4-(1,2-diarylbut-1-en-1-yl)isobutyranilide derivatives as inhibitors of topoisomerase II

Article abstract of DOI:10.1016/j.ejmech.2016.03.090

The synthesis and biological evaluation of a new library of 4-(1,2-diarylbut-1-en-1-yl)isobutyranilides is described. The new compounds were found to be cytotoxic in the micromolar range in two human tumor cell lines, MCF-7 (mammary gland adenocarcinoma)

Full text of DOI:10.1016/j.ejmech.2016.03.090

Accepted Manuscript
4-(1,2-diarylbut-1-en-1-yl)isobutyranilide derivatives as inhibitors of topoisomerase II
Michael S. Christodoulou, Mikel Zarate, Francesca Ricci, Giovanna Damia, Stefano
Pieraccini, Federico Dapiaggi, Maurizio Sironi, Leonardo Lo Presti, Aída Nelly García-
Argáez, Lisa Dalla Via, Daniele Passarella
PII:
S0223-5234(16)30269-0
10.1016/j.ejmech.2016.03.090
EJMECH 8517
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 December 2015
Revised Date: 15 March 2016
Accepted Date: 31 March 2016
Please cite this article as: M.S. Christodoulou, M. Zarate, F. Ricci, G. Damia, S. Pieraccini, F. Dapiaggi,
M. Sironi, L. Lo Presti, A.N. García-Argáez, L. Dalla Via, D. Passarella, 4-(1,2-diarylbut-1-en-1-
yl)isobutyranilide derivatives as inhibitors of topoisomerase II, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.03.090.
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ACCEPTED MANUSCRIPT
-(1,2-diarylbut-1-en-1-yl)isobutyranilide derivatives as inhibitors of
topoisomerase II
4
Michael S. Christodoulou*, Mikel Zarate, Francesca Ricci, Giovanna Damia, Stefano Pieraccini,
Federico Dapiaggi, Maurizio Sironi, Leonardo Lo Presti, Aída Nelly García-Argáez, Lisa Dalla
Via* and Daniele Passarella
Graphical Abstract

ACCEPTED MANUSCRIPT
-(1,2-diarylbut-1-en-1-yl)isobutyranilide derivatives as inhibitors of
topoisomerase II
4
a, *
a
b
b
Michael S. Christodoulou , Mikel Zarate , Francesca Ricci , Giovanna Damia ,
a
a
a
a, c, d
Stefano Pieraccini , Federico Dapiaggi , Maurizio Sironi , Leonardo Lo Presti
,
e
e, *
a
Aída Nelly García-Argáez , Lisa Dalla Via and Daniele Passarella
a
Dipartimento di Chimica – Università degli Studi di Milano – Via C. Golgi 19,
0133 Milano, Italy
Department of Oncology – IRCCS-Istituto di Ricerche Farmacologiche Mario Negri
Via La Masa 19 – 20156 Milano, Italy
2
b
–
c
CNR–ISTM, Istituto di Scienze e Tecnologie Molecolari, Via Golgi 19, 20133
Milano, Italy
d
Center for Materials Crystallography, Aarhus University, Langelandsgade 140,
8
000 Aarhus, Denmark
e
Dipartimento di Scienze del Farmaco – Università degli Studi di Padova – Via F.
Marzolo 5, 35131 Padova, Italy
*
Corresponding authors:
Tel.:
+39
02.50314081;
fax:
+39
02.50314078;
E-mail
address:
michalis.christ@gmail.com (M. Christodoulou); Via C. Golgi 19, 20133 Milano,
Italy, lisa.dallavia@unipd.it (L. Dalla Via); Via F. Marzolo 5, 35131 Padova, Italy.
Abstract: The synthesis and biological evaluation of a new library of 4-(1,2-
diarylbut-1-en-1-yl)isobutyranilides is described. The new compounds were found to
be cytotoxic in the micromolar range in two human tumor cell lines, MCF-7
(mammary gland adenocarcinoma) and HeLa (cervix adenocarcinoma) and two
human ovarian cancer cell lines (A2780 and OVCAR5). Detailed studies on the most
active compound 6g show that it was able to induce apoptosis and suggest
topoisomerase II as a possible intracellular target. The relevance of the interaction of
the most active compound with topoisomerase II is demonstrated and supported by
docking studies.
Keywords: Tamoxifen derivatives, topoisomerase I and II, McMurry reaction
1
. Introduction
Breast cancer is the second most common cancer in the world and ranks as the fifth
cause of death from cancer overall. In less developed regions it is the most frequent
cause of cancer death in women and the second cause in more developed regions after
1
lung cancer. Drugs that inhibit estrogen receptor (ER) or that block the production of
estrogens still remain frontline interventions in the treatment and management of
2
breast cancer. Among them, tamoxifen (1, Figure 1), the lead compound of the
selective estrogen receptor modulators (SERMs), is considered the gold-standard for

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3
the targeted therapy of all stages of breast cancer, including male breast cancer, and
4
is used in more than 120 countries in the world.
5
-8
However, the adverse side effects associated to this drug and the intrinsic or
acquired resistance to the therapy represents serious obstacles in the use of tamoxifen.
Indeed, a 30 % of ER-positive patients do not respond to tamoxifen therapy and the
emergence of resistance to the treatment has been observed in 30-40% of patients
2
,9
taking the drug more than 5 years.
The primary mechanism of action of tamoxifen and of its active metabolite 4-
1
0
hydroxytamoxifen involves a competitive inhibition of estrogens to ER, but high
concentrations of tamoxifen are also effective in the absence of ER expression in
1
1-13
breast cancer, melanoma, glioma, leukemia and pancreatic carcinoma.
Moreover,
it has been observed that besides acting as SERM, tamoxifen can induce apoptosis in
both ER-α-positive and ER-α-negative breast cancer cells through several distinct
pathways including production of oxidative stress, induction of mitochondrial
permeability transition, ceramide generation as well as changes in cell membrane
1
4
fluidity. Both ER-α-dependent and ER-α-independent pathways for tamoxifen-
induced programmed cell death are critical for successful therapy and interestingly, it
was also suggested that ER-α-independent induction of apoptosis could be a major
14,15
mechanism of the observed antitumor effect of tamoxifen.
Tamoxifen also inhibits
DNA topoisomerases and as a cationic drug is electrophoretically taken up by hepatic
mitochondria to achieve high concentrations able to inhibit both β-oxidation and
1
6
respiration.
In this context, we have previously demonstrated for some tamoxifen analogs, an
1
7
interesting cytotoxic activity on human breast cancer cell line MCF-7. Most of these
compounds exerted a significant antiproliferative effect also on two estrogen
independent human tumour cell lines, suggesting a molecular target different from the
estrogen receptor. In particular, the most interesting results were obtained for two
derivatives carrying an isobutyramide moiety as side chain and notably, the isomer
with the ethyl chain syn with respect to the anilide portion (Figure 1, Mod B) has
shown a 4 fold more potent cytotoxic activity than the reference drug on both estrogen
dependent (MCF-7) and estrogen independent (HeLa) human cell lines. Furthermore,
the ability of these compounds to inhibit the relaxation activity of supercoiled

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pBR322 DNA mediated by topoisomerase II was demonstrated, suggesting the
nuclear enzyme as a potential intracellular target.
On the basis of these results, we report here the synthesis of a new series of tamoxifen
analogs characterized by the triarylskeleton substituted with the isobutyramide
moiety. We describe the synthesis of twenty-one novel compounds bearing different
substituents in the rings A and B (Figure 1) and their antiproliferative activity on a
panel of tumour cell lines. For the most cytotoxic derivatives, the effect on
topoisomerases I and II relaxation is reported. Furthermore, cytofluorimetric analysis
was performed to investigate the mechanism of cell death. Docking studies suggest a
plausible explanation of the biological experimental evidences.
Figure 1. Structures of tamoxifen (1) and general structures of the obtained
compounds.
2
2
. Results and discussion
.1. Chemistry
The preparation of the compound’s library is based on the McMurry reaction (Scheme
1
7
1
) in agreement with our previous results.
Building blocks 4a was easily prepared by reaction of p-anisaldehyde (2) with the
lithium derivative of N-(4-bromophenyl)isobutyramide to provide the corresponding
hydroxyl-amide (Scheme 2) in good yield (60%). The latter was then oxidized in the
presence of Jones reagent to form the keto-amide 4a in very good yield (85%). Keto-

