Multimeric cyclic RGD peptides as potential tools for tumor targeting: Solid-phase peptide synthesis and chemoselective oxime ligation

Article abstract of DOI:10.1002/chem.200204304

The αvβ3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. Targeting this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Solid-phase peptide synthesis of multimeric cyclo(-RGDfE-)-peptides is described, which offer the possibility of enhanced integrin targeting due to polyvalency effects. These peptides contain an aminooxy group for versatile chemoselective oxime ligation. Conjugation with para-trimethylstannylbenzaldehyde results in a precursor for radioiododestannylation, which would allow them to be used as potential tools for targeting and imaging αvβ3-expressing tumor cells. The conjugates were obtained in good yield without the need of a protection strategy and under mild conditions.

Full text of DOI:10.1002/chem.200204304

FULL PAPER
Multimeric Cyclic RGD Peptides as Potential Tools for Tumor Targeting:
Solid-Phase Peptide Synthesis and Chemoselective Oxime Ligation
Georgette Thumshirn,[a] Ulrich Hersel,[a] Simon L. Goodman,[b] and Horst Kessler*[a]
Dedicated to Professor Dr. Peter Welzel on the occasion of his 65th birthday
Abstract: The avb3 integrin receptor
plays an important role in human meta-
stasis and tumor-induced angiogenesis.
Targeting this receptor may provide
information about the receptor status
of the tumor and enable specific ther-
apeutic planning. Solid-phase peptide
synthesis of multimeric cyclo(-RGDfE-)-
peptides is described,which offer the
possibility of enhanced integrin target-
ing due to polyvalency effects. These
peptides contain an aminooxy group for
versatile chemoselective oxime ligation.
Conjugation with para-trimethylstannyl-
benzaldehyde results in a precursor for
radioiododestannylation,which would
allow them to be used as potential tools
for targeting and imaging avb3-express-
ing tumor cells. The conjugates were
obtained in good yield without the need
of a protection strategy and under mild
conditions.
Keywords: chemoselective ligation
¥ integrin ¥ multimer ¥ peptides ¥
tumor targeting
RGD sequence have been developed as ligands for the avb3
integrin.[17] The first synthetic,highly active and selec-
tive antagonist of the avb3 receptor, cyclo(-RGDfV-) was
developed in our group.[12] Systematic derivatization of
this peptide resulted in the N-alkylated cyclopeptide
cyclo(-RGDf[NMe]V-),[18,19] which has entered phase II clin-
ical studies as an angiogenesis inhibitor (cilengitide,code
EMD 121974,Merck KGaA). Furthermore,regression of
primary tumors and eradication of micrometastases by
combining a specific antiangiogenic therapy with our cyclo-
peptide and an immunotherapy with a tumor-specific anti-
body interleukin 2 fusion protein have been reported.[20] In
addition,the highly active and selective cyclic pentapeptides
can be used for targeting and imaging avb3-expressing tumor
cells[15] as well as endothelial cells during neovasculariza-
tion[4,21] in research,diagnostics,and therapy. Since an
exchange of valine in the cyclic pentapeptide for another
amino acid has no significant influence on the activity and
selectivity,this position is valuable for molecular and func-
tional modifications.[22] Cyclo(-RGDfK-),which can easily be
functionalized because of the e-amino group on the lysine side
chain was therefore used for tumor targeting[23] and imaging[24]
as well as for stimulation of cell adhesion.[25,26]
Introduction
Cell cell and cell matrix interactions are involved in many
physiological processes including embryogenesis,cell differ-
entiation,hemostasis,wound healing,and immune re-
sponse.[1 They are also fundamental to tumor invasion and
3]
formation of metastases[4] as well as to tumor-induced angio-
genesis.[5 7] Integrins appear to be the major class of receptors
through which cells attach to the extracellular matrix. In
addition to these adhesive functions,integrins transduce
messages by classical signaling pathways and may influence
proliferation and apoptosis of tumor cells as well as activated
endothelial cells.[5,6,8,9] The tripetide sequence RGD (Arg-
Gly-Asp) is a common cell-recognition motif for a wide
variety of integrin receptors. The conformation of the RGD-
containing loop and its flanking amino acids in proteins are
mainly responsible for their different integrin affinity.[9 12] An
integrin with a well-characterized involvement in angiogen-
esis[13,14] and tumor invasiveness[15] is avb3,a vitronectin
receptor. This integrin is found on many cell types including
certain tumor cells,activated endothelial cells and smooth-
[16]
muscle cells,epithelial cells,platelets,and osteoclasts.
Cyclic peptides containing a conformationally restrained
The aim of our work is to develop improved ligands for
avb3 integrin targeting. We decided to mimic naturally
occurring examples to enhance the affinity of the receptor
ligand interactions using polyvalency.[27 35] Thus we developed
multimeric compounds in which the RGD sequence is locally
enriched and can bind polyvalently,that is,simultaneously,to
several integrins. The increased affinity of RGD ligands due
to multivalent interactions was shown for example by Kok
[a] Prof. Dr. H. Kessler,G. Thumshirn,U. Hersel
Institut f¸r Organische Chemie und Biochemie
Technische Universit‰t M¸nchen
Lichtenbergstrasse 4,85747 Garching (Germany)
Fax : (‡49)89-2891-3210
E-mail: horst.kessler@ch.tum.de
[b] Dr. S. L. Goodman
Oncology Research,Merck KGaA
64271 Darmstadt (Germany)
Chem. Eur. J. 2003, 9,2717 2725
DOI: 10.1002/chem.200204304
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2717

H. Kessler et al.
FULL PAPER
et al.[23] and Maheshwari et al.[36] To adapt the synthesis of our
multimeric compounds to solid-phase peptide synthesis
(SPPS) we used the MAP (multiple-antigen peptide) system
developed by Tam et al.[37,38] based on lysines as branching
units. Multiple copies of a ligand can be bound to this simple,
immunogenically inert branching core. The result is a large
macromolecule with a unique three-dimensional configura-
order to use them as tracers for
tumor targeting. The aminooxy
group for the chemical ligation
was introduced as N-b-(N-tert-
butyloxycarbonylaminooxyacetyl)-
l-diaminopropionic acid,and a
lysine core was used as branching
unit.
In order to prepare these com-
pounds by SPPS we synthesized a
heptaethylene glycol spacer with
an amino acid superstructure,that
is,a compound containing an
Fmoc-protected amino group
and a carboxyl group (Scheme 1).
oxime
ligation
radio label
precursor
lysine
core
tion. For further elucidation of the influence of core on the
[39,40]
immunogenicity of MAPs,Veprek et al.
prepared a
n = 1, 2
symmetrical core with b-Ala-Lys dipeptide as building unit.
However,no significant improvement of immunological
properties was detected.
RGD
RGD
n
Figure 1. General structure
of the target molecules.
For targeting it is necessary to use a radio label or a
fluorescence label. We chose oxime ligation,a chemoselective
reaction of an aldehyde group with an hydroxyl amino group.
The high efficiency and selectivity of the aminooxy aldehyde
coupling reaction has been demonstrated successfully by
The Fmoc heptaethylene glycol amino acid 4 was synthesized
in a similar manner to Boumrah et al.[57] in good yield without
the need for purification by chromatography. A report on the
attaching a variety of substances to proteins,[41 45] by chemical
50]
ligation of peptides,[46
by conjugation of peptides with
53]
carbohydrates[51 or oligonucleotides,[54] and by labeling of
oligonucleotides and RNA.[55] The hydroxylamine function-
ality can easily be implemented as a tert-butyloxycarbonyl-
protected aminooxy-functionalized amino acid building
block. The oxime ligation represents an elegant way to link
totally unprotected aminooxy-functionalized peptidic frag-
ments to any marker molecule. The label can be incorporated
site-specifically,that is,the conjugation does not interfere with
the RGD binding motif. The oxime ligation is compatible with
all standard amino-acid residues,with the exception of N-
terminal cysteine. Moreover,the oxime bond is known to be
reasonably stable both in vitro and in vivo. In order to
introduce radioactivity,we used a trimethylstannyl precursor
instead of a trimethylsilyl precursor,as suggested by Vaidya-
nathan et al.[56] The iododestannylation uses mild but high
yielding conditions. To take advantage of these results,we
decided to synthesize a trimethylstannylbenzaldehyde as the
carbonyl partner for the chemoselective oxime formation.
