Tacrine-ferulic acid-nitric oxide (no) donor trihybrids as potent, multifunctional acetyl- and butyrylcholinesterase inhibitors

Article abstract of DOI:10.1021/jm300106z

In search of multifunctional cholinesterase inhibitors as potential anti-Alzheimer drug candidates, tacrine-ferulic acid-NO donor trihybrids were synthesized and tested for their cholinesterase inhibitory activities, release of nitric oxide, vasodilator properties, cognition improving potency, and hepatotoxicity. All of the novel target compounds show higher in vitro cholinesterase inhibitory activity than tacrine. Three selected compounds (3a, 3f, and 3k) produce moderate vasorelaxation in vitro, which correlates with the release of nitric oxide. Compared to its non-nitrate dihybrid analogue (3u), the trihybrid 3f exhibits better performance in improving the scopolamine-induced cognition impairment (mice) and, furthermore, less hepatotoxicity than tacrine.

Full text of DOI:10.1021/jm300106z

Article
pubs.acs.org/jmc
Tacrine−Ferulic Acid−Nitric Oxide (NO) Donor Trihybrids as Potent,
Multifunctional Acetyl- and Butyrylcholinesterase Inhibitors
Yao Chen,†,‡,§ Jianfei Sun,∥ Lei Fang,‡ Mei Liu,∥ Sixun Peng,†,‡ Hong Liao,*,∥ Jochen Lehmann,*,§,⊥
and Yihua Zhang*,†,‡
†State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
‡Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
§Lehrstuhl fur Pharmazeutische/Medizinische Chemie, Institut fur Pharmazie, Friedrich-Schiller-Universitat Jena, Philosophenweg 14,
̈
̈
̈
D-07743 Jena, Germany
∥Neurobiology Lab, New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, P.R. China
⊥College of Pharmacy, King Saud University Riyadh, Saudi Arabia
S
* Supporting Information
ABSTRACT: In search of multifunctional cholinesterase
inhibitors as potential anti-Alzheimer drug candidates,
tacrine−ferulic acid−NO donor trihybrids were synthesized
and tested for their cholinesterase inhibitory activities, release
of nitric oxide, vasodilator properties, cognition improving
potency, and hepatotoxicity. All of the novel target compounds
show higher in vitro cholinesterase inhibitory activity than
tacrine. Three selected compounds (3a, 3f, and 3k) produce
moderate vasorelaxation in vitro, which correlates with the release of nitric oxide. Compared to its non-nitrate dihybrid analogue
(3u), the trihybrid 3f exhibits better performance in improving the scopolamine-induced cognition impairment (mice) and,
furthermore, less hepatotoxicity than tacrine.
palliative treatment.18 Because of the multifactorial nature of
AD, the traditional approach of single-target molecule can
INTRODUCTION
■
The cholinergic hypothesis of the pathogenesis of Alzheimer’s
disease (AD)1 asserts that dysfunction of cholinergic system,
generally only offer limited and transient benefits. Thus, a
strategy named multitarget-directed ligands (MTDLs)19 has
mainly decline of acetylcholine (ACh) level, leads to the
cognitive and memory deficits associated with AD, and
recently been applied in the research of ChEIs,20,21 which
means compounds with additional properties other than
cholinesterase inhibition have been focused. To extend this
strategy and further explore its potential is the topic of this
work.
sustaining or recovering cholinergic function is therefore
supposed to be clinically beneficial.2,3 ACh can be degraded
by two types of cholinesterases (ChEs), namely acetylcholi-
nesterase (AChE) and butyrylcholinesterase (BuChE). The
enzymatic cavity of human AChE has the shape of a nearly 20 Å
Nitric oxide (NO), a free radical gas, is a multifunctional
deep narrow gorge4,5 which is composed of two binding sites:
messenger molecule with diverse physiological roles, such as
dilation of blood vessels, immune responses, and potentiation
the catalytic active site (CAS) at the bottom and the peripheral
of synaptic transmission.22,23 Although the relation between
NO and AD is not yet clear, increasing recent evidence suggests
that NO may be beneficial for the treatment of AD also by
increasing blood supply24 and regulating the cerebral
circulation.25 In recent years, in the research of anti-AD
drugs, the NO-donating compound strategy,26 in which a NO-
donor is combined to a “native” molecule, has been proved to
be effective.27−29
anionic site (PAS) near the entrance of the gorge.6−8 PAS has
been proved to have close relation to both hydrolysis of ACh
and neurotoxic cascade of AD through AChE-induced β-
amyloid (Aβ) aggregation.9−11 Human BuChE has a similar
structure of the cavity but with a wider gorge.12,13 Compared to
BuChE, AChE is more active and can hydrolyze most of ACh
(80% according to Carolan, C. G., et al. and Arce, M. P., et al.)
in normal brains.14 However, in the case of AD, both the level
and the activity of AChE are found to be significantly reduced,
whereas BuChE is increased and modulates the ACh levels.15,16
Consequently, not only AChE but both enzymes are important
targets in the therapy of AD,17 and inhibitors of both, such as
tacrine (Figure 1) and rivastigmine, have been clinically applied.
Unfortunately, instead of curing or preventing the neuro-
degeneration, cholinesterase inhibitors (ChEIs) only enable a
Previously, we have reported on several tacrine−ferulic acid
hybrids (1a−d, Figure 1) as potent ChEIs which can block the
PAS30 and on highly active and nontoxic tacrine−organic
nitrate (2, Figure 1).28,31 To obtain new multifunctional ChEIs
Received: January 25, 2012
Published: April 18, 2012
© 2012 American Chemical Society
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Figure 1. Structures of tacrine, previous tacrine hybrid compounds 1a−d and 2, and tacrine trihydrid compounds 3a−t designed for this study.
endowed with additional properties at least such as vessel
relaxation activity, a nitrate moiety was conjugated to the
tacrine−ferulic acid hybrid compounds, via alkylene side chain,
leading to a series of novel trihybrids 3a−t.
In the present study, we describe the synthesis and
pharmacological evaluation of novel potential anti-Alzheimer
compounds. The pharmacological evaluation includes AChE
and BuChE inhibition, the kinetics of enzyme inhibition, NO-
releasing test, vascular relaxation study, in vivo behavioral study,
and hepatotoxicity studies.
showed better or comparable activity compared to the
corresponding parent compounds 1a−d and 4, indicating that
the introduction of nitrate group might be a contribution to the
inhibitory activity. Particularly, 3u, an analogue of 3f without
the nitrate group, showed lower activity (IC50 of AChE = 69.8
nM; IC50 of BuChE = 40.3 nM) than the parent compound 1b
(IC50 of AChE = 56.7 nM; IC50 of BuChE = 31.1 nM) and 3f
(IC50 of AChE = 10.9 nM; IC50 of BuChE = 17.7 nM), which
demonstrated that the alkane linker itself made no contribution
to the activity, highlighting the importance of the nitrate group
for the ChE inhibition.
RESULTS AND DISCUSSION
Kinetic Study of ChE Inhibition. To gain information on the
mechanism of inhibition, the potent inhibitor 3f was selected
for a kinetic study. This compound 3f was chosen not only for
kinetic studies but also for the further behavioral and
hepatotoxicity studies because of being the best candidate in
total performance with regard to cholinesterase inhibition,
release of NO, and vasodilatation. The type of inhibition was
elucidated from the analysis of Lineweaver−Burk reciprocal
plots, which were reciprocal rates versus reciprocal substrate
concentrations for the different inhibitor concentrations
resulting from the substrate−velocity curves for ChEs. For
■
Chemistry. The synthesis of the NO-donating tacrine−
ferulic acid hybrids and related compounds was depicted in
Scheme 1. 9-Aminoalkylamino-1,2,3,4-tetrahydroacridines
10a−e were synthesized starting from anthranilic acid, which
was condensed with cyclohexanone to yield chloro acridine 9.32
Treatment of 9 with corresponding alkylenediamines resulted
in 10a−e. Ferulic acid was esterified to give methyl ester 5,
which was treated with dibromoalkanes to give bromo
compounds 6a−d, followed by treatment of AgNO3 in dry
CH3CN to offer nitrates 7a−d. The nitrates were demethylated
under basic conditions to yield acids 8a−d, which were coupled
with 10a−e in the presence of DCC/DMAP to provide target
compounds 3a−t. Treatment of 5 with 1-bromopropane gave
propylated compound 6e and 8e by hydrolytic cleavage.
Condensation of 8e with 10b led to compound 3u. In addition,
ferulic acid was directly coupled with 10d to generate
compound 4.
Pharmacology. AChE and BuChE Inhibition. The
hybrids 3a−t and relative compounds were evaluated in vitro
as inhibitors of AChE from Electrophorus electricus (eeAChE)
and of BuChE from equine serum, following Ellman’s method33
(Table 1). The inhibitory activities of the hybrids were
compared to those of tacrine, 1a−d and 4, as well as to the
free amine precursors 10a−e and the ferulic acid derivatives
8a−d. All of the novel target compounds proved to be very
potent inhibitors of both cholinesterases. The results of 10a−e
showed a trend consistent with the target compounds, whereas
8a−d showed quite low affinity, indicating the tacrine analogue
moiety as the key structure for the ChE inhibitory activity. In
addition, the results revealed that the length of the diamine side
chain attached to the tacrine skeleton of the hybrids markedly
influenced the ability to inhibit ChE, and the optimal spacer
length was 6−8 atoms. Moreover, the target compounds 3a−t
AChE, the plot showed both increased slopes (decreased Vmax
)
and intercepts (higher Km) at increasing concentration of the
inhibitor (Figure 2). This pattern indicated a mixed-type
inhibition and therefore supported the dual site (CAS and
PAS) binding of these compounds. In contrast, a different plot
for BuChE was obtained, showing different Km and constant
Vmax in different inhibitor concentrations (Figure 3). This
suggested a competitive inhibition, revealing that these
compounds compete for the same binding site (CAS) as the
substrate acetycholine.
Antioxidant Activities. Target compounds (3a−t and 4)
were tested for the antioxidant activity using a radical
scavenging assay (DPPH assay).34 However, all the target
compounds (except compound 4) showed no antioxidant
activities (at concentration 2 mM, the free radical scavenging
activities in % of compounds 3a−t were less than 1%, while of
compound 4 was 64.7%, compared to reference ferulic acid
100%). Very probably, these results are due to the presence or
absence of the free phenolic hydroxy groups of ferulic acid in
these prodrug-hybrid compounds.
Correlation of NO Produced by the Hybrids with Their
Vascular Relaxation Activities. To investigate the potential
relationship between the NO generated or at least mimicked by
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a
Scheme 1. Synthesis of Compounds 3a−u and 4
a
Reagents: (a) POCl3, reflux, 3 h; (b) pentanol, NH2(CH2)mNH2, reflux, 18 h; (c) CH3OH, H2SO4, reflux, 3 h; (d) Br(CH2)nBr, DMF, K2CO3, 65
°C, 6 h; (e) Br(CH2)2CH3, DMF, K2CO3, 65 °C, 6 h; (f) AgNO3, CH3CN, 60 °C, 6 h; (g) LiOH, THF/CH3OH/H2O, room temp, 24 h; (h) DCC,
DMAP, anhydrous CH2Cl2, room temp, 24 h.
the target compounds and their vasodilator activities, the in
vitro reactivity of compounds 3a−t as NO-donating com-
pounds was first measured using the Griess reaction35 which
quantifies the inorganic nitrite produced from the degradation
of the organic nitrate (Table 1). Of course, all the compounds
showed higher levels of nitrite than tacrine, and compound 3f
and 3q produced levels of nitrite (0.31, 0.30 μg/mL,
respectively) near to the positive control isosorbide dinitrate
(ISDN, 0.38 μg/mL).
Compounds 1b, 3a, 3f, 3q, and 3u were evaluated in an ex
vivo organ bath (coronary arteries from rat) vascular relaxation
assay. ISDN and tacrine were taken as positive and negative
controls, respectively (Figure 4). All the tested compounds
(except 1b) showed higher activity than tacrine, and 3f showed
a comparable EC50 (34.3 μM) to ISDN (EC50 = 25.2 μM). The
activity of parent compound 1b was quite low due to the lack of
NO donor. The comparison between 3f and 3u confirms that
the nitrate group is crucial to the vascular relaxation potency.
