Design, synthesis and structure-activity relationships of a series of 9-substituted adenine derivatives as selective phosphodiesterase type-4 inhibitors

Article abstract of DOI:10.1016/S0223-5234(02)01446-0

Adenine derivatives substituted in position 9 have been demonstrated to have potent cyclic nucleotide phosphodiesterase (PDE) inhibition properties with high selectivity toward PDE-4. Starting from our initial lead compound 9-(2-fluorobenzyl)-N⁶-methyl-2-trifluoromethyladenine (4, NCS613), we designed and synthesized a new series of 9-substituted derivatives for developing structure-activity relationship studies. This new series of derivatives showed increased potencies and better selectivity profiles. Structural modifications were achieved in parallel on three different positions of the adenine ring, and led to the following observations: (i) introduction of a lipophilic substituent such as trifluoromethyl, n-propyl group or iodine in the C-2 position is favourable for both the PDE-4 inhibitory activity and the selectivity towards other isoenzymes; (ii) functionalization of the N9 benzyl group with a 2-methoxy substituent led to remarkably more active compounds; (iii) replacement of the N⁶-methylamino moiety by other amino groups is detrimental to the activity. Among all derivatives prepared, the 9-(2-methoxybenzyl)-N⁶-methyl-2-trifluoromethyladenine (9r), 9-(2-methoxybenzyl)-N⁶-methyl-2-n-propyladenine (9s), and the 2-iodo-9-(2-methoxybenzyl)-N⁶-methyladenine (13b) were found to be the most potent inhibitors within this series (PDE-4-IC₅₀=1.4, 7.0, and 0.096 nM, respectively). Compared to our reference compound 4, which showed an IC₅₀ of 42 nM, the derivative 13b was found 450-fold more potent. Moreover, 2-iodo-9-(2-methoxybenzyl)-N⁶-methyladenine (13b) and 9-(2-methoxybenzyl)-N⁶-methyl-2-trifluoromethyladenine (9r), were at least 50 000-150 000 times more selective for the PDE-4 than for the other PDE families. Additionally, these new derivatives showed improved efficiency in inhibiting the TNFα release from mononuclear cells from healthy subjects (e.g. adenines 7l, 9s and 13b). Thus, compounds 7l, 9r, 9s and 13b are among the most potent and selective PDE-4 inhibitors reported so far and represent very promising pharmacological tools for a better understanding of the signal transduction involving cyclic AMP within the cell: this pathway is implicated in the physiology and the pathophysiology of inflammation, asthma and autoimmune disorders.

Full text of DOI:10.1016/S0223-5234(02)01446-0

European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
www.elsevier.com/locate/ejmech
Original article
Design, synthesis and structureꢀ
/
activity relationships of a series of 9-
substituted adenine derivatives as selective phosphodiesterase type-4
inhibitors
Pierre Raboisson a,*,1, Claire Lugnier b, Christian Muller b, Jean-Marie Reimund b,
Dominique Schultz a, Guillaume Pinna b, Alain Le Bec b, He´le`ne Basaran b,
Laurent Desaubry a, Francois Gaudiot a, Mohamed Seloum a, Jean-
¸
Jacques Bourguignon a
a Laboratoire de pharmacochimie de la communication cellulaire, UMR 7081 du CNRS, Universite´ Louis Pasteur, faculte´ de pharmacie, 74, route du
Rhin, BP24, 67401 Illkirch cedex, France
b Laboratoire de pharmacologie et de physico-chimie des interactions cellulaires et mole´culaires, UMR 7034 du CNRS, Universite´ Louis Pasteur, faculte´
de pharmacie, 74, route du Rhin, BP24, 67401 Illkirch cedex, France
Received 29 September 2002; received in revised form 9 December 2002; accepted 11 December 2002
Abstract
Adenine derivatives substituted in position 9 have been demonstrated to have potent cyclic nucleotide phosphodiesterase (PDE)
inhibition properties with high selectivity toward PDE-4. Starting from our initial lead compound 9-(2-fluorobenzyl)-N6-methyl-2-
trifluoromethyladenine (4, NCS613), we designed and synthesized a new series of 9-substituted derivatives for developing structureꢀ
/
activity relationship studies. This new series of derivatives showed increased potencies and better selectivity profiles. Structural
modifications were achieved in parallel on three different positions of the adenine ring, and led to the following observations: (i)
introduction of a lipophilic substituent such as trifluoromethyl, n-propyl group or iodine in the C-2 position is favourable for both
the PDE-4 inhibitory activity and the selectivity towards other isoenzymes; (ii) functionalization of the N9 benzyl group with a 2-
methoxy substituent led to remarkably more active compounds; (iii) replacement of the N6-methylamino moiety by other amino
groups is detrimental to the activity. Among all derivatives prepared, the 9-(2-methoxybenzyl)-N6-methyl-2-trifluoromethyladenine
(9r), 9-(2-methoxybenzyl)-N6-methyl-2-n-propyladenine (9s), and the 2-iodo-9-(2-methoxybenzyl)-N6-methyladenine (13b) were
found to be the most potent inhibitors within this series (PDE-4-IC50
reference compound 4, which showed an IC50 of 42 nM, the derivative 13b was found 450-fold more potent. Moreover, 2-iodo-9-(2-
methoxybenzyl)-N6-methyladenine (13b) and 9-(2-methoxybenzyl)-N6-methyl-2-trifluoromethyladenine (9r), were at least 50 000ꢀ
ꢁ1.4, 7.0, and 0.096 nM, respectively). Compared to our
/
/
150 000 times more selective for the PDE-4 than for the other PDE families. Additionally, these new derivatives showed improved
efficiency in inhibiting the TNFa release from mononuclear cells from healthy subjects (e.g. adenines 7l, 9s and 13b). Thus,
compounds 7l, 9r, 9s and 13b are among the most potent and selective PDE-4 inhibitors reported so far and represent very
promising pharmacological tools for a better understanding of the signal transduction involving cyclic AMP within the cell: this
pathway is implicated in the physiology and the pathophysiology of inflammation, asthma and autoimmune disorders.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Adenine; Phosphodiesterase; Type-4; TNFa; Inflammation
1. Introduction
Cyclic nucleotide phosphodiesterases (PDEs), respon-
sible for the hydrolysis of key second messengers cyclic
AMP (cAMP) and cyclic GMP (cGMP), have been
* Corresponding author.
E-mail address: pierreraboisson@aol.com (P. Raboisson).
1
classified into at least 11 major families with respect to
their substrate specificity, sequence similarity, and
Present address: Department of Chemistry, 3-Dimensional
Pharmaceuticals, Inc., 665 Stockton Drive, Exton, PA 19341, USA.
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(02)01446-0

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P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
sensitivity to inhibitory drugs [1,2]. PDE-4 is a cAMP-
specific enzyme, separated in four human isoforms
(PDE-4 A, B, C, and D) [3], and represents a promising
potential molecular target for the development of new
antiasthmatic and antiinflammatory drugs. Further-
more, PDE-4 inhibitors have demonstrated efficacy in
models of dermatitis, rheumatoid arthritis, multiple
sclerosis, autoimmune diseases and various gastrointest-
stimulate the in vivo gastric acid secretion in rats
suggests that they may produce fewer gastrointestinal
side effects than other PDE-4 inhibitors, such as
compound 2 (Fig. 1) [17].
The aim of this work was to optimize our selective
PDE-4 lead inhibitor 9-(2-fluorobenzyl)-N6-methyl-2-
trifluoromethyladenine (4, Fig. 1). For this purpose a
systematic exploration of possible substitutions and
functionalizations on three different positions on the
adenine ring (positions 2, 6, and 9) was undertaken.
inal and neurological diseases [1,4ꢀ8]. Thus, several
/
PDE-4 inhibitors are under clinical development. Un-
fortunately, the development of PDE-4 inhibitors, such
as rolipram (1, Fig. 1) and structurally-related com-
pounds has been so far limited by various side effects,
such as nausea, emesis, gastric acid secretion, or central
nervous system activation [7,8,11,12]. Thus, the design
of novel, potent and selective second generation of
PDE-4 inhibitors with reduced side effects represent a
critical need and is still a challenge in the pharmaceutical
2. Chemistry
Three main synthetic approaches leading to adenine
derivatives are described in the literature, depending
upon the position of the substitution to be explored: (i)
syntheses started from a substituted pyrimidine nucleus
followed by imidazole ring construction (exploration of
position 8) [14]; (ii) or starting from the corresponding
imidazole ring, then construction of the pyrimidine
industry [5,8ꢀ10]. One way to increase both the potency
/
and the selectivity of PDE-4 inhibitors, and thus
improve the therapeutic index, is to develop new
compounds featuring original chemical structures, dif-
ferent than those of the known PDE-4 inhibitors [13], if
the side effects are not mechanism-based.
Several years ago, Kelley and Sokoro described the 9-
(2-fluorobenzyl)-N6-methyladenine (3) as a potent antic-
onvulsant [14]. We preliminarily demonstrated that this
compound presents potent anxiolytic and sedative
properties [15], and was a relatively potent PDE-4
nucleus (exploration of position 2) [19ꢀ
substitution of the preformed purine ring (exploration of
N-9 substitution) [16,23ꢀ27].
/22]; (iii) or direct
/
We previously demonstrated that within this series of
adenine PDE-4 inhibitors, no substitution is tolerated at
position 7. Thus, both alkylation of the purine ring and
inhibitor (IC50ꢁ
/
2.0 mM) [16]. Furthermore, initial
structureꢀactivity relationship (SAR) studies around 9-
/
substituted adenine derivatives allowed us to identify 9-
(2-fluorobenzyl)-N6-methyl-2-trifluoromethyladenine
(4), as a potent PDE-4 inhibitor (IC50ꢁ0.042 mM), with
/
a high selectivity vs PDE-3 [16]. Moreover, compound 4
elicited anti-inflammatory properties [17], and marked
dose-dependent inhibition of arachidonate release from
human mononuclear cells stimulated with N-formyl-
Metꢀ
/
LeuꢀPhe, a suitable model to investigate the in
/
vitro anti-inflammatory activity of PDE-4 inhibitors
[18]. In addition, compound 4 and several 9-substituted
adenine derivatives elicited a concentration-dependent
inhibition of the TNFa release from mononuclear cells
stimulated with lipopolysaccharide (LPS) [18]. More-
over, the fact that this series of PDE-4 inhibitors did not
Fig. 2. Access to the adenine derivatives by chloropurines and
adenines
alkylation.
Reagents:
(a)
R9Cl,
NaH;
(b)
PhCH(OH)COOMe, PPh3, DEAD; (c) HNR6R6?; (d) H2NCH3; (e)
NaBH4; (f) R9Cl, K2CO3, TBAI.
Fig. 1. Structures of PDE type-4 inhibitors.

