1,2,4-Oxadiazole-5-ones as analogues of tamoxifen: Synthesis and biological evaluation

Article abstract of DOI:10.1039/c9ob00651f

A series of 2,3,4-triaryl-substituted 1,2,4-oxadiazole-5-ones have been prepared as fixed-ring analogues of tamoxifen (TAM), a drug inhibitor of Estradiol Receptor (ER) used in breast cancer therapy, by an efficient synthetic protocol based on a 1,3-dipolar cycloaddition of nitrones to isocyanates. Some of the newly synthesized compounds (14d-f, 14h and 14k) show a significant cytotoxic effect in a human breast cancer cell line (MCF-7) possessing IC₅₀ values between 15.63 and 31.82 μM. In addition, compounds 14d-f, 14h and 14k are able to increase the p53 expression levels, activating also the apoptotic pathway. Molecular modeling studies of novel compounds performed on the crystal structure of ER reveal the presence of strong hydrophobic interactions with the aromatic rings of the ligands similar to TAM. These data suggest that 1,2,4-oxadiazole-5-ones can be considered analogues of TAM, and that their anticancer activity might be partially due to ER inhibition.

Full text of DOI:10.1039/c9ob00651f

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Chem., 2019, DOI: 10.1039/C9OB00651F.
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Takeharu Haino et al.
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DOI: 10.1039/C9OB00651F
1,2,4-OXADIAZOLE-5-ONES AS ANALOGUES OF TAMOXIFEN:
SYNTHESIS AND BIOLOGICAL EVALUATION
Maria A. Chiacchio,a Laura Legnani,a Agata Campisi,*a Bottino Paola,a Lanza
Giuseppe,a Daniela Iannazzo,b Lucia Veltri,c Salvatore Giofrèd and Roberto Romeod
a
Dipartimento di Scienze del Farmaco, Università di Catania, Viale A. Doria 6, 95125 Catania,
Italy.
b Dipartimento di Ingegneria, Università di Messina, Contrada Di Dio, 98166 Messina, Italy.
c
Dipartimento di Chimica e tecnologie chimiche, Università della Calabria,Via P. Bucci 12/C,
87036 Arcavacata di Rende, Italy.
d
Dipartimento di Scienze chimiche, biologiche, farmaceutiche ed ambientali, Università di
Messina, Via S.S. Annunziata, 98168 Messina, Italy.
Abstract. A series of 2,3,4-triaryl-substituted 1,2,4-oxadiazole-5-ones have been prepared as fixed-
ring analogues of tamoxifen (TAM), a drug inhibitor of Estradiol Receptor (ER) used in breast
cancer therapy, by an efficient synthetic protocol based on a 1,3-dipolar cycloaddition of nitrones to
isocyanates. Some of new synthesized compounds (14d-f, 14h and 14k) show a significant
cytotoxic effect in human breast cancer cell line (MCF-7) possessing IC50 values between 15.63 and
31.82 µM. In addition, compounds 14d-f, 14h and 14k are able to increase the p53 expression
levels, activating also the apoptotic pathway. Molecular modeling studies of novel compounds
performed on the crystal structure of ER, reveal the presence of strong hydrophobic interactions
with the aromatic rings of the ligands similar to TAM. These data suggest that 1,2,4-oxadiazole-5-
ones can be considered analogues of TAM, and that their anticancer activity might be partially due
to ER inhibition.
1. Introduction
Breast cancer is the most commonly diagnosed disease among women worldwide with over 2
million of new cases in 2018.1 Many experimental and clinical evidences point out that other than
genetic factors, steroid hormones play an important role in the etiology of this disease.2
Estrogen mediates its effects by binding to its receptors, ERα and ERβ, and support the
development and growth of the breast tumor cells and show also profound and carefully regulated
effects on other tissues, such as the endometrium, vagina, bone, liver and vessels of the
cardiovascular system.3

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ER, interacting with estrogen response elements (EREs) contained in the promoter region of
DOI: 10.1039/C9OB00651F
specific genes, modulates gene expressions that results in their biological effects. Extracellular
signals can also stimulate ERα-mediated transcription in the absence of estrogen.
In recent years, emerging evidence has revealed that a role for ERα is to affect gene expression in
the absence of direct DNA binding. In addition, ER activity in breast cancer is associated with drug
resistance, tumor growth, increased invasiveness, poor patient prognosis, and loss of p53 function.4
Tumor suppressor protein p53 plays a key role in regulation of several cellular processes, including
apoptosis, DNA repair and angiogenesis; it is a direct transcriptional target of ERα and modulates
DNA damage-induced growth in breast cancer cells.5
A great number of estrogens antagonists (antiestrogens) have been synthesized and tested with
regard to their binding affinity to the ER, a ligand-activated transcription factor that act by binding
to EREs.6 The assumption that the utilization of estrogen antagonists for the treatment of breast
cancers is capable of avoiding the progress of the disease has attracted considerable interest:7
pharmacological inhibition of the tumor stimulatory effects of physiological estrogens by an
antiestrogen therapy was reported since the 1940s.
Several findings demonstrated that synthetic efforts have led to the discovery and development of
various potent synthetic antiestrogens, many of them characterized by a stilbene-like skeleton such
as Tamoxifen (TAM).8
TAM 1, a non-steroidal, triphenylethylene-based compound (Fig. 1), has a complex pharmacology
consisting of both estrogenic and anti-estrogenic properties in several tissues.9 This drug has
revolutionized the treatment of breast cancer and it is considered the antiestrogen of choice in the
clinic to date. It inhibits proliferation and induces apoptosis in ER-positive breast cancer cell line
MCF-7, by selective estrogen receptor (ER)-dependent modulation of gene expression.10 After oral
administration, TAM is converted, by cytochrome P450 metabolism, into 4-hydroxytamoxifen 2 and
4-hydroxy-N-desmethyltamoxifen 3 (Fig. 1), potent antagonists of ERs and inhibitors of estrogen-
responsive gene transcription.11