Moreover, the levels of NO produced by the target compounds
are positively correlated with the vasorelaxation effects with the
exception of 3q.
To study the unexpected result of this compound, we
detected the levels of nitrite generated by 3f and 3q after
different times (10, 30, and 90 min, respectively). The results
(Figure 5) demonstrate that, the NO releasing rate of 3q lower
in the beginning, which should explain the lower vasorelaxation
effect of 3q because this activity was determined 5 min after the
test compounds were added.
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Table 1. Inhibition of AChE and BuChE (IC50 Values),
Selectivity Expressed as the Ratio of the Resulting IC50
Values and Release of nitrite from the organic nitrates
measured by Griess reaction
a
IC50 (nM) SEM
selectivity
nitrite (μg/
c
b
compd
AChE
BuChE
ratio
mL)
d
1a
1b
62.0 10.5
56.7 14.4
11.1 1.8
16.9 3.4
71.4 11.0
79.5 14.3
39.3 5.1
23.7 5.7
2.9 1.0
>10000
37.5 9.7
31.1 4.8
9.8 1.8
27.4 8.3
30.9 11.2
19.7 2.9
22.9 2.4
6.6 0.5
2.8 0.4
>10000
1.7
1.8
1.1
0.6
2.3
4.1
1.7
3.6
1.0
nd
nd
1c
nd
1d
10a
10b
10c
10d
10e
8a
nd
nd
nd
nd
nd
nd
Figure 2. Lineweaver−Burk plots resulting from subvelocity curves of
AChE activity with different substrate concentrations (25−450 μM) in
the absence and presence of 1, 2, 4, 10 nM 3f.
nd
8b
>10000
>10000
nd
8c
>10000
>10000
nd
8d
3a
>10000
>10000
nd
17.2 2.3
19.9 5.0
27.1 2.6
40.6 2.8
14.4 2.2
10.9 1.3
3.6 0.6
21.8 6.4
22.1 3.9
44.3 3.8
19.5 1.7
27.9 5.0
8.3 0.8
4.4 1.0
3.7 0.6
4.8 0.8
5.5 0.9
10.1 0.7
9.4 0.8
10.8 1.0
69.8 25.5
12.7 1.9
69.8 11.1
nd
10.5 1.1
8.4 1.1
7.6 0.9
21.8 1.7
24.9 2.4
17.1 1.2
6.2 0.5
19.3 2.8
20.2 3.0
23.6 4.8
4.7 0.6
11.7 4.6
5.2 0.6
5.6 0.7
1.4 0.4
2.0 0.8
1.6 0.3
1.0 0.3
1.8 0.3
1.9 1.8
40.3 5.2
26.3 5.7
10.6 1.1
nd
1.6
2.4
3.6
1.9
0.6
0.6
0.6
1.1
1.1
1.9
4.1
2.4
1.6
0.8
2.6
2.4
3.4
10.1
5.2
5.7
1.7
0.5
6.6
nd
0.182 0.003
0.071 0.003
0.042 0.003
0.221 0.002
0.243 0.001
0.314 0.001
0.069 0.003
0.070 0.006
0.173 0.005
0.074 0.004
0.052 0.001
0.044 0.007
0.223 0.002
0.081 0.004
0.114 0.003
0.012 0.007
0.301 0.004
0.102 0.004
0.139 0.001
0.193 0.003
nd
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
Figure 3. Lineweaver−Burk plots resulting from subvelocity curves of
BuChE activity with different substrate concentrations (25−450 μM)
in the absence and presence of 1, 2, 4, 10 nM 3f.
3s
3t
3u
4
nd
tacrine
ISDN
0.001 0.001
0.382 0.003
a
b
Data is the mean of at least three determinations. Selectivity ratio =
c
(IC50 of AChE)/(IC50 of BuChE). All values are the mean SEM.
d
nd means not determined.
Behavioral Studies. To gain more insight into the
therapeutic potential of the hybrid compounds, behavioral
studies were assayed using passive avoidance test.36 Scopol-
amine-induced cognition impaired adult mice were used as the
animal model to measure the cognitive improving effects of
compounds 1b, 3f, and 3u in comparison to tacrine. Measuring
was performed in a step-through passive avoidance apparatus,
which contained two symmetrical compartments, a dark and a
light chamber. Mice were first placed in the light chamber, and
they would spontaneously enter the dark chamber, however the
entry would be punished by low intensity electric foot shocks
(0.4 mA). After the mice were adapted to the apparatus, they
were treated with tacrine (1.978 μmol/100 g b wt) or test
compounds 1b, 3f, and 3u (equimolar dose), respectively. Fifty-
Figure 4. Concentration−response curves for the vasorelaxant effects
a
of tacrine, ISDN, and compounds 1b, 3a, 3f, 3q, 3u. Data are the
mean values of at least three determinations.
five min later, scopolamine (0.2 mg/100 g b wt), which
distinctly blocks the muscarinic cholinergic receptors, was
administrated to the animals. Then the first trial of 300 s for
training started, and 24 h later, a second trial in which the shock
was not delivered was carried out to test the retention. The
number of errors made entering from the light room to the
dark room in the first trial was recorded as the indication for
short time learning ability (Figure 6). In addition, the other
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Figure 5. Variable levels of NO produced by 3f and 3q at 10, 30, and 90 min post-treatment. aAll values are the mean SEM. Determined by Griess
reaction.
Figure 6. Effect of 1b, 3f, 3u, and tacrine (tac) on number of errors mice made entering from the light room to the dark room in the first trial. Values
are expressed as mean SEM (n = 8−9. t test, p** ≤ 0.01 compared to the control group, p# ≤ 0.05 compared to the scop group).
parameter, the transfer latency time (TLT in seconds) was
taken to determine the cognitive improving potency of the test
compounds (Figure 7).
non-nitrate analogue 3u. These findings are consistent with the
in vitro ChE inhibition activity and in vitro vasorelaxation
effect, which illustrates again the importance of the nitrate
group.
Hepatotoxicity Studies. The main disadvantage of tacrine is
the high hepatotoxicity and it elevates levels of liver
transaminase and decreases albumin concentration.37 It has
been reported that NO production in the liver is usually
increased in response to acute insult with hepatotoxicants.38
Moreover, some NO-donating drugs were found to exhibit
hepatoprotective activity.28,31,39 To determine whether our
nitrate containing test compounds can yield in decreased
hepatotoxicity in comparison to tacrine and the non-nitrate
parent compounds, 1b, 3f, and 3u were selected for the assay
with adult mice. After being treated with tacrine, 1b, 3f, and 3u
(equimolar doses), the heparinized serum of the mice was
obtained after different times and the levels of aspartate
aminotransferase (ASAT) and alanine aminotransferase (ALT)
were determined (Figure 8). In comparison to the control,
tacrine caused significant hepatotoxicity, as indicated by the
increased activity of ASAT and ALT. 1b and 3f possessed
higher safety than tacrine, and 3f showed the lowest
hepatotoxicity among all the compounds.
For histological studies, liver tissues taken from the animals
were stained with hematoxylin and eosin (HE). Paraffin
sections were observed 30 h after administration of tacrine,
1b, 3f, and 3u. For the tacrine treated section, complete
pericentral necrosis and distinct fatty degeneration of the
hepatocytes of the surrounding intermediate and periportal
zones could be seen (Figure 9B). Conversely, only minor
morphological changes were observed after the treatment with
test compounds (Figure 9C−E). All these findings give strong
evidence that the introduction of nitrate group could improve
the saftey of the parent compound.
Figure 7. Effect of 1b, 3f, 3u, and tacrine (tac) on transfer latency time
(TLT) in the PA test. Values are expressed as mean SEM (n = 8−9.
t test, p*** ≤ 0.001 compared to the control group, p## ≤ 0.01 p###
≤ 0.001 compared to the scop group).
The results in Figure 6 showed that the error numbers of
group treated with 1b, 3f, and 3u was lower than that of the
scopolamine group, indicating the test compounds could
benefit for short time learning ability. As for TLT (Figure 7),
a significant increase in the 1b, 3f, and 3u group was observed
in comparison to the scopolamine group, demonstrating that all
the test compounds were active in improving memory
impairment. As a consequence, 1b, 3f, and 3u all showed
cognitive improving potency. 3f showed highest in vivo activity,
compared with positive control tacrine and parent compound
1b. Furthermore, 3f possessed better performance than the
Molecular Modeling. A molecular modeling study using
CDOCKER within Discovery Studio (DS, Accelrys) was
performed to explore the binding mode of compound 1b, 3f,
and 3u with the enzyme eeAChE. The crystal structure of the
bis(7)-tacrine−AChE complex40 was taken as a model because
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Figure 8. ALT and ASAT activity after the administration of tacrine, 1b, 3f, or 3u. Values are expressed as mean SEM (n = 8−9. t test, compared
to control of the same time after administration, p* ≤ 0.05, p** ≤ 0.01, p*** ≤ 0.001).
of the similarity between the bis-tacrine and our compounds. A
tacrine fragment and a long flexible side chain were contained
in bis(7)-tacrine and our compounds. The proposed binding
model of 1b, 3f, and 3u with the key residues in the gorge site
was shown in Figure 10. The superimposition of the three
compounds with bis(7)-tacrine indicated that our compound
can cover the binding gorge in a satisfactory orientation and
conformation, thus leading to the high inhibitory potency. The
binding model suggested the tacrine fragment of all of the three
compounds was bound to near the bottom of the gorge in a
similar arrangement. Tacrine fragment showed strong parallel
π−π stacking against the indole ring of Trp84 and the
imidazole ring of His440 in all cases. In the middle of the gorge,
the constricted region, the aliphatic alkyl chains of 1b, 3f, and
3u were located as linkers and surrounded with phenyl rings of
Tyr121, Phe330, and Tyr334. The π−π stacking interaction
between the tacrine fragment and the benzene ring of Phe330
was observed. The cyclohexane ring of tacrine may have van
der Waals contact with Ser200 and Gly441 (distances between
the closest carbon atoms of 3.9 and 4.0 Å). The binding modes
of the three compounds and bis(7)-tacrine were found to be
very similar, which might explain why these compounds
exhibited very potent AChE inhibitory effects. At the mouth
of the gorge, the benzene ring of ferulic acid group in
compounds 1b, 3f, and 3u showed the hydrophobic interaction
with the residues Tyr70 (distance between the closest carbon
atoms was 3.7 Å, respectively), the key residue in PAS. It has
been estimated that the PAS site was about 20 Å deep from the
CAS.41 3f and 3u had a length of 21.0 and 19.1 Å, respectively,
which was enough to cover both the PAS and CAS, leading our
compounds acting as “dual binding site” inhibitors. The NO-
donating group in 3f could form a hydrogen bond with Asp276,
which stabilized the binding interaction with AChE, whereas in
3u and 1b, the corresponding interaction was missing due to
the lack of NO-donating group. This finding could explain the
higher potency of 3f compared to 3u and 1b. The
−CDOCKER energies for 1b, 3f, and 3u (37.23, 43.86, and
35.38, respectively) correlated well with their inhibitory effect.
As a further test, we have calculated the relative binding affinity
between the three compounds and AChE by means of MM/
PBSA method. The results indicated that binding of 3f to
AChE was more favored by near 15.0 and 22.8 kcal/mol
relative to 1b and 3u. Both the −CDOCKER energy and MM/
PBSA calculations confirmed the higher potency of 3f.
CONCLUSIONS
■
A series of NO-donating tacrine−ferulic acid hybrid com-
pounds have been designed and synthesized as novel
multifunctional potent ChEs inhibitors. All of the compounds
effectively inhibited ChEs in the nanomolar range in vitro.
