Synthesis and characterization of pseudopeptide bradykinin B2 receptor antagonists containing the 1,3,8-triazaspiro[4.5]decan-4-one ring system

Article abstract of DOI:10.1021/jm950676i

A series of pseudopeptides containing alkyl-, cycloalkyl-, aryl-, and aralkyl-substituted 1,3,8-triazaspiro[4.5]decan-4-one-3-acetic acids as amino acid surrogates to replace the Pro²-Pro³-Gly⁴-Phe⁵ section of the peptide bradykinin B2 receptor antagonist [Pro³, Phe⁵]HOE 140 (D-Arg⁰-Arg¹- Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-D-Tic⁷-Oic⁸-Arg⁹) were prepared. These psuedopeptides were examined in vitro for their B2 receptor affinities as well as for their ability to block bradykinin mediated actions in vivo. Two compounds in particular, NPC 18521 (I) and NPC 18688 (V) were quite potent in these latter assays, indicating that a significant portion of this prototypical second generation decapeptide antagonist can be replaced with a more compact nonpeptide molecule.

Full text of DOI:10.1021/jm950676i

J . Med. Chem. 1996, 39, 3169-3173
3169
Syn th esis a n d Ch a r a cter iza tion of P seu d op ep tid e Br a d yk in in B2 Recep tor
An ta gon ists Con ta in in g th e 1,3,8-Tr ia za sp ir o[4.5]d eca n -4-on e Rin g System
Babu J . Mavunkel,* Zhijian Lu, R. Richard Goehring, Songfeng Lu, Sarvajit Chakravarty, J ohn Perumattam,
Elizabeth A. Novotny, Maureen Connolly, Heather Valentine, and Donald J . Kyle
Scios Nova, Inc., 820 West Maude Avenue, Sunnyvale, California 94086
Received September 13, 1995X
A series of pseudopeptides containing alkyl-, cycloalkyl-, aryl-, and aralkyl-substituted 1,3,8-
triazaspiro[4.5]decan-4-one-3-acetic acids as amino acid surrogates to replace the Pro2-Pro3-
Gly4-Phe5 section of the peptide bradykinin B2 receptor antagonist [Pro3, Phe5]HOE 140 (D-
Arg0-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-D-Tic7-Oic8-Arg9) were prepared. These psuedopeptides were
examined in vitro for their B2 receptor affinities as well as for their ability to block bradykinin
mediated actions in vivo. Two compounds in particular, NPC 18521 (I) and NPC 18688 (V)
were quite potent in these latter assays, indicating that a significant portion of this prototypical
second generation decapeptide antagonist can be replaced with a more compact nonpeptide
molecule.
I. In tr od u ction
[Pro3, Phe5]HOE-1408,9 (D-Arg0-Arg1-Pro2-Pro3-Gly4-
Phe5-Ser6-D-Tic7-Oic8-Arg9 (Ki ) 0.21 ( 0.02 nM) as it
relates to the affinity for the bradykinin B2 receptor.10
In these sequences, D-Tic represents the D stereoisomer
of tetrahydroisoquinoline carboxylic acid and Oic cor-
responds to S,S,S-octahydroindolecarboxylic acid. It
was discovered that this tetrapeptide segment could be
replaced with simple nonpeptidic moieties while main-
taining affinities (Ki) to the receptor in the range of 40-
400 nM. Efforts to optimize these amino acid replace-
ments led to the development of several heterocyclic
tetrapeptide mimetics. Of those examined, one of the
most interesting series was comprised of a 1,3,8-
triazaspiro[4.5]decan-4-one-3-acetic acid motif. This
spirocyclic ring system was chosen as a tetrapeptide
(Pro2-Pro3-Gly4-Phe5) mimetic on the basis of compara-
tive computer modeling and solution conformations of
peptide agonists and antagonists containing the Pro-
Pro-Gly-Phe domain.11,12 Of particular use were com-
parisons made to a recently reported cyclic peptide kinin
antagonist (D-Arg-Arg-Cys-Pro-Gly-Phe-Cys-D-Tic-Oic-
Arg). The cyclic portion of the peptide encompassed only
five residues, one of which was Pro and two of which
were constrained by the requisite geometry of a disulfide
bond. These features rendered the cyclic domain of the
compound highly inflexible such that reasonable com-
putational models could be constructed.10 Ultimately,
the spirocyclic mimetic reported herein was devised on
the basis of its ability to mimic this cyclic domain of
the peptide precursor. Shown in Figure 1 is the 1-phen-
yl-substituted spirocyclic mimetic comparatively over-
layed with the cyclic pentamer domain of the previously
reported cyclic decapeptide antagonist.
