Identification of N-(quinolin-8-yl)benzenesulfonamides as agents capable of down-regulating NFκB activity within two separate high-throughput screens of NFκB activation

Article abstract of DOI:10.1016/j.bmcl.2007.10.100

We describe here a series of N-(quinolin-8-yl)benzenesulfonamides capable of suppressing the NFκB pathway identified from two high-throughput screens run at two centers of the NIH Molecular Libraries Initiative. These small molecules were confirmed in bot

Full text of DOI:10.1016/j.bmcl.2007.10.100

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 329–335
Identification of N-(quinolin-8-yl)benzenesulfonamides as agents
capable of down-regulating NFjB activity within two
separate high-throughput screens of NFjB activation
Yuli Xie,a ShiXian Deng,a Craig J. Thomas,b Yidong Liu,a Ya-Qin Zhang,b
Alison Rinderspacher,a Wenwei Huang,b Gangli Gong,a Michael Wyler,c Efithia Cayanis,c
Nathalie Aulner,c Udo To¨bben,c Caty Chung,d Sergey Pampou,c Noel Southall,b
d
d
c
e
´
Dusˇica Vidovic, Stephan Schurer, Lars Branden, R. Eric Davis,
¨
Louis M. Staudt,e James Inglese,b Christopher P. Austin,b
Donald W. Landry,a,* Deborah H. Smithc and Douglas S. Auldb,*
aDivision of Clinical Pharmacology & Experimental Therapeutics, Department of Medicine, Columbia University,
630 West 168th Street, New York, NY 10032, USA
bNIH Chemical Genomics Center, National Human Genome Research Institute, NIH, 9800 Medical Center Drive,
MSC 3370 Bethesda, MD 20892-3370, USA
cMLSCN Center at Columbia University, Columbia Genome Center, 1150 Saint Nicholas Avenue, New York, NY 10032, USA
dScientific Computing, The Scripps Research Institute, 5353 Parkside Drive, Jupiter, FL 33458, USA
eMetabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, 9000 Rockville Pike,
Building 10, Room 5A/02, Bethesda, MD 20892, USA
Received 3 October 2007; revised 19 October 2007; accepted 20 October 2007
Available online 30 October 2007
Abstract—We describe here a series of N-(quinolin-8-yl)benzenesulfonamides capable of suppressing the NFjB pathway identified
from two high-throughput screens run at two centers of the NIH Molecular Libraries Initiative. These small molecules were con-
firmed in both primary and secondary assays of NFjB activation and expanded upon through analogue synthesis. The series exhib-
ited potencies in the cell-based assays at as low as 0.6 lM, and several indications suggest that the targeted activity lies within a
common region of the NFjB pathway.
Ó 2007 Elsevier Ltd. All rights reserved.
Nuclear factor-jB (NFjB) is the designation for a col-
lection of transcription factors including homo- and het-
erodimers of the Rel family proteins (p50, p52, c-Rel,
RelA/p65, and RelB).1,2 First described3 in 1986 as an
enhancer of immunoglobulin-j, NFjB is now under-
stood to bind to the promoter region of more than
400 genes and play an important role in numerous phys-
iological events, most prominently as a primary regula-
tor of the immune and inflammatory responses.4 Several
pathways leading to activation of NFjB are currently
understood.5 Within the canonical pathway, the NFjB
heterodimer exists predominantly in the cytoplasm as
an inactive complex with the inhibitory IjB proteins.
Phosphorylation of the IjB proteins by an IjB kinase
(IKK) targets the IjBa protein for polyubiquitination
by an E3 ligase and degradation by the proteasome. This
permits the NFjB complex to translocate to the nucleus
and commence gene expression. The exploration of this
pathway has given the biomedical community several
potential targets for manipulation of NFjB activity
including IKK inhibition, proteasome regulation, and
transcriptional inhibitors of NFjB.6,7 Therefore, NFjB
has emerged as a highly studied therapeutic target and
small molecule regulation of signaling cascades associ-
ated with NFjB may provide novel approaches to alle-
viate numerous disease states.6,7
Keyword: NFjB inhibitor.