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amide 4b was easily prepared by acylation of commercially available 4-
aminobenzophenone (4c).
R2
R2
HN
HN
H
O
R3
N
R1
R2
R4
Zn, TiCl4
THF
O
R4
R3
R3
R1
4
4
4
a R = OCH , R = COiPr
5a R3, R4 = H
1
3
2
R1
b R = H, R = COiPr
5b R3 = H, R4 = OBn
5c R3, R4 = Cl
7
9
1
2
6
BBr3
BBr3
c R = H, R = H
1
2
8
R4
5
d R = H, R = OH
3 4
10
Mod A
Mod B
R
1
R
2
R
3
R
4
6
a/7a
H
COiPr
COiPr
COiPr
COiPr
COiPr
COiPr
COiPr
COiPr
COiPr
COiPr
H
H
H
6
b/7b
c/7c
d/7d
e/7e
f/7f
OCH
3
H
H
H
OBn
OBn
OH
Cl
6
H
6
OCH
OCH
H
3
H
6
3
H
6
Cl
Cl
H
6
8
g/7g
b/9b
OCH
OH
H
3
Cl
H
8
c/9c
H
OH
Cl
8
g/9g
OH
H
Cl
H
1
0
H
Scheme 1. Synthetic scheme for the obtained compounds, X-ray structure for
compound 6b.
Subsequently, keto-amides 4, were treated under McMurry olefination conditions with
various ketones (7), to provide a mixture of E/Z isomers (6 and 7) that were separated
by flash column chromatography or by crystallization from average to very good
yields (50 – 86%). Demethylation of compounds 6b, 7b, 6c, 7c, 6g and 7g with BBr3
at -78 °C afforded their corresponding derivatives 8b, 9b, 8c, 9c, 8g and 9g in very
good yields (75 – 80%) (Scheme 1). The stereochemical assignments of the E, Z
isomers were determined on the basis of 2D-NOESY experiments as previously
1
7
described. For three of the aforementioned compounds (6a, 6b and 6f), the
corresponding crystal structures were also obtained by single-crystal X-ray diffraction
experiments down to T = 100(2) K and were in accordance with the 2D-NOESY

ACCEPTED MANUSCRIPT
assignments (see Supporting Information). From the conformational viewpoint, in the
solid state the three derivatives adopt a similar ‘vane mill’ motif, with the aromatic
rings being significantly staggered with respect to the central double bond. Short
intermolecular Cl···Cl contacts are also set up in 6f, suggesting the possible presence
1
8
of T-shaped halogen bonds in the solid state for this structure. The interested reader
can find full details of the crystallographic analysis within the Supporting Information
(Section S2 SI). CCDC 1436893-1436895 contain the supplementary crystallographic
data for this paper. The data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/getstructures.
Scheme 2. Preparation of 4a.
A different approach to obtain the final products with a phenyl A ring was selected as
presented in Scheme 1. Although, in this case, when 4-aminobenzophenone (4c) was
reacted with propiophenone (5a) to furnish compound 10 only the Z stereoisomer was
obtained. Acylation of 10 in the presence of isobutyroyl chloride provided the final
compound 6a. In our hands, the formation of the final product 8c or 9c directly from
keto-amide 4b and 4-hydroxy propiophenone (5d) resulted unsuccessful.
2
.2. In vitro antiproliferative activity
The antiproliferative activity of the new tamoxifen derivatives was evaluated by an in
vitro assay performed on two human tumor cell lines, MCF-7 (mammary gland
adenocarcinoma) and HeLa (cervix adenocarcinoma) in comparison with the well-
known drug tamoxifen (1), taken as reference. The obtained results, expressed as GI50
values, i.e. the concentration of compound able to inhibit 50% cell growth with
respect to the control culture, are shown in Table 1.
Most of the synthesized tamoxifen analogues show an interesting antiproliferative
activity, with GI values in the micromolar range with a higher cytotoxic ability on
5
0
HeLa cells than on MCF-7 cells. An opposite behaviour, as expected, was found for

ACCEPTED MANUSCRIPT
tamoxifen, which shows on breast cancer cells a GI value about half with respect to
5
0
that obtained for HeLa.
Moreover, the comparison between the obtained results and the chemical structures of
the new derivatives, allowed some interesting structure-activity relationships to be
revealed. In detail, the analogues devoid of any aryl substituents (6a and 7a) such as
those characterized by a methoxy in R (6b and 7b) show a scarce antiproliferative
1
effect. Interestingly, the insertion of a hydroxyl function in the same position
(compounds 8b and 9b), renders the compounds more active, likely suggesting a role
for the hydrophilicity and/or for a hydrogen bond donor in the cytotoxic effect.
1
9
To validate the role of hydrophilicity clogP values for compounds 6-10 (Table 1)
were calculated. The partition coefficient (clogP) is used to estimate the lipophilicity
and solubility of chemical compounds, where a higher clogP value indicates greater
lipophilicity and poor aqueous solubility. Actually, the above assumptions appear
supported by the clogP values and by the presence of the hydroxyl in R . Indeed, as
4
regard the clogP, it drops from 6.28 and 6.33 for 6a, 7a and 6b, 7b, respectively, to
5
.80 for compounds 8b, 9b.
Moreover, a hydrogen bond donor in R leads to a significant antiproliferative effect
4
(8c and 9c), considerably higher than the one obtained for the analogues 6c and 7c
carrying a benzyloxy substituent in the same position, and even higher to those
obtained for the analogues 8b and 9b. Indeed, the GI values of 8c and 9c are about
5
0
half of those calculated for 8b and 9b highlighting for these latter isomers a
significantly lower cytotoxicity. This latter result suggests that, in addition to the
hydrogen bonding ability, also the aryl carrying the hydroxyl group could play an
important role for the cell effect.
The addition to 8c and 9c of a methoxy group in R position (6e and 7e) does not
1
appreciably affect the antiproliferative activity, thus indicating a more crucial role for
the hydroxyl that the methoxy substituent underlining once again the importance of
the occurrence of a hydrogen bond. Similarly, GI50 values of 6c and 7c appear
comparable to those obtained for the corresponding methoxy analogues, 6d and 7d.
Derivatives bearing a chloride in both R and R (6f, 7f, 6g, 7g, 8g and 9g), show an
3
4
interesting antiproliferative effect; indeed, compound 6g showed the lowest GI50
values in both cell lines. Among this latter series of analogues, neither the
hydrophilicity nor the hydrogen bond donor group seem to be important for the
cytotoxic effect, probably overhung by the prevailing effect of chloride.

ACCEPTED MANUSCRIPT
Finally, the intermediate 10 appears unable to exert any effect on cells as 6a and 7a,
thus substantiating a key role for linked functional groups to the triarylethylene
skeleton.
In addition, the antiproliferative activity of all the compounds was evaluated in A2780
(human ovarian carcinoma) and OVCAR5 (human ovarian epithelial carcinoma) cell
lines (Table 1). Most of compounds were less active than in MFC7 and HeLa cells
with IC values in the macromolar range.
5
0
a
GI50 (µM)
b
Compound
clogP
MCF-7
HeLa
A2780
OVCAR5
6
7
6
7
6
7
6
7
6
7
a
a
b
b
c
c
d
d
e
e
>30
>30
6.28
6.28
6.33
6.33
7.93
7.93
7.99
7.99
5.86
5.86
7.56
7.56
7.62
7.62
5.80
5.80
5.80
5.80
7.08
7.08
5.05
>
50
1.6±3.4
50
7.4±1.9
.8±0.5
50
>50
50
3.6±1.2
>50
>50
>50
>50
>50
>30
28.1±1.0
>30
3
1
>30
>
27.9±0.8
>30
10.1±0.5
10.2±2.3
>30
8
>30
>
>50
>50
29.0±1.0
>30
4.2±0.7
>30
>
>50
13.6±1.6
9.0±0.7
8.3±0.5
17.5±3.0
6.6±0.8
13.2±1.3
14.7±1.7
16.4±0.6
8.1±1.2
8.5±0.8
12.5±0.5
26.6±1.0
>30
5.8±0.7
3.7±0.1
3.3±0.5
8.2±0.4
2.2±0.7
5.5±2.0
9.9±0.5
9.9±1.7
3.8±0.9
5.1±0.8
8.7±0.4
20.3±0.2
>30
1
29.3±2.6
5
.7±0.5
23.6±2.5
>50
6
7
f
f
8
.4±0.7
>50
>50
>50
>50
6g
7g
8b
9b
8c
9c
8g
9g
10
3.8±0.5
8.2±0.6
3
4.0±4.0
2.6±3.2
42.0±4.6
>50
3
3
.7±0.6
.6±0.3
8.7±1.1
12.1±0.9
28.8±2.2
32.3±7.6
>50
4
1
2
5.7±2.0
8.1±2.7
>
50
2
0
1
6.5±0.8
12.0±0.9
6.1 (6.8)
3
0.3±5.0
32.0±2.8
Table 1. Cell growth inhibition in the presence of examined compounds and
a
tamoxifen as reference drug. Values are the mean ± SD of at least three independent
b
experiments. clogP = calculated octanol–water partition coefficient.
2
.3. Effect on topoisomerase relaxation activity

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In a previous paper, we demonstrated for some cytotoxic tamoxifen derivatives, the
1
7
ability to inhibit relaxation activity catalysed by topoisomerase II. To verify if this
enzyme could represent a possible cellular target also for the here reported
compounds, we assayed the ability of compound 6g, the most cytotoxic derivative,
especially on the non estrogenic HeLa cells, and of two less active 6e and 9c, to affect
the relaxation of pBR322 supercoiled DNA by topoisomerase II. Compound 1 was
also tested in the same experimental conditions for comparison. Figure 2 shows the
catalytic activity of the enzyme that converts plasmid DNA (DNA) into relaxed
topoisomers (topo II). The well-known topoisomerase II inhibitor drug m-amsacrine
(m-AMSA) was used as a reference at 10 µM concentration. In the presence of 6g, the
occurrence of a slight band corresponding to supercoiled DNA can be observed at 10
µM concentration, indicating a certain inhibition of the topoisomerase II activity. By
increasing the concentration of 6g up to 50 µM, a concurrent increase in the
supercoiled DNA can be observed, thus confirming the inhibitory effect toward the
enzyme. Compounds 6e and 9c, which show GI values appear approximately half
5
0
with respect to 6g (Table 1), demonstrate a similar effect also on the catalytic activity
of the enzyme. Indeed, for both 6e and 9c an inhibitory effect comparable to that
observed at 25 µM 6g can be obtained only at 50 µM. Finally, compound 1, which, as
expected, induces only a weak antiproliferative effect on non-estrogenic HeLa cells
causes a lower effect with respect to 6g, confirming ER as its main biological target.

