Pharmacophoric modifications lead to superpotent αvβ3 integrin ligands with suppressed α5β1 activity

Article abstract of DOI:10.1021/jm500092w

The selective targeting of the αvβ3 integrin subtype without affecting the structurally closely related receptor α5β1 is crucial for understanding the details of their biological and pathological functions and thus of great relevance for diagnostic and therapeutic approaches in cancer treatment. Here, we present the synthesis of highly active RGD peptidomimetics for the αvβ3 integrin with remarkable selectivity against α5β1. Incorporation of a methoxypyridine building block into a ligand scaffold and variation of different functional moieties led to αvβ3-antagonistic activities in the low nanomolar or even subnanomolar range. Furthermore, docking studies were performed to give insights into the binding modes of the novel compounds. The presented library comprises powerful ligands for specific addressing and blocking of the αvβ3 integrin subtype, thereby representing privileged tools for integrin-based personalized medicine.

Full text of DOI:10.1021/jm500092w

Article
pubs.acs.org/jmc
Pharmacophoric Modifications Lead to Superpotent αvβ3 Integrin
Ligands with Suppressed α5β1 Activity
Stefanie Neubauer,†,×,# Florian Rechenmacher,†,× Richard Brimioulle,† Francesco Saverio Di Leva,‡,§
Alexander Bochen,† Tariq R. Sobahi,∥ Margret Schottelius,⊥ Ettore Novellino,§ Carlos Mas-Moruno,†,∞
Luciana Marinelli,§ and Horst Kessler*,†,∥
†Department Chemie, Institute for Advanced Study (IAS) and Center of Integrated Protein Science (CIPSM), Technische
Universitat Munchen, Lichtenbergstrasse 4, 85747 Garching, Germany
̈
̈
‡Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego, 30, 16163 Genova, Italy
§Dipartimento di Farmacia, Universita
di Napoli “Federico II”, Via D. Montesano, 49-80131 Napoli, Italy
̀
∥Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
⊥Nuklearmedizinische Klinik und Poliklinik, Klinikum Rechts der Isar der Technische Universitat Munchen, Ismaninger Strasse 22,
̈
̈
81675 Munchen, Germany
̈
S
* Supporting Information
ABSTRACT: The selective targeting of the αvβ3 integrin
subtype without affecting the structurally closely related
receptor α5β1 is crucial for understanding the details of
their biological and pathological functions and thus of great
relevance for diagnostic and therapeutic approaches in cancer
treatment. Here, we present the synthesis of highly active RGD
peptidomimetics for the αvβ3 integrin with remarkable
selectivity against α5β1. Incorporation of a methoxypyridine
building block into a ligand scaffold and variation of different
functional moieties led to αvβ3-antagonistic activities in the
low nanomolar or even subnanomolar range. Furthermore,
docking studies were performed to give insights into the
binding modes of the novel compounds. The presented library
comprises powerful ligands for specific addressing and blocking of the αvβ3 integrin subtype, thereby representing privileged
tools for integrin-based personalized medicine.
development of linear and cyclic peptides, peptidomimetics,
or small molecules with affinity for RGD-based integrins as
promising drug candidates to inhibit pathological angio-
genesis.8−17 One of the most prominent integrin targeting
drug candidates is cilengitide,10,18,19 a cyclic pentapeptide with
affinity for αvβ3 in the subnanomolar range and for αvβ5 and
α5β1 in the low nanomolar range, which is currently
undergoing clinical phase II for treatment of several cancer
types, such as non-small-cell lung cancer (NSCLC) or prostate
cancer, but recently failed in a phase III study to cure
glioblastoma.20 However, the fact that the precise roles of αvβ3
and α5β1 integrins in tumor development are not yet fully
understood and that cyclic RGD peptides21,22 are lacking
selectivity to discriminate these two receptors has stimulated
extensive research to develop novel integrin subtype specific
compounds. Such a differentiation was early achieved to
discriminate αv and α5β1 integrins from the platelet integrin
αIIbβ3 in head-to-tail cyclic pentapeptides of the general
INTRODUCTION
■
The integrin family, which consists of 24 cell surface receptors,
represents a promising target for cancer therapy as well as
disease monitoring, since its members enable cell adhesion,
proliferation, and survival by interacting with proteins of the
extracellular matrix (ECM).1,2 Integrins are known to be
aberrantly up-regulated on various cancer cells and the
surrounding tissues compared to normal tissues and thus are
key players in tumor progression, pathological angiogenesis,
and vascularization.3 In this way, they are involved in the
formation of new blood vessels and guarantee supply of the
tumor with oxygen and nutrients. Furthermore, integrins are
involved in a plethora of other diseases, such as multiple
sclerosis, Crohn’s disease, or inflammation.1−4
An important subfamily comprises the arginine−glycine−
aspartate (RGD)5 binding integrins, including all αv integrins,
the α5β1 subtype, and the blood platelet integrin αIIbβ3.6 Over
the past 2 decades it has been pointed out that αvβ3, αvβ5, and
α5β1 are of high relevance in angiogenesis and tumor
development, which turned them into attractive targets for
medicinal chemistry.7 We and others have reported the
Received: January 16, 2014
Published: March 23, 2014
© 2014 American Chemical Society
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Figure 1. Structure of the αvβ3-selective compound 1 (left) and its fitting into the binding pocket of αvβ3 (right). The moieties modified in the
molecules of our library are highlighted in green. The metal cation at the MIDAS is represented as a magenta sphere.
α
Scheme 1. Synthesis Strategy of the αvβ3-Selective Compounds 2−6
α
Reagents and conditions: (a) 3-bromopropanol, DMF, 80 °C, 48 h; (b) K2CO3, DMF, 0 °C, 30 min, 3-bromopropanol, rt, 16 h; (c) Dess−Martin
periodinane, DCM, rt, 3.5 h; (d) 2-amino-4-methoxypyridine, MgSO4·H2O, DCM, rt, 1 h, NaBH(OAc)3, overnight; (e) TFA/DCM (1:1, v/v), rt,
1 h; (f) 4-isopropoxybenzoic acid, HATU, DIEA, DMF, rt, overnight; (g) 4-toluenesulfonyl chloride, DIEA, DMF, rt, overnight; (h) 4-fluorobenzoic
acid, HATU, DIEA, DMF, rt, overnight; (i) LiOH, MeOH/H2O (3:1, v/v), rt, overnight; (k) LiOH, NH2OHaq., THF, rt, overnight.
structure c(RGDfX), containing a D-Phe next to the aspartic
acid.8,23 However, the design of subtype specific molecules for
αvβ3 and α5β1 is of great interest, since it would allow selective
targeting and blocking of integrin-mediated signaling cascades
under pathological conditions. Furthermore, highly active and
selective integrin ligands would pave the way for developing
novel tools to investigate their biological roles in vitro as well as
in vivo. Nonetheless, the design of these antagonists is
challenging because of the similarity in the RGD binding
pockets of αvβ3 and α5β1 (>50%), respectively.24,25 In this
context, peptidomimetics offer certain advantages in compar-
ison to peptides because they enable incorporation of unnatural
amino acids and other building blocks to achieve a proper
fitting of the molecule into the desired RGD binding pocket.
