Potent and Selective Human Neutrophil Elastase Inhibitors with Novel Equatorial Ring Topology: In vivo Efficacy of the Polar Pyrimidopyridazine BAY-8040 in a Pulmonary Arterial Hypertension Rat Model

Article abstract of DOI:10.1002/cmdc.201500269

Human neutrophil elastase (HNE) is a key driver of inflammation in many cardiopulmonary and systemic inflammatory and autoimmune conditions. Overshooting high HNE activity is the consequence of a disrupted protease-antiprotease balance. Accordingly, there has been an intensive search for potent and selective HNE inhibitors with suitable pharmacokinetics that would allowing oral administration in patients. Based on the chemical probe BAY-678 and the clinical candidate BAY 85-8501 we explored further ring topologies along the equator of the parent pyrimidinone lead series. Novel ring systems were annulated in the east, yielding imidazolo-, triazolo-, and tetrazolopyrimidines in order to ensure additional inhibitor-HNE contacts beyond the S1 and the S2 pocket of HNE. The western annulation of pyridazines led to the polar pyrimidopyridazine BAY-8040, which combines excellent potency and selectivity with a promising pharmacokinetic profile. In vivo efficacy with regard to decreasing cardiac remodeling and amelioration of cardiac function was shown in a monocrotaline-induced rat model for pulmonary arterial hypertension. This demonstrated in vivo proof of concept in animals. Exploring new rings! Human neutrophil elastase (HNE) is a driver of inflammation in pulmonary arterial hypertension (PAH). Herein we describe the discovery of BAY-8040, a small-molecule inhibitor with nanomolar potency and good pharmacokinetics. BAY-8040 shows in vivo efficacy in a PAH rat model, thereby demonstrating proof of concept in animals.

Full text of DOI:10.1002/cmdc.201500269

DOI: 10.1002/cmdc.201500269
Full Papers
Potent and Selective Human Neutrophil Elastase Inhibitors
with Novel Equatorial Ring Topology: in vivo Efficacy of
the Polar Pyrimidopyridazine BAY-8040 in a Pulmonary
Arterial Hypertension Rat Model
Franz von Nussbaum,*[a] Volkhart M. Li,*[b] Daniel Meibom,*[c] Sonja Anlauf,[c]
Martin Bechem,[d] Martina Delbeck,[d] Michael Gerisch,[e] Axel Harrenga,[f] Dagmar Karthaus,[c]
Dieter Lang,[e] Klemens Lustig,[e] Joachim Mittendorf,[c] Martina Schäfer,[f] Stefan Schäfer,[d]
and Jens Schamberger[c]
Dedicated to Prof. Dr. Rolf Huisgen on the occasion of his 95th birthday
Human neutrophil elastase (HNE) is a key driver of inflamma-
tion in many cardiopulmonary and systemic inflammatory and
autoimmune conditions. Overshooting high HNE activity is the
consequence of a disrupted protease–antiprotease balance.
Accordingly, there has been an intensive search for potent and
selective HNE inhibitors with suitable pharmacokinetics that
would allowing oral administration in patients. Based on the
chemical probe BAY-678 and the clinical candidate BAY 85-
8501 we explored further ring topologies along the equator of
the parent pyrimidinone lead series. Novel ring systems were
annulated in the east, yielding imidazolo-, triazolo-, and tetra-
zolopyrimidines in order to ensure additional inhibitor–HNE
contacts beyond the S1 and the S2 pocket of HNE. The west-
ern annulation of pyridazines led to the polar pyrimidopyrida-
zine BAY-8040, which combines excellent potency and selectivi-
ty with a promising pharmacokinetic profile. In vivo efficacy
with regard to decreasing cardiac remodeling and amelioration
of cardiac function was shown in a monocrotaline-induced rat
model for pulmonary arterial hypertension. This demonstrated
in vivo proof of concept in animals.
