Substituted Pyridazin-3(2 H)-ones as Highly Potent and Biased Formyl Peptide Receptor Agonists

Article abstract of DOI:10.1021/acs.jmedchem.8b01912

Herein we describe the development of a focused series of functionalized pyridazin-3(2H)-one-based formyl peptide receptor (FPR) agonists that demonstrate high potency and biased agonism. The compounds described demonstrated biased activation of prosurvival signaling, ERK1/2 phosphorylation, through diminution of the detrimental FPR1/2-mediated intracellular calcium (Ca_i²⁺) mobilization. Compound 50 showed an EC₅₀ of 0.083 μM for phosphorylation of ERK1/2 and an approximate 20-fold bias away from Ca_i²⁺ mobilization at the hFPR1.

Full text of DOI:10.1021/acs.jmedchem.8b01912

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Brief Article
Substituted Pyridazin-3(2H)-ones as Highly Potent
and Biased Formyl Peptide Receptors Agonists
Girdhar Singh Deora, Chengxue Qin, Elizabeth Vecchio, Aaron Debono, Daniel Priebbenow, Ryan Brady, Julia
Beveridge, Silvia Teguh, Minh Deo, Lauren T. May, Guy Y Krippner, Rebecca Ritchie, and Jonathan B. Baell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01912 • Publication Date (Web): 30 Apr 2019
Downloaded from http://pubs.acs.org on May 1, 2019
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Journal of Medicinal Chemistry
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Substituted Pyridazin-3(2H)-ones as Highly Potent and Biased
Formyl Peptide Receptors Agonists
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†#
‡∆#
‡
†
Girdhar Singh Deora, Cheng Xue Qin, Elizabeth A. Vecchio, Aaron J. DeBono, Daniel L.
◊
†
†
†
†
‡
Priebbenow, Ryan M. Brady, Julia Beveridge, Silvia C. Teguh, Minh Deo, Lauren T. May, Guy
Krippner, Rebecca H. Ritchie,* Jonathan B. Baell*
§
†
‡
‡∆∏
§School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, People’s Republic of China
†Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia
‡Baker Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria 3004, Australia
∆Department of Pharmacology and Therapeutics, University of Melbourne, Melbourne, Victoria 3010, Australia
◊Drug Discovery Biology, Monash Institute of Pharmaceutical Science, Monash University, Parkville, Vic 3052, Australia
∏Department of Diabetes (Central Clinical School), Monash University, Melbourne, VIC 3004, Australia.
KEYWORDS: Biased GPCR agonist, Formyl peptide receptor, Biased agonism, Pyridazinones, ERK1/2
ABSTRACT: Herein we describe the development of a focused series of functionalized pyridazin-3(2H)-one-based formyl
peptide receptors (FPRs) agonists which demonstrate high potency and biased agonism. The compounds described
demonstrated biased activation of pro-survival signaling - ERK1/2 phosphorylation – through diminution of the detrimental
FPR1/2-mediated intracellular calcium (Cai2+) mobilization. Compound 50 showed an EC50 of 0.083 µM for phosphorylation
of ERK1/2 and an approximate 20-fold bias away from Cai2+ mobilization at the hFPR1.
the first proof-of-concept evidence that FPRs exhibit
INTRODUCTION
biased signaling, potentially favoring cardiomyocyte
survival, associated with beneficial outcomes in vivo.10
Formyl peptide receptors (FPR) are a group of Class A G-
protein-coupled receptors (GPCRs) involved in the
regulation and resolution of inflammation.1, 2 They have
attracted significant scientific attention as novel
therapeutic targets for a range of inflammatory diseases
including arthritis, myocardial infarction and Alzheimer’s
disease. There are 3 isoforms of human FPRs, hFPR1, hFPR2,
and hFPR3.3 These isoforms have been shown to interact
with a range of structurally−diverse ligands including
In cardiomyocytes, the reperfusion injury salvage kinase
pathways such as extracellular signal-regulated kinase 1/2
(pERK1/2) and Akt signaling are tightly linked to post-
ischemic cell survival.4,
In contrast, increased
11-13
intracellular calcium (Cai2+) mobilization in the context of
myocardial infarction (MI) injury promotes the influx of
damaging inflammatory cells and further cardiomyocyte
cell death.4 We have demonstrated14 that the small-
molecule FPR agonist compound 17b exhibits clear and
biased signaling, favoring cell survival mechanisms whilst
proteins,
peptides,
lipids
and
small-molecules.
