Solid-phase synthesis of a nonpeptide RGD mimetic library: New selective αvβ3 integrin antagonists

Article abstract of DOI:10.1021/jm0004953

The solid-phase synthesis of a low molecular weight RGD mimetic library is described. Activities of the compounds in inhibiting the interaction of ligands, vitronectin and fibrinogen, with isolated immobilized integrins αvβ3 and αIIbβ3 were determined in a screening assay. Highly active and selective nonpeptide αvβ3 integrin antagonists with regard to orally bioavailability were developed, based on the aza-glycine containing lead compound 1. An important variation is the substitution of the aspartic amide of 1 by an aromatic residue. Furthermore, different guanidine mimetics have been incorporated to improve the pharmacokinetic profile. Exchange of the β-amino acid NH by a methylene moiety in one set of RGD mimetics leads to the azacarba analogue compounds representing a novel peptidomimetic approach, which should increase the metabolic stability.

Full text of DOI:10.1021/jm0004953

1938
J . Med. Chem. 2001, 44, 1938-1950
Solid -P h a se Syn th esis of a Non p ep tid e RGD Mim etic Libr a r y: New Selective
rvâ3 In tegr in An ta gon ists
Ga´bor A. G. Sulyok,† Christoph Gibson,† Simon L. Goodman,‡ Gu¨nter Ho¨lzemann,§ Matthias Wiesner,§ and
Horst Kessler*,†
Institut fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨nchen, Lichtenbergstrasse 4, D-85747 Garching,
Germany, Department of Immunology/ Oncology, Merck KGaA, Frankfurter Strasse 250, D-64271 Darmstadt, Germany, and
Department of Medicinal Chemistry, Merck KGaA, Frankfurter Strasse 250, D-64271 Darmstadt, Germany
Received November 21, 2000
The solid-phase synthesis of a low molecular weight RGD mimetic library is described. Activities
of the compounds in inhibiting the interaction of ligands, vitronectin and fibrinogen, with
isolated immobilized integrins Rvâ3 and RIIbâ3 were determined in a screening assay. Highly
active and selective nonpeptide Rvâ3 integrin antagonists with regard to orally bioavailability
were developed, based on the aza-glycine containing lead compound 1. An important variation
is the substitution of the aspartic amide of 1 by an aromatic residue. Furthermore, different
guanidine mimetics have been incorporated to improve the pharmacokinetic profile. Exchange
of the â-amino acid NH by a methylene moiety in one set of RGD mimetics leads to the azacarba
analogue compounds representing a novel peptidomimetic approach, which should increase
the metabolic stability.
In tr od u ction
II studies as angiogenesis inhibitor (code EMD 121974,
Merck KGaA). Furthermore, regression of primary
tumors and eradication of micrometastases have been
reported by combining a specific antiangiogenic therapy
with our cyclopeptide and an immunotherapy with a
tumor-specific antibody-interleukin 2 fusion protein.29
Integrins are a family of heterodimeric transmem-
brane cell surface receptors that are involved in cell-
cell and cell-matrix adhesion processes. Currently,
more than 20 different integrin receptors have been
identified consisting of various combinations of at least
17 R- and 9 â-subunits.1 In the past decade the integrins
of the â3-subfamily have been the focus of drug dis-
covery research.2-4 At the beginning most of the atten-
tion was directed toward the platelet integrin RIIbâ3,
also known as the fibrinogen receptor.5 More recently,
the vitronectin receptor Rvâ3, which is expressed on
almost all cells originating from the mesenchyme, has
received increasing attention.6 This receptor represents
an interesting therapeutic target because of its impor-
tant role in pathologies as diverse as osteoporosis,7,8
restenosis following percutaneous transluminal coro-
nary angioplasty (PTCA),9,10 acute renal failure,6d,11
ocular diseases,12,13 tumor-induced angiogenesis,14,15 and
metastasis formation.16
The tripeptide sequence RGD (Arg-Gly-Asp) is a
common cell-recognition motif, which is part of integrin
binding ligands, like fibronectin, fibrinogen, and vitro-
nectin. It is responsible for the binding to the above-
mentioned integrin receptors.6a,17 This sequence has
been used as a lead for developing different integrin
antagonists. First, the RGD sequence was incorporated
into various linear and cyclic peptides.18-25 One decade
ago, we reported about the first selective small molecule
antagonist of the Rvâ3 receptor, the cyclic pentapeptide
c(-RGDfV-).18,23,26,27 Systematic derivatization of this
peptide resulted in the N-alkylated cyclopeptide
c(-RGDf[NMe]V-),28 which has entered clinical phase
More recently, we30,31 and other groups32-46 focused
on the development of selective nonpeptide Rvâ3
integrin antagonists. Because of enhanced metabolic
stability, bioavailability, and biological absorption of
aza-amino acid containing peptides,47,48 we developed
aza-glycine containing RGD mimetics. We have shown
that glycine can be replaced by aza-glycine in RGD-
containing cyclopeptides with preservation of the
biological activity and selectivity.49 A novel peptidomi-
metic approach is the substitution of the glycine or aza-
glycine amide bond into the azacarba analogue, which
is shown in Figure 1. This azacarba nomenclature
should not be mixed with the carbaza expression for aza-
peptides without one amide moiety, which was first
coined by Limal et al.50
In the past decade combinatorial chemistry has
received increasing attention as an important tool in
drug discovery and development.51 Here, we describe
the solid-phase synthesis of a low molecular weight RGD
mimetic library based on the aza-glycine containing
compound 1. The aza-RGD mimetic 1 was identified by
an on-bead screening assay of a combinatorial library
and shows already good affinity and selectivity toward
the Rvâ3 integrin receptor (IC50: 150 nM).52,31 Regard-
ing Pfizer’s “Rule of 5” for orally available drugs (M <
500 g/mol, cLogP < 5 (cLogP is the logarithm of the
partition coefficient for n-octanol/water), number of
H-bond donors < 5, number of H-bond acceptors < 10,
substrates of biological transporters are exceptions),53
the resorption profile of the hydrophilic compound 1 had
to be improved. To enhance the lipophilicity, we decided
to furnish our RGD mimetics with a lipophilic residue.
* To whom correspondence should be addressed. Phone: ++49-89-
2891 3301. Fax: ++49-89-2891 3210. E-mail: kessler@ch.tum.de.
† Institut fu¨r Organische Chemie and Biochemie.
‡ Department of Immunology/Oncology.
§ Department of Medicinal Chemistry.
10.1021/jm0004953 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/05/2001

Rvâ3 Integrin Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1939
Sch em e 1a
F igu r e 1. Comparison of peptide, aza-peptide, and azacarba
peptide.
a
Reagents: (a) NH4OAc, malonic acid, EtOH (74-88%); (b)
Fmoc-Cl, NaHCO3, dioxane (76-98%); (c) ethyl acetoacetate,
piperidine (cat.) (42-85%); (d) 20 M KOH, 85 °C (68-99%); (e)
acetic anhydride (62-89%); (f) N-Fmoc-hydrazine,58 THF (100%);
b
(g) TCP resin, DIEA, CH2Cl2. Compounds 3d , 3g, and 4a were
purchased from commercially available sources.
replaced by a methylene (X ) CH2). Thus, we decided
to use glutaric acid derivatives as carboxylic building
block A2 and hydrazine as building block B2 for the
azacarba RGD mimetics.
F igu r e 2. Aza-RGD mimetics with various aromatic â-amino
acids (X ) NH) or glutaric acids (X ) CH2) and different
guanidine mimetics derived from compound 1.
Building block C acts as an aromatic (3-aminobenzoic
acid) or aliphatic spacer (5-aminopentanoic acid) for the
guanidino building block D. Replacement of the guani-
dine by less basic functional groups is an alternative
strategy to obtain more lipophilic compounds. Therefore,
besides guanidine itself, methylamidine and 2-amino-
pyridine, which was shown to be a suitable guanidine
mimetic,39 were used as basic building block D. In the
library, guanidine and methylamidine were incorpo-
rated in combination with an aromatic spacer, whereas
aminopyridine was attached to an aliphatic spacer.
Synthesis of the RGD mimetics was performed on
solid support by the Fmoc strategy, which is commonly
used in solid-phase peptide synthesis.55 First, we syn-
thesized the different Fmoc-protected building blocks.
Since benzaldehydes are commercially available in rich
diversity, they are particularly suitable educts for the
syntheses of the carboxylic building blocks A. Further-
more, they can be used for both the syntheses of â-amino
acids (A1) and glutaric acids (A2) (Scheme 1).
F igu r e 3. Retrosynthetic analysis of the RGD mimetic library
obtaining four different building blocks: carboxylic acid A1 (â-
amino acids) and A2 (glutaric acids), B1 (aza-glycine) and B2
(hydrazine), spacer C, and basic building block D.
The synthesized RGD mimetics include various â-amino
acids (X ) NH) or glutaric acids (X ) CH2) and different
guanidine mimetics to improve the pharmacokinetic
profile (Figure 2).
Reaction of the benzaldehydes 2a -h with ammonium
acetate and malonic acid resulted in the corresponding
racemic â-amino acids, which could be transformed into
the Fmoc-protected derivatives 3a -h in high yields. The
3-phenyl-substituted glutaric acids 4a -e,h ,i were syn-
thesized by reaction of the benzaldehydes 2a -e,h ,i with
ethyl acetoacetate and catalytic amounts of piperidine,
followed by hydrolysis with aqueous KOH. The glutaric
acids 4a -e,h ,i were converted into the cyclic anhydrides
with acetic anhydride. Subsequently, ring opening with
Fmoc-protected hydrazine afforded the 5-(N′-Fmoc-
hydrazyl)-5-oxo-3-phenylpentanoic acids 5a -e,h ,i as a
racemic mixture.
Ch em istr y
Previous results confirm that the essential pharma-
cophores of RGD mimetics for the binding to the above-
mentioned integrins are the carboxylic acid and a
guanidine-like functional group fixed at a certain dis-
tance.6,54 We synthesized a library of RGD mimetics
combining various carboxylic acid building blocks and
different guanidine mimetics. Retrosynthetic analysis
suggests a modular design of the two sets of RGD
mimetics consisting of four building blocks A-D (Figure
3). For the carboxylic building block A1 of the first set
(X ) NH), different â-amino acids were used, which are
known to mimic the aspartic acid in Rvâ3 and RIIbâ3
antagonists.4 The aza-glycine is then a good choise as
building block B1. In the second set of RGD mimeticss
the azacarba analoguessthe NH of the â-amino acid is
The Fmoc-protected carboxylic building blocks 3a -h
and 5a -e,h ,i were attached to trityl chloride polystyrol
(TCP) resin using standard conditions. Different routes
have been reported for the following incorporation of an

1940 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Sulyok et al.
