Identification of aminopyridazine-derived antineuroinflammatory agents effective in an Alzheimer's mouse model

Article abstract of DOI:10.1021/ml3001769

Targeting neuroinflammation may be a new strategy to combat Alzheimer's disease. An aminopyridazine 1b previously reported as a novel antineuroinflammatory agent was considered to have a potential therapeutic effect for Alzheimer's disease. In this study, we further explored the chemical space to identify more potent antineuroinflammatory agents and validate their in vivo efficacy in an animal model. Compound 14 was finally identified as an effective agent with comparable in vivo efficacy to the marketed drug donepezil in counteracting spatial learning and working memory impairment in an Aβ-induced Alzheimer's mouse model.

Full text of DOI:10.1021/ml3001769

Letter
pubs.acs.org/acsmedchemlett
Identification of Aminopyridazine-Derived Antineuroinflammatory
Agents Effective in an Alzheimer's Mouse Model
Wei Zhou,† Guifa Zhong,† Xiurong Rao,† Hui Xie,† Shaogao Zeng,† Tianyan Chi,‡ Libo Zou,‡
Donghai Wu,† and Wenhui Hu*,†,§
†Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, People's Republic of China
‡Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China
§State Key Laboratory of Respiratory Disease, Guangzhou 510120, People's Republic of China
S
* Supporting Information
ABSTRACT: Targeting neuroinflammation may be a new strategy to combat Alzheimer's
disease. An aminopyridazine 1b previously reported as a novel antineuroinflammatory agent
was considered to have a potential therapeutic effect for Alzheimer's disease. In this study, we
further explored the chemical space to identify more potent antineuroinflammatory agents
and validate their in vivo efficacy in an animal model. Compound 14 was finally identified as
an effective agent with comparable in vivo efficacy to the marketed drug donepezil in
counteracting spatial learning and working memory impairment in an Aβ-induced
Alzheimer's mouse model.
KEYWORDS: Alzheimer's disease, neuroinflammation, antineuroinflammatory agent, animal model, Morris water maze
lzheimer's disease (AD)1 is considered one of the most
Adevastating, life-threatening diseases among the aged. It is
also the most common form of neurodegenerative senile
dementia, characterized by memory loss and progressive
impairment of cognitive functions.2 Until now, there have
been very few approved AD drugs on the market (e.g., Figure
1), and these only provide minimal symptomatic relief rather
than altering the disease progression. Thus, there is still a great
need for disease-modifying therapies for AD.
factors impose immunological insult to neurons and cause
neuronal damage or death, which can also induce microglial
activation, facilitating the propagation of a localized, detrimen-
tal cycle of neuroinflammation.8−10 Given the significant role of
neuroinflammation in variety of effects linked to AD
mechanisms, neuroinflammatory inhibition using small-mole-
cule inhibitors is an important and direct strategy for
therapeutic intervention in AD and other neurodegenerative
diseases.
Compound 1a (Figure 2) was reported to be a novel
antineuroinflammatory agent by the Watterson lab.11 It can
selectively block increased IL-1β, TNF-α, and nitric oxide
(NO) production by activated glia without inhibition of
Figure 1. Structures of marketed drugs to treat AD.
Accumulating evidence suggests that excessive microglial
activation and up-regulation of proinflammatory cytokine
production by activated microglia play a previously under-
appreciated role in the pathophysiology of AD.3−7 Neuro-
inflammation stimulated by β-amyloid (Aβ) aggregation and
intracellular neurofibrillary tangles leads to the overexpression
of proinflammatory factors such as interleukin 1 β (IL-1β) and
tumor necrosis factor α (TNF-α). These proinflammatory
Figure 2. Structures of reported antineuroinflammatory agents.
Special Issue: Alzheimer's Disease
Received: June 29, 2012
Accepted: September 3, 2012
Published: September 4, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
potentially beneficial glial functions. Although endowed with
this favorable biological function, the intrinsic deficiency of
druglikeness of compound 1a eliminated it from possible
clinical use because AD drugs must be orally available and able
to cross the blood−brain barrier. Follow-up studies that mainly
concentrated on the physical property-driven optimization led
to compound 1b (Minozac, under drug development, Figure
2), a water-soluble oral drug candidate.12,13 However, the
insufficient in vitro activity of compound 1b limits its potential
for anti-AD drug development, and its in vivo efficacy has not
been well established using a standard animal model.
