Discovery of triazines as potent, selective and orally active PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.05.099

Expanding on HTS hit 4 afforded a series of [1,3,5]triazine derivatives as novel PDE4 inhibitors. The SAR development and optimization process with the emphasis on ligand efficiency and physicochemical properties led to the discovery of compound 44 as a potent, selective and orally active PDE4 inhibitor.

Full text of DOI:10.1016/j.bmcl.2013.05.099

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4308–4314
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of triazines as potent, selective and orally active PDE4
inhibitors
⇑
Rainer Gewald , Christian Grunwald, Ute Egerland
BioCrea GmbH, Meissner Strasse 191, 01445 Radebeul, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Expanding on HTS hit 4 afforded a series of [1,3,5]triazine derivatives as novel PDE4 inhibitors. The SAR
development and optimization process with the emphasis on ligand efficiency and physicochemical
properties led to the discovery of compound 44 as a potent, selective and orally active PDE4 inhibitor.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 17 April 2013
Revised 27 May 2013
Accepted 31 May 2013
Available online 10 June 2013
Keywords:
PDE4 inhibitor
Phosphodiesterase
Triazine
Neutrophilia
COPD
Hydrolyzing the key secondary messengers adenosine and gua-
nosine 30,50-cyclic monophosphates (cAMP and cGMP) into their
corresponding 50-monophosphate nucleotides and, thus, decreas-
ing concentrations of cAMP and cGMP, phosphodiesterase en-
zymes (PDEs) play an important role in various biological
processes.1 Based on primary structure, substrate specificity and
sensitivity to cofactors or inhibitory drugs, eleven isoenzymes of
mammalian cyclic nucleotide phosphodiesterase have been identi-
fied so far.2 Among them, PDE4 is responsible for the degradation
of cAMP in many cell types and has been proposed as an attractive
target in various diseases including asthma and chronic obstructive
pulmonary disease (COPD).3
First generation PDE4 inhibitors represented by rolipram 1
(Fig. 1) demonstrated severe side effects of nausea and emesis at
effective anti-inflammatory doses. Whilst second generation
PDE4 inhibitors such as cilomilast 2 (Fig. 1) and roflumilast 3
(Fig. 1) have shown improved side effect profiles in clinical trials,
the maximum dose is still limited by adverse events.4 After numer-
ous compounds not having advanced past the clinic, roflumilast 3
was recently launched for the treatment of COPD.5 The side effect
profiles of many PDE4 inhibitors structurally related to compounds
1-3 have triggered interest in the development of novel, sometimes
sub-type selective chemotypes, that may exhibit an improved ther-
apeutic window, lacking the dialkoxyphenyl group and also avoid-
ing the aminodichloropyridine moiety that has recently come
under scrutiny by public health authorities because of its carcino-
genic metabolites (ADCP-N-oxide and its epoxide).4,6 As in many
instances an increased therapeutic index in animal models was
achieved only at the expense of accepting unfavorable physico-
chemical properties for the inhibitor molecule, our aim was there-
fore to control molecular weight and focus on ligand efficiency
during the optimization process.
In this context, screening of our in-house compound library re-
sulted in the identification of [1,3,5]triazine derivative 4 exhibiting
inhibitory activity in the sub-micromolar range (Fig. 2). PDE4
inhibitors based on the s-triazine scaffold, exemplified by 5
(Fig. 2) with an IC50 value of 140 nM, have been reported before
but were not tested in vivo.7 This communication describes our ef-
forts to optimize hit compound 4 into a potent, selective and orally
active PDE4 inhibitor.
The synthesis of the triazine derivatives, outlined in Scheme 1,
proceeded via sequential substitution of cyanuric chloride under
standard reaction conditions. Starting with the bulkier component
the two amino groups were introduced via a one-pot method with-
out isolating the intermediate. Replacement of the remaining
chloro atom at elevated temperatures led to the final products.
Some
a-aminonitriles used as building blocks were not commer-
cially available and prepared by treating the corresponding ke-
tones in the presence of ammonia or methylamine with
potassium cyanide.8 A different synthetic route was applied to ob-
tain 21 following an established procedure.9
The compounds described in this paper were assessed against
PDE4A, PDE4B and PDE4D isoforms.10 As no selectivity was ob-
served, only the PDE4A inhibitory activities will be presented
herein.
⇑
Corresponding author. Tel.: +49 351 40431552.
E-mail address: rainer.gewald@telecolumbus.net (R. Gewald).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.099

