Synthesis and evaluation as PDE4 inhibitors of pyrimidine-2,4-dione derivatives

Article abstract of DOI:10.1002/ddr.20395

A series of nitraquazone analogs with a pyrimidindione core was synthesized and tested for inhibitory activity on PDE4, selectivity versus PDE3 and PDE5 and for affinity towards the rolipram high-affinity binding site (HARBS). The 5-anilino derivatives 13

Full text of DOI:10.1002/ddr.20395

DRUG DEVELOPMENT RESEARCH 72 : 274–288 (2011)
Research Article
Synthesis and Evaluation as PDE4 Inhibitors
of Pyrimidine-2,4-dione Derivatives
Ã
Maria P. Giovannoni,1 Alessia Graziano,1 Rosanna Matucci,2 Marta Nesi,2
Nicoletta Cesari,1 Claudia Vergelli,1 Claudio Biancalani,1 Letizia Crocetti,1
Agostino Cilibrizzi,1 and Vittorio Dal Piaz1
1Dipartimento di Scienze Farmaceutiche, Firenze, Italy
2Dipartimento di Farmacologia Preclinica e Clinica ‘‘M.A. Mancini,’’ Firenze, Italy
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Toxicology, Formulation Phases I-III
Technology,
Genomics,
Proteomics
Preclinical
Research
Postmarketing
Phase IV
Drug Delivery,
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
A series of nitraquazone analogs with a pyrimidindione core was synthesized and tested
for inhibitory activity on PDE4, selectivity versus PDE3 and PDE5 and for affinity towards the rolipram
high-affinity binding site (HARBS). The 5-anilino derivatives 13–18 showed the best profile combining
appreciable PDE4 inhibitory activity (IC50 5 5–14 mM) with a good selectivity toward PDE3 and PDE5. The
same compounds demonstrate low affinity for the HARBS site with IC50 values of 12–69 mM (IC50 for
Rolipram 5 3.6 nM). Drug Dev Res 72:274–288, 2011.
r 2010 Wiley-Liss, Inc.
Key words: HARBS; PDE4; selectivity
INTRODUCTION
neutrophil responses responsible for pathological
inflammatory process, including the production of
IL-8, superoxide anions, degranulation, chemotaxis,
and adhesion [Houslay et al., 2005]. PDE4 inhibitors
could ameliorate the mucus hypersecretion that
characterizes asthma and COPD by reducing the
transcriptional activity of MUC5AC, a regulator gene
in mucin secretion [Mat et al., 2005]. Moreover, PDE4
inhibitors reduce superoxide anion production from
eosinophils as well as the adhesion and infiltration of
the latter, suggesting a potential role in allergic rhinitis,
asthma, and atopic dermatitis [Spina, 2004]. PDE4
inhibitors can also reduce tumor necrosis factor-a
(TNFa) production suggesting a role in rheumatoid
Phosphodiesterases are a superfamily of enzymes
responsible for the hydrolysis of cAMP and cGMP
to the corresponding nucleosides, AMP and GMP.
Eleven different families have been described
(PDE1–PDE11), but the role, the characteristics, as
well as the specific inhibitors of the newer discovered
types PDE8-PDE11 have not yet been widely defined
[Lugnier, 2006]. The PDE4 family, which selectively
hydrolyzes cAMP, is mainly present in inflammatory
cells, brain, and cardiovascular tissue [Tenor and
Schudt, 1996; Houslay et al., 1998; Stoclet et al.,
1995] and constitutes four subtypes, PDE4A–D
[Houslay, 2001]. The involvement of PDE4 in patho-
logical processes in these tissues indicates that
compounds able to modulate PDE4 activity could have
use in a variety of diseases [Houslay et al., 2005].
PDE4 inhibitors have been widely studied for the
Ã
Correspondence to: Maria P. Giovannoni, Dipartimento di
Scienze Farmaceutiche, Via Ugo Schiff 6, Sesto Fiorentino 50019,
Firenze, Italy. E-mail: mariapaola.giovannoni@unifi.it
Received 30 July 2010; Accepted 5 September 2010
treatment of asthma and chronic obstructive pulmon-
ary disease (COPD) [Lipworth, 2005; Dal Piaz and
Giovannoni, 2000] as they suppress many of the
Published online in Wiley Online Library (wileyonlinelibrary.
com). DOI: 10.1002/ddr.20395
ꢀc
2010 Wiley-Liss, Inc.

NITRAQUAZONE PDE4 INHIBITORS
275
arthritis, psoriasis, and Crohn’s disease [Seldon et al., compounds were further investigated [Martin et al.,
1995; Jimenez et al., 2001]. PDE4 inhibitors can also 2002; Pieretti et al., 2006].
induce cell cycle arrest and apoptosis in limphoblastic
In the present work, we have designed and
leukaemia cells [Ogawa et al., 2002] and block synthesized a new series of pyrimidine-2,4-diones as
angiogenic response of endothelial cells [Favot et al., simplified analogues of nitraquazone 8, a prototypic
2004], suggesting possible applications in oncology. The PDE4 inhibitor [Glaser and Traber, 1984; Russo et al.,
antidepressant effect of PDE inhibitors has been 1987] and report on their PDE4 inhibitory activity,
considered. Despite initial studies where PDE4 selectivity versus PDE3/PDE5, and affinity for
inhibitors had
a
potent antidepressant action HARBS.
[O’Donnel, 1993], no further progress has occurred
because of an incomplete understanding of the
functional importance of PDE4 subtypes and of low-
and high-affinity rolipram binding site (HARBS) for
pathology [O’Donnel and Zhang, 2004]. Finally, PDE4
MATERIALS AND METHODS
Chemistry
All melting points were determined on a Buchi
1
inhibitors have shown efficacy in the MPTP mouse apparatus and are uncorrected. H-NMR spectra were
model [Zhang et al., 2004], suggesting potential in recorded with Avance 400 instruments (Bruker Biospin,
Parkinson’s disease. Rolipram 1 (Fig. 1), the prototypic version 002 with SGU). Chemical shifts are reported in
PDE4 inhibitor, was extensively investigated as an ppm, using the solvent as internal standard. Extracts
antidepressant more than two decades ago but side were dried over Na2SO4 and the solvents were
effects including nausea, vomiting, and headache, related removed under reduced pressure. E. Merck (West
to binding to HARBS, limited its clinical potential Point, PA) F-254 commercial plates were used for
[Schneider et al., 1986; Sounes and Rao, 1997]. Many analytical TLC to follow the course of the reaction.
PDE4 inhibitors of different chemical classes have also Silica gel 60 (Merck 70-230 mesh) was used for column
been in clinical trials for the treatment of asthma, but chromatography.
they were discontinued because of side effects.
[3H]-cAMP, [3H]-cGMP, and [3H]Rolipram
The disconnect between HARBS affinity and were from Perkin-Elmer Life and Analytical Science
PDE4 inhibition found in more recent compounds (Waltham, MA); all other chemicals were purchased
allowed the selection of a number of molecules that are from Sigma-Aldrich (St. Louis, MO) (Fig. 1).
currently in clinical trials. Roflumilast 2 [Hatzeman and
The synthesis of the novel pyrimidine-2,4-diones
Schudt, 2001; Karish and Gagnon, 2006] is the most substituted at position 1, 3, and 5 is depicted in Figures
advanced PDE4 inhibitor, having been approved in the 2–4. In Figures 2 and 3, the procedure to afford the
EU for severe COPD associated with chronic bron- 5-substituted aniline derivatives 13a–d and 18a–c is
chitis area in 2010. Cilomilast 3 [Christensen et al., reported. In both instances, the starting material is
1998; Barnette et al., 1998] and tofimilast 4 [Duplantier represented by the previously described pyrimidine-
et al., 2007; Bayes et al., 2007] are selective PDE4 2,4-diones 9a,b [Winckelmann and Larsen, 1986;
inhibitors. The former is being studied for the Robin et al., 2006] and 14 [Winckelmann and Larsen,
treatment of emphysema and bronchitis while the 1986]. In Figure 2, the first step is a bromination at
latter was discontinued because of unacceptable side position 5 performed with bromine in acetic acid;
effects. Other PDE4 inhibitor molecules in clinical treatment of the intermediates 10a,b (10a) [Moltke-
trials include apremilast 5 [Khobzaoui et al., 2005] Leth and Joergensen, 1993] with concentrated ammo-
(CC10004), which is being evaluated in psoriatic nia afforded the 5-amino derivatives 11a,b, which were
arthritis and refractory psoriasis [http://clinicaltrial. alkylated in standard condition to give 12a,b. The last
gov/.], and tetomilast 6 [Bloomfield et al., 1997; step to obtain the final compounds 13a–d was
O’Mahony, 2005] (OPC-6535), which has been recently performed through a cross-coupling reaction with the
studied in patients with ulcerative colitis, Crohn’s appropriate aryl boronic acids using copper acetate as
disease, and COPD (Phase II) [http://clinicaltrial.gov/.]. catalyst and a weak base (triethylamine) in CH2Cl2
CC-1088 7 [Muller et al., 1998] is structurally related [Quach and Batey, 2003; Chiang and Olsson, 2004].
to 5.
Similarly, compounds 18a–c were obtained starting
In the last decade, we reported [Dal Piaz et al., from the 5-amino derivatives 17a,b, following the
1997, 1998; Giovannoni et al., 2007] on heterocyclic- above described cross-coupling reaction. In this synthetic
fused 3-(2H)-pyridazinones with appreciable activity as route, compound 15, obtained by alkylation from the
PDE-4 inhibitors, good selectivity toward PDE3 precursor 14, was nitrated with a mixture of potassium
isoenzyme, and, in some cases, low affinity for nitrate and sulfuric acid to afford compound 16.
[3H]rolipram binding site. The more interesting Treatment of compound 16 with stannous chloride in
Drug Dev. Res.

