New 'chemical probes' to examine the role of the hFPRL1 (or ALXR) receptor in inflammation

Article abstract of DOI:10.1016/j.bmcl.2007.09.043

We report the development of the novel N-substituted benzimidazole 11 as a potent and selective human formyl peptide receptor-like 1 (hFPRL1) agonist. This compound and its less active enantiomer 12 were identified as useful tools for studying receptor function in vitro.

Full text of DOI:10.1016/j.bmcl.2007.09.043

Bioorganic & Medicinal Chemistry Letters 17 (2007) 6633–6637
New ‘chemical probes’ to examine the role of the hFPRL1
(or ALXR) receptor in inflammation
Mike Frohn,a,* Han Xu,a Xiaoming Zou,b Catherine Chang,a Michele McElvaine,a
Matthew H. Plant,b Min Wong,b Philip Tagari,a Randall Hungatea and Roland W. Burlia
¨
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
Received 11 August 2007; revised 11 September 2007; accepted 11 September 2007
Available online 15 September 2007
Abstract—We report the development of the novel N-substituted benzimidazole 11 as a potent and selective human formyl peptide
receptor-like 1 (hFPRL1) agonist. This compound and its less active enantiomer 12 were identified as useful tools for studying recep-
tor function in vitro.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The human formyl-peptide receptor-like 1 (hFPRL1 or
ALXR) belongs to a family of Gi-protein-coupled recep-
tors that is predominantly expressed on neutrophils and
monocytes.1 Recent literature suggests that its role in
inflammation is complex; both pro- and anti-inflamma-
tory functions have been reported. hFPRL1 is modu-
lated by a wide variety of endogenous and exogenous
ligands; for instance, N-formylated peptides such as
the tripeptide N-formyl-Met-Leu-Phe (fMLP) are ago-
nists and chemo-attractants for the receptor.2 Since such
peptide ligands are primary products of bacterial (or
mitochondrial) translation, it has been postulated that
hFPRL1 evolved as part of a defense mechanism against
bacterial infections.3 hFPRL1 has also been reported to
mediate important pro-inflammatory processes in vari-
ous neurodegenerative states. The 42-amino acid form
of amyloid b (Ab42), which is believed to take part in
the pathogenesis of Alzheimer’s disease, can cause neu-
ronal damage via recruitment and activation of mono-
nuclear phagocytes (microglia) in the brain.4 Ab42 is
also a chemotactic agonist for hFPRL1 which suggests
that the receptor plays a role in this disease. Interest-
ingly, a 24-amino acid peptide called humanin blocks
the cytopathic effect of Ab42 in neuroblast cells by inter-
action with hFPRL1.5 In a different case serum amyloid
A, a precursor to the amyloid fibril deposits of amyloi-
dosis, is also a chemotactic hFPRL1 ligand (tested in
transfected HEK 293 cells).6 Moreover, the neurotoxic
prion protein fragment PrP106–126, which behaves in a
similar manner to the pathologic isoform of prion pro-
tein, chemoattracts and separately induces cytokine
secretion in human monocytes through interaction with
hFPRL1.7
Separate studies suggest an integral role of hFPRL1 in
host response to HIV-1 infection. Several synthetic pep-
tide domains of HIV-1 envelope proteins have been
shown to activate hFPRL1 in vitro, resulting in various
downstream events.8 Among these are the attenuation of
cell response to some chemokines, which likely occurs
via a cross-desensitization and down-regulation mecha-
nism of the receptors CCR5 and CXCR4 (both in hu-
man monocytes).
In contrast to the reported pro-inflammatory effects of
hFPRL1 modulators, Serhan and coworkers have
described anti-inflammatory effects of Lipoxin A4
(LXA4) and analogs, another class of hFPRL1 agonists.
The lipoxins, endogenously produced metabolites of
arachidonic acid, have been shown to promote resolu-
tion of acute inflammation through agonism of
hFPRL1.9 This interaction results in the attenuation of
immune cell chemotactic response towards pro-inflam-
matory mediators.10 There is also a growing body of evi-
dence suggesting that Annexin A1 (or Annexin-derived
metabolites), a glucocorticoid-regulated protein, inhibits
Keywords: hFPRL1; IL-6; Neutrophil migration; GPCR;
Benzimidazole.
