Synthesis of curcumin derivatives and analysis of their antitumor effects in triple negative breast cancer (TNBC) cell lines

Article abstract of DOI:10.3390/ph12040161

We analyzed antitumor effects of a series of curcumin analogues. Some of them were obtained by reaction of substitution involving the two phenolic OH groups of curcumin while the analogues with a substituent at C-4 was prepared following an original proce

Full text of DOI:10.3390/ph12040161

pharmaceuticals
Communication
Synthesis of Curcumin Derivatives and Analysis of
Their Antitumor Effects in Triple Negative Breast
Cancer (TNBC) Cell Lines
Paola Maria Bonaccorsi 1, Manuela Labbozzetta 2, Anna Barattucci 1,
Tania Maria Grazia Salerno 1, Paola Poma 2,* and Monica Notarbartolo 2
1
Department of Chemical, Biological, Pharmaceutical and Environmental Science, University of Messina,
98122 Messina, Italy; pbonaccorsi@unime.it (P.M.B.); abarattucci@unime.it (A.B.);
tsalerno@unime.it (T.M.G.S.)
Department of Biological, Chemical and Pharmaceutical Science and Technology (STEBICEF),
2
University of Palermo, 90133 Palermo, Italy; manuela.labbozzetta@unipa.it (M.L.);
monica.notarbartolo@unipa.it (M.N.)
*
Correspondence: paola.poma@unipa.it
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 October 2019; Accepted: 23 October 2019; Published: 26 October 2019
Abstract: We analyzed antitumor effects of a series of curcumin analogues. Some of them were
obtained by reaction of substitution involving the two phenolic OH groups of curcumin while the
analogues with a substituent at C-4 was prepared following an original procedure that regards the
condensation of benzenesulfenic acid onto the nucleophilic central carbon of the curcumin skeleton.
We analyzed cytotoxic effects of such derivatives on two TNBC (triple negative breast cancer) cell
lines, SUM 149 and MDA-MB-231, but only three of them showed an IC50 in a lower micromolar
range with respect to curcumin. We also focused on these three derivatives that in both cell lines
exhibited a higher or at least equivalent pro-apoptotic effect than curcumin. The analysis of molecular
mechanisms of action of the curcumin derivatives under study has highlighted that they decreased
NF-κB transcriptional factor activity, and consequently the expression of some NF-κB targets. Our data
confirmed once again that curcumin may represent a very good lead compound to design analogues
with higher antitumor capacities and able to overcome drug resistance with respect to conventional
ones, even in tumors difficult to treat as TNBC.
Keywords: antiproliferative activity; prooxidant activity; antioxidant activity; pro-apoptotic activity;
NF-κB inhibition; sulfenic acid
1. Introduction
Cancer can be described as uncontrolled DNA replication and cell division, evasion from
programmed cell death, breaking through normal tissue boundaries and invasion to new sites in the
body. Actually, the most promising anti-cancer therapies combine agents with different molecular
mechanisms such as specific drugs and chemo- or radiotherapies, resulting in a better efficacy and
longer survival [1].
In this scenario, polyphenols are to be considered molecules with antitumor action because of their
capacity to interfere with a large number of pathways that in the neoplastic cell are simultaneously
deregulated. Their role in improving bioavailability of drugs has been often underlined, perfecting
the response of cancer cell to different therapies [
polyphenols might be also used to sensitize tumor cells to chemo- and radiotherapy by inhibiting
pathways that lead to treatment resistance [ ]. For this reason, these compounds are often defined
as “privileged” structures [ ], the implications of which in human health are vast, including many
2]. Recently, researchers suggested that natural
3
1
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antitumor activities, neuroprotection, beneficial effects in cardiovascular dysfunctions, diabetes and
inflammatory processes.