Interestingly, these ligands can mediate opposing
biological responses. For example, bacterial-derived N-
formyl peptides mediate pro-inflammatory responses,
whereas annexin-A1 promotes anti-inflammatory signaling
and is cardioprotective.4 This cannot be explained by
receptor subtype selectivity, but rather is proposed to arise
from a new paradigm-shifting phenomenon called biased
signaling.5, 6
eschewing potentially deleterious pathways as
a
consequence of its ~30-fold bias away from intracellular
calcium (Cai2+) mobilization at both hFPR1 and hFPR2
relative to the reference ligand compound 43.10,
15
Interestingly, this biased signaling profile correlated with
superior in vitro and in vivo cardioprotection of compound
17b compared with the balanced, non-biased compound
43.10, 16
Biased agonism reflects the ability of a ligand to stabilize
different active GPCR conformational states, leading to the
engagement of an alternative subset of intracellular
effectors and distinct cellular outcomes.5-7 Thus, the
development of biased agonists offers the elusive
therapeutic opportunity to promote beneficial signal
transduction in the absence of on-target adverse effects,8
the utility of which is already apparent in the clinical
context of acute heart failure.9 Our recent work revealed
Biased signaling structure-activity relationship (SAR) for
the pyridazin-3(2H)-one chemotype has not previously
been investigated. Here we report our focused medicinal
chemistry efforts to establish clear SAR with the aim to
generate potent FPR agonists biased away from
intracellular calcium mobilization. This understanding will
ultimately facilitate the future development of biased FPR
agonists for the treatment of MI.
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Journal of Medicinal Chemistry
synthesis of the final compounds (9−42, 44−55), was
Page 2 of 7
RESULTS AND DISCUSSION
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4
5
6
7
8
achieved by two synthetic routes, both involving N-
alkylation at the 2-position of the pyridazinone core. The
first route involved the direct N-alkylation of LHS moiety
with the RHS moiety, while the second involved a 3-step
route where the N-alkylation was performed employing a
bromoester, which was then hydrolyzed to reveal the
carboxylic acid motif which was subsequently coupled
with an aniline under standard amide-coupling conditions.
The substituted pyridazin-3(2H)-ones (9−42 and 44−55)
were synthesized as shown in Scheme-1. Multi-step
synthetic procedures were employed, which involved
construction of RHS bromoanilides (3a−l) using a variety
of linkers and substituted anilines. For the synthesis of the
LHS benzyl pyridazinone scaffold (6a−j), a condensation
reaction between substituted aldehydes (4) and the 4,5-
dihydropyridazinone moiety (5) were performed.14 The
9
Scheme 1. Representative synthetic schemes for the intermediates and final compounds.a,b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
NH
OCH3
O
R3
R3
R3
N
NH
N
H
N
NH
N
R1
a
b
OH
Br
5
R2
c
O
Br
Br
R1
Br
R4
O
O
O
R2
6a
6a-j
1
2
3
4
e
R3
O
R3
OCH3
O
R3
O
O
OCH3
LHS
RHS
H
N
f
O
OH
g
3
N
N
Me/Et
N
N
NH
N
N
N
R4
R1
R1
O
O
O
d
R2
R2
7a-e
9-42, 44-55
8a-e
6a-j
aReagents and conditions. (a) i) COCl2, DMF (cat.), CH2Cl2, 0 oC, 1 h, ii) substituted anilines, Et3N, 0 oC–r.t., 1–2 h; (b)
substituted anilines, Et3N, 0 oC–r.t. 4–6 h; (c) KOH/ethanol (5% w/v), reflux 4–6 h, 22–89%; (d) K2CO3, CH3CN, 60 oC, 16
h, 58–99%; (e) BrCH(R3)CO2R, K2CO3, CH3CN, 60 oC, 16–20 h, 86–92%; (f) LiOH, THF/H2O (1:1), r.t., 16 h, 37–88%; (g)
substituted anilines, EDCI·HCl, HOBt, CH3CN 40 oC, 12–24 h, 4–87%. bDetailed experimental procedures and structures
of the intermediates are provided in the Supporting Information.
