Examining the structure-activity relationship of benzopyran-based inhibitors of the hypoxia inducible factor-1 pathway

Article abstract of DOI:10.1016/j.bmcl.2017.02.073

Many forms of solid tumor have a characteristic feature known as hypoxia, which describes a low or non-existent presence of oxygen in the cellular microenvironment. This decrease in oxygen causes activation of the hypoxia inducible factor (HIF) pathway, w

Full text of DOI:10.1016/j.bmcl.2017.02.073

Accepted Manuscript
Examining the Structure-activity Relationship of Benzopyran-based inhibitors
of the Hypoxia Inducible Factor-1 Pathway
Jalisa Ferguson, Zeus De Los Santos, Narra Devi, Erwin Van Meir, Sarah
Zingales, Binghe Wang
PII:
S0960-894X(17)30216-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.073
BMCL 24745
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
3 January 2017
26 February 2017
27 February 2017
Please cite this article as: Ferguson, J., De Los Santos, Z., Devi, N., Van Meir, E., Zingales, S., Wang, B., Examining
the Structure-activity Relationship of Benzopyran-based inhibitors of the Hypoxia Inducible Factor-1 Pathway,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.073
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Examining the Structure-activity Relationship of Benzopyran-based inhibitors of the
Hypoxia Inducible Factor-1 Pathway
Jalisa Fergusona, Zeus De Los Santosa, Narra Devib, Erwin Van Meirb, Sarah Zingalesa,c*, and Binghe
Wanga*
aDepartment of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303 USA
bLaboratory of Molecular Neuro-Oncology, Department of Neurosurgery, School of Medicine and Winship Cancer Institute, Emory University, Atlanta, GA
30322 USA
cDepartment of Chemistry and Physics, Armstrong State University, Savannah, GA 31419 USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Many forms of solid tumor have a characteristic feature known as hypoxia, which describes a
low or non-existent presence of oxygen in the cellular microenvironment. This decrease in
oxygen causes activation of the hypoxia inducible factor (HIF) pathway, which activates the
transcription of many genes that cause cell proliferation, metastasis, increased glycolysis and
angiogenesis. Increased HIF expression has been linked with poor patient prognosis, increased
malignancy, and therapeutic resistance. Previous work in our lab has identified 1 and 2 as
inhibitors of the HIF pathway, specifically as disrupters of the p300-HIF-1α complex formation.
A library of sulfonamide analogs has been designed and synthesized with the intent of
examining the SAR of this series of compounds and improving potency and physicochemical
properties as compared with lead compounds 1 and 2. At the end, we have achieved a thorough
understanding of the structural features critical for future optimization work.
Received in revised form
Accepted
Available online
Keywords:
Hypoxia Inducible Factor inhibitors
structure-activity relationship
molecular docking
2009 Elsevier Ltd. All rights reserved
.
Hypoxia is a condition in which the availability of oxygen to a
tissue is reduced from normal physiological levels. Many types
of solid tumors display regions with low partial oxygen pressure
or even anoxia due to abnormal vasculature and reduced blood
flow.1 Under these conditions, the hypoxia inducible factor (HIF)
pathway is activated and leads to the transcriptional activation of
a number of genes that mediate adaptive responses to hypoxia,
including cell metabolism, cell motility, and angiogenesis.2 One
of the main transcription factors driving these responses is
hypoxia-inducible factor 1 (HIF-1). HIF-1 is a basic helix-loop-
helix motif heterodimeric transcription factor comprised of two
subunits: HIF-1α and HIF-1β. Under normoxic conditions,
human HIF-1α is hydroxylated by the enzyme prolyl hydroxylase
2 (PHD2) on two Proline residues (402 and 564) that mediate the
interaction of the HIF-1α oxygen degradation domain with the
von Hippel-Lindau tumor suppressor protein (VHL), which
serves as an E3 ubiquitin ligase and marks the protein for
degradation by the proteasome.3
transferases are recruited, which opens the chromatin, recruits the
RNA polymerase machinery and activates the transcription of
HIF target genes, including VEGF (vascular endothelial growth
factor), EPO (erythropoietin), GLUT1 (glucose transporter 1),
LDH-A (lactate dehydrogenase), and NOS (nitric oxide
synthase).4 The activation of the HIF-1 pathway is associated
with several types of cancer and is also related to low success
rates of various treatment methods.5 Therefore, inhibition of the
HIF-1 pathway is recognized as a viable approach to the
development of anti-cancer agents.
