Phthalazine PDE4 inhibitors. Part 3: The synthesis and in vitro evaluation of derivatives with a hydrogen bond acceptor

Article abstract of DOI:10.1016/S0960-894X(01)00668-0

This communication describes the synthesis and in vitro evaluation of a novel and potent series of phthalazine phosphodiesterase type (IV) (PDE4) inhibitors. The interaction with two distinct polar binding sites allowed us to eliminate the cyclopentyloxy

Full text of DOI:10.1016/S0960-894X(01)00668-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 5–8
Phthalazine PDE4 Inhibitors. Part 3: The Synthesis and In Vitro
Evaluation of Derivatives with a Hydrogen Bond Acceptor
Mauro Napoletano,a,* Gabriele Norcini,c Franco Pellacini,a Francesco Marchini,b
Gabriele Morazzoni,a Raimondo Fattori,a Pierpaolo Ferlengab and Lorenzo Pradellab
aInpharzam Ricerche, Zambon Group, Via Ai Soi, 6807 Taverne, Switzerland
bR&D, Zambon Group SpA, Via Lillo Del Duca 10, 20091 Bresso, Milan, Italy
cLamberti SpA, Fine Chemical Lab., Via Piave 18, 21041 Albizzate, Italy
Received 12 March 2001;revised 10 September 2001;accepted 2 October 2001
Abstract—This communication describes the synthesis and in vitro evaluation of a novel and potent series of phthalazine phos-
phodiesterase type (IV) (PDE4) inhibitors. The interaction with two distinct polar binding sites allowed us to eliminate the cyclo-
pentyloxy substitution from rolipram-like analogues. # 2001 Elsevier Science Ltd. All rights reserved.
Phosphodiesterase type IV (PDE4) is a cAMP-specific
phosphodiesterase highly expressed in inflammatory
cells and in airway smooth muscle.1 The observation
that an elevation of cAMP in these cells can suppress
inflammatory effects and can induce muscle relaxation
has stimulated great interest in developing selective
PDE4 inhibitors as therapeutic agents for asthma and
other inflammatory diseases.2
other functionality.4 Among the most potent analogues
RP 73401 (2)5 and GW 3600 (3)6 present interesting and
distinct pharmacophores for the pyrrolidone ring. It has
been proposed that the acetyl carbonyl of 3 replaces the
lactam carbonyl in rolipram (1)6 and we wondered if the
carbamic group could mimic the pyridine ring of 2 at a
distinct binding site.
In order to explore this hypothesis we decided to add a
binding moiety in our recently described class of phtha-
lazine PDE4 inhibitors represented by 47 and 58 by means
of a carbonyl function or a suitable hydrogen bond
acceptor on the pyrazine ring as depicted in derivatives 6.
With the three-dimensional structure of the active site
only recently described,3 PDE4 inhibitors have been
designed mostly starting from the archetypal inhibitor
rolipram (1) by replacing the pyrrolidone of 1 with
*Corresponding author. Tel.: +41-91-9354519;fax: +41-91-9354512;e-mail: mauro.napoletano@zambongroup.com
0960-894X/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00668-0

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M. Napoletano et al. / Bioorg. Med. Chem. Lett. 12 (2002) 5–8
Chemistry
In fact, molecular modelling simulation of the overlay of
2, 3 and 6a (see Table 1) showed that proper substitution
in position 3 of the phthalazine nucleus could fit with
the carbonyl group of 3 (Fig. 1).9
The synthesis of the requisite phthalazines is illustrated
in Schemes 1 and 2. Catalytic hydrogenation of 4
(Scheme 1) gave dihydro derivative 7 chemo-selectively.
Compound 7 slowly decomposes on standing, and was
directly acylated with the proper reagent affording the
target products 6a,b.
In implementing this modification, we hoped to increase
enzyme inhibitory potency in order to eliminate the
cyclopentyloxy group from the aromatic ring. Such
substitution is commonly observed in rolipram-like
PDE4 inhibitors10 and its lipophilicity could contribute
to the poor pharmacokinetic and metabolic profile often
seen in this class of molecules.11
Scheme 2 describes the strategy adopted for the synth-
esis of 5-unsubstituted phthalazine 6c–p. Ortho-depro-
tonation of known protected acid 812 and subsequent
Table 1.
Compd
R
R1
PDE4 IC50 (nM)
Rolipram binding Ki (nM)
RBS/PDE4 ratio
1 (Rolipram)
2 (RP 73041)
Ariflo
1680
1
73
53
1.6
1.5
38
149
0.001
1.5
0.5
4
2.8
6a
6b
COCH3
SO2CH3
30
2
128
5
4.3
2.5
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
13
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
COH
COCH3
14% (10ꢀ7 M)
13% (10ꢀ6 M)
138
45% (10ꢀ7 M)
51
397
237
7.8
8.5
19.9
>20
>20
2.9
COCH2CH3
COCH(CH3)2
COPh
COCH2Ph
SO2CH3
COCOOCH2CH3
COOCH3
CONH2
CONHCH3
CON(CH3)2
CONHOH
28
25
30
12
21
113
72
498
17% (10ꢀ7 M)
36% (10ꢀ7 M)
60
212
199
63
235
473
1.9
2.8
1.7
1.8
36
131
35% (10ꢀ7 M)
85
208
42% (10ꢀ5 M)
2.4
0% (10ꢀ7 M)

