Could Dissecting the Molecular Framework of β-Lactam Integrin Ligands Enhance Selectivity?

Article abstract of DOI:10.1021/acs.jmedchem.9b01000

By dissecting the structure of β-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins α_vβ₃, α₅β₁, and α₄β₁. New selective ligands with a

Full text of DOI:10.1021/acs.jmedchem.9b01000

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Article
Could dissecting the molecular framework of #-
lactam integrin ligands enhance selectivity?
Giulia Martelli, Monica Baiula, Alberto Caligiana, Paola Galletti, Luca
Gentilucci, Roberto Artali, Santi Mario Spampinato, and Daria Giacomini
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01000 • Publication Date (Web): 31 Oct 2019
Downloaded from pubs.acs.org on November 8, 2019
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Page 1 of 13
Journal of Medicinal Chemistry
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Could dissecting the molecular framework of β-lactam
integrin ligands enhance selectivity?
Giulia Martelli,†≠ Monica Baiula,‡≠ Alberto Caligiana,‡ Paola Galletti,† Luca Gentilucci,† Roberto
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Artali,& Santi Spampinato, *‡ Daria Giacomini, *†
† Department of Chemistry "G. Ciamician", University of Bologna, Via Selmi 2, 40126 Bologna, Italy.
‡ Department of Pharmacy and Biotechnology, University of Bologna, Via Irnerio, 48, 40126, Bologna, Italy
&
Scientia Advice, 20832, Desio, Monza and Brianza, Italy
KEYWORDS Lactams, integrins, cell adhesion, agonist, antagonist, azetidinones, peptidomimetics.
ABSTRACT: By dissecting the structure of -lactam-based ligands, a new series of compounds was designed, synthesized, and
evaluated toward integrin αvβ3 α5β1, and α4β1. New selective ligands with antagonist or agonist activities of cell adhesion in the
nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells, as
a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseo-
integration and bone regeneration. Molecular modeling studies on prototypic compounds and v3 or 51 integrins supported that
ligand carboxylate fixing to the MIDAS in the -subunit can be sufficient for binding the receptors, while the aryl side chains play a
role in determining the selectivity as well as agonism vs antagonism.
H
N
COOH
COOH
COOH
INTRODUCTION
N
O
NH
N
H
N
Integrins are membrane receptors that play an important
role in the regulation of fundamental functions such as
development, cell adhesion, and migration.1,2 They also
participate in platelet adhesion3 and in the immune response,
considering their presence in leukocyte cells by modulating
specific intracellular signaling pathways.4 Integrins are also
implicated in the progression and metastasis of certain
tumors.5,6 Activation of integrins can occur by binding
extracellular ligands (outside-in signaling) or intracellular
activation of integrin tails (inside-out signaling). The
interaction of integrins with extracellular ligands could enable
or preclude cellular signaling that controls cell processes such
as cell shape, growth, and differentiation.7 Ligand binding may
influence integrin conformation from a low inactive state to
higher active states.8,9 Several studies have examined integrin
ligands acting as antagonists that block the interaction between
integrin and its endogenous ligands, such as antibodies,
peptides, or small organic molecules.10 Preclinical studies
suggest that several integrin antagonists might be useful to
suppress tumor angiogenesis and growth either alone, or in
combination with current cancer therapeutics.11 Less attention
has been paid to the ligands that activate integrins for the
possible activation of angiogenesis and tumor growth.
However, it has been recently recognized that integrin agonists
could lead to benefits by increasing integrin-dependent cell
adhesion rather than inhibiting it.12,13
O
O
O
NH
O
O
A
v3
40.9 ± 0.8
antagonist
C
41
B
51
9.9 ± 0.1
agonist
12.9 ± 0.6
agonist
O
COOH
O
COOH
COOH
N
H
H
H
N
N
N
N
N
O
O
D
O
NH
CF3COO
NH3
O
O
E
v3
F
41
1.39 ± 0.04
antagonist
51
active ligand
365.00 ± 0.05
agonist
Chart 1. -Lactam compounds previously reported (A-F, ref. 15)
and selected for the design of the new molecules.
We identified selective and potent ligands able to modulate
differently cell signaling pathways: some molecules acted as
agonists, hence promoting cell adhesion and intracellular
signaling activation, while others were antagonists inhibiting
integrin-dependent cell functions. Despite the selective
binding and valuable potency of some of the reported
molecules, it is relevant to recognize the structural
requirements to address the selectivity and the
agonist/antagonist behavior of new integrin ligands.
Our previous studies provided a novel series of -lactam-
based molecules that were designed to target different
integrins, mainly RGD-binding and leukocyte-integrins
(selected molecules A-F Chart 1).14, 15
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Journal of Medicinal Chemistry
Thus, a series of new ligands was designed and obtained
Page 2 of 13
1
2
3
4
5
6
7
8
by dissecting the structures of some compounds that were
previously synthesized (Chart 2).
Scheme 1. Synthesis of compounds 1-4, and 6a
OAc
ref.17
b
H
COOH
3
1
N
NH
NH
COOH
42%
COOH
O
O
O
N
O
N
O
NH
NH
O
O
O
COOBn
N
COOBn
O
3
1
H
4
2
c
a
C4 conformational
restriction
and lack of N-substituent
lack of N-substituent
6
NH
N
O
lack of COOH
lack of urea
91%
86%
O
O
9
12
COOH
COOH
NH
COOH
NH
COOH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
O
HN
O
HN
N
OAc
COOBn
O
COOBn
a
NH
ref.14
c
O
4
O
NH
N
NH
13
6
7
8
5
77%
99%
O
O
O
14
lack of urea
lack of imide
lack of beta lactam ring
O
and ring espansion
O
ref.14
COOH
H
N
COOBn
COOH
COOH
N
N
H
COOH
d
91%
c
N
O
N
O
2
NH
NH
O
O
O
NH
NH
NH
91%
O
O
15
O
16
O
9
10
11
CF3COO
NH3
a
4(R) configuration
4(R) configuration
4(R) configuration
Reagents and conditions: a) o-toluoylchloride, TEA, DMAP,
CH2Cl2, 0 °C then rt, 18 h; b) o-tolylisocyanate, NaHMDSA,
THF, -78 °C, 1 h; c) H2, Pd/C (10%), THF/CH3OH 1:1, rt, 2 h; d)
DCC, TEA, DMAP, glycine benzylester∙HCl, CH2Cl2, 0 °C to rt,
16 h.
Chart 2. Structure-based design of new molecules (1-11). The
arrows indicate the modified positions.
Scheme 2. Synthesis of compounds 5, 7, and 8a
The novel compounds were deprived of some functional
groups or destructured from their cyclic scaffold compared to
those already reported. The new series of molecules was tested
toward integrins αvβ3, α5β1, and α4β1, the three main receptors
targeted by the previously reported -lactam ligands through
cell adhesion assays, competitive solid-phase binding assays,
and integrin-mediated intracellular signaling analysis. Some
derivatives were very active ligands in the nanomolar range
despite the extent of chemical manipulation.
COOBn
COOH
COOBn
b
a
5
N
O
NBoc
ref.16
NH
17
99%
72%
NH
18
COOBn
COOBn
a
b
7
HN
O
97%
68%
NH2.HCl
NH
19
COOBn
RESULTS
COOBn
a
b
8
HN
O
NH3
PTSA
76%
99%
Synthesis of new compounds. The synthetic strategies
adopted for the new -lactams are reported in Schemes 1-3.
The synthesis of derivatives 1, 2 and 4 share the commercially
available 4-acetoxyazetidin-2-one as the starting material
(Scheme 1). Compound 1, characterized by a double bond
directly linked to the ring,16 was obtained as previously
described.17
NH
20
a Reagents and conditions: a) o-tolylisocyanate, TEA, CH2Cl2, rt,
4 h; b) H2, Pd/C 10%, THF/ CH3OH 1:1, rt, 2 h.
Compound 15 was obtained by hydrogenolysis of 13 and
further coupling with glycine benzylester to achieve 16. The
benzyl ester was finally removed to give compound 2. Starting
from the benzyl ester of the commercially available 4-
carboxylic-azetidin-2-one, intermediate 12 was obtained by
acylation with o-toluoylchloride, and subsequent ester
deprotection by hydrogenolysis to give carboxylic acid 6
(Scheme 1).
