A new class of pseudopeptide antagonists of the kinin B1 receptor containing alkyl spacers

Article abstract of DOI:10.1021/jm980495r

Four previously reported kinin receptor peptide antagonists, including the B₁ receptor-selective peptides desArg¹⁰-HOE 140 (H-D-Arg-Arg-Pro-Hyp- Gly-Thi-Ser-D-Tic-Oic-OH) and B-9858 (H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D- Igl-Oic-OH), have been modified by replacement of the central tetrapeptide Pro-Hyp-Gly-Xaa with linear alkyl spacers of variable length. The analogue of desArg¹⁰-HOE 140 containing the 11-aminoundecanoic acid as spacer, MEN 11575 [H-D-Arg-Arg-NH-(CH₂)₁₀-CO-Ser-D-Tic-Oic-OH], was found to be slightly more potent than the unmodified peptide (pA₂ = 7.1) as a kinin B₁ receptor antagonist in the rat ileum longitudinal smooth muscle assay. Moreover, MEN 11575 is devoid of residual agonist activity at the kinin B₁ receptor (rat ileum) and antagonist activity at the kinin B₂ receptor (guinea pig ileum longitudinal smooth muscle). Both these activities are displayed by the parent peptide desArg¹⁰-HOE 140. Therefore, despite its greatly simplified chemical structure, MEN 11575 shows an improved pharmacological profile in terms of both potency and selectivity, and it represents a good template for the development of new peptidomimetic kinin B₁ receptor antagonists. We also report an attempt to investigate the conformational role of the flexible, linear spacer of MEN 11575 and to design more constrained analogues, possibly locked in the bioactive conformation, using semirigid spacers based on C(α)-tetrasubstituted α-amino acids of the family of 1-aminocycloalkane-1-carboxylic acids (Ac(n)c).

Full text of DOI:10.1021/jm980495r

J . Med. Chem. 1999, 42, 409-414
409
A New Cla ss of P seu d op ep tid e An ta gon ists of th e Kin in B
Alk yl Sp a cer s
1
Recep tor Con ta in in g
§
†
§
§
†
†
Claudia Galoppini, Stefania Meini, Mariella Tancredi, Armida Di Fenza, Antonio Triolo, Laura Quartara,
†
‡
‡
3
3
Carlo A. Maggi, Fernando Formaggio, Claudio Toniolo, Silvia Mazzucco, Annamaria Papini, and
Paolo Rovero*,
§
Peptide Synthesis Laboratory, CNR, Institute of Mutagenesis and Differentiation, Via Svezia 2A, I-56124 Pisa, Italy,
Menarini Ricerche, Via Sette Santi 3, I-50131 Firenze, Italy, Department of Organic Chemistry, Biopolymer Research Center,
CNR, University of Padova, Via Marzolo 1, I-35131 Padova, Italy, and Department of Organic Chemistry “Ugo Schiff”,
University of Florence, Via G. Capponi 9, I-50121 Firenze, Italy
Received August 24, 1998
1
Four previously reported kinin receptor peptide antagonists, including the B receptor-selective
1
0
peptides desArg -HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-OH) and B-9858 (H-
Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-OH), have been modified by replacement of the
central tetrapeptide Pro-Hyp-Gly-Xaa with linear alkyl spacers of variable length. The analogue
1
0
of desArg -HOE 140 containing the 11-aminoundecanoic acid as spacer, MEN 11575 [H-D-
Arg-Arg-NH-(CH 10-CO-Ser-D-Tic-Oic-OH], was found to be slightly more potent than the
unmodified peptide (pA ) 7.1) as a kinin B receptor antagonist in the rat ileum longitudinal
smooth muscle assay. Moreover, MEN 11575 is devoid of residual agonist activity at the kinin
receptor (rat ileum) and antagonist activity at the kinin B receptor (guinea pig ileum
2
)
2
1
B
1
2
1
0
longitudinal smooth muscle). Both these activities are displayed by the parent peptide desArg -
HOE 140. Therefore, despite its greatly simplified chemical structure, MEN 11575 shows an
improved pharmacological profile in terms of both potency and selectivity, and it represents a
1
good template for the development of new peptidomimetic kinin B receptor antagonists. We
also report an attempt to investigate the conformational role of the flexible, linear spacer of
MEN 11575 and to design more constrained analogues, possibly locked in the bioactive
R
conformation, using semirigid spacers based on C -tetrasubstituted R-amino acids of the family
n
of 1-aminocycloalkane-1-carboxylic acids (Ac c).
