Design and synthesis of N-benzylpiperidine-purine derivatives as new dual inhibitors of acetyl- and butyrylcholinesterase

Article abstract of DOI:10.1016/j.bmc.2005.07.019

The synthesis and biological evaluation of N-benzyl-(piperidin or pyrrolidin)-purines are described. Compounds derived from N-benzylpiperidine and N-substituted purines showed moderate acetylcholinesterase inhibition. Preliminary structure-activity relati

Full text of DOI:10.1016/j.bmc.2005.07.019

Bioorganic & Medicinal Chemistry 13 (2005) 6795–6802
Design and synthesis of N-benzylpiperidine–purine derivatives
as new dual inhibitors of acetyl- and butyrylcholinesterase
*
Marıa Isabel Rodrıguez-Franco, Marıa Isabel Fernandez-Bachiller, Concepcion Perez,
´
´
´
´
Ana Castro and Ana Martınez
´
´
à
à
´
´ ´
Instituto de Quımica Medica (C.S.I.C.), Juan de la Cierva 3, 28006 Madrid, Spain
Received 6 April 2005; revised 12 July 2005; accepted 22 July 2005
Available online 23 September 2005
Dedicated to Prof. Carmen Ochoa on the occasion of her 65th birthday.
Abstract—The synthesis and biological evaluation of N-benzyl-(piperidin or pyrrolidin)-purines are described. Compounds derived
from N-benzylpiperidine and N-substituted purines showed moderate acetylcholinesterase inhibition. Preliminary structure–activity
relationships and a superimposition of the best compound with the active conformation of donepezil have revealed structural fea-
tures that have been used in the design of more potent N-benzylpiperidine inhibitors bearing an 8-substituted caffeine fragment and
a methoxymethyl linker. These new compounds are interesting dual inhibitors of acetylcholinesterase and butyrylcholinesterase and
have been chosen for further optimisation.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Tacrine, donepezil, rivastigmine and galantamine are
potent ChE-I. Some of these drugs selectively inhibit
acetylcholinesterase (AChE), but compounds that also
target butyrylcholinesterase (BuChE) may provide add-
ed benefits.12–14 BuChE is mainly associated with glial
cells and is found in much lower concentrations than
AChE in the healthy brain.15 However, over the course
of AD, AChE activity progressively decreases, while
BuChE activity increases, and BuChE may then act as
a compensatory mechanism for ACh metabolism.16
Consequently, as AD progresses, ACh regulation may
become increasingly dependent on BuChE and dual
inhibitors may provide more sustained efficacy than
AChE-selective agents.17 Moreover, a recent clinical
study has shown that patients whose drug treatment also
inhibited BuChE showed less cortical atrophic changes
than the subgroup treated with a selective AChE inhib-
itor, providing an empirical evidence of an additional ef-
fect of neuroprotection using dual cholinesterase
inhibitors in AlzheimerÕs disease.18
AlzheimerÕs disease (AD), the most common dementia
in elderly people, is a progressive neurodegenerative dis-
order characterised by three major pathological signs:
b-amyloid plaques, neurofibrillary tangles and loss of
central cholinergic function.1,2 In the last few years, sev-
eral rational pharmacological strategies have emerged,
including cholinergic and non-cholinergic interven-
tions.3,4 Among the cholinergic hypothesis, the first ap-
proved drugs for the management of the disease were
cholinesterase inhibitors (ChE-I) that increase neuro-
transmission at cholinergic synapses in the brain and
thereby improve cognition.5–7 Since recent research has
revealed that several ChE-I not only facilitate choliner-
gic transmission but also interfere with the synthesis,
deposition and aggregation of toxic amyloid-b-peptides
(Ab) slowing the neurodegenerative progression,8,9 the
current interest in these drugs has increased.10,11
Keywords: AlzheimerÕs disease; Benzylpiperidinylpurines; Acetylcho-
linesterase; Butyrylcholinesterase.
The crystal structures of human acetyl- and butyrylcho-
linesterase showed that the active catalytic triad is found
at the bottom of a deep gorge lined mostly with aromat-
ic residues that facilitate the recognition and guidance of
the quaternary ammonium ion of the natural substrate
by cation-p interactions.19–21 Continuing with our re-
search on different heterocyclic families of potential
*
Corresponding author. Tel.: +34 91 56 22 900; fax: +34 91 56 448
53; e-mail: isabelrguez@iqm.csic.es
´
This paper comprises a part of M. I. Fernandez-BachillerÕs Ph.D.
Thesis.
à
Present address: NeuroPharma, Avda. de la Industria 52, 28760 Tres
Cantos, Madrid, Spain.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.019

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M. I. Rodrıguez-Franco et al. / Bioorg. Med. Chem. 13 (2005) 6795–6802
6796
O
Me
O2C
HO
ii
i
N
N
6
7
Bn
Bn
N
MeO
MeO
N-Purine
Donepezil
Cl
Cl
N
H
8
Bn
N-Purine
Bn
n
iii
Bn
N
N
Purine
Purine
1, n = 1
3, n = 2
2
+
Bn
Bn
N
N
2a-c
1a-c
R
R'
O
Me N
O
Y
O
N
OMe
SMe
N
Me
N
N(7)
N
N
N
N
N
a =
b =
c =
N
N
N(9)
O
N
Me
N(9)
N
N
4, Y = CH2; R = R' = H
5, Y = CH2-O-CH2;
Me
Scheme 1. Reagents and conditions: (i) LiAlH4, THF, reflux, 5 h; (ii)
SOCl2, reflux, 3 h; (iii) purine, NaH, DME, reflux, 72 h.
