Ring-truncated deguelin derivatives as potent Hypoxia Inducible Factor-1α (HIF-1α) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.09.033

A series of fluorophenyl and pyridine analogues of 1 and 2 were synthesized as ring-truncated deguelin surrogates and evaluated for their HIF-1α inhibition. Their structure-activity relationship was systematically investigated based on the variation of th

Full text of DOI:10.1016/j.ejmech.2015.09.033

Accepted Manuscript
Ring-truncated Deguelin Derivatives as Potent Hypoxia Inducible Factor-1α (HIF-1α)
Inhibitors
Ho Shin Kim, Mannkyu Hong, Su-Chan Lee, Ho-Young Lee, Young-Ger Suh, Dong-
Chan Oh, Ji Hae Seo, Hoon Choi, Jun Yong Kim, Kyu-Won Kim, Jeong Hun Kim,
Joohwan Kim, Young-Myeong Kim, So-Jung Park, Hyun-Ju Park, Jeewoo Lee
PII:
S0223-5234(15)30277-4
10.1016/j.ejmech.2015.09.033
EJMECH 8130
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 July 2015
Revised Date: 24 September 2015
Accepted Date: 25 September 2015
Please cite this article as: H.S. Kim, M. Hong, S.-C. Lee, H.-Y. Lee, Y.-G. Suh, D.-C. Oh, J.H. Seo, H.
Choi, J.Y. Kim, K.-W. Kim, J.H. Kim, J. Kim, Y.-M. Kim, S.-J. Park, H.-J. Park, J. Lee, Ring-truncated
Deguelin Derivatives as Potent Hypoxia Inducible Factor-1α (HIF-1α) Inhibitors, European Journal of
Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.09.033.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Ring-truncated Deguelin Derivatives as Potent Hypoxia
Inducible Factor-1α (HIF-1α) Inhibitors
a
a
a
a
a
Ho Shin Kim , Mannkyu Hong , Su-Chan Lee , Ho-Young Lee , Young-Ger Suh , Dong-Chan
b
c
c
c
c,d
e
Oh , Ji Hae Seo , Hoon Choi , Jun Yong Kim , Kyu-Won Kim , Jeong Hun Kim , Joohwan
f
f
g
g
a,*
Kim , Young-Myeong Kim , So-Jung Park , Hyun-Ju Park , and Jeewoo Lee
a
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University,
Seoul 151-742, Korea
b
Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-
742, Korea
c
SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy, Seoul National
University, Seoul 151-742, Korea
d
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of
Convergence Science and Technology, Seoul National University, Seoul 151-742, Korea
e
College of Medicine, Seoul National University, Seoul 151-742, Korea
f
School of Medicine, Kangwon National University, Kangwon-do 200-701, Korea
g
School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea
*
To whom correspondence should be addressed.
Phone: 82-2-880-7846
Fax: 82-2-888-0649
E-mail: jeewoo@snu.ac.kr
1

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Key Words: Hypoxia Inducible Factor-1, HIF-1, Heat Shock Protein 90, HSP90, Antitumor,
Deguelin
Abstract
A series of fluorophenyl and pyridine analogues of 1 and 2 were synthesized as ring-
truncated deguelin surrogates and evaluated for their HIF-1α inhibition. Their structure-activity
relationship was systematically investigated based on the variation of the linker B-region moiety.
Among the inhibitors, compound 25 exhibited potent HIF-1α inhibition in a dose-dependent manner
and significant antitumor activity in H1299 with less toxicity than deguelin. It also inhibited in vitro
hypoxia-mediated angiogenic processes in HRMECs. The docking study indicates that 25 occupied
the C-terminal ATP-binding pocket of HSP90 in a similar mode as 1, which implies that the
anticancer and antiangiogenic activities of 25 are derived from HIF-1α destabilization by binding to
the C-terminal ATP-binding site of hHSP90.
