Structure-activity relationship study of pyridazine derivatives as glutamate transporter EAAT2 activators

Article abstract of DOI:10.1016/j.bmcl.2011.08.009

Excitatory amino acid transporter 2 (EAAT2) is the major glutamate transporter and functions to remove glutamate from synapses. A thiopyridazine derivative has been found to increase EAAT2 protein levels in astrocytes. A structure-activity relationship study revealed that several components of the molecule were required for activity, such as the thioether and pyridazine. Modification of the benzylthioether resulted in several derivatives (7-13, 7-15 and 7-17) that enhanced EAAT2 levels by >6-fold at concentrations <5 μM after 24 h. In addition, one of the derivatives (7-22) enhanced EAAT2 levels 3.5-3.9-fold after 24 h with an EC₅₀ of 0.5 μM.

Full text of DOI:10.1016/j.bmcl.2011.08.009

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5774–5777
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of pyridazine derivatives as glutamate
transporter EAAT2 activators
Xuechao Xing a, , Ling-Chu Chang b, , Qiongman Kong b, Craig K. Colton b, Liching Lai b,
a,
Marcie A. Glicksman a, Chien-Liang Glenn Lin b, , Gregory D. Cuny
⇑
⇑
a Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham & Women’s Hospital and Harvard Medical School, 65 Landsdowne St.,
Cambridge, MA 02139, USA
b Department of Neuroscience, The Ohio State University, 333 West 10th Ave, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Excitatory amino acid transporter 2 (EAAT2) is the major glutamate transporter and functions to remove
glutamate from synapses. A thiopyridazine derivative has been found to increase EAAT2 protein levels in
astrocytes. A structure–activity relationship study revealed that several components of the molecule
were required for activity, such as the thioether and pyridazine. Modification of the benzylthioether
resulted in several derivatives (7-13, 7-15 and 7-17) that enhanced EAAT2 levels by >6-fold at concentra-
Received 7 June 2011
Revised 28 July 2011
Accepted 1 August 2011
Available online 8 August 2011
tions <5
24 h with an EC50 of 0.5
l
M after 24 h. In addition, one of the derivatives (7-22) enhanced EAAT2 levels 3.5–3.9-fold after
M.
Keywords:
Excitatory amino acid transporter 2
Glutamate
l
Ó 2011 Elsevier Ltd. All rights reserved.
Pyridazines
Excitotoxicity
Glutamate is a major neurotransmitter in the mammalian cen-
tral nervous system (CNS) and essential for normal brain function
including cognition, memory, and learning. However, the extracel-
lular concentration of glutamate must remain below excitotoxic
In an effort to identify small molecules that can increase EAAT2
protein expression, a high-throughput screen of approximately
140,000 compounds was previously conducted in our laboratories
using a cell-based enzyme-linked immunosorbent assay.8 The hits
identified from this screening provide starting points for further
optimization in order to arrive at pharmacologically useful mole-
cules for studying the role of EAAT2 in excitotoxicity induced
neuronal injury and potentially as therapeutic agents.
The thiopyridazine 1 was confirmed to show a dose-dependent
increase in EAAT2 protein levels after 24 h exposure. Herein, we
report the structure–activity relationship (SAR) study of 1 for
elevating EAAT2 protein levels.
Many of the pyridazine analogues utilized in the SAR study
were prepared using the method depicted in Scheme 1. Treatment
of ketone 2 with glyoxylic acid (3) and K2CO3 gave 4, which was
used directly without purification. It was allowed to react with
hydrazine in acetic acid at 100 °C to yield the desired pyridazinone
5 as an off-white solid after recrystallization from ethyl acetate.9
Direct alkylation of 5 gave 8 in good yields. Intermediate 5 was also
converted into pyridazinethione 6 in the presence of P2S5 in pyri-
dine at 120 °C.10 Alkylation of 6 provided 7, which could be subse-
quently oxidized to sulfone 9 with 3-chloroperoxybenzoic acid
(m-CPBA) in CH2Cl2.11
levels (ꢀ1
lM) to avoid overstimulation of glutamate receptors,
leading to neuronal damage or death.1 Excitotoxicity has been
associated with multiple acute neurological conditions such as
ischemic stroke, epilepsy, and trauma, as well as chronic adult-
onset neurodegenerative disorders such as Alzheimer’s disease
and amyotrophic lateral sclerosis (ALS).2–6 One potential approach
to preventing excitotoxicity is to enhance glutamate reuptake.
