Acetylcholinesterase inhibitors: Synthesis and structure-activity relationships of ω-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)- methyl]aminoalkoxyheteroaryl derivatives

Article abstract of DOI:10.1021/jm9810046

Acetylcholinesterase (ACHE) inhibitors are one of the most actively investigated classes of compounds in the search for an effective treatment of Alzheimer's disease. This work describes the synthesis, AChE inhibitory activity, and structure-activity relationships of some compounds related to a recently discovered series of AChE inhibitors: the ω-[N-methyl-N-(3- alkylcarbamoyloxyphenyl)methyl]aminoalkoxyxanthen-9-ones. The influence of structural variations on the inhibitory potency was carefully investigated by modifying different parts of the parent molecule, and a theoretical model of the binding of one representative compound to the enzyme was developed. The biological properties of the series were investigated in some detail by considering not only the activity on isolated enzyme but the selectivity with respect to butyrylcholinesterase (BuChE) and the in vitro inhibitory activity on rat cerebral cortex as well. Some of the newly synthesized derivatives, when tested on isolated and/or AChE-enriched rat brain cortex fraction, displayed a selective inhibitory activity and were more active than physostigmine. In particular, compound 13, an azaxanthone derivative, displayed the best rat cortex AChE inhibition (190-fold higher than physostigmine), as well as a high degree of enzyme selectivity (over 60-fold more selective for AChE than for BuChE). When tested in the isolated enzyme, compound 13 was less active, suggesting some differences either in drug availability/biotransformation or in the inhibitor-sensitive residues of the enzyme when biologically positioned in rat brain membranes.

Full text of DOI:10.1021/jm9810046

3976
J . Med. Chem. 1998, 41, 3976-3986
Acetylch olin ester a se In h ibitor s: Syn th esis a n d Str u ctu r e-Activity
Rela tion sh ip s of ω-[N-Meth yl-N-(3-a lk ylca r ba m oyloxyp h en yl)-
m eth yl]a m in oa lk oxyh eter oa r yl Der iva tives
Angela Rampa,† Alessandra Bisi,† Piero Valenti,*,† Maurizio Recanatini,† Andrea Cavalli,† Vincenza Andrisano,†
Vanni Cavrini,† Lorena Fin,‡ Alessandro Buriani,‡,§ and Pietro Giusti‡
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Department of
Pharmacology, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy, and Cooperative Research & Innovation,
Via Leopardi 14, Sovizzo, VI, Italy
Received February 17, 1998
Acetylcholinesterase (AChE) inhibitors are one of the most actively investigated classes of
compounds in the search for an effective treatment of Alzheimer’s disease. This work describes
the synthesis, AChE inhibitory activity, and structure-activity relationships of some compounds
related to a recently discovered series of AChE inhibitors: the ω-[N-methyl-N-(3-alkylcarbam-
oyloxyphenyl)methyl]aminoalkoxyxanthen-9-ones. The influence of structural variations on
the inhibitory potency was carefully investigated by modifying different parts of the parent
molecule, and a theoretical model of the binding of one representative compound to the enzyme
was developed. The biological properties of the series were investigated in some detail by
considering not only the activity on isolated enzyme but the selectivity with respect to
butyrylcholinesterase (BuChE) and the in vitro inhibitory activity on rat cerebral cortex as
well. Some of the newly synthesized derivatives, when tested on isolated and/or AChE-enriched
rat brain cortex fraction, displayed a selective inhibitory activity and were more active than
physostigmine. In particular, compound 13, an azaxanthone derivative, displayed the best
rat cortex AChE inhibition (190-fold higher than physostigmine), as well as a high degree of
enzyme selectivity (over 60-fold more selective for AChE than for BuChE). When tested in
the isolated enzyme, compound 13 was less active, suggesting some differences either in drug
availability/biotransformation or in the inhibitor-sensitive residues of the enzyme when
biologically positioned in rat brain membranes.
Senile dementia of the Alzheimer type (SDAT) is a
neurodegenerative disease associated with neuronal
damage involving several neurotransmitter systems of
the brain. One of the most pronounced functional
deficits involves cholinergic neurons of the central
nervous system (CNS). For years cholinergic enhance-
ment has been considered a sensible therapeutic ap-
proach for improving brain functions in SDAT patients.
CNS cholinergic enhancement can be achieved with the
use of acetylcholine (ACh) precursors, muscarinic ago-
nists, or acetylcholinesterase (AChE) inhibitors.1
Unfortunately, efforts to develop drugs possessing this
It has long been postulated that the active site of
last activity have been so far hampered by the short
AChE contains an anionic as well as an esteratic site
persistency and/or low specificity of the pharmacological
effect.2 Many anticholinesterase agents, in fact, also
inhibit butyrylcholinesterase (BuChE) which is found
in both plasma and brain. This inhibition can lead to
adverse peripheral side effects.2 Further research is
thus needed to design new molecules with improved
activity, and much attention has been focused on AChE
inhibitors such as 1,2,3,4-tetrahydro-9-aminoacridine
(tacrine),2,3 physostigmine and its analogues,4 and N-
benzylpiperidines.5
and a hydrophobic binding site (HBS-l). However, from
the X-ray analysis of AChE from Torpedo californica
reported by Sussman et al.,6 it was shown that the
quaternary nitrogen of ACh binds strongly with the π
electrons of the Trp84 group. According to this model,
Chen et al.7 hypothesized that the carbonyl center of
physostigmine and other carbamates interacts with the
enzyme via the Ser-His-Glu catalytic triad and the
quaternary nitrogen interacts with neighboring aro-
matic residues. One of these residues might correspond
to the second hydrophobic binding site (HBS-2) proposed
by Ishihara et al.8 On this basis, in a previous paper,9
† University of Bologna.
‡ University of Padova.
§ Cooperative Research & Innovation.
S0022-2623(98)01004-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/12/1998

Synthesis of New Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3977
Sch em e 1a
a
Reagents: (i) Br(CH2)nCl, K2CO3 reflux, 24 h; (ii) NaI, methyl ethyl ketone, reflux 4 h; (iii) N-(3-hydroxybenzyl)alkylamine, reflux, 15
h; (iv) alkyl isocyanate, NaH, room temperature, 24 h.
we designed some xanthone analogues of general for-
mula
rationalize the structure-activity relationships of the
series was carried out, and a theoretical model of the
binding mode of the most active compound was devel-
oped. Some of the compounds displaying an activity
superior or equal to physostigmine were also examined
for enzymatic selectivity, by comparing their inhibitory
activity on isolated AChE with that exerted on isolated
BuChE. The AChE inhibitory activity of the most
interesting compounds was further analyzed on rat
brain cortex fractions enriched with AChE activity.
where the alkoxy chain occupies, alternatively, the 2,
3, and 4 positions of the xanthone moiety and has a
variable length. The compounds were then evaluated
for anti-AChE activity, and some were indeed more
active than physostigmine, taken as reference molecule.
Optimum activity was associated with the 3 side chain
position on the xanthone nucleus and with a three-
carbon chain length separating the xanthenonyloxy
moiety from the nitrogen atom of the benzylaminic
moiety. Encouraged by such results, we synthesized
several related compounds with the aim of identifying
key structural elements involved in the interactions
with AChE, which could endow the new molecules with
a better pharmacological activity.
Ch em istr y
The synthesis of the compounds was accomplished as
illustrated in Scheme 1. The selected hydroxy deriva-
tives were treated with 1-bromo-ω-chloroalkanes in the
presence of K2CO3 to afford ω-chloroalkoxy derivatives
1a -g, which were treated with NaI in refluxing methyl
ethyl ketone to give the corresponding ω-iodoalkoxy
derivatives 2a -g. Compounds 2a -g were condensed
with the selected N-(3-hydroxy)benzylalkylamine to
afford compounds 3a -j and then treated with the
selected alkyl isocyanate to give the studied compounds
4-33.
The anti-AChE activity of all the new compounds was
first tested using the isolated enzyme. An attempt to

3978 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Rampa et al.