Introduction
Neutrophil cells play a pivotal role in host defense (innate
immune response), inflammation, and tissue remodeling. A key
mediator of neutrophil-driven inflammation is human neutro-
phil elastase (HNE, EC 3.4.21.37), a member of the chymotryp-
sin-like family of serine proteases. HNE is stored in the azuro-
phil granules in the neutrophil cytoplasm and is liberated
upon the activation of neutrophils. HNE is able to degrade
1) scaffold proteins of the body’s own cellular matrix and
2) proteins foreign to the body such as cell-wall proteins of
Gram-negative bacteria. Degradation products and cleavage of
(bioactive) proteins elicit important pro-inflammatory respons-
es such as expression of the cytokine interleukin-8 (IL-8). Thus,
HNE orchestrates molecular and cellular cross-talk to protect
the body. Conversely, imbalanced activity of this highly active
protease might contribute to the onset and progression of
many inflammatory diseases with an impact on organ tissue in-
tegrity, especially cardiopulmonary diseases, such as chronic
obstructive pulmonary disease (COPD), bronchiectasis (BE), pul-
monary arterial hypertension (PAH), idiopathic pulmonary fib-
rosis (IPF),[1–16] as well as other systemic inflammatory and auto-
immune diseases such as rheumatoid arthritis (RA), inflamma-
tory bowel disease (IBD, Crohn’s disease), cystic fibrosis (CF),
[a] Dr. F. von Nussbaum
[e] Dr. M. Gerisch, Dr. D. Lang, Dr. K. Lustig
Medicinal Chemistry Berlin, Bayer HealthCare AG, 13353 Berlin (Germany)
E-mail: franz.nussbaum@bayer.com
DMPK Wuppertal, Bayer HealthCare AG, 42096 Wuppertal (Germany)
[f] Dr. A. Harrenga,+ Dr. M. Schäfer
[b] Dr. V. M. Li
Lead Discovery, Structural Biology Berlin
Lead Discovery Wuppertal
Bayer HealthCare AG, 13353 Berlin (Germany)
[+] Former affiliation and address during this project.
Bayer HealthCare AG, 42096 Wuppertal (Germany)
E-mail: volkhart.li@bayer.com
[c] Dr. D. Meibom, S. Anlauf, D. Karthaus,+ Prof. Dr. J. Mittendorf,
Dr. J. Schamberger
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500269.
This article is part of a Special Issue on Drug Discovery in the Field of
Autoimmune and Inflammatory Disorders. The complete issue can be
browsed via Wiley Online Library.
Medicinal Chemistry Wuppertal
Bayer HealthCare AG, 42096 Wuppertal (Germany)
E-mail: daniel.meibom@bayer.com
[d] Dr. M. Bechem, Dr. M. Delbeck, Prof. Dr. S. Schäfer+
Department of Cardiology Research Wuppertal
Bayer HealthCare AG, 42096 Wuppertal (Germany)
ChemMedChem 2016, 11, 199 – 206
199
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
and impaired wound healing (e.g., chronic wounds in venous,
arterial, diabetic, and pressure ulcers).[17–20]
high binding efficiency of the pyrimidinone class is based on
an induced-fit binding mode: Upon binding of these mole-
cules, the S2 pocket of HNE widens significantly to accommo-
date the northern para-cyanoaryl residue (e.g., with BAY-678).
In the case of C2’-substituted northern residues, the optimal
binding geometry is pre-locked, boosting potency into the pi-
comolar range (pre-adaptive pharmacophores;[21] e.g., with
BAY 85-8501).
Various players in academia and industry have sought to
identify efficient HNE inhibitors to tackle overshooting high
HNE activity.[21] To achieve sufficient biochemical potency, most
of these inhibitors were electrophiles that relied on a reactive
mode of action (suicide inhibitors, transition-state analogues).
Progression of these molecules into clinical testing was largely
unsuccessful. Reasons were intrinsic metabolic liabilities and
safety issues, potentially related to the reactive nature of these
inhibitors (acylators, electrophilic ketones).
Results and Discussion
Despite several decades of research, thus far only sivelestat
(1, Figure 1) has reached the market in Japan and South
Aim of this study
Due to the high impact of the pyrimidinone class[26] as HNE in-
hibitors, we wanted to expand the current understanding of
their structure–activity relationships (SAR). The northern and
the southern phenyl substituents of these systems such as 3
and 4 address the S2 and the S1 pocket of HNE. The SAR of
the northern and the southern sphere has been investigated in
detail. However, significantly less is known about their eastern
and western SAR. Upon binding to the target this equatorial
region is positioned along the natural substrate channel of
HNE, that is, the S4-S3-S1’-S2’-S3’ pocket sequence.[27] Focusing
on this equatorial region of pyrimidinone inhibitors appeared
promising, as it would potentially allow additional interactions
with HNE beyond the S1 and S2 pocket.