The spatial arrangement and separations of the RGD
moieties within one multimer for optimal cell targeting is not
known. Thus we introduced aminohexanoic acid and a longer
heptaethylene glycol moiety as spacer molecules. The use of
ethylene glycol spacers is desirable due to the solubility
problems of peptides containing long alkyl spacers.[57] Moreover,
polyethylene glycol spacers are nontoxic and unreactive. The
heptaethylene glycol spacer must be synthesized as a 9-fluo-
renylmethoxycarbonyl (Fmoc) -protected amino acid to make it
available as a building block in SPPS. In order to use SPPS for
coupling the RGD moiety to the branching core,we developed
the pentapeptide cyclo(-Arg[Pbf]GlyAsp[tBu]-d-PheGlu-). In
this work glutaminic acid was chosen as fifth amino acid that
offers a free carboxylic acid group for peptide coupling reactions.
The resulting general structure of our compounds is shown
in Figure 1.
O
a
O
O
HO
6 H
HO
O
6
1
2
O
O
b, c, d
e, f
O
Fmoc
O
H2N
O
N
H
6
6
3
4
Scheme 1. Synthesis of Fmoc heptaethylene glycol amino acid 4. a) Diazo-
acetic acid tert-butylester,BF 3 ¥ OEt2,DCM; b) TsCl,Et 3N; c) NaN3,
DMF; d) H2,Pd/C,EtOH,34% (4 steps); e) FmocCl,10% NaHCO
THF; f) 90% TFA/H2O,97% (2 steps).
,
3
same spacer was recently published by Dekker et al. whose
synthesis is one step longer than ours.[58] Starting from
hexaethylene glycol,we introduced the carboxyl moiety by
treatment with diazoacetic acid tert-butylester in the presence
of boron trifluoride etherate[59] with 82% yield. The resulting
heptaethylene glycol acid tert-butylester was first activated
with tosylchloride and then treated with sodium azide in dry
DMF. The corresponding amine 3 was obtained by catalytic
hydrogenation. Protection with an Fmoc group and final
deprotection of the tert-butyl ester gave 20-(N-Fmoc)-amino-
3,6,9,12,15,18-hexaoxaeicosanoic acid (Fmoc-Hegas) 4 in an
overall yield of 33%.
The cyclic pentapeptide cyclo(-Arg[Pbf]GlyAsp[tBu]-d-
PheGlu-) was synthesized according to the method already
developed in our group[22,60] by a combined solid-phase so-
lution methodology. Starting from Gly in order to prevent
racemization during cyclization later on,the linear peptide
was synthesized by SPPS by using an Fmoc strategy[61] on
tritylchloride polystyrene (TCP) resin[62,63] in N-methyl-2-
pyrrolidinon (NMP). The permanent protecting groups of the
amino acid side chains were 2,2,4,6,7-pentamethyldihydro-
benzofuran-5-sulfonyl (Pbf) for Arg, tert-butyl for Asp,and
benzyl (Bzl) for Glu. Amino acids (2.5 equiv) were coupled
stepwise with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-
uronium tetrafluoroborate (TBTU,2.5 equiv) and N-hydroxy-
benzotriazole (HOBt,2.5 equiv) as coupling reagents and
N,N-diisopropylethylamine (DIPEA,7 equiv) as a base. In
the coupling reaction of Fmoc-d-phenylalanine,collidine
Results and Discussion
Chemistry: A variety of conjugated cyclo(-RGDfE-) mono-
mers and multimers were synthesized for radioiodination in
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Chem. Eur. J. 2003, 9,2717 2725

Cyclic RGD Peptides
2717 2725
(25 equiv) was used instead of DIPEA to prevent pyrogluta-
mate formation. Cleavage from the resin occurred with
dichloromethane (DCM)/acetic acid/2,2,2-trifluoroethanol
(TFE) (3:1:1). Under these conditions the side chain protect-
ing groups are not removed. Cyclization of the linear
protected peptide was performed under high dilution in
N,N-dimethylformamide with diphenylphosphoryl azide
(DPPA)[64,65] and NaHCO3. The final benzyl deprotection of
the Glu side chain by hydrogenation releases a partially
deprotected cyclopeptide bearing a free carboxylic acid
group. This is suitable for fragment coupling in solid-phase
peptide synthesis.
The compounds containing the aminooxy linker for oxime
ligation were synthesized by SPPS[66] by using the TCP
resin[62,63] and applying the Fmoc strategy[61] starting from the
initiator unit N-a-Fmoc-N-b-(N-tert-butyloxycarbonylamino-
oxyacetyl)-l-diaminopropionic acid (DprAoa[Boc]) 5 as the
site for chemoselective oxime formation.
Dpr Aoa
s
p
a
c
e
r
6a: spacer: none
6b: spacer: Ahx
6c: spacer: Hegas
6d: spacer: (Hegas)2
c(RGDfE)
Figure 2. Monomeric compounds 6a d.
resin by using acetic acid and TFE in DCM and were purified
by HPLC prior to deprotection by treatment with trifluoro-
acetic acid (TFA). HPLC chromatography yielded the pure
precursors for oxime linkage.
In addition to the trimethylstannylbenzaldehyde conjugates
12a d and 13a d for radioiodination,the analogue non-
radioactive 127
I benzaldehyde conjugates 10a d and 11a d
were synthesized as a reference in the synthesis of the
radiolabeled compounds and for receptor-affinity assays. The
para-trimethylstannylbenzaldehyde (9) was synthesized by
starting from the corresponding iodobenzaldehyde 8 with
Stille coupling conditions (Scheme 3).
The monomers 6a
d (Fig-
ure 2) with varying spacer lengths
and diverse lipophilicity/hydro-
philicity were synthesized in the
same way as the multimeric com-
pounds (Scheme 2) but without
introducing
unit.
a lysine branching
Branching and spacer amino
acids were coupled stepwise with
TBTU (2.5 equiv) and HOBt
(2.5 equiv) as coupling reagents
and collidine (25 equiv) as a base.
The cyclic pentapeptide cyclo-
(-Arg[Pbf]GlyAsp[tBu]-d-Phe-
Glu-) is coupled as a fragment to
the aminooxy-lysine spacer core
by using 1.5 equiv cyclopeptide,
1.5 equiv O-(7-azabenzotriazole-
1-yl)-N,N,N',N'-tetramethyluroni-
um hexafluorophosphate (HA-
TU),1.5 equiv 1-hydroxy-7-aza-
benzotriazole
(HOAt)
and
15 equiv collidine. Finally the pep-
tides,except for the tetrameric
RGD moieties,were cleaved from
the resin with simultaneous de-
protection by using a mixture of
trifluoroacetic acid/water/triiso-
propylsilane (95:2.5:2.5). The tetra-
mers were cleaved from the
Scheme 2. Synthesis of the multimeric compounds 7a d. a) 20% piperidine in DMF; b) Fmoc-Lys(Fmoc),
HATU,HOAt,collidine,NMP; c) Fmoc spacer amino acid (i.e. Fmoc-Ahx or Fmoc-Hegas),HATU,HOAt,
collidine,NMP; d) c(-RGDfE-)* (i.e. c(-R[Pbf]GD[tBu]fE-)),HATU,HOAt,collidine,NMP; e) 95% TFA/
H2O,TIPS; f) DCM/HOAc/TFE ˆ 3:1:1; * indicates the protecting groups Pbf for Arg and tert-butyl for Asp.
Chem. Eur. J. 2003, 9,2717 2725
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
2719

H. Kessler et al.
FULL PAPER
sults of the screening assays show the retaining integrin
affinity (Table 1).
H
H
Pd(PPh3)4
Me6Sn2
O
O
The radioiodination and biological evaluation of the radio-
labeled compounds will be done in the Department of
Nuclear Medicine of the Technische Universit‰t M¸nchen.
In preliminary experiments,the in vivo stability of the oxime
bond in our type of compound was evaluated in a tumor nude
mouse model. Detailed studies will be published elsewhere.
We developed versatile compounds containing the amino-
I
SnMe3
8
9
Scheme 3. Synthesis of 4-trimethylstannylbenzaldehyde 9.
oxy group for general ligation that can be used for fluores-
The 4-iodobenzaldehyde 8 and the 4-trimethylstannylbenz-
aldehyde 9 react chemoselectively and quantitatively with the
aminooxy-functionalized RGD moieties 6a d, 7a d without
the need of a protection strategy and under mild conditions
(Scheme 4).
