Discovery of Highly Selective and Nanomolar Carbamate-Based Butyrylcholinesterase Inhibitors by Rational Investigation into Their Inhibition Mode

Article abstract of DOI:10.1021/acs.jmedchem.5b01674

Butyrylcholinesterase (BChE) is a promising target for the treatment of later stage cognitive decline in Alzheimer's disease. A set of pseudo-irreversible BChE inhibitors with high selectivity over hAChE was synthesized based on carbamates attached to tetrahydroquinazoline scaffolds with the 2-thiophenyl compound 2p as the most potent inhibitor of eqBChE (K_C = 14.3 nM) and also of hBChE (K_C = 19.7 nM). The inhibitors transfer the carbamate moiety onto the active site under release of the phenolic tetrahydroquinazoline scaffolds that themselves act as neuroprotectants. By combination of kinetic data with molecular docking studies, a plausible binding model was probed describing how the tetrahydroquinazoline scaffold guides the carbamate into a close position to the active site. The model explains the influence of the carrier scaffold onto the affinity of an inhibitor just before carbamate transfer. This strategy can be used to utilize the binding mode of other carbamate-based inhibitors.

Full text of DOI:10.1021/acs.jmedchem.5b01674

Article
pubs.acs.org/jmc
Discovery of Highly Selective and Nanomolar Carbamate-Based
Butyrylcholinesterase Inhibitors by Rational Investigation into Their
Inhibition Mode
Edgar Sawatzky,† Sarah Wehle,† Beata Kling,‡ Jan Wendrich,§ Gerhard Bringmann,§
Christoph A. Sotriffer,† Jorg Heilmann,‡ and Michael Decker*,†
̈
†Pharmazeutische und Medizinische Chemie, Institut fur Pharmazie und Lebensmittelchemie, Universitat Wurzburg, Am Hubland,
̈
̈
̈
D-97074 Wurzburg, Germany
̈
‡Lehrstuhl fur Pharmazeutische Biologie, Institut fur Pharmazie, Universitat Regensburg, Universitatsstraße 31, D-93053 Regensburg,
̈
̈
̈
̈
Germany
§Lehrstuhl fur Organische Chemie I, Institut fur Organische Chemie, Universitat Wurzburg, Am Hubland, D-97074 Wurzburg,
̈
̈
̈
̈
̈
Germany
S
* Supporting Information
ABSTRACT: Butyrylcholinesterase (BChE) is a promising
target for the treatment of later stage cognitive decline in
Alzheimer’s disease. A set of pseudo-irreversible BChE
inhibitors with high selectivity over hAChE was synthesized
based on carbamates attached to tetrahydroquinazoline
scaffolds with the 2-thiophenyl compound 2p as the most
potent inhibitor of eqBChE (KC = 14.3 nM) and also of
hBChE (KC = 19.7 nM). The inhibitors transfer the carbamate
moiety onto the active site under release of the phenolic
tetrahydroquinazoline scaffolds that themselves act as neuro-
protectants. By combination of kinetic data with molecular docking studies, a plausible binding model was probed describing how
the tetrahydroquinazoline scaffold guides the carbamate into a close position to the active site. The model explains the influence
of the carrier scaffold onto the affinity of an inhibitor just before carbamate transfer. This strategy can be used to utilize the
binding mode of other carbamate-based inhibitors.
NMDA. Besides, the cholinergic system is affected by the
acetylcholinesterase (AChE) inhibitors rivastigmine, donezepil,
and galantamine. Several clinical studies on these cholinesterase
inhibitors16−18 have proven their positive effectiveness with
regard to cognition, global cognitive functions, and daily
activities, but due to their (only) symptomatic mode of action,
no curative treatment is achieved.
The decline of cognition during AD is attributed to the
progressive loss of cholinergic neurons and therefore to a deficit
of ACh. This deficit is permanently enhanced through the
decomposition of available ACh in the synaptic gap of neuronal
cells by AChE that normally regulates the amount of ACh in
healthy brain. Therefore, inhibition of AChE can compensate the
lack of ACh and in consequence improve cognitive abilities. In
advanced AD the level of AChE drops down to 90% compared to
the healthy brain,19−21 making it impractical as a target for
therapeutic use in later stages of AD. Clinical trials are in striking
accordance with the lack of effectiveness of AChE inhibitors in
these stages of AD. On the other hand, several studies20−23 have
shown increased levels of the isoenzyme butyrylcholinesterase
INTRODUCTION
■
In 2010 the World Alzheimer Report1,2 estimated the worldwide
societal costs of dementia to a value of 604 billion dollars with an
increase of prevalence from 35 million people in 2010 to 115
million people in 2050. The most common form of dementia is
Alzheimer’s disease (AD), a progressive neurodegenerative
disorder of multifactorial nature characterized by the loss of
cognitive abilities through the death of central neuronal cells.
Although the initial pathophysiological reasons in AD are still
not yet fully understood, the pathogenesis is mainly determined
by several specific biochemical changes,3,4 namely, (i)
aggregation of toxic β-amyloid oligomers5,6 followed by
deposition of larger insoluble fibers in advanced amyloid
plaques,7 (ii) hyperphosphorylation of tau proteins and their
aggregation into toxic neurofibrillary tangles,8−10 (iii) oxidative
stress with subsequent cell death,11,12 and (iv) the imbalance of
the two major neurotransmitters acetylcholine (ACh) and
glutamate which are necessary for cognition.13−15 Although
these multifactorial changes provide a broad spectrum of possible
therapeutic targets, there are only four approved drugs on the
market, all targeting the neurotransmitter systems. The non-
competitive antagonist memantine targets the glutamatergic
system through binding at the ionotropic glutamate receptor
Received: October 26, 2015
© XXXX American Chemical Society
A
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(BChE) in AD patients in brain areas that are relevant for
cognition. Although the role of BChE for ACh hydrolysis in
healthy brain is only of minor impact, there is strong evidence
that BChE compensates the loss of neuronal AChE in progressed
AD and therefore takes over the function of AChE. This
hypothesis is supported by an AChE knockout mouse model24 in
which mice did not suffer on cholinergic hyperactivation in the
absence of AChE as ACh hydrolysis is controlled by BChE.
These data are in agreement with other reports18,25−28 showing a
positive correlation between selective BChE inhibition,
improved cognitive performance, and memory acquisition. In a
recent in vivo study with BChE knockout mice it was shown that
in contrast to wild-type mice, lipid peroxidation and ACh levels
were not changed after treatment with amyloid-β25−35 and
learning capacities were attenuated.28 Therefore, inhibition of
BChE might well constitute a therapeutic target for clinical use in
progressed AD, where AChE inhibitors fail.
And further, BChE might be of clinical interest not only for AD
treatment, as it was found to be involved in the regulation of the
serum metabolism in context with obesity,29,30 insulin resistance
and diabetes mellitus,30,31 and cardiovascular risk factors.32,33
Accordingly, for investigation into the role of BChE in these
research fields, the search for highly active and selective inhibitors
and their development are of high importance.
Selective targeting of BChE over AChE is a challenging task, as
sequence comparison of the two isoforms hAChE and hBChE
shows an identity of 49% and a similarity of 66% (see Supporting
Information). Although the catalytic active site (CAS), where
hydrolysis of the neurotransmitter is mediated,34 of both
enzymes is highly conserved, the two enzymes show differences
in the space they provide for a substrate or inhibitor, respectively.
These differences are notably seen in the amino acids forming the
gorge of the binding site and the acyl binding pocket (for a more
detailed comparison see ref 35).
However, selective inhibition of BChE can be achieved by
targeting the CAS with carbamate-based inhibitors. These
inhibitors generally feature a carrier scaffold guiding a carbamate
moiety into the correct position in the enzyme, successively
followed by the transfer of the carbamate moiety onto the serine
of the CAS under release of the carrier scaffold (see Figure 1). In
the literature the development of pseudo-irreversible ChE
inhibitors is in most cases limited to the modification of the
transferable carbamate moiety,36−39 but at the current stage only
very limited information with regard to the binding mode of the
carrier scaffold itself is available (Figure 1).40−43
For rational design of carbamate-based BChE inhibitors there
is a strong requirement for enhanced knowledge of the
interactions between the carrier scaffold and the enzyme. Only
a well bound carrier in the correct position offers the possibility
for carbamate transfer and thus for enzyme inhibition. Concise
knowledge about an appropriate experimental approach will of
course also be useful for the development of other types of
pseudo-irreversible inhibitors. Surprisingly, to our knowledge
such experimental investigations are lacking to a large degree. In
the present study we designed, synthesized, and investigated a set
of bicyclic compounds as selective BChE inhibitors to explore the
binding mode of the carrier scaffold into the active center of
BChE by chemical modifications of the carrier scaffold.
Subsequently and in part parallel to these efforts, a molecular
docking model was established that describes a suitable binding
mode.
Starting point of our investigation were tetrahydroquinazo-
line-derived scaffolds that can be used as templates for the
synthesis of potent AChE and BChE inhibitors.44−48 Further
incorporation of a carbamate moiety into these molecular
templates resulted in highly selective and nanomolar active
BChE inhibitors with the tetracyclic n-heptyl carbamate 1 as the
most potent one (Figure 2a).36,47 This inhibitor binds to the
active site of BChE with its tetrahydroquinazoline scaffold
followed by the pseudo-irreversible transfer of the carbamate
moiety onto the serine of the CAS (“pseudo” because the
carbamate moiety slowly hydrolyzes off from the serine and
enzyme activity is reconstituted). In consequence, this mode of
action leads to release of the heterocyclic carrier scaffold which
itself acts with its p-aminohydroquinoline moiety as an
antioxidant preventing oxidative-stress-induced cell death
(Figure 2b).36,47
Since the tetracyclic moiety of compound 1 is difficult to be
modified comprehensively and systematically in a chemical sense
with regard to SARs, the tetrahydroquinazoline-based scaffold
was “opened” to access compound series 2 and 3 (Figure 2a),
which enables the conduction of SARs. These compounds can be
modified at the aryl residue site, and they can also be further
exploited by introduction of suitable bulky N-site groups. In a
previous study,47 we had already investigated the role of the
carbamate moiety on BChE inhibition by introducing the
carbamate group of several known selective BChE inhibitors, like
eptastigmine bearing a n-heptyl residue, rivastigmine bearing an
ethylmethyl residue, or cymserine bearing a 4-i-Pr-phenyl residue
at the carbamate site. We found that a n-heptyl residue
incorporated into the carrier scaffold leads to the most affine
and BChE-selective inhibitor in this series (compound 1), and
therefore we decided for compound series 2 and 3 to keep this
residue constant for SAR investigations.47
With the target structures synthesized we performed kinetic
studies to evaluate the mode of action and computational studies.