2

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1. Introduction
Hypoxia Inducible Factor-1 (HIF-1) is the transcription factor that regulates the cellular
response for the survival of cells in hypoxia. HIF-1 protein is a heterodimer that consists of a
constitutively expressed β-subunit and an oxygen-regulated α-subunit. Under normoxia, HIF-1α is
degradable by the pVHL-mediated ubiquitin protease pathway, which includes hydroxylation by
prolyl hydroxylases (PHD), binding to the product of the von Hippel-Lindau (pVHL), being tagged
with polyubiquitin and proteasomal degradation. However, under hypoxia, HIF-1α becomes stable
from proline hydroxylation, accumulates and translocates to the nucleus. There, HIF-1α dimerizes
with HIF-1β to activate the HIF-1 complex, which binds to hypoxia-response elements (HRE) in the
HIF target genes to control transcription. This regulation can induce angiogenesis, proliferation,
1
,2
metastasis and invasion of cancer cells. Therefore, the HIF-1α inhibition can inhibit this
3
-9
angiogenesis, decrease the proliferation of cancer cells and reduce the chemotherapy resistance.
In addition, there has been growing interest in the biology of the HIF-1 pathway and its role in
human diseases that are associated with the hypoxic micro-environment, such as cancer, stroke and
1
0-15
heart disease.
Heat shock protein 90 (HSP90) is a molecular chaperone that regulates the post-
translational folding, stability and function of its client proteins such as ErbB2, Src, c-MET, AKT,
Raf-1, MMP-2 and HIF-1α. Because the chaperone inhibition can induce the decomposition of HIF-
1
α, HSP90 inhibition is considered a new and effective therapy against angiogenesis-associated
diseases such as cancer. Structurally, the HSP90 protein contains three functional domains: the
ATP-binding, protein-binding, and dimerizing domain. Most HSP90 inhibitors that were developed
as anticancer agents have been identified as the so-called N-term inhibitors, which bind to an ATP-
binding domain in the N-terminal. However, these inhibitors cause some problems, and one of them
induce a heat shock response (HSR), which ultimately leads to an increase in HSP90 and anti-
apototic proteins such as HSP70 and HSP27. Therefore, the inhibition of another ATP-binding site
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in the C-terminal may be an alternative strategy to discover clinically applicable HSP90
16-20
inhibitors.
Deguelin, which is a naturally occurring rotenoid, has been reported to prevent tobacco
carcinogen-induced lung carcinogenesis by blocking the Akt activation; it also exhibits potent
apoptotic and antiangiogenic activities against diverse transformed cells and cancer cells in vitro
2
1
(
Figure 1). It interferes with the chaperone function of HSP90 by inhibiting ATP binding, which
induces the destabilization of HIF-1α and consequent tumor growth reduction in xenograft models
2
2
of various human cancers.
Figure 1. Deguelin and its ring-truncated surrogates
Previously, Chang et al reported that two ring-truncated deguelin analogues, compounds 1
and 2, exhibited excellent HIF-1α suppression and potent cell growth inhibition in the human non-
2
3
small-cell lung carcinoma cell line, H1299 (Figure 1). In addition, their in vivo antiangiogenic
activities were observed in the zebrafish model in a dose-dependent manner. Jo et al also reported
that the destabilization of HIF-1α by both compounds suppressed hypoxia-mediated retinal
neovascularization and vascular leakage in diabetic retina without inducing a definite toxicity in the
2
4
oxygen-induced retinopathy mouse model. The results indicated that the new HSP90 inhibitors 1
and 2 were considered promising lead compounds for anti-proliferation and anti-angiogenesis.
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Structurally, the ring-truncated deguelin scaffold was divided into three pharmacophoric
parts: A-region (3,4-dimethoxyphenyl), B-region (linker), and C-region (2,2-dimethyl chromene
ring) (Figure 2). To further optimize the leads 1 and 2 as anticancer agents, we decided to
investigate their fluorophenyl (X=C-F) and pyridine (X=N) derivatives in the A-region to improve
the target binding for the HIF-1α inhibition and aqueous solubility. We presumed that polarizing the
3,4-dimethoxyphenyl group by incorporating a polar nitrogen or fluoro atom might provide better
binding interaction to HSP90 and pharmacokinetic profile. In addition, α-methyl carbonyl in the B-
region was modified with its bioisosteres, which include olefin, diol, alcohol, and acyl groups
(Figure 2).