A further goal of our endeavor was to dial in more polarity
into our HNE inhibitors with potential positive effects on their
ADME profile. We were aiming for compounds with logD
values below 3, potentially leading to better solubility. Polarity
should be increased by introduction of nitrogen atoms into
novel eastern and western ring topologies. In summary, our
project should lead to a compound that would permit an in
vivo proof of concept in an inflammation-driven animal model
related to PAH.
Exploring the east
Figure 1. Selected HNE inhibitors that have reached the clinic: Sivelestat (1)
reached the Japanese market in 2002. AZD9668 (2) has reached clinical Pha-
se II trials in COPD, CF, and BE. BAY-678 (3) has reached Phase I trials, while
BAY 85-8501 (4) is currently being investigated in clinical Phase II trials in BE.
Exploration of the eastern hemisphere SAR started by fusing
five-membered heterocycles of increasing nitrogen content to
the pyrimidine core (Figure 2). All three ring systems 5–7
showed excellent activities against HNE, in the lower double-
digit to single-digit nanomolar range.
Korea.[22] Sivelestat is a reactive active ester of pivalic acid with
only a moderate three-digit biochemical potency (Ki: 200 nm).
Recently, with AZD9668 (2) and BAY 85-8501 (4), two nonreac-
tive and significantly more potent molecules have reached
clinical Phase II testing. As clinical trials with AZD9668 (2) in
COPD and BE did not provide the desired outcome, this drug
is now available on an open-innovation platform.[23] BAY 85-
8501 (4) showed good tolerability and good pharmacokinetics
in human volunteers in early Phase I studies.[24] Data from
a Phase II trial in BE have not yet been reported.[25]
A co-crystal structure was determined to understand the
binding of imidazopyrimidine 5 with HNE: Compound 5
bound in a clamp-like fashion, forming only one hydrogen
bond between the imidazo nitrogen atom and the Val216
backbone amide of HNE. This hydrogen bond was analogous
to that found in the pyrimidinone series of HNE inhibitors.[26]
Also binding of inhibitor 5 was mainly driven by shape com-
plementarity in which both subsites of HNE, S1 and S2, were
perfectly filled with hydrophobic moieties (Figure 3). Despite
good potency, the imidazopyrimidine 5 had limitations with re-
spect to in vitro metabolic stability.
We recently outlined our medicinal chemistry program that
explored northern and southern structural variations in the
new pyrimidinone lead series of HNE inhibitors, ultimately
leading to the chemical probe BAY-678 (3, Figure 1) and the
’5th generation’[21] clinical candidate, BAY 85-8501 (4).[26] The
The more polar triazolopyrimidine 6 displayed a more favor-
able overall in vitro profile while retaining high potency (HNE
IC50: 15 nm). Based on metabolic stability data in rat hepato-
ChemMedChem 2016, 11, 199 – 206
www.chemmedchem.org
200
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
for R1 (compound 8) was already capable of enhancing anti-
HNE activity to 2.5 nm. Sub-nanomolar potency of 0.59 nm
against the human enzyme and excellent potency against the
rodent enzyme was achieved by the addition of a methylamino
group to the triazole ring (compound 10). In contrast, employ-
ing a methoxy group for R1 yielded an HNE inhibitor which
was less attractive (9, HNE IC50: 69 nm). There was little impact
upon potency when the nitrogen attached to the five-mem-
bered ring was less electron rich, such as in acetylated deriva-
tives 11, 13, and 14; all three compounds showed very good
IC50 values against HNE of <1 nm.
A co-crystal structure of 13 with HNE (Figure 4) revealed an
overall binding mode similar to that of imidazopyrimidine 5.
Both structures showed the key hydrogen bond between an
Figure 2. Exploration of eastern SAR with fused ring systems. Calculated oral
bioavailability (Fmax) based on in vitro half-life in rat hepatocytes.
Figure 4. Ligand 13 interacts with HNE via two hydrogen bonds between
a triazolo nitrogen atom/the carbamate nitrogen atom and the Val216 back-
bone atoms (3.6 and 2.8 Š, respectively). An additional water-mediated hy-
drogen bond is formed between the cyano nitrogen atom and the carbonyl
moiety of His57 (3.1 and 2.8 Š, respectively). See Figure 3 for color code.
Figure 3. The protease (HNE) is shown in a stick representation (white) with
transparent Connolly-like surface.[28] Ligand 5 (purple) is shown as balls and
sticks. Heteroatoms are colored: oxygen, red; nitrogen, blue; fluorine, cyan.
