Synthesis and anticancer mechanism investigation of dual Hsp27 and tubulin inhibitors

Article abstract of DOI:10.1021/jm4004736

Heat shock protein 27 (Hsp27) is a chaperone protein, and its expression is increased in response to various stress stimuli including anticancer chemotherapy, which allows the cells to survive and causes drug resistance. We previously identified lead comp

Full text of DOI:10.1021/jm4004736

Article
pubs.acs.org/jmc
Synthesis and Anticancer Mechanism Investigation of Dual Hsp27
and Tubulin Inhibitors
Bo Zhong,† Snigdha Chennamaneni,† Rati Lama,† Xin Yi,† Werner J. Geldenhuys,§ John J. Pink,∥
Afshin Dowlati,⊥ Yan Xu,† Aimin Zhou,†,‡ and Bin Su*,†,‡
†Department of Chemistry, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Avenue, Cleveland,
Ohio 44115, United States
‡Center for Gene Regulation in Health and Disease, College of Sciences and Health Professions, Cleveland State University, 2121
Euclid Avenue, Cleveland, Ohio 44115, United States
§Department of Pharmaceutical Sciences, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, Ohio 44272, United
States
∥Division of General Medical SciencesOncology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue,
Cleveland, Ohio 44106, United States
⊥Division of Hematology and Oncology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland,
Ohio 44106, United States
S
* Supporting Information
ABSTRACT: Heat shock protein 27 (Hsp27) is a chaperone
protein, and its expression is increased in response to various
stress stimuli including anticancer chemotherapy, which allows
the cells to survive and causes drug resistance. We previously
identified lead compounds that bound to Hsp27 and tubulin
via proteomic approaches. Systematic ligand based optimiza-
tion in the current study significantly increased the cell growth
inhibition and apoptosis inducing activities of the compounds.
Compared to the lead compounds, one of the new derivatives
exhibited much better potency to inhibit tubulin polymer-
ization but a decreased activity to inhibit Hsp27 chaperone function, suggesting that the structural modification dissected the dual
targeting effects of the compound. The most potent compounds 20 and 22 exhibited strong cell proliferation inhibitory activities
at subnanomolar concentration against 60 human cancer cell lines conducted by Developmental Therapeutic Program at the
National Cancer Institute and represented promising candidates for anticancer drug development.
properties and functions at multiple steps of the apoptotic
signaling pathway.
1. INTRODUCTION
The expression of heat shock proteins (Hsp) is increased under
lethal conditions, which is a typical response when cancer cells
are stressed by chemotherapies.1−3 Hsp27, a small heat shock
protein, shows a very tight correlation with anticancer drug
resistance.3,4 Its overexpression enhances the survival of
mammalian cells exposed to anticancer agents.5 The Hsp27
protective effects come from its molecular chaperone functions
at multiple control points of the apoptotic pathways to
eliminate anticancer agents induced programmed cell
death.6−8 Hsp27 negatively regulates the activation of pro-
caspase 9 via interaction with cytochrome c and therefore
blocks the formation of the apoptosome complex.7−9 In
addition, Hsp27 can inhibit caspase 3 activity via direct
interaction with the pro-caspase 3 molecule.10 Other evidence
has demonstrated that a significant pool of Hsp27 is located in
the mitochondrial fraction of thermotolerant Jurkat cells, where
it functions against apoptotic stimuli by blocking the release of
cytochrome c.11 Overall, Hsp27 has strong antiapoptotic
Mammalian Hsp27 has a molecular weight of 27 kDa;
however, it can form oligomeric complexes in the range 100
800 kDa.9,12,13 The oligomerization of Hsp27 is a highly
dynamic process that depends on the physiology of the cells,
phosphorylation status of the protein, and exposure to
stress.5,6,14 It has been well investigated that large oligomers
of Hsp27 are required for the chaperone activity, and the
oligomers protect cells from oxidative stress, whereas this
activity is down-regulated by phosphorylation-driven dissocia-
tion of the large oligomers.6,9Preventing the formation of
Hsp27 large oligomers can decrease the chaperone activity and
promote cell apoptosis, which has been demonstrated by the
investigation on some small molecules.15 Up-regulated
expression of Hsp27 leads to an overactive chaperone activity,
which in turn contributes to the anticancer drug resistance.1,2
Received: January 28, 2013
Published: June 16, 2013
© 2013 American Chemical Society
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Clinically, increased level of Hsp27 in a number of cancers such
as breast cancer, endometrial cancer, and leukemia has been
detected.1,2,16−20 The fact that Hsp27 is often highly expressed
in cells or tissues from a wide range of tumors supports the
hypothesis that this protein could limit the efficacy of cancer
therapy. It will be highly desirable to target Hsp27 as cancer
therapy. Hsp27 modulators can decrease the protective
function of the protein in cell apoptotic regulation.4,21,22
These agents exhibit promising antitumor activities in
preclinical models of a variety of tumors expressing high levels
of Hsp27 and may have significant clinical potential.16,21,22
So far, the most effective strategy to target Hsp27 is antisense
oligonucleotides (ASO) or siRNA, which can decrease Hsp27
expression in in vitro cell culture and in vivo xenograft as
well.4,22 The fourth generation ASO of Hsp27, OGX-427,16 has
shown clinical evidence of antitumor activity despite the
limitations associated with ASO administration. The results
further underline that Hsp27 is a good anticancer target. Small
molecule Hsp27 modulators without the administration
problems are highly possible to show even better clinical
outcomes. The difficulties of developing small molecule Hsp27
inhibitors include three critical factors: (1) Hsp27 does not
have endogenous ligand, and there is no information about the
binding site of the protein; (2) the dynamic oligomeric process
of Hsp27 is correlated to its function, and this dynamic
characteristic makes it difficult to evaluate small molecules
binding to Hsp27; (3) there is an absence of clearly
demonstrated Hsp27 pathway downstream proteins that are
directly affected by Hsp27 inhibitors. Therefore, it is difficult to
define a cellular biomarker for these small molecules.
Through biotinylated small molecular probe, affinity
chromatography, and proteomic approaches, we previously
identified potent anticancer agents that are dual ligands binding
to Hsp27 and tubulin.23,24 Systematic ligand based optimization
in the current study dramatically increased the cell proliferation
inhibition and apoptosis inducing activities of the compounds.
The preliminary mechanism study suggests that tubulin
inhibitory efficacy of the new derivatives was significantly
improved, and the inhibition of the Hsp27 chaperone activity
was decreased on one of the analogues. It seems that the
structural modification dissected the two activities. Some of the
new analogues became more potent and selective tubulin
inhibitors. The structure−activity relationship (SAR) summar-
ized from the study provided us with a foundation to further
dissect the two activities and develop pure Hsp27 or tubulin
inhibitors.
Figure 1. Structure of compound 48.
pounds.25,26 In the newly designed derivatives, we explored
other 2,5-disubstituted benzyl groups including 2,5-dimethox-
ybenzyl and 2-methyl-5-trifluoromethylbenzyl. Additionally, n-
hexyl group at the B position was also evaluated in order to
determine if the hydrophobic and flexible alkyl chain could be
well accommodated. Moiety A was modified to six substituted
aryl groups including 4-bromophenyl, 4-methoxyphenyl, 3,4-
dimethylphenyl, 4-iodophenyl, 2-naphthyl, and 3,4-methylene-
dioxyphenyl. These six substituted aryl groups were previously
demonstrated to be the best fit while keeping B position as 2,5-
dimethylbenzyl and C position as methylsulfonamide and D
position as methyl group.25 We anticipated that the new
combination of A and B moieties would generate more active
compounds. Additionally, the alternative to methylsulfonamide
at C position was investigated by substitution with the
trifluoromethylsulfonamide group. In drug design, the trifluor-
omethyl group is often used as a bioisostere to replace the
methyl group in drug candidates for improved pharmacological
activity. Trifluoromethyl is a very strong electron-withdrawing
group and can form strong interactions with target protein
through hydrogen bonds or polar contact. It is interesting to
know whether a trifluoromethylsulfonamide group in place of
the methylsulfonamide group or a trifluoromethyl group in
place of the methyl group of the benzyl moiety can improve the
molecular binding to target proteins and thereby increase the
biological activities.
Methanesulfonamide derivatives 1−30 were prepared
according to the published method (Scheme 1).25 The
alkylation of the hydroxyl group of 2-amino-5-nitrophenol
was followed by mesylation of the amino group by
methanesulfonyl chloride and sodium hydroxide mediated
hydrolysis of N,N-bismethanesulfonamide to afford the
monomethanesulfonamide intermediate. This intermediate
either underwent N-methylation of the sulfonamide group,
reduction of the nitro group, and then amidation of amino
group to yield compounds 1−24 or was directly subjected to
reduction and amidation to give compounds 25−30. Our first
attempt to react O-alkylated 2-amino-5-nitrophenol with
trifluoromethylsulfonyl chloride in the presence of sodium
hydride in dry N,N-dimethylformamide (DMF) failed to yield
trifluoromethanesulfonamide. So more reactive trifluorometha-
nesulfonic anhydride was used instead and DMF was replaced
with diethyl ether as solvent, since DMF is incompatible with
trifluoromethanesulfonic anhydride. The obtained trifluorome-
thanesulfonamide intermediate was subsequently subjected to
methylation, reduction, and amidation to yield the expected
compounds 31−42.
