Design, synthesis, and biological evaluation of coumarin derivatives tethered to an edrophonium-like fragment as highly potent and selective dual binding site acetylcholinesterase inhibitors

Article abstract of DOI:10.1002/cmdc.201000210

A large series of substituted coumarins linked through an appropriate spacer to 3-hydroxy-N,N-dimethylanilino or 3-hydroxy-N,N,N-trialkylbenzaminium moieties were synthesized and evaluated as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The highest AChE inhibitory potency in the 3-hydroxy-N,N-dimethylanilino series was observed with a 6,7-dimethoxy-3-substituted coumarin derivative, which, along with an outstanding affinity (IC₅₀=0.236 nm) exhibits excellent AChE/BChE selectivity (SI>300 000). Most of the synthesized 3-hydroxy-N,N,N- trialkylbenzaminium salts display an AChE affinity in the sub-nanomolar to picomolar range along with excellent AChE/BChE selectivities (SI values up to 138 333). The combined use of docking and molecular dynamics simulations permitted us to shed light on the observed structure-affinity and structure-selectivity relationships, to detect two possible alternative binding modes, and to assess the critical role of π-π stacking interactions in the AChE peripheral binding site.

Full text of DOI:10.1002/cmdc.201000210

MED
DOI: 10.1002/cmdc.201000210
Design, Synthesis, and Biological Evaluation of Coumarin
Derivatives Tethered to an Edrophonium-like Fragment as
Highly Potent and Selective Dual Binding Site
Acetylcholinesterase Inhibitors
Leonardo Pisani, Marco Catto, Ilenia Giangreco, Francesco Leonetti, Orazio Nicolotti,
Angela Stefanachi, Saverio Cellamare, and Angelo Carotti*[a]
A large series of substituted coumarins linked through an ap-
propriate spacer to 3-hydroxy-N,N-dimethylanilino or 3-hy-
droxy-N,N,N-trialkylbenzaminium moieties were synthesized
and evaluated as acetylcholinesterase (AChE) and butyryl-
cholinesterase (BChE) inhibitors. The highest AChE inhibitory
potency in the 3-hydroxy-N,N-dimethylanilino series was ob-
served with a 6,7-dimethoxy-3-substituted coumarin derivative,
which, along with an outstanding affinity (IC50 =0.236 nm) ex-
hibits excellent AChE/BChE selectivity (SI>300000). Most of
the synthesized 3-hydroxy-N,N,N-trialkylbenzaminium salts dis-
play an AChE affinity in the sub-nanomolar to picomolar range
along with excellent AChE/BChE selectivities (SI values up to
138333). The combined use of docking and molecular dynam-
ics simulations permitted us to shed light on the observed
structure–affinity and structure–selectivity relationships, to
detect two possible alternative binding modes, and to assess
the critical role of p–p stacking interactions in the AChE pe-
ripheral binding site.
Introduction
Senile dementia is a progressive neurodegenerative disorder
(ND) that affects memory, language, attention, emotions, and
problem-solving capabilities. Dementia may be caused by a va-
riety of diseases including Alzheimer’s disease (AD), stroke, and
other NDs. According to the World Health Organization, AD ac-
counts for two thirds of the cases of dementia observed in
nearly 6–10% of the population over 65 years of age in North
America.[1] Major clinical hallmarks of AD are the extracellular
deposits of b-amyloid peptide (Ab), derived from amyloid pre-
cursor protein (APP) upon cleavage by a- and b-secretases, the
intracellular formation of neurofibrillary tangles, constituted
mainly by hyperphosphorylated, microtubule-associated tau
protein, and the loss of basal forebrain cholinergic neurons.[2–4]
Unfortunately, despite great efforts by public and private re-
search institutions, the leading causes of the onset and pro-
gression of AD have not yet been discovered. The lack of this
pivotal information has therefore precluded the rational devel-
opment of a disease-modifying therapy, and indeed the phar-
macological approaches in current use are only palliative or
target the symptoms.[5] They are based on the restoration of
acetylcholine (ACh) through inhibition of acetylcholinesterase
(AChE) and on the antagonism the of N-methyl-d-aspartate re-
ceptor (NMDA), a glutamatergic receptor involved in the neu-
ronal neurotoxicity observed in AD. Five drugs have been mar-
keted for AD (Figure 1). Four are AChE inhibitors (AChEIs): ta-
crine (1993, Cognexꢀ, later withdrawn mainly because of hepa-
totoxicity), donepezil (1996, Ariceptꢀ), rivastigmine (2000,
Exelonꢀ), and galantamine (2001, Reminylꢀ);[5c] the remainder,
memantine (2003, Namendaꢀ), is an NMDA antagonist.[6] Many
alternative therapeutic approaches are currently being ad-
Figure 1. Marketed drugs for the treatment of Alzheimer’s disease.
dressed: these include immunotherapy,[7] neuronal cell regen-
eration,[8] blockade of Ab formation and amyloid plaque depo-
sition (with inhibitors of a- and b-secretase, and anti-aggregat-
ing and b-sheet-breaking agents),[9] induction of Ab cerebral
clearance,[10] and blockade of tau hyperphosphorylation and
aggregation (with kinase inhibitors and anti-aggregating
agents, respectively).[11] Despite such large-scale investigations,
results from recent AD clinical trials with new experimental
drugs have been quite discouraging.[5b,12–14] Less disappointing
[a] Dr. L. Pisani, Dr. M. Catto, Dr. I. Giangreco, Dr. F. Leonetti, Dr. O. Nicolotti,
Dr. A. Stefanachi, Prof. S. Cellamare, Prof. A. Carotti
Dipartimento Farmaco-chimico, Universitꢀ degli Studi di Bari “Aldo Moro”
via Orabona 4, 70125 Bari (Italy)
Fax: (+39)080-5442230
E-mail: carotti@farmchim.uniba.it
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Tethered Coumarin Derivatives as AChE Inhibitors
outcomes are expected from preclinical and clinical trials of
drugs that address multiple targets of AD.[15,16]
gands might serve as valuable molecular probes to gain in-
sight into the binding site regions of AChE and, to a lesser
extent, butyrylcholinesterase (BChE), the other ChE involved in
AD.[30] The designed amine derivatives may be regarded as po-
tential therapeutic agents for AD, whereas the corresponding
QASs, due to their inability to cross the blood–brain barrier,
may have therapeutic potential in different pathologies such
as glaucoma, neuromuscular blockade after surgical anesthesia,
and myasthenia gravis.[31] In a highly satisfactory manner, our
design led to the discovery of one amine derivative and many
QASs endowed with sub-nanomolar affinity toward AChE and
outstanding selectivity for AChE over BChE.
While working on the synthesis and biological evaluation of
dual AChE–monoamine oxidase inhibitors,[17,18] we also direct-
ed our attention to the design of new AChEIs, with the aim of
discovering original and more potent compounds and to clari-
fy the main interactions that occur at the molecular level in
the AChE binding sites. AChEIs may act at the primary/catalytic
binding sites (e.g., edrophonium and tacrine), at the secon-
dary/peripheral anionic (PAS) binding sites (e.g., propidium
and planar heterocyclic uncharged molecules), or at both (e.g.,
decamethonium and donepezil). In the latter case, efficient in-
teractions at both primary and peripheral binding sites must
be ensured by the presence of a linker of appropriate length
to tether two identical or different molecular groups for simul-
taneous binding at the two AChE sites. This simple yet efficient
design strategy led to the discovery of potent dual binding
site AChEIs, particularly homo-[19] and heterodimers[20a,b] of ta-
crine and (ꢀ)-huperzine A, as well as heterodimers bearing a
coumarinic scaffold.[20c] A similar valuable approach deals with
the design of multifunctional, chiefly chimeric or hybrid ligands
that simultaneously target two or more pathophysiological
processes underlying multifactorial illnesses such as NDs.[15a,21]
In this emerging and challenging scenario, many multifunc-
tional molecules have been reported in which AChE inhibitor
fragments (generally but not exclusively from tacrine, (ꢀ)-hu-
perzine A, and close analogues) were conjugated with a molec-
ular moiety profiling the adjunctive targeted activity (e.g., anti-
oxidant,[22] metal chelating,[23] neuroprotective,[17,18,24,25] and
anti-amyloidogenic[16c,26]). In the latter case, the design ration-
ale lies in the well-known implication of the PAS of AChE in
promoting the aggregation of Ab into senile plaques.[27] An al-
ternative approach combines the different pharmacological ac-
tivities into a single non-hybrid molecular structure that is able
to modulate different targets, as described by some in our re-
search group[17] and others.[15c,28]
Results
Chemistry
The synthesis of the 3-hydroxy-N,N-dimethylanilino derivatives
2–20 and their corresponding QASs required the preparation
of some common coumarinic building blocks. 6,7-Dimethoxy-
3-hydroxycoumarin was prepared from esculetin according to
a reported synthetic pathway,[32] with slight modifications; 5-
and 8-hydroxycoumarin were synthesized by a Wittig olefina-
tion of a suitable hydroxysalicylaldehyde with ethyl (triphenyl-
phosphoranylidene)acetate followed by intramolecular cycliza-
tion of the resulting cinnamate ester under very harsh condi-
tions according to a reported procedure,[33] with slight modifi-
cations. 7-Hydroxy-3,4-annulated coumarin intermediates 1a
and 1d were prepared according to the reaction pathway
shown in Scheme 1. Classical neat sulfuric acid catalyzed von
In a recent investigation[29] we reported the synthesis and
biological evaluation of a series of amines and quaternary am-
monium salts (QASs) bearing an edrophonium-like moiety and
a coumarin group as putative ligands at the AChE catalytic and
PAS binding sites, respectively, linked by an alkyl chain of ap-
propriate length. The interesting outcomes of that work
prompted us to extend the investigation to a new and larger
series of derivatives, the fundamental structure of which is il-
lustrated in Figure 2, to explore a greater molecular diversity at
the linker and, even more so at the coumarin fragment, with
the purpose of deepening our understanding of structure–af-
finity relationships (SAFIRs) and to discover new molecular en-
tities with therapeutic potential. Notably, all the designed li-
Scheme 1. Reagents and conditions: a) concd H2SO4 (cat), 1208C, 10 min;
b) CH3I, K2CO3, DMF (anhyd), RT, 24 h; c) DDQ, dioxane (anhyd), reflux, 24 h;
d) BBr3 (1.0n in CH2Cl2), CH2Cl2 (anhyd), RT, 36 h.
Pechmann reaction of resorcinol with ethyl 2-oxocyclohexane-
carboxylate afforded coumarin 1a, whose phenolic hydroxy
group was protected through methylation to avoid side reac-
tions in the subsequent oxidation reaction carried out by 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in dioxane at
reflux. Deprotection with boron tribromide in dichloromethane
unmasked the hydroxy group to afford the target coumarin in-
termediate 1d.
Compounds 2–7, 9, 10, and 12–17, all bearing a tetramethy-
lene linker, were prepared by a common synthetic pathway il-
lustrated in Scheme 2. The first step was the mono-protection
of 5-(dimethylamino)benzene-1,3-diol[34] as a silyl ether with
Figure 2. General structure of the synthesized coumarin derivatives.
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A. Carotti et al.
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Schemes 3 and 4 show the synthetic approaches taken to in-
troduce some molecular diversity into the tetramethylene
linker, leading to the 3-chloro-4-methyl-7-hydroxy- and 6-hy-
droxycoumarin derivatives 8, 11, and 18–20, respectively. The
starting hydroxycoumarins were reacted with trans-1,4-dibro-
mo-2-butene or 1,3-bis(bromomethyl)benzene under reflux in
tetrahydrofuran and with 1,5-dibromopentane under micro-
wave irradiation in acetonitrile. The reaction of 3-chloro-4-
methyl-7-hydroxycoumarin with excess 1,5-dibromopentane or
1,3-bis(bromomethyl)benzene yielded intermediate bromides
1i and 1j, respectively, which were then transformed into the
final compounds 8 and 11 by a nucleophilic substitution with
phenol 1e (Scheme 3). Amines 8 and 11 were transformed into
the corresponding trimethylbenzaminium iodides 8T and 11 T
by reaction with excess methyl iodide in dry acetonitrile at
room temperature. In the same manner, 6-hydroxycoumarin
was alkylated with excess trans-1,4-dibromo-2-butene and 1,5-
dibromopentane to afford intermediate bromides 1k and 1l,
respectively, which, in turn, were reacted with phenol 1e to
give compounds 18 and 19 (Scheme 4). As depicted in
Scheme 4, the synthesis of compound 20, bearing a basic
linker, started with the alkylation of 6-hydroxycoumarin with
1,2-dibromoethane in the presence of cesium carbonate and
potassium iodide as the catalyst under microwave irradiation.
The replacement of bromine in 1m with 2-methylaminoetha-
nol in THF at reflux afforded 1n, which was reacted with
phenol 1e under standard Mitsunobu conditions to yield inter-
mediate 1o. Final desilylation was carried out with tetra-n-bu-
tylammonium fluoride (TBAF) in THF to afford compound 20.
The N,N,N-trimethylbenzaminium iodides 18T–20T were ob-
tained by treating 3-hydroxy-N,N-dimethylanilino derivatives
18–20 with a large excess of methyl iodide in dry acetonitrile
at room temperature.
