Synthesis and structure-activity relationship of (E)-phenoxyacrylic amide derivatives as Hypoxia-Inducible Factor (HIF) 1α inhibitors

Article abstract of DOI:10.1021/jm301419d

A series of (E)-phenoxyacrylic amide derivatives were synthesized and evaluated as hypoxia inducible factor (HIF) 1α inhibitors. The present structure-activity relationship study on this series identified the morpholinoethyl containing ester 4p as a potent inhibitor of HIF-1α under hypoxic conditions (IC₅₀ = 0.12 μM in a cell-based HRE reporter assay) in HCT116 cells. The representative compound 4p suppressed hypoxia-induced HIF-1α accumulation and targeted gene expression in a dose-dependent manner. The effect of HIF-1α inhibition by 4p was further demonstrated by its inhibitory activity on in vitro tube formation and migration of cells, which may be valuable for development of novel therapeutics for cancer and tumor angiogenesis.

Full text of DOI:10.1021/jm301419d

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationship of (E)‑Phenoxyacrylic
Amide Derivatives as Hypoxia-Inducible Factor (HIF) 1α Inhibitors
Ravi Naik,†,∥ Misun Won,‡,∥ Bo-Kyung Kim,‡,∥ Yan Xia,† Hyun Kyung Choi,† Guanghai Jin,†
Youngjin Jung,# Hwan Mook Kim,§ and Kyeong Lee*,†
†College of Pharmacy, Dongguk UniversitySeoul, Seoul 100-715, Republic of Korea
‡Medical Genomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea
#Functional Genomics, Korea University of Science and Technology, Daejeon 305-350, Republic of Korea
§College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea
S
* Supporting Information
ABSTRACT: A series of (E)-phenoxyacrylic amide derivatives were
synthesized and evaluated as hypoxia inducible factor (HIF) 1α
inhibitors. The present structure−activity relationship study on this
series identified the morpholinoethyl containing ester 4p as a potent
inhibitor of HIF-1α under hypoxic conditions (IC50 = 0.12 μM in a
cell-based HRE reporter assay) in HCT116 cells. The representative
compound 4p suppressed hypoxia-induced HIF-1α accumulation and
targeted gene expression in a dose-dependent manner. The effect of
HIF-1α inhibition by 4p was further demonstrated by its inhibitory
activity on in vitro tube formation and migration of cells, which may
be valuable for development of novel therapeutics for cancer and
tumor angiogenesis.
Recently, considerable efforts have been directed toward the
discovery of HIF-1 inhibitors using chemical libraries and
natural products.7−15 HIF-1 inhibitors reportedly regulate the
HIF-1 signaling pathway through a variety of molecular
mechanisms, including transcriptional regulation, mRNA trans-
lation, nuclear translocation, and HIF-1α degradation.
Our group has focused on the development of small
molecule inhibitors targeting HIF-1α protein in solid tumors,
which has resulted in several synthetic leads, 1−3, as
summarized in Figure 1.10−12,16,17 To identify more potent
and efficient HIF-1α inhibitors, we performed structural
modifications on 1 and 2, including a phenyl ring substituted
with an adamantyl group (A-ring) and a N-aromatic ring (B-
ring) linked with oxyacetylamide. In the current work, we focus
on modifications that include replacement of the oxy-
acetylamide linker portion with a more conformationally
constrained oxyacrylic amide linker, as represented by 4. We
present data from the synthesis and evaluation of (E)-
phenoxyacrylic amide based compounds as potential HIF-1α
inhibitors.