276
GIOVANNONI ET AL.
CHF2
O
O
H3CO
Cl
NH
H
N
O
O
Rolipram 1
O
N
Cl
Roflumilast
2
H3CO
O
N
N
N
N
NC
S
N
COOH
Tofimilast 4
Cilomilast 3
O
OCH2CH3
OCH2CH3
SO2CH3
N
O
N
HOOC
N
NHCOCH3
S
OCH3
H3CH2CO
Tetomilast 6
Apremilast 5
O
O
N
CONH2
CH2CH3
N
N
O
O
O
OCH3
CC-1088 7
NO2
Nitraquazone 8
Fig. 1. PDE4 inhibitors.
hydrochloric acid using a 1:1 molar ratio selectively alkylated under standard conditions affording 19a–d,
reduced the nitro group at position 5 to give the which, in turn, were subjected to a cross-coupling
corresponding 5-amino derivative 17a. Reduction of reaction under classical Suzuki conditions using tetra-
both nitro groups at position 5 and in para position of kis(triphenylphosphine)palladium and the opportune
the phenyl ring was performed using a 1:5 molar ratio phenylboronic acid in toluene [Wang and Haseltine,
of substrate 16 and stannous chloride to yield 17b. In 1994] to give the final 5-(substituted)aryl pyrimidine-
Figure 4, the common precursors are again repre- 2,4-diones 20a–m. In the second route, the bromine at
sented by 5-bromo pyrimidine-2,4-diones 10a–c (10c) position 5 was displaced with 4-chloro-benzylamine to
[Gutschow et al., 2001], which, in the first route, were give compound 21, which, treated with ethyl bromide,
Drug Dev. Res.

NITRAQUAZONE PDE4 INHIBITORS
277
O
N
O
HN
HN
a
O
R
R
R
O
N
R
9a,b
10a,b
Br
b
Et
N
O
N
O
N
HN
c
O
R
O
11a,b
12a,b
H2N
H2N
d
Et
N
O
N
9-12
a
b
R
Cl
CH3
O
13a-d
HN
13
a
b
R
Cl
Cl
X
3-NO2
4-Cl
c
d
CH3
CH3
3-NO2
3-COOCH2CH3
X
Fig. 2. Synthesis of 5-(substituted)anilino pyrimidine-2,4-diones 13a–d. Reagents and conditions: (a) Br2, AcOH 100%, rt, 3h; (b) NH4OH 33%,
sealed tube, 1201C, 5 h; (c) EtBr, DMF, K2CO3, 901C, 1h; (d) X-C6H5-B(OH)2, CH2Cl2, Ac2Cu, Et3N, rt, 24 h.
afforded the final compound 22. Finally, several a sealed tube at 1201C for 4 h. After, the mixture was
molecules with pyridine-2-one core (24a–c) were concentrated in vacuo and the residue was treated with
synthesized starting from previously described pre- cold ethanol to afford a crude solid that was collected
cursors 23a,b [Bormann, 1981; Hagen et al., 1990], by suction.
performing an alkylation at position 1 (Fig. 5).
5-Amino-1-(4-chlorophenyl)-1H-pyrimidine-
2,4-dione (11a) Mp43001C; crystallization solvent 5
EtOH; yield 5 61%. H-NMR (DMSO), d: 4.25 (2H,
5-Bromo-1-p-tolyl-1H-pyrimidine-2,4-dione (10b)
1
To a stirred mixture of 9b (0.74 mmol) in glacial
acetic acid (7 mL), a solution of bromine (0.74 mmol) in
the same solvent (0.5 mL) was added in a dropwise
manner (over a 2-h period). The mixture was stirred for
1 h, concentrated in vacuo to afford a residue that,
when treated with cold water (15 mL), gave rise to a
crude precipitate that was recovered by suction.
Mp 5 202–2051C; crystallization solvent 5 EtOH;
yield 5 70%. 1H-NMR (CDCl3), d: 2.45 (3H, s),
7.20–7.35 (4H, m), 7.70 (1H, s), 8.50 (1H, exch br s).
exch br s), 6.85 (1H, s), 7.45 (2H, d, J 5 8.5 Hz), 7.55
(2H, d, 8.5 Hz), 11.50 (1H, exch br s).
5-Amino-1-p-tolyl-1H-pyrimidine-2,4-dione
(11b) Mp 5 227–2291C; crystallization solvent 5
EtOH; yield 5 80%. 1H-NMR (DMSO), d: 2.35
(3H, s), 4.20 (2H, exch br s), 6.80 (1H, s), 7.30
(4H, s), 11.45 (1H, exch br s).
General Procedure for Compounds (12a,b)
A mixture of the appropriate pyrimidin-2,4-dione
(11a or 11b, 0.5 mmol), anhydrous K2CO3 (1 mmol),
and ethyl bromide (0.75 mmol) in anhydrous DMF
General Procedure for Compounds (11a,b)
To the appropriate 5-bromopyrimidin-2,4-dione (2 mL) was heated under stirring for 40–120 min at
(10a or 10b, 0.76 mmol), 4 mL of concentrated 60–901C. After dilution with cold water, the crude
ammonia was added. The reaction was carried out in precipitate 12a was recovered by suction, while
Drug Dev. Res.