In the past, we have reported several αvβ3-active compounds
endowed with a satisfactory selectivity profile against the α5β1
integrin subtype.12 Out of these molecules, one of the most
active and selective ligands (compound 1, Figure 1) was
selected and functionalized for surface coating experiments as
well as for tumor imaging. However, the results of these studies
showed that the compounds were not potent enough to be
used for in vivo applications (data not shown).
Herein we report on a library of αvβ3 integrin antagonists
showing activities in the low nanomolar or even subnanomolar
range with remarkable selectivity against α5β1. These
compounds were designed based on pharmacophoric require-
ments obtained from previously described αvβ3-selective or
αvβ3/α5β1-biselective ligands (Figure 1).12,26,27 We incorpo-
rated a methoxypyridine residue and additionally modified
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distinct moieties to optimize the fitting of the ligands into the
αvβ3 integrin binding pocket. Because of the improved activity
profile and the possibility of functionalization without losing
affinity, we obtained powerful antagonists for coating of
surfaces28,29 or for positron emission tomography (PET)
imaging.30 Moreover, the binding modes of these compounds
at the αvβ3 and α5β1 receptors were carefully investigated
through molecular docking studies.
to obtain the deprotected carboxylic acids 2 and 3, respectively.
In a competitive integrin binding ELISA34 (see Supporting
Information) both compounds displayed outstanding subna-
nomolar activities for αvβ3, improved by more than 1 order of
magnitude in comparison to the reference compound 1 (Table
1). Besides their potent antagonistic activities, these com-
pounds also showed good selectivities against α5β1 and the
platelet integrin αIIbβ3 (>10000 nM).
RESULTS AND DISCUSSION
Table 1. Activity Profiles of the Synthesized αvβ3-Selective
Antagonists 1−6
■
a
In our previous studies we demonstrated that a β-homotyrosine
backbone in RGD peptidomimetics is essential to gain activity
toward the αvβ3 integrin and selectivity against α5β1 and
αIIbβ3.12 Besides the carboxylate group of tyrosine, which acts
as a mimic of the aspartate and coordinates a divalent metallic
cation at the metal-ion-dependent adhesion site (MIDAS) in
the RGD binding site, the basic moiety, the pyridine residue of
1, is also essential as an arginine surrogate (Figure 1). The
modification of this basic moiety is particularly important to
improve the affinity toward αvβ3 because of peculiar steric and
electrostatic demands imposed by the amino acid residues
present in the RGD binding region of the α-subunits. In this
regard, it has been already demonstrated that the substitution in
the para-position of the pyridine ring shows a greater influence
on the activity than in any other positions of the aromatic
system.12 In our design strategy, we aimed at introducing a
methoxy group in the para-position to enhance the pyridine
basicity, thus favoring the recognition by the RGD binding
region of the αv-subunits, which is more “acidic” than that of
α5β1.24 Thus, our first approach comprised the incorporation
of a p-methoxy group31,32 into the basic pyridine ring of 1.
Starting from the commercially available 2-amino-4-methoxy-
pyridine, we first performed a nucleophilic substitution with 3-
bromo-1-propanol to build up the new ligands according to a
reported strategy (Scheme 1A).12 However, the well-
established Mitsunobu reaction26 of the resulting alcohol 7
and the protected β-homotyrosine ester26 failed to yield the
scaffold 8. Extensive studies concerning the synthetic strategy
and reaction conditions (e.g., various nucleophilic substitutions
with improved leaving groups) finally led to a reverse synthetic
route starting with β-homotyrosine instead of the pyridine
derivative (Scheme 1B). First, a nucleophilic substitution was
performed with the protected β-homotyrosine ester and 3-
amino-1-propanol following a standard procedure with K2CO3
in DMF to obtain the alcohol 9 in excellent yields (Scheme
1B). Afterward, 9 was oxidized with Dess−Martin periodinane
in DCM33 to obtain the aldehyde 10, which was directly used in
the next step without further purification. To introduce the 2-
amino-4-methoxypyridine building block, we performed a
reductive amination of 10 in the presence of magnesium
sulfate and sodium triacetoxyborohydride in DCM33 in order to
yield 8 as backbone and lead structure for the development of
the new peptidomimetics 2−6.
IC50 (nM)
compd
b
αvβ3
α5β1
>10000
αIIbβ3
1
2
3
4
5
6
110 64
nd
0.86 0.06
0.65 0.05
638 124
75.1 8.6
1.6 0.17
0.54 0.02
126.5 25.6
108.0 27.5
>10000
>10000
>10000
nd
>10000
nd
64.2 13.1
nd
cilengitide
15.4
3
>1000
a
All IC50 values were determined in a solid phase integrin binding
ELISA34 and referenced to cilengitide.10,18 nd: not determined.
b
Compound 1 was synthesized according to the literature and
originally tested by the Jerini AG, Berlin, Germany, with a slightly
different method (13 nM for αvβ3 and 3946 nM for α5β1).12
Comparing compounds 2 and 3, the first one turned out to
be the most promising compound for further functionalization,
as it can be modified in a very straightforward manner through
the introduction of different functional moieties at the para-
position of the aromatic carboxylic acid building block. In order
to explore the affinity profile toward other RGD-binding
integrins, we determined the IC50 values of 2 toward the αvβ5
and αvβ6 subtypes. In comparison to the subnanomolar
antagonistic activity observed for αvβ3, compound 2 showed a
lower affinity for αvβ6 (IC50 of 20 nM) and a good selectivity
against αvβ5 (IC50 of 227 nM) (see Supporting Information,
Table S1). Moreover, we have already shown that this ligand
can be functionalized and used for coating gold and titanium-
based biomaterials in vitro and for selective tumor imaging in
vivo with outstanding results.28−30,35
After the incorporation of the methoxypyridine moiety, we
investigated the effects of changing the ligand scaffold length by
replacing the carboxylic acid moiety, responsible for the crucial
interaction with the metal cation at the MIDAS,36 with a larger
functional group. For this reason, we synthesized ligands with a
hydroxamic acid. This functionality, in contrast to carboxylic
acids, is not charged under neutral pH conditions and often has
shown better pharmacodynamic behaviors.37 Thus, the methyl
ester of the β-tyrosine scaffold was converted into the
hydroxamic acid following a modified procedure37,38 with
hydroxylamine to render compounds 4 and 5. This protocol
simultaneously favors both the saponification of the methyl
ester and the final conversion of the carboxylate to the
corresponding hydroxamic acid. To minimize saponification,
the addition of LiOH to the reaction mixture of THF/water
and hydroxylamine was reduced to less than 1 equiv of LiOH.
Although these compounds (4 and 5) display excellent
selectivities against α5β1, their activities toward αvβ3 were
significantly reduced compared to the analogues 2 and 3.