INTRODUCTION
■
Hypoxia inducible factor (HIF) 1 is a transcription factor that
functions as a mast regulator in the response of growing tumor
to hypoxia. HIF-1 activates genes that allow the cancer cells to
survive and grow in hypoxic tumor environment. HIF-1 is a
heterodimeric protein that consists of a constitutively expressed
β-subunit, also called aryl hydrocarbon nuclear translocator
(ARNT), and an α-subunit whose expression is regulated by O2
levels.1,2 Under normoxic condition, HIF-1α protein is
subjected to degradation via the von Hippel−Lindau tumor
suppressor gene product (pVHL) mediated ubiquitin−
proteosomal pathway. Under hypoxic conditions, HIF-1α
protein escapes proteolysis and rapidly accumulates in cells,
which allows dimerization of HIF-1α and -1β subunits and
subsequent binding to a cis-acting regulatory element referred
to as hypoxia response element (HRE, 5′-RCGTG-3′) in the
promoter region of a number of target genes, including vascular
epidermal growth factor (VEGF) and erythropoietin (EPO).3
Thus, HIF-1α plays a critical role during cancer development
by regulating the expression of various genes associated with
tumor angiogenesis, metastasis, invasion, proliferation, and
apoptosis. Overexpression of HIF-1α has been observed in
human cancers including brain, breast, colon, lung, ovary, and
prostate cancers,4 and HIF-1α has been implicated in resistance
to treatment and poor prognosis in the hypoxic region around
solid tumors. Accordingly, these findings have validated HIF-1α
as an attractive target for treatment of cancer and tumor
angiogenesis.5,6
RESULTS AND DISCUSSION
■
Chemistry. The key intermediate (E)-phenoxyacrylic acid
derivatives 9a−f were synthesized from the phenol 7a−d,
according to the procedures in the literature and shown in
Received: August 14, 2012
© XXXX American Chemical Society
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Figure 1. Structures of HIF-1α inhibitors.
a
Scheme 1. Synthesis of the Key Intermediates 9a−f
a
Reagents and conditions: (a) 1-methoxy-2-methylbenzene, Pd/C, K2CO3, reflux; (b) BBr3, MC, −10 °C; (c) PPh3, methyl propiolate, 115 °C; (d)
LiOH, THF/H2O; (e) (i) K2CO3, methyl 4-bromobutanoate, DMF; (ii) LiOH, THF/H2O; (f) (i) Pd/C, H2, MeOH; (ii) LiOH, THF/H2O.
a
Scheme 2. Synthesis of (E)-Phenoxyacrylic Amide Derivatives 4a−g and 4j−k
a
Reagents and conditions: (a) HATU, DIPEA, DMF.
Scheme 1.18 After the hydroxyl group of the phenol 7a−d was
subjected to alkylation with methyl propiolate in the presence
of PPh3 in toluene, the resulting (E)-phenoxyacrylic methyl
ester 8a−d was hydrolyzed with lithium hydroxide to form 9a−
d. The necessary 7b, which is not commercially available, was
prepared in two steps, including alkylation of 1-bromoada-
mantane 5 with 1-methoxy-2-methylbenzene in the presence of
palladium followed by demethylation of 6 using boron
tribromide with good yields.
The propionic acid 9e and the butanoic acid 9f were
synthesized from 7a or 8a, respectively (Scheme 1). Saturation
of the phenoxy acrylic portion of 8a was conducted by
palladium carbon hydrogenation to provide 9e. For the
preparation of 9f with three-carbon linker, the phenol 7a was
B
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a
Scheme 3. Synthesis of (E)-Phenoxyacrylic Amide Derivatives 4i and 4n−r
a
Reagents and conditions: (a) LiOH, THF/H2O for 4i; (b) alkyl halides, K2CO3 for 4n−q; amines, HATU, DIPEA, DMF for 4r.
a
Scheme 4. Synthesis of (E)-Phenoxyacrylic Amide Derivatives 4h, 4l,m, and 4s−v
a
Reagents and conditions: (a) MeOH, SOCl2 for 14c; (b) 1-bromo-2-methoxyethane, K2CO3, Cs2CO3 for 13e; amines, EDCI, HOBT, DIPEA for
13f−i; (c) Pd/C, MeOH for 14e−i; (d) 9a, HATU, DIPEA, DMF.
