrine, 1, and 2.
Scheme 1. General Method for the Synthesis of 3a-ha
Institute of Pharmacy, Institute of Pharmacology and Toxicology,
and Institute of Clinical Chemistry, Friedrich Schiller UniVersity
Jena, D-07743 Jena, Germany, Center of Drug DiscoVery, China
Pharmaceutical UniVersity, 210009 Nanjing, P. R. China, and
School of Pharmacy, Queen’s UniVersity Belfast,
Belfast, Ireland, U.K.
ReceiVed September 10, 2008
Abstract: A series of tacrine-NO donor hybrid compounds are
synthesized and evaluated for cholinesterase inhibitory activity, cogni-
tion improving activity, and hepatotoxicity. The pharmacological results
indicate that hybrid compounds 1, 2, and 3a potently inhibit cholinest-
erase in vitro and significantly improve the scopolamine-induced
cognition impairment, whereas an analogue (3h) of 2 without the NO
donor moiety does not. Compared to tacrine, 1 and 2 show much less
hepatotoxicity. Molecular modeling studies suggest that 2 may interact
with the catalytic and the peripheral anionic site of acetylcholinesterase.
Alzheimer’s disease (ADa) is one of the most common age-
related diseases in the world. Though much research effort has
been made, it is still incurable. Because of the complex
pathophysiology of the disease, developing novel agents with
multiple pharmacological effects has become a promising
strategy in today’s search for new treatment of AD.1,2 Previ-
ously, we have reported several NO donor-tacrine hybrid
compounds that simultaneously possess potent cholinesterase
(ChE) inhibitory activity, moderate vessel relaxant activity, and
hepatoprotective effects and that may be considered as novel
anti-AD drug candidates.3 Among the previously reported hybrid
compounds, the amide-linked nitrate-tacrine hybrid 1 (Figure
1) and the amine-linked nitrate-tacrine hybrid 2 (Figure 1)
showed the best activities in the in vitro evaluations.3 Therefore,
to investigate the influence of the alkylenediamine side chain
of the molecules on their activity, we have designed and
synthesized another eight nitrate-tacrine hybrid compounds
3a-h with shorter and longer diamine side chains. All the new
target compounds were evaluated for the ChE inhibitory activity
in vitro. Furthermore, 1, 2, and 3a were tested in vivo for their
ability to improve scopolamine-induced cognition impairment
and hepatotoxicity. In addition, with the help of a molecular
operating environment (MOE) software, the interaction of 2 with
a Reagents and conditions: (a) POCl3, reflux, 3 h; (b) pentanol,
NH2(CH2)mNH2, reflux, 18 h; (c) fuming HNO3, CH2Cl2, -5 °C; (d) K2CO3,
KI, CH2Cl2, 24 h, room temp.
the active sites of acetylcholinesterase (AChE) was simulated
and analyzed.
Similar to a previously described protocol,3 9-chloro-1,2,3,4-
tetrahydroacridine was reacted with different alkylenediamines
to produce the intermediate 9-aminoalkylamino-1,2,3,4-tetrahy-
droacridines 4a-d. The NO donor moiety was prepared by
treating bromoalkanols with HNO3 under ice cooling. The nitrate
intermediates 5a-d were reacted with amines 4a,c,d in CH2Cl2
in the presence of KI and K2CO3 to give the target compounds
3a-g. Compound 4b directly reacted with 1-bromopropane to
yield 3h (Scheme 1).
All the target compounds were screened on their ChE
inhibitory activity in vitro using Ellman’s assay.4 The results
(Table 1) showed that all the compounds generally retain the
ChE inhibitory effect of the parent compound tacrine. Compared
to tacrine (IC50 ) 45.1 nM), the AChE inhibitory activities of
2 (IC50 ) 5.6 nM), 3a (IC50 ) 9.1 nM), and 3e (IC50 ) 7.7
nM) are 5- to 6-fold improved while the butyrylcholinesterase
(BuChE) inhibitory activities of the target compounds, with the
IC50 varying from 7.2 to 18.1 nM, remaining at a comparable
level. BuChE inhibition has recently been regarded therapeuti-
cally beneficial for the treatment of AD, because BuChE,
contrary to AChE, increases in the course of the disease and
may compensate AChE.5 The target compounds potently inhibit
both of the enzymes more or less nonselectively, which could
be favorable since decreasing AChE and increasing BuChE have
met approximately at the same level when AD symptoms arise
and therapy sets in.6 Furthermore, no pronounced differences
were observed among the ChE inhibitory activities of the target
* To whom correspondence should be addressed. For Y.Z.: phone, +86-
25-86635503; fax, +86-25-86635503; e-mail, zyhtgd@sohu.com. For J.L.:
phone, +49-3641-94-9803; fax, +49-3641-94-9802; e-mail, j.lehmann@uni-
jena.de.
