Synthesis and biological evaluation of pyrethroid insecticide-derivatives as a chemical inducer for Bdnf mRNA expression in neurons

Article abstract of DOI:10.1016/j.bmc.2012.02.048

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in neuronal synaptic plasticity. A decrease of plasticity in the brain may be related to the pathogenesis of neurodegenerative or psychiatric disorders. Pyrethroid insecticides, which affect sodium channels in neurons, are widely used to control insect pests in agriculture and in the home. We previously found that deltamethrin (DM), a type II pyrethroid, increased Bdnf mRNA expression in cultured rat cortical neurons. However, the cyano group at the α-position of type II pyrethroids is likely susceptible to hydrolytic degradation and, its degraded product, hydrogen cyanide, could generate a cellular toxicity in the human body. To determine if the cyano group is required for the Bdnf exon IV-IX (Bdnf eIV-IX) mRNA expression induced by type II pyrethroids, for this study we synthesized a series of derivatives, in which the cyano group at the α-position was replaced with an ethynyl group. Then we added various substituents at the terminal position of the ethynyl group, and biologically evaluated the effects of these derivatives on Bdnf eIV-IX mRNA expression. These ethynyl derivatives induced the Bdnf eIV-IX mRNA expression in a concentration-dependent manner, at varying levels but lower levels than that evoked by DM. The mechanisms for the Bdnf induction and the morphological changes of neurons were the same whether the cyano or ethynyl group was included in the compounds.

Full text of DOI:10.1016/j.bmc.2012.02.048

Bioorganic & Medicinal Chemistry 20 (2012) 2564–2571
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of pyrethroid insecticide-derivatives
as a chemical inducer for Bdnf mRNA expression in neurons
Yuji Matsuya a, , Daisuke Ihara b, , Mamoru Fukuchi b, Daisuke Honma b, Kiyoshi Itoh b, Akiko Tabuchi b,
Hideo Nemoto a, Masaaki Tsuda b,
⇑
a Department of Organochemical Design and Synthesis, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
b Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Brain-derived neurotrophic factor (BDNF) plays a fundamental role in neuronal synaptic plasticity. A
decrease of plasticity in the brain may be related to the pathogenesis of neurodegenerative or psychiatric
disorders. Pyrethroid insecticides, which affect sodium channels in neurons, are widely used to control
insect pests in agriculture and in the home. We previously found that deltamethrin (DM), a type II pyre-
throid, increased Bdnf mRNA expression in cultured rat cortical neurons. However, the cyano group at the
Received 8 February 2012
Revised 20 February 2012
Accepted 21 February 2012
Available online 3 March 2012
a
-position of type II pyrethroids is likely susceptible to hydrolytic degradation and, its degraded product,
Keywords:
Pyrethroid
Deltamethrin
BDNF
hydrogen cyanide, could generate a cellular toxicity in the human body. To determine if the cyano group
is required for the Bdnf exon IV-IX (Bdnf eIV-IX) mRNA expression induced by type II pyrethroids, for this
study we synthesized a series of derivatives, in which the cyano group at the
a-position was replaced
with an ethynyl group. Then we added various substituents at the terminal position of the ethynyl group,
and biologically evaluated the effects of these derivatives on Bdnf eIV-IX mRNA expression. These ethynyl
derivatives induced the Bdnf eIV-IX mRNA expression in a concentration-dependent manner, at varying
levels but lower levels than that evoked by DM. The mechanisms for the Bdnf induction and the morpho-
logical changes of neurons were the same whether the cyano or ethynyl group was included in the
compounds.
Ethynyl substituent
Structure–activity relationship
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
(eIꢀVIII) and a common 30 exon (eIX), encoding a preproBDNF pro-
tein. Alternative splicing produces several types of transcripts be-
Brain-derived neurotrophic factor (BDNF) is a neurotrophin that
plays a fundamental role in a variety of neuronal functions, such as
cell survival, differentiation and synaptic plasticity. Recently, BDNF
has emerged as a key target in the treatment of several neuronal
disorders. Indeed, a decrease in BDNF-mediated trophic support
has been detected in patients with depression1 and Alzheimer’s
disease.2 Since BDNF has low permeability through the blood–
brain barrier,3 low-weight chemical compounds that induce its
expression in the brain are potentially useful in the treatment of
neuronal disorders. The BDNF gene (Bdnf) consists of eight 50 exons
tween each of the 50 exons and eIX.4 Transcripts containing eI or
eIV (eI-IX and eIV-IX mRNA) are expressed in an activity-dependent
manner.5 Specifically, Bdnf eIV-IX mRNA is the most abundantly
and widely expressed subtype in the rat brain.6
Pyrethroids are highly active synthetic derivatives of natural
pyrethrins which are toxins present in the flowers of Chrysanthe-
mum cinerariaefolium. Pyrethroids are used to control insect pests
particularly in the home. Pyrethroids are classified into two major
groups on the basis of chemical structure as shown in Figure 1:
type II pyrethroids have an O-acyl cyanohydrin substructure at
the benzylic position (a-position) (i.e., cypermethrin and delta-
methrin), whereas type I do not (i.e., permethrin).7 Both types of
pyrethroids prolong the opening of the voltage-sensitive sodium
channel (VSSC) and raise a prolonged sodium tail current in mam-
malian as well as invertebrate neurons.8,9
Abbreviations: APV,
D
-(ꢁ)-2-amino-5-phosphonopentanoic acid; BDNF, brain-
derived neurotrophic factor; DM, deltamethrin; DMEM, Dulbecco’s modified Eagle’s
medium; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; L-VDCC, L-type voltage-dependent calcium channel;
Previously, we found that deltamethrin (DM), a type II pyre-
throid, increased Bdnf eIV-IX mRNA expression not only in vitro
but also in vivo.10,11 Though their effects varied, type II pyrethroids
induced Bdnf eIV-IX mRNA expression, while type I pyrethroids
did not. However, type II pyrethroids possessing the cyano group
MAPK, mitogen-activated protein kinase; NMDA, N-methyl-D-aspartate; RT-PCR,
reverse transcription-polymerase chain reaction; TTX, tetrodotoxin; U0126, 1,4-
diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; VSSC, voltage-sensi-
tive sodium channel.
⇑
Corresponding author. Tel.: +81 76 434 7535; fax: +81 76 434 5048.
