Synthesis and characterization of bradykinin B2 receptor agonists containing constrained dipeptide mimics

Article abstract of DOI:10.1021/jm9901531

We have previously shown that substitution of the D-Tic-Oic dipeptide by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (D-BT) moiety in the bradykinin B₂ receptor antagonist HOE 140 resulted in a full potent and sel

Full text of DOI:10.1021/jm9901531

J . Med. Chem. 1999, 42, 4193-4201
4193
Syn th esis a n d Ch a r a cter iza tion of Br a d yk in in B2 Recep tor Agon ists Con ta in in g
Con str a in ed Dip ep tid e Mim ics
Muriel Amblard,† Isabelle Daffix,† Gilbert Berge´,† Monique Calme`s,† Pierre Dodey,‡ Didier Pruneau,‡
J ean-Luc Paquet,‡ J ean-Michel Luccarini,‡ Pierre Be´lichard,‡ and J ean Martinez*,†
Laboratoire des Aminoacides Peptides et Prote´ines, UMR CNRS 5810, Universite´s Montpellier I et II, Faculte´ de Pharmacie,
15 Av. C. Flahault, 34060 Montpellier Ce´dex, France, and Laboratoires de Recherche Fournier, Route de Dijon,
21121 Daix, France
Received March 30, 1999
We have previously shown that substitution of the D-Tic-Oic dipeptide by a (3S)-[amino]-5-
(carbonylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (D-BT) moiety in the bradykinin B2
receptor antagonist HOE 140 resulted in a full potent and selective bradykinin B2 receptor
agonist (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-D-BT-Arg-OH, J MV1116) exhibiting a high affinity
for the human receptor (Ki 0.7 nM). In the present study, we have investigated the effects of
replacement of the D-Tic-Oic moiety by various constrained dipeptide mimetics. The resulting
compounds were tested for their binding affinity toward the cloned human B2 receptor and for
their functional interaction with the bradykinin-induced contraction of isolated human umbilical
vein. Subsequently, we have designed novel bradykinin B2 receptor agonists which are likely
to be resistant to enzymatic cleavage by endopeptidases and which might represent interesting
new pharmacological tools. In an attempt to increase the potency of compound J MV1116, both
its N-terminal part and the D-BT moiety were modified. Substitution of the D-arginine residue
by a L-lysine residue led to a 10-fold more potent bradykinin B2 ligand [compound 22 (J MV1465)
(Ki 0.07 nM)], retaining full agonist activity on human umbilical vein. Substitution of the D-BT
moiety by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-8-methyl-1,5-benzothiazepin-4(5H)-
one [D-BT(Me)] moiety led to compound 23 (J MV1609) which exhibited a higher agonist activity
(pD2 ) 7.4) than J MV1116 (pD2 ) 6.8).
In tr od u ction
potent bradykinin agonists, resulted in binding with a
10 times higher affinity to the human than to rat
bradykinin B2 receptors.3 HOE 140 analogues contain-
ing various constrained and nonpeptide moieties replac-
ing the dipeptide -D-Tic-Oic- were synthesized to further
investigate the effect of this modification.
Since ACE is a major kinin-degrading enzyme5 which
cleaves bradykinin at the Pro7-Phe8 (and Phe5-Ser6)
amide bond, ACE inhibitors might display features
complementary to the bradykinin receptor. Therefore,
we used the core of ACE inhibitors as templates to
design HOE 140 analogues. In fact, the D-Tic-Oic
dipeptide was replaced by the core of various ACE
inhibitors including benzothiazepine and benzodiaz-
epine derivatives as well as five-, six-, and seven-
membered ring lactams.6,7 This strategy was success-
fully approached by Hoyer et al.8 for the design of
nonpeptide bradykinin B2 receptor antagonists.
In the design of peptidomimetics, incorporation of
nonpeptidic scaffolds into bioactive molecules has been
the focus of extensive research over the last years.1
Conformationally restrained surrogates have been used
in the design and synthesis of enzyme inhibitors and
antagonists of peptide hormone receptors.2 Backbone
conformational constraints are of interest in limiting the
number of conformations available to a peptide. Poten-
tial advantages resulting from such constrained com-
pounds include (i) increased potency by stabilizing a
biologically active conformation; (ii) enhanced stability
toward degradation by enzymes; (iii) improved biological
selectivity through elimination of bioactive conformers
that give unwanted biological responses. In addition,
some information can be obtained about the molecular
interactions between the ligand and the receptor through
the introduction of conformational constraints.
Spectroscopic studies have shown that the high af-
finity of bradykinin analogues (including HOE 140) for
B2 receptors is related to their high propensity to adopt
a C-terminal â-turn conformation9-11 spanning the
residues Ser6-Pro7-Phe8-Arg9. To follow this hypothesis
the D-Tic-Oic moiety was substituted by several â-turn
mimetics as well as by other constrained dipeptide
mimetics such as a benzodiazepine moiety12 and a
pyrrolidine-2,4-dione moiety.13
We have recently shown3 that replacement of the
dipeptide Pro7-Phe8 by the (3S)-[amino]-5-(carbonyl-
methyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (D-
BT) moiety in the bradykinin sequence resulted in a full
bradykinin agonist. The same modification applied to
the bradykinin antagonist HOE 1404 (H-D-Arg0-Arg1-
Pro2-Hyp3-Gly4-Thi5-Ser6-D-Tic7-Oic8-Arg9-OH), in which
replacement of the -D-Tic7-Oic8- dipeptide resulted in
* Correspondence to: J ean Martinez, UMR CNRS5810.
Tel: 04 67 04 01 83. Fax: 04 67 41 20 17. E-mail: martinez@
pharma.univ-montp1.fr.
Ch em istr y
All suitably protected constrained dipeptide mimetics
1-10 used in this study are reported in Table 1. The
† UMR CNRS 5810.
‡ Laboratoire de Recherche Fournier.
10.1021/jm9901531 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/21/1999

4194 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Ta ble 1. Structures of Protected Constrained Dipeptide Mimetics
Amblard et al.

BK Agonists Containing Dipeptide Mimics
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4195
Sch em e 1. Preparation of Constrained Dipeptide
Mimetic 2a
four dipeptide mimetics 5, 5′, 6, and 8 were prepared
according to Freidinger et al.17
Compounds 7 and 7′ were synthesized following
Scheme 4. The seven-membered lactam intermediates
15 and 16 were prepared by cyclization of Boc-(D or L)-
Lys-OH in DMF at a 10 mM concentration, with BOP
as coupling reagent in the presence of sodium bicarbon-
ate as “insoluble base”. This methodology, derived from
the procedure described by Brady et al.,18 led to the
lactams in fairly good yields. Alkylation of the amide
with benzyl bromoacetate afforded compounds 17 and
18. Despite the presence of a urethane function, only
alkylation of the lactam nitrogen occurred. Deprotection
of the benzyl group by hydrogenolysis produced com-
pounds 7 and 7′.
a
(i) 33% HBr in AcOH; (ii) Boc2O, 1 N NaOH, dioxane.
