Spiroquinazolinones as novel, potent, and selective PDE7 inhibitors. Part 2: Optimization of 5,8-disubstituted derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.07.010

The optimization of 5,8-disubstituted spirocyclohexane-quinazolinones into potent, selective, soluble PDE7 inhibitors with acceptable in vivo pharmacokinetic parameters is presented.

Full text of DOI:10.1016/j.bmcl.2004.07.010

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4627–4631
Spiroquinazolinones as novel, potent, and selective PDE7
inhibitors. Part 2: Optimization of 5,8-disubstituted derivatives
a
a
a
Patrick Bernardelli,a, Edwige Lorthiois, Fabrice Vergne, Chrystelle Oliveira,
*
Abdel-Kader Mafroud,a Emmanuelle Proust,a Nga Pham,a Pierre Ducrot,a
Franc¸ois Moreau,a Moulay Idrissi,a Anita Tertre,a Bernadette Bertin,a Magali Coupe,a
Eric Chevalier,a Arnaud Descours,a Franc¸oise Berlioz-Seux,a Patrick Bernaa and Mei Lib
aPfizer Global Research and Development, 3-9 rue de la Loge 94265Fresnes, France
bPfizer Global Research and Development, Eastern Point Road, Groton, CT 063409, USA
Received 23 January 2004; revised 17 June 2004; accepted 2 July 2004
Available online 27 July 2004
Abstract—The optimization of 5,8-disubstituted spirocyclohexane-quinazolinones into potent, selective, soluble PDE7 inhibitors
with acceptable in vivo pharmacokinetic parameters is presented.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Phosphodiesterases (PDE) are enzymes that degrade the
key secondary intracellular messengers adenosine and
guanosine 30,50-cyclic monophosphates (cAMP and
cGMP) into their corresponding 50-monophosphate nu-
cleotides.1 PDE7 is a high-affinity cAMP-specific phos-
phodiesterase for which two subtypes have been
identified PDE7A and PDE7B. Considering the func-
tional role of PDE7 reported in T-cells2 as well as the
Figure 1. Preliminary result with a 5,8-disubstituted spiroquinazoli-
none.
distribution of its mRNA in various tissues, the use of
PDE7 inhibitors may be relevant for the treatment of
T-cell related diseases,2 autoimmune diseases,2 airway
fied, the 5,8-dichloro-spirocyclohexane-quinazolinone 2
diseases,3 leukemia,4 CNS disorders,5 and fertility disor-
was mentioned as a very potent PDE7 inhibitor
ders.6 Although there have been several publications on
PDE7 inhibitors,7 there has been no report on a potent,
selective, and soluble compound with a suitable in vivo
pharmacokinetic profile that could be used as a tool to
validate this target in animal models.
(IC50 = 14nM).8 However, this compound is very
hydrophobic (clogP = 4.7).9 In this communication, we
present our preliminary results on the optimization of
the substituent at position-5 in this key lead structure to-
ward potent, selective, soluble PDE7 inhibitors with
acceptable pharmacokinetic properties.
In the preceding publication,8 we disclosed our prelimi-
nary SAR studies around the high throughput screening
hit 1 (Fig. 1). Among the spiroquinazolinones exempli-
2. Chemistry
The 5,8-disubstituted spirocyclohexane-quinazolinone
derivatives 2, 6, and 7 were synthesized by condensation
of the readily available ureas 3, 4, and 5 with cyclohexa-
none in polyphosphoric acid (PPA) at 80–100°C
(Scheme 1).10 It is worth mentioning that the
Keywords: PDE7; Phosphodiesterase; Pharmacokinetic; ADME.
*
ˆ
Corresponding author at present address: Aventis Pharma, Batiment
Grignard, 13 quai Jules Guesde, 94400 Vitry-sur-Seine, France. Tel.:
+33-1-58-932833; fax: +33-1-58-933450; e-mail: patrick.bernardelli@
aventis.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.010