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Figure 2. Effect of derivative 6g, 6e, 9c and 1 on relaxation of supercoiled plasmid
DNA by topoisomerase II. Supercoiled DNA (lane DNA) was incubated with
topoisomerase II in the absence (lane topo II) and in the presence of test compound at
indicated concentrations. m-AMSA was used as reference drug.
Based on the above results, it appeared of interest to establish if the effect on
topoisomerase could be due to a poisoning effect rather than to an aspecific inhibition
of the relaxation activity. In this connection, two of the most cytotoxic derivatives on
HeLa cells, 6f and 6g, were tested for the ability to stabilize the cleavable complex
and the obtained results are shown in Figure 3. Both compounds show the ability to
act as topoisomerase II poisons, as demonstrated by the occurrence of the band
corresponding to linear DNA.

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Figure 3. Effect of derivative 6f and 6g on the stabilization of covalent DNA-
topoisomerase II complex. Supercoiled DNA (lane DNA) was incubated with
topoisomerase II in the absence (lane topo II) and in the presence of test compounds at
indicated concentrations. m-AMSA was used as reference drug.
Otherwise, 6g appears unable to exert any effect on relaxation of supercoiled DNA
catalyzed by topoisomerase I: indeed there are not differences in topoisomers pattern
in the absence (topo I) or in the presence of 100 µM 6g (Figure 4). Overall, these data
point to the topoisomerase II as a possible intracellular target responsible for the
cytotoxicity of 6g, while exclude the involvement of topoisomerase I, in accordance
1
7
with previous results obtained for analogues of 4-OH tamoxifen.
Figure 4. Effect of derivative 6g on relaxation of supercoiled plasmid DNA by
topoisomerase I. Supercoiled DNA (lane DNA) was incubated with topoisomerase I

ACCEPTED MANUSCRIPT
in the absence (lane topo I) and in the presence of test compound at 100 µM
concentration.
2
.4. Determination of apoptosis
With the aim to investigate the mechanism of cell death induced by 6g, a flow
cytometric analysis was performed on HeLa cells labelled with Annexin V and
propidium iodide (PI). Compound 1 was taken as reference. The results, shown as dot
plots in Figure 5B, indicate that incubation of HeLa cells in the presence of 50 µM 6g
induces a significant decrease in viability, accomplished by a concurrent increase in
apoptotic cells. In particular, upon treatment with 6g, the percentage of viable cells
diminishes from 95.3% in the control (Fig. 5A) to 31.7% while the percentage of
apoptotic cells (both early and late fractions) increases from 3.8% to 66.2% (Figure
5
A and 5B) indicating that tamoxifen analogue induces cell death through the
activation of the apoptotic process. Also compound 1 causes apoptosis on cells
Figure 5C) and in these experimental conditions a slight difference in percentage of
(
cell death (57.9% vs 66.2%) occurs.
Figure 5. Dual parameter cytogram of FITC-labeled annexin V versus propidium
iodide staining. HeLa cells were incubated in the absence (A) and in the presence of
6
g (B) or 1 (C) at 50 µM for 28 hours before staining.
2
1-24
As previously demonstrated for several cytotoxic compounds,
mitochondria can
participate to intrinsic apoptosis process through the release of pro-apoptotic factors
following mitochondrial membrane depolarization. In this connection, the effect of 6g
and for comparison of 1, on mitochondria was investigated by staining whole HeLa

ACCEPTED MANUSCRIPT
2
5
cells with the green fluorescent monomeric dye JC-1. This lipophilic cationic
fluorochrome selectively enters in mitochondria driven by the mitochondrial
membrane potential, negative inside. The uptake in mitochondria, by increasing the
concentration of JC-1, induces the formation of aggregates characterized by a high
level of red fluorescence. A compound that induces a collapse in membrane potential
provokes the leak of the dye from mitochondria to cytoplasm as monomers, resulting
in a decrease of red fluorescence. The obtained results, shown in Figure 6 as dot plots,
demonstrate that HeLa cells incubated with 6g at 10, 15 and 25 µM undergoes a
notable concentration-dependent decrease in red fluorescence with respect to the
control. In particular, the percentage of cells with depolarized mitochondrial
membrane (i.e. JC-1 monomers) increases from 4.0% in the untreated sample
(control), to 80.3% following incubation for 28 hours in the presence of 6g at 25 µM,
the higher considered concentration. A dose-dependent mitochondrial depolarization
is observed also for 1, nevertheless for this latter compound a significant lower effect
is observed in accordance with the cytotoxicity data shown in Table 1.

ACCEPTED MANUSCRIPT
Figure 6. Mitochondrial membrane potential assessed by flow cytometry after JC-1
staining. HeLa cells were incubated in the absence (control) or in the presence of 1 or
6
g at indicated concentration for 28 hours before staining.
2
.5. Molecular modeling
In order to better understand the intermolecular interactions between the
Topoisomerase II/DNA complex and the ligands, molecular docking studies were
performed. All the docked structures share the same binding site, located between
2
6
four nucleobasis, namely G13, C8, A12 and T9 according to Wu et al. Although the
ligands exhibit many different binding modes, the main feature shared by all of them
is the intercalation between the nucleobasis of at least one aromatic ring. This
stacking interaction is likely to account for the inhibition of topoisomerase enzymatic
activity caused by the analyzed compounds.
The binding energies calculated with the Autodock scoring function (Section S60 SI)
are all largely negative, suggesting a strong binding affinity of tamoxifen derivatives
for the proposed binding site, although it was not possible to effectively rank the
compounds, due to the small binding energy differences observed among them, that
are in general lower than the suggested significance threshold (2.5 kcal/mol).
However, two of the most active compounds according to the biological studies, 6f
and 6g, share the same orientation in the active site, stacking with the nucleobases and
forming a H-bond with G13 (Figure 7). This orientation is also common to the
compound 8g.
Figure 7. Docking poses of compounds 6f (represented in blue) and 6g (represented
in orange). In both cases a H-bond between the amidic group of the molecules and
Guanine 13 is observed.

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3
. Conclusions
A new library of 4-(1,2-diarylbut-1-en-1-yl)isobutyranilide derivatives has been
described. The biological data on four different cancer cell lines showed interesting
activity for most of the described compounds. The presence of the OH group or Cl
moieties in the aromatic ring B seems to improve the cytotoxic activity while methoxy
or hydroxyl substituents in the aromatic ring A of the tamoxifen skeleton don’t affect
positively the cytotoxicity of the compounds. Further biological experiments point out
topoisomerase II as a possible intracellular target responsible for the cytotoxicity of
these compounds and interestingly a correlation between antiproliferative effect and
topoisomerase II inhibition is demonstrated for the most active derivatives. Moreover,
the occurrence of cell death through the activation of the intrinsic apoptotic pathway
is proved. Docking studies showed for the most active compounds the same
orientation in the shared binding site by stacking and forming H-bond. In conclusion,
interestingly, notwithstanding the common general triarylskeleton, the here reported
tamoxifen analogs show a different biological profile with respect to the reference
drug, being more cytotoxic on non estrogenic cell lines and affecting the catalytic
activity of the topoisomerase II. Altogether, these results show 4-(1,2-diarylbut-1-en-
1
-yl) isobutyranilide as an interesting scaffold for the obtainment of new anticancer
compounds.
4
4
. Experimental section
.1. General procedures
All reactions were carried out in oven-dried glassware and dry solvents under
nitrogen atmosphere. Unless otherwise stated, all solvents were purchased from
Sigma Aldrich and used without further purification. Substrates and reagents were
purchased from Sigma Aldrich and used as received. Thin layer chromatography
(TLC) was performed on Merck precoated 60F254 plates. Reactions were monitored
by TLC on silica gel, with detection by UV light (254 nm) or by a solution of p-
anisaldehyde with heating. Flash chromatography was performed using silica gel
(240-400 mesh, Merck). All tested compounds possessed a purity of > 98% confirmed
1
via elemental analyses (CHN) in Perkin Elmer 2400 instrument. H-NMR spectra

