An Agonist Radioligand for the Proinflammatory Lipid-Activated G Protein-Coupled Receptor GPR84 Providing Structural Insights

Article abstract of DOI:10.1021/acs.jmedchem.9b01339

The orphan G protein-coupled receptor (GPCR) GPR84 is expressed on immune cells mediating proinflammatory and immunostimulatory effects. In this study, we prepared the fully efficacious, nonbiased GPR84 agonist 6-hexylamino-2,4(1H,3H)-pyrimidinedione (6)

Full text of DOI:10.1021/acs.jmedchem.9b01339

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Article
An agonist radioligand for the proinflammatory lipid-activated
G protein-coupled receptor GPR84 providing structural insights
Meryem Köse, Thanigaimalai Pillaiyar, Vigneshwaran Namasivayam, Elisabetta De
Filippo, Katharina Sylvester, Trond Ulven, Ivar von Kügelgen, and Christa E Müller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01339 • Publication Date (Web): 13 Nov 2019
Downloaded from pubs.acs.org on November 14, 2019
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An agonist radioligand for the proinflammatory lipid-
activated G protein-coupled receptor GPR84
providing structural insights
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Meryem Köse,†,& Thanigaimalai Pillaiyar,†,& Vigneshwaran Namasivayam,†,& Elisabetta De
†
†
¥
%
†,
Filippo, Katharina Sylvester, Trond Ulven, Ivar von Kügelgen, and Christa E. Müller *
†PharmaCenter Bonn, Pharmaceutical Institute, Department of Pharmaceutical and Medicinal
Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
¥Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2,
DK-2100 Copenhagen, Denmark.
%Pharma Center Bonn, Department of Pharmacology and Toxicology, Sigmund-Freud-Str. 25, D-
5
3127 Bonn, Germany.
&Authors contributed equally
KEYWORDS: agonist, diindolylmethane, docking studies, G protein-coupled receptors, GPR84,
homology modeling, medium-chain fatty acid, orphan GPCR, radioligand, structure-activity
relationships, synthesis, uracil.
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Abstract
The orphan G protein-coupled receptor (GPCR) GPR84 is expressed on immune cells mediating
proinflammatory and immunostimulatory effects. In this study, we prepared the fully efficacious,
non-biased GPR84 agonist 6-hexylamino-2,4(1H,3H)-pyrimidinedione (6) in tritium-labeled form
0
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(
[3H]PSB1584) by hydrogenation of a hexenyl-substituted precursor with tritium gas. The
radioligand was characterized by kinetic, saturation, and competition assays using membranes of
3
Chinese hamster ovary cells recombinantly expressing the human GPR84. [ H]6 reversibly labeled
the receptor with high affinity (K 2.08 nM). Structurally diverse orthosteric and allosteric ligands,
D
including newly designed and synthesized compounds, were studied in competition binding assays.
A homology model of GPR84 was generated to perform docking studies rationalizing the
experimental data. The radioligand was additionally used for labeling GPR84 in native cells and
3
tissues. [ H]6 constitutes the first GPR84 agonist radioligand representing a powerful tool for this
poorly investigated GPCR, which has potential as a future drug target.
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Introduction
GPR84 is a proinflammatory G protein-coupled receptor (GPCR), which is highly expressed on
cells of the immune system (e.g. neutrophils, macrophages) and is upregulated under inflammatory
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conditions. GPR84 functions as an amplifier of inflammatory signaling by enhancing the release
7
of proinflammatory mediators, such as TNFα and various interleukins. Its activation results in G
i
4
signaling leading to a reduction in cAMP production via inhibition of adenylate cyclase. In
addition, GPR84 has been described to signal through G /G , linking receptor function to
1
2
13
Rho/Rac signaling and modulation of the cytoskeleton. In human macrophages, GPR84 activation
was found to mediate G protein-dependent Erk1/2 and Akt phosphorylation as well as G -βγ-
i
i
6
,8,9
dependent PI kinase activation and calcium mobilization.
So far, only limited data regarding
3
its functional significance is available. However, recent studies confirm the involvement of GPR84
in metabolic regulation and inflammation and indicate its clinical relevance in various diseases.
GPR84 was found to be involved in inflammatory processes relevant to gastroesophageal reflux
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0
11
5
12
disease, inflammatory bowel disease, multiple sclerosis, neuropathic pain and Alzheimer’s
1
3
14-17
disease. Moreover, GPR84 has been linked to obesity and diabetes.
Preliminary evidence
1
8
19
indicates that GPR84 might be associated with leukemogenesis, osteoclastogenesis, as well as
organ fibrosis, a pathological outcome of many inflammatory and metabolic diseases.20
GPR84 was identified as a fatty-acid sensing receptor, being activated by micromolar
4
concentrations of medium chain fatty acids (MCFAs) with chain lengths of C9-C14. Decanoic
acid (1) was described as the most potent MCFA with a moderate EC value of 4.5 µM. Several
5
0
laboratories confirmed GPR84 activation by MCFAs and additionally by hydroxylated MCFAs.7,21-
2
5
Nevertheless, to date, it is still not fully accepted by the scientific community that the weakly
potent MCFAs represent the endogenous agonists of GPR84. Due to the lack of potent ligands,
comprehensive studies were hampered. Only recently, potent synthetic agonists have been
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developed. The naturally occurring benzoquinone embelin (3-undecyl-2,5-dihydroxy-1,4-
benzoquinone, 2), a lipid-like molecule, also acts as a GPR84 agonist.21,26,27 Subsequent structure-
activity relationship study of 2 led to a new embelin analogue with 45-fold higher potency at human
8
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GPR84 than embelin itself. The synthetic compound 6-octylaminouracil (3) was discovered as
7
another surrogate GPR84 agonist by screening of a compound library. High-throughput screening
efforts by Zhang et al. resulted in the identification of 2-(hexylthio)-6-hydroxypyrimidin-4(3H)-
2
8
one (4). Further optimization of lead structure 4 resulted in the development of 6-nonylpyridine
29
derivative 5. Lead compound 3 was optimized leading to the development of potent GPR84
agonists (e.g. compound 6 (6-hexylamino-2,4(1H,3H)-pyrimidinedione) and 7 (6-((p-bromo-
2
4
phenylethyl)amino)-2,4(1H,3H)-pyrimidinedione). A structurally very different GPR84 agonist
is the natural product-derived 3,3'-diindolylmethane (DIM, 8),30 which had previously been
described to activate the estrogen receptor31 and the arylhydrocarbon receptor (AhR)32 at
micromolar concentrations (>5 µM). Subsequent studies confirmed the ability of 8 to activate
4
,22,24,25,28
human GPR84 with EC values between 0.5 to 5.9 µM.
