Phosphodiesterase inhibitors. Part 4: Design, synthesis and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic heteroaromatic-4,4-dimethylpyrazolones

Article abstract of DOI:10.1016/j.bmcl.2012.07.088

(-)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4, 5-dihydro-3-(2H)-pyridazinone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and anti-inflammatory activity. Here we show that a 4,4-dimethylpyrazolone subunit serves as an effective surrogate for the 5-methyl-4,5-dihydropyridazin-3(2H)-one ring of KCA-1490 whilst lacking a stereogenic centre. The 2- and 7-substituents in the pyrazolo[1,5-a]pyridine subunit markedly influence the PDE-inhibitory profile and can be adjusted to afford either potent PDE4-selective inhibitors or dual PDE3/4 inhibitors. A survey of bicyclic heteroaromatic replacements for the pyrazolo[1,5-a]pyridine allowed further refinement of the inhibitory profile and identified 3-(8-methoxy-2-(trifluoromethyl)imidazo[1,2-a]pyridin-5-yl)-4, 4-dimethyl-1H-pyrazol-5(4H)-one as an orally active, achiral KCA-1490 analog with well-balanced dual PDE3/4-inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2012.07.088

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5833–5838
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phosphodiesterase inhibitors. Part 4: Design, synthesis
and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic
heteroaromatic-4,4-dimethylpyrazolones
Koji Ochiai a, Satoshi Takita a, Akihiko Kojima a, Tomohiko Eiraku a, Naoki Ando a, Kazuhiko Iwase a,
Tetsuya Kishi a, Akira Ohinata a, Yuichi Yageta a, Tokutaro Yasue a, David R. Adams b, Yasushi Kohno a,
⇑
a Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., 2399-1, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan
b Chemistry Department, School of Engineering and Physical Sciences, Heriot-Watt University, Ricarton, Edinburgh EH14 4AS, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
(–)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazi-
none (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and
anti-inflammatory activity. Here we show that a 4,4-dimethylpyrazolone subunit serves as an effective
surrogate for the 5-methyl-4,5-dihydropyridazin-3(2H)-one ring of KCA-1490 whilst lacking a stereogen-
ic centre. The 2- and 7-substituents in the pyrazolo[1,5-a]pyridine subunit markedly influence the PDE-
inhibitory profile and can be adjusted to afford either potent PDE4-selective inhibitors or dual PDE3/4
inhibitors. A survey of bicyclic heteroaromatic replacements for the pyrazolo[1,5-a]pyridine allowed
further refinement of the inhibitory profile and identified 3-(8-methoxy-2-(trifluoromethyl)imidazo
[1,2-a]pyridin-5-yl)-4,4-dimethyl-1H-pyrazol-5(4H)-one as an orally active, achiral KCA-1490 analog
with well-balanced dual PDE3/4-inhibitory activity.
Received 7 June 2012
Revised 11 July 2012
Accepted 24 July 2012
Available online 31 July 2012
Keywords:
Dual PDE3/4 inhibitor
Pyrazolo[1,5-a]pyridine
Dimethylpyrazolone
Asthma
Ó 2012 Elsevier Ltd. All rights reserved.