Figure 2. Pharmcophoric regions of the ring-truncated deguelin analogues
In this paper, we investigated the structure activity relationships of the fluorophenyl and
pyridine analogues of 1 and 2 for HIF-1α inhibition using western blot assay. With selected potent
inhibitors in the series, we further characterized their cytotoxicities in a tumor cell line and anti-
angiogenesis in hypoxia-mediated angiogenic processes in human retinal microvascular endothelial
cells. In addition, we performed the docking study with HSP90 to determine its mode of action.
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2
. Result and discussion
.1. Chemistry
2
The final compounds were generally synthesized through a carbon-carbon bond formation
between the phenyl sulfonyl intermediates of the A-region and 2,2-dimethylchromene aldehyde of
the C-region.
To synthesize the phenyl sulfonyl intermediates (7, 8), fluorophenyl aldehyde 3 and
pyridine aldehyde 4 as the starting materials were prepared from commercially available o-fluoro
2
5,26
catechol and 2-bromo-3-pyridinol, respectively, according to the procedures in the literature.
The prepared aldehydes (3, 4) were reduced to the corresponding alcohols (5, 6), which were
halogenated and subsequently converted into the corresponding sulfonyl intermediates (7, 8),
respectively, using benzene sulfinic acid sodium salt (Scheme 1). Then, 2,2-Dimethylchromene C-
2
7
region aldehyde 9 was coupled with the sulfonyl intermediates (7, 8) using n-BuLi to provide two
diasteromeric mixtures, which were directly oxidized without separation and subsequently
desulfonylated to obtain ketones (12, 13). (Scheme 2)
Scheme 1. Synthesis of the A-region
Reagents and conditions: (a) NaBH , MeOH, 0 °C to rt; (b) SOCl , MC, 0 °C to rt; (c) PhSO Na,
4
2
2
DMF, rt
6

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O
O
O
O
O
O
X
O
X
O
O
H
a,b
c
O
R
O
O
O
9
1
1
2 X = C-F
3 X = N
1
1
0 R = SO Ph, X = C-F
2
1 R = SO Ph, X = N
2
Scheme 2. Synthesis of the ketone analogs
Reagents and conditions: (a) 7 or 8, n-BuLi, THF, -78 °C; (b) DMP, MC, rt; (c) (n-Bu) SnH, AIBN,
3
benzene, reflux
The final alcohol and acyl analogues were synthesized using the conventional routes from
the corresponding ketones (Scheme 3). Ketones (12, 13) were alkylated with the corresponding
alkyl halide to produce α-alkyl ketones (14-17), which were reduced by NaBH to provide alcohol
4
compounds (18-23). Under this condition, the syn diastereomer was found as a predominant isomer
1
(
syn:anti=ca. 20:1) when R was a monoalkyl group. For the structural analysis, only 25 (syn) and
1
2
6 (anti) were separated, and their stereochemistries were assigned based on the H-NMR analysis
2
8
according to a previous report. The alcohols were further acylated to obtain the acylated products
24-36, 41-43) or carbamoylated in two steps to obtain the carbamoylated products (37-40, 44-45).
(
Scheme 3. Syntheses of the alcohol and acyl analogues
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Reagents and conditions: (a) R-X, NaH, DMF 0 °C to rt; (b) NaBH , MeOH, rt; (c) R-COCl,
4
Pyridine, MC, 0°C; (d) i) CDI, MC, 0°C; ii) NH OH or NH Me or NHMe , water 40°C
4
2
2
To synthesize the olefinic and dihydroxyl analogues of the B-region, alcohol compounds
(18, 21) were dehydrated under acidic condition to provide only trans isomers (46, 47), which were
dihydroxylated using OsO to obtain the syn-diol compounds (48, 49). The oxime (50) and methyl
4
oxime (51) analogues of the pyridine A-region were obtained from ketone 13 (Scheme 4).