*
Corresponding authors. Tel.: +1 212 305 5838 (D.W.L.), tel.: +1 301
217 5722 (D.S.A.); e-mail addresses: dwl1@columbia.edu;
dauld@mail.nih.gov
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.100

330
Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 329–335
Several high-throughput campaigns in search of novel
inhibitors of the NFjB signal transduction pathway
have been undertaken by the Molecular Libraries Initia-
tive (MLI) of the NIH Roadmap for Medical Research.8
In one effort, a cell-based assay designed to identify
IjBa stabilization was undertaken by the NIH Chemical
Genomics Center in collaboration with the Metabolism
Branch of the NCI Center for Cancer Research.9 A sec-
ond effort was initiated by another center of the MLI lo-
cated at Columbia University, wherein a cell-based
assay was designed to identify inhibitors of TNFa-in-
duced translocation of NFjB. Importantly, both assays
were performed utilizing the same compound collection
provided to each center by the MLI. Despite the differ-
ing assay design, cell type, and signal readout a N-(quin-
olin-8-yl)benzenesulfonamide core scaffold was noted as
a primary lead in both screens.
inhibitors may be found that affect specific upstream
points in IKK activation.
To identify modulators of IjBa stability in ABC-
DLBCL lines, such as small molecules inhibiting IKK
or proteasome activity, we measured changes in the level
of an exogenous IjBa-luciferase fusion reporter
expressed by an NFjB-insensitive promoter.12,13 The
dual luciferase IjBa stabilization screen in OCI-Ly3
employed here was designed to be suitable for HTS,
by using IjBa fused to a green light-emitting beetle
luciferase, with a red light-emitting beetle luciferase
expressed in a native form to monitor cell uniformity
and non-specific effects.9 Fold-responsiveness was
further increased by having both reporters under the
control of inducible promoters regulated by doxycy-
cline. Upon doxycycline induction of both luciferase
reporters, compounds that increased green luminescence
with minimal effects on the red luminescence signal were
scored as IjBa stabilizers.9
The IjBa stabilization screen was performed using a
dual luciferase reporter system in the cell line OCI-
Ly3, an excellent model for primary tumors of the acti-
vated B-cell subtype (ABC) of diffuse large B-cell lym-
phoma (DLBCL) (Fig. 1a; PubChem AID: 445).9–11
Abnormally high constitutive NFjB activity levels have
been noted in ABC-DLBCL, as in several types of can-
cer, due to high levels of IKKb activity leading to ele-
vated expression of NFjB targeted genes.11 In this
setting, IKK and proteasome activity produce high deg-
radation of IjBa, liberating p50/65 and/or p50/c-Rel
heterodimers to translocate to the nucleus. Because lines
of ABC-DLBCL and other cancer types are dependent
on constitutive NFjB activity, the NFjB pathway is a
therapeutic target. This is particularly true for those
processes governing IjBa degradation, and the thera-
peutic potential of small molecules for this purpose
has been shown by studies in ABC-DLBCL lines using
a specific IKKb inhibitor. Furthermore, the use of an
ABC-DLBCL line for a small-molecule screen of IjBa
stabilization provides a context that is especially close
to that of the targeted disease, with the potential that
The translocation-based assay was a high-content screen
performed in human umbilical vein endothelial cells
(HUVEC) using TNFa to stimulate nuclear transloca-
tion of endogenous NFjB (Fig. 1b; PubChem AID:
438). NFjB is sequestered in the cytoplasm due to its
binding to IjBa, which blocks exposure of a nuclear
localization sequence. Activation by cytokines such as
TNFa results in proteasome degradation of IjBa and
subsequent translocation of NFjB from the cytoplasm
to the nucleus. In the assay, nuclear translocation of
the endogenous p65 RelA subunit of NFjB, at 30 min
post-stimulation, was monitored using fluorescent anti-
body detection and an automated imaging platform.14,15
NFjB inhibitors in this assay, such as IjBa stabilizers,
were detected as compounds that interfered with p65
translocation to the nucleus.
