Targeting integrins αvβ3 and α5β1 with new β-lactam derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.06.041

The α_vβ₃ and α₅β ₁ integrins are widely expressed in different cancer types and recognize the tripeptide Arg-Gly-Asp (RGD) motif present in several extracellular matrix proteins. We report here the design, synthesis and biological activity of some new β-lactam derivatives specifically designed to target integrins. The new molecules contain the azetidinone as the only cyclic framework armed with carboxylic acid and amine terminals spaced from 9 to 14 atoms to switch on recognition by integrins. All tested molecules showed a concentration-dependent enhancement in fibronectin-mediated adhesion of K562 and SK-MEL-24 cells; in particular 1, expressed a higher affinity towards α₅β₁ integrin (EC₅₀ of 12 nM) and 2 was more selective for integrin α_vβ₃ (EC ₅₀ of 11 nM).

Full text of DOI:10.1016/j.ejmech.2014.06.041

European Journal of Medicinal Chemistry 83 (2014) 284e293
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Targeting integrins avb3 and a5b1 with new b-lactam derivatives
,
Paola Galletti a *, Roberto Soldati a, Matteo Pori a, Margherita Durso a,
Alessandra Tolomelli a, Luca Gentilucci a, Samantha Deianira Dattoli b, Monica Baiula b,
,
, *
Santi Spampinato b *, Daria Giacomini a
a Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy
b Department of Pharmacy and Biotechnology, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The avb3 and a5b1 integrins are widely expressed in different cancer types and recognize the tripeptide
Received 8 November 2013
Received in revised form
17 June 2014
Accepted 18 June 2014
Available online 19 June 2014
Arg-Gly-Asp (RGD) motif present in several extracellular matrix proteins. We report here the design,
synthesis and biological activity of some new
b-lactam derivatives specifically designed to target
integrins. The new molecules contain the azetidinone as the only cyclic framework armed with car-
boxylic acid and amine terminals spaced from 9 to 14 atoms to switch on recognition by integrins. All
tested molecules showed a concentration-dependent enhancement in fibronectin-mediated adhesion of
K562 and SK-MEL-24 cells; in particular 1, expressed a higher affinity towards a5b1 integrin (EC50 of
12 nM) and 2 was more selective for integrin avb3 (EC50 of 11 nM).
Keywords:
Lactams
Integrins
© 2014 Elsevier Masson SAS. All rights reserved.
Azetidinones
Peptidomimetics
Agonists
Cell adhesion
1. Introduction
development, immune responses, leukocyte traffic, haemostasis,
and cancer, their potential as a therapeutic target is now widely
Integrins are heterodimeric
a
/b
transmembrane receptors that
recognized [8].
mediate dynamic adhesive cellecell and cellematrix interactions
[1e4]. Those receptors are able to sense and respond to different
kinds of extracellular cues, including the chemical, physical, and
topographical properties of the cell's microenvironment. The most
critical chemical signal transmitted via integrins is the specific
molecular composition of the extracellular matrix (ECM) [5].
The ability of integrins to bind and associate with various ECM
components such as proteins, transmembrane receptors and solu-
ble ligands largely depends on their structural conformation and
these distinct conformations are crucial for regulating both inside-
out and outside-in signalling [6]. The activation of intracellular
signalling pathways controls cell shape, motility, proliferation,
survival, and cell-type-specific gene expression.
Among the integrin superfamily, avb3 and a5b1 integrins play a
pivotal role in the formation of new blood vessels [9] and are
overexpressed on activated endothelial cells in physiological and
pathological angiogenesis [10]. The avb3 integrin is extensively
expressed on tumour cells. There are some evidences that this re-
ceptor is present in late stage glioblastomas, ovarian carcinoma,
melanomas; it also is an endothelial cell marker in breast cancer
and regulates melanoma cell proliferation, survival and metastases
[11]. The involvement of integrin avb3 in highly important pathol-
ogies induced the development of potential candidates able to
inhibit the functions of this receptor. Commonly, these candidates
are referred as antagonists, because the final physiological effect is
an inhibition of the usual function of avb3, but the mode of action of
these molecules has not been elucidated. This means that they
could be full or partial agonists, inverse agonists or antagonists;
they probably engage the integrin altering its conformation as
response, preventing intramolecular shape change or dragging a
conformational equilibrium towards the inactive form of the re-
ceptor [12].
This capacity of integrins to activate, integrate and distribute
information illustrates the potential of these receptors to serve as
functional distribution hubs in a bi-directional information transfer
[7]. Because of the important roles of integrins and their ligands in
* Corresponding authors.
E-mail addresses: paola.galletti@unibo.it (P. Galletti), santi.spampinato@unibo.it
(S. Spampinato), daria.giacomini@unibo.it (D. Giacomini).
The activation of integrin-mediated cell adhesion by antibodies
or small molecules has been recently reported: Vanderslice et al.
http://dx.doi.org/10.1016/j.ejmech.2014.06.041
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

P. Galletti et al. / European Journal of Medicinal Chemistry 83 (2014) 284e293
285
demonstrated that agonists enhanced the effects of stem cell-based
therapies by improving cell retention and engraftment [13a]; Yea
et al. found that a single agonist antibody against the alpha chains
of integrins can induce human stem cells to become dendritic cells
[13b]. It was thus recognized that integrin agonists could open up
novel opportunities for therapeutics which gain benefits to increase
rather than decrease integrin-dependent adhesion. For instance, a
significant factor in chemoresistance in melanoma is a loss of
integrin-mediated adhesion; in this case, stimulation of integrin
signalling by agonists significantly improved the response to
chemotherapy [14a]. Gupta et al. have reported that small molecule
mediated activation of integrins, rather than inhibition, reduced
leukocyte migration, tissue accumulation and inflammatory injury
[14b].
Fig. 2.
b-Lactams synthesized and tested.
replace the arginine residue. It is interesting to note the insertion of
a
b
-lactam ring as a rigid scaffold in cyclopeptides F and G.
In previous papers, some of us have identified a series of avb3
/
Up to now, a large number of peptidic and nonpeptidic ligands
for the avb3 receptor have been developed, which are all related to
the minimal recognition motif RGD (Arg-Gly-Asp) present on avb3
integrin ligands of the ECM such as fibronectin and vitronectin [11].
As an example, cilengitide (Fig. 1), a small RGD-containing cyclic
pentapeptide, is currently in clinical phase III for glioblastoma
multiform and in phase II for other types of cancers (e.g., prostate
a5b1 integrin ligands with unsaturated
[21b]. It was known that incorporation of a distinct
into a RGD-containing peptide resulted in the stabilization of spe-
cific conformations of the ligand [22]. It was argued that a restricted
conformation introduced by unsaturated
cyclic structure could give a favourable alignment of both the basic
and carboxylate moieties on the ligand, thus meeting the crucial
b
-amino acid fragment
b
-amino acid
b-amino acid and of a
cancer) due to its capability to antagonize avb3, avb5, and a5b1
requirements for integrin affinity and selectivity. The
(azetidinone) constitutes per se a site of conformational restriction
with a -amino acid moiety in a cyclic structure and constrained
features. As a part of our studies on design and synthesis of new
lactam derivatives [23] and of an on-going interdisciplinary project,
we would like to evaluate the ability of azetidinones, not inserted
into cyclopeptides, to target integrins. We report the synthesis and
the preliminary biological results on K562 (human eryth-
roleukemia expressing a5b1 integrin), SK-MEL-24 (human malig-
nant melanoma expressing avb3 integrin), and Jurkat E6.1 human T
b-lactam ring
integrins [15]. Recent research efforts have focused on improving
the pharmacological parameters mainly by altering the polarity and
rigidity of the scaffold and the nature of the basic moiety. Among
several heterocyclic structures able to antagonize integrins, lactam
derivatives found their own niche. In Fig. 1 are reported some lac-
tam derivatives active as integrin ligands.