Hydrogen bonds are depicted as broken yellow lines. Ligand 5 interacts with
HNE via a hydrogen bond (3.2 Š) formed between the imidazo nitrogen
atom and the Val216 backbone amide of HNE. The eastern imidazo ring is
depicted on the left-hand side. In addition, the S1 subsite is occupied by the
southern meta-(trifluoromethyl)phenyl moiety of ligand 5, whereas the S2
subsite is filled by the northern para-cyanophenyl residue of 5.
eastern nitrogen atom and the backbone Val216. However, tria-
zole 13 formed an additional hydrogen bond between the
eastern carbamate nitrogen atom (depicted on the left hand
side) and the Val216 oxygen atom. Moreover, the western
cyano moiety of 13 formed a water-mediated hydrogen bond
to His57 in the backbone of HNE. These two additional interac-
tions might explain the improved HNE affinity of 13 relative to
the imidazole 5.
On the other hand, the sulfonamides 15 and 16 were much
less active against HNE (12 and 50 nm, respectively; Table 1),
probably due to steric reasons. As previously shown, the addi-
tion of a substituent at the R2 position of the northern phenyl
moiety (’C2-north’) can boost potency, as it ensures preorgani-
zation of pyrimidinone-type binders in the bioactive conforma-
cytes, we calculated an intermediate bioavailability of 41% in
rats.
Adding another nitrogen atom to the eastern five-mem-
bered ring resulted in even better metabolic stability, as dem-
onstrated for the tetrazolopyrimidine 7 (rat Fmax: 59%). As tet-
razole 7 lacked a chemical attachment point to further explore
SAR in the eastern region, we turned our attention to triazole
6 as the most advantageous starting point.
Nitrogen-substituted triazolo rings were especially well
suited for improving potency (Table 1). A simple amino group
ChemMedChem 2016, 11, 199 – 206
www.chemmedchem.org
201
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
The carbamate 13 was chosen as candidate for rat
in vivo pharmacokinetic (PK) studies due to its excel-
lent potency and overall promising in vitro PK profile.
Unfortunately, the half-life after intravenous (i.v.) ad-
ministration was only 1 h (see Table 3 below), ham-
pering broad pharmacological in vivo testing in long-
term PAH models.
Table 1. Potency of triazoles: exploration of eastern and northern SAR.
In a Biginelli-type reaction 3,5-diamino-1,2,4-tria-
zole (19) could be directly used for the synthesis of
aminotriazolopyrimidine 20 (Scheme 1). The southern
m-(trifluoromethyl)phenyl unit was subsequently in-
troduced via Chan–Lam coupling (see Supporting In-
formation for further syntheses).
Compd
R1
H
R2
H
IC50 [nm][a]
Fmax [%][b]
CYP IC50 [mm][c]
2C9/3A4
HNE
RNE
6
15
–
41
–/–
In summary, the extraordinary effort required for
the synthesis of the fused systems 6–16 was not re-
imbursed by a significantly improved technical profile
relative to the simpler pyrimidinone base systems.
Accordingly, we decided to shift toward alternative
western ring topologies that seemed to be more
readily accessible.
8
NH2
H
2.5
153
28
50/50
9 (rac)
H
H
69
0.59
–
–
–/–
10
3.4
33
–/20
11
H
0.35
0.03
0.4
99
50/30
Exploring the west
We investigated the western hemisphere SAR by
evaluating pyridazinones annulated to the pyrimidi-
none core (Table 2, see Supporting Information for
synthetic procedures). N-Methyl analogue 24 (BAY-
8040; Scheme 2, Table 2) turned out to be a potent
HNE inhibitor with an IC50 value of 28 nm. Surprising-
ly, the X-ray co-crystal structure of 24 with HNE re-
vealed only one strong hydrogen bond, between the
pyrimidinone carbonyl group and Val216 (Figure 5).