Toward the goal of finding small-molecule inhibitors of the HIF
pathway, an HRE-alkaline phosphatase assay was designed to
screen
a
library of 10,000 compounds from
a
2,2-
dimethylbenzopyran combinatorial library.6 The HRE-alkaline
phosphatase assay used human glioblastoma cells (LN229-HRE-
AP cell line) stably transfected with an alkaline phosphatase
reporter under the control of a minimal CMV promoter and an
engineered hypoxia-activated enhancer constituted of 6 copies of
the HRE (hypoxia response element) from the VEGF gene.7 This
initial screening yielded a few promising hits, with the lead
compound identified as 1 (Figure 1a) having an IC50 of ~0.6
µM.8 1 was then taken to preliminary in vivo studies, where nude
mice were implanted with LN229 glioblastoma cells on their
hind flanks. After 1 week, the mice were either injected with 1
(60 mg/kg; 5 days/week) or vehicle (DMSO) for 10-12 weeks
Under hypoxic conditions, HIF-1α is not dihydroxylated, which
abrogates VHL binding and prevents its degradation; thus PHD2
acts as an oxygen sensor to stabilize HIF. Under hypoxia, HIF-1α
binds to HIF-1β, and the heterodimer translocates to the nucleus,
where it binds to specific DNA sequences in the promoters of
hypoxia-regulated genes. Following HIF binding to these
hypoxia response elements (HRE), p300/CBP histone acetyl

until the mice had to be terminated due to large size of tumors in
the control group. On average, a 6-fold difference in tumor size
was observed between the treatment and control groups, and
some of the tumors disappeared completely. The treatment group
did not appear to suffer negative side effects from 1 treatment,
suggesting that 1 is well tolerated.9
were then sulfonylated with various sulfonyl chlorides to yield
sulfonamides 3-7. Some of these sulfonamides were
hydrogenated to yield final products.
With 1 as the original lead compound, our laboratory began
synthesis of a library of over 100 analogs. This initial library of
analogs was screened against a human glioblastoma cell line
(LN229-HRE-Lux), with a luciferase reporter under the control
of the same hypoxia-inducible promoter as above. From this
initial study, N-cyclobutyl-N-((2,2-dimethyl-2H-pyrano[3,2-
Scheme 1. Synthesis of analogs. Reagents and conditions: (a) InCl3, NaBH4,
MeOH, room temperature, 20 minutes, 23-92%, or NaBH4, MeOH, room
temperature, overnight, and taken directly to the next step; (b) K2CO3, DCM,
room; (c) H2, Pd/C, MeOH, overnight, 70-99%.
b]pyridin-6-yl)methyl)-3,4-dimethoxybenzenesulfonamide
2
(Figure 1b) with IC50 of ~0.25 µM was identified as the
optimized lead for further study.10 With such promising results
from these initial studies, we report herein our further lead
optimization and broadening of the library in order to identify
more potent compounds and to further develop our SAR
(structure-activity relationship).
All 42 of the analogs were evaluated for their inhibitory effects
on HIF-1-mediated transcription under hypoxic conditions on
human glioma cells LN229-HRE-Lux. Each compound was
tested against 2 as positive control and IC50 values were
determined. The respective IC50 values for 1 and 2 were 0.59 and
0.28 µM.