Compound 3f was one of the most potent inhibitors that was 5-
fold and 2-fold more active than the parent compound 1b
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EXPERIMENTAL SECTION
■
Chemistry. Melting points are uncorrected and were measured in
open capillary tubes using a Gallenkamp melting point apparatus. H
1
and 13C NMR spectral data were obtained from a Bruker Advance 250
spectrometer (250 MHz). Considering the series containing closely
related compounds, the protons and carbons are assigned for instance
in 3a, 3u, and 4. HRMS data were recorded using Agilent technologies
LC/MSD TOF. TLC was performed on silica gel F254 plates
(Merck). Column chromatography was carried out with silica gel 60,
63−200 μm (Baker). Purities of the compounds were determined by
elemental analysis performed on a Heraeus Vario EL III apparatus
(Firma Elementar Analysensysteme GmbH, Germany). All values for
C, H, and N were found to be within 0.4% of the theoretical values.
All compounds showed >95% purity.
(E)-Methyl 3-[4-(Bromoalkoxy)-3-methoxyphenyl]acrylates
6a−6d: General Procedure I. A mixture of 5 and 3 molar equiv
of dibromoalkane in DMF (50 mL) was treated with a molar equiv of
K2CO3. After being stirred at 60 °C for 6 h, the reaction mixture was
cooled down to room temperature, poured into water, and extracted
with ethyl acetate (3 × 15 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and evaporated
under reduced pressure to give the crude product. The purification was
performed by column chromatography (ethyl acetate/petrol ether =
1/4, v/v).
(E)-Methyl 3-{3-Methoxy-4-[(nitrooxy)alkoxy]phenyl}-
acrylates 7a−7d: General Procedure II. Compounds 6a−6d and
1.2 molar equiv of AgNO3 in 50 mL of CH3CN were stirred at 60 °C
for 6 h under dark condition. Then the resulting mixture was cooled to
room temperature and filtered to give a brown filtrate, which was
evaporated under reduced pressure. The residue was purified by
recrystallization from CH3CN.
(E)-3-{3-Methoxy-4-[(nitrooxy)alkoxy]phenyl} Acrylic Acids
8a−8d: General Procedure III. Three molar equiv of LiOH·H2O
were added to a solution of compound 7 in a 15/5/5 mL mixture of
THF/MeOH/H2O, and the mixture was stirred at room temperature
for 24 h. After removing the solvent under reduced pressure, the
residue was diluted with H2O (30 mL), acidified to pH = 4 with 10%
HCl, and extracted with ethyl acetate (3 × 15 mL). The combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
and evaporated in vacuo to give the crude product, which was
recrystallized from ethyl acetate.
Figure 9. Histomorphological appearance of livers of male mice after
treatment with the solvent only (control) (A) or 30 h after
administration of tacrine (B), 1b (C), 3f (D), or 3u (E). HE, original
magnification ×200.
9-Aminoalkylamino-1,2,3,4-tetrahydroacridines 10a−10e:
General Procedure IV. To a solution of 9 (9-Chlorotetrahydroacri-
dine) in 10 mL of pentanol three molar equiv of diaminolkane were
added. After refluxing for 18 h under nitrogen, the solution was cooled
to 0 °C and then acidified to pH = 2 with HCl/ether solution. The
deposit was separated, dissolved in 10 mL of water, and then the
solution was basified to pH = 10 with a saturated aqueous solution of
Na2CO3 and extracted with CH2Cl2 (3 × 10 mL). The combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
and evaporated in vacuo. The residue was purified by column
chromatography (CH2Cl2/MeOH = 7/3, v/v, plus 5 mL triethylamine
per 1000 mL).
Figure 10. Representation of the binding mode of compound 3f
(purple), 1b (blue), and 3u (yellow) with AChE (PDB id: 2CKM).
The key residues that interacted with the three molecules were colored
in light green.
{2-Methoxy-4-[(1E)-3-oxo-3-((1,2,3,4-tetrahydroacridin-9-
ylamino)alkylamino)prop-1-enyl]phenoxy}alkyl Nitrates 3a−
3t: General Procedure V. Compound 8 and a molar equiv amount
of N,N′-dicyclohexylcarbodiimide (DCC) were dissolved in 20 mL of
dry CH2Cl2, and the mixture was stirred at room temperature for 0.5 h.
Then a solution of a molar equiv amount of 10 in 5 mL of dry CH2Cl2
was added, followed by a catalytic amount of 4-dimethylaminopyridine
(DMAP). After being stirred for 24 h at room temperature, the
reaction was quenched by adding 10 mL of H2O. Then the resulting
mixture was filtered, and the filtrate was extracted with CH2Cl2 (3 ×
15 mL). The combined organic phases were washed with brine, dried
over anhydrous Na2SO4, and evaporated in vacuo. The residue was
purified by column chromatography (CH2Cl2/MeOH = 10/1, v/v).
2-{2-Methoxy-4-[(1E)-3-oxo-3-(2-(1,2,3,4-tetrahydroacridin-
9-ylamino)ethylamino)prop-1-enyl]phenoxy}ethyl Nitrate (3a).
Following general procedure V, compound 10a (0.24 g, 1 mmol)
reacted with compound 8a (0.28 g, 1 mmol) to furnish 3a as a yellow
toward AChE and BuChE, respectively. In the vascular
relaxation assay, 3f possessed an activity, positively correlated
with the NO production and comparable to the activity of the
reference ISDN. Evaluated by the scopolamine-induced
cognition impairment animal model, 3f showed significant
cognition improving activity. In addition, hepatotoxicity studies
confirmed that 3f was much safer than tacrine as well as 1b.
The priority of 3f to 3u (the analogue of 3f without the nitrate
group) in all the tests demonstrated that the strategy of NO-
donating drugs was rational and beneficial. Altogether, the
multifunctional effects of the new hybrids qualified them as
potential anti-AD drug candidates and 3f might be considered
as a promising lead compound for further research.
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1
solid (0.15 g, 30%); mp 63−65 °C. H NMR (CDCl3): δ 8.00−7.87
Hz, 1H), 7.53−7.49 (t, 1H), 7.35−7.31 (t, 1H), 7.01−6.98 (m, 2H),
6.81−6.79 (d, 1H), 6.74−6.71 (t, 1H), 6.37 (d, J = 15.5 Hz, 1H), 5.02
(br, 1H), 4.81−4.79 (t, 2H), 4.28−4.26 (t, 2H), 3.80 (s, 3H), 3.55−
3.51 (m, 4H), 3.03 (br, 2H), 2.75 (br, 2H), 1.87−1.83 (m, 6H). 13C
NMR (CDCl3): δ 166.98, 158.04, 150.94, 150.03, 148.95, 146.68,
140.59, 129.47, 128.56, 127.85, 123.88, 122.76, 121.46, 120.08, 119.36,
116.11, 114.61, 110.06, 70.94, 65.67, 55.94, 45.36, 36.83, 33.51, 31.53,
(m, 2H, arom), 7.58 (d, J = 15.5 Hz, 1H, COCHCH), 7.50−7.44 (t,
1H, arom), 7.30−7.24 (t, 1H, arom), 7.04−7.00 (m, 3H, arom,
CONH), 6.82−6.79 (d, 1H, arom), 6.42 (d, J = 15.5 Hz, 1H,
COCHCH), 5.32 (br, 1H, NH), 4.83−4.80 (t, 2H, OCH2), 4.30−
4.26 (t, 2H, CH2ONO2), 3.82 (s, 3H, OCH3), 3.73 (br, 4H,
NHCH2CH2NHCO), 2.99 (br, 2H, C4−H2), 2.66 (br, 2H, C1−H2),
1.82 (br, 4H, C2−H2, C3−H2). 13C NMR (CDCl3): δ 167.69
(NHCO), 156.97 (arom), 151.49 (arom), 149.95 (arom), 149.02
(arom), 145.57 (arom), 141.05 (COCHCH), 129.21 (arom),
128.97 (arom), 126.79 (arom), 123.92 (arom), 122.98 (arom), 121.60
(arom), 119.15 (COCHCH), 118.93 (arom), 115.28 (arom),
114.34 (arom), 110.89 (arom), 70.89 (OCH2), 65.54 (CH2ONO2),
55.95 (OCH3), 50.02 (NHCH2), 40.55 (CH2NHCO), 32.84 (C4),
24.84 (C1), 22.81 (C3), 22.29 (C2). HRMS (ESI) m/z calcd for
C27H30N4O6 [M +] 506.2165; found 506.2170. Anal. Calcd for
(C27H30N4O6·3/4H2O): found C, 62.42; H, 6.17; N, 11.03; requires C,
62.36; H, 6.10; N, 10.77.
3-{2-Methoxy-4-[(1E)-3-oxo-3-(2-(1,2,3,4-tetrahydroacridin-
9-ylamino)ethylamino)prop-1-enyl] phenoxy}propyl Nitrate
(3b). Compound 10a (0.24 g, 1 mmol) reacted with compound 8b
(0.30 g, 1 mmol) to furnish 3b as a yellow solid (0.18 g, 35%); mp
60−62 °C. 1H NMR (CDCl3): δ 7.97−7.83 (m, 2H), 7.57 (d, J = 15.5
Hz, 1H), 7.49−7.43 (t, 1H), 7.30−7.24 (t, 1H), 7.03−6.97 (m, 3H),
6.80−6.77 (d, 1H), 6.36 (d, J = 15.5 Hz, 1H), 5.07 (br, 1H), 4.69−
4.64 (t, 2H), 4.11−4.07 (t, 2H), 3.80 (s, 3H), 3.68 (br, 4H), 2.97 (br,
2H), 2.66 (br, 2H), 2.27−2.18 (m, 2H), 1.81 (br, 4H). 13C NMR
(CDCl3): δ 167.68, 157.81, 151.04, 149.63, 149.56, 146.45, 141.20,
128.63, 128.36, 127.62, 123.81, 122.82, 121.73, 119.60, 118.49, 115.73,
+
25.07, 22.99, 22.59. HRMS (ESI) m/z calcd for C28H32N4O6 [M ]
520.2322; found 520.2325. Anal. Calcd for (C28H32N4O6·H2O): found
C, 62.57; H, 6.33; N, 10.23; requires C, 62.44; H, 6.36; N, 10.40.
3-{2-Methoxy-4-[(1E)-3-oxo-3-(3-(1,2,3,4-tetrahydroacridin-
9-ylamino)propylamino)prop-1-enyl]phenoxy}propyl Nitrate
(3f). Compound 10b (0.26 g, 1 mmol) reacted with compound 8b
(0.30 g, 1 mmol) to furnish 3f as a yellow solid (0.17 g, 32%); mp 53−
55 °C. 1H NMR (CDCl3): δ 8.06−7.89 (m, 2H), 7.59 (d, J = 15.5 Hz,
1H), 7.55−7.51 (t, 1H), 7.36−7.33 (t, 1H), 7.05−6.99 (m, 2H), 6.83−
6.81 (d, 1H), 6.47−6.44 (t, 1H), 6.34 (d, J = 15.5 Hz, 1H), 4.99 (br,
1H), 4.70−4.67 (t, 2H), 4.13−4.10 (t, 2H), 3.83 (s, 3H), 3.56−3.51
(m, 4H), 3.05 (br, 2H), 2.77 (br, 2H), 2.27−2.21 (m, 2H), 1.90−1.82
(m, 6H). 13C NMR (CDCl3): δ 166.98, 158.19, 150.82, 149.78,
149.58, 146.86, 140.91, 128.60, 128.49, 128.11, 123.88, 122.70, 121.67,
120.18, 118.80, 116.26, 113.53, 110.66, 69.89, 64.99, 55.90, 45.41,
36.88, 33.65, 31.58, 26.98, 25.10, 23.04, 22.66. HRMS (ESI) m/z calcd
for C29H34N4O6 [M +] 534.2478; found 534.2480. Anal. Calcd for
(C29H34N4O6·3/4H2O): found C, 63.72; H, 6.50; N, 10.22; requires C,
63.55; H, 6.53; N, 10.22.