P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ
/
214
201
cyclization of an appropriate 4-aminoimidazole-5-car-
boxamide were used to further explore substitutions and
functionalizations around positions 2 and 9 [16].
A classical route to prepare the 9-substituted adenines
7 and 9 deals with direct alkylation of the purine ring
with various amines provided the desired adenines 7
(Table 1) [24]. In the case of compound 7h, the
Mitsunobu procedure was used to afford the desired
product in 73% overall yield [26].
More recently, direct alkylation of adenines 8 under
phase-transfer catalyst conditions was reported to con-
stitute a powerful synthetic tool for the preparation of
N9 alkyl adenines [27]. This procedure was superior to
the previously described synthesis using the classical
alkylation of chloropurines, because it was assumed to
lead to regioselective alkylation at position 9 of the
purine ring (Fig. 2). However, in our hands, while the
N9-alkylated derivatives were obtained as major pro-
ducts, traces of N7-substituted compounds were also
using different experimental conditions [16,23ꢀ27].
/
Thus, different N9-alkyl adenine derivatives 7 were
readily prepared by alkylation of corresponding chlor-
opurines 5 [25] with the appropriate alkyl halides and
sodium hydride in dimethylformamide (Fig. 2) [16,24].
Under these conditions, alkylation occurred mainly at
position N9 (70ꢀ/85%), and the undesired N7 isomer
(15ꢀ30%) could be easily removed by silica gel column
/
chromatography. Subsequent treatment of compounds 6
Table 1
Physical constants and inhibition of PDE-4 by 6,9-disubstituted adenines
a
b
c
Compound Salt
m.p. (8C) Yield (%) Elemental analysis
R6
R6? R9
PDE-4-IC50 (mM) or
(%) of inhibition
d
7a
7b
7c
di-HCl
224
234
248
79
83
84
C12H12N6×
C12H11N5O×
C16H17N5×HCl×1H2O
/
2HCl
NH2
OH
H
H
Bn
Bn
Bn
30
32%
21
HCl
HCl
/
HCl×
/1H2O
/
/
7d
di-HCl
ꢀ/
300
53
C16H18N6×
/
2HCl
Bn
107
7e
7f
free base
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
221
248
266
204
182
220
258
200
210
230
252
212
232
205
274
287
257
222
205
215
244
64
78
72
73
68
80
73
76
51
57
20
40
30
46
37
38
25
20
56
40
73
C13H13N5O
C13H12ClN5×
C14H13N5O2×
15H15N5O2×
C15H16N6O×HCl×
C14H15N5O×HCl
C12H11N5×HCl×
C13H13N5×HCl×
10H15N5×HCl×
C11H15N5×HCl×
C11H17N5×HCl×
HCl
HCl×
HCl×
13H12ClN5×
13H12BrN5×
HCl×
HCl×
HCl×
HCl
C20H17N5O×HCl
OMe
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Bn
3-ClBn
90
4.0
0.89
12
/
HCl×
HCl
HCl×
2.1H2O
/
0.7H2O
7g
7h
7i
/
2,3-(methylenedioxy)Bn
(MeOCO)(Ph)CH
(MeNHCO)(Ph)CH
(HOCH2)(Ph)CH
Ph
C
/
/1.6H2O
/
/
91
7j
/
19
7k
9a
9b
9c
9d
9e
9f
/
/
0.2H2O
0.5H2O
3H2O
10
/
/
Bn
n-Bu
4.6
13
C
/
/
/
/2.3H2O
c-Pen
neopenthyle
dodecyle
2.9
6.6
61%
22
/
/1.5H2O
C
18H31N5×
/
C14H15N5×
/
/0.2H2O
Ph(CH2)2
Ph(CH2)3
9g
9h
9i
C15H17N5×
C
/
/1.5H2O
4.8
14
/
HCl×
/0.4H2O
4-ClBn
2-BrBn
C
/
HCl
1.0
0.50
8.8
27
9j
C14H15N5O×
C14H15N5O×
14H13N5O×
C21H21N5×
/
/
0.2H2O
0.4H2O
0.5H2O
2-(MeO)Bn
4-(MeO)Bn
PhCOCH2
(Ph)2CH(CH)2
4-(PhCO)PhCH2
9k
9l
/
/
C
/
/
9m
9n
/
11
13
/
a
For the final step.
C, H, N.
b
c
The IC50 was calculated by linear regression (correlation coefficient rꢁ0.95) and represents the mean value of three determinations. The
/
experimental error is about 15%.
d
At 100 mM of final drug concentration.

202
P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
observed. Despite the fact that the separation of these
polar adenine derivatives led to poor overall yields, this
synthetic method appeared to be particularly powerful
for an expeditious preparation of N9-substituted deri-
vatives in one-step procedure. Yields and physical
constants of adenine derivatives are listed in Table 1
and Table 2.
The presence of an amino moiety at position 2
constitutes another versatile synthetic route to access
at 2,9-disubstituted adenine derivatives. As shown in
Fig. 3, alkylation of the 2-amino-6-chloropurine (10)
gave 9-substituted intermediates 11a and 11b in a
regioselective manner [27]. The amino group was con-
verted to the key iodo intermediates (12a, b) [28], which
were subsequently displaced with methylamine to afford
adenines 13a and 13b.
iodine in position 2 could be easily displaced by sodium
methanethiolate leading to 14d.
3. Pharmacology, results and discussion
All N9-substituted adenine derivatives were tested for
their inhibitory capacities of bovine PDE-4 as described
in the Section 5. The concentration that inhibited 50% of
the enzymatic activity (IC50), or the percentage of
inhibition at a final concentration of 100 mM of each
drug were determined at 1 mM cAMP concentration.
Data are listed in Table 1 and Table 2. Isoenzyme
selectivity profile of the most potent inhibitors was
obtained by comparing the IC50 values of the com-
pounds against PDE-4 with their inhibitory activity
against PDE-1, -2, -3, and -5 (Table 3). Inhibition of
TNFa release from mononuclear cells stimulated with
LPS was also reported for the compounds 7l, 9s and 13b,
The last step of the synthesis involved palladium-
catalyzed cross-coupling reactions and led to 2-substi-
tuted derivatives 14aꢀc (Fig. 3) [29,30]. Finally, the
/
Table 2
Physical constants and inhibition of PDE-4 by 2,9-disubstituted N6-methyladenines
a
b
c
Compound Salt
m.p. (8C) Yield (%) Elemental analysis
R2
R9
PDE-4-IC50 (mM)
4, [16]
7l
free base 138
ꢀ
/
ꢀ
/
CF3
2-FBn
Bn
0.042
0.088
0.12
0.16
7.1
HCl
HCl
186
181
238
236
276
236
75
73
81
85
60
73
62
82
84
80
83
62
67
65
45
79
74
C
16H19N5×
C16H19N5×
C19H23N5×
/
HCl
HCl
HCl
HCl
HCl
2HCl
n-Pr
i-Pr
7m
7n
/
Bn
Bn
HCl
HCl
/
c-Hex
t-Bu
7o
7p
C
17H21N5×
/
Bn
Bn
HCl
di-HCl
C
21H19N5×
/
PhCHꢀ
NH2
CF3
Me
/
CH
9.0
6.9
7q
7r
C13H14N6×
C14H13F3N6O
/
Bn
free base 180
2-(MeO)-3-pyridylCH2
2-(MeO)Bn
4-(MeO)Bn
2-(MeO)Ph(CH2)2
CH3O(CH2)2O(CH2)2
Bn
0.10
0.061
0.92
54
7s
HCl
HCl
HCl
HCl
HCl
HCl
HCl
236
242
218
98
C15H17N5O×
15H17N5O×
C16H19N5O×
12H19N5O2×
C18H23N5×HCl
C21H21N5×HCl×
C22H23N5×HCl×
15H14F3N5O×
C17H21N5O×HCl×
23H23N5O×HCl×
/
HCl
HCl
HCl
7t
C
/
Me
7u
/
Me
7v
9o
C
/
HCl×
/
1.5H2O
Me
n-Pen
20
188
230
174
/
1.8
2.2
9p
/
/2H2O
Ph(CH2)2
Bn
9q
/
/1.3H2O
Ph(CH2)3
Bn
1.3
9r
Free base 169
C
/
0.2H2O
CF3
n-Pr
n-Pr
Me
I
2-(MeO)Bn
2-(MeO)Bn
4-(PhCO)PhCH2
2-FBn
0.0014
0.0070
0.92
0.19
0.030
0.000096
0.092
0.015
0.055
0.080
9s
HCl
HCl
119
241
/
/
0.5H2O
9t
C
/
/
0.5H2O
9u, [17]
13a
13b
14a
14b
14c
14d
MeSO3H 146
ꢀ/
ꢀ/
HCl
HCl
HCl
HCl
HCl
HCl
221
192
195
151
205
158
63
78
70
60
72
88
C
13H12IN5×
C14H14IN5O×
16H15N5×HCl
C17H17N5O×HCl×
C17H19N5O×HCl
C15H17N5OS×HCl
/
HCl
Bn
2-(MeO)Bn
Bn
/
HCl
I
C
/
CH3Cꢁ
CH3Cꢁ
CH3CHꢀ
CH3S
/C
/
/
1.6H2O
/C
2-(MeO)Bn
CH 2-(MeO)Bn
/
/
/
2-(MeO)Bn
a
For the final step.
C, H, N.
b
c
The IC50 was calculated by linear regression (correlation coefficient rꢁ0.95) and represents the mean value of three determinations. The
/
experimental error is about 15%.

P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ
/
214
203
Fig. 3. Access to the adenine derivatives by palladium cross-coupling reactions or aromatic nucleophilic substitution in position 2. Reagents: (a)
R9Cl, K2CO3, TBAI; (b) isoamyl nitrite, CH2I2; (c) H2NCH3; (d) CH3Cꢁ
/
CH, CuI, PdCl2, PPh3, TEA; (e)
Pd(PPh3)4, NaOH; (f)
MeSNa.
Table 3
Selectivity of adenine derivatives for PDE-4 vs PDE-1, -2, -3 and -5
a
b
Compound
R2
R9
IC50 (mM) or (%) of inhibition
PDE-1
39
PDE-2
41%
16%
16%
15
PDE-3
380
PDE-4
0.045
0.082
PDE-5
5
24
4 (NCS613)
7l (NCS675)
9r (NCS728)
9s (NCS700)
13b (NCS706)
CF3
n-Pr
CF3
n-Pr
I
2-FBn
Bn
33%
20%
62
43%
24%
54%
33%
2-(MeO)Bn
2-(MeO)Bn
2-(MeO)Bn
0.0014
0.0070
0.000096
74
41
49
26%
15
a
The IC50 was calculated by linear regression (correlation coefficient rꢁ/0.95) and represents the mean value of three determinations. The
experimental error is about 15%.
b
At 100 mM of final drug concentration.
which were found as three of the most potent PDE-4
inhibitors within this series.
out with the new series of adenine derivatives confirmed
these preliminary results. As shown in Table 1, replace-
ment of the methylamino group at position 6 with a
pyrrolidyl or a piperazinyl moiety induced dramatic
reductions in PDE-4 inhibitory activity (compare com-
pounds 7c and 7d with 9a). Other substitutions with an
amino, hydroxy or methoxy group were detrimental to
the activity (compare 7a, 7b and 7e with 9a).
The various substitutions and functionalizations on
the three different positions (2, 6 and 9) were designed to
complete the initial SAR studies, which already led to
the identification of the potent PDE-4 inhibitor 9-(2-
fluorobenzyl)-N6-methyl-2-trifluoromethyladenine (4,
IC50ꢁ45 nM) [16]. Improving PDE-4 activity and
/
Furthermore, a series of homologues of the 9-benzyl
derivative 9a was first tested to optimize the linker
between the adenine ring and the phenyl moiety at
position 9. Thus, a methylene group was found to be a
better spacer than an ethylene one (compare 9a and 9f).
When the linker is absent (9-phenyl substituent in 7k)
[31], the activity was 2-fold diminished. Surprisingly, the
phenylpropyl derivative 9g was as active as the reference
selectivity vs PDE-1, -2, -3 and -5 was also one of the
goods of the present study.
As reported in our previous works [16], a single
substitution with a methyl group on the exocyclic
nitrogen atom at position 6 of the adenine ring was
optimal. In particular, both N,N-dimethyl and N,N-
unsubstituted derivatives were 50- to 300-fold less active
than the N-monomethyl adenine 4. SAR studies carried