[41,68,69]
cence labeling,surface functionalization,
and cluster-
Table 1. Receptor-binding assay of RGD compounds 10a d, 11a d. The
peptides GRGSPK (IC50 ˆ 1000 nm) and cyclo(-RGDfV-) (IC50 ˆ 2.1 nm)
were used as references.
Scheme 5 shows an example of a synthesized precursor for
radioiodination,for example with the iodogen method.
compound
10a 10b 10c 10d 11a 11b 11c 11d
[67]
IC50 [nm] on avb3 10
10
20
7
0.9
3
5
0.2
Biology: We tested the activities of the multimeric RGD
compounds in inhibiting the interaction of ligands with
isolated immobilized integrins.
H
O
O
O
O
90% ACN
The ability of the RGD com-
pounds to inhibit the binding of
vitronectin to the isolated,im-
mobilized avb3 receptor was
compared with that of the
standard peptides GRGDSPK
and cyclo(-RGDfV-). The re-
pH 4 (TFA)
O
O
O
HO
N
N
HO
N
NH2
+
2h
H
H
HN
HN
X
R
X
R
6a-d, 7a-d
8 X = I
9 X = SnMe3
10a-d, 11a-d X = I
12a-d, 13a-d X = SnMe3
Scheme 4. Chemoselective oxime ligation.
SnMe3
O
N
O
NH
NH COOH
NH
13d
O
O
HN
O
HN
NH
HN
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HN
NH
O
HN
O
O
O
O
NH
O
O
O
HN
NH
NH
O
O
O
HN
O
NH
NH
HN
HN
O
NH
NH
O
HN
NH
NH
O
H
N
O
HO
O
HN
O
HN
O
O
O
H
N
NH
HN
H
N
HN
HO
H
N
HO
O
O
HO
O
O
NH
NH
NH2
NH2
O
HN
O
HN
NH
O
HN
NH2
NH2
Scheme 5. Tetrameric trimethylstannylbenzaldehyde conjugate with Hegas spacer 13d.
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Chem. Eur. J. 2003, 9,2717 2725

Cyclic RGD Peptides
2717 2725
ing studies of integrins.[36,70] Furthermore,since lysine is
commercially available with orthogonal protecting groups,it
offers the possibility of synthesizing chimeric multimers
containing further ligands besides RGD peptides.
tert-butylester (3) (10.8 g,27.3 mmol,39% over 4 steps) as a yellow oil.
1H NMR (250 MHz,CDCl 3): d ˆ 4.01 (s,2H; OC H2CO),3.62 3.71 (m,
20H; 10  CH2),3.51 (t, J ˆ 5.2 Hz,2H; OC H2CH2NH2),2.85 (t, J ˆ 5.2 Hz,
2H; CH2NH2),1.76 (brs,2H; NH 2),1.47 (s,9H; C(C H3)3); Rf (CHCl3/
MeOH 9:1) ˆ 0.1.
Amine 3 (10.8 g,27.3 mmol) in THF (100 mL) at 0 8C was treated with
sodium bicarbonate (10%,70 mL) and 9-fluorenyl chloroformate (7.4 g,
28.7 mmol) in THF (100 mL),added dropwise over 10 min,and the
resulting suspension was stirred at room temperature for 60 min. Volatiles
were removed,and the residue was dissolved in ethyl acetate (250 mL),
washed with brine,dried (Na 2SO4),filtered,and concentrated to give a
crude yellow oil that was used in the next step without further purification.
Rf (CHCl3/MeOH 9:1) ˆ 0.6.
Experimental Section
Chemistry
Materials and methods: All commercially available chemical reagents were
used without further purification. The solvents DMF,DCM,MeOH,and
Et2O were distilled before use. Thin-layer chromatography (TLC) was
performed on aluminium-backed plates Merck silica gel 60 F254. Com-
pounds were visualized by UV absorption at 254 nm,with 5% phospho-
molybdic acid solution in 95% EtOH or with 3% ninhydrin solution in
97% EtOH. Analytical reversed-phase (RP) HPLC was performed on an
Amersham Pharmacia Biotech instrument (system ækta Basic 10F with
autosampler A900,pump P-900,UV detector UV-900,software Unicorn)
by using a YMC ODS-A C18 column (120 ä,5 mm,250 mm  4.6 mm) with
a flow rate of 1 mLminÀ1. Semipreparative RP-HPLC separations were
performed on a Beckman instrument (system Gold,pump 125,UV
detector 166) by using an YMC ODS-A C18 column (120 ä,5 mm,
250 mm  20 mm) with a flow rate of 8 mLminÀ1. Preparative RP-HPLC
separations were performed on an Amersham Pharmacia Biotech instru-
ment (system ækta Basic 100F with pump P-900,UV detector UV-900,
software Unicorn) by using a YMC ODS-A C18 column The eluent was a
linear gradient from water (0.1% TFA) to acetonitrile (0.1% TFA) over
30 min. The retention time (Rt) of the analytical RP-HPLC is given in
minutes with the gradient in percentage of acetonitrile. The detection was
performed at 220 nm. Proton NMR spectra were obtained on a Bruker
AC250 and DMX500 at 300 K. Chemical shifts are reported in ppm on the
d scale relative to the solvent signal used. ESI-MS spectra were performed
on a LCQ Finnnigan and MALDI-TOF spectra on a Bruker BiflexIII
instrument.
The crude product was treated with 90% TFA for 30 min at room
temperature. After removal of the solvents under reduced pressure,the
residue was taken up in ethyl acetate (300 mL) and washed with brine (2 Â
70 mL) and saturated aqueous NaHCO3 (6 Â 70 mL) until the aqueous
layer became caustic. The combined aqueous layers were treated with
KHSO4 (5.4 g),acidified with HCl (6 n,approx. 20 mL) under vigorous
stirring,and diluted with a mixture of DCM (150 mL) and Et 2O (300 mL).
The whole procedure was repeated starting from washing with saturated
aqueous NaHCO3. Finally the organic phase was dried (Na2SO4),filtered,
and concentrated to give 20-(N-Fmoc)-amino-3,6,9,12,15,18-hexaoxaeico-
sanoic acid (4) (15.0 g,26.8 mmol,98% over 2 steps) as a yellow oil.
1H NMR (250 MHz,CDCl 3): d ˆ 9.72 (brs,1H; CO 2H),7.73 (d, J ˆ 7.0 Hz,
2H; Fmoc-H),7.58 (d, J ˆ 7.0 Hz,2H; Fmoc- H),7.17 7.35 (m,4H; Fmoc-
H),5.54 (brs,1H; N H),4.34 (d, J ˆ 7.0 Hz,2H; Fmoc- H),4.14 (t, J ˆ
7.0 Hz,1H; Fmoc- H),4.13 (s,2H; OC H2CO2H),3.45 3.62 (m,22H;
11CH2),3.24 3.37 (m,2H; C H2NH); 13C NMR (62.9 MHz,CDCl 3): d ˆ
172.4,156.7,143.9,141.2,127.6,127.0,125.0,119.9,77.2,71.1,70.2 70.5,69.9,
68.7,66.6,47.2,40.8; Rf (CHCl3/MeOH 4:1) ˆ 0.3; Rt ˆ 20.4 min (10 ! 90);
Mw C29H39NO10 calcd 561.3,found MS (ESI): m/z (%) ˆ 1146.5 (11)
‡
‡
‡
[2M‡H‡Na] ,600.2 (22) [ M‡K] ,584.3 (50) [ M‡Na] ,562.3 (100)
‡
‡
[M‡H] ,340.3 [ M À Fmoc‡H] .
