Identification of a new class of small molecule C5a receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.11.058

C5a is a terminal product of the complement cascade that activates and attracts inflammatory cells including granulocytes, mast cells and macrophages via a specific GPCR, the C5a receptor (C5aR). Inhibition of C5a/C5aR interaction has been shown to be eff

Full text of DOI:10.1016/j.bmcl.2009.11.058

Bioorganic & Medicinal Chemistry Letters 20 (2010) 662–664
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a new class of small molecule C5a receptor antagonists
a
a
b
a
a
Jack J. Chen a, , Derek C. Cole , Greg Ciszewski , Kimberly Crouse , John W. Ellingboe , Pawel Nowak ,
*
Gregory J. Tawa c, Gabriel Berstein b, Wei Li b
a Chemical Sciences, Wyeth Research, Pearl River, NY 10956, USA
b Inflammation Research, Wyeth Research, Cambridge, MA 02140, USA
c Structural Biology, Wyeth Research, Princeton, NJ 08543-8000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
C5a is a terminal product of the complement cascade that activates and attracts inflammatory cells
including granulocytes, mast cells and macrophages via a specific GPCR, the C5a receptor (C5aR). Inhibi-
tion of C5a/C5aR interaction has been shown to be efficacious in several animal models of autoimmune
diseases, including RA, SLE and asthma. This account reports the discovery of a new class of C5aR antag-
onists through high-throughput screening. The lead compounds in this series are selective and block C5a
binding, C5a-promoted calcium flux in human neutrophils with nanomolar potency.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 6 October 2009
Revised 12 November 2009
Accepted 16 November 2009
Available online 7 December 2009
Keywords:
Anaphylatoxin
C5a
G-protein coupled receptor
Anti-inflammation
Autoimmune diseases
The anaphylatoxin C5a is a 74 amino acid glycopeptide pro-
duced by complement activation under inflammatory conditions,1
or via complement cascade-independent mechanisms in certain
autoimmune disease models.2,3 C5a interacts with the C5a receptor
(C5aR),4 a 52 KD cell surface G-protein coupled receptor (GPCR)
present in common inflammatory cells to induce a series of innate
and adaptive immunological responses including (i) chemotaxis
and respiratory burst of neutrophils with up-regulation of CD11b
expression; (ii) chemotaxis and degranulation of basophils and
mast cells; (iii) production of GMCSF and IL-8 by eosinophils; (iv)
chemotaxis of monocytes and macrophages with up-regulation of
the activating FcRIII and downregulation of the inhibitory FcRIIB;
(v) myeloid and plasmacytoid dendritic cell balance in the lung;
and (vi) enhancement of proliferation, differentiation and viability
in T-cells.3,5–10
autoimmune diseases, and several peptidomimetic and small mole-
cule antagonists have been reported (Fig. 1).20–23
To discover novel C5aR small molecule antagonists, we carried
out a high-throughput screen (HTS) using recombinant CHO cells
stably transfected with human C5aR and Ga16, and recorded
C5a-dependent intracellular calcium mobilization on a fluorescent
imaging plate reader (FLIPR). Compounds with over 30% inhibition
O
N
H
N
HN
OMe
HN
O
O
N
O
O
HN
O
H
N
O
NH
The pro-inflammatory nature of C5a/C5aR interaction essentially
implicates the pathogenic role of C5aR in acute and chronic inflam-
matory disorders such as psoriasis, asthma, bullous pemphigoid,
and rheumatoid arthritis. In fact, patients with these conditions
often have elevated C5aR level in their diseased tissues.11–13
Pharmacological and genetic studies have also shown that blockade
or deficiency of C5aR has protective effects in animal models of sys-
temic lupus erythematosus, rheumatoid arthritis, antiphospholipid
syndrome and asthma.14–19 Thus, there is significant interest in
identification of C5aR antagonists as therapeutic agents for these
NH
N
NH
O
1
3
2
H2N
N
H
N
N
N
OMe
H
OMe
OMe
N
O
O
N
4
O
O
* Corresponding author. Tel.: +1 845 602 3470; fax: +1 845 474 4202.
