Exploration and optimization of substituted triazolothiadiazines and triazolopyridazines as PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.01.057

An expansion of structure-activity studies on a series of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine PDE4 inhibitors and the introduction of a related [1,2,4]triazolo[4,3-b]pyridazine based inhibitor of PDE4 is presented. The development of SAR included strategic incorporation of known substituents on the critical catachol diether moiety of the 6-phenyl appendage on each heterocyclic core. From these studies, (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (10) and (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-[1,2,4]triazolo[4,3-b]pyridazine (18) were identified as highly potent PDE4A inhibitors. Each of these analogues was submitted across a panel of 21 PDE family members and was shown to be highly selective for PDE4 isoforms (PDE4A, PDE4B, PDE4C, PDE4D). Both 10 and 18 were then evaluated in divergent cell-based assays to assess their relevant use as probes of PDE4 activity. Finally, docking studies with selective ligands (including 10 and 18) were undertaken to better understand this chemotypes ability to bind and inhibit PDE4 selectively.

Full text of DOI:10.1016/j.bmcl.2009.01.057

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3686–3692
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration and optimization of substituted triazolothiadiazines
and triazolopyridazines as PDE4 inhibitors
Amanda P. Skoumbourdis a, Christopher A. LeClair a, Eduard Stefan b, , Adrian G. Turjanski c,
William Maguire a, Steven A. Titus a, Ruili Huang a, Douglas S. Auld a, James Inglese a, Christopher P. Austin a,
Stephen W. Michnick b, Menghang Xia a, Craig J. Thomas a,
*
a NIH Chemical Genomics Center, National Human Genome Research Institute, NIH 9800 Medical Center Drive, MSC 3370 Bethesda, MD 20892-3370, USA
b Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7
c Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH Bethesda, MD 20892-3370, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An expansion of structure–activity studies on a series of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thi-
adiazine PDE4 inhibitors and the introduction of a related [1,2,4]triazolo[4,3-b]pyridazine based inhibitor
of PDE4 is presented. The development of SAR included strategic incorporation of known substituents
on the critical catachol diether moiety of the 6-phenyl appendage on each heterocyclic core. From
these studies, (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (10) and (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahy-
drofuran-3-yloxy)phenyl)-[1,2,4]triazolo[4,3-b]pyridazine (18) were identified as highly potent PDE4A
inhibitors. Each of these analogues was submitted across a panel of 21 PDE family members and was
shown to be highly selective for PDE4 isoforms (PDE4A, PDE4B, PDE4C, PDE4D). Both 10 and 18 were then
evaluated in divergent cell-based assays to assess their relevant use as probes of PDE4 activity. Finally,
docking studies with selective ligands (including 10 and 18) were undertaken to better understand this
chemotypes ability to bind and inhibit PDE4 selectively.
Received 1 December 2008
Revised 16 January 2009
Accepted 20 January 2009
Available online 23 January 2009
Keywords:
PDE4
PDE4 inhibitor
Triazolothiadiazines
Triazolopyridazines
Asthma
COPD
Ó 2009 Published by Elsevier Ltd.
The second messenger cyclic 3,5-adenosine monophosphate
(cAMP) is a key regulator of numerous signaling cascades.1,2 As
such, the production of cAMP by adenylate cyclase (AC) and the
degradation of cAMP by phosphodiesterases (PDEs) are highly reg-
ulated. There are 11 primary families of PDEs (designated PDE1–
PDE11) expressed throughout the human body, with several fami-
lies having multiple isoforms. Much effort has been devoted to
understanding the physiological role of each PDE isoform, and
the pharmacological inhibition of select PDE isoforms has proven
therapeutically beneficial for several indications.2–4 The PDE4 fam-
ily is comprised of 4 primary gene products (PDE4A, PDE4B, PDE4C,
PDE4D) and is highly expressed in neutrophils and monocytes, CNS
tissue and smooth muscles of the lung.2–4 Not surprisingly, the
clinical utility of PDE4 inhibitors has focused on asthma, chronic
obstructive pulmonary disease (COPD), memory enhancement
and as a general modulator of inflammation.4
in clinical settings.5 Reported molecules include rolipram (1), roflu-
milast (2) and cilomilast (3). It is notable that each of these small
molecules contains a common catechol diether motif. Crystallo-
graphic studies of PDE4 bound to several of these derivatives have
shown the catechol diether forming a key hydrogen bond with a
conserved glutamine residue (Gln443 in PDE4B and Gln369 in
PDE4D).4,6,7 The choice of alkyl, cycloalkyl and heterocyclic ether
moieties is presumably driven by improvements to the potency
of each molecule and selected DMPK properties. It is noteworthy
that methoxy, cyclopentyloxy, cyclopropylmethoxy and 2-difluo-
romethoxy are repeatedly incorporated into known PDE4 inhibitor
scaffolds. Another common ether substituent, an O-3-tetrahydrof-
uranyl such as in 4, is found in numerous published and patented
chemotypes.8–10 The frequency with which these moieties are
incorporated on small molecules intended to down-regulate
PDE4 is compelling. However, there are few reports that directly
compare these substituents on the same core chemotype to gain
an appreciation of each moieties potential and limitations.