Sch em e 2. Preparation of Constrained Dipeptide
Mimetic 3
The pyrrolidine-2,4-dione 19 was prepared according
to Pothion et al.13 from the N-(tert-butyloxycarbonyl)-
N-carboxyanhydride derivative of phenylalanine. Aci-
dolysis of compound 19 followed by N-tert-butyloxycar-
bonylation led to compound 20. Alkylation of the
pyrrolidine nitrogen with benzyl bromoacetate yielded
compound 21 which was deprotected by hydrogenolysis
to yield compound 9 (Scheme 5). Compound 10 was
purchased from Neosystem Laboratoire (Strasbourg,
France).
Sch em e 3. N-tert-Butyloxycarbonylation of 5(S and
R)-Amino-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indo-
4-one (S)-Carboxylic Acid Compounds
The above-mentioned dipeptide mimetics 1-10 were
used for the synthesis of the corresponding HOE 140
analogues 22-35 (Table 2). These compounds were
synthesized on a chloromethylated resin by the solid-
phase method with the first amino acid (Boc-Arg(Tos)-)
routinely bound to the resin.19 NR-tert-Butyloxycarbonyl
(Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc) protec-
tions were used as temporary protection of N-terminal
amino groups, and tosyl, nitro, and benzyl groups were
used for side chain protections. Couplings of protected
amino acids or dipeptide mimetics were carried out
using BOP20 reagent. Boc and Fmoc groups were
removed by TFA and piperidine, respectively. 22-35
were obtained according to Scheme 6. The two diaste-
reoisomers 34 and 35 were separated by HPLC, but the
determination of the absolute configuration was not
performed. Compound 33 was tested as a mixture of
diastereoisomers.
synthesis of Boc-D-BT-OH (mimetic 1) has been de-
scribed previously.3 Compound 11 was prepared accord-
ing to Slade et al.14 starting from D-acetylcysteine.
Acidolysis of 11 by HBr in acetic acid followed by N-tert-
butyloxycarbonylation afforded compound 2 (Scheme 1).
Saponification of compound 12 which was prepared
according to Nagel et al.15 yielded compound 3 (Scheme
2).
Compounds 4 and 4′ were obtained according to
Scheme 3. The intermediates 13 and 14 were prepared
following the synthetic route described by De Lombaert
et al.16 N-tert-Butyloxycarbonylation of 13 and 14 led
respectively to NR-Boc dipeptide mimetics 4 and 4′. The
HOE 140 analogues were obtained after cleavage of
the precursors from the resin using standard HF
a
Sch em e 4. Syntheses of Constrained Dipeptide Mimetics 7 and 7′
a
(i) BOP, NaHCO3, NMM; (ii) Br-CH2-COOBzl, NaH; (iii) H2/Pd/C.
Sch em e 5. Syntheses of Constrained Dipeptide Mimetic 9a
a
(i) TFA; (ii) Boc2O; (iii) Br-CH2-COOBzl, NaH; (iv) H2/Pd/C.

4196 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Amblard et al.
Ta ble 2. Binding Affinities of Bradykinin and HOE 140 Analogues to B2 Human Receptorsa
a
Results are means of at least three separate experiments in duplicate.

BK Agonists Containing Dipeptide Mimics
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4197
Sch em e 6. Syntheses of 22-35a
analogue without the benzyl group (28). None of these
HOE 140 analogues in the micromolar range were able
to recognize the human cloned B1 bradykinin receptor.
The most potent compounds, i.e. 22-24, were tested
for their ability to contract isolated human umbilical
vein in comparison with bradykinin, the bradykinin B2
receptor agonist RMP-7,21 and HOE 140. Results are
reported in Table 3.
As in the case of J MV1116, these three new deriva-
tives behaved as agonists. Effectively they contracted
human umbilical vein rings in a concentration-depend-
ent manner giving calculated pD2 values ranging from
5.1 to 7.4. Addition of a methyl group onto the benzothi-
azepinone moiety of J MV1116 led to 23, which con-
tracted HUV with a higher potency (pD2 7.4) than
J MV1116, and 22, although its affinity for the B2
receptor remained lower. Replacement of the benzothi-
azepinone in J MV1116 by a benzoxazepinone led to a
less potent derivative, i.e. 24.
Modification of the peptide moiety was approached
by replacing the N-terminal Arg in J MV1116 by a
L-lysine residue. The resulting compound (22) behaved
as a potent agonist (pD2 7.1). Although the pD2 value
for bradykinin in the presence of a cocktail of enzyme
inhibitors was about 5-8 times higher than that of 22
and 23 (7.9 vs 7.1 and 7.4, respectively), both compounds
developed a similar maximal contraction of the human
umbilical vein. The activity of compounds 22 and 23 was
not affected in the absence of protease inhibitors. HOE
140 did not stimulate contraction of the human umbili-
cal vein but was able to potently antagonize the brady-
kinin-, 22-, and 23-induced HUV contraction with the
same potency (pA2 of 8.2 nM) (Table 3).
In conclusion, we have synthesized bradykinin ana-
logues in which the dipeptide Pro-Phe (positions 7 and
8 in the bradykinin sequence) was replaced by â-turn
mimetics or lactams. Most of these compounds bound
to the human cloned B2 receptor. Introduction of ACE
inhibitor cores produced potent and selective agonists
of the B2 bradykinin receptor. These analogues are
apparently resistant to enzymatic (ACE) degradation.
Among the constrained structures that were studied,
the best mimetic of the Pro-Phe dipeptide was the D-BT
moiety. N-terminal modification (replacement of D-Arg
in position 1 by Lys) of D-BT-containing compounds
yielded even more potent agonists, i.e. 22. Introduction
of a methyl group in position 8 of the benzothiazepine
of J MV1116 produced 23 which was more potent than
the parent compound.
a
X ) constrained dipeptide mimetics 1-10. In compound 22,
the N-terminal residue is lysine.
procedures. Compounds 22-35 were purified by pre-
parative reverse-phase HPLC on a C18 column.
