Freezing the Bioactive Conformation to Boost Potency: The Identification of BAY85-8501, a Selective and Potent Inhibitor of Human Neutrophil Elastase for Pulmonary Diseases

Article abstract of DOI:10.1002/cmdc.201500131

Human neutrophil elastase (HNE) is a key protease for matrix degradation. High HNE activity is observed in inflammatory diseases. Accordingly, HNE is a potential target for the treatment of pulmonary diseases such as chronic obstructive pulmonary disease

Full text of DOI:10.1002/cmdc.201500131

DOI: 10.1002/cmdc.201500131
Full Papers
Freezing the Bioactive Conformation to Boost Potency:
The Identification of BAY 85-8501, a Selective and Potent
Inhibitor of Human Neutrophil Elastase for Pulmonary
Diseases
Franz von Nussbaum,*[a] Volkhart M.-J. Li,*[b] Swen Allerheiligen,[c] Sonja Anlauf,[c]
Lars Bärfacker,[c] Martin Bechem,[d] Martina Delbeck,[d] Mary F. Fitzgerald,[e] Michael Gerisch,[f]
Heike Gielen-Haertwig,[c] Helmut Haning,[c] Dagmar Karthaus,[c] Dieter Lang,[f]
Klemens Lustig,[f] Daniel Meibom,[c] Joachim Mittendorf,[c] Ulrich Rosentreter,[c]
Martina Schäfer,[g] Stefan Schäfer,[d] Jens Schamberger,[c] Leila A. Telan,[c] and
Adrian Tersteegen[b]
Dedicated to the memory of Ekkehard Winterfeldt.
Human neutrophil elastase (HNE) is a key protease for matrix
degradation. High HNE activity is observed in inflammatory dis-
eases. Accordingly, HNE is a potential target for the treatment
of pulmonary diseases such as chronic obstructive pulmonary
disease (COPD), acute lung injury (ALI), acute respiratory dis-
tress syndrome (ARDS), bronchiectasis (BE), and pulmonary hy-
pertension (PH). HNE inhibitors should reestablish the pro-
tease–anti-protease balance. By means of medicinal chemistry
a novel dihydropyrimidinone lead-structure class was identi-
fied. Further chemical optimization yielded orally active com-
pounds with favorable pharmacokinetics such as the chemical
probe BAY-678. While maintaining outstanding target selectivi-
ty, picomolar potency was achieved by locking the bioactive
conformation of these inhibitors with a strategically positioned
methyl sulfone substituent. An induced-fit binding mode al-
lowed tight interactions with the S2 and S1 pockets of HNE.
BAY 85-8501 ((4S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-3,6-di-
methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydro-
pyrimidine-5-carbonitrile) was shown to be efficacious in
a rodent animal model related to ALI. BAY 85-8501 is currently
being tested in clinical studies for the treatment of pulmonary
diseases.
Introduction
Human neutrophil elastase (HNE; EC 3.4.21.37) is a member of
the chymotrypsin-like family of serine proteases and is stored
in the azurophil granules in the neutrophil cytoplasm. This
highly active protease is able to break down mechanically im-
portant structures of the body’s own cellular matrix (e.g., pro-
teins such as elastin and collagen), as well as proteins foreign
to the body (e.g., outer cell wall proteins of Gram-negative
bacteria). Furthermore, the enzyme cleaves a variety of endog-
enous and exogenous proteins, tuning their biological activity
such as activation of other bioactive proteases (e.g., matrix
metalloproteinases, MMPs), liberation of growth factors, shed-
ding of cell-surface-bound receptors, and degradation of en-
dogenous proteinase inhibitors (e.g., tissue inhibitors of metal-
loproteases, TIMPs) or exogenous virulence factors.[1] Thus,
[a] Dr. F. von Nussbaum
[e] Dr. M. F. Fitzgerald+
Medicinal Chemistry Berlin
COPD Research, Bayer Healthcare AG, Slough SL2 4LY, Berkshire (UK)
Bayer HealthCare AG, 13353 Berlin (Germany)
E-mail: franz.nussbaum@bayer.com
[f] Dr. M. Gerisch, Dr. D. Lang, Dr. K. Lustig
DMPK Wuppertal, Bayer HealthCare AG, 42096 Wuppertal (Germany)
[b] Dr. V. M.-J. Li, Dr. A. Tersteegen
Lead Discovery Wuppertal
[g] Dr. M. Schäfer
Lead Discovery, Structural Biology Berlin
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. S. Allerheiligen, S. Anlauf, Dr. L. Bärfacker, Dr. H. Gielen-Haertwig,
Dr. H. Haning, D. Karthaus, Dr. D. Meibom, Prof. Dr. J. Mittendorf,
Dr. U. Rosentreter,+ Dr. J. Schamberger, Dr. L. A. Telan+
Medicinal Chemistry Wuppertal
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500131.
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
Bayer HealthCare AG, 42096 Wuppertal (Germany)
[d] Dr. M. Bechem, Dr. M. Delbeck, Prof. Dr. S. Schäfer+
Department of Cardiology Research Wuppertal
Bayer HealthCare AG, 42096 Wuppertal (Germany)
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human neutrophil elastase plays a pivotal role in tissue remod-
eling processes, as well as in the onset of inflammation and in
host defense (innate immune response).
The activity of the versatile protease HNE is tightly con-
trolled by: 1) channeling the potentially dangerous protease to
specialized compartments (e.g., storage granula and phagoly-
sosomes), and 2) the presence of extracellular neutralizing en-
dogenous serine protease inhibitors (SERPINs), for example,
a-1 antitrypsin (AAT, also known as a-PI) and elafin, which
maintain the crucial balance of the protease and its anti-pro-
teases.[2–4]
An imbalance in elastase activity might contribute to the
onset and progression of many inflammatory diseases
(Figure 1) with an impact on organ tissue integrity, especially
Figure 2. Selection of HNE inhibitors that have reached clinical development.
a) Biologicals such as elafin[11] were the first class of HNE-inhibiting drugs.
These compounds are only suitable for i.v. application. b) As a second promi-
nent class, covalent drugs that acylate serine in the active site of the
enzyme, such as sivelestat (1), or transition-state mimetics, such as fresele-
stat (2), were described. A tremendous effort was required to find the first
reversible and selective small-molecule drugs as a third class. A prominent
example is alvelestat (3).