204
P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
substituent in position 2. In each case the 2-methoxy
derivative was found more active (3 to 300-fold) than
the corresponding unsubstituted benzyl or 2-fluoroben-
zyl compounds (compare 9u and 7s, 4 and 9r, 7l and 9s,
13a and 13b or 14a and 14b). In particular, the 2-n-
propyl (9s) and 2-trifluoromethyl (9r) derivatives ex-
hibited high potencies with IC50 values equal to 7.0 and
1.4 nM, respectively. However, the iodo analogue 13b
was found to be the most potent PDE-4 inhibitor with
subnanomolar PDE-4 IC50 (0.096 nM). Moreover, these
structural optimizations around the positions 2 and 9
provided improved PDE-4 selectivity profiles vs PDE-1,
-2, -3, and -5 (Table 3). Thus, considering simulta-
neously inhibition of these five different isoenzymes
Fig. 4. Effects of compound 3, 7l, 9s and 13b on TNFa release by LPS-
activated peripheral blood mononuclear cells. The properties of
compounds 3, 7l, 9s and 13b on TNFa production by mononuclear
cells was evaluated by ELISA as described in Experimental Section in
(PDE-1ꢀ
/
5), compounds 9r and 13b were at least 50 000ꢀ
/
presence of 5 mg mLꢂ1 of LPS. Results (mean9
S.E. mean) express
/
150 000 times more selective toward the PDE-4.
TNFa production as the percentage of LPS activated control.
Finally, we examined the effects of compound 7l, 9s
and 13b on TNFa release by LPS-activated peripheral
blood mononuclear cells from healthy donors (Fig. 4).
These compounds elicited a strong concentration-de-
pendent inhibition of the TNFa release and were much
compound 9a. Except the 9-cyclopentyl derivative (9c),
all other lipophilic, non-aromatic substituents at the
same position led to less active compounds (see 9b, 9d
and 9e).
Different substituents were introduced on the aro-
matic carbocycle of derivative 9a. As shown in Table 1,
introduction of a substituent in ortho position of the
phenyl ring appeared to be especially beneficial. Thus,
the 2-bromobenzyl and 2-methoxybenzyl groups at
position 9 produced compounds 5- to 10-fold more
potent (compare 9i and 9j with 9a). Other 9-benzylade-
nines functionalized in positions 3 (7f), 4 (9h, 9k, 9n) or
more potent (7l-IC50ꢁ
13b-IC50ꢁ9 mM, respectively) than the reference com-
pound 3 (IC50ꢀ10 mM) [17]. Unexpectedly, we found
/
0.5 mM, 9s-IC50ꢁ0.7 mM and
/
/
/
no direct correlation between the activity of the com-
pounds 3, 7l, 9s and 13b in PDE-4 inhibition and TNFa
inhibition. Such a finding suggests that accumulation of
cAMP is a major way to decrease TNFa secretion but
certainly not the only one.
a (7hꢀ
/
j) of the benzyl ring and other N9-functionalized
products (7g, 9l, 9m) were either less active than the
unsubstituted benzyl derivative 9a or equipotent (7f).
The introduction of other methoxy-substituted side
chains led to less active compounds when compared to
the 2-methoxybenzyl 7s (see cpds 7u, 7v, Table 2).
Earlier SAR studies around position 2 established
that the introduction of a methyl or a trifluoromethyl
group strongly increase the potency of the resulting
compounds [16]. In a similar manner, when compared to
4. Conclusions
In summary, structural optimization within the 9-
substituted adenine series of PDE-4 inhibitors, led to
compounds with very potent PDE-4 inhibitory activities
(IC50 values in the nanomolar range) and PDE-4
selectivity vs PDE-1, -2, -3, and -5. In particular, the
9-(2-methoxybenzyl)-N6-methyl-2-n-propyladenine (9s)
and 9-(2-methoxybenzyl)-N6-methyl-2-trifluoromethyla-
denine (9r) derivatives showed high potencies with IC50
values of 7.0 and 1.4 nM, respectively. Surprisingly, the
iodo derivative 13b was found to be the most potent
PDE-4 inhibitor with subnanomolar PDE-4 IC50 value
9a (IC50ꢁ
/
4.6 mM), the n-propyl (7l) and propynyl
derivatives (14a) were found more potent (IC50:
nM), indicative of some degree of steric tolerance at
position 2 (Table 2). However, replacing the n-propyl or
/
90
propynyl groups by more bulky alkyl substituents (7mꢀ
/
(IC50ꢁ0.096 nM), 450-fold more active than our initial
/
lead compound 9-(2-fluorobenzyl)-N6-methyl-2-trifluor-
omethyladenine (4). Moreover, these structural optimi-
zations combining both positions 2 and 9 provided
improved PDE-4 selectivity vs PDE-1, -2, -3, and -5 with
o, 9o) or phenylalkyl groups (7p, 9p and 9q) always
produced less active compounds. Surprisingly, introdu-
cing an iodine atom in position 2 led to the most potent
compound within the 9-benzyl series (IC50 13aꢁ30
/
nM), whereas the 2-amino derivative 7q was less active
than the unsubstituted compound 9a.
an index of selectivity reaching 50 000ꢀ150 000 for
/
compounds 9r and 13b. Thus, compounds 9r, 9s and
13b appear as three of the most potent and selective
PDE-4 inhibitors so far known. Additionally, these new
derivatives showed improved efficacies in inhibiting the
TNFa release from mononuclear cells from healthy
subjects, as shown with compounds 7l, 9s, and 13b.
Finally, combining beneficial substituent effects in
both positions 2 and 9 led to new compounds with
increased potencies. Thus, synergistic effects were ob-
served when the 9-(2-methoxybenzyl) moiety was intro-
duced in derivatives with the most appropriate

P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ
/
214
205
This family of adenine derivatives that may compete
with cAMP within its binding site, differs from other
dialkoxyaryl-related compounds, and other known
PDE-4 inhibitors. This work thus highlights new potent
and selective PDE-4 inhibitors (e.g. compounds 9r, 9s
and 13b) as very promising pharmacological tools for a
better understanding of the signal transduction (includ-
ing cAMP) within the cell.
diluted with cold water (20 mL). The precipitate was
collected, washed with water and suspended in potas-
sium hydrogen carbonate (0.5 N, 140 mL). The resulting
suspension was heated to reflux for 4 h, concentrated
under reduced pressure, cooled to 0 8C, and adjusted to
pH 4 with 10% aqueous HCl. The solid was collected,
washed with water and EtOH, and dried in a vacuum
oven at 50 8C for 1 h. Then, phosphorus oxychloride
(10 mL, 107 mmol) and N,N-dimethylaniline (2 mL,
15.8 mmol) were added and the resultant mixture was
heated to reflux under nitrogen for 1 h, cooled down to
room temperature, concentrated under reduced pressure
and diluted with CH2Cl2. This solution was treated with
HCl gas at 0 8C. The precipitate solid was collected,
washed with CH2Cl2 and ether to give 5b as a white solid
which was identical in all respects with the product
reported in Ref. [16].
5. Experimental section
5.1. Chemical synthesis
5.1.1. General
Reagents used for the synthesis were purchased from
Sigma-Aldrich (Isle d’Abeau Chesnes, France) and
Lancaster (Bischheim-Strasbourg, France). [3H]-cAMP
(1.1ꢀ
/
1.85 TBq nmolꢂ1, TRK 498) was obtained from
5.1.3. 6-Chloro-2-n-propylpurine hydrochloride (5c)
The title compound was prepared (70%) from 5-
aminoimidazole-4-carboxamide hydrochloride and bu-
Amsterdam (Les Ulis, France). With the exception of
THF and Et2O, all solvents were obtained from
commercial suppliers and used without further purifica-
tion. These two solvents were freshly distilled from
sodium benzophenone ketyl. Flash chromatography was
1
tyryl chloride as described for 5b: H-NMR (200 MHz,
CD3OD) d 1.00 (t, Jꢁ
CH2), 3.03 (t, Jꢁ7.5, 2H, CH2), 9.46 (s, 1H, 8-H); m/z
197 (Mꢃ
H)ꢃ.
/
7.3, 3H, CH3), 1.81ꢀ1.95 (m, 2H,
/
/
performed on Geduran† Silica gel Si 60 (40ꢀ
Merck). Thin-layer chromatography was carried out
using plates Silica gel 60 F254 (Merck). The spots were
/
63 mm,
/
5.1.4. 6-Chloro-2-isopropylpurine hydrochloride (5d)
The title compound was prepared (63%) from 5-
aminoimidazole-4-carboxamide hydrochloride and 2-
methylpropionyl chloride as described for 5b: 1H-
visualized either under UV light (lꢁ
spraying with molybdate reagent (H2Oꢀ
H2SO4ꢀ(NH4)6Mo7O24×4H2Oꢀ(NH4)2Ce(SO4)4×
/
254 nm) or by
concentrated
2H2O,
/
/
/
/
/
90/10/25/1, v/v/w/w) and charring at 140 8C for a few
minutes. All chemical yields are unoptimized and
generally represent the result of a single experiment.
1H-NMR were recorded on a Bruker AC 200 (200
MHz) or a Bruker DPX 300 (300 MHz) spectrophot-
ometer at room temperature. Chemical shifts are given
in ppm (d), coupling constants (J) are in hertz (Hz) and
signals are designated as follows: s, singlet; d, doublet; t,
triplet; q, quadruplet; quint., quintuplet; m, multiplet; br
s, broad singlet.
NMR (200 MHz, CD3OD) d 1.39 (d, Jꢁ
2CH3), 2.96 (m, 1H, CH), 9.38 (s, 1H, 8-H); m/z 197
(Mꢃ
H)ꢃ.
/6.9, 6H,
/
5.1.5. 6-Chloro-2-cyclohexylpurine hydrochloride (5e)
The title compound was prepared (73%) from 5-
aminoimidazole-4-carboxamide hydrochloride and cy-
1
clohexanecarboxylyl chloride as described for 5b: H-
NMR (200 MHz, CD3OD) d 1.25ꢀ
5CH2), 2.92ꢀ3.08 (m, 1H, CH), 9.39 (s, 1H, 8-H); m/z
237 (Mꢃ
H)ꢃ.
/2.09 (m, 10H,
/
The mass spectra were obtained on a Mariner API-
TOF.
/
Melting points were determined with a Mettler FP62
apparatus and are uncorrected. Elemental analyses were
performed by the CNRS department of microanalysis
(CNRS, Vernaison, France) and are indicated only by
5.1.6. 6-Chloro-2-tert-butylpurine hydrochloride (5f)
The title compound was prepared (66%) from 5-
aminoimidazole-4-carboxamide hydrochloride and pi-
valoyl chloride as described for 5b: 1H-NMR (200 MHz,
CD3OD) d 1.48 (s, 9H, 3CH3), 9.40 (s, 1H, 8-H); m/z
the elemental symbols within 9
/
0.4% of the theoretical
values unless otherwise noted.
211 (Mꢃ
H)ꢃ.
/
5.1.2. 6-Chloro-2-methylpurine hydrochloride (5b)
To a solution of 5-aminoimidazole-4-carboxamide
hydrochloride (500 mg, 3.08 mmol), 4-(N,N-dimethyla-
mino)pyridine (12.5 mg, 0.10 mmol) in anhydrous
pyridine (25 mL) was slowly added acetyl chloride
(241 ml, 3.39 mmol). The mixture was stirred at 80 8C
for 8 h, concentrated under reduced pressure, then
5.1.7. 6-Chloro-2-trans-styrylpurine hydrochloride (5g)
The title compound was prepared (80%) from 5-
aminoimidazole-4-carboxamide hydrochloride and
trans-cinnamoyl chloride as described for 5b: 1H-
NMR (200 MHz, CD3OD) d 7.46 (d, Jꢁ
CH), 7.36ꢀ7.71 (m, 5H, 5CH), 7.85 (d, Jꢁ
CH), 9.45 (s, 1H, 8-H); m/z 257 (Mꢃ
H)ꢃ.
/
16.1, 1H,
16.1, 1H,
/
/
/