There were several common aspects of the two screens in
this study. Importantly, both screens were run as cell-
Figure 1. (a) The two-color dual luciferase based assay. IjBa is fused to a luciferase emitting green light (CBG68), while a red light-emitting
luciferase (CBR) is present in its native state. Both luciferases are regulated by binding of the tet repressor to the tet operator (TO) binding site (TO),
allowing simultaneous induction of the luciferases by the CMV promoter upon addition of doxycycline and test compounds. The CBR protein serves
to normalize for non-specific effects, while the levels of the IjBa–CBG68 protein increase uniquely with inhibition of the NFjB pathway leading to
IjBa degradation. (b) The nuclear translocation assay. TNFa stimulation results in nuclear translocation of NFjB, which is detected by
immunofluorescence using an anti-p65 antibody and a secondary antibody labeled with an Alexa 647 fluorophore (A647).

Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 329–335
331
based assays utilizing an identical set of small molecules.
Additionally, neither assay monitored NFjB gene tran-
scription, and therefore transcriptional inhibitors, a
potentially large source of non-specific positives, were
not likely to be detected by these assays. The assays were
validated using known inhibitors of the NFjB pathway
(Fig. 2). The proteasome inhibitor MG-132 (1)16 served
as a positive control for the IjBa stabilization assay.
The translocation assay used BAY 11-7082 (2), an agent
that inhibits the TNFa-induced phosphorylation of
IjBa.17–19 Additionally, a substituted 2-(thiophen-2-
yl)quinazoline 3 which acts as an inhibitor of NFjB
and AP1 mediated transcriptional activation20 was used
as a positive control in a secondary assay that used
TNFa to stimulate NFjB-dependent expression of a
b-lactamase reporter (NFjB-bla in Tables 1 and 2). This
compound also served as a negative control for the dual
luciferase IjBa stabilization assay.
by reduction of the corresponding nitro compounds
with stannous chloride in ethanol (Scheme 2). Over 40
novel analogues were prepared to establish preliminary
structure–activity relationships.
The synthesis of various analogues of 5 was more
detailed and the method has been recently reported.22
While several synthetic strategies had been attempted,
the construction of the sultam scaffold via a diazotiza-
tion-induced cyclization of accessible N-8-quinolinyl-
2-aminobenzenesulfonamides proved to be the most
efficient way of synthesizing this unique type of
C7-locked sulfonamide (Scheme 2).
Over 300 analogues of 4 and 5 were analyzed within
both screens. Results for selected analogues of 4 are
shown in Table 1, while the results for derivatives of 5
are shown in Table 2. In general, there was good agree-
ment in the relative potencies of selected analogues
tested in both screening assays, as well as the NFjB
b-lactamase reporter assay (although exceptions do
exist). We take this as an indication that the same
cellular target is being manipulated.
In addition to the required deposit of screening results
within the PubChem database, the collaborative nature
of the MLI allowed members of each research team to
recognize N-(quinolin-8-yl)benzenesulfonamide (4) and
the related C7-locked N-(quinolin-8-yl)benzenesulfon-
amide 5 as the two common lead structures that were
found within both screens (Fig. 2). To further confirm
that these agents interfered with the NFjB activation
in a genuine manner, a reporter assay obtained from
Invitrogen where induction of b-lactamase occurred in
an NFjB-dependent manner was also performed on
the active compounds. These data confirmed the ability
of these compounds to inhibit the NFjB pathway, and
the compounds were then advanced for further study
(Tables 1 and 2).
The results enabled a number of structure–activity rela-
tionships to be observed. In general, the presence of elec-
tron withdrawing groups upon the sulfonyl phenyl ring
produced a favorable potency response for analogues of
4. Interestingly, the presence of a 2-methyl substituent
on the quinoline ring often reduced the potency of the
response that is best illustrated by compounds 11 and
16. Heterocyclic rings in place of the sulfonyl phenyl
ring were well tolerated as indicated by potency values
associated with thiophene analogues 23–27.