At present, there are numerous chemotypes known to inhibit
PDE4 and several of these inhibitors are currently being evaluated
Recently, we introduced a series of substituted 7H-[1,2,4]triaz-
olo[3,4-b][1,3,4]thiadiazines as potent PDE4 inhibitors including
5.11 Our initial 77 member matrix library revealed that the 3,4-
catachol diether motif on the 6-phenyl appendage on the triazolo-
thiadiazine ring system played a key role in defining this chemo-
* Corresponding author. Tel.: +1 301 217 4079; fax: +1 301 217 5736.
E-mail address: craigt@nhgir.nih.gov (C.J. Thomas).
Present address: Institute of Biochemistry and Center for Molecular Biosciences,
University of Innsbruck, A-6020 Innsbruck, Austria.
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.01.057

A. P. Skoumbourdis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3686–3692
3687
F
MeO
O
cyclopentyloxy, cyclopropylmethoxy, 2-difluoromethoxy and O-
3-tetrahydrofuranyl moieties on to the 2-bromo-1-phenyletha-
none precursor, we relied upon 1-(3-hydroxy-4-methoxy-
phenyl)ethanone as an orthogonally protected starting reagent.
For the cyclopentyloxy and cyclopropylmethoxy substituents, we
utilized a nucleophilic displacement of the corresponding alkyl
bromides to ultimately provide the substitution pattern found in
derivatives 6 and 7 (Table 1). Reaction of 1-(3-(cyclopentyloxy)-
4-methoxyphenyl)ethanone with dodecane-1-thiol in sodium
methoxide/DMF at 100 °C provided demethylation in a mild man-
ner.12 Treatment of the resulting 1-(3-(cyclopentyloxy)-4-
hydroxyphenyl)ethanone with sodium 2-chloro-2,2-difluoroace-
tate in DMF at 100 °C afforded the incorporation of the 2-difluoro-
methoxy functionality on the C4 position of the catachol moiety
(found in derivative 8).13 Mitsonobu conditions were utilized to
condense tetrahydrofuran-3-ol (both racemic and R) with 1-(3-hy-
droxy-4-methoxyphenyl)ethanone to provide analogues 9 and 10.
The synthesis of [1,2,4]triazolo[4,3-b]pyridazines was based
upon the precedented works of Tišler and coworkers, Greenblatt
and coworkers and Street and coworkers.14–16 The general method
is outlined in Scheme 1 and begins with the coupling of commer-
cially available 2,5-dimethoxybenzoic acid (11) and 3-chloro-6-
hydrazinylpyridazine (12) to provide N0-(6-chloropyridazin-3-yl)-
2,5-dimethoxybenzohydrazide (13) in good yields. Direct treat-
ment of 13 with POCl3 at elevated temperature afforded the cycli-
zation to core [1,2,4]triazolo[4,3-b]pyridazine ring system
(analogue 14) and provided a common analogue for entry into
the convergent syntheses of multiple products via end-stage Suzu-
ki–Miyaura couplings. Here, we were primarily interested in exam-
ining the biochemical viability of this alternate heterocycle and
limited our explorations to the appropriately substituted O-3-tet-
rahydrofuranyl derivatives. Boronic acids 15 and 16 were synthe-
sized independently utilizing the aforementioned Mitsonobu
protocols and displacement of an aryl bromide with boronic acid.
Following purification of 15 and 16, standard Suzuki–Miyaura con-
ditions with microwave irradiation produced the appropriately
substituted [1,2,4]triazolo[4,3-b]pyridazines 17 and 18 in good
yields.
F
OCl
N
CN
O
O
O
N
MeO
O
NH
Cl
CO2H
1
2
3
(rolipram)
(roflumilast)
(cilomilast)
MeO
N
N
OMe
O
MeO
OMe
N
S
5
N
O
N
N
OMe
MeO
4
Figure 1. Structures of several known PDE4 inhibitors.
type’s pharmacophore. Here, we systematically incorporate the
methoxy, cyclopentyloxy, cyclopropylmethoxy, 2-difluoromethoxy
and O-3-tetrahydrofuranyl moieties onto the 6-phenyl-triazolothi-
adiazine core in an attempt to improve potency, but also better
understand these frequently utilized groups. The DMPK profiles
of a representative triazolothiadiazine showed low microsomal
stability (rat) [high intrinsic clearance (Clint >300 lL/min/mg pro-
tein) and short half-life (t1/2 ꢀ 3 min)]. The removal/alteration of
aromatic methoxy groups has been shown to positively affect
microsomal stability in several instances given the propensity for
demethylation and glucuronidation of this functional group. We
were further interested in transposing the substitution patterns
found on the triazolothiadiazine ring to the related triazolopyrid-
azine ring system in order to remove the lone sulfur atom and
eliminate S-oxidation as an additional mechanism of metabolism
and clearance (Fig. 1).
