Discovery of triazines as selective PDE4B versus PDE4D inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.06.002

In this study we report a series of triazine derivatives that are potent inhibitors of PDE4B. We also provide a series of structure activity relationships that demonstrate the triazine core can be used to generate subtype selective inhibitors of PDE4B versus PDE4D. A high resolution co-crystal structure shows that the inhibitors interact with a C-terminal regulatory helix (CR3) locking the enzyme in an inactive 'closed' conformation. The results show that the compounds interact with both catalytic domain and CR3 residues. This provides the first structure-based approach to engineer PDE4B-selective inhibitors.

Full text of DOI:10.1016/j.bmcl.2014.06.002

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4031–4034
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of triazines as selective PDE4B versus PDE4D inhibitors
b
c
c
a
b,d,
Timothy J. Hagen a, , Xuesheng Mo , Alex B. Burgin , David Fox 3rd , Zheng Zhang , Mark E. Gurney
⇑
⇑
a Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA
b Tetra Discovery Partners LLC, Grand Rapids, MI, USA
c Beryllium, 7869 NE Day Rd. West, Bainbridge Island, WA 98110, USA
d Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study we report a series of triazine derivatives that are potent inhibitors of PDE4B. We also provide
a series of structure activity relationships that demonstrate the triazine core can be used to generate sub-
type selective inhibitors of PDE4B versus PDE4D. A high resolution co-crystal structure shows that the
inhibitors interact with a C-terminal regulatory helix (CR3) locking the enzyme in an inactive ‘closed’
conformation. The results show that the compounds interact with both catalytic domain and CR3 resi-
dues. This provides the first structure-based approach to engineer PDE4B-selective inhibitors.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 28 January 2014
Revised 29 May 2014
Accepted 2 June 2014
Available online 12 June 2014
Keywords:
PDE4 inhibitor
PDE4B
PDE4D
Crystallography
Triazine
Phosphodiesterases (PDEs) hydrolyze cyclic adenosine mono-
phosphate (cAMP) and cyclic guanosine monophosphate (cGMP)
to their cognate 50-monophosphate derivatives. There are eleven
different PDE superfamily members (PDE1-11) and inhibitors
have been developed to prolong the effects of physiological pro-
cesses mediated by cAMP or cGMP. Phosphodiesterase 4 (PDE4)
is the primary enzyme that regulates the turnover of cAMP.1
PDE4 is comprised of four genes (PDE4A-D) and each gene has
multiple transcripts that can produce three isoforms of the pro-
tein termed long, short and super short. Long forms of PDE4 con-
tain two upstream regions known as UCR1 and UCR2. These
form a negative regulatory module that is activated by protein
kinase A (PKA) phosphorylation of UCR1. Burgin et al. were the
first to show that the negative regulation results from UCR2
closing over the active site, thereby preventing access of cAMP.2
A third C-terminal control region (CR3) is present in all PDE4
isoforms and was also shown to close over the active site by
weakly engaging inhibitors (e.g., PMNPQ; PDB ID: 3G58); how-
ever, the significance of this region was not well understood.2
Our recent structural studies reveal that potent and selective
PDE4B inhibitors bind CR3 thereby locking the enzyme in a
closed conformation.3 PDE4B selectivity is due to a single amino
acid polymorphism in CR3 that selects the helical registration of
the domain when it closes over the active site. Exchange of a
leucine in PDE4B CR3 for a glutamine in PDE4D causes a 70–
80-fold shift in inhibitor selectivity. In this report we describe
a series of triazine analogs that similarly bind to CR3 thereby
resulting in PDE4B selectivity.
PDE4B is a therapeutic target of high interest for central ner-
vous system (CNS), inflammatory and respiratory diseases;4,5 how-
ever, it has been extremely difficult to discover selective PDE4B
inhibitors due to the high amino sequence conservation of the
PDE4 active site.6
We initiated our chemistry effort for discovery of new PDE4B
inhibitors that engage PDE4 regulatory domains with in silico
screening and docking studies conducted with commercial
libraries against known PDE4 structures (3IAD, 3G45 & 3G58).2
Screening of the hits in enzymatic assays using PDE4B1 and
PDE4D7 resulted in identification of compound 1 as a novel PDE4
inhibitor (Fig. 1), and the 1,3,5-triazine series was selected to begin
our synthetic efforts. Although this initial hit does not show
PDE4B-subtype selectivity, we noted the similarity to 2, a com-
pound reported by Naganuma (A-33),7 which we confirmed to be
highly selective for PDE4B.8 In addition, a series of triazine analogs
has recently been reported by others to inhibit PDE4A.9
The general synthesis of the 1,3,5-triazine series is illustrated in
Scheme 1. Using the procedure of Harris10 a nitrile bearing the AR2
group is converted into the corresponding cyanoamidine. The R1
group is then introduced and the triazine ring formed by reaction
of the cyanoamidine with an N,N-dimethylamide. The chlorogroup
⇑
Corresponding authors. Tel.: +1 815 753 1463.
E-mail addresses: thagen@niu.edu (T.J. Hagen), mark@tetradiscovery.com
(M.E. Gurney).
http://dx.doi.org/10.1016/j.bmcl.2014.06.002
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