A significant number of N-(quinolin-8-yl)benzenesulf-
onamides (both open form and C7-locked form) are
commercially available, and the collection was expanded
quickly through vendor purchase. Further, the synthesis
of analogues of 4 was undertaken through a one-step
process involving the addition of unsubstituted or 2-
substituted, 6-substituted or 2,6-disubstituted quinolin-8-
amines and a variety of sulfonyl chlorides (Scheme 1).21
Amino-substituted analogues (19–22)18 were obtained
A more rigid structure was associated with compound 5,
because a bridge between the quinoline carbon 7 and the
ortho-position of the sulfonyl phenyl ring locks the core
scaffold in a rigid conformation. Two compounds (30
and 31) with the best overall potency values were identi-
fied here, although the efficacy in the IjBa stabilization
assay was sometimes lower in this series than the un-
fused series. Substituents at the 9 position (para to the
sulfonylamide moiety designated R000 in Table 2) are
O
N
O
O
HN
O
O
O
H
S
O
N
N
N
H
N
H
CHO
CN
O
S
N
1
2
3
O
O
O
S
HN
S
HN
O
N
N
4
5
Figure 2. Structures of MG-132 (1), BAY 11-7082 (2), a quinazoline based inhibitor of AP1 and NFjB mediated transcription (3), N-(quinolin-8-
yl)benzenesulfonamide; PubChem CID: 161167 (4), and C7-locked N-(quinolin-8-yl)benzenesulfonamide 5; PubChem CID: 659101.

Table 1. SAR surrounding the N-(quinolin-8-yl)benzenesulfonamides
Control compound #
IjBa stabilization
IjBa stabilization Translocation of
NFjB-bla
Cytotoxicity
EC50 (lM) and efficacya EC50 (lM) ratiob
NFjB IC50 (lM) IC50 (lM) IC50 (lM)
c
1
2
3
1.0, 100%
NA
Inactive
5.2, 100%
NA
Inactive
ND
3.0
0.6
ND
2
>10
ND
Inactive
Inactive
Analogue #
R
R0
Ra
4
Phenyl
4-Nitrophenyl
H
H
H
H
17, 350%
6.5, 560%
11, 620%
20
17
>10
1.5
8.0
5.0
Inactive
Inactive
Inactive
Inactive
>57
6
H
7
2-Nitrophenyl
2-Nitrophenyl
3-Nitrophenyl
H
12
3.2
3.8
10
6.0
8
H
Me 6.8, 200%
Me 6.0, 110%
8.0
13
9
H
0.9
2.1
1.0
6.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4-Methyl-2-nitrophenyl
2-Methyl-5-nitrophenyl
2-Nitro-4-(trifluoromethyl)phenyl
2-Nitro-4-(trifluoromethyl)phenyl
4-Methyl-2-nitrophenyl
4-Methoxy-2-nitrophenyl
2-Methyl-5-nitrophenyl
4-Methyl-2-nitrophenyl
4-Methylphenyl
H
H
H
H
6.8, 275%
1.0, 18%
3.6, 200%
10
>57
>57
H
>10
20
1.6
3.7
1.8
1.3
O
H
>57
>57
R
S
H
Me 10, 50%
6.8, 60%
3.6
7.6
ND
>57
>57
20
1.1
2.1
13
4.0
HN
O
OMe
H
H
>57
ND
R''
N
Me ND
Me >20
OMe Me >10
1.0
ND
>57
Inactive
>57
13.5
5.5
H
Inactive
Inactive
>10
2.0
Inactive
>57
>57
R'
OMe Me 20, 150%
2-Aminophenyl
2-Aminophenyl
H
H
11, 300%
Me 8.0, 250%
4.0, 90%
8.5
8.0
4.0
3.0
9.0
2.7
6.8
2.1
6.4
>57
Inactive
>57
H
OMe
2.8
1.0
2-Amino-4-methyl phenyl
2-Amino-4-methyl phenyl
Thiophen-2-yl
H
2.0
OMe Me 9.4, 23%
Inactive
3.6
1.3
>57
7.0
Inactive
Inactive
>57
H
H
H
H
H
H
H
H
5.3, 250%
2.7, 190%
5.9, 170%
5-Chlorothiophen-2-yl
5-Bromothiophen-2-yl
5-Chlorothiophen-2-yl
5-Bromothiophen-2-yl
1.0
3.4
1.1
1.0
Inactive
>10 lM
>57
Me 1.4, 60%
Me 6.1, 22%
1.3
5.0
1.4
a EC50 values from the IjBa stabilization assay shown for the green luminescence reporter along with the % efficacy.