The synthesis of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thi-
adiazines was accomplished via previously reported methods (for
full synthetic details associated with the synthesis of substituted
7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines see the Supporting
information).11 The key condensation reaction between appropri-
ately substituted 2-bromo-1-phenylethanone (ultimately the phe-
nyl ring at the C6 position of the heterocycle) and appropriately
PDE4A inhibition profile. With several new analogues now in
hand, we evaluated their inhibitory potency against PDE4A via a
previously reported, purified enzyme fluorescence polarization as-
say (IMAP; Molecular Devices, CA).11 The results for the newly syn-
thesized 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 5–10 and the
novel [1,2,4]triazolo[4,3-b]pyridazines 17 and 18 are shown in Ta-
ble 1. For 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 5–10, each
substituted
4-amino-3-phenyl-1H-1,2,4-triazole-5(4H)-thione
(ultimately the phenyl ring at the C3 position of the heterocycle)
was accomplished in ethanol at elevated temperatures. Utilizing
the known SAR from our previously reported matrix library, we
opted to maintain the 2,5-dimethoxy substitution pattern on the
3-phenyl appendage on the heterocyclic core. To incorporate the
Table 1
PDE4A inhibition by compounds 5–10, 17 and 18.*
Analogue #
R1
R2
PDE4A IC50 (nM)
5
6
7
8
–OCH3
–OCypent
–OCH2Cyprop
–OCH2Cyprop
–OCH3
–OCH3
–OCH3
–OCHF2
6.7 0.4
13 0.8
6.1 0.9
11 0.7
MeO
R1
R2
OMe
N
N
N
N
5-10
S
9
–O(3-THF)[rac]
–O(3-THF)[R]
–O(3-THF)[S]
–O(3-THF)[R]
–OCH3
–OCH3
–OCH3
–OCH3
3.4 0.4
3.0 0.2
7.3 3.8
1.5 0.7
MeO
R1
10
17
18
R2
OMe
N
N
N
N
17, 18
*
Data represents the results from three separate experiments. Definitions: OCH3 = methoxy, OCypent = cyclopentyloxy, OCH2Cyprop = cyclopropylmethoxy, OCHF2 = 2-
difluoromethoxy, O(3-THF) = O-3-tetrahydrofuranyl [racemic, RorS enantiomers].

3688
A. P. Skoumbourdis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3686–3692
Cl
OMe O
N
N
Cl
N
MeO
H
N
OH
ii
i
N
N
H
OMe
NH
OMe
H2N
O
13
11
12
O
O
O
MeO
MeO
O
MeO
B(OH)2
15
OMe
N
N
iii
MeO
N
N
17
OMe
Cl
N
O
N
N
O
O
MeO
N
MeO
O
MeO
14
B(OH)2
16
OMe
N
N
iii
N
N
18
Scheme 1. Reagents and conditions: (i) CDI, DMF, rt 2 h; (ii) POCl3, 105 oC, 2 h; (iii) 15 or 16, Pd(PPh3)4, 2 M aq. Na2CO3, DME, lwave, 90 °C 30 min.
substitution pattern yielded a molecule with potency in the low
nanomolar range. The enantiomerically pure O-(3-THF)[R] substi-
tution of 10 showed the best potency with an IC50 value of
3.0 nM. As a result, the enantiomerically pure O-(3-THF)[R] and
O-(3-THF)[S] substitutions were incorporated onto the [1,2,4]triaz-
olo[4,3-b]pyridazine core structure and the resulting constructs
were found to have excellent potencies for PDE4A inhibition (IC50
value of 7.3 3.8 nM for 17 and 1.5 0.7 nM for 18). Several ana-
logues were also explored with varying substitutions on the phenyl
ring attached to the C3 position of the 1,2,4-triazole ring system.
Substitutions included methoxy, fluoro, chloro and trifluoromethyl
groups on the ortho, meta and para positions of the phenyl ring (for
synthetic details and characterization of these analogues see Sup-
porting Information section). Analysis of these analogues con-
firmed that various substitutions at one of the ortho positions
were necessary to maintain potent PDE4A inhibition (see Support-
ing information). Additional substitutions have been shown to be
tolerated without effect on the inhibition of PDE4A.
