Structure-based design of six novel classes of nonpeptide antagonists of the bradykinin B2 receptor

Article abstract of DOI:10.1016/S0960-894X(00)00482-0

Six classes of nonpeptide bradykinin antagonists were designed using a template derived from structural studies of peptide antagonists. Several compounds from each class were synthesized and assayed for binding to the human bradykinin B₂ receptor. Each family showed compounds active at the level of the smallest template peptide; three classes contained compounds with K(d) < 8 μM. These results provide diverse leads for a medicinal chemistry-based optimization program. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00482-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2421±2425
Structure-Based Design of Six Novel Classes of Nonpeptide
Antagonists of the Bradykinin B2 Receptor
Dean R. Artis,*,y Christine Brotherton-Pleiss, Joseph H. B. Pease, Clara J. Lin,
Steve W. Ferla, Sherry R. Newman, Sunil Bhakta, Helene Ostrelich and Kurt Jarnagin{
Roche Bioscience, 3401 Hillview Avenue, Palo Alto, CA 94303, USA
Received 12 June 2000; accepted 15 August 2000
AbstractÐSix classes of nonpeptide bradykinin antagonists were designed using a template derived from structural studies of peptide
antagonists. Several compounds from each class were synthesized and assayed for binding to the human bradykinin B2 receptor. Each
family showed compounds active at the level of the smallest template peptide; three classes contained compounds with Kd<8mM. These
results provide diverse leads for a medicinal chemistry-based optimization program. # 2000 Elsevier Science Ltd. All rights reserved.
The nonapeptide hormone bradykinin (Arg-Pro-Pro-Gly-
Phe-Ser-Pro-Phe-Arg) acts as a mediator of in¯ammation
under conditions of tissue injury or trauma.1 Bradykinin is
produced by the cleavage of kininogens by certain kallik-
reins, and are potent e€ectors at a family of G-protein
coupled 7-transmembrane receptors. At the time this
work was begun, a large body of data concerning the
activities of peptide agonists and antagonists was avail-
able, but nonpeptide antagonists, of interest as novel anti-
in¯ammatory therapeutics, had not been reported.2 Sub-
sequent to the completion of this work, advances in the
pursuit of such compounds have recently been realized.3 5
receptor in a fashion distinct from bradykinin.10 In
order to develop the structural template from which to
design potential synthetic targets, we decided to center
our studies on three classes of peptide antagonists. As
representatives, we chose the linear compounds repre-
sented by HOE-140, and two cyclic peptide antagonists
containing a hexa- (I) and tetrapeptide (II) core.11
Compounds I and II bind with Kds in the 0.2±16 mM
range, and contain many of the features of HOE-140 in
a more constrained framework. The SAR of these cyclic
peptides had revealed a pharmacophore that could
contain ®ve features around the backbone core (labeled
at right). These included the hydrophobic groups at (1)
the dTic residue and (2) the Oic residue following 1; (3)
a positively charged residue immediately following 2 (4)
an aromatic residue proceeding 1 and (5) a second
positive charge, which could be displayed from di€erent
portions of the sca€old.
The structure-based design of bioactive small molecules
from peptides has proved to be a powerful technique in
new lead discovery.6 The derivation of small molecule
IIbIIIa antagonists from a structured peptide template
core has highlighted the utility of peptide structure±
function data in peptidomimetic design.7 The body of
available biological data for bradykinin-related peptides
suggested that, given an appropriate structural tem-
plate, such a strategy might well prove successful in the
creation of small nonpeptide bradykinin antagonists.
Mutagenesis data on the bradykinin B2 receptor had
indicated that peptide antagonists, such as HOE-1408,9
(d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-dTic-Oic-Arg), bind the
*Corresponding author. Tel.: +1-650-225-8085; fax: +1-650-225-
2061; e-mail: dra@gene.com
yPresent address: Genentech Inc., 1 DNA Way, South San Francisco,
CA 94080, USA.
{Present address: Iconix Pharmaceuticals Inc., 850 Maude Avenue,
Mountain View, CA 94043, USA.
We ®rst chose to examine the structure of HOE-140
using NMR spectroscopy in water, DMSO and in SDS
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00482-0