Excitatory amino acid transporter 2 (EAAT2) is the major gluta-
mate transporter and functions to remove glutamate from syn-
apses.7 An increase in EAAT2 protein expression and function
may provide a means to prevent insufficient glutamate reuptake
and consequently reduce neuronal damage.
A series of additional analogues 14–17 was prepared using the
methodology outlined in Scheme 2. Recently, 2-pyridyl N-methyli-
minodiacetic acid (MIDA) boronate (10) has been reported as an
air-stable slow-release reagent with high cross-coupling efficiency
even with heteroaryl chlorides.12 Therefore, this material was used
⇑
Corresponding authors. Tel.: +1 617 768 8640; fax: +1 617 768 8606 (G.D.C.);
tel.: +1 614 688 5433; fax: +1 614 688 8742 (C.-L.G.L.).
E-mail addresses: Chien-Liang.Lin@osumc.edu (C.-L.G. Lin), gcuny@rics.bwh.
harvard.edu (G.D. Cuny).
Equal contribution.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.009

X. Xing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5774–5777
5775
Scheme 1. Reagents and conditions: (a) K2CO3, H2O; (b) NH2NH2, AcOH, 100 °C
(20%); (c) P2S5, pyridine, 120 °C (80%); (d) R2Br, K2CO3, DMF (90%); (e) m-CPBA,
CH2Cl2 (80%).
Scheme 3. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, CH3CN/H2O (1/1), 75 °C,
4-benzylthiophenylboronic acid (54%); (b) Pd(PPh3)4, Na2CO3, CH3CN/H2O (1/1),
75 °C, 6-hydroxypyridine-3-boronic acid pinacol ester (70%); (c) P2S5, pyridine,
120 °C (71%); (d) 2-methylbenzyl bromide, K2CO3, DMF (76%).
in palladium-mediated cross-couple reactions with heteroaryl
chlorides 11–13 to obtain 14–16, respectively. In the case of 13
transesterification occurred during the cross-coupling reaction to
give the isopropyl ester 16, which was then allowed to react with
alkylamines in ethanol at 85 °C to generate amides 17.
Table 1
Effects of the modified pyridyl and benzyl substituent on EAAT2 protein levels
Finally, several additional analogues designed to evaluate
replacement of the pyridazine in 1 with phenyl and pyridyl moie-
ties were prepared using the methodology outlined in Scheme 3.
Suzuki coupling of 2-bromopyridine (18) with various boronic
acids afforded 19 and 20.13 Treatment of 20 with P2S5 in pyridine
at 120 °C provided 21 in 71% yield. Alkylation of 21 with 2-meth-
ylbenzyl bromide in the presence of K2CO3 in DMF generated 22.
All of the derivatives of 1 were initially evaluated in PA-EAAT2
cells3 (a primary astrocyte line stably expressing EAAT2 mRNAs)
Compound
R1
R2
Fold increase for EAAT2a
4 h
24 h
1
2-Py
3-Py
4-Py
4-Me-Ph
2-Py
3-Py
Ph
2-Py
3-Py
4-Py
2-Py
4-Py
2-Cl-6-F-Bn
2-Cl-6-F-Bn
2-Cl-6-F-Bn
2-Cl-6-F-Bn
2-Me-Bn
2-Me-Bn
2-Me-Bn
2-Cl-Bn
2-Cl-Bn
2-Cl-Bn
4-Me-Bn
4-Me-Bn
nab
na
na
2.0 0.8
2.3 0.8
1.9 0.9
2.3 0.7
3.5 0.3
na
2.0 0.4
4.0 0.3
1.2 0.3
1.7 0.3
2.6 0.4
2.4 0.6
7-1
7-2
7-3
7-4
7-5
7-6
7-7
7-8
7-9
7-10
7-11
following compound (10 lM) incubation for 4 and 24 h before har-
1.2 0.2
1.7 0.1
1.5 0.4
1.1 0.1
2.1 1.4
1.2 0.4
1.5 0.2
2.1 1.1
1.3 0.1
vesting and measuring EAAT2 levels by Western blot analysis. The
fold increases in EAAT2 protein levels relative to DMSO controls
are reported (Tables 1–4).
