Discovery of selective PDE4B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.121

In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.

Full text of DOI:10.1016/j.bmcl.2009.04.121

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3174–3176
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of selective PDE4B inhibitors
Kenji Naganuma, Akifumi Omura, Naomi Maekawara, Masahiro Saitoh, Naoto Ohkawa, Takashi Kubota,
Hiromitsu Nagumo, Toshiyuki Kodama, Masayoshi Takemura, Yuji Ohtsuka, Junji Nakamura,
*
Ryuichi Tsujita, Koh Kawasaki, Hirotsugu Yokoi, Masashi Kawanishi
Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni-shi, Shizuoka 410-2321, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine deriv-
atives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme.
With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects
in vivo and showed less emesis compared with Cilomilast.
Received 15 March 2009
Revised 22 April 2009
Accepted 24 April 2009
Available online 3 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PDE4B
Neutrophilia
2-Arylpyrimidine
Phosphodiesterase 4 (PDE4) is a hydrolytic enzyme responsible
for the degradation of the second messenger cAMP in many cell
types, and has been proposed as an attractive target in various dis-
eases including asthma and chronic obstructive pulmonary disease
(COPD).1,2 There are a number of PDE4 inhibitors including roflu-
milast3 and Cilomilast4 which have been applied in clinical trials,
however, extensive use of these compounds is limited because of
dose-limiting adverse effects such as nausea and emesis.2 The
PDE4 family consists of four isoforms; PDE4A, 4B, 4C, and 4D.
Knockout studies have revealed that PDE4B ablation suppresses
H
N
N
N
COOH
µ
IC50 = 0.19 M
1
The synthesis of the pyrimidine derivative is outlined in Scheme
1.9 The amidines used in this study were commercially available or
were prepared from nitriles via methyl imidates. Condensation of
amidines with b-ketoesters under basic conditions gave 4-oxopyr-
imidines, followed by chlorination with phosphorus oxychloride.
The resulting 4-chloropyrimidines were substituted with anilines
in the presence of acid to afford 4-aminopyrimidines. Carboxylic
acids were prepared by saponification of corresponding esters.
Firstly, the structure–activity relationships (SAR) of the alkyl
groups at 5- and 6-positions on the pyrimidine ring were investi-
gated (Table 1).10 The compounds bearing allyl ethyl, cyano, or for-
myl groups at 5-position were equipotent for PDE4B inhibition and
4B/4D selectivity (1, 3, 8 and 10). Bulkier groups at this position re-
duced the potency (4–7, 13 and 14). Compound 12 which had an
ethyl group on 6-position showed enhanced potency but less selec-
tivity compared with methyl (1) analog. Thus it seemed that the
steric effect was significant in these positions.
TNF-
a
production5, and PDE4D may be responsible for the occur-
rence of nausea and emesis,1a,6 heart failure and the risk of
arrhythmias.7 In this regard, a PDE4B inhibitor selective over
PDE4D is expected to be a useful anti-inflammatory agent and a
chemical probe is needed to investigate the role of PDE4B in detail.
However, high structural similarity between PDE4B and PDE4D has
made it difficult to develop a highly selective inhibitor which to
date has not been possible.8 In this letter, we describe the discov-
ery of the first PDE4B selective inhibitor with novel structural fea-
tures which has potential as an anti-inflammatory drug.
By screening our in-house compound library, a pyrimidine
derivative 1 was identified with potent inhibitory activity against
PDE4B. 1 has an IC50 of sub-micromolar range and a potential
10-fold selectivity over PDE4D. We focused on investigations on
the structure–activity relationships to optimize this lead
compound.
SAR exploration of the phenyl group at 2-position of the pyrim-
idine ring revealed substitution to 2- or 3-thienyl group (15 and 16
in Table 2, respectively) which could be suitable alternatives with-
out a loss of potency or selectivity. The introduction of a 2-methyl-
* Corresponding author. Fax: +81 558 76 5755.