Nearly all cells in most species express kinin receptors
which mediate the diverse physiological and patho-
physiological activities of bradykinin (Arg1-Pro2-Pro3-
Gly4-Phe5-Ser6-Pro7-Phe8-Arg9).1 Kinin receptors are
known to be G-protein coupled, and their activation
leads to relaxation of venular smooth muscle and
hypotension, increased vascular permeability, contrac-
tion of smooth muscle of the gut and airway leading to
increased airway resistance, stimulation of sensory
neurons, alteration of ion secretion of epithelial cells,
production of nitric oxide, release of cytokines from
leukocytes and the production of eicosanoids from
various cell types.2 Because of their broad spectrum of
activity, kinins have been implicated in many disorders
including pain, sepsis, asthma, rheumatoid arthritis,
pancreatitis, and a wide variety of other inflammatory
diseases. Moreover, a recent report demonstrated that
bradykinin B2 receptors on the surface of human
fibroblasts were upregulated 3-fold beyond normal in
patients with Alzheimer’s disease, implicating brady-
kinin as a participant in the peripheral inflammatory
processes associated with that disease.3
In contrast to the adverse physiologies associated with
bradykinin release, there is a growing body of literature
which implicates bradykinin as a protective agent
during periods of cardiac or renal stress.4-6 In this
regard there is substantial evidence that the cardiopro-
tective effects afforded by angiotensin-converting en-
zyme (ACE) inhibitor treatment are a result of meta-
bolically preserving bradykinin and are therefore
mediated by bradykinin B2 (and possibly B1) receptors.
These more recent results point to a possible therapeutic
role for a kinin receptor agonist.
In this paper we fully describe the synthetic proce-
dures required to prepare these novel pseudopeptide
bradykinin B2 receptor antagonists. In addition, through
the presentation of a series of differentially derivitized
spirocyclic mimetics, a structure-activity relationship
is described. Finally, in vivo activities for two of these
novel kinin antagonists are presented.
More than three decades have passed since the initial
discovery of bradykinin, yet there has only recently been
a single report of a nonpeptidic antagonist.7 However,
no nonpeptides have been described that are highly
potent, competitive and selective for the receptor. In
previous reports we have described the results of
systematic investigations into the role of the amino acid
sequence Pro2-Pro3-Gly4-Phe5 in the peptide antagonist
II. Meth od s
The preparation of the 1,3,8-triazaspiro[4.5]decan-
4-one-3-acetic acid structural fragment is outlined in
Scheme 1.15 1-Benzyl-4-piperidone (1) was subjected to
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
S0022-2623(95)00676-5 CCC: $12.00 © 1996 American Chemical Society

3170 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Mavunkel et al.
Sch em e 1a
a
Reagents: (a) RNH2, KCN, H+; (b) H2SO4; (c) (EtO)3CH, H+; (d) H2, H+, Pd/C; (e) (BOC)2O; (f) NaH, Br(CH2)CO2Me; (g) Na2CO3.
Strecker amino nitrile synthesis, followed by hydration
to give the amino amides 3 possessing the various R
substituents. In some cases (R ) n-propyl and cyclo-
hexyl) these amino amides were commercially available.
The spirocyclic ring system was then prepared by
heating 3 with triethyl orthoformate. Hydrogenation
over palladium on carbon reduced the carbon nitrogen
double bond with concomitant removal of the benzyl
group. Protection of the secondary amine with a Boc
group under standard conditions gave spirocyclic amide
5. Abstraction of the NH proton with sodium hydride
and alkylation of the resulting amide anion with methyl
bromoacetate gave the ester 6. Finally, standard sa-
ponification afforded the desired Boc-protected spirocy-
clic amino acid 7 (Scheme 1).
Ta ble 1. Psuedopeptide Structures of the Sequence
D-Arg-Arg-X-Ser-D-Tic-Oic-Arg with Corresponding Human B2
Kinin Receptor Affinity Dataa
The individual spirocyclic structural fragments were
incorporated into the pseudopeptides using standard
coupling procedures (see the Experimental Section).
Receptor binding assays were performed following
known procedures and made use of membrane prepara-
tions from a CHO cell line, engineered to express human
B2 receptors.
III. Resu lts
A series of pseudopeptide bradykinin B2 receptor
ligands were synthesized that contained differently
substituted 1,3,8-triazaspiro[4.5]decan-4-one systems.
