Biphenyls as potent vitronectin receptor antagonists

Article abstract of DOI:10.1016/S0960-894X(01)00717-X

Vitronectin receptor (α_Vβ₃) antagonism has been implicated as a mechanism for the treatment of restenosis following balloon angioplasty. In this work we present results from screening of a focused combinatorial library based on a biphenyl moiety. Our SAR studies led to the identification of compounds with subnanomolar activity, selectivity towards the related GPIIbIIIa receptor and functional activity on human smooth muscle cell migration.

Full text of DOI:10.1016/S0960-894X(01)00717-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 205–208
Biphenyls as Potent Vitronectin Receptor Antagonists
Klaus Urbahns,a,* Michael Harter,a Markus Albers,b Delf Schmidt,c
Beatrix Stelte-Ludwig,d Ulf Bruggemeier,c Andrea Vaupela and Christoph Gerdesd
aInstitute of Medicinal Chemistry, Pharma Research Centre, Bayer AG, D-42096Wuppertal, FRG
bCombinatorial Chemistry Group, Central Research, Bayer AG, D-51368 Leverkusen, FRG
cInstitute of Molecular Screening Technologies, Pharma Research Centre, Bayer AG, D-42096Wuppertal, FRG
dInstitute of Cardiovascular Research, Pharma Research Centre, Bayer AG, D-42096Wuppertal, FRG
Received 12 July 2001; revised 17 September 2001; accepted 22 October 2001
Abstract—Vitronectin receptor (aVb3) antagonism has been implicated as a mechanism for the treatment of restenosis following
balloon angioplasty. In this work we present results from screening of a focused combinatorial library based on a biphenyl moiety.
Our SAR studies led to the identification of compounds with subnanomolar activity, selectivity towards the related GPIIbIIIa
receptor and functional activity on human smooth muscle cell migration. # 2002 Elsevier Science Ltd. All rights reserved.
Coronary stentingand balloon angioplasty are the most
frequently used percutaneous revascularisation proce-
dures for the treatment of coronary heart disease and
myocardial infarction. Despite a high initial success rate
in restoringblood flow, percutaneous interventions are
plagued by arterial reocclusion within 6 months after
surgery, a clinical problem also referred to as restenosis.1
A critical element in the design of RGD mimetics is the
correct spatial presentation of arginine and aspartic acid
residues around a central core. The discovery of c-
RGDfV, a cyclic peptide with known 3-D structure
bodes well for the use of beta-turn mimetic templates in
the development of vitronectin receptor antagonists.4
Whereas biphenyls have been utilised as beta-turn
mimetics at several occasions,5 a recent molecular
dynamics simulation predicted a relatively poor beta-
turn stabilisation/imitation potential for biphenyl struc-
tures.6 Furthermore, a recent report of a combinatorial
biphenyl library screened against the vitronectin recep-
tor, furnished only disappointingresults with activities
in the micromolar range (Fig. 1).7
The vitronectin receptor aVb3 is a member of the inte-
grin superfamily of cellular adhesion receptors.2 Vitro-
nectin receptor-mediated migration of coronary smooth
muscle cells (SMC’s) into the neointima is regarded as a
key event of restenosis in both stented and non-stented
arteries. Specific antagonists of the related platelet
receptor GPIIbIIIa to date have failed to show anti-
restenotic properties. However, the longterm mortality
benefits observed after administration of a mixed aVb3/
GPIIbIIIa antibody support the relevance of vitronectin
receptor-mediated SMC migration inhibition for the
treatment of restenosis.3
Like many integrins, aVb3 is known to recognise a con-
tinous tripeptide epitope, the arginine-glycine-aspartic
acid (RGD) motif. We therefore investigated small
molecule peptidomimetics as aVb3 antagonists.
Figure 1. Design of biphenyl-based vitronectin receptor antagonists
from Kessler’s peptide c(RGDfV).
In this communication, we wish to describe a new
biphenyl series of small molecule RGD mimetics with
potent vitronectin receptor antagonism in the nano-
molar range.
*Correspondingauthor at present address: Bayer Yakuhin, Ltd,
Research Centre Kyoto, Department of Chemistry, 6-5-1-3 Kunimi-
dai, Kizu-cho, Soraku-gun, Kyoto 619-0126, Japan. Tel.: +81-774-75-
2480; fax: +81-774-75-2511; e-mail: klaus.urbahns.ku1@bayer.co.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00717-X

