Novel Selective Butyrylcholinesterase Inhibitors Incorporating Antioxidant Functionalities as Potential Bimodal Therapeutics for Alzheimer's Disease

Article abstract of DOI:10.3390/molecules21040440

Isosorbide-2-carbamates-5-aryl esters are highly potent and very selective butyrylcholinesterase inhibitors. The objective of the present work was to address the hypothesis that the isosorbide-aryl-5-ester group could be replaced with an antioxidant functionality while maintaining inhibitor effects and selectivity. We successfully incorporated ferulic acid or lipoic acid groups producing potent selective inhibitors of butyrylcholinesterase (BuChE). The hybrid compounds were non-toxic to the murine hippocampal cell line HT-22 and lipoate esters were neuroprotective at 10 and 25 μM when the cells were challenged with glutamate (5 mM) in a similar manner to the positive control quercetin. The benzyl carbamate 7a was a potent inhibitor of BuChE (IC₅₀ 150 nM) and it was effective in reducing glutamate toxicity to neuronal cells at >5 μM. Representative compounds exhibited an antioxidant effect in the oxygen radical absorbance capacity assay as the lipoate 7d was not active, whereas the ferulate 8a showed a weak, but significant, activity with 0.635 ± 0.020 Trolox Equivalent.

Full text of DOI:10.3390/molecules21040440

molecules
Communication
Novel Selective Butyrylcholinesterase Inhibitors
Incorporating Antioxidant Functionalities as
Potential Bimodal Therapeutics for
Alzheimer’s Disease
Mike Jones 1, Jun Wang 1, Shona Harmon 1, Beata Kling 2, Jörg Heilmann 2 and John F. Gilmer 1,
*
1
School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland;
mike_jone5@hotmail.com (M.J.); wangju@tcd.ie (J.W.); s.harmon@redxpharma.com (S.H.)
Institute of Pharmacy, University of Regensburg, 93053 Regensburg, Germany;
2
Beata.Kling@chemie.uni-regensburg.de (B.K.); Joerg.Heilmann@chemie.uni-regensburg.de (J.H.)
*
Correspondence: gilmerjf@tcd.ie; Tel.: +353-1-896-2795
Academic Editors: Michael Decker and Diego Muñoz-Torrero
Received: 2 March 2016; Accepted: 23 March 2016; Published: 1 April 2016
Abstract:
Isosorbide-2-carbamates-5-aryl esters are highly potent and very selective
butyrylcholinesterase inhibitors. The objective of the present work was to address the hypothesis
that the isosorbide-aryl-5-ester group could be replaced with an antioxidant functionality while
maintaining inhibitor effects and selectivity. We successfully incorporated ferulic acid or lipoic
acid groups producing potent selective inhibitors of butyrylcholinesterase (BuChE). The hybrid
compounds were non-toxic to the murine hippocampal cell line HT-22 and lipoate esters were
neuroprotective at 10 and 25
similar manner to the positive control quercetin. The benzyl carbamate 7a was a potent inhibitor of
BuChE (IC50 150 nM) and it was effective in reducing glutamate toxicity to neuronal cells at >5 M.
µM when the cells were challenged with glutamate (5 mM) in a
µ
Representative compounds exhibited an antioxidant effect in the oxygen radical absorbance capacity
assay as the lipoate 7d was not active, whereas the ferulate 8a showed a weak, but significant, activity
with 0.635 ˘ 0.020 Trolox Equivalent.
Keywords: neuroprotection; Alzheimer’s disease; antioxidant; cholinesterase inhibitor; hybrid
1. Introduction
Alzheimer’s disease (AD) is now the most common neurodegenerative disease, with more than
20 million cases worldwide [1]. AD is characterised by global cognitive decline and associated
neuropathological findings which may include neuronal loss, neurofibrillary tangles, neuritic plaques
and amyloid angiopathy. Cholinergic loss is the single most replicated neurotransmitter deficiency in
AD, and AChE has been a drug target for the treatment of AD since the emergence of this cholinergic
hypothesis over 30 years ago [2]. This followed recognition that the cognitive impairments in AD
correlated with cholinergic deficits such as reduced synaptic acetylcholine synthesis and choline
acetyltransferase (ChAT) activity. The introduction of the cholinesterase inhibitors (ChEIs)—first
tacrine then donepezil, rivastigmine, and galantamine—has made an important contribution to the
management and well-being of early stage AD patients. Selective inhibitors of acetylcholinesterase
(AChE; EC 3.1.1.7) e.g., donepezil and unselective inhibitors (ChEIs) e.g., rivastigmine, induce
dose-limiting adverse effects such as bradycardia, nausea, and diarrhea [3,4].
There are challenges facing this field in identifying new agents that have improved efficacy or
side effect profile. Medicinal chemistry themes that can be recognised as a response to this include
a switch in focus from AChE to butyrylcholinesterase (BuChE; EC 3.1.1.8) [5–7]. This is because
Molecules 2016, 21, 440; doi:10.3390/molecules21040440
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of the observations that BuChE assumes a more important role in the AD brain during disease
progression [ 10]. Another major theme has been the design of hybrids able to modulate
simultaneously several aspects of the complex Alzheimer pathobiology [11 12]. There is a lot of current
8
–
,
interest, for example, in the design of hybrid compounds able to inhibit cholinesterase and attenuate
neuronal apoptosis associated with tissue damage and reactive oxygen species [13]. The antioxidant
system loses effectiveness during the aging process and oxidative damage has been observed before
the formation of AD-specific pathological β-amyloid plaques [14]. α-Lipoic acid is an interesting
substance in this context because it plays an essential role in mitochondrial dehydrogenase reactions.