ACCEPTED MANUSCRIPT
were recorded on Bruker DRX-400 instruments and are reported relative to residual
13
CHCl and acetone-d . C-NMR spectra were recorded on the same instruments (100
3
6
MHz) and are reported relative to CDCl and acetone-d . Chemical shifts (δ) for
3
6
proton and carbon resonances are quoted in parts per million (ppm) relative to
tetramethylsilane (TMS), which was used as an internal standard. COSY, HSQC,
HMBC and NOESY experiments were used in the structural assignment. MS spectra
were recorded using electrospray ionization (ESI) technique on a Waters Micromass
Q-Tof micro mass spectrometer.
4
4
.2. Synthesis
.2.1. Synthesis of N-(4-(4-methoxybenzoyl)phenyl)isobutyramide (4a). To a cold
at -78 °C solution of N-(4-bromophenyl)isobutyramide (0.72 g, 3.0 mmol) in ΤΗF (17
mL), n-BuLi (5.6 mL, 1.6 M in Hexane, 9 mmol) was added dropwise and the new
solution was stirred for 10 minutes at the same temperature. Then, p-anisaldehyde (2)
(
0.36 mL, 3.0 mmol) was added and the resulted solution was stirred for another 1 h at
78 °C and 1 h at r.t. After the completion of the reaction, the solvent was evaporated
-
in vacuum, EtOAc was added and the organic layer was washed with sat. NH Cl,
4
water and brine, dried with Na SO , filtered and evaporated. The residue was purified
2
4
by flash column chromatography (Hex/EtOAc 6:4) to provide N-(4-(hydroxy(4-
methoxyphenyl)methyl)phenyl)isobutyramide as oil (60% yield). Rf = 0.36
1
(
(
(
Hex/EtOAc 6:4); H NMR (CDCl ) :δ = 7.51 – 7.48 (2H, m), 7.34 (1H, br. s), 7.29
3
2H, d, J 6.8 Hz), 7.24 (2H, d, J 8.8 Hz), 6.86 (2H, d, J 8.8 Hz), 5.31 (1H, s), 3.80
1
3
3H, s), 2.58 – 2.47 (1H, m), 1.26 (6H, d, J 6.8 Hz); C NMR (CDCl ): δ = 175.3,
3
1
58.9, 138.3, 137.2, 134.4, 128.5, 127.8, 119.8, 113.8, 79.06, 55.27, 36.66, 19.63;
+
MS: 300.37 (M+H ). To a cold at 0 °C solution of N-(4-(hydroxy(4-
methoxyphenyl)methyl)phenyl)isobutyramide (0.35 g, 1.2 mmol) in acetone (49 mL),
Jones reagent (9.1 mL) was added and the new solution was stirred for 30 minutes at
the same temperature. After the completion of the reaction, 2-propanol was added and
the solvent was evaporated in vacuum. Then, EtOAc was added and the organic layer
was washed with water and brine, dried with Na SO , filtered and evaporated in
2
4
vacuum till dryness to provide keto-amide 4a as white amorphous solid (85% yield).
1
R = 0.44 (Hex/EtOAc 1:1); H NMR (CDCl ): δ = 7.87 (1H, br. s), 7.80 (2H, d, J 8.8
f
3

ACCEPTED MANUSCRIPT
Hz), 7.77 (2H, d, J 8.8 Hz), 7.69 (2H, d, J 8.8 Hz), 6.97 (2H, d, J 8.8 Hz), 3.90 (3H,
1
3
s), 2.64 – 2.54 (1H, m), 1.27 (6H, d, J 6.8 Hz); C NMR (CDCl ): δ = 194.9, 176.0,
3
1
2
63.8, 142.4, 134.5, 132.8, 131.7, 131.3, 119.6, 114.3, 56.03, 37.35, 20.05; MS:
+
98.36 (M+H ).
4
.2.2. Synthesis of N-(4-benzoylphenyl)isobutyramide (4b). To a cooled at 0 °C
solution of 4-aminobenzophenone (4c, 5.0 g, 25.0 mmol) in t-butyl methyl ether (123
mL) and 40% NaOH (3 mL), isobutyryl chloride was added (3.2 mL, 30 mmol) and
the new solution was stirred for 30 minutes at r.t. After the completion of the reaction,
the solvent was evaporated in vacuum, EtOAc was added and the organic layer was
washed with water and brine, dried with Na SO , filtered and evaporated in vacuum
2
4
till dryness to provide compound 4b as white amorphous solid (85% yield). R = 0.25
f
1
(
Hex/EtOAc 7:3); H NMR (CDCl ): δ = 7.82 (2H, d, J 8.8 Hz), 7.81 (1H, br. s), 7.78
3
(2H, d, J 7.6 Hz), 7.71 (2H, d, J 8.8 Hz), 7.60 (1H, d, J 7.6 Hz), 7.49 (2H, d, J 7.6
13
Hz), 2.64 – 2.54 (1H, m), 1.28 (6H, d, J 7.2 Hz); C NMR (CDCl ): δ = 195.8, 175.8,
3
1
42.3, 137.9, 132.8, 132.2, 131.6, 129.8, 128.3, 118.8, 36.78, 19.55; MS: 268.34
+
(
M+H ).
4
4
.2.3. General procedure for the synthesis of analogues 6 and 7.
.2.3.1. Synthesis of (Z)-N-(4-(1,2-diphenylbut-1-en-1-yl)phenyl)isobutyramide
(6a) and (E)-N-(4-(1,2-diphenylbut-1-en-1-yl)phenyl)isobutyramide (7a). To a
cold at -10 °C suspension of Ζn (1.2 g, 18 mmol) in ΤΗF (18 mL), TiCl (0.82 mL,
4
7
.5 mmol) was added dropwise and the new mixture was stirred for another 10
minutes in the same temperature and after was refluxed for 2 h. Then, a solution of
keto-amide 4b (0.50 g, 1.9 mmol) and propiophenone (5a, 0.28 g, 2.1 mmol) in ΤΗF
(37 mL), was added to the cooled suspension of the titanium reagent at 0 °C, and the
reaction mixture was refluxed for 2.5 h. After the completion of the reaction, the
reaction mixture was cooled to r.t. and poured into a 10% aq K CO solution (15 mL).
2
3
Vigorous stirring was maintained for 5 min, and the dispersed insoluble material was
removed by vacuum filtration using celite. The organic layer was separated and the
aqueous layer was extracted 3 times with EtOAc. The combined EtOAc extracts were
washed with water and brine, dried with Na SO , filtered and evaporated. The residue
2
4
was purified by flash column chromatography (Hex/EtOAc 8:2) to provide a mixture
of the 2 diastereomers 6a and 7a (86% total yield). The 2 diastereomers were

ACCEPTED MANUSCRIPT
separated by crystallization from MeOH to provide compound 6a as crystals and
compound 9a as white amorphous solid in a ratio 6a/7a = 3. 6a: R = 0.30
f
1
(
Hex/EtOAc 8:2); H NMR (CDCl ): δ = 7.38 – 7.35 (2H, m), 7.31 – 7.25 (3H, m),
3
7
.20 (2H, d, J 8.4 Hz), 7.19 (2H, d, J 7.2 Hz), 7.15 – 7.14 (3H, m), 7.05 (1H, br. s),
.84 (2H, d, J 8.4 Hz), 2.49 (2H, q, J 7.6 Hz), 2.47 – 2.38 (1H, m), 1.21 (6H, d, J 7.2
6
1
3
Hz), 0.956 (3H, t, J 7.6 Hz); C NMR (CDCl ): δ = 175.3, 144.1, 143.0, 142.8, 139.7,
3
1
2
3
39.0, 136.4, 131.9, 130.3, 130.1, 128.7, 128.5, 127.2, 126.8, 119.4, 37.20, 29.61,
+
0.07, 13.94; MS: 370.52 (M+H ); Anal. Calcd for C H NO: C, 84.51; H, 7.37; N,
2
6
27
1
.79. Found: C, 83.65; H, 7.16; N, 3.65. . 7a: R = 0.30 (Hex/EtOAc 8:2); H NMR
f
(
acetone-d ): δ = 9.11 (1H, br. s), 7.72 (2H, d, J 8.8 Hz), 7.38 (2H, t, J 8.8 Hz), 7.30
6
(1H, t, J 8.8 Hz), 7.20 (2H, d, J 8.8 Hz), 7.03 – 6.99 (3H, m), 6.93 (2H, d, J 8.4 Hz),
6
.83 (2H, d, J 8.8 Hz), 2.68 – 2.61 (1H, m), 2.53 (2H, q, J 7.6 Hz), 1.19 (6H, d, J 7.2
13
Hz), 0.948 (3H, t, J 7.6 Hz); C NMR (acetone-d ): δ = 175.0, 143.3, 142.3, 138.7,
6
1
2
7
38.4, 138.3, 137.8, 130.8, 130.6, 129.5, 128.2, 127.8, 126.6, 125.7, 118.8, 35.72,
+
8.82, 19.02, 12.89; MS: 370.51 (M+H ); Anal. Calcd for C H NO: C, 84.51; H,
2
6
27
.37; N, 3.79. Found: C, 83.64; H, 7.14; N, 3.66.
4
.2.3.2.
Synthesis
of
(E)-N-(4-(1-(4-methoxyphenyl)-2-phenylbut-1-en-1-
yl)phenyl)isobutyramide (6b) and (Z)-N-(4-(1-(4-methoxyphenyl)-2-phenylbut-1-
en-1-yl)phenyl)isobutyramide (7b). According to the general procedure, compounds
6
b and 7b were obtained from keto-amide 4a and propiophenone (5a) after
purification by flash column chromatography (Hex/EtOAc 7:3) and evaporation of the
solvent as a mixture of diastereomers (74% yield). The 2 diastereomers were
separated by crystallization from MeOH to provide compound 6b as crystals and
compound 7b as white amorphous solid in a ratio 6b/7b = 0.15. 6b: R = 0.42
f
1
(
Hex/EtOAc 7:3); H NMR (CDCl ): δ = 7.28 – 7.21 (9H, m), 7.04 (1H, br. s), 6.90
3
(2H, d, J 8.4 Hz), 6.83 (2H, d, J 8.0 Hz), 3.85 (3H, s), 2.52 (2H, q, J 7.2 Hz), 2.47 –
1
3
2
1
1
.40 (1H, m), 1.21 (6H, d, J 6.8 Hz), 0.960 (3H, t, J 7.2 Hz); C NMR (CDCl ): δ =
3
75.4, 159.2, 143.2, 142.6, 140.1, 138.7, 136.7, 136.4, 131.9, 131.2, 130.3, 128.4,
+
26.6, 119.5, 114.4, 55.84, 37.21, 29.61, 20.05, 13.94; MS: 400.54 (M+H ); Anal.
Calcd for C H NO : C, 81.17; H, 7.32; N, 3.51. Found: C, 80.35; H, 7.11; N, 3.37.
2
7
29
2
1
7
b: R = 0.42 (Hex/EtOAc 7:3); H NMR (CDCl ): δ = 7.55 (2H, d, J 8.0 Hz), 7.28
f 3
(
1H, br. s), 7.23 – 7.13 (7H, m), 6.79 (2H, d, J 8.4 Hz), 6.56 (2H, d, J 8.4 Hz), 3.70
3H, s), 2.58 – 2.52 (1H, m), 2.50 (2H, q, J 7.8 Hz), 1.29 (6H, d, J 6.8 Hz), 0.947 (3H,
(