Ta ble 1. Physicochemical and Analytical Data of the Compounds Studied
l
compd
Ar
n
R
R1
Me
n-Hep
Me
n-Hep
Me
Et
mp (°C)a
yield (%)
formula
analysis
4
5
6
7
8
Z
Z
Z
Z
Z
Z
Z
Z
4
4
5
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
108-110
93-95
45
40
50
30
55
40
60
35
70
80
70
50
90
50
70
30
40
60
70
80
70
60
65
50
70
55
40
70
65
40
C
C
C
C
C
C
C
C
C
27H29ClN2O5
33H41ClN2O5
28H29N2O5
34H43ClN2O5
26H26N2O5
27H28N2O5
28H30N2O5
29H32N2O5
31H29ClN2O5
C25H26ClN3O5
C28H32ClN3O5
C31H38ClN3O5
C22H25ClN2O5
C25H31ClN2O5
C28H37ClN2O5
C22H25ClN2O5
C24H29ClN2O5
C28H37ClN2O5
C28H29ClN2O5
C31H35ClN2O5
C34H41ClN2O5
27H28N2O5
30H34N2O5
33H41ClN2O5
28H31ClN2O5
31H37ClN2O5
34H43ClN2O5
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
b
110-112
135-137
101-103
118-120
91-93
b
b
9
b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
n-Pr
n-Bu
Ph
b
67-68
Z
128-130
213-215
174-176
185-187
166-168
183-184
184-186
128-130
90-92
Z1
Z1
Z1
Z2
Z2
Z2
Z3
Z3
Z3
Z4
Z4
Z4
Z
Z
Z
Z
Z
Z
Z
Z
Z
Me
n-Bu
n-Hep
Me
n-Bu
n-Hep
Me
n-Pr
n-Hep
Me
n-Bu
n-Hep
Me
n-Bu
n-Hep
Me
n-Bu
n-Hep
Me
111-113
NDc
110-111
155-157
103-105
60-62
170-172
125-127
114-116
172-174
84-86
85-87
155-157
b
C
C
C
C
C
C
C
C
C
b
Et
Et
n-Pr
n-Pr
n-Pr
i-Pr
i-Pr
i-Pr
b
28H30N2O5
31H36N2O5
34H43ClN2O5
b
n-Bu
n-Hep
a
b
Crystallizing solvent: methanol-ether. Free base (crystallizing from ligroin). c Hygroscopic solid.
The N-(3-hydroxy)benzylalkylamines were prepared
by condensation of 3-hydroxybenzaldehyde and the
selected amine followed by NaBH4 reduction.
The structure and the physicochemical and analytical
data of compounds 4-33 are reported in Table 1.
ated with a three-carbon chain length (n ) 3, compound
8, IC50 ) 0.30 ( 0.01 nM), and increasing the chain
length to four carbons (n ) 4) results in a large
reduction of activity (compound 4). Interestingly, a
chain length of five carbons (n ) 5) leads to a recovery
of the inhibitory activity (compound 6). The association
of the best activity with n ) 3 is in agreement with what
we had seen previously,9 and for the following SAR
studies we proceeded maintaining n ) 3.
(b ) Th e Ca r b a m oyl Su b st it u en t (R1). Various
substituents on the carbamoyl moiety were examined
in order to study their influence on the activity. It is
known that the length of alkyl groups in that position
should influence the rate constant of AChE inhibition,10
and this aspect will be considered in a subsequent
section. Replacing the methyl group of 4 and 6 with a
n-heptyl group (compounds 5 and 7, respectively) strongly
decreases the inhibitory activity. When R1 is an alkyl
group and n ) 3, optimum activity is associated with a
methyl (compound 8 is 45-fold more active than phys-
ostigmine) and the anti-AChE activity decreases when
it is changed into an ethyl, n-propyl, or n-butyl (com-
pounds 9-11). A phenyl group in the carbamoyl moiety
gives a molecule (compound 12) which, though still less
Resu lts
Str u ctu r e-Activity Rela tion sh ip s of th e ACh E
In h ibit ion . Values for the inhibitory activity of the
compounds against isolated AChE were expressed as
IC50 ( SD and are reported in Table 2. In view of a
clearer presentation of the structure-activity relation-
ships (SAR) of the series, four structural features were
identified in the general structure of the compounds
studied: (a) the linker between the heteroaryloxy moiety
and the basic nitrogen (n); (b) the N-substituent on the
carbamoyl group (R1); (c) the heteroaryloxy moiety (Ar),
and (d) the N-substituent on the basic nitrogen (R).
(a ) Th e Lin k er (n ). Compounds 4-7 show a vari-
able chain (four or five carbon units) separating the
heteroaryloxy moiety from the nitrogen atom of benzyl-
aminic moiety. These compounds were prepared in
order to find the optimal length of this chain. Indeed,
the best inhibitory activity on isolated AChE is associ-

Synthesis of New Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3979
Ta ble 2. Inhibitory Activity on Isolated AChE and BuChE, Ratio of IC50, and Inhibitory Activity on Rat Brain AChE of the
Compounds Studied
compd
Ara
n
R
R1
Me
n-Hep
Me
n-Hep
Me
Et
IC50 (nM) AChE
IC50 (nM) BuChE
ratio IC50 (BuChE/AChE)
IC50 (nM) rat brain AChE
4
5
6
7
8
Z
Z
Z
Z
Z
Z
Z
Z
4
4
5
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
37 ( 5
100 ( 10
0.82 ( 0.07
100 ( 20
0.30 ( 0.01
13 ( 1
28 ( 4
36 ( 2
2.8 ( 0.2
1.1 ( 0.2
8.9 ( 0.4
42 ( 2
5.7 ( 0.5
14 ( 1
13 ( 0.2
8.9 ( 0.2
250 ( 10
21 ( 1
5.7 ( 0.7
31 ( 2
68 ( 3
0.56 ( 0.05
8.6 ( 0.05
160 ( 30
4.0 ( 1
70 ( 20
400 ( 60
0.7 ( 0.1
100 ( 10
170 ( 30
14 ( 0.2
83 ( 3
101
>100
48 ( 4
26 ( 3
160
2
20 ( 20
40 ( 20
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
phys.
n-Pr
n-Bu
Ph
Z
200 ( 10
76 ( 6
24 ( 2
71.4
69.1
2.7
>1000
Z1
Z1
Z1
Z2
Z2
Z2
Z3
Z3
Z3
Z4
Z4
Z4
Z
Z
Z
Z
Z
Z
Z
Z
Z
Me
0.009 ( 0.005
70 ( 30
n-Bu
n-Hep
Me
n-Bu
n-Hep
Me
n-Pr
n-Hep
Me
n-Bu
n-Hep
Me
n-Bu
n-Hep
Me
n-Bu
n-Hep
Me
3.6 ( 0.2
65 ( 3
0.6
5
1.5 ( 0.7
2 ( 2
47 ( 4
21 ( 2
8.2
0.6 ( 0.5
4 ( 3
37.5
Et
Et
n-Pr
n-Pr
n-Pr
i-Pr
i-Pr
i-Pr
n-Bu
n-Hep
23 ( 1
1.6
2 ( 1
a
See top of Table 1.
active than that with the methyl group, is more active
than those with longer alkyl chains. When the het-
eroaryl nucleus is different from the xanthone (com-
pounds 13-24), increasing the length of the carbamic
alkyl substituent generally leads to a decrease of activity
that not always depends on the number of carbon units.
In the coumarin subset (compounds 16-18), the n-butyl
and n-heptyl derivatives are equally potent, while the
chromone derivatives (compounds 19-21) show variable
activity, i.e., the n-propyl derivative is less active than
the n-heptyl one.
(c) Th e Heter oa r yloxy Moiety (Ar ). In the subset
of compounds 13-24, the xanthone nucleus was re-
placed by the bioisoster azaxanthone (compounds 13-
15) or by coumarin (compounds 16-18), chromone
(compounds 19-21), and flavone (compounds 22-24)
moieties. The rationale for these modifications is based
on the hypothesis that the xanthone moiety can be
successfully replaced with structurally related oxygen
heterocycles such as coumarin, chromone, and flavone.
In fact, in a number of cases this replacement allowed
us to obtain important results with regard to analep-
tics,11 adrenergic-â-blocking agents,12 clofibrate-like hy-
polipemic drugs,13 and more recently, bradycardic14 and
antitumor drugs.15-17 Some of the compounds of the
subset are more active than physostigmine in inhibiting
isolated AChE activity, and in all the cases, the best
activity is associated with a methyl group in the
carbamoyl fragment (compounds 13, 16, 19, and 22).