E-mail address: tsuda@pha.u-toyama.ac.jp (M. Tsuda).
at the
a-position are highly susceptible to hydrolytic degradation
These authors equally contributed to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.048

Y. Matsuya et al. / Bioorg. Med. Chem. 20 (2012) 2564–2571
2565
Me Me
Me Me
Cl
X
O
O
X
OPh
Cl
OPh
O
CN
O
X = Br: deltamethrin (1, DM)
permethrin (four diastereomers)
Type I
Type II
X = Cl: cypermethrin
Me Me
Br
R = H (2a)
R = Me (2e)
R = I (2b)
O
R = Et (2f)
Br
OPh
R = Br (2c)
R = Cl (2d)
R = CH2CF3 (2g)
O
R
Figure 1. Pyrethroids including deltamethrin (DM) and its ethynyl analogues.
compared with the type I pyrethroids. This degradation yields an
aldehyde and hydrogen cyanide, which brings about structural
instability. Hydrogen cyanide generates a cellular toxicity in the liv-
ing body because the cyanide ion halts cellular respiration by inhib-
iting the mitochondrial enzyme, cytochrome c oxidase. In this study,
route. That is, the hydroxyl group of 6a was protected as a TBS ether
(quant.) and the acetylide anion generated from 8 and n-BuLi was
treated with NCS to form the chlorinated product 9d (69%),14 which
was then deprotected by exposure to TBAF (92%). In addition,
methyl, ethyl, and trifluoroethyl substituents were introduced via
the same pathway. Thus, the alcohols 6b–g were synthesized
successfully. The target compounds 2b–g were obtained by esteri-
fication between the alcohols 6b–g and carboxylic acid 7 under
the same reaction conditions as for the synthesis of 2a. In this
way, the target ethynyl derivatives having various substituents
were synthesized by efficient pathways.
therefore, we aimed to determine if the cyano group at the a-posi-
tion in type II pyrethroids is required for the induction of Bdnf
eIV-IX mRNA expression and, if not, whether non-cyano derivatives
of DM could induce the expression via the same mechanisms as DM.
For this purpose, we synthesized several derivatives of DM, by
replacing the cyano group with an ethynyl group possessing various
substituents at its terminal to improve the chemical stability. These
changes also avoid a production of a harmful hydrolysate. We then
evaluated the effects of these derivatives on Bdnf eIV-IX mRNA
expression and the morphological changes in neurons.
2.2. Biology
2.2.1. The Bdnf eIV-IX mRNA expression induced by ethynyl
derivatives
2. Results and discussion
2.1. Chemistry
The inducibility of Bdnf eIV-IX mRNA expression in cultured rat
cortical neurons was evaluated using real-time RT-PCR with all the
derivatives synthesized here. Initially, we monitored the level of
Bdnf eIV-IX mRNA expression induced by the parental ethynyl
derivative, 2a. As shown in Figure 2, the expression of Bdnf eIV-IX
mRNA was increased by addition of 2a. While the optimum con-
Figure 1 shows the chemical structure of DM (1), a representa-
tive type II pyrethroid, which contains an O-acyl cyanohydrin sub-
structure at the benzylic position (a-position). This functional
centration of DM was 1
lM, the maximum induction by 2a was
group is likely to be susceptible to hydrolytic (or some other nucle-
ophilic) degradation to yield an aldehyde and hydrogen cyanide as
mentioned above. Here we envisaged that replacement of the cya-
no group (a carbon–nitrogen triple bond) with an ethynyl group (a
carbon–carbon triple bond) would increase the stability of the
compound, leading to more promising and safer candidates for
an agent to treat neuronal disorders. We used the ethynyl deriva-
tive 2a and its analogues, 2b–g, possessing various substituents at
the terminal of the ethynyl group, as targets in this study (Fig. 1).
3-Phenoxybenzaldehyde (3) was reacted with lithium trimeth-
ylsilylacetylide in THF to afford an adduct alcohol, which was then
oxidized with PDC in the presence of molecular sieves in CH2Cl2
(with a 94% yield in 2 steps). The resulting ketone 4 was subjected
to asymmetric reduction using (S)-2-methyl-CBS-oxazaborolidine
as a catalyst to give an optically active alcohol (5) with an 85%
yield. The TMS group in 5 was removed upon treatment with
K2CO3 in methanol quantitatively, and the alcohol 6a thus obtained
was condensed with a known chiral cyclopropanecarboxylic acid
(7) using carbonyldiimidazole and a catalytic amount of sodium
hydride to furnish the target ethynyl derivative 2a (Scheme 1).
Introduction of halogen or alkyl substituents into the terminal
alkyne was achieved according to Scheme 2. Iodination and bro-
mination were carried out through the reaction of 6a with I2/KOH
(39%)12 and the reaction of 5 with NBS/AgNO3 (70%),13 respectively.
On the other hand, chlorination involved a somewhat circuitous
obtained at 10 M (Fig. 2A), indicating that the inductive activity
l
of 2a is less than one tenth lower than that of DM. A time-depen-
dent increase in Bdnf eIV-IX mRNA expression was observed until
3 h after starting the incubation but declined at 6 h (Fig. 2B(a)).
However, the increased level of expression seen at 3 h returned
at 12 h and was maintained until 24 h (Fig. 2B(b)).
To examine whether or not the increase in Bdnf eIV-IX mRNA
expression induced by 2a is regulated similarly as that induced
by DM,10,11 we added several kinds of inhibitors to antagonize
the effect of 2a. The pretreatment of cortical cells with TTX, a po-
tent antagonist of VSSC, completely decreased the expression ob-
tained by 2a. The pretreatment of cells with nicardipine, a
blocker of L-type voltage-dependent calcium channels, but not
with APV, an antagonist for the NMDA receptor, also inhibited
the increase. The addition of U0126, a potent inhibitor of extracel-
lular signal-regulated kinase 1/2 (ERK1/2), also markedly de-
creased the expression, indicating the involvement of the ERK/
MAPK pathway in the increase in Bdnf eIV-IX mRNA expression.
These responses induced by 2a were consistent with those by
DM,11 though its inductive level was lower than DM’s. These data
are provided as Supplementary data.
To investigate the effect of hydrolysates on Bdnf eIV-IX mRNA
expression, we measured the mRNA levels using cyclopropanecar-
boxylic acid 7 or an aldehyde moiety 3 produced from pyrethroids.
These compounds did not increase the expression (data not

2566
Y. Matsuya et al. / Bioorg. Med. Chem. 20 (2012) 2564–2571
Me Me
Br
d
CHO
TMS
Br
CO2H
c
7
e
HO
a, b
2a
OPh
OPh
OPh
O
4
R = TMS (5)
R = H (6a)
3
R
Scheme 1. Reagents and conditions: (a) TMS-acetylene, n-BuLi, THF; (b) PDC, MS4A, CH2Cl2; (c) (S)-2-methyl-CBS-oxazaborolidine, BH3, THF; (d) K2CO3, MeOH; (e) (Im)2C@O,
cat. NaH, CH2Cl2.
Me Me
Br
a
HO
HO
Br
CO2H
OPh
b
7
OPh
2b–g
f
R = I (6b)
HO
6a
OPh
R = Br (6c)
R = Cl (6d)
R = Me (6e)
R = Et (6f)
R
e
5
c
TMS
R = CH2CF3 (6g)
d
TBSO
R = Cl (9d)
R = Me (9e)
R = Et (9f)
OPh
TBSO
OPh
8
R = CH2CF3 (9g)
R
Scheme 2. Reagents and conditions: (a) I2, KOH, MeOH/H2O (6b); (b) NBS, AgNO3, acetone (6c); (c) TBSOTf, 2,6-lutidine, CH2Cl2; (d) (i) n-BuLi, THF (ii) NCS (9d), MeI (9e), EtI
(9f), CF3CH2I (9g); (e) TBAF, THF (6d–g); (f) (Im)2C@O, cat. NaH, CH2Cl2.