Resu lts a n d Discu ssion
Analogues were evaluated for their binding properties
to cloned human B2 and B1 receptors expressed in CHO
and 293 cells, respectively. Compounds 22-35 inhibited
the binding of [3H]bradykinin to the cloned B2 brady-
kinin receptor with subnanomolar to micromolar affinity
(Table 2). The most potent compound (22) had a 10-fold
higher affinity for the B2 receptor than bradykinin (Ki
0.07 nM). Its methyl analogue (23) having a N-terminal
D-Arg bound to the B2 receptor with a lower affinity (Ki
6 nM). Compound 24 (J MV1442), having in its sequence
a (S)-benzoxazepine moiety, exhibited a fairly high
affinity for the B2 receptor (Ki 2.5 nM) as well as
interestingly the diastereoisomeric mixture of HOE
analogues (compounds 33) containing the pyrrolidine-
2,4-dione moiety (Ki 14 nM). Among the analogues
containing the benzodiazepine moiety, one isomer (e.g.
35, Ki 86 nM) was more potent at the B2 receptor than
the other (34, Ki 1040 nM). In general, the (S)-contain-
ing isomer analogues were more potent in recognizing
the B2 receptor than their (R)-counterparts with the
exception of 25 and 26 (Ki 160 and 140 nM, respectively)
for which both diastereoisomers showed low potencies.
A dramatic decrease in affinity for the human B2
receptor was observed with the five- and six-membered
lactam-containing analogues (27, 28, 29, 32, Ki varying
from 1030 to 18600 nM), while the seven-membered ring
lactams 30 and 31 had a slightly improved affinity. The
structure-activity relationship in the bradykinin series
pointed out the crucial role of an aromatic moiety at
position 8. We have synthesized the compound 29 (an
analogue of compounds 27 and 28) bearing a benzyl
group (Table 1). This analogue exhibited weak affinity
for the B2 human receptor (Ki 2070 nM) although it was
about 2.5 times more potent than its corresponding
Exp er im en ta l Section
All analogues were prepared by solid-phase synthesis on a
chloromethylated resin according to Barany and Merrifield19
with a manual apparatus. Boc-amino acids were obtained from
Bachem (Switzerland), and the chloromethylated resin of
Merrifield was from Pierce. The first amino acid was bound
to the resin according to the Gisin22 method. Peptides were
cleaved from the resin by HF:anisole (10:1) at 0 °C, 60 min.
HF was removed under reduced pressure at 0 °C, the product
was washed with ether, and the peptides were extracted with
a mixture of CH3CN:H2O:TFA (50:50:1) and lyophilized. The
crude products were purified by preparative reverse-phase
HPLC on
a Prep 4000 Waters system equipped with a
DeltaPrep cartridge (40 × 100 mm) filled with a C18 DeltaPrep
silica gel (15 µm, 100 Å) phase. Separation was performed with
a flow rate of 50 mL/min and UV detection at 220 nm using

4198 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Amblard et al.
Ta ble 3. Biological Activity of HOE 140 Analogues and HOE 140 on Human Umbilical Vein
a
See Experimental Section. Results are the means of at least three separate experiments in duplicate.
gradient condition in a solvent system of A (0.1% TFA in H2O)
and B (0.1% TFA in CH3CN). HPLC controls were run on a
Merck/Hitachi instrument on a DeltaPak C18 (5 µm), 150 ×
3.9 mm, 100 Å column, with UV detection at 220 nm and a
flow rate of 1 mL/min. 1H NMR spectra were recorded on a
Bruker AC 250 spectrometer. The HOE 140 analogues were
identified by FAB mass spectrometry on a J EOL J MS-DX-300
apparatus. Capillary zone electrophoresis (CZE) was per-
formed on a PACE 5000 Beckman instrument, using an
uncoated fused silica capillary. Melting points were taken on
a Bu¨chi apparatus in open capillary tubes. TLC was performed
on precoated plates of silica gel 60F254 (Merck) using the
following solvent systems (by volume): A, AcOEt/hexane, 1:5;
B, AcOEt/hexane, 3:7; C, AcOEt/hexane, 5:5; D, AcOEt/hexane,
7:3; E, chloroform/methanol/acetic acid, 180:10:5; F, chloroform/
methanol/acetic acid, 120:10:5; G, chloroform/methanol/acetic
acid, 60:10:5. Derivatives were located with UV light (254 nm),
charring reagent, or ninhydrin. All reagents and solvents were
of analytical grade. Abbreviations: DMF, dimethylformamide;
BOP, (benzotriazolyloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; NMM, N-methylmorpholine; TFA, tri-
fluoroacetic acid. Other abbreviations used were those recom-
mended by the IUPAC-IUB Commission (Eur. J . Biochem.
1984, 138, 9-37).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
22-35. The protected peptides were assembled starting with
the Boc-Arg(Tos)-Merrifield-resin (0.5 g, 0.24 mmol). The
dipeptide mimetics 1-10 (1.2 equiv) were first coupled to
H-Arg(Tos)-Merrifield-resin. The reaction time for complete
couplings was 60 min. The protected amino acids (3 equiv)
were then introduced in the following order: Boc-Ser(Bzl)-OH,
Boc-Thi-OH, Boc-Gly-OH, Boc-Hyp-OH, Boc-Pro-OH, Boc-Arg-
(Tos)-OH, Boc-D-Arg(NO2)-OH, or Boc-Lys(Boc)-OH. The reac-
tion times for complete couplings were 15 min for Boc-Ser(Bzl)-
OH, Boc-Thi-OH, and Boc-Gly-OH and and Boc-Hyp-OH and
45 min for Boc-Pro-OH and Boc-Arg(Tos)-OH. All amino acids
and constraint structures were coupled with BOP. Completion
of the reaction was checked by the ninhydrin test of Kaiser.23
NR-Boc deprotection was achieved with 40% TFA in CH2Cl2
for 5 min and then for 25 min, and NR-Fmoc deprotection was
achieved with a mixture of piperidine:DMF (20:80) for 10 min.
After incorporation of the constrained dipeptide mimetics 1,
2, 3, 4, 4′, and 10, deprotection was performed with a mixture
of TFA:CH2Cl2:ethanedithiol (40:60:2) and washings with
isopropyl alcohol, methylene chloride, and DMF were applied.
After coupling of the last amino acid, the Boc protecting group
was removed, and the cleavage and deprotection of the
compound linked to the resin were performed with HF:anisole
(10:1) at 0 °C. Analytical data are reported in Table 4.
3(S)-[(ter t-Bu tyloxycar bon yl)am in o]-5-(car boxym eth yl)-
2,3-d ih yd r o-8-m eth yl-1,5-ben zoth ia zep in -4(5H)-on e, Boc-
D-BT(Me)-OH (2). Compound 11 (850 mg, 1.98 mmol) was
deprotected with a 33% solution of HBr in acetic acid (15 mL).
