Potent Thiophene Antagonists of Human Complement C3a Receptor with Anti-Inflammatory Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b00927

Structure-activity relationships for a series of small-molecule thiophenes resulted in potent and selective antagonism of human Complement C3a receptor. The compounds are about 100-fold more potent than the most reported antagonist SB290157. A new compound JR14a was among the most potent of the new antagonists in vitro, assessed by (a) inhibition of intracellular calcium release (IC₅₀ 10 nM) induced in human monocyte-derived macrophages by 100 nM C3a, (b) inhibition of β-hexosaminidase secretion (IC₅₀ 8 nM) from human LAD2 mast cells degranulated by 100 nM C3a, and (c) selectivity for human C3aR over C5aR. JR14a was metabolically stable in rat plasma and in rat liver microsomes and efficacious in rats when given orally to suppress rat paw inflammation, macrophage and mast cell activation, and histopathology induced by intraplantar paw administration of a C3aR agonist. Potent C3aR antagonists are now available for interrogating C3a receptor activation and suppressing C3aR-mediated inflammation in mammalian physiology and disease.

Full text of DOI:10.1021/acs.jmedchem.9b00927

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Potent Thiophene Antagonists of Human Complement C3a
Receptor with Anti-Inflammatory Activity
Jessica A. Rowley,†,‡,∥ Robert C. Reid,*,†,‡,§,∥ Eunice K. Y. Poon,†,§,∥ Kai-Chen Wu,†,‡,§
Junxian Lim,†,‡,§ Rink-Jan Lohman,†,‡,§ Johan K. Hamidon,† Mei-Kwan Yau,† Maria A. Halili,†,‡
Thomas Durek,† Abishek Iyer,†,‡,§ and David P. Fairlie*,†,‡,§
‡
†Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, Australian Research Council Centre of
Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, and §Centre for Inflammation and Disease Research,
Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
S
* Supporting Information
ABSTRACT: Structure−activity relationships for a series of
small-molecule thiophenes resulted in potent and selective
antagonism of human Complement C3a receptor. The
compounds are about 100-fold more potent than the most
reported antagonist SB290157. A new compound JR14a was
among the most potent of the new antagonists in vitro,
assessed by (a) inhibition of intracellular calcium release (IC50
10 nM) induced in human monocyte-derived macrophages by
100 nM C3a, (b) inhibition of β-hexosaminidase secretion
(IC50 8 nM) from human LAD2 mast cells degranulated by 100 nM C3a, and (c) selectivity for human C3aR over C5aR. JR14a
was metabolically stable in rat plasma and in rat liver microsomes and efficacious in rats when given orally to suppress rat paw
inflammation, macrophage and mast cell activation, and histopathology induced by intraplantar paw administration of a C3aR
agonist. Potent C3aR antagonists are now available for interrogating C3a receptor activation and suppressing C3aR-mediated
inflammation in mammalian physiology and disease.
pathological roles in inflammatory and metabolic diseases6,7
INTRODUCTION
■
and cancers.8 The development of potent, selective, and
Complement is a network of over 40 immunological proteins
bioavailable small-molecule ligands for C3aR could lead to a
new therapy for treating inflammation-driven diseases,
that supplement the actions of antibodies and immune cells in
detecting, lysing, and removing microbes and infected/
including respiratory, metabolic, cardiovascular, gastrointesti-
damaged cells from an organism.1 The Complement system
nal, central nervous system (CNS), neurodegenerative
is activated1−3 by diverse stimuli, including pathogens,
diseases, and cancers.9
antigen−antibody complexes, carbohydrate-binding lectins,
proteolytic enzymes, as well as foreign surfaces, chemical and
physical injury, radiation, and neoplasia. These stimuli catalyze
complex multipathway cascades involving protein cleavages by
We have previously downsized the 77-residue C3a protein to
equipotent hexapeptides10 as well as small-molecule agonists
(Figure 1) featuring a heterocyclic constraint, such as oxazole 1
(EC50 7 nM, Ca2+, human monocyte-derived macrophage
serine proteases generated by Complement activation (and by
(HMDM))5,11 or imidazole 2 (BR103, EC50 15 nM, Ca2+,
other proteolytic cascades), culminating in inflammation,
HMDM).12 These agonists were equipotent with, and
recruitment of immune cells, tagging of microorganisms and
displayed the same functional properties as, human C3a across
infected or damaged cells, and assembly of a conglomerate of
multiple reporter and signaling cellular assays.5,13 Unlike C3a,
Complement proteins into a pore-forming membrane attack
which degrades in a few minutes in vivo, these agonists were
complex (MAC) that induces cell lysis. A pivotal protein in the
Complement cascade is C3,4 an intermediate at the
intersection of multiple Complement activation pathways.
This protein is cleaved into a small anaphylatoxin C3a and a
larger fragment C3b. The human Complement protein C3a
causes chemotaxis and degranulation of granulocytes and
phagocytes (e.g., neutrophils, monocytes, macrophages,
stable to proteases and metabolism. The first reported C3aR
antagonist, and most important to date, was SB290157 (3,
Figure 1)14 that was found to inhibit C3a-induced Ca2+ release
in human monocyte-derived macrophages with IC50 1.3 μM.12
A few analogues incorporating a heterocycle, such as furan 4,
oxazole 5, or thiazole 6, are reported to be weak antagonists of
eosinophils, mast cells, and dendritic cells).5 Human C3a
C3aR with inhibitory activity at micromolar concentra-
tions.15−17
binds to and activates a discrete G-protein-coupled receptor
(C3aR) expressed on innate immune and other cell types.1,2
Activation of this receptor stimulates important immune and
metabolic responses and is now believed to play important
Received: June 11, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.9b00927
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Article
analogues of thiazole 6 as prospective C3aR antagonists and
evaluate their function in human innate immune cells
(macrophages, mast cells) known to express the C3a receptor,
as well as their potential anti-inflammatory activity in a model
of C3aR agonist-induced paw inflammation in rats.
Synthesis of Thiophenes and Structure−Activity
Relationships. We synthesized two series of thiophene
carboxamides 10a−o and 14a−k (Scheme 1, Supporting
1
Information), purified by rpHPLC and characterized by H
and 13C NMR spectroscopy and mass spectrometry (Support-
ing Information), and evaluated their agonist and antagonist
activities on human macrophages (Table 1, Supporting Figures
1−4). Based on our earlier studies5,12,13 with small-molecule
ligands such as 1−6, an arginine residue contributes significant
binding affinity for C3aR, and so this residue was not modified
in this study. Antagonist 3 also comprises an arginine that is
connected to a diphenylmethyl substituent via a very flexible
linker. We sought to restrict this flexibility by introducing
thiophenes, resulting in C3aR antagonists (10a−o). We have
disclosed13 10a as an antagonist, measured by Ca2+
mobilization in human monocyte-derived macrophages
(HMDM; IC50 85 nM against 100 nM C3a). It represented
a 15-fold increase in antagonist potency relative to 3
(SB290157) and a 7-fold increase relative to thiazole 6
under the same assay conditions (Table 1). This suggested that
the potential hydrogen bond-accepting thiazole nitrogen
present in antagonist (6) is not required for antagonist
activity, and we suspected that it might even be detrimental for
antagonist activity. Antagonist 10a and agonist 2 compete in a
concentration-dependent manner with a fluorescent form of
the C3a protein,23 Eu-DTPA-C3a (IC50 2 nM), for binding to
HEK293-Gα16-C3aR transfected human cells (Supporting
Figure 5), supporting direct interaction with the target receptor
human C3aR. Importantly, while 10a has no agonist activity
below 10 μM, we do find that it has some agonist activity at
higher μM concentrations tested herein (Supporting Figure
1−4). From this baseline, we now develop structure−activity
relationships to identify improved thiophene derivatives as
potential C3aR antagonists with minimal or no agonist activity.
We modified the 5-diphenylmethyl moiety in 10a to explore
structure−activity relationships. Compounds (10a−o, Scheme
1a) were synthesized via a common route. The 5-position of
ethyl 2-thiophenecarboxylate 7a was deprotonated using
lithium diisopropylamide (LDA), then benzophenone deriva-
tives or other ketones were added under cooled, anhydrous
conditions. The resulting tertiary alcohols (8a−o) were then
dehydroxylated under ionic hydrogenation conditions using
triethylsilane and trifluoroacetic acid to afford 9a−o. These
reductions proceeded rapidly for diaryl derivatives 8a, 8h, and
8l−o because the reaction proceeds through the stabilized
doubly benzylic carbocation intermediate. However, analogues
8b−g and 8i−k containing alkyl groups adjacent to the
hydroxyl required longer reaction times and, in the case of 8e,
the expected phenyl isopropyl methyl group was not obtained.