We previously reported the biased nature of compound
17b, which demonstrated bias away from the potentially
detrimental FPR1/2-mediated Cai2+ mobilization, but
retained pro-survival pERK1/2 signaling.10, 15 These findings
initiated further SAR elucidation for compound 17b to
identify more potent compounds with biased agonistic
profiles. The synthesized compounds were assessed using
Cai2+ mobilization and pERK1/2 signal readouts. The
rational compound elaboration was primarily aimed at the
development of a biased agonist, which selectively targets
pERK1/2 whilst minimizing Cai2+ mobilization.
unless otherwise specified. The initial compounds of this
series had no substituents at the α-carbon of the amide
linker. Initially, the meta-methoxy substituent (as in
compound 17b) was preserved whilst we probed the RHS,
for which the effect of a chloro group at the ortho-, meta-
or para- positions on the RHS ring was simultaneously
determined. The para-chloro derivative (11) demonstrated
a substantial improvement in activity whilst the ortho- and
meta-chloro derivatives (9, 10) led to reduced activity with
pEC50values <4.00. Similarly, derivatives with electron-
withdrawing groups, including
a
nitrile (12−14),
trifluoromethoxy (15) and fluoro substituent (19, 20), gave
inactive compounds with pEC50 values <4.00. The activities
of derivatives with halogen-substituents at the para-
position of the RHS phenyl remained consistent with
compound 17b (with the LHS phenyl bearing a meta-
methoxy group); these analogues retained activity and
selectivity. Interestingly the incorporation of one or two
fluoro substituents on the RHS phenyl containing a para-
chloro group (34, 35) demonstrated a significant loss of
activity.
Lead optimization resulted in the synthesis of a focused
library of analogues exploring the SAR of this series (Table
1). The effects of a variety of substituents at the 2-and 4-
positions of the pyridazinone core were explored based on
the prototype compound 17b. The first generation
compounds explored the inclusion of various substituents
on both the left and right aryl rings. These substituents
include fluoro and methoxy groups at different positions
on the LHS ring, and halogens, cyano, nitro, methyl,
methoxy and ethoxy groups at various positions on the
RHS ring. These changes were subsequently introduced
with variations at the linker region, which demonstrated
significant impacts on the agonistic activity and biased
signaling of these compounds.
Methyl substituted linker. Compounds that
demonstrated improved activity from our initial SAR study
were further functionalized with the introduction of a
methyl substituent at the α-position of the linker. In all
cases, the compounds were tested as racemates.
Compounds with no substitutions on the linker. All
discussion on bioactivity refers to pERK1/2 signaling at the
hFPR1 and all activity comparisons are made with 17b
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Journal of Medicinal Chemistry
Table 1. Structures and FPR1/2 activity data of compounds.