Scheme 4. Syntheses of the olefin, dihydroxy and oxime analogs
Reagents and conditions: (a) p-TSA, toluene, reflux; (b) OsO , NMO, acetone, water, rt; (c)
4
NH OR-HCl, NaOAc, EtOH, water, reflux
2
To synthesize the chiral S-isomer of 16, the pyridine surrogate of compound 2, chiral (S)-
methyl aldehyde of A-region was prepared from alcohol 6 first (Scheme 5). Alcohol 6 was
converted to the nitrile and subsequently hydrolyzed to the corresponding acid 52. Acid 52 was
converted to the corresponding (S)-oxazolidinone, which was methylated to provide (S)-methyl
product 53. Further steps produced the key chiral aldehyde 54 to couple with the C-region. The C-
region iodide 56 was synthesized from the corresponding amine 55 by Sandmeyer reaction, which
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2
3
was prepared from resorcinol as shown in our previous report. The lithiation of iodo compound 56
and subsequent coupling with aldehyde 54 provided the corresponding alcohol, which was oxidized
to produce the final chiral (S)-methyl ketone 16S (Scheme 6).
Scheme 5. Synthesis of the chiral A-region of 16S
Reagents and conditions: (a) SOCl , MC, 0 °C to rt; (b) NaCN, DMSO, 60 °C; (c) KOH, EtOH,
2
water, reflux; (d) i) PivCl, TEA, THF, -78 °C; ii) Ax, n-BuLi, THF, -78 °C; (e) LiHMDS, CH3I,
THF, -78 °C; (f) LAH, Et O, 0 °C; (g) DMP, MC, rt
2
Scheme 6. Synthesis of the chiral 16S
Reagents and conditions: (a) NaNO , KI, c-HCl, H O, 0 °C to rt; (b) 54, n-BuLi, THF, -78 °C; (c)
2
2
DMP, MC, rt.
2.2. Structure activity relationship for HIF-1α inhibition
The synthesized compounds were evaluated for their HIF-1α inhibition using western blot
2
3
assay with H1299 cell lines. The H1299 cells were untreated or treated with deguelin and the
analogues at 100 nM for 72 h and subsequently incubated under hypoxic conditions for 12 h. The
resulting cell lysates were used for the western blot analysis with a monoclonal antibody against
HIF-1α. Because all of the compounds have fluorophenyl or pyridine A-region and 2,2-dimethyl
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2
H-chromene C-region, their inhibitory activities are represented by the structure of the B-region,
as shown in Tables 1-4.
First, we explored the ketone B-region analogues (Table 1). The fluorophenyl and pyridine
surrogates (12, 13) of parent 1 showed better HIF-1α inhibition than did parent 1. Interestingly, the
pyridine surrogate (16S) of parent 2 exhibited excellent inhibition, which was ca. 2-fold more
potent than 2 and its racemate 16. 16S also had better activity than deguelin did. This result
indicates that the corresponding fluorophenyl and pyridine surrogates are better HIF-1α inhibitors
than did the previous ring-truncated deguelin analogues. The analysis indicates that the inhibitory
activity increases in the order of no-substituent > α-methyl ≥ α,α’-dimethyl groups next to ketone.