Instead, elimination gave the stable tetrasubstituted alkene 9e
that was isolated and carried forward. The 2-pyridyl analogue
8g reacted extremely slowly, presumably because formation of
the required carbonium-ion intermediate was impeded by the
existing positive charge on the adjacent pyridine nitrogen atom
being protonated under the reaction conditions. The ethyl
esters of the disubstituted derivatives 9a−o were hydrolyzed
and the resulting carboxylic acids were coupled to L-arginine
Figure 1. Agonists (1, 2) and antagonists (3−6) of Complement C3a
receptor (C3aR).
RESULTS
■
The partial charges on the heterocyclic nitrogen atoms of the
oxazole in 1 and the imidazole in 2 were calculated using
Gaussian18,19 (Figure 2), with significant partial negative
Figure 2. Partial charges calculated (Gaussian 16, NBO 7.0)18,19 for
heteroatoms of heterocycles and an adjacent amide carbonyl oxygen,
with expected amide conformation due to electrostatic repulsion
(blue) or attraction (red).
electrostatic charge associated with the nitrogen atom common
to these two heterocycles. We have previously shown that this
nitrogen atom is a hydrogen-bond acceptor that is important
for C3aR receptor affinity and agonist activity.5,12 The furan in
4 and oxazole in 5 also have partial negative charge on the
oxygen heteroatom, and both of these oxygens have some,
albeit weak, hydrogen-bond-accepting properties.12,20 In
contrast, a thiazole (e.g., 6) or thiophene heterocycle has a
partial positive charge on the sulfur atom (Figure 2) that
should prevent it from being a hydrogen-bond acceptor, and
thus such compounds are less likely to be C3aR agonists.21,22
Here, we report the development of a series of thiophene
B
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a
Scheme 1. Synthesis of Thiophene Analogues
a
Reagents and conditions: (a) lithium diisopropylamide (LDA), tetrahydrofuran (THF), ketones, −78 °C; (b) trifluoroacetyl (TFA), Et3SiH,
room temperature (RT); (c) NaOH, H2O, MeOH, THF; (d) H-Arg-OMe, N,N-diisopropylethylamine (DIPEA), BOP, DMF; (e) phenylboronic
acid, K3PO4, Pd(PPh3)4, 1,4-dioxane; (f) BBr3, dichloromethane (DCM). For compounds 8a−o and 9a−o, the groups R1 and R2 are the same as
defined for 10a−o, except 8e and 9e, where R2 = isopropyl.
methyl ester using BOP. Hydrolysis of the arginine methyl
ester gave 10a−o in moderate to good yields (Scheme 1a).
Relative to 10a, there was a 100-fold loss in C3aR antagonist
activity for 10b and 10c (IC50 > 10 μM; Table 1), where one
of the phenyl groups was replaced with H or Me. Activity was
partially recovered when the aliphatic group was lengthened
from Me to Et (10d, IC50 2.3 μM). The isopropylidene
analogue (10e) was a 2-fold less active antagonist than the
ethyl derivative (10d), perhaps suggesting that its 3D shape
rather than a flat sp2 topology is preferred for interaction with
the receptor. Changing one of the phenyl groups to a
cyclohexyl group seemingly gave a more potent antagonist
(10f, IC50 4 nM); however, partial agonism up to ∼30%
response relative to that of C3a was also observed at higher
concentrations (EC50 70 nM), suggesting that receptor
desensitization10,12,24 is the cause of the apparent antagonist
activity. Replacing one of the phenyls with a 2-pyridyl group
introduced a potential hydrogen-bond acceptor into this
region, and this compound became a potent antagonist (10g,
IC50 40 nM).
To investigate permissible space around the phenyl groups,
the larger 2-naphthyl (10h) and dibenzyl (10i) substituents
were introduced, with both showing similar antagonist activity
(IC50 0.09 and 0.1 μM, respectively; Table 1). Combining one
benzyl group with an ethyl substituent reduced antagonist
activity 2-fold (10j, IC50 0.2 μM); however, this combination
was 12-fold more potent than the phenyl and ethyl
combination (10d) noted earlier. A single macrocyclic
cyclododecanyl ring (10k) in place of two separate
substituents abolished antagonist activity, suggesting the need
for aromaticity in this region or that its flexibility and number
of rotatable bonds made it entropically unfavorable for binding.
A para-chloro atom on each aromatic ring of the
diphenylmethyl segment increased antagonist activity 3-fold
(10l, IC50 0.03 μM), whereas the corresponding bis(4-
fluorophenyl) analogue did not increase antagonist activity
(10m, IC50 0.1 μM). This difference may be due to the greater
C
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Table 1. Thiophene Derivatives Modulate Ca2+ Release in
Human Monocyte-Derived Macrophages
the tertiary alcohols 11a−j. Reduction with Et3SiH/TFA gave
the ethyl 5-diphenylmethylthiophene-2-carboxylates 12a−j,
which were hydrolyzed to the carboxylic acids 13a−j that
were then coupled to L-arginine methyl ester and finally
hydrolyzed to give the series of analogues 14a−k (Scheme 1b).
Methylation at the thiophene-3-position had the most
beneficial effect on antagonist potency generally (Table 1),
with the 3-methyl derivative 14a (IC50 10 nM, JR14a) having a
3-fold higher antagonist potency over the bis(4-chlorophenyl)
analogue 10l. Likewise, the 3-methyl analogue 14b (IC50 7 nM,
Table 1) had a 10-fold improved activity over the
unsubstituted 10a, and there was no agonist activity observed
at concentrations up to 100 μM. Methylation of the thiophene-
3-position of the bis(4-fluorophenyl) compound also improved
the antagonist potency by approximately 3-fold (14c, IC50
0.035 μM). Methylation of the thiophene-3-position of the
bis(4-methoxyphenyl) derivative 14d resulted in a 3-fold
improvement in antagonist potency (14d, IC50 2 μM).
Thiophene-3-methylation of the bis(meta-chlorophenyl) ago-
nist derivative 10n resulted in 14e, having an even greater
partial agonist response of up to 80% that of C3a (14e, EC50
46 nM). The observation of some partial agonist responses,
especially at high μM concentrations, for 10f, 10g, 10n, and
14c, 14e, 14j (Supporting Figures 1−4) show how even subtle
changes to the structure can influence functional activity,
which can also change with different cell types as highlighted
by compound 3 having antagonist activity in some cell types
but agonist activity in others.25
The bis(4-hydroxyphenyl) analogue (14f, IC50 0.79 μM)
was an almost 3-fold more potent antagonist than the bulkier
bis(4-methoxyphenyl) analogue 14d, but still 80-fold less
potent than the similarly sized bis(4-chlorophenyl) analogue
14a (Table 1). Substituting the thiophene 4-position with a
methyl (14g, IC50 0.14 μM) or phenyl group (14h, IC50 0.14
μM) did not have a large effect on potency, with both being
only slightly less potent than the unsubstituted 10a. However,
placing a phenyl in the 3-position inactivated the compound
(14i, IC50 > 100 μM). The addition of a bromo substituent to
the 3-position also reduced antagonist potency (14j, IC50 0.51
μM). Finally, the 3,4-fused cyclohexyl ring reduced the activity
by over 20-fold (14k, IC50 2.4 μM), suggesting that a
substituent bulkier than a methyl group may not be tolerated
in the 3-position. The most promising analogues from this
series were the 3-methylthiophenes 14a (JR14a) and 14b.