Lipoate, or its reduced form, dihydrolipoate, reacts with reactive oxygen species such as superoxide
radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen [15]. α-Lipoate
is taken up and reduced in cells and tissues to dihydrolipoate. Dihydrolipoate is also exported to
the extracellular medium; therefore, protection is afforded to both intracellular and extracellular
environments. Thus, α-lipoate would seem an ideal substance in the treatment of oxidative brain and
neural disorders involving free radical processes. Lipoic acid has previously been linked to the ChI
compounds tacrine and quinazolinimines producing potential bimodal antioxidant cholinesterase
inhibitory therapeutics for AD [16
,17]. The introduction of LA into ChIs has also been used to alter
and considerably improve ChE inhibition and isoenzyme selectivity [16
,
17]. Ferulic acid (FA) and
its ester derivatives are also attractive candidates for incorporation into a hybrid design. These are
polyphenolic compounds with potent antioxidant activity in vitro, due to their ability to scavenge
radicals [18]. They have also been shown to up-regulate cytoprotective enzymes such as heme
oxygenase-1 and heat shock protein 70, and subsequently inhibit oxidative stress and cell death in
neurons treated with beta amyloid peptide [19]. Pretreatment with ferulic acid in a mouse model was
also shown to inhibit oxidative damage induced memory deficit in vivo [20]. This evidence, taken
together, suggested that ester derivatives of ferulic acid could be brain accessible, multifunctional
compounds for the treatment of AD. Several examples have been reported of successful incorporation
of ferulic acid into ChIs [21].
The present study concerns the design of neuroprotective hybrids that could selectively inhibit
BuChE over AChE and modulate ROS damage associated. Our design for accomplishing BuChE
specific inhibitors with additional anti-oxidant activity relies on observations around some carbamates
we have reported previously. Isosorbide-2-alkyl and aryl carbamates are selective and potent inhibitors
of BuChE (e.g., 1 in Figure 1, IC50 BuChE 4.3 nM), and they are selective for BuChE over AChE and
related carboxylesterases including CE1 and CE2 found in human liver and intestine [22]. Whereas the
2-carbamate is essential to activity, we showed that the 5-position of the isosorbide scaffold could
be modified in diverse ways producing more or less potent compounds [23]. We hypothesised that
it might be possible to retain the inhibitory isosorbide-2-carbamate functionality but substitute the
effective 5-benzoate in
1 with lipoic acid or ferulic acid esters, retaining cholinesterase sub-type
selectivity while introducing new disease modifying functionality as shown in Figure 1.
S
S
2
R NH
O
Maintain
2-carbamate
NH
O
O
Ph
O
COOH
COOH
H
H
H
H
2
O
O
2
MeO
HO
5
O
O
5
O
O
O
O
Replace with
antioxidant ester
3
1
R'
Ph
Figure 1. Isosorbide-based carbamates e.g.,
lipoic acid (2) and ferulic acid (3).
1 are potent selective inhibitors of BuChE; formulae for

Molecules 2016, 21, 440
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2. Results and Discussion
2.1. Synthesis of Isosorbide-Based Carbamate-Antioxidants
Isosorbide-2-carbamate-5-nitrates (5a
mononitrate with the appropriate isocyanate in pyridine. The 2-carbamates 6a
–
d
) were obtained by direct carbamylation of isosorbide
were obtained
–
d
by removal of the nitrate group under reductive conditions (H2, Pd/C). For this work, we chose
benzyl carbamate because, in the isosorbide-2-carbamate-5-ester compounds already reported [22],
benzyl carbamates were most potent irrespective of the identity of the 5-ester. Indeed the most potent
compound we reported was a 2-benzyl carbamate with a 5-salicylate group which had an IC50 for
BuChE of <150 pM and an estimated 500,000 fold selectivity over AChE [24]. To investigate the
present design hypothesis, we also chose butyl and ethyl carbamates because these are also active in
isosorbide-5-ester cases, depending on the 5-ester identity.
Isosorbide-based phenyl carbamates tend to be more AChE than BuChE selective, so we included
a phenyl carbamate in this series to see if the pattern would be maintained and to diversify the expected
pharmacology of the product hybrid compounds. Esterification of 6a–d with lipoic acid produced 7a–d;
esterification with ferulic acid afforded 8a
–
d. Esterification was achieved using standard EDCI/DMAP
coupling (Scheme 1).
