Highly selective bradykinin agonists and antagonists with replacement of proline residues by N-methyl-D- and L-phenylalanine

Article abstract of DOI:10.1021/jm9301954

For further studies on the structural and conformational requirements of positions 2, 3, and 7 in the bradykinin sequence, we replaced the proline residues by the more hydrophobic and conformationally restricted N-methyl-L- and D-phenylalanine (NMF). The biological activities of the new analogs were evaluated on rat uterus, guinea pig ileum, and guinea pig lung strip. Receptor binding of the analogs was studied in membranes from rat uterus and guinea pig ileum. Influence of bradykinin analogs on the release of cytokines from mouse spleen cell cultures was also measured. Bradykinin analogs were synthesized by the solid phase method, using Boc strategy on PAM or Merrifield resins. The best results in the formation of the N-methylamide bond were obtained with the coupling reagent PyBrop. In position 7 the substitution of D-Phe by D-NMF, retaining the configuration of the amino acid, converts bradykinin antagonists into agonists. The bradykinin analogs with D-NMF at position 7 gave the highest known tissue selectivity for rat uterus among agonists. [L-NMF²]bradykinin has moderate agonist activity on rat uterus but antagonist activity on guinea pig lung strip. It represents a new antagonist for B₂ receptors without any replacement at position 7. The same analog completely inhibits bradykinin-evoked cytokine expression by mononuclear cells.

Full text of DOI:10.1021/jm9301954

J . Med. Chem. 1996, 39, 929-936
929
High ly Selective Br a d yk in in Agon ists a n d An ta gon ists w ith Rep la cem en t of
P r olin e Resid u es by N-Meth yl-D- a n d L-p h en yla la n in e†
Siegmund Reissmann,*,‡ Carola Schwuchow,‡ Lydia Seyfarth,‡ Luis Felipe Pineda De Castro,‡ Claus Liebmann,‡
Inge Paegelow,§ Heinz Werner, and J ohn M. Stewart|
Institute of Biochemistry and Biophysics, Biological Faculty, Friedrich-Schiller-University-J ena, 07743 J ena, Germany,
Institutes of Pharmacology and Toxicology and Immunology, University of Rostock, 18057 Rostock, Germany, and
Department of Biochemistry, University of Colorado School of Medicine, Denver, Colorado 80262
Received J uly 12, 1994X
For further studies on the structural and conformational requirements of positions 2, 3, and 7
in the bradykinin sequence, we replaced the proline residues by the more hydrophobic and
conformationally restricted N-methyl-L- and D-phenylalanine (NMF). The biological activities
of the new analogs were evaluated on rat uterus, guinea pig ileum, and guinea pig lung strip.
Receptor binding of the analogs was studied in membranes from rat uterus and guinea pig
ileum. Influence of bradykinin analogs on the release of cytokines from mouse spleen cell
cultures was also measured. Bradykinin analogs were synthesized by the solid phase method,
using Boc strategy on PAM or Merrifield resins. The best results in the formation of the
N-methylamide bond were obtained with the coupling reagent PyBrop. In position 7 the
substitution of D-Phe by D-NMF, retaining the configuration of the amino acid, converts
bradykinin antagonists into agonists. The bradykinin analogs with D-NMF at position 7 gave
the highest known tissue selectivity for rat uterus among agonists. [L-NMF2]bradykinin has
moderate agonist activity on rat uterus but antagonist activity on guinea pig lung strip. It
represents a new antagonist for B2 receptors without any replacement at position 7. The same
analog completely inhibits bradykinin-evoked cytokine expression by mononuclear cells.
In tr od u ction 1
positions 7 and 8 of NPC-349 by D-tetrahydroisoquino-
line-3-carboxylic acid (Tic) and octahydroindole-2-car-
boxylic acid (Oic) to give the antagonist HOE-140. A
third series of potent antagonists was synthesized by
Kyle et al.13 They replaced proline in position 7 by
ethers of hydroxyproline, and phenylalanine in position
8 by Oic. One of the most active compounds, NPC-
17761, contains trans-4-(phenylthio)-D-proline at posi-
tion seven. Both HOE-140 and NPC-17761 are more
potent than the earlier antagonists containing 7-D-
phenylalanine.
Bradykinin (BK), an endogenous linear nonapeptide
hormone with the amino acid sequence Arg-Pro-Pro-Gly-
Phe-Ser-Pro-Phe-Arg, is involved in a variety of physi-
ological and pathophysiological processes. It stimulates
smooth muscle cells2,3 and sensory nerve endings,4,5
modulates the response of immunocompetent cells,3,6
and causes vasodilatation and microvascular leakage.2,3
Bradykinin contributes to the inflammatory response;
it produces pain, swelling, redness, and heat.3,4,7
Because of the pathophysiological role of bradykinin,
its antagonists are of great interest. Vavrek and
Stewart8 developed the first antagonists in 1984; their
key sequence alteration with respect to bradykinin was
the replacement of 7-proline with D-phenylalanine or
other D-aromatic amino acids. Following this key
substitution, they introduced other changes (particu-
larly addition of D-arginine to the amino end and
introduction of hydroxyproline at position 3 and thi-
enylalanine at positions 5 and 8) that gave the widely-
used “first generation” antagonists, especially D-Arg-
[Hyp3,Thi5,8,D-Phe7]BK, known as NPC-349.9 Since
then, many laboratories have attempted to develop more
potent antagonists. In particular, the antagonist D-Arg-
[Hyp3,D-Phe7,Leu8]BK10 was converted to the 6-cysteine-
containing analog, then cross-linked via bis(male-
imido)hexane, forming a dimeric peptide (known as CP-
0127).11 Knolle et al.12 replaced the amino acids in
In recent years various laboratories have attempted
to estimate the conformation of bradykinin agonists and
antagonists, especially the bioactive conformation. One
of the most interesting questions is a possible difference
in the conformation of agonists and antagonists. Be-
cause of the lack of strongly conformationally restricted
cyclic agonists and antagonists with high biological
activity and specificity, the proposed models give only
preliminary information about the bioactive conforma-
tion. From NMR measurements and calculations of the
dihedral angles φ, ψ, and ø, Kyle et al.14 estimated two
turns in bradykinin and bradykinin antagonists, one
turn in the N-terminal part and a â-turn in the
C-terminal part. Cyclization of the N-terminal part of
their antagonists14d led to large losses in potency. In
contrast to this model, NMR studies of agonist-
antagonist pairs by Liu et al. and Otter et al.15 showed
the C-terminal â-turn only in agonists while antagonists
had only one â-turn in the N-terminal part.
* To whom correspondence should be addressed.
† A preliminary account of this work was presented at the 21st
European Peptide Symposium, Interlaken, Switzerland, September
1992.
As shown for various di- and oligopeptides16-19 the
allowed dihedral angles in N-methyl amino acids are
very restricted. Generally, N-methylation is considered
to be one of the local and subtle modes of conformational
constraint. It reduces the energy barrier to rotation
around the N-methylated peptide bond and thus may
‡ Friedrich-Schiller-University-J ena.
§ Institute of Pharmacology and Toxicology, University of Rostock.
Institute of Immunology, University of Rostock.
| University of Colorado School of Medicine.
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-0929$12.00/0 © 1996 American Chemical Society

930 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Reissmann et al.