COPD
Cyclic 30,50-nucleotide phosphodiesterase (PDE) enzymes medi-
ate the hydrolytic degradation of key second messengers, cyclic
adenosine monophosphate (cAMP) and cyclic guanosine
monophosphate (cGMP), to their respective parent nucleoside 50-
monophosphates (AMP and GMP). There are 11 PDE gene families
and, of these, the PDE4 family in particular plays a pivotal role in
regulating a network of inflammatory mediators by controlling
intracellular levels and gradients of cAMP.1 Interest in PDE4 as a
therapeutic target stems, in part, from the pronounced anti-inflam-
matory activity exhibited by PDE4 inhibitors, with efforts to
harness this activity recently culminating in the launch of roflumi-
last for treatment of severe chronic obstructive pulmonary disease
(COPD).2 The efficacy of PDE4 inhibitors for treatment of respira-
tory disease may be augmented by a capacity to induce relaxation
of airway smooth muscle, although inhibition of PDE3 is more
effective in this regard.3 For this reason we have been exploring
the potential of dual PDE3/4 inhibitors as the basis for a new
approach to treating asthma and COPD, and have previously iden-
tified fused bicyclic heteroaromatic–dihydropyridazinones, typi-
fied by KCA-1490 (Fig. 1), as dual PDE3/4 inhibitors that exhibit
promising activity in pharmacological models of inflammation
and bronchoconstriction.4,5 Although the individual PDE3- and
PDE4-inhibitory components of KCA-1490 are modest, we attri-
bute the potent combined anti-inflammatory and bronchodilatory
activity of this compound and its analogs to their balanced dual
PDE3/4-inhibitory profile, where the combination of PDE3 and
PDE4 inhibition is thought to function additively or synergistically
to induce airway smooth muscle relaxation.6
In developing KCA-1490, we combined the pyrazolo[1,5-a]pyri-
dine core of our LTD4-antagonist and nonselective PDE inhibitor,
ibudilast (Fig. 1),7 with a 5-methyl-4,5-dihydropyridazin-3(2H)-
one subunit. The latter was originally introduced because the
dihydropyridazinone ring is a common structural component of
established PDE3 inhibitors such as imazodan8 and CI-930.9 Our
preliminary work revealed that replacement of the pyridazinone
ring of KCA-1490 with closely related subunits, for example 5,5-di-
methyl-4,5-dihydropyridazin-3(2H)-one and 5-methylpyridazin-
3(2H)-one, resulted in a loss of both PDE3 and PDE4 inhibitory
activity. The 5-methyl-4,5-dihydropyridazin-3(2H)-one was thus
found to be the most effective pyridazinone-based substituent
for the 4-position of the pyrazolo[1,5-a]pyridine core, albeit having
the drawback of introducing chirality into the structure.4
In order to eliminate the stereogenic center of KCA-1490, we
needed to identify alternative, achiral scaffolds that could replace
the 5-methyl-4,5-dihydropyridazin-3(2H)-one subunit whilst
maintaining the dual PDE3/4-inhibitory profile of the parent
⇑
Corresponding author.
E-mail address: yasushi.kohno@mb.kyorin-pharm.co.jp (Y. Kohno).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.088

5834
K. Ochiai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5833–5838
O
because pyrazolones are well established as ring contracted mim-
ics of dihydropyridazinone PDE3 inhibitors.10 Moreover, in recent
years Merck have actively developed a series of 3-aryl-4,4-di-
methyl-1H-pyrazol-5(4H)-one derivatives (Fig. 1) as potent and
selective PDE3B inhibitors.11 Here, we describe a novel series of
4,4-dimethylpyrazolone dual PDE3/4 inhibitors derived from
KCA-1490. A survey of potential bicyclic heteroaromatic replace-
ment subunits for the pyrazolo[1,5-a]pyridine core of this series
has also been undertaken and the activity of the resulting com-
pounds is disclosed.
R
NH
N
O
N
imazodan R = H
CI-930 R = Me
N
NH
N
Me, H
O
4
7
3
2
R2
N
N
N
N
Our general synthetic route to the pyrazolopyridine–pyrazolone
R1
target compounds is summarized in Scheme 1 (see Supplementary
ibudilast
12
data).
In brief, treatment of pyridine derivatives 1 with O-mes-
pyridazinone ring
optimisation
itylenesulfonylhydroxylamine (MSH) in CH2Cl2 gave N-aminopyrid-
inium salts 2. The pyrazolo[1,5-a]pyridine ring was constructed by
the reaction between 2 and appropriate alkynes under basic condi-
tions to afford 3. Synthesis of ketones 4 from 3 was performed by
three methods. For routes commencing with 3-methoxypyridine
starting materials (1, R1 = OMe), the sequence used for conversion
of 3 into 4 comprised demethylation of the 4-methoxy group, decar-
boxylation or deacylation at the 3-position, triflate derivatisation of
the resulting 4-hydroxypyrazolo[1,5-a]pyridine and finally Heck
reaction with n-butyl vinyl ether (Method A). For pyrazolopyridine
intermediates (3; R1 = CH2OH; R4 = OMe, OEt, OBn), synthesis of 4
was accomplished in a 5-step sequence consisting of ester hydroly-
sis, decarboxylation, MnO2 oxidation, and Grignard reaction fol-
lowed by Parikh–Doering oxidation or Swern oxidation. In cases
where a ketone moiety was preinstalled in pyridines 1, intermedi-
ates 4 were obtained by decarboxylation or deacylation to remove
the pyrazolopyridine 3-substituent in 3 (Method C). Construction
of the 4,4-dimethylpyrazolone ring was achieved by first converting
ketones 4 into pyrazolone precursors 5 through a 2-step sequence
beginning with introduction of a methoxycarbonyl group and
O
achiral replacement for top ring
O
NH
N
NH
∗
N
O
NH
R2
CF3
N
N
N
O
N
N
R1
OMe
R =
KCA-1490 (–)-form
Ar
PDE3A IC50 (μM): 0.36
PDE4B IC50 (μM): 0.042
NH
R
Merck PDE3B inhibitors
Figure 1. Discovery and development pathway for pyrazolopyridine-based dual
PDE3/4 inhibitor, KCA-1490. Activity data quoted here for KCA-1490 are for
inhibition of the isolated core catalytic domains of PDE3A and PDE4B.
