Design, synthesis of novel purin-6-one derivatives as phosphodiesterase 2 (PDE2) inhibitors: The neuroprotective and anxiolytic-like effects

Article abstract of DOI:10.1016/j.bmcl.2018.12.018

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders. Herein, based on the existing PDE2 inhibitors and their binding modes, a series of purin-6-one derivatives were designed, syn

Full text of DOI:10.1016/j.bmcl.2018.12.018

Accepted Manuscript
Design, synthesis of novel purin-6-one derivatives as phosphodiesterase 2
(PDE2) inhibitors: the neuroprotective and anxiolytic-like effects
Xian-Feng Huang, Yi-Jing Cao, Jing Zhen, Da-Wei Zhang, Ren Kong, Wen-
Tao Jiang, Ying Xu, Guo-Qiang Song, Heng-Ming Ke, Li Liu
PII:
S0960-894X(18)30952-1
https://doi.org/10.1016/j.bmcl.2018.12.018
BMCL 26187
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 October 2018
5 December 2018
8 December 2018
Please cite this article as: Huang, X-F., Cao, Y-J., Zhen, J., Zhang, D-W., Kong, R., Jiang, W-T., Xu, Y., Song, G-
Q., Ke, H-M., Liu, L., Design, synthesis of novel purin-6-one derivatives as phosphodiesterase 2 (PDE2) inhibitors:
the neuroprotective and anxiolytic-like effects, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.12.018
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Design, synthesis of novel purin-6-one derivatives as phosphodiesterase 2 (PDE2) inhibitors:
the neuroprotective and anxiolytic-like effects
Xian-Feng Huang 1, Yi-Jing Cao 1, Jing Zhen 1, Da-Wei Zhang 2, Ren Kong 2, Wen-Tao Jiang
1, Ying Xu 3, Guo-Qiang Song 1*, Heng-Ming Ke 4*, Li Liu 1*
1School of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou,
Jiangsu, 213164, PR China
2Institute of Bioinformatics and Medical Engineering, School of Electrical and Information
Engineering, Jiangsu University of Technology, Changzhou, Jiangsu, 213001, PR China
3Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center,
The University of North Carolina, Chapel Hill, NC 27599-7260, USA
4Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences,
the State University of New York at Buffalo, Buffalo, NY 14214, USA

*Correspondence:
Li Liu, Ph.D.
School of Pharmaceutical Engineering and Life Sciences
Changzhou University
Changzhou, Jiangsu, 213164, PR China
Tel: 086-519- 86334598
Email: czdx123@163.com
Guoqiang Song, Ph.D.
School of Pharmaceutical Engineering and Life Sciences
Changzhou University
Changzhou, Jiangsu, 213164, PR China
Tel: 086-519-86330600
Email: drugs@vip.sina.com
Hengming Ke, Ph.D.
Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center
The University of North Carolina
Chapel Hill, NC 27599-7260, USA
Tel: 1-919-966-2244
Email: hke@med.unc.edu

ABSTRACT: Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment
of the central nervous system (CNS) disorders. Herein, based on the existing PDE2 inhibitors and
their binding modes, a series of purin-6-one derivatives were designed, synthesized and evaluated
for PDE2 inhibitory activities, which led to the discovery of the best compounds 6p and 6s with
significant inhibitory potency (IC50: 72 and 81 nM, respectively). Docking simulation was
performed to insert compound 6s into the crystal structure of PDE2 at the active site to determine
the binding mode. Furthermore, compound 6s significantly protected HT-22 cells against
corticosterone-induced cytotoxicity and rescued corticosterone-induced decreases in cAMP and
cGMP levels. It also produced anxiolytic-like effect in the elevated plus-maze test and exhibited
favorable pharmacokinetic properties in vivo. These results might bring significant instruction for
further development of potent PDE2 inhibitors.
Keywords: PDE2 inhibitors; purin-6-one; anxiolytic-like effects
Phosphodiesterases (PDEs) are a class of enzymes degrading second messengers including
cAMP and cGMP, which play a key role in mediating cellular responses to various hormones and
neurotransmitters. Currently, eleven PDE families with distinct tissue distribution and selectivity for
cAMP and cGMP have been identified. PDEs 1, 2, 3, 10, 11 are considered dual specificity
enzymes, hydrolyzing both cAMP and cGMP. PDEs 4, 7, and 8 specifically hydrolyze cAMP,
whereas PDEs 5, 6, and 9 prefer to hydrolyze cGMP.1 PDEs are highly expressed in the human
brain and their inhibitors regulate neurodegenerative processes by increasing the concentration of
cAMP and cGMP in brain tissue and thereby modulate a variety of neuronal processes.2,3
Noticeably, inhibitors of PDEs 2, 3, 4, 5 and 9 have been shown to significantly enhance memory in
vitro and in vivo. 4-8 Modulation of cyclic nucleotide (cNT) levels by PDE inhibitors is becoming a
particularly attractive therapeutic approach to treating disorders of mood and emotion.
PDE2A is a dual-specificity phosphodiesterase controlling the cellular levels of both cAMP
and cGMP in the brain areas where mood signaling is regulated. Inhibition of PDE2A increases
cNT levels in brain to evoke antianxiety, antidepressant, and pro-cognitive effects in normal and
stressed rodents.6,9-12 In addition, PDE2 inhibition can promote procognitive activity, and restore
hippocampal function. For a growing body of preclinical research, PDE2A has recently received
intensive attention for its therapeutic potential to treat central nervous system (CNS) disorders,
including schizophrenia and Alzheimer’s disease. Although several PDE2 inhibitors have been
shown to be promising for treatment of mood and cognition disorders in the past two decades, none
of them have reached the market yet. EHNA [Figure 1] is a first generation of PDE2 inhibitors with
13
IC50 of 800 nM but low-selectivity over other PDEs.
Another PDE2 inhibitor, BAY60-7550,

