Discovery of novel PDE9A inhibitors with antioxidant activities for treatment of Alzheimer’s disease

Article abstract of DOI:10.1080/14756366.2017.1412315

Phosphodiesterase-9 (PDE9) is a promising target for treatment of Alzheimer’s disease (AD). To discover multifunctional anti-AD agents with capability of PDE9 inhibition and antioxidant activity, a series of novel pyrazolopyrimidinone derivatives, couplin

Full text of DOI:10.1080/14756366.2017.1412315

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Discovery of novel PDE9A inhibitors with
antioxidant activities for treatment of Alzheimer’s
disease
Chen Zhang, Qian Zhou, Xu-Nian Wu, Yat-sen Huang, Jie Zhou, Zengwei Lai,
Yinuo Wu & Hai-Bin Luo
To cite this article: Chen Zhang, Qian Zhou, Xu-Nian Wu, Yat-sen Huang, Jie Zhou, Zengwei Lai,
Yinuo Wu & Hai-Bin Luo (2018) Discovery of novel PDE9A inhibitors with antioxidant activities for
treatment of Alzheimer’s disease, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1,
260-270, DOI: 10.1080/14756366.2017.1412315
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Date: 24 December 2017, At: 17:58

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 260–270
https://doi.org/10.1080/14756366.2017.1412315
RESEARCH PAPER
Discovery of novel PDE9A inhibitors with antioxidant activities for treatment of
Alzheimer’s disease
a
a
a
a
a
Chen Zhang , Qian Zhou , Xu-Nian Wu , Yat-sen Huang , Jie Zhou , Zengwei Laia,b, Yinuo Wua and
ꢀ
ꢀ
Hai-Bin Luoa,c
b
aSchool of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, PR China; State Key Laboratory for Chemistry and Molecular
c
Engineering of Medicinal Resources, Guangxi Normal University, Guilin, PR China; Collaborative Innovation Center of High Performance
Computing, National University of Defence Technology, Changsha, PR China
ABSTRACT
ARTICLE HISTORY
Received 20 July 2017
Revised 28 October 2017
Accepted 29 November 2017
Phosphodiesterase-9 (PDE9) is a promising target for treatment of Alzheimer’s disease (AD). To discover
multifunctional anti-AD agents with capability of PDE9 inhibition and antioxidant activity, a series of novel
pyrazolopyrimidinone derivatives, coupling with the pharmacophore of antioxidants such as ferulic and lip-
olic acids have been designed with the assistance of molecular docking and dynamics simulations. Twelve
out of 14 synthesised compounds inhibited PDE9A with IC50 below 200 nM, and showed good antioxidant
capacities in the ORAC assay. Compound 1h, the most promising multifunctional anti-AD agent, had IC50
of 56 nM against PDE9A and good antioxidant ability (ORAC (trolox) ¼ 3.3). The selectivity of 1h over other
PDEs was acceptable. In addition, 1h showed no cytotoxicity to human neuroblastoma SH-SY5Y cells. The
analysis on structure-activity relationship (SAR) and binding modes of the compounds may provide insight
into further modification.
KEYWORDS
Phosphodiesterase-9; multi-
functional anti-AD agents;
antioxidant activity;
Alzheimer’s disease;
molecular docking
Introduction
development of multifunctional anti-AD agents which simultan-
eously hit more than single target in the AD pathophysiology, has
become an important research area in recent years6,7, especially
those hitting at non-Ab targets.
Alzheimer’s disease (AD) is the most common type of dementia,
which is characterised by progressive memory loss, decline in lan-
guage skills, and other neurodegenerative disorders1. According to
Alzheimer’s Association (USA), about 46.8 million people world-
wide are suffering from AD. The morbidity and mortality are high,
especially for elder persons above 60 years old. The global societal
economic cost for AD patients was estimated at $818 billion in
2015 and will rise to $1000 billion in 2018, and $2000 billion in
20302. Currently, AD has been one of the most challenges for pub-
lic health around the world, severely impacting lives of patients
and their families.
The pathogenic mechanism of Alzheimer’s disease is compli-
cated and still has not fully understood yet. Several factors, such
as low level of acetylcholine, deposition of b-amyloid (Ab), aggre-
gation of Tau-protein, and oxidative stress have been regarded as
important in the pathophysiology of AD3. Current drugs for
the treatment of AD include three acetylcholinesterase (AChE)
inhibitors (donepezil, rivastigmine, and galantamine) and one
N-methyl-d-aspartate receptor antagonist (memantine)4. However,
administration of these drugs only can improve cognition and
dementia degree of AD patients, but not reverse the underlying
progression. In recent decades, discovery of anti-AD candidate
drugs mainly focus on the anti-Ab pathology. However, most clin-
ical trials using these candidates failed5. One important reason for
this failure might be that the single targeted drugs were not able
Phosphodiesterases (PDEs) are a super-enzyme family in charge
of hydrolysing the second messengers cyclic adenosine mono-
phosphate (cAMP) and cyclic guanosine monophosphate
(cGMP)8,9. According to their structural and functional properties,
PDEs can be divided into 11 subfamilies (PDE1–PDE11). In brain,
the levels of cAMP and cGMP are increased via the inhibition of
PDEs, activating AC/cAMP/PKA, or NO/cGMP/PKG signalling path-
way, and thus increasing the level of the cAMP response element-
binding protein (CREB), enhancing synaptic transmission, and
reducing cognitive deficit10. PDEs inhibitors of PDE1, PDE2, PDE4,
PDE5, and PDE9 have been demonstrated to be effective in restor-
ing cognitive deficits in some preclinical models and clinical trials
of AD11.
Among all the PDE subfamilies, Phosphodiesterase 9 (PDE9) has
unique advantages on AD therapy for its highest affinity with
cGMP among all the PDE families and its high expression level in
the cortex, hippocampus, basal ganglia, and cerebellum of brain12.
Currently, the effects of PDE9 inhibitors in the AD animal models
have been deeply explored. In the social and object recognition
tasks of rodents, PDE9 inhibitor BAY73–6691 enhanced the ability
of acquisition, consolidation, and retention of long-term potenti-
ation (LTP), which also improved scopolamine-induced passive
to control the complicated pathogenic progression of AD. Thus, avoidance deficit and MK-801-induced short-term memory
CONTACT Yinuo Wu
wyinuo3@mail.sysu.edu.cn; Zengwei Lai
laizengw3@mail.sysu.edu.cn
School of Pharmaceutical Sciences, Sun Yat-sen University,
Guangzhou, PR China
ꢀ
These authors contribute equally.
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
261
deficits13,14. In comparison with AChE inhibitor donepezil that only from commercial suppliers and used directly without further
enhanced short memory in the early LTP, BAY73–6691 not only
increased both early and late LTP in rat hippocampal slices, but
also transformed the early LTP into late LTP15. In addition,
BAY73–6691 protected Ab25–35-induced oxidative damage in
hippocampus via the activation of the cGMP-related NO-depend-
ent signalling16. Besides BAY73–6691, PDE9 inhibitors PF-
04447943 also exhibited memory improvement in several AD
models17,18. Currently, PF-04447943 has already completed six
phase I clinical trials and one phase II trial of AD19. Despite good
effects on memory improvement obtained by PDE9 inhibitors in
the preclinical and clinical trials, the number of PDE9 inhibitors for
treatment of AD is very limited20,21, especially those combining
the PDE9 inhibition as well as hitting at other pathophysiology of
AD simultaneously.
purification.
Cyclopentyl-4-cyano-1h-pyrazol-5-amine (M-3)
To a solution of cyclopentylhydrazinedihydrochloride M-2 (2.7g,
20 mmol) in ethanol (20 ml), triethylamine (7.1g, 70 mmol) was
added slowly at 0 ꢁC. After stirring for 2 h, 2-(methoxymethylene)-
malononitrile M-1 (2.4 g, 22 mmol) was added to the mixture drop-
wise. The mixture was then stirred at room temperature for 3 h and
refluxed at 80ꢁC for 3 h. After the reaction was finished, ethanol
was evaporated to half of its volume. Water was added and the
resulted precipitate was collected as a brown solid. The brown solid
was purified by recrystallisation with ethanol as the solvent, provid-
ing compound M-320,27 as a yellow solid (3.0g, 85%). 1H NMR
(400 MHz, DMSO) d 8.11 (s, 1H), 5.51 (d, J ¼ 6.8 Hz, 1H), 4.50–4.38
(m, 1H), 3.39 (s, 1H), 2.03–1.93 (m, 2H), 1.88–1.79 (m, 2H), 1.76–1.68
(m, 2H), 1.63–1.53 (m, 2H). 13C NMR (101 MHz, DMSO) d 157.33,
134.01, 114.78, 76.01, 62.18, 31.82, 23.64. HRMS (ESI-TOF) m/z
[M þ H]þ calcd for C9H13N4 117.1143, found 117.1140.
