Lipase-catalysed synthesis of new acetylcholinesterase inhibitors: N- benzylpiperidine aminoacid derivatives

Article abstract of DOI:10.1016/S0968-0896(00)00020-1

New acetylcholinesterase inhibitors were synthetized via a lipase- mediated regioselective amidation using Candida antarctica lipase B as a biocatalyst in the key step. The new compounds have two different structural fragments: a N-benzylpiperidine moiety to anchor the enzyme active site and a dicarboxylic aminoacid to act as a biological carrier. Some analogues of N- benzylpiperazine were also synthesised and studied but they did not display AChE inhibitor activity. A preliminary structure-activity relationship study was performed employing some computational techniques as similarity indices and electrostatic potential maps. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00020-1

Bioorganic & Medicinal Chemistry 8 (2000) 731±738
Lipase-Catalysed Synthesis of New Acetylcholinesterase
Inhibitors: N-Benzylpiperidine Aminoacid Derivatives
Ana Martõnez,* Cristina Lanot, Concepcion Perez, Ana Castro, Paloma Lopez-Serrano
and Santiago Conde
Â
Instituto de Quõmica Medica (C.S.I.C.), Juan de la Cierva, 3, 28006 Madrid, Spain
Â
Received 22 April 1999; accepted 18 October 1999
AbstractÐNew acetylcholinesterase inhibitors were synthetized via a lipase-mediated regioselective amidation using Candida
antarctica lipase B as a biocatalyst in the key step. The new compounds have two di€erent structural fragments: a N-benzylpiperidine
moiety to anchor the enzyme active site and a dicarboxylic aminoacid to act as a biological carrier. Some analogues of N-benzyl-
piperazine were also synthesised and studied but they did not display AChE inhibitor activity. A preliminary structure±activity
relationship study was performed employing some computational techniques as similarity indices and electrostatic potential maps.
# 2000 Published by Elsevier Science Ltd. All rights reserved.
Introduction
and studied. The new designed compounds have two
di€erent structural fragments, coupled via regioselective
lipase-catalysed amidation: the N-benzylpiperidine
moiety present in donepezil7 to anchor in the AChE
active site and a dicarboxylic amino acid which acts as a
biological carrier. Two groups of compounds have been
studied: the a-amides of S-glutamic8 (structure I) and
the o-amides of S-a-aminoadipic (n=2) and R,S-a-
aminopimelic (n=3) acid derivatives (structure II).
Acetyl and carbobenzoxy (Cbz) were the N-protecting
groups used. The dicarboxylic amino acid derivatives
were selected because they have a free functional group
in their structure, which could be used for subsequent
transformations in hybrid compounds a€ording a dual
biological activity, i.e. by linkage with an m1 agonist.
The cholinergic hypothesis postulates the de®cit of cho-
linergic functions in the brain as one of the main causes
of memory impairments in Alzheimer's disease (AD)
patients.1 As a consequence, potentiation of central
cholinergic action has become an e€ective approach for
the palliative treatment of mild to moderate cases of
AD.2 One of the strategies to enhance cholinergic neu-
rotransmission is the inhibition of acetylcholinesterase
(AChE),3 the enzyme responsible for the metabolic
breakdown of acetylcholine (ACh). In fact, this
mechanism is the one that allowed the development of
drugs presently approved for the treatment of AD (i.e.
tacrine, donepezil, Fig. 1).4 However, there are many
other di€erent mechanisms acting on the neurodege-
nerative process. Many research e€orts are currently
focused on developing hybrid-compounds with dual
mechanism of action that potentiate the cognitive e€ect
and decrease side e€ects.5
Results and Discussion
Synthesis
Continuing with our work on AChE inhibitors,6 here
we report the synthesis, biological evaluation and
structure±activity relationship of N-benzylpiperidine
amino acid derivatives (Fig. 2; X=CH, R0=H). For
comparative purposes, some piperazine analogues
(X=N, R0=H, Cl, NO2 and Ph) were also synthetized
Candida antarctica lipase B (CAL from here on) cata-
lysed amidation of diesters of N-protected-S-glutamic
acid regioselectively a€ords a-monoamides.9 Amidation
with 4-(2-aminoethyl)-N-benzylpiperidine (obtained by
reduction of the corresponding nitrile)10 as nucleophile
yielded 1 and 2 (Scheme 1). Piperazine analogues 4±7
were obtained by using commercially available 1-(2-
aminoethyl)piperazine as the nucleophile of a doubly
regioselective, acyl donor and nucleophile, amidation
followed by the N-alkylation of the a-monoamide 3
*Corresponding author. Tel.: +34-91-562-2900; fax: +34-91-564-
4853.