4-Trimethylstannylbenzaldehyde:
A
mixture of p-iodobenzaldehyde
(0.40 g,1.72 mmol),hexamethyldistannane (0.46 mL,2.22 mmol),and
tetrakis(triphenylphosphine) palladium (0.20 g,0.17 mmol) in dry toluene
(15 mL) was heated under reflux for 3 h. After removal of the solvent and
the excess of hexamethyldistannane in vacuo,the residue was purified by
column chromatography (hexane/ethyl acetate 19:1). Aldehyde 9 was
obtained as a colorless oil (0.35 g,1.29 mmol,75%). 1H NMR (250 MHz,
CDCl3): d ˆ 9.95 (s,1H; C HO),7.77 (d, J ˆ 7.9 Hz,2H; 35, -Ar H),7.65 (d,
J ˆ 7.6 Hz,2H; 26,-Ar H),0.31 (s,9H; Sn(C H3)3 [d, J(117Sn,H) ˆ 53.4 Hz,
20-(N-Fmoc)-amino-3,6,9,12,15,18-hexaoxaicosanoic acid: Three drops of
freshly distilled boron trifluoride etherate (BF3 ¥ Et2O) were added to a
mixture of hexaethylene glycol (30 g,106.3 mmol) and ethyl diazoacetic
acid tert-butylester (10 g,70.3 mmol) in dry DCM (150 mL) under argon.
After 15 min 3 further drops of BF3 ¥ Et2O were added,and the resulting
mixture was stirred for 60 min. The reaction mixture was diluted with ethyl
acetate (450 mL) and washed with brine (1 Â 200 mL and 4 Â 50 mL),dried
(Na2SO4),filtered,and concentrated to give 2 as orange oil with 18% di-
tert-butyl ester as impurity. 1H NMR (250 MHz,CDCl 3): d ˆ 4.02 (s,2H;
OCH2CO),3.57 3.75 (m,24H; C H2 glycol),1.47 (s,9H; C(C H3)3); Rf
(CHCl3/MeOH 9:1) (2) ˆ 0.6,(di- tBu ester) ˆ 0.7. The crude product was
used in the next step without further purification.
117Sn(CH3)3,d, J(119Sn,H) ˆ 55.9 Hz, 119Sn(CH3)3]); 13C NMR (62.9 MHz,
117
CDCl3): d ˆ 192.40,152.14 (s,Sn- C [d, J(117Sn,C) ˆ 403.9 Hz, Sn C, d
À
J(119Sn,C) ˆ 422.5 Hz, Sn C),136.18 (s,Sn C [d, J ˆ 35.3 Hz, Sn C,
119
117
À
À
À
119
117
119
À
À
À
À
Sn C]),135.99,128.42 (s,Sn C [d, J ˆ 43.6 Hz, Sn C, Sn C]), À9.63
117
À
C
À
(s,Sn
[d, J(117Sn,C) ˆ 347.9 Hz,
Sn C,d,
J(119Sn,C) ˆ 348.1 Hz,
119
This crude mixture was coevaporated with toluene (3 Â 100 mL) and
dissolved in dry DCM (200 mL),then triethylamine (29.4 mL,210.9 mmol)
and trimethylammonium chloride (1.3 g,14.1 mmol) were added. Tosyl
chloride (13.4 g,70.3 mmol) in dry DCM (150 mL) was added dropwise
over a period of 10 min at 08C. After removal of the ice bath,the mixture
was stirred for 60 min,washed with H 2SO4 (1n, 2 Â 70 mL),saturated
aqueous NaHCO3 (1 Â 70 mL),and brine (1 Â 70 mL),dried (Na 2SO4),
filtered,and concentrated to give a dark yellow oil that was used in the next
step without further purification.
À
Sn C]).
Peptide Synthesis
General procedures for solid phase peptide synthesis
General procedure I: Attachment of N-Fmoc-amino acids to TCP resin:
After swelling with dry DCM (10 mL) for 20 min,the TCP resin (2.00 g,
theoretical 0.96 mmolgÀ1,1.92 mmol) was treated with a solution of Fmoc-
protected amino acids (1.2 equiv,2.3 mmol) in dry DCM (10 mL) and
DIPEA (980 mL,3 equiv,5.8 mmol) at room temperature for 2 h. MeOH
(2 mL) and DIPEA (0.4 mL) were added to cap the free sites,and the
reaction mixture was shaken for 15 min. The resin was washed with NMP
(3 Â 10 mL),DCM (5 Â 10 mL),and MeOH (3 Â 10 mL) and dried under
vacuo to give resin-bound N-Fmoc-amino acids.
The crude material was coevaporated with toluene (3 Â 100 mL) and
stirred overnight with NaN3 (4.6 g,70.3 mmol) in dry DMF (100 mL),and
the solvent was removed in vacuo. The residue was taken up in ethyl
acetate (300 mL),washed with brine (3 Â 100 mL),dried (Na 2SO4),
filtered,and concentrated to give a yellow oil that was used in the next
step without further purification. Rf (CHCl3/MeOH 9:1) ˆ 0.3.
This crude oil was dissolved in EtOH/CHCl3 (200:20 mL) and stirred
overnight with 5% Pd/C catalyst (2 g) under a hydrogen atmosphere. The
catalyst was removed by filtration over celite,and the filtrate was
concentrated under reduced pressure. The residue was taken up in ethyl
acetate (70 mL) and extracted with KHSO4 (1n, 1 Â 100 mL,2 Â 50 mL).
The combined aqueous layers were made caustic with Na2CO3 and
extracted with CHCl3 (1 Â 100 mL,2 Â 50 mL),dried (Na 2SO4),filtered,
and concentrated to give 20-amino-3,6,9,12,15,18-hexaoxaicosanoic acid
General procedure II. Fmoc deprotection: The Fmoc-protected resin was
suspended in a solution of 20% piperidine in NMP (2 Â 20 mL) and
agitated for 5 and 15 min. The resin was washed with NMP (6 Â 20 mL).
General procedure IIIa. Coupling with TBTU/HOBt: Fmoc-amino acid
(2.5 equiv),TBTU (2.5 equiv),HOBt (2.5 equiv),and DIPEA (7 equiv
(25 equiv sym-collidine instead of DIPEA in the case of Fmoc-d-phenyl-
alanine)) were dissolved in NMP to give a 0.2 mmolLÀ1 solution which was
added to the resin. The reaction mixture was shaken at room temperature
for 90 min and washed with NMP (6 Â 20 mL). The end of the coupling was
controlled by the Kaiser test.[71,72]
Chem. Eur. J. 2003, 9,2717 2725
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
2721

H. Kessler et al.
FULL PAPER
General procedure IIIb. Coupling with HATU/HOAt : Fmoc-amino acid
(1.5 equiv),HATU (1.5 equiv),HOAt (1.5 equiv),and 246,-collidine
(15 equiv) were dissolved in NMP to give a 0.2 mmolLÀ1 solution,which
was added to the resin. The reaction mixture was shaken at room
temperature for 90 min and washed with NMP (6 Â 20 mL). The end of the
coupling was controlled by the Kaiser test.[71,72]
The conjugation of 6a (3.0 mg,3.4 Â 10À3 mmol) with 4-trimethylstannyl-
benzaldehyde (2.7 mg,10.2 Â 10À3 mmol) yielded 12a as
a white
powder (1.2 mg,1.1  10À3 mmol,83%). Rt ˆ 22.5 min (5 ! 80%), Mw
C41H57N11O12Sn calcd 1015.3,found MS (ESI): m/z (%) ˆ 1016.3 (100)
‡
[M‡H] .
Monomeric compounds with aminohexanoic acid spacer: The monomeric
compound 6b was synthesized in
a similar manner to the general
General procedure IVa. Cleavage with acetic acid: The resin was washed
with DCM (4 Â 20 mL) and treated with a mixture of DCM,acetic acid,and
trifluoroethanol (3:1:1; 20 mL) for 60 min and (2 Â 20 mL) for 3 Â 10 min.