206
K. Urbahns et al. / Bioorg. Med. Chem. Lett. 12 (2002) 205–208
Chemistry8
in a migration assay with human coronary SMCs on
vitronectin-coated plates usinga horizontal measuring
arrangement. Half of a confluent SMC layer was
scraped off. After activation with PDGF-BB (1 nM)
migration from the leading edge of the scratch was
quantified, measuringmigration distance and cell den-
sity of migrated cells after 24 h incubation.10
For lead structure identification, we decided to synthe-
sise a targeted combinatorial biphenyl library on solid
support (Scheme 1). The acidic group is ideally suited
for traceless resin attachment and functions well as a
glutamate mimetic. Diversity has been obtained by using
a set of immobilised amino acids, synthesising3,3-, 3,4-,
4,3- and 4,4-biphenyls and exploitingthe penultimate
aniline for reductive aminations, amide couplings and
urea formations. This strategy gave rise to 3500 single
compounds, that were screened against the vitronectin
receptor.
Results
The vast majority of the biphenyl library members
turned out to be inactive, emphasisingthe hihgly
Whereas compounds 1–21, 24 and 27–32 were synthe-
sised on solid support, the residingderivatives were
obtained from liquid-phase chemistry, startingwith
amino acid esters. Final deprotection was achieved by
LiOH, in water/dimethoxyethane. Enantiomers were
synthesised separately usingchiral buildingblocks with
known configuration. Materials 20, 21 and 24 were
obtained using3-formyl-phenyl boronic acid under the
Suzuki-couplingconditions of Scheme 1. Compounds
22 and 23 were synthesised by heterocycle formation
startingfrom the correspondingthioureas II and IV.
HgO-mediated ring closure of II furnished benzimid-
azole III, whereas the aminohistidine VIIa was prepared
by cyclisingenamine formation of the corresponding
diethyl acetal precursor VI. The alternative aminohisti-
dine VIIb was obtained as a side product (Scheme 2).
Materials 25 and 26 were synthesised usingthe corre-
spondingpyrazine-5,10-diones as acylatingaengts
(THF, DMF, 4 equiv pyridine).
Biology
All compounds were evaluated for their in vitro activity
in an aVb3 scintillation bead assay using 125I labelled
echistatin as a radioligand. A GPIIbIIIa counterscreen
was performed similarly.9 Functional activity was tested
Scheme 2. Synthesis of heterocyclic biphenyl vitronectin receptor
antagonists: (a) (1) thiophosgene, toluene, reflux, 2 h; (2) o-phenyl-
endiamine, THF/toluene (1:1) 12 h rt 100%; (b) yellow HgO, CHCl3,
6 h, reflux, 68%; (c) (1) thiophosgene, toluene reflux, 1.5 h; (2) NH3,
THF/toluene (1:1), rt, 30 min, 92%; (d) iodomethane, MeOH, reflux
2 h, 98%; (e) aminoacetaldehyde–diethylacetal, n-propanol, reflux 6 h,
88%; (f) 6 N aq HCl, 40% (VIIa), 10% (VIIb).
Table 1. Structure–activity relationship of biphenyl vitronectin
receptor antagonistsa
Compd
Configuration
R1
R2
Ki (nM)
1
2
3
4
5
6
7
8
S
R
Me
Me
H
CH2Ph
CH2Ph
H
H
H
H
H
H
OMe
OMe
2000
>10,000
>10,000
30
>10,000
1500
1100
S
R
S
S
S
Scheme 1. Combinatorial synthesis of a biphenyl vitronectin receptor
antagonist library: (a) pyridine/DMF; (b) (Pd(PPh3)2Cl2, DMF/H2O,
K2CO3), 85 ꢀC; (c) R-CHO, HC(OMe)3, Bu4NBH4; (d) DIC, DMF;
(e) DIPEA, 4-nitrophenylchloroformate, THF/DCM (1:1), then: R-
NH2, DIPEA, DMF; (f) TFA/DCM (1:1); (*=Wangresin).
CH2-p-C6H4NO2
CH2-p-C6H4NH2
CH2Ph
13
aKi values are medians of three dose–response curves.

