Synthesis and cholinesterase activity of phenylcarbamates related to Rivastigmine, a therapeutic agent for Alzheimer's disease

Article abstract of DOI:10.1016/S0223-5234(01)01324-1

In order to develop new cholinesterase agents effective against Alzheimer's disease (AD) we synthesized some phenylcarbamates structurally related to Rivastigmine and evaluated their in vitro and in vivo biological activity. Among the compounds which displayed the most significant in vitro activity, 1-[1-(3-dimethylcarbamoyloxyphenyl)ethyl]piperidine (31b), in addition to a simple and cheaper synthesis, showed lower toxicity and very similar therapeutic index in comparison with Rivastigmine.

Full text of DOI:10.1016/S0223-5234(01)01324-1

Eur. J. Med. Chem. 37 (2002) 91–109
Original article
Synthesis and cholinesterase activity of phenylcarbamates related
to Rivastigmine, a therapeutic agent for Alzheimer’s disease
Carlo Mustazza a,*, Anna Borioni a, Maria Rosaria Del Giudice a, Franco Gatta a,
Rosella Ferretti a, Annarita Meneguz b, Maria Teresa Volpe b, Paola Lorenzini b
a Laboratorio di Chimica del Farmaco, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy
b Laboratorio di Farmacologia, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy
Received 11 December 237; received in revised form 1 October 2001; accepted 7 November 2001
Abstract
In order to develop new cholinesterase agents effective against Alzheimer’s disease (AD) we synthesized some phenylcarbamates
structurally related to Rivastigmine and evaluated their in vitro and in vivo biological activity. Among the compounds which
displayed the most significant in vitro activity, 1-[1-(3-dimethylcarbamoyloxyphenyl)ethyl]piperidine (31b), in addition to a simple
and cheaper synthesis, showed lower toxicity and very similar therapeutic index in comparison with Rivastigmine. © 2002
´
Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: acetylcholinesterase inhibitors; Alzheimer’s disease; rivastigmine; carbamates
1. Introduction
and nicotinic) by synthetical agonists and, finally, low-
ering acetylcholine metabolic breakdown by inhibition
of acetylcholinesterase (AChE), only the last approach
was successful as yet to palliative treatment of AD.
The first anticholinesterase drug for AD, Tacrine
(Cognex®) [10], suffered from bad though reversible
liver toxicity and low bioavailability [11,12], but the
new generation AChE inhibitors, Donezepil (Aricept®)
[13,14], Rivastigmine (Exelon®) [15] and Galantamine
(Reminyl®) [16], are endowed with a better pharmaco-
logical profile.
In addition, Rivastigmine showed interesting selectiv-
ity in the brain regions more affected by the neuronal
degeneration, inhibiting AChE more potently in human
cortex and hippocampus than in striatum and pons/
medulla [17]. The drug resulted also more effective
towards the monomeric form of the enzyme (G1),
which is present in relatively higher concentration in
these brain areas of AD patients [18,19].
Alzheimer’s disease (AD), one of the most diffuse
neurodegenerative pathology among the elderly [1,2],
causes a progressive impairment in functional perfor-
mances and continuous reduction in cognitive activities
and memory [3].
The loss of the basal forebrain cholinergic system is
one of the most significant aspects of neurodegenera-
tion in the brains of AD patients, and it is thought to
play a central role in producing cognitive impairments
[4–6].
Therefore, enhancement of cholinergic transmission
has been regarded as one of the most promising meth-
ods for treating AD patients. Among the following
possible strategies: use of acetylcholine precursors such
as choline [7], acetylcholine releasing agents such as
4-aminopyridine [8], stimulation of acetylcholine reup-
take [9], activation of cholinergic receptors (muscarinic
However, Rivastigmine has a short half-life [20] and
moreover, it is well known the difficulty of drug’s
intake in AD patients, so it would be wished to dispose
* Correspondence and reprints.
E-mail address: mustazza@iss.it (C. Mustazza).
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(01)01324-1

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C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
2. Chemistry
of drugs with longer half-life in order to obtain a better
patients compliance.
The synthetical pathways to compounds 1 are sum-
marized in Fig. 2.
Considering that Rivastigmine is usually adminis-
tered to AD patients for long periods, the high cost of
the drug is another considerable drawback. This is
mainly due to the employment of the very expensive
chemical intermediate N-ethyl-N-methylcarbamoyl
chloride in its synthesis.
The template which led to the formulation of Ri-
vastigmine was miotine, an AChE inhibitor which has
been used in therapy only as a miotic and which is
relatively unstable at physiologic pH [21]. A few carba-
mates related to miotine and Rivastigmine have been
synthesized and their pharmacological properties ex-
plored [22,23], but no data are reported about struc-
tural modifications of the aminoalkyl moiety of the
molecule.
On the basis of these findings and in connection with
our present studies directed towards the search of new
cholinesterase agents, we here describe the synthesis of
some dimethyl, diethyl and n-hexylcarbamates related
to Rivastigmine, containing a piperidine moiety instead
of the dimethylamino group, in order to evaluate their
in vitro an in vivo pharmacological properties and to
investigate the effects of such structural modifications
on activity (Fig. 1).
Compounds 1 (n=0, Fig. 1) were synthesized start-
ing from 6-(2- and 3-methoxyphenyl)-2-piperidones 4,
obtained according to a previously published procedure
[26] by heating in formamide at 190–200 °C the corre-
sponding 5-(2- and 3-methoxyphenyl)-5-oxovaleric
acids.
Piperidones 4 were methylated by methyl iodide–
sodium hydride in anhydrous dimethylformamide and
the resulting N-methylpiperidones 5 were first reduced
by lithium aluminum hydride to N-methylpiperidines 6,
then O-demethylated by refluxing in 48% hydrobromic
acid to afford the 1-methyl-2-(2- and 3-hydrox-
yphenyl)piperidines 7. These latter compounds were
carbamoylated either by treatment in refluxing benzene
with equimolar amount of n-hexyl isocyanate, to give
compounds 8, or by reaction with N,N-dimethyl- (and
N,N-diethyl)-carbamoylchloride in pyridine to give
compounds 9 and 10.
The 2-[(2- and 3-methoxyphenyl)methyl]-pyridines
11, prepared as reported in the literature [27,28] were
the key intermediates for the synthesis of compounds 1
(n=1, Fig. 1). By quaternizing the pyridine moiety
with methyl iodide and reducing the corresponding
pyridinium salts with sodium borohydride first, then
with nickel–aluminum alloy in aq. 1 M potassium
hydroxide solution [29], the desired 1-methyl-2-[(2- and
3-methoxyphenyl)methyl]piperidines 12 were obtained.
The O-demethylation with 48% hydrobromic acid fol-
lowed by the carbamoylation of the corresponding phe-
nols 13 with n-hexyl isocyanate and N,N-dimethyl-
(and N,N-diethyl)-carbamoylchloride to give com-
pounds 14, 15 and 16 were respectively accomplished
by the procedure previously described.
We chose the carbamic substituents on the basis of
literature reports:
(i) dimethylcarbamates of structures related to mi-
otine, are currently in use as AChE inhibitors; for
instance pyridostigmine and neostigmine are em-
ployed in the treatment of the myasthenia gravis;
besides the racemic dimethyl analogue of Ri-
vastigmine is highly active in vitro though short
lasting in vivo [23];
(ii) racemic diethylcarbamate analogue of Rivastig-
mine displays a low toxicity and a good therapeu-
tic ratio [23];
(iii) n-hexyl derivatives were prepared as the introduc-
tion of a long lipophilic substituent in some
AChE inhibiting compounds is reported to en-
hance their pharmacological properties (heptyl-
physostigmine [24], octyltacrine [25]).
Finally, the synthesis of compounds 1 (n=2, Fig. 2)
was achieved starting from the styrylquaternary salts 17
prepared by condensation of the appropriate anisalde-
hyde with 2-picoline methiodide [30]. The pyridinium
iodides 17 were reduced to 1-methyl-2-[(2- and 3-
methoxyphenyl)ethyl]piperidines 18 with sodium
Fig. 1. Structures of Rivastigmine and of the synthesized compounds.

C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
93
Fig. 2. Synthesis of 2-[(2- and 3-alkylcarbamoyloxyphenyl) and (2- and 3-alkylcarbamoyloxyphenyl)alkyl]-1-methylpiperidines.
borohydride and successively with nickel–aluminum
alloy in the same manner as described for compounds
12. O-demethylation and carbamoylation of the result-
ing phenols 19 proceeded as above described to give
compounds 20, 21 and 22.
The synthesis of compounds 2 is outlined in Fig. 3.
Compounds 23 (X=ꢀCH2ꢀ) and 28 (X=
ꢀCH(CH3)ꢀ) were synthesized by reaction of the appro-
priate benzyl chloride with piperidine, while
compounds 33 (X=ꢀ(CH2)2ꢀ) were obtained by reduc-
tion with lithium aluminum hydride of the correspond-
ing N-(phenylacetyl)piperidines. The O-demethylation
to give phenols 24, 29 and 34, followed by their car-
bamoylation to give compounds 25–27, 30–32 and
35–37 was accomplished in the same manner as re-
ported for the preparation of compounds 1.
3. Pharmacology
Synthesized compounds were evaluated for in vitro
inhibition of rat brain AChE and rat plasma butyryl-
cholinesterase (BuChE) by the spectroscopic method of
Ellman [31] (Table 1). The approximate LD50 of the
compounds that showed the most significant activity
(IC50 for AChEB100 nM), tested in vivo in mice after
oral administration, are reported in Table 2, while the
ED50 values and the time course of AChE of five
The unusually low values of ortho spin–spin cou-
1
pling constants in H-NMR spectra values of carba-
mates 8–10a are probably due to the steric hindrance
of the substituents, which may cause a distortion of the
benzene ring.
The chiral compounds were in vitro and in vivo
tested as racemates. For a pharmacological evaluation
of the pure enantiomers, studies are in progress
to perform their separation by chiral preparative
HPLC.
Fig.
3.
Synthesis
of
1-[(2-
and
3-alkylcarbamoyloxy-
phenyl)alkyl]piperidines.

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C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
Table 1
on brain globular tetrameric (G4) and monomeric
(G1) AChE molecular forms (Table 4).
No in vivo AChE inhibition was found for all se-
lected hexylcarbamates.
In vitro inhibition of brain AChE and plasma BuChE by hexyl- and
dimethylcarbamates
4. Results and discussion
As it is shown in Table 1, dimethylcarbamates
showed IC50 values ranging from 7 to 116 nM, unlike
hexylcarbamates whose IC50 did not result in so sharp
range (13\1000 nM), and showed a more selective
activity for central AChE respect to peripheral BuChE.
None of the diethylcarbamates displayed an IC50
lower than 1000 nM and therefore, they were not
further investigated.
Dimethylcarbamates showed a high activity for
AChE when a methylene bridge was inserted across the
piperidine moiety and the aromatic ring (compounds
15, 26, 31); a comparison between the more powerful
derivatives 26 and 31 points out that the introduction
of a methyl substituent on the methylene bridge re-
sulted in an enhancement of activity on both
cholinesterases so that compound 31b, whose structure
is reminiscent of Rivastigmine, was the most potent
inhibitor.
Varying the bridge across the piperidine and the
aromatic ring reduced activity as it is shown by the IC50
of compounds 9 (n=0, Table 1), 21 and 36 (n=2,
Table 1).
Hexylcarbamates tended to require the same struc-
tural features for activity enhancement as their
dimethyl analogues; compound 30b was indeed the
most potent in the hexyl derivatives series with an
IC50=13 nM for AChE and 16 nM for BuChE.
Table 2
AChE in vitro inhibition by carbamate derivatives and by Rivastig-
mine and corresponding approximate LD50
a
b
Compound
IC50 (nM)
LD50 (mg kg−1
)
9a
9b
14b
15b
47
64
47
14
\25
25BLD50B50
\50
\50
\50
a
8
, 14, 20, 25, 30, 35: RꢁH, R1=n-C6H13; 9, 15, 21, 26, 31, 36:
R=R1=ꢀCH3.
b Correlation coefficient was in all cases higher than 0.9.
c Selectivity calculated as IC50 for BuChE/IC50 for AChE.
20b
70
21b
50
\50
25b
13
25
26a
26b
30b
31a
10
31
13
15
\50
1BLD50B5
\25
\25
\50
selected compounds are reported in Table 3. For the
most interesting compound 31b, besides a BuChE time
course, a comparison with Rivastigmine was performed
by analysing:
31b
7
36b
Rivastigmine
40
\1000
\50
3BLD50B6
– the time course of AChE inhibition in total brain
(Fig. 4);
– the AChE inhibition in three different cerebral re-
gions, cortex, hippocampus and striatum (Fig. 5);
– the in vitro and in vivo properties including effects
a Correlation coefficient was in all cases higher than 0.9.
b Approximate LD50 was determined using a stepwise procedure.
Each dose was administered to five mice.