Table 1. Ketone analogues as ring-truncated deguelin
1
2
Code
X
R , R
WB (100 nM)
64
Deguelin
1
2
C
H, H
85.3
C
H, (S)-Me
H, H
82.1
1
1
1
1
1
1
1
2
C-F
C-F
C-F
N
67.8 (±37.03)
78.6 (±16.44)
74.9 (±6.17)
77.6 (±10.86)
89.3 (±11.54)
44.2 (±33.24)
99.9 (±10.10)
4
H, Me
5
Me, Me
H, H
3
6
N
H, Me
6S
7
N
H, (S)-Me
Me, Me
N
Second, we investigated the alcohol B-region analogues (Table 2). Compounds 18 and 19
without substitution at the α-position showed similar inhibitions with the corresponding ketone
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analogues (12, 13). However, unlike ketone, the inhibitory activity increased in the order of α,α’-
dimethyl > α-methyl > no-substituent next to alcohol.
Table 2. Alcohol analogues as ring-truncated deguelin
1
2
Code
X
R , R
WB (100 nM)
74.3 (±33.76)
75.0 (±24.20)
68.0 (±33.99)
75.2 (±36.33)
71.2 (±29.4)
69.2 (±19.9)
1
1
2
2
2
2
8
9
0
1
2
3
C-F
C-F
C-F
N
H, H
H, Me
Me, Me
H, H
N
H, Me
Me, Me
N
Third, we examined the O-acyl and O-carbamoyl analogues (Table 3). In this series, we
incorporated a methyl, ethyl, dimethyl or cyclopropyl group as an α-substituent and acetoxy,
propionyloxy, cyclopropanecarbonyloxy, carbamoyloxy, and dimethylcarbamoyloxy as the acyl and
carbamoyl groups. All of the α-monoalkyl analogues (25, 30, 33, 34, 36, 38-40, 42) were syn
isomers that were isolated as a predominant form in the B-region. For the SAR analysis, only anti-
isomer 26 was separated and found to be much less potent than syn 25. In this series, the four
compounds (25, 33, 38, 40) exhibited better activities than did deguelin. Interestingly, all of them
had α-methyl moiety next to the O-acyl/carbamate groups, which suggests that a methyl group
provided a favorable conformation for the HIF-1α inhibition. A more steric environment at the α-
position, such as the ethyl (34), dimethyl (27, 31, 35, 43, 45), and cyclopropyl (28) groups,
diminished the inhibitory activity.
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Table 3. Acyl analogues as ring-truncated deguelin
O
O
O
X
O
R3
1
2
R R
O
O
1
2
3
Code
X
R , R
R
WB (100 nM)
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
C-F
N
H, H
Me
69.3 (±49.85)
52.2 (±12.56)
79.4 (±5.01)
5 (Syn)
H, Me
H, Me
Me, Me
Me
6 (Anti)
Me
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Me
83.4 (±8.36)
-CH
H, H
-CH
-
Me
154.6 (±17.09)
77.3 (±39.35)
63.7 (±43.72)
88.2 (±16.37)
70.3 (±19.92)
35.0 (±6.05)
2
2
Et
H, Me
Me, Me
H, H
Et
Et
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclobutyl
H, Me
H, Et
87.9 (±20.53)
110.6 (±38.89)
69.5 (±18.25)
88.9 (±21.10)
48.8 (±2.99)
Me, Me
H, Me
H, H
NH
NH
2
H, Me
H, Me
H, Me
H, H
2
NHMe
76.5 (±28.37)
64.1 (±20.46)
114.0 (±27.98)
96.7 (±19.97)
113.2 (±6.43)
75.0 (±18.08)
107.7 (±25.14)
NMe
Me
2
N
H, Me
Me, Me
H, H
Me
N
Me
N
NH
NH
2
N
Me, Me
2
Finally, we examined olefin, diol, and oxime B-region analogues (Table 4). Although the
olefinic derivatives (46, 47) did not show any HIF-1α inhibition, the diol derivatives (48, 49)
exhibited good HIF-1α inhibition, which was comparable to that of deguelin. 48 and 49 had better
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activity than did their corresponding mono-hydroxyl surrogates (18, 21) and similar activity to the
ketone surrogates (12, 13). In addition, the methyl oxime derivative of pyridine A-region (51)
displayed excellent inhibition, which was better than deguelin, but its oxime isomers (50) showed
moderate inhibition.