DIM was proposed to bind to an
5
0
allosteric site distinct from the decanoic acid binding site and to act as an ago-allosteric modulator
activating the receptor by itself and also increasing the effects of lipidic and lipid-like agonists such
as decanoic acid.22,25,26 Starting from DIM as a lead structure, potent GPR84 agonists were
developed, such as the agonist 3,3'-di-(5,7-difluoro-1H-indole-3-yl)methane (9, PSB-16671).26
However, 9 appears to additionally interact with (an)other target(s).33
In contrast to agonists, the development of GPR84 antagonists has so far only been described in
34
the patent literature. In a recent study, antagonists from a patented series were reported as non-
competitive antagonists, binding to a different binding site than the fatty acids and also different
2
5
from that of 3,3'-diindolylmethane. Structures and EC values of selected standard GPR84
50
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agonists together with their potencies determined in different functional assay systems are
summarized in Table 1.
Table 1. Potencies of selected standard GPR84 agonists determined in different assay systems
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
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7
8
9
0
1
2
3
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9
0
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6
7
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0
O
O
OH
O
HN
HO
HO
O
N
H
N
H
O
1
Decanoic acid
2 Embelin
OH
3 6-Octylaminouracil
O
OH
N
HN
O
N
N
HO
N
S
HO
N
H
H
6
6-Hexylamino-2,4(1H,3H)-
pyrimidinedione (PSB-1584)
4
2-Hexylthiopyrimidine-4,6-diol
5 6-Nonylpyridine-2,4-diol
F
F
O
Br
HN
F
O
N
N
F
N
N
N
N
H
H
H
H
H
H
7
2
6-((p-Bromophenylethyl)amino)-
,4(1H,3H)-pyrimidinedione (PSB-17365)
8 3,3'-Diindolylmethane (DIM)
9 3,3'-Di-(5,7-difluoro-1H-indol-3-
yl)methane (PSB-16671)
Assay system
Calcium
assay
35
cAMP assay [ S]GTPγS
IP assay β-arrestin assay
Standard GPR84 agonists
EC (µM)
assay
EC (μM)
5
0
50
EC (μM)
EC (µM)
5
a,4
0
50
e,4
b,26
Decanoic acid (1)
4.5a, 4
4.6
48
30
n.d.
n.d.
6.08
10
b,26
j,25
f,21
c,21
7
20
4
.42
25
j,25
8d,22
c,8
0.63 j,25
0.63h,21
b,26
Embelin (2)
0.089
0.400
j,25
b,21
0
0
0
.2
5.0
a,21
b,26
.421
.795
b,24
0.512d,7
g,29
0.105h, 7 0.114
b,24
6-Octylaminouracil (3)
0.0167
0.653
1.25
c,28
g,28
i,28
0
0
.341
.438
1.75
c,29
j,25
c,28
c,29
g,29
0.597i,28
2
-Hexylthiopyrimidine
derivative (4)
0.001
0.134
0.00079 j,25
0.139
0.213
n.d.
g,28
0
.144
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6
(
6
-Nonylpyridine-2,4-diol
5)
-Hexylaminouracil (6)
0.000352c,29 n.d.
0.000189g,29 n.d.
n.d.
b,24
b,24
b,24
0.00493
0.00249
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.00318
0.104
b,24
6-(p-Bromophenyl-
ethylamino)uracil (7)
3
b,26
0.5a,4
1.64b,26
,3'-Diindolylmethane (8)
0.252
n.d.
n.d.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a,4
j,25
0
5
1
.7
.9
.11
1.0
d,22
c,28
d,30
11
j,25
1.26
b,26
5.47b,26
3,3'-Di-(5,7-difluoro-1H-
indol-3-yl)methane (9)
0.0413
n.d.
n.d.
n.d.
a
b
c
CHO/human GPR84 cells; CHO/ß-arrestin2/human GPR84 cells. HEK293/human GPR84 cells.
d
e
Sf9 insect/Gαi/human GPR84 cells. CHO/aequorin reporter/G , G , G , and G /human
α16
qs5
qo5
qi9
f
g
GPR84 cells. CHO/Gqi5/human GPR84 cells. HEK293/Gα16/human GPR84 cells.
h
i
j
HEK/Gqi5/human GPR84 cells. HEK293/β-arrestin2/human GPR84 cells. Flp-In TREx 293-
FLAG-human GPR84-eYFP cells. n.d., not determined.
As shown in Table 1, the determined agonist potencies in the different assay systems can vary
considerably. For example, determined EC values for agonist 3 range between 0.0167 and and
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.438 µM (26-fold difference). It is well known that functional assays can be influenced by various
factors. Receptor expression levels, ligand bias, activity of signaling cascades in specific cells, or
nonspecific, receptor-independent effects can affect the measured EC50 values. Thus, values
obtained in different functional test systems are not comparable.35,36
However, to date, only functional data are available, and so far, only two studies have been
performed attempting to investigate the binding mode of agonists.22,25 While Nicaido et al. created
22
a homology model of GPR84 based on the active-state structure of the β -adrenoceptor, Mahmud
2
et al. generated a homology model using the transmembrane domain architecture of the orexin
2
5
receptor type 1 (OX receptor). Moreover, mutagenesis studies were performed and the mutants
1
were investigated in GTPγS assays investigating G protein activation.22,25
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In addition to functional assays, radioligand binding studies would be an ideal basis for molecular
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modeling and docking studies. An antagonist GPR84 radioligand, [ H]G9543, whose preparation
3
4
was described in a patent, and which had been utilized by Milligan et al., could neither be
2
5
3
1
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6
0
1
2
3
4
5
6
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9
0
1
2
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7
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9
0
1
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9
0
1
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9
0
1
2
3
4
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6
7
8
9
0
displaced by decanoic acid nor by DIM. Consequently, [ H]G9543 was supposed to bind to a
third binding site distinct from the putative orthosteric binding site for fatty acids, and also different
from the allosteric binding site for DIM. Thus, to date, it has not been possible to directly assess
the binding affinities of standard GPR84 agonists. To address this issue, we prepared a high-affinity
agonist GPR84 radioligand, which binds to the putative orthosteric, fatty acid binding site. In a
previous study, we had shown that unlabeled 6 is a metabolically stable, highly potent and fully
efficacious, non-biased agonist at human GPR84 as determined in cAMP (EC 4.93 nM) and β-
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arrestin (EC 3.18 nM) assays. We selected this compound for tritium labeling to obtain the first
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agonist radioligand for GPR84. The radioligand was fully characterized and applied in competition
binding assays. The determined binding data served as a basis for analyzing interactions of ligands
with GPR84 studied by docking of compounds into a receptor homology model.