These are shown in Table 1. All pseudopeptides were
of the general sequence H-D-Arg-Arg-[1,3,8-triazaspiro-
[4.5]decan-4-one]acetyl-Ser-D-Tic-Oic-Arg-OH, where the
1-position of the spirocyclic fragment is substituted
variably to probe possible structure activity relation-
ships. The isolated C-terminal tetrapeptide, Ser-D-Tic-
Oic-Arg, contained within these pseudopeptides has
been previously shown by NMR to exist nearly exclu-
sively in a â-turn conformation in aqueous environ-
ment.4 Given the structural rigidity which was de-
scribed, the conformation reported previously was
presumed to remain similar when incorporated into the
current series, although definitive spectroscopic proof
is still required. The N-terminus of each contained the
dipeptide D-Arg-Arg, in accordance with the structure-
activity relationship associated with previously reported
first and second generation peptide antagonists.10 This
structure was derived by combining the known solution
conformation of the C-terminal tetrapeptide Ser-D-Tic-
Oic-Arg, with a model of the conformationally rigid
spirocyclic mimetic described herein. The N-terminal
D-Arg-Arg reflects an arbitrary conformation since there
is no experimental evidence for any preferred state. The
actual conformation(s) adopted by these new pseudopep-
tides remains to be eluciated, most likely via two-
dimensional NMR experiments.
a
Affinity to human B2 receptor in CHO cell membranes
determined by displacement of [3H]NPC 17731.
Receptor binding assays indicate that, in this series
of D-Arg-Arg-SPIROCYCLIC-Ser-D-Tic-Oic-Arg, all of
the substitutions which were made at position 1 on the
spirocyclic mimetic could be accommodated by the
receptor binding site as is reflected in the measured
affinities to the human B2 receptor (Table 1). Varying
the size of the alkyl substituent at the 1-position of the
spirocyclic mimetic revealed an interesting structure-
activity relationship. The highest affinity compound,
VI (Ki ) 0.15 nM), also contained the largest substituent
(phenethyl) at the 1-position of the mimetic. The lowest
affinity compound, IV (Ki ) 27 nM), corresponds to a
structure with the smallest substituent (n-propyl) tested
at the 1-position of the spirocyclic mimetic. This general
observation was consitent throughout the series where
the mimetics bearing large hydrophobic moieties at the
1-position had higher affinities to the B2 kinin receptor
than did those with smaller substituents. Aromatic

Bradykinin B2 Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3171
sponse in each species, 1 µg kg-1 bradykinin was needed
in the rabbit while 12 µg kg-1 bradykinin was needed
in the rat. In a related study published elsewhere,
compounds I and V were also shown to possess anti-
nociceptive activity in rodents.14
Ta ble 2. Pharmacological Profile for Pseudopeptides I and V
ED50 (mg/kg-1
I
)
(bradykinin induced) assay
V
hypotension in ratsa
400 (4)
22.1 (4)
600 (4)
8.6 (4)
hypotension in rabbitsb
a
b
BK challenge is 12 µg kg-1, iv. BK challenge is 1 µg kg-1, iv.
IV. Con clu sion s
In conclusion the 1,3,8-triazaspiro[4.5]decan-4-one
ring system, differentially substituted with alkyl and
aryl groups at position 1, can serve as a worthy
surrogate for the Pro-Pro-Gly-Phe segment of peptide
antagonists of the type D-Arg0-Arg1-Pro2-Pro3-Gly4-Phe5-
Ser6-D-Tic7-Oic8-Arg9. These new pseudopeptides bind
to B2 kinin receptors with high affinity and inhibit
bradykinin both in vitro and in vivo in several species.
The 1-position of the mimetic should ideally be a large
hydrophobic moiety such that, upon receptor complex-
ation, an appropriate interaction with a hydrophobic site
on the receptor can occur. We propose that this site is
the same site which accommodates the phenylalanine
side chain at position 5 and/or the side accommodating
Pro3 of known first and second generation decapeptide
kinin antagonists.
Finally, these new pseudopeptides represent a sig-
nificant reduction of the peptidic nature of their pre-
cursory second generation decapeptide antagonists.
Although one ultimately hopes for the discovery of novel
nonpeptidic compounds, pseudopeptides I-VI represent
an important milestone. They reveal that a significant
section of a known decapeptide antagonist can be
replaced by a nonpeptide moiety while maintaining good
biological activity in vivo and in vitro.