In a final approach, the scaffold 8 was modified with a
fluorine atom through coupling with p-fluorobenzoic acid
Previous studies on α5β1-selective compounds revealed that
introduction of a sulfonamide bond (Y = SO2, Scheme 1)
instead of a carboxamide (Y = CO, Scheme 1) at the amino
group of β-tyrosine-based peptidomimetics can enhance their
affinity because of different structural orientations of the
aromatic residue.12,26 On the basis of this observation, we
investigated this effect on αvβ3 antagonists. Hence, after Boc-
deprotection of 8 under acidic conditions, the free amine was
acylated with either 4-isopropoxybenzoic acid or 4-toluene-
sulfonyl chloride, followed by saponification of the methyl ester
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Figure 2. Binding modes of 2 (a, c) and 3 (b, d) at the αvβ3 and α5β1 integrins. 2 and 3 are shown as yellow and green sticks, respectively. The αv
and β3 subunits are depicted as pink and light blue surfaces (upper panels a and b), while the α5 and β1 subunits are depicted as red and blue
surfaces (lower panels c and d). Receptor amino acid side chains important for the ligand binding are represented as sticks. The metal cation at the
MIDAS is represented as a magenta sphere.
according to a standard procedure.39 Compound 6 showed
excellent activity for αvβ3 (1.6 nM) and good selectivity against
α5β1. Thus, labeling with 18F would be a feasible strategy to
selectively target and image αvβ3 integrins via PET.40
the (β3)-Arg214 side chain through its oxygen atoms and with
the backbone CO group of (β3)-Tyr122 through its NH group.
Overall, the docking poses predicted for 2 and 3 at the αvβ3
RGD binding site are useful to explain the subnanomolar
activities displayed by these ligands toward this integrin
subtype. Furthermore, it is worth noting that the substitution
of the 6-methyl group in our reference compound 1 with the
electron-donating 4-methoxy group enhances the basicity of the
pyridine ring and also reduces at minimum its steric clash with
(αv)-Thr212. These effects allow the basic moiety to properly
interact with (αv)-Asp218, increasing the affinity of the new
analogues for the αvβ3 receptor. For opposite reasons (i.e.,
enhanced steric clash with (αv)-Thr212), the elongation of the
ligand main chain through the replacement of the carboxylic
moiety with a hydroxamate group turned out to be detrimental
for the αvβ3 antagonism, as observed for compounds 4 and 5.
Indeed, the optimal distance between the acidic and the basic
moieties in αvβ3 integrin ligands is ∼13 Å,27 as in the case of
our carboxylic compounds, while this distance is higher in
hydroxamic derivatives which are too long for properly fitting in
the RGD binding cavity.12
Finally, the slightly lower affinity of the carboxylic derivative
6 toward αvβ3, if compared to 2, is mainly ascribable to the loss
of favorable lipophilic contacts with the (β3)-Met180 side chain
due to the replacement of the p-isopropoxy group at the phenyl
ring with a tiny and more polar fluorine atom.
The lower potencies of the newly synthesized peptidomi-
metics toward the α5β1 receptor with respect to αvβ3 are due
to single point mutations that differentiate the binding cavities
of these two integrin subtypes (Figure 2).12,24,27,37 In the α
subunit, the acidic (αv)-Asp150 is mutated to the nonpolar
(α5)-Ala159, and (αv)-Thr212 is replaced by the bulkier (α5)-
To rationalize the structure−activity relationships as well as
the selectivity profiles of the new αvβ3-binding peptidomi-
metics, molecular modeling studies were performed by docking
the most active compounds 2 and 3 into the binding site of
αvβ341 and α5β124 integrins (for docking methods, see the
Supporting Information). According to docking results,
compounds 2 and 3 bind to the αvβ3 RGD binding site in a
similar fashion (Figure 2). For both ligands, the carboxylate
group coordinates the metal ion at the MIDAS and also forms a
hydrogen bond with the backbone NH group of (β3)-Asn215.
On the opposite side, the positively charged 2-amino-4-
methoxypyridine moiety salt-bridges with (αv)-Asp218 and
establishes a π−π interaction with the (αv)-Tyr178 side chain.
Moreover, the β-homotyrosine scaffold is found in the
proximity of (αv)-Tyr178, thus establishing parallel-displaced
π−π interactions with the aromatic side chain of this residue.
The only difference observed in the binding modes of 2 and 3
resides in the interaction patterns between the p-isopropyloxy-
phenyl moiety of 2 and the p-methylbenzenesulfonamide group
of 3, respectively, owing to the distinctive structural features of
these substituents. The p-isopropyloxyphenyl moiety extends
toward the specificity determining loop (SDL) of the integrin
binding pocket, leading to a T-shape interaction with (β3)-
Tyr122, a cation−π interaction with (β3)-Arg214, and
lipophilic contacts with the (β3)-Met180 side chain. Alter-
natively, the p-methylbenzenesulfonamide group of 3 is
predicted to additionally establish a T-shape interaction with
the (β3)-Tyr122 side chain and to form a hydrogen bond with
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instrument (system gold, solvent delivery module 126, UV detector
166) using an YMC ODS-A column (20 mm × 250 mm, 5 μm), with a
flow rate of 8 mL/min. Linear gradients using H2O (0.1% (v/v) TFA)
and MeCN (0.1% (v/v) TFA) were run over varying periods of time.
HPLC−mass spectrometry (MS) analyses were performed on a
Hewlett-Packard series HP 1100 with a Finnigan LCQ mass
spectrometer using a YMC Hydrosphere C18 column (2.1 mm ×
125 mm, 3 μm). The system uses H2O (0.1% (v/v) formic acid) and
MeCN (0.1% (v/v) formic acid) as eluents. High resolution mass
spectrometry (HR-MS) was recorded on a Thermo Finnigan LTQ-FT
(ESI-ICR) spectrometer. 1H NMR and 13C NMR spectra were
recorded at 295 K on a 500 MHz Bruker DMX, 360 MHz Bruker AV,
or a 250 MHz Bruker AV spectrometer (Bruker, Karlsruhe, Germany).
Chemical shifts (δ) are given in parts per million (ppm). The
following solvent peaks were used as internal standards: DMSO-d6,
2.50 ppm (1H NMR) and 39.52 ppm (13C NMR); CHCl3, 7.26 ppm
(1H NMR) and 77.16 ppm (13C NMR).42 The yields of the syntheses
are not optimized. All synthesized compounds exhibit ≥95% purity
determined with HPLC−MS.
Boc-Deprotection and Acylation by Aromatic Benzylic Acids
(GP1a). The Boc-protected starting material was dissolved in a
mixture of TFA and DCM (1:1 (v/v), ∼0.1 M). After stirring for 1 h
at room temperature, the solvent was removed under reduced
pressure. The resulting deprotected amine was redissolved in DMF
(∼0.1 M) followed by addition of the corresponding aromatic acid
(1.3 equiv), HATU (1.3 equiv), and DIEA (5 equiv). The resulting
yellow solution was stirred overnight at room temperature and the
solvent was evaporated.
