Discovery of the Soluble Guanylate Cyclase Stimulator Vericiguat (BAY 1021189) for the Treatment of Chronic Heart Failure

Article abstract of DOI:10.1021/acs.jmedchem.7b00449

The first-in-class soluble guanylate cyclase (sGC) stimulator riociguat was recently introduced as a novel treatment option for pulmonary hypertension. Despite its outstanding pharmacological profile, application of riociguat in other cardiovascular indications is limited by its short half-life, necessitating a three times daily dosing regimen. In our efforts to further optimize the compound class, we have uncovered interesting structure-Activity relationships and were able to decrease oxidative metabolism significantly. These studies resulting in the discovery of once daily sGC stimulator vericiguat (compound 24, BAY 1021189), currently in phase 3 trials for chronic heart failure, are now reported.

Full text of DOI:10.1021/acs.jmedchem.7b00449

Article
Discovery of the Soluble Guanylate Cyclase Stimulator Vericiguat
BAY 1021189) for the Treatment of Chronic Heart Failure
(
Markus Follmann, Jens Ackerstaff, Gorden Redlich, Frank Wunder, Dieter Lang, Armin
Kern, Peter Fey, Nils Griebenow, Walter Kroh, Eva-Maria Becker-Pelster, Axel Kretschmer,
Volker Geiss, Volkhart Li, Alexander Straub, Joachim Mittendorf, Rolf Jautelat, Hartmut
Schirok, Karl-Heinz Schlemmer, Klemens Lustig, Michael Gerisch, Andreas Knorr, Hanna
Tinel, Thomas Mondritzki, Hubert Trübel, Peter Sandner, and Johannes-Peter Stasch
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2017
Downloaded from http://pubs.acs.org on May 30, 2017
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Discovery of the Soluble Guanylate Cyclase
Stimulator Vericiguat (BAY 1021189) for the
Treatment of Chronic Heart Failure
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Markus Follmann,* Jens Ackerstaff, Gorden Redlich, Frank Wunder, Dieter Lang, Armin Kern,
Peter Fey, Nils Griebenow, Walter Kroh, Eva-Maria Becker-Pelster, Axel Kretschmer, Volker
Geiss, Volkhart Li, Alexander Straub, Joachim Mittendorf, Rolf Jautelat, Hartmut Schirok, Karl-
Heinz Schlemmer, Klemens Lustig, Michael Gerisch, Andreas Knorr, Hanna Tinel, Thomas
Mondritzki, Hubert Trübel, Peter Sandner, and Johannes-Peter Stasch
Bayer AG, Drug Discovery, Aprather Weg 18a, 42113 Wuppertal, Germany
ABSTRACT: The firstꢀinꢀclass soluble guanylate cyclase (sGC) stimulator riociguat was
recently introduced as a novel treatment option for pulmonary hypertension. Despite its
outstanding pharmacological profile, application of riociguat in other cardiovascular indications
is limited by its short halfꢀlife, necessitating a three times daily dosing regimen. In our efforts to
further optimize the compound class, we have uncovered interesting structure–activity
relationships and were able to decrease oxidative metabolism significantly. These studies
resulting in the discovery of once daily sGC stimulator vericiguat (compound 24, BAY
1021189), currently in phase 3 trials for chronic heart failure, are now reported.
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INTRODUCTION
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The NO–sGC–cGMP axis belongs to the key signal transduction pathways involved in regulating
1, 2
the cardiovascular system. Central to this pathway is soluble guanylate cyclase (sGC), an
intracellular enzyme present in the smooth muscle cells of blood vessels and in platelets but also
in various other cell types like cardiomyocytes. sGC displays a high affinity for, and is activated
by, the first messenger signaling molecule nitric oxide (NO). Biosynthesis of NO is mediated by
three distinct isoforms of nitric oxide synthases, endothelial nitric oxide synthase (eNOS),
neuronal nitric oxide synthase (nNOS) and the inducible isoform of nitric oxide synthase (iNOS),
all synthesizing NO from Lꢀarginine. Within endothelial cells, NO is produced by eNOS and
diffuses rapidly to the underlying smooth muscle cells where it binds to its target enzyme sGC.
This binding leads to the stimulation of sGC resulting in increased production of intracellular
second messenger cGMP. cGMP interacts with three types of intracellular proteins such as
cGMPꢀdependent protein kinases, cGMPꢀregulated ion channels and phosphodiesterases. Further
downstream, these transduction cascades mediate various physiological and tissueꢀprotective
effects including smooth muscle relaxation and inhibition of smooth muscle proliferation,
leukocyte recruitment and platelet function. The pathogenesis of various diseases, especially
those of the cardiovascular system, has been related to insufficient bioavailability of NO and thus
3
ꢀ7
impaired stimulation of sGC and therefore decreased cGMP production.
sGC itself is a cytosolic, heterodimeric protein composed of an αꢀ and a βꢀsubunit with a
8
ꢀ13
prosthetic heme group located in the βꢀsubunit (hemeꢀbinding domain).
