Synthesis of (aryloxyacetylamino)-isonicotinic/nicotinic acid analogues as potent hypoxia-inducible factor (HIF)-1α inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.09.005

We report a new series of HIF-1α inhibitors which were obtained through structural modifications of previously reported lead 1. The in vitro inhibitory potencies of newly synthesized compounds were evaluated against hypoxia-induced HIF-1 activation using

Full text of DOI:10.1016/j.bmcl.2007.09.005

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6305–6310
Synthesis of (aryloxyacetylamino)-isonicotinic/nicotinic
acid analogues as potent hypoxia-inducible
factor (HIF)-1a inhibitors
Shanthaveerappa K. Boovanahalli,a Xuejun Jin,a Yinglan Jin,a Jin Hwan Kim,a
Nguyen Tien Dat,a Young-Soo Hong,a Jeong Hyung Lee,a,b Sang-Hun Jung,c
Kyeong Leea,* and Jung Joon Leea,*
aMolecular Cancer Research Center, KRIBB, Daejeon 305-806, Republic of Korea
bCollege of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
cCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
Received 18 May 2007; revised 14 August 2007; accepted 3 September 2007
Available online 7 September 2007
Abstract—We report a new series of HIF-1a inhibitors which were obtained through structural modifications of previously reported
lead 1. The in vitro inhibitory potencies of newly synthesized compounds were evaluated against hypoxia-induced HIF-1 activation
using cell-based reporter assay in three human cancer cell lines including SK-Hep-1, Hep3B, and AGS cells. Several compounds
displayed significant inhibitory activity in all the three tested cell lines. In particular, analogue 17 displayed potent inhibition of
hypoxia-induced accumulation of HIF-1a protein in Hep3B cell line, in addition to the dose-dependent inhibition of HIF-1 target
genes VEGF and EPO.
Ó 2007 Elsevier Ltd. All rights reserved.
Hypoxia-inducible factor (HIF)-1 is a key mediator for
adaptation and survival process of cells to hypoxia
(ꢀ1% O2). The bioactivity of HIF-1, a heterodimer
composed of HIF-1a and constitutively expressed
HIF-1b (also known as ARNT), depends on the
amount of HIF-1a protein.1–4 The levels of HIF-1a
are largely regulated on a post-translational level, by
the rate of protein synthesis and degradation. Under
normoxic conditions, HIF-1a protein is subjected to
degradation via the von Hippel–Lindau tumor suppres-
sor gene product (pVHL)-mediated ubiquitin–proteoso-
mal pathway. The association of HIF-1a and pVHL
under normoxia is triggered by the hydroxylation of
prolines and the acetylation of lysine within oxygen-
dependent degradation (ODD) domain.5–7 Hypoxic
conditions allow HIF-1a protein to escape proteolysis.
Upon activation, the HIF complex with co-activator,
such as p300/CBP, binds to HRE (Hypoxia Responsive
Element 50-RCGTG-30) sequence within target genes,
which leads to up-regulation of genes involved in angi-
ogenesis, glucose metabolism, and pH regulation. Thus
far, three isoforms of the a subunit have been cloned,
with the well characterized being HIF-1a and HIF-2a.
Although HIF-2a seems to be regulated in similar
fashion with HIF-1a, it is suggested that there is little
redundancy between two a subunits and that HIF-2a
exhibits more tissue-specific expression.8–11
In human tumors, HIF-1a is over-expressed as a result
of intratumoral hypoxia and genetic alterations affecting
key oncogenes (HER2, FRAP, H-RAS, and c-SRC) and
tumor suppressor genes (von Hippel–Lindau, PTEN,
and p53).6 Immunohistochemical analyses show that
HIF-1a is present at higher levels in human tumors than
in normal tissues.12 The expression of HIF-1a in various
solid tumors has been associated with tumor aggressive-
ness, vascularity, treatment failure, and mortality.13 In
addition, tumor growth and angiogenesis in xenograft
tumors also depends on HIF-1 activity and on the
expression level of HIF-1a.14 All of these activities make
the HIF-1 transcription factor an attractive target for
the development of new anticancer therapeutics.15–17
Keywords: Hypoxia; Hypoxia-inducible factor; HIF-inhibitor; HIF-1;
Isonicotinic/nicotinic acid analogues.
