Peptidomimetic C5a receptor antagonists with hydrophobic substitutions at the C-terminus: Increased receptor specificity and in vivo activity

Article abstract of DOI:10.1016/j.bmcl.2006.07.036

A new class of peptidomimetic C5a receptor antagonists characterized by C-terminal amino acids with hydrophobic side chains is presented. Systematic optimization of the first hits led to JPE1375 (36), which was intensively characterized in vitro and in vi

Full text of DOI:10.1016/j.bmcl.2006.07.036

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5088–5092
Peptidomimetic C5a receptor antagonists with hydrophobic
substitutions at the C-terminus: Increased receptor
specificity and in vivo activity
*
Karsten Schnatbaum, Elsa Locardi, Dirk Scharn, Uwe Richter,
Heiko Hawlisch, Jochen Knolle and Thomas Polakowski
Jerini AG, Invalidenstraße 130, 10115 Berlin, Germany
Received 1 June 2006; revised 11 July 2006; accepted 12 July 2006
Available online 28 July 2006
Abstract—A new class of peptidomimetic C5a receptor antagonists characterized by C-terminal amino acids with hydrophobic side
chains is presented. Systematic optimization of the first hits led to JPE1375 (36), which was intensively characterized in vitro and
in vivo. Compound 36 exhibits high microsomal stability and receptor specificity and is highly active in an immune complex
mediated peritonitis model (reverse passive Arthus reaction) in mice.
Ó 2006 Elsevier Ltd. All rights reserved.
The complement system (CS) is one central arm of the
innate immune system. However, besides its well-known
role for enhancing B-cell responses, the modulation of
2
T-cell responses has been described recently. Thus, it
by hydrophobic amino acids resulted in highly active
antagonists with increased microsomal stability and
enhanced specificity for CD88.
1
becomes increasingly evident, that the CS also interacts
with the adaptive immune system. One key player at the
interface between innate and adaptive immunity is the
anaphylatoxin C5a, a 74 amino acid protein, which is
generated during activation of the CS. C5a binds to
the G protein-coupled receptors CD88 (C5aR) and
The first step in a systematic approach towards new
6
a
C5aRAs was an alanine scan of 1 (PMX53) (Table
1). All compounds were synthesized by standard solid-
phase peptide synthesis, cleavage from the resin and
cyclization of the C-terminal carboxylate to the Orn side
chain. The antagonistic activity of all compounds was
determined using a functional assay system based on
the inhibition of C5a induced glucosaminidase release
3
C5L2 of which only CD88 is functionally active. C5a
is a potent chemotactic factor for monocytes, macro-
phages, neutrophils, activated T- and B-lymphocytes
8
stimulated from CD88 transfected RBL cells (RBL+).
4
and induces a strong pro-inflammatory response.
It turned out that the replacement of Phe (2), cha (4),
Trp (5) and Arg (6) by alanine led to a pronounced loss
of activity by a factor of >200. Apparently, the side
chains of all of these amino acids play an important role
in functional activity. Pro could be replaced by Ala
without loss of activity. It has been shown that the small
ring size of PMX53 furnishes a rigid secondary struc-
As C5a has been discussed to be involved in a number of
diseases, several peptidic and non-peptidic C5a receptor
antagonists (C5aRAs) have been described (reviewed in
Ref. 5). Of the peptide derived inhibitors nearly all com-
pounds reported have a positively charged Arg at the
6
6a
C-terminus, as it is also found in C5a itself. Peptides
lacking this Arg have significantly lower binding affini-
ture and this might overrule any effects of Pro on the
secondary structure (Fig. 1).
7
ty. Here the discovery of a new class of peptidomimetic
C5aRAs is reported. Replacement of the C-terminal Arg
It is interesting to note that compound 6 is distinctly
more active than 2, 4 and 5, revealing that Arg is the
least important of the four critical side chains. Due to
this observation, compounds with additional modifica-
tions at the Arg position were synthesized (Table 2). The
elongation of the Arg side chain by one methylene group
led to a 12-fold decrease in antagonistic activity (7).
Keywords: C5a receptor; Antagonist.
*
Corresponding author. Tel.: +49 30 97893 380; fax: +49 30 97893
05; e-mail: schnatbaum@jerini.de
1
Present address: Parexel International GmbH, Klinikum Westend,
Spandauer Damm 130, 14050 Berlin, Germany.
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.036

