Neuroprotective tri- and tetracyclic BChE inhibitors releasing reversible inhibitors upon carbamate transfer

Article abstract of DOI:10.1021/ml3001825

Tri- and tetracyclic nitrogen-bridgehead compounds were designed and synthesized to yield micromolar cholinesterase (ChE) inhibitors. Structure-activity relationships identified potent compounds with butyrylcholinesterase selectivity. These compounds were selected as starting points for the design and synthesis of carbamate-based (pseudo)irreversible inhibitors. Compounds with superior inhibitory activity and selectivity were obtained and kinetically characterized also with regard to the velocity of enzyme carbamoylation. Structural elements were identified and introduced that additionally showed neuroprotective properties on a hippocampal neuronal cell line (HT-22) after glutamate-induced intracellular reactive oxygen species generation. We have identified potent and selective pseudoirreversible butyrylcholinesterase inhibitors that release reversible inhibitors with neuroprotective properties after carbamate transfer to the active site of cholinesterases.

Full text of DOI:10.1021/ml3001825

Letter
pubs.acs.org/acsmedchemlett
Neuroprotective Tri- and Tetracyclic BChE Inhibitors Releasing
Reversible Inhibitors upon Carbamate Transfer
Fouad H. Darras, Beata Kling, Jorg Heilmann, and Michael Decker*
̈
Institut fur Pharmazie, Universitat Regensburg, Universitatsstraße 31, D-93053 Regensburg, Germany
̈
̈
̈
S
* Supporting Information
ABSTRACT: Tri- and tetracyclic nitrogen-bridgehead com-
pounds were designed and synthesized to yield micromolar
cholinesterase (ChE) inhibitors. Structure−activity relation-
ships identified potent compounds with butyrylcholinesterase
selectivity. These compounds were selected as starting points for the design and synthesis of carbamate-based
(pseudo)irreversible inhibitors. Compounds with superior inhibitory activity and selectivity were obtained and kinetically
characterized also with regard to the velocity of enzyme carbamoylation. Structural elements were identified and introduced that
additionally showed neuroprotective properties on a hippocampal neuronal cell line (HT-22) after glutamate-induced
intracellular reactive oxygen species generation. We have identified potent and selective pseudoirreversible butyrylcholinesterase
inhibitors that release reversible inhibitors with neuroprotective properties after carbamate transfer to the active site of
cholinesterases.
KEYWORDS: Alzheimer's disease, butyrylcholinesterase, carbamates, neuroprotection
Chart 1. Structures of the ChE Inhibiting Alkaloid
Physostigmine, the Tetracyclic Lead Structure (5a, cf.
Scheme 1), and the AD Drug Rivastigmine
lzheimer's disease (AD) is one of the most often occurring
Aand most devastating neurodegenerative diseases. Huge
efforts have been made to investigate the pathophysiology of
AD, but to make a long story short, the disease is still
incurable.1 The number of approved drugs is extremely limited
to one NMDA antagonist (memantine) and three acetylcho-
linesterase (AChE) inhibitors (rivastigmine, donepezil, and
galantamine). Despite their purely symptomatic mode of action
to improve cognition and memory, their clinical effectiveness
was proven in a number of studies.2 While galantamine and
donepezil are reversible cholinesterase (ChE) inhibitors,
rivastigmine (Exelon) represents an irreversible inhibitor
transferring a carbamate moiety to the serine unit in the active
center of AChE and butyrylcholinesterase (BChE), respectively,
the second cholinesterase in the human body.3,4 The
carbamates' mode of action is termed pseudoirreversible since
the carbamate moiety slowly hydrolyses from the enzyme,
yielding the active enzyme, again in contrast to irreversibly
inhibiting organophosphates. Rivastigmine, a compound
derived from the alkaloid physostigmine, is of special interest