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D. R. Artis et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2421±2425
studies provided several di€erent models of the `core'
structural unit of the peptide inhibitors. This structural
template provided a fairly high degree of con®dence in
the dTic-Oic-Arg or dTic-Oic-Dap regions, and sig-
ni®cantly higher variability in most other structural
aspects of the pharmacophore.
Table 1. CAVEAT-derived bradykinin antagonists
R1
R2
Kd
(mM)ÆS.E.
(1)
(2)
Ph
Ph
H
Inactive
Inactive
66Æ4.2
C(ˆNH)NH2
H
Our design strategy focused on replacing the structurally
well-de®ned `core' of our cyclic peptides with a new non-
peptide sca€old, preserving a degree of ¯exibility in the
areas of our molecule where we had little structural
information. The variable precision in our template
made it well suited for use with the program CAVEAT.13
This program may be used to search molecular databases
for compounds that can accommodate the geometrical
relationship between speci®c bonds in a template mole-
cule. In this case, the search templates were derived
from minimized structures extracted from low energy
regions of the molecular dynamics trajectory for each
peptide. These structures were used to de®ne a set of
bond vectors which were used to carry out a CAVEAT
search of the Cambridge Structural Database.14 We
ultimately utilized the bond vectors from the amide
nitrogen to the Ce or Cd for the dTic and Oic residues,
and from the Ca±Cb bond for the following residue in
the various peptides, leaving the incorporation of the
functionality of features 4 and 5 (see above) to the post-
search design process. This choice of vectors dramati-
cally enhanced the diversity of the compound classes
returned from the searches and avoided most of the less
desirable peptide and steroid systems normally found
using an all Ca±Cb vector search. Families of com-
pounds were extracted using the class module of the
CAVEAT suite, and examined for potential suitability
as organic linkers.
(3) b-Naphthyl
(4) b-Naphthyl
C(ˆNH)NH2
15Æ0.79
(5)
H
13Æ1.4
14Æ.63
(6) (CH2)3NH2
(7) C(ˆNH)NH2
CH2NHR1
CH2NH2
12Æ.37
4.3Æ.32
15Æ1.3
(8)
(9)
H
H
CH2CH2NH2
(10) C(ˆNH)NH2 CH2CH2NHR1 4.5Æ.41
micelles.10 These studies led to the conclusion that the
major structural motif of HOE-140 consisted of a type
II b-turn at residues 2±5 and a type II0 b-turn at residues
6±9, with a highly ¯exible `hinge' region separating the
turns. Cyclic peptides I and II were also examined in
DMSO solution via NMR, and found to retain a turn
centered on the dTic-Oic substructure identical to that
observed for HOE-140. The structures of HOE-140 and
peptides I and II were next subjected to runs of 50±100 ps
of unconstrained room temperature in vacuo dynam-
ics,12 in order to explore the areas of uncertainty in the
template. Two di€erent starting positions were used for
HOE-140, both with the experimentally observed b-
turns, but with di€erent extended structures in the hinge
regions. Although collapsed structures were often
observed, the type II0 b-turn centered on the dTic-Oic
pair persisted throughout the simulations, consistent
with the NMR data obtained for these molecules. These
Figure 1. Overlap of CAVEAT-derived compound 3 with template peptide I (relaxed stereoview).

D. R. Artis et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2421±2425
2423
Table 2. Model-derived bradykinin antagonists
Systems composed of a core of two or three rings, or
which could be simpli®ed down to one or two rings with-
out leaving the necessary vectors in a high-energy con-
formational state, were selected. These structures were
then simpli®ed by replacing saturated ring systems with
aromatic systems to eliminate unnecessary stereocenters,
especially when diequatorial substitution was present and
needed to be retained. Hybridization and conjugation
were also modi®ed to a€ect the in-plane/out-of-plane
conformation of particular functional groups. The initial
targets were then examined for ease of synthesis, chan-
ges in the structures were made as required, and the
compounds reanalyzed structurally to insure that rea-
sonable overlap with the template was maintained. Based
on these e€orts, three series of molecules were selected
for synthesis. The resulting compounds are shown in
Table 1.
R1
R2
Kd(mM)
ÆS.E
(11)
(12)
(13)
(14)
(CH2)4NH2 Inactive
(CH2)4NH2 28Æ2.6
CH2NH2
CH2NH2
101Æ12
17Æ2.7
(15)
(16)
(CH2)4NH2
(CH2)4NBu2
62Æ2.6
23Æ3.1
While these design e€orts were fundamentally structure-
based, the emphasis was placed on maximizing the diver-
sity and synthetic accessibility of the di€erent series of
molecules, rather than on displaying all of the template
features in any one compound. For instance, in Figure 1
the functionality associated with feature 1 of the phar-
macophore was omitted to facilitate a simple synthesis
of a compound class based on hydroxyproline, while
retaining substitution for features 2 and 3, and adding
the functionality of feature 4.
(17)
(18)
7.4Æ3.1
5.0Æ.29
(19)
(20)
Bn
Bn
3-CH2NH2 7.6Æ.3
2-CO-
Ornithine
Inactive
Nonpeptidic compounds were also designed from the
peptide templates based on a direct graphical analysis.
Novel organic linkers were created, each replacing a
small region of the peptide structure, in a fashion to
allow the display of some of the desired functional
groups. The iterative process of modifying the initial
design was similar to that employed with the
CAVEAT-derived sca€olds. Three series of com-
pounds were selected for synthesis, the results of
which are shown in Table 2. The synthetic approaches
to the molecules in Tables 1 and 2 are shown in
Scheme 1, save for the benzodiazepine class of com-
pounds. The SAR for these compounds was examined
in greater detail, and these results will be discussed in
a subsequent manuscript.15
Compounds were assayed for binding against the
human bradykinin B2 receptor expressed in CHO
cells.10 Each family yielded compounds with activity
close or comparable to the smallest peptide lead. In
addition, many families were found to contain both
Figure 2. Overlap of compound 17 with template peptide I (relaxed stereoview).