Table 4. Olefin, diol and oxime analogues as ring-truncated deguelin
Code
X
Y, Z
WB (100 nM)
133.8 (±40.03)
133.2 (±24.03)
68.1 (±4.23)
4
4
4
4
5
5
5
6
C-F
N
-CH=CH-
7
-CH=CH-
8
C-F
N
-CH(OH)-CH(OH)-
-CH(OH)-CH(OH)-
9
68.9 (±1.46)
0a
0b
1
N
-CH
-CH
-CH
2
2
2
-C(=NOH)-
-C(=NOH)-
78.5 (±25.36)
79.2 (±34.62)
64.8 (±19.78)
N
N
-C(=NOCH )-
3
Among the tested compounds, we selected 6 compounds with better HIF-1α inhibition than
deguelin and evaluated their inhibition at a low concentration (10 nM) to examine the dose-
dependent inhibition (Table 5). As expected, all of the selected compounds showed better inhibition
than deguelin at 10 nM. In particular, compound 25 showed the best inhibition.
Table 5. HIF-1α inhibition of the selected potent compounds
WB (10 nM)
94.0
WB (100 nM)
64
Deguelin
1
2
3
6S
88.4 (±11.53)
63.2 (±17.53)
81.6 (±22.07)
44.2 (±33.24)
52.2 (±12.56)
35.0 (±6.05)
5
3
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3
4
5
8
0
1
80.9 (±12.67)
48.8 (±2.99)
64.1 (±20.46)
64.8 (±19.78)
93.6 (±10.89)
74.0 (±4.75)
2
.3. Antitumor activity
Deguelin and its analogs exhibited profound antiproliferative effects on human malignant
bronchial epithelial cells and in vivo efficacy in suppressing the lung tumor formation in the mice
2
1,22
model, which indicates that they are promising antitumor candidates for lung cancer.
In this
regard, the two compounds 25 and 33, which were selected based on the primary HIF-1α inhibition
screening result, were tested for antitumor activity in the human non-small-cell lung carcinoma cell
(H1299) line using the MTT assay (Figure 3). As expected, both compounds showed promising
cytotoxicity in a concentration-dependent manner, where the viability of the cell was reduced to 50%
at 20 µM. The result indicates that the cytotoxicities are derived from their HIF-1α inhibition.
1
1
20
00
33
25
80
60
40
20
0
Control 5uM
5uM
10uM
10uM
20uM
20uM
Figure 3. Antitumor activity of 25 and 33 in H1299
Because the use of deguelin as anticancer agents is limited because of its potential
toxicities, we examined the toxicity of 25 by testing the effect of 25 on the viability of hippocampal
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cell (HT-22). Compared to deguelin, compound 25 showed significantly reduced cytotoxicity,
which indicates that 25 is a more potent and less toxic antitumor agent than deguelin (Figure 4).
Figure 4. Effect of deguelin and 25 on the viability of mouse hippcampus (HT-22)
2.4. Anti-angiogenesis activity
Angiogenesis is the formation of new blood vessels, which are a significant component of a
solid tumor for its proliferation and metastasis. The VEGF (vascular endothelial growth factor)
plays a pivotal role in tumor angiogenesis and is up-regulated in tumor cells under hypoxic
conditions.
The anti-angiogenesis activity of compound 25 was examined using proliferation and
migration assays in human retinal microvascular endothelial cells (HRMECs), which were
2
9
previously described , and compared to deguelin at each 100 nM (Figure 5). Although the VEGF
activated angiogenesis in the proliferation and migration assays, compound 25 effectively inhibited
the in vitro hypoxia-mediated angiogenic processes in both assays at a comparable level to deguelin,
which indicates that it can be used as a potential anti-angiogenic agent to suppress the retinal
neovascularization for the treatment of diabetic retinopathy.
