Role of side-chain bioisosteres in determining the binding affinity of click chemistry derived RGD peptidomimetics to αvβ3 integrin

Article abstract of DOI:10.1002/ejoc.201403129

Triazole-containing Arg-Gly-Asp (RGD) peptidomimetics capable of interacting with α_vβ₃ integrin were developed through click chemistry. The role of the diverse range of guanidine bioisosteres for the arginine mimetic moiety was evaluated in vitro together with different appendages on the aspartic acid side of the peptidomimetic. The corresponding azides and alkynes were constructed through the application of simple modular reactions. Integrin binding assays allowed the best guanidine bioisostere for binding activity to be selected; the aromatic ring at the azide fragment bearing the aspartic acid moiety proved to be unnecessary. Molecular modelling calculations of a hit compound showed that the triazole and 2-aminopyridine rings were involved in important π-stacking interactions with Tyr178.

Full text of DOI:10.1002/ejoc.201403129

FULL PAPER
DOI: 10.1002/ejoc.201403129
Role of Side-Chain Bioisosteres in Determining the Binding Affinity of Click
Chemistry Derived RGD Peptidomimetics to αvβ3 Integrin
Pierangelo Fabbrizzi,*[a] Gloria Menchi,[a,b] Silvia Raspanti,[c] Antonio Guarna,[a,b] and
Andrea Trabocchi*[a,b]
Dedicated to C.I.N.M.P.I.S. on the occasion of its 20th anniversary
Keywords: Peptidomimetics / Inhibitors / Amino acids / Structure-activity relationships / Click chemistry
Triazole-containing Arg-Gly-Asp (RGD) peptidomimetics
capable of interacting with αvβ3 integrin were developed
through click chemistry. The role of the diverse range of
guanidine bioisosteres for the arginine mimetic moiety was
evaluated in vitro together with different appendages on the
aspartic acid side of the peptidomimetic. The corresponding
azides and alkynes were constructed through the application
of simple modular reactions. Integrin binding assays allowed
the best guanidine bioisostere for binding activity to be se-
lected; the aromatic ring at the azide fragment bearing the
aspartic acid moiety proved to be unnecessary. Molecular
modelling calculations of a hit compound showed that the
triazole and 2-aminopyridine rings were involved in impor-
tant π-stacking interactions with Tyr178.
Starting from the mid 1990s, Kessler and co-workers re-
ported important contributions that allowed the basis of
molecular recognition of αvβ3 integrin to be defined.[6] The
recognition of the RGD sequence in the interaction between
this integrin and extracellular matrix protein ligands (i.e.,
vitronectin) was identified as a model for the design of inte-
grin antagonists, aimed at blocking integrin cell functions
correlated to the disease. Highly potent and selective inhibi-
tors were developed by systematically constraining the tri-
peptide RGD in cyclic motifs, and specifically into cyclic
penta- and hexapeptides. The “spatial screening” of a li-
brary of cyclic peptides differing in the amino acids com-
pleting the sequence together with the RGD motif allowed
for the selection of the cyclic pentapeptide c[RGDfV],
which showed the most potent binding affinity for this inte-
grin. The application of peptidomimetic strategy around
peptide hits is particularly effective in drug discovery and
development.[7] The development of linear nonpeptide ana-
logues of the RGD sequences retaining high affinity for
RGD-binding integrins consisted of a central scaffold, such
as aromatic rings, pyrimidones and benzodiazepines, bear-
Introduction
Among the large family of integrins that mediate cell–
matrix adhesion events, and cell differentiation, prolifera-
tion and migration,[1] Arg-Gly-Asp (RGD) sequence-bind-
ing receptors have attracted much attention in drug discov-
ery, because they are fundamental in several physiological
processes, such as platelet aggregation, αIIββ3,[2] angiogen-
esis, αvβ3, α5β1,[3] and in pathological processes such as car-
diovascular disorders, αIIββ3, and tumours, αvβ3, αvβ5,
α5β1.[4] One of the most important targeted events in cancer
therapy is tumor metastasis, in which αvβ3 integrin plays an
important role.[5] In fact, this integrin has a primary role in
cell–cell and cell–extracellular matrix adhesion events, and
regulates the organisation of tissue in tumour cells and their
migration; thus, it has been considered a therapeutic target
of interest. Another important biological process connected
to integrins is angiogenesis, which deals with the generation
of new capillaries starting from existing blood vessels.
[a] Department of Chemistry “Ugo Schiff”, University of
Florence,
Via della Lastruccia 13, 50019 Sesto Fiorentino, Florence, Italy ing bioisosteres[8] of carboxylic acid and guanidine at oppo-
site ends.[9] Structure-activity relationship (SAR) studies al-
E-mail: pfabbrizzi@gmail.com
andrea.trabocchi@unifi.it
http://www2.chim.unifi.it/index.php
lowed for the identification of three main pharmacophoric
regions (a carboxylic group, a guanidine-derived basic re-
gion and a hydrophobic group), that were useful for the
design of αvβ3 integrin inhibitors. It was only in 2002, when
the X-ray structure of the complex of αvβ3 integrin with
Kessler’s RGD-cyclopeptide c[RGDf(N-Me)V] (Cilengitide)
was published, that the molecular interactions of the RGD
[b] Interdepartmental Center for Preclinical Development of
Molecular Imaging (CISPIM), University of Florence,
Viale Morgagni 85, 50134 Florence, Italy
[c] Department of Biomedical, Experimental and Clinical Sciences
“Mario Serio”, University of Florence,
Viale Morgagni 50, 50134 Florence, Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201403129.
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P. Fabbrizzi, A. Trabocchi et al.
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motif with the binding site of the integrin receptor were
made clear.[10]
Our contribution to this field involved the application of
a “click chemistry” approach for the discovery of triazole-
based RGD mimetics, by CuI-catalysed 1,3-dipolar alkyne–
azide coupling (CuAAC), which showed binding affinity
towards αvβ3/αvβ5 integrins.[11] Biological assays revealed
that one such RGD peptidomimetic (1; Figure 1) can bind
αvβ3 integrin with nanomolar affinity according to a two-
sites model. In view of improving the capability of this class
of compounds as integrin ligands, we carried out a follow-
up study on the modulation of the binding affinity towards
the key αvβ3 integrin by introducing arginine mimetics and
different appendages on the aspartic acid side of the pepti-
domimetic.
Scheme 1. Synthetic strategy for the generation of triazole RGD
peptidomimetics by using click chemistry.
role of the phenyl ring of 1 (Scheme 2). According to the
proposed synthetic routes described for 5,[11] compound 6
was synthesised starting from β-alanine methyl ester
through amine acylation with bromoacetyl bromide, fol-
lowed by nucleophilic substitution with NaN3. No chroma-
tographic separation was performed after the acylation step,
and the resulting compound was obtained in a modest over-
all yield of 29%.
Figure 1. Reference click chemistry derived RGD peptidomimetic
for this study.
Results and Discussion
An array of RGD peptidomimetics based on the triazole
scaffold was studied with the aim of assessing the role of
the phenyl ring in the proximity of the aspartic acid moiety
and the effect of introducing guanidine bioisosteres in place
of arginine with respect to binding affinity towards αvβ3
integrin. To study the role of the phenyl ring, selected com-
pounds lacking such functionalisation, or presenting an ad-
ditional carboxylic group as a polar moiety were consid-
ered. Piperidine-4-carboxylic acid was also selected to study
the effect of ring constraints on the aspartic acid moiety
towards integrin binding affinity. With the aim of evaluat-
ing the modulation of basicity and of the hydrophobic con-
tent of the arginine mimetic moiety, 2-aminopyridine and
1H-benzimidazol-2-amine heterocyclic moieties as guanid-
ine bioisosteres were taken into account. The hit compound
1, resulting from the initial library screening, as previously
reported,[11] was taken as the reference compound for all
these comparative evaluations (Figure 1).