because in clinical and pharmacological investigations, it was
shown to be of high effectiveness, which some authors
attributed to its ability to inhibit also BChE with high efficiency
(Chart 1).4,5 There has been postulated a putative beneficial
role in the therapy of AD through BChE inhibition; for
example, it was found that the amount of AChE decreases in
later stages of AD (when AChE inhibitors become clinically
ineffective), whereas the amount of BChE stays the same or is
even increased.6,7 Also, it was shown that with a decreasing
amount of AChE, BChE seems to be able to compensate for
AChE loss, resulting in improved cognition.7 BChE's role in the
human body is not yet fully understood; it is responsible for
detoxification of xenobiotics, and it influences lipoprotein
metabolism and may also play a role in neuronal differ-
entiation.3−7
For the benefit of developing more effective AD drugs and to
investigate BChE's physiological functions, there have been
efforts in developing selective BChE inhibitors. Gilmer's group
has described a very interesting class of pseudoirreversible
carbamate inhibitors based on an isosorbide template with
remarkable potency and selectivity.8 Our group has used two
alkaloids (deoxyvasicine and dehydroevodiamine) that had
been described as moderately active and unselective ChE
inhibitors as lead structures to identify a series of N-bridgehead
tri- and tetracyclic compounds as reversible and competitive
inhibitors with improved inhibitory activities and BChE
selectivity (Chart 1).9,10 The greatest BChE selectivities were
achieved with quinazolinimine structures.10 By applying the
bivalent compound approach and the design of hybrid
molecules, their pharmacological activities were further
improved and enlarged.11−14
Special Issue: Alzheimer's Disease
Received: July 5, 2012
Accepted: August 16, 2012
© XXXX American Chemical Society
A
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Because even the best ChE inhibitors stay symptomatically
acting compounds, there has been considerable interest in
applying antioxidant compounds as disease-modifying drugs in
AD drug development. Oxidative stress and the formation of
reactive oxygen species (ROS) are key processes in AD and
result in neuronal damage and subsequent cell death.15,16 It was
therefore tried to combine AChE inhibitors with compounds
possessing antioxidant properties, for example, by forming
covalently connected hybrids or physiologically more labile
codrugs.11,12,17,18 Albeit highly promising compounds, hybrids
also suffer from some difficulties, such as their inherent high
molecular mass and therefore reduced bioavailability.11
For the investigation of SARs concerning the tri- and
tetracyclic N-bridgehead compounds, the six-membered ring
system containing compound 3,4-dihydroisoquinoline and the
seven-membered one 4,5-dihydro-3H-benzo[c]azepine were
reacted with isotoic acid anhydride, a reactive form of
anthranilic acid formed after reaction with triphosgene (Scheme
1). Preparation of isotoic acid anhydrides also provides an easy
Scheme 1. Synthesis of Tetra- and Tricyclic N-Bridgehead
a
Compounds
The vast majority of recent work on cholinesterase inhibitors,
mainly hybrids, has been done on the basis of a surprisingly
small number of chemical templates, especially tacrine.12 There
is a constant search for novel structural templates for ChE
inhibitors, especially from natural products.19−21 Our rationale
in this present work was to use a previously described
tetracyclic lead structure as a template for the development
of novel pseudoirreversible inhibitors in which the heterocyclic
structure serves as a kind of carrier into the active center of
ChEs. Such compounds would release reversible inhibitors after
transfer of the carbamate moiety to the enzymes, ideally
enabling a more pronounced efficiency of inhibition.