Figure 1. Elastase potentially plays key roles in many pulmonary diseases in
which the elastase–anti-protease balance is disrupted. Image: P. J. Lynch,
www.wikipedia.com.
hibitor AZD 9668 (3) in patients with pulmonary diseases: Here
a small four-week treatment study with 56 cystic fibrosis pa-
tients revealed modulation of some secondary endpoint bio-
markers (e.g., urinary desmosine concentration), but did not
show a significant improvement in primary endpoints (clinical
outcomes, e.g., lung function FEV1 or quality of life).[18] A fur-
ther small four-week treatment signal-searching study with 38
BE patients showed a promising improvement in lung function
(FEV1) and trends for decreases in sputum inflammatory mark-
ers.[19] For both indications, longer and larger studies would be
needed to confirm the initial findings. In two larger 12-week
treatment studies with nearly 1500 COPD patients in total, no
clinical benefit and no effect on biomarkers of inflammation or
tissue degradation could be demonstrated. This might have
been due to the rather short treatment period and the hetero-
geneity of this disease (a post-hoc analysis revealed an im-
provement in lung function in a subgroup of patients with
a chronic bronchitis phenotype).[20,21]
in cardiopulmonary diseases, such as chronic obstructive pul-
monary disease (COPD), bronchiectasis (BE), pulmonary arterial
hypertension (PAH), and pulmonary fibrosis. Elastase knock-out
in rodents or anti-protease transgenic animals reveal a signifi-
cant protection or resistance against experimental lung em-
physema,[5] pulmonary hypertension (PH),[6] pulmonary fibro-
sis,[7] and myocarditis.[8] Individuals with antitrypsin deficiency
(AATD) reveal dramatically lower levels of AAT and have an in-
creased risk of suffering from lung emphysema.[9] Notably, elas-
tase knock-out mice are vulnerable to infection with Gram-
negative bacteria.[10]
So far, various HNE inhibitors have been described;[12,13] how-
ever, only a few chemical entities have had an overall profile
suitable for clinical testing. The first potent elastase inhibitors
to reach the clinic were biologicals such as elafin[11] (Figure 2).
The first small-molecule inhibitors were electrophilic com-
pounds including serine acylators such as sivelestat (1)[14–16] or
transition-state mimetics such as freselestat (2).[17] Recently, As-
traZeneca reported phase II studies with the reversible HNE in-
In general, combining potency with selectivity is a large
hurdle for small-molecule serine protease inhibitors. Herein we
report the discovery of a new, highly potent and selective neu-
trophil elastase inhibitor, BAY 85-8501, with an unprecedented
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locked bioactive conformation. BAY 85-8501 is currently being
investigated in a safety and efficacy trial in BE patients.[22]
Table 2. Initial shift from core bicyclic to monocyclic 2-amino-1,4-dihy-
dropyridine systems: northern and southern SAR.
Results and Discussion
In the search for better HNE inhibitors, we performed a high-
throughput screen (HTS) of our small-molecule compound li-
brary. From the hit list, we identified hexahydroquinoline 4 as
the most promising and structurally unique starting point for
exploratory chemistry (Table 1). The racemic HTS hit 4 dis-
Compd
R1
R2
HNE
IC50 [nm][a]
Fmax [%][b]
CYP 2C9/3A4
IC50 [mm][c]
Table 1. Potency, lipophilic binding efficiency, metabolic stability, and
CYP inhibition data of the initial screening hit.
7
8
9
NO2
CF3
CF3
Br
Br
CN
200
200
20
15
31
57
–/–
0.6/0.5
0.5/25
[a] The inhibitory capacity of test compounds was assessed by applying
functional biochemical assays with the isolated enzyme (Supporting Infor-
mation); 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 (Sup-
porting Information). Clearance parameters and Fmax values were calculat-
ed 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 with pooled human liver micro-
somes as enzyme source and selective standard substrates (Supporting
Information); IC50 values were derived from enzyme activity data (pH 7.4)
in the presence/absence of various compound concentrations and diclo-
fenac/midazolam as selective CYP2C9/CYP3A4 substrate.
Compd
Stereo.
HNE
ClogD[b]
LipE[c]
CYP 2C9/3A4
IC50 [nm][a]
IC50 [mm][d]
4
5
6
rac
R
S
900
200
7000
–
4.2
4.2
–
2.5
1.0
–/–
2/10
8/0.4
[a] The inhibitory capacity of test compounds was assessed by applying
functional biochemical assays with the isolated enzyme (Supporting Infor-
mation); 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] ClogD
(pH 7.5) was calculated by using a highly predictive method developed at
Bayer, based on data points of experimentally determined logD values of
internal pharmaceutical compounds and the Simulations Plus pKa predic-
tor.[23] [c] Calculated as LipE=pIC50ÀlogD.[24] [d] The capacity of test com-
pounds to inhibit human CYP2C9 and CYP3A4 was investigated with
pooled human liver microsomes as enzyme source and selective standard
substrates (Supporting Information); IC50 values were derived from
enzyme activity data (pH 7.4) in the presence/absence of various com-
pound concentrations and diclofenac/midazolam as CYP2C9/CYP3A4
substrate.
Table 3. Shift from 2-aminodihydropyridines to dihydro-2-pyridones:
eastern and western SAR.
Compd
R1
R2
HNE
IC50 [nm][a]
CYP3A4
IC50 [mm][b]
played moderate in vitro potency (IC50: 0.9 mm). Upon separa-
tion of the enantiomers, stereospecific activity became evident
as the R enantiomer 5 proved to be fivefold more potent (IC50:
0.2 mm) than the nearly inactive S enantiomer 6 (7 mm). Due to
high lipophilicity and a molecular weight >500 Da, the lipo-
philic binding efficiency[24] (LipE=2.5) of the screening hit was
poor (Table 1). Unfortunately, both hexahydroquinolines 5 and
6 showed undesirable inhibition of human CYP2C9 and/or
CYP3A4.