C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
95
Table 3
ED50 and time course of AChE inhibition by carbamate derivatives and by Rivastigmine
a
Compound
ED50 (mg kg−1
)
% inhibition a
0.5 h
1 h
42.398.4
40.399.0
44.7912.5
53.090.6
43.0 92.6
54.094.4
2 h
8.394.9
16.594.9
16.394.0
19.095.7
32.394.6
32.297.6
3 h
9a
9b
15b
31a
31b
Rivastigmine
5.390.3
8.091.1
9.590.9
11.691.7
6.590.6
1.0 b
6097.8
41.0912.5
49.395.5
55.695.5
56.698.2
47.593.1
0
9.099.0
2.092.6
7.095.7
14.791.5
13.894.0
a The values are means9SD of three independent experiments.
b Lit. [23].
Fig. 4. Plot of observed time course of in vivo AChE inhibition.
systematic conformational search about rotable bonds
at increment of 30° using MME calculations. Spatial
CꢀN distances of the lowest energy conformations were
related to IC50 values for dimethylcarbamates (Fig. 6),
and hexylcarbamates (Fig. 7).
In the series of dimethyl derivatives the best activity
was found when CꢀN distance values were 4.3 and 4.5
The activity of long-chain carbamates strictly de-
pends on the molecular shape and on the ability to
reach a suitable conformation for inhibition. So, com-
pact structures as 8a,b, 14a, 25a and 30a displayed a
marked decrease of potency in comparison with the
more flexible 20a,b, 35a,b derivatives and the less hin-
dered 14b, 25b and 30b compounds (Table 1).
The in vitro activity of carbamates seems to be
related to the spatial distance between carbamic carbon
and piperidine nitrogen atoms (CꢀN). For AChE inter-
action, such a distance is considered a characteristic
parameter of each molecular structure depending on
the conformational features.
,
,
A (compounds 31a and 26a) and 6.6 A (compounds 15b
and 31b).
,
Hexylcarbamates showed an optimum at 5.8 A (30b),
a strong decrease of potency for shorter and a gradual
decrease of potency for longer CꢀN distances.
It should also be noted that similar activities corre-
spond to different ranges of distances. This multiplicity
suggests that our compounds inhibit the enzyme
through a covalent or non-covalent mechanism which
involves different binding sites [32]; moreover, in the
case of ligands interacting with the esteratic site, it is
In order to evaluate this geometric parameter, a
conformational analysis has been performed on a HP
computer with Chem-X (Chemical Design Ltd., July 97
version) molecular modelling software. Minimum en-
ergy conformations were determined by performing a

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C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
the peripheral anionic site (PAS) of the enzyme about
also possible that the inhibitor–enzyme adduct is stabi-
lized by aminoacids situated at different distances from
the esteratic serine [33].
,
20 A far from ES [34]. This interaction could be easily
lost during enzyme extraction from rat brain as re-
ported previously for other reversible inhibitors [35,36].
On the basis of the affinity for AChE, selectivity and
results of a preliminary screening, five compounds (9a,
9b, 15b, 31a, and 31b) were selected for in vivo activity
studies.
Compounds 31a and 26a whose CꢀN distances are
,
4.3 and 4.5 A, could undergo a mechanism similar to
that of acetylcholine, whose calculated CꢀN distance in
,
the trans active conformation is 4.4 A. These com-
pounds may interact by the carbamic portion with
serine esteratic locus (ES) and by the protonated aminic
As reported in Table 2, in comparison with Rivastig-
mine, their acute toxicity evaluated in mice, resulted
relatively lower. Death of animals occurred from 30
min to a maximum of 3 h after dosing, and the ob-
served clinical signs as mydriasis, tremor and fascicula-
tion, were indicative of a cholinergic system alteration.
However, peripheral cholinergic signs as sweating and
salivation, were less evident or minimal, indicating a
selective action on central nervous system.
,
portion with the anionic locus (AS), 4.7 A far from the
esteratic site. Compounds 15b and 31b, binding the
esteratic serine site, may instead extend towards the
,
hydrophobic region which is at about 7 A from ES [33].
On the contrary, CꢀN distance seems not to be a
critical parameter for activity of hexylcarbamates; in
vivo observation of typical symptoms of central cholin-
ergic inhibition was not associated with enzyme inhibi-
tion in vivo tests. Therefore, we suppose that these
compounds bind by a non-covalent interaction with
AChE, as reported for similar structures attaching to
The ED50 values for the five compounds range from
5.3 to 11.6 mg kg−1, in comparison to 1 mg kg−1 for
Rivastigmine [23], and the time course analysis of
Fig. 5. Inhibition of AChE activity by 31b and Rivastigmine in different regions of mouse brain.
Table 4
Comparison of in vitro and in vivo data of 31b and Rivastigmine
a
a
a
b
c
IC50 (nM)
G1 form IC50
G4 form IC50
G1/G4 LD50 (mg kg−1
)
ED50 (mg kg−1
)
T.I. LD50/ED50
(mM)
(mM)
31b
Rivastigmine
7
0.490.1
3490.5
0.290.1
390.5
2
11
25BDL50B50
3BDL50B6
6.590.6
4–8
3–6
\1000
1.0 d
a Correlation coefficient was in all cases higher than 0.9.
b Approximate LD50 was determined using a stepwise procedure. Each dose was administered to five mice.
c The values are means9SD of three independent experiments.
d Lit. [23].

C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
97
Fig. 6. Inhibitory activity of N,N-dimethyl carbamates related to spatial CꢀN distance of the lowest energy conformation.
Fig. 7. Inhibitory activity of n-hexyl carbamates related to spatial CꢀN distance of the lowest energy conformation.
AChE inhibition, illustrated in Table 3, indicated a
good absorption after oral administration with a maxi-
mum effect already present half an hour after treatment
for all compounds. Compound 31b, the most interes-
ting synthesized derivative, like Rivastigmine, displayed
significant AChE inhibition 2 h after dosing (about
30%) and 3 h after treatment still showed activity. The
time course of BuChE inhibition was analyzed in mice

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C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
treated with 31b at ED50 dose for brain AChE. BuChE
inhibition resulted lower than AChE inhibition with a
maximum effect (35%) at 0.5 h and a gradual decrease
to 13%, 3 h after administration. These data seem to
confirm a preferential activity of compound 31b on the
central nervous system.
Fig. 4 shows the AChE inhibition time course for 31b
and Rivastigmine at ED50 dose: half an hour after
treatment the latter compound induced the highest
inhibitory effect (PB0.05) while Rivastigmine in-
creased its effect during the first hour followed by a
slower decrease of anticholinesterase activity.
5. Conclusions
In summary, in vitro and in vivo studies have demon-
strated that 31b possesses a high affinity for brain
AChE, a relatively low acute toxicity, an ED50 of 6.5
mg kg−1 and a therapeutic index (T.I. LD50/ED50)
ranging from 4 to 8. Moreover, it is well absorbed after
oral administration and produces a consistent and se-
lective inhibition of AChE in brain areas where the
cholinergic impairment is particularly evident in AD.
Finally 31b binds to both AChE molecular forms,
though in a non-selective manner, and at the ED50 dose
produces minimal peripheral cholinesterase inhibition
and clinical signs.
So far, 31b appears a suitable anticholinesterase
agent for further pharmacological investigations in or-
der to test its ability to improve memory and cognitive
functions, especially taking into account that its synthe-
sis is very simple and cheap, aspect particularly relevant
for the treatment of AD very often associated with a
considerable cost burden [40].
Fig. 5 reports the values of in vivo AChE inhibition
in cerebral cortex, hippocampus and striatum after oral
31b and Rivastigmine administration in mouse. Both
compounds caused a significant (PB0.01) reduction of
AChE activity in the three regions in treated animals,
especially in cortex and hippocampus, while no anti-
cholinesterasic effect was seen in cerebellum, a non-
cholinergic region, (data not shown). There was a
trend, but not statistically significant, of a more pro-
nounced effect of Rivastigmine on hippocampus AChE.
On the basis of these results 31b, as already reported
for Rivastigmine [19], seems to be able to induce a
preferential inhibition of AChE in regions particularly
relevant in situation of cholinergic impairment like AD.
It is well known that AChE can be separated into
multiple molecular forms by means of their sedimenta-
tion coefficient in rodent and human brain [37–39], in
which the bulk of AChE is the G4 (10S) and G1 (4S)
forms. The evaluation of in vivo AChE molecular form
patterns in the three cerebral regions of mice treated
with 31b and Rivastigmine, indicated that G4 and G1
profiles are the same in control and treated animals
(data not shown). This result is not due to the lack of
binding to AChE G4 and G1 active sites, but probably
depends on the strength of the binding or on technical
dilution problems. Enzyme dilution in connection with
the time required for the separation (18 h), restores
almost completely the anticholinesterase activity. These
data are in agreement with those already published for
Tacrine [35] and confirm that 31b is a reversible in-
hibitor of AChE and interacts with active catalytic sites
by weak bindings. This phenomenon was never ob-
served using irreversible anticholinesterase compounds
like diisopropylfluorophosphate [38].
6. Experimental
6.1. Chemistry
Melting point (m.p.) were taken on a Ko¨fler hot
stage apparatus and are uncorrected. The 1H-NMR
spectra were obtained on a Varian Gemini 200 MHz
instrument; all values were reported in ppm (l) and
standard abbreviations were used (a, apparent; b,
broad; d, doublet; dd, doublet of doublets; m, multi-
plet; q, quadruplet; s, singlet; t, triplet); assignments
were also based on 1H-COSY experiments; electron
ionization mass spectra were recorded on a HP 59980 B
spectrometer operating at 70 eV.
Column chromatographic separations were accom-
plished on Merck silica gel (70–230 mesh) or on Merck
aluminium oxide 90.
The purity of each compound was checked on silica
gel C. Erba 60 F254 or Merck aluminium oxide 60 F254
(type E) plates and spots were located by UV light.
Sodium sulphate was used to dry organic solutions.
Analyses indicated by the symbols of the elements
were within 90.4% of the theoretical values.
The synthesis of 11a [27], 11b [28], 23a and 23b [41],
has been reported elsewhere.
Finally Table 4 illustrates the in vitro affinity of 31b
and Rivastigmine for AChE G1 and G4 molecular
forms from mouse brain, and summarizes the compari-
son between the two compounds. Compound 31b, like
physostigmine [39], shows no significant differences in
IC50 between G4 and G1 forms while Rivastigmine
seems to induce a selective effect on G4 form. This
result is different from published data which were ob-
tained from experiments carried out on human brain
AChE [17,19].
6.1.1. General procedure for the preparation of 6-(2-
and 3-methoxyphenyl)-2-piperidones (4)
These compounds were prepared starting from ethyl
3-(2- and 3-methoxyphenyl)-3-oxopropionates. The 2-
methoxy derivative was obtained in the same manner as
the known 3-methoxy compound [42], and had b.p.=
121–122 °C/0.3 Torr, 72%. Anal. C12H14O4 (C, H)