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DOI: 10.1039/C9OB00651F
Fig. 1 Tamoxifen and its metabolites with anti-estrogenic activity.
Since TAM 1 and its metabolites 2 and 3 contain a double bond, formation of E and Z isomers can
occur, with remarkably different binding affinities and effects at ER. For example, Z-TAM binds to
ER with a 100-fold greater affinity than E-TAM: Z-TAM acts as an ER antagonist, while E-TAM
acts as an ER agonist.12 Similar differences in affinity and activity have also been observed with
metabolites 2 and 3:13 (Z)-4-hydroxytamoxifen exhibits an 8-fold higher binding affinity to the ER
as compared to TAM, whereas the (E) isomer has only a 5% affinity to the ER.14 TAM also can
induce side effects, such as the uterine endometrial cancer and the non-alcoholic fatty liver disease.
These effects are a substantial concern for patients that have to continue TAM treatment for up to
10 years.15 Thus, after the approval of TAM for the treatment of breast cancer, considerable
research efforts have been devoted towards the design and the discovery of novel TAM analogs
with increased binding affinity for ER, able to combat the often reported TAM side effects and
acquired resistance.
In this context, a synthetic strategy exploits the synthesis of new non-steroidal fixed ring structures
derived from the stilbene skeleton in the aim to prevent the isomerization that occurs around the
double bond in the triphenylethylenes. These structures include benzothiophenes (Raloxifene 4,
SERM 5),16,17 naphthalenes (lasofoxifene 6, LY 326.315 7),18 benzopyrans (EM 800 8, acolbifene
9)19 and benzocycloheptenes 10 and 1120 that cannot isomerize (Fig. 2).

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DOI: 10.1039/C9OB00651F
N
N
O
O
O
O
OH
OH
S
S
HO
HO
4
Raloxifene
5
SERM 3
N
O
O
N
OH
HO
HO
7
6
Lasofoxifene
LY 326,315
O
OH
O
O
HO
O
O
O
N
N
O
O
9
Acolbifene
8
EM 800
O
N
O
N
10
11
Fig. 2 Non-steroidal fixed ring structures derived from the stilbene skeleton.
According to the same strategy, we wanted to explore if the substitution of the Z double bond with a
conformationally restricted cyclic C-N bond still keeps the biological features of TAM. Thus, we
have synthetized a series of new cis-restricted stilbenes 14 (Fig. 3), characterized by the presence of
an 1,2,4-oxadiazole-5-one system, where the C=C double bond has been replaced by a cis locked C-
N bond, as a potential anti-breast cancer agents. The choice of 1,2,4-oxadiazole unit as linker of the
two aromatic rings of stilbenes arises from the consideration that this heterocyclic nucleus itself
shows anticancer activity in different cancer cell lines.21
The new synthesized compounds were subsequently screened in MCF-7 cancer cell line, evaluating
their effect on cellular viability. The most active compounds were assessed on the expression levels

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of p53 and the ability to activate the apoptotic pathway by testing caspase-3 cleavagDeOwI: 1a0s.10d3e9/tCe9cOtBe0d0.651F
The effect of the compounds was also compared with normal human fibroblast primary cell cultures
(HLF), using TAM as drug control. Furthermore, molecular docking studies were assessed in order
to investigate the binding affinity of the synthesized compounds to ER.
2. Results and discussion
The synthetic approach leading to the formation of the differently substituted 1,2,4-oxazolidinyl-5-
ones 14a-k is based on the 1,3-dipolar cycloaddition of nitrones 12a-f to isocyanates 13a-d as
dipolarophiles (Fig. 3). Nitrones 12a-f, obtained by reaction of the corresponding aldehydes with N-
methyl or N-aryl hydroxylamine,22 were reacted with isocyanate 13a-d at room temperature in
dichloromethane dry (or dry acetone) and in nitrogen atmosphere for 24 h, leading to cycloadducts
14a-k in good yields ( 58-98%) (Table 1). Lower yields were observed for entries 4-6 underlying
the pivotal role of the substituent present at the nitrogen atom of the nitrone moiety in the control of
cycloaddition process efficiency (Table 1). In particular, the presence of the 4-MeO-C6H4-CH2-
group onto nitrone moiety (R1) increases the nucleophilicity of 12c, favoring the formation of by-
products and reducing the yields of the desired cycloadducts 14d-f.
Fig. 3 Reaction of nitrones 12a-f with isocyanates 13a-d.

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Table 1. Synthesis of 1,2,4-oxazolidinyl-5-ones 14a-k by 1,3-dipolar cycloaddition. DOI: 10.1039/C9OB00651F
R2
R1
N
N Ar
O
14a-k
O
Entry
R1
R2
Ar
Product
(yield (%))a
14a (70%)
14b (85%)
14c (98%)
1
2
3
Me
Ph
Ph
Ph
Ph
Ph
4-NO2-C6H4
4-NO2-C6H4
4-Cl-C6H4
4
5
6
7
8
9
10
11
4-MeO-C6H4-CH2
4-MeO-C6H4-CH2
4-MeO-C6H4-CH2
Ph-CH2
Ph
Ph
Ph
4-NO2-C6H4
4-Cl-C6H4
4-CF3-C6H4
4-NO2-C6H4
4-Cl-C6H4
4-NO2-C6H4
4-F-C6H4
4-NO2-C6H4
14d (68%)
14e (58%)
14f (71%)
14g (81%)
14h (79%)
14i (76%)
14j (77%)
14k (83%)
9-Phenanthryl
9-Phenanthryl
4-Py
Ph-CH2
Ph
Ph
4-F-C6H4
4-Py
4-Py
a Isolated yields.
The structure of the obtained adducts has been assigned on the basis of spectroscopic data. In
1
particular, the H NMR spectra of the synthesized compounds show the presence of the diagnostic
methine proton at C-3, which is located in the range 5.80-6.10 ppm, typical of a NCH(R)N system,
as in compounds 14. In the alternative regioisomers 15, the methine proton at C-2 should resonate at
higher fields as a consequence of O=CCH(R)N structure that involves an higher charge density on
these protons according to a reduced electronegative effect of the carbon respect to nitrogen one.
Similar trends have been obtained for the 13C of methine carbons of adducts 14 that fall to  values
between 77.5-86.9 ppm. 13C spectra of methine carbons for compounds 15 is expected to occur
between 67-70 ppm. As a further support to the assigned structure, the presence in the MS spectra
of the diagnostic peak at M+ -44, resulting from the loss of CO2 from the molecular ion, is only
amenable towards cycloadducts 14. Furthermore, the isomers 16 and 17, obtainable from a
alternative site selectivity of the nitrone 12 on the double C=O bond of isocyanate 13, should not
1
present these values of chemical shift. In particular the H NMR spectra of CH proton of
compounds 16 should resonate at 5.0-4.9 ppm according to a CHNO assemblage, while the same
signal into compounds 17 should resonate at lower fields and in the range between 6.7-6.5  due to
OCHN structure.
The obtained results indicate that the reaction proceeds with complete site- and regioselectivity
leading to only 1,2,4-oxadiazolidin-5-ones 14a-k with no traces of the alternative cycloadducts 15-
17 (Fig. 3).

