Discovery of effective phosphodiesterase 2 inhibitors with antioxidant activities for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2021.128016

The multi-target-directed-ligand (MTDL) strategy has been widely applied in the discovery of novel drugs for the treatment of Alzheimer's disease (AD) because of the multifactorial pathological mechanisms of AD. Phosphodiesterase-2 (PDE2) has been identified to be a novel and promising target for AD. However, MTDL combining with the inhibitory activity against PDE2A and other anti-AD factors such as antioxidants has not been developed yet. Herein, a novel series of PDE2 inhibitors with antioxidant capacities were designed, synthesized, and evaluated. Most compounds showed remarkable inhibitory activities against PDE2A as well as antioxidant activities. Compound 6d was selected, which showed good IC₅₀ of 6.1 nM against PDE2A, good antioxidant activity (ORAC (Trolox) = 8.4 eq.) and no cytotoxicity to SH-SY5Y cells. Molecular docking and dynamics simulations were applied for the rational design and explanation of structure–activity relationship (SAR) of lead compounds.

Full text of DOI:10.1016/j.bmcl.2021.128016

Bioorg. Med. Chem. Lett. 41 (2021) 128016
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of effective phosphodiesterase 2 inhibitors with antioxidant
activities for the treatment of Alzheimer’s disease
Mei-Yan Jiang, Chuan Han, Chen Zhang, Qian Zhou, Bei Zhang, Mei-Ling Le, Meng-Xing Huang,
Yinuo Wu*, Hai-Bin Luo
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The multi-target-directed-ligand (MTDL) strategy has been widely applied in the discovery of novel drugs for the
treatment of Alzheimer’s disease (AD) because of the multifactorial pathological mechanisms of AD.
Phosphodiesterase-2 (PDE2) has been identified to be a novel and promising target for AD. However, MTDL
combining with the inhibitory activity against PDE2A and other anti-AD factors such as antioxidants has not been
developed yet. Herein, a novel series of PDE2 inhibitors with antioxidant capacities were designed, synthesized,
and evaluated. Most compounds showed remarkable inhibitory activities against PDE2A as well as antioxidant
activities. Compound 6d was selected, which showed good IC50 of 6.1 nM against PDE2A, good antioxidant
activity (ORAC (Trolox) = 8.4 eq.) and no cytotoxicity to SH-SY5Y cells. Molecular docking and dynamics
simulations were applied for the rational design and explanation of structure–activity relationship (SAR) of lead
compounds.
Alzheimer’s disease (AD)
Phosphodiesterase-2 inhibitor
Antioxidant capacity
Multi-target-directed-ligand (MTDL)
Alzheimer’s disease (AD) is an age-related disease with irreversible
neurodegenerative disorder, which is characterized by progressive
memory loss, decline in language skills, and cognitive dysfunction.1
According to World’s Alzheimer Report in 2018,2 there were close to 50
million people suffering from Alzheimer’s disease in 2018. The number
will be doubled in every 20 years, reaching 75 million in 2030 and 131.5
million in 2050. In 2018, the treatment expenses for dementia patients
worldwide have already been above US$ 1 trillion. However, there are
still approximately three quarters of people with dementia that have not
received diagnosis yet.
failed.7 One important reason for the failure might be the complicated
pathogenic progression of AD. Thus, multi-target-directed-ligands
(MTDLs), which control more than one pathogenic factor of AD simul-
taneously, have drawn scientists’ attention and been widely developed
for the treatment of AD.