Scheme 2. Synthesis of selected azides containing the aspartic acid
mimetic.
The Asp carboxylate moiety of RGD ligands interacts
with the metal-ion-dependent adhesion site (MIDAS) of the
integrin consisting of Mn2+ ions. Considering the influence
of Mn2+ coordination on RGD ligands affinity, we wanted
to examine the binding affinity of a 1,5-dicarboxylic acid
moiety. Thus, compound 7, bearing a 1,5-dicarboxylic ester
moiety, was designed based on the original monocarboxylic
Synthesis
Selected compounds for this study were constructed by derivative (Scheme 2), being careful to keep the distance
taking advantage of the modular assembly afforded by click between the COOH group and the amido moiety of the
chemistry (Scheme 1), specifically by combining alkynes peptidomimetic backbone as similar as possible to the pre-
and azides bearing arginine and aspartic acid mimetics, viously optimised structure 1. The dicarboxylic amine 4 was
respectively.
readily synthesised through a Michael addition of benzyl-
Accordingly, efforts were focused on the synthesis of suit- amine to dimethyl glutaconate followed by debenzylation
able azide and alkyne precursors. Specifically, new azides of the amino group. The resulting amine was successively
differing in the appendage at the β-position of the carboxyl- transformed into the corresponding azide 7 by applying
ate moiety were designed and synthesised to evaluate the standard acylation/substitution reactions. Azide 9 was also
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Binding Affinity of RGD Peptidomimetics to αvβ3 Integrin
prepared in 46% overall yield from the corresponding N,N-dimethylformamide (DMF), reacted with the silyl-pro-
methyl piperidine-4-carboxylate by using similar reactions tected chloroalkyne, and the protecting group was removed
to those described above (Scheme 2, bottom).
with tetrabutylammonium fluoride (TBAF) in a one-pot
The role of guanidine bioisosteres in determining the procedure (Scheme 5). Although compound 17 was ob-
binding affinity was addressed by designing and synthesis- tained in 30% overall yield, possibly due to the instability
ing the corresponding functionalised alkynes. Moieties of the Boc group towards the reaction conditions, this result
differing in the basicity and lipophilicity were synthesised. was a threefold improvement over the 2-fluoropyridine-
Guanidine and 2-aminobenzimidazole derivatives (Schemes 3 based methodology.
and 4) were synthesised in good yield through the Mitsu-
nobu reaction starting from the corresponding carbamates
11 and 13, and pentyn-1-ol. Notably, carbamate 15 could
be conveniently obtained from the corresponding Boc-pro-
tected methylthiourea 14 (Scheme 4).[12] All reactions were
accelerated by using microwave heating in a sealed vessel
under controlled temperature to speed up the synthesis.
Scheme 5. Synthesis of alkyne precursor containing the 2-amino-
pyridine as the guanidine bioisostere.
Subsequently, to fully exploit the potential benefits of
CuAAC, a different set of reaction conditions were chosen.
A shift to full-organic conditions using CuP(OEt)3I as cata-
lyst, tetrahydrofuran (THF) as solvent, and microwave irra-
diation to accelerate the process led to a remarkable in-
Scheme 3. Synthesis of alkyne containing the 2-aminobenzimid-
crease in the average reaction yield and a dramatic decrease
in reaction times with respect to previously reported condi-
tions[11] (Schemes 6 and 7). Low yields were due to incom-
plete reaction, and varying amounts of reagents were reco-
vered from the reaction mixture. The final deprotection step
with 3 n HCl gave the free adducts 26–31 and 33 in quanti-
tative yield.
azole as the guanidine bioisostere.
Scheme 4. Synthesis of alkyne precursor containing the Cbz-pro-
tected 1-(2-aminoethyl)guanidine bioisostere.
Different synthetic routes were explored for the synthesis
of 2-aminopyridine derivatives 17 and 19. The first attempt
was an aromatic nucleophilic substitution of 2-fluoropyr-
idine with pent-4-ynylamine performed in a sealed vessel
under microwave heating (190 °C). Product 19 derived from
this reaction bears no protective groups and was used for
the synthesis of 22. However, the low reaction yield led us
to explore a different route (Scheme 5). Boc-protected 2-
Scheme 6. Synthesis of triazole-containing RGD peptidomimetics
aminopyridine was deprotonated with sodium hydride in possessing different side-chain mimetics.
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binding curves to be better described by a two-sites model,
as revealed by Hill coefficients differing largely from unity,
due to binding of the peptidomimetic compound with two
conformational states of integrins. In contrast, compounds
29 and 31 showed binding affinity values in the micromolar
range resulting from a binding curve profile described by a
one-site model, whereas compound 33 showed no binding
affinity. Such data showed the aspartic region was not affec-
ted by the lack of the aromatic ring. In fact, compounds 28
and 30 showed IC50 values in the nanomolar range, with
the former being the most potent of the series reported in
this work. IC50 value of 28 proved to be comparable to
those of their corresponding phenyl-containing compounds
1, 26 and 27, suggesting that any hydrophobic contact expe-
rienced by such moiety is not crucial for the receptor–ligand
interaction. The micromolar value of compound 29 and the
lack of binding affinity for 33 indicated the detrimental ef-
fect of the additional carboxylic moiety around the aspartic
acid region of the RGD moiety for the former, and of the
introduction of constraints in such part of the backbone for
the latter. Finally, the comparison between IC50 values of
1, 26 and 27, possessing guanidine, 2-aminobenzimidazole
and 2-aminopyridine, respectively, demonstrated the com-
parable role of guanidine and its bioisosteres towards bind-
ing affinity, whereas the introduction of an alkylated guan-
idine moiety containing an additional amino group, as in
compound 31, provoked a decrease of the binding activity
in the micromolar range.
Scheme 7. Synthesis of piperidine-containing RGD peptidomi-
metic.
Integrin Binding Assays
The ability of RGD peptidomimetics 26–31 and 33 to
compete with 125I-echistatin for binding to pure αvβ3 inte-
grin isolated from human placenta was evaluated in solid-
phase assays (Table 1).[11] Compounds 26–28 and 30
showed binding affinity values in the nanomolar range and
Table 1. Inhibition of 125I-echistatin specific binding to purified hu-
man integrin proteins αvβ3 by triazole-containing RGD peptidomi-
metics.
Molecular Modelling
The binding mode of bioactive RGD peptidomimetic 28
towards αvβ3 integrin was evaluated through a docking sim-
ulation to ascertain the role of chemical moieties of the li-
gand in determining the binding affinity. The crystal struc-
ture of the complex formed by c[RGDf(Me)V] and the ex-
tracellular fragment of αvβ3 integrin (PDB code: 1L5G)
provides a general mode of interaction between the integrin
and its ligands.[14] Asp carboxylate and Arg guanidinium
moieties of RGD-based ligands are key structural elements
for receptor recognition, in which the carboxylate group in-
teracts with the metal ion dependent adhesion site
(MIDAS) of the Mn2+ ion, and the Arg guanidinium group
is responsible for salt bridge interactions with Asp218 and
Asp150 side chains. Additional ligand–receptor hydro-
phobic π-stacking contacts occur with the Tyr122 side
chain.
The docking program Autodock 4.0[15] was used to eval-
uate the binding energies of minimum-energy conforma-
tions of RGD ligand 28. Docking calculations showed that
28 experiences key interactions with Asp218 and the
MIDAS site of the integrin, thus confirming that these in-
teractions are crucial for molecular recognition of Arg–
Gly–Asp-containing ligands (Figure 2). Specifically, the
COOH group was found in the MIDAS site, and the 2-
aminopyridine moiety formed part of a salt-bridge interac-
tion with Asp218. Regarding additional hydrophobic inter-
[a] Hi (high) and Lo (low) IC50 values refer to two-sites binding
model. [b] Data are shown as meanϮSD from three independent
experiments. [c] 0% inhibition at 1 μm concentration of 33, and
27% inhibition at 10 μm concentration of 33.