0968-0896/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00020-1

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A. Martõnez et al. / Bioorg. Med. Chem. 8 (2000) 731±738
732
Structural elucidation
The structures of all new compounds were elucidated
according to their analytical and spectroscopic data (1H
and 13C NMR) which are collected in the experimental
section and in Tables 1, 2 and 3.
The monoamidation position was determined using the
chemical shift displacement rule previously determined
by our group.12 It was observed that amide substitution
at position a produced an up®eld shift of the a-CH
proton of about 0.2 ppm, while no appreciable change
was observed for the g-CH2 resonances (Fig. 4). In
contrast, the g-monoamide derivatives show their g-
CH2 protons more shielded than the corresponding
diester (Ádꢀ0.2 ppm). Di€erences in the 13C NMR
spectra of the regioisomeric monoamide derivatives
were also found, as are depicted in Figure 4.
Figure 1.
Concerning the new synthetized glutamic derivatives
(compounds 1±7), higher chemical shift di€erences in
the a-CH than in o-CH2 were observed (Table 1),
pointing to a regioselective a-amidation. In addition,
the monoamidation site was con®rmed unequivocally
for compounds 1 and 3 by bidimensional experiments
such as HMQC for one bond correlation or HMBC
sequences for long-distance couplings. Thus, a-methyl-
ene protons (d=4.21 and 4.18 ppm respectively) corre-
lated exclusively with the a-CO (d=171.1 and 170.9
ppm), while o-CO (d=173.4 and 173.3 ppm) correlated
with the methylenic protons of the ethyl group (d=4.12
and 4.07 ppm). On the other hand, in aminoadipic
and aminopimelic ester derivatives (compounds 8±15),
higher chemical shift di€erences, both in 1H and 13C, on
the side chain methylene moiety were observed (Table 1)
which evidence a regioselective monoamidation on the
side chain.
Figure 2.
with a mixture of the selected benzyl halide and tri-
ethylamine (TEA).
The enzymatic amidation takes regioselectively place on
the o-ester group when the side chain is longer than that
of the glutamic acid.11 Thus, when the reactions,
showed in Scheme 1, were applied to S-a-aminoadipic
and R,S-a-aminopimelic derivatives, the corresponding
d- and E-monoamides were regioselectively obtained: N-
benzylpiperidines 8, 9, 12 and 13 and, via N-benzylation
of 10 and 14, N-benzylpiperazines 11 and 15 (Fig. 3).
Additionally, derivatives 3, 10 and 14, in which 4-(2-
aminoethyl)-piperazine was used as nucleophile in the
Scheme 1.

Â
A. Martõnez et al. / Bioorg. Med. Chem. 8 (2000) 731±738
733
Table 1. Measured di€erences in the chemical shifts between lipase-
catalyzed synthetised monoamide derivatives and the corresponding
diester and inhibitory activity on human erythrocytes isolated AChE
of the monoamide aminoacids derivatives 1±15
1H NMR
(d, ppm)a
13C NMR
(d, ppm)a
AChE inhb. IC50
b
Compound a-CH o-CH2 a-CH o-CH2
(mM)
1
2
3
4
0.21
0.15
0.18
Ð
0.02
0.04
0.03
Ð
0.8
1.3
1.1
Ð
0.2
0.2
0.4
Ð
7.1‹0.4
83‹5
>100
86‹2
5
6
7
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
>100
>100
>100
8
9
0.06
0.05
0.02
Ð
0.04
0.01
0.01
Ð
0.16
0.15
0.14
Ð
0.14
0.13
0.15
0.5
0.2
0.1
Ð
0.0
0.0
0.0
Ð
2.8
2.1
2.1
Ð
2.4
2.3
1.7
0.20‹0.02
0.52‹0.01
>100
2.5‹0.4
0.60‹0.02
1.7‹0.3
>100
10
11
12
13
14
15
Ð
Ð
3.6‹0.02
aÁd=ddiester±damide
.
bAll values are expressed as mean‹standard error of the mean of at
least four experiments. IC50: 50% inhibitory concentration of acetyl-
cholinesterase activity (mM). References values: tacrine, IC50
=
0.13‹0.02, donepezil,10 IC50=0.04‹0.01.
Figure 3.
cases were more potent than the glutamic ones (com-
pounds 8±15 versus 1±7). This fact could be related to
the monoamidation position more than to the length of
the aminoacid side chain.
enzymatic reaction, showed a broad triplet at dꢀ6.5
ppm interchangeable with deuterium dioxide, which
indicates a regioselective amidation on the primary ver-
sus secondary starting material amine group.
