Discovery of a new molecular probe ML228: An activator of the hypoxia inducible factor (HIF) pathway

Article abstract of DOI:10.1016/j.bmcl.2011.11.077

Hypoxia and ischemia are linked to several serious public health problems that affect most major organ systems. Specific examples include diseases of the cardiovascular, pulmonary, renal, neurologic, and musculoskeletal systems. The most significant pathw

Full text of DOI:10.1016/j.bmcl.2011.11.077

Bioorganic & Medicinal Chemistry Letters 22 (2012) 76–81
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a new molecular probe ML228: An activator of the hypoxia
inducible factor (HIF) pathway
Jimmy R. Theriault a, Andrew S. Felts b, Brittney S. Bates b, Jose R. Perez a, Michelle Palmer a,
Shawn R. Gilbert c, Eric S. Dawson b, Julie L. Engers b, Craig W. Lindsley b, Kyle A. Emmitte b,
⇑
a The Broad Institute Probe Development Center, Broad Institute, Cambridge, MA 02142, USA
b Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Hypoxia and ischemia are linked to several serious public health problems that affect most major organ
systems. Specific examples include diseases of the cardiovascular, pulmonary, renal, neurologic, and mus-
culoskeletal systems. The most significant pathway for cellular response to hypoxia is the hypoxia induc-
ible factor (HIF) pathway. HIFs are transcription factors responsible for the activation of genes which
encode proteins that mediate adaptive responses to reduced oxygen availability. A high-throughput
cell-based HIF-mediated gene reporter screen was carried out using the NIH’s Molecular Libraries Small
Molecule Repository to identify activators of the HIF pathway. This communication describes the subse-
quent medicinal chemistry optimization of a triazine scaffold that led to the identification of the new
molecular probe ML228. A discussion of HIF activation SAR within this chemotype as well as detailed
in vitro characterization of the probe molecule is presented here.
Received 19 October 2011
Revised 15 November 2011
Accepted 18 November 2011
Available online 28 November 2011
Keywords:
Hypoxia
Ischemia
Angiogenesis
HIF
Ó 2011 Elsevier Ltd. All rights reserved.
VEGF
Triazine
Hypoxia and ischemia are linked to several serious public health
problems that affect most major organ systems. Specific examples
include diseases of the cardiovascular, pulmonary, renal, neuro-
logic, and musculoskeletal systems. The hypoxia inducible factor
(HIF) pathway is the major pathway required for intracellular
adaptation initiated by lower oxygen availability in the blood-
stream. The HIF pathway is known to take part in the angiogenesis
processes enhancing blood supply required for tissue repair and
regeneration. A unique gene transcription program involving the
activation of multiple transcriptional factors known as the hypoxia
inducible factors (HIFs) is triggered in response to hypoxia.1 HIFs
mediated by three prolyl hydroxylase (PHD) isoforms known as
PHD1, PHD2, and PHD3.3 The presence of iron, oxygen, and 2-oxo-
glutarate (2-OG) are required for hydroxylation. Prolyl hydroxyl-
ation is blocked during hypoxia, which leads to the stabilization
of HIF-1
whereby hypoxia and HIF-1
Once HIF-1 is stabilized, it accumulates in the cells, dimerizes
with ARNT, and translocates into the nucleus. Inside the nucleus,
the HIF-1 /ARNT complex through interaction with its binding
a
and the establishment of a negative feedback loop
a
both upregulate PHD2 expression.4
a
a
partners initiates the transactivation of HIF-responsive genes, such
as the glucose transporter Glut1 and the angiogenic factor vascular
endothelial growth factor (VEGF).5 Several strategies have been
explored for manipulation of the HIF pathway at various points
to either promote or retard angiogenesis.6
To date, the most common strategy for HIF activation is through
inhibition of PHDs. General iron chelators such as desferrioxamine
(DFO) and the inorganic salt cobalt chloride (CoCl2) have been ex-
exists as a heterodimeric complex containing one of three
a sub-
units (HIF-1 , HIF-2 , or HIF-3 ) associated with the aryl hydro-
a
a
a
carbon receptor nuclear translocator (ARNT), also known as HIF-
1b. Although the hypoxic response requires multiple HIF subunits
to be functional, hypoxia only leads to changes in both the accu-
mulation and the activity of the HIF-1
In most cells the HIF pathway is primarily regulated by the inhi-
bition of the HIF-1
subunit degradation during hypoxia.2 In nor-
moxia, the intracellular level of HIF-1 protein is typically low
due to its on-going ubiquitination and proteasomal degradation.