Selectivity panel of PDE isoforms. Having arrived at several com-
pounds with good potency profiles and divergent core heterocy-
cles, it was of interest to confirm the selectivity of these agents
against a panel of PDE isoforms. BPS Bioscience (San Diego, CA)17
has available a panel of 21 PDE isoforms from all 11 primary PDE
families except PDE6 available for activity profiling. We submitted
5, 10, 18 and 1 for analysis across this panel and the resulting IC50
determinations are shown in Table 2.
However, not all active small molecules found in such screens find
utility in cell-based assays for a myriad of reasons. To evaluate
whether the substantial in vitro inhibition of PDE4 by 10 and 18
could be replicated in living cells we pursued two divergent, cell-
based assays of PDE4 activity.
Cyclic-nucleotide gated ion channel cell-based assay. The first cell-
based analysis of PDE4 activity utilized a recently reported assay
based on the coupling of a constitutively activated G-protein cou-
pled receptor (the thyroid-stimulating hormone receptor (TSHR))
and cyclic-nucleotide gated (CNG) ion channel coexpressed in
HEK293 cells.18 The read-out for this assay is based on measure-
ment of membrane electrical potential by a potential-sensitive
fluorophore (ACTOneTM dye kit). Inhibitors of PDE4 will interfere
with the native enzymatic conversion of cAMP to AMP resulting
in increased intracellular levels of the cyclic nucleotide due to con-
stitutive activity of the GPCR. In response to increased amounts of
cAMP, the CNG ion channel opens resulting in membrane polariza-
tion. The dye reacts to this alteration in membrane polarity with an
increase in fluorescence detectable by fluorescence spectroscopy of
whole cells read on a fluorescence microtitre plate reader.
Compounds 5, 10 and 18 were used in this analysis as along
with the common PDE4 inhibitor 1, and the results are shown in
Figure 2. In this assay, 1 was noted to produce an effective response
(EC50) (registered as % activity) of 131.5 nM. In comparison, the
triazolothiadiazine based inhibitors were found to be more potent
in this cell-based assay with 5 and 10 registering an EC50 value of
18.7 and 2.3 nM, respectively. The EC50 of the lone triazolopyrid-
azine 18 was 34.2 nM.
Protein-fragment complementation (PCA) cell-based assay. PCAs
take advantage of the ability of well-engineered protein fragments
to form a functional monomer with measurable enzymatic activity
when brought into suitable proximity by interacting proteins to
which the fragments are fused.20,21 For our purposes, we utilized
a previously described PCA based on the reporter enzyme Renilla
reniformis luciferase (Rluc) N- and C terminal fragments of Rluc
fused to the catalytic subunits (Cat) and inhibiting regulatory sub-
units (Reg) of protein kinase A (PKA).19 The signaling cascades ini-
tiated by GPCR activation is mediated by cAMP production and
activation of numerous protein kinases.1,20 Negative regulation of
It is apparent that both 10 and 18 represent novel inhibitors of
five isoforms of PDE4 with sub-nanomolar potencies found for
PDE4A1A. The modest activities found for PDE3B and PDE10A1
represent a sizeable enough difference in potency that cellular
phenotypes will not be complicated by dual activities. Interest-
ingly, PDE10 is considered to be an important modulator of cGMP
regulation in the brain. To date few reports of potent and selective
PDE10 inhibitors have surfaced. Analogues 10 and 18 may repre-
sent good starting points for optimizing novel compounds as po-
tent inhibitors of PDE10.
The identification of novel enzyme inhibitors via screening in
purified enzyme assays provides an important means for the iden-
tification of small molecules with a known mechanism of action.

A. P. Skoumbourdis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3686–3692
3689
Table 2
PDE isoform selectivity data for 1, 5, 10 and 18
PDE isoform*
1 IC50/% inh.
5 IC50/%inh.
10 IC50/%inh.
18 IC50/%inh.
PDE1A
PDE 1B
PDE1C
Inactive
Inactive
Inactive
Inactive
Inactive
26%
36%
52%
49%
32%
56%
74%
O
O
O
NH
1
PDE2A
PDE3A
PDE3B
PDE4A1A
PDE4B1
Inactive
Inactive
Inactive
102 nM
901 nM
41%
1.7 lM
720 nM
12.9 nM
48.2 nM
68%
56%
54%
54%
2.3 lM
0.6 nM
4.1 nM
MeO
OMe
4.6
l
M
MeO
0.26 nM
2.3 nM
OMe
N
S
N
N
N
N
N
5
PDE4B2
PDE4C1
PDE4D2
PDE5A1
PDE7A
534 nM
40%
403 nM
Inactive
Inactive
37.2 nM
452 nM
49.2 nM
60%
1.6 nM
46 nM
1.9 nM
58%
2.9 nM
106 nM
2.1 nM
51%
O
MeO
O
MeO
73%
48%
59%
OMe
N
N
N
S
10
PDE7B
Inactive
Inactive
Inactive
Inactive
Inactive
33%
57%
Inactive
823 nM
Inactive
43%
35%
O
PDE8A1
PDE9A2
PDE10A1
PDE11A4
Inactive
Inactive
632 nM
Inactive
Inactive
Inactive
388 nM
Inactive
MeO
O
MeO
OMe
N
N
N
18
*
Data generated by BPS Biosciences (CA) [http://www.bpsbioscience.com] and represents either the IC50 value or the % inhibition at 10
upon a fluorescence polarization assay using carboxyfluorescein (FAM)-cAMP as the binding probe.