*
Corresponding authors. Tel.: +82 42 8604382; fax: +82 42 8604595
(K.L.); tel.: +82 42 8604360; fax: +82 42 8604595(J.J.L.); e-mail
addresses: kaylee@kribb.re.kr; jjlee@kribb.re.kr
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.005

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S. K. Boovanahalli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6305–6310
Table 1. In vitro inhibition of HIF-1 transcriptional activity in cell-
based reporter assaya
In this context, recently amolecular targeted HTS method
was also reported, which coherently identifies small-mol-
ecule HIF-1 inhibitors.18,19 In recent years several anti-
cancer agents have been identified to inhibit HIF-1
O
R
Y
O
activity in tumor xenografts including
a soluble
O
guanylyl-cyclase stimulator YC-1 (3-(50-hydroxy-
methyl-20-furyl)-1-benzylindazole), 20the HSP90 inhibi-
tor 17-AAG (17-allyl-aminogeldanamycin),21–23 the
microtubule destabilizer 2-methoxyoestradiol (2ME2),24
the topoisomerase inhibitors camptothecin and topotec-
an,18the thioredoxin inhibitors pleurotin and 1-methyl-
propyl 2-imidazolyl disulfide,25 the mammalian target of
rapamycin (mTOR) kinase inhibitor CCI-779,26 a 2,2-
dimethylbenzopyran structural motif 103D5R,27 and a
small natural product chetomin.28 In addition, a number
of inhibitors targeting the HIF pathway with an emphasis
on the inhibition of HIF hydroxylase activity to prevent
HIF-1a degradation have also been reported.29–34
N
H
X
Compound R
X
Y
IC50 (lM)
SK-Hep1 Hep3B AGS
1
OCH3
OH
CH CH NAb
CH CH NA
2.6
0.4
1.2
1.0
0.7
0.35
2
2
11
12
OCH3
OH
N
N
CH 1.03
CH 2.0
0.8
H
N
13
14
N
N
CH >30
CH 3.4
>30
3.4
>30
5.2
O
H
N
As part of our ongoing research in the identification of
novel HIF-1 inhibitors, we have recently reported the
identification of a novel series of potent HIF-1a inhibi-
tors through closely related structural modifications of
a screening hit.35 Amongst them compounds 1 and 2
(Fig. 1) displayed potent HIF-1 inhibitory activity in hu-
man hepatocellular carcinoma Hep3B and human gastric
adenocarcinoma AGS cell lines, in addition compound 1
potently suppressed the HIF-1a protein accumulation
and its target gene expression under hypoxic conditions
in Hep3B cells. The structure–activity relationship study
of this series suggested that the presence of the adamantyl
group on ring A is essential for effective inhibition of
HIF-1 activation in hypoxia. We hypothesized that the
replacement of phenol in ring B of 1 with pyridine while
maintaining adamantyl moiety on ring A would be ex-
pected to modulate the inhibitory activity (Fig. 1) and
on the basis of this strategy we prepared a series of pyri-
dine-containing analogues and evaluated for their poten-
tial to inhibit HIF-1 in Hep3B, AGS, and SK-Hep-1 cell
lines. In this communication, we report the design, syn-
thesis, and HIF-1 inhibitory activity of a new series of
isonicotinic and nicotinic acid derivatives.
