Discovery of a new series of [1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.11.077

The synthesis, preliminary evaluation and structure-activity relationship (SAR) of a series of 1-aryl-4-methyl[1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors are described. From this investigation compound 31 was identified, showing good combined potency, acceptable brain uptake and high selectivity for both PDE2 and PDE10 enzymes. Compound 31 was subjected to a microdosing experiment in rats, showing preferential distribution in brain areas where both PDE2 and PDE10 are highly expressed. These promising results may drive the further development of highly potent combined PDE2/PDE10 inhibitors, or even of selective inhibitors of PDE2 and/or PDE10.

Full text of DOI:10.1016/j.bmcl.2012.11.077

Bioorganic & Medicinal Chemistry Letters 23 (2013) 785–790
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a new series of [1,2,4]triazolo[4,3-a]quinoxalines as dual
phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors
José-Ignacio Andrés a, , Peter Buijnsters b, Meri De Angelis a, Xavier Langlois c, Frederik Rombouts b,
⇑
Andrés A. Trabanco a, Greet Vanhoof d
a Janssen Research & Development, Medicinal Chemistry, Janssen-Cilag S.A., C/Jarama 75, 45007 Toledo, Spain
b Janssen Research & Development, Neuroscience-Medicinal Chemistry, Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium
c Janssen Research & Development, Neuroscience-Biology, Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium
d Janssen Research & Development, CREATe, Translational Sciences, Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, preliminary evaluation and structure–activity relationship (SAR) of a series of 1-aryl-4-
methyl[1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/
PDE10) inhibitors are described. From this investigation compound 31 was identified, showing good com-
bined potency, acceptable brain uptake and high selectivity for both PDE2 and PDE10 enzymes. Com-
pound 31 was subjected to a microdosing experiment in rats, showing preferential distribution in
brain areas where both PDE2 and PDE10 are highly expressed. These promising results may drive the fur-
ther development of highly potent combined PDE2/PDE10 inhibitors, or even of selective inhibitors of
PDE2 and/or PDE10.
Received 1 October 2012
Revised 19 November 2012
Accepted 20 November 2012
Available online 1 December 2012
Keywords:
Neurological disorders
PDE2 inhibitors
PDE10 inhibitors
Triazoloquinoxalines
Ó 2012 Elsevier Ltd. All rights reserved.
Phosphodiesterases (PDEs) are a family of enzymes encoded by
21 genes and subdivided into eleven distinct families according to
their structural and functional properties. These enzymes metabol-
ically inactivate the ubiquitous secondary messengers, 30,50-cyclic
adenosine monophosphate (cAMP) and 30,50-cyclic guanosine
monophosphate (cGMP), hydrolyzing their phosphodiester bonds
and converting them into the biologically inactive nucleotide 50-
monophosphates AMP and GMP, respectively.1 These two messen-
gers regulate a wide variety of biological processes, including pro-
inflammatory mediator production and action, ion channel func-
tion, muscle contraction, learning, differentiation, apoptosis, lipo-
genesis, glycogenolysis, and gluconeogenesis. In neurons, they
induce the activation of cAMP and cGMP-dependent kinases and
subsequent phosphorylation of proteins involved in acute regula-
tion of synaptic transmission as well as in neuronal differentiation
and survival.2 From the 11 identified mammalian PDE families
(PDE1–PDE11) three of them selectively hydrolyze cAMP (PDE4,
7, and 8), three are selective for cGMP (PDE5, 6, and 9), and five
families hydrolyze both cyclic nucleotides (PDE1, 2, 3, 10, and 11).3
Although the PDEs are in general expressed in various tissues
and cells throughout the body, the PDE2 and PDE10 enzymes are
preferentially expressed in the brain.4,5 The expression of PDE2
in limbic system and basal ganglia suggests that PDE2 may modu-
late neuronal signaling involved in emotion, perception, concentra-
tion, learning and memory.6 Additionally, PDE2 is expressed in the
nucleus accumbens, the olfactory bulb, the olfactory tubercle and
the amygdala, supporting the hypothesis that PDE2 may also be in-
volved in anxiety and depression.7 PDE10A is the single member
identified from the PDE10 family and, in the brain, mRNA and pro-
tein are highly expressed in a majority of striatal medium spiny
neurons (MSNs).8 This unique distribution of PDE10A in the brain,
together with its increased pharmacological characterization, indi-
cates a potential use of PDE10 inhibitors for treating neurological
and psychiatric disorders like schizophrenia.9 Thus, PDE10 inhibi-
tors may possess a pharmacological profile similar to that of the
current antipsychotics which mainly treat positive symptoms of
schizophrenia, but also having the potential to improve the nega-
tive and cognitive symptoms of the disease while lacking some
of the D2 related side effects, such as the extrapyramidal side ef-
fects (EPS) commonly observed with the currently available anti-
psychotics.10 Since PDE10 inhibitors can be used to raise levels of
cAMP and/or cGMP within cells that express the PDE10 enzyme,
for example neurons that comprise the basal ganglia, PDE10 inhib-
itors may be also useful in treating Parkinson’s disease, Hunting-
ton’s disease, addiction and depression.11
Abbreviations: CNS, central nervous system; cAMP, 30,50-cyclic adenosine
monophosphate; cGMP, 30,50-cyclic guanosine monophosphate; PDE2, phosphodi-
esterase 2; PDE10, phosphodiesterase 10; SAR, structure–activity relationship.
