Isosorbide-based cholinesterase inhibitors; replacement of 5-ester groups leading to increased stability

Article abstract of DOI:10.1016/j.bmc.2009.12.052

Isosorbide-2-carbamate-5-esters are highly potent and selective butyrylcholinesterase inhibitors with potential utility in the treatment of Alzheimer's Disease (AD). They are stable in human plasma but in mouse plasma they undergo hydrolysis at the 5-ester group potentially attenuating in vivo potency. In this paper we explore the role of the 5-position in modulating potency. The focus of the study was to increase metabolic stability while preserving potency and selectivity. Dicarbamates and 5-keto derivatives were markedly less potent than the ester class. The 2-benzylcarbamate-5-benzyl ether was found to be potent (IC₅₀ 52 nM) and stable in the presence of mouse plasma and liver homogenate. The compound produces sustained moderate inhibition of mouse butyrylcholinesterase at 1 mg/kg, IP.

Full text of DOI:10.1016/j.bmc.2009.12.052

Bioorganic & Medicinal Chemistry 18 (2010) 1045–1053
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Isosorbide-based cholinesterase inhibitors; replacement of 5-ester groups
leading to increased stability
Gerald P. Dillon a, Joanne M. Gaynor b, Denise Khan a, Ciaran G. Carolan a, Sheila A. Ryder a, Juan F. Marquez a,
Sean Reidy b, John F. Gilmer a,
*
a School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland
b School of Science, Athlone Institute of Technology, Westmeath, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Isosorbide-2-carbamate-5-esters are highly potent and selective butyrylcholinesterase inhibitors with
potential utility in the treatment of Alzheimer’s Disease (AD). They are stable in human plasma but in
mouse plasma they undergo hydrolysis at the 5-ester group potentially attenuating in vivo potency. In
this paper we explore the role of the 5-position in modulating potency. The focus of the study was to
increase metabolic stability while preserving potency and selectivity. Dicarbamates and 5-keto deriva-
tives were markedly less potent than the ester class. The 2-benzylcarbamate-5-benzyl ether was found
to be potent (IC50 52 nM) and stable in the presence of mouse plasma and liver homogenate. The com-
pound produces sustained moderate inhibition of mouse butyrylcholinesterase at 1 mg/kg, IP.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 9 October 2009
Revised 13 December 2009
Accepted 20 December 2009
Available online 6 January 2010
Keywords:
Butyrylcholinesterase inhibitor
Selectivity
Metabolic stability
1. Introduction
important role in neurotransmission, at least in the healthy brain.
The several drugs on the market for AD are therefore more or less
The cholinesterases are well established targets now for drugs
used to treat glaucoma, myasthenia gravis and Alzheimer’s Disease
(AD).1–3 Cholinesterase inhibitors also find application in chemo-
protection from organophosphate poisoning.4 There are two types
of cholinestesterase-AChE (3.1.1.7) and BuChE (3.1.1.8). Greatest
attention has been paid to AChE due to its classical actions in reg-
ulating synaptic acetylcholine and because of less clearly defined,
non-enzymatic or non-classical actions. The cholinesterase inhibi-
tion strategy in AD treatment arises from the recognition that the
cognitive deficits arising in the disease correlate with diminished
cholinergic neurotransmission. By inhibiting AChE the functional
pool of AChE in the cholinergic neurons is augmented.
selective for AChE. There has been a surge in interest in BuChE as a
potential target for AD treatment in recent years due to a number
of factors: (i) the nullizygote (AChE ꢀ/ꢀ) mouse while having a
clear phenotype appears to have normal CNS function, indicating
at least a compensatory role for BuChE in the CNS5; (ii) while syn-
aptic AChE levels decrease markedly in AD progression, there is a
corresponding increase in BuChE activity6; (iii) post mortem tissue
analysis on AD patients shows a high level of BuChE in the hall-
mark lesions of AD.7 In rats, the selective BChE inhibitor cymserine
causes elevation of acetylcholine and augments long term potenti-
ation and learning.8
Isosorbide-2-carbamate-5-esters (Fig. 1) are nanomolar BuChE
inhibitors with very high selectivity over AChE (up 60,000-fold).9
Although they possess a 5-ester group, they are stable in human
plasma because inhibitory interactions with BuChE (arising as a re-
sult of enzymatic interactions with the 2-carbamate functionality)
are preferred over substrate (5-ester) interactions. However, dur-
ing in vivo experiments in a mouse model of memory and learning
it became apparent that compound 1a (R0 = Ph) was undergoing
hydrolysis in plasma and liver tissue at the 5-ester position gener-
ating the less potent isosorbide-2-carbamate and benzoic acid.10
Mouse plasma possesses carboxylesterases which are absent in hu-
man plasma, and the undesirable degradation of the esters could
be attributed to these enzymes. In this paper we investigate the
relationship between 5-substituent and esterase inhibition in or-
der to better understand the SAR for the class and to find a more
BuChE has generally received less attention mainly because its
biological function is not as clearly defined as is that of AChE and
although it is more ubiquitous than AChE its distribution in the
CNS has not traditionally been recognized as consistent with an
Abbreviations: AChE, acetylcholinesterase; ATCI, acetylthiocholine iodide; BTCI,
butyrylthiocholine iodide; BuChE, butyrylcholinesterase; DCC, dicyclohexylcarbo-
diimide; DTNB, 5,50-dithiobis-(2-nitrobenzoic acid); huBuCHE, human butyrylcho-
linesterase; ISMN, isosorbide-5-mononitrate; ISMNA, isosorbide mononitrate
aspirinate; MAO, monoamino oxidase; SAR, structure–activity relationship, TBAF,
tetrabutylammonium fluoride; TBDMS, tertbutyldimethylsilyl; TLC, thin layer
chromatography; TMS, tetramethylsilane.
* Corresponding author. Tel.: +353 1 896 2795; fax: +353 1 896 2793.
E-mail address: gilmerjf@tcd.ie (J.F. Gilmer).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.052

1046
G. P. Dillon et al. / Bioorg. Med. Chem. 18 (2010) 1045–1053
H
H
H
R
N
O
BzN
O
BzN
O
O
H
H
O
H
H
O
H
H
O
O
O
2
O
O
N
O
O
O
5
O
O
1
2
O
R
3
R'
H
5-ketone and derivatives
5-ester
2,5-dicarbamates
vulnerable to murine carboxylesterases
Figure 1. General strategy for increasing metabolic stability towards rodent carboxylesterases.
stable but potent and selective BuChE inhibitor that could be used
to probe the role of the enzyme in the mouse.
using N-methylhydroxylamine in the presence of sodium acetate
generating lactam 11 (the intermediate methyl ketoxime arising
carried through without purification). Wittig reaction on 3 using
the commercially available ylide, carbethyloxymethylene triphen-
ylphosphorane yielded the alkenyl ester (12). The beta-amide 16
(Scheme 4) was accessed via a different strategy. Isoiodide (14)
was generated from the commercially available isomannide (13)
by bistosylation followed by SN2 substitution with sodium acetate
and hydrolysis. Monotosylation of isoiodide 14 was accomplished
by treating the diol with a single equivalent of tosyl chloride at
low temperature. The monotosylate was isolated by chromatogra-
phy and treated with sodium azide to afford the 5-azide, isosor-
bide-5-deoxyazide (15). The azide, which was easier to handle
than the corresponding amine was carbamylated at the 2-position
using benzyl isocyanate and a tertiary base. The 5-azido group was
then reduced to beta-amine and acylated with benzoyl chloride to
afford the beta-amide 16 (Scheme 5).
Lastly, a number of 5-ether compounds were prepared as the
ether functionality is resistant to esterase-induced hydrolysis.
Treatment of 6 with benzyl or phenylethyl bromide yielded insuf-
ficient target ether because of concomitant alkylation of the 2-car-
bamate nitrogen. Therefore, ISMN was TBDMS protected, the 5-
nitrate removed by reduction yielding 17 and the 5-OH group
alkylated. The TBDMS protection was then removed and the 2-po-
sition converted to benzylcarbamate resulting in the benzyl and
phenylpropyl ethers 18a and 18b.