Scheme 2. Reagents and conditions: a) TIPSCl, imidazole, CH2Cl2 (anhyd), RT,
2 h; b) HO(CH2)4OBz, PPh3, DIAD, THF (anhyd), RT, 15 h; c) LiOH·H2O, CH3OH/
THF 5:1, RT, 3.5 h; d) CBr4, PPh3, CH2Cl2 (anhyd), 08C!RT, 2 h; e) appropriate
hydroxycoumarin, Cs2CO3, DMF, H2O, RT, 24 h then 708C, 2 h; f) RI (excess),
CH3CN (anhyd), RT, 24–72 h.
triisopropylsilyl chloride (TIPSCl) in the presence of imidazole
as both the base and catalyst. Phenol 1e served as the nucleo-
philic component for a standard Mitsunobu coupling with 1,4-
butanediol, previously protected as a monobenzoate ester, in
the presence of diisopropyl azodicarboxylate (DIAD) and tri-
phenylphosphine. The hydrolysis of 1 f with lithium hydroxide
allowed selective removal of the more base-sensitive benzoate
protecting group under very mild conditions. Primary alcohol
1g underwent an Appel bromination with carbon tetrabro-
mide and triphenylphosphine to afford 1h, which in turn, was
treated with the appropriate hydroxycoumarin in N,N-dime-
thylformamide in the presence of excess cesium carbonate as
the base at room temperature. Subsequent heating at 708C
enabled a facile cleavage of the silyl protecting group to fur-
nish the targeted 3-hydroxy-N,N-dimethylanilino derivatives 2–
7, 9, 10, and 12–17. These derivatives were then transformed
into the corresponding N,N,N-trimethyl- and N,N-dimethyl-N-
ethylbenzaminium iodides 2T–7T, 9T, 10T, 12T–17T, 9D, and
17D by reaction with excess methyl or ethyl iodide, respective-
ly, using dry acetonitrile as the solvent.
Biological assays
3-Hydroxy-N,N-dimethylanilino derivatives 2–20 and the corre-
sponding quaternary trialkylammonium salts were tested as in-
hibitors of bovine AChE and equine serum BChE according to
the spectrophotometric method of Ellman et al.[35] The struc-
tures and inhibition data toward AChE and BChE, expressed as
IC50 values or percent inhibition at 10 mm, are listed in Tables 1
and 2. With the exception of the most potent AChE
inhibitor 13, the IC50 values of the 3-hydroxy-N,N-di-
methylanilino derivatives were not measured, as their
inhibition of BChE was very low (<50% at 10 mm).
Selectivity toward AChE is expressed as the selectivity
index SI, that is, the ratio of (IC50 BChE)/(IC50 AChE), where-
as the strong affinity increase generally observed in
moving from amines to QASs is expressed as the
(IC50 AChE-amine)/(IC50 AChE-QAS) affinity ratio, AR (Table 2).
Molecular modeling studies
Scheme 3. Reagents and conditions: a) for 1i: 1,5-dibromopentane, Cs2CO3, KI, CH3CN
(anhyd), 1608C, MW, 1 h; b) for 1j: 1,3-bis(bromomethyl)benzene, K2CO3, THF (anhyd),
reflux, 9 h; c) Cs2CO3, DMF, H2O, RT, 24 h then 708C, 2 h; d) CH3I (excess), CH3CN (anhyd),
RT, 24 h.
The program GOLD[36] was used in all the docking
simulations. In the present investigation, GOLD dem-
onstrated outstanding accuracy in docking donepezil
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Tethered Coumarin Derivatives as AChE Inhibitors
the 3,4-dimethylcoumarin derivative 6. Moreover, the
positive influence of substituent lipophilicity on activ-
ity may be appreciated by comparing the affinity of
the 4-trifluoromethyl congener 5 (IC50 =60 nm) with
the corresponding 4-methyl derivative
4 (IC50 =
349 nm). Surprisingly, the highly lipophilic 3,4-cyclo-
hexyl- and 3,4-benzo-fused derivatives 9 and 10, re-
spectively, were less active than the 3,4-dimethyl con-
gener 6; this might be ascribed to a likely steric
effect exerted by the 3,4-fused rigid ring.
The influence of the length and conformational
flexibility of the linker on inhibitory potency was ex-
amined with compounds 8, 11, 18, and 19. Inhibitors
8 and 19, bearing a pentamethylene linker, are signif-
icantly less active than the corresponding inhibitors 7
and 17, which are characterized by a shorter tetra-
methylene linker. Moreover, replacement of the cen-
tral methylene unit of inhibitor 19 with an N-methyl
group, probably protonated at pH 8 under the condi-
tions of the spectrophotometric biological assay, af-
forded the less active inhibitor 20. Unlike other re-
ported findings on homodimeric tacrine deriva-
tives,[39] the insertion of a protonatable nitrogen in
the linker, despite undergoing a favorable hydrogen
bond interaction with Tyr124, seemed to determine a
decrease in affinity for the occurrence of an inverted
binding mode. As a matter of fact, molecular docking
simulations showed the coumarin and the 3-hydroxy-
N,N-dimethylanilino moieties of 20 bound to the pri-
mary and PAS sites, respectively (data not shown). Fi-
nally, compounds 11 and 18, which bear the more
Scheme 4. Reagents and conditions: a) for 1k: (2E)-1,4-dibromo-2-butene, K2CO3, THF
(anhyd), reflux, 7 h; b) for 1l: 1,5-dibromopentane, Cs2CO3, KI, CH3CN (anhyd), 1608C,
MW, 1 h; c) for compounds 18 and 19: Cs2CO3, DMF, H2O, RT, 24 h then 708C, 2 h; d) 1,2-
dibromoethane, Cs2CO3, KI, CH3CN (anhyd), 1608C, MW, 1 h; e) 2-methylaminoethanol,
DIEA, THF (anhyd), reflux, 15 h; f) for compound 1o: PPh3, DIAD, THF (anhyd), 18 h;
g) TBAF (1.0n in THF), THF (anhyd), RT, 30 min; h) CH3I (excess), CH3CN (anhyd), RT, 24 h.
into the TcAChE active site, as the deviation from the binding
conformation observed in the crystal structure (PDB ID: 1EVE)
is very small (RMSD=0.99 ꢂ). Molecular dynamics (MD) simula-
tions were carried out by using a periodic box of TIP3P water
molecules extended for 18 ꢂ from the protein atoms, modeled
with the parm03 force field. Energy minimization was executed
by using the AMBER 10.0[37] suite, while a 5 ns MD simulation
was performed with NAMD.[38]
rigid meta-xylyl and 1,4-butenyl linkers, exhibited lower affinity
than the corresponding inhibitors 8 (or 7) and 17 (or 19), re-
spectively, which have more flexible polymethylene linkers.
The most dramatic decrease in affinity was observed with the
meta-xylyl linker, from 49 nm (compound 7), or 161 nm (com-
pound 8), to 33% inhibition at 10 mm (compound 11).
Taken together, the above findings coherently suggest that
the tetramethylene linker is the ideal spacer in terms of length
and conformational flexibility for connecting the 3-hydroxy-
N,N-dimethylanilino and coumarin moieties. Consequently, the
tetramethylene linker was kept unaltered in the design of new
compounds exploring all the possible anchoring points on the
coumarin ring: positions 3 (12), 4 (14), 5 (15), 6 (17), and 8
(16). The SAFIRs of this new series of inhibitors were evaluated
taking the 3,4-unsubstituted 7-alkoxycoumarin derivative 2 as
the reference compound.
Discussion
Our molecular design was initially based on the high inhibitory
potency toward AChE that was recently observed for com-
pound 6 (IC50 =275 nm, Table 1).[29] This compound was there-
fore taken as a reference for the evaluation of the SAFIRs.
While maintaining the tetramethylene linker at position 7 of
compound 6 unaltered, the substituent effects at positions 3
and 4 of the coumarin ring were explored by synthesizing the
unsubstituted, mono-, and disubstituted derivatives 2–7, 9,
and 10, respectively. Within this series, AChE inhibition data in-
dicate that the substituent lipophilicity plays an important role
in inhibitor binding. In fact, the 3-chloro-4-methyl congener 7,
the most lipophilic compound of the series, proved to be five-
fold more potent than congener 6 (IC50: 49 versus 275 nm),
whereas 3,4-unsubstituted derivative 2 and the two mono-
methyl-substituted derivatives 3 and 4 were less active than
Derivatives 14, 15, and 16, substituted at the 4-, 5-, and 8-
positions, exhibited lower affinity than the corresponding 3-,
6-, and 7-substituted regioisomers 12, 17, and 2, suggesting
that lateral substitution at the coumarin ring, i.e., at positions
3, 6, and 7, are preferred over substitutions at positions 4, 5,
and 8. The coumarin ring in the 3-, 6-, and 7-substituted deriv-
atives may likely assume a more favorable binding conforma-
tion at the PAS pocket of AChE than the corresponding 4-, 5-,
and 8-substituted derivatives.
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A. Carotti et al.
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selective AChE inhibitor with ele-
vated therapeutic potential in
AD.
Table 1. Structures and ChE inhibition data for 3-hydroxy-N,N-dimethylanilino derivatives 2–20.
Very low BChE affinities were
measured for all the N,N-dime-
thylanilino derivatives 2–20,
ranging from 2 to 49% enzyme
inhibition at a concentration of
10 mm (Table 1). The AChE and
BChE inhibition data suggest
that these compounds can be
regarded as selective, sub-micro-
molar AChE inhibitors.
Compd
Linker
C. S.[a]
R1
R2
R3
IC50 [nm][b]
AChE
456
463
349
60.0
275
49.0
161
333
415
33%[c]
143
0.236
BChE
2
3
4
5
6[29]
7[d]
8
9
10
11
12
13[e]
14
15
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)5–
–(CH2)4–
–(CH2)4–
7
7
7
7
7
7
7
7
7
7
3
3
4
5
8
6
6
6
6
H
Me
H
H
Me
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
MeO
H
H
H
H
H
H
32%[c]
24%[c]
38%[c]
23%[c]
25%[c]
2%[c]
Me
CF3
Me
Me
Me
3-Hydroxy-N,N-dimethylanilino
derivatives 2–20 were trans-
formed into their corresponding
Cl
3%[c]
–(CH2)4–
–(CH=CH)2–
Cl Me
–
–
H
H
H
H
H
H
H
15%[c]
12%[c]
2%[c]
N,N,N-trimethylammonium
(T)
and N,N-dimethyl-N-ethylammo-
nium salts (D), and their struc-
tures and AChE–BChE inhibitory
potencies are listed in Table 2.
The IC50 affinity ratios and selec-
tivity indexes are reported in
Table 2 as respective measures
of the affinity increase in moving
from amines to QASs, and of the
enzymatic selectivity.
m–CH2(C6H4)CH2–
–(CH2)4–
H
H
–
H
H
H
H
H
H
27%[c]
71.0ꢃ103
23%[c]
30%[c]
45%[c]
9%[c]
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
36.2ꢃ102
13.4ꢃ102
16
17
18
19
976
–(CH2)4–
103
391
164
492
trans–CH2CH=CHCH2–
–(CH2)5–
–(CH2)2N(CH3)(CH2)2–
15%[c]
7%[c]
20
H
49%[c]
2.25ꢃ103
donepezil[f]
5.33
[a] C.S.=coumarin substitution: linker anchoring positions at the coumarin ring. [b] Data represent the mean
value from 2–3 independent experiments (relative SEM<10%). [c] Percent inhibition at 10 mm (relative SEM<
10%). [d] IC50 on human AChE=194ꢁ8.2 nm. [e] IC50 on human AChE=39.7ꢁ5.90 nm. [f] IC50 on human
AChE=26.0ꢁ2.00 nm.
AChE inhibition data in Table 2
indicate that QASs are much
more active than the corre-
sponding amines, exhibiting out-
standing sub-nanomolar inhibi-
tion potencies, except for com-
With the aim of further optimizing the AChE binding affinity
of compound 12, two methoxy groups were introduced at po-
sitions 6 and 7. A highly satisfactory and dramatic increase in
affinity of more than two orders of magnitude was observed
with compound 13, from 143 to 0.236 nm. Although a consis-
tent increase in affinity was eagerly sought, its intensity was
rather unexpected when considering previous findings for re-
lated AChE inhibitors, such as donepezil.[40]
pounds 6T, 11 T, 14T, and 20T, the affinities of which remain in
the low nanomolar range. The most active AChE inhibitors are
compounds 13T and 17T, with IC50 values of 0.012 and
0.024 nm, respectively. AR values of QASs indicate an affinity
increase of approximately three orders of magnitude relative
to the corresponding amines, with the notable exception of
the bis-QAS 20T, the low AR of which derives mainly from its
relatively low AChE affinity (IC50 =17 nm).
Kinetics of bovine AChE inhibition by compound 13 re-
vealed a competitive-type inhibition, with Ki =2.1ꢁ0.4 nm.
This mechanism differs from that of donepezil and other inhib-
itors, which supposedly bind at both the catalytic and periph-
eral sites of AChE. In most cases, mixed or noncompetitive-
type inhibitory activity has been reported. Under our experi-
mental conditions, this last mechanism of inhibition was ac-
tually demonstrated for donepezil, exhibiting a Ki value of
4.5ꢁ0.5 nm. Notably, both 13 and donepezil showed IC50
values toward human AChE greater than those toward bovine
AChE: 39.7ꢁ5.90 and 26.0ꢁ2.00 nm, respectively.
The QASs listed in Table 2 were also evaluated for their affin-
ity toward BChE, and IC50 values in the range of 91–2030 nm
were measured. The most potent inhibitors are compounds
4T, 7T, and 17T, whereas the least potent compounds are 2T,
13T, and 14T. In contrast to the AChE inhibition data, no de-
fined SAFIR emerged from a comparative analysis of the BChE
affinities of the QASs.
The poor BChE inhibitory activity of QASs make them very
highly AChE-selective inhibitors, with SI values ranging from
515 (15T) to 138333 (13T). Two notable exceptions are com-
pounds 11 T and 20T, which exhibit SI values as low as 36 and
45, respectively. These rather low figures result mainly from
their comparatively weaker AChE inhibitory potency (IC50: 5.8
and 17 nm, respectively) confirming that the meta-xylyl and
positively charged linkers are unsuitable for favorable interac-
tions at the AChE binding gorge of our inhibitors.
The excellent AChE inhibitory potency of compound 13
prompted us to determine its IC50 toward BChE as well, to
assess its selectivity profile. As expected, a very low BChE affin-
ity was measured (IC50 =71000 nm), resulting in a SI >300000.