Oxidative stress is an important risk factor in the AD onset and
progression22. The abnormality of redox state may participate in
the neurodegenerative process, resulting in reactive oxygen spe-
cies (ROS) mediated and reactive nitrogen species (RNS) mediated
impairment in AD brain. Furthermore, the levels of oxidative
markers of biomolecules including proteins, lipids, carbohydrates,
and nucleic acids, as well as the antioxidant enzymes, were
observed to be changed. Numbers of antioxidants such as resvera-
trol, ferulic acid have been proved to be neuroprotectants.
However, results from the clinical trials suggested that only anti-
oxidant efficacy was not sufficient to modify AD progression23.
Antioxidants combining with additional pharmacological effects
might combat the complicated pathogenic mechanism of AD
more efficiently. Thus, numerous multifunctional anti-AD agents
with antioxidant activity and also hitting other targets in the AD
pathophysiology were developed. Some of them even went into
clinical trials24. Vinpocetine, a moderate PDE1 inhibitor with anti-
oxidant activity, significantly improved learning and memory in
the streptozotocin infused AD rat models. Further studies demon-
strated that both the regulation of cyclic nucleotide signalling and
antioxidant mechanism were responsible for the memory improve-
ment obtained by vinpocetine25. For unique advantages of PDE9
inhibition over other PDEs, development of PDE9 inhibitors with
antioxidant activity, to regulate both cGMP signalling and antioxi-
dant mechanism is of great importance20.
5-Amino-1-cyclopentyl-1H-pyrazole-4-carboxamide (M-4)
To a solution of compound M-3 (1.8 g, 10 mmol) in ethanol
(15 ml), 30% hydrogen peroxide (1.5 ml), and 25% ammonium
hydroxide (4.0 ml) were added sequentially. The mixture was
stirred at room temperature for 1 h. When the reaction was fin-
ished, saturated sodium thiosulfate was added and a precipitate
was formed. The precipitate was filtered, washed with water three
times, and dried over vacuum to give compound M-420,27 (1.7 g,
86%) as a white solide. 1H NMR (400 MHz, DMSO) d 7.63 (s, 1H),
7.16 (br s, 1H), 6.62 (br s, 1H), 6.13 (m, 1H), 4.57–4.45 (m, 1H), 3.39
(s, 1H), 2.00–1.87 (m, 2H), 1.87–1.72 (m, 4H), 1.63–1.50 (m, 2H). 13C
NMR (101 MHz, DMSO) d 165.73, 148.24, 136.27, 96.17, 55.30,
30.65, 23.48. HRMS (ESI-TOF) m/z [M þ H]þ calcd for C9H15N4O
195.1242, found 195.1246.
(R)-benzyl(1–(1-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl)carbamate (M-5)
With our continuing interest in discovery of PDE9 inhibitors26
,
we reported here the design, synthesis, and biological evaluation
of novel pyrazolopyrimidinone derivatives with both PDE9 inhib-
ition and antioxidant activity. Molecular docking and dynamics
simulation were applied at the beginning to choose potential lead
compounds with appropriate binding modes and binding free
energies, in order to reduce the next synthetic work and improve
the efficiency of lead discovery.
NaH (60% dispersion in mineral oil, 0.80 g, 30 mmol) was added to
the solution of M-4 (1.9 g, 10 mmol) and (R)-ethyl-2-(((benzyloxy)-
carbonyl)amino)propanoate (7.5 g, 30 mmol) in anhydrous THF
(40 ml). The mixture was stirred at room temperature overnight.
Water was added slowly to the mixture. The mixture was then
extracted with ethyl acetate three times, washed with brine, dried
over Na2SO4, and concentrated. The residue was purified by silica
gel chromatography (methanol/dichloromethane 1:100) to afford
compound M-520 as
a
yellow oil (2.0 g, 42%). 1H NMR
Materials and methods
(400 MHz,CDCl3) d 11.90 (s, 1H), 8.05 (s, 1H), 7.32–7.28 (m, 5H),
5.98 (d, J ¼ 6.1 Hz, 1H), 5.23–5.05 (m,3H), 4.98–4.83 (m, 1H), 2.10
(dd, J ¼ 13.6, 6.9 Hz, 4H), 2.02–1.92 (m, 2H), 1.79–1.69 (m, 2H), 1.60
(d, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) d 160.26, 159.79,
155.83, 151.90, 136.12, 134.56, 128.50, 128.18, 104.41, 67.18, 57.89,
50.43, 32.47, 24.78, 20.56. HRMS (ESI-TOF) m/z [M þ H]þ calcd for
C20H24N5O3 382.1880, found 382.1879.
Chemistry
1H NMR and 13C NMR spectra were recorded on a Bruker BioSpin
GmbH spectrometer (Bruker, Billerica, MA) at 400.1 and 100.6 MHz,
respectively. Coupling constants (J) were reported in Hertz (Hz).
Spectra were referenced internally to the residual proton reson-
ance in CDCl3 (d 7.26 ppm) or DMSO (d 2.50 ppm) as the internal
standard. High-resolution mass spectra (HRMS) were obtained on
an IT-TOF mass spectrometer. Flash column chromatography was
performed using silica gel (200–300 mesh). Thin-layer chromatog-
raphy was performed on precoated silica gel F-254 plates
(0.25 mm) and was visualised with UV light. Unless otherwise
(R)-6–(1-aminoethyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (M-6)
To a solution of compound M-5 (3.8 g, 10 mmol) in methanol, Pd/
noted, all the starting materials and reagents were purchased C (10%, 0.4 g) was added. The mixture was stirred at room

262
C. ZHANG ET AL.
temperature for 24 h under an atmospheric pressure of hydrogen. 1H), 5.22 (m, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 2.10 (m, 4H), 1.98 (m,
The catalyst was filtered and the filtrate was concentrated. The 2H), 1.73 (m, 2H), 1.66 (d, J ¼ 7.0 Hz, 3H). 13C NMR (101 MHz,
residue was purified by silica gel chromatography (methanol/ CDCl3) d 166.34, 160.21, 159.99, 151.99, 150.79, 149.08, 142.28,
dichloromethane 1:50) to afford compound M-620 as a yellow solid 134.25, 127.44, 122.21, 117.46, 111.02, 109.71, 104.46, 57.90, 55.92,
1
(1.3 g, 52%). H NMR (400 MHz, CDCl3) d 8.05 (s, 1H), 5.22–5.08 (m, 55.85, 49.03, 32.49, 24.80, 20.10. HRMS (ESI-TOF) m/z [M þ H]þ
1H), 4.12 (q, J ¼ 6.8 Hz, 1H), 2.20–2.03 (m, 4H), 2.01–1.91 (m, 2H), calcd for C23H27N5O4 438.2136, found 438.2143.
1.76–1.67 (m, 2H), 1.53 (d, J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz,
CDCl3) d 162.65, 158.53, 152.48, 134.49, 104.72, 57.81, 49.70, 32.44,
32.32, 24.75, 23.15. HRMS (ESI-TOF) m/z [M þ H]þ calcd for
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl)-3–(3-hydroxy-4- methoxyphenyl)
acrylamide (1d)
C12H17N5O 247.1438, found 247.1433.