The degradation of the HIF-1 subunit is initiated through
a subunit.
plored clinically. DFO activates HIF-1a by chelating iron, whereas
a
cobalt displaces iron from PHDs. Both compounds have been
shown to increase HIF activation in vitro and in vivo and have dem-
onstrated an ability to promote angiogenesis and produce a similar
response to ischemia.7 Although these agents have been investi-
gated clinically in certain disease settings, they have also been
associated with toxicity.8 As such, more recent approaches to
PHD inhibition have centered on small molecules.9 In many cases,
known PHD inhibitors contain functional groups that are isosteric
a
a
hydroxylation on a conserved proline residue, which is a process
⇑
Corresponding author.
E-mail address: kyle.a.emmitte@vanderbilt.edu (K.A. Emmitte).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.077

J. R. Theriault et al. / Bioorg. Med. Chem. Lett. 22 (2012) 76–81
77
analogs of 2-OG (Fig. 1). Common examples are N-acylated glycine
analogs such as 1 and 2.10 Most known PHD inhibitors contain a
carboxylic acid group, and in the case of compounds such as pyra-
zole 3, this functional group participates in a key interaction that
mimics that of 2-OG.11 PHD inhibitors without a carboxylic acid
group, such as 4 and 5, posses other acidic proton donors.12
In order to identify novel and interesting HIF activators, a phe-
notypic cell-based high-throughput screen was conducted on the
NIH’s Molecular Libraries Small Molecule Repository, which con-
tains more than 300 K compounds. The primary screening assay
utilized a previously described cell-based gene reporter assay with
a stably transfected human U2OS osteosarcoma cell line expressing
luciferase under control of hypoxia response elements (HREs).13
DFO (100
determined EC50 of 17.8
initially screened in the HRE-luciferase assay at a concentration
of 7.5 M. Using a cutoff at 10% or more of the activity obtained
l
M) was used as a positive control, with a previously
lM in the same assay. Compounds were
l
with the positive control DFO identified approximately 1200 pri-
mary hits. Complete dose–response curves were subsequently gen-
erated for these compounds using the primary HRE-luciferase
assay. These compounds were also tested in a high content imag-
ing assay examining the accumulation and nuclear translocation
Figure 1. Examples of PHD inhibitors.
of HIF-1a in USOS cells expressing HIF-1a fused to the green fluo-
rescent protein (GFP).14 Finally, each of these compounds was also
submitted to a commercially available cell-based proteasome inhi-
bition assay in order to eliminate any compounds acting as general
proteasome inhibitors.15
tested in a real time PCR assay for their ability to induce VEGF tran-
scription.14 Compounds were also subjected to the proteasome
inhibition assay and were found to be generally inactive. The activ-
ity of the isoxazole series in the HRE-luciferase assay was uni-
formly disappointing. Representative results with some of the
active compounds are shown here (Table 1). In fact, resynthesis
of hit 6 revealed that compound to be 3–4-fold less potent than
originally indicated. Although further work could certainly be done
Among the compounds emerging from the screening process,
isoxazoles 6 and 7 and triazines 8 and 9 represented particularly
interesting starting points for chemistry as they were dissimilar
to known PHD inhibitor scaffolds and lacked the acidic functional
groups common to such scaffolds ( Fig. 2). Such facts increased
the potential likelihood that these compounds may activate HIF
through alternative mechanisms to PHD inhibition, or if they did
act through PHD, may at the very least bind in a much different
manner. Furthermore, recent reports have demonstrated that HIF
activation increases cell viability and potentiates dopamine release
in dopaminergic cells, which may indicate a potential application
in certain neurodegenerative disorders such as Parkinson’s dis-
ease.16 Since highly acidic functional groups can often limit the
ability of small molecules to penetrate the blood–brain barrier,
new chemical starting points for HIF activators without such func-
tionality are considered interesting.