R NH
R NH
O
O
H
H
H
H
HO
2
H
H
O
O
O
O
(i)
(ii)
O
O
O
5
O
ONO2
OH
ONO2
6a
6b
, 6c
, 6d
R=Bn
R=Ph
R=Bu
R=Et
5a
5b
5c
5d
R=Bn,
R=Ph,
R=Bu
R=Et
4
R NH
O
H
O
O
Lipoate =
(iii)
S
S
O
H
O
O
R'
7a-d
8a-d
R' = Lipoate
R' = Ferulate
Ferulate =
OH
O
Scheme 1. Synthesis of hybrids 7a–d and 8a–d
. (i) RNCO, pyridine 100 ˝C, 2 h; (ii) Pd/C, H2, EtOAc,
12 h, RT; (iii) R’C(O)OH, EDCI, DMAP, DCM, RT, 16 h.
2.2. Cholinesterase Inhibition and Mechanism
Inhibitory activities of the isosorbide-2-carbamates bearing a lipoate ester (7a–d), a ferulate
ester (8a–d) ester or 5-unsubstituted compounds (6a–d) was measured using the standard Ellman
spectrophotometric method using AChE from electric eel or BuChE purified from human plasma
with acetylthiocholine or butyrylthiocholine as appropriate (Table 1). The most potent compounds
across the three series were the 5-lipoates 7a and 7c, which were 2-benzyl and -butyl carbamates.
These were potent, selective inhibitors of BuChE (IC50, 150 and 170 nM, respectively). The lipoate
esters were in each case more potent that the ferulate esters. The most potent 5-ferulate was
the butyl carbamate 8c (BuChE IC50, 430 nM). These values are in the potency range of clinically
used cholinesterase inhibitors. None of the hybrid compounds exhibited notable inhibitory effects
against AChE, apart from 8b, the phenyl carbamate which was a moderately potent inhibitor (IC50

Molecules 2016, 21, 440
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29.16 µM). In most cases, it was not possible to achieve a maximal inhibition of AChE because the
solubility threshold was lower than the concentration required for maximal inhibition. In previous
studies, we noticed that phenyl carbamates of isosorbide are relatively weak inhibitors of BuChE
compared with the benzyl and alkyl carbamates, but that AChE is relatively tolerant of isosorbide
phenyl carbamates. We published a paper showing that isosorbide-2,5-diaryl esters are moderately
potent substrate-inhibitors of AChE [25]. Both types of 5-ester in the present study—lipoate and
ferulate—were more potent than the corresponding unsubstituted compounds 6a–d. The effect of
(appropriate) 5-ester substitution in the series of isosorbide compounds already published is to increase
potency by promoting affinity in the pre-carbamylation state, reducing the pseudo Michaelis constant
for the carbamate substrate (referred to as Kc in carbamylation literature) [24]. When the intrinsic
affinity of the substrate carbamate is higher, there is a relatively greater proportion of the unimolecular
E. carbamate complex. In the present case, the anti-oxidant ester functionality directly contributes
to potency by increasing substrate affinity. The greater potency of the alkyl lipoate ester relative to
the aromatic ferulate was unexpected because in previous isosorbide-carbamate series, 5-aryl ester
carbamates were more potent. In the in vivo situation, the intact carbamate might be expected to exhibit
antioxidant properties, or the isosorbide-5-ferulate or lipoate product of carbamylation. Indeed, ester
hydrolysis at the isosorbide C-5, which is slow, would produce the ferulic acid or lipoic acid byproduct.
The compounds may therefore be classified as antioxidant acid prodrugs or, because they are more
active intact, carbamate-soft drugs.
Table 1. Carbamate–antioxidant conjugate inhibitory data for AChE and BuChE and selectivity (n = 3).
BChE IC50 µM
(pIC50 ˘ SEM)
AChE IC50 µM
(%I at 100 µM)
Selectivity †
Cpd
R
R’
7a
7b
7c
7d
8a
8b
8c
8d
6a
6b
6c
Bn
Ph
Bu
Et
Bn
Ph
Bu
Et
Bn
Ph
Bu
Et
Lipoate
Lipoate
Lipoate
Lipoate
Ferulate
Ferulate
Ferulate
Ferulate
H
0.15 (2.17 ˘ 0.03)
13.30 (4.12 ˘ 0.11)
0.17 (2.32 ˘ 0.04)
5.71 (3.76 ˘ 0.92)
1.82 (3.21 ˘ 0.02)
29.16 (4.42 ˘ 0.06)
0.43 (2.59 ˘ 0.05)
95.03 (1.98 ˘ 0.89)
25.78 (1.41 ˘ 0.03)
>100 µM
(30.75)
(18.75)
(68.70)
(84.10)
(25.20)
>667
>7.5
>55
0.92
27.1 ˘ 11.3 µM
ND ††
ND
37.5 ˘ 7.4 µM
1.45
H
H
H
(25.30)
27.98 (1.45 ˘ 0.02)
(30.0)
(22.0)
3.57
2.13
6d
46.86 (1.67 ˘ 0.03)
†
This was estimated from IC50 ratios for AChE/IC50 for BuChE: a value of 100
µM was used for AChE where
††
% inhibition at 100 µM was <50%. Not significantly different from zero.
