Discovery of BMS-986235/LAR-1219: A Potent Formyl Peptide Receptor 2 (FPR2) Selective Agonist for the Prevention of Heart Failure

Article abstract of DOI:10.1021/acs.jmedchem.9b02101

Formyl peptide receptor 2 (FPR2) agonists can stimulate resolution of inflammation and may have utility for treatment of diseases caused by chronic inflammation, including heart failure. We report the discovery of a potent and selective FPR2 agonist and its evaluation in a mouse heart failure model. A simple linear urea with moderate agonist activity served as the starting point for optimization. Introduction of a pyrrolidinone core accessed a rigid conformation that produced potent FPR2 and FPR1 agonists. Optimization of lactam substituents led to the discovery of the FPR2 selective agonist 13c, BMS-986235/LAR-1219. In cellular assays 13c inhibited neutrophil chemotaxis and stimulated macrophage phagocytosis, key end points to promote resolution of inflammation. Cardiac structure and functional improvements were observed in a mouse heart failure model following treatment with BMS-986235/LAR-1219.

Full text of DOI:10.1021/acs.jmedchem.9b02101

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Drug Annotation
Discovery of BMS-986235/LAR-1219: A Potent Formyl Peptide
Receptor 2 (FPR2) Selective Agonist for the Prevention of Heart Failure
Yoshikazu Asahina, Nicholas R. Wurtz, Kazuto Arakawa, Nancy Carson, Kiyoshi Fujii, Kazunori
Fukuchi, Ricardo A. Garcia, Mei-Yin Hsu, Junichi Ishiyama, Bruce Ito, Ellen K Kick, John Lupisella,
Shingo Matsushima, Kohei Ohata, Jacek Ostrowski, Yoshifumi Saito, Kosuke Tsuda, Francisco J
Villarreal, Hitomi Yamada, Toshikazu Yamaoka, Ruth R. Wexler, David A. Gordon, and Yasuchi Kohno
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02101 • Publication Date (Web): 14 May 2020
Downloaded from pubs.acs.org on May 15, 2020
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Discovery of BMS-986235/LAR-1219: A Potent
Formyl Peptide Receptor 2 (FPR2) Selective
Agonist for the Prevention of Heart Failure
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†
,‡
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Yoshikazu Asahina* , Nicholas R. Wurtz* , Kazuto Arakawa , Nancy Carson , Kiyoshi
†
†
‡
‡
†
#
Fujii , Kazunori Fukuchi , Ricardo Garcia , Mei-Yin Hsu , Junichi Ishiyama , Bruce Ito ,
‡
‡
†
†
‡
Ellen Kick , John Lupisella , Shingo Matsushima , Kohei Ohata , Jacek Ostrowski ,
†
†
#
†
Yoshifumi Saito , Kosuke Tsuda , Francisco Villarreal , Hitomi Yamada , Toshikazu
†
‡
‡
†
Yamaoka , Ruth Wexler , David Gordon , Yasushi Kohno .
†Discovery Research Laboratories, Kyorin Pharmaceutical Co. Ltd., 2399-1, Nogi, Nogi-
‡
Machi, Shimotsuga-Gun, Tochigi, 329-0114, Japan. Bristol-Myers Squibb Research
and Development, P.O. Box 5400, Princeton, New Jersey 08534, United States.
#Department of Medicine, University of California, San Diego, 9500 Gilman Drive La
Jolla, CA 92093.
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KEYWORDS: Formyl Peptide Receptor, Resolution of Inflammation, Heart Failure,
Conformational Restriction, GPCR Agonist
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ABSTRACT: Formyl Peptide Receptor 2 (FPR2) agonists can stimulate resolution of
inflammation and may have utility for treatment of diseases caused by chronic
inflammation, including heart failure. We report the discovery of a potent and selective
FPR2 agonist, and its evaluation in a mouse heart failure model. A simple linear urea with
moderate agonist activity served as the starting point for optimization. Introduction of a
pyrrolidinone core accessed a rigid conformation that produced potent FPR2 and FPR1
agonists. Optimization of lactam substituents led to the discovery of the FPR2 selective
agonist 13c, BMS-986235/LAR-1219. In cellular assays 13c inhibited neutrophil
chemotaxis and stimulated macrophage phagocytosis, key endpoints to promote
resolution of inflammation. Cardiac structure and functional improvements were
observed in a mouse heart failure model following treatment with BMS-986235/LAR-
1219.