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B
A
Figure 5. Anti-angiogenesis activity of 25 in HRMECs (A: proliferation assay; B: migration assay)
2.5. Molecular Modeling
We recently demonstrated that L80 (Figure 6D), which is a cyclized analogue of
compound 25 , strongly diminished the physical interaction between HIF-1α and HSP90 in H1299
cells by directly modulating the function of HSP90 using co-immunoprecipitation and pull-down
3
0
assays. In addition, we showed that L80 could bind to the C-terminal ATP-binding pocket of
HSP90 to hamper the HSP90 function when we studied recombinant HSP90 proteins, which
contain the full-length protein, N-terminal/middle domain, middle domain, and C-terminal domain.
Furthermore, our recent study confirmed that lead 1 also bound to the C-terminal ATP-binding
pocket of HSP90, disrupted the HSP90 function and caused a degradation of client proteins without
affecting the HSP70 expression (unpublished data). These data suggest that compound 25 inhibits
the HSP90 function by binding to the C-terminal ATP-binding pocket of HSP90.
To ensure the binding of 25 to the C-terminal ATP-binding pocket of HSP90 for the HIF-
1
α inhibition, we performed a docking study of 25 in the C-terminal ATP-binding pocket of
hHSP90 homodimer (Figure 6). The active site was determined based on the reported hHSP90 C-
3
1
terminal ATP-binding site. To examine the docking poses, we conducted a docking study of 25 in
the C-terminal ATP-binding pocket of the hHSP90 homodimer. The C-terminal ATP-binding site
was assigned as an active site at the dimerization interface (Figure 6C). After docking, we
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compared the docking pose and score of ATP with those of 25. Figure 6A shows that ATP (white
carbon) fits well into the ATP-binding pocket of chain A. The adenine ring is located deep into the
hydrophobic pocket. The phosphate moiety interacts with multiple hydrogen bonds with Glu611,
and 3´-hydroxy of the sugar moiety interacts with Ser677 in chain A via a hydrogen bond.
Compound 25 is partially superimposed with the sugar-phosphate moiety of ATP. However, 25
binds to the active site with a higher docking score (-logK = 7.80) than ATP (-logK = 6.29). 25
d
d
occupies the centre of the active site by forming multiple hydrogen bonds with both chains A and B
Figure 6B and 6C). The oxygen atom of the methoxy group that is attached to the 2,2-dimethyl
(
chromene ring forms a hydrogen bond with the side chain NH of Lys 615 in chain A, which is part
of the ATP-binding pocket. Two oxygen atoms of the acetoxy group form hydrogen bonds with the
side chain of Lys615 in chain B. Furthermore, the substituents of the 3,4-dimethoxy-2-fluorophenyl
group are involved in forming hydrogen bonding networks. The fluoro group and oxygen atom of 3-
methoxy form hydrogen bonds with Lys615 in chain B. The oxygen atom of 4-methoxy interacts
with sidechain NH of Asn 622 (chain B). Hugel and coworkers reported that the role of the C-
terminal inhibitors is to prohibit the global conformational changes, which include N-terminal
3
2-35
dimerization and the formation of the ATP binding pocket.
Our docking results demonstrate that
25 can compete with ATP in binding to the C-terminal ATP binding site and stabilize the open state
of C-terminal hHSP90 with the bridging hydrogen bond networks at the interface of the
homodimer.