We combined the kinetic data with the molecular docking studies
and established a model for binding of these inhibitors to the
enzyme. This model describes the binding mode of the
tetrahydroquinazoline scaffolds just before carbamate transfer
occurs onto BChE and therefore explains altered affinity and
potency of these inhibitors based on the carrier scaffold. The
scaffolds themselves take only little part in BChE inhibition, but
they are guiding the carbamate moiety into the correct position
Figure 1. Previously described carbamate-based structures investigated
as ChE inhibitors by altering the carrier scaffold40,42,43 (top) and by
altering the carbamate moiety.36−38 Carrier scaffolds are shown in black,
carbamate moieties in green.
B
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Figure 2. (a) Development of carbamate-based inhibitor 1 and modifications toward compounds 2 and 3 for SAR investigations of
tetrahydroquinazoline-based carbamates. (b) Mode of inhibition of BChE by tetrahydroquinazoline-based carbamate 1.47
a
Scheme 1. Synthesis of Aryl and N-Alkyl Substituted Target Compounds 2a−u and 3a−c
a
For substitution pattern of R1 and R2 see Table 1. Reagents and conditions: (i) CO(OCCl3)2, THF, 70 °C, 3.5 h; (ii) MeI, DIPEA, DMAc, 40 °C,
24 h; (iii) R1-NH2 or MeNH3Cl and Et3N, DMF, 40−120 °C, 4−5 h; (iv) R2-PhCHO, AcOH, 70 °C, 1−3 h; (v) LiAlH4, THF, reflux, 1−3 h; (vi)
(4-NO2)PhO(CO)NHn-Hept , NaH, THF, rt, 2 h or n-Hept-NCO, Et3N, rt, 6 h for 2o,p.
toward the active site to enable carbamate transfer and therefore
enzyme inhibition. By application of this general strategy, the
binding mode of carbamate based inhibitors bearing transferable
moieties can easily be investigated also for other biological targets
through the combination of kinetic data and computational
studies. This is possible as the general mechanism for carbamate-
based pseudo-irreversible inhibitors is similar for all targets.
6a−d in moderate yields. These amides were cyclized with either
benzaldehyde or substituted derivatives to yield dihydroquina-
zolinones 7a−u and 8a−c which were reduced with LiAlH4 to
give tetrahydroquinazolines 9a−u and 10a−c, respectively. In
the last step, the n-heptyl carbamate moiety bearing compounds
2a−u and 3a−c were synthesized using 4-nitrophenyl-n-
heptylcarbamate instead of the previously applied commercially
available n-heptyl isocyanate, as its use led in some cases to the
formation of the symmetric n-heptylurea which is difficult to
remove.
Besides the target compounds above, also the fully aromatic
compound 16 was synthesized in six steps (Scheme 2). 5-
Hydroxy-2-nitrobenzaldehyde was O-benzyl protected, then the
nitro moiety was reduced with iron using a catalytical amount of
conc HCl solution toward 12, and finally the amide bond of 13
was formed with benzoyl chloride. The cyclization was achieved
RESULTS AND DISCUSSION
■
Chemistry. Tetrahydroquinazoline substituted carbamates of
the series 2 and 3 were synthesized as outlined in Scheme 1. The
first two steps were described recently.49 Briefly, 3-hydroxyan-
thranilic acid and triphosgene were heated in THF to yield 6-
hydroxyisatoic anhydride 4 quantitatively. Subsequently, the
selective N-methylation yielded compound 5. Free amines or
corresponding hydrochlorides were used to synthesize amides
C
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a
Scheme 2. Synthesis of Quinazoline 16
a
Reagents and conditions: (i) BnBr, K2CO3, DMF, 40 °C, 24 h; (ii) Fe, HCl, EtOH/H2O, reflux, 1 h; (iii) Ph(CO)Cl, Et3N, DCM, rt, 4 h; (iv)
conc NH3, i-PrOH, MW, 90 °C, 6 h; (v) Pd/C, H2, MeOH, 50 °C, 3 h; (vi) (4-NO2)PhO(CO)NHn-Hept , NaH, THF, rt, 2 h.
a
Table 1. Cholinesterase Inhibition of the Synthesized Test Compounds
IC50 [μM] or % inhibition
IC50 [μM] or % inhibition
eqBChE hAChE
moiety
R1 = Me; R2 =
X = H
eqBChE
hAChE
X = (CO)NHn-Hept
d
H
9a
9b
9c
9d
9e
9f
39.9
13.8
2.1
327.0
235.6
242.4
2a
2b
2c
2d
2e
2f
0.106
4%
d
d
e
4-Cl-Ph-
0.115
0.096
0.474
0.231
0.199
0.251
0.875
0.208
0.238
0.044
2.7
24%
39%
48%
3-Cl-Ph-
b
2-Cl-Ph-
56.0
22.6
17.4
92.4
39.5
7.8
60%
d
4-Me-Ph-
3-Me-Ph-
2-Me-Ph-
4-MeO-Ph-
3-MeO-Ph-
2-MeO-Ph-
4-F-Ph-
109.9
437.0
143.4
103.8
9%
d
e
27%
18%
14%
10%
47%
1.61
59%
16%
18%
13%
9g
9h
9i
2g
2h
2i
d
d
e
b
61%
9j
9.9
192.8
143.4
2j
9k
9l
58.9
64.6
2.1
2k
2l
c
d
d
d
d
4-CF3-Ph-
4-pyridyl-
3-pyridyl-
3-thiophenyl-
2-thiophenyl
3-furyl-
nd
9m
9n
9o
9p
9q
9r
9s
9t
242.4
2m
2n
2o
2p
2q
2r
2s
2t
0.723
0.565
0.022
0.014, 0.013
0.083
0.023
36.2
b
70.7
193.7
63.2
196.1
15.3
2.8
61%
b
52%
f
d
225.8
341.7
115.9
341.7
0%
d
12%
3-pyrrolyl-
1-naphthyl-
2-naphthyl-
2,6-dichloro
0.852
e
8%
c
e
16.5
7.1
9%
0.374
0.531
5%
e
9u
13.5
2u
33%
R2 = Ph; R1 =
d
i-Pr-
10a
10b
10c
15
55.8
22.8
14.8
98.1
253.0
279.3
3a
0.021
0.040
0.034
1.8
33%
d
n-Pr-
3b
46%
c
e
benzyl-
16%
3c
17%
b
d
20%
16
22%
physostigmine
0.078
0.032
a
Phenols were incubated for 4.5 min and carbamates for 30 min. Experiments were performed in triplicate at AChE from human erythrocytes and
b−e
b
c
d
e
BChE from equine serum.
available in Supporting Information. Values determined at human BChE.
Percent inhibition at a concentration of 500 μM, 50 μM, 100 μM, 10 μM. Table with confidence intervals is
f
in a modified procedure50 by treatment of 13 with ammonia in a
sealed tube under microwave irradiation to obtain 14. In the last
steps the benzyl protection group was removed under standard
conditions using Pd/C and H2 and the carbamate moiety was
formed with 4-nitrophenyl-n-heptylcarbamate as mentioned
above to obtain the quinazoline-based carbamate 16.
Enzyme Inhibition and SARs. For evaluation and
quantification of enzyme inhibition, Ellman’s spectrophotomet-
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ric method45,51,52 was applied using AChE from human
erythrocytes (EC 3.1.1.7) and BChE from equine serum (EC
3.1.1.8). BChE from horse serum was chosen, as it shows a
sequence identity of 90% and a homology of 94% compared to
the human analog.53 Acetylthiocholine and butyrylthiocholine
were used as substrates for hAChE and eqBChE inhibition,
respectively. In short, the enzymes were preincubated with
different concentrations of the synthesized phenols for 4.5 min or
the corresponding carbamates for 30 min to enable time-
dependent carbamate transfer onto the serine of the active site,
before substrate was added to determine the remaining enzyme
activity.
Only few highly selective and potent BChE inhibitors are
described in the literature (and even fewer with the carbamate
structure; see Figure 1), and only limited information on SARs
regarding the carrier scaffold is reported. Therefore, the aromatic
system in the target compounds was systematically altered. The
unsubstituted target structure 2a was synthesized as the reference
compound and tested for the inhibition of hAChE and eqBChE
(Table 1). This structure is a highly potent inhibitor with an IC50
value of 106 nM on eqBChE and no significant inhibition on
hAChE, proving its eqBChE selectivity. By contrast, its
unsubstituted phenolic analog 9a shows weak inhibition on
hAChE with IC50 = 327 μM and a value of 39.9 μM for eqBChE
inhibition, corresponding to an approximately 400-fold decrease
in inhibition of eqBChE compared to the carbamate 2a. These
results already support the hypothesis that BChE inhibition
mainly occurs by carbamate transfer and that the tetrahydroqui-
nazoline scaffold is only the carrier for the carbamate moiety
lacking pronounced inhibition of the enzyme. Therefore, in the
subsequent SARs analysis only the carbamate based compounds
(right columns of Table 1) will be discussed, as their phenolic
carrier scaffolds showed no pronounced inhibition (left columns
of Table 1) in most cases on neither enzyme.