Compound 13 is the best of this series, and it is 13-fold
more active than physostigmine but 4-fold less active
than the xanthone analogue 8.
replacing the methyl group of the basic nitrogen with
ethyl, n-propyl, and i-propyl groups (compounds 25-
33). It is evident that these modifications, which
increase both lipophilicity and steric hindrance of the
molecules, are not favorable and, in some cases, even
detrimental for the inhibitory activity on isolated AChE.
Only compounds 25 and 31 show IC50 values compa-
rable with 8 (IC50 ) 0.30 ( 0.01 nM). In a recent
paper,18 the hypothesis was formulated that lipophilicity
that is too high might prevent the AChE inhibitors from
reaching their binding site, due to possible interactions
with the hydrophobic walls of the active site gorge. On
the other hand, steric effects might play a role in
determining the differences in activity, at least for
compounds with the same R1. For instance, when R1
is methyl or n-heptyl (compounds 25, 27, 28, 30, 31, 33),
the order of potency of the analogues with different R
substituents (ethyl = i-propyl > n-propyl) is in agree-
ment with the length of the groups as measured by the
Verloop’s parameter L:19 4.11, 4.11, and 4.92, respec-
tively.
To illustrate the SAR of the class, the binding mode
of the most active compound of the series was investi-
gated by building a three-dimensional model of the
quaternary complex formed between compound 8 and
AChE. The coordinates of the protein were taken from
the crystal structure of T. californica AChE determined
by Sussman et al.,6 and a slight modification was
introduced, according to the primary sequence of human
AChE.20 The identity of the active site residues of the
two enzymes was carefully checked, and Phe330 was
replaced by Tyr, which is present in the human AChE.
The inhibitor was then docked into the active site by
attaching it to the Ser200 Oγ in such a way as to
(d ) Th e Am in e Su bstitu en t (R). Finally, the sub-
stituent on the protonable amine group was studied by

3980 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Rampa et al.
F igu r e 1. Inhibitor 8 (green) docked into the AChE active site gorge. Only a few amino acid residues are shown for simplicity.
represent the tetrahedral intermediate of the carbam-
oylation reaction, and subsequently, a molecular dy-
namics simulation was carried out on the complex.
The results of the simulations are shown in Figure 1,
which illustrates the main features of the interactions
between some AChE active site residues and compound
8. The inhibitor (green) appears to be located in the
active site gorge so as to maximize the favorable
contacts, which might be the cause of its high potency.
The molecule is folded, and the xanthenonyl moiety
points toward the opening of the gorge, being caged
within a framework of aromatic residues. From inspec-
tion of the model, it results that compound 8 (bearing
an intermediate methylene chain of three carbon units)
can interact with Trp84, but its tricyclic ring is not able
to reach Trp279; the two mentioned residues have
recently been postulated to correspond to HBS-1 and
HBS-2, respectively.21
more potent than the corresponding derivatives bearing
different heterocyclic groups (Table 2). This might be
a consequence of a number of factors, like the optimal
fit in the lipophilic active site gorge and some favorable
interactions with specific residues. In the vicinity of the
xanthenonyl nucleus, there are eight aromatic residues,
some of which are shown in Figure 1. Moreover, the
arrangement of the phenyl ring of Tyr330 is optimal for
a stacking interaction with one benzene ring of the
tricycle (the distance between the centers of the two
rings is 3.44 Å), and the OH group of Tyr121 can form
a hydrogen bond with the ethereal O atom of the
xanthenonyl fragment (Figure 2c). On the other hand,
the Phe331 side chain, which is at a suitable distance
for a T-shaped interaction with the xanthone counter-
part (ca. 5 Å),22 does not show an ideal geometry.
En zym a tic Selectivity. Compounds displaying an
inhibitory activity similar to or better than that of
physostigmine were also tested for their inhibitory
activity on isolated BuChE. The ratio between the IC50
value for BuChE and that for AChE is positively
correlated with the specificity of the inhibitory effect on
AChE. The results are summarized in Table 2, and they
indicate that all the tested compounds, with the excep-
tion of compound 16, are more active on AChE than on
BuChE, the most selective being 8, 6, 12, and 13.
A more detailed description of the binding interactions
of compound 8 with the AChE active site gorge is given
in Figure 2.
Residues stabilizing the tetrahedral intermediate
group resulting from the nucleophilic attack of Ser200
onto the carbamoyl CdO are shown in Figure 2a. The
amide nitrogen atoms of Gly118 and Gly119 are hydro-
gen bonded to the oxyanion of the inhibitor, and the
methyl substituent on the carbamic nitrogen occupies
a lipophilic pocket formed mainly by Phe288, Phe290,
and Phe331.
In Figure 2b, residues possibly interacting with the
protonated amine group are shown. The proton is in a
position favorable for establishing a hydrogen bond with
the carbonyl of Gly441. The positive charge, which is
distributed over the methyl or methylene groups at-
tached to the basic N, can be stabilized by the carboxy-
lates of Glu199 and Glu445 and by the aromatic ring of
Trp84. The methyl substituent on the nitrogen inter-
acts with a lipophilic pocket determined by Met83,
Val129, and Tyr130 (Figure 1), which appears to be
large enough to contain small symmetrical alkyl groups
such as methyl, ethyl, and i-propyl.
Unlike the inhibitory activity, AChE selectivity does
not seem to be strictly related to the carbon chain length
(n) separating the xanthenonyloxy moiety from the
benzylaminic nitrogen. Compounds 8 and 6, with a
three- and a five-carbon chain length, respectively, are
both highly selective. Enzymatic selectivity is affected
by the size of the carbamoyl substituents (R1) with an
order of potency (methyl (8) > phenyl (12) > ethyl (9))
that is reflective of the order found for AChE inhibitory
activity. Moreover, replacement of the methyl group on
the nitrogen of the benzylaminic moiety with an ethyl
group decreases the selectivity (compound 25). The
xanthenonyloxy moiety confers the best selectivity to
the molecule. When the xanthone nucleus is replaced
by the bioisoster azaxanthone, the selectivity is de-
creased, though still high (compound 13). Selectivity
is reduced to modest levels or even inverted when the
The interactions of the xanthenonyl moiety of 8 with
the binding site are illustrated in Figure 2c. Com-
pounds containing this fragment are comparatively

Synthesis of New Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3981
F igu r e 3. Progressive recovery of AChE activity after incuba-
tion with inhibitors 16-18.
heterocycle is a coumarin or a flavone (compounds 18,
22, and 16).
Rever sibility of th e En zym a tic In h ibition . As
previously reported,23 the inhibition of AChE by car-
bamates involves a reversible complex formation fol-
lowed by carbamoylation of the hydroxyl of the catalytic
serine. To study the influence of the alkyl chain length
of the compounds under study on the extent of inhibi-
tion, the decarbamoylation reaction was studied for
three derivatives (16-18) bearing a methyl, n-butyl, and
n-heptyl substituent on the carbamic moiety, respec-
tively. The formation of the covalent bond was con-
firmed by the time dependence of inhibition showed by
the tested compounds. The enzyme inactivation was
found to increase with the inhibitor concentration and
with the enzyme-inhibitor time of incubation up to
nearly 20 min for the longer chain derivatives, slightly
less for the N-methyl-substituted derivative (16) at the
80% inhibition concentration.
The carbamoylated enzyme is then hydrolyzed to
regenerate the free enzyme. The velocity of this step
was found to be dependent on the length of the alkyl
chain on the carbamate group, as previously reported
for physostigmine derivatives;4c in particular, carbam-
ates substituted with a longer alkyl group were found
long-acting.
The results of the decarbamoylation kinetics experi-
ments are shown in Figure 3. From the graph, it
appears that the physostigmine-inhibited enzyme re-
covered its activity after 6 h, while in the cases of the
coumarin derivatives 16-18, the recovery times were
much longer. Actually, the enzyme inhibited with
compound 16 recovered its activity after 21 h, compound
17 after 44 h, and the n-heptyl derivative 18 was still
inhibited after 48 h.
To verify the reversibility of action also for the long-
acting compound 18, the heptylcarbamoylated enzyme
was isolated after 48 h dialysis and, after dilution, its
hydrolysis was followed to determine the decarbamoy-
lation rate constant.23a Under the described experi-
mental conditions, it was possible to follow the recovery
of the enzyme activity (up to 40%). During the experi-
ment, the stability of the reference enzyme was also
checked: the activity was maintained after the dialysis
and a 15% loss of activity was observed after the
dilution. The decarbamoylation rate constant k3 (5.05
× 10-3 min-1) is the slope of the linear plot (r2 ) 0.9780)
calculated as described in the Experimental Section.