E-mail address: kawanishi.mb@om.asahi-kasei.co.jp (M. Kawanishi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.121

K. Naganuma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3174–3176
3175
Table 2
H2N
HN
S
R5
R6
R5
R6
Inhibitory activity of pyrimidine derivatives 15–19
S
a,b
NC
H
N
R1
N
N
COOH
O
R2
O
R3
HN
NH2
COOR
c
d
R2
NH
+
R3
N
Compound R3
PDE4B IC50
(nM)
PDE4D IC50
(nM)
Ratio 4D/4B
IC50
R1
R3
R1 = Me, Et, Allyl, etc.
R
R3 = (substituted) phenyl,
(substituted) thienyl, etc.
1
Ph
190
1900
9.7
2 = Me, Et, nPr, iPr
S
15
120
1300
11
R1
R7
R1
H
N
R2
R2
Cl
16
17
18
68
990
15
e
N
S
N
N
N
N
R4
R3
R3
2800
3700
12,000
15,000
4.2
4.1
S
N
R
R
4 = COOMe, CH2COOEt
4 = COOH, CH2COOH
f
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH, rt; (b) NH4Cl, EtOH, 90 °C;
(c) NaH, MeOH, 55 °C; (d) POCl3, 100 °C; (e) aniline, HClaq, EtOH, 55 °C; (f) NaOHaq,
MeOH, rt.
N
19
860
3000
3.5
N
Table 3
thiazol-4-yl (17), 2-pyridyl (18), or pyradinyl (19) group resulted in
a decrease in potency.
Inhibitory activity of pyrimidine derivatives 20–30
During the course of the SAR work to enhance PDE4B selectivity
over the 4D isozyme, we found a suitable combination of substitu-
ents at 2-position and the carboxylic acid moiety at the phenyl
amino group, R3 and R4, respectively (Table 3). A substituted phe-
nyl or thienyl group at R3 did not improve selectivity when R4 was
a carboxy group (20–24), similar results were observed when R3
was substituted with phenyl and R4 with a carboxymethyl group
(25–28). However, compounds 29 and 30, bearing a substituted
thienyl group at R3 and a carboxymethyl group at R4 showed sig-
nificant selectivity greater than 25-fold.
H
N
N
N
R4
R3
R4
Compound R3
PDE4B
PDE4D
Ratio 4D/4B
IC50 (nM) IC50 (nM) IC50
1
Ph
–COOH
–COOH
190
220
1900
2800
9.7
13
20
CH3
CH3
Table 1
21
–COOH
210
1500
6.9
Inhibitory activity of pyrimidine derivatives 1–14
R1
H
22
23
24
25
26
–COOH
–COOH
–COOH
220
150
78
2000
8.9
8.6
9.8
n.d.
12
R2
N
O
N
N
S
S
1300
COOH
CH3
760
O
Compound
R1
R2
PDE4B IC50
PDE4D IC50
(nM)
Ratio 4D/
4B IC50
Ph
–CH2COOH 1800
–CH2COOH 940
>10,000
11,000
(nM)
Cilomilast
1
2
3
4
5
6
7
37
190
430
140
1300
>1000
>1000
>1000
120
>1000
300
540
27
0.7
9.7
7.9
15
5.6
n.d.
n.d.
n.d.
12
n.d.
5.2
15
2.4
n.d.
3.4
–CH2CH@CH2
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
1900
3400
2100
7600
n.d.
CH3
CH3
Et
n-Pr
i-Pr
n-Bu
Bn
–CN
–CH2NH2
–CHO
27
–CH2COOH 1200
9900
8.0
n.d.
n.d.
28
29
–CH2COOH 600
–CH2COOH 320
4800
8800
8.0
28
8
9
1500
n.d.