Optimization of the substitution pattern of the aromatic
moiety was conducted by various approaches starting with the
Topliss tree approach.54,55 Within this approach the hydro-
phobic, electronic, and steric properties of an aromatic system are
systematically altered by the introduction of different sub-
stituents with the aim to improve activity toward a biological
target. In our case the Topliss tree approach guided to the
synthesis of Cl- (2b−d), Me- (2e−g), and MeO- (2h−j)
substituted aromatic systems in ortho, meta, and para positions
and finally to the 4-fluorine substituted system 2k. Interestingly,
none of these groups improved affinity except for the fluorinated
compound 2k with the fluorine atom as the smallest substituent
in this series with IC50(BChE) = 44 nM. Compound 2l bearing a
strong electron withdrawing 4-CF3 group was synthesized
although not being recommended in the classical Topliss tree
approach and showed strongly decreased BChE inhibition by a
factor of 25 (IC50 = 2.7 μM). In addition to the Topliss tree
approach, bioisosteric replacements for the phenyl system were
investigated with pyridines 2m−n and thiophenes 2o−p.56,57
Surprisingly, the pyridyl residues as more polar bioisosteres of
the phenyl residue decreased inhibition and the thiophene
residues as less polar bioisosteres increased inhibition more than
5-fold to IC50(BChE) of 22 nM for 2o and 14 nM for 2p. With
regard to these results, further similarity replacements of the
thiophenyl system were investigated leading to the furyl
compound 2q with decreased activity (IC50(BChE) = 83 nM)
and the pyrrolyl compound 2r (IC50(BChE) = 23 nM) with
BChE inhibition similar to that of the thiophenyl compound 2o.
Finally, the phenyl system was replaced by sterically demanding
aromatic substituents; here the 1-naphthyl and the 2-naphthyl
substituted compounds 2s,t were investigated. Interestingly,
inhibition of BChE by the 1-naphthyl substituted compound 2s
dropped to IC50(BChE) = 36.2 μM (i.e., by a factor of
approximately 300 compared to reference compound 2a). To
further investigate steric influence on the affinity toward BChE,
the 2,6-disubstituted chlorine compound 2u was synthesized
which has to undergo a twist caused by its ortho-substitution
pattern, thus leading to a almost perpendicular arrangement of
the aryl ring and the bicyclic core. This compound shows an IC50
value of 531 nM, which is lower than for all three
monosubstituted chlorine compounds (2b,c,d).
Beyond the mentioned introduction of different substitution
patterns into the aromatic system, also the space close to the
tertiary nitrogen was exploited by the replacement of the methyl
group with other side groups (3a−c) with the best results for the
i-Pr group with IC50(BChE) = 21 nM. For the bicyclic aromatic
quinazoline 16 a strongly decreased inhibition was observed
meaning that either a bent system or a more basic nitrogen within
the core structure is necessary for binding, comparable to many
AChE inhibitors.58−61
As the 2-thiophenyl compound 2p was found to be the most
potent and selective compound toward eqBChE, its potency on
hBChE was also investigated. The IC50 value on hBChE was
determined to 13 nM and is comparable to the one on eqBChE
(14 nM), proving that the results obtained on eqBChE can be
used as an approximation for hBChE.
Even if the IC50 values of the compounds described in Table 1
(a respective table with confidence intervals is provided in the
Supporting Information) are a useful first hint to establish a
binding model for these inhibitors, the inhibition mode of
pseudo-irreversible inhibitors is much more complex. Therefore,
a more detailed view into the kinetic mode of action is necessary
to determine the influence of substituents on enzyme inhibition.
At this point, it should also be mentioned that the inhibition
curves of the weakest active compounds 2l, 2s, and 16 show a Hill
slope of around 0.5 (data not shown). These slopes are
remarkably different from those of all other inhibitors (slope of
≥1). This fact might be a strong hint for a different mode of
action for these three inhibitors.
Kinetic Investigations into the Mode of BChE Inhib-
ition. The mechanism of pseudo-irreversible enzyme inhibition
by carbamates can be described by three pivotal steps as shown in
Figure 3. First, the enzyme E forms a reversible enzyme−
Figure 3. Pseudo-irreversible inhibition of an enzyme E by carbamates
PC. For description see the main text.
inhibitor complex (EPC) with the carbamate-based inhibitor PC
(PC for phenol carbamate) comparable to reversible competitive
inhibitors. This reversible inhibition is quantified by KC which
describes the apparent affinity between the enzyme and the
inhibitor in an equilibrium state. In the second step, the
carbamate moiety itself is transferred onto the enzyme with
release of the carrier scaffold P (P for phenol), resulting in the
carbamoylated enzyme E−C. The constant k3 represents the
carbamolyation rate of the inhibitor from the reversible complex
(EPC) to the carbamoylated enzyme E−C. The last step is the
recovery of the enzyme through slow hydrolysis of E−C and
E
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a
Table 2. Kinetic Values for Carbamoylation and Decarbamoylation on eqBChE for Selected Compounds
a
Asterisks indicate the following: (∗) Values are measured for human BChE. (∗∗) Values are calculated under the assumption of a pseudo-
irreversible inhibition; they have to be carefully rated, as the inhibition mode might be different from that of the other inhibitors.
KC
k3 [PC]
1
kobs
1
1
k3
release of the carbamate C′ with k4 as the decarbamoylation rate
constant. Normally, k4 is significantly lower than k3 due to the fact
that carbamoylation occurs much faster than decarbamoylation
because of the high stability of E−C toward hydrolysis.
=
+
(4)
On the basis of this method, we investigated kinetic parameters
(Table 2) of a selected set of compounds including the reference
compound 2a and the highly potent inhibitors 2p and 2k as well
as those compounds that showed differences in the Hill slopes of
their IC50 curves compared to the other inhibitors (2l, 2s, and
16). The alkaloid physostigmine served as the external reference
compound. In this series the thiophene compound 2p was found
as the most affine inhibitor with KC = 14.3 nM and the one with
the highest carbamoylation efficacy (k3/KC = 9.79 μM−1 min−1).
The fluorine substituted compound 2k showed a comparably
high affinity and carbamoylation efficacy. Interestingly, the
twisted 2,6-dichloro compound 2u displayed the lowest affinity
to the enzyme with KC = 4118 nM. This finding supports the
hypothesis that a twist of the aromatic residue leads to decreased
affinity toward the binding pocket (for details see Computational
Studies). But even with this twist the carbamate is transferred to
the serine. The only major exception for IC50 and KC correlation
was measured for the 1-naphthyl compound 2s which showed a
high affinity to the enzyme with KC = 15.6 nM but an IC50 value
in the micromolar range. Similar to the curves for determination
of IC50 values, the shape of the obtained time-dependent
inhibition curves was much less steep for compounds 2l, 2s, and
16 compared to all other inhibitors, indicating a different binding
interaction.
The values of KC and k3 can be quantified applying the method
described by Hosie et al.62 and Feaster et al.63 Following this
approach, the carbamate-mediated inhibition described in Figure
3 is known to undergo apparent first order kinetics:
obst
A = A0 e−k + A∞
(1)
in which A is the activity of the enzyme at a specific time t, A0 the
activity at t = 0, A∞ the activity at t = ∞ and kobs represents the
apparent first order rate constant. Plotting of kobs against the
inhibitor concentration [PC] yields a hyperbolic curve described
by
k3[PC]
kobs
=
[S]
KC 1 +
+ [PC]
(
)
KM
(2)
Rearrangement of eq 2 into a double reciprocal form with 1/kobs
as a function of 1/[PC] results in a linear plot:
[S]
KM
KC 1 +
(
)
1
kobs
1
1
k3
=
+
k3
[PC]
(3)
in which k3 can be determined from the y-intercept and KC from
the slope. From these data also the second order rate constant k3/
KC can be calculated describing the overall carbamoylation
efficacy to evaluate differences in inhibitory potency. For the
case, that substrate is not added from the beginning of the
preincubation between inhibitor and enzyme, [S] becomes equal
to zero because substrate and inhibitor are not competing for the
binding pocket, and the substrate only adopts a function as
reporter for remaining enzyme activity. In this case, eq 3 is
simplified to
For all compounds high carbamoylation rate constants k3 were
observed with similar values between 0.13 and 0.24 min−1;
exceptions were, as mentioned, the 1-naphthyl substituted
derivative 2s and also the 4-CF3 analog 2l. Apart from the
exceptions, it can be assumed that similar values for k3 have their
origin in a similar orientation of the carbamate moiety with
respect to the serine of the CAS, supporting a conserved binding
mode of these inhibitors (see Computational Studies).
Enzyme recovery was measured exemplarily for the reference
compound 2a and the thiophenyl analog 2p. For this purpose,
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Figure 4. (a) Representation of the preferred binding mode for 2p as R-enantiomer (light green) and (b) S-enantiomer (light blue) in the BChE binding
site. Residues of the acyl pocket are shown in green, the oxyanion hole is in yellow, the CAS is in orange, the choline binding site (Trp82) is in turquoise,
and parts of the side cavity are in pink. Distances in Å are given in italics. The figure was created with Pymol.72
the derivatives were incubated for 1 h with the enzyme at a
concentration where complete inhibition was achieved and then
successively 1000-fold diluted to a concentration where no
carbamoylation was observed anymore. The recovery of the
enzyme through hydrolysis of the carbamate residue was
monitored after certain time points after dilution, showing first
order kinetics with k4 = 0.14 h−1 or a half-life for the
carbamoylated enzyme of approximately 5 h. This value is
identical for both compounds, as in both cases an n-heptyl
carbamate is cleaved from the serine.
Taken together, the kinetic parameters reveal that (a) most
inhibitors follow a conserved inhibition mode indicated by a
similar carbamoylation rate constant k3 and (b) they are only
differing in their affinity expressed as KC. It was observed that the
exceptions 2l, 2s, and 16 probably do not adhere to this
conserved binding mode, as shown by significant differences in
the slope of their IC50 curves and kinetic parameters. Therefore,
the results for these three inhibitors should be carefully rated, as
the kinetic parameters were determined under the assumption of
a pseudo-irreversible inhibition model, although the exact
inhibition mode might in fact be different. These observations
need to be further investigated.
