New orally active PDE4 inhibitors with therapeutic potential

Article abstract of DOI:10.1016/j.bmcl.2003.10.025

Structural optimization of pyrazolopyridine derivative 2, which is one of the newly discovered chemical leads for PDE4 inhibitors from our in-house library, was carried out successfully. The process of discovery of new orally active PDE4 inhibitors, which

Full text of DOI:10.1016/j.bmcl.2003.10.025

Bioorganic & Medicinal Chemistry Letters 14 (2004) 29–32
New orally active PDE4 inhibitors with therapeutic potential
Hiroshi Ochiai,a Akiharu Ishida,a Tazumi Ohtani,a Kensuke Kusumi,a
Katuya Kishikawa,b Takaaki Obata,a Hisao Nakaia,* and Masaaki Todaa
aMinase Research Institute, Ono Pharmaceutical Co., Ltd, 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan
bDevelopment Planning, Ono Pharmaceutical Co., Ltd, 2-1-5 Doshomachi, Chuo-ku, Osaka 541-8526, Japan
Received 11 September 2003; revised 10 October 2003; accepted 14 October 2003
Abstract—Structural optimization of pyrazolopyridine derivative 2, which is one of the newly discovered chemical leads for PDE4
inhibitors from our in-house library, was carried out successfully. The process of discovery of new orally active PDE4 inhibitors,
which are expected to possess therapeutic potential, is presented and their structure–activity relationships are discussed.
# 2003 Elsevier Ltd. All rights reserved.
Phosphodiesterase 4 (PDE4) is an enzyme that is
responsible for the inactivation of cyclic adenosine
monophosphate (cAMP).1,2 Inhibition of this enzyme
results in elevation of cAMP expression in inflammatory
cells such as eosinophils. The anti-inflammatory effects
of PDE4 inhibitors have been well documented both in
vitro and in vivo in a variety of animal models,3 but no
inhibitor has yet been used clinically because of dose-
limiting side effects such as nausea and vomiting that
restrict their therapeutic potential.4
apeutic potential, discovery and biological evaluation of
a new chemical lead with a completely different struc-
ture was considered to provide us with another
approach to the chemical modification of rolipram.
The pyrazolopyridines have been reported to be active
compounds for PDEs, adenosine and benzodiazepine
receptors.11À13 We identified compound 2 as a lead
compound by high throughput screening (HTS) of our
in-house library. As a result of structural optimization
of 2, an orally active PDE4 inhibitor, compound 12
(Fig. 2) was discoverd. The results of our SAR study are
also reported.
Two strategies have already been reported for the pro-
duction of efficient PDE4 inhibitors with less side
effects. ArifloTM 15 (Fig. 1) and the other second-gen-
eration PDE4 inhibitors were reported to be more
LPDE4-selective6 than (R)-rolipram and/or to be PDE4
subtype-selective.7,8
Synthesis of the test compounds listed in Tables is out-
lined in Scheme 1.12 Michael addition of 16 to an
acceptor 21 followed by the removal of an ethoxy moi-
ety provided 17, after which ring closure was accom-
plished with phosphorus oxychloride under reflux to
afford chloride 18. Alkaline hydrolysis of 18 gave car-
boxylic acid 19. Heating 19 with thionyl chloride followed
by treatment with aqueous ammonia resulted in the amide
20. Replacement of the chloro moiety of 20 with appro-
priate anilines gave us the test compounds 2–15.
In previous papers, we have described the design and
9
. .
synthesis of bicyclo[3 3 0]octane derivatives and piper-
idine derivatives10 as interesting new classes of PDE4
inhibitors with therapeutic potential. As we reported
previously, investigation of the structure–activity rela-
tionship (SAR) profile of PDE4 inhibitors based on the
structural features of Ariflo has revealed that modifi-
cation of Ariflo can give a series of PDE4 inhibitors
with an improved therapeutic index relative to the clas-
sical inhibitor, rolipram. To achieve the molecular
design of efficient new inhibitors with improved ther-
Keywords: PDE4; Inhibitor; Pyrazolopyrioline; Orally active.
* Corresponding author. Tel.:+81-75-961-1151; fax:+81-75-962-9314;
e-mail: hi.nakai@ono.co.jp
Figure 1. Structure of Ariflo 1.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.025