Introduction
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Formyl peptide receptors (FPRs) constitute a G-protein coupled chemotactic receptor
GPCR) family, which are mainly expressed in neutrophils and monocytes and play an
(
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important role in host defense and inflammation. In humans, three subtypes (FPR1, 2,
) have been identified.1−3 FPR1 initiates inflammatory responses such as neutrophil
3
chemotaxis, degranulation, respiratory burst, and cytokine release when activated with
ligands such as N-formyl-methionyl-leucyl-phenylalanine (fMLF) produced by bacteria.4
FPR2 is involved in both initiation and resolution of inflammation. FPR2 peptide ligands,
such as serum amyloid A (SAA), are associated with production of pro-inflammatory
5
cytokine interleukin (IL)-8, while ligands such as Lipoxin A4 and Annexin A1 have been
shown to inhibit migration of neutrophils, differentiate macrophages into pro-wound
healing phenotypes and upregulate anti-inflammatory cytokines such as IL-10.6,7 Thus,
FPR2 activates both inflammatory and pro-resolution responses depending on the ligand
and its binding regions.8,9
10
Since the first small-molecule FPR agonist was reported in 2004, research groups in
academia and industry have reported a diverse set of FPR agonists and antagonists.11−14
Representative FPR agonists include pyrazolone ureas (Compound 43),15,16
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benzimidazoles,17 bridged spiro[2.4]heptanes18 and aminotriazoles (ACT-389949)19
(Figure 1). Among these compounds, dual FPR2/FPR1 Compound 43 has been used for
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proof of concept in vivo studies, including the preservation of cardiac function and
prevention of adverse remodeling in rodent heart failure models.8,16,20 FPR2 agonist ACT-
389949 was dosed in Phase 1 clinical trials and induced plasma biomarker changes (e.g.
IL-10 and leukocyte levels) in humans after one dose; however, the authors reported
tachyphylaxis in the multiday dosing study.21 Although there are no compounds launched
as therapeutic agents to date, FPR2 remains a promising target to resolve chronic
inflammation and promote the wound healing processes.
Based on reported FPR biology, we focused on the discovery of FPR2 agonists with
pro-resolution activity and selectivity over FPR1. We selected simple urea derivative AG-
26 (1)22 as a promising starting point with multiple options for future structural
transformation.
Figure 1. Structures of selected reported FPR2 agonists
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Amgen (2006)
Pyrazolone Urea
(
Amgen (2007)
Benzimidazole
Me
Actelion (2013)
compound 43)
F
CH3
Bridged spiro[2,4]heptane
F
N
Cl
O
O
N
O
N
N
N
H
N
N
H
Me
N
H
O
Me
OMe
Actelion (2010)
Aminotriazole
ACT-389949)
AG-26 (1)
(
Investigation of substituents on the 2-(4-methoxypheny)ethyl moiety of compound 1
resulted in carbamoyl derivative 2d with enhanced FPR2 activity (Figure 2). In order to
investigate conformationally constrained analogs of 2d, four to six membered lactam
derivatives 3−7 were prepared. Among these, 5-membered pyrrolidinone derivatives 5
and 6 showed significantly improved activity. Of four optical isomers of 5 and 6, the
(3S,4R)-form was confirmed to be the most potent FPR2 agonist. Preliminary
2
investigation of the Ar group let to 4-fluorophenyl (3S,4R) analog 11b, which exhibited
1
better activity than the 4-chlorophenyl (3S,4R) analog. Optimization of the Ar group, led
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to 12g bearing the 2,6-difluoro-4-methoxyphenyl group which dramatically improved
selectivity over FPR1 without decreasing FPR2 activity. Finally, reoptimization of the Ar2
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group to further improve FPR2 selectivity resulted in the selection of clinical candidate
13c. Details of this research are described below.
