Design and in vitro activities of N -alkyl- N -[(8- R -2,2-dimethyl-2 H -chromen-6-yl)methyl]heteroarylsulfonamides, novel, small-molecule hypoxia inducible factor-1 pathway inhibitors and anticancer agents

Article abstract of DOI:10.1021/jm300752n

The hypoxia inducible factor (HIF) pathway is an attractive target for cancer, as it controls tumor adaptation to growth under hypoxia and mediates chemotherapy and radiation resistance. We previously discovered 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N- phenylbenzenesulfonamide as a novel, small-molecule HIF-1 pathway inhibitor in a high-throughput cell-based assay, but its in vivo delivery is hampered by poor aqueous solubility (0.009 μM in water; log P_7.4 = 3.7). Here we describe the synthesis of 12 N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl) methyl]heteroarylsulfonamides, which were designed to possess optimal lipophilicities and aqueous solubilities by in silico calculations. Experimental log P_7.4 values of 8 of the 12 new analogs ranged from 1.2-3.1. Aqueous solubilities of three analogs were measured, among which the most soluble N-[(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl) pyridine-2-sulfonamide had an aqueous solubility of 80 μM, e.g., a solubility improvement of ～9000-fold. The pharmacological optimization had limited impact on drug efficacy as the compounds retained IC₅₀ values at or below 5 μM in our HIF-dependent reporter assay.

Full text of DOI:10.1021/jm300752n

Article
pubs.acs.org/jmc
Design and in Vitro Activities of N-Alkyl-N-[(8-R-2,2-dimethyl-2H-
chromen-6-yl)methyl]heteroarylsulfonamides, Novel, Small-Molecule
Hypoxia Inducible Factor-1 Pathway Inhibitors and Anticancer
Agents
Jiyoung Mun,† Adnan Abdul Jabbar,‡ Narra Sarojini Devi,‡ Shaoman Yin,‡ Yingzhe Wang,∇ Chalet Tan,∇
Deborah Culver,⊥ James P. Snyder,⊥,# Erwin G. Van Meir,*,‡,§,∥ and Mark M. Goodman*,†,∥
†Department of Radiology and Imaging Sciences, Emory University CSI, Wesley Woods Health Center, 1841 Clifton Road,
NE, Atlanta, Georgia 30329, United States
§
∥
‡Department of Neurosurgery, Hematology and Medical Oncology, Emory University School of Medicine, Winship Cancer
⊥
#
Institute, Emory Institute for Drug Development, and Department of Chemistry, Emory University, Atlanta, Georgia 30322,
United States
∇Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, Atlanta, Georgia 30341, United States
S
* Supporting Information
ABSTRACT: The hypoxia inducible factor (HIF) pathway is
an attractive target for cancer, as it controls tumor adaptation
to growth under hypoxia and mediates chemotherapy and
radiation resistance. We previously discovered 3,4-dimethoxy-
N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzene-
sulfonamide as a novel, small-molecule HIF-1 pathway inhibitor
in a high-throughput cell-based assay, but its in vivo delivery
is hampered by poor aqueous solubility (0.009 μM in water;
log P7.4 = 3.7). Here we describe the synthesis of 12
N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)methyl]heteroarylsulfonamides, which were designed to possess optimal lip-
ophilicities and aqueous solubilities by in silico calculations. Experimental log P7.4 values of 8 of the 12 new analogs ranged from
1.2−3.1. Aqueous solubilities of three analogs were measured, among which the most soluble N-[(8-methoxy-2,2-dimethyl-2H-
chromen-6-yl)methyl]-N-(propan-2-yl)pyridine-2-sulfonamide had an aqueous solubility of 80 μM, e.g., a solubility improvement
of ∼9000-fold. The pharmacological optimization had limited impact on drug efficacy as the compounds retained IC50 values at or
below 5 μM in our HIF-dependent reporter assay.
(PHDs), which mediates recognition by the Von Hippel−
Lindau (VHL) E3 ubiquitin ligase complex and rapid
degradation by the proteasome. Under hypoxia, HIF-α subunits
are stabilized due to the inhibition of proline hydroxylation, and
a functional HIF transcriptional complex is assembled,
translocates to the nucleus, and transcribes genes that contain
DNA sequences called hypoxia response elements (HREs).4−7
Elevated levels of HIF-1α have been correlated with poor
prognosis of patients with glioblastoma (GBM), breast,
pancreatic, colon, and metastatic lung cancers.15−24
Hypoxic tumor and HIF-1 have been evaluated as targets for
anticancer therapy using a variety of approaches.25−30 While the
differential function of HIF-1 and HIF-2 isoforms is still under
investigation,7,31 both are associated with brain cancer stem-like
cells,13,14 and most studies suggest that one or both isoforms
need targeting, depending on tumor and cancer type. Therefore,
tumor cells overexpressing HIF represent an important target for
1. INTRODUCTION
The vasculature associated with fast proliferating solid tumors is
abnormal, which limits efficient oxygen supply and renders the
tumor tissue hypoxic.1−3 The presence of hypoxic areas in solid
cancers has been correlated with resistance to chemotherapy
and radiation treatment. Intratumoral hypoxia induces hypoxia
inducible factors (HIFs), transcription factors that activate
genes controlling mechanisms such as glycolysis, erythropoiesis,
angiogenesis, and cell motility, which can benefit the survival of
cancer cells.4−11 HIFs can also influence the self-renewal of
cancer stem-like cells (CSCs)12−14 and be activated in response
to growth factors, oncogenes, and inactivation of tumor sup-
pressor genes.4−7
HIFs are heterodimeric protein complexes, composed of
HIF-α and HIF-β subunits, which then associate with cofactors
such as p300 and CBP to form active transcription factors. The
regulation of HIFs largely occurs at the protein level, and is
dependent upon the synthesis and stability of the HIF-α
subunits. Under normoxia, HIF-α subunits are hydroxylated at
proline residues by oxygen-dependent prolyl hydroxylases
Received: January 3, 2012
Published: June 29, 2012
© 2012 American Chemical Society
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Figure 1. Design of N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)methyl]heteroarylsulfonamides.
antitumor therapy.31−33 A number of existing chemotherapeutics
can alter HIF activity as a result of their pleiotropic effects,
including 2ME2, 17-DMAG, 17-AAG, camptothecin, PX-478, and
YC-1.34−40 Most of the agents studied affect HIF indirectly via the
inhibition of microtubule polymerization, Hsp90, topoisomerase I,
thioredoxin 1, or other unknown mechanisms.41,42 A search for
more specific inhibitors used a screen targeting the interaction of
HIF with the key transcriptional coactivator p300. The small
molecule chetomin was identified but later found to act as a
general inhibitor of zinc ion binding proteins43 and was abandoned
due to unacceptable toxicity in mice.44 A recent study suggests that
acriflavine, an antitrypanocidal, antibacterial, and antiviral agent
interferes with HIF-1α and -1β dimerization and possibly other
signaling pathways such as NF-κB.45 It is too early to determine
which agent affecting the HIF pathway will have the best
antitumor efficacy and safety profile. It is also desirable to develop
several agents that can interfere with HIF transcription in different
ways so that we are prepared for the development of tumor
resistance against single targeted sites.
We recently discovered 3,4-dimethoxy-N-[(2,2-dimethyl-2H-
chromene-6-yl)methyl]-N-phenylbenzenesulfonamide (1)46 as
a novel, small-molecule HIF-1 pathway inhibitor in a high-
throughput screening among 10 000 molecular compounds that
were based on a 2,2-dimethyl-2H-chromene structure as a
naturally occurring biologically active lead moiety.40,47−49
Our ongoing investigations have identified the CH1 domain of
p300/CBP as the putative target of 1, and its binding is expected
to disrupt the formation of the transcriptional complex among
HIF-1α, HIF-1β, and p300/CBP under hypoxia.50,51 1 showed
anticancer activity in vivo in brain, eye, and pancreatic cancer
mouse models; however, it necessitated delivery in a formula-
tion (Cremophor:ethanol = 1:1) due to poor aqueous solubility
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Table 1. Molecular Weights (Mw) and log P and log Sw Values of 3,4-Dimethoxy-N-[(2,2-dimethyl-2H-chromene-6-yl)methyl]-
N-phenylbenzenesulfonamide (1),46 N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl)arylsulfonamides (2, 3, 4, 5),54
a
and Twelve N-Alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)methyl]heteroarylsulfonamides by in Silico Calculations
a
The values were calculated by online software, ALOGPS 2.1 (Virtual Computational Chemistry Laboratory, http://www.vcclab.org).57−62
(0.009 μM).52,53 To develop analogs of 1 with improved aqueous
solubility, we previously investigated structure−activity relation-
ships of 15 lipophilic analogs and selected N-[(2,2-dimethyl-
2H-chromene-6-yl)methyl]-N-(propan-2-yl)arylsulfonamides as
the molecular motifs for further modifications described in the
current study.54
Here we designed N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-
6-yl)methyl]heteroarylsulfonamides (abbreviated hereafter as
“heteroarylsulfonamides”) to possess molecular weights and
estimated log P and log Sw values optimal for lead compounds
in drug discovery.55,56 Twelve heteroarylsulfonamides were
synthesized, and their inhibition potency against the transcriptional
activity of HIF-1, effect on HIF-1α synthesis and stability,
physicochemical properties, metabolic stabilities, and cytotoxicities
in human glioma and fibroblast cells were measured.
chromen-6-yl)methyl]-N-(propan-2-yl)arylsulfonamides (2−5,
Figure 1). Heteroarylsulfonyl groups were used in region 1
instead of arylsulfonyl groups to increase aqueous solubility.
