Discovery of IWP-051, a Novel Orally Bioavailable sGC Stimulator with Once-Daily Dosing Potential in Humans

Article abstract of DOI:10.1021/acsmedchemlett.5b00479

In recent years, soluble guanylate cyclase (sGC, EC 4.6.1.2) has emerged as an attractive therapeutic target for treating cardiovascular diseases and diseases associated with fibrosis and end-organ failure. Herein, we describe our design and synthesis of

Full text of DOI:10.1021/acsmedchemlett.5b00479

Letter
pubs.acs.org/acsmedchemlett
Discovery of IWP-051, a Novel Orally Bioavailable sGC Stimulator
with Once-Daily Dosing Potential in Humans
Takashi Nakai,* Nicholas R. Perl, Timothy C. Barden, Andrew Carvalho, Angelika Fretzen,
Peter Germano, G-Yoon J. Im, Hong Jin, Charles Kim, Thomas W.-H. Lee, Kimberly Long, Joel Moore,
Jason M. Rohde, Renee Sarno, Chrissie Segal, Erik O. Solberg, Jenny Tobin, Daniel P. Zimmer,
and Mark G. Currie
Ironwood Pharmaceuticals, Inc. 301 Binney Street Cambridge, Massachusetts 02142 United States
S
* Supporting Information
ABSTRACT: In recent years, soluble guanylate cyclase (sGC, EC 4.6.1.2) has
emerged as an attractive therapeutic target for treating cardiovascular diseases and
diseases associated with fibrosis and end-organ failure. Herein, we describe our
design and synthesis of a series of 4-hydroxypyrimidine sGC stimulators starting
with an internally discovered lead. Our efforts have led to the discovery of IWP-051,
a molecule that achieves good alignment of potency, stability, selectivity, and
pharmacodynamic effects while maintaining favorable pharmacokinetic properties
with once-daily dosing potential in humans.
KEYWORDS: Soluble guanylate cyclase, sGC, NO-independent stimulators, heme-dependent sGC stimulators, nitric oxide, IWP-051
oluble guanylate cyclase (sGC, EC 4.6.1.2) is a signal-
Stransduction enzyme that binds nitric oxide (NO) and
catalyzes the conversion of guanosine-5′-triphosphate (GTP)
to the secondary messenger cyclic guanosine-3′,5′-mono-
phosphate (cGMP). The NO-sGC-cGMP signal-transduction
pathway is involved in the regulation of various physiological
processes, including smooth muscle relaxation, platelet
inhibition, and vasodilation.1 The NO-sGC-cGMP pathway
plays an important role in coordinating blood flow to tissues,
providing oxygen and nutrients, and removing waste products
in response to local demands.2 Impairment of sGC and/or
reduced NO bioavailability has been implicated in the
pathogenesis of cardiovascular, pulmonary, renal, and hepatic
diseases.3 The therapeutic benefit of NO-donors such as
organic nitrates is limited by lack of efficacy due to variable
biometabolism4 and the development of NO tolerance.5
Figure 1. Three examples of sGC stimulators.
namic profile allowing once-daily dosing and potential to
minimize the risk of hypotensive side effects.