4-{2-Methoxy-4-[(1E)-3-oxo-3-(3-(1,2,3,4-tetrahydroacridin-
9-ylamino)propylamino)prop-1-enyl]phenoxy}butyl Nitrate
(3g). Compound 10b (0.26 g, 1 mmol) reacted with compound 8c
(0.31 g, 1 mmol) to furnish 3g as a yellow solid (0.18 g, 33%); mp
113.24, 110.50, 69.87, 64.84, 55.86, 49.91, 40.64, 33.37, 26.88, 24.97,
22.91, 22.49. HRMS (ESI) m/z calcd for C28H32N4O6 [M
1
+
50−52 °C. H NMR (CDCl3): δ 8.06−7.88 (m, 2H), 7.62−7.49 (m,
]
2H), 7.37−7.31 (t, 1H), 7.05−6.97 (m, 2H), 6.82−6.78 (d, 1H),
6.38−6.28 (m, 2H), 4.95 (br, 1H), 4.57−4.53 (t, 2H), 4.07−4.03 (t,
2H), 3.82 (s, 3H), 3.54−3.51 (m, 4H), 3.04 (br, 2H), 2.76 (br, 2H),
1.96−1.80 (m, 10H). 13C NMR (CDCl3): δ 167.00, 158.35, 150.67,
149.82, 149.53, 147.03, 141.06, 128.40, 128.29, 128.03, 123.84, 122.66,
121.82, 120.25, 118.41, 116.36, 112.72, 110.24, 72.91, 68.14, 55.84,
45.35, 36.84, 33.80, 31.57, 25.35, 25.13, 23.91, 23.05, 22.71. HRMS
520.2322; found 520.2325. Anal. Calcd for (C28H32N4O6·H2O):
found C, 62.35; H, 6.33; N, 10.29; requires C, 62.44; H, 6.36; N,
10.40.
4-{2-Methoxy-4-[(1E)-3-oxo-3-(2-(1,2,3,4-tetrahydroacridin-
9-ylamino)ethylamino)prop-1-enyl]phenoxy}butyl Nitrate (3c).
Compound 10a (0.24 g, 1 mmol) reacted with compound 8c (0.31 g,
1 mmol) to furnish 3c as a yellow solid (0.19 g, 36%); mp 46−48 °C.
1H NMR (CDCl3): δ 7.97−7.84 (m, 2H), 7.57 (d, J = 15.5 Hz, 1H),
7.51−7.45 (t, 1H), 7.31−7.25 (t, 1H), 7.02−6.94 (m, 2H), 6.79−6.76
(d, 2H), 6.31 (d, J = 15.5 Hz, 1H), 4.85 (br, 1H), 4.56−4.51 (t, 2H),
4.03 (br, 2H), 3.79 (s, 3H), 3.66 (br, 4H), 2.99 (br, 2H), 2.68 (br,
2H), 1.93 (br, 4H), 1.82 (br, 4H). 13C NMR (CDCl3): δ 167.58,
158.42, 150.66, 149.89, 149.52, 147.11, 141.33, 128.39, 128.30, 127.91,
123.77, 122.66, 121.85, 119.93, 118.20, 116.13, 112.69, 110.27, 72.92,
68.13, 55.83, 49.84, 40.72, 33.82, 25.34, 25.06, 23.89, 23.00, 22.65.
HRMS (ESI) m/z calcd for C29H34N4O6 [M +] 534.2478; found
534.2483. Anal. Calcd for (C29H34N4O6·3/4H2O): found C, 63.68; H,
6.33; N, 10.14; requires C, 63.55; H, 6.53; N, 10.22.
+
(ESI) m/z calcd for C30H36N4O6 [M ] 548.2635; found 548.2639.
Anal. Calcd for (C30H36N4O6·3/4H2O): found C, 63.96; H, 6.63; N,
9.67; requires C, 64.10; H, 6.72; N, 9.97.
6-{2-Methoxy-4-[(1E)-3-oxo-3-(3-(1,2,3,4-tetrahydroacridin-
9-ylamino)propylamino)prop-1-enyl]phenoxy}hexyl Nitrate
(3h). Compound 10b (0.26 g, 1 mmol) reacted with compound 8d
(0.34 g, 1 mmol) to furnish 3h as a yellow solid (0.18 g, 31%); mp
1
54−56 °C. H NMR (CDCl3): δ 8.05−7.86 (m, 2H), 7.62−7.48 (m,
2H), 7.36−7.30 (t, 1H), 7.03−6.96 (m, 2H), 6.81−6.78 (d, 1H),
6.54−6.49 (t, 1H), 6.32 (d, J = 15.5 Hz, 1H), 4.92 (br, 1H), 4.46−4.41
(t, 2H), 4.03−3.98 (t, 2H), 3.80 (s, 3H), 3.53−3.51 (m, 4H), 3.02 (br,
2H), 2.75 (br, 2H), 1.87−1.71 (m, 10H), 1.49−1.48 (m, 4H). 13C
NMR (CDCl3): δ 167.07, 158.45, 150.61, 150.11, 149.44, 147.17,
141.04, 128.38, 128.33, 127.71, 123.79, 122.66, 121.66, 120.31, 118.30,
116.39, 112.53, 110.23, 73.20, 68.63, 55.90, 49.00, 45.35, 36.81, 31.88,
31.57, 28.82, 26.64, 25.57, 25.42, 23.06, 22.73. HRMS (ESI) m/z calcd
for C32H40N4O6 [M +] 576.2948; found 576.2955. Anal. Calcd for
(C32H40N4O6·1/2H2O): found C, 65.60; H, 7.27; N, 9.37; requires C,
65.62; H, 7.06; N, 9.57.
6-{2-Methoxy-4-[(1E)-3-oxo-3-(2-(1,2,3,4-tetrahydroacridin-
9-ylamino)ethylamino)prop-1-enyl]phenoxy}hexyl Nitrate
(10d). Compound 10a (0.24 g, 1 mmol) reacted with compound
8d (0.34 g, 1 mmol) to furnish 3d as a yellow solid (0.16 g, 28%); mp
50−52 °C. 1H NMR (CDCl3): δ 7.97−7.85 (m, 2H), 7.57 (d, J = 15.5
Hz, 1H), 7.52−7.46 (t, 1H), 7.32−7.26 (t, 1H), 7.04−6.97 (m, 2H),
6.82−6.79 (d, 1H), 6.56 (br, 1H), 6.29 (d, J = 15.5 Hz, 1H), 4.84 (br,
1H), 4.47−4.41 (t, 2H), 4.04−3.99 (t, 2H), 3.82 (s, 3H), 3.67 (br,
4H), 3.00 (br, 2H), 2.69 (br, 2H), 1.84−1.69 (m, 8H), 1.50−1.47 (m,
4H). 13C NMR (CDCl3): δ 167.57, 158.36, 150.66, 150.23, 149.46,
147.05, 141.473, 128.41, 128.31, 127.57, 123.79, 122.64, 121.98,
119.92, 117.96, 116.14, 112.51, 110.26, 73.20, 68.64, 55.93, 49.82,
40.72, 33.79, 28.81, 26.65, 25.58, 25.42, 25.05, 23.00, 22.65. HRMS
2-{2-Methoxy-4-[(1E)-3-oxo-3-(4-(1,2,3,4-tetrahydroacridin-
9-ylamino)butylamino)prop-1-enyl]phenoxy}ethyl Mitrate (3i).
Compound 10c (0.27 g, 1 mmol) reacted with compound 8a (0.28 g,
1 mmol) to furnish 3i as a yellow solid (0.17 g, 32%); mp 45−47 °C.
1H NMR (CDCl3): δ 7.94−7.88 (m, 2H), 7.56−7.50 (m, 2H), 7.36−
7.30 (t, 1H), 7.02−6.98 (m, 2H), 6.83−6.80 (d, 1H), 6.27 (d, J = 15.5
Hz, 1H), 6.11 (br, 1H), 4.84−4.80 (t, 2H), 4.30−4.27 (t, 2H), 4.03
(br, 1H), 3.82 (s, 3H), 3.51−3.49 (m, 2H), 3.45−3.40 (m, 2H), 3.03
(br, 2H), 2.68 (br, 2H), 1.88 (br, 4H), 1.69 (br, 4H). 13C NMR
(CDCl3): δ 166.13, 158.40, 150.57, 149.93, 148.85, 147.20, 140.46,
129.42, 128.49, 128.42, 123.80, 122.71, 121.47, 120.21, 119.34, 116.22,
114.39, 110.78, 70.89, 65.55, 55.92, 48.87, 39.27, 33.87, 28.99, 26.64,
27.22, 24.82, 22.97, 22.70. HRMS (ESI) m/z calcd for C29H34N4O6
+
(ESI) m/z calcd for C31H38N4O6 [M ] 562.2791; found 562.2796.
Anal. Calcd for (C31H38N4O6·3/4H2O): found C, 64.52; H, 6.93; N,
9.47; requires C, 64.62; H, 6.90; N, 9.72.
2-{2-Methoxy-4-[(1E)-3-oxo-3-(3-(1,2,3,4-tetrahydroacridin-
9-ylamino)propylamino)prop-1-enyl]phenoxy}ethyl Nitrate
(3e). Compound 10b (0.26 g, 1 mmol) reacted with compound 8a
(0.28 g, 1 mmol) to furnish 3e as a yellow solid (0.18 g, 35%); mp
59−61 °C. 1H NMR (CDCl3): δ 8.05−7.88 (m, 2H), 7.57 (d, J = 15.5
4316
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+
1
[M
]
534.2478; found 534.2483. Anal. Calcd for
45−47 °C. H NMR (CDCl3): δ 7.96−7.88 (m, 2H), 7.56−7.50 (m,
2H), 7.36−7.30 (t, 1H), 7.03−6.98 (m, 2H), 6.82−6.79 (d, 1H), 6.29
(d, J = 15.5 Hz, 1H), 6.00−5.96 (t, 1H), 4.70−4.65 (t, 2H), 4.13−4.08
(t, 2H), 4.01 (br, 1H), 3.82 (s, 3H), 3.47 (br, 2H), 3.39−3.31 (m,
2H), 3.04 (br, 2H), 2.69 (br, 2H), 2.28−2.18 (m, 2H), 1.90 (br, 4H),
1.67−1.61 (m, 2H), 1.57−1.52 (m, 2H), 1.40−1.39 (m, 4H). 13C
NMR (CDCl3): δ 166.11, 158.32, 150.81, 149.64, 149.35, 147.25,
140.44, 128.66, 128.49, 128.36, 123.64, 122.84, 121.57, 120.14, 119.10,
115.84, 113.33, 110.43, 69.89, 64.87, 55.85, 49.26, 39.46, 33.88, 31.59,
29.63, 26.91, 26.59, 26.52, 24.78, 23.01, 22.72. HRMS (ESI) m/z calcd
for C32H40N4O6 [M +] 576.2948; found 576.2953. Anal. Calcd for
(C32H40N4O6·3/4H2O): found C, 65.22; H, 6.69; N, 9.09; requires C,
65.12; H, 7.09; N, 9.49.
(C29H34N4O6·3/4H2O): found C, 63.38; H, 6.51; N, 10.31; requires
C, 63.55; H, 6.53; N, 10.22.
3-{2-Methoxy-4-[(1E)-3-oxo-3-(4-(1,2,3,4-tetrahydroacridin-
9-ylamino)butylamino)prop-1-enyl]phenoxy}propyl Nitrate
(3j). Compound 10c (0.27 g, 1 mmol) reacted with compound 8b
(0.30 g, 1 mmol) to furnish 3j as a yellow solid (0.17 g, 31%); mp 40−
42 °C. 1H NMR (CDCl3): δ 7.95−7.88 (m, 2H), 7.57−7.50 (m, 2H),
7.35−7.29 (t, 1H), 7.02−6.97 (m, 2H), 6.82−6.79 (d, 1H), 6.29 (d, J
= 15.5 Hz, 1H), 6.23−6.19 (t, 1H), 4.70−4.65 (t, 2H), 4.12−4.08 (t,
3H), 3.81 (s, 3H), 3.52−3.47 (m, 2H), 3.44−3.37 (m, 2H), 3.03 (br,
2H), 2.67 (br, 2H), 2.28−2.18 (m, 2H), 1.88−1.86 (m, 4H), 1.68 (br,
4H). 13C NMR (CDCl3): δ 166.27, 158.18, 150.73, 149.64, 149.40,
146.94, 140.58, 128.59, 128.51, 128.21, 123.82, 122.77, 121.62, 120.07,
118.98, 116.03, 113.31, 110.42, 69.88, 64.87, 55.86, 48.84, 39.23,
33.69, 28.96, 27.20, 26.90, 24.78, 22.94, 22.61. HRMS (ESI) m/z calcd
for C30H36N4O6 [M +] 548.2635; found 548.2641. Anal. Calcd for
(C30H36N4O6·1/2H2O): found C, 64.30; H, 6.57; N, 9.85; requires C,
64.62; H, 6.69; N, 10.05.
