Biological activity of 3-chloro-azetidin-2-one derivatives having interesting antiproliferative activity on human breast cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2013.09.054

Resveratrol (3,4′,5 tri-hydroxystilbene), a natural plant polyphenol, has gained interest as a non-toxic agent capable of inducing tumor cell death in a variety of cancer types. However, therapeutic application of these beneficial effects remains very lim

Full text of DOI:10.1016/j.bmcl.2013.09.054

Accepted Manuscript
Biological activity of 3-chloro-azetidin-2-one derivatives having interesting an‐
tiproliferative activity on human breast cancer cell lines
Adele Chimento, Marina Sala, Isabel M. Gomez-Monterrey, Simona Musella,
Alessia Bertamino, Anna Caruso, Maria Stefania Sinicropi, Rosa Sirianni,
Francesco Puoci, Ortensia Ilaria Parisi, Carmela Campana, Emilia Martire,
Ettore Novellino, Carmela Saturnino, Pietro Campiglia, Vincenzo Pezzi
PII:
S0960-894X(13)01128-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.09.054
BMCL 20906
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
30 July 2013
16 September 2013
18 September 2013
Please cite this article as: Chimento, A., Sala, M., Gomez-Monterrey, I.M., Musella, S., Bertamino, A., Caruso, A.,
Sinicropi, M.S., Sirianni, R., Puoci, F., Parisi, O.I., Campana, C., Martire, E., Novellino, E., Saturnino, C.,
Campiglia, P., Pezzi, V., Biological activity of 3-chloro-azetidin-2-one derivatives having interesting
antiproliferative activity on human breast cancer cell lines, Bioorganic & Medicinal Chemistry Letters (2013), doi:
http://dx.doi.org/10.1016/j.bmcl.2013.09.054
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Biological activity of 3-chloro-azetidin-2-one derivatives having interesting antiproliferative
activity on human breast cancer cell lines
Adele Chimentoa§, Marina Salab§, Isabel M. Gomez-Monterreyc, Simona Musellad, Alessia
Bertaminob, Anna Carusoa, Maria Stefania Sinicropia, Rosa Siriannia, Francesco Puocia, Ortensia
Ilaria Parisia, Carmela Campanaa, Emilia Martirea, Ettore Novellinoc, Carmela Saturninob*, Pietro
Campigliab and Vincenzo Pezzia*
aDepartment of Pharmacy, Health and Nutrition Sciences, University of Calabria, 87036
Arcavacata di Rende, Cosenza, Italy
bDepartment of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano,
Salerno, Italy
cDepartment of Pharmaceutical and Toxicological Chemistry, University of Naples “Federico II”,
8013 Napoli, Italy
dDepartment Pharmaco-Biological, University of Messina, 98168 Messina, Italy
AUTHOR INFORMATION
Corresponding Author
*
For Vincenzo Pezzi. Tel.: +39-0984-493148; Fax: +39-0984-493271. E-mail address:
v.pezzi@unical.it
*For Carmela Saturnino. Tel.: +39-089-969769; Fax: +39-089-969602. E-mail address:
saturnino@unisa.it
§ These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
Keywords: resveratrol analogs anticancer drugs, 3-chloro-azetidin-2-one derivatives, breast cancer,
MCF-7, SKBR3.

ABSTRACT
Resveratrol (3,4',5 tri-hydroxystilbene), a natural plant polyphenol, has gained interest as a non-
toxic agent capable of inducing tumor cell death in a variety of cancer types. However, therapeutic
application of these beneficial effects remains very limited due to its short biological half-life, labile
properties, rapid metabolism and elimination. Different studies were undertaken to obtain synthetic
analogues of resveratrol with major bioavailability and anticancer activity. We have synthesized a
series 3-chloro-azetidin-2-one derivatives, in which an azetidinone nucleus connects two aromatic
rings. Aim of the present study was to investigate the effects of these new 3-chloro-azetidin-2-one
resveratrol derivatives on human breast cancer cell lines proliferation. Our results indicate that some
azetidin-based resveratrol derivatives may become new potent alternative tools for the treatment of
human breast cancer.