An alternative to NO-donors are sGC stimulators, a class of
ligands that bind allosterically to the Fe(II) form of the heme-
containing enzyme and stimulate the formation of cGMP.6,7
sGC stimulators can act both independently and in synergy
with NO. In preclinical models, sGC stimulators have
demonstrated anti-inflammatory and antifibrotic effects, as
well as end-organ protections.8−11 The first marketed sGC
stimulator, Bayer’s riociguat (Adempas) (Figure 1), was
approved in 2013 by the FDA for the treatment of pulmonary
arterial hypertension (PAH) and inoperable chronic throm-
boembolic pulmonary hypertension (CTEPH) based on its
ability to improve exercise capacity and symptomatic profile
with disease severity defined by the World Health Organization
functional classification system.12 We sought to design an sGC
stimulator with a sustained pharmacokinetic and pharmacody-
Some known sGC stimulators, such as riociguat and BAY 41-
2272,13 feature a fused ring structure (Figure 1). We have
focused our discovery effort on sGC stimulators that utilize a
novel, biaryl pyrazole structure, as exemplified by 114 (Figure
1). 1 is a potent sGC stimulator as determined by production of
cGMP in a human embryonic kidney (HEK) cellular assay in
the presence of the NO-donor diethylenetriamine NONOate
(DETA-NO, 10 μM) (EC50 = 240 nM). When dosed at 1 and
10 mg/kg iv and po, respectively, 1 has a 26 min iv half-life and
is orally bioavailable (Fpo = 53%) in rat. An off-target screen
revealed that 1 binds to the κ-opioid (KOP) receptor (IC50
=
Received: December 11, 2015
Accepted: February 24, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00479
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ACS Medicinal Chemistry Letters
Letter
1.2 μM) and is a functional agonist (EC50 = 1.5 μM). This off-
target activity, coupled with a short half-life in rat, precluded the
advancement of 1 into additional preclinical models. We
therefore focused on designing sGC stimulators with cellular
potency comparable to or better than that of 1 with reduced
KOP binding and a better pharmacokinetic profile.
acrylates 3 in the presence of base such as 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine deliv-
ered the desired pyrimidines, which could then undergo further
transformations to access additional pyrazole analogs.22
Using the isoxazole-substituted pyrazole as a template,
various pyrimidine analogs were profiled (Table 1). The
With the goal of improving the selectivity and the
pharmacokinetic profile of 1, we sought to introduce
functionality at the pyrimidine 5-position. In the course of
this effort, amidine 2a was treated with cyanoacrylate 3a
(Scheme 1). In contrast to a previous report detailing a similar
Table 1. Pyrimidine Ring SAR
Scheme 1. Synthesis of Analogs 4 and 5
CLint,
RLM,
HLM
(μL/
min/mg
protein)
EC
50a
(nM)
Compound
R1
R2
R3
1
NH2
OH
OH
OH
OH
OH
NH2
OH
OH
OH
OH
OH
OH
OH
OH
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
240
26, 10
14, 8
15, 12
26, 14
8, 8
reaction,15 the desired 4-amino product 4 was obtained in low
yield (4%) with the hydroxypyrimidine 5 predominating as a
major product (38%). While 5 has attenuated cellular potency
(EC50 = 500 nM) relative to 1, the compound is more stable in
male rat liver microsomes (RLM, CLint = 14 versus 26 μL/min/
mg protein) and comparably stable in human liver microsomes
(HLM, CLint = 8 versus 10 μL/min/mg protein). When 5 was
dosed orally in rats, it exhibited a significantly higher maximum
plasma concentration (Cmax) and area under the curve (AUC)
compared with our previously synthesized 4-amino pyrimidine
compounds.16 On the basis of these encouraging data we
focused our efforts on expanding the 4-hydroxypyrimidine
series.
5
CN
H
500
b
8
ND (92)
ND (53)
ND (40)
290
9
-NHBz
10
11
12
13
14
15
16
17
18
19
20
21
-CMe2OH
F
6, 3
F
190
17, 14
5, 7
Cl
590
CF3
ND (54)
900
20, 19
13, 15
27, 18
15, 6
24, 8
58, 179
16,9
-SO2Me
-SO2Ph
350
-SO2(p-Cl-Ph)
-SO2N(Me)2
-SO2N(Me)(Ph)
F
160
ND (87)
730
ND (70)
320
The general synthesis of pyrimidine analogs is depicted in
Scheme 2.17−20 Starting with readily accessible methyl ketones
F
95, 20
Potency measured in human embryonic kidney (HEK) cells in the
a
presence of 10 μM DETA-NO. The geometric mean of at least two
Scheme 2. General Synthesis of Pyrimidine Analogs
b
repeated runs. Not determined due to incomplete concentration
response. Maximal efficacy (%Emax) at 30 μM shown in parentheses.