In tr od u ction
A first class of peptide antagonists selective for the
kinin B1 receptor was described in the late 1970s by
Two types of kinin receptors, termed B1 and B2, have
been proposed by Regoli and Barab e` on the basis of
the different rank order of potency of agonists and
antagonists. More recently, the human kinin B1 and B2
receptors have been cloned, and their genomic organi-
1
,7
1
Regoli and co-workers. The prototypical compound of
1
0
9
this family is an analogue of desArg -KD, with the Phe
1
0
9
residue replaced by Leu: desArg [Leu ]-KD (H-Lys-
2
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu-OH). This peptide,
9
3
4
although fairly potent (pA ) 8.37 vs desArg -BK in
zation was defined. The two kinins, bradykinin (BK,
2
9
H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) and kal-
lidin (KD, Lys-bradykinin), are generated as a result of
the activity of the proteolytic enzymes kallikreins on
kininogens. Once released, kinins exert a variety of
biological actions of pathophysiological relevance, in-
cluding pain, vasodilation, increased vascular perme-
isolated rabbit aorta; pA2 ) 7.99 vs desArg -BK in
7
,8
human umbilical vein), is rapidly metabolically de-
9
graded. A more stable peptide antagonist was reported
in 1991, following the observation that the potent kinin
1
0
B2 receptor antagonist HOE 140 can be converted to
a B1-selective antagonist upon removal of the C-terminal
5
9
3
5
7
8
ability, smooth muscle contraction, and secretion. Most
Arg residue: desArg -D-Arg[Hyp ,Thi ,D-Tic ,Oic ]-BK
1
0
-8
of these effects are mediated by the kinin B2 receptor,
which exhibits higher affinity for BK and KD. These
two kinins can be broken down by activity of carboxy-
peptidases to desArg -BK and desArg -KD, which in
turn display a greater affinity for the B1 receptor. The
expression of this receptor is induced in pathological
situations, particularly following tissue damage or the
inflammatory process. Consequently, the interest to-
ward the development of kinin B1 receptor-selective
antagonists is constantly increasing.
(desArg -HOE 140, 1; Table 1) has an IC50 of 1.2 × 10
9
11
vs desArg -BK in the rabbit aorta.
New antagonists have been obtained by further
9
10
9
modifications of both these peptides. Gobeil et al.
10
9
reported that the modification of desArg [Leu ]-KD by
replacement of Pro by D-âNal and Leu by Ile yields
an analogue (desArg [D-âNal ,Ile ]-KD, 2) which main-
tains the same antagonist potency as desArg [Leu ]-
8
9
10
8
9
6
10
9
KD at the rabbit kinin B1 receptor (pA2 ) 8.40 in the
9
rabbit aorta vs desArg -BK) and is more potent at the
human B1 receptor (pA2 ) 8.49 in the human umbilical
*
Corresponding author: Dr. Paolo Rovero. Tel: +39/50/574161.
9
Fax: +39/50/576661. E-mail: p.rovero@imd.pi.cnr.it.
vein vs desArg -BK). Most importantly, it is also par-
§
9
Institute of Mutagenesis and Differentiation.
Menarini Ricerche.
University of Padova.
University of Florence.
tially resistant to enzymatic degradation. Similarly,
†
1
2
‡
Stewart et al. modified 1 by introduction of Igl in the
3
place of Thi in position 5 and D-Igl in the place of D-Tic
1
0.1021/jm980495r CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/27/1999

4
10 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Galoppini et al.
Ta ble 1. Structure and Biological Activity of BK Antagonists
pA2d
structurea
RI (B1)
b
GPI (B2)
c
no.
1
H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-OH
6.8 ( 0.08
5.7 ( 0.13
1
0
(desArg -HOE 140)
2
3
Ac-Lys-Arg-Pro-Pro-Gly-Phe-Ser-D-âNal-Ile-OH
7.2 ( 0.24
8.1 ( 0.23
6.0 ( 0.20
5.5 ( 0.22
H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-OH
(
B-9858)
H-D-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-Arg-OH
B-9430)
4
7.7 ( 0.16
8.2 ( 0.11
(
5
6
7
H-D-Arg-Arg-Ahx-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-Aoc-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-Aun-Ser-D-Tic-Oic-OH
5.7 ( 0.50
5.4 ( 0.24
7.1 ( 0.13
nae
na
na
(MEN-11575)
8
9
H-D-Arg-Arg-Ado-Ser-D-Tic-Oic-OH
H-Lys-Arg-Aun-Ser-D-Tic-Oic-OH
Ac-Lys-Arg-Aun-Ser-D-Tic-Oic-OH
7.0 ( 0.19
6.0 ( 0.18
6.6 ( 0.17
6.1 ( 0.21
6.3 ( 0.40
6.3 ( 0.38
5.0 ( 0.30
7.0 ( 0.33
5.9 ( 0.24
5.2 ( 0.22
5.6 ( 0.18
5.7 ( 0.20
5.0 ( 0.20
5.5 ( 0.13
5.2 ( 0.20
5.4 ( 0.25
na
1
1
1
1
1
1
1
1
1
1
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
na
na
H-Lys-Lys-Arg-Aun-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-Aun-Ser-D-Igl-Oic-OH
H-Lys-Lys-Arg-Aun-Ser-D-Igl-Oic-OH
H-D-Arg-Arg-Aun-Ser-D-Igl-Oic-Arg-OH
Ac-Lys-Arg-Aun-Ser-D-âNal-Ile-OH
H-D-Arg-Arg-Ac7c-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-Gly-Ac7c-Gly-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-âAla-Ac7c-âAla-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-γ-Abu-Ac7c-γ-Abu-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-Ac6c-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-Ac8c-Ser-D-Tic-Oic-OH
H-D-Arg-Arg-Ac9c-Ser-D-Tic-Oic-OH
6.0 ( 0.22
5.5 ( 0.35
6.1 ( 0.11
5.9 ( 0.11
na
na
na
e5
na
e5
na
a
Axx ) -NH-(CH2)n-CO-, n ) 5 Ahx; n ) 7 Aoc; n ) 10 Aun; n ) 11 Ado; Acnc ) 1-aminocycloalkane-1-carboxylic acids, n ) 6-9;
see ref 4 for other amino acid abbreviations. RI, rat ileum longitudinal smooth muscle. c GPI, guinea pig ileum longitudinal smooth
muscle. pA2, negative log of the molar concentration of antagonist which produces an agonist dose ratio of 2. Values are the mean ( SE
of the mean of 4-15 experiments. na, no activity up to 10 µM concentration.