Compound 13 can therefore be considered a highly active and
1620
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ChemMedChem 2010, 5, 1616 – 1630

Tethered Coumarin Derivatives as AChE Inhibitors
Table 2. Structures and ChE inhibition data for alkylammonium deriva-
tives 2–20T, 9D, and 17D.
Compd[a]
R4
IC50 [nm][b]
BChE
AR[c]
SI[d]
AChE
2T
3T
4T
Me
Me
Me
Me
Me
Me
Me
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Et
0.131
0.090
0.122
0.127
1.00
0.129
0.114
0.208
0.373
0.304
5.80
0.0540
0.0120
1.65
0.650
0.310
0.0240
0.149
0.217
0.151
14.0ꢃ102
499
91.0
11.6ꢃ102
3481
5144
2681
472
275
380
10687
5544
746
9134
708
899
2272
928
2397
3783
36
3704
138333
1230
515
1948
5708
4564
2525
3775
45
5T
6T[29]
7T
708
116
259
193
894
8T
9T
9D
1412
1601
893
1365
ND[e]
2648
20
2194
2062
3148
4292
691
1802
1086
29
10T
11 T
12T
13T[f]
14T
15T
16T
17T
17D
18T
19T
20T[g]
11.5ꢃ102
206
200
16.6ꢃ102
20.3ꢃ102
335
604
137
680
548
570
762
Me
Me
Me
17.0
[a] N,N-Dimethyl-N-ethyl- and N,N,N-trimethylammonium salts of the cor-
responding coumarins 2–20 listed in Table 1 are indicated with capital let-
ters D and T, respectively; R1, R2, and R3 groups are listed in Table 1.
[b] Data represent the mean value from 2–3 independent experiments
(relative SEM<10%). [c] Affinity ratio: (IC50 AChE-amine)/(IC50 AChE-QAS). [d] Selec-
tivity index: (IC50 BChE)/(IC50 AChE). [e] Not determinable. [f] IC50 on human
AChE=31.0 nm. [g] Compound 20T is a bis-quaternary ammonium salt
bearing a central dimethylammonium group in the linker.
Figure 3. a) Top-scored docking poses of inhibitors 2, 12, and 17 (rendered
in yellow, cyan, and white capped-stick models, respectively) into the AChE
binding sites. Amino acid residues are represented by ball-and-stick models
colored according to atom type. Hydrogen bonds are indicated by dark-blue
dashed lines. b) Top-scored docking poses of inhibitors 14, 15, and 16 (ren-
dered in cyan, yellow, and white capped-stick models, respectively) into the
AChE binding sites. Amino acid residues are represented by ball-and-stick
models colored according to atom type.
To gain insight into the molecular determinants responsible
for high AChE binding affinity and selectivity, anticipated in
part in the previous SAFIR analysis, a molecular modeling
study was carried out. The objective of this study was three-
fold: 1) to understand how the remarkable change in AChE af-
finity is related to the various substituents on the coumarin
ring and to the various structural properties of the linker, 2) to
rationalize the high AChE affinity of compound 13 resulting
from the introduction of two methoxy groups at positions 6
and 7 of 3-substituted coumarin derivative 12, and 3) to eluci-
date the excellent enzymatic selectivity observed for com-
pound 13.
Both docking and molecular dynamics (MD) simulations
were performed on wild-type human AChE retrieved from the
RCSB Protein Data Bank (hAChE, PDB ID: 1B41),[41] used as a
target structure. The GOLD program, used in all docking simu-
lations, was first run on a number of structurally diverse amine
inhibitors, i.e., 2, 12, and 14–17 (Table 1). The binding mode of
inhibitors 12, 17, and 2, which carry the linker attached to the
coumarin ring at the lateral positions 3, 6, and 7, respectively,
and which exhibit higher affinity than the corresponding 4-, 5-,
and 8-substituted regioisomers 14, 15, and 16, was initially ad-
dressed. The top-scored docking solutions illustrated in Fig-
ure 3a indicate that the three inhibitors maintain a similar
binding topology irrespective of the anchoring position of the
linker to the coumarin ring. More specifically, the 3-hydroxy-
N,N-dimethylanilino moiety is engaged in a p–p stacking inter-
action with Trp86 located in the AChE catalytic site, and is fur-
ther stabilized by the formation of two hydrogen bonds be-
tween the phenolic hydroxy group and the carboxylate and
hydroxy groups of residues Asp74 and Thr83, respectively. The
differently bonded coumarin rings are situated in the large hy-
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1621

A. Carotti et al.
MED
drophobic gorge of the PAS. In comparison with the 3-, 6-, and
7-substituted derivatives 12, 17, and 2, molecular docking sim-
ulations of 4-, 5-, and 8-substituted regioisomers 14, 15, and
16 resulted in an unexpected inverted binding mode, with the
coumarin moiety facing Trp86 at the catalytic binding site and
the N,N-dimethylanilino moiety establishing hydrophobic inter-
actions with the side chain of Tyr341 in the PAS (Figure 3b).
The second step of our investigation was aimed at clarifying
the substantial improvement in AChE binding affinity observed
in moving from inhibitor 12 to 13, which differs only in the
6,7-dimethoxy substitution at the coumarin ring. Notably, an
increase in affinity, albeit to a much lower extent (nearly 20-
fold), had already been observed in going from an unsubstitut-
ed 1-indanone moiety to the 5,6-dimethoxy-1-indanone
moiety of donepezil.[40] As molecular docking failed to interpret
this significant increase in AChE binding affinity for inhibitor
13, an MD study was undertaken. The top-scored docking
poses of 12 and 13 were used as convenient starting geome-
tries for determining the structural variation over time of the
two AChE complexes through MD simulations. Both systems
marin ring within the PAS in an area largely exposed to the sol-
vent. The two methoxy groups facing the solvent enable inhib-
itor 13 to insert its coumarin ring into the open slot between
the indolic and phenolic rings of Trp286 and Tyr341, respec-
tively. This sandwich-like orientation was maintained through-
out the MD simulation and is likely due to a stabilizing p–p
stacking interaction (Figure 4). In the primary AChE binding
site, inhibitor 13 orients its 3-hydroxy-N,N-dimethylanilino
moiety orthogonally to Trp86 with the N,N-dimethylamino and
phenolic groups establishing a network of hydrogen bonds
with two solvent water molecules. Unlike inhibitor 13, the 6,7-
unsubstituted analogue 12 reveals a more pronounced ten-
dency to dive deeper into the AChE gorge to minimize its sol-
vent exposure. Moreover, the stabilizing sandwich-like interac-
tion of coumarin with the Trp286 and Tyr341 side chains was
not detected by MD studies, as the coumarin is exclusively en-
gaged in a T-shaped orthogonal p–p interaction with Trp286
or, alternatively, in a parallel p–p interaction with Tyr341 (data
not shown).
To interpret the pronounced molecular enzymatic selectivity
(SI=300847) observed for compound 13, docking studies on
BChE were performed with the human BChE crystal structure
(PDB ID: 1POI). The BChE target structure was prepared by fol-
lowing the same procedures described for AChE (see Experi-
mental Section). A comparison between the AChE and BChE
top-scored solutions of inhibitor 13 reveal that the 3-hydroxy-
N,N-dimethylanilino moiety is oriented orthogonally to Trp82,
as its phenolic and ethereal oxygen atoms interact through hy-
drogen bonds with Glu197 and Ser198 (BChE numbering) of
the catalytic triad, respectively. In addition, the lack of a PAS in
BChE does not allow the occurrence of p–p interactions, which
are often observed in AChE inhibition (data not shown). The
strong AChE/BChE selectivity can therefore be explained
mainly by the different molecular binding conformations ob-
served, and is supported, at least in part, by the different
energy scores (i.e., 57.27 versus 50.84 kJmolꢀ1).
were equilibrated according to standard MD protocols.[42]
A
visual inspection of the molecular frames suggests that the in-
troduction of the two methoxy substituents stabilize the cou-
Conclusions
Rational structural modifications of 3-hydroxy-N,N-dimethylani-
lino derivative 6 led to the discovery of highly potent AChE in-
hibitors that exhibit binding affinities in the sub-micromolar to
sub-nanomolar range (Table 1). SAFIRs clearly indicate that a
flexible tetramethylene linker has the optimum length and
conformational mobility for high AChE inhibitory potency. In-
hibitors equipped with longer or more rigid, or charged/pro-
tonated linkers, exhibited a lower potency than the corre-
sponding inhibitors with a tetramethylene linker. Moreover, the
anchoring position of the linker to the coumarin ring plays an
important role in binding affinity; the 3-, 6-, and 7-substituted
derivatives are more potent than the corresponding 4-, 5-, and
8-substituted regioisomers. The highest inhibitory potency in
the whole series of 3-hydroxy-N,N-dimethylanilines was ob-
served with the 6,7-dimethoxy-3-substituted regioisomer 13,
which, along with outstanding potency (IC50 =0.236 nm), ex-
hibits excellent AChE/BChE selectivity (SI=300847). This com-
Figure 4. a) Plot showing the absolute incremental variation (from the initial
conformations obtained from molecular docking) of the half-sum of distan-
ces calculated from the centers of mass of residues W286 and Y341, and the
centroid of the coumarin ring of inhibitor 13 over 5 ns MD simulations. b) A
representative sandwich-like binding conformation of 13 taken after 5 ns
MD simulations.
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ChemMedChem 2010, 5, 1616 – 1630

Tethered Coumarin Derivatives as AChE Inhibitors
6,7-Dimethoxy-2H-chromen-2-one: A Pyrexꢁ vessel was charged
with a magnetic stir bar and a WeflonTM heating bar. Esculetin
(0.855 g, 4.80 mmol) was added followed by anhydrous acetone
(15 mL), CH3I (2.40 mL, 38.4 mmol), and anhydrous K2CO3 (3.32 g,
24.0 mmol). The vessel was placed in a microwave apparatus and
irradiated at 1108C for 30 min. The heating cycle was repeated
twice. The mixture was cooled to room temperature, and the inor-
ganic residue was filtered off. The solution was concentrated under
reduced pressure to yield the desired product as a yellow solid of
pound can be considered a promising candidate for additional
preclinical studies using in vitro and in vivo AD models.
QAS inhibitors (Table 2) showed outstanding AChE inhibitory
potencies, from the sub-nanomolar to picomolar range, and an
excellent AChE versus BChE selectivity, with SIs generally
>1000. Compared with the corresponding 3-hydroxy-N,N-di-
methylanilino derivatives, the AChE inhibitory potency of QASs
is nearly three orders of magnitude higher. The most active
and selective QAS is the 6,7-dimethoxycoumarin derivative
13T, with picomolar potency (IC50 =12 pm) and outstanding
selectivity (SI=138333). Further studies of 13T are warranted
to assess its pharmacological activity in glaucoma, neuromus-
cular blockade after surgical anesthesia, and myasthenia gravis.
Finally, the combined use of docking and MD simulations has
shed light on the SAFIRs observed in our series of amine inhib-
itors.
1
satisfactory purity (0.95 g, 96%): H NMR (300 MHz, [D6]DMSO): d=
7.94 (d, J=9.6 Hz, 1H), 7.26 (s, 1H), 7.05 (s, 1H), 6.28 (d, J=9.6 Hz,
1H), 3.84 (s, 3H), 3.78 ppm (s, 3H).
3-Hydroxy-6,7-dimethoxy-2H-chromen-2-one: 3-Hydroxy-6,7-di-
methoxy-2H-chromen-2-one was prepared by following slight
modifications of a reported method.[32] 6,7-Dimethoxy-2H-chro-
men-2-one (0.474 g, 2.30 mmol) was dissolved in glacial acetic acid
(10 mL). Fuming HNO3 (0.190 mL) was added, and the mixture was
heated at reflux for 30 min. After cooling to room temperature, the
mixture was diluted with a mixture of acetic anhydride/acetic acid
(1:1 v/v, 20 mL), and freshly activated zinc powder was added
(2.0 g, 30.6 mmol). The reaction mixture was stirred at room tem-
perature for 24 h and then filtered through a CeliteTM pad, washing
with CHCl3. After evaporation of the solvent, the residue was sus-
pended in H2O (200 mL) and extracted with CHCl3 (3ꢃ80 mL). The
organic layers were collected, dried over anhydrous Na2SO4, and
concentrated to dryness to yield the crude N-(6,7-dimethoxy-2-
oxo-2H-chromen-3-yl)acetamide intermediate, which underwent
hydrolysis with 3.0n aqueous HCl (5 mL) at reflux for 6 h. The mix-
ture was cooled, diluted with cold H2O (80 mL), and then extracted
with EtOAc (3ꢃ50 mL). After drying the combined organic layers
over anhydrous Na2SO4, the removal of the solvent by rotary evap-
oration yielded a crude solid that was purified by flash chromatog-
raphy (gradient eluent: EtOAc in n-hexane 0!30%) to yield a
Experimental Section
Chemistry
Starting materials, reagents, and analytical-grade solvents were
purchased from Sigma–Aldrich (Europe). Microwave (MW) reactions
were performed in a Milestone MicroSynth apparatus, setting tem-
perature and hold times, fixing maximum irradiation power to
500 W and ramp times to 5 min. The purity of all intermediates,
1
1
checked by H NMR and HPLC, was always >95%. H NMR spectra
were recorded in the deuterated solvent specified, at 300 MHz on
a Varian Mercury 300 instrument. Chemical shifts (d) are expressed
in parts per million relative to the solvent signal, and the coupling
constants (J) are given in Hz. The following abbreviations are used:
s (singlet), d (doublet), t (triplet), q (quadruplet), h (heptet), dd
(double doublet), m (multiplet), br (broad signal); signals due to
OH protons were located by deuterium exchange with D2O. ESIMS
analyses were performed on an Agilent 1100 LC–MSD trap system
VL. Chromatographic separations were performed on a Biotage
SP1 purification system using flash cartridges pre-packed with KP-
SilTM 32–63 mm, 60 ꢂ silica. For reactions requiring the use of anhy-
drous solvents, glassware was flame-dried and then cooled under
a stream of dry argon before use. All reactions were routinely
checked by TLC using Merck Kieselgel 60 F254 aluminum plates and
visualized by UV light or iodine. 7-Hydroxycoumarin, 3-hydroxycou-
marin, 7-hydroxy-4-trifluoromethylcoumarin, 6,7-dihydroxycoumar-
in, 3-chloro-7-hydroxy-4-methylcoumarin, 6-hydroxycoumarin, and
4-hydroxycoumarin were obtained from commercial sources. 7-Hy-
droxy-4-methylcoumarin,[43] 7-hydroxy-3-methylcoumarin,[44] and
3,4-dimethyl-7-hydroxycoumarin[43] were prepared by following
published methods. The purity of all tested final products, deter-
mined by reversed-phase (RP) HPLC, was consistently >98%. RP
HPLC analyses were performed on a system equipped with auto-
matic injector and a Waters Breeze 1525 pump coupled with a
Waters 2489 UV detector using a Waters XTerra RP 5 mm C8 column
(150ꢃ3.0 mm i.d.). The UV detection was measured at l 254 and
280 nm. Each tested compound was analyzed by elution with two
different mobile phase systems: in system 1, compounds were
eluted with a mixture of CH3OH/H2O (80:20 v/v) at a flow rate of
0.5 mLminꢀ1; in system 2, compounds were eluted with a mixture
of CH3CN/H2O (65:35 v/v) at a flow rate of 0.5 mLminꢀ1. The syn-
thesis and spectroscopic data for compound 6 and its correspond-
ing QAS 6T have been previously reported.[45]
1
white solid (0.16 g, 31%): H NMR (300 MHz, [D6]DMSO): d=9.95 (s,
1H, dis. with D2O), 7.09 (s, 1H), 7.06 (s, 1H), 7.00 (s, 1H), 3.78 (s,
3H), 3.75 ppm (s, 3H).