Antagonist Activity, Receptor Specificity, and in Vitro
Stability. Compounds 14a and 14b were both approximately
10-fold more potent C3aR antagonists than 10a13 and 100-fold
more potent than SB29015714 (3) in inhibiting intracellular
Ca2+ (iCa2+) release in this assay after stimulation by human
C3a in human macrophages (Figure 3a). They were also
selective for the human C3a receptor over the closely related
C5a receptor, as they had no effect on the release of iCa2+
stimulated in the same cells by C5a (Figure 3b). The more
potent C3aR antagonists 10a, 10l, 14a, 14b, and 14f were
assessed for stability in rat plasma and in the presence of rat
liver microsomes. The thiophenes were all stable in rat plasma
over 4 h, whereas control tripeptide Boc-LAR-OH was rapidly
degraded (Figure 3c) at a rate similar to C3a.26,27 Both bis(4-
cholorophenyl)-substituted compounds 10l (JR10) and 14a
(JR14a), as well as the putative metabolite bis(4-hydrox-
yphenyl) analogue 14f, were metabolically stable to exposure
over 1 h to rat liver microsomes (Figure 3d). The
unsubstituted diphenylmethyl compounds 14b and 10a were
apparent antagonist
a
b
activity
agonist activity
EC50
(μM)
c
compound
pIC50 SEM IC50 (μM) pEC50 SEM
d
d
3
5.4 0.2
6.2 0.2
7.1 0.3
<5
3.8 (1.3 )
0.60
0.085
> 10
> 10
2.3
<4
<4
<4
<4
<4
<4
<4
7.2 0.2
<4
<4
<4
<4
<4
<4
<5
7.3 0.2
<4
<4
<4
<4
<4
7.3 0.2
<4
<4
<4
<4
<4
>100
>100
>100
>100
>100
>100
>100
0.07
>100
>100
>100
>100
>100
>100
>10
613,22
10a (BR111)13
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l (JR10)
10m
10n
e
e
<5
5.6 0.3
5.4 0.3
8.4 0.2
7.4 0.1
7.0 0.1
7.0 0.2
6.7 0.2
<4
4.4
(0.004)
e
e
e
0.04
0.09
0.10
0.20
> 100
0.03
0.1
(0.006)
6.1
0.010
0.007
0.035
2.0
7.5 0.2
7.0 0.1
8.2 0.2
5.2 0.2
8.0 0.1
8.2 0.1
7.5 0.1
5.7 0.1
8.5 0.2
6.1 0.6
6.9 0.3
6.9 0.2
<4
e
e
0.05
10o
>100
>100
>100
>100
>100
0.046
>100
>100
>100
>100
∼100
>100
14a (JR14a)
14b (JR14b)
14c
14d
14e
14f
14g
14h
14i
e
e
e
(0.003)
0.79
0.14
0.14
> 100
0.51
2.4
e
14j
14k
6.3 0.4
5.6 0.1
<4
a
Inhibition of intracellular Ca2+ release induced by C3a (100 nM) on
b
HMDM whole cells. All data n ≥ 3. Concentration to induce 50%
maximal Ca2+ release in HMDM relative to 100% induced by C3a at 1
c
μM. Compounds 10c−d, 10f−h, and 10j introduce a new chiral
center, but were tested as a mixture of diastereomers. SB290157.14
d
e
30−80% partial agonists at higher concentrations.
steric bulk of chloro versus fluoro substituents. The bis(3-
chlorophenyl) analogue resulted in apparently improved
antagonist potency (10n, IC50 6 nM), but it was also found
to have up to 60% partial agonist activity at higher
concentrations (EC50 50 nM) consistent with activation and
desensitization of the receptor, leading to apparent inhibition.
The bulkier 4-methoxy derivative (10o, IC50 6 μM) was 60-
fold less potent than 10a and 200-fold less potent than 10l
(JR10) as an antagonist. These data suggest that the p-chloro
substituent has the ideal size in this series of C3aR antagonists
and likely also blocks oxidative metabolism at this position (see
ahead).
Next, a series of compounds were synthesized to explore the
effect of placing substituents at the 3- and 4-positions of the
thiophene ring of compound 10a to generate 14a−k (Scheme
1b). Substituted thiophene-2-carboxylic acid ethyl esters 7b−g
were commercially available or were prepared by esterification
of the corresponding acids (Supporting Information), and the
3-bromo compound 7e was converted to the 3-phenyl
derivative 7g by Suzuki−Miyaura coupling with phenylboronic
acid. Lithiation at the thiophene 5-position with LDA at −78
°C, followed by addition to benzophenone derivatives, gave
D
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Journal of Medicinal Chemistry
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Figure 3. Evaluation of new C3aR antagonists in vitro. (a) C3aR antagonists inhibit iCa2+ release stimulated by C3a (100 nM) in HMDM in a
concentration-dependent manner. (b) C3a antagonists have no effect on iCa2+ release stimulated by C5a (30 nM) in HMDM, in contrast to the
known28,29 C5aR antagonist 3D53, cyclo-(2,6)-AcF[OP(dCha)WR] (black). (c) Potent thiophene antagonists 10a, 10l, 14a, and 14b were stable
to proteolytic degradation in rat plasma in vitro, whereas control tripeptide Boc-LAR-OH was rapidly degraded. (d) In the presence of rat liver
microsomes, the bis(4-chlorophenyl) analogues 10l and 14a showed minimal metabolism in vitro over 60 min, as did bis(4-hydroxyphenyl) 14f (a
potential metabolite of 14b; data not shown). Unsubstituted analogues 10a and 14b were approximately 40 and 60% degraded after 60 min. Data
are presented as mean standard error of the mean (SEM) (n = 3). Note: all points have error bars, but some are smaller than the data symbol.
Figure 4. Antagonism of β-hexosaminidase release induced by C3a in LAD2 mast cells in a concentration-dependent manner by: 3, 10a, 14a, and
a
14b. Concentration (IC50) and -Log(IC50) of ligand required to inhibit 50% of β-hexosaminidase release induced by C3a (100 nM) in human
LAD2 mast cells (n ≥ 3).
a
Table 2. Pharmacokinetic Properties of C3aR Antagonists in Wistar Rats
cmpd dose (mg/kg; route) AUC (0−8 h, ng h/mL) Vd (L) elimination half-life (min) clearance (mL/(min kg)) Tmax (min) Cmax (ng/mL)
10a
0.5 i.v.
10 p.o.
0.5 i.v.
10 p.o.
1 i.v.
10 p.o.
1 i.v.
10 p.o.
811
436
1802
814
3795
478
779
90
0.4
N/A
0.3
N/A
0.1
N/A
0.7
32
N/A
61
N/A
191
N/A
45
10.3
N/A
4.6
N/A
4.4
N/A
18.0
N/A
210
240
300
240
91
10l
143
88
14a
14b
N/A
N/A
23
a
n = 3 for each route and compound; time course, 0−8 h.
the least stable to rat liver microsomes, showing 40 and 60%
degradation, respectively, over 60 min (Figure 3d). In
summary, 14a (JR14a) was the most promising stable C3aR
antagonist of these series, containing a 3-methyl substituent on
the thiophene ring as well as a para-chloro substituent on both
aromatic rings of the diphenylmethyl group that may help
suppress P450 oxidative metabolism.
Activity in Human Mast Cells. Mast cells are key immune
cells that play major roles in regulating immunity, defense, and
disease.30 Activation of mast cells results in rapid degranulation
and release of various preformed inflammatory triggers (e.g.,
histamine, tryptase, β-hexosaminidase, lipid mediators, cyto-
kines, chemokines), and dysregulated activation of these cells
can lead to a range of acute and chronic human disease states,
including allergies, asthma, atopic dermatitis, arthritis, and
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many others.31 Here, we show that compounds 10a, 14a, and
14b were potent inhibitors of mast cell degranulation in vitro,
as measured by release of the lysosomal enzyme β-
hexosaminidase induced by hC3a (100 nM) in human LAD2
mast cells (Figure 4). By this measure, all three compounds
were around 100-fold more potent inhibitors of mast cell
degranulation than 3 (Figure 4).
Anti-Inflammatory Activity in Vivo. As a prelude to
investigating anti-inflammatory activity for C3aR antagonists,
the compounds 10a, 10l, 14a, and 14b were examined for
some pharmacokinetic parameters in male Wistar rats (Table
2). Compounds 10a, 10l, and 14a were detectable at higher
concentrations than 14b in rat plasma after oral administration,
10l and 14a had the lowest clearance rates in vivo (<5 mL/
(min kg)), but 14a had a longer elimination half-life (>3 h).
This supported advancement of 14a as an antagonist for
further studies in vivo.
14b (10 mg/kg p.o. 2 h prior) were evaluated for anti-
inflammatory activity in this acute rat paw model of
inflammation and edema (Figure 5a). Following oral
administration of C3aR antagonists via oral gavage in olive
oil, paw swelling was induced 2 h later by the C3a agonist 2
and monitored over a further period of 2 h. Compounds 10a,
10l, 14a, and 14b reduced paw swelling by 20, 45, 65, and
49%, respectively, over control at 30 min after agonist injection
(Figure 5a). In this model, compound 3 does not show
measurable anti-inflammatory activity below 30 mg/kg p.o.
The lower efficacy in vivo for 14b and 10a relative to 14a was
expected based on lower stability to P450 metabolism in vitro
in liver microsomes (Figure 3d). The improved efficacy of 14a
in vivo may be due to its dual advantages over 10a in being
both 10-fold more potent as a C3aR antagonist in vitro and
also having almost 6-fold greater metabolic stability in liver
microsomes.