Boc-Deprotection and Acylation by Aromatic Sulfonyl
Chlorides (GP1b). The Boc-protected starting material was dissolved
in a mixture of TFA and DCM (1:1; (v/v) ∼0.1 M). After the mixture
was stirred for 1 h at room temperature, the solvent was removed
under reduced pressure. The resulting deprotected amine was
redissolved in DMF (∼0.1 M) followed by addition of the 4-
toluenesulfonyl chloride (1.3 equiv) and DIEA (5 equiv). The
resulting yellow solution was stirred overnight at room temperature,
and the solvent was evaporated.
Saponification of Methyl Ester (GP2a). The residue was
dissolved in methanol/water (3:1, v/v), and LiOH (5 equiv) was
added to this solution, which was left under stirring at room
temperature overnight. The resulting deprotected compound was
purified using (semi)preparative reversed phase HPLC and lyophilized
to yield a colorless solid.
Gln221. Interestingly, the presence of the Gln residue reduces
the space available for the binding of the basic moiety, as clearly
shown by docking of 2 and 3 into the α5β1 RGD binding site,
thus leading to a decrease in affinity of these two ligands toward
this integrin subtype. This tendency is even more evident for
the hydroxamic derivatives 4 and 5, in which, as previously
reported, the distance between the metal-coordinating acidic
group and the basic moiety is increased. In addition, the
binding region on the α5 subunit is less acidic than that found
at the αv subunit due to the mutation of (αv)-Asp150 to (α5)-
Ala159. In this regard, the insertion of more basic groups
generally results in less potent α5β1 antagonists.12,24,25,27,37
Thus, the introduction of the electron-donating 4-methoxy
group, which increases the basicity of the pyridine ring, lowers
the affinity of the ligands toward the α5β1 integrin. In the β
subunit, the substitution of the (β3)-Arg214 residue with (β1)-
Gly217 is predicted to affect the binding affinity of both 2 and
3. In particular, in the α5β1 receptor the oxygen atoms of the
sulfonamide moiety of 3 cannot establish the same H-bonds as
observed in αvβ3, while the p-isopropyloxyphenyl ring of 2
misses an important cation−π interaction. In the case of 2, the
substitution of the lipophilic (β3)-Met180 with the acidic (β1)-
Asp184 might also contribute to reduce the ligand affinity
toward α5β1, since the p-isopropyloxyl substituent of the
phenyl ring cannot form any favorable hydrophobic contacts
with Asp, contrary to what is observed in αvβ3. Conversely, the
presence of the more polar fluorine atom on 6 allows this
ligand to retain satisfactory selectivity against α5β1.
CONCLUSION
■
We have developed a small library of RGD peptidomimetics
based on a β-homotyrosine scaffold as new αvβ3 integrin
antagonists. These compounds incorporate a basic methoxy-
pyridine moiety and present different metal-coordinating acidic
groups and aromatic building blocks. Competitive in vitro
assays have demonstrated that two of these ligands, namely, 2
and 3, are highly potent binders of the αvβ3 integrin, displaying
IC50 values in the subnanomolar range. Furthermore, they show
a high selectivity against the α5β1 receptor. Molecular docking
studies have identified the binding modes of 2 and 3 at both the
αvβ3 and α5β1 receptors, disclosing the molecular requisites
for achieving optimal activity and selectivity toward these
integrin subtypes. These studies therefore provide useful
insights to improve the design of new integrin-selective
peptidomimetics ligands, which are excellent candidates for
medical and biophysical applications.
Formation of Hydroxamic Acid (GP2b). The residue was
dissolved in THF, and then LiOH (<1 equiv) and hydroxylamine (50
wt % solution in water) were added to the mixture, which was left
under stirring at room temperature overnight. The resulting
deprotected compound was purified using (semi)preparative reversed
phase HPLC and lyophilized to yield a colorless solid.
(S)-3-Benzamido-4-(4-(3-((6-methylpyridin-2-yl)amino)-
propoxy)phenyl)butanoic Acid (1). Compound 1 was synthesized
according to the literature.12 1H NMR and 13C NMR spectra were
identical to those previously reported.12 HPLC (10−90%): tR = 14.95
min. MS (ESI): m/z (%) = 448.4 [M + H]+.
EXPERIMENTAL SECTION
■
(S)-3-(4-Isopropoxybenzamido)-4-(4-(3-((4-methoxypyridin-
2-yl)amino)propoxy)phenyl)butanoic Acid (2). The Boc-depro-
tection and acylation of the ligand precursor 8 (0.34 g, 0.71 mmol)
with 4-isopropoxybenzoic acid (0.17 g, 0.92 mmol) were done
according to GP1a. The crude product was saponified according to
GP2a. Purification yielded compound 2 (55.6 mg, 0.11 mmol, 15%) as
Chemistry. Solvents were purchased from Aldrich, Fluka, Merck,
and Prolabo. Reactions sensible to oxygen or water were performed in
flame-dried reaction vessels under an argon atmosphere (99.996%).
Coupling reagents were purchased from Iris Biotech GmbH and
Medalchemy. All other chemicals and organic solvents were purchased
from commercial suppliers at the highest purity available and used
without further purification. Flash chromatographic purification was
performed using silica gel 60 (40−63 μm) from Merck at 1−1.5 atm
pressure.
1
TFA salt. H NMR (500 MHz, DMSO): δ [ppm] = 12.87 (bs, 1H),
12.17 (bs, 1H), 8.28 (bs, 1H), 8.13 (d, 3J = 8.3 Hz, 1H), 7.80 (d, 3J =
7.1 Hz, 1H), 7.72 (d, 3J = 8.8 Hz, 2H), 7.13 (d, 3J = 8.5 Hz, 2H), 6.94
(d, 3J = 8.8 Hz, 2H), 6.83 (d, 3J = 8.6 Hz, 2H), 6.46 (dd, 3J = 7.0 Hz, 4J
Analytical high-performance liquid chromatography (HPLC) was
4
̈
= 2.1 Hz, 1H), 6.33 (d, J = 1.9 Hz, 1H), 4.72−4.65 (m, 1H), 4.44−
performed using an Amersham Pharmacia Biotech Akta Basic 10F
3
4.37 (m, 1H), 4.01 (t, J = 6.0 Hz, 2H), 3.86 (s, 3H), 3.46−3.42 (m,
equipment with a P-900 pump system, a reversed-phase YMC-ODS-A
C18 column (4.6 mm × 250 mm, 5 μm), and UV detection (UV-900,
220 and 254 nm). The system was run at a flow rate of 1 mL/min over
30 min using H2O (0.1% (v/v) TFA) and MeCN (0.1% (v/v) TFA)
as solvents. Semipreparative HPLC was carried out on a Beckmann
2H), 2.80 (dd, 2J = 13.6 Hz, 3J = 8.0 Hz, 1H), 2.73 (dd, 2J = 13.6 Hz,
3J = 5.8 Hz, 1H), 2.52−2.48 (m, 1H), 2.41 (dd, 2J = 15.4 Hz, 3J = 6.2
3
Hz, 1H), 2.02−1.97 (m, 2H), 1.27 (d, J = 6.0 Hz, 6H). 13C NMR
(125 MHz, DMSO): δ [ppm] = 172.5, 165.1, 159.7, 156.8, 130.9,
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130.1, 129.0, 126.5, 114.7, 114.1, 103.9, 99.4, 69.3, 64.6, 56.4, 48.3,
pound 10 (550 mg of crude product) was dissolved in absolute DCM.