Sch em e 1. Synthesis of Bradykinin Analogs with L-
reduce the predominance of the trans vs cis peptide
bond. With N-methylphenylalanine (NMF) the rate
constant for cis-trans interconversion is in the same
range as in proline.17,19 In this respect N-methyl amino
acids are able to simulate the situation of proline in
peptide bonds. On the one hand, the cis-trans inter-
conversion enhances the flexibility, while on the other
hand, significant conformational restriction is imposed
by the N-methyl group. Furthermore, N-methylation
of a peptide bond eliminates its ability to donate a
hydrogen bond. With respect to the biological activity,
in some cases N-methylation has given compounds with
improved pharmacological properties, such as enhanced
potency and selectivity.17,20,21
and D-NMF at Positions 5, 7, and 8, Using Boc Strategy
on Merrifield Resina
To study the influence of N-methylation on biological
activity we replaced the phenylalanine residue at sev-
eral positions in bradykinin antagonists by D- and
L-NMF. In order to help define conformational require-
ments about the critical position 7, we synthesized
analogs having D- or L-NMF at that position. Since
potency and tissue selectivity depend not only upon
replacement in position 7 but on the whole sequence,
we put D- and L-NMF into various positions in highly
potent and selective compounds from the [D-Phe7]BK
series. Continuing studies of one of the authors22 on
bradykinin analogs with L-NMF and N-methy-L-argin-
ine, we also investigated replacement of both the
phenylalanine residues at positions 5 and 8. Because
of the analogy of proline to N-methyl amino acids with
respect to the cis-trans conversion, we also replaced the
proline residues at positions 2 and 3 with L- and D-NMF.
Because it may be possible to find new types of brady-
kinin antagonists having their key modifications at
different positions in the sequence, one goal of these
studies was to find new classes of antagonists for
different pharmacological assay systems.
a
NG-Tos protection, PyBrop as coupling reagent for formation
of methylamide bonds, BOP for sterically hindered amino acids,
and DCC for normal coupling steps.
but also to develop highly potent and highly selective
agonists and antagonists. Enzymatic degradation of the
agonists in the pulmonary circulation was evaluated
from the difference in the hypotensive response follow-
ing intravenous and intraarterial administration. In
addition to these classical tests, we estimated the
influence of the bradykinin agonists and antagonists on
interleukin release from mononuclear cells.28 Binding
studies using displacement of 3H-labeled bradykinin
from membrane fractions of RUT and GPI were per-
formed to estimate the correlation between receptor
affinities and pharmacological activities. Binding pa-
rameters of the analogs were calculated by use of the
Tobler program.29 The radioligand binding experiments
combined with a sophisticated calculation of parameters
give additional and more detailed information about
receptor interactions than the pharmacological assays
alone.
The chemistry of bradykinin synthesis is very well
established. Starting from Boissonnas and Gutt-
mannn’s first synthesis23 in solution and Merrifield’s
synthesis24 on a solid phase, many synthetic variants
have been tested in the last three decades. We synthe-
sized the bradykinin analogs by different solid phase
strategies, using the Boc strategy on PAM or Merrifield
resin. One of the anticipated problems was coupling of
the sterically hindered N-methyl amino acid and the
formation of the N-methylamide bond at the following
step in the synthesis.
Resu lts a n d Discu ssion
Kinin receptors have been divided into three classes:
B1, B2, and B3.25,26 B1 receptors recognize primarily the
octapeptide bradykinin(1-8), and may be important in
chronic inflammation, while most responses to brady-
kinin are mediated by B2 receptors, which recognize
bradykinin and larger homologs and respond only very
weakly to bradykinin(1-8). Many laboratories have
attempted to subdivide B2 receptors on the basis of
relative potencies of bradykinin analogs in different
tissues.25 Thus Regoli27 suggested from differences in
agonist and antagonist activities three different sub-
types: B2A, B2B, and B2H. We have therefore estimated
the biological activities of the new series for contraction
of rat uterus (RUT), guinea pig ileum (GPI), and guinea
pig lung strip (LS) and for inhibition of bradykinin-
evoked cytokine release from mononuclear lymphocytes.
RUT, GPI, and LS are classified as tissues with B2
receptor subtypes.2,25,26 The goal of this structure-
activity study is not only to subdivide the B2 receptors
Syn th esis. Coupling of the hindered Boc-NMF re-
quires strong activation; BOP/HOBt was used. For
acylation of NMF residues, best results in formation of
the CO-NCH3 bond were obtained with the coupling
agent PyBrop. The synthetic routes used for Boc and
Fmoc syntheses of peptides containing NMF are shown
in Schemes 1 and 2.
The combination of two or three NMF residues in one
peptide caused the most difficulty. Thus, peptide 7,
containing three NMF residues, including the sequence
[NMF7,NMF8], was obtained only in low yield and with
a considerable amount of byproducts. It could be
purified only by a combination of CCD and preparative
HPLC. Unexpected difficulties were encountered in the
syntheses of [L-NMF3]- and [D-NMF3]BK, resulting in
impure products and low yields. In both cases the
coupling of proline at position 2 required two recoupling
steps with a total time of 72 h. Even a change of solvent

Bradykinin Agonists and Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 931
Sch em e 2. Synthesis of Bradykinin Analogs with L-and
D-NMF at Positions 2 and 7 Using Boc Strategy on PAM
Resina
The functional activities of compounds 3 to 7 (Table
1) show great differences in potency between the rat
uterus and the guinea pig ileum. The decrease in
potency between the key analog 3 and the other
compounds (4, 5, 6, and 7) in this series suggests that
there exist different structural requirements for ago-
nists and antagonists. We may draw this conclusion
from the finding that in contrast to results with an-
tagonists the replacements of phenylalanine at position
5 by thienylalanine and of proline at position 3 by
hydroxyproline in these agonist peptides do not increase,
as expected, but rather reduce their potency. The
replacement of phenylalanine residues by NMF in the
peptides enhances the stability against enzymatic in-
activation. The differences in the potencies for blood
pressure reduction between intraarterial and intrave-
nous administration of these agonists in comparison to
BK (3, ia 60%, iv 250%; 4, ia 40%, iv 160%; 5, ia 14%,
iv 760% of BK potency) demonstrate enhanced resis-
tance to degradation in the pulmonary circulation. This
degradation is principally due to angiotensin I convert-
ing enzyme (ACE).8 Furthermore, the peptide with
D-NMF in position 7 (3) is unable to interact with
isolated ACE (inhibition of ACE activity: 10-5 M, 0%;
10-4 M, 20%, IC50 . 10-4 M; W.-E. Siems, personal
communication). This finding is in good agreement with
the stability in vivo of [D-Phe7]BK analogs.32
a
NG-Mts protection, PyBrop as coupling reagent for formation
of methylamide bonds, TBTU for sterically hindered amino acids,
and DIC for normal coupling steps.
30
and addition of NaClO4 did not reduce the coupling
time. Standard HF cleavage and deprotection of the
peptides on Merrifield resin (Scheme 1) yielded a
considerable amount of incompletely deprotected pep-
tides. In contrast to this result, the use of the TFMSA
cocktail described for the syntheses on PAM resin
(Scheme 2) delivered peptides with better purity and
in higher yield.
Rep la cem en t of P r olin e Resid u es a t P osition s
2 a n d 3 by D- a n d L-NMF . Replacement of the imino
acid proline at positions 2 and 3 by the more hydropho-
bic and conformationally restricted imino acid N-meth-
ylphenylalanine decreases the biological activity strongly
in most cases (Table 1, compounds 11-13). Only
[L-NMF2]BK (10) shows moderate activity on RUT. This
fact emphasizes the different structural requirements
for positions 2 and 3 and the tissue differences in GPI
and RUT.