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DOI: 10.1039/C9OB00651F
Theoretical calculations
The observed site- and regioselectivity of the 1,3-dipolar cycloaddition process have been
rationalized using the Truhlar’s functional at the M06-2X23 level of theory with cc-pVTZ basis set.
The modeling study was performed on the reaction between nitrone 12b and isocyanate 13a.
In theory, four different approaches were expected to yield cycloadducts 14b-17b. In fact, the
nitrone 12b might attack the C=N site of isocyanate with a regioselectivity O1(1b)-C2(2b)/ C3(1b)-
N1(2b) giving 14b or O1(1b)-N1(2b)/ C3(1b)-C2(2b) with the obtainment of 15b or the C=O site
with the alternative regioselectivity O1(1b)-C2(2b)/ C3(1b)-O(2b) and O1(1b)-O(2b)/ C3(1b)-
C2(2b) that gives 16b and 17b, respectively. Experimentally, only compound 14b was obtained.
Fig. 4 Expected cycloadducts from reaction of 12b with 13a.
In order to rationalize the regioselectivity of the reaction, firstly, the reagents were optimized and
the electronic effects taken under consideration.24 Considering the HOMO-LUMO relative position,
the dominant electronic interaction involves the HOMO of the dipole and the LUMO of the
dipolarophile (ΔE HOMO12b-LUMO13a < ΔE HOMO13a-LUMO12b); so it is evident that the
reaction is HOMO-controlled dipole (Fig. 5).

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DOI: 10.1039/C9OB00651F
Fig. 5 3D-plots of the frontier molecular orbitals (HOMO and LUMO) of reagents 12b and 13a.
In Table 2 the energy values of the HOMO and LUMO orbitals, the electrophilicity index ω,25 and
the nucleophilicity index N26 of compounds 12b and 13a are reported.
Table 2. Computed data of the minimum energy conformation of 12b and 13a.
Ehomo Elumo
(eV) (eV)
ω
N
12b -7.14 -1.04 1.37 4.02
13a -8.86 -1.76 1.98 2.31
The electrophilicity ω value for 13a is 1.98 eV, corresponding to strong electrophiles. Nitrone 12b
presents a lower ω value (1.37 eV), still in the range of moderate electrophiles. On the other hand,
12b shows a high nucleophilicity index N of 4.02 eV, typical of strong nucleophiles, while
isocyanate 13a, showing N of 2.30 eV is a very moderate nucleophile. So, on the basis of the
calculated indices, it is possible to assert that in this cycloaddition the nitrone 12b is the
nucleophile, and isocyanate 13a is the electrophile.
Once the nucleophile and the electrophile of the reaction have been identified, we proceeded to
rationalize the regioselectivity of the reaction considering that, in a polar cycloaddition between
non-symmetrical compounds, the most favorable interaction involves the most nucleophilic center
of the nucleophile and the most electrophilic center of the electrophile.26 As well known, Parr
functions are very useful, in order to determine the local reactivity. So, the following step was to

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calculate nucleophilic Pk− Parr functions for nitrone 12b and electrophilic Pk+ Parr function for
DOI: 10.1039/C9OB00651F
isocyanate 13a.27 The calculated values are reported in Table 3.
Table 3. Values of nucleophilic Pk− Parr functions for nitrone 12b and electrophilic Pk+ Parr
function for isocyanate 13a.
12b
Pk+
13a
Pk+
Pk−
Pk−
N2
O1
C3
0.029
0.526
0.136
N1
C2
O3
-0.045
0.063
0.029
In the case of nitrone 12b, considering the nucleophilic Pk− Parr function values, O1 is the most
nucleophilically activated center. The electrophilic Pk+ Parr functions for isocyanate 13a indicate
that C2 is the most electrophilic center. Consequently, the most favorable electrophile-nucleophile
interaction along the reaction takes place between O1 atom of 12b and C2 atom of 13a. The reaction
could be considered completely regioselective, with the possibility to obtain only derivatives 14b
and 16b.
Then, in order to determine the site-selectivity of the reaction, and to support the obtained
cycloadducts, starting by data reported by Merino et al.,28 the transition states leading to 14b, 15b,
16b and 17b were constructed and located. Optimizations were performed in vacuo at the M06-
2X/cc-pVTZ level,29 as reported in literature.30
In Fig. 6 are shown the 3-D plots of the four transition states TS3, TS4, TS5, and TS6 for the
different reaction channels, with the forming bonds distances reported (Å).

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DOI: 10.1039/C9OB00651F
Fig. 6 3-D plots of the four preferred transition states TS3, TS5 and TS4,TS6 leading to expected
cycloadducts 14b, 15b, 16b and 17b. The forming bonds distances are reported in Å.
The IRC analysis allowed to verify that the reaction is concerted, albeit asynchronous as evident by
the bond distances. The transition state TS3 is favorite (ΔG= 19.83 kcal/mol; ΔE= 4.35 kcal/mol;
99% (408 K)) with respect to TS5 (ΔG= 25.73 kcal/mol; ΔE= 11.71 kcal/mol; 1% (408 K)) and in
particular to respect to TS4 (ΔG= 49.36 kcal/mol; ΔE= 34.84 kcal/mol) and TS6 (ΔG= 59.65
kcal/mol; ΔE= 44.56 kcal/mol) that are too high to be reached. So, computational data showed a
total site-selectivity with the exclusive obtainment of product 14b.
In conclusion, the modeling study, according to the experimental results, rationalized that the 1,3-
dipolar cycloaddition of 12b and 13a is regio- and site-selective.