R. Gewald et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4308–4314
4309
H
N
Cl
O
N
COOH
O
O
O
O
O
N
O
O
H
Cl
N
F
F
1
2
3
Figure 1. Structures of PDE4 inhibitors rolipram 1, cilomilast 2, and roflumilast 3.
At the outset of our SAR studies we focused on modifying the
tert-butyl group (Table 1). Increased steric bulkiness by extending
two of the methyl groups (6) or introducing a cyclohexyl ring (7)
led to an improvement in potency. Interestingly, the attachment
of a cyano function (8-10) yielded the first double digit nanomolar
inhibitors in the series. A smaller or larger size of the cycle and fur-
ther prolongation of the ethyl chains showed barely any influence
on inhibitory activity (11-13). The fourfold less active ethinyl
derivative 14 indicated a weak interaction of the cyano nitrogen
with the enzyme serving as an hydrogen bond acceptor.
substituent (23) was less pronounced, a ninefold increase in po-
tency was detected for methylthio derivative 24. But unfortu-
nately, despite achieving single digit nanomolar PDE4 inhibition,
Table 1
Inhibitory activity of triazine derivatives 4 and 6–14
Cl
N
N
Next we moved on to explore the structure-activity relationship
of the methylamino substituent (Table 2). While a sharp drop in
potency was caused by omitting the methyl group (15), adding size
at this position (16-18) was not well tolerated either. In addition,
the two hydrogen bond donor elements were identified as crucial
structural features required for potent inhibition (19 and 20).
Based on our initial lead 10 the SAR studies were finally cen-
tered on the chloro position (Table 3). At this point we also started
to pay attention to issues encountered during the course of our
investigations like a lack of selectivity over PDE7 and weak stabil-
ity in rat liver microsomes.
R
N
H
N
N
H
Compd
R
PDE4A410 IC50 (nM)
245
4
*
6
7
156
104
*
Small carbon linked groups slightly improved (21) or main-
tained (22) inhibitory activity. Although the impact of a methoxy
*
*
8
9
160
87
Cl
N
N
N
N
N
O
N
H
N
N
H
N
H
N
N
S
*
O
N
4
5
Figure 2. Structures of hit 4 and triazine 5.
10
11
40
54
*
*
N
N
R4
N
Cl
b
d
N
N
N
N
R3
R
R3
R
N
N
N
Cl
N
N
N
a
c
R2
R1
R2
R1
N
N
4, 6-20
22-47
Cl
Cl
12
44
*
*
*
N
N
N
N
N
Cl
N
N
H
N
H
N
N
H
N
N
13
14
60
21
N
Scheme 1. Reagents and conditions: (a) (i) RR1NH, THF, H2O, NaOH, 0 °C, 1 h, (ii)
R2R3NH, THF, H2O, NaOH, 10–15 °C, 1 h, 52–88% yield; (b) For 22–24: R4Na, THF,
50 °C, 6 h, 33–61% yield; For 25–30: R4H, THF, pyridine, reflux, 2 h, 50–67% yield;
For 31–47: R4H, DMF, pyridine, 35–62% yield; (c) (i) c-PrMgBr, THF, toluene, 10–
15 °C, 1 h, (ii) 1 N HCl, 0 °C, 1 h, (iii) 1-aminocyclohexylcarbonitrile, CH2Cl2, H2O,
K2CO3, 0 °C, 1 h, 25% yield; (d) methylamine, THF, reflux, 4 h, 74% yield.
149