†
Institute of Pharmacy, Friedrich Schiller University Jena.
China Pharmaceutical University.
Institute of Pharmacology and Toxicology, Friedrich Schiller University
‡
§
Jena.
b
Queen’s University Belfast.
Institute of Clinical Chemistry, Friedrich Schiller University Jena.
∞
a Abbreviations: AD, Alzheimer’s disease; ChE, cholinesterase; MOE,
molecular operating environment; AChE, acetylcholinesterase; BuChE,
butyrylcholinesterase; CAS, the catalytic active site; PAS, periphery anion
site; ASAT, aspartate aminotransferase; HE, hematoxylin and eosin.
10.1021/jm801131a CCC: $40.75
2008 American Chemical Society
Published on Web 11/21/2008

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Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7667
Table 1. Inhibition of AChE and BuChE (IC50 Values) and Selectivity
Expressed as the Ratio of the Resulting IC50 Values
IC50 ( SEMa (nM)
compd
AChEb
BuChEb
selectivity ratioc
tacrine
1
2
3a
3b
3c
3d
3e
45.1 ( 6.9
28.2 ( 4.0
5.6 ( 0.7
9.1 ( 3.8
13.0 ( 3.7
21.6 ( 7.4
15.4 ( 6.4
7.7 ( 0.9
17.1 ( 2.4
19.6 ( 0.7
23.4 ( 5.0
5.1 ( 1.0
13.5 ( 3.1
9.9 ( 1.1
7.3 ( 4.7
18.1 ( 9.4
13.3 ( 3.1
14.3 ( 3.2
17.0 ( 7.7
14.8 ( 1.9
12.9 ( 0.7
7.2 ( 1.1
8.8
2.1
0.6
1.4
0.7
1.6
1.1
0.4
1.2
1.5
3.2
3f
3g
3h
a Data are the mean values of at least three determinations. b AChE from
electric eel and BuChE from equine serum were used. c Selectivity ratio )
(IC50 of AChE)/(IC50 of BuChE).
compounds, indicating the length of the side chain does not
significantly influence the activity within the range of 4-14
atoms.
Using scopolamine and an eight-arm radial maze is an
accepted standard protocol in behavioral pharmacology for the
evaluation of ChE inhibitors as anti-AD drug candidates.7,8 To
determine the in vivo activity of the target compounds, we used
cognition impaired adult rats (after scopolamine administration)
as animal model to measure the cognition improving effects of
1, 2, and 3a. Measuring was performed in an eight-arm radial
maze. After the rats were adapted to the maze and trained to be
able to quickly find the bait at the end of each arm, they were
treated with scopolamine (0.05 mg/100 g body weight, ip).
Scopolamine distinctly blocks muscarinic cholinergic receptors
and thus impaires the animals’ cognition significantly. Then
tacrine (1.978 µmol/100 g body weight) and 1, 2, and 3a
(equimolar dose), which are supposed to compensate for the
cholinergic dysfunction in the central nervous system by
inhibiting ChEs, were administrated to the animals. To determine
whether the nitrate group of the target molecule has an influence
on the in vivo activity, 1-propylnitrate (5d) and 3h, which is
an analogue of 2 without the nitrate group, were also selected
for in vivo testing in the assay applied alone and in combination,
respectively. Two parameters, the number of errors made during
the exploration and the total exploration time, were recorded
before, 20, 60, and 180 min after scopolamine administration
to determine the cognition improving activity of the selected
compounds.
Figure 2. Number of errors rats made during the search for baits and
exploration time needed to find all food baits before, 20, 60, and 180
min after the administration of scopolamine, 1, 2, and 3a: (+) significant
difference to scopolamine (scop) (t test, p e 0.05).
The results show that, compared to the group treated with
scopolamine (scop) alone, the performance of groups 1, 2, and
3a was significantly improved, as indicated by decreased
numbers of errors and shorter exploration time (Figure 2).
Compared to tacrine (tac), 1 and 2 showed an approximately
equal activity. However, tacrine given without additional
scopolamine caused a significant slow-down of a rat’s perfor-
mance in the maze, whereas 1, 2, and 3a given alone had no
influence on a rat’s behavior at all. All these findings indicate
advantages of the NO-donating hybrid compounds in compari-
son to tacrine.
Interestingly, the nonhybrid 3h, though it showed comparable
ChE inhibitory activity to 2 in vitro, did not show any improving
effects in the in vivo assay in the dose range applied. This
suggests that the nitrate group of 2, which was reported to
possess vessel relaxant activity,3 may also contribute to the
overall procognitive effect of the hybrids, though the mechanism
is not clear yet. Administrating 3h with the nitrate 5d or
administrating 5d alone did not show significant activity in vivo,
Figure 3. Number of errors rats made during the search for baits and
exploration time needed to find all food baits before, 20, 60, and 180
min after the administration of scopolamine, 2, 3h, and 5d: ( +)
significant difference to scopolamine (scop) (t test, p e 0.05).
indicating that the administration of the combination of equimo-
lar parts of NO donor and tacrine in one hybrid molecule is
clearly superior to a simple mixture of both parts (Figure 3).