Small alkyl groups were used in region 2, to retain biological
activity without abruptly increasing lipophilicity. The 2,2-
dimethyl-2H-chromene structure in region 3 was diversified at
the C-8 position by adding a hydroxyl or methoxy group due to
synthetic feasibility. A hydroxyl group is advantageous in that it
can be an anchor for functional groups to further increase the
diversity and structural flexibility of the compounds.
Twelve heteroarylsulfonamides (6a−6l) were selected for
synthesis (Table 1) Their molecular weights range from 371
to 403 g/mol, log P values from 3.4 to 4.2, and log Sw values
from −4.2 to −3.2, which are optimal for lead compounds in
drug discovery.55,56
2.2. Synthesis of N-Alkyl-N-[(8-R-2,2-dimethyl-2H-
chromen-6-yl)methyl] heteroarylsulfonamides. The chro-
mene ring of 8-R-2,2-dimethyl-2H-chromene-6-carbaldehydes
(7a, 7b) was formed by Claisen cyclization of the propargyl
ether, 3-R-4-[(2-methylbut-3-yn-2-yl)oxy]benzaldehyde, as described
2. RESULTS
2.1. Design of N-Alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-
6-yl)methyl]heteroarylsulfonamides. The heteroarylsulfon-
amides were designed on the basis of N-[(2,2-dimethyl-2H-
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previously.54,63 The N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)-
methyl]alkylamines (8a−8g) were synthesized from 7a and 7b
by acid-catalyzed imine formation and then subsequent reduction
with diisobutylaluminum hydride (DIBAL).
Scheme 1. Synthesis of N-Alkyl-N-[(8-R-2,2-dimethyl-2H-
chromen-6-yl)methyl]heteroarylsulfonamides
a
The final N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)-
methyl]heteroarylsulfonamides (6a−6g, 6i−6k) were synthe-
sized from 8a−8g and heteroarylsulfonyl chlorides. Several
synthetic methods have been developed for the preparation of
heteroarylsulfonyl chlorides, which are unstable molecules.
These include low-temperature oxidative chlorination of
thioheterocycles with chlorine gas,64 chlorination of hetero-
aromatic methyl sulfides with sulfuryl chloride,65 oxidation of
heteroaromatic thiols in a cosolvent of methylene chloride and
1 N aqueous hydrochloric acid containing 25 wt % calcium
chloride with aqueous sodium hypochlorite,66 and oxidation of
heteroaromatic thiols in concentrated sulfuric acid with
aqueous sodium hypochlorite.67,68 We synthesized hetero-
arylsulfonyl chlorides by the last method due to facile reaction
conditions. Slow addition of aqueous sodium hypochlorite
solution to the reaction mixture was crucial for the high yield of
the reaction, since low temperature has to be maintained during
the highly exothermic reaction to prevent decomposition of the
resulting heteroarylsulfonyl chlorides. The final heteroarylsul-
fonamides were formed in the presence of triethylamine at
40 °C (refluxing methylene chloride) in the case of pyridine-2-
sulfonyl chloride, but in N,N-diisopropylethylamine (NIEA,
a
Reagents and conditions: (a) 3-chloro-3-methyl-1-butyne, 4 N aq
NaOH, DMF, 60 °C, overnight; (b) N-methylpyrrolidone (NMP),
reflux, overnight (15% in two steps); (c) alkylamine (propan-2-amine,
cyclopropanamine, 2-methylpropan-1-amine, 1-cyclopropylmethan-
amine, cyclobuthanamine, cyclopenthanamine), p-toluenesulfonic
acid monohydrate, methylene chloride, reflux, overnight; (d)
diisobutylaluminum hydride (DIBAL), toluene, overnight (39−89%
in two steps); (e) heteroarylsulfonyl chloride, triethylamine,
methylene chloride, reflux, overnight, or heteroarylsulfonyl chloride,
N,N-diisopropylethylamine, methylene chloride, 0 °C to room
temperature, overnight (17−77%).
Hunig’s base) at 0 °C for pyridine-4-sulfonyl chloride, 1-methyl-
̈
1H-imidazol-2-sulfonyl chloride, and 1,3-thiazol-2-sulfonyl chlo-
ride, since the latter heteroarylsulfonyl chlorides decomposed above
0 °C. For the reactions at 0 °C, the reaction mixture was gradually
concentrated by argon gas to accelerate the reactions (Scheme 1).
Insertion of a hydroxyl group at the C-8 position of the 2,2-
dimethyl-2H-chromene ring was accomplished by chromenyla-
tion of 3,4-dihydroxybenzaldehyde with 3-methylbut-2-enal, for
which the yield was low and not optimized.69−71 The hydroxyl
group at the C-8 position of the chromene ring was protected
with methyl chloromethyl ether (MOMCl) to form a
methoxymethyl ether, which was deprotected by 6 N aqueous
HCl mixed with 1 equiv of tetrahydrofuran (THF) at the end
of the synthesis (Scheme 2).
Region 2 of 6a−6f was varied with propan-2-yl, cyclopropyl,
2-methylpropan-1-yl, cyclopropylmethyl, cyclobutyl, and cyclo-
pentyl groups, among which cyclopropylmethyl of 6d showed
the strongest HIF reporter inhibition.
Region 3 of 6a and 6g was altered at the C-8 position of the
2,2-dimethyl-2H-chromene ring with a hydrogen and a methoxy
group, and 6a (hydrogen) showed the strongest inhibition. Region
3 of 6c and 6i was different at the same position with a hydrogen
and a hydroxyl group. In this case, 6i (hydroxyl) showed stronger
inhibition at low concentration; however, dose-dependent
inhibition increased more sharply for 6c (hydrogen). Region 3
of 6i and 6l was modified in the same way as 6c and 6g with a
hydrogen and a hydroxyl group, of which 6l (hydroxyl) showed
slightly stronger inhibition at low concentration; however, dose-
dependent inhibition increased more sharply for 6i (hydrogen).
2.4. Effect on HIF-1α Stability under Hypoxia. HIF-1
is a heterodimer of HIF-1α and HIF-1β subunits, which then
associates with p300 or CBP cofactors to form an active
transcriptional complex. We previously demonstrated that in
contrast to many prior HIF inhibitors, 1 does not antagonize
hypoxia-induced HIF-1α expression under concentrations at
which it blocks HIF transcription in the reporter assay.46 To
determine whether the heteroarylsulfonamides retain this
property, we determined the levels of HIF-1α protein under
hypoxia in LN229-V6R cells. For these experiments we selected
6a, 6g, and 6l because the three compounds exhibited different
degrees of HRE reporter inhibition and possess structural
diversities at the C-8 position of the chromene ring. We
investigated if the levels of HIF-1α protein would correlate
with transcriptional inhibition of the HRE reporter by the
2.3. Inhibition of HIF Transcriptional Activity. The
effects of the 12 heteroarylsulfonamides on HIF transcriptional
activity under hypoxia were measured by determining the
luciferase activity present in LN229-V6R cells, which contain a
stably integrated hypoxia/HIF inducible luciferase reporter
gene.40,46,54 As a control for the absence of inhibition on
luciferase enzyme per se, we also tested 1 and five hetero-
arylsulfonamides in LN229-Lux cells that contain a constitutive
luciferase reporter gene driven by a retroviral LTR promoter.
An Hsp90 inhibitor, 17-DMAG, and 1 were used as positive
controls. 17-DMAG inhibited both reporter cell lines due to its
known cytotoxicity; whereas 1 and heteroarylsulfonamides
reduced luciferase activity only in LN229-V6R cells under
hypoxia. The heteroarylsulfonamides decreased HIF-1 activity
in a dose-dependent fashion with IC50 values below (6a, 6b,
6d−6f, 6h, 6i, 6l) or close to 5 μM (6c, 6g, 6j, 6k) (Figure 2).
Region 1 of 6c, 6i, 6j, and 6k was diversified with pyridine-2-
sulfonyl, pyridine-4-sulfonyl, 1-methyl-1H-imidazole-2-sulfonyl,
and thiazole-2-sulfonyl groups, among which the pyridine-4-
sulfonyl group of 6i showed the strongest HIF reporter
inhibition. However, the pyridine-2-sulfonyl group of 6h
showed stronger inhibition than the pyridine-4-sulfonyl group of
6l, when a hydroxyl group replaces the C-8 hydrogen in region 3.
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compounds. LN229-V6R cells were treated with the com-
pounds at concentrations of 3, 6, 12, 25, 50, and 100 μM under
hypoxia for 24 h, and then HIF-1α protein levels in the cell
extracts were examined by Western blotting. 6a had no effect
on HIF-1α levels at all concentrations tested, except for a slight
reduction at the highest concentration (100 μM). 6g and 6l
decreased HIF-1α levels in a dose-dependent fashion starting
from 12 and 25 μM, respectively (Figure 3). In comparison, in
the above HRE-mediated luciferase assays HIF-1 transcriptional
activity was inhibited completely by 6a and by more than 60−
80% by 6g and 6l when tested at 10 μM (Figure 2). These
results indicate that 6a, 6g, and 6l most likely inhibit HIF tran-
scription in a protein stability independent way similar to 1.