206
P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
5.1.8. 6-Chloro-2-n-pentylpurine hydrochloride (5h)
The title compound was prepared (78%) from 5-
aminoimidazole-4-carboxamide hydrochloride and ca-
proyl chloride as described for 5b: 1H-NMR (200 MHz,
5.1.12. 9-Benzyl-6-hydroxylaminopurine hydrochloride
(7b)
The title compound was prepared from 6-chloropur-
ine, benzyl chloride and hydroxylamine following the
general procedure for the alkylation of 6-chloropurines
CD3OD) d 0.90 (t, Jꢁ
2CH2), 1.80ꢀ1.95 (m, 2H, CH2), 3.05 (t, Jꢁ
CH2), 9.48 (s, 1H, 8-H); m/z 225 (Mꢃ
H)ꢃ.
/
7.0, 3H, CH3), 1.30ꢀ
/
1.42 (m, 4H,
/
/
7.6, 2H,
(method A): 1H-NMR (200 MHz, DMSO-d6ꢃ
/
CD3OD)
7.49 (m, 5H, 5CH), 8.51 (s,
1H, 2-H), 8,80 (s, 1H, 8-H). Anal. Found: C12H11N5O×
HCl×1H2O (C, H, N).
/
d 5.62 (s, 2H, CH2), 7.42ꢀ
/
/
5.1.9. 6-Chloro-2-(3-phenylpropyl)purine hydrochloride
(5i)
/
The title compound was prepared (71%) from 5-
aminoimidazole-4-carboxamide hydrochloride and 4-
phenylbutyryl chloride as described for 5b: 1H-NMR
5.1.13. 9-Benzyl-6-(1-pyrrolidyl)purine hydrochloride
(7c)
The title compound was prepared from 6-chloropur-
ine, benzyl chloride and pyrrolidine following the
general procedure for the alkylation of 6-chloropurines
(200 MHz, CD3OD) d 2.13ꢀ
Jꢁ7.7, 2H, CH2), 3.02ꢀ3.11 (m, 2H, CH2), 7.08ꢀ
(m, 5H, 5CH), 9.45 (s, 1H, 8-H); m/z 295 (Mꢃ
Na)ꢃ.
/2.29 (m, 2H, CH2), 2.71 (t,
/
/
/
7.26
1
/
(method A): H-NMR (200 MHz, DMSO-d6) d 2.13ꢀ
/
2.20 (m, 4H, 2CH2), 3.86ꢀ
(m, 2H, CH2), 5.65 (s, 2H, CH2), 7.41ꢀ
5CH), 8.58 (s, 1H, 2-H), 8.80 (s, 1H, 8-H). Anal. Found:
C16H17N5×HCl×1H2O (C, H, N).
/
3.92 (m, 2H, CH2), 4.30ꢀ
/
4.36
5.1.10. General procedure for the alkylation of 6-
chloropurines (5) and subsequent displacement using
/
7.49 (m, 5H,
various amines provided adenines 7aꢀ
/
j, 7lꢀ
/
p and 7rꢀ
/
v
/
/
(method A)
To a stirring solution of 6-chloropurine hydrochloride
5 (0.976 mmol) in dry DMF (4 mL) was added at 0 8C
NaH (50% in mineral oil, 121 mg). After 5 min, the
reaction mixture was allowed warm up to room
temperature: the requisite alkyl halide (2.61 mmol) was
then added and stirring was continued for 24 h. The
mixture was diluted with water (15 mL), extracted with
ethyl acetate (60 mL), dried (Na2SO4), and concentrated
to dryness under reduced pressure. Chromatography on
silica (AcOEt) afforded the desired N9-alkylated pro-
duct 6. Sequential addition of ethanol (10 mL) and the
appropriate amine (5 mmol) was carried out under
argon atmosphere. The resulting solution was stirred for
24 h at room temperature. After evaporation of the
solvent, the residue was diluted with ethyl acetate (60
mL), washed with cold water (20 mL) and saturated
sodium bicarbonate (10 mL), dried (Na2SO4) and
concentrated under reduced pressure. Chromatography
5.1.14. 9-Benzyl-6-(1-piperazinyl)purine di
hydrochloride (7d)
The title compound was prepared from 6-chloropur-
ine, benzyl chloride and piperazine following the general
procedure for the alkylation of 6-chloropurines (method
1
A): H-NMR (200 MHz, DMSO-d6) d 3.34ꢀ
/
3.39 (m,
4.63 (m, 4H, 2CH2), 5.56 (s, 2H, CH2),
7.48 (m, 5H, 5CH), 8.48 (s, 1H, 2-H), 8.58 (s, 1H,
4H, 2CH2), 4.55ꢀ
7.39ꢀ
/
/
8-H), 9.57 (br s, 1H, NH). Anal. Found: C16H18N6×
2HCl (C, H, N).
/
5.1.15. 9-Benzyl-N6-methoxyadenine (7e)
The title compound was prepared from 6-chloropur-
ine, benzyl chloride and methoxylamine following the
general procedure for the alkylation of 6-chloropurines
1
(method A): H-NMR (200 MHz, CDCl3ꢃ
/
CD3OD) d
7.37 (m, 5H,
3.97 (s, 3H, CH3), 5.35 (s, 2H, CH2), 7.25ꢀ
5CH), 7.74 (s, 1H, 2-H), 8.17 (s, 1H, 8-H). Anal. Found:
C13H13N5O (C, H, N).
/
on silica (AcOEtꢀ
/
CH2Cl2ꢀEtOH, 50:40:10) afforded
/
adenines 7 as colorless solids, which were eventually
converted into the HCl salt. An analytically pure sample
was obtained by recrystallization from ethanol and
diethyl ether. Unoptimized percentage yields, referring
to the last step, are given for each product (Tables 1 and
2).
5.1.16. 9-(3-Chlorobenzyl)-N6-methyladenine
hydrochloride (7f)
The title compound was prepared from 6-chloropur-
ine, 3-chlorobenzyl chloride and methylamine following
the general procedure for the alkylation of 6-chloropur-
ines (method A): 1H-NMR (200 MHz, DMSO-d6) d
5.1.11. 9-Benzyl-6-hydrazinopurine di hydrochloride
(7a)
The title compound was prepared from 6-chloropur-
ine, benzyl chloride and hydrazine following the general
procedure for the alkylation of 6-chloropurines (method
A): 1H-NMR (200 MHz, DMSO-d6) d 5.62 (s, 2H,
3.13 (br s, 3H, CH3), 5.53 (s, 2H, CH2), 7.32ꢀ
4H, 4CH), 8.50 (s, 1H, 2-H), 8.66 (s, 1H, 8-H), 9.75 (br
s, 1H, NH). Anal. Found: C13H12ClN5×HCl×0.7H2O (C,
H, N).
/
7.47 (m,
/
/
5.1.17. N6-methyl-9-(2,3-methylenedioxybenzyl)adenine
hydrochloride (7g)
The title compound was prepared from 6-chloropur-
ine, 2,3-methylenedioxybenzyl chloride and methyla-
CH2), 7.40ꢀ
(s, 1H, 8-H), 10.21 (br s, 3H, NHꢃ
C12H12N6×2HCl (C, H, N).
/
7.49 (m, 5H, 5CH), 8.60 (s, 1H, 2-H), 8.77
/
NH2). Anal. Found:
/