2. Results and discussion
2.1. Chemistry
Dicarbamates (2a–2f) were prepared by heating isosorbide (4)
in dry pyridine in the presence of an excess of the appropriate iso-
cyanate for 1–2 h at 100 °C (Scheme 1). The excess isocyanate was
removed by adding methanol and the mixture poured into ice-
water. The target dicarbamates precipitated over a couple of hours.
The clinically used isosorbide mononitrate (ISMN) 5 is a conve-
nient material for exploring isosorbide chemistry because the dif-
ficult step of introducing regioselectivity has already been
achieved and the nitrate is easily removed by reduction after elab-
oration of the 2-position. As reported previously isosorbide benzyl
carbamates have optimal potency and selectivity for BuChE. There-
fore the 2-position of ISMN (5) was carbamylated using benzyl iso-
cyanate. Selective removal of the 5-nitrate was effected using Pd/C
over H2 and the triflate ester introduced using triflic anhydride. The
alkene 7 was obtained by elimination of the intermediate triflate
using DBU (Scheme 2). The 5,6-ene was distinguished from the po-
tential isomeric 4,5-ene compound by the collapse of the 6-meth-
ylene group in the proton NMR and by the presence of signals for
the 5- and 6-methine hydrogens.
The 5-ketone 3 was synthesised in order to determine the effect
of flattening the isosorbide ring on ligand–BuChE interactions, and
because the ketone is not susceptible to esterase-mediated metab-
olism. The ketone (3) was produced in good yield from isosorbide-
2-benzylcarbamate-5-OH (6) using PCC in DCM with silica gel
catalysis (Scheme 3). A number of classical or established ketone
transformations were then attempted (Scheme 3). The 5-oxime
(8) was generated by stirring with hydroxylamine hydrochloride
in aqueous solution at high temperature. The oxime benzoate ester
(9) was produced following acylation of 8 with benzoyl chloride
and DMAP catalysis. Baeyer Villiger oxidation was accomplished
by stirring 3 with mCPBA in DCM generating lactone 10. Rear-
rangements of isosorbide-based ketones proceed with the ex-
pected migration of the more substituted alpha 4-carbon. The
analogous Beckmann rearrangement was also performed on 3
2.2. Cholinesterase inhibition
Cholinesterase inhibition was determined using the Ellman
method11 with human plasma BuChE or electric eel AChE with bu-
tyryl- or acetyl-thiocholine as substrates as appropriate Table 1.
The test compounds were incubated in the presence of enzyme
for 30 min at 100 lM prior to addition of substrate. The inhibition
experiments were repeated at successively lower concentration to
determine the 30 min IC50 only where there was significant inhibi-
tion (>90%) at 100 lM. This corresponded to the solubility limit for
the compounds in the assay medium. The seven dicarbamates (2)
were tested first as these were the easiest to make. The diethyl
compound 2a was a moderately potent AChE inhibitor (IC50
6.5 lM) whereas the dibutyl compound 2c was a moderately po-
,
H
R
N
O
O
HO
2a R =ethyl
H
H
H
2b R= propyl
O
O
(i)
2c R=butyl
2d R=phenyl
O
O
O
2e R=4-nitrophenyl
2f R=4-chlorophenyl
2g R=benzyl
H
O
OH
N
R
4
2
H
Scheme 1. Preparation of the dicarbamates 2a–g: (i) isocyanate, pyr, 100 °C, 1–2 h, 50–70%.

G. P. Dillon et al. / Bioorg. Med. Chem. 18 (2010) 1045–1053
1047
H
H
BzN
BzN
O
HO
H
H
O
H
H
O
H
H
O
(i), (ii)
(iii), (iv)
O
O
O
O
O
O
7
ONO2
5
6
OH
Scheme 2. The preparation of alkene compound 7. Reagents and conditions: (i) benzyl isocyanate, Et3N, DMAP, DCM, 40 °C, 2 h; (ii) Pd/C, H2, EtOAc, 12 h, rt; (iii) Tf2O, Et3N,
DCM; (iv) DBU, Tol, 6 h, rt, 16%.
H
H
BzN
O
BzN
O
O
O
H
H
H
O
O
9
O
O
O
O
H
N
O
O
12
Ph
Et
(vi)
(iii)
H
H
H
BzN
BzN
O
O
O
H
H
(i)
(ii)
O
O
O
6
8
O
O
3
H
N
OH
O
(v)
(iv)
H
H
O
BzN
O
BzN
O
O
H
H
O
O
O
N
O
O
11
O
H
O
10
H
CH3
Scheme 3. The preparation of ketone derivatives. Reagents and conditions: (i) PCC, DCM, rt, 8 h; (ii) NH2OHꢁHCl, MeOH/H2O (2:1), 4 h, 85 °C; (iii) PhC(O)Cl, Et3N, DMAP, DCM,
4 h, rt, 78%; (iv) mCPBA, NaHCO3, DCM, 12 h, 0 °C, 39%; (v) MeNHOH, NaOAc, EtOH, N2, 4 h, rt then TsCl, Pyr, 6 h, 0 °C, 35%; (vi) Ph3PCHC(O)OEt, THF, 0 °C, 6 h, 45%.
H
H
BzN
O
BzN
O
HO
HO
O
H
H
H
H
H
O
H
H
O
O
O
(iii)
(iv)
(i)
O
(vi)
(ii)
(v)
O
O
O
(vii)
O
H
OH
OH
N
N3
16
15
13
14
O
H
Scheme 4. The preparation of 5- amide 16. Reagents and conditions: (i) 4 equiv TsCl, pyr, rt; (ii) NaOAc, MeOH, then NaOH, H2O; (iii) 1 equiv TsCl, Pyr, 24 h, 0 °C, 59%; (iv)
NaN3, DMSO, 125 °C, 24 h, 64%; (v) benzyl isocyanate, Et3N, DMAP, DCM, 40 °C, 2 h; (vi) Pd/C, H2, EtOAc/MeOH 24 h, rt; (vii) PhC(O)Cl, Et3N, DMAP, DCM, 4 h, rt, 20% from (14).
H
BzN
O
tBDMSO
i, ii
H
H
HO
H
H
O
O
H
H
O
iii, iv, v
O
18a R= Bn
O
18b R= (CH2)3Ph
O
O
OH
17
ONO2
18
OR
5
Scheme 5. The preparation of 5-ethers. Reagents and conditions: (i) tBDMSCl, imidazole, DMAP, DCM, 28 h, rt; (ii) H2, Pd/C, MeOH/EtOAc, 24 h, rt; (iii) NaH, RBr, 24 h, THF, rt,
56%; (iv) TBAF, THF, rt, 15 min; (v) benzyl isocyanate, pyr, 105 °C, 2 h.

1048
G. P. Dillon et al. / Bioorg. Med. Chem. 18 (2010) 1045–1053
Table 1
2.3. Mouse cholinesterase inhibition
Inhibition data for BuChE and AChE using human plasma BuChE or erythrocyte AChE
(n = 6 sd IC50 or mean% inhibition)
To confirm that the potent ether compound (18a) was stable in
mouse plasma and could effect BuChE inhibition in the mouse
model, the compound was administered to mice (n = 3) at 1 mg/
kg IP. As shown in Figure 2, administration was associated with
significant inhibition of peripheral plasma BuChE activity. More
crucially, this inhibition was apparent for more than 72 h, indicat-
ing that the compound was stable and not subject to carboxylest-
erase-mediated degradation as was the case for the ester
compounds described previously. This compound will be useful if
the mouse model is to be used for examining the consequences
of BuChE inhibition in vivo.