C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
99
Ketoesters were monocyanoethylated as reported in the
literature [43] to afford the ethyl 2-(2-cyanoethyl)-3-(2-
methoxyphenyl)-3-oxopropionate (oil, 55%) and the
corresponding 3-methoxyphenyl derivative (oil, 88%),
which were purified by silica gel column chromatogra-
phy, by eluting with a 1:3 EtOAc–n-C6H14 mixture.
The latter compounds were hydrolyzed in a 1:3:1 sul-
phuric acid–acetic acid–water mixture according to a
literature method [43], to afford the 5-(2-
methoxyphenyl)-5-oxovaleric acid (m.p.=63–65 °C,
EtOAc, 84%. Anal. C12H14O4 (C, H)) and the 5-(3-
methoxyphenyl)-5-oxovaleric acid (m.p.=87–89 °C,
EtOAc, 63%. Anal. C12H14O4 (C, H)), respectively. Cy-
clization to 2-piperidones 4 was carried out by heating
the oxoacids in formamide at 190–200 °C, according
to a previously described procedure [26].
Hz, J5eq,6=3.7 Hz), 3.83 (s, 3H, OCH3), 2.78 (s, 3H,
NꢀCH3), 2.44 (m, 2H, H-3), 2.02 (m, 1H, H-5eq), 1.90
(m, 1H, H-5ax), 1.63 (m, 2H, H-4); MS: m/z 219 [M+],
204, 190, 148. Anal. C13H17NO2 (C, H, N).
6.1.2.2. 6 - (3- Methoxyphenyl) - 1 - methyl - 2 - piperidone
(5b). Compound 5b was obtained in 94% yield, m.p.
60–62 °C (C6H6–n-C6H14); 1H-NMR (DMSO-d6): l
7.28 (t, 1H, H-5%, Jortho=8.1 Hz), 6.84 (dd, 1H, H-4%,
J
J
ortho=8.1 Hz, Jmeta=2.2 Hz), 6.73 (d, 1H, H-6%,
ortho=8.1 Hz), 6.70 (d, 1H, H-2%, Jmeta=2.2 Hz), 4.55
(t, 1H, H-6, Jvic=5.2 Hz), 3.74 (s, 3H, OCH3), 2.62 (s,
3H, NꢀCH3), 2.33 (m, 2H, H-3), 2.07 (m, 1H, H-5eq),
1.74 (m, 1H, H-5ax), 1.56 (m, 2H, H-4); MS: m/z 219
[M+], 204, 190, 148, 112. Anal. C13H17NO2 (C, H, N).
6.1.1.1. 6-(2-Methoxyphenyl)-2-piperidone (4a). Com-
pound 4a was obtained in 80% yield, m.p. 165–167 °C
(EtOAc) (lit. [26] m.p. 148–155 °C). 1H-NMR
(CDCl3): l 7.26 (d, 1H, H-6%, Jortho=7.6 Hz), 7.24 (t,
1H, H-4%, Jortho=7.4 Hz), 6.94 (t, 1H, H-5%, Jortho=7.4
Hz), 6.85 (d, 1H, H-3%, Jortho=7.5 Hz), 5.97 (s, 1H,
NH), 4.92 (bt, 1H, H-6, Jvic=6.0 Hz), 3.81 (s, 3H,
OCH3), 2.40 (t, 2H, H-3, Jvic=6.5 Hz), 2.07 (m, 1H,
H-5eq), 1.84–1.58 (m, 3H, H-4 and H-5ax); MS: m/z:
205 [M+], 177, 146, 134. Anal. C12H15NO2 (C, H, N).
6.1.3. General procedure for the preparation of 2-(2-
and 3-methoxyphenyl)-1-methylpiperidines (6)
A solution of each compound 5 (8.0 g, 39 mmol) in
anhydrous Et2O (80 mL) was added dropwise under
stirring and at r.t. to a suspension of LiAlH4 (2.0 g, 53
mmol) in anhydrous Et2O (60 mL). The mixture was
allowed to react for 2 h, then cooled in an ice–water
bath and treated with 20% aq. NaOH (5 mL). The
precipitated hydroxides were removed by filtration and
rinsed thoroughly with Et2O. The solvent was evapo-
rated to dryness and the crude products 6 were purified
by column chromatography on Al2O3 by eluting with
EtOAc.
6.1.1.2. 6-(3-Methoxyphenyl)-2-piperidone (4b). Com-
pound 4b was obtained in 76% yield, m.p. 105–107 °C
1
(EtOAc); H-NMR (DMSO-d6): l 7.73 (s, 1H, NH),
7.25 (t, 1H, H-5%, Jortho=7.5 Hz), 6.83 (overlapped
doublets, 2H, H-4% and H-6%, Jortho=7.5 Hz), 6.81 (d,
1H, H-2%, Jmeta=3.1 Hz), 4.46 (bt, 1H, H-6, Jvic=6.0
Hz), 3.74 (s, 3H, OCH3), 2.20 (bt, 2H, H-3, Jvic=6.7
Hz), 1.95 (m, 1H, H-5eq), 1.61 (m, 3H, H-4 and
H-5ax); MS: m/z 205 [M+], 174, 149, 134. Anal.
C12H15NO2 (C, H, N).
6.1.3.1. 2-(2-Methoxyphenyl)-1-methylpiperidine (6a).
Compound 6a was obtained as an oil in 64% yield, b.p.
120–123 °C/0.3 Torr (hydrochloride lit. [44] m.p.
147 °C); 1H-NMR (CDCl3): l 7.45 (dd, 1H, H-6%,
Jortho=7.5 Hz, Jmeta=1.7 Hz), 7.19 (dt, 1H, H-4%,
Jortho=7.5 Hz, Jmeta=1.8 Hz), 6.96 (t, 1H, H-5%,
Jortho=7.5 Hz), 6.86 (d, 1H, H-3%, Jortho=7.8 Hz), 3.82
(s, 3H, OCH3), 3.38 (dd, 1H, H-2, J2,3ax=10.4 Hz,
2,3eq=2,4 Hz), 3.05 (dt, 1H, H-6eq, Jgem=10.0 Hz),
2.15 (m, 1H, H-6ax), 2.02 (s, 3H, NꢀCH3), 1.74 (m, 4H,
H-5 and H-4), 1.47 (m, 2H, H-3); MS: m/z 205 [M+],
174, 167, 149. Anal. C13H19NO (C, H, N).
6.1.2. General procedure for the preparation of 6-(2-
and 3-methoxyphenyl)-1-methyl-2-piperidones (5)
J
To a stirred suspension of NaH (2.5 g of 60% oil
dispersion, 50 mmol) in anhydrous dimethylformamide
(100 mL) each compound 4 (8.0 g, 42 mmol) was added
in several portions. After 1 h stirring at room tempera-
ture (r.t.), MeI (3.2 mL, 51 mmol) was dropwise added
and the reaction mixture stirred for 1 h at r.t. The
inorganic salts were then filtered, the filtrate evaporated
to dryness and the residue purified by column chro-
matography on silica gel, by eluting with EtOAc.
6.1.3.2. 2-(3-Methoxyphenyl)-1-methylpiperidine (6b).
Compound 6b was obtained as an oil in 75% yield, b.p.
140–143 °C/0.3 Torr; 1H-NMR (CDCl3): l 7.20 (t, 1H,
H-5%, Jortho=8.2 Hz), 6.88 (d, 1H, H-6%, Jortho=8.0
Hz), 6.86 (s, 1H, H-2%), 6.75 (d, 1H, H-4%, Jortho=8.2
Hz), 3.79 (s, 3H, OCH3), 3.01 (ad, 1H, H-6eq, Jgem
11.3 Hz), 2.71 (dd, 1H, H-2, J2,3ax=10.4 Hz, J2,3eq
3.1 Hz), 2.08 (m, 1H, H-6ax), 1.99 (s, 3H, NꢀCH3), 1.69
(m, 4H, H-3eq, H-4eq and H-5), 1.53 (m, 1H, H-3ax),
1.36 (m, 1H, H-4ax); MS: m/z 205 [M+], 190, 164, 150,
134, 98. Anal. C13H19NO (C, H, N).
=
=
6.1.2.1. 6 - (2 - Methoxyphenyl) - 1 - methyl - 2 - piperidone
(5a). Compound 5a was obtained as an oil in 94% yield,
1H-NMR (CDCl3): l 7.24 (dt, 1H, H-4%, Jortho=8.0 Hz,
Jmeta=2.5 Hz), 6.96 (m, 2H, H-5% and H-6%), 6.87 (d,
1H, H-3%, Jortho=8.0 Hz), 4.88 (q, 1H, H-6, J5ax,6=5.6

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C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
6.1.4. General procedure for the preparation of
2-[(2-and 3-methoxyphenyl)methyl]-1-methylpiperidines
(12)
6.1.5.1. 2-[2-(2-Methoxyphenyl)ethenyl]pyridine methio-
dide (17a). Compound 17a was obtained in 74% yield,
m.p. 212–214 °C (dimethylformamide); MS: m/z 226
[M+−127], 211, 180, 127. Anal. C15H16INO (C, H, N).
A solution of each compound 11 [27,28] (9.9 g, 50
mmol) and MeI (9.3 mL, 149 mmol) in MeOH (50 mL)
was heated to reflux for 3 h. The solvent was evapo-
rated in vacuo and the residue, dissolved in water (200
mL), was treated with NaBH4 (2.0 g, 53 mmol) added
in small portions at r.t. The reaction mixture, moni-
tored by TLC on Al2O3 (1:2 EtOAc–n-C6H14 mixture),
was stirred at r.t. for about 2 h, then cooled in an
ice–water bath. Potassium hydroxide in pellets (11 g.
196 mmol) was carefully added, then the mixture was
heated while stirring at 60 °C. Nickel–Al alloy (20 g)
was added in portions over 1 h. After about 3 h at
60 °C, TLC showed that the reaction had gone to
completion so the mixture was filtered. The inorganic
solid residue and the filtrate were thoroughly extracted
with EtOAc. The organic phase was separated, washed
with water, dried and concentrated to afford a yellow–
brown oil, which was distilled.
6.1.5.2. 2-[2-(3-Methoxyphenyl)ethenyl]pyridine methio-
dide (17b). Compound 17b was obtained in 69% yield,
m.p. 256–257 °C (dimethylformamide); MS: m/z 226
[M+−127], 211, 180, 142, 127. Anal. C15H16INO (C,
H, N).
6.1.6. General procedure for the preparation of
2-[2-(2-and 3-methoxyphenyl)ethyl]-1-methylpiperidines
(18)
To a suspension of each compound 17 (10 g, 28
mmol) in MeOH (80 mL), NaBH4 (1.1 g, 29 mmol) was
added in portions while stirring at 0–5 °C, then the
mixture was stirred for 2 h at 50–60 °C. After cooling,
water (200 mL) was added and the mixture extracted
with EtOAc. Removal of the solvent gave an oil which
was dissolved in a 1:1 mixture of EtOH and 1 M aq.
KOH (200 mL) heated at 60 °C. Nickel–Al alloy (20 g)
was carefully added in portions during 1 h. The reac-
tion mixture was then stirred overnight at 60 °C,
whereupon TLC on Al2O3 (1:1 EtOAc–n-C6H14 mix-
ture) indicated that the reduction was complete. The
suspension was filtered while still hot and the inorganic
solid was rinsed with hot EtOH. Ethanol was evapo-
rated from the mixture under reduced pressure and the
resulting aq. suspension was extracted with CH2Cl2.
Removal of the solvent afforded an oily residue which
was distilled.
6.1.4.1. 2-[(2-Methoxyphenyl)methyl]-1-methylpiperidine
(12a). Compound 12a was obtained as an oil in 70%
1
yield; b.p. 110–112 °C/0.01 Torr; H-NMR (CDCl3): l
7.17 (dt, 1H, H-4%, Jortho=7.6 Hz, Jmeta=1.6 Hz), 7.08
(dd, 1H, H-6%, Jortho=7.5 Hz, Jmeta=1.6 Hz), 6.84 (t
and d, 2H, H-5%, Jortho=7.4 Hz and H-3%, Jortho=8.4
Hz), 3.79 (s, 3H, OCH3), 3.23 (dd, 1H, benzylic CH2,
J
J
gem=12.4 Hz, Jvic=3.4 Hz), 2.83 (bd, 1H, H-6eq,
gem=10.5 Hz), 2.41 (s, 3H, NꢀCH3), 2.34 (dd, 1H,
benzylic CH2, Jgem=12.5 Hz, Jvic=2.4 Hz), 2.13 (m,
2H, H-2 and H-6ax), 1.70–1.00 (m, 6H, H-3, H-5 and
H-4); MS: m/z 219 [M+], 198, 183, 167, 154, 121. Anal.
C14H21NO (C, H, N).
6.1.6.1. 2 - [2 - (2 - Methoxyphenyl)ethyl] - 1 - methylpipe-
ridine (18a). Compound 18a was obtained in 72% yield,
b.p. 111–114 °C/0.01 Torr (lit. [45] hydroiodide m.p.
1
146–147 °C); H-NMR (CDCl3): l 7.15 (t, 1H, H-4%,
J
ortho=7.7 Hz), 7.13 (d, 1H, H-6%, Jortho=7.7 Hz), 6.86
(t, 1H, H-5%, Jortho=7.8 Hz), 6.82 (d, 1H, H-3%, Jortho
7.8 Hz), 3.80 (s, 3H, OCH3), 2.85 (bd, 1H, H-6eq,
gem=10.6 Hz), 2.63 (m, 2H, benzylic CH2), 2.28 (s,
=
6.1.4.2. 2-[(3-Methoxyphenyl)methyl]-1-methylpiperidine
(12b). Compound 12b was obtained in 68% yield, b.p.
1
J
95–98 °C/0.01 Torr (lit. [28]: 102 °C/0.04 Torr); H-
NMR (CDCl3): l 7.17 (t, 1H, H-5%, Jortho=7.6 Hz),
6.73 (d, 1H, H-4%, Jortho=7.6 Hz), 6.72 (d, 1H, H-6%,
3H, NꢀCH3), 2.06 (m, 1H, H-6ax), 1.82 (m, 2H, pipe-
ridineꢀCH2), 1.80–1.10 (m, 7H, H-2, H-5, H-3 and
H-4); MS: m/z 233 [M+], 218. Anal. C15H23NO (C, H,
N).
Jortho=7.6 Hz), 6.69 (s, 1H, H-2%), 3.77 (s, 3H, OCH3),
3.15 (dd, 1H, benzylic CH2, Jgem=12.9 Hz, Jvic=3.9
Hz), 2.83 (bd, 1H, H-6eq, Jgem=10.5 Hz), 2.38 (s, 3H,
NꢀCH3), 2.29 (dd, 1H, benzylic CH2, Jgem=12.9 Hz,
6.1.6.2. 2 - [2 - (3 - Methoxyphenyl)ethyl] - 1 - methylpipe-
ridine (18b). Compound 18b was obtained in 66% yield,
J
vic=3.1 Hz), 2.12 (m, 2H, H-2 and H-6ax), 1.70–1.00
1
b.p. 125–127 °C/0.01 Torr; H-NMR (CDCl3): l 7.18
(m, 6H, H-3, H-5 and H-4); MS: m/z 219 [M+], 218,
(t, 1H, H-5%, Jortho=7.8 Hz), 6.76 (d, 1H, H-4%, Jortho
7.8 Hz), 6.73 (s, 1H, H-2%), 6.70 (d, 1H, H-6%, Jortho
=
=
198, 183, 154, 121. Anal. C14H21NO (C, H, N).
7.8 Hz), 3.76 (s, 3H, OCH3), 2.84 (bd, 1H, H-6eq,
gem=10.7 Hz), 2.60 (m, 2H, benzylic CH2), 2.26 (s,
3H, NꢀCH3), 2.06 (m, 1H, H-6ax), 1.80 (m, 2H, pipe-
ridineꢀCH2), 1.80–1.10 (m, 7H, H-2, H-3, H-4 and
H-5); MS: m/z 233 [M+], 218. Anal. C15H23NO (C, H,
N).
6.1.5. General procedure for the preparation of
2-[2-(2-and 3-methoxyphenyl)ethenyl]pyridine
methiodides (17)
These compounds were synthesized according to a
method reported in the lit. [30].
J