278
GIOVANNONI ET AL.
Et
N
O
N
O
N
HN
a
O
O
14
15
b
Et
N
Et
N
O
N
O
N
c
O
R
O
NO2
17a,b
16
H2N
O2N
d
17
a
b
R
NO2
NH2
Et
N
O
N
O
R
18
a
b
R
X
Cl
18a-c
NO2
NO2
NH2
HN
COOCH2CH3
COOCH2CH3
c
X
Fig. 3. Synthesis of 5-(substituted)anilino pyrimidine-2,4-diones 18a,b. Reagents and conditions: (a) EtBr, DMF, K2CO3, 901C, 1 h; (b) KNO3,
conc. H2SO4, rt, 8 h; (c) SnCl2, conc HCl, rt, 1 h; (d) ArB(OH)2, CH2Cl2, Ac2Cu, Et3N, rt, 24 h.
compound 12b was extracted (3 Â 20 mL) with ethyl The molecular sieves together with copper salts were
acetate (30–40 mL).
filtered off and the solution was extracted first with
5-Amino-1-(4-chlorophenyl)-3-ethyl-1H-pyri- concentrated ammonia (2 Â 5mL) and then with water
midine-2,4-dione (12a) Mp 5 1901C; crystallization (2 Â 5 mL). The organic layer was evaporated in vacuo
solvent 5 EtOH; yield 5 63%. 1H-NMR (DMSO), and the residue recovered by suction (compounds
d: 1.15 (3H, t, J 5 7.2 Hz), 3.90 (2H, q, J 5 7.2 Hz), 13c,d). For compounds 13a,b, evaporation of the solvent
4.30 (2H, exch br s), 6.90 (1H, s), 7.45 (2H, d, afforded an oil that was purified by column chromato-
J 5 8.9 Hz), 7.55 (2H, d, J 5 8.9 Hz).
graphy using cyclohexane/ethyl acetate 1:2 as eluent.
5-Amino-1-p-tolyl-3-ethyl-1H-pyrimidine-2,4-
1-(4-Chlorophenyl)-3-ethyl-5-(3-nitropheny-
lamino)-1H-pyrimidine-2,4-dione (13a) Mp 5
190–1931C; purified by column chromatography
(cyclohexane/ethyl acetate 1:2); yield 5 20%. 1H-
NMR (DMSO), d: 1.25 (3H, t, J 5 7.1 Hz), 3.90 (2H,
q, J 5 7.1 Hz), 7.20 (1H, d, J 5 8.8 Hz), 7.40 (1H, dd,
J 5 2.2, 8.8 Hz), 7.50 (1H, d, J 5 8.8 Hz), 7.55 (5H, m),
7.80 (1H, exch br s), 7.85 (1H, s). Anal. Calcd for
C18H15ClN4O4: C, 55.89; H, 3.91; N, 14.49. Found: C,
56.00; H, 3.92; N, 14.52.
dione (12b) Mp 5 157–1601C; crystallization solvent 5
1
EtOH; yield 5 64%. H-NMR (DMSO), d: 1.15 (3H, t,
J 5 7.2 Hz), 2.35 (3H, s), 3.90 (2H, q, J 5 7.2 Hz), 4.25
(2H, exch br s), 6.85 (1H, s), 7.30 (4H, s).
General Procedure for Compounds (13a–d)
A mixture of activated molecular sieves (0.8–1 g),
the substrates 12a or 12b (0.5 mmol), the appropriate
boronic acid (0.5 mmol) commercially available, copper
acetate (0.8 mmol), and triethylamine (1 mmol) in
CH2Cl2 (10 mL) were stirred for 6–24 h at rt À401C.
1-(4-Chlorophenyl)-5-(3-chlorophenylamino)-3-
ethyl-1H-pyrimidine-2,4-dione (13b) Mp 5117–1201C;
Drug Dev. Res.

NITRAQUAZONE PDE4 INHIBITORS
279
O
HN
10
a
b
R
Cl
CH3
H
O
a
N
R
c
10a-c
Br
for 10a
HN
c
R1
N
O
N
O
N
O
Cl
O
R
19a-d
21
HN
H2C
Br
b
R1
N
O
N
d
Cl
O
R
Et
N
O
N
20a-m
O
Cl
X
22
HN
20
a
b
c
d
e
f
g
h
i
R
Cl
Cl
Cl
Cl
Cl
Cl
CH3
CH3
CH3
H
X
H
3-NO2
R1
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
H2C
3-Cl
3-COOCH2CH3
3-CN
3,4-OCH3
3-NO2
3-Cl
3-COOCH2CH3
3-NO2
3-Cl
3-COOCH2CH3
3-Cl
Cl
j
k
l
19
R
Cl
R1
H
H
H
a
b
c
d
CH2CH3
CH2CH3
CH2CH3
m
cyclopropylmethyl
CH3
H
H
cyclopropylmethyl
Fig. 4. Synthesis of 5-(substituted)phenyl pyrimidine-2,4-diones 20a–m and of compound 22. Reagents and conditions: (a) R1Br, DMF, K2CO3, 901C,
1h; (b) X-C6H5-B(OH)2, Pd(PPh3)4, 2M Na2CO3, toluene, 901C, 16h; (c) 4-chlorobenzylamine, EtOH, 901C, 9h; (d) EtBr, DMF, K2CO3, 901C, 1h.
purified by column chromatography (cyclohexane/ crystallization solvent 5 EtOH; yield 5 17%.1H-NMR
1
ethyl acetate 1:2); yield 5 20%. H-NMR (CDCl3), d: (DMSO), d: 1.15 (3H, t, J 5 7.2 Hz), 2.35 (3H, s), 3.90
1.30 (3H, t, J 5 7.2 Hz), 4.15 (2H, q, J 5 (2H, q, J57.2 Hz), 7.20 (1H, d, J58.7 Hz), 7.35 (5H, m),
7.2 Hz), 6.85 (1H, d, J 5 9.0 Hz), 6.90 (1H, d, 7.50 (1H, d, J58.7 Hz), 7.55 (1H, s), 7.80 (1H, s), 7.85
J 5 9.0 Hz), 7.00 (1H, s), 7.20 (2H, m), 7.30–7.50 (1H, exch br s). Anal. Calcd for C19H18N4O4: C, 62.29; H,
(4H, m), 7.90 (1H, exch br s). Anal. Calcd for 4.95; N, 15.29. Found: C, 62.41; H, 4.96; N, 15.32.
C18H15Cl2N3O2: C, 57.46; H, 4.02; N, 11.17. Found:
C, 57.57; H, 4.03; N, 11.19.
3-(3-Ethyl-2,4-dioxo-1-p-tolyl-1,2,3,4-tetrahy-
dropyrimidin-5-yl-amino)benzoic acid ethyl ester
3-Ethyl-5-(3-nitrophenylamino)-1-p-tolyl-1H- (13d) Mp 5 130–1351C; crystallization solvent 5
pyrimidine-2,4-dione
(13c)
Mp 5 200–2031C; EtOH; yield 5 20%.1H-NMR (CDCl3), d: 1.30 (3H, t,
Drug Dev. Res.