10
11
12
13
COMe
COMe
CO2Et
CN
CO2Et cis
40
50
70
20
4
9
–
H
H
H
15
[a] The inhibitory capacity of test compounds was assessed by applying
functional biochemical assays with the isolated enzyme (Supporting Infor-
mation); 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 po-
tency of test compounds to inhibit human CYP3A4 was investigated with
pooled human liver microsomes as enzyme source (Supporting Informa-
tion); IC50 values were derived from enzyme activity data (pH 7.4) in the
presence/absence of various compound concentrations and midazolam
as CYP3A4 substrate.
To decrease the molecular weight and ring count, the corre-
sponding ring-opened dihydropyridine analogues 7–9 were
prepared (Table 2). Fortunately, the ring-opened analogue 7
was fivefold more potent than our screening hit 4. The poten-
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tially unfavorable nitro group of
7 was subsequently converted
into a trifluoromethyl group (in
8) without loss in potency. As
a second step toward an even
more drug-like molecule, we
exchanged the northern bromo
substituent for other, potential-
ly more stable, electron-with-
drawing groups. The linear
cyano substituent of 9 enabled
a 10-fold potency increase cou-
pled with a significant improve-
ment in the lipophilic binding
efficiency (LipE=4.0, ClogD=
3.7). Unfortunately, all 2-amino-
Table 4. Potency of 1,4-dihydropyrimidinones: exploration of eastern and western SAR.
Compd
R1
R2
X
IC50 [nm][a]
Fmax [%][b]
IC50 [mm][c]
CYP 2C9/3A4
HNE
RNE
14
15
16
17
18
19
20
21
22
23
CO2Et
CO2Et
CO2Et
H
CH2CONH2
CH2CO2H
H
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
27
1.0
192
15
43
276
–
44
800
49
1018
233
5
13
94
57
31
96
57
9
5/>50
>50/>50
>50/>50
2/>50
2.1
4.3
13
3.5
20
1
4.7
25
CO2CH2CH2OH
COCH3
COCH3
COCH3
COCH3
CN
H
7/>50
dihydropyridine-type
com-
CH2CO2H
H
CH2CH2NEt2
H
31/>100
12/>50
>50/>50
18/>50
>50/>50
pounds 7–9 revealed inhibitory
potency toward CYP2C9 and/or
CYP3A4.
35
77
CN
CH2CONH2
To further explore the central
ring system, dihydro-2-pyri-
dones 10–13 were pursued
next (Table 3). A shift from ni-
trogen to oxygen at C2 was
indeed possible, leading to 10
with an additional stereocenter
at C3, which was unfortunately
inclined to epimerize. Omitting
the ester functionality at C3
streamlined the system to only
one stereocenter at C4 (com-
pounds 11–13). In this series,
nearly unchanged target poten-
24
CN
CH
5
126
16
–/–
[a] The inhibitory capacity of test compounds was assessed by applying functional biochemical assays with the
isolated (HNE) or enriched (RNE) enzyme (Supporting Information); IC50 values were derived from enzyme activ-
ity data (pH 7.4) in the presence/absence of various compound concentrations by applying a suitable fluoro-
genic 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 inves-
tigated with pooled human liver microsomes as enzyme source and selective standard substrates; IC50 values
were derived from enzyme activity data (pH 7.4) in the presence/absence of various compound concentrations
and diclofenac/midazolam as selective CYP2C9/CYP3A4 substrate.
cy and overall less inhibitory potency toward CYP3A4 were ob-
served. A 5-cyano substituent yielded the most potent com-
pound 13. However, synthetic accessibility and stereochemical
integrity were potential limitations of this pyridone class. To
tackle these issues, we capitalized on the high SAR flexibility at
C3 that allowed another major structural transformation.
Introduction of a ring nitrogen at position 3 led to 1,4-dihy-
dropyrimidinones, which were especially potent against HNE
(Table 4). Enantiopure compounds of this type were readily
available by Biginelli chemistry[25] coupled with chiral chroma-
tography (Supporting Information). Impressive flexibility was
observed with respect to ligand polarity in the equatorial
sphere at N3. Either potentially anionic or cationic compounds,
such as carboxylic acids 16 and 19, or amines 21 and 24, inhib-
ited HNE with good to excellent potency. Furthermore, an
overall decreased inhibitory potency of the 1,4-dihydropyrimi-
dinones against CYP isoforms turned out to be a significant
improvement. For example, up to a concentration of 50 mm,
amide 23 elicited no observable inhibition toward CYP2C9 and
CYP3A4. Due to its overall balanced profile with respect to po-
larity and potency, BAY-678 (20) was selected for in-depth in
vitro and in vivo characterization as a chemical probe candi-
date for HNE (see below).
Apparently, all compounds from the 1,4-dihydropyrimidi-
nones series 14–24 showed significantly lower potency against
rat neutrophil elastase (RNE, Table 4). Obtaining compounds
with decent potency not only against HNE but against neutro-
phil elastase in rodents as well seemed more desirable for up-
coming in vivo studies with rats and mice. For this reason we
continued our efforts to further optimize potency versus HNE,
hoping that this would ultimately lead to compounds with im-
proved RNE inhibition as well.
X-ray crystallographic investigation of bound ligands
Ligand–protein co-crystallization experiments and modeling
were applied to design better HNE binders within our 1,4-dihy-
dropyrimidinone series: X-ray crystallography showed the an-
ticipated folding structure for HNE, typical for chymotrypsin-
like serine proteases. Upon binding, the equatorial C2 carbonyl
moiety of ligand 19 formed a strong hydrogen bond to
Val216[26] (Figure 3a). More significantly, binding of the inhibi-
tor 19 was driven by its shape complementarity with the bind-
ing subsites of HNE. The clamp-like ligand 19 fits perfectly into
the S1 and S2 subsites of HNE, with both phenyl moieties per-
pendicularly directed away from the central core (Figure 3b).
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Figure 3. Induced-fit binding mode. Protease (HNE) residues are shown in stick representation (white) with transparent Connolly-like surface.[28,29] Ligand 19
(purple) is shown in ball-and-stick model (oxygen: red, nitrogen: blue, fluorine: cyan); hydrogen bonds are depicted as broken yellow lines. a) Structure of
HNE in complex with 19. Ligand 19 interacts with HNE by a hydrogen bond (3.1 Š) formed between the C2 carbonyl oxygen atom of the central pyrimidine
ring and the Val216 backbone amide of HNE. b) Binding to the S1 and S2 subsites is governed by exact protein–ligand shape complementarity of the north-
ern and southern phenyl spheres of 19. c) Binding conformation of 19 overlaid on the binding site of apo-HNE. In the apo structure the S2 subsite next to
Leu99 is not large enough to accommodate the northern para-cyanophenyl ring. Binding is only possible through an induced-fit mechanism, in which
Leu99B is rotated toward the bulk solvent, thus expanding the lipophilic S2 pocket.