Table 1
SAR in isoxazole series
Entry
n
R
HRE luciferase HIF-1
a
nuclear RT-PCR
translocation VEGF EC50
M)
a
c
EC50 (lM)
b
EC50
7.72
3.04
20.0
16.6
(l
M)
(l
A 35 member amide library was rapidly prepared through stan-
dard coupling conditions with commercially available 5-phenylis-
oxazole-3-carboxylic acid and structurally diverse amines. In
addition to the aforementioned HRE-luciferase and high content
imaging nuclear translocation assays, the compounds were also
6
10
11
12
3
4
3
1
1-Imidazolyl
OH
1-Morpholinyl 23.9
Cyclopropyl 24.9
28.4
22.0
15.8
7.57
28.0
21.5
a
b
c
HRE-luciferase assay in U2OS cells (n P 2).
High content imaging assay for nuclear translocation of HIF-1
Real time PCR to evaluate VEGF transcription (n P 2).
a
(n P 2).
Scheme 1. Reagents and conditions: (a) NH2NH2ÁH2O, EtOH; (b) PhC(O)CO2Et,
EtOH; (c) POCl3,
w, 120 °C; (d) R1R2NH, THF.
Figure 2. HIF activator hit compounds.
l

78
J. R. Theriault et al. / Bioorg. Med. Chem. Lett. 22 (2012) 76–81
within this chemotype, early results in the triazine series were
more compelling and focus was directed there.
An initial library of 24 compounds (including resynthesized
batches of 8 and 9) focused on variation of the amine moiety
was prepared within the triazine series, and results there were
much more promising (Table 2). As with the isoxazole series, these
compounds were also inactive in the proteasome inhibition assay.
The potency of the resynthesized batches of 8 and 9 closely
matched those found during analysis of the initial hits. Not surpris-
ingly, cyclopropylmethanamine 17 was similar in potency to 8.
Increasing the size of the aliphatic group on the amine (18 and
19) led to improvements in potency relative to 8. Such modifica-
tions led to increasingly lipophilic compounds; however, incorpo-
ration of the more polar functional groups (20–22) was not
favorable. Tertiary amine analogs of hit 9 were also prepared, but
these analogs (23 and 24) failed to improve potency. Some hetero-
aryl secondary amine analogs such as 25 and 26 were also pre-
pared but were also not improvements. Although, the simple
benzyl amine analog 27 had similar activity to hit 8, it was noted
that methyl substitution of the benzyl group at any of the positions
on the aromatic ring (28–30) enhanced potency relative to 27. Hav-
ing an initial survey of the SAR in hand, attention was turned to the
synthesis of a second generation library that would refine the SAR
around the amine moiety. First, additional aliphatic amine analogs
would be prepared in an attempt to improve upon analogs 18 and
19. Second, more meta and para substituted analogs of 27 would be
prepared.
Selected aliphatic amine analogs of 18 and 19 are presented
here (Table 3). Introduction of a methyl group on the sp3 carbon
adjacent to the secondary amine of 18 afforded analogs 31 and
32. This modification had little effect on activity, and any prefer-
ence for one enantiomer over the other was minimal. Wanting to
continue to explore opportunities to reduce lipophilicity, tetrahy-
dropyran 33 was prepared; however, such a modification again
failed to yield positive results. Replacement of the t-butyl group
of 19 with phenyl gave analog 34, which was similarly potent in
the primary assay but less active in the secondary assays. Modifi-
cation of 34 by installation of a pyridyl ring (35) reduced activity.