30
H. Ochiai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 29–32
oxy groups resulted in 6, 7, and 8, respectively, which
all showed a marked decline of LPDE4 inhibitory
activity. Removal of the methyl portion of the meth-
oxy moiety of 4 afforded meta-hydroxy derivative 9,
which was nearly 19-fold less potent in its LPDE4
inhibitory activity, while 9 showed 40% inhibition of
TNF-a production in rats at an oral dose of 10 mg/
kg. Replacement of the methoxy group of 4 with a
nitrile group gave 10, which showed a nearyl 17-fold
decline of LPDE4 inhibitory activity and also failed to
inhibit TNF-a production in rats at an oral dose of 3
mg/kg.
Figure 2. Discovery of a new chemical lead 2 and identification of an
orally bioavailable PDE4 inhibitor 12.
Compounds 2 and 4 demonstrated very poor bioavail-
ability (BA) despite relatively good oral activity (the BA
of 2 and 4 was 8.2 and 1.7%, respectively). Design and
A series of pyrazolopyridine derivatives were synthe-
sized and evaluated for the ability to inhibit PDE4 pre-
pared from U937 cells14 (derived from human
monocytes). The results of the assays were expressed as
IC50 values, i.e., the test compound concentration that
achieved 50% inhibition relative to the vehicle. Test
compounds were also evaluated for the ability to inhibit
lipopolysaccharide (LPS)-induced production of tumor
necrosis factor-a (TNF-a) in rats.15 The results were
expressed as ID50 values, that is, the dose that resulted
in 50% inhibition relative to the vehicle.
Table 1. Activity profile of pyrazolopyridine derivatives 2–10
During the course of screening of PDE4 inhibitors,
compound 2 was found to demonstrate moderate
potency in both the in vitro and in vivo assays. As shown
in Table 1, design and synthesis of mono-substituted
aniline derivatives 3–10 was carried out. Introduction of
a ortho-methoxy group into the aniline moiety of 2
afforded 3, which was slightly less potent in vitro assay.
Introduction of a meta-methoxy group into the phenyl
moiety of 2 gave 4, which showed 10-fold greater potency
in vitro assay. Interestingly, para-methoxy derivative 5
did not show LPDE4 inhibitory activity up to a concen-
tration of 0.1 mM. Thus, meta-methoxy derivative 4
exhibited the most potent inhibitory activity in both the
in vitro and in vivo assays among the three isomers 3–5.
Further chemical modification of the meta-methoxy
moiety of 4 was subsequently carried out. Replacement
of the meta-methoxy group of 4 with meta-cyclo-
pentyloxy, meta-cyclohexyloxy, and meta-cyclobutyl-
Compd
X
Inhibition of LPDE4a Inhibition of TNF-ab
IC50 (mM)
ID50 (mg/kg, po)
2
3
4
5
6
7
8
9
10
H
2-OMe
3-OMe
4-OMe
3-cPentyloxy
3-cHexloxy
3-cButyloxy
3-OH
0.036Æ0.015
0.076Æ0.037
0.0050Æ0.0007
>0.1
(48%)c
(68%)c
2.4
NTe
>0.1
>0.1
(39%)c
NTe
>0.1
0.097Æ0.083
NTe
(40%)c
(2%)d
3-CN
0.085Æ0.005
a Inhibition of PDE4 prepared from U937 cells (a cell line derived
from human monocytes). IC50 represent a mean of n=2.
b ID50 for inhibition of LPS-induced TNF-a production in rats (n=7)
0.5 h after oral dosing of a test compound.
c Inhibition% at 10 mg/kg, po.
d Inhibition% at 3 mg/kg, po.
e Not tested.
Scheme 1. (a) 21, 120 ꢀC; (b) POCl3, 120 ꢀC; (c) KOHaq, dioxane; (d) SOCl2, 80 ꢀC; (e) NH3aq, THF; (f) Aniline derivatives, dioxane, reflux.