Sch em e 2a
a
Reagents: (a) TCP resin, DIEA, CH2Cl2; (b) 20% piperidine/DMF; (c) 5-(9H-fluoren-9-ylmethoxy)-1,3,4-oxadiazol-2(3H)-one (9);30 (d)
phosgene (1.9 M solution in toluene), sat. NaHCO3, CH2Cl2 (85%).30
Sch em e 3a
17 in low yield. After deprotection of the resin-bound
Fmoc-protected aza-compounds 6a -e,h ,i (X ) CH2) and
10a -h (X ) NH) with piperidine, the spacer building
blocks C and CD were coupled using standard solid-
phase coupling conditions (Scheme 4).
Solid-phase synthesis was completed by introduction
of the basic building block D. Syntheses of the guani-
dine-containing mimetics were performed by treatment
of the resin-bound amines 18a -e,h ,i (X ) CH2) and
19a -h (X ) NH) with N,N′-bis-Boc-1-guanylpyrazole59
at 50 °C. After guanylation the resulting resin-bound
aza-RGD mimetics were deprotected and cleaved from
the resin in one step using trifluoroacetic acid. Treat-
ment of the resin-bound amines 18a -e,h ,i (X ) CH2)
with S-2-naphthylmethyl thioacetimidate hydro-
bromide60 in the presence of the base DIEA and cleavage
from the resin with acetic acid yielded the corresponding
methylamidine containing aza-RGD mimetics 21a -e,h ,i
(X ) CH2). By use of the same cleavage procedure, the
aminopyridine-containing aza-RGD mimetics 23a -e,h ,i
(X ) CH2) and 24a -h (X ) NH) were obtained.
In addition, single compounds that are not included
in the library were synthesized. Compounds 25 and 26
represent other combinations of the building blocks C
and D, whereas compound 27 is an RGD mimetic
without the aromatic residue of the carboxylic building
block A.
a
Reagents: (a) 100 °C (23%); (b) 1 N NaOH (64%); (c) NaH,
DMF, 80 °C (10%).
aza-glycine on solid support.56,57 Recently, we reported
an improved approach for the synthesis of activated
Fmoc-protected aza-amino acids and their reaction with
resin-bound amines.30 N-Fmoc-hydrazine (8)58 was car-
bonylated with an excess of a solution of phosgene in
toluene to obtain the activated aza-glycine building
block 5-(9H-Fluoren-9-ylmethoxy)-1,3,4-oxadiazol-2(3H)-
one (9)30 (Scheme 2). After Fmoc deprotection the resin-
bound â-amino acids 7a -h were treated with excess
oxadiazolone (9) to yield the resin-bound Fmoc-protected
aza-Gly-â-amino acid derivatives 10a -h .
Synthesis of compound 25 was performed on a solid
support in analogy to compound 22a . Resin-bound
N-Fmoc-aza-Gly-â-phenylalanine (10a ) was treated with
5-(N-Fmoc)aminopentanoic acid instead of 3-(N-Fmoc)-
aminobenzoic acid as spacer building block C. After
Fmoc deprotection, guanylation, and cleavage from the
resin, compound 25 was achieved. Replacement of
â-phenylalanine with â-alanine in the synthesis of
compound 24a resulted in compound 26. Compound 27
was obtained by treating resin-bound 5-(N-Fmoc-
hydrazino)-3-(3,5-dichlorophenyl)-5-oxopentanoic acid
(6h ) with the combined building block CD, 3-(pyridine-
2-yl)aminobenzoic acid (17), and cleavage from the resin,
in analogy to compound 23h .
The following step of the solid-phase synthesis is the
coupling of the spacer building block C, where N-Fmoc-
protected 3-aminobenzoic acid and 5-aminopentanoic
acid were used. In contrast to guanidine or amidine as
building blocks D, which are coupled on solid support,
2-aminopyridine was first linked to the spacer molecule
C in solution (Scheme 3). 2-Amino-4-methylpyridine (11)
was treated with 5-bromopentanoic acid ethyl ester (12)
at 100 °C to yield 5-(4-methylpyridine-2-yl)aminopen-
tanoic acid ethyl ester (13). After hydrolysis with
aqueous NaOH, building block CD 5-(4-methylpyridine-
2-yl)aminopentanoic acid (14) was obtained by precipi-
tation. The combination of aminopyridine and an
aromatic spacer building block C was not used in library
synthesis but in further synthesis of single compounds.
Deprotonation of 3-aminobenzoic (16) acid with sodium
hydride followed by addition of 2-fluoropyridine (15)
afforded this building block combination CD, compound
Resu lts a n d Discu ssion
Biologica l Eva lu a tion . Screening assays were per-
formed by measuring the effect of RGD mimetics on the

Rvâ3 Integrin Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1941
Sch em e 4a
a
Reagents: (a) 20% piperidine/DMF; (b) 3-(N-Fmoc-amino)benzoic acid, HATU, collidine, DMF; (c) 5-(4-methylpyridine-2-yl)amino-
pentanoic acid (14), HATU, collidine, DMF; (d) AcOH/TFE/CH2Cl2 (1:1:3); (e) N,N′-bis-Boc-1-guanylpyrazole,59 CHCl3, 50 °C; (f) 95% TFA/
5% TIPS; (g) S-2-naphthylmethyl thioacetimidate hydrobromide,60 DIEA, NMP.
a
Ta ble 1. Effect of Substituent R and Basic Pharmacophore on IC50 of Aza-RGD Mimetics
compd
R
base
spacer
cLogP
Rvâ3 IC50 [nM]
RIIbâ3 IC50 [nM]
GRGDSPKb
1000
2
150
40
0.8
6
0.1
1
0.9
3
>104
850
c(RGDfV)b
1
(carboxamide)c
none
none
4-F
4-Cl
guanidine
guanidine
guanidine
guanidine
guanidine
guanidine
guanidine
guanidine
guanidine
guanidine
m-C6H4-
ω-C4H8
-2.42
-0.26
0.70
0.86
1.26
1.53
0.58
2.23
1.70
1.82
>104
1000
25
22a
22b
22c
22d
22e
22f
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
8500
1000
5500
>1000
>1000
>1000
>104
>1000
4-Br
4-OCH3
4-OCF3
(1-naphthyl)c
3-Cl,5-Cl
22g
22h
27
1
26
(H)c
none
4-F
4-Cl
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
-0.07
1.64
1.80
2.20
2.47
1.52
3.17
2.64
2.76
1400
37
48
0.3
7
42
130
140
80
n.d.
24a
24b
24c
24d
24e
24f
24g
24h
>104
>104
7000
n.d.
4-Br
4-OCH3
4-OCF3
(1-naphthyl)c
3-Cl,5-Cl
>104
n.d.
n.d.
>104
a
b
n.d. ) not determined. The peptides linear GRGDSPK and cyclo(RGDfV) were used as reference in the receptor binding assay.
c Instead of substituted phenyl ring.
interaction between immobilized integrin receptors
and biotinylated soluble ligands. The ability of RGD
mimetics to inhibit the binding of vitronectin and
fibrinogen to the isolated, immobilized Rvâ3 and RIIbâ3
receptors was compared with that of the standard
peptides GRGDSPK and c(RGDfV). The results of the
screening assays are shown in Tables 1 and 2.
Ch em istr y. The synthesized library is derived from
the structure of the aza-RGD mimetic 1, which has been
recognized by an on-bead screening assay.31 An impor-
tant variation of lead compound 1 was replacement of
the aspartic amide with an aromatic residue of a
â-amino acid (compounds 22a -h ). Different studies
have pointed out aromatic â-amino acids to be suitable

1942 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Ta ble 2. Effect of Substituent R and Basic Pharmacophore on IC50 of Azacarba RGD Mimetics
Sulyok et al.
a
compnd
R
base
spacer
cLogP
Rvâ3 IC50 [nM]
RIIbâ3 IC50 [nM]
20a
20b
20c
20d
20e
20h
20i
none
4-F
4-Cl
4-Br
4-OCH3
3-Cl, 5-Cl
4-phenyl
guanidine
guanidine
guanidine
guanidine
guanidine
guanidine
guanidine
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
1.13
1.28
1.68
1.95
1.00
2.24
2.80
15
4
2
2
1
>104
>1000
>1000
>1000
>1000
1000
2
0.7
1000
21a
21b
21c
21d
21e
21h
21i
none
4-F
4-Cl
4-Br
4-OCH3
3-Cl, 5-Cl
4-phenyl
methylamidine
methylamidine
methylamidine
methylamidine
methylamidine
methylamidine
methylamidine
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
m-C6H4-
1.44
1.60
2.00
2.27
1.31
2.55
3.11
23
160
160
40
37
6
>104
>1000
8000
>1000
>1000
n.d.
26
>1000
23a
23b
23c
23d
23e
23h
27
none
4-F
4-Cl
4-Br
4-OCH3
3-Cl,5-Cl
3-Cl,5-Cl
4-phenyl
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
2-NH2-pyridine
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
ω-C4H8-
m-C6H4-
ω-C4H8-
2.07
2.23
2.63
2.90
1.94
3.19
3.54
3.74
7
>104
35
15
6
22
9
n.d.
n.d.
9000
>104
>104
n.d.
210
3
23i
3000
a
n.d. ) not determined.
building blocks in potent RGD mimetics.4 It is known
that benzene interacts with amide bonds to form a
complex that results in the so-called ASIS (aromatic
solvent induced shift) effect.61 Thus, we assume that
aromatic residues substitute an amide bond in a favor-
able manner. The analogue compound 22a containing
â-phenylalanine shows a 200-fold increase in activity
to the vitronectin receptor Rvâ3 (IC50, 0.7 nM) while
preserving selectivity for the platelet receptor RIIbâ3.
Different substitutions, especially at the para position
of the phenyl ring, have been investigated (Table 1). All
of the synthesized guanidine-containing aza-RGD
mimetics 22a -h are Rvâ3 antagonists with excellent
IC50 values in the nanomolar or subnanomolar range
(compound 22c). Compound 22c with the highest affin-
ity (IC50, 0.1 nM) is about 2 orders of magnitude more
active than the naphthyl derivative 22g. Similar gly-
cine-containing compounds synthesized by Searle/Mon-
santo show comparable affinity to the vitronectin re-
ceptor, but the specifitiy toward the fibrinogen receptor
RIIbâ3 is about 1-2 log units smaller.8 The importance
of the meta-substituted aromatic spacer moiety could
be shown by exchange with an alkyl chain, compound
25. Because of higher flexibility of the alkyl spacer, a
50-fold decrease in Rvâ3 receptor activity was observed.
As an indicator for lipophilicity of the synthesized
compounds, we determined the calculated log P values
(cLogP: estimation of the logarithm of the partition
coefficient for n-octanol/water)62 using a computer
algorithm. Replacement of the lead compound amide
with an aromatic ring resulted in a 3 order increase of
cLogP. Despite enhancement of the lipophilicity by
incorporation of the aromatic ring, the guanidine-
containing compounds are still hydrophilic. In trying to
improve the resorption profile, the polar guanidino
moiety was replaced with a less basic aminopyridine
moiety, combined with the alkyl spacer, in another set
of aza-RGD mimetics (compounds 24a -h and 26). These
mimetics are still very potent and highly specific
antagonists of the Rvâ3 integrin receptor with IC50
values in the nanomolar range. In comparison with the
analogue guanidine-containing compounds, they show
a slight (5- to 50-fold) decrease in the activity, while
cLogP is increased by about 1 magnitude. The 4-chloro-
substituted compound 24c represents an exception with
subnanomolar activity as well (IC50, 0.3 nM). Since
replacement of the basic pharmacophore does not influ-
ence the activity toward the platelet receptor RIIbâ3,
further investigations of these selective RGD mimetics
regarding the pharmacokinetic profile are promising.