The reduction in HIF-1α levels afforded by all three com-
pounds at 100 μM may reflect nonspecific cytotoxicity.
2.5. Physicochemical Properties and Metabolic Stability.
Experimental determinations of log P7.4, aqueous solubility, and
metabolic stability of a selected number of the heteroarylsulfo-
namides were performed to further characterize the compounds
(Table 2).
Scheme 2. Synthesis of N-[(8-Hydroxy-2,2-dimethyl-2H-
chromen-6-yl)methyl]-N-(2-methylpropan-1-yl)pyridine-2-
sulfonamide (6h) and N-[(8-Hydroxy-2,2-dimethyl-2H-
chromen-6-yl)methyl]-N-(2-methylpropan-1-yl)pyridine-4-
a
sulfonamide (6l)
log P7.4 was measured by either the shake flask method (6a,
6g) or the HPLC method (1, 6b−6f, 6h−6l) according to
OECD guidlines.72 Most compounds followed the trends
predicted by in silico calculations, and the measured values
were smaller than the predicted values by 1.0−1.5. When a
methoxy group or hydroxyl group is placed at the C-8 position
of the chromene ring instead of a hydrogen, as in 6g, 6h, and 6l,
the log P7.4 value decreased by more than 2 from the predicted
values to result in 1.2−1.3. The presence of a 2-methylpropan-
2-yl group in region 2 conferred high log P7.4 values, and
heteroarylsulfonyl groups in region 1 also affected log P7.4
values, as shown for 6c, 6i, 6j, and 6k.
a
Reagents and conditions: (a) 3-methylbut-2-enal, pyridinium
trifluoromethanesulfonate, pyridine, reflux, 2 days (2.4%); (b)
chloromethyl methyl ether, N,N-diisopropylethylamine, tetrahydrofur-
an, reflux, overnight (38%); (c) 2-methylpropan-1-amine, p-
toluenesulfonic acid monohydrate, methylene chloride, reflux, over-
night; (d) 1 M diisobutylaluminium hydride (DIBAL) in toluene,
methylene chloride/toluene (1/2 (v/v)), overnight (13% in two
steps); (e) pyridine-2-sulfonyl chloride, triethylamine, methylene
chloride, reflux, overnight (for 6h), or pyridine-4-sulfonyl chloride,
N,N-diisopropylethylamine, methylene chloride, 0 °C to room
temperature, overnight (for 6l); (f) 6 N aqueous HCl, tetrahydrofuran,
3 h (48−49% in two steps).
Figure 2. Reporter assays measuring luciferase activity in extracts from cells containing hypoxia-inducible (A) or constitutive (B) luciferase reporter
genes following treatment with the indicated heteroarylsulfonamides. Results are plotted as percent of luciferase activity found in extracts of
untreated cells used as controls. (A) Luciferase activity in extracts of LN229-V6R cells, which contain a HIF-inducible luciferase reporter gene. The
cells were grown under hypoxia to activate HIF. (B) Luciferase activity in extracts of LN229-Lux cells, which contain a constitutively expressed
luciferase reporter gene. Results shown are from cells grown under normoxia; similar results were obtained under hypoxia.
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solubilities of 6a, 6d, and 6g are, respectively, 100, 20, and 9000
times better than that of 1, which are consistent with the log Sw
predictions for 6a and 6d. The substitution of hydrogen with a
methoxy group at the C-8 position of the chromene ring
increased the aqueous solubility by 90 times (compare 6a and
6g). Additional measurements of aqueous solubility of 1 and 6a
were performed by laser nephelometry at three different pHs
(3.0, 5.0, and 7.4). pH did not affect aqueous solubility of 1;
however, low pH increased the aqueous solubility of 6a by 50−
60 times (pH 5.0, pH 3.0) due to the presence of the basic
nitrogen in the pyridin-2-sulfonyl group in region 1.
Metabolic stabilities of 1, 6a, 6d, 6g, and 6l were measured
in mouse plasma and homogenates of mouse liver in PBS
[1:2 (w/v)]. The concentrations of all compounds did not
decrease by more than 1% when the compounds were incubated
in mouse plasma at 37 °C for 24 h, which indicated the absence
of degradation or metabolism in plasma. All compounds
underwent ex vivo hepatic metabolism with the half-lives shown
in Table 2, in which 1 showed the fastest and 6a the slowest
metabolism. (See a graph in the Supporting Information.)
2.6. Inhibition of Cell Viability/Proliferation. To
determine whether 6a, 6g, and 6l altered tumor cell growth in
culture, we performed sulforhodamine B (SRB) assays in LN229-
V6R glioma cells in 3 days, and to further examine the cell growth
inhibitory activity of 6a, 6g, and 6l in an independent biological
assay, we performed clonogenicity assays in LN229 human
glioblastoma cells and HFF-1 immortalized human fibroblasts,
over a period of 14 days. IC50 values (μM) of SRB and
clonogenicity assays are presented in Table 3.
Figure 3. Measurement of HIF-1α levels in LN229-V6R cells in
response to various doses of 6a, 6g, and 6l by Western blot analysis.
aControls contain vehicle only (1% DMSO).
Table 2. log P7.4, Aqueous Solubility, and Hepatic Metabolic
Stability of N-Alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)-
methyl]heteroarylsulfonamides
2.6.1. Sulforhodamine B (SRB) Assay.66 LN229-V6R cells
(5 × 103 cells/well) were seeded and cultured under normoxia
for 24 h, and cell densities were measured. The cells were then
treated with serially diluted 6a, 6g, and 6l (1.56−100 μM; 1%
DMSO final in culture medium) under normoxia or hypoxia for
72 h. The three heteroarylsulfonamides showed stronger
cytotoxicity under normoxia than under hypoxia. The IC50
values of cytotoxicity of the three heteroarylsulfonamides under
hypoxia were over 100 μM in 3 days, whereas all of them
decreased HRE-reporter activity by 60−95% at 10 μM in 1 day.
2.6.2. Clonogenicity Assays. 6a, 6g, and 6l showed stronger
inhibition of cell proliferation under normoxia than under
hypoxia in both of glioblastoma and fibroblast cell lines, which
was similar to results of SRB assays. The cytotoxicity profiles of
each compound against glioblastoma (LN229) and fibroblast
(HFF-1) cell lines were similar, suggesting nonspecific
cytotoxicities. 6l displayed the strongest cytotoxicity in both
of the two cell lines.
remaining
HIF-1 activity
at 2.5 μM
b
log
aqueous solubility (n = 3)
(μg/mL, μM)
half-life
(h)
% of
control rank
compd P7.4
a
1
3.7 0.003
0.001, 0.009 0.003
11
20
nd
nd
13
nd
nd
15
15
nd
nd
nd
nd
28
55
71
85
49
53
60
61
43
63
74
78
59
1
a
6a
6b
6c
6d
6e
6f
2.0 0.3
0.1, 1 0.1
5
c
3.0 nd
4.1 nd
10
13
3
a
3.1 0.1
3.0 nd
3.8 nd
0.02, 0.2 0.07
4
7
6g
6h
6i
1.3 22 16, 80 36
1.3 nd
8
2
3.6 nd
9
6j
3.1 nd
11
12
6
6k
6l
4.4 nd
1.2 nd
a
3. DISCUSSION AND CONCLUSIONS
Aqueous solubility by laser nephelometry was measured as follows. 1:
pH 7.4, <15 μg/mL; pH 5.0, <15 μg/mL; pH 3.0, <15 μg/mL. 6a: pH
7.4, <15 μg/mL; pH 5.0, 16 μg/mL; pH 3.0, 17 μg/mL. 6d: pH 7.4,
We previously discovered 1 as a potent HIF-1 pathway
inhibitor with a cell-based high-throughput assay.46 1 contains a
2,2-dimethyl-2H-chromene ring found in many natural
products. For example, 2,2-dimethyl-2H-chromene-based mol-
ecules isolated from Blepharispermum subsessile and the leaves of
Piper aduncum L. have antifungal, antibacterial, and trypanocidal
activities.73−75 The chromenes extracted from the leaves of
Melicope lunu-ankenda have antipyretic, analgesic, anti-inflam-
matory, and antioxidant activities.76 Further structure−activity
relationship (SAR) studies on 1 showed that N-[(2,2-dimethyl-
2H-chromen-6-yl)methyl]-N-(propan-2-yl)arylsulfonamides
possess strong HIF-1 pathway inhibition and are amenable for
further chemical optimization.54
b
<15 μg. Half-life was measured in homogenized mouse liver in PBS
c
[1:2 (w/v)]. The notation “nd” signifies that an experiment was not
done.