R = H, F, Me, NH ; R' = M
e
2
Figure 1. Purine–benzylpiperidine derivatives as new cholinesterase
inhibitors.
i
EtO2C
Cl
EtO2C
HO
application in AlzheimerÕs disease,22–24 and taking into
account that the purine ring is able to interact with aro-
matic aminoacid residues through p–p interactions,25,26
we have designed new purine–benzylpiperidine deriva-
tives 1–5 as potential cholinesterase inhibitors that could
establish additional interactions in the gorge of the en-
zymes (Fig. 1).
N
N
N
Bn
Bn
Bn
10
11
9
ii
iii
Cl
Bn
N
H
v
iv
12
O
H
Me
N
O
N
N
Bn
In previous papers, we described the synthesis of ben-
zylpiperidin–purines 1, together with the corresponding
benzylpyrrolidin–purines 2, as a result of ring contrac-
tion in the course of nucleophilic substitution.27,28 In
this work, we report on the synthesis of the N-substitut-
ed xanthine 3 derived from theophylline and benzylpi-
peridine with a dimethylene linker, the biological
evaluation of series 1–3 and a molecular modelling study
by comparing the more potent compound (3) with the
active conformation of donepezil, known for its ability
to interact along the active-site gorge of the AChE
through interactions with aromatic amino acid resi-
dues.29 On the basis of the above SAR study and molec-
ular modelling, we designed and synthesised two new
series bearing 8-substituted xanthines 4 and 5, with the
aim of increasing the potency of the previous series.
vi
N
13
(7)
N
Me
Me
N
O
O
N
N
N
Me
H
O
N
Bn
N
3
4
Bn
Scheme 2. Reagents and conditions: (i) H2 (35 psi), PtO2, EtOH, rt,
5 h; (ii) LiAlH4, THF, reflux, 5 h; (iii) SOCl2, reflux, 3 h; (iv)
theophylline, NaH, DME, reflux, 48 h; (v) (COCl)2, DMSO, Et3N,
CH2Cl2, ꢀ78 ꢁC (30 min) to rt; (vi) 5,6-diamino-1,3-dimethyl-1H-
pyrimidine-2,4-dione, H2O/acetic acid (3:1), reflux, 36 h.
sharp resonance signal at 5.59 ppm, attributable to the
exocyclic olefinic proton, and the lack of any triplet less
deshielded due to the endocyclic one. Catalytic hydroge-
nation of 9 over PtO2 in ethanol at room temperature
gave the aliphatic ester 10 in 81% yield. It is worth men-
tioning that where the above hydrogenation was carried
out using Pd/C as a catalyst, a mixture of 10 and the cor-
responding debenzylated derivative was obtained.
Reduction of 10 with lithium aluminium hydride gave
the required alcohol 11 which was then treated with
thionyl chloride, yielding the corresponding chloroderiv-
ative 12 in quantitative yield. Finally, the reaction of 12
and theophylline, using sodium hydride as base and
dimethoxyethane as solvent, afforded compound 3, in
which the position of alkylation was determined by
NMR using a combination of bidimensional 1H and
13C NMR experiments. The fact that the methylene pro-
tons bound to the purinic nitrogen showed a HMBC
correlation with carbon 5 of the purine, but not with
carbon 4, demonstrated that alkylation occurred at
nitrogen 7.
2. Results and discussion
2.1. Chemistry
N-(1-Benzylpiperidin-4-ylmethyl)-purines 1a–c and N-
[2-(1-benzylpyrrolidin-3-yl)ethyl]-purines 2a–c were syn-
thesised from 1-benzyl-4-chloromethylpiperidine and
commercially available purines according to Scheme 1,
as previously described by us.27,28
New theophylline derivatives 3 and 4 were obtained, as
shown in Scheme 2. Treatment of 1-benzyl-4-piperidone
with triethyl phosphonoacetate in the presence of sodi-
um hydride in dry tetrahydrofuran yielded 9 in 85%
yield. In contrast with the work of Gupta et al. where
both endocyclic and exocyclic olefins were formed,30 in
our case only one product was obtained, as shown by

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M. I. Rodrıguez-Franco et al. / Bioorg. Med. Chem. 13 (2005) 6795–6802
6797
Table 1. AChE inhibition activity of compounds derived from
N-substituted purines 1a–c, 2a–c, 3 and intermediates 6–12
O
O
Me
N
Me
N
Me
O
Me
O
N
i
N
Compound
IC50 (lM)a
N
N
OH
O
N
N
1a
1b
1c
2a
2b
2c
3
20 0.1
42 2.1
35 1.5
50 2.6
13 0.1
40 1.9
Me
Me
15
14
ii
N
R
Me
O
N
O
N
O
Br
N
N
2
0.1
25 1.1
0.2
Me
Me
N
Me
6
16c, R = Me
16d, R = NO2
16a, R = H
16b, R = F
7
8
8
40 2.6
100
9
iii
Me
N
R
10
11
12
30 1.3
100
10 0.5
O
N
O
N
N
N
Me
a Data are means standard deviation of three independent
experiments.