F igu r e 1. Stereoview of a putative model of compound I
overlayed with the cyclic portion of a known cyclic peptide
kinin antagonist, (D-Arg0-Arg1-Cys2-Pro3-Gly4-Phe5-Cys6-D-Tic7-
Oic8-Arg9). The cyclization is between residues 2 and 6. Shown
in dark gray are VDW radii of the Pro3 and Phe5 side chains
in the cyclic peptide. The remainder of the cyclic portion of
the peptide is shown as thin lines. The mimetic is drawn as
thick lines without showing the chemical structure of the
N-terminal D-Arg-Arg or the C-terminal Ser-D-Tic-Oic-Arg.
substituents showed similar pharmacology as nonaro-
matic substituents on the spirocyclic scaffold. One
possible exception to this generalization can be found
in a comparison of compounds I and V. Here, the only
chemical difference is that I has a phenyl ring attached
to the 1-position of the spirocyclic mimetic and V has
cyclohexyl. The latter was approximately 10-fold more
potent in the receptor binding assay than the former.
The rank order potency of these two compounds was
maintained in vivo in a model of bradykinin-induced
hypotension in rabbits. However, in live animals, the
ED50 values differed only by 2-fold. In a further study
(bradykinin-induced hypotension performed in rats)
these same two compounds (I and V) were effectively
equipotent (Table 2).
Although spectroscopically unproven, this data might
indicate that, when bound to the receptor, the 1-position
of the 1,3,8-triazaspiro[4.5]decan-4-one mimetic is ac-
commodated by some type of hydrophobic domain of the
B2 kinin receptor. On the basis of the results presented
herein, this domain is likely quite large and hydrophobic
given that the sterically demanding phenethyl substitu-
tion could be bound readily and with high affinity.
Given the overall structure of these new pseudopeptides,
it seems highly probable that the substituent on the
1-position of the spirocyclic mimetic used interacts with
the same hydrophobic site or sites on the receptor which
binds Pro3 and/or Phe5 in the parent decapeptide
antagonists. This hypothesis is depicted in Figure 1.
Of course only detailed crytallographic analysis of an
appropriate ligand-receptor complex could confirm this
experimentally.
V. Exp er im en ta l Section
A. Gen er a l. Reagents and chemicals were purchased from
Aldrich Chemical Co., Inc. (Milwaukee, WI) and used without
purification. Chromatography refers to flash chromatography
on silica gel. Melting points were determined in open capillary
tubes with a Thomas-Hoover melting point apparatus and are
uncorrected. 1H NMR spectra were recorded on a Bruker AC-
400 spectrometer. Combustion analyses were performed by
Atlantic Microlabs, Inc., Norcross, GA.
All peptides were synthesized manually according to stan-
dard solid phase techniques, using tert-butyloxycarbonyl (Boc)
amino acids. All amino acids were purchased from Bachem
Bioscience Inc. (King of Prussia, PA). Boc-Oic-OH was syn-
thesized by modifying previously reported procedures.16 The
unnatural amino acids used during this study were prepared
according to procedures reported below. Boc-Arg-(Tos)-PAM
resin (0.79 mmol/g) was purchased from Advanced ChemTech
(Louisville, KY) and used for the syntheses. Syntheses were
run at a 0.5 g resin-aa scale (0.4 mmol), and couplings were
monitored using qualitative ninhydrin determination of free
amine (Kaiser test). Amino acids were coupled using active
esters of the Boc-protected amino acids, generated by treating
individual amino acids with equimolar quantities of hydroxy-
benzotriazole hydrate and diisopropylcarbodiimide in meth-
ylene chloride:DMF (1:1) at 0 °C, prior to introduction into solid
phase synthesis. Couplings were continued until no further
free amine was detected using the Kaiser test, and in the event
of incomplete coupling, amino acids were recoupled. Routine
N-capping was performed by treating peptide-resins with
acetic anhydride and 10% diisopropylethylamine (1:1) in DMF
after every coupling to reduce the formation of deletion
peptides. The finished peptidyl-resin was washed and dried
to a constant weight and then treated with anhydrous hydro-
gen fluoride (in presence of anisole 10%) at 10 mL/g of peptidyl-
resin for 1 h at 0 °C. After removal of the hydrogen fluoride,
the resin peptide was washed extensively with anhydrous
diethyl ether (3 × 20 mL) and then extracted with 15%
aqueous acetic acid.
Antagonist character was confirmed by the assess-
ment of the ability of these pseudopeptides to inhibit
bradykinin induced hypotension in rats and rabbits.
