Phthalazine PDE4 inhibitors. Part 2: The synthesis and biological evaluation of 6-methoxy-1,4-disubstituted derivatives

Article abstract of DOI:10.1016/S0960-894X(00)00587-4

This communication describes the synthesis and in vitro evaluation of a novel and potent series of phosphodiesterase type IV (PDE4) inhibitors. The compounds described present substituents in position 4 of the phthalazine ring to replace the commonly obse

Full text of DOI:10.1016/S0960-894X(00)00587-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 33±37
Phthalazine PDE4 Inhibitors. Part 2: The Synthesis and
Biological Evaluation of 6-Methoxy-1,4-disubstituted Derivatives
Mauro Napoletano,a,* Gabriele Norcini,c Franco Pellacini,a Francesco Marchini,b
Gabriele Morazzoni,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 (Milano), Italy
cLamberti SpA, Fine Chemical Lab., Via Piave 18, 21041 Albizzate, Italy
Received 4 August 2000; revised 28 September 2000; accepted 11 October 2000
AbstractÐThis communication describes the synthesis and in vitro evaluation of a novel and potent series of phosphodiesterase
type IV (PDE4) inhibitors. The compounds described present substituents in position 4 of the phthalazine ring to replace the
commonly observed cyclopentyloxy moiety of rolipram analogues. Preliminary evidences of reduced side e€ects compared to
standards and improved pharmacokinetic properties for selected derivatives are also reported. # 2000 Elsevier Science Ltd. All
rights reserved.
Phosphodiesterase type IV (PDE4) metabolises cyclic
adenosine monophosphate in pulmonary in¯ammatory
and immune cells, and in pulmonary smooth muscle
cells, and thereby promotes bronchoconstriction and
airway in¯ammation.1 Selective PDE4 inhibitors would,
therefore, be expected to produce both bronchodilata-
tory and anti-in¯ammatory e€ects in patients with
asthma.2
a conformationally constrained analogue of RP 73401
(2), one of the most potent PDE4 inhibitors described
so far.4
Improved selectivity for the catalytic binding site over
the rolipram binding site, and preliminary evidences of
reduced side e€ects of 1 compared to 2, rolipram (3),
and Ari¯o (4) suggested an active role of the added
pyridazine nucleus to the observed activity.
In a previous paper we have described a novel series of
phthalazine PDE4 inhibitors represented by 1,3 which is
*Corresponding author. Tel.: +41-(0)91-9354519; fax: +41-(0)91-
9354512; e-mail: mauro.napoletano@zambongroup.com
0960-894X/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00587-4

34
M. Napoletano et al. / Bioorg. Med. Chem. Lett. 11 (2001) 33±37
Scheme 1. Reagents and conditions: (i) 37% aq HCHO (1.3 equiv), 37% HCl (1.5 equiv), acetic acid, 90 ꢀC, 14 h, 46%; (ii) NBS, cat AIBN,
chlorobenzene, 85 ꢀC, 0.7 h, 98%; (iii) triphenylphosphine, THF, re¯ux, 7 h, 86%; (iv) 3,5-dichloro-4-pyridinecarboxaldehyde, Et3N, THF, 1 h rt
then 2 h re¯ux, 95%; (v) hydrazine hydrate, MeOH, acetic acid, re¯ux, 2 h, 99%; (vi) POCl3, re¯ux, 4 h, 94%; (vii) for 5a: 2 atm H2, 10% Pd/C, 32%
NaOH (1.5 eq), DMF, rt, 2 h, 52%. For 5b±f: RH, DMF, 100 ꢀC, 20 h, 34±79%. For 5g±j: RH, NaH, DMF, 100 ꢀC, 20 h, 49±77%. For 5k: phenyl
lithium, ZnCl2, THF, rt, 1 h, then 12, cat PdAcO2/2PPh3, re¯ux, 24 h, 55%. For 5l: phenylacetylene, piperidine, cat PdCl2/2PPh3/CuI, DMF, rt,
20 h, 52%. For 5m: 2-methyl-3-butyn-2-ol, K2CO3, cat PdCl2/2PPh3/CuI, DMF, rt, 16 h, then cat NaH, toluene, re¯ux, 4 h, 61%.
Potent PDE4 inhibition was obtained for this class of
compounds indicating that a planar dihedral angle
between the phenyl ring and the linker region of roli-
pram-like analogues is allowed. However the derivative
1 showed a decreased potency compared to open
amide 2.
A possible reason for this behaviour could derive from
an unfavourable interaction between the cyclopentyloxy
and the peri-hydrogen in position 4 of the phthalazine
nucleus. In fact, studies carried out with rolipram ana-
logues having rigid heterocycles as surrogates for the
catechol substitution5 showed that the bulky alkoxy
Chemistry
group must adopt an orientation toward the region of
space occupied by the pyrazine ring in 1 to explicate
optimal PDE4 inhibition activity.
The synthesis of the requisite phthalazines is illustrated
in Schemes 1 and 2. Chloroformylation of 3-methoxy-
benzoic acid 6 (Scheme 1) and subsequently radical
bromination of 7 produced 8 in good yield. Treatment
with triphenylphosphine to give 9 and Wittig ole®nation
with 3,5-dichloro-4-pyridinecarboxaldehyde6 a€orded
compound 10 as a 7:3 mixture of diastereoisomers.
Hydrazine cyclization forming phthalazine 11 followed
by POCl3 chlorination gave 12, which upon treatment
with the proper reagents provided the target compounds
5a±m.
On the other hand, the presence of the added ring gave
us the possibility to evaluate if proper substituents in
position 4 of the phthalazine nucleus, as depicted in
derivatives 5, could replace the cyclopentyloxy moiety at
its important enzyme recognition site. In implementing
this modi®cation we hoped to improve the pharmaco-
kinetic pro®le of this class of molecules while retaining
the desired safety pro®le of 1.