After standing at room temperature for 2 h, the solution of
HBr/AcOH was concentrated under reduced pressure, and the
deprotected compound was precipitated in ether and hexane.
It was collected and dried in vacuo over KOH pellets. To a
solution of the deprotected material (684 mg, 1.98 mmol) in a
mixture of 1 N sodium hydroxide (4 mL) and dioxane (10 mL)
was added di-tert-butyl dicarbonate (518 g, 2.38 mmol). The
pH was continuously adjusted to 10 by addition of 1 N sodium
hydroxide. When no starting material could be detected by
TLC, the reaction mixture was diluted with water (30 mL) and
extracted with ether (2 × 20 mL). The aqueous phase was
acidified to pH 3 with 1 M potassium bisulfate and extracted
with ethyl acetate (3 × 20 mL). The combined organic layers
were washed with water (2 × 20 mL) and brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo to yield
the expected compound which precipitated upon addition of a
mixture of ether/hexane. It was collected by filtration and dried

BK Agonists Containing Dipeptide Mimics
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4199
2.41 (1 H, m), 3.02 (1 H, m), 3.11 (1 H, m), 3.27 (1 H, dd, J 1
5.6 Hz, J 2 ) 16.0 Hz), 3.49 (1 H, dd, J 1 ) 11.1 Hz, J 2 ) 16.0
Hz), 4.32 (1 H, m), 5.12 (1 H, dd, J 1 ) 6.1 Hz, J 2 ) 11.1 Hz),
5.88 (1 H, d, J ) 5.0 Hz), 7.02 (3H, m, H).
Ta ble 4. Analytical Characteristics of Bradykinin and HOE
140 Analogues
)
5(R)-[(ter t-Bu tyloxycar bon yl)am in o]-1,2,4,5,6,7-h exah y-
d r oa zep in o[3,2,1-h i]in d ol-4-on e 2-(S)-ca r boxylic a cid (4′)
was obtained as described for compound 4: yield 150 mg (70%);
mp 144-146 °C; Rf (E) 0.48; HPLC24 tR ) 22.9 min; FAB-MS
m/z 347 [M + H+]; 1H NMR (CDCl3, 250 MHz) δ 1.40 (9 H, s),
2.05 (1 H, m), 2.30 (1 H, m), 3.00 (1 H, m), 3.26 (1 H, m), 3.24
(1 H, dd, J 1 ) 2.9 Hz, J 2 ) 16.7 Hz), 3.41 (1 H, dd, J 1 ) 10.5
Hz, J 2 ) 16.7 Hz), 4.23 (1 H, m), 5.22 (1 H, dd, J 1 ) 2.9 Hz, J 2
) 10.5 Hz), 5.86 (1 H, d, J ) 5.6 Hz), 7.00 (3H, m, H).
3(S)-[(ter t-Bu t yloxyca r b on yl)a m in o]-2-oxo-1-p yr r o-
lid in ea cetic a cid (5) was synthesized according to Freidinger
et al.:17 yield (60%); mp 168-170 °C; Rf (G) 0.30; HPLC24 tR
)
1
20.80 min; FAB-MS m/z 259 [M + H+]; H NMR (DMSO-d6,
400 MHz) δ 1.38 (9 H, s), 1.81 (1 H, m), 2.22 (1 H, m), 3.30 (2
H, m), 3.81 (1H, d, J ) 17.5 Hz), 4.01 (1H, d, J ) 17.5 Hz),
4.09 (1 H, m), 7.14 (1 H, d, J ) 8.8 Hz).
3(R)-[(ter t-Bu t yloxyca r b on yl)a m in o]-2-oxo-1-p yr r o-
lid in ea cetic a cid (5′) was obtained by the synthetic route
described by Freidinger et al.17 starting from Boc-D-Met-Gly-
OMe: yield (57%); mp 170-172 °C; Rf (G) 0.30; HPLC24 tR
)
a
HPLC controls were run on a Merck/Hitachi instrument on a
1
20.80 min; FAB-MS m/z 259 [M + H+]; H NMR (DMSO-d6,
400 MHz) δ 1.38 (9 H, s), 1.81 (1 H, m), 2.22 (1 H, m), 3.30 (2
H, m), 3.81 (1H, d, J ) 17.5 Hz), 4.01 (1H, d, J ) 17.5 Hz),
4.09 (1 H, m), 7.14 (1 H, d, J ) 8.8 Hz).
DeltaPak C18 (5 mm), 150 × 3.9 mm, 100 Å column using gradient
conditions in a solvent system of A (0.1% TFA) and B (0.1% TFA
b
in CH3CN); gradient 0-100% B in 100 min. CZE were performed
on a PACE 5000 Beckman using an uncoated fused silica capillary
(75 µm × 50 cm × 800 µm aperture), pressure injection, run
conditions 15 min, 20 °C, 30 kV, 25 mM phosphate buffer (pH
2.75).
3(R)-[(ter t-Bu t yloxyca r b on yl)a m in o]-2-oxo-1-p yr r oli-
d in e-3(S)-p h en yl-2-p r op ion ic a cid (6) was obtained start-
ing from Boc-D-Met-Phe-OMe according to Freidinger et al.:17
yield (52%); foam; Rf (E) 0.32; HPLC24 tR ) 21.3 min; FAB-
1
MS m/z 349 [M + H+]; H NMR (CDCl3, 400 MHz) d 1.36 (9
in vacuo over phosphorus pentoxide: yield 700 mg (97%); mp
98-100 °C; Rf (F) 0.63; HPLC24 tR ) 23.5 min; FAB-MS m/z
367 [M + H+]; 1H NMR (CDCl3, 250 MHz) δ 1.39 (9 H, s), 2.34
(3 H, s), 2.83 (1H, t, J 1 ) J 2 ) 11.1 Hz), 3.72 (1H, dd, J 1 ) 6.8
Hz, J 2 ) 11.1 Hz), 4.14 (1H, d, J ) 17.4 Hz), 4.44 (1H, m),
4.82 (1H, d, J ) 17.4 Hz), 5.60 (1H, d, J ) 7.7 Hz), 7.23 (2H,
d), 7.45 (1H, s).
H, s), 1.77 (1 H, m), 2.38 (1 H, br), 2.96 (1 H, dd, J 1 ) 11.8 Hz,
J 2 ) 14.6 Hz), 3.16 (1 H, m), 3.31 (1 H, m), 3.38 (1 H, dd, J 1
)
4.3 Hz, J 2 ) 14.9 Hz), 3.88 (1H, m), 4.96 (1 H, dd, J 1 ) 4.6 Hz,
J 2 ) 11.4 Hz), 5.23 (1 H, br), 7.17 (5 H, m).