Compound 1d (0.19 g, 45%) was synthesised from 3-hydroxy-4-
General procedures for the preparation of compound 1a-1l, 2,
methoxycinnamic acid as a white solid. 1H NMR (400 MHz, CDCl3)
and 3
d 11.81 (s, 1H), 8.09 (s, 1H), 7.60 (d, J ¼ 15.4 Hz, 1H), 7.00 (d,
A mixture of compound M-6 (0.25 g, 1.0 mmol), EDC.HCl (0.15 g, J ¼ 6.7 Hz, 1H), 6.94 (s, 1H), 6.87 (m, 1H), 6.33 (d, J ¼ 15.1 Hz, 1H),
1.5 mmol), DMAP (0.002 g, 0.02 mmol), and the corresponding acid 5.99 (s, 1H), 5.30 (dd, J ¼ 15.1, 8.8 Hz, 1H), 5.20 (dd, J ¼ 16.8,
(1.0 mmol) was stirred at room temperature overnight28. Water 10.1 Hz, 1H), 3.85 (s, 3H), 2.15 (m, 4H), 1.98 (m, 2H), 1.78 (m, 2H),
was added to the mixture. The mixture was then extracted with 1.66 (d, J ¼ 4.5 Hz, 3H). 13C NMR (101 MHz, Acetone) d 166.53,
ethyl acetate three times, washed with brine, dried over Na2SO4, 160.67, 157.50, 151.74, 148.62, 147.75, 141.14, 133.78, 127.00,
and concentrated. The resulting residue was purified by silica gel 121.97, 117.79, 115.28, 110.54, 104.79, 57.67, 55.33, 48.40, 32.11,
chromatography and eluted with methanol and dichloromethane 24.51, 17.85. HRMS (ESI-TOF) m/z [M þ H]þ calcd for C22H25N5O4
(1:100) to afford the final product.
424.1979, found 424.1986.
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl)-3–(4-hydroxy-3-methoxyphenyl)
acrylamide (1a)
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl) -3–(3-hydroxy-2,4-dimethoxyphenyl)
acrylamide (1e)
Compound 1a (0.18 g, 42%) was synthesised from ferulic acid as a Compound 1e (0.23 g, 51%) was synthesised from 3-hydroxy-2,
white solid. 1H NMR (400 MHz, CDCl3) d 11.98 (s, 1H), 8.10 (s, 1H), 4-dimethoxycinnamic acid as a white solid. 1H NMR (400 MHz,
7.59 (dd, J ¼ 15.6, 2.9 Hz, 1H), 6.99 (d, J ¼ 8.2 Hz, 1H), 6.94 (t, CDCl3) d 11.66 (s, 1H), 8.08 (s, 1H), 7.58 (d, J ¼ 15.4 Hz, 1H), 6.77 (d,
J ¼ 5.3 Hz, 2H), 6.85 (d, J ¼ 8.2 Hz, 1H), 6.32 (t, J ¼ 14.4 Hz, 1H), 5.99 J ¼ 7.0 Hz, 1H), 6.72 (s, 1H), 6.35 (d, J ¼ 15.4 Hz, 1H), 5.78 (s, 1H),
(d, J ¼ 63.7 Hz, 1H), 5.35–5.26 (m, 1H), 5.26–5.17 (m, 1H), 3.84 (s, 5.24 (m, 2H), 3.87 (s, 6H), 2.11 (m, 4H), 1.99 (m, 2H), 1.73 (d,
3H), 2.22–2.11 (m, 2H), 2.10–2.01 (m, 2H), 2.00–1.90 (m, 2H), J ¼ 4.3 Hz, 2H), 1.67 (d, J ¼ 6.1 Hz, 4H). 13C NMR (101 MHz, CDCl3) d
1.79–1.69 (m, 2H), 1.66 (d, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, 166.41, 159.82, 159.68, 151.92, 147.18, 142.90, 136.89, 136.49,
DMSO) d 165.89, 162.06, 158.37, 152.03, 148.93, 148.29, 140.26, 134.27, 125.95, 117.31, 115.48, 104.93, 104.58, 57.96, 56.33, 48.91,
134.46, 126.71, 122.16, 118.75, 116.15, 111.27, 104.76, 57.62, 56.20, 32.51, 24.82, 19.60. HRMS (ESI-TOF) m/z [M þ H]þ calcd for
55.97, 48.39, 32.66, 32.44, 24.76, 19.78. HRMS (ESI-TOF) m/z C23H27N5O5 454.2085, found 454.2082.
[M þ H]þ calcd for C22H25N5O4 424.1979, found 424.1985.
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl)-3–(3,4-dihydroxyphenyl)acrylamide (1b)
d]pyrimidin-6-yl) ethyl)-3–(3-methoxyphenyl)acrylamide (1f)
Compound 1f (0.19 g, 47%) was synthesised from 3-methoxycin-
Compound 1b (0.19 g, 45%) was synthesised from caffeic acid as a namic acid as a white solid. 1H NMR (400 MHz, DMSO) d 8.60 (d,
white solid. 1H NMR (400 MHz, DMSO) d 9.31 (s, 2H), 8.52 (d, J ¼ 5.4 Hz, 1H), 8.03 (s, 1H), 7.49–7.28 (m, 2H), 7.22–7.06 (m, 2H),
J ¼ 6.6 Hz, 1H), 8.07 (s, 1H), 7.30 (d, J ¼ 15.7 Hz, 1H), 6.90 (d, 6.97 (d, J ¼ 7.2 Hz, 1H), 6.78 (d, J ¼ 15.7 Hz, 1H), 5.21–4.99 (m, 1H),
J ¼ 8.2 Hz, 1H), 6.80 (d, J ¼ 8.1 Hz, 1H), 6.52 (d, J ¼ 15.7 Hz, 1H), 4.95–4.77 (m, 1H), 3.79 (s, 3H), 2.04 (s, 2H), 1.90 (d, J ¼ 34.6 Hz, 4H),
5.20–5.08 (m, 1H), 4.91 (p, J ¼ 6.7 Hz, 1H), 2.09 (d, J ¼ 11.9 Hz, 2H), 1.64 (s, 2H), 1.46 (d, J ¼ 6.0 Hz, 3H). 13C NMR (101 MHz, DMSO) d
2.03–1.94 (m, 2H), 1.92 (d, J ¼ 16.4 Hz, 2H), 1.69 (s, 2H), 1.48 (t, 165.41, 161.86, 160.06, 158.39, 152.09, 139.76, 136.67, 134.44,
J ¼ 8.9 Hz, 3H). 13C NMR (101 MHz, DMSO) d 165.89, 162.06, 158.37, 130.49, 122.41, 120.43, 115.88, 113.13, 104.78, 57.67, 55.59, 48.42,
152.03, 148.93, 148.29, 140.26, 134.46, 126.71, 122.16, 118.75, 32.64, 32.43, 24.76. HRMS (ESI-TOF) m/z [M þ H]þ calcd for
116.15, 111.27, 104.76, 57.62, 56.20, 55.97, 48.39, 32.66, 32.44, C22H25N5O3 408.2030, found 408.2033.
24.76, 19.78. HRMS (ESI-TOF) m/z [M þ H]þ calcd for C21H23N5O4
410.1823, found 410.1827.
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl)-3–(4-hydroxyphenyl)acrylamide (1g)
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
Compound 1g (0.21 g, 52%) was synthesised from 4-hydroxycin-
d]pyrimidin-6-yl) ethyl)-3– (3,4-dimethoxyphenyl)acrylamide (1c)
namic acid as a white solid. 1H NMR (400 MHz, DMSO) d 12.08 (s,
Compound 1c (0.26 g, 59%) was synthesised from 3,4-dimethoxy- 1H), 9.90 (s, 1H), 8.46 (d, J ¼ 5.9 Hz, 1H), 8.02 (s, 1H), 7.50–7.35 (m,
cinnamic acid as a white solid. 1H NMR (400 MHz, CDCl3) d 12.12 2H), 7.34 (d, J ¼ 15.7 Hz, 1H), 6.80 (d, J ¼ 7.7 Hz, 2H), 6.55 (d,
(s, 1H), 8.09 (s, 1H), 7.62 (dd, J ¼ 15.6, 2.8 Hz, 1H), 7.11 (d, J ¼ 15.9 Hz, 1H), 5.23–4.96 (m, 1H), 4.94–4.68 (m, 1H), 2.03 (s, 2H),
J ¼ 7.8 Hz, 1H), 7.03 (d, J ¼ 8.3 Hz, 1H), 6.96 (s, 1H), 6.79 (d, 1.91 (d, J ¼ 37.4 Hz, 4H), 1.64 (s, 2H), 1.45 (d, J ¼ 6.4 Hz, 3H). 13C
J ¼ 8.4 Hz, 1H), 6.39 (d, J ¼ 15.6 Hz, 1H), 5.31 (dd, J ¼ 14.5, 7.2 Hz, NMR (101 MHz, DMSO) d 165.92, 162.08, 159.48, 158.39, 152.02,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
263
140.00, 134.44, 129.83, 126.20, 118.38, 116.25, 104.75, 60.24, 57.60, (R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
48.39, 32.65, 32.43, 24.76, 19.75. HRMS (ESI-TOF) m/z [M þ H]þ d]pyrimidin-6-yl)ethyl)-2–(3,4-dihydroxyphenyl)acetamide (1l)
calcd for C21H23N5O3 394.1874, found 394.1866.