4032
T. J. Hagen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4031–4034
Table 2
Inhibitory activity of Ar2 analogs
H
N
H
N
O
N
O
O
H
N
R1
N
N
S
N
N
N
O
OH
O
N
Ar2
N
OH
Cl
2
A-33
#
R1
Ar2
hPDE4B IC50
(nM)
hPDE4D IC50
(nM)
Ratio D/B
IC50
1
BAS 03374979
hPDE4B1 (IC50) = 55 nM
hPDE4D7 (IC50) = 1997 nM
hPDE4B1 (IC50) = 6700 nM
hPDE4D7 (IC50) = 2300 nM
S
Cl
Cl
7
8
251
237
1489
1181
5.9
5.0
Figure 1. Structure of lead compound 1 and A-33 (2).
9
3770
18755
1437
5611
22260
2107
1.5
1.2
1.5
NH
Cl
F
a, b
Cl
c
NC
NC
N
H
Ar2
Ar2
O
10 Me
11
H
N
R1
N
R1
N
Ar1
d
N
N
N
N
12 Et
13 Et
1460
2777
727
NT
0.5
F
Ar2
Ar2
O
Scheme 1. General synthesis of the 1,3,5-triazine series. Reagents and conditions:
(a) NaOMe, MeOH (b) NH2CN (c) R1CONMe2, POCl3 CH3CN (d) Ar1NH2, AcOH.
is then displaced to afford the desired Ar1 products. Detailed exper-
imental procedures are supplied in the Supplemental material.
The compounds were screened for their ability to inhibit long
isoforms of PDE4D and PDE4B in vitro (see Supplemental data),
and the results are shown in Tables 1–6. Generally the compounds
in this Letter are druglike and RO5 compliant.11 The compounds
have calculated polar surface areas ranging from 50 to 88 Å2 (see
Supplemental data).
Table 3
Inhibitory activity of linker modified analogs
N
N
L
O
N
N
OH
Ar2
Synthesis of various triazine analogs with aliphatic R1 groups
revealed ethyl and cyclopropyl substituents to be 4–5-fold more
active than methyl-, propyl-, or isopropyl-containing analogs
(Table 1). The cyclopropyl containing analog 7 displayed modest
selectivity, about six fold, for PDE4B versus PDE4D.
#
L
Ar2
hPDE4B IC50
(nM)
hPDE4D IC50
(nM)
Ratio D/B
IC50
Cl
8
NH
237
2282
845
1181
859
5.0
Varying Ar2 revealed that 2-chlorothiophene (7) and 3-chloro-
phenyl (8) were the preferred substituents for PDE4B selectivity
with the 2-chlorothiophene consistently displaying better selectiv-
ity. The NH linker between the triazine core and Ar1 is also
required for potency against both PDE4B and PDE4D. Analogs with
an aminomethylene (14 and 15) or oxygen linker (16) resulted in
14 NHCH2
15 NHCH2
0.38
0.86
1.2
Cl
Cl
726
Cl
16
O
1862
2268
Table 1
Inhibitory activity of R1 triazine analogs
H
R1
N
N
decreased potency with IC50 values >1
summarized in Tables 2 and 3.
lM. The inhibitory data is
O
N
N
S
As shown in Table 4 through 6, the Ar1 equal to CO2H was
required for PDE4B selectivity. Analogs containing a tetrazole,
which has similar acidity to a carboxylic acid, were potent but lost
selectivity for PDE4B, while sulfonamides and imidazolidin-2-ones
were inactive as was the methylester (Table 6). Substitution on the
benzylic position of Ar1 results in decreased potency (compounds
33 and 34).
OH
Cl
#
R1
hPDE4B IC50 (nM)
hPDE4D IC50 (nM)
Ratio D/B IC50
3
4
5
6
Me
i-Pr
Et
1287
1945
383
NT
3580
865
NT
1.8
2.3
To understand the basis for PDE4B selectivity, we pursued co-
crystallization studies with compound 8 and the catalytic domain
of PDE4B containing CR3. We identified ligand-dependent crystal-
lization conditions, obtained a complete diffraction dataset, and
n-Pr
2447
7
251
1489
5.9