Replacement of the 2-pyridyl with 3-pyridyl (7-1, 7-5 and 7-8),
4-pyridyl (7-2, 7-9 and 7-11) or phenyl (7-6) resulted in equal or
reduced activity (Table 1). Removing either one or both of the
nitrogen atoms in the pyridazine resulted in loss of activity (Table
2). Collectively, these results suggested that both the 2-pyridyl and
the pyridazine were required for enhancing EAAT2 protein
expression.
a
Compound concentration of 10
na = not active.
lM.
b
Table 2
Effects of modifications of the pyridazine on EAAT2 protein levels
Compound
X
Y
Z
R
Fold increase for EAATa
4 h
24 h
7-4
22
8-1
15
7-12
19
S
S
O
O
S
N
C
N
N
N
C
N
N
N
C
N
C
2-Me-Bn
2-Me-Bn
2-Me-Bn
2-Me-Bn
Bn
1.7 0.1
1.3 0.6
2.0 0.9
1.5 0.3
1.3 0.2
1.2 0.5
3.5 0.3
nab
2.5 0.5
na
3.0 0.4
1.1 0.4
S
Bn
a
Compound concentration of 10
na = not active.
lM.
b
Next, the sulfur linker was examined (Table 3). Replacing the
sulfur with oxygen generally yielded less active derivatives. An
Scheme 2. Reagents and conditions: (a) Pd2(dba)3, XPhos, Cu(OAc)2, K2CO3, DMF/i-
PrOH (4/1), 100 °C; (b) H2NR, ethanol, 85 °C (90%).

5776
X. Xing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5774–5777
Table 3
Table 4
Effects of modifications of the sulfur linker on EAAT2 protein levels
Effects of the benzyl group on EAAT2 protein levels
Compound
X
R
Fold increase for EAAT2a
Compound
R
Fold increase for EAAT2a
4 h
24 h
4 h
24 h
1
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
2-Cl-6-F-Bn
2-Cl-6-F-Bn
2-Me-Bn
nab
2.0 0.8
2.9 0.4
3.5 0.3
2.5 0.5
6.5 1.0
3.2 1.4
5.5 1.0
3.0 0.7
6.7 1.5
3.1 1.2
3.8 0.8
2.3 0.6
6.4 1.0
2.0 0.9
2.0 0.8
1.6 1.0
2.6 0.5
2.8 0.4
1.1 0.2
na
1
7-7
2-Cl-6-F-Bn
2-Cl-Bn
3-Cl-Bn
2,3-Di-Cl-Bn
2,5-Di-Cl-Bn
2,4-Di-Cl-Bn
2,6-Di-Cl-Bn
2-F-Bn
nab
2.1 1.4
2.1 0.3
na
2.0 0.8
4.0 0.3
4.1 0.5
2.4 0.4
2.5 0.4
na
8-2
7-4
8-1
7-13
8-3
7-14
8-4
7-15
8-5
7-16
8-6
7-17
8-7
6-1
5-1
14
1.1 0.3
1.7 0.1
2.0 0.9
na
2.1 1.2
1.7 0.9
1.6 0.2
1.9 0.7
2.2 0.4
2.4 1.5
na
1.9 0.8
na
na
1.5 0.5
1.7 1.1
1.4 0.3
2.3 0.4
1.6 0.3
na
7-18
7-19
7-20
7-21
7-22
7-23
7-24
7-16
7-25
7-26
7-4
7-27
7-28
7-29
7-17
7-13
7-30
7-15
7-31
7-32
7-33
7-14
7-34
7-35
7-12
7-36
6-1
2-Me-Bn
2,6-Di-Me-Bn
2,6-Di-Me-Bn
1-(2-Cl-6-F-phenyl)ethyl
1-(2-Cl-6-F-phenyl)ethyl
2-(2-Cl-6-F-phenylethyl)
2-(2-Cl-6-F-phenylethyl)
2,4-Di-F-Bn
2,4-Di-F-Bn
2,4-Di-Me-Bn
2,4-Di-Me-Bn
H
1.4 0.1
1.8 0.5
1.5 0.8
2.2 0.1
2.5 0.6
2.4 1.5
2.1 1.4
1.6 1.0
1.7 0.1
2.0 0.2
2.1 1.1
1.2 0.3
1.9 0.8
na
3.9 0.4
3.1 0.2
2.6 0.3
3.8 0.8
2.2 0.7
2.4 0.6
3.5 0.3
2.8 0.5
2.6 0.4
2.2 0.3
6.4 1.0
6.5 1.0
4.7 0.5
6.7 1.5