BuChE
% Inhibition at 100
AChE
Selectivitya
l
M or IC50 (lM)
2a
2b
2c
2d
2e
2f
55.12%
10.4 1.2
1.22 0.28
20.6%
17.4%
7.96%
6.5 1.1
28%
13%
77%
5.4 1.4
—
77%
10%
9.8%
83%
9%
10%
6.5%
4.9%
5%
46%
55%
51%
AChE 15.4
BuChE 9.6
BuChE 81
—
AChE 18.5
—
BuChE 250
—
BuChE 9.7
—
—
—
2g
3
0.4 0.06
46%
7
8
10.25 0.8
40%
9
64.8%
2.4. Discussion
10
11
12
15
16
18a
18b
56%
63%
4.8 0.9
50.4%
88%
—
BuChE 21
—
—
BuChE 1923
BuChE 497
At first glance the SAR of the isosorbide-based carbamates of
interest in this article appears complex. However, on close inspec-
tion, a number of patterns emerge.
0.052 0.007
0.201 0.043
Amongst the series as a whole, the compounds tend to have a
preference for BuChE inhibition over AChE inhibition—a pattern
that is in keeping with all isosorbide-based carbamate inhibitors
described to date.9,12 The di-carbamate compounds are the only
ones that show significant inhibition of AChE amongst those tested
herein, with both the di-ethyl and di-4-nitrophenyl carbamates
being most potent. This result is not entirely surprising when the
SAR of the compounds described previously is considered. Aside
from the di-carbamates, all other compounds described in this arti-
cle have a benzyl carbamate moiety at the 2-position of isosorbide.
Previous work has shown that the presence of a benzyl carbamate
at this position is optimal for BuChE inhibition, and in fact in-
creases selectivity over AChE inhibition to a reasonable extent,
helping to explain this first finding.
a
Calculated from the ratio of BuChE or AChE IC50 or using a value of 100 lM
where the IC50 value was not determined.
tent and selective BuChE inhibitor (IC50, 1.2 lM). The most potent
BuChE inhibitor in the series of di-carbamates tested was the di-
benzyl carbamate 2g (IC50, 0.4 V). Amongst AChE inhibitors, the
di-4-nitrophenyl carbamate 2e was most potent (IC50, 5.4 M).
Interestingly, the di-4-chlorophenyl carbamate 2f failed to inhibit
either cholinesterase enzyme to any significant extent.
l
l
The 5-ketone, 5-oxime and 5-oxime benzoate ester derivatives
of isosorbide were all poor inhibitors of both cholinesterase en-
zymes. This suggested that flattening of the functionality at the
5 position so that it was planar with the attached ring of isosor-
bide was detrimental to inhibition in both instances. In all other
instances tested and described previously as well as in this arti-
cle, the 5-group of active inhibitors was always positioned endo
to the V-shaped isosorbide ring, and this result confirms that this
orientation is important for activity. It was interesting that com-
pound 12, however, which included an ethyl ester group attached
to a planar alkene functionality—which itself would be planar
with the isosorbide ring in a similar manner to the ketone and
oxime compounds—was a moderately potent BuChE inhibitor
Modelling studies described previously help to explain this. The
cholinesterase enzymes are members of the
a/b-hydrolase fold
family of enzymes, and have their active sites located at the base
of a gorge some 20 Å deep.13,14 A number of important residues
and subsites that form this gorge have been described. Our own
work indicates that the functionality at the 2-position of isosorbide
extends into the acyl pocket of the cholinesterase enzymes upon
productive binding (as required for carbamylation and subsequent
prolonged inhibition of the enzyme). The pocket in BuChE, lined by
a leucine and valine residue (residues Leu286 and Val288 in hu-
BuChE) is larger than in AChE, in which the acyl pocket is occluded
by the presence of two large phenylalanine residues (Phe288 and
Phe290, numbering according to Torpedo californica enzyme). This
(IC50, 4.8 lM). For this particular compound, the replacement of
the nitrogen atom of the oxime functionality with an oxygen
means that the remainder of the functionality is at an angle to
the isosorbide core—this may allow for more avid binding as is
implied from the potency of inhibition—this is discussed further
below.
Modification of the isosorbide template by incorporation of lac-
tam and lactone rings in place of one of the five-membered rings
(compounds 10 and 11, respectively) also negatively affected
inhibitor potency. The lactam structure inhibited BuChE by just
110
100
90
63% at 100 lV and the lactone by 56% at the same concentration;
inhibition of AChE was even less apparent. Similarly, the 5-azide
derivative (15) was a relatively poor inhibitor of the cholinesteras-
es. The b-amide structure (16) showed more promise, inhibiting
80
BuChE by 88% at 100 lM.
70
The most potent inhibitors were the ether compounds. The
addition of a phenylpropyl ether moiety at the 5-position of isosor-
bide (18b) resulted in a potent BuChE inhibitor with an IC50 of
200 nM, while the benzyl ether derivative (18a) had an IC50 of
50 nM. This compound could be ranked alongside some of the most
potent ester derivatives of isosorbide described previously, but had
the added advantage that it was not susceptible to esterase-medi-
ated hydrolysis in vivo.
60
0.00
0.25
1
6
24
49
74
Time (hours)
Figure 2. The effect of a single ip dose of compound 18a at 1 mg/kg on mouse
plasma BuChE measured ex vivo (9 animals with n = 3 per time point).

G. P. Dillon et al. / Bioorg. Med. Chem. 18 (2010) 1045–1053
1049
means that larger groups such as the benzyl carbamate—as well as
4. Experimental
4.1. Chemistry
the butyl, and to a lesser extent, propyl carbamates now de-
scribed—occupy the BuChE pocket well, whereby they are too large
to be accommodated by AChE. Thus, AChE has a tendency to be
more selective for compounds with a smaller functionality at the
2-position, and this is apparent amongst our compounds where
the di-ethyl and di-phenyl carbamates are the most potent inhibi-
tors of the enzyme.
If we focus on BuChE inhibition, it is clear from the results that
the isosorbide moiety itself is also important for activity, as is the
presence of a suitable functionality at the 5-position of the sugar.
The lactam and lactone compounds were relatively poor inhibi-
tors, indicating that the fused five-membered ring structure of
isosorbide is probably more optimal. The poor inhibition apparent
for these compounds may also be due to the fact that they both
have carbonyl groups attached. Amongst the series of compounds
tested, all of those with flat ketone or oxime groups attached at
the 5-position were quite poor cholinesterase inhibitors. Once
again, modeling described previously could hint as to why this
might be the case—the moiety at the 5-position when the group
is endo to the V-shaped isosorbide ring extends upwards towards
Uncorrected melting points were obtained using a StuartÒ
melting point SMP11 melting point apparatus. Spectra were ob-
tained using a Perkin Elmer 205 FT Infrared Paragon 1000 spec-
trometer. Band positions are given in cmꢀ1. Solid samples were
obtained by KBr disk; oils were analyzed as neat films on NaCl
plates. 1H and 13C spectra were recorded at 27 °C on a Bruker Ad-
vance II 600 MHz spectrometer (600.13 MHz 1H, 150.91 MHz 13C)
and Bruker DPX 400 MHz FT NMR spectrometer (400.13 MHz 1H,
100.16 MHz 13C), in either CDCl3 or DMSO-d6, (tetramethylsilane
as internal standard). High resolution mass spectrometry (HRMS)
was performed on a Micromass mass spectrophotometer (EI
mode) at the Department of Chemistry, Trinity College. HPLC
was performed on a reverse phase 250 mm ꢂ 4.6 mm Waters
Spherisorb ODS-2, 5 lm column using a Waters Alliance 2695
chromatograph equipped with an autosampler, column oven
and dual wavelength detector. The flow rate was 1 mL/min with
a mobile phase consisting of 40% phosphate buffer pH 2.5 and
60% acetonitrile at time 0 and grading to 85% acetonitrile at
the gorge entrance, stacking vertically against the cation-
p site
(Trp82 in huBuChE) and extending towards the peripheral site
residues. Extension of a group in a different direction—that is,
in an exo orientation or indeed planar to the isosorbide ring—
means that groups are directed towards the gorge wall, where
there is little space for accommodation of functionalities of any
reasonable size. Amongst compounds without side-chains at-
tached directly to isosorbide in an endo orientation, only com-
4 min. Injection volume was 20 lL, and areas determined at
254 nm. The isocratic HPLC method was aqueous phosphate buf-
fer solution pH 2.5 40% and acetonitrile 60%. Flow rate was 1 mL/
min. Flash chromatography was performed on Merck Kieselgel 60
particle size 0.040–0.063 mm. TLC was performed on silica gel
Merck F-254 plates. Compounds were visually detected by absor-
bance at 254 nm and/or vanillin staining.
pound 12 was especially potent. However, it had
a freely-
rotatable ethyl-ester functionality immediately attached that
could, orientate itself in such a way to extend upwards towards
the gorge entrance, interacting favorably with residues in the
mid-gorge and upper-gorge regions.