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D. R. Artis et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2421±2425
active and inactive members, suggesting the presence of
a nascent SAR. The general synthetic strategy utilized in
this project allowed for the short synthesis of multiple
members of each family, and has provided the basis for
future synthetic e€orts. Both design approaches
employed in the study appeared to be equally successful,
and the six families of antagonists together form a
highly diverse set of compounds.
The design, synthesis, and assay phases of this project
were completed with a modest allocation of resources
over approximately nine months. These e€orts yielded a
variety of active compound classes with well-elaborated
synthetic approaches. The results described herein
highlight the bene®ts of a structure-based approach to
new lead discovery of peptidomimetics that can
complement existing screening techniques.
Scheme 1. Synthesis of bradykinin antagonists. I Reagents: (a) (i) NaH, (ii) benzylbromide, (iii) SOCl2, MeOH; (b) RCOCl, DCM/H2O; (c) LiBH4,
cat. B-OMe-9-BBN; (d) (i) Ph3P, DEAD, t-BuO2CNHP(OEt)2, (ii) HCl, toluene; (e) H2NC(ˆNH)SO3H. II Reagents: (a) (i) 4-nonanone, TsOH, (ii)
KMnO4, pyridine, H2O; (b) (PhO)2P(ˆO)N3, PhCH2OH; (c) H2, Pd/C; (d) (i) 4-(chloromethyl)benzoyl chloride, (ii) KNPht, DMF; (e) (i) NaH, (ii)
PhtN(CH2)3Br; (f) NH2NH2 H2O, MeOH. III Reagents: (a) D-N-b-Cbz-diaminopropionic acid, HOAc, NaOAc, 100 ꢀC; (b) (i) CDI, (ii) a,a0-di-
.
amino-p-xylene, (iii) H2, Pd/C. IV Reagents: (a) D-N-e-Cbz-lysine, HOAc, NaOAc, 100 ꢀC; (b) (i) CDI, (ii) a,a0-diamino-p-xylene; (c) (i) Cbz-Cl, (ii)
COCl2, toluene; (d) (i) PhCH2CH(R)NH2, DCC, (ii) H2, Pd/C. V Reagents: (a) (i) BuBr, Mg, ether, (ii) H+, (iii) NH2NH2, KOH, (iv) NaNH2,
cumene, 150 ꢀC, (v) Br2, 48% HBr, NaNO2, (vi) NaOH; (b) (i) n-BuLi, 78 ꢀC, (ii) C6H11CH2CON(OCH3)CH3, (iii) (R)-( )-2-phenylglycinol, (iv) H2,
Pd/C, (v) NaIO4, H2O, THF; (c) (i) BocNH(CH2)4CO2H, DCC, (ii) TFA; (d) (i) BocNHCH2(C6H4)CO2H, DCC, (ii) TFA; (d) n-PrCHO, NaCNBH3;
.
(e) NH2C(ˆNH)SO3H. VI Reagents: (a) (i) KH, (ii) PhCH2I; (b) (i) NaH, (ii) 3-(phthalimidomethyl)benzylbromide, (iii) NH2NH2 H2O, MeOH; (c) (i)
NaH, (ii) 2-(carbomethoxy)benzyl bromide, (iii) NaOH, (iv) N-@-Boc-l-ornithine methyl ester, Pybop, (v) TFA, (vi) LiOH.

D. R. Artis et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2421±2425
2425
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15. Benzodiazepine-based bradykinin antagonists with quite
di€erent substitution patterns have recently also been dis-
closed, see ref 5.