Benzimidazole-2-pyrazole HIF prolyl 4-hydroxylase inhibitors as oral erythropoietin secretagogues

Article abstract of DOI:10.1021/ml100198y

HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). Certain benzimidazole-2-pyrazole carboxylates were discovered to be PHD2 inhibitors using ligand- and structure-based methods and found to be potent, orally efficacious stimulators of erythropoietin secretion in vivo.

Full text of DOI:10.1021/ml100198y

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Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase
Inhibitors as Oral Erythropoietin Secretagogues
Mark D. Rosen,* Hariharan Venkatesan, Hillary M. Peltier, Scott D. Bembenek,
Kimon C. Kanelakis, Lucy X. Zhao, Barry E. Leonard, Frances M. Hocutt, Xiaodong Wu,
Heather L. Palomino, Theresa I. Brondstetter, Peter V. Haugh, Laurence Cagnon, Wen Yan,
Lisa A. Liotta, Andrew Young, Tara Mirzadegan, Nigel P. Shankley, Terrance D. Barrett, and
Michael H. Rabinowitz
Johnson & Johnson Pharmaceutical Research and Development, L.L.C, 3210 Merryfield Row, San Diego,
California 92121, United States
ABSTRACT HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate
key physiological responses to hypoxia by modulating the levels of hypoxia
inducible factor 1-R (HIF1R). Certain benzimidazole-2-pyrazole carboxylates were
discovered to be PHD2 inhibitors using ligand- and structure-based methods and
found to be potent, orally efficacious stimulators of erythropoietin secretion
in vivo.
KEYWORDS Prolyl 4-hydroxylase inhibitors, PHD inhibitors, hypoxia inducible factor,
HIF, erythropoietin, epo, erythropoietin stimulating agents, anemia
IF prolyl 4-hydroxylases (PHD1, PHD2, and PHD3)
are a family of highly conserved, iron-containing,
2-oxoglutarate- (2OG) and dioxygen-dependent
we report our efforts in this area, which led to the discovery
of novel PHD inhibitors that are potent, orally active erythro-
poietin secretagogues.
H
enzymes that play a crucial role in adaptation and survival
during oxygen-deficient conditions.1-3 Under normoxia, the
transcription factor hypoxia inducible factor 1-R (HIF1R) is
constitutively expressed and rapidly degraded, with a half-
life of less than 5 min.4 The degradation of HIF1R is regu-
lated in part by PHD enzymes, which hydroxylate HIF1R
proline residues 564 and 402, thereby enabling binding to
the von Hippel-Lindau protein and subsequent proteasomic
degradation.5 Under a sufficiently low partial pressure of
oxygen, the rate of PHD-mediated hydroxylation of HIF1R is
lessened such that the production of HIF1R outpaces its
degradation. The accumulated HIF1R then translocates to
the nucleus, leading to the formation of a HIF1R-HIF1β hetero-
dimer and the activation of genes involved in hypoxic responses
such as erythropoietin (epo) secretion and erythropoiesis,
anaerobic glycolysis, and angiogenesis.6,7 Therefore, posi-
tive modulation of HIF transcriptional activity holds promise
as a mode of treatment for a variety of debilitating ischemia-
associated diseases, including anemia, myocardial infarction,
stroke, and metabolic disorders, by conducting an orches-
trated response to hypoxic challenge.8,9
Screening of an internal compound library uncovered nu-
merous strongly metal-ligating molecules that, upon closer
investigation, appear to exert their inhibitory activity through
iron sequestration and subsequent enzyme deactivation.22
As this mechanism is unlikely to translate into a viable ther-
apeutic method, we undertook an alternative approach to
lead generation. A number of benzimidazole-2-glycinamides
emerged as targets for chemical synthesis and subsequent
evaluation as inhibitors, using a PHD2 enzymatic assay
previously reported from this laboratory.23 The synthesis
of this initial set of potential inhibitors began with known or
available benzimidazole-2-carboxylic acids 1a-h (Scheme 1).24
These acids were coupled to amino esters 2a-h, which were
then saponified to provide the corresponding benzimidazole
acids 3a-h.