l
M of compound. Assays are based
these events is the sole domain of the phosphodiesterase class of
enzymes.2 One ubiquitous pathway is activated when cAMP trig-
gers the disassociation of the PKA catalytic and regulatory sub-
units, which in turn, enables numerous signaling events. In the
Rluc PCA PKA reporter, the regulatory subunit II beta cDNA is fused
through a sequence coding for a flexible polypeptide linker of 10
amino acids (Gly.Gly.Gly.Gly.Ser)2 to the N-terminal fragment (Rluc
F[1])[amino acids 1–110 of Rluc] and the cDNA of the PKA catalytic
MeO
OMe
O
MeO
O
OMe
N
O
N
N
NH
N
S
1
5
1
5
Log [ ], M
Log [ ], M
O
O
MeO
MeO
O
O
MeO
MeO
OMe
OMe
N
N
N
N
N
N
N
N
S
18
10
Log [10], M
Log [18], M
Figure 2. Cell-based analysis of novel PDE4 inhibitors. Effect of PDE4 inhibition by 1, 5, 10 and 18 in a cell-based cyclic nucleotide-gated cation channel biosensor assay.
Specific EC50 values are as follows: (A) EC50 for 1 = 131.5 nM. (B) EC50 for 5 = 18.7 nM. (C) EC50 for 10 = 2.3 nM. (D) EC50 for 18 = 34.2 nM. The data represents the results from
four separate experiments.

3690
A. P. Skoumbourdis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3686–3692
subunit alpha is fused through the same flexible linker to the C-ter-
minal fragment (Rluc F[2])[amino acids 111–311 of Rluc]. The
resulting constructs Reg-F[1] and Cat-F[2] can reconstitute enzy-
matic activity of Rluc in the absence of cAMP. It has been recently
demonstrated that this assay could be used to detect the effects of
PDE4 inhibition on PKA activation downstream of basal b-2 adren-
ergic receptor (b2AR) activities.19
Here, we evaluated the effects of 1, 10 and 18 in HEK293 cells
stably expressing the b2AR and transiently transfected with the re-
quired PKA-Rluc fragments [Reg-F[1] and CatF-[2]]. It was con-
firmed that isoproterenol (19) activation of the b2AR is able to
reduce luminescence (indicating dissociation of the Rluc biosensor
complex and consequent activation of PKA catalytic activity)
(Fig. 3A). Further, pretreatment with the selective b2AR inverse
agonist ICI 118551 (20; decrease of basal b2AR activity) was capa-
ble of preventing the effects of 19 as was previously shown.19
These important controls confirm that alterations of the lumines-
cence signal are primarily mediated through the actions of the
b2AR signaling to PKA. Further, the effect of 1 confirms the respon-
siveness of the assay to PDE4 inhibition. Treatment with 10 and 18
PDE4B) that serves as an important binding residue for the purine
motif of cAMP and cGMP.
From the myriad of known PDE4 inhibitors, one common struc-
tural motif that continues to be exploited are catachol diether-
based small molecules.4 Indeed, the structure of 1 formed the basis
for numerous catachol diether based inhibitors of PDE4 and the
visualization of 1 bound to PDE4B provided the rational for its po-
tent inhibition profile. From numerous crystallographic analyses
and modeling efforts it is clear that the catachol diether based
inhibitors bind to the catalytic domain of PDE4 through specific
hydrogen bonds with the conserved glutamine residue. Our initial
SAR explorations of triazolothiadiazine based PDE4 inhibitors con-
firmed that a 3,4-dimethoxy phenyl moiety linked to the C6 posi-
tion of the 3,6-dihydro-2H-1,3,4-thiadiazine ring was a crucial
substitution pattern for potent PDE4 inhibition.11 Interestingly,
the phenyl ring attached to the C3 position of the 1,2,4-triazole
ring system was found to be more amendable to random substitu-
tions without loss of function. This formed the basis for our suppo-
sition that these novel PDE4 inhibitors bind in a similar way to 1.