Figure 2. The effects of the test compound 2a on Bdnf eIV-IX mRNA expression in cultured cortical neurons at 1–100 lM. (A) The cells were treated with DM or 2a and then
incubated for another 3 h before total RNA was extracted. One micromolar DM was used as a positive control. The ratio of mRNA expression relative to the control is shown.
Data are presented as the mean S.E.M. (number of experiments n = 3–4). ⁄⁄p <0.01 and ⁄⁄⁄p <0.001 compared with the control (CT). ⁄⁄⁄p <0.001 versus the control, using a
Student’s t-test with F test. ⁄⁄p <0.01 compared with the control, using a one-way ANOVA with the Scheffe’s F test. (B) Continuous induction of Bdnf eIV-IX mRNA expression
by 2a in rat cortical neurons. Ten micromolar 2a was added and the cells were incubated for the periods indicated before total RNA was prepared. The ratio of mRNA
expression relative to the control (2a (ꢁ)) at 0 h (a) or 12 h (b) is shown. The values represent the mean S.E.M (number of experiments n = 3–4). ⁄⁄p <0.01 compared with the
control at the same time point, using a one-way ANOVA with the Scheffe’s F test.
shown), indicating that pyrethroids are inactivated after their
hydrolysis.
possessing an alkyl group (2e–g) exhibited increases in Bdnf eIV-
IX mRNA expression with a maximum at 10 M (Fig. 3B). In partic-
l
ular, the methyl derivative 2e exhibited greater potency at 10
lM
2.2.2. The effect of substituents at the terminus of the ethynyl
group on Bdnf eIV-IX mRNA expression
Next, we modified the terminal hydrogen of the ethynyl group
with several substituents. The halogenated compounds (2b–d) sig-
nificantly increased Bdnf eIV-IX mRNA expression in a concentra-
tion-dependent manner (Fig. 3A). However, at 10 lM, the increase
induced by the halogenated compounds was lower than that in-
duced by 2a (Fig. 3A). On the other hand, the ethynyl derivatives
than 2a.
Based on these observations, a substituent containing a triple
bond at the -position effectively induces the expression of Bdnf
a
mRNA, with the cyano group having the greatest effect. In addition,
the size of the substituent at the terminal of the ethynyl group
affects the action toward sodium channels, resulting in the differ-
ence in the levels of Bdnf eIV-IX mRNA expression among the
derivatives.

Y. Matsuya et al. / Bioorg. Med. Chem. 20 (2012) 2564–2571
2567
Figure 3. The Bdnf eIV-IX mRNA-inducing effects of 2a and its derivatives 2b–g in cultured cortical neurons at 1–100
l
M. The cells were treated with 2a, 2b–d (A), or 2e–g (B)
and incubated for another 3 h before total RNA was extracted. The ratio of mRNA expression relative to the control is shown. The data are presented as the mean S.E.M.
(number of experiments n = 3–4). ⁄p <0.05 and ⁄⁄⁄p <0.001 comparing 2a with the control using the Student’s t-test with the F test. ⁄⁄p <0.01 compared the control with 2b, 2c,
2d, 2e, 2f and 2g, and p <0.01 compared 2a versus 2b, 2c, 2d and 2e at the same concentration (10 lM), using one-way ANOVA with the Scheffe’s F test.
2.2.3. Morphological changes of cortical neurons stimulated
with the ethynyl derivative 2a
Also, we examined the effect of 2a on the neuronal morphology.
First, we introduced the GFP expression vector into cultured corti-
the ethynyl group can stimulate neurons, although the efficiencies
on the inductive level were different. Furthermore, the same mech-
anisms for inducing Bdnf eIV-IX mRNA expression operate whether
the cyano or ethynyl group is present. Changing the substituent at
the terminus of the ethynyl group affected the extent of the
expression, suggesting that it is possible to control the inductive le-
vel of Bdnf eIV-IX mRNA in neurons by changing substituents. Thus,
the conversion from a cyano to an ethynyl group in the DM moiety
would increase the safety of the compound in the living body, lead-
ing to more promising candidates for an agent to treat neuronal
disorders.
cal neurons. Then, we cultured the cells with 10 lM 2a for 6 h, and
then, processed the culture for the fluorescence microscopic obser-
vation of GFP-positive cells (Fig. 4A) and immunostaining with
anti-MAP2 antibody (data shown in Supplementary data). To
examine the morphological changes in neurites, we performed
Sholl analysis to measure the neurite complexity (Fig. 4B(a)), and
also measured the length of neurites (Fig. 4B(b)).11 As shown in
Figure 4A, the addition of 2a increased the number of neurite
crossings and the length of neurites in cultures harvested 6 h after
the incubation in a manner similar to DM (Fig. 4B). The effect of 2a
on the morphological changes of neurites was detected at almost
the same level as that of DM, though their effects on the induction
of Bdnf eIV-IX mRNA expression were different (Fig. 2). These data
suggest that the level of BDNF expression induced by 2a is suffi-
cient to cause the morphological changes.
4. Experimental section
4.1. Chemistry
All reagents were purchased from commercial sources and used
as received. Anhydrous solvents were obtained from commercial
sources or prepared by distillation over CaH2 or P2O5. 1H and 13C
NMR spectra were obtained on a Varian Gemini 300 (300 MHz
for 1H and 75.46 MHz for 13C), using chloroform as an internal
reference. Mass spectra were measured on a JEOL D-200 or JEOL
AX 505 mass spectrometer, and the ionization method was via
electron impact (EI, 70 eV). IR spectra were recorded on a JASCO
FT/IR-460Plus spectrometer. Column chromatography was carried
out by employing Cica Silica Gel 60 N (spherical, neutral, 40–
3. Conclusion
In this study, we synthesized a series of ethynyl analogues of
DM, to confer less susceptibility to hydrolytic degradation than
DM. We found that the presence of a ethynyl group at the a-posi-
tion was sufficient to preserve the activity of type II pyrethroids to
induce Bdnf eIV-IX mRNA expression and morphological changes in
neurons. These changes indicate that not only the cyano but also
50
lm). The carboxylic acid 7 was prepared by the hydrolysis of
commercially available deltamethrin.