2. RESULTS AND DISCUSSIONS
2.1. Lead Optimization and Summarization of the
SAR. Our previous efforts to develop the potent anticancer
agents led to the discovery of compound 48 (NSC751382,25
Figure 1), benzo[1,3]dioxole-5-carboxylic acid [3-(2,5-dime-
thylbenzyloxy)-4-(methanesulfonylmethylamino)phenyl]amide,
which displayed potent antiproliferative activity against various
cancer cell lines with IC50 values in the range of 0.1−0.5 μM.
The molecular targets of this compound were subsequently
identified to be Hsp27 and tubulin.24 In the present study, a
total of 42 new derivatives based on compound 48 were
synthesized using combinatory strategy. First, we kept C and D
moieties as N-methyl methylsulfonamide, and changed A and B
moieties systematically (Table 1). Previous SAR study of A and
B moieties revealed that 2,5-dimethylbenzyl and 2,5-dichlor-
obenzyl-substituted B position led to more active com-
All the compounds were evaluated for the inhibition of
SKBR-3 breast cancer cell proliferation, and the results are
summarized in Tables 1 and 2. The IC50 values of lead
compound 48 and several previously synthesized 2,5-
dimethylbenzyl analogues assigned as compounds 43−49 are
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Table 1. SAR of A and B Moiety Methanesulfonamide
listed in Table 3 for comparison.25,27 The results suggest that
the B moiety is critical for the antiproliferative activity of this
series of compounds (Table 1). The general order of potency is
2,5-dimethoxybenzyl derivatives ≫ 2,5-dimethylbenzyl deriva-
tives > 2,5-dichlorobenzyl derivatives > 2-methyl-5-trifluor-
omethylbenzyl derivatives > n-hexyl derivatives. The electron-
donating groups on the 2,5-positions of the benzyl ring seemed
to be beneficial for the biological activity. Flexible alkyl chain
replacing benzyl moiety dramatically decreased the activity,
suggesting that the benzyl moiety is critical for the activity.
Additionally, 4-methoxybenzamide and 4-iodobenzamide ana-
logues showed better inhibition potency than the other four
amide derivatives including 4-bromobenzamide, 3,4-dimethox-
ybenzamide, 2-naphthylamide, and 3,4-methylenedioxybenza-
mide. This new combination of A and B moieties generated
several potent compounds 19−24 with IC50 values at
subnanomolar concentrations. Further SAR study of C and D
moieties revealed the importance of the methyl group and
methylsulfonylamide at the C and D positions, respectively.
Removal of the N-methyl group in compounds 19−22 and 24
to afford 25−28 and 30 resulted in 3- to 10-fold loss in
inhibitory potency, with the exception that compound 29
showed slightly better potency than 23. The trifluoromethyl-
sulfonamide group replacing the methylsulfonamide group
significantly impaired the cell proliferation inhibitory activity as
shown in Table 2. In the case of 2,5-dimethylbenzyl derivatives,
trifluoromethylsulfonamides 37−42 exhibited 35- to 207-fold
lower potency than the corresponding methylsulfonamides 43−
48. Similarly for 2,5-dimethoxybenzyl derivatives, trifluoro-
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a
Scheme 1. Synthesis of Compounds 1−42
a
Reagents and conditions: (a) R1X, DMF, X = Cl, Br, or I; (b) (1) MsCl, NaH, DMF, (2) NaOH, MeOH; (c) Tf2O, NaH, Et2O; (d) MeI, NaH,
DMF; (e) FeCl3, Zn, DMF/H2O; (f) R2COCl, K2CO3, 1,4-dioxane.
methyl sulfonamides 31−36 are 12- to 282-fold less potent
than the corresponding methyl sulfonamides 19-24.
2.2. Cell Cycle Studies. The lead compound 48 was
identified to be dual Hsp27 and tubulin binder24 and exhibited
significant cell cycle arrest activity in our previous studies.25
Some of the newly generated derivatives such as 19−24
displayed significantly improved cytotoxicity. It is necessary to
determine if the new potent derivatives still retain the
antimitotic activity of the lead compound, which is charac-
terized by G2/M phase cell accumulation.25 Two representative
compounds 20 and 22 were examined to elucidate the
anticancer mechanism. Both compounds inhibited SKBR-3
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Table 2. SAR of A and B Moiety
Trifluoromethylsulfonamides
Table 3. Previous Developed Dual Hsp27 and Tubulin
Binders
Table 4. Summary of Altered Cell Cycle Distribution in
a
Response to Treatment with 20 and 22
concn
DMSO
sub-G1 (%)
G1 (%)
S (%)
G2/M (%)
3.23
17.10
27.43
33.54
14.73
22.02
24.27
32.88
69.63
61.21
34.91
32.02
66.68
60.08
51.84
31.22
16.34
8.71
8.92
8.83
8.45
9.49
9.51
11.39
10.77
12.78
28.57
25.47
9.24
1.0 nM 20
2.5 nM 20
5.0 nM 20
1.0 nM 22
2.5 nM 22
5.0 nM 22
50.0 nM 22
8.10
13.76
24.32
a
cell proliferation with IC50 of around 2 nM. When the cells
were treated with 1 nM of the two compounds, a significant
amount of cells were halted at sub-G1 phase after 24 h,
suggesting cell apoptosis. When the dosage was increased to 2.5
and 5 nM for compound 20, cells started to accumulate at G2/
M phase as well as sub-G1 phase after 24 h (Table 4). However,
for compound 22, 2.5 and 5 nM treatment clearly induced sub-
G1 phase cell accumulation after 24 h, but a significant G2/M
phase cell accumulation was only observed at 50 nM. The
results suggest that compounds 20 and 22 displayed different
antimitotic potency, and compound 20 is possibly more active
to target tubulin. Apparently, the structural optimization
improved the antimitotic effect of the lead compound 48 that
affected the cell cycle at 200 nM.25 Particularly, compound 20
showed cell cycle arrest activity at a concentration as low as 2.5
nM. In addition, both compounds at subnanomolar concen-
trations clearly induced sub-G1 phase cell accumulation,
suggesting that both compounds exhibited potent cell apoptosis
SKBR-3 cells were treated for 24 h with the indicated concentrations
of compounds. Cells were processed for FACS using propidium iodide
staining as described. The percent distribution of cells in each cell
cycle phase is displayed.
inducing activity. To confirm this effect, we checked caspase 3
and its downstream protein poly ADP-ribose polymerase
(PARP) after the treatment with the two compounds. The
protein levels of cleaved caspase 3, and cleaved PARP were
remarkably increased after a 24 h treatment of SKBR-3 cells
with compounds 20 and 22 (Figure 2). The results indicate
that these small molecules can promote caspase 3 activation
and thereby induce cell apoptosis.
2.3. Inhibition of Tubulin Polymerization. Lead
compound 48 in a previous study inhibited tubulin polymer-
ization in the enzyme and live-cell assays.24 Compared to
compound 48 and several previously synthesized analogues, the
new derivatives 20 and 22 in this study displayed much more
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Figure 2. Compounds 20 and 22 induced SKBR-3 cell apoptosis. SKBR-3 cells were treated with DMSO and compounds 20 and 22 for 24 h.
Apoptosis was characterized by Western blot analysis of cleaved caspase 3 and cleaved PARP as described in Experimental Section.
Figure 3. Tubulin polymerization in the presence of different concentrations of compounds 20, 22, 44, 46, 49 and nimesulide.
potent antimitotic activity in subnanomolar concentrations. We
wonder if the cell cycle arrest activity is correlated with the
tubulin polymerization inhibitory potency. Therefore, com-
pounds 20 and 22 as well as their respective structural
analogues 44 and 46 were examined with the tubulin
polymerization assay to analyze the correlation. As shown in
Figure 3, all four compounds inhibited tubulin polymerization
in a dose dependent manner. In 15 min, compound 44
inhibited tubulin polymerization by 8% at 100 nM and 25% at 1
μM. Compared to 44, compound 20 displayed more potent
tubulin polymerization inhibition by 5% at 10 nM, 19% at 100
nM, and 66% at 1 μM. Compound 22 showed improved
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Figure 4. Hsp27 chaperone activity for the protection of DTT induced insulin aggregation in the presence of different concentrations of compounds
20, 22, 44, 46, 49 and nimesulide. The kinetics of the DTT reduction-induced insulin aggregation was monitored in the absence of Hsp27 or in the
presence of Hsp27 without or with compounds in triplicate.
activity compared to compound 46 as well. 22 and 46 at 1 μM
inhibited tubulin polymerization by 64% and 34%, respectively.
The results suggest that the structural modification by
substitution of 2,5-dimethylbenzyl group in compounds 44
and 46 with 2,5-dimethoxybenzyl group obviously increased
their tubulin inhibitory activity. Moreover, 4-methoxybenzox-
yamides at 100 nM showed more potent tubulin inhibition than
the corresponding 4-iodobenzoxyamides, which is demonstra-
ted by 20 versus 22 and 44 versus 46. The tubulin inhibitory
activities of compounds 20 and 22 match the cell cycle arrest
activities very well. To summarize the tubulin inhibitory activity
of the series of compounds, the earlier version lead compound
and the first generation compound 49 (JCC76)27,28 was also
examined, and it slightly inhibited tubulin polymerization by
5% at 5 μM. The very first lead compound cyclooxygenase 2
(COX-2) inhibitor nimesulide28 showed about 14% tubulin
inhibition at 100 μM (Figure 3). This suggests that along the
lead optimization, the tubulin targeting effects of the
compounds have significantly improved.