Since 14a (JR14a) had improved metabolic stability over
10a (BR111), we hypothesized that it may also be more
efficacious over a longer period of time. To investigate this, the
efficacy of JR14a was monitored in the paw edema model over
a period of 12 h and compared to 10a (Figure 5b vs 5c). Both
ligands were administered orally (p.o.) at 30 mg/kg to
maximize antagonism for an extended timeframe and paw
swelling was induced by agonist 2 (BR103) (350 μg/paw in
saline with 5% dimethyl sulfoxide (DMSO), 100 μL, i.pl.).
Thiophene antagonist 10a (Figure 5b) reduced paw swelling
by up to 60% for a period of up to 4 h following
administration. Thiophene 14a (Figure 5c) reduced paw
swelling by a greater margin (85% reduction) and maintained a
significant reduction in paw size for 6−8 h, an improvement in
both the duration of efficacy and potency over 10a.
A single intraplantar injection of the potent and selective
C3aR agonist 2 (BR103, 350 μg/paw in saline) into the hind
paws of Wistar rats caused an acute edema manifested by paw
swelling that peaked after 0.5 h and subsided within 4 h
(Figure 5a). C3aR antagonist compounds 10a, 10l, 14a, and
Next, we examined 14a (JR14a) for a dose−response
relationship and found that, when given orally 2 h prior to
BR103 challenge, this antagonist had a ceiling effect at 10 mg/
kg when monitored 30 min after agonist injection, with no
increase in anti-inflammatory activity in this assay when the
oral dose was elevated from 10 to 20 or 30 mg/kg (Figure
6a,b). At 10 mg/kg p.o., JR14a reduced paw swelling even
more effectively when given 4 h before i.pl. agonist
administration (Figure 6c). The antagonist also inhibited
BR103-induced expression (over 30 min) of a range of pro-
inflammatory rat cytokines, including Tnf, Il1b, Il6, Il10, Ccl2,
and Cd244 (Figure 6d−i).
Rat paw tissues were histologically analyzed after 30 min of
exposure to agonist (BR103) when paw inflammation had
maximized, and this coincided with activation and degranula-
tion of mast cells. Paw tissues of BR103-treated animals had
decreased numbers of intact mast cells (Figure 7a,d) and
tryptase-positive cells (Figure 7b,e), consistent with increased
histamine release (Figure 7f) compared to saline-treated
animals. This indicates agonist-induced activation/degranula-
tion of mast cells. There were also increased numbers of ED1
+ve macrophages (Figure 7c,g). Pretreatment with JR14a by
oral gavage (10 mg/kg) 2 h prior to intraplantar agonist
injection prevented these histological indications of rat paw
inflammation, by stabilizing mast cells and inhibiting their
degranulation/activation (Figure 7a,b,d,e,f) as well as
suppressing numbers of ED1 +ve macrophages (Figure 7c,g)
in these tissues.
Figure 5. Efficacy and duration of anti-inflammatory activity against
agonist-induced paw edema in male Wistar rats. (a) Paw edema
(swelling) induced by intraplantar injection of C3aR agonist BR103
(2, black circle) is suppressed by 10 mg/kg C3aR antagonists 10a
(gray square), 10l (black triangle), 14a (black inverse triangle), and
14b (gray diamond) given orally 2 h prior to agonist. Duration of
effect of (b) antagonist 10a (BR111) or (c) antagonist 14a (JR14a)
administered orally at 30 mg/kg, prior to challenge with an agonist 2
(BR103) injected intraplantar to induce paw edema in groups of rats
after different exposure times to antagonist (1−12 h). n = 3 per group.
Data are mean
SEM. Analysis by one-way analysis of variance
(ANOVA), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Figure 6. JR14a reduces rat paw inflammation induced by a C3aR agonist. (a) Oral pretreatment with JR14a (10 mg/kg p.o., 2 h prior) significantly
reduces paw swelling measured 30 min after intraplantar injection of C3aR agonist BR103 (350 μg/paw) into rat hind paw. (b) When given orally
2 h prior to BR103 (2) challenge, JR14a has a ceiling effect at 10 mg/kg. At higher doses (20 or 30 mg/kg p.o.), there is no additional reduction in
paw swelling after 30 min. (c) JR14a (10 mg/kg p.o.) maximally reduces paw swelling when given 4 h before i.pl. agonist administration, compared
to the BR103 control at 30 min (first bar). (a−c) All measurements are normalized to sham. (d−i) BR103 increases expression of inflammatory
genes at 30 min in the rat paw, including Il1b (d), Tnf (e), Il6 (f), Il10 (g), Ccl2 (h), and Cd244 (i). Pretreatment with JR14a (2 h before agonist)
attenuates increased expression of these genes. Target genes were normalized to housekeeping gene Hprt. Error bars = mean SEM. n = 3−5 male
Wistar rats (7−8 weeks old) per group. Analysis by two-tailed student’s t-test (A) or one-way ANOVA (B−G). *p < 0.05, **p < 0.01, ***p <
0.0001 compared to control; +p < 0.05, ++p < 0.01, +++p < 0.001 compared to sham.
like macrophages, neutrophils, and mast cells.33 C3a is known
DISCUSSION AND CONCLUSIONS
■
to cause degranulation and chemotaxis in vitro, but the
Complement protein C3 is synthesized by a range of cells4,32
physiological properties of C3a itself in vivo remain clouded
and is a central component of the classical, alternative, lectin,
due to mis-information arising from studies involving
and possibly extrinsic protease-mediated pathways of Comple-
administration of, metabolically unstable, C3a to mammals
ment activation.9 It is released into the circulation, is
and the unavailability of potent and metabolically stable
proteolytically cleaved at the membrane of microbial and
agonists and antagonists. For example, it does not seem to be
host innate immune cells by Complement convertases, and
commonly known that C3a undergoes extremely rapid
deposits C3b that contributes to and amplifies the Comple-
ment cascade leading to lysis of microorganisms and infected
or damaged cells.9 C3 cleavage to C3b also produces the
byproduct C3a, and this Complement protein is known to act
independently on a unique G-protein-coupled receptor C3aR
expressed on many human cells, including innate immune cells
proteolytic degradation to products that do not bind to
C3aR.13 Recent studies have identified potent and selective
small-molecule agonists,5,11,12 that have the same physiological
and molecular profiles as C3a in vitro but are much more
stable in vivo. However, no very potent small-molecule
antagonist of human C3aR has yet been reported.
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Preasugrel, and Ticlopidine, and there are many other
thiophene-containing compounds with demonstrated efficacy
in vitro or in animal models of diseases.34−38 The present study
has described structure−activity relationships centered around
thiophene-based scaffolds with diverse substitution that has
indicated some structural requirements for potent C3aR
antagonism. In vitro, thiophene compounds potently inhibited
intracellular Ca2+ release in human macrophages (e.g., 10a,
IC50 85 nM; 10l, 30 nM; 14a, 10 nM; 14b, 7 nM), without
inhibiting the agonist effects in the same concentration range
as the related Complement protein C5a. Among key findings
from structure−activity relationships in cells were: (i) for Ca2+
mobilization in human macrophages, a thiophene confers a 10-
to 15-fold increase in antagonist potency over a thiazole or no
heterocycle (e.g., 10 vs 6 or 3), indicating that a sulfur-
containing heterocycle is important for conferring antagonism
and that the hydrogen bond-accepting nitrogen of thiazole may
be detrimental for antagonist potency; (ii) in the same assay, a
chlorine at the para position of both aromatic rings of the
benzhydryl group confers a further 10-fold increase in
antagonist potency for thiophenes (e.g., 14a vs 10a); (iii) for
LAD2 human mast cells, the thiophene analogues were
similarly 100-fold more potent than 3 that has no heterocycle,
as antagonists of C3a-induced degranulation measured by
secretion of β-hexosaminidase; (iv) for rat liver microsomes in
vitro, there was a significant increase in metabolic stability for
14a and some analogues over other thiophenes (e.g., 10a, 10l,
14b).
Several of the thiophene derivatives from these series were
found to have anti-inflammatory activity in vivo, when
administered by oral gavage to rats subsequently injected
with a C3aR agonist into the paw to induce an acute local
inflammation. Among the compounds examined in vivo, orally
administered thiophene 14a (10 mg/kg) had the lowest
clearance from rats, consistent with the findings of its stability
to rat liver microsomes in vitro and its longer elimination half-
life in vivo after oral delivery. This increased metabolic stability
coupled with greater C3aR antagonist potency contributed to
superior anti-inflammatory activity and a longer duration of
action as assessed by C3aR agonist challenge to induce acute
rat paw edema. Compound 14a (JR14a) was the most potent
and effective C3aR antagonist from this study.