4.8 0.6
2.8 0.4
5.3 1.1
5.5 1.0
2.5 0.7
1.7 0.4
3.0 0.5
1.9 0.9
2.0 0.8
4-F-Bn
2,4-Di-F-Bn
2,6-Di-F-Bn
2,4,6-Rri-F-Bn
2-Me-Bn
3-Me-Bn
4-Me-Bn
2,4-Di-Me-Ph
2,4-Di-Me-Bn
2,6-Di-Me-Bn
2,4,6-Rri-Me-Bn
2-(2-Cl-6-F-phenylethyl)
2-(2-Cl-phenylethyl)
2-(2-Me-phenylethyl)
1-(1,2,3,4-Tetrahydronaphthalenyl) na
1-(2-Cl-6-F-phenyl)ethyl
2-Cl-4-F-Ph
2-Cl-4-F-Bn
Bn
Et
H
H
NH
SO2
SO2
SO2
CONH
CONH
2-Me-Bn
2-Cl-6-F-Bn
2-Me-Bn
2-Cl-Bn
n-Pr
9-1
9-2
9-3
17-1
17-2
1.7 0.3
1.9 0.7
1.7 0.2
1.2 0.3
1.2 0.2
1.4 0.2
2-Me-Bn
na
a
Compound concentration of 10
na = not active.
lM.
1.7 0.9
b
1.3 0.1
1.6 0.6
1.3 0.2
1.6 0.7
na
analogue containing a NH linker (14) showed weaker activity. Like-
wise, oxidation of the sulfur to a sulfone (9-1, 9-2, and 9-3) was
also detrimental. Finally, replacing the sulfur with an amide moiety
(17-1 and 17-2) was not tolerated. Collectively, these results indi-
cated that the sulfur linker was optimal.
a
Compound concentration of 10
na = not active.
lM.
b
Finally, the benzyl group was examined (Table 4). Compared to
1, 2-chloro, 3-chloro and 2,6-dichloro substitutions improved the
potency by 2-fold (7-7, 7-18 and 7-22), but 2,3-dichloro, 2,4-di-
chloro and 2,5-dichloro analogues (7-19, 7-21 and 7-20) were
equivalent to 1. In the case of fluorine substitutes, only the 2-fluoro
Table 5
Fold increase and EC50 values of EAAT2 protein levels of selected analoguesa
Compound
R
Fold increase for EAAT2
M, 24 h
EC50 (lM)
1
l
10 lM, 24 h
b-actinb
GAPDHc
b-actinb
GAPDHc
b-actinb
GAPDHc
7–4
7–7
2-Me-Bn
2-Cl-Bn
2,6-Di-Me-Bn
1-(2-Cl-6-F-phenyl)ethyl
2,4-Di-Me-Bn
1.0 0.8
1.1 0.5
2.0 0.2
1.5 0.5
2.0 0.3
3.0 0.1
1.0 0.2
1.5 0.4
1.2 0.3
1.0 0.7
2.4 0.3
4.0 0.4
1.0 0.3
1.6 0.4
2.0 0.4
1.8 0.4
2.2 0.6
3.5 0.0
1.0 0.5
1.6 0.4
1.2 0.2
1.0 0.1
2.8 0.4
4.2 0.1
3.5 0.3
4.0 0.3
6.5 1.0
5.5 1.0
6.4 1.0
3.9 0.4
3.8 0.8
4.7 0.5
6.7 1.5
4.8 0.6
5.3 1.1
4.1 0.5
3.1 0.2
4.5 0.6
6.4 0.9
5.2 1.2
6.0 0.9
3.5 0.5
3.7 0.6
4.8 0.6
5.3 1.2
4.5 0.6
4.5 0.6
3.8 0.5
2.6 0.8
3.3 0.5
2.6 0.2
3.9 0.5
2.7 0.3
0.5 0.1
2.6 0.2
2.4 0.4
3.3 0.3
4.7 0.8
1.9 0.3
1.7 0.4
1.8 0.3
3.2 0.5
2.4 0.4
2.4 0.4
1.8 0.6
0.5 0.0
2.6 0.5
2.1 0.4
3.3 0.2
5.1 0.1
1.7 0.4
0.5 0.1
7–13
7–14
7–17
7–22
7–16
7–30
7–15
7–31
7–33
7–18
2,6-Di-Cl-Bn
2,4-Di-F-Bn
2,4,6-Tri-Me-Bn
2-(2-Cl-6-F-phenylethyl)
2-(2-Cl-phenylethyl)
1-(1,2,3,4-Tetrahydronaphthalenyl)
3-Cl-Bn
a
b
c
Data represent the mean values of four separate experiments.
Data standardized by b-actin.
Data standardized by GAPDH.

X. Xing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5774–5777
5777