Obviously, no additional ligand–HNE interactions
were built up by the western pyridazinone ring. As
demonstrated with compounds 30 and 31, N-aryla-
tion was, in principle, tolerated by HNE; however, po-
tency was not improved relative to 24, despite the
introduction of five to six additional carbon atoms
which could potentially interact with the S2’ pocket
of HNE. Adding another carbon atom to the alkyl
chain of 24 slightly improved potency to 16 nm
(against the human enzyme), as shown with com-
pound 25. The oxygen-containing alkyl derivatives 27
and 28 displayed decreased inhibitory activities rela-
tive to 24. The most potent pyridazinone HNE inhibi-
tor was the cyclohexyl derivative 26 which, apart
from the human enzyme, also showed improved ac-
tivity against the rat enzyme (Table 2; HNE IC50:
5.5 nm, RNE IC50: 195 nm). On the other hand, 26 was
more lipophilic (clogD 3.54, LipE 4.7) than 24 and,
12
13
SO2Me
H
1.6
8.8
99
69
46/50
50/50
0.17
0.3
14
15
16
H
H
H
1.8
–
52
74
–
20/20
–/50
–/–
12
50
–
[a] The inhibitory capacity of test compounds was assessed by applying functional
biochemical assays with the isolated enzyme (Supporting Information); IC50 values
were derived from enzyme activity data (pH 7.4) in the presence/absence of various
compound concentrations by applying a suitable fluorogenic peptide substrate,
MeOSuc-AAPV-AMC. [b] The metabolic stability of test compounds was assessed in
the presence of rat hepatocytes by determination of the half-life of the compound
(Supporting Information). Clearance parameters and Fmax values were calculated from
this half-life, representing a measure of the phase 1 and phase 2 metabolism. [c] The
potency of test compounds to inhibit human CYP2C9 and CYP3A4 was investigated
using pooled human liver microsomes as enzyme source and selective standard sub-
strates (Supporting Information); IC50 values were derived from enzyme activity data
(pH 7.4) in the presence/absence of various compound concentrations and diclofe-
nac/midazolam as selective CYP2C9/CYP3A4 substrate.
tion, with a N3-C4-C1’-C2’ dihedral angle of ~1108.[26] We
wanted to determine whether this concept could also be
transferred into other ring topologies, e.g., compounds 5–7.
With an impressive HNE IC50 value of 0.03 nm, compound 12
demonstrated that a ‘frozen’ bioactive conformation indeed
also leads to picomolar biochemical potency within the triazo-
lopyrimidine series!
unfortunately, less metabolically stable in vitro (Fmax: 11% vs.
44%).
In summary, BAY-8040 (24) turned out to be the best in vivo
candidate from this series. This again demonstrated the princi-
ple that the most potent compound is seldom the best in vivo
candidate. As planned, with 24 the clogD value had been
pushed below 3 by introduction of two nitrogen atoms in the
ChemMedChem 2016, 11, 199 – 206
www.chemmedchem.org
202
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Scheme 2. Synthesis of pyridazinone 24 (BAY-8040). Reagents and conditions:
a) polyphosphoric acid ethylester, 1-[3-(trifluoromethyl)phenyl]urea, ethyl 3-
oxobutanoate, reflux, 18 h, 91%; b) preparative HPLC on chiral phase
(eluent: EtOAc/MeOH gradient), 99%; c) bromine, chloroform, 08C!RT, 1 h,
87%; d) methylhydrazine, 1,4-dioxane, reflux, 3 h, 66%.
Scheme 1. Synthesis of triazolopyrimidine 13. Reagents and conditions:
a) sodium 1-cyanoprop-1-en-2-olate, piperidine, acetic acid, CH2Cl2, reflux
(Dean–Stark trap), 1 d, 79%; b) d,l-proline, CH2Cl2/pyridine/DMF, RT, 8 d;
TsOH, toluene/pyridine, reflux, 1 h, 78%; c) phthalic anhydride, TEA, toluene/
pyridine, reflux, 1 d, 72%; d) 3-(trifluoromethyl)phenylboronic acid, molecular
sieves (4 Š), Cu(OAc)2, CH2Cl2/pyridine, TEA, RT, ~7 d, 19%; e) preparative
HPLC on chiral phase (eluent: EtOAc/isohexane 1:1), quant.; f) hydrazine hy-
drate, EtOH, 858C, 1 h, 97%; 4n HCl in 1,4-dioxane; g) methyl chloroformate,
THF/pyridine, 808C, 12 h, 91%. DMF=N,N-dimethylformamide;
Phth=phthaloyl; TsOH=p-toluenesulfonic acid; TEA=triethylamine;
THF=tetrahydrofuran.