There is a great need to develop new tactical approaches to
develop new modulators for the hundreds of membrane-
spanning G-protein-coupled receptors that mediate human
physiology.39,40 Many of the most interesting GPCR targets for
prospective drugs are expressed on innate immune cells, for
which we are still uncovering relationships between extrac-
ellular signals, intracellular signaling pathways, and physio-
logical responses in vivo. Studying the important interplay
between innate immune cells and their environment can
benefit from the availability of potent new compounds that can
modulate specific receptors and immune cell function. Here,
we present a new class of small-molecule modulators for the
human Complement protein C3a, an initiator of acute
inflammatory responses that need to be controlled in a diverse
range of disease indications. By virtue of their potency,
metabolic stability, and efficacy against different innate
immune cells, compounds reported here could prove to be
valuable new probes for investigating the detailed pharmacol-
ogy of C3a and its associated GPCR in vitro and in vivo in
mammalian physiology and disease.
Figure 7. JR14a reduces mast cell degranulation and macrophage
infiltration in BR103-induced rat paw edema. (a, d) Relative to sham
animals, intact/inactive mast cells are decreased 30 min after
intraplantar injection of agonist BR103 (350 μg/paw, i.pl.) into rat
paws. Oral pretreatment with JR14a (10 mg/kg, 2 h prior) prevents
this decrease. (d) Inactive mast cells (red) stained by safranin O.
Degranulated mast cells (blue) stained by alcian blue. Scale bar = 50
μm. (b, e) Tryptase-positive cells (green) stained by immunohis-
tochemistry are significantly reduced in control group compared to
sham, indicating activation/degranulation of mast cells (extracellular
tryptase undetectable by immunohistochemistry). This decrease is
inhibited by oral pretreatment with JR14a. Blue dye stains for cell
nuclei. Scale bar = 100 μm. (f) Immunohistochemistry staining of
histamine (green), cell nuclei stained blue. Sham paw is dominated by
intact/inactive histamine-containing mast cells. BR103 control paw
shows diffused extracellular histamine staining, indicating mast cell
degranulation and histamine release. JR14a pretreated paw contains
inactive mast cells with minimal mast cell activation and histamine
release. Scale bar = 200 μm. (c, g) ED1-positive macrophages (stained
red by immunohistochemistry) are significantly increased 30 min after
BR103 injection. This is prevented by oral pretreatment with JR14a.
Blue dye stains for cell nuclei. Scale bar = 50 μm. Error bars = mean
SEM, n = 5 male Wistar rats (7−8 weeks old) per group. Analysis by
one-way ANOVA. *p < 0.05, **p < 0.01 compared to control; +p <
0.05, ++p < 0.01 compared to sham.
Thiophenes are frequently occurring scaffolds in heterocyclic
drugs,34−38 for example, in the antidepressant Duloxetine, anti-
hypertensive Eprosartan, oral anticoagulant Rivaroxban,
antipsychotic Olanzapine, antiplatelet agents Clopidogrel,
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for 1 h, then at room temperature for 3 h. THF was evaporated and
the residue was dissolved in EtOAc and washed with 2 M HCl, 10%
NaHCO3, brine, and dried over MgSO4. The solvent was evaporated
and the solid residue was triturated with light petroleum ether and
collected on a filter giving 11a as a white solid (18.20 g, 95%).
Recrystallization from EtOAc/petroleum ether (1:1, 6 mL/g) gave
the pure product (15.7 g, 82%). Rf 0.29 (10% EtOAc/petroleum
EXPERIMENTAL SECTION
■
General. All reagent chemicals were purchased from Sigma-
Aldrich, Combi-Blocks Inc., Fluorochem Ltd., or Chem-Impex
International Inc. All final compounds were analyzed by NMR,
UPLC-MS, and HRMS, and the purity was determined to be >98%.
1H and 13C NMR spectra were recorded on a Bruker Avance III HD
600 spectrometer with cryoprobe at 298 K in the deuterated solvents
1
ether). H NMR (600 MHz, CDCl3): δ 1.33 (t, 3H, J = 7.1 Hz,
1
1
indicated. H NMR spectra were referenced to the residual H signal
DMSO-d5 2.49 ppm, except CDCl3 solutions were referenced to
internal TMS. 13C NMR resonances were referenced to the solvent
peak (DMSO-d6 39.51, CDCl3 77.0 ppm). The exact concentration of
the compounds was determined by the quantitative NMR integration
“PULCON” experiment.41 Thin-layer chromatography was performed
on Merck silica gel 60 TLC plates. Preparative-scale rpHPLC
separations were performed on a Phenomenex Luna C18 10 μm,
250 × 21.2 mm2 column. Standard conditions were used for elution of
all compounds unless otherwise indicated at a flow rate of 20 mL/
min: 100% A to 100% B linear gradient over 15 min followed by a
further 10 min at 100% B where solvent B was 90% MeCN, 10% H2O
+ 0.1% TFA and solvent A was H2O + 0.1% TFA. Detection was by
UV, and pure fractions were lyophilized. Analytical UPLC-MS was
used to assess compound purity using a Zorbax Eclipse plus C18
column (1.8 μm, 90 Å, 2.1 × 100 mm2) at 0.5 mL/min with detection
at three different wavelengths (λ 214, 230, and 254 nm) and
electrospray ionization-mass spectrometry (ESI-MS). High-resolution
mass spectra (ESI-HRMS) measurements were obtained on a Bruker
microTOF mass spectrometer equipped with a Dionex LC system
(Chromeleon) in positive-ion mode by direct infusion in MeCN at
100 μL/h using sodium formate clusters as an internal calibrant. Data
were processed using Bruker Daltonics DataAnalysis 3.4 software.
Mass accuracy was consistently better than 1 ppm error. All final
compounds were checked for PAINS and aggregators at http://
zinc15.docking.org/patterns/home, and no alerts were found. Full
experimental details and spectroscopic data including intermediates
are reported in the Supporting Information.
CH3CH2O), 2.47 (s, 3H, CH3), 2.94 (bs, 1H, OH), 4.29 (q, 2H, J =
7.1 Hz, CH3CH2O), 6.59 (s, 1H, C4 thiophene), 7.28−7.32 (m, 8H,
Ph). 13C NMR (150 MHz, CDCl3): δ 14.3, 16.1, 60.8, 79.3, 126.9,
128.4, 128.6, 131.1, 134.1, 143.8, 145.9, 155.1, 162.7. LC/MS m/z:
403.0 (M+ − OH).
Ethyl 5-(Bis(4-chlorophenyl)methyl)-3-methylthiophene-2-car-
boxylate 12a. A mixture of the tertiary alcohol 11a (2.27 g, 5.39
mmol), DCM (10 mL), and Et3SiH (1 mL, 6.25 mmol, 1.2 equiv) was
stirred at room temperature, then TFA (1 mL, 13 mmol) was added.
The solution became warm and refluxed spontaneously. After stirring
for a further 30 min, the mixture was diluted with DCM, washed with
excess 10% NaHCO3 and evaporated to a yellow oil. Purification by
flash chromatography (silica gel, eluting with 1−10% EtOAc/
petroleum ether) gave 12a as a colorless oil (2.11 g, 96%). Rf 0.52
(10% EtOAc/petroleum ether). 1H NMR (600 MHz, CDCl3): δ 1.32
(t, 3H, J = 7.1 Hz, CH3CH2O), 2.47 (s, 3H, CH3), 4.27 (q, 2H, J =
7.1 Hz, CH3CH2O), 5.54 (s, 1H, Ph2CH-thiophene), 6.52 (s, 1H, C4
thiophene), 7.11 (m, 4H, Ph), 7.29 (m, 4H, Ph). 13C NMR (150
MHz, CDCl3): δ 14.3, 16.0, 51.0, 60.6, 126.1, 128.8, 130.0, 131.3,
133.1, 140.8, 146.1, 151.3, 162.6. LC/MS m/z: 446.1 (MH+
+
CH3CN).