C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
101
1
6.1.7. General procedure for the preparation of 1-[1-(2-
and 3-methoxyphenyl)ethyl]piperidines (28)
115 °C/0.01 Torr; H-NMR (CDCl3): l 7.16 (m, 2H,
H-4% and H-6%), 6.85 (m, 2H, H-5% and H-3%), 3.80 (s,
3H, OCH3), 2.86–2.77 (m, 2H, benzylic CH2), 2.53–
2.44 (m, 6H, piperidineꢀCH2, H-2 and H-6), 1.61 (m,
4H, H-3 and H-5), 1.44 (m, 2H, H-4); MS: m/z 219
[M+], 217, 190, 134, 121. Anal. C14H21NO (C, H, N).
These compounds were synthesized by heating at
50 °C overnight a mixture of piperidine (9.9 mL, 100
mmol) and 1-chloro-1-(2-methoxyphenyl)ethane [46] (to
obtain 28a) or 1-chloro-1-(3-methoxyphenyl)ethane [47]
(to obtain 28b) (50 mmol). The reaction mixture was
treated with 1 M HCl and Et2O, shacked, the aq. layer
separated, alkalinized with K2CO3, then extracted with
Et2O. The solvent was evaporated and the residue
distilled.
6.1.8.2. 1-[2-(3-Methoxyphenyl)ethyl]piperidine (33b).
Compound 33b was obtained in 80% yield, b.p. 105–
110/0.01 Torr; H-NMR (CDCl3): l 7.10 (t, 1H, H-5%
1
J
ortho=7.6 Hz), 6.76 (d, 1H, H-4%, Jortho=7.6 Hz), 6.73
(s, 1H, H-2%), 6.70 (d, 1H, H-6%, Jortho=7.6 Hz), 3.78 (s,
3H, OCH3), 2.77 (m, 2H, benzylic CH2), 2.53 (m, 2H,
piperidineꢀCH2), 2.43 (m, 4H, H-2 and H-6), 1.59 (m,
4H, H-3 and H-5), 1.43 (m, 2H, H-4); MS: m/z 219
[M+], 190, 152, 134, 121. Anal. C14H21NO (C, H, N).
6.1.7.1. 1-[1-(2-Methoxyphenyl)ethyl]piperidine (28a).
Compound 28a was obtained in 41% yield, b.p. 90–
92 °C/0.01 Torr; 1H-NMR (CDCl3): l 7.16 (t, 1H,
H-4%, Jortho=7.5 Hz), 7.11 (d, 1H, H-6%, Jortho=7.6
Hz), 6.85 (t, 1H, H-5%, Jortho=7.6 Hz), 6.81 (d, 1H,
H-3%, Jortho=7.5 Hz), 3.79 (s, 3H, OCH3), 3.57 (q, 1H,
CHꢀCH3, Jvic=6.9 Hz), 2.32 (m, 4H, H-2 and H-6),
1.47 (m, 4H, H-3 and H-5), 1.37 (m, 2H, H-4), 1.33 (d,
3H, CHꢀCH3, Jvic=6.9 Hz); MS: m/z 219 [M+], 204.
Anal. C14H21NO (C, H, N).
6.1.9. General procedure for the preparation of phenol
deri6ati6es 7, 13, 19, 24, 29,34
A solution of each methoxyderivative 6, 12, 18, 23,
28 33 (10 g) in 48% hydrobromic acid (80 mL), was
heated at 125 °C for 3 h. The hydrobromic acid was
evaporated at reduced pressure and the residue, made
alkaline with aq. K2CO3 solution, was extracted with
EtOAc. Removal of the solvent gave a residue, which
upon chromatography over Al2O3 (EtOAc) afforded
the pure title compounds.
6.1.7.2. 1-[1-(3-Methoxyphenyl)ethyl]piperidine (28b).
Compound 28b was obtained in 70% yield, b.p. 110–
112 °C/0.01 Torr; 1H-NMR (CDCl3): l 7.19 (t, 1H,
H-5%, Jortho=7.8 Hz), 6.78 (d, 1H, H-4%, Jortho=7.8
Hz), 6.73 (s, 1H, H-2%), 6.69 (d, 1H, H-6%, Jortho=7.8
Hz), 3.77 (s, 3H, OCH3), 3.37 (q, 1H, CHꢀCH3, Jvic
=
6.1.9.1. 2-(2-Hydroxyphenyl)-1-methylpiperidine (7a).
Compound 7a was obtained as an oil in 75% yield;
1H-NMR (CDCl3): l 11.00 (bs, 1H, OH), 7.14 (dt, 1H,
H-4%, Jortho=8.0 Hz, Jmeta=1.8 Hz), 6.91 (dd, 1H,
H-6%, Jortho=7.4 Hz, Jmeta=1.8 Hz), 6.77 (m, 2H, H-3%
and H-5%), 3.03 (m, 2H, H-2 and H-6eq), 2.19 (s, 3H,
NꢀCH3), 2.09 (m, 1H, H-6ax), 1.73 (m, 5H, H-4, H-5
and H-3eq), 1.30 (m, 1H, H-3ax); MS: m/z 191 [M+],
162, 148, 134, 120, 98. Anal. C12H17NO (C, H, N).
6.9 Hz), 2.35 (m, 4H, H-2 and H-6), 1.52 (m, 4H, H-3
and H-5), 1.40 (m, 2H, H-4), 1.33 (d, 3H, CHꢀCH3,
J
vic=6.9 Hz); MS: m/z 219 [M+], 204. Anal.
C14H21NO (C, H, N).
6.1.8. General procedure for the preparation of 1-[2-(2-
and 3-methoxyphenyl)ethyl]piperidines (33)
The N-(2- and 3-methoxyphenylacetyl)piperidines
were obtained by adding dropwise a solution of the
corresponding phenylacetyl chloride (10 g, 55 mmol) in
anhydrous Et2O (50 mL) to a cooled and stirred solu-
tion of piperidine (12 mL, 120 mmol) in anhydrous
Et2O (50 mL). After 6 h stirring at r.t., water was
added. The organic phase was separated, washed with
diluted HCl and water, then evaporated and the residue
distilled.
N-(2-Methoxyphenylacetyl)piperidine was obtained
in 79% yield, b.p. 140–142 °C/0.01 Torr. Anal.
C14H19NO2 (C, H, N).
N-(3-Methoxyphenylacetyl)piperidine was obtained
in 56% yield, b.p. 133–135 °C/0.01 Torr (lit. [48] 166–
168 °C/0.2 Torr). Anal. C14H19NO2 (C, H, N).
The above amides were reduced with LiAlH4 accord-
ing to the procedure described for compounds 6.
6.1.9.2. 2-(3-Hydroxyphenyl)-1-methylpiperidine (7b).
Compound 7b was obtained in 80% yield, m.p. 144–
145 °C (EtOAc); (lit. [39] hydrobromide hemihydrate
1
m.p. 60 °C); H-NMR (CDCl3): l 7.32 (bs, 1H, OH),
7.13 (t, 1H, H-5%, Jortho=7.9 Hz), 6.97 (s, 1H, H-2%),
6.77 (d, 1H, H-6%, Jortho=7.9 Hz), 6.76 (d, 1H, H-4%,
J
ortho=8.0 Hz), 3.10 (bd, 1H, H-6eq, Jgem=11.5 Hz),
2.84 (t, 1H, H-2, Jvic=6.9 Hz), 2.18 (dt, 1H, H-6ax,
gem=11.5 Hz, J6ax,5ax=11.0 Hz, J6ax,5eq=2.8 Hz),
J
2.05 (s, 3H, NꢀCH3), 1.70 (m, 5H, H-3, H-4eq and
H-5), 1.41 (m, 1H, H-4ax); MS: m/z 191 [M+], 162,
148, 134, 98. Anal. C12H17NO (C, H, N).
6.1.9.3. 2-[(2-Hydroxyphenyl)methyl]-1-methylpiperidine
(13a). Compound 13a was obtained in 71% yield, m.p.
1
109–110 °C (EtOAc); H-NMR (CDCl3): l 7.34 (bs,
6.1.8.1. 1-[2-(2-methoxyphenyl)ethyl]piperidine (33a).
Compound 33a was obtained in 80% yield, b.p. 110–
1H, OH), 7.15 (t, 1H, H-4%, Jortho=7.6 Hz), 6.90 (d,
1H, H-6%, Jortho=7.5 Hz), 6.81 (d, 1H, H-3%, Jortho=7.6