east (clogD 2.46 at pH 7.5). The higher polarity translated into
sufficient binding efficiency (LipE 5.1) and acceptable solubility
(60 mgLÀ1 at pH 6.5, thermodynamic from DMSO solution) for
BAY-8040. The new eastern ring topology did not hamper
target specificity, as BAY-8040 (24) showed no significant inhib-
ition at a concentration of 10 mm toward 68 other pharmaco-
logically relevant receptors (Lead Profiling Screen at MDS
Pharma Services Ltd., Taiwan; Supporting Information). Also se-
lectivty was very good against a panel of related serine pro-
teases (IC50 >50 mm versus seven serine proteases; see Sup-
porting Information). In rats BAY-8040 (24) demonstrated favor-
able in vivo PK parameters (Table 3, t1/2: 3.2 h, F: ~100%). The
higher polarity translated into a high free fraction of 61% in
rat plasma (fraction unbound). Due to this overall good in vitro
and in vivo profile BAY-8040 (24) qualified for further explora-
tion within disease-relevant animal model studies.
Figure 5. BAY-8040 (24) forms a weak hydrogen bond between its pyrimidi-
none carbonyl group and the NH of Val216 (3.4 Š). No further water-mediat-
ed hydrogen bonds are observed. See Figure 3 for color code.
In vivo pharmacodynamic studies: monocrotaline (MCT) rat
model
The pathogenesis of PAH has been related to an excess of HNE
activity (see above). Therefore, we investigated the selective
ChemMedChem 2016, 11, 199 – 206
www.chemmedchem.org
203
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
a pyrrolizidine alkaloid from the plant Crotalaria spec-
tabilis, this model is characterized by an upregulation
of inflammatory cytokines, leukocyte infiltration, and
proliferation of pulmonary arterial smooth muscle
cells. MCT elicits pneumotoxicity, including pulmona-
ry endothelial cell injury in rats. Four weeks after the
single subcutaneous injection of MCT, rats in the con-
trol group displayed severely elevated right ventricu-
lar (RV) systolic pressure and marked RV hypertrophy
(Figure 6B).
Table 2. Potency of pyridazinones: exploration of western SAR.
However, treatment with BAY-8040 (50 mgkgÀ1
b.i.d. p.o., n=10–11) from days 14 to 28 significantly
decreased RV systolic pressure (Figure 7A) and RV hy-
pertrophy (Figure 7B) relative to placebo, with a sig-
nificant beneficial effect on RV dysfunction (Fig-
ure 7C) and preservation of systemic arterial pressure
(Figure 7D). Furthermore, 24 significantly improved
cardiac output (Figure 7E) and ameliorated arterial
oxygenation (Figure 7F) in rats with MCT-induced
PAH.
Compd
R
IC50 [nm][a]
Fmax [%][b]
CYP IC50 [mm][c]
2C9/3A4
HNE
RNE
24 BAY-8040
Me
28
16
558
44
50/50
–/50
25
Et
–
80
26
27
5.5
195
–
11
36
–/–
–/–
71
In this manner the treatment with BAY-8040 (24)
had been initiated at a time point at which the acute
inflammatory response due to the initial MCT chal-
lenge had already passed and the development of
the disease was already driven by a sustained inflam-
mation and subsequent tissue remodeling (Fig-
ure 6B). In this setting, BAY-8040 was seemingly able
to not only suppress inflammation but also to slow
down and/or reverse tissue remodeling in the PAH
setting.
28
29
52
32
–
–
79
11
50/50
–/–
30
31
74
29
–
–
12
13
42/50
–/–
[a] The inhibitory capacity of test compounds was assessed by applying functional
biochemical assays with the isolated enzyme (Supporting Information); IC50 values
were derived from enzyme activity data (pH 7.4) in the presence/absence of various
compound concentrations by applying a suitable fluorogenic peptide substrate,
MeOSuc-AAPV-AMC. [b] The metabolic stability of test compounds was assessed in
the presence of rat hepatocytes by determination of the half-life of the compound
(Supporting Information). Clearance parameters and Fmax values were calculated from
this half-life, representing a measure of the phase 1 and phase 2 metabolism. [c] The
potency of test compounds to inhibit human CYP2C9 and CYP3A4 was investigated
using pooled human liver microsomes as enzyme source and selective standard sub-
strates (Supporting Information); IC50 values were derived from enzyme activity data
(pH 7.4) in the presence/absence of various compound concentrations and diclofe-
nac/midazolam as selective CYP2C9/CYP3A4 substrate.