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DOI: 10.1039/C9OB00651F
BIOLOGICAL RESULTS
Cellular viability
To monitor cell viability MTT assays were used. MCF-7 cancer cell line and HLF primary cell
cultures were exposed to different concentrations (0.5–100 µM) of the tested compounds for 24 h.
In parallel, a group of cell cultures was treated with 10 µM of TAM for 24 h. The concentration of
compound required to cause 50% inhibition of cell proliferation (IC50) was calculated from
concentration–effect curves using Prism 5.0 (GrafPAD Software for Science) and a non-linear
regression analysis was used (Tab. 4).
The compounds 14d-f, 14h and 14k showed a significant degree of cytotoxic on the MCF-7 cell
line, showing a range of activity of IC50 between 15.63 and 31.82 µM. The most active compound
was 14d that showed an IC50 value of 15.63 µM. In contrast, the other compounds 14a-c, 14g, 14i,
and 14j displayed a low level of cytotoxicity having a range between 80.23 and 85.34 µM. The
exposure of MCF-7 cell line to 10 µM of TAM for 24 h induced a significant decrease of cellular
viability (IC50 = 10.38 µM). A low decrease in cellular viability in HLF cells, exposed for 24 h,
when were treated with 10 µM TAM or with the synthesized compounds at all concentrations was
observed.
Table 4. Concentrations (µM) of investigated compounds 14a–14k that induces for 24 h 50%
inhibition of cell proliferation (IC50) in MCF-7 human cancer cell line and HLF primary cell
cultures.
Compound
HLF
MCF-7
Untreated (control)
>100
>100
Tamoxifen
14a
82.14 ± 0.23
80.75 ± 0.32
81.23 ± 0.19
80.91 ± 0.21
82.01 ± 0.48
81.23 ± 0.15
81.93 ± 0.12
82.36 ± 0.24
81.99 ± 0.11
81.22 ± 0.27
81.48 ± 0.39
81.22 ± 0.31
10.38 ± 0.09*
85.34 ± 0.22
83.64 ± 0.17
80.23 ± 0.33
15.63 ± 0.26*
22.76 ± 0.08*
26.14 ± 0.21*
83.62 ± 0.22
17.80 ± 0.13*
80.97 ± 0.34
80.64 ± 0.28
31.82 ± 0.24*
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
Each data represents the mean ± SD from for independent experiments performed in triplicate.
Tamoxifen (10 µM) was uses as positive control. Values represent the mean ± S.D. of four
experiments performed in triplicate. *p<0.001vs control.

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Effect of the most active compounds (14d, 14h, 14e, 14f and 14k) on p53 expressiDoOnI: l1e0.v10e3l9s/C9OB00651F
Figure 7 shows representative immunoblots (Fig. 7A) and densitometric analysis (Fig. 7B) of p53
expression levels in total cell lysates from MCF-7 cell lines exposed to 10 µM 14d, 14h, 14e, 14f,
14k or TAM for 24 h. The treatment of the cells with these compounds led to a significant increase
of p53 protein expression levels, when compared with the untreated cells used as a control. The
effect appeared more evident when the cells were exposed to the compounds 14d, 14h, 14e and 14f.
Compound 14k showed a lower increase in p53 expression levels, when compared with the other
compounds. However, the levels of p53 expression were lower than observed in TAM exposed
cells.
Fig. 7: A) Representative Western blots and B) semiquantitative analyses of p53 expression levels
evaluated in total cell lysates from MCF-7 cell line unexposed (control) or exposed for 24 h to 10
µM 14d, 14h, 14e, 14f, 14k. Tamoxifen was used as drug control. Levels of protein were
normalized with β-tubulin. Values represent the mean ± S.D. of four separated experiments
performed in triplicate. *p<0.001 vs control.

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Effect of the most active compounds (14d, 14h, 14e, 14f and 14k) on caspase-3 cleavage
To elucidate whether 14d, 14h, 14e, 14f and 14k were able to activate the apoptotic pathway,
caspase-3 cleavage though Western Blot analysis was assessed (Fig. 8). Compounds 14d, 14h, 14e,
14f and 14k induced a significant increase of caspase-3 cleavage, when compared with the control.
The effect appeared more evident in 14d, 14h, 14e and 14f exposed cells, even if it appeared lower
than found cells treated with tamoxifen. Compound 14k was also able to activate caspase-3
cleavage but at lower levels than the other compounds.
Fig. 8: A) Representative Western blots and B) semiquantitative analyses of caspase-3 cleavage
evaluated in total cell lysates from MCF-7 cell line unexposed (control) or exposed for 24 h to 10
µM 14d, 14h, 14e, 14f, 14k. Tamoxifen was used as drug control. Cleaved caspase-3 levels were
normalized with β-tubulin. Values are the mean ± S.D. of four separated experiments performed in
triplicate. *p<0.001 vs control.

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Molecular modeling study
To explain the different activity of the novel compounds, a computational docking study was
undertaken for compounds 14a-k. The crystal structure of ER in complex with 4-OH-TAM (PDB
code: 3ERT) was selected based on a good experimental resolution (1.9 Å). The accuracy of the
docking protocol was validated by re-docking method of 4-OH-TAM in the binding site to
determine the lowest RMSD relative to the crystallographic pose. 4-OH-TAM was successfully re-
docked with a RMSD of 1.02 Å. The ERα has ligand-binding domain (LBD) which is
predominantly the hydrophobic cavity that composed by amino acid residues from helices 3, 6, 7, 8,
11, and 12. The agonist and antagonist activity of a ligand is determined by the helix-12 from
residues 536-544 in its macromolecule (ERα). When an antagonist for example 4-OHT binds to
LBD of ERα, the helix-12 will be closed and not binds to co-activator so it has the antagonist
activity based on the absence of hydrogen bond interaction with His524.32 Thus, the selective
binding of the potential anti-breast cancer agents will rely largely on contributions from
hydrophobic interactions. The docking of compounds 14a-k revealed a strong presence of
hydrophobic interactions and in a similar fashion as does 4-OH-TAM, nine out of eleven
derivatives, establish hydrogen bond with Arg394. Docking results are summarized in Table 5.
Table 5. Results of molecular docking of 14a-k in ligand binding domain of estrogen receptor alpha
(ERα)
Interactions with Amino Acids
Compound
Calculated Number
G
Ki (nM)
Kcal/mol
in Cluster
Hydrogen Bond van der Waals (Hydrophobic)
Met343,
Arg394, O (NO2) Trp383,
Leu346,
Leu384,
Ala350,
Met388,
14a
14b
-8.33
-9.77
789.90
100
Leu387, Leu391, Leu525
Met343,
Ala350,
Leu346,
Met388,
Thr347,
Leu391,
68.43
100
-
Met421, Ile424, Leu525,
Met343,
Leu349,
Leu387,
Leu346,
Ala350,
Leu391,
Thr347,
Trp383,
Phe404,
14c
14d
-8.85
325.69
100
51
Arg394, Cl
Met421, Ile424, Leu525,
-10.70 14.47
Arg394, O (OCH3)
Met343,
Leu346,
Thr347,