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Figure 2. Structure Activity Relationship (SAR) Summary of Pyrrolidinone Urea FPR2
Agonist
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Figure 2
H
O
N
Introduction of
substituent R
R
Conformational
restriction
H
N
(
Cl
O
NH2
O
O
O
O
)n
(+/-) 5
hFPR2 EC50: 0.91 nM
N
H
N
N
N
H
N
H
N
H
H
H
H
O
N
3
(cis), 4 (trans): n=0
OMe
2d
1
5 (cis), 6 (trans): n=1
7 (trans): n=2
hFPR2 EC50: 990 nM
hFPR2 EC50: 3.6 nM
(+/-) 6
hFPR2 EC50: 0.18 nM
Compounds 2-7 are racemate
Active stereoisomer of 5 and 6
Preliminary SAR of Ar2
Ar2
H
H
H
O
N
F
O
F
O
N
N
O
O
O
N
N
N
H
N
H
N
N
H
F
F
H
H
H
Final Ar2
Optimization
Ar SAR
1
F
F
OMe
OMe
1 OMe
Ar
13c
12g
11b
hFPR2 EC50: 0.41 nM
hFPR1 EC50: 2800 nM
hFPR2 EC50: 0.14 nM
hFPR1 EC50: 280 nM
hFPR2 EC50: 0.085 nM
hFPR1 EC50: 9.0 nM
Results and Discussions
All synthesized compounds were evaluated for agonistic activity to induce Ca2+
mobilization in HEK293 cells over-expressing hFPR2 and HEK293 cells over-expressing
hFPR1.
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First, the hFPR2 agonistic activity of acyclic urea derivatives 1 and 2a−d were evaluated
to determine the effect of substitution on the 4-methoxyphenylethyl moiety (Table 1).
Compound 1 showed weaker hFPR2 activity than that described in the literature, which
may be due to assay conditions and expression levels of the receptor in the reporter cell
lines. By contrast, compounds 2a−d exhibited 32- to 275-fold more potent hFPR2 activity
than 1. Carbamoyl derivative 2d exhibited the strongest hFPR2 activity (EC = 3.6 nM)
1
1
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50
and was selected as a template for structural modifications.
Table 1. Human FPR2 Agonist Activity for Acyclic Analogs
Cl
O
R
N
N
H
H
OMe
2
(racemate)
hFPR2
comp
R
EC50a (nM)
1
H
990
23
2
a
Me
2
b
CH OH
6.8
31
2
2
c
COOH
2
d
CONH2
3.6
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a
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EC values were determined by Ca mobilization in HEK293 cells over-expressing
50
hFPR2.
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We designed 4−6 membered lactam derivatives 3−7 to constrain the conformation of
2
d without changing the steric bulk of the carbamoyl moiety and the lipophilicity of the
molecule. In addition to the hFPR2 activity of compounds 3−7, the agonist activity for
hFPR1 was measured to evaluate receptor selectivity (Table 2). The template compound
2
d had excellent hFPR2 selectivity (hFPR1/2 ratio >2800). The hFPR2 activity of cis- and
trans-oriented azetidinone urea derivatives 3 and 4 were 3- to >28-fold less potent than
2
d. By contrast, cis- and trans-oriented pyrrolidinone urea derivatives and trans-oriented
piperidinone derivative 5−7 were 4- to 33-fold more potent for hFPR2 over 2d. However,
hFPR1 activity of 5−7 also increased, and hFPR1/2 selectivity of these compounds was
lower than that of 2d.
Table 2. Activity of 3−7 in HEK293 Cells Expressing Human FPR2 and FPR1
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H
Cl
O
N
O
( )n
N
N
H
H
1
1
1
1
1
1
1
1
1
1
2
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2
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5
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racemates
OMe
3-7
H
O
a
EC (nM)
N
50
(
)n
comp
2d
ratio
hFPR2 hFPR1
3.6 >10000 >2800
12 >10000 >830
O
O
NH
NH
3
4
5
6
7
>100 >10000
NCc
110
170
O
O
NH
0.91
0.18
0.11
100
31
NH
H
O
N
<10
<91
a
2+
EC values were determined by Ca mobilization in HEK293 cells over-expressing
50
c
hFPR2 or hFPR1. Not calculated.
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6
Based on the results described above, we selected pyrrolidinone derivatives 5 and 6
for further optimization since they offered the best combination of potency and selectivity.