Mechanistically, NO
2+
2+
stimulates sGC by binding to the Fe of the heme group, which induces cleavage of an Fe –
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histidine (His ) bond, likely resulting in conformational reorganizations which propagate into
the catalytic subunit, increasing cGMP production. cGMP interacts with three types of
intracellular proteins, namely cGMPꢀdependent protein kinases, cGMPꢀregulated ion channels
and phosphodiesterases. Further downstream, these transduction cascades mediate various
physiological and tissueꢀprotective effects, including smooth muscle relaxation and inhibition of
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smooth muscle proliferation, leukocyte recruitment and platelet function.
sGC can exist in two different states, a native hemeꢀcontaining or reduced form of sGC, which is
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, 14
the endogenous receptor for NO, and a hemeꢀfree form of sGC.
Under conditions of oxidative
stress, which is thought to be causal in the pathogenesis of many cardiovascular diseases,
reactive oxygen species (ROS) are produced. ROS are capable of oxidizing the heme iron of sGC
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+
3+
(
Fe → Fe ), resulting ultimately in heme loss from oxidized sGC. As a consequence, the
hemeꢀfree form of sGC is no longer binding NO, is not responsive to NO and thus termed
dysfunctional. During the last 20 years, two distinct compound classes have been discovered at
Bayer that are capable of activating sGC in a NOꢀindependent manner, the hemeꢀdependent sGC
stimulators and the hemeꢀindependent sGC activators. sGC stimulators display a dual mode of
action: they synergize with endogenous NO and, on top of this, are also able to directly stimulate
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5
the native form of the enzyme independently of NO, resulting in increased cGMP production.
In contrast, sGC activators are capable of activating the dysfunctional hemeꢀfree sGC, resulting
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in increased cGMP production even under conditions of reduced NO bioavailability.
The discovery of the sGC stimulators at Bayer, along with the efforts of several other
pharmaceutical companies to identify sGC stimulators and activators, has recently been
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reviewed. Riociguat (1) is the first sGC stimulator to have made a successful transition from
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animal experiments to controlled clinical studies in patients. In 2013 1 gained market approval
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for two lifeꢀthreatening diseases: pulmonary arterial hypertension (PAH) and chronic
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thromboembolic pulmonary hypertension (CTEPH).
However, application of 1 in other
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cardiovascular indications, such as heart failure, is limited by its short halfꢀlife.
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Herein, we report our recent efforts in optimizing the pharmacokinetic profile of sGC
stimulators, culminating in the discovery of vericiguat (compound 24, BAY 1021189), which has
been evaluated in phase 2 studies (SOCRATES trials) in heart failure patients with reduced
2
2, 23
(HFrEF) and preserved (HFpEF) ejection fraction.
Currently, 24 is being investigated in a
phase 3 outcome study (VICTORIA trial) in heart failure patients with HFrEF in coꢀdevelopment
24
with MSD (vericiguat code name: MKꢀ1242). That study is aimed at assessing whether 24, on
top of standard of care, is able to decrease mortality and morbidity in such HFrEF patients.
RESULTS AND DISCUSSION
Optimization Strategy. Our goal was to identify orally bioavailable sGC stimulators with a
longer duration of action than 1, in order to support a profile allowing for a once daily oral
dosing, and less oxidative metabolism in order to lower interaction potentials. 1 is a very potent
sGC stimulator in vitro and in vivo; however, it has a moderate halfꢀlife in different animal
2
5
species and this pharmacokinetic profile translated into a three times daily dosing regimen in
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6
patients. Nꢀdemethylation to compound 9, as described by Gnoth et al. in 2015, is the major
biotransformation of 1 and is mainly catalyzed by CYP1A1, and also by CYP3A4, CYP3A5 and
21, 26ꢀ31
CYP2J2.
Our strategy was to further optimize the metabolic stability of 1 and hence
reduce blood clearance to achieve a longer halfꢀlife. We began by optimizing the substituents on
the 5ꢀcarbamoyl residue on the pyrimidine ring, aiming to achieve metabolically more stable
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derivatives while maintaining good potency. Later, in a second optimization step, we focused on
variations of the central pyrazolopyridine scaffold.
Chemistry. Scheme 1 outlines the strategy for the synthesis of the 5ꢀcarbamoyl variations.
Starting from triamine 1c, which is available in two steps from amidine 1a and phenyldiazenylꢀ
substituted malonodinitrile 1b, selective carbamate formation at the 5ꢀamino group was achieved
either by treatment with the corresponding chloroformate or by addition of the respective alcohol
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preactivated with triphosgene, to yield compounds 9–14. Deprotonation of the carbamate NH
group and subsequent treatment with the corresponding halide or trichloromethanesulfonate
electrophile allowed further derivatization to the corresponding Nꢀalkylated carbamates (1, 2, 4–
8
). Alternatively, the alkyl side chain on the 5ꢀamino group could first be introduced via
reductive amination and the product 1d subsequently treated with methyl chloroformate, giving
access to the hydroxyethyl derivative 3. For the cyclic 5ꢀ(2ꢀoxoꢀ1,3ꢀoxazolidinꢀ3ꢀyl) analogues
1
5–18, the previously employed conditions for the selective formation of carbamates provided
e–g bearing a chloroꢀ or bromoꢀsubstituted alkyl chain, allowing for a baseꢀinduced
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intramolecular cyclization (NaHMDS).
