Ethynylphenyl carbonates and carbamates as dual-action acetylcholinesterase inhibitors and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2015.10.039

Novel ethynylphenyl carbonates and carbamates containing carbon- and silicon-based choline mimics were synthesized from their respective phenol and aniline precursors and screened for anticholinesterase and anti-inflammatory activities. All molecules were micromolar inhibitors of acetylcholinesterase (AChE), with IC₅₀s of 28-86 μM; the carbamates were two-fold more potent than the carbonates. Two of the most potent AChE inhibitors suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation by 40%. Furthermore, these molecules have physicochemical properties in the range of other CNS drugs. These molecules have the potential to treat inflammation; they could also dually target Alzheimer's disease through restoration of cholinergic balance and inflammation suppression.

Full text of DOI:10.1016/j.bmcl.2015.10.039

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ethynylphenyl carbonates and carbamates as dual-action
acetylcholinesterase inhibitors and anti-inflammatory agents
Jaya Saxena a, David Meloni a,b, Mou-Tuan Huang c, Diane E. Heck d, Jeffrey D. Laskin e, Ned D. Heindel a,
Sherri C. Young a,f,
⇑
a Department of Chemistry, Lehigh University, Bethlehem, PA 18015, United States
b Incyte Corporation, Wilmington, DE 19803, United States
c Department of Chemical Biology, Rutgers University, Piscataway, NJ 08854, United States
d Department of Environmental Health Science, New York Medical College, Valhalla, NY 10595, United States
e Department of Environmental and Occupational Medicine, Rutgers University, Piscataway, NJ 08854, United States
f Department of Chemistry, Muhlenberg College, Allentown, PA 18104, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel ethynylphenyl carbonates and carbamates containing carbon- and silicon-based choline mimics
were synthesized from their respective phenol and aniline precursors and screened for anticholinesterase
and anti-inflammatory activities. All molecules were micromolar inhibitors of acetylcholinesterase
Received 14 September 2015
Accepted 14 October 2015
Available online xxxx
(AChE), with IC50s of 28–86 lM; the carbamates were two-fold more potent than the carbonates. Two
of the most potent AChE inhibitors suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced
inflammation by 40%. Furthermore, these molecules have physicochemical properties in the range of
other CNS drugs. These molecules have the potential to treat inflammation; they could also dually target
Alzheimer’s disease through restoration of cholinergic balance and inflammation suppression.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Alzheimer’s disease
Acetylcholinesterase inhibitors
Anti-inflammatory drugs
Carbonates
Carbamates
The excessive release of proinflammatory cytokines contributes
to a multitude of disorders such as multiple sclerosis,1–3 diabetes,4
rheumatoid arthritis,5 Alzheimer’s disease (AD),6,7 and Parkinson’s
disease.8,9 While acetylcholinesterase inhibitors (AChEIs) have
been extensively explored for the treatment of glaucoma,10–12
myasthenia gravis,13 Lewy body dementia,14,15 and AD,16–18 there
has been an emerging interest in AChEIs for the treatment of
inflammatory disorders. Tracey’s work supports a physiological
link between inflammation and cholinergic imbalance termed the
cholinergic anti-inflammatory pathway.19 This pathway is medi-
and alleviated obesity-associated inflammation.23 Snorrason has
shown that ointments of AChEIs such as galantamine or donepezil
are useful in alleviating skin inflammations.24 Finally, locally
applied neostigmine suppressed inflammation in an inflamed
mouse knee model, and topical AChEIs have shown promise in
the treatment of hyperhidrosis.25–27 Clearly, inhibition of AChE is
a promising approach to treating inflammatory disorders. Further-
more, considering the well-documented role of both inflamma-
tion6,7 and cholinergic dysfunction16–18 in AD, anti-inflammatory
AChEIs represent a promising class of dual-action AD therapeutics.
In earlier work directed at the development of potential
pesticides, we reported the synthesis and development of a
mechanism-based, active-site directed, highly specific AChEI, cho-
line p-chloromethylphenyl carbonate iodide (1, Fig. 1).28,29 This
candidate was modeled after Bechet’s classic mechanism-based
albeit rather nonspecific chloromethyldihydrocoumarin (2, Fig. 1)
which inhibited a rather broad set of proteases and esterases.
Bechet proposed an electrophilic quinone methide metabolite as
the lethal transient which captured the active site nucleophile, pre-
sumably serine.30,31 The anticholinesterase activity of 1 was
assessed, and it performed as a classic mechanism-based inhibitor
with irreversible, time-dependent, pseudo first-order enzyme inac-
tivation. Additional reversibility and kinetic studies indicated that
the quinone methide electrophile was formed on, and remained in,
ated by the
a7 subunit of the nicotinic acetylcholine (ACh) receptor
(a7-nAChR) and involves regulation of systemic cytokine release
by the vagus nerve. ACh has been shown to decrease both central
and peripheral release of proinflammatory cytokines such as tumor
necrosis factor (TNF) and interleukin-6 (IL-6).20 Potentiation of ACh
via inhibition of AChE results in inflammation suppression in mod-
els of sepsis, obesity, and endotoxemia. For example, intraperi-
toneal administration of the AChEIs physostigmine and
neostigmine improved survival time in a murine model of sepsis.21
Galantamine, an AChEI on the market for AD treatment, also sup-
pressed the release of TNF and IL-6 in a model of endotoxemia22
⇑
Corresponding author. Tel.: +1 484 664 3271; fax: +1 484 664 4113.
E-mail address: young@muhlenberg.edu (S.C. Young).
http://dx.doi.org/10.1016/j.bmcl.2015.10.039
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Saxena, J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.039