Compounds I and V were capable of dose dependently
blocking bradykinin-induced hypotension in these spe-
cies. As shown in Table 2, both compounds (I and V)
block the hypotensive response of iv bradykinin. In
these assays, it was demonstrated that the rabbit was
considerably more sensitive to bradykinin than was the
rat in that, to achieve a comparable hypotensive re-

3172 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Mavunkel et al.
The crude material, obtained from lyophilization, was
purified by reverse phase high-performance liquid chromatog-
raphy, using a linear gradient of 5-60% water/acetonitrile
(both containing 0.2% acetic acid), on a C18 Vydac (22.5 mm
i.d. × 25 cm, 10 µm, 300 Å) column, at room temperature. The
homogeneous pooled pure fractions were lyophilized and the
purity of the white fluffy powders determined by analytical
reverse phase HPLC, using a linear gradient of 5-80% water/
acetonitrile (both containing 0.1% TFA), on a C18 Vydac (4.6
mm i.d. × 25 cm, 10 µm, 300 Å) column, at room temperature,
and fast atom bombardment mass spectroscopy.
wt %, 0.54 g, 14.42 mmol) was washed with hexane (3 × 5
mL), suspended in DMF (20 mL), and cooled to 0 °C.
A
solution of 5f (4.20 g, 11.68 mmol) in DMF (20 mL) was added
dropwise. After the mixture was stirred at 0 °C for 30 min,
methyl bromoacetate (2.24 g, 14.42 mmol) was added and the
mixture stirred at room temperature 3 h. After diluting with
water, the mixture was extracted with EtOAc (5 × 50 mL).
The organic extracts were washed with water (3 × 50 mL) and
saturated aqueous sodium chloride and dried over Na2SO4.
After filtration and removal of the solvent in vacuo, the residue
was chromatographed (10-50% EtOAc/hexane, gradient) to
give 4.42 g (88%) of 6f as a colorless oil: 1H NMR (CDCl3) δ
1.44 (s, 9H), 1.52-1.75 (m, 4H), 2.68-2.85 (m, 4H), 3.35-3.55
(m, 2H), 3.76 (s, 3H), 3.80-4.04 (m, 2H), 4.10 (s, 2H), 4.28 (s,
2H), 7.16-7.35 (m, 5H). Anal. (C23H33N3O5) C, H, N.
f. 1-(2-P h en yleth yl)-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -
4-on e-3-a cetic Acid (7f). A mixture of 6f (4.27 g, 9.78 mmol),
Na2CO3 (4.50 g, excess), MeOH (75 mL), and water (25 mL)
was refluxed for 3 h. After the MeOH was removed in vacuo,
the resulting aqueous solution was cooled to 0 °C and carefully
acidified (pH 4) with concentrated HCl. The product was
extracted with EtOAc (3 × 100 mL); these organic extracts
were washed with saturated aqueous sodium chloride, dried
over Na2SO4, and filtered; and the solvent was removed in
vacuo to give 3.80 g (93%) of 7f as a white foam: 1H NMR
(CDCl3) δ 1.44 (s, 9H), 1.64-1.80 (m, 2H), 1.80-1.92 (m, 2H),
2.82-2.96 (m, 4H), 3.27-3.62 (m, 2H), 3.80-4.22 (m, 6H), 4.46
(br s, 2H), 7.15-7.35 (m, 5H). Anal. (C22H31N3O5‚0.75H2O)
C, H, N.
P r ep a r a t ion of 8-Boc-1,3,8-t r ia za sp ir o[4.5]d eca n -4-
on e-3-a cetic Acid s (7). a . 1-Ben zyl-4-cya n o-4-[(2-p h en -
yleth yl)a m in o]p ip er id in e (2f). To a stirred mixture of
1-benzyl-4-piperidone (1) (37.87 g, 200 mmol), 2-phenylethyl-
amine (27.16 g, 224 mmol), and acetic acid (100 mL) at room
temperature was added dropwise a solution of potassium
cyanide (16.10 g, 247 mmol) in water (35 mL). After stirring
at room temperature for 48 h, the reaction mixture was poured
into a mixture of concentrated NH4OH (200 mL) and crushed
ice (200 g). The mixture was extracted with CHCl3 (5 × 100
mL). The organic extracts were washed with saturated sodium
chloride, dried over Na2SO4, and filtered, and the solvent was
removed in vacuo. Recrystallization from diisopropyl ether
1
gave 32.37 g (51%) of 2f as white crystals: mp 65-68 °C; H
NMR (CDCl3) δ 1.25 (br s, 1H), 1.73 (t, 2H, J ) 13.5 Hz), 1.98
(d, 2H, J ) 12.6 Hz), 2.34 (t, 2H, J ) 11.0 Hz), 2.70-2.90 (m,
2H), 2.82 (t, 2H, J ) 6.9 Hz), 3.01 (q, 2H, J ) 6.9 Hz), 3.53 (s,
2H), 7.18-7.35 (m, 10H). Anal. (C21H25N3) C, H, N.