2.4. Hsp27 Targeting Effect Investigation. Although the
lead compound 48 binds to Hsp27 and tubulin, it is still unclear
if the compound affects Hsp27 chaperone activity.24 It is well
documented that Hsp27 plays an important role in the
prevention of cell apoptosis and effectively prevents protein
aggregation.8,13 The cellular protective functions of Hsp27 in
the apoptotic pathway are regulated by its chaperone activity,
and this activity contributes to the protection of cells from
stress stimuli.14 Therefore, we examined how our new
derivatives and previous lead compounds regulate Hsp27
chaperone function in this study. Insulin is often used as a
model substrate protein to evaluate the chaperone activity of
small heat shock proteins. Dithiothreitol (DTT) reduction of
insulin can induce its B chain to aggregate. However in the
presence of Hsp27, the aggregation of insulin B chain can be
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attenuated or even completely prevented because of the
formation of stable complexes between Hsp27 and the
unfolded B chain.29−31 The capabilities of our compounds to
modulate the in vitro chaperone function of Hsp27 were
evaluated by monitoring the DTT-induced insulin aggregation
in the presence of Hsp27, with or without our compounds. In
the in vitro chaperone activity assays, Hsp27 exhibited strong
potency against DTT-induced insulin aggregation, which was
consistent with the results of other investigations.29−31 Our
compounds showed inhibitory activity against Hsp27 chaper-
one activity in the insulin aggregation assay (Figure 4). More
specifically, compound 22 at 10 μM significantly inhibited
Hsp27 functions by 27%. However, compound 20 only slightly
inhibited Hsp27 by 9% at 10 μM. The second generation
compounds 44 and 46 exhibited similar Hsp27 inhibition.
Compound 44 at 10 μM significantly inhibited Hsp27
chaperone activity by 30%. And compound 46 at 10 μM
inhibited Hsp27 function by 28%. The results suggest that the
structural modification dissected the tubulin and Hsp27
inhibition on some of the third generation compounds.
Compound 20 showed strong tubulin inhibition but weak
Hsp27 inhibition. The second-generation compounds 44 and
46 showed similar Hsp27 inhibition compared to the third
generation compound 22 but exhibited relatively weaker
tubulin inhibition. The compounds were much less active
than third generation compounds in the cell proliferation assay.
At 5 μM, first generation compound 49 showed strong Hsp27
inhibition by 29% and minimal tubulin inhibitory activity,
which led to the lower cytotoxicity with an IC50 of 3.17 μM. In
fact, compound 49 was the most potent Hsp27 inhibitor within
the study and showed selective cytotoxicity to human epidermal
growth factor receptor 2 (Her2) overexpressed breast cancer
cells in a previous investigation,32 which might be related to the
Hsp27 and Her2 pathway interaction.33,34 The parental
compound nimesulide did not show any Hsp27 inhibitory
activity at 10 μM. Moreover, all these compounds were
evaluated in this assay in the absence of Hsp27 and the results
demonstrated that the compounds exhibited no or minimal
direct chemical chaperone activity on DTT-induced insulin
aggregation.
Subsequently, we examined how the third generation
compounds regulated Hsp27 in cancer cells. We first checked
the levels of phosphorylated Hsp27 (pHsp27) and total Hsp27
in SKBR-3 breast cancer cells after a 24 h treatment with 20
and 22. pHsp27 levels were significantly increased by both
compounds at concentrations close to their IC50 values to
inhibit cell growth (Figure 5). Since compound 20 did not
show significant Hsp27 inhibition at 10 μM in the enzymatic
assay but still stimulated cellular pHsp27, we speculate that the
increased pHsp27 is not due to the Hsp27 direct targeting
effect. The tubulin inhibition of these compounds might be
responsible for the pHsp27 up-regulation, which has been
demonstrated by other tubulin inhibitors such as vinblastine
and paclitaxel.35 Cells activate pHsp27 as a defending
mechanism after being stressed by tubulin inhibitors. The
results suggest that compound 22 did not directly change
Hsp27 total protein, and the up-regulated Hsp27 phosphor-
ylation was due to the tubulin effect, even the compound
inhibited Hsp27 chaperone activity. Hsp27 has chaperone
activity and can stabilize its client proteins. As an Hsp27 binder,
compound 22 might affect the cellular Hsp27 chaperone
function and decrease the stability of the Hsp27 client proteins.
We checked several representative proteins including Her2,18
Figure 5. Effect of compounds 20 and 22 on Hsp27 and several
Hsp27 interactive proteins. SKBR-3 cells were treated with DMSO
and compounds 20 and 22 for 24 h. Levels of pHsp27, Hsp27, Her2,
Stat3, eIF4E, and Hsp70 were analyzed by Western blot of cell extracts
with specific antibodies as described in Experimental Section. The
bands for pHsp27 were quantified using ImageJ (NIH) and
normalized to β-actin.
eukaryotic translation initiation factor 4E (eIF4E),36 signal
transducer and activator of transcription 3 (Stat3),37 and 70
kDa heat shock protein (Hsp70),38 which have been reported
to interact with Hsp27. These proteins in SKBR-3 cells were
determined via Western blot after the treatment with
compounds 20 and 22. The results indicated that the protein
levels of Her2, eIF4E, Stat3, and Hsp70 remained nearly
constant after the treatment, which suggests that our Hsp27
inhibitor did not affect the stability of Her2, eIF4E, Stat3, and
Hsp70 in SKBR-3 cells. As a general chaperone protein, Hsp27
has many client proteins. It is very desirable to identify some of
these proteins that are sensitive to Hsp27 small molecule
inhibitors, which can be used as a biomarker for Hsp27
inhibitor characterization. Although we did not find such a
biomarker within the four Hsp27 client proteins, further
investigation will be executed when more Hsp27 client proteins
are discovered.
So far, our dual tubulin and Hsp27 inhibitor development
has gone through three major stages, which can be represented
by the three generations of compounds (Figure 6). The first
generation compound 49 (JCC76)27,28 was developed based on
the COX-2 inhibitor nimesulide and showed promising
antiproliferative activity with an IC50 of 3.17 μM. Examination
of 49 with tubulin and Hsp27 enzymatic assays revealed that
the compound mainly inhibited Hsp27 activity. The parental
compound nimesulide did not show significant activities in the
tubulin and Hsp27 assays. Apparently, the structural
modification based on nimesulide as a platform led to novel
tubulin and Hsp27 dual inhibitor, which was totally
independent of COX-2 activity. The further ligand-based
optimization of compound 49 resulted in the second
generation of derivatives including compounds 43−48, which
exhibited significantly improved cell growth inhibitory activity
with IC50 of 0.1−0.2 μM.25 The mechanism investigation
demonstrated that the tubulin inhibition of compounds 44 and
46 was greatly improved. And the Hsp27 chaperone inhibition
of 44 and 46 was decreased compared to 49. In the current
study, we further optimized the second generation of
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that the Hsp27 effect of compound 20 was almost diminished,
which was accompanied by a dramatic increase in tubulin
inhibition. However, compound 22 still retained similar Hsp27
inhibition and also obtained a significant increase in tubulin
inhibition compared to its second generation analogue 46. The
dual targeting effects of compound 22 have made it the most
active analogue in the current study. Unexpectedly, we
successfully dissected the two activities on compound 20.
2.5. Broad Anticancer Activities. Because of the higher
cytotoxic characteristics of compounds 20 and 22 on SKBR-3
breast cancer cells, they were submitted to the Developmental
Therapeutic Program at the National Cancer Institute for
screening against 60 human cancer cell lines, representing
leukemia, melanoma, and cancers of the lung, colon, CNS,
ovary, renal, prostate, and breast. Sixty cell lines from each class
of cancer cells were incubated for 48 h with compounds 20 and
22 of various concentrations ranging from 10 nM to 100 μM.
GI50 (50% of growth inhibition), TGI (total growth inhibition),
and LC50 (loss of 50% of the initial cell protein) were calculated
and summarized (Supporting Information). Compounds 20
and 22 exhibited extensive anticancer activity by inducing 50%
of growth inhibition of most cell lines at concentrations less
than 10 nM. These data (Supporting Information) clearly
demonstrate the in vitro efficacy of compounds 20 and 22.
2.6. Investigation of the Combination Treatment with
Hsp27 Inhibitor and Tubulin Inhibitor. Hsp27 modulators
exhibited synergistic anticancer effect when they were
combined with other chemotherapeutic agents.4,16,21,22,39,40
On the basis of our mechanism investigation, the first
generation compound 49 mainly targeted Hsp27 chaperone
activity, and the third generation compound 20 was almost a
pure tubulin inhibitor. It would be interesting to see if the
combination of compounds 49 and 20 could show an additive
effect to inhibit cancer cell proliferation. Compound 49 at 0.5,
1.0, and 2.0 μM significantly shifted the cell growth inhibition
curve of compound 20 to the left, suggesting that compound
49 could promote the activity of compound 20 (Figure 7A)
despite that compound 49 alone did not show any cytotoxicity
at 0.5 and 1.0 μM (Figure 7B). Similarly, when we combined
compound 20 at 1.0, 1.5, and 2.0 nM with various
concentrations of compound 49, the cell growth inhibition
curve of 49 was shifted to the left, suggesting that compound
20 could potentiate the activity of 49 as well (Figure 7B). The
results further demonstrated the synergistic anticancer
mechanism of the first generation compound 49 and third
generation compound 20.