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Ala350,
Leu38D7O,I: 10.10M39/eCt93O8B080,651F
Leu391, Phe404, Met421, Ile424,
Leu525
Met343,
Ala350,
Leu346,
Leu387,
Thr347,
Met388,
14e
-9.53
-9.16
103.70
192.83
43
37
34
47
100
Arg394, O (OCH3)
Leu391, Phe404, Met421, Ile424,
Leu525, Met528
Met343,
Arg394, O (OCH3)Leu387,
Thr347, F (CF3) Phe404, Met421, Ile424, Leu525,
Met528
Leu346,
Met388,
Ala350,
Leu391,
14f
14g
14h
14i
Met343,
Leu349,
Leu384,
Leu346,
Ala350,
Leu387,
Thr347,
Trp383,
Leu391,
-10.04 43.36
-10.64 15.77
-
Phe404, Ile424, Leu525, Met528,
Met343,
Ala350,
Met388,
Leu346,
Trp383,
Met421,
Thr347,
Leu387,
Ile424,
Arg394, O (NO2)
Leu525, Met528
Met343,
Trp383,
Met388,
Leu525
Leu346,
Leu384,
Leu391,
Ala350,
Leu387,
Met421,
-9.85
60.27
-
Met343,
Ala350,
Met388,
Leu346,
Leu384,
Phe404,
Leu349,
Leu387,
Met421,
14j
-8.55
-8.93
537.92
296.10
100
84
Arg394, F
Arg394, F
Ile424, Leu525
Met343,
Leu384,
Ala350,
Leu387,
Trp383,
Met388,
14k
Leu391, Met421, Ile424, Leu525
Met343,
Ala350,
Glu353, Arg394 Leu387,
Leu346,
Trp383,
Met388,
Leu349,
Leu384,
Leu391,
4-OH-TAM -11.48 3.85
100
Phe404, Met421, Ile424, Leu428,
Leu525,

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DOI: 10.1039/C9OB00651F
Fig. 9 2D interactions of compounds 14d, 14e, 14f and 14h in the estrogen receptor-α ligand
binding domain.
Compounds 14a-k shared a similar interaction constituting an extensive hydrophobic network and
in addition compounds 14a, 14c-f, 14h and 14j-k showed hydrogen bond with Arg394. The best
docking pose of the most active compounds (14d, 14e, 14f, 14h) were analyzed to highlight
important binding interaction of the drug candidates as anticancer agents. Compounds 14d, 14e, 14f
and 14h revealed a strong presence of hydrophobic interactions between Met343, Leu346, Ala350,
Leu387, Met388, Leu391, Met421, Ile424 and Leu525 with the aromatic rings of the ligands.
Compounds 14d, 14f and 14h presented an additional hydrophobic interaction with Thr347 and the
compounds 14e and 14f showed two additional hydrophobic bonds with Phe404 and Met528, while
compound 14h differed by van der Waals interaction with Trp383 as well as the compound 14d

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formed a unique T-shaped π-stacking interaction with Phe404. Each ligand showed DhOyI:d1r0o.1g03e9n/C9bOoBn0d0651F
with Arg394, similar to the 4-OH-TAM. Compounds 14d, 14e and 14f formed direct hydrogen
bond between the hydrogen bond donor Arg394 and the oxygen of the methoxy group, while
compound 14h established hydrogen bond between the hydrogen bond donor Arg394 and the
oxygen of nitro group. Moreover, compound 14f established an additional hydrogen bond with
Thr347 by a fluorine of the CF3 group. Additionally, compound 14d, the most active compound,
interact with the negatively charged residue Asp351 in the ligand binding domain of the ER by a
nitro group, suggesting that Asp351 plays a key role in antagonist binding.
5. Conclusion
A series of 3,4-diaryl-1,2,4-oxadiazolidin-5-ones, synthesized by 1,3-dipolar cycloaddition of
nitrones to isocyanates as fixed-ring analogues of TAM, a drug inhibitor of ER used in breast
cancer therapy, have been reported. The structure of the obtained adducts was investigated on the
basis of spectroscopic data. The observed site- and regioselectivity of the 1,3-dipolar cycloaddition
processes was rationalized by theoretical calculations using the Truhlar’s functional at the M06-2X
level of theory. Some of new synthesized compounds (14d-f, 14h and 14k) showed a significant
cytotoxic effect in MCF-7 human breast cancer cell line. In addition, compounds 14d-f, 14h and
14k were able to increase the p53 expression levels, activating also the apoptotic pathway, when
compared with the control. Molecular modeling studies of novel compounds, performed on the
crystal structure of ER, reveal the presence of strong hydrophobic interactions with the aromatic
rings of the ligands similar to TAM. The obtained results suggest that the 1,2,4-oxazolidin-5-one
ring system can replace the structurally more simple double bond present in TAM, and that the
effect of the compounds 14d-f, 14h and 14k in MCF-7 breast cancer cell line might due partially
via the inhibition of the ER. However, further studies are needed to better clarify the role played by
the new synthetized compounds in MCF-7 cancer cell line as inhibitor of ER. In addition, the effect
of the most active compound (14h) in breast cancer animal model will be the object of future
biological studies. The results reported in this study represent a starting point for future SAR and
docking studies to find new TAM analogues with a better pharmacological profile.
Experimental section
Materials and methods
Solvents and reagents are commercial. ESI-HRMS were determined with a Thermo Fischer
13
Scientific LTQ Orbitrap XL. NMR spectra (1H NMR at 500 MHz, C NMR at 126 MHz) were
recorded with Varian instruments and are reported in ppm relative to CDCl3 (7.26, and 77.0 ppm