In particular, the hydrophobic S1 subsite was fully occupied by
the southern meta-(trifluoromethyl)phenyl moiety of ligand 19.
In contrast, the S2 subsite found in the apo structure of HNE
was at best a shallow groove, clearly not providing sufficient
room for binding of the northern para-cyanophenyl moiety;
however, in the ligand-bound structure, Leu99B had been
moved toward the bulk solvent, thus expanding the lipophilic
S2 to a larger subsite, ideally suited to accommodate the large
northern cyanophenyl residue (Figure 3c). In our complete
series of co-crystallization structures we observed an induced-
fit binding mode.[27] All bound inhibitors from the dihydropyri-
midinone series were characterized by rotational dihedral
angles of the northern cyanophenyl moiety of ~908–1358
(Figure 4) with a preference of dihedral angles of ~1108 (mean:
111.68).
Strategy to tune conformational space
Locking the dihedral angle of the northern phenyl substituent
into the preferred bioactive conformation (Figure 4) was envis-
aged as a design strategy to improve binding efficiency. As
a base case, we assessed conformational freedom of the N3-
and C2’-unsubstituted system 22 with relaxed scan, density-
functional calculations.[30] Rotation appeared to be nearly un-
hindered, with a barrier of ~10 kJmolÀ1 and a lowest-energy
conformation at a (suboptimal) dihedral angle of ~1408
Figure 4. Conformational analysis of HNE-bound ligands based on X-ray crystallographic data (co-crystallization structures). a) Overlay of a series of ligands
bound to HNE. b) Rotational dihedral angles observed in this series of crystallized 1,4-dihydropyrimidinones in protein–ligand complexes. See reference [27]
for the X-ray data of 17. c) The dihedral angle was measured at the northern cyanophenyl moiety along N3ÀC4ÀC1’ÀC2’ (highlighted in red), as indicated for
compound 19.
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Synthesis and assessment of C2’-north substituted
systems
The synthesis of C2’-north-substituted systems
turned out to be challenging (Table 5). Benzalde-
hydes with +M substituents at the ortho position
proved to be less reactive starting materials for the
Biginelli reaction. Accordingly, electron-donating
substituents had to be avoided at the pyrimidine-
forming stage of the synthesis for compounds 25–
30 (Supporting Information).
Whereas N3 alkylation (22!25) only improved
potency twofold, trifluoromethylation at C2’-north
(22!26) advanced the IC50 by a factor of eight. Yet,
the combination of both substituents at N3 and C2’
(22!27) boosted potency by more than two orders
of magnitude in a synergistic fashion, validating our
design hypothesis. The double conformational lock
resulted in high lipophilic binding efficiency (LipE=
7.0). Still, compound 27 was not an ideal candidate,
with logD>3 (at pH 7.5). Therefore, we decided to
replace the lipophilic trifluoromethyl group by
a more polar, less lipophilic alternative while retain-
ing the double conformational lock.
Figure 5. Locking the bioactive conformation with substituents at N3 and C2’. Conforma-
tional analysis of free ligands based on modeling. Relaxed coordinate scan of the rota-
tion of the cyanophenyl moiety of 22 and 27 from 08 to 1808 in steps of 28. Depicted is
the dihedral angle along N3ÀC4ÀC1’ÀC2’. Whereas the unsubstituted system 22 has very
low rotation barriers around the ‘non-ideal’ dihedral angle of 1408, the substituted
system 27 is locked with significantly higher rotation barriers at 1208, which is close to
the ‘ideal’ dihedral binding angle of 1108. Energies were calculated using the B3LYP/6-
31G* density functional technique.[30]
(Figure 5).
Apparently,
this
Table 5. Conformational tuning at N3 and C2’ north: effect on lipophilic binding efficiency.
system in its free state was not
pre-oriented for HNE binding.
Next, the more hindered N3-
methylated and C2’-trifluorome-
thylated system 27 was ana-
lyzed. According to our protein
X-ray structures, substitution at
C2’ of the northern cyanophenyl
sphere (’C2’-north’) was consid-
ered to be feasible. This position
projected into the solvent and
accordingly was not likely to
compromise the binding mode;
instead, it was well suited to in-
troduce a large rotational barrier
at the C4ÀC1’ axis. Additional
substituents at N3 (and C5) of
the dihydropyrimidinone core
would reinforce that effect by
further locking the system at
the equatorial–northern inter-
phase. According to our calcula-
tions, ligand 27 was expected to
have significantly less rotational
freedom along the C4ÀC1’ axis,
pre-orienting the system with
Compd
R1
R2
HNE
logD[b]/ClogD[c]
LipE[d]
CYP 2C9/3A4
IC50 [nm][a]
IC50 [mm][e]
22
25
26
27
H
Me
H
Me
H
H
H
CF3
CF3
SO2Me
SO2Me
4.7
1.6
0.6
0.024
0.540
0.065
2.8[b]
3.6[c]
3.2[b]
3.7[b]
2.7[b]
3.0[b]
5.6
5.2
6.0
7.0
7.2
7.2
18/>50
23/40
>50/>50
–/>20
>50/>50
>50/>50
28
BAY 85-8501 (29)
Me
30
Me
0.250
3.3[c]
6.3
>50/>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] logD (pH 7.5) was determined by reversed-phase HPLC at physiological pH 7.5. A
series of standards were injected for which logD has already been determined using definitive analytical meth-
ods (a homologous series of n-alkanones). Plotting of the retention times against their logD generated a cali-
bration curve. The retention time of the test compound was then compared with the calibration curve leading
to its logD. [c] ClogD (pH 7.5) was calculated by using a highly predictive method developed at Bayer, based
on data points of experimentally determined logD values of internal pharmaceutical compounds and the Simu-
lations Plus pKa predictor.[23] [d] Calculated as LipE=pIC50ÀlogD.[24] [e] The potency of test compounds to inhib-
it human CYP2C9 and CYP3A4 was investigated with pooled human liver microsomes as enzyme source and
selective standard substrates (Supporting Information); IC50 values were derived from enzyme activity data
(pH 7.4) in the presence/absence of various compound concentrations and diclofenac/midazolam as selective
CYP2C9/CYP3A4 substrate.
a
rotational
barrier
of
>40 kJmolÀ1 at a dihedral angle
of ~1208, very close to the
‘ideal’ bioactive dihedral angle
of ~1108.