M. Napoletano et al. / Bioorg. Med. Chem. Lett. 12 (2002) 5–8
7
the pyridine ring and a polar substituent. In the rigid
phthalazine scaffold, the tetrahedral sulphonyl group is
probably better orientated to accept a hydrogen bond.
With these results in hand, we moved our attention to
the 5-unsubstituted phthalazine derivatives. Compound
13 and dihydro derivative 6c were devoid of activity
confirming the importance that the cyclopentyloxy sub-
stituent plays in the PDE4 inhibition. We were pleased
to verify that introducing a polar p-bond such as in 6e
and 6j, potent PDE4 inhibitory activity was restored
even in absence of the catechol cyclopentyl substitution.
It is worth noting the reduced difference in activity
between 6j and 6e compared to 6b and 6a. Probably the
absence of the sterically demanding cyclopentyloxy in 6e
makes it possible to better accommodate within the
binding pocket.
Figure 1. Overlay of 2 (blue), 3 (green), and 6a (red). The overlay is
based on RMS fitting of the respective catechol oxygens and the pyri-
dine nitrogens of 2 and 6a with the carbamic group of 3.
Following these results, a brief SAR study of substitu-
tion at R1 was performed varying the acetyl moiety.
Increasing the size and lipophilicity of the substituent,
from formyl to phenylacetyl, 6d–i gave gradual
improvement in PDE4 inhibitory activity. A similar
trend was observed for the selectivity for the catalytic
binding site over the rolipram-binding site (RBS/PDE4
ratio). Such selective binding is claimed to be a potential
property for overcoming the side effects often seen with
potent PDE4 inhibitors.16 Derivatives 6k–p showed that
other acyl substituents were allowed, with a drop of
PDE4 inhibition increasing the substitution in the urea
series (6m–o). No improvement of PDE inhibitory
activity was produced by hydroxamic derivative 6p in
contrast with what has been reported in other series of
rolipram analogues.17
formylation produced 9 in high yield. Acid hydrolysis
forming 10 followed by hydrazine cyclisation provided
phthalazinone 11 uneventfully. POCl3 chlorination gave
12, which upon treatment with the sodium salt of 3,5-
dichloro-4-methylpyridine furnished intermediate 13.
Optimised condition for the catalytic hydrogenation
resulted in stable 6c, followed by treatment with proper
reagents to access desired compounds 6d–p.
Biological Results and Discussion
Table 1 summarises the in vitro activity of phthalazines
with respect to human neutrophil PDE4 inhibition
(IC50, nM)13 and association the high affinity rolipram
binding site (Ki, nM).14 Activity of the three standards
was determined in-house using these procedures. Ariflo
was added in comparison because it has been recently
described as a second-generation inhibitor of PDE4 in
development.15
In conclusion, the synthesis and the in vitro evaluation
of a novel series of potent phthalazine PDE4 inhibitors
has been reported, demonstrating that two distinct
binding pockets exist in the PDE4 enzyme that accom-
modate the pyridine ring and a suitable hydrogen bond
donor. The interaction with these two binding sites
allowed us to eliminate the catechol substitution usually
observed in rolipram analogues. Moreover, the new
binding site can tolerate large lipophilic substituents,
which leads to a great improvement in the RBS/PDE4
ratio. Studies that further characterise this class of
molecules are in progress and the obtained result will be
reported in due course.
Disappointingly, only a slight variation in PDE4 cata-
lytic activity was observed passing from 4 to 6a but
changing from the acetyl to the methanesulphonyl sub-
stitution (6b) the expected activity improvement was
obtained. This result confirms the existence of two dis-
tinct binding sites in PDE4 enzyme that accommodate
Scheme 1. Reagents and conditions: (i) 4 atm H2, PtO2, THF, rt, 24 h, 75%;(ii) R 1Cl, Et3N, CH2Cl2, rt, 1 h, 60–80%.

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M. Napoletano et al. / Bioorg. Med. Chem. Lett. 12 (2002) 5–8
Scheme 2. Reagents and conditions: (i) BuLi, Et2O, 0 ꢁC, 4 h then DMF, rt, 8 h, 84%;(ii) 50% aq H 2SO4, EtOH, reflux, 20 h, 90%;(iii) hydrazine
hydrate, acetic acid, 80 ꢁC, 4 h, 82%;(iv) POCl 3, CH3CN, reflux, 3 h, 98%;(v) 3,5-dichloro-4-methylpyridine, NaH, DMF, rt, 20 h, 80%;(vi) 2 atm
H2, cat PtO2, THF, rt, 22 h, 99%;(vii) for 6d: formylimidazole, THF, rt, 24 h, 93%. For 6e–k: R1Cl, Et3N, THF, rt, 3 h, 66–86%. For 6l–p
(R1=R2CO): 1,10-carbonyldiimidazole, THF, rt, 1 h then R2H, rt, 3 h, 25–86%.
References and Notes
conformations used in the RMS fitting are within 1 Kcal/mol
of the lowest energy conformations.
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