The four optically active isomers 8−10 and 11a were assayed to ascertain the most active
stereoisomer (Table 3). In the cis-oriented isomers, (+)-enantiomer 9 had 5-fold more
potent hFPR2 activity than (-)- 8. In the trans-oriented isomers, (3S,4R)-enantiomer 11a
was 360-fold more potent for hFPR2 than the (3R,4S)-enantiomer 10. Comparing the
hFPR2 activity of diastereomers 9 and 11a, 11a was 10-fold more active than 9,
demonstrating that the (3S,4R)-isomer was the most active stereoisomer. However, 11a
was only moderately selective over FPR1 (42x). Compound 11b bearing a 4-fluorophenyl
group had similar hFPR2 activity and selectivity to 11a, so we selected 11b as the lead
compound for detailed structure activity relationship (SAR) studies to identify a candidate
compound.
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
Table 3. Activity of 8-11 in HEK293 Cells Expressing Human FPR2 and FPR1
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9
H
N
X
O
O
*
*
N
N
H
H
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
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
OMe
optically active 8-11
H
O
a
EC (nM)
N
50
comp
X
ratio
210
*
*
hFPR2 hFPR1
H
N
O
*
8
*
Cl 4.6
950
81
(
-)-cis
H
O
N
*
9
*
Cl 0.90
90
(
+)-cis
H
O
O
O
N
1
0
Cl 34
910
3.9
9.0
27
42
H
N
1
1a
Cl 0.092
H
N
1
1b
F
0.085
110
a
2+
EC values were determined by Ca mobilization in HEK293 cells over-expressing
50
hFPR2 or hFPR1.
After the core pyrrolidinone was selected, optimization of the 4-methoxyphenyl group
was carried out (Table 4). Introduction of a fluorine atom to the 2- or 3-position of the 4-
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5
6
7
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
methoxyphenyl group (12a, 12b) resulted in 5- to 10-fold decreased hFPR2 activity
relative to 11b and did not improve hFPR2 selectivity. Furthermore, 2-chloro (12c), 2-
methyl (12d) or 4-chloro (12e) analogs also had nanomolar hFPR2 activity. By contrast,
compounds 12f and 12g with an additional fluorine atom at position 5 or 6 of 12a
maintained picomolar hFPR2 activity. Remarkably, a dramatic decrease in FPR1 activity
was observed in the compound 12g bearing 2,6-difluoro-4-methoxyphenyl group as Ar1.
Compound 12g showed excellent hFPR2 activity (EC50 = 0.14 nM) and high hFPR2
selectivity (FPR1/2 ratio = 2000). Whereas the 3-fluoro-5-methoxypyridin-2-yl group
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
(12h) maintained FPR2 activity, the selectivity over hFPR1 was reduced relative to 12g.
Furthermore, replacement of the 4-methoxy group of 12g with an ethoxy (12i), an ethyl
(12j), a cyano (12k) or a methylamino (12l) group resulted in a 10- to 700-fold decreased
hFPR2 activity over 12g.
Table 4. Activity of 12 in HEK293 Cells Expressing Human FPR2 and FPR1
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5
6
7
8
9
H
F
O
N
O
N
N
H
H
Ar1
1
2
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
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
a
a
EC (nM)
EC (nM)
50
5
0
comp
Ar1
ratio comp
Ar1
ratio
FPR2 FPR1
FPR2 FPR1
F
1
1b
0.085
9.0
110 12f
0.15
17 110
OMe
OMe
F
F
F
1
2a
0.45
0.90
4.0
86
72
190 12g
80 12h
100 12i
0.14
0.36
14
280 2000
190 530
NTb NCc
OMe
F
F
F
OMe
OMe
N
F
1
2b
OMe
OMe
OMe
Cl
Cl
Me
F
1
2c
410
F
OEt
Et
F
F
1
2d
1.6
3.1
76
29
48 12j
9.4 12k
5.8
1500 260
F
12e
9.7 40000 4100
F
CN
F
1
2l
1.4
1900 1400
F
NHMe
a
2+
EC values were determined by Ca mobilization in HEK293 cells over-expressing
50
b
c
hFPR2 or hFPR1. Not tested. Not calculated.