It is generally thought that non-covalent forces dom-
inate the receptor drug interactions and these forces can
be described in terms of steric and electrostatic e€ects.
In an attempt to relate these e€ects with the biological
activity found in this work, some molecular modeling
studies have been additionally performed.
Biological results
The in vitro inhibition of AChE for the new synthetized
aminoacid monoamide derivatives 1±15 was determined
by the method of Ellman et al.13 using tacrine as refer-
ence. The results are presented in Table 1. They show
that some of the new compounds (nos. 8, 9 and 12)
inhibit the enzyme with IC50 values comprised between
0.2 and 0.6 mM.
Similarity indices measures are one of the most recent
techniques succesfully employed for this purpose.15
Here, compounds 8±11 have been selected for this
study. Molecular similarity indices were calculated using
donepezil as reference. Due to the quiral centre present
in donepezil both enantiomers were taken into account.
The best correlation was found with (S)-isomer. Simi-
larity indices for shape and electrostatic potential alone
did not reveal any correspondence with biological
activity, however, the combined index, based on an
equal contribution of shape and charge, showed a good
correlation with enzymatic inhibition ®nding similarity
around 35% for inhibitors 8, 9 and 11, in contrast to the
lower similarity found (24%) for the inactive derivative
10. These results could explain the indispensable
requirement of the N-benzyl moiety for AChE inhibition.
Some preliminary structure±activity relationships could
be derived. Firstly, all the compounds with a N-benzyl
moiety showed inhibition of AChE. However, substitu-
tion on its aromatic ring leads to completely inactive
compounds (nos. 5±7). The N-benzylpiperidine deriva-
tives inhibit more eciently the AChE than the N-ben-
zylpiperazine ones (compounds 1±2, 8±9, 12±13 versus
4, 11 and 15). Lipophilicity in the N-blocking group of
the aminoacid slightly increases the enzymatic inhibition
since compounds with the benzyloxycarbonyl group
were more active than the ones with the acetyl group
(compounds 1, 8, 12 versus 2, 9, 13 respectively). One
possible explanation for this result could be that the
gorge leading to the active site of AChE is lined with
aromatic residues,14 therefore, hydrophobic interactions
could stabilize compounds with a benzyloxycarbonyl
moiety through the aromatic channel. Finally, concern-
ing the aminoacid, the best results were obtained for the
aminoadipic and aminopimelic derivatives which in all
Moreover, the comparative study of the electrostatic
potential surfaces of the most active compound, the g-
monoamide 8, and (S)-donepezil showed a common
pattern for the electrostatic distribution (Fig. 5), with
the three previously established pharmacophoric areas
of charge in common.16 These data could explain the

Â
A. Martõnez et al. / Bioorg. Med. Chem. 8 (2000) 731±738
734
Figure 4.
good AChE inhibition found in the N-benzylpiperidine
aminoacid o-derivatives here synthetized.
using silica gel (E. Merck, Grade 60, particle size 0.040±
0.063 mm, 230±240 mesh ASTM) with the indicated
solvent as eluent. H NMR spectra were obtained on
1
The synthetic methodology here employed together with
the theoretical models proposed will be used for further
development of o-monoamide aminoacid derivatives as
AChE inhibitors. The free a-ethoxycarbonyl group will
be used for subsequent transformation in hybrid com-
pounds, i.e. by linkage with an m1 agonist or a radical
scavenger, searching a dual biological activity which
could improve the therapeutic pro®le of anti-Alzheimer's
drugs.
Varian XL-300 and Gemini-200 spectrometers working
at 300 and 200 MHz respectively. Typical spectral
parameters were: spectral width 10 ppm, pulse width
9 ms (57ꢁ), data size 32 K. 13C NMR experiments were
carried out on the Varian Gemini-200 spectrometer
operating at 50 MHz. The acquisition parameters were:
spectral width 16 kHz, acquisition time 0.99 s, pulse
width 9 ms (57ꢁ), data size 32 K. Chemical shifts are
reported in d values (ppm) relative to internal Me4Si. H
and C atoms are numbered as in Scheme 1. Analytical
HPLC was performed on a Beckman chromatograph
using a DeltaPak C18 column (3.9Â150 mm, 5 mm),
using di€erent rates of acetonitrile (CH3CN)-H2O (with
0.5 mL/L of ti¯uoroacetic acid) as eluent and an UV
detector. Elemental analyses were performed by the
analytical department at C.N.Q.O. (CSIC) and the
results obtained were within ‹0.4% of the theoretical
values. CAL was the Novo Nordisk's immobilized
preparation Novozym 435 and was used as received.