To explore this hypothesis, we conducted docking simulations
using the AutoDock software.28 We first retrieved the three-dimen-
sional coordinates for PDE4B from the Protein Data Bank (PDB ID:
1XMY). Protein and ligand structures were prepared in AutoDock28
and previously reported PDE4-inhibitor complexes were taken into
account when preparing the active site grid box. Flexibility was
granted to the active site glutamine and the ligand(s). Following
multiple docking simulations the most favorable binding confor-
mations were extracted based upon calculated binding constants
(reported as Ki values and found to be in the low nanomolar range
for favorable docking orientations). The primary docking modality
for 10 is shown in Fig. 4. Importantly, this docking orientation is
consistent with the idea that the catachol diether forms an integral
hydrogen bond with Q443 (right panel) and the aromatic moiety is
clearly positioned between the conserved isoleucine (I410) and
phenylalanine (F446). The remainder of the molecule is shown to
extend into the catalytic domain in close proximity to both the
Zn2+ and Mg2+ cations. Such an orientation would block the ap-
proach of cAMP to the catalytic domain and forms the basis for
inhibiting PDE4. This docking orientation is consistent with the
known SAR for this chemotype whereby the 3,4-dimethoxy phenyl
moiety at the C6 position of the 3,6-dihydro-2H-1,3,4-thiadiazine
ring is crucial for maintaining inhibition in the low nanomolar
range whereas the opposite phenyl ring is more amendable to
change without significant loss of potency. It also demonstrates
that alterations of the core heterocycle from the general triazolo-
thiadiazine structure to the triazolopyridazine structure will have
at 100 lM and 10 lM concentrations displayed marked loss of
luminescence suggesting a b2AR mediated increase of cAMP due
to inhibition of PDE4 (Fig. 3B). Next, we examined the real-time
kinetics of PKA subunit dissociation by administering 10 at a
10
on the control experiment of administering 10 following pretreat-
ment with 1 M of the inverse b2AR agonist 20. In four indepen-
lM concentration. The shown real-time kinetics are normalized
l
dent experiments, the presence of 10 reduced the luminescence
of the cell-based system by 25% to 50% within 2 min of administra-
tion (Fig. 3C).
Docking of 10 at PDE4B. Given the potency, selectivity and cell-
based PDE4 inhibition results for this chemotype, it was of interest
to examine its binding modality to the PDE4 structure. The PDE
classes of enzymes are comprised of an N-terminal domain, a cat-
alytic domain and a C-terminal domain. Crystallographic analyses
of several PDE isozymes have aided researchers in understanding
the divergent activities and pharmacology of this class of pro-
teins.6,22 Importantly, structures of PDE4 have been reported in
complex to AMP and several small molecule inhibitors.23–27
Through these efforts it was shown that the three domains of
PDE4 are coordinated through interactions with two metal cations
(Zn2+ and Mg2+).7 The residues that coordinate these metals are
highly conserved across the PDE family. Both the Zn2+ and Mg2+
play important roles in the catalytic mechanism of cAMP hydroly-
sis by coordinating the phosphate moiety. Other important insights
include the recognition of a conserved glutamine residue (Q443 in
RegF[1] + CatF[2]
RegF[1] + CatF[2]
µ
10
100
M
µ
M
+ 10 µM 10
18
10
control
19
20
1
Time (sec)
Figure 3. Effect of PDE inhibition on b2AR regulated PKA activities in cells. Bioluminescence (recorded in relative luminescence units or RLU) in A–C was detected from stable
b2AR-HEK293 (see reference19) cells transiently transfected with the PKA reporter Reg-F[1]:Cat-F[2]2 (from three independent experiments). (A) Effect of combinations of
pretreatment with the selective b2AR-antagonist 20 (1
from independent triplicates). (B) Concentration dependent effect of 18, 10 on the association of Reg-F[1]:Cat-F[2] (30 min, mean s.d. from independent triplicates).
Percentage of PKA activation is normalized based upon 20 (1 M) pretreated cells. (C) Real-time kinetics (normalized on the control experiment = pretreatment with 1
20) of Reg-F[1]:Cat-F[2] dissociation in response to 10 treatment (10 M, four independent samples).
lM) and treatment with 1 (100 lM; 30 min) or 19 (1 lM, 30 min) on the association of Reg-F[1]:Cat-F[2] (mean s.d.
l
lM
l

A. P. Skoumbourdis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3686–3692
3691
Figure 4. Model of PDE4B and docking of 10. The left panel details the entire PDE4B structure (N-terminal domain, a catalytic domain and a C-terminal domain) bound to 10.
