Discovery of small molecule human FPR1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.03.049

The identification of two novel series of formyl peptide receptor 1 (FPR1) antagonists are reported, represented by methionine benzimidazole 6 and diamide 7. Both series specifically inhibited the binding of labelled fMLF to hrFPR1 and selectively antagonized FPR1 function in human neutrophils, making them useful in vitro validation tools for the target.

Full text of DOI:10.1016/j.bmcl.2011.03.049

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2991–2997
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of small molecule human FPR1 receptor antagonists
John Unitt a, , Malbinder Fagura a, Tim Phillips a, Sarah King c, Matthew Perry c, Andrew Morley c,
⇑
⇑
,
Cathy MacDonald b, Richard Weaver b, Jadeen Christie c, Simon Barber c, Rukhsana Mohammed c,
Melanie Paul c, Andrew Cook c, Andrew Baxter c
a AstraZeneca R&D Charnwood, Bioscience, Bakewell Road, Loughborough LE11 5RH, UK
b DMPK, Bakewell Road, Loughborough LE11 5RH, UK
c Chemistry Departments, Bakewell Road, Loughborough LE11 5RH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The identification of two novel series of formyl peptide receptor 1 (FPR1) antagonists are reported, rep-
resented by methionine benzimidazole 6 and diamide 7. Both series specifically inhibited the binding of
labelled fMLF to hrFPR1 and selectively antagonized FPR1 function in human neutrophils, making them
useful in vitro validation tools for the target.
Received 14 February 2011
Revised 11 March 2011
Accepted 13 March 2011
Available online 21 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
FPR1 antagonist
Hit-to-lead
Small molecule
FPR1 is a member of the formyl peptide receptor family, a class
A G-protein coupled receptor (GPCR). It is functionally expressed
on a variety of cell phenotypes, especially neutrophils and macro-
phages, but also hepatocytes and platelets.1,2 Related receptors
FPR2 and FPR3 complete the FPR family and have significant se-
quence homology and overlapping pharmacology to FPR1.3
FPRs were originally identified by their ability to bind and be
stimulated by N-formyl peptides, such as N-formylmethionine,
produced by infecting bacteria.4,5 Subsequently, FPR1 was identi-
fied and cloned in 1990 from differentiated HL-60 cells.6 Recently
the FPR1 ligand repertoire has expanded with the inclusion of
mitochondrial genome-encoded N-formyl peptides/proteins, re-
leased by damaged host cell mitochondria.7,8
Agonism of FPR1 leads to activation of neutrophil and macro-
phage pro-inflammatory functions as part of the innate immune
response, leading to chemotaxis, superoxide generation and
degranulation required for efficient bacterial clearance. Consistent
with the pro-inflammatory action of FPR1 agonists, this receptor
and its ligands have been implicated in the pathogenesis of a range
of inflammatory disorders, such as COPD, where bacterial infection
and potentially smoke-driven lung damage (e.g., mobilisation of
mitochondrial N-formyl peptides) are linked to exacerbations of
symptoms and long term lung damage. Hence pharmaceutical
interest in this area is driven by the potential therapeutic develop-
ment of a FPR1 antagonist. We report in this paper the discovery of
two novel series of small molecule with such profiles.
There are only a few examples of FPR1 antagonists in the liter-
ature (see Fig. 1) including phenylbutazone analogues 1,9,10 group
E 211 cyclosporins 312 to t-Boc-peptides 4.13,14 Recently potent
FPR1 probes (e.g., 5) were identified by library screening by Young
et al.15, but their precise mechanism of action appears unclear.
However, all these chemical starting points are unattractive for
lead generation due to potentially chemically reactive functional-
ity, weak potency, no species cross-over, high lipophilicity, flat
SAR or having a complex peptidic structure.
To identify new chemical starting points, a High Throughput
Screen (HTS) of the corporate AstraZeneca collection (800K) was
run using Fluorometric Micro volume Assay Technology
(FMAT™).16 Compound inhibition of Alexa647-labelled fMLFK spe-
cific binding to HEK-293 cells expressing human recombinant FPR1
(hrFPR1) and hrG-protein G 16 was measured. A HTS active rate of
a
2.6% was obtained. As the target was categorised as a challenging
GPCR, in terms of known chemical tractability, a more lenient work
up than normal was undertaken. Compounds with an inhibition
>50% and a lipophilicity range À1.5 < c log P < 5.5 were evaluated
after the removal of unwanted chemotypes. Frequent HTS hitters
were also removed prior to testing for FMAT™ technology blockers
using a comparable parallel hrCCR1 FMAT™ assay.
The final set of active compounds were tested for FPR1 antago-
nism in a human neutrophil FPR1 Fluorescence Imaging Plate
Reader (FLIPR)17 assay and counter screened in the same assay
⇑
Corresponding author.
E-mail address: andy.morley@astrazeneca.com (A. Morley).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.049