Class 1A
Class 1A analogs were designed to probe the importance of the
features of the phenyl A ring to the activity of 1 (Table 2). The
first part of the A ring examined was the double bond in the
pyran ring. In many cases, double bonds are not preferred in
therapeutics because of the possibility of activation and/or
metabolism by cytochrome P450 (CYP) monooxygenases in the
Figure 1. a) lead compound 1 b) lead compound 2
Lead compounds 1 and 2 were divided into 4 parts (Figure 1):
the left-hand core (A, red), the N-substituent (B, green), the right-
hand ring (C, blue), and the linker (D, violet). Analogs were
designed and synthesized based off of modifications of 3 of these
parts, namely the A, B, and C rings. In total, 6 classes of
analogs, for a total of 42 compounds, were devised as described
in Table 1. Classes 1A-1C are analogs of 1: Class 1A has the A
ring modified (3a-g); Class 1B has both the B and C rings
modified (4a-f) and features one analogue with a hydrogenated
liver, which can lead to hepatotoxicity in vivo.11 For 3a (IC50
=
0.98 µM), the hydrogenated analog of 1, removal of the double
bond resulted in only a small decrease (1.5-fold) in activity,
which suggests that the double bond is not essential for HIF-1
inhibition. Next, various substituents were introduced on the
phenyl ring. Products 3b-3d (IC50 = 3.3, 3.2, 3.1 µM) had
decreased activities by about 5-fold, which suggests a somewhat
size constrained binding pocket. Next, the point of attachment of
the chromene ring was examined by changing it from carbon 6 to
carbon 8, 3e (IC50 = 1.8 µM), which resulted in a 3-fold decrease
in activity, confirming attachment at carbon 6 to be important.
Finally, two other fused ring systems were synthesized, 3f-3g,
with IC50 values greater than 5 µM, thus confirming the need for
the chromene moiety in the A ring.
2,2-dimethylpyranobenzene
A
ring (4f); Class 1C has
modifications to both the B and C rings (5a-n), with two
analogues, namely 5m and 5n, that have the hydrogenated 2,2-
dimethlpyranobenzene. Classes 2A-2C are analogs of 2: Classes
2A and 2B have, respectively, the A (6a-i) and both B and C (7a-
f) regions modified; while 7d and 7e have the hydrogenated A
ring.
Table 2. HIF Inhibition by Class 1A analogs
Table 1. Classes of compounds synthesized
Class 1B
Class 1A
Class 1C
[a]
[a]
Name
R1
IC50
Name
R1
IC50
3a
0.98
3.3
3d
3.1
1.8
>5
5
3
4
Class 2A
Class 2B
3e
3f
3b
7
6
A general 2- or 3-step reaction sequence was followed in the
synthesis of the analogs (Scheme 1). First, reductive amination
of aldehydes 8 (either commercially available or synthesized
from literature procedures, Schemes S1-S2) with various primary
amines yielded secondary amines 9. These secondary amines
3c
3.2
3g
>5
[a] IC50 values are in micromolar (µM).

Class 1B
Class 1B analogs were designed to probe the activity of the
moieties on the C ring (Table 3). In our previous work, it was
determined that removing the 3’-methoxy group from the C ring
resulted in little to no loss of activity (4e, IC50 = 0.6 µM).10 We
further probed the activity of this site by removing the 4’-
methoxy, 4a (IC50 = 1 µM), which resulted in a small 1.7-fold
decrease in activity. Next the 4’-monomethoxy was substituted
for a 4’-trifluoromethoxy moiety, 4b (IC50 = >5 µM), which
Table 4. HIF Inhibition by Class 1C analogues
[a]
Name
R2
R3
IC50
5a
0.75
>5
resulted in
a loss of activity. Other modifications that
incorporated electronegative atoms such as fluorine 4c (IC50 = >5
µM) or an electron-withdrawing group like cyano 4d (IC50 = >5
µM) led to a complete loss of activity Thus, the methoxy
substituent in the 4’ position seems to be needed for optimal
activity while the 3’-methoxy is not. This further supports our
model that the 4’-methoxy is likely involved in hydrogen
bonding to LYS350 of P300.12 The 4’-monomethoxy compound
4f (IC50 = 3.2 µM), in comparison with its non-hydrogenated
form 4e (IC50 = 0.6 µM), decreased in activity by 5-fold. This
initial result suggests the role of the double bond A ring is
somewhat important for activity.