2568
Y. Matsuya et al. / Bioorg. Med. Chem. 20 (2012) 2564–2571
Figure 4. The effect of 2a on neuronal morphology. At 4 days in culture, rat cortical neurons were transfected with a GFP plasmid for immunostaining. Two days after
transfection, the neurons were treated with 1 M DM or 10 M 2a and fixed after 6 h. Representative images of untreated or pretreated neurons are shown (A). A score for the
number of neurites was obtained for each cell by placing a 20–200 m-radius circle (20 m interval) over the center of the cell and counting the number of times neurites
emanating from the cell body intersected the circles (B(a)). The values represent the mean S.E.M (n = 3 experiments). ⁄p <0.05 control versus DM, #p <0.05 control versus 2a,
using a one-way ANOVA with the Scheffe’s F test. §§p <0.05 compared with the control, using a repeated measure ANOVA with the Bonferroni/Dunn test. Scale bar, 100
m.
m diameter (B(b)). The values represent the
l
l
l
l
l
The length of neurites was also measured by tracing all of the neurites starting from the cell body and extending within a 400
l
mean S.E.M (n = 3). ⁄p <0.05 and ⁄⁄p <0.01 versus the control, using a one-way ANOVA with the Scheffe’s F test.
4.1.1. 1-Phenoxy-3-(3-trimethylsilyl-2-propynoyl)benzene (4)
To a solution of trimethylsilylacetylene (982 mg, 12 mmol) in
anhydrous THF (25 mL) was added n-BuLi (1.6 M hexane solution,
6.25 mL, 10 mmol) at ꢁ78 °C and the mixture was stirred at the
same temperature for 15 min. A solution of 3-phenoxybenzalde-
hyde (2.023 g, 10 mmol) in anhydrous THF (25 mL) was added,
and the reaction mixture was warmed to room temperature for
20 min. The reaction was quenched by the addition of a sat. NH4Cl
solution and after extraction with CH2Cl2, the organic layer was
dried over MgSO4. Evaporation of the solvent gave a crude adduct
alcohol (3.096 g, quant.) in an almost pure form. The crude alcohol
(100 mg, 0.337 mmol) was dissolved in anhydrous CH2Cl2 (2 mL),
and MS4A (100 mg) and PDC (389 mg, 1.01 mmol) were added.
After 2 h of stirring at room temperature, the precipitates were
filtered off through a celite pad, and the filtrate was concentrated.
The residue was subjected to column chromatography to afford the
ketone 4 (93 mg, 94%) as a yellow oil. 1H NMR (300 MHz, CDCl3): d
7.88–7.04 (9H, m), 0.27 (9H, s); 13C NMR (75 MHz, CDCl3): d
176.67, 157.78, 155.94, 137.90, 129.84, 129.81, 124.03, 123.92,
119.44, 118.42, 100.77, 100.49, 53.43, ꢁ0.69; IR (neat): 2152
(C„C), 1649 cmꢁ1 (C@O); MS (EI): m/z 294 (M+); HRMS (EI) Calcd
for C18H18O2Si: 294.1094 (M+), found: 294.1076.
with optically pure 7 afforded ester 2a in high yield (94%) without
any other diastereomers, although the specific rotation value of 6a
was rather low compared with that reported, estimated as 65% ee
(vide infra). 1H NMR (300 MHz, CDCl3): d 7.61–6.90 (9H, m), 5.42
(1H, s), 2.13 (1H, br), 0.17 (9H, s); 13C NMR (75 MHz, CDCl3): d
157.36, 156.57, 142.07, 129.71, 129.63, 123.35, 121.20, 119.07,
118.32, 116.64, 91.58, 64.46, 53.43, ꢁ0.16; IR (neat): 3354 (–OH),
2173 cmꢁ1 (C„C); MS (EI): m/z 296 (M+); HRMS (EI) Calcd for
C
18H20O2Si: 296.1238 (M+), found: 296.1233;
(c = 0.780, CHCl3).
[
a]
ꢁ9.41
D
4.1.3. (ꢁ)-1-(1-Hydroxy-2-propynyl)-3-phenoxybenzene (6a)
To a solution of the alcohol 5 (120 mg, 0.404 mmol) in methanol
(6 mL) was added K2CO3 (111 mg, 0.808 mmol) and the reaction
was continued at room temperature for 0.5 h. The mixture was
diluted with water and extracted with CH2Cl2, and the organic
layer was dried over MgSO4. Evaporation of the solvent gave a ter-
minal ethynyl product 6a (93 mg, quant.) as an almost pure yel-
lowish oil, which was identified by 1H NMR spectroscopy. 1H
NMR (270 MHz, CDCl3): d 7.30–6.88 (9H, m), 5.36 (1H, s), 2.57
(1H, s), 2.07 (1H, s); MS (EI): m/z 224 (M+); [
benzene) (lit.15
ꢁ16 (1% in benzene)).
a
]
D
ꢁ10.36 (c = 2.15,
[a]
D
4.1.2. (ꢁ)-1-Phenoxy-3-(3-trimethylsilyl-1-hydroxy-2-
4.1.4. (ꢁ)-1-(3-Phenoxyphenyl)-2-propynyl 2-(2,2-
propynyl)benzene (5)
dibromoethenyl)-3,3-dimethylcyclopropanecarboxylate (2a)
To a solution of the carboxylic acid 7 (57 mg, 0.19 mmol) in
anhydrous CH2Cl2 (5 mL) was added a solution of 1,1-carbonyldi-
imidazole (45 mg, 0.29 mmol) in anhydrous CH2Cl2 (0.6 mL) at
room temperature, and the mixture was stirred for 50 min. A solu-
tion of the alcohol 6a (129 mg, 0.574 mmol) in anhydrous CH2Cl2
(2 mL), and then a small amount of NaH were added. After
30 min, the reaction mixture was diluted with CH2Cl2 and washed
with 10% HCl, sat. NaHCO3, and brine successively. The organic
layer was dried over MgSO4. Evaporation of the solvent gave a res-
idue, which was chromatographed on silica gel to afford the ester
2a (90 mg, 94%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3): d
7.38–6.98 (9H, m), 6.64 (1H, d, J = 8.4 Hz), 6.41, (1H, d, J = 1.8 Hz),
A solution of BH3-THF complex (1 M in THF, 5.1 mL, 5.1 mmol)
was diluted with anhydrous THF (8 mL), and (S)-2-methyl-CBS-
oxazaborolidine (1 M in THF, 0.34 mL, 0.34 mmol) was added at
0 °C. After 15 min of stirring at 0 °C, a solution of 4 (1 g, 3.4 mmol)
in anhydrous THF (9 mL) was added dropwise, and the mixture was
stirred at 0 °C for 70 min. Methanol (10 mL) was added and the
reaction mixture was diluted with Et2O. The mixture was washed
with 10% HCl and brine successively, and the organic layer was
dried over MgSO4. Evaporation of the solvent gave a residue, which
was chromatographed on silica gel to afford the alcohol 5 (859 mg,
85%) as a yellow oil. We consider that the optical purity of 5 would
be practically high because condensation of 6a (desilylation of 5)

Y. Matsuya et al. / Bioorg. Med. Chem. 20 (2012) 2564–2571
2569
2.66 (1H, d, J = 2.4 Hz), 1.99 (1H, t, J = 8.4 Hz), 1.90 (1H, d,
J = 8.4 Hz), 1.23 (3H, s), 1.20 (3H, s); 13C NMR (75 MHz, CDCl3): d
168.92, 157.40, 156.52, 138.27, 132.98, 129.89, 129.70, 123.48,
122.13, 122.06, 118.97, 117.71, 89.77, 79.85, 75.67, 64.77, 35.95,
31.68, 28.32, 28.07, 15.11; IR (neat): 2358 (C„C), 1732 cmꢁ1
(C@O); MS (EI): m/z 502, 504, 506 (M+); HRMS (EI) Calcd for
(for 9g, excess amount) was added, and the mixture was warmed
to room temperature. After 1.5 h of stirring, the reaction was
quenched by addition of a sat. NH4Cl solution, and the aqueous
solution was extracted with CH2Cl2 and dried over MgSO4. Evapo-
ration of the solvent gave a residue, which was chromatographed
on silica gel to afford the products 9d–g as pale yellow oils.