4310
R. Gewald et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4308–4314
Table 2
So far, all derivatives were rapidly metabolized in rat liver
Inhibitory activity of triazine derivatives 10 and 15–20
microsomes. Discovering reasonable potency for compound 30
bearing a pyrrolidinyl residue prompted us to investigate several
nitrogen linked azoles (31-36). Among the two strong inhibitors
with sufficient metabolic stability 31 and 34 the latter compound
exhibited all features of an advanced lead with an IC50 value of
1.7 nM, more than 1000-fold selectivity over PDE7 and no signifi-
cant hERG or CYP inhibition.
Cl
N
N
R3
N
N
N
R2
R1
N
With metabolite ID data of 34 indicating the involvement of the
cylohexyl moiety as a metabolic liability, we finally revisited
opportunities from our first SAR series while also slightly expand-
ing on the structural scope (Table 4). This resulted in three com-
pounds (38-40) reaching subnanomolar activity levels. Stepwise
reduction of the chain lengths (42-45) led through an affinity max-
imum for derivative 44.
In general, triazines bearing an alicyclic moiety (34, 37-41) be-
haved metabolically less stable in rat liver microsomes than their
open-chain counterparts (42-47). Although 46 exhibited the high-
est metabolic stability compound 44 was selected for further
in vivo profiling because of its superior aqueous solubility.
Compd
R1
R2
R3
PDE4A410 IC50 (nM)
10
15
16
17
18
19
20
H
H
H
H
H
H
Me
H
H
H
H
H
Me
H
Et
MeO
c-Pr
Me
H
40
1040
232
165
126
668
563
Me
H
selectivity over PDE7 collapsed. Advancing from primary via sec-
ondary to tertiary amino groups a clear trend towards an increas-
ing activity was established (25-29).
Table 3
Inhibitory activity and metabolic stability in rat liver microsomes of triazine derivatives 10 and 21–36
R4
N
N
N
H
N
N
H
N
Compd
R4
PDE4A410 IC50 (nM)
PDE7A110 IC50 (nM)
Intrinsic clearancea (mL/min/kg)
10
21
22
23
24
25
26
27
28
29
Cl
c-Pr
–CN
–OMe
–SMe
40
19
38
19
4.4
>1000
153
59
56
18
1070
539
>1000
521
46
ND
>1000
>5000
>5000
>5000
867
276
122
196
579
ND
210
ND
ND
–NH2
–NHMe
–NHc–pentyl
–NHPh
–NMe2
276
30
31
N
38
>5000
1050
>276
52
*
N
6.6
N
*
N
32
12
>1000
ND
N
*
N
N
33
34
3.6
1.7
>5000
>5000
103
52
*
N
N
N
*
N
N
N
35
1.0
>5000
>276
*

R. Gewald et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4308–4314
4311
Table 3 (continued)
Compd
R4
PDE4A410 IC50 (nM)
PDE7A110 IC50 (nM)
Intrinsic clearancea (mL/min/kg)
N
N
36
4.9
>5000
210
N
N
*
a
Incubation with rat liver microsomes at 37 °C.
Table 4
Inhibitory activity, metabolic stability in rat liver microsomes and aqueous solubility of triazine derivatives 34 and 37–47
N
N
N
N
N
R
N
H
N
N
H
Compd
R
PDE4A410 IC50 (nM)
Intrinsic clearancea (mL/min/kg)
Aqueous solubility @pH7.4 (mg/L)
34
1.7
52
11
*
N
N
37
38
1.4
43
165
*
0.42
276
9.0
*
*
N
39b
0.15
0.20
86
96
25
N
40c
ND
*
*
*
*
*
N
N
N
N
N
41c
42
1.6
37
32
29
26
8.0
6.5
ND
43d
1.1
76
44
0.81
165
(continued on next page)

4312
R. Gewald et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4308–4314
Table 4 (continued)
Compd
R
PDE4A410 IC50 (nM)
Intrinsic clearancea (mL/min/kg)
Aqueous solubility @pH7.4 (mg/L)
50
45d
46
2.4
32
*
N
N
0.89
2.7
3.7
22
*
*
47
22
ND
a
Incubation with rat liver microsomes at 37 °C.
Diastereomeric mixture.
cis/trans Isomeric mixture.
Racemate.
b
c
d
Tyr233
Tyr403
Tyr329
Tyr159
Asn395
Asn321
Thr407
Thr333
His234
His160
3 02
Ile410
Ile336
2 80
2 89
2 91
Hoh4
Asp392
2 88
Hoh46
3 29
Gln443
Gln369
3 33
3 20
2 88
3 23
3 02
Leu393
Ser442
Met347
Phe414
Phe340
Phe446
Phe372
Met431
Met357
Figure 3. Ligplot+ representation of interactions of compound 44 with amino acid residues of PDB4B binding pocket (PDB ID 4KP6: colored foreground) compared with (R)-
rolipram (R)-1 (PDE4D, chain A, PDB ID 1q9m: grey background). Equivalent amino acid residues are arranged in similar positions and encircled. Hydrogen bonds are
symbolized by green dashed lines including their distance in Å. Non-ligand residues involved in hydrophobic contacts and corresponding ligand atoms are marked with red
spikes. Cyan balls symbolize water molecules.
To prove our conclusions derived from SAR and support further
syntheses, compound 44 was crystallized in complex with PDE4B
(core catalytic domain construct, PDB ID 4KP6).Interactions be-
tween enzyme and ligand were analyzed by means of LigPlot+11

