The synthesis and biological evaluation of a novel series of phthalazine PDE4 inhibitors I

Article abstract of DOI:10.1016/S0960-894X(00)00449-2

This communication describes the synthesis and in vitro evaluation of a novel and potent series of phosphodiesterase type IV (PDE4) inhibitors. The compounds described represent conformationally constrained analogues of RP 73401, Piclamilast. Preliminary evidences of reduced side effects of 11 compared to standards are also reported. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00449-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2235±2238
The Synthesis and Biological Evaluation of a Novel Series of
Phthalazine PDE4 Inhibitors I
Mauro Napoletano,a,* Gabriele Norcini,a,y Franco Pellacini,a Francesco Marchini,b
Gabriele Morazzoni,a Pierpaolo Ferlengab and Lorenzo Pradellab
aInpharzam Ricerche, Zambon Group, Via Industria 1, 6814 Cadempino, Switzerland
bR&D, Zambon Group SpA, Via Lillo Del Duca 10, 20091 Bresso, Milan, Italy
Received 8 May 2000; revised 1 August 2000; accepted 7 August 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 represent conformationally constrained analogues of RP 73401, Piclamilast. Preliminary
evidences of reduced side e€ects of 11 compared to standards are also reported. # 2000 Elsevier Science Ltd. All rights reserved.
PDE4 is a cAMP-speci®c phosphodiesterase highly
expressed in in¯ammatory cells and in airway smooth
muscle. Inhibition of PDE4 results in an elevation of cAMP
in these cells, which mediates anti-in¯ammatory e€ects and
muscle relaxation in a variety of animal models.1 The
potential use of PDE4 inhibitors as anti-in¯ammatory
agents for the treatment of asthma and other in¯ammatory
disorder has generated great interest in this area.2
Subsequently, many e€orts have focused on ®nding
suitable isosteres for the 3,4-dialkoxyphenyl moiety of 1
(or its analogues).6 8
Until now, no studies have been carried out to evaluate
the possible conformations that the phenyl ring can
adopt during the interaction with the enzyme. In order
to gather information on this aspect, we designed rigid
analogues of 2 where the carbonyl function is replaced
by a p-bond of an aromatic ring.
The archetypal PDE4 inhibitor rolipram (1) has been
the starting point for the majority of medicinal chem-
istry e€orts2 and from which derives the commonly
observed 4-methoxy-3-cyclopentyloxy substitution on
the aromatic ring.3
In implementing this modi®cation, we also hoped to
retain the necessary recognition elements for enzyme
inhibition while reducing the unwanted side e€ects, such
as emesis9 and gastrointestinal acid secretion.10 The
phthalazine nucleus was chosen in order to increase the
polarity of 2.
Initially, most of the research in this area dealt with the
replacement of the pyrrolidinone of 1 with other func-
tionality4 and from this work RP 73401 (2)5 emerged as
one of the most potent PDE4 inhibitors.
Chemistry
The synthesis of the requisite phthalazines is illustrated
in Schemes 1±3. Oxidation of aldehyde 3 (Scheme 1) to
4, followed by amidation a€orded intermediate 5. Low
temperature deprotonation and formylation gave deriva-
tive 6.
Acid hydrolysis of both the benzyl ether and amide
bond produced 7 in high yield. Hydrazine cyclization
forming 8 followed by O-alkylation with selected mesyl-
ates a€orded compounds 9. POCl3 chlorination gave 10,
which upon treatment with the sodium salt of 3,5-
dichloro-4-methylpyridine or 4-amino-3,5-dichloropyr-
idine provided the target compounds 11±15. Selective
*Corresponding author. Tel.: +41-(0)91-9354519; fax: +41-(0)91-
9354512; e-mail: mauro.napoletano@zambongroup.com
yPresent address: Lamberti SpA, Fine Chemical Lab., Via Piave 18,
21041 Albizzate, Italy.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00449-2