Figure 6. Development of potent anticancer agents from COX-2
inhibitor nimesulide to compound 20.
compounds and generated a series of extremely potent
antiproliferative agents such as compounds 20 and 22 with
IC50 of around 2 nM. The mechanism investigation revealed
Figure 7. First generation compound 49 and third generation compound 20 combination treatment on SKBR-3 breast cancer cell proliferation. (A)
Compound 49 at 0.5, 1.0, and 2.0 μM shifted compound 20 cell proliferation inhibition curve to the left, suggesting the additive effect of the two
compounds. (B) Compound 20 at 1, 1.5, and 2.0 nM shifted compound 49 cell proliferation inhibition curve to the left.
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3. CONCLUSION
Article
mm × 150 mm, 5 μm) from Phenomenex (Torrance, CA). Two
mobile phases (H2O/CH3OH, H2O/CH3CN) were employed for
isocratic elution with a flow rate of 0.2 mL/min. The injection volume
was 20 μL, and the UV detector was set up at 256 and 290 nm.
Intermediate compounds 1a−e, 2a−d, 3a−d, 3d′, and 4a−d were
prepared according to the previously published procedures.25
We developed a series of potent anticancer agents with IC50
values at subnanomolar levels to inhibit the proliferation of
various cancer cell lines. Several new compounds such as 19−
22 are 57- to 230-fold more active than previous lead
compound 48. The significantly increased anticancer activity
is mainly attributed to the structural modification by
substitution of 2,5-dimethylbenzyl moiety in the lead
compound with a 2,5-dimethoxybenzyl group.
Trifluoromethanesulfonamides 2e−f were prepared from aryl-
substituted 2-amino-5-nitrophenols 1d−e according to a modified
procedure. NaH (95% powder, 0.211g, 8.81 mmol) was added to a
solution of aryl-substituted 2-amino-5-nitrophenol 1d−e (3.67 mmol)
in anhydrous Et2O (70 mL) at room temperature. After being stirred
for 20 min, the mixture was cooled to 0 °C and Tf2O (1.24 g, 4.4
mmol) was slowly added. The resulting mixture was continuously
stirred for 1 h at 0−5 °C. Water (15 mL) was added to quench the
reaction. Ether was evaporated under vacuum, and then 3 N HCl (4
mL) was added to acidify the residue. The intermediates 2e−f
precipitated as a yellow solid. It was collected by filtration, washed with
water, dried in air, and then used for the next reaction directly.
According to the same procedures used for preparation of
intermediates 4a−d, 2e−f was subjected to N-methylation to afford
3e−f followed by reduction of nitro group to give 4e−f.
N-Methyl-N-[2-(2,5-dimethoxybenzyloxy)-4-nitrophenyl]-
trifluoromethanesulfonamide (3e). Yellow solid, 59% yield for the
two steps of sulfonylamidation and N-methylation; H NMR (400
MHz, CDCl3) δ 8.038 (1H, d, J = 2.4 Hz), 7.849 (1H, dd, J = 2.4, 8.8
Hz), 7.493 (1H, d, J = 8.8 Hz), 7.036 (1H, s), 6.876 (2H, m), 5.293
(2H, s), 3.889 (3H, s), 3.775 (3H, s), 3.414 (3H, s).
N-Methyl-N-[2-(2,5-dimethylbenzyloxy)-4-nitrophenyl]-
trifluoromethanesulfonamide (3f). Yellow solid, 78% yield for the
two steps of sulfonylamidation and N-methylation; H NMR (400
MHz, CDCl3) δ 7.974 (1H, d, J = 2.4 Hz), 7.895 (1H, dd, J = 2.4, 8.4
Hz), 7.531 (1H, d, J = 8.4 Hz), 7.242 (1H, s), 7.143 (2H, m), 5.195
(2H, s), 3.365 (3H, s), 2.374 (3H, s), 2.345 (3H, s).
N-[4-Amino-2-(2,5-dimethoxybenzyloxy)phenyl]-N-
methyltrifluoromethanesulfonamide (4e). Pale white solid, 96%
yield; 1H NMR (400 MHz, CDCl3) δ 7.115 (1H, s), 7.062 (1H, d, J =
8.4 Hz), 6.827 (2H, m), 6.315 (1H, d, J = 2.4 Hz), 6.217 (1H, dd, J =
2.4, 8.8 Hz), 5.101 (2H, s), 3.822 (3H, s), 3.817 (2H, br), 3.783 (3H,
s), 3.359 (3H, s).
N-[4-Amino-2-(2,5-dimethylbenzyloxy)phenyl]-N-methyltrifluoro-
methanesulfonamide (4f). Pale white solid, 98% yield; 1H NMR (400
MHz, CDCl3) δ 7.257 (1H, s), 7.084 (3H, m), 6.319 (1H, d, J = 2.4
Hz), 6.234 (1H, dd, J = 2.4, 8.4 Hz), 5.006 (2H, s), 3.847 (2H, br),
3.312 (3H, s), 2.332 (3H, s), 2.315 (3H, s).
The final compounds 1−42 were prepared from the reaction of the
corresponding substituted anilines (4a−f and 3d′) with each
arylcarbonyl chloride including 4-bromobenzoyl chloride, 4-methox-
ybenzoyl chloride, 3,4-dimethoxybenzoyl chloride, 4-iodobenzoyl
chloride, 2-naphthoyl chloride, and piperonyloyl chloride.
These compounds mainly targeted tubulin polymerization to
inhibit cancer cell proliferation. Their Hsp27 inhibition also
contributed to their significant anticancer activity. The
conclusion is based on the results of tubulin and Hsp27
enzymatic assays. In the tubulin polymerization inhibition and
Hsp27 chaperone activity assays, two pairs of compounds, 20
versus 44 and 22 versus 46, were evaluated. The only structural
difference in each pair of compounds is the 2,5-dimethox-
ybenzyl moiety in the former and the 2,5-dimethylbenzyl
moiety in the latter compounds. The results indicated that the
new analogues 20 and 22 exhibited much better tubulin
inhibition compared to 44 and 46, which led to the dramatic
increase in their anticancer activity. Unexpectedly, the structural
modification significantly decreased the Hsp27 inhibition of
compound 20. We fortunately dissected the dual anticancer
activities on one of the third generation of compounds.
Compound 22 inhibited Hsp27 at a similar level as 46, and the
dual targeting effect made this compound the most potent one
in this series of new derivatives, even if it was less active than
compound 20 to inhibit tubulin polymerization. The
combination of the 4-methoxylbenzamide moiety and the 2,5-
dimethoxybenzyl moiety of compound 20 improved the tubulin
inhibitory activity and decreased the Hsp27 chaperone function
inhibition. The SAR summarized in the study is very important
for further drug optimization to generate more potent and
selective inhibitors. Particularly, potent Hsp27 inhibitors can
provide a solution to solve the drug resistance issues of many
chemotherapeutic agents16,22,36 and have great clinical
application potential. Future study will focus on discerning
the structural fragments, which are important for Hsp27 effects,
developing more selective and potent Hsp27 inhibitors, and
investigating the consequence of Hsp27 modulation. The
investigation of how Hsp27 inhibitors regulate Hsp27
oligomerization, its chaperone functions, and the activity/
stability of its client proteins could provide important
information to elucidate their anticancer mechanisms.
1
1
N-[3-(2,5-Dichlorobenzyloxy)-4-(methanesulfonylmethylamino)-
phenyl]-4-bromobenzamide (1). White solid, 65% yield; H NMR
1
(400 MHz, CDCl3) δ 8.153 (1H, s), 7.795 (1H, s), 7.778 (2H, d, J =
8.4 Hz), 7.635 (2H, d, J = 8.4 Hz), 7.553 (1H, d, J = 2 Hz), 7.365 (1H,
d, J = 8.4 Hz), 7.303 (2H, m), 6.946 (1H, dd, J = 2, 8.4 Hz), 5.086
(2H, s), 3.252 (3H, s), 2.878 (3H, s). ESI-MS calculated for
C22H20BrCl2N2O4S [M + H]+, 556.97; found, 556.94.
4. EXPERIMENTAL SECTION
4.1. Chemistry. Chemicals were commercially available and used
as received without further purification unless otherwise noted.
Moisture sensitive reactions were carried out under a dry argon
atmosphere in flame-dried glassware. Solvents were distilled before use
under argon. Thin-layer chromatography was performed on precoated
silica gel F254 plates (Whatman). Silica gel column chromatography
was performed using silica gel 60 Å (Merck, 230−400 mesh), and
hexane/ethyl acetate was used as the elution solvent. Mass spectra
were obtained on a Micromass quadrupole time-of-flight (QTOF)
electrospray mass spectrometer at Cleveland State University MS
facility center. All the NMR spectra were recorded on a Bruker 400
MHz instrument in either DMSO-d6 or CDCl3. Chemical shifts (δ) for
1H NMR spectra are reported in parts per million to residual solvent
protons. The purity of the final compounds was determined via HPLC
(Beckman) analysis with different mobile phases (Supporting
Information). All the final compounds exhibited purities above 95%.