1
2
3
4
5
6
7
8
9
pEC50 (FPR1)
pEC50 (FPR2)
Entry
R1
R2
R3
R4
Cai2+ (SEM) pERK1/2 (SEM) Cai2+ (SEM)
pERK1/2 (SEM)
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
3-OCH3
3-OCH3
2-OCH3
2-OCH3
2-F
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-F
3-OCH3
3-OCH3
4-OCH3
4-F
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CF3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
2-Cl
3-Cl
4-Cl
<4.00
<4.00
5.91 (0.13)
<4.00
<4.00
<4.00
<4.00
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
44
45
46
47
48
49
50
51
52
53
54
55
17b
43
2-CN
3-CN
4-CN
4-OCF3
4-OCH3
4-C2H5
2,4-di-Cl
2-F,4-Cl
2,6-di-F, 4-Cl
4-Cl
<4.00
<4.00
<4.00
<4.00
<4.00
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
<4.00
<4.00
<4.00
<4.00
<4.00
5.1 (0.23)
5.3 (0.12)
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.76 (0.23)
<4.00
<4.00
<4.00
4-Cl
4-Br
4-Cl
4-Br
4-Br
2-Cl
3-Cl
4-Cl
2-F
3-F
4-F
4-Br
5.65 (0.13)
5.22 (0.13)
5.86 (0.16)
6.07 (0.11)
5.75 (0.12)
<4.00
<4.00
5.47 (0.12)
<4.00
<4.00
4.60 (0.63)
<4.00
5.12 (0.38)
5.07 (0.18)
5.68 (0.16)
5.25 (0.13)
<4.00
5.58 (0.14)
5.74 (0.13)
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
2-F, 4-Cl
2,6-di-F, 4-Cl
4-Br
H
CH3
CH3
H
H
H
H
H
CH3
Et
nPr
iPr
4-Br
4-F
4-F
4-Br, 2-NO2
4,6-di-F, 2-NO2
4-Cl
<4.00
<4.00
2,3-di-OCH3 CH3
2,5-di-OCH3 CH3
2,6-di-OCH3 CH3
2,3-di-OCH3 CH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
3-OCH3
3-OCH3
2-OCH3
6.77 (0.05)
5.0 (0.37)
5.58 (0.02)
6.84 (0.21)
5.73 (0.26)
5.2 (0.19)
5.55 (0.15)
<4.00
6.65 (0.50)
5.2 (0.22)
5.55 (0.08)
6.81 (0.22)
<4.00
5.01 (0.06)
<4.00
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Br
4-Cl
4-Br
4-Cl
4-Cl
4-Cl
4-Br
4-Cl
-
n.d
n.d
n.d
n.d
n.d
n.d
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
CH3
CH3
CH3
CH3 cycloPr
<4.00
CH3
CH3
CH3
CH3
CF3
nBu
nBu
nBu
nBu
nBu
nBu
nBu
CH3
-
6.48 (0.21)
7.08 (0.03)
6.83 (0.12)
6.90 (0.14)
<4.00
7.03 (0.08)
6.61 (0.06)
6.22 (0.18)
7.29 (0.08)
<4.00
n.d
7.15 (0.25)
n.d
6.05 (0.21)
4.94 (0.23)
5.28 (0.32)
<4.00
7.26 (0.14)
8.19 (0.24)
5.21 (0.14)
7.49 (0.17)
7.29 (0.15)
n.d.
n.d
n.d.
n.d.
n.d
7.21 (0.06)
n.d
2,3-di-OCH3 CH3
2,3-di-OCH3 CH3
3-OCH3
-
7.28 (0.05)
n.d.
CH3
-
5.02(0.16)
7.39 (0.05)
<4.00
7.02 (0.15)
These methyl-substituted compounds were synthesized
with either ortho-, subsm eta- or para-methoxy
substituents on the LHS phenyl ring whilst maintaining a
halogen substituent on the RHS phenyl ring. Similar to the
first generation of analogues, compounds presenting a
meta-methoxy group on the LHS ring and a meta-/ortho-
chloro (27, 28) or fluoro substitution on the RHS (30, 31)
ring were found to be inactive. While, a para-chloro or
para-bromo substituent with an ortho-methoxy group on
the LHS ring, i.e. compounds 24 and 25, retained pERK1/2
potency in the micromolar range. Replacement of the
pyridazinone methyl in 11 with a trifluoromethyl group (21)
resulted in a dramatic loss of activity. Further substitutions
at the para-position on LHS ring i.e. methoxy (36) or fluoro
(37), generated compounds with low micromolar potency.
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Journal of Medicinal Chemistry
Longer alkyl chain substitutions on the linker.