P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ
/
214
207
mine following the general procedure for the alkylation
of 6-chloropurines (method A): 1H-NMR (200 MHz,
DMSO-d6) d 3.10 (br s, 3H, CH3), 5.45 (s, 2H, CH2),
(Na2SO4), and concentrated to dryness under reduced
pressure. Chromatography on silica and elution
(AcOEtꢀEtOH, 80:20) afforded the desired product as
/
6.03 (s, 2H, CH2), 6.71ꢀ
2-H), 8.51 (s, 1H, 8-H), 9.72 (br s, 1H, NH). Anal.
Found: C14H13N5O2×HCl (C, H, N).
/
6.91 (m, 3H, 3CH), 8.46 (s, 1H,
a colorless solid, which was converted to the HCl salt 7j.
Recrystallization from ethanol and diethyl ether yielded
compound 7j (171 mg, 80%) as analytically pure sample:
1H-NMR (200 MHz, CDCl3) d 3.20 (br s, 3H, CH3),
/
5.1.18. Methyl R,S-(9
2-phenylacetate hydrochloride (7h)
/
)-2-(6-methylaminopurin-9-yl)-
3.63 (s, 1H, OH), 4.17ꢀ
(m, 1H, CH), 7.32ꢀ7.42 (m, 5H, 5CH), 8.35 (s, 1H, 2-
H), 8.58 (s, 1H, 8-H). Anal. Found: C14H15N5O×HCl
(C, H, N).
/
4.67 (m, 2H, CH2), 5.78ꢀ
/
5.99
/
To a solution containing chloropurine (1.00 g, 6.45
mmol), methyl mandelate (2.15 g, 12.9 mmol), and
triphenyl phosphine (2.55 g, 9.70 mmol) in THF (100
mL) was slowly added a solution of diethyl azodicar-
boxylate (1.53 mL, 9.70 mmol) in THF (50 mL) under
argon atmosphere. The mixture was stirred at room
temperature for 12 h. Volatiles were removed, and the
residue was directly purified by chromatography
/
5.1.21. N6-methyl-9-phenyladenine hydrochloride (7k)
The title compound was prepared following the
1
literature procedure [36]: H-NMR (200MHz, DMSO-
d6) d 3.21 (s, 3H, CH3), 7.63ꢀ
1H, 2-H), 8.89 (s, 1H, 8-H), 9.20 (br s, 1H, NH). Anal.
Found: C12H11N5×HCl×0.2H2O (C, H, N).
/8.00 (m, 5H, 5CH), 8.56 (s,
(CH2Cl2ꢀ
/
hexaneꢀ
/
Et2O, 60:30:10) to give compound
1
/
/
6d (1.27 g, 65%) as a colorless syrup: H-NMR (300
MHz, CDCl3) d 3.85 (s, 3H, CH3), 6.57 (s, 1H, CH),
5.1.22. 9-Benzyl-N6-methyl-2-n-propyladenine
hydrochloride (7l)
7.39ꢀ
2-H); m/z 303 (Mꢃ
/
7.53 (m, 5H, 5CH), 8.23 (s, 1H, 8-H), 8.76 (s, 1H,
H)ꢃ. A stirring solution of 6d (1.00
/
The title compound was prepared from 5c, benzyl
chloride and methylamine following the general proce-
dure for the alkylation of 6-chloropurines (method A):
g, 3.30 mmol) in EtOH (5 mL) was treated with
methylamine (1.0 M in THF, 3.47 mL) at room
temperature for 5 h. After evaporation of the solvent,
the residue was chromatographed on silica and eluted
1H-NMR (200 MHz, CDCl3) d 1.03 (t, Jꢁ
/
7.3, 3H,
7,5, 2H,
5.5, 3H, CH3), 5.36 (s, 2H, CH2),
7.40 (m, 5H, 5CH), 7.84 (s, 1H, 8-H), 9.44 (br s,
1H, NH). Anal. Found: C16H19N5×HCl (C, H, N).
CH3), 1.85ꢀ
CH2), 3.59 (d, Jꢁ
7.28ꢀ
/
2.04 (m, 2H, CH2), 2.94 (t, Jꢁ
/
(AcOEtꢀ
/
CH2Cl2ꢀ/EtOH, 4:5:1), to afford a solid. Re-
/
crystallization from ethanol and diethyl ether gave 7h
(804 mg, 73%) as a colorless solid: 1H-NMR (200 MHz,
1
CDCl3) d: H-NMR (300 MHz, CDCl3) d 3.62 (s, 3H,
/
/
CH3), 3.91 (s, 3H, CH3), 6.48 (s, 1H, CH), 7.38ꢀ
5H, 5CH), 8.06 (s, 1H, 8-H), 8.22 (s, 1H, 2-H), 9.53 (br
s, 1H, NH). Anal. Found: C15H15N5O2×HCl×1.6H2O
(C, H, N).
/7.50 (m,
5.1.23. 9-Benzyl-2-isopropyl-N6-methyladenine
hydrochloride (7m)
The title compound was prepared from 5d, benzyl
chloride and methylamine following the general proce-
dure for the alkylation of 6-chloropurines (method A):
/
/
5.1.19. R,S-(9
/
)-N-methyl-2-(6-methylaminopurin-9-
1H-NMR (200 MHz, CDCl3) d 1.46 (d, Jꢁ
/
6.9, 6H,
5.5, 3H,
yl)-2-phenyl acetamide hydrochloride (7i)
2CH3), 3.21ꢀ
CH3), 5.37 (s, 2H, CH2), 7.29ꢀ
(s, 1H, 8-H), 9.60 (br s, 1H, NH). Anal. Found:
C16H19N5×HCl (C, H, N).
/
3.24 (m, 1H, CH), 3.57 (d, Jꢁ
/
Prepared from 6d (500 mg, 1.65 mmol) and methyla-
mine (1.0 M in THF, 8.00 mL) using the procedure
described for compound 7h. Compound 7i (379 mg,
69%) was obtained as a colorless solid: H-NMR (200
MHz, CD3OD) d 2.80 (s, 3H, CH3), 3.20 (s, 3H, CH3),
/7.45 (m, 5H, 5CH), 7.86
/
1
5.1.24. 9-Benzyl-2-cyclohexyl-N6-methyladenine
hydrochloride (7n)
The title compound was prepared from 5e, benzyl
chloride and methylamine following the general proce-
dure for the alkylation of 6-chloropurines (method A):
6.53 (s, 1H, CH), 7.42ꢀ
8-H), 8.42 (s, 1H, 2-H). Anal. Found: C15H16N6O×
2.1H2O (C, H, N).
/
7.56 (m, 5H, 5CH), 8.21 (s, 1H,
/
HCl×
/
5.1.20. R,S-(9
phenylethanol hydrochloride (7j)
To a solution of methyl R,S-(9
purin-9-yl)-2-phenylacetate hydrochloride (7h) (207 mg,
0.70 mmol) in anhydrous THF (5 mL) was added
sodium borohydride (130 mg, 3.45 mmol) at 0 8C under
argon atmosphere. The reaction mixture was allowed to
warm up to room temperature and refluxed for 1 h.
After cooling, volatiles were removed, and the residue
was diluted with ethyl acetate, washed with water, dried
/
)-2-(6-methylaminopurin-9-yl)-2-
1H-NMR (200 MHz, CDCl3) d 1.37ꢀ
5CH2), 2.88ꢀ
CH3), 5.36 (s, 2H, CH2), 7.29ꢀ
(s, 1H, 8-H), 9.67 (br s, 1 H, NH). Anal. Found:
C19H23N5×HCl (C, H, N).
/
2.10 (m, 10H,
2.94 (m, 1H, CH), 3.59 (d, Jꢁ5.1, 3H,
7.41 (m, 5H, 5CH), 7.83
/
/
/
)-2-(6-methylamino-
/
/
5.1.25. 9-Benzyl-2-tert-butyl-N6-methyladenine
hydrochloride (7o)
The title compound was prepared from 5f, benzyl
chloride and methylamine following the general proce-

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P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
dure for the alkylation of 6-chloropurines (method A):
1H-NMR (200 MHz, CDCl3) d 1.55 (s, 9H, 3CH3), 3.57
general procedure for the alkylation of 6-chloropurines
(method A): 1H-NMR (200 MHz, CDCl3) d 2.74 (s, 3H,
(d, Jꢁ
5H, 5CH), 7.88 (s, 1H, 8-H), 10.34 (br s, 1 H, NH).
Anal. Found: C17H21N5×HCl (C, H, N).
/
5.5, 3H, CH3), 5.36 (s, 2H, CH2), 7.27ꢀ
/
7.40 (m,
CH3), 3.60 (d, Jꢁ
(s, 2H, CH2), 6.95ꢀ
9.26 (br s, 1H, NH). Anal. Found: C15H17N5O×
H, N).
/
4.9, 3H, CH3), 3.82 (s, 3H, CH3), 5.30
7.25 (m, 4H, 4CH), 7.82 (s, 1H, 8-H),
HCl (C,
/
/
/
5.1.26. 9-Benzyl-N6-methyl-2-trans-styryladenine
hydrochloride (7p)
5.1.31. 9-(2-Methoxyphenethyl)-2,N6-dimethyladenine
hydrochloride (7u)
The title compound was prepared from 5b [16], 2-
methoxyphenethyl chloride and methylamine following
the general procedure for the alkylation of 6-chloropur-
ines (method A): 1H-NMR (200 MHz, CDCl3) d 2.67 (s,
The title compound was prepared from 5g, benzyl
chloride and methylamine following the general proce-
dure for the alkylation of 6-chloropurines (method A):
1H-NMR (200 MHz, CDCl3) d 3.33 (d, Jꢁ
/
5.1, 3H,
7.13 (m, 8H, 8CH), 7.10ꢀ
7.78 (m, 4H, 4CH), 7.81 (s, 1H, 8-H), 9.10 (br s, 1H,
NH). Anal. Found: C21H19N5×HCl (C, H, N).
CH3), 5.17 (s, 2H, CH2), 7.08ꢀ
/
/
3H, CH3), 3.17 (t, Jꢁ
3H, CH3), 3.75 (s, 3H, CH3), 4.48 (t, Jꢁ
6.85ꢀ7.24 (m, 4H, 4CH), 7.48 (s, 1H, 8-H), 9.20 (br s,
1H, NH). Anal. Found: C16H19N5O×HCl (C, H, N).
/
6.6, 2H, CH2), 3.59 (d, Jꢁ
/4.8,
/
/
6.6, 2H, CH2),
/
5.1.27. 2-Amino-9-benzyl-N6-methyladenine di
hydrochloride (7q)
/
A solution of 11a (200 mg, 0.77 mmol) and methy-
lamine (1.0 M in THF, 5 mL) in absolute ethanol (5 mL)
was stirred for 24 h at room temperature. After
evaporation of the solvent, the residue was chromato-
5.1.32. 9-[2-(2-Methoxyethoxy)ethyl]-2,N6-
dimethyladenine hydrochloride (7v)
The title compound was prepared from 5b [16], 2-(2-
methoxyethoxy)ethyl chloride and methylamine follow-
ing the general procedure for the alkylation of 6-
chloropurines (method A): 1H-NMR (200 MHz,
graphed on silica (AcOEtꢀ
/
CH2Cl2ꢀEtOH, 4:5:1), then
/
recrystallized from ethanol and ether to give 7q which
1
was converted to the HCl salt (184 mg, 73%): H-NMR
(200 MHz, CD3OD) d 3.21 (s, 3H, CH3), 5.38 (s, 2H,
CDCl3) d 2.69 (s, 3H, CH3), 3.38 (s, 3H, CH3), 3.49ꢀ
6.68 (m, 7H, 2CH2ꢃCH3), 3.82 (t, Jꢁ4.8, 2H, CH2),
4.41 (t, Jꢁ4.8, 2H, CH2), 8.10 (s, 1H, 8-H), 9.21 (br s,
HCl×1.5H2O (C,
/
/
/
CH2), 7.34ꢀ
Found: C13H14N6×
/
7.40 (m, 5H, 5CH), 8.52 (s, 1H, 8-H). Anal.
2HCl (C, H, N).
/
/
1H, NH). Anal. Found: C12H19N5O2×
/
/
H, N).
5.1.28. -9[(2-Methoxy-3-pyridyl)methyl]-N6-methyl-2-
trifluoromethyladenine (7r)
5.1.33. N6-methyladenine (8a)
The title compound was prepared from 6-chloro-2-
trifluoromethyladenine 5h [16], and methylamine fol-
lowing the general procedure for the alkylation of 6-
chloropurines (method A): 1H-NMR (200 MHz, CDCl3)
d 3.25 (br s, 3H, CH3), 4.03 (s, 3H, CH3), 5.36 (s, 2H,
A solution of 6-chloropurine (5a) (263 mg, 1.70
mmol) and methylamine (1.0 M in THF, 5 mL) in
absolute ethanol (5 mL) was heated at 120 8C in a
sealed tube for 2 h. After evaporation of the solvent, the
residue was diluted with cold water (5 mL) then, the
precipitate was filtered, sequentially washed with cold
water, ethanol and Et2O, then recrystallized from EtOH
to yield 5a (246 mg, 97%) as a white powder. This
sample was identical in all respects with the product
reported in Ref. [32].
CH2), 5.90 (br s, 1H, NH), 6.87ꢀ/6.91 (m, 1H, CH),
7.64ꢀ7.67 (m, 1H, CH), 7.96 (s, 1H, 8-H), 8.14ꢀ
/
/8.16 (m,
1H, CH). Anal. Found: C14H13F3N6O (C, H, N).
5.1.29. 9-(2-Methoxybenzyl)-2,N6-dimethyladenine
hydrochloride (7s)
The title compound was prepared from 5b [16], 2-
methoxybenzyl chloride and methylamine following the
general procedure for the alkylation of 6-chloropurines
(method A): 1H-NMR (200 MHz, CDCl3) d 2.73 (s, 3H,
5.1.34. N6-methyl-2-n-pentyladenine (8b)
The title compound was prepared from 5h following
1
the procedure described for 8a: H-NMR (200 MHz,
CH3), 3.59 (d, Jꢁ
(s, 2H, CH2), 6.91ꢀ
9.19 (br s, 1H, NH). Anal. Found: C15H17N5O×
H, N).
/
4.9, 3H, CH3), 3.88 (s, 3H, CH3), 5.34
7.33 (m, 4H, 4CH), 7.97 (s, 1H, 8-H),
HCl (C,
CD3OD) d 0.90 (t, Jꢁ
2CH2), 1.72ꢀ1.83 (m, 2H, CH2), 2.74 (t, Jꢁ
CH2), 3.13 (s, 3H, CH3), 7.96 (s, 1H, 8-H); m/z 220
(Mꢃ
H)ꢃ.
/
6.6, 3H, CH3), 1.31ꢀ/1.39 (m, 4H,
/
/
/7.5, 2H,
/
/
5.1.30. 9-(4-Methoxybenzyl)-2,N6-dimethyladenine
hydrochloride (7t)
The title compound was prepared from 5b [16], 4-
methoxybenzyl chloride and methylamine following the
5.1.35. N6-methyl-2-trans-styryladenine (8c)
The title compound was prepared from 5g following
1
the procedure described for 8a: H-NMR (200 MHz,