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Binding Affinity of RGD Peptidomimetics to αvβ3 Integrin
Figure 2. Peptidomimetic 28 docked into the binding region of αvβ3 integrin highlighting the protein residues (green) that form the key
interactions: Asp218 vs. Arg, and Mn2+ vs. Asp. The reference ligand c[RGDf(Me)V] (coordinates from PDB: 1L5G) is shown in red,
and reference compound 1 in black. Nonpolar hydrogen atoms are omitted for clarity.
actions, the best-binding pose of 28 showed two π-stacking aminopyridine as arginine mimetic fragments showed com-
interactions, which were experienced by the triazole ring parable binding affinity in competition studies in vitro, they
facing Tyr178, similar to that evidenced for reference com- may give different in vivo profiles as a consequence of their
pound 1,[11] together with the 2-aminopyridine moiety in differing basicity and hydrophobic content, thus influencing
proximity to the Tyr178 side-chain (Figure 2). This binding the suitability of such ligands for use in drugs. In vivo stud-
mode accounts for the high binding affinity observed for ies are ongoing to assess this issue.
the compound with the 2-aminopyridine moiety, irrespec-
tive of the lack of π-stacking interaction with Tyr122, as
found for Cilengitide (Figure 2, red structure) and also for
Experimental Section
General Remarks: H and 13C NMR spectra were recorded with a
1
reference compound 1 of this class of peptidomimetics.
Varian Gemini 200 (1H: 200 MHz, 13C: 50 MHz) or a Varian Gem-
Nevertheless, the increased potency of 28 with respect to 27,
ini 300 (1H: 300 MHz, 13C: 75 MHz), with tetramethylsilane as in-
both containing the 2-aminopyridine bioisostere, suggested
ternal reference. The chemical shifts (δ) and coupling constants (J)
are expressed in ppm and Hertz, respectively. Flash column
that the absence of the aromatic ring allows better accom-
modation of the ligand in the RGD integrin site.
chromatography (FCC) purifications were performed manually by
using glass columns [Merck silica gel (0.040–0.063 mm)] or with a
Biotage Isolera system (SNAP silica cartridges). TLC analyses were
performed on Merck silica gel 60 F254 plates. Elemental analyses
were recorded with a Perkin–Elmer 240 C,H,N analyzer. IR spectra
were recorded with a Perkin–Elmer 881 FTIR spectrophotometer.
ESI mass spectra were recorded with a Thermo LCQ-Fleet. Tetra-
hydrofuran (THF) was distilled from Na/benzophenone, dichloro-
Conclusions
The role of guanidine as a mimic for the arginine moiety
and a study of the effect of a range of appendages or ring
constraints on the aspartic acid moiety of the peptidomi-
metic towards the integrin binding affinity were evaluated methane (CH2Cl2) was distilled from CaH2. Microwave-heated re-
actions were performed in sealed vessels by using a single-mode
Biotage Initiator instrument. All commercially available reagents
and solvents were purchased from Sigma–Aldrich and used as re-
ceived, unless otherwise specified. Analytical HPLC purity tests
were performed with a Dionex UltiMate 3000 (columns: Phe-
nomenex Synergi 4 μm Fusion-RP 80A, 150ϫ4.6 mm and Phe-
nomenex Synergi 4 μm Hydro-RP 80A, 150ϫ 4.6 mm), using
CH3CN (0.1% TFA)/H2O (0.1% TFA), 5:95 for 5 min, then 5:95
to 95:5 over 12 min as gradient. All tested compounds were Ͼ95%
pure. Other abbreviations: DPPA: diphenylphosphoryl azide;
by synthesising a pool of compounds through click chemis-
try from the corresponding azides and alkynes. Integrin
binding assays showed the importance of the guanidine
bioisostere for securing high binding activity, whereas the
aromatic ring at the azide fragment proved to be unneces-
sary. Insight into the binding mode of hit compound 28 was
achieved through molecular modelling calculations, which
showed that the triazole and 2-aminopyridine rings engage
in π-stacking interactions with Tyr178. Although com-
pounds possessing guanidine, 2-aminobenzimidazole or 2- DIAD: diisopropyl azodicarboxylate.
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Methyl (3S)-(2-Azidoacetylamino)-3-phenylpropionate (5): Synthe-
solution was washed with saturated NaHCO3 and brine, the or-
ganic layer was dried with Na2SO4 and concentrated under reduced
pressure to afford the pure product (0.89 g, 89% yield) as a white
sised according to a reported procedure.[11]
Methyl 3-(2-Azidoacetylamino)propionate (6): β-Alanine methyl es-
ter hydrochloride (1.12 g, 8 mmol) was dissolved in CH2Cl2
(30 mL) and NEt3 (3.5 mL) and cooled to –10 °C. Bromoacetyl
bromide (1.60 g, 7.9 mmol) was slowly added, and the solution was
stirred for 15 min. The mixture was allowed to reach room temp.
and stirred for an additional 30 min, then washed with NaHCO3
and brine, the organic layer was dried with Na2SO4, and the solvent
was evaporated. The crude product was dissolved in anhydrous
DMF (12 mL), NaN3 (0.9 g, 13.8 mmol) was added and the solu-
tion was heated at 80 °C overnight. Water (50 mL) was added and
the product was extracted with EtOAc and diethyl ether. The mixed
organic layers were dried with Na2SO4, and the solvents were evap-
orated. The crude product was purified by FCC with a Biotage
Isolera system [TLC: Rf = 0.4 (EtOAc/PE, 2:1)] to give 6 (0.4 g,
29% overall yield) as a pale-yellow liquid. 1H NMR (300 MHz,
CDCl3): δ = 3.96 (s, 2 H), 3.71 (s, 3 H), 3.56 (q, J = 6 Hz, 2 H),
2.56 (t, J = 6 Hz, 2 H) ppm. 13C NMR (50 MHz, CDCl3): δ =
1
solid. H NMR (200 MHz, CDCl3): δ = 7.76–7.60 (m, 2 H), 7.40–
7.25 (m, 2 H), 1.74 (s, 9 H), 1.57 (s, 9 H) ppm. 13C NMR (50 MHz,
CDCl3): δ = 150.8, 149.5, 146.1, 141.7, 129.4, 124.7, 122.9, 119.4,
114.1, 87.2, 82.2, 28.1 ppm. MS (ESI): m/z (%) = 355.9 (100) [M +
Na]+, 333.9 (72) [M + 1]+. IR (CH Cl ): ν = 3320 (m), 3052 (m),
˜
2
2
2982 (m), 1752 (m), 1728 (s), 1525 (s), 1456 (m), 1357 (s), 1139
(s) cm–1. C17H23N3O4 (333.39): calcd. C 61.25, H 6.95, N 12.60;
found C 61.17, H 6.91, N 12.53.
tert-Butyl 2-(tert-Butoxycarbonylpent-4-ynylamino)benzimidazole-1-
carboxylate (12): Compound 11 (0.4 g, 1.2 mmol) and tri-
phenylphosphine (0.346 g, 1.32 mmol) were dissolved in THF
(10 mL). 4-Pentyn-1-ol (0.101 g, 111 μL, 1.2 mmol) was added, and
the solution was cooled to –10 °C. DIAD (0.267 g, 260 μL,
1.32 mmol) was added dropwise whilst stirring, and the solution
was stirred at –10 °C for 20 min. The solution was allowed to reach
room temp., transferred to a microwave system and heated at
110 °C for 1 h. Solvent was evaporated under reduced pressure and
the crude product was purified by FCC with a Biotage Isolera sys-
tem (TLC: PE/EtOAc, 3:1; Rf = 0.66). Yield: 310 mg (65%); colour-
172.5, 165.5, 52.7, 52, 34.9, 33.7 ppm. IR (CH Cl ): ν = 3419 (m),
˜
2
2
3052 (m), 2952 (m), 2109 (s), 1733 (s), 1678 (s), 1604 (w), 1530 (m),
1436 (m), 1367 (m), 1204 (m), 1179 (m) cm–1. MS (ESI): m/z (%)
= 187.1 (100) [M + H]+. C6H10N4O3 (186.17): calcd. C 38.71, H
5.41, N 30.09; found C 38.82, H 5.45, N 30.11.