a
Scheme 1. Synthesis of Compounds 1–18
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c
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:R2 = methyl
1:R = methyl
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10:R = isopropyl
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1:R = cyclobutyl
4:R = 2,2-difluoroethyl
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2:R = 3-oxetanyl
5:R = 2,2,2-trifluoroethyl
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13:R = 2,2,2-trifluoroethyl
4:R2 = cyclopropylmethyl
6:R = 2-fluorobenzyl
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7:R = 3-fluorobenzyl
:R1 = 4-fluorobenzyl
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e:R2 = -(CH ) Cl
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8:R3 = H, R4 = CF3
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1
a
Reagents and conditions: (a) NaOMe, DMF, 110 °C. (b) H (65 bar), Raney nickel, DMF. (c)
R OCOCl, pyridine, or i. triphosgene, pyridine, ii. R OH, pyridine. (d) R X (X = halide or
trichloromethanesulfonate), LiHMDS. (e) glycolaldehyde, NaBH CN, AcOH, MeOH, 0 °C to rt.
f) methyl chloroformate, pyridine, rt. (g) NaHMDS, THF, 0 °C to rt.
2
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1
3
(
The synthesis of the compounds with a core variation is outlined in Schemes 2–5. For the 1Hꢀ
pyrazolo[4,3ꢀb]pyridine derivative 19, the synthesis started from 3,5ꢀdichloropyridineꢀ2ꢀcarbonyl
chloride (2a) which was converted into 3ꢀ(2ꢀfluorobenzyl)ꢀ1Hꢀpyrazolo[4,3ꢀb]pyridine (2b) in
four steps. The fluoro derivative 2d was synthesized in a similar manner from the corresponding
difluoropyridine analogue 2c. Both key intermediates 2b and 2d were then reacted with 2ꢀchloroꢀ
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ꢀnitropyrimidineꢀ4,6ꢀdiamine, followed by reduction of the pyrimidine 5ꢀnitro group and
methyl carbamate formation, to give the 1Hꢀpyrazolo[4,3ꢀb]pyridine core analogues 19 and 20
Scheme 2).
(
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Scheme 2. Synthesis of Compounds 19 and 20
a
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Reagents and conditions: (a) methyl 2ꢀ(2ꢀfluorophenyl)acetate, LiHMDS. (b) aq NaCl
solution, DMSO, microwave, 150 °C. (c) hydrazine hydrate, pyridine, reflux. (d) H , 10% Pd/C,
Et N, EtOH, THF. (e) NaH, DMF, 80 °C. (f) H , 10% Pd/C, pyridine. (g) methyl chloroformate,
2
3
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pyridine.
A different route was used for the preparation of 1Hꢀpyrazolo[3,4ꢀc]pyridazine 21 (Scheme 3),
which began with the cyclization of methyl 2ꢀaminothiopheneꢀ3ꢀcarboxylate (3a) with hydrazine
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hydrate to give 1Hꢀpyrazolo[3,4ꢀc]pyridazinꢀ3ꢀol (3b). Subsequent bromination afforded
intermediate 3c which was alkylated with 2ꢀfluorobenzyl bromide to yield 3d. Then, conversion
of the bromide into the cyanide 3e and Pinnerꢀtype reaction of the cyano group with sodium
methoxide and ammonium chloride afforded key intermediate 3f. Further modifications,
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analogous to the formation of carbamate 9 from 1a (Scheme 1), yielded the methyl carbamate
derivative 21.
a
Scheme 3. Synthesis of Compound 21
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Reagents and conditions: (a) hydrazine hydrate. (b) POBr , sulfolane, 150 °C. (c) 2ꢀ
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fluorobenzyl bromide, Cs CO , DMF, rt. (d) CuCN, DMSO, 150 °C. (e) NaOMe, NH Cl, AcOH,
2
3
4
MeOH. (f) 1b, DMF, Et N, 100 °C. (g) H (1 bar), 10% Pd/C, DMF. (h) methyl chloroformate,
3
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pyridine, DCM.
The preparation of imidazo[1,5ꢀa]pyrimidine 22 (Scheme 4) started with condensation of the
readily available thioimide 4a and amine 4b to access aminoimidazole 4c. Further orthoester
condensation then gave the bicyclic imidazopyrimidine derivative 4d bearing an ethyl ester
functionality. Subsequent saponification, amidation and dehydration yielded nitrile 4g as a key
intermediate. Pinnerꢀtype reaction with sodium methoxide and ammonium chloride delivered the
corresponding amidine 4h which could be converted into the final compound 22 in analogy to
the previous examples.
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a
Scheme 4. Synthesis of Compound 22
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e Y = CO H
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Reagents and conditions: (a) MeI, K CO , acetone. (b) TBAI, KOtꢀBu, DCM, then aq HCl,
Et O. (c) dioxane, 60 °C. (d) 1,1,3,3ꢀtetramethoxypropane, aq HCl, MeOH, EtOH, reflux. (e)
NaOH. (f) HATU, NH Cl, Hünig’s base, DMF. (g) POCl , 120 °C. (h) NaOMe, NH Cl, MeOH.