Phosphodiesterase-2 (PDE2), belonging to the phosphodiesterase
(PDE) super-enzyme family, could hydrolyze both cyclic adenosine
monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP).8
PDE2A, the only isoform of PDE2, is highly expressed in brain, but keeps
a low level in most peripheral tissues.9 It has been reported that
compared with normal persons, the level of PDE2A increased in the
brain of AD patients. PDE2 inhibitors can increase the concentrations of
cAMP and cGMP via the inhibition of PDE2, and thus activating protein
kinase A (PKA), protein kinase G (PKG). The cAMP-responsive element
binding protein (CREB),10,11 a molecular switch in charge of regulating
the learning and memory, could be phosphorylated by the cAMP/ PKA
or/and cGMP/PKG signaling. Thus, PDE2 has been regarded as a novel
and promising therapeutic target for AD.12 Some PDE2 inhibitors have
entered the preclinical and clinical studies (Fig. 1).13 For example, BAY-
60-7550, a PDE2 inhibitor developed by Bayer company, could enhance
the long-term potentiation of synaptic transmission without altering
basal synaptic transmission.14 PF-999 improved short-term synaptic
The pathogenesis of Alzheimer’s disease has been widely studied in
these years but the exact etiology is still unclear.3 Factors such as low
levels of acetylcholine, deposition of β-amyloid (Aβ) plaques, aggrega-
tion of Tau protein, oxidative stress, and the accumulation of biometals
have been considered to be important in the pathophysiology of AD.4,5
Currently, three acetylcholine inhibitors (Donepezil, Rivastigmine, and
Galantamine) and one N-methyl-D-aspartate (NMDA) receptor antago-
nist (Memantine) been used in the clinical practice.6 However, these
drugs could only improve AD patients’ cognition and the degree of de-
mentia but not reverse the progression of AD. On the other hand,
numerous anti-AD bioactive compounds were developed based on the
factors such as deposition of β-amyloid (Aβ) plaques but most of them
* Corresponding author.
E-mail address: wyinuo3@mail.sysu.edu.cn (Y. Wu).
https://doi.org/10.1016/j.bmcl.2021.128016
Received 7 February 2021; Received in revised form 30 March 2021; Accepted 2 April 2021
Available online 8 April 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

M.-Y. Jiang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128016
Fig. 1. Current PDE2 inhibitors for the treatment of AD.
Q812
F862
Y655
N
N
H
Zn2+
N
Q812
I866
L774
Y827
N
O
N
L774
F862
Mg2+
L770
Y655
N
Zn2+
F830
L770
Co-crystal Structure
N
N
N
1
Mg2+
R
H
N
I826
OH
Y827
Cl
F830
Molecular docking
O
OH
O
HO
OH
OH
HO
OH
HO
OMe
OMe
OMe
Ferulic acid
OH
Resveratrol
Curcumin
Fig. 2. The design strategy of the MTDLs with PDE2 inhibition as well as antioxidant capacities.
plasticity by regulating the hydrolysis of cAMP to accommodate changes
in cGMP levels associated with presynaptic short-term plasticity.15,16
Two PDE2 inbihitors ND-7001 and TAK-915 have already finished
several phase I clinical trials.17,18 However, compared with other PDE
families such as PDE4 and PDE5, the developments of PDE2 inhibitors
are relative limited.19 MTDLs combining the PDE2 inhibition as well as
hitting at other pathophysiology of AD simultaneously have not been
reported yet.
forming hydrophobic interactions with the lipophilic residues Leu770
and Leu774, providing rooms for the replacement of substituents with
antioxidant activities (Fig. 2). In addition, the oxygen atom in the amide
group forms a hydrogen bond with a fourth water molecule that is
unconservative. This alternative amide group is then replaced by an
amino one in our design as amide derivatives may have low brain
uptake.27
The phenol structure exists in a variety of antioxidants such as
resveratrol, ferulic acid and curcumin.28,29 We speculated that intro-
ducing the fragment of phenol at the [1,2,4]triazolo[4,3-a]quinoxaline
cores might result in a series of potential anti-AD agents with inhibitory
activities against PDE2 and antioxidant activities. Thus, a series of
[1,2,4]triazolo[4,3-a]quinoxaline derivatives with different substituted
phenyl were designed and synthesized (Fig. 2 and Scheme 1). Molecular
docking and dynamics simulations results suggested that the phenol
group did not impact inhibitory activities of targeted compounds against
PDE2 (Fig. S1). For further prediction of the interaction strength be-
tween the inhibitors and PDE2, binding free energies were performed
based on the molecular dynamics simulation results (Table 1). The
calculated binding free energies for targeted compounds to PDE2A are
all negative than ꢀ 21.8 kcal/mol (ranging from ꢀ 21.88 to –23.80 kcal/
mol), suggesting that all these compounds have strong interactions with
PDE2A.
