Design, synthesis, and evaluation of 2-aryl-7-(3′,4′-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidines as novel PDE-4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.12.070

Described herein is design, synthesis, and biological evaluation of novel series of 2-aryl-7-(3′,4′-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidines acting as inhibitors of type 4 phosphodiesterase (PDE4) which is known as a good target for the treatment of asthma and COPD. For this purpose, structure optimization was conducted with the aid of structure-based drug design using the known X-ray crystallography. Also, biological effects of these compounds on the target enzyme were evaluated by using in vitro assays, leading to the potent and selective PDE-4 inhibitor (IC₅₀ < 10 nM).

Full text of DOI:10.1016/j.bmcl.2009.12.070

Bioorganic & Medicinal Chemistry Letters 20 (2010) 922–926
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of 2-aryl-7-(30,40-dialkoxyphenyl)-pyrazolo
[1,5-a]pyrimidines as novel PDE-4 inhibitors
Ikyon Kim a, Jong Hwan Song a, Chang Min Park a, Joon Won Jeong a, Hyung Rae Kim a, Jin Ryul Ha a,
a,
Zaesung No b, Young-Lan Hyun c, Young Sik Cho d, Nam Sook Kang e, , Dong Ju Jeon
*
*
a Medicinal Chemistry Research Center, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
b Institut Pasteur Korea, Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea
c Crystalgenomics, Inc. Seoul 138-736, Republic of Korea
d Pharmacology Research Center, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
e Drug Discovery Platform Technology Team, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Described herein is design, synthesis, and biological evaluation of novel series of 2-aryl-7-(30,40-dialkoxy-
phenyl)-pyrazolo[1,5-a]pyrimidines acting as inhibitors of type 4 phosphodiesterase (PDE4) which is
known as a good target for the treatment of asthma and COPD. For this purpose, structure optimization
was conducted with the aid of structure-based drug design using the known X-ray crystallography. Also,
biological effects of these compounds on the target enzyme were evaluated by using in vitro assays, lead-
ing to the potent and selective PDE-4 inhibitor (IC50 < 10 nM).
Article history:
Received 6 October 2009
Revised 16 December 2009
Accepted 17 December 2009
Available online 23 December 2009
Keywords:
PDE-4 inhibitor
SBDD
Ó 2009 Elsevier Ltd. All rights reserved.
COPD
Asthma
Phosphodiesterases (PDEs), a super family of 11 isozymes, are
responsible for the hydrolysis of cAMP and c-GMP.1 Cyclic nucleo-
tides are important intracellular secondary messengers in cell func-
tion, relaying the signals from hormones at specific cell-surface
receptors. An increase of cAMP due to the stimulation of adenylyl cy-
clase or the inhibition of PDEs affects the activity of immune system
and inflammatory cells.2 Thus, PDE4, a cAMP specific PDE, received
much attention as a target for the treatment of the diseases such
as asthma and Chronic Obstructive Pulmonary Disease (COPD).3
Since the first report of rolipram as a selective inhibitor of
PDE4,4 a number of compounds have been studied to increase
the activity and to reduce the side effects such as nausea, vomiting,
psychotropic activity, and increased gastric secretion (Fig. 1).5
In this communication, we wish to report the potent and selec-
tive PDE4D inhibitors. First of all, using the crystal structures of the
ligand-bound PDE4D (PDB entry; 1XOQ),6,7 we hoped to design
molecules to bind the S1 and S3 sites while not to directly bind
the metal binding S2 site as shown in Figure 2. Initially, while con-
F
F
F
F3C CF3
OH
O
NH
O
O
NC
F
MeO
MeO
CO2H
F
O Cl
HN
F
O
O
O
N
O
N
O
Cl
Roflumilast
Me
Rolipram
Cilomilast
L-826,141
Figure 1. Structures of catechol-type PDE4 inhibitors.
* Corresponding authors. Tel.: +82 42 860 7452; fax: +82 42 860 7456 (N.S.K).
E-mail address: nskang@krict.re.kr (N. Sook Kang).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.070

I. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 922–926
923
As shown in Scheme 1, these pyrazolo[1,5-a]pyrimidines8 were
envisaged to derive from condensation of b-ketoaldehydes 3 or
b-enaminoketones 4 with aminopyrazoles 2. Two types of products
(L-type and linear type) were anticipated depending on the mode
of cyclization.
The key intermediate, aminopyrazoles 2 were prepared in two
steps (Scheme 2). Thus, reaction of ethyl benzoate with the anion
of acetonitrile gave cyanoacetophenones 6, which were converted
to aminopyrazoles 2 upon exposure to hydrazine hydrate in
60–80% yields.9 The requisite arylacetoaldehydes were synthesized
from the reaction of enolates derived from acetophenones with
ethyl formate in moderate yields,10 whereas b-enaminoketones 4
were easily prepared by treatment of acetophenones with DMF-
DMA (Scheme 2).11
Indeed, 5-aminopyrazoles 2 were coupled with arylacetoalde-
hydes 3 in acetic acid at room temperature to give pyrazolopyrim-
idines,
1 and 5. Alternatively, 1 and 5 were obtained by
condensation of 2 with 4. Interestingly, whereas dehydrative cycli-
zation of aminopyrazoles 2 with arylacetoaldehydes 3 gave a reg-
ioisomeric mixture of pyrazolo[1,5-a]pyrimidines, 1 and 5 in an
approximately 1:1 ratio, 112 was obtained as a major isomer by
employing b-enaminoketone 4 as a coupling partner.13 Regioisom-
er 5 was observed in a small portion in the latter case, and could be
separated by silica gel column chromatography. As shown in
Figure 4, structure assignment of 1n was unambiguously con-
firmed by X-ray crystallographic analysis.14 This structure takes
an L-shape and three aromatic moieties are nearly on the same
plane.
Figure 2. The crystallographic structure of roflumilast (PDB entry; 1XOQ, yellow)
and docking structure of our compound (gray).
OR1
OR2
These derivatives were assayed against purified human PDE4D
and their inhibitory effects are shown in Table 1. For 1a–1j where
R1 is methyl and R2 is cyclopentyl, it was found that the com-
pounds bearing meta-substituted phenyl group exhibited more po-
tent inhibitory activity than the compounds having ortho- or para-
substituted phenyl group. To validate their activity, the level of
R3
N
N
N
1
Figure 3. New scaffold proposed by SBDD.
cAMP was measured using U937 cell at 20 lM. Disappointingly,
however, the levels of cAMP were lower compared with their
PDE4D inhibition activity.
serving the S1 site of roflumilast, we tried to introduce new linker
fragment positioning at side chains of L319 and M273 to bridge S1
and S3 sites from our in-house fragment library, consisting of het-
ero-cyclic compounds. Through the virtual fragment exploration
using docking method for potent PDE4 inhibitors, 2,7-diphenylpy-
razolo[1,5-a]pyrimidine scaffold 1 as the bridging moiety between
S1 and S3 sites was selected (Fig. 3). For docking by LigandFit8
interfaced with Accelrys Discovery Studio2.0, NH2 group of side
chain in Q369 residue was defined as the hydrogen donor interac-
tion site and the default parameters used. The predicted docking
mode for roflumilast was well generated showing RMSD value,
0.33 Å, compared the crystallographic structure. Through the
docking study for roflumilast and scaffold 1 (R1 = CHF2, R2 = cyclo-
propylmethyl), Dock Score were 63.648 and 64.845 kcal/mol,
respectively.
We further evaluated PDE-4 enzyme inhibition of a series of 2-
phenyl-7-(30-cyclopropylmethoxy-40-difluoromethoxyphenyl)pyr-
azolo[1,5-a]pyrimidines carrying meta-substituted phenyl groups
(compounds 1k–1p in Table 1).15 At first, we checked the level of
cAMP using U937 cell at 10 lM for compounds 1k and 1m, which
indicated that the derivatives of 2-phenyl-7-(30-cyclopropylmeth-
oxy-40-difluoromethoxyphenyl)pyrazolo[1,5-]pyrimidines increase
the level of cAMP. With these results in hand, a wide variety of
derivatives were synthesized and tested for their in vitro activi-
ties.16 Although relatively bulky groups containing polar moiety
are required for higher activity, introduction of diverse functional
groups for R has kept the inhibitory activity against PDE4D. These
results can be rationalized by our hypothesis that R group occupy-
ing S3 binding site is exposed to outward of PDE4D binding pocket
O
O
H
OR1
R3
N
R1O
a
OR2
N
R3
3
N
N
or
OR2
N
+
O
R1O
R3
NH2
N
N
N
OR2
5 (linear type)
Scheme 1. Reagents and conditions: (a) AcOH, rt, 12 h, 60–90%.
H
N
2
R1O
4
1 (L-type)
OR2