Page 4 of 7
Given that the prototype molecule 17b activates both
hFPR1 and hFPR2 in engineered CHO cells,10, 15 we then
evaluated the two most active novel compounds (50, 54) at
the hFPR2 subtype. Similar to activity at hFPR1, both 50, 54
demonstrated significant improvement in ERK1/2 potency
(pEC50, 7.15, 7.21 respectively), and in stimulating Cai2+
mobilization (pEC50, 7.29, 7.28 respectively) (Table 1). In
contrast to 17b, which elicited minimal activity with
respect to Cai2+ mobilization at hFPR2,10, 15 both 50 and 54
displayed a non-significant trend for a bias towards Cai2+
mobilization in hFPR2-CHO cells (Figure 2F; Table S1,
supporting information). This is in contrast to the bias
profile observed at the hFPR1 subtype, whereby 50 is biased
away from Cai2+ mobilization and 54 behaves as a non-
biased (i.e. balanced) agonist. These divergent signaling
profiles will hopefully enable 50 and 54 to be used as
complementary tool compounds to further interrogate the
biological relevance of FPR signaling bias in disease
settings in our future studies.
1
2
3
4
5
6
7
8
Compounds with both ortho- or meta-methoxy groups on
the LHS and a para-chloro/bromo group on the RHS ring
were further modified. Longer alkyl chains at the α-
position of the linker region were investigated.
Interestingly the propyl derivative (46) demonstrated no
marked improvement in activity or pathway selectivity,
while the butyl derivatives resulted in a significant
improvement in pERK1/2 potency relative to Cai2+
mobilization. Compounds with a n-butyl-substituted
linker, and either an ortho- or a meta-methoxy group on
the LHS, and a para-chloro group on the RHS (50, 52),
exhibited a pERK1/2 pEC50 of 7.08 and 6.90 respectively,
and 50 demonstrated an approximate 20-fold bias away
from Cai2+ signaling at the FPR1 (Table S1, supporting
information). In addition, 50 behaves as a partial agonist
with reduced maximal response in Cai2+ mobilization at the
FPR1 (Figure 2B). Moreover, replacement of this para-
chloro group in 50 and 52 with a para-bromo group (49 and
51 respectively) resulted in a slight reduction in pERK1/2
potency with considerably good selectivity over Cai2+
mobilization.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In summary, exploration of the SAR of 17b-related
compounds focused on four distinct regions of the
compound structure: the RHS and LHS aromatic rings,
pyridazinone core, and modification of the linker between
the pyridazinone core and the RHS ring. Figure 1
represents the regions of the pharmacophore and the key
structural changes that are either favorable or unfavorable.
This SAR study suggests that optimal pERK1/2 potency is
achieved by having a methoxy substituent on the LHS
phenyl, combined with a para-chloro or para-bromo
substituent on the RHS phenyl. Replacement of the methyl
on the pyridazinone core with a trifluoromethyl group was
Cyclic or branched substitutions on the linker.
Apart from the methyl and longer alkyl chain substituents
on the linker, compounds possessing branched and cyclic
moieties were also synthesized and biologically evaluated.
However, activity data revealed that these substitutions
were not tolerated within the linker region. Compound
with branched (47) or cyclic (48) substitution as a part of
the linker showed a pERK1/2 pEC50 of 5.55 and <4.00,
respectively.
detrimental
and
pERK1/2
potency
diminished.
Dimethoxy substitutions on the LHS ring.
Compounds pertaining a 2,3-dimethoxy substitution on
the LHS ring with or without a methyl substituent at the α-
position of the linker (40, 44) improved the potency of the
agonists. Similarly, 2,3-dimethoxy substitution along with
a butyl substituent at the α-position of the linker (54, 55)
showed significant improvement in ERK1/2 potency.
Surprisingly, unlike the other active compounds in the
series, 54 and 55 potently stimulates Cai2+ mobilization
(pEC50, 7.26 and 8.19, respectively), whilst, 2,5 or 2,6-di-
methoxy derivatives (41, 42) did not show any
improvement in potency or pathway selectivity.
Functionalization on the α-position of the linker resulted
in substantial improvements of both the potency and
biased activity of these compounds. Compounds with a
methylene or methyl group α to the amide linker with a
mono-substituted LHS phenyl were found to be inactive
(pEC50<4.0), whilst longer alkyl chains at the
aforementioned position resulted in a great improvement
in both activity and selectivity, producing a novel lead (50)
for further biological assessment. In addition, this novel
lead (50) display similar signaling bias away to Cai2+
mobilization at hFPR1, and distinct signaling bias towards
Cai2+ mobilization at hFPR2, in comparison to 17b.