Compound 1l (0.22 g, 56%) was synthesised from 3, 4-dihydroxy-
phenylacetic acid as a white solid. 1H NMR (400 MHz, DMSO) d
12.06 (s, 1H), 8.74 (d, J ¼ 19.1 Hz, 2H), 8.34 (d, J ¼ 6.2 Hz, 1H), 8.00
(s, 1H), 6.85–6.56 (m, 2H), 6.53 (d, J ¼ 7.5 Hz, 1H), 5.09–4.84 (m, 1H),
4.84–4.53 (m, 1H), 3.33–3.24 (m, 2H), 2.03 (s, 2H), 1.88 (s, 4H), 1.68
(s, 2H), 1.39 (d, J ¼ 6.7 Hz, 3H). 13C NMR (101 MHz, DMSO) d 171.07,
161.94, 158.36, 151.92, 145.39, 144.33, 134.42, 127.12, 120.22,
117.10, 115.63, 104.63, 57.39, 48.00, 41.90, 32.81, 32.38, 24.84,
19.86. HRMS (ESI-TOF) m/z [M þ H]þ calcd for C20H23N5O4
398.1823, found 398.1826.
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl)-3–(3-hydroxyphenyl)acrylamide (1h)
Compound 1h (0.15 g, 39%) was synthesised from 3-hydroxycin-
namic acid as a white solid. 1H NMR (400 MHz, DMSO) d 12.08 (s,
1H), 9.90 (s, 1H), 8.46 (d, J ¼ 5.9 Hz, 1H), 8.02 (s, 1H), 7.50–7.35 (m,
2H), 7.34 (d, J ¼ 15.7 Hz, 1H), 6.80 (d, J ¼ 7.7 Hz, 2H), 6.55 (d,
J ¼ 15.9 Hz, 1H), 5.23–4.96 (m, 1H), 4.94–4.68 (m, 1H), 2.03 (s, 2H),
1.91 (d, J ¼ 37.4 Hz, 4H), 1.64 (s, 2H), 1.45 (d, J ¼ 6.4 Hz, 3H). 13C
NMR (101 MHz, DMSO) d 165.92, 162.08, 159.48, 158.39, 152.02,
140.00, 134.44, 129.83, 126.20, 118.38, 116.25, 104.75, 60.24, 57.60,
48.39, 32.65, 32.43, 24.76, 19.75. HRMS (ESI-TOF) m/z [M þ H]þ
calcd for C21H23N5O3 394.1874, found 394.1871.
N-((1N)-1–(1-cyclopentyl-4-oxo-3a, 4, 5, 6-tetrahydro-1H-
pyrazolo[3,4-d]pyrimi–din-6-yl)ethyl)-5-((R)-1,2-dithiolan-3-
yl)pentanamide (2)
Compound 2 (0.21 g, 48%) was synthesised from (R)-a-lipoic acid
1
as a white solid. H NMR (400 MHz, DMSO) d 12.03 (s, 1H), 8.32 (d,
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
J ¼ 6.7 Hz, 1H), 8.02 (s, 1H), 5.11 (s, 1H), 4.73 (s, 1H), 3.60 (dd,
J ¼ 8.4, 5.9 Hz, 1H), 3.14 (dd, J ¼ 13.5, 7.2 Hz, 2H), 2.38 (s, 1H), 2.20
(s, 2H), 2.04 (s, 2H), 2.01–1.75 (m, 5H), 1.75–1.61 (m, 3H), 1.59–1.45
(m, 3H), 1.38 (d, J ¼ 7.0 Hz, 5H). 13C NMR (101 MHz, DMSO) d
172.71, 162.26, 158.36, 152.07, 134.44, 104.70, 57.54, 56.50, 48.06,
38.55, 35.32, 34.55, 32.55, 28.74, 25.38, 24.78, 19.57. HRMS (ESI-
TOF) m/z [M þ H]þ calcd for C20H29N5O2S2 436.1835, found
436.1830.
d]pyrimidin-6-yl)ethyl)-3–(4-(dimethylamino)phenyl)acrylamide
(1i)
Compound 1i (0.27 g, 63%) was synthesised from 4-hydroxycin-
namic acid as a white solid. 1H NMR (400 MHz, DMSO) d 12.10 (s,
1H), 9.62 (s, 1H), 8.60 (d, J ¼ 6.7 Hz, 1H), 8.02 (s, 1H), 7.34 (d,
J ¼ 15.8 Hz, 1H), 7.21 (d, J ¼ 7.8 Hz, 1H), 7.00 (d, J ¼ 7.7 Hz, 1H),
6.95 (s, 1H), 6.80 (dd, J ¼ 8.0, 1.8 Hz, 1H), 6.70 (d, J ¼ 15.8 Hz, 1H),
5.11 (m, 1H), 4.89 (m, 1H), 2.51 (d, J ¼ 1.6 Hz, 2H), 2.05 (m, 2H),
1.94 (m, 2H), 1.86 (m, 2H), 1.64 (m, 2H), 1.46 (d, J ¼ 7.0 Hz, 3H).
13C NMR (101 MHz, DMSO) d 165.47, 161.95, 158.37, 158.18,
152.01, 140.01, 136.49, 134.44, 130.45, 121.82, 119.27, 117.32,
114.20, 104.77, 57.68, 48.43, 32.63, 32.42, 24.75, 19.72. HRMS (ESI-
TOF) m/z [M þ H]þ calcd for C23H28N6O2 421.2347, found
421.2350.
N-((R)-1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl) -6-hydroxy-2,5,7,8-tetramethylchromane-
2-carboxamide (3)
Compound 3 (0.17 g, 35%) was synthesised from trolox as a white
1
solid. H NMR (400 MHz, CDCl3) d 8.02 (d, J ¼ 17.0 Hz, 1H), 5.16 (m,
1H), 4.98 (m, 1H), 2.81 (s, 3H), 2.63 (dd, J ¼ 16.2, 9.2 Hz, 2H), 2.35
(dd, J ¼ 13.2, 6.7 Hz, 1H), 2.25 (s, 1H), 2.18 (m, 3H), 2.12 (s, 3H), 2.05
(d, J ¼ 3.7 Hz, 3H), 1.98 (m, 2H), 1.73 (m, 2H), 1.61 (s, 6H), 1.59 (s,
1H). 13C NMR (101 MHz, CDCl3) d 174.78, 159.43, 151.72, 145.88,
144.19, 134.71, 121.77, 119.52, 119.34, 117.86, 104.55, 99.99, 57.71,
47.94, 38.66, 32.57, 29.72, 24.83, 23.72, 20.43, 19.64, 12.39, 12.02,
11.45. HRMS (ESI-TOF) m/z [M þ H]þ calcd for C26H33N5O4
480.2605, found 480.2615.
(R, E)-N-3–(3-chlorophenyl)-N-(1–(1-cyclopentyl-4-oxo-4,5-
dihydro-1H-pyrazolo[3,4-d] pyrimidin-6-yl)ethyl)acrylamide (1j)
Compound 1j (0.19 g, 45%) was synthesised from 3-chlorolcin-
namic acid as a white solid. 1H NMR (400 MHz, DMSO) d 8.65 (d,
J ¼ 6.6 Hz, 1H), 8.02 (s, 1H), 7.65 (s, 1H), 7.56 (d, J ¼ 4.9 Hz, 1H), 7.43
(t, J ¼ 11.7 Hz, 3H), 6.83 (d, J ¼ 15.9 Hz, 1H), 5.15–5.02 (m, 1H),
4.94–4.80 (m, 1H), 2.04 (d, J ¼ 12.2 Hz, 2H), 1.98–1.74 (m, 4H), 1.63
(s, 2H), 1.46 (d, J ¼ 6.9 Hz, 3H). 13CNMR (101 MHz, DMSO) d 165.17,
161.76, 158.45, 151.96, 138.34, 137.47, 134.45, 134.16, 131.30,
129.75, 127.70, 126.58, 123.64, 104.73, 57.71, 48.46, 32.61, 32.41,
24.75, 19.72. HRMS (ESI-TOF) m/z [M þ H]þ calcd for C21H22ClN5O2
412.1535, found 412.1540.
