Synthesis and biological evaluation of 2,5-dihydropyrazolo[4,3-c]quinolin-3-ones, a novel series of PDE 4 inhibitors with low emetic potential and antiasthmatic properties

Article abstract of DOI:10.1016/S0960-894X(00)00548-5

A novel series of 2,5-dihydropyrazolo[4,3-c]quinolin-3-ones has been prepared. These compounds showed good PDE 4 inhibitory activity and weak affinity for rolipram's binding site. They also exhibited a good anti-inflammatory profile without emetic side effects. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00548-5

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2661±2664
Synthesis and Biological Evaluation of
2,5-Dihydropyrazolo[4,3-c]quinolin-3-ones, a Novel Series
of PDE 4 Inhibitors with Low Emetic Potential
and Antiasthmatic Properties
Marõa I. Crespo,* Jordi Gracia, Carles Puig, Armando Vega, Josep Bou, Jordi Beleta,
Teresa Domenech, Hamish Ryder, Võctor Segarra and Jose M. Palacios
Almirall Prodesfarma, Research Centre, Cardener 68-74, 08024 Barcelona, Spain
Received 18 May 2000; revised 7 September 2000; accepted 18 September 2000
AbstractÐA novel series of 2,5-dihydropyrazolo[4,3-c]quinolin-3-ones has been prepared. These compounds showed good PDE 4
inhibitory activity and weak anity for rolipram's binding site. They also exhibited a good anti-in¯ammatory pro®le without
emetic side e€ects. # 2000 Published by Elsevier Science Ltd.
Introduction
Figure 1 shows the interaction pattern of the two mol-
ecules derived from the GRID computation. Although
the carbonyl group on the right-hand side is situated in
di€erent positions, both the maps of interactions and
the aromatic rings show good overlap.
Selective PDE 4 inhibitors, lacking adverse e€ects such
as emesis, have potential utility in asthma therapy.1
Following our initial strategy based on the pharmaco-
phore of compounds structurally related to nitraqua-
zone2 and arofylline,3
a
new series of 2,5-
dihydropyrazolo[4,3-c]quinolin-3-ones (DHPQ) has
Synthesis
been designed and synthesised.
The DHPQs were synthesised according to literature
procedures5 (Scheme 1). Condensation of aniline with
ethoxymethylenemalonate diethyl ester yielded the
quinolone 26, which was ®rst treated with phosphorous
oxychloride and then with the corresponding alkylated
hydrazine, to give DHPQ 14. Alkylation of 26 a€orded
the N-substituted quinolones 27, which reacted with
phosphorous pentasul®de to yield the thioderivatives
28. The ®nal DHPQs 1±13, 15±25 were obtained by
direct reaction of 28 with the corresponding alkylated
hydrazine or via initial condensation with hydrazine
and subsequent N-alkylation.
The synthesis, structure±activity relationship (SAR),
and the antiasthmatic potential of these new PDE 4
inhibitors are described.
Molecular Modelling Studies
In order to evaluate the 2,5-dihydropyrazolo[4,3-c]quin-
olin-3-ones in the nitraquazone-related pharmacophore,
the possible areas of interaction as hydrogen bond
acceptors for nitraquazone (represented in purple) and a
representative DHPQ (displayed in green, compound 3
in Table 1) have been studied by means of the pro-
gramme GRID.4
The maps have been obtained using the N1 probe (NH
¯at amide) and contoured at the level of 4 kcal/mol.
*Corresponding author. Fax: +34-932-913-420; e-mail: macrespo
@almirallprodesfarma.com
0960-894X/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00548-5

