Potent hFPRL1 (ALXR) agonists as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2006.04.068

We report the discovery of potent agonists for the human formyl-peptide-like 1 receptor (hFPRL1). These compounds did not act at a closely related receptor denoted human formyl peptide receptor (hFPR) up to 10 μM concentration. Recent studies have indicat

Full text of DOI:10.1016/j.bmcl.2006.04.068

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3713–3718
Potent hFPRL1 (ALXR) agonists as potential
anti-inflammatory agents
Roland W. Burli,a,* Han Xu,a Xiaoming Zou,b Kristine Muller,a Jennifer Golden,a
¨
Mike Frohn,a Matthew Adlam,b Matthew H. Plant,b Min Wong,b Michele McElvain,a
Kelly Regal,c Vellarkad N. Viswanadhan,d Philip Tagaria and Randall Hungatea
aChemistry Research and Discovery, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
bInflammation, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
cPharmacokinetics and Drug Metabolism, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
dMolecular Structure, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
Received 27 January 2006; revised 20 April 2006; accepted 20 April 2006
Available online 11 May 2006
Abstract—We report the discovery of potent agonists for the human formyl-peptide-like 1 receptor (hFPRL1). These compounds
did not act at a closely related receptor denoted human formyl peptide receptor (hFPR) up to 10 lM concentration. Recent studies
have indicated that agonizing this receptor may promote resolution of inflammation. In an exploratory study, a novel hFPRL1
agonist showed efficacy in a mouse ear inflammation model following oral administration.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Inappropriate inflammatory processes play a central
role in many diseases such as asthma and rheumatoid
arthritis. Many anti-inflammatory therapeutic agents
have been developed; however, most have some limita-
tions.1 Most of the currently used anti-inflammatory
drugs interfere with the action of pro-inflammatory
mediators, whereas less is understood about the
biochemical processes that resolve inflammation.
Principally, selective activation of such a pathway might
lead to an alternative treatment for inflammation.1 For
instance, Lipoxin A4 (LXA4) is an endogenously occur-
ring metabolite of arachidonic acid, which is formed in
response to inflammation.2 LXA4 and related structures
have been shown to promote resolution of inflammatory
processes, and it has been postulated that their anti-in-
flammatory effect is related to selective agonism of the
G-protein-coupled receptor hFPRL1 (also known as
ALXR or LXA4R).3–8 Recent studies suggest that this
receptor might also be involved in neuro-inflammatory
diseases.9 It is possible that an application of exogenous
Lipoxin A4 (or close analogs)10–12 for the treatment of
inflammation is hampered by the inherent physicochem-
ical properties of the poly-olefinic natural product.
Therefore, we set out to investigate whether a small-
molecule agonist for hFPRL1 would have potential as
an anti-inflammatory therapeutic agent. First non-pep-
tidic hFPRL1 agonists have recently been described.13,14
Using a cell-based assay in a high-throughput screen of
our compound library,15 we identified the pyrazolone 24
as a potent hFPRL1 agonist (Table 1). In absence of
structural information about the receptor, our next goal
was to establish a pharmacophore model by preparing
and testing analogs of 24. The synthesis of these com-
pounds is shown in Schemes 1 and 2. Condensation of
phenylhydrazine and various b-keto-esters (1–3),16 fol-
lowed by N-methylation of the cyclic intermediates,
led to the corresponding pyrazolones on multi-gram
scales. Subsequently, these pyrazolones were selectively
nitrosylated or nitrated at C(4), and the crude products
were reduced by hydrogenation to provide the respective
amines. Without further purification, the amines were
treated with a variety of aryl isocyanates in CH2Cl2.
Collection of the precipitated products by filtration,
followed by washing with CH2Cl2 or Et2O, provided
the final ureas 24–48. The N-ethyl derivative 49 was pre-
pared by analogy to this reaction sequence using ethyl
tosylate for the N-alkylation. The acetamide 14 and
Keywords: Inflammation; G-protein-coupled receptor; hFPRL1;
ALXR; Pyrazolones.