b
d
e
in position 8, with a concomitant N-terminal elongation
Ch em istr y
We previously reported that the C-terminal dipep-
by two Lys residues. The resulting peptide 3 (B-9858)
1
7
1
2
shows a pA2 of 9.5 for B1 receptor in the human ileum,
1
0
tide sequence of desArg -HOE 140 (D-Tic-Oic-OH) is
particularly prone to a well-known side reaction en-
countered in solid-phase peptide synthesis: diketopip-
erazine (DKP) formation by intramolecular aminolysis
of the C-terminal dipeptide, with consequent cleavage
and its action is long lasting in vivo.13 Interestingly, the
introduction of the same modifications in the sequence
of the B2 receptor-selective antagonist HOE 140 yields
an analogue (4, B-9430) showing high antagonist po-
tency at both B1 and B2 kinin receptors [pA2(B1) ) 7.3,
pA2(B2) ) 8.6 in the human ileum].12
18
of the peptide chain from the resin. We also showed
that the rate of DKP formation from the resin-bound
D-Tic-Oic dipeptide is very high, due to the N-alkyl-
amino acid structure of both these residues and the
D-configuration of the first one. Therefore, it was not
possible to suppress DKP formation by increasing the
rate of deprotection of the Fmoc residue at the dipeptide
stage, using a continuous flow synthetic strategy.19
However, we obtained good results by the use of a
Recently, our group reported a new class of B1-
selective pseudopeptide agonists, where the central
1
0
tetrapeptide Pro-Pro-Gly-Phe of desArg -KD was re-
placed by a flexible alkyl spacer.14 The same approach
was previously used by Kyle and co-workers15 to modify
the kinin B2 receptor antagonist HOE 140, on the
assumption that the minimal requirements for the
interaction of agonist peptides with the kinin B2 recep-
tor are “an intact C-terminal â-turn structure with
appropriate side chains in place and N-terminal amino
and guanidine groups”. The resulting pseudopeptide
H-D-Arg-Arg-NH-(CH2)11-CO-Ser-D-Tic-Oic-Arg-OH, char-
acterized by the presence of the 12-aminododecanoic
acid in the place of residues 3-6 of HOE 140, was
2
0
hindered tertiary alcohol linker. Accordingly, all of the
peptides characterized by a C-terminal D-Tic-Oic dipep-
tide described in the present paper have been prepared
2
1
on the 2-chlorotrityl resin, according to the previously
1
7
described protocol. Briefly, the 2-chlorotrityl chloride
resin was loaded with Fmoc-Oic-OH following the
2
1c
procedure published by Barlos et al. The amino acid
was used in equimolar amount in order to obtain a
substitution level in the range 0.3-0.4 mmol/g. Unre-
acted trityl groups were capped with methanol. Syn-
theses were performed manually, using a 2.5-fold excess
of protected amino acid derivatives and HATU/NMM
3
reported to have a pKi of 6.4 against [ H]BK in the
classical guinea pig ileum B2 binding assay and to be a
functional antagonist against BK-induced contractions
in the same tissue (pA2 ) 5.5).15
We describe here the application of this approach to
several first- and second-generation B1-selective peptide
antagonists, by using both flexible and conformationally
constrained alkyl spacers, the latter being based on the
22
activation. Each coupling was checked by the Kaiser
2
3
test and repeated if the test gave a positive result.
After cleavage (reagent K, 1.5 h at room temperature)
the crude peptide was precipitated by addition of cold
diethyl ether, filtered, and lyophilized. Crude products
were purified to homogeneity by semipreparative RP-
R
exploitation of C -tetrasubstituted R-amino acids of the
1
6
family of 1-aminocycloalkane-1-carboxylic acids (Acnc).

New Pseudopeptide Antagonists of Kinin B
1
Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 411
Ta ble 2. Analytical Data of BK Antagonists
In a first series of analogues (compounds 5-8) we
MW (ES-MSa)
calcdb
introduced the same modification proposed by Kyle et
HPLCa
tR (min)
1
5
foundc
al. and subsequently exploited by us for the prepara-
no.