compound. To this end we first chose to explore a 4,4-dimethylpy-
razolone replacement for the pyridazinone ring of KCA-1490
Table 1
SAR survey for optimization of the pyrazolopyridine 7-substituent
O
O
R1
R2
R1
R2
R1
R4
R3
O
HN
N
HN
N
a
b
N+
N
N
-
N
SO3
NH2
R2
3
2
1
N
N
N
N
R1 = OMe,CH2OH, COMe, COEt
R3 = specified in Table 2
R4 = OMe, OEt, OBn, Me, Et
R
OMe
R2 = specified in Table 1
6a-6h
7
c
O
O
OMe
N
HN
N
O
O
R5
Cmpd
R
Inhibition IC50
(
l
M)a
e
d
PDE3A
PDE4B
R3
R3
R3
7b
11.3
2.37
2.00
9.21
9.01
75.55
1.59
1.63
2.50
0.47
0.16
0.017
0.41
0.079
0.015
0.035
0.0034
0.0075
N
N
N
N
N
6a
6b
6c
6d
6e
6f
Me
Et
OMe
R2
R2
R2
4
5
6
R5 = Me, Et
SMe
NHMe
CH2OH
CH(OH)Me
Ac
Scheme 1. Reagents and conditions: (a) MSH / DCM, rt; (b) R3–C„C–COR4, K2CO3
/
EtOH, rt; (c) (Method A: R1 = OMe): (i) 48% HBr aq reflux or BBr3 / DCM, 0 °C then
50% H2SO4 aq, 150 °C, (ii) Tf2O, Et3N / DCM, 0 °C, (iii) n-butyl vinyl ether, Pd(OAc)2,
DPPP, Et3N / DMF, 80 °C. (Method B: R1 = hydroxymethyl): (i) KOH aq. / EtOH,
reflux, (ii) PhCH3, reflux, (iii) MnO2 / CHCl3, 50 °C, (iv) EtMgBr / THF, 0 °C to rt, (v)
SO3-pyridine, Et3N / DMSO, rt or TFAA, DMSO / DCM, ꢀ78 °C then Et3N, ꢀ78 °C to rt.
(Method C: R1 = acetyl, propionyl) 48% HBr aq, reflux or 50% H2SO4 aq, 150 °C or (i)
KOH aq / EtOH, reflux, (ii) PhCH3, reflux; (d) (i) NaH, dimethyl carbonate, reflux, (ii)
NaH, MeI / DMF, rt; (e) hydrazine, AcOH / EtOH, reflux or t-butyl carbazate, PPTS /
xylene, reflux.
6gc
6h
a
Enzyme assays were performed using the core catalytic domains from the
PDE3A and PDE4B isoforms according to previously procedures.7b Data reported are
the mean of at least three experiments.
b
6-(2-Ethyl-7-methoxypyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-
(2H)-pyridazinone (racemic form).4
c
6g was tested in racemic form.