N
H
N
N
15
16
N
CH 0.9
11.2
>30
11
N
H
N
N
N
CH 2.5
CH 0.6
18.6
Cl
17
20
21
NH2
OCH3
OH
3.1
5.9
1.8
5
CH N >30
CH N >30
>30
>30
H
N
22
23
CH N 4.1
CH N >30
>30
>30
30
O
H
N
1.3
N
H
N
N
24
CH N 1.9
>10
>10
N
26
YC-1
NH2
CH N 11.9
>30
>10
13.8
0.9
2.0
a Values are means of three experiments.
b NA, data is not available.
The compounds described in this paper (Table 1) were
prepared as outlined in Schemes 1–3.36 As shown in
Scheme 1, reaction of 4-adamantyl phenol 3 with ethyl
chloroacetate followed by alkaline hydrolysis afforded
the corresponding 4-adamantyl phenoxy acetic acid 4
in quantitative yield.
O
OH
a
O
OH
3
4
During our initial medicinal chemistry efforts we pre-
pared intermediates 9 and 10 by following the chemistry
described in Scheme 2.
Scheme 1. Reagents: (a) i—Ethyl chloroacetate, K2CO3, DMF; ii—
LiOHÆH2O, dioxane, H2O.
O
R
O
NH2
O
O
B
B
N
O
O
N
N
A
A
H
H
OH
1: R = OCH3,
17
2
: R = OH
Figure 1. Structural modification of phenol to pyridine.

S. K. Boovanahalli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6305–6310
6307
CH3
O
OH
CH3
O
b
X
a
c
O
O
12
N
H
N
4
H2N
N
H3C
N
H
N
7
6
5
d
O
R1
O
O
OCH3
O
OH
O
N
H
N
f
d
11: R1 = OCH3
4
H2N
N
H2N
N
g
9
8
12: R1 = OH
h
e
13-16: R1 = NHR
O
NH2
O
NH2
O
i
O
N
H
N
4
H2N
N
10
17
Scheme 2. Reagents: (a) EDC, DIPEA, DMF; (b) SeO2, dioxane or KMnO4, NaOH, H2O; (c) i—Ac2O, DMAP; ii—KMnO4, NaOH, H2O; (d)
SOCl2, MeOH; (e) aqueous ammonia; (f) PyBOP, DMAP, DMF; (g) LiI, pyridine; (h) arylamine, PyBOP, DMAP, DMF; (i) PyBOP, DMAP, DMF.
O
NH2
N
O
O
NH2
N
O
O
OH
f
O
+
N
H
H2N
4
25
26
e
O
R1
N
O
OH
N
O
OCH3
O
a
O
4
N
H
N
b
H2N
H2N
20: R1 = OCH3
19
c
18
21: R1= OH
d
22-24: R1 = NHR
Scheme 3. Reagents: (a) SOCl2, MeOH; (b) PyBOP, DMAP, DMF; (c) LiI, pyridine; (d) arylamine, PyBOP, DMAP, DMF; (e) aqueous ammonia;
(f) PyBOP, DMAP, DMF.
Commercially available picoline 5 was utilized to syn-
thesize the requisite intermediates. Thus, acetylation of
5 followed by subsequent oxidation using KMnO4
gave 2-acetylamino isonicotinic acid 7,37 which upon
without further purification, subjected to esterification
in the presence of thionyl chloride and methanol. This
reaction affected both esterification along with deacet-
ylation of acetylmino moiety to yield 2-amino isonicot-
inic acid methyl ester 9 in modest yield. Alternatively,
this ester 9 was also prepared by the reaction of com-
mercially available 2-amino isonicotinic acid 8 with
thionyl chloride in refluxing methanol. Further reac-
tion of this ester 9 with ammonium hydroxide pro-
vided the required 2-amino isonicotinamide 10 in
good yield.38
Analogue 6 possessing picoline moiety was readily ob-
tained by the reaction of 2-amino-4-picoline 5 with 4 in
the presence of EDC and DIPEA. Attempting subsequent
oxidation of the methyl group utilizing SeO2 or KMnO4
failed to give the desired acid 12, consequently a two-step
method was adopted as follows. 2-Amino isonicotinic
acid methyl ester 9 was coupled with 4 to obtain the cor-
responding ester derivative 11, which upon subsequent
hydrolysis with LiI furnished the desired acid 12. Subse-
quent coupling of acid 12 with the appropriate amines
in the presence of PyBOP and DMAP afforded the corre-
sponding amide analogues 13–16 in good yields.