(i) 1b, DMF, Et N, 100 °C. (j) H (1 bar), 10% Pd/C, DMF, 0 °C. (k) methyl chloroformate,
2
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2
pyridine.
The synthesis of the related imidazo[1,5ꢀb]pyridazine derivative 23 (Scheme 5) started from
methyl 5ꢀaminolevulinate hydrochloride (5a). Addition of hydrazine hydrate yielded the
dihydropyridazinone 5b which was reacted with the readily available acid chloride 5c under
basic conditions. The resulting amide was oxidized with bromine to provide the corresponding
pyridazinone 5d which underwent an intramolecular condensation in the presence of phosphoryl
chloride to give the bicyclic intermediate 5e. Dechlorination of the pyridazine core catalyzed by
palladium on charcoal and subsequent bromination of the imidazo core with Nꢀ
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bromosuccinimide gave access to 5f. Further modifications, as outlined previously, then yielded
amidine 5g as a key intermediate which was converted into the final target compound 23.
a
Scheme 5. Synthesis of Compound 23
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23
a
Reagents and conditions: (a) hydrazine hydrate, Et N, reflux. (b) Et N, MeCN. (c) Br , AcOH,
3
3
2
5
0 °C. (d) POCl , DCE, reflux. (e) H , 5% Pd/C, Et N, EtOAc. (f) NBS, DCM. (g) CuCN. (h)
3 2 3
NaOMe, NH Cl, AcOH. (i) 1b, DMF, Et N, 100 °C. (j) H (1 bar), 10% Pd/C, DMF, MeOH. (k)
4
3
2
methyl chloroformate, pyridine.
The synthesis of the 1Hꢀpyrazolo[3,4ꢀb]pyridine derivative (24) (Scheme 6), involved initial
activation of 2,2,3,3ꢀtetrafluoroꢀ1ꢀpropanol (6a) with trifluoromethanesulfonic anhydride and
treatment with morpholine to yield 6b after aqueous workup and distillation. At 135 °C, 6b was
alkylated with methyl methanesulfonate leading to quaternized derivative 6c which was
converted into 6d using sodium hydroxide. Further reaction with morpholine and triethylamine
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9
yielded 6e after crystallization from toluene. Acrylaldehyde derivative 6e was reacted with ethyl
2
5
5
ꢀaminoꢀ1ꢀ(2ꢀfluorobenzyl)ꢀ1Hꢀpyrazoleꢀ3ꢀcarboxylate in ethanol yielding ethyl ester
intermediate 6f. Ester 6f was then converted in three steps into the corresponding amidine 6i via
amide 6g and nitrile 6h in analogy to the chemistry outlined in Scheme 4. The synthesis of 24
was completed as described for compound 9 (Scheme 1) via the diazenyl derivative 6j and
triamine 6k.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Scheme 6. Synthesis of 24
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
F
F
F
a
b
N
F
N+
F
H O
F
O
F
F
O
F
F
-
F
F
CH SO
3 3
6
b
6c
6
a
F
O
F
c
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N+
F
CH SO
N
O
F
F
-
3
3
O
6
d
6e
F
F
H N
N
N
N
O
F
2
e
f
N
N
N
N
N
+
F
N
O
O
O
F
6
e
O
6f
O
6g
O
H N
2
F
F
F
N
N
N
F
g
h
i
N
N
N
N
N
N
N
N
N
NH2
F
F
H N
NH
2
N
N
H N
x HCl
2
6
h
6i
6j
F
F
N
N
N
N
N
j
k
N
F
F
N
N
N
H N
N
NH2
O
NH2
NH2
2
N
H
H N
2
O
2
4
6
k
a
Reagents and conditions: (a) Tf O, 70 °C, then morpholine, 5 °C, then 40 °C. (b) MeSO Me,
35 °C, then 100 °C. (c) 45% aq NaOH, 50 °C. (d) morpholine, Et N, reflux. (e) MsOH, LiCl,
EtOH, reflux. (f) formamide, NaOMe, MeOH, EtOH, 95–125 °C. (g) POCl , sulfolane, 107 °C.
2
3
1
3
3
(
h) NaOMe, NH Cl, MeOH, EtOH, 65 °C. (i) 1b, DMF, Et N, 100 °C. (j) H (60 bar), 5% Pd/C,
4 3 2
DMF, 60 °C. (k) methyl chloroformate, iꢀPrOH, MeOH, then Et N, 50 °C.
3
SAR and DMPK Optimization. The SAR of the series of novel Nꢀsubstituted methyl
carbamates 1–8 was explored using a cGMP formation assay with sGCꢀoverexpressing Chinese
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34
hamster ovary (CHO) cells. In addition, the metabolic stability of the compounds was assessed
in vitro by incubation with rat hepatocytes. Initially, different carbamate Nꢀsubstituents were
tested with the aim of achieving higher metabolic stability than 1 while maintaining high potency
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
Table 1). Increasing steric bulk as in the ethyl derivative 2 had no beneficial effect on either
potency or metabolic stability. The introduction of polarity (e.g., a hydroxyethyl functionality, as
in 3) led to a significant loss of potency relative to 1, along with decreased metabolic stability.