K. Urbahns et al. / Bioorg. Med. Chem. Lett. 12 (2002) 205–208
207
defined structural prerequisites of the aVb3/ligand inter-
action. However, a small set of the urea sublibrary
(Scheme 1, condition e) tended to demonstrate both,
useful bindingaffinities and first SAR trends. This class
was therefore selected for further investigations.
related 3-picolyl derivative 18 turned out to be equally
potent as 8, the most effective derivative of the alpha-
phenylalanine series.
We also examined whether omission of the urea frag-
ment would still be compatible with vitronectin receptor
affinity (Table 3). Indeed, the benzimidazole compounds
20–22 showed bindingaffinities in the submicromolar
range. In particular, aminobenzimidazole 22 had a 4 nM
affinity to aVb3. The correspondingaminoimidazole
The first active compound identified from our library
was the alanine derivative 1, displayingan encouraging
Ki of 2000 nM to the vitronectin receptor (Table 1).
The activity resided exclusively on the S-enantiomer;
the R-enantiomer 2 was practically inactive. The novel
benzimidazolyl urea motif has to our knowledge not
been described as an arginine mimetic hitherto. The lack
of basicity of this novel guanidine mimetic is attractive
for the development of vitronectin receptor antagonists
with druglike physicochemical properties.
Table 3. Structure–activity relationship of biphenyl vitronectin
receptor antagonistsa
Substituents in close vicinity to the carboxylic group of
the RGD motif have been reported to afford improved
bindingaffinities to the related GPIIbIIIa receptor.
Similar observations have been made for aVb3.11 As the
Compound
R1
R2
H
Ki (nM)
20
90
correspondinglgycine derivative
3 was inactive, we
speculated, that further substitutions of the methyl
group could result in similar activity enhancements in
our series.
21
22
23
24
25
26
27
OMe
H
60
4
Indeed, the S-phenylalanin derivative 4 displayed a 100-
fold improvement in bindingaffinity. Additional meth-
oxy substitution on the biphenyl nucleus further
enhanced activity, furnishing 8. Interestingly, further
enlargement of the phenyl ring by para-substitutions led
to decreased activity (6,7).
H
180
360
350
300
280
OMe
OMe
OMe
OMe
Whereas alpha-phenylalanine derivatives showed activ-
ity exclusively in combination with benzimidazolyl
ureas, the SAR of beta-phenylalanine derived com-
pounds was more tolerant towards urea substitutions
(Table 2). The unsubstituted urea (9) was itself inactive,
but even small aliphatic substituents improved the
activity to submicromolar levels. Methoxy substitution
of benzyl derivative (12) gave rise to a 10-fold
improvement of bindingaffinity ( 15). The closely
Table 2. Structure–activity relationship of biphenyl vitronectin
receptor antagonistsa
aThe beta-amino acids were racemic. Ki values are medians of three
dose–response curves.
Table 4. Structure–activity relationship of biphenyl vitronectin
receptor antagonistsa
Compd
R1
R2
Ki (nM)
9
H
Me
c-Prop
CH2Ph
n-Prop
i-Bu
CH2–Ph
3-Pyridyl
2-Picolyl
3-Picolyl
4-Picolyl
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
>10,000
2000
700
220
100
90
10
11
12
13
14
15
16
17
18
19
Compd
Z
R2
Config.
Ki (nM)
20
60
200
14
28
29
30
31
32
NH
NH
NH
NH
S
H
R/S
R/S
R
S
R/S
4
1.4
0.7
>2000
>2000
OMe
OMe
OMe
OMe
25
aThe beta-amino acids were racemic. Ki values are medians of three
dose–response curves.
aKi values are medians of three dose–response curves.

208
K. Urbahns et al. / Bioorg. Med. Chem. Lett. 12 (2002) 205–208
showed a 45-fold drop in activity, suggesting 22’s addi-
tional benzene ringto interact favourably with aVb3. This
observation is consistent with the SAR for benzodiaze-
pine-based vitronectin receptor antagonists.12 A direct
comparison of 22 and 28 (Table 4) indicates that the
urea moiety in 28 can be omitted without loss of act-
ivity. As observed in the alpha-phenylalanine series,
methoxy substitution confers enhanced activity result-
ingin 29, which was chosen for further biological
characterisation.
3. (a) Brown, S. L.; Lundgren, C. H.; Nordt, T.; Fujii, S.
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Albers, M.; Vaupel, A.; Schmidt, D.; Stelte-Ludwig, B.;
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Replacingthe benzimidazole moiety of 29 by a benzo-
thiophene resulted in total loss of activity (32), again
confirming the importance of NH groups as arginine
mimetics. The racemate 29 showed a eudismic ratio of
at least 2000, the R-enantiomer 30 representingthe
active configuration. Compound 30 showed a >300
fold selectivity to the related platelet GPIIbIIIa receptor
(Ki=300 nM). When it was applied to SMC’s freshly
isolated from human aorta, 30 inhibited PDGF-induced
migration with an IC50 of 30 nM. The drop in activity
by two orders of magnitude upon moving from binding
affinity determination to functional SMC migration
inhibition in a cellular context has also been observed in
other series.12
In summary, we have discovered a potent series of
vitronectin receptor antagonists based on a biphenyl
motif. The compounds exhibit clear SAR, which led to
the identification of 30 with selectivity to GPIIbIIIa and
functional activity on SMC migration.
12. (a) Benzodiazepine-based aVb3 inhibitors: Keenan, R. M.;
Miller, W. H.; Lago, M. A.; Ali, F. E.; Bondinell, W. E.;
Callahan, J. F.; Calvo, R. R.; Cousins, R. D.; Hwang, S. M.;
Jakas, D. R.; Ku, T. W.; Kwon, C.; Nguyen, T. T.; Reader,
V. A.; Rieman, D. J.; Ross, S. T.; Takata, D. T.; Uzinskas,
I. N.; Yuan, C. C. K.; Smith, B. R. Bioorg. Med. Chem. Lett.
1998, 8, 3165. (b) Keenan, R. M.; Lago, M. A.; Miller, W. H.;
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D. R.; Kwon, C.; Louden, C.; Nguyen, T. T.; Ohlstein, E. H.;
Rieman, D. J.; Ross, S. T.; Samanen, J. M.; Smith, B. R.;
Stadel, J.; Takata, D. T.; Vickery, L.; Yuan, C. C. K.; Yue,
T. L. Bioorg. Med. Chem. Lett. 1998, 8, 3171.
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