Breast cancer is a major cause of death in women, even if disease-free survival and overall survival
of patients with breast cancer have been improved through intensive treatment.1 Major breast cancer
treatment methods consist, both separately and in combination with surgery, radiotherapy and
chemotherapy. In particular, the anti-estrogen tamoxifen is widely used in the prevention and
treatment of estrogen receptor positive breast cancer.2 Inherent or acquired tumor drug resistance
limits many agents that could be used to treat this disease and are often associated with severe,
dose-limiting and systemic toxicities. Therefore, new agents acting on novel targets in breast cancer
are currently under investigation. Interest in the pharmacological effects of bioactive compounds on
cancer treatment and prevention has increased dramatically in recent years. A great number of
natural agents derived from plants are studied as agents potentially useful in combined therapy for
cancer patients. As well, there is a need to develop new more powerfully active drugs with reduced
side effects that can substitute current pharmacological therapies.
Several studies confirmed that increasing vegetable and fruit consumption might reduce the risk of
breast cancer.3 Also, a lower incidence of breast cancer is associated with a high consumption of
phytoestrogens,4 which are biologically active plant-derived phenolic compounds that structurally
mimic the mammalian estrogen, 17β-estradiol.5
Among many bioactive compounds, basic and preclinical researches on resveratrol, a non-flavonoid
polyphenolic compound abundant in grapes, peanuts and other foods that are commonly consumed
as part of human diet,6 have shown a broad range of advantageous biological actions, including
cardioprotection7 and prolongation of lifespan in several species.8 Resveratrol is a phytoalexin that
in nature protects the plant from injury, ultraviolet (UV) irradiation, and fungal attack.9 Resveratrol
exists as cis- and transisomeric forms, with trans to cis isomerization facilitated by UV exposure. Its
stilbene structure is related to the synthetic estrogen diethylstilbestrol. Two phenol rings are linked
by a styrene double bond to generate 3, 4’,5- trihydroxystilbene (Fig. 1).

Figure 1. Chemical structure of resveratrol (RSV)
The biological properties of resveratrol are attributed to its ability to inhibit the oxidation of human
low-density lipoprotein, while suppression of cycloxygenase-2 and inducible nitric oxide synthase
activities also contribute to its anti-inflammatory and antioxidant effects.10 Furthermore, the
chemopreventive effect of resveratrol is thought to be due to inhibition of quinone reductase 2
activity, which in turn up-regulates the expression of cellular antioxidant and detoxification
enzymes to improve cellular resistance to oxidative stress.11 Resveratrol also increases the activity
of SIRT (a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent
deacetylases), resulting in improved cellular stress resistance and longevity,8,10,12 Jang et al.13 have
suggested that it inhibits all three phases of tumor development: initiation, promotion, and
progression in various cancers such as prostate14, colon15, endometrial16, hepatocarcinoma17 and
breast cancer.18,19 Resveratrol contains strong anti-proliferative properties in many cultured cancer
cell lines, and acts both by arresting cell cycle and by inducing apoptosis,20,21,22,23,24 but the
apoptosis inducing effects of resveratrol seemed diverse on different tumor cells.20,21,22 Resveratrol
has been shown to interfere with signal transduction pathways, to modulate cell cycle regulating
proteins and to induce apoptosis in multiple cancer cell lines with various mechanisms, including
through a p53-dependent pathway and PKC/Akt pathway.25
However, therapeutic application of these beneficial effects of resveratrol remains very limited due
to its short biological half-life, labile properties, and rapid metabolism and elimination.10 In fact, in
human and rodent, three metabolic pathways have been identified, i.e., sulfate and glucuronic acid
conjugation of the phenolic groups and hydrogenation of the aliphatic double bond, the latter likely