2
We next evaluated the substituent effect of Ar for a series of 4-(2,6-difluoro-4-
methoxyphenyl)pyrrolidinone derivatives 13a−q (Table 5). The 3-fluorophenyl and 2-
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6
7
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
fluorophenyl derivative 13a and 13b showed 7- and 310-fold less hFPR2 activity than
12g. On the other hand, the phenyl, 4-chorophenyl, and 4-bromophenyl derivatives 13c−e
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
showed comparable hFPR2 activity to 12g. Among these compounds, phenyl derivative
13c showed higher hFPR2 selectivity (FPR1/2 = 6800) than 12g. Other 4-position
substituents, including cyano, methyl and methoxy derivatives 13f−h were 6- to 23-fold
less active than 12g. The 3,4-disubstituted phenyl derivatives 13i−l showed comparable
hFPR2 activity to 12g. Among these compounds 3,4-difluorophenyl derivative 13i and 3-
hydroxy-4-methylphenyl derivative 13l showed higher hFPR2 selectivity (hFPR1/2 = 5100
and 13000, respectively) than 12g. Although, the aromatic heterocycle derivatives 13m−q
showed 2- to 32-fold reduced activity compared to 12g, 5-chlorothiophen-2-yl derivative
1
3n showed good hFPR2 activity (EC = 0.29 nM), and higher hFPR2 selectivity (FPR1/2
50
= 4100) than 12g.
Table 5. Activity of 13a-q in HEK293 Cells Expressing Human FPR2 and FPR1
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7
8
9
H
O
N
O
Ar2
N
N
H
F
F
H
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
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
OMe
13
a
a
EC (nM)
EC (nM)
50
50
comp
Ar2
ratio
2000
comp
Ar2
ratio
FPR2 FPR1
FPR2 FPR1
0.16 820
F
F
F
F
1
1
1
1
2g
3a
3b
3c
0.14
0.95
43
280
13i
5100
830
F
1700 1800
13j
13k
13l
0.087 72
0.050 40
0.078 1000
HO
Cl
NTb
NCc
800
HO
F
Me
HO
0.41
0.11
2800 6800
13000
1500
Cl
Br
Cl
N
13d
13e
120
96
1100
510
13m
4.5
6900
1200
NTb
Cl
S
0.19
13n
0.29
4100
Cl
NC
Me
S
NCc
1
3f
0.78
2.0
350
450
13o
13p
13q
1.9
N
N
S
Me
13g
3200 1600
1000 310
0.32
0.76
290
910
MeO
S
1
3h
3.2
3000
3900
a
2+
EC values were determined by Ca mobilization in HEK293 cells over-expressing
50
b
c
hFPR2 or hFPR1. Not tested. Not calculated.
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7
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
Finally, to select a candidate compound, in vivo efficacy and CYP3A4 inhibition were
evaluated for 5 analogs, 12g, 13c, 13i, 13l, and 13n, which had potent in vitro hFPR2
activity (EC < 0.50 nM) and high hFPR2 selectivity (hFPR1/2 > 2000). In vivo efficacy
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
50
was evaluated in a mouse inflammation model by measuring inhibition of neutrophil
infiltration into lung after oral administration at 1 mg/kg (Table 6). The 4-fluorophenyl
derivative 12g, phenyl derivative 13c and 5-chlorothiophen-2-yl derivative 13n showed
excellent in vivo efficacy (inhibition >80%), while the disubstituted phenyl derivatives 13i
and 13l showed weaker in vivo efficacy.
Among the three compounds that inhibited neutrophil infiltration in mice, selection for
advanced in vivo studies relied on several factors. Analog 13n was excluded as a
candidate compound due CYP3A4 inhibition. Higher FPR2 selectivity in the Ca2+ flux
assay was measured for 13c versus 12g (6800 vs 2000 fold, Table 5); in addition, only
oxidative metabolites were observed after hepatocyte incubation with 13c, while oxidative
defluorination was observed with 12g (data not provided). Based on these differences,
13c was progressed to additional studies.
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Table 6. Effect of Selected Compounds on LPS-induced Neutrophil Infiltration in Mouse
Lungs
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
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
H
O
N
O
Ar2
N
N
F
H
H
F
OMe
12g, 13c,i,l,n
a
FPR2 EC (nM)
In vivo
CYP inhibition
5
0
Ar2
compd
inhibition (%)b
IC (μM)
c
human
0.14
mouse
50
F
12g
1.5
97
>20
1
1
3c
3i
0.41
0.16
3.4
8.5
88
>20
>20
F
4.9
F
Me
HO
13l
0.078
0.29
0.63
55
84
>20
5.6
Cl
S
13n
1.4
a
2+
EC values were determined by Ca mobilization in HEK293 cells over-expressing
50
b
hFPR2 or mFPR2. ꢀ Inhibition (%) was determined by measurement of neutrophils in
c
BALF of LPS-dosed mice after oral dosing of 1 mg / kg. IC values were determined
50
with recombinant human CYP3A4.