4.1.1. Isosorbide-di-(ethylcarbamate) (2a)
General procedure for dicarbamates (2): Isosorbide (0.7304 g,
5 mmol) was dissolved in dry pyridine (5 mL), the appropriate iso-
cyanate (15 mmol) was added and the mixture heated at 100 °C for
1.5 h. The mixture was cooled and methanol (7 mL) added to re-
move excess isocyanate. It was heated for 10 min at 100 °C, cooled
to room temperature, poured into ice-water, precipitate filtered
and recrystallised twice from hot methanol to yield a white crys-
The ether compounds were the most potent BuChE inhibitors
synthesized in this study. It is hardly surprising that the benzyl
ether compound, 18a, was the most potent, as it closely resembles
the benzyl ester compounds that were found to be optimal for
BuChE inhibition in previous studies,9 differing only in its lack of
an ester carbonyl group. Clearly, it can orientate itself in a similar
manner in the BuChE active site and form many of the same inter-
actions, thus favoring carbamylation of the catalytic serine. With
an IC50 of 4 nM, isosorbide 2-benzyl carbamate 5-benzoate is a
more potent inhibitor of BuChE; the difference may be attributed
in large part to the stacking interactions that can form between
the double bond of the carbonyl group and the indole ring of
Trp82. However, the loss of affinity is warranted for our purposes
as the ether compound is stable in the presence of carboxylesterase
enzymes in mouse plasma.
talline product (18%): mp 169.9 °C. IR
(KBr): 1693 (C@O),
m
max
3333 (2° NH) cmꢀ1 1H NMR d (CDCl3): 1.05–1.09 (m, 6H,
.
2 ꢂ CH3), 3.11–3.19 (m, 4H, 2 ꢂ CH2), 3.61–3.95 (2m, 4H, IsH-6,
IsH-60, IsH-1, IsH-10), 4.42–4.43 (d, 1H, J = 4 Hz, IsH-3), 4.68–4.70
(t, 1H, J = 5.2 Hz, IsH-4), [4.90–4.97 (2s, 1H N–H), 5.02–5.06 (m,
4H, IsH-2, IsH-5)]. 13C NMR ppm (CDCl3): 15.45, 15.50 (CH3),
36.27, 36.35 (CH2), 70.33 (IsC-6), 74.22–74.47 (IsC-1), 77.16–
77.80 (IsC-5), 78.65 (IsC-2), 81.38 (IsC-4), 86.23 (IsC-3), 155.51,
155.82 (CO). C12H20N2O6 requires C, 49.99; H, 6.99; N, 9.7. Found:
C, 50.08; H, 7.05; N, 9.46.
4.1.2. Isosorbide-di-(propylcarbamate) (2b)
A white crystalline product (28%): mp 137 °C. IR max (KBr): 1699
m
3. Conclusions
(C@O), 3327 (2° NH) cmꢀ1. 1H NMR d (DMSO): 0.8–0.9 (t, 6H),1.35–
1.44 (m, 4H), 2.90–2.95 (q, 4H, J = 6.8 Hz), 3.61–3.64 (m, 1H), 3.78–
3.89 (m, 3H), 4.36–4.44 (d, 1H), 4.66–4.68 (t, 1H), 4.91–4.92 (d,
1H), 4.95–4.99 (q, 1H), 7.28–7.31 (t, 1H), 7.34–7.36 (t, 1H). 13C
NMR ppm (DMSO): 11.51, 11.55, 22.90, 22.92, 42.34, 42.36,
70.33, 73.18, 73.65, 77.85, 81.12, 85.93, 155.58, 155.81.
Some potent isosorbide 2-carbamate 5-ester inhibitors of
BuChE are degraded rapidly by carboxylesterase enzymes found
in mouse plasma, meaning that their use as agents to probe the
effects of BuChE inhibition in the important mouse model is inap-
propriate. We have created a series of compounds in which the
labile 5-ester group has been modified and tested their potency
as cholinesterase inhibitors. It was found that isosorbide 2-carba-
mate 5-ether compounds are nanomolar inhibitors of BuChE with
negligible effects on AChE. One of these 18a is stable in mouse
plasma and that its administration can effect enzyme inhibition
for sustained periods of time. We expect that this compound will
be useful for probing BuChE roles and effects in mice going
forward.
4.1.3. Isosorbide-di-(butylcarbamate) (2c)
A white crystalline product (72%). IR
(KBr): 1691 (C@O),
m
max
3329 (2° NH) cmꢀ1 1H NMR d (CDCl3): 0.91–0.95 (m, 6H), 1.30–
.
1.1.38 (m, 4H), 1.45–1.53 (m, 4H), 3.16–3.22 (m, 4H), 3.68–3.3.72
(m, 1H), 3.97–4.03 (m, 3H), 4.49–4.51 (d, 1H), 4.75–4.79 (m, 2H),
4.89 (t, 1H), 5.09–5.14 (m, 2H). 13C NMR ppm (CDCl3): 13.25,
19.41, 31.41–31.45, 40.32–40.39, 69.45, 73.45, 73.68, 77.87,

1050
G. P. Dillon et al. / Bioorg. Med. Chem. 18 (2010) 1045–1053
80.56, 85.38, 154.75. C16H2N2O6 requires C, 55.79; H, 8.20; N, 8.14;
found: C, 55.65; H, 8.29; N, 8.11.
(20 mL) saturated brine solution (20 mL) and dried (Na2SO4). Puri-
fication by column chromatography over silica gel using hexane
and ethyl acetate (3:1) as eluent, afforded the title compound as
4.1.4. Isosorbide-di-phenylcarbamate (2d)
white crystalline solid (14.8 mg, 15%). Mp 92 °C. IR
(KBr):
m
max
A white crystalline product (51%): mp 178.8 °C, IR
(KBr):
max
1055.8 (C–O–C), 1611.0 (C@C), 1719.3 (C@O), 2859.2, 2924.4 (C–
m
1702 (C@O), 3309 (2° NH) cmꢀ1
.
1H NMR d (DMSO): 3.8–3.9 (2m,
H stretching), and 3328.0 (2° amine) cmꢀ1 1H NMR d (CDCl3):
.
2H), 4.0 (m, 2H), 4.5 (d, 1H, J = 4.8 Hz), 4.8–4.9 (t, 1H, J = 5.2 Hz),
5.1–5.2 (m, 2H), 7.0 (m, 2H), 7.3 (t, 4H), 7.5 (t, 4H), 9.8–9.9 (2s).
13C NMR ppm (DMSO): 70.62, 73.08, 74.14, 78.19, 81.33, 86.05,
126.67, 126.97, 118.48, 118–59, 122.79–122.97, 129.11, 139.20–
139.41, 152.89, 152.07. C20H20N2O6 requires C, 62.49; H, 5.24; N,
7.29. Found: C, 62.13; H, 5.08; N, 7.09.
3.58 (dd, 1H, J = 2.51 and 10.56 Hz, IsH-1), 3.99 (d, 1H,
J = 6.02 Hz, IsH-10), 4.40 (d, 2H, J = 6.02 Hz, CH2), 4.87 (d, 1H,
J = 6.53 Hz, IsH-3), 5.06 (t, 1H, J = 2.51 Hz, IsH-4), 5.13 (m, 1H,
NH), 5.20 (d, 1H, J = 2.01 and 6.03 Hz, IsH-2), 6.56 (d, 1H,
J = 3.01 Hz, IsH-6), 7.28–7.40 (m, 5H, ArH).