produced by the intestinal microflora.26 Extremely rapid sulfate conjugation by the intestine/liver
appears to be the rate-limiting step in resveratrol’s bioavailability.27 Analysis of recent literature
reveals an increasing number of formulations under study, which reflects the major interest in
developing pharmaceutical forms able to improve resveratrol bioavailability as a step towards
applying its therapeutic potential in vivo.27
Several studies were undertaken to obtain resveratrol synthetic derivatives with potent anticancer
activity, enhanced structural rigidity and major bioavailability.28 Structure-activity studies have
revealed crucial elements of the parental components that are required for specific effects. To give
one example, the 4-hydroxy group in the trans conformation on the 4- and 4’-positions of the
stilbenic backbone and the methoxy groups added to the trihydroxystilbene scaffold of resveratrol
have been identified as crucial for antiproliferative29 or cytotoxic30 resveratrol effects respectively.
In addition, the structural replacement of the stilbene moiety of resveratrol has been found to be a
promising strategy for thegeneration of synthetic analogues with improved pharmacokinetic
parameters. Mayhoub et al.31 described a series of trans and cis 2,3-thiadiazol analogs of resveratrol
using as design strategy the replacement of the alkene linker between the two aromatic rings with a
heterocyclic system. Starting from this approach, recently we have synthesized 2,3-thiazolidin-4-
one resveratrol analogues that have increased structural rigidity and potent activity (I Fig. 2) fixing
the cis-conformation of resveratrol, hypothesizing that also using cis stilbene template could have
active derivatives. We demonstrated that some derivatives displayed stronger antiproliferative
effects than resveratrol in human breast cancer cells.28
In the present work, we prepared a new series of 1,4-diaryl-3-chloro-azetidin-2-one derivatives in
which a azetidin-2-one nucleus connects two aromatic rings (II Fig. 2); also these new compounds
have structural rigidity, major bioavailability and antiproliferative activity.

O
O
R'
Cl
R'
S
N
N
R
R
II
I
Figure 2. Structures of 2,3-thiazolidin-4-one derivatives (I) and of 3-chloro-azetidin-2-one derivatives (II).
The designed compounds (4a-f, Table 1)32 were prepared by the following Staudinger reaction.33
Cycloaddition of imines (3) with 2-chloro-acetylchloride in the presence of triethylamine afforded
beta-lactams 4a-f in high yields (47-93%). The imines were obtained by condensation of aldehydes
(1) and amines (2) in toluene (Scheme 1).
O
H
NH2
O
Cl
O
R'
Cl
Cl
N
Toluene
+
N
TEA
CH2Cl2
-78°C
R
R'
R
1
R
R'
3
4
2
4a R= R'= CH3
4b R= R'= H
4c R= Cl; R'= H
4d R= R'= Br
4e R= H; R'= Cl
4f R= Cl; R'= I
Scheme 1. Synthesis of 3-chloro-azetidin-2-one derivatives (4).
The bioavailability of the new synthesized 3-chloro-azetidin-2-one resveratrol derivatives was
measured by dialysis tubing procedure,34,35 a quick and low cost method that represents a good
model to evaluate the bioavailability of different kinds of molecules.
In most cases, the oral route represents the most convenient one for drug administration and, thus, it
is important to choose an appropriate model to investigate absorption and bioavailability of
therapeutic agents in the gastrointestinal system.
Bioavailability was defined as the percentage of tested compounds recovered in the bioavailable
fraction, after in vitro digestion, in relation to the original non-digested samples.
This value can be calculated by the following equation (1):
(bioavailable content/total content) x 100
(1).

The obtained results, after six hours, were reported in Table 1.
Table 1: Bioaccessibility (%) of 4a-f compounds.
SAMPLE
BIOACCESSIBILITY (%)
Resveratrol
31 0.9
79 0.8
78 0.9
83 1.1
87 0.7
80 1.0
70 0.9
4a
4b
4c
4d
4e
4f
As it is possible to note, all the synthesized compounds showed a higher bioavailability compared
to resveratrol, which was more than 2 times less bioavailable, and the best results were observed for
4d with about 87% of the initial content recovered in the bioavailable fraction.
The higher bioavailability of these novel 3-chloro-azetidin-2-one resveratrol derivatives could be
due to their modified structures making these molecules more lipophilic compared to resveratrol.
Starting from bioavailability results of 4a-f compounds, we evaluated its effects on cell proliferation
against estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER−) SKBR3
human breast cancer cell line (Fig. 3) at different concentration (1, 5, 10, 20 µM) using the MTT
assay.36 Therefore, we treated for 72 h MCF-7 and SKBR3 cells with each compounds and also
with RSV in order to compare the antiproliferative effects of the chemicals used to this well-known
anticancer agent.36-44