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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
Additional in vitro assays examined more detailed cellular signaling of FPR2 agonist
1
3c (Table 7). Inhibition of cAMP levels was observed in cells over-expressing either
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
hFPR2 or hFPR1, consistent with G coupling, with an 80x selectivity for hFPR2. -Arrestin
i
recruitment was also observed. Potency was right shifted in both assays compared to calcium
mobilization, which could be due to different assay conditions, including the level of receptor
expression in the cell lines. Confirming cross species selectivity, 13c demonstrated 1000x
selectivity for FPR2 over FPR1 in the cAMP assay using CHO cells over-expressing the mouse
receptors. The impact on inflammatory cells was measured in two key cellular assays: Compound
1
3c inhibited chemotaxis of a neutrophil-like cell line against a gradient of SAA and stimulated
phagocytosis by mouse macrophages at low nanomolar potency. These assays show the potential
of FPR2 agonists to prevent pro-inflammatory neutrophil infiltration, and the ability to promote a
wound healing phenotype in macrophages, which are key characteristics of compounds that
stimulate resolution of inflammation.
Table 7. Mechanistic in vitro assays 13c
EC or
IC50 (nM)
5
0
Assay
a
hFPR2 cAMP
hFPR1 cAMP
mFPR2 cAMP
mFPR1 cAMP
5.0
400
0.5
500

-Arrestin Recruitment
130
Chemotaxis
57
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Phagocytosis
<1
a
EC values for cAMP were determined in Chinese Hamster Ovary cells (CHO) over-
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
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
50
expressing hFPR1, hFPR2, mFPR1 or mFPR2 receptors. -Arrestin values were
®
measured in a DiscoverX Pathhunter cell line. Phagocytosis assays used mouse
peritoneal macrophages. IC value for the chemotaxis assays used a HL-60 cell line.
50
In vivo pharmacokinetic (PK) data and in vitro intrinsic clearance values in liver
microsomes and hepatocytes for compound 13c are listed in Table 8. Compound 13c
had high clearance in rat and mouse, but moderate clearance in dog and monkey with
good bioavailability.
The clearance values in human microsomes and human
hepatocytes were about 10-fold lower than those of rat, dog and monkey, suggesting that
13c is more metabolically stable in humans than in these animal species.
Table 8. Cross Species Comparison of In Vivo PK Parameters and In Vitro Intrinsic CL of
13c
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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
Liver
microsome
int.CL
i.v.
p.o.
Hepatocyte
int.CL
Species
a
d
e
g
a
b
c
Dose Vdss
CLtot
Dose
C
max
T
1/2 (h) AUC0-inf BA (%)
f
h
h
Mouse
Rat
0.5
1.0
1.0
3.5
3.0
0.98
1.2
90
49
1.0
1.0
1.0
3.0
160
240
820
800
NT
0.68
120
640
24
67
50
49
NT
NT
NT
15
3.8
2.5
5.0
0.021
0.042
0.030
0.0033
Dog
6.6
5700
50
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
Monkey 1.0
12
5800
28
h
h
h
h
h
h
h
h
Human
NT
NT
NT
NT
NT
NT
3.5
a
b
c
Dose is expressed in mg/kg. Vdss is expressed in L/kg. CL is expressed in
tot
d
e
f
mL/min/kg. C
is expressed in nmol/L. AUC
is expressed in nmol/L • h. CL is
max
0-inf
g
h
expressed in mL/min/mg protein. CL is expressed in mL/min/kg. Not tested
A major cause of heart failure is adverse cardiac remodeling post myocardial infarction
(
MI) during a period characterized by dysregulated chronic inflammation. FPR2 agonists
have the potential to resolve this inflammation and improve cardiac structure and function.