3-Hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (1a):
Ethyl 2-oxocyclohexanecarboxylate (1.61 mL, 10.0 mmol) and resor-
cinol (1.10 g, 10.0 mmol) were heated at 1208C for 10 min with vig-
orous magnetic stirring after the addition of a few drops of concd
H2SO4. The solid formed after cooling was washed with EtOAc and
filtered to yield 1a as a white solid (1.54 g, 71%): 1H NMR
(300 MHz, [D6]DMSO): d=10.30 (s, 1H, dis. with D2O), 7.51 (d, J=
8.8 Hz, 1H), 6.73–6.77 (m, 1H), 6.66–6.67 (m, 1H), 2.70–2.71 (m,
2H), 2.36–2.38 (m, 2H), 1.67–1.74 ppm (m, 4H).
3-Methoxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (1b):
Coumarin derivative 1a (1.30 g, 6.0 mmol) was dissolved in anhy-
drous DMF (20 mL). Anhydrous K2CO3 (1.25 g, 9.0 mmol) and CH3I
(0.374 mL, 6.0 mmol) were then added. The mixture was stirred at
room temperature for 24 h and poured onto crushed ice (200 g).
The resulting precipitate was then filtered and washed with H2O to
yield O-methylated coumarin derivative 1b as a white solid (1.35 g,
98%): 1H NMR (300 MHz, [D6]DMSO): d=7.60 (d, J=8.5 Hz, 1H),
6.93 (s, 1H), 6.89–6.94 (m, 1H), 3.82 (s, 3H), 2.72–2.75 (m, 2H),
2.36–2.39 (m, 2H), 1.66–1.78 ppm (m, 4H).
3-Methoxy-6H-benzo[c]chromen-6-one
(1c):
DDQ
(2.45 g,
10.8 mmol) was suspended in anhydrous dioxane (30 mL), and
then coumarin intermediate 1b (1.24 g, 3.60 mmol) was added.
After holding at reflux for 24 h, the mixture was cooled to room
temperature, and the solvent was removed under vacuum. The
crude solid residue was dissolved in a mixture of H2O/EtOAc (3:1
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1623

A. Carotti et al.
MED
v/v, 600 mL) and extracted with EtOAc (3ꢃ150 mL). The organic
layers were combined, dried over Na2SO4, and concentrated under
vacuum. Purification of the resulting solid by flash chromatography
(gradient eluent: EtOAc in n-hexane 0!10%) yielded coumarin de-
49%): 1H NMR (300 MHz, [D6]DMSO): d=10.17 (s, 1H, dis. with
D2O), 7.99 (d, J=9.6 Hz, 1H), 7.06–7.16 (m, 3H), 6.44 ppm (d, J=
9.6 Hz, 1H).
1
3-(Dimethylamino)-5-[(triisopropylsilyl)oxy]phenol (1e): 5-(Dime-
thylamino)benzene-1,3-diol[34] (4.60 g, 30.0 mmol) was dissolved in
anhydrous CH2Cl2 (100 mL), and imidazole (5.11 g, 75.0 mmol) was
added to the clear solution. The mixture was cooled to 0–58C
through an external ice bath, and TIPSCl (6.42 mL, 30.0 mmol) was
added dropwise by syringe. The solution was slowly allowed to
warm to room temperature and then kept under magnetic stirring
for 2 h. H2O (450 mL) was added, and the aqueous layer was ex-
tracted with CH2Cl2 (3ꢃ200 mL). The organic phases were com-
bined, dried over anhydrous Na2SO4, and concentrated under
vacuum. The resulting crude yellow oil was purified by flash chro-
matography (gradient eluent: EtOAc in n-hexane 0!20%) to yield
a yellow oil that solidified upon standing (8.82 g, 95%): 1H NMR
(300 MHz, [D6]DMSO): d=8.95 (s, 1H, dis. with D2O), 5.70–5.71 (m,
1H), 5.63–5.66 (m, 2H), 2.77 (s, 6H), 1.19 (h, J=6.6 Hz, 3H),
1.04 ppm (d, J=6.6 Hz, 18H).
rivative 1c (0.56 g, 69%): H NMR (300 MHz, [D6]DMSO): d=8.32 (d,
J=8.3 Hz, 1H), 8.26 (d, J=9.1 Hz, 1H), 8.18–8.21 (m, 1H), 7.87–7.92
(m, 1H), 7.56–7.61 (m, 1H), 7.00–7.01 (m, 1H), 6.97–6.98 (m, 1H),
3.85 ppm (s, 3H).
3-Hydroxy-6H-benzo[c]chromen-6-one (1d): Coumarin derivative
1c (0.450 g, 1.98 mmol) was dissolved in anhydrous CH2Cl2 (15 mL),
and a 1.0n solution of BBr3 in CH2Cl2 (0.590 mL, 5.90 mmol) was
added dropwise with simultaneous cooling to 08C with an external
ice bath. The reaction was slowly warmed to room temperature
and then stirred for 36 h. H2O (50 mL) was added with cooling and
stirred for 1 h. The obtained suspension was extracted with CH2Cl2
(3ꢃ80 mL). The organic layers were collected, dried over anhy-
drous Na2SO4, concentrated under vacuum, and finally purified by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
20%) to yield the desired O-demethylated coumarin intermediate
1
1d as a red solid (0.223 g, 53%): H NMR (300 MHz, [D6]DMSO): d=
4-Hydroxybutyl benzoate: 1,4-Butanediol (7.44 mL, 84.0 mmol)
was suspended in anhydrous CH3CN (180 mL), and DIPEA (4.70 mL,
27.0 mmol) was added. Benzoyl chloride (3.13 mL, 27.0 mmol), pre-
viously dissolved in anhydrous CH3CN (20 mL), was added drop-
wise while maintaining the solution at 0–58C through an external
ice bath. The mixture was stirred for 15 min with cooling and then
brought to room temperature and stirred for additional 3 h. The
solvent was removed under reduced pressure, and the crude oil
was purified by flash chromatography (gradient eluent: EtOAc in n-
hexane 0!30%) to yield the desired product as a colorless oil
(4.82 g, 92%): 1H NMR (300 MHz, CDCl3): d=8.02–8.06 (m, 2H),
7.53–7.58 (m, 1H), 7.41–7.47 (m, 2H), 4.36 (t, J=6.3 Hz, 2H), 3.73 (t,
J=6.3 Hz, 2H), 1.80–1.92 (m, 2H), 1.68–1.77 (m, 2H), 1.66 ppm
(brs, 1H, dis. with D2O).
10.35 (s, 1H, dis. with D2O), 8.23–8.26 (m, 1H), 8.16–8.18 (m, 1H),
8.14–8.15 (m, 1H), 7.84–7.89 (m, 1H), 7.52–7.57 (m, 1H), 6.81–6.84
(m, 1H), 6.73–6.74 ppm (m, 1H).
2,6-Dihydroxybenzaldehyde:
2,6-Dimethoxybenzaldehyde
(0.831 g, 5.0 mmol) was stirred in anhydrous CH2Cl2 (50 mL) until
complete dissolution. The solution was then cooled to 08C, and a
1.0n solution of BBr3 in CH2Cl2 (20.0 mL, 20.0 mmol) was added
dropwise over 30 min. After slow warming to room temperature,
the mixture was stirred for 36 h, during which a precipitate
formed. The mixture was poured onto crushed ice (500 g) and
acidified by the careful addition of a 10% aqueous solution of HCl.
The aqueous solution was extracted with EtOAc (3ꢃ200 mL), and
the organic layers were combined, dried over Na2SO4, and concen-
trated under vacuum. Subsequent purification by flash chromatog-
raphy (gradient eluent: EtOAc in n-hexane 0!20%) gave the de-
sired aldehyde (0.387 g, 56%): 1H NMR (300 MHz, [D6]DMSO): d=
11.25 (s, 2H, dis. with D2O), 10.23 (s, 1H), 7.34 (t, J=8.3 Hz, 1H),
6.35 ppm (d, J=8.3 Hz, 2H).
4-{3-(Dimethylamino)-5-[(triisopropylsilyl)oxy]phenoxy}butyl
benzoate (1 f): 4-Hydroxybutyl benzoate (4.27 g, 22.5 mmol) was
dissolved in anhydrous THF (63 mL), and then phenol 1e (8.36 g,
27.0 mmol) was added followed by PPh3 (7.08 g, 27.0 mmol). The
solution was then cooled to 08C through an external ice bath, and
a solution of DIAD (5.31 mL, 27.0 mmol) in anhydrous THF (27 mL)
was added dropwise. The reaction was slowly warmed to room
temperature and stirred for 15 h. The solvent was removed under
reduced pressure, and the resulting crude mixture was purified by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
5%), thus obtaining the desired product as a dark-brown oil
(10.27 g, 94%): 1H NMR (300 MHz, CDCl3): d=8.03–8.06 (m, 2H),
7.53–7.58 (m, 1H), 7.41–7.46 (m, 2H), 5.84–5.91 (m, 3H), 4.39 (t, J=
6.1 Hz, 2H), 3.98 (t, J=5.5 Hz, 2H), 2.91 (s, 6H), 1.92–1.98 (m, 4H),
1.21–1.32 (m, 3H), 1.10 ppm (d, J=6.6 Hz, 18H).
General procedure A for the synthesis of 6- and 8-hydroxycou-
marin: The suitable aldehyde (1.0 equiv) was dissolved in N,N-di-
ethylaniline (20 mL) and ethyl (triphenylphosphoranylidene)acetate
(1.5 equiv) was added. The mixture was stirred at room tempera-
ture for 2 h and then held at reflux for 1–3 h until TLC monitoring
showed disappearance of the starting aldehyde and cinnamate in-
termediate. The reaction mixture was then cooled and diluted with
6.0n HCl (150 mL) before extracting with EtOAc (3ꢃ100 mL). The
organic layers were combined, dried over anhydrous Na2SO4, and
concentrated under vacuum.
5-Hydroxy-2H-chromen-2-one: Prepared according to procedur-
e A, starting from 2,3-dihydroxybenzaldehyde (0.276 g, 2.0 mmol)
and ethyl (triphenylphosphoranylidene)acetate (1.05 g, 3.0 mmol).
Purification by flash column chromatography (gradient eluent:
EtOAc in n-hexane 0!20%) yielded the desired aldehyde (0.292 g,
90%): 1H NMR (300 MHz, [D6]DMSO): d=10.75 (s, 1H, dis. with
D2O), 8.09 (d, J=9.6 Hz, 1H), 7.38 (t, J=8.3 Hz, 1H), 6.78 (d, J=
8.3 Hz, 1H), 6.74 (d, J=8.3 Hz, 1H), 6.31 ppm (d, J=9.6 Hz, 1H).
4-{3-(Dimethylamino)-5-[(triisopropylsilyl)oxy]phenoxy}butan-1-
ol (1g): Benzoate 1 f (7.29 g, 15.0 mmol) was suspended in a mix-
ture of CH3OH/THF (5:1 v/v, 60 mL) and stirred until complete dis-
solution. LiOH·H2O (3.15 g, 75.0 mmol) was added, and the reaction
was stirred at room temperature for 3.5 h. H2O (7.5 mL) was added
dropwise, and the resulting suspension was kept under stirring for
1 h. The mixture was further diluted with the addition of H2O
(150 mL) and extracted with Et2O (3ꢃ90 mL). The organic layers
were collected and washed with a solution of 1.0n NaOH in H2O
(1ꢃ60 mL). The organic phase was dried over Na2SO4, and the sol-
vent was removed under vacuum to yield the desired product as a
8-Hydroxy-2H-chromen-2-one: Prepared according to procedure
A, starting from 2,6-dihydroxybenzaldehyde (0.276 g, 2.0 mmol)
and ethyl (triphenylphosphoranylidene)acetate (1.05 g, 3.0 mmol).