Table 1. Structures and the IL-1β Synthesis Inhibition
Activity of Compounds 4−21
In this paper, we further investigate the unexplored chemical
space of Minozac, attempt to identify inhibitors with higher in
vitro activity, and validate in vivo efficacy using the Morris
water maze AD animal model. Previous structural modifications
focused on the improvement of molecular properties by
diversifying the C-3 and C-4 positions of the aminopyridazine
while leaving C-5 and C-6 unchanged.4,5,11−14 The preliminary
structure−activity relationship (SAR) study also showed that
the in vitro function might be sensitive to substituents at C-6
position, prompting us to study this SAR further. This series of
neuroinflammation suppressors have been reported to have a
potential therapeutic effect in neurodegenerative disorders such
as AD, but there is no concrete animal model evidence revealed
in the literature to date. Several clinical candidates to treat AD
had been discontinued because of the absence of data on
behavioral effects in AD animal models,15 suggesting that such
studies are important criteria for the evaluation of AD drug
candidates. Thus, we decided to employ the Morris water maze,
a frequently used laboratory tool in behavioral neuroscience to
investigate spatial learning and working memory16−18 and
assess the behavioral effects of our compounds in an AD mouse
model.
To validate our hypothesis, a series of pyridazine compounds
(Table 1) based on compound 1b with changes at the C-6
position were designed and synthesized. The benzene ring was
replaced with wide range of substituents. All synthesized
compounds were screened for their inhibition of IL-1β
production in lipopolysaccharide (LPS)-stimulated BV-2
mouse microglia cells. The synthetic approach to construct
target compounds 5−21 is presented in Scheme 1. Briefly, the
commercially available starting material 3,6-dichloro-4-methyl-
pyridazine was treated with acetic acid19 to afford 6-chloro-4-
methylpyridazin-3(2H)-one (2) in 57% yield. Compound 2 was
then transformed to the triflate intermediate 3. Condensation
of 3 with 1-(2-pyrimidyl) piperazine in dimethylformamide
provided the key intermediate 4. Hydrogen reduction with Pd/
C converted compound 4 to the unsubstituted analogue 5.
Compounds 6 and 7 were finally obtained via iron-catalyzed
cross-coupling reactions20 with acceptable yields. Compounds
8−21 (Scheme 1 and Table 1) were prepared using Suzuki−
Miyaura cross-coupling21 reactions with intermediate 4.
To retain druglikeness, all of the compounds designed here
(Table 1) strictly obey Lipinski's rules of five. The IC50 of
Minozac 1b in our BV-2 cell-based assay was 11.4 μM. As
compared with this positive control, the replacement of 6-
phenyl with H (5), Cl (4), CH3 (6), and even isopropyl (7) led
to a loss in activity, unsurprisingly, confirming that in vitro
activity is sensitive to the C-6 position and may require larger
or aromatic functional groups. Thus, we decided to modify the
6-phenyl ring with different substituents, such as fluoro, methyl,
and methoxy groups. The 3-methoxy analogue 8 exhibited
superior activity over Minozac 1b. The attachment of an
a
Concentration (μM) for 50% inhibition of IL-1β release in BV-2
cells. The IC50 values are the mean
experimental determinations.
SEM for at least three
aromatic heterocyclic ring (15−21) at the C-6 position led to a
great loss in activity, whereas compound 14 also exhibited
higher activity than Minozac (2.4 and 11.4 μM, respectively).
Because our focus was to validate in vivo efficacy of these
aminopyridazine-derived neuroinflammation suppressors
against AD, further SAR studies were not pursued in the
paper, and compound 14 was immediately selected to probe in
vivo efficacy in the AD animal model.
The Morris water maze test was employed to evaluate the in
vivo function of compound 14 in an AD mouse model by
examining the effect of attenuating spatial learning and working
memory impairment. The dosage was set at 2.5 mg/kg based
on the previous study by Watterson, and the top marketed AD
drug donepezil served as a reference at its regular dosage (0.65
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a
Scheme 1. Synthesis of Target Compounds 5−21
Figure 3. Effect of compound 14 on escape latency in the Morris
water maze test for mice with icv Aβ1−42 (n = 7−8, mean SD). ###
P
< 0.001 vs sham group; *P < 0.05 or ***P < 0.001 vs model group.
Day 1 (F3,83 = 1.278, P = 0.288), day 2 (F3,83 = 0.466, P = 0.707), day 3
(F3,83 = 8.121, P = 0.001), and day 4 (F3,83 = 16.989, P = 0.0001).
a
Reagents and conditions: (a) Acetic acid, reflux (57%). (b)
Trifluoromethanesulfonic anhydride, triethylamine, DCM, −10 to 0
°C. (c) 1-(2-Pyrimidyl) piperazine, DMF, 0 °C (77% over two steps).