5b
5c
>5
5d
5e
>5
4.6
4.4
1.5
>5
Table 3. HIF Inhibition by Class 1B analogues
5f
5g
[a]
[a]
Name
R3
IC50
Name
R3
IC50
4a
1
4d
>5
5h
5i
4b
4c
>5
>5
4e
0.610
3.0
0.5
4.0
>5
4f[b]
3.2
5j
[a] IC50 values are in micromolar (µM). [b] A ring is hydrogenated 2,2-
dimethylpyranobenzene.
5k
5l[b]
5m[b]
Class 1C
Class 1C analogs were designed to further probe the activity of
the B and C rings and to try to introduce more polar groups in
order to lower the clogP (Table 4). Although 5k (IC50 = 4 µM)
had a clogP of 3.7, it demonstrated a significant loss of activity.
These results support our previous computational model, that
suggests that the B ring points into a hydrophobic pocket.12 Some
of these analogues are hybrid 1/2 compounds, with the A ring
from 1 and the B ring from 2 (5a-f). In this combination, only 5a
(IC50 = 0.75 µM) demonstrated activity below 1 µM. None of
these hybrid compounds were as potent as 2. Analogues 5g-j
were developed as analogs that contain the A ring from 1 and an
N-cyclopentyl substituent, with varying C rings. Two of these
compounds, 5g (IC50 = 1.5 µM) and 5i (IC50 = 3.0 µM) exhibited
some activity in the luciferase assay, but with 3- and 6-fold
decreases in activity compared to the 3,4-dimethoxysulfonyl
compound 5j (IC50 = 0.5 µM), which we reported previously (Ref
10). The activity of 5l (IC50 = >5 µM) decreased in comparison
with its non-hydrogenated counterpart 5k (IC50 = 4 µM).
However, hybrid 5m (IC50 = 0.39 µM), was more potent than its
non-hydrogenated counterpart 5a (IC50 = 0.75 µM). The role of
the double bond in the A ring is still unclear and may be studied
further in the future.
0.39
[a] IC50 values are in micromolar (µM). [b] A ring is hydrogenated 2,2-
dimethylpyranobenzene.
Class 2A
Class 2A analogs were designed to probe the importance of the
features of the A ring to the activity of 2 (Table 5). The first
strategy was to remove the fused ring feature altogether and
determine whether a simple phenyl ring with various substituents
would have activity. Unfortunately, only one such compound, 6b
(IC50 = 3.9 µM), with a 4-methoxyphenyl A ring had any activity,
and this activity was diminished by almost 14-fold from 2. 6a, c-f
(IC50 = >5 µM) were considered inactive, which demonstrates the
importance of the fused ring system to the activity. However, 6g
(IC50 = 1 µM), an analog containing a morpholine substituent,
which can increase water solubility, was active. It is highly

possible that increased solubility is responsible for the activity of
this compound. Next, other fused ring systems that contained
oxygen were examined, 6h-i, (IC50 = 1.3, 3.5 µM). Both of these
compounds showed decreased activity (by 2.5-fold to 6.0-fold
when compared to 2). Overall, no suitable replacement for the A
ring of 2 was found.
7c
4
7d[b]
0.25
1.2
Table 5. HIF Inhibition by Class 2A analogs
7e[b]
7f
>5
[a]
[a]
Name
R1
IC50
Name
R1
IC50
6a
>5
6f
>5
[a] IC50 values are in micromolar (µM). [b] A ring is hydrogenated 2,2-
dimethylpyridinylbenzene.