17

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Figure 6. Molecular modeling of 25
A) Overlay of the docking pose of 25 (purple carbon) and ATP (white carbon) in the active site of
(
the hHsp90 C-terminal. The key amino acid residues of the binding site are represented by the grey
carbon capped stick. The active site is shown as a lipophilicity property surface map (brown colour:
hydrophobic; blue colour: hydrophilic) (B) Docked pose of 25. The dashed ellipsoids are hydrogen-
bonding interactions (<2.8 Å). A and B indicate the chain names. (C) Binding site for 25 in the
dimerization interface of the open state hHsp90. 25 is represented by the purple CPK model. Chain
A is shown as an orange ribbon, and chain B is a cyan ribbon. (D) Structure of L-80
3. Conclusion
A series of fluorophenyl and pyridine analogues of 1 and 2, which are lead HIF-1α
inhibitors through HSP90 inhibition as ring-truncated deguelin surrogates, were investigated for
their HIF-1α inhibition. Their structure-activity relationship was systematically examined by
varying the B-region moiety. Among the studied inhibitors, 6 compounds showed better HIF-1α
inhibition than deguelin. The two selected inhibitors, 25 and 33, exhibited promising antitumor
activity in human non-small cell lung carcinoma (H1299). In addition, compound 25 inhibited in
vitro hypoxia-mediated angiogenic processes in human retinal microvascular endothelial cells
(
HRMECs). The docking study of 25 with the hHSP90 C-terminal ATP-binding site indicated that
5 snugly bound to the C-terminal ATP-binding pocket of the hHSP90 homodimer with a high
docking score, as found in its cyclized analog L80. Overall, compound 25 is a potential anticancer
2
18

ACCEPTED MANUSCRIPT
and antiangiogenic agent with an HIF-1α inhibition mechanism through binding to the C-terminal
ATP-binding site of hHSP90.
Acknowledgments
This work was supported by grants from the National Research Foundation of Korea (NRF), the
Ministry of Science, Republic of Korea (NRF-20110019400).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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Legends
Figure 1. Deguelin and its ring-truncated surrogates
Figure 2. Pharmcophoric regions of the ring-truncated deguelin analogues
Scheme 1. Synthesis of the A-region
Scheme 2. Synthesis of the ketone analogs
Scheme 3. Syntheses of the alcohol and acyl analogues
Scheme 4. Syntheses of the olefin, dihydroxy and oxime analogs
Scheme 5. Synthesis of the chiral A-region of 16S
Scheme 6. Synthesis of the chiral 16S
Table 1. Ketone analogues as ring-truncated deguelin
Table 2. Alcohol analogues as ring-truncated deguelin
Table 3. Acyl analogues as ring-truncated deguelin
Table 4. Olefin, diol and oxime analogues as ring-truncated deguelin
Table 5. HIF-1α inhibition of the selected potent compounds
Figure 3. Antitumor activity of 25 and 33 in H1299
Figure 4. Effect of deguelin and 25 on the viability of mouse hippcampus (HT-22)
Figure 5. Anti-angiogenesis activity of 25 in HRMECs
Figure 6. Molecular modeling of 25
25

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Figure 1. Deguelin and its ring-truncated surrogates
Figure 2. Pharmcophoric regions of the ring-truncated deguelin analogues
26

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Scheme 1. Synthesis of the A-region
Reagents and conditions: (a) NaBH , MeOH, 0 °C to rt; (b) SOCl , MC, 0 °C to rt; (c) PhSO Na,
4
2
2
DMF, rt
O
O
O
O
O
H
X
O
O
X
O
O
a,b
c
O
O
R
O
O
9
1
1
2 X = C-F
3 X = N
1
1
0 R = SO Ph, X = C-F
2
1 R = SO Ph, X = N
2
Scheme 2. Synthesis of the ketone analogs
Reagents and conditions: (a) 7 or 8, n-BuLi, THF, -78 °C; (b) DMP, MC, rt; (c) (n-Bu) SnH, AIBN,
3
benzene, reflux
27

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Scheme 3. Syntheses of the alcohol and acyl analogues
Reagents and conditions: (a) R-X, NaH, DMF 0 °C to rt; (b) NaBH , MeOH, rt; (c) R-COCl,
4
Pyridine, MC, 0°C; (d) i) CDI, MC, 0°C; ii) NH OH or NH Me or NHMe , water 40°C
4
2
2
Scheme 4. Syntheses of the olefin, dihydroxy and oxime analogs
Reagents and conditions: (a) p-TSA, toluene, reflux; (b) OsO , NMO, acetone, water, rt; (c)
4
NH OR-HCl, NaOAc, EtOH, water, reflux
2
28