1
4
tion of pseudopeptide B1 receptor agonists, namely, the
replacement of the central tetrapeptide by a series of
linear alkyl spacers of variable length. We chose as a
1
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
12.7
8.8
1147.4
1283.8
1338.7
840.5
868.5
910.6
924.6
882.6
924.7
1010.7
924.6
1024.7
1080.7
924.6
866.5
980.6
1008.6
1036.6
852.5
880.5
894.5
1147.8
1284.0
1338.2
840.5
868.6
910.8
924.5
882.6
924.7
1010.9
924.4
1024.6
1080.6
924.6
866.7
980.6
1008.6
1036.7
852.6
880.8
894.5
13.6
13.0
12.0
14.8
13.7
13.2
13.5
12.9
14.4
13.7
12.9
14.5
13.4
12.9
12.9
13.0
12.9
13.8
14.3
1
0
template the B1-selective receptor antagonist desArg -
11
3
4
5
HOE 140 (1) in which the tetrapeptide Pro -Hyp -Gly -
6
Thi was replaced by 6-aminohexanoic acid (Ahx, 5),
8-aminooctanoic acid (Aoc, 6), 11-aminoundecanoic acid
(Aun, 7), and 12-aminododecanoic acid (Ado, 8), respec-
tively. All these analogues are devoid of any agonist
1
1
1
1
1
1
1
1
1
1
2
2
2
-
5
activity at the B1 receptor up to 10 M concentration
and behave as competitive antagonists. Interestingly,
a first effect related to the introduction of the alkyl
10
spacer into the sequence of desArg -HOE 140 is the
removal of the slight agonist effect displayed by this
2
7
antagonist. The two analogues containing the shorter
spacers, i.e., Ahx (5) and Aoc (6), are less potent than
the unmodified peptide, while those containing Aun (7)
and Ado (8) are slightly more potent. Similarly, the
a
10
See Experimental Section for HPLC and MS methods.
corresponding agonist analogues of desArg -KD previ-
b
c
Monoisotopic relative molecular mass. Quasi-molecular ion
ously described showed a roughly linear correlation
between the length of the spacer and their agonist
minus 1 (m/z).
HPLC and characterized by analytical RP-HPLC and
mass spectrometry (Table 2).
14
activity at the kinin B1 receptor. Interestingly, the
pseudopeptides 5-8 show very weak antagonist activity,
if any, at the B2 receptor, while the reference compound
The Fmoc derivatives of Aoc and Aun, not com-
mercially available, have been prepared by the Fmoc-
OSu method.24 The use of these derivatives in solid-
phase peptide synthesis does not give any peculiar
problem, under the conditions reported above. The poor
water solubility of the Acnc residues with large rings (n
g 6) hampers the introduction of N-protecting groups
under standard conditions (aqueous/organic solvent
mixtures). We extended the methodology proposed by
J ohnson and co-workers25 to the Fmoc N-protection of
such hydrophobic residues. The use of the lipophilic base
tetramethylammonium hydroxide was, nevertheless, not
sufficient to completely solubilize the Acnc residues in
dioxane or acetonitrile, as also reported by J ohnson for
Ac5c, but solubilization was easily achieved upon addi-
tion of a small amount of water. The yields were
acceptable, despite the need of purifying the derivatives
by means of a chromatographic technique.
1
5
has a clear-cut B2 receptor antagonist activity (pA2 )
11,28
.7).
Taken together, these data indicate that the
10
replacement of the central tetrapeptide of desArg -HOE
40 by a linear alkyl spacer containing 10 (Aun) or 11
Ado) methylene units improves the pharmacological
1
(
profile of this B1 receptor antagonist in terms of both
potency and selectivity: in fact, introduction of the
spacer enhanced the antagonist potency at the kinin B1
receptor and abolished both the residual agonist activity
at the B1 receptor and, at least in the case of Aun, the
weak antagonist activity at the B2 receptor. Thus,
compound 7 (termed MEN 11575) appears to be a good
lead for the rational development of B1-selective pseudo-
peptide antagonists.
Subsequently, we studied the structure-activity re-
lationship (SAR) of the two residual peptide portions of
MEN 11575 in order to optimize its activity. Initially,
we focused our attention on the two positively charged
R
In the past, the synthesis of peptides containing C -
tetrasubstituted amino acids was regarded as difficult
because it requires the use of very reactive reagents or
harsh reaction conditions. More recently, the develop-
ment of new coupling reagents enabled the use of these
1
0
N-terminal residues of desArg -HOE 140 (D-Arg-Arg).
Since literature data indicate that the presence of Lys
residue(s) at the N-terminus is favorable to antagonism
3
,9,12
2
6
at the B1 receptor,
we replaced the N-terminal D-Arg
amino acids also in solid-phase peptide synthesis.
residue in MEN 11575 by Lys (9), Ac-Lys (10), and Lys-
Lys (11), respectively. However, these three pseudopep-
tides are less potent by 0.5-1 log unit at the B1 receptor
in the rat ileum, as compared to the unmodified
compound 7.
Accordingly, Fmoc-Acnc-OH were easily incorporated
into our synthetic peptides using the standard condi-
tions previously optimized for the preparation of
1
0
14
desArg -HOE 140, i.e., 2.5-fold excess of N-protected
amino acid and HATU/NMM activation.
Further modifications were performed in the C-
terminal portion of MEN 11575, following recent litera-
ture reports on new classes of B1 receptor antago-
Resu lts a n d Discu ssion
The biological activity of the pseudopeptides was
tested in two functional assays: the rat ileum longitu-
dinal smooth muscle, a kinin B1 receptor assay, and the
guinea pig ileum longitudinal smooth muscle, a prepa-
ration endowed only with the kinin B2 receptor. The
results, expressed in terms of pA2 (the negative log of
the molar concentration of antagonist which produces
an agonist dose ratio of 2), are listed in Table 1.