Next, 2-amino-4-methoxypyridine (0.21 mg, 1.73 mmol) and
magnesium sulfate monohydrate (0.87 mg, 6.27 mmol) were added.
After the suspension was stirred for 1 h at room temperature, sodium
triacetoxyborohydride (1.71 g, 8.07 mmol) was added, and stirring was
continued overnight. After addition of 50 mL of a saturated NaHCO3
solution and further 30 min of stirring, the organic layer was extracted
twice with DCM (50 mL) and dried over Na2SO4. After filtration, the
solution was concentrated to dryness and the crude product was
purified by flash chromatography (DCM/EtOAc 1:1) to give 8 as a
colorless oil (0.42 mg, 0.89 mmol, 62% over two steps). 1H NMR (500
MHz, DMSO): δ [ppm] = 7.77 (d, 3J = 5.8 Hz, 1H), 7.07 (d, 3J = 8.5
Hz, 2H), 6.86−6.84 (m, 3H), 6.49 (t, 3J = 5.5 Hz, 1H), 6.11 (dd, 3J =
5.8 Hz, 4J = 2.2 Hz, 1H), 5.94 (d, 4J = 2.2 Hz, 1H), 3.99 (t, 3J = 6.3 Hz,
2H), 3.92−3.85 (m, 1H), 3.69 (s, 3H), 3.54 (s, 3H), 3.36−3.32 (m,
40.1, 39.8, 38.7, 27.9, 21.7. HPLC (10−90%): tR = 17.63 min. MS
+
(ESI): m/z (%) = 522.4 [M + H]+. HR-MS (ESI) (C29H36N3O6 )
calcd, 522.259 86; found, 522.257 95.
(S)-4-(4-(3-((4-Methoxypyridin-2-yl)amino)propoxy)phenyl)-
3-(4-methylphenylsulfonamido)butanoic Acid (3). The Boc-
deprotection and acylation of the ligand precursor 8 (0.34 g, 0.71
mmol) with 4-toluenesulfonyl chloride (0.16 g, 0.92 mmol) followed
GP1b. The crude product was deprotected according to GP2a.
Purification yielded compound 3 (47.4 mg, 92.2 μmol, 13%) as TFA
salt. 1H NMR (500 MHz, CDCl3): δ [ppm] = 9.42 (bs, 1H), 7.64 (d,
3J = 8.2 Hz, 2H), 7.59 (d, 3J = 7.1 Hz, 1H), 7.22 (d, 3J = 8.0 Hz, 2H),
6.92 (d, 3J = 8.5 Hz, 2H), 6.71 (d, 3J = 8.5 Hz, 2H), 6.28 (dd, 3J = 7.1
4
4
3
Hz, J = 2.3 Hz, 1H), 6.01 (d, J = 2.1 Hz, 1H), 5.41 (d, J = 7.9 Hz,
1H), 4.03 (t, 3J = 5.5 Hz, 2H), 3.84 (s, 3H), 3.74−3.67 (m, 1H), 3.48−
3.43 (m, 2H), 2.78−2.68 (m, 2H), 2.41−2.37 (m, 5H), 2.18−2.13 (m,
2H). 13C NMR (125 MHz, CDCl3): δ [ppm] = 171.2, 163.5, 163.2,
157.8, 156.3, 143.7, 138.0, 130.8, 130.0, 129.4, 127.3, 114.9, 103.5,
89.4, 64.8, 56.5, 51.9, 40.1, 39.7, 37.3, 28.4, 21.8. HPLC (10−90%): tR
= 15.95 min. MS (ESI): m/z (%) = 514.4 [M + H]+. HR-MS (ESI)
(C26H32N3O6S+) calcd, 514.200 63; found, 514.199 02.
3
2H), 2.65−2.56 (m, 2H), 2.37 (d, J = 6.9 Hz, 2H), 1.96−1.90 (m,
2H), 1.32 (s, 9H). 13C NMR (125 MHz, DMSO): δ [ppm] = 171.4,
166.0, 160.7, 157.1, 154.9, 148.7, 130.4, 130.2, 114.2, 100.9, 90.7, 77.7,
65.3, 54.6, 51.4, 49.5, 37.8, 28.9, 28.3. HPLC (10−90%): tR = 19.15
t
min. MS (ESI): m/z (%) = 474.2 [M + H]+, 418.3 [M − Bu + H]+,
(S)-N-(4-(Hydroxyamino)-1-(4-(3-((4-methoxypyridin-2-yl)-
amino)propoxy)phenyl)-4-oxobutan-2-yl)-4-isopropoxybenza-
mide (4). The Boc-deprotection and acylation of 8 (32.0 mg, 67.6
μmol) with 4-isopropoxybenzoic acid (14.6 mg, 81.1 μmol) followed
GP1a. After formation of the hydroxamic acid according to GP2b,
purification yielded compound 4 (2.35 mg, 4.72 μmol, 7%) as TFA
salt. HPLC (10−90%): tR = 17.93 min. MS (ESI): m/z (%) = 537.3
374.3 [M − Boc + H]+.
(S)-Methyl 3-((tert-Butoxycarbonyl)amino)-4-(4-(3-
hydroxypropoxy)phenyl)butanoate (9). A solution of N-Boc-β-
tyrosine methyl ester26 (0.52 mg, 1.68 mmol) and K2CO3 (0.58 mg,
4.20 mmol) in absolute DMF was stirred for 30 min at 0 °C. Next, 3-
bromo-1-propanol (161 μL, 1.85 mmol) was added, and the mixture
was stirred for 16 h while warming to room temperature. The solvent
was evaporated in vacuo. The residue was taken up with 1 N HCl and
extracted with EtOAc. The combined organic layers were treated with
saturated NaCl, dried over Na2SO4, and concentrated to dryness. The
crude product was purified by flash chromatography (DCM/EtOAc
3:1) to give 9 as a colorless oil (0.56 mg, 1.53 mmol, 91%). 1H NMR
(500 MHz, DMSO): δ [ppm] = 7.06 (d, 3J = 8.5 Hz, 2H), 6.82 (d, 3J =
8.3 Hz, 2H), 6.79 (d, 3J = 8.6 Hz, 1H), 4.51 (bs, 1H), 3.98 (t, 3J = 6.4
Hz, 2H), 3.93−3.85 (m, 1H), 3.54 (s, 3H), 3.33−3.29 (m, 2H), 2.63−
+
[M + H]+. HR-MS (ESI) (C29H37N4O6 ) calcd, 537.270 76; found,
537.269 13.