electronic nature of the substituent at the pyrimidine 5-position
has a significant influence on sGC potency. A reduction in
cellular potency is observed when the electron-withdrawing
nitrile of 5 (EC50 = 500 nM) is replaced with a proton (8, EC50
= ND), an electron-donating benzoyl-protected amino group
(9, EC50 = ND), or a carbinol (10, EC50 = ND). Alternatively,
replacement of the nitrile with other electron-withdrawing
groups generally results in similarly potent compounds. For
instance, 5-fluoro analog 11 (EC50 = 290 nM) is more potent
than 5 and is also stable in the presence of RLM (CLint = 6 μL/
min/mg protein) and HLM (CLint = 3 μL/min/mg protein).
Not all compounds featuring electron withdrawing groups at
the 5-position are potent stimulators. While chloro-substituted
derivative 13 (EC50 = 590 nM) performs comparably to the
nitrile analog 5, the increased electron withdrawing nature of
the trifluoromethyl group is detrimental to cellular sGC
potency (14, EC50 = ND).
of general structure 6, Claisen condensation with diethyl
oxalate in the presence of lithium hexamethyldisilazide
(LHMDS) at low temperature, followed by acetic acid-
mediated cyclization with benzyl and alkyl hydrazines gave
intermediate pyrazole esters 7. Conversion of the pyrazole
esters to amidines 2 was accomplished by treatment of the
esters with excess trimethylaluminum and ammonium chloride
at elevated temperatures.21 Condensation of amidines 2 with
A number of 5-substituted sulfones and sulfonamides possess
moderate-to-good cellular potency. While methyl sulfone 15 is
moderately potent (EC50 = 900 nM), replacing the methyl
group with a phenyl moiety increases the potency (16, EC50
=
B
DOI: 10.1021/acsmedchemlett.5b00479
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ACS Medicinal Chemistry Letters
Letter
350 nM). Additionally, the 4-chlorophenyl sulfone 17 is the
most potent compound in the series (EC50 = 160 nM).
Sulfonamides are less potent than their sulfone counterparts
(18, EC50 = ND and 19, EC50 = 730 nM) and 19 is also
metabolically unstable in vitro (RLM CLint = 58 μL/min/mg
protein, HLM CLint = 179 μL/min/mg protein). Introduction
of the methyl group to the pyrimidine 6-position of 11
attenuates cellular potency (20, EC50 = ND). While O-
methylation of 11 has no effect on potency (21, EC50 = 320
nM), the metabolic stability is decreased presumably due to
oxidative O-demethylation. Finally, the addition of a fluorine
atom at the pyrimidine 5-position imparts a greater improve-
ment in potency for hydroxyprimidine 8 (8, EC50 = ND vs 11,
With the 5-fluoro-4-hydroxypyrimidine and isoxazole moi-
eties established at the pyrazole 3- and 5-positions, respectively,
we next optimized the pyrazole nitrogen substitution (Table 3).
Table 3. SAR of the R5 Group
EC50 = 290 nM) compared to aminopyrimidine 1 (1, EC50
240 nM vs 12, EC50 = 190 nM).
=
Holding the 5-fluoro-4-hydroxyprimidine moiety constant,
we next explored modifications to the pyrazole 5-position
(Table 2). Adjustments to the isoxazole moiety significantly
Table 2. SAR of the R4 Group
Potency measured in human embryonic kidney (HEK) cells in the
a
presence of 10 μM DETA-NO. The geometric mean of at least two
b
repeated runs, unless stated otherwise. Not determined due to
incomplete concentration response. Maximal efficacy (%Emax) at 30
μM shown in parentheses.
The 2-fluoro benzyl substituent confers sGC potency, as 2-H
benzyl analog 30 (EC50 = 880 nM) is less potent than 11. sGC
activity is maintained with the introduction of a halogen atom
(31, EC50 = 370 nM and 32, EC50 = 380 nM) or a methyl
group (33, EC50 = 240 nM) adjacent to the benzyl fluorine.