K. Ochiai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5833–5838
5835
followed by methylation (for 4, R5 = Et) or dimethylation (for 4,
R5 = Me) at the
-position next to the carbonyl group. Closure of
compound 6f, is conserved in the pyrazolone series, because a
favorable supplementary hydrogen bond from the pyrazolone car-
bonyl group to a distal histidine residue may also be formed
(Fig. 2A). However, pyridazinone ring contraction will have an
important impact on the position occupied by the connected
pyrazolopyridine subunit. In particular, fitting the pyrazolone ser-
ies compounds to this model positions the pyrazolopyridine core
more deeply within the catalytic pocket, close to the proximal his-
tidine (a residue adjacent to the metal centers that is conserved
across all PDEs and that functions as a proton donor during sub-
strate hydrolysis). Our analysis of this binding mode suggests that
unfavorable steric compression will arise in the case of 7-substitu-
ents that adopt a conformation coplanar with the pyrazolopyri-
dine. Indeed loosening of the hydrogen bonds that anchor the
pyrazolone ring in the distal region of the pocket may be necessary
to accommodate such groups on the inhibitor. We therefore pro-
pose that the weak PDE3-inhibitory activity for 7-NHMe derivative
(6e) in the pyrazolone series may be due to a particularly strong
bias for such a conformation with the methylamino group. In con-
trast, heteroatom-linked groups of similar size (6c, 7-OMe; 6d,
SMe) that have a less pronounced preference for coplanarity with
the pyrazolopyridine are expected to be more readily accommo-
dated, as are carbon based substituents linked through an sp3 cen-
tre [6b, 7-Et; 6f, 7-CH2OH; 6f, 7-CH(Me)OH] or an sp2 centre (6h,
7-Ac), but where a degree of twist on the connecting bond to the
pyrazolopyridine is likely.
SAR analysis in our foundational pyridazinone compound ser-
ies,4,5,15 has suggested a PDE4-binding mode that differs pro-
foundly from that invoked for PDE3. As illustrated in Figure 2B
for compound 6f, it is proposed that the bound orientation of the
inhibitor is reversed so that the pyrazolopyridine engages the pur-
ine-scanning glutamine. In the case of 7-methoxy pyrazolopyri-
dines, such as KCA-1490 and its ethyl analogue (7), both the
pyrazolopyridine N(1) center and the methoxy group are expected
to hydrogen bond to the purine scanning glutamine in a manner
that is very well established from PDE4 X-ray co-crystal structures
with catechol ethers such as roflumilast. 14b,18 Such a binding mode
would position the 7-OMe group within the confined PDE4 Q1 sub-
pocket, as defined by Card et al.,18 with the pyrazolopyridine
2-substituent directed into the Q2 subpocket (labelled in Fig. 2B)
at the opening of catalytic pocket and adjacent to the purine-scan-
ning glutamine. The 7-OMe derivative (6c) in the pyrazolone series
was essentially identical in it its PDE4-inhibitory activity to the
a
the pyrazolone ring was then accomplished by the reaction of 5 with
hydrazine to afford the target compounds (6). In the case of the 7-
methoxy-2-trifluoromethyl derivative, t-butyl carbazate was em-
ployed instead of hydrazine because the high nucleophilicity of
hydrazine resulted in an undesired side reaction at the pyrazolo-
pyridine 2-position.13 For a number of the analogs introduction of
the pyrazolopyridine 7-substituent was accomplished after initial
formation of the core bicycle. This was typically achieved by acetal
protection of the ketone in 4 followed by lithiation, iodination,
deprotection of the acetal and iodide substitution with an appropri-
ate nucleophile (e.g., methoxide, methanethiolate, methylamine). A
similar lithiation strategy followed by formylation (using ethyl for-
mate) was used to develop some of the carbon-based substituents at
the pyrazolopyridine 7-position. (Full details are presented in the
Supplementary data).