Aqueous solubilities of 1, 6a, 6d, and 6g were quantified by
HPLC coupled with a UV detector67 on saturated aqueous
suspensions after filtration with a polytetrafluoroethylene
(PTFE) filter (pore size 0.2 μm). 6a and 6d were chosen
due to their strong activity in the HRE-reporter assay, and 6g
was picked to evaluate the influence of the methoxy group
attached to the C-8 position of the chromene ring. Aqueous
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We developed novel heteroarylsulfonamides to optimize
pharmacological properties of 1, and investigated their
structure−activity relationships to improve HIF-1 inhibitory
activity simultaneously. Six-membered heteroaryl groups in
region 1, a propan-2-amine in region 2, and a (6-hydroxy-2,2-
dimethyl-2H-chromen-6-yl)methyl group in region 3 were the
best selections to increase the aqueous solubility and HIF-1
inhibition and decrease lipophilicity. 1-Cyclopropylmethan-
amine and cyclobutanamine in region 2 showed better HIF-1
inhibition than propan-2-amine; however, lipophilicity, aqueous
solubility, and metabolic stability made the latter group the
superior choice. A functional group at the C-8 of the chromene
ring significantly affected physicochemical properties and HIF-1
inhibition, so a further investigation of derivatization is
required.
In conclusion, the novel heteroarylsulfonamides we synthe-
sized have dramatically improved aqueous solubility, less
lipophilicity, and slightly better stability, while retaining much
of the bioactivity of the parent compound.
The next step toward the clinical translation of the
heteroarylsulfonamides will be to optimize their pharmaco-
logical properties in vivo and test their antitumor properties in
animal models for cancer.
Table 3. IC50 (μM) Values of SRB and Clonogenecity
Assays
a
(A) SRB Assay
compd
condition
IC50 (μM)
6a
normoxia
hypoxia
100
126
73
6g
6l
normoxia
hypoxia
146
92
normoxia
hypoxia
113
(B) Clonogenecity Assays
compd
cell line
condition
IC50 (μM)
6a
6g
6l
LN229
HFF-1
LN229
HFF-1
LN229
HFF-1
normoxia
hypoxia
82
134
62
normoxia
hypoxia
93
normoxia
hypoxia
69
>100
55
normoxia
hypoxia
>100
23
normoxia
hypoxia
30
normoxia
hypoxia
22
4. EXPERIMENTAL SECTION
40
a
IC50 values were calculated by fitting the data to exponential or
4.1. Synthesis. Reagents were purchased from Sigma-Aldrich, Alfa
Aesor, and Fisher Scientific. Anhydrous solvents were purchased from
Sigma-Aldrich. Glassware was oven-dried at 200 °C and cooled under
argon to room temperature. All reactions were performed under argon
gas. Column chromatography was performed with silica gel 60
(particle size 40−63 μm) with less than 4 psi of argon pressure.
Thin-layer chromatography was performed with TLC silica gel 60
F254, and organic compounds were visualized by UV light (254 nm),
iodine vapor, phosphomolybdic acid [10% (w/v) in ethanol] staining
with heating, and ninhydrin solution with heating for amines. NMR
spectroscopy was recorded by Varian INOVA 400, Mercury 300, and
VNMRS 400 spectrometers. Mass spectroscopy was obtained by a
JEOL JMS-SX102/SX102A/E instrument. HPLC was accomplished
with a Waters 1525 binary HPLC pump coupled with a Waters 2487
Dual λ absorbance detector, and data were processed by Waters Breeze
GPC Software. Dual wavelengths of 254 and 280 nm were used for
UV absorbance measurements. The running time of HPLC for each
compound was 20 min, and the peaks were integrated after 3.5 min.
The purity of 6a−6l was over 95%, which was determined by the
percent area of its UV absorbance peak at 254 nm by two different
HPLC systems. In the first HPLC system, a symmetry C18 column
(WAT045905, 5 μm, 4.6 × 150 mm) was used with the eluent
70:30:0.1 = methanol:water:triethylamine at a flow rate of 2 mL/min.
In the second HPLC system, a Nova-Pak C18 column (WAT011695,
4 μm, 3.9 × 300 mm) was used with the eluent 60:40:0.1 = (90%
ethanol, 5% methanol, 5% 2-propanol):water:triethylamine at a flow
rate at 0.8 mL/min. An alcohol mix of 90% ethanol, 5% methanol, and
5% 2-propanol was used instead of pure ethanol due to an institutional
regulation limiting ethanol purchase.
2,2-Dimethyl-2H-chromene-6-carbaldehyde or 8-Methoxy-2,2-
dimethyl-2H-chromene-6-carbaldehyde (7a, 7b). An aqueous
sodium hydroxide solution (4 N, 5 mL) was added to a solution of
4-hydroxybenzaldehyde (2.38 g, 19.5 mmol) and 3-chloro-3-methyl-1-
butyne (1 g, 9.75 mmol) in 8 mL of dimethylformamide (DMF), to
form a binary solution. The reaction mixture was stirred vigorously at
60 °C overnight. After cooling, 20 mL of water was added to the
reaction mixture, which was extracted with diethyl ether (30 mL × 3).
The combined organic phase was washed with 40 mL of 1 N aqueous
sodium hydroxide and then brine, dried with anhydrous magnesium
sulfate, and concentrated in vacuo. Formation of crude 4-[(2-
methylbut-3-yn-2-yl)oxy]benzaldehyde was confirmed by TLC with
ethyl acetate/hexane (1/8) and NMR. 1H NMR (CDCl3): δ 1.73
polinominal equations with R2 ≥ 0.8. The graphs are presented in the
Supporting Information (S5).
Here, we improved the prior compounds by designing a
series of 2,2-dimethyl-2H-chromene-based heteroarylsulfon-
amides with the purpose of decreasing lipophilicity and
increasing aqueous solubility. We modified the N-[(2,2-
dimethyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl)-
arylsulfonamides by substituting the arylsulfonyl group in
region 1 with heteroarylsulfonyl groups such as pyridine-2-
sulfonyl, pyridine-4-sulfonyl, 1-methyl-1H-imidazole-2-sulfonyl,
and thiazole-2-sulfonyl groups; by replacing the propan-2-yl
group in region 2 with short alkyl groups that mainly consist of
small cyclic alkyl groups; and by derivatizing the chromene
moiety at the C-8 position in region 3. Twelve hetero-
arylsulfonamides were designed to possess optimal log P and
log Sw values by in silico calculations and subsequently
synthesized. All 12 heteroarylsulfonamides show inhibition of
HIF-1 transcription in a reporter assay at low micromolar con-
centrations, and the mechanism of action appeared independ-
ent of alteration in HIF-1α protein expression. Our ongoing
studies suggest that the mechanism of action of 1 involves the
disruption of the interaction between HIF-1α and its cofactors
p300/CBP by antagonizing the CH1 domain of p300.50,51
We expect that heteroarylsulfonamides will possess similar
biological activities; however, this will need further exper-
imental validation.
Eight heteroarylsulfonamides showed lower log P7.4 values
ranging from 1.2 to 3.1 versus 3.7 for 1. Increased aqueous
solubilities were achieved for compounds 6a, 6d, and 6g, which
were 100, 20, and 9000 times more soluble than 1, respectively.
The stabilities of 6a, 6d, 6g, and 6l in mouse plasma were
similar to that of the parent compound 1; however, they
exhibited longer half-lives in homogenized mouse liver, ranging
from 13 to 20 h, compared with 11 h for 1, which suggests that
they will likely be more resistant to in vivo hepatic metabolism
than 1.
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(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).
(m, 2H), 0.47−0.53 (m, 2H), 0.97−1.05 (m, 1H), 1.42 (s, 6H), 2.51
(d, J = 6.74 Hz, 2H), 3.73 (s, 2H), 5.61 (d, J = 9.67 Hz, 1H), 6.32 (d,
J = 9.67 Hz, 1H), 6.73 (d, J = 8.20 Hz, 1H), 6.98 (d, J = 2.05 Hz, 1H),
7.07 (dd, J = 2.05, 8.20 Hz, 1H).
Crude 4-[(2-methylbut-3-yn-2-yl)oxy]benzaldehyde was dissolved
in 10 mL of N-methylpyrrolidone (NMP, bp = 202−204 °C) and
refluxed overnight. After cooling to room temperature, 80 mL of
water was added to the solution, which was extracted with diethyl
ether (100 mL × 3). The combined organic phase was washed with
brine, dried with anhydrous magnesium sulfate, and concentrated in
vacuo. 7a was purified by silica gel chromatography with ethyl acetate/
hexane (1/8) as a viscous yellowish liquid (565 mg, 15% yield).
1H NMR (CDCl3): δ 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). HRMS (m/z): M+ calcd
189.09101, found 189.09071.
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]cyclobutanamine
1
(8e). Yield = 60% in two steps. H NMR (CDCl3): δ 1.42 (s, 6H),
1.59−1.75 (m, 5H), 2.19−2.27 (m, 2H), 3.25−3.35 (m, 1H), 3.60 (s,
2H), 5.60 (d, J = 9.67 Hz, 1H), 6.31 (d, J = 9.67 Hz, 1H), 6.72 (d, J =
8.20 Hz, 1H), 6.95 (d, J = 2.05 Hz, 1H), 7.04 (dd, J = 2.05, 8.20 Hz, 1H).
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]cyclopentanamine
1
(8f). Yield = 67% in two steps. H NMR (CDCl3): δ 1.36−1.42 (m,
3H), 1.42 (s, 6H), 1.51−1.56 (m, 2H), 1.68−1.73 (m, 2H), 1.84−1.89
(m, 2H), 3.12 (quintet, J = 6.74 Hz, 1H), 3.67 (s, 2H), 5.60 (d, J =
9.67 Hz, 1H), 6.31 (d, J = 9.96 Hz, 1H), 6.72 (d, J = 8.20 Hz, 1H),
6.96 (d, J = 2.05 Hz, 1H), 7.05 (dd, J = 2.05, 8.20 Hz, 1H).