The time dependence of BuChE inhibition associated with the pseudoirreversible carbamylation
phase was investigated for the most potent BuChE inhibitor 7a. Solutions of human BuChE were
treated with 7a in the range of 1–50
µM. At successive time intervals, aliquots were removed
and the carbamylation reaction stopped by dilution. Residual enzyme activity at each time
point and each inhibitor concentration level was estimated by adding the sample to a solution
containing butyrylthiocholine substrate. As expected, enzyme activity decayed time and concentration
dependently with pseudo first order rate constants at 50 µ µM
M of 0.198 min´1 (r2 > 0.99) and at 10
0.068 min´1 (r2 > 0.99). These are in the expected range for cholinesterease carbamylation indicating
that pseudo irreversible covalent bond formation occurred.
2.3. Neuroprotection and Antioxidant Activity
The neuroprotective properties of the compounds were assessed using the HT-22 murine
hippocampal cell line. This is an immortalized cell line which has become widely used in recent
years to study oxidative stress induced neuronal cell death. The cell line lacks ionotropic glutamate

Molecules 2016, 21, 440
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receptors but is sensitive to glutamate cytotoxicity via non-receptor mediated pathway(s) that are
to a significant extent propagated by reactive oxygen species [26 27]. The cell line appeared to be
,
a good cellular model for measuring an anti-oxidant mediated neuroprotective in vitro effect of the
compounds [26–28].
Test compounds in DMSO solution (0.1%) were added to HT-22 cell culture for (24 h), using four
repeats in three independent experiments. Some solubility issues were observed at 50
medium with the ferulate ester series. These compounds also caused a small reduction in cell viability
in the range 1–25 M which was significant only in the case of 8d at 25 M (data not shown). The
µM in culture
µ
µ
lipoates did not manifest solubility problems and did not cause any reduction in cell viability when
tested by themselves, indicating a lack of intrinsic neurotoxicity. Lipoic acid and ferulic acid were also
non-toxic in the same concentration range. Next, the neuronal cell culture was treated with glutamate
(5 mM) which caused about 90% cell death as reflected in the MTT assay. Quercetin served as positive
control and caused rescue of cell death to around 80% cell viability at 25
with test compounds 7a and 8a at 1, 5, 10 and 25 M in four repeats and three independent
experiments. Lipoic and ferulic acid were tested in parallel experiments in the same concentration
range. Lipoic acid was active in the range 10–25 M but ferulic acid was not active (data not shown).
Several compounds in the lipoate series (7a ) produced a concentration dependent effect as shown in
Figure 2. The most effective compound was the benzyl carbamate 7a, which had also been the most
potent and selective BuChE inhibitor. This was effective in the range 5–25 M, slightly more effective
than lipoic acid, which was not effective at 5 M and similar in efficacy to the positive control quercetin
at 25 M. Surprisingly, a neuroprotective effect could not be observed during treatment with the AChE
µM. Cells were co-treated
–
d
–
d
µ
µ
–
d
µ
µ
µ
phenyl carbamate 7b. The butyl and ethyl carbamates 7c and 7d showed some effects but they were
not as potent or efficacious as the benzyl carbamate. The reasons for this unexpected variation in
efficacy is not clear but may be due to (intra)cellular access. Ferulic acid and the isosorbide-based
ferulate ester series 8a–d did not exhibit any protective effects in this model in the range 1–25 µM.
120
7b
120
7a
100
100
***
***
***
80
60
40
20
0
***
80
60
40
20
0
**
+ Glu 5mM
+ Glu 5mM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
7d
7c
***
***
***
***
***
+ Glu 5mM
+ Glu 5mM
Figure 2. Neuroprotection was investigated by treating HT-22 cells in culture with glutamate (5 mM)
and lipoates 7a in the range 1–25 M (n = 3, means SD); quercetin (25 M) was used as positive
–
d
µ
˘
µ
control. One-way ANOVA followed by Dunnett’s multiple comparison post-test using GraphPad
Prism 5 Software. (Levels of significance ** p < 0.01; *** p < 0.001).

Molecules 2016, 21, 440
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The antioxidant capacity of 7d and 8a was assessed directly using the oxygen radical absorbance
capacity (ORAC) assay, in which the physicochemical ability of the test compounds to scavenge radicals
and quench fluorescein absorbance is quantified and related to so-called trolox equivalents (TE = 1
represents the antioxidant equivalence to trolox, a water-soluble Vitamin E derivative) [
6,16,29,30].
Compound 7d was 0.163 0.146, 8a, 0.635 0.020, thus showing a very weak ORAC for the lipoate
˘
˘
and a weak, but significant, effect for the ferulic acid derivative. The lack of activity of the lipoate in
the non-cellular ORAC assay is not surprising as its anti-oxidant effects are mainly related to cellular
or mitochondria related processes.
3. Materials and Methods
3.1. General Chemistry
1H and 13C spectra were recorded at 27 ˝C on a Bruker DPX 400 MHz FT NMR spectrometer
(400.13 MHz 1H, 100.61 MHz 13C) or a Bruker AV600 (600.13 MHz 1H, 150.6 MHz 13C, Bruker, Coventry,
UK). In CDCl3, 1H spectra were assigned relative to the TMS peak at 0.0 ppm, and 13C spectra were
assigned relative to the middle CDCl3 triplet at 77.00 ppm. Coupling constants were reported in hertz
(Hz). HRMS (positive mode) was performed using a Micromass mass spectrophotometer (Waters,
Herdfordshire, UK) with electrospray ionization at the School of Chemistry, Trinity College Dublin
(Dublin, Ireland).