In vitro assay for PDE9 inhibitors
Protein expression and purification
The recombinant pET-PDE9A2 plasmid (catalytic domain,
181–506) was subcloned and purified in the same protocols
we reported previously26 and then transferred into the E. coli
strain BL21 (Codonplus, Stratagene, San Diego, CA). The E. coli
cells carrying pET-PDE9A2 was grown in LB medium (containing
100 lg/ml ampicillin and 0.4% glucose) at 37 ꢁC until
OD600 ¼ 0.6–0.8. 0.1 mM iso-propyl-b-D-thiogalactopyranoside was
added to induce the PDE9A2 protein expression at 15 ꢁC for
20 h. PDE9A was purified on the nickel nitriloacetic acid (Ni-NTA)
column (Qiagen, Hilden, Germany) and eluted with 0.25 M imid-
azole. The concentration of PDE9A was estimated by the absorb-
ance at 280 nm (calculated using ProtParam software, http://web.
expasy.org/protparam). A typical batch of purification yielded
30–60 mg PDE9A2 protein from a 0.5 l cell culture. The PDE9A2
proteins had purity greater than 90% as shown by sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
(R, E)-N-(1–(1-cyclopentyl-4-oxo-4, 5-dihydro-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)ethyl)-3–(4-(trifluoromethyl)phenyl)acrylamide
(1k)
Compound 1k (0.17 g, 39%) was synthesised from 4-trifluorome-
thylcinnamic acid as a white solid. 1H NMR (400 MHz, DMSO) d
12.13 (s, 1H), 8.71 (d, J ¼ 6.8 Hz, 1H), 8.03 (s, 1H), 7.80 (s, 4H), 7.52
(d, J ¼ 15.9 Hz, 1H), 6.92 (d, J ¼ 15.9 Hz, 1H), 5.18–5.04 (m, 1H),
4.96–4.83 (m, 1H), 2.13–2.00 (m, 2H), 1.95 (dd, J ¼ 17.8, 11.5 Hz,
2H), 1.86 (dd, J ¼ 9.8, 6.9 Hz, 2H), 1.64 (s, 2H), 1.47 (d, J ¼ 7.0 Hz,
3H). 13C NMR (400 MHz, DMSO) d 166.67, 163.65, 159.42, 156.07,
149.85, 141.77, 135.38, 130.64, 126.91, 121.90, 116.23, 112.07,
61.59, 48.95, 36.17, 25.81, 22.00. HRMS (ESI-TOF) m/z [M þ H]þ
calcd for C22H22F3N5O2 446.1798, found 446.1794.

264
C. ZHANG ET AL.
Enzymatic assays against PDE9A
at 570 nm using EnSpire-2300 Multimode Reader (PerkinElmer,
Waltham, MA).
The enzymatic activities of the catalytic domain of PDE9A2 were
3
measured by using H-cGMP as the substrate and the assay buffer
of 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, and 1 mM DTT. Each
assay used 3H-cGMP 20,000–30,000 cpm. The reaction was carried
out at room temperature (25 ꢁC) for 15 min and then terminated
by addition of 0.2 M ZnSO4. The reaction product was precipitated
by 0.2 N Ba(OH)2, whereas unreacted 3H-cGMP remained in the
supernatant. The radioactivity in the supernatant was measured in
2.5 ml of Ultima Gold liquid scintillation cocktails (PerkinElmer,
Waltham, MA) using a PerkinElmer 2910 liquid scintillation counter.
At least eight concentrations of inhibitors were measured parallel
in each measurement, resulting an inhibition curve to calculate
the IC50 value of inhibitors by nonlinear regression. Each measure-
ment was repeated at least three times. The mean and standard
deviation statistics of these three IC50 values were used as the
final IC50 value and the corresponding SD. BAY73–6691 pur-
chased from SIGMA (Mendota Heights, MN) was used as the refer-
ence compound.
Molecular docking
The crystal structure of PDE9A in the complex with a highly select-
ive inhibitor 28 (PDB ID:4GH6) was used in this study34. Surflex-
dock35 embedded in the software Tripos Sybyl version 2.0 (Tripos
Software, Inc., El Cerrito, CA) was used for molecular docking. Two
metal ions in this crystal structure were kept as they were crucial
for the PDE’s catalytic activity. Most of the water molecules in this
crystal structure were removed except the ones near these two
metal ions. Hydrogen atoms were added and the ionisable resi-
dues were protonated at the neutral pH. The protomol was gener-
ated using a ligand-based approach and the bound ligand was
used for protomol generation. The proto_thresh and proto_bloat
parameters represent how much the protomol can be buried in
the protein and how far the protomol extends outside the cavity,
respectively. The proto_thresh was selected as 0.5 and proto_bloat
was selected as 0. After the preparation of the protomol, molecu-
lar docking was performed. All molecules designed were docked
to the prepared structure of PDE9A and 20 molecules with the
highest docking scores and appropriate binding modes went into
the following MD simulations and binding free energy
calculations.
In vitro assay for inhibition of the lead compounds on PDE1B
and PDE8A
The catalytic domains of PDE1B (10–516) and PDE8A (480–820)
were purified with the published protocol29,30. The enzymatic
activities of PDE1B and PDE8A were assayed by using 3H-cGMP
and 3H-cAMP as the substrates, respectively, following the same
procedure for PDE9A.
Molecular dynamics simulation
The MM-PBSA approach36–38 was applied for the binding-free-
energy calculations using the 100 snapshots isolated from the final
1.0 ns period of each 8 ns MD simulations trajectory. The electro-
static contribution to the solvation-free energy was determined
using the PBSA program in the Amber 16 suite39. A grid size of
0.5 Å was used. The interior and exterior dielectric constants for
the solute and solvent were set to 1 and 80, respectively. The opti-
mised atomic radii set in Amber 16 were used. To speed up calcu-
lations without much decrease in accuracy, the entropy
contributions are omitted.
Antioxidant activity assay
The antioxidant activity was determined by the modified oxygen
radical absorbance capacity fluorescein (ORAC-FL) method31,32. All
the assays were diluted with 75 mM phosphate buffer (pH 7.4).
The final reaction mixture was 200 ll. Test compound (20 ll) and
fluorescein (120 ll, 150 nM final concentration) were placed in the
wells of a black 96-well plate. After the mixture was incubated at
37 ꢁC for 15 min, AAPH solution (60 ll, 12 mM final concentration)
was added. The plate was placed in a Spectrafluor Plus plate
reader (Tecan, Crailsheim, Germany) and the fluorescence was
recorded every minute for 4 h with excitation at 485 nm and emis-
sion at 535 nm. Trolox was used as standard (1–8 lM, final concen-
tration). A blank (FL þ AAPH) in phosphate buffer instead of test
compounds and trolox calibration were performed in each assay.
The samples were measured at different concentration (1–10 lM).
All the reaction mixture was prepared fourfold and at least three
independent assays were performed for each sample.
Fluorescence in time course was normalised on basis of the blank
(without antioxidants). The ORAC-FL values were calculated as the
reported method. ORAC-FL values were expressed as trolox
equivalents.
Result and discussion
The design of novel PDE9A inhibitors with assistance of
molecular docking and dynamics simulation
The crystal structures of PDE9A-inhibitors showed that two con-
served residues Gln453 and Phe456 play essential roles for binding
of the inhibitors11,18,27,34 (Figure 1). Most PDE9A inhibitors contain
a pyrazolopyrimidinone scaffold to form hydrogen bond with
Gln453 and p–p interactions with Phe456. A narrow and long
pocket next to pyrazolopyrimidinone is available in the PDE9
structure for introducing an extra fragment with antioxidant activ-
ity. In addition, Tyr424 in this narrow pocket, which is unique to
PDE8 and PDE9, has been shown to be important for selectivity of
PDE9 inhibitors over other PDEs27,34. Thus, we took pyrazolopyri-
midinone as the scaffold of our designed compounds. Functional
fragments from common antioxidants such as ferulic acid, caffeic
acid, lipolic acid were attached to 6-position of pyrazolopyrimidi-
none with various linkers for antioxidant activities (compounds
In vitro cell viability assay
SH-SY5Y cells were used for the cytotoxicity test by the MTT
(3–(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide)
method33. After incubation of the SH-SY5Y cells (5 ꢂ 103) in a 96-
well plate for 24 h, compounds at concentrations of 10, 20, 40, 60,
80, and 100 lM, were added. After incubation for 48 h, MTT solu-
tion was added to each cell well and the system was further ino-
structure shown in Figure S1)28,40,41
.