Diethyl N-protected dicarboxylic amino acids were
obtained from the commercial free amino acids follow-
ing standard procedures.17
Experimental
Chemical procedures
Melting points were determined with a Reichert±Jung
Thermovar apparatus and are uncorrected. Flash column
chromatography was carried out at medium pressure
General method for enzymatic synthesis
To a solution of the corresponding N-blocked amino-
acid diethyl esther (20 mM) and amine (50 mM) in
diisopropylether, CAL (50 mg/mL) and 4 A molecular
sieves (50 mg/mL) were added. The reaction mixture
was stirred in an orbital shaker at 60 ꢁC. After that time,
the enzyme and molecular sieves were ®ltered o€ and
washed with methanol. The combined organic solution
was evaporated to dryness. In the case of piperazine
derivatives, 3, 10 and 14, the residue was dissolved in
dichloromethane (60 mL) and washed with water (3Â25
mL). The organic phase was dryed (MgSO4), the solvent
was eliminated under reduced pressure and the residue
was used in the next step without further puri®cation. In
piperidine derivatives, the residue was puri®ed by silica
gel column chromatography using EtOAc:MeOH as
eluent.
N-Benzylpiperidine derivatives
These derivatives were obtained when N-benzyl-4-(2-
aminomethyl)piperidine was the amine used as nucleo-
phile in the enzymatic amidations, following the general
procedure.
Figure 5. Electrostatic potential maps of inhibitor 8 and (S)-donepezil
showing the pharmacophoric charge areas in common.

Â
A. Martõnez et al. / Bioorg. Med. Chem. 8 (2000) 731±738
735
1
Table 2. Signi®cant H NMR data of monoamide derivatives 1±15 (200 MHz, CDCl3 d ppm)
No.
n
P.G.
Cbz
R
R0
a
b-CH2
g-CH2
d-CH2
E-CH2
a-NH
R
R0
3.19 (1-CH2, NHPd)
6.53 (NH, NHPd)
3.43 (CH2, NBn)
3.26 (1-CH2, NHPd)
6.45 (NH, NHPd)
3.47 (CH2, NBn)
3.33 (1-CH2, NHPz)
6.67 (CH, NHPz)
3.31 (1-CH2, NHPz)
6.61 (NH, NHPz)
3.47 (CH2, NBn)
3.31 (1-CH2, NHPz)
6.56 (NH, NHPz)
3.55 (CH2, NBn)
3.26 (1-CH2, NHPz)
6.57 (NH, NHPz)
3.37 (CH2, NBn)
3.30 (1-CH2, NHPz)
6.60 (NH, NHPz)
3.50 (CH2, NBn)
3.22 (1-CH2, NHPd)
1.23 (CH3, Et)
1
1
NHPda
OEt
4.18
1.84±
2.12
2.27±
2.48
Ð
Ð
5.88
1.19 (CH3, Et)
4.07 (CH2, Et)
2
3
1
1
Ac
NHPd
OEt
OEt
4.41
4.21
1.88±
2.55
1.89±
2.17
2.30±
2.57
2.33
2.54
Ð
Ð
Ð
Ð
6.52
5.67
1.26 (CH3, Et)
4.14 (CH2, Et)
1.24 (CH3, Et)
4.12 (CH2, Et)
Cbz
NHPzb
4
5
6
7
8
1
1
1
1
2
Cbz
Cbz
Cbz
Cbz
Cbz
NHPz
NHPz
NHPz
NHPz
OEt
OEt
OEt
4.18
4.18
4.15
4.19
4.35
1.90±
2.32
2.33±
2.45
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
5.65
5.65
5.64
5.63
5.35
1.23 (CH3, Et)
4.11 (CH2, Et)
1.90±
2.20
2.30
2.57
1.23 (CH3, Et)