3.1.1. General Procedure for Synthesis of Isosorbide-2-carbamates-5-nitrates
The 2-carbamate-5-nitrates were prepared as described.22 Isosorbide mono-nitrate (ISMN), (0.5 g,
2.6 mmol, 1 eq.) was dissolved in anhydrous pyridine (5 mL). Appropriate isocyanate (2 eq.) was
˝
added and the reaction mixture was heated to 100 C for 1 h, in a round bottom flask equipped with a
reflux condenser. Methanol (3.5 mL) was then added and the reaction was continued for ten minutes
to remove excess isocyanate. After this time, the reaction mixture was cooled to room temperature and
dropped onto ice-water. The resulting precipitate was removed by filtration, washed with ice-water
and recrystallised using ethanol to yield pure isosorbide-2-carbamate-5-nitrate. Spectroscopic and
chemical properties of 5a–d were as reported [22].
3.1.2. General Procedure for the Preparation of Isosorbide-2-carbamates (GP2)
Isosorbide-2-benzylcarbamate (6a). Isosorbide-2-benzylcarbamate-5-nitrate (0.5 g, 1.5 mmol) was
dissolved in 100 mL of a 1:1 mixture of ethyl acetate and methanol in a round bottom flask and
palladium on activated carbon (50 mg, 10% w/w) was added. Air was expelled from the flask and the
reaction mixture was stirred under an atmosphere of hydrogen for 24 h. After this time palladium
removed by filtration through a pad of celite, solvent was removed under reduced pressure and pure
˝
(5) was recrystallised from ethanol as white crystals (0.428 g, 100%). mp 76.0 C. Spectroscopic features
were as described previously [22].
Isosorbide-2-phenylcarbamate (6b). Yield 100%. 1H-NMR (400 MHz, CDCl3)
δ 7.4 (4H, m), 7.1 (1H,
t, J = 7.5 Hz), 6.8 (1H, br s), 5.3 (1H, d, J = 3 Hz), 4.7 (1H, t, J = 5 Hz), 4.5 (1H, d, J = 3 Hz), 4.3
(1H, q, J = 6 Hz), 4.1 (1H, d, J = 10 Hz), 4.0 (1H, dd, J = 3.5, 10 Hz), 3.9 (1H, q, J = 6 Hz), 3.6 (1H,
m).13C-NMR (100 MHz, CDCl3) ppm 151.7, 136.9, 128.7, 123.4, 118.2, 85.2, 81.5, 78.5, 73.2, 73.1, 71.9.
HRMS C13H15NO5 [M + Na]+ requires 288.0842, found 288.0891.
1
Isosorbide-2-butylcarbamate (6c). Yield 100%. H-NMR (400 MHz, CDCl3)
δ 5.2 (1H, d, J = 3 Hz), 4.8
(1H, br s), 4.6 (1H, t, J = 5 Hz), 4.5 (1H, d, J = 3 Hz), 4.3 (1H, q, J = 6 Hz), 4.1 (1H, d, J = 10 Hz), 4.0 (1H,
dd, J = 3.5, 10 Hz), 3.9 (1H, q, J = 6 Hz), 3.6 (1H, m), 3.2 (2H, m), 1.6 (2H, m), 1.4 (2H, m) 0.95 (3H, t,
J = 7.5 Hz). 13C-NMR (100 MHz, CDCl3) ppm. 154.6, 85.3, 81.5, 78.1, 73.4, 73.1, 71.9, 40.6, 31.4, 19.4,
13.3. HRMS C11H20NO5 [M + H]+ requires 246.1336, found 248.1366.

Molecules 2016, 21, 440
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Isosorbide-2-ethylcarbamate (6d). Yield 100%.1H-NMR (400 MHz, CDCl3)
δ
5.2 (1H, d, J = 3 Hz), 4.6 (1H,
t, J = 5 Hz), 4.5 (1H, d, J = 3 Hz), 4.3 (1H, q, J = 6 Hz), 4.1 (1H, d, J = 10 Hz), 4.0 (1H, dd, J = 3.5, 10 Hz),
3.9 (1H, q, J = 6 Hz), 3.6 (1H, m), 3.2 (2H, t, J = 6 Hz), 2.5 (1H, br s), 1.1 (3H, t, J = 7.5 Hz). 13C-NMR
(100 MHz, CDCl3) ppm 154.5, 85.3, 81.4, 78.0, 73.4, 73.0, 71.9, 35.5, 14.7. HRMS C9H16NO5 [M + H]+
requires 218.1083, found 218.1104.