Both of the two basic pharmacophores, guanidine and
aminopyridine, used in this set of RGD mimetics are
suitable arginine mimetics when combined with the
appropriate spacers.
Deletion of the aromatic residue of the â-amino acid,
compound 26, led to a 3 order decrease of activity to
the Rvâ3 receptor (IC50, 1400 nM). This observed effect
confirms the importance of the aromatic residue (or
amide) in proximity to the carboxylic acid for a high
binding affinity to the vitronectin receptor Rvâ3.
Another series of compounds, the azacarba set, was
synthesized to investigate the influence of the â-amino
acid NH, which was replaced with a methylene moiety.
We hope that this exchange of the amide bond will
induce an improved metabolic stability of the RGD
mimetics. The IC50 values of the obtained azacarba set
of RGD mimetics (X ) CH2) are shown in Table 2.
Analogue azacarba RGD mimetics have been synthe-
sized with similar phenyl substitution patterns to allow
comparison with the above-mentioned corresponding

Rvâ3 Integrin Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1943
aza-RGD mimetics. The guanidine-containing com-
pounds 20a -e,h ,i have excellent affinities to the Rvâ3
receptor in the nanomolar and subnanomolar ranges
(compound 20i) and high selectivities (low affinities)
toward the platelet receptor RIIbâ3. Except for the
unsubstituted compound 20a showing a 200-fold de-
crease in activity, only small differences in IC50 values
are observed in comparison with those of aza- and the
analogous azacarba RGD mimetics (22a -e,h and 20a -
e,h ), while cLogP values are slightly increased.
with promising properties for oral bioavailability start-
ing from the known linear RGD sequence. Our approach
combines rational and combinatorial drug design to
inhibit protein-protein interactions presented in Table
3. A peptidic anticancer drug (clinical studies phase II)
was obtained by cyclization and optimization of the
linear RGD sequence.28 Further investigations led to
aza-glycine-containing cyclic peptides and a library of
aza-RGD mimetics. Optimization of the lead compound
1 with regard to an improved resorption profile is
described in this work. The key step was the substitu-
tion of an amide bond by an aromatic residue, resulting
in a 2 orders of magnitude increase in activity toward
the vitronectin receptor Rvâ3.
On the basis of the lead compound 1, a library of
RGD mimetics consisting of four building blocks A-D
has been synthesized. Two sets of mimetics have been
investigated. In the first set, â-amino acids have
been used as carboxylic building block A1, whereas the
second setsthe azacarba analoguessrepresents a novel
peptidomimetic approach with glutaric acids as car-
boxylic building block A2. These azacarba RGD mimetics
with the amine of the â-amino acid substituted by a
methylene moiety were designed to improve metabolic
stability.
Reduction of basicity by replacement of guanidine
with methylamidine, compounds 21a -e,h ,i, led to a
further slight increase of cLogP while losing activity to
the Rvâ3 receptor (20- to 100-fold). These results suggest
a binding contribution of the guanidine NH2 moiety to
the vitronectin receptor Rvâ3. Only the high affinity of
the 3,5-dichloro-substituted compound 21h (IC50, 6 nM)
could be preserved. Exchange of the basic building block
D by an aminopyridine, compound 27, resulted in a 40-
to 100-fold decrease in activity (IC50, 210 nM) compared
with the analogue 3,5-dichloro-substituted compounds
20h (IC50, 2 nM) and 21h (IC50, 6 nM). By analogy to
the aza-RGD mimetics, aminopyridine has been used
as a basic pharmacophore in combination with the
pentanoic acid spacer. The obtained compounds 23a -
e,h ,i also show high affinities to the Rvâ3 receptor (IC50,
4-35 nM) like the guanidine-containing azacarba RGD
mimetics. The compounds are slightly more active than
the corresponding aza-RGD mimetics 24a -h (X ) NH),
except for the 4-chloro-substituted derivative 23c (IC50,
15 nM) with a 50-fold smaller activity compared to 24c.
Although the aromatic spacer C is an important factor
in guanidine-containing compounds, a decrease in activ-
ity resulted in the case of aminopyridine-containing
RGD mimetics (compound 27). The principle of addi-
tivity, which is often used in drug design, fails in this
case. The reason might be the nonplanarity of the
pyridine-benzene system, preventing the simple addi-
tion of individual effects. Therefore, the best combina-
tions of building blocks C and D are arylguanidine and
alkylaminopyridine.
When the two different sets of RGD mimetics (aza and
azacarba) are compared, small differences in activity on
the vitronectin receptor Rvâ3 and in selectivity toward
the platelet receptor RIIbâ3 can be observed. The amine
of the â-amino acid appears to be less important for
binding to the Rvâ3 receptor. The aminopyridine-
containing azacarba compounds show the highest cLogP
values in the range 1.6-3.2 and are supposed to be
compounds with enhanced metabolic stability, suitable
for further resorption studies. Though nearly all com-
pounds are very potent Rvâ3 integrin antagonists (nano-
molar IC50 values), the best results were achieved by
4-chloro substitution, compounds 22c (IC50, 0.1 nM) and
24c (IC50, 0.3 nM), as well as 4-phenyl substitution,
compound 20i (IC50, 0.7 nM).
All of the guanidine-containing compounds show
excellent affinities to the Rvâ3 receptor in the nano-
molar range. To increase lipophilicity of the RGD
mimetics, the guanidino moiety has been substituted
by methylamidine and aminopyridine as basic building
block D. In particular, incorporation of the less basic
aminopyridine led to RGD mimetics with promising
properties for oral bioavailability while keeping nano-
molar activity. Pharmacokinetic studies of these com-
pounds are in progress. All compounds show little or
no activities to the platelet receptor RIIbâ3 (IC50
>
10 000 nM), so we have obtained highly specific Rvâ3
integrin antagonists (selectivities > 1000). Further
investigations of activities and selectivities toward other
integrin receptors, like Rvâ5 and Rvâ6, will be done. In
summary, we have synthesized a library of highly active
and selective RGD mimetics with promising properties
for further pharmacokinetic investigations.
Exp er im en ta l Section
Gen er a l. Trityl chloride polystyrol (TCP) resin (0.94 mmol/
g) was purchased from PepChem. Coupling reagents and
amino acid derivatives were purchased from Advanced
ChemTech, Perseptive Biosystems GmbH, Neosystem, and
PurChem. All other reagents and solvents were purchased
from Merck-Schuchardt, Lancaster, Aldrich, and Fluka and
were used as received. The solvents ethyl acetate, ethanol, and
methanol were distilled before use. Standard syringe tech-
niques were applied for transferring dry solvents. Reactions
on solid support were performed in filter columns (5 and 10
mL) from Abimed. Melting points (mp) were determined on a
1
Bu¨chi 510 apparatus and were uncorrected. H spectra were
obtained in CDCl3 or DMSO-d6 as solvent and internal
Since all compounds of the library were synthesized
as racemic mixtures, the activities of the RGD mimetics
can be further improved by synthesis of the pure
S-enantiomers as shown by several groups.4
reference on
a Bruker AC250 or DMX500 spectrometer.
Chemical shifts (δ) are reported in parts per million (ppm),
and coupling constants (J values) are given in hertz (Hz). Thin-
layer chromatography was performed on silica gel 60 F254
plates from Merck. Flash chromatography was performed on
silica gel 60 (230-400 mesh ASTM) from Merck. Reversed-
phase HPLC analyses and separations were conducted on
Amersham Pharmacia Biotech instruments using Omnicrom
YMC columns (analytical, 4.6 mm × 25 mm, 5 µm C18, 1 mL/
Con clu sion s
This work represents a further step in the develop-
ment of highly specific, small nonpeptidic compounds

1944 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Sulyok et al.
Ta ble 3. Combination of Rational and Combinatorial Drug Design to Inhibit Protein-Protein Interactions
a
The selectivity designates the quotient of affinities to integrins Rvâ3 and RIIbâ3.
min; preparative, 30 mm × 25 mm, 10 µm C18, 25 mL/min)
with a 30 min linear gradient from water (0.1% TFA) and CH3-
CN (0.1% TFA) and detection at 220 nm. Mass spectra (ESI)
were performed on a LCQ Finnigan instrument. Calculations
of log P (cLogP) were performed with ChemDraw Ultra (ver-
sion 6.0) using the Crippen’s fragmentation algorithm.62
Gen er a l P r oced u r es. 3-[N-(9H-F lu or en -9-ylm eth oxy-
ca r bon yl)-a m in o]-3-p h en ylp r op ion ic Acid s (3a -c,e,f,h ).
Benzaldehyde (30 mmol), malonic acid (3.06 g, 30 mmol), and
ammonium acetate (4.63 g, 60 mmol) were refluxed in ethanol
(30 mL) for 6 h. The amino acid precipitated from the reaction
mixture as a white solid and was isolated by filtration. Fmoc-
chloride (3.24 g, 12.5 mmol) in dioxane (20 mL) was slowly
added to a stirred suspension of the 3-amino-3-phenylpropionic
acid (12.0 mmol) in aqueous sodium carbonate (10%, 25 mL)
and dioxane (12 mL) at 0 °C. After being additionally stirred
for 1 h at 0 °C and 1 d at room temperature, the reaction
mixture was poured into ice water (300 mL) and extracted with
diethyl ether (2 × 100 mL). The pH of the aqueous layer was
adjusted to 1 by addition of aqueous HCl (2 N). The precipitate
was filtered off and dried under vacuo to yield product 3 as a
white solid.
3-P h en ylglu t a r ic Acid s (4b -e,h ,i). A mixture of benz-
aldehyde (30 mmol) and ethyl acetoacetate (7.8 g, 60 mmol)
was cooled to 0 °C, and piperidine (0.5 mL) was added
dropwise. The mixture was left at room temperature for 3 days.
The resulting solid was dissolved in ethanol (60 mL) and
refluxed. After cooling to room temperature, the crystalline
product was filtered off and dried under vacuo. The precipitate
was added stepwise to an aqueous solution of KOH (20 M),
and the mixture was stirred at 80 °C for 3 h. Ice (50 mL) and
ethyl acetate (50 mL) were added. The aqueous phase was
separated, and the pH was adjusted to 1 by cautious addition
of concentrated aqueous HCl. The precipitate was filtered off,
washed with water, and dried under vacuo to yield product 4
as a white solid.