Compound 9d: Yield 69%; 1H NMR (300 MHz, CDCl3): d 7.38–6.92
(9H, m), 5.46 (1H, s), 0.91 (9H, s), 0.16 (3H, s), 0.12 (3H, s); 13C NMR
(75 MHz, CDCl3): d 157.36, 156.72, 143.06, 129.65, 129.54, 123.34,
120.46, 119.10, 117.87, 116.04, 69.85, 64.64, 64.38 25.82, 18.35,
ꢁ4.43, ꢁ4.90; IR (neat): 2236 cmꢁ1 (C„C); MS (EI): m/z 372, 374
(M+); HRMS (EI) Calcd for C21H2535ClO2Si: 372.1312 (M+), found:
C
23H20Br2O3: 501.9781 (M+), found: 501.9780, [
benzene).
a] 7.06 (c = 1.15,
D
4.1.5. (ꢁ)-1-(1-Hydroxy-3-iodo-2-propynyl)-3-phenoxybenzene
(6b)
A mixture of the alkyne 6a (100 mg, 0.446 mmol), KOH (1 M
aqueous solution, 1.12 mL, 1.12 mmol), and I2 (125 mg,
0.491 mmol) in methanol (2.2 mL) was stirred for 80 min at room
temperature. Water was added, and the aqueous mixture was ex-
tracted with CH2Cl2 and dried over MgSO4. Evaporation of the sol-
vent gave a residue, which was chromatographed on silica gel to
afford the iodide 6b (62 mg, 39%) as a pale yellow oil. 1H NMR
(300 MHz, CDCl3): d 7.39–6.94 (9H, m), 5.55 (1H, s), 2.29 (1H,
br); 13C NMR (75 MHz, CDCl3): d 157.45, 156.59, 141.85, 129.89,
129.70, 123.44, 121.11, 119.03, 118.53, 116.80, 93.73, 65.64,
4.78; IR (neat): 3375 (–OH), 2184 cmꢁ1 (C„C); MS (EI): m/z 350
(M+); HRMS (EI) Calcd for C15H11IO2: 349.9804 (M+), found:
372.1278; [
a
]
D ꢁ8.06 (c = 0.555, benzene).
Compound 9e: Yield 89%; 1H NMR (300 MHz, CDCl3): d7.37–6.90
(9H, m), 5.45 (1H, d, J = 1.8 Hz), 1.86 (3H, d, J = 2.1 Hz), 0.92 (9H, s),
0.16 (3H, s), 0.13 (3H, s); 13C NMR (75 MHz, CDCl3): d 157.17,
156.88, 144.34, 129.58, 129.34, 123.18, 120.60, 119.03, 117.56,
116.22, 81.92, 79.91, 64.57, 25.91, 18.41, 3.80, ꢁ4.34, ꢁ4.84; IR
(neat): 2228 cmꢁ1 (C„C); MS (EI) m/z 352 (M+); HRMS (EI) Calcd
for C22H28O2Si: 352.1859 (M+), found: 352.1841; [
a
]
D
ꢁ4.92
(c = 0.62, benzene).
Compound 9f: Yield 54%; 1H NMR (300 MHz, CDCl3): d 7.36–6.89
(9H, m), 5.45 (1H, s), 2.22 (2H, qd, J = 7.5, 2.1 Hz), 1.13 (3H, t,
J = 7.4 Hz), 0.90 (9H, s), 0.15 (3H, s), 0.13 (3H, s); 13C NMR
349.9799; [
a]
ꢁ6.03 (c = 1.41, benzene).
D
(75 MHz, CDCl3):
d 157.17, 156.86, 144.28, 129.57, 129.31,
4.1.6. (ꢁ)-1-(3-Bromo-1-hydroxy-2-propynyl)-3-
123.16, 120.62, 119.05, 117.53, 116.22, 87.74, 80.07, 64.54, 25.92,
18.40, 13.75, 12.59, ꢁ4.24, ꢁ4.79; IR (neat): 2230 cmꢁ1 (C„C);
MS (EI): m/z 366 (M+); HRMS (EI) Calcd for C23H30O2Si: 366.1966
phenoxybenzene (6c)
A mixture of the alkyne 5 (40.2 mg, 0.136 mmol), NBS (29 mg,
0.163 mmol), and AgNO3 (1.6 mg, 0.009 mmol) in acetone
(1.25 mL) was stirred for 70 min at room temperature in a light-
omitted flask. Water was added, and the aqueous mixture was ex-
tracted with Et2O and dried over MgSO4. Evaporation of the solvent
gave a residue, which was chromatographed on silica gel to afford
the bromide 6c (28.6 mg, 70%) as a yellow oil. 1H NMR (300 MHz,
CDCl3): d 7.39–6.95 (9H, m), 5.44 (1H, s), 2.32 (1H, br); 13C NMR
(M+), found: 366.2015; [
a
]
D
ꢁ20.12 (c = 0.52, benzene).
Compound 9g: Yield 82%; 1H NMR (300 MHz, CDCl3): d7.37–6.91
(9H, m), 5.57 (1H, s), 0.90 (9H, s), 0.15 (3H, s), 0.12 (3H, s); 13C NMR
(75 MHz, CDCl3): d 157.33, 156.70, 143.08, 129.63, 129.50, 123.32,
120.52, 119.12, 119.07, 117.84, 116.07, 94.83, 65.79, 64.19, 25.84,
18.40, 3.14, ꢁ4.42, ꢁ4.87; IR (neat): 2182 cmꢁ1 (C„C); MS (EI):
m/z 420 (M+); HRMS (EI) Calcd for C23H27F3O2Si: 420.1733 (M+),
(75 MHz, CDCl3):
d
157.48, 156.62, 141.72, 129.91, 129.70,
found: 420.1602; [
a
]
ꢁ22.24 (c = 0.505, benzene).