2992
J. Unitt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2991–2997
Ph
N
N
O
Ph
N
O
O
O
O
Ph
N
COOH
S
2
1
Group E
pIC50 5.3
S
Suphinpyrazone
pIC50 5.0
S
O
O
N
O
O
O
N
4
O
N
O
O
N
N
N
N
N
O
N
O
O
O
t-Boc-MLF
pIC50 5.6
O
N
O
O
N
O
O
O
O
O
N
N
N
O
N
O
N
3
5
Cyclosporin H
pIC50 7.0
3570-0208
pKi 7.0
Figure 1. A selection of known FPR1 antagonists.
against FPR2 (also called FPRL1) (see Table 5), where >30-fold
selectivity was the required progression criteria. This led to the
identification a small number of chemical series, two of which
are described in this Letter.
The diamide SAR was rapidly explored utilising Ugi related
chemistry, which allowed multiple variations of R1–4 to be incorpo-
rated, generating broad data sets. Key results are highlighted in
Table 1.
Substitution at R[2] and R[3] are tolerated. R[2] does appear to
have the greatest scope for modification. Polar heterocycles do im-
prove lipophilic ligand efficiencies (LLE)20, but the most potent
analogues identified (i.e. pIC50 >7) possessed more lipophilic R[2]
substituents. Variation of the R[3] group was the least explored re-
gion and there appears to be scope to vary this further. Replace-
ment of hydrogen as the R[4] motif with methyl resulted in a
loss of FPR1 potency. For R[1] the ortho phenol was essential to
maintain FPR1 activity and despite varying this motif broadly, only
compound 17 showed any sign of FPR1 activity without R[1] being
a specific 2-hydroxyphenyl analogue. Even in this case there is still
a strong similarity to a phenolic type pharmacophore. This initial
SAR suggested that in order to generate good FPR1 biological pro-
files, the series would require a number of parameters that would
be challenging to successfully remove/modify in latter phases of
the drug discovery process. Accordingly further work, including
testing of enantiomers, was not undertaken.
Spot test screening of available near neighbours of benzimid-
azole 6 identified additional active analogues, suggesting chemical
scope for broader SAR evaluation. Overall the compounds are lipo-
philic, leading to unfavourable metabolic stability and modest LLE.
Therefore, the main priority in expanding the series was to reduce
lipophilicity whilst attempting to increase potency.
Preliminary exploration of the SAR of R[1] was achieved
through parallel synthesis, with profiles for key compounds high-
lighted in Table 2. Modification of the benzofuran suggests no spe-
cific part dominates potency for FPR1. Removal of the ethoxy group
reduces activity, whilst maintaining ligand efficiency. Disrupting
the planarity of the amide/aromatic ring may have some benefit,
but initial data was inconclusive. The R[1] substituent does not
need to be aromatic, with the cyclohexyl motif being equiactive
to phenyl. Insertion of an alkyl linker between the amide and cyclic
motif did however reduce potency. The lipophilicity of the
aromatic group can be reduced with only limited impact on po-
tency and ligand efficiency, allowing LLEs to be increased to >3.
S
S
O
H
N
O
N
N
H
NH OH
N
O
O
O
7
6
Benzimidazole 6 was a member of a small set (3) of external
compounds, derived from methionine, whereas diamide 7 was
from an Ugi library and subsequently formed part of a large cluster,
providing early hints of Structure–Activity Relationship (SAR). The
similarity of both these series to known FPR1 modulators is easy to
visualise (e.g., fMLF, fMLFF, tBocMLF). This raises the distinct pos-
sibility that these compounds interact with FPR1 in a similar man-
ner to the known peptidic modulators.
The syntheses of both templates are straightforward and shown
in Schemes 1 and 2. The diamides were isolated in acceptable
yields using a one pot multi-component reaction in methanol.18
The benzimidazoles were prepared from the corresponding aro-
matic diamines employing standard amide coupling chemistry, fol-
lowed by dehydration under acidic conditions to generate the
imidazole ring.
R2
O
O
H
N
a
NH2
R4
CHO
R2
R3 N+
R1
OH
R3
N
R1
+
+
+
O
R4
Scheme 1. Synthesis of diamides. Reagent and condition: (a) methanol 40 °C.

J. Unitt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2991–2997
2993
NHBoc
Y
Y
R3
N
R3
Y
Y
R3
CO2H
Y
Y
H
N
NH2
Y
Y
Y
N
NHBoc
Y
R4
Y
NHBoc
NHR4
O
Y
a
b
NHR4
R3
Y
R1COX
Y
Y
Y
N
N
Y
R3
Y
Y
Y
N
O
N
d
c
R4
NH2
R4
N
H
R1
Y = CH or N
Scheme 2. Synthesis of benzimidazoles. Reagents and conditions: (a) 2-propanephosphonic acid anhydride (T3P), dimethylfomamide; (b) acetic acid; (c) trifluoroacetic acid,
dichloromethane; (d) X = OH, T3P, dimethylformamide, X = Cl, dichloromethane, triethylamine.
Table 1
Diamide SAR
H
N
R2
O
R3
N
R4
R1
R4
H
O
R1
R2
R3
FPR1 pIC50
6.5
c log P
LEb
LLEc
2.6
a
Compound
OH
OH
OH
OH
OH
OH
OH
OH
OMe
S
7
3.9
0.23
8
H
H
H
H
H
H
H
6.1
7.1
5.9
5.8
6.1
5.3
5.6
4.5
4.7
3.6
1.6
4.6
4.8
5.1
0.21
0.24
0.19
0.21
0.22
0.19
0.2
1.6
2.4
2.3
4.2
1.5
0.5
0.5
Cl
9
10
11
12
13
14
N
N
Cl
Cl
Cl
Cl
15
16
H
H
NA
NA
4.0
4.0
––
––
––
––
(continued on next page)

2994
J. Unitt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2991–2997
Table 1 (continued)
a
Compound
R1
R2
R3
R4
H
FPR1 pIC50
c log P
LEb
LLEc
2.4
O
NH
Cl
17
5.6
3.2
0.16
N
OH
18
Me
5.0
3.3
0.17
1.7
a
Inhibition of fMLF stimulated intracellular calcium mobilisation in human neutrophils. pIC50 values are the means of at least two experiments. NA: Not active (<25%
inhibition) at 50 lM.
b
LE (Ligand efficiency). 19Values are calculated as pIC50/Heavy atom count.
c
LLE (Lipophilic ligand efficiency). 20Values are calculated as pIC50–c log P.
Key modifications around the benzimidazole motif are shown in
Table 3. Limited SAR for substitution of the heterocyclic nitrogen
was available. Lipophilic motifs were generally equipotent to
hydrogen, irrespective of the size of substituent. Introduction of a
cyano group (31) interestingly did give a log unit increase in po-
tency compared to 30, but this compound still has a c log P >4.
Incorporation of heteroatoms into the phenyl ring of the benzimid-
azole had variable effects on FPR1 profiles depending on the substi-
tution pattern. Whilst all reduced lipophilicity and gave superior
LLEs to 6, potency and ligand efficiency (LE) [19] were reduced
and for 32 stimulation of intracellular calcium mobilisation was
observed. This apparent agonism (pEC50 5.8, intrinsic activity (IA)
0.81) was shown to be mediated by FPR1 as 7 antagonized the
compound’s effects in the neutrophil (pA2 6.3). This finding is not
without precedent as benzimidazole-based agonists of FPR1 and
FPR2 have been recently described.21 Replacement of the benz-
imidazole could be achieved, with the 4-phenylimidazole ana-
logue, 35, showing slightly reduced FPR1 activity compared to
27, although reductions in LE and LLE were more pronounced.
The SAR for the amino acid substituent is summarised in Table
4. The ethylthiomethyl side chain (R[3]) appears to be required for
FPR1 potency, with the (S) isomer preferentially inhibiting intracel-
lular calcium mobilisation. Replacement of the side chain with a
butyl group (40) maintained FPR1 potency, but this analogue
mobilised intracellular calcium, suggesting it was an FPR1 agonist.
This is supported by the finding that its potency as an agonist
(pEC50 6.3, IA 0.7) was in good accordance with the pIC50 value
estimated from its blockade of the effect of fMLF. Other variants,
such as aromatic substituents were all inactive, whilst the sul-
phone analogue showed reduced potency and LE, though LLE was
improved.
Table 2
Benzimidazole R1 SAR
S
N
O
H
N
R1
N
H
Compound
R1
FPR1 pIC50
6.3
c log P
LE
LLE
1.8
O
O
O
6
4.5
0.21
OEt
19
20
5.4
5.4
3.9
2.5
0.2
1.5
2.9
0.23
21
22
5.3
NA
3.1
3.3
0.23
––
2.2
––
MeO
HO
23
5.4
3.8
0.23
1.6
N
N
24
25
5
2
0.22
0.24
3
5.6
3.5
2.1
The initial data shows SAR in this region is likely to be limited.
This is in agreement with data generated for formylated methio-
nine peptide analogues, where modification of the thioether side
chain hasn’t been tolerated.22 Compounds 38 and 41 show
improvement in metabolic stability in rodent and human systems
compared to 6 (human microsomal intrinsic clearances 3, 14,
O
26
27
5
1.5
3
0.22
0.25
3.5
2.7
N
S
37 lL/min/mg, respectively) demonstrating the thioether, as ex-
5.7
pected, to be the major metabolic liability. Finding a suitable
replacement would be essential if the series were to have any
chance to progress towards Lead Optimisation.
Once preliminary SAR had been established, more specific fol-
low up rounds of synthesis were undertaken as shown in Figure
2, looking to expand on the initial observations.
Cl
28
29
5
3.7
3.6
0.21
––
1.3
––
NA
These campaigns failed to generate suitable FPR1 potency or LLE
improvements. A large number of these analogues possessed
c log P values in the range of 2–3.5, generating compounds with
acceptable Lead Identification in vitro DMPK and physicochemical
However, despite making a number of heterocyclic R[1] analogues,
none were more potent than 6.

J. Unitt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2991–2997
2995
Table 3
Benzimidazole R2 SAR
S
O
R2
N
R1
H
R1
R2
FPR1 pIC50
5.6
FPR1 agonism pEC50
c log P
LE
LLE
a
Compound
Ph
N
N
30
NA
4.7
0.19
0.9
2.4
N
CN
31
6.5
NA
4.1
0.2
N
N
O
O
H
N
32
33
6.0
5.5
5.8 (IA = 0.8)
3.6
3.6
0.2
2.4
1.9
OEt
OEt
N
H
N
NA
0.19
N
N
O
S
H
N
34
5.3
5.2
NA
NA
2.8
3.5
0.18
0.21
2.5
1.7
N
OEt
N
N
H
N
Ph
35
N
Cl
Apparent stimulation of intracellular calcium mobilisation in human neutrophils antagonized by a selective FPR1 antagonist, 7.
a
substituents for both benzimidazole and diamide series (R[1] and
R[3] in particular) suggesting possible differences in how they
interact with the FPR1 receptor.
Table 4
Benzimidazole R3 SAR
Although reduction of lipophilicity could be achieved in certain
regions around the benzimidazole template, reviewing the overall
data demonstrated a lipophilicity/potency correlation with a clear
leading edge for the more potent compounds (Fig. 3). This indicated
that consistently achieving LLEs >3 where FPR1 pIC50 >6 would be
a challenge. The trend was similar for the diamides (see Supple-
mentary data) leading to the conclusion that options to improve
FPR1 profiles for either series in subsequent phases would be
difficult.
R3
O
H
N
O
N
H
N
O
Compound
R3
FPR1 pIC50
<4.6
c log P
LE
––
LLE
––
S
S
Complete profiles of 6 and 7 are highlighted in Table 5. Both
were shown to be reversible antagonists and exhibited no FPR1
agonism in the human neutrophil FLIPR assay. Additional selectiv-
36
4.5
(R)
37
38
39
6.2
5
4.5
2.6
5.1
0.21
0.16
––
1.8
2.4
––
(S)
O
ity profiling, showed these compounds to be inactive at 30 lM
O
against other human GPCRs, such as FPR2, C5aR and CXCR2. Spe-
cies cross-over was examined in cell calcium mobilisation assays
expressing recombinant rat and mouse FPR1. Both series demon-
strated limited cross-over to these rodent receptors. FPR1 agonism
was only observed in the previously described benzimidazole ana-
logues and not by 6 or 7 or in any other diamides tested (data not
shown).
S
NA
S
40
41
6b
5.5
5
0.2
––
0.5
––
(S)
NA
Ph
In order to further characterise the nature of the receptor inter-
action and functional antagonism of both series, exemplars were
tested and shown to inhibit [[3]H] fMLF binding to hrFPR1 in par-
allel with attenuation of fMLF-driven neutrophil functions such as
degranulation. Both 6 and 7 are potent inhibitors of fMLF binding
to FPR1 suggesting the compounds interact at the receptor level
All compounds were tested in an FPR1 agonism assay.
b
Compound 40 was shown to possess equivalent activity in both assays.
parameters, but the majority possessed inadequate FPR1 potency.
The expanded data set clearly showed a disconnection in preferred

2996
J. Unitt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2991–2997
Table 5
Hit profiles for 6 and 7
Parameter
Lead criteria23
Compound 6
Compound 7
S
S
O
H
N
H
N
O
NH OH
N
Structure
H
O
N
O
O
a
FPR1
D
[Ca2+
]
i
pIC50 >6
pIC50 >7
6.3
NA
7.5
NA
NA
NA
5.5
5.0
NA
6.8
4.0
6.4
NA
7.0
NA
NA
NA
5.8
5.9
4.6
6.4
4.1
FPR1 agonismb
hrFPR1bindingc
FPR2d
CXCR2e
C5aRf
[Ca2+
g
hrFPR1D ]
i
Rat rFPR1h
Mouse rFPR1i
Degranulationj
Cytotoxicityk
pIC50 >6
c log P
Log D
Mol Wt
LE
<3.0
<3.0
<450
4.5
4.8
423
0.21
1.8
17
37
27
>260
3.9
4.1
395
0.23
2.5
68
43
70
>260
LLE
Solubilityl
Hu Micsm
Rat Hepsn
>10
<30
<15
lM
Chem stability26
t
>100 h
½
pIC50 values are the means of at least three experiments. NA: not active (<25% inhibition) at 50
l
M.
a
b
c
Inhibition of fMLF stimulated intracellular calcium mobilisation in human neutrophils.
Apparent stimulation of intracellular calcium mobilisation in human neutrophils.
Inhibition of [3H]fMLF binding to CHO cell membranes expressing hrFPR1.
d–f
f
Inhibition of dSHAAGtide, ehrGRO
a
and hrC5a-stimulated intracellular calcium mobilisation in human neutrophils.
g–i
i
i
Inhibition of g,hfMLF or fMLFF-stimulated intracellular calcium mobilisation in HEK cells expressing ghuman, hrat or mouse recombinant FPR1.
j
Inhibition of fMLF-stimulated human neutrophil degranulation.
Inhibition of THP-1 cell viability.
k
l
Solubility protocol (see Supplementary data).
Human microsome metabolism intrinsic clearance (l
L/min/mg).24
m
n
Rat Sprague–Dawley hepatocyte metabolism intrinsic clearance (
l
L/min/106 cells).25
to antagonize FPR1-driven cell function. Additionally the com-
pounds were shown not to inhibit degranulation per se as they
had no effect on PMA-stimulated neutrophil degranulation. Finally
to identify any innate cytotoxicity in the hit series, compounds
were shown to be weakly active (pIC50 ꢀ4) in a standard 24-hour
cell viability assay. Even though the measured thermodynamic sol-
ubility of 6 and 7 was below the top test concentrations employed,
no evidence was observed for these compounds being insoluble
under assay conditions.
S
R3
S
O
O
H
N
R5
N
H
N
O
R1
N
H
Ph
N
H
R4
N
N
H
Ph
N
O
In summary, HTS has provided two novel FPR1 antagonist hit
series that are superior to any known antagonists. These are use-
ful tools to help understand the role of FPR1 in various disease
settings. More detailed studies of these compounds will subse-
quently be reported elsewhere. Literature data and our own find-
ings suggest that identification of drug like FPR1 antagonists will
be a challenge.
Figure 2. Follow up libraries.
Acknowledgements
We acknowledge Becky Holford and Kathy Dodgson for devel-
oping the FPR1 FMAT assay and running the HTS and Iain Dougall
for helpful discussions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.049.
Figure 3. Plot of c log P vs FPR1 pIC50
.

J. Unitt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2991–2997
2997
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