*
Corresponding author. Tel.: +1 805 447 2761; fax: +1 805 480
1337; e-mail: mfrohn@amgen.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.043

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M. Frohn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6633–6637
leukocyte migration/activation through agonism of
hFPRL1.11 In summary, hFPRL1 is clearly involved
in inflammatory and pathogenic processes and therefore
is a target of interest for drug discovery. Further studies
will be required to understand how interference with
hFPRL1 signaling might be of therapeutic benefit. We
believe that small molecules, which selectively interact
with hFPRL1, will provide valuable tools for such
investigations.
hFPR (no activity up to 10 lM) and potential for struc-
tural diversification in a modular fashion.
Compounds were prepared according to the synthetic se-
quences outlined in Schemes 1 and 2. The benzimidazole
core was constructed via SNAr-type chemistry. For com-
pounds 4–9, ethyl 4-aminopiperidine-1-carboxylate was
added to 1-fluoro-2-nitrobenzene by heating in the pres-
ence of potassium carbonate, which resulted in the corre-
sponding
2-aminonitrobenzene
derivative.
This
intermediate was converted to bis-aniline I under modi-
Recently, we reported the discovery of pyrazolone 1 as a
potent and selective agonist of hFPRL1 (Fig. 1).12 This
molecule showed dose-dependent inhibition of polymor-
phonuclear human neutrophil (PMN) chemotaxis,
whereas the hFPRL1-inactive isomer 2 did not have
an effect.
´
fied Bechamp conditions (Feꢁ, AcOH). Mono-acylation
of this bis-aniline followed by dehydration of the result-
ing amide under microwave irradiation resulted in the
corresponding benzimidazoles. The carbamate function
was subsequently removed under basic conditions to give
the unprotected piperidines (generic structure II) and
amide coupling under standard conditions led to the final
compounds. Pyrrolidine-containing compounds 10–18
were prepared using an analogous strategy according to
Scheme 2.14 Final compounds were in excess of 95% pur-
ity as measured by HPLC and 1H NMR.15
Compound 1 also demonstrated promising efficacy in a
murine model of inflammation. Given that structurally
diverse agonists of hFPRL1 induce divergent down-
stream effects (as outlined above), we continued to study
the pyrazolones and started to develop a second series of
agonists. The goal was to generate another pair of active
and inactive molecules, which are structurally related to
each other, but diverse from the pyrazolones.
The effect of structural modifications to the benzimi-
dazolone moiety of compound 3 was investigated first.
Formal replacement of this group by a benzimidazole
resulted in a negligible increase in potency (compound
4, Table 1). However, the transition from benzimidazo-
lone to the benzimidazole core allowed modifications at
C(2), which proved beneficial for potency. As shown in
Table 1, benzimidazoles with small alkyl substituents
such as ethyl at C(2) exhibited submicromolar activity.
Increasing the size of this group from an ethyl group
Our screening campaign using an aequorin-based biolu-
minescence assay (Ca2+ flux) revealed compound 3
(Fig. 1) to be a relatively weak agonist of hFPRL1
(EC50 = 6.39 lM).13 Despite its moderate potency, this
molecule seemed an attractive starting point for further
optimization, since it showed modest selectivity over
i
to Bu, CF3 or Ph did not result in further improve-
ments. The ethyl-substituted benzimidazole fragment
was therefore selected for subsequent efforts.
HN
O
OMe
N
NH
N
We then turned our attention to the piperidine linker
element (Table 2) and observed that contraction of the
piperidine ring of 6 to a pyrrolidine 10 (racemic) resulted
in a five fold increase in potency. Application of the syn-
thetic sequence (Scheme 2) utilizing enantiomerically
pure 3-aminopyrrolidines yielded the individual antipo-
des 11 and 12, of which the R-isomer 11 proved signifi-
cantly more potent than S-enantiomer 12.
N
O
R2
HN
O
N
O
N
H
R1
1: R1 = Cl; R2 = H
3: hFPRL1 (EC50) = 6.39 μM
hFPRL1 (EC50) = 0.044 μM
2: R1 = H; R2 = Cl
hFPRL1 (EC50) >10 μM
The effects of compounds with modified indole groups
on hFPRL1-mediated Ca2+ mobilization are summa-
Figure 1.
