Structure-activity relationship of 2,2-dimethyl-2H-chromene based arylsulfonamide analogs of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl) methyl]-N-phenylbenzenesulfonamide, a novel small molecule hypoxia inducible factor-1 (HIF-1) pathway inhibitor and anti-cancer agent

Article abstract of DOI:10.1016/j.bmc.2012.04.064

We have discovered that 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl) methyl]-N-phenylbenzenesulfonamide, a novel small molecule HIF-1 pathway inhibitor, can antagonize tumor growth in animal models of cancer, but the treatment necessitates its delivery in a formulation, due to poor water solubility (<15 μg/mL; pH 7.4), evidencing that the chemotype needs further exploration of its amenability to additional chemical modifications for ultimate optimization of function and pharmacology. As a first step towards this goal we investigated the structure-activity relationships of 15 lipophilic 2,2-dimethyl-2H-chromene based arylsulfonamide analogs of 3,4-dimethoxy-N-[(2,2- dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide to find out strategies of modification. A 3,4-dimethoxybenzenesulfonyl group in region 1 showed the strongest inhibition among five arylsulfonyl groups tested. The presence of propan-2-amine in region 2 conferred the strongest inhibitory effect of the compound on HIF-1 activated transcription in a reporter assay. These findings are important as they help define the structural motifs where the 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N- phenylbenzenesulfonamide can be chemically modified to improve its pharmacological properties towards development as a cancer therapeutic.

Full text of DOI:10.1016/j.bmc.2012.04.064

Bioorganic & Medicinal Chemistry 20 (2012) 4590–4597
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship of 2,2-dimethyl-2H-chromene based
arylsulfonamide analogs of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-
yl)methyl]-N-phenylbenzenesulfonamide, a novel small molecule hypoxia
inducible factor-1 (HIF-1) pathway inhibitor and anti-cancer agent
Jiyoung Mun a, Adnan Abdul Jabbar c, Narra Sarojini Devi b, Yuan Liu d,e, Erwin G. Van Meir b,c,d,
,
⇑
Mark M. Goodman a,d,
⇑
a Department of Radiology and Imaging Sciences, Emory University CSI, Wesley Woods Health Center, 1841 Clifton Road NE, Atlanta, GA 30329, USA
b Department of Neurosurgery, Emory University School of Medicine, 1365B Clifton Road NE, Atlanta, GA 30322, USA
c Department of Hematology and Medical Oncology, Emory University School of Medicine, 1365C Clifton Road NE, Atlanta, GA 30322, USA
d Winship Cancer Institute, Emory University, 1365C Clifton Road NE, Atlanta, GA 30322, USA
e Department of Biostatistics and Bioinformatics, Rollins School of Public Health, 1518 Clifton Road NE, Atlanta, GA 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have discovered that 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzene-
sulfonamide, a novel small molecule HIF-1 pathway inhibitor, can antagonize tumor growth in animal
models of cancer, but the treatment necessitates its delivery in a formulation, due to poor water solubility
(<15 lg/mL; pH 7.4), evidencing that the chemotype needs further exploration of its amenability to addi-
tional chemical modifications for ultimate optimization of function and pharmacology.
Received 17 February 2012
Revised 19 April 2012
Accepted 27 April 2012
Available online 14 May 2012
As a first step towards this goal we investigated the structure–activity relationships of 15 lipophilic 2,
2-dimethyl-2H-chromene based arylsulfonamide analogs of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-
Keywords:
Hypoxia inducible factor-1
Small molecule HIF-1 transcription factor
inhibitors
chromen-6-yl)methyl]-N-phenylbenzenesulfonamide to find out strategies of modification.
A 3,4-
dimethoxybenzenesulfonyl group in region 1 showed the strongest inhibition among five arylsulfonyl
groups tested. The presence of propan-2-amine in region 2 conferred the strongest inhibitory effect of
the compound on HIF-1 activated transcription in a reporter assay. These findings are important as they
help define the structural motifs where the 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-
N-phenylbenzenesulfonamide can be chemically modified to improve its pharmacological properties
towards development as a cancer therapeutic.
Tumor hypoxia
3,4-Dimethoxy-N-[(2,2-dimethyl-2H-
chromen-6-yl)methyl]-N-
phenylbenzenesulfonamide
Structure–activity relationships
Anti-cancer drugs
Ó 2012 Elsevier Ltd. All rights reserved.
Brain cancer
1. Introduction
elements (HREs) in target genes that encode proteins regulating a
variety of biological functions, including erythropoiesis, angiogene-
Fast tumor cell proliferation in the context of a dysfunctional
vasculature generates a hypoxic microenvironment in solid tumors,
which hinders the efficacy of chemo- and radiotherapies. Therefore,
developing novel therapeutics to target hypoxic tumor is an active
area of investigation.1–3 Tumor cells respond to the hypoxic stress
by activating the hypoxia inducible factor (HIF) family of transcrip-
tion factors, which bind to DNA sequences called hypoxia response
sis, glycolysis, and metastasis. Hypoxia inducible factors (HIF-1 and
HIF-2), are heterodimers consisting of an oxygen-sensitive subunit
(HIF-1a or -2a), and a constitutively expressed subunit HIF-1b. HIF-
a
subunits are stabilized under hypoxic conditions, while they un-
dergo proteasomal degradation in an oxygenated environment.4
The best-studied family member in cancer is HIF-1. High
expression of HIF-1
a in solid tumors has been associated with
treatment failure and mortality,5 which prompted the develop-
ment of HIF-1 inhibitors as anti-cancer drugs, either alone or in
combination with chemo- or radiotherapy.6–16
Abbreviations: HIF-1, hypoxia inducible factor-1; HREs, hypoxia response
elements; HRE-AP assay, hypoxia response element-mediated alkaline phosphatase
assay; HRE-Luc assay, hypoxia response element-mediated luciferase assay.
We previously screened a library of 10,000 small molecules,
containing 2,2-dimethyl-2H-chromene as a structural motif,17,18 for
inhibition of HIF activity using a cell-based reporter assay.14,15,19–22
The inhibition of HIF-1 transcriptional activation under hypoxia
was measured by the activity of a genetically engineered reporter
⇑
Corresponding authors. Tel.: +1 404 778 5563; fax: +1 404 778 5550 (E.G.V.M.);
tel.: +1 404 727 9366; fax: +1 404 712 5689 (M.M.G.).