F igu r e 2. Detailed view of the putative binding interactions
of 8 (dark) with some active site residues (light); H-bonds are
shown as dashed lines. (a) Residues involved in the binding
of the tetrahedral complex between Ser200 Oγ and the
carbamic group. (b) Residues involved in the binding of the
protonated amine group. (c) Residues involved in the binding
of the xanthenonyloxy moiety.

3982 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Rampa et al.
In Vitr o In h ibition of ACh E Activity in Ra t
Cer ebr a l Cor tex. The AChE inhibitory activity of
some of the most interesting compounds of the present
series was tested in AChE-enriched preparations ob-
tained from partial fractionation of rat brain cortex
homogenates (see Experimental Section). The results
are shown in Table 2, and they indicate that, while most
of the SAR established with the purified enzyme assays
still hold, some significant differences are seen for
specific compounds. As found with the isolated enzyme,
a good inhibitory activity requires a three-carbon chain
length separating the xanthenonyloxy moiety from the
benzylaminic nitrogen (compounds 8 and 9). The activ-
ity decreases with the size of the carbamoyl substituents
(R1), but opposite to what is found when using the
purified enzyme, the phenyl substitution leads to a
dramatic loss of activity (compound 12). Variation of
the heteroaryloxy moiety (Ar) leads to a ranking of
activities, which is strikingly different from that ob-
tained with isolated AChE, with the xanthone moiety
being the least active (compound 8). Replacing the
xanthone nucleus with the bioisoster azaxanthone
(compound 13) greatly enhances the inhibitory activity,
followed by the flavone (compound 22) and the coumarin
(compound 16) substitutions. With regard to the amine
substituent (R), the inhibitory activity is slightly higher
with the ethyl group (compound 25) than with the
methyl group (compound 8).
new ones. On the other hand, the linker chain does not
seem to be responsible for the enzymatic selectivity.
(b) A small size of the carbamic substituent is critical
for both inhibitory potency and enzymatic selectivity.
Both parameters are best when the substituent is a
methyl group. The loss of activity occurring with larger
alkyl groups is most likely due to a bad fitting of this
part of the molecule in a small lipophilic pocket of the
enzyme (the AChE esteratic site). On the other hand,
the rate of enzyme regeneration is inversely correlated
with the length of the alkyl substituent, even if interest-
ing results can also be obtained with a methyl group.
In fact, the complex formed between AChE and com-
pound 16 has a much longer stability than that formed
by physostigmine, and the duration of action increases
dramatically by increasing the chain length. The longer
recovery time of the former complex with respect to that
of the enzyme-physostigmine complex might be due to
different residual affinities of the hydrolyzed moieties.
The benzylaminic fragment of 16 might contribute to
the longer lasting inhibition, having a higher affinity
for the enzyme than the eseroline fragment of physo-
stigmine.
(c) The inhibitory activity requires the presence of a
methyl group on the basic nitrogen. Slightly larger
substituents (up to i-propyl) are tolerated without
seriously decreasing activity because they can occupy a
hydrophobic cavity lined by the residues Met83, Val129,
and Tyr130 (see Figure 1).
(d) Replacement of the xanthone moiety of the lead
molecule 8 with either the bioisoster azaxanthone or the
analogues coumarin or flavone reduces in most cases
both the inhibitory activity on isolated enzyme and the
selectivity. This might be a consequence of the relative
stereoelectronic dissimilarity of the former heteroaryl-
oxy groups with respect to the xanthone parent struc-
ture. However, the working hypothesis at the basis of
the modifications still seems valid, as the activity is
maintained at quite acceptable levels.
In these experimental conditions, the inhibitory activ-
ity of physostigmine is higher than that found for the
purified enzyme. As a result, the only compounds which
are still more active than physostigmine are those with
modifications in the heteroaryloxy moiety and namely
13, 16, and 22.
Inhibition experiments conducted in the presence of
a saturating concentration of acetylcholine (2 mM)
resulted in higher IC50 values for the compounds (data
not shown), an underestimation expected when working
with competitive inhibitors.24
When tested on the rat cerebral cortex AChE, the
order of potency of the various heteroaryloxy-substituted
compounds is totally different from that noticed with
the isolated enzyme, and the azaxanthone derivative
(compound 13) is the most active. This difference might
be related to the steric complexity of biological environ-
ment, which can cause structural changes in the con-
formation of enzymes with respect to the native form
of the isolated protein. Moreover, tissue-bound enzy-
matic activities can affect the chemical structure of
drugs, making them more or less available for the
enzyme to inhibit. If present, such differences between
the isolated and the tissue-bound preparations can
affect the enzymatic activities as well as sensitivities
to modulating drugs. AChE can indeed be present in
different molecular forms. Such forms are known to
exhibit virtually the same enzymatic activity; however,
a contribution to the inhibitory potencies of the com-
pounds examined cannot be ruled out.
Discu ssion
The AChE inhibitory activity of a series of xanthone
carbamoyloxy derivatives was examined. Four different
parts of the molecule were modified and analyzed for
determining the structure-activity relationships, using
both isolated AChE and rat cerebral cortex AChE.
Enzymatic selectivity was determined comparing the
inhibitory activity of the compounds on BuChE with
that exerted on AChE. Reversibility of the inhibition
was also verified.
Several structural clues have been highlighted from
this study with regard to the AChE inhibition:
(a) In accordance with earlier reports,9 optimum
inhibitory activity requires a three-carbon-atom chain
separating the heteroaryloxy moiety from the basic
nitrogen. In light of the model of Figure 1, one can note
that the length of the intermediate methylene chain is
critical in influencing the position of the heterocyclic
moiety and, consequently, in allowing the very specific
interactions responsible for the global high affinity to
the enzyme. Increasing n of one carbon unit might
disrupt the optimal array of contacts, while lengthening
the chain up to five C units might reconstitute the
framework or compensate for the lost interactions with
Con clu sion s
In conclusion, this study led to the identification of a
series of compounds showing an inhibitory potency on
AChE up to the subnanomolar level. The enzyme-
inhibitor binding mode and the structure-activity

Synthesis of New Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3983
(DMSO-d6) 2.25 (m, 2H), 3.45 (t, 2H), 4.2 (t, 2H), 7.1-8.8 (m,
6H, Ar). Anal. (C15H12INO3): C, H, N.
relationships were determined, and a detailed investi-
gation of the biological properties was carried out. One
derivative was identified as possessing all the structural
requirements established for best activity and selectivity
for AChE inhibition. This molecule is compound 13, an
azaxanthone derivative 190-fold more potent than phy-
sostigmine on rat brain AChE and over 60-fold more
selective for AChE than for BuChE. This compound has
thus been selected for further development.
7-(3-Iod op r op oxy)-2H-1-ben zop yr a n -2-on e (2e). Using
the previous procedure and starting from 2.38 g (0.01 mol) of
1
1e, 2.5 g (75%) of 2e was obtained: mp 98-100 °C; H NMR
δ 2.3 (m, 2H), 3.35 (t, 2H), 4.1 (t, 2H), 6.25 (d, 1H, H-3), 6.8-
7.4 (m, 3H, Ar), 7.65 (d, 1H, H-4). Anal. (C12H11IO3): C, H.
7-(3-Iod op r op oxy)-4H-1-ben zop yr a n -4-on e (2f). Using
the previous procedure and starting from 2.38 g (0.01 mol) of
1
1f, 3 g (90%) of 2f was obtained: mp 121-122 °C; H NMR δ
2.3 (m, 2H), 3.35 (t, 2H), 4.1 (t, 2H), 5.8 (d, 1H, H-2), 6.4-7.7
(m, 3H, Ar), 8.1 (d, 1H, H-3). Anal. (C12H11IO3): C, H.
7-(3-Iodopr opoxy)-2-ph en yl-4H-1-ben zopyr an -4-on e (2g).
Using the previous procedure and starting from 3.14 g (0.01
mol) of 1g, 3.25 g (80%) of 2g was obtained: mp 131-133 °C;
1H NMR δ 2.35 (m, 2H), 3.4 (t, 2H), 4.2 (t, 2H), 6.8-8.2 (m,
9H, Ar and H-3). Anal. (C18H15IO3): C, H.