3-phenylglutaric acid 4 (10 mmol) in acetic anhydride (2.8 mL,
30 mmol) was refluxed at 130 °C until complete dissolution
occurred. After the mixture was cooled to room temperature,
diethyl ether (2 mL) was added dropwise. The precipitate was
isolated by filtration, washed with cold ether, and dried under
vacuo to yield the anhydride as a gray solid. A solution of the
anhydride (2.0 mmol) and Fmoc-hydrazine (0.51 g, 2.0 mmol)
in anhydrous THF (30 mL) was heated to reflux for 16 h. After
evaporation of the solvent product 5 was obtained.
5-[N-(4-Meth ylp yr id in -2-yl)-a m in o]p en ta n oic
Acid
Eth yl Ester (13). 5-Bromopentanoic acid ethyl ester (12) (12
mL, 100 mmol) and 2-amino-4-methylpyridine (11) (21.6 g, 200
mmol) were heated together at 100 °C for 12 h. After cooling
to room temperature, the mixture was treated with saturated
sodium hydrogen carbonate solution (100 mL) and extracted
with ether (3 × 50 mL). The combined organic layers were
dried over Na2SO4, filtered, and concentrated under reduced
pressure to yield the crude ester 13 as yellow oil. After
purification by flash chromatography (ethyl acetate/hexane
2:1) product 13 was obtained as a white solid (3.17 g, 23%).
1H NMR (DMSO-d6, 250 MHz): δ 7.78 (d, 2H, J ) 7.0 Hz),
6.25-6.28 (m, 2H), 6.22 (s, 1H), 3.165 (m, 2H), 2.20 (m, 2H),
2.11 (s, 3H), 1.50 (m, 4H). MS(ESI): m/z 237.1 [M + H+].
5-[N-(4-Meth ylp yr id in -2-yl)a m in o]p en ta n oic Acid (14).
Ester 13 (3.17 g, 13.4 mmol) was dissolved in methanol (5 mL)
and treated with an aqueous NaOH solution (2 N, 10 mL) for
20 h at room temperature. The mixture was concentrated in
vacuo and acidified with HCl (1 N) to pH 4.5. The precipitate
was collected by filtration, washed with water, and dried under
vacuo to yield the product 14 as a white solid (1.78 g, 64%).
1H NMR (DMSO-d6, 250 MHz): δ 7.79 (d, 1H, J ) 5.2 Hz),
6.26-6.30 (m, 2H), 6.22 (s, 1H), 3.17 (m, 2H), 2.21 (t, 2H, J )
7.0 Hz), 2.11 (s, 3H), 1.41-1.58 (m, 4H). MS(ESI): m/z 209.1
[M + H+].
3-[N-(P yr id in -2-yl)a m in o]ben zoic Acid (17). Sodium
hydride (0.88 g, 55% dispersion in mineral oil, 20.0 mmol, 2.0
equiv) was suspended in DMF (20 mL) and cooled to 0 °C. After
slow addition of 3-aminobenzoic acid (16) (1.37 g, 10.0 mmol)
5-[N′-(9H-F lu or en -9-ylm eth oxyca r bon yl)h yd r a zin o]-5-
oxo-3-p h en ylp en ta n oic Acid s (5a -e,h ,i). A suspension of

Rvâ3 Integrin Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1945
the suspension was stirred for 2 h at room temperature.
2-Fluoropyridine (15) (0.86 mL, 10.0 mmol) was added, and
stirring was continued for 60 h at 80 °C. After addition of water
(5 mL), the mixture was extracted with NaHCO3 (30 mL) and
ethyl acetate (2 × 30 mL). The aqueous layer was acidified
with HCl (1 N) to pH 3 and extracted with ethyl acetate (2 ×
30 mL). Evaporation of the solvent afforded the product 17 as
a red solid (0.21 g, 10%). 1H NMR (DMSO-d6, 250 MHz): δ
9.65 (s, 1H), 8.12 (d, 1H, J ) 6.5 Hz), 7.99 (s, 1H), 7.82 (d, 1H,
J ) 8.0 Hz), 7.72 (t, 1H, J ) 7.5 Hz), 7.46 (d, 1H, J ) 7.5 Hz),
7.41 (m, 1H), 6.95 (d, 1H, J ) 8.5 Hz), 6.85 (t, 1H, J ) 6.0
Hz). MS(ESI): m/z 215.2 [M + H+].
Gen er a l P r oced u r es for Solid -P h a se Syn th esis. Gen -
er a l P r oced u r e Ia . Atta ch m en t of 3-(N-F m oc)a m in o-3-
p h en ylp r op ion ic Acid s 3a -h to TCP Resin . After swelling
with CH2Cl2 (10 mL) for 20 min, TCP resin (2.00 g, theoretical
0.96 mmol/g, 1.92 mmol) was treated with a solution of Fmoc-
protected â-amino acids (3a -h ; 2.40 mmol) in 10 mL of CH2-
Cl2 and DIEA (514 µL, 3.00 mmol) at room temperature for 2
h. MeOH (1 mL) was added to cap the free sites, and the
reaction mixture was shaken for 15 min. The resin was washed
with CH2Cl2 (5 × 10 mL) and MeOH (3 × 10 mL) and dried
under vacuo to give resin-bound 3-(N-Fmoc)amino-3-phenyl-
propionic acids (7a -h ).
Gen er a l P r oced u r e Ib. Atta ch m en t of 5-(N′-F m oc)-
h yd r a zyl-5-oxo-3-p h en ylp en ta n oic Acid s 5a -e,h ,i to TCP
Resin . After swelling with CH2Cl2 (10 mL) for 20 min, the
TCP resin (2.00 g, theoretical 0.96 mmol/g, 1.92 mmol) was
treated with a solution of Fmoc-protected pentanoic acids (5a -
e,h ,i; 2.40 mmol) in 10 mL of CH2Cl2 and DIEA (514 µL, 3.00
mmol) at room temperature for 2 h. MeOH (1 mL) was added
to cap the free sites, and the reaction mixture was shaken for
15 min. The resin was washed with CH2Cl2 (5 × 10 mL) and
MeOH (3 × 10 mL) and dried under vacuo to give resin-bound
5-(N′-Fmoc)hydrazyl-5-oxo-3-phenylpentanoic acids (6a -e,h ,i).
Gen er a l P r oced u r e II. Cou p lin g of Aza -glycin e to
Resin -Bou n d 3-(N-F m oc)a m in o-3-p h en ylp r op ion ic Acid s
7a -h . Resin (0.20 g, 0.4-0.6 mmol/g, 0.08-0.12 mmol) was
washed with CH2Cl2 (2 mL) and DMF (2 mL) and treated with
a solution of 20% piperidine in DMF (2 × 2 mL) for 5 and 15
min. Resin was washed with DMF (6 × 2 mL) and CH2Cl2 (3
× 2 mL), and a solution of 9 (0.24-0.36 mmol) in CH2Cl2 (2
mL) was added. The reaction mixture was shaken at room
temperature for 90 min and washed with CH2Cl2 (6 × 2 mL)
and DMF (4 × 2 mL) to give resin-bound 3-[N-(N′-Fmoc)-
hydrazinocarbonyl]-amino-3-phenylpropionic acids (10a-e,h ,i).
Gen er a l P r oced u r e Va . Clea va ge w ith Tr iflu or oa cetic
Acid (TF A). The resin was treated with a solution of 5%
triisopropylsilane (TIPS) in TFA (2 mL) for 1.5 h and (2 × 2
mL) for 2 × 15 min. After removal of the resin by filtration,
the filtrates were combined and solvent was removed in vacuo
to yield the product as yellow oil. Purification was carried out
by preparative reversed-phase chromatography (C18, 20 f 45%
ACN/H2O).
Gen er a l P r oced u r e Vb . Clea va ge w it h Acet ic Acid .
Resin was washed with CH2Cl2 (4 × 2 mL) and treated with
a mixture of CH2Cl2, acetic acid, and trifluoroethanol (3:1:1; 2
mL) for 1.5 h and (2 × 2 mL) for 2 × 15 min. After removal of
the resin by filtration, the filtrates were combined and solvent
was removed in vacuo to yield the product as yellow oil.
Purification was carried out by preparative reversed-phase
chromatography (C18, 20 f 45% ACN/H2O).
5-[N′-(3-Gu a n id in oben zoyl)h yd r a zin o]-3-p h en yl-5-oxo-
p en ta n oic Acid Tr iflu or oa ceta te (20a ). The procedure is
similar to the general procedures for Ib, IIIa , IVa , and Va (6
mg, oil). 1H NMR (DMSO-d6, 500 MHz): δ 11.98 (s, 1H), 10.33
(s, 1H), 9.95 (s, 1H), 9.78 (s, 1H), 7.76 (d, 1H, J ) 7.8 Hz),
7.69 (s, 1H), 7.54 (t, 1H, J ) 7.8 Hz), 7.49 (s, 4H), 7.41 (d, 1H,
J ) 7.8 Hz), 7.24-7.30 (m, 5H), 3.50 (m, 1H), 2.77 (dd, 1H, J
) 5.5/16.0 Hz), 2.49-2.58 (m, 3H). FAB-HRMS: calcd, 384.1672;
found, 384.1668 [M + H+].
5-[N ′-(3-Gu a n id in ob e n zoyl)h yd r a zin o]-3-(4-flu or o-
p h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ceta te (20b). The
procedure is similar to the general procedures for Ib, IIIa , IVa ,
1
and Va (17 mg, oil). H NMR (DMSO-d6, 500 MHz): δ 12.05
(s, 1H), 10.32 (s, 1H), 9.97 (s, 1H), 9.95 (s, 1H), 7.76 (d, 1H, J
) 7.8 Hz), 7.70 (s, 1H), 7.59 (s, 4H), 7.55 (t, 1H, J ) 7.8 Hz),
7.41 (d, 1H, J ) 7.8 Hz), 7.32 (m, 2H), 7.11 (m, 2H), 3.50 (m,
1H), 2.77 (dd, 1H, J ) 5.5/16.0 Hz), 2.48-2.58 (m, 3H). MS-
(ESI): m/z 402.2 [M + H+].
5-[N ′-(3-Gu a n id in ob e n zoyl)h yd r a zin o]-3-(4-ch lor o-
p h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ceta te (20c). The
procedure is similar to the general procedures for Ib, IIIa , IVa ,
and Va (9 mg, oil). 1H NMR (DMSO-d6, 500 MHz): δ 12.05 (s,
1H), 10.31 (s, 1H), 9.96 (s, 1H), 9.93 (s, 1H), 7.75 (d, 1H, J )
7.8 Hz), 7.69 (s, 1H), 7.57 (s, 4H), 7.54 (t, 1H, J ) 7.8 Hz),
7.41 (d, 1H, J ) 7.8 Hz), 7.30-7.37 (m, 4H), 3.48 (m, 1H), 2.75
(dd, 1H, J ) 5.7/16.0 Hz), 2.45-2.56 (m, 3H). FAB-HRMS:
calcd, 418.1282; found, 418.1294 [M + H+].