P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ
/
214
209
DMSO-d6) d 3.20 (s, 3H, CH3), 7.29ꢀ
/
7.70 (m, 7H,
7.30ꢀ
/
7.39 (m, 5H, 5CH), 8.38 (s, 1H, 2-H), 8.40 (s, 1H,
7CH), 8.00 (s, 1H, 8-H); m/z 274 (Mꢃ
/
Na)ꢃ.
8-H). Anal. Found: C13H13N5×
/
HCl×0.5H2O (C, H, N).
/
5.1.36. N6-methyl-2-phenethyladenine (8d)
A mixture of 8c (210 mg, 0.83 mmol) and 10% Pdꢀ
(30 mg) in absolute methanol (100 mL) was shaken in a
hydrogenation apparatus at room temperature for 15 h.
The catalyst was removed by filtration, washed with
methanol and the filtrate was concentrated to dryness to
give compound 8d (187 mg, 88%) as a colorless solid:
5.1.42. 9-n-Butyl-N6-methyladenine hydrochloride (9b)
The title compound was prepared from 8a and n-
butyl chloride following the general procedure for the
/C
1
alkylation of N6-methyladenines (method B): H-NMR
(200 MHz, CD3OD) d 1.00 (t, Jꢁ
1.49 (m, 2H, CH2), 1.86ꢀ1.97 (m, 2H, CH2), 3.24 (s, 3H,
CH3), 4.37 (t, Jꢁ7.1, 2H, CH2), 8.39 (s, 1H, 2-H), 8.44
(s, 1H, 8-H). Anal. Found: C10H15N5×HCl×0.3H2O (C,
H, N).
/
7.3, 3H, CH3), 1.31ꢀ
/
/
1H-NMR (200 MHz, CD3OD) d 2.98ꢀ
2CH2), 3.14 (s, 3H, CH3), 7.10ꢀ7.22 (m, 5H, 5CH), 7.96
(s, 1H, 8-H); m/z 276 (Mꢃ
Na)ꢃ.
/
3.11 (m, 4H,
/
/
/
/
/
5.1.37. N6-methyl-2-(3-phenylpropyl)adenine (8e)
The title compound was prepared (89%) from 5i
following the procedure described for 8a: 1H-NMR
5.1.43. 9-Cyclopentyl-N6-methyladenine hydrochloride
(9c)
The title compound was prepared from 8a and benzyl
chloride following the general procedure for the alkyla-
(200 MHz, CD3OD) d 2.03ꢀ
Jꢁ7.3, 2H, CH2), 2.78 (t, Jꢁ
3H, CH3), 7.05ꢀ7.27 (m, 5H, 5CH), 7.95 (s, 1H, 8-H);
m/z 290 (Mꢃ
Na)ꢃ.
/2.21 (m, 2H, CH2), 2.71 (t,
/
/7.4, 2H, CH2), 3.13 (s,
1
tion of N6-methyladenines (method B): H-NMR (200
/
MHz, CD3OD) d 1.78ꢀ
CH3), 8.40 (s, 1H, 2-H), 8.44 (s, 1H, 8-H). Anal. Found:
C11H15N5×HCl×2.3H2O (C, H, N).
/
2.40 (m, 8H, 4CH2), 3.22 (s, 3H,
/
/
/
5.1.38. N6-methyl-2-trifluoromethyladenine (8f)
The title compound was prepared (95%) from 6-
chloro-2-trifluoromethyladenine 5h [16], following the
procedure described for 8a. A colorless solid which was
identical in all respects with the product reported in the
literature was obtained [33].
5.1.44. N6-methyl-9-neopentyladenine hydrochloride
(9d)
The title compound was prepared from 8a and
neopentyl chloride following the general procedure for
the alkylation of N6-methyladenines (method B): 1H-
NMR (200 MHz, CD3OD) d 0.90 (s, 9H, 3CH3), 3.11 (s,
3H, CH3), 4.04 (s, 2H, CH2), 8.19 (s, 1H, 2-H), 8.28 (s,
5.1.39. N6-methyl-2-n-propyladenine (8g)
The title compound was prepared from 5c following
1
the procedure described for 8a: H-NMR (200 MHz,
1H, 8-H). Anal. Found: C11H17N5×
/
HCl×
/
1.5H2O (C, H,
DMSO-d6) d 0.97 (t, Jꢁ
2H, CH2), 2.72 (t, Jꢁ7.7, 2H, CH2), 3.13 (s, 3H, CH3),
7.95 (s, 1H, 8-H); m/z 192 (Mꢃ
H)ꢃ.
/
7.3, 3H, CH3), 1.72ꢀ/1.91 (m,
N).
/
/
5.1.45. 9-Dodecyl-N6-methyladenine hydrochloride (9e)
The title compound was prepared from 8a and
dodecyl chloride following the general procedure for
the alkylation of N6-methyladenines (method B): 1H-
5.1.40. General procedure for the alkylation of N6-
methyladenines (8) provided adenines 9aꢀt (method B)
/
To a solution of N6-methyladenine 8 (11.8 mmol),
K2CO3 (1.63 g, 11.8 mmol), and tetra-n-butylammo-
nium iodide (200 mg, 0.54 mmol) in dry DMF (16.0 mL)
was added dropwise the requisite alkyl chloride (13
mmol). The resulting suspension was stirred for 24 h at
room temperature. After evaporation of the solvent, the
residue was diluted with cold water (50 mL) and
extracted three times with ethyl acetate (60 mL).
Organic layers were subsequently dried (Na2SO4) and
concentrated to dryness under reduced pressure. Chro-
NMR (200 MHz, CD3OD) d 0.89 (t, Jꢁ
1.23ꢀ1.34 (m, 18H, 9CH2), 1.82 (m, 2H, CH2), 3.20 (s,
3H, CH3), 4.31 (t, Jꢁ 7.1, 2H, CH2), 8.32 (s, 1H, 2-H),
8.38 (s, 1H, 8-H). Anal. Found: C18H31N5×HCl (C, H,
N).
/
6.4, 3H, CH3),
/
/
/
5.1.46. 9-Phenethyl-N6-methyladenine hydrochloride
(9f)
matography on silica and elution (AcOEtꢀ
afforded compounds 9 as a colorless solid.
/
EtOH, 9:1)
The title compound was prepared from 8a and
phenethyl chloride following the general procedure for
the alkylation of N6-methyladenines (method B): 1H-
NMR (200 MHz, DMSO-d6) d 2.95 (br s, 3H, CH3),
5.1.41. 9-Benzyl-N6-methyladenine hydrochloride (9a)
The title compound was prepared from 8a and benzyl
chloride following the general procedure for the alkyla-
3.14 (t, Jꢁ
7.12ꢀ7.26 (m, 5H, 5 CH), 7.61 (br s, 1H, NH), 7.91 (s,
1H, 2-H), 8.23 (s, 1H, 8-H). Anal. Found: C14H15N5×
HCl×0.2H2O (C, H, N).
/
7.3, 2H, CH2), 4.39 (t, Jꢁ7,3, 2H, CH2),
/
/
1
tion of N6-methyladenines (method B): H-NMR (200
/
MHz, CD3OD) d 3.20 (s, 3H, CH3), 5.51 (s, 2H, CH2),
/

210
P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
5.1.47. N6-methyl-9-(3-phenylpropyl)adenine
hydrochloride (9g)
5.1.52. N6-methyl-9-(2-oxo-2-phenethyl)adenine
hydrochloride (9l)
The title compound was prepared from 8a and 3-
phenylpropyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
The title compound was prepared from 8a and a-
chloro acetophenone following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
NMR (200 MHz, DMSO-d6) d 3.11 (s, 3H, CH3), 6.03
NMR (200 MHz, CD3OD) d 2.19ꢀ
2.68 (t, Jꢁ7.7, 2H, CH2), 3.21 (s, 3H, CH3), 4.35 (t, Jꢁ
7.1, 2H, CH2), 7.06ꢀ7.27 (m, 5H, 5CH), 8.34 (s, 1H, 2-
HCl×
/2.33 (m, 2H, CH2),
/
/
(s, 2H, CH2), 7.58ꢀ
2H, 2CH), 8.39 (s, 1H, 2-H), 8.41 (s, 1H, 8-H), 9.60 (br
s, 1H, NH). Anal. Found: C14H13N5O×HCl×0.5H2O (C,
H, N).
/
7.79 (m, 3H, 3 CH), 8.07ꢀ8.12 (m,
/
/
H), 8.39 (s, 1H, 8-H). Anal. Found: C15H17N5×
/
/
/
/
1.2H2O (C, H, N).
5.1.48. 9-(4-Chlorobenzyl)-N6-methyladenine
hydrochloride (9h)
5.1.53. N6-methyl-9-(3,3-diphenylpropyl)adenine
hydrochloride (9m)
The title compound was prepared from 8a and 4-
chlorobenzyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
The title compound was prepared from 8a and 3,3-
diphenylpropyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
NMR (200 MHz, DMSO-d6) d 2.94 (d, Jꢁ
CH3), 5.41 (s, 2H, CH2), 7.21ꢀ7.29 (m, 4H, 4CH), 8.35
(s, 1H, 2-H), 8.89 (s, 1H, 8-H), 9.78 (q, Jꢁ4.8, 1H,
NH). Anal. Found: C13H12ClN5×HCl×0.4H2O (C, H,
N).
/
4.8, 3H,
NMR (200 MHz, CD3OD) d 2.68ꢀ
3.18 (s, 3H, CH3), 3.92ꢀ4.03 (m, 1H, CH), 4.31 (t, Jꢁ
7.1, 2H, CH2), 7.10ꢀ7.25 (m, 10H, 10CH), 8.17 (s, 1H,
HCl
/2.75 (m, 2H, CH2),
/
/
/
/
/
/
/
2-H), 8.35 (s, 1H, 8-H). Anal. Found: C21H21N5×
/
(C, H, N).
5.1.49. 9-(2-Bromobenzyl)-N6-methyladenine
hydrochloride (9i)
The title compound was prepared from 8a and 2-
bromobenzyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
5.1.54. 9-[(4-Benzoyl)benzyl]-N6-methyladenine
hydrochloride (9n)
The title compound was prepared from 8a and 4-
benzoylbenzyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
NMR (200 MHz, DMSO-d6) d 3.10 (br s, 3H, CH3),
NMR (200 MHz, CDCl3) d 3.21 (d, Jꢁ
5.47 (s, 2H, CH2), 5.97 (br s, 1H, NH), 7.09ꢀ
3H, 3CH), 7.58ꢀ7.63 (m, 1H, CH), 7.80 (s, 1H, 8-H),
8.44 (s, 1H, 2-H). Anal. Found: C13H12BrN5×HCl (C, H,
N).
/4.8, 3H, CH3),
/
7.29 (m,
5.61 (s, 2H, CH2), 7.43ꢀ
2-H), 8.69 (s, 1H, 8-H), 9.92 (br s, 1H, NH). Anal.
Found: C20H17N5O×HCl (C, H, N).
/7.71 (m, 9H, 9CH), 8.46 (s, 1H,
/
/
/
5.1.55. 9-Benzyl-N6-methyl-2-n-pentyladenine
hydrochloride (9o)
5.1.50. 9-(2-Methoxybenzyl)-N6-methyladenine
hydrochloride (9j)
The title compound was prepared from 8b and benzyl
chloride following the general procedure for the alkyla-
The title compound was prepared from 8a and 2-
methoxybenzyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
NMR (200 MHz, DMSO-d6) d 3.03 (br s, 3H, CH3),
1
tion of N6-methyladenines (method B): H-NMR (200
MHz, CD3OD) d 0.93 (t, Jꢁ
(m, 4H, 2CH2), 1.83ꢀ1.91 (m, 2H, CH2), 2.84ꢀ
Jꢁ7.7, 2H, CH2), 3.22 (s, 3H, CH3), 5.47 (s, 2H, CH2),
7.29ꢀ7.39 (m, 5H, 5CH), 8.30 (s, 1H, 8-H). Anal.
Found: C18H23N5×HCl (C, H, N).
/
6.8, 3H, CH3), 1.32ꢀ
/1.48
/
/3.18 (t,
3.77 (s, 3H, CH3), 5.35 (s, 2H, CH2), 6.81ꢀ
4CH), 8.39 (s, 2H, 2-Hꢃ8-H), 9.59 (br s, 1H, NH).
Anal. Found: C14H15N5O×HCl×0.2H2O (C, H, N).
/7.31 (m, 4H,
/
/
/
/
/
/
5.1.51. 9-(4-Methoxybenzyl)-N6-methyladenine
hydrochloride (9k)
The title compound was prepared from 8a and 4-
methoxybenzyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
5.1.56. 9-Benzyl-N6-methyl-2-phenethyladenine
hydrochloride (9p)
The title compound was prepared from 8d and benzyl
chloride following the general procedure for the alkyla-
1
NMR (200 MHz, DMSO-d6) d 3.06 (d, Jꢁ
CH3), 3.66 (s, 3H, CH3), 5.36 (s, 2H, CH2), 6.88ꢀ
(m, 4H, 4 CH), 8.43 (s, 1H, 2-H), 8.60 (s, 1H, 8-H), 9.85
(br s, 1H, NH). Anal. Found: C14H15N5O×HCl×0.4H2O
(C, H, N).
/
4.0, 3H,
tion of N6-methyladenines (method B): H-NMR (200
/
7.28
MHz, CD3OD) d 2.89 (s, 3H, CH3), 4.90ꢀ
2CH2), 5.47 (s, 2H, CH2), 7.15ꢀ7.28 (m, 5H, 5CH),
7.29ꢀ7.40 (m, 5H, 5CH), 8.32 (s, 1H, 8-H). Anal.
Found: C21H21N5×HCl×2H2O (C, H, N).
/5.01 (m, 4H,
/
/
/
/
/
/