D
123.44, 121.07, 119.02, 118.57, 116.79, 79.49, 65.09, 47.63; IR
(neat): 3358 (–OH), 2213 cmꢁ1 (C„C); MS (EI): m/z 302, 304
(M+); HRMS (EI) Calcd for C15H1179BrO2: 301.9942 (M+), found:
4.1.9. General procedure for the synthesis of 6d–g
To a solution of the TBSether 9d–g (0.14 mmol) in anhydrous THF
(2 mL) was added TBAF (1 M THF solution, 0.28 mL, 0.28 mmol), and
the mixture was stirred at 0 °C for 0.5 h. The reaction mixture was
diluted with CH2Cl2 and washed with brine, and the organic layer
was dried over MgSO4. Evaporation of the solvent gave a residue,
which was chromatographed on silica gel to afford the alcohols
6d–g as yellow oils.
301.9925; [
a]
ꢁ6.91 (c = 1.26, benzene).
D
4.1.7. (ꢁ)-1-(1-tert-Butyldimethylsilyloxy-2-propynyl)-3-
phenoxybenzene (8)
To a solution of the alcohol 6a (263 mg, 1.17 mmol) in anhy-
drous CH2Cl2 (7 mL) were added 2,6-lutidine (252 mg, 2.34 mmol)
and then TBSOTf (465 mg, 1.76 mmol), and the resulting mixture
was stirred at room temperature for 1 h. The reaction mixture
was diluted with CH2Cl2, and washed with 10% HCl, sat. NaHCO3,
and brine successively, and the organic layer was dried over
MgSO4. Evaporation of the solvent gave a residue, which was chro-
matographed on silica gel to afford the TBS ether 8 (397 mg,
quant.) as a yellow oil. 1H NMR (300 MHz, CDCl3): d 7.36–6.91
(9H, m), 5.45 (1H, s), 2.55 (1H, d, J = 2.1 Hz), 0.90 (9H, s), 0.16
(3H, s), 0.12 (3H, s); 13C NMR (75 MHz, CDCl3): d 157.32, 156.78,
143.19, 129.62, 129.50, 123.27, 120.52, 119.07, 117.85, 116.14,
84.33, 73.75, 64.19, 25.82, 18.37, ꢁ4.43, ꢁ4.89; IR (neat): 3307
(C„C–H), 2124 cmꢁ1 (C„C); MS (EI): m/z 338 (M+); HRMS (EI)
Compound 6d: Yield 92%; 1H NMR (300 MHz, CDCl3):
d
7.39–6.95 (9H, m), 5.43 (1H, s), 2.27 (1H, br); 13C NMR (75 MHz,
CDCl3): d 157.45, 156.60, 141.80, 129.91, 129.70, 123.44, 121.06,
118.99, 118.55, 116.77, 77.21, 68.75, 65.72, 64.49; IR (neat):
3335 (–OH), 2238 cmꢁ1 (C„C); MS (EI): m/z 258, 260 (M+); HRMS
(EI) Calcd for C15H1135ClO2: 258.0448 (M+), found: 258.0424; [
a]
D
ꢁ6.63 (c = 0.975, benzene).
Compound 6e: Yield quant.; 1H NMR (300 MHz, CDCl3): d
7.38–6.92 (9H, m), 5.40 (1H, d, J = 2.1 Hz), 2.11 (1H, br), 1.90 (3H,
d, J = 2.1 Hz); 13C NMR (75 MHz, CDCl3): d 157.30, 156.80, 143.08,
129.73, 129.65, 123.29, 121.19, 118.94, 118.27, 116.93, 83.28,
78.86, 64.46, 3.82; IR (neat): 3381 (–OH), 2228 cmꢁ1 (C„C); MS
(EI): m/z 238 (M+); HRMS (EI) Calcd for C16H14O2: 238.0994 (M+),
Calcd for
C [a]
21H26O2Si: 338.1702 (M+), found: 338.1704;
D
ꢁ4.72 (c = 1.00, benzene).
found: 238.0974; [
a
]
ꢁ6.03 (c = 1.36, benzene).
D
Compound 6f: Yield 92%; 1H NMR (300 MHz, CDCl3): d 7.37–6.93
(9H, m), 5.41 (1H, s), 2.23 (2H, qd, J = 7.5, 1.9 Hz), 1.15 (3H, dd,
J = 7.2, 7.8 Hz); 13C NMR (75 MHz, CDCl3): d 157.30, 156.73,
143.06, 129.70, 129.63, 123.29, 121.20, 118.99, 118.24, 116.83,
83.04, 78.97, 64.41, 13.77, 12.57; IR (neat): 3366 (–OH), 2229 cm
4.1.8. General procedure for the synthesis of 9d–g
A solution of the alkyne 8 (100 mg, 0.296 mmol) in anhydrous
THF (2 mL) was cooled to ꢁ78 °C, n-BuLi (1.6 M hexane solution,
0.277 mL, 0.444 mmol) was added, and the mixture was stirred
at the same temperature for 15 min. NCS (for 9d, 2 equiv), MeI
(for 9e, excess amount), EtI (for 9f, excess amount), or CF3CH2I
(C„C); MS (EI): m/z 252 (M+); HRMS (EI) Calcd for C17H16O2:
ꢁ1
252.1150 (M+), found: 252.1128; [
a]
ꢁ8.47 (c = 0.41, benzene).
D

2570
Y. Matsuya et al. / Bioorg. Med. Chem. 20 (2012) 2564–2571
Compound 6g: Yield 78%; 1H NMR (300 MHz, CDCl3): d 7.39–
(EI): m/z 503 (M+–CH2CF3), 505, 507; HRMS (EI) Calcd for
23H2179Br2O3: 502.9858 (M+–CH2CF3), found: 502.9860;
ꢁ19.92 (c = 0.42, benzene).
6.94 (9H, m), 5.55 (1H, s), 2.26 (1H, br); 13C NMR (75 MHz, CDCl3):
d 157.49, 156.62, 141.86, 129.91, 129.71, 123.45, 121.11, 119.05,
118.55, 116.80, 93.75, 65.69, 4.73; IR (neat): 3363 (–OH),
2184 cmꢁ1 (C„C); MS (EI): m/z 306 (M+); HRMS (EI) Calcd for
C
[a]
D
4.2. Biological activity
C
17H13F3O2: 306.0868 (M+), found: 306.0901;
[
a]
ꢁ4.61
D
(c = 0.89, benzene).
4.2.1. The preparation of the test compound solutions
The test compounds were dissolved in dimethylsulfoxide
(DMSO) and then diluted with suitable medium until the final con-
centration of DMSO was 0.1%.
4.1.10. Synthesis of 2b–g
According to the procedure used for the synthesis of 2a, the car-
boxylic acid 7 was reacted with the alcohols 6b–g (3 equiv) to af-
ford compounds 2b–g as pale yellow oils.