(S)-N-Hydroxy-4-(4-(3-((4-methoxypyridin-2-yl)amino)-
propoxy)phenyl)-3-((4-methylphenyl)sulfonamido)-
butanamide (5). The Boc-deprotection and acylation of 8 (43.0 mg,
90.8 μmol) with 4-toluenesulfonyl chloride (20.8 mg, 0.11 mmol)
followed GP1b. After formation of the hydroxamic acid according to
GP2b, purification yielded compound 5 (4.36 mg, 8.25 μmol, 9%) as
TFA salt. HPLC (10−90%, 30 min): tR = 13.78 min. MS (ESI): m/z
(%) = 529.3 [M + H]+. HR-MS (ESI) (C26H33N4O6S+) calcd,
529.211 53; found, 529.210 55.
3
2.59 (m, 2H), 2.38 (d, J = 6.9 Hz, 2H), 1.86−1.80 (m, 2H), 1.32 (s,
9H). 13C NMR (125 MHz, DMSO): δ [ppm] = 171.3, 157.1, 154.8,
130.2, 130.1, 114.1, 77.6, 64.4, 57.3, 51.3, 49.4, 38.8, 32.1, 28.2. HPLC
(10−100%): tR = 18.34 min. MS (ESI): m/z (%) = 757.1 [2M + Na]+,
390.2 [M + Na]+, 268.3 [M − Boc + H]+.
(S)-3-(4-Fluorobenzamido)-4-(4-(3-((4-methoxypyridin-2-yl)-
amino)propoxy)phenyl)butanoic Acid (6). The deprotection and
acylation of the ligand precursor 8 (50 mg, 0.11 mmol) with 4-
fluorobenzoic acid (18.2 mg, 0.13 mmol) followed GP1a. The crude
product was deprotected according to GP2a. Purification yielded
(S)-Methyl 3-(tert-Butoxycarbonyl)amino)-4-(4-(3-
oxopropoxy)phenyl)butanoate (10). Compound 9 (0.53 mg,
1.44 mmol) was dissolved in absolute DCM, and Dess−Martin
periodinane (1.83 g 4.32 mmol) was added in three portions within
1.5 h. The suspension was stirred for an additional 2 h at room
temperature and extracted twice with a mixture of 10% sodium
thiosulfate solution and saturated NaHCO3 solution (1:1). The
organic layers were combined, washed with H2O and saturated NaCl,
and dried over Na2SO4. The solvent was evaporated in vacuo, and
compound 10 was obtained as a yellow oil (550 mg). The compound
1
compound 6 (7.9.mg, 16.4 μmol, 15%) as TFA salt. H NMR (500
MHz, DMSO): δ [ppm] = 12.77 (bs, 1H), 12.19 (bs, 1H), 8.34 (d, 3J
3
3
= 8.2 Hz, 1H), 8.18 (bs, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.80 (d, J =
7.1 Hz, 1H), 7.29 (d, 3J = 8.8 Hz, 2H), 7.13 (d, 3J = 8.5 Hz, 2H), 6.83
3
3
3
(d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 6.46 (d, J = 6.3 Hz,
1H), 6.31 (s, 1H), 4.45−4.38 (m, 1H), 4.01 (t, 3J = 5.9 Hz, 2H), 3.85
(s, 3H), 3.47−3.41 (m, 2H), 2.81−2.74 (m, 2H), 2.53−2.48 (m, 1H),
2.43 (dd, J = 15.4 Hz, J = 6.1 Hz, 1H), 2.03−1.96 (m, 2H). 13C
NMR (125 MHz, DMSO): δ [ppm] = 172.4, 169.0, 164.7, 162.8,
158.0, 157.1, 156.8, 131.1, 130.8, 130.1, 129.8, 115.2, 114.2, 103.9,
91.5, 64.6, 56.3, 48.5, 38.6, 27.9. HPLC (10−90%): tR = 15.58 min.
MS (ESI): m/z (%) = 482.4 [M + H]+. HR-MS (ESI)
2
3
1
was used directly in the next step without further purification. H
NMR (360 MHz, DMSO): δ [ppm] = 9.72 (t, 3J = 1.7 Hz, 1H), 7.08
3
3
3
(d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 8.9 Hz,
1H), 4.24 (t, 3J = 5.9 Hz, 2H), 3.94−3.82 (m, 1H), 3.54 (s, 3H), 2.87−
2.83 (m, 2H), 2.64−2.35 (m, 2H), 2.44−2.33 (m, 2H), 1.32 (s, 9H).
13C NMR (90 MHz, DMSO): δ [ppm] = 201.8, 171.3, 156.7, 154.8,
130.7, 130.1, 114.2, 77.6, 61.5, 51.3, 49.3, 42.7, 28.2. HPLC (10−
90%): tR = 21.91 min. MS (ESI): m/z (%) = 388.1 [M + Na]+, 332.3
+
(C26H29FN3O5 ) calcd, 482.208 58; found, 482.206 65.
3-((4-Methoxypyridin-2-yl)amino)propan-1-ol (7). 2-Amino-4-
methoxypyridine (0.40 g, 3.22 mmol, 1 equiv) and 3-bromopropanol
(1.34 g, 9.67 mmol, 3 equiv) were dissolved in DMF and heated up in
a sealed glass tube to 80 °C for 48 h. The reaction mixture was purified
by flash chromatography (DCM/MeOH 9:1 + 1% TEA) to give 7 as a
t
[M − Bu + Na]+, 266.1 [M − Boc + H]+.
1
pale yellow solid (0.54 g, 2.95 mmol, 92%). H NMR (250 MHz,
CDCl3): δ [ppm] = 8.11 (s, 1H), 7.92 (d, 3J = 7.5 Hz, 1H), 6.63−6.59
(dd, 3J = 5.0, Hz, 3J = 2.5 Hz, 1H), 6.40 (d, 3J = 2.5 Hz, 1H), 4.76 (bs,
1H), 4.13 (t, 3J = 7.5 Hz, 2H), 3.89 (s, 3H), 3.46−3.40 (m, 2H), 1.88−
1.80 (m, 2H). HPLC (0−30%, 30 min): tR = 15.05 min. MS (ESI): m/
z = 183.1 [M + H]+.
ASSOCIATED CONTENT
■
S
* Supporting Information
Competitive solid-phase integrin binding assay, activity profile
of compound 2, and docking methods. This material is available
free of charge via the Internet at http://pubs.acs.org.
(S)-Methyl 3-(tert-Butoxycarbonyl)amino)-4-(4-(3-((4-me-
thoxypyridin-2-yl)amino)propoxy)phenyl)butanoate (8). Com-
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Article
(10) Dechantsreiter, M. A.; Planker, E.; Matha, B.; Lohof, E.;
̈
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 (0)89 289 13300. Fax: +49 (0)89 289 13210. E-
mail: kessler@tum.de.
■
Holzemann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. N-
̈
Methylated cyclic RGD peptides as highly active and selective αvβ3
integrin antagonists. J. Med. Chem. 1999, 42, 3033−3040.
