S.-L. Zhang et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 2. The PDC activation rates were measured by the primary PDC enzymatic activity assay in the presence of the prepared compounds at 1 µM. The PDC activation
was 36.6% for DAP at 80 µM. PDK1 kinase activity was measured by the Kinase-Glo® luminescent kinase assays (Promega). The PDC activation and PDK1 inhibition
assays were three independent experimental measurements, the values were reported as the average
S.D.
and 11–2).
With the dichloroacetophenone scaffold, diverse substituents were
at 1 µM) and DAP.
Inspired by these observations, we then turned to meta-nitro and
para-substitution with different electron-donating groups to form a 3,4-
disubstituted dichloroacetophenones, with the aim to further improve
their PDK inhibitory potencies. As expected, the designed compounds
(53–72) displayed improved activities as compared with their pre-
cursors (18–28). For example, the PDKs inhibition rates were 19.7,
59.9, and 44.0% for 18–20 at 1 µM, and were 50.5, 66.6, and 75.1% for
53–55 at 1 µM, respectively, which appeared to be a significant im-
provement. For compounds 53–57, with the increment of the lengths of
alkyl chains in the para-position, the antiproliferative activity and PDKs
inhibitory potency improved, but not for compounds 56 and 57, sug-
gesting the length of alkyl chain is important. As for the alkoxyl-sub-
stitutions 61–72, all of these compounds displayed improved PDC ac-
tivation and PDK1 kinase inhibition potency, as well as antiproliferative
activity as compared with other compounds mentioned above. In par-
ticular 64 displayed the best potency in PDKs inhibition (PDKs inhibi-
tion rate: 84.3%).
introduced into the para-, ortho-, and meta-positions of benzene ring to
scope out the SARs. For meta-substituted dichloroacetophenones
(32–39), all of which gave satisfactory improvements in anti-
proliferative activity in contrast to DAP, especially for 35, the IC50
value found to be 200 nM against NCI-H1975 cells. However, the ac-
tivation of PDC by the compound was moderate (PDKs inhibition rate is
15.1% at 1 µM, see Fig. S1), indicating other mechanisms could be in-
volved in its anti-tumor effect. For ortho-substituted compounds 40–46,
both anticancer activity and PDKs inhibitory potency were not good.
To our delight, the introduction of electron-donating groups such as
ethyl, isopropyl, alkoxyl groups into the para-position of di-
chloroacetophenone offered some potent compounds (18–28), which
not only showed strong antiproliferative potencies against the three
cancer cell lines, but also displayed good PDKs inhibitory effects (PDKs
inhibitory rates ranging from 19.7 to 67.9%). The IC50 values of some
potential compounds (21, 24–28, 50, 54, 55, and 64) reached around
2 µM against NCI-H1975 cells, and 4 µM for A549 cells, respectively
(Figs. 3A and B). Meanwhile, they reduced the PDC phosphorylation in
DAP showed a moderate antiproliferative activity, but displayed a
very weak potency against PDKs, which led us to speculate that DAP
might not be a selective PDKs inhibitor. It has been reported that DAP
inhibited the AKT phosphorylation and EGFR / ERK signaling path-
ways.21 We aimed to develop PDK-selective anticancer di-
chloroacetophenones by minimizing their activities on AKT, and EGFR
/ ERK pathways. Potent compounds (19–21, 24–28, 50, 54, 55, 62,
64–66, 68, 70) with good anticancer activity and PDKs inhibitory po-
tency were chosen to assess their effect on EGFR, AKT, and ERK
phosphorylation through western blotting. As shown in Fig. 4 and Fig.
S2 (see SI), DAP at 10 µM strongly reduced the EGFR, AKT, and ERK
phosphorylation, and so did most of the prepared compounds. Inter-
estingly, three compounds, namely 54, 55, and 64 at the same tested
concentration exerted a minimal effect on the EGFR, AKT, and ERK
S1). A great enhancement of the inhibitory potency could be achieved
via replacement of the chloro group with a nitro- moiety (60, PDKs
inhibition rate: 41.7%) in meta-position of benzene ring, but not by the
methyl group (49, PDKs inhibition rate: 10.2%). This observation
suggested that nitro substitution in meta-position of benzene ring con-
tributed to the PDC activity improvement. Compound 50 was derived
from compound 47 by replacing the chloro group with a methoxyl
moiety in the para-position of dichloroacetophenone. It displayed pro-
mising PDC activation potency with apparent PDKs inhibitory rate of
47.1% at 1 µM, a substantial improvement from 47 (no PDKs inhibition
Fig. 3. A, B: IC50 values of some potential compounds against A549 and NCI-H1975 cell lines; (C) Compounds reduced the PDC phosphorylation in NCI-H1975 cells.
3