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C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
Hz), 6.68 (t, 1H, H-5%, Jortho=7.5 Hz), 2.65 (dd, 1H,
benzylic CH2, Jgem=12.4 Hz, Jvic=3.3 Hz), 2.57 (bd,
1H, H-6eq, Jgem=10.5 Hz), 2.46 (s, 3H, NꢀCH3), 2.44
(dd, 1H, benzylic CH2, Jgem=12.4 Hz, Jvic=2.5 Hz),
2.25 (m, 2H, H-2 and H-6ax), 1.70–1.00 (m, 6H, H-3,
H-5 and H-4); MS: m/z 205 [M+], 184, 160, 146, 133.
Anal. C13H19NO (C, H, N).
6.1.9.8. 1-[(3-Hydroxyphenyl)methyl]piperidine (24b).
Compound 24b was obtained in 90% yield, m.p. 135–
137 °C (EtOH); (lit. [49] m.p. 136–137 °C); H-NMR
1
(CDCl3): l 7.84 (bs, 1H, OH), 7.09 (t, 1H, H-5%,
J
ortho=7.6 Hz), 6.75 (d, 1H, H-4%, Jortho=7.6 Hz),
6.71–6.67 (m, 2H, H-2% and H-6%), 3.43 (s, 2H, benzylic
CH2), 2.46 (t, 4H, H-2 and H-6), 1.59 (m, 4H, H-3 and
H-5), 1.41 (m, 2H, H-4); MS: m/z 191 [M+], 190, 162,
148, 134, 107. Anal. C12H17NO (C, H, N).
6.1.9.4. 2-[(3-Hydroxyphenyl)methyl]-1-methylpiperidine
(13b). Compound 13b was obtained as an oil in 82%
1
6.1.9.9. 1-[1-(2-Hydroxyphenyl)ethyl]piperidine (29a).
Compound 29a was obtained as an oil in 75% yield;
1H-NMR (CDCl3): l 7.12 (dt, 1H, H-4%, Jortho=7.0 Hz,
yield (lit. [27] hydrobromide m.p. 78–79 °C); H-NMR
(CDCl3): l 7.20 (bs, 1H, OH), 7.10 (t, 1H, H-5%,
J
ortho=7.6 Hz), 6.68 (s, 1H, H-2%), 6.64 (overlapped
doublets, 2H, H-4%, Jortho=7.6 Hz and H-6%, Jortho=7.5
Hz), 3.12 (dd, 1H, benzylic CH2, Jgem=12.8 Hz, Jvic
Jmeta=1.7 Hz), 6.95 (dd, 1H, H-6%, Jortho=7.2 Hz,
Jmeta=0.9 Hz), 6.77 (dd, 1H, H-3%, Jortho=7.3 Hz,
Jmeta=1.2 Hz), 6.75 (dt, 1H, H-5%, Jortho=7.3 Hz,
Jmeta=1.2 Hz), 3.65 (q, 1H, CHꢀCH3, Jvic=6.8 Hz),
=
3.9 Hz), 2.89 (bd, 1H, H-6eq, Jgem=11.7 Hz), 2.40 (s,
3H, NꢀCH3), 2.39 (dd, 1H, benzylic CH2, Jgem=12.8
Hz, Jvic=3.1 Hz), 2.26 (m, 2H, H-2 and H-6ax), 1.70–
1.00 (m, 6H, H-3, H-5 and H-4); MS: m/z 204 [M+−
1], 184, 167, 160, 133. Anal. C13H19NO (C, H, N).
2.54 (m, 4H, H-2 and H-6), 1.61 (m, 4H, H-5 and H-3),
1.47 (m, 2H, H-4), 1.35 (d, 3H, CHꢀCH3, Jvic=6.8 Hz);
MS: m/z 205 [M+], 190, 122, 107. Anal. C13H19NO (C,
H, N).
6.1.9.5. 2 - [2 - (2 - Hydroxyphenyl)ethyl] - 1 - methylpipe-
ridine (19a). Compound 19a was obtained as an oil in
61% yield; H-NMR (CDCl3): l 7.25 (s, 1H, OH), 7.09
6.1.9.10. 1-[1-(3-Hydroxyphenyl)ethyl]piperidine (29b).
Compound 29b was obtained in 92% yield, m.p. 143–
145 °C (EtOAc) (lit. [50] m.p. 142–143 °C) H-NMR
1
1
(t, 1H, H-4%, Jortho=7.7 Hz), 7.04 (d, 1H, H-6%, Jortho
=
(CDCl3): l 7.97 (bs, 1H, OH), 7.10 (t, 1H, H-5%,
7.7 Hz), 6.86 (d, 1H, H-3%, Jortho=7.7 Hz), 6.79 (t, 1H,
H-5%, Jortho=7.7 Hz), 2.94 (bd, 1H, H-6eq, Jgem=10.6
Hz), 2.73 (m, 1H, benzylic CH2), 2.59 (m, 1H, benzylic
CH2), 2.34 (overlapped m and s, 4H, H-6ax and
NꢀCH3), 1.90–1.10 (m, 9H, piperidineꢀCH2, H-2, H-3,
H-4 and H-5); MS: m/z 219 [M+], 190, 162, 146, 131,
107. Anal. C14H21NO (C, H, N).
J
ortho=8.1 Hz), 6.86 (s, 1H, H-2%), 6.72 ( two over-
lapped d, 2H, H-4% and H-6%, Jortho=8.2 Hz), 3.50 (q,
1H, CHꢀCH3, Jvic=6.9 Hz), 2.51 (m, 4H, H-2 and
H-6), 1.57 (m, 4H, H-3 and H-5), 1.41 (d, 3H,
CHꢀCH3, Jvic=6.9 Hz), 1.34 (m, 2H, H-4); MS: m/z
205 [M+], 190, 112, 107. Anal. C13H19NO (C, H, N).
6.1.9.11. 1-[2-(2-Hydroxyphenyl)ethyl]piperidine (34a).
Compound 34a was obtained in 70% yield, hydrobro-
mide m.p. 225–228 °C (EtOH); H-NMR (CDCl3): l
6.1.9.6. 2 - [2 - (3 - Hydroxyphenyl)ethyl] - 1 - methylpipe-
ridine (19b). Compound 19b was obtained as an oil in
69% yield; 1H-NMR (CDCl3): l 7.66 (bs, 1H, OH), 7.09
(t, 1H, H-5%, Jortho=7.8 Hz), 6.62 (overlapped d and s,
1
13.27 (bs, 1H, OH), 7.10 (dt, 1H, H-4%, Jortho=7.8 Hz,
Jmeta=1.5 Hz), 6.97 (dd, 1H, H-6%, Jortho=7.4 Hz,
Jmeta=1.5 Hz), 6.86 (dd, 1H, H-3%, Jortho=7.8 Hz,
Jmeta=1.2 Hz), 6.71 (dt, 1H, H-5%, Jortho=7.5 Hz,
Jmeta=1.3 Hz), 2.80 (m, 2H, benzylic CH2), 2.63 (m,
3H, H-4%, H-6% and H-2%), 2.89 (bd, 1H, H-6eq, Jgem
=
11.9 Hz), 2.55 (m, 2H, benzylic CH2), 2.29 (s, 3H,
NꢀCH3), 2.17 (m, 1H, H-6ax), 1.98 (m, 2H, pipe-
ridineꢀCH2), 1.85–1.10 (m, 7H, H-2, H-3, H-4 and
H-5); MS: m/z 219 [M+], 190, 162, 148, 133, 107. Anal.
C14H21NO (C, H, N).
6H, piperidineꢀCH2, H-2 and H-6), 1.69 (m, 4H, H-3
and H-5), 1.51 (m, 2H, H-4); MS: m/z 205 [M+], 190,
174, 146, 121. Anal. C13H19NO (C, H, N).
6.1.9.7. 1-[(2-Hydroxyphenyl)methyl]piperidine (24a).
Compound 24a was obtained as an oil in 74% yield;
1H-NMR: l 9.20 (bs, 1H, OH), 7.13 (t, 1H, H-4%,
6.1.9.12. 1-[2-(3-Hydroxyphenyl)ethyl]piperidine (34b).
Compound 34b was obtained in 88% yield, m.p. 134–
135 °C (EtOAc); 1H-NMR (CDCl3): l 7.12 (t, 1H,
H-5%, Jortho=7.9 Hz), 6.80 (bs, 1H, OH), 6.69 (s, 1H,
H-2%), 6.65 (m, 2H, H-4% and H-6%), 2.78 (m, 2H,
benzylic CH2), 2.69 (m, 2H, piperidineꢀCH2), 2.56 (m,
4H, H-2 and H-6), 1.68 (m, 4H, H-3 and H-5), 1.47 (m,
2H, H-4). MS: m/z 205 [M+], 204, 190, 146, 121. Anal.
C13H19NO (C, H, N).
J
ortho=7.3 Hz), 6.93 (d,1H, H-6%, Jortho=7.3 Hz), 6.77
(partially overlapped d and t, 2H, H-3% and H-5%,
ortho=7.3 Hz), 3.64 (s, 2H, benzylic CH2), 2.47 (m,
J
4H, H-2 and H-6), 1.62 (m, 4H, H-3 and H-5), 1.48 (m,
2H, H-4); MS: m/z 191 [M+]. Anal. C12H17NO (C, H,
N).

C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
103
6.1.10. General procedure for the preparation of n-hexyl-
carbamoyloxyphenyl deri6ati6es 8, 14, 20, 25, 30, 35
Each compound 7, 13, 19, 24, 29, 34 (20 mmol) was
added to a stirred solution of n-hexylisocyanate (20
mmol), prepared in situ from heptanoyl azide by Cur-
tius rearrangement in refluxing C6H6 [51]. The mixture
was refluxed for 2 h, the solvent was evaporated and
the residue purified by column chromatography on
Al2O3 by eluting with an 1:2 EtOAc–n-C6H14 mixture.
carbamic H-1, Jvic=6.7 Hz), 3.17 (dd, 1H, benzylic
CH2, Jgem=12.8 Hz, Jvic=3.9 Hz), 2.84 (bd, 1H, H-
6eq, Jgem=11.5 Hz), 2.37 (s, 3H, NꢀCH3), 2.33 (d, 1H,
benzylic CH2, Jgem=12.8 Hz), 2.10 (m, 2H, H-2 and
H-6ax), 1.70–1.00 (m, 14H, H-3, H-4, H-5 and car-
bamic H-2–H-5), 0.88 (bt, 3H, carbamic H-6); MS: m/z
333 [M++1], 228, 199, 185. Anal. C20H32N2O2 (C, H,
N).
6.1.10.5. 2-[2-(2-n-Hexylcarbamoyloxyphenyl)ethyl]-1-
methylpiperidine (20a). Compound 20a was obtained as
an oil in 63% yield; 1H-NMR (CDCl3): l 7.20–7.02
(overlapped d and t, 4H, phenyl protons), 5.10 (bt, 1H,
6.1.10.1. 2-(2-n-Hexylcarbamoyloxyphenyl)-1-methyl-
piperidine (8a). Compound 8a was obtained as an oil in
30% yield; 1H-NMR (CDCl3): l 7.52 (dt, 1H, H-4%,
J
ortho=5.9 Hz, Jmeta=3.6 Hz), 7.28 (t, 1H, NH, Jvic=
NH, Jvic=6.7 Hz), 3.22 (bq, 2H, carbamic H-1, Jvic
=
6.7 Hz), 7.23 (d, 1H, H-3%, Jortho=5.9 Hz), 7.20 (d, 1H,
H-6%, Jortho=5.9 Hz), 6.94 (dt, 1H, H-5% Jortho=5.9
Hz), 3.01 (bd, 1H, H-6eq, Jgem=11.4 Hz), 2.91 (dd,
1H, H-2, J2,3ax=10.7 Hz, J2,3eq=3.0 Hz), 2.58 (t, 2H,
carbamic H-1, Jvic=7.3 Hz), 2.03 (m, 1H, H-6ax), 1.94
(s, 3H, NꢀCH3), 1.68 (m, 6H, H-3, H-4 and H-5), 1.36
(m, 8H, carbamic H-2–H-5), 0.90 (t, 3H, carbamic
H-6); MS: m/z 318 [M+], 303, 288, 190, 168, 134. Anal.
C19H30N2O2 (C, H, N).
6.7 Hz), 2.82 (bd, 1H, H-6eq, Jgem=10.6 Hz), 2.55 (m,
2H, benzylic CH2), 2.23 (s, 3H, NꢀCH3), 2.05 (m, 1H,
H-6ax), 1.90–1.35 (m, 9H, piperidineꢀCH2, H-2, H-3,
H-4 and H-5), 1.29 (bs, 8H, carbamic H-2-H-5), 0.87
(bt, 3H, carbamic H-6); MS: m/z 347 [M++1], 275,
219, 190, 112, 107. Anal. C21H34N2O2 (C, H, N).
6.1.10.6. 2-[2-(3-n-Hexylcarbamoyloxyphenyl)ethyl]-1-
methylpiperidine (20b). Compound 20b was obtained as
1
an oil in 52% yield; H-NMR (CDCl3): l 7.19 (t, 1H,
6.1.10.2. 2-(3-n-Hexylcarbamoyloxyphenyl)-1-methyl-
piperidine (8b). Compound 8b was obtained as an oil in
35% yield; 1H-NMR (CDCl3): l 7.26 (t, 1H, H-5%,
H-5%, Jortho=7.6 Hz), 6.95 (d, 1H, H-4%, Jortho=7.6
Hz), 6.91 (s, 1H, H-2%), 6.88 (d, 1H, H-6%, Jortho=7.6
Hz), 5.30 (t, 1H, NH, Jvic=6.4 Hz), 3.17 (bq, 2H,
carbamic H-1, Jvic=6.4 Hz), 2.79 (bd, 1H, H-6eq,
J
ortho=7.7 Hz), 7.11 (d, 1H, H-4%, Jortho=7.8 Hz), 7.08
(s, 1H, H-2%), 6.98 (d, 1H, H-6% Jortho=8.0 Hz), 5.01 (t,
1H, NH, Jvic=6.6 Hz), 3.23 (q, 2H, carbamic H-1,
Jgem=11.2 Hz), 2.59 (m, 2H, benzylic CH2), 2.22 (s,
3H, NꢀCH3), 2.03 (m, 1H, H-6ax), 1.90–1.35 (m, 9H,
piperidineꢀCH2, H-2, H-3, H-4 and H-5), 1.26 (bs, 8H,
carbamic H-2–H-5), 0.85 (bt, 3H, carbamic H-6); MS:
m/z 347 [M++1], 275, 218, 190, 162, 120,107. Anal.
C21H34N2O2 (C, H, N).
Jvic=6.7 Hz), 2.99 (bd, 1H, H-6eq, Jgem=11.4 Hz),
2.72 (dd, 1H, H-2, J2,3ax=10.6 Hz, J2,3eq=2.6 Hz),
2.08 (m, 1H, H-6ax), 1.98 (s, 3H, NꢀCH3), 1.79–1.51
(m, 6H, H-3, H-4 and H-5), 1.30 (bs, 8H, carbamic
H-2–H-5), 0.88 (t, 3H, carbamic H-6); MS: m/z 318
[M+], 191, 162, 134, 120. Anal. C19H30N2O2 (C, H, N).
6.1.10.7. 1-[(2-n-Hexylcarbamoyloxyphenyl)methyl]pi-
peridine (25a). Compound 25a was obtained as an oil in
46% yield; 1H-NMR (CDCl3): l 7.39 (dd, 1H, H-3%,
6.1.10.3. 2-[(2-n-Hexylcarbamoyloxyphenyl)methyl]-1-
methylpiperidine (14a). Compound 14a was obtained as
1
Jortho=7.3 Hz, Jmeta=2.0 Hz), 7.27 (t, 1H, NH), 7.25–
an oil in 49% yield; H-NMR (CDCl3): l 7.20–7.05 (m,
7.16 (overlapped 2 dt, 2H, H-4% and H-5%, Jortho=7.3
Hz, Jmeta=1.6 Hz), 6.98 (dd, 1H, H-6%, Jortho=7.4 Hz,
4H, phenyl protons), 5.04 (bt, 1H, NH, Jvic=6.7 Hz),
3.24 (q, 2H, carbamic H-1, Jvic=6.7 Hz), 3.13 (dd, 1H,
benzylic CH2, Jgem=12.9 Hz, Jvic=3.5 Hz), 2.83 (bd,
1H, H-6eq, Jgem=11.5 Hz), 2.38 (s, 3H, NꢀCH3), 2.33
(d, 1H, benzylic CH2, Jgem=12.9 Hz), 2.09 (m, 2H, H-2
and H-6ax), 1.70–1.00 (m, 14H, H-3, H-4, H-5 and
carbamic H-2–H-5), 0.88 (bt, 3H, carbamic H-6); MS:
m/z 333 [M++1], 228, 206, 168. Anal. C20H32N2O2 (C,
H, N).
Jmeta=1.6 Hz), 3.35 (s, 2H, benzylic CH2), 2.55 (t, 2H,
carbamic H-1, Jvic=7.3 Hz), 2.31 (m, 4H, H-2 and
H-6), 1.74 (m, 2H, carbamic H-2), 1.48 (m, 4H, H-3
and H-5), 1.42–1.24 (m, 8H, H-4, carbamic H-3–H-5),
0.88 (t, 3H, carbamic H-6); MS: m/z 318 [M+], 261,
247, 190, 162, 134. Anal. C19H30N2O2 (C, H, N).
6.1.10.8. 1-[(3-n-Hexylcarbamoyloxyphenyl)methyl]pi-
peridine (25b). Compound 25b was obtained as an oil in
60% yield; 1H-NMR (CDCl3): l 7.25 (t, 1H, H-5%,
6.1.10.4. 2-[(3-n-Hexylcarbamoyloxyphenyl)methyl]-1-
methylpiperidine (14b). Compound 14b was obtained as
an oil in 75% yield; H-NMR (CDCl3): l 7.22 (t, 1H,
H-5%, Jortho=7.6 Hz), 6.97 (d, 1H, H-4%, Jortho=7.6
Hz), 6.93 (d, 1H, H-6%, Jortho=7.5 Hz), 6.91 (s, 1H,
H-2%), 4.98 (t, 1H, NH, Jvic=6.7 Hz), 3.24 (q, 2H,
1
J
J
ortho=7.6 Hz), 7.11 (overlapped d and s, 2H, H-4%,
ortho=7.6 Hz and H-2%), 6.98 (d, 1H, H-6%, Jortho=7.6
Hz), 5.04 (bt, 1H, NH, Jvic=6.7 Hz), 3.43 (s, 2H,
benzylic CH2), 3.23 (q, 2H, carbamic H-1, Jvic=6.7