5-[N ′-(3-Gu a n id in ob e n zoyl)h yd r a zin o]-3-(4-b r om o-
p h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ceta te (20d ). The
procedure is similar to the general procedures for Ib, IIIa , IVa ,
and Va (15 mg, white powder), mp 114-118 °C. 1H NMR
(DMSO-d6, 500 MHz): δ 12.05 (s, 1H), 10.31 (s, 1H), 9.97 (s,
1H), 9.95 (s, 1H), 7.74 (d, 1H, J ) 7.8 Hz), 7.69 (s, 1H), 7.59
(s, 4H), 7.53 (t, 1H, J ) 7.8 Hz), 7.46 (d, 2H, J ) 8.2 Hz), 7.40
(d, 1H, J ) 7.8 Hz), 7.23 (d, 2H, J ) 8.2 Hz), 3.49 (m, 1H),
2.76 (dd, 1H, J ) 5.7/16.0 Hz), 2.47-2.58 (m, 3H). FAB-
HRMS: calcd, 462.0777; found, 462.0757 [M + H+].
5-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]-3-(4-m et h oxy-
p h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ceta te (20e). The
procedure is similar to the general procedures for Ib, IIIa , IVa ,
and Va (9 mg, white powder), mp 107-111 °C. 1H NMR
(DMSO-d6, 500 MHz): δ 11.99 (s, 1H), 10.33 (s, 1H), 9.94 (s,
1H), 9.95 (s, 1H), 7.76 (d, 1H, J ) 8.0 Hz), 7.71 (s, 1H), 7.59
(s, 4H), 7.55 (t, 1H, J ) 7.8 Hz), 7.41 (d, 1H, J ) 7.8 Hz), 7.18
(d, 2H, J ) 8.2 Hz), 6.85 (d, 2H, J ) 8.2 Hz), 3.72 (s, 3H), 3.47
(m, 1H), 2.74 (dd, 1H, J ) 5.5/15.4 Hz), 2.44-2.55 (m, 3H).
FAB-HRMS: calcd, 414.1778; found, 414.1774 [M + H+].
5-[N′-(3-Gu a n id in oben zoyl)h yd r a zin o]-3-(3,5-d ich lor o-
p h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ceta te (20h ). The
procedure is similar to the general procedures for Ib, IIIa , IVa ,
and Va (12 mg, white powder), mp 104-107 °C. 1H NMR
(DMSO-d6, 500 MHz): δ 12.15 (s, 1H), 10.32 (s, 1H), 9.95 (s,
1H), 9.91 (s, 1H), 7.74 (d, 1H, J ) 7.8 Hz), 7.69 (s, 1H), 7.56
(s, 4H), 7.53 (t, 1H, J ) 7.8 Hz), 7.41 (s, 1H), 7.40 (d, 1H, J )
7.8 Hz), 7.35 (s, 2H), 3.50 (m, 1H), 2.77 (dd, 1H, J ) 5.9/16.4
Hz), 2.56-2.66 (m, 3H). FAB-HRMS: calcd, 452.0892; found,
452.0890 [M + H+].
Gen er a l P r oced u r e IIIa /b. Dep r otection a n d Cou p lin g
w ith HATU. The Fmoc protecting group was removed using
a solution of 20% piperidine in DMF (2 × 2 mL) for 5 and 15
min. The resin was washed with DMF (6 × 2 mL) and CH2Cl2
(3 × 2 mL), and a solution of Fmoc-3-aminobenzoic acid (a ,
0.16-0.24 mmol) or 5-[N-(4-methyl)pyridin-2-yl]aminopen-
tanoic acid 14 (b, 0.16-0.24 mmol), HATU (0.16-0.24 mmol),
and collidine (1.60-2.40 mmol) in DMF (2 mL) was added to
the resin. The reaction mixture was shaken at room temper-
ature for 90 min and washed with DMF (6 × 2 mL).
Gen er a l P r oced u r e IVa . Gu a n yla tion of Resin -Bou n d
Am in es. The Fmoc protecting group was removed using a
solution of 20% piperidine in DMF (2 × 2 mL). The resin was
washed with DMF (6 × 2 mL) and CHCl3 (3 × 2 mL), and a
solution of N,N′-bis-Boc-1-guanylpyrazole57 (0.48-0.72 mmol,
3 equiv) in CHCl3 (1.5 mL) was added. The reaction mixture
was shaken at 50 °C for 16 h and washed with CH2Cl2 (6 × 2
mL) to give the resin-bound Boc-protected guanidine.
Gen er a l P r oced u r e IVb . Am id a t ion of R esin -Bou n d
Am in es. The Fmoc protecting group was removed with a
solution of 20% piperidine in DMF (2 × 2 mL). The resin was
washed with NMP (6 × 2 mL) and a solution of S-2-
naphthylmethyl thioacetimidate hydrobromide58 (0.16-0.24
mmol, 2 equiv), and DIEA (0.20-0.30 mmol) in NMP (3 mL)
was added. The reaction mixture was shaken at room tem-
perature for 16 h and washed with NMP (6 × 2 mL) and CH2-
Cl2 (3 × 2 mL) to give the resin-bound amidines.

1946 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Sulyok et al.
5-[N′-(3-Gu a n id in oben zoyl)h yd r a zin o]-3-(4-bip h en yl)-
5-oxop en ta n oic Acid Tr iflu or oa ceta te (20i). The procedure
is similar to the general procedures for Ib, IIIa , IVa , and Va
and Vb (5 mg, oil): 1H NMR (DMSO-d6, 500 MHz): δ 11.23
(s, 1H), 10.39 (s, 1H), 10.00 (s, 1H), 9.51 (s, 1H), 8.63 (s, 1H),
7.90 (d, 1H, J ) 7.8 Hz), 7.77 (s, 1H), 7.61-7.68 (m, 3H), 7.58
(d, 2H, J ) 8.5 Hz), 7.48 (d, 1H, J ) 7.8 Hz), 7.45 (m, 2H),
7.31-7.39 (m, 3H), 3.56 (m, 1H), 2.79 (m, 1H), 2.51-2.63 (m,
3H), 2.32 (s, 3H). MS(ESI): m/z 459.1 [M + H+].
3-{N-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]ca r b on yl}-
a m in o-3-p h en ylp r op ion ic Acid Tr iflu or oa ceta te (22a ).
The procedure is similar to the general procedures for Ia , II,
IIIa , IVa , and Va (6 mg, oil): 1H NMR (DMSO-d6, 500 MHz):
δ 12.21 (s, 1H), 10.19 (s, 1H), 9.89 (s, 1H), 8.09 (s, 1H), 7.76
(d, 1H, J ) 8.0 Hz), 7.70 (s, 1H), 7.48-7.57 (m, 5H), 7.40 (d,
1H, J ) 8.0 Hz), 7.28-7.35 (m, 4H), 7.22 (t, 1H, J ) 7.0 Hz),
7.01 (d, 1H, J ) 8.5 Hz), 5.08 (m, 1H), 2.67-2.77 (m, 2H). FAB-
HRMS: calcd, 385.1625; found, 385.1634 [M + H+].
1
(17 mg, white powder), mp 102-104 °C. H NMR (DMSO-d6,
500 MHz): δ 12.05 (s, 1H), 10.35 (s, 1H), 9.99 (s, 1H), 9.93 (s,
1H), 7.76 (d, 1H, J ) 8.2 Hz), 7.70 (s, 1H), 7.64 (d, 2H, J ) 7.8
Hz), 7.55-7.60 (m, 6H), 7.54 (t, 1H, J ) 7.8 Hz), 7.45 (t, 2H,
J ) 7.5 Hz), 7.41 (d, 1H, J ) 8.2 Hz), 7.36 (d, 2H, J ) 7.8 Hz),
7.34 (d, 2H, J ) 7.0 Hz), 3.57 (m, 1H), 2.80 (dd, 1H, J ) 5.5/
16.0 Hz), 2.55-2.63 (m, 3H). FAB-HRMS: calcd, 460.1986;
found, 460.1968 [M + H+].
5-[N ′-(3-Ac e t im id o y la m in o b e n zo y l)h y d r a zin o ]-3-
p h en yl-5-oxop en ta n oic Acid Tr iflu or oa ceta te (21a ). The
procedure is similar to the general procedures for Ib, IIIa , IVb,
and Vb (15 mg, oil): 1H NMR (DMSO-d6, 500 MHz): δ 12.10
(s, 1H), 10.26 (s, 1H), 9.92 (s, 1H), 7.82 (s, 3H), 7.43 (m, 2H),
7.36 (t, 1H, J ) 8.0 Hz), 7.28-7.34 (m, 4H), 7.22 (m, 1H), 7.14
(d, 1H, J ) 8.0 Hz), 3.55 (m, 1H), 2.82 (dd, 1H, J ) 5.5/16.0
Hz), 2.47-2.62 (m, 6H). FAB-HRMS: calcd, 383.1719; found,
383.1735 [M + H+].
3-{N-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]ca r b on yl}-
a m in o-3-(4-flu or op h en yl)p r op ion ic Acid Tr iflu or oa ce-
ta te (22b). The procedure is similar to the general procedures
for Ia , II, IIIa , IVa , and Va (7 mg, white powder), mp 116-
1
120 °C. H NMR (DMSO-d6, 500 MHz): δ 12.26 (s, 1H), 10.19
5-[N′-(3-Ace t im id oyla m in ob e n zoyl)h yd r a zin o]-3-(4-
flu or oph en yl)-5-oxopen tan oic Acid Tr iflu or oacetate (21b).
The procedure is similar to the general procedures for Ib, IIIa ,
IVb, and Vb (8 mg, white powder), mp 81-85 °C. 1H NMR
(DMSO-d6, 500 MHz): δ 12.05 (s, 1H), 11.25 (s, 1H), 10.37 (s,
1H), 9.98 (s, 1H), 9.52 (s, 1H), 8.63 (s, 1H), 7.89 (d, 1H, J )
7.8 Hz), 7.77 (s, 1H), 7.63 (t, 1H, J ) 8.0 Hz), 7.48 (d, 1H, J )
8.0 Hz), 7.30 (m, 2H), 7.10 (m, 2H), 3.55 (m, 1H), 2.76 (dd,
1H, J ) 5.5/16.0 Hz), 2.49-2.59 (m, 3H), 2.33 (s, 3H). FAB-
HRMS: calcd, 401.1626; found, 401.1621 [M + H+].
5-[N′-(3-Ace t im id oyla m in ob e n zoyl)h yd r a zin o]-3-(4-
ch lor op h en yl)-5-oxop en t a n oic Acid Tr iflu or oa cet a t e
(21c). The procedure is similar to the general procedures for
Ib, IIIa , IVb, and Vb (9 mg, oil): 1H NMR (DMSO-d6, 500
MHz): δ 12.05 (s, 1H), 11.24 (s, 1H), 10.37 (s, 1H), 9.98 (s,
1H), 9.51 (s, 1H), 8.63 (s, 1H), 7.88 (d, 1H, J ) 7.8 Hz), 7.76
(s, 1H), 7.64 (t, 1H, J ) 7.8 Hz), 7.48 (d, 1H, J ) 7.8 Hz), 7.23-
7.38 (m, 4H), 3.56 (m, 1H), 2.76 (dd, 1H, J ) 5.5/16.0 Hz),
2.50-2.60 (m, 3H), 2.33 (s, 3H). FAB-HRMS: calcd, 417.1330;
found, 417.1342 [M + H+].
