Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.03.104

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC ₅₀ = 150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrim

Full text of DOI:10.1016/j.bmcl.2013.03.104

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3325–3328
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of the fused bicyclic 4-amino-2-phenylpyrimidine
derivatives as novel and potent PDE4 inhibitors
b
a
c
a
Taiji Goto a,d, , Akiko Shiina , Toshiharu Yoshino , Kiyoshi Mizukami , Kazuki Hirahara ,
Osamu Suzuki a, Yoshitaka Sogawa a, Tomoko Takahashi a, Tsuyoshi Mikkaichi a, Naoki Nakao a,
Mizuki Takahashi a, Masashi Hasegawa a, Shigeki Sasaki d
⇑
a R&D Division, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Quality & Safety Management Division, Daiichi Sankyo Co., Ltd, 3-5-1 Nihonbashihoncho, Chuo-ku, Tokyo 103-8426, Japan
c Corporate Strategy Division, Daiichi Sankyo Co., Ltd, 3-5-1 Nihonbashihoncho, Chuo-ku, Tokyo 103-8426, Japan
d Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 4 April 2013
2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4
inhibitor with moderate PDE4B activity (IC50 = 150 nM). A number of derivatives with a variety of 4-
amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihy-
dro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC50 = 25
nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B
Keywords:
PDE4
PDE4 inhibitor
PDE4B
cAMP
(IC50 = 7.5 nM) and TNF-
a production in mouse splenocytes (IC50 = 9.8 nM) and showed good in vivo
anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID50 = 18 mg/kg). The
binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray
crystal structure of the ligand–enzyme complex.
COPD
Anti-inflammatory agent
Crystallography
2-Phenylpyrimidine
Ó 2013 Elsevier Ltd. All rights reserved.
Phosphodiesterases (PDEs)1 catalyze the hydrolysis of cyclic
adenosine monophosphate (cAMP) and cyclic guanosine mono-
phosphate (cGMP) to the corresponding nucleoside 50-monophos-
phates. PDEs are responsible for the regulation of intracellular
levels of cAMP and cGMP, mediating various biological responses.
PDE4 specifically catalyzes the hydrolysis of cAMP and is a pre-
dominant isozyme expressed in airway smooth muscle cells as
well as immune and inflammatory cells.2 Elevation of cAMP by
inhibition of PDE4 contributes to bronchodilation and suppression
of inflammatory responses. Therefore PDE4 inhibitors are expected
to be promising anti-inflammatory agents for the treatment of
asthma, chronic obstructive pulmonary disease (COPD) and other
inflammatory diseases.3
number of autoimmune and inflammatory diseases, suppression
of TNF-a production is thought to be a key effect of anti-inflamma-
tory activity of PDE4 inhibitors.8 In this Letter, we report our explo-
ration of novel PDE4 inhibitors that have sulfur containing bicyclic
structures (Fig. 1).
Initially a modification of the piperidine side chain of 2 was
conducted. Preparation of derivatives is shown in Scheme 1. Start-
ing material 3 was condensed with benzamidine 4 to give 5. The
conversion of the hydroxyl group of 5 to the chlorine substituent
was performed by treatment with phosphoryl chloride to give 4-
chloropyrimidine 6. The chlorine atom of 6 was substituted with
ethyl 2-(4-piperidinyl)acetate or ethyl 2-(4-aminophenyl)acetate,
Roflumilast (1)4 is a selective PDE4 inhibitor and the only one
that has been launched as an anti-inflammatory drug for treating
COPD.5 The initial compound for our investigation is 2-phenyl-4-
Cl
OH
S
O
N
N
O
F
N
H
Cl
N
N
F
O
piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine
which moderately inhibited human
(IC50 = 150 nM)6 and lipopolysaccharide (LPS)-induced tumor
derivative
2,
O
PDE4B activity
necrosis factor alpha (TNF-a) production in mouse splenocytes
1
2
Roflumilast (DaxasTM
)
(IC50 = 620 nM).7 As overproduction of TNF-
a is associated with a
hPDE4B (IC50) = 0.49 nM
hPDE4B (IC50) = 150 nM
mTNF-α (IC50) = 1.2 nM
mTNF-α (IC50) = 620 nM
⇑
Corresponding author. Tel.: +81 334923131.
E-mail address: goto.taiji.kt@daiichisankyo.co.jp (T. Goto).