A single-step coupling of 4 with amides 10 or 25
yielded the corresponding amide derivatives 17 and 26,

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S. K. Boovanahalli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6305–6310
respectively. Esterification of 3-amino-5-nicotinic acid
18 in the presence of thionyl chloride and methanol fur-
nished the corresponding ester 19 in satisfactory yield,
which upon subsequent aminolysis using aqueous
ammonia afforded the desired amide analogue 25. A
similar sequence of reactions described for analogues
11–16 was followed to obtain nicotinic acid derivatives
20–24 in moderate to high yields (Scheme 3).
and rest of the compounds were found to be inactive or
poor inhibitors. Of note, none of these nicotinic acid
analogues exhibited inhibitory activity in all the three
cell lines, suggesting that the position of nitrogen atom
at pyridine ring is important for potency. Accordingly,
a simple modification of phenol to pyridine resulted in
a new series of potent HIF-1 inhibitors.
To confirm the HIF-1 inhibitory activity of these com-
pounds, a representative analogue 17 which demon-
strated significant inhibitory activity of HIF activation
in all the three cell lines was chosen for further
evaluation. Accordingly, this analogue was evaluated
by Western blot analysis for its potential to inhibit
hypoxia-induced accumulation of HIF-1a protein in
Hep3B cell line.
The in vitro inhibitory potencies of all the newly synthe-
sized compounds were evaluated against hypoxia-in-
duced HIF-1 activation in three cell lines including
human hepatocellular carcinoma SK-Hep-1, human
hepatocellular carcinoma Hep3B, and human gastric
adenocarcinoma AGS cell lines. The inhibitory poten-
cies were obtained using a HRE-mediated cell-based re-
porter assay under hypoxic conditions (1% O2, 94% N2,
and 5% CO2) and the results are tabulated as IC50 values
in Table 1. All the assays were performed under stan-
dard assay conditions by employing hypoxic condition
and following the previously described assay protocol.39
Cell viability, as measured by the MTT assay, showed
that all of the tested compounds had no significant
cytotoxicity at their effective concentrations for the inhi-
bition of HIF-1 activation. YC-1, a known small-mole-
cule HIF-1 inhibitor,20 was used as a reference
compound for comparison, which showed IC50 13.8
and 2.0 lM in Hep3B and AGS cells, respectively.
As shown in Figure 2, analogue 17 blocked the accumu-
lation of HIF-1a protein in a dose-dependent manner
without affecting the expression level of HIF-1b. Thus,
confirming the HIF-1a inhibitory property of this
analogue.
HIF-1a responds to the hypoxia by binding to the HRE
of target genes, including VEGF and EPO. Moreover,
expression of these HIF-1 target genes, such as VEGF,
stimulates new blood vessel formation from existing vas-
culature, increasing tumor oxygenation and, ultimately,
tumor growth and metastasis.40 Therefore, compounds
17 possessing potent HIF-1a accumulation inhibitory
profile was further tested for its ability to inhibit the
expression of HIF-1 target genes VEGF and EPO in
Hep3B cells by RT-PCR analysis (Fig. 3). Interestingly,
this inhibitor significantly suppressed the expression of
VEGF and EPO in a dose-dependent manner without
affecting mRNA expression level of HIF-1a and GAP-
DH, wherein potent inhibition was observed at the
The isonicotinic acid analogues 11 and 12, which were
prepared based on compound 1 as a starting point, dis-
played highly potent inhibition of hypoxia-induced
HIF-1 activity in all the three tested cell lines with
IC50 values ranging from 1 to 2 lM. Encouraged by this
we further extended the analogue series characterized by
an amide substitution at ring B. This derivatization pro-
duced amide derivatives 13–16, comprising furfuryl,
ethyl pyridine, propyl imidazole, and p-chloro phenyl
moieties linked through an amide bond. It is worth not-
ing that, while analogue 14 comprising ethyl pyridine
moiety exhibited good inhibitory activities in all the
three cell lines, derivatives 15 and 16 demonstrated high
inhibitory potency against SK-Hep-1 cell line. In con-
trast, compound 13 in which it was linked to furfuryl
ring was found to be a poor inhibitor.