7b was synthesized by the same method as 7a. Vanillin (3 g,
20 mmol) and 3-chloro-3-methyl-1-butyne (1 g, 10 mmol) were used
as starting materials. 7b was purified by silica gel chromatography with
ethyl acetate/hexane (1/6) as a white solid (204 mg, 5% in two steps)
3-Methoxy-4-[(2-methylbut-3-yn-2-yl)oxy]benzaldehyde. 1H
NMR (CDCl3): δ 1.75 (s, 6H), 2.63 (s, 1H), 3.90 (s, 3H), 7.43−
7.40 (m, 2H), 7.64 (d, J = 8.79 Hz, 1H).
N-[8-Methoxy-(2,2-dimethyl-2H-chromen-6-yl)methyl]propan-2-
1
amine (8g). Yield = 39% in two steps. H NMR (CDCl3): δ 1.11 (d,
J = 6.15 Hz, 6H), 1.46 (s, 6H), 2.88 (septet, J = 6.15 Hz, 1H), 3.68 (s,
2H), 3.86 (s, 3H), 5.61 (d, J = 9.67 Hz, 1H), 6.28 (d, J = 9.96 Hz, 1H),
6.60 (s, 1H), 6.78 (s, 1H).
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl)-
pyridine-2-sulfonamide (6a). Pyridine-2-sulfonyl Chloride. 2-Mer-
captopyridine (126 mg, 1 mmol) was dissolved in 5 mL of concentrated
sulfuric acid to form a yellow solution, which was cooled to around
−15 °C with a sodium chloride/ice (1/3) bath. Aqueous sodium
hypochlorite solution (10−15%, 11 mL, 15−20 mmol) was added to the
solution slowly enough to maintain the internal temperature of the
reaction mixture below 10 °C, with vigorous stirring. The reaction mixture
was stirred at 0 °C for 1 hour and then 10 mL of water was added, which
was then extracted with methylene chloride (20 mL × 3). The combined
organic phase was washed with water, dried with anhydrous magnesium
sulfate, and then concentrated in vacuo. Pyridine-2-sulfonyl chloride (145
mg, 72%) was produced as a yellowish viscous liquid. 1H NMR (CDCl3):
δ 7.684−7.729 (m, 1H), 8.043−8.143 (m, 2H), 8.84 (d, J = 4.10 Hz, 1H).
Freshly prepared pyridine-2-sulfonyl chloride (100 mg, 0.6 mmol)
was added to a solution of 8a (25 mg, 0.1 mmol) and triethylamine
(0.5 mL, 3.6 mmol) in 0.8 mL of methylene chloride. The reaction
mixture was refluxed overnight and then concentrated in vacuo. 6a
(31 mg, 77%) was purified by silica gel chromatography with ethyl
acetate/hexane (1/1). 1H NMR (CDCl3): δ 1.03 (d, J = 6.74 Hz, 6H),
1.42 (s, 6H), 4.27 (septet, J = 6.74 Hz, 1H), 4.45 (s, 2H), 5.61 (d, J =
9.67 Hz, 1H), 6.30 (d, J = 9.67 Hz, 1H), 6.68 (d, J = 8.21 Hz, 1H),
7.04 (d, J = 2.05 Hz, 1H), 7.10 (dd, J = 2.05, 8.20 Hz, 1H), 7.38−7.47
(m, 1H), 7.81−7.96 (m, 2H), 8.7 (d, J = 4.40 Hz, 1H). HRMS (m/z):
[M + Na]+ calcd 395.13999, found 395.14017. HPLC-1: tR = 4.5 min,
purity = 98%. HPLC-2: tR = 6.5 min, purity = 99%.
8-Methoxy-2,2-dimethyl-2H-chromene-6-carbaldehyde (7b). 1H
NMR (CDCl3): δ 1.51 (s, 6H), 3.93 (s, 3H), 5.70 (d, J = 9.78 Hz,
1H), 6.37 (d, J = 9.78 Hz, 1H), 7.17 (d, J = 1.96 Hz, 1H), 7.32 (d, J =
1.96 Hz, 1H), 9.81 (s, 1H). HRMS (m/z): M+ calcd 219.10157, found
219.10124.
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]propan-2-amine (8a).
7a (102 mg, 0.54 mmol) was dissolved in 2 mL of anhydrous
methylene chloride, to which p-toluenesulfonic acid monohydrate
(20 mg, 0.1 mmol) and propan-2-amine (1 mL, 12 mmol) were added.
The reaction mixture was refluxed overnight and then concentrated in
vacuo. The NMR of the crude product confirmed a complete conver-
sion of the aldehyde, 7a, to the corresponding imine by disappearance
of the aldehyde proton peak, 9.83 ppm (s, 1H), and appearance of the
imine proton peak, 8.19 ppm (s, 1H).
The crude imine was suspended in 3 mL of toluene, to which 1 M
diisobutylaluminum hydride in toluene (DIBAL, 2.5 mL, 2.5 mmol)
was added slowly to control vigorous bubbling. The reaction mixture
was stirred overnight at room temperature, and then 20 mL of 1 N
aqueous hydrochloric acid was added to the reaction mixture slowly,
to quench the reduction. The resulting emulsion was basified with
concentrated aqueous sodium carbonate solution. The reaction
mixture was extracted with ethyl acetate (20 mL × 3). The combined
organic phase was washed with brine, dried with anhydrous
magnesium sulfate, and then concentrated in vacuo. 8a was purified
by silica gel chromatography with triethylamine/methanol/methylene
chloride (1/1/100) as a viscous oil (111 mg, 89% in two steps).
1H NMR (CDCl3): δ 1.1 (d, J = 6.15 Hz, 6H), 1.4 (s, 6H), 2.85
(septet, J = 6.45 Hz, 1H), 3.66 (s, 2H), 5.6 (d, J = 9.96 Hz, 1H), 6.3
(d, J = 9.67 Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 2.05 Hz,
1H), 7.04 (dd, J = 2.34, 7.91 Hz, 1H).
Other heteroarylsulfonamides containing a pyridine-2-sulfonyl
group were synthesized by the same method as that of 6a.
N-Cyclopropyl-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-
pyridine-2-sulfonamide (6b). Purification was by silica gel chroma-
tography with ethyl acetate/hexane (2/3). Yield = 24%. 1H NMR
(CDCl3): δ 0.725 (t, J = 7.33 Hz, 2H), 1.35−1.43 (m, 1H), 3.23
(dd, J = small as broadening of peaks, 7.62 Hz, 2H), 4.44 (s, 2H), 5.61
(d, J = 9.96 Hz, 1H), 6.27 (d, J = 9.97 Hz,1H), 6.68 (d, J = 8.20 Hz,
1H), 6.93 (d, J = 1.76 Hz, 1H), 7.01 (dd, J = 2.05, 8.20 Hz, 1H), 7.47
(ddd, J = 0.88,4.69, 7.32 Hz, 1H), 7.85−7.99 (m, 2H), 8.70 (d, J =
4.69 Hz, 1H). HRMS (m/z): [M + Na]+ calcd 393.12488, found
Other amines (8b−8g) were synthesized by the same method as 8a.
The imine formation was confirmed by disappearance of the aldehyde
proton peak [9.83 ppm (7a) or 9.81 ppm (7b)] and appearance of the
corresponding imine peak [8.1−8.3 ppm (s, 1H)]. The amines were
synthesized by reduction of the corresponding imines.
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]cyclopropanamine
1
395.14008. HPLC-1: tR = 5.1 min, purity = 100%. HPLC-2: tR
=
(8b). Yield = 37% in two steps. H NMR (CDCl3): δ 0.93 (t, J =
6.8 min, purity = 97%.
7.33 Hz, 2H), 1.43 (s, 6H), 1.56 (sextet, J = 7.33 Hz, 1H), 2.61 (t, J =
7.33 Hz, 2H), 3.70 (s, 2H), 5.61 (d, J = 9.67 Hz, 1H), 6.31 (d, J = 9.67
Hz, 1H), 6.73 (d, J = 8.20 Hz, 1H), 6.97 (d, J = 2.05 Hz, 1H), 7.06
(dd, J = 2.05, 8.20 Hz, 1H).