Longer alkyl substitutions favored
Branched or cyclic substitutions not favored
LHS
O
R3
H
Methoxy at 2 or 3 favored
2,3-Dimethoxy favoured but reverts to non-biased ligand
Halogen substituents not favored
N
Br or Cl at 4-position favored
N
N
R4
R1
Multiple halogen substituents not favored
Other electron withdrawing group (-CN, -OCF3) not favored
O
2,5-/2,6-Dimethoxy not favored
R2
RHS
Methyl favored
Trifluoromethyl not favored
Figure 1. SAR summary of substituted pyridazin-3(2H)-ones analogues.
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Journal of Medicinal Chemistry
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Figure 2. Agonist-stimulated pERK1/2 and Cai2+ mobilization in CHO cells stably expressing hFPR1 (A, B; top panel) and hFPR2
(D, E; bottom panel). Bias factors at the hFPR1 (C) and hFPR2 (F). Compound 43 is used as a reference compound that is equipotent
at both the pERK1/2 and Cai2+ mobilization pathways.16 Bias factors of 50, 54 and 17b, quantified relative to the reference ligand,
compound 43 and the reference pathway, Cai2+ mobilization, demonstrate that, like 17b, compound 50 exhibits a significant bias
towards pERK1/2 over Cai2+ at hFPR1 (C), but toward Cai2+ over pERK1/2 at hFPR2 (F). Compound 54 has a bias factor of 1, indicating
it is non-biased towards the two pathways at hFPR1, but toward Cai2+ over pERK1/2 at hFPR2. Bias of 17b at the hFPR2 couldn’t be
calculated due to incomplete curve in Cai2+ mobilization **P < 0.01, unpaired t-test on ΔLog(τ/KA). Data represent the mean ±
SEM from 4 independent experiments, performed in duplicate.
Biased agonism. An extension of the Black-Leff
operational model of agonism was used to estimate agonist
transduction coefficients (Log(τ/KA)) for each signaling
output.17 Transduction coefficients were normalized to a
reference ligand, compound 43, and a reference pathway,
Cai2+ mobilization, as described in the Supporting
Information to generate normalized transduction
coefficients (ΔLog(τ/KA)) and Log(bias factors). This
provides a quantification of the relative bias of each agonist
at a given pathway. Almost all of the compounds in the
series were more potent in stimulating pERK1/2 than Cai2+
mobilization (Table 1). The most active compound of the
series, 50 showed a similar degree of bias (approx. 20-fold;
Table S1, supporting information) to compound 17b at the
hFPR1, but conversely a trend towards bias favoring Cai2+
mobilization at the hFPR2. This biased activity was
abrogated by the introduction of the dimethoxy
substituents at the ortho- and meta-position of the LHS
phenyl ring. Similar to compound 43, these dimethoxy
analogues (40, 42 and 54) were roughly equipotent for both
pathways.
important role in the resolution of inflammation, as both
we4 and others18-20 have reviewed. Much of the literature to
date has focussed on FPR2 activation (rather than both FPR
subtypes) as contributing to the resolution of
inflammation. On this basis, FPR2 agonists (and perhaps
also certain FPR1 ligands, both agonists and antagonists)
have been postulated as potential therapeutic approaches
for diseases associated with inflammation.19-21 Prior to our
observations of bias at this receptor family,10 the ability of
FPRs to interact with a diverse range of agonists capable of
stimulating opposing cellular responses downstream of
receptor activation (specific to cell type and ligand, but not
FPR subtype selectivity), a known distinguishing feature of
FPRs, was not well understood. It would be of interest for
future studies to confirm the signaling bias profile in
primary immune cells, in addition to receptor selectivity.
Whether or not the signaling bias contributes to the extent
of inflammatory response warrants further investigation
especially given that FPRs are known to be “promiscuous”
receptors, interacting with a range of structurally diverse
ligands.2, 4 Our studies provides a better understanding of
the key pharmacophores that are critical for mediating
signaling bias at the FPRs. Whether specific disease
settings trigger phenotypic change of immune cells such as
macrophages (e.g. from a pro-inflammatory, M1-like
phenotype to a pro-resolving, M2-like phenotype), as a
result of altered cytokine and lipid mediator profiles18 and
how this impacts on the signaling fingerprint of each
ligand, also warrants investigation in future studies.