The next strategy, the introduction of fluorine atoms at the terminal position of the Nꢀsubstituent,
was designed to block metabolism. Interestingly, both the 2,2ꢀdifluoroethyl and 2,2,2ꢀ
trifluoroethyl derivatives, 4 and 5, exhibited high potency in the cGMP assay but had a 2–4.5ꢀ
fold higher clearance in rat hepatocytes. NꢀFluorobenzyl substitution revealed that the orthoꢀ
fluoro derivative 6 is significantly more potent (MEC = 0.1 ꢀM) than the meta and para isomers,
7
and 8 (MEC = 0.2 and 0.7 ꢀM, respectively). Nevertheless, all the benzylic derivatives had a
high clearance in rat hepatocytes. These results and the testing of many further derivatives (data
not shown) suggested that improving the metabolic stability by altering the Nꢀsubstitution might
be very difficult to achieve. Hence, optimization efforts were focused on the main metabolite of
1
, the Nꢀdesmethyl derivative 9. This compound is less potent than 1 but displayed a somewhat
higher metabolic stability in the rat hepatocyte assay, with a clearance of 0.1 L/h/kg. When tested
in vivo in rats (iv dosing), compound 9 had a moderate clearance of 1.2 L/h/kg and a short halfꢀ
life of only 1.2 hours.
Table 1. Properties of the NꢀSubstituted Methyl Carbamates 1–8
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
F
N
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
NH2
H N
2
N
R
O
O
3
5
Compd
R
cGMP formation
ClogD
In vitro clearance (rat hepatocytes)
a
MEC [ꢀM]
pH 7.5
1.99
CL
b
[L/h/kg]
0.2
1
2
3
0.03
0.2
Me
2.29
1.52
0.7
3.7
Et
OH
0.3
F
4
5
F
0.1
0.1
2.21
2.48
0.4
0.9
FF
F
6
7
0.1
0.2
3.15
3.20
3.2
3.2
F
F
F
8
0.7
3.29
2.4
a
MEC: Minimal effective concentration to achieve stimulation of cGMP formation (≥3ꢀfold
increase in basal luminescence) in a recombinant sGCꢀoverexpressing cell line.
3
4
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9
Therefore, variations in the carbamate alkoxy group while leaving the Nꢀposition unsubstituted
were examined next, with the goal of improving the halfꢀlife and other properties relative to
compound 9 (Table 2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The introduction of steric bulk in the form of an isopropyl residue (compound 10) led to a strong
decrease in potency relative to 9, as well as decreased metabolic stability. Interestingly, there
was increased potency with cyclobutyl derivative 11; however, 11 was also unfavorable with
respect to rat hepatocyte clearance. The oxetanyl group is well known in medicinal chemistry for
3
6, 37
improving metabolic stability
over related alkyl derivatives. Remarkably, the exchange of
cyclobutyl for oxetanyl (compound 12) led to a 14ꢀfold higher stability in rat hepatocytes, but
sGC stimulation was weaker than with derivative 9 or 11. Since the permeability across Cacoꢀ2
cell monolayers was also very low (Papp A–B = 2 nm/s), combined with a high efflux ratio of 74,
compound 12 was not pursued further. Additional efforts to improve the overall profile, by the
introduction of fluorine or steric bulk (compounds 13 and 14), led to a slight increase in potency
(MEC = 0.2 ꢀM vs 0.3 ꢀM for 9); however, metabolic stability was dramatically reduced.
Table 2. Properties of the NꢀH Alkyl Carbamates 9–14
F
N
N
N
N
N
N
NH2
H N
2
H
O
O
R
3
5
Compd
R
cGMP
ClogD
In vitro clearance (rat
Cacoꢀ2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
formation
pH 7.5
hepatocytes)
Papp A–B [nm/s]
(Efflux ratio)
a
MEC [ꢀM]
CL
b
[L/h/kg]
0.1
9
0.3
0.8
1.49
2.10
79 (5)
Me
iPr
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
10
0.9
1.4
n.d.
1
1
1
1
0.2
2.3
0.2
2.05
1.21
2.05
23 (25)
2 (74)
O
2
3
4
0.1
2.1
1.4
FF
F
b
n.d.
b
1
0.2
2.09
n.d.
a
MEC: Minimal effective concentration to achieve stimulation of cGMP formation (≥3ꢀfold
3
4 b
increase in basal luminescence) in a recombinant sGCꢀoverexpressing cell line.
determined.
n.d.: not
The oxazolidinone derivatives 15–18, as conformationally fixed versions of the foregoing
carbamates, were also studied to assess the influence of the constrained nature on sGC
stimulation (Table 3). The parent oxazolidinone 15 was as equipotent as carbamate 9 and
exhibited slightly decreased stability in rat hepatocytes. Intravenous dosing of compound 15 to
rats resulted in a moderate clearance of 1.9 L/h/kg and a short halfꢀlife of about 1.0 hour, and
thus 15 was not profiled further. Other attempts to increase metabolic stability by the
38
introduction of steric bulk were unsuccessful, probably due to the more lipophilic character of
the resulting compounds 16–18.