Table 1. In Vitro HIF-1 Inhibitory Activity and Cytotoxicity
of Linker Modified Derivatives
alkylated with methyl 4-bromobutanoate in the presence of
potassium carbonate in DMF and then hydrolyzed to give 9f.
With preparation of the key intermediates 9a−f, the (E)-
phenoxyacrylic amide derivatives were prepared by the methods
shown in Schemes 2−4. Compounds with a variety of
substitution patterns on the A- and B-rings or with different
linkers (4a−g and 4j−k) were synthesized by direct amide
coupling of the requisite anilines 10a−d with the newly
synthesized acids 9a−f using HATU as coupling agent (Scheme
2). Additionally, (E)-4-(1-adamantyl)phenoxyacrylic amide 4c
was converted to the corresponding carboxylic acid 4i using
lithium hydroxide and then further reacted with alkyl halides or
anilines to provide the various ester and amide analogues 4n−r,
as shown in Scheme 3.
More esters and amides with different substitution patterns
on the B-ring (4h, 4l,m, and 4s−v) were prepared starting from
5-amino-2-hydroxybenzoic acid 11 or 3-nitrobenzoic acid 12 as
described in Scheme 4. 2-Amino-5-hydroxybenzoic acid 11 was
transformed to 14c in the presence of thionyl chloride and
methanol, whereas 3-nitrobenzoic acid 12 was converted to the
substituted ester 13e using 1-bromo-2-methoxyethane and to
amides 13f−i using various amines under standard coupling
conditions, which were further reduced at the nitro group to
produce anilines 14e−i. With the addition of the commercially
available 3-aminobenzoic acid ethyl ester 14d, a series of
anilines 14c−i were coupled with the carboxylic acid 9a in the
presence of HATU and DIPEA to produce the final (E)-4-(1-
a
Values are the mean of three experiments.
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Table 2. In Vitro HIF-1 Inhibitory Activity and Cytotoxicity of (E)-Phenoxyacrylic Amide Derivatives
a
Values are the mean of three experiments.
adamantyl)phenoxyacrylic amide derivatives 4h, 4l,m, and 4s−
v, respectively.
a more rigid conformation in the linker, resulted in an apparent
increase in inhibitory activity (IC50 = 0.74 μM), as shown in
Table 1. Encouraged by this result with 4c, we further extended
the synthesis of the series characterized by the (E)-4-(1-
adamantyl)phenoxyacrylic amide moiety.
We previously defined the key structural requirements for
HIF inhibition of aryloxyacetylbenzoic acid derivatives that
involve the adamantyl group in the A-ring.10 Because structural
changes in the linker portion may affect those requirements,
our next approach was to confirm the importance of the
adamantyl group attached to the A-ring. The replacement of
the adamantane ring with the smaller tert-butyl (4d) or octyl
(2,4,4-trimethylpentan-2-yl) moiety (4e) on the 4-phenoxy
group resulted in reduction or maintenance of HIF-1 activity
(IC50 of 6.03 and 1.04 μM, respectively), indicating the
importance of the lipophilic group such as the adamantyl on the
A-ring.