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(2-methylpropyl)-
pyridine-2-sulfonamide (6c). Purification was by silica gel chromatog-
raphy with ethyl acetate/hexane (1/4). Yield = 60%. 1H NMR
(CDCl3): δ 0.76 (d, J = 6.74 Hz, 6H), 1.42 (s, 6H), 1.74 (septet, J =
7.03 Hz, 1H), 3.11 (d, J = 7.325 Hz, 2H), 4.42 (s, 2H), 5.60 (d, J =
9.67 Hz, 1H), 6.24 (d, J = 9.67 Hz, 1H), 6.65 (d, J = 8.20 Hz, 1H),
6.87 (d, J = 2.05 Hz, 1H), 6.96 (dd, J = 2.05, 8.20 Hz, 1H), 7.45 (ddd,
J = 1.17, 4.69, 7.62 Hz, 1H), 7.865 (td, J = 1.76, 7.62 Hz,1H), 7.95 (d,
J = 7.62 Hz, 1H), 8.69 (d, J = 4.69 Hz, 1H). HRMS (m/z): [M + Na]+
calcd 409.15564, found 409.15649. HPLC-1: tR = 6.8 min, purity =
100%. HPLC-2: tR = 8.4 min, purity = 97%.
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-2-methylpropan-1-
1
amine (8c). Yield = 30% in two steps. H NMR (CDCl3): δ 0.92 (d,
J = 6.45 Hz, 6H), 1.43 (s, 6H), 1.79 (septet, J = 6.45, 6.74 Hz, 1H),
2.44 (d, J = 7.03 Hz, 2H), 3.68 (s, 2H), 5.61 (d, J = 9.96 Hz, 1H), 6.32
(d, J = 9.96 Hz, 1H), 6.73 (d, J = 8.20 Hz, 1H), 6.96 (d, J = 2.05 Hz,
1H), 7.05 (dd, J = 2.05, 7.91 Hz, 1H).
1-Cyclopropyl-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-
methamine (8d). Yield = 77% in two steps. 1H NMR (CDCl3): δ 0.13
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N-(Cyclopropylmethyl)-N-[(2,2-dimethyl-2H-chromen-6-yl)-
(d, J = 9.96 Hz, 1H), 6.29 (d, J = 9.67 Hz, 1H), 6.71 (d, J = 8.20 Hz,
1H), 6.93−6.94 (m, 2H), 7.04 (dd, J = 2.34, 8.20 Hz, 1H), 7.08 (d, J =
0.88 Hz, 1H). HRMS (m/z): [M + Na]+ calcd 412.16654, found
412.16699. HPLC-1: tR = 5.8 min, purity = 100%. HPLC-2: tR = 8.7 min,
purity = 97%.
methyl]pyridine-2-sulfonamide (6d). Purification was by silica gel
1
chromatography with ethyl acetate/hexane (1/2). Yield = 24%. H
NMR (CDCl3): δ 0.0115 (d, J = 5.27 Hz, 2H), 0.301 (d, J = 7.62 Hz,
2H), 0.67−0.825 (m, 1H), 1.425 (s, 6H), 3.17 (d, J = 6.74 Hz, 2H),
4.58 (s, 2H), 5.61 (d, J = 9.67 Hz, 1H), 6.28 (d, J = 9.67 Hz, 1H), 6.69
(d, J = 8.21 Hz, 1H), 6.975 (s, 1H), 7.04 (d, J = 7.91 Hz, 1H), 7.47
(dd, J = 4.98, 7.62 Hz, 1H), 7.88 (t, J = 7.62 Hz, 1H), 7.99 (d, J =
7.62 Hz, 1H), 8.71 (d, J = 4.69 Hz, 1H). HRMS (m/z): [M + Na]+
calcd 407.13999, found 407.14046. HPLC-1: tR = 5.7 min, purity =
100%. HPLC-2: tR = 7.5 min, purity = 95%.
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(2-methylpropyl)-
1,3-thiazole-2-sulfonamide (6k). Purification was by silica gel
1
chromatography with ethyl acetate/hexane (1/2). Yield = 17%. H
NMR (CDCl3): δ 0.79 (d, J = 6.45 Hz, 6H), 1.42 (s, 6H), 1.77−1.81
(m, 1H), 3.12 (d, J = 7.62 Hz, 2H), 4.43 (s, 2H), 5.62 (d, J = 9.96 Hz,
1H), 6.26 (d, J = 9.67 Hz, 1H), 6.68 (d, J = 8.20 Hz, 1H), 6.87 (d, J =
2.34 Hz, 1H), 6.97 (dd, J = 2.34, 8.20 Hz, 1H), 7.58 (d, J = 3.22 Hz,
1H), 7.95 (d, J = 3.22 Hz, 1H). HRMS (m/z): [M + Na]+ calcd
415.11206, found 415.11253. HPLC-1: tR = 8.0 min, purity = 100%.
HPLC-2: tR = 10 min, purity = 89%.
N-(Cyclobutyl)-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-
pyridine-2-sulfonamide (6e). Purification was by silica gel chromatog-
raphy with ethyl acetate/hexane (1/2). Yield = 65%.
1H NMR (CDCl3): δ 1.42 (s, 6H), 1.42−2.04 (m, 6H), 4.31−4.39
(m, 1H), 4.51 (s, 2H), 5.61 (d, J = 9.96 Hz, 1H), 6.30 (d, J = 9.67 Hz,
1H), 6.69 (d, J = 8.20 Hz, 1H), 7.00 (s, 1H), 7.06 (dd, J = 2.05, 8.205 Hz,
1H), 7.43−7.48 (m, 1H), 7.82−7.94 (m, 2H), 8.69 (d, J = 3.81 Hz, 1H).
HRMS (m/z): [M + Na]+ calcd 407.13999, found 407.14034. HPLC-1:
tR = 5.6 min, purity = 100%. HPLC-2: tR = 7.1 min, purity = 96%.
N-(Cyclopentyl)-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-
pyridine-2-sulfonamide (6f). Purification was by silica gel chromatog-
raphy with ethyl acetate/hexane (1/4). Yield = 16%. 1H NMR
(CDCl3): δ 1.26−1.59 (m, 8H), 1.43 (s, 6H), 4.31−4.40 (m, 1H),
4.48 (s, 2H), 5.61 (d, J = 9.67 Hz, 1H), 6.30 (d, J = 9.67 Hz, 1H), 6.69
(d, J = 8.21 Hz, 1H), 7.03 (s, 1H), 7.07 (d, J = 8.50 Hz, 1H), 7.44−
7.48 (m, 1H), 7.83−7.94 (m, 2H), 8.71 (d, J = 4.69 Hz, 1H). HRMS
(m/z): [M + Na]+ calcd 421.15564, found 421.15577. HPLC-1: tR =
7.3 min, purity = 100%. HPLC-2: tR = 8.4 min, purity = 96%.
N-[(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)methyl]pyridine-2-
sulfonamide (6g). Purification was by silica gel chromatography with
8-Hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde (9). 3,4-
Dihydroxybenzaldehyde (2.3 g, 17 mmol) and 3-methylbut-2-enal
(1 mL, 10 mmol) were dissolved in 3 mL of pyridine and refluxed
overnight. Pyridine was evaporated under vacuum, and the crude
reaction mixture was dissolved in methylene chloride. 9 (103 mg,
2.4%) was purified with silica gel chromatography with ethyl acetate/
1
hexane (1/50). H NMR (CDCl3): δ 1.50 (s, 6H), 5.70 (d, J = 10.1
Hz, 1H), 6.38 (d, J = 10.1 Hz, 1H), 7.15 (d, J = 2.14 Hz, 1H), 7.32
(d, J = 1.83 Hz, 1H), 9.78 (s, 1H).
8-(Methoxymethoxy)-2,2-dimethyl-2H-chromene-6-carbalde-
hyde (10). Chloromethyl methyl ether (4 mL, 5 mmol) was added
to a solution of 9 (103 mg, 0.5 mmol) and N,N-diisopropylethylamine
(0.2 mL, 1 mmol) in 4 mL of methylene chloride. The reaction mixture
was refluxed overnight and concentrated in vacuo. 10 (48 mg, 38%) was
purified with ethyl acetate/hexane (1/50). 1H NMR (CDCl3): δ 1.52 (s,
6H), 3.54 (s, 3H), 5.25 (s, 2H), 5.71 (d, J = 9.67 Hz, 1H), 6.38 (d, J =
9.96 Hz, 1H), 7.26 (d, J = 1.76 Hz, 1H), 7.52 (d, J = 1.76 Hz, 1H), 9.80
(s, 1H).
N-[(8-Methoxymethoxy-2,2-dimethyl-2H-chromen-6-yl)methyl]-
2-methylpropan-1-amine (11). 2-Methylpropan-1-amine (0.5 mL, 5
mmol) and p-toluenesulfonic acid monohydrate (110 mg, 0.6 mmol)
were added to a solution of 10 (143 mg, 0.6 mmol) in 5 mL of
methylene chloride. The reaction mixture was refluxed overnight
and concentrated in vacuo. Disappearance of the aldehyde peak at
9.80 ppm (s, 1H) and appearance of the imine peak at 8.07 ppm (s, 1H)
were confirmed by NMR.
The crude product was dissolved in 3 mL of a mixture of methylene
chloride and toluene [1/2 (v/v)], to which 1 M DIBAL in toluene
(3 mL, 3 mmol) was added slowly to generate gentle bubbling. The
reaction mixture was stirred for 5 h and 20 mL of 1 N aqueous HCl
was added. The aqueous phase was extracted by methylene chloride
(40 mL × 3). The combined organic layer was washed by brine, dried
with magnesium sulfate, and then concentrated in vacuo.