5a, R = H
5b, R = F
5c, R = Me
5d, R = NH2
O
Scheme 3. Reagents and conditions: (i) 4-chloromethylpyridine, NaH,
DMF, reflux, 3 h; (ii) RC6H4CH2Br, CH3CN, reflux, 3 h; (iii) H2
(20 psi), PtO2, EtOH, rt, 6–7 h.
diates 6–12, towards bovine AChE (Table 1). From
these data, some preliminary structure–activity relation-
ships could be derived. The N-benzylpiperidine deriva-
tives (1a–c and 3) inhibited the AChE better than the
N-benzylpyrrolidine ones (2a–c). As regards the nature
of heterocycle, xanthine derivatives were better than
purine ones (compound 1a vs 1b and 1c). Finally, com-
paring the length of the linker between theophylline and
benzylpiperidine moieties, the dimethylene chain in-
creased the enzymatic inhibition in 1 order of magnitude
Moreover, Swern oxidation31 of alcohol 11 using oxalyl
chloride and dimethylsulfoxide afforded aldehyde 13,
with no carboxylic acid being produced. This aldehyde
was then condensed with 5,6-diamino-1,3-dimethyl-1H-
pyrimidine-2,4-dione affording a new 8-substituted the-
ophylline derivative 4 in one-step cyclization, using a
mixture of water:acetic acid as solvent.32
in relation to the methylene one, compound
3
Finally, compounds with a methoxymethyl spacer were
synthesised according to Scheme 3. Following the
described methods, 8-hydroxymethylcaffeine (14) was
obtained by condensation and ring closure reaction of
5,6-diamino-1,3-dimethyl-1H-pyrimidine-2,4-dione with
glycolic acid,33 followed by alkylation with methyl
iodide in the presence of potassium carbonate. The
reaction of 14 with sodium hydride and 4-chloromethyl-
pyridine yielded 15, which was then treated with several
benzyl bromides without any base to yield the corre-
sponding benzylpyridinium salt 16a–d. Hydrogenation
of 16a–c over platinum (IV) oxide in ethanol generated
the corresponding benzylpiperidine derivative 5a–c in
47–49% yields. In the case of 16d, the nitro group was
also reduced to the expected amino derivative, rendering
compound 5d in 41% yield.
(IC50 = 2 lM) being the best among these N-substituted
purines.
With regard to tested intermediates 6–12, it is worth
mentioning that the methanol derivative 7 showed an
interesting AChE inhibition (IC50 = 8 lM), in contrast
with the value exhibited by its homologous 11
(IC50 = 100 lM), derived from ethanol. This fact could
be related to stabilizing interactions between the enzyme
and the hydroxylic group of 7 located to one methylene
group from the piperidine fragment.
With the aim of obtaining some information about the
possible interactions of the new molecules with the en-
zyme, which, in turn, could be useful in the design of
more potent inhibitors, a molecular modelling study
was performed comparing 3 with donepezil, a nanomo-
lar AChE inhibitor. Using the semi-empirical method
AM1, compound 3 was optimised by rotating the tor-
sional angles of the linear alkyl fragments and the min-
imum was then superimposed with the active
conformation of donepezil, whose 3D coordinates were
obtained from the crystal structure of the Torpedo cali-
fornica AChE:donepezil complex37 (PDB entry:
1EVE). As it can be seen in Figure 2, the benzylpiperi-
dine fragments correctly superimposed in both com-
pounds, whereas the xanthine ring was located near
the indanone fragment but did not fit it exactly. From
this study none can deduce that a better superimposition
could be reached with 8-substituted xanthines, instead of
N-substituted ones.
2.2. Biological and molecular modelling studies
The in vitro inhibition of AChE for the new synthesised
compounds, as well as for intermediates, was deter-
mined by the method of Ellman et al.34 using a commer-
cially available AChE from bovine erythrocytes. We
also used butyrylcholinesterase (BuChE) from equine
serum to determine the selectivity profile of selected
inhibitors. Both bovine AChE and equine BuChE show
a high degree of homology (>90%) with the correspond-
ing human enzymes.35,36
Initially, we tested compounds derived from N-substi-
tuted purines 1a–c, 2a–c and 3, as well as their interme-

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6798
The AChE/BuChE selectivity of the 8-substituted xanth-
ines was investigated by measuring the BuChE inhibito-
ry activity of 16a–d and 5a–d, which are the most potent
AChE inhibitors of the series. From the IC50 data
reported in Table 2, it becomes evident that all tested
compounds did not show remarkable selectivity towards
one of the two enzymes, and only the benzylpyridinium
derivative 16a showed moderate selectivity towards
AChE (20-fold up compared to BuChE). The opposite
selectivity was found in the 3-methylbenzylpiperidine
derivative, 5c (IC50 = 0.40 lM) being the best BuChE
inhibitor of this work.
Figure 2. Superimposition of 3 (blue) and donepezil (yellow).