Results and discussion
3
Chemistry. The radioligand [ H]6 was prepared as depicted in Scheme 1. N-Hexenylphthalimide
(12) was synthesized by Mitsunobu reaction from commercially available phthalimide (10) and
E)-hex-2-enol (11) in the presence of diethyl azodicarboxylate (DEAD) and triphenylphosphine.37
(
Compound 12 was converted to the amine 13 using hydrazine hydrate under reflux conditions.
3
8
Amine 13 was subsequently reacted with 6-chlorouracil (14) under reflux to afford the desired
radioligand precursor (E)-6-(hex-2-en-1-ylamino)pyrimidine-2,4(1H,3H)-dione (15). The catalytic
hydrogenation procedure was initially investigated with non-radioactive hydrogen gas to prepare
non-radioactive 6, before radiolabeling was approached. In procedure A, the reaction was
performed at 25 psi (1.7 bar) in methanol (MeOH) : tetrahydrofuran (THF) (1:1) in the presence of
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1
0% palladium on activated charcoal for 2 h at room temperature. In procedure B, the reaction was
performed at 35 psi (2.4 bar) in N,N’-dimethylformamide (DMF) : THF (1:1) as a solvent instead.
Under both conditions, 15 was completely hydrogenated to product 6. The alkene 15 was
subsequently custom-labeled via hydrogenation of the double bond with tritium gas to obtain the
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3
desired radioligand [ H]6, whose identity was confirmed by mass spectrometry. [ H]6 displayed a
specific activity of 60 Ci/mmol (2.22 TBq/mmol) and a radiochemical purity of 99.8% as
determined by HPLC−UV (254 nm). (see Figures S1-S3).
3
a
Scheme 1. Preparation of the GPR84 agonist radioligand [ H]6
O
O
(i)
(ii)
NH + HO
N
CH3
CH3
H N
2
CH3
O
O
10
11
12
13
O
O
O
HN
HN
O
HN
13, (iii)
(iv)
O
N
H
Cl
N
H
N
H
CH3
O
N
H
N
H
CH3
6
14
15
(v)
O
HN
O
*
*radiolabel
N
H
N
H
CH3
*
[3
3
H]6 ([ H]PSB-1584)
a
°
Reagents and conditions: (i) PPh , EtO CN=NCO Et, THF, 0 C to rt, 12 h, 95%; (ii) hydrazine
3
2
2
°
o
hydrate solution (~80% in H O), 100 C, 3 h, 75.6%; (iii) 1-butanol, 125 C, 12 h, 91%; (iv)
2
procedure A: MeOH:THF (1:1), Pd/C (10%), H , 1.7 bar, 2 h, 98%; procedure B: N,N’-
2
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dimethylformamide (DMF):THF (1:1), Pd/C (10%), H , 2.4 bar, 2 h, 97.5%; (v) custom labeling
2
in THF as a solvent (for details see SI).
A large number of previously described GPR84 ligands4,8,24,25,26,27 were subsequently studied in
binding assays using the new radioligand. In addition, we synthesized three series of new lipid-like
compounds (Scheme 2). Pyrimidine derivatives 4, 62-68, 73-77 and 79-85 were prepared from 6-
hydroxy-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (61), 4-hydroxy-pyrimidine-2(1H)-thione
derivatives (69-71), or 2-thiopyrimidine-4-carboxylic acid (72), respectively, by alkylation with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(aryl)alkyl bromides in the presence of potassium carbonate in methanol under reflux conditions.
Carboxylic acid ester 76 was hydrolyzed in the presence of 2-N sodium hydroxide to produce the
corresponding carboxylic acid 77. Orotic acid ester and amide derivatives were synthesized from
orotic acid (78) as depicted in Scheme 2. For preparing the orotic acid esters 79 and 80, compound
7
8 was dissolved in butanol or hexanol and refluxed for 12 h in the presence of catalytic amounts
of concentrated sulfuric acid. Orotic acid amides were obtained by condensation of 78 with various
amines in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-
hexafluorophosphate (HATU) and triethylamine (TEA) in DMF.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 2. Synthesis of 4, 62-68, 73-77 and 79-85a
1
4
: R = CH , n = 4
3
OH
1
OH
NH
62
:
R = C H n = 2
6
5,
1
63: R = C H n = 3
(i)
N
6 5,
1
R1
6
4: R = 4-F-C H n = 1
6 5,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
S
n
1
O
N
S
65: R = 4-Cl-C H n = 1
6 5,
H
H
1
4
,62-68
66: R = 4-Br-C H n = 1
6 5,
61
1
6
7: R = 4-CH -C H n = 1
3 6 5,
1
6
8: R = 4-CF -C H n = 1
3
6 5,
OH
1
OH
73: R = CH , n = 4
3
1
2
7
4: R = CH , R = CH , n = 4
3 3
N
(i)
N
1
2
R1
3
7
3
7
5: R = C H , R = CH , n = 4
R2
N
S
R2
N
S
76: R = CO CH , R = CH , n = 4
2 3 3
1
2
n
H
1
2
7
7: R = CO H, R = CH , n = 4
73-76
2 3
69-72
(ii)
77
1
O
O
79: R = CH , n = 2
3
1
8
0: R = CH , n = 4
3
HN
HN
1
(iii)
81: R = C6H5, n = 2
R1 82: R = CH , n = 3
OH
X
1
O
N
H
O
N
H
3
n
1
O
O
83: R = CH , n = 4
3
1
8
4: R = CH , n = 6
78
3
7
8
9, 80: X = O
1-85: X = NH
1
8
5: R = CH , n = 7
3
a
Reagents and conditions: (i) K CO , methanol, reflux, 12 h; (ii) from 76, 2 N NaOH, methanol,
2
3
o
8
0 C, 1 h; (iii) for esters (X = O): alcohols, H SO , reflux, 12 h; for amides (X = NH): amines,
2 4
HATU, TEA, DMF, 12 h, rt.