In order to improve the efficiency and reduce synthetic work,
molecular docking was performed on the designed compounds to
culated for 4 h at 37 ꢁC. The purple formazan dye and 120 ll predict their binding patterns. This is quite different from the
DMSO were added to the cells and the absorbance was measured usual drug discovery in which molecular docking was used to

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
265
Figure 1. The design of PDE9 inhibitors with antioxidant activities.
predict the binding mode after lead compounds were identified. benzoyl group of M-5 was removed by hydrogenation in hydro-
Furthermore, binding free energies between designed compounds gen atmosphere and the presence of the Pd/C catalyst, producing
and protein in atomic scales, which can improve the hit rate sig- M-6 in high yield20. Compounds 1a-1l were prepared in moderate
nificantly but are not available in the experiments sometimes were to high yield by condensation reaction of M-6 with the acids such
obtained by the molecular dynamics simulation for a more precise as ferulic acid28. Compounds 2 and 3 were prepared in the same
prediction. According to the calculation results, we found that the procedure by using lipoic acid and trolox28.
compounds with an amide group as the linker had stable MD sim-
ulations trajectories and high binding free energies (compounds
structure in Figure 1, Binding free energies in Table 1, Binding
Inhibition of designed compounds on PDE9A in vitro
mode in SP), indicating that they might be potential PDE9A
inhibitors.
The inhibitory activities of compounds 1a–1l, 2, and 3 against
PDE9A were assayed with the procedure previously reported26.
The results are summarised in Table 1. BAY73–6691, a PDE9
inhibitor developed by Bayer company serves as the reference in
the assay (Lit: IC50¼55nM)43.
In order to filter possible false hits, pan-assay interfering com-
pound substructures (PAINS) screening was performed using the
online program PAINS-Remover (http://cbligand.org/PAINS)42. All
the compounds with an amide group as the linker (Figure 1)
passed this test and were then synthesised.
Compounds 1a–1l, which contain the fragment of ferulic acid
derivatives, have the IC50 values of 56–190 nM against PDE9A.
The substitutions on the phenyl ring showed remarkable impacts
on the inhibitory activities against PDE9A. Compounds (1a, 1b,
1d, 1g, 1h) with a hydroxyl group on the phenyl ring have bet-
Chemistry
As outlined in Scheme 1, condensation of 2-(methoxymethylene)- ter inhibitory activities than other substitutions. Among all the
malononitrile (M-1) with cyclopentylhydrazine (M-2) afforded M-3 compounds in series 1, compound 1h with the 3-hydroxyl on
in the presence of triethylamine. Hydrolysis of the cyano group of the phenyl ring gave the best IC50 value of 56 nM, which is as
M-3 in the presence of ammonium hydroxide and hydrogen per- effective as BAY73–6691 (55 nM). Compounds 1b with two
oxide produced M-4 with an amide group27. M-5 was synthesised hydroxyl group and 1c with two methoxy groups gave compar-
by the reaction of M-4 with (R)-ethyl-2-(((benzyloxy)carbonyl)ami- able inhibitory activities. Introduction of an additional methoxy
no)propanoate under basic condition at room temperature. The group from compound 1a to 1e makes the IC50 drop to

266
C. ZHANG ET AL.
Table 1. The Inhibitory activities against PDE9A, binding free energies, and oxy-
gen radical absorbance capacity of compounds 1a–1l, 2, and 3.
be essential for the affinity with PDE9A. Compound 2 having lip-
olic acid linked to 6-position of pyrazolopyrimidinone gave the
best IC50 of 25 nM, in comparison with IC50>500 nM of com-
pound 3 with trolox group. We speculated that the trolox group
might be too large to fit into the binding pocket of PDE9A. The
linear correlation between docking score values and IC50 values
of compound 1a–1l, 2, and 3 was observed with a moderate R2
value of 0.58 (Table S1 and Figure S2). Compound 3 and 1e
with the lowest docking score values, gave the lowest inhibitory
activities against PDE9A. However, for some compounds such as
1i and 1f, there are some deviations between the docking score
values and IC50 values.
O
HN
N
N
N
NH
R
O
Compound
R
IC50 SD (nM)a GBTOT (kcalꢃmolꢄ1) ORAC
HO
1a
126 15
ꢄ31.09 2.64
ꢄ29.78 2.33
ꢄ29.53 2.82
ꢄ35.41 3.09
ꢄ35.77 2.64
2.4
MeO
HO
1b
1c
1d
1e
64
65
2
4
2.5
Antioxidant activity test by the ORAC method
HO
MeO
0.23
1.26
0.50
Oxygen radical absorbance capacity assay (ORAC-FL) has been
widely used to evaluate antioxidant activities of hydrophilic antiox-
idants including ferulic acid and phenolic derivatives. Thus, the
antioxidant activities of compounds 1a–1l and compound 2 were
evaluated by ORAC-FL method31,32. The results were expressed as
trolox equivalents and summarised in Table 1. Ferulic acid was
used as the positive reference compound, showing an ORAC-FL
value of 1.6 trolox equivalents. The feruic acid derivatives with the
hydroxyl group on the phenyl ring (1a, 1b, 1d–1h, 1l) showed
good ORAC-FL values in a range of 1.3–3.3 fold of trolox equiva-
lents, while compounds without the hydroxyl group (1c, 1f, 1j,
and 1k) showed low ORAC-FL values except for compound 1i
with dimethylamino group substituted. These results are consist-
ent with the previous reports that the hydroxyl-phenyl groups of
the ferulic acid are the main source of antioxidant activity41. To
our surprise, compound 2 with the fragment of lipolic acid
showed low antioxidant activity.
MeO
MeO
83 11
HO
HO
OMe
>200
MeO
1f
190
63
7
3
ꢄ27.39 3.05
ꢄ35.71 2.81
0.12
2.9
MeO
HO
1g
1h
1i
56
7
ꢄ38.16 3.08
ꢄ33.80 3.08
ꢄ35.52 2.72
ꢄ43.39 2.53
3.3
HO
Me2N
133 22
1.00
0.08
0.02
1j
186
130
7
6
Selectivity of compounds 1h and 2 over PDE1 and PDE8
Cl
F3C
1k
The PDE super-family of enzymes are divided into 11 subfamilies
according to the differences in structure and distribution. Each
PDE subfamily is in charge of different biological process. In
order to reduce drug adverse effects, the selectivity over other
PDEs is important for potential PDE inhibitors. Since the binding
pocket of PDE subfamily is structurally similar, selective PDE
inhibitors are not easy to be obtained. For the PDE9A inhibitors,
the selectivity over PDE1 and PDE8 are usually difficult to
obtain18,20,34. Thus, compound 1h with PDE9A inhibition and
good antioxidant activity, compound 2 with the best inhibition
towards PDE9A were chosen for exploration of selectivity
over PDE1 and PDE8 (Table 2). Compounds 1h and 2 at 1 lM
inhibited 13 and 59% activity of PDE1B, thus estimating 25 and
40-fold selectivity, respectively. The selectivity is better than
BAY73-6691 but not PF-0444794318,43. The inhibitory activities
of compounds 1h and 2 against PDE8A at 10 lM were 10 and
9%, respectively, implying the 250–400 fold selectivity of these
compounds towards PDE8A. Thus, both compounds 1h and 2
HO
HO
1l
57
25
4
4
ꢄ28.79 2.93
2.6
2
3
ꢄ40.01 3.74
ꢄ31.98 2.52
0.28
0.24
S
S
>500
HO
O
BAY73-6691
Ferulic acid
–
–
46
–
–
n.a.c
1.6
n.d.b
aIC50 values are given as the means of three independent determinations.
bn.d. means not determined.
cn.a. means not active.
>200 nM, suggesting steric hinder in the binding pocket is
important. Using other substitution groups such as dimethyl- are good selective PDE9 inhibitors.
amine, trifluoromethyl, and chloro to instead the methoxy or
hydroxyl group of ferulic acid formed compounds 1i–1k. The
Effects of compounds 1h and 2 on cell viability
inhibitory activities of these compounds against PDE9A dropped
to 130–180nM. We concluded that the hydroxyl group on the
phenyl ring might form interactions with residues in the binding
pocket of PDE9A, improving the inhibitory activity against
PDE9A. Compared with 1b derived from caffeic acid, compound
1l which does not contain a double bond, gave a comparable
The cytotoxicity of compounds 1h and 2 were tested using human
neuroblastoma cell line SH-SY5Y by the MTT method33. As shown
in Figure 2, after incubation for 48 h, the cellular viability was
about 90% at 40 lM of compound 1 h and 70% at 100 lM.