To investigate the role of FPR2 agonism in the setting of heart failure, the selective FPR2
agonist 13c was evaluated in a mouse model of myocardial infarction (MI), which leads
to heart failure develpment. In these studies, the left anterior descending (LAD) artery
was permanently occluded with a surgical suture. Non-infarcted surgical sham mice had
sutures placed around the LAD artery but not tightened. The lowest dose of 13c that
showed a significant effect on IL-10 stimulation (0.3 mg/kg) in mice treated with LPS was
selected for testing in the myocardial infarction model. Vehicle and 13c were dosed daily
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9
by oral gavage for 24 days starting 4 days post MI. The angiotensin converting enzyme
inhibitor (ACEi), captopril, was provided in the drinking water (~ 100 mg/kg/day) to
compare a standard of care therapy for acute MI.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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After 28 days, infarct scar expansion and left ventricular chamber remodeling were
evaluated. The passive mechanics of the left ventricle (LV) were analyzed using a
modified Langendorff apparatus. A deflated balloon attached to a pressure transducer
was inserted into the LV cavity. The passive compliance of the left ventricle was evaluated
following three inflation and deflation cycles of the balloon. Figure 4A shows mean left
ventricular pressure–volume (P–V) curves obtained from the various treatment groups as
well as surgical sham mice. Treatment with vehicle yielded a right-shifted P-V curve
relative to non-infarcted sham hearts shown at the left of the plot. The results indicate that
infarcted mouse hearts treated with vehicle have dilated LV chambers at each measured
pressure. By contrast, the P-V curve obtained with the FPR2 agonist 13c is left shifted
relative to vehicle, indicating smaller LV chambers with treatment. Treatment with
captopril also yielded a left shifted P-V curve relative to vehicle, but to a lesser degree
than that obtained with compound 13c. The findings suggest that LV chamber remodeling
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is attenuated with both compound 13c and captopril treatments post MI. Heart weights
for compound 13c and captopril treatment groups were reduced relative to the vehicle
group (Figure 4B). These findings indicate that compound 13c can attenuate left ventricle
and global cardiac remodeling.
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Figure 4: Effect of 13c and captopril (ACEi) treatment on myocardial structure and
function post LAD in mice. A) Left ventricular pressure-volume curves. B) Heart weights
28 days post MI. C) Representative heart cross-sections by histology depicting the degree
of myocardial infarction. Non-infarcted surgical sham animals are shown for comparisons.
D) Scar length 28 days post MI. Doses: 13c, 0.3 mg/kg/d; captoril, 100 mg/kg/d.
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Cross sections of the left ventricle were analyzed for histomorphometric assessments
of infarct structure. Representative images showing the size of the MI and LV chambers
are shown for all groups (Figure 4C). Infarct length was quantified for all groups as an
indicator of overall scar expansion. As shown in Figure 4D, vehicle-treated mice yielded
the largest scar lengths. By contrast, treatment with compound 13c reduced infarct length
by 39% relative to vehicle (P<0.05). Despite the decreases in heart weight and decreased
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LV chamber areas noted in Figure 4A and B, treatment with captopril was unable to
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Chemistry
The synthesis of racemic cis- and trans-oriented azetidine urea derivatives 3 and 4 are
shown in Scheme 1. Reaction of imine 14 with 2-(1,3-dioxoisoindolin-2-yl)acetyl chloride
in the presence of triethylamine, gave 1523 as the mixture of cis and trans isomers.
Removal of the 4-methoxyphenyl group of 15 with cerium (IV) ammonium nitrate (CAN)
provided cis isomer 16. Treatment of 16 with hydrazine monohydrate, followed by
treatment with HCl, gave 17. Reaction of 17 with 4-chlorophenyl isocyanate in the
presence of saturated aqueous sodium hydrogen carbonate provided cis-oriented 3.
Azidation of a hydroxyl group of cis-isomer 18,24 followed by deprotection of the 4-
methoxyphenyl group of trans-19 with CAN, gave 20. A Staudinger reaction followed by
treatment with 4-chlorophenyl isocyanate, provided trans-oriented 4. The stereochemistry
of the C-3 and C-4 positions of all compounds was determined by comparison of coupling
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constant of the C-3 and C-4 hydrogen atom in the H NMR (J >4.0 Hz for the cis isomer,
3,4
25
J <3.0 Hz for the trans isomer).