2236
M. Napoletano et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2235±2238
Scheme 1. Reagents and conditions: (i) Bu4N+KMnO4 , pyridine, rt, 3 h, 88%; (ii) SOCl2, re¯ux, 2 h; then Et2NH, CH2Cl2, 10 ꢀC, 1 h, 94%; (iii)
s-BuLi, TMEDA, DMF, THF, 78 ꢀC, 4 h, 32%; (iv) 10% HCl in acetic acid, re¯ux, 18 h, 100%; (v) hydrazine hydrate, EtOH, 60 ꢀC, 5 min, 72%;
(vi) R1O2SCH3, Na2CO3, cat KI, DMF, 90 ꢀC, 16 h, 66±88%; (vii) POCl3, re¯ux, 1 h, 95±98%; (viii) 3,5-dichloro-4-methylpyridine or 4-amino-3,5-
dichloropyridine, NaH, DMF, rt, 1.5 h, 22±60%; (ix) 11, sodium p-thiocresolate, DMF, 90 ꢀC, 2 h, 40%; (x) CHClF2, K2CO3, cat KI, DMF, 75 ꢀC,
4.5 h, 21%; (xi) 11, 55% m-chloroperbenzoic acid, CH2Cl2, rt, 1 h, 64%.
Scheme 2. Reagents and conditions: (i) 32% aq HCHO (1.2 equiv), 20% HCl, 60 ꢀC, 48 h, 60%; (ii) NBS, cat benzoyl peroxide, CCl4, re¯ux, 2 h,
then 5% HCl, re¯ux, 4 h, 60%; (iii) hydrazine hydrate, EtOH, 60 ꢀC, 5 min, 93%; (iv) POCl3, re¯ux, 1 h, 98%; (v) 3,5-dichloro-4-methylpyridine or
4-amino-3,5-dichloropyridine, NaH, DMF, rt, 1.5 h; (24: 59%; 25: 22%).
demethylation of 11 to give 16 and di¯uorocarbene
insertion resulted in the methoxy bioisostere derivative
17. Regiochemical oxidation of 11 to its corresponding
N-oxide 18 proceeded smoothly with m-CPBA.11
formation to chloride 28 and subsequent alkylation
provided 29 as described above. Sequential catalytic
hydrogenation a€orded 30 and 31, respectively.
In Scheme 2 is described the strategy adopted for the
synthesis of phthalazines bearing the cyclopentyloxy
substitution in position 7. Chloroformylation of known
acid 195 and subsequent radical oxidation of 20 pro-
duced 21 uneventfully. Following the same pathway
utilized for the 5,6-disubstituted phthalazines, the desired
derivatives 24 and 25 were obtained.
Biological Results and Discussion
Table 1 summarizes the in vitro activity of phthalazines
with respect to human neutrophil PDE4 inhibition (IC50,
nM),12 association with the high anity rolipram binding
site (Ki, nM)13 and human monocytes TNFa synthesis
inhibition (IC50, nM).14 Activity of the three standards
was determined in-house using these procedures. SB
207499 (Ari¯o) was added for comparison because it
has been recently described as a second-generation
inhibitor of PDE4 with a decreased potential for side
e€ects.15
Intermediate 8 (Scheme 3) formed tri¯ate 26 that was
suciently reactive to give Pd0 cross-coupling with
alkynyl derivatives. This behaviour gave us the possi-
bility to prepare compounds such as 27. Usual trans-

M. Napoletano et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2235±2238
2237
Scheme 3. Reagents and conditions: (i) (CF3SO2)2O, pyridine, CH2Cl2, 5 ꢀC, 1 h, 73%; (ii) pent-4-ynylbenzene, 5% bis(triphenylphosphine)PdCl2,
Et3N, DMF, 90 ꢀC, 3 h, 65%; (iii) POCl3, 80 ꢀC, 0.5 h, 100%; (iv) 3,5-dichloro-4-methylpyridine, NaH, DMF, rt, 3 h, 51%; (v) H2, 10% Pd/C, THF,
rt, 1 h, 43%; (vi) 4 atm H2, 10% Pd/C, THF, rt, 48 h, 17%.
Table 1.
Compound
X
R1
R2
R3
n
PDE4
IC50 (nM)
Rolipram binding
Ki (nM)
TNFa
IC50 (nM)
1 (Rolipram)
2 (RP 73041)
SB 207499 (Ari¯o)
11
1680
1
73
53
1.6
1.5
38
149
225
1.3
158
254
CH2
NH
Ð
Ð
Ð
CH3
CH3
CH3
0
0
0
12
13
59
30
95
16
187
252
CH2
14
15
CH2
CH2
O(CH2)5Ph
Ð
Ð
CH3
CH3
0
0
57
13
83
Ð
153
286
17
CH2
Ð
Ð
CHF2
0
39
46
167
18
24
CH2
CH2
CH3
CH3
1
0
42
121
Ð
139
Ð
Ð
Ð
186
7
25
29
NH
CH3
CH3
0
0
7% (10 M)
Ð
71
Ð
30
CH2
Ð
10
30
31
CH2
CH2
Ð
Ð
CH3
CH3
0
0
19
30
164
320
52
(CH2)5Ph
103