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Table 3. Properties of the Oxazolidinones 15–18
F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
N
N
N
N
NH2
H N
2
R
3
5
Compd
R
cGMP formation ClogD
In vitro clearance (rat hepatocytes)
a
MEC [ꢀM]
pH 7.5
1.60
CL
b
[L/h/kg]
O
O
N
15
0.3
0.4
O
O
N
1
1
6
7
0.2
0.2
2.29
2.25
1.3
O
N
b
n.d.
O
O
F3C
O
N
18
0.03
2.25
1.6
F3C
a
MEC: Minimal effective concentration to achieve stimulation of cGMP formation (≥3ꢀfold
increase in basal luminescence) in a recombinant sGCꢀoverexpressing cell line.
determined.
3
4 b
n.d.: not
In summary, although further optimization of the carbamate motif of 1 could not be achieved, a
number of interesting and novel SAR observations were made, with the Nꢀunsubstituted
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
carbamate 9 being identified as the most promising path forward. Thus, the focus of the
optimization strategy was shifted away from the carbamate moiety and our efforts were directed
to the central scaffold and the identification of alternative cores that could lead to superior
overall pharmacokinetic profiles of the corresponding compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Our studies with respect to central scaffold modifications are summarized in Table 4. Efforts
were concentrated on novel core systems which had not been tested before in combination with
4,6ꢀdiaminopyrimidinꢀ5ꢀylcarbamates.
Firstly, changes were made to the pyrazolo portion of the molecule, leading to the 1Hꢀ
pyrazolo[4,3ꢀb]pyridine derivative 19. In addition to the benefit of a shorter synthetic route than
that for compound 9, 19 proved to be a moderately potent sGC stimulator with an MEC of 1.2
ꢀM and had high metabolic stability when tested in rat hepatocytes. The introduction of
substituents at the 6ꢀposition of the 1Hꢀpyrazolo[4,3ꢀb]pyridine core was, in general, not well
tolerated leading to a dramatic loss of potency, with the exception of the 6ꢀfluoro derivative 20
which exhibited an MEC of 0.5 ꢀM and good metabolic stability. To our surprise, compounds 19
and 20 had very different in vivo clearances when compared in a rat pharmacokinetic experiment
(iv dosing): fluoro derivative 20 had a low clearance of 0.3 L/h/kg versus 1.0 L/h/kg for
compound 19. As metabolite identification did not point to metabolism occurring at the pyridine
core, the rationale for this threefold reduction in blood clearance remains unclear.
Table 4. Properties of the Core Variation Compounds 19–24
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9
core
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compd
Core
cGMP
formation
In vitro clearance (rat
hepatocytes)
In vivo clearance (rat)
CL [L/h/kg]
a
b
MEC [ꢀM]
CL
b
[L/h/kg]
19
1.2
0.5
2.0
1.7
0.7
<0.1
1.0
0.3
3.8
1.8
0.9
2
0
1
0.1
2
<0.1
b
22
n.d.
23
<0.1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4
0.3
0.1
0.3
a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MEC: Minimal effective concentration to achieve stimulation of cGMP formation (≥3ꢀfold
increase in basal luminescence) in a recombinant sGCꢀoverexpressing cell line.
determined.
3
4 b
n.d.: not
Novel 1Hꢀpyrazolo[3,4ꢀc]pyridazine 21 proved to be a reasonably potent sGC stimulator but
exhibited a high clearance of 3.8 L/h/kg when tested in vivo in rats and thus was not further
profiled, along with imidazo[1,5ꢀa]pyrimidine 22 for similar reasons. In contrast, imidazo[1,5ꢀ
b]pyridazine 23 exhibited potent sGC stimulator properties (MEC = 0.7 ꢀM), good metabolic
stability in rat hepatocytes and a low to moderate clearance of 0.9 L/h/kg after intravenous
dosing to rats. Finally, revisiting 1Hꢀpyrazolo[3,4ꢀb]pyridine 9 with an additional fluorine at the
5ꢀposition resulted in the potent sGC stimulator 24 (MEC = 0.3 ꢀM) with good metabolic
stability in rat hepatocytes and a surprisingly low clearance of 0.3 L/h/kg after intravenous
dosing to rats (Table 4). Thus, the blood clearance of derivative 9 in rats (1.2 L/h/kg) was
reduced fourfold by fluorination at position 5.
Finally, compounds 20, 23 and 24 were selected for further pharmacokinetic profiling across
species (Table 5). Fluoropyrazolo[3,4ꢀb]pyridine derivative 24 exhibited the best overall
pharmacokinetic profile by far, with a low clearance and long halfꢀlife in rats and dogs after
intravenous dosing, as well as high oral bioavailability. In addition, 24 had no inhibitory effects
on major CYP isoforms (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4), as indicated by IC50
values of >50 ꢀM. Metabolite profiling in human hepatocytes was characterized by a low
turnover, with glucuronide 25 (major) and debenzylated compound 26 (minor) being the only
metabolites identified (Figure 1). Thus, the main biotransformation pathway shifted from
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9
predominantly phase I oxidative CYPꢀmediated metabolism (1) to primarily phase II UGTꢀ
mediated conjugation with glucuronic acid.