We further investigated the most potent compound 2p by
performing enantiomeric resolution with HPLC on a chiral phase
and chiropical analysis by CD spectroscopy. Although both
enantiomers could be obtained after preparative chiral HPLC in
their pure form (revealed by CD spectroscopy), they underwent
rapid isomerization back into the racemic mixture (conditions
and chromatograms are provided in the Supporting Informa-
tion). It seems that racemization at the chiral center occurs by
opening of the ring system into an imine structure which in turn
reacts nonstereoselectively back to the ring system, thereby
producing the racemate. Even though resolution of enantiomers
was achieved, it can be assumed that isomerization of pure
enantiomers into the racemate will occur at least in aqueous
media during the biological testing of the compound. Assuming
that the postulated binding modes (see Computational Studies)
are correct, conformational energies and intermolecular scores of
the respective binding poses suggest that in general the S-
enantiomers might be more active than the R-enantiomers (see
Supporting Information for further details).
inhibitory activity on hBChE (as reported above for the IC50
values) compared to eqBChE with slightly decreased affinity (Kc
= 19.7 nM), a higher carbamoylation rate (k3 = 0.32 min−1), and a
higher carbamoylation efficacy (k3/KC = 16.24 μM−1 min−1).
With regard to the high similarity in inhibition and kinetic values
of 2p on hBChE and eqBChE, it can be assumed that all
compounds synthesized will bind on hBChE and eqBChE in a
similar manner.
Computational Studies. Modeling studies were performed
in a combined docking and minimization approach with
GOLD64 and MiniMuDS.65 For this purpose, the human
BChE crystal structure 1P0I was used,66 which possesses the
highest resolution (2.0 Å) of BChE structures in the PDB.67
Docking studies of reversible ligands of BChE had also been
carried out by other groups.43,68−70
As the inhibitors were tested as racemic mixtures in the assay,
both enantiomeric forms were built up and used for docking. All
ligands of the series examined by detailed kinetic measurements
(Table 2) as well as ligand 1 were used for docking studies.
For pseudo-irreversible inhibition, three different states may
be addressed with classical docking methods: (1) A covalent
docking can be carried out representing the carbamoylated serine
(corresponding to E−C in Figure 3). As the carbamate stays the
same throughout the data set, this approach was not performed
here. (2) The tetrahedral transition state that occurs during the
carbamoylation can be docked covalently to the serine. This
method had been chosen by Carolan et al.43 However, in this case
a generally applicable interpretation for a set of compounds in
light of the inhibition and kinetic data was not possible.
Therefore, (3) the inhibitor structures were docked non-
covalently to BChE to mimic reversible inhibition by formation
of the initial noncovalent complex ((EPC) in Figure 3). These
docking data can be analyzed in relation to the experimentally
determined KC values.
Docking poses of inhibitor 1 showed the same binding mode
for both enantiomeric forms on the top (S-enantiomer) and the
second-best rank (R-enantiomer) (see Experimental Section for
a detailed description of pose selection). These poses were taken
as reference poses to model the binding mode of the other
inhibitor structures by deletion and addition of the correspond-
ing functional groups and subsequent local minimization in the
binding site using MiniMuDS. The methyl group on the benzylic
nitrogen can adopt a pseudo-equatorial or pseudo-axial
We also investigated the kinetic properties of 2p on human
BChE (Table 2). We found this compound to have similar
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orientation. The herein displayed structures show the methyl
group in the pseudo-equatorial position for the R-enantiomer
and in the pseudo-axial position for the S-enantiomer, as these
orientations were found to be preferred.
formed by the 1-naphthyl group (2s), which might overall lead to
the similarly favorable affinity as observed for 2p. However,
because of the different kinetic characteristics described above,
caution is warranted in interpreting the results for compound 2s
(as well as for 2l). This also applies to the achiral compound 16,
which served as a test compound to confirm the binding mode
without the uncertainty of enantiomeric forms. However,
docking showed that the planar shape of the ligand cannot be
accommodated in the common binding mode adopted by the
other inhibitors. This could indicate a different mode of action, as
suggested also by the kinetic data.
Antioxidant Capacity, Neuroprotection, and Neuro-
toxicity. Neuronal cell death induced by reactive oxygen species
(ROS) is prevented by antioxidants that contain radical
scavenging structures. The determination of these antioxidant
capacities can be achieved by the oxygen radical absorbance
capacity (ORAC) assay in which the ability of an antioxidant to
reduce the amount of induced peroxylradicals is determined.
This test therefore describes direct physiochemical radical
scavenging properties of compounds but does not necessarily
directly relate to results from cell-based methods,36,47 although
often claimed in the literature. Results in this assay are expressed
in relation to the radical scavenging properties of 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (also called Tro-
lox, a water-soluble vitamin E analog) yielding the Trolox
equivalents (TE) unit.
In this work, the synthesized carbamates release the
corresponding carrier phenols after carbamate transfer onto
BChE (compare Figure 2b),36,47 which can act as potent radical
scavengers with their p-aminohydroquinone moiety. Therefore,
we chose pairs of compounds (carbamates and corresponding
phenols) to investigate and correlate their antioxdative proper-
ties, including the unsubstituted compound 2a, the thiophene
bearing compound 2p as a highly potent BChE inhibitor, the 4-
MeO bearing compound 2h as a relatively weak BChE inhibitors,
and the N-benzyl containing compound 3c as well as their
phenolic analogs 9a,p,h and 10c. The results for the ORAC assay
are summarized in Table 3. All of the tested carbamates show by
The binding mode of the compounds is characterized by the
following features: The n-heptyl chain is placed over the acyl
binding site (mainly formed by Trp231, Leu286, and Val288).
The carbamate moiety is positioned near His438 and Ser198
(together with Glu325 the CAS comprising amino acids),
showing distances of the carbamate carbon to the serine oxygen
of 2.9−3.0 Å and 3.6−4.1 Å for the R- and S-enantiomers,
respectively. The oxygen of the carbamate moiety is oriented
toward Gly117-NH in hydrogen bond distances of 2.6−2.9 Å and
2.6−3.0 Å for the R- and S-enantiomers, respectively. Gly116,
Gly117, and Ala119 form the oxyanion hole. These residues are
important in stabilizing the negative charge formed at the
carbonyl oxygen during substrate hydrolysis.71
The tetrahydroquinazoline is placed “above” the oxyanion
hole, and the aromatic ring is in π−π interaction distances of 3.4
and 3.8 Å to His438 (measured for S- and R-2p, respectively).
The aryl rings (the substituents R2 of Table 2) are placed in a
cavity “below” Trp82, formed by Asn68, Asp70, and Thr120
(colored in pink in Figure 4).
The ligand poses derived from ligand 1 show the aryl ring “in
plane” with the tetrahydroquinazoline (more precisely, the bond
between the aryl ring and the tetrahydroquinazoline shows a
dihedral angle of approximately 0°). However, a twisted
conformation with a ∼90° dihedral, similar as in ortho-
substituted biaryls,73 would also be possible. To test whether
the “in plane” or “twisted” orientation of the aryl ring is favored,
compound 2u, a 2,6-dichloro derivative, was therefore
synthesized. Due to steric hindrance of the ortho-chloro
substituents and the methyl groups on the quinazoline core,
the aryl ring needs to adopt the twisted conformation. As the
affinity of this compound (KC = 4118 nM) is the lowest in the
tested series, the twisted conformation is apparently not
favorable for high-affinity binders. As the modeled ligand pose
shows, without a displacement from the generally preferred
binding mode, this conformation would lead to clashes with the
protein. Thus, except for 2u, the aryl ring can be assumed to be
“in plane” with the tetrahydroquinazoline core as shown in
Figure 4.
Table 3. Antioxidant Capacities of Target Compounds
Expressed as Trolox Equivalents (TE)
Although a quantitative correlation between the docking
scores and the activity of the compounds cannot be expected
(primarily due to the well-known accuracy limitations of scoring
functions and the uncertain contribution of the enantiomers to
the experimentally measured affinities of the racemic com-
pounds), some differences in affinity (in terms of KC) can
qualitatively be explained by the structural differences of the
inhibitors in this binding model. Compared to the thiophene
derivative 2p as the smallest compound, the significantly weaker
affinity of compounds with sterically demanding para-substitu-
ents (2h, 2l) is in line with the steric restrictions imposed by the
side cavity (formed by Ile69, Asn83, and Pro84) to which these
para-substituents point. Especially the para-CF3 compound 2l
leads to clashes with the protein when forcing it into a similar
binding mode as identified for thiophene derivative 2p (see
Figure S5 in the Supporting Information). This is in agreement
with the different kinetic profile and supports the hypothesis of a
different binding mode for this compound. Although not forming
particularly favorable interactions, meta-substituents (cf. 2c, 2i)
are better tolerated because they point “upward” to a more
accessible area. Additional interactions in this region can be
themselves radical scavenging properties with antioxidant
capacities between 0.2 and 0.5 TE. A remarkable difference can
be observed for the N-benzyl bearing compound 3c with an
antioxidant capacity of 1.7 which might be explained by the
possibility to form stable benzyl radicals, thereby acting as a
strong antioxidant exceeding Trolox already in its carbamate
form.
As expected, the antioxidant capacities of the hydroxy
compounds 9 and 10 were significantly higher than those of
their corresponding carbamates and of the positive control and
reference Trolox, itself ranging from 3.0 to 3.9 TE. The N-benzyl
bearing tetrahydroquinazoline 10c shows the highest antioxidant
capacity with 3.9 TE in this series, as it might be able to trap
radicals with its p-aminohydroquinone core and additionally with
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Figure 5. (a) Neurotoxicity tests for target compounds on HT-22 cells. (b) Neuroprotection tests against glutamate induced oxidative stress for target
compounds on HT-22 cells.
the benzyl group as mentioned before for the carbamate 10c. In
summary, it was observed that the carbamates by themselves
show already antioxidant capacities, albeit only moderate ones,
and their corresponding phenols released after carbamate
transfer onto the active site of BChE represent highly potent
radical scavengers with 3- to 4-fold higher antioxidant capacities
than Trolox.
to determine self-toxicity toward the cells (Figure 5). In general,
all tested hydroxy compounds (series 9 and 10) showed similar
effects on HT-22 cells with no or only little toxicity at
concentrations of 1 and 5 μM (Figure 5a). The carbamates
(series 2 and 3) are even less neurotoxic compared to the
corresponding phenols. The cell viability was maintained above
80% for all carbamates up to a concentration of 10 μM.