selectively inhibited PDE2 with IC50 of 4.7 nM, increased cGMP in neuronal culture and
hippocampal slice. Besides, this compound also could improved acquisition and consolidation in
14
the tests of object recognition and social recognition of young adult and aged rats. However, the
clinical application of BAY60-7550 was limited due to moderate pharmacokinetic properties. A
pyrazolopyrimidine PDE2 inhibitor, PF-05180999 reported by Pfizer in 2012, was in phase I
9,15,16
clinical trial for the treatment of schizophrenia and migraine, but there was no further report.
Overall, development of novel PDE2 inhibitors for treatment of neurodiseases is still urgently
needed.
In the previous studies, we had reported a selective PDE2 inhibitor, Hcyb1, with purin-6-one
structure, which was a moderate PDE2 inhibitor with an IC50 value of 0.57 μM and produced
neuroprotective and antidepressant-like effects most likely mediated by cAMP/cGMP-CREB-
17
BDNF signaling in vitro and in vivo. As the continuing work for searching more potent PDE2
inhibitors to treat CNS disorders, herein, further optimization of Hcyb1 was carried out and 20
derivatives were designed, synthesized and tested with biological assays.
Figure 1. Structures of known PDE2 inhibitors
The X-ray crystal structure of PDE2 in complex with BAY60-7550 revealed that its
9,18,19
heterocyclic core of is anchored via -stacking against Phe862 and Phe830
and the
pyrimidone forms two hydrogen bonds respectively with Gln859 and Gln812. The latter of
hydrogen bond is unique to PDE2, while the hydrogen bond with Gln859 is common for all PDEs.
Interestingly, the propylphenyl group of BAY60-7550 interacts with a hydrophobic pocket that was

thought to play an important role in the enhancement of binding affinity and selectivity for PDE2.18
BAY60-7550 also interacts with the surface hydrophobic patch through the dimethoxybenzyl group.
Other PDE2 inhibitors reported recently bear various heterocyclic cores such as purin-6-one of
Hcyb1 may have a similar binding pattern of stacking and hydrogen-bonding to that of BAY60-
7550 or ENHA. We therefore reasoned that replacement of the imidazo[1,5-f][1,2,4]triazin-4(3H)-
one core in BAY60-7550 with purin-6-one core will lead to a novel type of PDE2 inhibitors.
Molecular docking suggest that the 5,6-fused heteroaromatic rings of Hcyb1 and BAY60-7550 have
similar interactions with PDE2 and the phenyl and other substitution groups (we denote as R1 and
R2) can be further modified to lead more potent PDE2 inhibitors. Therefore, we designed and
synthesized 20 derivatives with purin-6-one scaffold.
The general routes for synthesizing derivatives 6a–6t are shown in Scheme 1.
Diaminomaleonitrile 1 was refluxed with triethylorthoformate in dioxane, to afford compound 2.
Compound 2 was reacted with the amines containing various desired substitutions to generate 3,
which was further treated with an aqueous solution of potassium hydroxide to obtain molecules 4.
The remaining cyano group was oxidized with 30% peroxide in aqueous ammonium hydroxide to
yield 5. Cyclization by reacting with esters that contain the desired substitutions in a solution of
dioxane/sodium hydride and heating generated the targeted derivatives 6a-6t.
Scheme 1. Synthesis of compounds 6. Reagents and conditions: (a) HC(OC2H5)3, 1, 4-dioxane,
reflux, 2 h; (b) R2NH2,EtOH, 30 °C overnight; (c) 1 mol/L KOH, rt, 0.5 h (65–75%); (d) NH4OH,
50% H2O2, EtOH, 30 °C 6 h; (e) R1COOC2H5, NaH, 1,4-dioxane.
The in vitro inhibitory activity of compounds 6a-6t against PDE2 is listed in Table 1. Our
initial exploration was focused on introducing different substituents in R2 of the purin-6-one