*
Corresponding author. Tel.: +1 805 447 9740; fax: +1 805 480
1337; e-mail: rburli@amgen.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.068

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R. W. Bu¨rli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3713–3718
Table 1. Influence of substituents on hFPRL1-mediated Ca2+ mobi-
lization (generic structure shown in Scheme 1, EC50 values in [lM])
Table 1 (continued)
Compound R1
R2
R3
hFPRL1a
Compound R1
R2
R3
hFPRL1a
EC50 [lM]
EC50 [lM]
43
CH3
CH3
0.044 0.005
>10
Cl
Cl
24
25
26
27
28
29
30
31
32
33
34
35
36
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3 0.03 0.01
CH3 0.09 0.03
CH3 0.49 0.09
CH3 4.85 1.16
CH3 >10
I
Cl
44
Br
Cl
F
45
46
CH3
0.023 0.006
0.31 0.12
F
CH2CH3 CH3
F
F
F
47
48
49
CH3
CH3
0.081 0.023
0.083 0.015
CH3 1.26 0.53
CH3 0.77 0.30
CH3 12.3 6.37
CH3 0.10 0.05
CH3 2.73 1.74
CH3 3.70 0.80
CH3 0.17 0.03
CH3 >10
CH3
CH2CH3 0.10 0.02
a EC50 values (n P 4) have been determined by Ca2+ flux in CHO
recombinant cells co-expressing hFPRL1, Ga15 protein, and
aequorin.
CF3
O
the thiourea 16 were synthesized from the commercially
available aniline 13 applying standard amide and thio-
urea formation conditions, whereas the N,N-dimethy-
lated urea 15 was obtained from urea 24 (MeI, NaH,
DMF).17 Compounds with purities below 95% (HPLC)
were purified by crystallization or trituration. Coupling
of the known hydroxypyrazole 1718 with various aryl
bromides under Buchwald-type amidation conditions
gave the pyrazolones 18–20 in modest yields, as well as
O-alkylated pyrazoles as major side products.19 The
intermediates 18–20 were converted to the final ureas
50–58 as described above. All compounds that were
tested for biological activity showed purities in excess
of 95% as evidenced by HPLC and 1H NMR
spectroscopy.20
CN
S
Cl
Cl
37
38
CH3
CH3
CH3 >10
F
As the 4-iodo-substituent in molecule 24 may constitute
a potential liability (e.g., homolytic C–I bond cleavage),
we started to investigate alternative groups at position 4
of the phenyl group (Table 1). Replacement of this io-
dide with other halides resulted in decreasing hFPRL1
potency, correlating well with the size of the halo-substi-
tuent. In fact, the unsubstituted phenyl urea 28 was
completely inactive up to at least 10 lM. The 4-ethyl
compound 30 was slightly more active than the methyl
derivative 29. In contrast, the sterically more demanding
isopropyl compound 31 lost significant potency
(12.3 lM). A more detailed survey of substituent chang-
es pending off the phenyl-urea moiety demonstrated that
the steric and electronic nature of the para-substituent
on the phenyl group has incisive effects on hFPRL1
activity. For instance, the 4-CF3–phenyl compound 32
CH3 0.98 0.50
39
40
CH3
CH3
CH3 >10
F
O
CH3 5.88 2.48
O
N
S
41
42
CH3
CH3 >10
CH2CH3 CH3 0.14 0.03
Cl

R. W. Bu¨rli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3713–3718
3715
R3
R2
R2
R2
R2
NH2
O
1
5
N
R1
O
N
N
N
N
N
N
N
N
(a)
(b)
(c)
(d)
N
Ph
cis conformation (W)
R3
COOEt
R2
H
H
Ph
Ph
Ph
O
O
O
O
R2: Ethyl
1
2
3
4
5
6
7
8
9
10
11
12
iPropyl
R2
NH
tButyl
N
13 R2: CH3
N
I
I
Ph
O
S
N
N
N
O
O
N
N
N
X
Ph
N
Ph
N
H
H
R
H
R1
24-48 R3 = CH3
49 R3 = CH2CH3
O
O
14 R = H, X = CH2
16
trans conformation (U)
15 R = CH3, X = NCH3
Scheme 1. Synthesis of 24–49. Reagents and conditions: (a) PhNHNH2 (1 equiv), 50% aq AcOH, 115 ꢁC, 3 h, 4 (53%), 5 (>95%), 6 (38%); (b)
CH3OTos (2 equiv), 160 ꢁC, 2 h, 7 (66%), 8 (23%), 9 (53%); (c) NaNO2, AcOH, aq HCl, 5 ꢁC, 1 h or 70% aq HNO3 (5 equiv), CF3COOH, 50 ꢁC,
30 min, then H2, Pd (black), AcOEt, MeOH, crude; (d) CH2Cl2, R-NCO (1.1 equiv), ca. 1 h, 24 ꢁC, 40–80%.