and 2,4-difluoro derivatives (7-23 and 7-16) demonstrated im-
proved activity. 4-Fluoro, 2,6-difluoro, and 2,4,6-trifluoro ana-
logues (7-24, 7-25, and 2-26) did not result in significant
improvement. Replacement of the halogens with methyls (7-4,
7-13, 7-17, and 7-30) gave increased potency. Compounds contain-
ing 2-methyl substituted benzyl groups significantly increased
EAAT2 protein level, where as the 3- or 4-methylbenzyl derivatives
(7-27 and 7-28) were less potent. Increasing the tether length be-
tween the phenyl and the sulfur to an ethylene generally yielded
more potent analogues (7-15 and 7-31 vs 1 and 7-7, respectively)
with one noted exception (compare 7-32 to 7-4). However, trun-
cating the linker resulting in a diarylthioether decreased potency
(7-29 vs 7-17). Interestingly, adding substitutes on the carbon lin-
ker (7-14 and 7-33) increased activity about 3-fold. Finally, other
changes gave compounds with essentially the same potency as 1
(7-12, 7-36, and 6-1).
may also be beneficial in determining the biological mechanisms
for regulating EAAT2 levels.
Acknowledgment
We are grateful to the National Institutes of Health (U24
NS04933901 and R01 NS064275), the Harvard NeuroDiscovery
Center, ALS Therapy Alliance, Inc and Project ALS for financial
support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.009.
References and notes
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The fold increases at 1 and 10 M, as well as the EC50 values are
l
summarized in Table 5. In addition, these values were determined
relative to two unrelated proteins (e.g., b-actin and GAPDH). Sev-
eral compounds (7-13, 7-17, 7-31 and 7-18) were able to increase
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EAAT2 levels >6-fold at 10
with EC50 values in the 2.6–3.3
strated the lowest EC50 of 0.5
EAAT2 levels of 3- to 4-fold.
l
M compared to b-actin or GAPDH, but
M range. Analog 7-22 demon-
M with a maximum increase of
l
l
In summary, a series of pyridazine derivatives were synthesized
and evaluated for increasing EAAT2 protein levels. Several com-
pounds were found to significantly increase EAAT2 protein levels
(>6-fold), such as 7-13, 7-15 and 7-17. Derivative 7-22 increased
EAAT2 protein levels to a lower extent (3.5–3.9-fold), but with a
lower EC50 value (0.5 lM). These compounds will provide useful
tools for further assessing the role of glutamate excitotoxicity in
cellular systems and potentially in animal models of acute and
chronic neurodegeneration. In addition, these probe molecules