E-mail addresses: evanmei@emory.edu (E.G. Van Meir), mgoodma@emory.edu
(M.M. Goodman).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.064

J. Mun et al. / Bioorg. Med. Chem. 20 (2012) 4590–4597
4591
gene, which was stably transfected in a human glioma cell line. The
library screening with this bioassay revealed 3,4-dimethoxy-N-[(2,2-
dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide
(6e, Fig. 1), which showed strong inhibition of reporter activity under
hypoxia.19–21 Further testing of 6e revealed that it has potent anti-
cancer properties in animal models for brain cancer (glioblastoma),
eye cancer (uveal melanoma) and pancreatic cancer (manuscripts
in preparation). However, the treatment necessitated delivery of 6e
give an insight into the potential behavior of future compounds
with a heteroarylaniline such as pyridin-2-amine and pyridin-4-
amine in region 2. Benzenesulfonyl (5a, 6a, 7a), 2,5-dic-
hlorobenzenesulfonyl (5b, 6b, 7b), 4-fluorobenzenesulfonyl (5c,
6c, 7c), 4-nitrobenzenesulfonyl (5d, 6d, 7d), and 3,4-dim-
ethoxybenzenesulfonyl (5e, 6e, 7e) groups were used to investigate
the effect of substituents on a benzene ring of the arylsulfonyl
group in region 1 (Table 1).
in a formulation, due to poor water solubility (<15 lg/mL; pH7.4),
suggesting that further pharmacological optimization is necessary
before this family of compounds can be translated to the patient. This
structural optimization requires the exploration of chemical routes
for modification of the compounds.
2.2. Syntheses of analogs of 6e
Fifteen analogs of 6e, N-[(2,2-dimethyl-2H-chromen-6-yl)-
methyl]-N-R-arylsulfonamides, were synthesized from N-[(2,2-di-
As the first step, we synthesized a series of lipophilic analogs of
6e and compared their activities. We focused on lipophilic congen-
ers that contain a privileged structural motif, 2,2-dimethyl-2H-
chromene group, and systematically diversified the arylsulfon-
amides. The goal was to discover effect of variations in sulfonamide
groups on hypoxia-induced reporter activity, which would provide
useful information for further modification. The inhibition of HIF-1
transcriptional activity under hypoxia by each compound was
measured independently in an HRE-luciferase reporter cell-based
assay in a dose-dependent manner. Finally, to probe the molecular
mechanisms underlying the biological action of the inhibitors, we
methyl-2H-chromen-6-yl)methyl]amine
available arylsulfonyl chlorides (Scheme 1).
and
commercially
4-[(2-Methylbut-3-yn-2-yl)oxy]benzaldehyde (1) was synthe-
sized from an alkylation of 4-hydroxybenzaldehyde with 3-
chloro-3-methyl-1-butyne using sodium hydroxide as a base in
aqueous dimethylformamide (DMF) (water/DMF (v/v) = 8:5). 4-
Hydroxybenzaldehyde was used in excess, since it was the less
expensive reagent, and easily eliminated from the crude reaction
mixture in diethyl ether by washing with aqueous potassium
hydroxide solution and then performing column chromatography.
2,2-Dimethyl-2H-chromene-6-carbaldehyde (2) was formed from
investigated the HIF-1
a
protein levels under hypoxia in human
1 by Claisen cyclization in refluxing N-methyl-2-pyrrolidone
glioblastoma cells in the presence of analogs of 6e by a Western
blot analysis.
(NMP). NMP was selected as a Claisen cyclization solvent due to
its high boiling point (202 °C), and ease of removal from a diethyl
ether solution by washing with water.23–25 N-[(2,2-dimethyl-2H-
chromen-6-yl)methyl]propan-2-amine (4a) was synthesized from
2 and propan-2-amine by imine formation, and then reduction
with diisobutylaluminum hydride (DIBAL). The final sulfonamide,
N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl)ben-
zenesulfonamide (5a), was synthesized by combining 4a with a
benzenesulfonyl chloride in the presence of triethylamine. All
other sulfonamides were synthesized in the same manner. The
reaction yields were not optimized.
2. Results and discussion
2.1. Design of analogs of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-
chromen-6-yl)methyl]-N-phenylbenzenesulfonamide
Fifteen N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-R-aryl-
sulfonamides were designed, based on the structure of 6e. Analogs
of 6e were designed with subtle variations with the hope that they
would not change its presumable molecular pose. The 2,2-di-
methyl-2H-chromene motif (region 3) was preserved in all com-
2.3. HRE-mediated luciferase reporter assays
pounds as
a privileged structure, while the arylsulfonamide
We measured the inhibition of HIF-1 transcriptional activity un-
der hypoxia by analogs of 6e using a cell-based reporter assay. A
hypoxia/HIF-responsive luciferase gene reporter plasmid driven
by a mini cytomegalovirus (CMV) gene promoter and an enhancer
element made of 6 tandem repeats of the hypoxia response
group was diversified to investigate structure–activity relation-
ships (Fig. 1). Propan-2-amine (5a–5e), aniline (6a–6e) and 4-fluo-
roaniline (7a–7e) were chosen as alternate amines in region 2. This
strategy allowed us to investigate the effect of substituting aniline
with an alkylamine or a functionalized aniline on the inhibition of
hypoxia/HIF-activated reporter expression. 2-Methylpropan-1-
amine in region 2 produced five potent analogs in our previous
study,19 so we further investigated propan-2-amine in region 2
to see if the potency would be preserved with minor chain length
change. 4-Fluoroaniline was selected due to small size and the
electron-withdrawing property of fluorine; the latter would also
Table 1
Structures of 15 analogs of 6e
Region 2 (N–R) Region 1 (SO2-Ar)
Cl
F
NO2
SO2
O
O
Cl
SO2
SO2
O
Region 1
SO2
SO2
: Arylsulfonyl Group
: SO2-Ar
O
N
N
N
5a
6a
7a
5b
6b
7b
5c
6c
7c
5d
6d
7d
5e
6e
7e
O
O
O
Arylsulfonamide
S
N
Region 3
Region 2
: Amine Group
: N-R
F
: Privileged Structure
: N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]
Propan-2-amine (5), aniline (6), and 4-fluoroaniline (7) in region 2, were combined
with benzenesulfonyl (a), 2,5-dichlorobenzenesulfonyl (b), 4-fluorobenzenesulfo-
nyl (c), 4-nitrobenzenesulfonyl (d), and 3,4-dimethoxybenzenesulfonyl (e) group in
region 1 to generate 15 different arylsulfonamide analogs. Alterations in region 1
are shown horizontally, while alterations in region 2 are shown vertically.
Figure 1. Chemical structure of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-
yl)methyl]-N-phenylbenzenesulfonamide (6e) and design of N-[(2,2-dimethyl-2H-
chromen-6-yl)methyl]-N-R-arylsulfonamides. Regions were defined according to
Ref. 19.