(d) H2, Pd/C, MeOH, rt, 24 h (64%). (e) R1 = Me (6), MeMgCl,
NMP, ferric acetylacetonate, Et2O, THF, 0.5 h, rt (77%); R1 = i-Pr (7),
i-PrMgCl, NMP, ferric acetylacetonate, Et2O, THF, 0.5 h, rt (84%). (f)
R2B(OH)2, Pd(PPh3)4, K2CO3, DME (32−80%), R2 as defined in
Table 1 (8−21).
Figure 4. Effect of compound 14 on swimming distances in the Morris
water maze test for mice with icv Aβ1−42 (n = 7−8, mean SD). #P <
0.05, ###P < 0.001 vs sham group; *P < 0.05 or ***P < 0.001 vs model
group. Day 1 (F3,83 = 2.080, P = 0.109), day 2 (F3,83 = 0.848, P =
0.472), day 3 (F3,83 = 4.216, P = 0.008), and day 4 (F3,83 = 24.142, P =
0.0001).
mg/kg). The Aβ model mouse group received interacere-
broventricular (icv) injection with aggregated Aβ1−42 (equiv-
alent to 410 pmol/mouse). As a control, the sham-operated
group mice were injected icv with the vehicle (saline) only. The
Aβ + compound 14 group and the Aβ + donepezil group
received daily single dose per oral (po) administration of
compound 14 at 2.5 mg/kg and donepezil at 0.65 mg/kg,
respectively. The Aβ model group and sham-operated group
received po administration of the same volume of 0.5% CMC−
saline solution. All compounds were systemically administrated
in a volume of 0.1 mL/10 g body weight from the day of icv Aβ
until the end of behavioral testing. The Morris water maze was
performed as previously described.22 The reference memory
test was conducted three times a day for 4 consecutive days.
After the 12th training trial of the reference memory test on day
13 following the Aβ1−42 injection, probe tests were carried out,
and the swimming percentage of path length and the time spent
in the target quadrant where the platform was located during
training were recorded.
swimming percentage of path length (p < 0.05; Figure 5) and
the swimming time (p < 0.05; Figure 6) spent in the target
Figure 5. Effect of compound 14 on swimming percentage of path
length spent in the target quadrant in the probe test for mice with icv
Aβ1−42 (n = 7−8, mean SD). #P < 0.05 vs sham group; *P < 0.05 vs
model group.
Changes of escape latency and swimming distance are shown
in Figures 3 and 4. Starting from day 3 of the reference memory
test, Aβ1−42 model mice showed significantly prolonged
swimming distance (p < 0.05) and escape latency to the safe
platform (p < 0.001) as compared with the sham-operated
group. Treatment with compound 14 significantly reversed the
impairments in spatial recognition memory for Aβ1−42 model
quadrant. Compound 14 treatment significantly increased the
swimming percentage of path length (F3,26 = 4.574, P = 0.025)
and the swimming time (F3,26 = 4.576, P = 0.012) spent in the
target quadrant. This effect was also reversed by donepezil
treatment, suggesting the improvement of spatial memory.
In the experiment, a single icv injection of a picomolar dose
of Aβ1−42 effectively impaired learning and memory behavior in
mice. Compound 14 prevented Aβ1−42-induced spatial
recognition memory impairment in the Morris water maze
test. This study demonstrates that the antineuroinflammatory
mice at day 3 (F3,83 = 8.121, P = 0.001) and day 4 (F3,83
=
16.989, P = 0.0001). The donepezil group exhibited a pattern
similar to the compound 14 group in terms of swimming time
and escape latency.
One day after the last training trial, we performed a probe
test to measure the maintenance of memory function. The
results of the probe test showed significant memory impairment
in the Aβ1−42 model mice. As compared with the sham-
operated group, Aβ1−42 model mice decreased in both the
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ACS Medicinal Chemistry Letters
Letter
ACKNOWLEDGMENTS
■
We are grateful to Hong Li for valuable technical support.
ABBREVIATIONS
■
AD, Alzheimer's disease; Aβ, β-amyloid; TNF-α, tumor
necrosis factor α; IL-1β, interleukin 1 β; SAR, structure−
activity relationship; LPS, lipopolysaccharide; NO, nitric oxide;
icv, interacerebroventricular; po, per oral; CNS, central nervous
system
Figure 6. Effect of compound 14 on the swimming time spent in the
target quadrant in the probe test for mice with icv Aβ1−42 (n = 7−8,
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for the synthesis, characterization, and
in vitro assay protocols of compounds 1b and 4−21 as well as
the in vivo study and the brain uptake assay of compound 14.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +862032015211. Fax: +862032015299. E-mail: hu_
wenhui@gibh.ac.cn.
■
Author Contributions
The manuscript was written through contributions of all
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Funding
This work is supported by Natural Science Foundation of
Guangdong Province, China (10251066302000000).
Notes
The authors declare no competing financial interest.
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