1
less oil. H NMR (200 MHz, CDCl3): δ = 7.83 (m, 1 H), 7.63 (m,
1 H), 7.45–7.30 (m, 2 H), 2.35–2.25 (m, 2 H), 2.02–1.85 (m, 3 H),
1.69 (s, 9 H), 1.36 (s, 9 H) ppm. 13C NMR (50 MHz, CDCl3): δ =
147.7, 140, 132.1, 124.7, 124.1, 119.8, 114.8, 85.4, 83.4, 81.6, 68.8,
48.4, 28, 27.4, 15.9 ppm. MS (ESI): m/z (%) = 422.0 (100) [M +
Dimethyl 3-(2-Azidoacetylamino)pentanedioate (7): Amine 4[16]
(0.68 g, 3.88 mmol) and NEt3 (1.18 g, 11.64 mmol) were dissolved
in CH2Cl2 (20 mL) then cooled to –10 °C. Bromoacetyl bromide
(0.784 g, 3.88 mmol) was slowly added, and the solution was stirred
for 15 min. The mixture was allowed to reach room temp. and
stirred for 30 min, then washed with 5% HCl and brine, the organic
layer was dried with Na2SO4, and the solvent was evaporated. The
crude product was dissolved in anhydrous DMF (4 mL), NaN3
(0.46 g, 7.08 mmol) was added, and the solution was heated at
80 °C overnight. Water was added, and the product was extracted
with diethyl ether. The organic phase was dried with Na2SO4, and
the solvents were evaporated. The crude product was purified by
FCC with a Biotage Isolera system [TLC: Rf = 0.23 (EtOAc/PE,
Na]+, 400.0 (98) [M + H]+. IR (CH Cl ): ν = 3305 (m), 2977 (m),
˜
2
2
2937 (m), 1755 (s), 1715 (s), 1552 (s), 1453 (s), 1369 (s), 1335 (s),
1157 (bs) cm–1. C22H29N3O4 (399.49): calcd. C 66.14, H 7.32, N
10.52; found C 66.42, H 7.45, N 10.30.
N,NЈ-Bis(tert-butoxycarbonyl)-N-pent-4-ynyl-S-methylisothiourea
(14): N,NЈ-Bis(tert-butoxycarbonyl)-S-methylisothiourea (13)[17]
(0.5 g, 1.72 mmol), triphenylphosphine (0.54 g, 2.06 mmol) and 4-
pentyn-1-ol (0.145 g, 160 μL, 1.72 mmol) were dissolved in THF
(20 mL). The solution was cooled to –10 °C, and DIAD (0.416 g,
405 μL, 2.06 mmol) was added dropwise. The mixture was stirred
at –10 °C for 15 min, then allowed to reach room temp. and trans-
ferred into a microwave system and heated at 50 °C for 30 min.
Solvent was evaporated under reduced pressure, and the product
was purified by FCC with a Biotage Isolera system (TLC: PE/
EtOAc, 8:1; Rf = 0.45). Yield: 613 mg (quantitative); yellow oil. 1H
NMR (300 MHz, CDCl3): δ = 3.63 (t, J = 7.5 Hz, 2 H), 2.39 (s, 3
H), 2.24 (td, J = 6.9, 2.4 Hz, 2 H), 1.96 (t, J = 2.4 Hz, 1 H), 1.92–
1.85 (m, 2 H), 1.52 (s, 9 H), 1.48 (s, 9 H) ppm. 13C NMR (50 MHz,
CDCl3): δ = 162.7, 157.8, 151.8, 83.1, 82.4, 81.8, 68.9, 48, 28.4, 28,
27.6, 15.9, 15.5 ppm. MS (ESI): m/z (%) = 379.0 (78) [M + Na]+,
356.9 (100) [M + 1]+. C17H28N2O4S (356.48): calcd. C 57.28, H
7.92, N 7.86; found C 57.54, H 7.99, N 7.64.
1
1:1)] to give 7 (0.32 g, 32% overall yield) as a pale-yellow oil. H
NMR (200 MHz, CDCl3): δ = 7.21 (m, 1 H, NH), 4.63 (m, 1 H),
3.95 (s, 2 H), 3.69 (s, 6 H), 2.81–2.57 (m, 4 H) ppm. 13C NMR
(50 MHz, CDCl3): δ = 171.2, 166.3, 52.2, 51.6, 43.1, 37.3 ppm. MS
(ESI): m/z (%) = 281.1 (30) [M + Na]+, 259 (100) [M + H]+. IR
(CH Cl ): ν = 3399 (bm), 3052 (m), 2992 (w), 2952 (m), 2853 (w),
˜
2
2
2109 (s), 1733 (s), 1683 (s), 1520 (m), 1441 (m) cm–1. C9H14N4O5
(258.23): calcd. C 41.86, H 5.46, N 21.70; found C 41.63, H 5.32,
N 21.49.
Ethyl 1-(2-Azidoacetyl)piperidine-4-carboxylate (9): As described
for 7, compound 9 was obtained from ethyl isonipecotate 8 (0.61 g,
3.88 mmol), and the resulting crude bromide was treated with
NaN3 (0.46 g, 7.08 mmol) to give the product (0.43 g, 46% overall
yield) as a pale-yellow oil after FCC purification (EtOAc/PE, 1:1;
NЈ-[2-(Benzyloxycarbonylamino)ethyl]-N,NЈЈ-bis(tert-butoxycarbon-
yl)-N-pent-4-ynylguanidine (15): Compound 14 (0.535 g, 1.5 mmol),
benzyl (2-aminoethyl)carbamate hydrochloride[18] (0.519 g,
2.25 mmol) and NEt3 (0.455 g, 4.4 mmol) were suspended in THF
(10 mL) and heated at 110 °C under microwave irradiation for 1 h.
The solvent was evaporated, and the residue was dissolved in
EtOAc and washed with NaHCO3 and brine. The organic phase
was dried with Na2SO4, concentrated to dryness, and the crude
product was purified by FCC with a Biotage Isolera system (TLC:
PE/EtOAc, 2:1; Rf = 0.28). Yield: 302 mg (40%); colourless oil. 1H
NMR (300 MHz, CDCl3): δ = 7.43–7.29 (m, 5 H), 5.12 (s, 2 H),
3.72 (d, J = 7.2 Hz, 2 H), 3.49–3.35 (m, 4 H), 2.25–2.14 (m, 2 H),
1.95 (t, J = 2.4 Hz, 1 H), 1.84–1.72 (m, 2 H), 1.49 (s, 9 H), 1.47 (s,
1
Rf = 0.60). H NMR (200 MHz, CDCl3): δ = 4.17 (m, 1 H), 4.10
(q, J = 7.2 Hz, 2 H), 4.06 (s, 2 H), 3.84 (m, 1 H), 3.62 (m, 1 H),
3.10 (m, 1 H), 2.52 (m, 1 H), 2.01–1.58 (m, 4 H), 1.22 (t, J = 7.2 Hz,
3 H) ppm. 13C NMR (50 MHz, CDCl3): δ = 173.6, 165.3, 60.8,
50.8, 44.5, 41.6, 40.8, 28.4, 27.8, 14.4 ppm. MS (ESI): m/z (%) =
241.1 (100) [M + H]+. C10H16N4O3 (240.26): calcd. C 49.99, H
6.71, N 23.32; found C 50.11, H 6.83, N 23.48.