5-[N′-(3-Ace t im id oyla m in ob e n zoyl)h yd r a zin o]-3-(4-
b r om op h en yl)-5-oxop en t a n oic Acid Tr iflu or oa cet a t e
(21d ). The procedure is similar to the general procedures for
Ib, IIIa , IVb, and Vb (5 mg, white powder), mp 98-102 °C.
1H NMR (DMSO-d6, 500 MHz): δ 12.08 (s, 1H), 11.23 (s, 1H),
10.34 (s, 1H), 9.98 (s, 1H), 9.51 (s, 1H), 8.63 (s, 1H), 7.89 (d,
1H, J ) 8.0 Hz), 7.76 (s, 1H), 7.64 (t, 1H, J ) 8.0 Hz), 7.48 (d,
1H, J ) 8.0 Hz), 7.47 (d, 2H, J ) 8.2 Hz), 7.23 (d, 2H, J ) 8.2
Hz), 3.55 (m, 1H), 2.76 (dd, 1H, J ) 5.5/16.0 Hz), 2.50-2.61
(m, 3H), 2.33 (s, 3H). FAB-HRMS: calcd, 461.0825; found,
461.0823 [M + H+].
5-[N′-(3-Ace t im id oyla m in ob e n zoyl)h yd r a zin o]-3-(4-
m eth oxyp h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ceta te
(21e). The procedure is similar to the general procedures for
Ib, IIIa , IVb, and Vb (5 mg, oil): 1H NMR (DMSO-d6, 500
MHz): δ 12.03 (s, 1H), 11.25 (s, 1H), 10.37 (s, 1H), 9.99 (s,
1H), 9.53 (s, 1H), 8.63 (s, 1H), 7.89 (d, 1H, J ) 7.8 Hz), 7.77
(s, 1H), 7.65 (t, 1H, J ) 7.8 Hz), 7.47 (d, 1H, J ) 7.8 Hz), 7.20
(d, 2H, J ) 8.5 Hz), 6.85 (d, 2H, J ) 8.2 Hz), 3.72 (s, 3H), 3.55
(m, 1H), 2.77 (dd, 1H, J ) 5.5/16.0 Hz), 2.50-2.60 (m, 3H),
2.33 (s, 3H). MS(ESI): m/z 413.3 [M + H+].
(s, 1H), 9.95 (s, 1H), 8.12 (s, 1H), 7.76 (d, 1H, J ) 7.8 Hz),
7.70 (s, 1H), 7.56 (s, 4H), 7.53 (t, 1H, J ) 7.8 Hz), 7.33-7.42
(m, 3H), 7.13 (t, 2H, J ) 8.8 Hz), 7.05 (d, 1H, J ) 8.8 Hz),
5.06 (m, 1H), 2.65-2.77 (m, 2H). FAB-HRMS: calcd, 403.1530;
found, 403.1537 [M + H+].
3-{N-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]ca r b on yl}-
a m in o-3-(4-ch lor op h en yl)p r op ion ic Acid Tr iflu or oa ce-
ta te (22c). The procedure is similar to the general procedures
for Ia , II, IIIa , IVa , and Va (7 mg, white powder), mp 124-
1
126 °C. H NMR (DMSO-d6, 500 MHz): δ 12.29 (s, 1H), 10.19
(s, 1H), 9.87 (s, 1H), 8.14 (s, 1H), 7.77 (d, 1H, J ) 7.8 Hz),
7.70 (s, 1H), 7.48-7.57 (m, 5H), 7.40 (d, 1H, J ) 8.4 Hz), 7.33-
7.38 (m, 4H), 7.06 (d, 1H, J ) 8.4 Hz), 5.06 (m, 1H), 2.66-
2.78 (m, 2H). FAB-HRMS: calcd, 419.1235; found, 419.1230
[M + H+].
3-{N-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]ca r b on yl}-
a m in o-3-(4-br om op h en yl)p r op ion ic Acid Tr iflu or oa ce-
ta te (22d ). The procedure is similar to the general procedures
for Ia , II, IIIa , IVa , and Va (5 mg, white powder), mp 147-
1
152 °C. H NMR (DMSO-d6, 500 MHz): δ 12.18 (s, 1H), 10.19
(s, 1H), 9.83 (s, 1H), 8.13 (s, 1H), 7.76 (d, 1H, J ) 7.8 Hz),
7.70 (s, 1H), 7.53 (t, 1H, J ) 7.8 Hz), 7.50 (d, 2H, J ) 8.5 Hz),
7.49 (s, 4H), 7.40 (d, 1H, J ) 7.8 Hz), 7.30 (d, 2H, J ) 8.5 Hz),
7.06 (d, 1H, J ) 8.2 Hz), 5.03 (m, 1H), 2.71 (m, 2H). MS(ESI):
m/z 463.1 [M + H+].
3-{N-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]ca r b on yl}-
a m in o-3-(4-m eth oxyp h en yl)p r op ion ic Acid Tr iflu or oa ce-
ta te (22e). The procedure is similar to the general procedures
for Ia , II, IIIa , IVa , and Va (5 mg, oil): 1H NMR (DMSO-d6,
500 MHz): δ 11.89 (s, 1H), 10.20 (s, 1H), 9.78 (s, 1H), 8.06 (s,
1H), 7.78 (d, 1H, J ) 7.8 Hz), 7.71 (s, 1H), 7.55 (t, 1H, J ) 7.8
Hz), 7.48 (s, 4H), 7.41 (d, 1H, J ) 7.8 Hz), 7.26 (d, 2H, J ) 8.5
Hz), 6.93 (d, 1H, J ) 8.5 Hz), 6.88 (d, 2H, J ) 8.5 Hz), 5.04
(m, 1H), 3.74 (s, 3H), 2.65-2.77 (m, 2H). FAB-HRMS: calcd,
415.1730; found, 415.1690 [M + H+].
3-{N-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]ca r b on yl}-
a m in o-3-(4-tr iflu or om eth oxyp h en yl)p r op ion ic Acid Tr i-
flu or oa ceta te (22f). The procedure is similar to the general
procedures for Ia , II, IIIa , IVa , and Va (8 mg, oil): 1H NMR
(DMSO-d6, 500 MHz): δ 12.29 (s, 1H), 10.19 (s, 1H), 9.80 (s,
1H), 8.15 (s, 1H), 7.77 (d, 1H, J ) 7.8 Hz), 7.70 (s, 1H), 7.53 (t,
1H, J ) 7.8 Hz), 7.46 (d, 2H, J ) 8.2 Hz), 7.48 (s, 4H), 7.40 (d,
1H, J ) 7.8 Hz), 7.31 (d, 2H, J ) 8.2 Hz), 7.08 (d, 1H, J ) 8.4
Hz), 5.10 (m, 1H), 2.75 (m, 2H). FAB-HRMS: calcd, 469.1447;
found, 469.1435 [M + H+].
5-[N′-(3-Acet im id oyla m in ob en zoyl)h yd r a zin o]-3-(3,5-
d ich lor op h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ceta te
(21h ). The procedure is similar to the general procedures for
Ib, IIIa , IVb, and Vb (18 mg, oil): 1H NMR (DMSO-d6, 500
MHz): δ 12.15 (s, 1H), 11.23 (s, 1H), 10.38 (s, 1H), 10.00 (s,
1H), 9.55 (s, 1H), 8.63 (s, 1H), 7.88 (d, 1H, J ) 8.0 Hz), 7.76
(s, 1H), 7.63 (t, 1H, J ) 8.0 Hz), 7.48 (d, 1H, J ) 8.0 Hz), 7.42
(s, 1H), 7.35 (s, 1H), 3.50 (m, 1H), 2.77 (dd, 1H, J ) 5.5/16.0
Hz), 2.53-2.69 (m, 3H), 2.33 (s, 3H). FAB-HRMS: calcd,
451.0941; found, 451.0930 [M + H+].
3-{N-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]ca r b on yl}-
a m in o-3-(1-n a p h t h yl)p r op ion ic Acid Tr iflu or oa cet a t e
(22g). The procedure is similar to the general procedures for
Ia , II, IIIa , IVa , and Va (5 mg, white powder), mp 151-154
1
°C. H NMR (DMSO-d6, 500 MHz): δ 12.28 (s, 1H), 10.21 (s,
5-[N′-(3-Acetim id oyla m in oben zoyl)h yd r a zin o]-3-(4-bi-
p h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ceta te (21i). The
procedure is similar to the general procedures for Ib, IIIa , IVb,
1H), 9.94 (s, 1H), 8.14 (s, 1H), 8.09 (s, 1H), 7.94 (d, 1H, J )
8.0 Hz), 7.83 (d, 1H, J ) 8.2 Hz), 7.76 (d, 1H, J ) 7.5 Hz),
7.70 (s, 1H), 7.50-7.60 (m, 7H), 7.49 (t, 1H, J ) 8.2 Hz), 7.39

Rvâ3 Integrin Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1947
(d, 1H, J ) 7.5 Hz), 7.20 (d, 1H, J ) 7.5 Hz), 5.91 (m, 1H),
2.74-2.91 (m, 2H). MS(ESI): m/z 435.2 [M + H+].
1H), 1.54-1.66 (m, 4H). FAB-HRMS: calcd, 481.1410; found,
481.1402 [M + H+].
3-{N-[N′-(3-Gu a n id in ob en zoyl)h yd r a zin o]ca r b on yl}-
a m in o-3-(3,5-d ich lor op h en yl)p r op ion ic Acid Tr iflu or o-
a ceta te (22h ). The procedure is similar to the general
procedures for Ia , II, IIIa , IVa , and Va (5 mg, white powder),
mp 143-147 °C. 1H NMR (DMSO-d6, 500 MHz): δ 12.05 (s,
1H), 10.21 (s, 1H), 9.71 (s, 1H), 8.24 (s, 1H), 7.78 (d, 1H, J )
7.8 Hz), 7.71 (s, 1H), 7.56 (s, 1H), 7.54 (t, 1H, J ) 7.8 Hz),
7.43 (s, 4H), 7.41 (d, 1H, J ) 7.8 Hz), 7.40 (s, 2H), 7.11 (d, 1H,
J ) 8.2 Hz), 5.04 (m, 1H), 2.76 (m, 2H). MS(ESI): m/z 453.1
[M + H+].