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GIOVANNONI ET AL.
R
CH3
CH3
HN
N
X
X
a
O
O
23a,b
24a-c
NC
23
NC
X
H
24
a
R
X
a
CH2CH3
3,4 OCH3
b
3,4-OCH3
b
c
cyclopropylmethyl
cyclopropylmethyl
H
3,4 OCH3
Fig. 5. Synthesis of 1-alkylpyrimidones 24a–c. Reagents and conditions: (a) RBr, DMF (or CH3CN), K2CO3, 901C, 1h.
J 5 7.2 Hz), 1.40 (3H, t, J 5 7.2 Hz), 2.40 (3H, s), 4.15 (2H, exch br s), 7.00 (1H, s), 7.75 (2H, d, J 5 8.9 Hz),
(2H, q, J 5 7.2 Hz), 4.40 (2H, q J 5 7.2 Hz), 7.15 (1H, 8.35 (2H, d, J 5 8.9 Hz).
d, J 5 8.8 Hz), 7.30 (6H, m), 7.60 (1H, d, J 5 8.8 Hz),
5-Amino-3-ethyl-1-(4-aminophenyl)-1H-pyri-
7.70 (1H, s), 7.85 (1H, exch br s).
midine-2,4-dione (17b) MpZ3001C; crystallization
1
Anal. Calcd for C22H23N3O4: C, 67.16; H, 5.89; solvent 5 EtOH; yield 5 40%. H-NMR (DMSO), d:
N, 10.68. Found: C, 67.29; H, 5.90; N, 10.70.
1.10 (3H, t, J 5 7.2 Hz), 3.87 (2H, q, J 5 7.2 Hz), 4.14
(2H, exch br s), 5.27 (2H, exch br s), 6.56 (2H, d,
J 5 8.3 Hz), 6.75 (1H, s), 6.95 (2H, d, J 5 8.3 Hz r).
3-Ethyl-1-phenyl-1H-pyrimidine-2,4-dione (15)
Compound 15 was obtained from compound 14
following the general procedure described for 13a–d.
After dilution with cold water, the suspension was
extracted with ethyl acetate (3 Â 20 mL) and the
solvent was evaporated in vacuo to afford 15.
Mp 5 119–1211C; crystallization solvent 5 EtOH;
yield 5 20%. 1H-NMR (CDCl3), d: 1.30 (3H, t,
J 5 7.0 Hz), 4.10 (2H, q, J 5 7.0 Hz), 5.90 (d, 1H,),
7.30–7.55 (6H, m).
3-Ethyl-5-nitro-1-(4-nitrophenyl)-1H-pyrimi-
dine-2,4-dione (16) A mixture of pyrimidine-2,4-
dione 15 (0.46 mmol) KNO3 (1.84 mmol) and 0.1 mL
of concentrated H2SO4 (1.84 mmol) was heated at
1001C in a sealed tube for 3 h. After cooling, cold water
was added and the precipitate filtered off. Mp 5 1121C
dec.; crystallization solvent 5 EtOH; yield 5 90%.
1H-NMR (CDCl3), d: 1.35 (3H, t, J 5 7.0 Hz), 4.15
(2H, q, J 5 7.0 Hz), 7.65 (2H, d, J 5 8.8 Hz), 8.45 (2H,
d, J 5 8.8 Hz), 8.80 (s, 1H, 6-CH).
General Procedure for Compounds (18a–c)
Compounds 18a–c were obtained starting from
17a,b following the general procedure described for
13a–d. Evaporation of the solvent afforded 18a as a
crude precipitate and 18b,c as oil. The latter were
purified by column chromatography using cycloexane/
ethyl acetate 2:1 as eluent.
5-(3-Chlorophenylamino)-3-ethyl-1-(4-nitrophe-
nyl)- 1H-pyrimidine-2,4-dione (18a) Mp 5166–1681C;
crystallization solvent 5EtOH; yield 55%. 1H-NMR
(CDCl3), d: 1.40 (3H, t, J57.2 Hz), 4.15 (2H, q,
J57.2 Hz), 6.90 (1H, d, J58.8 Hz), 6.95 (1H, d,
J58.8 Hz), 6.95 (3H, m), 7.25 (2H, m), 7.65 (2H, d, J5
9.0 Hz), 7.90 (1H, exch br s), 8.40 (2H, d, J59.0 Hz).
Anal. Calcd for C18H15ClN4O4: C, 55.89; H, 3.91; N,
14.49. Found: C, 56.00; H, 3.92; N, 14.52.
3-(3-Ethyl-2,4-dioxo-1-(4-nitrophenyl)-1,2,3,4-
tetrahydropyrimidin-5-yl-amino)benzoic acid ethyl
ester (18b) Mp 5 129–1311C; purified by column
chromatography (cyclohexane/ethyl acetate 2:1);
General Procedure for Compounds (17a,b)
To a mixture of compound 16 (0.30 mmol) and yield 5 30%. 1-NMR (DMSO), d: 1.30 (3H, t, J 5
concentrated HCl (0.8 mL), a solution of SnCl2 7.2 Hz), 1.40 (3H, t, J 57.2 Hz), 4.20 (2H, q, J 5 7.2 Hz),
(0.3 mmol to obtain compound 17a and 1.5 mmol to 4.40 (2H, q, J 57.2 Hz), 7.10 (1H, m), 7.25 (2H, m), 7.50
obtain compound 17b) in 1 mL of concentrated HCl (1H, m), 7.80 (3H, m), 7.90 (1H, exch br s), 8.35 (2H, d,
was added. The reaction was carried out at room J 58.9 Hz). Anal. Calcd for C21H20N4O6: C, 59.43; H,
temperature for 1 h and then 6N NaOH was added 4.75; N, 13.20. Found: C, 59.31; H, 4.74; N, 13.17.
until pH 5 8. The crude precipitate was recovered by
3-(1-(4-aminophenyl)-3-ethyl-2,4-dioxo-1,2,3,4-
suction.
tetrahydropyrimidin-5-yl-amino)benzoic acid ethyl
5-Amino-3-ethyl-1-(4-nitrophenyl)-1H-pyrimi- ester (18c) Mp 5105–1081C; purified by column
dine-2,4-dione (17a) MpZ3001C; crystallization chromatography (cyclohexane/ ethyl acetate 2:1);
solvent5 EtOH; yield 5 75%. 1H-NMR (DMSO), d: yield 520%.1H-NMR (DMSO), d: 1.10 (3H, t,
1.15 (3H, t, J 5 7.1 Hz), 3.90 (2H, q, J 5 7.1 Hz), 4.40 J57.1 Hz), 1.27 (3H, t, J 57.2 Hz), 3.89 (2H, q,
Drug Dev. Res.

