K. Xu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2772–2774
2773
Table 1
synthesis, and biological evaluation of a series of 1,4-napthquinone
derivatives.
Previous docking and structure–activity relationship (SAR)
Inhibitory activities of selected compounds against tumor cell growtha
Compounds
IC50
(l
M)
studies8 suggested a hypothetical pharmacophore for 1,4-napth-
quinone proteasome inhibitors. The 1,4-napthquinone scaffold is
assumed to be crucial for proteasome inhibition, and its two oxy-
gen atoms form hydrogen bonds with Thr21 and Gly47 of 20S pro-
teasome, respectively. Similarly, a hydrogen bond was observed
between the sulfonamide group in PI-083 and Asp114 in the b6
subunit of 20S proteasome, which implies that future structural
design of new inhibitors should incorporate a hydrogen bond
acceptor in this molecular area. Accordingly, 1,4-napthquinone
derivatives with the general formula illustrated in Figure 1b were
designed and synthesized, and structural variations on linker,
hydrogen bonding acceptor, and tailing group were introduced to
allow sufficient SAR study.
As shown in Scheme 1, compound 1 was designed to investigate
the effect of substituting a carbonyl group for the sulfonyl group in
PI-083, and the pyridyl group was also replaced with different tail-
ing groups (compounds 2–4) to explore the effect of this molecular
region on antiproliferative activity. Compounds 5–7 were intro-
duced according to the reversed amide strategy. Compounds
(8–11) with diverse substituents bearing hydrogen bond acceptors,
were intended to further examine SAR in this molecular area. In
compounds 12 and 13, the phenyl linker was replaced with an ali-
phatic linker to expand the SAR exploration and improve solubility.
Compound 14 contains a weak hydrogen bond acceptor, the sulfur
atom in a thienyl group, to verify the necessity for hydrogen bond-
ing interaction.
A549
DU145
KB
9.57 3.16
10.43 2.89
13.99 3.05
16.55 2.25
12.88 0.62
8.12 1.20
16.17 4.66
KBvin
PI-083
2
4
8
9
10
13
12.19 1.09
9.64 3.37
12.45 0.71
23.18 3.71
18.76 4.64
2.90 0.27
20.40 3.70
10.82 1.36
10.46 0.99
13.68 1.79
18.28 2.16
15.83 1.93
4.55 2.21
18.12 2.40
11.66 3.93
11.26 0.84
14.49 2.39
18.34 2.98
14.23 1.71
1.30 0.53
19.14 4.72
a
The remaining compounds did not show significant inhibition at 10 lg/mL.
tive activities comparable or superior to that of the phenotype
structure PI-083 (Table 1), and the most active compound (10)
exhibited significant inhibition against all four tested tumor cell
lines, especially KBvin cells. As expected, these compounds were
generally effective against KBvin cell growth, since they presum-
ably act through a molecular mechanism other than inhibiting
tubulin polymerization. Preliminary SAR on the antitumor activity
of these 1,4-napthquinone derivatives could also be deduced from
the data listed in Table 1. Replacing the sulfonamide group in PI-
083 with an amide group decreased the antitumor activity (1, 5
vs PI-083). The structure of the tailing group could have a consid-
erable effect on the activity, and incorporation of additional hydro-
gen bond forming atoms in this group might be beneficial for the
antitumor activity (2, 4 vs 1, 3). However, the activity of compound
8 (R = p-nitrophenyl) also indicated that a tailing group might be
dispensable. In addition, an ether extended with a bulky and
hydrophobic tailing group provided a favorable effect on the activ-
ity (e.g. compound 10, R = p-phenoxyphenyl). The results for com-
pound 13 implied that an appropriately substituted aliphatic
linkage could also be accommodated with retention of activity.
The six most active compounds were further examined for their
proteasome inhibition activity. There are generally two types of as-
says for proteasome inhibition, in vitro and cell-based assays.
These assays vary in their applicability, and inconsistent results be-
tween in vitro and cell-based assays were observed previously.13
Therefore, it is necessary to verify the underlying mechanism of
proteasome inhibition with both in vitro and cell-based assays,
so as to identify those active in both assays.
All synthesized compounds were tested for their inhibitory
activities against the growth of A549, DU145, KB, and KBvin cell
lines.12 Six compounds (2, 4, 8–10, and 13) showed antiprolifera-
O
O
O
O
Cl
Cl
Cl
RNH2
anhydrous ethanol
reflux, 3 days
NH
R
O
O
O
O
2: R=
4: R=
6: R=
A cell-based assay was first performed with HeLa-GFP cells14
and lactacystin was tested in parallel as a positive control. Surpris-
ingly, at the tested concentration of 10 g/mL, only compound 8
,
N
1: R=
HN
HN
l
O
N
O
showed noticeable activity, while PI-083 and the remaining five
compounds did not exhibit any evident activity. The lack of activity
in a cell-based assay could be attributed to poor cellular perme-
ability or susceptibility to cellular metabolism of this compound
class. The discrepancy between antitumor activity and cell-based
proteasome inhibitory activity also implicates that a molecular
mechanism other than proteasome inhibition may be involved.
Compound 8 was further evaluated with the in vitro assay14 for
inhibition against the chymotrypsin-like activity of 20S protea-
some, which is considered as a primary hallmark for proteasome
3: R=
5: R=
HN
O
NH
O
N
NH
O
O
O
NO2
8: R=
7: R=
9: R=
NH
O
N
O
10: R=
O
Table 2
Inhibition of the chymotrypsin-like activity of 20S proteasome
O
O
Compounds
8
PI-083
Lactacystin
10.09
N
O
11: R=
13: R=
12: R=
14: R=
a
IC50
(lM)
3.65
18.56b
a
S
The IC50 values are for inhibition against the chymotrypsin-like activity of 20S
O
proteasome, which are the averages from two independent experiments with
N
purified 20S human proteasome.
b
The IC50 value for PI-083 was 1.0 lM in Ref. 8 However, purified 20S rabbit
proteasome was used for the measurement therein.
Scheme 1. Synthesis of compounds 1–14.
Xu, Kai