⇑
Corresponding author. Tel.: +34 925 245 780; fax: +34 925 245 771.
E-mail address: iandres@its.jnj.com (J.-I. Andrés).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.077

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The preferential distribution of PDE2 and PDE10 in the brain, to-
29–40 were obtained in moderate yields (25–60%). The synthesis
of compound 31 starting from the commercially available reagent
2b is described as an example to illustrate the synthetic
methodology.15
gether with the preclinical evidence described with the use of
selective PDE2 and PDE10 inhibitors for the treatment of multiple
CNS disorders, make this combination of potential interest for ther-
apeutic use. It has recently been reported that blockade of PDE2,
PDE4 and PDE10 with the atypical anxiolytic drug Tofisopam could
improve the negative symptoms of psychosis.12 Although a series
of combined PDE2/PDE10 inhibitors have recently been described,
their pharmacological characterization has been limited to in vitro
experiments, without any reported pharmacokinetic or pharmaco-
dynamic studies.13
The in vitro inhibitory activity against hPDE2 and rPDE10 en-
zymes was evaluated for final compounds 1a, 7b, 8–26 and 29–
40 (Table 1).16 Our initial exploration focused on introducing dif-
ferent substituents in the phenyl ring at position C-1 of the triazo-
loquinoxaline scaffold, while keeping the ethoxycarbonyl moiety
fixed at position C-8. From a previous exploration (data not shown)
we found that the o-chloro-phenyl derivative 7b seemed to be the
most potent PDE2/PDE10 inhibitor, suggesting that distorting
coplanarity between the aryl ring and the triazoloquinoxaline core
enhanced the inhibitory activity for both enzymes. Replacement of
the ethyl ester by the corresponding ethyl-amide analog led to a
fivefold increase in potency for PDE2 and to a lesser extent for
PDE10 (compare results of 8 with 1a). Combination of o-chloro-
phenyl at C-1 and ethylamide at C-8 resulted in a 20-fold increase
in PDE2 potency and a 12-fold increase in PDE10 potency, increas-
ing its selectivity versus PDE10 to 72-fold (Table 1, compounds 8
and 9). A small exploration on the C-1 aryl was done while keeping
the ethyl-amide fixed at C-8. Replacing the chlorine atom in 9 by
other ortho-substituents such as methoxy (10) or fluorine (11)
was somewhat detrimental for activity, whereas the introduction
of a second chlorine atom at position 6 of the C-1 phenyl ring
(12) increased the PDE10A inhibition while maintaining the PDE2
activity. Introduction of pyridyl rings at C-1 (13–15) was found
to be detrimental especially for PDE10 activity. However, the 3-
and 4-pyridyl derivatives 14 and 15 showed acceptable PDE2 inhi-
bition and a remarkable selectivity versus PDE10 (14, 35-fold and
15, 63-fold selectivity). Introduction of substituents in the 3-pyri-
dyl ring (16–19) resulted in compounds with comparable selectiv-
ity range. From these 3-pyridyl analogs the most interesting one
proved to be the 2-methyl-3-pyridyl derivative 16, with substan-
tially better PDE2 and PDE10 activity when compared to 14,
although it was significantly less potent than the o-chlorophenyl
compound 9 (20-fold decrease in PDE2 potency and eightfold de-
crease in PDE10 potency).
