Linear and Cyclic Depsipeptidomimetics with β-Lactam Cores: A Class of New αvβ3 Integrin Receptor Inhibitors

Article abstract of DOI:10.1002/cbic.201600642

The α_vβ₃ integrin receptor plays an important role in tumor metastasis and tumor-induced angiogenesis. The inhibition of this receptor with diverse ligands, antibodies, or cyclic peptides is a promising research field for the treatme

Full text of DOI:10.1002/cbic.201600642

Accepted Article
Title: Lineal and Cyclic Depsipeptidomimetics with a beta-Lactam Core:
A Class of new alphavbeta3 Integrin Receptor Inhibitors
Authors: José Ignacio Ganboa, Nerea Zabala, José Ignacio Miranda,
Antonio Laso, and Claudio Palomo
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To be cited as: ChemBioChem 10.1002/cbic.201600642
Link to VoR: http://dx.doi.org/10.1002/cbic.201600642
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Lineal and Cyclic Depsipeptidomimetics with a -Lactam Core: A
Class of new v3 Integrin Receptor Inhibitors
Nerea Zabala-Uncilla,[a] José I. Miranda, [b] Antonio Laso, [c] Jose I. Ganboa,*[a] and Claudio Palomo*[a]
Abstract: The αvβ3 integrin receptor plays an important role in tumor
metastasis and tumor-induced angiogenesis. The inhibition of
this receptor using diverse ligands, antibodies or cyclic
peptides, is a promising research field for the treatment of a
variety of tumors. The replacement of Phe-(Me)Val dipeptide
by a -lactam ring in Cilengitide leads to new products that
show higher inhibitory activity respect to parent cyclopeptide.
In particular, substitution of a peptide bond -lactam-NH–Asp
by one -lactam-O–Asp ester linkage, increases the activity of
the new cyclodepsipeptide. In the same way it has been found
that open-chain compounds, Asp–-lactam–Arg, can interact
with the receptor and inhibit its activity moderately. The
integrin inhibitory activity of synthesized compounds has been
established by using CGH array, a method that appears to be
more reliable trial than the classical adhesion test.
The most common integrins, including v3, recognize the
tripeptide Arg-Gly-Asp (RGD) sequence found in many
extracellular matrix adhesive proteins.10 The spatial layout of the
RGD (rigidity, 3D disposal, possibility of interaction with integrin
binding pockets…) determines the specificity and efficacy of
interaction. 11 Whilst cyclic RGD peptides 12 and several RGD
cyclic lactam mimetics13 have been developed, the product EMD
121974 called Cilengitide14 seems to be one of the most potent
15
inducers (Scheme 1) of endothelial apoptosis,
angiogenesis and blocks metastasis16.
inhibits
Introduction
The discovery that tumor growth and metastasis are
codependent on the formation of neovascularization revealed
many potential protein targets for cancer treatment.1 In other
pathological states as diabetic retinopathy and chronic
inflammatory disorders abnormal angiogenesis is observed too.2
Scheme 1. EMD 121974 (Cilengitide®) structure.
In an effort to found new bioactive compounds a prior work from
this laboratory has documented that -lactam based cyclic RGD
peptides are promising candidates.17 In particular cyclic product
1, which is readily available from the -amino -lactam 2, shows
antagonist activity against v3 somewhat higher than
Cilengitide.27 Herein we report that cyclodepsipeptides 3, which
can be prepared from the respective -hydroxy -lactam 4, are
also promising new candidates for a greater inhibitory capacity.
The angiogenic process depends on
a family of highly
conserved adhesion molecules, known as integrins, central
compounds for regulation of these processes.3,4 In particular,
integrin v3 is one of the most well-characterized integrin
heterodimers and it is one of the several heterodimers that have
been implicated in tumor-induced angiogenesis.5 While sparingly
expressed in mature blood vessels, is significantly upregulated
during angiogenesis “in vivo”. 6 The overexpression of v3
correlates with the aggressiveness of the tumor.7 The inhibition
of this v3-receptor is important, induces apoptosis of the
proliferative vascular cells 8 and prevent tumor growth and
metastasis.9
[a]
[b]
[c]
Prof. C.Palomo, Prof J.I. Ganboa, Dr N. Zabala-Uncilla Title(s),
Departamento de Química Orgánica-I, Facultad de Química
Universidad del País Vasco UPV/EHU
Paseo Manuel Lardizabal-3, 20018 San Sebastian, Spain
E-mail: claudio.palomo@ehu.eus ; joseignacio.ganboa@ehu.eus
Dr. J.I. Miranda
SGIKer NMR Facility
Universidad del País Vasco UPV/EHU
Joxe Mari Korta R&D Center
Avda Tolosa-72, 20018 San Sebastian, Spain
Dr. A. Laso
Genetadi Biotech A. G.,
Scheme 2. RGD mimetics from -lactams as constraining elements
.
Edificio 502, Parque Tecnológico de Bizkaia, 48160 Derio, Spain
It has been reported that the replacement of an amide bond of
the peptidomimetic by an ester linkage can modify the internal
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/cbic.201600642
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hydrogen bond interactions as well as the preferential torsion
angles18 and therefore the active conformations19 of the native
peptide. This approach, called “Ester-Scan” approach, has been
applied to several cyclopeptidomimetics to modify the
conformational ensemble and populations distribution, changing
H-bonding pattern and torsional preferences.20 Moreover, whilst
increasing the structural flexibility of a given cyclopeptide a
weaker interaction with the receptor can be produced21, it has
been proposed that a certain degree of flexibility is needed to
adopt the bioactive conformation22, in this latter case appropriate
modulation of the architecture of depsipeptidomimetics is
facilitated in order to achieve high receptor affinity.19 Given
Asp(OtBu)-F 24 and the resulting products were subjected to
desilylation and subsequent oxidation with BAIB/TEMPO to
furnish the carboxylic acids 9a and 9b, respectively. Coupling of
-lactams
9a,b
with
H2N-Arg(Pbf)-Gly-OBn
provided
pseudopeptides 10a,b. In a similar way 13a,b were prepared
from 7a,b via coupling of carboxylic acids 12a,b with H2N-Arg-
(Pbf)-OtBut.
Next, after deprotection of the Cbz and benzyloxy groups by
hydrogenolysis, intramolecular HATU/HOBt25 mediated peptide
coupling between the glycine carboxy terminus and the aspartic
amino group under high dilution conditions afforded the
corresponding cyclized products 14a,b, which were purified by
column chromatography and isolated in yields between 45‒65%
range. The purity of cyclic and acyclic compounds was about
98% as determined using the UPLC/MS technique. After
examining several conditions to perform complete deprotection
in a single pot operation, a modification of the method described
by Mehta et al26. was found to be the most appropriate. The
resulting 15a,b were isolated27 by successive precipitations with
diisopropyl ether, centrifugation and a final washing of the
resulting white solid compound with more diisopropyl ether.
these
observations
we
decided
to
prepare
the
cyclodepsipeptides 15a and 15b, the first members of 3 type
cyclodepsipeptides, to evaluate their activity profiles.
For comparative purpouses open-chain compounds, 16a,b,
were also prepared as shown in Scheme 6. These compounds
(Scheme 6) as well as the cyclized products, 15a,b, were then
subjected to biological tests. The absence of recognition RGD
sequence in open chain depsipeptides led us to expect poor or
no activity. These compounds showed appreciable activity28, as
confirmed by qRT-PCR, and can be considered as DGR mimetic
units, where the β-lactam is an extra residue.
Scheme 3. Cyclodepsipeptides 15a,b starting from a 3-HO--lactam.
Results and Discussion.
Synthesis of cyclopeptides.
The starting -lactams 6a and 6b were prepared as essentialy
single diastereomers by using the standard cycloaddition
between benzyloxyacetyl chloride and imines 5a and 5b in the
presence of triethyamine followed by hydrogenolysis of the
benzyloxy group 23 . With -hydroxy--lactams 6a-b in hand
cyclodepsipeptides 15a,b were synthesized as shown in
Schemes 5 and 6. Each azetidin-2-one was coupled with Cbz-
Scheme 4. DGR compound with -lactam as an extra residue.
Scheme 5. Preparation of macrocycle linear precursors. Reagents and conditions: i) BnOCH2COCl, TEA, ‒78 °C → r.t., 16 h; ii) HCO2NH4, Pd/C (10%), MeOH
reflux, 1 h; iii) Cbz-Asp(OtBu)-F, TEA, CH2Cl2, r.t., 30 min; iv) PyrHF, THF, 0 °C → r.t., 3 h; v) TEMPO, BAIB, MeCN/H2O, r.t., 4 h; vi) H2N-Arg(Pbf)-Gly-OBn (17),
HOBt, EDC·HCl, TEA, CH2Cl2, 0 °C → r.t., 16 h; vii) Boc-Asp(OtBu)-F, Pyridine, THF, 0 °C → r.t.; viii) H2N-Arg(Pbf)-OtBu, EDC·HCl, HOBt, TEA, CH2Cl2, 0 °C →
r.t.
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receptors, overexpressed in HUVEC cells. Through analysis of
the whole 20,500 human genes, the microarray assay provided
us a two-color output image of normalized gene-expression
data.32 Genes that were at least 1.5 fold differentially expressed
on 3 of 4 arrays were scored as significant. From these
transcriptomics analysis we could identify up to 230/217
activated genes and 90/62 inhibited genes in analyzed
compounds after the RGD mimetic cell-treatment. Among all the
up or down-regulated genes, we selected for a more detailed
analysis 17 genes, which can be seen in Table 1, known to be
related to angiogenesis.33 Ten of these genes were activated
and seven were blocked after treatment with Cilengitide. The
pattern of gene-expression was mainly conserved (17/15 genes)
for the remaining compounds. On the other hand, the lack of
correlation in the expression of 2/3 genes (CDC7, ADAM6 and
FAIM3) demonstrated the highly specific “in-vivo” cell activity
after ligand-receptor binding.
Table 1. Angiogenesis-related gene regulation of DNA samples extracted from
HUVEC cells after treatment with Cilengitide® C, RGD β-lactam ligands 15a,b
and open chain compounds 16a-d. Numbers refer to a binary logarithmic scale,
and colors refer to activation (red) or inhibition (blue). For numerical values,
gene hierarchical clustering and gene function see ref. 29.
Scheme 6. Preparation of cyclodepsipeptidomimetics and depsipeptidomi
metics. Reagents and conditions: i) H2 (1 atm), Pd/C (10%), THF/EtOAc (1:1),
r.t., 16 h; ii) HATU, HOAt, KHCO3, DMF, ‒15 °C, 16 h; iii) Et3SiH, TFA., H2O,
4 °C, 16 h.