Curcumin, extracted from rhizomes of Curcuma longa L. and used for thousands of years in
traditional eastern medicine, is certainly one of the most studied natural polyphenols, despite its poor
bioavailability, rapid in vivo metabolism and low cellular uptake that limit their use in therapy [
Curcumin is still explored for its antioxidant and anti-inflammatory properties and especially for its
antitumor properties [ ] exerted towards many types of cancers, such as breast cancer, hepatocellular
cancer, multiple melanoma, osteosarcoma, hematological malignances, lung cancer, head and neck
squamous cell carcinoma, prostate cancer and brain tumors [ 22]. Research on curcumin over the
4].
5–8
9–
years focused on the development of derivatives or delivery systems that could bypass the critical
issues of such polyphenols and emphasize its potential health benefits [23–33].
In this context, we recognized the interest in the synthesis of curcumin derivatives that could act
against a targeted typology of cancer. Therefore, we describe synthetic procedures of some curcumin
derivatives and their antitumor capacities in two triple negative breast cancer (TNBC) cellular models,
MDA-MB-231 and SUM 149, in comparison with curcumin. We have chosen to study these molecules
on TNBC cell line models because TNBC represents one of the most aggressive malignant neoplasms,
characterized by molecular aspects as the lack of the respective receptor targets, that limit therapeutic
possibility because it does not respond to conventional hormonal interventions. Moreover, another
peculiar characteristic is the over-expression and hyperactivation of the transcription factor NF-
κB,
of which curcumin is an inhibitor [34,35].
2. Results and Discussion
2.1. Chemistry
Extensive structure-activity studies have shown that the phenolic OHs and the
di-keto groups are essential for the antioxidant properties exerted by curcumin and the
α
,
β
-unsaturated
α,β-unsaturated
di-keto moiety, in particular, is a key for its anticancer activity. On the other hand, it is believed that the
poor bioavailability and stability of curcumin in physiological media depend on these groups [36 37].
,
Taking into account such observations, we have chosen to synthesize and study curcumin derivatives
where the phenolic OH groups are completely or partially substituted, and a curcumin derivative,
the unsaturated di-keto chain of which has been substituted on C-4. Five curcumin derivatives were
chosen to carry out this investigation and their structures are shown in Figure 1. Compounds
and
were known in the literature, and their synthesis (1H and 13C NMR spectra in Materials and
Methods) was conducted by modification of an already described procedure [38]. Compounds and
were obtained from curcumin and dibromo-p-xylene 6 in the presence of K2CO3, separated and
1
2
4
5
purified by column chromatography (Scheme 1). The substitution reaction of the phenolic function of
curcumin on the di-bromo derivative 6 was complicated by the formation of an insoluble precipitate
that was nearly avoided by the addition of p-xylene derivative 6 and a base in two tranches. Finally,
Compound
3
(1H and 13C NMR spectra in Materials and Methods) was synthesized, taking into
account the highly selective reaction reported by Allison [39] (Scheme 1) in which the electrophilic
sulfenic function reacts with the nucleophilic central carbono f a 1,3-diketone, producing a thioether
with loss of water. The mechanism of the reaction is believed to be concerted. Sulfenic acids are
usually transient intermediates with a dual electrophilic/nucleophilic nature and for this reason they
are generated in situ from suitable precursors [40,41]. In Scheme 1 the thermolysis of sulfoxide 7,
the precursor of the corresponding sulfenic acid, is shown, together with its formation from thiophenol.
The reaction was carried out in 1,2-dichloroethane (DCE) at reflux, without the presence of any base or
acid [42], so avoiding any possible decomposition of the labile curcumin.