5-(Bis(4-chlorophenyl)methyl)-3-methylthiophene-2-carboxylic
Acid 13a. The ester 12a (2.00 g, 4.93 mmol) was dissolved in THF
(25 mL) and MeOH (25 mL), then a solution of NaOH (1 g in 25
mL of water) was added. The solution was stirred at room
temperature for 3 h, then diluted with water and washed with diethyl
ether/petroleum ether 1:1. The aqueous layer was acidified with HCl,
and the precipitate was extracted with ether. The ether extracts were
washed with brine, dried over MgSO4 and evaporated to give the acid
Chemical Synthesis. Human C3a was chemically synthesized,
purified, and characterized as described.42 This material has been
shown to have the identical structure, affinity, and functional
properties as recombinant C3a.42 The agonist compound 2
(BR103)12 and antagonist compound 3 (SB290157)14 were
synthesized, purified by RP-HPLC, characterized by NMR and MS,
and functionally profiled for affinity and functions, as described
elsewhere.12,13 The syntheses and characterization of most com-
pounds are reported in the Supporting Information.
1
13a as a white solid (1.85 g, 99%). H NMR (600 MHz, CDCl3): δ
2.47 (s, 3H, Me), 5.55 (s, 1H, Ph2CH), 6.56 (s, 1H, C4 thiophene),
7.11 (m, 4H, o-Ph), 7.29 (m, 4H, m-Ph). 13C NMR (150 MHz,
CDCl3): δ 16.2, 51.1, 125.4, 128.9, 130.0, 131.7, 133.2, 140.6, 148.0,
153.4, 167.9. LCMS m/z: 377.1 (MH+).
(5-(Bis(4-chlorophenyl)methyl)-3-methylthiophene-2-carbonyl)-
L-arginine Hydrochloride 14a (JR14a). The acid 13a (1.85 g, 4.91
mmol) was dissolved in DMF (20 mL) with stirring, then EDC (1.00
g, 5.21 mmol), L-arginine methyl ester dihydrochloride (1.93 g, 7.39
mmol, 1.5 equiv), and DIPEA (2.5 mL, 14.4 mmol, 3 equiv) were
added. The mixture was stirred at room temperature for 17 h then the
DMF was removed on a rotary evaporator at 0.1 mbar. The residue
was dissolved in MeOH (40 mL), then a solution of NaOH (2 g in
water 20 mL) was added. The solution was stirred at room
temperature for 30 min, then 1-butanol (200 mL) was added and
the solution was washed with 1 M HCl (100 mL, pH 1) and
evaporated to dryness. The residue was dissolved in 30% MeCN 70%
water and lyophilized to give the crude product as a white powder.
Further purification was achieved by preparative reversed-phase
HPLC, and the product was lyophilized as the TFA salt (3.20 g).
Finally, the TFA salt was dissolved in 30% MeCN and 70% water (50
mL) and treated with Dowex-1 × 8 chloride in 200−400 mesh (5 g)
for 30 min. The resin was filtered off and washed with 30% MeCN/
water (20 mL), and the filtrate was lyophilized to give 14a as the
hydrochloride salt (2.80 g, 78%). 1H NMR (600 MHz, DMSO-d6): δ
1.57−1.43 (m, 2H), 1.75−1.65 (m, 1H), 1.86−1.76 (m, 1H), 2.33 (s,
3H), 3.14−3.02 (m, 2H), 5.86 (s, 1H), 4.26 (m, 1H), 6.62 (s, 1H),
7.26−7.22 (m, 4H), 7.43−7.39 (m, 4H), 7.70 (t, J = 5.5 Hz, 1H),
8.03 (d, J = 7.7 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δ 15.4,
25.4, 27.6, 40.3, 49.3, 52.2, 128.6, 130.1, 130.3, 130.8, 131.7, 140.4,
141.8, 147.8, 156.8, 162.3, 173.3. HRMS m/z calcd for
C25H27Cl2N4O3S+ 533.1175 found 533.1175.
(5-(Bis(4-chlorophenyl)methyl)thiophene-2-carbonyl)-L-arginine
10l (JR10). Compound 9l (1.14 g, 2.92 mmol) was hydrolyzed to
produce the carboxylic acid (939 mg, 2.59 mmol, 89%) as a white
solid, then an aliquot (200 mg, 0.55 mmol) was converted to (5-
(bis(4-chlorophenyl)methyl)thiophene-2-carbonyl)-L-arginine 10l
(TFA salt, 133 mg, 34% over three steps) using a procedure
1
analogous to that for the preparation of 14a shown below. H NMR
(600 MHz, DMSO-d6): δ 1.47−1.57 (m, 2H, NHCH2CH2CH2CH),
1.66−1.72 (m, 1H, NHCH2CH2CH2CH), 1.80−1.86 (m, 1H,
NHCH2CH2CH2CH), 3.07−3.11 (m, 2H, NHCH2CH2CH2CH),
4.29−4.33 (m, 1H, α-CH), 5.92 (s, 1H, Ph2CH-thiophene), 6.78 (dd,
1H, J = 3.7 Hz, J = 0.7 Hz, C4 thiophene), 7.23 (d, 4H, J = 8.50 Hz, o-
Ph), 7.41 (d, 4H, J = 8.4 Hz, m-Ph), 7.53 (t, 1H, J = 5.6 Hz,
NHCH2), 7.73 (d, 1H, J = 3.90 Hz, C3 thiophene), 8.62 (d, 1H, J =
8.1 Hz, NH). 13C NMR (150 MHz, DMSO-d6): δ 25.5, 27.7, 40.3,
49.5, 52.0, 127.3, 128.67, 128.74, 130.3, 131.7, 138.2, 141.9, 151.8,
156.6, 161.2, 173.4. HRMS: C24H25Cl2N4O3S+ expected 519.1019
found 519.1019.
Ethyl 5-(Bis(4-chlorophenyl)(hydroxy)methyl)-3-methylthio-
phene-2-carboxylate 11a. A solution of ethyl 3-methylthiophene-
2-carboxylate 7b (7.74 g, 45.5 mmol) in dry THF (75 mL) was stirred
at −78 °C under N2, then a solution of LDA (2 M, 25 mL, 50 mmol,
1.1 equiv) was added over 5 min. After a further 10 min, a solution of
4,4′-dichlorobenzophenone (11.50 g, 45.8 mmol) in THF (100 mL)
was added over 10 min at −78 °C. The solution was stirred at −78 °C
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(5-Benzhydryl-3-methylthiophene-2-carbonyl)-L-arginine 14b
(JR14b). Compound 12b (228 mg, 0.68 mmol) was hydrolyzed to
produce the carboxylic acid (153 mg, 0.50 mmol, 74%) as a white
solid, then an aliquot (80 mg, 0.26 mmol) was converted to (5-
benzhydryl-3-methylthiophene-2-carbonyl)-L-arginine TFA salt 14b
(114 mg, 0.20 mmol, 77%) using a procedure analogous to that used
measured at 405 nm using FLUOstar Optima (BMG LabTechnol-
ogies). Responses to 100 nM C3a were comparable to degranulation
induced by 10 μg/mL substance P or 30 μg/mL 48/80 in LAD2 mast
cells.
Plasma and Metabolic Stability. Compounds (10 μL, 1 mM in
DMSO) were added to rat plasma (190 μL) and incubated at 37 °C
in a water bath. Aliquots (20 μL) were collected at different time
points (0, 2, 5, and 10 min for Boc-LAR-OH; 0, 5, 15, 30, 60, 120,
and 240 min for other compounds) and added directly to MeCN/
H2O (80 μL, 9:1). Samples were centrifuged (13 000g, 3 min) before
isolating supernatants and analyzing for compound degradation via
LC-MS. Commercially available rat liver microsomes (Life Tech-
nologies, GIBCO) stored at −80 °C were slowly defrosted on ice.
Test compounds (183 μL, 1 μM in 100 mM phosphate buffer pH 7.4
containing 0.1% DMSO) were added to microsomes (5 μL) and
NADPH (2 μL, 20 mM) and incubated (37 °C, 5 min) before adding
cofactor NADPH (10 μL, 20 mM). Aliquots (20 μL) at time points 0,
5, 15, 30, and 60 min after adding NADPH were added to MeCN/
H2O (80 μL, 9:1), centrifuged (13 000g, 3 min), and supernatants
were analyzed by LC-MSMS. Concentrations were plotted as a
function of time on a semilogarithmic graph. Rat plasma and
microsomal activity was confirmed by rapid degradation of SLIGRL-
NH2 (t1/2 < 5 min) in a parallel experiment.