4-{2-Methoxy-4-[(1E)-3-oxo-3-(6-(1,2,3,4-tetrahydroacridin-
9-ylamino)hexylamino)prop-1-enyl]phenoxy}butyl Nitrate
(3o). Compound 10d (0.30 g, 1 mmol) reacted with compound 8c
(0.31 g, 1 mmol) to furnish 3o as a yellow solid (0.19 g, 32%); mp
1
37−39 °C. H NMR (CDCl3): δ 7.96−7.88 (m, 2H), 7.56−7.50 (m,
2H), 7.35−7.29 (t, 1H), 7.02−6.98 (m, 2H), 6.80−6.77 (d, 1H), 6.28
(d, J = 15.5 Hz, 1H), 6.01−5.97 (t, 1H), 4.54 (br, 2H), 4.04 (br, 3H),
3.81 (s, 3H), 3.46 (br, 2H), 3.39−3.31 (m, 2H), 3.04 (br, 2H), 2.68
(br, 2H), 1.93−1.89 (m, 8H), 1.66−1.61 (m, 2H), 1.57−1.52 (m, 2H),
1.39−1.38 (m, 4H). 13C NMR (CDCl3): δ 166.17, 158.36, 150.78,
149.66, 149.51, 147.31, 140.53, 128.52, 128.33, 128.21, 123.63, 122.83,
121.70, 120.16, 118.86, 115.86, 112.74, 110.20, 72.92, 68.14, 55.83,
4-{2-Methoxy-4-[(1E)-3-oxo-3-(4-(1,2,3,4-tetrahydroacridin-
9-ylamino)butylamino)prop-1-enyl]phenoxy}butyl Nitrate
(3k). Compound 10c (0.27 g, 1 mmol) reacted with compound 8c
(0.31 g, 1 mmol) to furnish 3k as a yellow solid (0.16 g, 28%); mp
1
39−41 °C. H NMR (CDCl3): δ 7.94−7.87 (m, 2H), 7.57−7.49 (m,
2H), 7.35−7.29 (t, 1H), 7.01−6.96 (m, 2H), 6.80−6.76 (d, 1H), 6.27
(d, J = 15.5 Hz, 1H), 6.21−6.18 (t, 1H), 4.54 (br, 2H), 4.03 (br, 3H),
3.80 (s, 3H), 3.48−3.39 (m, 4H), 3.02 (br, 2H), 2.67 (br, 2H), 1.93−
1.87 (m, 8H), 1.68 (br, 4H). 13C NMR (CDCl3): δ 166.30, 158.37,
150.58, 149.71, 149.51, 147.19, 140.66, 128.44, 128.40, 128.14, 123.77,
122.73, 121.74, 120.19, 118.73, 116.17, 112.72, 110.18, 72.92, 68.13,
55.82, 48.86, 39.24, 33.85, 28.98, 27.22, 25.34, 24.81, 23.89, 22.96,
22.67. HRMS (ESI) m/z calcd for C31H38N4O6 [M +] 562.2791; found
562.2798. Anal. Calcd for (C31H38N4O6·1/2H2O): found C, 64.84; H,
6.98; N, 9.59; requires C, 65.13; H, 6.88; N, 9.80.
49.26, 39.46, 33.91, 31.59, 29.63, 26.60, 26.53, 25.35, 24.79, 23.90,
+
23.01, 22.73. HRMS (ESI) m/z calcd for C33H42N4O6 [M
]
590.3104; found 590.3108. Anal. Calcd for (C33H42N4O6 ·3/4H2O):
found C, 65.47; H, 7.30; N, 9.14; requires C, 65.60; H, 7.26; N, 9.27.
6-{2-Methoxy-4-[(1E)-3-oxo-3-(6-(1,2,3,4-tetrahydroacridin-
9-ylamino)hexylamino)prop-1-enyl]phenoxy}hexyl Nitrate
(3p). Compound 10d (0.30 g, 1 mmol) reacted with compound 8d
(0.34 g, 1 mmol) to furnish 3p as a yellow solid (0.19 g, 31%); mp
1
38−40 °C. H NMR (CDCl3): δ 7.96−7.88 (m, 2H), 7.56−7.50 (m,
6-{2-Methoxy-4-[(1E)-3-oxo-3-(4-(1,2,3,4-tetrahydroacridin-
9-ylamino)butylamino)prop-1-enyl]phenoxy}hexyl Nitrate (3l).
Compound 10c (0.27 g, 1 mmol) reacted with compound 8d (0.34 g,
1 mmol) to furnish 3l as a yellow solid (0.19 g, 32%); mp 43−45 °C.
1H NMR (CDCl3): δ 7.94−7.87 (m, 2H), 7.57−7.49 (m, 2H), 7.35−
7.29 (t, 1H), 7.03−6.97 (m, 2H), 6.81−6.78 (d, 1H), 6.26 (d, J = 15.5
Hz, 1H), 6.18−6.13 (t, 1H), 4.46−4.41 (t, 2H), 4.03−3.98 (t, 3H),
3.82 (s, 3H), 3.49 (br, 2H), 3.44−3.36 (m, 2H), 3.03 (br, 2H), 2.67
(br, 2H), 1.89−1.86 (m, 6H), 1.81−1.68 (m, 6H), 1.49−1.45 (m, 4H).
13C NMR (CDCl3): δ 166.35, 158.41, 150.58, 150.04, 149.43, 147.22,
140.76, 128.49, 128.39, 127.79, 123.78, 122.72, 121.85, 120.22, 118.51,
116.20, 112.52, 110.17, 73.20, 68.63, 55.91, 48.88, 39.25, 33.87, 28.99,
28.82, 27.23, 26.64, 25.57, 25.42, 24.81, 22.97, 22.68. HRMS (ESI) m/
z calcd for C33H42N4O6 [M +] 590.3104; found 590.3112. Anal. Calcd
for (C33H42N4O6·1/2H2O): found C, 66.07; H, 7.20; N, 9.26; requires
C, 66.09; H, 7.23; N, 9.34.
2-{2-Methoxy-4-[(1E)-3-oxo-3-(6-(1,2,3,4-tetrahydroacridin-
9-ylamino)hexylamino)prop-1-enyl]phenoxy}ethyl Nitrate
(3m). Compound 10d (0.30 g, 1 mmol) reacted with compound 8a
(0.28 g, 1 mmol) to furnish 3m as a yellow solid (0.20 g, 36%); mp
2H), 7.35−7.30 (t, 1H), 7.02−6.98 (m, 2H), 6.82−6.78 (d, 1H), 6.28
(d, J = 15.5 Hz, 1H), 5.97 −5.93 (t, 1H), 4.46−4.41 (t, 2H), 4.03−
3.98 (t, 3H), 3.83 (s, 3H), 3.49−3.44 (t, 2H), 3.39−3.31 (m, 2H), 3.05
(br, 2H), 2.69 (br, 2H), 1.90−1.81 (m, 6H), 1.76−1.71 (m, 2H),
1.67−1.61 (m, 2H), 1.57−1.47 (m, 6H), 1.40−1.38 (m, 4H). 13C
NMR (CDCl3): δ 166.20, 158.29, 150.82, 149.98, 149.44, 147.23,
140.61, 128.47, 128.36, 127.87, 123.64, 122.84, 121.80, 120.12, 118.67,
115.81, 112.54, 110.18, 73.20, 68.63, 55.91, 49.25, 39.45, 33.88, 31.58,
29.64, 28.83, 26.65, 26.59, 26.52, 25.57, 25.42, 24.78, 23.00, 22.72.
HRMS (ESI) m/z calcd for C35H46N4O6 [M +] 618.3417; found
618.3423. Anal. Calcd for (C35H46N4O6·1/4 H2O): found C, 67.30; H,
7.69; N, 9.03; requires C, 67.45; H, 7.52; N, 8.99.
2-{2-Methoxy-4-[(1E)-3-oxo-3-(8-(1,2,3,4-tetrahydroacridin-
9-ylamino)octylamino)prop-1-enyl]phenoxy}ethyl Nitrate (3q).
Compound 10e (0.31 g, 1 mmol) reacted with compound 3a (0.28 g,
1 mmol) to furnish 3q as a yellow solid (0.18 g, 31%); mp 40−42 °C.
1H NMR (CDCl3): δ 7.97−7.89 (m, 2H), 7.56−7.50 (m, 2H), 7.36−
7.30 (t, 1H), 7.02−6.99 (m, 2H), 6.82−6.79 (d, 1H), 6.33 (d, J = 15.5
Hz, 1H), 6.10−6.06 (t, 1H), 4.82−4.79 (t, 2H), 4.29−4.26 (t, 2H),
4.00 (br, 1H), 3.82 (s, 3H), 3.50−3.45 (t, 2H), 3.38−3.30 (m, 2H),
3.05 (br, 2H), 2.69 (br, 2H), 1.89 (br, 4H), 1.66−1.61 (m, 2H), 1.58−
1.48 (m, 2H), 1.28 (br, 8H). 13C NMR (CDCl3): δ 166.00, 158.25,
150.96, 149.92, 148.74, 147.21, 140.13, 129.56, 128.39, 123.60, 122.92,
121.39, 120.06, 119.67, 115.66, 114.42, 110.78, 70.92, 65.56, 55.89,
49.35, 39.65, 33.85, 31.69, 29.60, 29.09, 26.73, 24.74, 23.00, 22.72.
HRMS (ESI) m/z calcd for C33H42N4O6 [M +] 590.3104; found
590.3110. Anal. Calcd for (C33H42N4O6·1/2H2O): found C, 65.99; H,
7.33; N, 9.24; requires C, 66.09; H, 7.23; N, 9.34.
1
50−52 °C. H NMR (CDCl3): δ 7.97−7.89 (m, 2H), 7.56−7.50 (m,
2H), 7.36−7.30 (t, 1H), 7.03−7.00 (m, 2H), 6.84−6.81 (d, 1H), 6.29
(d, J = 15.5 Hz, 1H), 5.94−5.90 (t, 1H), 4.84−4.80 (t, 2H), 4.31−4.27
(t, 2H), 4.03 (br, 1H), 3.83 (s, 3H), 3.50−3.45 (m, 2H), 3.40−3.32
(m, 2H), 3.05 (br, 2H), 2.69 (br, 2H), 1.91−1.89 (m, 4H), 1.68−1.62
(m, 2H), 1.58−1.50 (m, 2H), 1.41−1.39 (m, 4H). 13C NMR
(CDCl3): δ 166.00, 158.25, 150.85, 149.95, 148.80, 147.17, 140.30,
129.51, 128.41, 123.66, 122.85, 121.42, 120.09, 119.51, 115.79, 114.44,
110.80, 70.90, 65.58, 55.92, 49.24, 39.47, 33.84, 31.58, 29.62, 26.58,
3-{2-Methoxy-4-[(1E)-3-oxo-3-(8-(1,2,3,4-tetrahydroacridin-
9-ylamino)octylamino)prop-1-enyl]phenoxy}propyl Nitrate
(3r). Compound 10e (0.31 g, 1 mmol) reacted with compound 3b
(0.30 g, 1 mmol) to furnish 3r as a yellow solid (0.19 g, 31%); mp 39−
41 °C. 1H NMR (CDCl3): δ 7.99−7.93 (m, 2H), 7.58−7.51 (m, 2H),
7.38−7.32 (t, 1H), 7.06−7.02 (m, 2H), 6.85−6.82 (d, 1H), 6.32 (d, J
= 15.5 Hz, 1H), 5.86−5.82 (t, 1H), 4.71−4.66 (t, 2H), 4.15−4.10 (t,
3H), 3.85 (s, 3H), 3.55−3.50 (t, 2H), 3.39−3.31 (m, 2H), 3.08 (br,
26.51, 24.77, 23.00, 22.70. HRMS (ESI) m/z calcd for C31H38N4O6
+
[M
] 562.2791; found 562.2799. Anal. Calcd for
(C31H38N4O6·3/4H2O): found C, 64.33; H, 6.90; N, 9.44; requires
C, 64.62; H, 6.91; N, 9.72.