Combining conclusions extracted from both the struc-
ture–activity relationships and the molecular modelling,
we planned to obtain new series bearing an 8-substituted
xanthine, 4 and 5a–d (see Fig. 1). Compound 4 was de-
signed to explore the effect of substitution in the xan-
thine fragment, and 5a was designed to confirm if a
longer linker with an oxygen atom located to one meth-
ylene group from the piperidine fragment could be
favourable for activity. Further substitutions of 5a on
the benzyl moiety at the meta position (F, CH3 and
NH2) were chosen on the basis of their higher inhibitory
activities in the donepezil series, as previously
described.38,39
3. Conclusions
In summary, we have reported the synthesis and preli-
minary results for acetylcholinesterase and butyrylcho-
linesterase inhibition activity of a series of N-benzyl-
(piperidin or pyrrolidin)-purines. Compounds derived
from N-benzylpiperidine and N-substituted purines 1–3
showed only moderate AChE inhibition (IC50 = 20–
50 lM). Preliminary structure–activity relationships
and a comparison between the optimised structure of 3
and the active conformation of donepezil have envis-
aged that an 8-substituted caffeine substructure and a
methoxymethyl spacer could increase enzymatic inhibi-
tion of these N-benzylpiperidine derivatives. As a result,
1-benzyl-4-[(xanthin-8-yl)-methoxymethyl]-pyridinium
16a–d and 1-benzyl-4-[(xanthin-8-yl)-methoxymethyl]-
piperidine derivatives 5a–d exhibited better AChE inhi-
bition (IC50 = 0.1–1.0 lM) by 2 orders of magnitude
compared with the preceding structures. Since these
derivatives also inhibited BuChE (IC50 = 0.4–4.0 lM),
they can be considered as interesting dual inhibitors in
the search of new therapies for AlzheimerÕs disease.
The AChE inhibition of these new compounds is given
in Table 2. As the previous modelling study envisaged,
compound 4 bearing an 8-substituted caffeine is more
potent (IC50 = 2.50 lM) than its homologue derived
from 7-substituted theophylline 1a (IC50 = 20 lM),
pointing out that the correct orientation of the xanthine
improves inhibition by 1 order of magnitude. In addi-
tion, elongation of the chain and presence of an oxygen
atom increased the potency of benzylpyridinium and
benzylpiperidine derivatives by another order of magni-
tude: 16a and 5a showed sub-micromolar inhibition of
AChE (IC50 = 0.10 and 0.75 lM, respectively). In con-
trast with the bibliographic data,38,39 the introduction
of any substituent in the position 3 of the benzyl frag-
ment (such as fluor, methyl, nitro, or amino) did not im-
prove the enzymatic activity (compounds 16b–d and 5b–
d vs 16a and 5a, respectively). Finally, benzylpyridinium
cations 16a–d showed better inhibition than the corre-
sponding benzylpiperidine derivatives 5a–d, revealing
that an aromatic fragment with a positive charge could
establish favourable interactions with the aromatic resi-
dues from the gorge of the AChE.
4. Experimental
4.1. Chemistry
Reagents and solvents were purchased from common
commercial suppliers and were used without further puri-
fication. Tetrahydrofuran (THF) and dimethoxyethane
(DME) were freshly distilled from LiAlH4 prior to their
use. Chromatographic separations were performed on sil-
ica gel, using flash column chromatography (Kieselgel 60
Merck of 230–400 mesh), and compounds were detected
with UV light (254 nm), iodine chamber, or ninhydrin.
HPLC analyses were performed on a Waters 6000 equip-
ment, with UV detector (214–274 nm), using a Delta Pak
Table 2. AChE and BuChE inhibition data (IC50, lM)a of compounds
derived from 8-substituted xanthines 4, 5a–d and 16a–d
Compound
AChE-I
BuChE-I
4
5a
2.50 0.10
0.75 0.03
1.00 0.05
1.00 0.04
1.00 0.04
0.10 0.01
0.30 0.01
0.50 0.02
0.30 0.01
n.d.
˚
C18.5 lm, 300 A (3.9 nm · 150 nm) column, eluted with
2.50 0.09
2.00 0.08
0.40 0.01
1.00 0.05
2.00 0.10
1.00 0.04
3.00 0.10
4.00 0.10
mixtures of CH3CN (solvent A) and H2O with 0.05%
H3PO4 and 0.04% Et3N (solvent B), as indicated in each
case, at a flow rate of 1.0 mL/min.
5b
5c
5d
16a
16b
16c
16d
Melting points were determined with a Reichert–Jung
Thermovar apparatus. Nuclear magnetic resonance
spectra were recorded in CDCl3 solutions, using Varian
Unity-500, Varian Unity Inova-400 and Varian XL-300
spectrometers. Typical spectral parameters for 1H NMR
were: spectral width 10 ppm, pulse width 9 ls (57ꢁ) and
n.d.: not determinded.
a Data are means standard deviation of three independent
experiments.