Our foundational studies leading to KCA-1490 had revealed key
roles for the pyrazolopyridine 2- and 7-substituents in shaping the
PDE3/4-inhibitory profile of the compounds.4 We therefore under-
took a focused structure activity relationship (SAR) survey for sub-
stituents at these positions in our pyrazolone series. To explore the
impact of substituents at the 7-position of the pyrazolo[1,5-a]pyr-
idine (Table 1) we selected ethyl as the invariant group at C-2 in
order to make a direct comparison with the activity of the corre-
sponding pyridazinone analogs in our previously published work
leading to KCA-1490.4 For this purpose, compounds were assayed
for inhibitory activity against the core catalytic domains from
PDE3A and PDE4B using previously reported protocols.7b 14
,
All compounds other than the 7-NHMe derivative (6e) exhibited
comparable levels of PDE3 inhibition to the corresponding pyridaz-
inone derivatives in the KCA-1490 series.4 Surprisingly, 6e showed
very weak PDE3 inhibitory activity. In contrast the activity of the
7-OMe derivative (6c) was essentially the same as the correspond-
ing pyridazinone (7, included in Table 1 for reference). SAR analysis
of our own pyridazinone series,4,5,15 corroborated by the protein
cocrystal structure for a PDE3-selective 5-methyl-4,5-dihydropy-
ridazinone inhibitor from Merck,16 have implicated a binding
mode in which the pyridazinone ring docks to a region of the sub-
strate-binding pocket distal to the catalytic metal centers, engag-
ing the conserved purine-scanning glutamine (a residue that
hydrogen bonds the cAMP adenine during catalytic turnover). It
is likely that this mode of interaction, illustrated in Figure 2 A for
Figure 2. Interaction models for compound 6f (green stick) with PDE3 (panel A) and PDE4 (panel B). The PDE3-binding model is templated on the co-crystal structure (PDB:
1SO2) of (R)-6-(4-{[2-(3-iodobenzyl)-3-oxocyclohex-1-en-1-yl]amino}phenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one with the core catalytic domain from PDE3B.16
Residues lining the catalytic pocket of PDE3B are fully conserved in the core catalytic domain of PDE3A (used in the enzyme inhibition assays of the present study). In this
binding pose the pyrazolone ring hydrogen bonds (dotted lines) the PDE3 purine-scanning Gln and His residues distal to the catalytic metal centers (large spheres). The PDE4
binding model is templated on the co-crystal structure of zardaverine with the core catalytic domain of PDE4D (PDB: 1XOR, 1MKD).17 Residues lining the catalytic pocket are
fully conserved between PDE4D and PDE4B (the latter used in the assays of the present study). The profound difference in orientation of bound inhibitor between the two
models is a consequence of differences between PDE3 and PDE4 in residues lining the distal region of the catalytic pocket. The Q1 subpocket in PDE4 is more restricted and
lacks the hydrogen bonding feature afforded by the distal His residue in PDE3.

5836
K. Ochiai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5833–5838
corresponding analog (7) in the pyridazinone series, and it is prob-
able that both compounds adopt this catechol ether-like binding
mode.
subpocket. Given the confines of that site, a difference in activity
between the two enantiomers of 6g is clearly to be expected. We
have not, however, assessed the two enantiomers separately to
determine the extent to which the activity resides with one or
the other.
The data summarised in Table 1 highlight the importance of the
pyrazolopyridine 7-substituent in controlling the levels of PDE4
inhibition exhibited by members of the compound series. Although
our primary focus is the development of dual PDE3/4 inhibitors,
the data demonstrate that appropriate selection of that substituent
can deliver both very potent PDE4 inhibitors (6g and 6h,
IC50 = 3.4 nM and 7.5 nM respectively) and switch the inhibitory
profile from dual PDE3/4 inhibition to selective PDE4 inhibition
(6e, IC50 = 15 nM with 5000-fold selectivity over PDE3).
Our SAR survey of pyrazolopyridine 2-substituents (Table 2)
was undertaken with methoxy as the invariant group in the 7-po-
sition. We opted for the methoxy group rather than substituents
that confer more potent PDE4 inhibition because we had attributed
the potent combined bronchodilatory and anti-inflammatory
activity of KCA-1490 to its balanced dual PDE3/4-inhibitory profile.
The activity balance of both the 7-OMe and 7-Me derivatives (6c
and 6a respectively, Table 1) was similar to KCA-1490.4 However,
in the pyridazinone series the 7-Me analog failed to show activity
in vivo, thus dictating our selection of the 7-OMe group for the pyr-
azolone series SAR survey presented here.4 Guided by our work
with the pyridazinones, we focused on a limited population of 2-
substitutents that we had previously found to show promise. All
of these compounds (6i–6l) had similar levels of inhibitory activity
against PDE3 (within a threefold range) and were less potent than
the cognate pyridazinone analogs including KCA-1490.4 The con-
sistently weaker PDE3-inhibitory activity in this pyrazolone series
likely reflects changes in the enzyme-bound position and orienta-
tion of the pyrazolopyridine subunit associated with the ring con-
traction (vide supra).
A notable feature in the PDE4-inhibitory activity of the Table 2
compound set was the pronounced loss of activity for the 2-H
derivative (6i). A similar activity loss was observed in the pyridaz-
inone series,4 and this highlights the importance of a suitably sized
substituent targeted to the Q2 subpocket for conferring effective
PDE4 inhibition. Although the 2-CHF2 and 2-CF3 compounds (6k
and 6l) possessed comparable levels of PDE4 inhibition to KCA-
1490, neither compound was as effective in inhibiting PDE3. This
prompted us to explore replacement fused bicyclic heteroaromatic
subunits for the pyrazolopyridine.