Figure 3. Effects of different doses of 3-chloro-azetidin-2-one derivatives on MCF-7 (a) and SKBR3 (b) cell
proliferation. Cells were treated for 72 h with the indicated concentrations of compounds 4a-f. Cell viability was
evaluated by MTT assay. Statistically significant differences are indicated. Columns, mean of three independent
experiments each performed with triplicate samples expressed as percent of basal. bars, SD; (*P<0.01 compared with
basal).
As showed in figure 3, in MCF7 cells (Fig. 3a) 4d-f compounds, determined a clear dose-dependent
inhibitory effect on cell growth. Higher doses of 4a compound elicited an stimulation while only

low dose of 4b compound favored it. However, 4c compound decreased cell viability only at 20 µM
concentration.
Among all tested compounds in SKBR3 cells (Fig. 3b), higher concentrations of 4b, 4c, 4e
derivatives elicited a negative inhibitory effect. Remarkably, a 72 h treatment with 4a compound
(20 µM) was able to prevent SkBr3 cell growth. It should be pointed out that 4d and 4f compounds
inhibited the proliferation in a dose dependent manner in both estrogen dependent MCF-7 and in
estrogen independent SKBR3 cell lines suggesting that these compounds could be potentially active
in different breast cancer subtypes.
As showed in Figure 3, 4d, 4f compounds are more active, as also evidenced by its half-maximal
inhibitory concentration (IC50) values (Table 2) that are much more relevant respect to IC50 value of
RSV.
Table 2. IC50 of resveratrol and its derivatives 4d and 4f for MCF7 and SKBR3 cells on cell viability.
Compounds
MCF7
SKBR3
IC50
95% confidence IC50
95% confidence
interval
(µM)
interval
(µM)
4d
4f
16.72
11.77
28.38
13.42-20.84
8.93-15.52
25.71-31.33
11.09
9.51
8.37-14.68
7.69-11.75
34.21-49.67
RSV
41.22
In fact, it is evident that the dose at which RSV shows antiproliferative effects on MCF7 and
SKBR3 cells (Supplementary data Fig. S1),is relatively higher (>20 µM) (Table 2)45 compared to
that of the tested compounds.
On the basis of the aforementioned promising results obtained in two different model systems of
breast cancer cells, the capability of 4d and 4f compounds to elicit strong repressive effects on
breast cancer cell growth, could be determined by presence, as electron- attractor atoms, of two
halogens on aromatic rings.
In addition, a control experiment using 3T3 mouse embryonic fibroblast cells has been performed;
no effects on cell viability was obtained using all azetidin-based resveratrol derivatives of 5 µM
from to 20 µM after 72 h of treatment (Supplementary data Fig. S2), suggesting that these
compounds have specific inhibitory effect on breast cancer cells.

