Identification of potent pyrimidine inhibitors of phosphodiesterase 7 (PDE7) and their ability to inhibit T cell proliferation

Article abstract of DOI:10.1016/j.bmcl.2009.02.060

A series of pyrimidine based inhibitors of PDE7 are discussed. The synthesis, structure-activity relationships (SAR) and selectivity against several other PDE family members as well as activity in T cells are presented. These compounds were found to have effects on T cell proliferation, however it is not clear whether the mechanism is related to PDE7 inhibition.

Full text of DOI:10.1016/j.bmcl.2009.02.060

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1935–1938
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of potent pyrimidine inhibitors of phosphodiesterase 7
(PDE7) and their ability to inhibit T cell proliferation
*
Junqing Guo , Andrew Watson, James Kempson, Marianne Carlsen, Joseph Barbosa, Karen Stebbins,
*
Deborah Lee, John Dodd, Steven G. Nadler, Murray McKinnon, Joel Barrish, William J. Pitts
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrimidine based inhibitors of PDE7 are discussed. The synthesis, structure–activity relation-
ships (SAR) and selectivity against several other PDE family members as well as activity in T cells are pre-
sented. These compounds were found to have effects on T cell proliferation, however it is not clear
whether the mechanism is related to PDE7 inhibition.
Received 27 October 2008
Revised 13 February 2009
Accepted 13 February 2009
Available online 21 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Pyrimidine
Phosphodiesterase 7
T cell
Phosphodiesterases (PDEs) play
a
critical role in various
molecule inhibitor of PDE7 has continued to be of interest in order
to complete our understanding of the relationship between PDE7
inhibition and T cell function. Recently there have been reports
of selective PDE7 inhibitors which have failed to demonstrate sig-
nificant effects on T cells.8 We report herein on selective inhibitors
of PDE7 which were found to suppress T cell proliferation and evi-
dence which suggests that this activity is not related to PDE7
inhibition.
Our initial SAR focused on bicyclic systems such as 1a and 1b. In
an effort to understand the minimal phamacophore we prepared
pyrimidine 2a. This compound displayed a 22-fold loss in PDE7
potency compared to 1a. However we postulated that addition of
a second group at the 6-position would increase the potency
against PDE7 similar to that observed with the constrained analog
1b (Fig. 1).
The synthesis of pyrimidine analogues 2b–m is shown in
Scheme 1. Thiazole guanidine 5 was prepared through the conden-
sation of 2-imino-4-thiobiurea 3 with 2-chloroacetoacetate 4 in
the presence of pyridine. Guanidine 5 was condensed with com-
mercially available diethyl malonate under basic conditions in
refluxing ethanol to give the desired pyrimidone 6 in ꢀ100% yield.
Dichloropyrimidine 7 was formed in 80–95% yield after treatment
of 6 with POCl3. The chlorine atom at the 4 position of dichloropy-
rimidine could be readily displaced with amines to produce inter-
mediate 8. Subjecting intermediate 8 to a second displacement
with a variety of amines at higher temperature using a microwave
reactor produced compounds 2b–m.
biological processes by hydrolyzing the key second messengers
cAMP and cGMP to the corresponding 50-monophosphate nucleo-
tides. In the immune system, cAMP is the primary regulatory cyclic
nucleotide and it is believed that cAMP broadly suppresses the func-
tions of immune and inflammatory cells. The reduction of cAMP lev-
els is mediated principally by the action of cell-specific
phosphodiesterases (PDEs) and as such, an approach to sustain
cAMP levels through PDE-inhibition would provide a strategy to
treat a variety of immune and inflammatory diseases.1
A functional role of PDE7A in activation and/or proliferation of T
cells has been reported.2 Resting T lymphocytes express mainly
PDE3 and PDE4. However, upon activation, T cells dramatically
up-regulate PDE7A1 and appear to principally rely on this isozyme
for regulation of cAMP levels. Suppression of PDE7 up-regulation
by anti-sense oligonucleotides inhibited T cell proliferation and
decreased IL-2 production, and maintained elevated levels of intra-
cellular cAMP in CD3xCD28 stimulated T cells. PDE7A3, a splice
variant of PDE7A1, is also reported to be up-regulated in activated
CD4+ T cells.3 This expression profile suggests inhibitors of PDE7A
would have broad application as an immunosuppressant. To this
end, several groups have reported the preparation of potent inhib-
itors of PDE74 with optimization of pharmacokinetic properties.5
We recently reported chemistry efforts around our purine-based
deck hit.6 However, since our report that PDE7A deficient mice
show no deficiencies in T cell function,7 the identification of a small
In vitro data for the diaminopyrimidine series (2b–m) is
presented in Table 1. We were gratified to find that compared to
* Corresponding authors.
E-mail address: junqing.guo@bms.com (J. Guo).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.060