1500
8000
82
O
10
11
12
13
14
S
S
–CH2OH
–CH2CH@CH2
–CH2CH@CH2
–CH2CH@CH2
CH3
34
>1000
690
i-Pr
n-Pr
n.d.
2400
30
–CH2COOH 190
5300
29
O
n.d. = Not determined.

3176
K. Naganuma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3174–3176
Table 4
Compound 33 was subjected to further biological evaluation.
Table 6 shows a summary of the results compared with Cilomilast.
Compound 33 had the same potency as Cilomilast in inhibiting
Compounds with improved inhibitory activity and selectivity
R7
H
TNF-a production in mouse peripheral blood mononuclear cells
N
(PBMC) in vitro, reflecting the same degree of activity against
PDE4B, or had an additional effect due to the PDE4D inhibiting
N
N
COOH
activity of Cilomilast. Oral activity against LPS-induced TNF-a pro-
S
duction in mice was evaluated, and it was found that compound 33
showed potential activity in this assay. We further evaluated the
inhibitory activity against neutrophil accumulation in lung in-
duced by LPS, and the emetic adverse effect in ferrets. Cilomilast
showed an effect on neutrophilia at a dose of 10 mg/kg, and vom-
iting was observed at a dose of 1 mg/kg, indicating a rather narrow
therapeutic window. Compound 33 had the same potency on neu-
trophilia, and a significantly lower emetic effect with no vomiting
at a dose of 100 mg/kg. The greater safety index of 33 is possibly
due to PDE4B selectivity over PDE4D. To the best of our knowledge,
this is the first disclosure of PDE4B inhibitors which show both
strong potency and high selectivity (>100-fold) against PDE4D. A
further exploration of these PDE4B inhibitors and their application
in COPD and other therapeutic areas is now ongoing.
R5
R6
Compound R5
R6 R7 PDE4B IC50
(nM)
PDE4D IC50
(nM)
Ratio 4D/4B
IC50
31
32
33
34
35
SCH3
Br
Cl
CH3
F
H
H
H
Br
H
H
H
H
H
F
34
19
15
6.8
15
1500
1600
1700
2900
3100
43
87
114
420
205
Table 5
Pharmacokinetic dataa for compound 33 and 35
Compound
Mice
g h/mL)
Ferret
g h/mL)
In summary, a novel class of potent, selective, and orally avail-
able PDE4B inhibitors has been developed. Biological evaluation of
the compound identified as 33 had anti-inflammatory therapeutic
potential and a wider safety margin regarding emesis.
AUCb
(
l
Cmax
(lg/mL)
AUCb
(l
Cmax (lg/mL)
33
35
52.3
21.9
8.7
10.9
33.5
340.9
4.5
14.8
a
Dosed at 2.0 mg/kg po as suspension in 0.5% methylcellulose.
AUC of 0–1.
b
Acknowledgments
The authors thank the analytical groups in the Research Center,
Asahi Kasei Pharma Corporation for analytical and spectroscopic
data. The authors thank Naoko Yamaguchi for biological
evaluation.
Table 6
Biological evaluation of compound 33
Compound
Inhibition of TNF-
production
Neutrophilia
inhibitionc
Vomiting testd
References and notes
In vitro
In vivo
In vivo, ferret
inhibition% @
Dose in ferret
(mg/kg, po),
Emetic responders
(vomiting /tested)
mice PBMCa miceb ID50
1. (a) Zhang, K. Y. J.; Ibrahim, P. N.; Gillette, S.; Bollag, G. Exp. Opin. Ther. Targets
2005, 9, 1283; (b) Burnouf, C.; Pruniaux, M.-P. Curr. Pharm. Design 2002, 8,
1255; (c) Kodimuthali, A.; Jabaris, S. S. L.; Pal, M. J. Med. Chem. 2008, 51, 5471.