Compound 3 was obtained starting from the commercially
available 2-azetidinone by o-tolylisocyanate acylation at low
temperature (Scheme 1). Compound 17 was obtained from N-
Boc-L-proline following a previously reported procedure:18
acylation with o-tolylisocyanate gave 18 and
a final
hydrogenolysis quantitatively yielded target compound 5
(Scheme 2). Linear derivatives 7 and 8 were obtained similarly
starting from glycine and -alanine
2
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Journal of Medicinal Chemistry
Table 1. Effects of -lactam compounds 1-11 on RGD-binding- and leukocyte-integrin-mediated cell adhesion. Data are presented
as EC50 for agonists, and as IC50 for antagonists (nM).a,b
1
2
3
4
comp.
number
SK-MEL-24/ FN
K562/FN
Jurkat/ VCAM-1
comp.
number
SK-MEL-24/FN
K562/FN
Jurkat/VCAM-1
αvβ3
α5β1
α4β1
αvβ3
α5β1
α4β1
5
6
7
8
1
75.6 ± 7.8
agonist
>5000
255 ± 26
agonist
10
11
>5000
>5000
96.9 ± 2.3
antagonist
>5000
>5000
2
3
4
>5000
>5000
>5000
1110 ± 111
antagonist
>5000
>5000
9
>5000
976 ± 54
agonist
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
419 ± 31
antagonist
>5000
A
40.9 ± 0.8
antagonist
1031 ± 35
agonist
> 5000
> 5000
5
6
7
8
9
2880 ± 69
agonist
140 ± 15
agonist
10.0 ± 2.1
agonist
B
C
D
E
F
> 5000
> 5000
9.9 ± 0.1
agonist
>5000
41.5 ± 2.3
antagonist
>5000
>5000
>5000
>5000
> 5000
12.9 ± 0.6
agonist
84.8 ± 4.5
agonist
3512 ± 48
antagonist
352 ± 7
158 ± 4
1.39 ± 0.04
antagonist
antagonist
antagonist
active ligand
> 5000
29.9 ± 5.9
agonist
>5000
>5000
>5000
>5000
>5000
365.00 ± 0.05
agonist
> 5000
aCell adhesion mediated by v3 was measured by assaying SK-MEL-24 cells adhesion to FN; by 51 assaying K562 cell adhesion
to FN; by 41 evaluating Jurkat cell adhesion to VCAM-1. bValues represent the mean ± SD, n=3.
benzyl esters through the acylated intermediates 19 and 20,
respectively, and their subsequent hydrogenolysis (Scheme 2).
Scheme 3. Synthesis of compounds 9, 10, and 11a
Azetidinone benzyl ester 21 was obtained from D-aspartic acid
in a two-step procedure: formation of the di-benzylester19 and
O
cyclization with tert-butylmagnesium chloride20 (Scheme 3).
COOBn
ref.17,18
HO
OH
-Lactam 21 was then exploited as a starting material for the
NH
O
O
NH2
D--lactam derivatives 9-11. Acylation of 21 with o-
tolylisocyanate gave 22, and subsequent hydrogenolysis
yielded 9; then, coupling with -alanine benzylester gave 23,
followed by a final hydrogenolysis to obtain 10 (Scheme 3).
Finally, intermediate 24 was obtained from 21 by
condensation with tert-butyl(4-isocyanatobenzylcarbamate) 26
prepared in situ with triphosgene, TEA and 4-
21
O
COOBn
H
N
COOBn
N
a
c
H
a
N
9
N
10
H
97%
O
50%
O
23
97%
N
22
O
b
O
85%
60%
21
aminobenzylamine.14, Hydrogenolysis of 24 gave acid 25
and the final deprotection of the N-Boc group yielded 11
(Scheme 3).
17
d
COOBn
NH
COOH
NH
a
e
N
N
11
Pharmacology. The ability of the new ligands to modulate
cell adhesion was assayed using different cell lines: SK-MEL-
24 (expressing αvβ3),21 K562 (mainly expressing α5β1),22 and
Jurkat E6.1 (expressing α4β1)15. The results of the cell adhesion
assays with compounds 1-11 are summarized in Table 1.
Ligands that inhibit the cell adhesion promoted by fibronectin
(FN) or VCAM-1 are referred to as antagonists whereas
compounds that increase the cell adhesion are defined
agonists. For comparison purposes, cell adhesion data of
selected -lactam ligands A-F previously studied15 have been
added to Table 1. Further characterization to evaluate the
integrin affinity of the most interesting compounds in cell
adhesion assays was performed with a solid-phase competitive
O
O
88%
94%
O
O
24
25
NHBoc
NHBoc
a Reagents and conditions: a) H2, Pd/C (10%), THF/CH3OH 1:1,
rt, h; b) o-tolylisocyanate, TEA, CH2Cl2, rt, h; c)
2
4
oxalylchloride, TEA, CH2Cl2, -alanine benzylester∙PTSA,
DMAP, rt, 16 h; d) NaHMDSA, tert-butyl 4-
isocyanatobenzylcarbamate 26, THF, -78 °C, 1 h; e) TFA,
CH2Cl2, 0 °C then rt, 6 h.
3
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Page 4 of 13
integrin binding assay for the integrins αvβ3 and α5β1 and with
nM), although they displayed a lower potency than those
obtained in SK-MEL-24 cells.
a scintillation proximity-binding assay (SPA) for α4β115 (Table
1
2
3
4
5
6
7
8
9
2).
Table 2. IC50 values (nM)a of compounds 1, 4-8, and 10 on
αvβ3, α5β1, and α4β1 integrins.
Proline derivative 5 showed the most potent agonist
activity toward α4β1 (EC50 = 10.0 nM) and displayed an
excellent affinity for this integrin as measured by SPA (Table
2). In addition, compound 5, which was previously coated to
the wells by passive absorption, slightly increased Jurkat cell
adhesion compared to VCAM-1 (Figure 1A). Neutralizing
antibodies to the α4 integrin subunit (10 µg/mL) reduced the
cell adhesion mediated by compound 5 (10 µg/mL) to Jurkat
cells (data not shown). Regarding integrin α5β1, compounds 6
and 10 behaved as selective antagonists (IC50 of 41.5 and 96.9
nM, respectively). In contrast, compound 5 favored α5β1-
mediated cell adhesion with an EC50 in the submicromolar
range, and this result was confirmed by the affinity of
compound 5 for this integrin (Table 2). Compounds 1, 7, and 8
increased cell adhesion mediated by integrin αvβ3 with
excellent potency (EC50 75.6, 84.8, and 29.9 nM,
Compd.
αvβ3
29.0 ± 2.6
>1000
α5β1
α4β1
294 ± 28
>1000
1
4
>1000
409 ± 33
136 ± 16
59.3 ± 7.1
>1000
5
>1000
18.9 ± 2.1
>1000
6
>1000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
>1000
3.4 ± 0.8
9.5 ± 1.9
>1000
8
>1000
>1000
10
>1000
11.9 ± 2.5
a
IC50 for αvβ3, α5β1 was evaluated by competitive solid-
phase binding assay to FN. IC50 for α4β1 was determined by
SPA. Values are the mean ± SEM (n=3).
respectively), whereas
Furthermore, 7 and 8 emerged as selective ligands for the αvβ3
integrin.
5 behaved as a weak agonist.
Moreover, 1, 7 and 8, which had been previously coated to
the wells by passive absorption, induced significant adhesion
of SK-MEL-24 and Saos-2 cells in the absence of FN (Figure
1 B and C, respectively).
50000
40000
30000
20000
10000
0
50000
40000
30000
20000
10000
0
A 40000
B
C
***
***
***
30000
The addition of a neutralizing antibody to the αv subunit
added to SK-MEL-24 or Saos-2 cells 10 min prior to the
addition of compounds 1, 7 and 8 (10 µg/mL) strongly
reduced the adhesion (data not shown), indicating that these
compounds specifically mediate the process through αvβ3
integrins.
***
***
***
20000
10000
0
**
***
***
***
1
-
5
1
7
8
1
7
8
A
A
N
F
A
S
B
N
F
S
S
M
B
B
A
C
V
To further characterize the most effective compounds
evaluated in the integrin-mediated cell adhesion assays,
intracellular signaling activation was investigated.
Figure 1. Jurkat (A), SK-MEL-24 (B) and Saos-2 (C) cell
adhesion to wells coated with VCAM-1, FN, the most effective
agonists (1, 5, 7 and 8) or BSA as controls. Values as mean ±
SEM from three independent experiments carried out in
quadruplicate. **p<0.01; ***p<0.001 vs to BSA-coated wells
(Newman−Keuls test after ANOVA).
Extracellular signal-regulated kinase 1 and 2 (ERK1/2)
phosphorylation was quantified as evidence of the intracellular
signal derived by integrin-extracellular matrix component
interactions.15 As ERK1/2 signaling pathway is impaired in
SK-MEL-24 cells,26 we transfected HEK293 cells, which do
not express αvβ3 integrin,27-29 with plasmids coding for αv and
β3 subunits (HEK293+ αvβ3) in order to study αvβ3-mediated
ERK1/2 phosphorylation.
The most active compounds 1, 4-8, and 10 were further
evaluated by solid-phase competitive binding assays for αvβ3
and α5β1 integrins, or with SPA for α4β1 (Table 2). The affinity
data are in complete agreement with the data obtained in the
adhesion tests, and confirmed that compounds 7 and 8 were
the best and most selective ligands for αvβ3, compound 10 was
the best and most selective for α5β1, and compound 5 was the
best ligand for α4β1 but not selective.
Positive controls for signaling activation were FN for αvβ3
and α5β1 and VCAM-1 for α4β1; as expected both endogenous
agonists were able to induce ERK1/2 phosphorylation when
compared with vehicle-treated cells or with not transfected
HEK293 cells (adopted as negative control for HEK293+αvβ3
cells) (Figure 2).
To better characterize the αvβ3 agonists 1, 7 and 8, we
investigated their ability to modulate adhesion of Saos-2 cells,
a human osteoblast-like cell expressing αvβ3 and α5β1
integrins, considered a valuable in vitro model to study
osteoblast adhesion and osseointegration of implants in
dentistry and orthopedics.23-25
A significant increase in ERK1/2 phosphorylation was
induced by compounds 1, 7, and 8 in HEK+αvβ3 cells (Figure
2A); however the compounds were not able to activate
ERK1/2 signaling in not transfected HEK293 cells (data not
shown). Regarding integrin α4β1, compound 5 confirmed its
agonist effect by increasing the integrin-mediated intracellular
signaling activation in a concentration-dependent manner in
Jurkat cells (Figure 2B). In contrast, antagonist compound 6,
active towards α5β1, prevented FN-induced ERK1/2
phosphorylation in K562 cells (Figure 2C). On the basis of
these data, we can conclude that compounds acting as agonists
increase integrin-mediated cell adhesion, promote integrin
The use of αvβ3 and α5β1 integrin agonists has been
described as a feasible and powerful strategy to improve
osteoblast adhesion because it can mimic the extracellular
matrix of the bone.23-25 -Lactams 1, 7 and 8 were able to
increase Saos-2 cell adhesion to FN in the nanomolar range (1
EC50= 154 ± 35 nM; 7 EC50=299 ± 54 nM; 8 EC50 = 984 ± 72
4
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Journal of Medicinal Chemistry
conformation,31 and 3VI4 for 51 integrin in its bent-inactive
mediated-intracellular signaling, mimicking the behavior of
endogenous agonists, like fibronectin or VCAM-1.
conformation.32 The ligand-receptor structures were obtained
by a systematic conformer search, followed by geometry
optimization. The best-scoring poses are shown in Figure 3
and Figure S1 and S2 (Supporting Information).
1
2
3
4
5
6
7
8
7
8
1
f
f
A
s
s
n
a
r
n
e
l
c
i
a
r
t
e
l
t
t
c
i
FN 10-7M 10-8
M
10-9
M
t
h
h
o
n
o
e
e
FN 10-7
M
10-8
M
10-9
M
10-7
M
10-8
M
10-9
M
n
v
v
 44 kDa
 42 kDa
pERK1/2
totERK1/2
 44 kDa
 42 kDa
Figure 3A shows the carboxylate group of compound 7
closely coordinated to the MIDAS of  subunit, as expected.
The ligand shows its urea core in a S-shaped cis,trans
configuration (Figure 3A), which allows to fit the cavity
delimited by the residues Tyr122, Arg214, Asn215, Asp217,
Ala218, Glu220, Ser121, Ser123 in the  subunit. The
receptor keeps in place the carboxylate also by means of three
hydrogen bonds, with Tyr122NH (2.09 Å), with Ser123NH
(1.55 Å), and with Ser123OH (1.92 Å). In addition, Asn215
contributes to the stabilization of the complex with a hydrogen
bond between its carbonyl oxygen and ligand urea NH (2.88
Å). The o-tolyl group occupies a hydrophobic cavity delimited
by the residues Tyr166, Arg216, Arg214 of the -subunit
(Figure 3A), and by Tyr178 of the -subunit. Obviously, this
short ligand occupies a much smaller portion of the RGD-
recognition site of the receptor compared to RGD itself
(Figure S1).
1000
800
600
400
200
0
600
400
200
0
***
***
***
**
**
***
**
**
**
*
*
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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f
f
e
l
e
l
c
N
s
7
8
9
7
8
9
N
M
M
M
M
M
M
s
7
8
9
-
0
M
M
M
-
-
-
-
-
-
-
-
c
i
n
F
n
a
F
i
a
h
0
0
0
0
0
0
h
0
0
r
r
t
t
e
1
1
1
1
1
1
e
v
1
1
1
v
n
n
o
o
N
N
7
8
1
5
FN+6
B
C
h
1
e
l
-
M
e
l
c
i
c
i
A
h
10-7
M
10-8
M
10-9
M
10-7
M
10-8
M M
10-9
e
e
C
FN
v
v
V
 44 kDa
 42 kDa
pERK1/2
totERK1/2
 44 kDa
 42 kDa
200
150
100
50
200
150
100
50
***
*
**
*
#
#
The best pose of the ligand 6 involves only residues
belonging to the 1 subunit (Figure 3B and Figure S2 in the
Supporting Information). The -lactam core is framed within a
cavity delimited by the residues Ser227, Glu229, Ser134,
Tyr133, Asn224. Besides the expected ionic bond between
ligand carboxylate and MIDAS (Figure 3B), the carboxylate is
stabilized by three hydrogen bonds with the residues
Ser134NH (2.99 Å), Asn224NH (1.51 Å), and Asn215CONH
(1.60 Å). Finally, the carbonyl oxygen at the C2 position of
the -lactam is hydrogen-bonded to Tyr133NH (2.67 Å). The
0
0
e
l
e
1
-
N
l
M
M
M
9
M
M
M
9
7
8
7
8
c
i
-
-
-
c
i
-
-
-
F
M
h
h
0
0
0
1
0
0
0
A
C
e
e
1
1
1
1
1
v
v
V
5
FN+6
Figure 2. Concentration dependent effects of new β-lactam
ligands on integrin-mediated intracellular signaling activation. (A)
Compounds 8, 1, and 7 increased ERK1/2 phosphorylation in
HEK293+αvβ3 cells; (B) compound 5 activated intracellular
signaling in Jurkat cells expressing α4β1; (C) compound 6
prevented FN-induced ERK1/2 phosphorylation in K562 cells
expressing α5β1. Not transf: not transfected HEK293 cells.
Densitometric analysis of the bands is reported (mean ± SEM;
n=5). *p<0.05, **p<0.01, ***p<0.001 vs vehicle; #p<0.05 vs FN
(Newman-Keuls test after ANOVA).
o-tolyl group appears well inserted in
a wide cavity
establishing interactions with defined residues of the
1 subunit, i.e. Gly223, Pro186, Cys187, Cys187, and the
phenol ring of Tyr133.
DISCUSSION AND CONCLUSIONS
Conversely, ligands acting as antagonists bind specifically
to integrin under investigation, reduce cell adhesion and
prevent the activation of integrin-mediated intracellular
signaling by endogenous agonists.
To discuss how the structural changes modified the
biological activities, the new compounds were compared with
the previously reported -lactams A-F (Chart 1 and 2, and
Figure S3).
Molecular Modeling. It is apparent that the large majority
of the bioactive compounds in Chart 2 interacts with RGD-
binding integrins with nM affinity albeit showing atypical
structures as compared to the classic peptide or
peptidomimetic ligands. Hence, we performed Molecular
Modeling studies to explore the binding modes of
representative compounds into integrins whose X-ray
structures have been disclosed, namely integrins v and
51. For its high affinity and selectivity, and for the atypical
structure lacking the lactam ring, compound 7 was chosen as a
model agonist ligand for v integrin, and the very small -
lactam 6, lacking the urea group, was selected as a model
antagonist for 51 integrin.
Compound 1 showed to increase the adhesion of both
model cell lines for αvβ3 and α4β1 integrins (Table 1 and Figure
1). Competitive binding assays or SPA using isolated
receptors confirmed that this compound binds to both integrins
with moderate affinity (Table 2). Due to the very small size, it
seems plausible that this ligand could interact only with the -
subunits, thanks to the ionic bond between its carboxylate
group and the MIDAS. The dual efficacy of 1 would be the
result of the minimal structure of
1 lacking of any
pharmacophores to specifically address the 3- or 1-subunits.
Compared to compound A, the constraint due to the C=C bond
on the ring in 1 had no effect on the affinity for αvβ3 but it
reversed the functional activity, from antagonism of A to
agonism of 1. It is possible that the less flexible ligand 1 could
establish better non-covalent interactions with the receptor in
Docking simulations were performed with Autodock 4.030
using the receptor models derived from the deposited X-ray
structures, i.e. 4MMX for v integrin in its extended-active
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the coordination of the Mg++ held in the metal-ion dependent
adhesion site (MIDAS) of the 3 subunit.
1
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6
7
8
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11
12
13
Figure 3. Side views of the calculated binding poses: A) compound 7 into the v3 integrin (pdb code 4MMX); B) compound 6
into the 51 integrin (pdb code 3VI4). The ligands are rendered in sticks, the protein backbone is represented as a solid ribbon, the
 subunit being highlighted in yellow. The relevant receptor residues are rendered in thick lines and indicated by the one-letter
code. Dashed green lines represent hydrogen bonds, cation- interactions are rendered in brown,  interactions in pink,
hydrophobic interactions in white. Divalent cations are shown as gray spheres.
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It could be due to a greater directionality because of the
conformational restriction along with the increased acidity of
the ,-unsaturated acid compared to the saturated carboxylic
acid in compound A. Both these effects could cooperate in the
interaction of this small molecule with the 3 subunit thus
promoting an active open-extended integrin conformation
leading to the agonism of 1.19 As previously demonstrated, in
fact, there is an autonomous regulation of integrin
conformations by the 3 subunit that suggests its major role in
integrin activation.33 It could be also observed that the lack of
a N-substituent as in compounds 1 and A compared to C, leads
to a shift in integrin selectivity from α4β1 to αvβ3, instead. The
significance of the two structural elements, COOH and N-
substituent, was confirmed by the behavior of compound 3:
the lack of the COOH group completely deactivated the ligand
towards αvβ3 and a poor residual activity for α4β1 remained due
to the N-o-tolyl-group. However, the presence of a COOH is
not still sufficient for integrin recognition, as demonstrated by
the inactivity of compound 2 that has a longer C4 side chain
than A. A shorter distance between the C4 carboxylate and the
-lactam could favor a cooperative interaction with the very
close ADMIDAS metal site of the 3 subunit thus enforcing
the integrin recognition,34 not possible in a longer chain as in
compound 2.
αvβ3, but it maintained the activity toward α5β1 and α4β1.
Interestingly in this case there is a complete switch in the
ligand behavior: from antagonism of the -lactam D to
agonism of proline derivative 5.
The complete absence of a cyclic scaffold, such as in
compounds 7 and 8, had a strong effect on increasing the
agonist selectivity toward αvβ3 compared to the corresponding
cyclic ligands D and C, which were more active or only active
toward α4β1, respectively. In particular, the linear compound 8
and -lactam C turned out to be both agonists but with a
switch in selectivity from α4β1 for C to αvβ3 for 8. This
selectivity of C for α4β1 could be tentatively attributed to
favorable interactions with the ligand-binding domain  of
the α4β1 integrin,36-37 which is lost in linear compound 8. As a
general consideration, a linear molecule could be useful to
influence the selectivity, but the cyclic -lactam analog has a
higher potency, probably because of better side chain
alignment on the receptor due to the cyclic core.
The configuration at the C-4 position of the -lactam ring
turned to be very important. A change from the (S)
configuration in compounds D and F, to the (R) configuration
in compounds 9 and 11 switched off the activity. This
stereopreference in integrin recognition is consistent with
results that were recently reported.38 Compound 10 with an (R)
C-4 configuration interestingly was a selective antagonist of
α5β1, whereas its (S) enantiomer E was a selective ligand for
αvβ3. In general, it could be observed that (S)-enantiomers are
preferred for receptor recognition, but (R) enantiomers should
be carefully evaluated.38
A lack of the ureidic NH group as in compound 4 and 6
leads to a deactivation toward α4β1, compared with C and D
that are good α4β1 ligands. In contrast, 4 and especially 6 are
selective antagonist toward α5β1. The selectivity switch from
α4β1 to α5β1 could be attributed to more favorable interactions
with the 5 subunit due to an increased basicity of the imide
group compared to the urea, and with a hydrogen bond
acceptor character. The absence of the classical guanidinium
group, as in the RGD sequence, would not be crucial to gain
recognition by α5β1 or αvβ3, as demonstrated by small non-
peptidic molecules without the guanidinium group previously
developed as selective ligands of integrin α5β1.35
Molecular docking computations suggested useful hints to
explain the preference of compounds 7 and 6 towards integrins
v3 and 51, respectively. In 51 integrin, the MIDAS is
topped by a large hydrophobic pocket capable to host the o-
tolyl group of -lactam 6 (Figure 3B). In contrast, in the v3
integrin this hydrophobic cavity is considerably narrowed for
the presence of large residues, especially Arg214, Asn215, and
Arg216,39 thus preventing ligand 6 to fit the receptor.
The proline derivative 5 with a larger ring than that of -
lactam D and without the imide group was a poor ligand for
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On the other hand, ligand 7, but not ligand 6, is capable to atmosphere. Anhydrous TEA was added dropwise, followed
1
2
3
4
5
6
7
8
skirt these obstacles and to insert its o-tolyl moiety into the
large pocket delimited by hydrophobic residues of the - and
-subunits (Figure 3A), thanks to the comparatively longer
and more flexible S-shaped urea backbone.
by a dropwise addition of the commercially available o-
tolylisocyanate. The mixture was stirred at room temperature
until a complete consumption of the starting beta-lactam (4h,
TLC monitoring) and then quenched with a saturated aqueous
solution of NH4Cl. The mixture was then extracted with
CH2Cl2 (3 x 10mL), the organic layers were collected, dried
over anhydrous Na2SO4, concentrated in vacuum and purified
by flash-chromatography affording the desired N-acylated
compounds.
Furthermore, compound 7 was shown to behave as an
agonist of v3 integrin, despite of the lack of relevant
interactions with the v-subunit. The docking pose clearly
showed that this ligand occupies only a small fraction of the
RGD-binding site (Figure S1). Previously, small molecules,
such as the thyroid hormone,40 were shown to bind integrin
αvβ3 mainly by interacting with the -subunit. More recent
evidence strongly supported that ligand carboxylate binding to
the MIDAS can be sufficient to open the headpiece, and hence
to activate the integrin, while Arg (or any mimetics) might not
be required.34
9
General procedure for hydrogenolysis (GP2). In a 25
mL two-neck flask the starting benzyl ester (1 equiv) was
dissolved in a 1:1 mixture of THF and CH3OH (22 mL/mmol)
and Pd/C (10% w/w) was added. The solution was then stirred
under a H2 atmosphere (1 atm) at room temperature. After a
complete consumption of the starting material (TLC
monitoring, 2h) the reaction mixture was filtered through
celite and concentrated in vacuum. The crude was then
triturated with few drops of pentane to afford the desired
carboxylic acids.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Intriguingly, ligand 6 was shown to act as a pure
antagonist of 51 integrin. Extensive investigations on IIb3
and v3 integrins showed that the mechanism of extension
and activation requires a specific reorganization of pre-
existing interaction networks around Y122 of the -subunit, in
the proximity of the ligand recognition site.41, 31
2-(4-Oxoazetidin-2-yl)acetyl)glycine (2). Following GP2,
compound 16 (68 mg, 0.25 mmol) yielded compound 2 as a
1
waxy solid (42 mg, 91%). H NMR (400 MHz, CD3OD) δ
In this perspective, the peculiar structure of compound 6
appears perfectly adequate to block the position of Tyr122 in
the inactive conformation. Antagonism would be determined
by the presence of the bulky, o-methylbenzoyl aromatic group
that optimally packs against Tyr122, therefore freezing hinge
opening and domain traslocation.31, 42
(ppm) 3.98 – 3.94 (m, 1H), 3.93 (d, J = 18.0, 1H), 3.89 (d, J =
18.0 Hz, 1H), 3.10 (dd, J = 15.0, 4.9 Hz, 1H), 2.69 (dd, J =
15.0, 1.9 Hz, 1H), 2.66 – 2.55 (m, 2H); 13C NMR (100 MHz,
CD3OD) δ (ppm) 173.23, 173.21, 170.4, 45.9, 43.6, 42.1, 41.8;
IR (film, cm-1) 3316, 2927, 1730, 1636, 1534, 1476, 1414,
1379, 1193.
In summary, this study contributes to
a
better
2-Oxo- -( -tolyl)azetidine-1-carboxamide (3). In a 25
comprehension of the structural requirements relevant to
confer agonist or antagonist behavior toward integrins, and,
moreover, new potent and selective integrin agonists were
discovered. In recent decades, many efforts have been made to
develop integrin antagonists, whereas integrin agonists have
been considered as potential therapeutics only very recently.
Agonists for αvβ3 could be useful for functionalizing titanium
surfaces to foster bone regeneration on implant materials.22 In
addition, agonists for α4β1 have been proposed to enhance
stem cell therapy when coadministered with progenitor cells
by increasing cell adhesion,43 or enhance vascularization in
regenerative medicine.44
mL
two-neck
flask,
a
solution
of
sodium
bis(trimethylsilyl)amide(NaHMDSA) (1.0 M in THF, 875 μL,
1.25 equiv) was added dropwise to a solution of 2-azetidinone
(50 mg, 0.7 mmol, 1 equiv) in anhydrous THF (6.2 mL) at −78
°C under a nitrogen atmosphere. The mixture was stirred for
15 min, then o-tolylisocyanate (108 μL, 0.875 mmol, 1.25
equiv) was added dropwise. After completion (TLC
monitoring, 30 min), the mixture was quenched with a
saturated aqueous solution of NH4Cl and extracted with
CH2Cl2 (3 × 10 mL). The combined organic extracts were
dried over anhydrous Na2SO4, concentrated in vacuum, and
purified by flash chromatography (cyclohexane/AcOEt 3:2),
1
affording 3 as a colorless oil (60 mg, 42%). H NMR (400
EXPERIMENTAL SECTION
MHz, CDCl3) δ (ppm) 8.44 (bs, 1H), 7.95 (d, J = 8.0 Hz, 1H),
7.23 – 7.17 (m, 2H), 7.04 (t, J = 7.4 Hz, 1H), 3.72 (t, J = 4.8
Hz, 2H), 3.12 (t, J = 4.8 Hz, 2H), 2.30 (s, 3H); 13C NMR (100
MHz, CDCl3) δ (ppm) 167.4, 147.9, 135.3, 130.3, 127.4,
126.7, 124.3, 121.0, 37.3, 36.0, 17.6; HPLC-MS (ESI+) Rt=
6.8 min, m/z=205 [M+H]+; IR (film, cm-1) 3298, 2919, 1762,
1710, 1614, 1552, 1459, 1314, 1297, 1191
General methods. Compounds 1, 13, 15, and 26 were
synthesized according to our previously reported procedures.14,
17
Compounds 17 and 21 are known and were synthesized
according to reported procedures.18-20 Structure and purity of
1
known compounds was assessed by H NMR and HPLC-MS
analysis: spectroscopic data are in accordance to those
reported in literature. Target compounds were determined to
be ≥ 95% pure by analytical HPLC analyses (Supporting
information). Optical purity of compounds synthetized from
D-aspartic acid was assessed by chiral-HPLC analysis on
compound 22 chosen as a model using a Chiralpack IA
column, eluent: isopropanol/n-hexane 50:50, flow= 0.5
mL/min, temperature = 40°C.
2-(1-(2-Methylbenzoyl)-4-oxoazetidin-2-yl)acetic acid
(4). Following GP2, compound 14 (32 mg, 0.09 mmol)
yielded compound 4 as a waxy solid (22 mg, 99%). H NMR
1
(400 MHz, CD3OD) δ (ppm) 7.45 (d, J = 7.7 Hz, 1H), 7.39 (t,
J = 7.5 Hz, 1H), 7.24 (dd, J = 14.4, 7.3 Hz, 2H), 4.47 (ddd, J =
10.2, 7.6, 3.6 Hz, 1H), 3.30 (dd, J = 16.1, 6.4 Hz, 1H), 3.13
(dd, J = 16.3, 3.7 Hz, 1H), 3.01 (dd, J = 16.3, 3.7 Hz, 1H),
2.85 (dd, J = 16.3, 8.3 Hz, 1H), 2.38 (s, 3H); 13C NMR (100
MHz, CD3OD) δ (ppm) 174.0, 168.7, 165.6, 137.7, 135.0,
132.0, 131.6, 129.4, 126.5, 48.6, 43.0, 37.2, 19.5; HPLC-MS
General procedure for N-acylation (GP1) In a 25 mL
two neck flask the starting compound (1 equiv ) was dissolved
in anhydrous CH2Cl2 (11 mL/mmol) under a nitrogen
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(ESI+) Rt=5.1 min, m/z=248 [M+H]+; IR (film, cm-1) 2960,
2857, 1795, 1704, 1677, 1430, 1327, 1224, 1184
1H), 7.83 – 7.81 (m, 2H), 7.21 – 7.18 (m, 2H), 7.07 (t, J = 7.4
1
2
3
4
5
6
7
8
Hz, 1H), 4.59 (dd, J = 6.0, 3.1 Hz, 1H), 3.63 – 3.52 (m, 2H),
3.44 (dd, J = 16.2, 3.1 Hz, 1H), 3.24 (dd, J = 16.2, 6.0 Hz,
1H), 2.59 (t, J = 5.6 Hz, 2H), 2.29 (s, 3H); 13C NMR (100
MHz, CD3OD) δ (ppm) 175.3, 171.0, 168.0, 149.4, 136.4,
131.5, 129.9, 127.6, 126.0, 123.0, 51.6, 42.1, 36.7, 34.5, 17.7;
HPLC-MS (ESI+) Rt=4.3 min, m/z=320 [M+H]+, 342
[M+Na]+; IR (film, cm-1) 3352, 1779, 1734, 1708, 1648, 1595,
( -Tolylcarbamoyl)-L-proline (5). Following GP2,
compound 18 (60 mg, 0.18 mmol) yielded compound 5 as a
1
waxy solid (44 mg, 99%). H NMR (400 MHz, CD3OD) δ
(ppm) 7.38 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.12
(t, J = 7.4 Hz, 1H), 7.02 (t, J = 7.3 Hz, 1H), 4.31 (t, J = 5.9 Hz,
1H), 3.61 – 3.56 (m, 2H), 2.27 (s, 3H), 2.17 – 1.91 (m, 4H);
13C NMR (100 MHz, CD3OD) δ (ppm) 177.0, 157.6, 138.1,
134.9, 131.3, 127.5, 127.1, 126.6, 60.8, 47.5, 31.0, 25.4, 18.2;
HPLC-MS (ESI+) Rt= 3.3 min, m/z=249 [M+H]+; IR (film,
cm-1) 3423, 2927, 1720, 1709, 1639, 1527, 1457, 1377, 1254,
1199, 1124; [α]D20 = - 46.6 (c=10.0 mg/mL, CH3OH)
1550, 1458, 1311, 1254, 1203; [α]D = +56.7 (c=7.0 mg/mL,
20
CH3OH)
9
( )-(4-(2-carboxy-4-oxoazetidine-1-carboxamido)
phenyl)methanaminium 2,2,2-trifluoroacetate (11). In a 25
mL two-neck flask, compound 25 (72 mg, 0.19 mmol, 1
equiv) was dissolved in CH2Cl2 (3.5 mL) under a nitrogen
atmosphere and trifluoroacetic acid (TFA) was added
dropwise at 0°C. New TFA aliquots were added each 30 mins
at 0°C until a complete conversion (261 μL, 3.42 mmol, 18
equiv in total, 8 h, TLC monitoring). The solvent was removed
under reduced pressure and the crude was triturated with few
drops of pentane, yielding compound 11 (67 mg, 94%) as a
waxy solid. 1H NMR (400 MHz, CD3OD) δ (ppm) 7.57 (d, J =
8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 4.61 – 4.59 (m, 1H),
4.08 – 4.06 (m, 2H), 3.50 (dd, J = 15.7, 5.6 Hz, 1H), 3.12 (d, J
= 15.7 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ (ppm) 171.9,
167.1, 148.8, 139.4, 130.9, 130.2, 121.5, 43.8, 43.6, 42.1; 19F
NMR (375 MHz, CD3OD) δ (ppm) -76.7 ppm; HPLC-MS
(ESI+) Rt=1.3 min, m/z=247 [M+H]+; IR (film, cm-1) 3417,
3301, 2969, 1781, 1743, 1708, 1666, 1604, 1549, 1421, 1320,
1184; [α]D20 = +96.4 (c=11.3 mg/mL, CH3OH).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
( )-1-(2-Methylbenzoyl)-4-oxoazetidine-2-carboxylic
acid (6). Following GP2, compound 12 (80 mg, 0.25 mmol)
1
yielded compound 6 as a waxy solid (53 mg, 91%). H NMR
(400 MHz, CD3OD) δ (ppm) 7.49 (d, J = 7.7 Hz, 1H), 7.39 (t,
J = 7.5 Hz, 1H), 7.27 – 7.21 (m, 2H), 4.59 (dd, J = 7.0, 3.4 Hz,
1H), 3.41 (dd, J = 16.1, 7.0 Hz, 1H), 3.05 (dd, J = 16.1, 3.4
Hz, 1H), 2.40 (s, 3H); 13C NMR (100 MHz, CD3OD) δ (ppm)
172.9, 167.7, 164.0, 138.1, 134.4, 132.2, 131.7, 129.4, 126.5,
50.4, 41.5, 19.5; HPLC-MS (ESI+) Rt= 6.3 min, m/z=234
[M+H]+; IR (film, cm-1) 2967, 2930, 1806, 1728, 1686, 1603,
1324, 1259, 1184; [α]D20 = - 80.7 (c= 10 mg/mL, CH2Cl2)
( -Tolylcarbamoyl)-glycine
(7).
Following
GP2,
compound 19 (70 mg, 0.23 mmol) yielded compound 7 as a
1
waxy solid (47 mg, 97%). H NMR (400 MHz, CD3OD) δ
(ppm) 7.50 (d, J = 7.9 Hz, 1H), 7.18 – 7.11 (m,, 2H), 7.01 (t, J
= 7.4 Hz, 1H), 3.92 (s, 2H), 2.26 (s, 3H); 13C NMR (100 MHz,
CD3CN) δ (ppm) 172.8, 157.2, 136.6, 130.4, 130.0, 126.0,
124.0, 123.5, 35.3, 16.6; HPLC-MS (ESI+) Rt= 2.5 min,
m/z=209 [M+H]+; IR (film, cm-1) 3411, 3263, 2988, 1707,
1614, 1598, 1459, 1390, 1239, 1114.
Benzyl
carboxylate (12). In
commercially available
( )-1-(2-methylbenzoyl)-4-oxoazetidine-2-
10 mL two-neck flask, the
benzyl (S)-4-oxoazetidine-2-
a
carboxylate (62 mg, 0.3 mmol, 1 equiv) was dissolved in
anhydrous CH2Cl2 (1.5 mL) under nitrogen. TEA (135 μL,
0.96 mmol, 3.2 equiv) and DMAP (4 mg, 0.03 mmol, 0.1
equiv) were then added. O-toluylchloride (78 μL, 0.6 mmol, 2
equiv) was then added dropwise at 0°C. After 10 minutes, the
solution was warmed to rt and left under stirring. After
complete consumption of the starting material (6 h), the
mixture was quenched with a saturated aqueous solution of
NH4Cl and extracted with CH2Cl2 (3 × 10 mL). The combined
organic extracts were dried over anhydrous Na2SO4,
concentrated in vacuum, and purified by flash chromatography
(cyclohexane/AcOEt 7:3), affording 12 as a colorless oil (83
3-(3-( -Tolyl)ureido)propanoic acid (8). Following GP2,
compound 20 (45 mg, 0.14 mmol) yielded compound 8 as a
1
waxy solid (30 mg, 99%). H NMR (400 MHz, CD3OD) δ
(ppm) 7.48 (d, J = 7.9 Hz, 1H), 7.16 – 7.11 (m, 2H), 7.00 (t, J
= 7.8 Hz, 1H), 3.45 (t, J = 6.3 Hz, 2H), 2.53 (t, J = 6.3 Hz,
2H), 2.23 (s, 3H); 13C NMR (100 MHz, CD3OD) δ (ppm)
175.8, 158.8, 138.1, 131.9, 131.4, 127.4, 125.4, 125.0, 36.8,
35.6, 18.0; HPLC-MS (ESI+) Rt= 2.6 min, m/z=223 [M+H]+;
IR (film, cm-1) 3303, 3032, 2954, 1715, 1632, 1573, 1418,
1296, 1222, 1106.
1
( )-4-Oxo-1-(o-tolylcarbamoyl)azetidine-2-carbo xylic
mg, 86%). H NMR (400 MHz, CDCl3) δ (ppm) 7.52 (d, J =
acid (9). Following GP2, compound 22 (105 mg, 0.31 mmol)
7.5 Hz, 1H), 7.44 – 7.35 (m, 6H), 7.26 (t, J = 7.2 Hz, 2H), 5.30
(d, JAB = 12.2 Hz, 1H), 5.26 (d, JAB = 12.1 Hz, 1H), 4.68 (dd, J
= 6.8, 3.5 Hz, 1H), 3.34 (dd, J = 16.1, 6.8 Hz, 1H), 3.07 (dd, J
= 16.1, 3.5 Hz, 1H), 2.41 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ (ppm) 169.0, 165.8, 161.3, 137.4, 134.7, 132.1,
131.6, 130.8, 128.7, 128.64, 128.57, 128.4, 125.4, 67.7, 48.7,
40.4, 19.5; HPLC-MS (ESI+) Rt= 10.2 min, m/z=324 [M+H]+;
IR (film, cm-1) 3031, 2962, 2928, 1805, 1746, 1684, 1490,
1
yielded compound 9 as a white solid (74 mg, 97%). H NMR
(400 MHz, CDCl3) δ (ppm) 8.37 (bs, 1H), 8.19 (bs, 1H), 7.86
(d, J = 8.4 Hz, 1H), 7.18 – 7.15 (m, 2H), 7.05 (t, J = 7.4 Hz,
1H), 4.59 (dd, J = 6.2, 2.2 Hz, 1H), 3.37 (dd, J = 16.0, 6.2 Hz,
1H), 3.20 (dd, J = 16.0, 2.2 Hz, 1H), 2.28 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ (ppm) 172.0, 165.7, 147.9, 134.6, 130.5,
128.0, 126.8, 125.1, 121.5, 49.7, 41.3, 17.6; HPLC-MS (ESI+)
Rt=5.9 min, m/z=249 [M+H]+; IR (film, cm-1) 3344, 3024,
2966, 2962, 1775, 1717, 1615, 1593, 1459, 1308, 1252; m.p.
121-123 °C; [α]D20 = +114.3 (c=11.5 mg/mL, CH2Cl2)
1386, 1288, 1207, 1145; [α]D = - 78.2 (c=11.6 mg/mL,
20
CH2Cl2)
Benzyl
2-(1-(2-methylbenzoyl)-4-oxoazetidin-2-yl)
( )-3-(4-oxo-1-( -tolylcarbamoyl)azetidine-2-car
boxamido)propanoic acid (10). Following GP2, compound
23 (46 mg, 0.11 mmol) yielded compound 10 as a waxy solid
acetate (14). In a 10 mL two-neck flask, compound 13 (50
mg, 0.23 mmol, 1 equiv) was dissolved in anhydrous CH2Cl2
(1.2 mL) under nitrogen. TEA (103 μL, 0.73 mmol, 3.2 equiv)
and DMAP (3 mg, 0.023 mmol, 0.1 equiv) were then added.
1
(34 mg, 97%). H NMR (400 MHz, CDCl3) δ (ppm) 8.46 (bs,
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O-toluylchloride (60 μL, 0.46 mmol, 2 equiv) was then added
1524, 1455, 1369, 1254, 1169; [α]D20 = - 55.5 (c=11.0 mg/mL,
CH2Cl2).
Benzyl
1
2
3
4
5
6
7
8
dropwise at 0°C. After 10 minutes, the solution was warmed to
rt and left under stirring overnight. After 18 h, the mixture was
quenched with a saturated aqueous solution of NH4Cl and
extracted with CH2Cl2 (3 × 10 mL). The combined organic
extracts were dried over anhydrous Na2SO4, concentrated in
3-(3-(o-tolyl)ureido)propanoate
(20).
Commercially available beta alanine benzylester p-
toluenesulfonate salt (70 mg, 0.2 mmol, 1 equiv) was treated
with TEA (56 μL, 0.4mmol, 2 equiv) and o-tolylisocyanate
(27 μL, 0.22 mmol, 1.1 equiv) following GP1.
Chromatography (cyclohexane/AcOEt 3:2) yielded 20 as a
vacuum,
and
purified
by
flash
chromatography
(cyclohexane/AcOEt 7:3), affording 14 as a colorless oil (60
1
mg, 77%). H NMR (400 MHz, CDCl3) δ (ppm) 7.44 – 7.32
1
colorless oil (47 mg, 76%). H NMR (400 MHz, CD3CN) δ
(m, 7H), 7.25 – 7.22 (m, 2H), 5.19 (d, JAB = 12.3 Hz, 1H),
5.15 (d, JAB = 12.2 Hz, 1H), 4.56 – 4.50 (m, 1H), 3.37 – 3.25
(m, 2H), 2.94 (dd, J = 16.6, 3.6 Hz, 1H), 2.85 (dd, J = 16.3,
8.4 Hz, 1H), 2.40 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
(ppm) 169.7, 166.8, 163.0, 137.0, 135.3, 132.7, 131.3, 130.8,
128.6, 128.58, 128.45, 128.36, 125.4, 66.8, 46.6, 42.1, 36.8,
19.6; HPLC-MS (ESI+) Rt=7.6 min, m/z=338 [M+H]+; IR
(film, cm-1) 2958, 2929, 1796, 1734, 1676, 1511, 1456, 1289,
1184.
(ppm) 7.63 (d, J = 8.0 Hz, 1H), 7.37 – 7.31 (m, 5H), 7.16 –
7.10 (m, 2H), 6.96 (t, J = 7.0 Hz, 1H), 6.79 (bs, 1H), 5.68 (bs,
1H), 5.11 (s, 2H), 3.43 (q, J = 6.4 Hz, 2H), 2.56 (t, J = 6.4 Hz,
2H), 2.25 (s, 3H); 13C NMR (100 MHz, CD3CN) δ (ppm)
173.0, 156.7, 138.5, 137.4, 131.2, 130.2, 129.5, 129.04,
128.95, 127.2, 124.4, 123.6, 66.9, 36.6, 35.7, 18.2; HPLC-MS
(ESI+) Rt= 8.4 min, m/z=313 [M+H]+; IR (film, cm-1) 3308,
3066, 2954, 1727, 1632, 1566, 1456, 1419, 1253, 1186.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benzyl
( )-4-oxo-1-(o-tolylcarbamoyl)azetidine-2-
Benzyl (2-(4-oxoazetidin-2-yl)acetyl)glycinate (16). In a
25 mL two-neck flask, compound 15 (76 mg, 0.59 mmol, 1
equiv) was dissolved in a mixture of CH2Cl2 (6.6 mL) and
CH3CN (1.3 mL) under nitrogen. Dicyclohexylcarbodiimide
(DCC) (16 mg, 0.79 mmol, 1.1 equiv) was then added at 0°C.
It was followed by the dropwise addition of a previously
prepared solution of glycine benzylester p-chlorohydrate salt
(179 mg, 0.89 mmol, 1.5 equiv) and TEA (132 μL, 0.94 mmol,
1.6 equiv) in CH2Cl2 (7.4 mL). After addition of catalytic
DMAP (14 mg, 0.12 mmol, 0.2 equiv), the solution was
warmed to rt and left under stirring overnight. After complete
consumption of the starting material (16 h), the mixture was
quenched with H2O and extracted with CH2Cl2 (3 × 10 mL).
The collected organic layers were dried on anhydrous Na2SO4
and filtered. The crude was suspended in AcOEt, and the solid
residual dicyclohexylurea was eliminated by filtration. The
organic layer was concentrated in vacuum and purified by
flash chromatography (CH2Cl2/AcOEt from 3:2 to 100%
AcOEt), yielding compound 16 (68 mg, 91%) as a colorless
oil. 1H NMR (400 MHz, CDCl3) δ (ppm) 7.37 – 7.31 (m, 5H),
6.99 (bs, 1H), 6.72 (bs, 1H), 5.16 (d, J = 12.3 Hz, 1H), 5.12 (d,
J = 12.3 Hz, 1H), 4.15 (dd, J = 18.1, 6.1 Hz, 1H), 3.97 – 3.95
(m, 1H), 3.91 (dd, J = 18.1, 5.0 Hz, 1H), 3.09 (dd, J = 14.8,
3.2 Hz, 1H), 2.68 – 2.55 (m, 2H), 2.45 (dd, J = 14.4, 9.4 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 170.7, 170.1,
167.5, 134.9, 128.6, 128.5, 128.3, 67.2, 44.8, 43.3, 41.5, 41.1;
HPLC-MS (ESI+) Rt=10.5 min, m/z=277 [M+H]+, 294
[M+H2O]+; IR (film, cm-1) 3300, 2954, 1742, 1657, 1562,
1411, 1192, 1126.
carboxylate (22). Compound 21 (80 mg, 0.39 mmol, 1 equiv)
was treated with TEA (66 μL, 0.47 mmol, 1.2 equiv) and o-
tolylisocyanate (58 μL, 0.47 mmol, 1.2 equiv) following GP1.
Chromatography (CH2Cl2/Et2O 95:5) yielded 22 as a waxy
1
solid (112 mg, 85%). H NMR (400 MHz, CDCl3) δ (ppm)
8.29 (bs, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.41 – 7.31 (m, 5H),
7.25 – 7.15 (m, 2H), 7.07 (t, J = 7.4 Hz, 1H), 5.30 (d, JAB =
12.2 Hz, 1H), 5.25 (d, JAB = 12.2 Hz, 1H), 4.62 (dd, J = 6.2,
2.9 Hz, 1H), 3.40 (dd, J = 15.8, 6.2 Hz, 1H), 3.11 (dd, J =
15.8, 2.9 Hz, 1H), 2.31 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
(ppm) 168.8, 165.2, 146.8, 135.0, 134.7, 130.4, 128.65,
128.61, 128.3, 127.7, 126.8, 124.7, 121.2, 67.8, 48.9, 41.2,
17.6; HPLC-MS (ESI+) Rt=10.5 min, m/z=339 [M+H]+; IR
(film, cm-1) 3346, 3031, 2924, 1776, 1751, 1718, 1614, 1593,
1387, 1252, 1187; [α]D20 = + 96.4 (c=10.0 mg/mL, CH2Cl2).
Benzyl (R)-3-(4-oxo-1-(o-tolylcarbamoyl)azetidine -2-
carboxamido)propanoate (23). In a 25 mL two-neck flask,
compound 9 (69 mg, 0.28 mmol, 1 equiv) was dissolved in
anhydrous CH2Cl2 (2.8 mL) under nitrogen. Oxalyl chloride
(29 μL, 0.34 mmol, 1.2 equiv) was then added dropwise. The
mixture was left under stirring for 30 minutes, then beta
alanine benzylester p-toluenesulfonate salt (98 mg, 0.28 mmol,
1 equiv) was added, followed by the dropwise addition of
TEA (157 μL, 1.12 mmol, 4 equiv) at 0°C and DMAP (7 mg,
0.056 mmol, 0.2 equiv). After 10 minutes, the solution was
warmed to rt and left under stirring overnight. After complete
consumption of the starting material (16 h), the mixture was
quenched with a saturated aqueous solution of NH4Cl and
extracted with CH2Cl2 (3 × 10 mL). The combined organic
extracts were dried over anhydrous Na2SO4, concentrated in
Benzyl (o-tolylcarbamoyl)-L-prolinate (18). Compound
17 (82 mg, 0.26 mmol, 1 equiv) was treated with TEA (73 μL,
0.52 mmol, 2 equiv) and o-tolylisocyanate (36 μL, 0.29 mmol,
vacuum,
and
purified
by
flash
chromatography
(cyclohexane/AcOEt 3:2), affording 23 as a colorless oil (56
mg, 50%). 1H NMR (400 MHz, CDCl3) δ (ppm) 8.46 (bs, 1H),
7.89 (d, J = 8.4 Hz, 1H), 7.73 (bs, 1H), 7.35 – 7.29 (m, 5H),
7.22 – 7.18 (m, 2H), 7.07 (t, J = 7.4 Hz, 1H), 5.11 (s, 2H),
4.54 (dd, J = 6.1, 3.1 Hz, 1H), 3.66 – 3.54 (m, 2H), 3.46 (dd, J
= 16.3, 3.1 Hz, 1H), 3.26 (dd, J = 16.3, 6.1 Hz, 1H), 2.63 (t, J
= 6.2 Hz, 2H), 2.30 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
(ppm) 171.6, 167.5, 166.8, 148.6, 135.5, 134.7, 130.5, 128.5,
128.22, 128.16, 128.0, 126.7, 124.9, 121.4, 66.5, 51.7, 40.3,
35.3, 33.9, 17.6; HPLC-MS (ESI+) Rt=9.2 min, m/z=410
[M+H]+; IR (film, cm-1) 3339, 2957, 1773, 1730, 1678, 1615,
1.1
equiv)
following
GP1.
Chromatography
(cyclohexane/AcOEt 1:1) yielded 18 as a colorless oil (63 mg,
1
72%). H NMR (400 MHz, CDCl3) δ (ppm) 7.73 (d, J = 8.0
Hz, 1H), 7.36 – 7.29 (m, 5H), 7.18 – 7.12 (m, 2H), 6.99 (t, J =
7.4 Hz, 1H), 6.34 (bs, 1H), 5.22 (d, JAB = 12.4 Hz, 1H), 5.14
(d, JAB = 12.4 Hz, 1H), 4.55 (dd, J = 8.1, 2.3 Hz, 1H), 3.61 –
3.48 (m, 2H), 2.20 (s, 3H), 2.16 – 1.99 (m, 4H); 13C NMR
(100 MHz, CDCl3) δ (ppm) 172.6, 154.1, 136.7, 135.5, 130.1,
128.4, 128.24, 128.16, 128.0, 126.5, 123.7, 122.4, 66.8, 59.3,
46.1, 29.6, 24.4, 17.6; HPLC-MS (ESI+) Rt= 9.4 min,
m/z=338 [M+H]+; IR (film, cm-1) 3313, 2971, 1743, 1640,
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Journal of Medicinal Chemistry
1593, 1459, 1253, 1174; [α]D = +101.8 (c=9.2 mg/mL,
CH2Cl2).
Funding Sources
20
1
2
3
4
5
6
7
8
This research was supported by University of Bologna (RFO2017
and RFO2018), FARB (FFBO 125290), Fondazione Cassa di
Risparmio in Bologna (2018/0347) and Ministero dell'Università
e della Ricerca (PRIN2015 project 20157WW5EH).
Benzyl
(R)-1-((4-(((tert-butoxycarbonyl)amino)
methyl)phenyl)carbamoyl)-4-oxoazetidine-2-carboxy late
(24). In a 25 mL two-neck flask, compound 21 (70 mg, 0.34
mmol, 1 equiv) was dissolved in anhydrous CH2Cl2 (3.2 mL)
under nitrogen. TEA (84 μL, 0.60 mmol, 1.5 equiv) was then
added dropwise. The mixture was stirred for 15 minutes, then
a solution of freshly prepared isocyanate 26 (1.5 equiv) in
anhydrous CH2Cl2 (1.6 mL) was added dropwise. The mixture
was stirred at room temperature until a complete consumption
of the starting beta-lactam (4h, TLC monitoring) and then
quenched with a saturated aqueous solution of NH4Cl. The
mixture was extracted with CH2Cl2 (3 x 10mL), the organic
layers were collected, dried over anhydrous Na2SO4,
concentrated in vacuum and purified by flash chromatography
(cyclohexane/AcOEt 1:1), affording 24 as a colorless oil (110
mg, 60%). 1H NMR (400 MHz, CDCl3) δ (ppm) 8.27 (bs, 1H),
7.42 (d, J = 8.4 Hz, 2H), 7.33 – 7.28 (m, 5H), 7.23 (d, J = 8.4
Hz, 2H), 5.28 (d, JAB = 12.2 Hz, 1H), 5.23 (d, JAB = 12.2 Hz,
1H), 4.86 (bs, 1H), 4.59 (dd, J = 6.2, 2.9 Hz, 1H), 4.27 – 4.25
(m, 2H), 3.38 (dd, J = 15.8, 6.2 Hz, 1H), 3.08 (dd, J = 15.8,
2.9 Hz, 1H), 1.45 (s, 9H); 13C NMR (400 MHz, CDCl3) δ
(ppm) 168.8, 165.0, 155.8, 146.6, 135.8, 135.1, 134.7, 128.64,
128.62, 128.3, 128.2, 119.9, 79.4, 67.8, 48.9, 44.1, 41.2, 28.4;
HPLC-MS (ESI+) Rt=10.4 min, m/z=471 [M+H]+; IR (film,
cm-1) 3344, 2975, 1777, 1751, 1708, 1603, 1542, 1317, 1244,
1170; [α]D20 = +62.2 (c=8.3 mg/mL, CH2Cl2)
ACKNOWLEDGMENT
DG would like to thank Mr. Riccardo Pedrazzani for his technical
assistance. ACS Authoring Service is acknowledged for the
English revision of the manuscript.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ABBREVIATIONS
THF, tetrahydrofuran; TEA, triethylamine; TLC, thin layer
chromatography; FN, fibronectin; VCAM-1, vascular cell
adhesion molecule-1; SPA, scintillation proximity-binding assay;
ERK1/2, extracellular signal-regulated kinases 1 and 2.
REFERENCES
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Res. 2010, 339, 269-280.
(2) Hynes, R. O. Integrins: bidirectional, allosteric signaling
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(3) Kuriri, F. A; O'Malley, C. J.; Jackson, D. E. Molecular
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(4) Rose, D. M.; Han, J.; Ginsberg, M. H. Alpha4 integrins and the
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progression and metastasis. Nat. Rev. Cancer 2018, 18, 532-547.
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(7) Humphries, J. D.; Byron, A.; Humphries, M. J. Integrin ligands at
a glance. J. Cell Sci. 2006, 119, 3901-3903.
(8) Luo, B. H.; Springer, T. A. Integrin structures and conformational
signaling. Curr. Opin. Cell Biol. 2006, 18, 579-586.
(9) Moore, T. I.; Aaron, J.; Chew, T.-L.; Springer, T. A. Measuring
integrin conformational change on the cell surface with super-
resolution microscopy. Cell Rep. 2018, 22, 1903-1912.
(10) Pandolfi, F.; Franza, L.; Altamura, S.; Mandolini, C.; Cianci, R.;
Ansari, A.; Kurnick, J. T. Integrins: integrating the biology and
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(11) Ley, K.; Rivera-Nieves, J.; Sandborn, W. J.; Shattil, S. Integrin-
based therapeutics: biological basis, clinical use and new drugs. Nat.
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(12) Tolomelli, A.; Galletti, P.; Baiula, M.; Giacomini, D. Can
integrin agonists have cards to play against cancer? A literature
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(13) Humphries, M. J. Integrin cell adhesion receptors and the
concept of agonism. Trends Pharmacol. Sci. 2000, 21, 29-32.
(14) Galletti, P.; Soldati, R.; Pori, M.; Durso, M.; Tolomelli, A.;
Gentilucci, L.; Dattoli, S.D.; Baiula, M.; Spampinato, S. M.;
Giacomini, D. Targeting integrins v3 and 51 with new -lactam
derivatives. Eur. J. Med. Chem. 2014, 83, 284-293.
(15) Baiula, M.; Galletti, P.; Martelli, G.; Soldati, R.; Belvisi, L.;
Civera, M.; Dattoli, S. D.; Spampinato S. M.; Giacomini, D. New β-
lactam derivatives modulate cell adhesion and signaling mediated by
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9742.
( )-1-((4-(((tert-butoxycarbonyl)amino)methyl)
phenyl)carbamoyl)-4-oxoazetidine-2-carboxylic acid (25).
Following GP2, compound 24 (110 mg, 0.24 mmol) yielded
1
compound 25 as a waxy solid (78 mg, 88%). H NMR (400
MHz, CD3OD) δ (ppm) 7.43 (d, J = 8.4 Hz, 2H), 7.23 (d, J =
8.4 Hz, 2H), 4.51 (dd, J = 6.4, 2.9 Hz, 1H), 4.19 – 4.17 (m,
2H), 3.45 (dd, J = 15.8, 6.4 Hz, 1H), 3.08 (dd, J = 15.8, 2.9
Hz, 1H), 1.45 (s, 9H); 13C NMR (100 MHz, CD3OD) δ (ppm)
173.3, 167.3, 158.4, 149.0, 137.2, 137.0, 128.8, 121.2, 80.2,
50.8, 44.5, 42.1, 28.8; HPLC-MS (ESI+) Rt=7.1 min, m/z=381
[M+H2O]+, 386 [M+Na]+; IR (film, cm-1) 3341, 2978, 2932,
1776, 1707, 1604, 1544, 1417, 1321, 1244, 1167; [α]D
=
20
+44.9 (c=10.0 mg/mL, CH2Cl2)
ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
compounds 1-25. Assays procedures and methods. Additional
pharmacological assays. Molecular modelling and HPLC traces.
AUTHOR INFORMATION
Corresponding Author
*D.G.: e-mail, daria.giacomini@unibo.it.
*S.S. : e-mail, santi.spampinato@unibo.it
(16) Cainelli, G.; Giacomini, D.; Galletti, P.; Quintavalla, A.
Synthesis of novel 4-(2-oxoethylidene)azetidin-2-ones by a Lewis
acid mediated reaction of acyldiazo compounds. Eur. J. Org. Chem.
2003, 9, 1765-1774.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript. ≠G.M. and M.B. contributed equally.
(17) Pori, M.; Galletti, P.; Soldati, R.; Calza, L.; Mangano, C.;
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Giacomini, D. Azetidinone-retinoid hybrids: synthesis and
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Q.; Chen, Y.; Soni R.; Scott, C.; Ross, D. T.; Guo, H.; Howorth, P.;
Valentine, H.; Liang, S.; Spicer, D.; Fuller, M.; Steine, J.; Hamilton,
G. S. Use of parallel-synthesis combinatorial libraries for rapid
identification of potent FKBP12 inhibitors. Bioorg. Med. Chem. Lett.
2002, 12, 1421–1428.
(19) Bolchi, C.; Valoti, E.; Fumagalli, L.; Straniero, V.; Ruggeri, P.;
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COOH
51 antagonist
IC50 = 41.5 nM
N
O
O
41 agonist
EC50 = 12.9 nM
COOH
COOH
NH
COOH
N
H
N
41 agonist
EC50 = 10.0 nM
HN
O
O
V3 agonist
EC50 = 29.9 nM
N
O
NH
O
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