scaffold, while keeping the benzyl group (R1) fixed. As mentioned above, The propylphenyl group
of classic PDE2 inhibitor BAY60-7550 is accommodated very well through a hydrophobic induced
binding pocket located under L770 of the protein. Thus, for purin-6-one derivatives in this report,
only by bearing certain length and lipophilicity can the R2 group interact properly with the pocket.
Based on this rule, various R2 groups in Table 1 were selected to evaluated their effects on PDE2
inhibitory activities. Most of the derivatives have appropriate values of CLogP (2.0-5.0), indicating
good blood-brain barrier penetration. Compounds 6a-6k were tested for in vitro PDE2 inhibitory
activities and showed that R2 group remarkably affected the PDE2 inhibitory activities. The
substitutions with hydrophilic groups significantly sacrified the inhibitory potency, as seen in the
examples of compounds 6f and 6g that have the IC50 values of 17 μM and 40 μM, respectively.
Compound 6c did not inhibit PDE2 with IC50> 50μM. Then we introduced 4-benzyloxy (6l-6q)
group to the 4-position of the benzene of R1 and varies R2. Overall, most of these compounds
displayed good PDE2 inhibitory activities. Especially, compound 6p showed an IC50 of 72nM. And
substitution of R2 with 3,4-Dimethoxyphenethyl group (compound 6o) almost abolish the
inhibitory activity on PDE2, as shown by IC50 > 50 μM [Table 1]. The data showed that both of R1
and R2 groups can significantly affect the PDE2 inhibitory activities of the purin-6-one scaffold.
Replacement with the methoxybenzyl led to compounds 6r-6t, of which 6s also showed high
inhibitory potency against PDE 2 (IC50 = 81 nM).
The in vitro inhibitory selectivity of compounds 6p and 6s over other PDEs (PDE1, 4, 5, 9 and
10) was evaluated [Table 2]. The two compounds displayed > 5000-fold selectivity with IC50 > 500
μM against PDE 1, 4, 5, 10, and inhibited moderately PDE9 with IC50 values of 13.9 and 32.3 μM,
respectively. Although inhibition of 6p and 6s on PDE2 is less potent than BAY60-7550, they
achieved significant selectivity for closely-related PDE enzymes. For example, > 5000-fold
selectivity of 6p and 6s over PDE1, in comparison with 50-fold selectivity of BAY-7550 over
12
PDE1 represents a significant progress, and the poor selectivity over PDE1 for PDE2 inhibitors
could often lead to side effect of headache.
Table 1. In vitro PDE2 inhibitory activity of compounds 6a-6t
O
N
HN
N
R1
N
R2

PDE2 IC50
Compounds
R1
R2
CLogP
2.85
(μM)a
6a
0.31±0.023
3.99±0.29
6b
6c
3.41
2.81
> 50
6d
6e
5.94±0.47
4.32±0.32
3.36
2.31
S
6f
16.98±1.23
2.46
H3CO
H3CO
6g
6h
40.35±3.89
7.27±0.58
2.47
2.81
6i
1.24±0.08
4.29
6j
4.28±0.51
4.42
2.73
6k
11.02±1.33
H3CO
BnO
6l
6m
6n
3.91±0.36
5.46±0.60
5.05
3.99
4.50
BnO
BnO
5.8±0.57

