Discovery of potent nonpeptide vitronectin receptor (α(v)β3) antagonists

Full text of DOI:10.1021/jm970205r

© Copyright 1997 by the American Chemical Society
Volume 40, Number 15
J uly 18, 1997
Communications to the Editor
Ta ble 1. Binding Activity of Constrained RGD Analogs
Discover y of P oten t Non p ep tid e
Vitr on ectin Recep tor (rVâ3) An ta gon ists
Richard M. Keenan,*,† William H. Miller,*,†
Chet Kwon,*,† Fadia E. Ali,† J ames F. Callahan,†
Raul R. Calvo,† Shing-Mei Hwang,‡
Kenneth D. Kopple,§ Catherine E. Peishoff,§
J ames M. Samanen,† Angela S. Wong,‡
Chuan-Kui Yuan,† and William F. Huffman†
Departments of Medicinal Chemistry, Cellular Biochemistry,
and Physical & Structural Chemistry, Research &
Development Division, SmithKline Beecham
Pharmaceuticals, 709 Swedeland Road, P. O. Box 1539,
King of Prussia, Pennsylvania 19406-0939
Received March 26, 1997
The vitronectin receptor (RVâ3) is a member of the
integrin family of transmembrane heterodimeric glyco-
protein complexes, which function in cell-cell and cell-
substrate adhesion and communication.1 The integrin
RVâ3 is expressed on the surface of a variety of cell types,
including osteoclasts, vascular smooth muscle cells,
endothelial cells, and tumor cells, and has been shown
to mediate several biologically relevant processes, in-
cluding adhesion of osteoclasts to the bone matrix,2
vascular smooth muscle cell migration,3 and angiogen-
esis.4 As a result, RVâ3 antagonists are expected to have
utility in the treatment of osteoporosis, restenosis
following balloon angioplasty, and diseases involving
neovascularization, such as macular degeneration,5
diabetic retinopathy,5 and cancer.4b,6
we designed small-molecule RGD mimetics based on the
1,4-benzodiazepine ring system which provided highly
potent and selective RIIbâ3 antagonists.11-14 In the
present communication, we describe the application of
a similar strategy to the identification of a series of
potent and selective nonpeptide RVâ3 antagonists.
Our initial studies on RVâ3 antagonists focused on
constrained RGD analogs, and identified several key
compounds (Table 1). We found that the cyclic pen-
tapeptide cyclo(RGDfV) (2), as reported by Kessler and
co-workers,15 had high affinity for RVâ3 and showed
substantial selectivity for RVâ3 relative to RIIbâ3.16,17 The
reported conformation of the RGD sequence in 2, based
on 1H NMR studies, invoked a turn in the Gly region.
The cyclic hexapeptide cyclo(RGDSPG)18 (3) showed
similar affinity and selectivity and, by analogy to other
cyclic hexapeptides,8 could be expected to also adopt a
conformation of the RGD sequence containing a turn
in the Gly region. Since our previous studies had
indicated that an extended conformation about Gly is
optimal for RIIbâ3, the results suggested that selectivity
for RVâ3 may be related to a conformation that contains
a turn about Gly. Figure 1 depicts an overlay compari-
son of the cyclic peptide 1, in which the RGD sequence
The vitronectin receptor (RVâ3) is related to the
platelet fibrinogen receptor (RIIbâ3) in that both integrins
possess the â3 subunit. Furthermore, both RVâ3 and
RIIbâ3 are known to recognize the Arg-Gly-Asp (RGD)
tripeptide sequence.1 A number of groups have reported
the discovery of potent nonpeptide RIIbâ3 antagonists
based on mimetics of the RGD sequence.7 In our
previous investigations into RGD peptidomimetics, a
detailed evaluation of cyclic RGD-containing peptides
revealed that, in addition to the key Asp and Arg
residues, a turn-extended-turn conformation for the
RGD sequence was crucial for potent RIIbâ3 peptide
antagonists.8-10 Guided by these cyclic peptide studies,
S0022-2623(97)00205-7 CCC: $14.00 © 1997 American Chemical Society