K. Schnatbaum et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5088–5092
5089
Table 1. Alanine scan of 1
the charged guanidine group was completely removed
by the introduction of Nva leading to a compound with
surprisingly high activity (12). Compound 12 and addi-
tional compounds described below are the first C5aRAs
with hydrophobic substitutions at the C-terminus which
show activities in the low nanomolar range.
a
b
Compound
Sequence
Inhib. GAR
IC50 (lM)
1
2
3
4
5
6
(PMX53)
Ac-Phe-[Orn-Pro-cha-Trp-Arg]
Ac-Ala-[Orn-Pro-cha-Trp-Arg]
Ac-Phe-[Orn-Ala-cha-Trp-Arg]
Ac-Phe-[Orn-Pro-ala-Trp-Arg]
Ac-Phe-[Orn-Pro-cha-Ala-Arg]
Ac-Phe-[Orn-Pro-cha-Trp-Ala]
0.029
23
0.025
55
100
Subsequently, the C-terminal position was analyzed by a
variety of hydrophobic amino acid substitutions (13–
6.9
a
2
6). It turned out that a number of straight chain (13),
cha, D-cyclohexylalanine; ala, D-Ala.
Inhib. GAR, inhibition of glucosaminidase release.
b
branched (14–16) and cyclic (17–18) alkyl groups lead
to compounds with high antagonistic activity. Nva and
Nle are the best residues tested (Fig. 2). The space for
hydrophobic substituents seems to be limited though,
since the sterically more demanding cyclohexylethyl
O
N
(
19) and n-octyl (20) led to decreased activity compared
N
H
H
O
NH
to 1. This was also the case for nle (21) and 2Ni (26).
With small unsaturated or aromatic groups (22–25) high
activity could be achieved as well. The most active com-
pounds contain Eag, Phe or Thi (Fig. 2).
N
NH
HN
O
O
O
O
H
N
NH
NH
NH2
O
N
The effect of Arg replacements by amino acids with
hydrophobic side chains was also explored for linear
peptidomimetics. Two Arg containing compounds de-
rived from 1 by ring opening were synthesized (27, 28,
Table 3). It turned out that this modification is not well
tolerated and leads to a significant decrease in activity to
0.535 and 0.190 lM, respectively. Surprisingly the car-
boxamide is more active than the carboxylate, even
though C5a has got a C-terminal carboxylate and the
group has been proposed to be an essential feature of
H
Figure 1. PMX53 (Ac-Phe-[Orn-Pro-cha-Trp-Arg], 1).
The loss of affinity was even more pronounced when
Arg was replaced by Lys (8). In contrast to that Orn
(
that the appropriate orientation of the charged groups is
extremely important for a high antagonistic activity.
9) led to an activity similar to Arg. These results suggest
9
the pharmacophore of peptidomimetic C5aRAs.
The replacement of Arg by its bioisostere Cit (10) and by
Arg(NO ) (11), which are polar but uncharged under
Replacement of Arg by Nle (29) results in lower activity
compared to 28, suggesting that the SAR of the C-termi-
nus is different in linear peptidomimetics compared to
cyclic compounds. However, high activity can be
obtained by incorporating aromatic groups like in Phe
(30, 0.031 lM).
2
physiological conditions, leads to compounds with 17–
1
8 times lower activity compared to 1. This dramatic
loss of activity suggests that the positive charge of Arg
is very important for functional activity. In spite of that
Table 2. Antagonistic activity of Ac-Phe-[Orn-Pro-cha-Trp-Xxx] (7–26)
a
a
Compound
Xxx
Inhib. GAR IC50 (lM)
Compound
Xxx
Inhib. GAR IC50 (lM)
7
8
9
Har
Lys
0.360
8.700
0.018
0.490
0.570
0.018
0.025
0.050
0.054
0.057
17
18
19
20
21
22
23
24
25
26
Chg
Cha
Hch
Ocg
nle
0.038
0.045
0.146
0.300
0.107
0.019
0.026
0.022
0.051
0.076
Orn
Cit
1
1
1
1
1
1
1
0
1
2
3
4
5
6
Arg(NO
Nva
Nle
2
)
Eag
Phe
Thi
1Ni
2Ni
Ile
Leu
Hle
a
Har, homoarginine; Orn, ornithine; Cit, citrulline; Nva, norvaline; Nle, norleucine; Hle, homoleucine; Chg, cyclohexylglycine; Cha, cyclohexyl-
alanine; Hch, homocyclohexylalanine; Ocg, n-octylglycine; nle, D-norleucine; Eag, propargylglycine; Thi, 2-thienylalanine; 1Ni, 1-naphthylalanine;
2Ni, 2-naphthylalanine.
O
O
O
O
O
H N
2
H N
2
H N
H N
2
H N
2
OH
OH
2
OH
OH
OH
S
Nva
Nle
Eag
Phe
Thi
Figure 2. Structures of selected amino acids Xxx in Ac-Phe-[Orn-Pro-cha-Trp-Xxx].