Animals. All in vivo experimental procedures and animal handling
were conducted with approval approval from the Animal Ethics
Committee at the University of Queensland (UQ), and adhere to The
Australian Code of Practice for Use Of Animals for Scientific
Purposes (2013) and reported in accordance with the ARRIVE
guidelines.44 Male Wister rats (8 weeks old, 250−300 g) were bred at
the Animal Resources Centre (Canning Vale, WA) and air-
transported to the University of Queensland Biological Resources
Animal Facility (UQBR) at the Institute for Bioengineering and
Nanotechnology (AIBN). Animals were housed at room temperature
and kept in 12 h light/dark cycles. Standard chow and water were
provided ad libitum. The rats were acclimatized for 48 h before
experiments. Experiments were terminated by CO2 inhalation as
stipulated by approved ethical agreements.
Pharmacokinetic Studies. Male Wistar rats were surgically
implanted with a jugular vein catheter and fasted overnight, as
described.45,46 Blood samples (200 μL) were collected from the
indwelling catheter of an unanesthetized unrestrained rat, 5 min prior
to compound administration (10 or 8 mg/kg p.o. in olive oil, or 1 or
0.5 mg/kg i.v. in 50% DMSO) by oral gavage (75 mm soft-tipped
polyethylene 18G) or i.v. (directly via the indwelling catheter using a
250 μL glass Hamilton syringe). Further blood samples were collected
5, 15, and 30 min and at given time points from 1 to 8 h post
administration. Volumes collected were replaced with sterile hepari-
nized saline (20 U/mL). Blood samples were centrifuged (8000 rpm,
5 min), plasma-extracted, and snap-frozen on dry ice. The rats were
fed after the 4 h sample was collected. Compounds were quantitated
as described in the Supporting Information.
Rat Paw Edema. Hind paw swelling was induced by i.pl. injection
of BR103 (350 μg in 100 μL of 5% DMSO). Vehicle only was given
to sham paws. Treated animals were given an oral dose of a C3aR
antagonist at 10 mg/kg dissolved in olive oil 2 h prior to agonist
challenge. Sham and control rats received olive oil only. Paw thickness
and width were measured using digital calipers (WPI) at 0 (baseline),
0.5, 1, and 2 h after agonist BR103 administration. In all experiments,
hind paw swelling was expressed as % change in cross-sectional area
from baseline, normalized to sham against maximal swelling induced
by agonist alone. The magnitude of paw swelling and inflammatory
responses induced by the C3aR agonist BR103 were comparable to
responses induced by both a PAR2 agonist47 and L-carrageenan
(Supporting Figure 3).
1
for the preparation of 14a (57% over three steps). H NMR (600
MHz, DMSO-d6): δ 1.47−1.53 (m, 2H, NHCH2CH2CH2CH), 1.65−
1.72 (m, 1H, NHCH2CH2CH2CH), 1.77−1.83 (m, 1H,
NHCH2CH2CH2CH), 2.33 (s, 3H, CH3), 3.06−3.09 (m, 2H,
NHCH2CH2CH2CH), 4.25−4.29 (m, 1H, α-CH), 5.77 (s, 1H,
Ph2CH-thiophene), 6.62 (s, 1H, C4 thiophene), 7.24−7.26 (m, 6H,
Ph), 7.32−7.35 (m, 4H, Ph), 7.52 (t, 1H, J = 5.5 Hz, NHCH2), 8.04
(d, 1H, J = 8.1 Hz, NH). 13C NMR (150 MHz, DMSO-d6): δ 15.4,
25.5, 27.6, 40.3, 51.0, 52.1, 126.9, 128.5, 128.6, 129.7, 130.5, 140.3,
143.2/143.2, 149.0, 156.6, 162.5, 173.4. HRMS: C25H29N4O3S+
expected 465.1955 found 465.1955.
Isolation of Primary Human Monocyte-Derived Macro-
phages. HMDMs were harvested from buffy coat (of anonymous
human donors) provided by the Australian Red Cross Blood Service,
Brisbane. Monocytes were isolated using Ficoll-Paque Premium
density centrifugation (GE Healthcare Bio-Science, Uppsala, Sweden)
and repeatedly washed with ice-cold water to remove erythrocytes,
and CD14+ monocytes were then positively selected using human
CD14 MicroBeads and LS Columns (Miltenyi Biotec, Auburn).
CD14+ monocytes were then differentiated to HMDMs using 100 ng/
mL recombinant human macrophage colony-stimulating factor
(PeptroTech, Rocky Hill) for 7 days in Iscove’s modified Dulbecco’s
media supplemented with 10% fetal bovine serum, penicillin (50 U/
mL), streptomycin (50 μg/mL), and GlutaMAX (2 mM). HMDMs
were resupplemented after 5 days with fresh medium containing 10
ng/mL recombinant human macrophage colony-stimulating factor
and collected by gentle scraping on day 7.
Intracellular Calcium Release Assay. Intracellular calcium
release was monitored in real time at 1 s intervals using a fluorescence
imaging plate reader (FLIPR, Molecular Devices, CA) for at least 200
s (excitation 495 nm, emission 520 nm). HMDMs were seeded
overnight in 96-well clear-bottom black-wall plates at a density of 5 ×
104 cells/well. Prior to assay, the cells were incubated with dye-
loading buffer (Hank’s balance salt solution (HBSS) buffer, 4 μM
Fluo-3 AM, 0.04% Pluronic acid F-127, and 1% fetal bovine serum)
for 1 h at 37 °C. The cells were then washed once with assay buffer
(HBSS supplemented with 2.5 mM probenecid and 20 mM N-(2-
hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), pH 7.4).
For agonist measurements, HBSS (50 μL/well) was added first into
the assay plate and agonist was dispensed (50 μL/well 2×
concentration) using FLIPR. For antagonist measurements, antago-
nist (50 μL/well 2× concentration) was preincubated for 30 min at
37 °C followed by human full-length synthetic 100 nM C3a (50 μL/
well 2× concentration). It was important not to reuse and just wash
FLIPR tips to prevent carryover or cross-contamination of
compounds in the calcium release assay. The percent response was
plotted against log concentration of test compound.
LAD2 Mast Cell Culture. LAD2 human mast cells (kindly
provided by Dr. Dean Metcalfe, National Institute of Allergy and
Infectious Diseases, National Institutes of Health) were cultured in
StemPro-34 Serum Free Media (Invitrogen, Australia) supplemented
with StemPro-34 nutrient, 100 U/mL penicillin, 100 μg/mL
streptomycin, 2 mM GlutaMAX, and 100 ng/mL human stem cell
factor (SCF). Cells were hemidepleted weekly and were not allowed
to grow beyond a density of 5 × 105/mL. This cell line was
authenticated by expression of tryptase and histamine granules.
β-Hexosaminidase Release Assay. LAD2 human mast cells
were seeded overnight in 96-well plates at a density of 1 × 104 cells/
well, and a β-hexosaminidase release assay was performed as
previously described.43 Briefly, LAD2 cells were presensitized with
IgE (100 ng/mL) overnight before experiment. In the antagonist
assay, LAD2 cells were preincubated with compounds 3, 10a, 14a, or
14b for 30 min at 37 °C in 0.1 M HEPES buffer (pH 7.4) before the
addition of C3a (1 μM), and release of β-hexosaminidase was
For duration of action experiments, a separate group of rats
received 30 mg/kg of either BR111 or JR14a dissolved in olive oil via
oral gavage at 1, 2, 4, 6, 8, and 12 h prior to i.pl. injection of agonist
BR103 into the hind paws. Control rats received olive oil 2 h prior to
agonist challenge. Paw thickness and width were measured using
digital calipers at 0 and 0.5 h after BR103 injection. To further probe
J
DOI: 10.1021/acs.jmedchem.9b00927
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the molecular changes that occur after JR14a administration, a group
of sham, BR103 control, and JR14a-treated (10 mg/kg) rats were
terminated at 0.5 h after agonist challenge, and their paw tissues were
collected for further analyses (see next sections). To further assess the
anti-inflammatory property of JR14a, two separate experiments were
performed. In the first experiment, JR14a was administered to a group
of rats at four different doses (5, 10, 20, and 30 mg/kg) dissolved in
olive oil 2 h prior to i.pl. BR103 challenge. Sham and control rats
received olive oil only. Paw thickness and width were measured using
digital calipers at 0, 0.25, 0.5, and 1 h after BR103 injection. In the
second experiment, another group of rats received JR14a at 10 mg/kg
dissolved in olive oil at 0.5, 1, 2, and 4 h prior to i.pl. agonist
challenge. Again, sham and control rats received olive oil only. Paw
thickness and width were measured using digital calipers at 0 and 0.5
h after BR103 injection.