3.1.3. General Procedure for Synthesis of Lipoic Acid Esters (GP3)
Isosorbide-2-benzylcarbamate-5-lipoic acid ester (7a). Isosorbide-2-benzylcarbamate (0.3 g, 1.07 mmol),
lipoic acid (0.266 g, 1.28 mmol, 1.2 eq.) and dimethyl amino pyridine (0.157, 1.28 mmol, 1.2 eq.) were
dissolved in anhydrous DCM (150 mL) and stirred for 10 min at room temperature in the presence of
3 Å molecular sieves. N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI, 0.245 g,
1.28 mmol, 1.2 eq.) was then added and the reaction mixture was stirred in the dark for 12 h, filtered
and concentrated under reduced vacuum. The concentrated reaction mixture was purified by column
chromatography using silica gel without further preparation. Methanol (5%) in chloroform was used
1
as eluent to give (7a) as a pale yellow solid (0.145 g, 31%). H-NMR (CDCl3)
δ
ppm: 7.34 (5H, m, n),
5.15 (2H, m, m), 4.82 (1H, d, J = 5 Hz, k), 4.53 (1H, d, J = 4.5 Hz), 4.40 (2H, d, J = 6 Hz), 4.05–3.95 (4H,
m,), 3.6 (1H, m), 3.15 (2H, m,), 2.55 (1H, m), 2.47 (2H, m), 1.94 (1H, m), 1.68 (4H, m), 1.52 (2H, m).
13C-NMR (CDCl3): 172.4, 154.8, 137.6, 128.3, 127.2, 127.1, 85.6, 80.2, 78.0, 76.8, 73.2, 69.9, 55.9, 44.7, 39.8,
38.0, 34.1, 33.4, 33.3, 30.5, 28.2, 25.1. HRMS C22H29NO6S2 [M + Na]+ requires 488.1536 found 488.1531.
1
Isosorbide-2-phenylcarbamate-5-lipoic acid ester (7b). Yield 45%. Pale yellow crystals. H-NMR (CDCl3)
δ
ppm: 7.4 (4H, m), 7.1, (1H, t, J = 7.5 Hz), 5.15 (2H, m), 4.82 (1H, d, J = 5 Hz), 4.53 (1H, d, J = 4.5 Hz),
4.05–3.95 (4H, m), 3.65 (1H, m), 3.15 (2H, m), 2.55 (1H, m), 2.47 (2H, m), 1.94 (1H, m), 1.68 (2H, m).
13C-NMR (CDCl3): 172.4, 151.7, 136.9, 128.7, 123.4, 118.2, 85.2, 81.5, 78.5, 73.2, 73.2, 71.9, 55.9, 39.8, 38.0,
34.1, 33.4, 28.2, 25.1. HRMS: C21H27NO6S2 [M + H]+ requires 454.1353, found 454.1358.
1
Isosorbide-2-butylcarbamate-5-lipoic acid ester (7c). Yield 60%. H-NMR (CDCl3)
δ ppm: 5.15 (2H, m),
4.82 (1H, d, J = 5 Hz), 4.53 (1H, d, J = 4.5 Hz), 4.05–3.93 (4H, m), 3.60 (1H, m), 3.25–3.15 (4H, m), 2.55
(1H, m), 2.47 (2H, m), 1.94 (1H, m), 1.68 (4H, m), 1.59 (2H, m), 1.52 (2H, m), 1.45 (2H, m), 0.95 (3H, t,
J = 7.5 Hz). 13C-NMR (CDCl3): 172.4, 154.6, 85.3, 81.5, 78.1, 73.4, 73.1, 71.9, 55.8, 40.6, 39.8, 38.0, 34.1,
33.4, 31.4, 28.2, 25.1, 19.4, 13.3. HRMS: C19H31NO6S2 [M + H]+ requires 434.1666, found 434.1670.
1
Isosorbide-2-ethylcarbamate-5-lipoic acid ester (7d). Yield 56%. H-NMR (CDCl3)
δ
ppm: 5.15 (2H, m), 4.82
(1H, d, J = 5 Hz), 4.53 (1H, d, J = 4.5 Hz), 4.05–3.93 (4H, m), 3.60 (1H, m) 3.25 (2H, m), 3.15 (2H, m), 2.55
(1H, m), 2.47 (2H, m), 1.94 (1H, m), 1.68 (4H, m), 1.52, (2H, m), 1.10 (3H, t, J = 7.5 Hz). 13C-NMR (MHz
100.16, CDCl3): 172.4, 85.3, 81.4, 78.0, 73.4, 73.0, 71.9, 55.9, 39.8, 38.0, 35.6, 34.1, 33.4, 28.2, 25.1, 14.7.
HRMS: C17H27NO6S2 [M + H]+ requires 406.1353, found 406.1358.