4.2.2. Cells
Compound 2b: Yield 87%; 1H NMR (300 MHz, CDCl3): d 7.39–
6.96 (9H, m), 6.75 (1H, d, J = 8.7 Hz), 6.51 (1H, s), 1.99 (1H, t,
J = 8.4 Hz), 1.89 (1H, d, J = 8.7 Hz), 1.22 (3H, s), 1.18 (3H, s); 13C
NMR (75 MHz, CDCl3): d 168.88, 157.46, 156.49, 138.45, 132.98,
129.92, 129.75, 123.57, 122.06, 119.07, 118.94, 117.66, 90.35,
89.86, 66.14, 36.02, 31.67, 28.33, 28.11, 15.13, 6.02; IR (neat):
3055 (C@C–H), 2191 (C„C), 1731 cmꢁ1 (C@O); MS (EI): m/z 628,
630, 632 (M+); HRMS (EI) Calcd for C23H1979Br2IO3: 627.8746
A primary culture of rat cortical cells was prepared from
Sprague–Dawley rats (Japan SLC, Hamamatsu, Japan) at embry-
onic day 17 (E17), as described previously.10,11 In brief, small
pieces of cerebral cortex were dissected by enzymatic treatment
with trypsin and DNase I and suspended by pipetting, and the
cells were seeded at 2.5 ꢂ 106 cells in a 35 mm-diameter poly-
ethylenimine-coated dish (Iwaki, Tokyo, Japan). The cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invit-
rogen, Carlsbad, CA) containing 10% fetal calf serum (FCS), 1 mM
(M+), found: 627.8723; [
a
]
D
ꢁ19.05 (c = 0.365, benzene).
Compound 2c: Yield 61%; 1H NMR (300 MHz, CDCl3): d 7.39–
6.96 (9H, m), 6.75 (1H, d, J = 8.1 Hz), 6.41 (1H, s), 1.99 (1H, dd,
J = 8.7, 8.1 Hz), 1.89 (1H, d, J = 8.7 Hz), 1.22 (3H, s), 1.19 (3H, s);
13C NMR (75 MHz, CDCl3): d 168.90, 157.46, 156.51, 138.27,
132.95, 129.94, 129.73, 123.57, 122.01, 119.05, 118.95, 117.64,
89.88, 76.39, 65.63, 48.61, 36.02, 31.65, 28.33, 28.12, 15.13; IR
(neat): 3055 (C@C–H), 2221 (C„C), 1731 cmꢁ1 (C@O); MS (EI):
m/z 580, 582, 584, 586 (M+); HRMS (EI) Calcd for C23H1979Br3O3:
sodium pyruvate, 100 U/mL penicillin, and 100 lg/mL streptomy-
cin for 3 days, and then the medium was replaced with serum-
free DMEM.
4.2.3. RNA isolation and reverse transcription
Total cellular RNA was extracted by the acid guanidine phenol-
chloroform method. The isolation of RNA from cultured cells was
described in detail previously.10,11 In brief, total cellular RNA was
extracted using TRIsure (BIOLINE, London, UK) and treated with
RNase-free DNase I (TaKaRa, Shiga, Japan) to prevent contamina-
tion by genomic DNA. After the removal of DNase I, total RNA
was quantified with a Beckman spectrophotometer (Beckman
Coulter, Fullerton, CA). One microgram of RNA was used for reverse
transcription with SuperScript II (Invitrogen).
579.8884 (M+), found: 579.8863;
benzene).
[a]
ꢁ12.83 (c = 0.395,
D
Compound 2d: Yield 65%; 1H NMR (300 MHz, CDCl3): d 7.39–
6.97 (9H, m), 6.76 (1H, d, J = 8.1 Hz), 6.40 (1H, s), 2.00 (1H, dd,
J = 8.1, 8.7 Hz), 1.89 (1H, d, J = 8.4 Hz), 1.23 (3H, s), 1.19 (3H, s);
13C NMR (75 MHz, CDCl3): d 168.92, 157.48, 156.51, 138.34,
132.95, 129.96, 129.75, 123.57, 122.00, 119.05, 118.95, 117.76,
117.63, 89.88, 65.77, 65.16, 36.02, 31.67, 28.35, 28.12, 15.13; IR
(neat): 3056 (C@C–H), 2245 (C„C), 1732 cmꢁ1 (C@O); MS (EI):
m/z 536, 538, 540 (M+); HRMS (EI) Calcd for C23H1979Br235ClO3:
4.2.4. Quantitative PCR analysis
Quantitative PCR was performed using the Stratagene Mx3000p
Real-Time PCR system (La Jolla, CA, USA) as described previ-
535.9390 (M+), found: 535.9378; [
a
]
ꢁ5.55 (c = 1.305, benzene).
ously.10,11 In brief, PCR was performed in 20
l
L of a reaction mix-
ture containing 1 ꢂ SYBR Green QPCR master mix (Stratagene),
L cDNA solution, and 0.2 M primer pairs. The thermal profile
D
Compound 2e: Yield 84%; 1H NMR (300 MHz, CDCl3): d7.38–6.95
(9H, m), 6.78 (1H, d, J = 8.4 Hz), 6.38 (1H, d, J = 2.4 Hz), 1.97 (1H, dd,
J = 8.7, 8.1 Hz), 1.91–1.87 (4H, m), 1.22 (3H, s), 1.19 (3H, s); 13C
NMR (75 MHz, CDCl3): d 169.13, 157.30, 156.65, 139.47, 133.19,
129.75, 129.66, 123.40, 122.08, 118.97, 118.68, 117.74, 89.51,
84.22, 75.59, 65.55, 35.89, 31.83, 28.36, 27.93, 15.13, 3.95; IR
(neat): 3055 (C@C–H), 2240 (C„C), 1730 cmꢁ1 (C@O); MS (EI):
m/z 516, 518, 520 (M+); HRMS (EI) Calcd for C24H2279Br2O3:
2
l
l
for real-time PCR was as follows: initial denaturation at 95 °C for
10 min, followed by 40 cycles of denaturation at 95 °C for 45 s,
annealing at 57–59 °C (dependent on Tm °C of primers) for 45 s,
and extension at 72 °C for 1 min. Standard curves were generated
for each gene using a plasmid DNA dilution series containing the
target sequences. The Bdnf mRNA levels were computed from the
threshold cycle (Ct) value and normalized to the concentration of
internal control Gapdh mRNA. The primer sequences for measuring
each mRNA were rat eIV-containing Bdnf mRNA sense, 50-TCGGCCA
CCAAAGACTCG-30 and antisense, 50-GCCCATTCACGCTCTCCA-30;
Gapdh mRNA sense, 50-TCCATGACAACTTTGGCATCGTGG-30 and
antisense, 50-GTTGCTGTTGAAGTCACAGGAGAC-30.
515.9936 (M+), found: 515.9955; [
a
]
ꢁ9.38 (c = 0.67, benzene).