Interestingly the N-benzyl carbamate 3c showed no significant
decrease in cell viability even at the highest concentration of 25
μM.
When glutamate was added to induce oxidative stress (Figure
5b), the phenols very effectively counteracted glutamate-induced
neurotoxicity already at a concentration of 5 μM as the cell
viability was maintained. Decreasing viability at higher
concentrations relates to neurotoxic effects for compounds like
9h and 10c but is only significant at 25 μM. Furthermore, the N-
benzyl containing phenol 10c maintained cell viability at already
1 μM, proving its high neuroprotectivity already at low
concentrations.
It is remarkable that also the carbamate-based structures
(series 2 and 3) showed at concentrations above 5 μM that cell
viability is comparable to the one of the reference compound
quercetin at 25 μM, proving their neuroprotective effect. This
finding is quite of interest, since hydrolysis to the phenol is not
necessary for cell-based neuroprotective effects.
However, neuroprotection assays suffer from the drawback
that the concentrations needed to be applied in such assays are
much higher than what is expected for a ligand/inhibitor to be
effective in vivo. This is because respective assays are not
As mentioned above, evaluation of compounds in a cellular
neuronal assay provides additional information with respect to
therapeutic applications, since it enables assessment of how well
physicochemical properties translate into biological settings. In
fact correlating both methods is most meaningful. Therefore, to
evaluate the ability of the target compounds to prevent
oxycytotic cell death after glutamate exposure, the compounds
were applied onto a model system with the glutamate sensitive
murine hippocampal HT-22 cells which lack an ionotropic
glutamate receptor. These cells contain a glutamate−cysteine
antiporter that is necessary for the cysteine uptake from the
extracellular side under physiological conditions and at the same
time transports glutamate out of the cytosol.74 When HT-22 cells
are exposed to high glutamate concentrations, this transporter is
blocked by extracellular glutamate. In consequence, no cysteine
is uptaken for glutathione (GSH) synthesis, the antioxidant
protecting from intracellular ROS induced cell death.75−77
The same compounds evaluated in the ORAC assay were
tested for neuroprotection on HT-22 cell line with the natural
product quercetin as the antioxidant reference. Cells were
incubated with the compounds in different concentrations either
with 5 mM glutamate to induce oxycytosis or without glutamate
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with iodine. Nuclear magnetic resonance spectra were performed with a
Bruker AV-400 NMR instrument (Bruker, Karlsruhe, Germany) in
DMSO-d6 or CDCl3. Chemical shifts are expressed in ppm relative to
sensitive enough and the data therefore have to be correlated to
the positive control (in our case Trolox in the ORAC assay and
quercetin in the HT-22 assay). Compared to quercetin (applied
at 25 μM) the compounds are more active even at lower
concentrations than for quercetin.
In summary, the compounds described are not only very
potent and selective BChE inhibitors but also act as highly active
neuroprotectants both by themselves and after release of the
phenolic carrier.
CDCl3 or DMSO-d6 (7.26/2.50 and 77.16/39.52 ppm for 1H and 13
C
NMR, respectively). Melting points were determined in open capillaries
on a Buchi B-540 without any further correction except for the phenolic
̈
tetrahydroquinazolines, as they undergo rapid decomposition during
heating. For purity of target compounds, analytic HPLC was performed
on a system from Shimadzu equipped with a DGU-20A3R controller,
LC20AB liquid chromatograph, and a SPD-20A UV/vis detector.
Stationary phase was a Synergi 4U fusion-RP (150 mm × 4.6 mm)
column (Phenomenex, Aschaffenburg, Germany). As mobile phase,
water (phase A) and MeOH (phase B) were used with 1 mL/min.
(Method A: conc B, 5% → 90% from 0 to 8 min; 90% from 8 to 13 min;
90% → 5% from 13 to 15 min; 5% from 15 to 18 min. Method B: conc B,
10% → 80% from 0 to 8 min; 80% from 8 to 13 min; 80% → 10% from
13 to 15 min; 10% from 15 to 18 min. Method C: conc B, 30% → 90%
from 0 to 8 min; 90% from 8 to 13 min; 90% → 30% from 13 to 15 min;
30% from 15 to 18 min) Target compounds were ≥95% pure. Purities of
9r and 10a were determined by CHN analysis on a Vario MICRO
CUBE (Elementar Analysesysteme GmbH, Hanau, Germany) because
of their low stability on HPLC. ESI mass spectral data were acquired on a
Simadzu LCMS-2020.
CONCLUSION
■
In this report we identified a set of pseudo-irreversible
carbamate-based inhibitors of BChE (2a−u, 3a−c, and 16) by
applying rational approaches to conduct SARs of the
tetrahydroquinazoline carrier scaffold. Although the correspond-
ing phenolic tetrahydroquinazolines 9a−u, 10a−c, and 15
showed BChE inhibition in the micromolar range lacking
significant selectivity over hAChE, they are the source of the
modulated affinity of the carbamate series. In this series we found
thiophene compound 2p as the most potent inhibitor with regard
to both affinity (KC = 14.3 nM) and carbamoylation efficacy (k3/
KC = 9.79 μM−1 min−1) toward the enzyme. To our knowledge,
this compound represents by far one of the most active and
selective inhibitors of BChE described to date.
General Reactions. General Amide Formation Procedure
(GP1). 6-Hydroxy-1-methyl-2H-benzo[d][1,3]oxazine-2,4(1H)-dione
5 was dissolved in dry DMF (30 mL) and treated with the corresponding
amine (5 equiv) or a mixture of the amine hydrochloride (5 equiv) and
triethylamine (5 equiv). The mixture was heated to 40−120 °C
(depending on the amine) for 4−5 h. For workup, the mixture was
poured into water (100 mL) and the product was extracted with ethyl
acetate (5 × 100 mL). The combined organic layers were washed with
brine (50 mL), dried over Na2SO4, and evaporated to dryness. The
crude product was purified by column chromatography to obtain 5-
hydroxy-N-methyl-2-(alkylamino)benzamides 6a−d.
General Cyclization Procedure (GP2). 5-Hydroxy-N-methyl-2-
(alkylamino)benzamides 6a−d were dissolved in glacial acetic acid (20
mL). The mixture was treated with the corresponding aldehyde (1.2
equiv) and heated to 70 °C for 1−3 h. Then the mixture was poured
onto ice−water (20 mL), basified with a NaOH solution (2 M) and the
pH was adjusted to 9 with sat. NH4Cl solution. The product was
extracted with ethyl acetate (3 × 40 mL), the combined organic layers
were washed with brine (30 mL), dried over Na2SO4, and the solvent
was evaporated under reduced pressure. The crude product was either
recrystallized or purified by column chromatography to obtain
dihydroquinazolinones 7a−u and 8a−c.
General Reduction Procedure (GP3). Dihydroquinazolinones
7a−u and 8a−c were dissolved in dry THF (30 mL) at 0 °C, and LiAlH4
(4 equiv) was added. The mixture was allowed to reach rt and was then
heated to reflux temperature for 1−3 h. After cooling to rt, the mixture
was poured into ice−water (50 mL) followed by the addition of
saturated NH4Cl solution until pH = 9. The aqueous phase was then
extracted with ethyl acetate (3 × 80 mL). The combined organic layers
were washed with brine (50 mL), dried over Na2SO4, and the solvent
was evaporated under reduced pressure. The crude product was purified
by column chromatography to obtain the corresponding tetrahydro-
quinazolines 9a−u and 10a−c.
By combining kinetic investigations with computational
studies, we established a model for binding of the tetrahy-
droquinazoline scaffold and therefore for modulated affinity of
the inhibitors toward the enzyme. In the computational studies,
the heterocyclic inhibitors place their carbamate moieties in close
proximity to the serine of the CAS. This accounts for appropriate
preorientation for the subsequent transfer of the carbamate.
Substituents on the aryl ring that are responsible for affinity of
this set of compounds bind to a side cavity of BChE’s active site
located next to the entrance of the binding site. Both kinetic data
and computational studies support a conserved binding mode for
the described inhibitors, with the exception of the 4-CF3-
substituted compounds 2l, 1-naphthyl compound 2s, and the
planar quinazoline 16. Combining computational studies with
kinetic values can be considered as a general approach to
investigate the binding mode of carbamate-based pseudo-
irreversible inhibitors, as the binding mode of these inhibitors
is similar.
Neuroprotective abilities for a subset of carbamates and their
corresponding phenols were determined in an ORAC assay and a
neuronal cell-based one and revealed high radical scavenging
properties of the phenolic heterocycle carrier scaffold formed
after transfer of the carbamate moiety to the enzyme.
Pronounced neuroprotection comparable to quercetin was
seen when intracellular oxidative stress was triggered through
glutamate exposure in neuronal cells.
In addition to the development of improved and neuro-
protective BChE inhibitors, our results might be of relevance for
a general methodology to be applied for development of other
pseudo-irreversible enzyme inhibitors.
General Carbamate Formation Procedure (GP4). A solution of
tetrahydroquinazolines 9a−n, 9q−u, and 10a−c in dry THF (5 mL) was
treated with NaH in paraffin oil (60%, 1.2 equiv). The mixture was
stirred until the formation of gas stopped. Then, a solution of 4-
nitrophenyl-n-heptylcarbamate (1.2 equiv) in dry THF (3 mL) was
added at once. The mixture was stirred for 2 h. For workup, the mixture
was diluted with ethyl acetate (30 mL), washed with water (10 mL), and
washed with brine (10 mL). The organic phase was dried over Na2SO4,
and the solvent was evaporated under reduced pressure. The crude
product was purified by column chromatography to obtain the
corresponding n-heptylcarbamate 2a−n, 2q−u, and 3a−c.