Having in mind these results, we investigated the effect on
PDE2/PDE10 inhibition of different substituents at C-8, while
keeping the o-chloro-phenyl group at C-1 fixed. Firstly, a diverse
set of different amides, exemplified by compounds 20–26, was
synthesized. A wide variety of amide moieties was tolerated
and proved to be very potent PDE2 inhibitors, with IC50 values
in the single digit nM range, showing significant combined
PDE10A inhibitory activity. Aromatic rings with shorter (20, 21)
or longer (22) spacers retained good combined activities. Note-
worthy is compound 21, having a tertiary amide at position C-
8, which showed a marked decrease in activity for both PDE2
and PDE10A when compared to its direct analogue 20, indicating
that secondary amides are preferred for activity. Interestingly,
amides bearing a basic center (23–26) were also potent inhibitors
of both enzymes, with compound 26 showing the most balanced
profile among this set in terms of potency (PDE2 IC50 0.42 nM and
PDE10 IC50 8.9 nM). The most potent PDE2 inhibitors from this
amide series were the ethyl-amides 9 and 12, whereas the most
selective PDE2 inhibitor proved to be the benzyl-amide derivative
20 that showed 32-fold selectivity PDE2 versus PDE10 (IC50 0.38
and 13 nM, respectively).
We recently started a medicinal chemistry program towards the
identification of selective PDE2 inhibitors and/or molecules with
combined PDE2/PDE10 activity. A high throughput screen (HTS)
of our compound collection identified
methyl[1,2,4]triazolo[4,3-a]quinoxaline derivatives
a
series of 1-aryl-4-
of which
A
compound 1a (Fig. 1) showed interesting dual activity, with IC50
30 nM for PDE2 and 325 nM for PDE10. The corresponding regio-
isomer 1b presented also dual activity, however the potency was
significantly lower. We considered this chemotype and particularly
compound 1a as an interesting starting point for further optimiza-
tion. This Letter summarizes the preliminary in vitro structure–
activity relationships (SAR) and brain penetration results obtained
from our exploration focusing on positions C-1 and C-8 of the tria-
zoloquinoxaline scaffold.14
The synthesis of the hit 1a and the key intermediates 7a–m is
depicted in Scheme 1. Condensation of commercially available
1,2-diaminophenyl derivatives 2a and 2b with pyruvic acid 3a or
ethyl pyruvate 3b afforded the two possible regioisomers in a 1:1
ratio, which could be separated by column chromatography yield-
ing intermediates 4a, b. In view of the difference in in vitro PDE2
potency between the initial hits 1a and 1b only compounds 4a, b
were used for the next reaction step. Intermediates 4a, b were hal-
ogenated by means of phosphorous oxychloride affording deriva-
tives 5a and 5b and cyclized using the appropriate hydrazido
compounds 6 yielding 1a and key intermediates 7a–m in moderate
to good yields (60–85%).
The target amides 8–26 were prepared by a HATU mediated
reaction of the appropriate amines and the acids obtained follow-
ing hydrolysis of esters 7a–l in good overall yields (Scheme 2).
All the aminomethyl derivatives, 29–40, depicted in Scheme 3
were synthesized starting from the 8-bromo-substituted interme-
diate 7m. The key step of the synthesis was the preparation of
aldehyde 28 that was synthesized in a two-step reaction proce-
dure. Firstly, palladium-catalyzed Stille cross-coupling reaction of
7m with tributyl vinyl tin led to the vinyl derivative 27, which
was subsequently oxidized by means of osmium tetroxide and so-
dium periodate to the target aldehyde intermediate 28. The reduc-
tive amination of primary amines with 28 was performed at room
temperature using sodium borohydride as reducing agent, whereas
sodium triacetoxyborohydride and microwave irradiation condi-
tions were needed for secondary amines. The target compounds
To expand the SAR knowledge, a series of C-8 aminomethyl
derivatives containing secondary (29, 32, 34 and 35) or tertiary
amines (30, 31, 33 and 36–40) were prepared. In general, the ami-
nomethyl derivatives were weaker PDE2 inhibitors than the com-
pounds bearing the amide substitution. Thus, compounds 29 and
32 showed 65- and 40-fold less PDE2 inhibition than their corre-
sponding amide pairs 9 and 20. The PDE10A activity was less
Figure 1. Triazoloquinoxaline hits 1a and 1b.