M. Napoletano et al. / Bioorg. Med. Chem. Lett. 11 (2001) 33±37
35
Scheme 2. Reagents and conditions: (i) RLi or RMgX, Et2O, 78 ꢀC, 0.75 h, 38±65%; (ii) hydrazine hydrate, MeOH, acetic acid, re¯ux, 2 h, 85±98%;
(iii) 55% m-chloroperbenzoic acid, CHCl3, re¯ux, 18 h, 55%; (iv) hydrazine hydrate, MeOH, acetic acid, re¯ux, 18 h, 68%.
In Scheme 2 is described the strategy adopted for the
synthesis of phthalazine 5n±q. Low temperature alkyla-
tion with selected lithium derivatives or Grignard
reagents of intermediate 10 to give 13 and subsequent
treatment with hydrazine furnished derivatives 5n±p
uneventfully. Finally 13 (R=thiazolyl) was selectively
oxidised to the corresponding N-oxide 14 using m-
CPBA and transformed into 5q by means of usual
hydrazine cyclisation.
that present substituents with diverse physico-chemical
characteristics giving us the possibility to modulate their
pharmacokinetic pro®le. The introduction of a polar
tertiary amine gave potent derivatives 5d and 5f, which
were soluble in water as hydrochloride salts. Phenyl sub-
stituted 5k and its heterocyclic analogues 5i and 5p showed
both potent PDE4 activity and whole cell activity.
For most compounds, improved TNFa inhibition com-
pared to PDE4 inhibition was obtained by increasing
the polarity of the substituent in position 4 (for instance
5f versus 5k). This is probably due to an augmented
capability for more polar derivatives to penetrate into
the cells, even if a di€erent anity toward PDE4 sub-
types can not be ruled out.1 It is worth noting the PDE4
inhibitory potency of pyridine N-oxide 5q in accordance
with what has been reported in literature.4,10
Biological Results and Discussion
Table 1 summarises the in vitro activity of phthalazines
with respect to human neutrophil PDE4 inhibition
(IC50, nM),7 association with the high anity roli-
pram binding site (Ki, nM)8 and human monocytes
TNFa synthesis inhibition (IC50, nM).9 Activity of the
three standards was determined in-house using these
procedures.
For all active derivatives an excellent selectivity for the
catalytic binding site over the rolipram binding site
compared to standards was retained with the bulky
lipophilic 5l as the most selective in this series. Such
selective binding is claimed to be a potential property
for overcoming the side e€ects often seen with potent
PDE4 inhibitors.9
As expected, phthalazine 5a, which is unsubstituted
in positions 4 and 5, was devoid of PDE4 inhibitory
activity con®rming the importance that a substituent in
that space region plays in the binding interaction.
Introducing a substituent in position 4 a great activity
enhancement was observed for several derivatives.
Whether the added substitution improves the anity for
the PDE4 enzyme by mimicking the commonly
observed cyclopentyloxy moiety of rolipram-like inhib-
itors or it interacts with a hitherto unexplored binding
site remains to be clari®ed.
Preliminary studies to evaluate activity, potential side
e€ects and pharmacokinetic properties of this novel
series were performed comparing phthalazines 5k and 5i
to standard. Ari¯o (4) was chosen because it has been
recently described as a second-generation inhibitor of
PDE4 with a good bioavailability and a decreased
potential for side e€ects.11 Their in vitro metabolic
stability,12 oral bioavailability in the rat,13 inhibition of
eosinophil in®ltration in the guinea pig,14 ability to
increase acid secretion in isolated whole rat stomach15
and to induce emesis in the dog16 are reported in
Table 2.
The inhibition of PDE4 catalytic activity proved to be
quite sensitive to substituent modi®cation in position 4
of the phthalazine ring rendering a structure±activity
relationship dicult to rationalise. However, potent
inhibition for PDE4 has been obtained for derivatives