Bioactivity of the -lactam based
cyclopeptides
In order to determine the angiogenesis antagonist activity
towards integrin v3, many authors have used a binding test
based on the capacity to inhibit the extracelular adhesion of a
29
set of cellular line, typically HUVEC.
Recently we have
observed that the inhibition assay of extracelular adhesion is not
always directly related to the activation/deactivation of the genes
responsible for controlling the inhibition of a specific integrin.
Specifically, it was found a tetrapeptidomimetic-lactam30 that
markedly inhibits the extracellular adhesion in a culture of
HUVEC cells and, in its turn, acts as an angiogenic agonist of
v3 integrin. From these data it appears that quantification of
the extracellular adhesion phenomenon is not enough to
determine the inhibitory activity of a given compound. To
evaluate the intracellular activity of genes involved in
angiogenesis and inhibition of integrin v3 the most accurate
path would be the use of the comparative genomic hybridization
(CGH)31 technique. This technique enables the determination of
the complete genome of HUVEC cells and the quantitative
analysis of the activation/deactivation date in specific genes to
compare these results with integrin inhibition related genes
found in literature. Thus HUVEC cells were treated separately
with a 10‒2 mM concentration of compounds 15a,b/16a,b and
Cilengitide was used as a reference standard. After 48 h of
incubation the cells were removed from culture and RNA was
extracted. For data analysis of gene expression an Agilent
G2505-B Array scanner was used.
As positive control we chose the EMD 121974 (Cilengitide®), the
magnitude of the activation or inhibition in each gene is
determined by direct comparison with the pattern obtained from
Cilengitide® (see supporting). Array gene expression qualitative
data were reconfirmed using gene-specific quantitative mRNA
assays, qRT-PCR, analyzing the pattern expression in a
selected gene set (TGFBR2, TRIP12, MMP1, ITGA9, FOXC1),
over each tested compound, using specific amplification primers
and hybridization probes. Data consistency between quantitative
qRT-PCR analysis and CGH array validates the process and
results.32 In addition, no significant changes were observed in
the gene expression of the apoptosis associated tyrosine kinase
(AATK)34 and the apoptotic regulator BAX (BL2 associated X
protein)35 genes, suggesting that apoptosis was not induced
after 72 h of treatment.
As can be seen in Table 2, Ki values obtained by theoretical
calculations, Autodock 4.2 release 4.2.5, 36 show very similar
values for cyclic compounds, 15a and 15b, indicating an
The simultaneous analysis of all human gene expression by
CGH array allowed us to identify the genes affected after the
adhesion of compounds to be then tested on v3 integrin
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effective interaction of the substrates with the receptor in
docking experiment. Cilengitide shows the best value for Ki, but
this excellent data is not corroborated by IC50 and gene
expression experimental data. Moreover open chain compounds
show very low values of Ki that do not fit with the moderated
experimental values of activity. We can conclude that
experimental model of docking based on the use of extracelular
domain of v3 protein crystallized with Cilengitide37 as receptor
model is relatively effective with cyclic depsipeptides and has
some predictive value on their activity but in open chain
compounds docking predictions are far from experimental data
and not allow anticipate real activity.
These differences are obvious using CGH Array technique that
allows easily distinguish the most active compound. In both
types of structures, peptidomimetics and depsipeptidomimetics
16b and 16d, which present higher chain length, are the most
active.
To rationalize this result we analyzed the bioactive
conformations of the compounds 15a,b, 16a,b, in their
interaction with the extracellular domain of integrin receptor v3
using VMD program. 38 Figure 1 shows four representative
structures of the major conformational clusters in cyclic and
open chain compounds, although it should be noted that the
clusters of different compounds have very different populations.
In both cyclic compounds 15a,b, the interaction of the aspartic
carboxylic group with the Ca2+ ion and guanidino group of
arginine with a pocket of the β domain stabilizes the interaction,
resulting in a distribution of low-energy clusters with abundant
populations.
For comparative purposes we prepared the corresponding open
chain cyclopeptidomimetics 16c and 16d from the respective 3-
amino--lactams32.
Scheme 7. Cyclopeptidomimetics 16c and 16d starting from a 3-NH--lactam.
As Table 2 shows IC50 values in compounds 16a-d are very
similar and adhesion test does not allow distinguish activity
differences between them.
Table 2. Docking, adhesion test data and genic expression results in cyclic
compounds 15a,b; 16a–d and Cilengitide.
Theoretical data
Experimental results32
Adhesion test
IC50 (M)
Order
genic
of
Binding
energy
(Kcal)
Torsion
energy
(Kcal)
Ki
(M)
expressiona
1b
–7.91
–10.70
–8.04
‒8.18
–5.94
–4.86
–5.48
–5.18
+2.98
+ 2.68
+2.98
+2.98
+5.37
+5.67
+5.07
+5.37
1.58
0.014
1.27
3.1 ±0.3
4.0 ±0.1
0.6 ±0.1
2.5 ±0.3
6.3 ±0.4
5.0 ±0.3
5.0 ±0.4
6.3 ±0.2
3
4
1
2
6
5
8
7
C
15a
15b
16a
16b
16c
16d
1.01
44.60
272.59
95.45
159.16
a
The order is obtained comparing the relative values of each analyzed gene.
b Only for comparative purpouses.30
.
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we relate the low Ki values with an inadequate docking receptor
model for acyclic compounds, as previously mentioned.
Evaluating the results jointly we can conclude that application of
an amide-ester substitution or “Ester scan” in designed
cyclopeptides can be used to overpass the bioactivity of the
synthesized compounds by fine conformational adjustment.
Thus, compound 15a seems to be the best one in terms of
inhibitory activity and also shows to be relatively more potent
than Cilengitide.
Conclusions
We have described an approach to v3 inhibitors based on the
generation of conformational restrictions introduced through the
incorporation of a -lactam framework into a RGD macrocycle.
The effect AspCO–NH--lactam replacement by AspCO–O--
lactam has also been studied and an increase in biological
activity has been observed resulting in products that are
appreciably more potent than Cilengitide, the reference
compound in this field. Another interesting aspect emerged from
this research, is the significant activity we have found in open-
chain compounds, leading to a new family of open-chain RGD
mimetics, which may be optimized very easily. However, the
concept of an amide-ester substitution or “Ester scan” can be
used in design of cyclopeptides to overpass the bioactivity of the
synthesized compounds by fine conformational adjustment. This
observation could be translated to another type of cyclic or
acyclic structures, opening a new field of synthetic possibilities.
In addition to these observations we also have stablished, for
the first time, the CGH array pattern of Cilengitide, an aspect of
this investigation that may be considered for further
developments in the area.
Experimental Section
General Information. All reactions were carried out under an
atmosphere of nitrogen in oven or flame-dried glassware with magnetic
stirring. Solvents were distilled prior to use. Tetrahydrofuran (THF) was
dried by Pure solv column and acetonitrile (MeCN) and methylene
chloride (CH2Cl2) were dried by distillation from calcium hydride.
Purification of reaction products was carried out by flash chromatography
using silicagel 60 (230-400 mesh, from Merck 60F PF254). Analytical thin
layer chromatography was performed on 0.25 mm silica gel 60-F plates.
Visualization was accomplished with UV light and phosphomolybdic acid-
ammonium cerium (IV) nitrate sulfuric acid-water reagent, followed by
heating. Analytical high performance liquid chromatography (HPLC) was
performed on a Waters Acquity UPLC chromatograph equipped with a
diode array UV detector (210-400 nm), using the analytical column BEH
C18 1.7 un 2.1 x 50 mm with flow rates of 0.2 mL/min and 0.3 mL/min.
Solvent: 99.9 (water, 0.1% HCO2H) / 0.1 (MeCN, 0.1% HCO2H). Infrared
spectra were recorded on a Bruker Alpha 360 FT-IR spectrophotometer.
Optical rotations were measured at 25 ± 0.2 °C in a Perkin-Elmer 243-B
polarimeter using methylene chloride as solvent unless otherwise stated.
1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance500
spectrometer using a BBI probe at 500 MHz and 300 MHz respectively
and are reported as  values (ppm) relative to residual CHCl3 H (7.26
ppm) and CDCl3 C (77.16 ppm) as internal standards, respectively.
Exact mass values were obtained by a Waters LCT Premier XE (TOF)
after direct injection (HRMS) of sample. qRT-PCR analysis were
recorded in a Roche Lightcycler 480, 96 well plate version equipment.
Basic silicagel was prepared by mechanical stirring of the commercial
Figure 1. Docking simulation of the interaction of compounds 16a,b with v3
receptor. Only the major conformational clusters are represented (The
calculations were performed with Xplor 2.35 program, and the dihedral angle
restriction, determined by the J coupling of NH-C for different amino acids
present on the macrocycle, was the only previous condition).32 For easy
viewing we only represent, in white, the conformation of majority cluster. For
more details see supporting information.
In both open-chain compounds 16a,b, the mentioned
interactions are weak and cluster majority populations are not
abundant. In compound 16a MIDAS interaction is significant,
with three structures interacting with the Ca2+ ion, but only the
guanidino group of one of the clusters is able to access the
pocket of the
β domain. Regarding compound 16b few
structures show both interactions and consequently the
interconnection between protein domains is weak; also the
interaction with Ca2+ metal ion occurs with an atom located at a
different position to 15a, 15b and 16a MIDAS. These results are
reflected in low interaction energies and greater Ki values, unlike
what happens in cyclic compounds, resulting from the low
interaction with the two domains (α and β) of the receptor.
However experimental values show moderate activity therefore
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acidic silica in saturated sodium bicarbonate solution (300 ml of solution
per 100g of silicagel), followed by filtration and evaporation of the
wastewater in a calefactor at 80 °C for 72h.