3.1.4. General Procedure for Synthesis of Carbamate Ferulates 8a–d (GP4)
Isosorbide-2-benzylcarbanate-5-ferulate (8a). Isosorbide-2-benzylcarbamate (0.3 g, 1.07 mmol), ferulic
acid (0.266 g, 1.28 mmol, 1.2 eq.) and dimethyl amino pyridine (0.157, 1.28 mmol, 1.2 eq.) were
dissolved in anhydrous DCM (150 mL) and stirred for 10 min at room temperature in the presence
of 3 Å molecular sieves. N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI,
0.245 g, 1.28 mmol, 1.2 eq.) was then added and the reaction mixture was stirred in the dark for 12 h,
filtered and concentrated under vacuum. The concentrated reaction mixture was purified by column
chromatography using silica gel without further preparation. Methanol (5%) in chloroform was used
1
as eluent to give (
J = 16.1 Hz), 7.40
6
´
) as a pale yellow solid (0.145 g, 31%). H-NMR
δ
(400 MHz, CDCl3): 7.56 (1H, d,
7.23 (m, 5H), 7.0–6.83 (3H, m) 6.22 (1H, d, J = 16.1 Hz), 5.84 (1H, s), 5.22 (d, 1H,
J = 3.01 Hz), 5.17 (m, 1H), 4.61 (t, 1H, J = 4.77 Hz), 4.51 (d, 1H, J = 4.01 Hz), 4.38 (d, 2H, J = 6.03 Hz),
4.31 (m, 1H), 4.10 (d, 1H, J = 10.45 Hz), 4.00 (dd, 1H, J = 3.51, 10.54 Hz), 3.90 (4H, m), 3.57 (dd, 1H,
J = 5.53, 8.54 Hz), 2.67 (d, 1H, J = 7.03 Hz). 13C-NMR ppm (100.16 MHz., CDCl3): 44.7, 55.8, 71.8, 73.0,

Molecules 2016, 21, 440
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73.4, 78.4, 81.5, 85.3, 109.3, 114.6, 114.8, 122.8, 126.6, 127.1, 127.2, 128.3, 137.6, 144.8, 146.6, 147.8,9 154.7,
167.6. HRMS: C24H25NO8 [M + H]+ requires 456.1653, found 456.1670.
1
Isosorbide-2-phenylcarbanate-5-ferulate (8b). Yield 65%. H-NMR (400 MHz, CDCl3)
δ ppm: 7.56 (1H,
d, J = 16.1 Hz), 7.41 (4H, m), 7.11 (1H, t, J = 7.5 Hz), 7.0–6.85 (3H, m), 6.80 (1H, br s), 6.22 (1H, d,
J = 16.1 Hz), 5.84, (1H, s) 5.31 (1H, d, J = 3 Hz), 4.72 (1H, t, J = 5 Hz), 4.53 (1H, d, J = 3 Hz), 4.33 (1H, q,
J = 6 Hz), 4.10 (1H, d, J = 10 Hz), 4.00 (1H, dd, J = 3.5, 10 Hz), 3.95 (1H, s), 3.90 (1H, q, J = 6 Hz), 3.61
(1H, m). 13C-NMR (100 MHz, CDCl3) ppm. 167.6, 151.7, 147.8, 146.6, 144.8, 136.9, 128.7, 126.6, 123.4,
118.2, 114.8, 114.7, 109.6 85.2, 81.5, 78.5, 73.2, 73.1, 71.9, 55.8. HRMS: C23H23NO8 [M + H]+ requires
442.1496, found 442.1495.
1
Isosorbide-2-butylcarbanate-5-ferulate (8c). Yield 60%. H-NMR (400 MHz, CDCl3)
δ
ppm: 7.60 (1H, d,
J = 16.1 Hz), 7.12 – 6.90 (3H, m), 6.25 (1H, d, J = 16.1 Hz), 5.88 (1H, s), 5.23 (1H, d, J = 3 Hz), 4.82 (1H,
br s), 4.61 (1H, t, J = 5 Hz), 4.53 (1H, d, J = 3 Hz), 4.39 (1H, q, J = 6 Hz), 4.11 (1H, d, J = 10 Hz), 4.01
(1H, dd, J = 3.5, 10 Hz), 3.91 (2H, m), 3.60 (1H, m), 3.21 (2H, m), 1.61 (2H, m), 1.40 (2H, m), 0.95 (3H, t,
J = 7.5 Hz). 13C-NMR (100.16 MHz, CDCl3) ppm. 167.8, 154.6, 147.7, 146.7, 144.9, 126.5, 114.9, 114.6,
109.6, 85.3, 81.5, 78.1, 73.4, 73.1, 71.9, 55.79, 40.6, 31.4, 19.4, 13.3. HRMS: C21H27NO8 [M + H]+ requires
422.1809, found 422.1799.
1
Isosorbide-2-ethylcarbanate-5-ferulate (8d). Yield 60%. H-NMR (CDCl3)
δ ppm: 7.56 (1H, d, J = 16.1 Hz),
7.09–6.83 (3H, m), 6.22 (1H, d, J = 16.1 Hz), 5.87 (1H, s) 5.21 (1H, d, J = 3 Hz), 4.60 (1H, t, J = 5 Hz), 4.53
(1H, d, J = 3 Hz), 4.33 (1H, q, J = 6 Hz), 4.06 (1H, d, J = 10 Hz), 4.0 (1H, dd, J = 3.5, 10 Hz), 3.91 (2H, m),
3.60 (1H, m), 3.21 (2H, t, J = 6 Hz), 2.51 (1H, br s), 1.11 (3H, t, J = 7.5 Hz). 13C-NMR (100 MHz, CDCl3)
ppm 167.6, 154.5, 147.9, 146.6, 144.8, 126.6, 114.8, 114.7, 109.6, 85.3, 81.4, 78.0, 73.4, 73.0, 71.9, 55.8, 35.5,