Evaluation of compounds 3a-h showed several to possess
measurable inhibitory activity in our PHD2 enzymatic assay
(Table 1). Dichloride analogue 3b proved to be the most
potent, with a pIC50 value of 4.9. Representative members
of this set were found to have low iron affinity in solution
relative to literature reference compounds, suggesting that
the observed inhibitory activity does not result from metal
center depletion.25 However, these particular benzimidazole
inhibitors failed to significantly stimulate epo release from
Because of their key role in the regulation of HIF levels,
inhibition of PHD enzymes is an attractive strategy by which
to potentiate the transcriptional activity of HIF in a thera-
peutically relevant manner. Indeed, during the last several
years, intense efforts to discover PHD inhibitors have been
adumbrated primarily in the patent literature,10 and to a much
lesser degree in the peer-reviewed literature,11 -19 leading
to four compounds entering clinical trials.20,21 In this letter,
Received Date: August 20, 2010
Accepted Date: September 29, 2010
Published on Web Date: October 05, 2010
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DOI: 10.1021/ml100198y ACS Med. Chem. Lett. 2010, 1, 526–529
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Scheme 1. Synthesis of Benzimidazole Amides 3a-ha
a Reagents and conditions: (a) HATU, amino acid methyl ester hydrochloride, DIEA, DMF, 23 °C, 16 h. (b) LiOH, THF/H2O, 23 °C, 2-16 h. For R1-3
groups, see Table 1.
Table 1. PHD2 Enzyme Inhibition and Epo Release in Hep3B Cells
for Compounds 3a-h
Table 2. PHD2 Enzyme Inhibition, Epo Release, and HIF1R Accu-
mulation for Compounds 6a-h
R1
R2
R3
pIC50
epo (%)b
HIF1R (%)c
a
compd
a
compd
R1
R2
R3
R4
pIC50
epo release (%)b
6a
6b
6c
6d
6e
6f
H
H
H
H
F
H
H
H
H
H
Br
H
H
6.0
6.4
6.4
7.1
6.5
6.8
7.1
7.0
<20
105 ( 6
<20
<20
70 ( 3
<20
3a
3b
3c
3d
3e
3f
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
<4
4.9
4.6
<4
4.3
4.7
4.3
4.1
<20
<20
<20
<20
<20
<20
<20
<20
Br
Cl
H
OMe
Cl
Br
H
129 ( 11
142 ( 10
56 ( 4
72 ( 7
63 ( 2
21 ( 1
92 ( 4
92 ( 5
F
H
OCF3
OCF3
CF3
CF3
H
H
Cl
F
OMe
CF3
CF3
CF3
H
CF3
CF3
CF3
6g
6h
176 ( 7
28 ( 9
3g
3h
(S)-Me
(R)-Me
a Negative logarithm of the concentration required to achieve 50%
enzyme inhibition; the value is (0.3 log units. b Percent increase in
erythropoietin release from Hep3B cells at 100 μM concentration relative
to positive control at 24 h. c Percent increase in HIF1R accumulation in
Hep3B cells at 100 μM concentration relative to positive control measured at
24 h. See the Supporting Information for assay details.
a Negative logarithm of the concentration required to achieve 50%
enzyme inhibition; the value is (0.3 log units. b Percent increase in epo
release from Hep3B cells at 100 μM concentration relative to positive control
measured at 24 h. See the Supporting Information for assay details.
Scheme 2. Synthesis of Benzimidazole-2-pyrazoles 6a-ha
Removal of the protective group under acidic conditions
provided ethyl esters 5a-h, which were saponified to afford
the desired carboxylic acids 6a-h.
Benzimidazole-2-pyrazoles 6a-e proved to be potent
inhibitors of PHD2 relative to the benzimidazole-2-carbox-
amides 3a-h (Table 2). For example, compounds 6a-c each
show an increase in enzyme inhibitory activity of about 2
orders of magnitude relative to their direct comparators 3a,
3c, and 3e, while several other compounds display pIC50
values of 7.0 or more. As with the glycine amide compounds,
selected members of this set also displayed low iron chelation
ability in solution, indicating that the increased inhibitory
activity was not a result of iron sequestration.25 Of greater
significance, these compounds effected a robust increase
of epo secretion from Hep3B cells measured after 24 h of
incubation. To demonstrate that the observed increase in
epo secretion was a consequence of PHD inhibition, 6a-e
were tested in a HIF1R accumulation assay also using Hep3B
cells, and compounds from this group were found to pro-
mote increases in HIF1R accumulation by as much as 92 (
4% (e.g., 6g, Table 2).
a Reagents and conditions: (a) SEMCl or MEMCl, DIEA or NaH, THF.