6b
6c
6d
6e
3.9
6g
6h
6i
1.0
Previously, 1 was computationally observed, as expected, to
have several satisfactory interactions on two sites interfering with
the p300-HIF-1α complex formation, disrupting the structure of
p300 and inhibiting the binding of HIF-1α.12 In the studies
presented here, the lowest energy conformation of 1 binds in the
same site, making similar interactions with the amino acid
residues of interest. For that reason, it is sensible to use this site
for comparison of 1 with the remaining analogs previously
discussed. In order to gain a better understanding of the effect of
molecular interactions between the 1 analogs and p300 on the
HIF-1 pathway inhibitory activity demonstrated in vitro, 10 of
the analogs were subjected to flexible docking using AutoDock
Vina,13 including the previously discovered lead 2. The first
model of the NMR structure of p300-CH1/HIF-1α C-TAD
protein complex (PDB entry code: 1L3E) was prepared using
AutoDock by removal of the HIF-1α chain and Zn ions. The
optimal ligand-binding site on p300 was highlighted in a grid box
>5
>5
>5
1.3
3.5
[a] IC50 values are in micromolar (µM).
Class 2B
Class 2B analogs (Table 6) were designed to further probe the
importance of the B and C rings to the activity of 2. None of the
compounds with the conserved reduced A ring of 2 (7a-c)
showed improved activity over 2; the best compound 7a (IC50
=
1.8 µM), with the 3’-methoxy removed, still lost potency by 6.4-
fold, whereas a similar analog to 1 (4e) did not lose activity. It is
interesting that 7d (IC50 = 0.25 µM), the hydrogenated form of 2,
demonstrated slightly better activity compared to 2. Additionally,
7e (IC50 = 1.2 µM) demonstrated slightly better activity than its
non-hydrogenated counterpart 7a (IC50 = 1.8 µM). This suggests
that the double bond in the A ring may not be required for
potency, and could possibly be eliminated with no loss of activity
for analogs of 2. This suggests that 1 and 2 may bind to different
sites or in different poses. Analogue 7f is the only example in this
class that does not include a cyclobutyl ring, but instead offers a
3,4-dimethoxybenzeneethyl in the B position. As a result, the
activity plummets compared to its fellow class members.
Table 6. HIF Inhibition by Class 2B analogs
Figure 2. Molecular docking image of 1 bound to p300
based on the lowest energy conformation of 1. Specifically, the
interactions between p300 and 1 are most profound via several
key residues that are supported by previous studies.12 The A ring,
which consists of a benzopyran moiety, not only has favorable
hydrophobic interactions with Ile400 but also has hydrogen
bonding between its oxygen and Ser401. As illustrated from
Figure 2, the N-phenyl B ring is burrowed into the hydrophobic
pocket, interacting with Ile400 and several Leucine residues.
Finally, the 3,4-dimethoxybenzene of the D ring interacts with
Leu376, Thr380 and Gln352 residues; and Gln352 interacts
specifically with the 4-methoxy group of 1, which we have
previously reported as an important component for ligand
[a]
Name
R3
IC50
1.8
7a
7b
2.5

binding in these sulfonamide analogs10. Two active (IC50 value
less than 5 µM) ligands were further examined due to their
structural similarity to 1 and observed for any favorable or
unfavorable interactions that might affect binding and,
ultimately, activity in the binding site of p300. These compounds,
along with 2, and their respective IC50 values, and calculated
binding affinities are summarized in Table 7.
analogues of 2 were docked in the same manner, none of which
showed any significant trend between binding affinity and IC50
value (Table S1, Figures S1-18). This suggests that these
analogs do not bind with this protein in the same manner as
analogues of 1.
Table 7 Docking results of selected 1, 2, and selected analogs
[a]
Name
IC50
0.59
0.98
1.8
Binding Affinity[b]
1
-7.6
-7.5
-6.7
-6.5
3a
3e
2
0.28
[a]IC50 values are in micromolar (µM), [b]binding affinity values are in
kcal/mol.