Oxidative stress is a condition produced by the imbalance between
oxidants and antioxidants because of the excessive generation of reac-
tive oxygen species (ROS) and reactive nitrogen species (RNS).20 The
levels of some oxidation markers were found to be increased in AD pa-
tients brains. Thus, oxidative stress has been considered to be involved
in the pathological mechanisms of AD and antioxidants have been
studied for the treatment of AD.21 However, the clinical trials suggested
that only antioxidant was not an effective therapeutic treatment for AD.
MTDLs combing antioxidants with other pharmacological effects might
be more efficient.22,23
With our continuing interests in the discovery of MTDLs for the
treatment of AD,23,24 herein, we report the design, synthesis, and eval-
uation of a novel series of [1,2,4]triazolo[4,3-a]pyrazines compounds
with good inhibitory activities against PDE2 as well as antioxidant ac-
tivities. Molecular docking and dynamics simulations were used at the
beginning for the rational design and the explanation of structur-
e–activity relationship of lead compounds, greatly increasing the effi-
ciency of lead discovery.
The synthetic routes for targeted compounds 6a-6j, 7a-7d, 8a and 8b
were outlined in Scheme 1. 4-nitrobenzene-1,2-diamine was used as the
starting material, which reacted with methyl pyruvate to yield inter-
mediate 2 in the hydrochloric acid solution. Intermediate 2 converted
into intermediate 3 in the presence of POCl3. Intermediate 3 reacted
with 2-chlorobenzhydrazide, giving intermediate 4 in good yield, which
was then reduced by Raney nickel and H2 to afford 5. The targeted
compounds 6a-6j and 7a-7d were obtained by Borch reduction in the
presence of sodium triacetoxyborohydride. Compounds 8a and 8b were
obtained by aldimine condensation without reducer.
A series of effective PDE2 inhibitors possessing pyrido[4,3-e][1,2,4]
triazolo[4,3-a]pyrazines cores were reported by Rombouts and cow-
orkers.25–27 According to the X-ray co-crystal structure of PDE2A with
one lead compound 1,25 the triazole core is sandwiched by Phe830 and
Phe862 in the hydrophobic clamp, forming proper
π-π stacking.
Hydrogen bond interactions can be observed between the [1,2,4]tri-
azolo[4,3-a]quinoxaline core and Tyr655/Gln812 intermediated by two
conservative water molecules. Moreover, N5-postion of the core also
forms a hydrogen bond with Tyr827 through a third water molecular. N-
benzylamide moiety stretches outward from the catalytic pocket,
The inhibitory activities of compounds 6a-6j, 7a-7d, 8a and 8b
against PDE2A were tested according to the reported methods. BAY-60-
7550, a PDE2 inhibitor developed by Bayer company works as the
2

M.-Y. Jiang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128016
Scheme 1. The synthetic routes of targeted compounds. Reagents and conditions: (a) methyl pyruvate, HCl (20%), rt; (b) POCl3, 100 ◦C; (c) 2-Chlorobenzhydrazide,
BuOH, 160 ◦C; (d) Raney nickel, H2, rt; (e) substituted benzaldehyde, NaBH(OAc)3, 1,2-DCE, 40–80 ◦C; (f) substituted 2-pyridylaldehyde, NaBH(OAc)3, 1,2-DCE,
40–80 ◦C; (g) substituted benzaldehyde, MeOH, 40 ◦C.
reference in the assay (Lit: IC50 = 4.7 nM).14 All the results are sum-
marized in Table 1.