The synthetic route for the synthesis of triazine HIF activators
such as 8 and 9 was relatively straightforward (Scheme 1).17 Reac-
tion of 2-cyanopyridine 13 with hydrazine afforded imidohydraz-
ide 14, which was subsequently reacted with ethyl 2-oxo-2-
phenylacetate in a condensation reaction to provide triazin-
5(4H)-one 15. Treatment with phosphorous oxychloride under
thermal conditions provided the chloride 16, which was directly
reacted with commercially available amines in an SNAr reaction
to afford the final compounds.
Table 2
Initial SAR in triazine series
Entry
R
HRE
luciferase
EC50
HIF-1
a
nuclear
RT-PCR
VEGF
EC50
translocation
a
b
c
(
l
M) EC50
(l
M)
(lM)
8
9
10.8
16.1
12.7
4.62
6.15
9.06
6.21
9.82
4.27
17
18
4.74
2.46
3.82
3.45
2.48
19
20
2.97
25.0
10.5
10.6
10.0
21
22
20.8
14.2
9.08
6.27
Table 3
SAR in analogs of 18 and 19
8.12
23
24
22.9
21.9
11.6
12.1
10.2
8.69
Entry
R
HRE
luciferase
EC50
HIF-1
a
nuclear
RT-PCR
VEGF EC50
(lM)
c
translocation
a
b
(lM)
EC50
(l
M)
25
26
22.4
5.41
7.55
3.26
31
32
4.17
2.06
2.55
1.88
7.64
4.46
6.73
1.20
27
28
29
30
7.74
6.67
5.41
4.58
4.80
9.54
2.39
1.19
4.48
3.08
2.91
2.60
33
34
29.5
7.67
9.82
8.38
8.72
3.63
35
17.5
9.84
9.60
a
b
c
a
b
c
HRE-luciferase assay in U2OS cells (n P 3).
High content imaging assay for nuclear translocation of HIF-1
Real time PCR to evaluate VEGF transcription (n P 2).
HRE-luciferase assay in U2OS cells (n P 3).
High content imaging assay for nuclear translocation of HIF-1
Real time PCR to evaluate VEGF transcription (n P 2).
a
(n P 2).
a
(n P 2).

J. R. Theriault et al. / Bioorg. Med. Chem. Lett. 22 (2012) 76–81
79
Table 4
SAR in analogs of 27
38 and 39 were less active than 36 and 37. Substitution of 27 with
a para-t-butyl group (40) gave one of the most potent compounds
tested. As was demonstrated in other analogs, attempts to intro-
duce polarity through installation of pyridine rings (41 and 42) sig-
nificantly reduced activity. Installation of a phenyl ring at either
the meta (43) or para (ML228) position of the benzyl amine 27,
on the other hand, quite enhanced potency.
ML228 was selected for further characterization since it showed
the most improved potency within the chemotype and was viewed
as an interesting tool compound. The probe compound ML228 was
among the top active compounds in each of the three HIF-specific
activity assays and was inactive in the proteasome inhibition
counterscreen assay (Fig. 3). In order to gain an understanding of
the broad pharmacology of this compound, ML228 was submitted
to a radioligand binding assay panel of 68 GPCRs, ion channels, ki-
nases, and transporters.18 ML228 was found to inhibit greater than
75% of radioligand binding in six assays when tested at a concen-
Entry
R
V
Q
HRE
luciferase
EC50
HIF-1
a
nuclear RT-PCR
VEGF EC50
M)
c
trans-location
a
b
(
l
M) EC50
(l
M)
(l
36
37
38
39
40
41
42
43
3,4-di-Me CH CH
2.96
2.30
1.49
3.73
6.27
0.60
5.02
7.62
0.50
1.40
1.85
2.77
3.67
3.92
1.12
4.35
6.45
2.57
1.63
3-Cl
4-OMe
3-OMe
4-tBu
H
CH CH
CH CH
CH CH
CH CH
N
CH
2.33
4.74
4.15
1.71
CH 26.0
27.8
H
3-Ph
N
tration of 10 lM (Table 5). An additional ten molecular targets
CH CH
CH CH
2.49
1.23
were identified where binding inhibition was moderate (50–75%
inhibition),19 while there was no significant binding (<50% inhibi-
tion) at the remaining 52 targets. Lipophilic compounds such as
ML228 sometimes exhibit non-specific binding in assays such as
these; however, this binding assay profile provides some basic
information for future users of the probe compound to consider.