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M. I. Rodrıguez-Franco et al. / Bioorg. Med. Chem. 13 (2005) 6795–6802
6799
data size 32 K. The acquisition parameters in decoupled
13C NMR spectra were: spectral width 16 kHz, acquisi-
tion time 0.99 s, pulse width 9 ls (57ꢁ) and data size
32 K. Chemical shifts are reported in d values (ppm), rel-
ative to internal Me4Si, and J values are reported in
Hertz. Other experiments, such as HSQC (Heteronucle-
ar Single Quantum Coherence) and HMBC (Heteronu-
clear Multiple Bond Correlation), were obtained in
standard conditions. Mass spectra (MS) were obtained
by electronic impact (EI) at 70 eV in a Hewlett-Packard
5973 spectrometer (with direct insertion probe) or by
electron spray ionization (ESI) in positive mode using
a Hewlett-Packard MSD 1100 spectrometer. Elemental
analyses were carried out in a Perkin-Elmer 240C equip-
solution and washed with ethyl acetate (3· 50 mL). The
organic extracts were treated with water (3· 30 mL), brine
(3· 30 mL), dried (Na2SO4) and evaporated to dryness,
yielding a syrup that was purified by flash chromatogra-
phy on silica gel column using CH2Cl2/MeOH/NH4OH,
90:10:1 as eluent. Fractions of Rf 0.6 were evaporated to
dryness, yielding 4 (66.2 mg, 18%) as a pure yellow solid
(mp 203–205 ꢁC). 1H NMR (DMSO-d6, 400 MHz) d
13.0 (br s, 1H), 7.25 (m, 5H), 3.37 (s, 3H), 3.35 (s, 2H),
3.17 (s, 3H), 2.72 (br d, 2H, J = 11.5 Hz), 2.56 (d, 2H,
J = 7.0 Hz), 1.84 (br d, 2H, J = 11.5 Hz), 1.69 (m, 1H),
1.50 (br d, 2H, J = 11.5 Hz), 1.16 (br d, 2H,
J = 11.5 Hz). 13C NMR (DMSO-d6, 100 MHz) d 154.3,
153.2, 151.5, 148.5, 138.9, 129.0, 128.4, 127.1, 106.3,
62.7, 54.0, 53.3, 35.4, 30.1, 29.0, 27.9. MS (ESI) m/z 368
(M+H)+. HPLC analysis (A:B, 50:50) tR = 3.83 min.
Anal. Calcd for C20H25N5O2 (367.20): C, 65.37; H, 6.86;
N, 19.06. Found: C, 65.35; H, 6.80; N, 19.00.
´
´
ment in the Centro de Quımica Organica ÔManuel Lora-
TamayoÕ (CSIC) and the results are within 0.4% of the
theoretical values.
N-(1-Benzylpiperidin-4-ylmethyl)-purines 1a–c, N-[2-(1-
benzylpyrrolidin-3-yl)-ethyl]-purines 2a–c were obtained
as previously described by us,27,28 and intermediates 6–
14 according to literature methods.30,40–43
4.4. 4-[(1,3,7-Trimethyl-2,6-dioxo-1,2,3,6-tetrahydropurin-
8-yl)-methoxymethyl]-pyridine (15)
A mixture of 14 (80.0 mg, 0.36 mmol) and NaH
(17.3 mg, 0.36 mmol, oil free) in dry DME (20 mL)
was refluxed for 30 min under N2. Then, a solution of
4.2. 7-[2-(1-Benzyl-piperidin-4-yl)-ethyl]-1,3-dimethyl-2,6-
dioxo-1,2,3,6-tetrahydropurine (3)
4-chloromethylpyridine
hydrochloride
(59.0 mg,
Under N2 to a stirred suspension of NaH (53.0 mg,
1.1 mmol, oil free) in dry DME (10 mL), a solution of
theophylline (43.0 mg, 0.24 mmol) in DME (10 mL)
was added and the mixture was refluxed for 30 min.
Then, a solution of 12 (85.0 mg, 0.31 mmol) in dry
DME was slowly added and the reaction mixture was re-
fluxed for an additional 48 h. Then, it was cooled to
room temperature and then treated with a 15% aq solu-
tion of NH4Cl until pH 8 and evaporated to dryness.
The residue was dissolved in CH2Cl2 (25 mL), washed
with H2O (3· 25 mL), brine (25 mL), dried (Na2SO4)
and evaporated to dryness, yielding a syrup that was
purified by flash chromatography on a silica gel column.
Fractions of Rf 0.7 (CH2Cl2/CH3OH, 5:1) afforded unre-
acted theophylline (4.5 mg, 10%). Fractions of Rf 0.5
(CH2Cl2/CH3OH, 5:1) yielded compound 3 (23.8 mg,
23%) as a pure colourless syrup. 1H NMR (CDCl3,
400 MHz) d 7.50 (s, 1H), 7.29 (m, 5H), 4.29 (t, 2H,
J = 7.4 Hz), 3.57 (s, 3H), 3.48 (s, 2H), 3.39 (s, 3H),
2.87 (br d, 2H, J = 11.4 Hz), 1.93 (dt, 2H, J = 2.2,
11.5 Hz), 1.81 (q, 2H, J = 7.4 Hz), 1.67 (br d, 2H,
J = 11.4 Hz), 1.60 (m, 1H), 1.35 (m, 2H). 13C NMR
(CDCl3, 100 MHz) d 151.1, 151.7, 148.9, 140.6, 138.5,
129.2, 128.2, 127.0, 106.9, 63.3, 53.5, 45.0, 37.6, 33.1,
31.9, 29.7, 28.0. MS (EI) m/z 91 (100), 397 (25, M+).
Anal. Calcd for C22H31N5O2 (397.51): C, 66.47; H,
7.86; N, 17.62. Found: C, 66.10; H, 7.59; N, 18.00.
0.36 mmol) in dry DME (10 mL) was slowly added
and the mixture was refluxed for an additional 5 h. After
cooling to room temperature, the mixture was neutral-
ized with a 15% aq solution of NH4Cl and evaporated
to dryness. The residue was dissolved in CH2Cl2
(25 mL), washed with H2O (3· 25 mL), brine (25 mL),
dried (Na2SO4) and evaporated to dryness, yielding a
syrup that was purified by flash chromatography on a
silica gel column, using CH2Cl2/CH3OH, 7:1 as eluent.