104
C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
Hz), 2.33 (m, 4H, H-2 and H-6), 1.54 (m, 6H, H-3, H-5
and carbamic H-2), 1.29 (m, 8H, H-4 and carbamic
H-3–H-5), 0.88 (t, 3H, carbamic H-6) MS: m/z 318
[M+], 261, 247, 216, 190, 162, 134. Anal. C19H30N2O2
(C, H, N).
H-6); MS: m/z 332 [M+], 261, 204, 112. Anal.
C20H32N2O2 (C, H, N).
6.1.11. General procedure for the preparation of
N,N-dimethyl- and N,N-diethylcarbamoyloxyderi6ati6es
9, 15, 21, 26, 31, 36 and 10, 16, 22, 27, 32, 37
6.1.10.9. 1-[1-(2-n-Hexylcarbamoyloxyphenyl)ethyl]pi-
peridine (30a). Compound 30a was obtained as an oil in
55% yield; 1H-NMR (CDCl3): l 7.44 (dd, 1H, H-3%,
To a cooled and stirred solution of each compound
7, 13, 19, 24, 29, 34 (20 mmol) in a mixture of anhy-
drous pyridine (50 mL) and triethylamine (3.5 mL, 25
mmol), N,N-dimethyl- or N,N-diethylcarbamoyl chlo-
ride [52] (25 mmol) was dropwise added. The mixture
was stirred overnight at r.t., then evaporated to dryness
under reduced pressure (maximum bath temperature
40 °C). After addition of water, the mixture was ex-
tracted with EtOAc, the solvent removed and the
residue chromatographed on Al2O3 by eluting with an
1:2 EtOAc–n-C6H14 mixture.
Jortho=6.7 Hz, Jmeta=2.7 Hz), 7.24–7.17 (three par-
tially overlapped t, 3H, NH, H-4% and H-5%, Jortho=6.8
Hz, Jmeta=2.7 Hz), 6.97 (dd, 1H, H-6%, Jortho=6.9 Hz,
Jmeta=2.4 Hz), 3.47 (q, 1H, CHꢀCH3, Jvic=6.7 Hz),
2.56 (t, 2H, carbamic H-1, Jvic=7.3 Hz), 2.28 (m, 4H,
H-2 and H-6), 1.76 (m, 2H, carbamic H-2), 1.49 (m,
4H, H-3 and H-5), 1.35–1.24 (m, 8H, H-4 and car-
bamic H-3–H-5), 1.29 (d, 3H, CHꢀCH3, Jvic=6.7 Hz),
0.90 (t, 3H, carbamic H-6); MS: m/z 317 [M+−15],
302, 190, 149, 121. Anal. C20H32N2O2 (C, H, N).
6.1.11.1. 2-(2-Dimethylcarbamoyloxyphenyl)-1-methyl-
piperidine (9a). Compound 9a was obtained as an oil in
73% yield; 1H-NMR (CDCl3): l 7.51 (t, 1H, H-4%,
6.1.10.10. 1-[1-(3-n-Hexylcarbamoyloxyphenyl)ethyl]pi-
peridine (30b). Compound 30b was obtained as an oil in
68% yield; 1H-NMR (CDCl3): l 7.25 (t, 1H, H-5%,
J
ortho=5.1 Hz), 7.20 (partially overlapped doublets,
2H, H-3% and H-6%, Jortho=5.1 Hz), 7.02 (t, 1H, H-5%,
ortho=5.1 Hz), 3.12 (s, 3H, carbamic NꢀCH3), 3.01
J
ortho=7.6 Hz), 7.07 (d, 1H, H-4%, Jortho=7.6 Hz), 7.04
J
(s, 1H, H-2%), 6.97 (d, 1H, H-6%, Jortho=7.6 Hz), 5.03
(bs, 1H, NH), 3.37 (q, 1H, CHꢀCH3, Jvic=6.9 Hz),
3.22 (q, 2H, carbamic H-1, Jvic=6.9 Hz), 2.32 (m, 4H,
H-2 and H-6), 1.49 (m, 6H, H-3, H-5 and carbamic
H-2), 1.34 (d, 3H, CHꢀCH3, Jvic=6.9 Hz), 1.32 (m, 8H,
H-4 and carbamic H-3–H-5), 0.87 (t, 3H, carbamic
H-6); MS: m/z 332 [M+], 317, 261, 205, 190, 112. Anal.
C20H32N2O2 (C, H, N).
(bs, 5H, carbamic NꢀCH3, H-6eq and H-2), 2.06 (m,
1H, H-6ax), 1.99 (s, 3H, piperidine NꢀCH3), 1.85–1.18
(m, 6H, H-3, H-4 and H-5); MS: m/z 262 [M+], 247,
202, 174, 134. Anal. C15H22N2O2 (C, H, N).
6.1.11.2. 2-(3-Dimethylcarbamoyloxyphenyl)-1-methyl-
piperidine (9b). Compound 9b was obtained as an oil in
45% yield; 1H-NMR (CDCl3): l 7.27 (t, 1H, H-5%,
J
ortho=7.9 Hz), 7.12 (d, 1H, H-4%, Jortho=7.8 Hz), 7.08
6.1.10.11. 1-[2-(2-n-Hexylcarbamoyloxyphenyl)ethyl]pi-
peridine (35a). Compound 35a was obtained as an oil in
63% yield; 1H-NMR (CDCl3): l 7.24–7.11 (m, 4H,
phenyl protons), 5.23 (t, 1H, NH, Jvic=6.1 Hz), 3.23
(q, 2H, carbamic H-1, Jvic=6.1 Hz), 2.75 (m, 2H,
piperidineꢀCH2), 2.51 (m, 2H, benzylic CH2), 2.45 (t,
4H, H-2 and H-6), 1.59 (m, 6H, H-3, H-5 and carbamic
H-2), 1.43 (m, 2H, H-4), 1.30 (m, 6H, carbamic H-3–
H-5), 0.88 (t, 3H, carbamic H-6); MS: m/z 333 [M++
1], 289, 261, 228, 205, 190, 133, 121, 112, 107. Anal.
C20H32N2O2 (C, H, N).
(s, 1H, H-2%), 6.99 (d, 1H, H-6%, Jortho=7.8 Hz), 3.07
(bs, 4H, carbamic NꢀCH3 and H-6eq), 2.99 (s, 3H,
carbamic NꢀCH3), 2.77 (dd, 1H, H-2, J2,3ax=10.4 Hz,
J
2,3eq=2.8 Hz), 2.11 (m, 1H, H-6ax), 2.01 (s, 3H,
piperidine NꢀCH3), 1.88–1.20 (m, 6H, H-3, H-4 and
H-5); MS: m/z 262 [M+], 247, 205, 190, 134. Anal.
C15H22N2O2 (C, H, N).
6.1.11.3.
2-(2-Diethylcarbamoyloxyphenyl)-1-methyl-
piperidine (10a). Compound 10a was obtained as an oil
in 53% yield; H-NMR (CDCl3): l 7.51 (t, 1H, H-4%,
1
6.1.10.12. 1-[2-(3-n-Hexylcarbamoyloxyphenyl)ethyl]pi-
peridine (35b). Compound 35b was obtained as an oil in
72% yield; 1H-NMR (CDCl3): l 7.24 (t, 1H, H-5%,
J
ortho=5.0 Hz), 7.21 (partially overlapped doublets,
2H, H-3% and H-6%, Jortho=5.0 Hz), 7.02 (t, 1H, H-5%,
ortho=5.0 Hz), 3.42 (partially overlapped quartets, 4H,
J
J
ortho=7.8 Hz), 7.00 (d, 2H, H-4%, Jortho=7.8 Hz), 6.95
carbamic NꢀCH2), 3.00 (overlapped dd and m, 2H,
H-2, J2,3ax=10.6 Hz, J2,3eq=2.5 Hz and H-6eq), 2.05
(m, 1H, H-6ax), 1.97 (s, 3H, NꢀCH3), 1.70 (m, 5H,
H-4, H-5 and H-3eq), 1.50 (m, 1H, H-3ax), 1.23 (par-
tially overlapped triplets, 6H, carbamic CH3); MS: m/z
290 [M+], 275, 202, 174, 134. Anal. C17H26N2O2 (C,
H, N).
(s, 1H, H-2%), 6.93 (d, 1H, H-6%, Jortho=7.8 Hz), 5.01 (t,
1H, NH, Jvic=6.1 Hz), 3.25 (q, 2H, carbamic H-1,
J
piperidineꢀCH2), 2.44 (m, 4H, H-2 and H-6), 1.59 (m,
6H, H-3, H-5 and carbamic H-2), 1.43 (m, 2H, H-4),
1.29 (m, 6H, carbamic H-3–H-5), 0.87 (t, 3H, carbamic
vic=6.1 Hz), 2.74 (m, 2H, benzylic CH2), 2.55 (m, 2H,