Table 5. In vivo Pharmacokinetic Properties of 20, 23, 24 in comparison with 1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compd
Species
V
ss [L/kg]
CL [L/h/kg]
t [h]
Bioavailability
%]
b
1/2
[
2
0
3
rat
dog
rat
0.5
1.0
0.3
2.0
1.0
1.4
1.2
0.7
0.3
0.2
1.5
4.1
0.5
1.8
3.4
6.2
1.4
2.4
26
56
2
0.9
37
a
b
dog
rat
0.9 (Cl
0.3
p
)
n.d.
65
24
1
dog
rat
0.2
75
1.3
46
a
dog
0.3 (Cl
p
)
79
a
b
Cl : Plasma clearance. n.d.: not determined.
p
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
F
H
N
N
N
N
HO
N
N
OH
COOH
human hepatocytes
F
F
HO
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Figure 1. Biotransformation products of 24 in human hepatocytes.
Pharmacology. 24 was extensively profiled preclinically in vitro, on the isolated sGC enzyme
and on isolated vessels, ex vivo in isolated hearts and in vivo in a rat model of cardiovascular
disease associated with cardiorenal syndrome.
Highly Purified Recombinant sGC. Studies on the in vitro effects of 24 on highly purified sGC
revealed that 24 (0.01 ꢀM to 100 ꢀM) stimulates recombinant sGC concentrationꢀdependently,
by 1.7ꢀfold to 57.6ꢀfold (Figure 2). When combined with the NO donor diethylamine/nitric oxide
complex (DEA/NO), 24 and DEA/NO had a synergistic effect on the enzyme activity over a
wide range of concentrations. At highest concentrations of 24 (100 ꢀM) and DEA/NO (100
nM), the specific activity of sGC was 341.6ꢀfold above baseline (Figure 2). Moreover, the effects
of 24 in the presence of the sGC inhibitor 1Hꢀ[1,2,4]oxadiazolo[4,3ꢀa]quinoxalinꢀ1ꢀone (ODQ)
were evaluated. The sGC stimulation induced by 24 was nearly completely blocked by ODQ,
and reduced to 18ꢀfold (Figure 2). Thus, 24 exhibits all the characteristics of a potent and
selective sGC stimulator, stimulating sGC NOꢀindependently and in synergy with NO. In
addition, 24 predominantly acts on the hemeꢀcontaining nonoxidized form of sGC.
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Figure 2. Effects of 24 and NO on the stimulation of highly purified sGC, and blocking effects
of the sGC inhibitor ODQ.
sGC-Overexpressing Cells. The stimulation of sGC by 24 was examined with a recombinant
CHO cell line overexpressing rat sGC. 24 stimulated the sGC reporter cell line concentrationꢀ
dependently, with an EC of 1005 ± 145 nM. In the presence of the NO donor SꢀnitrosoꢀNꢀ
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0.6 ± 1.7 nM, respectively. In the presence of ODQ, pretreatment of the sGC reporter cell line
with 10 ꢀM ODQ for 3 hours resulted in a significantly reduced efficacy of 24, with an EC of
D,Lꢀpenicillamine (SNAP) (30 and 100 nM), the EC value shifted to 39.0 ± 5.1 nM and
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Isolated Vessels and Tolerance. 24 inhibited phenylephrineꢀinduced contractions of rabbit
saphenous artery rings, rabbit aortic rings and canine femoral vein rings concentrationꢀ
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dependently, with IC values of 798 nM, 692 nM and 3072 nM, respectively. In addition, 24
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dependently, with an IC of 956 nM.
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Chronic administration of organic nitrates leads to the rapid development of nitrate tolerance.
Thus, the vasorelaxant effect of 24 on isolated saphenous artery rings taken from normal and
nitrateꢀtolerant rabbits was examined. Treatment with isosorbide dinitrate for 3–4 days resulted
in a marked inhibition of glycerol trinitrate (GTN) mediated vasodilation. GTN inhibited
phenylephrineꢀinduced contractions with an IC of 1.9 nM in control vessels and an IC of 9.6
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nM in tolerant vessels, confirming the presence of nitrate tolerance. In contrast to GTN, 24 is a
potent inhibitor of phenylephrineꢀinduced contractions both in normal and nitrateꢀtolerant
saphenous artery rings, with IC values of 5.6 nM and 5.8 nM, respectively.
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Langendorff-Perfused Hearts. In rat heart Langendorff preparations, ex vivo, 24 reduced the
coronary perfusion pressure in a concentrationꢀdependent manner (Figure 3). Up to the highest
concentration tested, 24 had no effect on heart rate, left ventricular diastolic pressure and
contractility.
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Figure 3. Effects of 24 on rat heart Langendorff preparations.
Long-Term Study with L-NAME-Treated Renin Transgenic Rats. The potential impact on
cardiovascular health of the stimulation of sGC was evaluated by determining the longꢀterm
effects of 24 on hemodynamic and hormonal parameters in renin transgenic rats (RenTG)
carrying the additional mouse renin gene (mRenR2)27. These RenTG(mRenR2)27 rats were
ă
additionally treated with the NO synthase inhibitor N ꢀnitroꢀLꢀarginine methyl ester (LꢀNAME)
in the drinking water. This wellꢀestablished rodent disease model is characterized by
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hypertension, heart and kidney failure, and increased mortality. The RenTG(mRenR2)27/Lꢀ
NAMEꢀsupplemented rats were chronically treated with either placebo (2 mL/kg q.d.), which
served as the control group, or 24 (3 or 10 mg/kg q.d.).