Con ver sion of An ta gon ists in to Agon ists by
Rep la cem en t a t P osition 7. Table 1 shows that all
analogs having D-NMF at position 7 are agonists on
RUT and GPI. Thus, the simple replacement of D-Phe
at position 7 by D-NMF converts the anticipated an-
tagonists into agonists in all analogs tested. Analog 8
with L-NMF in position 7 is an agonist as well. In good
agreement with the studies of Mazur et al.31 on the
biological activity of bradykinin analogs containing
L-NMF, the replacement of Phe at positions 5 and 8 in
our compounds 6 and 7 reduced the agonist potency and
stabilized the peptide against enzymatic degradation in
the circulation. The strongly conformationally re-
stricted compounds 6 and 7 are interesting objects for
conformational studies.
Summarizing these results from the viewpoint of
amino acid configuration, we can conclude that on the
one hand at position 7 NMF in both configurations gives
agonists, whereas on the other hand at position 2, only
the L-configuration provides moderate agonist potency.
A New Typ e of An ta gon ist for th e B2 Recep tor .
Both the ileum and lung of guinea pigs have B2
receptors with similar structural requirements for BK
and analogs, although the lung may be more complex.33
A significant difference between these tissues is indi-
cated by peptide 10, which has L-NMF at position 2; this
analog is an antagonist on the lung strip. This analog
is one of very few antagonists26 for B2 receptors without
any change at position 7.
Given the state of our present knowledge, it is
impossible to explain the conversion of antagonists with
D-amino acids at position 7 into agonists with D-NMF.
We compared the calculated conformational energy
maps for N-methyl amino acids, especially D- and
L-NMF, with those for BK and the amino acid combina-
tions D-Phe-Oic, D-Tic-Oic, and trans-D-Hyp-Oic in an-
tagonists. The conformational energy maps for N-meth-
yl amino acids with the peptide bond in the cis or trans
configuration were taken from the literature16-18 and
estimated for di- and oligopeptides. The dihedral angles
for the dipeptides in the C-terminal part were estimated
by Kyle and co-workers.14 Neither D-NMF nor L-NMF
is able to have the same allowed areas for dihedral
angles as has been estimated for position 7 in antago-
nists, but the conformational φ, ψ energy maps also do
not correspond to that of proline. Nevertheless, the
analogs with D-NMF at position 7 act as agonists.
On the basis of differences in potencies of agonists
and antagonists, Farmer et al.33 proposed that guinea
pig lungs contain two receptor subtypes: B2 and B3.
With our analogs we found only small differences
between the agonist activities on the GPI and LS. The
only exception is compound 10. Thus, we would suggest
that [L-NMF2]BK is a new antagonist for the B2 receptor
type. This compound is a weak antagonist on LS but
the strongest known antagonist on mononuclear cells,
as shown below. This new antagonist and the B2A
selective antagonists DArg-[Hyp3,Leu8]BK and DArg-
[Hyp3,Gly6,Leu8]BK developed by Regoli et al.27 indicate
different structural requirements for different types of

932 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Reissmann et al.
Ta ble 1. Biological Activities of Bradykinin Analogs Containing N-Methylphenylalaninea
activity (% of bradykinin)
GPI
no.
peptide structure
RUT
LS
1
2
3
4
5
6
7
8
9
BK
100
100
100
0.9 ( 0.7
[D-Phe7]BK
1.7 ( 0.03
136 ( 23
89 ( 4.7
86 ( 14.4
40 ( 2.5
15 ( 5.1
0.2 ( 0.04
0.5 ( 0.04
35 ( 4
pA2 5.56
0.04 ( 0.01
0.02*
0.006*
0.01*
[D-NMF7]BK
1.4 ( 0.03
0.04 ( 0.007
0.04 ( 0.01
0.45 ( 0.04
0.5 ( 0.05
0.1 ( 0.02
0.08 ( 0.02
1.4 ( 0.01
pA2 5.59 ( 0.47
0.05 ( 0.01
0.03 ( 0.002
0.03*
[Hyp3,Thi5,D-NMF7]BK
D-Arg-[Hyp3,Thi5,D-NMF7]BK
D-Arg-[Hyp3,D-NMF7,L-NMF8]BK
D-Arg-[Hyp3,D-NMF7,L-NMF5,8]BK
[L-NMF7]BK
0.005*
0.03 ( 0.004
0.07 ( 0.01
4 ( 0.8
D-Arg-[L-NMF7]BK
10
[L-NMF2]BK
11
12
13
[D-NMF2]BK
[L-NMF3]BK
[D-NMF3]BK
0.15 ( 0.02
0.03 ( 0.009
0.02 ( 0.008
0.15 ( 0.09
0.05 ( 0.005
0.03 ( 0.004
a
Assays were performed as described in the Experimental Section: RUT, rat uterus; GPI, guinea pig ileum; LS, guinea pig lung strip.
Antagonist activities of peptides 2 and 10 are given as the pA2. The pD2 of BK is 7.9 in RUT and 7.4 in GPI. The results are means (
SEM of three to seven separate determinations. Data marked with an asterisk (*) are the means of duplicate determinations that varied
by less than 10%.
this assay and receptor binding of several series of
bradykinin analogs will be published separately.
Tissu e Selectivity. One of our goals is the develop-
ment of highly selective bradykinin agonists and an-
tagonists. Table 2 shows the tissue selectivity of
bradykinin agonists for RUT and GPI. The quotient
RUT/GPI indicates the selectivity of various bradykinin
analogs for the rat uterus. In comparison with the most
selective agonists from the literature,9,34,35 some of our
analogs with D-NMF at position 7 considerably exceed
their selectivity quotients and are the most selective
agonists known for rat uterus.
Receptor Bin din g P ar am eter s. For both rat uterus
F igu r e 1. Effect of HOE-140 and [L-NMF2]BK on the BK-
induced secretion of charge-changing cytokines in mouse
spleen cell cultures. Spleen cells (106/mL) were incubated for
4 h, 37 °C with BK alone (10-7 M) (- - -) or with this BK dose
after preincubation (0.5 h, 37 °C) with graded concentrations
of HOE-140 (b) or [L-NMF2]BK (0). Cell-free supernatants
were incubated with target cells (sulfosalicylic acid stabilized,
tanned sheep erythrocytes) for 1 h, 23 °C, and the migration
time of the targets was estimated in a cytopherometer. The
slowing effect was calculated using targets and targets plus
peptides (to exclude side effects) as controls. Values are mean
(SD, taken from three experiments in duplicate cultures. The
mean of all SD was calculated as residual variance.
and guinea pig ileum there are contradictory results
postulating a single binding site36 or two binding sites,
one with picomolar and one with nanomolar affinity.37
The biphasic displacement curves (Figure 2) obtained
for most, but not all, analogs tested here indicate an
interaction with both of the previously described brady-
kinin binding sites. There is no obvious correlation
between the recognition of one or two bindings sites and
the functional activities. However, the functional po-
tencies of most analogs relative to bradykinin give a
closer correlation with the low-affinity site (Table 3),
thus confirming our assumption, based on previous
evidence, that the smooth muscle contracting effect of
bradykinin seems to be mediated by the site with
nanomolar affinity38 that is linked to phospholipase C.39
It has recently been shown that the high-affinity (pico-
molar) binding site on guinea pig ileum correlates with
inhibition of adenylate cyclase in that tissue.40 It should
be noted that [L-NMF2]BK showed a higher biological
activity but lower binding affinity on the rat uterus
compared with that on the guinea pig ileum. It may be
assumed, therefore, that there exists a higher signal
amplification in the rat uterus, or that there are
different signal transduction pathways in the two tis-
sues.
antagonists and show that the various analogs with
replacements of proline at position 7 represent only one
type.