3,4
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Scheme 1. Synthesis of Azetidinone Urea Derivatives 3 and 4a
OMe
OMe
O
O
O
O
Cl
O
O
N
NH
NH
N
N
R
N
H
N
a
b
d
H
MeO
OMe
OMe
R=phthalimido
OMe
14
15
(cis:trans=5:1)
16
3
:
c
7: R=NH +Cl-
1
3
OMe
e
OMe
O
O
O
O
Cl
O
N
N
NH
NH
HO
N3
N3
N
N
H
f
g
H
OMe
OMe
OMe
OMe
18
19
20
4
Compounds 3, 4, 15-20 are racemic.
a
Reagents and conditions: (a) 2-(1,3-dioxoisoindolin-2-yl)acetyl chloride, Et N, CH Cl ,
3
2
2
28%; (b) CAN, MeCN-THF-H O, 62%; (c) (1) hydrazine monohydrate, dioxane, (2) HCl,
2
5
3%; (d) 4-chlorophenyl isocyanate, sat NaHCO aq., AcOEt, 63%; (e) (1) MsCl, Et N,
3
3
(
2) NaN , DMF, 89%; (f) CAN, MeCN, 85%; (g) (1) PPh , THF (2) H O (3) 4-chlorophenyl
3
3
2
isocyanate, 48% (three steps) .
The synthesis of racemic cis- and trans-oriented pyrrolidinone urea derivatives 5 and 6
26
(
Scheme 2) began with the reaction of 21 and tert-butyl (2,2,2-trifluoroacetyl)glycinate
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in the presence of lithium bis(trimethylsilyl)amide (LHMDS) to give compound 22. After
removal of the tert-butyl ester group with trifluoroacetic acid (TFA), treatment of 23 with
iodomethane in the presence of potassium hydrogen carbonate provided 24. Reduction
of the nitro group using zinc powder and ammonium chloride, followed by treatment with
titanium(III) chloride and sodium acetate,27 gave pyrrolidinone 25 as a cis and trans
mixture. Removal of the trifluoroacetyl group under basic conditions and subsequent
treatment of 26 with 4-chlorophenyl isocyanate, was followed by separation of the
diastereomers by column chromatography, to provide cis-oriented 5 and trans-oriented
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6.
Scheme 2. Synthesis of Pyrrolidinone Urea Derivatives 5 and 6a
H
H
H
NO2
O
COOR
O
R
Cl
O
Cl
O
N
N
N
O
O
F3C
N
NO2
H
N
N
H
N
N
H
H
H
d
f
a
+
OMe
OMe
22: R=C(CH3)3
OMe
OMe
OMe
21
25: R=NHCOCF3
(cis:trans=1:2)
26: R=NH2
5
6
b
23: R=H
e
c
2
4: R=CH3
Compounds 5, 6, 22-26 are racemic
aReagents and conditions: (a) tert-butyl (2,2,2-trifluoroacetyl)glycinate, LHMDS, THF,
8
8%; (b) TFA, 58%.; (c) MeI, KHCO , DMF, 92%; (d) (1) Zn, NH Cl, MeOH-H O, (2) 20%
3
4
2
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TiCl , NaOAc, MeOH-H O 63% (two steps); (e) 1 M aq. NaOH, MeOH; (f) 4-chlorophenyl
3
2
isocyanate, EtOAc, 4% (two steps for 5), 44% (2 steps for 6).
Racemic trans-oriented piperidinone urea derivative 7 (Scheme 3) was synthesized by
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conjugate addition of dimethyl malonate to compound 27 in the presence of sodium
28
methoxide, followed by reduction of the cyano group in 28 using nickel boride to give
piperidinone 29. After hydrolysis of ester 29 under basic conditions, treatment of 30 with
diphenylphosphoryl azide (DPPA) in the presence of triethylamine, followed by the
reaction with 4-chloroaniline provided piperidinone 7.
Scheme 3. Synthesis of Piperidinone Urea Derivative 7a
H
N
H
N
O
Cl
O
CN
COOMe
O
MeOOC
CN
ROOC
N
N
H
H
a
b
d
OMe
27
OMe
OMe
OMe
28
29: R=Me
30: R=H
7
c
Compounds 7, 28-30 are racemic
aReagents and conditions: (a) Dimethyl malonate, NaOMe, MeOH, 24%; (b) NaBH4,
NiCl ∙6H O, MeOH, 65%; (c) 1 M NaOH, MeOH, 92%; (d) DPPA, Et N, toluene then 4-
2
2
3
chloroaniline (6.5%).
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