ViiA 7 Real-Time PCR System (Applied Biosystems). All samples
were duplicates, and target genes were normalized to housekeeping
gene Hprt. Fold change was calculated relative to sham. Sequences of
primers (Sigma-Aldrich) are listed in Supporting Table 1.
Statistical Analysis. Experimental results were expressed as mean
standard error (SEM). Graphs were plotted and data were analyzed
using GraphPad Prism7. Statistical differences were assessed using
Student’s t-tests (two-tailed) for data sets of two, one-way ANOVA
for data sets of three or more with Bonferroni post-hoc tests, or two-
way repeated measures ANOVA with Tukey post-hoc tests for
temporal data sets, as appropriate. Significance was set at +p < 0.05, +
+p < 0.01, +++p < 0.001, ++++p < 0.0001 compared to sham, and *p
< 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to
disease control.
Histopathology and Immunohistochemistry. After euthanasia
of the rats by CO2 inhalation, plantar soft tissues of the left paw were
dissected out with a scalpel blade and placed in 10% neutral-buffered
formalin for 2 h at 4 °C for fixing, before being transferred to 25%
sucrose solution in PBS overnight at 4°C. Tissues were then
embedded with Optimal Cutting Temperature compound (OCT,
Sakura Finetek, CA). Frozen sections of 5 μm were cut using a
cryostat (Leica Biosystems, Germany). Tissue sections were briefly
washed with distilled water before staining with 1% alcian blue
(Sigma-Aldrich, pH 2.5) for 30 min and 0.5% safranin O (Sigma-
Aldrich) for 5 min. They were then dehydrated and mounted in
dibutylphthalate polystyrene xylene (DPX, Sigma-Aldrich).
For immunohistochemistry, paw tissue sections were briefly rinsed
with PBS to remove the OCT. Except for tryptase staining, antigen
retrieval was performed by incubating tissue sections in citrate buffer
(0.01 M, pH 6) at 95 °C for 20 min. They were then cooled down
briefly and incubated with blocking medium (PBS, 0.1% triton X-100,
10% horse serum) for 1 h at room temperature in a humidity
chamber. Samples were then incubated with the respective primary
antibodies (PBS, 0.1% Triton X-100, 4% horse serum; 1:100 primary
antibody, including histamine, neutrophil elastase, or ED1 antibodies)
overnight at 4 °C. The next day, the samples were washed with PBS
and incubated with the secondary antibody (PBS, 0.1% Triton X-100,
4% horse serum, detection antibody 1:200) for 2 h at room
temperature. For tryptase staining, tissue sections were incubated with
primary antibody (as above with tryptase antibody at 1:100 dilution)
for 4 h, then secondary antibody (at 1:200 dilution) for 2 h. Finally,
tissue sections were dried, counterstained with 4′,6-diamidino-2-
phenylindole (DAPI, Invitrogen, Australia), and sealed with clear nail
polish. Primary antibodies for tryptase (AA1, ab2378, Abcam,
Australia), histamine (H7403, Sigma-Aldrich), neutrophil elastase
(ab21595, Abcam, Australia), and ED1 (ab31630, Abcam, Australia)
were purchased from commercial sources. Secondary detection
antibody was purchased from Abcam, Australia. All microscopic
images were obtained using an Olympus BX-51 upright microscope
with Olympus DP-71 12Mp color camera, utilizing DP Capture and
DP Manager software packages (Olympus, Tokyo, Japan). Trypatse-,
neutrophil elastase-, and ED1-positive cells were quantified using
FIJI/ImageJ 1.42q software.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b00927.
■
S
Experimental descriptions for synthesis and character-
ization (1H and 13C NMR, LCMS, HRMS) of
compounds 7a−e, 8a−o, 9a−o, 10a−o, 11a−j, 12a−j,
1
and 14a−k; 1D H NMR spectra, HPLC traces, and
concentration−response curves for intracellular calcium
release in HMDM for 10a−o and 14a−k; competitive
binding curves for human C3a, BR103 (2), and BR111
(10a) against Eu-DTPA-C3a on HEK293-Gα16-C3aR
cells; and pharmacokinetics description and primer
sequences for PCR (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: r.reid@imb.uq.edu.au (R.C.R.).
*E-mail: d.fairlie@imb.uq.edu.au (D.P.F.).
ORCID
Robert C. Reid: 0000-0002-0829-239X
Junxian Lim: 0000-0002-8309-0704
David P. Fairlie: 0000-0002-7856-8566
■
Author Contributions
∥J.A.R., R.C.R., and E.K.Y.P. joint first authors.
Notes
The authors declare the following competing financial
interest(s): M.-K.Y., R.C.R., and D.P.F. are named inventors
on a patent (US14356,821), involving complement C3a
receptor agonists and antagonists, that is owned by the
University of Queensland. The remaining authors declare no
competing interests.
RNA Isolation and Gene Analysis. Following euthanasia of the
rats, plantar soft tissues from their right hind paws were dissected out
with a scalpel blade, snap-frozen with liquid nitrogen, and kept at −80
°C until RNA extraction was performed. Paw tissues were
homogenized in TRIsure (Bioline, Australia, 1 mL per sample)
using a Bullet Blender tissue homogenizer (Next Advance, NY) at 4
°C. Afterward, 200 μL of chloroform was added and the mixture was
shaken vigorously. Samples were incubated at RT for 5 min before
centrifugation (11 000g, 15 min, 4 °C). The clear supernatant from
each sample was collected for RNA isolation using ISOLATE II RNA
Mini Kit (Bioline, Australia) according to manufacturer’s instructions.
RNA (500 ng) was converted to cDNA using SuperScript III Reverse
Transcriptase (Invitrogen, Australia) and Oligo(dT) 12−18 primer
(Invitrogen, Australia) in a Veriti 96-well thermal cycler (Applied
Biosystems). Real-time polymerase chain reaction (PCR) was
conducted using SYBR Green (Life Technologies, Australia) in a
ACKNOWLEDGMENTS
■
The authors thank the National Health and Medical Research
Council for grant (1145372, 1084018, 1028423) and fellow-
ship (Senior Principal Research Fellowships 1027369,
1117017) support. They also acknowledge support from the
Australian Research Council (grant DP130100629,
DP180103244), the Australian Research Council Centre of
Excellence in Advanced Molecular Imaging (CE140100011),
and the Queensland Government (CIF grant). They thank the
University of Queensland for PhD scholarships to J.A.R.,
E.K.Y.P., and M.-K.Y. and a UQ Postdoctoral Research
Fellowship to A.I. The authors thank Dr. Alun Jones (Institute
for Molecular Bioscience) for mass spectrometry advice and
K
DOI: 10.1021/acs.jmedchem.9b00927
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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ABBREVIATIONS USED
■
ANOVA, analysis of variance; Boc, tert-butyloxycarbonyl;
BOP, benzotriazol-1-yloxytris(dimethylamino)-phosphonium
hexafluorophosphate; C3a, Complement protein component
3; C3aR, Complement C3a receptor; Ccl2, Cd244, cluster of
differentiation 244; CCL2, chemokine (C−C motif) ligand 2;
dCha, D-cyclohexylalanine; CNS, central nervous system;
DCM, dichloromethane; DTPA, diethylenetriaminepentaacetic
acid; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dime-
thylformamide; DMSO, dimethylsulfoxide; DPX, dibutylph-
thalate polystyrene xylene; ED1, monoclonal antibody clone
directed against the rat CD68 protein; Et3SiH, triethylsilane;
GPCR, G-protein-coupled receptor; H-bond, hydrogen bond;
hC3a, human Complement protein C3a; HBSS, Hank’s
balanced salt solution; HEK-293, human embryonic kidney
293 cells; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesul-
fonic acid); HMDMs, human monocyte-derived macrophages;
Hprt, hypoxanthine-guanine phosphoribosyltransferase;
HRMS, high-resolution mass spectrometry; iCa2+, intracellular
calcium; Ig, immunoglobulin; IL, interleukin; LAD2, labo-
ratory of allergic diseases 2; LDA, lithium diisopropylamide;
LPS, lipopolysaccharide; MAC, membrane attack complex;
NBO, natural bond orbital; OCT, optimal cutting temperature;
PCR, polymerase chain reaction; PBS, phosphate-buffered
saline; rp-HPLC, reversed-phase high-performance liquid
chromatography; RT, room temperature; SCF, stem cell
factor; SEM, standard error of mean; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; TMS, trimethilsilane; TNF, tumor
necrosis factor α; TOF, time of flight
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C3a receptor agonists derived from oxazole amino acids: Structure-
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