Compound 2 at 100 lM showed no significant effect on cell viabil-
IC50 value of 57nM, suggesting that the double bond may not ity. Control experiments were performed, eliminating the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
267
O
O
O
H
NC
Cl
NH2
O
N
H2N
H2N
HN
OEt
HN
NC
CN
N
N
N
b
O
a
N
+
N
N
H2N
N
c
OMe
NHCbz
M-1
M-5
M-2
M-3
M-4
O
N
1g: R = 4-OH
1h: R = 3-OH
1a
: R = 3-OMe, 4-OH
1b: R = 3, 4- 2OH
HN
N
R
N
OH
1i
1c
: R = 4-NMe2
: R = 3, 4- 2OMe
1d
R
NH
1j : R= 3-Cl
1k: R= 4-CF3
O
: R = 3-OH, 4-OMe
1e: R = 3, 5 - 2OMe, 4-OH
1f
O
1a-1k
: R = 3-OMe
O
N
HN
HO
HO
OH
N
N
O
HO
HO
NH
O
N
O
HN
e
1l
N
d
N
O
N
NH2
HN
S
N
OH
S
N
M-6
O
S
NH
S
O
2
O
HO
OH
HN
O
N
HO
O
N
N
NH
O
O
3
Scheme 1. Reagents and conditions: (a) Triethylamine, 0 ꢁC, r.t., 3 h, then reflux 3 h, EtOH; (b) NH3ꢃH2O,H2O2, EtOH, r.t.; (c) (R)-ethyl-2-(((benzyloxy)carbonyl)amino)pro-
panoate, 60% NaH, dry THF, r.t.; (d) Pd/C, MeOH, H2, r.t.; (e) EDCꢃHCl, DMAP, DMF, r.t.
According to the difference in the binding modes, the
designed compounds in series 1 can be divided into three classes.
Compounds 1a, 1c, 1d, and 1e adopted a similar binding mode
(Figure S3) and form two hydrogen bonds with invariant Gln453
and p–p interactions with conserved Phe456. Compounds 1b, 1j,
and 1k formed an additional hydrogen bond with Ala452 in add-
ition to the interactions with Gln453 and Phe456 (Figure S4).
Compounds 1f, 1g, 1h, and 1i formed an additional hydrogen
bond with Tyr424 besides the interactions with Gln453 and
Phe456 (Figure S5). As is mentioned before, the interactions with
Gln453 and Phe456 are the main reasons for the affinity of inhibi-
tors against PDE9. Thus, all these compounds should have good
inhibitory activities against PDE9A. Further biological tests demon-
strated that most compounds exhibited the inhibition activity
against PDE9 with IC50 below 200 nM except compound 1e.
Compound 1h, which has IC50 of 57 nM against PDE9 and the
highest ORAC value of 3.3 trolox, belongs to the class II, forming
an additional hydrogen bond with Tyr424 (Figure 3). Since Tyr424
is unique to PDE9 and PDE8, the interaction with Tyr424 will ren-
der high selectivity of PDE9 inhibitors. This is supported by the
good selectivity of compound 1h over PDE1B and PDE8A.
Compound 2, which has IC50 of 25 nM against PDE9 and good
selectivity over PDE1 and PDE8 showed no interaction with
Tyr424, implying a new way to design selective PDE9 inhibitors
(Figure 3).
Table 2. Inhibitory activities of compounds 1h and 2 with PDE1 and PDE8.
IC50 (nM)
of PDE9A
Inhibition of
PDE1B at 1000 nM
Inhibition of
PDE8A at 10 lM
Compound
1h
2
56
25
1
4
13% (>25)a
10% (>250)a
59% (40)a
9% (>400)a
BAY73-6691
PF-04447943
55b
1400 (25)b
1394 (174)c
–
–
8c
aThe numbers in parentheses are the fold of selectivity of inhibitors against
PDE9 over other PDEs (IC50, nM).
bData was obtained from the reported literature43
.
cData was obtained from the reported literature18
.
possibility that compounds 1h and 2 interfered with the MTT
assay (Figure S8). These results suggested that both 1h and 2 are
not toxic to SH-SY5Y cells and potential candidates for further
development as therapeutics for AD.
Analysis of the structure-activity relationship by molecular
docking and dynamics simulation
In order to improve the efficiency and reduce synthetic work in
the process of identifying PDE9 inhibitors, the binding mode, and
binding free energies of all these compounds were performed by
molecular docking and dynamics simulation.

268
C. ZHANG ET AL.
1h
2
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Figure 2. Cell viability of compounds 1h and 2.
The binding free energy calculations were performed by the
MM-PBSA method. The results of compounds 1h and 2 are shown
in Table 3, suggesting that van der Waals(DEvdw) and electrostatic
components (DEele) are two important factors for the high affinity
of both compounds 1h and 2 while polar component to solvation
(DEpol,solv) is less important.
Table 3. Components of the binding free energy (kcal/mol) for PDE9A in com-
plex with 1h or 2 by using the MM–PBSA method.
Compound
1h
2
DEvdw
DEele
DEpol,solv
DEnonpol,solv
DGgas
ꢄ50.32 3.38
ꢄ33.66 5.38
50.40 4.08
ꢄ4.57 0.10
ꢄ83.99 5.79
45.83 4.05
38.16 3.08
ꢄ50.86 3.14
ꢄ25.46 3.95
41.05 2.28
–4.74 0.13
ꢄ76.32 4.04
36.31 2.30
ꢄ40.01 3.74
DGsolv
DGbind
Conclusion
In summary, a series of novel pyrazolopyrimidinone derivatives
have been designed, synthesised and evaluated as multifunctional
anti-AD ligands, in combination of PDE9A inhibition and antioxi-
dant activities. With the assistance of molecular docking and
dynamics simulation, high selective PDE9 inhibitors with antioxi-
dant activities have been successfully developed as shown by
compounds 1h (IC50 of 56 and 3.3 trolox of ORAC). These PDE9
inhibitors do not disturb cell viability of SH-SY5Y and thus are
worth for further development as therapeutics for treatment of
AD. The structure-activity relationship was explained by the bind-
ing modes of inhibitors with PDE9 protein, providing evidence for
further structural modification.
MM–PBSA: molecular mechanical and Poisson–Boltzmann/surface accessible;
DEele: electrostatic interactions calculated by the MM force field; DEvdw: van der
Waals’ contributions from MM; DEnonpol, sol: the nonpolar contribution to solv-
ation; DEele, sol: the polar contribution to solvation; TDS: entropic contribution by
using N-mode methods; DE: the predicted binding free energies with entropic
contribution omitted; DGbind, pred: the predicted binding free energies with
entropic contribution.
Acknowledgements
We cordially thank Prof. H. Ke from Department of Biochemistry
and Biophysics at the University of North Carolina, Chapel Hill, for
his help with molecular cloning, expression, purification, crystal
structure, and bioassay of PDEs.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Natural Science Foundation of
China (81522041, 81373258, 21572279, 21402243 and 81602968),
Science Foundation of Guangdong Province (2014A020210009,
2016A030310144), Guangdong Province Higher Vocational
Colleges & Schools Pearl River Scholar Funded Scheme (2016),
Foundation of Traditional Chinese medicine Bureau of Guangdong
Province (20171049) and State Key Laboratory for Chemistry and
Molecular Engineering of Medicinal Resources (CMEMR2016-B03).
We thank Special Program for Applied Research on Super
Computation of the NSFC-Guangdong Joint Fund (the second
Figure 3. The binding modes of compounds 1h and 2.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
269
phase) under Grant No.U1501501 for providing the supercomput-
ing service.
d]pyrimidin-4-one (PF-04447943), a selective brain penetrant
PDE9A inhibitor for the treatment of cognitive disorders. J
Med Chem 2012;55:9045–54.
19. Schwam EM, Nicholas T, Chew R, et al. A multicenter, dou-
ble-blind, placebo-controlled trial of the PDE9A inhibitor, PF-
04447943, in Alzheimer’s disease. Curr Alzheimer Res
2014;11:413–21.
20. Su T, Zhang TH, Xie SS, et al. Discovery of novel PDE9 inhibi-
tors capable of inhibiting A beta aggregation as potential
candidates for the treatment of Alzheimer’s disease. Sci Rep
2016;6:21826.