CHS[dioxol]); 4.19 (1H, dd, CH2[dioxol], J1 = 8.8 Hz, J2 = 7.1 Hz); 4.00
(1H, dd, HβS[β-lact], J1 = 8.8 Hz, J2 = 5.1 Hz); 3.90‒3.83 (1H, m,
CH2[dioxol]); 3.79‒3.71 (2H, m, CH2-O-Si); 3.66 (1H, dt, N-CH2, J1 = 14.0
Hz, J2 = 4.8 Hz); 3.39 (1H, d, HO, J = 8.5 Hz); 3.24 (1H, dt, N-CH2, J1
=
14.0 Hz, J2 = 4.8 Hz); 1.46 (3H, s, CH3[dioxol]); 1.35 (3H, s, CH3[dioxol]);
General procedure for the synthesis of β-lactams 6a,b. Step 1. To a
stirred solution of (R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde (0.79 g,
t
0.88 (9H, s, Bu); 0.05 (6H, s, CH3-Si-CH3). 13C-NMR (, ppm, CDCl3):
170.8, 109.7, 76.6, 75.3, 66.7, 62.1, 60.2, 43.6, 26.8, 25.9, 25.1, 18.2, –
6.1 mmol) cooled to
0 °C in CH2Cl2 (30 mL) was added the
5.3, ‒5.4.
corresponding silylated aminoalcohol (6.0 mmol) and molecular sieves (4
Å). The mixture was stirred over 60 minutes at same temperature, the
solids were filtered off and the solvent was evaporated to give the
intermediate imine 5a,b. Step 2. The crude product was dissolved in
CH2Cl2 (30 mL) and the solution was cooled to ‒78 °C. Then, Et3N (1.7
mL, 12.0 mmol) was added at the same temperature, followed by a
solution of the corresponding acid chloride (6.1 mmol) in CH2Cl2 (20 mL).
The reaction mixture was stirred overnight, while slowly warming to room
temperature. The resulting solution was washed successively with water
(30 mL), 0.1 M HCl (2 x 30 mL) and saturated aqueous solution of
NaHCO3 (2 x 30 mL), it was dried (MgSO4) and evaporated in “vacuo”.
Purification of the resulting crude oil was performed by flash column
chromatography on basic silicagel (hexane/EtOAc 95/5) to afford the
product 6a,b.
(3R,4S)-3-Hydroxy-1-[3-(tert-butyldimethylsilyloxy)propyl]-4-((S)-2,2-
dimethyl-1,3-dioxolan-4-yl)azetidin-2-one (7b). The general procedure
was followed starting with the β-lactam 2b (1.50 g, 3.30 mmol). Yield:
1.14 g (96%). []D = +13.83 (c = 1, CH2Cl2). IR (, cm‒1, CH2Cl2
25
solution): 3308, 2985, 2929, 2894, 2858, 1719. 1H-RMN (, ppm, CDCl3):
4.79 (1H, dd, HαR[β-lact], J1 = 7.5 Hz, J2 = 4.9 Hz); 4.34 (1H, dt,
CHS[dioxol], J1 = 6.5 Hz, J2 = 5.5 Hz); 4.21 (1H, dd, CH2[dioxol], J1 = 8.8
Hz, J2 = 5.5 Hz); 3.97 (1H, d, OH, J = 7.5 Hz); 3.81 (1H, dd, CH2[dioxol],
J1 = 8.8 Hz, J2 = 5.5 Hz); 3.71 (1H, dd, HβS[β-lact], J1 = 6.5 Hz, J2 = 4.9
Hz); 3.63 (2H, t, CH2-O-Si, J = 6.1 Hz); 3.55 (1H, ddd, N-CH2, J1 = 14.4
Hz, J2 = 8.1 Hz, J3 = 6.8 Hz); 3.25 (1H, ddd, N-CH2, J1 = 14.4 Hz, J2 = 8.0
Hz, J3 = 6.1 Hz); 1.96‒1.66 (2H, m, CH2-CH2-CH2); 1.45 (3H, s,
CH3[dioxol]); 1.35 (3H, s, CH3[dioxol]); 0.89 (9H, s, Si-tBu); 0.04 (6H, s,
CH3-Si-CH3). 13C-NMR (, ppm, CDCl3): 170.6, 109.6, 76.9, 75.0, 66.9,
61.8, 60.8, 39.0, 30.6, 26.9, 26.0, 25.2, 18.3, –5.3.
(3R,4S)-3-Benzyloxy-1-[2-(tert-butyldimethylsilyloxy)ethyl]-4-((S)-2,2-
dimethyl-1,3-dioxolan-4-yl)azetidin-2-one (6a). The general procedure
was followed by coupling of benzyloxyacetyl chloride (1 mL, 6.1 mmol)
with the imine 1a. Overall yield: 1.88 g (72%). IR (, cm‒1, CH2Cl2
solution): 2985, 2953, 2928, 2856, 1761. 1H-RMN (, ppm, CDCl3): 7.43–
7.25 (5H, m, arom); 4.91 (1H, d, CH2-Ph, J = 11.7 Hz); 4.72‒4.53 (2H, m,
CH2-Ph y HαR[β-lact]); 4.32 (1H, dt, CHS[dioxol], J1 = 8.9 Hz, J2 = 6.5 Hz);
4.14 (1H, dd, CH2[dioxol], J1 = 8.7 Hz, J2 = 6.5 Hz); 3.79 (2H, t, CH2-O-Si,
J = 6.0 Hz); 3.71 (1H, dd, HβS[β-lact], J1 = 8.9 Hz, J2 = 5.1 Hz); 3.63 (1H,
dd, CH2[dioxol], J1 = 8.7 Hz, J2 = 6.5 Hz); 3.60–3.49 (1H, m, N-CH2); 3.35
(1H, dt, N-CH2, J1 = 13.7 Hz, J2 = 6.0 Hz); 1.43 (3H, s, CH3[dioxol]); 1.34
(3H, s, CH3[dioxol]); 0.90 (9H, s, Si-tBu); 0.07 (6H, s, CH3-Si-CH3). 13C-
NMR (, ppm, CDCl3): 167.7, 137.0, 128.4, 127.9, 127.7, 109.4, 80.5,
77.2, 72.8, 66.8, 60.9, 59.9, 43.5, 26.8, 25.8, 25.1, 18.1, –5.4.
General procedure for the acoplation of aspartic. Compounds 8a,b
and 11a,b. Step 1. The corresponding L-aspartic acid-derived (2.4
mmol) was dissolved in CH2Cl2 (7 mL) at ‒15 °C and pyridine (0.2 mL,
2.4 mmol) and 2,4,6-trifluoro-1,3,5-triazine (0.3 mL, 3.6 mmol) were
added slowly. The solution was stirred during one hour at same
temperature and a white precipitate was formed. The mixture was
quenched with water/ice solution (10 mL) and extracted with CH2Cl2 (3 x
10 mL). The organic layer was washed with water (3 x 20 mL), dried with
MgSO4, and the solvent was evaporated under reduced pressure to give
the activated L-aspartic derived fluoride and after checking by NMR was
used without further purification. Step 2. The HO-β-lactam 7a,b (2.07
mmol) was dissolved in CH2Cl2 (20 mL) and Et3N (0.58 mL, 4.14 mmol)
was added. A solution of L-aspartic fluoride (0.66 g, 2.28 mmol) in
CH2Cl2 (20 mL) was added slowly. The mixture was stirred for 30
minutes at room temperature. After this time the solution was washed
with 0.1 M HCl (2 x 30 mL), saturated solution of NaHCO3 (2 x 30 mL)
and brine (2 x 30 mL). The organic layer was dried with MgSO4 and
evaporated to give 8a,b, which were used in the next step without further
purification.
(3R,4S)-3-Benzyloxy-1-[3-(tert-butyldimethylsilyloxy)propyl]-4-((S)-
2,2-dimethyl-1,3-dioxolan-4-yl)azetidin-2-one (6b). The general
procedure was followed by couplig of imine 1b with benzyloxyacetyl
chloride (1 mL, 6.1 mmol). Overall yield: 1.50 g (55%). IR (, cm‒1
,
CH2Cl2 solution): 2985, 2929, 2884, 2857, 1755. 1H-RMN (, ppm,
CDCl3): 7.41–7.20 (5H, m, arom.); 4.90 (1H, d, CH2-Ph, J = 11.8 Hz);
4.63 (1H, d, CH2-Ph, J = 11.8 Hz); 4.57 (1H, d, HαR[β-lact], J = 5.1 Hz);
4.30 (1H, dt, CHS[dioxol], J1 = 8.9 Hz, J2 = 6.5 Hz); 4.14 (1H, dd,
CH2[dioxol], J1 = 8.7 Hz, J2 = 6.5 Hz); 3.74–3.58 (4H, m, 2H CH2-O-Si,
HβS[β-lact], 1H CH2[dioxol]); 3.50 (1H, ddd, N-CH2, J1 = 13.9 Hz, J2 = 8.3
Hz, J3 = 6.7 Hz); 3.30 (1H, ddd, N-CH2, J1 = 13.9 Hz, J2 = 8.1 Hz, J3 = 6.0
Hz); 2.02–1.60 (2H, m, CH2-CH2-CH2); 1.42 (3H, s, CH3[dioxol]); 1.32
(3H, s, CH3[dioxol]); 0.89 (9H, s, Si-tBu); 0.04 (6H, s, CH3-Si-CH3). 13C-
NMR (, ppm, CDCl3): 167.6, 137.1, 128.5, 128.0, 127.8, 109.5, 80.3,
77.2, 72.8, 66.9, 60.8, 60.5, 38.8, 30.6, 26.9, 25.9, 25.2, 18.3, –5.3.
Cbz-Asp(OtBu)-[(3R,4S)-3-oxy-1-(2-(tert-butyldimethylsilyl)ethyl)-4-
((S)-2,2-dimethyl-1,3-dioxolan-4-yl)azetidin-2-one] (8a). Following the
general procedure the title compound was obtained by coupling the HO-
β-lactam 7a (0.71 g, 2.07 mmol) with Cbz-L-aspartic acid-tert-butyl ester
(0.78 g, 2.4 mmol). Overall yield: 1.18 g (88%). IR (, cm‒1, CH2Cl2
solution): 3442, 2984, 2931, 2856, 1752, 1726. 1H-NMR (, ppm, CDCl3):
7.40‒7.29 (5H, m, arom.); 5.77‒5.68 (2H, m, NH[Asp], HαR[β-lact]); 5.13
(2H, s, CH2-Ph); 4.65 (1H, dt, HαS[Asp], J1 = 8.9 Hz, J2 = 4.4 Hz);
4.14‒4.00 (2H, m, CH2[dioxol], CHS[dioxol]); 3.86 (1H, dd, HβS[β-lact], J1
= 8.3 Hz, J2 = 4.9 Hz); 3.78 (2H, dt, CH2-O-Si, J1 = 5.6 Hz, J2 = 1.7 Hz);
3.68‒3.58 (2H, m, N-CH2, CH2[dioxol]); 3.34 (1H, dt, N-CH2, J1 = 13.8 Hz,
J2 = 5.6 Hz); 2.97 (1H, dd, Hβ[Asp], J1 = 17.2 Hz, J2 = 4.4 Hz); 2.79 (1H,
General procedure for the synthesis of β-lactams 7b,e. Deprotection.