The right panel shows the catalytic domain bound to 10 including interactions with conserved glutamine (Q443) isoleucine (I410) and phenylalanine (F446) and the Zn2+
(grey) and Mg2+ (green) cations.
limited affect on the inhibitory profile of these reagents. These
docked structures may allow future SAR explorations to focus on
improved pharmacokinetic and drug metabolism properties with-
out compromising potency, efficacy and selectivity for this
chemotype.
the potential of substituted triazolothiadiazines and introduce tria-
zolopyridazines as potent and selective inhibitors of this important
cellular target. Not only are selected analogues of this novel chem-
otype capable of down-regulating purified isozymes of PDE4, but
they maintain excellent cell-based activity as well. Their binding
modality is predicted to mimic known catachol diether based
inhibitors of PDE4. Importantly, both computational and structure
activity studies suggest that the phenyl ring at the C3 position of
the 1,2,4-triazole ring system could be modified providing a mech-
anism for advanced SAR considerations.
Numerous chemotypes exist for the potent, selective inhibition
of PDE4. Here, we expand upon our discovery of substituted 6-(3,4-
dialkoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and
introduce 6-(3,4-dialkoxyphenyl)-[1,2,4]triazolo[4,3-b]pyridazines
as novel inhibitors of PDE4. In this study, we examined the com-
mon structural substitutions found on other catachol diether based
inhibitors of PDE4 and several of the resulting analogues are
among the most potent inhibitors of this important cellular target.
These include methoxy, cyclopentyloxy, cyclopropylmethoxy, 2-
difluoromethoxy and O-3-tetrahydrofuranyl moieties. It was found
that the chirally pure R-O-3-tetrahydrofuranyl substitution main-
tained the best potency in this study. Further, these reagents pos-
sess impressive selectivity for PDE4 versus other PDE family
members. However, these chemotypes, like others, do not posses
subtype selectivity across PDE4 isozymes.4
Acknowledgments
We thank Ms. Allison Peck for critical reading of this manu-
script. This research was supported by the Molecular Libraries Ini-
tiative of the National Institutes of Health Roadmap for Medical
Research, the Intramural Research Program of the National Human
Genome Research Institute and the National Institute of Dental and
Craniofacial Research.
Our initial analysis of the compounds and their activity was
based upon an in vitro analysis using purified PDE4 protein. It is
critical to examine chemical probes discovered via purified-protein
assays within cell-based contexts to confirm activity and establish
that they are relevant for cell-based experimentation. Here, we
examine selected analogues (5, 10 and 18) in two different cell-
based assays. One assay is based upon the ability of PDE4 to reduce
cAMP levels in a CNG cell line while the other utilizes a PCA repor-
ter for PKA activity. Both analyses demonstrated the utility of these
novel reagents as cell-based chemical probes of PDE4 activity.
Finally, with the myriad of structural data surrounding PDE4
and PDE4 complexes with selected inhibitors, it was important to
explore the binding modality of these compounds. Docking studies
demonstrated that these agents utilize the conserved binding
mode whereby the catachol diether functionality forms a strong
interaction with a conserved glutamine residue. This docking ori-
entation further provides a roadmap for additional SAR around
the seemingly modifiable phenyl ring attached to the 1,2,4-triazole
moiety of the core heterocycle. This key aspect of these reagents
may be of importance during attempts to modify ADME properties
of these compounds without altering the affinity or selectivity for
PDE4.
Supplementary data
Experimental methods for the cyclic nucleotide-gated cation
channel assay, protein-fragmentation complementation assays
and molecular docking and the full synthetic procedures and char-
acterization of reported compounds are detailed. This material is
available free of charge via the Internet. Supplementary data asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.01.057.
References and notes
1. Beavo, J. A.; Brunton, L. L. Nat. Rev. Mol. Cell. Biol. 2002, 3, 710.
2. Bender, A. T.; Beavo, J. A. Pharmacol. Rev. 2006, 58, 488.
3. McKenna, J. M.; Muller, G. W. In Cyclic Nucleotide Phosphodiesterases in Health
and Disease; Beavo, J. A., Francis, S. H., Houslay, M. D., Eds.; CRC Press, 2006; p
667.
4. Zhang, K. Y. J.; Ibrahim, P. N.; Gillette, S.; Bollag, G. Expert Opin. Ther. Targets
2005, 9, 1283.
5. Huang, Z.; Ducharme, Y.; Macdonald, D.; Robichaud, A. Curr. Opin. Chem. Biol.
2001, 5, 432.
6. Xu, R. X.; Rocque, W. J.; Lambert, M. H.; Vanderwall, D. E.; Luther, M. A.; Notle,
R. T. J. Mol. Biol. 2004, 337, 355.
7. Card, G. L.; England, B. P.; Suzuki, Y.; Fong, D.; Powell, B.; Lee, B.; Luu, C.;
Tabrizizad, M.; Gillette, S.; Ibrahim, P. N.; Artis, D. R.; Bollag, G.; Milburn, M. V.;
Kim, S.-H.; Schlessinger, J.; Zhang, K. Y. J. Structure 2004, 12, 2233.
8. Dunn, R. F.; Kuester, E. M.; Conticello, R. D.; Hopper, A. T. WO 2007123953,
2007; PCT Int. Appl.