P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ
/
214
211
5.1.57. 9-Benzyl-N6-methyl-2-(3-phenylpropyl)adenine
hydrochloride (9q)
dryness under reduced pressure. Chromatography on
silica (AcOEt) afforded compound 11a (930 mg, 60%) as
1
a colorless solid: H-NMR (200 MHz, CDCl3) d 5.00
The title compound was prepared from 8e and benzyl
chloride following the general procedure for the alkyla-
(br s, 2H, NH2), 5.31 (s, 2H, CH2), 7.30ꢀ
/
7.32 (m, 5H,
1
tion of N6-methyladenines (method B): H-NMR (200
5CH), 8.09 (s, 1H, 8-H).
MHz, CDCl3) d 2.19ꢀ
7.5, 2H, CH2), 3.02 (t, Jꢁ
5.1, 3H, CH3), 5.34 (s, 2H, CH2), 7.11ꢀ
10CH), 7.88 (s, 1H, 8-H), 9.35 (br s, 1 H, NH). Anal.
Found: C22H23N5×HCl×1.3H2O (C, H, N).
/
2.34 (m, 2H, CH2), 2.75 (t, Jꢁ
7.5, 2H, CH2), 3.59 (d, Jꢁ
7.42 (m, 10H,
/
A solution of 11a (270 mg, 1 mmol), isoamyle nitrite
(2.69 mL, 20 mmol) and diiodomethane (10 mL, 124
mmol) was stirring at 85 8C under argon for 1 h. After
cooling, volatiles were evaporated, and the residue was
taken up in AcOEt. The organic layer was washed with
brine, dried (Na2SO4), and concentrated. The residue
was purified by chromatography on silica. Elution with
/
/
/
/
/
5.1.58. 2-Trifluoromethyl-9-(2-methoxybenzyl)-N6-
methyladenine (9r)
CH2Cl2ꢀAcOEt (90:10) delivered 12a as an amorphous
/
The title compound was prepared from 8f and 2-
methoxybenzyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
NMR (200 MHz, CDCl3) d 3.19 (br s, 3H, CH3), 3.83
(s, 3H, CH3), 5.34 (s, 2H, CH2), 5.85 (br s, 1H, NH),
solid (260 mg, 70%) which was used in the next step
without further purification: 1H-NMR (200 MHz,
CDCl3) d 5.40 (s, 2H, CH2), 7.26ꢀ
/
7.41 (m, 5H, 5CH),
7.98 (s, 1H, 8-H).
A solution of 12a (200 mg, 0.54 mmol) and methy-
lamine (1.0 M in THF, 4 mL) in absolute ethanol (4 mL)
was stirred for 24 h at room temperature. After
evaporation of the solvent, the residue was chromato-
6.85ꢀ
/
6.94 (m, 2H, 2CH), 7.23ꢀ
/
7.33 (m, 2H, 2CH), 7.84
0.2H2O (C,
(s, 1H, 8-H). Anal. Found: C15H14F3N5O×
/
H, N).
graphed on silica (AcOEtꢀ
recrystallized from ethanol to give 13a (137 mg, 63%) as
1
a white solid: H-NMR (200 MHz, CD3OD) d 3.15 (s,
/
CH2Cl2ꢀEtOH, 4:5:1), then
/
5.1.59. 9-(2-Methoxybenzyl)-N6-methyl-2-n-
propyladenine hydrochloride (9s)
The title compound was prepared from 8g and 2-
methoxybenzyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
3H, CH3), 5.51 (s, 2H, CH2), 7.36ꢀ
8.96 (s, 1H, 8-H). Anal. Found: C13H12IN5×
N).
/
7.44 (m, 5H, 5CH),
/
HCl (C, H,
NMR (200 MHz, CDCl3) d 1.03 (t, Jꢁ
1.85ꢀ2.06 (m, 2H, CH2), 2.92 (t, Jꢁ7.5, 2H, CH2), 3.47
(d, Jꢁ4.7, 3H, CH3), 3.88 (s, 3H, CH3), 5.34 (s, 2H,
CH2), 6.90ꢀ6.99 (m, 2H, 2CH), 7.27ꢀ7.39 (m, 2H,
2CH), 7.97 (s, 1H, 8-H), 9.28 (br s, 1H, NH). Anal.
Found: C17H21N5O×HCl×0.5H2O (C, H, N).
/
7.3, 3H, CH3),
/
/
/
5.1.62. 2-Iodo-9-(2-methoxybenzyl)-N6-methyladenine
hydrochloride (13b)
Prepared from 10 and 2-methoxybenzyl chloride using
the procedure described for compound 13a: H-NMR
(200 MHz, CDCl3) d 3.86 (s, 3H, CH3), 5.36 (s, 2H,
/
/
1
/
/
5.1.60. 9-[(4-Benzoyl)benzyl]-N6-methyl-2-n-
propyladenine hydrochloride (9t)
The title compound was prepared from 8g and 4-
benzoylbenzyl chloride following the general procedure
for the alkylation of N6-methyladenines (method B): 1H-
CH2), 6.88ꢀ
/
7.00 (m, 2H, 2CH), 7.26ꢀ7.38 (m, 2H,
/
2CH), 8.05 (s, 1H, 8-H). Anal. Found: C14H14IN5O×
/
HCl (C, H, N).
NMR (200 MHz, CDCl3) d 1.03 (t, Jꢁ
1.86ꢀ2.01 (m, 2H, CH2), 2.95 (t, Jꢁ7.5, 2H, CH2), 3.62
(d, Jꢁ5.1, 3H, CH3), 5.48 (s, 2H, CH2), 7.40ꢀ7.84 (m,
9H, 9CH), 7.98 (s, 1H, 8-H), 9.45 (br s, 1H, NH). Anal.
Found: C23H23N5O×HCl×0.5H2O (C, H, N).
/
7.3, 3H, CH3),
5.1.63. 9-Benzyl-N6-methyl-2-prop-1-ynyladenine
hydrochloride (14a)
Methyl acetylene (2 mL) was condensed in a sealed
/
/
/
/
tube at ꢂ78 8C. Then 13a (200 mg, 0.55 mmol), CuI (10
/
/
/
mg, 0.055 mmol), PdCl2 (6 mg, 0.034 mmol), triphenyl-
phosphine (20 mg, 0.076 mmol), triethylamine (2 mL,
14.3 mmol) and acetonitrile (4 mL) were added. The
solution was stirred at room temperature for 24 h, the
solvent was then removed under vacuum, and the
residue was purified by flash chromatography
5.1.61. 9-Benzyl-2-iodo-N6-methyladenine hydrochloride
(13a)
To a solution of 2-amino-9-chloropurine 10 (1.0 g,
5.90 mmol), K2CO3 (815 mg, 5.90 mmol), and tetra-n-
butylammonium iodide (100 mg, 0.27 mmol) in dry
DMF (8.0 mL) was added dropwise benzyl chloride (815
ml, 7.08 mmol). The resulting suspension was stirred for
12 h at room temperature. After evaporation of the
solvent, the residue was diluted with cold water (20 mL)
and extracted three times with ethyl acetate (30 mL).
Organic layers were dried (Na2SO4) and concentrated to
(CH2Cl2ꢀAcOEt, 80:20) and converted to an HCl salt.
/
Recrystallization from ethanol and diethyl ether yielded
compound 14a (121 mg, 70%): 1H-NMR (200 MHz,
CDCl3) d 2.20 (s, 3H, CH3), 3.61 (d, Jꢁ
5.37 (s, 2H, CH2), 7.27ꢀ7.44 (m, 5H, 5CH), 7.88 (s, 1H,
8-H), 9.82 (br s, 1H, NH). Anal. Found: C16H15N5×HCl
(C, H, N).
/5.5, 3H, CH3),
/
/