Among the compounds based on the (E)-4-(1-adamantyl)-
phenoxyacrylic amide structure, the methyl carboxylic ester
containing analogue 4c at the meta-position of ring B was more
active (IC50 = 0.74 μM) than the corresponding para-position
analogue 4f (IC50 = 2.17 μM), which suggested that the ester at
the meta-position positively affected HIF inhibition. Addition
of a methyl substituent on phenyl ring A, as represented by 4g,
Cell-Based HRE Reporter Assay. The newly synthesized
compounds were evaluated for their potential to inhibit
hypoxia-induced accumulation of HIF-1α using a cell-based
HRE reporter assay in the HCT116 cell line (Tables 1−3). All
of the assays were performed under standard assay conditions
by employing hypoxic conditions (1% O2, 94% N2, and 5%
CO2) and following the previously described assay protocol.19
Cell viability, as measured by the sulforhodamine B assay,
showed that most of the compounds had no significant
cytotoxicity at the concentrations at which they effectively
inhibited HIF-1 activation (>20 μM). Compound 1 (LW6, IC50
= 2.44 μM) was used as a positive control.10,13
We first examined the effect of increasing the length of the
carbon linker between rings A and B, which may affect the
overall conformation of the prototype compound, i.e., methyl
3-(2-(4-adamantylphenoxy)acetamido)benzoate 15 (Table
1).10 Addition of one carbon resulted in a 2-fold increase in
potency, as shown by the IC50 values (15 vs 4a giving 5.03 vs
2.54 μM, respectively). However, addition of two carbons
resulted in a loss of potency, as shown in the case of 4b.
Additionally, introduction of a double bond linking the
phenoxy group to the NH-phenyl portion (4c), which creates
D
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Because 4c displayed more inhibitory potency than LW6, we
explored the effects of altering the methyl group of the ester in
the B-ring with ethyl or substituted ethyl groups having a
phenoxtacrylamide core 4l−p. In general, the resulting ester
compounds, including the ethyl (4l), methoxyethyl (4m),
dimathylaminoethyl (4n), pyrrolininoethyl (4o), and morpho-
linoethyl ester (4p) compounds, possessed good to potent
activity, with IC50 in the low micromolar range. The
morpholine-substituted ethyl ester 4p was the most potent
(IC50 = 0.12 μM). It is noteworthy that 4p has a secondary
amine that helps improve its solubility in water. One of the
shortcomings of the previous lead compounds (e.g., LW6) was
their poor water solubility, which is a common issue with
synthetic chemicals and often leads to difficulties in the further
study of such compounds. Accordingly, the kinetic solubilities
of LW6, 4c, and 4p were determined using an established
nephlometry method,20,21 and the results are provided in Table
4. As expected, the secondary amine-containing 4p showed
much better water solubility at the three different pH
conditions than LW6 or 4a, which merits further evaluation
of this compound as a validated lead for the development of a
novel anticancer agent.
Table 3. In Vitro HIF-1 Inhibitory Activity and Cytotoxicity
of (E)-Phenoxyacrylic Amide Derivatives
In a similar fashion, a variety of benzamide derivatives with
the (E)-phenoxyacrylic amide core structure were also prepared
(4q−v) and evaluated for their HIF inhibitory activity. The free
benzamide 4q or secondary amide with a one or no carbon
spacer (4r,s) displayed poor activity (Table 3). Of interest, the
secondary amides on the B-ring, which feature insertion of two
or three carbons between heterocycles/amines and NH in the
amide portion (4t−v), restored the potency of the compound.
Compound 4u in which the secondary amide was substituted
with an ethylene morpholine chain (IC50 = 1.07 μM) was of
equal potency compared with the corresponding methyl ester
4c. The most active analogues, including 4p and 4u, have a
common substitution pattern with a morpholinoethyl group in
the ester or amide in the B-ring, which seems to contribute to
better biological activity in terms of the steric and electrostatic
aspects of the pharmacophore, as well as to increased water
solubility.
Inhibition of HIF-1α Accumulation by 4p. The
representative compound 4p was evaluated for its inhibitory
effect on HIF-1α accumulation under hypoxic conditions in
HCT116 colon cancer cells. As demonstrated by Western blot
analysis, 4p blocked HIF-1α accumulation in a dose-dependent
manner (Figure 2A). Compared to LW6, 4p inhibited hypoxia-
induced accumulation of HIF-1α protein at a lower
concentration of 50 nM. However, the expression of HIF-1β
was not significantly affected by the presence of 4p. The effects
of 4p on inhibition of HIF-1α accumulation in other cancer
cells such as colorectal cancer LoVo cells, lung cancer A549
cells, cervical cancer HeLa cells, and prostate cancer PC-3 cells
were investigated. As shown in Figure 2B, 4p blocked HIF-1α
accumulation in a dose-dependent manner in all of the cell lines
tested. Of note, 4p showed a greater effect on HIF-1α
expression in LoVo and A549 cells, which warrants further
mechanistic studies on the cell line specificity of this
compound.
a
Values are the mean of three experiments.