Most compounds that are known to activate HIF act through
metal chelation. In order to provide some further insight into the
possible mechanisms by which ML228 acts on the HIF pathway,
the role of metal chelation was investigated (Fig. 4). To achieve this
ML228 4-Ph
a
b
c
HRE-luciferase assay in U2OS cells (n P 3).
High content imaging assay for nuclear translocation of HIF-1
Real time PCR to evaluate VEGF transcription (n P 2).
a
(n P 2).
Clearly, polar functional groups, whether installed in saturated or
aromatic systems, were not advantageous. Additional meta and
para substituted analogs of 27 are also shown here (Table 4). Both
3,4-dimethylphenyl analog 36 and 3-chlorophenyl analog 37 were
noted improvements over both 27 and previously prepared meth-
ylphenyl analogs 29 and 30. Conversely, methoxyphenyl analogs
task, excess iron or zinc (50
lM) were added in the media with the
probe and tested in the primary HRE-luciferase assay. Results were
Figure 3. Complete response curves for ML228. (A) HRE luciferase assay, (B) proteasome inhibition assay, (C) HIF-1a nuclear translocation high content imaging assay and
(D) RT-PCR VEGF transcription assay. The relative activity of ML228 was compared to the neutral (DMSO, 0% activity) and the stimulator control (DFO 100 lM, which equals
to 100% activity).

80
J. R. Theriault et al. / Bioorg. Med. Chem. Lett. 22 (2012) 76–81
Table 5
Binding assay profile for ML228
Radioligand binding assay
Species
% Inhibition
Adenosine A3
Dopamine Transporter (DAT)
Human
Human
Human
Human
Human
Rat
80
87
85
86
92
Opiate
l
(OP3, MOP)
Potassium channel hERG
Serotonin (5-hydroxytryptamine) 5-HT2B
Sodium Channel, site 2
105
compared to those with only media and probe. The presence of
iron produced a dramatic effect, shifting the dose–response curve
to the right and substantially decreasing the magnitude of re-
sponse. The addition of zinc produced a much smaller rightward
shift and the magnitude of response was similar to compound
alone. These results suggest that ML228 can chelate iron, which
is consistent with some known mechanisms of HIF activation. A
cell toxicity assay20 was also performed in the same cell line and
ruled out that the changes in activity observed with the addition
of these metals was due to cell death (data not shown).
Recent publication of PHD inhibitor 3 and related analogs dis-
closed X-ray co-crystal structures of some inhibitors from this
chemotype bound to the PHD2 active site.11 Although ML228 has
not been shown to act through PHD, molecular modeling of
ML228 was deemed an expeditious method for determining
whether it could reasonably bind at this known binding site. Flex-
ible superposition of an ensemble of 30 low energy structures of
ML228 with a structural hypothesis taken from a mutual flexible
alignment of all ligands bound to the published PHD2 active site
Figure 5. Shape based molecular graphics aligned with the SurflexSim conforma-
tional ensemble illustrate the large dissimilarity in shape and volume of ML228
(green surface) with the composite shape of the published PHD2 ligands.11
using SurflexSim21 gave a rather low shape based similarity score
of 5.5 (normalized similarity on a 1–10 scale) (Fig. 5). Generally,
compounds with a higher likelihood to bind a similar protein site
based on shape and surface considerations typically score above
7.0 in a SurflexSim shape-based similarity score. Furthermore,
manual docking of ML228 positioned to chelate iron in the active
site revealed multiple negative steric interactions and a lack of
favorable contacts. While not conclusive, molecular modeling sug-
gests a low probability that ML228 shares a similar mode of bind-
ing to PHD2 as those reported for analogs of 3.