4592
J. Mun et al. / Bioorg. Med. Chem. 20 (2012) 4590–4597
Table 2
IC50 values of HIF reporter gene inhibition under hypoxia for 15 N-[(2,2-dimethyl-2H-
chromen-6-yl)methyl]-N-R-arylsulfonamides
OH
O
O
a
b
Compound
name
IC50
M)
Number of
repeats
Standard
error
95% Confidence
interval
O
(
l
H
5a
5b
5c
5d
5e
6a
6b
6c
6d
6ea
7a
7b
7c
7d
7e
2.2
16.8
3.8
3.0
1.5
2.7
3.3
4.8
4.2
1.6
7.2
8.0
6.6
4.8
3.2
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.7
9.0
0.8
1.0
0.3
0.6
0.6
1.0
1.0
0.2
1.9
2.2
1.9
1.8
0.7
0.7, 3.6
À0.9, 34.5
2.2, 5.4
1.1, 4.9
0.9, 2.1
1.6, 3.9
2.1, 4.6
2.9, 6.7
2.1, 6.3
1.2, 1.9
3.5, 10.8
3.7, 12.4
2.9, 10.4
1.3, 8.3
1.8, 4.6
H
O
H
O
2
1
O
O
c
d
H
N
N
R
R
H
N-R = Propan-2-amine (3a),
N-R = Propan-2-amine (4a),
Aniline (3b), 4-Fluoroaniline (3c)
Aniline (4b), 4-Fluoroaniline (4c)
Ar
O
O
O
R
e
S
N
5a~5e, 6a~6e, 7a~7e
a
We previously reported the IC50 values for 6e in the ꢀ0.6
In the current experimental set the IC50 value of 6e (1.6
l
M range (n = 42).19,21
N-R = Propan-2-amine (5), Aniline (6), 4-Fluoroaniline (7)
SO2-Ar = Benzenesulfonyl (a), 2,4-Dichlorobenzenesulfonyl
(b), 4-Fluorobenzenesulfonyl (c), 4-Nitrobenzenesulfonyl (d),
3,4-Dimethoxybenzenesulfonyl (e)
l
M) was larger than the
previous values; this variation might be related to the particular batch of cells used
and the smaller number of repeats (n = 6). Because this compound was used as an
internal standard we used the latter value for comparison with the other com-
pounds tested under the same conditions.
Scheme 1. Synthesis of 15 analogs of 6e. Reagents and conditions: (a) 3-chloro-3-
methyl-1-butyne, aq NaOH, DMF, 60 °C, 8 h; (b) N-methyl-2-pyrrolidone, reflux, 8 h
(15% in two steps); (c) amine (propan-2-amine, aniline, 4-fluoroaniline), p-
toluenesulfonic acid monohydrate, methylene chloride, reflux, overnight; (d) DIBAL,
toluene, overnight (89% in two steps); (e) arylsulfonyl chloride, triethylamine,
methylene chloride, reflux, overnight (12%–71%).
statistical analysis was conducted in R statistical software, as we
previously described.19
Fifteen analogs of 6e showed inhibition of HIF-1 transcriptional
activity under hypoxia in a dose-dependent fashion with IC50 values
ranging from 1.5 to 17 lM. Among the three amines tested for re-
element (HRE) of the vascular endothelial growth factor gene
(VEGF) was stably transfected in LN229 human glioblastoma cells
to generate LN229-V6R cells (Fig. 2).19,27 LN229-V6R cells were
cultured in an oxygenated ambient air environment (21% O2, nor-
moxia) for 24 h, the analogs of 6e added and after 1 hour, the cell
culture flasks transferred to a hypoxic cell culture incubator (1%
O2) for 24 h, after which a cell extract was prepared. Hypoxia in-
gion 2, propan-2-amine showed the strongest inhibitory activity
when combined with four arylsulfonyl groups: benzenesulfonyl
(5a), 4-fluorobenzenesulfonyl (5c), 4-nitrobenzenesulfonyl (5d)
and 3,4-dimethoxybenzenesulfonyl (5e). Among five sulfonyl
groups tested for region 1, the 3,4-dimethoxybenzenesulfonyl
group showed the strongest HIF-1 inhibitions when combined with
all three amines: propan-2-amine (5e), aniline (6e) and 4-fluoroan-
iline (7e).
duced the intracellular stabilization of the HIF-1a subunit, HIF
transcriptional complex assembly, binding to the HRE on genomic
DNA in the nucleus and transcription of the luciferase reporter
gene. To measure the amount of luciferase enzyme synthesized
in the cells following HIF activation in the presence or absence of
inhibitors, we used a chemiluminescence assay as we previously
described.14,15 Briefly, the amount of luciferase present in the cell
extracts was quantified by exposing it to luciferine substrate
in vitro and measuring the light emitted in the reaction with a
luminometer. Analogs of 6e were diluted serially to measure repor-
ter inhibition in a dose-dependent fashion at concentrations of 0.5,
2.4. HIF-1a Western blot analyses
The activity of the HIF transcription factor is tightly regulated
under normoxia. In the presence of oxygen, the HIF-1 subunit is
a
constitutively hydroxylated at proline -402 and -564 by oxygen-
dependent prolyl hydroxylases (PHDs), which triggers the interac-
tion of HIF-1
a with the VHL tumor suppressor protein, an E3
ubiquitin-protein ligase that targets it for proteosomal degrada-
tion.4,5,28 Under hypoxic conditions, as occurs in solid tumors, the
1, 2, 5, 10 and 25 lM. Three independent measurements for each
HIF-1a subunit becomes stabilized due to lack of PHD binding,
concentration of each compound were performed and the experi-
ment was repeated twice independently to obtain six measure-
ments in total. The average percent of remaining luciferase
activity in extracts from treated cells versus that found in extracts
from untreated hypoxic cells as a control was plotted for each com-
pound in a dose-dependent fashion. The IC50 values of each com-
pound were obtained by fitting the data of luciferase activities
and concentrations by a four-parameter nonlinear regression mod-
el, in which the IC50 value was one of the parameters (Table 2). The
binds to HIF-1b and a stable HIF transcriptional complex is formed
in the nucleus with co-factors p300/CBP.28–30 To determine
whether analogs of 6e may block HIF activity by preventing HIF-
1a
protein accumulation under hypoxia or destabilizing the
protein, HIF-1 protein levels under hypoxia were measured by
a
Western blotting in the presence of analogs of 6e (Fig. 3). 5e, 6e,
and 7e showed the strongest HIF-1 inhibition for each amine series
in region 2 (less than 5% remaining activity at 10 lM), and 5c
6X HRE
Poly A
Luciferase
CMV mini
CMV mini
Lac Z
Poly A
Figure 2. pBI-GL HRE V6R plasmid structure. The pBI-GL HRE V6R plasmid was generated from the mammalian expression vector pBI-GL (TET) and is redrawn here from
Figure 1 in Ref. 29. The arrows indicate the direction of transcription of both genes (luciferase and LacZ) that are under the control of the 6 copies of the VEGF gene HREs.