Physicochemical, plasma protein binding (PPB)
and metabolic properties. Compound 17b and 50 were
further evaluated for their physicochemical, PPB and
metabolic stability in liver microsomes. The observed
poorer aqueous solubility of 50 compared with compound
17b may be due to the increased lipophilicity as a result of
the longer alkyl substituent in the linker region.
Conversely, 50 showed slightly better metabolic stability
compared with compound 17b although the rate of
microsomal metabolism remained relatively high (Table
2).
CONCLUSION
A pyridazin-3(2H)-one based series of FPR agonists was
synthesized and evaluated at pERK1/2 and Cai2+
mobilization signaling pathways. The most active
Involvement of FPRs in inflammation and its
resolution. There is clear evidence that FPRs play an
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Journal of Medicinal Chemistry
compound (50) in the series was significantly more potent
than the original molecule 17b (EC50 in pERK1/2 of 0.083
µM and 0.603 µM, away from Cai2+ mobilization at hFPR1.
Interestingly, this compound had an opposing bias profile
with a trend towards Cai2+ mobilization at hFPR2.
Compounds with a flexible butyl group attached to the
linker moiety were found to be the most active compounds
Page 6 of 7
in the series, and 50 is the most potent biased FPR1 agonist
reported to date. Interestingly, dimethoxy substitution at
the ortho- and meta-position (54) increased potency in
Cai2+ mobilization and abolished ligand bias at FPR1, but
retained modest bias towards Cai2+ mobilization over
pERK1/2 at the hFPR2.
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9
Table 2. Physicochemical, PPB and metabolic properties of 50 and compound 17b.
Physicochemical properties
cPPBc
in vitro CLintd
(μL/min/mg protein) predicted EH
microsome-
Entry
cLogD
at pH 7.4
gLogD
at pH 7.4
Sol2.0
Sol6.5
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cLogPa
(%)
(µg/mL)b
(µg/mL)b
17b
50
4.0
5.2
4.0
4.3
< 1.6
< 1.6
97.4
98.4
477e
0.92
0.83
5.2
5.1
< 0.78
< 0.78
233
aMeasured chromatographically. bKinetic solubility determined by nephelometry. cHuman plasma protein binding estimated using a
chromatographic method. dMouse liver microsome. eFor human liver microsomes, value was 273 μL/min/mg protein
Although metabolic stability would need to be improved to
impart future therapeutic relevance, we promote 50 as an
improved tool compound compared with compound 17b
for in vitro investigations focused on pharmacological
intervention with FPR biased agonists. In addition, our
data supports assessment of receptor signaling profile as
well as receptor subtype selectivity in the rational drug
design at FPRs.
and HOAt (1.3 eq) were added. After 15 min the amine was
o
added and the reaction stirred at 40 C for 16 h. After
cooling to room temperature, the reaction was quenched
by the addition of NH4Cl (sat. aq. solution) and extracted
with ethyl acetate (3 × 10 mL). The combined organic
phases were dried over MgSO4, filtered, then concentrated
under reduced pressure. Purification by column
chromatography afforded the corresponding amide
products (yields and spectral data are reported in the
Supporting Information).
EXPERIMENTAL SECTION
Pyridazinone N-alkylation (7a-e, 9−11, 15−23, 25−46,
51−55). Method A: Pyridazin-3(2H)-one in MeCN
(ml/mmol) was treated with K2CO3 (3.0 eq), followed by
the bromo anilide (1.3 eq). The reaction was warmed to
reflux over 4-6 h. After this time, the mixture was cooled
to rt and the solvent was removed in vacuo. The residual
material was taken up in EtOAc and washed with water.