N-R-Boc-L-R-a m in o-ꢀ-ca p r ola cta m (15). To a solution of
Boc-Lys-OH (1 g, 4 mmol) in DMF (400 mL) were added BOP
(1.8 g, 4 mmol) and sodium bicarbonate (1.7 g, 20 mmol). After
12 h stirring at room temperature, the mixture was concen-
trated to a small volume (5 mL). Ethyl acetate (100 mL) was
then added, and the solution was washed with a saturated
sodium bicarbonate solution (3 × 100 mL), water, 1 M
potassium hydrogenosulfate solution (3 × 100 mL), and brine,
dried over magnesium sulfate, filtered, and concentrated under
reduced pressure to afford a residue that crystallized upon
trituration in hexane: yield (85%); mp 110 °C dec; Rf (C) 0.27;
Rf (D) 0.44.
3(S)-[(ter t-Bu tyloxycar bon yl)am in o]-5-(car boxym eth yl)-
2,3-d ih yd r o-1,5-ben zoxa zep in -4(5H)-on e, Boc-D-BO-OH
(3). 1 M sodium hydroxide (3.3 mL, 3.3 mmol) was added to a
solution of Boc-D-BO-OEt (12) (1 g, 2.75 mmol) in 95% EtOH
(10 mL). After stirring at room temperature for 2 h, the solvent
was partially concentrated under reduced pressure and the
resulting solution was diluted with water (20 mL) and
extracted with ether (2 × 10 mL). The aqueous phase was
acidified to pH 3 with 1 M potassium bisulfate and extracted
with ethyl acetate (3 × 10 mL). The combined organic layers
were washed with water (2 × 10 mL) and brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo to give
the expected compound which precipitated upon addition of a
mixture of ether/hexane. It was collected by filtration and dried
in vacuo over phosphorus pentoxide: yield 830 mg (90%); mp
60-65 °C; Rf (E) 0.44; HPLC24 tR ) 19.7 min; FAB-MS m/z
337 [M + H+]; 1H NMR (CDCl3, 400 MHz) d 1.33 (9 H, s), 4.13
(1H, t, J 1 ) J 2 ) 10 Hz), 4.22 (1H, d, J ) 17.6), 4.54 (1H, t, J
) 10 Hz), 4.66 (1H, m), 4.68 (1H, d, J ) 17.6), 5.46 (1H, d, J
) 7.1), 7.13 (4H, m).
5(S)-[(ter t-Bu tyloxycar bon yl)am in o]-1,2,4,5,6,7-h exah y-
d r oa zep in o[3,2,1-h i]in d ol-4-on e 2-(S)-Ca r b oxylic Acid
(4). To a solution of compound 13 (150 mg, 0.6 mmol) in a
mixture of 0.1 M aqueous solution hydroxide (6 mL) and
dioxane (6 mL) was added di-tert-butyl dicarbonate (157 mg,
7.2 mmol), and the pH was continuously adjusted to 10 by
addition of 1 M sodium hydroxide. When no starting material
could be detected by TLC, the reaction mixture was diluted
with water (40 mL) and extracted with ether (2 × 20 mL). The
expected compound precipitated upon addition of the aqueous
phase into 1 M potassium hydrogenosulfate (40 mL). It was
collected, thoroughly washed with water, and dried in vacuo
over phosphorus pentoxide: yield 180 mg (84%); mp 170-172
°C; Rf (E) 0.41; HPLC24 tR ) 22.5 min; FAB-MS m/z 347 [M +
H+]; 1H NMR (CDCl3, 250 MHz) δ 1.40 (9 H, s), 2.06 (1 H, m),
N-R-Boc-D-R-a m in o-ꢀ-ca p r ola cta m (16) was obtained as
described for compound 15 from Boc-D-Lys-OH: yield (90%);
mp 105 °C dec; Rf (C) 0.27; Rf (D) 0.44.
1-N-[(Ben zyloxyca r bon yl)m eth yl]-3(S)-[(ter t-bu tyloxy-
ca r bon yl)a m in o]a zep a n -2-on e (17). A solution of N-R-Boc-
L-R-amino-ꢀ-caprolactam (15) (740 mg, 3.2 mmol) in 5 mL of
THF was added to a suspension of sodium hydride (147 mg,
6.4 mmol) in 5 mL of THF. The reaction was stirred at room
temperature for 15 min, and benzyl bromoacetate was added
(613 mL, 3.2 mmol). After 5 h stirring at room temperature,
ethyl acetate (100 mL) was added followed by water (100 mL).
The organic phase was washed with brine, dried over magne-
sium sulfate, filtered and concentrated under reduced pressure
to afford an oily compound: yield 1.1 g (95%); Rf (B) 0.40; Rf
(C) 0.62.
N-[(Ben zyloxyca r b on yl)m et h yl]-3(R)-[(ter t-b u t yloxy-
ca r bon yl)a m in o]a zep a n -2-on e (18) was obtained as de-
scribed for compound 17 from the corresponding D-caprolactam
16: yield (82%); oil; Rf (B) 0.40; Rf (C) 0.0.62.
3(S)-[(ter t-Bu tyloxyca r bon yl)a m in o]-2-oxo-1-a zep in e-
a cetic Acid (7). Compound 17 (1.1 g, 3.2 mmol) was hydro-
genated overnight at room temperature in 95% EtOH (100 mL)
in the presence of a 10% Pd/C catalyst. The catalyst was then
removed by filtration, and the filtrate was concentrated in
vacuo to give a residue which crystallized upon trituration in

4200 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
hexane: yield 780 mg (96%); mp 125 °C dec; Rf (E) 0.40;
Amblard et al.
from Calbiochem (La J olla, CA). All molecular biology and cell
culture reagents were purchased from Life Technologies
(Cergy-Pontoise, France). Other chemicals were from Sigma
Chemical Co. (St Louis, MO). [3H]Bradykinin (80-120 mCi/
mmol, 1 Ci ) 37 GBq) and [3H]des-Arg10-[Leu9]-kallidin (74-
77 Ci/mmol) were purchased from New England Nuclear.
1
HPLC24 tR ) 16.8 min; FAB-MS m/z 287 [M + H+]; H NMR
(CDCl3, 250 MHz) δ 1.46 (9 H, s), 1.71 (4 H, m), 2.01 (2 H, m),
3.21 (1 H, dd, J 1 ) 4.6 Hz, J 2 ) 15.4 Hz), 3.70 (1 H, dd, J 1
)
11.4 Hz, J 2 ) 15.4 Hz), 4.20 (2 H, AB system, J ) 7.5 Hz),
4.45 (1 H, m), 5.91 (1H, d, J ) 6.2 Hz).