Figure 1. Structure of Roflumilast (1) and lead compound 2.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.104

3326
T. Goto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3325–3328
S
R1
N
R2
c
e
N
N
S
S
2, 7d
OH
Cl
H2N
NH
S
a
b
N
N
N
N
O
O
S
O
+
R1
N
O
O
S
O
O
Cl
R2
c
e
d
N
N
N
N
3
4
5
6
8
9a-f
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH; (b) POCl3 (55% from 3); (c) amine, DMF; (d) mCPBA, CH2Cl2 (53%); (e) 1 N NaOH, EtOH.
Table 1
In vitro inhibitory activity of compounds 2, 7d, and 9a–f
S
O
S
O
N
N
N
N
hPDE4B IC50 (nM)
150
hPDE4B IC50 (nM)
190
OH
OH
2
9a
9b
N
N
O
O
N
—
—
—
—
5000
3000
H
N
O
9c
N
OH
H
N
O
O
7d
29
—
9d
9e
140
OH
OH
H
N
—
2500
H
N
—
—
9f
800
OH
O
and the ester group of the resulting compound was hydrolyzed to
afford compounds 2 and 7d, respectively. Intermediate 6 was oxi-
dized to give sulfone 8. The 4-position of pyrimidine was substi-
tuted with amines and the esters were hydrolyzed to afford
compounds 9a–f.
tives, benzopyrimidine 14, pyridopyrimidine 15 and thienopyrim-
idine 16, showed lower activities than 7d.
Further exploration of the sulfide ring moiety is shown in Table 3.
Six-membered cyclic sulfide 17, sulfone 18, and sulfone 19 exhib-
ited equipotent PDE4B inhibitory activity to 7d. However, 19 did
The results of in vitro assay are shown in Table 1. Compound 9a,
the oxidized analogue of sulfide 2, sustained moderate PDE4B
inhibitory activity, however, introduction of basic side chains
was not favorable (9b,c). On the other hand, 9d with the 2-(4-ami-
nophenyl)acetic acid side chain showed moderate PDE4B inhibi-
tion, and sulfide 7d exhibited good PDE4B inhibitory activity. The
2-(3-aminophenyl)acetic acid side chain and the 3-(4-aminophe-
nyl)propanoic acid side chain decreased the potency (9e,f). These
results indicated that the 2-(4-aminophenyl)acetic acid side chain
is appropriate for PDE4B inhibition.
Next, we optimized the sulfide ring moiety of 7d. The PDE4B
inhibitory activities of the bicyclic derivatives are shown in Table 2.
Six-membered ring compound 11 exhibited the highest PDE4B
inhibitory activity in the aliphatic ring compounds (10–12).
Replacement of the sulfur atom with the oxygen atom decreased
the PDE4B inhibitory potency (13). The aromatic bicyclic deriva-
not show activity against LPS-induced TNF-a production in mouse
splenocytes, probably due to its low permeability.9 Although five-
membered cyclic sulfone 20 exhibited moderate PDE4B inhibitory
potency, it was inactive against the TNF-
a production. Six-mem-
bered cyclic sulfone 21, which is an isomer of 18 and 19, showed
only poor PDE4B inhibition. As a result, we could acquire the new
templates, 7d, 17, and 18 for further investigation.
Although these new carboxylic acid compounds exhibited good
PDE4B potency, their inhibitory activities against LPS-induced
TNF-
that of Roflumilast (IC50 = 1.2 nM). While investigating more potent
compounds against TNF- production, we found that the conver-
sion of the carboxylic acid moiety to primary amide was effective
(Fig. 2). Amide 22 strongly inhibited TNF- production with im-
a production in mouse splenocytes were much weaker than
a
a
proved permeability. From this result, we conducted further explo-
ration of amide derivatives.

T. Goto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3325–3328
3327
Table 2
Table 3 (continued)
In vitro inhibitory activity of compounds 7d and 10–16
hPDE4B
IC50 (nM) IC50 (nM) (pH
7.4)
mTNF-
a
LogDa
PAMPA Peb
(10ꢀ6 cm/sec) (pH
7.4)
H
N
O
N
N
OH
O
S
O
N
280
>10
NT
lM
NT
NT
NT
NT
20
21
N
S
N
hPDE4B IC50
(nM)
hPDE4B IC50
(nM)
O
O
S
O
N
7900
29
2200
480
N
7d
10
13
14
N
N
N
N
N
a
Distribution coefficient.6
Permeability coefficient in parallel artificial membrane permeability assay.9
b
610
430
N
N
N
O
O
N
S
O
N
S
O
N
14%a
H
N
H
N
11
12
15
16
N
N
N
N
N
O
O
N
N
OH
NH2
S
35%a
230
N
N
18
22
hPDE4B (IC50) = 25 nM
mTNF-α (IC50) = 390 nM
hPDE4B (IC50) = 16 nM
mTNF-α (IC50) = 8.5 nM
a
inhibition % at 10 lM.