In conclusion, considering the widespread chemopreventive and chemotherapeutic applications of
resveratrol, a strong demand exists to search for other pharmacologically active resveratrol analogs
with enhanced bioavailability, potency and selectivity. For such reason, a series of new 3-chloro-
azetidin-2-one derivatives has been synthesized and evaluated for their growth regulatory effects in
MCF7 and SkBr3 human breast cancer cells. Among these compounds, that showed moderate to
high antitumor activity and displayed a more in vitro bioavailability respect to resveratrol, the
strongest antiproliferative activity against human breast cancer cells tested was displayed especially
by 4d and 4f. Hence, the capability of these compounds to have greater bioavailability than
resveratrol and to elicit selective inhibitory effects on breast cancer cell growth could be taken into
account towards novel pharmacological approaches in breast cancer therapy. Experiments useful to
investigate the molecular mechanism involved and to evaluate in vivo bioavailability and
chemotherapeutic potential of compounds are in progress.
Acknowledgments
This work was supported by grants from the Italian Ministry of Education (MIUR) (PRIN n°
20098SJX4F) and by Associazione Italiana per la Ricerca sul Cancro (AIRC) project n. IG10344.
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32. Experimental section: Reagents, starting material and solvents were purchased from Sigma-Aldrich (Milano,
Italy) and used as received. Analytical TLC was performed on plates coated with a 0.25 mm layer of silica gel
60 F254 Merck and preparative TLC on 20x20 cm glass plates coated with a 2 mm layer of silica gel PF254
Merck. Silica gel 60 (300–400 mesh, Merck) was used for flash chromatography. Melting points were measured
1
with a Köfler apparatus and are uncorrected. H and 13C NMR spectra were recorded with a Bruker 300
spectrometer operating at 300 and 75 MHz, respectively. Chemical shifts are reported in δ values (ppm) relative
to internal Me4Si and J values are reported in Hz. Mass spectra were obtained using a ESI mass spectrometer:
Finnigan LCQ Advantage max (Thermo Finnigan; San Jose, CA, USA).
General Procedure for the synthesis of imine (3). To a solution of the arylamine (10 mmol) in toluene (40 mL)
was added aryl-aldehyde (12 mmol) and the mixture was refluxed overnight using a Dean–Stark water
separator (monitored by TLC). When the reaction was over, toluene was evaporated under reduced pressure,
and the crude product was used as such for the next reaction.
General Procedure for the synthesis of β-lactam (4). A solution consisting of 2-chloro-acetylchloride (1.5
mmol) in dichloromethane (10 mL) was added drop wise to a stirred solution containing imine (1 mmol) and
distilled triethylamine (3 mmol) in dry dichloromethane (10 mL) at -78 °C. The reaction mixture was then
stirred overnight at room temperature, washed with saturated sodium bicarbonate solution (10 mL), dilute
hydrochloric acid (10%, 10 mL), brine (10 mL), dried with anhydrous sodium sulfate, and evaporated to obtain
the crude product. The pure product (48–68%) was then isolated via column chromatography over silica gel
using ethyl acetate : hexanes (1:4) as the solvent.
1
3-Chloro-1,4-di-p-tolylazetidin-2-one (4a). Yellow solid. Yield 51%. Mp 103-104 ºC. H NMR (300 MHz,
CDCl3): δ 2.29 (d, 6H), 5.27 (d, J = 6 Hz, 1H), 5.39 (d, J = 6 Hz, 1H), 7.07 (m, 2H), 7.22-7.26 (m, 4H), 7.28
(m, 2H). 13C NMR (75 MHz, CDCl3): δ 21.3, 62.0, 68.1, 125.3, 129.2, 133.4, 136.4, 136.5, 136.8, 140.5,
162.2. ESI m/z calcd for 285.77; found: 286.04. Anal Calcd for C17H16ClNO (%): C, 71.45; H, 5.64. Found
(%): C, 71.48; H, 5.62.
1
3-Chloro-1,4-diphenylazetidin-2-one (4b). White solid. Yield 61%. Mp 189-190 ºC. H NMR (300 MHz,
CDCl3): δ 5.30 (d, J = 3 Hz,1H), 5.43 (d, J =6 Hz, 1H), 7.13 (m, 2H), 7.26-7.40 (m, 6H), 7.43 (m, 2H).13C