3(R)-[(ter t-Bu tyloxyca r bon yl)a m in o]-2-oxo-1-a zep in e-
a cetic a cid (7′) was obtained as described for compound 7:
yield (95%); mp 85 °C dec; Rf (E) 0.40; HPLC24 tR ) 16.8 min;
FAB-MS m/z 287 [M + H+]; 1H NMR (CDCl3, 250 MHz) δ 1.46
(9 H, s), 1.71 (4 H, m), 2.01 (2 H, m), 3.21 (1 H, dd, J 1 ) 4.5
Hz, J 2 ) 15.4 Hz), 3.70 (1 H, dd, J 1 ) 11.3 Hz, J 2 ) 15.4 Hz),
4.20 (2 H, AB system, J ) 7.5 Hz), 4.45 (1 H, m), 5.91 (1H, d,
J ) 6.2 Hz).
3(S)-[(ter t-Bu t yloxyca r b on yl)a m in o]-2-oxo-1-p ip er i-
d in ea cetic a cid (8) was obtained according to Freidinger et
al.:17 yield 800 mg (98%); mp 110-113 °C; Rf (E) 0.70; HPLC24
tR ) 24.65 min; FAB-MS m/z 273 [M + H+]; 1H NMR (CDCl3,
250 MHz) δ 1.46 (9 H, s), 1.77 (1 H, m), 2.02 (2 H, m), 2.45 (1
H, m), 3.40-3.60 (2 H, m), 3.92 (1 H, d, J ) 15 Hz), 4.18 (1 H,
m), 4.37 (1 H, d, J ) 15 Hz), 5.70 (1H, m).
3-[(ter t-Bu tyloxyca r bon yl)a m in o]-3,5-d iben zyl-2,4-d i-
oxop yr r olid in e (20). 1-(tert-Butyloxycarbonyl)-3-[(tert-butyl-
oxycarbonyl)amino]-3,5-dibenzyl-2,4-dioxopyrrolidine13 (19) (1
g, 2.1 mmol) was deprotected with TFA (7 mL). After standing
30 min at room temperature, the expected compound precipi-
tated upon addition of ether (50 mL). It was collected,
thoroughly washed with ether, and dried in vacuo over KOH.
To a solution of this deprotected compound (700 mg, 1.7 mmol)
in a mixture of 0.1 M sodium hydroxide (17 mL) and dioxane
(34 mL) was added di-tert-butyl dicarbonate (370 mg, 1.7
mmol), and the pH was continuously adjusted to 10 by addition
of 1 M aqueous sodium hydroxide solution. When no starting
material could be detected by TLC, the reaction mixture was
diluted with water (40 mL) and extracted with ether (2 × 20
mL). The expected compound precipitated upon addition of the
aqueous phase into 1 M potassium hydrogenosulfate (40 mL).
It was collected, thoroughly washed with water, and dried in
vacuo over phosphorus pentoxide: yield 600 mg (90%); yield
1.2 g (85%); mp 235-237 °C; Rf (B) 0.20; Rf (C) 0.60.
1-[(Ben zyloxyca r b on yl)m et h yl]-3-[(ter t-b u t yloxyca r -
bon yl)a m in o]-3,5-d ib en zyl-2,4-d ioxop yr r olid in e (21). A
solution of compound 20 (190 mg, 0.5 mmol) in 5 mL of THF
was added to a suspension of sodium hydride (24 mg, 1 mmol)
in 5 mL of THF. The reaction was stirred at room temperature
fo 15 min, and benzyl bromoacetate was added (80 mL, 0.5
mmol). After 5 h stirring at room temperature, ethyl acetate
(20 mL) was added followed by water (20 mL). The organic
phase was washed with brine, dried over magnesium sulfate,
filtered, and concentrated under reduced pressure to afford
an oily compound: yield 260 mg (90%); mp 125-127 °C; Rf
(A) 0.12; Rf (B) 0.60.
2. Clon in g of Hu m a n a n d Ra t Br a d yk in in Recep tor s.
As previously described25 the coding region of the human B2
receptor was isolated by PCR from genomic DNA of HepG2
cells using specific primers. The PCR product was subcloned
into the EcoRI and XbaI sites of the vector pBlueScript SK-
(Stratagene). The recombinant plasmid was digested with
EcoRI and XbaI, and the insert was subcloned into the
eukaryotic expression vector pcDNA3 (Invitrogen). The human
B1 receptor was cloned as previously described26 and expressed
in 293 cells.
3. Cell Cu ltu r e a n d Tr a n sfection . CHO and 293 cells
were maintained in HAM F12 containing 10% fetal calf serum,
4.5 g/L glucose, 100 mg/L streptomycin, and 105 units/L
penicillin. Human embryonic kidney 293 cells were grown in
Dulbecco’s modified Eagles medium containing 10% fetal calf
serum, 4.5 g/L glucose, 1% Glutamax (v/v), 1% nonessential
amino acids (v/v), 1 mM sodium pyruvate, 100 mg/mL penicil-
lin, and 100 mg/mL streptomycin. Cells were transfected with
the different cDNA containing vectors (10 µg/plate of 150 mm
in diameter) using the calcium phosphate precipitation method.
After 48-72 h of recovery, the selection of transfectants was
performed using 500 µg/mL Geneticin. Cell clones were
isolated by dilution plating, screened for receptor expression,
and then propagated.
4. Bin d in g Stu d ies. Stably transfected CHO and 293 cells
were scrapped from dishes in 5 mL of binding buffer containing
20 mM TES (pH 6.8), 1 mM 1,10-phenanthroline, 140 µg/mL
bacitracine, and 0.1% bovine serum albumin; 293 cells stably
transfected with the B1 receptor were treated as described
above except that TES was used at 25 mM and pH 7.4. Cell
membranes were obtained by centrifugation (40000g for 15
min). Competition binding experiments were carried out by
incubating membranes with the competitor ligands (brady-
kinin and analogues) and 400 pM [3H]bradykinin for the B2
receptor (0.5 mL final volume for 90 min) or 1 nM [3H]des-
Arg10-[Leu9]-kallidin for the B1 receptor (0.5 mL final volume
for 60 min). Nonspecific binding was determined in the
presence of 10 µM bradykinin or des-Arg10-[Leu9]-kallidin.