b. 1-Ben zyl-4-[(2-p h en yleth yl)a m in o]p ip er id in e-4-ca r -
boxa m id e (3f). To 90% H2SO4 (200 mL) was added 2f (32.00
g, 100 mmol) and the mixture stirred for 20 h. After this
mixture was cautiously poured into a mixture of concentrated
NH4OH (750 mL) and crushed ice (750 g), the resulting
alkaline mixture was extracted with CHCl3 (5 × 100 mL). The
organic extracts were washed with saturated sodium chloride,
dried over Na2SO4, and filtered, and the solvent was removed
in vacuo. Recrystallization from 2-propanol gave 27.03 g (80%)
of 3f as an off-white powder: mp 89-92 °C; 1H NMR (CDCl3)
δ 1.57 (d, 2H, J ) 11.3 Hz), 1.95-2.06 (m, 2H), 2.06-2.17 (m,
2H), 2.62-2.80 (m, 6H), 3.42 (s, 2H), 5.29 (s, 1H), 6.85 (s, 1H),
7.16-7.35 (m, 10H). Anal. (C21H27N3O) C, H, N.
1-P h en yl-8-Boc-1,3,8-tr iazaspir o[4.5]decan -4-on e-3-ace-
tic a cid (7a ): mp 200-201 °C; 1H NMR (CDCl3) δ 1.50 (s, 9H),
1.63-1.83 (m, 2H), 2.38-2.56 (m, 2H), 3.43-3.70 (m, 2H),
3.90-4.15 (m, 2H), 4.23 (s, 2H), 4.79 (s, 2H), 6.68-7.05 (m,
3H), 7.20-7.35 (m, 2H). Anal. (C20H27N3O5) C, H, N.
1-(4-Meth ylp h en yl)-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -
1
4-on e-3-a cetic a cid (7b): mp 173-175 °C; H NMR (CDCl3)
δ 1.45 (s, 9H), 1.64-1.84 (m, 2H), 2.20-2.40 (m, 5H), 3.42-
3.60 (m, 2H), 3.88-4.10 (m, 2H), 4.20 (s, 2H), 4.75 (s, 2H), 6.78
(d, 2H, J ) 11.4 Hz), 7.08 (d, 2H, J ) 11.4 Hz). Anal.
(C21H29N3O5) C, H, N.
1-(Cycloh exylm eth yl)-8-Boc-1,3,8-tr iazaspir o[4.5]decan -
1
4-on e-3-a cetic a cid (7c): mp 148-149 °C; H NMR (CDCl3)
c. 1-(2-P h en yleth yl)-8-ben zyl-1,3,8-tr ia za sp ir o[4.5]d ec-
2-en -4-on e (4f). A mixture of 3f (11.80 g, 34.97 mmol),
triethyl orthoformate (23 mL, excess), acetic acid (5 mL), and
toluene (75 mL) was refluxed for 20 h. The mixture was cooled
to room temperature and diluted with water (100 mL) and
CHCl3 (100 mL). The mixture was made basic with aqueous
sodium hydroxide, the layers were separated, and the aqueous
layer was extracted with CHCl3 (3 × 50 mL). The combined
organic extracts were dried over Na2SO4 and filtered, and the
solvent was removed in vacuo. Chromatography (1-15%
MeOH/CH2Cl2, gradient) gave 8.79 g (73%) of 4f as a pale
yellow oil: 1H NMR (CDCl3) δ 1.57 (d, 2H, J ) 12.5 Hz), 1.90-
2.22 (m, 2H), 2.79 (br s, 2H), 3.07 (br s, 2H), 3.61 (t, 2H, J )
7.1 Hz), 3.68 (br s, 2H), 7.15 (d, 2H, J ) 6.9 Hz), 7.20-7.42
(m, 8H), 7.80 (s, 1H). Anal. (C22H25N3O‚0.5H2O) C, H, N.
d . 1-(2-P h en yleth yl)-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -
4-on e (5f). A mixture of 4f (8.75 g, 25.22 mmol), MeOH (150
mL), and concentrated HCl (30 mL) was hydrogenated at 45
psi of H2 in the presence of 5% Pd/C (10 g) for 4 days. The
catalyst was filtered off and the MeOH removed in vacuo. The
remaining acidic aqueous solution was made basic by the
addition of solid Na2CO3. This alkaline mixture was diluted
with 1,4-dioxane (150 mL) and cooled to 0 °C, and di-tert-butyl
dicarbonate (10.90 g, 50.0 mmol) was added. After stirring
at room temperature for 20 h, the reaction mixture was diluted
with water (250 mL) and extracted with ether (5 × 100 mL).