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Figure 1. Structural skeletons of curcumin analogues 1–5.
Scheme 1. Synthetic procedures for curcumin analogues 3–5.
2.2. Biological Studies
2.2.1. Antiproliferative Activity
We analyzed cytotoxic effects of curcumin derivatives 1–5 on the two TNBC cell lines SUM 149
and MDA-MB-231, and also in a normal cell line, 1-7HB2. In Table 1 the IC50 of Compounds and
curcumin are reported and only Compounds showed an IC50 lower than curcumin (Figures 2
1–5
1–3
and 3), with SUM 149 cells being the most sensitive to the action of these derivatives. This result led us
to focus our studies on Compounds 1–3.

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Table 1. Cell growth inhibitory effects of Compounds 1–5 and curcumin evaluated after 72 h of
treatment by MTS assays.
SUM149
IC50 (µM)
MDA-MB-231
IC50 (µM)
1-7HB2
IC50 (µM)
Compounds
Curcumin (curc)
14.0 ± 0.29
11.2 ± 1.30
13.2 ± 1.59
13.5 ± 0.88
not reached
not reached
25.5 ± 0.35
18.0 ± 0.41
20.0 ± 0.00
15.0 ± 0.85
not reached
not reached
37.5 ± 0.51
20.0 ± 0.22
not reached
12.5 ± 0.43
not reached
not reached
1
2
3
4
5
Data are expressed as the concentrations which inhibit 50% (IC50) cell growth and are means
separate experiments.
±
SE of at least three
Figure 2. Cytotoxic activity of curcumin and its derivatives 1–3 on SUM 149 cells. Cell viability
was assessed by MTS assay. Data are expressed as the mean of at least three different experiments
performed in triplicate. Different letters represent significant differences in cytotoxic activity among the
concentration (Tukey test, p < 0.05).
Figure 3. Cytotoxic activity of curcumin and its derivatives 1–3 on MDA-MB-231 cells. Cell viability was
assessed by MTS assay. Data are expressed as mean of at least three different experiments performed in
triplicate. Different letters represent significant differences in cytotoxic activity among the concentration
(Tukey test, p < 0.05).

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In order to verify if these analogues are characterized by a specificity of action towards the
cancer model examined, we performed a cell growth assay in non-TNBC cells, in particular, in a cell
line of acute promyelocytic leukemia HL60 and in its multidrug resistant variant (HL60: curcumin
IC50 = 47.5 µM ± 3.3, 1 IC50 = 14.0
IC50 = 40.5 2.1, 1 IC50 = 7.0
results confirmed that Compounds
µ
M
M
±
1.7, 2 IC50 = 17.0
0.8, 2 IC50 = 9.0
1–3 do not have specific effects on TNBC cell lines, on the contrary,
µ
M
±
1.2, 3 IC50 < 1
µ
M; HL60 R: curcumin
µM
±
µ
±
µM
±
2.8, 3 IC50 = 6.7
µM
±
1.1). These
they show a strong action also in the non-TNBC cancer model analyzed and for this are even more
interesting, because they can be used in different types of cancer. In line with the results obtained in the
TNBC cells, also in the non-TNBC cells, Analogues
1–3 are more active than curcumin. Noteworthy
the IC50 of Compounds and are lower in the multidrug resistant variant, HL60R, than the parental
1
2
HL60; these results are worthy to further experimental analyses to verify if these analogues are capable
to bypass the wide problem of acquired multidrug resistance.
2.2.2. Pro- and Antioxidant Activity
We investigated the mechanism by which compounds could exert any anticancer activity with
the study of their pro- or antioxidant properties, due to the well-known role that oxidative stress
plays in the progression of a number of cancers. Therefore, our investigation on the mechanism by
which Compounds
antioxidant properties.
In order to assess whether Analogues
cells were treated with the antioxidant N-acetyl-L-cysteine (NAC), which is a well-known scavenger of
reactive oxygen intermediates, at 2mM for 1h, before exposure to curcumin and Compounds at the
corresponding IC50 values. As shown in Table 2, both for SUM 149 cells (A) and MDA-MB-231 cells
(B), the addition of NAC reduced the cytotoxic activity of Compounds and as well as of curcumin,
whereas the same results were not obtained for Compound . In our study, the analysis carried out
1–3 could exert any anticancer activity started with the study of their pro- or
1–3 could behave as pro-oxidant substances, the two TNBC
1–3
1
2
3
with DPPH (2,2-diphenyl-1-picrylhydrazyl) reduction assay indicated that Analogues 1 and 2 do not
possess antioxidant activity, since the efficient dose (ED50) of these was not identified (Table 3). From a
chemical point-of-view, these results are in line with previous observations showing that the protection
of the phenolic groups causes the complete abolition of the antioxidant activity [43]. From a biological
point of view, they indicated that the mechanism of antitumor activity against TNBC can involve an
at least partially pro-oxidant effect for Compounds
methylene site of Compound showed a scavenging activity lower then curcumin [43], reaching an
ED50 at a concentration of 19.2 µM (Table 3).
1 and 2. Surprisingly, substitution in the active
3