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s. All melting points were
determined in open glass capillaries using a Bu¨chi apparatus
and are uncorrected. 1H NMR spectra were recorded in CDCl3
solution on a Varian Gemini 300 spectrometer with Me4Si as
the internal standard. Wherever analyses are only indicated
with element symbols, analytical results obtained for those
elements are within 0.4% of the theoretical values.
3-(5-Ch lor op en toxy)xa n th en -9-on e (1c). A stirred sus-
pension of 2.12 g (0.01 mol) of 3-hydroxyxanthen-9-one, 1.85
g (0.01 mol) of 1-bromo-5-chloropentane, and 1.4 g (0.01 mol)
of K2CO3 in dry acetone was refluxed for 24 h. The reaction
was monitored by TLC. The hot reaction mixture was filtered
and evaporated to dryness. The residue was crystallized from
toluene to give 2.2 g (70%) of 1c: mp 128-130 °C (EtOH); 1H
NMR δ 1.7 (m, 2H), 1.9 (m, 4H), 3.6 (t, 2H), 4.1 (t, 2H), 6.85-
8.4 (m, 7H, Ar). Anal. (C18H17ClO3): C, H.
3-[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]p r op oxyxa n -
th en -9-on e (3a ). A solution of 1.9 g (0.005 mol) of 2a and
1.37 g (0.01 mol) of N-(3-hydroxybenzyl)methylamine in 100
mL of toluene was refluxed for 15 h. After cooling, the reaction
mixture was washed (H2O), dried (Na2SO4), and evaporated
to dryness. The residue, on crystallizing from toluene, afforded
1
1.13 g (60%) of 3a : mp 148-150 °C; H NMR δ 2.05 (m, 2H),
2.3 (s, 3H), 2.6 (t, 2H), 3.5 (s, 2H), 4.15 (t, 2H), 6.7-8.4 (m,
11H, Ar). Anal. (C24H23NO4): C, H, N.
3-[N-Met h yl-N-(3-h yd r oxyb en zyl)a m in o]b u t oxyxa n -
th en -9-on e (3b). Using the previous procedure and starting
from 0.98 g (0.0025 mol) of 2b, 0.6 g (60%) of 3b was obtained:
1
3-(3-Ch lor op r op oxy)-5-a za xa n t h en -9-on e (1d ). Using
the previous procedure and starting from 2.13 g (0.01 mol) of
3-hydroxy-5-azaxanthen-9-one and 1.57 g (0.01 mol) of 1-bromo-
3-chloropropane, 2.1 g (70%) of 1d was obtained: mp 174-
mp 96-98 °C (toluene); H NMR δ 1.7-1.9 (m, 4H), 2.25 (s,
3H), 2.5 (t, 2H), 3.5 (s, 2H), 4.05 (t, 2H), 6.7-8.4 (m, 11H, Ar).
Anal. (C25H25NO4): C, H, N.
3-[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]p en toxyxa n -
th en -9-on e (3c). Using the previous procedure and starting
from 3.22 g (0.0064 mol) of 2c, 1.35 g (50%) of 3c was obtained:
mp 101-103 °C (toluene); 1H NMR δ 1.4-1.65 (m, 4H), 1.8
(m, 2H), 2.2 (s, 3H), 2.4 (t, 2H), 3.45 (s, 2H), 4.05 (t, 2H), 6.7-
8.4 (m, 11H, Ar). Anal. (C26H27NO4): C, H, N.
1
175 °C (toluene); H NMR (DMSO-d6) δ 2.25 (m, 2H), 3.85 (t,
2H), 4.3 (t, 2H), 7.1-8.8 (m, 6H, Ar). Anal. (C15H12ClNO3):
C, H, N.
7-(3-Ch lor op r op oxy)-2H-1-ben zop yr a n -2-on e (1e). Us-
ing the previous procedure and starting from 1.62 g (0.01 mol)
of 7-hydroxy-2H-1-benzopyran-2-one, 1.78 g (75%) of 1e was
obtained: mp 105-108 °C (toluene); H NMR δ 2.3 (m, 2H),
3.75 (t, 2H), 4.2 (t, 2H), 6.25 (d, 1H, H-3), 6.8-7.4 (m, 3H, Ar),
7.65 (d, 1H, H-4). Anal. (C12H11ClO3): C, H.
7-(3-Ch lor op r op oxy)-4H-1-ben zop yr a n -4-on e (1f). Us-
ing the previous procedure and starting from 1.62 g (0.01 mol)
of 7-hydroxy-4H-1-benzopyran-4-one, 2.14 g (90%) of 1f was
obtained: mp 73-75 °C (ligroin); 1H NMR δ 2.3 (m, 2H), 3.75
(t, 2H), 4.2 (t, 2H), 5.8 (d, 1H, H-2), 6.4-7.7 (m, 3H, Ar), 8.1
(d, 1H, H-3). Anal. (C12H11ClO3): C, H.
7-(3-Ch lor op r op oxy)-2-p h e n yl-4H -1-b e n zop yr a n -4-
on e (1g). Using the previous procedure and starting from 2.38
g (0.01 mol) of 7-hydroxy-4H-1-benzopyran-4-one, 2.2 g (70%)
of 1g was obtained: mp 118-121 °C (toluene); 1H NMR δ 2.35
(m, 2H), 3.8 (t, 2H), 4.25 (t, 2H), 6.8-8.2 (m, 9H, Ar and H-3).
Anal. (C18H15ClO3): C, H.
3-[N-Meth yl-N-(3-h ydr oxyben zyl)am in o]pr opoxy-5-aza-
xa n th en -9-on e (3d ). Using the previous procedure and
starting from 1.9 g (0.005 mol) of 2d , 1.17 g (60%) of 3d was
1
1
obtained: mp 162-164 °C (toluene); H NMR δ 2.0 (m, 2H),
2.2 (s, 3H), 2.5 (t, 2H), 3.4 (s, 2H), 4.15 (t, 2H), 6.7-8.7 (m,
10H, Ar). Anal. (C23H22N2O4): C, H, N.
7-[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]p r op oxy-2H-
1-ben zop yr a n -2-on e (3e). Using the previous procedure and
starting from 1.65 g (0.005 mol) of 2e, 1.19 g (70%) of 3e was
obtained: mp 90-92 °C (toluene); 1H NMR δ 2.0 (m, 2H), 2.23
(s, 3H), 2.55 (t, 2H), 3.45 (s, 2H), 4.1 (t, 2H), 6.25 (d, 1H, H-3),
6.7-7.75 (m, 8H, Ar and H-4). Anal. (C20H21NO4): C, H, N.
7-[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]p r op oxy-4H-
1-ben zop yr a n -4-on e (3f). Using the previous procedure and
starting from 1.65 g (0.005 mol) of 2f, 0.85 g (50%) of 3f was
obtained: mp 62-65 °C (toluene); 1H NMR δ 2.0 (m, 2H), 2.25
(s, 3H), 2.55 (t, 2H), 3.5 (s, 2H), 4.0 (t, 2H), 5.8 (d, 1H, H-2),
6.3-8.15 (m, 8H, Ar and H-3). Anal. (C20H21NO4): C, H, N.
7-[N-Me t h yl-N-(3-h yd r oxyb e n zyl)a m in o]p r op oxy-2-
p h en yl-4H-1-ben zop yr a n -4-on e (3g). Using the previous
procedure and starting from 2.03 g (0.005 mol) of 2g, 1.04 g
(50%) of 3g was obtained: mp 117-118 °C (toluene); 1H NMR
δ 2.0 (m, 2H), 2.25 (s, 3H), 2.55 (t, 2H), 3.5 (s, 2H), 4.1 (t, 2H),
6.7-8.1 (m, 13H, Ar and H-3). Anal. (C26H25NO4): C, H, N.
3-[N-Eth yl-N-(3-h ydr oxyben zyl)am in o]pr opoxyxan th en -
9-on e (3h ). Using the previous procedure and starting from
2.5 g (0.0065 mol) of 2a and 2 g (0.013 mol) of N-(3-
hydroxybenzyl)ethylamine, 1.53 g (70%) of 3h was obtained:
3-(3-Iod op r op oxy)xa n t h en -9-on e (2a ). A mixture of
2.88 g (0.01 mol) of 3-(3-chloropropoxy)-xanthen-9-one9 and 1.5
g (0.01 mol) of NaI in 30 mL of methyl ethyl ketone was
refluxed for 4 h. After cooling, the separated solid was
collected by filtration and 2.66 g (70%) of 2a was obtained:
1
mp 119-121 °C; H NMR δ 2.35 (m, 2H), 3.45 (t, 2H), 4.3 (t,
2H), 6.9-8.4 (m, 7H, Ar). Anal. (C16H13IO3): C, H.