4.1.10. 2-(Benzylaminocarbonyloxy)-5-ketoxime-1,4:3,6-
4.1.5. Isosorbide-di-(4-nitrophenyl carbamate) (2e)
dianhydro-D-glucitol (8)
A yellow powdered product (40%): mp 262.2 °C, IR
(KBr):
max
To a solution of 3 (250 mg, 0.9016 mmol) in methanol/water
(2:1, 6 mL) was added 2 mol equiv of hydroxylamine hydrochlo-
ride (125.3 mg, 1.80 mmol,) and 5 mol equiv of NaOAc
(4.508 mmol, 369.8 mg). The reaction mixture was heated to
105 °C for 2 h. The reaction mixture was evaporated to dryness un-
der vacuum and the residue was dissolved in chloroform. Any ex-
cess water was dried (Na2SO4) and the solution was filtered and
evaporated. Purification by column chromatography yielded
m
1744 (C@O), 3383 (2° NH) cmꢀ1 1H NMR d (DMSO): 3.89–3.91
.
(m, 2H), 4.02–4.04 (m, 2H), 4.56–4.57 (d, 1H), 4.88–4.91 (t, 1H),
5.18–5.23 (dq, 2H), 7.68–7.72 (m, 4H), 8.18–8.21 (m, 4H), 10.58
(s, 2H). 13C NMR ppm (DMSO): 70.69, 72.94, 74.77, 78.68, 81.36,
85.94, 118.00, 118.11, 125.38, 125.41, 142.08, 142.17, 145.69,
145.87, 152.63, 152.82. C20H18O10N4 requires C, 50.6; H, 3.8; N,
11.8. Found: C, 50.49; H 3.68; N, 12.05.
176 mg (66%) of a clear oil. IR
(NaCl): 1697.2, 1720.1 (C@O),
m
max
1H NMR
4.1.6. Isosorbide-di-(4-chlorophenylcarbamate) (2f)
2876.4, 2928.3, (C–H stretching), and 3332.5 (OH) cmꢀ1
.
d
A white powdered product (37%): mp 221.2 °C, IR
(KBr):
max
(CDCl3): 3.90–4.10 (m, 2H, IsH-1, IsH-10), 4.31 (d, 2H, J = 6.02 Hz,
CH2), 4.50 (d, 1H, J = 15.56 Hz, IsH-3), 4.54–4.70 (m, 2H, IsH-6,
IsH-60), 4.96 (d, 1H, J = 3.01 Hz, IsH-4), 5.14 (s, 1H, IsH-2), 7.20–
7.39 (m, 5H, 5ArH). 13C NMR ppm (CDCl3): 44.42, 69.13, 72.21,
78.53, 80.32, 86.50, 127.17, 127.28, 128.48, 139.14, 156.58,
159.85. HRMS (M+23); C14H16N2O5Na requires 315.0959; found,
315.0957.
m
1705 (C@O), 3318 (2° NH) cmꢀ1 1H NMR d (DMSO): 3.80–3.91
.
(m, 2H), 3.99 (s, 2H), 4.52–4.53 (d, 1H, J = 4.8 Hz), 4.83–4.86 (t,
1H, J = 5.2 Hz), 5.13–5.18 (dq, 2H, J = 3.2 Hz, J = 5.2 Hz), 7.32–7.36
(m, 4H), 7.48–7.52 (m, 4H), 10.00, 10.05 (2s, 2H). 13C NMR ppm
(DMSO): 70.61, 73.02, 74.29, 78.3, 81.31, 85.99, 119.94, 120.16,
126.44, 126.61, 128.98, 129.03, 138.22, 138.4, 152.82.
C20H18N2O6Cl2 requires C, 52.99; H, 4.00; N, 6.18. Found: C,
52.73; H, 3.88; N, 5.74.
4.1.11. 2-(Benzylaminocarbonyloxy)-5-(phenylcarbonyloxyimi-
no-)-1,4:3,6-dianhydro-D-glucitol (9)
4.1.7. Isosorbide-di-(benzylcarbamate) (2g)
Compound 8 (0.3421 mmol, 100 mg,) was dissolved in DCM
(10 mL). Triethylamine (0.376 mmol, 38.0 mg, 0.0524 mL), benzoyl
chloride (0.3763 mmol, 0.0437 mL) and DMAP (10%, 5 mg) were
added. The reaction mixture was stirred overnight. Chloroform
(10 mL) was added to the reaction vessel and the mixture was
washed with 1 M HCl (20 mL), 5% NaHCO3 (20 mL) saturated brine
solution (20 mL) and dried (Na2SO4). The solution was filtered and
evaporated to give a white crystalline solid, which was recrystal-
lised from MeOH to yield 106.0 mg (78%) of a white crystalline
A white crystalline product (58%): mp 160.4 °C, IR
(KBr):
max
m
1552 (benzene), 1690 (C–O), 3328 (2° amine) cmꢀ1 1H NMR d
.
(DMSO): 3.68–3.95 (2m, 4H,), 4.19–4.22 (m, 4H), 4.40–4.41 (d,
1H, J = 4.8 Hz), 4.72–4.74 (t, 1H, J = 5.2 Hz), 4.97–5.03 (dq, 2H,
J = 2.8 Hz), 7.22–7.35 (m, 10H), 7.87, 7.90 (2t, 2H). 13C NMR ppm
(DMSO): 44.02, 44.11, 70.55, 73.19, 73.95, 78.1, 81.27, 86.01,
127.09, 127.4, 128.58, 128.65, 139.88–140.09, 155.89, 156.15.
C22H24N2O6 requires C, 64.07; H, 5.87; N, 6.79. Found: C, 63.99;
H, 5.97; N, 6.74.
product. Mp 91 °C, IR
(KBr): 1710.3, 1725.9 (C@O), 2875.6,
m
max
2979.5 (C–H stretching), and 3325.5 (2° NH) cmꢀ1 1H NMR d
.
4.1.8. 2-(Benzylaminocarbonyloxy)-5-keto-1,4:3,6-dianhydro-
(CDCl3): 4.07–4.25 (m, 2H, IsH-1, IsH-10), 4.40 (d, 2H, J = 5.52 Hz,
CH2), 4.70 (m, 2H, IsH-6, IsH-60), 4.87 (d, 1H, J = 16.57 Hz, IsH-3),
5.20 (m, 2H, IsH-4, NH), 5.35 (s, 1H, IsH-2), 7.23–7.43 (m, 5H,
5Ar1H), 7.51 (t, 2H, J = 7.53 Hz, Ar2H-3/Ar2H-5), 7.65 (t, 1H,
J = 7.28 Hz, Ar2H-4), 8.03 (d, 2H, J = 7.53 Hz, Ar2H-2/Ar2H-6). 13C
NMR ppm (CDCl3): 44.75, 69.47, 72.72, 77.60, 79.74, 86.30,
127.16, 127.27, 127.57, 128.25, 128.33, 129.27, 133.37, 137.46,
154.53, 162.61, 168.02. C21H20N2O6 requires C, 63.63; H, 5.09; N,
7.07. Found: C, 63.69; H, 4.92; N, 6.77.
D-glucitol (3)
A white crystalline compound (67%). Mp 107 °C, IR
(KBr):
max
m
1599 (benzene), 1725.0, 1774 (C@O), and 3326.4 (2° NH) cmꢀ1
.
1H NMR d (CDCl3): 3.97 (d, 1H, J = 17.57 Hz, IsH-1), 4.05–4.20 (m,
3H, IsH-10, IsH-6, IsH-60), 4.30 (d, 1H, J = 4.02 Hz, IsH-4), 4.39 (d,
2H, J = 6.02 Hz, CH2), 4.85 (d, 1H, J = 4.01 Hz, IsH-3), 5.19 (s, 1H,
NH), 5.36 (d, 1H, J = 2.51 Hz, IsH-2), 7.22–7.40 (m, 5H, ArH). 13C
NMR ppm (CDCl3): 44.73, 69.63, 72.67, 78.29, 84.57, 127.14,
127.29, 128.33, 137.42, 154.53, 209.28. C14H15NO5 requires C,
60.64; H, 5.45; N, 5.05. Found: 60.73; H, 5.43; N, 4.67.