9
,12
nists,
as outlined in the Introduction. Accordingly,
we synthesized peptides 12 and 13, bearing a D-Igl
residue, as analogues of B-9858 (4). More precisely,
compound 13 is a true analogue of B-9858, with the
central tetrapeptide Pro-Hyp-Gly-Igl replaced by the
undecanoic spacer (Aun), while 12 maintains the same
N-terminal portion as MEN 11575, i.e., D-Arg-Arg, in
2
7

4
12 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Galoppini et al.
the presence of a D-Igl residue in place of D-Tic.
However, both these peptides are less potent as kinin
B1 receptor antagonists as compared to MEN 11575, and
they also show a residual B2 antagonist activity. Simi-
larly, peptide 14 is an analogue of the B1/B2 mixed
antagonist B-9430, containing Aun in place of the
central tetrapeptide. In this case, the modification
brings about an almost total loss of kinin B1 receptor
antagonist activity along with a great reduction of
activity also at the kinin B2 receptor. Taken together,
these data indicate that the D-Igl modification is not
fully compatible with the introduction of an alkyl spacer
in the central portion of these peptides.
both potency and selectivity. This peptide (7), termed
MEN 11575, is characterized by a linear, flexible alkyl
spacer connecting two peptide portions. Preliminary
attempts to investigate the role of this spacer indicate
that it cannot be effectively mimicked by the conforma-
R
tionally restrained alicyclic C -tetrasubstituted R-amino
acids. In any case, MEN 11575 represents a good
template for the development of new peptidomimetic
kinin antagonists.
Exp er im en ta l Section
Gen er a l. Protected standard amino acid and Fmoc-Ahx-
OH were purchased from Novabiochem (Bubendorf, Switzer-
land); Fmoc-D-Tic-OH, Fmoc-Thi-OH, Fmoc-Oic-OH, Fmoc-
âAla-OH, Fmoc-D-âNal-OH, and Fmoc-Hyp(tBu)-OH were from
Chem-Impex International (Wood Dale, IL); Fmoc-Ado-OH was
from Advanced Chemtech (Louisville, KY), and Fmoc-D-Igl-
OH was from Synthetech (Albany, OR). Fmoc-Ile-Novasyn
TGA and 2-chlorotrityl chloride resins were purchased from
Novabiochem. HATU was from PerSeptive Biosystem (Framing-
Peptide 15 is based on the antagonist described by
9
Gobeil et al. (2), with the central portion replaced by
Aun. In this case the effect of the substitution is more
favorable: the resulting pseudopeptide 15 has the same
activity as MEN 11575 at the kinin B1 receptor,
although it is also weakly active at the kinin B2 receptor
3
ham, MA). Analytical grade CH CN, DMF, and MeOH were
(pA2 ) 5.9).
obtained from Lab-Scan (Dublin, Ireland), and all other
reagents were from Aldrich (Milwaukee, WI) and Acros (Geel,
Belgium). Peptide 2 (Ac-Lys-Arg-Pro-Pro-Gly-Phe-Ser-D-âNal-
Ile-OH) was a kind gift of Prof. D. Regoli (University of
Ferrara, Italy).
We subsequently addressed our attention to the alkyl
spacer, in an attempt to investigate its role and to
design more constrained analogues, possibly locked in
the bioactive conformation. As previously observed in
TLC was performed on silica gel 60-F254 aluminum sheets
1
4
the case of the agonists, the higher activity of the
peptides containing the longer spacers (cf. 7 and 8 vs 5
and 6) could be explained by two hypotheses: (a) the
optimal distance between the two termini of the ligand
requires the longer spacers in an extended conforma-
tion; (b) the overall hydrophobicity of the central portion
of the ligand plays a crucial role, and the spacers are in
a coiled conformation. However, the high flexibility of
the alkyl spacers enables their easy interconversion
among various conformations. As a consequence, the
activity of our pseudopeptide antagonists could result
from a combination of both the above-mentioned effects.
A first, very simple attempt to design a semirigid spacer
(
)
Merck, Darmstadt, Germany), using the following eluants: 1
CHCl COOH/H O, 3:1:1; 3 )
3
/EtOH, 9:1; 2 ) 1-BuOH/CH
3
2
toluene/EtOH, 7:1; 4 ) AcOEt/petroleum ether, 1:3. Melting
points (mp) were determined in open capillaries and are
1
uncorrected. H NMR: Varian Gemini 200 MHz. MS analysis
was perfomed on a VG Quattro mass spectrometer (Altricham,
U.K.) equipped with standard electrospray (ES) ion source. The
samples (about 50 µg/mL) were dissolved in a 1:1 mixture of
3
CH CN/10 mM ammonium acetate containing 1% acetic acid
and introduced by infusion at 5 µL/min using a Harvard
Apparatus 11 pump (South Natik, MA). Ionization was ob-
tained using positive ion pneumatically assisted ES, at a
nebulizer voltage of 4 kV and a cone voltage of 50-70 V.