Structure−activity relationships (SARs) were investigated for
the heterocyclic template, in particular to find out whether the
introduction of a phenolic hydroxyl group to enable connection
with a carbamate unit is tolerated. Additionally, SARs of the
resulting carbamates with the aim to achieve higher selectivity
and inhibitory activity toward BChE were investigated. A
second rationale of this work was to incorporate antioxidant
properties into our target compounds, but not by connecting
them covalently to antioxidant moieties as it is the case for
hybrid molecules, but to use the heterocyclic phenolic structure
of the reversible inhibitor itself as an antioxidant. Weinstock
and Groner have used such an approach by combining
carbamate structures with MAO-B inhibitors.22 Phenolic
structures are key factors for antioxidant and neuroprotective
properties in a number of natural products, such as quercetin,
ferulic acid, and silibinin.17,18
We assumed that the compounds released after carbamate
transfer represent antioxidants (since, for example, cyclic
quinonimines could be formed after oxidation) and could
therefore enlarge the biological spectrum of the compounds
without increasing their molecular mass. For assaying
antioxidant properties, we used two different assays: the first
one, the oxygen radical absorbance capacity (ORAC) assay,
determines the ability of compounds to protect fluorescein
from destruction by peroxyl radicals and, therefore, directly
assesses their antioxidant physicochemical properties.14,18 The
second assay is cell-based and uses murine HT-22 hippocampal
cells that lack ionotropic glutamate receptors to study
intracellular oxidative stress-induced neurotoxicity.17,23,24 In
this neuronal oxidative stress-induced toxicity (oxytosis), cell
death is induced by extracellular glutamate challenge in a
nonreceptor-mediated “oxidative” pathway. Glutamate toxicity
is mediated by the cystine/glutamate antiporter system,
whichafter exposure to high extracellular glutamate concen-
trationsresults in inhibition of cystine uptake, leading to
intracellular cysteine and therefore glutathione depletion, which
induce ROS accumulation and cell injuries.23−25 Administration
of antioxidants such flavonoids can effectively prevent oxidative
neuronal death in this cell line.26,27
a
Reagents: (i) (Cl3CO)2CO, dry THF, 40−50 °C, 3 h. (ii) CH3I,
N,N-diisopropylethylamine, N,N-dimethylacetamide, 40 °C, 24 h. (iii)
Toluene/reflux, 24 h. (iv) LiAlH4, THF, 70 °C, 3 h. (v)
Ethyl(methyl)carbamic chloride, NaH, THF, rt or isocyanate, Et3N,
CH2Cl2, rt. (vi) Pyrrolidin-2-one, MW 100−200 W, 130 °C, 1 h. (vii)
Benzylbromide, K2CO3, acetone, 70 °C, 24 h. (viii) CH3I, dioxane, 90
°C, 24 h. (ix) LiAlH4, THF, 70 °C, 3 h. (x) H2, Pd/C, ethanol, rt, 24 h.
(xi) Isocyanate, CH2Cl2, Et3N, rt, 1−3 h.
method for selective monomethylation of anthranilic acid's
amino group. Seven- and six-membered rings were synthesized
to investigate their influence on BChE selectivity (previous
work on related quinazolinimines showed higher selectivity
with increasing ring size).10,13,14
Reduction of the amide group of compounds 3a−c and 4a,c
by lithium aluminum hydride afforded the desired tetracyclic
quinazoline compounds 5a−c and 6a,c, respectively. Com-
pound 6b was synthesized by demethylation of 6c using boron
tribromide. Compound 5b was treated with ethyl(methyl)-
carbamic chloride in the presence of sodium hydride to afford
8a in 60% yield. The reaction of n-heptylisocyanate with 6b in
the presence of sodium hydride gave 7 in 75% yield.
Carbamoylation of 5b with the appropriate isocyanate in
dichloromethane in the presence of triethylamine gave the
carbamates 8b−e in good yields (72−88%) (Scheme 1).
Unsubstituted tetracyclic compounds 5a and 6a were prepared,
and compounds bearing a methoxy (5c and 6c) and a hydroxyl
group (5b and 6b) to test whether substitution in this position
is tolerated. Determination of IC50 values at AChE and BChE
B
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ACS Medicinal Chemistry Letters
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confirmed that the introduction of −OCH3 or −OH groups has
no influence on the inhibitory activities and, therefore, proved
that this position of the heterocycle is ideal for introduction of
the carbamoyl unit (Table 1). Interestingly, for the seven-
membered ring-containing compounds, the inhibition data
were considerably modified: As expected from the results with
tricyclic quinazolinimines, activity at BChE was slightly higher,
whereas inhibitory activities at AChE were slightly lower,
resulting in increased selectivity toward BChE (>8).10 Again,
introduction of a methoxy or hydroxyl group did not result in a
significant alteration of binding behavior. It has to be stated
though that all compounds are only 1−2 digit micromolar
inhibitors of BChE, and selectivities were never higher than 10.