The corresponding β-lactam 6a,b (4.32 mmol) was dissolved in methanol
(40 mL) under nitrogen atmosphere. Ammonium formate (1.63 g, 25.9
mmol) and 10% Pd/C (%15) were added and the solution was stirred
under hydrogen atmospheric pressure during one hour. The catalyst was
removed by filtration over celite and the resulting solution was washed
with 0.1 M HCl (2 x 60 mL). The organic layer was dried with MgSO4 and
evaporated to give the product 7a,b.
dd, Hβ[Asp], J1 = 17.2 Hz, J2 = 4.4 Hz); 1.43 (12H, s, Bu, CH3[dioxol]);
t
1.30 (3H, s, CH3[dioxol]); 0.90 (9H, s, Si-tBu); 0.07 (6H, s, CH3-Si-CH3).
13C-NMR (, ppm, CDCl3): 169.7, 163.8, 155.8, 136.1, 128.4, 128.0,
127.8, 109.5, 81.9, 75.7, 75.1, 66.9, 66.4, 60.6, 59.7, 50.4, 43.9, 37.1,
27.8, 26.6, 25.7, 24.8, 18.0, –5.5, ‒5.6.
(3R,4S)-3-Hydroxy-1-[2-(tert-butyldimethylsilyloxy)ethyl]-4-((S)-2,2-
dimethyl-1,3-dioxolan-4-yl)azetidin-2-one (7a). The general procedure
was followed starting with the β-lactam 2a (1.88 g, 4.32 mmol). Yield:
1.43 g (96%). []D25 = ‒4.07 (c = 1, CH2Cl2). IR (, cm‒1, CH2Cl2 solution):
3333, 2984, 2952, 2929, 2884, 2856, 1735. 1H-RMN (, ppm, CDCl3):
4.82 (1H, dd, HαR[β-lact], J1 = 8.5 Hz, J2 = 5.1 Hz); 4.40‒4.30 (1H, m,
Cbz-Asp(OtBu)-[(3R,4S)-3-oxy-1-(3-(tert-butyldimethylsilyloxy)
propyl)-4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)azetidin-2-one]
Following the general procedure the title compound was obtained by
coupling the HO-β-lactam 7b (0.85 g, 2.38 mmol) with Cbz-L-aspartic
acid-tert-butyl ester (0.88 g, 2.73 mmol). Overall yield: 1.39 g (88%). IR
(8b).
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10.1002/cbic.201600642
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(, cm‒1, CH2Cl2 solution): 3324, 2931, 2886, 2857, 1768, 1724. 1H-NMR
(, ppm, CDCl3): 7.45‒7.25 (5H, m, arom.); 5.77‒5.64 (2H, m, NH[Asp],
HαR[β-lact]); 5.13 (2H, m, CH2-Ph); 4.65 (1H, dt, HαS[Asp], J1 = 9.1 Hz, J2
= 4.7 Hz); 4.20‒3.93 (2H, m, CH2[dioxol], CHS[dioxol]); 3.78 (1H, dd,
HβS[β-lact], J1 = 8.6 Hz, J2 = 4.8 Hz); 3.69‒3.48 (4H, m, 2H CH2-O-Si, 1H
N-CH2, 1H CH2[dioxol]); 3.32 (1H, ddd, N-CH2, J1 = 13.9 Hz, J2 = 8.2 Hz,
J3 = 5.9 Hz); 2.95 (1H, dd, Hβ[Asp], J1 = 17.3 Hz, J2 = 4.7 Hz); 2.78 (1H,
dd, Hβ[Asp], J1 = 17.3 Hz, J2 = 4.7 Hz); 1.97‒1.68 (2H, m, CH2CH2CH2);
1.43 (12H, s, tBu, CH3[dioxol]); 1.30 (3H, s, CH3[dioxol]); 0.89 (9H, s, Si-
tBu); 0.04 (6H, s, CH3-Si-CH3). 13C-NMR (, ppm, CDCl3): 170.0, 163.8,
156.0, 136.2, 128.7, 128.4, 128.2, 109.9, 82.4, 76.1, 75.3, 67.3, 66.8,
60.9, 60.4, 50.6, 38.8, 37.4, 30.6, 28.2, 27.0, 26.0, 25.1, 18.4, –5.3.
1.82 mmol). Overall yield: 0.79 g (80%). IR (, cm‒1, CH2Cl2 solution):
3339, 2982, 2934, 1719. 1H-NMR (, ppm, CDCl3): 7.41‒7.29 (5H, m,
arom.); 5.83‒5.68 (2H, m, NH[Asp], HαR[β-lact]); 5.13 (2H, s, CH2-Ph);
4.66 (1H, dt, HαS[Asp], J1 = 8.8 Hz, J2 = 4.3 Hz); 4.35 (1H, d, N-CH2, J =
18.2 Hz); 4.20‒3.94 (4H, m, CH2[dioxol], CHS[dioxol], HβS[β-lact], N-CH2);
3.65 (1H, dd, CH2[dioxol], J1 = 9.1 Hz, J2 = 4.4 Hz); 2.99 (1H, dd, Hβ[Asp],
J1 = 17.5 Hz, J2 = 4.6 Hz); 2.79 (1H, dd, Hβ[Asp], J1 = 17.5 Hz, J2 = 4.6
Hz); 1.43 (9H, s, tBu); 1.39 (3H, s, CH3[dioxol]); 1.28 (3H, s, CH3[dioxol]).
13C-NMR (, ppm, CDCl3): 171.5, 170.1, 169.9, 164.3, 156.1, 136.1,
128.7, 128.3, 128.2, 109.9, 82.5, 75.8, 75.6, 67.4, 66.6, 60.6, 50.5, 43.0,
28.1, 26.8, 25.1.
Cbz-Asp(OtBu)-[(3R,4S)-3-oxy-1-carboxyethylen-4-((S)-2,2-dimethyl-
1,3-dioxolan-4-yl)azetidin-2-one] (9b). The general procedure was
followed starting with the Cbz-Asp(OtBu)-O-β-lactam 8b (1.39 g, 2.09
mmol). Overall yield: 0.84 g (71%). IR (, cm‒1, CH2Cl2 solution): 3327,
3310, 3064, 3034, 2982, 2937, 1719. 1H-NMR (, ppm, CDCl3):
7.42‒7.28 (5H, m, arom.); 5.77 (1H, d, NH[Asp], J = 9.3 Hz); 5.71 (1H, d,
HαR[β-lact], J = 4.8 Hz); 5.13 (2H, s, CH2-Ph); 4.66 (1H, dt, HαS[Asp], J1 =
9.3 Hz, J2 = 4.7 Hz); 4.17‒3.99 (2H, m, CH2[dioxol], CHS[dioxol]); 3.84
Boc-Asp(OtBu)-[(3R,4S)-3-oxy-1-(2-(tert-butyldimethylsilyloxy)ethyl)-
4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)azetidin-2-one] (11a). Following
the general procedure the title compound was obtained by coupling the
HO-β-lactam 7a (0.36 g, 1.04 mmol) with Boc-L-aspartic acid-tert-butyl
ester (0.34 g, 1.19 mmol). Overall yield: 0.58 g (90%). IR (, cm‒1
,
CH2Cl2 solution): 3443, 3346, 2979, 2930, 2857, 1774, 1719. 1H-NMR (,
ppm, CDCl3): 5.72 (1H, d, HαR[β-lact], J = 4.8 Hz); 5.43 (1H, d, NH[Asp],
J = 9.0 Hz); 4.59 (1H, dt, HαS[Asp], J1 = 9.0 Hz, J2 = 4.6 Hz); 4.19‒4.03
(2H, m, CH2[dioxol], CHS[dioxol]); 3.85 (1H, dd, HβS[β-lact], J1 = 8.4 Hz,
J2 = 4.8 Hz); 3.77 (2H, dt, CH2-O-Si, J1 = 5.6 Hz, J2 = 1.4 Hz); 3.68‒3.53
(2H, m, N-CH2, CH2[dioxol]); 3.34 (1H, dt, N-CH2, J1 = 13.9 Hz, J2 = 5.6
Hz); 2.92 (1H, dd, Hβ[Asp], J1 = 17.0 Hz, J2 = 4.6 Hz); 2.76 (1H, dd,
(1H, dd, HβS[β-lact], J1 = 8.8 Hz, J2 = 4.8 Hz); 3.74 (1H, dt, N-CH2, J1
=
14.0 Hz, J2 = 6.9 Hz); 4.67‒3.47 (2H, m, N-CH2, CH2[dioxol]); 2.95 (1H,
dd, Hβ[Asp], J1 = 17.1 Hz, J2 = 4.7 Hz); 2.86‒2.61 (3H, m, Hβ[Asp], CH2-
t
COOH); 1.44 (12H, s, Bu, CH3[dioxol]); 1.31 (3H, s, CH3[dioxol]). 13C-
NMR (, ppm, CDCl3): 175.3, 170.0, 169.9, 164.2, 156.1, 136.2, 128.6,
128.3, 128.1, 110.0, 82.4, 75.8, 75.1, 67.3, 66.6, 64.5, 60.6, 50.6, 37.9,
37.4, 31.9, 28.1, 26.8, 25.0.
t
Hβ[Asp], J1 = 17.0 Hz, J2 = 4.6 Hz); 1.44 (21H, s, 2 x Bu, CH3[dioxol]);
1.31 (3H, s, CH3[dioxol]); 0.88 (9H, s, Si-tBu); 0.05 (6H, s, CH3-Si-CH3).
13C-NMR (, ppm, CDCl3): 170.2, 169.9, 164.0, 155.2, 109.7, 82.0, 80.1,
75.9, 75.1, 66.6, 60.7, 59.8, 50.0, 44.0, 37.4, 28.2, 28.0, 26.8, 25.8, 25.0,
18.1, –5.4, ‒5.5.
Boc-Asp(OtBu)-[(3R,4S)-3-oxo-1-carboxymethylen-4-((S)-2,2-
dimethyl-1,3-dioxolan-4-yl)azetidin-2-one]
(12a).