NITRAQUAZONE PDE4 INHIBITORS
281
J 57.1Hz), 4.24 (2H, q, J 57.2 Hz), 5.40 (2H, exch br s),
1-(4-Chlorophenyl)-3-ethyl-5-(3-nitrophenyl)-
6.60 (2H, d, J 58.3 Hz), 7.05 (2H, d, J58.3 Hz), 7.35 1H-pyrimidine-2,4-dione (20b) Mp 5 145–1471C;
(5H m), 7.85 (exch br s, 1H, NH), 8.40 (2H, d, purified by column chromatography (toluene/ethyl
1
J 58.9 Hz). Anal. Calcd for C21H22N4O4: C, 63.95; H, acetate 8:2); yield5 21%. H-NMR (CDCl3), d: 1.35
5.62; N, 14.20. Found: C, 63.82; H, 5.61; N, 14.17.
(3H, t, J 5 7.2 Hz), 4.15 (2H, q, J 5 7.2 Hz), 7.40 (2H, d,
J 5 8.4Hz), 7.50–7.65 (4H, m), 8.00–8.20 (2H, m), 8.40
(1H, s). Anal. Calcd for C18H14ClN3O4: C, 58.15; H,
3.80; N, 11.30. Found: C, 58.03; H, 3.79; N, 11.28.
1-(4-Chlorophenyl)-5-(3-chlorophenyl)-3-ethyl-
1H-pyrimidine-2,4-dione (20c) Mp 563–651C; puri-
fied by column chromatography (toluene/ethyl acetate
General Procedure for Compounds (19a–d)
Compounds 18a–d were obtained starting from
10a–c following the general procedure described for
12a,b.
5-Bromo-1-(4-chlorophenyl)-3-ethyl-1H-pyri-
1
8:2); yield 517%. H-NMR (CDCl3), d: 1.35 (3H, t,
midine-2,4-dione (19a) Mp 5 195–1971C; crystalliza-
J57.2 Hz), 4.15 (2H, q, J57.2 Hz), 7.35–7.55 (9H, m).
Anal. Calcd for C18H14Cl2N2O2: C, 59.85; H, 3.91; N,
7.76. Found: C, 59.73; H, 3.90; N, 7.74.
1
tion solvent 5 EtOH; yield 5 90%. H-NMR (CDCl3),
d: 1.30 (3H, t, J 5 7.1 Hz), 4.10 (2H, q, J 5 7.1 Hz),
7.30 (2H, d, J 5 8.8 Hz), 7.50 (2H, d, J 5 8.8 Hz), 7.65
(1H, s).
3-[1-(4-Chlorophenyl)-3-ethyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl]benzoic acid ethyl ester
(20d) Mp 5102–1051C; purified by column chromato-
graphy (cyclohexane/ethyl acetate 3:1); yield 516%.1H-
NMR (CDCl3), d: 1.35 (3H, t, J57.1 Hz), 1.40 (3H, t,
J57.2Hz), 4.15 (2H, q, J57.1Hz), 4.40 (2H, q,
J57.2 Hz), 7.40 (2H, d, J58.4 Hz), 7.50–7.55 (4H, m),
7.65 (1H, d, J58.4 Hz), 7.85 (1H, d, J58.4 Hz), 8.15
(1H, m). Anal. Calcd for C21H19ClN2O4: C, 63.24; H,
4.80; N, 7.02. Found: C, 63.37; H, 4.81; N, 7.03.
5-Bromo-3-ethyl-1-p-tolyl-1H-pyrimidine-2,4-
dione (19b) Mp 5 176–1771C; crystallization solvent5
1
EtOH; yield 5 81%. H-NMR (CDCl3), d: 1.30 (3H, t,
J 5 7.3 Hz), 2.40 (3H, s), 4.15 (2H, q, J 5 7.3 Hz), 7.20
(d, 2H, J 5 8.8 Hz), 7.30 (d, 2H, J 5 8.8 Hz), 7.65 (1H, s).
5-Bromo-3-ethyl-1-phenyl-1H-pyrimidine-2,4-
dione (19c) Mp 5 184–1851C; crystallization solvent5
EtOH; yield 5 100%.1H-NMR (CDCl3), d, ppm: 1.30
(3H, t, J 5 7.2 Hz), 4.15 (2H, q, J 5 7.2 Hz), 7.40 (d, 2H,
J 5 8.8 Hz), 7.45-7.55 (3H, m), 7.70 (1H, s).
5-Bromo-3-cyclopropylmethyl-1-phenyl-1H-
pyrimidine-2,4-dione (19d) Mp 5 150–1521C; crys-
tallization solvent 5 EtOH; yield 5 96%. 1H-NMR
(CDCl3), d: 0.45 (4H, m), 1.30 (1H, m), 3.95 (2H, d,
J 5 6.8 Hz), 7.35 (2H, d, J 5 8.0 Hz), 7.40–7.55 (3H, m),
7.70 (1H, s).
3-[1-(4-Chlorophenyl)-3-ethyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl]benzonitrile (20e) Mp 5
163–1641C; purified by column chromatography (to-
1
luene/ethyl acetate 8:2); yield 520%. H-NMR (CDCl3),
d: 1.35 (3H, t, J57.2 Hz), 4.15 (2H, q, J57.2 Hz), 7.40
(2H,d, J58.5 Hz), 7.45–7.55 (4H, m), 7.65 (1H, d,
J58.5 Hz), 7.80 (1H, d, J58.5 Hz), 8.00 (1H, s). Anal.
Calcd for C19H14ClN3O2: C, 64.87; H, 4.01; N, 11.94.
Found: C, 65.06; H, 4.02; N, 11.98.
General Procedure for Compounds (20a–m)
1-(4-Chlorophenyl)-5-(3,4-dimethoxyphenyl)-
A mixture of the appropriate 5-bromo derivative 3-ethyl-1H-pyrimidine-2,4-dione
(20f)
Mp 5
19 (0.3–0.6 mmol), tetrakis(triphenyl phosphine) 66–681C; purified by column chromatography (to-
palladium(0) (0.017–0.025 mmol), toluene (2–3 mL), luene/ethyl acetate 8:2); yield 5 19%. 1H-NMR
the commercially available phenylboronic acid (CDCl3), d: 1.35 (3H, t, J 5 7.2 Hz), 3.90 (3H, s), 3.95
(0.6–1.2 mmol) dissolved in 1–2 mL of EtOH and 2M (3H, s), 4.15 (2H, q, J 5 7.2 Hz), 6.90 (1H, m), 7.05
Na2CO3 (2–3 mL), was heated at 60–901C for 3–18 h. (1H, d, J 5 7.9 Hz), 7.20 (1H, s), 7.40–7.50 (5H, m).
After cooling, the mixture was concentrated in vacuo, Anal. Calcd for C20H19ClN2O4: C, 62.10; H, 4.95; N,
diluted with water (10 mL), and extracted with CH2Cl2 7.24. Found: C, 62.29; H, 4.96; N, 7.26.
(3 Â 10 mL). Evaporation of the solvent afforded an oil
3-Ethyl-5-(3-nitrophenyl)-1-p-tolyl-1H-pyrimi-
that was purified by column chromatography. Com- dine-2,4-dione (20g) Mp 5 162–1641C; purified by
pound 20j was purified by crystallization from EtOH. column chromatography (toluene/ethyl acetate 8:2);
1-(4-Chlorophenyl)-3-ethyl-5-phenyl-1H-pyr- yield 5 34%. 1H-NMR (CDCl3), d: 1.35 (3H, t,
imidine-2,4-dione (20a) Mp 5 48–511C; purified by J 5 7.2 Hz), 2.45 (3H, s), 4.20 (2H, q, J 5 7.2 Hz),
column chromatography (toluene/ethyl acetate 8:2); 7.25–7.40 (4H, m), 7.60 (2H, m), 8.00 (1H, d,
yield 5 19%. 1H-NMR (CDCl3), d: 1.30 (3H, t, J 5 8.9 Hz), 8.20 (1H, d, J 5 8.9 Hz), 8.40 (1H, s). Anal.
J 5 7.2 Hz), 4.15 (2H, q, J 5 7.2 Hz), 7.35–7.45 (6H, Calcd for C19H17N3O4: C, 64.95; H, 4.88; N, 11.96.
m), 7.50 (2H, d, J 5 8.4 Hz), 7.55 (2H, d, J 5 8.4 Hz). Found: C, 65.14; H, 4.89; N, 12.00.
Anal. Calcd for C18H15ClN2O2: C, 66.16; H, 4.63; N,
5-(3-Chlorophenyl)-3-ethyl-1-p-tolyl-1H-pyri-
8.57. Found: C, 66.25; H, 4.64; N, 8.59.
midine-2,4-dione (20h) Mp 5 40–421C; purified by
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GIOVANNONI ET AL.
column chromatography (cyclohexane/ethyl acetate (2 mL) and cold water was added (25 mL) and the
1
3:1); yield 5 31%. H-NMR (CDCl3), d: 1.35 (3H, t, mixture was extracted with ethyl acetate (3 Â 20).
J 5 7.2 Hz), 2.45 (3H, s), 4.15 (2H, q, J 5 7.2 Hz), Evaporation of the solvent afforded a residue that
7.30–7.60 (9H, m). Anal. Calcd for C19H17ClN2O2: C, was crystallized by EtOH. Mp 5 243–2461C; crystal-
66.96; H, 5.03; N, 8.22. Found: C, 67.16; H, 5.05; N, lization
solvent 5 EtOH;
yield 5 17%.1H-NMR
8.24.
(CDCl3), d: 4.10 (2H, d), 5.30 (1H, exch br t), 6.45
3-(3-Ethyl-2,4-dioxo-1-p-tolyl-1,2,3,4-tetrahy- (1H, s), 7.30–7.40 (6H, m), 7.50 (2H, d, J 5 8.6 Hz).
dropyrimidin-5-yl)benzoic acid ethyl ester (20i)
5-(4-Chlorobenzylamino)-1-(4-chlorophenyl)-
Mp 5 97–1001C; purified by column chromatography 3-ethyl-1H-pyrimidine-2,4-dione (22) Mp 5 186–1871C;
1
(cyclohexane/ethyl acetate 3:1); yield 5 15%.
crystallization solvent 5 EtOH; yield 5 60%. H-NMR
1H-NMR (CDCl3), d: 1.35 (3H, t, J 5 7.2 Hz), (DMSO), d: 1.10 (3H, t, J 5 7.2 Hz), 3.90 (2H, q, J 5
1.40 (3H, t, J 5 7.3 Hz), 2.45 (3H, s), 4.20 (2H, q, 7.2 Hz), 4.10 (2H, d), 5.35 (1H, exch t), 6.50 (1H, s),
J 5 7.2 Hz), 4.40 (2H, q, J 5 7.3 Hz), 7.20–7.70 (6H, 7.35 (6H, m), 7.50 (2H, d, J 5 8.6 Hz).
m), 7.90 (1H, d, J 5 8.2 Hz), 8.05 (1H, d, J 5 8.2 Hz),
Anal. Calcd for C19H17Cl2N3O2: C, 58.47; H,
8.15 (1H, s). Anal. Calcd for C22H22N2O4: C, 69.83; H, 4.39; N, 10.77. Found: C, 58.65; H, 4.40; N, 10.80.
5.86; N, 7.40. Found: C, 69.69; H, 5.85; N, 7.39.
General Procedure for Compounds (24a–c)
3-Ethyl-5-(3-nitrophenyl)-1-phenyl-1H-pyri-
midine-2,4-dione (20j) Mp 5 151–1541C; crystal-
Compounds 24a–c were obtained starting from
lization solvent 5 EtOH; yield 5 26%. 1H-NMR 23a,b following the general procedure described for
(CDCl3), d: 1.30 (3H, t, J 5 7.2 Hz), 4.15 (2H, q, 12a,b. For compounds 24a,c, the reaction was carried
J 5 7.2 Hz), 7.35–7.55 (5H, m), 7.65–7.75 (2H, m), out in anhydrous DMF, the suspension was diluted
8.00 (1H, d, J 5 8.5 Hz), 8.30 (1H, d, J 5 8.5 Hz), 8.55 with cold water and the precipitate 24a was recovered
(1H, s). Anal. Calcd for C18H15N3O4: C, 64.09; H, by suction and recrystallized by ethanol. Compound
4.48; N, 12.46. Found: C, 63.96; H, 4.47; N, 12.44.
24c was obtained by extraction with CH2Cl2
5-(3-Chlorophenyl)-3-ethyl-1-phenyl-1H-pyr- (3 Â 15 mL) and was purified by column chromatography
imidine-2,4-dione (20k) Mp 5 42–441C; purified by using toluene/ethyl acetate 8:2, as eluent. For compound
column chromatography (cyclohexane/ethyl acetate 24b, the reaction was carried out in anhydrous CH3CN
2:1); yield 5 19%. 1H-NMR (CDCl3), d, ppm: 1.35 and suspension was diluted with cold water and extracted
(3H, t, J 5 7.2 Hz), 4.20 (2H, q, J 5 7.2 Hz), 7.30–7.60 with CH2Cl2 (3 Â 15 mL) to afford a residue that was
(10H, m). Anal. Calcd for C18H15ClN2O2: C, 66.16; H, purified by column chromatography using cyclohexane/
4.63; N, 8.57. Found: C, 66.29; H, 4.64; N, 8.59.
ethyl acetate 2:1, as eluent.
3-(3-Ethyl-2,4-dioxo-1-phenyl-1,2,3,4-tetra-
5-(3,4-Dimethoxyphenyl)-1-ethyl-2-oxo-1,2-
hydropyrimidin-5-yl)benzoic acid ethyl ester dihydropyridine-3-carbonitrile (24a) Mp 5 112–1141C;
1
(20l) Mp 5 50–521C; purified by column chromato- crystallization solvent 5 EtOH; yield 5 60%. H-NMR
graphy (cyclohexane/ethyl acetate 3:1); yield 5 17%.
(CDCl3), d: 1.50 (3H, t, J 5 7.2 Hz), 2.50 (3H, s), 3.90
1H-NMR (CDCl3), d: 1.35 (3H, t, J 5 7.2 Hz), (3H, s), 3.95 (3H, s), 4.55 (2H, q, J 5 7.2 Hz), 6.75 (1H,
1.40 (3H, t, J 5 7.0 Hz), 4.20 (2H, q, J 5 7.2 Hz), 4.40 s), 6.85–6.95 (2H, m), 7.70 (1H, s). Anal. Calcd for
(2H, q, J 5 7.0 Hz), 7.40–7.60 (7H, m), 7.85 (1H, d, C17H18N2O3: C, 68.44; H, 6.08; N, 9.39. Found: C,
J 5 8.3 Hz), 8.05 (1H, d, J 5 8.3 Hz), 8.15 (1H, s). Anal. 68.65; H, 6.10; N, 9.42.
Calcd for C21H20N2O4: C, 69.22; H, 5.53; N, 7.69.
1-Cyclopropylmethyl-2-oxo-5-phenyl-1,2-dihy-
Found: C, 69.36; H, 5.54; N, 7.71.
dropyridine-3-carbonitrile (24b) Mp 5117–1181C;
5-(3-Chlorophenyl)-3-cyclopropylmethyl-1- purified by column chromatography (cyclohexane/ethyl
1
phenyl-1H-pyrimidine-2,4-dione (20m) Mp 5 acetate 2:1); yield 5 30%. H-NMR (CDCl3), d: 0.60
47–481C; purified by column chromatography (cy- (4H, m), 1.15 (1H, m), 2.45 (3H, s), 4.15 (2H, d,
1
clohexane/ethyl acetate 3:1); yield 5 23%. H-NMR J 56.9 Hz), 7.20 (2H, m), 7.40 (3H, m), 7.70 (1H, s).
(CDCl3), d: 0.4–0.5 (4H, m), 1.35 (1H, m), 4.00 (2H,
Anal. Calcd for C17H16N2O: C, 77.25; H, 6.10; N,
d, J 5 6.9 Hz), 7.30–7.55 (10H, m). Anal. Calcd for 10.60. Found: C, 77.10; H, 6.09; N, 10.59.
C20H17ClN2O2: C, 68.09; H, 4.86; N, 7.94. Found: C,
67.95; H, 4.85; N, 7.92.
1-Cyclopropylmethyl-5-(3,4-dimethoxyphenyl)-
2-oxo-1,2-dihydropyridine-3-carbonitrile (24c) Mp 5
5-(4-Chlorobenzylamino)-1-(4-chlorophenyl)- 107–1091C; purified by column chromatography (to-
1H-pyrimidine-2,4-dione (21) A mixture of 5-bromo luene/ethyl acetate 8:2); yield 530%.
derivative 10a (0.5 mmol) and 4-chlorobenzylamine
1H-NMR (CDCl3), d: 0.45 (2H, m), 0.65 (2H, m),
(4.0 mmol) was heated in a sealed tube at 901C for 1.40 (1H, m), 2.45 (3H, s), 3.90 (3H, s), 3.95 (3H, s),
9 h. After cooling, the residue was treated with EtOH 4.30 (2H, d, J56.8 Hz), 6.75 (1H, s), 6.85–6.95 (2H, m),
Drug Dev. Res.