Purification by flash column chromatography (gradient eluent:
EtOAc in n-hexane 0!20%) yielded the desired aldehyde (0.159 g,
1
colorless oil (5.15 g, 90%): H NMR (300 MHz, CDCl3): d=5.86–5.92
(m, 3H), 3.96 (t, J=6.1 Hz, 2H), 3.72 (t, J=5.8 Hz, 2H), 2.91 (s, 6H),
1624
www.chemmedchem.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1616 – 1630

Tethered Coumarin Derivatives as AChE Inhibitors
1.82–1.91 (m, 2H), 1.70–1.79 (m, 2H), 1.19–1.31 (m, 3H), 1.11 ppm
(d, J=6.9 Hz, 18H), 1 OH not detected.
butene (0.749 g, 3.50 mmol) and 6-hydroxycoumarin (0.162 g,
1.0 mmol). Purification by flash chromatography (gradient eluent:
EtOAc in n-hexane 0!20%) afforded 1k as a yellow solid (0.186 g,
63%): 1H NMR (300 MHz, [D6]DMSO): d=7.98 (d, J=9.4 Hz, 1H),
7.33 (d, J=8.8 Hz, 1H), 7.28 (d, J=2.8 Hz, 1H), 7.21 (dd, J1 =2.8 Hz,
J2 =8.8 Hz, 1H), 6.48 (d, J=9.4 Hz, 1H), 6.06–6.09 (m, 2H), 4.62–
4.63 (m, 2H), 4.12–4.19 (m, 2H).
3-(4-Bromobutoxy)-N,N-dimethyl-5-[(triisopropylsilyl)oxy]aniline
(1h): A solution of alcohol 1g (4.54 g, 11.9 mmol) in anhydrous
CH2Cl2 (34 mL) was cooled to 08C and CBr4 (4.34 g, 13.1 mmol) was
added. While keeping the reaction mixture at 08C, a solution of
PPh3 (3.74 g, 14.3 mmol) in anhydrous CH2Cl2 (8.5 mL) was added
dropwise. The reaction was slowly allowed to warm to room tem-
perature and stirred for additional 2 h. The resulting crude mixture
was then evaporated to dryness and purified by flash chromatog-
raphy (gradient eluent: EtOAc in n-hexane 0!10%) to afford the
desired product 1h (4.34 g, 82%): 1H NMR (300 MHz, CDCl3): d=
5.83–5.88 (m, 3H), 3.94 (t, J=6.5 Hz, 2H), 3.48 (t, J=6.6 Hz, 2H),
2.90 (s, 6H), 2.01–2.09 (m, 2H), 1.86–1.95 (m, 2H), 1.19–1.31 (m,
3H), 1.11 ppm (d, J=6.9 Hz, 18H).
General procedure D for the synthesis of coumarins 2–7, 9, 10,
12–17: A suitable hydroxycoumarin derivative (0.350 mmol), com-
mercially available or prepared by following the procedures de-
scribed above, was dissolved in DMF (3.5 mL) followed by the addi-
tion of H2O (70.0 mL) and Cs2CO3 (0.326 g, 1.0 mmol). The mixture
was stirred for 30 min, then bromide 1h (0.187 g, 0.420 mmol) was
added, and the reaction was stirred for 24 h at room temperature
and then heated at 708C for 2 h. The solvent was removed by
rotary evaporation.
General procedure B for the preparation of bromides 1i, 1l: A
Pyrexꢁ vessel was charged with a magnetic stir bar and a WeflonTM
heating bar. The appropriate coumarin (2.0 mmol) was added and
dissolved in anhydrous THF (10 mL). Then 1,5-dibromopentane
(1.63 mL, 12.0 mmol), Cs2CO3 (0.651 g, 2.0 mmol), and KI (0.033 g,
0.2 mmol) were added, and the vessel mixture was placed in the
microwave reactor and irradiated at 1608C for 1 h. After cooling,
the mixture was concentrated to dryness.
7-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-2H-chro-
men-2-one (2): Prepared by following general procedure D, start-
ing from 7-hydroxycoumarin (0.057 g, 0.35 mmol). Purification by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
1
60%) afforded 2 as a white solid (0.084 g, 65%): H NMR (300 MHz,
[D6]acetone): d=7.90 (d, J=9.4 Hz, 1H), 7.58 (d, J=8.5 Hz, 1H),
6.94 (dd, J1 =2.5 Hz, J2 =8.5 Hz, 1H), 6.90 (d, J=2.5 Hz, 1H), 6.21
(d, J=9.4 Hz, 1H), 5.85–5.87 (m, 3H), 4.22 (t, J=6.3 Hz, 2H), 4.01 (t,
J=6.3 Hz, 2H), 2.86 (s, 6H), 1.92–2.02 ppm (m, 4H), 1 OH not de-
tected; ESIMS m/z: 392 [M+Na]+, 368 [MꢀH]ꢀ.
7-[(5-Bromopentyl)oxy]-3-chloro-4-methyl-2H-chromen-2-one
(1i): Prepared by following general procedure B, starting from 3-
chloro-7-hydroxy-4-methylcoumarin (0.421 g, 2.0 mmol). Purifica-
tion by flash chromatography (gradient eluent: EtOAc in n-hexane
0!20%) afforded 1i as a white solid (0.626 g, 87%): 1H NMR
(300 MHz, CDCl3): d=7.53 (d, J=8.8 Hz, 1H), 6.89 (dd, J1 =2.5 Hz,
J2 =8.8 Hz, 1H), 6.83 (d, J=2.5 Hz, 1H), 4.09 (t, J=6.1 Hz, 2H), 3.41
(t, J=6.1 Hz, 2H), 2.55 (s, 3H), 1.94–2.13 (m, 4H), 1.40–1.45 ppm
(m, 2H).
7-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-3-methyl-
2H-chromen-2-one (3): Prepared by following general proced-
ure D, starting from 7-hydroxy-3-methylcoumarin (0.062 g,
0.35 mmol). Purification by flash chromatography (gradient eluent:
EtOAc in n-hexane 0!60%) afforded 3 as a white solid (0.105 g,
1
78%): H NMR (300 MHz, [D6]acetone): d=7.69 (s, 1H), 7.49 (d, J=
8.5 Hz, 1H), 6.88–6.93 (m, 2H), 5.82–5.86 (m, 3H), 4.19 (t, J=6.1 Hz,
2H), 4.01 (t, J=6.1 Hz, 2H), 2.83 (s, 6H), 2.09 (d, J=1.4 Hz, 3H),
1.90–2.01 ppm (m, 4H), 1 OH not detected; ESIMS m/z: 406
[M+Na]+, 382 [MꢀH]ꢀ.
6-[(5-Bromopentyl)oxy]-2H-chromen-2-one (1l): Prepared by fol-
lowing general procedure B, starting from 6-hydroxycoumarin
(0.324 g, 2.0 mmol). Purification by flash chromatography (gradient
eluent: EtOAc in n-hexane 0!25%) afforded 1l as a yellow solid
(0.523 g, 84%): 1H NMR (300 MHz, [D6]DMSO): d=7.98 (d, J=
9.6 Hz, 1H), 7.31 (d, J=9.1 Hz, 1H), 7.27 (d, J=3.0 Hz, 1H), 7.17
(dd, J1 =3.0 Hz, J2 =8.8 Hz, 1H), 6.47 (d, J=9.6 Hz, 1H), 3.99 (t, J=
6.3 Hz, 2H), 3.55 (t, J=6.6 Hz, 2H), 1.85 (q, J=6.6 Hz, 2H), 1.74 (q,
J=6.6 Hz, 2H), 1.46–1.59 ppm (m, 2H).
7-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-4-methyl-
2H-chromen-2-one (4): Prepared by following general proced-
ure D, starting from 7-hydroxy-4-methylcoumarin (0.062 g,
0.35 mmol). Purification by flash chromatography (gradient eluent:
EtOAc in n-hexane 0!60%) afforded 4 as a off-white solid
(0.074 g, 55%): 1H NMR (300 MHz, [D6]acetone): d=7.93 (s, 1H),
7.67 (d, J=8.8 Hz, 1H), 6.95 (dd, J1 =2.5 Hz, J2 =8.8 Hz, 1H), 6.89
(d, J=2.5 Hz, 1H), 6.12 (d, J=1.4 Hz, 1H), 5.80–5.84 (m, 3H), 4.22
(t, J=6.1 Hz, 2H), 4.01 (t, J=6.1 Hz, 2H), 2.83 (s, 6H), 2.44 (d, J=
1.4 Hz, 3H),1.91–2.02 ppm (m, 4H); ESIMS m/z: 406 [M+Na]+, 382
[MꢀH]ꢀ.
General procedure C for the synthesis of bromides 1j, 1k: A
suitable bromide (3.5 mmol) and anhydrous K2CO3 (0.278 g,
2.0 mmol) were mixed in anhydrous THF (10 mL) at reflux. The ap-
propriate coumarin (1.0 mmol) was then added portion-wise over
4 h. After completion of the addition, the reaction was heated for
an additional 5 h and then cooled to room temperature. The inor-
ganic precipitate was filtered off and washed with THF. The solvent
was evaporated under vacuum.
7-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-4-(trifluoro-
methyl)-2H-chromen-2-one (5): Prepared by following general
procedure D, starting from 7-hydroxy-4-trifluoromethylcoumarin
(0.081 g, 0.35 mmol). Purification by flash chromatography (gradi-
ent eluent: EtOAc in n-hexane 0!50%) afforded 5 as a dark-pink
7-{[3-(Bromomethyl)benzyl]oxy}-3-chloro-4-methyl-2H-chromen-
2-one (1j): Prepared according to procedure C, starting from 1,3-
bis(bromomethyl)benzene (0.924 g, 3.5 mmol) and 3-chloro-7-hy-
droxy-4-methylcoumarin (0.211 g, 1.0 mmol). Purification by flash
chromatography (gradient eluent: EtOAc in n-hexane 0!30%) af-
1
solid (0.107 g, 70%): H NMR (300 MHz, [D6]DMSO): d=9.00 (s, 1H,
dis. with D2O), 7.58–7.61 (m, 1H), 7.13–7.14 (m, 1H), 7.03–7.06 (m,
1H), 6.84 (s, 1H), 5.68–5.70 (m, 3H), 4.14–4.18 (m, 2H), 3.89–3.92
(m, 2H), 2.78 (s, 6H), 1.81–1.85 (m, 4H); ESIMS m/z: 460 [M+Na]+,
436 [MꢀH]ꢀ.
1
forded 1j as an off-white solid (0.299 g, 76%): H NMR (300 MHz,
[D6]DMSO): d=7.78 (d, J=9.1 Hz, 1H), 7.54 (s, 1H), 7.40–7.41 (m,
3H), 7.13 (d, J=2.5 Hz, 1H), 7.08 (dd, J1 =2.5 Hz, J2 =9.1 Hz, 1H),
5.22 (s, 2H), 4.71 (s, 2H), 2.52 ppm (d, J=9.1 Hz, 3H).
3-Chloro-7-{4-[3-(dimethylamino)-5-hydroxyphenoxy]butoxy}-4-
methyl-2H-chromen-2-one (7): Prepared by following general pro-
cedure D, starting from 3-chloro-7-hydroxy-4-methylcoumarin
6-{[(2E)-4-Bromobut-2-en-1-yl]oxy}-2H-chromen-2-one (1k): Pre-
pared according to procedure C, starting from (2E)-1,4-dibromo-2-
ChemMedChem 2010, 5, 1616 – 1630
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1625

A. Carotti et al.
MED
(0.074 g, 0.35 mmol). Purification by flash chromatography (gradi-
ent eluent: EtOAc in n-hexane 0!30%) afforded 7 as a yellow
solid (0.097 g, 66%): 1H NMR (300 MHz, CDCl3): d=7.51 (d, J=
8.8 Hz, 1H), 6.89 (dd, J1 =2.2 Hz, J2 =8.8 Hz, 1H), 6.83 (d, J=2.2 Hz,
1H), 5.84–5.88 (m, 3H), 4.10 (t, J=6.1 Hz, 2H), 4.00 (t, J=5.8 Hz,
2H), 2.90 (s, 6H), 2.55 (s, 3H), 1.94–2.02 ppm (m, 4H), 1 OH not de-
tected; ESIMS m/z (%): 440 (100) [M+Na]+, 442 (36), 416 (100)
[MꢀH]ꢀ, 418 (35).
60%) afforded 15 as a off-white solid (0.089 g, 69%): 1H NMR
(300 MHz, [D6]DMSO): d=9.00 (s, 1H, dis. with D2O), 8.08 (d, J=
9.6 Hz, 1H), 7.53 (t, J=8.5 Hz, 1H), 6.92–6.95 (m, 2H), 6.34 (d, J=
9.6 Hz, 1H), 5.67–5.70 (m, 3H), 4.17 (t, J=6.1 Hz, 2H), 3.93 (t, J=
5.8 Hz, 2H), 2.78 (s, 6H), 1.87–1.91 ppm (m, 4H); ESIMS m/z: 392
[M+Na]+, 368 [MꢀH]ꢀ.
8-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-2H-chro-
men-2-one (16): Prepared by following general procedure D, start-
ing from 8-hydroxycoumarin (0.057 g, 0.35 mmol). Purification by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
3-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-7,8,9,10-tet-
rahydro-6H-benzo[c]chromen-6-one (9): Prepared by following
general procedure D, starting from 1a (0.076 g, 0.35 mmol). Purifi-
cation by flash chromatography (gradient eluent: EtOAc in n-
hexane 0!40%) afforded 9 as a brown solid (0.086 g, 58%):
1H NMR (300 MHz, [D6]DMSO): d=8.99 (s, 1H, dis. with. D2O), 7.60
(d, J=8.5 Hz, 1H), 6.90–6.94 (m, 2H), 5.67–5.70 (m, 3H), 4.10 (t, J=
5.8 Hz, 2H), 3.90 (t, J=5.8 Hz, 2H), 2.78 (s, 6H), 2.71–2.75 (m, 2H),
2.38–2.40 (m, 2H), 1.78–1.86 (m, 4H), 1.68–1.75 ppm (m, 4H);
ESIMS m/z: 446 [M+Na]+, 422 [MꢀH]ꢀ.