We also synthesized analogous tricyclic quinazoline com-
pounds to investigate their binding profiles. Their synthesis was
accomplished as illustrated in Scheme 1. The fusion of isatoic
anhydrides 1a,b with pyrrolidin-2-one using a microwave
reactor at 100−200 W power and 100 °C temperature afforded
9a,b in good yields (79−80%). Benzylation of 9b by
benzylbromide in the presence of K2CO3 afforded 9c.
Quaternization reaction of 9a,c with methyl iodide gave
10a,c. Reduction of 10a,c by lithium aluminum hydride
afforded the desired tricyclic quinazoline compounds 11a,c in
good yields (74−78%). Debenzylation reaction of 11c gave 12.
Finally, carbamoylation of 12 to yield the different carbamates
13a−d was accomplished as reported for the preparation of
8b−e in good yields (68−83%) (Scheme 1).
Table 1. AChE and BChE Inhibition Results, Resulting ChE
Selectivity, and Antioxidant Capacities Expressed as Trolox
Equivalents (Concentration Range, 0.5−7.5 μM)
For investigation into the SARs of this class of compounds,
several structurally diverse carbamate groups were introduced
at the phenolic hydroxyl group. Several aliphatic and aromatic
moieties were used in previous work on carbamates: the methyl
ethyl carbamate occurs in rivastigmine (Chart 1) and, therefore,
served as our first structure. Previous medicinal research has
been done using a physostigmine or rivastigmine-based
heterocyclic core:28,29 The compounds synthesized inhibited
both ChEs in the nanomolar range. A heptyl group was
identified that led to high acitivity, in the case of conforma-
tionally restricted rivastigmine analogues with 16-fold selectivity
[heptylphysostigmine (eptastigmine) shows 4-fold selectiv-
ity].29 The synthesis of phenylcarbamates has been described,
again connected to a physostigmine-related heterocyclic core.29
In this case, modulation of selectivity could be achieved by
fairly minor structural modifications, for example, a methyl
group in the o-position led to AChE selectivity, whereas an
isobutyl substituent in the p-position led to BChE selectivity.28
We therefore also applied these carbamate structures to our
heterocyclic templates to investigate SARs.
Tri- as well as tetracyclic target compounds bear a chiral C
atom attached to two N atoms, but at this stage, no
enantioseparation was performed. The stability of carbamoy-
lated compounds under assay conditions was investigated by
incubation of 8a and 8b in buffer and control by LC-ESI-MS,
and no decomposition was observed.
The tetracyclic carbamate compound 8a bearing methyl and
ethyl substituents at the carbamate group (like rivstigmine)
shows significant improvement in binding activities over
noncarbamate compounds, moderately pronounced at AChE
(6-fold more potent as compared to the unsubstituted
compound 5a) and very pronounced at BChE (100-fold
more potent as compared to the unsubstituted compound 5a),
leading to submicromolar activities at BChE and 54-fold
selectivity over AChE (Table 1). The introduction of a heptyl
substituent as in heptylphysostigmine for compounds 7 and 8b
a
AChE from electric eel and BChE from equine serum; data are the
b
means of at least three independent determinations. Selectivity ratio
c
[IC50(AChE)/IC50(BChE)]. Inhibition at 100 μM, 26% inhibition.
d
e
f
21% inhibition. 33% inhibition. Concentration range, 1.0−5.0 μM.
g
h
i
j
8% inhibition. 18% inhibition. 1% inhibition. 24% inhibition.