Log D (pH 7.4) = 0.43
Log D (pH 7.4) = 2.9
PAMPA (pH 7.4)
PAMPA (pH 7.4)
Pe (10-6 cm/sec) = 5.8
Pe (10-6 cm/sec) > 50
Table 3
Figure 2. Comparison of 18 and 22.
In vitro profile of compounds 7d, 9d, and 17–21
The amide derivatives (22, 31a–h) were prepared as shown in
H
N
Scheme 2. Starting material 23 was reacted with methyl thioglyco-
late 24 to give 25. Dieckmann condensation of 25 afforded a mixture
of desired 26a and its isomer 26b. The mixture of 26a and 26b was
condensed with benzamidine to give the pyrimidine intermediate
27a and 27b (27a:27b = 8:1). After conversion to the triflate 28a
and 28b, these two isomers were separated. Oxidation of 28a affor-
ded the sulfone 29, which reacted with ethyl 2-(4-aminophe-
nyl)acetate to give 30. Hydrolysis of 30, followed by condensation
with various amines provided the amide derivatives (22, 31a–h).
As shown in Table 4, Linear alkyl, branched alkyl and aryl
substituted compounds were synthesized (31a–h). These amide
derivatives achieved significant enhancement of activities against
O
N
N
OH
hPDE4B
mTNF-
a
LogDa
PAMPA Peb
(10ꢀ6 cm/sec) (pH
7.4)
IC50 (nM) IC50 (nM) (pH
7.4)
S
29
220
970
90
1.3
13.3
<2.0
15.5
5.8
7d
9d
17
LPS-induced TNF-a production in mouse splenocytes. Particularly,
31f showed higher potency than Roflumilast.
N
N
O
S
Some of these amide derivatives were evaluated in in vivo
inhibitory activity against LPS-induced pulmonary neutrophilia in
mice.10 In this model, 31b exhibited high efficacy with an
ID50 = 18 mg/kg (ip), and 31c was also effective with an
ID50 = 23 mg/kg (ip). On the other hand, 31f was not efficacious,
although the reason is unclear.
For further consideration of SAR, an X-ray crystal structure of
31e in PDE4B was obtained (Fig. 3).11 The nitrogen atom of pyrim-
idine interacts with Glu443 and the oxygen atoms of the sulfone
form the hydrogen bonding network to Tyr233 and Asn395 via
water molecules. The aliphatic moiety of the sulfone ring occupies
a lipophilic pocket similar to the difluoromethoxy moiety of Roflu-
milast.12 The side chain of 31e forms a bending conformation and
the tertiary butyl group occupies a lipophilic pocket composed of
Phe414, Met431 and Phe506. These interactions presumably con-
tribute to high PDE4B inhibitory activity of 31e.
O
140
29
0.016
1.4
N
N
S
N
N
O
S
O
N
25
390
0.43
18
19
N
O
S
O
28
>10
l
M
ꢀ0.45
<2.0
N
N
In summary, we identified a series of novel PDE4 inhibitors that
have a 4-amino-2-phenylpyrimidine core fused with sulfur con-
tained rings. These compounds showed high in vitro PDE4B

3328
T. Goto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3325–3328
S
S
S
S
OH
OH
OTf
OTf
SH
Cl
S
S
S
d
a
b
c
O
N
N
N
N
N
N
N
N
O
O
O
O
O
+
+
+
+
O
O
O
O
O
O
O
O
23
24
25
26a
26b
27a
27b
28a
28b
O
S
O
S
O
S O
S
O
N
O
H
H
OTf
OTf
N
N
O
O
g
e
f
h
R1
N
N
N
N
N
N
N
O
N
R2
28a
29
30
22, 31a-h
Scheme 2. Reagents and conditions: (a) NaOMe, NaI, MeOH; (b) NaOMe, toluene; (c) benzamidine, NaOMe, MeOH; (d) Tf2O, NEt3, DMAP, CH2Cl2 (43% for 28a, 5% for 28b, 4
steps); (e) mCPBA, CH2Cl2 (98%); (f) ethyl 2-(4-aminophenyl)acetate, DMF; (g) 1 N NaOH, EtOH; (h) amine, WSC, HOBt, DIPEA, DMF.