2238
M. Napoletano et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2235±2238
Table 2
this class are being produced and the progress in this
area will be reported in the near future.
Compound
Acid secretion
IC50 (ꢀM)
Emesis (dog model)
ED50 (ꢀmol/kg iv)
1 Rolipram
2 (RP 73041)
SB 207499 (Ari¯o)
11
0.04
0.07
1
0.3
1
10
References and Notes
1. Torphy, T. J. Am. J. Respir. Crit. Care Med. 1998, 157,
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7
>10 (0/8)
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Potent inhibition for PDE4 has been obtained for most
of the phthalazines synthesized and these data well cor-
relate with TNFa inhibition observed. These activities
strongly indicate that a planar dihedral angle between
the phenyl ring and the linker region of rolipram-like
PDE4 inhibitors is allowed. Of the two possible planar
conformations, that represented by phthalazines 11 and
12 (cyclopentyloxy substitution in position 5) is clearly
preferred compared with the substitution in position 7
(compound 24 and 25). The replacement of the nitrogen
with a methylene linker between the phthalazine nucleus
and the pyridine (11 vs 12) does not a€ect or improves (24
vs 25) the activity, suggesting only a spacer role for this
part of PDE4 inhibitors, at least in this series.
In general, the role of the alkoxy substituents con®rmed
the known structure±activity relationship of rolipram-
derived PDE4 inhibitors.3,16 It is interesting to note the
increased anity for the rolipram-binding site shown by
the bulky, more lipophilic derivatives 13 and 14 compared
to 11. On the contrary, it is worth noting the excellent
selectivity of compounds 29±31, with the alkyl sub-
stituents directly on the aromatic ring, for the catalytic
binding site over the rolipram-binding site. Such selec-
tive binding is a potential property for overcoming the
side e€ects often seen with potent PDE4 inhibitors.14
10. Schmeichen, R.; Schneider, H. H.; Watchtel, H. Psycho-
pharmacology 1990, 102, 17.
11. The oxidation regiochemistry was assigned on the basis of
the down-shift signal shown by the proton in position 4 in the
1H NMR spectra.
12. Nielson, C. P.; Vestal, R. E.; Sturm, R. J.; Heaslip, R. J.
All. Clin. Immunol. 1990, 86, 801.
Preliminary studies to evaluate the potential side e€ects
of this novel series were performed comparing phthala-
zine 11 to standards. Their ability to increase acid
secretion in isolated whole rat stomach17 and to induce
emesis in dog18 is reported in Table 2.
13. Schneider, H. H.; Schmiecher, R.; Brezinski, M.; Seidler,
J. Eur. J. Pharmacol. 1986, 127, 105.
14. Barnette, M. S.; Bartus, J. O.; Burman, M.; Christensen,
S. B.; Cieslinski, L. B.; Esser, K. M.; Prabhakar, U. S.; Rush,
J. A.; Torphy, T. J. Biochem. Pharmacol. 1996, 51, 949.
15. 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.;
Cieslinski, 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.;
Torphy, T. J. J. Med. Chem. 1998, 41, 821.
16. Cheng, J. B.; Cooper, K.; Duplantier, A. J.; Eggler, J. F.;
Kraus, K. G.; Marshall, S. C.; Marfat, A.; Masamune, H.;
Shirley, T. J.; Tickner, J. E.; Umland, J. P. Bioorg. Med.
Chem. Lett. 1995, 5, 1969.
17. Brunce, K. T.; Parsons, M. E. J. Physiol. 1976, 453.
18. Emesis method: Male and female beagle dogs (10±12 kg)
were used. Tests compounds were dissolved in PEG200/
DMSO 85/15 and administered by iv injection (3 mL) with an
ascending dose regiment, waiting 1 h between two successive
treatments. The dose that induced emesis in at least four out
eight animals has been determined.
Both the in vitro and the in vivo assays suggested an
improved therapeutic potential for 11. We were very
pleased to verify that, at the maximum solubility
obtainable for 11 in the vehicle, no sign of emesis was
detected. Studies are in progress to verify if this favour-
able behaviour of 11 is the consequence of the improved
selectivity for the catalytic over the high anity roli-
pram-binding site or is due to a preferential anity
toward the PDE4 subtypes.1
In conclusion, the synthesis and in vitro evaluation of a
novel series of potent PDE4 inhibitors has been repor-
ted, demonstrating that the phthalazine nucleus is an
e€ective, polar sca€old to design rolipram-like PDE4
inhibitors. Preliminary results on ecacy and safety of