3-(4-Iod obu toxy)xa n th en -9-on e (2b). Using the previous
procedure and starting from 3 g (0.01 mol) of 3-(4-chlorobu-
toxy)xanthen-9-one,9 2.76 g (70%) of 2b was obtained: mp
113-115 °C; 1H NMR δ 2.05 (m, 4H), 3.3 (t, 2H), 4.15 (t, 2H),
6.85-8.4 (m, 7H, Ar). Anal. (C17H15IO3): C, H.
1
3-(5-Iod op en toxy)xa n th en -9-on e (2c). Using the previ-
ous procedure and starting from 3.16 g (0.01 mol) of 1c, 3.3 g
(80%) of 2c was obtained: mp 129-131 °C; 1H NMR δ 1.7 (m,
2H), 1.9 (m, 4H), 3.3 (t, 2H), 4.15 (t, 2H), 6.85-8.4 (m, 7H,
Ar). Anal. (C18H17IO3): C, H.
3-(3-Iod op r op oxy)-5-a za xa n th en -9-on e (2d ). Using the
previous procedure and starting from 2.89 g (0.01 mol) of 1d ,
3 g (80%) of 2d was obtained: mp 165-166 °C; 1H NMR δ
mp 206-208 °C (toluene); H NMR δ 1.1 (t, 3H), 2.0 (m, 2H),
2.5-2.7 (m, 4H), 3.55 (s, 2H), 4.1 (t, 2H), 6.65-8.35 (m, 11H,
Ar). Anal. (C25H25NO4): C, H, N.
3-[N-n -P r op yl-N-(3-h yd r oxyb en zyl)a m in o]p r op oxyx-
a n th en -9-on e (3i). Using the previous procedure and starting
from 2 g (0.005 mol) of 2a and 1.6 g (0.01 mol) of N-(3-
hydroxybenzyl)-n-propylamine, 1.04 g (50%) of 3i was obtained:
1
mp 142-144 °C (toluene); H NMR δ 0.8 (t, 3H), 1.8 (m, 2H),

3984 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Rampa et al.
2.3 (m, 2H), 2.75 (m, 2H), 3.05 (m, 2H), 3.9-4.1 (m, 4H), 6.7-
3H), 1.35 (m, 2H), 1.5 (m, 2H), 1.95 (m, 2H), 2.5-2.7 (m, 4H),
3.2 (m, 2H), 3.6 (s, 2H), 4.1 (t, 2H), 4.95 (broad, 1H, NH), 6.8-
8.4 (m, 11H, Ar). 27: δ (DMSO-d6) 0.8 (t, 3H), 1.1-1.3 (m,
11H), 1.4 (m, 2H), 2.2 (m, 2H), 2.95-3.4 (m, 6H), 4.1-4.4 (m,
4H), 6.95-8.2 (m, 12H, Ar and NH). 28: δ (DMSO-d6) 0.9 (t,
3H), 1.8 (m, 2H), 2.3 (m, 2H), 2.7 (d, 3H), 3.05 (m, 2H), 3.25
(m, 2H), 4.25 (s, 2H), 4.2-4.5 (m, 4H), 7.0-8.2 (m, 12H, Ar
and NH). 29: δ (DMSO-d6) 0.9 (m, 6H), 1.3 (m, 2H), 1.45 (m,
2H), 1.75 (m, 2H), 2.25 (m, 2H), 3.05 (m, 4H), 3.25 (m, 2H),
4.2-4.5 (m, 4H), 7.0-8.2 (m, 12H, Ar and NH). 30: δ (DMSO-
d6) 0.85 (m, 6H), 1.2 (m, 8H), 1.4 (m, 2H), 1.75 (m, 2H), 2.25
(m, 2H), 3.05 (m, 4H), 3.25 (m, 2H), 4.2-4.4 (m, 4H), 7.0-8.2
(m, 12H, Ar and NH). 31: δ (CDCl3) 1.0 (d, 6H), 1.9 (m, 2H),
2.6 (t, 2H), 2.85 (d, 3H), 2.95 (m, 2H), 3.55 (s, 2H), 4.1 (t, 2H),
4.95 (broad,1H, NH), 6.8-8.4 (m, 11H, Ar). 32: δ (CDCl3) 0.9
(t, 3H), 1.0 (d, 6H), 1.35 (m, 2H), 1.55 (m, 2H), 1.9 (m, 2H),
2.65 (t, 2H), 3.0 (m, 1H), 3.25 (m, 2H), 3.6 (s, 2H), 4.1 (t, 2H),
5.0 (broad, 1H, NH), 6.8-8.4 (m, 11H, Ar). 33: δ (DMSO-d6)
0.8 (t, 3H), 1.2 (m, 8H), 1.35 (d, 6H), 2.2 (m, 2H), 2.95-3.25
(m, 4H), 3.6 (m, 1H), 4.1-4.5 (m, 4H), 6.95-8.2 (m, 12H, Ar
and NH).
8.2 (m, 11H, Ar). Anal. (C26H27NO4): C, H, N.
3-[N-Isop r op yl-N-(3-h yd r oxyben zyl)a m in o]p r op oxyx-
a n th en -9-on e (3j). Using the previous procedure and starting
from 2 g (0.005 mol) of 2a and 1.6 g (0.01 mol) of N-(3-
hydroxybenzyl)-isopropylamine, 1.25 g (60%) of 3j was ob-
1
tained: mp 122-124 °C (toluene); H NMR δ 1.0 (s, 3H), 1.05
(s, 3H), 1.9 (m, 2H), 2.65 (t, 2H), 2.95 (m, 1H), 3.55 (s, 2H),
4.1 (t, 2H), 6.65-8.35 (m, 11H, Ar). Anal. (C26H27NO4): C,
H, N.
Gen er a l Meth od for th e P r ep a r a tion of Ca r ba m a tes
(4-33). A mixture of the selected ω-[N-alkyl-N-(3-hydroxy)-
benzyl]aminoalkoxy derivative (0.001 mol), selected alkyl
isocyanate (0.001 mol), and 10 mg of sodium hydride in toluene
was stirred at room temperature for 24 h, quenched with
water, and then extracted with methylene chloride. The
organic layer was washed with water, dried and evaporated
to dryness. The residue was purified by crystallization or by
flash chromatography.