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M.I. Crespo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2661±2664
Results and Discussion
Table 1 summarises the in¯uence of substitution at the
N2 position (R0) for a series of compounds with a benzyl at
N5. Consistent with the nitraquazone pharmacophore,2 a
lipophilic group is required for activity. The best results
are obtained with cyclopentyl or cyclobutylmethyl
groups (compounds 10 and 12).
Structure±activity relationships
Compounds were tested for their inhibitory potencies
against isolated guinea pig ventricular PDE 4. Selectiv-
ity versus PDE 3, isolated from the same tissue, was also
determined. A representative group of compounds was
also evaluated for their ability to displace [3H]rolipram
from its high-anity binding site (HARBS) in rat brain
membranes. Interaction with this site has been proposed
as responsible for the unwanted CNS6 and emetic side
e€ects7 of PDE 4 inhibitors. The results are shown in
Tables 1 and 2.
Table 2 shows the e€ect on activity of the lipophilic
group at the N5 position (R). Interesting potencies and
selectivities are observed with compound 17, with a
cyclohexylmethyl group, and particularly with com-
pound 25 (R: 2-thienylmethyl, R0: cyclopentyl), which
has a PDE 4 activity comparable to that of rolipram.
With regard to the rolipram binding site, all DHPQs
evaluated have an improved HARBS/PDE 4 ratio with
respect to the reference compounds. For example, com-
pound 25 has an improvement in ratio of >100-fold and
>10-fold relative to rolipram and nitraquazone, respec-
tively. Further SAR and molecular modelling studies
will be carried out in order to ascertain the cause of this
improvement.
Table 1.
Activity in asthma models
Table 3 summarises the in vivo results obtained for a
selected group of DHPQs. All compounds tested show
anti-in¯ammatory activity in the guinea pig eosinophilia
model.8 By way of contrast, they are only moderately
e€ective in the histamine-induced bronchoconstriction
in guinea pig.9 In order to evaluate the side-e€ect pro®le
of this new series, the selected DHPQs were tested in an
emesis model.10 Consistent with their favourable
HARBS/PDE 4 ratio, these compounds fail to provoke
emesis in dog.
Compound
R0
PDE 3a PDE 4a HARBSa
1
2
3
4
5
6
7
8
9
10
11
12
H
Methyl
Ethyl
Isopropyl
Butyl
tert-Butyl
Pentyl
Benzyl
48
67
10
86
37
14
3
n.t.b
n.t.
n.t.
2
1.2
31
2.1
2.1
6.4
7
4.7
5.6
n.t.
n.t.
n.t.
35
6
5
>100
120
25
>100
26
>20
>200
102
242
>200
Phenethyl
Cyclopentyl
Norbornyl
Cyclobutylmethyl
Cyclohexylmethyl
10
0.7
1.4
0.8
24
0.32
0.05
In conclusion, we have identi®ed a new series of 2,5-
dihydropyrazolo[4,3-c]quinolin-3-ones which show PDE
4 inhibitory activities comparable to rolipram and
reduced anity for its high-anity binding site. They
also present a good anti-in¯ammatory pro®le and no
emetic e€ects. Compound 25 has been selected for
further evaluation.
13
Rolipram
Nitraquazone
n.t.
0.006
0.01
aIC50 (mM).
bn.t., not tested.
Figure 1. Interaction maps obtained with the N1 probe (NH ¯at amide) of GRID software for nitraquazone (purple) and DHPQ 3 (green).