Some of the ligands include the RGD tripeptide framework (in
red, A [15], C [16], and F [17]), others still hold the carboxylic acid
and guanidine tails B [18] and G [19], whereas lactams D [20] and E
[21a] demonstrated that a phenylamine portion could successfully
b
b
-
cells (expressing a4b1 and aLb2 integrins) cells of some new
b-
Fig. 1. Cilengitide and lactams as integrin ligands, highlighted in red the RGD sequence and in blue the b-lactam ring. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

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P. Galletti et al. / European Journal of Medicinal Chemistry 83 (2014) 284e293
lactam derivatives. The approach for the design of the new mole-
cules was based on rationalization of known ligands structures: we
set out to explore molecules containing the azetidinone as a rigid
cyclic framework, armed with carboxylic acid and amine terminus
spaced from 9 to 14 atoms to activate a conceivable recognition by
integrins (Fig. 2). A 4-amidobenzylamine residue was chosen as the
basic terminus directly linked to the b-lactam nitrogen atom thus
resulting as imido function. The carboxylic acid was on the C-4 side
chain of the azetidinone and differently spaced from the ring: in
compound 1 the beta-lactam ring together with the C4 side chain
could be considered a sort of beta-glutamic acid derivative, which
was elongated with a glycine residue in compound 2, in the aze-
tidinone 3 the carboxylic acid is directly linked to the beta-lactam
ring thus resembling an aspartic cyclic amide. The C-3 position
was not substituted in order to mimic the methylene residue of
glycine in the RGD peptide. Here we report our discovery of aze-
tidinones as new cell adhesion ligands and their characterization as
avb3 and a5b1 agonists.
Scheme 1. Synthesis of compound 1.
2. Results and discussion
2.1. Chemistry
Compounds 1 and 2 were both obtained starting from the
commercially available 4-acetoxy-azetidin-2-one. The introduction
of the carboxylic function in the C-4 side chain was achieved
through a Reformatsky reaction followed by N-acylation of the b-
lactam ring to give compound 1. The C-4 side chain was eventually
converted into a longer peptidic chain through insertion of a
glycine unit to give compound 2 (Fig. 3).
Compound 3 was prepared starting from 4-carboxylic-azetidin-
2-one, easily obtained in enantiomerically pure form by ring-
Scheme 2. Synthesis of compound 2.
closure of an
A careful protecting group strategy for the C-4 and N-1 side
chains of azetidinones was developed to preserve the -lactam ring
L-aspartic acid diester.
b
throughout the synthesis, in particular in the final deprotection
step, and to enable specific combination of temporary or perma-
nent protecting groups to achieve a full or partial deprotection
depending on the synthetic strategy requirements. In Schemes 1e3,
the synthetic steps are described in details.
As outlined in Scheme 1, tert-butylbromoacetate and benzyl-
bromoacetate were treated with an excess of metallic Zn in THF to
furnish the corresponding Reformatsky reagents which were then
coupled with 4-acetoxyazetidin-2-one 4 to give 4-acetate-azetidin-
2-one esters 5 and 6 in good overall yields after purification by flash
column chromatography (Scheme 1).
Scheme 3. Synthesis of compound 3.
The N-1-side chain was constructed starting from 4-
aminobenzylamine 7 selectively protected with a Boc group on
the benzylic amine and then transformed into the corresponding
isocyanate 8 with triphosgene. Treatment of azetidinones 5 and 6
with NaHMDSA (Sodium bis(trimethylsilyl)amide) followed by
addition of isocyanate 8 furnished compound 9 and 10 in good
yields after purification by flash column chromatography. Com-
pound 9 was then deprotected (both C-4 and N-1 side chains) by
treatment with trifluoroacetic acid to give compound 1 as tri-
fluoroacetate salt (Scheme 1).
The benzyl ester of compound 10 was in turn selectively
deprotected to give acid 11 so that a DCC mediated coupling with
benzylglycine 12 could be accomplished to deliver compound 13
(Scheme 2). Compound 13 was subsequently deprotected in a two
steps procedure furnishing 14 by hydrogenolysis and then treating
Fig. 3. Synthetic strategy for compounds 1e3.

P. Galletti et al. / European Journal of Medicinal Chemistry 83 (2014) 284e293
287
with TFA; azetidinone 2 was successfully isolated as trifluoro
acetate salt (Scheme 2).
with an EC50 of 12 nM; it was zfive times less potent vs avb3
integrin. -Lactam 2 was the most effective toward avb3 integrin
b
As to the synthesis of 3, the starting 4-carboxylic-azetidin-2-one
16 was obtained by a Grignard mediated cyclization of L-aspartic
acid dibenzylester 15 (Scheme 3). Enantiomerically pure 16 was
then N-acylated to give 17 and deprotected in two steps with the
same procedures exploited above furnishing 18, and finally, 3 was
isolated as trifluoroacetate salt.
(EC50 ¼ 11 nM) and 3 was z1000 less effective. These cell models
were validated demonstrating that the avb3 and a5b1 agonist
fibronectin produces a concentration-dependent elevation of cell
adhesion in SK-MEL-24 and K562 cells (data not shown). Reference
antagonists were capable to displace cell adhesion in SK-MEL-
24 cell line (Ac-Asp-ArgdLeu-Asp-Ser-OH, IC50 ¼ 25 nM) and in
K562 cell line (cyclo-Arg-Gly-Asp-D-Phe-Val, IC50 ¼ 34.7
mM).
In another set of cell adhesion assays, 1 and 2 were ineffective to
modify Jurkat E6.1 cell adhesion mediated by a4b1- (toward
vascular cell adhesion molecule-1, VCAM-1) and aLb2- (toward
2.2. Pharmacology
The ability of 1, 2 and 3 to modulate the adhesion of K562
(expressing a5b1 integrin) or SK-MEL-24 (expressing avb3 integrin)
cells to immobilized fibronectin (10 mg/mL) was evaluated. These
cell models are widely used to investigate potential ligands capable
of influencing cell adhesion mediated by the above mentioned
integrins [24].
intercellular adhesion molecule-1, ICAM-1) integrin (IC50 ꢀ 100
mM,
data not shown), thus demonstrating selective interactions by the
new beta-lactam ligands.