Interestingly the 7-Et derivative (6b) was some 25-fold more
effective as a PDE4 inhibitor than the 7-OMe analog (6c), revealing
that strict catechol ether mimicry is not required and indeed
potentially detrimental to PDE4-inhibitory activity. In the case of
6c it is likely that slight adjustment in the orientation of the pyraz-
olopyridine allows geometrical optimization of a single, strong
hydrogen bond between the pyrazolopyridine N(1) center and
the purine-scanning glutamine NH2 whilst simultaneously posi-
tioning the 7-Et group more favorably in the Q1 subpocket. Poten-
tially, the 7-SMe derivative (6d) might be rather similar in that
regard. The 10-fold enhancement in activity of the 7-Et analog
(6b) compared to the 7-Me analog (6a) suggests that optimal occu-
pancy of the Q1 subpocket is particularly important for achieving
potent PDE4 inhibition. We did not examine the activity of the
7-Et analog in the pyridazinone series.4 However, the 7-Me deriv-
ative in that series exhibited similar PDE3- and PDE4-inhibitory
activity to the cognate compound (6b) presented in Table 1.
Our PDE3-binding model for compounds 6a–6h does not invoke
a hydrogen bonding interaction between the protein and func-
tional groups at the pyrazolopyridine 7-position. This is consistent
with the rather narrow PDE3-inhibitory activity range observed
across this compound set (with the exception of 6e, discussed
above). In contrast, hydrogen bonding features in the pyrazolopyri-
dine 7-substituent profoundly influence the PDE4-inhibitory activ-
ity of the compounds. Moreover, the activity data in Table 1
suggest that appropriate hydrogen donor or acceptor features
may both support potent PDE4-inhibitory activity, and some vari-
ation in the hydrogen bonding pattern between protein and ligand
is conceivable. Thus, with the 7-acetyl derivative (6h) the pyrazol-
opyridine N(1) center and carbonyl oxygen may deliver a pincer-
like engagement of the glutamine NH2 whilst positioning the acet-
yl methyl group effectively in the Q1 subpocket. On the other hand,
with the 7-hydroxymethyl derivative (6f) the side chain hydroxy
group may target the glutamine O(
e) centre, as illustrated
(Fig. 2B). Interestingly branched methyl extension of the
a
hydroxymethyl side chain in 6f (to give 6g) affords a 10-fold
enhancement in PDE4-inhibitory potency, similar to the enhance-
ment seen with the chain extension from methyl to ethyl (6a to
6b). The particularly potent PDE4-inhibitory activity of 6g, which
was assessed in racemic form, probably arises therefore from a
combination of optimal hydrogen bonding to the purine-scanning
glutamine and fit of the 1-hydroxyethyl group within the Q1
We had previously evaluated5 a set of pyrazolopyridine replace-
ment subunits in the pyridazinone series derived from KCA-1490
and found that both PDE3- and PDE4-inhibitory performance could
be modestly improved by optimization of interactions within the
‘hydrophobic clamp’19 of the catalytic pocket, a feature provided
by the purine-stacking phenylalanine residue and the surface of
helix-14 (marked in Fig. 2). We therefore decided to evaluate a
similar set of analogs in the pyrazolone series (Table 3) and se-
lected CF3 (or Et) and OMe as groups corresponding, respectively,
to the 2- and 7-substituents of the pyrazolopyridine, as this combi-
nation had delivered promising compounds in the pyridazinone
series. Synthesis of the required compound set (6m–6s) was
undertaken by adaptation of the routes developed for the pyridaz-
Table 2
SAR survey for optimization of the pyrazolopyridine 2-substituent
O
HN
N
R
N
N
OMe
M)a
5,20
Cmpd
R
Inhibition IC50
(l
inone series (see Supplementary data).
In the case of PDE3 a likely alteration in the bound position of
the heterobicycle allied to pyridazinone ring contraction (vide
supra) made it difficult to predict whether PDE3-inhibitory activity
in the pyrazolone series would parallel that in the pyridazinone
series, where 4-methoxy-2-(trifluoromethyl)benzo[d]thiazol-7-yl
and 8-methoxy-2-(trifluoromethyl)quinolin-5-yl analogs had
performed better than the parent pyrazolopyridine whilst the
PDE3
PDE4
KCA-1490
6c
6i
6j
6k
6l
0.36
9.21
10.84
6.17
3.28
4.13
0.042
0.41
2.93
0.15
0.05
0.11
Et
H
c-Pr
CHF2
CF3
a
See footnotes to Table 1.