M.I. Crespo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2661±2664
2663
Scheme 1. Reagents: (a) 100 ꢀC then PPA; (b) POCl3, re¯ux; (c) R0-NHNH2, dioxane, re¯ux; (d) R-X, KI, K2CO3, DMF; (e) P4S10, pyridine, re¯ux;
(f) NH2NH2, ethanol, re¯ux; (g) R0-NHNH2. C2O4H2, 2 N NaOH, pyridine, re¯ux; (h) R0-X, 2 N NaOH, DMF, 50 ꢀC.
Table 2.
Compound
R
R0
PDE 3a
PDE 4a
HARBSa
14
15
5
16
17
18
19
20
21
22
23
24
H
Phenyl
Benzyl
Phenethyl
Butyl
Butyl
Butyl
Butyl
Butyl
136
68
31
>200
111
>20
>20
>20
>200
>20
>20
>20
>20
242
90
18
n.t.b
n.t.
4.7
n.t.
1.5
1.2
0.9
n.t.
n.t.
14
50
2
1.0
0.006
0.01
2.1
160
0.7
0.5
0.4
11
Cyclohexylmethyl
Cyclohexylmethyl
Cyclohexylmethyl
m-Nitrobenzyl
m-Aminobenzyl
m-Chlorobenzyl
2-Thienylmethyl
2-Thienylmethyl
2-Thienylmethyl
tert-Butyl
Cyclopentyl
Butyl
Butyl
Butyl
Butyl
tert-Butyl
Cyclopentyl
12
2.3
2.7
0.8
0.4
0.32
0.05
25
Rolipram
Nitraquazone
>200
aIC50 (mM).
bn.t., not tested.

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M.I. Crespo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2661±2664
7. Barnette, M. S.; Torphy, T. J.; Christensen, S. B. Com-
pounds WO 00139, 1995.
Table 3.
Compound
Eosinophiliaa
Bronchodilationb
Emetic e€ectc
8. Guinea pig airway eosinophil in®ltration: Male Dunkin±
Hartley guinea-pigs (400Æ50 g) were sensitised with an injec-
tion of ovalbumin (OA, 3 mg/kg, 1 mL/kg ip) plus 5% Freund
incomplete adjuvant on days 0 and 3. On day 21 the animals
were fasted and the compounds were dosed by oral route
(1 mL/kg), 2 h prior to and 6 h after aerosolised antigen expo-
sure (1.5 mg/mL OA, 75 seg in 5 min) to pyrilamine protected
animals. Under urethane (1.5 g/kg ip) anaesthesia, 72 h later, a
bronchoalveolar lavage was done with saline (4 mL, twice)
through an intratracheal polyethylene cannula. Cellular sus-
pensions were centrifuged and extended for microscopic
observation after staining (Sangodi€1, Merck). Di€erent leu-
cocyte subtypes and proportions were determined under
1000Â magni®cation. Results are expressed as percentage
inhibition of OA-induced eosinophil in®ltration.
5
6
17
22
23
52
51
60
53
56
62
58
>100
n.t.
>100
62
>3 (iv)
>10 (po)
>1 (iv)
>10 (po)
>10 (po)
>3 (iv)
37
83
4
25
Rolipram
0.03 (iv)
aGuinea pig eosinophilia percentage inhibition at 10 mg/kg po.
bInhibition of histamine induced bronchoconstriction in guinea pig,
ED50 (mg/kg) iv.
cMinimum emetic dose in dogs (mg/kg).
9. Bronchodilation in anaesthetised guinea pigs: Male Dunkin±
Hartley guinea pigs (450Æ50 g) were anaesthetised with
sodium pentobarbitone (60 mg/kg ip) and externally ventilated
(10 mL/kg, 60 strokes/min) through a tracheal tube with a side
branch for measuring airway resistance. After stabilisation,
the left jugular vein was infused with histamine (10±20 mg/kg/
min) in order to achieve a sustained increase of about 150% in
in¯ation pressure. Compounds were dosed cumulatively
through a catheter in the right jugular vein. Results are
expressed as percentage of inhibition of histamine-induced
bronchospasm.
10. Emetic e€ects in conscious dogs: Trained Beagle dogs (10±
15 kg) of either sex were fasted overnight. On the day of the
experiment the animals were placed in slings and a Te¯on
catheter was inserted in the right cephalic vein and connected
to a perfusion pump. The compounds were dissolved and
administered through a 0.22 mM Millipore ®lter. Cumulative
doses of compounds were infused for 3 min and subsequent
observation periods of 30 min were allowed before increasing
the dose. This procedure was repeated until any emetic episode
occurred. Results are expressed as the minimum emetic dose.
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