b EC50 values from the ratio of the green and red luminescent values for the IjBa stabilization. Data are averages from two to three experiments where each experiment consisted of concentration-titration
for each compound performed in duplicate and fitting concentration–response curves to the response after the bioassay. NA, not applicable; the compound only showed a strong inhibitory response in
the original qHTS (IC50 = 2.5 lM, 95% inhibition) in the non-specific (red luminescence) dataset.
c IC50 values are derived from curve-fitting to data from a single experiment performed in triplicate. ND, not determined. All compounds showed >90% efficacy in the translocation assay except
compounds 15 (78%) and 27 (75%). The cytotoxicity assay was performed in OCI-Ly3 cells using a 4 h endpoint.

Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 329–335
Table 2. SAR surrounding the C7-locked N-(quinolin-8-yl)benzenesulfonamides
333
Analogue #
R
R0
R00
R000
IjBa
IjBa
Translocation NFjB-bla Cytotoxicity
IC50 (lM) IC50 (lM)
IC50 (lM)
stabilization stabilization of NFjB
c
EC50 (lM)
EC50 (lM)
and efficacya ratiob
5
28
29
30
31
32
33
34
35
36
37
38
39
40
H
H
H
H
H
H
H
H
OH
2.2, 120%
ND
2.6
ND
1.4
0.8
1.0
1.6
12
1.0
3.4
ND
1.3
0.8
1.2
2.0
1.8
1.9
ND
8.1
1.1
6.7
3.2
3.6
Inactive
ND
H
H
Inactive
1.0
0.6
H
H
OMe 1.3, 20%
Me 0.5, 60%
CF3 0.7, 32%
Inactive
>57
H
H
O
O
H
H
1.0
1.2
Inactive
Inactive
Inactive
Inactive
ND
S
HN
H
H
OMe Me
1.3, 34%
4.1, 122%
1.1, 66%
ND
N
R
H
H
H
H
H
H
F
1.7
1.1
R'''
H
H
Cl
H
H
11
R'
H
OMe
Me
H
ND
10
2.5
R''
H
7.0, 32%
1.4, 300%
8.0, 120%
ND
0.9
Inactive
>57
>57
Me
Me
Me
Me
OMe Me
2.3
11
H
H
H
H
OH
10
2.5
H
OMe 3.7, 200%
CF3 8.9, 700%
3.4
7.2
>57
Inactive
H
1.2
a EC50 values from the IjBa stabilization assay shown for the green luminescence reporter along with the % efficacy.
b EC50 values from the ratio of the green and red luminescent values for the IjBa stabilization. Data are averages from two to three experiments
where each experiment consisted of concentration-titration for each compound performed in duplicate and fitting concentration–response curves to
the response after the bioassay.
c IC50 values are derived from curve-fitting to data from a single experiment performed in triplicate. ND, not determined. All compounds showed
>90% efficacy in the translocation assay. The cytotoxicity assay was performed in OCI-Ly3 cells using a 4 h endpoint.
dal aggregation as the most deleterious reason.23 How-
O
R
NH2
S
ever, these N-(quinolin-8-yl)benzenesulfonamides were
evaluated in a recent profile of the MLSCN compound
collection for persistent, small molecule colloidal aggre-
gators and were not noted as being ‘aggregation-like’.24
Further, the colloidal aggregation phenomenon is most
significant in purified protein assays and is likely to be
less prevalent in cell-based assays. Additionally, for
cell-based assays measuring signal transduction path-
ways, Hill slopes often differ from unity and values of
1000 are theoretically possible.25 Also, while the poten-
cies of these compounds were generally similar, the effi-
cacies varied in the IjBa stabilization assay.