J. Mun et al. / Bioorg. Med. Chem. 20 (2012) 4590–4597
4593
in inhibiting reporter activity among the five sulfonyl groups tested
in the sulfonamide group.
To probe the molecular mechanisms underlying the inhibition
of HIF transcriptional activity by the compounds, we measured
control
10 μM
C
C
5c
5e
7e
6e
6e*
HIF-1
HIF-1
a
a
protein levels under hypoxia. A modest 30% decrease in
protein level was observed with 5e and little decrease with
HIF-1α
5c, 6e, and 7e in comparison to the almost complete abrogation in
transcriptional activity of the reporter at 10 M. This result indi-
cates that the mechanism of HIF inhibition by the analogs of 6e
is not by preventing accumulation of HIF-1 protein or destabiliz-
l
Actin
a
ing it under hypoxia. Inhibition of HIF activity may occur at any
step in its activation cascade, in which hypoxia-stabilized cytoplas-
Normoxia
Hypoxia
mic HIF-1a translocates to the nucleus where it binds to HIF-1b
and p300/CBP to form a stable HIF transcriptional complex, which
can bind to the HREs of target genes and activate their transcrip-
tion. Our ongoing investigations with 6e are focused on the
hypothesis that 6e might bind the CH1 domain of the p300/CBP
co-factors and thereby antagonize their interaction with the C-ter-
minal activation domain of HIF-1a.
In summary, N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-R-
arylsulfonamides are novel small molecules that inhibit the func-
tion of an important transcription factor necessary for cancer cell
growth under hypoxia. These compounds have molecular weights
around 400 g/mole, and can be synthesized in 4–5 steps with rea-
sonable yields. The structure–activity relationship studies in this
Letter, demonstrated that we could modify 3,4-dimethoxy-N-
[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesul-
fonamide (6e), using either of two strategies. One is to introduce
hydrophilic amines in region 2 with a 3,4-dimethoxybenzenesulfo-
nyl group in region 1, which is suggested from results with 5e, 6e,
and 7e. In this strategy, simple electron withdrawing heteroaryl
amines, such as pyridine-2-amine, pyridine-4-amine, might de-
crease HIF-1 pathway inhibition, if they behave the same way as
4-fluoroaniline. The other strategy is to introduce hydrophilic aryl-
sulfonyl groups in region 1 with a propan-2-amine group in region
2 as suggested from results with 5a, 5c, 5d, and 5e. In a follow-up
study, we plan on using the latter strategy to introduce hetero-
arylsulfonyl groups in region 1 with simple alkyl groups in region
2. Since the molecules can be diversified at the last step of a synthe-
sis, this approach is more advantageous than the former strategy.
Figure 3. Protein immunoblot (Western blot) analysis of HIF-1
a
expression under
hypoxia. 6e⁄ is previously synthesized 6e 19. Densitometry of the Western blot was
processed by ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA,
http://imagej.nih.gov/ij), and band intensity expressed as percent of untreated
control cells under hypoxia normalized for b-actin.
contains a 4-fluorobenzenesulfonyl group in region 1 that could
predict the behavior of electron-withdrawing heteroarylsulfonyl
groups (e.g. pyridine-4-sulfonyl group). HIF-1
ses were performed on cell extracts from LN229-V6R cells incu-
bated with 10 M of the compounds under hypoxia for 24 h. A
modest 30% reduction in the level of HIF-1 protein was observed
a Western blot analy-
l
a
in extracts from hypoxic cells treated with 5e, whereas little change
was observed for 5c, 6e, and 7e treated cells. The batch-to-batch
variation between 6e and 6e⁄ was minimal as well. While this mod-
est reduction in HIF-1
ute to the strong inhibition of HIF transcriptional activity, it is
unlikely to be the primary cause, because at 10 M 5e almost en-
tirely abolished HIF-1 activity in the luciferase reporter assay (less
than 5% remaining activity, see Supplementary data 4). Overall,
the western blot analysis showed that the compounds had little
a protein level induced by 5e might contrib-
4. Experimental
4.1. Chemistry
l
Glassware was oven-dried (200 °C) overnight and cooled under
argon, and all reactions were performed under argon. All reagents
were purchased from commercial sources. Column chromatogra-
activity on the synthesis and stability of HIF-1a.
phy was performed with Silica Gel 60 (particle size 40–63 lm) with
3. Conclusions
less than 4 psi of argon pressure. TLC was performed with TLC Silica
gel 60 F254 and visualized by UV (254 nm) and staining with 10% (w/
w) of phosphomolybdic acid hydrate solution in ethanol.
NMR spectroscopy was recorded by Varian INOVA 400, Mercury
300 and VNMRS 400 spectrometers. Mass spectroscopy was
obtained by JEOL JMS-SX102/SX102A/E. HPLC was measured with
a Waters 1525 Binary HPLC pump coupled with a Waters 2487 Dual
k Absorbance Detector. HPLC data was processed by Waters Breeze
GPC Software. Two HPLC systems were used to measure the purity
of each analog by % area of a UV absorbance peak at 254 nm:
Fifteen analogs of 6e, N-[(2,2-dimethyl-2H-chromen-6-yl)-
methyl]-N-R-arylsulfonamides, were synthesized to possess 2,2-di-
methyl-2H-chromene as
sulfonamide group composed of three different amines and five dif-
ferent sulfonyl groups. The inhibition of HIF-1 transcriptional activ-
ity mediated by the 15 analogs was measured in LN229-V6R, a
human glioblastoma cell line containing a hypoxia/HIF activated
luciferase reporter gene. All 15 N-[(2,2-dimethyl-2H-chromen-6-
yl)methyl]-N-R-arylsulfonamides inhibited the reporter gene
expression under hypoxia in a dose-dependent fashion with IC50
a privileged structural motif, and a
WAT045905 (Symmetry C18,
methanol/water/triethylamine, 2 mL/min of
WAT011695 (Nova-Pak C18, 4
ethanol, 5% methanol, 5% isopropanol/water/triethylamine (5a–
5d, 6a–6d, and 7a–7d) or 60:40:0.1 = 90% ethanol, 5% methanol,
5% isopropanol: water: triethylamine (5e, 6e, 7e), 0.8 mL/min of a
5
l
m, 4.6 Â 150 mm), 70:30:0.1 =
a
flow rate;
values in the range of 1.5–17 lM. The substitution of propan-2-
lm, 3.9 Â 300 mm), 70:30:0.1 = 90%
amine in region 2 of 6e, showed the strongest inhibition among
three tested amines in the sulfonamide group. The presence of a
3,4-dimethoxybenzenesulfonyl group in region 1 was most potent

4594
J. Mun et al. / Bioorg. Med. Chem. 20 (2012) 4590–4597
flow rate. The peaks after 3.5 min were considered as peaks of com-
pounds. As an eluent, we used a mixture of 95% ethanol, 5% metha-
nol, and 5% isopropanol instead of 100% of ethanol due to an
institutional regulation.