Table 4. In Vitro Water Solubility of the Representative
Compounds at pH 3, 7.4, and 9
a
solubility (μM)
compd
4c
pH 2
pH 7.4
pH 9
4.25 2.12
45.12 2.64
68.15 2.12
6.56 0.66
113.09 0.85
6.85 1.00
12.21 0.77
61.33 1.68
44.91 3.10
4p
1 (LW6)
a
Values are the mean of three experiments.
resulted in a 6-fold decrease in activity (IC50 = 4.12 μM) in
comparison with 4c. Similarly, an additional hydroxyl
substituent on the B-ring also did not positively affect activity,
as shown in 4h. Of note, introducing a sulfonic ester (4j) to the
aromatic ring B resulted in comparable potency (IC50 = 1.18
Previously, we presented evidence that LW6 promotes HIF-
1α degradation via up-regulation of VHL, which interacts with
prolyl-hydroxylated HIF-1α, leading to its ubiquitination and
subsequent proteasomal degradation.13 To examine whether 4p
has the same HIF-1α degradation signaling as LW6, a cell-based
HRE reporter assay was carried out in the presence of dipyridol
μM), whereas the sulfonamide 4k showed less activity (IC50
=
2.38 μM) than 4c. This result indicated that the sulfonic ester
group is a good isosteric replacement for the carboxylic ester
and yielded comparable potency.
E
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Figure 2. Effects of 4p on hypoxia-induced HIF-1α accumulation. (A) The accumulation of HIF-1α was examined in the cells treated with 4p at
various concentrations under hypoxia. LW6 was used as a control. (B) The effect of 4p on HIF-1α accumulation in various human cancer cell lines,
LoVo, A549, HeLa, and PC-3, was determined by Western blot analysis.
Figure 3. VHL-dependent HIF-1α degradation by 4p. (A) Effect of 4p on HRE-luciferase activity in the presence of DIP (dipyridol). The cells co-
transfected with pGL3-HRE-luciferase and pRL-SV40 were incubated for 24 h. After incubation of cells under hypoxic conditions for 4 h, they were
treated with 5 μM 4p or 0.4 μM 17-AAG in the presence of 200 μM DIP for 12 h under hypoxia. Luciferase activity was measured using Renilla as a
control. (B) Effect of 4p on VHL expression. HCT116 cells treated with 4p (0, 2, or 5 μM) under hypoxia were lysed with the RIPA buffer, and total
lysates were prepared.
(DIP), a prolyl hydroxylase inhibitor. Compound 4p did not
reduce HRE activity when prolyl hydroxylation of HIF-1α was
inhibited by 200 μM DIP, but 4p did reduce HRE activity in
the absence of DIP. However, 17-AAG, which leads to O2/
PHD/VHL-independent degradation of HIF-1α, reduced HRE
activity regardless of whether DIP was present (Figure 3A).
Furthermore, VHL expression was clearly increased in both
normoxic and hypoxic conditions (Figure 3B). This result
indicated that 4p can abolish the accumulation of HIF-1α in a
similar manner compared to LW6 by increasing VHL
expression, which leads to the subsequent ubiquitination and
proteasomal degradation of HIF-1α.
HIF-1α regulates transcription of target genes including
VEGF, EPO, and MMP-9, which are associated with an
aggressive tumor phenotype. To assess whether inhibition of
HIF-1α by 4p results in reduced expression of its target genes,
mRNA levels of VEGF, EPO, and MMP9 were examined
(Figure 4A). Compound 4p suppressed the hypoxia-induced
mRNA expression of VEGF, EPO, and MMP-9 in a dose-
dependent manner without affecting expression of HIF-1α
mRNA. Of particular interest, mRNA expressions of VEGF,
EPO, and MMP-9 were significantly suppressed by 2 μM 4p.