N
N
N
N
N
N
N
N
(a)
(b)
(c)
NH2
R1
NH
R1
R1
O
O
O
OH
O
HN
R1
17
18
19
20
21
22
23
N
N
N
R2
50-58
N
S
Scheme 2. Synthesis of 50–58. Reagents and conditions: (a) R1Br, CuI (0.05 equiv), 1,10-phenanthroline (0.11 equiv), Cs2CO3 (1.4 equiv), dioxane
(for 20, DMF), 110 ꢁC (sealed tube), 48 h, 18 (44%), 19 (28%); (b) 70% aq HNO3 (5 equiv), CF3COOH, 50 ꢁC, 30 min, then H2, Pd (black), AcOEt,
MeOH, 21 (34%), 22 (30%), 23 (5%, from 17, 3 steps); (c) CH2Cl2, R-NCO (1.1 equiv), ca. 1 h, 24 ꢁC, 50–90%.
was about as active as the one bearing the SMe group 35
or bromide 25 (100, 170, and 90 nM, respectively),
whereas the methoxy analog 33 showed potency only
in the micromolar range. Likewise, nitrile 34 was only
moderately active (3.7 lM). Alternating the position of
the substituent resulted in dramatic loss of potency, as
exemplified by the meta- and ortho-substituted chlorides
36 and 37, respectively. Various disubstituted ring
systems were investigated and compounds 38–40 stand
as representative examples: as above, subtle modifica-
tions in the substitution pattern at the phenyl moiety
were found to alter hFPRL1 activity dramatically; for
example, the 4-methyl-3-fluoro-compound 38 showed
low micromolar potency, while its isomer 39 was not
active below 10 lM. Replacing the phenyl group by a
thiazole ring (41) resulted in reduced activity as well,
suggesting that heterocyclic moieties at this position
might not be well tolerated.
activity (<10 lM), we reasoned that the bioactive
conformation of the ureas described herein might be
U-shaped. Quantum mechanic calculations suggest that
the urea 24 prefers a U-shaped conformation by
ꢀ1.4 kcal.22 Interestingly, for thioureas such as 16, the
U-shaped conformation is predicted to be more stable
than the W-conformer by about 4.6 kcal. Compound
16 demonstrated roughly equal potency as compared
to the urea analog 24 (26 nM vs 30 nM). Similarly to
the acetamide 14, the N,N-dimethylated derivative 15
did not show increased Ca2+ mobilization below
10 lM concentration. Next, we modified the substituent
at C(5) of the pyrazolone ureas and synthesized several
ethyl, isopropyl, and tertiary butyl derivatives (42–48).
We found that the potency of compounds with bulkier
C(5)-substituents was consistently enhanced [e.g., for
26 (Me) 490 nM; for 42 (Et) 140 nM; for 43 (iProp) 44
nM; for 45 (tButyl) 23 nM]. The observation that
increased steric demand of the group at C(5) gives rise
to enhanced activity may be a result of a shift in the
conformational equilibrium between the U- and
W-shaped conformation and/or additional van der
Waals interactions.
Generally, ureas are well known to adopt a cis or trans
conformation or to exist as a mixture of both conform-
ers.21 For molecules like 24, it is conceivable that an
intramolecular hydrogen bond stabilizes the trans or
U-shaped conformation (illustrated in Scheme 1). Such
a conformation is not expected to be highly populated
for the acetamide 14, since the H-bond donor has been
removed. As the acetamide 14 did not show hFPRL1
As above, the ortho-chloro substituted compound 44,
an isomer of the potent pyrazolone 43, did not show
activity up to 10 lM. Replacing the methyl group at

3716
R. W. Bu¨rli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3713–3718
N(1) with the larger ethyl substituent had no major im-
pact on potency (see 49).
EC50 values determined for Ca2+ mobilization, which
may be due to high protein binding of the compounds
and/or different levels of receptor expression in neutro-
phils versus transfected CHO cells. Interestingly,
16-phenoxy-Lipoxin,10 a well-documented stable analog
of Lipoxin A4 (prepared according to Phillips et al.),12
did not demonstrate blockage of PMN migration under
these conditions.
We also studied the influence of the N(2)-substituent of
the pyrazolone core on hFPRL1 activity (Table 2) and
found that modifications within this portion of the
molecule are relatively well tolerated. For example, the
N(2)-pyrimidines (50, 53, and 56) and quinolines (51,
54, and 57) were roughly equally or more potent than
the corresponding N(2)-phenyl derivatives, while thiaz-
oles (52, 55, and 58) showed somewhat reduced activity.