C
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lead to a further 10-fold increase in activity at BChE and a very
remarkable decrease in activity at AChE. For compound 8b, no
activity at all could be observed even at 100 μM. So, two-digit
nanomolar inhibitors of BChE were obtained lacking any
activity at AChE. The tricyclic heptyl carbamate 13a only
showed moderate activity and selectivity with IC50 (AChE) =
32.3 μM and IC50 (BChE) = 2.0 μM.
We also studied the effect of phenylcarbamates on ChE
inhibition activity and selectivity. Tetracyclic 3-methoxyphenyl-,
2-methylphenyl-, and 4-isopropylphenylcarbamates 8c, 8d, and
8e, respectively, showed submicromolar activities at BChE with
900−1500-fold selectivity over AChE (Table 1). Tricyclic 3-
methoxyphenyl-, 2-methylphenyl-, and 4-isopropylphenyl
carbamates 13b, 13c, and 13d, respectively, only showed
moderate activity and selectivity with IC50 (AChE) ranging
from 40 to lower than 200 μM and IC50 (BChE) ranging from
3 to 8 μM (Table 1).
the most potent compounds with antioxidant capacities ranging
from 2.5 to 3.4 trolox equivalents, meaning that they are far
more potent than the very active positive control trolox. The
methoxy compounds 5c and 6c show lower potencies with 0.5
and 1.2 trolox equivalents, respectively. The unsubstuituted
compound 5a is among the least potent compounds. Finally,
the carbamates tested show only very moderate antioxidant
properties (0.65 trolox equiv for the ethyl methyl carbamate 8a,
0.30 trolox equiv for the tetracyclic heptyl carbamate 8b, and
1.03 equiv for the tricyclic heptyl carbamate 13a). None of
these compounds lacks radical scavenging properties com-
pletely, though, and for structurally related compounds, there
can be significant differences in their capacities.14 As a general
rule, it can be seen that the hydroxyl compounds released after
carbamate transfer to the enzymes' serine unit represent highly
potent antioxidants with slightly higher capacities for the
tricyclic compound (Table 1).
Kinetic studies were performed to get more detailed
information about the time course of inhibition and therefore
more precise data about the equilibrium constant of inhibition
(for experimental details, cf. Supporting Information). The
equilibrium constant of the inhibitor−ChE complexes (KC) and
the carbamoylation rate constants (k3) of two representative
target compounds 8a and 8b and physostigmine as a positive
control were determined (Figure 1). The KC value calculated
In a second assay assessing the target compounds'
antioxidant properties, their ability to prevent oxycytotic cell
death of murine hippocampal HT-22 cells after glutamate
exposure and subsequent intracellular ROS formation was
evaluated. The six- and seven-membered ring tetracyclic
hydroxyl compound 5b and 6b, as well as the tricyclic
hydroxyl-compound 12 formed after carbamate transfer to the
enzyme, showed very pronounced neuroprotective properties
(tested at 10 μM) to the same extent as the potent positive
control quercetin (tested at 25 μM) and much stronger than
the flavolignan silibinin.17 The six-membered ring heterocyclic
methoxy compound 5c showed activity in the same range,
although performing these experiments at various concen-
trations revealed that the hydroxy compound exhibits neuro-
protection already at 5 μM, when the methoxy compound is
not active (cf. Supporting Information for data at 5 μM). The
unsubstituted compound 5a showed no activity. Surprisingly,
all tested carbamates 7, 8b, and 13a showed potent antioxidant
capacities to the same extent as quercetin does, and only the
tricyclic compound 13a showed slightly lower activities at a
concentration of 5 μM. The methyl ethyl-substituted carbamate
8a did not show neuroprotective activity at 10 μM (Figure 2).