Interestingly, some analogues showed efficacies more
than threefold higher than that noted for MG-132 while
others showed efficacies of less than 50% of the MG-132
control levels (all potency values reported here for the
IjBa stabilization are relative to the observed efficacy
of the particular compound). For example, compound
11 showed a low efficacy in both the IjBa stabilization
assay (18%) and the NFjB b-lactamase assay (30% inhi-
bition) with similar potency, although this compound
showed an efficacy of 97% in the NFjB translocation.
Finally, the current series shows lower potencies when
serum concentrations are increased to levels between
HN
N
R-SO2Cl
O
R''
N
R''
pyridine
R. T.
24 h
R'
R'
6-18, 23-28
Scheme 1.
generally well tolerated as are 6-methoxy and 2-methyl sub-
stitutions (analogues 32 and 37–40, respectively). Unfortu-
nately, despite numerous modifications made to both core
structures of 4 and 5, we were unsuccessful in identifying
compounds with potencies equivalent to or better than
the proteasome inhibitor MG-132 in these assays.
Several points should be made regarding the behavior of
these compounds. First, the concentration–response
curves associated with these compounds were generally
steep (Hill slope showed an average = 4 in the b-lacta-
mase reporter and IjBa stabilization assays, data not
shown). Shoichet has recently provided an evaluation
of such phenomena and provides rationales for why
steep curves emerge, pointing to the formation of colloi-
R'''
R'''
O
S
O
O
O
S
S
HN
HN
HN
O
SnCl2
O
NO2
tBuONO
NH2
R
R
N
N
R
N
R'''
ethanol
reflux
2 h
acetic acid
10 oC to R.T.
10 min.
R''
R'
R''
R'
R''
R'
28-40
19-22
Scheme 2.

334
Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 329–335
9. Davis, R. E.; Zhang, Y.-Q.; Southall, N.; Staudt, L. M.;
Austin, C. P.; Inglese, J.; Auld, D. S. Assay Drug Dev.
Technol. 2007, 5, 85.
10. Davis, R. E.; Brown, K. D.; Siebenlist, U.; Staudt, L. M.
J. Exp. Med. 2001, 184, 1861.
10% and 20% (data not shown). We attempted to alter
the water solubility profile of these compounds to allevi-
ate this issue with no effect.
Selected analogues were submitted to numerous addi-
tional assays in an attempt to isolate the target region
in the NFjB pathway. The compounds were inactive
in biochemical assays of IKKb using recombinant
kinase. Additionally, a full screen for proteasome inhibi-
tion (PubChem AID: 526) against the library that
included the leads described here did not yield any
actives other than MG-132. As well, selected
compounds were screened versus a panel of kinases
including IKKa, IRAK1, IRAK4, JNK1a1, JNK2a2,
JNK3, MAPK1, MAPK2, PKBa, RIPK2, SAPK2a,
TAK1, and TBK1 without any inhibition. An E3 ligase
target was ruled out by treating HUVEC with compound
5 and staining for phosphorylated IjBa. Treatment of
HUVEC with Ro106-9920, a known E3 ligase inhibitor,26
but not compound 5, blocked IjBa ubiquitination
and subsequent proteasome degradation, resulting in
detectable staining of IjBa (data not shown). Thus, the
target of this class of small molecule NFjB pathway
inhibitors has not been identified at present.
11. Okamoto, T.; Sanda, T.; Asamitsu, K. Curr. Pharm. Des.
2007, 13, 447.
12. Lam, L. T.; Davis, R. E.; Pierce, J.; Hepperle, M.; Xu, Y.;
Hottelet, M.; Nong, Y.; Wen, D.; Adams, J.; Dang, L.;
Staudt, L. M. Clin. Cancer Res. 2005, 11, 28.