Reactions were terminated by filtration with a Brandel cell
harvester through Whatman GF/B filters presoaked for 2 h
in poly(ethylenimide) 0.1% (w/v). Filters were washed three
times with ice-cold 50 mM TES or Tris, and the radioactivity
retained on the filters was counted with a Beckman liquid
scintillation counter. Protein concentration was measured by
the method of Bradford.27
5. F u n ction a l Exp er im en ts. Human umbilical cords were
collected postdelivery and immediately placed in a Krebs
solution of the following composition (in mM): NaCl 119, KCl
4.7, KH2PO4 1.18, MgSO4 1.17, NaHCO3 25, CaCl2 2.5,
ethylenediaminetetraacetic acid (EDTA) 0.026, glucose 5.5,
bubbled with 95% O2 plus 5% CO2 and maintained at 4 °C.
Vein rings (3-4 mm in length) without endothelium were set
up in 8-mL jacketed organ baths containing Krebs solution
and maintained at 37 °C. The resting tension was 1 g. After a
maximal contraction obtained with a high-potassium-contain-
ing Krebs solution (KPSS) in which NaCl was replaced by KCl
and return to the baseline following repetitive washings, the
following compounds were added in the organ bath: mepyr-
amine (1 µM), atropine (1 µM), indomethacin (3 µM), NG-nitro-
L-arginine (L-NOARG, 30 µM), captopril (10 µM), thiorphan
(1 µM), DL-2 -(mercaptomethyl)-3-(guanidinoethylthio)-
propanoic acid (MERGETPA, 5 mM), and nifedipine (0.1 µM).
Thirty minutes later the concentration-response curve to
bradykinin or bradykinin analogues was obtained. At the end
of the experiments, after washing and return to the baseline
level, the maximal contraction of each vein segment was
obtained by adding the thromboxane A2 mimetic, U46619 (1
µM).
3-[(ter t-Bu tyloxyca r bon yl)a m in o]-3,5-d iben zyl-2,4-d i-
oxo-1-p yr r olid in ea cetic Acid (9). Compound 21 (260 mg,
0.47 mmol) was hydrogenated overnight at room temperature
in 95% EtOH (100 mL) in the presence of a 10% Pd/C catalyst.
The catalyst was then removed by filtration, and the filtrate
was concentrated in vacuo to leave a residue which crystallized
upon trituration in hexane: yield 200 mg (95%); mp 90-92
°C; Rf (E) 0.62; HPLC24 tR ) 30.28 min; FAB-MS m/z 453 [M
1
+ H+]; H NMR (CDCl3, 400 MHz) δ 1.30 (4.5 H, s), 1.34 (4.5
H, s), 1.41 (0.5 H, dd, J 1 ) 9.2 Hz, J 2 ) 14.5 Hz), 2.59 (0.5 H,
dd, J 1 ) 3.8 Hz, J 2 ) 14.7 Hz), 2.69 (0.5 H, d, J ) 12.7 Hz),
2.90 (0.5 H, dd, J 1 ) 8.4 Hz, J 2 ) 15 Hz), 2.92 (0.5 H, d, J )
13.1 Hz), 2.98 (0.5 H, d, J ) 12.8 Hz), 3.05 (0.5 H, d, J ) 12.8
Hz), 3.36 (0.5 H, dd, J 1 ) 4 Hz, J 2 ) 15.2 Hz), 3.58 (0.5 H, d,
J ) 17.7 Hz), 3.60 (0.5 H, d, J ) 18.02 Hz), 3.67 (0.5 H, dd, J 1
) 4 Hz, J 2 ) 8.9 Hz), 3.98 (0.5 H, d, J ) 18.9 Hz), 4.03 (0.5 H,
d, J ) 18.5 Hz), 4.28 (0.5 H, dd, J 1 ) 3.8 Hz, J 2 ) 9.2 Hz),
5.22 (0.5 H, s), 5.33 (0.5 H, s), 6.88 (1 H, d), 7.15 (3 H, m), 7.22
(5 H, m), 7.28 (1 H, m).
P h a r m a cologica l Stu d ies. 1. Ma ter ia ls. HOE 140 was
synthesized in our laboratory. MERGETPA (DL-2-(mercapto-
methyl)-3-(guanidinoethylthio)propanoic acid) was obtained

BK Agonists Containing Dipeptide Mimics
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4201
(11) 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 receptor antagonists
adopt C-terminal â-turns. J . Med. Chem. 1993, 36, 1450-1460.
(12) Evans, B. E.; Rittle, M. G.; Bock, M. G.; DiPardo, R. M.;
Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.;
Anderson, P. S.; Chang, R. S. L.; Lotti, V. J .; Cerino, D. J .; Chen,
T. B.; Kling, P. J .; Kuntel, K. A.; Springer, J . P.; Hirshfield, J .
Methods for drug discovery: development of potent, selective,
orally effective cholecystokinin antagonists. J . Med. Chem. 1988,
31, 2235-2246.
(13) Pothion, C.; Fehrentz, J . A.; Aumelas, A.; Loffet, A.; Martinez,
J . Synthesis of pyrrolidine-2,4-diones from urethane N-carboxy-
anhydrides (UNCAs). Tetrahedron Lett. 1996, 37, 1027-1030.
(14) Slade, J .; Stanton, J . L.; Ben-David, D.; Mazzenga, G. C.
Angiotensin converting enzyme inhibitors: 1,5-benzothiazepine
derivatives. J . Med. Chem. 1985, 28, 1517-1521.
Female Sprague-Dawley rats weighing 250-300 g (Iffa
Credo, L’Arbresles, France) were pretrated with diethylstil-
bestrol at 0.1 mg/kg subcutaneously; 18 h later, rats were
sacrified by a CO2 intoxication, and the uterus was dissected
out and immediately placed in a J alon solution of the following
composition (in mM): NaCl 154, KCl 5.6, NaHCO3 1.7, MgCl2
1.4, glucose 5.5, and CaCl2 0.3. Four segments (approximately
10 mm in length) were prepared and suspended in jacketed
organ baths containing 8 mL of J alon’s solution maintained
at 37 °C and bubbled with 95% O2 and 5% CO2. The resting
tension was 1 g. After a 90-min resting period, captopril (1
µM), atropine (1 µM), indomethacin (3 µM), mepyramine (1
µM), and DL-thiorphan (1 µM) were added to the bath. The B2
receptor antagonist HOE 140 was added at various concentra-
tions 15 min before cumulative addition of bradykinin or
analogues. A single concentration-response curve to brady-
kinin or the tested analogues was obtained for each uterus
segment. At the end of the experiment, after washing and
return to the baseline, angiotensin II (3 µM) was added in
order to obtain the maximal contractile response of each
segment.
(15) Nagel, A. A. Benzothiazepine and benzoxazepine derivatives as
cholecystokinin receptor antagonists. International Patent Ap-
plication WO 94/01421, J an 20, 1994.