The chromatographic separation was performed on a C18 column (2.0
N-[3-(2,5-Dichlorobenzyloxy)-4-(methanesulfonylmethylamino)-
1
phenyl]-4-methoxybenzamide (2). White solid, 70% yield; H NMR
(400 MHz, CDCl3) δ 7.959 (1H, s), 7.919 (1H, d, J = 2 Hz), 7.860
(2H, d, J = 9.2 Hz), 7.550 (1H, d, J = 2.4 Hz), 7.311 (3H, m), 6.988
(2H, d, J = 8.8 Hz), 6.903 (1H, dd, J = 2.4, 8.4 Hz), 5.164 (2H, s),
3.884 (3H, s), 3.249 (3H, s), 2.850 (3H, s). ESI-MS calculated for
C23H23Cl2N2O5S [M + H]+, 509.07; found, 509.11.
N-[3-(2,5-Dichlorobenzyloxy)-4-(methanesulfonylmethylamino)-
phenyl]-3,4-dimethoxybenzamide (3). White solid, 86% yield; 1H
NMR (400 MHz, CDCl3) δ 8.047 (1H, s), 7.925 (1H, d, J = 2.4 Hz),
7.548 (1H, d, J = 2.4 Hz), 7.502 (1H, d, J = 2 Hz), 7.435 (1H, dd, J =
2, 8.4 Hz), 7.334 (3H, m), 6.911 (2H, m), 5.152 (2H, s), 3.960 (3H,
s), 3.955 (3H, s), 3.250 (3H, s), 2.858 (3H, s). ESI-MS calculated for
C24H25Cl2N2O6S [M + H]+, 539.08; found, 539.12.
5315
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Article
N-[3-(2,5-Dichlorobenzyloxy)-4-(methanesulfonylmethylamino)-
phenyl]-4-iodobenzamide (4). White solid, 85% yield; 1H NMR (400
MHz, DMSO-d6) δ 7.941 (2H, m), 7.773 (1H, d, J = 2.4 Hz), 7.745
(2H, m), 7.681 (1H, d, J = 2.4 Hz), 7.593 (1H, d, J = 8.4 Hz), 7.501
(1H, dd, J = 2.8, 8.4 Hz), 7.464 (1H, dd, J = 2.4, 8.4 Hz), 7.329 (1H, d,
J = 8.4 Hz), 5.215 (2H, s), 3.139 (3H, s), 2.931 (3H, s). ESI-MS
calculated for C22H20Cl2IN2O4S [M + H]+, 604.96; found, 604.95.
N-[3-(2,5-Dichlorobenzyloxy)-4-(methanesulfonylmethylamino)-
= 8 Hz), 7.330 (1H, d, J = 8.4 Hz), 6.835 (1H, dd, J = 2.4, 8.4 Hz),
5.160 (2H, s), 3.228 (3H, s), 2.783 (3H, s), 2.457 (3H, s). ESI-MS
calculated for C24H23BrF3N2O4S [M + H]+, 571.05; found, 571.04.
N-[3-(2-Methyl-5-trifluoromethylbenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-4-methoxybenzamide (14). White solid, 97%
yield; 1H NMR (400 MHz, CDCl3) δ 8.081 (1H, d, J = 2.4 Hz), 7.959
(1H, s), 7.860 (2H, m), 7.702 (1H, s), 7.543 (1H, d, J = 8 Hz), 7.362
(1H, d, J = 8 Hz), 7.330 (1H, d, J = 8.8 Hz), 6.990 (2H, m), 6.815
(1H, dd, J = 2, 8.4 Hz), 5.163 (2H, s), 3.883 (3H, s), 3.224 (3H, s),
2.770 (3H, s), 2.455 (3H, s). ESI-MS calculated for C25H26F3N2O5S
[M + H]+, 523.15; found, 523.15.
1
phenyl]-2-naphthalenecarboxamide (5). White solid, 98% yield; H
NMR (400 MHz, CDCl3) δ 8.413 (1H, s), 8.264 (1H, s), 7.948 (5H,
m), 7.579 (3H, m), 7.360 (2H, m), 7.291 (1H, dd, J = 2.8, 8.8 Hz),
6.988 (1H, dd, J = 2, 8.4 Hz), 5.164 (2H, s), 3.262 (3H, s), 2.861 (3H,
s). ESI-MS calculated for C26H23Cl2N2O4S [M + H]+, 529.08; found,
529.07.
N-[3-(2-Methyl-5-trifluoromethylbenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-3,4-dimethoxybenzamide (15). White solid,
1
91% yield; H NMR (400 MHz, CDCl3) δ 8.087 (1H, s), 8.032 (1H,
s), 7.707 (1H, s), 7.534 (2H, m), 7.432 (1H, d, J = 8 Hz), 7.345 (2H,
m), 6.922 (1H, d, J = 8 Hz), 6.826 (1H, d, J = 8.4 Hz), 5.161 (2H, s),
3.965 (3H, s), 3.955 (3H, s), 3.227 (3H, s), 2.775 (3H, s), 2.453 (3H,
s). ESI-MS calculated for C26H28F3N2O6S [M + H]+, 553.16; found,
553.15.
N-[3-(2,5-Dichlorobenzyloxy)-4-(methanesulfonylmethylamino)-
phenyl]-1,3-benzodioxole-5-carboxamide (6). White solid, 84%
1
yield; H NMR (400 MHz, CDCl3) δ 7.953 (1H, s), 7.864 (1H, d,
J = 2.4 Hz), 7.548 (1H, d, J = 2.4 Hz), 7.428 (1H, dd, J = 1.6, 8 Hz),
7.370 (2H, m), 7.311 (2H, m), 6.898 (2H, m), 6.067 (2H, s), 5.143
(2H, s), 3.247 (3H, s), 2.860 (3H, s). ESI-MS calculated for
C23H21Cl2N2O6S [M + H]+, 523.05; found, 523.09.
N-[3-(2-Methyl-5-trifluoromethylbenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-4-iodobenzamide (16). White solid, 87%
1
yield; H NMR (400 MHz, CDCl3) δ 8.064 (1H, s), 8.018 (1H, d,
N-[3-(n-Hexyloxy)-4-(methanesulfonylmethylamino)phenyl]-4-
1
bromobenzamide (7). White solid, 60% yield; H NMR (400 MHz,
J = 2.4 Hz), 7.855 (2H, d, J = 8.4 Hz), 7.703 (1H, s), 7.616 (2H, d, J =
8.8 Hz), 7.546 (1H, d, J = 8.4 Hz), 7.363 (1H, d, J = 7.6 Hz), 7.314
(1H, d, J = 8.4 Hz), 6.833 (1H, dd, J = 2.4, 8.4 Hz), 5.149 (2H, s),
3.225 (3H, s), 2.781 (3H, s), 2.451 (3H, s). ESI-MS calculated for
C24H23F3IN2O4S [M + H]+, 619.04; found, 619.02.
CDCl3) δ 8.042 (1H, s), 7.836 (1H, s), 7.766 (2H, d, J = 8.4 Hz),
7.644 (2H, d, J = 8.4 Hz), 7.277 (1H, d, J = 8 Hz), 6.755 (1H, d, J = 8
Hz), 4.060 (2H, t, J = 6.4 Hz), 3.269 (3H, s), 2.936 (3H, s), 1.817
(2H, m), 1.467 (2H, m), 1.349 (4H, m), 0.911 (3H, t, J = 6.8 Hz).
ESI-MS calculated for C21H28BrN2O4S [M + H]+, 483.10; found,
483.13.
N-[3-(2-Methyl-5-trifluoromethylbenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-2-naphthalenecarboxamide (17). White
1
solid, 97% yield; H NMR (400 MHz, DMSO-d6) δ 10.580 (1H, s),
N-[3-(n-Hexyloxy)-4-(methanesulfonylmethylamino)phenyl]-4-
methoxybenzamide (8). White solid, 58% yield; 1H NMR (400 MHz,
CDCl3) δ 7.889 (2H, m), 7.850 (2H, d, J = 8.8 Hz), 7.303 (1H, d, J =
8.4 Hz), 6.990 (2H, d, J = 8.4 Hz), 6.742 (1H, dd, J = 2, 8.4 Hz), 4.076
(2H, t, J = 6.4 Hz), 3.885 (3H, s), 3.271 (3H, s), 2.931 (3H, s), 1.821
(2H, m), 1.472 (2H, m), 1.350 (4H, m), 0.911 (3H, t, J = 6.8 Hz).
ESI-MS calculated for C22H31N2O5S [M + H]+, 435.20; found, 435.22.
N-[3-(n-Hexyloxy)-4-(methanesulfonylmethylamino)phenyl]-3,4-
8.592 (1H, s), 8.070 (4H, m), 7.925 (1H, s), 7.833 (1H, d, J = 2 Hz),
7.654 (3H, m), 7.506 (2H, m), 7.351 (1H, d, J = 8.4 Hz), 5.264 (2H,
s), 3.146 (3H, s), 2.909 (3H, s), 2.470 (3H, s). ESI-MS calculated for
C28H26F3N2O4S [M + H]+, 543.16; found, 543.15.