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Table 2. Cell counting analysis in SUM 149 cells (A) and in MDA-MB-231 cells (B) after treatment with
antioxidant NAC at 2mM before exposure to the curcumin and its analogues at the IC50 values. Data
are expressed as mean ± standard error (SE).
Cell Lines and Treatments
Cell Viability (%)
SUM 149
+ NAC 2 mM a,
*
*
100.0 ± 9.5
32.0 ± 2.1
28.0 ± 3.2
36.6 ± 1.8
34.0 ± 2.1
74.5 ± 0.0
100.0 ± 20.5
74.4 ± 1.4
14.0 ± 4.6
+ curc. 14 µM bc,
A
+ 1. 11 µM bc,
*
+ 2. 13 µM bd,
+ 3. 14 µM bc,
*
*
+ NAC + curc de,
*
+ NAC + 1 ae,
+ NAC + 2 ae,
*
*
+ NAC + 3 c,
*
MDA-MB-231
+ NAC 2 mM a,
*
*
87.0 ± 3.9
45.0 ± 4.6
23.0 ± 1.4
44.0 ± 4.9
25.6 ± 0.7
60.0 ± 2.1
53.5 ± 0.7
78.0 ± 1.8
+ curc. 25µM ab,
+ 1. 18µM b,
+ 2. 20µM ab,
+ 3. 15µM b,
*
*
*
B
+ NAC + curc abc,
*
+ NAC + 1 ab,
+ NAC + 2 ac,
*
*
Significant differences among the treatments of each cell line are represented by different letters (a, b, c, d and e) in
the column (Cell lines and treatments). * Differences when treatments are compared to the control, p < 0.05.
Table 3. DPPH free radical scavenging activity. The results were expressed as the antiradical capacity
(ARC), which is the inverse of the ED50
.
ARC
(1/ED50
ED50
)
Trolox
Curcumin
12.0 µM
7.5 µM
5
8
1
2
3
>100 µM
>100 µM
19.2 µM
-
-
3.1
2.2.3. Pro-Apoptotic Activity
In the two TNBC cell lines, Compounds
1–3 showed increased or at least equivalent cell death
induction than curcumin. The cells were treated for 24 h with curcumin and its derivatives at
concentrations close to the corresponding IC50 values. The flow cytometry analysis with propidium
iodide revealed that in SUM 149 cells only Compound 1 determined a significant block in a pre-G0-G1
position, while in MDA-MB-231 cells the order of potency in inducing cell death was Compound
Compound > curcumin (Figures 4 and 5). Anyway, the results appeared to be in agreement with the
cytotoxicity data.
1 >
3

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Figure 4. Representative example of flow cytometry analysis with propidium iodide. SUM 149 cells
were treated with curcumin and its analogues at 15
of the events in the preG0–G1 position.
µM for 24 h. Numbers in the panels indicate the %
Figure 5. Representative example of flow cytometry analysis with propidium iodide. MDA-MB-231
cells were treated with curcumin and its analogues at 25 µM for 24 h. Numbers in the panels indicate
the % of the events in the preG0–G1 position.
2.2.4. NF-κB Inhibition
Since curcumin is an inhibitor of the nuclear activation of NF-
curcumin derivatives to inhibit NF- B DNA-binding activity by TransAM assays. Each cell line
was treated for 8 h and 24 h with curcumin and its derivatives, at the relative IC50 values. In the
SUM 149 cell line we observed a high reduction of NF- B DNA-binding activity for Compounds
in particular, Compound exhibited a stronger decrease than curcumin, only after 24 h of treatment
(Figure 6). In MDA-MB-231 cells Compounds and were NF- B inhibitors but only Compound
showed a greater effect than curcumin, after 8 h of treatment (Figure 7).
κB, we analyzed the capacity of
1–3
κ
κ
1–3,
3
1
3
κ
1