The ability of the new b-lactams to increase cell adhesion was
then tested in the absence of fibronectin. In a second set of ex-
periments, adhesion of K562 and SK-MEL-24 cells to wells previ-
Interestingly,
b-lactams 1, 2 and 3 showed a concentration-
ously coated by passive adsorption with the most active
and 2, fibronectin as comparison, and BSA as a control, were tested
(Fig. 5). Both -lactams 1 and 2 produced a significant adhesion of
b-lactams 1
dependent enhancement in fibronectin-mediated adhesion of
K562 and SK-MEL-24 cells (Fig. 4). With regards to a5b1 integrin
expressing cells, 1 was the most potent in enhancing cell adhesion
b
Fig. 4. Compounds 1, 2 and 3 enhance fibronectin-mediated adhesion to K562 cells (right column, expressing a5b1 integrin) and SK-MEL-24 cells (left column, expressing avb3
integrin). Concentrationeresponse curves showing the effects of 1, 2 and 3 on cell adhesion are reported. Cells were incubated for 30 min at room temperature with each compound
(B) or with the vehicle (:) as described in the experimental section. Results are expressed as the number of cells attached S.E.M from quadruplicate wells and repeated at least
three times.

288
P. Galletti et al. / European Journal of Medicinal Chemistry 83 (2014) 284e293
that both a5b1 and aIIbb3 integrins may contribute to mediate cell
adhesion induced by compound 1 and 2 in K562 cells.
These data support the hypothesis that the novel
b
-lactam de-
rivatives 1 and 2 possess an interesting activity as avb3 and a5b1
integrin agonists. Compound 1 displays a higher affinity toward
a5b1 integrin whereas 2 is more selective for integrin avb3. Both
integrins are targeted by the RGD sequence [9] and it is conceivable
to presume that these novel compounds could mimic this
sequence. Interestingly, Aizpurua et al. [19,27] have already
described substituted cyclic peptides bearing a
b-lactam moiety
(Fig. 1) that bind to avb3 integrin: the cyclic tetrapeptide with a
deleted glycine residue behaves as an agonist and displays opposite
angiogenic gene-regulation activity of the parent RGD peptidomi-
metic. Agonists have also been described for the b2 family of
integrins [28] and for aMb2 [29]. These latter compounds are
thought to stabilize the high affinity conformation of aMb2 integrin
and may function as anti-inflammatory drugs through a novel
mechanism of action (perturbation of integrin de-adhesion).
Recently, Vanderslice et al. [13], have reported that a small mole-
cule agonist of a4b1 integrin induces progenitor cell adhesion and
may be an adjunct to cell-based therapy.
Until now, small molecules acting as integrin antagonists have
been developed and proposed as novel drugs [8]. However, it would
be advisable to develop novel small molecules that behave as
integrin agonists and increase rather than decrease integrin-
dependent cell adhesion. Further studies will better address the
mechanism of agonism of these ligands and how they may influ-
ence cell signalling.
Fig. 5. K562 and SK-MEL-24 cell adhesion to wells coated with 10 mg/mL of fibronectin
(FN), compound 1, or 2 as described under Materials and Methods. Controls were cells
plated in wells coated with 10 mg/mL bovine serum albumin (BSA). Each value is the
mean S.E.M. from four separate experiments carried out in duplicate. **P < 0.001
compared to BSA-coated wells (NewmaneKeuls test after ANOVA).
K562 and SK-MEL-24 cells, comparable to fibronectin, on the con-
trary, both cell lines did not adhere to wells coated with bovine
serum albumin alone.
Neutralizing antibodies to the b1 or av integrin subunit (10
mL), added to the cells 10 min in advance, blocked the adhesion
mediated by compounds 1 and 2 (10 g/mL) to K562 and SK-MEL-
mg/
m
3. Conclusion
24 cells, respectively (data not shown). This result strengthened the
evidence that the cell adhesion was effectively and specifically
mediated on avb3 and a5b1 integrins by the new beta-lactam
ligands.
Wandzik et al. have reported that the protein kinase C (PKC)
activator PMA (Phorbol myristate acetate) induces megakaryocytic
differentiation of K562 cells upregulating aIIbb3 integrin expression,
Three new b-lactam derivatives targeting integrins have been
designed and synthesized as founders of a new family of molecules.
The azetidinone is the only cyclic framework in these new com-
pounds armed with carboxylic acid and amine terminals spaced
from 9 to 14 atoms to switch on recognition by integrins. The ability
of these new derivatives to modulate the adhesion of specific
integrin expressing cells to immobilized fibronectin have been
which may represent a target of the assayed b-lactams [25]. We
ascertained, by flow cytometry analysis, that K562 cells exposed to
PMA (25 ng/mL for 40 h) express this integrin subtype, in addition
to a5b1 (Fig. 6A). Interestingly, cell adhesion of K562 cells mediated
by compounds 1 and 2 was partially blocked by the selective aIIbb3
antagonist tirofiban (Fig. 6B) [26]. These data seem to be indicative
tested; all molecules showed
enhancement in fibronectin-mediated adhesion of K562 and SK-
MEL-24 cells. In particular, -lactam 1 has a higher affinity towards
a5b1 integrin (EC50 of 12 nM) and -lactam 2 is more selective for
integrin avb3 (EC50 of 11 nM). The novel -lactam derivatives
a
concentration-dependent
b
b
b
Fig. 6. Integrin aIIbb3 partially mediates K562 cells adhesion to compound 1 and 2. A: PMA (25 ng/mL for 40 h) induces upregulation of aIIbb3 integrin expression on K562 cell
surface in comparison to untreated cells (Ctrl). A representative experiment repeated three times with the same result is shown. B: The selective aIIbb3 antagonist tirofiban (TF, 5, 10
or 50
mM) partially blocks K562 cell adhesion to wells coated with compound 1, or 2. Each value is the mean S.E.M. from four separate experiments carried out in duplicate.
*p < 0.05 compared to 2, **p < 0.01 compared to 1 (NewmaneKeuls test after ANOVA).

P. Galletti et al. / European Journal of Medicinal Chemistry 83 (2014) 284e293
289
described here may represent an interesting tools that increase
rather than decrease avb3 and a5b1 integrin-dependent cell
adhesion.
Work is still in progress on developing a library of compounds to
assess a structure-activity analysis which could pick out the
structural conditions to agonize specific classes of integrins.
raised to 30÷35 ꢂC and a solution of benzylbromoacetate (2.46 mL,
15.52 mmol) in THF (19.4 mL) was slowly added in 30 min. After
30 min of stirring the mixture was cooled to rt and decanted,
providing a limpid grey supernatant that was slowly added drop-
wise to a 100 mL flask under nitrogen containing a solution of 4
(500 mg, 3.88 mmol) in anhydrous THF (22 mL) at 0 ꢂC. The mixture
was stirred at rt for 3 h, quenched with ice and a saturated Seig-
nette salt (potassium sodium tartrate) solution and extracted with
AcOEt. The organic layers were dried on Na2SO4, filtered and
concentrated in vacuum. Flash-chromatography (cyclohexane/
AcOEt, 1/1) gave 6 (552 mg) as a white solid in 65% yield.