ACCEPTED MANUSCRIPT
1
3
t, J 6.8 Hz); C NMR (CDCl ): δ = 175.7, 158.4, 143.3, 142.3, 140.6, 138.6, 137.3,
3
1
36.2, 132.4, 130.7, 130.3, 128.4, 126.6, 120.3, 113.6, 55.63, 37.26, 29.59, 20.12,
+
1
3.97; MS: 400.53 (M+H ); Anal. Calcd for C H NO : C, 81.17; H, 7.32; N, 3.51.
27
29
2
Found: C, 80.34; H, 7.12; N, 3.38.
4
.2.3.3. Synthesis of (Z)-N-(4-(2-(4-(benzyloxy)phenyl)-1-phenylbut-1-en-1-
yl)phenyl) isobutyramide (6c) and (E)-N-(4-(2-(4-(benzyloxy)phenyl)-1-
phenylbut-1-en-1-yl)phenyl) isobutyramide (7c). According to the general
procedure, compounds 6c and 7c were obtained from keto-amide 4b and 1-(4-
(benzyloxy)phenyl)propan-1-one (5b) after purification by flash column
chromatography (Hex/EtOAc 7:3) and evaporation of the solvent as white amorphous
1
solids in a ratio 6c/7c = 0.33 (62% yield). 6c: R = 0.41 (Hex/EtOAc 7:3); H NMR
f
(
acetone-d ): δ = 9.08 (1H, br. s), 7.71 (2H, d, J 8.8 Hz), 7.47 (2H, br. d, J 6.8 Hz),
6
7
.39 (2H, t, J 6.4 Hz), 7.28 (1H, t, J 6.8 Hz), 7.18 (2H, d, J 8.8 Hz), 7.09 (2H, d, J 8.8
Hz), 7.05 – 6.98 (3H, m), 6.94 (2H, dd, J 8.4 Hz J 1.6 Hz), 6.84 (2H, d, J 8.8 Hz),
.05 (2H, s), 2.69 – 2.59 (1H, m), 2.50 (2H, q, J 7.6 Hz), 1.19 (6H, d, J 6.8 Hz), 0.950
5
13
(
3H, t, J 7.6 Hz); C NMR (acetone-d ): δ = 175.7, 158.0, 144.2, 142.1, 139.2, 139.0,
6
1
1
38.8, 138.2, 135.2, 131.4, 131.3, 130.2, 130.0, 129.0, 128.4, 128.0, 126.3, 120.0,
+
14.8, 70.15, 36.44, 29.24, 19.72, 13.71; MS: 476.64 (M+H ); Anal. Calcd for
C H NO : C, 83.33; H, 6.99; N, 2.94. Found: C, 82.49; H, 6.79; N, 2.81. 7c: R =
33
33
2
f
1
0
.45 (Hex/EtOAc 7:3); H NMR (acetone-d ): δ = 8.89 (1H, br. s), 7.48 (2H, br. d, J
6
6
.8 Hz), 7.43 – 7.30 (8H, m), 7.27 (2H, d, J 8.0 Hz), 7.13 (2H, d, J 8.8 Hz), 6.88 (2H,
d, J 8.8 Hz), 6.84 (2H, d, J 8.8 Hz), 5.07 (2H, s), 2.60 – 2.51 (1H, m), 2.46 (2H, q, J
1
3
7
1
1
.2 Hz), 1.13 (6H, d, J 6.8 Hz), 0.933 (3H, t, J 7.6 Hz); C NMR (acetone-d ): δ =
6
74.8, 157.4, 143.9, 141.1, 138.2, 137.5, 134.6, 130.9, 130.8, 129.3, 128.4, 128.1,
27.7, 127.6, 126.5, 118.2, 114.2, 69.52, 35.70, 28.44, 18.98, 12.98; MS: 476.63
+
(
M+H ); Anal. Calcd for C H NO : C, 83.33; H, 6.99; N, 2.94. Found: C, 82.48; H,
33
33
2
6
.80; N, 2.82.
4
.2.3.4. Synthesis of (E)-N-(4-(2-(4-(benzyloxy)phenyl)-1-(4-methoxyphenyl)but-
-en-1-yl)phenyl) isobutyramide (6d) and (Z)-N-(4-(2-(4-(benzyloxy)phenyl)-1-(4-
1
methoxyphenyl)but-1-en-1-yl)phenyl) isobutyramide (7d). According to the
general procedure, compounds 6d and 7d were obtained from keto-amide 4a and 1-(4-
(benzyloxy)phenyl)propan-1-one (5b) after purification by flash column