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quently achieved by HPLC on chiral phase. The
cyano group at the dihydropyrimidinone was instal-
led from carboxylic acid 37 via amide 38 by dehydra-
tion with the Burgess reagent.
For a better understanding of the binding mode
with our novel conformationally locked systems, 28
was co-crystallized with HNE (Figure 6), which re-
vealed a binding mode nearly identical to that of
ligand 19 (Figure 3). The N3ÀC4ÀC1’ÀC2’ dihedral
angle of 109.58 was very close to the assumed opti-
mum of 1108 (Supporting Information). The sulfone
moiety pointed outward from the active site while
one of its oxygen atoms was hydrogen bonded to
a water molecule, gaining further binding energy.
In-depth in vitro testing of BAY-678 and BAY 85-
8501
We ran the biochemical inhibition assay for BAY 85-
8501 also in the presence of 1 mm hydrogen perox-
ide to mimic in vivo conditions of oxidative stress in
an inflammatory environment. Under these harsh cir-
cumstances, the IC50 only shifted by a factor of two
toward 140 pm, indicating good oxidative stability.
To confirm the exceptionally high potency of BAY-
678 (20) and BAY 85-8501 (29), we measured enzyme
reaction velocities with different substrate concentra-
tions at various inhibitor concentrations and extrapo-
lated the inhibition constants (Ki) from Dixon plots
(Supporting Information). Both compounds revealed
(substrate) competitive inhibition, further confirming
their binding into the active site of the enzyme.
However, the Ki values toward rodent orthologous
enzymes were about two orders of magnitude
higher than toward HNE (Table 6). Beneficially, BAY-
678 and BAY 85-8501 revealed no inhibition against
21 related serine proteases, up to an inhibitor con-
centration of 30 mm (Table 6).
Scheme 1. Synthesis of BAY 85-8501 (29). Reagents and conditions: a) mCPBA, CH2Cl2,
108C!RT, 93%; b) N,N-dimethylformamide dimethyl acetal, DMF, 1408C, 98% (crude
product); c) sodium periodate, H2O/THF (1:1), RT, 65%; d) triethyl phosphate, phosphorus
pentoxide, 508C, then 1-[3-(trifluoromethyl)phenyl]urea, allyl acetoacetate, reflux, 64%;
e) preparative HPLC, eluent: isohexane/isopropanol (1:1), selector poly(N-methacryloyl-d-
leucine-dicyclopropylmethylamide), 69%; f) morpholine, tetrakis(triphenylphosphine)pal-
ladium(0) (0.05 equiv), THF, RT, 81%; g) HATU, DMF, NH4Cl, N,N-diisopropylethylamine,
08C!RT, 88%; h) Burgess reagent [(methoxycarbonylsulfamoyl)triethylammonium hy-
droxide], THF, RT, 87%; i) LiHMDS, CH3I, THF, À788C!RT, 96%. DMF=N,N-dimethylforma-
mide; LiHMDS=lithium bis(trimethylsilyl)amide; THF=tetrahydrofuran; HATU = 1-[bis(di-
methylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate.
Next we investigated the binding kinetics of
BAY 85-8501 (Table 7). BAY 85-8501 showed a long
residence time of ~17 min. According to published
data,[33] 29 binds to HNE as rapidly as the endoge-
Indeed, with a sulfone group, potency could again be ad-
vanced by a factor of ten (22!28). Combination of the C2’-
sulfone with a methyl group at N3 enhanced potency by
nearly two orders of magnitude relative to 22, yielding BAY 85-
8501 (29, HNE IC50: 65 pm) with a formidable lipophilic binding
efficiency (LipE 7.2). The C2’-north position also tolerated the
slightly basic sulfoximine[31] residue, yielding compound 30
with improved solubility (Table 5). Due to its overall balanced
technical profile, BAY 85-8501 (29) was selected for in-depth in
vitro and in vivo testing (see below).
nous a-proteinase inhibitor (aPI). The latter, however, being
a protein, shows a much longer residence time, leading to
pseudo-irreversible binding characteristics.
Pharmacokinetic studies
Various dihydropyrimidinones showed overall promising phar-
macokinetic data in rodents (Table 8). While both early dihy-
dropyrimidinones 17 and 20 revealed medium clearance in ro-
dents as a result of oxidation at C4, the additional electron-
withdrawing effect of the sulfone C2’-north substituent in
BAY 85-8501 resulted in metabolic stabilization of the drug.
BAY 85-8501 (29) displayed low clearance and improved half-
life in rats, and no inhibitory potency toward CYP isoforms
(Tables 5 and 8).
BAY 85-8501 was synthesized in a nine-step sequence, with
deliberate introduction of the electron-withdrawing sulfone
substituent prior to the Biginelli reaction in order to increase
electrophilicity and reactivity of the corresponding benzalde-
hyde 34 (Scheme 1). Separation of enantiomers 35 was subse-
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Table 8. Pharmacokinetic profile of selected dihydropyrimidinones in
rats.[a]
[b]
[c]
Compd
CLp [LhÀ1 kgÀ1
]
VSS [LkgÀ1
]
t1/2 [h]
F [%][d]
17
20
29
2.1
2.0
0.5
4.7
3.9
5.8
2.6
1.3
8.5
43
83
63[e]
[a] Mean values were derived by intravenous (0.25–2 h infusion) and oral
(gavage) administration of 0.3 mgkgÀ1 in EtOH/PEG400/H2O vehicles.
[b] Total plasma clearance. [c] Apparent steady-state volume of distribu-
tion. [d] Oral bioavailability. [e] From Tylose (0.5%) suspension.