The serious hepatotoxicity of tacrine is the main limitation
for its clinical use.9 To determine the hepatotoxicity, rats were
treated with the highest tolerated dose of tacrine (5.93 µmol/
100 g body weight) or equimolar doses of 1 and 2. The
determination of ASAT and albumin concentration in serum
samples was performed before (intraindividual control), 24, and
36 h after administration using an Abbot Architect ci 16200
analyzer according to the instructions of the manufacturer. The

7668 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Letters
Figure 4. ASAT activity, serum albumin concentration, and total
protein in the liver before and after the administration of tacrine, 1, or
2: (/) significant difference to values before administration (t test, p e
0.05).
results (Figure 4) demonstrate significant hepatotoxic effects
caused by tacrine concerning ASAT activity and albumin
concentration in serum and protein content in liver tissue. In
contrast, none of these parameters are affected significantly by
the hybrid compounds 1 and 2.
For morphological studies liver tissue was stained with
hematoxylin and eosin (HE). Complete pericentral necrosis and
distinct fatty degeneration of the hepatocytes of the surrounding
intermediate and periportal zones were seen 24 h after admin-
istration of tacrine (compare parts A and B of Figure 5). In
contrast, after administration of 2 no major histopathological
changes were seen (Figure 5C). These findings give further
evidence for the absence of hepatotoxic effects of 2.
It is well-known that AChE has a dumbbell-shape active site
gorge composed of two active sites: the catalytic active site
(CAS) at the bottom and the peripheral active site (PAS) at
the lip.10 It has been shown that PAS not only contributes to
the hydrolysis of ACh but also is responsible for the AChE-
induced ꢀ-amyloid aggregation.11 Dual inhibitors toward CAS
and PAS are regarded as improved agents for the treatment of
AD, expanding the therapeutic spectrum of AChE inhibitors.
Some previous studies have revealed that a protonatable
nitrogen-containing side chain connected to the tacrine template
may be helpful for the parent drug to obtain a PAS inhibitory
effect.12 To determine whether our target compounds can
interact with both active sites of AChE, 2 was selected to
Figure 5. Histomorphological appearance of livers of female rats after
treatment with the solvent only (controls) (A) or 24 h after administra-
tion of tacrine (B) or 2 (C): HE, original magnification × 200.
perform the docking study. The crystal structure of AChE
(Protein Data Bank code 2CKM) was obtained from the Protein
Data Bank. The active site was calculated with the “site finding”
function of MOE. The docking results showed that 2 can adopt
different binding modes within the active gorge of AChE (Figure
6). A potent interaction between the heterocycle moiety and
residues Trp84 and Phe330 was observed through a π-π
interaction. Besides, 2 also presented a direct interaction with
residue Asp72 through the formation of an H-bond between
the secondary amine group at the side chain and the corre-
sponding residue. Interestingly, with regard to the PAS, it was
found that the end of the side chain is exactly located at the lip
of the gorge, directly toward the PAS. Moderate interaction is
therefore gained through a hydrophobic action of the side chain
and the residue Trp279, indicating that the target compound
might act as a dual inhibitor toward CAS and PAS. The side

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Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7669
as a dual-site inhibitor. Altogether, the distinct ChE inhibitory
activity and cognition enhancing effects of the new nitrate-tacrine
hybrid substances and the absence of hepatotoxicity qualify them
as potential novel anti-AD drug candidates.
Acknowledgment. The support of “DAAD Sandwich Pro-
gram” Scholarship (August 2006 to September 2008) is very
gratefully acknowledged.
Supporting Information Available: Experimental procedures
for the synthesis of 3a-h, detailed procedures of pharmacological
investigations, and elemental analysis results. This material is
available free of charge via the Internet at http://pubs.acs.org.
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In summary, we have designed and synthesized a series of
amine-linked tacrine-NO donor hybrid compounds. All of the
compounds effectively inhibited ChEs in vitro. Evaluated by
the scopolamine-induced cognition impairment animal model,
1, 2, and 3a showed significant cognition improving activity
whereas the analogue 3h, which is without the nitrate group,
did not. This result indicates that the nitrate group of 2 not only
may contribute to the vessel relaxant activity as previously
reported3 but also is essential for the ChE inhibitory effect.
Additional hepatotoxicity studies confirmed the tacrine-induced
liver damage whereas the hybrid compounds 1 and 2 did not
show obvious signs of hepatotoxicity. Furthermore, the docking
study showed that 2 may block the CAS and the PAS, acting
JM801131A