F
H
CO2Et
N
HN
O
N
OMe
H
NO2
O
N
N
+
(f)
(a), (b)
(c), (d), (e)
+
N
N
HO
CO2Et
N
OMe
NH
R
N
N
NH2
R
I
II
III
NH2
R = H, Me, Ethyl, iButyl, CF3, Ph
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 100 ꢁC, 96%; (b) Feo, THF/H2O/AcOH (5:5:1), 68%; (c) RCOCl, NEt3, CH2Cl2, 0 ꢁC or
trifluoroacetic anhydride, pyridine, CH2Cl2; (d) CH3COOH, 150 or 180 ꢁC, microwave irradiation, 15 min; (e) H2NNH2, KOH, 2-propanol, 85 ꢁC or
2 N NaOH, reflux, 29–87% for 3 steps; (f) EDC, DMF or CH2Cl2, 29–88%.

M. Frohn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6633–6637
6635
O
H
NP
H2N
NH
N
NP
H
N
O
N
+
(a), (b)
(c), (d) or (e)
(f)
X
+
N
N
F
NH
X
HO
R
N
N
Et
R
Et
NH2
NO2
P = Bn for rac
P = Boc for R- and S-enantiomers
X = CH, N
R = OMe, F, Cl, Br, OCF3, Et
Scheme 2. Reagent and conditions: (a) K2CO3, DMF, 60 ꢁC, 46–65%; (b) Pd/C, H2, MeOH, 90–97%; (c) propionyl chloride, NEt3, CH2Cl2, 0 ꢁC to
room temperature, 59% for rac; (d) Pd/C, NH4CO2H, EtOH, reflux, 70% for rac; (e) HCl (2 equiv), dioxane, 80 ꢁC, 80% over 2 steps for ‘R’, 97%
over 2 steps for ‘S’; (f) EDC, DMF or CH2Cl2, 32–86%.
Table 1. Influence of benzimidazoles 4–9 (general structure III,
Scheme 1) on hFPRL1-mediated Ca2+ mobilization
Table 3. Influence of indole substituents/replacement on hFPRL1-
mediated Ca2+ mobilization
a
a
Compound
R
hFPRL1 EC50 (lM) hFPR EC50 (lM)
O
H
N
4
5
6
7
8
9
H
5.10 0.73
1.02 0.27
0.69 0.28
0.83
>10
>10
>10
>10
>10
>10
N
Me
Et
iBu
CF3
Ph
X
N
N
X
R
1.52
2.71
Et
a EC50 values have been determined by Ca2+ flux in CHO recombinant
cells co-expressing hFPRL1, Ga15 protein, and aequorin. Values
lacking standard deviation are the average of two data points.
a
a
Compound
R
hFPRL1 EC50
(lM)
hFPR EC50
(lM)
11
13
14
15
16
17
18
OMe
F
Cl
CH
CH
CH
CH
CH
CH
N
0.034 0.010
1.49
14.1
>10
>10
>10
>10
>10
>10
>10
rized in Table 3. A brief survey centering around the
nature of the C(5)-substituent revealed that C(5) OMe
is optimal for in vitro potency. The C(5)-bromo and
-ethyl substituted analogs 15 and 17 were the most
Br
0.81
12.2
OCF3
Et
OMe
0.27
1.87
potent alternatives, but lost significant activity (EC50
=
a EC50 values have been determined by Ca2+ flux in CHO recombinant
cells co-expressing hFPRL1, Ga15 protein, and aequorin. Values
lacking standard deviation are the average of two data points.
Table 2. Influence of amine ring modification on hFPRL1-mediated
Ca2+ mobilization
HN
O
0.81 and 0.27 lM, respectively) compared to 11.