Conclusions
Exploration of new equatorial ring topologies has al-
lowed access to novel HNE inhibitor classes related
to the parent pyrimidinone lead series. By annulation
of a nitrogen-rich five-membered ring system in the
east, highly potent inhibitors such as 13 were possi-
ble. Some of these compounds showed a promising
in vitro DMPK profile; however, they had limitations
with respect to the half-life in rodents after i.v. appli-
cation. Also in this series the C2-north substitution al-
lowed for pre-adaptive pharmacophores with pico-
molar biochemical potency (e.g., 12).
On the other hand, modifications in the west of
Table 3. Pharmacokinetic profile of selected compounds in rats.[a]
our inhibitors led us to the new in vivo candidate BAY-8040
(24). Based on a pyrimidopyridazine core, this compound
showed good potency and good in vivo pharmacokinetics.
Most importantly, an in vivo proof of concept was demonstrat-
ed in a rat model for PAH. BAY-8040 decreased chronic inflam-
mation and thus slowed or even reversed tissue remodeling of
the pulmonary artery. Upon treatment with BAY-8040 (24),
a statistically significant effect on cardiopulmonary parameters
such as cardiac output and oxygen saturation was observed.
Thus, BAY-8040 turned out to be a valuable chemical in vivo
tool in rodents that might help to further foster the under-
standing of elastase biology and pharmacology.
[b]
[c]
Compd
CLp [LhÀ1 kgÀ1
]
VSS [LkgÀ1
]
t1/2 [h]
F [%][d]
13
24
1.8
1.5
2.1
7.5
1.0
3.2
99
~100
[a] Values were derived by i.v. (0.25 h infusion) and oral (gavage) adminis-
tration of 0.3 mgkgÀ1 in EtOH/PEG400/H2O vehicles. [b] Total plasma
clearance. [c] Apparent volume of distribution at steady state. [d] Oral
bioavailability.
HNE inhibitor BAY-8040 (24) in a monocrotaline (MCT) rat
model of PAH (Figure 6A). Triggered by administration of MCT,
ChemMedChem 2016, 11, 199 – 206
www.chemmedchem.org
204
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Experimental Section
All experimental data are provided in the Supporting Information.
Acknowledgements
We thank Sandra Korthals and Kay Greenfield for valuable tech-
nical support with the manuscript. We also thank Susanne
Wegner for continuous support of this project.
Keywords: Biginelli reaction · biological activity · elastase
inhibitors · medicinal chemistry · pyrimidinones
[1] A. Belaaouaj, R. McCarthy, M. Baumann, Z. Gao, T. J. Ley, S. N. Abraham,
S. D. Shapiro, Nat. Med. 1998, 4, 615–618.
[2] R. A. Stockley, Am. J. Respir. Crit. Care Med. 1999, 160, S49–S52.
[3] S. H. E. Zaidi, C.-C. Hui, A. Y. L. Cheah, X.-M. You, M. Husain, M. Rabino-
vitch, J. Clin. Invest. 1999, 103, 1211–1219.
[4] W. L. Lee, G. P. Downey, Am. J. Respir. Crit. Care Med. 2001, 164, 896–
904.
[5] R. A. Stockley, Chest 2002, 121, 151S–155S.
[6] S. H. E. Zaidi, X.-M. You, S. Ciura, M. Husain, M. Rabinovitch, Circulation
2002, 105, 516–521.
[7] S. D. Shapiro, N. M. Goldstein, A. M. Houghton, D. K. Kobayashi, D.
Kelley, A. Belaaouaj, Am. J. Pathol. 2003, 163, 2329–2335.
[8] B. G. Zeiher, A. Artigas, J. L. Vincent, A. Dmitrienko, K. Jackson, B. T.
Thompson, G. Bernard, Crit. Care Med. 2004, 32, 1695–1702.
[9] F. Chua, S. E. Dunsmore, P. H. Clingen, S. E. Mutsaers, S. D. Shapiro, A. W.
Segal, J. Roes, G. J. Laurent, Am. J. Pathol. 2007, 170, 65–74.
[10] B. Korkmaz, T. Moreau, F. Gauthier, Biochimie 2008, 90, 227–242.
[11] C. M. Greene, N. G. McElvaney, Br. J. Pharmacol. 2009, 158, 1048–1058.
[12] B. Korkmaz, M. S. Horwitz, D. E. Jenne, F. Gauthier, Pharmacol. Rev. 2010,
62, 726–759.
[13] S. S. Pullamsetti, R. Savia, W. Janssen, B. K. Dahal, W. Seeger, F. Grim-
minger, H. A. Ghofrani, N. Weissmann, R. T. Schermuly, Clin. Microbiol.