NMR (75 MHz, CDCl3): 62.0, 68.1,117.7, 126.7, 126.9, 128.0, 128.5, 129.9, 139.5, 143.5,162.2. ESI m/z calcd
for C15H12ClNO: 257.71; found: 257.95. Anal Calcd for C15H12ClNO (%): C, 69.91; H, 4.69. Found (%): C,
69.94; H, 4.71.
1
3-Chloro-4-(4-chlorophenyl)-1-phenylazetidin-2-one (4c). White solid. Yield 48%. Mp 178-179 ºC. H NMR
(300 MHz, CDCl3): δ 5.33 (d, J = 12 Hz, 1H), 5.41 (d, J = 6 Hz, 1H), 7.14 (m, 2H), 7.26-7.31 (m, 5H), 7.40
(m, 2H). 13C NMR (75 MHz, CDCl3): 62.0, 68.1, 117.7, 127.2, 128.6, 128.0, 128.9, 132.3, 139.5, 141.6, 162.2.
ESI m/z calcd for C15H11Cl2NO: 292.16; found: 292.08. Anal Calcd for C15H11Cl2NO (%): C, 61.67; H, 3.79.
Found (%): C, 61.70; H, 3.81.
1
1,4-Bis(4-bromophenyl)-3-chloroazetidin-2-one (4d). White solid. Yield 59%. Mp 163-164 ºC. H NMR (300
MHz, CDCl3): δ 5.30 (d, J = 3 Hz, 1H), 5.39 (d, J = 6 Hz, 1H), 7.17-7.20 (m, 4H), 7.44 (m, 2H), 7.56 (m,
2H).13C NMR (75 MHz, CDCl3):62.0, 68.1, 121.1, 122.3, 127.2, 131.4, 131.8, 136.7, 138.5, 142.5, 162.2. ESI
m/z calcd for C15H10Br2ClNO: 415.51; found: 415.03. Anal Calcd for C15H10Br2ClNO (%): C, 43.36; H, 2.43.
Found (%): C, 43.34; H, 2.40.
1
3-Chloro-1-(4-chlorophenyl)-4-phenylazetidin-2-one (4e). White solid. Yield 65%. Mp 156-157 ºC. H NMR
(300 MHz, CDCl3): δ 5.30 (d, J = 3 Hz, 1H), 5.41(d, J = 6Hz, 1H), 7.31-7.44 (m, 7H), 7.54 (m, 2H).13C NMR
(75 MHz, CDCl3): 62.0, 68.1, 86.1, 123.2, 127.2, 128.6, 132.3, 137.8, 138.4, 141.6, 162.2. ESI m/z calcd for
C15H11Cl2NO: 292.17; found: 292.04. Anal Calcd for C15H11Cl2NO (%): C, 61.67; H, 3.79. Found (%): C,
61.70; H, 3.77.
3-Chloro-4-(4-chlorophenyl)-1-(4-iodophenyl)azetidin-2-one (4f). Yellow solid. Yield 68%. Mp 191-193 ºC.
1H NMR (300 MHz, CDCl3): δ 5.30 (d, J = 6 Hz, 1H), 5.40 (d, J = 6 Hz,1H), 7.06 (m, 2H), 7.25 (m, 2H), 7.40
(m, 2H), 7.60 (m, 2H). 13C NMR (75 MHz, CDCl3):62.0, 68.1, 86.1, 123.2, 127.2, 128.6, 132.3, 137.8, 138.4,
141.6, 162.2. ESI m/z calcd for C15H10Cl2INO: 418.06; found: 418.10. Anal Calcd for C15H10Cl2INO (%): C,
43.10; H, 2.41. Found (%): C, 43.08; H, 2.43.
33. Banik, B.K.; Becker, F.F.; Banik, I. Bioorg. Med. Chem. 2004, 12, 2523.
34. In vitro bioavailability studies: In vitro bioavailability studies in simulated gastric and intestinal fluids were
carried out by performing a slight modified version of the dialysis tubing procedure.35
The dialysis tubing method is characterized by two consecutive enzymatic digestions: pepsin and pancreatin
digestion, respectively. These steps are described as follows.
Pepsin Digestion. 100 μL of each sample (4a-f, 10 mM in DMSO) were mixed with 1.0 mL of a 0.85 N HCl
solution containing 24000 U of porcine pepsin per mL and 3 mL of a sodium cholate solution (2% w/v in
distilled water). The obtained mixture was introduced into a dialysis bag (Spectrum Laboratories Inc., MWCO:
12-14,000 Dalton, USA) which was then carefully closed and immersed inside a flask containing 10 mL of a