Organic & Biomolecular Chemistry
Page 18 of 25
13
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respectively). Furthermore, C NMR spectra were recorded using the Attached ProDtoOnI: 1T0.1e0s3t9/(CA9OPBT00)651F
technic. Merck silica gel 60-F254 precoated aluminum plates have been used for thin-layer
chromatographic separations. Flash chromatography was performed on Merck silica gel (200–400
mesh). Preparative separations were carried out by a MPLC Büchi C-601 by using Merck silica gel
0.040–0.063 mm. All compounds were determined to have purity >95% using Shimadzu
LC/MS/MS-8040 system (C18 column; eluting gradient 10-90% acetonitrile in water).
General procedure.
1.1 To nitrone 12 (1 mmol) in dichloromethane or acetone dry (5 ml) at room temperature, is added
the corresponding aryl isocyanate 13 (1 mmol). The solution is stirred at rt for 24 h and then
concentrated in vacuo to afford a yellow solid which was triturated with 10 ml of methanol for 3 h.
The resulting suspension was filtered off, and the solid was washed with hexane and then purified
by silica gel column chromatography to yield final desired compounds 14a-k.
2.1.
2-methyl-4-(4-nitrophenyl)-3-phenyl-1,2,4-oxadiazolidin-5-one
(14a).
Eluent
cyclohexane/ethyl acetate 9:1; yellow solid (yield 70%); mp = 107-110 °C. max/ cm-1: 1746
1
(C=O). H NMR (500 MHz, CDCl3):  8.14 ( d, J = 9.0 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 7.40—
7.42 (m 5 H), 5.72 (s, 1H), 3.06 (s, 3H). 13C NMR (125 MHz, CDCl3):  153.8, 143.9, 142.0, 139.2,
131.8, 130.5, 129.4, 127.1, 124.8, 119.5, 85.0, 53.0. HRMS-EI (M/z) calcd for C15H13N3O4
299.0906 found 299.0901.
2.2 4-(nitrophenyl)-2,3-diphenyl-1,2,4-oxadiazolidin-5-one (14b). Eluent cyclohexane/ethyl acetate
1
9:1; yellow solid (yield 85%); mp = 125-128 °C. max/ cm-1 : 1765 (C=O). H NMR (500 MHz,
CDCl3):  8.15 ( d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.50 Hz, 2H), 7.47—7.42 (m, 5H), 7.43-7.40 (m,
13
2H), 7.26-7.25 (m, 3H), 6.23 (s, 1H). C NMR (125 MHz, CDCl3):  153.8, 148.8, 146.3, 141.4,
134.5, 130.5, 129.6, 127.0, 124.9, 119.3, 117.5, 85.9. HRMS-EI (M/z) calcd for C21H16N2O4
360.1110 found 360.0999.
2.3. 4-(4-chlorophenyl)-2,3-diphenyl-1,2,4-oxadiazolidin-5-one (14c). Eluent cyclohexane/ethyl
acetate 8:2; white solid (yield 98%); mp =120-122 . max/ cm-1: 1754 (C=O). 1H NMR (200 MHz,
CDCl3):  7.49-7.48 ( m, 2H), 7.44-7.43 (m, 3), 7.41-7.38 (m, 2H), 7.24-7.21 (m, 7), 6.10 (s, 1H).
13C NMR (125 MHz, CDCl3):  154.1, 149.2, 135.3, 134.2, 130.3, 129.5, 129.4, 129.3, 128.1,
127.2, 125.9, 122.4, 117.8, 86.5. HRMS-EI (M/z) calcd for C20H15ClN2O2 350.0822 found
350.0829.
2.4. 2-(4-methoxybenzyl)-4-(4-nitrophenyl)-3-phenyl-1,2,4-oxadiazolidin-5-one (14d). Eluent
cyclohexane/ethyl acetate 4:1, yellow solid (yield 68%); mp = 129-132 °C. max/ cm-1: 1747
(C=O). 1H NMR (500 MHz, CDCl3):  8.14 (d, J = 9.0 Hz, 2H), 7.51 (d, J = 9.0 Hz, 2H), 7.37-7.30
( m, 5H), 7.16. (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.0 Hz, 2H), 5.87 (s, 1H), 4.42 ( d, J = 14 Hz, 1 H),
4.14 ( d, J = 14 Hz, 1 H), 3.82 ( s, 3H). 13C NMR (125 MHz, CDCl3):  160.0, 154.3, 142.8, 142.0,
134.3, 131.1, 130.1, 129.3, 126.5, 124.9, 124.8, 118.6, 114.4, 79.9, 62.0, 55.3. HRMS-EI (M/z)
calcd for C22H19N3O5 405.1325 found 405.1330.