4-methoxy-2-(trifluoromethyl)-1H-benzo[d]imidazol-7-yl
and

K. Ochiai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5833–5838
5837
Table 3
SAR survey for optimization of the fused bicylic heteroaromatics
O
HN
N
R
Cmpd
KCA-1490
6a
R
Inhibition IC50
PDE3A
(
l
M)a
Cmpd
6o
R
Inhibition IC50
PDE3A
(l
M)a
PDE4B
PDE4B
0.36
9.21
4.13
0.042
0.54
14.12
0.14
0.063
0.41
0.11
6p
0.2
6l
6q
0.15
6m
6n
1.1
0.36
0.69
6r
6s
10.08
6.65
2.58
1.07
5.36
a
See footnotes to Table 1.
7-methoxy-2-(trifluoromethyl)benzofuran-4-yl counterparts had
performed approximately 10-fold less well. With the pyrazolones,
Table 3, significant PDE3-inhibitory activity enhancement was
seen with benzothiazole 6o and benzimidazole 6p did prove to
be the weakest inhibitor, although it was only 3.4-fold less potent
than the parent pyrazolopyridine (6l). However, in other respects
PDE3-inhibitory activity trends between the two compound series
broke down. Thus, in the pyrazolone series benzofuran 6m showed
a modest activity enhancement relative to pyrazolopyridine 6l (in-
stead of activity loss) and the imidazopyridine (6q) showed a strik-
ing 30-fold gain in inhibitory activity.
As with the pyridazinones, the PDE4-inhibitory activity of the
benzothiazole analog (6o) in the pyrazolone series was also im-
proved relative to the parent pyrazolopyridine (6l), albeit that
the activity gain was modest. Other replacements for the pyrazol-
opyridine caused varying degrees of activity loss to a maximal 10-
fold reduction in performance in the case of quinoline 6s. The
activity loss was marginal for imidazopyridine 6q, however, and
this resulted in a compound with almost equipotent inhibitory
activity against PDE3 and PDE4 (IC50 150 nM).
Given the promising dual PDE3/4-inhibitory activity exhibited
by benzothiazole 6o and imidazopyridine 6q, both compounds
were further evaluated for their capacity to suppress histamine-in-
duced bronchoconstriction in guinea pig according to an estab-
lished procedure.21 Testing was initially conducted with iv dosing
at 0.3 mg/kg. As we expected, both 6o and 6q displayed potent
bronchodilatory activity (96% and 100% suppression of broncho-
constriction vs control respectively). We next evaluated the bron-
chodilatory performance of 6o and 6q under po administration at
0.1 mg/kg in guinea pig. Unfortunately, benzothiazole derivative
6o was not effective for suppression of histamine-induced bron-
choconstriction under these conditions. This finding was consis-
tent with our previous work in the pyridazinone series,4 where
the benzothiazole derivative was only weakly active under po
administration, and is probably due to the vulnerability of the ben-
zothiazole scaffold to metabolism under po administration.22 On
the other hand, imidazopyridine 6q exhibited potent bronchodila-
tory potency under po administration (>70% inhibition of hista-
mine-induced bronchoconstriction at 0.1 mg/kg).
In summary, we have demonstrated that an achiral 4,4-dim-
ethylpyrazolone scaffold is an effective surrogate for the
5-methyl-4,5-dihydropyridazin-3(2H)-one subunit of our previ-
ously reported pyrazolopyridine-linked pyridazinone PDE inhibi-
tors. Selection of an appropriate pyrazolopyridine 7-substituent
can engender potent and, as in the case of 7-NHMe analog (6e),
highly selective inhibitory activity against PDE4. However, the
4,4-dimethylpyrazolone also supports the development of well-bal-
anced dual PDE3/4-inhibitory compounds, as with analog 6q, where
an isosteric imidazopyridine replacement was made for the pyraz-
olopyridine subunit of our original series. Imidazopyridine 6q
showed potent bronchodilatory activity under po administration
with efficacy comparable to our chiral lead compound, KCA-1490.
Acknowledgments
We are grateful to Dr S. J. MacKenzie, Dr S. Hastings and Dr R. A.
Clayton of the Kyorin Scotland Research Laboratory for their many
valuable suggestions and performing the PDE inhibition assays.

5838
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