E-mail address: chenj24@wyeth.com (J.J. Chen).
Figure 1. Peptidomimetic and small molecule C5aR antagonists.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.058

J. J. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 662–664
663
at 10 lM concentration were structurally prioritized and subjected
to IC50 determination. The confirmed hits were further character-
ized in a radioligand binding assay to measure their binding
affinity.
This process led to identification of bis-sulfonamide 5 as a po-
tent and selective C5aR antagonist. Substructure and similarity
searches were performed to investigate the SAR related to the
structure. The results are illustrated in Table 1.
The study revealed constrained SAR around the terminal methyl
groups of the tosyl moiety as their removal rendering complete
loss of activity (entry 6). The two N-methyl groups could be homol-
ogated (entry 7), but cyclizing them with linkers of various lengths
caused complete loss of activity (entries 8 and 9). Connection of
the two tosyl units through piperazine (10) or bisphenyl-diamino-
propane (11) (Fig. 2) was attempted as illustrated by Figure 2, but
did not produce active molecules.
A low energy pharmacophore model (Fig. 3) suggested the pos-
sibility of replacing one of the sulfonamide moieties to minimize
the symmetric nature of the lead molecule.26 A series of amide
and benzophone sulfonamides were therefore synthesized using
the scheme outlined in Scheme 1 (Table 2). Sulfonylation of N-
methyl anthranilic acid 12 yielded the intermediate 13 which
was then converted to the indoline amide 14, or alternatively benz-
ophone 16 in two steps via the Weinreb amide 15.
Figure 3. Superimposition of bis-sulfonamide
sulfonamide 17 (purple).
5
(green) with
a
benzophone
Replacement of the tolyl sulfonamide with an indoline amide
(entry 14) led to loss of binding and FLIPR activity. The activity
was restored in thienyl ketone 17, suggesting the lipophilic prefer-
ence of pharmacophores in this region. Halogenations of the cen-
tral phenyl ring resulted in much weaker antagonists (entries 15
and 16). And substitution of the aryl ketone with acetyl group
led to loss of activity (entry 20).
Three lead compounds were further characterized for functional
activity in primary cells, mouse C5a receptor cross-reactivity, and
selectivity relative to other GPCRs (Table 3). Potency in FLIPR
O
O
S
NH
ii
R=H
S
i
R1
N
O
N
O
O
O
OH
OH
N
R1
O
12
13
14
iii
O
O
S
S
O
N
O
N
O
O
iv
O
R2
N
R1
R1
15
16
Scheme 1. Reagents and conditions: (i) tosyl chloride, Na2CO3, water; (ii) oxalyl
chloride, indoline; (iii) oxalyl chloride, CH3NHOCH3ÁHCl, TEA, DCM; (iv) aryl
bromide, n-BuLi, THF, À78 °C.
Table 1
Binding affinity and functional activity of bis-sulfonamide analogsa
R2R2
O
S
O
S
R1
N
N
R1
O
O
Table 2
Activity of benzophone sulfonamide analogsa
Entry
R1
R2
CHO-cell FLIPR
CHO cell binding
24
25
IC50
(l
M)
IC50
(lM)
R2
O
5
6
7
8
p-Tol
Ph
p-Tol
p-Tol
Me
Me
0.2
>60
0.3
0.2
>60
0.3
1
N
2
3
S
O
CH2@CH–CH2–
–CH2CH2–
O
5
>60
>60
R1
4
Entry
R1
R2
CHO-cell FLIPR
CHO cell binding
9
p-Tol
>60
>60
24
25
IC50
(l
M)
IC50
(lM)
a
All active compounds showed full antagonism and were inactive as C5aR ago-
14
17
H
H
38.3
44.5
N
nists with EC50 >60
lM.
S
0.6
1.7
18
19
20
3-Cl
3-Br
H
Ph
Ph
Me
>60
>60
>60
8.7
14.6
>60
O
O
S
O
O
N
S
S
O
a
N
N
All active compounds showed full antagonism and were inactive as C5aR ago-
nists with EC50 >60 M.