H. Ochiai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 29–32
31
synthesis of PDE4 inhibitors with a better oral BA was
attempted, and compound 12 was discovered to show
both good potency and a good BA (33%). Thus, further
chemical modification of the pyrolidine moiety of 12
was carried out as illustrated in Table 2. Removal of the
methoxycarbonyl moiety of 12 afforded 11, which
showed complete loss of LPDE4 inhibitory activity at
0.3 mM. Therefore, the N-methoxycarbonyl moiety of
12 seems to be essential for its potent inhibitory activity.
Surprisingly, the R-enantiomer of 12 exhibited less than
50% inhibition (ꢁ20%) at 0.1 mM. Replacement of the
methoxycarbonyl moiety of 12 with a tert-butoxy-
carbonyl moiety gave 13, which also showed loss
of activity at 0.3 mM. The N-acetyl pyrolidinyloxy
derivative 14 exhibited 41% inhibition in LPDE4 assay
at a concentration of 0.3 mM. LPDE4 inhibitory
activity was restored in the case of N-sulfonylmethyl
derivative 15, which had an IC50 value of 0.25 mM. As
a result, compounds with good potency and good BA
such as 12 were obtained by very limited chemical
modification.
LPS-induced TNF-a production assay, was carried out
as shown in Table 3. These compounds were evaluated
for their ability to inhibit slow reacting substance of
anaphylaxis (SRS-A) mediated bronchoconstric-
tion.16,17 The results were expressed as ID50 values, that
is, the dose that resulted in 50% inhibition relative to
the vehicle. These compounds were also evaluated for
the ability to inhibit TNF-a production in human whole
blood (HWB)18 to estimate their clinical potential. The
results of the assays were expressed as IC50 values, that
is, the test compound concentration that resulted in
50% inhibition relative to the vehicle. The potency of
these compounds for inhibiting in SRS-A-mediated
bronchoconstriction in actively sensitized guinea pigs
was not always consistent with their potency in inhibit-
ing LPS-induced TNF-a production in rats, probably
because of differences in pharmacokinetics due to cross-
species comparison. Compounds 4 and 12 demonstrated
50% inhibition of SRS-A-induced bronchoconstriction
at ID50 values of 6.8 mg/kg, po and 45% inhibition at
10 mg/kg, po, respectively, while their ID50 values for
TNF-a production were 2.4 and 1.0 mg/kg, respectively.
With respect to inhibition of gastric emptying in rats,19
the ID50 values of 4 and 12 were higher than that of
Ariflo 1, while their IC50 values for LPS-induced TNF-a
production in HWB were lower than that of Ariflo 1.
Based on the above-mentioned biological data, 4 and
12 were estimated to be likely to have improved
therapeutic potential and an improved side effect
profile. Both of the compounds were also evaluated
in the in vivo ferret emesis model. According to our
evaluation, they did not cause emesis up to the oral dose
of 10 mg/kg.
Further biological evaluation of compounds 4 and 12,
which were selected based on their oral activity against
Table 2. Activity profile of pyrazolopyridine derivatives 11–15
Design and synthesis of efficient new PDE4 inhibitors
with improved therapeutic potential was accomplished
by the structural optimization of chemical lead 2, which
was found in our in-house library. Among the com-
pounds tested, 4 and 12 could have improved thera-
peutic potential with an improved side effect profile
based on biological data obtained using both cross-spe-
cies comparison and same-species comparison. Full
details will be reported in due course.
Compd
X
Inhibition of LPDE4a Inhibition of TNF-aa
IC50 (mM)
ID50 (mg/kg, po)
11
12
13
14
15
H
>0.3
0.010Æ0.005
>0.3
>0.3
0.25Æ0.01
NTa
1.0
NTa
NTa
NTa
COOMe
COOtBu
COMe
SO2Me
a See corresponding footnotes from Table 1.
Table 3. Activity profile of pyrazolopyridine derivatives 4, 12 and Ariflo 1
Compd
Inhibition of
bronchoconstrictiona
Inhibition of
TNF-a productionb
Inhibition of
gastric emptyingc
Inhibition of TNF-a
Ferret emesise
(vomiting/tested)
production in HWBd
ID50 (mg/kg, po)
ID50 (mg/kg, po)
ID50 (mg/kg, po)
IC50 (mM)
3
10
(mg/kg, po)
30
1(Ariflo)
4
12
4.5
6.8
45%f
1.7
2.4
1.0
5.7
23%f
15
18
3.0
4.8
NTg
0/2
NTg
0/2
NTg
1/2
0/2
0/3
2/2
a Inhibition of SRS-A-mediated bronchoconstriction and airway microvascular leakage in actively sensitized guinea pigs (n=3–6); OVA challenge
0.15 mg/kg 1 h after oral dosing of a test compound.
b See corresponding footnotes from Table 1.
c Inhibition of gastric emptying in rats (n=5).
d Inhibition of LPS-induced TNF-a production in human whole blood. IC50 represent a mean of n=3.
e Vomiting test in fasted ferrets.
f Inhibition% at 10 mg/kg, po.
g Not tested.