Table 4
In vitro inhibitory activity of 22 and 31a–h
O
S
O
N
H
N
O
R1
N
N
R2
R1
R2
hPDE4B IC50 (nM) mTNF-a IC50 (nM)
22
31a
31b
31c
31d
31e
31f
31g
H
H
H
H
H
H
H
H
H
16
27
7.5
22
6.6
11
4.7
43
8.5
35
9.8
13
4.7
8.5
0.80
18
Methyl
n-Propyl
n-Butyl
i-Butyl
t-Butyl
Neopentyl
Phenyl
Methyl
31h
Roflumilast
Methyl 33
0.49
17
1.2
inhibitory activity. Furthermore, we found that the amidation of
the 2-(4-aminophenyl)acetic acid moiety strongly enhanced inhib-
itory activity against LPS-induced TNF-
a production in isolated
mouse splenocytes. Among these compounds, 31b and 31c exhib-
ited high in vivo efficacy in the lung inflammation model in mice.
Further investigation of this series of potent PDE4 inhibitors is now
underway.
Figure 3. X-ray crystal structure of 31e with human PDE4B catalytic binding site.11
3. (a) Huang, Z.; Mancici, J. A. Curr. Med. Chem. 2006, 13, 3253; (b) Houslay, M.;
Schafer, P.; Zhang, K. Y. J. Drug Discovery Today 2005, 10, 1503.
4. (a) Rabe, K. F. Br. J. Pharmacol. 2011, 163, 53; (b) Cazzola, M.; Picciolo, S.;
Matera, M. G. Expert Opin. Pharm. 2010, 11, 441; (c) Giembycz, M. A.; Field, S. K.
Drug Des. Dev. Ther. 2010, 4, 147.
Acknowledgement
We thank Professor Soichi Wakatsuki of the Institute of Materi-
als Structure Science for the use of the facilities at the Photon Fac-
tory and technical staff for their excellent support in the use of the
beamlines.
5. (a) Pages, L.; Gavalda, A.; Lehner, M. D. Expert Opin. Ther. Pat. 2009, 19, 1501; (b)
Spina, D. Br. J. Pharmacol. 2008, 155, 308.
6. hPDE4B(152–564) was cloned, expressed and purified according to the
reported protocols: (a) Zhang, K. Y. J.; Card, G. L.; Suzuki, Y.; Artis, D. R.;
Fong, D.; Gillette, S.; Hsieh, D.; Neiman, J.; West, B. L.; Zhang, C.; Milburn, M. V.;
Kim, S. H.; Schlessinger, J.; Bollag, G. Mol. Cell 2004, 15, 279; (b) hPDE4B assay:
See the Supplementary data.
Supplementary data
7. Mouse TNF-a production assay: See the Supplementary data.
8. Castro, A.; Jerez, M. J.; Gil, C.; Martinez, A. Med. Res. Rev. 2005, 25, 229.
9. The membrane permeability coefficients were assayed using PAMPA
Evolution™ (pION Inc., MA, USA). See the Supplementary data.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03. 104.
10. In vivo assay: See the Supplementary data.
11. The X-ray crystallographic studies were accomplished based on the procedure
described in Ref. 6a The coordinate and statistics are available from the protein
data bank using accession code 3W5E (complex with compound 31e).
12. Card, G. L.; England, B. P.; Suzuki, Y.; Fong, D.; Powell, B.; Lee, B.; Luu, C.;
Tabrizuzad, M.; Gillette, S.; Ibrahim, P. N.; Artis, D. R.; Bollag, G.; Milburm, M.
V.; Kim, S. H.; Schlessinger, J.; Zhang, K. Y. J. Structure 2004, 12, 2223.
References and notes
1. (a) Bender, A. T.; Beavo, J. A. Pharmacol. Rev. 2006, 58, 488; (b) Torphy, T. J. Am. J.
Respir. Crit. Care Med. 1998, 157, 351.
2. Kodimuthali, A.; Jabaris, S. L.; Pal, M. J. Med. Chem. 2008, 51, 5471.