J.-I. Andrés et al. / Bioorg. Med. Chem. Lett. 23 (2013) 785–790
787
Scheme 1. Reagents and conditions: (i) for 3a CH3CO2H/H2O, rt, 7 h (42%), for 3b, toluene, reflux, 3 h (40%); (ii) for 5a POCl3, reflux, 2 h (65–75%); for 5b, POCl3, 1,2-
dichloroethane, reflux, 4 h (23%); (iii) EtOH, microwaves, 160 °C, 15–25 min (60–85%).
tion (31, IC50 2.8 nM vs 35 nM; 36, IC50 5.0 nM vs 19 nM). In both
the amide and the amine series a similar trend was observed:
when the potency for PDE2 increased or decreased, a similar in-
crease or reduction was also observed in PDE10 inhibition.
Several compounds (1a, 9, 17, 18, 22, 31, 33–35 and 37) were
tested for PDE10 inhibition using isolated recombinant expressed
human enzyme (hPDE10A) and showed comparable PDE10 inhibi-
tion to the one found with the rat PDE10 enzyme (rPDE10A), prov-
ing that there are no large differences in inhibition between both
species (Table 1).
Scheme 2. Reagents and conditions: (i) LiOH, H2O/THF, rt, 3 h, (quant. yield); (ii)
R3NH2, Et3N, HATU, DMF/CH2Cl2, rt, 3 h (60–85%).
Representative examples from the amide (9, 17, 19, 24 and 25)
and aminomethyl (29, 31 and 40) series were selected to evaluate
their potential to cross the blood–brain barrier (BBB). The studies
were conducted in rats to measure brain and plasma concentra-
tions 1 h after subcutaneous (sc) or oral (p.o.) administration of a
10 mg/kg dose. The results obtained are shown in Table 2. Unfortu-
nately, all the amidic derivatives tested had a very low brain up-
take, with 9 and 24 being the only compounds that showed
some significant brain penetration (brain/plasma (B/P) ratios of
0.11 and 0.14, respectively).
To our delight most compounds from the aminomethyl subser-
ies showed acceptable distribution to the brain. Thus, compounds
29 and 31 showed B/P ratios higher than 0.5 (B/P 0.73 and 0.62,
respectively) with acceptable absolute brain levels. On the con-
trary, compound 40, having a terminal hydroxyl substituent,
showed limited brain uptake (B/P 0.18). From these studies it could
be concluded that aminomethyl derivatives, although less potent,
had better brain uptake than the more potent amide derivatives.
We decided to further profile and test compound 31 in a micro-
dosing experiment17 to verify whether the distribution of 31 in the
different brain regions would match the PDE2/PDE10 brain expres-
sion. The microdosing study was conducted in rats after a 0.03 mg/
kg intravenous (iv) dose, in a 2 to 30 min timeframe. As can be ob-
served in Figure 2, compound 31 reached the brain in good levels
and showed the highest uptake in the striatum, which is the region
where both PDE2 and PDE10 are mainly expressed. The compound
also showed higher uptake in cortex and hippocampus (where only
PDE2 is highly expressed) compared to cerebellum,4,5 a region in
which expression of both PDE2 and PDE10 is almost negligible.
In addition, compound 31 showed good in vitro selectivity over
the other members of the PDE family. A full PDE profile for 31 is
shown in Figure 3.
Scheme 3. Reagents and conditions: (i) LiCl, tributylvinyl tin, Pd(PPh3)4, toluene,
120 °C, 1 h (93%); (ii) NaIO4, OsO4, 1,4-dioxane/H2O, rt, 2 h (75%); (iii) for
compounds 29, 32, 34 and 35: primary amine, NaBH4, MeOH, rt (25–45%); (iv) for
compounds 30, 31, 33, and 36–40: secondary amine, NaBH(OAc)3, 1,2-dichloroeth-
ane, microwaves, 80 °C, 10–20 min (35–60%).
affected between both series, and in this case 29 and 32 were
approximately 8- and 4-fold weaker PDE10A inhibitors than 9
and 20. It is remarkable that compounds showing the most bal-
anced profile regarding dual activity reside within the amino-
methyl subseries; the most potent aminomethyl PDE2 inhibitors
were 31 and 36, both compounds showing also good PDE10 inhibi-

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Table 1
In vitro hPDE2 and rPDE10 inhibitory activity of final compounds 1a, 7b, 8–26 and 29–40
N
N
1
R1
R2
4
N
N
8
a
a,b
Compd
R1
R2
hPDE2 IC50 (nM SD)
rPDE10A IC50 (nM SD)
O
1a
30 (n = 1)
325 39
O
O
O
O
O
O
O
O
O
O
O
Cl
7b
8
4.4 1.3
35 12(33 6.5)
50 1.6
6.2 2.5
N
H
Cl
9
0.6 0.32
5.9 4.2
74 36 (29 7.2)
51 22 (33, n = 1)
52 17 (41 9)
19 5.4
N
H
OCH3
F
10
11
12
13
14
N
H
2.8 1.6
N
H
Cl
Cl
0.28 (n = 1)
263 142
39 5.7
N
H
N
N
39 13 (20, n = 1)
39 1.9
N
H
N
H
15
29 6.6
6.3 1.5
21 11
N
H
N
O
O
O
O
16
17
18
47 18
N
H
N
N
21
28
2
5
113 39
N
H
N
O
35 12(33 6.5)
N
H
19
8.9 2.6
50 1.6
N
H
N
O
O
O
Cl
20
21
0.38 0.14
19 1.0
74 36 (29 7.2)
51 22 (33, n=1)
N
H
Cl
N

J.-I. Andrés et al. / Bioorg. Med. Chem. Lett. 23 (2013) 785–790
789
Table 1 (continued)
a
a,b
Compd
R1
R2
hPDE2 IC50 (nM SD)
rPDE10A IC50 (nM SD)
O
Cl
N
H
22
23
24
n
1.7 1.09
1.4 0.31
2.2 0.57
52 17 (41 9)
n=2
O
O
Cl
Cl
N
19 5.4
N
H
N
N
N
H
n
n
39 13 (20, n = 1)
n=2
O
O
Cl
Cl
N
H
n=2
25
26
0.47 0.11
0.42 0.05
39 1.9
21 11
O
N
N
H
Cl
Cl
Cl
Cl
Cl
Cl
29
30
31
32
33
34
24 16
20 3.2
2.8 1.0
15 1.7
7.1 2.5
7.3 1.7
47 18
NH
N
113 39
O
35 12(33 6.5)
50 1.6
N
N
H
N
O
N
74 36 (29 7.2)
51 22 (33, n = 1)
N
H
OH
Cl
35
12 3.6
52 17 (41 9)
N
H
Cl
Cl
Cl
Cl
Cl
OH
OH
N
N
36
37
38
39
5.0 2.5
9.7 2.4
15 11
6.3 3.9
24 13
19 5.4
39 13 (20, n = 1)
39 1.9
O
N
N
21 11
OH
OH
40
47 18
N
a
Values are mean SD of at least two experiments unless specified.
Values in brackets refer to data generated using isolated recombinant human PDE10A enzyme.
b
In summary, we have identified and synthesized a series of 1-
(sub)nanomolar range. Substituted amides were not brain pene-
trant and therefore we synthesized a series of related aminomethyl
derivatives that nicely showed combined PDE2/PDE10 inhibitory
activity. Among them, we selected compound 31 in view of its
good potency (IC50 2.8 nM for PDE2 and 35 nM for PDE10) for a
aryl-4-methyl[1,2,4]triazolo[4,3-a]quinoxaline derivatives as novel
combined PDE2/PDE10 inhibitors, selective over all other PDEs.
From this investigation we identified several highly potent com-
pounds possessing IC50 values for PDE2 and PDE10 in the

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J.-I. Andrés et al. / Bioorg. Med. Chem. Lett. 23 (2013) 785–790
Table 2
ments of compound concentration in the rat brain samples. They
also thank members of the analysis and purification group of Jans-
sen R&D, Spain.