Biological experiments
Pharmacological characterization of the new radioligand. Membrane preparations of a CHO
cell line stably transfected with the human GPR84 (CHO-hGPR84) were used to establish a
3
radioligand binding assay. In preliminary experiments, specific binding of 3-10 nM of [ H]6 to
GPR84-expressing cells was detected, while only negligible binding to non-transfected CHO cells
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was observed under the same conditions (see Figure S4). Subsequently, we investigated the effects
3
of the buffer composition on [ H]6 binding (1 nM). The addition of 0.05 % of fatty acid-free bovine
serum albumin (BSA) to tris(hydroxylmethyl)aminomethane (TRIS) as an incubation buffer
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
significantly increased specific binding (8-fold, 0.0024, **). The addition of MgCl (10 mM) also
2
increased specific binding by 3-fold. Therefore, we combined both, MgCl and BSA, which led to
2
a further significant increase in specific binding (Figure 1). In contrast, the addition of NaCl led to
a significant decrease in specific binding while the IC value of agonist 6 was not affected (Figure
5
0
1
and Figure S5). Sodium ions are known to act as negative allosteric modulators of many
3
8
2+
GPCRs. In contrast, Mg ions may promote the formation of a high-affinity agonist-receptor-G
protein complex acting as a positive allosteric modulator. The same effect had been observed in
previous studies with agonist radioligands, e.g. for GPR35 and GPR17.39-42
Thus, 50 mM TRIS buffer, pH 7.4 containing 10 mM MgCl and 0.05 % fatty acid-free BSA was
2
found to be the optimal assay buffer resulting in low non-specific binding of only 15%, and these
conditions were therefore used in all subsequent binding assays.
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1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
4
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5
5
5
5
5
5
6
2
2
1
1
1
1
250
000
750
500
250
000
**
*
*
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Specific binding
Nonspecific binding
1
2
3
: 50 mM Tris
: 50 mM Tris + 0.05 % BSA
: 50 mM Tris + 10 mM MgCl2
**
4
: 50 mM Tris + 10 mM MgCl + 0.05 % BSA
2
7
50
500
250
0
1
2
3
4
3
Figure 1. Effects of BSA and cations on [ H]6 binding. The experiments were performed at 25 °C
3
(
150 min) using 1 nM [ H]6 and 10 µg of a membrane preparation of CHO cells expressing the
human GPR84 (CHO-hGPR84 cells). Data represent means of two independent experiments
performed in duplicate; dpm, decays per minute. The unpaired t-test was used for statistical
comparisons (**, p<0.01).
As a next step, kinetic studies with CHO-hGPR84 cell membranes (10 µg of protein) were
3
performed using 1 nM [ H]6 in 50 mM TRIS buffer, pH 7.4, supplemented with 10 mM MgCl and
2
0
.05 % BSA. Both association and dissociation appeared monophasic (Figure 2A/B). In the
association experiments (t = 21 min) an equilibrium was reached within less than 120 min which
1
/2
remained stable for at least 6 h indicating that an incubation time of 150 min is sufficient to ensure
3
equilibrium binding of [ H]6. The binding was reversible after the addition of a high concentration
2
4
(100 µM) of the potent GPR84 agonist 7 with a dissociation half-life of 32 min (Figure 2B). A
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kinetic K value of 2.04 nM was calculated. In saturation experiments using 12 different
D
radioligand concentrations ranging from 0.04 to 10 nM, a K value of 2.08 nM and an apparent
D
Bmax value of 1.87 pmol/mg protein were determined (Figure 2C). We subsequently transferred the
assay to a 96-well plate format, which allows a higher throughput than the originally used single
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
vials. A radioligand concentration of 2 nM [ H]6 was found to be suitable for performing
competition binding assays in a 96-well plate format. The nonspecific binding at that concentration
amounted less than 7 % of total binding. Homologous competition experiments with the unlabeled
ligand 6 were performed using a high concentration of GPR84 ligand 7 (10 µM) to determine non-
specific binding. Homologous competition assays for agonist 6 revealed a K value of 2.98 nM
i
(Figure 2D). Thus, all three determined affinity values, K for the unlabeled agonist 6, the kinetic
i
3
K value, as well as the K value of [ H]6 obtained in saturation assays, were virtually identical.
D
D
125
100
75
A
120
100
B
8
6
0
0
5
2
0
5
0
40
t1/2 = 21 min
t1/2 = 32 min
2
0
0
0
100
200
300
400
500
0
30 60 90 120 150 180 210 240 270
Time (min)
Time (min)
150
125
00
5
000
D
C
total binding (dpm)
4000
specific binding (dpm)
nonspecific binding (dpm)
1
3
2
1
000
000
000
0
7
5
2
5
0
5
0
0
2
4
6
8
10
10-11 10-10 10-9 10-8 10-7 10-6 10-5
[6], M
[3
H]6 (nM)
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
Figure 2. (A) Association and (B) dissociation curve of [ H]6 binding (1 nM) at 25 °C to membrane
preparations of CHO-human GPR84 cells (10 µg protein/vial). Dissociation was achieved by
addition of the GPR84 agonist 7 (100 μM) after 150 min of pre-incubation. A kinetic K value of
D
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.04 nM was determined. (C) Saturation curve of binding of increasing concentrations of [ H]6;
experiments were performed at 25 °C with 10 µg of protein per vial. For the determination of
nonspecific binding, 10 µM of 7 was used. The following binding parameters were calculated:
K = 2.08 ± 0.68 nM, B = 1.87 ± 0.19 pmol/mg protein. (D) Homologous competition
D
max
experiments. A K value of 2.98 ± 0.38 nM was determined for 6 (see Table 2). Mean values ±
i
SEM from 3-6 independent experiments performed in duplicates are shown.