Fractions of Rf 0.5 afforded 15 (80 mg, 71%) as a yellow
1
solid (mp 106–108 ꢁC). H NMR (CDCl3, 400 MHz) d
8.59 (d, 2H, J = 6.0 Hz), 7.20 (d, 2H, J = 6.0 Hz), 4.67
(s, 2H), 4.59 (s, 2H), 3.98 (s, 3H), 3.52 (s, 3H), 3.35 (s,
3H). 13C NMR (CDCl3, 100 MHz) d 155.3, 151.5,
150.1, 149.4, 147.3, 145.9, 121.7, 108.6, 70.9, 64.0,
32.2, 29.6, 27.9. MS (EI) m/z 208 (94), 315 (100, M+).
HPLC analysis (A:B, 50:50) tR = 1.35 min. Anal. Calcd
for C15H17N5O3 (315.33): C, 57.13; H, 5.43; N, 22.20.
Found: C, 56.99; H, 5.23; N, 22.00.
4.5. General procedure for the synthesis of benzyl pyridini-
um salts 16 a–d
Under N2 atmosphere, to a solution of 15 (1 equiv) in
dry CH3CN (10 mL) was slowly added a solution of
the appropriate benzyl bromide derivative (2 equiv) in
CH3CN (2 mL). The mixture was stirred at room tem-
perature for 5 h and evaporated to dryness, affording
syrups that were treated with Et2O (40 mL), yielding
solids that were collected by filtration.
4.3. 8-[(1-Benzylpiperidin-4-yl)-methyl-1,3-dimethyl-3,7-
dihydro-purine-2,6-dione (4)
A misture of 25% aq acetic acid solution and 5,6-diamino-
1,3-dimethyluracil hydrate (174.5 mg, 0.99 mmol) was re-
fluxed for 1 h. Then, a solution of 13 (215 mg, 0.99 mmol)
in ethanol (4 mL) was added dropwise over 30 min and
then refluxed for 48 h. After cooling to room temperature,
the mixture was neutralized with 2 N sodium hydroxide
4.6. 1-Benzyl-4-[(1,3,7-trimethyl-2,6-dioxo-1,2,3,6-tetra-
hydropurin-8-yl)-methoxymethyl]-pyridinium bromide (16a)
Following the general method, 15 (70 mg, 0.222 mmol)
and benzyl bromide (50 lL, 0.444 mmol) afforded 16a
(107 mg, 99%) as a pure yellow solid (mp 177–179 ꢁC).

´
M. I. Rodrıguez-Franco et al. / Bioorg. Med. Chem. 13 (2005) 6795–6802
6800
1H NMR (CDCl3, 500 MHz) d 9.42 (d, 2H, J = 6.5 Hz),
7.97 (d, 2H, J = 6.5 Hz), 7.62 (dd, 2H, J = 2.1 Hz,
J = 4.9 Hz), 7.40–7.20 (m, 3H), 6.24 (s, 2H), 4.90 (s,
2H), 4.81 (s, 2H), 4.01 (s, 3H), 3.52 (s, 3H), 3.37 (s,
3H). 13C NMR (CDCl3, 125 MHz) d 157.9, 155.4,
151.5, 147.5, 147.4, 144.5, 132.7, 130.1, 129.7, 129.6,
125.1, 108.2, 69.5, 64.4, 63.8, 32.4, 29.8, 27.9. MS
(ESI) m/z 406 (M+ꢀBr). HPLC analysis (A:B, 60:40)
tR = 4.46 min. Anal. Calcd for C22H24N5O3Br (485.11):
C, 54.33; H, 4.97; N, 14.40. Found: C, 54.30; H, 4.97;
N, 14.37; Br, 16.42.
(d, 1H, J = 8.3 Hz), 7.93 (t, 1H, J = 8.3 Hz), 6.17 (s,
2H), 5.18 (s, 2H), 5.10 (s, 2H), 4.20 (s, 3H), 3.70 (s,
3H), 3.51 (s, 3H). 13C NMR (CD3OD, 100 MHz) d
161.2, 156.8, 152.3, 150.3, 148.7, 145.8, 136.6, 136.2,
132.1, 126.9, 125.7, 125.1, 109.8, 70.7, 65.2, 63.9, 32.8,
30.1, 28.3. MS (ESI) m/z 451 (M+ꢀBr). HPLC analysis
(A:B, 50:50) tR = 1.41 min. Anal. Calcd for
C22H23N6O5Br (530.09): C, 49.73; H, 4.36; N, 15.82.
Found: C, 49.72; H, 4.33; N, 15.80.
4.10. General procedure for the hydrogenation of pyrid-
inium salts
4.7. 1-(3-Fluorobenzyl)-4-[(1,3,7-trimethyl-2,6-dioxo-1,2,3,
6-tetrahydropurin- 8-yl)-methoxymethyl]-pyridinium bromide
(16b)
To a solution of the corresponding pyridinium salt
(1.0 mmol) and platinum dioxide (0.6 mmol) in purged
ethanol (75 mL) was added triethylamine (1.3 mmol).