4628
P. Bernardelli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4627–4631
Scheme 1. Reagents and conditions: (a) KNCO, AcOH, H2O; (b) cyclohexanone, PPA, 80–100°C; (c) BBr3, CH2Cl2, 0°C to rt; (d) R2Cl or R2Br,
K2CO3 or NaH, DMF, 100°C.
PPA-mediated cyclization was much more efficient with
the electron-rich phenyl ureas 5 and 4 (78% and 60%
yield, respectively), than with 3 (7% yield). Phenol 8
was prepared by demethylation of 7 with boron tribro-
mide. Alkylation of phenol 8 with various alkyl halides
directly provided the 5-alkoxy-8-chloro-spirocyclohex-
ane-qinazolinone derivatives 11, 18, 20, 23, and 24.
shown in Scheme 2, carboxylic acid 9 was synthesized
by alkylation of 8 with ethyl bromoacetate followed by
ester hydrolysis. Similarly, furoic acid 19 resulted from
the ethyl ester hydrolysis of 18. Tetrazole 10 and hyd-
roxy-oxadiazole 13 were obtained from nitrile 11 follow-
ing usual protocols.11 Alcohol 14 was derived from
phenol 8 by alkylation with 2-(2-bromoethoxy)tetra-
hydro-2H-pyran followed by deprotection of the tetra-
hydropyran group under acidic conditions. The
resulting alcohol was also used to prepare amines 16,12
21, and 22 by transformation into the corresponding
The more functionalized 5-alkoxy-8-chloro-qinazoli-
none derivatives presented in this publication were pre-
pared in several steps from phenol intermediate 8. As
Scheme 2. Reagents and conditions: (a) ethyl bromoacetate, K2CO3, DMF, 100°C; (b) KOH, THF, H2O; (c) bromoacetonitrile, K2CO3, DM F,
100°C; (d) Me3SnN3, toluene, reflux; (e) NH2OHÆHCl, NaOH, EtOH, reflux; (f) ClCO2Et, CHCl3, Et3N; (g) DBU, CH3CN, reflux; (h) 2-(2-
bromoethoxy)tetrahydro-2H-pyran, K2CO3, DMF, 100°C; (i) HCl, THF, H2O, reflux; (j) methanesulfonyl chloride, Et3N, CH2Cl2, 0°C to rt;
(k) morpholine, EtOH, 70°C; (l) ethyl glycinate, CH3CN, reflux; (m) HCl (6N), 1,4-dioxane, 90°C.

P. Bernardelli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4627–4631
4629
mesylate 15 followed by treatment with morpholine,
ammonia, and methylamine, respectively. Similarly,
compound 17 was obtained by addition of ethyl glyci-
nate to mesylate 15 and hydrolysis of the resulting
ethyl ester. Compound 25 was prepared by alkylation
of phenol
8
with 2-[2-(2-chloroethoxy)ethyl]-1H-
isoindole-1,3(2H)-dione and subsequent removal of the
phthalamido group by treatment with hydrazine.
Finally, aminoalcohol derivatives 26 and 27 were syn-
thesized by alkylation of phenol 8 with epibromhydrin
followed by condensation with methylamine and
dimethylamine, respectively.
Figure 2. Structures, DHf calculations (in kcal/mol), and correspond-
ing differences in DHf (DDHf in kcal/mol) for the chair conformers of 1,
2, 6, 7, and 8.
3. Biological results and discussion
The initial results in the replacement of the 5-chloro sub-
stituent in the key lead structure 2 were quite promising
(Table 1). Since no selectivity for PDE7A versus PDE7B
enzyme subtypes was observed, only PDE7A results are
presented herein.
explain the higher inhibitory activity of 2, 7, and 8
versus 1.
The 5-methoxy and 5-hydroxy substituted analogs 7 and
8, respectively, were equipotent to 2 in inhibiting the
PDE7 enzyme. However, the 5-methyl derivative 6 ap-
peared to be less potent than 2, 7, and 8 and showed
similar level of activity than 1.
There may not be a simple rationale for the observed
activities and, perhaps, these compounds have different
binding mode in the enzyme catalytic site. Further stud-
ies, like cocrystallization experiments, would help to
rationalize these data.
Considering the diverse nature of these substituents, nei-
ther an electronic effect on the aromatic ring (electron-
donating OMe vs electron-withdrawing Cl)13 or a
lipophilic interaction (OMe vs OH) could account for
these results.
Although compounds 7 and 8 are very potent PDE7
inhibitors, their aqueous solubility (<4lg/mL in pH7.4
buffer solution for both) and metabolic stability in
human microsomes (t1/2 = 28.4 and 68.4min for 7 and
8, respectively), can be improved.
The effect of the substituent at position 5 on the confor-
mation of the spirocyclohexyl ring was evaluated. The
enthalpies of formation (DHf) of the two cyclohexyl
chair conformers a and b for the non-substituted deriv-
ative 1 and the 5-substituted analogs were calculated with
MOPAC14 in Sybyl15 using the semi-empiric method
AM1 without minimization16 (Fig. 2). Regardless of
the nature of the substituent at position 5, the very high
difference in enthalpy of formation between the two
cyclohexyl conformations a and b (D(DHf) P 14.8kcal/
mol) indicates that all the spiroquinazolinones predom-
inantly adopt the conformation b in which the less hin-
dered urea group is axially oriented. The rigid structure
b probably represents the active conformation for all
these PDE7 inhibitors in the enzyme binding site. There-
fore, there is no specific influence of the substituent
at position 5 on the cyclohexyl conformation that could
In order to improve drug-like characteristics, we ex-
plored substitution of the 5-OH group with various side
chains containing polar substituents thereby reducing
the overall hydrophobicity of the spirocyclohexane-
quinazolinone core.
Neutral, acidic, and basic side chains with various
lengths between the spiroquinazolinone core and the po-
lar or ionizable group as well as different lipophilicity
and pKa for the ionizable group were selected (Table
2). These three classes of compounds with diverse
physicochemical properties were designed in an attempt
to obtain tools with varied tissue distribution profiles
that could be used in the appropriate animal disease
model depending on the targeted location of the PDE7
enzyme. Beside their effect on solubility, the influence
of the ionizable side chains on potency and selectivity
was investigated.
Among the neutral derivatives 11, 14, and 18, alcohol 14
shows the best balance between potency, selectivity, and
solubility. Although compound 14 (IC50 = 55nM ) is a
less potent PDE7 inhibitor than 7 (IC50 = 14nM), it
was selected for further in vivo evaluation due to its im-
proved solubility (18lg/mL in pH7.4 buffer solution for
14 vs <4lg/mL for 7) and metabolic stability in human
microsomes (t1/2 = 220.7min for 14 vs 28.4min for 7)
probably resulting from its lower hydrophobicity (clog
P = 3.04 and 3.91 for 14 and 7, respectively).
Table 1. PDE7A1 inhibitory activity for compounds 1, 2, and 6–8
Compd
R1
PDE7A1 IC50 (lM)a
1
2
6
7
8
H
Cl
0.17
0.014
0.1
Me
OMe
OH
0.014
0.013
a Measured against the human full length enzyme produced in bacu-
lovirus infected sf9 cells. Values are means of three experiments.