3-{2-Methoxy-4-[(1E)-3-oxo-3-(6-(1,2,3,4-tetrahydroacridin-
9-ylamino)hexylamino)prop-1-enyl]phenoxy}propyl Nitrate
(3n). Compound 10d (0.30 g, 1 mmol) reacted with compound 8b
(0.30 g, 1 mmol) to furnish 3n as a yellow solid (0.19 g, 33%); mp
4317
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Article
2H), 2.70 (br, 2H), 2.30−2.20 (m, 2H), 1.91 (br, 4H), 1.69−1.61 (m,
2H), 1.52−1.49 (m, 2H), 1.31 (br, 8H). 13C NMR (CDCl3): δ 166.03,
157.76, 151.31, 149.67, 149.34, 146.62, 140.39, 128.71, 127.94, 123.73,
122.98, 121.57, 119.77, 119.17, 115.33, 113.35, 110.41, 69.88, 64.88,
7.09−7.08 (d, 1H, arom), 6.98−6.94 (m, 1H, arom), 6.78−6.75 (d,
1H, arom), 6.40 (d, J = 15.5 Hz, 1H, COCHCH), 5.38−5.33 (t, 1H,
CONH), 3.78 (s, 3H, OCH3), 3.41−3.36 (m, 2H, NHCH2), 3.15−
3.07 (m, 2H, CH2NHCO), 2.88−2.85 (m, 2H, C4−H2), 2.72−2.79
(m, 2H, C1−H2), 1.79−1.75 (m, 4H, C2−H2, C3−H2), 1.54−1.51
(m, 2H,NHCH2CH2), 1.41−1.36 (m, 2H, CH2CH2NHCO), 1.28−
1.22 (m, 4H, NHCH2CH2CH2CH2). 13C NMR (DMSO-d6): δ 165.65
(NHCO), 158.41 (arom), 150.74 (arom), 148.63 (arom), 148.25
(arom), 147.41 (arom), 139.15 (COCHCH), 128.78 (arom),
128.25 (arom), 126.90 (arom), 123.62 (arom), 123.47 (arom), 121.89
(arom), 120.76 (COCHCH), 116.33 (arom), 116.09 (arom),
111.15 (arom), 55.96 (OCH3), 48.37 (NHCH2), 33.99 (CH2NHCO),
31.04 (NHCH2CH2), 26.67 (CH2CH2NHCO), 26.51 (C4), 25.54
(C1, NHCH2CH2CH2), 23.21 (NHCH2CH2CH2CH2), 22.92 (C3,
C2). HRMS (ESI) m/z calcd for C29H35N3O3 [M +] 473.2678; found
473.2674. Anal. Calcd for (C29H35N3O3·1/2H2O): found C, 72.13; H,
7.71; N, 8.47; requires C, 72.17; H, 7.52; N, 8.71.
Cholinesterase Inhibition Assay in Vitro. The assay followed
the method of Ellman et al.,33 using a Shimadzu 160 spectropho-
tometer. AChE (EC3.1.1.7, Type VI−S, from Electric Eel) and BuChE
(EC3.1.1.8, from equine serum), 5,5′-dithiobis(2-nitrobenzoic acid)
(Ellman’s reagent, DTNB), acetylthiocholine (ATC), and butyrylth-
iocholine (BTC) iodides were purchased from Sigma-Aldrich
(Steinheim, Germany). AChE/BuChE stock solution was prepared
by adjusting 500 units of the enzyme and 1 mL of gelatin solution (1%
in water) to 100 mL with water. This enzyme solution was further
diluted before use to give 2.5 units/mL in 1.4 mL aliquots. ATC/BTC
iodide solution (0.075 M) was prepared in water. DTNB solution
(0.01 M) was prepared in water containing 0.15% (w/v) sodium
bicarbonate. For buffer preparation, potassium dihydrogen phosphate
(1.36 g, 10 mmol) was dissolved in 100 mL of water and adjusted with
KOH to pH = 8.0 0.1. Stock solutions of the test compounds were
prepared in ethanol, 100 μL of which gave a final concentration of
10−4 M when diluted to the final volume of 3.32 mL. For each
compound, a dilution series of at least five different concentrations
(normally 10−4−10−9 M) was prepared.
For measurement, a cuvette containing 3.0 mL of phosphate buffer,
100 μL of the respective enzyme, and 100 μL of the test compound
solution was allowed to stand for 5 min before 100 μL of DTNB were
added. The reaction was started by addition of 20 μL of the substrate
solution (ATC/BTC). The solution was mixed immediately, and
exactly 2 min after substrate addition the absorption was measured at
25 °C at 412 nm. For the reference value, 100 μL of water replaced the
test compound solution. For determining the blank value, additionally
100 μL of water replaced the enzyme solution. Each concentration was
measured in triplicate at 25 °C. The inhibition curve was obtained by
plotting percentage enzyme activity (100% for the reference) versus
logarithm of test compound concentration. Calculation of the IC50
values was performed with Graph Pad Prism 4.
Kinetic measurements were performed in the same manner, while
the substrate (ATC/BTC) was used in concentrations of 25, 50, 90,
150, 226, and 452 μM for each test compound concentration and the
reaction was extended to 4 min before measurement of the absorption.
Vmax and Km values of the Michaelis−Menten kinetics were calculated
by nonlinear regression from substrate−velocity curves using Graph
Pad Prism 4. Linear regression was used for calculating the
Lineweaver−Burk plots.
55.87, 49.31, 39.64, 33.45, 31.64, 29.62, 29.05, 26.93, 26.70, 24.65,
+
22.93, 22.58. HRMS (ESI) m/z calcd for C34H44N4O6 [M
]
604.3261; found 604.3264. Anal. Calcd for (C34H44N4O6): found C,
67.29; H, 7.73; N, 8.89; requires C, 67.53; H, 7.33; N, 9.26.
4-{2-Methoxy-4-[(1E)-3-oxo-3-(8-(1,2,3,4-tetrahydroacridin-
9-ylamino)octylamino)prop-1-enyl]phenoxy}butyl Nitrate (3s).
Compound 10e (0.31 g, 1 mmol) reacted with compound 8c (0.31 g,
1 mmol) to furnish 3s as a yellow solid (0.19 g, 31%); mp 35−37 °C.
1H NMR (CDCl3): δ 7.97−7.88 (m, 2H), 7.56−7.50 (m, 2H), 7.35−
7.29 (t, 1H), 7.02−6.98 (m, 2H), 6.79−6.76 (d, 1H), 6.32 (d, J = 15.5
Hz, 1H), 6.13−6.08 (t, 1H), 4.56−4.51 (t, 2H), 4.03 (br, 3H), 3.80 (s,
3H), 3.50−3.44 (t, 2H), 3.38−3.30 (m, 2H), 3.04 (br, 2H), 2.69 (br,
2H), 1.92−1.90 (m, 8H), 1.66−1.57 (m, 2H), 1.50−1.48 (m, 2H),
1.29 (br, 8H). 13C NMR (CDCl3): δ 166.15, 158.26, 150.94, 149.61,
149.50, 147.25, 140.36, 128.43, 128.36, 128.28, 123.59, 122.92, 121.66,
120.09, 119.03, 115.68, 112.73, 110.18, 72.93, 68.13, 55.81, 49.35,
39.64, 33.87, 31.69, 29.62, 29.09, 26.74, 25.35, 24.74, 23.90, 23.01,
22.72. HRMS (ESI) m/z calcd for C35H46N4O6 [M +] 618.3417; found
618.3422. Anal. Calcd for (C35H46N4O6·H2O): found C, 65.91; H,
7.62; N, 8.75; requires C, 66.02; H, 7.60; N, 8.80.
6-{2-Methoxy-4-[(1E)-3-oxo-3-(8-(1,2,3,4-tetrahydroacridin-
9-ylamino)octylamino)prop-1-enyl]phenoxy}hexyl Nitrate (3t).
Compound 10e (0.31 g, 1 mmol) reacted with compound 8d (0.34 g,
1 mmol) to furnish 3t as a yellow solid (0.21 g, 33%); mp 33−35 °C.
1H NMR (CDCl3): δ 7.97−7.89 (m, 2H), 7.57−7.51 (m, 2H), 7.36−
7.30 (t, 1H), 7.04−7.00 (m, 2H), 6.82−6.79 (d, 1H), 6.30 (d, J = 15.5
Hz, 1H), 5.97−5.92 (t, 1H), 4.47−4.41 (t, 2H), 4.04−3.99 (t, 3H),
3.84 (s, 3H), 3.48 (br, 2H), 3.38−3.30 (m, 2H), 3.05 (br, 2H), 2.69
(br, 2H), 1.90 (br, 6H), 1.85−1.72 (m, 2H), 1.69−1.61 (m, 2H), 1.50
(br, 6H), 1.29 (br, 8H). 13C NMR (CDCl3): δ 166.17, 158.21, 150.98,
149.94, 149.41, 147.18, 140.53, 128.40, 127.90, 123.61, 122.92, 121.78,
120.05, 118.75, 115.64, 112.50, 110.13, 73.21, 68.63, 55.90, 49.36,
39.63, 33.83, 31.69, 29.63, 29.09, 28.83, 26.74, 26.65, 25.58, 25.42,
+
24.74, 23.01, 22.71. HRMS (ESI) m/z calcd for C37H50N4O6 [M ]
646.3730; found 646.3735. Anal. Calcd for (C37H50N4O6·1/2H2O):
found C, 68.01; H, 8.16; N, 8.18; requires C, 67.76; H, 7.84; N, 8.54.
(E)-3-(3-Methoxy-4-propoxyphenyl)-N-[3-(1,2,3,4-tetrahy-
droacridin-9-ylamino)propyl]acrylamide (3u). Following general
procedure V, compound 10b (0.26 g, 1 mmol) reacted with
compound 8e (0.24 g, 1 mmol) to furnish 3u as a yellow solid
(0.17 g, 36%); mp 70−72 °C. 1H NMR (CDCl3): δ 8.06−8.03 (d, 1H,
arom), 7.90−7.87 (d, 1H, arom), 7.59 (d, J = 15.5 Hz, 1H, COCH
CH), 7.55−7.49 (t, 1H, arom), 7.36−7.30 (t, 1H, arom), 7.04−6.96
(m, 2H, arom), 6.82−6.78 (d, 1H, arom), 6.55−6.50 (t, 1H, CONH),
6.32 (d, J = 15.5 Hz, 1H, COCHCH), 5.02−4.96 (t, 1H, NH),
4.00−3.94 (t, 2H, OCH2), 3.81 (s, 3H, OCH3), 3.55−3.51 (m, 4H,
NHCH2CH2CH2NHCO), 3.03 (br, 2H, C4−H2), 2.75 (br, 2H, C1−
H2), 1.89−1.81 (m, 8H, C2−H2, C3−H2, NHCH2CH2, OCH2CH2),
1.05−0.99 (t, 3H, OCH2CH2CH3). 13C NMR (CDCl3): δ 167.13
(NHCO), 158.28 (arom), 150.72 (arom), 150.29 (arom), 149.43
(arom), 146.99 (arom), 141.10 (COCHCH), 128.41 (arom),
128.22 (arom), 127.53 (arom), 123.82 (arom), 122.70 (arom), 121.95
(arom), 120.22 (COCHCH), 118.19 (arom), 116.28 (arom),
112.48 (arom), 110.27 (arom), 70.44 (OCH2), 55.96 (OCH3), 45.30
(CH2NHCO), 36.78 (NHCH2), 33.78 (C4), 31.57 (NHCH2CH2),
Detection of Nitrite (Griess Reaction). A solution of the
appropriate compound (80 μL) in DMSO was added to 320 μL of
phosphate buffer (pH 7.4) containing 5 mM L-cysteine. The final
concentration of the compound was 10−4 M. After incubation for 1.5 h
at 37 °C, 120 μL of the reaction mixture was treated with 25 μL of
Griess reagent (2 g of sulfanilamide, 0.1 g of N-naphthylethylenedi-
amine dihydrochloride, 85% phosphoric acid [5 mL] in distilled water
[final volume, 50 mL]). After 30 min in darkness at room temperature,
the absorbance was measured at 540 nm using a microplate reader
TECAN Safire2 (Tecan Group Ltd., Maennedorf, Switzerland). Each
compound was measured in triplicate. Standard sodium nitrite
solutions (0, 0.01, 0.03, 0.05, 0.07, 0.09, 0.20, and 0.40 μg/mL)
25.13 (C1), 23.04 (C3), 22.69 (C2), 22.36 (OCH2CH2CH3), 10.38
+
(OCH2CH2CH3). HRMS (ESI) m/z calcd for C29H35N3O3 [M
]
473.2678; found 473.2681. Anal. Calcd for (C29H35N3O3·1/2H2O):
found C, 72.03; H, 7.40; N, 8.46; requires C, 72.17; H, 7.52; N, 8.71.