The organic layer was collected, dried over MgSO4 and
concentrated in vacuo. The product was then purified by
flash chromatography. Method B: To a solution of the
pyridazinone in dry THF, sodium hydride (3 eq, 60% in
mineral oil) was added, followed by the alkyl halide (1.3 eq),
and the reaction mixture heated at 50 oC for 5 h, monitored
by TLC and/or LC-MS. The reaction was quenched by the
addition of NH4Cl (sat. aq. solution) and extracted with
ethyl acetate (3 × 10 mL). The combined organic phases
were dried over MgSO4, filtered then evaporated under
reduced pressure. The product was then purified by
column chromatography. Some alkylation reactions
resulted in partial hydrolysis of the ester, in which case the
crude material was progressed to the hydrolysis reaction
without further purification (yields and spectral data are
reported in the Supporting Information).
The identity and purity (>95% pure) of the target
compounds were confirmed using HRMS, NMR and HPLC.
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acs.jmedchem.XXXXXXX.
Synthetic experimental procedures, spectroscopic
data for the intermediates and final compounds,
biological (in vitro and quantification of biased
agonism) assay methods and some data, Molecular
formula strings and some data.
AUTHOR INFORMATION
Corresponding Author
*For J.B.B: E-mail, jonathan.baell@monash.edu; phone, +61 3
9903 9044.
*For R.H.R: E-mail, Rebecca.Ritchie@baker.edu.au; phone,
+61 3 8532 1392.
Author Contributions
#G.S.D. and C.X.Q. contributed equally to this work.
ORCID
Ester hydrolysis (8a-e). The ester was dissolved in a 1:1
mixture of THF and H2O (5 mL/mmol) and treated with
LiOH (10 eq). The suspension was stirred at room
temperature overnight. The reaction was quenched by the
addition of 1M HCl and extracted with EtOAc. The organic
layers were collected, dried using MgSO4 and reduced in
vacuo.
Girdhar Singh Deora: 0000-0001-9857-101X
Daniel L. Priebbenow: 0000-0002-7840-0405
Rebecca H. Ritchie: 0000-0002-8610-0058
Jonathan B. Baell: 0000-0003-2114-8242
ACKNOWLEDGMENT
Amide coupling (12−14, 24, 47−50). To a solution of
carboxylic acid in anhydrous CH3CN, EDCI·HCl (1.3 eq)
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Journal of Medicinal Chemistry
Receptor Agonist Compound 17b Protects Against Myocardial
Ischaemia-reperfusion Injury in Mice. Nat. Commun. 2017, 8,
National Health and Medical Research Council (NHMRC) of
Australia (#1045140, #1020411, #1117602). Australian
Translational Medicinal Chemistry Facility (ATMCF)
acknowledges the support of the Australian Government's
National Collaborative Research Infrastructure Strategy
(NCRIS) program via Therapeutic Innovation Australia (TIA).
1
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14232.
11.
Datta, K.; Bellacosa, A.; Chan, T. O.; Tsichlis, P. N. Akt
is a Direct Target of the Phosphatidylinositol 3-kinase. Activation
by Growth Factors, v-src and v-Ha-ras, in Sf9 and Mammalian
Cells. J. Biol. Chem. 1996, 271, 30835-30839.
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Hausenloy, D. J.; Tsang, A.; Yellon, D. M. The
ABBREVIATIONS
Reperfusion Injury Salvage Kinase Pathway: a Common Target for
Both Ischemic Preconditioning and Postconditioning. Trends
Cardiovasc. Med. 2005, 15, 69-75.
Cai2+, intracellular calcium; ERK1/2, extracellular signal-
regulated kinase 1/2; FPR, formyl peptide receptor; GPCRs, G-
protein coupled receptors; LHS, left hand side; MI, myocardial
infarction; RHS, right hand side.
13.
Yue, T. L.; Wang, C.; Gu, J. L.; Ma, X. L.; Kumar, S.; Lee,
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J. C.; Feuerstein, G. Z.; Thomas, H.; Maleeff, B.; Ohlstein, E. H.
Inhibition of Extracellular Signal-Regulated Kinase Enhances
Ischemia/Reoxygenation-Induced Apoptosis in Cultured Cardiac
Myocytes and Exaggerates Reperfusion Injury in Isolated Perfused
Heart. Circ. Res. 2000, 86, 692-699.
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Table of Contents artwork
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