5090
K. Schnatbaum et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5088–5092
Table 3. Antagonistic activity, stability against degradation by human microsomes and agonistic activity of compounds 27–36
a
b
Stab. micr. (%)
Compound
Sequence
Inhib. GAR IC50 (lM)
Agonism % at 286 lM
27
28
29
30
31
32
33
34
35
36
Ac-Phe-Orn-Pro-cha-Trp-Arg-OH
Ac-Phe-Orn-Pro-cha-Trp-Arg-NH
0.535
0.190
0.463
0.031
0.063
0.027
0.116
0.060
0.071
0.039
n.d.
n.d.
n.d.
34
n.d.
n.d.
n.d.
n.d.
0
2
Ac-Phe-Orn-Pro-cha-Trp-Nle-NH
Ac-Phe-Orn-Pro-cha-Trp-Phe-NH
2
2
Ac-Phe-Orn-Pro-hle-Trp-Phe-NH
Ac-Phe-Orn-Pro-hle-Bta-Phe-NH
Ac-Phe-Orn-Pro-hle-Dcf-Phe-NH
Ac-Phe-Orn-Pro-hle-Mcf-Phe-NH
Ac-Phe-Orn-Pro-hle-Pff-Phe-NH
Hoo-Phe-Orn-Pro-hle-Pff-Phe-NH
2
37
1
2
0
2
49
62
7.2
1.2
0
2
2
2
65
80
0
n.d. = not determined.
a
Nle, norleucine; hle, D-homoleucine; Bta, benzothienylalanine; Dcf, Phe(3,4-Cl); Mcf, Phe(3-Cl); Pff, Phe(4-F); Hoo, L-hydroorotic acid.
Human microsomes, % remaining after 1 h.
b
Table 4. In vitro properties of JPE1375 (36) and PMX53 (1)
The next goal was to optimize the stability in the pres-
ence of human liver microsomes. This stability is often
used for predicting hepatic clearance. Compound 30
shows a modest stability of 34% (Table 3). The replace-
ment of cha by hle (31) leads to a similar stability (37%).
The most important determinant for microsomal stabil-
ity is the Trp position though. The substitution of Trp
by Dcf, Mcf or Pff improved stabilities to 49%, 62%
and 65%, while retaining functional activity (33–35).
Among the latter compounds 35 is preferred, since unde-
sired agonistic activity has been found for 33 and 34,
which have bulkier side chains. This means that the ami-
no acid at the Trp position determines agonistic or
antagonistic activity which is in accordance with results
Assay
JPE1375 PMX53
Inhib. GAR, RBL+ cells, IC50 (lM)
Inhib. GAR, hPMN cells, IC50 (lM)
Binding, HEK293+ cells, IC50 (lM)
0.039
0.041
0.111
100
80
0.029
0.031
0.104
100
10
a
Plasma stability (hum, 1 h, 37 °C) (%)
Microsomal stability (hum, 1 h, 37 °C) (%)
b
CYP3A4 Inhibition at 10 lM (%)
38
2
77
4
No. of receptor interactions >50% at 10 lM
Binding NK2, IC50 (lM)
Binding MC4, IC50 (lM)
Binding V1a, IC50 (lM)
Binding ORL1, IC50 (lM)
0.71
13
0.087
0.40
0.74
2.20
7.1
0.74
> 50
Chemotaxis, mouse J774A.1 cells, IC50 (lM) 0.42
a
1
0
Inhibition of C5a binding to human C5aR on HEK293 cells.
Inhibition of CYP3A4 activity with BFC as substrate.
for other C5aRAs and could be a suitable starting
point for the development of agonists.
b
Finally the antagonistic activity of 35 was increased
about 2-fold by substitution of Ac for hydroorotic acid
(Hoo). This led to JPE1375 (36, Table 3, Fig. 3), a new
C5a receptor antagonist featuring high in vitro activity
binding site 1 is not effectively inhibited. Compound
6 exhibits high plasma stability (100% after 1 h) and
a higher microsomal stability compared to 1 (80% vs
3
1
1
0%). The inhibition of CYP3A4 is negligible (14% at
0 lM) which reduces the risk of undesired interactions
(0.039 lM), high stability against human microsomes
(80%, 1 h) and complete functional antagonism (0%
with the metabolism of other drugs.
agonism up to 286 lM).
For the determination of the receptor specificity a screen
with 44 different receptors, four ion channels and two
transporter proteins (ExpresSProfile, Cerep, Poitiers,
France) was performed at a concentration of 10 lM.
For 36 only two receptors with an inhibition of >50%
were identified, whereas 1 inhibits four (NK2, MC4,
V1a and ORL1). Subsequently, the IC50 values for these
four receptors were determined (Table 4). Compound 36
had a selectivity of at least 18 (factor between IC50 value
on NK2 receptor and IC50 on C5aR) while for 1 the low-
est selectivity factor was 3. Thus, 36 exhibits a markedly
improved receptor specificity.
Compound 36 was further characterized in vitro for
activity, stability and specificity (Table 4). Compounds
3
6 and 1 exhibit similarly high antagonism for human
C5aR expressed on RBL cells and human PMNs. The
inhibition of C5a binding to C5aR is slightly weaker
than the antagonistic potency. This can be explained
by binding of the compounds to the transmembrane
binding site 2 of the receptor as it was proposed for
1
1
other C5aRAs. It is expected that binding in this re-
gion is sufficient for the inhibition of the agonistic activ-
ity of C5a, but the binding of C5a to the extracellular
The in vivo efficacy of JPE1375 was tested in a mouse
model of the reverse passive Arthus reaction (RPAR),
an immune complex (IC) mediated disease which is driv-
en by activation of complement and Fc-receptors. The
role of C5a in the RPAR was described by K o¨ hl and
F
O
O
H
H
N
N
O
N
N
NH2
H
N
H
N
H
O
O
O
N
O
12
Gessner. Here the inhibitory activity of 1 and 36 on
H
HN
O
the neutrophil influx into the mouse peritoneum after
iv challenge with the OVA (chicken ovalbumin) peptide
and ip challenge with an anti-OVA-antibody was tested.
The compounds (1 mg/kg iv, dissolved 0.15 mg/mL in
O
NH2
2
Figure 3. JPE1375 (Hoo-Phe-Orn-Pro-hle-Pff-Phe-NH , 36).