As part of an exploratory profiling, the active and inac-
tive iodo-pyrazolones 24 (30 nM) and 15 (>10 lM) were
studied in a mouse ear inflammation model (topi-
cal).25,26 For this, mice (10 animals per group) were
treated topically with test compound 15 min prior to
inducing an ear edema with a combination of prosta-
glandin E2 and leukotriene B4 (5 lg each). Ear thickness
was measured 3 h post induction and compared to the
thickness prior to the inflammatory insult. As illustrated
in Figure 1, the active pyrazolone 24 caused reduction of
ear edema, whereas the inactive analog 15 did not show
a significant effect. Under these conditions (applied
topically), 16-phenoxy-Lipoxin demonstrated consider-
able reduction of ear swelling (at 6.2 lg/ear: 22%, at
12.5 lg/ear: 33%, at 25 lg/ear, 69% reduction of
swelling).
Notably, the compounds described above did not show
significant activity (up to 10 lM) for the human formyl
peptide receptor (hFPR, counter-screen),23 which is the
closest homolog of hFPRL1.
Two pyrazolones with good hFPRL1-mediated Ca2+
flux activity (24, 43) were tested in a functional cell-
based assay side by side with two inactive analogs (15,
44): polymorphonuclear human neutrophils (PMN)
were preincubated for 45 min with compound or vehicle.
Subsequently, their migration against a gradient of
chemo-attractant (either IL-8 or N-formyl-methyl-leu-
cyl-phenylalanine = fMLP) was determined by fluores-
cence according to a standardized protocol by Martin
and coworkers (Table 3).24 In essence, the agonists 24
and 43 showed a dose-dependent inhibition of PMN
migration, irrespective of the nature of the stimulant,
while their inactive analogs 15 and 44 had no impact.
The concentrations required for inhibition of PMN
migration are somewhat higher than the respective
The poor solubility characteristics of the iodides 15 and
24 did not allow formulation necessary for advanced
in vivo studies. Therefore, we investigated the pharma-
cokinetic behavior and efficacy of the more soluble
pyrazolone 43. Following iv administration to rats
(0.5 mg/kg), the compound showed relatively low
Table 2. hFPRL1 activity of compounds 50–58a
R1
R2
Cl
SMe
CF3
N
50 0.25 0.07
53 0.043 0.005
56 0.15 0.02
N
51 0.080 0.013
54 0.030 0.003
55 5.13 1.75
57 0.044 0.006
58 1.47 0.68
N
N
52 0.54 0.46
S
EC50 values in [lM] (generic structure shown in Scheme 2).
a EC50 values (n P 4) have been determined by Ca2+ flux in CHO recombinant cells co-expressing hFPRL1, Ga15 protein, and aequorin.
Table 3. Inhibition of neutrophil (PMN) migration by pyrazolones (IC50 in [lM])
Compound
IC50 [lM]
hFPRL1b EC50 [lM]
fMLPa
IL-8
24
15
43
44
ꢀ2c
ꢀ2c
0.03 0.01
>10
>10
0.64 0.20
>10
>10
0.24 0.05
>10
0.044 0.005
>10
a Mean IC50 values determined using PMNs from at least three donors. IL-8 or fMLP were used as chemo-attractants (each 10 nM). Final
[DMSO] = 0.1%.
b hFPRL1 activity for comparison.
c Approximate IC50 as determined by mean value of two donors. PMNs of one donor did not show activity below 10 lM.

R. W. Bu¨rli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3713–3718
3717
350
300
2%
11%
8%
8%
**
31%
***
**
39%
35%
***
***
***
58%
54%
52%
***
71%
0
0
dosing [mg/kg]
dosing (μg/ear)
Figure 2. Reduction of ear swelling in mice: oral administration of
agonist 43. (X-axis: vehicle alone, 43 (po, 50, 16.6, 5 mg/kg),
dexamethasone (Dex) at 1 mg/kg (iv). Y-axis: ear thickness [lm], 3 h
post induction. Statistical significance: ***P < 0.005.)