(E)-3-(4-Hydroxy-3-methoxyphenyl)-N-[6-(1,2,3,4-tetrahy-
droacridin-9-ylamino)hexyl]acrylamide (4). Following general
procedure V, compound 10d (0.30 g, 1 mmol) reacted with ferulic
acid (0.19 g, 1 mmol) to furnish 4 as a yellow solid (0.12 g, 25%); mp
172−174 °C. H NMR (DMSO-d6): δ 9.39 (br, 1H, OH), 8.11−8.08
(d, 1H, arom), 7.90−7.85 (t, 1H, arom), 7.70−7.66 (m, 1H, arom),
7.52−7.46 (m, 1H, arom), 7.35−7.25 (m, 2H, arom, COCHCH),
1
4318
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Article
were used to construct the calibration curve, from which the amount
of nitric oxide release (quantitated as nitrite ion) was calculated.
Vascular Relaxation Study. Aortic rings isolated from rat heart
(3−5 mm long) were suspended between two L-shaped platinum
hooks and mounted under a passive tension (1−1.1 g) in a 10 mL
organ bath filled with modified Krebs−Henseleit solution, which was
kept at 37 °C. Preparation was connected to isometric force transducer
JZ100 (New Channel Instruments, Gaobeidian, China). After an
equilibration period of 60 min, contractions were induced by
norepinephrine (NE, final concentration 10−5 M) to detect the
activity of the vessel. Endothelial integrity was further assessed by the
acetylcholine (10−5 M)-induced relaxation of NE-precontracted
vessels.The ring segments were initially made to contract with KCl
(60 mM) and subsequently with L-cysteine (10−3 M). The relaxation
response to addition of test compounds was studied after the
contractions became constant by constructing a cumulative concen-
tration−response curve. Relaxation responses were expressed as a
percentage of the KCl (60 mM)-induced contraction.
Behavioral Studies. The experiments were performed with adult
male ICR mice (weighing 18−22 g), which were purchased from
Comparative Medicine Centre, Yangzhou University (certificate no.
2023793). Scopolamine hydrobromide was obtained from Aladdin
Reagents, Shanghai, China. Tacrine hydrochloride hydrate was
purchased from BioTrend, Wangen, Switerland.
Scopolamine (0.2 mg/100 g b wt) were dissolved in saline and
administered intraperitoneally (ip) 5 min before the training trial.
Tacrine was dissolved in CMC-Na solution (0.5 g CMC-Na in 100 mL
distilled water) and 0.5 mg/100 g b wt, corresponding to 1.978 μmol/
100 g b wt, were given in intragastric (id) administration 1 h before
the training trial. The new substances were dissolved in CMC-Na
solution and a equimolar dose corresponding to tacrine was
administered id 1 h before the training trial. Mice were divided into
six groups. (i) vehicle, (ii) scopolamine, (iii) compound 1b plus
scopolamine, (iv) compound 3f plus scopolamine, (v) compound 3u
plus scopolamine, and (vi) tacrine plus scopolamine were given to
each group, respectively.
The mice were subjected to the passive avoidance test by using a
step-through passive avoidance apparatus for mice RD1105-RA-M-1
(Mice Doctor, Shanghai, China). The apparatus contained two
symmetrical compartments, a dark and a light chamber, which were
separated by a dark board that had a door near the floor. The grid
floors of the dark chamber consisted of copper bars. To begin with, the
dark compartment was electrified to generate low intensity foot shocks
(0.4 mA). A trial of 300 s started as soon as one animal was gently put
inside the light compartment, with the head facing away from the door.
After entering to the dark compartment, the mouse would return to
the light one. Afterward, the mouse would repeat the actions. The
number of enterings from the light room to the dark room was
recorded as the indication for short time learning ability. Besides, a
transfer from light chamber into the dark chamber was recorded as
transfer lantency time (TLT in seconds). The first trial was for
training, and the retention was tested by a second trial 24 h later, in
which the shock was not delivered. The criterion for improvement of
memory impairment was taken as significant increase in the TLT on
the test compounds groups comparing to the scopalamine group in
second trial.
Hepatotoxicity Studies. The experiments were carried out on
adult male ICR mice (weighing 18−22 g), which were purchased from
Comparative Medicine Centre, Yangzhou University (certificate no.
2023793). Tacrine hydrochloride hydrate (BioTrend, Wangen,
Switerland) was dissolved in CMC-Na solutiuon (0.5 g CMC-Na in
100 mL of distilled water) and 3 mg/100 g b wt, corresponding to
11.86 μmol/100 g b wt, were given in id. Test compounds were
dissolved in CMC-Na solutiuon, and the equimolar dose correspond-
ing to tacrine was administered id. Heparinized serum was obtained 8,
20, and 30 h after dosing from the retrobulbar plexus to determine
aspartate aminotransferase (ASAT) and alanine aminotransferase
(ALT) activity, two indicators of a liver damage, using routine
methods.
Histology. Tacrine and tested compounds treated rats were
sacrificed in ether anesthesia 30 h after dosing, and livers were
harvested. Two 3 mm sections of each liver extending from the hilus to
the margin of the left lateral lobe were immediately placed in 10%
buffered formaldehyde, fixed for two days, and embedded together in
one paraffin block. Subsequently, 5 μm sections were prepared from
these paraffin blocks. Paraffin sections of each block were
deparaffinated and stained with hematoxylin and eosin or by means
of the periodic acid−Schiff procedure for glycogen.
Statistics. The number of animals investigated per group for the
histological and biochemical investigations comprised n = 3−12. For
behavioral studies, 8−9 rats per group were used. Results are expressed
as arithmetic means SEM. For statistical analysis, Student’s t test (p
≤ 0.05) was applied.
Molecular Modeling. The docking study was performed by using
CDOCKER module inbuilt in DS. CDOCKER was an in-house
docking method as described previously.42,43 In general, CDOCKER
generated ligand “seeds” to populate the binding pocket. Each seed
was then subjected to high temperature molecular dynamics (MD)
using a modified version of CHARMm force field. Each of the
structures from the MD run were then located and fully minimized.
The solutions were then clustered according to position and
conformation and ranked by energy. CHARMm charges were used
for the protein structure. The cocrystal structure of bis(7)-tacrine with
Torpedo Californica AChE (TcAChE, PDB id: 2CKM) was selected for
molecular modeling. Residues around bis(7)-tacrine in AChE (in 9 Å
radius) was set as binding site. The heating step, cooling steps, and
cooling temperature were set to 5000, 5000, and 310, respectively.
Other parameters were kept as default. The MM/PBSA calculations
were carried out by using “binding energy with MM/PBSA” protocol
within Pipeline Pilot 8.0. All the parameters were kept as default.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed synthetic procedures as well as physical, analytical, and
spectral data for intermediary compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For H.L.: phone, +86-25-83271043; fax, +86-25-83271142; E-
mail, hliao@cpu.edu.cn. For J.L.: phone, +49-3641949803; Fax,
+49-3641949802; E-mail, j.lehmann@uni-jena.de. For Y.Z.:
phone, +86-25-83271015; fax, +86-25-83271015; E-mail,
zyhtgd@hotmail.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The support of “DAAD Sandwich Program” Scholarship (April
2010 to March 2012) is very gratefully acknowledged. We
thank the National Science Foundation of China (NSFC, no.
30973608), the National Natural Science Foundation of China
(no. 81070967), and the Natural Science Foundation of Jiangsu
Province of China (no. BK2009296) for funding. We also thank
Yinquan Fang, Zhe Zhang, and Jianing Wang for their valuable
help concerning this project.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; ACh, acetylcholine; ChEs, cholines-
terases; AChE, acetylcholinesterase; BuChE, butyrylcholinester-
ase; CAS, catalytic active site; PAS, peripheral anionic site; Aβ,
β-amyloid; ChEIs, cholinesterase inhibitors; MTDLs, multi-
target-directed ligands; NO, nitric oxide; DCC, dicyclohex-
ylcarbodiimide; DMAP, 4-dimethylaminopyridine; DMF, di-
4319
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Journal of Medicinal Chemistry
Article
(16) Chianella, C.; Gragnaniello, D.; Delser, P. M.; Visentini, M. F.;
Sette, E.; Tola, M. R.; Barbujani, G.; Fuselli, S. BCHE and CYP2D6
Genetic Variation in Alzheimer’s Disease Patients Treated with
Cholinesterase Inhibitors. Eur. J. Clin. Pharmacol. 2011, 67, 1147−
1157.
methylformamide; THF, tetrahydrofuran; ISDN, isosorbide
dinitrate; TLT, transfer latency time; ASAT, aspartate amino-
transferase; ALT, alanine aminotransferase; HE, hematoxylin
and eosin
(17) Ballard, C. G. Advances in the Treatment of Alzheimer’s
Disease: Benefits of Dual Cholinesterase Inhibition. Eur Neurol. 2002,
47, 64−70.
REFERENCES
■
(1) Scarpini, E.; Scheltens, P.; Feldman, H. Treatment of Alzheimer’s
Disease: Current Status and New Perspectives. Lancet Neurol. 2003, 2,
539−547.
(18) Terry, A. V., Jr.; Buccafusco, J. J. The Cholinergic Hypothesis of
Age and Alzheimer’s Disease-Related Cognitive Deficits: Recent
Challenges and Their Implications for Novel Drug Development. J.
Pharmacol. Exp. Ther. 2003, 306, 821−827.
(2) Perry, E. K.; Tomlinson, B. E.; Blessed, G.; Bergman, K.; Gibson,
P. H.; Perry, R. H. Correlation of Cholinergic Abnormalities with
Senile Plaques and Mental Test Scores in Senile Dementia. Br. Med. J.
1978, 25, 1457−1459.
(19) Youdim, M. B.; Buccafusso, J. J. Multifunctional Drugs for
Various CNS Targets in the Treatment of Neurodegenerative
Disorders. Trends. Pharmacol. Sci. 2005, 26, 27−35.
́
(3) Holzgrabe, U.; Kapkova, P.; Alptuzun, V.; Scheiber, J.;
̈ ̈
(20) Bolognesi, M. L.; Cavalli, A.; Valgimigli, L.; Bartolini, M.; Rosini,
M.; Andrisano, V.; Recanatini, M.; Melchiorre, C. Multi-Target-
Directed Drug Design Strategy: From a Dual Binding Site
Acetylcholinesterase Inhibitor to a Trifunctional Compound Against
Alzheimer’s Disease. J. Med. Chem. 2007, 50, 6446−6449.