Binding affinity of standard GPR84 ligands. With the optimized radioligand-binding assay, we
initially investigated the standard GPR84 agonists 1-4 and 6-9 in competition experiments (for Ki
values see Table 2-5). As expected, compounds 1-4, 6 and 7 with a lipid-mimetic structure were
able to completely displace the radioligand (Figure 3). The determined K values were lower than
i
the EC values previously determined in functional assays (for previously determined EC values
5
0
50
see Table 1). Decanoic acid (1) and embelin (2) both showed a higher affinity (K 1780 nM and
i
23.6 nM, respectively) in binding assays compared to the previously determined potencies in
functional assays (3- to 14-fold for 1 and 8- to 212-fold for 2). Agonist 3 displayed a K value of
i
0
.626 nM in the binding studies, which is 27- to 2800-fold lower than the respective EC values
50
determined in functional assays (see Table 1). Similarly, 4 (K 0.219 nM) displayed a 5- to 2730-
i
fold higher affinity in binding studies compared to the potency measured in functional studies.
Interestingly, the K value for 6 (2.98 nM) corresponded very well with the EC values previously
i
50
determined in cAMP and β-arrestin assays (4.93 nM and 3.18 nM, respectively). In contrast, the Ki
value of 7 (0.317 nM) was 8- to 330-fold lower than the published EC values for 7.
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0
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8
9
DIM (8) did not lead to an inhibition but to a concentration-dependent increase in the specific
3
binding of [ H]6 by up to 100 % compared to the control in the absence of DIM (Figure 3).
Although previous studies had suggested that 8 may be an ago-allosteric modulator,22,25,26 it has
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
not been possible so far to study direct receptor-ligand interaction for DIM. Here we directly show
3
for the first time that 8 indeed binds to a different site than the lipid mimetic [ H]6. The observed
3
increase in specific binding of [ H]6 further suggests that 8 not only activates the receptor, but at
the same time, it positively modulates the binding of the lipid mimetic 6, which is presumed to act
as an orthosteric GPR84 agonist. This observation is in agreement with previously reported cAMP
data showing an increase in efficacy and potency for decanoic acid with increasing concentrations
of 8.26
250
200
150
100
1
2
3
4
7
8
9
5
0
0
1
0-12
10-10
10-8
10-6
10-4
[GPR84 agonist], M
Figure 3. Competition binding experiments for standard agonists versus the agonist radioligand
[3
H]6. The experiments were performed as described in the Experimental section. K values for
i
lipid-like agonists were as follows: 1780 ± 285 nM for 1 (decanoic acid), 23.6 ± 6.8 nM for 2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
embelin), 0.626 ± 0.205 nM for 3 (6-octylaminouracil), 0.219 ± 0.054 nM for 4 (2-
hexylthiopyrimidine
derivative),
and
0.317
±
0.033
nM
for
7
(6-(p-
bromophenylethylamino)uracil). For positive allosteric modulators, EC values were as follows:
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
68 ± 42 nM for 8 (diindolylmethane) and 41.5 ± 7.0 nM for 9 (5,5’,7,7’-tetrafluoro-DIM). Data
points represent means ± SEM from 3-6 independent experiments, each performed in duplicates.
Binding affinities of medium-chain (hydroxy)fatty acids. Next, we investigated several
medium-chain (hydroxy)fatty acids (C8-C14, (hydroxy)MCFAs) in binding assays at GPR84
(Table 2). In parallel, we also evaluated these MCFAs in functional assays (cAMP and β-arrestin
assays).
Table 2. Binding affinities and functional potencies and efficacies of (hydroxy)fatty acids at the
human GPR84
O
O
O
OH
O
R
R
HO
HO
R
HO
HO
OH
OH
Structure A
1, 16-20)
Structure B
(21, 22)
Structure C
(23-26)
Structure D
(
(27)
Human GPR84
Radioligand binding
assay
cAMP assay
β-Arrestin assay
EC ± SEM (µM)
(or percent receptor
activation at
5
0
Compound
K ± SEM (µM)
vs. [ H]6
EC ± SEM (µM)
(or percent receptor
activation at 100
i
50
R
3
(or percent inhibition ±
100 µM)
c
SEM at 100 µM)
µM) [efficacy]
b
[
efficacy]
a
a
(n=3)
(n=3)
a
(n=3)
Embelin (2)
for structure see Table 1)
0.0236 ± 0.0068
0.795 [86 %]26
0.400 [100 %]26
(
Structure A: Fatty acids
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Octanoic acid (16)
C H
19.6 ± 6.8
> 100 (36 %)
> 100 (2 %)d
6
13
17
19
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Decanoic acid (1)
C H
8
1.78 ± 0.29f
0.108 ± 0.024
2.17 ± 0.24
1.93 ± 0.80
2.64 ± 0.63
7.42 [100 %]26
6.08 [92%]26
Undecanoic acid (17)
Dodecanoic acid (18)
Tridecanoic acid (19)
Tetradecanoic acid (20)
C H
9
1.60 ± 0.14 [93 %]e
8.87 ± 0.75 [94 %]
9.61 ± 1.25 [51 %]
11.4 ± 1.6 [63 %]
4.36 ± 1.51 [82 %]e
C H
3.49 ± 0.56 [92 %]e
1
0
21
23
25
0
1
2
3
4
5
6
7
1.70 ± 0.64 [77 %]d
C H
1
1
7.01 ± 3.20 [61 %]e
C H
1
2
Structure B: 2-Hydroxy-fatty acids
R,S)-2-Hydroxy-
(
C H
≈ 100 (50 %)
0.318 ± 0.070
> 100 (17 %)
> 100 (5%)
5
11
8 octanoic acid (21)
9 (R,S)-2-Hydroxy-tetra-
C H
3.86 ± 1.00 [102 %]
4.36 ± 1.42 [101 %]
1
1
23
0
1
decanoic acid (22)
Structure C: 3-Hydroxy-fatty acids
2
3
4
5
6
7
(
R,S)-3-Hydroxy-
decanoic acid (23)
R,S)-3-Hydroxy-do-
decanoic acid (24)
C H
3.31 ± 0.81
31.8 ± 4.67 [97 %]
1.31 ± 0.15 [112 %]
1.57 ± 0.47 [88 %]
1.75 ± 0.62 [91 %]e
4.05 ± 1.35 [54 %]
3.25 ± 1.34 [125 %]
0.432 ± 192 [78 %]d
0.452 ± 0.158 [77 %]
6
13
(
C H
0.110 ± 0.021
0.511 ± 0.107e
0.472 ± 0.120
8
17
8 (R,S)-3-Hydroxy-tetra-
C H
1
0
21
9
0
1
2
decanoic acid (25)
R)-3-Hydroxy-tetra-
decanoic acid (26)
(
C H
1
0
21
3 Structure D: 12-Hydroxydodecanoic acid
4
(R,S)-12-Hydroxy-do-
See struct.
