Communications
ever, the molecular mechanisms that underlie stem-cell-fate
antibodies, the morphology of positively stained cells was
analyzed, and the number of cells with the desired phenotypes
were counted.
specification are still poorly understood, and we lack robust
and selective methods to control the differentiation programs
of NPCs. Although several agents, including retinoic acid
A class of 4-aminothiazoles, which induces specific neuro-
nal differentiation in a dose-dependent manner, was identi-
fied. To improve the potency and specificity of these
compounds further, a sublibrary of 4-aminothiazole com-
pounds was generated by the reaction of a-chloroacetylani-
lines and thiobenzamides (Scheme 1) and their activity was
assayed. This structure–activity relationship (SAR) study
revealed that: 1) aminomethylation (i.e., R3 = Me) signifi-
cantly enhanced activity relative to other substituents at the
R3 position, 2) substitution of R1 with 4-Cl and 4-CF3 leads to
a decrease in activity, and 3) R2 substitution at the meta
position or the presence of a hydrogen-bond donor dramat-
ically decreases activity. Compound 1, neuropathiazol
(Scheme 1), has the highest activity among the analogues
tested (representative analogues and their relative activity are
provided in the Supporting Information).
(RA),[1] leukemia inhibitory factor (LIF),[5] and insulin-like
[6]
growth factor-1(IGF-1),
have been found to direct neuro-
nal, astroglial, and oligodendrocytic differentiation, they
either are pleiotropic or have poor in vivo activities. More-
over, relatively little is known about the endogenous mole-
cules that control stem-cell fate. Consequently, the generation
of small molecules that can direct differentiation of adult
NPCs could provide useful chemical tools to probe signaling
pathways that control neuronal specification and could
ultimately facilitate therapeutic application of NPCs.
Herein, we report the identification and preliminary charac-
terization of a novel small molecule, neuropathiazol, that
selectively induces neuronal differentiation of multipotent
hippocampal neural progenitor cells.
To screen for small-molecule inducers of neuronal differ-
entiation, a primary neural progenitor (HCN) cells isolated
from adult rat hippocampus were used. Although it has been
shown that HCN cells can differentiate into neurons, astro-
cytes, and oligodendrocytes in vitro and can functionally
integrate into existing neuronal networks in vivo, HCN cells
very rarely differentiate into neuronal and astroglial lineages
without treatment with exogenous factors, for example, with
RA or LIF in the absence of basic fibroblast growth factor
(bFGF): In contrast, a relatively large percentage of HCN
cells are induced to differentiate into oligodendrocytes at high
cell density by the insulin present in N2 supplemented
medium. The extremely low frequency of ꢀspontaneousꢁ
(cell autonomous) neuronal and astroglial differentiation of
HCN cells makes them a good in vitro model system to screen
for novel chemical regulators.
HCN cells were expanded and maintained in an undiffer-
entiated state on a polyornithine/laminin-coated dish in a
defined serum-free growth medium (DMEM/F12 supple-
mented with N2 and bFGF (20 ngmLÀ1)) in a homogeneous
monolayer. Neuronal and astroglial differentiation were
analyzed directly with double immunofluorescence staining
of bIII tubulin (TuJ1) as a neuronal marker and of glial
fibrillary acidic protein (GFAP) as an astroglial marker. A
combinatorial heterocycle library of 50000 compounds was
screened by using undifferentiated, early passage HCN cells,
which were plated onto polyornithine/laminin-coated 384-
well plates at a density of 6000 cells/well in complete growth
medium. After overnight incubation, the medium was
exchanged with bFGF-free basal medium, and the com-
pounds (final concentration of 5 mm) were added to each well.
After treatment with the compounds for four days, the cells
were fixed and stained with bIII tubulin/TuJ1and GFAP
Treatment of HCN cells with neuropathiazol significantly
slowed cell proliferation (Figure 1) without visible cytotoxic
Figure 1. BrdU proliferation assay. Neuropathiazol inhibits proliferation
of HCN neural progenitor cells. HCN cells treated with RA (1 mm,
0.5 mm, and 0.25 mm) or neuropathiazol (NPT) (15 mm, 10 mm, and
5 mm) for 5 h in bFGF-free N2 medium were labeled with BrdU for 2 h,
and BrdU-positive cells were counted.
effects; more than 90% of the cells differentiated into
neuronal cells as determined by immunostaining with TuJ1
and the characteristic neuronal morphology. RA, however,
showed weaker antiproliferation activity and significant
cytotoxicity over 2 mm in growth factor (bFGF or LIF/
BMP2)-free basal medium. GFAP-positive cells were very
rarely detected after neuropathiazol treatment, indicating
that neuropathiazol specifically induces a neuronal lineage
(Figure 2B). In contrast, relatively large numbers of both
neuronal and astroglial cells were found with RA-induced
differentiation of HCN cells, which indicates that RA is not
very specific (Figure 2 A). Longer treatment of HCN cells
(7 days) with neuropathiazol led to positive staining for later-
Scheme 1. Synthesis of 4-aminothiazole analogues. Reagents: a) DMF (or EtOH), 808C, overnight; b) NaH, RX, room temperature, 2 h.
www.angewandte.org ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2006, 45, 591 –593
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Warashina, Masaki