Nitrogen was used as the nebulization and desolvation gas.
The temperature of the ion source was 65 °C. For each sample,
either singly or doubly charged quasi-molecular ions were
obtained. The experimental and calculated molecular weights
in Table 2 are expressed as monoisotopic molecular weights.
R
was made by the use of C -tetrasubstituted R-amino
acids of the family of 1-aminocycloalkane-1-carboxylic
acids (Acnc).16 Accordingly, Ac7c (1-aminocycloheptane-
2
9
1
-carboxylic acid) was used to replace the flexible Aun
Syn th esis of F m oc-Aoc-OH a n d F m oc-Au n -OH. The
amino acid (1 equiv) was suspended in water and dissolved
spacer either by itself (peptide 16) or flanked by two
residues of Gly (17), â-Ala (18), or γ-Abu (19), which
were introduced in order to modulate the overall length
of the spacer. The best results in terms of antagonist
potency were obtained when Ac7c was used alone to
replace Aun (peptide 16), even if the potency as kinin
B1 receptor antagonist is lower by more than 1 log unit.
This finding would suggest that hydrophobicity, as well
as the conformational behavior of the linear alkyl spacer
of MEN 11575, plays an important role in the activity
of this antagonist. However, the conformationally con-
strained Ac7c residue does not mimic effectively the
bioactive conformation of the Aun-containing peptide.
In addition, the ring size of the Acnc residue is not a
key determinant for the antagonist activity of these
compounds. Indeed, smaller (Ac6c, peptide 20) and
larger (Ac8c, 21, and Ac9c, 22) rings roughly maintain
the same activity.
2 3
under reflux, and pH was adjusted to 9 with a 10% Na CO
solution. A solution of Fmoc-OSu (1 equiv) in dioxane was then
added dropwise over 20 min, and the mixture was stirred
under reflux for 24 h. The reaction mixture was then diluted
with water, acidified to pH 3 with HCl, and extracted twice
with CH
dried over Na
2
Cl
2
. The organic layer was washed with water and
SO , and the solvent was removed to dryness.
2
4
F m oc-Aoc-OH: yield 85%; mp 118-119 °C (from AcOEt/
petroleum ether); Rf1 ) 0.50, Rf2 ) 0.90, Rf3 ) 0.30, Rf4 ) 0.05;
1
-2
H NMR (1 × 10 M CDCl
3
) δ 7.80-7.26 (m, 8H, Fmoc
), 4.22
aromatic CH), 4.79 (s, 1H, NH), 4.41 (d, 2H, Fmoc CH
2
(t, 1H, Fmoc 9-CH), 3.16 (m, 2H, Aoc RCH ), 2.35 (t, 2H, Aoc
2
âCH
2
2
), 1.68-1.24 (m, 10H, Aoc ωCH ).
F m oc-Au n -OH: yield 92%; mp 117-118 °C (from AcOEt/
petroleum ether); Rf1 ) 0.50, Rf2 ) 0.95, Rf3 ) 0.30, Rf4 ) 0.10;
1
-2
H NMR (1 × 10 M CDCl
3
) δ 7.80-7.20 (m, 8H, Fmoc
), 4.20
), 1.80-1.20 (m,
aromatic CH), 4.80 (s, 1H, NH), 4.40 (d, 2H, Fmoc CH
(
1
2
t, 1H, Fmoc 9-CH), 2.30 (t, 2H, Aun RCH
2
8H, Aun ωCH
Syn th esis of F m oc-Ac
c-OH (1 equiv) and tetramethylammonium hydroxide
2
).
n
c-OH: Typ ica l P r oced u r e. H-
Ac
n
In conclusion, we have obtained a new pseudopeptide
antagonist selective for the kinin B1 receptor which,
despite the greatly simplified chemical structure, as
(TMAH; 1 equiv) were dissolved under stirring in a 9:1 mixture
of dioxane (or CH CN)/water. A solution of Fmoc-OSu (1 equiv)
3
in dioxane was then added. After 2 days at room temperature
an excess of Fmoc-OSu (0.3 equiv) was added. Stirring was
continued for 2 days; then the reaction mixture was concen-
1
0
compared to the reference compound desArg -HOE 140,
shows an improved pharmacological profile in terms of

New Pseudopeptide Antagonists of Kinin B
1
Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 413
trated under reduced pressure, acidified with 10% KHSO
solution, and extracted twice with AcOEt. The organic layer
was washed with water and dried over Na SO , and the solvent
was removed to dryness. The desired product was purified by
means of silica column chromatography.
4
(c) P u r ifica tion . Crude peptides were purified by semi-
preparative RP-HPLC on a Beckman System Gold apparatus,
under the following conditions: Vydac C18 column, 1 × 25 cm;
2
4
2 3
eluant A, 0.1% TFA in H O; eluant B, 0.1% TFA in CH CN;
gradient from 15% to 45% B over 200 min; flow 4 mL/min;
UV detection at 210 nm.