212
P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
5.1.64. 9-(2-Methoxybenzyl)-N6-methyl-2-prop-1-
ynyladenine hydrochloride (14b)
Prepared from 13b using the procedure described for
the two-step assay previously described [35] at a [3H]-
cAMP or [3H]-cGMP concentration of 1 mM as
substrate in a buffer solution of 50 mM of TrisꢀHCl,
/
1
compound 14a: H-NMR (200 MHz, CDCl3) d 2.20 (s,
at pH 7.5, containing 2 mM of magnesium acetate, 1
mg mLꢂ1 of BSA. PDE-1 was assayed at 1 mM cGMP
in calmodulin activated state (18 nM calmodulin with 10
3H, CH3), 3.59 (d, Jꢁ
5.35 (s, 2H, CH2), 6.92ꢀ
(m, 2H, 2CH), 7.99 (s, 1H, 8-H), 9.77 (br s, 1H, NH).
Anal. Found: C17H17N5O×HCl×1.6H2O (C, H, N).
/5.5, 3H, CH3), 3.88 (s, 3H, CH3),
/
7.04 (m, 2H, 2CH), 7.34ꢀ7.55
/
mM CaCl2). PDE-2 was evaluated at 1 mM cAMPꢃ
/
1
/
/
mM EGTA in activated state (in presence of 5 mM
cGMP). PDE-3 and PDE-4 were assayed at 1 mM
cAMPꢃ1 mM EGTA. To prevent the influence of
/
5.1.65. trans-9-(2-Methoxybenzyl)-N6-methyl-2-prop-1-
enyladenine hydrochloride (14c)
reciprocal cross-contamination between PDE-3 and
PDE-4, the studies were always carried out in the
presence of 50 mM rolipram for PDE-3 and in the
presence of 50 mM cGMP for PDE-4. PDE-5 activity
was measured at 1 mM cGMP in the presence of 1 mM
of EGTA. PDE inhibitors were dissolved in dimethyl
sulfoxide (DMSO) at a final concentration of 1%. At
this concentration, DMSO had no significant effects on
PDE activity. The concentration of drugs that produced
50% inhibition of substrate hydrolysis (IC50) was
calculated by nonlinear regression analysis from con-
To a solution of 13b (150 mg, 0.35 mmol), tetrakis-
triphenylphosphine palladium (4 mg, 3.46 10ꢂ3 mmol)
in CH3CN (10 mL) was added 6 N sodium hydroxide
(400 ml) and (E)-prop-1-enylcatecholborane [38] (320
mg, 2 mmol). The reaction mixture was refluxed under
argon for 4 h. After cooling, the solvent was removed
under vacuum and the residue was partitioned between
ethyl acetate and water. The organic layer was washed
with brine, dried (Na2SO4), and concentrated. The
residue was purified by flash chromatography (AcOEt)
and converted to an HCl salt. Recrystallization from
ethanol and diethyl ether gave 14c (87 mg, 72%) as
centrationꢀresponse curves and included different con-
/
centrations of inhibitors. The results represent the mean
of three determinations obtained for three different
enzymatic preparations. The experimental error is about
15%.
1
colorless prisms: H-NMR (200 MHz, CDCl3) d 2.05
(dd, Jꢁ
CH3), 3.89 (s, 3H, CH3), 5.34 (s, 2H, CH2), 6.45ꢀ
(m, 1H, CH), 6.92ꢀ7.01 (m, 2H, 2CH), 7.33ꢀ7.41 (m,
1H, CH), 7.46ꢀ7.65 (m, 2H, 2CH), 7.96 (s, 1H, 8-H),
HCl (C,
/
6.9, Jꢁ
/
1.7, 3H, CH3), 3.59 (d, Jꢁ
/
5.1, 3H,
/
6.54
/
/
5.2.2. Subjects
/
Healthy donors who had no clinical history were
selected for this study. This work was approved by the
local ethics committee (CCPPRB d’Alsace no. 1), and
carried out according national guidelines.
9.55 (br s, 1H, NH). Anal. Found: C17H19N5O×
/
H, N).
5.1.66. 9-(2-Methoxybenzyl)-N6-methyl-2-
methylmercaptoadenine hydrochloride (14d)
A solution of 13b (43 mg, 0.10 mmol), sodium
methanethiolate (350 mg, 0.50 mmol) in dimethylforma-
mide (3 mL) and ethanol (3 mL) was stirring at 120 8C
under argon in a sealed tube for 6 h. After cooling, the
solvent was removed under vacuum, and the residue was
5.2.3. Peripheral blood mononuclear cells
Healthy subjects PBMCs were separated as previously
described [36]. Briefly, peripheral blood diluted with
Hank’s balanced salt solution, Ca2ꢃ and Mg2ꢃ free,
containing 100 IU heparin mLꢂ1 was layered over
Histopaque-1077 (Sigma, St. Louis, MO), and centri-
fuged for 30 min at 400ꢄ
from the interface were washed 3 times in HBSS-CMF
106
/
g (20 8C). Cells harvested
purified by flash chromatography (AcOEtꢀEtOH,
/
90:10). Transformation into the HCl salt and recrystal-
lization from EtOH and diethyl ether gave 14d (31 mg,
1
88%) as colorless prisms: H-NMR (200 MHz, CDCl3)
and resuspended at a final concentration of 2ꢄ
/
mlꢂ1 in a culture medium. Human PBMCs were
incubated with increasing doses of the tested drugs
ranging from 10ꢂ8 to 10ꢂ5 M, with or without
activation by lipopolysacharide from Salmonella abortus
equi (Sigma, L’Isle d’Abeau Chesnes, France) in 24 well
culture plates (Falcon, Poly Labo, Strasbourg, France)
d 2.62 (s, 3H, CH3), 3.14 (d, Jꢁ
3H, CH3), 5.41 (s, 2H, CH2), 6.93ꢀ
7.33ꢀ7.51 (m, 2H, 2CH), 8.38 (s, 1H, 8-H), 8.56 (br s,
1H, NH). Anal. Found: C15H17N5OS×HCl (C, H, N).
/4.8, 3H, CH3), 3.90 (s,
/
7.04 (m, 2H, 2CH),
/
/
for 24 h at 37 8C in a humidified 5% CO2ꢀ
atmosphere. After incubation, supernatants were re-
moved and stored at ꢂ80 8C until ELISA. Cell viability
/
95% air
5.2. Pharmacology
/
was assessed by the Trypan blue exclusion test.
5.2.1. PDE inhibition
PDE-4 was isolated from the media layer of bovine
aorta according to a modification of the previously
reported method [34]. PDE activities were measured by
5.2.4. Immunoassays for TNFa
Culture supernatants were assayed with two-site
ELISAs specific for human interleukin: TNFa antibo-

P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ
/
214
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Appendix A. Supporting information
Product Formula
C (%)
H (%)
N (%)
Found Calculated
Calculated
Found Calculated
Found
7a
7b
7c
7d
7e
7f
C12H12N6×
C12H11N5O×
C16H17N5×HCl×
C16H18N6×2HCl
C13H13N50
C13H12ClN5×
C14H13N5O2×
C15H15N5O2×
/
2HCl
HCl×
1H2O
46.02
48.74
57.57
52.32
61.17
48.37
52.59
49.68
46.18
48.89
57.39
52.42
61.34
48.42
52.61
49.68
4.50
4.77
6.04
5.48
5.13
4.50
4.41
4.89
4.51
4.49
5.87
5.45
5.04
4.40
4.38
4.78
26.83
23.68
20.98
22.88
27.43
21.69
21.90
19.31
26.69
23.64
21.13
22.67
27.31
21.43
21.81
19.42
/
/
1H2O
/
/
/
/
HCl×
HCl
HCl×
/
0.7H2O
7g
7h
/
/
/
1.6H2O

214
P. Raboisson et al. / European Journal of Medicinal Chemistry 38 (2003) 199ꢀ214
/
Appendix (Continued)
Product Formula
C (%)
H (%)
N (%)
Found Calculated
Calculated
Found Calculated
Found
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
C15H16N6O×
C14H15N5O×
C12H11N5×HCl×
C16H19N5×
C16H19N5×
C19H23N5×
C17H21N5×
C21H19N5×
C13H14N6×
/
HCl×
HCl
0.2H2O
/
2.1H2O
48.61
51.41
54.70
60.46
60.46
63.77
61.52
66.75
47.70
49.71
56.33
56.33
57.57
43.84
54.83
48.60
44.76
46.73
61.08
57.32
55.36
49.20
44.03
54.35
53.73
53.76
66.40
63.24
62.50
60.64
63.31
52.84
57.22
64.11
38.88
38.95
55.80
51.32
59.04
51.20
48.61
51.14
54.63
60.41
60.32
63.98
61.24
66.53
47.98
49.62
56.31
56.25
57.72
43.71
54.98
48.56
44.80
46.46
60.74
57.14
55.24
49.49
43.80
54.20
53.89
53.72
66.70
63.00
62.11
60.54
63.35
52.93
57.20
64.08
39.10
39.15
55.73
51.13
58.87
51.16
5.19
5.27
4.30
6.34
6.32
6.76
6.68
5.33
4.93
3.87
5.67
5.67
6.04
7.05
5.31
6.36
7.03
7.48
9.11
5.63
6.32
4.38
3.69
5.34
5.41
4.83
5.84
4.77
6.70
6.30
6.18
4.26
6.44
5.85
3.26
3.50
5.67
5.57
5.83
5.16
5.14
5.40
4.62
6.29
6.35
6.74
6.63
5.12
5.03
3.67
5.81
5.50
6.28
7.30
5.27
6.57
7.03
7.52
9.13
5.57
6.32
4.32
3.77
5.38
5.30
4.59
5.94
4.81
6.99
6.48
6.01
4.24
6.59
5.72
3.35
3.62
5.66
5.52
5.78
5.03
22.67
22.90
26.41
22.03
22.03
19.57
21.10
18.53
25.68
24.84
21.90
21.90
20.98
21.30
24.59
28.34
23.73
24.76
19.79
23.87
21.52
22.07
19.75
22.64
22.38
22.39
18.44
18.43
20.24
16.83
16.78
20.54
19.62
16.25
17.43
16.22
20.33
17.60
20.25
19.90
22.43
22.62
26.11
22.37
22.17
19.75
20.99
18.78
25.70
25.98
21.79
21.69
21.12
21.42
24.81
28.33
23.69
24.60
19.88
23.47
21.60
21.97
19.78
22.63
22.51
22.60
18.45
18.28
20.16
16.84
16.99
20.33
19.73
16.38
17.08
16.15
20.24
17.37
20.13
19.98
/
/
/
/
HCl
HCl
HCl
HCl
HCl
2HCl
/
/
/
/
/
C14H13F3N6O
C15H17N5O×HCl
C15H17N5O×
C16H19N5O×
7s
7t
/
/
HCl
HCl
7u
7v
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
/
C12H19N5O2×
C13H13N5×HCl×
C10H15N5×HCl×
C11H15N5×HCl×
C11H17N5×HCl×
C18H31N5×HCl
C14H15N5×HCl×
C15H17N5×HCl×
C13H12ClN5×
C13H12BrN5×
C14H15N5O×HCl×
C14H15N5O×HCl×
C14H13N5O×HCl×
C21H21N5×HCl
C20H17N5O×HCl
C18H23N5×HCl
C21H21N5×HCl×
C22H23N5×HCl×
C15H14F3N5O×
C17H21N5O×HCl×
C23H23N5O×HCl×
C13H12IN5×HCl
C14H14IN5O×HCl
C16H15N5×HCl
C17H17N5O×HCl×
C17H19N5O×HCl
C15H17N5OS×HCl
/
HCl×
/
1.5H2O
/
/
0.5H2O
0.3H2O
2.3H2O
1.5H2O
/
/
/
/
/
/
/
/
/
0.2H2O
1.2H2O
/
/
/
HCl×
/0.4H2O
/
HCl
/
/
0.2H2O
0.4H2O
0.5H2O
/
/
/
/
/
/
/
/
/
2H2O
1.3H2O
/
/
/0.2H2O
9s
9t
/
/
0.5H2O
0.5H2O
/
/
13a
13b
14a
14b
14c
14d
/
/
/
/
/
1.6H2O
/
/