4.1.12. 2-(Benzylaminocarbonyloxy)-5-deoxy, 5-dehydro-
((ethyloxycarbonyl)ene-)-1,4:3,6-dianhydro-D-glucitol (12)
4.1.9. 2-(Benzylaminocarbonyloxy)-5-deoxy-
(7)
L
-xylohex-6-enitol
A solution of 3 (0.902 mmol, 250 mg) in DCM/THF (1:1, 10 mL)
was cooled to 0 °C on ice in a Dewar flask and kept under an atmo-
sphere of N2 gas. Carbethoxymethylene(triphenylphosphorane)
(0.9918 mmol, 314.6 mg) was added to the solution and the mix-
ture was stirred for 6 h. The reaction mixture was evaporated to
dryness under vacuum and the residue purified by column chro-
matography using hexane and ethyl acetate (2:1) as eluent yielded
141.7 mg of the title compounds as a white crystalline solid (45%).
To
a
solution of 2-(Benzylaminocarbonyloxy)-5-O-trifluoro-
-glucitol (0.3647 mmol,
methanesulfonyl-1,4:3,6-dianhydro-
D
150 mg) in anhydrous toluene (10 mL) was added 1,5-diazabicy-
clo[4.3.0]non-5-ene (0.4012 mmol, 61.2 mg, 0.06 mL) and the mix-
ture was stirred for 6 h at room temperature. The organic solvent
was removed under vacuum and the crude mixture was diluted
with DCM and washed with 1 M HCl (20 mL), 5% NaHCO3
Mp 121 °C. IR
(KBr): 1721.4 (C@O), and 3324.8 (2° NH) cmꢀ1
.
m
max

G. P. Dillon et al. / Bioorg. Med. Chem. 18 (2010) 1045–1053
1051
1H NMR d (CDCl3): 1.30 (t, 3H, J = 7.31 Hz, CH3), 4.00 (m, 2H, IsH-1),
4.20 (m, 2H, –CH2CH3), 4.38 (d, 2H, J = 6.02 Hz, –NCH2Ar), 4.55 (d,
1H, J = 4.10 Hz, IsH-3), 4.74 (dd, 1H, J = 2.92 and 17.54 Hz, IsH-4),
4.97 (m, 2H, IsH-6, IsH-60), 5.15 (s, 1H, NH), 5.25 (s, 1H, IsH-2),
6.11 (s, 1H, –C@CH–), 7.23–7.40 (m, 5H, 5ArH). 13C NMR ppm
(CDCl3): 13.79 (CH3), 44.69 (ꢀNCH2Ar), 60.16 (–CH2CH3), 70.92
(IsC-1), 71.77 (IsC-6), 78.24 (IsC-2), 83.28 (IsC-4), 84.74 (IsC-3),
116.12 (–C@CH–), 127.12 (ArC-4), 127.22 (Ar-2/Ar-6), 128.29
(ArC-3/ArC-5), 137.59 (ArC-1), 154.71 (–NC(O)O–), 158.13 (ArC-
5), 165.30 (–C(O)CH2CH3). C18H21NO6 requires C, 62.24; H, 6.09;
N, 4.03. Found: C, 62.20; H, 6.08, N, 3.98.
21.69 (CH3), 72.03 (IsC-6), 74.72 (IsC-1), 75.71 (IsC-2), 83.08 (IsC-
5), 84.81 (IsC-4), 87.57 (IsC-3), 127.84 (ArC-2/ArC-6), 130.09
(ArC-3/ArC-5), 133.10 (ArC-1), 145.37 (ArC-4).
4.1.16. 5-Deoxy-5-azido-1,4:3,6-dianhydro-D-glucitol
5-O-(Toluenesulfonyl)-1,4:3,6-dianhydro-L-iditol (4.16 mmol,
1.25 g) was dissolved in a minimum volume of DMSO (7.5 mL). Ex-
cess sodium azide (20.8 mmol, 1.36 g) was added to form a slurry.
The mixture was heated to 125 °C and stirred for 24 h. The mixture
was partitioned between ethyl acetate and water and the organic
extract washed several times. The crude product was purified by
flash chromatography giving a white solid, which was recrystal-
lised to afford the title compound as a white crystalline product
(514.4 mg, 72%). 1H NMR d (CDCl3): 3.13 (d, 1H, J = 4.52 Hz, OH),
3.64 (m, 1H, IsH-5), 3.85–4.00 (m, 4H, IsH-1, IsH-6), 4.32 (s, 1H,
IsH-2), 4.42 (d, 1H, J = 4.02 Hz, IsH-3), 4.82 (d, 1H, J = 4.52 Hz,
IsH-4). 13C NMR ppm (CDCl3): 61.80 (IsC-5), 69.25 (IsC-6), 75.43
(IsC-1), 75.86 (IsC-2), 82.04 (IsC-4), 88.21 (IsC-3).
4.1.13. 7-exo-(Benzylaminocarbonyloxy)-3-oxo-2,5,9-
trioxabicyclo [4.3.0] nonane (10)
A solution of 3 (0.9016 mmol, 250 mg) in DCM was stirred and
cooled to 0 °C. Chloroperbenzoic acid (500 mg) and NaOAc
(6.0953 mmol, 500 mg) were added and the mixture stirred over-
night. The reaction was monitored by TLC. The solvent was evapo-
rated and the crude product was purified by flash chromatography
(DCM/ethyl acetate, 9:1) to give 102 mg (38%) of the title product
4.1.17. 2-(Benzylaminocarbonyloxy)-5-deoxy-5-phenylcarbon-
as a clear oil. IR
(NaCl): 1719.5, 1725.1 (C@O), 2953.0 (C–H
ylamino-1,4:3,6-dianhydro-
D-glucitol (16)
m
max
stretching), and 3328.3 (2° NH) cmꢀ1
.
1H NMR d (CDCl3): 4.03
To a solution of 5-deoxy-5-azido-1,4:3,6-dianhydro-D-glucitol
(m, 2H, IsH-1, IsH-10), 4.28-4.43 (m, 5H, IsH-6, IsH-60, IsH-3, CH2),
5.24 (m, 2H, IsH-2, NH), 5.86 (d, 1H, J = 3.52 Hz, IsH-4), 7.22–7.42
(m, 5H, 5ArH). 13C NMR ppm (CDCl3): 44.76 (CH2), 62.87 (IsC-1),
72.05 (IsC-6), 77.04 (IsC-2), 77.40 (IsC-3), 100.89 (IsC-4), 127.15
(ArC-4), 127.37 (Ar-2/Ar-6), 128.36 (ArC-3/ArC-5), 137.29 (ArC-1),
154.41 (CO), 165.29 (ArC-5). HRMS (M+23); C14H15NO6Na requires
316.0798; found, 316.0881.
(2.92 mmol, 500 mg), in DCM was added triethylamine
(3.21 mmol, 324.6 mg, 0.447 mL), benzyl isocyanate (3.21 mmol)
and DMAP (0.4092 mmol, 50 mg). The reaction was monitored by
TLC. The mixture was cooled upon completion of the reaction
and methanol (10 mL) was added to remove excess isocyanate.
The mixture was heated for a further 15 min at 105 °C and cooled
to room temperature. All organic solvent was removed giving a
clear oil which was purified by column chromatography using hex-
ane and ethyl acetate (3:1 and 1:1) as eluent to isolate the primary
product as a white crystalline solid. The white crystalline solid was
dissolved in ethyl acetate/methanol (1:1, 15 mL). A spatula tip-full
of 10% palladium on activated carbon was added to the solution.
Air was expelled from the flask and the mixture was kept under
an atmosphere of hydrogen gas and stirred for 24 h. The palladium
catalyst was removed by filtration and the filtrate was collected
and evaporated under vacuum to give a clear oil. This oil was di-
luted with DCM (10 mL) and excess triethylamine (3.59 mmol,
363.0 mg, 0.5 mL), benzoyl chloride (3.59 mmol, 505.2 mg,
0.4172 mL) and DMAP (0.4092 mmol, 50 mg) were added to the
reaction vessel. The reaction mixture was stirred for 4 h. DCM
(10 mL) was added to the reaction vessel and the mixture was
washed with 1 M HCl (20 mL), 5% NaHCO3 (20 mL) saturated brine
solution (20 mL) and dried over anhydrous sodium sulfate (1 g).