Similarly, the indole core appeared to be necessary for
efficient downstream signaling; for example, the corre-
sponding benzimidazole analog 18 proved less active
(EC50 = 1.87 lM). Other indole replacements have been
studied in a slightly different series, but no equipotent
replacement of the 5-methoxy-indole unit has been iden-
tified (data not shown). None of the tested compounds
showed significant activity against hFPR below 10 lM
concentrations.16
OMe
N
n = 0, 1
N
Et
N
a
a
Compound
Amine ring
hFPRL1 EC50
(lM)
hFPR EC50
(lM)
6
0.69 0.28
0.13
>10
>10
In conclusion, optimization of the initial benzimidazo-
lone 3 (EC50 = 6.39 lM) for hFPRL1-mediated activity
N
N
in
a cell-based system yielded benzimidazole 11
10
(EC50 = 0.034 lM) and its enantiomer 12, which was
roughly 100-fold less active (EC50 = 3.50 lM). Our next
goal was to utilize these two molecules alongside the ini-
tially reported pyrazolones (1 and 2) in order to interro-
gate the functional consequence of hFPRL1 activation.
We first tested the impact of these compounds on poly-
morphonuclear human neutrophil (PMN) chemotaxis.
N
11
0.034 0.010
>10
N
12
3.50
>10
a EC50 values have been determined by Ca2+ flux in CHO recombinant
cells co-expressing hFPRL1, Ga15 protein, and aequorin. Values
lacking standard deviation are the average of two data points.
For this, freshly collected human PMN’s were preincu-
bated for 45 min with compound or vehicle and subse-

6636
M. Frohn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6633–6637
Table 4. Comparison of Ca2+ mobilization and inhibition of neutro-
phil (PMN) migration by compounds 1, 2, 11, and 12
hFPRL1 had no effect. The previously reported, structur-
ally diverse pyrazolones (1 and 2) showed the same trend
in the migration and cytokine secretion assays. From
these results, we conclude that the anti-inflammatory
inhibition of neutrophil migration of compounds 1 and
11 as well as their pro-inflammatory stimulation of IL-6
secretion are likely to be hFPRL1-mediated. Clearly,
more studies will be required to understand the complex
role of hFPRL1 in vivo and how it might serve as a ther-
apeutic target. We believe that the two pairs of structur-
ally distinct active and ‘inactive’ hFPRL1 agonists
(benzimidazoles 11 and 12; pyrazolones 1 and 2) will
provide excellent tools to study this receptor in more
detail.
Compound
Ca2+ flux
Migrationa
hFPRL1
b
EC50 (lM)
fMLP
IC50 (lM)
IL-8
IC50 (lM)
11
12
1
0.034 0.010
3.50
0.22 0.22
>10
1.71 2.19
>10
0.044 0.005
>10
0.64 0.20
>10
0.24 0.05
>10
2
aMean IC50 (lM) values determined using PMNs from at least three
donors. fMLP or IL-8 were used as chemo-attractants (each 10 nM).
Final [DMSO] = 0.1%.
b hFPRL1 activity for comparison.
quent migration against a gradient of chemo-attractant
(either IL-8 or fMLP) was followed by fluorescence
measurement according to Martin and coworkers.17
The result of this experiment is summarized in Table
4. For both structural series, it is clear that the hFPRL1
agonist inhibits neutrophil migration, whereas the struc-
turally related isomer/enantiomer, which does not mod-
ulate hFPRL1, had no effect on migration. These results
strongly suggest that the observed inhibition of neutro-
phil migration, which is anti-inflammatory in nature, is
indeed hFPRL1-mediated.
References and notes
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We then tested the effect of the same four molecules on
secretion of IL-6 from human whole blood.18 In this
experiment, 50% human blood was incubated with IL-
1b both in the presence and absence of test compound
for 18 h. The secretion of IL-6 was then detected by fluo-
rescence measurement. As exemplified in Figure 2, we ob-
served that the hFPRL1 agonists 1 and 11 stimulate IL-6
secretion above levels induced by IL-1b alone (defined as
100 POC). The less potent analogs did not cause an effect.
This stimulatory effect is clearly pro-inflammatory in
nature.
4. Le, Y.; Gong, W.; Tiffany, H. L.; Tumanov, A.; Nedo-
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In summary, optimization of a weakly potent hFPRL1
agonist 3 resulted in a structurally novel, optically active
hFPRL1 agonist 11 with an in vitro activity well below
micromolar concentrations (EC50 = 34 nM). Its enantio-
mer 12 was about 100-fold less active in the Ca2+ mobili-
zation assay. Subsequently, the pair of enantiomers was
studied in functional cell-based assays of inflammation.