The
general
procedure was followed starting with the Boc-Asp(OtBu)-O-β-lactam 11a
(0.58 g, 0.94 mmol). Overall yield: 0.36 g (74%). IR (, cm‒1, CH2Cl2
solution): 3338, 2979, 2933, 1760, 1717, 1502. 1H-NMR (, ppm, CDCl3):
5.79 (1H, d, HαR[β-lact], J = 4.7 Hz); 5.47 (1H, d, NH[Asp], J = 10.0 Hz);
4.57 (1H, dt, HαS[Asp], J1 = 10.0 Hz, J2 = 4.9 Hz); 4.33 (1H, d, N-CH2, J =
18.1 Hz); 4.24‒4.15 (1H, m, CH2[dioxol]); 4.12‒3.97 (3H, m, CHS[dioxol],
HβS[β-lact], N-CH2); 3.64 (1H, dd, CH2[dioxol], J1 = 9.0 Hz, J2 = 4.5 Hz);
2.92 (1H, dd, Hβ[Asp], J1 = 17.2 Hz, J2 = 4.9 Hz); 2.75 (1H, dd, Hβ[Asp],
J1 = 17.2 Hz, J2 = 4.9 Hz); 1.44 (21H, s, 2 x tBu, CH3[dioxol]); 1.38 (3H, s,
CH3[dioxol]). 13C-NMR (, ppm, CDCl3): 172.1, 171.5, 170.3, 164.3, 155.5,
109.9, 82.4, 80.6, 75.7, 66.7, 60.6, 50.1, 42.9, 37.6, 28.4, 28.2, 20.2,
26.9, 25.1.
Boc-Asp(OtBu)-[(3R,4S)-3-oxy-1-(3-(tert-butyldimethylsilyloxyl)
propyl)-4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)azetidin-2-one]
(11b).
Following the general procedure the title compound was obtained by
coupling the HO-β-lactam 7b (0.28 g, 0.79 mmol) with Boc-L-aspartic
acid-tert-butyl ester (0.26 g, 0.90 mmol). Overall yield: 0.44 g (88%). IR
(, cm‒1, CH2Cl2 solution): 3433, 3347, 2978, 2953, 2929, 2894, 2856,
1770, 1718. 1H-NMR (, ppm, CDCl3): 5.70 (1H, d, HαR[β-lact], J = 4.8
Hz); 5.43 (1H, d, NH[Asp], J = 9.1 Hz); 4.59 (1H, dt, HαS[Asp], J1 = 9.1 Hz,
J2 = 4.7 Hz); 4.18‒4.03 (2H, m, CH2[dioxol], CHS[dioxol]); 3.78 (1H, dd,
HβS[β-lact], J1 = 8.6 Hz, J2 = 4.8 Hz); 3.69‒3.49 (4H, m, 2H CH2-O-Si, 1H
N-CH2, 1H CH2[dioxol]); 3.32 (1H, ddd, N-CH2, J1 = 14.0 Hz, J2 = 8.2 Hz,
J3 = 5.9 Hz); 2.92 (1H, dd, Hβ[Asp], J1 = 17.0 Hz, J2 = 4.7 Hz); 2.75 (1H,
dd, Hβ[Asp], J1 = 17.0 Hz, J2 = 4.7 Hz); 1.94‒1.74 (2H, m, CH2-CH2-CH2);
1.44 (21H, s, 2 x tBu, CH3[dioxol]); 1.31 (3H, s, CH3[dioxol]); 0.88 (9H, s,
Si-tBu); 0.04 (6H, s, CH3-Si-CH3). 13C-NMR (, ppm, CDCl3): 170.3, 170.0,
163.8, 155.3, 109.8, 82.2, 80.3, 76.1, 75.0, 66.7, 60.8, 60.3, 50.1, 39.6,
37.5, 30.5, 28.3, 28.1, 26.9, 26.1, 25.0, 18.3, –5.3.
Boc-Asp(OtBu)-[(3R,4S)-3-oxo-1-carboxyethylen-4-((S)-2,2-dimethyl-
1,3-dioxolan-4-yl)azetidin-2-one] (12b). The general procedure was
followed starting with the Boc-Asp(OtBu)-O-β-lactam 11b (0.44 g, 0.69
mmol). Overall yield: 0.27 g (74%). IR (, cm‒1, CH2Cl2 solution): 3334,
2979, 2933, 1762, 1718. 1H-NMR (, ppm, CDCl3): 9.79 (1H, s, COOH);
5.70 (1H, d, HαR[β-lact], J = 5.0 Hz); 5.50‒5.36 (1H, m, NH[Asp]);
4.67‒4.52 (1H, m, HαS[Asp]); 4.26‒4.00 (2H, m, CH2[dioxol], CHS[dioxol]);
3.79 (1H, dd, HβS[β-lact], J1 = 8.8 Hz, J2 = 5.0 Hz); 3.75‒3.49 (3H, m, 2H
N-CH2, 1H CH2[dioxol]); 3.03‒2.66 (4H, m, 2H Hβ[Asp], CH2-COOH);
1.46 (21H, s, 2 x tBu, CH3[dioxol]); 1.30 (3H, s, CH3[dioxol]). 13C-NMR (,
ppm, CDCl3): 199.7, 174.7, 170.3, 164.1, 155.5, 110.0, 82.3, 80.5, 75.9,
75.1, 66.7, 60.7, 50.1, 41.7, 37.9, 37.6, 28.4, 28.2, 26.9, 25.1.
General procedure to get de acid 9a,b. Deprotection and oxidation.
Step 1. The desilylation of TBS group was conducted dissolving the
corresponding Asp-β-lactam 8a‒h (3.47 mmol) in THF (17 mL) at 0 °C
and adding hydrogen fluoride pyridine (0.27 mL, 13.88 mmol). The
solution was stirred at room temperature about 3 hours and was
neutralized with a saturated aqueous solution of NaHCO3. The product
was extracted with diethyl ether (3 x 15 mL) and the organic layer was
washed with brine (3 x 10 mL) and dried with MgSO4. The solvent was
eliminated in “vacuo” to afford the terminal alcohol. Step 2. BAIB (2.24 g,
7.26 mmol), TEMPO (0.13 g, 0.82 mmol) and the corresponding alcohol
(3.33 mmol) were added to a mixture of MeCN/water (8/8 mL). The
reaction mixture was stirred for 4 hours. After this time the compound
was extracted several times from the water solution using CH2Cl2 (3 x 15
mL). The organic layer was dried with MgSO4 and evaporated under
reduced pressure.
General procedure for the synthesis of the linear precursors 10a,b
and 13a,b. To a stirred solution of Asp--lactam-(CH2)x-COOH (X = 1,2)
compounds 9a,b (1.26 mmol) cooled at 0 °C in dry CH2Cl2 (60 mL) 17
(1.39 mmol), EDC·HCl (0.38 g, 2.01 mmol), HOBt (0.23 g, 1.76 mmol)
and Et3N (0.37 mL, 2.52 mmol) were added. The mixture was stirred for
20 hours at room temperature. After this time, the reaction was washed
with HCl 0.1 M (2 x 40 mL) and saturated solution of NaHCO3 (2 x 40
mL). The organic layer was dried with MgSO4 and evaporated and the
crude product was purified by flash chromatography MeOH/CH2Cl2 1/25.
Cbz-Asp(OtBu)-[(3R,4S)-3-oxy-1-carboxymethylen-4-((S)-2,2-
dimethyl-1,3-dioxolan-4-yl)azetidin-2-one] (9a). The general procedure
was followed starting with the Cbz-Asp(OtBu)-O-β-lactam 8a (1.18 g,
Cbz-Asp(OtBu)-[(3R,4S)-3-oxy-4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-
1-methylen
carbonyl-azetidin-2-one]-Arg(Pbf)-Gly-OBn
(10a).
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10.1002/cbic.201600642
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Following the general procedure the title compound was obtained by
coupling the Cbz-Asp(OtBu)-O--lactam-CH2-COOH 9a (0.39 g, 0.72
mmol) with H2N-Arg(Pbf)-Gly-OBn 12 (0.45 g, 0.79 mmol). Yield: 0.60 g
(76%). IR (, cm‒1, CH2Cl2 solution): 3443, 3328, 2975, 2932, 1728, 1700,
1547. 1H-NMR (, ppm, CDCl3): 7.63 (1H, t, NH[Gly], J = 5.8 Hz);
7.40‒7.26 (11H, m, arom., NH[Arg]); 6.35‒6.17 (2H, m, NH-(C=NH)-NH);
6.05 (1H, b.s., NH-(C=NH)-NH); 5.86‒5.74 (2H, m, HαR[β-lact], NH[Asp]);
5.20‒5.01 (4H, m, 2 x CH2-Ph); 4.69‒4.53 (2H, m, HαS[Asp], HαS[Arg]);
4.33‒4.13 (2H, m, CHS[dioxol], N-CH2); 4.13‒3.91 (5H, m, HβS[β-lact], 1H
CH2[dioxol], 1H N-CH2, 2H Hα[Gly]); 3.61 (1H, dd, CH2[dioxol], J1 = 9.1
Hz, J2 = 4.6 Hz); 3.32‒3.14 (2H, m, Hδ[Arg]); 2.99‒2.86 (3H, m, CH2[Pbf],
Hβ[Asp]); 2.78 (1H, dd, Hβ[Asp], J1 = 17.3 Hz, J2 = 4.8 Hz); 2.56 (3H, s,
CH3[Pbf]); 2.49 (3H, s, CH3[Pbf]); 2.08 (3H, s, CH3[Pbf]); 2.02‒1.81 (1H,
m, Hβ[Arg]); 1.78‒1.64 (1H, m, Hβ[Arg]); 1.64‒1.51 (2H, m, Hγ[Arg]); 1.45
(6H, s, CH3-C-CH3[Pbf]); 1.41 (9H, s, tBu); 1.40 (3H, s, CH3[dioxol]); 1.36
(3H, s, CH3[dioxol]). 13C-NMR (, ppm, CDCl3): 172.3, 170.1, 170.0,
169.9, 167.2, 165.2, 159.0, 156.6, 156.1, 138.5, 136.2, 135.4, 132.8,
132.4, 128.7, 128.6, 128.4, 128.3, 128.2, 124.8, 117.7, 110.1, 86.6, 82.4,
75.8, 75.6, 67.4, 67.2, 66.7, 61.0, 53.0, 50.6, 45.3, 43.4, 41.4, 40.6, 37.5,
30.0, 29.4, 28.7, 28.2, 26.9, 25.2, 19.4, 18.1, 12.6.