D
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
> 100 (-17 %)d
>
10 (5 %)
> 100 (27 %)
decanoic acid (27)
a
Unless otherwise noted
Efficacy [Emax] relative to the max. effect of decanoic acid (100 μM) (= 100%).
b
c
Efficacy [Emax] relative to the max. effect of embelin (10 μM) (= 100%).
d
e
f
n=2; n=4; n=7.
Undecanoic acid showed the highest binding affinity among the tested MCFAs with a K value of
i
1
1
08 nM (see Table 2 and Figure S6). It was also the most potent fatty acid in cAMP assays (EC50
.60 µM). Fatty acids with a shorter or a longer chain length displayed lower binding affinities
ranging between 2-20 µM. We detected a K value of 19.6 µM for octanoic acid although no activity
i
in cAMP and β-arrestin assays at concentrations up to 100 µM could be observed for this fatty acid.
4,7
In previous studies, octanoic acid had also been shown to be inactive in functional assays. In
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
general, we observed a high correlation between K values determined in binding studies and the
i
EC values determined in cAMP accumulation studies using the same cell line (see Table 2 and
5
0
2
Figure 4, R = 0.906), while correlation with EC values obtained from -arrestin assays appeared
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
to be much lower (R = 0.621). Previously, it had been difficult to predict the true binding affinities
of GPR84 agonists. The herein determined binding affinity for decanoic acid (1.78 µM), for
example, is 100-300 higher than the predicted affinity (K 170-530 µM) based on functional data.25
A
4
7
For tetradecanoic acid, a very high EC value of 93.2 µM or even no activity had been reported
5
0
in previous studies. In contrast, we determined a K value of 2.64 µM in binding assays. Using the
i
same CHO cell line that overexpresses the human GPR84, the EC values were 11.4 µM (cAMP
50
assay) and 7.01 µM (β-arrestin assay), respectively. These data illustrate, that functional data for
agonists are highly dependent on the test system, e.g. on receptor expression levels, whereas
binding data are largely independent and provide “true” affinities.
Medium-chain free fatty acids
6-(Ar)alkylamino-substituted
uracil derivatives
2
cAMP assay, R =0.906
2
2
cAMP assay, R =0.775

-arrestin assay, R =0.621
2
-arrestin assay, R =0.476
1
0
9
8
7
6
10
9
6.5
5.5
4.5
3.5
6.5
5.5
4.5
3.5
8
7
6
3.5
4.5
5.5
6.5
6
7
8
9
10
pK values, radioligand binding assay
pK values, radioligand binding assay
i
i
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7
8
9
Figure 4. Correlation of pEC values of test compounds determined in cAMP assays (●) or β-
5
0
arrestin assays (■) versus their respective pK values determined in radioligand binding assays. The
i
2
same cell line (CHO-β-arrestin-human GPR84 cells) was used for all assays. R was determined in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a two-tailed Pearson test. A. fatty acid derivatives; B. 6-(ar)alkylamino-substituted uracil
derivatives.
All hydroxyfatty acids, except for (R,S)-3-hydroxy-decanoic acid (23), showed higher binding
affinities than the respective non-hydroxylated fatty acids confirming previous results from
7
functional assays reported by Suzuki et al. The position of the hydroxyl group, at C2 or C3, did
not significantly affect the binding affinity (compare 22 and 25).
Binding affinities of 6-(ar)alkylamino-substituted uracil derivatives. Next, a series of 6-
2
4
(
ar)alkylamino-substituted uracil derivatives (28-60) previously synthesized in our group were
tested for their binding affinity to the human GPR84 (for selected curves see Figure 5A, for Ki
values see Table 3). As depicted in Figure 4B, the binding affinities correlated with the agonist
2
potencies previously determined in cAMP assays (r =0.775), but much less with pEC values
5
0
2
determined in β-arrestin assays (r =0.476) (for the previously obtained functional data see Table
S2). As seen for the standard agonists 1-4, 6 and 7, binding affinities were up to 57-fold (cAMP
assay, on average 17-fold) or up to 803-fold (β-arrestin assay, on average 161-fold) higher
compared to their respective potencies in functional assays.
Table 3. Affinities of agonists at the human GPR84 determined in radioligand binding assays.
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2
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
O
O
O
4
5
6
Br
N O
HN3
2
HN
HN
HN
HN
HN
1
R1
R1
R1
R1
O
N
N
H
O
N
H
N
O
N
H
N
O
N
H
N
O
N
H
N
H
O
N
H
N
H
H
CH3
N
R1
R1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Structure A
6,28-49)
Structure B
(50,51)
Structure C
Structure D
(53-55)
Structure E
(56,57)
Structure F
(58-60)
(
(52)
Human GPR84
Radioligand binding
assays
R1
a
Compd.