11 (22 mg, 13%) was purified by silica gel chromatography with
triethylamine/methanol/methylene chloride (1/3/100). 1H NMR
(CDCl3): δ 0.91 (d, J = 3.13 Hz, 6H), 1.46 (s, 6H), 1.75−1.82
(m, 1H), 2.43 (d, J = 7.04 Hz, 2H), 3.53 (s, 3H), 3.66 (s, 2H), 5.20
(s, 2H), 5.62 (d, J = 9.78 Hz, 1H), 6.30 (d, J = 9.78 Hz, 1H), 6.69 (d,
J = 1.96 Hz, 1H), 6.96 (d, J = 1.96 Hz, 1H).
N-[(8-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)methyl]-N-(prop-
an-2-yl)pyridine-2-sulfonamide (6h). 11 (30 mg, 0.1 mmol),
pyridine-2-sulfonyl chloride (57 mg, 0.3 mmol), and N,N-diisopropy-
lethylamine (0.2 mL, 1 mmol) were dissolved in 1 mL of methylene
chloride and stirred at room temperature overnight. A completion of
the reaction was confirmed by disappearance of the amine spot by
TLC with triethylamine/methanol/methylene chloride (1/3/100) and
appearance of the product spot by TLC with ethyl acetate/hexane
(1/1). N-[(8-Methoxymethoxy-2,2-dimethyl-2H-chromen-6-yl)-
methyl]-N-(propan-2-yl)pyridine-2-sulfonamide (31 mg, 70%) was
purified by silica gel chromatography with ethyl acetate/hexane (1/1).
1H NMR (CDCl3): δ 0.78 (d, J = 6.45 Hz, 6H), 1.45 (s, 6H), 1.72−
1
ethyl acetate/hexane (2/1). Yield = 23%. H NMR (CDCl3): δ 1.05
(d, J = 6.74 Hz, 6H), 1.47 (s, 6H), 3.84 (s, 3H), 4.27 (septet, J = 6.74
Hz, 1H), 4.45 (s, 2H), 5.61 (d, J = 9.96 Hz, 1H), 6.27 (d, J = 9.96 Hz,
1H), 6.62 (s, 1H), 6.88 (s, 1H), 7.43−7.48 (m, 1H), 7.82−7.93 (m,
2H), 8.70 (d, J = 4.40 Hz, 1H). HRMS (m/z): [M + Na]+ calcd
425.15055, found 425.15089. HPLC-1: flow rate 2 mL/min, t = 2.9
R
min, purity = 97%; flow rate 1 mL/min, tR = 5.6 min, purity = 99%.
HPLC-2: tR = 5.1 min, purity = 100%.
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(2-methylpropyl)-
pyridine-4-sulfonamide (6i). Pyridine-4-sulfonyl Chloride. Pyridine-
4-sulfonyl, 1,3-thiazole-2-sulfonyl, and 1-methyl-1H-imidazole-2-
sulfonyl chloride were synthesized by the same method as that of
pyridine-2-sulfonyl chloride. They were used immediately after
synthesis due to their instabilities at temperatures above 0 °C. Ten
theoretical equivalents of the sulfonyl chloride were synthesized, which
were extracted with 10 mL of cold methylene chloride, washed with
cold brine, and then dried with anhydrous magnesium sulfate. The
sulfonyl chloride solution in methylene chloride was immersed in ice−
water bath and concentrated with argon flow until the volume was
reduced to 3 mL.
8a (25 mg, 0.1 mmol) was dissolved in 0.8 mL of methylene
chloride, to which 0.5 mL of N,N-diisopropylethylamine (DIEA,
0.5 mL, 3 mmol) was added and cooled to 0 °C. Ten equivalents of
freshly prepared pyridine-4-sulfonyl chloride was added to the reaction
mixture slowly, which was stirred at 0 °C for 3 h with slow argon flow.
6i (25 mg, 63%) was purified by silica gel chromatography with ethyl
acetate/hexane (1/5). 1H NMR (CDCl3): δ 0.78 (d, J = 6.74 Hz, 6H),
1.43 (s, 6H), 1.72−1.82 (m, 1H), 2.96 (d, J = 7.62 Hz, 2H), 4.27 (s,
2H), 5.62 (d, J = 9.96 Hz, 1H), 6.21 (d, J = 9.96 Hz, 1H), 6.68 (d, J =
7.91 Hz, 1H), 6.77 (d, J = 2.05 Hz, 1H), 6.91 (dd, J = 2.05, 8.50 Hz,
1H), 7.63 (d, J = 5.86 Hz, 1H), 8.82 (d, J = 6.15 Hz, 1H). HRMS
(m/z): [M + Na]+ calcd 386.16641, found 387.17447. HPLC-1: tR =
6.5 min, purity = 98%. HPLC-2: tR = 8.4 min, purity = 96%.
6j and 6k were synthesized by the same method as 6i.
N-[(2,2-Dimethyl-2H-chromen-6-yl)methyl]-N-(2-methylpropyl)-
1-methyl-1H-imidazole-2-sulfonamide (6j). Purification was by silica
gel chromatography with ethyl acetate/hexane (1/2). Yield = 58%.
1H NMR (CDCl3): δ 0.77 (d, J = 6.45 Hz, 6H), 1.43 (s, 6H), 1.63−1.70
(m, 1H), 3.20 (d, J = 7.62 Hz, 1H), 3.90 (s, 3H), 4.47 (s, 2H), 5.62
1.76 (m, 1H), 3.11 (d, J = 7.62 Hz, 2H), 3.50 (s, 3H), 4.40 (s, 2H),
5.14 (s, 2H), 5.62 (d, J = 9.96 Hz, 1H), 6.23 (d, J = 9.67 Hz, 1H), 6.60
(d, J = 1.76 Hz, 1H), 6.87 (d, J = 1.76 Hz, 1H), 7.43−7.47 (m, 1H),
7.84−7.96 (m, 2H), 8.68−8.7 (m, 1H).
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N-[(8-Methoxymethoxy-2,2-dimethyl-2H-chromen-6-yl)methyl]-N-
(propan-2-yl)pyridine-2-sulfonamide (31 mg, 0.07 mmol) was
dissolved in a cosolvent of tetrahydrofuran (1 mL) and 6 N aqueous
HCl (1 mL), and the reaction stirred for 3 h. Ten milliliters of water
was added to the reaction mixture, and then tetrahydrofuran was
evaporated. The remaining aqueous phase was extracted by methylene
chloride (10 mL × 3), washed with brine, dried with anhydrous
magnesium sulfate, and concentrated in vacuo. 6h (19 mg, 49%) was
purified by silica gel chromatography with ethyl acetate/hexane (1/1).
1H NMR (CDCl3): δ 0.77 (d, J = 6.45 Hz, 6H), 1.44 (s, 6H), 1.72−
1.79 (m, 1H), 3.11 (d, J = 7.62 Hz, 2H), 4.39 (s, 2H), 5.61 (d, J = 9.67
Hz, 1H), 6.25 (d, J = 9.96 Hz, 1H), 6.50 (d, J = 1.76 Hz, 1H), 6.65 (d,
J = 1.76 Hz, 1H), 7.43−7.47 (m, 1H), 7.84−7.97 (m, 2H), 8.69 (d, J =
4.40 Hz, 1H). HRMS (m/z): [M + Na]+ calcd 425.15055, found
reference compounds to obtain coefficients of a linear regression. log
P7.4 values of all other compounds were obtained by the linear
regression equation constructed from the reference compounds.
4.2.2. Aqueous Solubility Measurements. 1 (1−2 mg) was placed
in a 10 mL glass tube, to which 2 mL of distilled water was added. The
suspension was sonicated for 30 min at room temperature and then
centrifuged at 1500g at 20 °C for 5 min. The liquid was transferred to a
clean glass tube and then filtered through a polytetrafluoroethylene
(PTFE) filter (pore size 0.2 μm) slowly. Then 500 μL or 1 mL of the
filtered clear solution was injected into the HPLC system, and peak areas
at 254 nm were quntified using the standard curves described above.
Three independent experiments were performed, and the average value
was calculated. The same procedure was used for 6a, 6d, and 6g.77
The aqueous solubilities of 1, 6a, and 6d, were also independently
measured by laser nephelometry. The compounds and an internal
control were dissolved in 100% of DMSO to obtain a final
concentration of 30 mg/mL to make stock solutions. A stock solution
was serially diluted (concentration profile 30, 20, 15, 10, 7.5, 5, 2.5,
1.25, 0.63, 0.31, and 0.15 mg/mL) in test tubes with 100% of DMSO.
The concentration profile was transferred to 96-well microplates
(Costar black clear bottom) and serially diluted to a final
concentration of 1% of DMSO and a final drug concentration of
300, 200, 150, 100, 75, 50, 25, 12, 6, 3, and 1.5 μg/mL with phosphate-
buffered saline (pH 7.4, 5.0, or 3.0). The microplates were incubated
for 90 min at ambient temperature. Laser nephelometry (NEPH-
ELOstar, BMG Lab Technologies) was used to determine the point at
which the solute began to precipitate out of solution.
425.15091. HPLC-1: flow rate 2 mL/min, t = 2.9 min, purity =
R
100%; flow rate 1 mL/min, tR = 5.6 min, purity = 100%. HPLC-2: tR =
5.172 min, purity = 100%.