13. Ngo, V. N.; Davis, R. E.; Lamy, L.; Yu, X.; Zhao, H.;
Lenz, G.; Lam, L. T.; Dave, S.; Yang, L.; Powell, J.;
Staudt, L. M. Nature 2006, 441, 106.
14. Mayer, T.; Jagla, B.; Wyler, M.; Kelly, P.; Aulner, N.;
Beard, M.; Barger, G.; Toebben, U.; Smith, D. H.;
Branden, L.; Rothman, J. Methods Enzyme 2006, 414, 266.
15. The NFjB translocation assay was performed essentially
as described.14 HUVEC grown for fewer than 8 passages
were seeded at 3000 cells/well in 384-well view plates. For
the concentration–response curves, one of two alternate
protocols was used: compounds were added to a final
concentration of 10 lM in media, 0.33% DMSO, and
serially diluted by 1:3, or compounds were added to a final
concentration of 8 lM and serially diluted by 1:2. In either
case, this produced eight data points. After a 3.5-h
incubation with compound, cells were stimulated using
10 lM TNFa for 30 min. Fixed and permeabilized cells
were stained with Hoechst to visualize the nuclei, and with
mouse anti-p65 (Invitrogen) followed by a secondary
antibody conjugated to Alexa Fluor 647 (Invitrogen).
Images were scanned using the In Cell Analyzer 3000 (GE
Healthcare) and analyzed using the Nuclear Trafficking
module.
16. Lee, D. H.; Goldberg, A. L. J. Biol. Chem. 1996, 271,
27280.
17. Kamthong, P. J.; Wu, M.-C. Biochem. J. 2001, 356, 525.
18. Izban, K. F.; Ergin, M.; Qin, J.-Z.; Martinez, R. L.;
Pooley, R. J.; Saeed, S.; Alkan, S. Hum. Pathol. 2000, 31,
1482.
19. Pierce, J. W.; Schoenleber, R.; Jesmok, G.; Best, J.;
Moore, S. A.; Collins, T.; Gerritsen, M. E. J. Biol. Chem.
1997, 272, 21096.
20. Palanki, M. S. S.; Erdman, P. E.; Ren, M.; Suto, M.;
Bennett, B. L.; Manning, A.; Ransone, L.; Spooner, C.;
Desai, S.; Ow, A.; Totsuka, R.; Tsao, P.; Toriumi, W.
Bioorg. Med. Chem. Lett. 2003, 13, 4077.
In summary, we have identified a series of N-(quinolin-
8-yl)benzenesulfonamides that inhibit NFjB activation
with good potency, and selected derivatives maintained
high efficacy levels. Lead compounds 4 and 5 provided
templates for optimization where novel analogues were
obtained with improved potencies (<1 lM). This series
provides a starting point for more detailed selectivity
and mechanistic studies.
Acknowledgments
The authors would like to thank Jeremy Smith, Adam
Yasgar, and Paul Shinn for assistance in compound
management at the NIH Chemical Genomics Center,
and Darryl Leja of the National Human Genome
Research Institute for graphical artwork. We thank
Ms. Allison Peck for critical reading of this manuscript.
This research was supported by the Molecular Libraries
Initiative of the National Institutes of Health Roadmap
for Medical Research and the Intramural Research
Program of the National Human Genome Research
Institute, National Institutes of Health.
21. General procedure for analogues of 4 (procedure for
compound 27): To a solution of 2-methylquinolin-8-amine
(0.16 g, 1.0 mmol) in pyridine (5 mL) was added 5-
bromothiophene-2-sulfonyl chloride (0.26 g, 1.0 mmol).