Figure 4. Effects of metals on HRE luciferase activity of ML228. (A) Medium only, EC50 = 1.12
lM, (B) 50 lM iron, EC50 = 15.6 lM and (C) 50 lM zinc, EC50 = 5.01 lM. The
relative activity of ML228 is only compared to neutral control (DMSO) since the activity of DFO is reduced with the presence of iron and zinc.

J. R. Theriault et al. / Bioorg. Med. Chem. Lett. 22 (2012) 76–81
81
11. Rosen, M. D.; Venkatesan, H.; Peltier, H. M.; Bembenek, S. D.; Kanelakis, K. C.;
Zhao, L. X.; Leonard, B. E.; Hocutt, F. M.; Wu, X.; Palomino, H. L.; Brondstetter, T.
I.; Haugh, P. V.; Cagnon, L.; Yan, W.; Liotta, L. A.; Young, A.; Mirzadegan, T.;
Shankley, N. P.; Barrett, T. D.; Rabinowitz, M. H. ACS Med. Chem. Lett. 2010, 1,
526.
12. (a) Warshakoon, N. C.; Wu, S.; Boyer, A.; Kawamoto, R.; Sheville, J.; Renock, S.;
Xu, K.; Pokross, M.; Zhou, S.; Winter, C.; Walter, R.; Mekel, M.; Evdokimov, A. G.
Bioorg. Med. Chem. Lett. 2006, 16, 5517; (b) Clements, M. J.; Debenham, J. S.;
Hale, J. J.; Madsen-Duggan, C. B.; Walsh, T. F. PCT Int. Patent Appl. WO2009/
117269, 2009.
ML228 represents a novel chemotype available to the research
community for the study of HIF activation and its therapeutic po-
tential. Not only is the compound substantially different in struc-
ture from known HIF activators, ML228 lacks the acidic
functional group almost universally present in PHD inhibitors,
which may be important for certain disease applications. ML228
was demonstrated to potently activate HIF in vitro as well as its
downstream target VEGF. Further biological evaluation of ML228
is ongoing and will be reported in due course.
13. Chau, N. M.; Rogers, P.; Aherne, W.; Carroll, V.; Collins, I.; McDonald, E.;
Workman, P.; Ashcroft, M. Cancer Res. 2005, 65, 4918.
14. (a) Wang, Y.; Wan, C.; Deng, L.; Liu, X.; Cao, X.; Gilbert, S. R.; Bouxsein, M. L.;
Faugere, M. C.; Guldberg, R. E.; Gerstenfeld, L. C.; Haase, V. H.; Johnson, R. S.;
Schipani, E.; Clemens, T. L. J. Clin. Invest. 2007, 117, 1616; (b) Wan, C.; Gilbert, S.
R.; Wang, Y.; Cao, X.; Shen, X.; Ramaswamy, G.; Jacobsen, K. A.; Alaql, Z. S.;
Eberhardt, A. W.; Gerstenfeld, L. C.; Einhorn, T. A.; Deng, L.; Clemens, T. L. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 686.
15. Proteasome-Glo™ Chymotrypsin, www.promega.com.
16. Johansen, J. L.; Sager, T. N.; Lotharius, J.; Witten, L.; Mork, A.; Egebjerg, J.;
Thirstrup, K. J. Neurochem. 2010, 115, 209.
Acknowledgments
We thank NIH and the Molecular Libraries Probe Production
Centers Network (R03 MH082355-01A2) for their generous sup-
port of this work. The Vanderbilt Specialized Chemistry Center
for Accelerated Probe Development (1U54MH084659-01) and
The Broad Institute Probe Development Center (1U54HG005032-
01) also thanks NIH and the MLPCN. We also thank Stuart L. Schre-
iber of The Broad Institute for his support of this work.
