9-Benzyladenines: Potent and selective cAMP phosphodiesterase inhibitors [2]

Full text of DOI:10.1021/jm960827x

1768
J . Med. Chem. 1997, 40, 1768-1770
9-Ben zyla d en in es: P oten t a n d Selective
cAMP P h osp h od iester a se In h ibitor s
J ean-J acques Bourguignon,*,† Laurent De´saubry,†
Pierre Raboisson,† Camille-Georges Wermuth,† and
Claire Lugnier‡
Laboratoire de Pharmacochimie Mole´culaire, UPR 421 du
CNRS, Centre de Neurochimie, 5, rue Blaise Pascal, 67084
Strasbourg Cedex, France, and Laboratoire de
Pharmacologie Cellulaire et Mole´culaire, Faculte´ de
Pharmacie, 74, route du Rhin, BP 24,
67401 Illkirch Cedex, France
Received December 6, 1996
Adenine derivatives substituted in position 9 consti-
tute an important class of pharmacologically active
compounds for which different targets can be postulated.
Because they resemble the structure of adenosine, they
may compete with the neuromodulator at its specific
pharmacological receptors,1 transport proteins, or meta-
bolic enzymes. Particularly erythro-9-(2-hydroxy-3-
nonyl)adenine (EHNA) is known as a potent inhibitor
of adenosine deaminase2 (Figure 1).
However, 9-substituted adenines also constitute pos-
sible competitors for adenosine-deriving endogenous
substances, such as adenosine monophosphate and its
corresponding cyclic nucleotide, diphosphate, and tri-
phosphate (AMP, cAMP, ADP, ATP) respectively. Thus
EHNA was found to inhibit a specific phosphodiesterase
(PDE) isozyme found in heart3 and vascular endothelial
cells4 with micromolar IC50 values.
F igu r e 1. Phosphodiesterase inhibition of two known phar-
macologically active 9-substituted adenines.
ent akyl halides, afforded the corresponding 9-substi-
tuted isomers 3 as major compounds. The 6-chloro-4,5-
diaminopyrimidines 4, obtained as described earlier,5
were reacted with methyl orthoacetate in presence of
catalytic amounts of methanesulfonic acid to yield the
corresponding 8-methylchloropurines 5. Treatment of
the latter compounds 3 or 5 with various amines
provided the desired adenines 6 (NCS compounds). The
most basic adenines were analyzed and tested as their
hydrochlorides or their more water-soluble methane-
sulfonate salts.
The efficiencies of a first series of differently substi-
tuted 9-alkyladenines have been evaluated as PDE
inhibitors via their IC50 values on PDE3 and PDE4 from
vascular smooth muscle. The concentration of each
drug that inhibited 50% of the enzymatic activity was
determined at 1 µM substrate concentration. Data are
listed in Table 1.
Several years ago, Kelley et al. described a 9-benzyl-
adenine derivative (BWA78U) possessing potent anti-
convulsant effects.5 As this compound also presents
potent anxiolytic and sedative properties,6 an interac-
tion with benzodiazepine receptors has been first sus-
pected, but found to be very weak7,8 ([3H]diazepam, IC50
) 13 µM). Moreover, a pretreatment of the rats with
the benzodiazepine receptor antagonist flumazenil did
not block the potent anticonvulsant properties of this
compound (Marescaux and Bourguignon, unpublished
results). As BWA78U did not bind to A1 and A2
Resu lts a n d Discu ssion
PDE1-PDE5 are well-known and pharmacologically
characterized. PDE1 is activated by calcium-calmodu-
lin and preferentially hydrolyzes cGMP in brain and in
vascular smooth muscle. The PDE2 which hydrolyzes
both cAMP and cGMP is activated by cGMP. Activated
PDE2 is specifically inhibited by EHNA. This isoform
is present in brain and vascular endothelial cells. PDE3
which preferentially hydrolyzes cAMP is inhibited by
cGMP and specific cardiotonic agents (SKF 94120).
PDE3 is present in platelets, cardiac tissue, and vas-
cular smooth muscle but is absent in brain. PDE4
specifically hydrolyzes cAMP, is potently and specifically
inhibited by rolipram, and is present in brain, cardiac
tissue, and vascular smooth muscle. PDE5 specifically
hydrolyzes cGMP and is preferentially inhibited by
zaprinast. PDE5 is present in vascular and airway
smooth muscle tissues but is absent in the brain and
heart.
adenosine receptors ([3H]CHA and [3H]NECA, IC50
>
100 µM), we evaluated its capacity to inhibit the
different phosphodiesterases (PDE), particularly those
hydrolyzing cAMP. Following the classification pro-
posed by Beavo,9 the different PDE isoforms can be
grouped into seven families according to the related
gene, their substrate specificity, their modulation by
endogenous regulators (calcium-calmodulin, cGMP),
and their selective inhibitions by typical inhibitors. In
this paper, we report the behavior of BWA78U and some
of its analogues toward PDE3 and PDE4 preparations.
It appears that some of these compounds exhibit potent
