N-Aryl benzenesulfonamide inhibitors of [3H]-thymidine incorporation and β-catenin signaling in human hepatocyte-derived Huh-7 carcinoma cells

Article abstract of DOI:10.1016/j.bmcl.2015.07.040

Abstract Structure-activity relationships (SAR) in 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535) were examined as part of a program to identify agents that inhibit the Wnt/β-catenin signaling pathway that is frequently upregulated in hepatocellular carcinoma (HCC). FH535 was reported as an inhibitor of both β-catenin in the Wnt signaling pathway and the peroxisome proliferator-activated receptor (PPAR). A β-catenin/T-cell factor (TCF)/Lymphoid-enhancer factor (LEF)-dependent assay (i.e., luciferase-based TOPFlash assay) as well as a [³H]-thymidine incorporation assay were used to explore SAR modifications of FH535. Although replacing the 2,5-dichlorophenylsulfonyl substituent in FH535 with a 2,6-dihalogenation pattern generally produced more biologically active analogs than FH535, other SAR modifications led only to FH535 analogs with comparable or slightly improved activity in these two assays. The absence of a clear SAR pattern in activity suggested a multiplicity of target effectors for N-aryl benzenesulfonamides.

Full text of DOI:10.1016/j.bmcl.2015.07.040

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3897–3899
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Aryl benzenesulfonamide inhibitors of [3H]-thymidine
incorporation and b-catenin signaling in human hepatocyte-derived
Huh-7 carcinoma cells
Liliia M. Kril a,b, Valery Vilchez c, Jieyun Jiang d,e, Lilia Turcios c, Changguo Chen c, Vitaliy M. Sviripa a,b
,
Wen Zhang a,e, Chunming Liu a,e, Brett Spear d,e, David S. Watt a,b,e, , Roberto Gedaly c,e,
⇑
⇑
a Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY 40536-0509, United States
b Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0596, United States
c Department of Surgery, College of Medicine, University of Kentucky, Lexington, KY 40536-0596, United States
d Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky, Lexington, KY 40536-0093, United States
e Lucille Parker Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0093, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationships (SAR) in 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide
(FH535) were examined as part of a program to identify agents that inhibit the Wnt/b-catenin signaling
pathway that is frequently upregulated in hepatocellular carcinoma (HCC). FH535 was reported as an
inhibitor of both b-catenin in the Wnt signaling pathway and the peroxisome proliferator-activated
Received 24 May 2015
Revised 15 July 2015
Accepted 17 July 2015
Available online 26 July 2015
receptor (PPAR).
A b-catenin/T-cell factor (TCF)/Lymphoid-enhancer factor (LEF)-dependent assay
(i.e., luciferase-based TOPFlash assay) as well as a [3H]-thymidine incorporation assay were used to
explore SAR modifications of FH535. Although replacing the 2,5-dichlorophenylsulfonyl substituent in
FH535 with a 2,6-dihalogenation pattern generally produced more biologically active analogs than
FH535, other SAR modifications led only to FH535 analogs with comparable or slightly improved activity
in these two assays. The absence of a clear SAR pattern in activity suggested a multiplicity of target
effectors for N-aryl benzenesulfonamides.
Keywords:
FH535
Inhibitor
b-Catenin
Wnt/b-catenin signaling pathway
N-Aryl benzenesulfonamides
Huh-7 carcinoma cells
Ó 2015 Elsevier Ltd. All rights reserved.
Hepatocellular carcinoma (HCC) is the most common primary
malignancy of the liver and represents a significant health care
problem as the third most common cause of cancer-related deaths
worldwide.1,2 The prevalence of HCC differs greatly by geographi-
cal location, reflecting variations in the main risk factors. Most
cases of HCC (80%) arise in the Asian Pacific and sub-Saharan
African regions, where the dominant risk factor is chronic infection
with hepatitis B virus.3 Elsewhere, the incidence reflects increasing
infection with hepatitis C virus (HCV) and alcohol use, although
obesity-related non-alcoholic fatty liver disease (NAFLD) is becom-
ing an important risk factor in developed countries.4,5 HCC involves
the disregulation of multiple signaling pathways including the
RAS/RAF/MAPK, PI3K/AKT/mTOR, HGF/c-MET, IGF, VEGF, PDGF
and Wnt/b-catenin pathways. Among them, disregulation of the
Wnt/b-catenin pathway is by far the most difficult to treat.6–9
Chemical agents that target the Wnt signaling pathway provide
the means for probing the intricacies of this pathway and
ultimately providing drugs for treating liver, pancreatic and col-
orectal cancers that frequently exhibit upregulated Wnt signaling.
The reported activity10,11 of 2,5-dichloro-N-(2-methyl-4-nitro-
phenyl)benzenesulfonamide (FH535, Fig. 1) as an inhibitor of both
b-catenin in the Wnt signaling pathway and the peroxisome prolif-
erator-activated receptor (PPAR) prompted our investigation of the
specific molecular target or targets interdicted by FH535. To
address to this problem, we required a biologically active, biotiny-
lated version of FH535, and in the absence of published, structure–
activity relationships, we explored modifications of FH535 that
inhibited both [3H]-thymidine incorporation and activation of a
b-catenin-dependent reporter gene in human hepatocyte-derived
carcinoma Huh-7 cells and that provided suitable, chemical
‘handles’ for biotin incorporation.
In defining the modifications to FH535 compatible with biolog-
ical activity, we began by removing, replacing or relocating various
substituents with comparable electron-donating or withdrawing
capability. We synthesized numerous analogs of FH535 given the
availability of variously substituted sulfonyl chlorides and anilines
(Fig. 1).
⇑
Corresponding authors. Tel.: +1 859 323 4661 (R.G.), +1 859 576 7373 (D.S.W.).
E-mail addresses: dwatt@uky.edu (D.S. Watt), rgeda2@uky.edu (R. Gedaly).
http://dx.doi.org/10.1016/j.bmcl.2015.07.040
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