In other words, from these and other recent findings
we might assume that there exists more than one
position in the sequence where alterations may give
antagonists, but nothing is known about the conforma-
tional requirements at these positions. Cyclization and
subsequent conformational analysis of the new analogs
by different physical and computational methods will
be required.
Cytok in e Relea se fr om Mon on u clea r Cells. The
TEEM (tanned erythrocyte electrophoretic mobility) test
was used to characterize the cytokines released from
mononuclear cells by the peptides.28 Bradykinin re-
leases cytokines from mouse spleen cells, mainly inter-
leukins 1, 2, and 6. Figure 1 shows inhibition of
cytokine release by the bradykinin antagonists HOE-
140 and [L-NMF2]BK. The latter compound completely
inhibited cytokine expression at a concentration of 10-13
M. Other antagonists are less potent, e.g. [DPhe7]BK
with an inhibitory concentration of 10-10 M and HOE-
140 with 10-12 M. Structure-activity relationships in
While most of the bradykinin analogs reported here
bind to both the high-affinity and low-affinity sites on
ileal membranes, several did not (Table 3); this group
of peptides showed only one affinity. This has previ-
ously been observed for some other agonist analogs.41
Another puzzle is the very large difference between
binding affinity and functional potency of certain of the
analogs, especially in ileum (Table 1). This difference
is apparent for bradykinin itself, since the pD2 (the

Bradykinin Agonists and Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 933
Ta ble 2. Tissue Selectivity for Smooth Muscle Contraction of Bradykinin Agonistsa
no.
compound
RUT
GPI
RUT/GPI
1
bradykinin (BK)
100%
12
0.001
0.2
55
100%
0
2
Lys-Lys-[∆Phe7]BKb
[p-GuanidoPhe1,9]BKc
[Lys9]BKc
0.001
0.01
0.13
0.291
0.30
0.442
5.0
13
25
30
100
200
300
4000
10000
4000
3000
20
[Aib7]BKb
418
426
321
226
200
40
11
7
0.3
0.1
[D-Phe6,Aib7]BKb
124
97
[D-Phe(Cl)6,Aib7]BKb
[D-Trp6,Aib7]BKb
100
1000
500
271
237
31
15
136
89
[Thi5,8]BKb
[Thi8]BKb
D-Ile-Ser-BKb
D-Arg-[Hyp3,Thi5,8]BKb
[Gly6,D-Phe7]BKb
[Thi5,8,∆Phe7]BKb
[D-NMF7]BK
3
4
5
6
7
0.4
[Hyp3,Thi5,D-NMF7]BK
D-Arg-[Hyp3,Thi5,D-NMF7]BK
D-Arg-[Hyp3,D-NMF7,NMF8]BK
D-Arg-[Hyp3,NMF5,8,D-NMF7]BK
0.02
0.006
0.01
0.005
86
40
15
a
b
Activities are given as percent of BK activity. Reference 35. c Reference 34. The ratio RUT/GPI indicates the selectivity of the
analogs for rat uterus.
F igu r e 2. Inhibition of specific [3H]BK (30 pM) binding to membranes of guinea pig ileum by BK (A) and [D-NMF7]BI (B) and
membranes of rat myometrium by BK (C) and [D-NMF7]BK (D). Plots from the Tobler program29 are shown. Calculations were
from untransformed data (total binding values). Nonspecific binding is reflected by the curve shape at BK concentrations higher
than 1 µM and can differ between several experiments. Locations of the vertical bars indicate the IC50 values of the binding
sites. Their height represents the relative concentrations of the binding sites. The affinity spectra (continuous curves) allow for
visual differentiation between statistically valid and spurious peaks. Data points are means from duplicate determinations in a
representative experiment.
negative logarithm of the ED50) for bradykinin is 7.9
on rat uterus and 7.4 on guinea pig ileum, indicating
an EC50 between 10 and 100 nM, whereas the binding
IC50 is about 1 nM for the low-affinity site in both
tissues. With the peptides reported here, the dissocia-
tion between binding and functional potency is particu-
larly impressive on ileum. Lower potencies on ileum
are often attributed to the known greater activity of
various kininase enzymes in ileum. Binding studies are
done in low ionic strength medium, at low temperature,
and in the presence of enzyme inhibitors, whereas the
functional assays are carried out at physiological tem-
perature and ionic strength. Physiological ionic strength
media have been reported to inhibit bradykinin receptor
binding.39 Internalization of agonist-receptor com-
plexes, which may contribute in an undefined way to
the lower functional potencies of agonists in various
tissues, is inhibited in binding studies by use of low
temperature.
At this time only one bradykinin B2 receptor amino
acid sequence has been identified for any given species,
although from very early in bradykinin antagonist
research peptides were obtained which discriminated
between tissues within a species as well as between
species.9 As mentioned above, several subclasses of B2
receptors have been proposed on the basis of pharma-
cological responses to different antagonists.27 The mo-
lecular basis of these differences remains an open
question, although different post-translational process-
ing of receptors in different tissues is a likely cause. A
recent study of the rat B2 receptor gene identified
differential processing leading to two different mRNAs

934 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Reissmann et al.
Ta ble 4. Analytical Characteristics of Bradykinin Analogs
Ta ble 3. Binding Affinities of Bradykinin Analogs to
Membranes from Rat Uterus and Guinea Pig Ileuma
TLCa Rf values
electrophoresisb HPLCc
route of
IC50(nM)
no.
A
B
RArg
K′
synthesisd
RUT
HA LA HA
0.01 1.00 0.01 1.75
6.6 3.5 14
132
GPI
3
4
5
6
7
8
9
10
11
12
13
0.53
0.5
0.45
0.5
0.5
0.53
0.5
0.60
0.60
0.60
0.60
0.35
0.30
0.13
0.15
0.15
0.35
0.15
0.38
0.38
0.35
0.35
0.95
0.95
1.0
1.0
1.0
0.95
1.0
0.95
0.95
0.95
0.95
14.20
13.50
13.10
14.30
14.60
13.60
13.50
13.30
13.30
13.10
13.20
A,B
A
A
A
A
B
B
B
B
B
no.
peptide structure
LA
1
2
3
4
5
6
8
9
BK
[D-Phe7]BK
[D-NMF7]BK
0.11 4.80 1.0
0.49 65
[Hyp3,Thi5,D-NMF7]BK
D-Arg-[Hyp3,Thi5,D-NMF7]BK
29
38
33
0.30 21
D-Arg-[Hyp3,D-NMF7,L-NMF8]BK 0.33 18
[L-NMF7]BK
500
33
700
1.0
D-Arg-[L-NMF7]BK
B
10 [L-NMF2]BK
11 [D-NMF2]BK
12 [L-NMF3]BK
13 [D-NMF3]BK
3.5
232 1.0
100 57
25
3780
8000
a
Silica gel F254, 250 µm (Merck 5715) glass plates were used.
Solvent systems: (A) pyridine/ethyl acetate/acetic acid/H2O: 5/5/
1/3 (v/v/v/v); (B) 1-butanol/acetic acid/water: 48/18/24 (v/v/v).