After removal of the resin by filtration,the filtrates were combined,and the
solvent was removed in vacuo and lyophilized out of tert-BuOH/H2O to
yield the protected peptide as a yellow powder.
procedures I,II,IIIa,IIIb,IVa,and VII and yielded a white powder
(44 mg,0.04 mmol,42%) after purification by semipreparative HPLC
1
(10 ! 50). H NMR (500 MHz,DMSO): d ˆ 8.30 8.28 (m,1H; HN-Gly),
8.07 7.96 (m,5H; HNa-Dpr, HN-Glu, HNb-Dpr, HN-d-Phe, HN-Asp),7.75
(d, J ˆ 8.3 Hz,1H; HNa-Arg),7.69 (t, J ˆ 5.5 Hz,1H; HN-Ahx),7.46 7.45
(m,1H; HNe-Arg),7.26 7.10 (m,5H; DÀPheC-H),6.82 (brs,2H; N H2-Arg),
4.65 4.60 (m,1H; Ha-Asp),4.45 (q, J ˆ 7.1 Hz,1H; Ha-d-Phe),4.36 4.31
(m,1H; Ha-Dpr),4.16 4.12 (m,3H; Ha-Arg,C H2-Aoa),4.05 (dd, J ˆ
15.2/7.7 Hz,1H; Ha-Gly),4.01 3.96 (m,1H; Ha-Glu),3.51 3.32 (m,2H;
Hb-Dpr),3.24 (dd, J ˆ 15.2/3.9 Hz,1H; Ha-Gly),3.10 3.07 (m,2H; Hd-
Arg),3.00 2.93 (m,3H; He-Ahx, Hb-d-Phe),2.78 (dd, J ˆ 13.4/6.3 Hz,1H;
Hb-d-Phe),2.69 (dd, J ˆ 15.8/8.5 Hz,1H; Hb-Asp),2.35 (dd, J ˆ 15.8/
5.7 Hz,1H; Hb-Asp),2.10 (t, J ˆ 7.4 Hz,2H; Ha-Ahx),2.01 1.90 (m,2H;
Hg-Glu),1.83 1.62 (m,3H; Hb-Glu, Hb-Arg),1.51 1.33 (m,7H; Hb-Arg,
Hb-Ahx, Hg-Arg, Hd-Ahx),1.26 1.20 (m,2H; Hg-Ahx); Rt ˆ 12.9 min
(5 ! 60%); Mw C37H56N12O13 calcd 876.4,found MS (ESI): m/z (%) ˆ 877.5
General procedure IVb. Cleavage with trifluoroacetic acid: The resin was
washed with DCM (4 Â 20 mL) and treated with a solution of TFA,H O,
2
and triisopropylsilane (TIPS) (90:4.75:4.75; 20 mL) for 3 Â 10 min. After
removal of the resin by filtration,the filtrates were combined and stirred
for another 2.5 h. The solvent was removed in vacuo and lyophilized out of
tert-BuOH/H2O to yield the product as a yellow powder.
General procedure V. Cyclization of the linear pentapeptide: The linear
pentapeptide was dissolved in high dilution (c ˆ 5  10À3 m) in DMF. After
addition of DPPA (3 equiv) and NaHCO3 (5 equiv) as solid base,the
suspension was stirred for 48 h at room temperature. The solid base was
removed by filtration and,after the solution had been concentrated in
vacuo,the cyclopeptide was precipitated and washed several times with
H2O.
‡
(100) [M‡H] .
The conjugation of 6b (1.9 mg,1.9 Â 10À3 mmol) with 4-iodobenzaldehyde
(1.3 mg,5.7 Â 10À3 mmol) according to the general procedure VIII yielded
10b as a white powder (1.2 mg,1.9  10À3 mmol,99%). Rt ˆ 18.9 min (5 !
80%), Mw C44H59IN12O13 calcd 1090.3,found MS (ESI): m/z (%) ˆ 1091.4
General procedure VI. Bzl deprotection: The cyclic pentapeptide (1 mmol)
was dissolved in N,N-dimethylacetamide (30 mL) and stirred for 4 h with
5% Pd/C catalyst (0.5 1 g) under a hydrogen atmosphere. After removal
of the solvent in vacuo,the residue was taken up in MeOH (50 mL) and
filtered over celite,and the filtrate was concentrated to precipitate the
peptide with Et2O.
‡
(100) [M‡H] .
The conjugation of 6b (3.7 mg,3.8 Â 10À3 mmol) with 4-trimethylstannyl-
benzaldehyde (3.1 mg,11.4 Â 10À3 mmol) yielded 12b as
a white
powder (1.3 mg,1.1  10À3 mmol,28%). Rt ˆ 22.4 min (5 ! 80%), Mw
C47H68N12O13Sn calcd 1128.4,found MS (ESI): m/z (%) ˆ 1129.4 (100)
General procedure VII. Pbf and OtBu deprotection with trifluoroacetic acid
(TFA): The protected peptide was dissolved in TFA/H2O (95:5). After 3 h,
the solvent was removed under reduced pressure,and the residue was
lyophilized out of tBuOH/H2O to yield the product as white powder.
‡
[M‡H] .
Monomeric compounds with Hegas spacer: The monomeric compound 6c
was synthesized in a similar manner to the general procedures I,II,IIIa,
IIIb,IVa,and VII as a white powder (57 mg,0.04 mmol,44%) after
purification by semipreparative HPLC (10 ! 50). 1H NMR (500 MHz,
General procedure VIII. Conjugation with benzaldehyde: Peptide (1 mmol)
was dissolved in ACN/H2O (1 mL,90%) and TFA was added until the pH
reached 4. After addition of benzaldehyde (3 mmol) the mixture was
stirred for 2 h,and the solvent was removed in vacuo. The residue was
washed with Et2O (4 Â 10mL) and lyophilization out of tBuOH/H2O
yielded the product as a white powder.
DMSO): d ˆ 8.30 (dd, J ˆ 4.2/7.5 Hz,1H; HN-Gly),8.06 8.04 (m,2H; HN
-
a
Dpr, HN-Glu),8.00 7.97 (m,2H; HN-d-Phe, HN-Asp),7.92 (d, J ˆ 7.8 Hz,
1H; HNb-Dpr),7.78 7.73 (m,2H; HN-Hegas, HNa-Arg),7.44 7.43 (m,1H;
HNe-Arg),7.26 7.13 (m,5H; DÀPheC-H),6.78 (brs,2H; N H2-Arg),4.64
4.60 (m,1H; Ha-Asp),4.45 (q, J ˆ 7.2 Hz,1H; Ha-d-Phe),4.40 4.36 (m,
1H; Ha-Dpr),4.16 4.11 (m,1H; Ha-Arg),4.07 4.02 (m,3H; C H2-Aoa,
Cyclo(-R[Pbf]GD[tBu]fE-): The procedure is similar to the general
procedures for I,II,IIIa,IVa,V,and VI (4.22 g,4.62 mmol,79%,white
powder). Rt ˆ 22.2 min (5 ! 90%), Mw C43H60N8O12S calcd 912.4,found MS
Ha-Gly),4.00 3.96 (m,1H;
Ha-Glu),3.90 (s,2H; OC H2CO-Hegas),
3.60 3.38 (m,24H; Hb-Dpr,CH 2-Hegas),3.24 (dd, J ˆ 15.2/3.9 Hz,1H;
Ha-Gly),3.19 3.16 (m,2H; C H2NH-Hegas),3.10 3.06 (m,2H; Hd-Arg),
2.95 (dd, J ˆ 13.4/7.6 Hz,1H; Hb-d-Phe),2.79 (dd, J ˆ 13.4/6.3 Hz,1H; Hb-
d-Phe),2.69 (dd, J ˆ 16.3/8.7 Hz,1H; Hb-Asp),2.35 (dd, J ˆ 16.2/5.7 Hz,
1H; Hb-Asp),2.02 1.90 (m,2H; Hg-Glu),1.83 1.61 (m,3H; Hb-Glu, Hb-
Arg),1.51 1.32 (m,3H; Hb-Arg, Hg-Arg); Rt ˆ 14.5 min (5 ! 60%); Mw
C45H72N12O19 calcd 1084.5,found MS (ESI): m/z (%) ˆ 1085.6 (100)
‡
‡
(ESI): m/z (%) ˆ 951.3 (9) [M‡K] ,935.3 (14) [ M‡Na] ,913.3 (100)
[M‡H] ,857.4 (32) [ M À isobuten‡H] ,476.3 (14) [( M À K‡H)/2]2‡,448.4
‡
‡
(15) [(M À isobuten‡K‡H)/2]2‡
.
Monomeric compounds without spacer: The monomeric compound 6a was
synthesized in a similar way to the general procedures I,II,IIIb,IVa,and
VII as a white powder (41 mg,0.05 mmol,55%) after purification by
semipreparative HPLC (10 ! 50). 1H NMR (500 MHz,DMSO): d ˆ 8.30
(dd, J ˆ 4.2/7.5 Hz,1H; HN-Gly),8.06 8.00 (m,3H; HNa-Dpr, HN-Glu,
HN-Asp),7.96 7.94 (m,2H; HN-d-Phe, HNb-Dpr),7.77 (d, J ˆ 7.9 Hz,1H;
‡
[M‡H] .