(21) Tumiatti, V.; Milelli, A.; Minarini, A.; Rosini, M.; Bolognesi, M.
L.; Micco, M.; Andrisano, V.; Bartolini, M.; Mancini, F.; Recanatini,
M.; Cavalli, A.; Melchiorre, C. Structure−Activity Relationships of
Acetylcholinesterase Noncovalent Inhibitors Based on a Polyamine
Backbone. 4. Further Investigation on the Inner Spacer. J. Med. Chem.
2008, 51, 7308−7312.
(22) Furchgott, R. F.; Zawadzki, J. V. The Obligatory Role of
Endothelial Cells in the Relaxation of Arterial Smooth Muscle by
Acetylcholine. Nature 1980, 288, 373−376.
(23) Moncada, S.; Higgs, A. The L-Arginine-Nitric Oxide Pathway. N.
Engl. J. Med. 1993, 329, 2002−2012.
(24) Cooke, J. P.; Dzau, V. J. Nitric Oxide Synthase: Role in the
Genesis of Vascular Disease. Annu. Rev. Med. 1997, 48, 489−509.
(25) Faraci, F. M. Role of Endothelium-Derived Relaxing Factor in
Cerebral Circulation: Large Arteries vs Microcirculation. Am. J. Physiol.
1991, 261, 1038−1042.
(26) Megson, I, L.; Webb, D. J. Nitric Oxide Donor Drugs: Current
Status and Future Trends. Expert Opin. Invest. Drugs 2002, 11, 587−
601.
(27) Thatcher, G. R. J.; Bennett, B. M.; Reynolds, J. N. Nitric Oxide
Mimetic Molecules as Therapeutic Agents in Alzheimer’s Disease.
Curr. Alzheimer Res. 2005, 2, 171−182.
(28) Fang, L.; Appenroth, D.; Decker, M.; Kiehntopf, M.; Lupp, A.;
Peng, S. X.; Fleck, C.; Zhang, Y. H.; Lehmann, J. NO-Donating
Tacrine Hybrid Compounds Improve Scopolamine-Induced Cogni-
tion Impairment and Show Less Hepatotoxicity. J. Med. Chem. 2008,
51, 7666−7669.
(29) Schiefer, I. T.; Abdul-Hay, S.; Wang, H. L.; Vanni, M.; Qin, Z.
H.; Thatcher, G. R. J. Inhibition of Amyloidogenesis by Nonsteroidal
Anti-inflammatory Drugs and Their Hybrid Nitrates. J. Med. Chem.
2011, 54, 2293−2306.
(30) Fang, L.; Kraus, B.; Lehmann, J.; Heilmann, J.; Zhang, Y. H.;
Decker, M. Design and Synthesis of Tacrine−Ferulic Acid Hybrids as
Multi-Potent Anti-Alzheimer Drug Candidates. Bioorg. Med. Chem.
Lett. 2008, 18, 2905−2909.
(31) Fang, L.; Appenroth, D.; Decker, M.; Kiehntopf, M.; Roegler,
C.; Deufel, T.; Fleck, C.; Peng, S. X.; Zhang, Y. H.; Lehmann, J.
Synthesis and Biological Evaluation of NO-Donor−Tacrine Hybrids as
Hepatoprotective Anti-Alzheimer Drug Candidates. J. Med. Chem.
2008, 51, 713−716.
(32) Carlier, P. R.; Han, Y. F.; Chow, E. S.-H.; Li, C.P.-L.; Wang, H.;
Lieu, T. X.; Wong, H. S.; Pang, Y.-P. Evaluation of Short-Tether Bis-
THA AChE Inhibitors. A Further Test of the Dual Binding Site
Hypothesis. Bioorg. Med. Chem. 1999, 7, 351−357.
Kugelmann, E. Targeting Acetylcholinesterase to Treat Neuro-
degeneration. Expert Opin. Ther. Targets 2007, 11, 161−179.
(4) Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A. L;
L., T.; Silman, I. Atomic Structure of Acetylcholinesterase from
Torpedo californica: A Prototypic Acetylcholine-Binding Protein.
Science 1991, 253, 872−879.
(5) Axelsen, P. H.; Harel, M.; Silman, I.; Sussman, J. L. Structure and
Dynamics of the Active Site Gorge of Acetylcholinesterase: Synergistic
Use of Molecular Dynamics Simulation and X-ray Crystallography.
Protein Sci. 1994, 3, 188−197.
(6) Munoz-Ruiz, P.; Rubio, L.; García-Palomero, E.; Dorronsoro, I.;
̃
́ ́
del Monte-Millan, M.; Valenzuela, R.; Usan, P.; de Austria, C.;
Bartolini, M.; Andrisano, V.; Bidon-Chanal, A.; Orozco, M.; Luque, F.
J.; Medina, M.; Martínez, A. Design, Synthesis, and Biological
Evaluation of Dual Binding Site Acetylcholinesterase Inhibitors: New
Disease-Modifying Agents for Alzheimer’s Disease. J. Med. Chem.
2005, 48, 7223−7233.
́
(7) Bourne, Y.; Taylor, P.; Radic, Z.; Marchot, P. Structural Insights
into Ligand Interactions at the Acetylcholinesterase Peripheral Anionic
Site. EMBO J. 2003, 22, 1−12.
(8) Butini, S.; Campiani, G.; Borriello, M.; Gemma, S.; Panico, A.;
Persico, M.; Catalanotti, B.; Ros, S.; Brindisi, M.; Agnusdei, M.;
Fiorini, I.; Nacci, V.; Novellino, E.; Belinskaya, T.; Saxena, A.;
Fattorusso, C. Exploiting Protein Fluctuations at the Active-Site Gorge
of Human Cholinesterases: Further Optimization of the Design
Strategy to Develop Extremely Potent Inhibitors. J. Med. Chem. 2008,
51, 3154−3170.
(9) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. β-Amyloid
Aggregation Induced by Human Acetylcholinesterase: Inhibition
Studies. Biochem. Pharmacol. 2003, 65, 407−416.
́
(10) Alvarez, A.; Opazo, C.; Alarcon, R.; Garrido, J.; Inestrosa, N. C.
Acetylcholinesterase Promotes the Aggregation of Amyloid-Beta-
Peptide Fragments by Forming a Complex with the Growing Fibrils.
J. Mol. Biol. 1997, 272, 348−361.
(11) Inestrosa, N. C.; Sagal, J. P.; Colombres, M. Acetylcholinesterase
Interaction with Alzheimer Amyloid Beta. Subcell. Biochem. 2005, 38,
299−317.
(12) Saxena, A.; Redman, A. M.; Jiang, X.; Lockridge, B. P.; Doctor,
B. P. Differences in Active Site Gorge Dimensions of Cholinesterases
Revealed by Binding of Inhibitors to Human Butyrylcholinesterase.
Biochemistry 1997, 36, 14642−14651.
(13) Conejo-García, A.; Pisani, L.; del Nunez, M. C.; Catto, M.;
́
̃
Nicolotti, O.; Leonetti, F.; Campos, J. M.; Gallo, M. A.; Espinosa, A.;
Carotti, A. Homodimeric Bis-Quaternary Heterocyclic Ammonium
Salts as Potent Acetyl- and Butyrylcholinesterase Inhibitors: A
Systematic Investigation of the Influence of Linker and Cationic
Heads over Affinity and Selectivity. J. Med. Chem. 2011, 54, 2627−
2645.
(14) Darvesh, S.; Hopkins, D. A.; Geula, C. Neurobiology of
(33) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R.
M. A New and Rapid Colorimetric Determination of Acetylcholines-
terase Activity. Biochem. Pharmacol. 1961, 7, 88−95.
(34) Reddy, K. K.; Shanker, K. S.; Ravinder, T.; Prasad, R. B. N.;
Kanjilal, S. Chemo-enzymatic Synthesis and Evaluation of Novel
Butyrylcholinesterase. Nature Rev. Neurosci. 2003, 4, 131−138.
(15) Arendt, T.; Bruckner, M. K.; Lange, M.; Volker, B. Changes in
̈
Acetylcholinesterase and Butyrylcholinesterase in Alzheimer’s Disease
Resemble Embryonic DevelopmentA Study of Molecular Forms.
Neurochem. Int. 1992, 21, 381−396.
4320
dx.doi.org/10.1021/jm300106z | J. Med. Chem. 2012, 55, 4309−4321

Journal of Medicinal Chemistry
Article
Structured Phenolic Lipids as Potential Lipophilic Antioxidants. Eur. J.
Lipid Sci. Technol. 2010, 112, 600−608.
(35) Sorba, G.; Medana, C.; Fruttero, R.; Cena, C.; Di Stilo, A.; Galli,
U.; Gasco, A. Water Soluble Furoxan Derivatives as NO Prodrugs. J.
Med. Chem. 1997, 40, 463−469.
(36) Das, A.; Kapoor, K.; Sayeepriyadarshini, A. T.; Dikshit, M.; Palit,
G.; Nath, C. Immobilization Stress-Induced Changes in Brain
Acetylcholinesterase Activity and Cognitive Function in Mice.
Pharmacol. Res. 2000, 42, 213−217.
(37) Gracon, S. I.; Knapp, M. J.; Berghoff, W. G.; Pierce, M.; de Jong,
R.; Lobbestael, S. J.; Symons, J.; Dombey, S. L.; Luscombe, F. A.;
Kraemer, D. Safety of Tacrine: Clinical Trials, Treatment IND, and
Postmarketing Experience. Alzheimer Dis. Assoc. Disord. 1998, 12, 93−
101.
(38) Liu, J.; Waalkes, M. P. Nitric Oxide and Chemically Induced
Hepatotoxicity: Beneficial Effects of the Liver-Selective Nitric Oxide
Donor, V-PYRRO/NO. Toxicology 2005, 208, 289−297.
(39) Futter, L. E.; al-Swayeh, O. A.; Moore, P. K. A Comparison of
the Effect of Nitroparacetamol and Paracetamol on Liver Injury. Br. J.
Pharmacol. 2001, 132, 10−12.
(40) Rydberg, E. H.; Brumshtein, B.; Greenblatt, H. M.; Wong, D.
M.; Shaya, D.; Williams, L. D.; Carlier, P. R.; Pang, Y. P.; Silman, I.;
Sussman, J. L. Complexes of Alkylene-Linked Tacrine Dimers with
Torpedo californica Acetylcholinesterase Binding of Bis(5)-tacrine
Produce a Dramatic Rearrangement in the Active-Site Gorge. J. Med.
Chem. 2006, 49, 5491−5500.
́ ́
(41) Bolea, I.; Juarez-Jimenez, J.; de los Ríos, C.; Chioua, M.;
Pouplana, R.; Luque, F. J.; Unzeta, M.; Marco-Contelles, J.; Samadi, A.
Synthesis, Biological Evaluation and Molecular Modeling of Donepezil
and N-[(5-(Benzyloxy)-1-methyl-1H-indol-2-yl)methyl]-N-methyl-
prop-2-yn-1-amine Hybrids as New Multipotent Cholinesterase/
Monoamine Oxidase Inhibitors for the Treatment of Alzheimer’s
Disease. J. Med. Chem. 2011, 54, 8251−8270.
(42) Wu, G.; Robertson, D. H.; Brooks, C. L., III; Vieth, M. Detailed
Analysis of Grid-Based Molecular Docking: A Case Study of
CDOCKER-A CHARMm-Based MD Docking Algorithm. J. Comput.
Chem. 2003, 24, 1549−1562.
(43) Sun, H. P.; Zhu, J.; Chen, Y. D.; Sun, Y.; Zhi, H. J.; Li, Hao.;
You, Q. D. Docking Study and Three-Dimensional Quantitative
Structure−Activity Relationship (3D-QSAR) Analyses and Novel
Molecular Design of a Series of 4-Aminoquinazolines as Inhibitors of
Aurora B Kinase. Chin. J. Chem. 2011, 29, 1785−1799.
4321
dx.doi.org/10.1021/jm300106z | J. Med. Chem. 2012, 55, 4309−4321