0.2
0.3
375 2.6
57
1400
b
Paper electrophoresis in 6% aqueous acetic acid. Rf values are
a
Conditions for assays and analysis of results are given in the
Experimental Section. IC50 values were calculated using the
program of Tobler and Engel.29 Data are means of duplicate
determinations, which did not differ by more than 5%. Most
analogs interact with both high- (HA) and low-affinity (LA)
bindings sites. Some analogs cannot discriminate between the HA
and LA sites; values for these are given in the middle column for
each tissue: RUT, rat uterus; GPI, guinea pig ileum.
relative to Arg. c Capacity factor for Bioselect 300-5 C18 reversed
phase columm (250 × 4 mm), elution A, 0.1% aqueous TFA; B,
0.1% TFA in acetonitrile; gradient 0-50% B in 50 min, flow rate
d
1 mL/min, monitored at 220 nm. A: synthesis on Merrifield resin
according to Scheme 1 and example A. B: synthesis on PAM resin
according to Scheme 2 and example B.
Cleavage and deprotection of the peptide-resin was done with
HF:anisole (10:1) at 0 °C, 40 min. HF was removed by vacuum
at 0 °C, the product was washed with ethyl ether, and the
peptide was extracted with 16 mL of glacial acetic acid and
lyophilized. The crude product was dissolved in 8 mL of a
mixture of 1-butanol and 1% aqueous TFA (1:1) and purified
by 100-step CCD in the same solvent system. The distribution
was monitored by the quantitative Sakaguchi test. Fractions
were pooled, evaporated, and evaluated by TLC and analytical
HPLC. Analytical data are presented in Tables 4 and 5.
Yield: 75 mg of white lyophilized powder; mp 145-160 °C;
for the receptor, both of which contained the same
coding sequence.42 These messages did contain alter-
nate translation start codons, which could lead to
alternate protein sequences. Definitive statements
about receptor heterogeneity must await actual receptor
protein sequence information.
Exp er im en ta l Section
P ep tid e Syn th esis. All of the analogs were synthesized
by solid phase methods, using Boc strategy. Boc-Arg(Tos)-
Merrified resin (0.234 mequiv/g, Bachem, CA) and Boc-Arg-
(Mts)-PAM resin (0.5 mequiv/g, Bachem, Switzerland), were
purchased. Boc-amino acids were obtained from Orpegen,
Bachem, CA and Bachem, Switzerland. Boc-D-NMF was
synthesized by the procedure of Olsen.43
[R]D ) -34.8 ( 2 (c ) 1, H2O).
20
Exa m p le B: Syn th esis of [D-NMF 7]Br a d yk in in on P AM
Resin (Sch em e 2). Boc-Arg(Mts)-PAM-resin (0.57 g, 0.285
mmol) was used with DIC/HOBt as coupling agent, except that
the couplings of Boc-Phe at position 8 and Boc-Arg(Mts) at
position 1 were done with TBTU/HOBt and Boc-NMF was
coupled with PyBrop. The internal monitoring of the PSS-80
synthesizer showed that under these conditions couplings of
more than 99.5% were attained. Only [Boc-Pro2] required
recoupling to reach this value. Cleavage from the resin and
deprotection was carried out with a cocktail containing 1.5 mL
of TFA, 0.15 mL of TFMSA, 0.11 mL of thioanisole, and 0.055
mL of EDT for 200 mg of dry resin. The reaction mixture was
stirred for 10 min at 0 °C and for 60 min at room temperature,
filtered, and poured into ethyl ether. The precipitate was
isolated by centrifugation. The crude peptide was dissolved
in 20 mL of tert-butyl alcohol and lyophilized (yield 350 mg).
The product was dissolved in 2% aqueous acetic acid (1 mg/5
mL), passed through an ion exchange resin (Amberlite IRA
410; 12 mL of resin/mequiv of peptide), and relyophilized. The
crude product was purified by HPLC as described above. The
fractions were collected, evaporated, lyophilized, and evaluated
by TLC and analytical HPLC (for analytical data see Tables 4
and 5).
The first strategy (Scheme 1) with Boc-Arg(Tos) resin was
carried out on the Beckman 990 synthesizer, and the synthetic
route with Boc-Arg(Mtr) resin (Scheme 2) on the PSS-80
synthesizer. With the PSS-80, coupling reactions were moni-
tored by the internal automatic monitoring system with
dimethoxytrityl chloride. With the Beckman Synthesizer and
as an additional control on the PSS-80 we used the Kaiser test.
Peptides were purified by CCD, by gel filtration on Biogel
P2 in 2% acetic acid and by RP-HPLC (Shimadzu LC-8A, SPD-
6A) on a Nucleosil 100, C18, 5 µm reversed phase columm (25
cm × 1.6 cm) and 30% CH3CN in 0.1% aqueous trifluoracetic
acid at a flow rate of 6 mL/min with UV detection at 233 nm.
Purity of the free peptides was evaluated by TLC in two
solvent systems on silica gel, by paper electrophoresis at pH
2.3, and by analytical HPLC (see details in Table 4). Amino
acid analyses were performed on a Beckman 6300 amino acid
analyzer and gave acceptable results for all peptides (Table
5). The (M + H)+ molecular ions and fragmentation patterns
were obtained by FAB-MS and were in good agreement with
the calculated molecular weights for each peptide (Table 5).
Exa m p le A: Syn th esis of [D-NMF 7]Br a d yk in in on Mer -
r ifield Resin (Sch em e I). Boc-Arg(Tos)-Resin (0.85 g, 0.2
mmol) was used, and the following protected amino acids were
coupled to the growing peptide chain in stepwise fashion: Boc-
Phe, Boc-D-NMF, Boc-Ser(Bzl), Boc-Phe, Boc-Pro, Boc-Pro, and
Boc-Arg(Tos). All amino acids except Boc-D-NMF and Boc-
Ser(Bzl) were coupled with DCC. Boc-D-NMF was coupled
with BOP/HOBt and Boc-Ser(Bzl) with PyBrop. Reaction
times for complete couplings were 2 h with DCC and PyBrop
and 12 h with BOP/HOBt. The Boc group was removed with
25% TFA in DCM with indole as scavenger in two steps: 1.5
min prewash and 30 min deprotection. After coupling of the
last amino acid, the Boc protecting group was removed, and
the resin was washed with DCM and ethanol and dried.
Ra d ioliga n d Bin d in g. Binding assays on rat myometrial
and guinea pig ileum membranes were carried out as described
previously44 with minor modifications. Briefly, membranes
(0.3-0.5 mg of protein per assay, always freshly prepared)
were incubated in a total volume of 1 mL containing 25 mM
TES buffer, pH 6.8, 1 µM captopril, 1 mM DTE, bacitracin (140
mg/L), bovine serum albumin (1 g/L), [3H]BK (0.05 nM,
obtained from New England Nuclear, specific activity 102 Ci/
mmol), and the various peptides in increasing concentrations.
After incubation (4 °C, 30 min), the samples were filtered
through Whatman GF/C glass fiber filters pretreated with
0.1% (w/v) aqueous polyethylenimine solution, using a Brandel
harvester. The filters were washed three times with 5 mL of
TES buffer (10 mM, pH 6.8), transferred into scintillation vials,
dried, and counted for radioactivity in 6 mL of a toluene-based
scintillator. Nonspecific binding was determined in the pres-
ence of 1 µM BK. The IC50 values were calculated using the

Bradykinin Agonists and Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 935
FAB-MS (M + H)
Ta ble 5. Amino Acid Compositions and Molecular Weights of the Peptides
no.