Because 4p inhibited the expression of VEGF and EPO,
which are involved in angiogenesis, an in vitro tube formation
assay was carried out using human umbilical vascular
endothelial cells (HUVECs). When HUVECs were grown on
a Matrigel surface, capillary-like structures such as those seen in
angiogenesis were observed (Figure 4B). Cells treated with the
positive control SU11248, a VEGFR inhibitor, did not form
capillary-like tube structures, which was indicative of the
inhibition of in vitro tube formation by HUVECs. Compound
4p also inhibited in vitro tube formation, suggesting that 4p has
inhibitory activity against angiogenesis. It was clear that the
growth of HUVECs was not affected by the presence of 4p or
SU11248 at the tested concentrations. Furthermore, a wound-
healing assay was carried out to determine whether 4p could
inhibit the migration of HCT116 cells (Figure 4C). In the
absence of 4p, the scratch wound was almost completely
covered by migrating cells after incubation for 24 h. However,
the scratch wound was not filled with cells in the presence of
4p, indicating that cell migration was inhibited by 4p (Figure
4C). SU11248, a positive control, inhibited migration of cells
and the scratch wound remained open, as expected.
CONCLUSIONS
■
We have synthesized a new series of (E)-phenoxyacrylic amide
derivatives as potential HIF-1α inhibitors. Among the
synthesized compounds, the morpholinoethyl ester analogue
4p exhibited the highest activity in a cell-based HRE reporter
assay in HCT116 cells and improved the solubility of the
compound in water. The representative compound 4p
exhibited an inhibitory effect on hypoxia-induced HIF-1α
accumulation via VHL-dependent HIF-1α degradation. Fur-
ther, 4p reduced target gene expression in a dose-dependent
manner, which was consistent with its inhibitory effect on in
vitro tube formation and migration of cells. The current work
F
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Figure 4. Effect of 4p on the mRNA levels of HIF-1 target genes. (A) The mRNA levels of HIF-1α and its target genes were determined by RT-PCR
in the cells treated with 4p. (B) Inhibition of in vitro tube formation assay. The 96-well plates were coated with ice-cold BD Matrigel matrix gel
solution, and human umbilical vein endothelial cells (HUVECs) were seeded. HUVECs were incubated on the Matrigel in the presence of 4p or
SU11248 under 1% O2 for 18 h, and tubule branches were photographed. At the same time, growth inhibition of HUVECs by 4p or SU11248 was
examined with the SRB assay. (C) A wound-healing assay was carried out using cell culture inserts. Migration of HCT116 cells treated with 4p or
SU11248 was observed for 24 h.
(3H, m, aromatic-H), 7.02 (2H, d, J = 8.7 Hz, aromatic-H), 5.68 (1H,
d, J = 11.7 Hz, CH), 3.90 (3H, s, CH3), 2.11 (3H, m, adamantly-H),
1.90 (6H, m, adamantly-H), 1.77 (6H, m, adamantly-H); MS (EI) m/z
431 (M+); HRMS (EI) m/z calcd for C27H29NO4 [M+] 431.2097,
found 431.2101.
provides a valuable platform for development of small molecule
inhibitors of HIF-1α-dependent tumor progression and angio-
genesis.