C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
105
6.1.11.4.
2-(3-Diethylcarbamoyloxyphenyl)-1-methyl-
H-5%, Jortho=7.6 Hz), 6.97 (d, 1H, H-4%, Jortho=7.6
Hz), 6.93 (d, 1H, H-6%, Jortho=7.6 Hz), 6.91 (s, 1H,
H-2%), 3.39 (partially overlapped quartets, 4H, carbamic
NꢀCH2), 3.19 (dd, 1H, benzylic CH2, Jgem=13.1 Hz,
piperidine (10b). Compound 10b was obtained as an oil
in 48% yield; H-NMR (CDCl3): l 7.26 (t, 1H, H-5%,
1
Jortho=7.8 Hz), 7.10 (d, 1H, H-4%, Jortho=7.9 Hz), 7.08
(s, 1H, H-2%), 6.99 (d, 1H, H-6%, Jortho=7.9 Hz), 3.38
(partially overlapped quartets, 4H, carbamic NꢀCH2),
3.01 (bd, 1H, H-6eq, Jgem=10.3 Hz), 2.74 (dd, 1H,
H-2, J2,3ax=10.5 Hz, J2,3eq=2.8 Hz), 2.08 (m, 1H,
H-6ax), 1.99 (s, 3H, NꢀCH3), 1.85–1.25 (m, 6H, H-3,
H-4 and H-5), 1.19 (partially overlapped triplets, 6H,
carbamic CH3); MS: m/z 290 [M+], 275, 233, 190, 162.
Anal. C17H26N2O2 (C, H, N).
J
vic=3.7 Hz), 2.84 (bd, 1H, H-6eq, Jgem=11.7 Hz),
2.39 (t, 3H, NꢀCH3), 2.33 (dd, 1H, benzylic CH2,
gem=13.1 Hz, Jvic=3.4 Hz), 2.11 (m, 2H, H-2 and
J
H-6ax), 1.80–1.10 (m, 6H, H-3, H-5 and H-4), 1.22
(partially overlapped triplets, 6H, carbamic CH3); MS:
m/z 304 [M+], 277, 220, 192, 107. Anal. C18H28N2O2
(C, H, N).
6.1.11.9. 2-[2-(2-Dimethylcarbamoyloxyphenyl)ethyl]-1-
methylpiperidine (21a). Compound 21a was obtained as
an oil in 50% yield; 1H-NMR (CDCl3): l 7.21–7.02
(overlapped d and t, 4H, phenyl protons), 3.11 (s, 3H,
carbamic NꢀCH3), 3.00 (s, 3H, carbamic NꢀCH3), 2.83
(bd, 1H, H-6eq, Jgem=10.4 Hz), 2.56 (m, 2H, benzylic
CH2), 2.24 (s, 3H, piperidine NꢀCH3), 2.05 (m, 1H,
H-6ax), 1.95–1.10 (m, 9H, piperidineꢀCH2, H-2, H-3,
H-4 and H-5); MS: m/z 291 [M++1], 275, 261, 218,
162, 146, 124, 112, 107. Anal. C17H26N2O2 (C, H, N).
6.1.11.5. 2-[(2-Dimethylcarbamoyloxyphenyl)methyl]-1-
methylpiperidine (15a). Compound 15a was obtained as
an oil in 89% yield; H-NMR (CDCl3): l 7.20–7.02 (m,
1
3H, H-3%, H-4% and H-5%), 7.03 (d, 1H, H-6%, Jortho=7.5
Hz), 3.15 (dd, 1H, benzylic CH2, Jgem=13.1 Hz, Jvic
=
3.7 Hz), 3.12 (s, 3H, carbamic NꢀCH3), 2.99 (s, 3H,
carbamic NꢀCH3), 2.85 (bd, 1H, H-6eq, Jgem=11.5
Hz), 2.38 (s, 3H, piperidine NꢀCH3), 2.33 (d, 1H,
benzylic CH2, Jgem=13.1 Hz), 2.10 (m, 2H, H-2 and
H-6ax), 1.80–1.10 (m, 6H, H-3, H-4 and H-5); MS: m/z
277 [M++1], 275 [M+−1], 230, 186, 132, 107. Anal.
C16H24N2O2 (C, H, N).
6.1.11.10. 2-[2-(3-Dimethylcarbamoyloxyphenyl)ethyl]-
1-methylpiperidine (21b). Compound 21b was obtained
as an oil in 55% yield; 1H-NMR (CDCl3): l 7.23 (t, 1H,
H-5%, Jortho=7.6 Hz), 7.00 (d, 1H, H-4%, Jortho=7.6
Hz), 6.93 (s, 1H, H-2%), 6.90 (d, 1H, H-6%, Jortho=7.6
Hz), 3.08 (s, 3H, carbamic NꢀCH3), 2.99 (s, 3H, car-
bamic NꢀCH3), 2.83 (bd, 1H, H-6eq, Jgem=11.5 Hz),
2.62 (m, 2H, benzylic CH2), 2.25 (s, 3H, piperidine
NꢀCH3), 2.05 (m, 1H, H-6ax), 1.95–1.10 (m, 9H, pipe-
ridineꢀCH2, H-2, H-3, H-4 and H-5); MS: m/z 291
[M++1], 290 [M+], 289 [M+−1], 273, 247, 218, 178,
132, 126, 111. Anal. C17H26N2O2 (C, H, N).
6.1.11.6. 2-[(3-Dimethylcarbamoyloxyphenyl)methyl]-1-
methylpiperidine (15b). Compound 15b was obtained as
1
an oil in 62% yield; H-NMR (CDCl3): l 7.19 (t, 1H,
H-5%, Jortho=7.9 Hz), 6.93 (d, 1H, H-4%, Jortho=7.9
Hz), 6.88 (d, 1H, H-6%, Jortho=8.0 Hz), 6.86 (s, 1H,
H-2%), 3.13 (dd, 1H, benzylic CH2, Jgem=13.1 Hz,
Jvic=3.9 Hz), 3.03 (s, 3H, carbamic NꢀCH3), 2.94 (s,
3H, carbamic NꢀCH3), 2.78 (bd, 1H, H-6eq, Jgem=11.7
Hz), 2.33 (s, 3H, piperidine NꢀCH3), 2.30 (d, 1H,
benzylic CH2, Jgem=13.1 Hz), 2.09 (m, 2H, H-2 and
H-6ax), 1.53 (m, 4H, H-5, H-3eq and H-4eq), 1.10 (m,
2H, H-3ax and H-4ax); MS: m/z 275 [M+−1], 233,
188, 160, 147, 133, 111. Anal. C16H24N2O2 (C, H, N).
6.1.11.11. 2-[2-(2-Diethylcarbamoyloxyphenyl)ethyl]-1-
methylpiperidine (22a). Compound 22a was obtained as
an oil in 60% yield; 1H-NMR (CDCl3): l 7.22–7.01
(overlapped d and t, 4H, phenyl protons), 3.40 (par-
tially overlapped quartets, 4H, carbamic NꢀCH2, Jvic
=
6.1.11.7.
2-[(2-Diethylcarbamoyloxyphenyl)methyl]-1-
7.3 Hz), 2.80 (bd, 1H, H-6eq, Jgem=11.5 Hz), 2.55 (m,
2H, benzylic CH2), 2.23 (s, 3H, NꢀCH3), 2.05 (m, 1H,
H-6ax), 1.90–1.20 (m, 9H, piperidineꢀCH2, H-2, H-3,
H-4 and H-5), 1.21 (partially overlapped triplets, 6H,
carbamic CH3, Jvic=7.3 Hz); MS: m/z 319 [M++1],
303, 219, 200, 190, 112. Anal. C19H30N2O2 (C, H, N).
methylpiperidine (16a). Compound 16a was obtained as
an oil in 72% yield; H-NMR (CDCl3): l 7.16 (over-
1
lapped d and t, 4H, phenyl protons), 3.42 (partially
overlapped quartets, 4H, carbamic NꢀCH2), 3.18 (dd,
1H, benzylic CH2, Jgem=13.1 Hz, Jvic=3.9 Hz), 2.85
(bd, 1H, H-6eq, Jgem=11.7 Hz), 2.38 (s, 3H, NꢀCH3),
2.35 (d, 1H, benzylic CH2, Jgem=13.1 Hz), 2.13 (m,
2H, H-2 and H-6ax), 1.70–1.10 (m, 6H, H-3, H-5 and
H-4), 1.24 (partially overlapped triplets, 6H, carbamic
CH3); MS: m/z 304 [M+], 289, 232, 190, 168, 107. Anal.
C18H28N2O2 (C, H, N).
6.1.11.12. 2-[2-(3-Diethylcarbamoyloxyphenyl)ethyl]-1-
methylpiperidine (22b). Compound 22b was obtained as
1
an oil in 49% yield; H-NMR (CDCl3): l 7.19 (t, 1H,
H-5%, Jortho=7.1 Hz), 6.99 (d, 1H, H-4%, Jortho=7.1
Hz), 6.94 (s, 1H, H-2%), 6.91 (d, 1H, H-6%, Jortho=7.1
Hz), 3.39 (partially overlapped quartets, 4H, carbamic
NꢀCH2, Jvic=7.6 Hz), 2.85 (bd, 1H, H-6eq, Jgem=11.4
Hz), 2.62 (m, 2H, benzylic CH2), 2.27 (s, 3H, Nꢀ
CH3), 2.08 (m, 1H, H-6ax), 2.00–1.20 (m, 9H,
6.1.11.8.
2-[(3-Diethylcarbamoyloxyphenyl)methyl]-1-
methylpiperidine (16b). Compound 16b was obtained as
an oil in 67% yield; H-NMR (CDCl3): l 7.24 (t, 1H,
1