Arg
Pro
Hyp
Gly
Phe
Thi
Ser
NMF
calcd
found
3
4
5
6
7
8
9
10
11
12
13
1.9 (2)
2.0 (2)
3.0 (2)
3.0 (2)
3.0 (2)
2.0 (2)
2.9 (3)
1.7 (2)
1.7 (2)
1.9 (2)
1.9 (2)
2.0 (2)
1.0 (1)
1.0 (1)
0.85 (1)
0.75 (1)
1.9 (2)
2.0 (2)
1.7 (2)
1.7 (2)
1.8 (2)
1.9 (2)
1.0 (1)
1.0 (1)
1.0 (1)
1.2 (1)
0.95 (1)
1.0 (1)
0.95 (1)
1.1 (1)
1.0 (1)
1.0 (1)
0.95 (1)
2.1 (2)
0.97 (1)
0.97 (1)
1.0 (1)
0.85 (1)
0.83 (1)
0.87 (1)
1.2 (1)
0.8 (1)
0.9 (1)
0.85 (1)
0.85 (1)
0.85 (1)
0.9 (1)
1.3 (1)
1.15 (1)
1.15 (1)
2.3 (2)
3.2 (3)
1.1 (1)
1.15 (1)
1.0 (1)
0.9 (1)
0.9 (1)
1.1 (1)
1124.2
1146.0
1302.2
1310.2
1324.1
1124.2
1280.3
1124.2
1124.2
1124.2
1124.2
1124.5
1146.5
1302.6
1310.7
1324.7
1124.4
1280.7
1124.5
1124.8
1124.5
1124.9
0.95 (1)
0.95 (1)
0.75 (1)
0.75 (1)
1.0 (1)
1.0 (1)
2.1 (2)
2.0 (2)
2.0 (2)
2.0 (2)
2.0 (2)
2.0 (2)
0.87 (1)
program of Tobler and Engel.29 The Tobler program was
supplemented with a special data input program and combined
with subsequent nonlinear regression analysis as recently
described by Schnittler et al.45 The calculation program of
Tobler and Engel was not altered.
Opton cytopherometer. The slowing effect (percent) was
calculated using targets or targets plus peptides (to exclude
side effects) as controls. In order to test antagonist activities
of selected BK analogs, an inhibition variant of the TEEM test
was used: spleen cells of mice were preincubated (0.5 h, 37
°C) with the potential antagonists followed by incubation with
the optimal agonist dose of BK (10-7 M) for 4 h at 37 °C.
Interleukin activity in the cell-free supernatant was deter-
mined as described above.
Estr u s Ra t Uter u s Assa y. Female Wistar rats weighing
180-200 g were injected with diethylstilbestrol (100 µg/kg sc)
16 h before the uterus was used. The distal parts of the
uterine horns were suspended at 28 °C in de J alon solution.
Isotonic contractions were recorded under a resting tension
of 0.5 g. BK (half-maximal dose) was added to the organ bath
at 4 min intervals and left in the organ bath for 45 s. To
investigate inhibition, analogs were added to the organ bath
1 min before the BK dose. Peptides without antagonist action
were tested for agonist activity by means of the 4-point method
of Schild.46 In some cases cumulative dose-response curves
for bradykinin were made to estimate the antagonist activities,
which are expressed as pA2 values (negative log of the
concentration of the antagonist that reduces the effect of a
double dose of BK to that of a single dose).47
Gu in ea P ig Ileu m Assa y. Food was withheld from
animals for 16 h before use. Terminal ileum (1.5 cm) was
suspended in Tyrode solution at 37 °C. Isotonic contractions
were recorded under a resting tension of 0.5 g. BK was applied
in a cumulative way, reaching a maximum contraction at a
BK concentration of 0.3 µM. Antagonist analogs were added
to the bath 5 min before the cumulative dose-response curve
for BK was made. The pA2 value of antagonists was estimated
as described above. BK analogs without inhibitory action were
tested for agonist action by the 4-point method of Schild46 using
BK doses of 1 and 2 nM.
In h ibition of Isola ted An gioten sin Con ver tin g En -
zym e. Inhibitory activities of some peptides were determined
with an enzyme preparation from pig lung, using benzoyl-Gly-
His-Leu as substrate. The method was described elsewhere.48
Ack n ow led gm en t . We thank B. Schilling, C.
Mertens, R. Rothe, and R. Reed for skillful technical
assistance. We gratefully acknowledge the assistance
of G. Eberhardt in the preparation of the manuscript.
Some of the pharmacological activities were determined
by F. Shepperdson. Dr. W.-E. Siems tested the influ-
ence of the bradykinin analogs on angiotensin convert-
ing enzyme. The work was performed with financial
support from the Bundesministerium fu¨r Forschung und
Technologie (BEO 21/19992A), from Hoechst AG, and
from NIH Grant HL-26284.
Refer en ces
(1) Symbols and abbrevations are in accord with the recommenda-
tions of the IUPAC-IUB Commission on Nomenclature (Eur.
J . Biochem. 1984, 138, 9-37). Other abbreviations: ACE,
angiotensin I converting enzyme; Aib, R-aminoisobutyric acid;
BK, bradykinin; Boc, tert-butyloxycarbonyl; BOP, (benzotriazol-
1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate;
Bzl, benzyl; CCD, countercurrent distribution; DCC, dicyclo-
hexylcarbodiimide; DCM, dichloromethane; DIEA, diisopropyl-
ethylamine; DIC, diisopropylcarbodiimide; DMF, N,N-dimeth-
ylformamide; DTE, dithioerithritol; EDT, ethanedithiol; GPI,
guinea pig ileum; HOBt, 1-hydroxybenzotriazole; HOE-140,
Darg-[Hyp3,Thi5,DTic7,Oic8]BK; ia, intraarterial; iv, intravenous;
LS, lung strip; Mts, mesitylene-2-sulfonyl; NMF, N-methyl-Phe;
Oic, octahydroindole-2-carboxylic acid; DPhe(Cl), p-chloro-D-Phe;
∆Phe, 2,3-dehydro-Phe; PyBrop, bromotrispyrrolidinophospho-
nium hexafluorophosphate; RUT, rat uterus; TBTU, 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate;
TEEM, tanned erythrocyte electrophoretic mobility; TES, N-
[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; TFA,
trifluoroacetic acid; TFMSA, trifluoromethanesulfonic acid; Tic,
tetrahydroisoquinoline-3-carboxylic acid; Thi, â-2-thienylalanine;
Tos, p-toluenesulfonyl.
Gu in ea P ig Lu n g Str ip Assa y. Male or female guinea
pigs weighing 400-600 g were used. The lungs were dissected
out and placed in a modified Krebs solution. Sections of lung
tissue about 2-3 mm wide were cut quickly from the lower
lobes parallel to the margin. The isolated strips, loaded with
1 g tension, were placed in an organ bath containing Krebs-
Henseleit solution at 37 °C. Subsequent changes in the
tension were measured isometrically. The tissues were al-
lowed to equilibrate for 1 h with solution changes at 15 min
intervals. Cumulative dose-response curves for BK were
made in the concentration range 0.01-10 µM BK. To examine
antagonist activity the peptides were incubated with the lung
strip 5 min before addition of an ED50 dose of BK. The pA2
values were calculated by the method of van Rossum et al.47
In ter leu k in Relea se Assa y. Ta n n ed Er yth r ocyte Elec-
tr op h or etic Mobility (TEEM) Test. Mouse spleen cells
were incubated with the different BK analogs in a serum-free
short-time (5 h, 37 °C) culture to evoke interleukin release.28
The supernatants were prepared by centrifugation and par-
tially purified by gel filtration (AcA54, column 0.9 × 60 cm;
25 mL/min; fractions of 1 mL).