1H NMR for Com p ou n d s 4-33. 4: δ (DMSO-d6) 1.7-1.9
(m, 4H), 2.6 (s, 3H), 2.7 (d, 3H), 3.0 (m, 2H), 4.05-4.4 (m, 4H),
6.9-8.25 (m, 11H, Ar). 5: δ (DMSO-d6) 0.85 (t, 3H), 1.2 (m,
8H), 1.4 (m, 2H), 1.7-1.9 (m, 4H), 2.65 (s, 3H), 2.9-3.2 (m,
4H), 4.1-4.4 (m, 4H), 7.0-8.2 (m, 12 H, Ar and NH). 6: δ
(CDCl3) 1.4-1.65 (m, 4H), 1.85 (m, 2H), 2.2 (s, 3H), 2.4 (t, 2H),
2.85 (d, 3H), 3.45 (s, 2H), 4.05 (t, 2H), 4.95 (broad, 1H, NH),
6.8-8.4 (m, 11H, Ar). 7: δ (DMSO-d6) 0.8 (t, 3H), 1.25 (m,
8H), 1.45 (m, 4H), 1.8 (m, 4H), 2.65 (s, 3H), 2.9-3.2 (m, 4H),
4.1-4.4 (m, 4H), 7.0-8.2 (m, 12H, Ar and NH). 8: δ (CDCl3)
2.0 (m, 2H), 2.25 (s, 3H), 2.55 (t, 2H), 2.85 (d, 3H), 3.5 (s, 2H),
4.15 (t, 2H), 4.95 (broad, 1H, NH), 6.8-8.4 (m, 11H, Ar). 9:
δ (CDCl3) 1.2 (t, 3H), 2.05 (m, 2H), 2.3 (s, 3H), 2.6 (t, 2H), 3.3
(m, 2H), 3.55 (s, 2H), 4.2 (t, 2H), 5.0 (broad, 1H, NH), 6.9-8.4
(m, 11H, Ar). 10: δ (CDCl3) 0.95 (t, 3H), 1.6 (m, 2H), 2.05 (m,
2H), 2.3 (s, 3H), 2.6 (t, 2H), 3.2 (m, 2H), 3.55 (s, 2H), 4.2 (t,
2H), 5.0 (broad, 1H, NH), 6.85-8.4 (m, 11H, Ar). 11: δ (CDCl3)
0.95 (t, 3H), 1.35 (m, 2H), 1.5 (m, 2H), 2.05 (m, 2H), 2.3 (s,
3H), 2.6 (t, 2H), 3.2 (m, 2H), 3.55 (s, 2H), 4.15 (t, 2H), 5.05
(broad, 1H, NH), 6.85-8.4 (m, 11H, Ar). 12: δ (DMSO-d6) 2.3
(m, 2H), 2.7 (s, 3H), 3.2 (m, 2H), 4.2-4.5 (m, 4H), 7.0-8.2 (m,
16H, Ar), 10.32 (s, 1H, NH). 13: δ (DMSO-d6) 2.25 (m, 2H),
2.6-2.75 (m, 6H), 3.15 (m, 2H), 4.2-4.5 (m, 4H), 7.0-8.8 (m,
10H, Ar). 14: δ (DMSO-d6) 0.85 (t, 3H), 1.25-1.5 (m, 4H),
2.3 (m, 2H), 3.7 (s, 3H), 3.05 (m, 2H), 3.2 (m, 2H), 4.2-4.5 (m,
4H), 7.0-8.8 (m, 11H, Ar and NH). 15: δ (DMSO-d6) 0.85 (t,
3H), 1.25 (m, 8H), 1.45 (m, 2H), 2.25 (m, 2H), 2.7 (s, 3H), 3.0
(m, 2H), 3.2 (m, 2H), 4.2-4.5 (m, 4H), 7.0-8.8 (m, 11H, Ar
and NH). 16: δ (DMSO-d6) 2.25 (m, 2H), 2.6 (s, 3H), 2.65 (s,
3H), 3.2 (m, 2H), 4.1-4.5 (m, 4H), 6.3 (d, 1H), 6.9-8.05 (m,
8H, Ar and H-4 coumarin). 17: δ (CDCl3) 0.95 (t, 3H), 1.4
(m, 2H), 1.6 (m, 2H), 2.0 (m, 2H), 2.25 (s, 3H), 2.5 (t, 2H), 3.3
(m, 2H), 3.5 (s, 2H), 4.05 (t, 2H), 5.25 (broad, 1H, NH), 6.25
(d, 1H), 6.8-7.2 (m, 8H, Ar and 4-H coumarin). 18: δ (DMSO-
d6) 0.8 (t, 3H), 1.25 (m, 8H), 1.4 (m, 2H), 2.25 (m, 2H), 2.65 (s,
3H), 3.0 (m, 2H), 3.2 (m, 2H), 4.15 (s, 2H), 4.3 (m, 2H), 6.3 (d,
N-Meth yl-N-[(3-h yd r oxyp h en yl)m eth yl]im in e. Methy-
lamine solution (30%, 3 mL) was added rapidly to a solution
of 3-hydroxybenzaldehyde (2 g, 0.016 mol) in ethanol (10 mL)
at about 40 °C. The mixture was allowed to stand at 0-5 °C
for 2 h. The resulting precipitate was collected by filtration,
washed successively with 40% ethanol and water, and dried
in vacuo to give the Schiff base (2.16 g, 100%): mp 157-159
1
°C; H NMR δ 3.4 (d, 3H), 6.8-7.3 (m, 4H), 7.85 (d, 1H).
N-Eth yl-N-[(3-h yd r oxyp h en yl)m eth yl]im in e. Using the
previous procedure and starting from ethylamine solution
(70%, 1 mL) and 1.22 g of 3-hydroxybenzaldehyde, 1.3 g (88%)
was obtained: mp 128-129 °C (lit.8 mp 127-129 °C); 1H NMR
δ 1.3 (t, 3H,), 3.6 (m, 2H), 6.9-7.3 (m, 4H, 7.85 (s, 1H).
N-P r op yl-N-[(3-h yd r oxyp h en yl)m eth yl]im in e. A solu-
tion of n-propylamine (5.02 g, 0.085 mol) and 3-hydroxyben-
zaldehyde (10.3 g, 0.085 mol) was stirred for 1 h and then was
refluxed until 90-95% of the theoretical amount of H2O was
collected in a Dean-Stark trap. After cooling, the precipitate
was collected by filtration to give 11.7 g (90%) of product: mp
1
123-125 °C; H NMR δ 0.9 (t, 3H), 1.7 (m, 2H), 3.55 (t, 2H),
6.9-7.4 (m, 5H), 8.1 (s, 1H).
N-Isop r op yl-N-[(3-h yd r oxyp h en yl)m eth yl]im in e. Us-
ing the previous procedure and starting from 10.3 g of
3-hydroxybenzaldehyde, 11.7 g (90%) of product was obtained:
mp 84-86 °C (petroleum ether); 1H NMR δ 1.2 (s, 3H), 1.3 (s,
3H), 3.55 (m, 1H), 6.8-7.3 (m, 4H), 7.45 (broad, 1H), 8.15 (s,
1H).
N-Meth yl-N-[(3-h yd r oxyp h en yl)m eth yl]a m in e. Sodium
borohydride (0.5 g) was added portionwise to a solution of the
Schiff base (1.25 g) in ethanol (20 mL) at 0-5 °C. The mixture
was stirred for 20 h and quenched with water. Ethanol was
evaporated, and the remaining aqueous solution was extracted
with CH2Cl2. The organic solution was dried over Na2SO4, and
the solvent was evaporated. The residue was crystallized from
ligroin to give 1.2 g (80%) of product: mp 112-113 °C; 1H NMR
δ 2.45 (s, 3H), 3.6 (s, 2H), 5.4 (broad, 1H), 6.6-7.2 (m, 4H).
Anal. (C7H9NO): C, H, N.
1H), 6.85-8.0 (m, 9H, Ar, NH and H-4 coumarin). 19:
δ
(DMSO-d6) 2.2 (m, 2H), 2.6-2.7 (m, 6H), 3.2 (m, 2H), 4.05-
4.5 (m, 4H), 6.25 (d, 1H), 7.0-8.2 (m, 8H, Ar and H-3). 20: δ
(DMSO-d6) 0.85 (t, 3H), 1.45 (m, 2H), 2.2 (m, 2H), 2.75 (s, 3H),
3.0 (m, 2H), 3.2 (m, 2H), 4.1-4.5 (m, 4H), 6.25 (d, 1H), 6.9-
8.2 (m, 9H, Ar, NH and H-3). 21: δ (DMSO-d6) 0.85 (t, 3H),
1.25 (m, 8H), 1.5 (m, 2H), 2.25 (m, 2H), 2.7 (s, 3H), 3.05 (m,
2H), 3.2 (m, 2H), 4.1-4.5 (m, 4H), 6.3 (d, 1H), 7.0-8.2 (m, 9H,
Ar, NH and H-3). 22: δ (DMSO-d6) 2.3 (m, 2H), 2.6-2.75 (m,
6H), 3.2 (m, 2H), 4.1-4.5 (m, 4H), 6.95-8.15 (m, 13H, Ar and
H-3). 23: δ (DMSO-d6) 0.85 (t, 3H), 1.2-1.5 (m, 4H), 2.3 (m,
2H), 2.7 (s, 3H), 3.0 (m, 2H), 3.2 (m, 2H), 4.15-4.5 (m, 4H),
6.9-8.1 (m, 14 H, Ar, NH and H-3). 24: δ (DMSO-d6) 0.8 (t,
3H), 1.2 (m, 8H), 1.4 (m, 2H), 2.25 (m, 2H), 2.7 (s, 3H), 3.0 (m,
2H), 3.2 (m, 2H), 4.15-4.5 (m, 4H), 6.95-8.1 (m, 14H, Ar, NH
and H-3). 25: δ (CDCl3) 1.05 (t, 3H), 1.95 (m, 2H), 2.5-2.7
(m, 4H), 2.85 (d, 3H), 3.55 (s, 2H), 4.1 (t, 2H), 4.95 (broad, 1H,
NH), 6.8-8.4 (m, 11H, Ar). 26: δ (CDCl3) 0.9 (t, 3H), 1.1 (t,
N-Eth yl-N-[(3-h ydr oxyph en yl)m eth yl]am in e. Using the
previous procedure and starting from 1.49 g (0.01 mol) of Schiff
base, 1.36 g (90%) of product was obtained: mp 106-109 °C
(lit.8 mp 107-111 °C); 1H NMR δ (CDCl3) 1.15 (t, 3H), 2.75
(m, 2H), 3.7 (s, 2H), 5.15 (broad, 1H), 6.6-7.2 (m, 4H).