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Figure 6. NF-
were treated for 24 h with curcumin and derivatives
experiments carried out in duplicate) are expressed as arbitrary units/
κ
B (p65 subunit) DNA binding capacity in nuclear extracts of SUM 149 cells. The cells
. Results (mean standard error of two
g protein of cells nuclear extracts.
1–
3
±
µ
Different letters (a and b) in the column of the cell lines and treatments represent significant differences
among the different treatments. * Differences when treatments are compared to the control, p < 0.01.
Figure 7. NF-
cells were treated for 8 h with curcumin and derivatives
experiments carried out in duplicate) are expressed as arbitrary units/
κ
B (p65 subunit) DNA binding capacity in nuclear extracts of MDA-MB-231 cells. The
. Results (mean standard error of two
g protein of cells nuclear extracts.
1–
3
±
µ
Different letters (a and b) in the column of the cell lines and treatments represent significant differences
among the different treatments. Differences when treatments are compared to the control: ** p < 0.05,
* p < 0.01.
In order to verify if the three analogues alter the expression of some targets of NF-
western blot analysis was used. The two cell lines were treated with curcumin and derivatives
same conditions. In line with the results of NF- B DNA-binding activity inhibition, only Compounds
and caused a decrease of expression of some targets in the same cell lines. In particular, in SUM
149 cells only Compound caused a decreased expression of Survivin (a 55% of reduction respect to
κ
B, like curcumin,
1–3
in the
κ
1
3
3
control) and Bcl-2 (a 48% of reduction respect to control), while in MDA-MB-231 cells only Compound
1 caused a reduction of IAP1 expression (a 32% of reduction with respect to the control) (Figures 8
and 9).

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Figure 8. Western blot analysis of the levels of Survivin and Bcl-2 in SUM 149 cells treated for 24 h
with Compound . On the left the results are expressed as mean standard error (SE) of two different
3
±
experiments; on the right, the results of a representative experiment. * Differences when treatments are
compared to the control, p < 0.01.
Figure 9. Western blot analysis of the levels of IAP1 in MDA-MB-231 cells treated for 8 h with
Compound
1
. On the left are the results expressed as mean
±
standard error (SE) of two different
experiments; on the right, the results of a representative experiment. * Differences when treatments are
compared to the control, p < 0.01.
3. Conclusions
In summary, the synthesis of three new curcumin derivatives has been described, one of which
has been prepared by an original procedure via sulfenic acid condensation. Five curcumin derivatives
have been analyzed in a study on TNBC cell lines, of which Compounds
pro-apoptotic activity towards the cellular models studied. Among the three compounds,
seem to be able to inhibit the activation of NF- B and the expression of some of its targets in a stronger
1
–
3
showed cytotoxic and
1
and
3
κ
way than curcumin and in a mode which is peculiar with respect to the two TNBC cell lines examined.
This difference could be due to some differences, both phenotypic and molecular, between the two cell
lines studied. In fact, SUM 149 is a model of inflammatory breast cancer, a basal-like subtype, while
MDA-MB-231 is claudin-low subtype. Other authors observe different responses to drugs between
these two cell lines though both are TNBC models [44,45].
We thought that SUM 149 and MDA-MB-231 cells could represent the enormous clinical
heterogeneity which is found in patients. In fact, although both cell lines belong to the triple
negative type, they represent a good study model to compare the response to drugs just for their
different characteristics.
Even though Compound
comparable or lower effects to those of curcumin (respectively in SUM 149 cells and in MDA-MB-231
cells). Its lower efficacy on the inhibition of the targets could depend precisely on the effect on NF-
2 has an IC50 inferior than curcumin in both cell lines, on NF-κB it has
κ
B