17. Synthesis of ML228: (a) Compound 14: Ethanol was added dropwise to 2-
cyanopyridine 13 (10 g, 96 mmol, 1.0 equiv) and hydrazine hydrate (3.08 g,
96.1 mmol, 1.00 equiv) until a clear brown solution was obtained. The solution
was allowed to sit at room temperature for 24 h. Additional hydrazine hydrate
(1.54 g, 48.0 mmol, 0.50 equiv) was added, and the solution was allowed to sit
for an additional 24 h. The solvent was removed in vacuo, and the solid was
washed with ether to give the title compound as an orange solid that was used
without further purification. ES-MS [M+1]+: 137.3. (b) Compound 15:
Compound 14 (5.0 g, 37 mmol, 1.0 equiv) was dissolved in ethanol (367 mL)
and cooled to 0 °C. Ethyl benzoylformate (5.83 mL, 36.7 mmol, 1.00 equiv) was
added, and the reaction was allowed to warm to room temperature and stirred
for 72 h. The solvent was removed in vacuo to give a yellow solid that was used
without further purification. ES-MS [M+1]+: 251.2. (c) Compound 16: A dry,
argon-purged vial was charged with compound 15 (180 mg, 0.72 mmol,
1.0 equiv) and phosphorus oxychloride (1.8 mL) and subjected to microwave
irradiation at 120 °C for 10 min. The resulting solution was added dropwise to
cold, saturated NaHCO3 and then extracted (3Â) with cold CH2Cl2. The
combined organics were filtered via phase separation, concentrated in vacuo,
and further dried under vacuum to afford a yellow oil that was carried forward
without purification. ES-MS [M+1]+: 269.0. (d) ML228: Compound 16 (180 mg,
0.67 mmol, 1.0 equiv) and 4-phenyl benzylamine (200 mg, 1.1 mmol,
1.6 equiv) were dissolved in anhydrous THF (6.5 mL) and stirred at rt for
48 h under argon. The reaction mixture was concentrated in vacuo. The
concentrate was then dissolved in ethyl acetate, washed (2Â) with 1 N HCl, and
washed (2Â) with 1 N NaOH. The aqueous layers were back-extracted (2Â).
The combined organics were dried (MgSO4), filtered, and concentrated in
vacuo. Purification by flash chromatography on silica gel afforded 115 mg
(41%) of the title compound as a yellow solid: 1H NMR (400 MHz, DMSO-d6) d
8.77 (d, J = 4.0 Hz, 1H), 8.31 (d, J = 7.9 Hz, 1H), 8.16 (dd, J = 5.9, 5.9 Hz, 1H), 7.97
(m, 1H), 7.75 (dd, J = 7.7, 1.7 Hz, 2H), 7.70 (m, 1H), 7.63–7.51 (m, 8H), 7.43 (dd,
J = 7.6, 7.6 Hz, 2H), 7.33 (dd, J = 7.4, 7.4 Hz, 1H), 4.74 (d, J = 5.9 Hz, 2H), 4.14 (m,
1H); ES-MS [M+1]+:416.2.
References and notes
1. Semenza, G. L. Curr. Opin. Cell. Biol. 2001, 13, 167.
2. (a) Huang, L. E.; Arany, Z.; Livingston, D. M.; Bunn, H. F. J. Biol. Chem. 1996, 271,
32253; (b) Huang, L. E.; Gu, J.; Schau, M.; Bunn, H. F. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 7987; (c) Kallio, P. J.; Wilson, W. J.; O’Brien, S.; Makino, Y.; Poellinger,
L. J. Biol. Chem. 1999, 274, 6519; (d) Sutter, C. H.; Laughner, E.; Semenza, G. L.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4748.