N-P r op yl-N-[(3-h yd r oxyp h en yl)m eth yl]a m in e. Using
the previous procedure and starting from 1.63 g (0.01 mol) of
Schiff base, 1.5 g (90%) of product was obtained: mp 75-77
°C; 1H NMR δ 0.9 (t, 3H), 1.55 (m, 2H), 2.6 (t, 2H), 3.7 (s, 2H),
5.9 (broad, 1H), 6.6-7.2 (m, 4H). Anal. (C9H13NO): C, H, N.
N-Isop r op yl-N-[(3-h yd r oxyp h en yl)m eth yl]a m in e. Us-
ing the previous procedure and starting from 1.63 g (0.01 mol)
of Schiff base, 1.5 g (90%) of product was obtained: mp 128-
1
130 °C; H NMR δ 1.1 (s, 3H), 1.2 (s, 3H), 2.9 (m, 1H), 3.7 (s,
2H), 5.15 (broad, 1H), 6.7-7.2 (m, 4H). Anal. (C9H13NO): C,
H, N.

Synthesis of New Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 3985
In h ibition of Isola ted ACh E a n d Bu Ch E. The method
of Ellman et al.25 was followed. The assay solution consisted
of a 0.1 M phosphate buffer pH 8.0 with the addition of 340
µM 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman’s re-
agent), 0.035 unit/mL AChE derived from human erythrocytes
(Sigma Chemical Co.), and 550 µM acetylthiocoline iodide. The
final assay volume was 1 mL.
Test compounds were added to the assay solution and
preincubated with the enzyme for 20 min, the addition of
substrate following. The time dependence of inhibition was
assessed by testing enzyme activities with inhibitor IC50
concentration at 5 min intervals up to 60 min of preincubation
time at 37 °C. Changes in absorbances at 412 nm were
recorded for 5 min, the reaction rates were compared, and the
percent inhibition due to the presence of test compounds was
calculated.
Inhibition of BuChE was measured as described above,
substituting 0.035 unit/mL BuChE from human serum (Sigma
Chemical Co.) and 550 µM butyrylthiocholine for enzyme and
substrate, respectively.
Deca r ba m oyla tion Kin etics. Stock solutions in replicates
were prepared as following: the free enzyme (0.65 units in 3
mL of 0.1 M phosphate buffer pH 8.0), the same amount of
enzyme plus 0.9 nmol of physostigmine, and the same amount
of enzyme plus 0.3 nmol of the tested inhibitors 16, 17, and
18, respectively.
The solutions were gently stirred and incubated at 35 °C
for 20 min. They were then loaded into 12.000 MW cutoff
dialysis bags and dialyzed at 4 °C against 2 L of 0.1 M
phosphate buffer pH 8.0. The dialysate was replaced four
times during the first 6 h. Dialysis was interrupted after
6-12-18-24-48 h. The solutions were five times diluted
with the same buffer and kept at 20-22 °C under magnetic
stirring. Aliquots of 0.95 mL of each solution were sampled,
and the enzyme activity was spectrophotometrically tested
after addition of 0.033 mL of DTNB and 0.017 mL of acetylth-
iocholine as above-described. Plots of enzyme activity for each
compound expressed as percentage of the dialyzed free enzyme
activity against time of dialysis were built for each inhibitor.
As the enzyme incubated with 18 was still inhibited after
48 h of dialysis, after dilution a linear plot was obtained for
up to 5 h, according to the following expression
addition of a mixture of isoamylic alcohol/toluene to the
reaction tube, the amount of 3H-acetate formed was measured
in a liquid scintillation counter as the amount of radioactivity
present in the organic phase. Blanks and positive controls
were performed in the presence or absence of physostigmine
(10 µL), respectively. Data were obtained from at least three
determinations conducted in duplicate and the IC50 values
determined with the use of a computed-assisted curve-fitting
program (EBDA).30
3H-acetylcholine was from Dupont NEN, all the other
reagents were from Sigma Chemical Co.
Com pu tation al Meth ods. Molecular dynamics (MD) simu-
lations were performed using the united-atom AMBER* force
field implemented in the MacroModel Ver. 5.5 program,31
running on a Silicon Graphics Indigo2 workstation. The
coordinates of the protein were obtained from the X-ray
structure of AChE isolated from T. californica6 and retrieved
from the Brookhaven Protein Data Bank (entry 1ace); Phe330
was replaced by Tyr with the same conformation.
Polar and aromatic hydrogens were added to the protein,
while the aliphatic portions of the macromolecule were treated
using the united-atom model of AMBER.32 The inhibitor 8 was
docked into the enzyme by building a tetrahedral complex
between the Ser200 Oγ and the carbamate group; the proto-
nated amine group was placed at van der Waals distance from
Trp84, and the xanthenonyloxyalkyl substituent was posi-
tioned within the gorge in such a way as to avoid unfavorable
contacts.
In setting up the criteria for building the molecular model,
we defined a core of atoms around the active site and a shell
surrounding the core, on which the minimizations and the
dynamics simulations were carried out. The core was made
up by any atom of the protein within 10 Å from any atom of
the inhibitor; the shell contained any atom within 3 Å from
any atom of the core. All the atoms within the core were
unconstrained, while atoms in the shell were constrained by
applying an energy penalty force constant of 100 kJ /Å2 mol-1
.
Atoms beyond the shell were maintained at the X-ray coordi-
nates.
On this system, an initial minimization (1500 steps, steepest
descent) and a subsequent temperature constant MD simula-
tion (80 ps, 298 K, 1.5 fs time step) were carried out. An
equilibration time of 30 ps was allowed before starting the data
collection. The SHAKE algorithm33 was used to constrain all
bonds involving hydrogens at their equilibrium values. The
last conformation sampled by MD was energy minimized first
by steepest descent (1500 steps) and then by conjugate
gradient with a derivative convergence criterion of 0.05 kJ /Å2
ln(v∞ - v0) - ln(v∞ - vt) ) k3t
were v∞ is the activity of the free reference enzyme submitted
to dialysis and v0 and vt correspond to the residual carbam-
oylated enzyme activity at t ) 0 and t. The decarbamoylation
constant (k3) was calculated by the slope of the plot.
mol-1
.
Ack n ow led gm en t. Investigation supported by Uni-
versity of Bologna (funds for selected research topics)
and by MURST.
In h ibition of ACh E in Ra t Cer ebr a l Cor tex. Assays for
in vitro inhibition of AChE activity in rat cortex were con-
ducted using a method modified from previously published
ones.25-29 Briefly, brain cortical tissue was dissected out from
adult wistar rats and stored frozen for several weeks. Frozen
tissues were then suspended in phosphate buffer (25 mM, pH
7.4) to a ratio of 1:200 wet weight/vol and homogenized. The
homogenates were incubated at 37 °C for 5 min and then
centrifuged at 50000g for 15 min at 4 °C. The pellets were
then suspended in the initial volume of phosphate buffer and
used for AChE assays. This partial fractionation allows
elimination of endogenous substrate, which would interfere
with the assay, and leads to an enrichment of AChE activity.
For enzymatic assay, 100 µL of the AChE-enriched fraction
was added to a tube containing 50 µL of test compound in PBS
(pH 7.4) and 50 µL of tritiated ACh (specific activity 4.56 mCi/
mmol, final ACh concentration 0.4 mM, in PBS, pH 7.4). Such
conditions were found to be optimal to study competitive
inhibition. Experiments conducted at the saturating concen-
tration of substrate required a dilution of tritiated ACh with
cold ACh to a specific activity of 124 µCi/mmol (final ACh
concentration 2 mM). The enzymatic reaction was allowed to
proceed for 15 min at 37 °C and stopped with 200 µL of
blocking solution (94.5 g of chloroacetic acid and 116.88 g of
sodium chloride in 1 L of a 0.5 M NaOH solution). After the
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