4.11 (CH2, Et)
OEt
1.82±
2.10
2.28±
2.44
1.18 (CH3, Et)
4.05 (CH2, Et)
OEt
1.87±
2.14
2.32±
2.54
1.21 (CH3, Et)
4.09 (CH2, Et)
NHPd
1.58±
1.97
1.58
1.77
2.11±
2.19
Ð
Ð
5.55 (NH, NHPd)
3.45 (CH2, NBn)
4.11 (CH2, Et)
3.26 (1-CH2, NHPd)
1.28 (CH3, Et)
4.20 (CH2, Et)
1.25 (CH3, Et)
4.17 (CH2, Et)
9
2
2
Ac
OEt
OEt
NHPd
NHPz
4.57
4.31
1.62±
1.87
1.62±
1.97
1.62±
1.87
1.62±
1.73
2.10±
2.32
2.18
Ð
Ð
6.22
5.55
5.56 (NH, NHPd)
3.49 (CH2, NBn)
3.29 (1-CH2, NHPz)
6.09 (NH, NHPz)
3.44 (1-CH2, NHPz)
5.99 (NH, NHPz)
3.55 (CH2, NBn)
3.22 (1-CH2, NHPd)
5.42 (NH, NHPd)
3.46 (CH2, NBn)
3.19 (1-CH2, NHPd)
5.77 (NH, NHPd)
3.50 (CH2, NBn)
3.25 (1-CH2, NHPz)
6.06 (NH, NHPz)
3.27 (1-CH2, NHPz)
6.07 (NH, NHPz)
3.39 (CH2, NBn)
10
Cbz
11
12
2
3
Cbz
Cbz
OEt
OEt
NHPz
NHPd
4.27
4.31
1.59±
1.88
1.59±
1.69
2.06±
2.22
Ð
5.46
5.33
1.20 (CH3, Et)
4.12 (CH2, Et)
1.52±
2.00
1.52±
1.72
1.38
2.10
1.24 (CH3, Et)
4.16 (CH2, Et)
13
14
3
3
Ac
OEt
OEt
NHPd
NHPz
4.51
4.26
1.50±
1.86
1.54±
1.84
1.50±
1.86
1.54±
1.63
1.18±
1.49
1.31
2.10
2.10
6.31
5.42
1.22 (CH3, Et)
4.13 (CH2, Et)
1.20 (CH3, Et)
4.12 (CH2, Et)
Cbz
15
3
Cbz
OEt
NHPz
4.29
1.53±
1.90
1.53±
1.90
1.34
2.11
5.42
1.22 (CH3, Et)
4.14 (CH2, Et)
aPd=2-(4-benzylpiperidinyl)ethyl.
bPz=2-(1-piperazinyl)ethyl.
1-(N-Benzylpiperidin-4-yl-2-ethylamido)-N-benzyloxy-
carbonyl-S-glutamic acid 5-ethyl ester (1). Reaction
time, 5 h. Chromatographic conditions: HPLC, CH3CN:
H2O 40:60, l=215 nm; column, EtOAc:MeOH
60:1. Yield, 61%. Colorless solid mp 107±108 ꢁC, [a]d:
3.7ꢁ (c=1, CHCl3). Anal. calcd for C29H39N3O5: C,
68.34; H, 7.71; N, 8.25. Found: C, 68.38; H, 7.85; N,
8.30.
CHCl3). Anal. calcd for C23H35N3O4: C, 66.16; H, 8.45;
N, 10.06. Found: C, 66.27; H, 8.28; N, 9.94.
6-(N-Benzylpiperidin-4-yl-2-ethylamido)-N-benzyloxy-
carbonyl-S-ꢀ-amino-adipic acid 1-ethyl ester (8). Reac-
tion time, 9 h. Chromatographic conditions: HPLC,
CH3CN:H2O 50:50, l=215 nm; column, EtOAc:MeOH
30:1. Yield, 70%. Colorless solid mp 85±86 ꢁC, [a]d:
+2.4ꢁ (c=1, CHCl3). Anal. calcd for C30H41N3O5: C,
68.81; H, 7.89; N, 8.02. Found: C, 69.02; H, 7.84; N,
7.96.
1-(N-Benzylpiperidin-4-yl-2-ethylamido)-N-acetyl-S-glut-
amic acid 5-ethyl ester (2). Reaction time, 14 h. Chro-
matographic conditions: HPLC, CH3CN:H2O 20:80,
l=200 nm; column, EtOAc:MeOH 15:1. Yield, 68%.
6-(N-Benzylpiperidin-4-yl-2-ethylamido)-N-acetyl-S-ꢀ-
aminoadipic acid 1-ethyl ester (9). Reaction time, 7 h.
Colorless solid mp 99±100 ꢁC, [a]d:
9.4ꢁ (c=1,

Â
A. Martõnez et al. / Bioorg. Med. Chem. 8 (2000) 731±738
736
Table 3. Signi®cant 13C NMR data of monoamide derivatives 1±15 (50 MHz, CDCl3, d ppm)
No.
n
P.G.