0.85 N HCl solution (pH 1.0). The flask was then incubated into a shaking water bath at 37°C to simulate the
human body conditions of temperature for 2 h.
Pancreatin Digestion. At the end of the 2 h pepsin digestion, the dialysis bag was opened and 11 mg of
amylase, 11 mg of esterase and 1.3 mL of a 0.8 M NaHCO3 solution containing 22.60 mg porcine
pancreatin/mL were added to the peptic digesta. After the digesta and enzyme solution were well-mixed, the
dialysis bag was sealed on each end with clamps and placed into a flask with 10 mL of buffer solution at pH
7.0. The flask was incubated into the shaking water bath at 37°C for further 4 h.
In the aim to evaluate the bioavailability of the different samples, 2 mL of the medium were withdrawn from
the flask for sample analysis at the time points of two and six hours, after pepsin and pancreatin digestions
respectively. The concentration of the samples was determined by UV/VIS spectroscopy (UV/VIS spectrometer
V-530, Jasco – U.S.) and the percentages were calculated by using the equations obtained from the calibration
curves of five standard solutions, for each tested compound, at pH 1.0 and 7.0 respectively.
For this purpose, all the prepared standard solutions were analysed by UV-Vis spectrophotometer and the
correlation coefficient (R2), slope and intercept of the regression equations obtained by the method of least
square were calculated at pH 1.0 and 7.0, respectively. Each experiment was performed in triplicate.
35. Bollinger, D.W.; Tsunoda, A.; Ledoux, D.R.; Ellersieck, M.R.; Veum, T.L. J. Agric. Food Chem. 2005, 53,
3287.
36. Cell culture and treatments. MCF-7 breast cancer cells (a ER positive breast cancer cells, obtained from
American Type Culture Collection (ATCC), Manassas, VA, USA) were maintained as previously described.37
SKBR3 breast cancer cells (a ER negative breast cancer cells, obtained from American Type Culture Collection
(ATCC), Manassas, VA, USA) were maintained in RPMI1640 without phenol red supplemented with 10% fetal
bovine serum (FBS), 1% glutamine and 1% penicillin/streptomycin (Sigma-Aldrich, Milano, Italy) (complete
medium).38 3T3 mouse embryonic fibroblast cells, obtained from American Type Culture Collection (ATCC,
Manassas, VA, USA), were maintained in DMEM with phenol red supplemented with 10% fetal bovine serum
(FBS), 1% glutamine and 1% penicillin/streptomycin (Sigma-Aldrich, Milano, Italy) (complete medium). Cells
were maintained at 37˚C in a humidified atmosphere of 95% air and 5% CO2 and were screened periodically for
Mycoplasma contamination. All 3-chloro-azetidin-2-one derivatives compounds were dissolved in
dimethylsulfoxide (DMSO) (Sigma, St. Louis, Missouri, USA) at a concentration of 10 mM and diluted in
DMEM/F12 (for MCF-7 cells), in DMEM (for 3T3 cells) or in RPMI (for SKBR3 cells) medium supplemented