D
Compound 2f: Yield 52%; 1H NMR (300 MHz, CDCl3): d 7.37–6.95
(9H, m), 6.77 (1H, d, J = 8.1 Hz), 6.41 (1H, s), 2.27 (2H, qd, J = 7.6,
2.4 Hz), 1.97 (1H, t, J = 8.4 Hz), 1.89 (1H, d, J = 8.4 Hz), 1.21 (3H,
s), 1.19 (3H, s), 1.14 (3H, dd, J = 7.8, 7.2 Hz); 13C NMR (75 MHz,
CDCl3): d 169.13, 157.33, 156.64, 139.49, 133.24, 129.75, 129.68,
123.42, 122.13, 119.03, 118.71, 117.69, 89.92, 89.46, 75.71, 65.55,
35.89, 31.89, 28.38, 27.93, 15.13, 13.61, 12.65; IR (neat): 3055
(C@C–H), 2239 (C„C), 1729 cmꢁ1 (C@O); MS (EI): m/z 530, 532,
534 (M+); HRMS (EI) Calcd for C25H2479Br2O3: 530.0093 (M+),
4.2.5. Immunostaining and morphological analysis
The procedure was performed as described previously.11 To
monitor the effect of DM on the morphology of cortical neurons,
found: 530.0059; [
a
]
ꢁ11.22 (c = 0.45, benzene).
a green fluorescent protein (GFP) expression vector (1 lg/well)
D
Compound 2g: Yield 74%; 1H NMR (300 MHz, CDCl3): d 7.39–
6.96 (9H, m), 6.75 (1H, d, J = 8.4 Hz), 6.52 (1H, s), 1.99 (1H, t,
J = 8.4 Hz), 1.89 (1H, d, J = 8.4 Hz), 1.22 (3H, s), 1.18 (3H, s); 13C
NMR (75 MHz, CDCl3): d 168.88, 157.46, 156.49, 138.45, 132.97,
129.92, 129.73, 123.55, 122.06, 119.07, 118.94, 117.77, 117.66,
90.35, 89.85, 66.14, 36.02, 31.67, 29.77, 28.33, 28.11, 15.13, 5.99;
IR (neat): 3055 (C@C–H), 2191 (C„C), 1731 cmꢁ1 (C@O); MS
was transfected at 4 days in culture using the calcium phosphate
precipitation method. At 6 days in culture, after treatment with
the drugs for 6 h, cells were fixed by placing them in PBS contain-
ing 4% formaldehyde and 4% sucrose for 15 min. Then the cells
were permeabilized by placing them in PBS containing 0.3% Tri-
ton X-100, 3% bovine serum albumin, and 3% normal goat serum
for 1 h. The cells were incubated with anti-GFP and microtubule-

Y. Matsuya et al. / Bioorg. Med. Chem. 20 (2012) 2564–2571
2571
associated protein 2 (MAP2) primary antibodies in the presence
of blocking solution (PBS with 3% BSA and 3% normal goat serum)
at 4 °C overnight. After being washed in PBS, the cells were incu-
bated for 1 h at room temperature with Alexa488- and Alexa594-
conjugated secondary antibodies against rabbit or mouse IgG
diluted in blocking solution. After another wash, nuclei were
counter-stained with 300 nM 40,6-diamidino-2-phenylindole
(DAPI) (Invitrogen). Finally, coverslips were mounted with Fluoro-
mount (Diagnostic BioSystems, Pleasanton, CA). The rabbit poly-
clonal antibodies used were anti-GFP (1:500; Invitrogen) and
Alexa 488-conjugated anti-rabbit IgG (1:1000, Invitrogen). The
mouse monoclonal antibodies used were anti-MAP2 (1:1000,
Sigma) and Alexa 594-conjugated anti-mouse IgG (1:1000,
Invitrogen).
with the Scheffe’s F test, or repeated measure ANOVA with the
Bonferroni/Dunn test (see figure captions).
Acknowledgments
This study was supported in part by a Grant-in-aid for Scientific
Research from the Ministry of Education, Science, Sports and Cul-
ture, Japan (Project Number 20390023, M.T.), and the Mitsubishi
Foundation (M.T.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.02.048.
After immunostaining, the cells were processed for microscopy
(BX50-34-FLA-1, Olympus, Tokyo, Japan), as described previously
(Sholl analysis). A series of concentric circles at 20-lm intervals
References and notes
were centered on the cell body and the number of intersections
with GFP-positive processes was recorded as an index of neurite
complexity11; that is, the increase in the number of the intersec-
tions represents the increase in neurite complexity. The length of
neurites was measured by tracing all of the neurites starting from
1. Tsankova, N. M.; Berton, O.; Renthal, W.; Kumar, A.; Neve, R. L.; Nestler, E. J. Nat.
Neurosci. 2006, 9, 465.
2. Garzon, D.; Yu, G.; Fahnestock, M. J. Neurochem. 2002, 82, 1058.
3. Pardridge, W. M. Adv. Exp. Med. Biol. 2002, 513, 397.
4. Aid, T.; Kazantseva, A.; Piirsoo, M.; Palm, K.; Timmusk, T. J. Neurosci. Res. 2007,
85, 525.
5. Tabuchi, A.; Nakaoka, R.; Amano, K.; Yukimine, M.; Andoh, T.; Kuraishi, Y.;
Tsuda, M. J. Biol. Chem. 2000, 275, 17269.
6. Timmusk, T.; Palm, K.; Metsis, M.; Reintam, T.; Paalme, V.; Saarma, M.; Persson,
H. Neuron 1993, 10, 475.
7. Miyamoto, J.; Kaneko, H.; Tsuji, R.; Okuno, Y. Toxicol. Lett. 1995, 82–83, 933.
8. Narahashi, T. Neurotoxicology 1985, 6, 3.
9. Vijverberg, H. P.; van den Bercken, J. Crit. Rev. Toxicol. 1990, 21, 105.
10. Imamura, L.; Yasuda, M.; Kuramitsu, K.; Hara, D.; Tabuchi, A.; Tsuda, M. J.
Pharmacol. Exp. Ther. 2006, 316, 136.
the cell body and extending within a 400-lm diameter using
Image J software. More than 96 neurons were evaluated from
three independent experiments (n = 3). We confirmed that the
GFP-positive cells used for the morphological analysis were
MAP2-positive cells (Supplementary data). Notably, we measured
all kinds of neurons without any selection of cells for these
measurements.
11. Ihara, D.; Fukuchi, M.; Honma, D.; Takasaki, I.; Ishikawa, M.; Tabuchi, A.; Tsuda,
M. Neuropharmacology 2012, 62, 1091.
4.2.6. Statistics
12. Antunes, L. M.; Organ, M. G. Tetrahedron Lett. 2003, 44, 6805.
13. Nishikawa, T.; Shibuya, S.; Hosokawa, S.; Isobe, M. Synlett 1994, 485.
14. Murray, R. E. Synth. Commun. 1980, 10, 345.
15. Martel, J. J.; Demoute, J. P.; Tèche, A. P.; Tessier, J. R. Pestic. Sci. 1980, 11,
188.
All data were expressed as the mean S.E.M. for the separate
experiments performed in duplicate, as indicated in the figure cap-
tions. Statistical analyses were performed using the Student’s t-test
followed by the F-test or a one-way analysis of variance (ANOVA)