B.-C. Ge et al. / European Journal of Medicinal Chemistry 141 (2017) 440e445
441
brominated using NBS as a brominating reagent. All the bromides
were used in all subsequent reactions without further purification,
owing to their instability. Compounds 8a-f and 9a-f were synthe-
sized by the nucleophilic substitution of the bromides with the
corresponding pyridazin-3(2H)-ones. Pyridazin-3(2H)-ones 18a
(R
¼
H) and 18b (R
¼
Cl) were synthesized from 3,6-
dichloropyridazine and 4,5-dichloropyridazin-3(2H)-one, respec-
tively [18].
The antiproliferative activities of 8a-f and 9a-f were assessed
against three human cancer cell lines, including human neuro-
blastoma (SH-SY5Y), human myelogenous leukemia (K562), and
gastric cancer (AGS), using the MTT method [19]. Fluorouracil (5-
FU), which is one of the most effective anticancer agents, was
included in the experiments as a reference cytotoxic compound for
the three cell lines. The results were expressed as growth inhibitory
concentration (GI50) values, which represent the compound con-
centrations required to produce 50% growth inhibition of cell
growth after 48 h of incubation compared with untreated controls
(Table 1).
As shown in Table 1, compounds 8a-f and 9a-f displayed
different antiproliferative activities against different cancer cell
lines, with the GI50 values ranging from 6.3 to >100 mM. For com-
pounds 8a-f bearing a 2-(2-methoxyphenoxy)ethylamino moiety
at the 6 position of the pyridazin-3(2H)-one ring, their anti-
proliferative activities toward different cancer cell lines vary
significantly. Compounds 8c and 8f were inactive in all test cells.
Compound 8d exhibited no activity toward SH-SY5Y and AGS cells,
but exhibits moderate inhibitory activity against K562. Further-
more, compounds 8a, 8b, and 8e showed moderate to good anti-
proliferative activity against the tested cancer cell lines and were
more active toward SH-SY5Y cells. Of these, compound 8b exhibi-
ted the best antiproliferative activity against SH-SY5Y cells with a
Fig. 1. Pyridazinone derivatives with antitumor activities.
GI50 value of 6.3
(GI50 ¼ 11.9 M). Compounds 8a and 8e exhibited slightly
decreased inhibitory activity against SH-SY5Y cells with the GI50
mM, which was higher than that of 5-FU
m
values of 9.3 and 10.5
that of 5-FU.
mM, respectively, which were comparable to
By contrast, most of the compounds 9a-f, which bear a 2-(2-
methoxyphenoxy)ethylamino moiety at the 5 position of the pyr-
idazin-3(2H)-one ring showed remarkably decreased inhibitory
activity against the test cells. Compounds 9a and 9f were inactive
against all the test cells, while compound 9b exhibited weak
inhibitory activity against K562. However, compounds 9c-e showed
selective inhibitory activities for SH-SY5Y with moderate GI50
value.
These results indicated that most of these compounds exhibited
weak (or even zero) to moderate activity against the tested cell
lines. However, compounds 8a, 8b, and 8e were more active against
SH-SY5Y cells, exhibiting good inhibitory activity. Moreover, com-
pound 8b with low-micromolar GI50 value displayed selectivity for
SH-SY5Y cells over other two test cell lines. These findings provide
useful information regarding the structural requirements for better
potency and will help in designing more potent small molecules
that selectively target SH-SY5Y cells.
Several studies have indicated that inhibition of PDE4 reduces
proliferation, inhibits brain tumor cell growth [20,21], and causes
selective apoptosis of malignant cells without affecting normal
healthy cells [22]. Thus, the inhibitory activities (Table 1) of com-
pounds 8a-f and 9a-f were evaluated against PDE4 according to
reported protocols [17,23] using rolipram as a positive control (see
Fig. S1 in supporting information). All compounds were tested at
nine concentrations (10ꢀ8e10ꢀ4 M) and their IC50 values were
determined by the nonlinear regression analysis of their inhibition
curves. As shown in Table 1, most compounds exhibited moderate
PDE4 inhibition activity. Among these compounds, compounds 8a-
Fig. 2. Design of aminopyridazin-3(2H)-one derivatives as potential antitumor agents.
as potential antitumor agents (Scheme 1). The preliminary evalu-
ation of antiproliferative activity against three human cancer cell
lines (K562, SH-SY5Y, and AGS), PDE4 inhibitory activities (the core
catalytic domains of human PDE4), cell cycle analysis and apoptosis
assay of the synthesized compounds were also performed. (See
Fig. 3).
The route adopted for the preparation of (2-(2-
methoxyphenoxy) ethyl)aminopyridazin-3(2H)-one derivatives is
depicted in Scheme 1. As shown in Scheme 1, 3-alkoxy-4-
difluoromethoxybenzyl alcohols 16 were obtained by the reduc-
tion of 3-alkoxy-4-difluoromethoxybenzaldehydes 15, which were
prepared from 3,4-dihydroxybenzaldehyde using our prior syn-
thetic methodology [17]. Compounds 16 were brominated using
PBr3 as a brominating reagent, whereas compounds 14 were
Ge, Bing-Chen