4.1.3.3. 1-(2,2-Dimethyl-2H-chromen-6-yl)-N-(4-fluorophenyl)
methanimine (3c).
1H NMR (CDCl3): d 1.48 (s, 6H), 5.69 (d,
J = 9.96 Hz, 1H), 6.40 (d, J = 9.96, 1H), 6.60–7.62 (m, 8H), 8.32 (s,
1H).
4.1.1. 4-[(2-Methylbut-3-yn-2-yl)oxy]benzaldehyde (1)
A solution of 2.4 g (20 mmole) of 4-hydroxybenzaldehyde and
1 g (10 mmole) of 3-chloro-3-methyl-1-butyne in 8 mL of DMF,
was mixed with 5 mL of 4 N aqueous sodium hydroxide solution.
The bi-phase mixture was stirred vigorously at 60 °C for 8 h. After
cooling, 20 mL of water was added to the reaction mixture, which
was extracted with 30 mL of diethyl ether three times. The com-
bined organic layer was washed with 40 mL of 1 N aqueous sodium
hydroxide solution and brine. The organic portion was then dried
with magnesium sulfate and concentrated in vacuo. A pale yellow
viscous liquid was produced as a crude product. A small portion of
the crude product was purified by column chromatography using
ethyl acetate/hexane (1:8) as an eluent, and the rest of the crude
product was used in the next reaction.
4.1.4. Amine synthesis (4a, 4b, 4c)
The crude 3a from the previous reaction was dissolved in 3 mL
of toluene, to which 1 mL (1 mmole) of diisobutylaluminum hy-
dride (1 M solution in toluene) was added slowly. The reaction
mixture was stirred overnight and poured slowly into 10 mL of
1 N aqueous hydrochloric acid solution with stirring. The mixture
was extracted with 20 mL of ethyl acetate three times. The com-
bined organic layer was washed with brine, dried with magnesium
sulfate, and then concentrated in vacuo. N-[(2,2-dimethyl-2H-
chromen-6-yl)methyl]methanamine was purified by column chro-
matography using triethylamine/methanol/methylene chloride
(1:3:100) as an eluent to afford 111 mg (0.5 mmole) of a pure
product (4a) (89% in two steps). N-[(2,2-Dimethyl-2H-chromen-
6-yl)methyl]aniline (4b) (85% in two steps) and N-[(2,2-
dimethyl-2H-chromen-6-yl)methyl]-4-fluoroaniline (4c) (80% in
two steps) were synthesized by the same method.
1H NMR (CDCl3): d 1.73 (s, 6H), 2.67 (s, 1H), 7.34 (d, J = 8.8 Hz,
1H), 7.83 (d, J = 8.8 Hz 1H), 9.91 (s, 1H).
4.1.2. 2,2-Dimethyl-2H-chromene-6-carbaldehyde (2)
A crude 1 from the previous reaction was dissolved in 20 mL of
N-methyl-2-pyrrolidone, NMP (bp 202 °C), and refluxed for 8 h.
After cooling, a small portion of the crude mixture was extracted
with diethyl ether to confirm complete conversion to the desired
product by NMR. Forty milliliters of water was added to the reac-
tion mixture, and then extracted with 60 mL of diethyl ether three
times. The combined organic layer was washed with brine, dried
with magnesium sulfate, and then concentrated in vacuo. The
crude product was purified by column chromatography using ethyl
acetate/hexane (1:8) as an eluent, which afforded 565 mg
(3 mmole) of 2,2-dimethyl-2H-chromen-6-carboxaldehyde as a
pale-yellow viscous liquid (15% in two steps, based on 20 mmole
of 4-hydroxybezaldehyde).
4.1.4.1. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]propan-2-
amine (4a).
1H NMR (CDCl3): d 1.10 (d, J = 6.35 Hz, 6H), 1.42
(s, 6H), 2.86 (septet, J = 6.35 Hz, 1H), 3.67(s, 3H), 5.60(d,
J = 9.85 Hz, 1H), 6.31(d, J = 9.85 Hz, 1H), 6.72(d, J = 8.26 Hz, 1H),
6.95(d, J = 2.22 Hz, 1H), 7.04(dd, J = 7.94, 1.9 Hz, 1H).
HRMS m/z M+ calcd 232.16959, found 232.17035.
4.1.4.2.
(4b).
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]aniline
1H NMR (CDCl3): d 1.44 (s, 6H), 4.21 (s, 2H), 5.63 (d,
J = 9.67 Hz, 1H), 6.31 (d, J = 9.67 Hz), 6.64–6.77 (m, 4H), 7.00–
7.22 (m, 4H).
HRMS m/z M+ calcd 266.15394, found 266.15385.
4.1.4.3. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-4-fluoroan-
1H NMR (CDCl3): d 1.46 (s, 6H), 5.68 (d, J = 9.9 Hz, 1H), 6.36 (d,
J = 9.9 Hz, 1H), 6.85 (d, J = 8.05 Hz, 1H), 7.50 (d, J = 2.37 Hz, 1H),
7.63 (dd, J = 8.52, 1.89 Hz, 1H), 9.83 (s, 1H).
iline (4c).
1H NMR (CDCl3): d 1.44 (s, 6H), 4.16 (s, 2H), 6.31 (d,
J = 9.96 Hz, 1H), 6.54–6.61 (m, 2H), 6.76 (d, J = 8.21 Hz, 1H), 6.85–
7.12 (m, 4H).
HRMS m/z M+ calcd 189.09101, found 189.09071.
HRMS m/z M+ calcd 284.14452, found 284.14434.