The mixture was stirred at room temperature overnight
and was precipitated with water. The crude product was
filtered and recrystallized from ethanol to afford 27
(0.33 g, 87%) as white crystals. 1H NMR (300 MHz,
CDCl3) d 9.35 (br, 1H), 8.00 (d, 1H), 7.82 (d, 1H), 7.55–
7.39 (m, 2H), 7.35–7.21 (m, 2H), 6.94 (d, 1H), 2.69 (s, 3H);
13C NMR (75 MHz, CDCl3) d 158.3, 140.7, 138.2, 136.5,
132.9, 132.6, 130.3, 126.6, 123.3, 122.9, 120.3, 115.7, 25.5;
ESI-MS (M++H): 384.0.
References and notes
1. Gilmore, T. D. Oncogene 2006, 25, 6680.
2. Perkins, N. D. Nat. Rev. Mol. Cell Biol. 2007, 8, 49.
3. Sen, R.; Baltimore, D. Cell 1986, 47, 921.
4. Aggarwal, B. B. Expert Opin. Ther. Targets 2007, 11, 109.
5. Hoffmann, A.; Natoli, G.; Ghosh, G. Oncogene 2006, 25,
6706.
6. D’Acquisto, F. D.; Ianaro, A. Curr. Opin. Pharmacol.
2006, 6, 387.
7. Gilmore, T. D.; Herscovitch, M. Oncogene 2006, 25, 6887.
8. Austin, C. P.; Brady, L. S.; Insel, T. R.; Collins, F. S.
Science 2004, 306, 1138.
General procedure for analogues of 4 (amino-substituted
analogues) (Procedure for compound 21): To a suspension
of N-(6-methoxyquinolin-8-yl)-4-methyl-2-nitrobenzene-
sulfonamide (0.37 g, 1.0 mmol) in ethanol (5 mL), stan-
nous chloride (0.38 g, 2.0 mmol) was added slowly. The
mixture was refluxed for 2 h. After removal of ethanol,
the residue was treated with 1 M sodium hydroxide. The
aqueous solution was extracted with methylene chloride.
The combined organic phases were washed with brine,
dried over anhydrous sodium sulfate, and concentrated in

Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 329–335
335
vacuo to give a brown solid which was recrystallized from
ethanol to yield 22 (0.29 g, 85%) as a tan solid. H NMR
23. Shoichet, B. K. J. Med. Chem. 2006, 49, 7274.
1
24. Feng, B. Y.; Simeonov, A.; Jadhav, A.; Babaoglu, K.;
Inglese, J.; Shoichet, B. K.; Austin, C. P. J. Med. Chem.
2007, 50, 2385.
25. Inglese, J.; Johnson, R. L.; Simeonov, A.; Xia, M.; Zheng,
W.; Austin, C. P.; Auld, D. S. Nat. Chem. Biol. 2007, 3,
466.
26. Swinney, D. C.; Xu, Y.-Z.; Scarafia, L.; Lee, I.; Mak,
A.; Gan, Q.-F.; Ramesha, C.; Mulkins, M.; Dunn, J.;
So, O.-Y.; Biegel, T.; Dinh, M.; Volkel, P.; Barnett, J.;
Dalrymple, S.; Lee, S.; Huber, M. J. Biol. Chem. 2002,
277, 23573.
(300 MHz, CDCl3) d 9.40 (br, 1H), 8.54 (s, 1H), 7.90 (d,
1H), 7.67 (d, 1H), 7.37–7.26 (m, 2H), 6.63 (s, 1H), 6.48 (d,
1H), 6.38 (s, 1H), 4.95 (br, 2H), 3.84 (s, 3H), 2.13 (s, 3H);
13C NMR (75 MHz, CDCl3) d 158.1, 146.1, 145.7, 145.6,
135.1, 135.0, 134.9, 130.2, 129.3, 122.5, 118.8, 118.1, 118.0,
106.8, 99.6, 55.9, 21.8; ESI-MS (M++H): 344.1. For
general procedure for analogues of 5, see Ref. 22.
22. Xie, Y.; Gong, G.; Liu, Y.; Deng, S.; Rinderspacher, K.
A.; Branden, L.; Landry, D. W. Tetrahedron Lett.,
submitted for publication.