Effects on Blood Pressure and on the Heart. Chronic oral treatment with 3 or 10 mg/kg 24 q.d.
resulted in a significant attenuation of blood pressure increase during the course of the study.
However, the overall rise of blood pressure increase was not halted in the 3 and 10 mg/kg
treatment groups (Figure 4).
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Figure 4. Increase in systolic blood pressure in mmHg during the course of the study with Lꢀ
NAMEꢀtreated renin transgenic rats.
In addition, a significant and doseꢀdependent reduction of heart hypertrophy, in both the right
and left ventricle, was found in the 3 and 10 mg/kg treatment groups compared to the placebo
group (Figure 5). Furthermore, plasma ANP levels decreased significantly in both treatment
groups, also suggesting a functional improvement of the heart.
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Figure 5. Right and left ventricle weight normalized on body weight (left/middle) and plasma
atrial natriuretic peptide levels [in pg/mL] at the study end, after 3 weeks of treatment.
Effects on the Kidneys. With respect to kidney damage, 24 treatment at 3 or 10 mg/kg led to a
significant reduction in kidney injury molecule Kimꢀ1 and osteopontin expression which are
used as biomarkers for renal injury and dysfunction (data not shown). In addition, proteinuria
was significantly and doseꢀdependently decreased in the treatment groups, also suggesting a
functional improvement of the kidneys (Figure 6).
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Figure 6. Effects on proteinuria at the study end, after 3 weeks of treatment.
Effects on Mortality. Treatment with 24 resulted in a significant and doseꢀdependent increase in
survival rates. In the 3 and 10 mg/kg q.d. treatment groups, the rat survival rate was 70% and
90%, respectively, at the study end. In contrast, the survival rate in the placebo group was only
25% after 21 days (Figure 7).
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Figure 7. Kaplan–Meier survival curves.
These in vivo data strongly suggest that the sGC stimulator 24 can maintain heart and kidney
function in a model of hypertensionꢀinduced endꢀorgan damage, with substantially reduced
overall mortality, strongly suggesting a beneficial role of 24 for the treatment of cardiovascular
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diseases associated with cardiorenal syndrome.
CONCLUSION
In summary, we have identified 24 as a potent, orally available stimulator of sGC. Our
optimization work starting from 1 led to the identification of compounds with superior in vitro
and in vivo pharmacokinetic and metabolic profiles. 24, the compound with the best overall
profile from these preclinical studies, was selected as clinical candidate and proved to have a
pharmacokinetic profile in humans suitable for once daily dosing. Additional in vivo studies in
animal models of hypertension, heart failure and kidney disease have revealed doseꢀdependent
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antifibrotic and organꢀprotective properties in line with the sGC stimulator mode of action. 24 is
currently being investigated in a phase 3 clinical trial in HFrEF patients.
EXPERIMENTAL SECTION
Chemistry
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General Procedures. Unless otherwise noted, all nonaqueous reactions were carried out under
an argon atmosphere with commercialꢀgrade reagents and solvents. All final products were at
least 95% pure, as determined by analytical HPLC.
1
H NMR spectra were recorded on Bruker Avance spectrometers operating at 400 or 500 MHz.
Chemical shifts (δ) are reported in ppm relative to TMS as an internal standard and coupling
constants (J) are given in hertz (Hz). Spin multiplicities are reported as s = singlet, br s = broad
singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet. LCꢀMS
analysis was performed using the respective method a–d, as noted. Method a: instrument:
Micromass TOFꢀMUX interface 4× parallel injection with Waters 600 HPLC; column:
Phenomenex Synergi 2 ꢀm HydroꢀRP Mercury 20 × 4 mm; mobile phase A: H O (1 L) + 50%
2
formic acid (0.5 mL), mobile phase B: MeCN (1 L) + 50% formic acid (0.5 mL); gradient: 0.0
min 90% A → 2.5 min 30% A → 3.0 min 5%A → 4.5 min 5% A; oven: 20 °C; flow: 1 mL/min;
UV detection: 210 nm. Method b: instrument: Micromass Quattro Premier with Waters Acquity
UPLC; column: Thermo Hypersil GOLD 1.9 ꢀm, 50 × 1 mm; mobile phase A: H O (1 L) + 50%
2
formic acid (0.5 mL), mobile phase B: MeCN (1 L) + 50% formic acid (0.5 mL); gradient: 0.0
min 90% A → 0.1 min 90% A → 1.5 min 10% A → 2.2 min 10% A; oven: 50 °C; flow: 0.33
mL/min; UV detection: 210 nm. Method c: instrument: Waters Micromass Quattro Micro with
Agilent 1100 series HPLC; column: Thermo Hypersil GOLD 3 ꢀm, 20 × 4 mm; mobile phase A:
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