Interleukin standards (IL-1R, IL-1â, IL-3, IL6, TNFR) were
all from Genzyme, Cambridge, MA. Culture supernatants or
fractions with or without monoclonal antibodies (hamster anti-
mouse IL-1R, rat anti-mouse IL-2, rat anti-mouse IL-3, rat
anti-mouse IL-6, hamster anti-mouse TNFR/â, all from Gen-
zyme; anti-IL-2 receptor, mouse, Boehringer Mannheim, Ger-
many) were added to the targets (sulfosalicylic acid-stabilized
tanned sheep red blood cells) and incubated for 1 h at 23 °C.
The migration time of the target cells was estimated in an
(2) Regoli, D.; Barabe, J . Pharmacology of bradykinin and related
kinins. Pharmacol. Rev. 1980, 32, 1-46.
(3) Bhoola, K. D.; Figueroa, C. D.; Worthy, K. Bioregulation of
kinins: kallikrein, kininogens, and related kininases. Pharma-
col. Rev. 1992, 44, 1-78.
(4) Dray, A.; Perkins, M. Bradykinin and inflammatory pain.
Trends Neurosci. 1993, 16, 99-104.
(5) Chau, T. T.; Lewin, A. C.; Walter, T. L.; Carlson, R. P.;
Weichmann, B. M. Evidence for a role of bradykinin in experi-
mental pain models. Agents Actions 1991, 34, 235-238.
(6) Burch, R. M.; Kyle, D. J . Minireview: Recent developments in
the understanding of bradykinin receptors. Life Sci. 1992, 50,
829-838.

936 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Reissmann et al.
(7) (a) Steranka, L. R.; Manning, D. C.; de Haas, C. J .; Ferkany, J .
W.; Borosky, S. A.; Connor, J . R.; Vavrek, R. J .; Stewart, J . M.;
Snyder, S. H. Bradykinin as pain mediator: receptors are
localized to sensory neurons and antagonists have analgesic
effects. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 3245-3249. (b)
Stewart, J . M. The kinin system in inflammation. Agents
Actions 1993, 42S, 145-147.
(27) Regoli, D.; J ukic, D.; Toussignant, C.; Rhaleb, N.-E. Kinin
receptor classification. In Recent Progress on Kinins; Fritz, H.,
Mu¨ller-Esterl, W., J ochum, M., Roscher, A., Luppertz, K., Eds.;
Birkha¨user Verlag: Basel, 1992; pp 475-486. (Agents Actions
38/SI).
(28) (a) Paegelow, Il; Werner, H.; Reissmann, S. Effects of bradykinin
and bradykinin analogues on spleen cells of mice. Eur. J .
Pharmacol. 1995, 279, 211-216. (b) Werner, H. Investigations
of interleukins (IL-1, IL-2) using particle electrophoretic mobil-
ity. In Electrophoresis 86; Dunn, M. J ., Ed.; VCH Ver-
lagsgesellschaft: Weinheim, 1986; pp 61-64.
(29) Tobler, H. J .; Engel, G. Affinity spectra: a novel way for the
evaluation of equilibrium binding experiments. Naunyn-
Schmiedebergapos Arch. Pharmacol. 1983, 322, 183-192.
(30) Stewart, J . M.; Klis, W. A. Polystyrene-based solid phase peptide
synthesis: the state of the art. In Solid Phase Synthesis; Epton,
R., Ed.; SPCC (UK) Ltd.: Birmingham, 1990; pp 1-9.
(31) Mazur, R. H.; J ames, P. A.; Tyner, D. A.; Hallinan, E. A.; Sanner,
J . H.; Schulze, R. Bradykinin analogues containing N-methyl
amino acids. J . Med. Chem. 1980, 23, 758-763.
(32) Togo, J .; Burch, R. M.; DeHaas, C. J .; Connor, J . R.; Steranka,
L. R. D-Phe7-substituted peptide bradykinin antagonists are not
substrates for kininase II. Peptides 1989, 10, 109-112.
(33) (a) Farmer, S. G.; Burch, R. M.; Meeker, S. A.; Wilkins, D. E.
Evidence for a pulmonary B3 bradykinin receptor. Mol. Phar-
macol. 1989, 36, 1-8. (b) Mak, J . C. W.; Barnes, P. J .
Autoradiographic visualization of bradykinin receptors in human
and guinea pig lung. Eur. J . Pharmacol. 1991, 194, 37-43.
(34) Schro¨der, E. Structure-activity relationships in kinins. In
Handbook of Experimental Pharmacology; Erdo¨s, E. G., Ed.;
Springer-Verlag: Heidelberg, 1970; Vol. XXV, pp 324-350.
(35) Stewart, J . M. Chemistry and biological activity of peptides
related to bradykinin. In Handbook of Experimental Pharma-
cology; Erdo¨s, E. G., Ed.; Springer-Verlag: Heidelberg, 1979; Vol.
XXV (Suppl.), pp 227-272.
(36) Farmer, S. G.; Burch, R. M.; DeHaas, C. J .; Togo, J .; Steranka,
L. R. [Arg1-D-Phe7]-substituted analogs of bradykinin inhibit
vasopressin- and bradykinin-induced contraction of uterine
smooth muscle. J . Pharmacol. Exp. Ther. 1989, 248, 677-681.
(37) (a) Manning, D. C.; Vavrek, R.; Stewart, J . M.; Snyder, S. H.
Two bradykinin binding sites with picomolar affinities. J .
Pharmacol. Exp. Ther. 1986, 237, 504-512. (b) Liebmann, C.;
Offermanns, S.; Spicher, K.; Hinsch, K.-D.; Schnittler, M.;
Morgat, J .-L.; Reissmann, S.; Schultz, G.; Rosenthal, W. A high-
affinity bradykinin receptor in membranes from rat myometrium
is coupled to pertussis toxin-sensitive G-proteins of the Gi-family,
Biochem. Biophys. Res. Commun. 1990, 167, 910-917.
(38) Liebmann, C.; Schnittler, M.; Stewart, J . M.; Reissmann, S.
Antagonist binding reveals two heterogeneous B2 bradykinin
receptors in rat myometrial membranes. Eur. J . Pharmacol.
1991, 199, 363-365.
(39) Ransom, R. W.; Goodman, C. B.; Young, G. S.; Bradykinin
stimulation of phosphoinositide hydrolysis in guinea-pig ileum
longitudinal muscle. Br. J . Pharmacol. 1992, 105, 919-924.
(40) Liebmann, C.; Mammery, K.; Graness, A. Bradykinin inhibits
adenylate cyclase activity in guinea pig ileum membranes via a
separate high-affinity bradykinin B2 receptor. Eur. J . Pharma-
col. 1994, 288, 35-43.
(41) Liebmann, C.; Bosse, R.; Escher, E. Discrimination between
putative bradykinin B2 receptor subtypes in guinea pig ileum
smooth muscle membranes with a selective, iodinatable, brady-
kinin analogue. Mol. Pharmacol. 1994, 46, 949-956.
(42) Pesquero, J . B.; Lindsey, C. J .; Zeh, K.; Paiva, A. C. M.; Ganten,
D.; Bader, M. Molecular structure and expression of rat brady-
kinin B2 receptor gene. Evidence for alternative splicing. J .
Biol. Chem. 1994, 269, 26920-26925.
(43) Olsen, R. K. A convenient synthesis of protected N-methylamino
acid derivatives. J . Org. Chem. 1970, 35, 1912-1915.
(44) Liebmann, C.; Nawrath, St.; Ludwig, B.; Paegelow, I. Pharma-
cological and molecular actions of the bradykinin B2 receptor
antagonist, Hoe 140, in the rat uterus. Eur. J . Pharmacol. 1993,
235, 183-188.
(8) Vavrek, R. J .; Stewart, J . M. Competitive antagonists of brady-
kinin. Peptides 1985, 6, 161-164.