Purification by column chromatography, using hexane and ethyl
acetate (2:1) as eluent afforded title compound as a white crystal-
4.1.14. N-Methyl-7-exo-(benzylaminocarbonyloxy-)-3-oxo-5,9-
dioxa-2-azabicyclo[4.3.0] nonane (11)
To a solution of 3 (0.9016 mmol, 250 mg) in anhydrous ethanol
(6 mL) was added 4 mol equiv of N-methylhydroxylamine HCl
(3.606 mmol, 301.2 mg) and 6 mol equiv of sodium acetate
(5.4094 mmol, 443.7 mg). The reaction mixture was stirred for
4 h. The reaction mixture then filtered and evaporated. The crude
intermediate product was purified by flash chromatography yield-
ing a yellow viscous oil, which was diluted in pyridine (7 mL) and
cooled to 0 °C. The reaction mixture was stirred and kept under an
atmosphere of N2 gas. p-Toluenesulfonyl chloride (1.56 mmol,
300 mg) was added to the mixture and stirred for 6 h. The crude
product was purified by flash chromatography (DCM/ethyl acetate,
9:1) to give 96.5 mg (34%) of the title product as a clear oil. IR
m
max
(NaCl): 1606.7 (C–N), 1718.7, 1721.4 (C@O), 3321.5 (2° NH) cmꢀ1
.
1H NMR d (CDCl3): 3.06 (s, 3H, CH3), 3.90 (d, 1H, J = 10.36 Hz, IsH-1)
4.05 (d, 1H, J = 16.06 Hz, IsH-6), 4.19 (d, 1H, J = 2.51 Hz, IsH-3) 4.25
(d, 1H, J = 16.57 Hz, IsH-60) 4.30–4.40 (m, 3H, IsH-10, CH2), 4.98 (d,
1H, J = 2.51 Hz, IsH-4), 5.17 (d, 1H, J = 4.02, IsH-2), 5.26–5.37 (s, 1H,
NH), 7.23–7.40 (m, 5H, ArH). 13C NMR ppm (CDCl3): 32.01 (CH3),
45.19 (CH2), 66.24 (IsC-1), 71.17 (IsC-6), 77.28 (IsC-2), 77.43 (IsC-
3), 87.47 (IsC-4), 127.14 (ArC-4), 127.31 (Ar-2/Ar-6), 128.33 (ArC-
3/ArC-5), 137.43 (ArC-1), 154.45 (CO), 166.12 (ArC-5). HRMS
(M+23); C15H18N2O6Na requires 329.1115; found, 329.1212.
line (218.5 mg, 19%). Mp 164 °C. IR
(KBr): 1698.6, 1720.1
m
max
(C@O), and 3326.5, 3350.6 cmꢀ1 1H NMR d (CDCl3): 3.45 (t, 1H,
.
J = 8.79 Hz, IsH-6), 4.02–4.11 (m, 2H, IsH-1, IsH-10), 4.32 (t, 1H,
J = 8.03 Hz, IsH-60), 4.39 (d, 2H, J = 6.02 Hz, CH2), 4.55–4.77 (m,
3H, IsH-3, IsH-4, IsH-5), 5.15 (s, 1H, NH), 5.24 (s, 1H, IsH-2), 6.74
(d, 1H, J = 7.02 Hz, NH-benzylamide), 7.23–7.40 (m, 5H, 5Ar1H),
7.47 (m, 2H, Ar2H-3/Ar2H-5), 7.53 (m, 1H, Ar2H-4), 7.81 (m, 2H,
Ar2H-2/Ar2H-6). 13C NMR ppm (CDCl3): 44.73 (CH2), 52.83 (IsC-
5), 70.77 (IsC-6), 73.44 (IsC-1), 78.55 (IsC-2), 81.36 (IsC-4), 85.37
(IsC-2), 126.59 (Ar2C-2/Ar2C-6), 127.14 (Ar1C-4), 127.26 (Ar1C-2/
Ar1C-6), 128.19 (Ar2C-3/Ar2C-5), 128.32 (Ar1C-3/Ar1C-5), 131.36
(Ar2C-4), 133.35 (Ar2C-1), 137.53 (Ar1C-1), 154.65 (–Ar1CH2-
NC(O)O–), 166.33 (–NC(O)Ar2). HRMS (M+23); C21H22N2O5Na re-
quires 405.1427; found, 405.1367.
4.1.15. 5-O-Toluenesulfonyl-1,4:3,6-dianhydro-L-iditol
Isoiodide 14 (6.84 mmol, 1 g) and p-toluenesulfonyl chloride
(6.84 mmol, 1.30 g) were dissolved in acetone (20 mL). The reac-
tion vessel was cooled to 0 °C following the method outlined for
the preparation of esters. Recrystallisation from ethanol yielded
the title compound as a white crystalline product (1.27 g, 62%).
1H NMR d (CDCl3): 2.48 (s, 3H, CH3), 3.76–3.90 (m, 4H, IsH-6,
IsH-1, IsH-10), 3.98 (d, 1H, J = 11.04 Hz, IsH-60), 4.34 (s, 1H, IsH-2),
4.56 (d, 1H, J = 4.31 Hz, IsH-3), 4.65 (d, 1H, J = 3.51 Hz, IsH-4),
4.90 (m, 1H, J = 3.52 Hz, IsH-5), 7.39 (d, 2H, J = 8.03, ArH-3/ArH-
5), 7.82 (d, 2H, J = 8.03, ArH-2/ArH-6). 13C NMR ppm (CDCl3):
4.1.18. 2-(Benzylaminocarbonyloxy)-5-O-benzyl-1,4:3,6-
dianhydro-
2-O-(t-(Butyl)-dimethylsilyl-)-1,4:3,6-dianhydro-
(1.1521 mmol, 300 mg) and excess NaH (100 mg of 65% NaH in
D-glucitol 18a
D
-glucitol 17

1052
G. P. Dillon et al. / Bioorg. Med. Chem. 18 (2010) 1045–1053
an oil suspension) was placed under an atmosphere of N2 gas.
Anhydrous THF was added to the flask and the mixture was stirred
for 30 min. Excess benzyl bromide (3.363 mmol, 575 mg, 0.4 mL)
was added dropwise over 15 min and the reaction mixture was
stirred for a further 24 h. The mixture was diluted with water
(25 mL), extracted with ethyl acetate (3 ꢂ 25 mL) and dried over
anhydrous sodium sulfate. The organic portions were collected
and evaporated to dryness to give a clear oil. The remaining excess
benzyl bromide was eliminated from the mixture by column chro-
matography, which yielded a clear oil. The oil was diluted with THF
(10 mL). Excess 0.1 M tetrabutylammonium fluoride (1.2 mL) was
added and the mixture was stirred at room temperature for
15 min. The organic solvent was evaporated to dryness under vac-
uum giving a brown oil. DCM (10 mL) was added to the reaction
vessel and the solution was stirred at room temperature. To the
mixture was added excess Et3N (1.79 mmol, 181.5 mg, 0.250 mL),
benzyl isocyanate (2.0240 mmol, 269.5 mg, 0.25 mL) and DMAP
(0.245 mmol, 30 mg). The mixture was heated to 105 °C for 2 h.