2. Houslay, M. D.; Schafer, P.; Zhang, K. Y. Drug Discovery Today 2005, 10, 1503.
3. Timmer, W.; Leclerc, L.; Birraux, G.; Neuhauser, M.; Hatzelmann, A.; Bethke, T.;
Wurst, W. J. Clin. Pharmacol. 2002, 42, 297.
IC50 (M)
(mg/kg, po) dose (po)
Cilomilast
0.20
2.2
35% @ 10 mg/kg
0.3, 0/6
1, 1/6
4. Christensen, S. B.; Guider, A.; Forster, C. J.; Gleason, J. G.; Bender, P. E.;
Karpinski, J. M.; DeWolf, W. E., Jr.; 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.; Bartus, J. O.; Ryan, M. D.; Eggleston, D. S.;
Haltiwanger, R. C.; Torphy, T. J. J. Med. Chem. 1998, 41, 821.
10, 2/3
33
0.50
14
44% @ 12.5 mg/kg 100, 0/4
46% @ 25 mg/kg
200, 2/2
a
Inhibitory concentration on LPS-induced TNF-
a
production from mouse
5. Jin, S.-L. C.; Conti, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7628.
6. Robichaud, A.; Stamatiou, P. B.; Jin, S.-L. C.; Lachance, N.; MacDonald, D.;
Laliberte, F.; Liu, S.; Huang, Z.; Conti, M.; Chan, C. C. J. Clin. Invest. 2002, 110,
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R. D.; Richter, W.; Jin, S.-L. C.; Conti, M.; Marks, A. R. Cell 2005, 123, 25.
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3854.
peripheral blood mononuclear cells (PBMC).
b
Inhibitory dose on LPS-induced (ip) TNF-
The percentage inhibition for a change in the number of neutrophils in lung
a
production in mice.
c
lavage after LPS inhalation in ferrets.
d
Occurrence of vomiting episode after administration for 6 h.
9. Alternatively, compounds 8–11 having functionalized substituent at 5-position
of pyrimidine ring were synthesized via 1,6-dihydro-4-methyl-6-oxo-2-
phenyl-5-pyrimidine-carbonitrile described in Ellingboe, J.; Alessi, T.; Millen,
J.; Sredy, J.; King, A.; Prusiewicz, C.; Guzzo, F.; VanEngen, D.; Bagli, J. J. Med.
Chem. 1990, 33, 2892.
We then focused on optimization of 2-thienyl derivatives. After
extensive investigations to introduce a wide variety of functional
groups on the thiophene moiety and a combination with substitu-
ents on the other position, a series of compounds with highly im-
proved potency and selectivity were found. Compounds 33–35 in
Table 4 showed excellent potency on 4B and >100-fold selectivity
over 4D.11
These compounds generally showed good pharmacokinetic
properties. Examples are compounds 33 and 35 in Table 5. Bioavail-
ability of 33 and 35 in mice was 85% and 73%, respectively (data not
10. Recombinant human PDE4B1 (catalytic domain, a.a. 324–736) and human
PED4D3 (catalytic domain, a.a. 244–673) were used. Inhibitory activity of the
compounds were assayed using
10 min. The reaction was stopped by adding 25
mixture and the solution was charged on
3
l
M
cAMP with 0.83
L of trichloroacetic acid to the
neutral alumina column
l
Ci [3H]-cAMP for
l
a
equilibrated with 0.1 M of 2-[[tris(hydroxylmethyl)methyl]-amino]-1-
ethanesulfonic acid (TES) buffer (pH 8.0). After washing the column with a
sufficient amount of 0.1 M TES buffer solution, the column was eluted with 2 N
NaOH, and the radioactivity of [3H]-50AMP product was measured.
shown). Compounds 33 and 35 were not inhibitors (IC50 > 10 lM)
of CYP1A2, CYP3A4, CYP2C9, and CYP2D6 (data not shown).
11. Compound 33 showed no inhibition (IC50 > 2 lM) toward PDE 1–3, 5 and 6
(data not shown).