tert-Butyl 2-(tert-Butoxycarbonylamino)benzimidazole-1-carboxyl-
ate (11):[13] 2-Aminobenzimidazole (0.4 g, 3 mmol), triethylamine
(0.835 mL, 1.17 g, 11 mmol), and Boc2O (1.26 g, 6 mmol) were dis-
solved in CH2Cl2 (10 mL) and stirred at room temp. overnight. The
7600
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 7595–7604

Binding Affinity of RGD Peptidomimetics to αvβ3 Integrin
9 H) ppm. 13C NMR (50 MHz, CDCl3): δ = 156.6, 153.2, 136.4,
Yield: 449 mg; colourless oil. 1H NMR (300 MHz, CDCl3): δ =
128.4, 128, 83.2, 82.5, 79.4, 69, 66.7, 46.7, 43.7, 40.3, 28.1, 27.7,
7.89 (m, 1 H), 7.74 (m, 1 H), 7.57 (s, 1 H), 7.40–7.11 (m, 7 H), 5.37
16 ppm. MS (ESI): m/z (%) = 525.1 (9) [M + Na]+, 503 (100) [M (m, 1 H), 5.02 (s, 2 H), 3.56 (s, 3 H), 2.94–2.85 (m, 2 H), 2.84–2.76
+ H]+. IR (CH Cl ): ν = 3439 (m), 3300 (m), 3062 (m), 2972 (m), (m, 2 H), 2.18–2.06 (m, 2 H), 1.68 (s, 9 H), 1.58–1.44 (m, 2 H), 1.35
˜
2
2
2942 (m), 1718 (s), 1619 (bs), 1367 (s) cm–1. C26H38N4O6 (502.61): (s, 9 H) ppm. Minor peaks and peak broadenings due to carbamate
calcd. C 62.13, H 7.62, N 11.15; found C 61.87, H 7.57, N 11.03.
rotamers are clearly visible in CDCl3; such effects disappear after
acidic deprotection (see compound 26). 13C NMR (100 MHz,
CDCl3): δ = 170.9, 164.6, 147.7, 139.9, 132.0,128.7, 127.8, 126.3,
124.9, 124.3, 123.1, 85.7, 53.4, 52.9, 51.8, 50.2, 39.9, 28.1, 22.7 ppm.
MS (ESI): m/z (%) = 684.2 (100) [M + Na]+, 662.1 (38) [M + H]+.
tert-Butyl Pent-4-ynylpyridin-2-ylcarbamate (17): N-Boc-2-amino-
pyridine (16)[19] (0.5 g, 2.57 mmol) was dissolved in anhydrous
DMF under N2 and cooled to 0 °C. To the vigorously stirred solu-
tion, NaH (60% in mineral oil, 0.129 g) was slowly added, keeping
the temperature below 5 °C. The mixture was stirred under these
conditions for 20 min, then (5-chloro-1-pentynyl)trimethylsilane
(0.52 g, 530 μL, 2.96 mmol) was added dropwise, and stirring was
continued at Յ5 °C for 30 min. The solution was allowed to reach
room temp. and stirred for an additional 1 h, then water (1.33 mL)
was added. TBAF (1 m in THF, 5.15 mL, 5.15 mmol) was added,
and the solution was stirred at room temp. overnight. Finally, the
mixture was diluted with water (6 mL) and extracted with diethyl
ether (2ϫ 40 mL), the organic layer was washed with 0.1 m HCl,
saturated NaHCO3 and brine, and dried with Na2SO4. The solvent
was evaporated under reduced pressure, and the product was puri-
fied by FCC (PE/EtOAc, 10:1; Rf = 0.43). Yield: 0.2 g (30 %);
colourless oil. 1H NMR (200 MHz, CDCl3): δ = 8.36 (m, 1 H),
7.65–7.55 (m, 2 H), 7.00 (m, 1 H), 4.03 (t, J = 7.1 Hz, 2 H), 2.22
(td, J = 7.4, 2.6 Hz, 2 H), 1.94–1.80 (m, 3 H), 1.51 (s, 9 H) ppm.
13C NMR (50 MHz, CDCl3): δ = 147.6, 136.9, 119.9, 119.5, 83.9,
81.1, 68.3, 46, 28.3, 27.8, 16.2 ppm. MS (ESI): m/z (%) = 283.0
IR (CH Cl ): ν = 3409 (bs), 3052 (s), 2972 (s), 2942 (m), 1747 (s),
˜
2
2
1718 (s), 1604 (m), 1550 (m), 1451 (m), 1372 (m), 1154 (s) cm–1.
C34H43N7O7 (661.76): calcd. C 61.71, H 6.55, N 16.92; found C
61.68, H 6.53, N 16.88.
Methyl (S)-3-(2-{4-[3-(tert-Butoxycarbonylpyridin-2-ylamino)prop-
yl][1,2,3]triazol-1-yl}acetylamino)-3-phenylpropionate (21): Synthe-
sised according to general procedure A starting from alkyne 17 and
azide 5. FCC with a Biotage Isolera system (TLC: EtOAc; Rf =
1
0.35). Yield: 99 mg; pale-yellow oil. H NMR (300 MHz, CDCl3):
δ = 8.34 (m, 1 H, NH), 7.65–7.52 (m, 2 H), 7.48 (s, 1 H), 7.32–7.15
(m, 6 H), 7.00 (m, 1 H), 5.33 (m, 1 H), 5.01 (s, 2 H), 4.00 (t, J =
7.2 Hz, 2 H), 3.56 (s, 3 H), 2.86–2.71 (m, 4 H), 2.09–1.94 (m, 2 H),
1.48 (s, 9 H) ppm. 13C NMR (50 MHz, CDCl3): δ = 171.0, 164.8,
154.5, 154.1, 148.2, 147.6, 139.8, 136.9, 128.7, 127.8, 126.2, 122.5,
120.1, 119.6, 81.1, 52.8, 51.8, 50.1, 46.1, 39.8, 28.3, 23.0 ppm. MS
(ESI): m/z (%) = 545.2 (100) [M + Na]+, 523.1 (34) [M + H]+, 478.3
(16) [M – (CH ) ]+. IR (CH Cl ): ν = 3404 (bm), 3057 (s), 2977
˜
3 3
2
2
(100) [M + Na]+, 260.9 (58) [M+]. IR (CHCl ): ν = 3680 (m), 3595
˜
(w), 2947 (w), 1696 (s), 1602 (s), 1473 (m), 1389 (m), 1364 (m),
1152 (s) cm–1. C27H34N6O5 (522.60): calcd. C 62.05, H 6.56, N
16.08; found C 61.99, H 6.56, N 15.98.
3
(m), 3052 (s), 2985 (m), 2252 (m), 1607 (m), 1422 (m) cm–1
.
C15H20N2O2 (260.34): calcd. C 69.20, H 7.74, N 10.76; found C
69.31, H 7.76, N 10.73.
Methyl 3-(2-{4-[3-(Pyridin-2-ylamino)propyl][1,2,3]triazol-1-
yl}acetylamino)propionate (22): Synthesised according to general
procedure A starting from alkyne 17 and azide 6. FCC with a
Biotage Isolera system (TLC: CH2Cl2/MeOH, 15:1; Rf = 0.15).