The organic extracts were dried over Na2SO4 and filtered, and
the solvent was removed in vacuo. Chromatography (1-5%
MeOH/CH2Cl2, gradient) gave 8.47 g (93%) of 5f as white
crystals: mp 138-140 °C; 1H NMR (CDCl3) δ 1.44 (s, 9H),
1.53-1.70 (m, 4H), 2.68-2.84 (m, 4H), 3.30-3.55 (m, 2H),
3.78-4.05 (m, 2H), 4.21 (s, 2H), 6.68 (br s, 1H), 7.10-7.34 (m,
5H). Anal. (C20H29N3O3) C, H, N.
δ 0.75-0.90 (m, 2H), 1.10-1.30 (m, 4H), 1.50 (s, 9H), 1.60-
1.80 (m, 8H), 2.30 (d, 2H, J ) 7.2 Hz), 3.40-3.50 (m, 2H),
3.80-4.00 (m, 2H), 4.05-4.15 (m, 2H), 4.20 (s, 2H). Anal.
(C21H35N3O5) C, H, N.
1-n -P r op yl-8-Boc-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e-3-
a cetic a cid (7d ): mp 180-183 °C dec; 1H NMR (CDCl3) δ 0.93
(t, 3H, J ) 7.1 Hz), 1.46 (s, 9H), 1.50-1.90 (m, 6H), 2.55 (t,
2H, J ) 6.5 Hz), 3.30-3.67 (m, 2H), 3.95-4.20 (m, 2H), 4.08
(br d, 2H, J ) 7.2 Hz), 4.31 (brs, 2H). Anal. (C17H29N3O5) C,
H, N.
1-Cycloh exyl-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -4-on e-
3-a cetic a cid (7e): mp 172-173 °C; 1H NMR (CDCl3) δ 1.20-
1.42 (m, 8H), 1.47 (s, 9H), 1.58-1.85 (m, 6H), 2.64-2.74 (m,
1H), 3.42-3.70 (m, 2H), 3.70-3.95 (m, 2H), 3.95-4.15 (m, 2H),
4.39 (s, 2H). Anal. (C20H33N3O5) C, H, N.
B. Com p u ta tion a l P r oced u r es. All modeling work was
done using the programs CHARMm and Quanta13 on Silicon
Graphics computers. Energy minimizations were terminated
when the drop in energy after 50 steps of minimization was
less than 0.2 kcal mol-1
. The spirocyclic mimetic has no
rotatable bonds of structural consequence; hence no confor-
mational search was performed. The structure was optimized
using second-derivative minimization techniques. The com-
parative modeling entailed achieving an optimal end to end
distance wherein the N- and C-terminal heteroatoms of the
spirocyclic mimetic could closely match the corresponding
heteroatoms of Pro2 and Phe5. Moreover, the hydrophobic side
chains of Phe5 and Pro3 were accounted for in the mimetic by
a bulky substituent at the 1-position of the spirocycle. These
features are represented in Figure 1 where the thick bonds
correspond to the mimetic, the thin bonds are of the cyclic
peptide (D-Arg-Arg-Cys-Pro-Gly-Phe-Cys-D-Tic-Oic-Arg), and
the Pro3, Phe5 hydrophobic domain is shown as dark gray
spheres.
e. 1-(2-P h en yleth yl)-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -
4-on e-3-a cetic Acid Meth yl Ester (6f). Sodium hydride (60

Bradykinin B2 Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3173
C. P h ar m acology. 1. Receptor Bin din g Assay. CHO/K
cells (ATCC, Rockville, MD) were transfected with plasmid
DNA containing the human bradykinin B2 receptor using
Transfectase Reagent (Life Technologies, Gaithersburg, MD).