The antioxidant activities of targeted compounds were evaluated by
oxygen radical absorbance capacity assay (ORAC-FL).30 The values were
expressed as Trolox equivalents. Melatonin was used as the positive
reference compound, showing an ORAC value of 2.0 Trolox equivalents.
Most targeted compounds showed excellent antioxidant activities, with
ORAC values ranging from 0.2 to 8.6 Trolox equivalents. Compared with
compound 6a, compounds with substituted phenol group at R position
gave enhanced antioxidant activities except compound 6j with a bromo
atom. Compounds 6d-6f with fluorine on the phenol groups gave the
best antioxidant activities, with the ORAC values of 8.4, 8.1 and 8.6
Trolox equivalents, respectively. Compounds 7a with pyridyl group at R
position gave the ORAC value of 5.5 Trolox equivalent while compound
7b with an additional chloro atom gave a decreased ORAC value of 1.9
Trolox equivalent.
As expected, most designed compounds were proved to be effective
PDE2A inhibitors with the IC50 below 10 nM. The substitutions on the
phenol ring didn’t show significant impact on the inhibitory activities
against PDE2A. Compared with compound 6a with no substitution at the
R position, Compounds 6b-6h with different substituted phenol groups
showed comparable inhibitory activities against PDE2A. However,
compound 6c with CF3O, compound 6i with two chloro atoms and
compound 6j with a bromo atom on the phenol groups, showed relative
weaker inhibitory activities possible due to the steric hindrance effects.
Compounds 7a and 7b with pyridyl groups were also synthesized and
both of them gave the IC50 value of 11 nM against PDE2A. We also
synthesized compounds 7c and 7d with phenyl groups at the R position,
which gave comparable IC50 values as compounds 6d and 6f. These
results are in accordance with our molecular docking results that the
phenolic hydroxyl groups have no significant effects on the inhibitory
activities against PDE2. The two imine compounds 8a and 8b gave
excellent inhibitory activities with the IC50 of 11 and 4.8 nM,
respectively.
As is mentioned above, the hydrogen of the phenol group might be
the main source for the antioxidant activity. The results of compounds
7c and 7d confirmed this speculation. Compared with compounds 6d
and 6f with the hydrogen groups, the antioxidant activities of com-
pounds 7c and 7d almost disappeared. However, the two imine com-
pounds 8a and 8b, which have the hydrogen groups, also gave low
3

M.-Y. Jiang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128016
ORAC value of 0.3 and 0.2 Trolox equivalents, respectively. We specu-
lated that the hydrogen groups of compounds 8a and 8b might form
intramolecular hydrogen bonds with the nitrogen atom on the imine
double bonds, which cannot act as the active groups for antioxidant
activities.
Table 1
The predicted binding free energies, inhibitory activities against PDE2A and
oxygen radical absorbance capacity of compounds 6a-6j, 7a-7d, 8a and
N
R
N
N
The cytotoxicity of compounds 6d and 6f were tested using human
neuroblastoma cell line SH-SY5Y by the CCK8 method.31 As shown in
Fig. 3, after incubation for 48 h, the cellular viability of compound 6d
8b.
N
Cl
was about 53% at 50
μ
M and 39% at 100
μ
M. The cellular viability of
M. These results
Compound
R
Binding Free
Energy (kcal/
mol) a
PDE2A
ORAC c
compound 6f was about 23% at 50
μ
M and 10% at 100
μ
IC50 (nM) b
suggested that the cell viability of compound 6d is better than that of 6f.
Combining with the excellent inhibitory activity against PDE2 and
antioxidant activity, compound 6d is suitable for the further investiga-
tion for the treatment of AD.