1936
J. Guo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1935–1938
SO2NH2
H
N
N
Me
HN
N
N
N
Et
Me
EtO2C
N
N
N
OMe
S
N
N
N
H
N
EtO2C
S
N
H
OMe
OMe
1a
PDE7 IC50 10nM
1b
PDE7 IC50 6nM
SO2NH2
HN
Me
4
N
N
EtO2C
6
S
N
H
N
2a
PDE7 IC50 220nM
R1
N
R2
N
N
Me
N
R3
EtO2C
N
S
N
H
R4
2 b-m
Figure 1. Proposed target 2b–m.
Me
NH
S
O
O
Pyridine, EtOH
N
NH
+
EtO2C
H2N
EtO
N
NH2
OEt
100C (75%)
S
H
N
NH2
Cl
4
H
5
3
O
O
OH
N
Me
OEt
POCl3
100oC (80-95%)
N
EtO2C
NaOEt, EtOH
reflux (100%)
S
N
H
N
OH
6
Cl
R1
N
R2
Me
N
N
N
HNR1R2
N
EtO2C
Me
N
S
S
DIPEA, n-
BuOH, 100°C,
60-91%
N
H
N
Cl
N
H
Cl
EtO2C
8
R1
R2
N
N
HNR3R4
N
Me
N
R3
n-BuOH, 120°C
MW, 30-80%
N
H
N
R4
S
EtO2C
2b-m
Scheme 1.

J. Guo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1935–1938
1937
Table 1
NR1R2
N
N
EtO2C
NR3R4
S
N
H
N
Compd –NR1R2
–N R3R4
PDE7 IC50
M)
PDE1 IC50
M)
PDE3 IC50
M)
PDE4 IC50
M)
PDE5 IC50
M)
T cell IC50
M)
Cytotoxicity IC50
M)
(
l
(
l
(
l
(
l
(
l
(
l
(l
N
H
N
O
O
2b
0.10
>10
>50
0.030
0.065
0.50
>25
S
H2N
H
N
H
O
O
2c
0.010
0.031
0.010
0.039
0.056
4.1
>10
7.9
4.1
27
2.8
8.0
22
27
19
0.22
2.3
3.2
5.1
9.4
0.072
0.32
7.6
7.2
>25
12
N
NH
NH
NH
N
S
H2N
OMe
N
H
2d
1.2
N
OMe
OMe
N
N
2e
0.45
0.69
2.2
14
N
OMe
OMe
OMe
OMe
2f
5.2
>25
15
N
OMe
N
N
2g
>10
N
N
N
N
2h
0.083
0.13
>50
15
46
13
>10
9.5
>10
>10
2.5
2.0
15
N
N
O
N
N
2i
>25
N
N
N
O
NH2
2j
0.082
0.12
23
11
>50
>50
8.0
9.3
6.5
3.1
5.0
>25
>25
N
N
N
NH
OH
2k
>10
N
N
2l
0.076
0.063
23
>50
20
>10
3.2
>10
1.5
>25
>25
N
N
N
OH
OH
N
2m
4.9
0.70
0.12
N
OH
2a all compounds with a second substituent showed improved
potency against PDE7. Compounds 2e and 1b which contain the
same substitution pattern in different chemotypes were essentially
equipotent. This compound also possessed excellent selectivity
against PDE1 and PDE3-5. Interestingly, we observed the phenyl
ring was not a requirement for PDE7 activity (2g–m). Compounds
2c–l all had excellent aqueous solubility at acidic pH, and had
aqueous solubility in the range of >20 lg/mL <100 lg/mL at pH
6.5. In general CACO2 permeability for these compounds was high
(data not shown) suggesting these compounds had acceptable