We have found that the hFPRL1-active enantiomer 11
inhibits neutrophil migration in a dose-dependent man-
ner, while its ‘hFPRL1-inactive’ analog 12 did not show
an effect. In contrast, we observed that the active molecule
significantly stimulates IL-6 secretion in human whole
blood; again, the compounds that did not modulate
6. Su, S. B.; Gong, W.; Gao, J.-L.; Shen, W.; Murphy, P. M.;
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1g
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9. For a recent review, see (a) Chiang, N.; Serhan, C. N.;
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Figure 2. Stimulation of IL-6 secretion by hFPRL1 agonists in 50%
human whole blood.

M. Frohn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6633–6637
6637
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15. HPLC (Phenomenex, MAX RP, 4l, 50· 2.0 mm, 1 mL/
min, A: 0.1% TFA in H2O, B: 0.1% TFA in MeCN, 10–
100% B in 10 min), 1H NMR (Bruker, 400 MHz) in
DMSO-d6 or CDCl3.
16. For the counter screen, CHO cells were stably transfec-
ted with human formyl peptide receptor (hFPR),
aequorin, and Ga15 protein. The assay was performed
as described.
17. Frevert, C. W.; Wong, V. A.; Goodman, R. B.; Goodwin,
R.; Martin, T. R. J. Immunol. Methods 1998, 213, 41.
18. Heparinized whole blood (0.10 mL) was plated in a 96-
well polystyrene flat bottom micro titer tissue culture plate
and pre-incubated with 4· compound for 1 h at 37 ꢁC, 5%
CO2 in a tissue culture incubator. 4· IL-1b (300 pM final
concentration) or control was then added and the plates
were incubated at 37 ꢁC, 5% CO2 in a tissue culture
incubator for 18 h. The final percent concentration whole
blood was 50%. The final DMSO concentration was 0.1%.
The total volume for all assays was 200 lL. After 18 h,
supernatant/plasma was collected and assayed immedi-
ately or frozen at À80 ꢁC using IL-6 detection MSD ECL
based plates. Fifty microliters of supernatant was added to
MSD plates and incubated for 1 h on a shaker. The plates
were then washed once with 200 lL of PBS/0.1% Tween.
Twenty-five microliters of detection antibody diluted in
antibody diluent (1 lg/mL), and 125 lL of 2· Read Buffer
P were added, and the plates were incubated for 1 h on a
shaker, protected from light. ECL was measured using the
SECTOR HTS Imager (MSD, Gaithersburg, MD).
11. (a) Gavins, F. N. E.; Yona, S.; Kamal, A. M.; Flower, R.
J.; Perretti, M. Blood 2003, 101, 4140; (b) Perretti, M.;
Chiang, N.; La, M.; Fierro, I. M.; Marullo, S.; Getting, S.
J.; Solito, E.; Serhan, C. N. Nat. Med. 2002, 8, 1296.
12. Burli, R. W.; Xu, H.; Zou, X.; Muller, K.; Golden, J.;
¨
Frohn, M.; Adlam, M.; Plant, M. H.; Wong, M.;
McElvain, M.; Regal, K.; Viswanadhan, V.; Tagari, P.;
Hungate, R. Bioorg. Med. Chem. Lett. 2006, 16, 3713.
13. Chinese hamster ovarian cells (CHO) were stably co-
transfected with aequorin, Ga15 protein, and hFPRL1. In
this assay, receptor activation by an agonist induces release
of cytosolic Ca2+ storage. The increased cytosolic [Ca2+
was monitored by luminescence emitted by aequorin.
]
14. All indole 2-carboxylates were obtained from commercial
sources. The benzimidazole 2-carboxylate was prepared
from 4-methoxy-o-phenylenediamine dihydrochloride:
H
H2N
1. TFA, 80 oC, 99%
O
N
2HCl
2. NaOH, dioxane/H2O (3:1),
200 oC, microwave irradiation
34%
N
HO
H2N
OMe
OMe
.