The mixture was stirred under hydrogen atmosphere
(20 psi) at room temperature for 15 min. The suspension
was filtered and the solvent was removed in vacuo to
give a yellow oil that was dissolved in dichloromethane
(50 mL) and washed with water (3· 25 mL) and brine
(3· 25 mL). The organic phase was dried and evapored
under reduced pressure and the resulting syrups were
purified by flash chromatography eluting with mixtures
of CH2Cl2/MeOH (13:1).
According to the general method, from 15 (70 mg,
0.222 mmol) and 3-fluorobenzyl bromide (54 lL,
0.444 mmol) compound 16b (102 mg, 91%) was ob-
tained as a pure yellow solid (mp 185–187 ꢁC). 1H
NMR (CDCl3, 500 MHz) d 9.48 (d, 2H, J = 6.2 Hz),
3
7.95 (d, 2H, J = 6.2 Hz), 7.47 (d, 1H, JF,H = 7.7 Hz),
7.40–7.30 (m, 2H), 7.15–7.06 (m, 2H), 6.27 (s, 2H),
4.88 (s, 2H), 4.77 (s, 2H), 3.96 (s, 3H), 3.48 (s, 3H),
3.31 (s, 3H). 13C NMR (CDCl3, 125 MHz) d 162.9 (d,
1JC,F = 249.5 Hz), 158.2, 155.3, 151.5, 147.4, 147.3,
144.6,
134.9
(d,
3JC,F = 8.2 Hz),
131.5
(d,
4.11. 1-Benzyl-4-[(1,3,7-trimethyl-2,6-dioxo-1,2,3,6-tetra-
hydropurin-8-yl)-methoxymethyl]-piperidine (5a)
3JC,F = 8.2 Hz), 125.4, 125.2, 117.2 (d, JC,F = 20.6 Hz),
116.4 (2JC,F = 21.9 Hz), 108.7, 69.5, 64.3, 62.6, 32.4,
29.7, 27.9. MS (ESI) m/z 424 (M+ꢀBr). HPLC analysis
(A:B, 60:40) tR = 4.46 min. Anal. Calcd for
C22H23N5O3BrF (503.10): C, 52.39; H, 4.60; N, 13.89.
Found: C, 52.36; H, 4.60; N, 13.86.
2
Following the general method, using 16a (150 mg,
0.31 mmol) as starting material, benzylpiperidine deriv-
ative 5a (60 mg, 47%) was obtained as a white solid
(mp 106–108 ꢁC). Rf 0.5 (CH2Cl2/MeOH, 7:1). 1H
NMR (CDCl3, 400 MHz) d 7.25 (m, 5H), 4.58 (s, 2H),
3.98 (s, 3H), 3.55 (s, 3H), 3.50 (s, 2H), 3.39 (s, 3H),
3.33 (d, 2H, J = 6.4 Hz), 2.90 (d, 2H, J = 11.3 Hz),
1.97 (t, 2H, J = 11.3 Hz), 1.67 (d, 2H, J = 11.3 Hz),
1.60 (m, 1H), 1.30 (m, 2H). 13C NMR (CDCl3,
100 MHz) d 155.4, 151.6, 149.4, 147.3, 129.3, 128.4,
128.2, 127.2, 108.5, 75.9, 64.7, 63.1, 53.1, 35.9, 32.2,
29.7, 28.7, 27.9. MS (ESI) m/z 412 (M+H)+. HPLC anal-
ysis (A:B, 35:65) tR = 1.88 min. Anal. Calcd for
C22H29N5O3 (411.23): C, 64.21; H, 7.10; N, 17.02.
Found: C, 63.98; H, 7.31; N, 16.87.
4.8. 1-(3-Methylbenzyl)-4-[(1,3,7-trimethyl-2,6-dioxo-1,2,3,
6-tetrahydropurin-8-yl)-methoxymethyl]-pyridinium bromide
(16c)