phosphodiesterase inhibition properties with high se-
lectivity toward PDE4.
Ch em istr y. The 2-substituted 6-chloropurines 2a -c
were obtained from their corresponding hypo-
xanthines10-12 by treatment with POCl3 in presence of
N,N-dimethylaniline (Scheme 1).13 Deprotonation of
6-chloropurines 2 by means of potassium carbonate in
DMF or DMSO, and subsequent alkylation with differ-
We found that BWA78U was able to significantly
inhibit several PDE isoforms from vascular smooth
muscle, particularly its Ki value of 2 µM for PDE4 could
be compared with that of rolipram (Ki ) 0.8 µM), the
reference inhibitor for this isoform (Figure 1). Unex-
pectedly, when considering Ki values, it presented some
* To whom all correspondence should be addressed.
† Laboratoire de Pharmacochimie Mole´culaire.
‡ Laboratoire de Pharmacologie Cellulaire et Mole´culaire.
S0022-2623(96)00827-8 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1769
Sch em e 1. Access to Differently Substituted Adenine Derivativesa
a
(a) POCl3; (b) RCl (or RBr), K2CO3, DMSO; (c) HNR1R2, EtOH; (d) RNH2, BuOH, Et3N, reflux; (e) Me-C(OMe)3, H+, room temperature.
Ta ble 1. Inhibition of PDE’s by 9-Alkyladenines
a
IC50 (µM)
compd
salt
mp, °C
R1
R2
Me
n-Bu
-(CH2)5-
R3
R4
R
PDE3
PDE4
BWA78Ua
MeSO3H
HCl
HCl
195
186
177
146
138
188 dec
176
233
156
175
109
155
195
H
H
H
H
H
CH3
CF3
Ph
H
H
H
CF3
CF3
CF3
CF3
H
H
H
H
H
H
H
H
Me
H
H
H
H
2-FC6H4
2-FC6H4
2-FC6H4
2-FC6H4
2-FC6H4
2-FC6H4
C6H5
C6H5
2-FC6H4
C6H5
121
ns
ns
250
380
ns
339
ns
ns
101
61
ns
331
3
53
6a
6b
6c
6d
6e
6f
6g
6h
6i
121
0.2
0.04
0.5
4.6
160
54
1.8
11
0.75
11
MeSO3H
free base
MeSO3H
MeSO3H
free base
MeSO3H
free base
free base
free base
free base
H
H
H
H
H
H
H
Me
H
H
Me
Me
Me
Me
H
Me
H
Me
Me
Me
6j
C6H5
CH2C6H5
H
6k
6l
a
The IC50 was calculated by linear regression (correlation coefficient r ) 0.95) and represents the mean value of three determinations.
The experimental error is about 15%. ns: IC50 > 200 µM.
selectivity profile, as it is significantly less active on
PDE2 and PDE3 (Ki ) 122 and 35 µM, respectively).
Thus we focused our attention on both PDE4 and PDE3
and checked substituent effects on different positions
of the adenine ring of BWA78U.
dramatic loss in activity (compare 6l and 6d ). The
unsubstituted N-benzyladenine 6g with an IC50 of 160
µM clearly highlights the necessity for potency of the
presence of a methyl group on exocyclic nitrogen of the
adenine.
It is noteworthy that the fluorine in position 2 of the
phenyl ring is not needed for PDE4 inhibition (compare
BWA78U and compound 6f). Methyl substituent effects
allowed a first evaluation of minimal structural pertur-
bations tolerated. Thus the presence of a methyl group
in position 8 (6h ) led to a more than 1 order of
magnitude decrease in potency of this compound, when
compared to BWA78U.
Introducing a methyl group in position 2 in compound
6c significantly increased its potency. Thus the 2-meth-
yl group was replaced by a trifluoromethyl (6d ) or a
phenyl group (6e). Strong beneficial effects were ob-
Cyclic AMP analogues have been recently tested as
PDE inhibitors.14 Some of them, with limited structural
modifications such as 2′-deoxy-cAMP, showed micromo-
lar IC50 values on PDE3 and PDE4. However, it was
unexpected to discover cAMP PDE inhibitors derived
from ribose-free adenines, and with same micromolar
IC50 values. First SAR analysis indicated a high
potency of adenines in the presence of (i) a secondary
amidine bearing a methyl group in position 6, (ii) a
benzyl group in position 9, and (iii) a trifluoromethyl
group in position 2. Compound 6d (NCS 613) was found
to be highly selective in inhibiting the PDE4 (IC50 values
of 40, 380, 0.04, and 5 µM for PDE1, PDE3, PDE4, and
PDE5, respectively) and thus constitutes an efficient tool
for the study of PDE4 inhibition. When compared with
the high-affinity rolipram binding sites, another site of
action in PDE4 can be postulated for BWA78U and
adenine derivatives. This site probably partially over-
laps the cAMP binding site.14 Further work including
served with the trifluoromethyl derivative 6d (IC50
)
40 nM). This compound served as reference for further
SAR studies. The N,N-dimethyl derivative 6j was found
to be 300 times less potent and less selective than 6d ,
whereas the N,N-unsubstituted derivative 6i was equi-
potent with BWA78U (micromolar range IC50 value).
Suppressing the phenyl ring at position 9 led to a

1770 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Communications to the Editor
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better the mechanism of action of this novel class of
PDE4 inhibitors, in relation with their in vivo pharma-
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