EXPERIMENTAL SECTION
■
Chemistry. General. Common reagents and solvents were obtained
from commercial suppliers and used without any further purification.
Tetrahydrofuran (THF) was distilled from sodium/benzophenone
under argon atmosphere. Reaction progress was monitored using
analytical thin layer chromatography (TLC) on precoated silica gel
Synthesis and Spectral Data. Syntheses of 4 and 5 were
GF254 plates (Macherey-Nagel GmbH & Co. KG, Duren, Germany),
̈
and spots were detected under UV light (254 nm) or through staining
performed as described.49 The synthesis of target compound 2a is
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exemplarily given in the following. For synthetic procedures and spectral
data of 2b−u and 3a−c see Supporting Information.
2-Amino-5-(benzyloxy)benzaldehyde 12. 5-(Benzyloxy)-2-ni-
trobenzaldehyde 11 (2.83 g, 10.44 mmol, 1 equiv) was dissolved in
EtOH/H2O (4:1, 50 mL) and treated with elementary iron (5.85 g,
104.4 mmol, 10 equiv) and a catalytic amount of conc HCl solution (50
μL). The mixture was heated to reflux temperature for 1 h. The product
was then extracted with ethyl acetate (3 × 100 mL), and the combined
organic layers were washed with brine (50 mL) and dried over Na2SO4.
After removal of the solvent under reduced pressure, 2-amino-5-
(benzyloxy)benzaldehyde 12 (1.44 g, 61%) was obtained as a yellow
solid; mp 83 °C. 1H NMR (400 MHz, CDCl3): δ = 9.74 (d, J = 0.5 Hz,
1H), 7.38−7.21 (m, 5H), 6.99 (dd, J = 8.7, 2.9 Hz, 1H), 6.96 (d, J = 2.8
5-Hydroxy-N-methyl-2-(methylamino)benzamide 6a. Accord-
ing to GP1, 6-hydroxy-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione 5
(500 mg, 2.59 mmol, 1 equiv), methylamine hydrochloride (874 mg,
12.95 mmol, 5 equiv), and triethylamine (1.79 mL, 12.95 mmol, 5 equiv)
were used to obtain 5-hydroxy-N-methyl-2-(methylamino)benzamide
6a (321 mg, 69%) after column chromatography (petroleum ether/
EtOAc = 1:4) as a yellow-brown solid; mp 164−166 °C. 1H NMR (400
MHz, DMSO-d6): δ = 8.59 (s, OH), 8.15 (q, J = 4.2 Hz, NH), 6.92 (d, J
= 2.8 Hz, 1H), 6.85 (q, J = 4.3 Hz, NH), 6.80 (dd, J = 8.8, 2.8 Hz, 1H),
6.49 (d, J = 8.8 Hz, 1H), 2.71 (d, J = 1.3 Hz, 3H), 2.70 (d, J = 2.0 Hz, 3H)
ppm. 13C NMR (101 MHz, DMSO-d6): δ = 169.4, 146.5, 143.3, 119.6,
116.7, 114.6, 111.6, 30.0, 26.0 ppm. ESI-MS: m/z calcd, 180.09; found,
181.2 [M + H]+.
Hz, 1H), 6.54 (d, J = 8.7 Hz, 1H), 5.77 (s, NH2), 4.95 (s, 2H) ppm. 13
C
NMR (101 MHz, CDCl3): δ = 193.5, 149.9, 145.0, 137.0, 128.6 (2C),
128.1, 127.5 (2C), 125.5, 118.7, 118.5, 117.6, 71.1 ppm. ESI-MS: m/z
calcd, 227.09; found, 228.10 [M + H]+.
N-(4-(Benzyloxy)-2-formylphenyl)benzamide 13. A mixture of
2-amino-5-(benzyloxy)benzaldehyde 12 (1022 mg, 4.49 mmol, 1 equiv)
and triethylamine (934 μL, 4.63 mmol, 1.2 equiv) was dissolved in DCM
(50 mL) and treated dropwise with benzoyl chloride (784 μL, 6.75
mmol, 1.5 equiv). After stirring for 4 h, the reaction mixture was diluted
with DCM (100 mL) and washed with 2 M HCl (50 mL), sat. NaHCO3
(50 mL), and brine (50 mL). The solvent was removed under reduced
pressure and the crude product was purified by column chromatography
(DCM/petroleum ether = 1:1) to obtain N-(4-(benzyloxy)-2-
formylphenyl)benzamide 13 (986 mg, 66%) as a yellow solid; mp
131−133 °C. 1H NMR (400 MHz, CDCl3): δ = 11.84 (s, NH), 9.94 (d, J
= 0.6 Hz, 1H), 8.92 (d, J = 9.1 Hz, 1H), 8.12−8.03 (m, 2H), 7.60−7.50
(m, 3H), 7.48−7.38 (m, 4H), 7.38−7.34 (m, 1H), 7.32 (dd, J = 9.0, 3.0
Hz, 1H), 7.29 (d, J = 2.9 Hz, 1H), 5.14 (s, 2H) ppm. 13C NMR (101
MHz, CDCl3): δ = 195.5, 165.8, 154.3, 136.3, 135.2, 134.5, 132.0, 128.9
(2C), 128.7 (2C), 128.3, 127.5 (2C), 127.4 (2C), 123.3, 122.9, 121.8,
121.1, 70.7 ppm. ESI-MS: m/z calcd, 331.12; found, 332.15 [M + H]+.
6-(Benzyloxy)-2-phenylquinazoline 14. According to the
literature,50 N-(4-(benzyloxy)-2-formylphenyl)benzamide 13 (980
mg, 2.96 mmol, 1 equiv) was suspended in i-PrOH/conc ammonia
(1:1, 30 mL) and heated to 90 °C for 6 h under microwave irradiation.
Then the product was extracted with ethyl acetate (2 × 100 mL), the
combined organic layers were washed with brine (50 mL) and dried over
Na2SO4. After removal of the solvent, 6-(benzyloxy)-2-phenylquinazo-
line 14 (889 mg, 96%) was obtained as a beige solid; mp 119−120 °C.
1H NMR (400 MHz, CDCl3): δ = 9.38 (d, J = 0.7 Hz, 1H), 8.63−8.56
(m, 2H), 8.04 (d, J = 9.2 Hz, 1H), 7.66 (dd, J = 9.2, 2.8 Hz, 1H), 7.58−
7.51 (m, 5H), 7.51−7.43 (m, 2H), 7.43−7.36 (m, 1H), 7.27 (d, J = 2.8
Hz, 1H), 5.25 (s, 2H) ppm. 13C NMR (101 MHz, CDCl3): δ = 159.6,
158.9, 157.3, 147.1, 138.2, 136.1, 130.3, 130.2, 128.8 (2C), 128.6 (2C),
128.4, 128.2 (2C), 127.6 (2C), 127.5, 124.5, 105.3, 70.6 ppm. ESI-MS:
m/z calcd, 312.13; found, 313.20 [M + H]+.
2-Phenylquinazolin-6-ol 15. 6-(Benzyloxy)-2-phenylquinazoline
14 (870 mg, 2.79 mmol, 1 equiv) was dissolved in MeOH (50 mL), and
Pd/C (87 mg, 10 wt %) was added. The atmosphere of the reaction
vessel was removed under reduced pressure, and then the vessel was
purged with hydrogen. The mixture was heated to 50 °C for 3 h. Then
the mixture was filtered over a short silica column to obtain 2-
phenylquinazolin-6-ol 15 (435 mg, 70%) as a white solid; mp 233−235
°C. 1H NMR (400 MHz, DMSO-d6): δ = 10.44 (br, OH), 9.50 (d, J =
0.7 Hz, 1H), 8.58−8.43 (m, 2H), 7.95 (d, J = 9.1 Hz, 1H), 7.61−7.48 (m,
4H), 7.32 (d, J = 2.6 Hz, 1H) ppm. 13C NMR (101 MHz, DMSO-d6): δ
= 159.7, 157.7, 156.9, 145.4, 138.3, 130.6, 130.1, 129.1 (2C), 128.1 (2C),
127.7, 125.2, 108.2 ppm. ESI-MS: m/z calcd, 222.08; found, 223.15 [M
+ H]+. HPLC (method A): 100%.
6-Hydroxy-1,3-dimethyl-2-phenyl-2,3-dihydroquinazolin-
4(1H)-one 7a. According to GP2, 5-hydroxy-N-methyl-2-
(methylamino)benzamide 6a (170 mg, 0.94 mmol, 1 equiv) and
benzaldehyde (114 μL, 1.13 mmol, 1.2 equiv) were used to obtain 6-
hydroxy-1,3-dimethyl-2-phenyl-2,3-dihydroquinazolin-4(1H)-one 7a
(167 mg, 66%) after column chromatography (petroleum ether/
EtOAc = 1:2) as a yellow solid; mp 205−207 °C. 1H NMR (400 MHz,
DMSO-d6): δ = 8.99 (s, OH), 7.35−7.27 (m, 3H), 7.22 (d, J = 2.9 Hz,
1H), 7.19−7.09 (m, 2H), 6.80 (dd, J = 8.7, 3.0 Hz, 1H), 6.46 (d, J = 8.7
Hz, 1H), 5.67 (s, 1H), 2.90 (s, 3H), 2.73 (s, 3H) ppm. 13C NMR (101
MHz, DMSO-d6): δ = 161.7, 149.6, 139.4, 136.8, 128.6, 128.5 (2C),
126.2 (2C), 120.9, 117.6, 114.1, 113.4, 78.6, 35.7, 32.1 ppm. ESI-MS: m/
z calcd, 268.12; found, 559.25 [2M + Na]+.
1,3-Dimethyl-2-phenyl-1,2,3,4-tetrahydroquinazolin-6-ol 9a.