H3CO
H3CO
BnO
BnO
6o
6p
>50
4.16
5.98
0.072±0.006
BnO
6q
6r
2.63±0.11
3.79±0.35
6.10
3.28
H3CO
H3CO
H3CO
6s
0.081±0.009
4.12
6t
1.94±0.20
2.22
2.64
Bay60-7550
4.8±0.20nM
a Results are expressed as the mean of at least three experiments.
Table 2. PDE selectivity of compounds 6p and 6s
PDE IC50 (μM)a
Compounds
PDE2
PDE1
> 500
> 500
PDE4
> 500
> 500
PDE5
> 500
> 500
PDE9
PDE10
> 500
> 500
6p
6s
0.072±0.006
0.081±0.009
13.9±1.5
32.3±3.9
a Results are expressed as the mean of at least three experiments.
Compound 6s was docked into the binding pocket of PDE2 by using Discovery Studio 2017.
The simulation indicated that the compound has a lower CDOCKER interaction energy when its R2
group bears the (R)-stereochemistry. As predicted, this compound binds to the catalytic domain in a
cGMP-like binding mode shown by the orientation of Gln859 [Figure 2]. Two hydrogen bonds
were observed between the carbonyl and lactam groups of compound 6s and Gln859. As shown in
Figure 2, the aromatic ring of the purin-6-one is anchored in the active binding site through a face-
face p-stacking interaction with Phe862 and Phe830, and these interactions are commonly referred
to in PDE nomenclature as the hydrophobic clamp region. The isooctyl group is situated in a

binding-induced hydrophobic pocket between Ile866 and Leu770 which is unique to PDE2 and has
been described in earlier publications.18 Also, the methoxybenzyl moiety of compound 6s interacts
with the surface hydrophobic patch of the binding pocket of PDE2, which can enhance the PDE2
inhibitors’ potency and selectivity. 18
Figure2. Binding mode of compound 6s (R: green, S: ) and BAY60-7550 (yellow) within the active
pocket of PDE2 (PDB ID: 4HTX). Hydrogen bonds are represented by dashed lines (blue).
HT-22 cells, which are immortalized mouse hippocampal neuronal precursor cells, were used
20
to understand cellular potency of 6p and 6s relevant to the hippocampus activity. To determine
the neuroprotective effect against corticosterone-induced toxicity, HT-22 cell viability in the
presence of 100 μM CORT (positive control) or absence of CORT (vehicle) and presence of various
concentrations of 6p and 6s was assayed. As shown in Figure 3, the cell viability was significantly
decreased (about 60% survived) when HT-22 cell was treated with 100 μM CORT (p < 0.001).
Compound 6s protected HT-22 cells from corticosterone-induced death at all the tested
concentrations between 0.01 and 1 μM, and the best concentration was 0.1 μM (p < 0.01). In
contrast, compound 6p did not show the similar effects. We speculated that the lack of activity of
6p in vitro may be attributed to its low cell membrane penetration resulting from higher molecular
weight compared with compound 6s, but it need more detailed study.

Figure 3. Compounds 6s and 6p protected cells against corticosterone (CORT)-induced
cytotoxicity in a concentration-dependent manner. (A) HT-22 cells were treated with 100 M CORT
for 30 min and Compound 6s was added for 24 hr. (B) HT-22 cells were treated with 100 M CORT
for 30 min and Compound 6p was added for 24 hr. Cell viability was measured by MTS assay.
Results are expressed as the mean ± standard error of the mean (SEM) of six independent
#
experiments performed in triplicates. ***p < 0.001, compared to control group. p < 0.05 and ##p <
0.01, compared to vehicle-treated CORT group.
To explore whether its cell protection activity is achieved via inhibition on PDEs, the effects of
compound 6s on accumulation of cGMP and cAMP in HT-22 cells were further evaluated. Cyclic
nucleotide (cAMP and cGMP) signaling in brain is fundamentally involved in mechanisms that
require for the neuronal activity and energy production, metabolic processes and synaptic
21,22
physiology.
A decrease in cGMP-dependent signal transduction has been demonstrated in
hippocampus during aging and Alzheimer’s disease. 23-25 Our results revealed a significant decrease
in cGMP expression when HT-22 cells were exposed to 100 μM corticosterone (p < 0.01). The
decrease of cGMP level was rescued by treatment with compound 6s at concentrations of 0.01, 0.1
and 1.0 μM, when compared to corticosterone-treated group (p < 0.05; p < 0.01; p < 0.05).
However, cAMP levels did not show significant changes even though there was slight increase.
These effects were similar to those of the positive control drug at concentration of 1.0 μM. The
present results may indicate that compound 6s probably protected HT-22 cells from corticosterone-
induced death by activation of cAMP and cGMP-dependent signaling.