3898
L. M. Kril et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3897–3899
Cl
A
halogenated analog 1j also possessed activities that exceeded that
of FH535 (1a). Other 2,6-difluorinated analogs 1l–1m, 2,5-difluori-
nated analogs 1n, 2,6-dichlorinated analogs 1o–1r, or the
non-halogenated analogs 1s–1u with varied, representative sub-
stituents on the N-aryl ring, which were among many that were
explored (data not shown), were at best equipotent or diminished
in activity relative to FH535 (Table 1).
2
CH3
'
2
SO2NH
6
Cl
B
'
4
NO2
1a
The liver tumor microenvironment12 is a complex mixture of
tumor cells within the extracellular matrix as well as stromal cells,
infiltrating immune cells and secreted proteins, all of which con-
tribute to and participate in the carcinogenic process. Prior work
in our groups established that FH535 affected, at the very least,
b-catenin in the Wnt signaling pathway.10,11 The use of an assay
relying on [3H]-thymidine incorporation was problematic in the
sense that thymidine incorporation in DNA occurs far downstream
from the presumed and desired FH535 protein targets in the Wnt
pathway. The absence of an unmistakable SAR pattern among the
analogs in Table 1 as well as many others synthesized in the course
of this study (data not shown) suggested that FH535 has targets
other than b-catenin and PPAR, or at the very least, differential
effects on these two targets.
We also employed a b-catenin/T-cell factor (TCF)/Lymphoid-
enhancer factor (LEF)-dependent assay (i.e., luciferase-based
TOPFlash assay) to evaluate these FH535 analogs. In this case, we
assessed TCF/LEF-dependent promoter activation due to b-catenin
translocation to the nucleus. This assay monitors the ability of
FH535 to decrease luciferase levels as a surrogate for b-catenin
inhibition and represents a more direct assay than [3H]-thymidine
incorporation for Wnt pathway regulation. As expected and as
shown in Table 1, the [3H]-thymidine incorporation and the
TOPFlash assays exhibited only partial agreement. For example,
methyl 2-(2,5-dichlorophenylsulfonamido)-5-fluorobenzoate (1c)
showed little activity in the [3H]-thymidine incorporation assay
but activity comparable to FH535 in the TOPFlash assay. The con-
verse was also true: N-4-hydroxymethyl-2-methylphenyl 2,6-
dichlorobenzenesulfonamide (1h) showed enhanced activity in
the [3H]-thymidine incorporation assay relative to FH535 but min-
imal activity in the TOPFlash assay. In summary, we developed
a
SO2Cl
SO2NHAr
X
X
1
Figure 1. FH535 (1a) and synthesis of N-aryl benzenesulfonamides 1. Reagents: (a),
ArNH2, NaH, THF (Method A) or ArNH2, Py (Method B).
With respect to the substituents in the N-aryl ring, replacement
of the C-4 nitro group or the C-2 methyl group in FH535 (1a) with
combinations of a carbomethoxy group and either a methyl or
halogen as in 1b, 1c, and 1d, respectively, diminished biological
activity at 10 l
M concentrations relative to FH535 in the [3H]-thy-
midine incorporation assay (Table 1). On the other hand, reduction
of the carbomethoxy group in 1b to the primary alcohol 1e or
replacement of the N-(2-methyl-4-nitrophenyl) group in FH535
with a 4-nitro-1-naphthyl group as in 1f led to more active com-
pounds than FH535 by a factor of 2.5 and 1.6, respectively, at this
same concentration.