50%) afforded 16 as
a
white solid (0.075 g, 58%): 1H NMR
(300 MHz, [D6]DMSO): d=9.00 (s, 1H, dis. with D2O), 8.03 (d, J=
9.6 Hz, 1H), 7.24–7.32 (m, 3H), 6.48 (d, J=9.6 Hz, 1H), 5.69 (brs,
3H), 4.15 (t, J=6.1 Hz, 2H), 3.92 (t, J=5.8 Hz, 2H), 2.78 (s, 6H),
1.82–1.93 ppm (m, 4H); ESIMS m/z: 392 [M+Na]+, 368 [MꢀH]ꢀ.
6-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-2H-chro-
men-2-one (17): Prepared by following general procedure D, start-
ing from 6-hydroxycoumarin (0.057 g, 0.35 mmol). Purification by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
3-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-6H-ben-
zo[c]chromen-6-one (10): Prepared by following general proced-
ure D, starting from 1d (0.074 g, 0.35 mmol). Purification by flash
chromatography (gradient eluent: EtOAc in n-hexane 0!30%) af-
forded 10 as a red solid (0.103 g, 70%): 1H NMR (300 MHz,
[D6]acetone): d=8.26–8.29 (m, 2H), 8.20 (d, J=9.1 Hz, 1H), 7.87–
7.93 (m, 1H), 7.57–7.63 (m, 1H), 7.01 (dd, J1 =2.5 Hz, J2 =8.8 Hz,
1H), 6.95 (d, J=2.5 Hz, 1H), 5.82–5.85 (m, 3H), 4.23 (t, J=6.1 Hz,
2H), 4.02 (t, J=6.1 Hz, 2H), 2.86 (s, 6H), 1.91–2.02 ppm (m, 4H),
1 OH not detected; ESIMS m/z: 442 [M+Na]+, 418 [MꢀH]ꢀ.
70%) afforded 14 as
a
white solid (0.079 g, 61%): 1H NMR
(300 MHz, [D6]DMSO): d=8.99 (s, 1H, dis. with D2O), 7.97 (d, J=
9.6 Hz, 1H), 7.32 (d, J=9.1 Hz, 1H), 7.27 (d, J=2.8 Hz, 1H), 7.18
(dd, J1 =2.8 Hz, J2 =9.1 Hz, 1H), 6.47 (d, J=9.6 Hz, 1H), 5.67–5.70
(m, 3H), 4.05 (t, J=6.1 Hz, 2H), 3.90 (t, J=5.8 Hz, 2H), 2.78 (s, 6H),
1.79–1.87 ppm (m, 4H); ESIMS m/z: 392 [M+Na]+, 368 [MꢀH]ꢀ.
General procedure E for the synthesis of coumarin derivatives 8,
11, 18–19: Phenol 1e (0.108 g, 0.350 mmol), was dissolved in DMF
(3.5 mL) followed by the addition of H2O (70.0 mL) and Cs2CO3
(0.326 g, 1.0 mmol). The mixture was stirred for 30 min at room
temperature, then a suitable bromide 1i,j or 1k,l (0.420 mmol) was
added, and the reaction was stirred for additional 24 h and then
heated at 708C for 2 h. The solvent was removed by rotary evapo-
ration.
3-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-2H-chro-
men-2-one (12): Prepared by following general procedure D, start-
ing from 3-hydroxycoumarin (0.057 g, 0.35 mmol). Purification by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
30%) afforded 12 as
a
white solid (0.081 g, 63%): 1H NMR
(300 MHz, CDCl3): d=7.31–7.40 (m, 3H), 6.84–6.91 (m, 2H), 5.80–
5.88 (m, 3H), 4.10 (t, J=5.8 Hz, 2H), 4.01 (t, J=5.8 Hz, 2H), 2.89 (s,
6H), 1.96–2.17 ppm (m, 4H), 1 OH not detected; ESIMS m/z: 392
[M+Na]+, 368 [MꢀH]ꢀ.
3-Chloro-7-({5-[3-(dimethylamino)-5-hydroxyphenoxy]pentyl}-
oxy)-4-methyl-2H-chromen-2-one (8): Prepared by following
general procedure E, starting from bromide 1i (0.151 g,
0.42 mmol). Purification by flash chromatography (gradient eluent:
EtOAc in n-hexane 0!30%) afforded 8 as a white solid (0.083 g,
3-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-6,7-dime-
thoxy-2H-chromen-2-one (13): Prepared by following general pro-
cedure D, starting from 3-hydroxy-6,7-dimethoxycoumarin ob-
tained as previously described (0.078 g, 0.35 mmol). Purification by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
1
55%): H NMR (300 MHz, [D6]DMSO): d=8.98 (s, 1H, dis. with D2O),
7.74 (d, J=8.8 Hz, 1H), 7.03 (d, J=2.5 Hz, 1H), 6.99 (dd, J1 =2.5 Hz,
J2 =8.8 Hz, 1H), 5.67–5.69 (m, 3H), 4.09 (t, J=6.6 Hz, 2H), 3.85 (t,
J=6.3 Hz, 2H), 2.78 (s, 6H), 2.52 (s, 3H), 1.67–1.80 (m, 4H),1.50–
1.58 ppm (m, 2H); ESIMS m/z: 454 (100) [M+Na]+, 456 (34), 430
(100) [MꢀH]ꢀ, 432 (35).
60%) afforded 13 as
a
white solid (0.096 g, 64%): 1H NMR
(300 MHz, [D6]DMSO): d=9.00 (s, 1H, dis. with D2O), 7.28 (s, 1H),
7.11 (s, 1H), 7.02 (s, 1H), 5.70 (m, 3H), 4.02 (t, J=5.8 Hz, 2H), 3.92
(t, J=5.8 Hz, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 2.78 (s, 6H), 1.81–
1.89 ppm (m, 4H); ESIMS m/z: 452 [M+Na]+, 428 [MꢀH]ꢀ.
3-Chloro-7-[(3-{[3-(dimethylamino)-5-hydroxyphenoxy]methyl}-
benzyl)oxy]-4-methyl-2H-chromen-2-one (11): Prepared by fol-
lowing general procedure E, starting from bromide 1j (0.165 g,
0.42 mmol). Purification by flash chromatography (gradient eluent:
EtOAc in n-hexane 0!40%) afforded 11 as a white solid (0.092 g,
57%): 1H NMR (300 MHz, [D6]acetone): d=7.96 (s, 1H, dis. with
D2O), 7.77 (d, J=8.8 Hz, 1H), 7.62 (s, 1H), 7.43–7.48 (m, 3H), 7.09
(dd, J1 =2.5 Hz, J2 =8.8 Hz, 1H), 7.04 (d, J=2.5 Hz, 1H), 5.85–5.91
(m, 3H), 5.31 (s, 2H), 5.07 (s, 2H), 2.86 (s, 6H), 2.59 ppm (s, 3H);
ESIMS m/z: 488 (100) [M+Na]+, 490 (37), 464 (100) [MꢀH]ꢀ, 466
(35).
4-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-2H-chro-
men-2-one (14): Prepared by following general procedure D, start-
ing from 4-hydroxycoumarin (0.057 g, 0.35 mmol). Purification by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
70%) afforded 14 as
a
white solid (0.093 g, 72%): 1H NMR
(300 MHz, [D6]DMSO): d=9.00 (s, 1H, dis. with D2O), 7.77–7.80 (m,
1H), 7.61–7.67 (m, 1H), 7.30–7.40 (m, 2H), 5.89 (s, 1H), 5.69–5.70
(m, 3H), 4.26 (t, J=6.1 Hz, 2H), 3.94 (t, J=6.3 Hz, 2H), 2.78 (s, 6H),
1.84–1.97 ppm (m, 4H); ESIMS m/z: 392 [M+Na]+, 368 [MꢀH]ꢀ.
6-({(2E)-4-[3-(Dimethylamino)-5-hydroxyphenoxy]but-2-en-1-
yl}oxy)-2H-chromen-2-one (18): Prepared by following general
procedure E, starting from bromide 1k (0.124 g, 0.42 mmol). Purifi-
cation by flash chromatography (gradient eluent: EtOAc in n-
5-{4-[3-(Dimethylamino)-5-hydroxyphenoxy]butoxy}-2H-chro-
men-2-one (15): Prepared by following general procedure D, start-
ing from 5-hydroxycoumarin (0.057 g, 0.35 mmol). Purification by
flash chromatography (gradient eluent: EtOAc in n-hexane 0!
1
hexane 0!60%) afforded 18 as a red solid (0.080 g, 62%): H NMR
1626
www.chemmedchem.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1616 – 1630

Tethered Coumarin Derivatives as AChE Inhibitors
(300 MHz, [D6]DMSO): d=9.02 (s, 1H, dis. with D2O), 7.97 (d, J=
9.6 Hz, 1H), 7.33 (d, J=8.8 Hz, 1H), 7.28 (d, J=3.0 Hz, 1H), 7.21
(dd, J1 =3.0 Hz, J2 =8.8 Hz, 1H), 6.47 (d, J=9.6 Hz, 1H), 6.01–6.04
(m, 2H), 5.71 (m, 3H), 4.62–4.63 (m, 2H), 4.48 (brs, 2H), 2.78 ppm
(s, 6H); ESIMS m/z: 390 [M+Na]+, 366 [MꢀH]ꢀ.
6-{2-[{2-[3-(Dimethylamino)-5-hydroxyphenoxy]ethyl}-
(methyl)amino]ethoxy}-2H-chromen-2-one (20): Intermediate 1o
(0.222 g, 0.40 mmol) was dissolved in anhydrous THF (2.8 mL), and
a 1.0n solution of TBAF (1.20 mL, 1.20 mmol) in THF was slowly
added. The mixture was stirred for 30 min at room temperature
and then quenched by the addition of saturated aqueous NH4Cl
(5 mL) under stirring for 30 min. After dilution with H2O (40 mL),
the mixture was extracted with Et2O (3ꢃ60 mL). The organic layers
were collected, dried over Na2SO4, and the solution was concen-
trated to dryness. The final product was then isolated by flash
chromatography (gradient eluent: CH3OH in EtOAc 0!10%) to
6-({5-[3-(Dimethylamino)-5-hydroxyphenoxy]pentyl}oxy)-2H-
chromen-2-one (19): Prepared by following general procedure E,
starting from bromide 1l (0.131 g, 0.42 mmol). Purification by flash
chromatography (gradient eluent: EtOAc in n-hexane 0!50%) af-
forded 19 as a white solid (0.064 g, 48%): 1H NMR (300 MHz,
[D6]DMSO): d: 8.97 (s, 1H, dis. with D2O), 7.97 (d, J=9.6 Hz, 1H),
7.31 (d, J=9.1 Hz, 1H), 7.26 (d, J=2.8 Hz, 1H), 7.18 (dd, J1 =2.8 Hz,
J2 =9.1 Hz, 1H), 6.46 (d, J=9.6 Hz, 1H), 5.68–5.70 (m, 3H), 4.01 (t,
J=6.3 Hz, 2H), 3.85 (t, J=6.1 Hz, 2H), 2.78 (s, 6H), 1.67–1.82 (m,
4H), 1.53–1.58 ppm (m, 2H); ESIMS m/z: 406 [M+Na]+, 382
[MꢀH]ꢀ.
obtain
a
colorless oil (0.145 g, 91%): 1H NMR (300 MHz,
[D6]acetone): d=7.90 (d, J=9.4 Hz, 1H), 7.19–7.24 (m, 3H), 6.39 (d,
J=9.6 Hz, 1H), 5.80–5.85 (m, 3H), 4.17 (t, J=6.1 Hz, 2H), 4.03 (t,
J=5.8 Hz, 2H), 2.86–2.94 (m, 4H), 2.83 (s, 6H), 2.43 ppm (s, 3H),
1 OH not detected; ESIMS m/z: 399 [M+Na]+.
General procedure F for the preparation of ammonium salts
2T–20T, 9D, and 17D: The appropriate amine 2–20 (0.070 mmol)
was dissolved in anhydrous CH3CN, and CH3I (0.044 mL, 0.70 mmol)
or CH3CH2I (0.056 mL, 0.70 mmol) was added. After stirring for 24 h
(for reaction with CH3I) or 72 h (for reactions with CH3CH2I) at
room temperature, the starting material disappeared (TLC monitor-
ing). The solvent and excess CH3I or CH3CH2I were then removed
under vacuum, and the resulting crude was treated with Et2O and
filtered to give the desired salts.
6-(2-Bromoethoxy)-2H-chromen-2-one (1m): In a Pyrexꢁ vessel
charged with a magnetic stir bar and a WeflonTM heating bar, 6-hy-
droxycoumarin (0.973 g, 6.0 mmol) was dissolved in anhydrous THF
(15 mL), and then 1,2-dibromoethane (3.10 mL, 36.0 mmol), Cs2CO3
(1.95 g, 6.0 mmol), and KI (0.100 g, 0.60 mmol) were added. The
mixture was heated by microwave irradiation to 1508C for 1 h,
then cooled, and the irradiation cycle was repeated. The mixture
was cooled, filtered, and the solution was concentrated and puri-
fied by flash chromatography (gradient eluent: EtOAc in n-hexane
0!25%) to give the desired coumarin derivative 1m as a white
solid (0.727 g, 45%): 1H NMR (300 MHz, CDCl3): d=7.65 (d, J=
9.6 Hz, 1H), 7.28 (d, J=9.1 Hz, 1H), 7.13 (dd, J1 =3.0 Hz, J2 =9.1 Hz,
1H), 6.95 (d, J=3.0 Hz, 1H), 6.44 (d, J=9.6 Hz, 1H), 4.32 (t, J=
6.1 Hz, 2H), 3.66 ppm (t, J=6.1 Hz, 2H).
3-Hydroxy-N,N,N-trimethyl-5-{4-[(2-oxo-2H-chromen-7-yl)oxy]bu-
toxy}benzenaminium iodide (2T): Prepared by following general
procedure F, starting from amine 2 (0.026 g, 0.070 mmol). Treat-
ment with Et2O yielded a white solid (0.032 g, 89%): 1H NMR
(300 MHz, [D6]acetone): d=9.56 (s, 1H, dis. with D2O), 7.91 (d, J=
9.6 Hz, 1H), 7.59 (d, J=8.5 Hz, 1H), 7.24–7.25 (m, 1H), 7.04 (s, 1H),
6.94 (dd, J1 =2.5 Hz, J2 =8.5 Hz, 1H), 6.89 (d, J=2.5 Hz, 1H), 6.71–
6.73 (m, 1H), 6.22 (d, J=9.6 Hz, 1H), 4.23 (t, J=6.1 Hz, 2H), 4.18 (t,
J=6.1 Hz, 2H), 3.82 (s, 9H), 2.00–2.06 ppm (m, 4H); ESIMS m/z:
384 [M]+.