(11) Dayam, R.; Aiello, F.; Deng, J.; Wu, Y.; Garofalo, A.; Chen, X.;
Neamati, N. Discovery of small molecule integrin αvβ3 antagonists as
novel anticancer agents. J. Med. Chem. 2006, 49, 4526−4534.
(12) Heckmann, D.; Meyer, A.; Marinelli, L.; Zahn, G.; Stragies, R.;
Kessler, H. Probing integrin selectivity: rational design of highly active
and selective ligands for the α5β1 and αvβ3 integrin receptor. Angew.
Chem., Int. Ed. 2007, 46, 3571−3574.
Present Addresses
#S.N.: Department of New Materials and Biosystems, Max
Planck Institute for Intelligent Systems, Heisenbergstrasse 3,
70569 Stuttgart, Germany.
∞C.M.-M.: Biomaterials, Biomechanics and Tissue Engineering
Group, Department of Materials Science and Metallurgical
Engineering, Technical University of Catalonia (UPC), Av.
Diagonal 647, E08028 Barcelona, Spain.
(13) Zanardi, F.; Burreddu, P.; Rassu, G.; Auzzas, L.; Battistini, L.;
Curti, C.; Sartori, A.; Nicastro, G.; Menchi, G.; Cini, N.; Bottonocetti,
A.; Raspanti, S.; Casiraghi, G. Discovery of subnanomolar arginine-
glycine-aspartate-based αVβ3/αVβ5 integrin binders embedding 4-
aminoproline residues. J. Med. Chem. 2008, 51, 1771−1782.
(14) Gentilucci, L.; Cardillo, G.; Spampinato, S.; Tolomelli, A.;
Squassabia, F.; De Marco, R.; Bedini, A.; Baiula, M.; Belvisi, L.; Civera,
M. Antiangiogenic effect of dual/selective α5β1/αvβ3 integrin
antagonists designed on partially modified retro-inverso cyclo-
tetrapeptide mimetics. J. Med. Chem. 2010, 53, 106−118.
(15) Corti, A.; Curnis, F. Tumor vasculature targeting through NGR
peptide-based drug delivery systems. Curr. Pharm. Biotechnol. 2011, 12,
1128−1134.
(16) Bianchini, F.; Cini, N.; Trabocchi, A.; Bottoncetti, A.; Raspanti,
S.; Vanzi, E.; Menchi, G.; Guarna, A.; Pupi, A.; Calorini, L. 125I-
Radiolabeled morpholine-containing arginine-glycine-aspartate (RGD)
ligand of αvβ3 integrin as a molecular imaging probe for angiogenesis.
J. Med. Chem. 2012, 55, 5024−5033.
(17) Colombo, R.; Mingozzi, M.; Belvisi, L.; Arosio, D.; Piarulli, U.;
Carenini, N.; Perego, P.; Zaffaroni, N.; De Cesare, M.; Castiglioni, V.;
Scanziani, E.; Gennari, C. Synthesis and biological evaluation (in vitro
and in vivo) of cyclic arginine-glycine-aspartate (RGD) peptidomi-
metic-paclitaxel conjugates targeting integrin αVβ3. J. Med. Chem. 2012,
55, 10460−10474.
Author Contributions
×S.N. and F.R. contributed equally.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank CompInt (Materials Science of Complex Interfaces)
of the Elite Network of Bavaria, IGSSE (International Graduate
School of Science and Engineering) and Bund der Freunde der
TU Munchen e.V. for funding, IAS (Institute for Advanced
̈
Study) of Technische Universitat Munchen, CIPSM (Center
̈
̈
for Integrated Protein Science Munich), and Max Planck
Society for financial support. We gratefully thank King
Abdulaziz University (KAU) for technical and financial support
(Grant HiCi/25-3-1432).
ABBREVIATIONS USED
■
(18) Mas-Moruno, C.; Rechenmacher, F.; Kessler, H. Cilengitide: the
first anti-angiogenic small molecule drug candidate. Design, synthesis
and clinical evaluation. Anti-Cancer Agents Med. Chem. 2010, 10, 753−
768.
(19) Reardon, D. A.; Cheresh, D. Cilengitide: a prototypic integrin
inhibitor for the treatment of glioblastoma and other malignancies.
Genes Cancer 2011, 2, 1159−1165.
ECM, extracellular matrix; ELISA, enzyme-linked immuno-
sorbent assay; MIDAS, metal-ion-dependent adhesion site;
HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-
nium hexafluorophosphate
REFERENCES
■
(20) Merck KGaA press release, February 25, 2013: http://www.
merckgroup.com/en/media/extNewsDetail.html?newsId=
BE2FE07AD630830EC1257B1D001F007B&newsType=1.
(21) Bochen, A.; Marelli, U. K.; Otto, E.; Pallarola, D.; Mas-Moruno,
C.; Di Leva, F. S.; Boehm, H.; Spatz, J. P.; Novellino, E.; Kessler, H.;
Marinelli, L. Biselectivity of isoDGR peptides for fibronectin binding
integrin subtypes α5β1 and αvβ6: conformational control through
flanking amino acids. J. Med. Chem. 2013, 56, 1509−1519.
(22) Mondal, G.; Barui, S.; Chaudhuri, A. The relationship between
the cyclic-RGDfK ligand and αvβ3 integrin receptor. Biomaterials
2013, 34, 6249−6260.
(1) Goodman, S. L.; Picard, M. Integrins as therapeutic targets.
Trends Pharmacol. Sci. 2012, 33, 405−412.
(2) Kapp, T. G.; Rechenmacher, F.; Sobahi, T. R.; Kessler, H.
Integrin modulators: a patent review. Expert Opin. Ther. Pat. 2013, 23,
1273−1295.
(3) Desgrosellier, J. S.; Cheresh, D. A. Integrins in cancer: biological
implications and therapeutic opportunities. Nat. Rev. Cancer 2010, 10,
9−22.
(4) Shattil, S. J.; Kim, C.; Ginsberg, M. H. The final steps of integrin
activation: the end game. Nat. Rev. Mol. Cell Biol. 2010, 11, 288−300.
(5) Pierschbacher, M. D.; Ruoslahti, E. Cell attachment activity of
fibronectin can be duplicated by small synthetic fragments of the
molecule. Nature 1984, 309, 30−33.
(6) Hynes, R. O. A reevaluation of integrins as regulators of
angiogenesis. Nat. Med. 2002, 8, 918−921.
(7) Marelli, U. K.; Rechenmacher, F.; Sobahi, T. R.; Mas-Moruno, C.;
Kessler, H. Tumor targeting via integrin ligands. Front. Oncol. 2013, 3,
222.
(23) Pfaff, M.; Tangemann, K.; Muller, B.; Gurrath, M.; Muller, G.;
̈
̈
Kessler, H.; Timpl, R.; Engel, J. Selective recognition of cyclic RGD
peptides of NMR defined conformation by αIIbβ3, αVβ3, and α5β1
integrins. J. Biol. Chem. 1994, 269, 20233−20238.