The reaction of 15 (70 mg, 0.222 mmol) and 3-methylb-
enzyl bromide (62 lL, 0.444 mmol), following the gener-
al method, afforded 16c (98.8 mg, 89%) as a pure yellow
1
solid (mp 175–177 ꢁC). H NMR (CDCl3, 400 MHz) d
9.29 (d, 2H, J = 6.6 Hz), 7.98 (d, 2H, J = 6.6 Hz),
7.28–7.20 (m, 4H), 6.15 (s, 2H), 4.90 (s, 2H), 4.81 (s,
2H), 4.03 (s, 3H), 3.55 (s, 3H), 3.39 (s, 3H), 2.33 (s,
3H); 13C NMR (CDCl3, 100 MHz) d 157.9, 155.3,
151.5, 147.4, 147.1, 144.5, 139.8, 132.4, 130.9, 130.1,
129.6, 126.6, 125.3, 108.7, 77.2, 69.7, 64.2, 32.6, 29.9,
28.0, 21.3. MS (ESI) m/z 420 (M+ꢀBr). Anal. Calcd
for C23H26N5O3Br (499.12): C, 55.21; H, 5.24; N,
14.00. Found: C, 55.20; H, 5.23; N, 14.00.
4.12. 1-(3-Fluorobenzyl)-4-[(1,3,7-trimethyl-2,6-dioxo-1,2,
3,6-tetrahydropurin -8-yl)-methoxymethyl]-piperidine (5b)
Following the general procedure, from 16b (150 mg,
0.30 mmol), the corresponding piperidine derivative 5b
(62 mg, 49%) was obtained as a pure yellow solid (mp
1
118–120 ꢁC). Rf 0.4 (CH2Cl2: MeOH, 10:1). H NMR
4.9. 1-(3-Nitrobenzyl)-4-[(1,3,7-trimethyl-2,6-dioxo-1,2,3,
6-tetrahydropurin -8-yl)-methoxymethyl]-pyridinium bro-
mide (16d)
(CDCl3, 400 MHz) d 7.24 (m, 1H), 7.06–7.02 (m, 2H),
6.91 (m, 1H), 4.60 (s, 2H), 4.00 (s, 3H), 3.56 (s, 3H),
3.46 (s, 2H), 3.39 (s, 3H), 3.34 (d, 2H, J = 5.8 Hz),
2.85 (d, 2H, J = 11.2 Hz), 1.95 (t, 2H, J = 11.2 Hz),
1.67 (d, 2H, J = 11.2 Hz), 1.60 (m, 1H), 1.30 (m, 2H).
According to the general method, the reaction of 15
(70 mg, 0.222 mmol) with 3-nitrobenzyl bromide
(95.9 mg, 0.444 mmol) produced compound 16d
(74.0 mg, 63%) as a pure yellow solid (mp 183–
13C
NMR
(CDCl3,
100 MHz)
d
162.8
(1JC,F = 245.6 Hz), 155.4, 151.6, 149.4, 147.3, 141.3,
129.5 (3JC,F = 8.4 Hz), 124.4 (3JC,F = 3.1 Hz), 115.6
(2JC,F = 21.4 Hz), 113.7 (2JC,F = 21.4 Hz), 108.5, 76.0,
64.7, 62.7, 53.3, 36.1, 32.2, 29.7, 29.1, 27.9. MS (ESI)
m/z 430 (M+H)+. HPLC analysis (A:B, 60:40)
1
185 ꢁC). H NMR (CD3OD, 400 MHz) d 9.26 (d, 2H,
J = 6.8 Hz), 8.62 (d, 1H, J = 1.2 Hz), 8.52 (dd, 1H,
J = 1.2 Hz, J = 8.3 Hz), 8.31 (d, 2H, J = 6.8 Hz), 8.10

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M. I. Rodrıguez-Franco et al. / Bioorg. Med. Chem. 13 (2005) 6795–6802
6801
tR = 1.40 min. Anal. Calcd for C22H28FN5O3 (429.22):
C, 61.52; H, 6.57; N, 16.31. Found: C, 61.50; H, 6.47;
N, 16.28.
of the linear alkyl fragments in 60 ꢁC increments. Semi-
empirical calculations were carried out using the AM1
method45 in MOPAC v5.0 program package46 and full
geometry optimisations were performed with the Fletch-
er-Power algorithm.
4.13. 1-(3-Methylbenzyl)-4-[(1,3,7-trimethyl-2,6-dioxo-1,2,
3,6-tetrahydropurin-8-yl)-methoxymethyl]-piperidine (5c)
According to the general method, 16c (150 mg,
0.30 mmol) was converted into 5c (61 mg, 48%) as a
white solid (mp 135–137 ꢁC). Rf 0.5 (CH2Cl2/MeOH,
7:1). 1H NMR (CDCl3, 400 MHz) d 7.12 (t, 1H,
J = 7.5 Hz), 7.05 (s, 1H), 7.02 (d, 1H, J = 7.5 Hz), 6.99
(d, 1H, J = 7.5), 4.53 (s, 2H), 3.93 (s, 3H), 3.50 (s,
3H), 3.38 (s, 2H), 3.34 (s, 3H), 3.27 (d, 2H,
J = 6.3 Hz), 2.82 (d, 2H, J = 11.5 Hz), 2.27 (s, 3H),
1.86 (t, 2H, J = 11.5 Hz), 1.60 (d, 2H, J = 13.0 Hz),
1.54 (m, 1H), 1.22 (m, 2H). 13C NMR (CDCl3,
100 MHz) d 155.5, 151.6, 149.4, 147.3, 137.8, 129.9,
128.0, 127.7, 126.3, 108.5, 76.1, 64.7, 63.4, 53.3, 36.1,
32.2, 29.7, 27.9, 21.4. MS (ESI) m/z 426 (M + H)+.
HPLC analysis (A:B, 50:50) tR = 1.43 min. Anal. Calcd
for C23H31N5O3 (419.20): C, 64.92; H, 7.34; N, 16.46.
Found: C, 64.90; H, 7.30; N, 16.50.
Acknowledgments
The authors gratefully acknowledge the financial sup-
ports of CICYT (SAF 2003/2262) and Comunidad de
Madrid (8.5/53.1/2003) and the fellowship to M. I. Fern-
´
andez-Bachiller from CSIC—NeuroPharma, S.A.
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´
Using the pyridinium salt 16d (70 mg, 0.13 mmol), com-
pound 5d (25 mg, 41%) was obtained as a pure syrup. Rf
0.4 (CH2Cl2/MeOH, 8:1). 1H NMR (CD3OD,
400 MHz) d 7.33 (t, 1H, J = 8.0 Hz), 6.97 (d, 1H,
J = 2.0 Hz), 6.94 (dd, 1H, J = 2.0 Hz, J = 8.0 Hz), 6.92
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