In vivo pharmacodynamic studies: acute lung injury (ALI)
A main role of elastase inhibitors in lung diseases could be the
prevention of lung injury driven by chronic inflammation
(Figure 1). We set up a rapid, preventive in vivo model that
was designed to reflect basic aspects of lung diseases, such as
ALI, by combining an exogenous ‘noxa’ [triggered by HNE or
porcine pancreatic elastase (PPE)] with an endogenous inflam-
mation that developed over time and was driven by murine
neutrophil elastase (MNE).
Figure 6. Co-crystallization of 28 with HNE. Protease residues are shown in
stick representation with transparent Connolly-like surface; ligand 28 is
shown in ball-and-stick representation (oxygen: red, nitrogen: blue, fluorine:
cyan, sulfur: yellow). Ligand 28 has a dihedral angle of 109.58 at the north-
ern cyanophenyl moiety along N3ÀC4ÀC1’ÀC2’. The image was generated
with PyMOL.[29]
Intratracheal instillation of HNE into the lungs of mice
caused severe injury, leading to lung hemorrhage and inflam-
mation that were quantified 1 h after the HNE challenge by
measuring hemoglobin concentrations and neutrophil count in
the bronchoalveolar lavage fluid (BALF; Figure 7a, 1st). In this
model the exogenous HNE noxa was the primary cause of
injury and lung hemorrhage. Accordingly, the degree of pri-
mary injury was directly dependent on the amount of HNE
given. However, the subsequent inflammation that was primar-
ily driven by the endogenous MNE also contributed to secon-
dary injury effects, developing over time. Based on picomolar
potency against HNE as well as single digit potency versus
MNE, BAY 85-8501 (29) completely prevented the development
of lung injury and subsequent inflammation when adminis-
tered 1 h prior to the HNE noxa. In the 0.01 mgkgÀ1 dose
group, hemoglobin concentration was already significantly de-
creased (Figure 7b). At a dose of 0.1 mgkgÀ1, a significant
effect on neutrophil count was observed (Figure 7c). In this
setup, efficacy was predominantly driven by potency against
HNE (Ki =0.08 nm).
Table 6. Species selectivity: in vitro potency of BAY-678 (20) and BAY 85-
8501 (29).
Compd
Ki [nm][a]
RNE
Ser protease
panel IC50 [nm][c]
HNE
MNE[b]
20
29
15
0.08
600
8.0
700
6.0
>30000
>30000
[a] Ki values were extrapolated from Dixon plots (Supporting Information).
As expected, Ki values showed good correlation to the IC50 values.[24]
[b] Murine neutrophil elastase. [c] IC50 values for 21 related serine proteas-
es, including porcine pancreatic elastase (PPE), were determined by ap-
plying functional biochemical assays with the respective isolated enzyme
and the appropriate fluorogenic peptide substrate (Supporting Informa-
tion).
Table 7. Enzyme binding kinetics of BAY 85-8501 (29) and the endoge-
nous a-proteinase inhibitor (aPI).
Next we changed the experimental setup to an exogenous
PPE noxa (Figure 7a, 2nd): Intratracheal instillation of PPE
caused severe lung hemorrhage and inflammation which were
quantified by measuring hemoglobin concentrations and neu-
trophil count in BALF 1 h after the noxa. As the highly HNE-se-
lective inhibitor BAY 85-8501 had no effect on PPE, BAY 85-
8501 could not prevent the primary lung injury in this setup.
Nevertheless, BAY 85-8501 could inhibit MNE, the endogenous
driver of inflammation and secondary injury, although with de-
creased potency. Consequently, the effects of BAY 85-8501 on
inflammation and secondary injury were weaker at this point,
and only observed at 30-fold higher doses (Figure 7d,e). Effica-
cy was predominantly driven by potency against MNE (Ki =
6 nm) in this second setup. Ultimately, in vitro potency against
[a]
[b]
Compd
k
on [106 mÀ1 sÀ1
]
koff [10À3 sÀ1
]
Residence
time [h]
29
aPI
12.6
14.5[c]
1.0
0.0002[d]
~0.3
~1900[e]
[a] The on-rates at which elastase inhibitors bind to the target were de-
termined by applying a functional biochemical assay using a substrate
with a modified fluorescent label, MeOSuc-AAPV-umbelliferyl; this allows
very sensitive detection of substrate hydrolysis on the millisecond time-
scale, in the presence or absence of elastase inhibitor. Using nonlinear re-
gression of the reaction progress curves, the observed rate constant of
the onset of inhibition (kobs) was obtained and plotted against the inhibi-
tor concentration. The slope of the linear regression revealed the estimat-
ed kon value (Supporting Information). [b] Calculated from kon and Ki ac-
cording to the equation koff =kon ”Ki. [c] Data from Sinden et al.[33] [d] Cal-
culated with Ki ~1”10À14 m, taken from Beatty et al.[32] [e] Calculated ac-
cording to target residence time, 1/koff
.
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Figure 7. Acute lung injury (ALI) in vivo model in mice. a) Schematic representation of the experimental rationale. 1st: HNE-induced ALI model: BAY 85-8501
(29) has a strong inhibitory effect on the exogenous noxa from HNE (Ki for HNE=0.08 nm), which primarily triggers injury. BAY 85-8501 has only a medium
effect on murine neutrophil elastase (MNE; Ki for MNE=6 nm), which is the primary endogenous driver of inflammation and only a secondary driver of lung
injury. 2nd: Porcine pancreatic elastase (PPE)-induced ALI model: BAY 85-8501 has no inhibitory effect on the exogenous noxa from PPE, which primarily trig-
gers injury in this second setup. Again, BAY 85-8501 has a medium effect on MNE, which is the primary endogenous driver of inflammation and a secondary
driver of injury. b) Hemoglobin concentration after HNE noxa as a primary quantification of injury; data are the mean ÆSEM, n=4–13, *p<0.05. c) Neutrophil
count after HNE noxa as a primary quantification of inflammation; data are the mean ÆSEM, n=4–13, *p<0.05. d) Hemoglobin concentration after PPE noxa
as a primary quantification of injury; data are the mean ÆSEM, n=4–13, *p<0.05. e) Neutrophil count after PPE noxa as a primary quantification of inflamma-
tion; data are the mean ÆSEM, n=4–13, *p<0.05.