Modification of the halogenation pattern in the 2,5-
dichlorobenzenesulfonyl portion of FH535 revealed that either
the 2,6-difluorosulfonyl or 2,6-dichlorosulfonyl structures exhib-
ited enhanced activity in the 3–10
only for 10 M concentrations). For example, among the 2,6-
dichloro analogs of FH535, the N-2-methyl-4-hydrox-
ymethylphenyl 2,6-dichlorobenzenesulfonamide (1h) exhibited
an activity 2 times that of the activity displayed by FH535 (1a).
In the same vein, the 2,6-difluorinated analog 1i and non-
lM range (Table 1; data shown
l
Table 1
Percentage inhibition of [3H]-thymidine incorporation in Huh-7 cells by FH535 analogs at 3
l
M concentrations
FH535
analog
C-2 C-3 C-4 C-5 C-6 C-20
C-30
C-40
C-50
C-60
[3H]-thymidine % Inhibition Ratio of %
TOPFlash
assay
(10 lM)
TOPFlash
assay as a
percentage
decrease
relative to
control
incorporation
ratio in Huh-7
at 10
lM
inhibition of
analog to %
inhibition by
FH535 at
cells at 10
relative to
DMSO
lM
10 lM
Control
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
100 10
64 3.7
82 4.2
93 5.6
106 14
33 4.8
52 6.4
31.2 4.5
8.5 2.1 73
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
H
F
F
Cl
F
Cl
Cl
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
F
CH3
CH3
CO2CH3
CO2CH3
CH3
H
H
H
H
H
NO2
CO2CH3
F
H
H
H
H
H
H
H
H
H
H
36
18
7
1.0
0.5
0.2
0.0
1.9
1.3
0.8
2.0
1.1
1.5
0.9
1.2
0.9
0.9
1.1
0.9
0.9
0.7
0.8
1.1
1.2
12.1 0.9 61
Cl
0
CH2OH
1-(4-NO2)C10H6
CO2CH3
CH2OH
CH2OH
CH2OH
H
67
48
29
73
38
53
34
43
33
31
38
31
33
26
29
38
42
CH3
CH3
CH3
CH3
OC6H5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
71
1
27 5.1
62 1.5
47 2.3
66 3.2
57 21
67 12
69 3.51
62 10
69 6.9
67 5.9
74 27
71 12
62 2.3
58 6.9
27.3 3.5 13
H
F
F
OCH2C6H5
H
H
Cl
H
Cl
Cl
F
Cl
H
H
H
H
COC6H5
1-(4-NO2)C10H6
H
11.2 1.0 64
17.2 0.9 45
14.8 0.7 53
OC6H5
H
H
H
H
H
H
H
H
H
OCH2C6H5
H
H
COC6H5
Cl
H
H
H
1-(4-NO2)C10H6
16.6 1.1 47
1s
1t
1u
H
H
OCH2C6H5
H
H
COC6H5
1-(4-NO2)C10H6
H
H
H
H
16.8 0.9 46
8.7 0.1 72

L. M. Kril et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3897–3899
3899
analogs of FH535 with activities in a [3H]-thymidine incorporation
Supplementary data
assay and/or a TOPFlash assay that were equipotent or slightly
increased relative to the activity of FH535. Replacing the 2,5-
dichlorophenylsulfonyl substituent in FH535 with a 2,6-dihalo-
genation pattern generally produced more biologically active
analogs than FH535, but none of the modifications made in the
N-phenyl substituent, including replacement with various hetero-
cycles, led to FH535 analogs with improved potency presumably
because FH535 and its analogs may have either a multiplicity of
target effectors or a few effectors that do not have robust structural
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07.
040.
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the
N-aryl
benzenesulfonamide
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C.L. and D.S.W. were supported by R01 CA172379 from the NIH
– United States and by the Kentucky Lung Cancer Research
Program. B.T.S. and J.J. were supported by R01 DK074816 from
the NIH – United States. D.S.W. was supported by the Office of
the Dean of the College of Medicine and by NIH Grant number
P30 RR020171 from the National Institute of General Medical
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