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that was more limited compared to Analogues
1
and
3. This result would not seem to depend on the
chemical structure of Compound
different analogues on the inhibition of the transcription factor. Of all three compounds, Compound
appears to be the most promising, given that in both cell lines it shows strong inhibitory effects of cell
proliferation and pro-apoptotic as well as a downregulation of NF- B activity. For these biological
2, rather than on the peculiarity of the mechanism of action of the
3
κ
effects, this compound appears to be worthy for further analysis, as in vivo assays, to demonstrate the
real efficacy as a new anticancer agent.
Taken together, our data corroborate potentiality of curcumin as a lead compound in cancer
diseases. In particular, in this work we highlighted Analogues
towards triple negative cancer cell lines. Their cytotoxic and pro-apoptotic capacities with ability to
interfere with NF- B pathway can contribute to paving the way to their use as new anticancer agents.
1, 2 and 3 were effective molecules
κ
4. Materials and Methods
4.1. Synthesis
Curcumin from Curcuma longa (C1386, purity > 65%, Sigma-Aldrich) was purified by column
chromatography on silica gel using CHCl3/hexane 90:10 as eluent; the other commercial reagents and
solvents were used without further purification. The reactions were monitored by TLC on commercially
available precoated plates (silica gel 60 F254) and the products were visualized with vanillin (1 g
dissolved in MeOH (60 mL) and conc. H2SO4 (0.6 mL)) and/or by a UV lamp. Silica gel 60 was used
for column chromatography. 1H and 13C NMR spectra were recorded in CDCl3 with a Varian 500
spectrometer (at 500 MHz for 1H and 125 MHz for 13C). Chemical shifts are given in parts per million
(ppm) referenced to the residual protons in CDCl3 (δ δ C
= 7.27 ppm for 1H NMR and = 77.0 ppm for 13
NMR) solvent. Multiplicities of the signals are abbreviated as follows: s = singlet; bs = broad singlet;
d = doublet; t = triplet; and m = multiplet. NMR peak assignments are supported by homonuclear
1
(COSY, Correlation Spectroscopy) and heteronuclear correlation H-13C spectroscopy (HSQCAD).
Coupling constants (J) are given in Hertz. We have chosen to give the same number at two equivalent
carbon atoms of Compounds 3–5, as shown in the structures reported below (equivalent carbons are
colored blue). Combustion analyses were carried out on a FISONS EA1108 elemental analyzer.
4.1.1. (1E,6E)-1-(4-hydroxy-3-methoxyphenyl)-7-(3-methoxy-4-propargyloxyphenyl)hepta-1,6-diene
-3,5-dione (1) and (1E,6E)-1,7-bis(3-methoxy-4-propargyloxyphenyl)hepta-1,6-diene-3,5-dione (2)
Curcumin (300 mg, 0.81 mmol) was dissolved in 15 mL of dry acetone, then propargyl bromide
(61 mg, 0.40 mmol) and K2CO3 (56 mg, 0.40 mmol) were added. The mixture was stirred under
argon at reflux temperature for 24 h. After that, a second addition of propargyl bromide (61 mg,
0.40 mmol) and K2CO3 (56 mg, 0.40 mmol) was made. The reaction was monitored for another
24 h, using TLC on silica gel (CHCl3/hexane 9:1). The solvent was removed under reduced pressure.
The residue was suspended in water and extracted with ethyl acetate. The combined organic layers
were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude was
purified by column chromatography (CHCl3/hexane 9:1) to obtain Compound
1
as an orange solid
in 47% yield, Rf 0.6 (CHCl3/hexane 9:1). The same column provided
2 as a yellow solid, 34% yield,
Rf 0.8 (CHCl3/Hexane 9:1). The 1H and 13C NMR data are consistent with the one reported in the
literature [38].
4.1.2. 4-phenylsulfanyl-(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (3)
Curcumin (643 mg, 1.77 mmol) and methyl 3-(phenylsulfinyl)propanoate 7 [43] (366 mg, 1.77 mmol)
were dissolved in DCE (45 mL) and refluxed (83 ◦C) for 48 h. The reaction was monitored by TLC
(100% DCM), until disappearance of sulfoxide 7. The solvent was removed under reduced pressure
and the crude was purified by column chromatography (hexane/DCM 2:8) to obtain Compound
3
(Figure 10) as an orange solid in 65% yield. Rf 0.3 (hexane/DCM 2:8); 1H (CDCl3):
δ
7.73 (2H, d, J =