ACCEPTED MANUSCRIPT
chromatography (Hex/EtOAc 6:4) and evaporation of the solvent as white amorphous
1
solids in a ratio 6d/7d = 1 (69% yield). 6d: R = 0.50 (Hex/EtOAc 6:4); H NMR
f
(
acetone-d ): δ = 8.87 (1H, br. s), 7.48 (2H, br. d, J 6.8 Hz), 7.48 – 7.38 (4H, m), 7.34
6
(1H, t, J 7.2 Hz), 7.17 (2H, d, J 8.8 Hz), 7.11 (2H, d, J 8.8 Hz), 6.95 (2H, d, J 8.8 Hz),
6
.86 (2H, d, J 8.8 Hz), 6.83 (2H, d, J 8.8 Hz), 5.06 (2H, s), 3.83 (3H, s), 2.60 – 2.53
13
(
1H, m), 2.49 (2H, q, J 7.6 Hz), 1.13 (6H, d, J 7.2 Hz), 0.940 (3H, t, J 7.6 Hz); C
NMR (acetone-d ): δ = 174.8, 158.6, 157.3, 140.8, 138.5, 137.8, 137.6, 137.4, 136.1,
6
1
3
8
6
7
34.8, 130.9, 130.8, 130.4, 128.4, 127.7, 127.6, 118.1, 114.2, 113.5, 69.51, 54.60,
+
5.71, 28.82, 18.99, 13.03; MS: 506.67 (M+H ); Anal. Calcd for C H NO : C,
3
4
35
3
0.76; H, 6.98; N, 2.77. Found: C, 79.93; H, 6.78; N, 2.65. 7d: R = 0.46 (Hex/EtOAc
f
1
:4); H NMR (acetone-d ): δ = 9.08 (1H, br. s), 7.70 (2H, d, J 8.4 Hz), 7.48 (2H, d, J
6
.2 Hz), 7.40 (2H, t, J 7.2 Hz), 7.34 (1H, t, J 7.2 Hz), 7.16 (2H, d, J 8.4 Hz), 7.10 (2H,
d, J 8.8 Hz), 6.86 (2H, d, J 8.8 Hz), 6.83 (2H, d, J 8.8 Hz), 6.61 (2H, d, J 8.8 Hz),
5
6
1
1
.07 (2H, s), 3.69 (3H, s), 2.68 – 2.61 (1H, m), 2.48 (2H, q, J 7.2 Hz), 1.19 (6H, d, J
13
.8 Hz), 0.935 (3H, t, J 7.2 Hz); C NMR (acetone-d ): δ = 175.6, 158.5, 158.0,
6
41.3, 139.6, 138.9, 138.6, 138.3, 136.5, 135.7, 132.3, 131.4, 130.1, 128.9, 128.2,
28.1, 119.7, 115.0, 113.5, 70.37, 55.08, 36.42, 29.42, 19.57, 13.53; MS: 506.66
+
(
M+H ); Anal. Calcd for C H NO : C, 80.76; H, 6.98; N, 2.77. Found: C, 79.94; H,
34
35
3
6
.77; N, 2.67.
4
.2.3.5. Synthesis of (E)-N-(4-(2-(4-hydroxyphenyl)-1-(4-methoxyphenyl)but-1-
en-1-yl)phenyl) isobutyramide (6e) and (Z)-N-(4-(2-(4-hydroxyphenyl)-1-(4-
methoxyphenyl)but-1-en-1-yl)phenyl) isobutyramide (7e). According to the
general procedure, compounds 6e and 7e were obtained from keto-amide 4a and 1-(4-
hydroxyphenyl)propan-1-one (5d) after purification by flash column chromatography
(Hex/EtOAc 6:4) and evaporation of the solvent as oils in a ratio 6e/7e = 1 (56%
1
yield). 6e: R = 0.26 (Hex/EtOAc 6:4); H NMR (acetone-d ): δ = 8.86 (1H, br. s),
f
6
8.16 (1H, br. s), 7.37 (2H, d, J 8.4 Hz), 7.16 (2H, d, J 8.4 Hz), 7.00 (2H, d, J 8.4 Hz),
6.94 (2H, d, J 8.8 Hz), 6.82 (2H, d, J 8.8 Hz), 6.68 (2H, d, J 8.8 Hz), 3.83 (3H, s),
2.59 – 2.52 (1H, m), 2.47 (2H, q, J 7.2 Hz), 1.12 (6H, d, J 6.8 Hz), 0.940 (3H, t, J 7.2
1
3
Hz); C NMR (acetone-d ): δ = 174.8, 158.5, 155.7, 141.1, 138.7, 137.3, 136.2,
6
1
33.3, 130.9, 130.7, 130.4, 118.1, 114.8, 113.4, 54.59, 35.70, 28.83, 18.99, 13.06;
+
MS: 416.55 (M+H ); Anal. Calcd for C H NO : C, 78.04; H, 7.03; N, 3.37. Found:
2
7
29
3
1
C, 77.28; H, 6.84; N, 3.23. 7e: R = 0.28 (Hex/EtOAc 6:4); H NMR (acetone-d ): δ =
f
6

ACCEPTED MANUSCRIPT
9
.13 (1H, br. s), 8.25 (1H, br. s), 7.70 (2H, d, J 8.4 Hz), 7.15 (2H, d, J 8.4 Hz), 6.99
(
2H, d, J 8.8 Hz), 6.82 (2H, d, J 8.8 Hz), 6.68 (2H, d, J 8.8 Hz), 6.61 (2H, d, J 8.8
Hz), 3.68 (3H, s), 2.68 – 2.61 (1H, m), 2.46 (2H, q, J 7.6 Hz), 1.19 (6H, d, J 6.8 Hz),
1
3
0
1
2
7
.932 (3H, t, J 7.6 Hz); C NMR (acetone-d ): δ = 175.6, 158.5, 156.4, 141.7, 139.8,
6
38.8, 137.8, 136.7, 134.2, 132.2, 131.3, 130.1, 119.9, 115.5, 113.5, 55.09, 36.42,
+
9.08, 19.54, 13.47; MS: 416.56 (M+H ); Anal. Calcd for C H NO : C, 78.04; H,
2
7
29
3
.03; N, 3.37. Found: C, 77.28; H, 6.83; N, 3.24.
4
.2.3.6.
Synthesis
of
(Z)-N-(4-(2-(2,4-dichlorophenyl)-1-phenylbut-1-
(6f) and (E)-N-(4-(2-(2,4-dichlorophenyl)-1-
enyl)phenyl)isobutyramide
phenylbut-1-enyl)phenyl)isobutyramide (7f). According to the general procedure,
compounds 6f and 7f were obtained from keto-amide 4b and 1-(2,4-
dichlorophenyl)propan-1-one (5c) after purification by flash column chromatography
(Hex/EtOAc 8:2) and evaporation of the solvent as white amorphous solids in a ratio
1
6
8
–
f/7f = 2.2 (50% yield). 6f: R = 0.30 (Hex/EtOAc 8:2); H NMR (acetone-d ): δ =
f
6
.89 (1H, br. s), 7.42 – 7.39 (5H, m), 7.34 – 7.25 (5H, m), 6.92 (2H, d, J 8.8 Hz), 2.57
2.51 (1H, m), 2.45 (2H, q, J 7.6 Hz), 1.11 (6H, d, J 6.8 Hz), 0.949 (3H, t, J 7.6 Hz);
1
3
C NMR (acetone-d ): δ = 175.5, 143.1, 142.0, 140.4, 138.8, 138.3, 137.7, 134.7,
6
1
1
33.1, 134.1, 130.3, 129.7, 129.5, 128.9, 127.6, 127.4, 118.8, 36.35, 28.40, 19.59,
+
2.91; MS: 439.41 (M+H ); Anal. Calcd for C H Cl NO: C, 71.23; H, 5.75; N, 3.20.
2
6
25
2
1
Found: C, 70.50; H, 5.59; N, 3.06. 7f: R = 0.24 (Hex/EtOAc 8:2); H NMR (acetone-
f
d6): δ = 9.15 (1H, br. s), 7.74 (2H, d, J 8.8 Hz), 7.39 (1H, d, J 1.6 Hz), 7.29 – 7.24
(4H, m), 7.06 – 7.00 (4H, m), 6.92 (1H, t, J 8.4 Hz), 2.68 – 2.61 (1H, m), 2.49 (2H, q,
1
3
J 7.6 Hz), 1.18 (6H, d, J 6.8 Hz), 0.968 (3H, t, J 7.6 Hz); C NMR (acetone-d ): δ =
6
1
1
75.6, 143.2, 142.1, 140.5, 139.4, 138.7, 137.7, 134.7, 133.1, 131.1, 129.9, 129.4,
+
28.0, 127.2, 126.9, 119.8, 36.43, 28.24, 19.58, 12.82; MS: 439.41 (M+H ); Anal.
Calcd for C H Cl NO: C, 71.23; H, 5.75; N, 3.20. Found: C, 70.48; H, 5.60; N,
2
6
25
2
3
.05.
4
.2.3.7. Synthesis of (E)-N-(4-(2-(2,4-dichlorophenyl)-1-(4-methoxyphenyl)but-1-
enyl)phenyl) isobutyramide (6g) and (Z)-N-(4-(2-(2,4-dichlorophenyl)-1-(4-
methoxyphenyl)but-1-enyl)phenyl) isobutyramide (7g). According to the general
procedure, compounds 6g and 7g were obtained from keto-amide 4a and 1-(2,4-
dichlorophenyl)propan-1-one (5c) after purification by flash column chromatography