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2.5. 4-(4-chlorophenyl)-2-(4-methoxybenzyl)-3-phenyl-1,2,4-oxadiazolidin-5-one (14e). Eluent
DOI: 10.1039/C9OB00651F
cyclohexane/ethyl acetate 9:1; pale yellow solid (yield 58%); mp= 100-103 °C. max/ cm-1: 1739
1
(C=O). H NMR (500 MHz, CDCl3):  7.34-7.15 (m, 11H), 6.89 (d, J = 8.8 Hz, 2H), 5.74 (s, 1H),
13
4.38 (d, J =12.8 Hz, 1H), 4.14 (d, J = 12.8 Hz, 1H), 3.79 (s, 3H). C NMR (125 MHz, CDCl3):
 159.9, 151.6, 134.9, 131.1, 130.6, 130.5, 129.7, 129.6, 126.8, 126.1, 125.3 121.2, 114.3, 80.5,
61.8, 55.3 HRMS-EI (M/z) calcd for C22H19ClN2O3 394.1084 found 394.1100.
2.6. 2-(4-methoxybenzyl)-3-phenyl-4-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazolidin-5-one (14f).
1
Eluent cyclohexane/ethyl acetate 9:1; yellow oil (yield 71%). max/ cm-1: 1728 (C=O). H NMR
(500 MHz, CDCl3):  7.51 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 7.37-7.28 ( m, 5H), 7.21.
(d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 5.81 (s, 1H), 4.40 ( d, J = 14 Hz, 1 H), 4.12 ( d, J = 14
13
Hz, 1 H), 3.79 ( s, 3H). C NMR (125 MHz, CDCl3):  159.9, 154.6, 139.5, 135.0, 131.1, 129.8,
129.2, 126.7, 126.6, 125.1, 124.9, 122.7, 119.0, 114.4, 80.0, 61.5, 55.3. HRMS-EI (M/z) calcd for
C23H19F3N2O3 428.1348 found 428.1353.
2.7. 2-benzyl-4-(4-nitrophenyl)-3-(phenanthren-9-yl)-1,2,4-oxadiazolidin-5-one (14g). Eluent
cyclohexane/ethyl acetate 4:1; beige solid (yield 81%); mp = 196-198 °C cyclohexane/ethyl acetate
4:1. max/ cm-1: 1757 (C=O). 1H NMR (500 MHz, CDCl3):  8.74 d, J = 8.5 Hz, 1H), 8.63 (d, J =
8.5 Hz, 1H), 8.12 (d, J = 9.0 Hz, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.68-7.65 (m, 2H), 7.65-7.34 (m, 10
13
H), 7.11 (m, 1H), 6.67 (s, 1H), 4.64 (d, J = 12 Hz, 1H), 4.42 (d, J =12 Hz, 1H). C NMR (125
MHz, CDCl3):  155.5, 143.8, 142.1, 131.5, 131. 0, 130.6, 130.4, 130.3, 129.4, 129.2, 128.7, 128.6,
128.2, 127.2, 126.9, 126.8, 127.3, 125.8, 125.2, 123.8, 122.7, 122.4, 118.1, 77.5, 63.1. HRMS-EI
(M/z) calcd for C29H21N3O4 475.1532 found 475.1530.
2.8. 2-benzyl-4-(4-chlorophenyl)-3-(phenanthren-9-yl)-1,2,4-oxadiazolidin-5-one (14h). Eluent
cyclohexane/ethyl acetate 4:1; orange solid (yield 79%); mp = 132-136 °C . max/ cm-1 : 1752
(C=O). 1H NMR (500 MHz, CDCl3):  8.71 (d, J = 8.0 Hz, 1H), 8.61 (d, J = 8.0 Hz, 1H), 7.77 (d, J
= 7.5 Hz, 1H), 7.67-7.64 (m, 3H), 7.61-7.56 (m, 1H), 7.53-7.41 (m, 6 H), 7.33 (d, J= 10 Hz, 2H),
7.21 (d, J = 8.5, 2H), 7.14-7.08 (m, 1 H), 6.55 (s, 1H), 4.59 (d, J = 12 Hz, 1H), 4.38 (d, J =12 Hz,
1H). 13C NMR (125 MHz, CDCl3):  157.2, 135.2, 133.2, 131.4, 131.0, 130.8, 130.6, 130.4, 129.8,
129.4, 129.0, 128.6, 127.9, 127.4, 127.1, 126.9, 126.8, 123.0, 122.7, 122.4, 122.3, 122.2, 120.2,
77.8, 62.9. HRMS-EI (M/z) calcd for C29H21ClN2O2 464.1292 found 464.1290.
2.9
4-(4-nitrophenyl)-2-phenyl-3-(pyridin-4-yl)-1,2,4-oxadiazolidin-5-one
(14i).
Eluent
1
cyclohexane/ethyl acetate 3:1; yellow sticky oil (yield 76%); max/ cm-1: 1730 (C=O). H NMR
(500 MHz, CDCl3):  8.63 (d, J = 7.0 Hz, 2H), 8.05 (d, J = 7.5 Hz, 2H), 7.47 ( d, J = 7.5 Hz, 2H),
13
7.41(d, J = 7.0 Hz, 2H), 7.41- 7.40 (m, 3H), 7.25 (d, J = 8.0 Hz, 2H), 6.25 (s, 1H). C NMR (125
MHz, CDCl3)  153.4, 151.0, 148.3, 143.2, 141.0, 136.8, 129.7, 125.0, 121.5, 121.4, 121.2, 117.4,
83.9. HRMS-EI (M/z) calcd for C19H14N4O4 362.1015 found 362.1019.
2.10
4-(4-fluorophenyl)-2-phenyl-3-(pyridin-4-yl)-1,2,4-oxadiazolidin-5-one
(14j).
Eluent
cyclohexane/ethyl acetate 4:1; yellow sticky oil (yield 77%); max/ cm-1 : 1748 (C=O). H NMR
(500 MHz, CDCl3):  8.52 (d, J = 6.3 Hz, 2H), 7.50-7.48 (m, 2H), 7.43 (d, J = 8.5 Hz, 2H), 8-7.37 (
m, 2H), 7.23-7.21(m, 3H), 6.97-6.94 (m, 2) 6.06 (s, 1H). 13C NMR (125 MHz, CDCl3   d,
JCF = 245.6 Hz), 154.3, 150.6, 149.2, 135.5, 131.4 (d, JCF = 3.4 Hz), 129.4, 127.3, 125.8 (d), 123.9
1

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(d, JCF = 8.2 Hz), 117.3, 116.2 (d, JCF = 22.7 Hz), 86.9. HRMS-EI (M/z) calcd foDrOCI: 1109.H103194/FCN9O3BO002651F
335.1070 found 335.1075.
2.11 2-(4-fluorophenyl)-4-(4-nitrophenyl)-3-(pyridin-4-yl)-1,2,4-oxadiazolidin-5-one (14k). Eluent
1
cyclohexane/ethyl acetate 3:1; yellow sticky oil (yield 83%). max/ cm-1 : 1740 (C=O). H NMR
(500 MHz, CDCl3):  8.75 (d, J = 6.0 Hz, 2H), 8.21 (dd, J = 2.0 and 9.0 Hz, 2H), 7.54 ( dd, J = 2.0
and 9.5 Hz, 2H), 7.38 (d, J = 6.0 Hz, 2H), 7.27- 7.25 (m, 2H), 7.16-7.12 (m, 2H), 6.18 (s, 1H). 13C
NMR (125 MHz, CDCl3)   d, JCF = 248.7 Hz), 153.3, 151.1, 142.9 (d, JCF = 3.2 Hz), 140.8,
136.7, 125.2, 123.7, 122.1, 119.2 (d, JCF = 8.3 Hz), 116.6 (d, JCF = 22.7 Hz), 84.2. HRMS-EI (M/z)
calcd for C19H13FN4O4 380.0921 found 380.0924.
Computational Methods
All of the calculations were performed in vacuo using the Gaussian09 program package.30
Optimizations were performed with the M06-2X functional in conjunction with cc-pVTZ basis
set.29 The reaction pathways were confirmed by IRC analyses performed at the same level of
calculation. Vibrational frequencies were computed at the same level of theory to define the
optimized structures as minima or transition states, that all present an imaginary frequency
corresponding to the forming bonds. The polar nature of the reactions has been evaluated through
the analysis of the reactivity indices. The electronic chemical potential (μ), chemical hardness (η),
global electrophilicity (ω), and global nucleophilicity (N), were computed at the same level as
above. The local electrophilicity and nucleophilicity power were determined by the calculation of
Parr functions.
Biological evaluation
Materials
MCF-7 human breast cancer cell line was purchased from Cell Bank Interlab Cell Line Collection
(Genova, Italy). Human Lymphatic Fibroblasts (HLF), Basal Medium and Fibroblast Growth
Supplement (FGS) were provided by Innoprot (Derio Bizkaia, Spain). Dulbecco’s Modified Eagle
Medium (DMEM) containing 2 mM GlutaMAX, heat inactivated-Foetal Bovine Serum (FBS,
GIBC), streptomycin and penicillin antibiotics, Trypsin-EDTA 0,05% solution, SuperSignal West
Pico Plus was from Pierce were from ThermoFisher Scientific (Milan, Italy). Poly-L-lysine
(1mg/mL), 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide salts (MTT), Tamoxifen
free base, Dimethyl Sulfoxide (DMSO), horseradish peroxidase-conjugated anti-mouse IgG and
other analytical chemicals were obtained from Sigma-Aldrich (Milano, Italy). Mouse monoclonal
antibody against p53 was from Abcam (Milan, Italy). Mouse monoclonal antibody against caspase-
3 was from Becton Dickinson (Milan, Italy). Mouse monoclonal antibody against β-tubulin was
from Cell Signaling, EuroClone (Milan. Italy). SuperSignal West Pico Plus was from Pierce
(ThermoFisher Scientific, Milan, Italy).
Cell Cultures
MCF7 cells were suspended in DMEM containing 10% (v/v) FBS, 2 mM L-glutamine, 50 μg/mL),
penicillin (50 U/mL), plated in a final density of 2x106 cells. HLF primary cells were suspended in
Basal Medium containing 10% (v/v) FBS, streptomycin (50 μg/mL), penicillin (50 U/mL) and 5 mL
of FGS added to 500 mL of Basal Medium, and seeded at the density of 5,000 cells/cm2 of poly-L-
lysine-coated flasks. Cell cultures were then incubated at 37°C in humidified atmosphere containing