Figure 1. Reduction of ear swelling in mice: topical administration of
agonist 24 and inactive analog 15. (X-axis: vehicle alone, topical
administration of 25, 2.5, and 0.25 lg/ear of 24 (white blocks), 15 (grey
blocks), and 30 lg of dexamethasone (Dex). Y-axis: ear thickness [lm],
3 h post induction. Statistical significance: **P < 0.01, ***P < 0.005.)
functional cell-based assay and in vivo (topical), while
its inactive derivative did not have an impact, indi-
cates that the observed anti-inflammatory effect is tar-
get-mediated. The differential effects of the
pyrazolones and 16-phenoxy-Lipoxin on PMN migra-
tion may suggest that the signal transduction events
triggered by these different classes of agonists differ
from each other. Such differences between synthetic
and endogenous agonists have been precedented.29
Studies to further address the mode of action of these
molecules are ongoing.
clearance (0.126 L/h/kg) and a half-life of 2.78 h. Signif-
icant levels of 43 were detected in the systemic circula-
tion such that the bioavailability following po
administration (2 mg/kg) was 91%. Encouraged by these
data, we tested 43 in the mouse ear inflammation model
described above following oral administration (Fig. 2):25
mice (10 animals per group) were treated orally with
compound 43 60 min prior to induction of edema and
the ear thickness was analyzed 3 h post insult. At the
50 mg/kg dose, a reduction in edema by 58% was ob-
served, which is similar to that of the positive control
(dexamethasone, 1 mg/kg iv).27 At this dose, the plasma
concentration of 43 at the end of the experiment (free
fraction, corrected for plasma protein binding) was
185 42 nM, that is, about four times higher than the
EC50 value for Ca2+ mobilization and in the range of
the IC50 value determined for IL-8 induced PMN migra-
tion.28 At the lower doses (16.6 and 5 mg/kg, respective-
ly), which resulted in only partial or no efficacy, the
endpoint concentration of free 43 was below the EC50
value (Ca2+ flux).
Importantly, a prototypic hFPRL1 agonist and PMN
migration inhibitor (43) with improved properties and
very good oral bioavailability showed a dose-dependent
inhibition of ear swelling in mice following oral
administration.
Acknowledgments
The authors thank Dr. Silvia Hu, Dr. Hai Yan, and
Michelle Shen for providing the hFPR cell line used in
the counter screen and for helpful discussions.
Notably, compound 43 did not show significant
cross-interaction with a series of unrelated protein
targets including the mitogen-activated P38a kinase,
the chemokine receptor CXCR2, the histamine receptor
1, and the vanilloid receptor 1.
References and notes
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In summary, a high-throughput screen and subsequent
medicinal chemistry effort resulted in potent and novel
hFPRL1 agonists. Two active compounds (24, 43)
inhibited fMLP and IL-8 induced human neutrophil
migration, whereas closely related structures with no
hFPRL1-mediated Ca2+ mobilization activity (15, 44)
did not show inhibition. Under these conditions,
16-phenoxy-Lipoxin did not regulate neutrophil migra-
tion. In a mouse model of acute inflammation (topical
application), compound 24 and 16-phenoxy-Lipoxin
were efficacious. Again, an inactive analog 15 did
not have an effect. The observation that a potent
hFPRL1 agonist showed inhibitory activity in a
3. Fiore, S.; Maddox, J. F.; Perez, H. D.; Serhan, C. N.
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transfected with aequorin, Ga15 protein, and hFPRL1.
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release of cytosolic Ca2+ storage. The increased cytosolic
[Ca2+] was monitored by luminescence emitted by aequo-
rin. For experimental details, see Refs.30 and 31.
6-31 G basis set at B3LYP/6-31 G level, were carried
* *
out on compounds 16 and 24.
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aequorin, and Ga15 protein. The assay was performed
as described.15
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R.; Martin, T. R. J. Immunol. Methods 1998, 213, 41.
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26. A stock solution of compound (50 lg/lL) was diluted to
2.5, 0.25, and 0.025 lg/lL using acetone/DMSO (95:5).
Ten microliters of these dilutions was topically applied
to the ear dermis 15 min prior to induction of ear
edema.
27. Dexamethasone (positive control) was administered as an
iv bolus. For the analysis, the 3-h time point was found to
provide an optimal window as the ear swelling slowly
declines afterwards (data not shown).
28. Compound 43 is 97.9% bound to mouse plasma as
determined by equilibrium dialysis. Analysis of blood
samples taken at the endpoint of the study revealed that at
the highest dose (50 mg/kg), the concentration of free
agent 43 was 185 42 nM (corrected for plasma protein
binding). Lower concentrations of unbound 43 were found
in blood samples from mice treated at lower dose ([43]
corrected for plasma protein binding: 17 7.0 nM at
16.6 mg/kg, 14 3.2 nM at 5 mg/kg).
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DMSO-d6.
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