According to GP3, starting from 6-hydroxy-1,3-dimethyl-2-phenyl-2,3-
dihydroquinazolin-4(1H)-one 7a (160 mg, 0.6 mmol, 1 equiv) the title
compound 1,3-dimethyl-2-phenyl-1,2,3,4-tetrahydroquinazolin-6-ol 9a
(79 mg, 52%) was obtained as a pale yellow solid. 1H NMR (400 MHz,
DMSO-d6): δ = 8.42 (s, OH), 7.34−7.27 (m, 2H), 7.27−7.21 (m, 1H),
7.18−7.08 (m, 2H), 6.56 (dd, J = 8.6, 2.7 Hz, 1H), 6.51 (d, J = 8.7 Hz,
1H), 6.32 (d, J = 2.6 Hz, 1H), 4.83 (s, 1H), 3.50 (d, J = 16.2 Hz, 1H),
3.27 (d, J = 16.1 Hz, 1H), 2.84 (s, 3H), 2.36 (s, 3H) ppm. 13C NMR (101
MHz, DMSO-d6): δ = 147.8, 141.1, 136.7, 128.1 (2C), 127.3, 126.9
(2C), 119.1, 114.1, 113.9, 110.1, 80.7, 49.2, 41.6, 36.8 ppm. ESI-MS: m/
z calcd, 254.14; found, 255.2 [M + H]+. HPLC (method A): 98%.
1,3-Dimethyl-2-phenyl-1,2,3,4-tetrahydroquinazolin-6-yl n-
Heptylcarbamate 2a. According to GP4, starting from 1,3-
dimethyl-2-phenyl-1,2,3,4-tetrahydroquinazolin-6-ol 9a (120 mg, 0.47
mmol, 1 equiv) the title compound 1,3-dimethyl-2-phenyl-1,2,3,4-
tetrahydroquinazolin-6-yl n-heptylcarbamate 2a (95 mg, 51%) was
obtained after column chromatography (DCM/MeOH = 95:5) as white
solid; mp 93−95 °C. 1H NMR (400 MHz, DMSO-d6): δ = 7.51 (t, J =
5.7 Hz, NH), 7.36−7.29 (m, 2H), 7.29−7.23 (m, 1H), 7.15 (d, J = 7.1
Hz, 2H), 6.82 (dd, J = 8.7, 2.7 Hz, 1H), 6.62 (d, J = 8.9 Hz, 1H), 6.60 (d,
J = 2.7 Hz, 1H), 4.96 (s, 1H), 3.56−3.49 (m, 1H), 3.36−3.30 (m, 1H),
3.01 (dd, J = 13.0, 6.7 Hz, 2H), 2.92 (s, 3H), 2.40 (s, 3H), 1.50−1.38 (m,
2H), 1.27 (s, 8H), 0.87 (t, J = 6.8 Hz, 3H) ppm. 13C NMR (101 MHz,
DMSO-d6): δ = 155.1, 141.0, 140.9, 140.6, 128.2 (2C), 127.5, 126.7
(2C), 120.6, 120.1, 118.0, 108.8, 80.5, 48.5, 41.6, 40.4, 36.7, 31.2, 29.2,
28.4, 26.2, 22.0, 13.9 ppm. ESI-MS: m/z calcd, 395.26; found, 396.3 [M
+ H]+. HPLC (method B): 100%.
5-(Benzyloxy)-2-nitrobenzaldehyde 11. A solution of 5-
hydroxy-2-nitrobenzaldehyde (2 g, 11.98 mmol, 1 equiv) in dry DMF
(40 mL) was treated with benzyl bromide (2.85 mL, 23.95 mmol, 2
equiv) and potassium carbonate (3.3 g, 23.95 mmol, 2 equiv). The
reaction mixture was stirred for 48 h at 40 °C. Then ice cold water (100
mL) was added and the mixture was stirred for further 15 min. The
precipitated solid was filtered and washed with petroleum ether (20 mL)
to obtain 5-(benzyloxy)-2-nitrobenzaldehyde 11 (2.86 g, 88%) as a
yellow solid; mp 68−70 °C. 1H NMR (400 MHz, CDCl3): δ = 10.48 (s,
1H), 8.16 (d, J = 9.1 Hz, 1H), 7.46−7.30 (m, 6H), 7.21 (dd, J = 9.1, 2.9
Hz, 1H), 5.20 (s, 2H) ppm. 13C NMR (101 MHz, CDCl3): δ = 188.4,
163.1, 142. 5, 135.0, 134.3, 128.9 (2C), 128.7, 127.6 (2C), 127.3, 119.3,
114.2, 71.1 ppm.
2-Phenylquinazolin-6-yl n-Heptylcarbamate 16. According to
GP4, starting from 2-phenylquinazolin-6-ol 15 (60 mg, 0.27 mmol, 1
equiv) the title compound 2-phenylquinazolin-6-yl heptylcarbamate 16
(35 mg, 36%) was obtained after column chromatography (petroleum
ether/EtOAc = 3:1) as white powder; mp 141−144 °C. 1H NMR (400
MHz, CDCl3): δ = 9.41 (s, 1H), 8.64−8.55 (m, 2H), 8.07 (d, J = 9.1 Hz,
1H), 7.71 (d, J = 2.4 Hz, 1H), 7.66 (dd, J = 9.1, 2.6 Hz, 1H), 7.58−7.48
(m, 3H), 5.20 (t, J = 5.6 Hz, NH), 3.29 (dd, J = 13.3, 7.0 Hz, 2H), 1.67−
1.49 (m, 2H), 1.44−1.23 (m, 8H), 0.90 (t, J = 6.9 Hz, 3H) ppm. 13C
NMR (101 MHz, CDCl3): δ = 160.8, 160.0, 154.0, 149.3, 148.5, 137.9,
K
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Journal of Medicinal Chemistry
Article
130.6, 130.0, 129.7, 128.6 (2C), 128.5 (2C), 123.7, 117.3, 41.4, 31.7,
29.8, 28.9, 26.7, 22.6, 14.1 ppm. ESI-MS: m/z calcd, 363.19; found,
364.25 [M + H]+. HPLC (method B): 100%.
protonated at the acridinic N at pH = 8, according to a calculation with
MoKa.79 Fifty independent docking runs were carried out, with the
number of operations set to 1 million to improve docking pose
clustering. Among the four tested scoring functions, Goldscore80,81
found the pose with the lowest rmsd (0.45 Å) to the crystal structure on
higher ranks (rank 14) than the other functions (rank >40) and in a
cluster containing 21 poses. The top pose showed an rmsd of 0.76 Å and
was in a cluster with 27 poses. The Goldscore scoring function had
previously proven to be applicable also in docking studies with
AChE.45,48
Docking was performed with the same parameters (as used in the
redocking test) for the kinetically investigated ligands listed in Table 2
(i.e., 2a, 2c, 2h, 2i, 2k, 2l, 2p, 2s, 2u, 3a, and 16) as well as for ligand 1,
which possesses the most rigid structure. Chiral ligands were handled in
both enantiomeric forms. The Goldscore value provided the first
ranking criterion for the poses of a given ligand. In addition, rescoring
with DSX82 and CSD potentials were used as a second evaluation
criterion. Moreover, docking poses were visually inspected, in particular
the position of the carbamate moiety with respect to the catalytic serine
and the orientation of the CO group with respect to the oxyanion
hole. Only poses showing a suitable orientation for the transfer of the
carbamate to the serine were further considered.83
Ligand 1 as the most rigid compound showed well-clustered results
and reasonable positions for carbamate transfer in both enantiomeric
forms. The top rank for the S-enantiomer (second best in rescoring the
top-five poses with DSX/CSD) and the second-best rank for the R-
enantiomer (top rank in rescoring the top-five poses with DSX/CSD)
showed the carbonyl oxygen of the carbamate pointing toward the
oxyanion hole with distances of 2.7 and 2.8 Å between CO and
Gln117-N for the S- and the R-enantiomer, respectively. These poses
were chosen as a reference for the binding modes of the studied class of
compounds.
Because of the large size of the BChE binding pocket and the higher
flexibility of the compounds in comparison to 1, docking of all other
investigated ligands resulted in more diverse and energetically rather
degenerate poses besides the reference binding mode. To generate a
more consistent binding-mode hypothesis, a modeling approach was
followed for these structurally very similar ligands, in which the
reference poses of compound 1 were used to construct the ligands in the
binding site. Essentially, this was done with MOE 2014.0901 by deleting
the methylene bridge of the tetracyclic structure of 1 and adding the
required substituents to the aryl ring. The obtained structures were
relaxed in the binding pocket with MiniMuDS65 and scored with DSX/
CSD.
Enzyme Inhibition. AChE (EC 3.1.1.7, from human erythrocytes)
and BChE (EC 3.1.1.8, from equine serum and from human serum)
were purchased from Sigma-Aldrich (Steinheim, Germany). DTNB
(Ellman’s reagent), ATC and BTC iodides were obtained from Fluka
(Buchs, Switzerland). The stock solutions of the test compounds were
prepared in ethanol with a concentration of 33.3 mM (1 mM in assay)
and stepwise diluted with ethanol to a concentration of 33.3 nM (1 nM
in assay). For assay buffer preparation, an amount of 3.12 g of potassium
dihydrogen phosphate was dissolved in 500 mL of water and adjusted
with a NaOH solution (0.2 M) to pH = 8.0. Enzyme solutions were
prepared with buffer to give 2.5 units/mL and stabilized with 2 mg of
albumin bovine (SERVA, Heidelberg, Germany) per mL of enzyme
solution. An amount of 396 mg of DTNB was dissolved in 100 mL of
buffer to give a 10 mM solution (0.3 mM in assay). ATC and BTC
solutions were prepared in buffer with a concentration of 75 mM (452
μM in assay). The assay was performed at 25 °C as described in the
following: Into a cuvette containing 1.5 mL of buffer, 50 μL of the
respective enzyme, and 50 μL of DTNB solution, the test compound
solution (50 μL) was added, and the mixture was mixed. The mixture
was incubated for 4.5 min (phenols) or 30 min (carbamates), before an
amount of 10 μL of ATC or BTC (depending on the enzyme) was
added. The mixture was incubated for further 2.5 min before the
absorption at 412 nm was determined with a Shimadzu UVmini-1240
spectrophotometer. To measure full enzyme activity, the compound
solution was replaced by ethanol. Each compound concentration was
tested three times. The enzyme activity in percentage was plotted
against the logarithm of the compound concentrations from which the
IC50 values were calculated by the software GraphPad Prism 4.