NITRAQUAZONE PDE4 INHIBITORS
283
7.70 (1H, s). Anal. Calcd for C19H20N2O3: C, 70.35; H, 10 min. In order to separate and quantify the amounts
6.21; N, 8.64. Found: C, 70.49; H, 6.22; N, 8.66.
of nucleotides and adenine, aliquots of 40 mL of each
sample were deposed on 60 F254 silica gel plates
(eluent ethanol:H2O, 70:30 v/v). Under UV light, three
different lanes were identified, corresponding to cAMP,
AMP, and adenine, which have the same ratio frontis of
the standard molecules comigrated on the same plate.
Then the three different lanes were separated by
scraping, the silica powder collected and placed into
plastic vials; the radioactivity was extracted with 1 mL
water, added with 6 mL of InstagelPlus scintillation
liquid (Perkin-Elmer Life and Analytical Science,
Boston, MA) and counted in a TRICARB 1900TR
Scintillation Counter (Perkin-Elmer Life and Analyti-
cal Science, Boston, MA).
Pharmacology
PDE4 activity assay
Experiments were conducted using undifferen-
tiated U937 cells. As previously demonstrated [Mackenzie
and Houslay, 2000], this human monocytic cell line
express a range of cAMP-specific phosphodiesterase
(PDE) isoenzymes: PDE4A, PDE4B, and PDE4D,
which provide around 76% of the total cAMP PDE
activity of U937 cells. Only approximately 9% of the
cAMP PDE activity in U937 cells was due to PDE3.
Cell culture
U937 cells were grown in plastic dishes in RPMI-
1640 medium supplemented with 10% FCS,
L-glutamine 2 mM and 50 U/mL of penicillin G and
0.05 mg/mL streptomycin at 371C in a humidified
atmosphere of 95% air-5% CO2 and culture medium
changed every two days. Cells were harvested at a
density of approximately 1.0 Â 106 cells per mL; in all
experiments, the medium was removed by centrifuga-
tion at 300 g for 5 min at room temperature and cells
washed in PBS buffer (pH 5 7.4) and re-centrifuged.
The cells were resuspended in 50 mM ice-cold TRIS-
HCl buffer (pH 5 7.4) containing 1 mM PMSF and
sonicated on ice four times for 25 sec. The sonically
disrupted cells were centrifuged at 12,000g for 10 min;
aliquots of the supernatant fraction were assayed for
PDE activity using a modification of the method of
Morelli et al. [2004] as described below. Protein
concentration was determined using the Bradford
method [Bradford, 1976].
Enzymatic activity was measured as percentage of
substrate into product conversion as follows: conver-
sion
(%) 5 [products
count/(substrate1products
counts)] Â 100. At the same time, 40 mL of the sample
were placed into vials with scintillation cocktail and
counted for radioactivity in order to obtain the recovery
of each sample: in these experimental conditions, the
total recovery was about 90%.
[3H] Rolipram Binding
Membrane preparation
Male Sprague-Dawley rats (Harlan, Italy) were
housed in a temperature- and light-controlled room
and were allowed free access to food and water. Their
experimental use was conducted in accordance with
the European Communities Council Directive of 24
November 1986 (86/609/EEC) for experimental animal
care.
Rats were killed by decapitation and the cerebral
cortex was dissected on ice and homogenized in buffer
(50 mM Tris-HCl, 5 mM MgCl2, pH 7.5) using an
Utraturrax homogenizer at 16,000 rpm. Separation of
supernatant and particulate fractions was achieved by
centrifugation at 15,000g for 15 min at 41C and the
pellet was then resuspended in the same buffer.
cAMP assay
Aliquots of supernatant fraction of fresh cell
lysates (50 mL) were incubated in a final volume of
200 mL for 20 min at 301C with 50 nM [3H]-cAMP,
1 mM cold cAMP in TRIS-HCl buffer containing 1 mM
PMSF with or without PDE4 inhibitors. To inhibit the
activity of the small fraction of PDE3, all samples were Binding studies
added with 1 mM cilostamide. The total PDE activity of
[3H]Rolipram binding was measured following a
the cell preparation was measured in the absence of modification of the method of Schneider et al. [1986].
inhibitors and total PDE4 activity was measured For heterologous competition assays, 2 nM [3H]Roli-
adding the selective inhibitor, rolipram (10 mM). All pram was incubated with membrane preparations
compounds were evaluated at a single final concentra- containing 0.2–0.3 mg of protein at 301C in a final
tion of 10 mM. Compounds that exhibited activity in volume of 0.25 mL in the presence of increasing
single-dose testing were further evaluated to generate concentrations of the newly synthesized compounds.
dose response curves, which were used to determine For mixed homologous saturation binding assays,
IC50 values. Reactions were terminated by boiling for [3H]Rolipram was present at 0.2 nM in tubes contain-
1 min, each sample was then supplemented with a ing increasing concentrations of unlabeled Rolipram
mixture of carriers (60 mL of a solution containing (0.03–1000 nM) and at 0.075–0.2 nM in tubes without
3 mM cAMP, AMP, and adenine), and placed on ice for unlabeled ligand. Reactions were stopped after 1 h by
Drug Dev. Res.