6a
6b
6c
6d
6e
6f
ꢀ 22.91 ± 3.08
ꢀ 23.80 ± 3.07
ꢀ 22.59 ± 2.62
ꢀ 23.18 ± 2.49
ꢀ 22.94 ± 2.74
ꢀ 23.45 ± 3.07
7.3 ± 0.4
6.4 ± 0.5
15 ± 0.7
6.1 ± 0.1
9.1 ± 0.9
4.2 ± 0.6
3.7 ±
N
0.3
H
OH
As is mentioned above, the predicted binding patterns, and binding
free energies of all these PDE2 inhibitors were performed by molecular
docking and dynamics simulations (Fig. S1 and Table 1). The predicted
results are in accordance with the experimental results.
8.0 ±
MeO
N
0.7
H
OH
5.2 ±
F3CO
N
0.3
H
Compound 6f gave the best IC50 value against PDE2A among all the
targeted compounds. From the docking pattern of 6f with PDE2A
(Fig. 4), the [1,2,4]triazolo[4,3-a]quinoxaline group functions as the
scaffold to make indirect hydrogen bond interactions with residues
Gln859/Tyr827/Gln812/Tyr655 via three water molecules. Besides,
OH
8.4 ±
N
1.7
H
F
OH
8.1 ±
F
N
0.2
direct π-π/π-σ interactions between Phe862/Ile826/Phe830 and 6f are
H
OH
also observed. The 2-chlorophenyl group is nearly perpendicular to the
triazoloquinoxaline core, which forms hydrophobic interactions with
neighboring residues such as Leu770 and Tyr655. The benzylamine
group at 8-position of the triazoloquinoxaline core stretches outward
from the catalytic pocket, possessing a similar pose to 1 in the co-crystal
structure. It is worth mentioning that the 6-F substituted phenolic group
is suitable for a small pocket formed by four hydrophobic residues
Phe862/Ile866/Leu770/Lue774.
8.6 ±
F
0.4
N
H
OH
6g
6h
ꢀ 23.07 ± 2.84
ꢀ 22.98 ± 2.48
7.6 ± 0.4
9.2 ± 2
6.9 ±
N
0.3
H
Cl
OH
3.9 ±
Cl
N
0.1
H
Compared with compound 6f, 6i gave a relatively poor IC50 value
against PDE2A. The binding free energies between 6i-PDE2A and 6f-
PDE2A complexes are –22.40 and –23.45 kcal/mol, respectively, with a
difference of 1.05 kcal/mol in accordance with the 4-fold enhanced
PDE2A inhibitory activity from 6i to 6f (IC50 values from 22 nM to 4.2
nM). The docking results showed that compound 6i has a similar binding
pattern to 6f for its triazoloquinoxaline core (Fig. 4). However, the 3,5-
Cl substituted phenolic group of 6i stretches in another direction
because of steric hindrance, which fails to make interactions with the
small hydrophobic pocket, thus resulting in decreased PDE2A inhibition.
The effect of steric hindrance also explains the overall decreased
PDE2A inhibitions such as 6b, 6c, 6e, 6h and 6j with different m-
substituted benzylamine groups, or 6d, 6g and 7c with p-substituted
ones (Fig. S1). Polar pyridine ring of compound 7a or 7b weakens the
hydrophobic interactions with the small pocket and leads to decreased
PDE2A inhibition in contrast to 6f. Compounds 8a and 8b possess
different binding patterns in their planar imine groups compared with
all other compounds, which also fail to make interactions with the small
pocket (Fig. S1). The better inhibition of 8b than 8a on PDE2A might be
due to additional interactions with Ile826 and Phe830.