O
N
S
l
O
11
FLIPR IC50 (µM) >60
O
10
assays correlated well between CHO cells and primary human neu-
trophils (PMN), indicating that compound activity on recombinant
C5aR translated to the naturally expressed receptor. Consistent
with this observation, 5 and 7 blocked C5a-promoted chemotaxis
FLIPR IC50 (µM) >60
Figure 2. Bis-sulfonamides with variations of the benzo backbone.

664
J. J. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 662–664
16. Ji, H.; Ohmura, K.; Mahmood, U.; Lee, D. M.; Hofhuis, F. M. A.; Boackle, S. A.;
Table 3
Takahashi, K.; Holers, V. M.; Walport, M.; Gerard, C.; Ezekowitz, A.; Carroll, M.
C.; Brenner, M.; Weissleder, R.; Verbeek, J. S.; Duchatelle, V.; Degott, C.; Benoist,
C.; Mathis, D. Immunity 2002, 16, 157.
Functional activity of lead compounds in various cell lines
Assay
5
7
17
17. Wenderfer, S. E.; Ke, B.; Hollmann, T. J.; Wetsel, R. A.; Lan, H. Y.; Braun, M. C. J.
Am. Soc. Nephrol. 2005, 16, 3572.
18. Bao, L.; Osawe, I.; Puri, T.; Lambris, J. D.; Haas, M.; Quigg, R. J. Eur. J. Immunol.
2005, 35, 2496.
19. Girardi, G.; Berman, J.; Redecha, P.; Spruce, L.; Thurman, J. M.; Kraus, D.;
Hollmann, T. J.; Casali, P.; Caroll, M. C.; Wetsel, R. A.; Lambris, J. D.; Holers, V.
M.; Salmon, J. E. J. Clin. Invest. 2003, 112, 1644.
20. Finch, A. M.; Wong, A. K.; Paczkowski, N. J.; Wadi, S. K.; Craik, D. J.; Fairlie, D. P.;
Taylor, S. M. J. Med. Chem. 1999, 42, 1965.
27
PMN FLIPR IC50
(
l
M)
0.067
5.7
27
>60
>60
>60
>60
0.059
2.5
0.4
Human PMN chemotaxis27
(
lM)
>60
>60
3.1
>60
>60
>60
Human PMN Shape Change, whole blood28
(l
M)
29
RAW cell FLIPR IC50
CXCR3 FLIPR IC50
CXCR4 FLIPR IC50
(
M)
M)
l
M)
>60
>60
>60
>60
(l
l
(
P2Y2 FLIPR IC50 (lM)
21. Sumichika, H.; Sakata, K.; Sato, N.; Takeshita, S.; Ishibuchi, S.; Nakamura, M.;
Kamahori, T.; Ehara, S.; Itoh, K.; Ohtsuka, T.; Ohbora, T.; Mishina, T.; Komatsu,
H.; Naka, Y. J. Biol. Chem. 2002, 277, 49403.
22. Brodbeck, R. M.; Cortright, D. N.; Kieltyka, A. P.; Yu, J.; Baltazar, C. O.; Buck, M.
E.; Meade, R.; Maynard, G. D.; Thurkauf, A.; Chien, D.-S.; Hutchison, A. J.;
Krause, J. E. J. Pharmacol. Exp. Ther. 2008, 327, 898.
in human PMN. Furthermore, compound 5 also inhibited C5a-med-
iated human PMN shape change in whole blood. The diminished
potency of the compounds in these assays may be due to longer
incubation time in the chemotaxis assay and non-specific binding
of compounds to serum proteins in the whole blood assay. In con-
trast to the lack of rodent cross-reactivity reported for other C5aR
small molecule antagonists,20–23 compound 17 showed antagonis-
tic activity for the mouse receptor (RAW cells) which would enable
in vivo studies. All three compounds were founded inactive to-
wards other GPCRs, such as human lymphocyte CXCR3 and CXCR4
in human T cells, and P2Y2 in CHO cells, suggesting that these com-
pounds are selective C5aR antagonists.
In summary, a new class of small molecular C5a antagonists has
been identified through HTS and a hit-to-lead process. The lead
molecules exhibit promising binding and functional activities with
human and mouse cell lines. Further optimization of this series will
be the subject of future communication.