4.1.3. Imine synthesis (3a, 3b, 3c)
4.1.5. Sulfonamide synthesis (5a–5e, 6a–6e, 7a–7e)
A mixture of 102 mg (0.54 mmole) of 2, 20 mg of p-toluenesul-
fonic acid monohydrate and 1 mL of propan-2-amine (3a) (0.1 mL
of aniline (3b) or 0.1 mL of 4-fluoroaniline (3c)) in 2 mL of methy-
lene chloride, was refluxed overnight. After cooling, a small portion
of the crude product was concentrated in vacuo to confirm com-
plete conversion to the desired imine by NMR. The indication was
the disappearance of the aldehyde peak (9.83 ppm) and the appear-
ance of the imine peak (8.18 ppm). After confirming the complete
conversion to an imine, the crude mixture was concentrated in va-
cuo and then, used for the next reaction without purification.
A mixture of 22 mg (0.093 mmole) of 4a, 0.1 mL (164 mg,
0.93 mmole, 10 equiv) of benzenesulfonyl chloride, and 1 mL of tri-
ethylamine in 2 mL of methylene chloride was refluxed overnight.
After cooling, the reaction mixture was concentrated in vacuo, and
purified by column chromatography using ethyl acetate/hexane
(1:6) as an eluent to afford 9 mg (0.024 mmole) of N-[(2,2-di-
methyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl)benzenesulfon-
amide (5a, 26%). All 15 sulfonamides were synthesized by the same
method.
4.1.5.1. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(propan-
4.1.3.1.
methanimine (3a).
1-(2,2-Dimethyl-2H-chromen-6-yl)-N-(propan-2-yl)
1H NMR (CDCl3): d 1.24 (d, J = 6.15 Hz,
2-yl)benzenesulfonamide (5a).
matography eluent: ethyl acetate/hexane (1:6).
Yield: 26%, a column chro-
6H), 1.43 (s, 6H), 3.48 (septet, J = 6.1 Hz, 1H), 5.63 (d, J = 5.6 Hz,
1H), 6.35 (d, J = 10 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 7.39 (dd,
J = 8.2, 2.1 Hz, 1H), 7.45 (d, J = 1.76 Hz, 1H), 8.18 (s, 1H).
13C NMR (CDCl3): d 24.4, 24.5, 28.3, 61.7, 77.0, 77.3, 77.5, 116.5,
121.5, 122.2, 122.3, 125.6, 125.6, 129.6, 130.0, 130.0, 131.3, 155.3,
157.9, 158.0.
1H NMR (CDCl3): d 0.97 (d, J = 7.03 Hz, 6H), 1.43 (s, 6H), 4.12–
4.19 (m, 1H), 4.30 (s, 2H), 5.61 (d, J = 9.67 Hz, 1H), 6.30 (d,
J = 9.96 Hz, 1H), 6.70 (d, J = 8.20 Hz, 1H), 7.02 (s, 1H), 7.08 (d,
J = 8.20 Hz, 1H), 7.46–7.56 (m, 3H), 7.79–7.82 (m, 2H).
HRMS m/z (M+Na)+ calcd 394.14474, found 394.14566.
HPLC (1) RT = 10.246 min, Purity = 97%.
HPLC (2) RT = 5.614 min, Purity = 91%.
4.1.3.2. 1-(2,2-Dimethyl-2H-chromen-6-yl)-N-phenylmethani-
mine (3b).
1H NMR (CDCl3):
d
1.49 (s, 6H), 5.69 (d,
4.1.5.2.
methyl]-N-(propan-2-yl)benzenesulfonamide (5b).
12%, a column chromatography eluent: ethyl acetate/hexane (1:6).
2,5-Dichloro-N-[(2,2-dimethyl-2H-chromen-6-yl)
J = 9.67 Hz, 1H), 6.41 (d, J = 9.67 Hz, 1H), 6.70–7.61 (m, 8H), 8.35
(s, 1H).
Yield:

J. Mun et al. / Bioorg. Med. Chem. 20 (2012) 4590–4597
4595
1H NMR (CDCl3): d 1.16 (d, J = 6.74 Hz, 6H), 1.41 (s, 6H), 4.23–
4.28 (m, 1H), 4.39 (s, 2H), 5.58 (d, J = 9.96 Hz, 1H), 6.22 (d,
J = 9.96 Hz, 1H), 6.60 (d, J = 8.20 Hz, 1H), 6.87 (d, J = 2.05 Hz, 1H),
6.99 (d, J = 8.20 Hz, 1H), 7.38 (d, J = 8.50 Hz, 1H), 7.54–7.63 (m, 2H).
HRMS m/z (M+Na)+ calcd 496.05114, found 496.05160.
HPLC (1) RT = 19.756 min, Purity = 96%.
1H NMR (CDCl3): d 1.39 (s, 6H), 4.62 (s, 2H), 5.58 (d, J = 9.67 Hz,
1H), 6.23 (d, J = 9.67 Hz, 1H), 6.60 (d, J = 8.50 Hz, 1H), 6.86–7.24 (m,
9H), 7.66 (dd, J = 8.79, 5.27 Hz, 2H).
HRMS m/z (M+Na)+ calcd 446.11967, found 446.11967.
HPLC (1) RT = 9.330 min, Purity = 98%.
HPLC (2) RT = 5.164 min, Purity = 98%.
HPLC (2) RT = 7.325 min, Purity = 96%.
4.1.5.9. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-4-nitro-N-
4.1.5.3. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-4-fluoro-N-
phenylbenzenesulfonamide (6d).
Yield: 23%, a column chro-
(propan-2-yl)benzenesulfonamide (5c).
Yield: 17%, a col-
matography eluent: ethyl acetate/hexane (1:6).
1H NMR (CDCl3): d 1.45 (s, 6H), 4.72 (s, 2H), 5.65 (d, J = 9.96 Hz,
1H), 6.28 (d, J = 9.96 Hz, 1H), 6.67 (d, J = 7.91 Hz, 1H), 6.91–7.02 (m,
4H), 7.30–7.33 (m, 3H), 7.88 (d, J = 9.08 Hz, 2H), 8.38 (d, J = 8.79 Hz,
2H).
umn chromatography eluent: ethyl acetate/hexane (1:6).
1H NMR (CDCl3): d 1.12 (d, J = 7.03 Hz, 6H), 1.55 (s, 6H), 4.17–
4.32 (m, 1H), 4.40 (s, 2H), 5.74 (d, J = 9.67 Hz, 1H), 6.41 (d,
J = 9.67 Hz, 1H), 6.82 (d, J = 8.20 Hz, 1H), 7.11 (s, 1H), 7.18 (d,
J = 8.50 Hz, 1H), 7.27 (t, J = 8.50 Hz, 2H), 7.88–7.93 (m, 2H).
HRMS m/z (M+Na)+ calcd 412.13532, found 412.13553.
HPLC (1) RT = 9.684 min, Purity = 99%.
HRMS m/z (M+Na)+ calcd 450.12494 found 450.12551.