5-{N′-[5-(4-Meth ylp yr id in -2-yla m in o)p en ta n oyl]h yd r a -
zin o}-3-p h en yl-5-oxop en t a n oic Acid Tr iflu or oa cet a t e
(23a ). The procedure is similar to the general procedures for
Ib, IIIb, and Vb (5 mg, oil): 1H NMR (DMSO-d6, 500 MHz):
δ 12.91 (s, 1H), 11.98 (s, 1H), 9.71 (s, 1H), 9.65 (s, 1H), 8.37
(s, 1H), 7.78 (d, 1H, J ) 6.5 Hz), 7.20-7.28 (m, 4H), 7.17 (t,
1H, J ) 6.2 Hz), 6.79 (s, 1H), 6.68 (d, 1H, J ) 6.5 Hz), 3.46
(m, 1H), 3.26 (m, 2H), 2.77 (dd, 1H, J ) 5.5/16.0 Hz), 2.39-
2.48 (m, 3H), 2.31 (s, 3H), 2.13 (m, 2H), 1.57 (m, 4H). FAB-
HRMS: calcd, 413.2190; found, 413.2187 [M + H+].
5-{N′-[5-(4-Meth ylp yr id in -2-yla m in o)p en ta n oyl]h yd r a -
zin o}-3-(4-flu or op h en yl)-5-oxop en ta n oic Acid Tr iflu or o-
a ceta te (23b). The procedure is similar to the general
procedures for Ib, IIIb, and Vb (9 mg, oil): 1H NMR (DMSO-
d6, 500 MHz): δ 13.07 (s, 1H), 12.07 (s, 1H), 9.71 (s, 1H), 9.65
(s, 1H), 8.59 (s, 1H), 7.80 (m, 1H), 7.38-7.47 (m, 1H), 7.25 (m,
1H), 7.01 (t, 1H, J ) 8.5 Hz), 6.84 (s, 1H), 6.70 (d, 1H, J ) 6.5
Hz), 3.46 (m, 1H), 3.27 (m, 2H), 2.91 (m, 1H), 2.70 (dd, 1H, J
) 5.5/16.0 Hz), 2.37-2.52 (m, 2H), 2.32 (s, 3H), 2.13 (m, 2H),
1.57 (m, 4H). FAB-HRMS: calcd, 431.2095; found, 431.2110
[M + H+].
5-{N′-[5-(4-Meth ylp yr id in -2-yla m in o)p en ta n oyl]h yd r a -
zin o}-3-(4-ch lor op h en yl)-5-oxop en ta n oic Acid Tr iflu or o-
a ceta te (23c). The procedure is similar to the general
procedures for Ib, IIIb, and Vb (23 mg, oil): 1H NMR (DMSO-
d6, 500 MHz): δ 13.11 (s, 1H), 9.72 (s, 1H), 9.65 (s, 1H), 8.60
(s, 1H), 7.80 (s, 1H), 7.40 (m, 2H), 7.19-7.34 (m, 2H), 6.84 (s,
1H), 6.71 (d, 1H, J ) 6.5 Hz), 3.45 (m, 1H), 3.28 (m, 2H), 3.00-
3.18 (m, 1H), 2.88-2.98 (m, 1H), 2.70 (m, 1H), 2.39-2.53 (m,
1H), 2.31 (s, 3H), 2.13 (m, 2H), 1.52-1.70 (m, 4H). FAB-
HRMS: calcd, 447.1800; found, 447.1807 [M + H+].
5-{N′-[5-(4-Meth ylp yr id in -2-yla m in o)p en ta n oyl]h yd r a -
zin o}-3-(4-br om op h en yl)-5-oxop en ta n oic Acid Tr iflu or o-
a ceta te (23d ). The procedure is similar to the general
procedures for Ib, IIIb, and Vb (16 mg, oil): 1H NMR (DMSO-
d6, 500 MHz): δ 13.20 (s, 1H), 12.08 (s, 1H), 9.72 (s, 1H), 9.65
(s, 1H), 8.50 (s, 1H), 7.80 (d, 1H, J ) 6.5 Hz), 7.44 (d, 2H, J )
8.0 Hz), 7.19 (d, 2H, J ) 8.0 Hz), 6.81 (s, 1H), 6.68 (d, 1H, J
) 6.5 Hz), 3.44 (m, 1H), 3.26 (m, 2H), 2.91 (m, 1H), 2.70 (dd,
1H, J ) 5.5/16.0 Hz), 2.37-2.54 (m, 2H), 2.31 (s, 3H), 2.12 (m,
2H), 1.57 (m, 4H). FAB-HRMS: calcd, 491.1295; found,
491.1296 [M + H+].
5-{N′-[5-(4-Meth ylp yr id in -2-yla m in o)p en ta n oyl]h yd r a -
zin o}-3-(4-m et h oxyp h en yl)-5-oxop en t a n oic Acid Tr i-
flu or oa ceta te (23e). The procedure is similar to the general
procedures for Ib, IIIb, and Vb (5 mg, oil): 1H NMR (DMSO-
d6, 500 MHz): δ 13.14 (s, 1H), 9.69 (s, 1H), 9.65 (s, 1H), 8.61
(s, 1H), 7.80 (d, 1H, J ) 6.5 Hz), 7.13 (d, 2H, J ) 8.5 Hz), 6.84
(s, 1H), 6.81 (d, 2H, J ) 8.5 Hz), 6.69 (d, 1H, J ) 6.5 Hz), 3.70
(s, 3H), 3.41 (m, 1H), 3.24 (m, 2H), 2.67 (dd, 1H, J ) 5.5/16.0
Hz), 2.35-2.47 (m, 2H), 2.32 (s, 3H), 2.13 (m, 2H), 1.54-1.66
(m, 4H). FAB-HRMS: calcd, 443.2294; found, 443.2279 [M +
H+].
5-{N′-[5-(4-Meth ylp yr id in -2-yla m in o)p en ta n oyl]h yd r a -
zin o}-3-(3,5-d ich lor op h en yl)-5-oxop en ta n oic Acid Tr i-
flu or oa ceta te (23h ). The procedure is similar to the general
procedures for Ib, IIIb, and Vb (21 mg, oil): 1H NMR (DMSO-
d6, 500 MHz): δ 13.06 (s, 1H), 9.73 (s, 1H), 9.67 (s, 1H), 8.59
(s, 1H), 7.80 (m, 1H), 7.39 (s, 1H), 7.30 (s, 2H), 6.84 (m, 1H),
6.71 (d, 1H, J ) 6.5 Hz), 3.45 (m, 1H), 3.27 (m, 2H), 3.06-
3.20 (m, 1H), 2.95 (m, 1H), 2.71 (dd, 1H, J ) 5.5/16.5 Hz), 2.58
(dd, 1H, J ) 9.8/16.5 Hz), 2.32 (s, 3H), 2.28 (m, 1H), 2.12 (m,
5-{N′-[5-(4-Meth ylp yr id in -2-yla m in o)p en ta n oyl]h yd r a -
zin o}-3-(4-bip h en yl)-5-oxop en ta n oic Acid Tr iflu or oa ce-
ta te (23i). The procedure is similar to the general procedures
for Ib, IIIb, and Vb (18 mg, white powder), mp 176-177 °C.
1H NMR (DMSO-d6, 500 MHz): δ 12.00 (s, 1H), 9.73 (s, 1H),
9.65 (s, 1H), 7.79 (d, 1H, J ) 6.5 Hz), 7.63 (d, 2H, J ) 7.5 Hz),
7.56 (d, 2H, J ) 8.2 Hz), 7.44 (m, 2H), 7.30-7.36 (m, 3H), 6.35
(m, 1H), 6.28 (d, 1H, J ) 6.5 Hz), 6.25 (s, 1H), 3.52 (m, 1H),
3.17 (m, 2H), 2.75 (dd, 1H, J ) 5.5/16.5 Hz), 2.55 (dd, 1H, J )
9.8/16.5 Hz), 2.44-2.51 (m, 2H), 2.31 (s, 3H), 2.09 (m, 2H),
1.46-1.58 (m, 4H). FAB-HRMS: calcd, 489.2503; found,
489.2522 [M + H+].
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h yd r a zin o]ca r bon yl}a m in o-3-p h en ylp r op ion ic Acid Tr i-
flu or oa ceta te (24a ). The procedure is similar to the general
procedures for Ia , II, IIIb, and Vb (17 mg, oil): 1H NMR
(DMSO-d6, 500 MHz): δ 13.15 (s, 1H), 9.47 (s, 1H), 8.60 (s,
1H), 7.86 (s, 1H), 7.78 (d, 1H, J ) 6.5 Hz), 7.26-7.32 (m, 4H),
7.18-7.23 (m, 1H), 6.88 (d, 1H, J ) 8.5 Hz), 6.83 (s, 1H), 6.70
(d, 1H, J ) 6.2 Hz), 5.03 (m, 1H), 3.27 (m, 2H), 2.63-2.74 (m,
2H), 2.31 (s, 3H), 2.13 (t, 2H, J ) 6.5 Hz), 1.54-1.62 (m, 4H).
FAB-HRMS: calcd, 414.2141; found, 414.2145 [M + H+].
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h y d r a zin o ]c a r b o n y l}a m in o -3-(4-flu o r o p h e n y l)p r o -
p ion ic Acid Tr iflu or oa ceta te (24b). The procedure is
similar to the general procedures for Ia , II, IIIb, and Vb (5
mg, white powder), mp 49-51 °C. 1H NMR (DMSO-d6, 500
MHz): δ 13.00 (s, 1H), 12.37 (s, 1H), 9.46 (s, 1H), 8.48 (s, 1H),
7.87 (s, 1H), 7.78 (d, 1H, J ) 6.5 Hz), 7.33 (m, 2H), 7.11 (m,
2H), 6.90 (d, 1H, J ) 8.5 Hz), 6.80 (s, 1H), 6.68 (d, 1H, J ) 6.5
Hz), 5.02 (m, 1H), 3.26 (m, 2H), 2.63-2.75 (m, 2H), 2.30 (s,
3H), 2.12 (t, 2H, J ) 6.5 Hz), 1.54-1.61 (m, 4H). FAB-HRMS:
calcd, 432.2047; found, 432.2041 [M + H+].
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h y d r a zin o ]c a r b o n y l}a m in o -3-(4-c h lo r o p h e n y l)p r o -
p ion ic Acid Tr iflu or oa ceta te (24c). The procedure is simi-
lar to the general procedures for Ia , II, IIIb, and Vb (25 mg,
1
white powder), mp 47-49 °C. H NMR (DMSO-d6, 500 MHz):
δ 13.07 (s, 1H), 9.47 (s, 1H), 8.56 (s, 1H), 7.89 (s, 1H), 7.78 (d,
1H, J ) 6.5 Hz), 7.30-7.36 (m, 4H), 6.92 (d, 1H, J ) 8.5 Hz),
6.82 (s, 1H), 6.69 (d, 1H, J ) 6.5 Hz), 5.01 (m, 1H), 3.26 (m,
2H), 2.63-2.75 (m, 2H), 2.31 (s, 3H), 2.12 (t, 2H, J ) 6.5 Hz),
1.54-1.61 (m, 4H). FAB-HRMS: calcd, 448.1752; found,
448.1769 [M + H+].