The conjugation of 6c (1.8 mg,1.5 Â 10À3 mmol) with 4-iodobenzaldehyde
(1.0 mg,4.5 Â 10À3 mmol) according to general procedure VIII yielded 10c
as a white powder (1.5 mg,1.1  10À3 mmol,72%). Rt ˆ 18.2 min (5 !
80%), Mw C52H75IN12O19 calcd 1298.4,found MS (ESI): m/z (%) ˆ 1299.5
DÀPhe
HNa-Arg),7.44 7.43 (m,1H; HNe-Arg),7.27 7.14 (m,5H;
C H),6.78
À
(brs,2H; N H2 Arg),4.62 (m,1H; Ha-Asp),4.48 (q, J ˆ 7.1 Hz,1H; Ha-d-
Phe),4.33 (m,1H; Ha-Dpr),4.14 4.03 (m,5H; Ha-Arg,C H2-Aoa, Ha-
Gly, Ha-Glu),3.53 3.38 (m,2H; Hb-Dpr),3.24 (dd, J ˆ 15.5/4.2 Hz,1H;
Ha-Gly),3.10 3.07 (m,2H; Hd-Arg),2.96 (dd, J ˆ 13.6/7.1 Hz,1H; Hb-d-
Phe),2.79 (dd, J ˆ 13.6/6.5 Hz,1H; Hb-d-Phe),2.68 (dd, J ˆ 16.2/8.6 Hz,
1H; Hb-Asp),2.35 (dd, J ˆ 15.9/5.6 Hz,1H; Hb-Asp),2.04 2.01 (m,2H;
Hg-Glu),1.85 1.72 (m,2H; Hb-Glu, Hb-Arg),1.70 1.61 (m,1H; Hb-Glu),
À
‡
(100) [M‡H] .
The conjugation of 6c (3.5 mg,3.0 Â 10À3 mmol) with 4-trimethylstannyl-
benzaldehyde (2.4 mg,9.0 Â 10À3 mmol) yielded 12c as
a white
powder (1.5 mg,1.0  10À3 mmol,35%). Rt ˆ 22.6 min (5 ! 80%), Mw
C55H84N12O19Sn calcd 1336.5,found MS (ESI): m/z (%) ˆ 1337.5 (100)
‡
[M‡H] .
1.51 1.32 (m,3H;
Hb-Arg, Hg-Arg); Rt ˆ 9.8 min (5 ! 80%); Mw
Monomeric compounds with (Hegas)2 spacer: The monomeric compound
6d was synthesized in a similar manner to general procedures I,II,IIIa,
IIIb,IVa,and VII as a white powder (45 mg,0.03 mmol,27%) after
purification by semipreparative HPLC (20 ! 50). 1H NMR (500 MHz,
‡
C31H45N11O12 calcd 763.3,found MS (ESI): m/z (%) ˆ 764.4 (100) [M‡H] .
The conjugation of 6a (1.5 mg,1.7 Â 10À3 mmol) with 4-iodobenzaldehyde
(0.5 mg,2.1 Â 10À3 mmol) according to the general procedure VIII yielded
10a as a white powder (1.8 mg,1.6  10À3 mmol,94%). Rt ˆ 19.0 min (5 !
80%), Mw C38H48IN11O12 calcd 977.3,found MS (ESI): m/z (%) ˆ 978.3
DMSO): d ˆ 8.30 (dd, J ˆ 3.8/7.4 Hz,1H; HN-Gly),8.10 8.09 (m,1H; HN
-
a
Dpr),8.05 (d, J ˆ 7.2 Hz,1 H ; HN-Glu),8.00 7.97 (m,2H; HN-d-Phe, HN-
‡
(100) [M‡H] .
Asp),7.92 (d, J ˆ 7.9 Hz,1H; HNb-Dpr),7.78 7.73 (m,2H; HN-Hegas, HN
-
a
2722
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9,2717 2725

Cyclic RGD Peptides
2717 2725
Arg),7.64 7.62 (m,1H; HN-Hegas),7.44 7.42 (m,1H; HNe-Arg),7.26
7.13 (m,5H; DÀPheC-H),6.77 (brs,2H; N H2-Arg),4.65 4.60 (m,1H; Ha-
Asp),4.45 (q, J ˆ 7.2 Hz,1H; Ha-d-Phe),4.41 4.37 (m,1H; Ha-Dpr),
4.16 4.12 (m,3H; Ha-Arg,C H2-Aoa),4.05 (dd, J ˆ 15.2/7.7 Hz,1H; Ha-
Gly),4.00 3.96 (m,1H; Ha-Glu),3.90 (s,2H; OC H2CO-Hegas),3.86 (s,
2H; OCH2CO-Hegas),3.59 3.37 (m,46H; Hb-Dpr,CH 2-Hegas),3.27
3.22 (m,3H; C H2NH-Hegas, Ha-Gly),3.19 3.15 (m,2H; C H2NH-Hegas),
3.10 3.06 (m,2H; Hd-Arg),2.95 (dd, J ˆ 13.4/7.7 Hz,1H; Hb-d-Phe),2.79
(dd, J ˆ 13.4/6.3 Hz,1H; Hb-d-Phe),2.69 (dd, J ˆ 16.2/8.6 Hz,1H; Hb-
Asp),2.38 2.33 (m,1H; Hb-Asp),2.02 1.90 (m,2H; Hg-Glu),1.85 1.61
Arg, He-Lys),2.95 (dd, J ˆ 14.4/7.1 Hz,2H; Hb-d-Phe),2.79 (dd, J ˆ 13.7/
6.4 Hz,2H; Hb-d-Phe),2.69 (dd, J ˆ 16.3/8.7 Hz,2H; Hb-Asp),2.35 (dd,
J ˆ 16.3/5.4 Hz,2H; Hb-Asp),2.02 1.90 (m,4H; Hg-Glu),1.83 1.63 (m,
7H; Hb-Glu, Hb-Arg, Hb-Lys),1.57 1.36 (m,9H; Hb-Lys, Hb-Arg, Hg-
Arg, Hd-Lys),1.26 1.22 (m,2H; Hg-Lys); Rt ˆ 16.9 min (5 ! 90%); Mw
C98H148IN23O35 calcd 2334.0,found MS (ESI): m/z (%) ˆ 1179.0 (8)
[(M‡Na‡H)/2]2‡,1168.7 (100) [( M‡2H)/2]2‡,792.2 (13) [( M‡2Na‡H)/
3]3‡,786.9 (10) [( M‡Na‡2H)/3]3‡,779.5 (7) [( M‡3H)/3]3‡
.
The conjugation of 7b (2.0 mg,0.9 Â 10À3 mmol) with 4-trimethylstannyl-
benzaldehyde (0.8 mg,2.8 Â 10À3 mmol) according to the general proce-
dure VIII yielded 13b as a white powder (2.2 mg,0.9 Â 10À3 mmol,99%).
Rt ˆ 19.0 min (5 ! 90%); Mw C101H157N23O35Sn calcd 2372.0,found MS
(m,3H; Hb-Glu, Hb-Arg),1.51 1.32 (m,3H;
Hb-Arg, Hg-Arg); Rt ˆ
16.2 min (5 ! 60%); Mw C59H99N13O26 calcd 1405.7,found MS (ESI): m/z
‡
(%) ˆ 1406.8 (100) [M‡H] .
(ESI): m/z (%) ˆ 1197.6 (12) [(M‡Na‡H)/2]2‡,1187.2 (100) [( M‡2H)/2]2‡
,
The conjugation of 6d (3.0 mg,2.0 Â 10À3 mmol) with 4-iodobenzaldehyde
(1.4 mg,6.0 mmol) according to general procedure VIII yielded 10d as a
white powder (1.8 mg,1.0  10À3 mmol,53%). Rt ˆ 18.7 min (5 ! 80%),
Mw C66H102IN13O26 calcd 1619.6,found MS (ESI): m/z (%) ˆ 1620.6 (100)
804.7 (18) [(M‡2Na‡H)/3]3‡,799.2 (17) [( M‡Na‡2H)/3]3‡
.
Tetrameric compounds with amino hexanoic acid spacer: The tetrameric
compound 7c was synthesized in similar manner to the general
a
procedures I,II,IIIa,IIIb,IVb,and VII with additional preparative HPLC
(60 ! 100) purification between steps IVb and VII. After purification by
semipreparative HPLC,(20 ! 50) 7c was yielded as a white powder
(73 mg,0.02 mmol,18%). 1H NMR (500 MHz,DMSO): d ˆ 8.32 8.30 (m,
‡
[M‡H] .