(24) Marinelli, L.; Meyer, A.; Heckmann, D.; Lavecchia, A.;
Novellino, E.; Kessler, H. Ligand binding analysis for human α5β1
integrin: strategies for designing new α5β1 integrin antagonists. J. Med.
Chem. 2005, 48, 4204−4207.
(8) Aumailley, M.; Gurrath, M.; Muller, G.; Calvete, J.; Timpl, R.;
̈
(25) Smallheer, J. M.; Weigelt, C. A.; Woerner, F. J.; Wells, J. S.;
Daneker, W. F.; Mousa, S. A.; Wexler, R. R.; Jadhav, P. K. Synthesis
and biological evaluation of nonpeptide integrin antagonists containing
spirocyclic scaffolds. Bioorg. Med. Chem. Lett. 2004, 14, 383−387.
(26) Heckmann, D.; Meyer, A.; Laufer, B.; Zahn, G.; Stragies, R.;
Kessler, H. Rational design of highly active and selective ligands for the
α5β1 integrin receptor. ChemBioChem 2008, 9, 1397−1407.
Kessler, H. Arg-Gly-Asp constrained within cyclic pentapeptides.
Strong and selective inhibitors of cell adhesion to vitronectin and
laminin fragment P1. FEBS Lett. 1991, 291, 50−54.
(9) Haubner, R.; Finsinger, D.; Kessler, H. Stereoisomeric peptide
libraries and peptidomimetics for designing selective inhibitors of the
αvβ3 integrin for a new cancer therapy. Angew. Chem., Int. Ed. 1997, 36,
1374−1389.
3416
dx.doi.org/10.1021/jm500092w | J. Med. Chem. 2014, 57, 3410−3417

Journal of Medicinal Chemistry
Article
(27) Marinelli, L.; Lavecchia, A.; Gottschalk, K.-E.; Novellino, E.;
Kessler, H. Docking studies on αvβ3 integrin ligands: pharmacophore
refinement and implications for drug design. J. Med. Chem. 2003, 46,
4393−4404.
(28) Rechenmacher, F.; Neubauer, S.; Polleux, J.; Mas-Moruno, C.;
De Simone, M.; Cavalcanti-Adam, E. A.; Spatz, J. P.; Fassler, R.;
̈
Kessler, H. Functionalizing αvβ3- or α5β1-selective integrin
antagonists for surface coating: a method to discriminate integrin
subtypes in vitro. Angew. Chem., Int. Ed. 2013, 52, 1572−1575.
(29) Rahmouni, S.; Lindner, A.; Rechenmacher, F.; Neubauer, S.;
Sobahi, T. R.; Kessler, H.; Cavalcanti-Adam, E. A.; Spatz, J. P.
Hydrogel micropillars with integrin selective peptidomimetic function-
alized nanopatterned tops: a new tool for the measurement of cell
traction forces transmitted through αvβ3- or α5β1-integrins. Adv. Mater.
2013, 25, 5869−5874.
(30) Neubauer, S.; Rechenmacher, F.; Beer, A. J.; Curnis, F.; Pohle,
K.; D’Alessandria, C.; Wester, H.-J.; Reuning, U.; Corti, A.; Schwaiger,
M.; Kessler, H. Selective imaging of the angiogenic relevant integrins
α5β1 and αvβ3. Angew. Chem., Int. Ed. 2013, 52, 11656−11659.
(31) Wang, J.; Breslin, M. J.; Coleman, P. J.; Duggan, M. E.; Hunt, C.
A.; Hutchinson, J. H.; Leu, C.-T.; Rodan, S. B.; Rodan, G. A.; Duong,
L. T.; Hartman, G. D. Non-peptide αvβ3 antagonists. Part 7: 3-
Substituted tetrahydro-[1,8]naphthyridine derivatives. Bioorg. Med.
Chem. Lett. 2004, 14, 1049−1052.
(32) Stragies, R.; Osterkamp, F.; Zischinsky, G.; Vossmeyer, D.;
Kalkhof, H.; Reimer, U.; Zahn, G. Design and synthesis of a new class
of selective integrin α5β1 antagonists. J. Med. Chem. 2007, 50, 3786−
3794.
(33) Knor, S.; Khrenov, A. V.; Laufer, B.; Saenko, E. L.; Hauser, C. A.
̈
E.; Kessler, H. Development of a peptidomimetic ligand for efficient
isolation and purification of factor VIII via affinity chromatography. J.
Med. Chem. 2007, 50, 4329−4339.
(34) Frank, A. O.; Otto, E.; Mas-Moruno, C.; Schiller, H. B.;
Marinelli, L.; Cosconati, S.; Bochen, A.; Vossmeyer, D.; Zahn, G.;
Stragies, R.; Novellino, E.; Kessler, H. Conformational control of
integrin-subtype selectivity in isoDGR peptide motifs: a biological
switch. Angew. Chem., Int. Ed. 2010, 49, 9278−9281.
(35) Rechenmacher, F.; Neubauer, S.; Mas-Moruno, C.; Dorfner, P.
M.; Polleux, J.; Guasch, J.; Conings, B.; Boyen, H.-G.; Bochen, A.;
Sobahi, T. R.; Burgkart, R.; Spatz, J. P.; Fassler, R.; Kessler, H. A
̈
molecular toolkit for the functionalization of titanium-based
biomaterials that selectively control integrin-mediated cell adhesion.
Chem.Eur. J. 2013, 19, 9218−9223.
(36) Humphries, M. Integrin structure. J. Biochem. Soc. Trans. 2000,
28, 311−339.
(37) Heckmann, D.; Laufer, B.; Marinelli, L.; Limongelli, V.;
Novellino, E.; Zahn, G.; Stragies, R.; Kessler, H. Breaking the
dogma of the metal-coordinating carboxylate group in integrin ligands:
introducing hydroxamic acids to the MIDAS to tune potency and
selectivity. Angew. Chem., Int. Ed. 2009, 48, 4436−4440.
(38) Ho, C. Y.; Strobel, E.; Ralbovsky, J.; Galemmo, R. A. Improved
solution- and solid-phase preparation of hydroxamic acids from esters.
J. Org. Chem. 2005, 70, 4873−4875.
(39) Hackel, B. J.; Kimura, R. H.; Miao, Z.; Liu, H.; Sathirachinda, A.;
Cheng, Z.; Chin, F. T.; Gambhir, S. S. 18F-Fluorobenzoate-labeled
cystine knot peptides for PET imaging of integrin αvβ6. J. Nucl. Med.
2013, 54, 1101−1105.
(40) Schottelius, M.; Laufer, B.; Kessler, H.; Wester, H.-J. Ligands for
mapping αvβ3-integrin expression in vivo. Acc. Chem. Res. 2009, 42,
969−980.
(41) Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.;
Goodman, S. L.; Arnaout, M. A. Crystal structure of the extracellular
segment of integrin αvβ3 in complex with an Arg-Gly-Asp ligand.
Science 2002, 296, 151−155.
(42) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts
of common laboratory solvents as trace impurities. J. Org. Chem. 1997,
62, 7512−7515.
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