21. Yu YF, Huang YD, Zhang C, et al. Discovery of novel pyrazo-
lopyrimidinone derivatives as PDE9A inhibitors capable of
inhibiting BuChE for treatment of Alzheimer’s disease. ACS
Chem Neurosci 2017;8:2522–34.
22. Rosini M, Simoni E, Milelli A, et al. Oxidative stress in
Alzheimer’s disease: are we connecting the dots? J Med
Chem 2014;57:2821–31.
23. Mecocci P, Polidori MC. Antioxidant clinical trials in mild
cognitive impairment and Alzheimer’s disease. Biochim
Biophys Acta 2012;1822:631–8.
24. Zhang HY, Yang DP, Tang GY. Multipotent antioxidants: from
screening to design. Drug Discov Today 2006;11:749–54.
25. Deshmukh R, Sharma V, Mehan S, et al. Amelioration of
intracerebroventricular streptozotocin induced cognitive dys-
function and oxidative stress by vinpocetine–a PDE1 inhibi-
tor. Eur J Pharmacol 2009;62:49–56.
26. Li Z, Lu X, Feng LJ, et al. Molecular dynamics-based discov-
ery of novel phosphodiesterase-9A inhibitors with non-pyra-
zolopyrimidinone scaffolds. Mol Biosyst 2015;11:115–25.
27. Claffey MM, Helal CJ, Verhoest PR, et al. Application of struc-
ture-based drug design and parallel chemistry to identify
selective, brain penetrant, in vivo active Phosphodiesterase
9A inhibitors. J Med Chem 2012;55:9055–68.
28. Rosini M, Simoni E, Bartolini M, et al. Exploiting the lipoic
acid structure in the search for novel multitarget ligands
against Alzheimer’s disease. Eur J Med Chem 2011;46:
5435–42.
References
1. Stopschinski BE, Diamond MI. The prion model for progres-
sion and diversity of neurodegenerative diseases. Lancet
Neurol 2017;16:323–32.
2. Alzheimer’s Association (USA). World Alzheimer Report. 2015.
Available from: http://news.medlive.cn/uploadfile/20150902/
14411602338388.pdf
3. Goedert M, Spillantini MG. A century of Alzheimer’s disease.
Science 2006;314:777–81.
4. Herrmann N, Chau SA, Kircanski I, et al. Current and emerg-
ing drug treatment options for Alzheimer’s disease: a sys-
tematic review. Drugs 2011;71:2031–65.
5. Terry AV, Callahan PM, Hall B, et al. Alzheimer’s disease and
age-related memory decline (preclinical). Pharmacol Biochem
Behav 2011;99:190–210.
6. Rosini M, Simoni E, Caporaso R, et al. Multitarget strategies
in Alzheimer’s disease: benefits and challenges on the road
to therapeutics. Future Med Chem 2016;8:697–711.
7. Anand R, Gill KD, Mahdi AA. Therapeutics of Alzheimer’s dis-
ease: past, present and future. Neuropharmacology
2014;76:27–50.
8. Azevedo MF, Faucz FR, Bimpaki E, et al. Clinical and molecu-
lar genetics of the phosphodiesterases (PDEs). Endocr Rev
2014;35:195–233.
9. Maurice DH, Ke HM, Ahmad F, et al. Advances in targeting
cyclic nucleotide phosphodiesterases. Nat Rev Drug Discov
2014;13:290–314.
10. Ricciarelli R, Fedele E. Phosphodiesterase 4D: an enzyme to
remember. Br J Pharmacol 2015;172:4785–9.
11. Garcia-Osta A, Cuadrado-Tejedor M, Garcia-Barroso C, et al.
Phosphodiesterases as therapeutic targets for Alzheimer’s
disease. ACS Chem Neurosci 2012;3:832–44.
12. Reyes-Irisarri E, Ittersum MMV, Mengod G, et al. Expression
of the cGMP-specific phosphodiesterases 2 and 9 in normal
29. Wang H, Yan Z, Yang S, et al. Kinetic and structural studies
of phosphodiesterase-8A and implication on the inhibitor
selectivity. Biochemistry 2008;47:12760–8.
and Alzheimer’s disease human brains. Eur
2007;25:3332–8.
J Neurosci
13. van der Staay FJ, Rutten K, Barfacker L, et al. The novel
selective PDE9 inhibitor BAY 73-6691 improves learning and
memory in rodents. Neuropharmacology 2008;55:908–18.
14. Kroker KS, Mathis C, Marti A, et al. PDE9A inhibition rescues
amyloid beta-induced deficits in synaptic plasticity and cog-
nition. Neurobiol Aging 2014;35:2072–8.
30. Reed TM, Browning JE, Blough RI, et al. Genomic structure
and chromosome location of the murine PDE1B phospho-
diesterase gene. Mamm Genome 1998;9:571–6.
31. Ou BX, Hampsch-Woodill M, Prior RL. Development and val-
idation of an improved oxygen radical absorbance capacity
assay using fluorescein as the fluorescent probe. J Agr Food
Chem 2001;49:4619–26.
32. Davalos A, Gomez-Cordoves C, Bartolome B. Extending
applicability of the oxygen radical absorbance capacity
(ORAC-fluorescein) assay. J Agric Food Chem 2004;52:48–54.
33. Denizot F, Lang R. Rapid colorimetric assay for cell growth
and survival. Modifications to the tetrazolium dye procedure
15. Kroker KS, Rast G, Giovannini R, et al. Inhibition of acetyl-
cholinesterase and phosphodiesterase-9A has differential
effects
on
hippocampal
early
and
late
LTP.
Neuropharmacology 2012;62:1964–74.
16. Li J, Liu CN, Wei N, et al. Protective effects of BAY 73-6691, a
selective inhibitor of phosphodiesterase 9, on amyloid-beta
peptides-induced oxidative stress in in vivo and in vitro mod-
els of Alzheimer’s disease. Brain Res 2016;1642:327–35.
giving improved sensitivity and reliability.
J Immunol
Methods 1986;89:271–7.
17. Hutson PH, Finger EN, Magliaro BC, et al. The selective
phosphodiesterase 9 (PDE9) inhibitor PF-04447943 (6-[(3S,
4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetra-
hydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimi-
din-4-one) enhances synaptic plasticity and cognitive
function in rodents. Neuropharmacology 2011;61:665–76.
18. Verhoest PR, Fonseca KR, Hou XJ, et al. Design and discovery
of 6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-
1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-
34. Meng F, Hou J, Shao YX, et al. Structure-based discovery of
highly selective phosphodiesterase-9A inhibitors and impli-
cations for inhibitor design. J Med Chem 2012;55:8549–58.
35. Jain AN. Surflex: fully automatic flexible molecular docking
using a molecular similarity-based search engine. J Med
Chem 2003;46:499–511.
36. Massova I, Kollman PA. Combined molecular mechanical and
continuum solvent approach (MM-PBSA/GBSA) to predict lig-
and binding. Perspect Drug Discov 2000;18:113–35.

270
C. ZHANG ET AL.
37. Hou TJ, Wang JM, Li YY, et al. Assessing the performance of
the MM/PBSA and MM/GBSA methods. 1. The accuracy of
treatment of Alzheimer’s disease. Eur
2017;130:379–92.
J Med Chem
binding free energy calculations based on molecular dynam- 41. Chen ZW, Digiacomo M, Tu YL, et al. Discovery of novel riva-
ics simulations. J Chem Inf Model 2011;51:69–82.
stigmine-hydroxycinnamic acid hybrids as multi-targeted
38. Xu L, Sun HY, Li YY, et al. Assessing the performance of MM/
PBSA and MM/GBSA methods. 3. The impact of force fields
agents for Alzheimer’s disease. Eur
2017;125:784–92.
J
Med Chem
and ligand charge models.
8408–21.
J
Phys Chem
B
2013;117: 42. Baell JB, Holloway GA. New substructure filters for removal
of pan assay interference compounds (PAINS) from screen-
39. Case DA, Betz RM, Cerutti DS, et al. Amber 2016. San
Francisco, CA: University of California; 2016.
ing libraries and for their exclusion in bioassays. J Med
Chem 2010;53:2719–40.
40. Sang ZP, Pan WL, Wang KR, et al. Design, synthesis 43. Wunder F, Tersteegen A, Rebmann A, et al. Characterization
and evaluation of novel ferulic acid-O-alkylamine deriva-
tives as potential multifunctional agents for the
of the first potent and selective PDE9 inhibitor using a
cGMP reporter cell line. Mol Pharmacol 2005;68:1775–81.