(9) Stewart, J . M.; Vavrek, R. J . Chemistry of peptide B2 bradykinin
antagonists. In Bradykinin Antagonists. Basic and Clinical
Research; Burch, R. M., Ed.; Marcel Dekker, Inc.: New York,
1991; pp 51-96.
(10) Regoli, D.; Rhaleb, N. E.; Dion, S.; Drapeau, G. New selective
bradykinin antagonists and bradykinin B2 receptor characteriza-
tion. Trends Pharmacol. Sci. 1990, 11, 156-161.
(11) Cheronis, J . C.; Whalley, E. T.; Nguyen, K. T.; Eubanks, S. R.;
Allen, L. G.; Duggan, M. J .; Loy, S. D.; Bonham, K. A.; Blodgett,
J . K. A new class of bradykinin antagonists: synthesis and in
vitro activity of bissuccinimidoalkane peptide dimers. J . Med.
Chem. 1992, 35, 1563-1572.
(12) Lembeck, F.; Griesbacher, T.; Eckhardt, M.; Henke, St.; Breipohl,
G.; Knolle, J . New, long-acting, potent bradykinin antagonists.
Br. J . Pharmacol. 1991, 102, 297-304.
(13) Kyle, D. J .; Martin, J . A.; Burch, R. M.; Carter, J . P.; Lu, S.;
Meeker, S.; Prosser, J . C.; Sullivan, J . P.; Noronha-Blob, J . T.
L.; Sinsko, J . A.; Walters, R. F.; Whaley, L. W.; Hiner, R. N.
Probing the bradykinin receptor: mapping the geometric topog-
raphy using ethers of hydroxyproline in novel peptides. J . Med.
Chem. 1991, 34, 2649-53.
(14) (a) Kyle, D. J .; Hicks, R. P.; Blake, P. R.; Klimkowski, V. J .
Conformational properties of bradykinin and bradykinin an-
tagonists. In Bradykinin Antagonists: Basic and Clinical Re-
search; Burch, R. M., Ed.; Marcel Dekker: New York, 1990; pp
131-146. (b) Kyle, D. J .; Martin, J . A.; Farmer, St. G.; Burch,
R. M. Design and conformational analysis of several highly
potent bradykinin receptor antagonists. J . Med. Chem. 1991,
34, 1230-1233. (c) Kyle, D. J .; Blake, P. R.; Smithwick, D.;
Green, L. M.; Martin, J . A.; Sinsko, J . A.; Summers, M. F. NMR
and computational evidence that high-affinity bradykinin recep-
tor antagonists adopt C-terminal â-turns. J . Med. Chem. 1993,
36, 1450-1460. (d) Chaturvedi, S.; Wilkens, D.; Kyle, D. J .
Design of potent, cyclic peptide bradykinin receptor antagonists
from conformationally constrained linear peptides. J . Med.
Chem. 1993, 36, 2569-2571.
(15) (a) Liu, X.; Stewart, J . M.; Gera, L.; Kotovych, G. Proton
magnetic resonance studies of bradykinin antagonists. Biopoly-
mers 1993, 33, 1237-1247. (b) Otter, A.; Bigler, P.; Stewart, J .
M.; Kotovych, G. A proton magnetic resonance study of two
synthetic agonist-antagonist pairs of bradykinin analogues.
Biopolymers 1993, 33, 769-780.
(16) Momany, F. A. Conformational analysis and polypeptide drug
design. Top. Curr. Phys. 1981, 26, 41-79.
(17) Penkler, L. J .; van Rooyen, P. H.; Wessels, P. L. Conformational
analysis of µ-selective [D-Ala2,MePhe4]-enkephalins. Int. J . Pept.
Protein Res. 1993, 41, 261-274.
(18) (a) Tonelli, A. E.; The effects of isolated N-methylated residues
on the conformational characteristics of polypeptides. Biopoly-
mers. 1976, 15, 1615-1622. (b) Manavalan, P.; Momany, F. A.
Conformational energy studies on N-methylated analogs of
thyrotropin releasing hormone, enkephalin, and luteinizing
hormone-releasing hormone. Biopolymers 1980, 19, 1943-1973.
(19) Vitoux, B.; Aubry, A.; Cung, M. T.; Marraud, M. N-methyl
peptides. Int. J . Pept. Protein Res. 1986, 27, 617-632.
(20) Ron, D.; Gilon, C.; Hanani, M.; Vromen, A.; Selinger, Z.; Chorev,
M. N-methylated analogs of Ac-[Nle28,31]-CCK(26-33): Synthe-
sis, activity, and receptor selectivity. J . Med. Chem. 1992, 35,
2806-2811.
(21) Haviv, F.; Fitzpatrick, T. D.; Swenson, R. E.; Nichols, C. J .; Mort,
N. A.; Bush, E. N.; Diaz, G.; Bammert, G.; Nguyen, A.; Rhutasel,
N. S.; Nellans, H. N.; Hoffmann, D. J .; J ohnson, E. S.; Greer, J .
Effect of N-methyl substitution of the peptide bonds in lutein-
izing hormone-releasing hormone agonists. J . Med. Chem. 1993,
36, 363-389.
(45) Schnittler, M.; Liebmann, C.; Opfermann, J . Experimentelles
Design and Auswertung von Radioliganden-Bindungsstudien.
(Experimental design and evaluation of radioligand-binding
studies.) Pharmazie 1991, 46, 2-8.
(22) Filatova, M. P.; Krit, N. A.; Beschastnaya, N. V.; Reissmann, S.
Synthesis of new bradykinin analogs with modified peptide
chain. Bioorg. Khim. 1979, 5, 1445-1454.
(23) Boissonnas, R. A.; Guttmann, S.; J aquenoud, P.-A. Synthese de
la L-arginyl-L-prolyl-L-prolyl-glycyl-L-phenylanyl-L-seryl-L-pro-
lyl-L-phenylalanyl-L-arginine, un nonapeptide presentant les
propreietes de la bradykinine. (Synthesis of L-arginyl-L-prolyl-
L-prolyl-glycyl-L-phenylalanyl-L-seryl-L-prolyl-L-phenylalanyl-
L-arginine, a nonapeptide presenting the properties of brady-
kinin.) Helv. Chim. Acta 1960, 43, 1349-1358.
(24) Merrifield, R. B. Solid phase peptide synthesis: II. The synthesis
of bradykinin. J . Am. Chem. Soc. 1964, 86, 304-305.
(25) Farmer, S. G.; Burch, R. M. Biochemical and molecular phar-
macology of kinin receptors. Annu. Rev. Pharmacol. Toxicol.
1992, 32, 511-36.
(26) Hall, J . M. Bradykinin receptors: pharmacological properties
and biological roles. Pharm. Ther. 1992, 56, 131-190.
(46) Schild, H. O. A method of conducting a biological assay on a
preparation giving repeated graded responses illustrated by the
estimation of histamine. J . Physiol. 1942, 101, 115.
(47) Van Rossum, J . M.; van Hurkmans, J . A. Th. M.; Wolters, C. J .
J . Cumulative dose-response curves. II. Technique for the
making of dose-response curves in isolated organs and the
evaluation of drug parameters. Arch. Int. Pharmacodyn. 1963,
143, 299-330.
(48) Siems, W.-E.; Heder, G.; Komissarova, N. W. Verfahren zur
Bestimmung des Angiotensin-Converting Enzyms. (Procedure
for measurement of angiotensin-converting enzyme.) Z. Med.
Lab. Diagn. 1985, 26, 230.
J M9301954