4. Experimental section
4.1. General information
Commercial reagents were used as received without additional
purification. 1H and 13C NMR spectra were recorded with an INOVA
400 or a GEMINI 200 instrument with a 5 mm probe. All chemical
M.p. 92e95 ꢂC; Rf 0.42 (cyclohexane/tAcOEt, 1/4); 1H NMR
(400 MHz, CDCl3):
d
¼ 2.64 (dd, J ¼ 9.2, 16.8 Hz, 1H, CHHCO2Bn),
2.66 (ddd, J ¼ 1.2, 2.4, 15.2 Hz, 1H, CHHCHCH2CO2Bn), 2.78 (dd,
J ¼ 4.8, 16.8 Hz, 1H, CHHCO2Bn), 3.15 (ddd, J ¼ 2.4, 4.8, 15.2 Hz, 1H,
CHHCHCH2CO2Bn), 3.96 (dddd, J ¼ 2.4, 4.8, 4.8, 9.2 Hz, 1H,
CHHCHCH2CO2Bn), 5.15 (s, 2H, CH2Ph), 6.22 (bs, 1H, NH), 7.34e7.39
shifts are quoted relative to deuterated solvent signals (d in ppm
and J in Hz). Polarimetric Analyses were conducted on Unipol L
1000 “ShcmidteHaensch” Polarimeter at 598 nm. FTIR spectra:
Thermo Nicolet 380 instrument, measured as films between NaCl
plates; wave numbers are reported in cmꢁ1. TLC: Merck 60 F254
plates. Column chromatography: Merck silica gel 200e300 mesh.
HPLCeMS: Agilent Technologies HP1100 instrument, equipped
with a ZOBRAX-Eclipse XDB-C8 Agilent Technologies column;
mobile phase: H2O/CH3CN, 0.4 mL/min, gradient from 30 to 80% of
CH3CN in 8 min,80% of CH3CN until 25 min, coupled with an Agilent
Technologies MSD1100 single-quadrupole mass spectrometer, full
scan mode from m/z ¼ 50 to 2600, scan time 0.1 s in positive ion
mode, ESI spray voltage 4500 V, nitrogen gas 35 psi, drying gas flow
11.5 mL/min, fragmentor voltage 20 V. Elemental analysis were
performed on a Thermo Flash 2000 CHNS/O Analyzer.
(m, 5H, Ph) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 39.8, 43.4, 43.8,
66.8, 128.3, 128.5, 128.7, 135.3, 166.9, 170.7 ppm. IR:
n
¼ 3238, 2961,
1738, 1460, 1372, 1262 cmꢁ1. HPLCeMS: Rt ¼ 4.82 min, m/z ¼ 220
[MþH]þ, 237 [MþH2O]þ, 439 [2MþH]þ.
4.2.3. tert-Butyl 4-isocyanatobenzylcarbamate (8)
In a 50 mL 2-neck flask with a reflux condenser under nitrogen
atmosphere, a solution of (7) (149 mg, 0.67 mmol) and TEA (188 mL,
1.34 mmol) in anhydrous CH2Cl2 (7.0 mL) was cooled to 0 ꢂC and
after was added with bis(trichloromethyl) carbonate (398 mg,
1.34 mmol) in one portion. The mixture was warmed to rt and then
refluxed for 3e4 h. The reaction was monitored through the in-
crease of isocyanate IR signal (2274 cmꢁ1). The solvent was evap-
orated under vacuum avoiding any air exposure. The crude was
extracted five times with anhydrous Et2O under nitrogen, concen-
trated in vacuum, stored under nitrogen atmosphere and imme-
diately used.
4.2. Synthesis
Azetidinone 4 is commercial, compounds 7 [30], 12 [31], and 15
[32] were synthesized following already reported procedures.
4.2.1. tert-Butyl 2-(4-oxo-azetidin-2-yl) acetate (5)
In a 50 mL 3-neck flask under nitrogen, Zn powder (2.0 g,
31 mmol) and THF (10 mL) were introduced followed by TMSCl
4.2.4. tert-Butyl 2-(1-(4-Boc-aminomethyl-phenylcarbamoyl)-4-
oxoazetidin-2-yl) acetate (9)
In a 25 mL 2-neck flask under nitrogen at ꢁ78 ꢂC, a solution of
(200
mL, 1.55 mmol). After 30 min of stirring the temperature was
NaHMSA (Sodium bis(trimethylsilyl)amide) 1.0 M in THF (500 mL,
raised to 30 ÷ 35 ꢂC and a solution of tert-butylbromoacetate
(1.43 mL, 15.5 mmol) in THF (20 mL) was slowly added in 30 min.
After 30 min of stirring the mixture was cooled to rt and decanted,
providing a limpid grey supernatant that was slowly added drop-
wise to a 100 mL flask under nitrogen containing a solution of 4
(500 mg, 3.88 mmol) in anhydrous THF (22 mL) at 0 ꢂC. The mixture
was stirred at rt for 3 h, quenched with ice and a saturated Seig-
nette salt solution and extracted with AcOEt. The organic layers
were dried on Na2SO4, filtered and concentrated in vacuum. Flash-
chromatography (cyclohexane/AcOEt, 6/4) gave 5 (545 mg) as a
white solid in 76% yield.
0.5 mmol) was added dropwise to a solution of 5 (83 mg,
0.45 mmol) in anhydrous THF (4 mL). After 15 min of stirring, a
solution of isocyanate 8 (166 mg, 0.67 mmol) in THF (2 mL) was
added dropwise. After completion (TLC monitoring, 30 min) the
mixture was quenched with a saturated solution of NH4Cl and
extracted with AcOEt and then CH2Cl2. The combined organic ex-
tracts were dried over Na2SO4, concentrated in vacuum and puri-
fied by flash-chromatography (CH2Cl2/Et2O, 95/5) affording 9
(162 mg) as a white solid in 83% yield.
Rf 0.67 (cyclohexane/AcOEt, 2/3); 1H NMR (400 MHz, CDCl3):
d
¼ 1.45 (s, 9H, tBu), 1.46 (s, 9H, tBu), 2.68 (dd, J ¼ 8.8, 16.0 Hz, 1H,
M.p. 83e84 ꢂC; Rf 0.34 (cyclohexane/AcOEt, 1/4); 1H NMR
CHCHHCO2tBu), 2.96 (dd, J ¼ 2.8, 16.0 Hz, 1H, CHCHHCO2tBu), 3.24
(dd, J ¼ 4.0, 16.4 Hz, 1H, CHHCHCHHCO2tBu), 3.35 (dd, J ¼ 5.6,
(400 MHz, CDCl3):
d
¼ 1.49 (s, 9H, tBu), 2.50 (dd, J ¼ 8.8, 16.0 Hz, 1H,
CHCHHCO2tBu), 2.67 (dd, J ¼ 4.8, 16.0 Hz, 1H, CHHCO2tBu), 2.69
(ddd, J ¼ 1.2, 2.4, 14.8 Hz, 1H, CHHCHCH2CO2tBu), 3.19 (ddd, J ¼ 2.4,
4.8, 14.8 Hz, 1H, CHHCHCH2CO2tBu), 3.95 (dddd, J ¼ 2.4, 4.8, 4.8,
8.8 Hz, 1H, CHHCHCH2CO2tBu), 6.40 (bs, 1H, NH) ppm. 13C NMR
16.4 Hz, 1H, CHHCHCHHCO2tBu), 4.28 (d,
J
¼
5.6 Hz, 2H,
CH2NHCO2tBu), 4.39 (m, 1H, CHHCHCHHCO2tBu), 4.80 (bs, 1H, NH),
7.25 (d, J ¼ 8.8 Hz, 2H, arom), 7.43 (d, J ¼ 8.8 Hz, 2H, arom), 8.47 (bs,
1H, NH) ppm. 13C NMR (100 MHz, CDCl3):
d
¼ 28.0, 28.3, 37.9, 42.6,
(100 MHz, CDCl3):
d
¼ 28.0, 41.0, 43.3, 43.9, 81.4, 167.2, 170.1 ppm.
44.2, 47.7, 81.7 (2 ꢃ C), 119.8, 128.2, 134.9, 136.1, 140.3, 147.7, 166.9,
IR:
n
¼
3238, 2964, 1763, 1735, 1398, 1261 cmꢁ1
.
GCeMS:
169.0 ppm. IR:
n
¼ 3337, 2976, 2926, 2852, 1769, 1712, 1603, 1543,
Rt ¼ 13.5 min, m/z (%) ¼ 170 (7), 129 (13), 112 (13), 101 (10), 86 (12),
1367, 1162 cmꢁ1. HPLCeMS: Rt ¼ 9.71 min, m/z ¼ 456 [MþNa]þ, 472
[MþK]þ.
70 (36), 57 (100).
4.2.2. Benzyl 2-(4-oxo-azetidin-2-yl) acetate (6)
In a 50 mL 3-neck flask under nitrogen, Zn powder (2.0 g,
31 mmol) and THF (10 mL) were introduced followed by TMSCl
4.2.5. Benzyl 2-(1-(4-Boc-aminomethyl-phenylcarbamoyl)-4-
oxoazetidin-2-yl) acetate (10)
In a 25 mL 2-neck flask under nitrogen at ꢁ78 ꢂC, a solution of
(200
mL, 1.55 mmol). After 30 min of stirring the temperature was
NaHMSA (Sodium bis(trimethylsilyl)amide) 1.0 M in THF (500 mL,

290
P. Galletti et al. / European Journal of Medicinal Chemistry 83 (2014) 284e293
0.5 mmol) was added dropwise to a solution of 6 (100 mg,
0.45 mmol) in anhydrous THF (4 mL). After 15 min of stirring, a
solution of 8 (166 mg, 0.67 mmol) in THF (2 mL) was added drop-
wise. After completion (TLC monitoring, 30 min) the mixture was
quenched with a saturated solution of NH4Cl and extracted with
AcOEt and then CH2Cl2. The combined organic extracts were dried
over Na2SO4, concentrated in vacuum and purified by flash-
chromatography (CH2Cl2/Et2O, 95/5) affording 10 (187 mg) in 89%
yield as a colourless oil.
4.2.8. Benzyl 2-(2-(1-(4-Boc-aminomethyl-phenylcarbamoyl)-4-
oxoazetidin-2-yl) acetamido) acetate (13)
In
a 25 mL 2-neck flask, dicyclohexylcarbodiimide (DCC)
(45.4 mg, 0.22 mmol) was added to a solution of 11 (51 mg,
0.135 mmol) in anhydrous CH2Cl2 (2.5 mL) and CH3CN (0.5 mL) at
0
ꢂC. Then a previously prepared solution of 12 (106.6 mg,
0.3 mmol) and stoichiometric TEA in CH2Cl2 (2 mL) was immedi-
ately added dropwise, followed by DMAP (4.9 mg, 0.04 mmol). The
solution was warmed to rt and after complete consumption of the
starting material (16 h) the mixture was quenched with H2O
(10 mL) and extract with CH2Cl2 (3 ꢃ 10 mL). The organic layers
were dried on Na2SO4 and filtered. The crude was suspended in
AcOEt, the residual urea by-product of DCC remained solid and was
eliminated by filtration, the organic layer was concentrated in
vacuum and purified by flash-chromatography (CH2Cl2/CH3CN, 90/
10) to afford 13 (45 mg) in 63% yield.
Rf 0.73 (cyclohexane/AcOEt, 2/3); 1H NMR (400 MHz, CDCl3):
d
¼ 1.47 (s, 9H, tBu), 2.80 (dd, J ¼ 8.8, 16.4 Hz, 1H, CHCHHCO2Bn),
2.94 (dd, J ¼ 3.2, 16.4 Hz, 1H, CHCHHCO2Bn), 3.34 (dd, J ¼ 2.8,
16.4 Hz, 1H, CHHCHCH2CO2Bn), 3.35 (dd, J ¼ 4.0, 16.4 Hz, 1H,
CHHCHCH2CO2Bn), 4.27 (d, J ¼ 5.2 Hz, 2H, CH2NHCO2tBu), 4.44
(dddd, J ¼ 2.8, 3.2, 4.0, 8.8 Hz, 1H, CH2CHCH2CO2Bn), 4.88 (bs, 1H,
NHCO2tBu), 5.14 (d, JAB ¼ 12.4 Hz, 1H, PhCHH), 5.18 (d, JAB ¼ 12.4 Hz,
1H, PhCHH), 7.23e7.43 (m, 9H, arom), 8.42 (s, 1H, NCONH) ppm. 13
C
Light yellow waxy solid. Rf 0.75 (AcOEt), 0.79 (CH2Cl2/CH3CN, 1/
NMR (100 MHz, CDCl3):
119.7, 128.1, 128.2, 128.3, 128.5, 134.9, 135.2, 135.9, 147.6, 155.8,
166.5, 169.6 ppm. IR:
¼ 3337, 2976, 1769, 1732, 1709, 1603, 1543,
9.84 min, m/z 412
d
¼ 28.3, 36.8, 42.5, 44.0, 47.4, 66.7, 79.3,
1); 1H NMR (400 MHz, CDCl3):
d
¼ 1.46 (s, 9H, tBu), 2.74 (dd, J ¼ 8.4,
15.2 Hz, 1H, CHCHHCONH), 3.16 (m, 2H, CHCHHCONH,
CHHCHCHHCONH), 3.32 (dd, 5.6, 16.4 Hz, 1-H,
n
J
¼
1244, 1168 cmꢁ1
.
HPLCeMS: Rt
¼
¼
CHHCHCHHCONH), 4.02 (dd, J ¼ 5.2, 18.0 Hz, 1H, NCHHCO2), 4.10
(dd, J ¼ 5.6, 18.4 Hz, 1H, NCHHCO2), 4.27 (d, J ¼ 5.6 Hz, 2H, tBuO-
COCH2N), 4.42 (m, 1-H, CHHCHCHHCONH), 4.85 (bs, 1H,
NHCO2tBu), 5.16 (s, 2H, CH2Ph), 6.50 (t, J ¼ 5.2 Hz, CONH), 7.22e7.42
(m, 9H, arom), 8.51 (s, 1H, NCONH) ppm. 13C NMR (50 MHz,
[MꢁtBuþ2H]þ, 485 [MþH2O]þ.
4.2.6. 4-(2-Carboxymethyl-4-oxoazetidine-1-carboxamido)
phenylmethanaminium 2,2,2-trifluoroacetate (1)
CD3OD):
d
¼ 28.3, 38.1, 41.2, 42.6, 44.1, 48.3, 67.1, 79.5, 120.0, 128.1,
In a 10 mL 2-neck flask under nitrogen at 0 ꢂC, TFA (34
457 mmol) was added dropwise to a solution of 9 (33 mg, 76 m
m
L,
mol)
in anhydrous CH2Cl2 (1.4 mL). The mixture was stirred at rt for 21 h,
cooled at 0 ꢂC and then treated with another aliquot of TFA (68
L,
910 mol). At starting material disappearing, the mixture was
128.2, 128.3, 128.5, 128.6, 135.0, 135.9, 148.2, 155.9, 167.0, 169.2,
169.4 ppm. IR:
n
¼ 3326, 2978, 2921, 1761, 1741, 1708, 1687, 1663,
1601, 1548, 1413, 1319, 1246, 1164, 1119, 1050 cmꢁ1 HPLCeMS:
Rt ¼ 10.51 min, m/z ¼ 525 [MþH]þ, 542 [MþH2O]þ, 547 [MþNa]þ.
m
m
evaporated under vacuum yielding 1 (29 mg) in 97% yield as a light
yellow waxy oil.
4.2.9. 2-(2-(1-(4-Boc-aminomethyl-phenylcarbamoyl)-4-
oxoazetidin-2-yl) acetamido) acetic acid (14)
1H NMR (400 MHz, CD3OD):
d
¼ 2.85 (dd, J ¼ 8.8, 16.4 Hz, 1H,
In a 10 mL 2-neck flask, Pd on C (10%w/w) (3 mg) was added to a
CHCHHCO2H), 3.05 (dd, J ¼ 3.2, 16.4 Hz, 1H, CHCHHCO2H), 3.22 (dd,
J ¼ 3.2, 16.4 Hz, 1H, CHHCHCHHCO2H), 3.40 (dd, J ¼ 5.6, 16.4 Hz, 1H,
CHHCHCHHCO2H), 4.12 (s, 2H, CH2N), 4.45 (m, 1H,
CHHCHCHHCO2H), 7.45 (d, J ¼ 8.8 Hz, 2H, arom), 7.63 (d, J ¼ 8.8 Hz,
solution of 13 (31 mg, 59
mmol) in anhydrous THF (350 mL) and
MeOH (350 L). The mixture was stirred under H2 atmosphere at
m
room temperature (1 atm) and after complete starting material
consumption (2 h) it was filtered and concentrated in vacuum. The
crude was then triturated with a few drops of chloroform to afford
14 after evaporation as a white solid (25 mg) in 98% yield.
2H, arom) ppm. 13C NMR (50 MHz, D2O):
d
¼ 35.9, 41.4, 42.2, 47.4,
115.9 (q, J1CF ¼ 290 Hz), 121.7, 129.0, 129.4, 136.3, 149.1, 162.5 (q,
J2CF ¼ 35 Hz), 168.4, 174.0 ppm. IR: ¼ 3334, 2958, 2921, 1769, 1677,
n
Rf 0.12 (CH3CN/MeOH, 4/1); 1H NMR (400 MHz, CD3OD):
d
¼ 1.48
1610, 1544, 1422, 1335, 1203 cmꢁ1. HPLCeMS: Rt ¼ 1.10 min, m/
(s, 9H, tBu), 2.75 (dd, J ¼ 8.4, 14.8 Hz; 1H, CHCHHCONH), 3.10 (dd,
J ¼ 3.2, 16.0 Hz, 1H, CHHCHCHHCONH), 3.17 (dd, J ¼ 4.4, 14.8 Hz, 1H,
CHCHHCONH), 3.34 (dd, J ¼ 5.6, 16.0 Hz, 1H, CHHCHCHHCONH),
3.91 (d, J ¼ 17.6 Hz, 1H, NCHHCO2), 3.97 (d, J ¼ 17.6 Hz, 1H,
NCHHCO2), 4.21 (s, 2H, CH2NHCO2tBu), 4.45 (m, 1H,
CHHCHCHHCONH), 7.26 (d, J ¼ 8.4 Hz, 2H, arom), 7.46 (d, J ¼ 8.4 Hz,
2H, arom), 8.39 (bs, 1H, NH), 8.77 (s, 1-H, NH) ppm. 13C NMR
z
¼
261 [MꢁTFAꢁNH3þH]þ, 296 [MꢁTFAþH2OþH]þ, 555
[2Mꢁ2TFAþH]þ, 577 [2MꢁTFAþNa]þ. Found C, 46.32; H, 4.21; N,
10.43%; C15H16F3N3O6 requires C, 46.04; H, 4.12; N, 10.74%.
4.2.7. 2-(1-(4-Boc-aminomethyl-phenylcarbamoyl)-4-oxoazetidin-
2-yl) acetic acid (11)
In a 25 mL 2-neck flask a mixture of 10 (100 mg, 0.214 mmol),
anhydrous THF (2.5 mL), MeOH (2.5 mL) and Pd/C (10% w/w)
(10 mg) was stirred under H2 atmosphere (1 atm) at room tem-
perature. At starting material consumption (2 h) the mixture was
filtered and concentrated in vacuum. The desired product 11 was
obtained as a light yellow solid (76 mg) in 94% yield after trituration
with CH2Cl2.
(100 MHz, CD3OD):
129.7, 137.8, 138.3, 150.6, 159.4, 169.4, 173.2, 173.7 ppm. IR:
2974, 2925, 1757, 1712, 1683, 1638, 1609, 1556, 1422, 1319,
1172 cmꢁ1. HPLCeMS: Rt ¼ 2.16 min, m/z ¼ 457 [MþNa]þ, 473
[MþK]þ.
d
¼ 29.6, 39.7, 42.6, 43.9, 45.4, 50.5, 81.0, 122.0,
n
¼ 3314,
4.2.10. 4-(2-(2-(Carboxymethylamino)-2-oxoethyl)-4-
oxoazetidine-1-carboxamido) phenylmethanaminium 2,2,2-
trifluoroacetate (2)
M.p. 162e164 ꢂC, dec.; Rf 0.69 (CH2Cl2/MeOH/NH4OH, 30/10/1);
1H NMR (400 MHz, CDCl3):
d
¼ 1.47 (s, 9H, tBu), 2.81 (dd, J ¼ 8.4,
16.8 Hz, 1H, CHCHHCO2H), 2.98 (dd, J ¼ 3.2, 16.8 Hz, 1H,
CHCHHCO2H), 3.33e3.42 (m, 2H, CH2CHCH2CO2H), 4.28 (m, 2H,
CH2N), 4.46 (m, 1H, CH2CHCH2CO2H), 4.83 (bs, 1H, NHCO2tBu), 7.25
(d, J ¼ 8.4 Hz, 2H, arom), 7.43 (d, J ¼ 8.8 Hz, 2H, arom), 8.46 (s, 1-H,
In a 10 mL 2-neck flask under nitrogen at 0 ꢂC, TFA (12
m
L,
mol)
in anhydrous CH2Cl2 (1.4 mL). The mixture was stirred at rt for 21 h,
cooled at 0 ꢂC and then treated with another aliquot of TFA (16
L,
220 mol). At starting material disappearing, the mixture was
165 mmol) was added dropwise to a solution of 14 (24 mg, 55 m
m
NH) ppm. 13C NMR (50 MHz, CD3OD):
d
¼ 29.6, 38.6, 44.2, 45.4, 46.9,
m
81.0, 122.0, 129.7, 137.8, 138.3, 150.7, 159.4, 169.5, 179.7 ppm. IR:
evaporated under vacuum, the crude was triturated with a drop of
MeOH and pentane to provide 2 as a white solid (16 mg) in 65%
yield.
n
¼ 3338, 2977, 2929, 1767, 1699, 1650, 1539, 1333, 1246, 1168 cmꢁ1
.
HPLCeMS: Rt ¼ 4.77 min, m/z ¼ 395 [MþH2O]þ, 400 [MþNa]þ.

P. Galletti et al. / European Journal of Medicinal Chemistry 83 (2014) 284e293
291
1H NMR (400 MHz, CD3OD):
d
¼ 2.78 (dd, J ¼ 8.4, 14.8 Hz, 1-H,
2H, arom), 7.47 (d, J ¼ 8.4 Hz, 2H, arom), 8.69 (bs, 1H, NH) ppm. 13
C
CHCHHCONH), 3.13 (m, 2-H, CHCHHCONH þ CHHCHCH2CONH),
3.35 (m, 1H, CHHCHCH2CONH), 3.90e3.98 (m, 2H, NHCH2COOH),
4.11 (s, 2H, CH2NHþ3 ), 4.46 (m, 1H, CH2CHCH2CONH), 7.44 (d,
J ¼ 8.8 Hz, 2H, arom), 7.61 (d, J ¼ 8.8 Hz, 2H, arom), 8.90 (s, 1H, NH)
NMR (50 MHz, CD3OD):
d
¼ 29.6, 42.9, 45.4, 51.1, 81.1, 122.2, 129.7,
138.0, 138.1, 149.8, 159.4, 167.9, 173.6 ppm. IR:
n
¼ 3342, 2978, 2929,
1773, 1704, 1667, 1605, 1540, 1417, 1319,1242, 1164 cmꢁ1. HPLCeMS:
Rt ¼ 2.03 min, m/z ¼ 280 [MꢁCO2tBuþH2O]ꢁ.
ppm. 13C NMR (100 MHz, CD3OD):
d
¼ 39.6, 42.6, 43.9, 44.7, 50.5,
116.8 (q, J1CF ¼ 286 Hz), 122.2, 130.8, 131.7, 140.5, 150.5, 162.3 (q),
169.4, 173.2, 173.7 ppm. IR:
¼ 3387, 2949, 2921, 2839, 1772, 1707,
HPLCeMS:
4.2.14. (S)-(2-carboxy-4-oxoazetidine-1-carboxamido)
n
phenylmethanaminium 2,2,2-trifluoroacetate (3)
1678, 1654, 1621, 1560, 1458, 1417, 1020 cmꢁ1
.
In a 10 mL 2-neck flask under nitrogen at 0 ꢂC a, TFA (13
mL,
Rt ¼ 1.13 min, m/z ¼ 318 [MꢁTFAꢁNH3þH]þ, 357 [MꢁTFAþNa]þ,
373 [MꢁTFAþK]þ, 669 [2Mꢁ2TFAþH]þ, 691 [2Mꢁ2TFAþNa]þ.
Found C, 45.86; H, 4.34; N, 12.38%; C17H19F3N4O7 requires C, 45.54;
H, 4.27; N, 12.50%.
165 mmol) was added dropwise to a solution of 18 (24 mg, 66 m
mol)
in anhydrous CH2Cl2 (1.2 mL). After stirring at rt for 15 h, the
mixture was again cooled to 0 ꢂC and treated with another aliquot
of TFA (20
mL, 265 mmol). After starting material complete dis-
appearing (24 h in all), the crude was evaporated under vacuum
and triturated with a drop of CH2Cl2 and pentane to provide 3 as a
white solid (22 mg) in 88% yield.
4.2.11. (S)-Benzyl 4-oxoazetidine-2-carboxylate (16)
Commercially available, it has been prepared starting from
dibenzylester of
ported in Ref. [28] M.p.137e140 ꢂC. Rf 0.6 (cyclohexane/AcOEt, 1/4);
1H NMR (400 MHz, CDCl3):
L-aspartic acid 15 according to the procedure re-
[a
]
¼ ꢁ41.8 (c ¼ 0.25, CH2Cl2); 1H NMR (200 MHz, CD3OD):
D
d
¼ 3.15 (dd, J ¼ 3.0, 16.2 Hz, 1-H, CHHCH), 3.54 (dd, J ¼ 6.4, 16.2 Hz,
d
¼ 3.05e3.11 (m, 1H, CHCHH), 3.33 (dd,
1H, CHHCH), 4.11 (s, 2H, CH2NHþ3 ), 4.58 (dd, J ¼ 3.0, 6.4 Hz, 1H,
J ¼ 6.0, 14.8 Hz, 1H, CHCHH), 4.22 (dd, J ¼ 2.8, 6.0 Hz, 1H, CHCHH),
CHHCH), 7.45 (d, J ¼ 8.4 Hz, 2H, arom), 7.63 (d, J ¼ 8.4 Hz, 2H, arom)
5.21 (s, 2H, CH2Ph), 6.32 (bs, 1H, NH), 7.36e7.38 (m, 5H, Ph) ppm.
ppm. 13C NMR (100 MHz, CD3OD):
d
¼ 43.0, 44.7, 51.3, 116.8 (q),
13C NMR (100 MHz, CDCl3):
d
¼ 43.5, 47.3, 67.4, 128.5, 128.6, 128.7,
122.4, 131.0, 131.7, 140.3, 149.7, 163.7 (q), 167.9, 173.6 ppm. IR:
¼ 3411, 2954, 2921, 2851, 1777, 1691, 1625, 1552, 1462, 1323,
134.8, 166.3, 170.8 ppm. HPLCeMS: Rt ¼ 4.29 min, m/z ¼ 206
n
[MþH]þ, 223 [MþH2O]þ, 228 [MþNa]þ, 433 [2MþNa]þ.
1204 cmꢁ1
.
HPLCeMS: Rt
¼
1.07 min, m/z
¼
247
[MꢁTFAꢁNH3þH]þ, 527 [2Mꢁ2TFAþH]þ, 549 [2Mꢁ2TFAþNa]þ.
Found C, 44.78; H, 3.95; N, 10.88%; C14H14F3N3O6 requires C, 44.57;
H, 3.74; N, 11.14%.
4.2.12. (S)-Benzyl 1-(4-Boc-aminomethyl-phenylcarbamoyl)-4-
oxoazetidine-2-carboxylate (17)
In a 10 mL 2-neck flask under inert atmosphere at ꢁ78 ꢂC,
NaHMSA (Sodium bis(trimethylsilyl)amide) 1.0 M in THF (190
mL,
4.3. Pharmacology
0.190 mmol) was added dropwise to a solution of 16 (35 mg,
0.17 mmol) in anhydrous THF (1.5 mL) followed after 30 min by a
solution of isocyanate 8 (64 mg, 0.26 mmol) in THF (1 mL); after
starting material disappearing (30 min), the mixture was quenched
with a saturated solution of NH4Cl and extracted with AcOEt and
CH2Cl2. The organic extracts were dried over Na2SO4, concentrated
in vacuum and purified by flash-chromatography (CH2Cl2, then
CH2Cl2/Et2O 95:5) to afford 17 as a colourless viscous oil (69 mg) in
90% yield.
4.3.1. Cell culture
SK-MEL-24 cells (expressing avb3 integrin) were from American
Type Culture Collection, ATCC, Rockville, MD, USA and were
routinely grown in minimum essential medium (MEM, Cambrex,
Walkersville, MD, USA) supplemented with 10% foetal bovine
serum (FBS; Life technologies, Carlsbad, CA, USA), non-essential
aminoacids and sodium pyruvate.
K562 cells (expressing a5b1 integrin) and Jurkat E6.1 human T
cells (expressing a4b1 and aLb2 integrins) were also from ATCC; they
were maintained as a stationary suspension culture in RPMI-1640
and glutamine with 10% FBS. Cells were kept at 37 ꢂC under a 5%
CO2 humidified atmosphere; 40 h prior to experiments, K562 cells
were treated with 25 ng/mL PMA (Phorbol 12-myristate 13-acetate,
SigmaeAldrich SRL, Milan, Italy) to induce differentiation and to
increase expression of integrin cell surface [33].
Rf 0.6 (cyclohexane/AcOEt, 4/1); [
a
]
¼ ꢁ64.1 (c ¼ 0.7, CH2Cl2);
D
1H NMR (400 MHz, CDCl3):
d
¼ 1.47 (s, 9-H, tBu), 3.10 (dd, J ¼ 2.8,
16.0 Hz, 1H, CHHCH), 3.40 (dd, J ¼ 6.0, 16.0 Hz, 1H, CHHCH), 4.28 (d,
J ¼ 5.2 Hz, 2H, CH2NH), 4.60 (dd, J ¼ 2.8, 6.4 Hz, 1H, CHHCH), 4.85
(bs, 1H, NHCO2tBu), 5.24 (d, JAB ¼ 12.4 Hz, 1H, PhCHH), 5.29 (d,
JAB ¼ 12.4 Hz, 1H, PhCHH), 7.25 (d, J ¼ 8.4 Hz, 2H, arom), 7.35e7.38
(m, 5H, arom), 7.43 (d, J ¼ 8.4 Hz, 2H, arom), 8.28 (s, 1H, NH) ppm.
13C NMR (100 MHz, CDCl3):
d
¼ 28.3, 41.2, 44.1, 48.9, 67.8, 79.4,
119.9, 128.2, 128.3, 128.6, 128.7, 134.7, 135.1, 135.8, 146.6, 155.8,
4.3.2. Cell adhesion assays
165.0, 168.8. IR:
n
¼ 3424, 3342, 3060, 2974, 2921, 1777, 1744, 1708,
The assay was performed as described in Refs. [24, 34]. Briefly,
for adhesion assay on SK-MEL-24 or K562 cells 96-well plates
(Corning Costar, Celbio, Milan, Italy) were coated by passive
1683, 1601 cmꢁ1. HPLCeMS: Rt ¼ 11.14 min, m/z ¼ 471 [MþH2O]þ,
476 [MþNa]þ.
adsorption with fibronectin (10 m
g/mL) overnight at 4 ꢂC. Cells were
4.2.13. (S)-1-(4-Boc-aminomethyl-phenylcarbamoyl)-4-
oxoazetidine-2-carboxylic acid (18)
counted with a haemocytometer and pre-incubated with various
concentrations of each compound or with the vehicle (water) for
30 min at room temperature to reach ligandereceptor equilibrium.
At the end of the incubation time, the cells were plated
(50,000 cells per well) and incubated at room temperature for 1 h
in fibronectin-coated plates. All the wells were then washed with
PBS (phosphate-buffered saline) to remove non-adherent cells, and
50 mL hexosaminidase [4-nitrophenyl-N-acetyl-b-d-glucosaminide
dissolved at a concentration of 7.5 mM in 0.09 M citrate buffer (pH
5) and mixed with an equal volume of 0.5% Triton X-100 in H2O]
In a 10 mL 2-neck flask, Pd/C10%w/w (3 mg) was added to a
solution of 17 (33 mg, 73
mmol) in anhydrous THF (400 mL) and
MeOH (400 L). The mixture was stirred under H2 atmosphere
m
(1 atm) at room temperature and after starting material complete
consumption (2 h), it was filtered and concentrated in vacuum. The
crude was then triturated with a few drops of CH2Cl2 and pentane
to afford the desired product as a white lumpy solid (25 mg) in 95%
yield.
Rf 0.03 (cyclohexane/AcOEt, 1/4); [
a
]
¼ ꢁ51.5 (c ¼ 0.4, CH3OH);
was added. This product is a chromogenic substrate for b-N-ace-
tylglucosaminidase, whereby it is transformed into 4-nitrophenol;
absorbance was measured at 405 nm. The reaction was blocked by
D
1H NMR (200 MHz, CD3OD):
d
¼ 1,48 (s, 9-H, tBu), 3.13 (dd, J ¼ 3.2,
16.0 Hz, 1-H CHHCH), 3.52 (dd, J ¼ 6.2, 16.0 Hz, 1H, CHHCH), 4.21 (s,
2H, CH2N), 4.56 (dd, J ¼ 3.2, 6.2 Hz, 1H, CHHCH), 7.27 (d, J ¼ 8.4 Hz,
the addition of 100 mL of a stopping solution [50 mM glycine and

292
P. Galletti et al. / European Journal of Medicinal Chemistry 83 (2014) 284e293
5 mM EDTA (pH 10.4)], and the plates were read in a Victor2 Mul-
tilabel Counter (PerkinElmer, Waltham, MA, USA). Reference an-
tagonists (Ac-Asp-Arg-Leu-Asp-Ser-OH and cyclo-Arg-Gly-Asp-D-
Phe-Val) were purchased from Bachem. For adhesion assay on
Jurkat E6.1 cells black 96-well plates were coated overnight at 4 ꢂC
c) M.A. Arnaout, B. Mahalingam, J.P. Xiong, Integrin structure, allostery, and
bidirectional signaling, Annu. Rev. Cell Dev. Biol. 21 (2005) 381e410.
[5] J.D. Humphries, A. Byron, M.J. Humphries, Integrin ligands at a glance, J. Cell
Sci. 119 (2006) 3901e3903.
[6] a) F. Ye, C. Kim, M.H. Ginsberg, Molecular mechanism of inside-out integrin
regulation, J. Thromb. Haemost. 9 (Suppl. 1) (2011) 20e25;
b) B.-H. Luo, C.V. Carman, T.A. Springer, Structural basis of integrin regulation
and signaling, Ann. Rev. Immunol. 25 (2007) 619e647;
with VCAM-1 or ICAM-1 (both 5
mg/mL). Jurkat E6.1 cells were
c) J. Takagi, T.A. Springer, Integrin activation and structural rearrangement,
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