6-{2-[(2-Hydroxyethyl)(methyl)amino]ethoxy}-2H-chromen-2-one
(1n): A solution of 1m (0.457 g, 1.70 mmol) in anhydrous THF
(15 mL) was cooled to 08C, and then DIPEA (1.78 mL, 10.2 mmol)
was added followed by 2-(methylamino)ethanol (0.410 mL,
5.10 mmol). The reaction was warmed to room temperature and
then held at reflux for 15 h. The solvent was removed under re-
duced pressure, and the crude oil was purified by flash chromatog-
raphy (gradient eluent: CH3OH in EtOAc 0!10%) to yield coumar-
in intermediate 1n as a colorless oil (0.412 g, 92%): 1H NMR
(300 MHz, [D6]DMSO): d=7.98 (d, J=9.4 Hz, 1H), 7.32 (d, J=9.1 Hz,
1H), 7.28 (d, J=2.8 Hz, 1H), 7.18 (dd, J1 =3.0 Hz, J2 =9.1 Hz, 1H),
6.47 (d, J=9.6 Hz, 1H), 4.36 (t, J=5.2 Hz, 1H, dis. with D2O), 4.06 (t,
J=5.8 Hz, 2H), 3.43–3.49 (m, 2H), 2.75 (t, J=5.8 Hz, 2H), 2.48 (t,
J=6.3 Hz, 2H), 2.26 ppm (s, 3H).
3-Hydroxy-N,N,N-trimethyl-5-{4-[(3-methyl-2-oxo-2H-chromen-7-
yl)oxy]butoxy}benzenaminium iodide (3T): Prepared by following
general procedure F, starting from amine 3 (0.027 g, 0.070 mmol).
1
Treatment with Et2O yielded a white solid (0.035 g, 96%): H NMR
(300 MHz, [D6]acetone): d=9.48 (s, 1H, dis. with D2O), 7.70 (s, 1H),
7.50 (d, J=8.5 Hz, 1H), 7.23–7.24 (m, 1H), 7.02–7.06 (m, 1H), 6.92
(dd, J1 =2.5 Hz, J2 =8.5 Hz, 1H), 6.87 (d, J=2.5 Hz, 1H), 6.73–6.74
(m, 1H), 4.07–4.23 (m, 4H), 3.83 (s, 9H), 2.09 (s, 3H), 1.99–2.03 ppm
(m, 4H); ESIMS m/z: 398 [M]+.
6-{2-[(2-{3-(Dimethylamino)-5-
3-Hydroxy-N,N,N-trimethyl-5-{4-[(4-methyl-2-oxo-2H-chromen-7-
yl)oxy]butoxy}benzenaminium (4T): Prepared by following gener-
al procedure F, starting from amine 4 (0.027 g, 0.070 mmol). Treat-
ment with Et2O yielded a white solid (0.033 g, 90%): 1H NMR
(300 MHz, [D6]acetone): d=9.78 (s, 1H, dis. with D2O), 7.69 (d, J=
8.8 Hz, 1H), 7.26–7.27 (m, 1H), 6.99–7.02 (m, 1H), 6.96 (dd, J1 =
2.5 Hz, J2 =8.8 Hz, 1H), 6.88 (d, J=2.5 Hz, 1H), 6.66–6.67 (m, 1H),
6.13 (s, 1H), 4.17–4.23 (m, 4H), 3.80 (s, 9H), 2.44 (s, 3H), 2.00–
2.03 ppm (m, 4H); ESIMS m/z: 398 [M]+.
[(triisopropylsilyl)oxy]phenoxy}ethyl)(methyl)amino]ethoxy}-2H-
chromen-2-one (1o): Intermediate alcohol 1n (0.263 g, 1.0 mmol)
was dissolved in anhydrous THF (3 mL), and then phenol 1e
(0.371 g, 1.20 mmol) was added followed by PPh3 (0.315 g,
1.20 mmol). The solution was then cooled to 08C through an exter-
nal ice bath, and a solution of DIAD (0.236 mL, 1.20 mmol) in anhy-
drous THF (2 mL) was added dropwise. The reaction was slowly
warmed to room temperature and stirred for 18 h. The solvent was
removed under reduced pressure, and the resulting crude mixture
was purified by flash chromatography (gradient eluent: EtOAc in n-
hexane 0!80%) to yield compound 1o as a dark-brown oil
(0.372 g, 67%): 1H NMR (300 MHz, [D6]acetone): d=7.90 (d, J=
9.4 Hz, 1H), 7.19–7.24 (m, 3H), 6.39 (d, J=9.6 Hz, 1H), 5.80–5.85
(m, 3H), 4.17 (t, J=6.1 Hz, 2H), 4.03 (t, J=5.8 Hz, 2H), 2.86–2.94
(m, 4H), 2.83 (s, 6H), 2.43 (s, 3H), 1.15–1.32 (m, 3H), 1.11 ppm (d,
J=6.6 Hz, 18H).
3-Hydroxy-N,N,N-trimethyl-5-(4-{[2-oxo-4-(trifluoromethyl)-2H-
chromen-7-yl]oxy}butoxy)benzenaminium iodide (5T): Prepared
by following general procedure F, starting from amine 5 (0.031 g,
0.070 mmol). Treatment with Et2O yielded a red solid (0.036 g,
88%): 1H NMR (300 MHz, [D6]acetone): d=7.67–7.70 (m, 1H), 7.32
(t, J=2.2 Hz, 1H), 7.08 (dd, J1 =2.5 Hz, J2 =8.8 Hz, 1H), 7.04 (d, J=
2.5 Hz, 1H), 6.97 (t, J=2.2 Hz, 1H), 6.72 (s, 1H), 6.66 (t, J=2.2 Hz,
ChemMedChem 2010, 5, 1616 – 1630
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1627

A. Carotti et al.
MED
1H), 4.29 (t, J=5.8 Hz, 2H), 4.17 (t, J=5.8 Hz, 2H), 3.79 (s, 9H),
1.96–2.01 ppm (m, 4H), 1 OH not detected; ESIMS m/z: 452 [M]+.
3-Hydroxy-N,N,N-trimethyl-5-{4-[(2-oxo-2H-chromen-3-yl)oxy]bu-
toxy}benzenaminium iodide (12T): Prepared by following general
procedure F, starting from amine 12 (0.026 g, 0.070 mmol). Treat-
ment with Et2O yielded a white solid (0.031 g, 87%): 1H NMR
(300 MHz, [D6]acetone): d=9.90 (s, 1H, dis. with D2O), 7.57–7.60
(m, 1H), 7.41–7.47 (m, 1H), 7.28–7.32 (m, 4H), 6.96–6.99 (m, 1H),
6.67–6.68 (m, 1H), 4.19–4.20 (m, 4H), 3.79 (s, 9H), 2.00–2.03 ppm
(m, 4H); ESIMS m/z: 384 [M]+.
3-{4-[(3-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]butoxy}-5-
hydroxy-N,N,N-trimethylbenzenaminium iodide (7T): Prepared
by following general procedure F, starting from amine 7 (0.029 g,
0.070 mmol). Treatment with Et2O yielded a white solid (0.036 g,
93%): 1H NMR (300 MHz, [D6]acetone): d=7.77 (d, J=8.8 Hz, 1H),
7.38–7.39 (m, 1H), 7.02 (dd, J1 =2.5 Hz, J2 =8.8 Hz, 1H), 6.93–6.95
(m, 2H), 6.64–6.66 (m, 1H), 4.25 (t, J=5.8 Hz, 2H), 4.17 (t, J=
5.8 Hz, 2H), 3.78 (s, 9H), 2.59 (s, 3H), 2.01–2.04 ppm (m, 4H), 1 OH
not detected; ESIMS m/z: 432 (100) [M]+, 434 (36).
3-{4-[(6,7-Dimethoxy-2-oxo-2H-chromen-3-yl)oxy]butoxy}-5-hy-
droxy-N,N,N-trimethylbenzenaminium iodide (13T): Prepared by
following general procedure F, starting from amine 13 (0.030 g,
0.070 mmol). Treatment with Et2O yielded a white solid (0.037 g,
92%): 1H NMR (300 MHz, [D6]acetone): d=7.31 (t, J=2.5 Hz, 1H),
7.25 (s, 1H), 7.13 (s, 1H), 6.96 (t, J=2.5 Hz, 1H), 6.92 (s, 1H), 6.69 (t,
J=2.5 Hz, 1H), 4.12–4.20 (m, 4H), 3.89 (s, 3H), 3.84 (s, 3H), 3.79 (s,
9H), 1.99–2.01 ppm (m, 4H), 1 OH not detected; ESIMS m/z: 444
[M]+.
3-({5-[(3-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]pentyl}-
oxy)-5-hydroxy-N,N,N-trimethylbenzenaminium iodide (8T): Pre-
pared by following general procedure F, starting from amine 8
(0.030 g, 0.070 mmol). Treatment with Et2O yielded a white solid
1
(0.037 g, 91%): H NMR (300 MHz, [D6]acetone): d=9.41 (s, 1H, dis.
with D2O), 7.77 (d, J=8.8 Hz, 1H), 7.15 (t, J=2.2 Hz, 1H), 7.05 (t,
J=2.2 Hz, 1H), 7.01 (dd, J1 =2.5 Hz, J2 =8.8 Hz, 1H), 6.93 (d, J=
2.5 Hz, 1H), 6.71 (t, J=2.2 Hz, 1H), 4.19 (t, J=6.3 Hz, 2H), 4.12 (t,
J=6.3 Hz, 2H), 3.84 (s, 9H), 2.59 (s, 3H), 1.85–1.98 (m, 4H), 1.66–
1.73 ppm (m, 2H); ESIMS m/z: 446 (100) [M]+, 448 (34).
3-Hydroxy-N,N,N-trimethyl-5-{4-[(2-oxo-2H-chromen-4-yl)oxy]bu-
toxy}benzenaminium iodide (14T): Prepared by following general
procedure F, starting from amine 14 (0.026 g, 0.070 mmol). Treat-
ment with Et2O yielded a yellow solid (0.033 g, 91%): 1H NMR
(300 MHz, [D6]acetone): d=7.85–7.88 (m, 1H), 7.62–7.67 (m, 1H),
7.28–7.37 (m, 2H), 7.11–7.14 (brm, 1H), 7.00–7.02 (m, 1H), 6.70–
6.72 (m, 1H), 5.78 (s, 1H), 4.39 (t, J=6.1 Hz, 2H), 4.22 (t, J=6.1 Hz,
2H), 3.80 (s, 9H), 2.09–2.17 ppm (m, 4H), 1 OH not detected; ESIMS
m/z: 384 [M]+.
3-Hydroxy-N,N,N-trimethyl-5-{4-[(6-oxo-7,8,9,10-tetrahydro-6H-
benzo[c]chromen-3-yl)oxy]butoxy}benzenaminium iodide (9T):
Prepared by following general procedure F, starting from amine 9
(0.030 g, 0.070 mmol). Treatment with Et2O yielded a yellow solid
(0.036 g, 92%): 1H NMR (300 MHz, [D6]acetone): d=7.62 (d, J=
8.8 Hz, 1H), 7.18 (t, J=1.9 Hz, 1H), 7.05 (t, J=1.9 Hz, 1H), 6.93 (dd,
J1 =2.5 Hz, J2 =8.8 Hz, 1H), 6.85 (d, J=2.5 Hz, 1H), 6.71 (t, J=
1.9 Hz, 1H), 4.18–4.21 (m, 4H), 3.84 (s, 9H), 2.46–2.48 (m, 4H),
2.01–2.03 (m, 4H), 1.77–1.85 ppm (m, 4H), 1 OH not detected;
ESIMS m/z: ESIMS m/z: 438 [M]+.
3-Hydroxy-N,N,N-trimethyl-5-{4-[(2-oxo-2H-chromen-5-yl)oxy]bu-
toxy}benzenaminium iodide (15T): Prepared by following general
procedure F, starting from amine 15 (0.026 g, 0.070 mmol). Treat-
ment with Et2O yielded a white solid (0.034 g, 95%): 1H NMR
(300 MHz, [D6]acetone): d=9.41 (s, 1H, dis. with D2O), 8.18 (d, J=
9.9 Hz, 1H), 7.54 (t, J=8.5 Hz, 1H), 7.22 (t, J=2.2 Hz, 1H), 7.08 (t,
J=2.2 Hz, 1H), 6.95 (d, J=8.5 Hz, 1H), 6.89 (d, J=8.5 Hz, 1H), 6.79
(t, J=2.2 Hz, 1H), 6.32 (d, J=9.9 Hz, 1H), 4.26–4.30 (m, 2H), 4.19–
4.23 (m, 2H), 3.84 (s, 9H), 2.01–2.04 ppm (m, 4H); ESIMS m/z: 384
[M]+.