The mixture was cooled upon completion of the reaction and
methanol (10 mL). The mixture was heated for a further 15 min
at 105 °C and cooled to room temperature. The reaction mixture
was evaporated to dryness giving a clear oil to which was added
DCM (20 mL) and washed with 1 M HCl (20 mL), 5% NaHCO3
(20 mL), saturated brine solution (20 mL) and dried with anhy-
drous sodium sulfate. The solution was filtered into a round bot-
tom flask and the organic solvent was evaporated to dryness
under vacuum giving a white crystalline compound. Purification
by flash chromatography afforded the title compound as a white
tion of 100 mM]. Butyrylthiocholine iodide (BTCI): BTCI (15.9 mg)
was dissolved in 10 mL of phosphate buffer pH 8.0. [25 L of this
solution in 250 L of test solution will give a concentration of
0.5 mM] [25 L of this solution in 250 L of test solution will give
a concentration of 0.5 mM]. Acetylthiocholine iodide (ATCI): ATCI
(14.5 mg) was dissolved in 10 mL of phosphate buffer pH 8.0.
l
l
l
l
[25 lL of this solution in 250 lL of test solution will give a concen-
tration of 0.5 mM]. For Human plasma BuChE, Human blood sam-
ples were collected by venipuncture into Li-Heparin Sarstedt
Monovette tubes (9 mL). Plasma was obtained by centrifugation
at 10,000 rpm for 5 min. Plasma was stored at 2–6 °C. A plasma
solution for the activity/inhibition assay was prepared by diluting
1 mL of plasma to 20 mL with phosphate buffer pH 8.0.
AChE: 10 lL of electric eel AChE was diluted to 10 mL with
phosphate buffer pH 8.0. HuBuChE activity was measured in repli-
cate samples using a 96-well plate reader. The total volume of test
solution in each well was 250
solution, 175 L of phosphate buffer pH 8.0, 25
tion [0.5 mM] and 25 L of acetonitrile/distilled water (1:1). The
96-well plate was incubated for 30 min before 25 L of BTCI solu-
lL. This consisted of 25
lL of plasma
l
l
L of DTNB solu-
l
l
tion [0.5 mM] was added and the reaction was measured at
412 nm over 5 min using an Anthos bt2 plate reader. For the deter-
mination of AChE activity, 25
solution were used instead of the plasma and BTCI solutions. For
determination of the inhibition of enzymes, 25 L of an inhibitor
lL of AChE solution and 25 lL of ATCI
l
solution was added to the test solution instead of the acetoni-
trile/water (1:1) solution. The uninhibited enzyme solution acted
as a positive control during experimentation; negative controls
were also run in which enzyme was left out of the mixture in order
to account for spontaneous substrate hydrolysis.
crystalline product (108.6 mg, 25%). Mp 107 °C, IR
(KBr):
m
max
1697.2 (C@O), and 3349 cmꢀ1 1H NMR d (CDCl3): 3.65 (t, 1H,
.
J = 8.28 Hz, IsH-6), 3.90 (dd, 1H, J = 6.53 and 8.54 Hz, IsH-60),
4.04–4.15 (m, 3H, IsH-5, IsH-1, IsH-10), 4.39 (d, 2H, J = 6.02 Hz,
CH2), 4.53 (d, 1H, J = 4.02 Hz, IsH-3), 4.59 (d, 1 h, J = 12.04 Hz,
OCH2Ar1), 4.68 (t, 1H, J = 4.27 Hz, IsH-4), 4.78 (d, 1H, J = 12.04 Hz,
OCH2Ar2), 5.08 (s, 1H, NH), 5.19 (d, 1H, J = 3.01 Hz, IsH-2), 7.23–
7.43 (m, 10H, 5Ar1H, 5Ar2H). 13C NMR ppm (CDCl3): 45.06 (CH2),
69.82 (IsC-6), 72.05 (OCH2Ar2), 73.55 (IsC-1), 78.58 (IsC-2), 78.68
(IsC-5), 80.06 (IsC-4), 85.54 (IsC-3), 127.12 (Ar1C-4), 127.20
(Ar1C-2/Ar1C-6), 127.52 (Ar2C-2/Ar2C-6/Ar2C-4), 128.06 (Ar2C-3/
Ar2C-5), 128.29 (Ar1C-3/Ar1C-5), 137.19 (Ar2C-1), 137.64 (Ar1C-1),
154.79 (CO). C21H23NO5 requires C, 68.28; H, 6.28; N, 3.79. Found:
C, 68.22; H, 6.27; N, 3.69.
4.3. Monitoring of BuChE inhibition in mouse plasma by
compound 18a
Healthy experimentally naïve male CD-1 mice (35–40 g) were
obtained from the Bioresources Unit, Trinity College Dublin. The
animals were housed in a specific pathogen free (SPF) environment
with controlled temperature and humidity in individual cages with
isopad bedding (Harlan, UK). The mice had ad libitum access to
food and water. A 12 h light/dark cycle (lights on 7 a.m.–7 p.m.
G.M.T.) was in operation. All experiments were performed during
the light cycle and carried out in a sound-proof room. All experi-
ments were carried out in accordance with the European (Amend-
ment of Cruelty to Animals Act 1876) Regulations 2002. A single
dose of 1 mg/kg compound 18a, in a volume of 0.1 mL per 10 g
body weight, was administered to mice (n = 9 per group subdi-
vided into groups A, B, C to permit regular sampling) via intraperi-
toneal (ip) injection into the left lower quadrant of the abdomen
using a sterile 21 gauge needle (BD Microlance, UK). Approxi-
4.1.19. 2-(Benzylaminocarbonyloxy)-5-O-(phenylpropyloxy)-
1,4:3,6-dianhydro-D-glucitol 18b
Method was as for 18a: (88.4 mg, 19%). Mp 97 °C. IR
(KBr):
max
m
1691.2 (C@O), and 3328.5 (2° amine) cmꢀ1. 1H NMR d (CDCl3): 1.97
(m, 2H, OCH2CH2CH2Ar2), 2.73 (t, 2H, J = 7.78 Hz, OCH2CH2CH2Ar2),
3.48 (m, 1H, OCH2CH2CH2Ar2), 3.63 (t, 1H, J = 7.78 Hz, IsH-6), 3.71
0
(m, 1H, OCH2CH2CH2Ar2 ), 3.92–4.15 (m, 4H, IsH-60, IsH-5, IsH-1,
mately 50
lL of submandibular venous blood was collected in
IsH-10), 4.38 (d, 2H, J = 6.02 Hz, CH2), 4.54 (d, 1H, J = 4.02 Hz, IsH-
3), 4.64 (t, 1H, J = 4.02 Hz, IsH-4), 5.11 (s, 1H, NH), 5.19 (d, 1H,
J = 3.51 Hz, IsH-2), 7.17–7.40 (m, 10H, 5Ar1H, 5Ar2H). 13C NMR
ppm (CDCl3): 31.21 (OCH2CH2CH2Ar2), 32.11 (OCH2CH2CH2Ar2),
45.06 (CH2), 69.94 (OCH2CH2CH2Ar2), 70.06 (IsC-6), 73.94 (IsC-1),
78.96 (IsC-2), 80.33 (IsC-5), 80.41 (IsC-4), 85.95 (IsC-3), 125.77
(Ar2C-4), 127.51 (Ar1C-4), 127.59 (Ar1C-2/Ar1C-6), 128.29 (Ar1C-
3/Ar1C-5), 128.41 (Ar2C-2/Ar2C-6), 128.68 (Ar2C-3/Ar2C-5), 138.05
(Ar1C-1), 141.70 (Ar2C-1), 155.21 (CO). C23H27NO5 requires C,
69.50; H, 6.85; N, 3.52. Found: C, 69.46; H, 6.88; N, 3.39.
200 L lithium heparinized Microvette capillary tubes (Sarstedt,
l
Germany) and placed on ice. Following blood sampling the mice
were returned to their home cages. Prior to terminal blood sam-
pling mice were anaesthetized by administration of ketamine/
xylazine ip in a volume of 0.1 mL/10 g.15 Terminal blood samples
were obtained via cardiac puncture using a lithium heparinized
sterile 25 gauge needle (Sterican, Braun, Germany). Blood samples
were centrifuged for 10 min at 4 °C at 3000 rpm using a Sorvall
centrifuge RT6000B (GMI, USA). The plasma was removed using a
Pasteur pipette and pipetted into labelled 1.5 mL Eppendorfs
(Braun, Germany). The Ellman assay was carried out as described.
4.2. Determination of cholinesterase activity and inhibition
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