HPLC (1) RT = 10.179 min, Purity = 98%.
HPLC (2) RT = 5.202 min, Purity = 97%.
HPLC (2) RT = 5.019 min, Purity = 99%.
4.1.5.10.
3,4-Dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)
Yield: 31%, a
4.1.5.4. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-4-nitro-N-
methyl]-N-phenylbenzenesulfonamide (6e).
(propan-2-yl)benzenesulfonamide (5d).
Yield: 26%, a col-
column chromatography eluent: ethyl acetate/hexane (1:3).
1H NMR (CDCl3): d 1.39 (s, 6H), 3.80 (s, 3H), 3.97 (s, 3H), 4.57 (s,
2H), 5.58 (d, J = 9.96 Hz, 1H), 6.23 (d, J = 9.67 Hz, 1H), 6.60 (d,
J = 7.91 Hz, 1H), 6.85–6.95 (m, 8H), 7.00 (d, J = 2.05 Hz, 1H), 7.32
(dd, J = 8.50, 2.05 Hz, 1H).
umn chromatography eluent: ethyl acetate/hexane (1:6).
1H NMR (CDCl3): d 1.074 (d, J = 6.74 Hz, 6H), 1.43 (s, 6H), 4.21–
4.26 (m, 1H), 4.31 (s, 2H), 5.62 (d, J = 9.96 Hz, 1H), 6.24 (d,
J = 9.96 Hz, 1H), 6.68 (d, J = 8.20 Hz, 1H), 6.91 (s, 1H), 7.04 (d,
J = 8.20 Hz, 1H), 7.88 (d, J = 8.79 Hz, 2H), 8.28 (d, J = 8.50 Hz, 2H).
HRMS m/z (M+Na)+ calcd 439.12982, found 439.13048.
HPLC (1) RT = 9.791 min, Purity = 99%.
HRMS m/z (M+Na)+ calcd 488.15022 found 488.15029.
HPLC (1) RT = 7.372 min, Purity = 99%.
HPLC (2) RT = 7.730 min, Purity = 97%.
HPLC (2) RT = 4.976 min, Purity = 99%.
4.1.5.11. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(4-fluo-
4.1.5.5.
3,4-Dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)
Yield:
rophenyl)benzenesulfonamide (7a).
Yield: 61%, a column
methyl]-N-(propan-2-yl)benzenesulfonamide (5e).
chromatography eluent: ethyl acetate/hexane (1:6).
1H NMR (CDCl3): d 1.39 (s, 6H), 4.59 (s, 2H), 5.58 (d, J = 9.67 Hz,
1H), 6.23 (d, J = 9.67 Hz, 1H), 6.60 (d, J = 8.50 Hz, 1H), 6.84–6.90 (m,
6H), 7.51–7.68 (m, 5H).
45%, a column chromatography eluent: ethyl acetate/hexane (1:3).
1H NMR (CDCl3): d 1.05 (d, J = 6.74 Hz, 6H), 1.47 (s, 6H), 3.94 (s,
3H), 3.99 (s, 3H), 4.14–4.24 (m, 1H), 4.33 (s, 2H), 5.66 (d,
J = 9.67 Hz, 1H), 6.35 (d, J = 9.67 Hz, 1H), 6.76 (d, J = 8.20 Hz, 1H),
6.96 (d, J = 8.50 Hz, 1H), 7.07 (s, 1H), 7.144 (dd, J = 8.20, 2.05 Hz,
1H), 7.28(d, J = 2.05 Hz, 1H), 7.47 (dd, J = 8.50, 2.05 Hz, 1H).
HRMS m/z (M+Na)+ calcd 454.16587, found 454.16570.
HPLC (1) RT = 6.042 min, Purity = 98%.
HRMS m/z (M+Na)+ calcd 446.11967, found 446.11995.
HPLC (1) RT = 8.941 min, Purity = 96%.
HPLC (2) RT = 5.082 min, Purity = 88%.
4.1.5.12.
2,5-Dichloro-N-[(2,2-dimethyl-2H-chromen-6-yl)
Yield:
a column chromatography eluent: ethyl acetate/hexane
HPLC (2) RT = 6.367 min, Purity = 94%.
methyl]-N-(4-fluorophenyl)benzenesulfonamide (7b).
22%,
4.1.5.6. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-phenyl-
(1:6).
benzenesulfonamide (6a).
Yield: 71%, a column chromatog-
1H NMR (CDCl3): d 1.44 (s, 6H), 4.16 (s, 2H), 5.63 (d, J = 9.96 Hz,
1H), 6.31 (d, J = 9.96 Hz, 1H), 6.58 (dd, J = 9.08, 4.40 Hz, 2H), 6.75
(d, J = 8.21 Hz, 1H), 6.89 (t, J = 8.79 Hz, 2H), 6.98 (d, J = 2.05 Hz,
1H), 7.10 (dd, J=7.91, 1.76, 1H).
raphy eluent: ethyl acetate/hexane (1:6).
1H NMR (CDCl3): d 1.39 (s, 6H), 4.63 (s, 2H), 5.57 (d, J = 9.67 Hz,
1H), 6.23 (d, J = 9.96 Hz, 1H), 6.59 (d, J = 8.79 Hz, 1H), 6.87–6.98 (m,
4H), 7.21–7.23 (m, 3H), 7.47–7.68 (m, 5H).
HRMS m/z (M+Na)+ calcd 518.38138, found 518.38149.
HPLC (1) RT = 8.951 min, Purity = 98%.
HPLC (2) RT = 5.723 min, Purity = 91%.
HRMS m/z (M+Na)+ calcd 428.12909, found 428.12915.
HPLC (1) RT = 8.910 min, Purity = 93%.
HPLC (2) RT = 4.974 min, Purity = 97%.
4.1.5.13. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-4-fluoro-
4.1.5.7.
2,5-Dichloro-N-[(2,2-dimethyl-2H-chromen-6-yl)
Yield: 28%, a
N-(4-fluorophenyl)benzenesulfonamide (7c).
column chromatography eluent: ethyl acetate/hexane (1:6).
1H NMR (CDCl3):
1.39 (s, 6H), 4.59 (s, 2H), 5.59 (d,
Yield: 54%, a
methyl]-N-phenylbenzenesulfonamide (6b).
column chromatography eluent: ethyl acetate/hexane (1:6).