PDE4 inhibitors are highly sought after as probes of selected cell
signalling pathways and as potential therapeutics in diverse areas
including memory enhancement and COPD. Here, we expand on

3692
A. P. Skoumbourdis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3686–3692
9. Napoletano, M.; Norcini, G.; Pellacini, F.; Marchini, F.; Morazzoni, G.; Ferlenga,
P.; Pradella, L. Bioorg. Med. Chem. Lett. 2000, 10, 2235.
17. BPS Bioscience Inc.; 11526 Sorrento Valley Rd. Ste. A2; San Diego, CA 92121.
www.bpsbioscience.com.
10. Andrés, J. I.; Alonso, J. M.; Diaz, A.; Fernández, J.; Iturrino, L.; Martinez, P.;
Matesanz, E.; Freyne, E. J.; Deroose, F.; Boeckx, G.; Petit, D.; Diels, G.; Megens,
A.; Somers, M.; Van Wauwe, J.; Stoppie, P.; Cools, M.; De Clerck, F.; Peeters, D.;
de Chaffoy, D. Bioorg. Med. Chem. Lett. 2002, 12, 653.
18. Titus, S. A.; Xiao, L.; Southall, N.; Lu, J.; Inglese, J.; Brasch, M.; Austin, C. P.;
Zheng, W. J. Biomol. Screen. 2008, 13, 609.
19. Stefan, E.; Aquin, S.; Berger, N.; Landry, C. R.; Nyfeler, B.; Bouvier, M.; Michnick,
S. W. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16916.
11. Skoumbourdis, A. P.; Huang, R.; Southall, N.; Leister, W.; Guo, V.; Cho, M.-H.;
Inglese, J.; Nirenberg, M.; Austin, C. P.; Xia, M.; Thomas, C. J. Bioorg. Med. Chem.
Lett. 2008, 18, 1297.
12. Katoh, T.; Awasaguchi, K.-i.; Mori, D.; Kimura, H.; Kajimoto, T.; Node, M. Synlett
2005, 19, 2919.
13. Hall, A.; Brown, S. H.; Chessell, I. P.; Chowdhury, A.; Clayton, N. M.; Coleman, T.;
Giblin, G. M. P.; Hammond, B.; Healy, M. P.; Johnson, M. R.; Metcalf, A.; Michel,
A. D.; Naylor, A.; Novelli, R.; Spalding, D. J.; Sweeting, J.; Winyard, L. Bioorg.
Med. Chem. Lett. 2007, 17, 916.
14. Pollak, A.; Tišler, M. Tetrahedron 1966, 22, 2073.
15. Albright, J. D.; Moran, D. B.; Wright, W. B., Jr.; Collins, J. B.; Beer, B.; Lippa, A. S.;
Greenblatt, E. N. J. Med. Chem. 1981, 24, 592.
16. Carling, R. W.; Madin, A.; Guiblin, A.; Russell, M. G. N.; Moore, K. W.;
Mitchinson, A.; Sohal, B.; Pike, A.; Cook, S. M.; Ragan, I. C.; McKernan, R. M.;
Quirk, K.; Ferris, P.; Marshall, G.; Thompson, S. A.; Wafford, K. A.; Dawson, G. R.;
Atack, J. R.; Harrison, T.; Castro, J. L.; Street, L. J. Med. Chem. 2005, 48, 7089.
20. Michnick, S. W.; Ear, P. H.; Manderson, E. N.; Remy, I.; Stefan, E. Nat. Rev. Drug
Discov. 2007, 6, 569.
21. Remy, I.; Michnick, S. W. BioTechniques 2007, 42, 137.
22. Xu, R. X.; Hassell, A. M.; Vanderwall, D.; Lambert, M. H.; Holmes, W. D.; Luther,
M. A.; Rocque, W. J.; Milburn, M. V.; Zhao, Y.; Ke, H.; Nolte, R. T. Science 2000,
288, 1822.
23. Lee, M. E.; Markowitz, J.; Lee, J.-O.; Lee, H. FEBS Lett. 2002, 530, 53.
24. Huai, Q.; Colicelli, J.; Ke, H. Biochemistry 2003, 42, 13220.
25. Huai, Q.; Wang, H.; Sun, Y.; Kim, H.-Y.; Liu, Y.; Ke, H. Structure 2003, 11, 865.
26. Huai, Q.; Wang, H.; Zhang, W.; Colman, R. W.; Robinson, H.; Ke, H. Proc. Nat.
Acad. Sci. U.S.A. 2004, 101, 9624.
27. Huai, Q.; Liu, Y.; Francis, S. H.; Corbin, J. D.; Ke, H. J. Biol. Chem. 2004, 279,
13095.
28. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E. J. Comput.
Chem. 1998, 19, 1639.