3. (a) Ivan, M.; Kondo, K.; Yang, H.; Kim, W.; Valiando, J.; Ohh, M.; Salic, A.; Asara,
J. M.; Lane, W. S.; Kaelin, W. G., Jr. Science 2001, 292, 464; (b) Jaakkola, P.; Mole,
D. R.; Tian, Y. M.; Wilson, M. I.; Gielbert, J.; Gaskell, S. J.; Kriegsheim, A.;
Hebestreit, H. F.; Mukherji, M.; Schofield, C. J.; Maxwell, P. H.; Pugh, C. W.;
Ratcliffe, P. J. Science 2001, 292, 468.
4. (a) Berra, E.; Benizri, E.; Ginouves, A.; Volmat, V.; Roux, D.; Pouyssegur, J. EMBO
J. 2003, 22, 4082; (b) Marxsen, J. H.; Stengel, P.; Doege, K.; Heikkinen, P.;
Jokilehto, T.; Wagner, T.; Jelkmann, W.; Jaakkola, P.; Metzen, E. Biochem. J. 2004,
381, 761; (c) Metzen, E.; Stiehl, D. P.; Doege, K.; Marxsen, J. H.; Hellwig-Burgel,
T.; Jelkmann, W. Biochem. J. 2005, 387, 711.
5. Kallio, P. J.; Okamoto, K.; O’Brien, S.; Carrero, P.; Makino, Y.; Tanaka, H.;
Poellinger, L. EMBO J. 1998, 17, 6573.
6. (a) Giaccia, A.; Siim, B. G.; Johnson, R. S. Nat. Rev. Drug Disc. 2003, 2, 803; (b)
Hewitson, K. S.; McNeill, L. A.; Schofield, C. J. Curr. Pharm. Des. 2004, 10, 821; (c)
Yeo, E. J.; Chun, Y. S.; Park, J. W. Biochem. Pharmacol. 2004, 68, 1061.
7. (a) Chekanov, V. S.; Nikolaychik, V.; Maternowski, M. A.; Mehran, R.; Leon, M.
B.; Adamian, M.; Moses, J.; Dangas, G.; Kipshidze, N.; Akhtar, M. Ann. Thorac.
Surg. 2003, 75, 184; (b) Hirsila, M.; Koivunen, P.; Xu, L.; Seeley, T.; Kivirikko, K.
I.; Myllyharju, J. FASEB J. 2005, 19, 1308; (c) Xi, L.; Taher, M.; Yin, C.; Salloum, F.;
Kukreja, R. C. Am. J. Physiol. Heart Circ. Physiol. 2004, 287, H2369; (d) Yuan, Y.;
Hilliard, G.; Ferguson, T.; Millhorn, D. E. J. Biol. Chem. 2003, 278, 15911.
8. Bentur, Y.; McGuigan, M.; Koren, G. Drug Saf. 1991, 6, 37.
9. Rabinowitz, M. H.; Barrett, T. D.; Rosen, M. D.; Venkatesan, H. Ann. Rep. Med.
Chem. 2010, 45, 123.
10. (a) Allen, J. R.; Burli, R.; Bryan, M. C.; Cao, G.-Q.; Neira, S. C.; Reed, A. B. PCT Int.
Patent Appl. WO2008/130600, 2008.; (b) Guiadeen, D.; Hale, J. J.;
Kothandaraman, S. PCT Int. Patent Appl. WO2008/130527, 2008.
18. LeadProfilerScreenÒ, www.ricerca.com.
19. Targets where moderate binding displacement was noted (human unless
otherwise noted): adrenergic a1D (53%), adrenergic a2A (57%), rat calcium
channel
L-type, dihydropyridine (56%), dopamine D1 (54%), dopamine D3 (67%),
histamine H1 (65%), melatonin MT1 (51%), norepinephrine transporter (67%),
opiate
j (71%), prostanoid EP4 (53%).
20. CellTiter-GloÒ, www.promega.com.
21. Jain, A. N. J. Med. Chem. 2004, 47, 947.