However, the introduction of a methyl group introduces a
Potency measured in human embryonic kidney (HEK) cells in the
a
presence of 10 μM DETA-NO. The geometric mean of at least two
b
repeated runs. Not determined due to incomplete concentration
response. Maximal efficacy (%Emax) at 30 μM shown in parentheses.
affect potency in the cellular sGC assay. Introduction of
nitrogen into the isoxazole framework of 11 to give 1,2,4-
oxadiazole 22 (EC50 = ND) dramatically reduces sGC activity,
although the metabolic stability is maintained. Oxazoles 23
(EC50 = 1500 nM) and 24 (EC50 = ND) also suffer a reduction
in potency. The limited space afforded in this vector is
highlighted by 5-methylisoxazole 25, in which the addition of a
single methyl group to 11 drastically reduces the potency.
When the isoxazole is replaced by a larger isothiazole (26, EC50
= 180 nM) cellular potency is retained, albeit at the expense of
microsomal stability (RLM CLint = 129 μL/min/mg protein,
HLM CLint = 169 μL/min/mg protein). Beyond 5-membered
metabolic site that results in decreased RLM stability (CLint =
79 μL/min/mg protein). While the sGC activity of the 3-
fluorothiophene bioisostere 34 is maintained (EC50 = 280 nM),
the thiophene is less stable (RLM CLint = 13 μL/min/mg
protein, HLM CLint = 11 μL/min/mg protein) than the parent
11.
As with the pyrazole 5-position, electronic manipulations to
the 2-fluorobenzyl group can result in a significant loss of
potency in this particular series. Introduction of a nitrogen
atom (36, EC50 = ND) is not tolerated. Incorporation of
perfluorinated alkyl groups23 also adversely affects sGC activity
(37, EC50 = ND and 38, EC50 = 1500 nM) in the cellular assay.
Based on the exploration of the pyrazole N-1 functionality, we
determined that 2-fluorobenzyl substitution was optimal for this
5-fluoro-4-hydroxypyrimidine scaffold.
heteroarenes, 2-pyridine 27 (EC50 = ND), nitrile 28 (EC50
=
ND) and cyclopropane 29 (EC50 = ND) all fail to reach full
efficacy in this particular series.
C
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ACS Medicinal Chemistry Letters
Letter
In purified recombinant human sGC enzyme assay (Enzo
Life Sciences) and whole cell (HEK) assays, 11 (hereafter
referred to as IWP-051) as well as other compounds of this
class stimulate cGMP production in the absence of NO and
demonstrate synergy with NO in a concentration responsive
manner. In the absence of NO, IWP-051 is less potent and it
shows a lower degree of sGC stimulation as measured by
maximal efficacy (%Emax). This profile is consistent with the
stimulator class of sGC agonist compounds and provides
evidence that IWP-051 is a direct sGC stimulator.24
lowering of mean arterial pressure (MAP), with a 10 mmHg
drop in MAP at the minimum effective dose of 1 mg/kg
(Figure 2).28 The reduction in MAP approaches −30 mmHg at
the 30 mg/kg dose. At the 10 mg/kg dose and above, the
reduction in MAP is maintained through 24 h.
IWP-051 is highly protein bound in both rat (99.9% bound)
and human (99.4% bound) plasma. Albumin, a major
component of plasma, binds acidic compounds,25 and IWP-
051 is mildly acidic with a pKa of 5.75. The acidic
hydroxypyrimidine proton also appears to affect the aqueous
solubility, as the thermodynamic solubility of IWP-051
increases with increasing pH (3 μg/mL at pH = 7.4 and 142
μg/mL at pH = 9.2). IWP-051 is readily absorbed in the gut
with high permeability (Papp, A‑B = 21.8 × 10−6 cm/s). Further,
the compound does not inhibit cytochrome P450 (CYP)
isozymes (<20% inhibition at 10 μM of CYP 3A4, 2D6, 2C9,
1A2, and 2C19). With regard to off-target activity, it has
minimal activity in a screen of over 60 potential targets,
including the KOP receptor (21% binding at 10 μM). For a
representative panel of phosphodiesterases (PDE1B, PDE2A1,
PDE3A, PDE4D2, PDE5, PDE10A1) less than 30% inhibition
was observed at 10 μM of IWP-051. The lack of inhibition of
phosphodiesterases further supports the mechanism of the
direct stimulation of sGC.