F m oc-Ac
EtOH, 99:1; yield 48%; mp 171-172 °C (from Et
ether); Rf1 ) 0.75, Rf2 ) 0.95, Rf3 ) 0.50, Rf4 ) 0.30; H NMR
6
c-OH: eluant for column chromatography, CHCl
3
/
2
O/petroleum
Ra t a n d Gu in ea P ig Ileu m Lon gitu d in a l Sm ooth
Mu scle Assa ys. Longitudinal muscle-myenteric plexus prepa-
rations of rat or guinea pig ileum were prepared as previously
described27 and put in organ baths filled with oxygenated (95%
1
-
2
(
1 × 10 M CDCl
3
) δ 7.77-7.28 (m, 8H, Fmoc aromatic CH),
.96 (s, 1H, NH), 4.45 (d, 2H, Fmoc CH ), 4.23 (t, 1H, Fmoc
-CH), 2.03 and 1.87 (2m, 4H, Ac c âCH ), 1.63 and 1.40 (2m,
H, Ac c γ and δCH ).
F m oc-Ac c-OH: eluant for column chromatography, petro-
4
9
6
2
6
2
2 2
O , 5% CO ) normal Krebs’ solution containing indomethacin,
6
2
guanethidine (3 µM each), chlorpheniramine, and atropine (1
µM each). Cumulative concentration-response curves to BK
7
9
or to desArg -BK were constructed in the guinea pig or rat
leum ether/AcOEt/EtOH, 70:25:5; yield 51%; mp 193-194 °C
ileum, respectively, in the presence of the peptidase inhibitors
thiorphan, bestatin, and captopril (1 µM each). We first
investigated the possible agonist activity of all the peptides
(
0
8
from AcOEt/petroleum ether); Rf1 ) 0.80, Rf2 ) 0.95, Rf3
)
1
-2
.50, Rf4 ) 0.35; H NMR (1 × 10 M CDCl
3
) δ 7.77-7.27 (m,
H, Fmoc aromatic CH), 5.02 (s, 1H, NH), 4.43 (d, 2H, Fmoc
1 2
at B and B receptors as follows: Those peptides which did
CH
âCH
Fm oc-Ac
2
), 4.21 (t, 1H, Fmoc 9-CH), 2.15 and 2.00 (2m, 4H, Ac
), 1.57 (m, 8H, Ac c γ and δCH ).
c-OH: eluant for column chromatography, toluene/
7
c
not produce any motor effect (contraction) in the two prepara-
tions used, up to 10 µM concentration, were considered inactive
as agonists. Those peptides which, at the highest concentration
tested (10 µM), produced a contraction lower than 50% of the
2
7
2
8
EtOH, 26:1; yield 44%; mp 188-189 °C (from AcOEt/petroleum
1
ether); Rf1 ) 0.85, Rf2 ) 0.95, Rf3 ) 0.50, Rf4 ) 0.35; H NMR
9
maximal response produced by the full agonist (BK or desArg -
-
2
(
4
9
ꢀ
1 × 10 M CDCl
3
) δ 7.77-7.27 (m, 8H, Fmoc aromatic CH),
BK), were considered as very weak agonists. The latter
compounds were expected to produce 50% of the maximal
response at concentrations exceeding 10 µM and hence to have
.95 (s, 1H, NH), 4.42 (d, 2H, Fmoc CH
-CH), 2.05 (m, 4H, Ac c âCH ), 1.54 (m, 10H, Ac
).
Fm oc-Ac
EtOH, 26:1; yield 45%; mp 186-187 °C (from CH
2
), 4.21 (t, 1H, Fmoc
8
2
8
c γ, δ and
CH
2
pD
2
lower than 5. Those peptides which produced a contraction
9
c-OH: eluant for column chromatography, toluene/
Cl
>
50% of the maximal response at 10 µM or lower concentra-
2
2
/
tions were assayed for their agonist activity in further experi-
ments in which complete concentration-response curves were
constructed. For the characterization of the antagonist activity,
the compounds under study were administered 15 min before
repetition of the agonist curve, at concentrations 1-10 µM,
and were tested for their ability to block the contractile
response to the agonist. Antagonist potency was expressed in
petroleum ether); Rf1 ) 0.85, Rf2 ) 0.95, Rf3 ) 0.50, Rf4 ) 0.40;
1
-2
H NMR (1 × 10 M CDCl
3
) δ 7.78-7.27 (m, 8H, Fmoc
), 4.21
), 1.54 (m, 12H,
aromatic CH), 4.90 (s, 1H, NH), 4.44 (d, 2H, Fmoc CH
2
(
t, 1H, Fmoc 9-CH), 1.97 (m, 4H, Ac
Ac c γ, δ and ꢀCH ).
Gen er a l P r oced u r e for th e Solid -P h a se P ep tid e Syn -
9
c âCH
2
9
2
t h esis (SP P S) w it h t h e F m oc St r a t egy. (a ) Ma n u a l
Syn th esis. All of the peptides containing a C-terminal Oic
residue were synthesized manually, in a mechanically agitated
reactor, using DMF as solvent. Fmoc-Oic-OH was linked to
the 2-chlorotrityl resin following the procedure described by
terms of pA (the negative log of the molar concentration of
antagonist which produces an agonist dose ratio of 2), while
2
agonist activity was expressed in terms of pD (negative log
2
of the agonist concentration which produces 50% of the
maximal effect). All the values reported are mean ( SE of the
mean.