1938
J. Guo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1935–1938
is the case for 2 m since the effects observed in cells are at a level
PDE7 IC50 vs T cell proliferation IC50
0.2
0.15
0.1
below the enzyme IC50 for PDE4. Further results from this lab
regarding the activity of these and similar compounds will be
presented in due course.
References and notes
0.05
0
1. Francis, S. H.; Turko, I. V.; Corbin, J. D. Prog. Nucleic Acid Res. Mol. Biol. 2001, 65,
1.
2. Li, L.; Yee, C.; Beavo, J. A. Science 1999, 283, 848.
0
1
2
3
4
5
6
T cell proliferation IC50 (uM)
3. Smith, S. J.; Brookes-Fazakerley, S.; Donnelly, L. E.; Barnes, P. J.; Barnette, M. S.;
Giembycz, M. A. Am. J. Physiol. Lung Cell Mol. Physiol. 2003, 284, L279.
4. (a) Martnez, A.; Castro, A.; Gil, C.; Miralpeix, M.; Segarra, V.; Domenech, T.;
Beleta, J.; Palacios, J. M.; Ryder, H.; Miro, X.; Bonet, C.; Casacuberta, J. M.;
Azorin, F.; Pina, B.; Puigdomenech, P. J. Med. Chem. 2000, 43, 683; (b) Barnes, M.
J.; Cooper, N.; Davenport, R. J.; Dyke, H. J.; Galleway, F. P.; Galvin, F. C. A.;
Gowers, L.; Haughan, A. F.; Lowe, C.; Meissner, J. W. G.; Montana, J. G.; Morgan,
T.; Picken, C. L.; Watson, R. J. Bioorg. Med. Chem. Lett. 2001, 11, 1081.
5. Vergne, F.; Bernadelli, P.; Lorthiois, E.; Pham, N.; Proust, E.; Oliveira, C.;
Magroud, A.-K.; Ducrot, P.; Wrigglesworth, R.; Berlioz-Seux, F.; Coleon, F.;
Chevalier, E.; Moreau, F.; Idrissi, M.; Tertre, A.; Descours, A.; Berna, P.; Li, M.
Bioorg. Med. Chem. Lett. 2004, 14, 4615.
Figure 2.
biopharmaceutical properties.9 Compound 2m also maintained
comparable aqueous solubility (20 g/ml at pH 6.5) even in the
absence of a basic amine. The compounds displayed PDE7 IC50’s
comparable to our earlier reported series together with excellent
PDE family selectivity profiles (e.g., 2e–l).
l
6. (a) Pitts, W. J.; Vaccaro, W.; Huynh, T.; Leftheris, K.; Roberge, J. Y.; Barbosa, J.;
Guo, J.; Brown, B.; Watson, A.; Donaldson, K.; Starling, G. C.; Kiener, P. A.; Poss,
M. A.; Dodd, J. H.; Barrish, J. C. Bioorg. Med. Chem. Lett. 2004, 14, 2955; (b)
Kempson, J.; Pitts, W. J.; Barbosa, J.; Guo, J.; Omotoso, O.; Watson, A.; Stebbins,
K.; Starling, G. C.; Dodd, J. H.; Barrish, J. C.; Felix, R.; Fischer, K. Bioorg. Med.
Chem. Lett. 2005, 15, 1829.
Since the compounds had sufficient solubility they were exam-
ined in an in vitro T cell proliferation assay.10 Compounds 2b–m
were found to inhibit T cell proliferation in a range of IC50’s from
0.12 lM to 7.2 lM. A graph of the PDE7 IC50’s versus the T cell pro-
liferation IC50’s for the most selective compounds (2e–l) suggests a
rough correlation, although the data set is limited (Fig. 2). The inhi-
bition of T cell proliferation by these compounds is surprising since
we had seen minimal effects on T cell proliferation between wild
type and PDE7 KO animals. This discrepancy could result from a
number of potential compensatory mechanisms in KO animals.
Alternatively the reports of selective PDE7 inhibitors which are
devoid of effects in T cells could result from a lack of cell penetra-
tion. In an effort to put our results in context we examined com-
pound 2m in a proliferation assay using splenocytes derived
from both wild type and PDE7 KO animals. The percent inhibition
values obtained with compound 2m for both wild type and PDE7
7. Yang, G.; McIntyre, K. W.; Townsend, R. M.; Shen, H. H.; Pitts, W. J.; Dodd, J. H.;
Nadler, S. G.; McKinnon, M.; Watson, A. J. J. Immunol. 2003, 171, 6414.
8. (a) Smith, S. J.; Cieslinski, L. B.; Newton, R.; Donnelly, L. E.; Fenwick, P. S.;
Nicholson, A. G.; Barnes, P. J.; Barnette, M. S.; Giembycz, M. A. Mol. Pharmacol.
2004, 66, 1679; (b) Nueda, A.; Garcia-Roger, N.; Domenech, T.; Godessart, N.;
Cardenas, A.; Santamaria-Babi, L. F.; Beleta, J. Cell. Immun. 2006, 242, 31.
9. Lennernaes, H.; Abrahamsson, B. In Comprehensive Medicinal Chemistry II; Testa,
B., Waterbeemd, H. van de, Eds.; Elsevier: Vol. 5, 2006; pp 971–998.
10. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by
density gradient centrifugation over Lymphoprep, 1.077. Cells were plated into
96 well U-bottom plates at 2.5 ꢁ 105 cells/well in 10% FBS RPMI 1640 (Life
Technologies/Gibco-BRL) containing 10
lg/ml anti-CD3 (G19-4, Bristol-Myers
Squibb P.R.I., Princeton, NJ) and 1 g/ml anti-CD28 (9.3, Bristol-Myers Squibb
l
P.R.I.) in the presence and absence of inhibitors. DMSO (used as a solvent for
inhibitors) was added to the medium at 0.2% final concentration. The total
volume per well was 200
which time 0.5 Ci of 3H-thymidine was added to each well. Six hours
following the addition of 3H-thymidine, the plates were harvested onto filter
plates, 30 l EcoLite scintillant (ICN, Costa Mesa, CA) was added per well, and
ll. Cells were incubated at 37C 5% CO2 for 3 days, at
KO animals were virtually identical (wild type IC50 = 0.21
lM,
l
PDE7 KO IC50 = 0.15 M). This result suggests that the inhibition
l
reported in Table 1 is not directly related to inhibition of PDE7.
There have been reports in the literature of dual inhibitors of
PDE4 and PDE7 showing greater potency in assays than might be
expected from inhibition of a single PDE.11 It is not clear that this
l
plates read on a Top Count-NXT scintillation counter.
11. (a) Giembycz, M. A. Proc. Am. Thoracic Soc. 2005, 2, 326; (b) Yamamoto, S.;
Sugahara, S.; Ikeda, K.; Shimizu, Y. Eur. J. Pharmacol. 2007, 559, 219.