23. Gong, Y.; Barbay, J. K.; Buntinx, M.; Li, J.; Wauwe, J. V.; Claes, C.; Lommen, G. V.;
Hornby, P. J.; He, W. Bioorg. Med. Chem. Lett. 2008, 18, 3852.
24. FLIPR assay conditions: CHO-G
well in 384 well black clear bottom TC plates (BD 353962) in media (R1
(invitrogen) + 1% FBS + 1 mM -glutamine) 24 h before the assay. On the day of
a16-hC5aR cells were seeded at 15,000 cells/
L
experiment, cells were washed and loaded with Ca3 dye (molecular devices
R8090) for 2 h (1 h 37 °C followed by 1 h at rt). Compounds were added to the
cells by FLIPR3 (molecular devices). After 10 min incubation with compound,
hC5a was added at the concentration of 2 Â EC50 (0.1 nM). C5a induced
intracellular calcium accumulation was recorded on FLIPR3.
25. Binding assay conditions: CHO-G
a16-C5a cells were seeded in growth media
(R1 + 10% FBS + 1 mM -glutamine) at 50,000/well in 96 well white-clear bottom
L
TC plate (Perkin–Elmer 1450-517) the day before the assay. On the day of
experiment, culture media was replaced by assay buffer (1 Â HANKS + 20 mM
HEPES + 0.1% BSA). Compounds were then added to the cells followed by 10 min
incubation at rt. 125I-hC5a (0.6 nM) (Perkin–Elmer, NEX250) was then added to
the cells. After 1 h incubation at room temp, the cell plate was washed eight
times with wash buffer (PBS + 1 mM CaCl2 + 0.1% BSA) on a harvester (Packard).
Excess buffer was removed by blotting with paper. 40 ml of Microscint 40
(Perkin–Elmer) was added to each well. The plates were then counted on Trilux
(Perkin–Elmer).
Acknowledgements
26. To create the alignment, each compound was expanded into multiple low
conformations using OMEGA (OpenEye Scientific Software, Santa Fe, NM. http://
www.eyesopen.com). Then pairwise alignments between the conformations of
(5) and the conformations of (17) were constructed using ROCS (OpenEye
Scientific Software). The alignment with the most consistent molecular shape
and 3D chemistry orientation was chosen for analysis.
We thank the CPG team and DAC group at Wyeth for their ana-
lytical supports to this project.
References and notes
27. Neutrophil assay conditions: Human neutrophils (PMN) were purified from
human whole blood using a Ficoll gradient protocol. The cells were then re-
suspended in 1 Â Ca3 dye and seeded at 50,000 cells/well in 384 well black
clear bottom TC plates (BD 353962) in assay buffer (1 Â HANKS + 20 mM
HEPES + 0.1% BSA). After 1 hr incubation at 37 °C, 5% CO2, Compounds were
added to the cells by FLIPR3. 10 nM hC5a was added after 10 min incubation
with compound. C5a induced intracellular calcium accumulation was recorded
on FLIPR3.
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incubated at 37 °C for 10 min. Then 250 ll ice-cold 1% (v/v) paraformaldehyde
was added to fix cells, followed by addition of ice-cold hypotonic buffer and
incubation on ice for 40 min to lyse red blood cells. Neutrophils were identified
and gated by flow cytometry on FACSCalibur (Becton Dickinson) using forward
and side scatter. Increase in neutrophil autofluorescence forward scatter on
FL2-H channel, which reflects shape change of neutrophils due to cytoskeletal
rearrangement stimulated by the C5a chemoattractant, was measured by
Flowjo software.
29. RAW assay conditions: RAW264.7 cells were seeded at 50,000 cells/well in 384
well black clear bottom TC plates (BD 353962) in media (DME (invitrogen) + 1%
FBS + 2 mM L-glutamine) the day before. On the day of experiment, cells were
washed and loaded with Ca3 dye for 1 h at 37 °C. Compounds were added to
the cells by FLIPR3. After 10 min incubation with compound, mC5a was added
at the concentration of 2 Â EC50 (10 nM). C5a induced intracellular calcium
accumulation was recorded on FLIPR3.