HNE and MNE translated nicely into in vivo efficacy in our
acute lung injury model.
induced-fit binding mode. Electronic modulation of the north-
ern hemisphere improved in vitro and in vivo pharmacokinet-
ics data significantly. BAY 85-8501 (29) has shown in vivo effica-
cy in various preclinical animal models, results that will be pub-
lished in due course. BAY 85-8501 is currently in clinical testing
for the treatment of pulmonary diseases.
Conclusions
In summary, we have evolved a sub-micromolar lipophilic
screening hit into a highly selective picomolar HNE inhibitor
with lower molecular weight (Scheme 2). Boosting of the lipo-
philic binding efficiency by nearly five was possible by locking
the bioactive conformation of our pyrimidinone lead series on
the basis of a thorough conformational understanding of the
Experimental Section
Allyl (rac)-4-[4-cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-
1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-car-
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layer was dried over Na2SO4 and concentrated. The residue was pu-
rified by preparative HPLC (column: GromSil C18, 10 mm; eluent:
CH3CN/H2O+0.1% TFA 10:90!90:10). Yield: 696 mg (94%);
1H NMR (400 MHz, [D6]DMSO): d=2.14 (s, 3H), 3.45 (s, 3H), 6.35 (d,
1H), 7.14 (d, 1H), 7.72 (m, 2H), 7.80 (m, 1H), 7.86 (s, 1H), 8.11 (d,
1H), 8.27 (d, 1H), 8.36 (s, 1H), 12.64 (brs, 1H) ppm; MS (ESI+) m/z:
480.1 [M+H]+; MS (ESIÀ) m/z: 478.1 [MÀH]À.
(4S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-1-[3-
(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carboxa-
mide (38): The reaction was carried out under argon. 37 (696 mg,
1.45 mmol) and HATU (1.1 g, 2.9 mmol, 2 equiv) were dissolved in
anhydrous DMF (35 mL) at 08C. After 20 min, NH4Cl (388 mg,
7.26 mmol, 5 equiv) and N,N-diisopropylethylamine (1.31 g,
10.16 mmol, 7 equiv) were added. The mixture was stirred at room
temperature for 4 h, and was then concentrated. The residue was
purified by preparative HPLC (column: GromSil C18, 10 mm; eluent:
CH3CN/H2O+0.1% TFA 10:90!90:10). Yield: 612 mg (88%);
1H NMR (400 MHz, [D6]DMSO): d=1.80 (s, 3H), 3.40 (s, 3H), 6.35 (s,
1H), 7.20 (s, 1H), 7.25 (brs, 1H), 7.45 (brs, 1H), 7.65–7.80 (m, 4H),
8.10 (d, 1H), 8.30 (s, 1H), 8.35 (d, 1H) ppm; MS (ESI+) m/z: 479.1
[M+H]+; MS (ESIÀ) m/z: 477 [MÀH]À.
Scheme 2. Summary of the optimization path.
boxylate (35): The reaction was carried out under argon. Triethyl
phosphate (22.98 g, 126 mmol) and phosphorus pentoxide
(11.94 g, 84.1 mmol) were stirred at 508C overnight. The mixture
was diluted with tert-butyl methyl ether (MTBE; 450 mL). 4-Formyl-
3-(methylsulfonyl)benzonitrile (22.00 g, 105 mmol), 1-[3-(trifluoro-
methyl)phenyl]urea (21.47 g, 105 mmol) and allyl acetoacetate
(22.42 g, 158 mmol) were added. The reaction mixture was heated
at reflux overnight. MTBE (350 mL) was removed by distillation.
The residue was heated at reflux for a further 4 h. The organic sol-
vents were removed. The residue was suspended in Et2O and fil-
tered. The solid was washed with H2O (350 mL) and Et2O (50 mL).
Yield: 34.74 g (64%); 1H NMR (400 MHz, [D6]DMSO): d=2.15 (s,
3H), 3.45 (s, 3H), 4.45 (m, 2H), 4.95 (d, 1H), 5.05 (d, 1H), 5.65 (m,
1H), 6.40 (d, 1H), 7.20 (d, 1H), 7.70 (m, 2H), 7.80 (m, 1H), 7.85 (brs,
1H), 8.10 (brd, 1H), 8.25 (d, 1H), 8.35 (s, 1H) ppm; MS (ESI+) m/z:
520.2 [M+H]+.
Method A: (4S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-
oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-
5-carbonitrile (28): The reaction was carried out under argon. 38
(560 mg, 1.17 mmol) was dissolved in anhydrous THF (35 mL). (Me-
thoxycarbonylsulfamoyl)triethylammonium hydroxide (Burgess re-
agent; 1115 mg, 4.68 mmol, 4 equiv) was added, and the mixture
was stirred at room temperature for 90 min. The solvent was re-
moved in vacuo. The residue was purified by preparative HPLC
(column: GromSil C18, 10 mm; eluent: CH3CN/H2O+0.1% TFA
10:90!90:10). Yield: 470 mg (87%).
Method B: (4S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-
oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-
5-carbonitrile (28): Compound 38 (10.4 g, 21.7 mmol) and trime-
thylamine (5.63 g, 55.6 mmol) were dissolved in anhydrous THF
(50 mL). Trifluoroacetic anhydride (11.69 g, 55.6 mmol) was added
dropwise, ensuring a temperature <358C. The reaction mixture
was stirred at room temperature for 15 min. A saturated aqueous
solution of NaHCO3 (250 mL) was added dropwise. The mixture
was extracted twice with EtOAc. The combined organic layers were
washed with brine and dried over MgSO4. Silica gel (30 g) was
added, and the mixture was concentrated. The residue was purified
by column chromatography on silica gel (CH2Cl2/EtOAc 2:1). Yield:
6.46 g (65%); mp: 258–2598C; [a]D20 =À222.0 (c=0.480 in DMF);
1H NMR (400 MHz, [D6]DMSO): d=1.80 (s, 3H), 3.40 (s, 3H), 6.45 (s,
1H), 7.70–7.85 (m, 3H), 7.95 (brs, 1H), 8.30–8.40 (m, 4H) ppm; MS
(ESI+) m/z: 461.1 [M+H]+; MS (ESIÀ) m/z: 459.2 [MÀH]À.