Brain and plasma levels for dual PDE2/PDE10 inhibitors 1 h after administrationa
Compd
Route
(B)
(P)
B/P ratio
9b
p.o.
sc
p.o.
sc
sc
sc
185 44
98 22
1880 805
2800 537
467 83
1073 64
2540 295
845 78
0.11 0.03
0.036 0.01
0.034 0.01
0.14 0.04
0.034 0.005
0.73 0.17
0.62 0.03
0.18 0.02
17c
Supplementary data
19c,d
24b,e
25b
29c
16
2
143 36
88 22
628 204
189 78
Supplementary data (the experimental details of the synthesis
of key intermediates required for the preparation of final com-
pound 31, and the protocols for in vitro PDE’s inhibition are pro-
vided) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.077.
31c,e
40b
sc
sc
299 120
569 93
99
8
a
(B): brain levels in ng/g; (P): plasma levels in ng/mL. Compounds formulated in
20% HP-b-CD at pH 4. Data are expressed as geometric mean values of at least two
runs the standard error measurement (SEM).
References and notes
b
Study in male Wistar rats dosed at 10 mg/kg.
Study in male Sprague Dawley rats dosed at 10 mg/kg.
Formulated in 0.5% methocel suspension.
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Figure 2. Microdosing experiment for compound 31.
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published. Lankau, H.-J.; Langen, B.; Grunwald, C.; Höfgen, N.; Stange, H.;
Dost, R.; Egerland, U. PCT Int. Appl. WO2012104293, August 9, 2012.
15. Synthesis of 1-(2-chlorophenyl)-4-methyl-8-(morpholin-4-ylmethyl)[1,2,4]
triazolo[4,3-a]-quinoxaline (31). The synthesis of intermediates 4b, 5b, 7k, 27,
and 28 are provided as Supporting Information. Morpholine (1.37 mL,
15.67 mmol) was added to
a stirred solution of intermediate 28 (2.3 g,
7.12 mmol) dissolved in 1,2-dichloroethane (50 mL) and the mixture was
heated at 80 °C for 15 min under microwave irradiation (the reaction was
divided in three batches). Then sodium triacetoxyborohydride (1.81 g,
8.55 mmol) was added portionwise and the mixture was heated again at the
same conditions as before for 20 min. The mixture was then quenched with H2O
and extracted with CH2Cl2. The organic layer was separated, dried (Na2SO4),
filtered and the solvent evaporated in vacuo. The crude compound was purified
by chromatography (silica, MeOH in EtOAC 2/98 to 10/90) the desired fractions
were collected and the solvent evaporated to yield final compound 31 as pale
yellow solid that was further triturated with diethyl ether/DIPE and dried in
vacuo (1.6 g, 57%). 1H NMR (400 MHz, CDCl3) d ppm 2.24–2.41 (m, 4 H), 3.08 (s, 3
H), 3.42 (s, 2 H), 3.53–3.69 (m, 4 H), 7.37 (d, J = 1.2 Hz, 1 H), 7.49 (dd, J = 8.3,
1.6 Hz, 1 H), 7.54–7.62 (m, 1 H), 7.64–7.75 (m, 3 H), 7.99 (d, J = 8.3 Hz, 1 H). Mp
160.4 °C.
Figure 3. PDE profile of compound 31.
microdosing study to ascertain its ability to enter the brain and
bind specifically to both enzymes. The results obtained with 31
proved that this derivative possessed acceptable brain uptake
and was able to bind selectively to PDE2 and PDE10 enzymes.
These promising results make 31 a valuable tool compound to fur-
ther study the implication of dual PDE2/PDE10 inhibitors on sev-
eral neurological pathologies, opening the way to the synthesis of
new more potent and drug-like compounds.
16. Protocols for in vitro PDE’s inhibition are provided as Supplementary data.
17. Male Wistar rats (180–200 g) were injected intravenously with 0.03 mg/kg of
compound 31 and sacrificed at 2, 5, 10 and 30 min after injection (3 rats per
time point). Brains were rapidly removed and dissected. Striatum,
hippocampus, whole cortex and cerebellum were homogenized using an
ultrasonic dismembrator probe (Branson) in 4 volumes of 75% acetonitrile.
After centrifugation, drug concentration was measured in each tissue using
liquid chromatography coupled to mass spectroscopy.
Acknowledgments
The authors thank Ilse Lenaerts for the in vivo experimental
work of the microdosing study, and Lieve Dillen for the measure-