106
C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
piperidineꢀCH2, H-2, H-3, H-4 and H-5), 1.20 (partially
overlapped triplets, 6H, carbamic CH3, Jvic=7.6 Hz);
MS: m/z 318 [M+], 290, 258, 204, 174, 126. Anal.
C19H30N2O2 (C, H, N).
overlapped dt, 2H, H-4% and H-5%, Jortho=7.8 Hz,
J
J
meta=2.2 Hz), 7.04 (dd, 1H, H-6%, Jortho=7.8 Hz,
meta=2.2 Hz), 3.57 (q, 1H, CHꢀCH3, Jvic=6.7 Hz),
3.10 (s, 3H, carbamic NꢀCH3), 2.99 (s, 3H, carbamic
NꢀCH3), 2.32 (m, 4H, H-2 and H-6), 1.47 (m, 4H, H-3
and H-5), 1.37 (m, 2H, H-4), 1.33 (d, 3H, CHꢀCH3,
6.1.11.13. 1-[(2-Dimethylcarbamoyloxyphenyl)methyl]pi-
peridine (26a). Compound 26a was obtained as an oil in
90% yield; 1H-NMR (CDCl3): l 7.37 (dd, 1H, H-3%,
J
vic=6.7 Hz); MS: m/z 276 [M+], 261, 233, 204, 188.
Anal. C16H24N2O2 (C, H, N).
Jortho=7.3 Hz, Jmeta=1.9 Hz), 7.24 (dt, 1H, H-5%,
Jortho=7.3 Hz, Jmeta=2.1 Hz), 7.13 (dt, 1H, H-4%,
Jortho=7.3 Hz, Jmeta=1.5 Hz), 7.07 (dd, 1H, H-6%,
Jortho=7.3 Hz, Jmeta=1.5 Hz), 3.41 (s, 2H, benzylic
6.1.11.18. 1-[1-(3-Dimethylcarbamoyloxyphenyl)ethyl]-
piperidine (31b). Compound 31b was obtained as an oil
in 64% yield; H-NMR (CDCl3): l 7.26 (t, 1H, H-5%,
1
CH2), 3.10 (s, 3H, carbamic NꢀCH3), 2.99 (s, 3H,
carbamic NꢀCH3), 2.33 (t, 4H, H-2 and H-6), 1.52 (m,
4H, H-3 and H-5), 1.43 (m, 2H, H-4); MS: m/z 262
[M+], 233, 219, 190, 174, 144. Anal. C15H22N2O2 (C, H,
N).
J
ortho=7.7 Hz), 7.05 (d, 1H, H-4%, Jortho=7.7 Hz), 7.03
(s, 1H, H-2%), 6.97 (d, 1H, H-6%, Jortho=7.8 Hz), 3.37 (q,
1H, CHꢀCH3, Jvic=6.7 Hz), 3.08 (s, 3H, carbamic
NꢀCH3), 2.99 (s, 3H, carbamic NꢀCH3), 2.35 (m, 4H,
H-2 and H-6), 1.52 (m, 4H, H-3 and H-5), 1.40 (m, 2H,
H-4), 1.33 (d, 3H, CHꢀCH3, Jvic=6.7 Hz); MS: m/z
276 [M+], 261, 193, 178, 134, 112. Anal. C16H24N2O2
(C, H, N).
6.1.11.14. 1-[(3-Dimethylcarbamoyloxyphenyl)methyl]pi-
peridine (26b). Compound 26b was obtained as an oil in
80% yield; 1H-NMR (CDCl3): l 7.26 (t, 1H, H-5%,
J
ortho=7.6 Hz), 7.11 (d, 1H, H-4%, Jortho=7.6 Hz), 7.06
6.1.11.19. 1-[1-(2-Diethylcarbamoyloxyphenyl)ethyl]pi-
peridine (32a). Compound 32a was obtained as an oil in
54% yield; 1H-NMR (CDCl3): l 7.43 (dd, 1H, H-3%,
(s, 1H, H-2%), 6.97 (d, 1H, H-6%, Jortho=7.6 Hz), 3.43 (s,
2H, benzylic CH2), 3.07 (s, 3H, carbamic NꢀCH3), 2.99
(s, 3H, carbamic NꢀCH3), 2.35 (t, 4H, H-2 and H-6),
1.54 (t, 4H, H-3 and H-5), 1.40 (m, 2H, H-4); MS: m/z
262 [M+], 261, 233, 219, 178, 135, 107. Anal.
C15H22N2O2 (C, H, N).
Jortho=6.7 Hz, Jmeta=2.7 Hz), 7.17 (two partially over-
lapped dd, 2H, H-4% and H-5%, Jortho=6.8 Hz, Jmeta
=
2.7 Hz), 7.03 (dd, 1H, H-6%, Jortho=6.9 Hz, Jmeta=2.6
Hz), 3.56 (q, 1H, CHꢀCH3, Jvic=6.7 Hz), 3.39 (m, 4H,
carbamic NꢀCH2), 2.33 (m, 4H, H-2 and H-6), 1.50 (m,
4H, H-3 and H-5), 1.28 (m, 11H, H-4, CHꢀCH3 and
carbamic CH3); MS: m/z 304 [M+], 289, 261, 204, 188,
112. Anal. C18H28N2O2 (C, H, N).
6.1.11.15. 1-[(2-Diethylcarbamoyloxyphenyl)methyl]pi-
peridine (27a). Compound 27a was obtained as an oil in
86% yield; 1H-NMR (CDCl3): l 7.39 (dd, 1H, H-3%,
J
J
J
J
ortho=7.2 Hz, Jmeta=1.9 Hz), 7.23 (dt, 1H, H-5%,
ortho=7.3 Hz, Jmeta=2.0 Hz), 7.13 (dt, 1H, H-4%,
ortho=7.3 Hz, Jmeta=1.7 Hz), 7.05 (dd, 1H, H-6%,
ortho=7.3 Hz, Jmeta=1.7 Hz), 3.41 (m, 6H, benzylic
6.1.11.20. 1-[1-(3-Diethylcarbamoyloxyphenyl)ethyl]pi-
peridine (32b). Compound 32b was obtained as an oil in
68% yield; 1H-NMR (CDCl3): l 7.26 (t, 1H, H-5%,
CH2 and carbamic NꢀCH2), 2.33 (t, 4H, H-2 and H-6,
J
ortho=7.6 Hz), 7.22 (d, 1H, H-4%, Jortho=7.6 Hz), 7.03
J
vic=4.4 Hz), 1.52 (m, 4H, H-3 and H-5), 1.40 (m, 2H,
H-4), 1.22 (m, 6H, carbamic CH3); MS: m/z 290 [M+],
(s, 1H, H-2%), 6.95 (d, 1H, H-6%, Jortho=7.6 Hz), 3.38
(m, 5H, CHꢀCH3 and carbamic NꢀCH2), 2.34 (m, 4H,
H-2 and H-6), 1.53 (m, 4H, H-3 and H-5), 1.33 (m, 5H,
H-4 and CHꢀCH3), 1.24 (m, 6H, carbamic CH3), MS:
m/z 304 [M+], 289, 248, 220, 185, 154. Anal.
C18H28N2O2 (C, H, N).
206, 190, 174, 146. Anal. C17H26N2O2 (C, H, N).
6.1.11.16. 1-[(3-Diethylcarbamoyloxyphenyl)methyl]pi-
peridine (27b). Compound 27b was obtained as an oil in
60% yield; 1H-NMR (CDCl3): l 7.28 (t, 1H, H-5%,
J
J
ortho=7.8 Hz), 7.09 (overlapped d and s, 2H, H-4%,
ortho=7.6 Hz and H-2%), 7.00 (d, 1H, H-6%, Jortho=7.9
6.1.11.21. 1-[2-(2-Dimethylcarbamoyloxyphenyl)ethyl]-
piperidine (36a). Compound 36a was obtained as an oil
in 74% yield;1H-NMR (CDCl3): l 7.20 (overlapped t
and d, 2H, H-4% and H-6%, Jortho=7.3 Hz), 7.16 (d, 1H,
H-3%, Jortho=7.3 Hz), 7.06 (t, 1H, H-5%, Jortho=7.2 Hz),
3.11 (s, 3H, carbamic NꢀCH3), 3.00 (s, 3H, carbamic
NꢀCH3), 2.76 (m, 2H, piperidineꢀCH2), 2.53 (m, 2H,
benzylic CH2), 2.46 (m, 4H, H-2 and H-6), 1.60 (m, 4H,
H-3 and H-5), 1.44 (m, 2H, H-4); MS: m/z 277 [M++
1], 276 [M+], 230, 192, 132, 118. Anal. C16H24N2O2 (C,
H, N).
Hz), 3.44 (s, 2H, benzylic CH2), 3.39 (m, 4H, carbamic
NꢀCH2), 2.35 (t, 4H, H-2 and H-6, Jvic=5.3 Hz), 1.58
(m, 4H, H-3 and H-5), 1.41 (m, 2H, H-4), 1.23 (m, 6H,
carbamic CH3); MS: m/z 290 [M+], 263, 234, 207, 192.
Anal. C17H26N2O2 (C, H, N).
6.1.11.17. 1-[1-(2-Dimethylcarbamoyloxyphenyl)ethyl]-
piperidine (31a). Compound 31a was obtained as an oil
in 59% yield; H-NMR (CDCl3): l 7.41 (dd, 1H, H-3%,
1
J
ortho=7.9 Hz, Jmeta=2.5 Hz), 7.24–7.14 (two partially

C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
107
6.1.11.22. 1-[2-(3-Dimethylcarbamoyloxyphenyl)ethyl]-
piperidine (36b). Compound 36b was obtained as an oil
in 70% yield; H-NMR (CDCl3): l 7.24 (t, 1H, H-5%,
nmoles of AcThCh and BuThCh per hydrolyzed min−1
g−1 of tissue and the IC50 values were determined
graphically from log concentration–inhibition curve
from 20 to 80%. Correlation coefficients were in all
cases greater that 0.95.
1
Jortho,=7.8 Hz), 7.00 (d, 1H, H-4%, Jortho=7.8 Hz), 6.93
(overlapped s and d, 2H, H-2% and H-6%), 3.08 (s, 3H,
carbamic NꢀCH3), 2.99 (s, 3H, carbamic NꢀCH3), 2.77
(m, 2H, benzylic CH2), 2.53 (m, 2H, piperidineꢀCH2),
2.43 (t, 4H, H-2 and H-6), 1.59 (m, 4H, H-3 and H-5),
1.43 (m, 2H, H-4), MS: m/z 277 [M++1], 276 [M+],
275 [M+−1], 247, 204, 174, 132, 121. Anal.
C16H24N2O2 (C, H, N).
6.2.2. In 6i6o inhibition of AChE from different brain
regions
White Swiss male mice (20–25 g body weight from
Charles River Calco, Como, Italy), were housed on
polypropylene cages at 22 °C and 50% humidity, with
lighting from 7:00 to 22:00 h and were given standard
chow and drinking water ad libitum. Mice were killed
by decapitation, the cerebral cortex, hippocampus and
striatum were dissected [53] and were treated separately
according to the procedure described for total brain
AChE.
6.1.11.23.
1-[2-(2-Diethylcarbamoyloxyphenyl)ethyl]-
piperidine (37a). Compound 37a was obtained as an oil
1
in 58% yield; H-NMR (CDCl3): l 7.24–7.04 (m, 4H,
phenyl protons), 3.43 (m, 4H, carbamic NꢀCH2), 2.75
(m, 2H, benzylic CH2), 2.51 (m, 2H, piperidineꢀCH2),
2.43 (t, 4H, H-2 and H-6), 1.59 (m, 4H, H-3 and H-5),
1.42 (m, 2H, H-4), 1.24 (m, 6H, carbamic CH3); MS:
m/z 304 [M+], 277, 232, 220, 192, 130, 112. Anal.
C18H28N2O2 (C, H, N).
6.2.3. In 6itro inhibition of AChE G1 and G4
molecular forms
AChE was extracted from control mouse brains fol-
lowing the same procedure already described for the in
vitro assay. The molecular forms of AChE were sepa-
rated by ultracentrifugation in sucrose gradient as pre-
viously described [54]. Briefly, 200 mL of a fresh 100,000
g supernatant were layered on 4.8 mL of 5–20% linear
sucrose gradient and centrifuged at 38,000 rpm in a
SW55B rotor in a Centrikon T-1055 (Kontron) ultra-
centrifuge for 18 h. In some experiments, catalase (11.3
S) and bovine serum albumin (4.3 S) were used as
sedimentation standards. Ten drops fractions were col-
lected from the bottom of each centrifuge tube and
AChE determined in each fraction. The distribution
profiles of individual molecular forms were evaluated.
In vitro inhibition of AChE forms G4 and G1 was
determined by Ellman’s spectrophotometric method
using acetylthiocholine (AcThCh) as already described
for the in vitro assay. Results are expressed as nmol of
AcThCh per hydrolyzed min−1 g−1 of tissue and the
IC50 values were determined graphically from log con-
centration–inhibition curve from 20 to 80%. Correla-
tion coefficients were in all cases greater that 0.95.
6.1.11.24.
1-[2-(3-Diethylcarbamoyloxyphenyl)ethyl]-
piperidine (37b). Compound 37b was obtained as an oil
1
in 58% yield; H-NMR (CDCl3): l 7.24 (t, 1H, H-5%,
Jortho=7.4 Hz), 7.02 (d, 1H, H-4%, Jortho=7.5 Hz), 6.95
(s, 1H, H-2%), 6.94 (d, 1H, H-6%, Jortho=7.4 Hz), 3.39
(m, 4H, carbamic NꢀCH2), 2.79 (m, 2H, benzylic CH2),
2.55 (m, 2H, piperidineꢀCH2), 2.44 (m, 4H, H-2 and
H-6), 1.60 (m, 4H, H-3 and H-5), 1.44 (m, 2H, H-4),
1.20 (m, 6H, carbamic CH3); MS: m/z 304 [M+], 277,
220, 192, 112. Anal. C18H28N2O2 (C, H, N).
6.2. Pharmacology
6.2.1. In 6itro assays on total brain AChE and on
blood BuChE
Sprague–Dawley male rats (200–250 g body weight
from Charles River Calco, Como, Italy), were housed
on polypropylene cages at 22 °C and 50% humidity,
with lighting from 7:00 to 22:00 h and were given
standard chow and drinking water ad libitum. Rats
were killed by decapitation, the whole brain minus
cerebellum rapidly removed, weighted and homoge-
nized in 10 volumes of cold 0.038 M Tris–HCl buffer,
pH 8.5, containing 0.25% Triton X-100 using a Braun
Potter S for 2 min. The homogenates were then cen-
trifuged at 100,000×g for 1 h in a Centrikon T-1055
(Kontron) ultracentrifuge, and supernatant used as
AChE source divided into aliquots and stored at
−20 °C. The same animals supplied blood as butyryl-
cholinesterase (BuChE) source.
6.2.4. Acute toxicity study
Acute toxicity was evaluated in male mice (20–25 g
body weight from Charles River Calco, Como, Italy)
which were housed on polypropylene cages at 22 °C
and 50% humidity, with lighting from 7:00 to 22:00 h
and were given standard chow and drinking water ad
libitum.
The method used was a stepwise procedure, similar
to OCSE guideline no. 423, and is not intended to allow
the calculation of a precise LD50 value, but to afford
the determination of a range of exposure where lethal-
ity is expected. According to our National rules on
behalf of laboratory animals, as little as possible num-
ber of animals was used to give acceptable data.
Inhibition of AChE and BuChE was determined by
Ellman’s spectrophotometric method using acetylthio-
choline (AcThCh) and butyrylthiocholine (BuThCh) as
already described [37,38]. Results are expressed as

108
C. Mustazza et al. / European Journal of Medicinal Chemistry 37 (2002) 91–109
Moreover, taking into account that anticholinesterase
compounds are known to cause signs of severe and
enduring distress to animals, the maximum dose used in
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Acknowledgements
The authors wish to thank Dr L. Turchetto for mass
spectra and Mr R. Lecce for mycroanalyses and are
grateful to Novartis for kindly providing a Rivastig-
mine sample.

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