OH
6i
6j
ꢀ 22.40 ± 2.62
ꢀ 22.67 ± 2.24
22 ± 3
15 ± 4
6.8 ±
Cl
N
0.1
H
OH
Cl
2.6 ±
Br
N
0.1
H
OH
7a
7b
ꢀ 21.88 ± 2.13
ꢀ 22.58 ± 2.29
11 ± 4
5.5 ±
N
H
0.2
N
11 ± 0.7
1.9 ±
N
0.2
H
N
Cl
7c
7d
8a
8b
ꢀ 22.80 ± 2.14
ꢀ 23.19 ± 2.41
ꢀ 22.16 ± 2.26
ꢀ 23.16 ± 2.95
11 ± 2
5.6 ± 0.1
11 ± 5
0.2 ±
N
0.1
H
F
0.6 ±
N
H
0.1
F
0.3 ±
N
0.1
In summary, a series of 1-aryl-4-methyl[1,2,4]triazolo[4,3-a]qui-
noxaline derivatives were designed as novel multi-target-directed-
ligands with both inhibition against PDE2 and antioxidant activities.
Compound 6d exhibited excellent inhibitory activities against PDE2
(IC50 = 6.1 nM) and antioxidant capacity (ORAC value = 8.4 Trolox
equivalent), which is suitable for the further development of in vivo
study of AD. Molecular docking and dynamics simulations were used at
the beginning for the rational design and the explanation of structur-
e–activity relationship of lead compounds. The predicted results are in
accordance with the experimental data, which gave evidence for the
further rational design.
OH
4.8 ± 0.1
0.2 ±
N
0.1
F
OH
1
1.7 d
4.7 e
BAY-60-
7550
Melatonin
2.0 ±
0.3
a The binding free energies were estimated by MM-GBSA method. b The means ±
SD of at least two independent experiments. c The data are expressed as
μmol of
Trolox equiv/
μ
mol tested compound. Trolox serves as a standard.28,29 d Data was
obtained from the reported literature.14 e Data was obtained from the reported
literature.25
4

M.-Y. Jiang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128016
6d
6f
120
100
80
60
40
20
0
120
100
80
60
40
20
0
***
**
****
****
****
****
****
****
Control3.125 6.25 12.5
25
50
100
Control3.125 6.25 12.5
25
50
100
Concentration (μM)
Concentration (μM)
Fig. 3. SH-SY5Y cell viability of compounds 6d and 6f with various concentrations by CCK8 assay. Data were shown as mean ± SEM of three independent ex-
periments (**P < 0.01, ***P < 0.001, ****P < 0.0001, compared to control group).
Q812
F862
Q812
F862
I866
I866
L774
L774
Y655
Y655
Zn2+
L770
Mg2+
Zn2+
L770
Mg2+
I826
I826
Y827
Y827
F830
F830
Q812
F862
Q812
F862
I866
I866
L774
L774
Y655
Y655
Zn2+
L770
Mg2+
Zn2+
L770
Mg2+
I826
I826
Y827
Y827
F830
F830
Fig. 4. (A) The binding pattern of compound 6f with PDE2A by molecular docking. (B) Superposition of 6f (green) over the structure of the 1 (yellow) in PDE2A
(PDB ID: 4D09). (C) The binding pattern of compound 6i with PDE2A by molecular docking. (D) Superposition of 6i (pink) over the structure of the 6f (green) in
PDE2A. Hydrogen bonds are depicted in dashed green lines.
Declaration of Competing Interest
(2019A1515011883), Natural Science Foundation of China (21977127
and 82003652), China Postdoctoral Science Foundation funded project
(2020T130152ZX). We cordially thank Prof. H. Ke from Department of
Biochemistry and Biophysics at University of North Carolina, Chapel
Hill, for his assistance with the molecular cloning, expression, purifi-
cation, determination of the crystal structures, and bioassay of PDEs.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Appendix A. Supplementary data
We cordially thank anonymous reviewers’ valuable comments and
kind proofreading of the manuscript. This work was supported by the
Guangzhou Pearl River New Star Fund Science and Technology Planning
Project (201806010190), Science Foundation of Guangdong Province
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128016.
5

M.-Y. Jiang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128016
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