Figure 4. The effects of Compound 6s on CORT-induced cGMP and cAMP reduced levels in
concentration-dependent manners in the HT-22 cells. HT-22 cells were treated with 100 M CORT
**
for 30 min and added compound 6s for 24 hr. The results represent the mean ± SEM, n = 6. p <
#
0.01, compared to control group. p < 0.05 and ##p < 0.01, compared to vehicle-treated CORT
group.
To detect if compound 6s has physiological effects in vivo, we examined the effects of
compound 6s in vivo and evaluated behavioral changes of chronic stress-induced mice using the
elevated plus-maze test. As shown in Figure 5, chronic stress induced a significant decrease in the
open-arm entry time (p < 0.001), and compound 6s reversed the effects in dose-dependent manner
in comparison to vehicle group. The maximum increases were observed at a dose of 2.0 mg/kg
(p.o.) [F(4, 32) = 16.23, p<0.01; F(4, 32) = 10.87, p<0.01]. The classical drug diazepam showed
similar anxiolytic-like effects, but Bay 60-7550 did not exhibited the effects at doses of 0.5 mg/kg
via i.p. Their locomotion counts were measured 10 minutes after a single treatment to rule out
whether compound 6s had any CNS stimulating or inhibiting effects on mice. The results showed
that none of doses of compound 6s affected locomotor activity, indicating the anxiolytic-like effects
of compound 6s were not due to central stimulation or inhibition. Due to the promising activity in
vivo, compound 6s was evaluated for pharmacokinetic properties [Table 3]. A preliminary
pharmacokinetic analysis revealed that compound 6s has Cmax of 511 ng/mL, Tmax of 0.6 h and T1/2
of 1.7 h in the oral administration mode. Especially, the compound achieved good brain exposure
(AUC of 646 h·ng/ml) and acceptable B/P ratio (B/P of 0.56). From these studies it could be
concluded that compound 6s, although less potent, had better pharmacokinetic profile than BAY60-
7550.

Figure 5. The effects of compound 6s on chronic stress-induced anxiety-like behaviors and
cognitive impairment in EPM (A and B) and LA (C) tests. The results represent the mean ± SEM, n
#
= 6. **p < 0.05, compared to control group. p < 0.05, ##p < 0.01, compared to vehicle-treated
stressed group. DZP (diazepam, 0.5 mg/kg, i.p.).
Table 3. Pharmacokinetic profile of compound 6s in micea
Compound
Parameters
O
T1/2 (h)
Tmax (h)
1.4 ± 0.16
0.53± 0.02
511 ± 83
646 ± 83
H3CO
N
N
HN
Cmax (ng/ml)
N
AUC0-∞ (h·n g /ml)
B/P ratiob
0.56 ± 0.03
a
b
Dose: 2 mg/kg, p.o. (oral administration). B/P ratio: Brain to plasma ratio at 0.5 h after oral
administration.
In summary, a series of purin-6-one derivatives with a new scaffold were designed and
synthesized, of which compounds 6p and 6s showed high inhibitory activities with the IC50 values
of 72 and 81 nM, respectively, and > 5000 fold selectivity over PDEs 1, 4, 5 and 10. Compound 6s
protected the HT-22 cells from the corticosterone-induced death and rescued corticosterone-induced
decrease of cAMP and cGMP. The in vivo study suggested that compound 6s dose-dependently
ameliorated acute stress-induced anxiety impairment and possessed acceptable brain uptake. Thus,

our study provided a basis for the rational design of novel PDE2 inhibitors with high affinity, and
subsequent efforts on further optimization of this structural class would lead to more potent and
selective PDE2 inhibitors with favorable pharmacokinetic properties.
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Design, synthesis of novel purin-6-one derivatives as phosphodiesterase 2 (PDE2) inhibitors:
the neuroprotective and anxiolytic-like effects
Xian-Feng Huang 1, Yi-Jing Cao 1, Jing Zhen 1, Da-Wei Zhang 2, Ren Kong 2, Wen- Tao Jiang 1,
Ying Xu 3, Guo-Qiang Song 1*, Heng-Ming Ke 4*, Li Liu 1*
1School of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou,
Jiangsu, 213164, PR China
2Institute of Bioinformatics and Medical Engineering, School of Electrical and Information
Engineering, Jiangsu University of Technology, Changzhou, Jiangsu, 213001, PR China
3Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The
University of North Carolina, Chapel Hill, NC 27599-7260, USA
4Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, the
State University of New York at Buffalo, Buffalo, NY 14214, USA