1H NMR (CDCl3): d 1.41 (s, 6H), 4.91 (s, 2H), 5.59 (d, J = 9.96 Hz,
1H), 6.26 (d, J = 9.96 Hz, 1H), 6.63 (d, J = 8.79 Hz, 1H), 6.88–7.49 (m,
9H), 7.82 (d, J = 2.05 Hz, 1H).
d
J = J = 9.96 Hz, 1H), 6.23 (d, J = 9.96 Hz, 1H), 6.61 (d, J = 7.91 Hz,
1H), 6.84–6.92 (m, 6H), 7.18 (t, J = 8.50 Hz, 2H), 7.67 (dd, J = 8.79,
4.98 Hz, 2H).
HRMS m/z (M+Na)+ calcd 496.05114, found 496.05160.
HPLC (1) RT = 22.911 min, Purity = 99%.
HRMS m/z (M+Na)+ calcd 464.11024, found 464.11058.
HPLC (1) RT = 10.205 min, Purity = 91%.
HPLC (2) RT = 4.985 min, Purity = 99%.
HPLC (2) RT = 5.102 min, Purity = 99%.
4.1.5.8. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-4-fluoro-N-
4.1.5.14. N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(4-fluo-
phenylbenzenesulfonamide (6c).
Yield: 62%, a column chro-
rophenyl)-4-nitrobenzenesulfonamide (7d).
Yield: 33%, a
matography eluent: ethyl acetate/hexane (1:6).
column chromatography eluent: ethyl acetate/hexane (1:6).

4596
J. Mun et al. / Bioorg. Med. Chem. 20 (2012) 4590–4597
1H NMR (CDCl3): d 1.40 (s, 6H), 4.63 (s, 2H), 5.60 (d, J = 9.96 Hz,
incubated under normoxia for one hour, and then transferred into
a hypoxic incubator at 37 °C for 24 h. The cells were lysed with
1 Â loading buffer (62.5 mM Tris–HCl (pH 6.8 at 25 oC), 2% (w/v)
1H), 6.23 (d, J = 9.67 Hz, 1H), 6.62 (d, J = 8.21 Hz, 1H), 6.82––6.97
(m, 6H), 7.83 (d, J = 8.79 Hz, 2H), 8.34 (d, J = 9.08 Hz, 2H).
HRMS m/z (M+Na)+ calcd 469.12280, found 469.12403.
HPLC (1) RT = 10.882 min, Purity = 91%.
SDS, 50 lM 2-mercaptoethanol, 10% glycerol, 0.01% bromophenol
blue) buffer, and the proteins were separated by electrophoresis
in a 12.5% Tris–HCl polyacrylamide gel, before transfer onto a nitro-
cellulose membrane.14,15 The membranes were immunoblotted
HPLC (2) RT = 5.325 min, Purity = 95%.
using primary antibodies against HIF-1a (1:500 dilution, BD biosci-
4.1.5.15.
methyl]-N-(4-fluorophenyl)benzenesulfonamide
(7e). Yield: 34%, a column chromatography eluent: ethyl ace-
3,4-Dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)
ences, San Diego, CA) and b-actin (1:3000 dilution, Santa Cruz Bio-
technologies, Santa Cruz, CA). The secondary horseradish peroxide-
conjugated antibodies were added and horseradish peroxide
activities were measured by enhanced chemiluminescence as
previously described.15 Densitometry of the Western blot was
processed by ImageJ 1.44p. (Wayne Rasband, National Institutes
of Health, USA, http://imagej.nih.gov/ij)
tate/hexane (1:3).
1H NMR (CDCl3): d 1.37 (s, 6H), 3.74 (s, 3H), 3.95 (s, 3H), 4.59 (s,
2H), 5.55 (d, J = 9.96 Hz, 1H), 6.21 (d, J = 9.96 Hz, 1H), 6.58 (d,
J = 9.08 Hz, 1H), 6.86–7.01 (m, 6H), 7.19–7.34 (m, 5H).
HRMS m/z (M+Na)+ calcd 506.14079, found 506.14080.
HPLC (1) RT = 6.565 min, Purity = 99%.
Acknowledgements
HPLC (2) RT = 7.219 min, Purity = 95%.
This research was supported by the National Cancer Institute
Grants P50 CA 128301-01A1 Emory Molecular and Translational
Imaging Research Center (EMTIC)/In vivo Cellular and Molecular
Imaging Centers (ICMIC) (to Goodman, Hu, Meltzer), P50 CA
128301-01A1 Pilot Project #2 (to J.M.), the P30 CA 138292 Cancer
Center grant (to the Winship Cancer Institute; W Curran, PI), R01
CA116804 and CA86335 (to E.G.V.M.), and the V Foundation, and
Max Cure/Samuel Waxman cancer research foundation grants (to
E.G.V.M.). Data analyses were performed in part through use of
the Biostatistics and Bioinformatics shared Resource of the Win-
ship Cancer Institute of Emory University.
4.2. Biology
4.2.1. Generation of hypoxia/HIF reporter cell line (LN229-V6R
cells)
The LN229-V6R cell line was constructed by stably transfecting
LN229 human glioblastoma cells28 with a vector, pBI-GL VEGF HRE
V6R, which was engineered by modifying the commercial pBI-GL
(TET) mammalian expression vector, so as to replace the tet-
responsive elements driving reporter gene expression with 6 tan-
dem copies of the VEGF gene HRE.26 pBI-GL VEGF HRE V6R contains
luciferase and lacZ reporter genes under a bi-directional hypoxia/
HIF-responsive promoter. These cells synthesize luciferase and b-
galactosidase under hypoxic (1% oxygen) conditions and can be
used as a bioassay for the detection of HIF transcription inhibitors.19
The LN229-V6R cells were cultured in DMEM medium supple-
mented with 10% fetal bovine serum and penicillin/streptomycin
at 37 °C under normoxic (21% O2, 5% CO2, 74% N2) or hypoxic (1%
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.064.
O2, 5% CO2, 74% N2) conditions.
References and notes
1. Post, D. E.; Devi, N. S.; Li, Z.; Brat, D. J.; Kaur, B.; Nicholson, A.; Olson, J. J.; Zhang,
Z.; Van Meir, E. G. Clin. Cancer Res. 2004, 10, 8603.
2. Kim, H.-S.; Peng, G.-Y.; Hicks, J. M.; Weiss, H. L.; Van Meir, E. G.; Brenner, M. K.;
Yotnda, P. Mol. Ther. 2008, 16, 599.
3. Post, D. E.; Sandberg, E. M.; Devi, S. N.; Brat, D. J.; Xu, Z.; Tighiouart, M.; Van
Meir, E. G. Cancer Res. 2007, 67, 6872.
4. Harris, A. L. Nat. Rev. Cancer. 2002, 2, 38.
4.2.2. Assays to measure HIF-1 transcriptional activity using
HRE-driven luciferase reporter gene expression (luciferase
assays)
LN229-V6R cells were seeded in 48-well plates (3 Â 104 cells per
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