N,NЈ-Di-Boc-NЈЈ-(pent-3-ynyl)guanidine (18): Synthesised accord-
ing to a reported procedure.[11]
2-(Pent-4-ynylamino)pyridine (19): 2-Fluoropyridine (0.175 g,
1.8 mmol) and pent-4-ynylamine (0.3 g, 3.6 mmol) were dissolved
in anhydrous pyridine (1 mL) and heated in a sealed vessel at
190 °C by microwave irradiation for 1 h. The solvent was evapo-
rated, and the product was isolated by FCC with a Biotage Isolera
1
Yield: 75 mg (56%); pale-yellow oil. H NMR (300 MHz, CDCl3):
δ = 8.02 (m, 1 H, NH), 7.49 (s, 1 H), 7.34 (m, 1 H), 6.91 (m, 1 H),
6.53 (m, 1 H), 6.37 (d, J = 8.4 Hz, 1 H), 4.99 (s, 2 H), 3.63 (s, 3
H), 3.51 (q, J = 6.3 Hz, 2 H), 3.34 (t, J = 6.6 Hz, 2 H), 2.83 (t, J
= 7.2 Hz, 2 H), 2.52 (t, J = 6.3 Hz, 2 H), 2.09–1.95 (m, 2 H) ppm.
13C NMR (50 MHz, CDCl3): δ = 172.4, 165.4, 158.6, 157.9, 147.6,
137.6, 122.6, 112.6, 106.9, 52.8, 51.8, 41.3, 35.2, 33.4, 28.8,
23.0 ppm. MS (ESI): m/z (%) = 347.1 (100) [M + H]+. IR (CH2Cl2):
1
system (TLC: PE/EtOAc, 1:1; Rf = 0.62). Yield: 29 mg (10%). H
NMR (300 MHz, CDCl3): δ = 8.11 (m, 1 H), 7.41 (m, 1 H), 6.55
(m, 1 H), 6.39 (d, J = 8.1 Hz, 2 H), 4.66 (br. s, 1 H), 3.40 (q, J =
6.6 Hz, 2 H), 2.30 (td, J = 6.9, 2.7 Hz, 2 H), 1.99 (t, J = 2.7 Hz, 2
H), 1.91–1.76 (m, 2 H) ppm. 13C NMR (100 MHz, CDCl3): δ =
158.7, 148.1, 137.4, 112.7, 106.5, 83.6, 69, 41, 28.1, 16 ppm. MS
ν = 3404 (m), 3057 (s), 2977 (w), 1696 (s), 1602 (s), 1520 (m), 1475
˜
(m), 1389 (m), 1368 (m), 1216 (m), 1152 (s) cm–1. C16H22N6O3
(346.39): calcd. C 55.48, H 6.40, N 24.26; found C 55.36, H 6.37,
N 24.19.
(ESI): m/z (%) = 161.1 (100) [M + H]+. IR (CH Cl ): ν = 3429 (m),
˜
2
2
3310 (s), 3042 (m), 2933 (bm), 2863 (m), 2119 (w), 1599 (s), 1569
(m), 1505 (s), 1441 (m), 1327 (m) cm–1. C10H12N2 (160.22): calcd.
C 74.97, H 7.55, N 17.48; found C 74.82, H 7.48, N 17.44.
Dimethyl 3-[(2-{4-[3-(tert-Butoxycarbonylpyridin-2-yl-amino)prop-
yl][1,2,3]triazol-1-yl}acetylamino)methyl]pentanedioate (23): Syn-
thesised according to general procedure A starting from alkyne 19
and azide 7. FCC with a Biotage Isolera system (PE/EtOAc, 1:1,
then EtOAc/MeOH, 95:5. TLC: EtOAc; Rf = 0.42). Yield: 48 mg
General Procedure A. CuAAC Reaction: Azide (1.1 equiv.) was dis-
solved in THF (0.15 m solution), and alkyne (1 equiv.) and
CuP(OEt)3I (0.11 equiv.) were added. The mixture was heated un-
der microwave irradiation at 120 °C for 1 h. THF was evaporated,
the crude material was dissolved in either EtOAc or CH2Cl2,
washed with concentrated ammonia solution (2ϫ 4 mL) and brine.
The organic layer was dried with Na2SO4, concentrated under re-
duced pressure, and the product was purified by FCC.
1
(50%). H NMR (200 MHz, CDCl3): δ = 8.35 (m, 1 H), 7.68–7.50
(m, 2 H), 7.47 (s, 1 H), 7.01 (m, 1 H), 6.88 (d, J = 8.8 Hz, 1 H),
4.97 (s, 2 H), 4.58 (m, 1 H), 4.00 (t, J = 7.2 Hz, 2 H), 3.63 (s, 6 H),
2.76 (t, J = 7.8 Hz, 2 H), 2.69–2.50 (m, 4 H), 2.11–1.92 (m, 2 H),
1.48 (s, 9 H) ppm. 13C NMR (50 MHz, CDCl3): δ = 171.1, 164.9,
154.5, 154.1, 148.1, 147.6, 136.9, 122.4, 120.1, 119.6, 81.0, 52.7,
tert-Butyl 2-{tert-Butoxycarbonyl[(S)-3-{1-[(2-methoxycarbonyl-1- 51.7, 46.1, 43.4, 37.3, 28.2, 23.0 ppm. MS (ESI): m/z (%) = 541.2
phenylethylcarbamoyl)methyl]-1H-[1,2,3]triazol-4-yl]propyl)-
amino}benzimidazole-1-carboxylate (20): Synthesised according to
general procedure A, starting from alkyne 12 and azide 5. FCC
with a Biotage Isolera system (TLC: EtOAc/PE, 2:1; Rf = 0.32).
(100) [M + Na]+, 519.2 (33) [M + H]+. IR (CH Cl ): ν = 3404
˜
2 2
(bm), 3057 (m), 2977 (m), 2947 (m), 1730 (s), 1696 (s), 1592 (s),
1369 (m), 1295 (m), 1206 (m), 1152 (s) cm–1. C25H36N6O7 (532.60):
calcd. C 56.38, H 6.81, N 15.78; found C 56.22, H 6.75, N 15.69.
Eur. J. Org. Chem. 2014, 7595–7604
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7601

P. Fabbrizzi, A. Trabocchi et al.
FULL PAPER
Methyl N-({4-[3-({Bis[(tert-butoxycarbonyl)amino]methylene}- 2 H), 3.17 (t, J = 6.6 Hz, 2 H), 2.79 (d, J = 7.2 Hz, 2 H), 2.69 (t,
amino)propyl]-1H-1,2,3-triazol-1-yl}acetyl)-β-alaninate (24): Syn- J = 7.2 Hz, 2 H), 1.94–1.82 (m, 2 H) ppm. 13C NMR (50 MHz,
thesised according to general procedure A starting from alkyne 18
and azide 6. FCC with a Biotage Isolera system (EtOAc/PE gradi-
ent; TLC: EtOAc; Rf = 0.45). Yield: 55 mg (35%); colourless oil.