Boc-Asp(OtBu)-[(3R,4S)-3-oxy-4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-
1-ethylencarbonyl-azetidin-2-one]-Arg(Pbf)-OtBu (13b). Following the
general procedure the title compound was obtained by coupling the
commercial NH2-Arg(Pbf)-OtBu (0.13 g, 0.28 mmol) with Boc-Asp(OtBu)-
O--lactam-(CH2)2-COOH 12b (0.13 g, 0.25 mmol). Yield: 0.17 g (67%).
IR (, cm‒1, CH2Cl2 solution): 3443, 3333, 2976, 2932, 1761, 1722, 1662,
1618, 1545. 1H-NMR (, ppm, CDCl3): 6.47‒6.32 (1H, m, NH[Arg]);
6.20‒6.13 (2H, m, NH-(C=NH)-NH); 5.81 (1H, b.s., NH-(C=NH)-NH);
5.67 (1H, d, HαR[β-lact], J = 4.9 Hz); 5.50 (1H, d, NH[Asp], J = 8.8 Hz);
4.57 (1H, dt, HαS[Asp], J1 = 8.8 Hz, J2 = 4.5 Hz); 4.50‒4.41 (1H, m,
HαS[Arg]); 4.20 (1H, dt, CHS[dioxol], J1 = 9.0 Hz, J2 = 4.2 Hz); 4.11 (1H, d,
CH2[dioxol], J1 = 8.8 Hz, J2 = 4.2 Hz); 3.99 (1H, dd, HβS[β-lact], J1 = 9.0
Hz, J2 = 4.9 Hz); 3.78‒3.67 (1H, m, N-CH2); 3.63 (1H, dd, CH2[dioxol], J1
= 8.8 Hz, J2 = 4.2 Hz); 3.60‒3.47 (1H, m, N-CH2); 3.37‒3.12 (2H, m,
Hδ[Arg]); 2.95 (3H, m, CH2[Pbf]); 2.87 (1H, dd, Hβ[Asp], J1 = 17.2 Hz, J2
= 4.5 Hz); 2.73 (1H, dd, Hβ[Asp], J1 = 17.2 Hz, J2 = 4.5 Hz); 2.60 (3H, m,
CH3[Pbf]); 2.57‒2.49 (5H, m, CH3[Pbf], N-CH2-CH2); 2.09 (3H, s,
CH3[Pbf]); 1.94‒1.76 (1H, m, Hβ[Arg]); 1.71‒1.51 (3H, m, 1H Hβ[Arg], 2H
Hγ[Arg]); 1.51‒1.37 (36H, m, CH3-C-CH3[Pbf], 3 x tBu, CH3[dioxol]); 1.34
(3H, s, CH3[dioxol]). 13C-NMR (, ppm, CDCl3): 171.4, 170.6, 170.2,
170.1, 164.9, 158.6, 156.3, 155.4, 138.4, 133.6, 132.4, 124.6, 117.4,
110.1, 86.4, 82.7, 82.4, 80.6, 76.0, 75.1, 66.7, 60.0, 52.1, 50.1, 43.4,
40.5, 39.4, 37.6, 33.8, 29.7, 28.7, 28.4, 28.2, 28.1, 26.9, 25.2, 24.6, 19.4,
18.1, 12.6.
Cbz-Asp(OtBu)-[(3R,4S)-3-oxy-4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-
1-ethylencarbonyl-azetidin-2-one]-Arg(Pbf)-Gly-OBn (10b). Following
the general procedure the title compound was obtained by coupling the
Cbz-Asp(OtBu)-O--lactam-(CH2)2-COOH 9b (0.42 g, 0.74 mmol) with
H2N-Arg(Pbf)-Gly-OBn 12 (0.47 g, 0.81 mmol). Yield: 0.56 g (68%). IR (,
cm‒1, CH2Cl2 solution): 3434, 3319, 2974, 2934, 1757, 1729, 1541. 1H-
NMR (, ppm, CDCl3): 7.63 (1H, t, NH[Gly], J = 5.7 Hz); 7.43‒7.23 (10H,
m, arom.); 7.21‒7.10 (1H, m, NH[Arg]); 6.39‒6.25 (2H, m, NH-(C=NH)-
NH); 6.02 (1H, b.s., NH-(C=NH)-NH); 5.88 (1H, d, NH[Asp], J = 9.2 Hz);
5.68 (1H, d, HαR[β-lact], J = 5.1 Hz); 5.19‒5.01 (4H, m, 2 x CH2-Ph); 4.65
(1H, dt, HαS[Asp], J1 = 9.2 Hz, J2 = 5.0 Hz); 4.59‒4.51 (1H, m, HαS[Arg]);
4.17‒4.09 (1H, m, CHS[dioxol]); 4.09‒3.96 (3H, m, 1H CH2[dioxol], 2H
Hα[Gly]); 3.92 (1H, dd, HβS[β-lact], J1 = 9.0 Hz, J2 = 5.1 Hz); 3.76 (1H, dt,
N-CH2, J1 = 13.6 Hz, J2 = 6.1 Hz); 3.65‒3.58 (1H, m, CH2[dioxol]); 3.50
(1H, dt, N-CH2, J1 = 13.6 Hz, J2 = 6.1 Hz); 3.30‒3.18 (2H, m, Hδ[Arg]);
3.00‒2.83 (3H, m, CH2[Pbf], Hβ[Asp]); 2.77 (1H, dd, Hβ[Asp], J1 = 17.2 Hz,
J2 = 5.0 Hz); 2.67‒2.53 (5H, m, 2H N-CH2-CH2, CH3[Pbf]); 2.50 (3H, s,
CH3[Pbf]); 2.09 (3H, s, CH3[Pbf]); 2.00‒1.86 (1H, m, Hβ[Arg]); 1.75‒1.64
(1H, m, Hβ[Arg]); 1.64‒1.50 (2H, m, Hγ[Arg]); 1.46 (6H, s, CH3-C-
CH3[Pbf]); 1.43 (3H, s, CH3[dioxol]); 1.41 (9H, s, tBu); 1.31 (3H, s,
CH3[dioxol]). 13C-NMR (, ppm, CDCl3): 172.6, 170.9, 170.1, 170.0, 169.9,
164.6, 158.8, 156.6, 156.0, 138.4, 136.1, 135.4, 133.0, 132.3, 128.7,
128.5, 128.4, 128.3, 128.2, 124.8, 117.6, 110.0, 86.5, 82.4, 75.8, 75.2,
67.3, 67.1, 66.6, 60.1, 52.7, 50.5, 43.3, 41.4, 40.5, 39.2, 37.4, 33.7, 29.7,
28.7, 28.1, 26.9, 25.1, 19.4, 18.1, 12.6.
General procedure for the cyclation. Step 1. A suspension of the
linear precursor 10a,b (0.49 mmol) and Pd/C (0.08 g, 15%) in MeOH (or
THF/EtOAc 1/1 v/v) (20 mL) was stirred for 16 hours in a H2 atmosphere,
generated by bubbling gas through the mixture. After this time the
mixture was filtered through celite. The solvent was evaporated in
“vacuo”. Step 2. The intermediate N,C-deprotected pseudopeptide (yield
> 95 %) was immediately dissolved under N2 atmosphere in dry DMF
(180 mL) and the solution was cooled to ‒15 °C. Anhydrous KHCO3 (0.46
g, 4.6 mmol), HOAt (0.10 g, 0.74 mmol) and HATU (0.23 g, 0.60 mmol)
were added at ‒15 °C, and the mixture was stirred for 20 hours at the
same temperature. Evaporation of the solvent under vacuum provided a
crude product which was dissolved in EtOAc (60 mL), and the solution
was washed consecutively with 1 M HCl (40 mL) and 5% aqueous
NaHCO3 (40 mL), dried and evaporated. The crude product was purified
by preparative TLC (EtOAc/acetone 4/6).
Cyclo-{[(3R,4S)-3-oxy-4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-1-
methylencarbonyl-azetidin-2-one]-Arg(Pbf)-Gly-Asp(OtBu)}
(14a).
Following the general procedure the title compound was obtained starting
with the Cbz-Asp(OtBu)-(O-β-lactam-1-methylencarbonyl)-Gly-Arg(Pbf)-
Gly-OBn 10a (0.42 g, 0.38 mmol). Overall yield: 0.20 g (60%). IR (, cm‒1
CH2Cl2 solution): 3440, 3337, 3063, 2971, 2924, 2853, 1763, 1662, 1545.
1H-NMR (, ppm, CDCl3): 7.99‒7.92 (1H, m, NH[Gly]); 7.88 (1H, d,
NH[Asp], J = 9.1 Hz); 7.08 (1H, d, NH[Arg], J = 8.5 Hz); 6.37‒6.24 (2H,
m, NH-(C=NH)-NH); 6.13 (1H, b.s., NH-(C=NH)-NH); 5.85 (1H, d, HαR[β-
lact], J = 4.2 Hz); 4.90 (1H, dt, HαS[Asp], J1 = 9.1 Hz, J2 = 6.1 Hz); 4.54
(1H, dt, HαS[Arg], J1 = 8.5 Hz, J2 = 5.5 Hz); 4.40 (1H, d, N-CH2, J = 15.5
Hz); 4.20‒4.04 (3H, m, CH2[dioxol], Hα[Gly], CHS[dioxol]); 3.88 (1H, dd,
,
Boc-Asp(OtBu)-[(3R,4S)-3-oxy-4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-
1-methylencar bonyl-azetidin-2-one]-Arg(Pbf)-OtBu (13a). Following
the general procedure the title compound was obtained by coupling the
commercial NH2-Arg(Pbf)-OtBu (0.19 g, 0.38 mmol) with Boc-Asp(OtBu)-
O--lactam-CH2-COOH 12a (0.18 g, 0.35 mmol). Yield: 0.26 g (70%). IR
(, cm‒1, CH2Cl2 solution): 3430, 3335, 2978, 2945, 1773, 1719, 1617,
1543. 1H-NMR (, ppm, CDCl3): 6.87 (1H, d, NH[Arg], J = 7.8 Hz);
6.17‒6.00 (2H, m, NH-(C=NH)-NH); 5.87 (1H, b.s., NH-(C=NH)-NH);
5.78 (1H, d, HαR[β-lact], J = 4.9 Hz); 5.51 (1H, d, NH[Asp], J = 8.8 Hz);
4.59 (1H, dt, HαS[Asp], J1 = 8.8 Hz, J2 = 4.6 Hz); 4.45 (1H, dt, HαS[Arg], J1
= 7.8 Hz, J2 = 4.7 Hz); 4.24 (1H, ddd, CHS[dioxol], J1 = 9.2 Hz, J2 = 6.3
Hz, J3 = 4.7 Hz); 4.19‒4.01 (3H, m, 2H N-CH2, CH2[dioxol]); 3.98 (1H, dd,
HβS[β-lact], J1 = 9.2 Hz, J2 = 4.9 Hz); 3.64 (1H, dd, CH2[dioxol], J1 = 9.0
Hz, J2 = 4.7 Hz); 3.28‒3.17 (2H, m, Hδ[Arg]); 2.99‒2.84 (3H, m, CH2[Pbf],
Hβ[Asp]); 2.76 (1H, dd, Hβ[Asp], J1 = 17.0 Hz, J2 = 4.6 Hz); 2.59 (3H, m,
CH3[Pbf]); 2.52 (3H, s, CH3[Pbf]); 2.09 (3H, s, CH3[Pbf]); 1.96‒1.83 (1H,
m, Hβ[Arg]); 1.77‒1.50 (3H, m, 1H Hβ[Arg], 2H Hγ[Arg]); 1.49‒1.42 (33H,
m, CH3-C-CH3[Pbf], 3 x tBu); 1.39 (3H, s, CH3[dioxol]); 1.30 (3H, s,
CH3[dioxol]). 13C-NMR (, ppm, CDCl3): 170.9, 170.1, 170.0, 166.9,
164.9, 158.6, 156.3, 155.3, 138.2, 133.0, 132.2, 124.5, 117.4, 109.8,
86.3, 82.2, 82.0, 80.2, 75.5, 75.5, 66.5, 60.8, 52.6, 50.0, 44.7, 43.2, 40.6,
37.4, 29.5, 28.6, 28.2, 28.0, 27.9, 26.7, 25.2, 25.0, 19.2, 17.9, 12.4.