Figure 2. IWP-051 (1, 10, and 30 mg/kg in PEG400) and Bay 41-
2272 (10 mg/kg in 0.5% methylcelluolose, Sigma-Aldrich) were dosed
orally to male, normotensive Sprague−Dawley rats. Vehicle-subtracted
mean arterial pressure (ΔvMAP) in mmHg was monitored for 12−24
h via a conscious, tethered rat model. Data is represented as the
change from their own vehicles as 1 h averages + SEM, n = 5−9.
In summary, IWP-051 is a novel, potent, orally bioavailable
sGC stimulator that was discovered through the structural
manipulation of a 1,3,5-substituted pyrazole scaffold. IWP-051
exhibit minimal off-target liabilities, including CYP and
phosphodiesterase activities. Across species, IWP-051 has
high protein binding, low clearance, long half-life, and slow
absorption. It also exhibits sustained and dose-dependent
reduction in MAP in rats. Pharmacokinetic modeling suggests
that IWP-051 may have once-daily dosing potential in humans.
Based on its in vitro and in vivo profile, IWP-051 is a useful
molecule for probing pharmacologic utility of sustained sGC
stimulation in preclinical models of human disease.
Pharmacokinetic data associated with IWP-051 in multiple
species are represented in Table 4. IWP-051 has a low volume
Table 4. Pharmacokinetic Parameters of IWP-051 in Mouse,
a
Rat, and Dog
parameter
mouse
rat
dog
CL (mL/min/kg)
t1/2 iv (h)
5.0 0.1
3.4 0.5
1300 100
1.7 1.0
1000 300
270 75
>100
0.6 0.1
4.1 0.5
180 60
5.3 1.2
1500 400
1500 280
96 26
0.2 0.01
5.1 0.9
95 12
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00479.
■
S
Vdss (mL/kg)
Tmax (h)
3.3 1.2
3500 600
2500 290
Cmax (ng/mL)
AUC0‑∞ (min·μg/mL)
Fpo (%)
Synthetic procedures, compound characterization data,
description of the cGMP generation assay, determination
of cGMP concentrations using LC-MS/MS, error
propagation for potency, description of the rat liver
microsomal and human liver microsomal stability assays,
pharmacokinetic data of select compounds, and
procedures for measuring hemodynamic parameters.
(PDF)
45
3
a
Oral doses of compound administered as solutions in PEG400 at 1
mg/kg; iv doses administered in 10% DMI, 35% propylene glycol, 15%
EtOH, 40% D5W (5% dextrose in water) at 0.1 mg/kg. Please see the
Supporting Information for additional detail.
of distribution (Vdss = 180 mL/kg), low clearance (CL = 0.6
mL/min/kg), and long half-life (t1/2 = 4.1 h) in rat, all of which
are consistent with high plasma protein binding and a small free
fraction in plasma. When dosed orally at 1 mg/kg, IWP-051 is
slowly absorbed and reaches a Cmax of over 4 μM (free drug
Cmax ∼ 80 nM) at 5 h, with high bioavailability (Fpo = 96
AUTHOR INFORMATION
Corresponding Author
* E-mail: tnakai@ironwoodpharma.com.
Notes
The authors declare the following competing financial
interest(s): All authors own stock/stock options in Ironwood
Pharmaceuticals, Inc.
■
26%). In dog and mouse, the profile of IWP-051 is similar to
low clearance, moderate half-life, and high Cmax
26
.