1H NMR (200 MHz, CDCl3): δ = 7.80 (s, 1 H), 6.47 (br. s, 1 H),
D2O): δ = 174.4, 166.2, 152.4, 143.4, 139.8, 134.6, 129.3, 128.9,
128.0, 126.9, 126.2, 113, 112.3, 52.6, 50.7, 40.6, 39.8, 26.5,
21.2 ppm. MS (ESI): m/z (%) = 409.2 (100) [M + H]+, 359 (8). IR
(CH Cl ): ν = 3598 (s), 3062 (bs), 1658 (m), 1604 (s), 1421 (w)
˜
2
2
5.01 (s, 2 H), 3.99–3.88 (m, 2 H), 3.67 (s, 3 H), 3.51 (q, J = 6.2 Hz, cm–1. C21H25ClN6O3 (444.92): calcd. C 56.69, H 5.66, N 18.89;
2 H), 2.81 (t, J = 7.2 Hz, 2 H), 2.52 (t, J = 6.2 Hz, 2 H), 2.14–1.92 found C 56.48, H 5.82, N 18.69.
(m, 2 H), 1.52 (s, 9 H), 1.51 (s, 9 H) ppm. 13C NMR (100 MHz,
3-(2-{4-[3-(Pyridin-2-ylamino)propyl][1,2,3]triazol-1-yl}acetyl-
CDCl3): δ = 172.2, 165.4, 163.7, 160.6, 154.8, 148.0, 122.9, 83.9,
amino)propionic Acid HCl Salt (28): Synthesised according ot gene-
78.8, 52.7, 51.7, 43.9, 35.1, 33.4, 28.3, 27.9, 27.6, 22.8 ppm. MS
ral procedure B starting from 22. Yield: 77 g (quantitative). 1H
(ESI): m/z (%) = 512 (100) [M + H]+. IR (CH Cl ): ν = 3429 (m),
˜
2
2
NMR (300 MHz, D2O): δ = 7.83 (s, 1 H), 7.73–7.66 (m, 1 H), 7.62–
7.57 (m, 1 H), 6.81 (d, J = 9 Hz, 1 H), 6.69 (m, 1 H), 5.11 (s, 2 H),
3.36 (t, J = 6.6 Hz, 2 H), 3.26 (t, J = 6.6 Hz, 2 H), 2.76 (t, J =
7.2 Hz, 2 H), 2.47 (t, J = 6.3 Hz, 2 H), 2.00–1.87 (m, 2 H) ppm.
3389 (m), 3062 (s), 2972 (m), 1708 (m), 1604 (s), 1367 (m), 1149
(m) cm–1. C22H37N7O7 (511.57): calcd. C 51.65, H 7.29, N 19.17;
found C 51.89, H 7.22, N 19.21.
3-[2-(4-{3-[NЈ-(2-Amino-Cbz-ethyl)-N,NЈЈ-bis(tert-butoxycarbonyl)- 13C NMR (100 MHz, D2O): δ = 175.9, 167.8, 152.5, 147.2, 143.3,
guanidino]propyl}[1,2,3]triazol-1-yl)acetyl]-β-alaninate (25): Synthe-
sised according to general procedure A starting from alkyne 15 and
azide 6. FCC with a Biotage Isolera system (gradient: EtOAc 100%
to EtOAc/MeOH, 92:8. TLC: EtOAc/MeOH, 50:1; Rf = 0.29).
134.5, 124.6, 113.1, 112.2, 51.9, 48.8, 40.7, 35.3, 33.2, 26.8,
21.7 ppm. MS (ESI): m/z (%) = 333.3 (100) [M + H]+. IR (CH2Cl2):
ν = 3598 (s), 3062 (s), 1663, 1604 (s), 1530 (w), 1416 (w), 1159 (w)
˜
cm–1. C15H21ClN6O3 (368.82): calcd. C 48.85, H 5.74, N 22.79;
found C 48.62, H 5.94, N 22.66.
1
Yield: 314 mg (76%); colourless oil. H NMR (300 MHz, CDCl3):
δ = 7.50 (s, 1 H), 7.36–7.29 (m, 5 H), 5.08 (s, 2 H), 4.95 (s, 2 H),
3.69–3.63 (m, 4 H), 3.53–3.44 (m, 2 H), 3.41 (s, 3 H), 2.74 (t, J =
7.2 Hz, 2 H), 2.54–2.46 (m, 2 H), 2.00–1.84 (m, 4 H), 1.48 (s, 9 H),
1.44 (s, 9 H) ppm. 13C NMR (50 MHz, CDCl3): δ = 172.3, 165.5,
156.7, 153.1, 147.4, 136.5, 128.4, 128.0, 127.8, 122.8, 82.4, 79.4,
66.6, 52.5, 51.7, 46.9, 35.2, 33.4, 28.1, 22.8 ppm. MS (ESI): m/z (%)
= 689.3 (100) [M + H]+. C32H48N8O9 (688.78): calcd. C 55.80, H
7.02, N 16.27; found C 55.97, H 7.08, N 16.41.
3-[(2-{4-[3-(Pyridin-2-ylamino)propyl][1,2,3]triazol-1-yl}acet-
ylamino)methyl]pentanedioic Acid HCl Salt (29): Synthesised ac-
cording to general procedure B starting from 23. Yield: 36 mg
(quantitative); pale-yellow solid. 1H NMR (200 MHz, D2O): δ =
7.78 (s, 1 H), 7.68 (m, 1 H), 7.57 (m, 1 H), 6.80 (d, J = 9.2 Hz, 1
H), 6.68 (m, 1 H), 5.07 (s, 2 H), 4.45 (m, 1 H), 3.25 (t, J = 6.6 Hz,
2 H), 2.82–2.67 (m, 2 H), 2.66–2.36 (m, 4 H), 2.04–1.79 (m, 2 H)
ppm. 13C NMR (50 MHz, D2O): δ = 174.4, 165.8, 152.4, 143.5,
Ethyl 1-({4-[3-({(Z)-[(tert-Butoxycarbonyl)amino][(tert-butoxycarb- 134.7, 113, 112.4, 53.1, 43.8, 40.7, 37.9, 26.4, 20.8 ppm. MS (ESI):
onyl)imino]methyl}amino)propyl]-1H-1,2,3-triazol-1-yl}acetyl)piper-
idine-4-carboxylate (32): Synthesized according to general pro-
m/z (%) = 405.2 (100) [M + H]+. IR (CH Cl ): ν = 3599 (s), 3056
˜
2 2
(s), 2957 (m), 2928 (m), 1730 (m), 1607 (s), 1419 (m), 1172 (w)
cedure A starting from alkyne 18 and azide 9. FCC (CH2Cl2/ cm–1. C18H25N6O5 (405.43): calcd. C 49.04, H 5.72, N 19.06; found
MeOH, 80:1 to 10:1). Yield: 61 mg (35%); colourless oil. 1H NMR C 48.82, H 5.87, N 18.96.
(200 MHz, CDCl3): δ = 7.67 (s, 1 H), 7.26 (s, 2 H), 4.30 (m, 1 H),
3-{2-[4-(3-Guanidinopropyl)[1,2,3]triazol-1-yl]acetylamino}propionic
4.19 (q, J = 7.2 Hz, 2 H), 4.00–3.80 (m, 3 H), 3.26 (m, 1 H), 2.92
Acid HCl Salt (30): Synthesised according to general procedure B
(m, 1 H), 2.82–2.72 (m, 2 H), 2.57 (m, 1 H), 2.02–1.90 (m, 3 H),
1
starting from 24. Yield: 33 mg (quantitative); pale-yellow solid. H
1.88–1.60 (m, 3 H), 1.50 (s, 18 H), 1.24 (t, J = 7.2 Hz, 3 H) ppm.
NMR (200 MHz, D2O): δ = 7.84 (s, 1 H), 5.10 (s, 2 H), 3.36 (t, J
MS (ESI): m/z (%) = 566.3 (100) [M + H]+. C26H43N7O7 (565.67):
= 6.4 Hz, 2 H), 3.08 (t, J = 6.4 Hz, 2 H), 2.75–2.62 (m, 2 H), 2.47
calcd. C 55.21, H 7.66, N 17.33; found C 55.32, H 7.78, N 17.46.
(t, J = 6.4 Hz, 2 H), 1.86–1.82 (m, 2 H) ppm. 13C NMR (50 MHz,
General Procedure B. Deprotection Step: The protected adduct was
suspended in 3 m HCl (10 mL/mmol) and stirred at 30 °C over-
night. The solution was concentrated to dryness in vacuo to afford
the pure product.