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h y d r a zin o ]c a r b o n y l}a m in o -3-(4-b r o m o p h e n y l)p r o -
p ion ic Acid Tr iflu or oa ceta te (24d ). The procedure is
similar to the general procedures for Ia , II, IIIb, and Vb (16
mg, yellow powder), mp 58-62 °C. 1H NMR (DMSO-d6, 500
MHz): δ 13.03 (s, 1H), 9.47 (s, 1H), 8.50 (s, 1H), 7.90 (s, 1H),
7.78 (d, 1H, J ) 6.5 Hz), 7.48 (d, 2H, J ) 8.0 Hz), 7.26 (d, 2H,
J ) 8.0 Hz), 6.93 (d, 1H, J ) 8.5 Hz), 6.81 (s, 1H), 6.68 (d, 1H,
J ) 6.5 Hz), 5.00 (m, 1H), 3.26 (m, 2H), 2.62-2.77 (m, 2H),
2.31 (s, 3H), 2.12 (t, 2H, J ) 6.5 Hz), 1.52-1.65 (m, 4H). FAB-
HRMS: calcd, 492.1247; found, 492.1249 [M + H+].
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h yd r a zin o]ca r b on yl}a m in o-3-(4-m e t h oxyp h e n yl)p r o-
p ion ic Acid Tr iflu or oa ceta te (24e). The procedure is simi-
lar to the general procedures for Ia , II, IIIb, and Vb (22 mg,
1
white powder), mp 43-45 °C. H NMR (DMSO-d6, 500 MHz):
δ 13.06 (s, 1H), 12.22 (s, 1H), 9.46 (s, 1H), 8.56 (s, 1H), 7.82
(s, 1H), 7.78 (d, 1H, J ) 6.5 Hz), 7.21 (d, 2H, J ) 8.7 Hz), 6.84
(d, 2H, J ) 8.7 Hz), 6.82 (s, 1H), 6.79 (d, 1H, J ) 8.5 Hz), 6.69
(d, 1H, J ) 6.5 Hz), 4.97 (m, 1H), 3.71 (s, 3H), 3.26 (m, 2H),
2.60-2.72 (m, 2H), 2.31 (s, 3H), 2.12 (t, 2H, J ) 6.5 Hz), 1.55-
1.61 (m, 4H). FAB-HRMS: calcd, 444.2247; found, 444.2239
[M + H+].
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h ydr azin o]car bon yl}am in o-3-(4-tr iflu or om eth oxyph en yl)-
p r op ion ic Acid Tr iflu or oa ceta te (24f). The procedure is
similar to the general procedures for Ia , II, IIIb, and Vb (24
mg, oil): 1H NMR (DMSO-d6, 500 MHz): δ 12.89 (s, 1H), 9.46

1948 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Sulyok et al.
(s, 1H), 8.43 (s, 1H), 7.89 (s, 1H), 7.77 (d, 1H, J ) 6.5 Hz),
7.42 (d, 2H, J ) 8.5 Hz), 7.28 (d, 2H, J ) 8.5 Hz), 6.93 (d, 1H,
J ) 8.5 Hz), 6.80 (s, 1H), 6.68 (d, 1H, J ) 6.5 Hz), 5.05 (m,
1H), 3.26 (m, 2H), 2.66-2.77 (m, 2H), 2.31 (s, 3H), 2.12 (m,
2H), 1.53-1.62 (m, 4H). FAB-HRMS: calcd, 498.1964; found,
498.1968 [M + H+].
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h ydr azin o]car bon yl}am in o-3-(1-n aph th yl)pr opion ic Acid
Tr iflu or oa ceta te (24g). The procedure is similar to the
general procedures for Ia , II, IIIb, and Vb (28 mg, white
powder), mp 103-106 °C. 1H NMR (DMSO-d6, 500 MHz): δ
13.00 (s, 1H), 9.49 (s, 1H), 8.53 (s, 1H), 8.11 (d, 1H, J ) 8.5
Hz), 7.94 (d, 1H, J ) 7.5 Hz), 7.86 (s, 1H), 7.82 (d, 1H, J ) 8.0
Hz), 7.76 (d, 1H, J ) 6.5 Hz), 7.57 (t, 1H, J ) 7.2 Hz), 7.50-
7.55 (m, 2H), 7.46 (t, 1H, J ) 7.5 Hz), 7.05 (d, 1H, J ) 8.5
Hz), 6.81 (s, 1H), 6.66 (d, 1H, J ) 7.5 Hz), 5.87 (m, 1H), 3.26
(m, 2H), 2.86 (dd, 1H, J ) 6.5/16.2 Hz), 2.75 (dd, 1H, J ) 7.5/
16.2 Hz), 2.29 (s, 3H), 2.13 (m, 2H), 1.58 (m, 4H). FAB-
HRMS: calcd, 464.2299; found, 464.2284 [M + H+].
2. Recep tor Bin d in g Assa y. Inhibitory effects of aza- and
azacarba RGD mimetics were quantified by measuring their
effect on the interactions between immobilized integrin and
biotinylated soluble ligands. Purified vitronectin or fibrinogen
(1 mg/mL; pH 8.2) was biotinylated with N-hydroxysuccin-
imidobiotin (100 µg/mL; 1 h, 20 °C) before dialysis into PBS.
Integrins diluted to 1 µg/mL (∼1:1000 dilution of stock) were
adsorbed to 96-well nontissue culture treated microtiter plates.
After free protein binding sites were blocked, the biotinylated
ECM ligand at 1 µg/mL was added and incubation continued
at 37 °C for 3 h. Unbound ligand was washed away, and bound
biotin was detected with alkaline-phosphate-conjugated anti-
biotin antibodies. For competition assays, compounds were
serially diluted before addition to integrin-coated wells, fol-
lowed by co-incubation with biotinylated ligand and detection
as described above. Assays were performed in triplicate, the
mean binding values were fitted to a sigmoid, and the IC50
was derived. Values shown in Tables 1-3 represent the mean
of at least three separate determinations of IC50. External
standards of linear GRGDSPK and cyclo(RGDfV) were rou-
tinely included to monitor the dynamic range and the stability
of the assay. Intra-assay variation, the relationship between
IC50 values of standard inhibitors and the test substances, was
typically less than (5%, and inter-assay variation in absolute
IC50 was typically a factor of less than (2.
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h y d r a zin o ]c a r b o n y l}a m in o -3-(3,5-d ic h lo r o p h e n y l)-
p r op ion ic Acid Tr iflu or oa ceta te (24 h ). The procedure is
similar to the general procedures for Ia , II, IIIb, and Vb (26
1
mg, white powder), mp 150-152 °C. H NMR (DMSO-d6, 500
MHz): δ 13.05 (s, 1H), 9.50 (s, 1H), 7.95 (s, 1H), 7.76 (d, 1H,
J ) 5.5 Hz), 7.42 (m, 1H), 7.37 (m, 2H), 7.13 (s, 1H), 6.81 (s,
1H), 6.31 (m, 2H), 4.99 (m, 1H), 3.17 (m, 2H), 2.67-2.75 (m,
2H), 2.14 (s, 3H), 2.11 (m, 2H), 1.59 (m, 2H), 1.53 (m, 2H).
FAB-HRMS: calcd, 482.1363; found, 482.1371 [M + H+].
Ack n ow led gm en t. The authors gratefully acknowl-
edge technical assistance from M. Kranawitter, B.
Cordes, and A. Schro¨der. We thank D. Hahn (Merck
KGaA) for performing the screening assays and Dr. J .
Osterodt (Merck KGaA) for the FAB-HRMS analyses
of the RGD mimetics. Financial support was provided
by the Deutsche Forschungsgemeinschaft and the Fonds
der Chemischen Industrie.
3-{N-[N′-(5-Gu a n id in op en ta n oyl)h yd r a zin o]ca r bon yl}-
a m in o-3-p h en ylp r op ion ic Acid Tr iflu or oa ceta te (25). The
procedure is similar to the general procedures for Ia , II, III
(5-(N-Fmoc)-aminopentanoic acid), and Vb (22 mg, oil): 1H
NMR (DMSO-d6, 500 MHz): δ 12.21 (s, 1H), 9.46 (s, 1H), 7.85
(s, 1H), 7.53 (m, 1H), 7.27-7.32 (m, 5H), 7.21 (m, 1H), 6.86
(d, 1H, J ) 8.7 Hz), 5.03 (m, 1H), 3.90 (s, 4H), 3.08 (m, 2H),
2.69 (m, 2H), 2.10 (t, 2H, J ) 6.5 Hz), 1.40-1.56 (m, 4H). FAB-
HRMS: calcd, 365.1859; found, 365.1939 [M + H+].
Su p p or tin g In for m a tion Ava ila ble: Additional synthesis
procedures and table of analytical data. This material is
available free of charge via the Internet at http://pubs.acs.org.
3-{N-[N′-(3-(N-P yr id in -2-yl)-a m in oben zoyl)h yd r a zin o]-
ca r bon yl}a m in o-3-(3,5-d ich lor op h en yl)p r op ion ic Acid
Tr iflu or oa ceta te (26). The procedure is similar to the general
procedures for Ia , II, III (3-(N-pyridin-2-yl)-aminobenzoic acid
(17)), and Va (16 mg, white powder), mp 110-114 °C. 1H NMR
(DMSO-d6, 500 MHz): δ 12.03 (s, 1H), 10.22 (s, 1H), 9.89 (s,
1H), 9.65 (s, 1H), 8.12 (d, 1H, J ) 6.5 Hz), 7.99 (s, 1H), 7.82
(d, 1H, J ) 8.0 Hz), 7.72 (t, 1H, J ) 7.5 Hz), 7.46 (d, 1H, J )
7.5 Hz), 7.41 (m, 1H), 7.39 (s, 1H), 7.35 (s, 2H), 6.95 (d, 1H, J
) 8.5 Hz), 6.85 (t, 1H, J ) 6.0 Hz), 3.51 (m, 1H), 2.78 (dd, 1H,
J ) 5.7/16.2 Hz), 2.63 (dd, 1H, J ) 9.8/16.3 Hz), 2.57 (m, 2H).
FAB-HRMS: calcd, 487.0941; found, 487.0945 [M + H+].
3-{N-[N′-(5-(4-Met h ylp yr id in -2-yla m in o)p en t a n oyl)-
h yd r a zin o]ca r b on yl}a m in op r op ion ic Acid Tr iflu or o-
a ceta te (27). The procedure is similar to the general proce-
dures for Ia (3-(N-Fmoc)-aminopropionic acid), II, IIIb, and
Vb (27 mg, white powder), mp 107-110 °C. 1H NMR (DMSO-
d6, 500 MHz): δ 13.17 (s, 1H), 9.43 (s, 1H), 8.62 (s, 1H), 7.80
(d, 1H, J ) 6.8 Hz), 7.78 (s, 1H), 6.84 (s, 1H), 6.70 (d, 1H, J )
6.5 Hz), 6.33 (t, 1H, J ) 5.8 Hz), 3.27 (m, 2H), 3.19 (m, 2H),
2.34 (t, 2H, J ) 6.8 Hz), 2.32 (s, 3H), 2.12 (m, 2H), 1.52-1.62
(m, 4H). FAB-HRMS: calcd, 338.1828; found, 338.1823 [M +
H+].
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