(16) De Lombaert, S.; Blanchard, L.; Stamford, L. B.; Sperbeck, D.
M.; Grim, M. D.; J enson, T. M.; Rodriguez, H. R. Practical
syntheses of a novel tricyclic dipeptide mimetic based on a [6H]-
azepino indole nucleus: Application to angiotensin-converting
enzyme inhibition. Tetrahedron Lett. 1994, 35, 7513-7516.
(17) Freidinger, R. M.; Perlow, D. S.; Veber, D. F. Protected lactam-
bridged dipeptides for use as conformational constraints in
peptides. J . Org. Chem. 1982, 47, 104-109.
(18) Brady, S. F.; Paleveda, W. J .; Arison, B. H.; Freidinger, R. M.;
Nutt, R. F.; Veber, D. F. An improved procedure for peptide
cyclization. In Peptides: Structure and Function; Proceedings
of the Eighth American Peptide Symposium; Hruby, V. J ., Rich,
D. H., Eds.; Pierce Chemical Co.: Rockford, IL, 1983; pp 127-
130.
Refer en ces
(1) For reviews see: (a) Olson, G. L.; Bolin, D. R.; Bonner, M. P.;
Bo¨s, M.; Cook, C. M.; Fry D. C.; Graves, B. J .; Hatada, M.; Hill,
D. E.; Kahn, M.; Madison, V. S.; Rusiecki, V. K.; Sarabu, R.;
Sepinwall, J .; Vincent, G. P.; Voss, M. E. Concepts and progress
in the development of peptide mimetics. J . Med. Chem. 1993,
36, 3039-3049. (b) Fairlie, D. P.; Abbenante, G.; March, D. R.
Macrocyclic peptidomimetics - Forcing peptides into bioactive
conformations. Curr. Med. Chem. 1995, 2, 654-686.
(2) Rich, D. H. Peptidase inhibitors. In Comprehensive medicinal
chemistry: The rational design, mechanistic study and thera-
peutic application of chemical compounds; Hansch, C., Sammes,
P. G., Taylor, J . B., Eds.; Pergamon Press: New York, 1990; pp
391-44 and references therein.
(3) Amblard, M.; Daffix, I.; Bedos, Ph.; Berge´, G.; Pruneau, D.;
Paquet, J . L.; Luccarini, J . M.; Be´lichard, P.; Dodey, P.; Martinez,
J . Design and synthesis of potent bradykinin agonists containing
a benzothiazepine moiety. J . Med. Chem. 1999, 42,
(19) Barany, G.; Merrifield, R. B. Solid-phase peptide synthesis. In
The Peptides, Analysis, Synthesis and Biology; Gross, E., Meien-
hofer, J ., Eds.; Academic Press: New York, 1979; Vol. 2, pp
3-284.
(20) Castro, B.; Dormoy, J . R.; Evin, G.; Selve, C. Peptide coupling
reagents. N-[oxytris(dimethylamino)phosphoniumbenzotriazole
hexafluorophosphate. Tetrahedron Lett. 1975, 1219-1222.
(21) Inamura, T.; Nomura, R.; Black, K. L. Intracarotid infusion of
RMP-7, a method for selective drug delivery to brain tumors. J .
Neurosurg. 1994, 81, 752-758.
4185-4192.
(4) 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.
(22) Gisin, B. F. The preparation of Merrifield-resins through total
esterification with cesium salts. Helv. Chim. Acta 1973, 56,
1476-1482.
(5) Dorer, F. E.; Ryan, J . W.; Steward, J . M. Hydrolysis of brady-
kinin and it’s higher homologues by angiotensine-converting
enzyme. Biochem. J . 1974, 14, 915-917.
(23) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color
test for detection of free terminal amino groups in the solid-phase
synthesis of peptides. Anal. Biochem. 1970, 34, 595-598.
(24) HPLC controls were run on a Merck/Hitachi instrument on a
Waters DeltaPak C18 (5 µm), 150 × 3.9 mm, 100 Å column using
gradient conditions in a solvent system of A (0.1% TFA) and B
(0.1% TFA in CH3CN); gradient 0-100% B in 50 min.
(25) Paquet, J .-L.; Luccarini, J .-M.; Fouchet, C.; Defreˆne, E.; Loillier,
B.; Robert, C.; Be´lichard, P.; Cremers, B.; Pruneau, D. Pharma-
cological characterization of the bradykinin B2 receptor: inter-
species variability and dissociation between binding and func-
tional responses. Br. J . Pharmacol. 1999, 126, 1083-1090.
(26) Bastian, S.; Loillier, B.; Paquet, J . L.; Pruneau, D. Stable
expression of human kinin B1 receptor in 293 cells: pharma-
cological and functional characterization. Br. J . Pharmacol.
1997, 122, 393-399.
(6) Freidinger, R. M.; Perlow, D. S.; Veber, D. F. Protected lactam-
bridged dipeptides for use as conformational constraints in
peptides. J . Org. Chem. 1982, 47, 104-109.
(7) Thorsett, E. D.; Harris, E. E.; Aster, S. D.; Peterson, E. R.;
Snyder, J . P.; Springer, J . P.; Hirshfield, J .; Tristam, E. W.;
Patchett, A. A.; Ulm, E. H.; Vassil, T. C. Conformationally
restricted inhibitors of angiotensin converting enzyme: synthesis
and computations. J . Med. Chem. 1986, 29, 251-260.
(8) Hoyer, D.; Awad, M. M. A.; Salvino, J . M.; Seoane, P. R.; Dolle,
R. E.; Houck, W. T.; Sawutz, D. G. ACE inhibitors as a template
of bradykinin B2 receptor antagonists. Bioorg. Med. Chem. Lett.
1995, 5, 367-370.
(9) Kyle, D. J .; Martin, J . A.; Farmer, S. G.; Burch, R. M. Design
and conformational analysis of several highly potent bradykinin
receptor antagonists. J . Med. Chem. 1991, 34, 1230-1233.
(10) Kyle, D. J .; Martin, J . A.; Burch, R. M.; Carter, J . P.; Lu, S.;
Meeker, S.; Prosser, J . C.; Sullivan, J . P.; Togo, J .; Noronha-
Blob, L.; Sinsko, J . A.; Walters, R. F.; Whaley, L. W.; Hiner, R.
N. Probing the bradykinin receptor: mapping the geometric
topography using ethers of hydroxyproline in novel peptides. J .
Med. Chem. 1991, 34, 2649-2653.
(27) Bradford, M. M. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 1976, 721, 248-254.
J M9901531