ACCEPTED MANUSCRIPT
(Hex/EtOAc 6:4) and evaporation of the solvent as white amorphous solids in a ratio
1
6
8
7
2
g/7g = 1 (58% yield). 6g: R = 0.54 (Hex/EtOAc 6:4); H NMR (acetone-d ): δ =
f
6
.90 (1H, br. s), 7.41 – 7.38 (3H, m), 7.29 (1H, d, J 8.4 Hz), 7.26 (1H, d, J 2.0 Hz),
.23 (2H, d, J 8.8 Hz), 6.97 (2H, d, J 8.8 Hz), 6.89 (2H, d, J 8.4 Hz), 3.83 (3H, s),
.58 – 2.52 (1H, m), 2.48 (2H, q, J 7.6 Hz), 1.11 (6H, d, J 6.8 Hz), 0.956 (3H, t, J 7.2
1
3
Hz); C NMR (acetone-d ): δ = 175.5, 159.5, 141.6, 140.7, 138.7, 138.1, 137.9,
6
1
2
35.2, 134.7, 134.7, 132.9, 131.0, 130.4, 129.5, 127.4, 118.8, 114.2, 55.30, 36.37,
+
8.46, 19.63, 13.01; MS: 469.42 (M+H ); Anal. Calcd for C H Cl NO : C, 69.23;
2
7
27
2
2
H, 5.81; N, 2.99. Found: C, 68.52; H, 5.65; N, 2.86. 7g: R = 0.51 (Hex/EtOAc 6:4);
f
1
H NMR (acetone-d ): δ = 9.10 (1H, br. s), 7.73 (2H, d, J 8.8 Hz), 7.40 – 7.39 (1H,
6
m), 7.29 (1H, d, J 8.4 Hz), 7.26 (1H, d, J 2.0 Hz), 7.23 (2H, d, J 8.4 Hz), 6.90 (2H, d,
J 8.8 Hz), 6.63 (2H, d, J 8.8 Hz), 3.68 (3H, s), 2.68 – 2.61 (1H, m), 2.48 (2H, q, J 7.6
13
Hz), 1.19 (6H, d, J 6.8 Hz), 0.954 (3H, t, J 7.2 Hz); C NMR (acetone-d ): δ = 175.1,
6
1
1
58.2, 141.0, 140.1, 138.7, 137.3, 137.1, 134.8, 134.1, 133.5, 132.3, 130.6, 129.4,
+
28.8, 126.7, 118.9, 112.8, 54.41, 35.78, 27.82, 19.04, 12.36; MS: 469.43 (M+H );
Anal. Calcd for C H Cl NO : C, 69.23; H, 5.81; N, 2.99. Found: C, 68.51; H, 5.64;
2
7
27
2
2
N, 2.85.
4
.2.3.8. Synthesis of (Z)-4-(1,2-diphenylbut-1-en-1-yl)aniline (10). According to the
general procedure, compound 10 was obtained from 4-aminobenzophenone (4c) and
propiophenone (5a) after purification by flash column chromatography (Hex/EtOAc
8
:2) and evaporation of the solvent as white amorphous solid (40% yield). R = 0.24
f
1
(
Hex/EtOAc 8:2); H NMR (CDCl ): δ = 7.37 (2H, t, J 7.2 Hz), 7.31 – 7.27 (3H, m),
3
7
.23 – 7.13 (5H, m), 6.69 (2H, d, J 8.8 Hz), 6.40 (2H, d, J 8.8 Hz), 2.48 (2H, q, J 7.6
1
3
Hz), 0.950 (3H, t, J 7.6 Hz); C NMR (CDCl ): δ = 144.7, 144.0, 143.5, 141.4, 139.4,
3
1
3
8
34.7, 132.3, 130.4, 130.1, 128.6, 128.4, 127.0, 126.5, 115.2, 29.55, 13.99; MS:
+
00.42 (M+H ); Anal. Calcd for C H N: C, 88.25; H, 7.07; N, 4.68. Found: C,
2
2
21
7.35; H, 6.87; N, 4.50.
4
4
.2.4. General procedure for the demethylation of compounds 6 and 7.
.2.4.1. Synthesis of (E)-N-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-
enyl)phenyl)isobutyramide (8b). To a cold at -78 °C solution of compound 6b
0.050 g, 0.125 mmol) in CH Cl (8.9 mL), BBr (0.625 mmol, 1.0 M in CH Cl , 5
(
2
2
3
2
2
equiv per bond, 0.625 mL) was added dropwise and the reaction was kept for another

ACCEPTED MANUSCRIPT
1
h at the same temperature. Then, the solution was cooled at 0 °C, neutralized with
MeOH and HCl 1 N was added. The organic phase was extracted twice with EtOAc.
The combined organic extracts were washed with water and brine, dried with Na SO ,
2
4
filtered and evaporated. The residue was purified by flash column chromatography
(
Hex/EtOAc 6:4) to provide compound 8b as oil (75% yield). R = 0.34 (Hex/EtOAc
f
1
6
7
:4); H NMR (CDCl ): δ = 7.28 (1H, br. s), 7.20 – 7.11 (9H, m), 6.83 (4H, br. d, J
3
1
3
.6 Hz), 2.53 – 2.45 (3H, m), 1.21 (6H, d, J 6.8 Hz), 0.951 (3H, t, J 7.6 Hz); C NMR
(
CDCl ): δ = 175.4, 155.2, 143.2, 142.3, 140.6, 138.5, 136.7, 136.3, 131.9, 131.4,
3
+
1
30.3, 128.4, 126.6, 119.6, 115.8, 37.26, 29.62, 20.06, 13.95; MS: 386.50 (M+H );
Anal. Calcd for C H NO : C, 81.01; H, 7.06; N, 3.63. Found: C, 80.17; H, 6.83; N,
2
6
27
2
3
.47.
4
.2.4.2.
Synthesis
of
(Z)-N-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-
enyl)phenyl)isobutyramide (9b). According to the general procedure, compound 9b
was obtained from compound 7b after purification by flash column chromatography
(
(
(
Hex/EtOAc 6:4) and evaporation of the solvent as oil (75% yield). R = 0.34
f
1
Hex/EtOAc 6:4); H NMR (CDCl ): δ = 7.53 (2H, d, J 8.0 Hz), 7.29 (1H, br. s), 7.21
3
2H, d, J 8.4 Hz), 7.18 (2H, d, J 8.0 Hz), 7.14 – 7.11 (3H, m), 6.72 (2H, d, J 8.4 Hz),
6
.50 (2H, d, J 8.4 Hz), 2.59 – 2.53 (1H, m), 2.49 (2H, q, J 7.2 Hz), 1.29 (6H, d, J 6.4
1
3
Hz), 0.942 (3H, t, J 6.8 Hz); C NMR (CDCl ): δ = 175.5, 153.8, 142.4, 141.5, 140.0,
3
1
1
3
37.6, 136.4, 135.4, 132.1, 130.2, 129.7, 127.9, 126.0, 119.6, 114.4, 36.79, 29.04,
+
9.67, 13.62; MS: 386.53 (M+H ); Anal. Calcd for C H NO : C, 81.01; H, 7.06; N,
2
6
27
2
.63. Found: C, 80.16; H, 6.84; N, 3.51.
4
.2.4.3.
Synthesis
of
(Z)-N-(4-(2-(4-hydroxyphenyl)-1-phenylbut-1-en-1-
yl)phenyl)isobutyramide (8c). According to the general procedure, compound 8c
was obtained from compound 6c after purification by flash column chromatography
(
Hex/EtOAc 6:4) and evaporation of the solvent as oil (80% yield). R = 0.28
f
1
(
Hex/EtOAc 6:4); H NMR (acetone-d ): δ = 8.90 (1H, br. s), 8.23 (1H, s), 7.40 – 7.37
6
(
4H, m), 7.29 (1H, t, J 7.2 Hz), 7.25 (2H, d, J 8.4 Hz), 7.01 (2H, d, J 8.8 Hz), 6.82
2H, d, J 8.8 Hz), 6.69 (2H, d, J 8.4 Hz), 2.58 – 2.52 (1H, m), 2.43 (2H, q, J 7.6 Hz),
(
1
3
1
.12 (6H, d, J 6.8 Hz), 0.930 (3H, t, J 7.6 Hz); C NMR (acetone-d ): δ = 175.0,
6
1
56.4, 144.7, 142.2, 138.9, 138.0, 134.0, 131.3, 129.9, 128.6, 127.0, 119.0, 115.4,

ACCEPTED MANUSCRIPT
+
3
8
6.36, 29.24, 19.48, 13.41; MS: 386.53 (M+H ); Anal. Calcd for C H NO : C,
2
6
27
2
1.01; H, 7.06; N, 3.63. Found: C, 80.19; H, 6.85; N, 3.48.
4
.2.4.4.
Synthesis
of
(E)-N-(4-(2-(4-hydroxyphenyl)-1-phenylbut-1-en-1-
yl)phenyl)isobutyramide (9c). According to the general procedure, compound 9c
was obtained from compound 7c after purification by flash column chromatography
(
Hex/EtOAc 6:4) and evaporation of the solvent as oil (80% yield). R = 0.36
f
1
(
Hex/EtOAc 6:4); H NMR (acetone-d ): δ = 9.08 (1H, br. s), 8.18 (1H, s), 7.70 (2H,
6
d, J 8.8 Hz), 7.17 (2H, d, J 8.8 Hz), 7.06 – 7.00 (3H, m), 6.99 (2H, d, J 8.8 Hz), 6.93
(2H, d, J 8.0 Hz), 6.66 (2H, d, J 8.8 Hz), 2.70 – 2.60 (1H, m), 2.48 (2H, q, J 7.2 Hz),
1
3
1
1
1
.19 (6H, d, J 6.8 Hz), 0.951 (3H, t, J 7.2 Hz); C NMR (acetone-d ): δ = 174.8,
6
55.8, 143.7, 141.8, 138.7, 138.3, 137.8, 133.1, 130.7, 130.6, 129.6, 127.3, 125.5,
+
18.9, 114.8, 35.75, 28.62, 19.02, 13.01; MS: 386.52 (M+H ); Anal. Calcd for
C H NO : C, 81.01; H, 7.06; N, 3.63. Found: C, 80.18; H, 6.86; N, 3.49.
26
27
2
4
.2.4.5. Synthesis of (E)-N-(4-(2-(2,4-dichlorophenyl)-1-(4-hydroxyphenyl)but-1-
en-1-yl)phenyl) isobutyramide (8g). According to the general procedure, compound
g was obtained from compound 6g after purification by flash column
8
chromatography (Hex/EtOAc 6:4) and evaporation of the solvent as oil (80% yield).
1
R = 0.32 (Hex/EtOAc 6:4); H NMR (acetone-d ): δ = 8.90 (1H, br. s), 8.43 (1H, br.
f
6
s), 7.40 – 7.38 (3H, m), 7.28 (1H, d, J 8.0 Hz), 7.25 (1H, d, J 2.0 Hz), 7.14 (2H, d, J
.4 Hz), 6.89 (2H, d, J 8.8 Hz), 6.88 (2H, d, J 8.4 Hz), 2.58 – 2.52 (1H, m), 2.49 (2H,
8
1
3
q, J 7.6 Hz), 1.11 (6H, d, J 6.8 Hz), 0.953 (3H, t, J 7.6 Hz); C NMR (acetone-d ): δ
6
=
175.6, 157.2, 141.9, 140.8, 138.6, 138.3, 137.6, 134.7, 134.2, 132.9, 131.0, 130.4,
+
1
29.4, 127.4, 118.7, 115.6, 36.37, 28.45, 19.63, 13.04; MS: 455.41 (M+H ); Anal.
Calcd for C H Cl NO : C, 68.73; H, 5.55; N, 3.08. Found: C, 68.03; H, 5.40; N,
2
6
25
2
2
2
.95.
4
.2.4.6. Synthesis of (Z)-N-(4-(2-(2,4-dichlorophenyl)-1-(4-hydroxyphenyl)but-1-
en-1-yl)phenyl) isobutyramide (9g). According to the general procedure, compound
g was obtained from compound 7g after purification by flash column
9
chromatography (Hex/EtOAc 6:4) and evaporation of the solvent as oil (80% yield).
1
R = 0.32 (Hex/EtOAc 6:4); H NMR (acetone-d ): δ = 9.13 (1H, br. s), 8.18 (1H, br.
f
6
s), 7.72 (2H, d, J 8.4 Hz), 7.40 – 7.38 (1H, m), 7.27 (1H, d, J 8.0 Hz), 7.26 (1H, d, J