K ± SEM (µM)
i
3
b
vs. [ H]6 (n=3)
Structure A: 6-(Aryl)alkylaminouracils
-CH (CH ) -CH
2
8
0.0910 ± 0.0080
2
2 3
3
3
6 (PSB-1584)
-CH (CH ) -CH
0.00298 ± 0.00038c
2
2 4
29
-CH (CH ) -CH
0.000866 ± 0.000204
0.000626 ± 0.000205
2
2 5
3
3
3 (6-octylaminouracil)
-CH (CH ) -CH
2 2 6
30
-CH (CH ) -CH
0.00112 ± 0.00007
0.000726 ± 0.000076
0.267 ± 0.016
2
2 7
3
3
3
3
1
2
-CH (CH ) -CH
2 2 8
-CH (CH ) -OH
2
2 6
3
3
3
4
0.0108 ± 0.0022
0.000580 ± 0.000176
CH3
35
36
0.00125 ± 0.00021
0.0114 ± 0.0008
F
Cl
Br
3
7
7
0.000650 ± 0.000038
0.000317 ± 0.000033
(PSB-17365)
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3
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5
6
7
8
9
3
3
4
8
9
0
0.000582 ± 0.000063
0.00127 ± 0.00043
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.00948 ± 0.00201
0.00475 ± 0.00079
0.00399 ± 0.00061
F
41
Cl
Br
4
4
2
3
0.0356 ± 0.0090
0.0360 ± 0.0032
F
4
4
4
4
5
6
Cl
Cl
0.00203 ± 0.00018
0.497 ± 0.032
Cl
NH
47
0.0494 ± 0.0072
S
O
4
4
8
9
0.00312 ± 0.00071
0.00210 ± 0.00018
0.00694 ± 0.00031
O
6
Structure B: N -Methyl-substituted uracil derivatives
-CH (CH ) -CH
5
0
2
2 4
3
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
1 Y-364
-CH (CH ) -CH
0.00360 ± 0.00047
0.0187 ± 0.0021
2
2 6
3
Structure C
5
2 Y-573
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Structure D
5
3
Cl
0.0444 ± 0.0049
0.0147 ± 0.0028
54 Y-617
55
0.0627 ± 0.0019
Structure E
5
5
6
0.225 ± 0.011
CH3
7 Y-373
0.0347 ± 0.0100d
CH3
Structure F
CH3
5
8
0.0586 ± 0.0140
0.0370 ± 0.0129
59 Y349
CH3
60
0.189 ± 0.052
a
3
Affinities were determined in competition binding experiments using 2 nM [ H]6 and
b
membrane preparations of CHO cells recombinantly expressing hGPR84. Unless
otherwise noted. n=6; n=4.
c
d
Interestingly, for compound 6, previously determined as the most potent unbiased GPR84 agonist
2
4
of the series, no significant difference between the K value and the EC values in both functional
i
50
assays was observed (K 2.98 nM and EC 4.93 nM, respectively). In contrast, 37, previously
i
50
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7
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9
determined as the most potent G -biased GPR84 agonist of the series, showed the highest difference
i
(
803-fold) between its K value from radioligand binding (0.650 nM) and its EC value determined
i
5
0
in β-arrestin assays (EC 522 nM).
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B
A
125
6
2
1
1
25
00
63
64
100
6
5
6
8
7
5
7
5
2
5
0
5
7
4
3
5
5
5
5
4
1
4
2
9
77
50
25
0
8
1
82
83
0
1
57
0-12
10-10
10-8
10-6
10-10
10-8
10-6
10-4
[Agonist], M
[6-(Ar)alkylamino-substituted
uracil derivatives], M
Figure 5. Competition binding experiments at membrane preparations of recombinant CHO cells
expressing human GPR84. A. Competition binding curves for selected 6-(ar)alkylamino-
substituted uracil derivatives. B. Competition binding curves for synthesized lipid-like compounds
62-65, 68, 74, 77, 81-83. Data points represent means ± SEM from 3 independent experiments,
each performed in duplicates. For K values see Table 4.
i
Binding affinities of thiopyrimidones and tetrahydropyrimidine carboxylic acid esters and
28
amides. Taking the potent GPR84 agonist 4 (2-(hexylthio)-6-hydroxypyrimidin-4(3H)-one). as a
lead structure, we prepared a small series of derivatives and analogues and evaluated them in
radioligand binding assays for their GPR84 affinity (for selected curves see Figure 5B, for K values
i
see Table 4). Agonist 4 itself displayed a K value of 0.219 nM and was actually the compound
i
with the highest affinity of all investigated GPR84 ligands. 6-Nonylpyridine-2,4-diol (5) previously
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
28
described to be even more potent than 4, was found to display 7-fold lower affinity as compared
to 4 in binding studies (K 1.56 nM). 6-Octylaminouracil (3, 0.626 nM) and 6-p-
i
bromophenethylaminouracil (7, 0.317 nM) belong to the compounds with the highest GPR84
affinity being similarly potent as 4, while the affinity of 6-hexylaminouracil (6, 2.98 nM) is slightly
lower. The rank order of potency of lipophilic agonists was generally somewhat different in
radioligand binding assays as compared to functional assays. One reason could be due to
difficulties in handling highly lipophilic, poorly water-soluble molecules. In binding assays
employing cell membrane preparations, a higher percentage of DMSO can be used than in cellular
systems, and predilutions can all be made in DMSO rather than aqueous buffer. Predilution in
buffer could lead to a loss of compound resulting in reduced concentrations in the assay. Therefore,
according to our experience, these data obtained in binding studies are most reliably representing
true affinities.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Introducing aromatic residues into the alkyl chain of lead structure 4 was well tolerated leading to
analogs with similarly potent affinity, e.g. 6-phenylethylthiopyrimidine-2,4-diol (62, K 0.700 nM),
i
6
-phenylpropylthiopyrimidine-2,4-diol (63, 0.324 nM), and p-chlorobenzylthiopyrimidine-2,4-
diol (65, 2.63 nM).
Deletion or replacement of the 4-hydroxy/oxo-function by hydrogen, alkyl or carboxylate in
compounds 73-77 abolished or dramatically reduced GPR84 affinity. Replacement of the amino
group in 6-(ar)alkylaminouracils by an ester or amide linker (compounds79-85) was also not well
tolerated (Table 4).