2290 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
Communications to the Editor
F igu r e 1. Overlay of cyclic peptides 1 (cyan) and 2 (magenta)
revealing the difference in the distance between the Arg and
Asp centers.
F igu r e 2. Overlay of the benzodiazepine nucleus (green) on
the cyclic peptide 2 (magenta) indicating the 7-position as the
optimal point of attachment for arginine mimetics.
is in the turn-extended-turn conformation with the
arginine side chain extended such that the guanidine
group lines up with the cationic residues present in
potent nonpeptide RIIbâ3 antagonists,14 and the cyclic
pentapeptide 2 in the conformation containing a turn
Ta ble 2. Binding Activity of Small Molecule RVâ3 Antagonist
Leads
1
in the Gly region derived from H NMR studies. This
comparison suggested that an important manifestation
of the turn about Gly present in 2 is that the overall
length for preferred binding to RVâ3 is shorter than for
R
IIbâ3 (Figure 1).19
Analysis of semipeptide 4,9 which contains a γ-turn
mimetic about Asp, provided additional insight into
structural features which might be tolerated by RVâ3.
This compound has comparable affinity for both RVâ3
and RIIbâ3, suggesting that the γ-turn about Asp imposed
by the seven-membered ring of 4 may be accommodated
by RVâ3 as well as by RIIbâ3. Taken together, these data
suggested that a conformation of the RGD sequence
containing turns about both Gly and Asp, resulting in
a reduced overall separation between the Arg and Asp
centers, may be favorable for binding to RVâ3.
Since a γ-turn about Asp appeared to be tolerated by
RVâ3, we felt that appropriate functionalization of our
1,4-benzodiazepine system, in which the seven-mem-
bered diazepine ring is a γ-turn mimetic, might afford
potent and selective nonpeptide RVâ3 antagonists. Com-
parison of our 1,4-benzodiazepine nucleus with cyclic
peptide 2 (Figure 2) suggested that attachment of an
arginine mimetic to the 7-position of the 1,4-benzodi-
azepine nucleus would be optimal to mimic the turn
about Gly. To quickly test this hypothesis, we screened
our collection of 7-substituted 1,4-benzodiazepine-based
RGD mimetics for binding to RVâ3 and identified two
important leads 5 and 7 (Table 2).
The m-benzamidine derivative 520 had submicromolar
affinity for RVâ3 and low micromolar affinity for RIIbâ3.
The corresponding p-benzamidine 6, however, was a
potent RIIbâ3 antagonist8 and had much lower affinity
for RVâ3. Since compound 5 is shorter than compound
6 in the distance from the carboxyl terminus to the
amidine terminus, these results appeared to support our
modeling hypothesis. The arylpiperazine derivative 7,
although clearly a potent and selective RIIbâ3 antagonist,
also showed low micromolar affinity for RVâ3. We
speculated, based on our modeling hypothesis, that the
central anilino nitrogen was responsible for the RVâ3
activity, and the distal pyridyl nitrogen was responsible
for the RIIbâ3 activity. To test this hypothesis, we
prepared derivative 8, which lacks the pyridyl nitrogen.
Compound 8 had RVâ3 affinity comparable to that of 7
but had drastically reduced affinity for RIIbâ3, providing
additional evidence that the length required for optimal
binding to RVâ3 is shorter than for RIIbâ3. These data
also demonstrated that the 1,4-benzodiazepine system
could be employed to generate nonpeptides selective for
the vitronectin receptor.
Using the information gleaned from these preliminary
studies, we evaluated a wide range of potential Arg
mimetics in our 7-substituted 1,4-benzodiazepine series
and identified the benzimidazole-containing derivative
9 as a potent and selective RVâ3 antagonist (Table 3).
In this compound, the benzimidazole unit presumably
functions as an Arg mimetic, although the exact nature
of the interactions between the benzimidazole moiety
and RVâ3 are not entirely clear at this time. Analogs of
9 which were one methylene unit longer (10) or one
methylene unit shorter (11) were much less active,
confirming that the overall length in 9 was optimized
for interaction with RVâ3. Subsequently, we evaluated
the resolved enantiomers of 9 and found the activity to
reside almost exclusively in the (S)-enantiomer (12), the
same as the natural configuration of Asp, supporting