The conjugation of 6d (6.0 mg,3.9 Â 10À3 mmol) with 4-trimethylstannyl-
benzaldehyde (3.1 mg,11.7 Â 10À3 mmol) yielded 12d as
a white
powder (5.0 mg,2.8  10À3 mmol,71%). Rt ˆ 22.5 min (5 ! 80%), Mw
4H; HN-Gly),8.12 (d, J ˆ 7.1 Hz,3H; HNa-Lys),8.07 8.05 (m,5H; HN
-
a
Dpr, HN-Glu),8.00 7.98 (m,8H; HN-d-Phe, HN-Asp),7.88 7.80 (m,4H;
HNb-Dpr, HNe-Lys),7.73 7.68 (m,8H; HNa-Arg, HN-Ahx),7.45 7.44 (m,
C69H111N13O26Sn calcd 1657.7,found MS (ESI): m/z (%) ˆ 1658.7 (100)
‡
[M‡H] .
4H; HNe-Arg),7.25 7.13 (m,20H; DÀPheC-H),6.81 (brs,8H; N H2-Arg),
4.62 (dd, J ˆ 8.6 Hz,4H; Ha-Asp),4.45 (dd, J ˆ 7.2 Hz,4H; Ha-d-Phe),
4.33 4.29 (m,3H; Ha-Lys),4.23 3.97 (m,8H; He-Lys, Ha-Dpr, Ha-Arg)
4.07 3.96 (m,10H; Ha-Gly,C H2-Aoa, Ha-Glu),3.51 3.39 (m,2H; Hb-
Dpr),3.25 (dd, J ˆ 15.0/3.6 Hz,4H; Ha-Gly),3.08 (m,8H; Hd-Arg),2.98
2.93 (m,15H; He-Ahx, He-Lys, Hb-d-Phe),2.79 (dd, J ˆ 6.4 Hz,4H; Hb-d-
Dimeric compounds with aminohexanoic acid spacer: The dimeric com-
pound 7a was synthesized in a similar manner to the general procedures I,
II,IIIa,IIIb,IVa,and VII as a white powder (66 mg,0.04 mmol,31%) after
purification by semipreparative HPLC (20 ! 50). 1H NMR (500 MHz,
DMSO): d ˆ 8.31 8.29 (m,2H; HN-Gly),8.16 (d, J ˆ 5.7 Hz,1H; HNa-Lys)
8.07 8.05 (m,3H; HNa-Dpr, HN-Glu),8.00 7.97 (m,4H; HN-d-Phe, HN-
Asp),7.90 7.89 (m,1H; HNb-Dpr),7.74 7.69 (m,5H; HNa-Arg, HN-Ahx,
HNe-Lys),7.46 7.45 (m,2H; HNe-Arg),7.25 7.14 (m,10H; DÀPheC-H),6.81
(brs,4H; N H2-Arg),4.63 4.60 (m,2H; Ha-Asp),4.46 4.41 (m,2H; Ha-
d-Phe),4.32 4.29 (m,2H; Ha-Lys, He-Lys),4.21 3.97 (m,9H; Ha-Dpr,
Ha-Arg,C H2-Aoa, Ha-Gly, Ha-Glu),3.51 3.38 (m,2H; Hb-Dpr),3.24 (d,
2H; J ˆ 14.6 Hz, Ha-Gly),3.09 2.94 (m,11H; He-Lys, Hd-Arg, He-Ahx,
Hb-d-Phe),2.80 2.77 (m,2H; Hb-d-Phe),2.71 2.66 (m,2H; Hb-Asp),
2.38 2.34 (m,2H; Hb-Asp),2.11 2.10 (m,4H; Ha-Ahx),2.02 1.93 (m,
6H; Hg-Glu, Hb-Lys),1.78 1.62 (m,6H; Hb-Glu, Hb-Arg),1.46 1.21 (m,
22H; Hb-Arg, Hb-Ahx, Hg-Arg, Hd-Ahx, Hd-Lys, Hg-Ahx, Hg-Lys); Rt ˆ
Phe),2.69 (dd, J ˆ 8.2 Hz,4H; Hb-Asp),2.38 2.34 (m,4H;
Hb-Asp),
2.13 1.89 (m,22H; Ha-Ahx, Hg-Glu, Hb-Lys),1.83 1.62 (m,12H; Hb-
Glu, Hb-Arg),1.47 1.35 (m,34H; Hb-Arg, Hb-Ahx, Hg-Arg, Hd-Ahx, Hd-
Lys),1.22 1.20 (m,14H; Hg-Ahx, Hg-Lys); Rt ˆ 13.0 min (5 ! 90%); Mw
C151H227N45O43 calcd 3358.7,found MS (ESI): m/z (%) ˆ 1680.7 (11)
[(M‡2H)/2]2‡,1121.2 (56) [( M‡3H)/3]3‡,841.4 (100) [( M‡4H)/]4‡; MS
‡
(MALDI-TOF): m/z ˆ 3359.5 [M‡H] .
The conjugation of 7c (1.0 mg,0.3 Â 10À3 mmol) with 4-iodobenzaldehyde
(0.2 mg,0.9 Â 10À3 mmol) according to general procedure VIII yielded 11c
as a white powder (0.8 mg,0.2  10À3 mmol,77%). Rt ˆ 15.5 min (5 !
90%); Mw C158H230IN45O43 calcd 3572.6,found MS (ESI): m/z (%) ˆ
1205.0 (5) [(M‡2Na‡H)/3]3‡,1192.5 (14) [( M‡3H)/]3‡,904.2 (12)
12.3 min (5 ! 90%); Mw C75H113N23O23 calcd 1703.8,found MS (ESI): m/z
‡
(%) ˆ 1704.8 (22) [M‡H] ,853.4 (100) [( M‡2H)/2]2‡
.
The conjugation of 7a (1.0 mg,0.6 Â 10À3 mmol) with 4-iodobenzaldehyde
(0.4 mg,1.8 Â 10À3 mmol) according to the general procedure VIII yielded
11a as a white powder (1.1 mg,0.6  10À3 mmol,97%). Rt ˆ 16.5 min (5 !
[(M‡2Na‡2H)/4]4‡,894.9
(100)
[(
M‡4H)/4]4‡,723.7
(8)
[(M‡2Na‡3H)/5]5‡
.
The conjugation of 7c (3.3 mg,1.0 Â 10À3 mmol) with 4-trimethylstannyl-
benzaldehyde (0.8 mg,3.0 Â 10À3 mmol) according to general procedure
VIII yielded 13c as a white powder (3.2 mg,0.9  10À3 mmol,90%). Rt ˆ
17.0 min (5 ! 90%); Mw C161H239N45O43Sn calcd 3610.7,found MS (ESI):
m/z (%) ˆ 1205.0 (7) [(M‡3H)/3]3‡,913.5 (9) [( M‡2Na‡2H)/4]4‡,904.0
90%), Mw C82H116IN23O23 calcd 1917.8,found MS (ESI): m/z (%) ˆ 1918.7
(5) [M‡H] ,960.5 (100) [( M‡2H)/2]2‡
.
‡
The conjugation of 7a (1.7 mg,1.0 Â 10À3 mmol) with 4-trimethylstannyl-
benzaldehyde (0.8 mg,3.0 Â 10À3 mmol) yielded 13a as
a white
powder (1.6 mg,0.8  10À3 mmol,83%). Rt ˆ 18.8 min (5 ! 90%), Mw
(100) [(M‡4H)/4]4‡,730.7 (8) [( M‡2Na‡3H)/5]5‡
.