Table 4. Affinities of agonists at the human GPR84 determined in radioligand binding assays
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6
7
8
9
O
OH
OH
6
4
HN
1
3
5
N
5
N
X
O
N
H
R2
2
R2
2
R2
n
O
N
S
R1
4
n
N
S
3
6
n
O
H
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Structure A
4, 62-68
Structure C
9,80: X = O
1-85: X = NH
Structure B
3-77
7
8
7
Human GPR84
a
Radioligand binding assays
K ± SEM (µM) vs. [ H]6
n=3)
Compd.
n
R1
R2
3
i
(
Structure A
(Decanoic acid, also see Table 1))
1
1.78 ± 0.29
4b (2-(Hexylthio)-6-hydroxypyrimidin-4(3H)-one), also
0.000219 ± 0.000054
see Table 1)
5
(6-Nonylpyridine-2,4-diol, also see Table 1)
0.00156 ± 0.00037
6
2
2
3
1
-
-
-
0.000700 ± 0.000322
6
6
329
0.000324 ± 0.000090
0.00377 ± 0.00107
4
F
6
6
6
6
5
6
7
8
1
1
1
1
-
-
-
-
0.00263 ± 0.00050
0.00317 ± 0.00119
0.00328 ± 0.00043
0.0367 ± 0.0087
Cl
Br
CH3
CF3
Structure B
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
3
4
4
4
4
4
H
3.47 ± 0.65
3.63 ± 1.56
≥100 (51 %)
>10 (9 %)
CH3
CH3
CH3
74
CH3
7
7
5
6
C H
3 7
CO CH
2
3
CH3
CH3
0.145 ± 0.052c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
77
CO H
2
Structure C
7
9
2
4
2
-
-
-
> 10 (-3 %)
1.21 ± 0.22
1.59 ± 0.43c
CH3
CH3
80
8
8
1
2
1.56 ± 0.25c
3
4
6
7
-
-
-
-
CH3
CH3
83
0.469 ± 0.197c
>10 (12 %)
>10 (26 %)
8
8
4
5
CH3
CH3
a
3
Affinities were determined in competition binding experiments using 2 nM [ H]6 and membrane
preparations of CHO cells recombinantly expressing human GPR84. Compound 4 was
resynthesized and the synthetic procedure is described in the experimental section. n=2.
b
c
Binding affinities of diindoylmethane derivatives. We further selected a series of
diindolylmethane derivatives (compound 86-103) that were previously shown to activate GPR84
2
6
in cAMP accumulation and/or -arrestin recruitment assays. Like DIM (8) itself (Figure 3), most
of the substituted DIM derivatives and analogs also showed a concentration-dependent increase in
3
specific binding of [ H]6 (for selected curves see Figure 6, for K values see Table 5).
i
Table 5. Affinity of 3,3’-diindolylmethane derivatives at the human GPR84
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3
4
5
6
7
8
9
5
4' 5'
R1
6'
7'
R1
3
R1
6
4
R2
R2
R1
3
3'
2'
10
7
2
N
R3
N1'
N
2
2
N
N
1
R R
3
H
R
H
H
Structure A
8,9 86-98)
Structure C
(102,103)
Structure B
99-101)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(
Human GPR84
radioligand binding assaysa
b
EC (or IC ) ± SEM (µM)
5
0
50
R1
R2
R3
Compd.
3
vs. [ H]6
[
max. stimulation of radioligand
c
binding]
(n=3)d
Structure A: DIM or symmetric DIMs
8
9
8
8
8
8
9
9
H
5-F,7-F
4-F
0.368 ± 0.042 [100 %]
0.0415 ± 0.007 [87 %]d
(PSB-16671)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
6 (PSB-16357)
1.52 ± 0.19 [56 %]
7
4-Cl
H
0.175 ± 0.016 [94 %]
8 (PSB-16105)
5-OCH3
5-F
H
0.103 ± 0.063 [46 %]
0.199 ± 0.049 [125 %]d
9 (PSB-15160)
H
0.250 ± 0.080 [90 %]d
0
1
5-Cl
H
5-Br
H
0.0591 ± 0.0145 [69 %]
0.302 ± 0.059 [57 %]d
(43.7 ± 9.6)b
92 (PSB-16358)
93
6-F
H
6-Cl
H
94 (PSB-16381)
7-F
H
0.0618 ± 0.0136 [153 %]
0.129 ± 0.009 [36 %]
0.0836 ± 0.0167 [67 %]
0.0524 ± 0.0068 [55 %]
(14.0 ± 4.2)b
9
9
9
9
5
5-F,6-Cl
4-F,5-F
5-F,6-F
H
H
6
H
7 (PSB-16586)
H
8
CH3
27
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Journal of Medicinal Chemistry
Page 28 of 68
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
9
9 (PSB-16359)
-
-
1.93 ± 0.42 [59 %]
N
H
Structure B: unsymmetrically substituted diindolylmethane derivatives
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
CH3
100
-
-
-
-
0.495 ± 0.096 [53 %]
N
H
F
0.276 ± 0.065 [55 %]d
1
01(PSB-16244)
N
H
Structure C: Substitution of the methylene linker (C10)
102
03
CH3
CH3
–
–
(46.3 ± 14.9)b
(
22.4 ± 9.60)b
1
a
3
Affinities were determined in competition binding experiments using 2 nM [ H]6 and membrane
preparations of CHO cells recombinantly expressing hGPR84. Screenings were performed at a
b
c
concentration of 10 μM. IC values (inhibition of radioligand binding). Max. stimulation (%) of
5
0
3
radioligand binding relative to the max. binding of [ H]6 in absence of test compound (DMSO-
d
control (=100 %)). n=4.
28
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Page 29 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
3
2
2
50
00
50
00
9
9
94
3
9
98
102
103
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
150
1
00
50
0
1
0-10
10-8
3,3'-Diindolylmethane
derivatives], M
10-6
10-4
[
Figure 6. Competition binding experiments at membrane preparations of recombinant CHO cells
expressing human GPR84. Allosteric modulation of radioligand binding by 3,3’-diindolylmethane
derivatives. Data points represent means ± SEM from 3-7 independent experiments, each
performed in duplicates. For EC and IC values (B) see Table 5.
5
0
50
There was a certain correlation of EC values determined in cAMP accumulation assays with EC
50
5
0
2
values determined in radioligand binding studies (R = 0.433, see Figure 7). For example, the most
potent GPR84 agonist from this series, the tetrafluoro-DIM 9, showed almost the same EC value
5
0
in cAMP assays as in the binding study (41.3 nM, Table 1, as compared to 41.5 nM, Table 5).
29
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