2
J. Saxena et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
A series of ortho- and para-substituted ethynylphenyl carbon-
ates (4–7) and carbamates (8–11) was synthesized from commer-
cially available phenol and aniline precursors (see Supporting
information). The carbonates were isolated via
a three-step
process, with yields ranging from 9% to 57%. The carbamates were
synthesized in a single step, resulting in 37% to 65% yields. The
identities of these novel molecules were confirmed by nuclear
magnetic resonance (NMR) spectroscopy and elemental analysis.
When screened in an Ellman assay,37 all compounds inhibited
AChE in the micromolar range, with IC50s ranging from 28 to
Figure 1. Choline p-chloromethylphenyl carbonate iodide (1),28,29 Bechet’s
chloromethyldihydrocoumarin antiprotease (2),30,31 and a generic ynenol furyl
lactone (3).35
86 lM (Table 1). Out of the series, carbamate 8 was the most
potent inhibitor and represents a promising lead for further study.
The carbamates proved to be approximately two times more
potent AChEIs than the carbonates. The mode of AChE binding of
many known carbamate AChEIs (e.g., rivastigmine, physostigmine)
involves carbamoylation of the active site serine residue followed
by restoration of the active enzyme.38 A critical hydrogen bonding
interaction between the nitrogen atom of the carbamate and the
histidine residue of the catalytic triad could therefore be responsi-
ble for the enhanced potency of the carbamates relative to the car-
bonates.38 When all other structural elements are conserved,
changing the choline mimic from a 3,3-dimethylbutyl to a 2-
(trimethylsilyl)ethyl moiety does not have a significant impact
on AChE inhibition. It is interesting to note that the para-substi-
tuted ethynyls were slightly more potent than the ortho-ethynyls
in the carbonate series, perhaps due to steric constraints in the
active site gorge of the enzyme.39
Following incubation of AChE with each inhibitor and subse-
quent gel filtration, 80–100% of enzyme activity was recovered
(Table 1). Interestingly, a higher percentage of enzyme activity
was recovered following incubation of AChE with the carbamates
(85–100%) compared to the carbonates (60–83%), with the excep-
tion of carbamate 11. This difference may be because the more
hydrophobic carbonates lead to formation of an acylated enzyme
complex which is slower to reactivate than the corresponding car-
bamylated enzyme complex.40 Slow reactivation of other lipophilic
acyl complexes of various esterases has been reported
previously.40–42
the active site of the enzyme.28,29 Considering the structural and
functional similarities between 1 and 2, it was proposed that the
AChE inactivation mechanism by 1 involves a quinone methide
intermediate.28 Unfortunately, 1 proved ineffective when screened
against fully-developed adult insects, and it was only modestly
effective against immature larvae. Furthermore, in control experi-
ments, 1 was unable to cross biological membranes and proved
unstable both in solution and in the solid state, an occurrence
traced to its chloromethyl moiety.28,29 Despite the limitations of
this molecule as a pesticide, it has served as a structural platform
for the synthesis and optimization of selective and potent AChEIs
by our group.32–34
It is well known in other mechanism-based inhibitors that a ter-
minal unsubstituted ethynyl group on an aromatic ring or on an
sp2 carbon can act as an electrophilic surrogate for a halomethyl.
For example, ynenol furyl lactones (3, Fig. 1) serve as irreversible
inhibitors of human leukocyte elastase apparently through tran-
sient formation of an electrophilic allenone.35 Considering the doc-
umented similarities in reactivity between conjugated ethynyls
and halomethyls, we chose to design a new class of ethynylphenyl
candidate inhibitors (Fig. 2).
Uncharged isosteres of choline have moderate affinities for
AChE, with inhibition constants (Ki) of 7.5 mM and 3.3 mM
reported for 3,3-dimethyl-1-butanol and 2-(trimethylsilyl)ethanol,
respectively.36 As such, many of the molecules designed by our
group, including the molecules presented herein (Fig. 2), contain
one of these moieties to provide cholinesterase recognition.32–34
Substitution of the nitrogen-based choline analog with a carbon-
or silicon-based analog may also address the inherent instability
and poor membrane permeability of carbonate 1, while maintain-
ing potent anticholinesterase activity. Furthermore, we decided to
replace the p-chloromethyl moiety of 1 with an ethynyl group
(vide infra) because the chloromethyl proved to be a source of
instability in other studies.28,29
Table 1
IC50 values, percent recoveries of AChE activities, percent reductions of TPA-induced
edema, cLogP, and PSA values for ethynylphenyl carbonates and carbamates
a
#
IC50
(
lM)
% AChE
recovery
% red.
(TPA)b
cLogPe
PSAh
(Å2)
Ethynylphenyl carbonates
Presented herein is the synthesis of a series of ethynylphenyl
carbonates and carbamates linked to lipophilic ACh mimics. Not
only do these molecules have the potential to target inflammation,
they may also dually target AD through AChE inhibition and
inflammation suppression, potentially through the cholinergic
anti-inflammatory pathway.
4
5
6
7
86
59
80
62
1
2
3
2
83
83
78
8
5
4
18
22
nd
nd
4.41
4.41
24.1
25.1
24.2
25.2
f
—
—
f
60 11
Ethynylphenyl carbamates
8
9
10
11
Tacrine
28.4
34.3
38.1
34.4
9
1
1
1
100
95
96
85
95
9
39c
nd
3.73
3.73
27.5
29.4
27.6
29.5
28.4g
3
5
5
f
Irritant
—
—
40d
f
0.058 0.005
nd
2.91g
a
IC50 values represent mean S.D. Individual experiment is performed at least in
duplicate; source of AChE is electric eel (Electrophorus electricus); enzyme was
incubated for 30 min with 52
l
M inhibitor at 25 °C.
b
nd: not determined.
c
Value differs from positive control (TPA only) based on one-way ANOVA, with P
<0.05.
d
Value differs from positive control (TPA only) based on one-way ANOVA, with P
<0.10.
e
cLogP values calculated using ChemBioDraw Ultra 14.0.
f
g
h
cLogP values could not be calculated.
Calculations performed on free base.
PSA values calculated using Spartan’14 V1.1.4.
Figure 2. Ethynylphenyl carbonates (4–7) and carbamates (8–11).
Please cite this article in press as: Saxena, J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.039