N-Ethyl-3-hydroxy-N,N-dimethyl-5-{4-[(6-oxo-7,8,9,10-tetrahydro-
6H-benzo[c]chromen-3-yl)oxy]butoxy}benzenaminium
iodide
(9D): Prepared by following general procedure F, starting from
amine 9 (0.030 g, 0.070 mmol). Treatment with Et2O yielded a
1
white solid (0.026 g, 65%): H NMR (300 MHz, [D6]acetone): d=7.62
(d, J=8.8 Hz, 1H), 7.06–7.09 (m, 1H), 7.01–7.03 (m, 1H), 6.93 (dd,
J1 =2.5 Hz, J2 =8.8 Hz, 1H), 6.85 (d, J=2.5 Hz, 1H), 6.74–6.80 (m,
1H), 4.17–4.23 (m, 4H), 4.16 (q, J=7.2 Hz, 2H), 3.78 (s, 6H), 2.79–
2.81 (m, 2H), 2.44–2.48 (m, 2H), 1.99–2.04 (m, 4H), 1.74–1.89 (m,
4H), 1.23 ppm (t, J=7.2 Hz, 3H), 1 OH not detected; ESIMS m/z:
452 [M]+.
3-Hydroxy-N,N,N-trimethyl-5-{4-[(2-oxo-2H-chromen-8-yl)oxy]bu-
toxy}benzenaminium iodide (16T): Prepared by following general
procedure F, starting from amine 16 (0.026 g, 0.070 mmol). Treat-
ment with Et2O yielded a off-white solid (0.033 g, 93%): 1H NMR
(300 MHz, [D6]acetone): d=9.41 (s, 1H, dis. with D2O), 7.99 (d, J=
9.6 Hz, 1H), 7.21–7.34 (m, 2H), 7.16–7.20 (m, 1H), 7.12–7.14 (brm,
1H), 7.03–7.05 (m, 1H), 6.71–6.72 (m, 1H), 6.45 (d, J=9.6 Hz, 1H),
4.22–4.28 (m, 4H), 3.83 (s, 9H), 1.96–2.02 ppm (m, 4H); ESIMS m/z:
384 [M]+.
3-Hydroxy-N,N,N-trimethyl-5-{4-[(6-oxo-6H-benzo[c]chromen-3-
yl)oxy]butoxy}benzenaminium iodide (10T): Prepared by follow-
ing general procedure F, starting from amine 10 (0.029 g,
0.070 mmol). Treatment with Et2O yielded a yellow solid (0.037 g,
3-Hydroxy-N,N,N-trimethyl-5-{4-[(2-oxo-2H-chromen-6-yl)oxy]bu-
toxy}benzenaminium iodide (17T): Prepared by following general
procedure F, starting from amine 17 (0.026 g, 0.070 mmol). Treat-
ment with Et2O yielded a white solid (0.034 g, 84%): 1H NMR
(300 MHz, CD3OD): d=7.91 (d, J=9.6 Hz, 1H), 7.27–7.30 (m, 1H),
7.15–7.20 (m, 2H), 6.84–6.85 (m, 1H), 6.78–6.79 (m, 1H), 6.52–6.53
(m, 1H), 6.43 (d, J=9.6 Hz, 1H), 4.10–4.12 (m, 4H), 3.58 (s, 9H),
1.98–2.02 ppm (m, 4H); ESIMS m/z: 384 [M]+.
1
95%): H NMR (300 MHz, [D6]acetone): d=8.20–8.30 (m, 3H), 7.88–
7.93 (m, 1H), 7.58–7.63 (m, 1H), 7.37–7.39 (m, 1H), 7.11–7.18 (m,
1H), 7.02 (dd, J1 =2.5 Hz, J2 =8.8 Hz, 1H), 6.94–6.95 (m, 1H), 6.66–
6.67 (m, 1H), 4.19–4.41 (m, 4H), 3.78 (s, 9H), 1.98–2.01 ppm (m,
4H), 1 OH not detected; ESIMS m/z: 434 [M]+.
3-[(3-{[(3-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl}-
benzyl)oxy]-5-hydroxy-N,N,N-trimethylbenzenaminium
iodide
(11T): Prepared by following general procedure F, starting from
amine 11 (0.033 g, 0.070 mmol). Treatment with Et2O yielded a
white solid (0.038 g, 90%): H NMR (300 MHz, [D6]acetone): d=7.79
N-Ethyl-3-hydroxy-N,N-dimethyl-5-{4-[(2-oxo-2H-chromen-6-
yl)oxy]butoxy}benzenaminium iodide (17D): Prepared by follow-
ing general procedure F, starting from amine 17 (0.026 g,
1
(d, J=8.8 Hz, 1H), 7.67 (s, 1H), 7.47–7.51 (m, 3H), 7.38–7.40 (m,
1H), 7.10 (dd, J1 =2.5 Hz, J2 =8.8 Hz, 1H), 7.01–7.04 (m, 2H), 6.75–
6.76 (m, 1H), 5.33 (s, 2H), 5.21 (s, 2H), 3.78 (s, 9H), 2.59 ppm (s,
3H), 1 OH not detected; ESIMS m/z: 480 (100) [M]+, 482 (37).
0.070 mmol). Treatment with Et2O yielded a off-white solid
(0.034 g, 92%): 1H NMR (300 MHz, CD3OD): d=7.91 (d, J=9.6 Hz,
1H), 7.27–7.30 (m, 1H), 7.15–7.20 (m, 2H), 6.75–6.76 (m, 1H), 6.67–
6.70 (m, 1H), 6.52–6.54 (m, 1H), 6.43 (d, J=9.6 Hz, 1H), 4.09–4.88
1628
www.chemmedchem.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1616 – 1630

Tethered Coumarin Derivatives as AChE Inhibitors
(m, 4H), 3.87 (q, J=7.2 Hz, 2H), 3.52 (s, 6H), 1.98–2.01 (m, 4H),
1.13 ppm (t, J=7.2 Hz, 3H); ESIMS m/z: 398 [M]+.
Computational methods
Docking simulations
3-Hydroxy-N,N,N-trimethyl-5-({(2E)-4-[(2-oxo-2H-chromen-6-yl)-
oxy]but-2-en-1-yl}oxy)benzenaminium iodide (18T): Prepared by
following general procedure F, starting from amine 18 (0.026 g,
0.070 mmol). Treatment with Et2O yielded a white solid (0.031 g,
87%): 1H NMR (300 MHz, [D6]acetone): d=7.94 (d, J=9.6 Hz, 1H),
7.30–7.34 (m, 1H), 7.21–7.29 (m, 3H), 6.94–6.97 (m, 1H), 6.68–6.69
(m, 1H), 6.42 (d, J=9.6 Hz, 1H), 6.14–6.16 (m, 2H), 4.70–4.73 (m,
4H), 3.78 ppm (s, 9H), 1 OH not detected; ESIMS m/z: 382 [M]+.
GOLD (v. 4.1.2), a genetic-algorithm-based software, was used for
the docking study, and GoldScore was chosen as a fitness function.
Parameters used in the fitness function (hydrogen bond energies,
atomic radii and polarizabilities, torsion potentials, hydrogen bond
directionalities, etc.) were taken from the GOLD parameter file. In
the present study, the 3D coordinates of hAChE (PDB ID: 1B41)
and hBChE (PDB ID: 1P0I) were retrieved from the RCSB Protein
Data Bank. Rather than the more frequently used Torpedo californi-
ca form (TcAChE), hAChE was chosen because it has almost identi-
cal amino acid residues at both the catalytic and peripheral bind-
ing sites, apart from the substitution of Tyr337 (hAChE) with
Pro330 (TcAChE).[46] This single residue change was not observed
when analyzing primary sequences of either human (PDB ID: 1B41)
or bovine (NCBI entry: AA123899.1) AChEs.
3-Hydroxy-N,N,N-trimethyl-5-({5-[(2-oxo-2H-chromen-6-yl)oxy]-
pentyl}oxy)benzenaminium iodide (19T): Prepared by following
general procedure F, starting from amine 19 (0.027 g, 0.070 mmol).
1
Treatment with Et2O yielded a yellow solid (0.035 g, 95%): H NMR
(300 MHz, [D6]acetone): d=7.95 (d, J=9.6 Hz, 1H), 7.25–7.28 (m,
2H), 7.22 (dd, J1 =2.8 Hz, J2 =8.8 Hz, 1H), 7.06–7.14 (brs, 1H), 6.99
(t, J=2.2 Hz, 1H), 6.70 (t, J=2.2 Hz, 1H), 6.40 (d, J=9.6 Hz, 1H),
4.08–4.12 (m, 4H), 3.81 (s, 9H), 1.83–1.98 (m, 4H), 1.63–1.73 ppm
(m, 2H), 1 OH not detected; ESIMS m/z: 398 [M]+.
The reliability of the docking procedure was validated by the accu-
rate reproduction of the binding conformation of the well-known
AChE inhibitor donepezil, whose Cartesian coordinates were ex-
tracted from its complex with the TcAChE (PDB ID: 1EVE).[47] Given
that the role of water molecules in the AChE binding site is not
completely understood, docking simulations were performed with-
out taking water molecules into account.[48]
3-[2-(Dimethyl{2-[(2-oxo-2H-chromen-6-yl)oxy]ethyl}ammonio)e-
thoxy]-5-hydroxy-N,N,N-trimethylbenzenaminium diiodide (20T):
Prepared by following general procedure F, starting from amine 20
(0.028 g, 0.070 mmol). Treatment with Et2O yielded a white solid
(0.030 g, 63%): 1H NMR (300 MHz, CD3OD): d=7.94 (d, J=9.6 Hz,
1H), 6.68–7.20 (m, 6H), 6.43 (d, J=9.6 Hz, 1H), 4.21–4.25 (m, 4H),
3.76 (s, 4H), 3.59 (s, 9H), 3.22 ppm (s, 6H).
The target proteins were prepared by adding hydrogen atoms,
completing and optimizing missing residues, removing water and
the co-crystallized molecules. The basic amino groups were pro-
tonated, aromatic amino functional groups were left uncharged,
and carboxylic groups were considered to be deprotonated. Molec-
ular docking resulted in 10 poses per inhibitor in a sphere of 10 ꢂ
radius centered on the centroid atom of donepezil co-crystallized
with TcAChE (PDB ID: 1EVE) previously aligned to hAChE.
Biological assays
The inhibition assays of AChE, either from bovine erythrocytes
(0.36 Umgꢀ1) or human recombinant (2770 Umgꢀ1), and BChE,
from equine serum (13 Umgꢀ1), were run in phosphate buffer
(0.1m, pH 8.0). Acetyl- and butyrylthiocholine iodides were used as
substrates and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) was used
as the chromophoric reagent.[35] Inhibition assays were carried out
on an Agilent 8453E UV/Vis spectrophotometer equipped with a
cell changer. Solutions of tested compounds were prepared by
starting from 10 mm stock solutions in DMSO, which were diluted
with aqueous assay medium to a final content of organic solvent
always <1%. AChE inhibitory activity was determined in a reaction
mixture containing 200 mL of a solution of AChE (0.415 UmLꢀ1 in
0.1m phosphate buffer, pH 8.0), 100 mL of a 3.3 mm solution of
DTNB in 0.1m phosphate buffer (pH 7.0) containing 6 mm NaHCO3,
100 mL of a solution of the inhibitor (six to seven concentrations
ranging from 1ꢃ10ꢀ12 to 1ꢃ10ꢀ4 m), and 500 mL of phosphate
buffer, pH 8.0. After incubation for 20 min at 258C, acetylthiocho-
line iodide (100 mL of 5 mm aqueous solution) was added as the
substrate, and AChE-catalyzed hydrolysis was followed by measur-
ing the increase in absorbance at l 412 nm for 3.0 min at 258C.
The concentration of compound that effected 50% inhibition of
AChE activity (IC50) was calculated by nonlinear regression of the
response–log(concentration) curve, using GraphPad Prismꢀ v. 5.
BChE inhibitory activity was assessed similarly using butyrylthio-
choline iodide as the substrate. Kinetic studies were performed
under the same incubation conditions, using six concentrations of
substrate (from 0.033 to 0.2 mm) and four concentrations of inhibi-
tor (0 to 0.9 nm for compound 13 and 0 to 5 nm for donepezil).
Apparent inhibition constants and kinetic parameters were calcu-
lated within the “enzyme kinetics” module of Prism.
Molecular dynamics
Top-scored solutions from molecular docking of compounds 12
and 13 were used as starting points for MD simulations. Com-
plexes of AChE–12 and AChE–13 were immersed in cubic TIP3P
water boxes that extended 18 ꢂ from the protein atoms and were
neutralized by the addition of Na+ counterions using the AMBER
Leap module. This led to simulation systems of 114008 and
114013 atoms for the AChE–12 and AChE–13 complexes, respec-
tively. The parm03 version of the all-atom AMBER force field was
used to model the system. The solvent molecules were initially re-
laxed by means of energy minimizations and 30 ps of MD. Subse-
quently, the full system was minimized to remove bad contacts in
the initial geometry and heated gradually to 310 K over 600 ps of
MD. The SHAKE algorithm was employed to constrain all RꢀH
bonds, and periodic boundary conditions were applied to simulate
a continuous system. A nonbonded cutoff of 12 ꢂ was used,
whereas the particle mesh Ewald (PME) method was employed to
include the contributions of long-range interactions. The pressure
(1 atm) and temperature (310 K) of the system were controlled
during the MD simulations by the Langevin method. A 5 ns trajec-
tory was computed for each model with a time-step of 1.5 fs. Co-
ordinates were collected every 2 ps and saved for analysis.
Acknowledgements
The authors are grateful to MIUR, Rome (Italy) for financial sup-
port (PRIN, 2008).
ChemMedChem 2010, 5, 1616 – 1630
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1629

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Keywords: acetylcholinesterase
coumarin derivatives · glaucoma · inhibitors
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Published online on August 2, 2010
1630
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1616 – 1630