N-[3-(2-Methyl-5-trifluoromethylbenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-1,3-benzodioxole-5-carboxamide (18). White
solid, 84% yield; 1H NMR (400 MHz, CDCl3) δ 8.032 (1H, s), 7.943
(1H, s), 7.701 (1H, s), 7.542 (1H, d, J = 8 Hz), 7.371 (4H, m), 6.888
(1H, d, J = 8 Hz), 6.812 (1H, d, J = 8 Hz), 6.065 (2H, s), 5.153 (2H,
s), 3.223 (3H, s), 2.780 (3H, s), 2.453 (3H, s). ESI-MS calculated for
C25H24F3N2O6S [M + H]+, 537.13; found, 537.17.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-4-bromobenzamide (19). White solid, 91%
yield; 1H NMR (400 MHz, CDCl3) δ 7.933 (1H, s), 7.875 (1H, d, J =
2 Hz), 7.759 (2H, m), 7.647 (2H, m), 7.327 (1H, d, J = 8.4 Hz), 7.012
(1H, d, J = 2.8 Hz), 6.882 (1H, d, J = 2 Hz), 6.857 (2H, m), 5.130
(2H, s), 3.783 (3H, s), 3.779 (3H, s), 3.234 (3H, s), 2.816 (3H, s).
ESI-MS calculated for C24H26BrN2O6S [M + H]+, 549.07; found,
549.12.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-4-methoxybenzamide (20). White solid, 60%
yield; 1H NMR (400 MHz, CDCl3) δ 7.932 (1H, d, J = 2.4 Hz), 7.863
(3H, m), 7.327 (1H, d, J = 8.4 Hz), 7.004 (3H, m), 6.853 (3H, m),
5.138 (2H, s), 3.886 (3H, s), 3.782 (6H, s), 3.232 (3H, s), 2.807 (3H,
s). ESI-MS calculated for C25H29N2O7S [M + H]+, 501.17; found,
501.21.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-3,4-dimethoxybenzamide (21). White solid,
80% yield; 1H NMR (400 MHz, CDCl3) δ 7.942 (1H, d, J = 2.4 Hz),
7.925 (1H, s), 7.504 (1H, d, J = 2 Hz), 7.411 (1H, dd, J = 2, 8.4 Hz),
7.332 (1H, d, J = 8.4 Hz), 7.016 (1H, d, J = 2.4 Hz), 6.928 (1H, d, J =
8.4 Hz), 6.861 (3H, m), 5.138 (2H, s), 3.968 (3H, s), 3.961 (3H, s),
3.783 (3H, s), 3.781 (3H, s), 3.236 (3H, s), 2.810 (3H, s). ESI-MS
calculated for C26H31N2O8S [M + H]+, 531.18; found, 531.23.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-4-iodobenzamide (22). White solid, 62%
yield; 1H NMR (400 MHz, CDCl3) δ 7.940 (1H, s), 7.872 (3H,
m), 7.608 (2H, m), 7.323 (1H, d, J = 8.4 Hz), 7.011 (1H, d, J = 2.8
Hz), 6.879 (1H, d, J = 2.4 Hz), 6.854 (2H, m), 5.127 (2H, s), 3.782
(3H, s), 3.778 (3H, s), 3.232 (3H, s), 2,814 (3H, s). ESI-MS calculated
for C24H26IN2O6S [M + H]+, 597.06; found, 597.11.
1
dimethoxybenzamide (9). White solid, 75% yield; H NMR (400
MHz, CDCl3) δ 7.914 (2H, m), 7.503 (1H, d, J = 2 Hz), 7.406 (1H,
dd, J = 2, 8.4 Hz), 7.313 (1H, d, J = 8.4 Hz), 6.929 (1H, d, J = 8.4 Hz),
6.751 (1H, dd, J = 2.4, 8.4 Hz), 4.084 (2H, t, J = 6.8 Hz), 3.966 (3H,
s), 3.961 (3H, s), 3.277 (3H, s), 2.937 (3H, s), 1.828 (2H, m), 1.476
(2H, m), 1.353 (4H, m), 0.913 (3H, t, J = 6.8 Hz). ESI-MS calculated
for C23H33N2O6S [M + H]+, 465.21; found, 465.23.
N-[3-(n-Hexyloxy)-4-(methanesulfonylmethylamino)phenyl]-4-
iodobenzamide (10). Yellowish solid, 36% yield; 1H NMR (400 MHz,
CDCl3) δ 8.055 (1H, s), 7.851 (3H, m), 7.617 (2H, d, J = 8.4 Hz),
7.271 (1H, d, J = 8.4 Hz), 6.749 (1H, dd, J = 2.4, 8.4 Hz), 4.056 (2H,
t, J = 6.4 Hz), 3.267 (3H, s), 2.933 (3H, s), 1.814 (2H, m), 1.467 (2H,
m), 1.348 (4H, m), 0.911 (3H, t, J = 7.2 Hz). ESI-MS calculated for
C21H28IN2O4S [M + H]+, 531.08; found, 531.13.
N-[3-(n-Hexyloxy)-4-(methanesulfonylmethylamino)phenyl]-2-
naphthalenecarboxamide (11). White solid, 83% yield; 1H NMR
(400 MHz, CDCl3) δ 8.412 (1H, s), 8.297 (1H, s), 7.950 (5H, m),
7.594 (2H, m), 7.296 (1H, d, J = 8.4 Hz), 6.822 (1H, dd, J = 2, 8.4
Hz), 4.066 (2H, t, J = 6.8 Hz), 3.274 (3H, s), 2.922 (3H, s), 1.801
(2H, m), 1.458 (2H, m), 1.332 (4H, m), 0.909 (3H, t, J = 6.8 Hz).
ESI-MS calculated for C25H31N2O4S [M + H]+, 455.20; found, 455.23.
N-[3-(n-Hexyloxy)-4-(methanesulfonylmethylamino)phenyl]-1,3-
benzodioxole-5-carboxamide (12). Yellowish solid, 76% yield; 1H
NMR (400 MHz, CDCl3) δ 7.969 (1H, s), 7.839 (1H, d, J = 2.4 Hz),
7.425 (1H, dd, J = 2, 8.4 Hz), 7.370 (1H, d, J = 2 Hz), 7.269 (1H, d, J
= 8.4 Hz), 6.884 (1H, d, J = 8 Hz), 6.735 (1H, dd, J = 2.4, 8.4 Hz),
6.065 (2H, s), 4.055 (2H, t, J = 6.8 Hz), 3.265 (3H, s), 2.934 (3H, s),
1.811 (2H, m), 1.464 (2H, m), 1.346 (4H, m), 0.909 (3H, t, J = 6.8
Hz). ESI-MS calculated for C22H29N2O6S [M + H]+, 449.17; found,
449.20.
N-[3-(2-Methyl-5-trifluoromethylbenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-4-bromobenzamide (13). White solid, 78%
yield; 1H NMR (400 MHz, CDCl3) δ 8.026 (2H, m), 7.759 (2H, m),
7.704 (1H, s), 7.653 (2H, m), 7.548 (1H, d, J = 8 Hz), 7.365 (1H, d, J
5316
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N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-2-naphthalenecarboxamide (23). White
solid, 83% yield; 1H NMR (400 MHz, CDCl3) δ 8.402 (1H, s),
8.153 (1H, s), 7.947 (5H, m), 7.607 (2H, m), 7.355 (1H, d, J = 8.4
Hz), 7.028 (1H, d, J = 2.8 Hz), 6.940 (1H, dd, J = 2.4, 8.4 Hz), 6.855
(2H, m), 5.159 (2H, s), 3.786 (3H, s), 3.782 (3H, s), 3.248 (3H, s),
2.818 (3H, s). ESI-MS calculated for C28H29N2O6S [M + H]+, 521.17;
found, 521.22.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonyl-
methylamino)phenyl]-1,3-benzodioxole-5-carboxamide (24). White
solid, 66% yield; 1H NMR (400 MHz, CDCl3) δ 7.890 (1H, d, J = 2.4
Hz), 7.857 (1H, s), 7.412 (1H, dd, J = 1.6, 8 Hz), 7.368 (1H, d, J = 1.6
Hz), 7.318 (1H, d, J = 8.4 Hz), 7.012 (1H, d, J = 2.4 Hz), 6.891 (1H,
d, J = 8 Hz), 6.852 (3H, m), 6.070 (2H, s), 5.128 (2H, s), 3.781 (6H,
s), 3.230 (3H, s), 2.810 (3H, s). ESI-MS calculated for C25H27N2O8S
[M + H]+, 515.15; found, 515.20.
