ACS Medicinal Chemistry Letters
Letter
retained (9−11). Encouragingly, the results showed that
additional substitutions were beneficial for the activity, and
the 4-methyl derivative 10 showed 4-fold higher activity than
(Table 3), among which PC3 prostate cancer and HT29 colon
adenocarcinoma cells possess only low levels of activated
STAT3 (Figure S9). Compound 15 exhibited much improved
efficacies compared with NTZ in most of the cell lines, except
for PC3 and HT29. These data suggest again that STAT3
inhibition plays an important role in the antitumor function of
thiazolides.
TIZ (10: IC50 = 2.3
0.08 μM; Table 1). However,
introduction of a hydroxyl group instead of a methyl group at
the 4-position eliminated the activity (10 vs 12).
Inspired by the above results, we subsequently focused on
modifications at the 3- or 4-position. We found that removing
the −OH while introducing −Cl at the 3- or 4-position
generated 3−6-fold increased activity, as in 4-Cl derivative 14
(IC50 = 1.5 0.3 μM; Table 1). Electron-donating groups (2,
OH; 17, OCH3), bulky groups (18, 4-methylpiperazin-1-yl;
19, morpholino; 20, tert-butyl; 23, N-phenylsulfamoyl), or
strongly polar groups (21, methylsulfonyl; 22, sulfamoyl) at
the 4-position were all harmful to the activity. By contrast,
small lipophilic substitutions at the 4-position, such as −Cl,
−CF3, or −N3 (14−16), generated significantly improved
potency. In particular, 15 exhibited a submicromolar IC50 (0.7
0.1 μM) and lower cytotoxicity (Table 1). The 3,4-dichloro
analogue 24 was more potent than TIZ (IC50 = 0.8 0.04
μM; Table 1). However, the 3,4-dihydroxy analogue 25 was
not active. In addition, the effect of amide −NH on the activity
seemed indispensible because replacing the hydrogen atom
with propynyl group resulted in a total loss of STAT3 activity
(15 vs 26).
We further analyzed the effect of 15 on STAT3 activation
and transcription using Western blot assays. A time-course
study revealed that 15 could significantly inhibit Tyr705
phosphorylation within 15 min and keep it at a very low level
for a long term. There was no obvious change in Ser727
phosphorylation (Figure 4A). 15 dose-dependently decreased
the protein level of p-STAT3 (Y705), whereas the p-STAT3
(S727) protein level remained stable after incubation for 24 h
(Figure 4B). Additionally, 15 did not affect the total STAT3
protein level in either a time- or dose-dependent manner
(Figure 4A,B). EMSA analysis revealed that similar to NTZ, 15
could also significantly block the interaction between STAT3
and DNA at both the molecular and cellular levels (Figure S6),
facilitating the dephosphorylation of p-STAT3 (Y705) (Figure
S7). The transcriptional activity of STAT3 was negatively
regulated, resulting in an obvious decrease of downstream
target proteins, as indicated by cyclin D1 and survivin (Figure
4C).
As an ester prodrug intended to treat intestinal pathogens,
NTZ can be partially taken up though passive absorption in the
gastrointestinal tract, but its oral bioavailability is poor.37 Upon
oral absorption, NTZ is immediately deacetylated to give TIZ,
which is subsequently metabolized as tizoxanide−glucuronide
in the liver and rapidly eliminated via urine.38 This
pharmacokinetic property is perfect for an anthelmintic drug
but much less desirable for a systemic antitumor agent. Finally,
we selected 15 and further compared the in vivo
pharmacokinetic properties in rats with those of NTZ. The
plasma concentration−time curves are shown in Figure S10,
and the key pharmacokinetic parameters are summarized in
Table 4. After a single dose of oral administration, NTZ
displayed a high clearance rate (6.3 L h−1 kg−1) and low
absolute bioavailability (5.7%). By comparison, 15 exhibited
more desirable pharmacokinetic properties, with a significantly
longer half-life for elimination (t1/2β) (11.1 vs 0.8 h), greater
absolute bioavailability (F) (87.4% vs 5.7%), and higher
maximum plasma concentration (Cmax) (20.7 vs 1.0 mg/L).
In conclusion, we report that the FDA-approved antipar-
asitic drug NTZ can effectively inhibit the STAT3 pathway
independent of interfering with the activities of the upstream
kinases JAK2 and Src. In contrast to most reported SH2
binders, NTZ relies more on blocking the STAT3−DNA
interaction. Furthermore, we performed preliminary structural
deviations and structure−activity relationship analysis toward
improved inhibitory activities against STAT3 pathway and
identified eight thiazolides with potencies greater than that of
NTZ. These derivatives exhibited increased antitumor
potencies that were closely associated with their STAT3
inhibitory activities. Compared with WP1066, the optimized
derivative 15 showed better inhibitory activity against both the
STAT3 pathway and cancer cell proliferation, while its
cytotoxicity was significantly lower. In addition, 15 exhibited
greatly improved pharmacokinetic parameters compared with
NTZ and showed broad-spectrum antitumor activities in
multiple cancer cell lines, which indicates that 15 deserves
further in vivo evaluation as an antitumor agent and that
To further investigate the antitumor potency of the
compounds with STAT3 pathway inhibitory activities higher
than those of NTZ and TIZ, the antiproliferative activities
were evaluated in HeLa cells. A significant correlation between
the antitumor activity and the individual STAT3 inhibitory
activity was observed, with a Pearson correlation coefficient of
better than 0.99 (Figure S8). All of these compounds
demonstrated satisfactory safety, as none of them induced
cell death higher than 40% at a concentration as high as 50 μM
in human normal embryonic kidney HEK 293T cells (Table
2). Notably, 15 and 24 exhibited even better activities than the
positive control WP1066 in both STAT3 pathway activation
and HeLa cell proliferation, while the cytotoxicities were much
lower, indicating good potential of these compounds as
antitumor agents. We selected 15 and measured its
antiproliferative activities against diverse types of cancer cells
Table 2. Growth-Inhibitory Activities of Thiazolides in the
a
HEK 293T and HeLa Cell Lines
HEK 293T cell growth
inhibition % (48 h)
IC50 (μM, 20 h)
STAT3 pathway
IC50 (μM,
in HEK-Blue IL- 48 h) HeLa
compd
6 cells
cell growth
50 μM
10 μM
NTZ
TIZ
9
10
11
13
14
15
16
9.8 0.2
8.9 0.3
4.6 0.3
2.3 0.08
3.6 0.3
3.0 1.1
1.5 0.3
0.7 0.1
2.5 0.5
0.8 0.04
2.5 0.7
35.0 0.1
29.8 1.5
20.5 4.4
16.4 1.5
17.7 6.4
11.1 1.7
7.9 2.1
2.7 1.1
9.9 1.7
3.3 0.8
4.2 0.2
24.4 1.0
21.6 3.8
21.2 1.8
24.9 03
25.8 4.1
−0.2 3.4
21.0 2.4
38.3 0.9
30.8 3.0
19.1 3.7
97.2 0.2
−3.3 4.6
−5.7 4.3
−1.0 5.8
11.0 1.8
−4.3 2.9
−5.5 3.3
8.5 3.6
12.8 2.7
5.1 3.0
8.0 4.6
24
WP1066
79.9 6.8
a
All of the values are averages of three independent experiments.
700
ACS Med. Chem. Lett. 2021, 12, 696−703