2
1c
Barlos et al. The amino acid, in equimolar amount, was
dissolved in CH Cl , and the resin was added together with 1
equiv of DIEA. The reaction mixture was stirred for 5 min,
then 1.5 equiv of DIEA in CH Cl (1:1) was added, and the
2
2
Refer en ces
2
2
(
1) Regoli, D.; Barab e` , J . Pharmacology of Bradykinin and Related
Kinins. Pharmacol. Rev. 1980, 32, 1-46.
mixture was stirred again for 30 min. Subsequently, an excess
of methanol was added, to end-cap the remaining trityl group
of the resin. The substitution level was measured by UV
determination of the 9-fluorenylmethylpiperidine formed after
cleavage of the Fmoc group with piperidine. Fmoc deprotection
was obtained by treatment with 20% piperidine in DMF for
(
2) (a) Hess, J . F.; Borkowski, J . A.; Young, G. S.; Strader, C. D.;
Ransom, R. W. Cloning and Pharmacological Characterization
of a Human Bradikinin (BK-2) Receptor. Biochem. Biophys. Res.
Commun. 1992, 184, 260-268. (b) Menke, J . G.; Borkowski, J .
A.; Bierilo, K. K.; MacNeil, T.; Derrik, A. W.; Schneck, K. A.;
Ransom, R. W.; Strader, C.; Linemeyer, D.; Hess, J . Expression
2
0 min. Couplings activation was achieved with a 2.5-fold
and Cloning of a Human B
994, 269, 21583-21586.
1
Bradykinin Receptor. J . Biol. Chem.
excess of HATU, the same excess of protected amino acids,
and a 5-fold excess of NMM for 40-180 min. Completion of
each coupling was monitored by the Kaiser test. At the end of
1
(
3) (a) Bacharov, D. R.; Hess, F.; Menke, J . G.; Larrivee, J . F.;
Marceau, F. Structure and Genomic Organization of the Human
the synthesis the resin was washed with DMF, MeOH, CH
and diethyl ether and dried under vacuum. After cleavage with
reagent K (TFA/phenol/thioanisol/H O/ethanedithiol, 82.5:5:
:5:2.5, 120 min at room temperature) the crude peptides were
2
Cl
2
,
Bradykinin B
996, 33, 374-381. (b) Kammerer, S.; Braun, A.; Arnold, N.;
Roscher, A. A. The Human Bradykinin B Receptor Gene: Full
1 1
Receptor Gene for Kinins (BDKRB ). Genomics
1
2
2
Length cDNA, Genomic Organization and Identification of the
Regulatory Region. Biochem. Biophys. Res. Commun. 1995, 211,
226-233.
5
precipitated by addition of cold diethyl ether, filtered, dissolved
in water, and lyophilized. Crude peptides were analyzed by
analytical RP-HPLC on a Beckman System Gold apparatus
(4) Abbreviations are in accord with the recommendations of the
IUPAC-IUB Commission on Biochemical Nomenclature (J . Biol.
Chem. 1972, 247, 977). All amino acids are in the L-configuration
unless otherwise specified. Other abbreviations: γ-Abu, 4-amino-
(
SanRamon, CA) under the following conditions: Vydac C18
column, 0.46 × 15 cm; eluant A, 0.1% TFA in H O; eluant B,
.1% TFA in CH CN; gradient from 5% to 65% B over 20 min;
flow 1 mL/min; UV detection at 210 nm.
b) Au tom a ted Syn th esis. All other syntheses were per-
2
n
butyric acid; Ac c, 1-aminocycloalkane-1-carboxylic acids; Ado,
0
3
12-aminododecanoic acid; Ahx, 6-aminohexanoic acid; Aoc, 8-ami-
nooctanoic acid; Aun, 11-aminoundecanoic acid; BK, bradykinin;
Boc, tert-butyloxycarbonyl; DIEA, diisopropylethylamine; DMF,
dimethylformamide; ES-MS, electrospray ionization mass spec-
trometry; Fmoc, 9-fluorenylmethoxy-carbonyl; HATU, O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate; Hyp, 4-hydroxy-L-proline; Igl, R-(2-indanyl)-L-glycine;
KD, kallidine; âNal, â-3-(2-naphthyl)alanine; NMM, N-methyl-
morpholine; NMP, N-methylpyrrolidone; Oic, (3aS,7aS)-octahy-
droindole-2-carboxylic acid; PEG-PS, poly(ethylene glycol) poly-
styrene; RP-HPLC, reverse-phase high-performance liquid
chromatography; TFA, trifluoroacetic acid; Thi, â-(2-thienyl)-L-
alanine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
(
formed on a Milligen 9050 peptide synthesizer (Bedford, MA),
using the high-flow protocol on a PEG-PS resin. Coupling cycle
of 20 min was carried out with 3-fold excess of amino acids
and HATU as the activating reagent. Fmoc group removal was
obtained by treatment with 2% piperidine and 2% DBU in
DMF for 5 min. After completion of the synthesis, the resin
was washed and the crude peptide was cleaved and analyzed
on RP-HPLC as reported in the previous section.

4
14 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
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(
(
(
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Receptors: a Review. Immunopharma-
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