Cbz
R
R0
a-CH
b-CH2
g-CH2
d-CH2
E-CH2
R
R0
37.20 (1-CH2, NHPd)
35.90 (2-CH2, NHPd)
63.30 (CH2, NBn)
1
1
NHPda
OEt
54.10
28.00
30.30
Ð
Ð
14.00 (CH3, Et)
60.60 (CH2, Et)
36.00 (1-CH2, NHPd)
37.20 (2-CH2, NHPd)
63.40 (CH2, NBn)
35.90 (1-CH2, NHPz)
56.70 (2-CH2, NHPz)
36.10 (1-CH2, NHPz)
56.20 (2-CH2, NHPz)
62.90 (CH2, NBn)
36.10 (1-CH2, NHPz)
56.20 (2-CH2, NHPz)
62.00 (CH2, NBn)
36.00 (1-CH2, NHPz)
56.20 (2-CH2, NHPz)
62.10 (CH2, NBn)
2
3
1
1
Ac
NHPd
OEt
OEt
52.40
54.40
27.90
28.30
30.50
30.50
Ð
Ð
Ð
Ð
14.10 (CH3, Et)
60.70 (CH2, Et)
14.20 (CH3, Et)
60.80 (CH2, Et)
Cbz
NHPzb
4
5
6
7
8
1
1
1
1
2
Cbz
Cbz
Cbz
Cbz
Cbz
NHPz
NHPz
NHPz
NHPz
OEt
OEt
OEt
54.20
54.30
54.20
54.20
54.10
28.20
28.30
28.30
28.30
32.90
30.30
30.40
30.30
30.40
22.10
Ð
Ð
Ð
Ð
Ð
Ð
Ð
14.20 (CH3, Et)
60.70 (CH2, Et)
14.20 (CH3, Et)
60.70 (CH2, Et)
OEt
Ð
14.20 (CH3, Et)
60.70 (CH2, Et)
36.00 (1-CH2, NHPz)
56.20 (20CH2, NHPz)
62.60 (CH2, NBn)
OEt
Ð
14.20 (CH3, Et)
60.70 (CH2, Et)
37.10 (1-CH2, NHPd)
38.00 (2-CH2, NHPd)
64.10 (CH2, NBn)
NHPd
36.40
14.80 (CH3, Et)
62.20 (CH2, Et)
36.30 (1-CH2, NHPd)
37.20 (2-CH2, NHPd)
63.50 (CH2, NBn)
35.60 (1-CH2, NHPz)
56.10 (2-CH2, NHPz)
35.80 (1-CH2, NHPz)
56.50 (2-CH2, NHPz)
62.90 (CH2, NBn)
9
2
2
Ac
OEt
OEt
NHPd
NHPz
51.60
53.70
31.90
32.00
21.40
21.50
35.50
35.70
Ð
Ð
14.20 (CH3, Et)
61.60 (CH2, Et)
14.20 (CH3, Et)
61.50 (CH2, Et)
10
Cbz
11
12
2
3
Cbz
Cbz
OEt
OEt
NHPz
NHPd
53.60
53.70
31.90
32.40
21.40
24.80
35.60
25.10
Ð
14.20 (CH3, Et)
61.50 (CH2, Et)
36.30 (1-CH2, NHPd)
37.20 (2-CH2, NHPd)
63.40 (CH2, NBn)
36.30
14.10 (CH3, Et)
61.50 (CH2, Et)
36.10 (1-CH2, NHPd)
37.00 (2-CH2, NHPd)
63.00 (CH2, NBn)
36.20 (1-CH2, NHPz)
57.10 (2-CH2, NHPz)
35.80 (1-CH2, NHPz)
56.50 (2-CH2, NHPz)
62.90 (CH2, NBn)
13
14
3
3
Ac
OEt
OEt
NHPd
NHPz
52.00
53.70
32.00
32.30
24.80
24.80
25.00
25.10
36.10
35.60
14.10 (CH3, Et)
61.30 (CH2, Et)
14.10 (CH3, Et)
61.40 (CH2, Et)
Cbz
15
3
Cbz
OEt
NHPz
53.80
32.40
24.80
25.10
36.20
14.10 (CH3, Et)
61.40 (CH2, Et)
aPd=2-(4-benzylpiperidinyl)ethyl.
bPz=2-(1-piperazinyl)ethyl.
Chromatographic conditions: HPLC, CH3CN:H2O
20:80, l=200 nm; column, EtOAc:MeOH 15:1. Yield,
73%. Colorless solid mp 110±111 ꢁC, [a]d: +2.8ꢁ (c=1,
CHCl3). Anal. calcd for C24H37N3O4: C, 66.79; H, 8.64;
N, 9.74. Found: C, 66.79; H, 8.53; N, 9.57.