284
GIOVANNONI ET AL.
addition of 5 mL of ice-cold buffer and rapid vacuum performed as described above. For PDE5 activity, the
filtration through glass fiber filters (Whatman GF/C, mixture of carriers used was composed by a solution
Florham Park, NJ) presoaked in a 0.3% polyethyleni- containing 3 mM cGMP, GMP, and guanine.
mine (PEI) solution. The filters were washed twice
RESULTS AND DISCUSSION
with 5 mL of ice-cold buffer, 6 mL of scintillation liquid
(FilterCount, Perkin-Elmer Life and Analytical
Experiments to measure the inhibitory activity on
Science, Boston, MA) was added and radioactivity PDE4 isoenzyme of the new synthesized compounds
measured in a scintillation beta counter. Binding data were assessed in fresh cell lysates of human U937
were evaluated quantitatively with the weighted least- monocytic cells. The PDE4 selective inhibitor, roli-
squares iterative curve fitting LIGAND program pram (10 mM), and the PDE3 selective inhibitor,
[Munson and Rodbard, 1980], to obtain the Ki values. cilostamide (1 mM), were used to determine the activity
of these enzyme families in U937 cell lysates. This
PDE3 and PDE5 activity assay
method allowed us to determine that the majority of
Platelets contain three classes of PDEs: PDE2, the cAMP PDE activity in U937 monocytic cells was
PDE3, and PDE5 [Roma et al., 2007]. PDE2 due to the actions of these two PDE families. PDE
hydrolyzes both cAMP and cGMP. The PDE3 isoform activity in U937 cell lysate attributable to PDE4 was
is the most abundant and preferentially hydrolyzes defined experimentally as that inhibited by 10 mM
cAMP, while PDE5 is a specific inactivator of cGMP.
rolipram. PDE4 was found to be the predominant
isoform (ꢁ80% of the total cAMP PDE activity) as
previously reported [Torphy et al., 1992]. Moreover, to
Preparation of disrupted platelet suspension
Human blood was freshly drawn from healthy evaluate the isoenzyme selectivity of the newly
volunteers, who gave informed consent and had not synthesized PDE4 inhibitors, their effects on PDE3
taken medication for at least 2 weeks. Blood was and PDE5 activities were examined. For PDE5,
collected into ACD (2.5% sodium citrate, 1.5% citric sildenafil was used as a reference drug. The source of
acid, and 2% glucose) solution (6:1 [v/v]) and these two PDE isoforms was human platelets, and in
centrifuged at 200g for 20 min to generate platelet- order to characterize the differences between the PDE
rich plasma (PRP). The latter was then centrifuged at isoforms present in platelets, the inhibitory effects of
1,000g for 10 min, resuspended in Tyrode’s buffer various PDE inhibitors were evaluated. The reference
(138 mmol/L NaCl, 2.7 mmol/L KCl, 2 mmol/L MgCl2, PDE2-selective inhibitor, EHNA, was used in all
0.42 mmol/L NaH2PO4, 5 mmol/L glucose, 10 mmol/L experiments in order to exclude the activity of this
HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfo- isoform.
nic acid], and 0.2% bovine serum albumin, pH 7.4)
Biological data on the newly synthesized com-
and re-centrifuged. Platelets were resuspended in pounds are shown in Tables 1 and 2. For most
50mM ice-cold TRIS-HCl buffer (pH 5 7.4) containing compounds, we inserted an ethyl group at position 3
1 mM PMSF and sonicated on ice four times for 30 sec. of the pyrimidindione nucleus according to the
The disrupted platelet suspension was centrifuged at requirement for nitraquazone analogs. Starting the
12,000g for 20min. The soluble fraction contains about analysis from 5-anilino substituted pyrimidindiones
70–80% of the cAMP PDE activity, as described [Grant (compounds 13–18, Table 1) we observed that
and Colman, 1984]. This fraction was assayed both for compounds 13a,b and 18a–c, in which the phenyl
cAMP and cGMP PDE activity.
ring at position 1 was substituted in para with a
chlorine (13a,b), a nitro group (18a,b), or an amino
group (18c), demonstrated appreciable activity as
cAMP and cGMP assay
PDE activity was measured in TRIS-HCl buffer PDE4 inhibitors with IC50 values in the micromolar
containing 1 mM PMSF. Aliquots of fresh platelet range. In particular, compound 13a, bearing a nitro
lysate suspension (50 mL) were incubated in a final group in the meta position of the aniline moiety, had an
volume of 200 mL for 15 min at 301C with substrates IC50 5 6.54 mM, being 2-fold more potent than the
([3H]-cAMP or [3H]-cGMP 50 nM, and cold cAMP or corresponding para chlorine derivative 13b. In deri-
cold cGMP 1 mM), with or without inhibitors. All vatives 18a,b, the anilino compound substituted with
experiments were performed in the presence of 10 mM an ethoxy carbonyl group (18b) was approximately
erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to 3-fold more potent with respect to the corresponding
selectively inhibit PDE2 activity. Total PDE3 activity 3-Cl anilino derivative 18a (IC50 values of 5.72 and
was determined in the presence of 10 mM cilostamide 17.54 mM, respectively). Compound 18c was also active
and total PDE5 activity was measured in the presence in the micromolar range. When the phenyl ring at
of 10 mM sildenafil. cAMP and cGMP assays were position 1 was substituted with a methyl group, no
Drug Dev. Res.