36
M. Napoletano et al. / Bioorg. Med. Chem. Lett. 11 (2001) 33±37
Both the in vitro and the in vivo assays suggested an
improved therapeutic potential for 5k and 5i. Stability
in rat hepatocytes and promising bioavailability in the
rat were encouraging for obtaining oral activity. More-
over 5k and 5i con®rmed their activity in a guinea pig
model of late-phase response to antigen. We were also
very pleased to verify that, at the maximum dose tested,
no sign of emesis was detected. Studies are in progress
to verify if this favourable behaviour of 5k and 5i is
shared among this series.
In conclusion, the synthesis and biological evaluation of
a novel series of potent phthalazine PDE4 inhibitors has
been reported, demonstrating that it is possible to
Table 1.
Compound
R
n
PDE4 IC50 (nM)
Rolipram binding Ki (nM)
TNFa IC50 (nM)
3 (Rolipram)
2 (RP 73041)
4 (Ari¯o)
1
5a
5b
5c
1680
1
73
1.6
1.5
225
1.3
158
254
Ð
38
149
53
H
0
0
0
0% (10 7 M)
19% (10 7 M)
39% (10 7 M)
42% (10 5 M)
nPrNH
(CH3)2N
Ð
Ð
Ð
Ð
5d
5e
5f
0
0
0
132
16% (10 7 M)
38
452
Ð
62
Ð
9
89
5g
0
6% (10 7 M)
Ð
Ð
5h
5i
0
0
0
0
0
0
0
0
0
17% (10 7 M)
Ð
Ð
72
241
383
5j
132
436
166
46
5k
5l
Ph
37
271
48
93
21% (10 6 M)
273
87
5m
5n
5o
5p
204
Ð
C2H5
23% (10 7 M)
19% (10 7 M)
14
Ð
Ph(CH2)4
Ð
Ð
34
8
5q
1
4
17
3
Table 2.
Compound
Met. stab. Cl.int
1
Bioav.
(rat)
F%
Eosinophilia
(guinea pig)
% inhibition at 30 mmol/kg ip
Acid secretion
IC50 (mM)
Emesis
(dog model) ED50
(mmol/kg iv)
1
(mLÂmin Â106cells
)
4 (Ari¯o)
5k
5i
9.2
5.8
1.5
35
27
27
17
34
37
1
10
>30 (0/8)
>30 (0/8)
4
>30 (33%)

M. Napoletano et al. / Bioorg. Med. Chem. Lett. 11 (2001) 33±37
37
replace the cyclopentyloxy moiety of rolipram with a
proper substituent in position 4 of the phthalazine ring.
Further results on ecacy and safety of this class are
being produced and the progress in this area will be
reported in the near future.
9. Barnette, M. S.; Bartus, J. O.; Burman, M.; Christensen, S.
B.; Cieslinski, L. B.; Esser, K. M.; Prabhakar, U. S.; Rush, J.
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10. Hulme, C.; Mathew, R.; Moriarty, K.; Miller, B.;
Ramanjulu, M.; Cox, P.; Souness, J.; Page, K. M.; Uhl, J.;
Travis, J.; Labaudiniere, R.; Huang, F.; Djuric, S. W. Bioorg.
Med. Chem. Lett. 1988, 8, 3053.
11. Christensen, S. B.; Guider, A.; Forster, C. J.; Gleason, J.
G.; Bender, P. E.; Karpinski, J. M.; DeWolf, W. E., Jr.;
Barnette, M. S.; Underwood, D. C.; Griswold, D. E.; Cie-
slinski, L. B.; Burman, M.; Bochnowicz, S.; Osborn, R. R.;
Manning, C. D.; Grous, M.; Hillegas, L. M.; O'Leary Bartus,
J.; Ryan, M. D.; Eggleston, D. S.; Haltiwanger, R. C.; Tor-
phy, T. J. J. Med. Chem. 1998, 41, 821.
12. Metabolic stability method: Fresh rat hepatocytes were
obtained by in situ perfusion of the liver. The cellular vitality
was >80%. Hepatocytes were incubated at 37 ꢀC for up to
120 min in either Williams' E medium or Hanks' balanced salt
solution at a concentration of 3±4Â106 cells/mL of suspension.
Compounds were dissolved in DMSO and used at di€erent
concentrations 1±100 mM (®nal concentration of DMSO=
1%). After liquid±liquid extraction or deproteinisation samples
were submitted to HPLC/UV analysis. The metabolic stability
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