Membranes were prepared from the transfected cells, which
had been grown to confluency in Ham’s F12 medium contain-
ing 10% fetal bovine serum and 500 µg/mL G418 (Life
Technologies). Monolayers were washed one with Ca2+- and
Mg2+-free Dulbecco’s phosphate-buffered saline (DPBS).
Cells were scraped in DPBS and centrifuged at 500g for 10
min at 4 °C. Pellets were resuspended in 25 mM N-[tris-
(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) and
1 mM 1,10-phenanthroline pH 6.8 buffer and then homog-
enized using a Brinkmann Instrument Polytron (Westbury,
NY) at setting 5 for 10 s. Membranes were isolated by
centrifugation at 48000g for 10 min at 4 °C. Pellets were
resuspended in TES buffer with 140 µg/mL bacitracin and 0.1%
BSA, flash frozen in liquid nitrogen, and stored at -80 °C.
Competition binding assays were carried out in a 3 mL volume
using [3H]NPC-17731 (Du Pont NEN, Boston, MA) at final
concentrations of 15 pM. Nonspecific binding was determined
in the presence of 1 µM bradykinin and represented less than
10% of the total binding. After a 90 min incubation at 25 °C,
the assay was terminated by rapid vacuum filtration through
Whatman GF/B filters (Maidstone, U.K.) presoaked in 0.2%
polyethylenimine for 3 h followed by three washes with Tris
(50 mM, pH 7.4, 0 °C). Radioactivity was counted with a
Beckman scintillation counter (Beckman Instruments, Ful-
lerton, CA).
2. F u n ction a l Assa y. a . In Vivo In h ibition of Br a d y-
k in in -In d u ced Hyp oten sion in Ra bbits. Animals were
maintained at a constant temperature of 20-22 °C on a 12 h
light/dark cycle prior to experimentation. Food and water
were provided ad libitum. Male New Zealand white rabbits
weighing 1.5-2.0 kg were administered acepromozine (1 mg
kg-1, im) 30 min prior to anesthetizing with sodium pento-
barbital (15-30 mg kg-1, iv). Animals were placed on a
temperature control unit (Narco Bio-Systems, Houston, TX).
Mean systemic arterial blood pressure (MABP) was measured
continuously using carotid catheter (PE-50 polyethylene tub-
ing) filled with heparanized saline (20 units mL-1). The
catheter was connected to a physiological pressure transducer
(P-50, Gould Statham). The jugular vein was cannulated (PE-
10 polyethylene tubing) for the administration of bradykinin
(BK) and bradykinin antagonist (BKA). A tracheotomy was
performed, but animals were allowed to breath spontaneously.
The data was recorded on a physiograph (Gould 4 channel,
Model 30V8404).
lowed by an additional bradykinin injection given 5 min later.
MABP was measured to determine if the test compounds
would prevent bradykinin-induced hypotension. Successive
increasing doses of test compounds were administered to
generate cumulative dose response.
Su p p or tin g In for m a tion Ava ila ble: Analytical data for
pseudopeptides (Table 3) (1 page). Ordering information can
be found on any current masthead page.
Refer en ces
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Molecular Biology Intelligence Unit; R. G. Landes Co.: Austin,
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After surgery, each animal was allowed a period of stabi-
lization (15 min), after which three bolus doses of BK (1 µg
kg-1, iv) were administered at intervals of 5 min and the effect
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(0.03-1000 µg kg-1, iv) followed by a bolus of BK (1 µg kg-1
,
iv) 5 min after each dose of antagonist (the 5 min period allows
for time to observe agonist effects). The decrease in MABP
(mmHg) was calculated following each bolus of BK. The
animal was then allowed to stabilize for 10 min before the next
dose of antagonist was given. Each animal served as its own
control.
Animals were individually assessed for percent inhibition
of BK-induced hypotension and then averaged at each dose
as mean ( sem ED50’s were individually calculated using
Probit analysis.
b. In h ibition of Br a d yk in in -In d u ced Hyp oten sion in
Ra ts. Anesthetized Sprague-Dawley rats were cannulated
via the right carotid artery (for measurement of mean arterial
blood pressure (MABP)) and the jugular vein (for administra-
tion of bradykinin and/or test agent). The airway was kept
open via tracheotomy. Animals were stabilized for 30 min,
after which hypotension was induced by bradykinin (12 µg
kg-1, iv). The baseline hypotensive effect was confirmed by
two additional injections of bradykinin given at 5 min intervals
after the first injection. Test agents to be evaluated for
antagonist activity were administered 10-15 min (return to
baseline MABP) after the third injection of bradykinin, fol-
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J M950676I