HβS[β-lact], J1 = 8.5 Hz, J2 = 4.2 Hz); 3.67‒3.42 (3H, m, CH2[dioxol], N-
CH2, Hα[Gly]); 3.43‒3.31 (1H, m, Hδ[Arg]); 3.29‒3.16 (1H, m, Hδ[Arg]);
2.95 (2H, s, CH2[Pbf]); 2.79 (1H, d, Hβ[Asp], J = 6.1 Hz); 2.57 (3H, s,
CH3[Pbf]); 2.49 (3H, s, CH3[Pbf]); 2.08 (3H, s, CH3[Pbf]); 2.01‒1.88 (1H,
m, Hβ[Arg]); 1.86‒1.69 (1H, m, Hβ[Arg]); 1.67‒1.53 (2H, m, Hγ[Arg]); 1.46
(6H, s, CH3-C-CH3[Pbf]); 1.44 (9H, s, tBu); 1.43 (3H, s, CH3[dioxol]); 1.31
(3H, s, CH3[dioxol]). 13C-NMR (, ppm, CDCl3): 172.8, 169.4, 169.2,
169.1, 169.0, 164.9, 159.0, 156.6, 138.5, 132.7, 132.4, 124.8, 117.7,
110.5, 86.6, 82.4, 76.2, 74.3, 66.6, 62.0, 53.7, 48.8, 47.2, 44.7, 43.4,
40.5, 36.7, 29.5, 28.7, 28.2, 26.9, 25.6, 24.9, 19.4, 18.1, 12.6.
Cyclo-{[(3R,4S)-3-oxy-4-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-1-
ethylencarbonyl-azetidin-2-one]-Arg(Pbf)-Gly-Asp(OtBu)}
(14b).
Following the general procedure the title compound was obtained starting
with the Cbz-Asp(OtBu)-(O-β-lactam-1-methylencarbonyl)-Arg(Pbf)-Gly-
OBn 10b (0.39 g, 0.35 mmol). Overall yield: 0.14 g (45%). IR (, cm‒1
,
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10.1002/cbic.201600642
ChemBioChem
FULL PAPER
CH2Cl2 solution): 3564, 3435, 3325, 3075, 2954, 2922, 2852, 1759, 1652,
1546. 1H-NMR (, ppm, CDCl3): 7.97 (1H, d, NH[Asp], J = 8.9 Hz); 7.82
(1H, t, NH[Gly], J = 3.2 Hz); 7.38 (1H, d, NH[Arg], J = 7.6 Hz); 6.44‒6.15
(3H, m, NH-(C=NH)-NH); 5.76 (1H, d, HαR[β-lact], J = 4.7 Hz); 4.80 (1H,
dt, HαS[Asp], J1 = 8.9 Hz, J2 = 5.8 Hz); 4.55 (1H, ddd, HαS[Arg], J1 = 7.6
Hz, J2 = 5.6 Hz, J3 = 4.9 Hz); 4.17‒4.09 (3H, m, CH2[dioxol], Hα[Gly],
CHS[dioxol]); 3.72 (1H, dd, HβS[β-lact], J1 = 8.4 Hz, J2 = 4.7 Hz);
3.69‒3.53 (3H, m, 2H N-CH2, 1H Hα[Gly]); 3.47‒3.39 (1H, m,
CH2[dioxol]); 3.33‒3.09 (3H, m, 2H Hδ[Arg], 1H N-CH2-CH2); 2.94 (2H, s,
CH2[Pbf]); 2.81 (2H, d, Hβ[Asp], J = 5.8 Hz); 2.56 (3H, s, CH3[Pbf]);
2.53‒2.41 (4H, m, CH3[Pbf], N-CH2-CH2); 2.08 (3H, s, CH3[Pbf]);
1.95‒1.79 (1H, m, Hβ[Arg]); 1.77‒1.47 (3H, m, 1H Hβ[Arg], 2H Hγ[Arg]);
1.45 (6H, s, CH3-C-CH3[Pbf]); 1.43 (9H, s, tBu); 1.40 (3H, s, CH3[dioxol]);
1.33 (3H, s, CH3[dioxol]). 13C-NMR (, ppm, CDCl3): 173.0, 171.3, 169.7,
169.6, 169.30, 164.8, 158.9, 156.6, 138.4, 132.9, 132.4, 124.8, 117.6,
110.1, 86.5, 82.2, 76.0, 74.7, 66.3, 61.5, 52.8, 48.9, 44.6, 43.4, 41.0,
40.6, 36.8, 34.2, 29.5, 28.7, 28.2, 26.9, 25.7, 25.2, 19.4, 18.1, 12.6.
Asp-[(3R,4S)-3-oxy-4-((S)-1,2-dihidroxy-ethyl)-1-methylencarbonyl-
azetidin-2-one]-Arg trifluoroacetic salt (16a). Following the general
procedure the title compound was obtained starting with the product 13a
(35 mg, 0.036 mmol). Yield: 14 mg (80%). IR (, cm‒1, solid): 3370‒2939
(b.s.), 1771, 1659, 1416. 1H-NMR (, ppm, D2O): 6.13 (1H, d, HαR[β-lact],
J = 5.0 Hz); 4.60 (1H, t, HαS[Asp], J = 4.6 Hz); 4.42 (1H, dd, HαS[Arg], J1
= 8.6 Hz, J2 = 5.0 Hz); 4.35‒4.22 (2H, m, HβR[β-lact], N-CH2); 4.18 (1H, d,
N-CH2, J = 17.3 Hz); 4.02‒3.91 (1H, m, CHSOH); 3.69 (1H, dd, CH2OH,
J1 = 12.2 Hz, J2 = 3.6 Hz); 3.58 (1H, dd, CH2OH, J1 = 12.2 Hz, J2 = 5.4
Hz); 3.29‒3.17 (3H, m, Hδ[Arg], Hβ[Asp]); 3.09 (1H, dd, Hβ[Asp], J1 = 18.3
Hz, J2 = 4.6 Hz); 2.04‒1.89 (1H, m, Hβ[Arg]); 1.89‒1.74 (1H, m, Hβ[Arg]);
1.74‒1.61 (2H, m, Hγ[Arg]). 1H-RMN (, ppm, D2O/H2O (1/9)): 8.73 (1H, d,
NH[Arg], J = 7.2 Hz); 7.27‒7.21 (1H, m, NH-(C=NH)-NH2); 6.70 (3H, b.s.,
NH-(C=NH)-NH2). 13C-NMR (, ppm, D2O): 175.2, 172.6, 169.6, 167.6,
167.0, 156.5, 74.9, 68.9, 63.4, 60.1, 52.4, 48.9, 44.8, 40.2, 33.4, 27.4,
24.1. HRMS (ESI): m/z calcd for C17H28N6O10 (M + H)+ 477.1945, found
477.1942.
General procedure for the deprotection of the macrocycles 15a,b
and 16a,b. The final deprotection was carried out dissolving the
macrocycle 14a,b or 13a,b (0.035 mmol) in (CF3CO2H/H2O/Et3SiH (1.8
mL/ 0.2 mL/ 0.3 mL) at 4 °C and stirring the mixture at same temperature
during 16 hours. After this time the product was precipitated adding
diisopropylether (10 mL) at 0 °C and centrifuging at 2.2 RCF. The solid
was washed again with diisopropylether (4 x 2 mL) and dried under
Asp-[(3R,4S)-3-oxo-4-((S)-1,2-dihidroxy-ethyl)-1-ethylencarbonyl-
azetidin-2-one]-Arg trifluoroacetic salt (16b). Following the general
procedure the title compound was obtained starting with the product 13b
(35 mg, 0.035 mmol). Yield: 14 mg (80%). IR (, cm‒1, solid): 3359‒2500
(b.s.), 1742, 1654, 1559, 1388. 1H-NMR (, ppm, D2O): 5.96 (1H, d,
HαR[β-lact], J = 4.5 Hz); 4.55 (1H, t, HαS[Asp], J = 4.9 Hz); 4.36 (1H, dd,
HαS[Arg], J1 = 8.7 Hz, J2 = 5.0 Hz); 4.21‒4.18 (1H, m, HβR[β-lact]);
3.98‒3.92 (1H, m, CHSOH); 3.91‒3.82 (1H, m, N-CH2); 3.66 (1H, dd,
CH2OH, J1 = 12.1 Hz, J2 = 3.8 Hz); 3.59 (1H, dd, CH2OH, J1 = 12.1 Hz, J2
= 6.6 Hz); 3.53 (1H, td, N-CH2, J1 = 13.9 Hz, J2 = 5.6 Hz); 3.24 (2H, t,
Hδ[Arg], J = 6.8 Hz); 3.17 (1H, dd, Hβ[Asp], J1 = 18.1 Hz, J2 = 4.9 Hz);
3.05 (1H, dd, Hβ[Asp], J1 = 18.1 Hz, J2 = 4.9 Hz); 2.68 (2H, t, N-CH2-CH2,
J = 6.6 Hz); 2.00‒1.87 (1H, m, Hβ[Arg]); 1.85‒1.73 (1H, m, Hβ[Arg]);
1.73‒1.61 (2H, m, Hγ[Arg]). 1H-RMN (, ppm, D2O/H2O (1/9)): 8.53 (1H, d,
NH[Arg], J = 7.6 Hz); 7.20 (1H, t, NH-(C=NH)-NH2, J = 5.0 Hz); 6.67 (3H,
b.s., NH-(C=NH)-NH2). 13C-NMR (, ppm, D2O): 175.7, 173.7, 173.1,
168.0, 166.4, 156.4, 74.3, 69.0, 63.0, 57.7, 52.5, 49.2, 40.1, 38.7, 34.1,
32.5, 27.5, 24.1. HRMS (ESI): m/z calcd for C18H30N6O10 (M + H)+
491.2101, found 491.2103.