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5% CO2 (95%-5%) and the medium was replaced every 2 or 3 days. When the cultures were about
DOI: 10.1039/C9OB00651F
80-85% confluent, cells were trypsinized by 0.05% trypsin and 0.53 mM EDTA at 37°C in
humidified atmosphere containing 5% CO2 for 5 min. Trypsinization was stopped by adding 20%
FBS, resuspended and plated in flasks fed with fresh basic complete media. Cells were seeded again
at 1:4 density ratio and incubated at 37°C in humidified atmosphere containing 5% CO2.
Treatment of Cells
MCF-7 and HLF primary cells were placed at the final density of 60x104 cells/well of a 96-
multiwell flat-bottomed 200-μl microplates to assess MTT test or in 75 cm2 flasks to perform
Western Blot analysis and untreated or treated with different concentrations of all synthesized
compounds (0.1, 0.5, 1, 5 μM) for 24, 48 and 72 h. A group of cell was treated with TAM, a well
know ER inhibitor in breast cancer, as control drug was used.
MTT bioassay
To monitor cell viability MTT test bioassay was used. MCF-7 and HLF cell lines were set up 6 x
105 cells per well of a 96-multiwell, flat-bottomed, 200-μL microplate, and maintained at 37°C in a
humidified 5% CO2/95% air mixture.33 At the end of treatment time, 20 μL of 0.5% MTT in (pH
7.4) PBS were added to each microwell. After 2 h of incubation with the reagent, the supernatant
was removed and replaced with 100 μL of DMSO and incubated at 37°C for 1 h. The optical
density of each well was measured with a microplate spectrophotometer reader (Titertek Multiskan;
Flow Laboratories, Helsinki, Finland) at λ=570 nm. Results were normalized with DMSO control
(0.05%) and expressed as a percentage of cell viability inhibition. Interactions of compounds and
media were estimated on the basis of the variations between the drug-containing medium and drug-
free medium to control for false-positive or false negative results. The half maximal inhibitory
concentration (IC50) values were obtained graphically from dose–effect curves using Prism 5.0
(GraphPad Software Inc.).34 We found that for the all cultures the optimal concentration of all
synthesized compounds was 10 μM and the exposure time was 24 h. No significant toxic effect of
the compound in HLF cell line cultures was found.
Western Blot Analysis
To identified the expression levels of p53 and caspase-3 cleavage Western Blot Analysis was
performed in MCF-7 cell line cultures untreated or treated with the most active compounds (14d,
14h, 14e, 14f and 14k) or TAM at the concentration of 10 µM for 24h. Briefly, untreated and
treated MCF-7 cell line cultures were harvested in cold PBS, collected by centrifugation, and
resuspended in a homogenizing buffer with 50 mM Tris-HCl (pH 6.8), 150 mM NaCl, 1 mM
EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), and 10 μg/ml of aprotinin,
leupeptin, and pepstatin and sonicated on ice.33, 35, 36 The protein concentration of the homogenates
was then diluted to 1 mg/ml with reducing stop buffer (0.25 M Tris-HCl, 5 mM EGTA, 25 mM
dithiothreitol, 2 % SDS, and 10 % glycerol with bromophenol blue as the tracking dye). Proteins
were separated on 8 % SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blots
were blocked overnight at 48 °C with 5 % non-fat dry milk dissolved in 20 mM Tris-HCl (pH 7.4),
150 mM NaCl, and 0.5 % Tween 20. p53 expression levels and caspase-3 cleavage were detected
through incubation for 1 h with monoclonal anti-mouse against the p53 or monoclonal anti-mouse
against (1:1000 in PBS), followed by incubation for 1 h with horseradish peroxidase-conjugated
anti-mouse IgG (1:1500 in PBS). p53 expression and caspase-3 cleavage were visualized using a

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SuperSignal West Pico Plus after autoradiography film exposure. Densitometric analysis was
DOI: 10.1039/C9OB00651F
performed after normalization with anti-rabbit β-tubulin.
Statistical analysis
Data were statistically analysed using one-way analysis of variance (one-way ANOVA) followed
by post hoc Holm–Sidak test to estimate significant differences among groups. Data were reported
as mean ± SD of four experiments in duplicate, and differences between groups were considered
tobe significant at *p< 0.05.
Conflict of Interest
There are no conflicts to declare.
Acknowledgements
We gratefully acknowledge the Universities of Catania and Messina, Interuniversity Research
Centre on Pericyclic Reactions and Synthesis of Hetero and Carbocycles Systems and the
Interuniversity Consortium for Innovative Methodologies and Processes for Synthesis (CINMPIS)
for partial financial support. The authors are very grateful to Professor Pierluigi Caramella for
helpful discussion and suggestions.
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