1% O2, 17 (μM)
1
10
30
DMSO
3
HIF-1
HIF-1ß
TOPO1
We also investigated the effect of the free amide moiety
at ring B on the HIF-inhibition. This modification re-
sulted in the highly potent inhibitor represented by com-
pound 17, which demonstrated sub-micromolar
inhibitory potencies in all the three tested cell lines.
Additionally, 17 exhibited better potency (IC50
3.1 lM) than the corresponding phenol compound28
(IC50 > 30 lM) in Hep3B cell lines. From these results
it is evident that pyridine moiety serves as a good bio-
isosteric replacement for phenol portion of 1 (Fig. 1).
Further to confirm this hypothesis, we also prepared
various pyridine analogues, wherein nitrogen atom was
introduced at different position of ring B which resulted
in nicotinic acid analogues 20–24 and 26. Amongst
these, compounds 20, 23, and 26 displayed significant
inhibition in AGS cell line, while analogues 22 and 24
were found to be potent inhibitors in SK-Hep-1 cell line
Figure 2. Western blot analysis of the effect of compound 17 on the
hypoxia-induced HIF-1a protein level in Hep3B cells.
1% O2, 17 (μM)
1
10
30
DMSO
3
VEGF
EPO
HIF-1α
GAPDH
Figure 3. RT-PCR analysis of the effect of compound 17 on the
hypoxia-induced expression of VEGF and EPO in Hep3B cells.

S. K. Boovanahalli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6305–6310
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Acknowledgments
This study was supported by a grant from KRIBB Re-
search Initiative Program, Korea, and the Molecular
and Cellular BioDiscovery Research Program
(M10601000155), Korea.
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Supplementary data
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McDonnell, T. J.; Golub, T. R.; Loda, M.; Lane, H. A.;
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.09.005.
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(1H, d, J = 5.1 Hz, pyridine-H), 7.26 (2H, d, J = 9.3 Hz,
aromatic-H), 6.89 (2H, d, J = 8.4 Hz, aromatic-H), 4.78
(2H, s, OCH2), 2.03 (3H, br s, adamantyl-H), 1.82 (6H, s,
adamantyl-H), 1.71 (6H, s, adamantyl-H); MS (ESI) m/z
406 (M+H)+, 428 (M+Na)+, 404 (MꢁH)ꢁ; Pur-
ity = >99.9% (as determined by RP-HPLC, Method C,
tR = 17.2 min).
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36. All of the newly synthesized compounds were character-
ized by 1H NMR and ESIMS, and purified to a minimum
purity of 96% as determined by RP-HPLC, either by flash
column chromatography or by preparative thin layer
chromatography: 2-[2-(4-Adamantan-1-yl-phenoxy) ace-
tylamino]-isonicotinamide (17). 1H NMR (DMSO-d6,
300 MHz) d 10.62 (1H, s, CONH), 8.43 (2H, m, CONH2),
8.19 (1H, s, pyridine-H), 7.67 (1H, s, pyridine-H), 7.49
38. Deardy, L. W.; Korytsky, O. L.; Rowe, J. E. Aust.
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39. All the assays were performed under standard assay
conditions by employing hypoxic condition as
described by Cai, X. F.; Jin, X.; Lee, D.; Yang, Y.
T.; Lee, K.; Hong, Y.-S.; Lee, J. H.; Lee, J. J. J. Nat.
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