vacuum to provide the corresponding RGD cyclic mimetic as
trifluoracetate salt.
a
Cyclo-{[(3R,4S)-3-oxy-4-((S)-1,2-dihidroxy-ethyl)-1-
methylencarbonyl-azetidin-2-one]-Arg-Gly-Asp} trifluoroacetic salt
(15a). Following the general procedure the title compound was obtained
starting with the macrocycle 14a (30 mg, 0.035 mmol). Yield: 15 mg
(80%). IR (, cm‒1, solid): 3277, 2921, 1735, 1648, 1542. 1H-NMR (,
ppm, D2O): 5.94 (1H, d, HαR[β-lact], J = 4.4 Hz); 4.92‒4.79 (1H, m,
HαS[Asp]); 4.53 (1H, d, N-CH2, J = 15.3 Hz); 4.42 (1H, dd, HαS[Arg], J1
=
8.3 Hz, J2 = 6.5 Hz); 4.16 (1H, dd, HβS[β-lact], J1 = 7.9 Hz, J2 = 4.4 Hz);
4.07 (1H, d, Hα[Gly], J = 14.7 Hz); 3.86 (1H, ddd, CHSOH, J1 = 8.2 Hz, J2
= 6.2 Hz, J3 = 4.0 Hz); 3.79 (1H, d, Hα[Gly], J = 14.7 Hz); 3.70‒3.61 (2H,
m, CH2OH, N-CH2); 3.57 (1H, dd, CH2OH, J1 = 12.1 Hz, J2 = 6.1 Hz);
3.26 (2H, t, Hδ[Arg], J = 6.8 Hz); 3.12 (1H, dd, Hβ[Asp], J1 = 17.3 Hz, J2 =
6.0 Hz); 2.95 (1H, dd, Hβ[Asp], J1 = 17.3 Hz, J2 = 7.3 Hz); 1.99‒1.73 (2H,
m, Hβ[Arg]); 1.73‒1.58 (2H, m, Hγ[Arg]). 1H-RMN (, ppm, D2O/H2O
(1/9)): 8.86 (1H, d, NH[Asp], J = 8.1 Hz); 8.06 (2H, m, NH[Arg], NH[Gly]);
7.23 (1H, t, NH-(C=NH)-NH2, J = 6.1 Hz) 6.79‒6.53 (3H, b.s, NH-
(C=NH)-NH2). 13C-NMR (, ppm, D2O): 174.4, 173.1, 170.6, 170.2, 169.8,
165.7, 156.8, 76.8, 69.7, 62.6, 60.2, 54.2, 49.4, 46.3, 43.0, 40.5, 34.3,
27.4, 24.4. HRMS (ESI): m/z calcd for C19H29N7O10 (M + H)+ 516.2054,
found 516.2051.
NH2-Arg(Pbf)-Gly-Obn (17). Step 1. The commercial Boc-L-Arg(Pbf)-OH
(4 g, 7.6 mmol) was dissolved in dry CH2Cl2 (180 mL) at 0 °C and Et3N (2
mL, 15.2 mmol), EDC·HCl (2.33 g, 12.16 mmol), HOBt (1.44 g, 10.6
mmol) and NH2-Gly-OBn·HCl (1.76 g, 8.4 mmol) was added. The mixture
was stirred at room temperature about 16 hours. After this time, the
reaction was washed with HCl 0.1 M (2 x 80 mL) and saturated solution
of NaHCO3 (2 x 80 mL). The organic layer was dried with MgSO4,
evaporated and the crude product was purified by flash chromatography
(hexane/EtOAc 1/2). Step 2. The deprotection of Boc group was carried
out dissolving the compound in HCOOH (40 mL) at room temperature
during 5 hours getting the desired product. This product was used in the
next step without purification process. Overall yield: 4.23 g (84%). IR (,
cm‒1, CH2Cl2 solution): 3433, 3334, 2972, 1748, 1669, 1616, 1548. 1H-
NMR (, ppm, CDCl3): 8.00 (1H, t, NH[Gly], J = 5.3 Hz); 7.42‒7.21 (5H,
m, arom.); 6.42‒6.22 (3H, m, NH(C=NH)NH); 5.13 (2H, s, CH2-Ph); 4.03
(2H, dd, Hα[Gly], J1 = 5.3 Hz, J2 = 2.7 Hz); 3.51‒3.37 (1H, m, HαS[Arg]);
3.30‒3.12 (2H, m, Hδ[Arg]); 3.00 (2H, s, CH2[Pbf]); 2.60 (3H, s,
CH3[Pbf]); 2.52 (3H, s, CH3[Pbf]); 2.10 (3H, s, CH3[Pbf]); 1.95‒1.74 (2H,
m, Hβ[Arg]); 1.74‒1.53 (2H, m, Hγ[Arg]); 1.48 (6H, s, CH3-C-CH3). 13C-
NMR (, ppm, CDCl3): 176.1, 170.0, 158.7, 156.5, 138.2, 135.3, 133.0,
132.2, 128.6, 128.4, 128.3, 128.2, 124.7, 117.5, 86.4, 67.0, 54.3, 43.2,
41.1, 40.6, 32.0, 28.6, 25.4, 19.2, 17.9, 12.4.
Cyclo-{[(3R,4S)-3-oxo-4-((S)-1,2-dihidroxy-ethyl)-1-ethylencarbonyl-
azetidin-2-one]-Arg-Gly-Asp} trifluoroacetic salt (15b). Following the
general procedure the title compound was obtained starting with the
macrocycle 14b (30 mg, 0.034 mmol). Yield: 15 mg (80%). IR (, cm‒1
,
solid): 3500‒2700, 1743, 1654, 1542. 1H-NMR (, ppm, D2O): 5.91 (1H, d,
HαR[β-lact], J = 4.7 Hz); 4.96‒4.86 (1H, m, HαS[Asp]); 4.60 (1H, dd,
HαS[Arg], J1 = 8.2 Hz, J2 = 6.1 Hz); 4.13 (1H, d, Hα[Gly], J = 14.9 Hz);
4.02 (1H, dd, HβS[β-lact], J1 = 8.9 Hz, J2 = 4.7 Hz); 3.81 (1H, d, Hα[Gly], J
= 14.9 Hz); 3.76‒3.60 (4H, m, 2H N-CH2, CHSOH, CH2OH); 3.55 (1H, dd,
CH2[dioxol], J1 = 12.2 Hz, J2 = 6.0 Hz); 3.30 (2H, t, Hδ[Arg], J = 6.7 Hz);
3.19 (2H, ddd, N-CH2-CH2, J1 = 15.7 Hz, J2 = 12.4 Hz, J3 = 5.1 Hz);
3.09‒2.98 (2H, m, Hβ[Asp]); 2.66 (1H, dt, N-CH2-CH2, J1 = 15.7 Hz, J2 =
3.1 Hz); 2.02‒1.80 (2H, m, Hβ[Arg]); 1.77‒1.60 (2H, m, Hγ[Arg]). 1H-RMN
(, ppm, D2O/H2O (1/9)): 8.70 (1H, d, NH[Asp], J = 8.1 Hz); 8.58 (1H, t,
NH[Gly], J = 8.3 Hz); 8.37 (1H, d, NH[Arg], J = 8.6 Hz); 7.10 (3H, m, NH-
(C=NH)-NH2). 13C-NMR (, ppm, D2O): 175.3, 173.5, 173.4, 167.9, 166.5,
156.8, 74.8, 69.9, 63.3, 58.1, 52.4, 49.2, 40.5, 39.0, 33.7, 32.8, 27.7,
24.4. HRMS (ESI): m/z calcd for C20H31N7O10 (M + H)+ 530.2210, found
530.2210
“In vitro” HUVEC Cell Adhesion Inhibition Test.
Cell lines and reagents Human umbilical endothelial cell (HUVEC) was
purchased from Cambrex BioScience (USA). Cells were grown on the
0.5% gelatin coated plate in CS-C Complete Medium (Sigma) and were
used for experiments after three passages. Human vitronectin was from
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Sigma. Cyclopentapeptides were dissolved in double-distilled water at a
concentration of 10 mg/mL and stored in the dark at –80°C.
Endothelial cell adhesion assay HUVEC cells were grown to
subconfluent state and then harvested by 0.025% trypsin/EDTA (Sigma)
in one minute. Cells were preincubated with various concentrations of
peptides on ice for 15 min in CS-C Medium. Same volume of vehicle was
Acknowledgements
We thank the Ministerio de Economía y Competividad (MEC,
Spain) (Project: IPT-010000-2011-2014), UPV/EHU (UFI
QOSYC 11/22) and Gobierno Vasco/Eusko Jaurlaritza (GAITEK
IG-2011/0000489, GV grant IT-628-13) for financial support and
SGIker UPV/EHU for NMR facilities.
added to cells as
a control. Peptide-treated cells (5 x
104 cells
/100μL/well) were plated onto a 96-well microplate which was precoated
with vitronectin (10 mg/mL in PBS), and incubated for 1h at 37°C, 5%
CO2/95% air to allow cell attachment. Cells were washed gently with PBS
for three times to remove detached cells. Number of adherent cells was
measured by Fluorescent Cell counting Kit (FCCK) at 535 nm (excitation
at 485 nm) using a fluorescent plate reader (Bioscan). Experiments were
done in triplicate wells and repeated four times.
Keywords cyclopeptidomimetics, cyclodepsipeptidomimetics, -
lactams, αvβ3 integrin, CGH array
Gene Regulation Assay
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