K. Schnatbaum et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5088–5092
5091
PBS buffer) were administered 15 min before the induc-
tion of the RPAR. After 6 h, the number of neutrophils
in the peritoneal lavage was determined. Results are
shown in Figure 4.
Compound 36 is significantly more active in the RPAR
model than 1. This effect can be explained by improved
pharmacokinetic properties due to increased microsom-
al stability, but it is unlikely that this is the only expla-
nation. Therefore, the inhibitory activity of the
compounds on the C5a induced chemotaxis of mouse
J774A.1 cells was measured (Table 4). Compound 36
(
0.42 lM) is 17 times more active on the murine CD88
compared to 1 (7.1 lM). However, the antagonistic
activity of the two molecules for human cell lines (Table
Figure 5. Homology model of CD88 with bound 36.
4
experiments with 1 to C5aRAs of different species.
) is similar. This is in accordance with reported binding
1
3
Due to the activity of 36 on mouse cells this molecule
gives a new option for performing in vivo disease models
in this species.
Trp (Pff in 36) is located between Val286 and Ile116
1
0,14
(TM3) as was described before.
Phe binds close to
the position of the retinal head group in the rhodopsin
structure. It is surrounded by a number of aromatic
and hydrophobic residues. D-Cha (hle in 36) binds to a
patch of aliphatic amino acids from TM3-5 and EL2
(Leu167, Pro170, Val190 and Val202), which explains
the importance of this side chain for activity. The in-
creased affinity observed when exchanging Ac by Hoo
is due to the interaction with neighbouring polar resi-
dues (Ser266, Arg206).
In order to elucidate the SAR described, a homology
model of the C5a receptor, containing the seven trans-
membrane helices and the second extracellular loop
(
bovine rhodopsin. Compound 1 was constructed with-
EL2), was built on the basis of the X-ray structure of
1
4
in the interior of the receptor binding pocket with vari-
1
5
ous orientations. Subsequently, a multiple simulated
annealing protocol was employed to relax the system
and to define the best ligand receptor complex. Com-
pound 1 was mutated to the linear antagonist 36 and
In conclusion, new C5aRAs with hydrophobic substitu-
tions at the C-terminus are presented. Optimization of
activity and early ADME parameters led to JPE1375
1
6
the complex relaxed again via MD simulations.
(36), which shows favourable in vitro parameters and
high in vivo activity in mice.
One of the main features of the preferred model (Fig. 5)
is that Arg of 1 does not penetrate deeply into the bind-
ing pocket of the receptor, as it was suggested by Gerber
1
0
et al. for Me-FKPdChaWdR. Instead, Arg forms a
salt-bridge to Asp282 (TM7) as it was described in the
closed-loop binding model for 1 by Higginbottom
Acknowledgements
We thank Bianka Knopp, Rocco Hunds, Anja Rosen-
berg, Anja Mueller-Heyn, Seike Gericke and Doreen
Stern for their skillful technical assistance and J o¨ rg K o¨ hl
for helpful discussions.
1
7
et al. Alternatively, binding of a completely extended
Arg6 side chain to Asp37 (TM1) is conceivable. Due
to the proximity of Phe93, Val97 (TM2), Val186,
Ile187 (EL2) and Val286 (TM7), the same receptor re-
gion is also well suited to accommodate a hydrophobic
ligand. This perfectly corresponds to our observation
that Arg6 can be substituted by hydrophobic amino
acids without loss of activity.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.07.036.
RPAR in Mice
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