R. Gewald et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4308–4314
4313
Table 5
Biological and physicochemical comparison of PDE4 inhibitors 1–3 and 44
Compd PDE4A IC50
(nM)
Neutrophilia inhibition in ferret (po) a (inhibition% First emetic dose in ferret
@dose in mg/kg)
MW Aqueous solubility
@pH7.4 (mg/l)
Protein
PSA
(po)b (mg/kg)
bindingc (%)
1d
2
3
95
62
0.35
0.81
NTe
0.3f
16a
1
275
343
403
287
171
17
1.3
165
58
91
97
51
57
81
64
35 @106a
65 @0.01
66 @0.1
33 @0.01
44
3
115
a
b
c
d
e
f
The percentage inhibition for a change in the number of neutrophils in BAL-fluid after LPS inhalation in ferrets.
Occurrence of vomiting episode after administration for 6 h.
Human serum albumin binding.
Racemate.
NT: not tested.
iv Administration.
in comparison to 1 could possibly explain the superiority of 44
describing a new path forward in the development of a new gener-
ation of PDE4 inhibitors.
In summary, a novel class of potent, selective, highly ligand effi-
cient, synthetically easily accessible and orally active PDE4 inhibi-
tors for the treatment of COPD has been discovered. Thus,
compound 44 was selected for further in vivo safety studies in
animals.
PDE4A: IC50 = 0.81 nM; LE = 0.61; LLE = 8.6
PDE4B: IC50 = 0.87 nM;
PDE4D: IC50 = 1.18 nM
N
N
N
N
N
Non-PDE4 PDEs: IC50 > 1000 nM
N
Rat PK (3 mg/kg): F = 41 % (po)
Cl = 3.3 mL/min/kg (iv)
T
1/2 = 1.7 h (iv)
N
H
N
H
Cmax = 669 ng/ml (po)
Vd = 0.5 l/kg
N
Microsomal clearance: 26 mL/min/kg (rat)
< 1 mL/min/kg (dog, human)
hERG (binding): 6 % inhibition @ 10 µM
Inhibition of TNF-a production:
human PBMC: IC50 = 22 nM
MW = 287
PSA = 115
LogD = 1.5
Acknowledgments
HWB: IC50 = 55 nM
Aqu. solubility (pH7.4) = 165 mg/l
The authors thank Drs. Antje Gasparic and Hildegard Kuss for
pharmacokinetic and pharmacological evaluation of compound
44. The project was supported by the European Fund for Regio-
nal Development (EFRE) and the Free State of Saxony (SAB
8093).
Figure 4. In vitro and rat PK profile of 44.
as depicted in Figure 3. The interaction pattern includes features
well known from ligands of various PDE subtypes12 including
hydrophobic interactions with Ile410, Phe414, and Phe446 (known
as hydrophobic clamp13) and a hydrogen bond towards Gln443, a
residue strictly conserved in all phosphodiesterases. Apart from
additional hydrophobic contacts there are direct and water-medi-
ated hydrogen bonds with Asn395, Tyr233, and Asp392. These
hydrogen bonds formed by one of the triazine ring nitrogens and
the nitrogen atoms of two adjacent amino groups enhance the
affinity in accordance with SAR findings. This interpretation was
confirmed by comparison with (R)-rolipram (R)-1 (Fig. 3, grey
background drawing). This PDE4 inhibitor lacks hydrogen bond
interactions with the exception of two hydrogen bonds to
PDE4D/Gln369 (analog of PDE4B/Gln443) and displays an about
90-fold weaker affinity towards PDE4A. In contrast to our initial
perception, the cyano group is not involved in polar interactions
with amino acid residues of the binding pocket but sits at the en-
trance of the pocket and seems to mediate between the more
hydrophobic inside and the polar solvent. The same applies to
the triazole substituent in addition to its interaction with Gln443.
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CYP1A1/2, 2A6, 2C9/19, 2D6 and 3A4B/D/Q and was found to be
highly selective over all other PDEs.14 Preliminary pharmacokinetic
testing demonstrated oral bioavailability in rats with acceptable
clearance and half-life (Fig. 4).
Inhibitory activity of 41% against LPS-induced neutrophil accu-
mulation in lung was observed at an oral dose of 0.1 mg/kg in rats
(data not shown). The therapeutic index of 44 was assessed by
applying this COPD animal model to ferrets while determining
the emetic adverse effect in the same species (Table 5). With a
safety margin close to that of 3 but rolipram-like physicochemical
properties, the triazine derivative 44 avoids joining the general
trend hampering the transition from first (1) to second generation
(2 and 3) PDE4 inhibitors characterized by increasing molecular
weight and plasma protein binding combined with lower aqueous
solubility. Higher PSA value and number of hydrogen bond donors

4314
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