NITRAQUAZONE PDE4 INHIBITORS
285
TABLE 1. Inhibitory Effect of Newly Synthesized Compounds on PDE4, PDE3, PDE5 Activities and Displacement of [3H] Rolipram From Its
Binding Sites (HARBS)
Et
N
R1
N
Et
N
O
O
O
O
N
R
O
N
R
O
N
Cl
HN
HN
H2C
X
X
22
13-18
20a-m
Cl
Compd.
R
R1
X
PDE4a,b
PDE3a,c
PDE5a,c
[3H] Roliprama
13a
13b
13c
13d
18a
18b
18c
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
20k
20l
Cl
Cl
–
–
–
–
–
–
–
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
3-NO2
4-Cl
3-NO2
3-COOEt
3-Cl
3-COOEt
3-COOEt
H
3-NO2
3-Cl
3-COOEt
3-CN
3,4-OMe
3-NO2
3-Cl
3-COOEt
3-NO2
3-Cl
6.5471.38
13.3272.75
14.1770.32 (10)
11.7071.74 (10)
17.5472.82
N.A.
N.A.
–
N.A.
N.A.
–
67.07714.11
12.2571.42
Me
Me
NO2
NO2
NH2
Cl
Cl
Cl
Cl
Cl
–
–
N.A.
5.179.17(10)
N.A.
1.29711.95 (10)
69.53711.20
25.5077.32
24.1671.99
5.7270.80
10.4672.19
N.A.
–
–
–
–
–
–
–
–
N.A.
–
–
–
–
–
–
–
12.1270.88 (10)
0.6279.92 (10)
N.A.
18.5772.50 (10)
12.9875.63 (10)
23.4970.72 (10)
11.2573.09 (10)
14.34710.84 (10)
10.3279.08 (10)
N.A.
–
–
–
–
–
–
–
–
Cl
Me
Me
Me
H
H
H
–
–
–
–
–
–
42.92710.92
39.1474.49
N.A.
N.A.
2.9473.24 (10)
N.A.
25.9176.30
26.7375.91
3-COOEt
20m
22
Rolipram
Cilostamide
Sildenafil
H
–
3-Cl
–
4.8771.37
22.7773.36 (10)
0.3170.06
–
N.A.
–
N.A.
N.A.
–
0.6170.60 (10)
N.A.
12.5571.16
–
–
0.003670.0001
70.3276.61(10)
N.A.
–
–
N.A.
79.3975.94 (10)
aData are expressed as IC50 (mM)7SEM or inhibition percentage at indicated concentration (mM).
bIn the presence of cilostamide 1 mM.
cIn the presence of EHNA 10 mM. N.A. 5 not active.
PDE4 inhibitory activity was observed (compounds 5-phenyl pyrimidindiones (20a–m), it is clear that a
13c,d). All active compounds exhibited good selectivity methyl or a chlorine substitution at the phenyl ring
toward PDE3, and PDE5 isoenzymes and demon- linked at position 1 of the pyrimidindione nucleus led
strated a low affinity for HARBS with IC50 values to inactive compounds independent of the nature of
between 12 and 69 mM (IC50 for Rolipram 5 3.6 nM). substituent X present in the phenyl at position 5
The observed selectivity versus HARBS (HARBS/ (compounds 20a–i). When R 5 H and R1 5 ethyl
PDE4) is appreciable for compound 13a (ꢁ10) and (compounds 20j–l), moderate activity was dependent
decreased
for
other
compounds
(HARBS/ on the substituent X, since 20j (X 5 3-NO2) is inactive,
PDE4 5 0.9–4.5). The introduction of a methylenic while 20k and 20l (3-Cl and 3-COOEt, respectively)
spacer between the amino group at position 5 of the had IC50 values of approximately 40 mM. Replacement
pyrimidindione system and the phenyl ring in com- of the ethyl at position 3 in compound 20k with
pound 13b was detrimental for the activity (compound cyclopropylmethyl (compound 20m) increased PDE4
22) (PDE4 inhibition 5 23% at 10 mM). Analysing the inhibitory activity by an order of magnitude
Drug Dev. Res.

286
GIOVANNONI ET AL.
TABLE 2. Inhibitory Effect of Newly Synthesized Compounds on PDE4, PDE3, PDE5 Activities and Displacement of [3H] Rolipram From Its
Binding Site (HARBS)
R
CH3
N
X
O
NC
24
R
X
PDE4a,b
PDE3a,c
PDE5a,c
[3H] Roliprama
a
Et
3,4-OMe
H
22.0670.46 (10)
15.4770.93
–
–
–
b
N.A.
N.A.
37.2275.10
c
3,4-OMe
15.5074.83
0.3170.06
–
N.A.
N.A.
70.3276.61 (10)
N.A.
0.6170.60 (10)
N.A.
4500
0.003670.0001
Rolipram
Cilostamide
Sildenafil
–
–
N.A.
N.A.
79.3975.94 (10)
aData are expressed as IC50 (mM)7SEM or inhibition percentage at indicated concentration (mM).
bIn the presence of cilostamide 1 mM.
cIn the presence of EHNA; 10 mM. N.A. 5 not active.
(IC50 54.87 mM). Analogous to the 5-anilino series
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