Allyl (4S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-
1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-car-
boxylate (36): Compound 35 (2.33 g) was separated into enantio-
mers by preparative chiral-phase HPLC [column: chiral silica gel
phase based on the selector poly(N-methacryloyl-d-leucine-dicyclo-
propylmethylamide), 600 mm”30 mm, 10 mm; sample preparation:
each 1 g of the sample was dissolved in THF/EtOAc/isohexane
(20:25:25, 70 mL); injection volume: 8 mL; eluent: isohexane/iso-
propanol 1:1; flow: 60 mLminÀ1; T: 248C; UV detection: l=
260 nm]. Yield: 0.8 g (69%, >99.5% ee); LC–MS (method 14, Sup-
porting Information): tR =2.11 min; chiral analytical HPLC [column:
chiral silica gel phase based on the selector poly(N-methacryloyl-d-
leucine-dicyclopropylmethylamide),
250 mm”4.6 mm,
5 mm;
eluent: isohexane/EtOAc 1:1; flow: 2 mLminÀ1
;
UV detection:
l 260 nm]: tR =1.45 min; [a]D20 = +41.6 (c=0.485 in MeOH);
1H NMR (400 MHz, [D6]DMSO): d=2.10 (s, 3H), 3.45 (s, 3H), 4.45 (m,
2H), 4.95 (d, 1H), 5.05 (d, 1H), 5.65 (m, 1H), 6.40 (d, 1H), 7.20 (d,
1H), 7.70 (m, 2H), 7.80 (m, 1H), 7.85 (brs, 1H), 8.10 (brd, 1H), 8.25
(d, 1H), 8.35 (s, 1H) ppm; MS (ESI+) m/z: 520.1 [M+H]+; MS
(ESIÀ) m/z: 518.2 [MÀH]À.
Method A: (4S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-3,6-dimeth-
yl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimi-
dine-5-carbonitrile (BAY 85-8501, 29): The reaction was carried
out under argon. 28 (75 mg, 163 mmol) was dissolved in THF
(2 mL), and NaH suspension in mineral oil (60%; 9.2 mg, 228 mmol)
was added. After stirring at room temperature for 20 min, CH3I
(32.4 mg, 14.2 mL, 228 mmol) was added. The reaction mixture was
stirred at room temperature for 120 min. The mixture was purified
directly by preparative HPLC (column: Kromasil-100A C18,
250 mm”4.6 mm, 5 mm; eluent: CH3CN/H2O+0.1% TFA 10:90!
80:20). Yield: 18 mg (23%).
(4S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-6-methyl-2-oxo-1-[3-
(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbox-
ylic acid (37): The reaction was carried out under argon. 36
(800 mg, 1.54 mmol) and morpholine (201 mg, 2.31 mmol,
1.5 equiv) were dissolved in anhydrous THF (25 mL) at room tem-
perature.
Tetrakis(triphenylphosphine)palladium(0)
(89 mg,
0.077 mmol, 0.05 equiv) was added. The mixture was stirred at
room temperature for 90 min, then concentrated. The residue was
dissolved in EtOAc (500 mL) and washed with saturated aqueous
NH4Cl solution (50 mL), H2O (50 mL) and brine (50 mL). The organic
Method B: (4S)-4-[4-Cyano-2-(methylsulfonyl)phenyl]-3,6-dimeth-
yl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimi-
dine-5-carbonitrile (BAY 85-8501, 29): The reaction was carried
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[14] B. G. Zeiher, S. Matsuoka, K. Kawabata, J. Repine, Crit. Care Med. 2002,
30, S281–S287.
[15] S. Tamakuma, M. Ogawa, N. Aikawa, T. Kubota, H. Hirasawa, A. Ishizaka,
N. Taenaka, C. Hamada, S. Matsuoka, T. Abiru, Pulm. Pharmacol. Ther.
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[16] B. G. Zeiher, A. Artigas, J.-L. Vincent, A. Dmitrienko, K. Jackson, B. T.
Thompson, G. Bernard, Crit. Care Med. 2004, 32, 1695–1702.
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out under argon. 28 (460.4 mg, 1 mmol) was dissolved in absolute
THF (10 mL) and cooled to À788C. A solution of LiHMDS (1m in
THF; 1 mL, 1 mmol, 1 equiv) was added dropwise. After stirring for
20 min, CH3I (710 mg, 5 mmol, 5 equiv) was added. The mixture
was allowed to warm to room temperature over 60 h. The mixture
was purified directly by preparative HPLC (column: GromSil C18,
10 mm; eluent: CH3CN/H2O+0.1% TFA 10:90!90:10). Yield:
1
454 mg (96%); H NMR (400 MHz, [D6]DMSO): d=1.80 (s, 3H), 2.65
(s, 3H), 3.40 (s, 3H), 6.45 (s, 1H), 7.65–8.40 (m, 6H), 8.45 (s, 1H)
ppm; MS (ESI+) m/z: 475.0 (100) [M+H]+; MS (ESIÀ) m/z: 473.2
[MÀH]À.
The crystallographic data for the four protein co-crystallization
structures with 19, 20, 24, and 28 have been deposited at the
RCSB Protein Data Bank (PDB) with the respective access codes
5A09, 5A0A, 5A0B, and 5A0C. All in vivo procedures conformed to
European Community directives and national legislation (German
law for the protection of animals) for the use of animals for scien-
tific purposes and were approved by the competent regional au-
thority. All other experimental data including chemical procedures
and analytics are provided in the Supporting Information.
[23] R. Fraczkiewicz, M. Lobell, A. H. Gçller, U. Krenz, R. Schoenneis, R. D.
Clark, A. Hillisch, J. Chem. Inf. Model. 2015, 55, 389–397.
[24] We calculated the lipophilic binding efficiency as LipE=pIC50ÀlogD, as-
1
suming that according to the Cheng–Prusoff equation Ki ꢀ = IC50 if
2
[S]=Km. See also: M. D. Shultz, Bioorg. Med. Chem. Lett. 2013, 23, 5980–
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Keywords: biginelli reaction · biological activity · elastase
inhibitors · proteases · pyrimidinones
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Published online on June 17, 2015
ChemMedChem 2015, 10, 1163 – 1173
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1173 ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim