Discovery of pyrazoles as novel FPR1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.08.085

A series of pyrazole inhibitors of the human FPR1 receptor have been identified from high throughput screening. The compounds demonstrate potent inhibition in human neutrophils and attractive physicochemical and in vitro DMPK profiles to be of further interest.

Full text of DOI:10.1016/j.bmcl.2011.08.085

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6456–6460
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of pyrazoles as novel FPR1 antagonists
Andrew D. Morley a, , Andrew Cook a, Sarah King a, Bryan Roberts a, Sarah Lever a, Richard Weaver b,
⇑
Cathy MacDonald b, John Unitt c, Malbinder Fagura c, Tim Phillips c, Richard Lewis a, Mark Wenlock a
a AstraZeneca R&D Charnwood, Chemistry, Bakewell Road, Loughborough LE11 5RH, UK
b DMPK, Bakewell Road, Loughborough LE11 5RH, UK
c BioScience, 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:
A series of pyrazole inhibitors of the human FPR1 receptor have been identified from high throughput
screening. The compounds demonstrate potent inhibition in human neutrophils and attractive
physicochemical and in vitro DMPK profiles to be of further interest.
Received 18 July 2011
Revised 15 August 2011
Accepted 17 August 2011
Available online 6 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
FPR1 antagonist
Hit-to-lead
Small molecule
N-Formyl peptide receptors (FPR1, 2 and 3) represent a family
of G protein-coupled receptors that form a key element in mount-
ing an innate immune response and in response to products of cell
damage.1–3 FPR1 is functionally expressed on a variety of cell types
ranging from inflammatory cells like neutrophils and macrophages
to nonhematopoietic cells, such as lung epithelial cells, platelets,
osteoblasts, and hepatocytes,4–7 suggesting a wider role of FPR1
beyond antibacterial host defense. Most importantly activation of
FPR1 leads to stimulation of proinflammatory neutrophil functions,
critical to the detection, and efficient clearance of bacterial
pathogens. Indeed the presence of N-formyl peptides and FPR1
expressing cells has been associated with inflammatory disease
progression, in particular neutrophilic diseases like chronic
obstructive pulmonary disease (COPD), where bacterial infection
and potentially smoke-driven lung cell damage (e.g., mobilisation
of host mitochondrial N-formyl peptides8–10) maybe linked to
exacerbation of symptoms and chronic lung damage. Hence there
is a strong therapeutic desire to antagonise FPR1 receptor function
to potentially reduce inflammatory lung function and long term
damage in diseases such as COPD.
S
N
S
O
H
N
N
O
N
H
NH OH
O
O
O
1
2
pIC50 = 6.3
clogP 4.5
pIC50 = 6.4
clogP 3.9
HN
O
O
O
S
N
N
N
H
3
We have previously reported the characterised two hit chemical
series (i.e., benzimidazole 1 and diamide 2) as suitable tool com-
pounds for exploring FPR1 biology.11 These compounds provided
useful in vitro tools but tended to be too lipophilic to possess the
desired physicochemical and DMPK characteristics to be of value
for a drug discovery programme. In addition to these series, we
also identified a series of pyrazoles (e.g., 3) from the HTS.
pIC50 = 5.7
clogP 4.8
Compound 3 was part of a collection enhancement library of
ꢀ350 compounds having two points of diversity at the amide
and sulphonamide. Some of the SAR from this library is shown in
Table 1. Compounds were tested for FPR1 antagonism in a human
neutrophil FPR1 Fluorescence Imaging Plate Reader (FLIPR)12 assay.
⇑
Corresponding author.
E-mail address: andymorley@live.com (A.D. Morley).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.085

A. D. Morley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6456–6460
6457
Table 1
Historic Library SAR
SAR exploration of the substituent attached via the nitrogen of
the pyrazole was limited, with no improvements in potency
observed. Ortho substitution (e.g., 18) reduces FPR1 activity and
introduction of polar groups (e.g., 19) has a detrimental impact
on biological activity. Substitution with heteroaromatics (20) had
a similar effect, though improved LLEs were observed for some of
these examples. Direct N-aryl attachment to the pyrazole is not
essential for potency, with the benzyl analogue (21) showing a
similar profile to phenyl. Smaller aliphatic substituents such as
isopropyl (22) were tolerated, but less active, showing slightly
reduced LE but marginally increased LLE. The N-methyl substituent
(23) was inactive. Broader expansion of the sulphonamide substi-
tuent showed SAR to be quite flat. Potency and LLEs could be
significantly improved through introduction of polar substituents
at the 4 position of the aryl ring, such as compound 25, but these
observations were only with lipophilic tertiary amide analogues
(compound 24 being significantly less active) and also came at
the expense of increasing molecular weight to >500. These results
are summarised in Table 2.
R
O
O
O
R1
S
N
N
N
H
Ph
R1
Compd
R
FPR1
pIC50
clogP LEb
LLEc
a
3
4
5
6
7
8
9
NHCH(Et)Et
NHMe
NHc-Hexyl
NMec-Hexyl
NHCH2Ph
4-MePh
4-MePh
4-MePh
4-MePh
4-MePh
4-MePh
4-MePh
5.7
NA
5.4
5.8
5
4.8
2.9
4.9
4.2
4.9
5.8
5.9
0.19
—
0.17
0.18
0.16
0.18
0.2
0.9
—
0.6
1.6
0.1
0.3
0.6
NHCH2(2,4-Me)Ph
NHCH(nPr)Et
6.1
6.5
10
4-MePh
NA
3.0
—
—
N
H
NMe
Introduction of a methyl substituent at the 3-position of the
pyrazole scaffold produced a modest increase in potency (26 vs
3). Despite detailed evaluation, attempts to introduce polar motifs
in this region were not compatible with FPR1 activity. Analysis
indicated that lipophilic substituents were still required, but
increasing the steric bulk close to the amide nitrogen gave potency
improvements. This SAR wasn’t observed to the same extent when
the 3-substituent is hydrogen as can be seen when comparing 30
and 31. This substitution pattern also brought about improvements
in LLE. Effects appear specific with a need for both lipophilic com-
ponents in order to achieve potent FPR1 activity, as shown by the
significantly weaker potency of 27 and 33 compared with 34 and
35. These latter motifs appeared to be useful amide substituents
for FPR1 with enhanced potency, LE and LLEs. In both these cases
the individual enantiomers were prepared and tested. No signifi-
cant enantiomeric FPR1 potency differences were observed for
34, where as for the isomers of 35, the (S)-enantiomer (36) was
preferentially active over the (R)-enantiomer (37) by ꢀsevenfold.
This data is summarised in Table 3.
In order to try and understand the variation in SAR by introduc-
tion of the 3-methyl substituent, NMR evaluation of the DMSO-d6
spectra of matched pairs were undertaken. These studies showed
almost identical chemical shift for the regions of interest, suggest-
ing the introduction of the 3-methyl group is not affecting internal
hydrogen bonding capabilities between the amide and sulphona-
mide units under these conditions. Preliminary molecular
modelling studies and analysis alongside the NMR spectra failed
to support any particular hypothesis and subtle conformation
effects can not be ruled out in effecting SAR. Additional substitu-
ents were introduced at the 3-position of the pyrazole, but with
the exception of a CF3 motif (e.g., 38) all other variations resulted
in reduced FPR1 potency.
11
4-MePh
5
2.5
0.16
2.5
N
H
O
12
13
14
NHCH(Et)Et
NHCH(Et)Et
NHCH(Et)Et
2-MePh
Ph
c-Pentyl
5.2
5.3
5.3
4.8
4.3
4.1
0.17
0.18
0.19
0.4
1
1.2
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.
LE, ligand efficiency.13 Values are calculated as pIC50/heavy atom count
LLE, lipophilic ligand efficiency.14 Values are calculated as pIC50-clogP
b
c
Data suggests FPR potency correlates with lipophilicity for R and
although some analogues had pIC50 values of ꢀ6.5, their ligand
efficiencies (LE)13 and lipophilic ligand efficiencies (LLE)14,15 were
modest. The sulphonamide substituents (R1) that were included
in the original library were all relatively simple and contained only
simple functional groups as substituents, but both aliphatic and
aromatic analogues showed similar levels of potency, indicating
some scope to explore further.
Attempts to modify or replace the sulphonamide and amide
motifs were largely unsuccessful. Alkylation of the sulphonamide
completely abolishes FPR1 activity, as did replacement of the
sulphonamide with amide. This suggests the acidic group is neces-
sary for FPR potency, although conformational effects of these
changes can not be ruled out. The amide group can be successfully
modified to maintain FPR1 activity, but only by incorporating less
polar alternatives. In the cases where potency is maintained the
physicochemical and in vitro DMPK profiles are significantly
inferior to 3, offering no overall advantage. Other heterocyclic
scaffolds were evaluated as part of the SAR exploration for FPR1
and as a broader assessment of overall profiles. Triazole 15 and
imidazole 16, with the same substitution vectors maintained
potency, whereas exploration of different regions/vectors (e.g.,
17) resulted in a loss of FPR1 activity.
HN
HN
HN
O
O
O
O
O
N
O
N
O
O
O
S
S
N
S
N
N
N
Ph
N
N
N
H
H
N
H
Ph
Ph
15
16
17
pIC50 = 5.4
clogP 4.3
pIC50 = 5.8
clogP 5.6
pIC50 = 5.0
clogP 4.8

6458
A. D. Morley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6456–6460
Table 2
Sulphonamide and pyrazole substituent SAR
R
O
O
O
R1
S
N
N
N
H
R2
Compd
R
R1
R2
FPR1 pIC50
clogP
LE
LLE
18
19
20
21
22
23
NHCH(Et)Et
NHCH(Et)Et
NHCH(Et)Et
NHCH(Et)Et
NHCH(Et)Et
NHCH(Et)Et
4-MePh
4-MePh
4-MePh
4-MePh
4-MePh
4-MePh
2-ClPh
4-CNPh
2-Pyridyl
CH2Ph
i-Pr
5
4.8
4.7
5.3
5
5.1
4.4
3.6
4.8
3.7
2.8
0.16
0.15
0.16
0.17
0.18
—
À0.1
0.4
1.1
0.5
1.3
—
Me
NA
N
N
24
25
NHCH(Et)Et
Ph
Ph
5.3
7.1
4.1
4
0.16
0.19
1.2
3.1
O
O
NEtc-hexyl
Table 3
Amide SAR and comparison of effect of 3-substituent
R
R3
O
O
O
S
N
N
N
Ph
H
Compd
R
R3
FPR1 pIC50
clogP
LE
LLE
3
26
27
28
29
NHCH(Et)Et
NHCH(Et)Et
NHi-Pr
NHc-hexyl
NMec-hexyl
H
5.7
6.3
5.2
6.1
5.5
4.8
4.7
3.7
4.9
4.4
0.19
0.20
0.18
0.19
0.17
0.9
1.6
1.5
1.2
1.1
Me
Me
Me
Me
a
30
H
6.0
5.3
0.19
0.7
N
H
a
31
32
33
34
35
Me
Me
Me
Me
Me
6.8
5.3
5.7
6.8
6.9
5.4
4.1
4.7
4.2
5.0
0.21
0.16
0.19
0.23
0.22
1.2
1.2
1.0
2.6
1.9
N
H
N
H
O
N
H
N
H
N
H
N
H
36
37
Me
Me
7.1
6.3
7.6
5.0
5.0
4.9
0.22
0.20
0.21
2.1
1.3
2.7
N
H
N
H
38
CF3
a
Diasterisomeric mixture

A. D. Morley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6456–6460
6459
Table 4
gave the best opportunity to improve FPR1 activity, with com-
pounds 42 and 43 being the most potent examples identified in
this phase and subsequently possess some of the best LE and LLEs
for the series.
Broader evaluation of the profiles of 36 and 42 are shown in Ta-
ble 5. The compounds show good potency, behaving as FPR1 antag-
onists, with no effects in an artefact assay. They also demonstrate
good potency in a CD11 whole blood assay. The series, in general,
showed no activity against a broad panel of receptors, kinases
Follow up sulphonamide and pyrazole substituent SAR
HN
O
O
O
R1
S
N
N
N
R2
H
and enzymes (<20% effect @ 10 lM). Compounds shows little cross
over for inhibiting rat, mouse, or guinea pig FPR1, which is consis-
tent with our findings with other unrelated series for this receptor.
Physicochemical and in vitro DMPK parameters are generally good,
giving confidence that the series could provide a foundation for
broader evaluation.
Compd R1
R2
FPR1
pIC50
clogP LE
LLE
—
N
39
Ph
NA
4.2
—
O
A generic synthetic route to the analogues is shown in Scheme
1. The key pyrazole core 46 was synthesized either by reaction of
substituted hydrazines with 44, or by reaction of hydrazidoyl ha-
lides 45 with ethyl cyanoacetate according to literature proce-
dures.21,22 Intermediate 46 was then sulfonylated with the
appropriate sulfonyl chloride using sodium hydride to deprotonate
the amino-pyrazole. Target compounds 48 were obtained either by
hydrolysis of the carboxylic acid ester 47 to the corresponding car-
boxylic acid and subsequent coupling of the acid with amines
using standard coupling agents or more conveniently by direct
reaction of 47 with an excess of the aminomagnesium halide pre-
pared by pre-mixing amine and iso-propyl magnesium chloride in
THF (Bodroux reaction).23 It should be noted that using the later
method yields were generally significantly higher.
We have identified a novel series of antagonists, for a challeng-
ing GPCR target and evaluated SAR to generate significant increases
in potency from the original HTS hit. The associated profiles of
these initial compounds make the series significantly more attrac-
tive than other published FPR1 antagonists and provide a useful
start point for more detailed evaluation of the series, which will
subsequently be reported.
40
41
c-hexyl
Ph
4-
FPh
4-
FPh
4-
6.0
6.0
4.9
4.2
0.19 1.1
0.19 1.8
3-pyridyl
4-CNPh
4-CF3Ph
42
43
7.6
7.9
4.7
6.0
0.22 2.9
0.22 1.9
FPh
As the introduction of the 3-methyl group had altered the
amide SAR, we revisited evaluation of the N-pyrazole and sulph-
onamide groups. In the case of analogues derived from 34, no
improvements in potency were generated. Expansion of 35 showed
SAR around the N-pyrazole aryl group appeared broadly to track
with previous observations, whereas the sulphonamide SAR now
became more specific with aryl analogues being significantly more
potent than alkyl (e.g., 36 vs 40). Some key observations are shown
in Table 4. Despite extensive efforts, attempts to introduce basic
functions at any site around the pyrazole template resulted in
completely inactive analogues, with compounds 39 and 10 exem-
plifying these observations. The para position of both aryl groups
Table 5
Profiles of 36 and 42
O
O
N
H
O
S
N
H
N
O
N
N
F
N
H
N
N
N
H
S
O
O
36
42
Parameter
Lead criteria16
pIC50 >7
36
42
FPR1 pIC50
FPR1 artefect
Whole blood CD11 pA2
clogP/logD
Mol Wt
7.1
NA
6.4
5/1.4
455
7.6
NA
6.9
4.7/0.5
483
<3.0 (logD)
<450
LE
LLE
Solubility
0.22
2.1
0.22
2.9
l
M17
>100
% Free
<30
<15
t1/2 >100 hr
pIC50 <5
>1000
1.1/4.2
40/53
7/12
>1000
<5 (5/5)
>1000
6/10
5/13
<3/4
>1000
<5 (5/5)
Hu/Rat Prot Binding
Hu/Rat Micsa
Hu/Rat Hepsb
(l
l/min/mg)
(l
l/min/106cells)
Chem stability18
c
Cyp inhib pIC50
pIC50 and pA2 values are the means of at least three experiments.
a
Human microsome metabolism intrinsic clearance (
l
L/min/mg).19
Rat Sprague–Dawley hepatocyte metabolism intrinsic clearance (
Inhibition of cytochrome P450 isoforms: 1A2, 2C9, 2C19, 2D6, and 3A4
b
c
l
L/min/106 cells).20

6460
A. D. Morley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6456–6460
CO2Et
CN
R1
EtO
R5
O
a
R1
CO2Et
R1
R1
CO2Et
NH2
N
c
R4
NHSO2R3
(d,e) or f
NHSO2R3
N
N
44
N
N
N
N
R2
R2
b
R2
R1
Br
47
48
46
N
NH
R2
45
Scheme 1. Synthesis of target derivatives. Reagents and conditions: (a) R2NHNH2, EtOH/water (20:1), reflux, 3–15 h. (b) Ethyl cyanoacetate, EtOH, NaOEt, 25 °C, 3–12 h. (c)
R3SO2Cl, THF, NaH, 25 °C, 3–12 h. (d) LiOHÁH2O, EtOH, 70 °C, 5–12 h. (e) HNR4R5, HATU, DIEA, DCM or DMF, 25 °C, 4–20 h. (f) HNR4R5, iso-propyl magnesium chloride, THF,
25 °C, 1–3 h.
11. Unitt, J.; Fagura, M.; Phillips, T.; King, S.; Perry, M.; Morley, A.; MacDonald, C.;
Cook, A.; Baxter, A. Bioorg. Med. Chem. Lett. 2011, 21, 2991.
12. Schroeder, K. S.; Neagle, B. D. J. Biomol. Screen. 1996, 1, 75.
Acknowledgments
We acknowledge Becky Holford and Kathy Dodgson for devel-
oping the FPR1 FMAT assay and running the HTS and Jadeen Chris-
tie, Matthew Perry and Andrew Baxter and chemistry input.
13. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430. Values
in this manuscript are calculated and reported as pIC50/Heavy atom count.
14. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881.
15. clogP, Version 4.3, BioByte (www.biobyte.com).
16. Baxter, A.; Cooper, A.; Kinchin, E.; Moakes, K.; Unitt, J.; Wallace, A. Bioorg. Med.
Chem. Lett. 2006, 16, 960.
17. Wenlock, M. C.; Barton, P.; Potter, T.; Austin, R. P. J. Assoc. Lab. Autom. 2011, 5,
1–10. doi:10.1016/j.jala.2010.10.002.
18. MacFaul, P. A.; Ruston, L.; Wood, J. M. Med. Chem. Commun. 2011, 2, 140.
19. McGinnity, D. F.; Parker, A. J.; Soars, M.; Riley, R. J. Drug Metab. Dispos. 2000, 28,
1327.
20. Soars, M. G.; Grime, K.; Sproston, J. L.; Webborn, P. J. H.; Riley, R. J. Drug Metab.
Dispos. 2007, 35, 859.
21. (a) Tanaka, K.; Suzuki, T.; Maeno, S.; Mitsuhashi, K. J. Het. Chem. 1986, 23, 1535;
(b) Abunada, N. M.; Hassaneen, H. M.; Kandile, N. G.; Miqdad, O. A. Molecules
2008, 13, 1501.
22. (a) Sutherland, H. S.; Blaser, A.; Kmentova, I.; Franzblau, S. G.; Wan, B.; Wang,
Y.; Ma, Z.; Palmer, B. D.; Denny, W. A.; Thompson, A. M. J. Med. Chem. 2010, 53,
855; (b) Das, J.; Moquin, R. V.; Dyckman, A. J.; Li, T.; Pitt, S.; Zhang, R.; Shen, D.
R.; McIntyre, K. W.; Gillooly, K.; Doweyko, A. M.; Newitt, J. A.; Sack, J. S.; Zhang,
H.; Kiefer, S. E.; Kish, K.; McKinnon, M.; Barrish, J. C.; Dodd, J. H.; Schieven, G. L.;
Leftheris, K. Bioorg. Med. Chem. Lett. 2010, 20, 6886; (c) Beck, J. R.; Gajewski, R.
P.; Lynch, M. P.; Wright, F. L. J. Het. Chem. 1987, 24, 267.
References and notes
1. Ye, R. D.; Boulay, F.; Wang, J. M.; Dahlgren, C.; Gerard, C.; Parmentier, M.;
Serhan, C. N.; Murphy, P. M. Pharmacol. Rev. 2009, 61, 119.
2. Schiffmann, E.; Corcoran, B. A.; Wahl, S. M. Proc. Natl. Acad. Sci. U.S.A. 1975, 72,
1059.
3. Schiffmann, E.; Showell, H. V.; Corcoran, B. A.; Ward, P. A.; Smith, E.; Becker, E.
L. J. Immunol. 1975, 114, 1831.
4. McCoy, R.; Haviland, D. L.; Molmenti, E. P.; Ziambaras, T.; Wetsel, R. A.;
Perlmutter, D. H. J. Exp. Med. 1995, 182, 207.
5. Czapiga, M.; Gao, J.; Kirk, A.; Lekstrom-Himes, J. Exp. Hematol. 2005, 33, 73.
6. Rescher, U.; Danielczyk, A.; Markoff, A.; Gerke, V. J. Immunol. 2002, 169, 1500.
7. Shin, M. K.; Jang, Y. H.; Yoo, H. J.; Kang, D. W.; Park, M. H.; Kim, M. K.; Song, J.
H.; Kim, S. D.; Min, G.; You, H. K.; Choi, K. Y.; Bae, Y. S.; Min do, S. J. Biol. Chem.
2011, 286, 17133.
8. Rabiet, M. J.; Huet, E.; Boulay, F. Eur. J. Immunol. 2005, 35, 2486.
9. Carp, H. J. Exp. Med. 1982, 155, 264.
10. Zhang, Q.; Raoof, M.; Chen, Y.; Sumi, Y.; Sursal, T.; Junger, W.; Brohi, K.; Itagaki,
K.; Hauser, C. J. Nature 2010, 464, 104.
23. (a) Bodroux, F. Bull. Soc. Chim. Fr. 1905, 33, 831; (b) Bodroux, F. Bull. Soc. Chim.
Fr. 1906, 35, 519.