EXPERIMENTAL SECTION
(E)-3-[3-(4-Adamantan-1-ylphenoxy)acrylamido]benzoic
Acid Morpholinoethyl Ester (4p). 4p was obtained as a white solid
(9.3 mg, 18.6% yield) from commercially available 4-(2-chloroethyl)-
■
General Procedures. All of the commercial chemicals and
solvents were of reagent grade and were used without further
purification. All reactions were carried out under an atmosphere of
dried argon in flame-dried glassware. Melting points were determined
in open capillary tubes on an electrothermal apparatus and were
uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra
were determined on a Varian (300 MHz) spectrometer. Chemical
shifts are provided in parts per million (ppm) downfield from
tetramethylsilane (internal standard) with coupling constants in hertz
(Hz). Multiplicity is indicated by the following abbreviations: singlet
(s), doublet (d), doublet of doublet (dd), triplet (t), pseudo triplet
(ps-t), quartet (q), multiplet (m), broad (br). Mass spectra were
recorded on a Finnigan ESI mass spectrometer, and HRMS (ESI-MS)
was conducted with a Mariner instrument (Perseptive Biosystem).
Products from all reactions were purified to a minimum purity of 96%
as determined by high-pressure liquid chromatography (HPLC) either
by flash column chromatography using silica gel 60 (230−400 mesh
Kieselgel 60) or by preparative thin layer chromatography using glass-
backed silica gel plates (1 mm thickness) unless otherwise indicated.
Additionally, thin-layer chromatography on 0.25-mm silica plates (E.
Merck, silica gel 60 F254) was used to monitor reactions. The
chromatograms were visualized using ultraviolet illumination, exposure
to iodine vapors, dipping in PMA, or Hanessian’s solution. Purity was
determined by HPLC analysis. See Supporting Information for details.
(E)-3-[3-(4-Adamantan-1-ylphenoxy)acryloylamino]benzoic
Acid Methyl Ester (4c). 4c was obtained as a white solid (236.1 mg,
88.5% yield) from (E)-3-(4-adamantan-1-ylphenoxy)acrylic acid 9a
1
morpholine chlorohydride. Mp 166.1−166.5 °C; H NMR (DMSO-
d6, 300 MHz) δ 10.13 (1H, s, NH), 8.27 (1H, m, aromatic-H), 7.86−
7.89 (1H, m, aromatic-H), 7.76 (1H, d, J = 11.7 Hz, CH), 7.62 (1H, d,
J = 7.8 Hz, aromatic-H), 7.41−7.48 (3H, m, aromatic-H), 7.14 (2H, d,
J = 9.0 Hz, aromatic-H), 5.81 (1H, d, J = 12.0 Hz, CH), 4.38 (2H, t, J
= 6.0 Hz, CH2), 3.56 (4H, t, J = 5.1 Hz, CH2), 2.68 (2H, t, J = 6.0 Hz,
CH2), 2.47 (4H, t, J = 4.8 Hz, CH2), 2.06 (3H, m, adamantly-H), 1.86
(6H, m, adamantly-H), 1.74 (6H, m, adamantly-H); MS (EI) m/z 530
(M+); HRMS (EI) m/z calcd for C32H38N2O5 [M+] 530.2781, found
530.2783.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis procedures and compound characterization, tables
reporting the HPLC purity results of the final products,
screening methods, and the biological experiments on the HIF
inhibitory activity of the compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Address: College of Pharmacy, Dongguk UniversitySeoul,
26, Pil-dong 3-ga, Jung-gu, Seoul 100-715, Republic of Korea.
Phone: +82-31-961-5214. Fax: +82-31-961-5206. E-mail:
kaylee@dongguk.edu.
1
and 3-aminobenzoic acid methyl ester 10a. Mp 138.5−139.0 °C; H
NMR (CDCl3, 300 MHz) δ 8.06 (1H, s, aromatic-H), 7.89−7.95 (2H,
m, aromatic-H, CH), 7.77 (1H, d, J = 7.5 Hz, aromatic-H), 7.33−7.42
G
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Author Contributions
∥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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This study was supported by a NRF Grants 2012-0006336 and
2012-0007275 from MEST and KRIBB Initiative Program,
Republic of Korea. We gratefully acknowledge the assistance of
Jung Eun Kang, Song-Kyu Park, Kiho Lee.
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