4630
P. Bernardelli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4627–4631
Table 2. In vitro potency, selectivity, and solubility for neutral compounds 11, 14, 18; acids 9, 10, 13, 19, 20; bases 16, 21–27, and amino acid 17
Compd
R2
PDE7A1
IC50
(lM)a
PDE1
IC50
(lM)b
PDE3A3
IC50
(lM)a
PDE4D3
IC50
(lM)a
PDE5
IC50
(lM)c
Solubility
pH7.4
(lg/mL)d
Solubility
pH1
(lg/mL)e
11
14
18
9
–CH2CN
–CH2CH2OH
0.028
0.055
0.011
0.046
0.004
0.0035
0.016
0.015
0.038
1.04
5.86
10.5
2.98
94
>73
1.7
12.2
1.02
15
>101
>91
80
4
18
4
11
–CH2–(5-CO2Et–Furan-2-yl)
–CH2CO2H
78
>68
4.76
0.32f
0.611
1.6
>101
>88
>101
>101
60
>500
345
113
100
>500
4
1
<1
10
13
19
20
16
21
22
23
24
25
26
27
17
–CH2–(1H-Tetrazol-5-yl)
–CH2–(5-OH-[1,2,4]Oxadiazol-3-yl)
–CH2–(5-CO2H–Furan-2-yl)
–CH2CH2CH2SO3H
–CH2CH2–(4-Morpholino)
–CH2CH2NH2
11.8
21.9
10.8
12.7
>101
>101
>101
>101
>101
>101
>101
>101
>101
0.328
0.823
1.13
2.41
7.35
10.5
53.2
3.28
23.7
99
<1
<1
0.65
>500
>500
>500
>500
>500
29
33
>101
73.2
>101
74
190
>500
195
–CH2CH2NHMe
–CH2CH2NMe2
0.967
0.157
1.24
72.4
19
–CH2CH2CH2NMe2
–CH2CH2OCH2CH2NH2
–CH2CH–(OH)–CH2NHMe
–CH2CH–(OH)–CH2NMe2
–CH2CH2NHCH2CO2H
>101
98
1.62
>101
>101
>101
>101
>101
>101
>101
>500
>500
137
>500
>500
>500
>500
0.213
0.4
0.042
12
55
29.7
14.7
230
a Measured against the human full length enzyme produced in baculovirus infected sf9 cells. Values are means of three experiments.
b Measured against the human full length enzyme partially purified from THP-1 cell pellets. Values are means of three experiments.
c Measured against the human full length enzyme partially purified from MCF-7 cell pellets. Values are means of three experiments.
d Determined by stirring in pH7.4 Na2HPO4/NaH2PO4 buffer for 24h.
e Determined by stirring in pH1 HCl/KCl buffer for 24h.
f Measured against the human full length PDE1A enzyme produced in baculovirus infected sf9 cells.
As one could expect, the acidic derivatives 9, 10, 13, 19,
and 20 were more soluble than the neutral derivatives at
pH7.4 but not at pH1, except for sulfonic acid 20. Inter-
estingly, the bioisosteric substitution of the carboxylic
group in 9 (IC50 = 46nM) for a presumably less acidic
tetrazole (10) or hydroxy-oxadiazole (13) led to more
potent PDE7 inhibitors (IC50 = 4 and 3.5nM, respec-
tively). However, the latter were less selective versus
PDE1 and PDE4 than 9. In addition, since carboxylic
acid 9 was found to be more selective versus other PDEs
than 19 and sulfonic acid 20, it was chosen as a repre-
sentative of the class of acids for further in vivo
evaluation.
selective than the acids and to have good solubility in
both pH buffers.
The rat in vivo pharmacokinetic profiles were deter-
mined for the selected compounds 9, 14, 16, and 17
(Table 3).
The acid 9 and the neutral derivative 14 displayed more
favorable pharmacokinetic parameters (t1/2 = 1.4h,
F = 21%, and t1/2 = 1.5h, F = 27%, respectively), than
16 and 17. The longer half-life for 9 and 14 compared
to 16 resulted mainly from a lower clearance. Besides,
oral bioavailabilities for 9 and 14 were superior to those
obtained for 16 and 17. Regarding 17, since its clearance
is acceptable, this probably reflects insufficient intestinal
absorption, which could be explained by its zwitterionic
nature. It is worth noting that, although 9 is acidic and
The basic spiroquinazolinones 16 and 21–27 were found
to be generally less potent than the neutral or acidic
derivatives but more selective versus other PDEs.
Despite its lower solubility in pH7.4 buffer solution,
the least basic morpholine 16 was found to be soluble
at pH1 and potent enough (IC50 = 38nM) to be selected
for further evaluation.
Table 3. Rat pharmacokinetic profile for compounds 9, 14, 16, and 17
Compd
Cl (mL/min/kg)a
Vd (L/kg)a
t1/2 (h)b
F (%)b
In order to combine the advantages of acidic derivatives
in terms of potency and solubility at pH7.4 with the
advantages of basic compounds in terms of selectivity
and solubility at pH1, the amino acid analog 17 was
tested. As expected, it was demonstrated to be more po-
tent (IC50 = 42nM) than most basic compounds, more
9
39.4
33.6
62.8
39.9
1.04
1.41
1.02
0.47
1.4
1.5
0.32
2.7
21
27
14
16
17
3.7
6.5
a Dose 0.5mg/kg iv.
b Dose 2.5mg/kg po.