ACCEPTED MANUSCRIPT
2
.0 Hz), 7.22 (2H, d, J 8.8 Hz), 6.80 (2H, d, J 8.8 Hz), 6.54 (2H, d, J 8.8 Hz), 2.70 –
.60 (1H, m), 2.47 (2H, q, J 7.6 Hz), 1.18 (6H, d, J 6.8 Hz), 0.947 (3H, t, J 7.2 Hz);
2
1
3
C NMR (acetone-d ): δ = 175.7, 156.6, 141.9, 140.9, 139.3, 138.1, 137.4, 134.7,
6
1
1
3
34.4, 134.2, 132.8, 131.3, 130.1, 129.4, 127.4, 119.5, 114.9, 36.40, 28.40, 19.67,
+
3.03; MS: 455.40 (M+H ); Anal. Calcd for C H Cl NO : C, 68.73; H, 5.55; N,
2
6
25
2
2
.08. Found: C, 68.05; H, 5.41; N, 2.94.
.3. Biological evaluation
.3.1. Cell cultures
4
4
HeLa (human cervix adenocarcinoma cells) and MCF-7 (human mammary gland
adenocarcinoma cells) were grown in Nutrient Mixture F-12 [HAM] (Sigma
Chemical Co.) and Dulbecco’s Modified Eagle’s Medium (Sigma Chemical Co.),
respectively. 10% Heat-inactivated fetal calf serum (Invitrogen), 100 U/mL penicillin,
1
00 µg/mL streptomycin and 0.25 µg/mL amphotericin B (Sigma Chemical Co.) were
added to both media. Ovarian cancer cell lines A2780 and OVCAR5 were cultured in
RPMI1640 (Lonza), with the addition of 10% Fetal Bovine Serum (FBS, Lonza) and
2
5
mM Glutamine (Lonza). The cells were cultured at 37°C in a moist atmosphere of
% carbon dioxide in air.
4
.3.2. Inhibition growth assay
4
HeLa and MCF-7 (4-5×10 ) cells were seeded into each well of a 24-well cell culture
plate. After incubation for 24 h various concentrations of the test agents and
tamoxifen (1, T-5648 Sigma-Aldrich) were added and the cells were then incubated in
standard conditions for a further 72 h. A trypan blue assay was performed to
determine cell viability. Cytotoxicity data were expressed as GI50 values, i.e. the
concentration of the test agent inducing 50% reduction in cell number compared with
control cultures. A2780 and OVCAR5 were plated at a concentration of 15 000
cell/mL. 72 h later cells were treated with the different compounds, after 72 h cell
survival was assessed by MTS assay and dose-response curves were plotted.
4
.3.3. Topoisomerase-Mediated DNA Relaxation

ACCEPTED MANUSCRIPT
Supercoiled pBR322 plasmid DNA (0.25 µg, Fermentas Life Sciences) was incubated
with 1U topoisomerase II (human recombinant topoisomerase II α, USB Corporation)
or 2U topoisomerase I (human recombinant topoisomerase I, TopoGen) and the test
compounds as indicated, for 60 min at 37 °C in 20 µL reaction buffer.
Reactions were stopped by adding 4 µL stop buffer (5% sodium dodecyl sulfate
(
SDS), 0.125% bromophenol blue, and 25% glycerol), 50 µg/mL proteinase K
Sigma) and incubating for a further 30 min at 37 °C. The samples were separated by
(
electrophoresis on a 1% agarose gel at room temperature. The gels were stained with
ethidium bromide 1 µg/mL in TAE buffer (0.04 M Tris-acetate and 0.001 M EDTA),
transilluminated by UV light, and fluorescence emission was visualized by a CCD
camera coupled to a Bio-Rad Gel Doc XR apparatus.
4
.3.4. Topoisomerase II-mediated DNA cleavage
Supercoiled pBR322 plasmid DNA (0.25 µg) was incubated with 10 U topoisomerase
II (human recombinant topoisomerase II α, USB) and the test compounds, as
indicated, for 60 min at 37 °C in 20 µL reaction buffer.
Reactions were stopped by adding 4 µL stop buffer (5% SDS, 0.125% bromophenol
blue and 25% glycerol), 50 µg/mL proteinase K (Sigma) and incubating for a further
3
0 min at 37 °C. The samples were separated by electrophoresis on a 1% agarose gel
containing ethidium bromide 0.5 µg/mL in TBE buffer (0.09 M Tris-borate and 0.002
M EDTA), transilluminated by UV light, and fluorescence emission was visualized by
a CCD camera coupled to a Bio-Rad Gel Doc XR apparatus.
4
.3.5. Evaluation of apoptotic cell death by Annexin V-FITC and propidium
iodide staining
To detect phosphatidylserine translocation from the inner face to the outer surface of
plasma membrane a FITC Annexin V Apoptosis Detection Kit I (BD Pharmigen) was
used.
5
HeLa cells (2.0 x 10 ) were seeded into each cell culture plate. After incubation for 24
h the test agents were added to the complete medium at the indicated concentrations
and cells were incubated for a further 28 h. After treatment, cells were centrifuged and
6
resuspended at 10 cells/mL in binding buffer. Cell suspensions (100 µL) were added
with Annexin V-FITC and propidium iodide (PI) as indicated by the supplier’s

ACCEPTED MANUSCRIPT
instructions, and incubated in the dark for 15 min at room temperature. The
populations of Annexin V-positive/PI-negative cells (early apoptosis) and Annexin V-
positive/PI-positive cells (late apoptosis) were evaluated by FACSCanto II flow
cytometer (Becton-Dickinson, Mountain View, CA).
4
.3.6. Determination of mitochondrial membrane potential on whole cells
The mitochondrial membrane potential was evaluated by using the BD™ MitoScreen
Kit (BD Pharmigen) containing the membrane-permeable lipophilic cationic
fluorochrome
5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine
25
5
iodide (JC-1), according to Cossarizza et al. HeLa cells (2.0 x 10 ) were seeded into
each cell culture plate. After incubation for 24 h the test agents were added to the
complete medium at the indicated concentrations and cells were incubated for a
further 28 h. After treatment, cells were centrifuged, resuspended in JC-1 Working
Solution and incubated for 30 min at 37°C in CO incubator. Following incubation,
2
cells are washed twice, resuspended in Assay Buffer and analyzed by a FACSCanto II
flow cytometer (Becton-Dickinson, Mountain View, CA).
4
.4. Molecular modeling
All the synthesized compounds were docked in the etoposide binding site in the
2
6
crystal structure of the topoisomerase II beta/DNA complex (3QX3.pdb) using
2
7
28
AutoDock 4.2 software. A Lamarkian genetic algorithm was employed for the
docking simulation, performing 100 independent runs per molecule. In each run, a
population of 50 individuals evolved along 27000 generations and a maximum
number of 25 million energy evaluations was performed. The best fit (lowest docked
energy) solutions of the 100 independent runs were stored for subsequent analysis.
2
9
The visual inspection of docked structures was carried out using VMD.
ACKNOWLEDGEMENTS
This research has been developed under the umbrella of CM1106 COST Action
Chemical Approaches for Targeting Drug Resistance in Cancer Stem Cells”. G.
Damia and F. Ricci acknowledge the generous contributions of The Italian
“

ACCEPTED MANUSCRIPT
Association for Cancer Research (AIRC, IG14536 to G.D.). The authors express their
gratitude to Ms. Ioana Stupariu for the revision of the manuscript.
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