14.7. HRMS: C19H23NO8 [M + H]+ requires 394.1496, found 394.1501.
3.2. Biochemical Studies
3.2.1. Ellman Assay for Measuring Inhibition of Cholinesterase Activity
Cholinesterase activity was measured using the Ellman spectrophotometric method. HuBuChE
activity was measured in replicate samples using a 96-well plate reader. The total volume of test
solution in each well was 250
pH 8.0, 25 L of DTNB solution (0.5 mM), and 25
plate was incubated for 30 min before 25 L of butyrylthiocholine iodide (BTCI) solution (0.5 mM) was
added, and the reaction was measured at 405 nm over 5 min using an Anthos bt2 plate reader. For the
determination of AChE activity, 25 L of electric eel AChE solution and 25 L of acetylthiocholine
iodide (ATCI) solution were used instead of the plasma solution and BTCI solution. For determination
of enzyme inhibition, 25 L of an inhibitor solution was added to the test solution instead of the
µL. This consisted of 25
µL of plasma solution, 150 µL of phosphate buffer
µ
µL of acetonitrile:distilled water (1:1). The 96-well
µ
µ
µ
µ
acetonitrile:water (1:1) solution. Inhibitors were added 30 min before determination of remaining
enzyme activity. IC50 values were calculated using GraphPad Prism 4.02 software (GraphPad Software
Inc., La Jolla, CA, USA).
3.2.2. Inhibitor Kinetics
In order to monitor enzyme carbamylation over time, a 96-well plate was used, with all assay
mixtures being made up to a final concentration of 250
At time 0, 150 L of inhibitor, at various concentrations, was added to an equal volume of enzyme.
Thereafter, at successive time points, 50 L aliquots were removed from these wells and added to
wells containing 200 L DTNB and BTCI in phosphate buffer pH 8.0. Hydrolysis of substrate was
µL for monitoring of absorbance at 412 nm.
µ
µ
µ
monitored by examining the changes in absorbance in each well at 412 nm over the following 30 s.
Negative controls were run alongside the test solutions to take account of non-enzymatic hydrolysis,
while positive controls gauged the rate of the uninhibited reaction. At least two replicate wells were
run for each concentration of inhibitor. Plots of percentage inhibition over time were prepared using

Molecules 2016, 21, 440
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GraphPad Prism 4.03. A first order inhibition constant, kobs was obtained for each concentration of
inhibitor by non-linear regression using an exponential decay function.
3.2.3. MTT Assay and Neuroprotection of HT-22 Neurons
This test was performed as reported before. Briefly, HT-22 cells were seeded in 96-well plates at a
density of 5
ˆ
103 per well and cultured for 24 h. Subsequently, cells were incubated for another 24 h
either with medium or test compounds with potential cytoprotective activity either in the absence (to
test for toxic effects) or the presence (to test for the protective potential against glutamate induced
oxidative stress) of 5 mM glutamate. MTT solution (4 mg/mL in PBS) was diluted 1:10 with medium
and the mixture was added to the wells after removal of the previous medium. The plates were then
incubated for another 3 h. Then, the supernatant was removed and 100 µL of lysis buffer (10% SDS,
pH 4.1) were added to the wells. Absorbance at 560 nm was determined on the next day using a
multiwell plate photometer (Spectra Fluor Plus, Tecan, Crailsheim, Germany). The results of these cell
viability assays were expressed as percentages relative to untreated control cells. All compounds were
dissolved in DMSO and diluted with fresh medium, with the DMSO concentration always below 0.1%
(v/v).
3.2.4. Statistics
Data of all MTT tests were expressed as means
quadruplicates). Data were subjected to one-way ANOVA followed by Dunnett’s multiple comparison
˘
SD of at least three independent experiments (in
post-test using GraphPad Prism 5 Software. (Levels of significance ** p < 0.01; *** p < 0.001).
3.2.5. ORAC Assay
Compounds were assessed for antioxidant capacity using the standard ORAC assay performed
as described previously [29–31].
4. Conclusions
Ferulate and lipoate esters as potential antioxidants were successfully introduced into the
isosorbide-2-carbamate scaffold, which has previously been shown to be useful in preparation of
highly potent and selective cholinesterase inhibitors. The resultant compounds were shown to inhibit
BuChe with potency and selectivity. Moreover, in both lipoate and ferulate series, the integrated
anti-oxidant promoted carbamylation or increased potency relative to the unsubstituted compounds
by enforcing hydrophobic interactions with the cholinesterase active site. Compound 7a is a potent
(IC50 150 nM) and selective inhibitor (667 fold) of BuChe over AChE, and, in the neuroprotection assay,
it was efficacious in preventing glutamate induced cytotoxicity in a murine hippocampal cell line in the
range of 10–25 µM. The ferulate esters and parent compound were not active in this model. This work
shows the versatility of the isosorbide scaffold in the design of esterase directed compounds.
Acknowledgments: This work was funded by Science Foundation Ireland.
Author Contributions: John F. Gilmer directed the compound design and synthesis and cholinesterase assay,
Mike Jones carried out synthesis, Jun Wang and Shona Harmon performed cholinesterase assays, Jörg Heilmann
directed neuroprotection and antioxidant assays, Beata Kling carried these out.
Conflicts of Interest: The authors declare no conflict of interest.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).