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2291
J . Bone Mineral Res. 1994, 9, 1021-1028. (b) Helfrich, M. H.;
Nesbitt, S. A.; Dorey, E. L.; Horton, M. A. Rat Osteoclasts Adhere
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Ta ble 3. Binding Activity of Benzimidazole-Containing RVâ3
Antagonists
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the hypothesis that the 1,4-benzodiazepine acts as a
Gly-Asp mimic. In our work on both peptide and
nonpeptide RIIbâ3 antagonists, we had found that amide
N-methylation resulted in an increase in activity.12
Application of the same tactic in the present investiga-
tion afforded SB 223245 (14),20 which had 2 nM affinity
for RVâ3 and 30 µM affinity for RIIbâ3sgreater than
10000-fold selectivity.
The discovery of 14 demonstrated that highly potent
and selective nonpeptide RVâ3 antagonists could be
designed by consideration of the conformations of con-
strained RGD peptides and peptidomimetics in an
approach similar to that used in the identification of
highly potent and selective nonpeptide RIIbâ3 antago-
nists. Further evaluation in functional assays has
revealed that 14 is a potent inhibitor of both human
osteoclast-mediated bone resorption21 and vitronectin-
induced haptotaxis of human endothelial cells,22 show-
ing the potential therapeutic utility of compounds in this
series. This work has also revealed the remarkable
versatility of the benzodiazepine nucleus as a Gly-Asp
mimetic, as potent nonpeptide antagonists from the
benzodiazepine series can be designed with selectivity
14
for either RIIbâ3 or RVâ3 simply by altering the length
and nature of the Arg mimetic.
Su p p or tin g In for m a tion Ava ila ble: Characterization
data (1H NMR, MS, and elemental analyses) for compounds
5-14 (10 pages). Ordering information is given on any current
masthead page.
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Synthetic Arg-Gly-Asp Peptides During Osteoclast Formation.

2292 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
Communications to the Editor
(14) Samanen, J . M.; Ali, F. E.; Barton, L. S.; Bondinell, W. E.;
Burgess, J . L.; Callahan, J . F.; Calvo, R. R.; Chen, W.; Chen, L.;
Erhard, K.; Feuerstein, G.; Heys, R.; Hwang, S.-M.; J akas, D.
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P.; Miller, W. H.; Newlander, K. A.; Nichols, A.; Parker, M.;
Peishoff, C. E.; Rhodes, G.; Ross, S.; Shu, A.; Simpson, R.;
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(20) Compounds 5-14 were synthesized by coupling of the appropri-
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synthesis of 14 is shown as an example:
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(16) Binding affinity for human RVâ3 was determined in a competitive
binding assay employing [3H]SK&F 107260 as the displaced
ligand. The Ki values represent the means of values deter-
mined in two to three separate experiments. See: Wong,
A.; Hwang, S. M.; McDevitt, P.; McNulty, D.; Stadel, J . M.;
J ohanson, K. Studies on RVâ3/Ligand Interactions Using a [3H]-
SK&F-107260 Binding Assay. Mol. Pharmacol. 1996, 50, 529-
537.
(17) Binding affinity for human RIIbâ3 was determined in a competi-
tive binding assay employing [3H]SK&F 107260 as the displaced
ligand. The Ki values represent the means of values determined
in two to three separate experiments (see ref 10).
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(19) For related studies on cyclic RGD peptides which come to a
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B.; Gurrath, M.; Mu¨ller, G.; Kessler, H.; Timpl, R.; Engel,
J . Selective Recognition of Cyclic RGD peptides of NMR
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Chem. 1994, 269, 20233-20238. (b) Bach, A. C.; Espina, J . R.;
J ackson, S. A.; Stouten, P. F. W.; Duke, J . L.; Mousa, S. A.;
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W. H.; Ku, T. W.; Ali, F. E.; Bondinell, W. E.; Calvo, R. R.; Davis,
L. D.; Erhard, K. F.; Hall, L. B.; Huffman, W. F.; Keenan, R.
M.; Kwon, C.; Newlander, K. A.; Ross, S. T.; Samanen, J . M.;
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214857, a Potent, Orally Active, Nonpeptide Fibrinogen Receptor
Antagonist. Tetrahedron Lett. 1995, 36, 9433-9436. The TFA
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(23) A preliminary account of this work was presented at the 211th
American Chemical Society National Meeting, New Orleans, LA,
March 24-28, 1996.
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