T. J. Hagen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4031–4034
4033
Table 4
Table 6
Inhibitory activity of Ar1 modified analogs
Inhibitory activity of Ar1 substitution
H
H
N
R1
N
N
N
N
N
S
N
N
R3
R3
Cl
Cl
#
R3
hPDE4B IC50 (nM) hPDE4D IC50 (nM) Ratio D/B IC50
#
R1
R3
hPDE4B IC50
(nM)
hPDE4D IC50
(nM)
Ratio D/B
IC50
26 CH2CO2H
27 –CO2H
28 CN
781
241
12
681
213
13
0.9
0.9
1.1
0.04
3
17
5
18
19
4
20
21
Me
Me
Et
Et
Et
i-Pr
i-Pr
i-Pr
–CH2CO2H
–CO2H
–CH2CO2H
–CO2H
–F
–CH2CO2H
–CO2H
–F
1287
4048
383
NT
NT
29 CH2CN
165
7
865
251
207
3580
219
886
2.3
1.0
0.15
1.8
1.0
261
H
N
1394
1945
220
30
126
132
1.0
N
N
N
H
8815
N
31
32
33
34
69
402
63
160
96
0.9
N
N
N
O
NH2
S
Table 5
0.4
N
Inhibitory activity of Ar1 substitution
O
H
O
H
N
N
N
1090
1574
0.09
0.4
OH
OH
N
R3
O
600
Ar2
#
Ar2
R3
hPDE4B IC50 hPDE4D IC50 Ratio D/B
(nM)
(nM)
IC50
Cl
Cl
8
–CH2CO2H
–CO2H
237
1181
5.0
22
9
46
20
0.4
7.3
2.0
–CH2CO2H
770
135
5611
268
Cl
23
24
–CO2H
Cl
Cl
135
268
2.0
O
N
H
Cl
O
25
Inactive
Inactive
N
NH
Figure 2. X-ray co-crystal structure of 8 in PDE4B (PDB: 4NW7).
solved the resulting structure by molecular replacement. Analysis
of the refined structure model shows that the central triazine ring
stacks between Phe618 and Ile582 (P clamp) and makes a hydro-
gen bond to Gln615 (Q switch) in the active site demonstrating
how the triazine core can function as a general PDE4 inhibitor
(Fig 2).6 The cyclopropyl group has good shape complimentarily
and fills the Q1 hydrophobic pocket in the active site explaining
why substituents at this position are important for potency
(Table 1). The amine in the Ar1 linker region is in position to make
a hydrogen bond to a conserved water molecule explaining why
modifications at this position also affect potency (Table 3). A sim-
ilar water-bridge was observed in the A-33 PDE4B structure (PDB
ID: 4MYQ).3 Also consistent with the A-33 structure, both Ar1
and Ar2 groups, which we show above modulate PDE4B vesus
PDE4D selectivity in the triazine series (Tables 2, 4 and 5), reach
out of the active site and engage the CR3 regulatory helix. The
chlorophenyl Ar2 group on the triazine is in position to make
hydrophobic interactions with CR3 Phe678 and Leu674, and the
carboxylic acid group of Ar1 hydrogen bonds to multiple water
molecules which engage CR3.
The new triazine co-crystal structure is similar to 4MYQ with
CR3 clearly captured over the active site locking the enzyme in
an inactive ‘closed’ conformation.3 In both cases, the ligand pro-
vides good shape complementarity allowing the CR3 helix to be
stabilized over the active site. The structure activity relationships
(SAR) of a series of triazine PDE4 inhibitors in combination with
co-crystal structure data revealed that the CR3 region can be
exploited to generate PDE4B-selective inhibitors.

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T. J. Hagen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4031–4034
2. Burgin, A. B.; Magnusson, O. T.; Singh, J.; Witte, P.; Staker, B. L.; Bjornsson, J. M.;
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This study was funded by the National Institute of Mental
Health award 1R43MH091791 to M.E.G.
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Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.06.
002.
References and notes
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