32
H. Ochiai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 29–32
T.; Nakai, H.; Toda, M. Eur. J. Med. Chem., submitted
for publication.
References and notes
10. Ochiai, H.; Ohtani, T.; Ishida, A.; Kusumi, K.; Kato, M.;
Kohno, H.; Kishikawa, K.; Obata, T.; Nakai, H.; Toda,
M. Bioorg. Med. Chem. Lett., in press.
1. Houslay, M. D. Prog. Nucleic Acid Res. Mol. Biol. 2001,
69, 249.
2. Essayan, D. M. J. Allergy Clin. Immunol. 2001, 108, 671.
3. Teixeira, M. M.; Gristwood, R. W.; Cooper, N.; Helle-
well, P. G. Trends Pharmacol. Sci. 1997, 18, 164.
4. Burnouf, C.; Pruniaux, M. P. Curr. Pharm. Des. 2002, 8,
1255.
5. Christensen, S. B.; Guider, A.; Forster, C. F.; Gleason,
J. G.; Bender, P. E.; Karponski, J. M.; Dewolf, W. E.;
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.; Bar-
tus, J. O.; Ryan, M. D.; Eggleston, D. S.; Haltiwanger,
R. C.; Torphy, T. J. J. Med. Chem. 1998, 41, 821.
6. Duplantier, A. J.; Biggers, M. S.; Chambers, R. J.; Cheng,
J. B.; Cooper, K.; Damon, D. B.; Eggler, J. F.; Kraus,
K. G.; Marfat, A.; Masamune, H.; Pillar, J. S.; Shirley,
J. T.; Umland, J. P.; Watson, J. W. J. Med. Chem. 1996,
39, 120.
7. Kleinman, E. F.; Campbell, E.; Giordano, L. A.; Cohan,
V. L.; Jenkinson, T. H.; Cheng, J. B.; Shirley, J. T.; Pet-
tipher, E. R.; Salter, E. D.; Hibbs, T. A.; Dicapua, F. M.;
Bordner, J. J. Med. Chem. 1998, 41, 266.
8. Hersperger, R.; Dawson, J.; Mueller, T. Bioorg. Med.
Chem. Lett. 2002, 12, 233.
9. Ochiai, H.; Ohtani, T.; Ishida, A.; Kishikawa, K.; Obata,
11. Shi, D.; Padgett, W. L.; Hutchinson, K. D.; Moore, S. P.;
Daly, J. W. Drug Dev. Res. 1997, 42, 41.
12. Bare, T. M.; McLaren, C. D.; Campbell, J. B.; Firor,
J. W.; Resch, J. F.; Walters, C. P.; Salama, A. I.; Meiners,
B. A.; Patel, J. B. J. Med. Chem. 1989, 32, 2561.
13. Hohn, H.; Polacek, I.; Schulze, E. J. Med. Chem. 1973,
16, 1340.
14. Torphy, T. J.; Zhou, H.; Cieslinski, L. B. J. Pharmacol.
Exp. Ther. 1992, 263, 1195.
15. Tracey, K. J.; Cerami, A. Annu. Rev. Med. 1994, 45, 491.
16. Nakagawa, N.; Obata, T.; Kobayashi, T.; Okada, Y.;
Nambu, F.; Terawaki, T.; Aishita, H. Jpn. J. Pharmacol.
1992, 60, 217.
17. Nakagawa, N.; Obata, T.; Kobayashi, T.; Okada, Y.;
Nambu, F.; Terawaki, T.; Furuya, T.; Muryobayashi, K.;
Sawada, M.; Aishita, H. Eur. J. Pharmacol. 1993, 235, 211.
18. Brideau, C.; Staden, C. V.; Sthyler, A.; Rodger, I. W.;
Chan, C. C. Br. J. Pharmacol. 1999, 126, 979.
19. Andres, J. I.; Alonso, J. M.; Diaz, A.; Fernandez, J.;
Iturrino, L.; Martinez, P.; Matesanz, E.; Freyne, E. J.;
Deroose, F.; Boeckx, G.; Petit, D.; Diels, G.; Megens, A.;
Somers, M.; Wauwe, J. V.; Stoppie, P.; Cools, M.; Clerck,
F. D.; Peeters, D.; Chaffoy, D. D. Bioorg. Med. Chem.
Lett. 2002, 12, 653.