7.110 (1H, s), 7.024 (1H, dd, J = 2, 8.4 Hz), 6.834 (1H, s), 6.831 (1H,
s), 5.156 (2H, s), 3.810 (3H, s), 3.779 (3H, s), 3.388 (3H, s). ESI-MS
calculated for C24H23BrF3N2O6S [M + H]+, 603.04; found, 603.00.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-4-methoxybenzamide (32). White solid, 72%
yield; 1H NMR (400 MHz, CDCl3) δ 7.831 (3H, m), 7.727 (1H, d, J =
2 Hz), 7.276 (1H, m), 7.129 (1H, s), 6.991 (3H, m), 6.837 (1H, s),
6.833 (1H, s), 5.183 (2H, s), 3.879 (3H, s), 3.815 (3H, s), 3.791 (3H,
s).3.387 (3H, s). ESI-MS calculated for C25H26F3N2O7S [M + H]+,
555.14; found, 555.13.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-3,4-dimethoxybenzamide (33). White solid,
1
61% yield; H NMR (400 MHz, CDCl3) δ 7.875 (1H, s), 7.750 (1H,
d, J = 2 Hz), 7.485 (1H, d, J = 2 Hz), 7.385 (1H, dd, J = 2, 8.4 Hz),
7.283 (1H, d, J = 8.4 Hz), 7.131 (1H, s), 7.007 (1H, dd, J = 2, 8.4 Hz),
6.908 (1H, d, J = 8.4 Hz), 6.835 (2H, m), 5.184 (2H, s), 3.959 (3H, s),
3.952 (3H,s), 3.813 (3H, s), 3.794 (3H, s), 3.393 (3H, s). ESI-MS
calculated for C26H28F3N2O8S [M + H]+, 585.15; found, 585.13.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-4-iodobenzamide (34). White solid, 74%
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonylamino)-
1
phenyl]-4-bromobenzamide (25). White solid, 80% yield; H NMR
(400 MHz, DMSO-d6) δ 10.37 (1H, s), 9.02 (1H, s), 7.91(2H, d, J =
8.8 Hz), 7.76 (2H, d, J = 8.8 Hz), 7.68 (1H, 2.4 Hz), 7.41 (1H, dd, J =
2.4, 8.8 Hz), 7.28 (1H, d, J = 3.2 Hz), 7.22 (1H, d, J = 8.4 Hz), 6.98
(1H, d, J = 9.2 Hz), 6.88 (1H, dd, J = 3.2, 8.8 Hz), 5.08 (2H, s), 3.79
(3H, s), 3.73 (3H, s), 2.87 (3H, s). ESI-MS calculated for
C23H24BrN2O6S [M + H]+, 535.05; found, 535.04.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonylamino)-
phenyl]-4-methoxybenzamide (26). White solid, 46% yield; 1H NMR
(400 MHz, DMSO- d6) 10.159 (1H, s), 9.009 (1H, s), 7.966 (2H, d, J
= 8.8 Hz), 7.705 (1H, d, J = 2.4 Hz), 7.415 (1H, dd, J = 2, 8.8 Hz),
7.292 (1H, d, J = 3.2 Hz), 7.208 (1H, d, J = 8.8 Hz), 7.073 (2H, d, J =
8.8 Hz), 6.981 (1H, d, J = 9.2 Hz), 6.883 (1H, dd, J = 3.2, 8.8 Hz),
5.077 (2H, s), 3.845 (3H, s), 3.789 (3H, s), 3.730 (3H, s), 2.863 (3H,
s). ESI-MS calculated for C24H27N2O7S [M + H]+, 487.15; found,
487.14.
1
yield; H NMR (400 MHz, CDCl3) δ 7.910 (1H, s), 7.832 (2H, d,
J = 8.4 Hz), 7.662 (1H, s), 7.570 (2H, d, J = 8.4 Hz), 7.273 (1H, d, J =
8.4 Hz), 7.107 (1H, s), 7.017 (1H, dd, J = 2, 8.4 Hz), 6.828 (2H, s),
5.148 (2H, s), 3.806 (3H, s), 3.777 (3H, s), 3.386 (3H, s). ESI-MS
calculated for C24H23F3IN2O6S [M + H]+, 651.03; found, 651.00.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-2-naphthalenecarboxamide (35). White
1
solid, 92% yield; H NMR (400 MHz, DMSO-d6) δ 10.645 (1H, s),
8.604 (1H, s), 8.074 (4H,m), 7.829 (1H, s), 7.658 (2H, m), 7.583
(1H, d, J = 6.4 Hz), 7.389 (1H, d, J = 8.4 Hz), 7.127 (1H, d, J = 3.2
Hz), 7.012 (1H, d, J = 8.8 Hz), 6.918 (1H, dd, J = 3.2, 9.2 Hz), 5.151
(2H, s), 3.803 (3H, s), 3.727 (3H, s), 3.367 (3H, s). ESI-MS calculated
for C28H26F3N2O6S [M + H]+, 575.15; found, 575.13.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonylamino)-
N-[3-(2,5-Dimethoxybenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-1,3-benzodioxole-5-carboxamide (36). White
solid, 69% yield; 1H NMR (400 MHz, CDCl3) δ 7.779 (1H, s), 7.694
(1H, d, J = 2 Hz), 7.389 (1H, dd, J = 1.6, 9.6 Hz), 7.348 (1H, d, J = 1.6
Hz), 7.279 (1H, d, J = 8.4 Hz), 7.122 (1H, s), 7.001 (1H, dd, J = 2, 8.4
Hz), 6.883 (1H, d, J = 8 Hz), 6.839 (1H, s), 6.835 (1H, s), 6.070 (2H,
s), 5.183 (2H, s), 3.819 (3H, s), 3.792 (3H, s), 3.386 (3H, s). ESI-MS
calculated for C25H24F3N2O8S [M + H]+, 569.12; found, 569.10.
N-[3-(2,5-Dimethylbenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-4-bromobenzamide (37). White solid, 81%
1
phenyl]-3,4-dimethoxybenzamide (27). White solid, 36% yield; H
NMR (400 MHz, CDCl3) δ 10.14 (1H, s), 8.99 (1H, s), 7.70 (1H, d, J
= 2.4 Hz), 7.63 (1H, dd, J = 2, 8 Hz), 7.53 (1H, d, J = 2 Hz), 7.39 (1H,
dd, J = 2.4, 8.4 Hz), 7.29 (1H, d, J = 2.8 Hz), 7.22 (1H, d, J = 8.8 Hz),
7.09 (1H, d, J = 8.4 Hz), 6.98 (1H, d, J = 8.4 Hz), 6.88 (1H, dd, J =
3.2, 8.8 Hz), 5.08 (2H, s), 3.85 (3H, s), 3.84 (3H, s), 3.79 (3H, s), 3.73
(3H, s), 2.87 (3H, s). ESI-MS calculated for C25H29N2O8S [M + H]+,
517.16; found, 517.16.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonylamino)-
phenyl]-4-iodobenzamide (28). White solid, 56% yield; 1H NMR
(400 MHz, DMSO-d6) δ δ 10.355 (1H, s), 9.031 (1H, s), 7.933 (2H,
d, J = 8.4 Hz), 7.749 (2H, d, J = 8.4 Hz), 7.678 (1H, d, J = 2.4 Hz),
7.411 (1H, dd, J = 2, 8.8 Hz), 7.282 (1H, d, J = 3.2 Hz), 7.223 (1H, d,
J = 8.4 Hz), 6.979 (1H, d, J = 9.2 Hz), 6.881 (1H, dd, J = 2.8, 8.8 Hz),
5.077 (2H, s), 3.785 (3H, s), 3.726 (3H, s), 2.868 (3H, s). ESI-MS
calculated for C23H24IN2O6S [M + H]+, 583.04; found, 583.02.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonylamino)-
phenyl]-2-naphthalenecarboxamide (29). White solid, 85% yield; 1H
NMR (400 MHz, CDCl3) δ 10.50 (1H, s), 9.03 (1H, s), 8.59 (1H, s),
8.06 (4H, m), 7.75 (1H, d, J = 2 Hz), 7.65 (2H, m), 7.50 (1H, dd, J =
2, 8.4 Hz), 7.30 (1H, d, J = 3.2 Hz), 7.25 (1H, d, J = 8.8 Hz), 6.98
(1H, d, J = 8.8 Hz), 6.89 (1H, dd, J = 3.2, 8.8 Hz), 5.11 (2H, s), 3.80
(3H, s), 3.74 (3H, s), 2.88 (3H, s). ESI-MS calculated for
C27H27N2O6S [M + H]+, 507.16; found, 507.15.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(methanesulfonylamino)-
phenyl]-1,3-benzodioxole-5-carboxamide (30). White solid, 46%
yield; 1H NMR (400 MHz, CDCl3) δ 10.12 (1H, s), 9.00 (1H, s), 7.69
(1H, d, J = 2 Hz), 7.58 (1H, dd, J = 2, 8 Hz), 7.51 (1H, d, J = 1.6 Hz),
7.40 (1H, dd, J = 2.4, 8.8 Hz), 7.28 (1H, d, J = 3.2 Hz), 7.21 (1H, d, J
= 8.8 Hz), 7.07 (1H, d, J = 8.4 Hz), 6.98 (1H, d, J = 9.2 Hz), 6.88 (1H,
dd, J = 3.2, 9.2 Hz), 6.14 (2H, s), 5.07 (2H, s), 3,79 (3H, s), 3.73 (3H,
s), 2.86 (3H, s). ESI-MS calculated for C24H25N2O8S [M + H]+,
501.13; found, 501.13.
N-[3-(2,5-Dimethoxybenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-4-bromobenzamide (31). White solid, 73%
yield; 1H NMR (400 MHz, CDCl3) δ 7.895 (1H, s), 7.724 (2H, d, J =
8.8 Hz), 7.662 (1H, s), 7.623 (2H, d, J = 8.4 Hz), 7.280 (1H, m),
1
yield; H NMR (400 MHz, CDCl3) δ 7.874 (1H, s), 7.835 (1H, s),
7.741 (2H, d, J = 8.8 Hz), 7.647 (2H, d, J = 8.8 Hz), 7.292 (2H, m),
7.098 (2H, m), 6.953 (1H, dd, J = 2.4, 8.4 Hz), 5.103 (2H, s), 3.344
(3H, s), 2.343 (3H, s), 2.335 (3H, s). ESI-MS calculated for
C24H23BrF3N2O4S [M + H]+, 571.05; found, 571.04.
