Journal of Medicinal Chemistry
Brief Article
refluxed with stirring for 2 h. After cooling, water and 0.5 M NaOH
were added to the mixture. The mixture was stirred at rt for 1 h and
extracted with EtOAc. The extract was washed with satd NaCl, dried
over MgSO4, and concentrated under reduced pressure. The residue
was purified by silica gel chromatography (hexane−EtOAc, 90:10 to
75:25) and successively recrystallized from EtOAc/hexane to give the
title compound (1.60 g, 83%) as colorless crystals; mp 152−155 °C.
1H NMR (CDCl3): δ 1.46 (6H, s), 2.08 (3H, s), 2.19 (3H, s), 2.24
(3H, s), 2.91 (2H, s), 3.06−3.34 (8H, m), 3.78 (3H, s), 6.81−6.90
(2H, m), 6.92−7.01 (2H, m). ESI MS m/z 381 [M + H]+. Anal. Calcd
for C24H32N2O2: C, 75.75; H, 8.48; N, 7.36. Found: C, 75.50; H, 8.52;
N, 7.15.
10 ng/mL) in neuronal survivability. This effect was observed
to be concentration-independent in the concentration range
from 3 to 100 ng/mL. Compound 8 (1 μmol/L) alone
increased the neuronal survival rate 3-fold compared with that
of the control; in addition, in the presence of 1 μmol/L of 8,
depending on the concentration, IGF-1 enhanced the neuro-
protecitive effect by approximately 6-fold (main effect of 8, p ≤
0.05; main effect of IGF-1, p ≤ 0.05; interaction of compound 8
and IGF-1, p ≤ 0.05 (two-way ANOVA)) (Figure 2). In
contrast, in the presence of 100 ng/mL of IGF-1, which is a
concentration relevant to physiological conditions, 8 increased
the neuronal survival rate in a concentration-dependent
manner, with an EC50 value of 0.15 μM; this effect plateaued
at 0.33 μM, where the neuronal survival rate increased
approximately 3-fold (Figure 3). In conclusion, treatment
with IGF-1 or 8 induced a 1.3- or 3-fold increase in the
neuronal survival rate, respectively, and cotreatment further
enhanced it as much as 6-fold. These results clearly
demonstrate that, although IGF-1 and 8 individually exhibit
the neuroprotective effect, they act in a collaborative manner to
produce greater neuroprotective activity.
ASSOCIATED CONTENT
* Supporting Information
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S
The Supporting Information is available free of charge on the
Experimental information and methods; chemistry,
ADME-tox, and PK studies; biology (PDF)
Molecular formula strings (CSV)
We have not yet elucidated how these compounds interact
with the IGF-1 signaling to sensitize the cells to IGF-1.
Although the underlying mechanism needs to be clarified,
exploring the pharmacological effects of the “IGF-1 sensitizer”
in vivo would be interesting. For this purpose, 8 was selected as
an appropriate chemical probe among the three potent
compounds 8, 13e, and 18j because 13e exhibited a faster
metabolic clearance than 8 (8, 2 μL/min/mg; 13e, 30 μL/min/
mg) and 18j caused a slight decrease in cellular ATP content at
100 μM in a general cytotoxicity assay (8, 93.3%; 18j, 68.0%).
Moreover, 8 has been observed to be brain-penetrable in rats
(Kp was 2.9 at 1 h after iv administration). At present, 8 is being
evaluated in several in vivo tests, including disease models, to
search for possible indications of an “IGF-1 sensitizer.”
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-466-32-1284. Fax: +81-466-29-4451. E-mail:
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We are grateful to Dr. Shigenori Ohkawa and Masahiro Okura
for their pioneering works on this research. We thank Yasuhiko
Kawano and Akihisa Maeda for their support with the synthesis,
Dr. Hideo Fujiwara, Masahiko Hattori, and Masumi Sagayama
for their support with the biological experiments, Keiko
Higashikawa and Mitsuyoshi Nishitani for their assistance
with the X-ray measurements, and Dr. Shinji Fujimoto, Akira
Oda, and Yuhei Miyanohana for their valuable discussions. Drs.
Takanobu Kuroita and Takashi Miki are also acknowledged for
their guidance and for proofreading of the manuscript.
CONCLUSION
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A series of benzofuran derivatives with neuroprotective activity
based on their collaborative effect with IGF-1 was discovered.
Among the three scaffolds tested, 2,3-dihydro-1-benzofuran
derivatives exhibited potent activity without any adverse effects
such as CYP2C9 inhibition or photoinduced cytotoxicity. A
subsequent SAR study of the dihydrobenzofuran derivatives
revealed (1) an advantage of lipophilic substituents at the 2-
position, (2) the importance of the molecular framework
defined by the linker, and (3) the effect of the substituent on
the terminal aryl ring. As a result, we observed 8, as a
representative compound, to exhibit potent neuroprotective
activity with brain penetrability. An in vitro pharmacological
study demonstrated that, although IGF-1 and 8 individually
exhibited neuroprotective effects, 8 acted in collaboration with
IGF-1 to exhibit greater neuroprotective activity. Compound 8
is being evaluated in several tests to explore its potential
therapeutic applications; the results of the pharmacological
characterization and mechanistic study will be reported
elsewhere.
ABBREVIATIONS USED
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DMF, N,N-dimethylformamide; EtOAc, ethyl acetate; EtOH,
ethanol; KOt-Bu, potassium tert-butoxide; MeCN, acetonitrile;
MEK, methyl ethyl ketone; NaOt-Bu, sodium tert-butoxide;
Pd/C, palladium on carbon; PhNEt2, diethylaniline; rt, room
temperature; TFA, trifluoroacetic acid; THF, tetrahydrofuran
REFERENCES
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EXPERIMENTAL SECTION
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1-(4-Methoxyphenyl)-4-(2,2,4,6,7-pentamethyl-2,3-dihydro-
1-benzofuran-5-yl)piperazine (8). To an ice-cooled suspension of
LiAlH4 (577 mg, 15.2 mmol) in THF (20 mL) was added AlCl3 (2.03
g, 15.2 mmol). After stirring at 0 °C for 10 min, a solution of 1a (2.00
g, 5.07 mmol) in THF (25 mL) was added and the mixture was
(6) Guan, J.; Williams, C.; Gunning, M.; Mallard, C.; Gluckman, P.
The effects of IGF-1 treatment after hypoxic-ischemic brain injury in
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