924
I. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 922–926
R3
R3
R3
a
b
CN
CO2Et
N
NH2
R1O
O
N
H
6
2
O
O
O
O
d
c
H
N
R1O
R1O
OR2
OR2
OR2
3
4
Scheme 2. Reagents and conditions: (a) CH3CN, NaH, THF, rt, 40–80%; (b) NH2NH2, EtOH, reflux, 60–80%; (c) ethyl formate, NaH, THF, rt, 12 h, 60–90%; (d) DMF-
dimethylacetal, toluene, reflux, 60–90%.
which directly contacts with solvent molecules as anticipated
through modeling study.
Finally, we introduced pyridine moiety instead of benzene ring
(compounds 1o and 1p in Table 1), which resulted in maintaining
Table 2
PDE isozyme selectivity profile of 1l, 1o, and 1p
Compd
IC50(lM)
PDE-4
PDE-3
PDE-5
PDE-7
1l
1o
1p
0.006
0.009
0.026
2.0
>5
>5
>5
3.5
>5
>5
3.0
>5
Figure 4. Crystal structure of 1n.
Table 1
OMe
O
OCHF2
O
OCHF2
O
OCHF2
O
N
N
N
R
N
N
N
N
R
N
N
N
O
N
N
N
N
1a-1j
1k-1n
1o
1p
Compd
R
IC50 (nM)
cAMP
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
H
2-Br
2-OMe
4-Cl
4-Br
4-OMe
3-Cl
3-Br
3-OMe
2,5-Cl2
3-Br
3-I
3-OMe
3-COOMe
Pyridine
N-oxide
70
140
70
90
130
60
20
30
40
30
13
6
NAa
NA
NA
NA
NA
NA
19.7b
11.4b
20.1b
10.4b
57.1c
19.8c
44.4c
NA
30
37
27
42
66.9
1.61 l
Md
a
b
c
NA: not available.
% cAMP level relative to control at 20
% cAMP level relative to control at 10
l
l
M in U937 cells.
M in U937 cells.
d
EC50 of 1p for cAMP accumulation in U937 cells.

I. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 922–926
925
Figure 5. The Inhibitory effects of compound 1p on OVA-induced airway hyperresponsiveness and eosinophil peroxidase activity. AHR was measured in mice 48 h after OVA
inhalation (A). Results are mean SD, n = 7–10 per group. *p <0.05 and **p <0.01 versus. vehicle control (0.5% CMC). Also, EPO activity was measured in BALF 62 h after OVA
inhalation. Results are mean SD, n = 9 to 10 per group. *p <0.05 and **p <0.001 versus. vehicle control.
3. (a) Giembycz, M. A.; Dent, G. Clin. Exp. Allergy 1992, 22, 337; (b) Torphy, T. J.;
the in vitro activity for PDE4D. The treatment of cells with com-
pound 1p led to the considerable accumulation of cAMP with
EC50 of 1.61 lM. Also, our scaffold exhibited isozyme selectivity
as shown in Table 2.
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further evaluated in ovalbumin-induced asthma animal model.
Penh values to 25 and 50 mg/mg in vehicle group were about
two fold higher than those in controls, but as a reference, adminis-
tration of 30 mg/kg (mpk) roflumilast suppressed completely the
responsiveness to Mch. In mice treated with compound 1p
(100 mpk), a significant decrease in airway responsiveness to
50 mg/ml Mch was observed (Fig. 5A). Moreover, to examine the
preventive effect of 1p on pulmonary inflammation, eosinophil
peroxidase (EPO) activity in bronchoalveolar lavage fluid (BALF)
was evaluated as an indicator of infiltrated eosinophils. As shown
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compared with control while either 1p or roflumilast treatment
diminished the EPO activity as low as about 50% of vehicle group.
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tective effect on OVA-induced AHR and its beneficial effect results
partly from the suppression of eosinophil infiltration.
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In conclusion, we have identified a novel series of 2-aryl-7-
(30,40-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidines which show
good PDE4 inhibitory activities. Further evaluation of selectivity
to other PDEs and anti-inflammatory activity in the animal model
are now underway and will be reported in due course.
Acknowledgements
This research was supported by the Center for Biological Mod-
ulators of the 21st Century Frontier R&D program, the Ministry
of Science and Technology, Korea.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.12.070.
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12. For NMR data of 1, see the Supplementary data.
13. In addition, these b-ketoaldehydes are usually difficult to handle compared
with b-enaminoketones.
14. CCDC 741294 contains the supplementary crystallographic data for compound
1n. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via ww.ccdc.cam.ac.uk/data_request/cif.
15. Compound 1p was prepared by mCPBA oxidation of compound 1o.
16. For the full in vitro data, see the Supplementary data.