C.-S. Jiang et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Scheme 1. Synthesis of 2a and 2b. Reagents and conditions: (i) NH2OHÁHCl, pyridine, EtOH, rt, overnight; (ii) [Ru2Cl2(p-cymene)]2, CH3CN, reflux, 3 h; (iii) NH2OHÁHCl, K2CO3,
EtOH, reflux, 2 h; (iv) (1) indole-3-acetic acid (8), HATU, DIPEA, CH2Cl2, (2) NaOAc, EtOH, reflux, 2 h; (v) Pd(PPh3)4, 2-aminopyrimidine-5-boronic acid pinacol ester (for 2a)/4-
aminophenylboronic acid pinacol ester (for 2b), K2CO3, dioxane, H2O, 10 h.
Scheme 2. Synthesis of 11a and 11b. Reagents and conditions: (i) (1) HATU, DIPEA, CH2Cl2, (2) NaOAc, EtOH; (ii) Pd(PPh3)4, 4-aminophenylboronic acid pinacol ester, K2CO3,
dioxane, H2O.
As mentioned in the introduction section, in the course of our
early studies the compound 2b with the 4-(furan-2-yl)aniline as
side chain was found to exhibit promising neuroprotective activity
against Ab25–35- and OGD-induced neurotoxicity in SH-SY5Y cells,
however compound 2a with 5-(furan-2-yl)pyrimidin-2-amine as
side chain was inactive. The preliminary study indicated that the
substituent group attached to the furan played an important role
in neuroprotective activity.
Before a systematic examination of the different substituent
groups attached to the furan was undertaken, our initial effort
was focused on screening different ring systems to replace the
furan ring to optimize the neuroprotective activity. Therefore, the
analogs 11a and 11b with a thiofuran or benzene ring were synthe-
sized. The result of bioassay indicated that the introduction of thi-
ofuran ring in 11a completely led to the loss of neuroprotective
activity, while the compound 11b bearing a benzene ring showed
good neuroprotective activity against Ab25–35-induced neurotox-
result of MTT may reflect either the number of viable cells or the
activity of mitochondrion. In order to better determine the changes
of cell numbers, we performed SRB assay, which is based on the
measurement of protein content. After 24 h of incubation with
SH-SY5Y cells, the SRB values of 2k and 2r groups were 96% and
94% of control, respectively, which suggested that neither of them
promote the cell proliferation.
Compared with 4-amino compound (2b), installation of dimeth-
ylamino (2c), morpholine (2d), and 4-methylpiperazino (2e) led to
the loss of neuroprotective effect due to the increased steric hin-
drance. Replacement of the 4-amino group with H, or 2-,3-,4-methyl
group gave compounds 2f–2i, of which only compound 2i with
4-methyl showed activity (cell viability: 80.3% at 10 lM) indicating
that the substituent at 4-position in the benzene ring was important
for the activity. Among the compounds 2i–2k, the activity was found
to increase with the increase of the alkyl chain. The similar phenom-
enon was observed when the 4-hydroxyl (2o, inactive at 10
alkylated to give 2p (91.5% at 10 M), 2q (89.1% at 10 M), and 2r
(115.5%, at 10 M; 90% at 1 M). However, the introduction of
lM) was
icity with cell viability of 106% at 10
lM. However, it was disap-
l
l
pointing that compound 11b did not display neuroprotective
activity against OGD-induced neurotoxicity. Consequently, the
furan ring was considered to be optimal among the ring systems
explored here. Thus, optimization of the substituent groups on
the terminal benzene rings attached at 5-position of the furan ring
was performed with the series of 1,2,4-oxadiazoles 2c–2v.
The results of the neuroprotective activity of 2c–2v against
Ab25–35-induced neurotoxicity in SH-SY5Y cells are shown in
Table 1. Most of compounds showed bioactivity with the range from
l
l
isopropyl (2l), and tert-butyl (2m) with increased steric hindrance
decreased the activity in some degree by comparison with 2j and
2k. Interestingly, the replacement of electron-releasing groups
(such as methyl and methoxyl groups) with electron-withdrawing
groups (nitrile or halogen groups) provided the corresponding
2s–2v, which still showed effective neuroprotective activity (cell
viability: 83.5–93.3% at 10 lM), indicating electronic effect of the
substituents in 4-position might not be a key factor in their bioactiv-
80.3% to 115.5% of cell viability at 10
and 2r could further significantly increase the cell viability (89.7%
and 90%, respectively) at 1 M. Among all these tested derivatives,
compound 2r bearing propoxy group at the 4-position of the
benzene ring showed the highest activity (115.5% at 10 M), much
lM, and two compounds 2k
ity against Ab25–35-induced neurotoxicity in SH-SY5Y cells.
All target compounds were further subjected to bioassay to test
their in vitro neuroprotective effects against H2O2-induced damage
in SH-SY5Y cells. The results are shown in Figure 3. Although only
four compounds (2c and 2p–2r) were active with 98.0–99.7% of
l
l
better than the positive control EGGC. Although MTT reduction
activities of compounds 2k, 2r and 11b were over 100%, only 2k
and 2r are significant as compared with control group, which indi-
cated that these two compounds may promote the cell proliferation.
However, it is noticeable that MTT reduction is dependent on
NAD(P)H-dependent oxidoreductase enzyme, which is often used
to determine the cell viability rather than the cell number, so the
cell viability at 10
with the positive control L-NAC (89.2% at 100
these compounds did not show significant activity at 1
Further, the above compounds 2c and 2p–2r were selected to be
evaluated for their activity against ODG-induced injury in SH-SY5Y
cells (as an in vitro stoke or AD model). Among these compounds,
l
M, they still exhibited higher activity compared
M). Unfortunately,
M.
l
l
2q (76.2%) and 2r (84.1%) were active at 10 lM (shown in Fig. 4),