D2O): δ = 177.8, 166.9, 156.7, 146, 126.1, 53.0, 42.2, 38.9, 31.0,
27.0, 21.0 ppm. MS (ESI): m/z (%) = 298.2 (100) [M + H]+. IR
(CH Cl ): ν = 3593 (s), 3057 (s), 1605 (s), 1419 (m), 1155 (w), 900
˜
2
2
(w), 895 (w) cm–1. C11H20ClN7O3 (333.78): calcd. C 39.58, H 6.04,
N 29.38; found C 39.22, H 6.25, N 29.19.
(S)-3-(2-{4-[3-(1H-Benzimidazol-2-ylamino)propyl][1,2,3]triazol-1-
yl}acetylamino)-3-phenylpropionic Acid HCl Salt (26): Synthesised
according to general procedure B starting from 20. Yield: 305 g
(quantitative); pale-yellow solid. 1H NMR (300 MHz, D2O): δ =
3-[2-(4-{3-[N-(2-Aminoethyl)guanidino]propyl}[1,2,3]triazol-1-yl)acet-
ylamino]propionic Acid HCl Salt (31): Synthesised according to a
modified general procedure B, starting from 25, adding 3 m HCl
7.33 (s, 1 H), 7.18–7.01 (m, 5 H), 6.97 (s, 4 H), 5.15–5.02 (m, 3 H), (20 mL/mmol) and heating at 50 °C. Yield: 154 g (quantitative);
3.16–3.04 (m, 2 H), 2.79–2.58 (m, 4 H), 1.95–1.80 (m, 2 H) ppm.
pale-yellow solid. 1H NMR (300 MHz, D2O): δ = 8.12 (s, 1 H),
13C NMR (100 MHz, [D6]DMSO): δ = 171.9, 185.1, 142.3, 130.2, 5.25 (s, 2 H), 3.28 (t, J = 6 Hz, 2 H), 3.02 (t, J = 6.9 Hz, 2 H),
129.1, 128.8, 128.1, 127.6, 127.0, 124.2, 123.4, 111.7, 52.0, 50.3,
2.98–2.91 (m, 4 H), 2.68 (t, J = 8.4 Hz, 2 H), 2.50 (t, J = 6.6 Hz,
2 H), 1.80–1.67 (m, 2 H) ppm. 13C NMR (50 MHz, D2O): δ =
174.1, 168.3, 155.7, 143.6, 127.8, 52.9, 40.3, 38.5, 38.1, 35.1, 30.8,
42.7, 41.1, 28.8, 22.6 ppm. MS (ESI): m/z (%) = 448.3 (100) [M +
H]+. IR (CH Cl ): ν = 3593 (s), 3057 (s), 2987 (m), 1602 (s), 1419
˜
2
2
(m) cm–1. C23H26N7O3 (448.50): calcd. C 57.08, H 5.42, N 20.26; 26.5, 20.2 ppm. MS (ESI): m/z (%) = 341.2 (100) [M + H]+. IR
found C 56.89, H 5.65, N 20.01.
(CH Cl ): ν = 3598 (s), 3052 (s), 1604 (s), 1421 (m), 1159 (w) cm–1.
˜
2 2
C13H25N8O3 (341.39): calcd. C 41.43, H 6.69, N 29.73; found C
41.29, H 6.89, N 29.52.
(S)-3-Phenyl-3-(2-{4-[3-(pyridin-2-ylamino)propyl][1,2,3]triazol-1-
yl}acetylamino)propionic Acid HCl Salt (27): Synthesised according
to general procedure B starting from 21. Yield: 84 g (quantitative);
1-{2-[4-(3-Guanidinopropyl)[1,2,3]triazol-1-yl]acetyl}piperidine-4-
carboxylic Acid HCl Salt (33): Synthesised according to general
1
pale-green solid. H NMR (300 MHz, D2O): δ = 7.67–7.56 (m, 2
H), 7.51 (d, J = 7.2 Hz, 1 H), 7.36–7.12 (m, 5 H), 7.15 (d, J = 9 Hz,
1 H), 6.63 (t, J = 6.6 Hz, 1 H), 5.12 (m, 1 H), 5.05 (d, J = 1.8 Hz, yellow solid. H NMR (200 MHz, D2O): δ = 7.83 (s, 1 H), 5.44 (s,
procedure B starting from 32. Yield: 37 mg (quantitative); pale-
1
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 7595–7604

Binding Affinity of RGD Peptidomimetics to αvβ3 Integrin
2 H), 4.09 (m, 1 H), 3.74 (m, 1 H), 3.18 (m, 1 H), 3.06 (t, J = Supporting Information (see footnote on the first page of this arti-
1
6.8 Hz, 2 H), 2.90–2.48 (m, 4 H), 1.65–1.36 (m, 2 H) ppm. 13C cle): H and 13C NMR spectra.
NMR (50 MHz, D2O): δ = 178.8, 164.9, 156.8, 145.8, 126.2, 52.0,
44.4, 42.0, 40.2, 40, 27.6, 27.1, 21.1 ppm. MS (ESI): m/z (%) =
338.2 (100) [M + H]+. IR (CH Cl ): ν = 3593 (s), 3057 (s), 2977
˜
2
2
Acknowledgments
(m), 1607 (s), 1419 (m), 1157 (w) cm–1. C14H24ClN7O3 (373.84):
calcd. C 44.98, H 6.47, N 26.23; found C 44.71, H 6.71, N 26.02.
Financial support from the University of Florence (MIUR
PRIN2010-2011, cod. 2010NRREPL) and the Regione Toscana
Council (POR CRO FSE 2007-2013 and UNIFI_FSE2012) is ac-
knowledged.
Solid-Phase Integrin Binding Assay: 125I-Echistatin, labelled by the
lactoperoxidase method[20] to a specific activity of 2000 Ci/mmol,
was purchased from Perkin–Elmer, and integrin αvβ3 from human
placenta was purchased from Millipore. Purified αvβ3 integrin was
diluted in coating buffer [20 mm Tris (pH = 7.4), 150 mm NaCl,
2 mm CaCl2, 1 mm MgCl2, 1 mm MnCl2] at concentrations of 500
or 1000 ng/mL. An aliquot of diluted integrin (100 μL/well) was
added to a 96-well microtiter plate (Optiplate-96 HB, Perkin–Elmer
Life Sciences, Boston, MA), and plates were incubated overnight
at 4 °C. Plates were washed once with blocking/binding buffer
[20 mm Tris (pH = 7.4), 150 mm NaCl, 2 mm CaCl2, 1 mm MgCl2,
1 mm MnCl2, 1% BSA] and incubated at room temp. for an ad-
ditional 2 h. Plates were rinsed twice with the same buffer, and
then competition binding assays were performed with a constant
concentration of 125I-echistatin (0.05 nM). Concentrations of tested
compound ranged from 0.01 to 100 nM. All assays were performed
in triplicate in a final volume of 0.2 mL, each containing the follow-
ing species: 0.05 mL of 125I-echistatin, 0.04 mL of tested com-
pound, and 0.11 mL of blocking/binding buffer. Nonspecific bind-
ing was defined as 125I-echistatin bound in the presence of an excess
(1 μm) of unlabelled echistatin. After incubation at room temp. for
3 h, plates were washed three times with blocking/binding buffer
and counted in a Top-Count NXT microplate scintillation counter
(PerkinElmer Life Sciences, Boston, MA) using 200 μL/well of
MicroScint-40 liquid scintillation (PerkinElmer Life Sciences, Bos-
ton, MA). Data are shown as meanϮSD from three independent
experiments. IC50 values were determined by fitting binding inhibi-
tion data by nonlinear regression using GraphPad Prism 4.0 Soft-
ware Package (GraphPad Prism, San Diego, CA).
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