Infect. 2011, 17, 7–14.
Figure 6. MCT rat model of PAH: A) Schematic representation of the experi-
mental rationale. B) Treatment schedule.
[14] M. Rabinovitch, J. Clin. Invest. 2012, 122, 4306–4313.
Figure 7. Effect of treatment with BAY-8040 (24; 50 mgkgÀ1 p.o.) on MCT-induced pulmonary hypertension in rats. A) Right ventricular systolic pressure (RVP).
B) RV to LV weight ratio [RV/(LV+S)]. C) Right ventricular end-diastolic pressure (RVEDP). D) Systemic arterial pressure (SAP). E) Cardiac output (CO). F) Arterial
oxygen saturation (SaO2). Data are the mean ÆSEM (n=10–11/group); *p<0.05 versus MCT/placebo day 28.
ChemMedChem 2016, 11, 199 – 206
www.chemmedchem.org
205
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[15] M. Rabinovitch, C. Guignabert, M. Humbert, M. R. Nicolls, Circ. Res.
2014, 115, 165–175.
Aquino, C. D. O’Brien, J. Perret, K. Gunawardena, COPD 2012, 9, 111 –
120.
[16] J. Fu, Y.-F. Chen, X. Zhao, J. R. Creighton, Y. Guo, F. G. Hage, S. Oparil,
D. D. Xing, Arterioscler. Thromb. Vasc. Biol. 2014, 34, 1539–1547.
[17] B. M. Fournier, C. A. Parkos, Mucosal Immunol. 2012, 5, 354–366.
[18] M. J. Kaplan, Arthritis Res. Ther. 2013, 15, 219–228.
[19] J. C. McDaniel, S. Roy, T. A. Wilgus, Wound Repair Regen. 2013, 21, 339–
351.
[20] H. L. Wright, R. J. Moots, S. W. Edwards, Nat. Rev. Rheumatol. 2014, 10,
593–601.
[21] F. von Nussbaum, V. M.-J. Li, Bioorg. Med. Chem. Lett. 2015, DOI:
10.1016/j.bmcl.2015.08.049.
[24] J. Nagelschmitz, M. Becka, D. van der Mey, S. Schwers, F. von Nussbaum,
V. Li, G. Wensing, Eur. Respir. J. 2014, 44, P1510.
[25] J. Nagelschmitz, D. Kaufel, S. Schwers, F. von Nussbaum, V. Li, M. Del-
beck, K. Lustig, T. Bandel, H. Watz, Eur. Respir. J. 2014, 44, 3416.
[26] F. von Nussbaum, V. Li, S. Allerheiligen, S. Anlauf, L. Bärfacker, M.
Bechem, M. Delbeck, M. F. Fitzgerald, M. Gerisch, H. Gielen-Haertwig, H.
Haning, D. Karthaus, D. Lang, K. Lustig, D. Meibom, J. Mittendorf, U.
Rosentreter, M. Schäfer, S. Schäfer, J. Schamberger, L. A. Telan, A. Ter-
steegen, ChemMedChem 2015, 10, 1163–1173.
[27] Y. Odagaki, K. Ohmoto, S. Matsuoka, N. Hamanka, H. Nakai, M. Toda, Y.
Katsuya, Bioorg. Med. Chem. 2001, 9, 647–651.
[28] Figures 3–5 were generated with PyMOL: The PyMOL Molecular Graph-
ics System, Version 1.5.0.4, Schrçdinger LLC.
[22] Ono Pharmaceutical Co. Ltd., Press Release, December 19, 2006:
https://www.ono.co.jp/eng/cn/contents/sm_cn_061219.pdf (accessed
June 6, 2015).
[23] a) J. P. Elborn, J. Perret, K. Forsman-Semb, J. Marks-Konczalik, K. Guna-
wardena, Eur. Respir. J. 2012, 40, 969–976; b) R. Stockley, A. De Soyza,
K. Gunawardena, J. Perret, K. Forsman-Semb, N. Entwistle, N. Snell,
Respir. Med. 2013, 107, 524–533; c) P. Kuna, M. Jenkins, C. D. O’Brien,
W. A. Fahy, Respir. Med. 2012, 106, 531–539; d) C. Vogelmeier, T. O.
Received: June 21, 2015
Published online on September 3, 2015
ChemMedChem 2016, 11, 199 – 206
www.chemmedchem.org
206
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim