Azetidinone-retinoid hybrids: Synthesis and differentiative effects

Article abstract of DOI:10.1016/j.ejmech.2013.09.057

As a part of a systematic investigation on the synthesis and biological activities of new β-lactam compounds, we examined β-lactam candidates 1, 2E and 2Z and their ability to induce cell proliferation or differentiation. Azetidinone 1 was chosen for its

Full text of DOI:10.1016/j.ejmech.2013.09.057

European Journal of Medicinal Chemistry 70 (2013) 857e863
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Azetidinoneeretinoid hybrids: Synthesis and differentiative effects
,
,
,
,
c
,
,2
Matteo Pori a 1, Paola Galletti a 1, Roberto Soldati a 1, Laura Calzà b 2, Chiara Mangano c
,
Daria Giacomini a
,
*
a Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy
b Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Via Tolara di Sopra 50, I-40064
Ozzano Emilia, Bologna, Italy
c Department of Veterinary Medicine, University of Bologna, Via Tolara di Sopra 50/A, 40064 Ozzano Emilia, Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 May 2013
Received in revised form
12 September 2013
Accepted 29 September 2013
Available online 12 October 2013
As a part of a systematic investigation on the synthesis and biological activities of new
pounds, we examined -lactam candidates 1, 2E and 2Z and their ability to induce cell proliferation or
differentiation. Azetidinone 1 was chosen for its activity (previously evaluated) as selective HDAC8 in-
hibitor, whereas -lactams 2E and 2Z were designed to have a hybrid retinoid-azetidinone structure, de
novo synthesized and fully characterized. Biological activities were determined in cellular assays on
neuroblastoma cells SH-SY5Y. Azetidinone 1, 2E and 2Z led to a moderate effect in decreasing SH-SY5Y
b-lactam com-
b
b
cell proliferation and b-lactams 2E and 2Z induced an early stage differentiation.
The present results uncovered a new role of specifically designed b-lactam compounds in epigenetic
regulation.
Keywords:
b
-Lactams
Retinoids
Synthesis
Ó 2013 Elsevier Masson SAS. All rights reserved.
Cell differentiation
HDAC inhibitors
Epigenetic regulation
1. Introduction
appeared on
b
-lactam compounds as anti-tumor drugs. Some aryl-
b
-lactam derivatives have been evaluated for cytotoxicity against a
number of human tumor and normal cell lines [8]. In particular N-
thiolated- -lactams were found to induce DNA damage, cell growth
arrest, and apoptosis in cultured human cancer cells, and anti-
proliferative effects on human breast cancer cells of some new
b
-Lactams have historically been viewed as an important class of
antimicrobials since the discovery of naturally occurring penicillins
and cephalosporins [1]. Recognition of the azetidin-2-one moiety
as the key pharmacophoric component of
b
b-lactam antibiotics
initiated a flurry of synthetic activity to develop new structures,
and nowadays new variants of monocyclic compounds (azetidi-
nones) are showing new and specific biological activities [2].
In the last few decades their role as enzyme inhibitors has been
expanded. b-Lactams are well-known as potent inhibitors of some
enzymes that contain serine as the catalytic residue, including the
bacterial penicillin binding proteins (PBPs), and Class A and Class C
azetidinones were evaluated [9]. Some bicyclic b-lactams were
recently reported as inhibitors of Histone Deacetylases (HDACs), a
family of proteins involved in the pattern of acetylation of chro-
matin proteins and thus in the regulation of gene expression [10].
We recently reported the synthesis of some azetidinone derivatives
which showed good affinity and specificity towards histone
deacetylases HDAC8 and HDAC6 [11].
b
-lactamases [3].
b
-Lactams have also been appropriately modified
Histone deacetylases are epigenetic regulators playing an
important role in modulating gene transcription as well as protein
function. Eighteen classical HDAC isoforms, divided into IV classes
based on yeast deacetilases homology, are identified to date. HDAC
inhibitors constitute a new class of promising anticancer drugs
targeting one or more HDAC isoforms. HDAC6 overexpression was
found in carcinoma of oral squamous tissue [12], although in breast
cancer it correlates with better survival [13]. In lung, colon, and
cervical cancer cell line the knockout of HDAC8 gene reduces cell
proliferation [14]; in leukemia cell HDAC8 inhibitors leads to
apoptosis [15]. The expression of HDAC8 (class I member) has been
to develop active site-directed and mechanism-based inhibitors of
Human Leukocyte Elastase (HLE) [4], and in this field some new
monocyclic b-lactams have shown important activities as HLE in-
hibitors [5], as new antibiotics towards resistant bacteria [6], and as
inhibitors of platelet aggregation [7]. Recently, relevant studies
* Corresponding author. Tel.: þ39 051 209 9528; fax: þ39 051 209 9456.
E-mail address: daria.giacomini@unibo.it (D. Giacomini).
1
Tel.: þ39 051 209 9450; fax: þ39 051 209 9456.
2
Tel.: þ39 051 2097 947; fax: þ39 051 2097 953.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.057

858
M. Pori et al. / European Journal of Medicinal Chemistry 70 (2013) 857e863
correlated with poor outcome in neuroblastoma [15], and in such
cell line selective HDAC8 inhibitors induces differentiation and
proliferation arrest [16].
Nowadays, there is great interest in the development of small
molecules that regulate or modulate protein function and chemical
genetic offers a valuable approach in this field [17].
processes to mimic the action of retinoic acid (RA). The design of
b-
lactam 2 was based on a 4-alkylidene- -lactam scaffold with
b
demonstrated enhanced reactivity in protein inhibition [2a,5], and
functionalized with a retinoid-amide side chain. The strategy is
outlined in Scheme 1 and consists of a convergent synthesis with
the coupling between the 4-alkylidene-azetidinone carboxy acid 3
and the retinoid amine 4 as the key step.
4-Alkyliden-azetidin-2-ones can be obtained using an original
and well established protocol developed in our laboratory starting
from 4-acetoxy-azetidinones and diazoesters in the presence of
Lewis acids [30]. Given that 4-acetoxy azetidinone is commercially
Retinoids, as derivatives of Vitamin A, regulate key develop-
mental pathways throughout life, by modulating the expression of
many genes involved in cell proliferation, migration, differentiation
and apoptosis, though the molecular mechanisms are not
completely understood [18e21]. The effects of retinoic acid (RA) are
mediated by the heterodimerization of nuclear RAR (Retinoic Acid
Receptor), a family of ligand-dependent transcription factors, and
RXR (Retinoid X Receptors), the 9-cis RA binding receptor [18]. To
date, retinoids are known to activate or inhibit MAPK cascade,
which is involved in both proliferation and growth arrest [22].
Some synthetic RA-derived molecules exert differentiative effect
rather than apoptosis, blocking tumor progression in cancer cell
lines [23,24]. In fact, retinoids are currently used as potential
chemotherapeutic drugs in the treatment of several cancer types
(neuroblastoma, breast cancer, lung cancer, rhabdomyosarcoma,
and many others) [25e28]. For this reason, there is great interest in
developing new synthetic molecules targeting retinoic acid
receptors.
available,
b
-lactam 3 was obtained with benzyldiazoacetate and
-lactams 5 were
TiCl4 as outlined in Scheme 2. The 4-alkyliden-
b
obtained in 43% yields and in a E/Z diastereomeric ratio of 41/59.
The diastereomeric mixture 5Z/5E was easily separated by flash
chromatography. Hydrogenolysis of the isolated pure 5Z with Pd on
carbon in THF-methanol 1:1 mixture gave 3 in quantitative yield.
Retinoid amine 4 was obtained starting from the easily available
b-ionone in a three step reaction (Scheme 3). Addition of lithium
acetonitrile to -ionone in THF at low temperature gave 6 [30],
b
which underwent water elimination under ptoluensulfonic acid
catalysis [31]. The nitrile 7 obtained as a E/Z mixture, was then
reduced to give the amine 4EZ in 79% yield.
Finally, with a carbodiimide coupling method, amides 2E and 2Z
were obtained as a mixture and easily separated by flash chroma-
tography. The 2E,4E and 2Z,4E configurations were assigned in the
two isomers by NOE 1D analysis on the separated products 2E and
2Z (Scheme 4, and Supplementary material). In isomer 2E, the
methyl group in the chain, chosen as the irradiated site, showed
two NOE correlations with methylene and methine, respectively,
As part of an interdisciplinary project on the development of
new b-lactam derivatives with a modular structure designed for
specific biological activities, the effect of the azetidinone 1 (Fig. 1),
previously evaluated by us as specific HDAC8 inhibitor [11], was
investigated on the proliferative activity of a neuron-like cell line.
Moreover, we developed the synthesis of new b-lactams 2 with a
hybrid retinoid-azetidinone structure and we investigated the
retinoic acid-dependent differentiation of a neuron-like cell line. To
our knowledge, only two example of b-lactams were reported in
the literature with a differentiation-inducing activity on leukemia
cells [29]. We report here the synthesis of the new hybrid retinoid-
indicating a close spatial relationship whereas for b-lactam 2Z NOE
enhancements of the two vinylic CH were observed. The 2E,4E
configuration for 2E, and the 2Z,4E configuration for 2Z were thus
attributed.
b-lactams and preliminary results in biological evaluation of the
2.2. Biology
three molecules on neuroblastoma cell line SH-SY5Y.
As said before, HDAC inhibitors are emerging as anti cancer
drugs. In particular, HDAC8 inhibitors have shown to regulate
proliferation in neuroblastoma cells [16]. As potential HDAC in-
hibitors, we evaluated the antiproliferative effect of azetidinones 1,
2. Results and discussion
2.1. Compound design and synthesis
2E, and 2Z on undifferentiated SH-SY5Y. Moreover, as
and 2Z have hybrid retinoid-azetidinone structures, we performed
a set of experiments aiming to evaluate differentiative action of
b-lactams 2E
As mentioned above, we have previously reported an efficient
synthesis of b-lactam 1 [11]. Its design was based on the pharma-
b
-
lactams 2E and 2Z compared to azetidinone 1 and retinoic acid
treated cells. In fact, retinoids are currently used as potential
chemotherapeutic drugs in the treatment of several cancer types
including neuroblastoma due to their differentiative action [25e
28].
In order to assess azetidinone 1, 2E, and 2Z cytotoxicity, we
calculated first the IC50 (50% inhibiting concentration) using MTT
assay (Fig. 2). The MTT assay is a colorimetric assay for measuring
the activity of cytosolic enzymes that reduce tetrazolium dye (MTT)
in formazan salt in living cells. IC50 values were calculated and are
reported above the curves in Fig. 2. The concentration used for
subsequent experiments was chosen accordingly in order to obtain
cytostatic effects rather than apoptosis or necrosis (Fig. 2).
cophore model of HDAC inhibitors which consists in a modular
structure with a cap-group that interacts with residues in the active
site of the receptor and a linker group that sits in a hydrophobic
channel and positions a metal-binding group which coordinates to
the zinc ion in the active site. We demonstrated that the azetidi-
none ring can efficiently work as the zinc-binding group. Moreover,
the substituent on the nitrogen atom allowed for the specificity
against HDAC8 or HDAC6 inhibition. Azetidinone 1 was therefore
chosen in the present study for its specific HDAC8 activity.
Once the
binding group for HDAC, we developed a new hybrid structure
retaining the -lactam scaffold to target HDACs and functionalized
with a retinoic side chain which could activate differentiative
b-lactam ring was confirmed as an effective zinc-
b
2.2.1. Effect on proliferation
In a preliminary analysis of biological effect on proliferation,
undifferentiated SH-SY5Y cells were exposed to 7.5 microM of
azetidinone 1, 10 microM of b-lactams 2E and 2Z for 24, 48, 72 and
96 h, the number of cells was counted daily and compared to
control (vehicle treated). Fig. 3 shows the proliferation curve of
Fig. 1.
b-Lactam derivatives.
control cells (ctrl, open circle), azetidinone 1 (black square), b-

M. Pori et al. / European Journal of Medicinal Chemistry 70 (2013) 857e863
859
Scheme 1. Retro-synthetic route to retinoid b-lactam 2.
Scheme 2. Synthesis of 4-alkyliden-azetidinone 3.
Scheme 3. Synthesis of retinoid amine 4 and retinoid b-lactams 2E and 2Z.
lactam 2E (black triangle) and 2Z treated cell (black rhombus).
Significant differences were observed between the groups (two-
way ANOVA, Dunnet’s multiple comparisons test).
and 2Z determine a significative reduction in cell proliferation after
48 h of treatment (a: ctrl vs -lactam 2E; b: ctrl vs -lactam 2Z). All
the compounds tested show an antiproliferative effect compared to
control from 72 to 96 h (c: ctrl vs azetidinone 1 and -lactam 2E, d:
ctrl vs -lactam 2Z, e: ctrl vs azetidinone 1 and -lactam 2E
p < 0.0001, f: ctrl vs -lactam 2Z). Although the reduction is stable,
b-lactams 2E
b
b
b
b
b
Scheme 4. NOE 1D correlations in new retinoid-lactams for configuration
assignments.
b

860
M. Pori et al. / European Journal of Medicinal Chemistry 70 (2013) 857e863
Sample micrographs of cell SH-SY5Y exposed to the vehicle
(control), RA, azetidinone 1, and -lactams 2E and 2Z are shown in
Fig. 4, where panels AeE refers to -tubulin-IR, and panels FeJ
b
b
refers to neurofilament-IR, graph K and L to respective quantifica-
tion of IR area/number of cells. While a differentiative effect of RA is
evident in both bIII-tubulin and NF200 immunostaining (panels A
vs panel B and panel F vs panel G, respectively) and azetidinone 1
does not display any differentiative effect (panels A vs panel C and
panel F vs panel H),
panel I) and 2Z (panels A vs panel E and panel F vs panel J) induce a
moderate differentiation, which is statistically significant in III-
b-lactams 2E (panels A vs panel D and panel F vs
b
tubulin staining (panels K and L, statistical analysis was performed
with one-way ANOVA, Tukey post test). This effect could be
possibly related to moderate structural analogies among RA and
lactams 2E and 2Z. The data indicate a differentiative effect of
b
b
-
-
lactams 2E and 2Z on SH-SY5Y, even though smaller when
compared to RA. The absence in the -lactams 2E and 2Z of an
Fig. 2. Cytotoxic evaluation of azetidinone 1, b-lactams 2E and 2Z on undifferentiated
SH-SY5Y; IC50 ranges are reported above the curves. Data are shown as mean
b
ionizable function, as like the carboxylic acid in RA, can explain the
observed lower activity, as reported for some non-ionizable RA
derivatives such as RA-esters or amides [32]. To bind effectively to
the retinoic acid receptor (RAR), the polar terminus of the retinoid
must be capable of efficient interactions, as observed in the crystal
(normalized to control) ꢁSD.
doubling time calculated in 24e72 h range (Td) is affected exclu-
sively in cells treated with
Td2 ¼ 39.78, Td8 ¼ 51,25), whereas doubling time calculated in 24e
96 h range is not affected. These results indicated a slight effect of
azetidinone 1, 2E and 2Z on SH-SY5Y proliferation.
b
-lactam 2Z (TdCTRL ¼ 34,9 h; Td1 ¼ 39 h,
structure of RA-RAR complex [33]. The
b-lactam scaffold, while it
would not be considered a strong “polar terminus” in anchoring
RAR, shows a positive effect on SH-SY5Y cell differentiation (b-
lactams 2E and 2Z), and it deserves further investigation.
2.2.2. Differentiation effect
As they are derived from neuroblastoma, SH-SY5Y may be
3. Conclusions
differentiated toward neural-like lineage. Retinoic acid is
a
powerful differentiating molecule inducing growth inhibition,
neurites lengthening and branching. A six-days RA treatment in-
duces neural-like differentiation as shown by neurites outgrowth
As part of an interdisciplinary research project involving the
synthesis of new monocyclic -lactams specifically designed to
b
target epigenetic regulators, we presented here an evaluation of
three monocyclic azetidinones candidates 1, 2E and 2Z and their
ability to interfere with cell proliferation or differentiation. Azeti-
dinone 1 was chosen because of its specific inhibition of HDAC8,
whereas 2E and 2Z were designed as innovative hybrid
azetidinone-retinoid structures. Their de novo synthesis allowed
for the anchoring of an alkylidenecarboxyl-side chain on the C4
position of the b-lactam core which was further condensed with a
retinoid-amine. The new hybrids 2E and 2Z and the azetidinone 1
were tested in cellular assays on neuroblastoma cells SH-SY5Y.
(Fig. 4, panel B vs panel A; panel G vs panel F) visualized by
tubulin and NF200. Quantitative analysis was performed measuring
the III-tubulin and NF200 network normalized according to the
cells number. As hybrids retinoid-azetidinones, we investigated a
possible differentiative effect of -lactams 2E and 2Z compared to
azetidinone 1 and RA treated SH-SY5Y. III-Tubulin is an early
marker for differentiation toward neuronal lineage and the increase
of III-tubulin area may be related to an early stage of differentia-
bIII-
b
b
b
b
tion. Neurofilament play important structural roles and influence
axonal transport, neurite outgrowth and cell survival.
Azetidinone 1,
liferation rate in SH-SY5Y neuroblastoma cells, possibly due to the
HDAC inhibition properties. The hybrid azetidinone-retinoids
b-lactams 2E and 2Z slightly decrease the pro-
b
-
lactams 2E and 2Z have a differentiative effect, even if smaller when
compared to RA.
These preliminary results are worthy of a further evaluation and
prompt us to currently work on the synthesis of a library of new
hybrid azetidinone-retinoid molecules.
4. Experimental section
4.1. Chemistry
4.1.1. General
All reactions were performed under an inert atmosphere (N2).
Commercial reagents (reagent grade, >99%) were used as received
without additional purification. Anhydrous solvents (CH3CN,
CH2Cl2, THF) were obtained commercially.
Fig. 3. Effect of azetidinones 1, 2E, and 2Z on SH-SY5Y proliferation. The rate of cells
proliferation was determined as total cell number. Cells were incubated in the presence
of 7.5 microM of azetidinone 1, 10 microM of 2E and 2Z and counted at 24, 48, 72, 96 h.
Significant differences between groups were observed after 48 h of treatment. Sta-
tistical analysis was performed with two-way ANOVA, Dunnet’s multiple comparisons
1H and 13C NMR values were recorded on a INOVA 400, or a
GEMINI 200 instrument with a 5 mm probe. All chemical shifts
have been quoted relative to deuterated solvent signals,
d in ppm
and J in Hz. FT-IR: Nicolet 380 measured as films between NaCl,
plates wavenumbers reported in cmꢀ1. TLC: Merck 60 F254. Column
chromatography: Merck silica gel 200e300 mesh. GCeMS: Agilent
test (a: ctrl vs
b
-lactam 2E, p < 0.05; b: ctrl vs
-lactam 2E p < 0.001, d: ctrl vs
-lactam 2E p < 0.0001, f: ctrl vs b
b
-lactam 2Z, p < 0.01; c: ctrl vs azeti-
-lactam 2Z p < 0.0001, e: ctrl vs aze-
-lactam 2Z p < 0.001).
dinone 1 and
b
b
tidinone 1 and
b

M. Pori et al. / European Journal of Medicinal Chemistry 70 (2013) 857e863
861
Fig. 4. Effect of azetidinone 1,
intermediate filaments rearrangement. Panels A and F refer to vehicle treated cells; B and G to cells treated for 6 days with RA; C and F correspond to cells treated for 6 days with
7.5 microM of azetidinone 1, D and I correspond to cells treated for 6 days with 10 microM of -lactam 2E; E and J correspond to cells treated for 6 days with 10 microM of -lactam
2Z (Scale bar ¼ 10 micron). Graphs K and L refer to III-tubulin and neurofilament immunoreactive area, respectively, normalized for number of cells (mean ꢁ SD). Statistical
analysis was performed with one-way ANOVA, Tukey post test, **p < 0.01, ***p < 0.001, ****p < 0.0001.
b-lactams 2E and 2Z on SH-SY5Y differentiation. bIII-tubulin (AeC) and neurofilament (DeF) were used to investigate a possible microtubule and
b
b
b
Technologies, column HP5 5% Ph-Me Silicon MS: Agilent Technol-
ogies MSD1100 single-quadrupole mass spectrometer, EI voltage
70 eV, gradient from 50 ꢂC to 280 ꢂC in 30 min. HPLC-MS, HPLC:
Agilent Technologies HP1100, column ZOBRAX-Eclipse XDB-C8
Agilent Technologies, mobile phase: H2O/CH3CN, gradient from 30%
to 80% of CH3CN in 8 min, 80% of CH3CN until 25 min, 0.4 mL/min
MS: Agilent Technologies MSD1100 single-quadrupole mass spec-
trometer, full-scan mode from m/z 50 to m/z 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.
A Shimadzu UV-1601 PC spectrophotometer was used for spec-
trophotometric measurements.
4.1.2.1. (Z)-2-(4-Oxoazetidin-2-ylidene)acetic acid (3). To a solution
of 5Z (220 mg, 1.01 mmol) in a 10 mL mixture of THF/Methanol 1:1
was added Pd 10% wt on activated Carbon (44 mg). The solution was
treated with H2 (1 atm) and after 2 h was filtered on celite and
concentrated to give 3 (100 mg, 78%).
Yellow solid: mp ¼ 162e163; 1H NMR (400 MHz, (DMSO d6):
d
¼ 3.57 (s, 2H), 4.98 (s, 1H), 10.47 (brs, 1H), 11.94 (brs, 1H); 13C NMR
(100 MHz, CD3OD):
d
¼ 46.0, 92.2, 153.3, 169.7, 171.0; IR (neat):
ṽ ¼ 3289, 2919, 1828, 1185 cmꢀ1; HPLC-MS (ESI): Rt ¼ min 1.66, m/z
(%): 128 [M þ H]þ, 150 [M þ Na]þ, 679 (70) [2M þ Na]þ; Found C,
47.42; H, 3.92; N, 10.87%; C5H5NO3 requires C, 47.25; H, 3.97; N,
11.02%.
4.1.2.2. (E)-3-Hydroxy-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1-
4.1.2. Synthesis
yl)pent-4-enenitrile (6). To a stirred solution of acetonitrile (330 mL,
Azetidinone 5Z was prepared as previously reported [6b].
6.24 mmol) in dry THF (5 mL) at ꢀ78 ꢂC under inert atmosphere

862
M. Pori et al. / European Journal of Medicinal Chemistry 70 (2013) 857e863
were added dropwise nButyllithium 2.5 M in THF (6.86 mmol,
2.74 mL) and thereupon a solution of -ionone (1 g, 5.2 mmol) in
3H), 1.86 (s, 3H), 2.01 (m, 2H), 3.51 (s, 2H), 4.06 (m, 2H), 5.02 (s, 1H),
5.30 (brs, 1H, NH), 5.43 (m, 1H), 6.01 (d, J ¼ 16 Hz, 1H), 6.13 (d,
J ¼ 16 Hz, 1H), 8.75 (brs, 1H, NH); 13C NMR (100 MHz, CDCl3):
b
dry THF (4 mL) were added over a period of 10 min. The solution
was allowed to warm at room temperature and after 3 h the re-
action was quenched with aqueous NH4Cl (10 mL) and extracted
with EtOAc (3 ꢃ 10 mL). The organic extracts were dried over
Na2SO4, filtered and concentrated in vacuo. Flash chromatography
(cyclohexane/EtOAc: 80/20) of the extracts gave 6 (1.031 g, 85%).
d
¼ 12.5, 19.2, 21.6, 28.8, 29.7, 32.9, 34.2, 37.3, 39.5, 44.9, 92.2, 124.9,
127.0, 129.0, 136.6, 137.5, 137.6, 146.6, 165.3, 166.3; IR (neat):
ṽ ¼ 3367, 3250, 2958, 2922, 2853, 1815, 1692 cmꢀ1; HPLC-MS (ESI):
Rt ¼ min 12.72, m/z (%): 329 [M þ H]þ; Found C, 72.98; H, 8.65; N,
8.41%; C20H22N2O2 requires C, 73.14; H, 8.59; N, 8.53%.
Pale yellow oil: 1H NMR (400 MHz, CDCl3):
d
¼ 0.92 (s, 6H,
2Z: pale yellow syrup: Rf ¼ 0.5 (cyclohexane/EtOAc, 5/5); 1H
C(CH3)2), 1.35e1.38 (m, 2H, C(CH3)2CH2CH2CH2), 1.41 (s, 3H,
CH3C(OH)), 1.49e1.55 (m, 2H, C(CH3)2CH2CH2CH2), 1.58 (s, 3H,
CH3C]C), 1.88e1.91 (m, 2H, C(CH3)2CH2CH2CH2), 2.56 (s, 2H,
CH2CN), 3.34 (brs, 1H, OH), 5.45 (d, 1H, J ¼ 16.0 Hz, CH]CHC(OH),
6.11 (d, 1H, J ¼ 16.0 Hz, CH]CHC(OH)); 13C NMR (100 MHz, CDCl3):
NMR (400 MHz, CDCl3):
d
¼ 1.26 (s, 6H), 1.47 (m, 2H), 1.63 (m, 2H),
1.70 (s, 3H), 1.91 (s, 3H), 2.02 (m, 2H), 3.51 (m, 2H), 4.04 (m, 2H),
4.99 (s, 1H), 5.25 (brs, 1H, NH), 5.36 (m, 1H), 6.22 (d, J ¼ 16 Hz, 1H),
6.38 (d, J ¼ 16 Hz, 1H), 8.72 (brs, 1H, NH); 13C NMR (100 MHz,
CDCl3):
d
¼ 19.2, 20.3, 21.7, 28.9, 29.7, 32.9, 34.1, 36.4, 39.5, 44.9,
d
¼ 18.8, 20.9, 27.4, 28.3 (2 ꢃ C), 31.6, 32.3, 33.6, 38.9, 70.8, 117.3,
92.3, 123.1, 129.0, 129.6, 129.7, 136.7, 137.7, 146.4, 165.3, 166.3; IR
(neat): ṽ ¼ 3327, 2954, 2923, 2853, 1810, 1683 cmꢀ1; HPLC-MS
(ESI): Rt ¼ min 11.21, m/z (%): 329 (65) [M þ H]þ, 351 (100)
[M þ Na]þ, 679 (70) [2M þ Na]þ; Found C, 72.88; H, 8.65; N, 8.38%;
127.1,128.5,136.0,136.6; IR (neat); ṽ ¼ 3444, 3023, 2925, 2256,1722,
1651, 1644, 1114 cm-1; GCeMS (EI): Rt ¼ 16.81 min, m/z: 233 [M]þ,
218 [M ꢀ CH3]þ, 193 [M ꢀ CH2CN]þ, 135, 109, 91, 69; Found C, 77.01;
H, 9.98; N, 5.92%; C15H23NO requires C, 77.21; H, 9.93; N, 6.00%.
C20H22N2O2 requires C, 73.14; H, 8.59; N, 8.53%.
4.1.2.3. (2ZE,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1-enyl)penta-
2,4-dienenitrile (7EZ). A solution of 6 (400 mg, 1.72 mmol) and
pToluensulfonic acid (33 mg, 0.172 mmol) in toluene (17 mL) was
refluxed and the reaction was followed by TLC. After the completion
of the reaction the solution was cooled at 0 ꢂC and washed with a
solution of NaHCO3 5% and after with water. The organic extracts
were dried over Na2SO4, filtered and concentrated in vacuo
obtaining 352 mg (95%) of 7 as a E/Z:54/46mixture. Characteriza-
tion resulted in agreement with the reported data [34].
4.2. Cell culture and treatments
Human neuroblastoma SH-SY5Y cell line were cultured in
DMEM/F12 (Gibco-Invitrogen, Milan, Italy) supplemented with 10%
fetal bovine serum (Euroclone, Milan, Italy), 1X MEM/NEAA (Gibco-
Invitrogen), 1X Penicillin-Streptomycin (Gibco-Invitrogen), NaOH
10 mM (Carlo Erba) and maintained at 37 ꢂC in a humidified 5% CO2
incubator.
Azetidinone 1, b-Lactams 2, b-Lactams 2Z, and all-trans retinoic
acid (RA, Sigma Aldrich) were dissolved in dimethylsulfoxide
(DMSO, Sigma Aldrich) to an intermediate concentration of
100 mM (1 and 2E), and 16.6 mM (RA). Final dilutions (10 microM
for b-lactam 2E, b-Lactams 2Z and RA, 7.5 microM for azetidinone 1)
were done in the culture medium. A corresponding volume of
DMSO was added to control cells (vehicle treated). To evaluate
4.1.2.4. (2EZ,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl)
penta-2,4-dien-1-amine (4). Amine 4 was prepared as reported in
Ref. [35]; for a complete characterization:
E/Z: 59/41. pale yellow oil, 1H NMR (400 MHz, CDCl3):
d
¼ 0.97(s,
6H, C(CH3)2, trans), 0.98 (s, 6H, C(CH3)2, cis) 1.41e1.48 (m, 2H,
CH2CH2C(CH3)2), 1.48e1.64 (m, 2H, CH2CH2C(CH3)2), 1.68 (s, 3H,
CH2CH2C(CH3)]C, trans),1.72 (s, 3H, CH2CH2C(CH3)]C, cis), 1.81 (s,
3H, CH3C]CH, trans), 1.88 (s, 3H, CH3C]CH, cis), 1.95e1.97 (m, 2H,
CH2CH2C(CH3)]C), 2.47 (bs, 2H, NH2), 3.40 (d, 2H, J ¼ 6.8 Hz,
CH2NH2), 5.37 (dd, 1H, J ¼ 6.4, 7.6 Hz, C]CHCH2NH2, cis), 5.45 (dd,
1H, J ¼ 6.4 Hz, 7.2 Hz, C]CHCH2NH2, trans), 5.94e6.01 (m, 2H, CH]
CH, trans), 6.12 (d, 1H, J ¼ 15.6 Hz, CH]CH, cis), 6.30 (d, 1H,
citotoxic effect exerted by Azetidinone 1, b-Lactams 2E and b-Lac-
tams 2Z, SH-SY5Y cells were seeded at a density of 1 ꢃ104 cells/well
in 96 platic multiwells/dish; 24 h after seeding were treated for
24 h with Azetidinone 1, b-Lactams 2E and b-Lactams 2Z in the
concentration range 1 microM to 1 mM and was performed MTT
assay [36]. Cytotoxicity of compounds 1 and 2E and 2Z on SHSY5Y
(IC50) was assessed in order to define the working concentration
(IC501 ¼ 10e28 microM R2 ¼ 0.973; IC502E ¼ 38e65 microM
R2 ¼ 9988; IC50 2Z ¼ 35e97 microM R2 ¼ 0.954) (Fig. 2).
J ¼ 15.6 Hz, CH]CH, cis); 13C (100 MHz, CDCl3):
¼ 12.2, 19.1, 21.6,
d
28.4 (2 ꢃ C), 32.7, 34.1, 38.4, 39.3, 126.0, 128.5, 128.6, 129.0, 129.3,
130.1,133.7,135.0,137.1,137.5,137.8; IR (neat); ṽ ¼ 3373, 2928, 2866,
1454, 1266 cmꢀ1; GCeMS (EI): Rt ¼ 16.09 (cis), 16.57 (trans) min, m/
z: 219 [M]þ, 204 [M ꢀ CH3]þ, 187 [M ꢀ CH2CN]þ, 162, 131,119, 91, 83.
4.2.1. Proliferation effect
To evaluate the effect of Azetidinone 1, b-Lactams 2E and 2Z on
proliferation, cells were seeded at a density of 1 ꢃ104 cells/cm2 in 6
plastic multiwells dish and allowed to grow for 24 h before treat-
ment. Twenty-four h after seeding, the medium was removed and
fresh medium containing the drugs was added for additional 24, 48,
72 and 96 h. Rate of cells growth was determined counting viable
cells using trypan blue exclusion assay. Doubling time (Td) was
calculated during logarithmic growth phase. The experiment was
repeated twice. Statistical analysis were performed with two-way
ANOVA, Dunnet’s multiple comparisons test.
4.1.2.5. (2Z)-N-((2EZ,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1-
enyl)penta-2,4-dienyl)-2-(4-oxoaze tidin-2-ylidene)acetamide (2E
and 2Z). To a solution of b-lactam 3 (55 mg, 0.43 mmol) in dry DCM
(4.5 mL) under inert atmosphere at 0 ꢂC were added amine 4EZ
(94 mg, 0.43 mmol), DMAP (53 mg, 0.43 mmol) and after 10 min
EDCl (82.5 mg, 0.43 mmol). The solution was stirred at 0 ꢂC for
45 min and after was allowed to warm at room temperature. The
reaction was followed by TLC and after the disappearing of the
starting materials was quenched with aqueous NH4Cl (10 mL) and
extracted with DCM (3 ꢃ 5 mL). The organic extracts were dried
over Na2SO4, filtered and concentrated in vacuo. The E/Z mixture
was separated by flash chromatography (cyclohexane/EtOAc 70/30)
affording 2E (62 mg) and 2Z (44 mg) in a overall yield of 75%.
Configurations of the two diastereoisomers was assigned by NOE
1D studies.
4.2.2. Differentiative effect
Indirect immunofluorescence was used to investigate possible
morphological changes of cytoskeleton due to Azetidinone 1,
b-
Lactams 2E and 2Z exposure. For this experiment, SH-SY5Y were
seeded on glass coverslips coated with CultrexÒ Basement Mem-
brane Extract (BME) (TrevigenÒ, Gaithersburg, MD, USA) at a
density of 1 ꢃ 104 cells/cm2 and cultured in the previously
described culture media. After 24 h of seeding, the medium was
2E: white syrup: Rf ¼ 0.4 (cyclohexane/EtOAc, 5/5); 1H NMR
(400 MHz, CDCl3):
d
¼ 1.00 (s, 6H),1.46 (m, 2H), 1.62 (m, 2H), 1.68 (s,

M. Pori et al. / European Journal of Medicinal Chemistry 70 (2013) 857e863
863
(b) G. Cainelli, P. Galletti, S. Garbisa, D. Giacomini, L. Sartor, A. Quintavalla,
Bioorg. Med. Chem. 13 (2005) 6120e6132.
[6] (a) F. Broccolo, G. Cainelli, G. Caltabiano, C.E.A. Cocuzza, C.G. Fortuna,
P. Galletti, D. Giacomini, G. Musumarra, R. Musumeci, A. Quintavalla, J. Med.
Chem. 49 (2006) 2804e2811;
(b) P. Galletti, C.E.A. Cocuzza, M. Pori, A. Quintavalla, R. Musumeci,
D. Giacomini, ChemMedChem 6 (2011) 1919e1927.
[7] M. Pavanetto, A. Zarpellon, D. Giacomini, P. Galletti, A. Quintavalla, G. Cainelli,
A. Folda, G. Scutari, R. Deana, Platelets 18 (2007) 357e364.
[8] (a) N.M. O’Boyle, L.M. Greene, N.O. Keely, S. Wang, T.S. Cotter, D.M. Zisterer,
M.J. Meegan, Eur. J. Med. Chem. 62 (2013) 705e721;
removed and was added fresh medium containing 7.5 microM
azetidinone 1,10 microM of -lactam 2E,10 microM of -Lactams 2Z
and 10 microM of RA. Cells were treated for additional 6 days and
medium was replaced every 48 h. Untreated cells and cells treated
with retinoic acid (RA) 10 microM were used respectively as non-
differentiated and differentiated controls [37].
b
b
After treatments, cells were washed in PBS and fixed with 4%
paraformaldehyde in 0.1 M Sørensen phosphate buffer for 20 min at
RT. Subsequently, fixed cells were blocked in PBS containing 0.3%
Triton-X 100 (Merck, Darmstadt, Germany), 5% Donkey Normal
Serum (Sigma Aldrich) for 1 h at RT, then incubated overnight
at þ4 ꢂC in humid atmosphere with anti-neurofilament 200 (Sigma
(b) P. Pérez-Faginas, M.T. Aranda, M.T. García-López, A. Francesch, C. Cuevas,
R. González-Muñiz, Eur. J. Med. Chem. 46 (2011) 5108e5119;
(c) N.M. O’Boyle, M. Carr, L.M. Greene, N.O. Keely, A.J.S. Knox, T. McCabe,
D.G. Lloyd, D.M. Zisterer, M.J. Meegan, Eur. J. Med. Chem. 46 (2011) 4595e
4607;
Aldrich) and anti- bIII-tubulin (R&D) antibodies diluted in blocking
(d) B.K. Banik, I. Banik, F.F. Becker, Eur. J. Med. Chem. 45 (2010) 846e848;
(e) B.K. Banik, F.F. Becker, I. Banik, Bioorg. Med. Chem. 12 (2004) 2523e2528.
[9] D. Chen, S.C. Falsetti, M. Frezza, V. Milacic, A. Kazi, Q.C. Cui, T.E. Long, E. Turos,
Q.P. Dou, Cancer Lett. 268 (2008) 63e69.
[10] S. Oha, J.-C. Jung, Z. Naturforsch., B: J. Chem. Sci. 62b (2007) 1459e1464.
[11] P. Galletti, A. Quintavalla, C. Ventrici, G. Giannini, W. Cabri, S. Penco, G. Gallo,
S. Vincenti, D. Giacomini, ChemMedChem 4 (2009) 1991e2001.
[12] T. Sakuma, K. Uzawa, T. Onda, M. Shiiba, H. Yokoe, T. Shibahara, H. Tanzawa,
Int. J. Oncol. 29 (2006) 117e124.
[13] Z. Zhang, H. Yamashita, T. Toyama, H. Sugiura, Y. Omoto, Y. Ando, K. Mita,
M. Hamaguchi, S. Hayashi, H. Iwase, Clin. Cancer Res. 10 (2004) 6962e6968.
[14] A. Vannini, C. Volpari, G. Filocamo, E.C. Casavola, M. Brunetti, D. Renzoni,
P. Chakravarty, C. Paolini, R. De Francesco, P. Gallinari, C. Steinkuhler, S. Di
Marco, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 15064e15069.
[15] S. Balasubramanian, J. Ramos, W. Luo, M. Sirisawad, E. Verner, J.J. Buggy,
Leukemia 22 (2008) 1026e1034.
[16] I. Oehme, E. Deubzer, D. Wegener, D. Pickert, J.P. Linke, B. Hero, A. Kopp-
Schneider, F. Westermann, S.M. Ulrich, A. von Deimling, M. Fischer, O. Witt,
Clin. Cancer Res. (2009) 91e99.
solution 1:400 and 1:1000 respectively. After rinsing twice in PBS
for 10 min, cells were incubated for 30 min at 37 ꢂC with Dylight
488-labeled secondary antisera (Thermo Scientific) diluted in 0.3%
Triton-X 100/PBS. For nuclear staining, cells were first washed in
PBS then incubated 15 min with 1 mg/ml Hoechst 33258 in 0.2%
Triton-X 100/PBS. After rinsing in PBS, coverslips containing cells
were finally mounted in 1,4-phenylendiamine (Sigma Aldrich)/
glycerol 0.1%. Negative controls were performed by primary anti-
body omission.
Images of SH-SY5Y cells were taken by Nikon Eclipse 600 mi-
croscope equipped with motorized z-stage control and F-view II
CCD Cameras with 20ꢃ objective. Immunofluorescence staining
was analyzed using the Nis-Elements Software 3.21.03 (Nikon).
Analysis of
bIII-tubulin and neurofilament 200 immunoreactive
[17] (a) C.A. Lyssiotis, L.L. Lairson, A.E. Boitano, H. Wurdak, S. Zhu, P.G. Schultz,
Angew. Chem. Int. Ed. 50 (2011) 200e242;
areas (IR) were executed applying “sharpen” and “detect edges”
filters. For each image, nuclei were counted and IR/nꢂ of nuclei were
calculated. The experiment was repeated twice and 3 samples for
each experiment (at least 100 cells) were analyzed. Statistical
analysis were performed with one-way ANOVA.
(b) M. Fisher, A. Nelson, New Frontiers in Chemical Biology, in: RSC Drug
Discovery Series, 2011, pp. 1e32;
(c) D.P. Walsh, Y.-T. Chang, Chem. Rev. 106 (2006) 2476e2530;
(d) M. Biel, V. Wascholowski, A. Giannis, Angew. Chem. Int. Ed. 44 (2005)
3186e3216.
[18] D.J. Mangelsdorf, R.M. Evans, Cell 83 (1995) 841e850.
[19] A.A. Farooqui, P. Antony, W.Y. Hong, L.A. Horrocks, L. Freysz, Brain Res. Rev. 45
(2004) 179e195.
[20] F.M. Tsai, R.Y. Shyu, S.C. Lin, C.C. Wu, S.Y. Jiang, BMC Cell Biol. 10 (2009) 15,
http://dx.doi.org/10.1186/1471-2121-10-15.
Representative images were collected using a Nikon A1R
confocal laser-scanning microscope, equipped with a Nikon Pla-
nApo 40ꢃ lens.
[21] S. Ueki, G. Mahemuti, H. Oyamada, H. Kato, J. Kihara, M. Tanabe, W. Ito,
T. Chiba, M. Takeda, H. Kayaba, J. Chihara, J. Immunol. 181 (2008) 7689e
7698.
Acknowledgments
[22] D.L. Crowe, R. Kim, R.A.S. Chanfraratna, Mol. Cancer Res. 1 (2003) 532e540.
[23] B. Meister, F.M. Fink, A. Hittmair, C. Marth, M. Widschwendter, Anticancer Res.
18 (1998) 1777e1786.
[24] B.J. Maurer, L.S. Metelitsa, R.C. Seeger, M.C. Cabott, C.P. Reynolds, J. Natl.
Cancer Inst. 91 (1999) 1138e1146.
[25] A.L. Sabichi, M.R. Modiano, J.J. Lee, Y.M. Peng, M.J. Xu, H. Villar, Clin. Cancer
Res. 9 (2003) 2400e2405.
[26] A. Garaventa, R. Luksch, M.S. Lo Piccolo, E. Cavadini, P.G. Montaldo,
M.R. Pizzitola, Clin. Cancer Res. 9 (2003) 2032e2039.
The authors DG, PG, and MP would like to thank Mrs Jessica
Giacoboni for technical assistance. We are grateful to Dr. Francesca
Paradisi (senior lecturer at the UCD School of Chemistry and
Chemical Biology, University College Dublin) for revision of the En-
glish manuscript. This work was supported by MIUR (PRIN project),
University of Bologna and POR-FESR Regione Emilia Romagna.
[27] R. Lotan, J. Natl. Cancer Inst. 87 (1995) 1655e1657.
[28] L.A. Hansen, C.C. Sigman, F. Andreola, S.A. Ross, G.J. Kelloff, L.M. De Luca,
Carcinogenesis 7 (2000) 1271e1279.
[29] (a) H. Morioka, M. Takezawa, H. Shibai, T. Okawara, M. Furukawa, Agric. Biol.
Chem. 50 (1986) 1757e1764;
(b) C. Li, E.L. Schwartz, S.L. Mella, L.S. Rittmann, A.C. Sartorelli, J. Med. Chem.
24 (1981) 1092e1094.
[30] G. Cainelli, G. Cardillo, M. Contento, P. Grasselli, A. Umani Ronchi, Gazz. Chim.
Ital. 103 (1973) 117e125.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.057.
References
[31] E. Brenna, C. Fuganti, P. Grasselli, S. Serra, Eur. J. Org. Chem. (2001) 1349e
1357.
[1] C. Walsh, Antibiotics: Actions, Origins, Resistance, American Society for
Microbiology (ASM) Press, Washington DC, 2003.
[32] J.H. Barnard, J.C. Collings, A. Whiting, S.A. Przyborski, T.B. Marder, Chem. Eur. J.
15 (2009) 11430e11442.
[33] (a) J.P. Renaud, N. Rochel, M. Ruff, V. Vivat, P. Chambon, H. Gronemeyer,
D. Moras, Nature 378 (1995) 681e689;
(b) F. Sussman, A.R. de Lera, J. Med. Chem. 48 (2005) 6212e6219.
[34] (a) A. Valla, B. Valla, R. Le Guillou, D. Cartier, L. Dufosse, R. Labia, Helv. Chim.
Acta 90 (2007) 512e520;
[2] (a) P. Galletti, D. Giacomini, Curr. Med. Chem. 18 (2011) 4265e4283;
(b) P.D. Mehta, N.P.S. Sengar, A.K. Pathak, Eur. J. Med. Chem. 45 (2010) 5541e
5560;
(c) M.I. Konaklieva, B.J. Plotkin, T. Herbert, Anti-Infect. Agents Med. Chem. 8
(2009) 28e35.
[3] J.C. Powers, J.L. Asgian, O.D. Ekici, K.E. James, Chem. Rev. 102 (2002) 4639e
4750.
(b) Y.L. Bennani, J. Org. Chem. 61 (1996) 3542e3544.
[4] (a) B. Xing, J. Rao, R. Liu, Mini-Rev. Med. Chem. 8 (2008) 455e471;
(b) S. Gérard, M. Galleni, G. Dive, J. Marchand-Brynaert, Bioorg. Med. Chem. 12
(2004) 129e138.
[5] (a) D. Giacomini, A. Quintavalla, F. Calabrese, C. Giacometti, E. Brunetta,
F. Piazza, C. Agostini, S. Garbisa, J. Pharmacol. Exp. Ther. 316 (2006) 539e546;
[35] M.B. Li, Y. Wang, S.K. Tian, Angew. Chem. 51 (2012) 2968e2971.
[36] I. Mickuviene, V. Kirveliene, B. Juodka, Toxicol. In Vitro 18 (2004) 639e648.
ꢀ
[37] L. Agholme, T. Lindstrsm, K. Kagedal, J. Marcusson, M. Hallbeck, J. Alzheimers
Dis. 20 (2010) 1069e1082.