N-[3-(2,5-Dimethylbenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-4-methoxybenzamide (38). White solid, 66%
yield; 1H NMR (400 MHz, CDCl3) δ 7.892 (1H, d, J = 2.4 Hz), 7.844
(3H, m), 7.291 (2H, m), 7.096 (2H, m), 6.994 (2H, m), 6.928 (1H,
dd, J = 2, 8.4 Hz), 5.114 (2H, s), 3.887 (3H, s), 3.339 (3H, s), 2.347
(3H, s), 2.340 (3H, s). ESI-MS calculated for C25H26F3N2O5S [M +
H]+, 523.15; found, 523.14.
N-[3-(2,5-Dimethylbenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-3,4-dimethoxybenzamide (39). White solid,
73% yield; 1H NMR (400 MHz, CDCl3) δ 7.894 (2H, m), 7.493 (1H,
d, J = 2 Hz), 7.393 (1H, dd, J = 2, 8 Hz), 7.297 (2H, m), 7.096 (2H,
m), 6.921 (2H, m), 5.113 (2H, s), 3.966 (3H, s), 3.959 (3H, s), 3.343
(3H, s), 2.341 (6H, s). ESI-MS calculated for C26H28F3N2O6S [M +
H]+, 553.16; found, 553.15.
N-[3-(2,5-Dimethylbenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-4-iodobenzamide (40). White solid, 78%
1
yield; H NMR (400 MHz, CDCl3) δ 7.891 (1H, s), 7.855 (2H, d,
J = 8.4 Hz), 7.829 (1H, m), 7.587 (2H, d, J = 8.8 Hz), 7.293 (2H, m),
7.092 (2H, m), 6.949 (1H, dd, J = 2, 8.4 Hz), 5.092 (2H, s), 3.340
(3H, s), 2.335 (6H, s). ESI-MS calculated for C24H23F3IN2O4S [M +
H]+, 619.04; found, 619.01.
N-[3-(2,5-Dimethylbenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-2-naphthalenecarboxamide (41). White
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dx.doi.org/10.1021/jm4004736 | J. Med. Chem. 2013, 56, 5306−5320

Journal of Medicinal Chemistry
Article
solid, 60% yield; 1H NMR (400 MHz, CDCl3) δ 8.386 (1H, s), 8.098
(1H, s), 7.944 (5H, m), 7.606 (2H, m), 7.312 (2H, m), 7.097 (2H, m),
7.013 (1H, dd, J = 2, 8.4 Hz), 5.133 (2H, s), 3.356 (3H, s), 2.354 (3H,
s), 2.338 (3H, s). ESI-MS calculated for C28H26F3N2O4S [M + H]+,
543.16; found, 543.15.
N-[3-(2,5-Dimethylbenzyloxy)-4-(trifluoromethanesulfonyl-
methylamino)phenyl]-1,3-benzodioxole-5-carboxamide (42). White
solid, 66% yield; 1H NMR (400 MHz, CDCl3) δ 7.851 (1H, s), 7.802
(1H, s), 7.384 (2H, m), 7.298 (2H, m), 7.095 (2H, m), 6.902 (2H, m),
6.074 (2H, s), 5.105 (2H, s), 3.337 (3H, s), 2.344 (3H, s), 2.339 (3H,
s). ESI-MS calculated for C25H24F3N2O6S [M + H]+, 537.13; found,
537.12.
4.2. Biological Studies. 4.2.1. Cell Culture. SKBR-3 cells were
obtained from ATCC (Rockville, MD). The cells were maintained in
RPMI 1640 medium supplemented with 10% fetal bovine serum
(FBS), 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, and 100
U/mL penicillin−streptomycin. FBS was heat inactivated for 30 min in
a 56 °C water bath before use. Cell cultures were grown at 37 °C in a
humidified atmosphere of 5% CO2 in a VWR CO2 incubator
(Bridgeport NJ).
A(Hsp27+compound+DTT) − A(Hsp27+DTT)
ADTT − A(Hsp27+DTT)
where A(Hsp27+compound+DTT) − A(Hsp27+DTT) represents an increase of
insulin aggregation level in the presence of Hsp27 with compounds
compared to that in the presence of Hsp27 without compounds and
ADTT − A(Hsp27+DTT) represents a decrease of insulin aggregation level
caused by Hsp27.
4.2.6. Western Blot. SKBR-3 cells were treated with 5, 10, and 15
nM 20 and 22 for 24 h. The cells were lysed, briefly sonicated, and
centrifuged at 12000g for 10 min. Then 30 μg of protein for each
sample was boiled with 4× loading buffer for 5 min, electrophoresed
on a 10% SDS−polyacrylamide gel, and transferred onto poly-
vinylidene difluoride (PVDF) membrane. For cleaved caspase 3 and
PARP examination, 60 μg of protein was used. The membrane was
blocked for 1 h with 5% nonfat milk in PBST and then incubated with
specific primary antibody (Cell Signaling). After being washed, the
membrane was incubated with horseradish-conjugated secondary
antibody (Cell Signaling). The bands were visualized by chemilumi-
nescence with ECL reagent (Thermo Scientific).
4.2.2. Cell Viability Analysis. The effects of the new derivatives on
SKBR-3 cell viability were assessed using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-2H-tetrazolium bromide assay in six replicates. Cells
were grown in RPMI 1640 medium in 96-well, flat-bottomed plates for
24 h and were exposed to various concentrations of the compounds
dissolved in DMSO (final concentration of ≤0.1%) in medium for 48
h. Controls received DMSO vehicle at a concentration equal to that in
drug-treated cells. The medium was removed, replaced by 200 μL of
0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide in fresh medium, and cells were incubated in the CO2
incubator at 37 °C for 2 h. Supernatants were removed from the wells,
and the reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazo-
lium bromide dye was solubilized in 200 μL/well DMSO. Absorbance
at 570 nm was determined on a plate reader. Statistical and graphical
information was determined using GraphPad Prism software (Graph-
Pad Software Incorporated) and Microsoft Excel (Microsoft
Corporation). IC50 values were determined using nonlinear regression
analysis.
4.2.3. Cell Cycle Study. For all the assays, cells were treated for the
indicated time. To analyze the cell cycle profile, treated cells were fixed
overnight with 70% EtOH at −20 °C and stained with propidium
iodide buffer [38 mM sodium citrate (pH 7.5), 69 μM propidium
iodide, and 120 μg/mL RNase A]. Samples were mixed gently and
incubated at room temperature in the dark for 15 min. Before the
analysis by flow cytometry, 400 μL of binding buffer was immediately
added to each sample. A total of 1.2 × 104 cells were acquired for each
sample, and a maximum of 1 × 104 cells within the gated region were
analyzed.
ASSOCIATED CONTENT
* Supporting Information
Results from HPLC analysis for compounds 1−42 and the
results of Developmental Therapeutic Program at the National
Cancer Institute screening against 60 human tumor cell lines
with compounds 20 and 22. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 216-687-9219. Fax: 216-687-9298. E-mail: B.su@
csuohio.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by a startup fund from Cleveland
State University and also partially supported by Grant R15AI
103889 (B.S.), Center for Gene Regulation in Health and
Disease of CSU, and National Science Foundation Major
Research Instrumentation Grants CHE-0923398 and CHE-
1126384. We thank the Developmental Therapeutic Program at
the National Cancer Institute for screening the compounds
against 60 human cancer cell lines.
4.2.4. Tubulin Polymerization Assay. A mixture of 100 μL of
microtubule-associated protein-rich tubulin (2 mg/mL, bovine brain,
Cytoskeleton) in buffer containing 80 mM PIPES (pH 6.9), 2 mM
MgCl2, 0.5 mM EGTA, and 5% glycerol was mixed with DMSO (as
control) or various concentrations of 20, 22, 44, 46, 49, and
nimesulide in DMSO and incubated at 37 °C. Then 1 μL of 100 mM
GTP was added to the mixture to initiate the tubulin polymerization
and the absorbance at 340 nm was monitored over 20 min using a
Varian Cary 50 series spectrophotometer.
4.2.5. Hsp27 Chaperone Activity Assay. A mixture of 0.24 mg/mL
human recombinant insulin (Life Technologies), 0.02 mg/mL Hsp27
(Cell Sciences), and compounds 20, 22, 44, 46, 49, and nimesulide in
98 μL of sodium phosphate buffer, pH 7.4, was incubated at 37 °C for
5 min, whereupon 2 μL of 1 M DTT in the same assay buffer was
added to initiate the insulin aggregation. The absorbance (A) at 400
nm was monitored over 30 min using a Varian Cary 50 series
spectrophotometer. A mixture of 0.24 mg/mL insulin in the absence
or presence of 0.02 mg/mL Hsp27 with DMSO was used as control.
The Hsp27 inhibition potency (%) of compounds at 30 min were
determined by
ABBREVIATIONS USED
■
COX-2, cyclooxygenase 2; Hsp27, heat shock protein 27;
MAPK, mitogen-activated protein kinase; ASO, antisense
oligonucleotide; siRNA, small interfering RNA; SAR, struc-
ture−activity relationship; Her2, human epidermal growth
factor receptor 2; eIF4E, eukaryotic translation initiation factor
4E; Stat3, signal transducer and activator of transcription 3;
Hsp70, 70 kDa heat shock protein; PARP, poly ADP-ribose
polymerase; FBS, fetal bovine serum; PVDF, polyvinylidene
difluoride; DTT, dithiothreitol
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