Qualitative analysis of the docking images reveals some
interesting commonality among many of the active analogues.
Unsurprisingly, analog 3a, the hydrogenated version of 1, binds
very similarly, conserving the Ser401 hydrogen bond and the
other major interactions already discussed (Figure 3).
Figure 4. Structure-Activity Relationship of analogs.
In conclusion, 42 analogs were synthesized. As illustrated in
Figure 4, a qualitative structure-activity relationship (SAR) was
developed. For the A ring, the 2,2-dimethyl chromene with either
a N or C in the 5 position is important to the activity. Open ring
structures are not well tolerated, with the exception of some 4-
position moderately polar substituents. The double bond is not
crucial for activity of compounds and can be eliminated with the
result of better or only slightly decreased activity. For the B ring,
only hydrophobic groups, such as aromatics or small aliphatic
rings or chains are acceptable. Introduction of polar moieties in
this position dramatically decreases the activity. For the C ring,
3’,4’-dimethoxy is still the best, with the 4’-methoxy more
crucial to activity than the 3’-methoxy. Molecular docking
studies revealed that analogs more closely related in structure to
1 demonstrated better binding affinity, whereas 2 and other
structures with the N-phenyl as the B ring, seem to bind
elsewhere. This is based on the poor binding affinities observed
when binding is limited to the site of 1. Such information will be
important for the synthesis of future analogues.
Figure 3. Molecular docking image of 1 (magenta), 3a (gray), 3e (cyan),
and 2 (brown) bound to p300.
Alternatively, 3e is a structural isomer of 1, with the chromene
ring attachment point at the 8-position instead of the 6-position.
The docking study revealed that the two analogues share many of
the same interactions with p300 amino acid residues, but are
without the important hydrogen bonding with Ser401 (Figure 3).
This could explain the weaker inhibition of 3e (IC50 = 1.8 µM)
compared with 1 (IC50 = 0.59 µM), in addition to the lower
calculated binding affinity.
Acknowledgments
Financial support from the NIH (CA180805, CA116804) is
gratefully acknowledged. We also thank the GSU MBD and CDT
programs for fellowships to SKZ, and a Department of Education
GAANN grant (P200A120122) with Barbara Baumstark as the
Principal Investigator in support of JF.
References and Notes
Surprisingly, 2 has a low in silico binding affinity compared with
1, even though it has better in vitro activity in the luciferase
assay. In the studies performed here, the lowest energy
conformation of 2 was longitudinally flipped such that the
benzopyran ring interacted with Leu376 and Met379 and formed
a hydrogen bond with Thr380 (Figure 3). This leaves the
dimethoxyphenyl group to interact with the Ile400 and His349.
Having a relatively poor binding affinity of -6.5 kcal/mol, in spite
of its extraordinary ability to inhibit HIF transcriptional activity,
suggests that 2, and perhaps some of its more closely related
analogs, do not bind in specifically the same way 1 does. To
further explore this idea, nine of the active analogs (IC50 <5 uM)
that are more structurally similar to 2 were subjected to the same
molecular docking as discussed above. Seven randomly selected
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Examining the Structure-activity
Relationship of Benzopyran-based inhibitors
of the Hypoxia Inducible Factor-1 Pathway
Jalisa Ferguson,[a] Zeus A. De los Santos,[a] Narra
Sarojini Devi,[b] Erwin G. Van Meir,[b] Sarah K.
Zingales,[a],[c],* and Binghe Wang [a],*
aDepartment of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303 USA
bLaboratory of Molecular Neuro-Oncology, Department of Neurosurgery, School of Medicine and Winship Cancer Institute, Emory University, Atlanta,
GA 30322 USA
cDepartment of Chemistry and Physics, Armstrong State University, Savannah, GA 31419 USA