N-[(8-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)methyl]-N-(prop-
an-2-yl)pyridine-4-sulfonamide (6l). 6l was synthesized by the same
method as 6h. Yield in two steps = 48%. 1H NMR (CDCl3): δ 0.80 (d,
J = 6.74 Hz, 6H), 1.45 (s, 6H), 1.75−1.82 (m, 1H), 2.98 (d, J = 7.62
Hz, 2H), 4.24 (s, 2H), 5.62 (d, J = 9.67 Hz, 1H), 6.22 (d, J = 9.67 Hz,
1H), 6.39 (s, 1H), 6.60 (s, 1H), 7.62 (d, J = 5.86 Hz, 1H), 8.81 (d, J =
4.98 Hz, 1H). HRMS (m/z): [M + Na]+ calcd 402.16133, found
403.16924. HPLC-1: flow rate 2 mL/min, tR = 2.7 min, purity = 98%;
flow rate 1 mL/min, tR = 5.3 min, purity = 98%. HPLC-2: tR = 5.532 min,
purity = 100%.
4.2. Physicochemical Property Measurements. HPLC was
measured with a Waters 1525 binary HPLC pump coupled with a
Waters 2487 Dual λ absorbance detector, and the data were processed
by Waters Breeze GPC Software; 254 and 280 nm were used for
UV absorbance measurements. A Symmetry C18 (WAT045905, 5 μm,
4.6 × 150 mm) column was used with the eluent (70:30:0.1 =
methanol:water:triethylamine) and a flow rate of 2 mL/min. Standard
curves of 1, 6a, 6d, and 6g were constructed by injection of 20, 40, 60,
80, and 100 μL of standard solutions in 100% of methanol (0.01 mg/
mL, 100% of methylene chloride for 1) into the HPLC system. 1-
Octanol (99%) was purchased from Alfa Aesar and phosphate buffer
(0.2 M, pH 7.4) was purchased from Electron Microscopy Sciences.
4.2.1. log P7.4 Measurements.66 log P7.4 values of 6a and 6g were
measured according to the “OECD Guideline for Testing of Chemicals
107 adopted by the Council on July 27, 1995” (Method 1), and they
were used as two reference materials for the rest of the compounds
for which log P7.4 values were measured by “OECD Guideline for
Testing of Chemicals 117 adopted by the Council on April 13, 2004”
(Method 2). The first method was more direct but too complicated to
be applied for all compounds, so it was used only for 6a and 6g, which
were then used as reference materials with aniline, benzene, toluene,
and chlorobenzene in method 2 to increase its reliability.
Method 1. 1-Octanol (70 mL) was stirred vigorously with 30 mL
of phosphate buffer at room temperature for 24 h, and the aqueous
layer was discarded. The 1-octanol layer was washed with 30 mL of
phosphate buffer. The same procedure was applied to 70 mL of
phosphate buffer and 30 mL of 1-octanol. One to two milligrams of 6a
(or 6g) was dissolved in 2 mL of 1-octanol saturated with phosphate
buffer in 20 mL glass tubes in duplicate. Two milliliters of phosphate
buffer saturated with 1-octanol was added to each tube, which were then
tightly sealed with a cover. The mixtures were mechanically shaken for
15 min. The two layers were separated by centrifugation at 1500g for
10 min, 1 mL of each layer was transferred to a glass vial, and 0.1 mL
of the 1-octanol layer was mixed with 5 mL of water and 10 mL of
HPLC eluent (70:30:0.1 = methanol:water:triethylamine) to eliminate the
1-octanol effect on the retention time of HPLC, of which 0.5 mL was
injected into the HPLC system. One-half milliliter of the aqueous layer
was injected into the HPLC system. Peak areas at 254 nm were quantified
by standard curves described above and used for the log P7.4 calculations.
Method 2. All 13 compounds (1, 6a−6l), aniline, benzene, toluene,
and chlorobenzene were injected into the HPLC system described
above. Retention times for solvent and each compound were obtained.
Aniline, benzene, toluene, chlorobenzene, 6a, and 6g were used as
4.3. Metabolism. 1, 6a, 6d, 6g, and 6h (20 μM) were individually
incubated with fresh mouse plasma or liver homogenate in phosphate-
buffered saline (pH 7.4) [1/2 (w/v)] at 37 °C with constant shaking.
At predetermined time points, 50 μL of plasma or liver homogenate
sample was collected and extracted with 100 μL of acetonitrile. The
supernatant was reduced to dryness under vacuum overnight and
reconstituted in the HPLC mobile phase. The concentrations of each
compound were individually determined using reverse phase HPLC
methodology. The HPLC system (Waters, Milford, MA) consisted
of a Waters 2795 pump, a Phenomenex (Torrance, CA) C8 column
(5 μm, 4.6 × 150 mm), and a Waters 996 photodiode array detector.
As an internal standard, α-naphthoflavone was used at a concentration
of 2 μM. The detection wavelengths for these compounds were as
follows: 1 at 250 nm, both 6a and 6d at 264 nm, both 6g and 6h at
268 nm, and α-naphthoflavone at 281 nm. The linear range for the
standard curves of all compounds was between 1 and 50 μM. All
studies were done in triplicates.
4.4. HIF-Dependent Luciferase Reporter Assays. The LN229-
V6R and LN229-Lux cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) (Mediatech, Herdon, VA) with 10% serum
and antibiotics as previsouly described.46 The cells (3 × 104 in 300 μL
of DMEM medium) were seeded in each well of 48-well plates, and
cultured under normoxia (21% O2, 5% CO2, and 74% N2) for 24 h.
Subsequently, the medium of each well was replaced with 300 μL of
DMEM medium with 1% of DMSO, without (control) or with
heteroarylsulfonamides, 6a, 6g, and 6l (at 2.5, 5.0, and 10.0 μM); the
plates were further incubated at 37 °C for 1 h and then transferred to a
Steri-cycle CO2 incubator (Thermo Forma, Model 370) under hypoxia
(1% O2, 5% CO2, and 74% N2) at 37 °C for another 24 h. The cells
were washed with PBS buffer, and a cell extract was prepared after
lysing them with 40 μL/well of lysis buffer (Promega, part# E1944) for
10 min. Protein suspensions (20 μL) from each well were mixed with
25 μL of luciferase assay substrate (Promega, Part# E151A), and
luminescence was measured using a 20/20n luminometer (Turner
BioSystems Inc.).46 Assays were performed in triplicates.
4.5. Detection of Cellular HIF-1α Protein Levels by Western
Blot Analyses. Western blots were prepared as previously
described.46 Briefly, LN229-V6R cells (2 × 105 in 2 mL of DMEM)
were seeded in 35 mm cell culture dishes and cultured under normoxia
for 24 h, and then new DMEM medium was added with 1% of
DMSO and heteroarylsulfonamides 6a, 6g, and 6l (at 3, 6, 12, 25, 50,
or 100 μM). Dishes were incubated under normoxia at 37 °C for 1 h
and then transferred to hypoxia (1% O2) for 24 h.
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Cells were lysed with 1× loading buffer [62.5 mM Tris-HCl (pH 6.8
at 25 °C), 2% (w/v) SDS, 50 μM 2-mercaptoethanol, 10% glycerol,
0.01% bromophenol blue], and proteins were separated by electro-
phoresis in a 4−12.5% Tris-HCl gradient gel (Bio-Rad, Richmond,
CA). The proteins were transferred onto a nitrocellulose membrane
and immunoblotted using anti-HIF-1α antibody (1:500 dilution BD
biosciences, San Diego, CA) and anti-β-actin antibody (1:3000
dilution, Santa Cruz Biotechnologies, Santa Cruz, CA). The secondary
horseradish peroxide-conjugated antibodies were added and horse-
radish peroxide activities were measured by enhanced chemilumines-
cence (Pierce, Rockford, IL).
The authors greatly appreciate Dr. Ronald Voll and Dr. Stefan
Kaluz for helpful discussions.
ABBREVIATIONS USED
■
HIF-1, hypoxia inducible factor-1; HRE, hypoxia response
element; LN229-V6R, LN229 cells transfected with a vector,
pBI-GL HRE V6R that contains a HRE-driven promoter and a
luciferase reporter gene; DMSO, dimethyl sulfoxide; NMP,
N-methyl-2-pyrrolidone
Western blotting of each heteroarylsulfonamide (6a, 6g, or 6l) was
repeated at least two times.
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ASSOCIATED CONTENT
■
S
* Supporting Information
NMR, HPLC, TLC, metabolic stability curve, and cytotoxicity
curves of 12 N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)-
methyl]heteroarylsulfonamides. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*M.G.: phone, (404) 727-9366; fax, (404) 712-5689; e-mail,
mgoodma@emory.edu. E.G.V.M.: phone, (404) 778-5563; fax,
(404) 778-5550; e-mail, evanmei@emory.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institute of Health
grants P50 CA128301-01A1, Emory Molecular and Transla-
tional Imaging Research Center (EMTIC)/In vivo Cellular and
Molecular Imaging Centers (ICMIC) (to M.M.), P50
CA128301-01A1 Pilot Project #2 (to J.M.), R01 CA116804
(to E.G.V.M.), and V Foundation, Max Cure, and Samuel
Waxman Cancer Research foundation grants (to E.G.V.M.).
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