(b) Ethyl pyrazole-4-carboxylate, Cs2CO3, DMF, 80 °C, 1-5 h. (c) 4 M
HCl/dioxane, EtOH, reflux, 1 h. (d) LiOH, THF/H2O. For R1-3 groups,
see Table 2.
Hep3B cells at concentrations up to 100 μM, while efforts to
optimize the biopharmaceutical properties of the benzimid-
azole-2-carboxamide compounds proved challenging. As
part of a larger search for suitable isosteric glycine replace-
ment groups, we prepared a number of benzimidazole-2-
pyrazoles as shown in Scheme 2.26
The synthesis of benzimidazole-2-pyrazoles 6a-h began
from known or available 2-chlorobenzimidazoles 4a-h.
Protection of the benzimidazole nitrogen atom as a MEM
or SEM aminal facilitates displacement of the chloride atom
by ethyl pyrazole-4-carboxylate in the presence of Cs2CO3.
Having discovered compounds that potently inhibit PHD2
with concomitant increases in both HIF1R accumulation and
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epo release in Hep3B cells, we evaluated 6g and 6h in an in
vivo, orally dosed epo release model. Measured 6 h after a
single oral dose of 100 μmol/kg, compounds 6g and 6h elicited
increases in epo levels of 56- and 18-fold, respectively (Figure 1).
This robust increase corresponds to plasma epo levels of
1410 ( 410 pg/mL versus the vehicle group value of 25 (
8 pg/mL for the compound 6g cohort and 1270 ( 760 pg/mL
versus the vehicle group value of 72 ( 20 pg/mL for the 6h
cohort. For compound 6h, this pharmacodynamic response
occurred with 1 h plasma concentrations of 14 μM and 6 h
concentrations of 0.1 μM.
With these novel and structurally distinct PHD2 inhibitors
in hand, we undertook structural studies to elucidate their
mode of inhibition. Schofield, Syed, and co-workers, as well
as Evodokimov and co-workers, have independently reported
X-ray cocrystal structures of a truncated form of PHD2 with
two structurally related isoquinoline inhibitors, both of which
clearly mimic the endogenous cofactor 2OG in the active site
of the enzyme.27,28 To supplement these data for our own
studies, we obtained the X-ray cocrystal structure of PHD2
with its natural ligand, 2OG (Figure 2).29 In agreement with
the previously published structures, the carboxylic acid
group is clearly participating in a salt bridge interaction with
R383, while the R-keto acid moiety is ligated to the iron atom
in the active site. Furthermore, an ordered water cascade
suggests the possibility that the R-ketocarboxylic acid moiety
may be involved in a water-bridged interaction with Y303,
analogous to the direct interaction observed between Y303
and the isoquinoline hydroxyl groups of the previously reported
structures. Taken together, these X-ray crystal structures
illustrate several key interactions between PHD2 and these
known ligands.
Figure 1. Epo levels vs vehicle for compounds 6g and 6h after
a single oral dose of 100 μmol/kg in the mouse.
In the case of benzimidazole glycine amide 3b and pyrazole
6d, the iron atom is engaged by the two sp2 nitrogen atoms
of the benzimidazole and pyrazole rings, respectively, while
the carboxylic acid group appears well poised to interact with
Arg383 (Figure 3).29 Once again, the benzimidazole NH
group is proximal to an ordered water molecule, suggesting
a possible water-bridged interaction with Y303. These struc-
tures clearly demonstrate that compounds 3b and 6d act as
2OG mimetics and that they adopt a similar binding mode to
that of the endogenous ligand.
In summary, we disclose the discovery and preliminary
pharmacological profiling data for novel benzimidazole-based
PHD2 inhibitors. The X-ray cocrystal structures of PHD2 with
2OG, 3b, and 6d are reported, and these structures elucidate
key protein interactions and verify target engagement.
Several compounds were identified that were efficacious in
a series of assays, including biochemical enzyme inhibition,
cell-based epo release and HIF1R accumulation, and in vivo
Figure 2. X-ray cocrystal structure of PHD2 and the endogenous
ligand 2OG.
Figure 3. X-ray cocrystal structures of (a) PHD2/3b and (b) PHD2/6d.
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SUPPORTING INFORMATION AVAILABLE Experimental
details for the synthesis and characterization of 3a-h and 6a-h
as well as assay details. This material is available free of charge via
the Internet at http://pubs.acs.org.
ACKNOWLEDGMENT We thank Dr. Jiejun Wu and Heather
McAllister for analytical assistance.
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