7-(N-Benzylpiperidin-4-yl-2-ethylamido)-N-acetyl-R,S-
ꢀ-aminopimelic acid 1-ethyl ester (13). Reaction time,
6 h. Chromatographic conditions: HPLC, CH3CN:H2O
25:75, l=200 nm; column, EtOAc:MeOH 20:1. Yield,
71%. Colorless syrup. Anal. calcd for C25H39N3O4: C,
67.39; H, 8.82; N, 9.43. Found: C, 67.40; H, 8.89; N, 9.35.
7-(N-Benzylpiperidin-4-yl-2-ethylamido)-N-benzyloxy-
carbonyl-R,S-ꢀ-amino-pimelic acid 1-ethyl ester (12).
Reaction time, 7 h. Chromatographic conditions: HPLC,
CH3CN:H2O 50:50, l=215 nm; column, EtOAc:MeOH
15:1. Yield, 72%. Colorless syrup. Anal. calcd for
C31H43N3O5: C, 69.25; H, 8.06; N, 7.81. Found: C,
69.08; H, 8.12; N, 7.74.
N-Benzylpiperazine derivatives. These compounds were
obtained in a two step process: following the general
procedure, the enzymatic amidation of the diesters using
2-(1-piperazinyl)ethylamine as nucleophile and the sub-
sequent N-benzylation of the N-free piperazine inter-
mediates 3, 10 and 14 obtained.

Â
A. Martõnez et al. / Bioorg. Med. Chem. 8 (2000) 731±738
737
1-(Piperazinyl-2-ethylamido)-N-benzyloxycarbonyl-S-
glutamic acid 5-ethyl ester (3). Reaction time, 5 h.
Chromatographic conditions: HPLC, CH3CN:H2O
40:60, l=215 nm. Yield, 83%. Colorless syrup, [a]d:
78%. Eluent EtOAc:MeOH 20:1. Colorless solid mp
80±81 ꢁC, [a]d: +4.4ꢁ (c=1, CHCl3). Anal. calcd for
C29H40N4O5: C, 66.39; H, 7.68; N, 10.68. Found: C,
66.24; H, 7.51; N, 10.52.
+3.8ꢁ (c=1, CHCl3). Anal. calcd for C21H32N4O5:
C
59.98; H, 7.67; N, 13.32. Found: C, 60.18; H, 7.52; N,
13.60.
7-(N-Benzylpiperazinyl-2-ethylamido)-N-benzyloxycar-
bonyl-R,S-ꢀ-amino-pimelic acid 1-ethyl ester (15). Yield
61%. Eluent EtOAc:MeOH 20:1. Colorless syrup. Anal.
calcd for C30H42N4O5: C, 66.89; H, 7.86; N, 10.40.
Found: C, 67.02; H, 7.89; N, 10.29.
6-(Piperazinyl-2-ethylamido)-N-benzyloxycarbonyl-S-ꢀ-
aminoadipic acid 1-ethyl ester (10). Reaction time, 5 h.
Chromatographic conditions: HPLC, CH3CN:H2O 50:50,
l=215 nm. Yield, 75%. Colorless syrup, [a]d: +3.2ꢁ
(c=1, CHCl3). Anal. calcd for C22H34N4O5: C, 60.81; H,
7.89; N, 12.89. Found: C, 61.11; H, 7.97; N, 12.93.
Modeling methods
Semiempirical methods. All compounds were built in
SPARTAN.18 Each molecule were fully optimised using
the semi-empirical method AM1.19 Partial atomic charges
required for calculation of electrostatic potential were
evaluated by ®tting point to electrostatic potential.
6-(Piperazinyl-2-ethylamido)-N-benzyloxycarbonyl-R,S-
ꢀ-aminopimelic acid 1-ethyl ester (14). Reaction time,
6 h. Chromatographic conditions: HPLC, CH3CN:H2O
50:50, l=215 nm. Yield, 81%. Colorless syrup. Anal.
calcd for C23H36N4O5: C, 61.59; H, 8.09; N, 12.49.
Found: C, 62.70; H, 7.78; N, 12.53.