Standard
allometric scaling using mouse, rat and dog PK parameters
predicts a human elimination half-life of about 8 h, consistent
with once-daily dosing.27 The slow absorption of IWP-051 may
be related to its pH-dependent solubility profile with solubility-
limited absorption in the upper GI.
When dosed orally as a PEG400 solution in normotensive
Sprague−Dawley rats, IWP-051 elicits a dose dependent
ACKNOWLEDGMENTS
■
We thank our colleagues at Ironwood Pharmaceuticals,
especially, Dr. Vishnu Karnati and Dr. Song Xu, for providing
starting materials and intermediates. We thank Dr. James
D
DOI: 10.1021/acsmedchemlett.5b00479
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ACS Medicinal Chemistry Letters
Letter
potent, oral stimulator of soluble guanylate cyclase for the treatment of
pulmonary hypertension. ChemMedChem 2009, 4, 853−865.
(14) Additional pharmacokinetic parameters of 1 are provided in the
Supporting Information.
Sheppeck II and Dr. Paul Renhowe for helpful discussions. We
also thank Sam Rivers for his technical assistance.
ABBREVIATIONS
(15) Straub, A.; Benet-Buchholz, J.; Frode, R.; Kern, A.; Kholsdorfer,
̈
■
C.; Schmitt, P.; Schwarz, T.; Siefert, H.-M.; Stasch, J.-P. Metabolites of
orally active NO-independent pyrazolopyridine stimulators of soluble
guanylate cyclase. Bioorg. Med. Chem. 2002, 10, 1711−1717.
(16) Additional pharmacokinetic parameters of 5 are provided in the
Supporting Information. Also: Nakai, T.; Perl, N. R.; Im, G-Y. J.; Lee,
T. W.-H.; Rohde, J. M.; Kim, C.; Moore, J.; Barden, T. C.; Fretzen, A.;
Butler, C.; Long, K.; Sarno, R.; Germano, P.; Jin, H.; Carvalho, A.;
Solberg, E. O.; Zimmer, D. Discovery of a novel, orally bioavailable
soluble guanylate cyclase stimulator (IWP-051). Presented at 12th
Winter Conference on Medicinal & Bioorganic Chemistry, Steamboat
Springs, Colorado, January 24−29, 2015.
sGC, soluble guanylate cyclase; PAH, pulmonary arterial
hypertension; CTEPH, chronic thromboembolic pulmonary
hypertension; HEK, human embryonic kidney; DETA-NO,
diethylenetriamine NONOate; KOP, κ-opioid; Cmax, maximum
plasma concentration; AUC, area under the curve; EtOH,
ethanol; LHMDS, lithium hexamethyldisilazide; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; RLM, rat liver microsome;
HLM, human liver microsome; CYP, cytochrome P450
isozymes; MAP, mean arterial pressure; SEM, the standard
error of the mean
(17) Kim, C.; Nakai, T.; Moore, J.; Perl, N. R.; Im, G-Y. J.; Barden, T.
C.; Iyengar, R. R.; Zimmer, D. P.; Fretzen, A.; Renhowe, P. A. 2-
Benzyl,3-(pyrimidin-2-yl) substituted pyrazoles useful as sgc stim-
ulators. WO 2013/101830A1.
(18) Kim, C.; Nakai, T.; Lee, T. W.-H.; Moore, J.; Perl, N. R.; Rhode,
J. SGC stimulators. WO 2012/064559 A1.
(19) Im, G-Y. J.; Iyengar, R.; Moore, J.; Fretzen, A. SGC stimulators.
WO 2014/047111A1.
(20) Nakai, T.; Moore, J.; Perl, N. R.; Iyengar, R. R.; Mermerian, A.;
Im, G-Y. J.; Lee, T. W.-H.; Hudson, C.; Rennie, G. R.; Jia, J.; Renhowe,
P. A.; Barden, T. C.; Yu, X. Y.; Sheppeck, J. E.; Iyer, K.; Jung, J. SGC
stimulators. WO 2014/144100 A2.
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