with 1% DCC-FBS (dextran-coated charcoal-treated newborn calf serum serum) to obtain the working
concentration.
Assessment of Cell Viability. MCF-7, SKBR3 and 3T3 cells were seeded on twenty-four well plates (0.2x105
cells/well) and grown for 48h in complete medium. Before being treated, cells were starved in DMEM/F12 (for
MCF-7 cells), in DMEM (for 3T3 cells) or in RPMI (for SKBR3 cells) serum free medium for 24 h to the
purpose of cell cycle synchronization. The effect of the different doses of 3-chloro-azetidin-2-one derivatives
was measured using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) assay as
previously described 28,38-44. Seventy two hours after treatments, fresh MTT (Sigma), re-suspended in PBS, was
added to each well (final concentration (0.33 mg/ml). After 3h incubation, cells were lysed with 1 ml of DMSO.
Each experiment was performed in triplicate and the optical density was measured at 570 nm in a
spectrophotometer. Statistical analyses. All experiments were conducted at least three times and the results
were from representative experiments. Data were expressed as mean values standard deviation (SD), and the
statistical significance between control (basal) and treated samples was analyzed using the GraphPad Prism 5
software program. The unpaired Student’s t -test was used to compare two groups. P < 0.05 was considered
statistically significant.
37 Sirianni, R.; Capparelli, C.; Chimento, A.; Panza, S.; Catalano, S.; Lanzino, M.; Pezzi, V.; Andò, S. Mol Cell
Endocrinol. 2012, 363, 100.
38 Chimento, A.; Casaburi, I.; Rosano, C.; Avena, P.; De Luca, A.; Campana, C.; Martire, E.; Santolla, M.F.;
Maggiolini, M.; Pezzi, V.; Sirianni, R. Mol Nutr Food Res. 2013 Sep 9,doi: 10.1002/mnfr.201300323.
39 Sirianni, R.; Zolea, F.; Chimento, A.; Ruggiero, C.,Cerquetti, L.; Fallo, F.; Pilon, C.; Arnaldi, G.; Carpinelli, G.;
Stigliano, A.; Pezzi, V. J Clin Endocrinol Metab. 2012, 97, E2238.
40 Caruso, A.; Chimento, A.; El-Kashef, H.; Lancelot, J.C.; Panno, A.; Pezzi, V.; Saturnino, C.; Sinicropi, M.S.;
Sirianni, R.; Rault S. J. Enzym. Inhib. Med. Chem. 2012, 27, 60.
41 Sinicropi, M.S.; Caruso, A.; Conforti, F.; Marrelli, M.; El Kashef, H.; Lancelot, J.C.; Rault, S.; Statti, G.A.;
Menichini, F. J. Enzym. Inhib. Med. Chem. 2009, 24, 1148.
42 Caruso, A.; Chimento, A.; El-Kashef, H.; Lancelot, J.C.; Panno, A.; Pezzi, V.; Saturnino, C.; Sinicropi, M.S.;
Sirianni, R.; Rault S. J. Enzym. Inhib. Med. Chem. 2012, 27, 60.
43 Sinicropi, M.S.; Caruso, A.; Conforti, F.; Marrelli, M.; El Kashef, H.; Lancelot, J.C.; Rault, S.; Statti, G.A.;
Menichini, F. J. Enzym. Inhib. Med. Chem. 2009, 24, 1148.
44 Sirignano, E.; Saturnino, C.; Botta, A.; Sinicropi, M.S.; Caruso, A.; Pisano, A.; Lappano, R.; Maggiolini, M.;
Longo P. Bioorg. Med. Chem. Lett. 2013, 23, 3458.
45 (a) Pozo-Guisado, E.; Lorenzo-Benayas, M.J.; Fernández-Salguero P.M. Int. J. Cancer. 2004, 109, 167; (b)
Pozo-Guisado, E.; Alvarez-Barrientos, A.; Mulero-Navarro, S.; Santiago-Josefat, B.; Fernandez-Salguero, P.M.
Biochem. Pharmacol. 2002, 64, 1375.

Graphical Abstract
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Biological activity of 3-chloro-azetidin-2-one
derivatives having interesting antiproliferative
activity on human breast cancer cell lines
Leave this area blank for abstract info.
Adele Chimento, Marina Sala, Isabel M. Gomez-Monterrey, Simona Musella, Alessia Bertamino, Anna Caruso,
Maria Stefania Sinicropi, Rosa Sirianni, Francesco Puoci, Ortensia Ilaria Parisi, Carmela Campana, Emilia
Martire, Ettore Novellino, Carmela Saturnino, Pietro Campiglia and Vincenzo Pezzi.
O
O
R
H
R'
NH2
O
R'
Cl
N
Cl
Toluene
Cl
+
N
TEA
CH2Cl2
-78°C
R'
R
R