C85H125N23O23Sn calcd 1955.8,found MS (ESI): m/z (%) ˆ 1956.9 (6)
Tetrameric compounds with Hegas spacer: The tetrameric compound 7d
was synthesized in a similar manner to the general procedures I,II,IIIa,
IIIb,IVb,and VII with additional preparative HPLC (60 ! 100) purifica-
tion between steps IVb and VII (Protected: 117 mg,17%,pale yellow
powder, Rt ˆ 22.6 min (30 ! 100%)). After final purification by semi-
preparative HPLC (20 ! 50) 7d was yielded as a white powder (43 mg,
0.01 mmol,8%). 1H NMR (500 MHz,DMSO): d ˆ 8.31 8.30 (m,4H; HN-
Gly),8.14 (d,3H; J ˆ 8.3 Hz, HNa-Lys),8.08 8.06 (m,5H; HNa-Dpr, HN-
Glu),7.99 7.98 (m,8H; HN-d-Phe, HN-Asp),7.90 7.89 (m,1H; HNb-Dpr),
7.82 7.73 (m,6H; HN-Hegas, HNa-Arg),7.65 7.59 (m,5H; HNe-Lys HN-
Hegas),7.54 7.49 (m,4H; HNe-Arg),7.25 7.13 (m,20H; DÀPheC-H),6.89
(brs,8H; N H2-Arg),4.65 4.60 (m,4H; Ha-Asp),4.47 4.43 (m,4H; Ha-
d-Phe),4.33 4.12 (m,11H; Ha-Lys, Ha-Dpr, He-Lys, Ha-Arg),4.07 3.96
(m,10H; C H2-Aoa, Ha-Gly, Ha-Glu),3.91 (s,4H; OC H2CO-Hegas),3.84
[M‡H] ,979.2 (100) [( M‡2H)/2]2‡
.
‡
Dimeric compounds with Hegas spacer: The dimeric compound 7b was
synthesized in a similar manner to the general procedures I,II,IIIa,IIIb,
IVb as a white powder (53.3 mg,0.03 mmol,23%) after purification by
semipreparative HPLC (20 ! 50). Rt ˆ 12.9 min (5 ! 90%); Mw
C91H145N23O35 calcd 2120.0,found MS (ESI): m/z (%) ˆ 1061.5 (100)
‡
[(M‡2H)/2]2‡; MS (MALDI-TOF): m/z ˆ 2120.7 [M‡H] .
The conjugation of 7b (1.0 mg,0.5 Â 10À3 mmol) with 4-iodobenzaldehyde
(0.3 mg,1.4 Â 10À3 mmol) according to general procedure VIII yielded 11b
as a white powder (1.1 mg,0.5 Â 10À3 mmol,96%). 1H NMR (500 MHz,
DMSO): d ˆ 8.31 8.29 (m,3H; HN-Gly, HNa-Lys) 8.08 8.04 (m,3H; HN
-
a
Dpr, HN-Glu),8.00 7.97 (m,4H; HN-d-Phe, HN-Asp),7.90 7.89 (m,1H;
HNb-Dpr),7.77 7.73 (m,3H; HN-Hegas, HNa-Arg),7.64 7.60 (m,2H; HN
-
e
Lys HN-Hegas),7.45 7.44 (m,2H; HNe-Arg),7.25 7.13 (m,10H; DÀPheC-
H),6.79 (brs,4H; N H2-Arg),4.65 4.60 (m,2H; Ha-Asp),4.47 4.43 (m,
2H; Ha-d-Phe),4.34 4.28 (m,3H; Ha-Lys, He-Lys, Ha-Dpr),4.16 4.12
(m,2H; Ha-Arg) 4.07 3.96 (m,6H; C H2-Aoa, Ha-Gly, Ha-Glu),3.91 (s,
2H; OCH2CO-Hegas),3.84 (s,2H; OC H2CO-Hegas),3.55 3.49 (m,46H;
Hb-Dpr,CH 2-Hegas),3.39 3.37 (m,2H; C H2NH-Hegas),3.27 3.22 (m,
2H; Ha-Gly),3.18 3.16 (m,2H; C H2NH-Hegas),3.09 3.04 (m,5H; Hd-
(s, 4H; OCH2CO-Hegas),3.54 3.39 (m,94H;
Hb-Dpr,CH 2-Hegas,
CH2NH-Hegas),3.25 (d,4H; J ˆ 13.5 Hz, Ha-Gly),3.18 3.17 (m,4H;
CH2NH-Hegas),3.09 3.04 (m,11H; Hd-Arg, He-Lys),3.00 2.92 (m,4H;
Hb-d-Phe),2.80 2.76 (m,4H; Hb-d-Phe),2.69 (dd,4H; J ˆ 16.2/8.9 Hz,
Hb-Asp),2.35 (dd,4H; J ˆ 16.2/5.7 Hz, Hb-Asp),2.00 1.90 (m,8H; Hg-
Glu),1.82 1.72 (m,8H; Hb-Glu, Hb-Arg),1.67 1.64 (m,8H; Hb-Glu, Hb-
Lys),1.48 1.22 (m,32H; Hb-Lys, Hb-Arg, Hg-Arg, Hd-Lys, Hg-Lys); Rt ˆ
Chem. Eur. J. 2003, 9,2717 2725
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
2723

H. Kessler et al.
FULL PAPER
13.4 min (5 ! 90%); Mw C183H291N45O67 calcd 4191.1,MS (MALDI-TOF):
[15] B. Felding-Habermann,B. M. Mueller,C. A. Romerdahl,D. A.
‡
m/z ˆ 4191.9 [M‡H] .
Cheresh, J. Clin. Invest. 1992, 89,2018 2022.
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Kessler in Peptides 1996 (Proc. 24th European Peptide Symposium,
September 8 13, 1996, Edinburgh, Scotland) (Eds.: R. Ramage,R.
Epton),Mayflower Scientific,England, 1998,pp. 329 330.
[19] M. A. Dechantsreiter,E. Planker,B. Math‰,E. Lohof,G. Holzemann,
The conjugation of 7d (1.1 mg,0.3 Â 10À3 mmol) with 4-iodobenzaldehyde
(0.2 mg,0.8 Â 10À3 mmol) according to general procedure VIII yielded 11d
as a white powder (1.1 mg,0.3  10À3 mmol,99%). Rt ˆ 14.9 min (5 !
90%); Mw C190H294IN45O67 calcd 4405.0,found MS (ESI): m/z (%) ˆ
1470.0 (12) [(M‡3H)/3]3‡,1113.6 (7) [( M‡2Na‡2H)/4]4‡,1103.1
(100) [(M‡4H)/4]4‡,894.9 (14) [( M‡3Na‡2H)/5]5‡,890.3 (14)
[(M‡2Na‡3H)/5]5‡
.
The conjugation of 7d (3.8 mg,0.9 Â 10À3 mmol) with 4-trimethylstannyl-
benzaldehyde (0.7 mg,2.7 Â 10À3 mmol) according to general procedure
A. Jonczyk,S. L. Goodman,H. Kessler,
3033 3040.
J. Med. Chem. 1999, 42,
VIII yielded 13d as a white powder (3.9 mg,0.9 Â 10À3 mmol,95%). R
ˆ
t
17.3 min (5 ! 90%); Mw C193H303N45O67Sn calcd 4443.1,found MS (ESI):
m/z (%) ˆ 1482.7 (15) [(M‡3H)/3]3‡,1121.9 (11) [( M‡2Na‡2H)/4]4‡
1118.0 (7) [(M‡Na‡3H)/4]4‡,1112.4 (100) [( M‡4H)/4]4‡,897.5 (18)
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.
Biology
Isolated integrin ligand binding assays: The production of recombinant
human avb3 has been described elsewhere.[73] The inhibitory activity of the
compounds described here was tested in ligand binding inhibition assays,[19]
by using immobilized integrin as the target,and biotinylated human serum
vitronectin as ligand. Purified vitronectin (1 mg mLÀ1
; pH 8.2) was
biotinylated with N-hydroxysuccinimidobiotin (100 mgmLÀ1; 1 h,20 8C)
before dialysis into phosphate buffer saline. In brief,96-well ELISA plates
were coated with 1 mg mLÀ1 integrin. After blocking residual sites on the
plate with bovine serum albumin,biotinylated ligands (1 mg mLÀ1) were
added in the presence or absence of serial dilutions of inhibitor,and after
incubation and washing,bound biotin was detected with a peroxidase-
coupled antibiotin antibody and N,N,N',N'-tetramethylbenzene substrate.
IC50,the concentration of inhibitor needed to inhibit ligand binding in the
absence of inhibitor by 50%,was established by curve fitting,and the
values presented are usually the mean of three or more such independent
determinations. External standards of linear GRGDSPK and cyclo-
(-RGDfV-) were routinely 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 Æ5%,and interassay variation in absolute IC 50 was typically a factor
of less than Æ2.
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Chem. Eur. J. 2003, 9,2717 2725
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
2725