P. Bernardelli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4627–4631
4631
322–334; (b) Miro, X.; Perez-Torres, S.; Palacios, J. M.;
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14 neutral, both compounds turn out to have similar
volumes of distribution.
4. Conclusion
6. (a) Kotera, J. PDE7-news and views, William Harvey
Research Conferences, Porto, Portugal, December 5–7,
2001; (b) Kluxen, F. W. PCT int. Appl. WO 0183772 A1,
2001.
A novel series of 5-substituted 8-chloro-spirocyclohex-
ane-quinazolinones have been developed as potent,
selective, and soluble PDE7 inhibitors. The neutral,
basic, and acidic compounds 14, 16, and 9, respectively,
are useful inhibitors to reveal the functional role of the
enzyme PDE7 in vitro. More importantly, derivatives
9 and 14 are the first reported selective PDE7 inhibitors
that display in vivo pharmacokinetic parameters suita-
ble to validate this target in rat models.
7. (a) Barnes, M. J.; Cooper, N.; Davenport, R. J.; Dyke, H.
J.; Galleway, F. P.; Galvin, F. C. A.; Gowers, L.;
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Mafroud, A.-K.; Proust, E.; Heuze, L.; Moreau, F.;
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Acknowledgements
The authors are grateful to the chemists at Evotec OAI
(Oxford, UK), in particular to Ajith Manage, for their
assistance in preparing some of these products and to
the members of the PGRD Fresnes Analytical Support
team for their help in compound characterization. We
also thank Alexis Denis for fruitful discussions in the
preparation of the manuscript.
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