Enzyme Kinetics. Carbamoylation kinetics were measured
following the enzyme inhibition protocol. For this purpose, the enzyme
was 5, 10, 20, 30, and 40 min preincubated with different inhibitor
concentrations before the addition of substrate. Plotting the enzyme
activity in percentage as a function of time for each inhibitor
concentration gave nonlinear time dependent inhibition curves from
which kobs was determined by eq 1. Double reciprocal plot of 1/kobs
against 1/c(inhibitor) gave a linear plot from which k3 and KC were
determined by eq 4. Decarbamoylation kinetics were measured by
incubating the enzyme with an inhibitor concentration that fully inhibits
the enzyme for 1 h. This mixture was then diluted 1:1000 with assay
buffer to prevent further enzyme inhibition. The assay was then
performed as described in the enzyme inhibition protocol at different
time points after the dilution to determine recovery of enzyme activity.
Uninhibited enzyme was treated by the same procedure to access
control values for enzyme activity after dilution. Plotting the enzyme
activity against different time points after dilution gave an exponential
first order kinetics from which k4 was calculated. All kinetic values were
determined using the software GraphPad Prism 4.
For both enantiomers, the methyl group at the benzylic nitrogen was
initially modeled in both the pseudo-equatorial and the pseudo-axial
form. All R-enantiomers showed unfavorable distances of 2.1−2.4 Å
between the axial methyl group and Gly116-Cα and were, hence, scored
lower than the poses with the equatorial methyl. Thus, the R-
enantiomers are presented with the methyl group in equatorial
orientation. For the S-enantiomers, the methyl group in the axial
position is much more favorable because it points to a small cavity
between Trp82 of the choline binding site and His438 of the CAS. The
conformer with the equatorial methyl group also fits to the binding site
but corresponds to an energetically less favorable conformation, as
investigated by force field calculations with the MMFF94s force field84
in MOE. Accordingly, the S-enantiomers are presented with the methyl
group in an axial orientation.
Molecular Modeling Studies. To derive a common binding-mode
hypothesis, a combined docking and minimization approach was
applied.
Docking studies were conducted with the program GOLD, version
5.2.2 (CCDC Software, http://www.ccdc.cam.ac.uk)64 using the crystal
structure PDB code 1P0I of human BChE (resolution 2.0 Å).66 Prior to
docking, all water molecules and non-protein atoms were removed and
the amino acids were protonated with Protonate 3D in MOE78 (version
2013.0801) at pH = 8 as the assay pH value. The binding site
corresponding to the search region in the docking was defined by the
following residues: Asn68, Asp70, Ser79, Trp82, Asn83, Gly115,
Gly116, Gly117, Gln119, Thr120, Gly121, Thr122, Leu125, Tyr128,
Glu197, Ser198, Ala199, Trp231, Leu286, Val288, Glu325, Ala328,
Phe329, Tyr332, Trp430, His438, Gly439, Tyr440, Ile442.
Ligand structures were built in MOE or extracted from the crystal
structure (ligand for redocking experiment, see below). The ligands
were energy minimized in MOE with the MMFF94x force field to an
rms gradient of 0.001 kcal/(mol·Å).
A redocking test was performed with the BChE crystal structure
4BDS (resolution, 2.10 Å) and tacrine as a reversible ligand. Tacrine was
All figures of docking poses and enzyme structures were made with
Pymol.72
Antioxidant Capacities (ORAC Assay). The antioxidant activity
was determined by the oxygen radical absorbance capacity fluorescein
(ORAC-FL) assay, modified by Dav
́
alos et al.85 The ORAC assay
measures antioxidant scavenging activity against peroxyl radicals, their
formation induced by 2,2′-azobis(2-amidinopropane) dihydrochloride
(AAPH) at 37 °C. The reaction was carried out in 75 mM phosphate
buffer (pH 7.4), and the final reaction mixture was 200 μL. Antioxidant
(20 μL) and fluorescein (120 μL; 300 nM final concentration) were
placed in the wells of a 96-well plate, and the mixture was incubated for
15 min at 37 °C. Then AAPH (Sigma, Steinheim, Germany) solution
L
DOI: 10.1021/acs.jmedchem.5b01674
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(60 μL; 12 mM final concentration) was added rapidly. The plate was
immediately placed into a SpectraFluor Plus plate reader (Tecan,
Crailsheim, Germany) and fluorescence measured every 60 s for 90 min
with excitation at 485 nm and emission at 535 nm. 6-Hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox, Sigma, Steinheim,
Germany) was used as a standard (1−8 μM, final concentration). A
blank (FL + AAPH) using phosphate buffer instead of antioxidant and
Trolox calibration were carried out in each assay. The samples were
measured at different concentrations (1−5 μM). All reaction mixtures
were prepared 4-fold, and at least four independent runs were performed
for each sample. Fluorescence measurements were normalized to the
curve of the blank (without antioxidant). From the normalized curves,
the area under the fluorescence decay curve (AUC) was calculated as
enantiomeres, the postulated binding mode of 2l,
synthetic procedures and spectral data for 6b−d, 7b−u,
8a−c, 9b−u, 10a−c, 2b−u, and 3a−c (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
■
Corresponding Author
*M.D.: phone, +0049-931 31-89676; fax, +0049-931 31-85494;
e-mail, Michael.Decker@uni-wuerzburg.de.
Author Contributions
The manuscript was written with contributions of all authors.
E.S. performed the synthesis and biological testing on AChE and
BChE as well as the kinetic studies on BChE. S.W., under the
supervision of C.S., was responsible for the molecular docking
studies on BChE. B.K., under the supervision of J.H., tested the
antioxidant capacity, neuroprotection, and toxicity of the
synthesized compounds. J.W. performed the separation of
enantiomers of 2p under the supervision of G.B. M.D. was
responsible for the supervision and development of the whole
project.
i=90
AUC = 1 +
f /f
i 0
∑
i=1
where f 0 is the initial fluorescence at 0 min and fi is the fluorescence at
time i. The net AUC for a sample was calculated as follows:
net AUC = AUC antioxidant − AUC blank
The ORAC-FL values were calculated:
⎡
⎢
⎤
⎡
⎢
⎤
⎥
(AUC sample) − (AUC blank)
⎣ (AUC Trolox) − (AUC blank) ⎦ concentration of sample
concentration of Trolox
⎥
⎦
⎣
Notes
The authors declare no competing financial interest.
and expressed as Trolox equivalents by using the standard curve
calculated for each assay. Final results were in μM of Trolox equivalent/
μM of compound.
ACKNOWLEDGMENTS
■
Neurotoxicity and Neuroprotection. Neurotoxicity and neuro-
protection were done as described before.47 HT 22 cells were derived
from murine hippocampal tissue, kindly provided by the Max Planck
Institute of Psychiatry, Munich, and were grown in high glucose
Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Karlsruhe,
Germany) supplemented with 10% (v/v) heat inactivated fetal calf
serum (FCS) (Biochrom, Berlin, Germany). Cells were kept under
standard cell culture conditions at 37 °C under 5% CO2 in a humidified
incubator and were subcultured every 2 d. Cell viability was determined
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. Briefly, cells were seeded in 96-well plates at a densitiy
of 5 × 10 per well and cultured for 24 h. Subsequently cells were
incubated for another 24 h with medium, compounds, or solvent in
presence (neuroprotection assay) or absence (neurotoxicity assay) of 5
mM glutamate (monosodium L-glutamate, Merck, Darmstadt, Ger-
many). Quercetin (Sigma, Steinheim, Germany) in a concentration of
25 μM served as the positive control in the neuroprotection assay. MTT
(Sigma, Steinheim, Germany) solution (4 mg/mL in PBS) was diluted
1:10 with medium, and the mixture was added to the wells after removal
of previous medium. The plates were then incubated for another 3 h.
The supernatants were removed, and 100 μL of lysis buffer (10% SDS,
pH 4.1) was added to the wells. Absorbance at 560 nM was determined
on the next day with a multiwall plate photometer (Spectra Fluor Plus,
Crailsheim, Germany). Results of cell viability are expressed as
percentage to untreated control cells. All compounds were dissolved
in DMSO and diluted with fresh medium. DMSO concentration in final
dilutions was ≤0.1%. Data are expressed as the mean SD of at least
three different independent experiments. Data were subjected to one
way ANOVA followed by Dunnett’s multiple comparison post-test
using GraphPad Prism 4 software (levels of significance: (∗) p < 0.05;
(∗∗) p < 0.01; (∗∗∗) p < 0.001).
We gratefully acknowledge the German Science Foundation
(DFG) for financial support (Grant DFG DE1546/6-1) as well
as the German Academic National Foundation (Studienstiftung
des Deutschen Volkes) for awarding a Ph.D. scholarship to S.W.
We thank Gabriele Brunner for technical assistance on
performing the ORAC assay.
ABBREVIATIONS USED
■
AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; ATC,
acetylthiocholine iodide; BTC, butyrylthiocholine iodide; CAS,
catalytic active site; CD, circular dichroism; DIPEA, N,N-
diisopropylethylamine; DMAc, dimethylacetamide; DTNB, 5,5′-
dithiobis(2-nitrobenzoic acid); EC, Enzyme Commission
number; eqBChE, equine butyrylcholinesterase; FL, fluorescein;
GSH, glutathione; hAChE, human acetylcholinesterase; hBChE,
human butyrylcholinesterase; MW, microwave; ORAC, oxygen
radical absorbance capacity; rmsd, root mean square deviation;
SAR, structure−activity relationship; TE, Trolox equivalent;
Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
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