J. Saxena et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Although it was predicted that these molecules would exhibit
suicide-type inhibition similar to that of chloromethyl carbonate
1, all molecules in this series exhibited either reversible or pseu-
doirreversible AChE inhibition (Table 1).43 It is important to note
that the compound after which this class of molecules was mod-
eled, carbonate 1, exhibited irreversible, time-dependent, suicide-
like AChE inhibition.28,29 The irreversible modification of AChE,
for example by organophosphates, results in a build-up of ACh at
neuromuscular junctions and can lead to adverse effects such as
headache, nausea, seizures, paralysis, and death.44,45 As such,
reversible (or pseudoirreversible) enzyme-inhibitor interactions
are desired when exploring therapeutic AChEIs; several reversible
(e.g., galantamine) and pseudoirreversible (e.g., rivastigmine) car-
bamate AChEIs have already been explored for the treatment of
AD.46,47 Because of the non-permanent nature of the action of
these molecules, they could be safely used to treat cholinergic
and inflammatory disorders.
stabilities and toxicities of these molecules, as well as their specific
mode of binding to AChE.
Acknowledgements
This work was funded in part by the National Institutes of
Health CounterACT Program through the National Institute of
Arthritis and Musculoskeletal and Skin Diseases (award
#U54AR055073). Its contents are solely the responsibility of the
authors and do not necessarily represent the official views of the
federal government. Testing methods employed herein are
described in detail in the doctoral dissertation (Lehigh University,
2012) of Sherri C. Young.
Supplementary data
A mouse ear inflammation model (MEIM)48 using 12-O-tetrade-
canoylphorbol-13-acetate (TPA), a phorbol ester, as the proinflam-
matory agent was used to investigate the anti-inflammatory
properties of the ethynylphenyl carbonates and carbamates (see
Supporting information). The results shown in Table 1 are reported
as % suppression of TPA-induced edema. Carbamates 8 and 11, two
of the most potent AChEIs of this series, exhibited statistically sig-
nificant inflammation suppression in the MEIM, suggesting that
this inflammation suppression may be occurring through the
cholinergic anti-inflammatory pathway.19–23 Since inflammation
involves a host of mediators, a direct correlation between AChE
inhibition and inflammation suppression is typically not observed.
Past work in our group on non-steroidal anti-inflammatory drug
(NSAID)–AChEI conjugates suggests that topical anti-inflammatory
activities depend upon drug stabilities, lipophilicities, and AChE
inhibitory potencies.33 Carbonates 4 and 5 did not exhibit signifi-
cant inflammation suppression, either because of their less potent
AChE inhibition or because of their lower stabilities compared to
the carbamates (or both).40
(NMR spectra, melting point ranges, and elemental analysis for
all ethynylphenyls, details for the clogP and PSA calculations, and
protocols for the synthesis, Ellman assay, reversibility study, and
MEIM).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
039.
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