Figure 1. (A) Time-dependent pattern of inhibition of BChE by
compound 8b (10−50 nM). (B) Stability constants of the inhibitor−
ChE complex (KC) and rate constants of carbamoyl−ChE formation
(k3) of 8a,b and physostigmine, respectively.
for physostigmine as positive control is in agreement with the
value reported in the literature.29 For compound 8a at AChE,
the actual KC value is even smaller than determined as the IC50
values with KC = 1.1 μM and at BChE with KC = 274 nM,
resulting in 4-fold selectivity. The rate constants of 0.31 min−1
(AChE) and 0.18 min−1 (BChE) show rapid carbamoylation,
albeit slower than for physostigmine (Figure 1). For compound
8b, KC (BChE) is even lower as for determination of the IC50
value with KC = 12.7 nM, and no carbamoylation could be
observed at AChE for the concentrations tested. The velocity of
carbamoylation was fairly rapid with k3 = 0.15 min−1.
Compound 8b shows a time-dependent pattern of inhibition
characterized by an increase until a steady state after 60 min
(Figure 1).
Figure 2. Evaluation of neuroprotection of unsubstituted, phenolic,
methoxy-substituted, and selected carbamyolated compounds at 10
μM against glutamate-induced oxidative stress on HT-22 cells (cf. the
Supporting Information for details).
The tetracyclic heptyl carbamate 8b represents a highly
potent pseudoirreversible BChE inhibitor with KC = 12.7 nM
without any activity at AChE and fast onset of action.
For evaluation of antioxidant potencies, in a first assay, the
target compounds' radical scavenging capacities were deter-
mined by means of their ability to reduce the amount of peroxyl
radicals (ORAC assay). Results are expressed in “trolox” (6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-
soluble vitamin E derivative) equivalents.14 As can be seen from
Table 1, the phenolic compounds (5b, 6b, and 12) are by far
The phenolic hydroxyl group, which is also formed after
carbamate transfer to the enzyme, led to potent ROS
scavenging molecules exhibiting up to 3.5 trolox equiv in a
physicochemical ORAC assay. Interestingly, evaluation of the
antioxidant properties of the target compounds on glutamate-
challenged neuronal HT-22 cells (and therefore increased
D
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(4) Giacobini, E. Cholinergic function and Alzheimer's disease. Int. J.
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protective properties not only for the hydroxyl compounds but
also for the carbamates in the same or higher range than for the
positive control quercetin. These results are surprising since we
expected high neuroprotection only for the hydroxyl
compounds, but obviously, in the cell-based assay also, the
carbamate compounds are very potent neuroprotectants in
their own regard.
Into a novel heterocyclic template consisting of basic tri- and
tetracyclic N-bridgehead compounds that inhibit ChEs, a
phenolic hydroxyl group was introduced, and SARs showed
that the −OH group as well as respective phenol ethers are
tolerated without loss of activity. Introduction of carbamate
units not only increased inhibitory activities into the
submicromolar range, but SARs on the carbamate moiety led
to nanomolar inhibitors with ≫15000-fold selectivity toward
BChE over AChE. Kinetic studies on selected carbamates
proved pseudoirreversible inhibition anddepending on the
concentration of inhibitorsa comparatively fast onset of
action. Kinetic studies revealed an even higher stability constant
(KC) for compounds 8a and 8b as reflected by the IC50 value
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We are currently performing computational and docking
studies to explain the very pronounced changes in enzyme
inhibitory activity and selectivity by only minor changes in the
chemical structure, and results will be presented at a later stage.
ASSOCIATED CONTENT
* Supporting Information
■
S
Elemental analysis and spectral data, detailed synthetic and
pharmacological procedures, and additional figures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 0049-941-943-3289. Fax: 0049-941 943-4990. E-mail:
michael.decker@chemie.uni-regensburg.de.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A Ph.D. scholarship of the German Academic Exchange Service
(DAAD) for F.H.D. is gratefully acknowledged. Appreciation is
expressed to Gabriele Brunner for technical assistance, to J.
Kiermeier for MS measurements, and to Professor S. Elz for
scientific mentoring.
ABBREVIATIONS
■
AD, Alzheimer's disease; AChE, acetylcholinesterase; BChE,
butyrylcholinesterase; ROS, reactive oxygen species; ORAC,
oxygen radical absorbance capacity
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■
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