Similarity calculations. Molecular alignment and mole-
cular similarity indices were carried out using TSAR
package.20 Molecular alignment were carried out using
a function based on a mixture of shape, electrostatic
charge and lipophilicity. The combined property (cp)
for atom i is calculated as follows:
N-Benzylation general procedure. A solution of the cor-
responding benzylhalide (0.25 mmol) in anhydrous
CH2Cl2 (5 mL) was added dropwise to a stirred ice-
cooled solution of the monoamide 3, 10 or 14 (0.23
mmol) and triethylamine (0.24 mmol) in anhydrous
CH2Cl2 (15 mL). The mixture was stirred 12 h (TLC,
CHCl3:acetone 1:1) at room temperature and then
washed with HCL 0.1 N (3Â10 mL) and water. The
organic solution was dried (MgSO4), evaporated and
the remaining residue eluted on a silicagel column.
cpꢀi† ˆ 1:0wS ‡ qiwQ ‡ liwL
where wS, wQ and wL are user-de®ned weights (shape,
charge and lipophilicity) and qi and li are the partial
charge and lipophilicity for atom i respectively. The best
results were obtained when wS=1, wQ=0, wL=0.
Molecular similarity indices were calculated without
optimisation of previous alignment.
1-(N-Benzylpiperazinyl-2-ethylamido)-N-benzyloxycar-
bonyl-S-glutamic acid 5-ethyl ester (4). Yield 60%. Elu-
ent EtOAc:MeOH 20:1. Colorless solid mp 100±101 ꢁC,
[a]d: +2.8ꢁ (c=1, CHCl3). Anal. calcd for C28H38
N4O5: C, 65.86; H, 7.50; N, 10.97. Found: C, 65.98; H,
7.72; N, 10.92.
Biological methods
The method of Ellman et al.12 was followed. The assay
solution consisted of 0.1 M phosphate bu€er pH 8, 200
mM 5,50-dithiobis(2-nitrobenzoic acid) (DTNB, Ellan's
reagent), 0.02 unit/mL AChE (Sigma Chemical Co.,
from human erythrocytes), and 400 mM acethylthio-
choline iodide as the substrate of the enzymatic reac-
tion. The compounds tested were added to the assay
solution and pre incubated with the enzyme for 10 min
at 30 ꢁC. After that period, the substrate was added. The
absorbance changes at 412 nm were recorded for 5 min
with a Perkin±Elmer 550 SE UV/VIS spectrometer, the
reaction rates were compared, and the percent inhibi-
tion due to the presence of test compounds was calcu-
lated. The IC50 is de®ned as the concentration of each
compound that reduces a 50% the enzymatic activity
with respect to that without inhibitors.
1-[N-(4-Nitrophenylmethyl)piperazinyl-2-ethylamido]-N-
benzyloxycarbonyl-S-glutamic acid 5-ethyl ester (5).
Yield 91%. Eluent, EtOAc:MeOH 15:1. Reddish syrup,
[a]d: +2.1ꢁ (c=1, CHCl3). Anal. calcd for C28H37N5O7:
C, 60.53; H, 6.71; N, 12.60. Found: C, 60.86; H, 6.75; N,
12.45.
1-[N-(4-Chlorophenylmethyl)piperazinyl-2-ethylamido]-
N-benzyloxy-carbonyl-S-glutamic acid 5-ethyl ester (6).
Yield 58%. Eluent, EtOAc:MeOH 20:1. Colorless solid
mp 93±94 ꢁC, [a]d: +3.0ꢁ (c=1, CHCl3). Anal. calcd for
C28H37N4ClO5: C, 61.70; H, 6.84; N, 10.28. Found: C,
61.93; H, 6.75; N, 10.08.
1-[N-(4-Biphenylylmethyl)piperazinyl-2-ethylamido]-N-
benzyloxycarbonyl-S-glutamic acid 5-ethyl ester (7).
Yield 44%. Eluent EtOAc:MeOH 30:1. Colorless solid
mp 122±123 ꢁC, [a]d: +3.7ꢁ (c=1, CHCl3). Anal. calcd
for C34H42N4O5: C, 69.60; H, 7.22; N, 9.55. Found: C,
69.35; H, 7.23; N, 9.31.
Acknowledgements
This work was ®nancially supported by CICYT (pro-
jects nos. SAF 96/107 and SAF 99/98). Generous gifts
of CAL and N-benzyl-4-cianomethylpiperidine are
kindly acknowledged to Novo Nordisk Bioindustrial
S.A. and Janssens Pharmaceuticals respectively.
6-(N-Benzylpiperazinyl-2-ethylamido)-N-benzyloxycar-
bonyl-S-ꢀ-amino-adipic acid 1-ethyl ester (11). Yield

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A. Martõnez et al. / Bioorg. Med. Chem. 8 (2000) 731±738
738
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