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15.6 Hz, H-8), 7.52 (2H, d, J = 15.6 Hz, H-9), 7.28-7.22 (4H, m, H-13,14), 7.14-7.08 (3H, m, H-3,15), 6.97
(2H, d, J = 1.5 Hz, H-1), 6.89 (2H, d, J = 8.3 Hz, H-4), 5.89 (2H, s, OH), 3.895 (6H, s, H-7); 13C NMR
(CDCl3):
δ 188.0 (C-10), 148.2 (Cq), 146.7 (Cq), 143.5 (C-8), 139.1 (Cq), 129.1 and 125.6 (C-13 and C-14),
127.8 (Cq), 125.3 (C-15), 123.5 (C-3), 118.8 (C-9), 114.7 (C-4), 110.0 (C-1), 101.9 (C-11), 55.9 (C-7). Anal.
Calcd for C27H24O6S (476,54): C, 68.05; H, 5.08. Found: C, 67.91; H, 5.07. Figures 11 and 12 show
respectively 1H NMR spectrum and 13C NMR spectrum of Compound 3 in CDCl3 as solvent.
Figure 10. Structure of Compound 3.
Figure 11. 1H NMR spectrum of Compound 3 in CDCl3 as solvent.

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Figure 12. 13C NMR spectrum of Compound 3 in CDCl3 as solvent.
4.1.3. (1E,6E)-1-(4-hydroxy-3-methoxyphenyl)-7-[3-methoxy-4(4-methylbromobenzyl)oxyphenyl]
hepta-1,6-diene-3,5-dione (4) and (1E,6E)-1,7-bis [3-methoxy-4(4-methylbromobenzyl)
oxyphenyl]hepta-1,6-diene-3,5-dione (5)
Dibromo-p-xylene 6 (363, 1.37 mmol) was dissolved in a solution of curcumin (1 g, 2.74 mmol)
in dry acetone (100 mL), under argon. K2CO3 (190 mg, 1.37 mmol) was then added and the mixture
stirred at reflux for 12 h. After that, a second addition of dibromo-p-xylene 6 (363, 1.37 mmol) and
K2CO3 (190 mg, 1.37 mmol) was made and the reaction was maintained at reflux for 24 h. The reaction
was monitored using TLC (hexane/ethyl acetate 1:1). The crude was filtered, and the solvent was
removed from the solution under reduced pressure. The residue was suspended in water and extracted
with ethyl acetate. The crude was purified by column chromatography (hexane/ethyl acetate 7:3) to
obtain Compound
(CDCl3): 7.58 (2H, d, J = 15.6 Hz, H-8), 7.40 (8H, bs, H-14,15), 7.09 (2H, d, J = 1.5 Hz, H-1), 7.07 (2H,
dd, J = 8.3 Hz, J = 1.5 Hz, H-3), 6.86 (2H, d, J = 8.3 Hz, H-4), 6.48 (2H, d, J = 15.6 Hz, H-9), 5.80 (1H, s,
H-11), 5.17 (4H, s, H-12), 4.49 (4H, s, H-17), 3.93 (6H, s, H-7); 13C NMR (CDCl3):
183.2 (C-10), 149.9
5
(Figure 13) as an orange solid in 40% yield. Rf 0.7 (hexane/EtOAc 7:3); 1H NMR
δ
δ
(Cq), 149.7 (Cq), 140.2 (C-8), 137.5 (Cq), 136.9 (Cq), 129.3 and 127.5 (C-14 and C-15), 128.5 (Cq), 122.3
and 122.2 (C-3 and C-9), 113.4 (C-4), 110.4 (C-1), 101.3 (C-11), 70.4 (C-12), 56.0 (C-7), 33.1 (C-17). Anal.
Calcd for C37H34Br2O6 (734,47): C, 60.51; H, 4.67; Found: C, 60.70; H, 4.68. Figures 14 and 15 show
respectively 1H NMR spectrum and 13C NMR spectrum of Compound 5 in CDCl3 as solvent.
Figure 13. Structure of Compounds 4 and 5.

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Figure 14. 1H NMR spectrum of Compound 5 in CDCl3 as solvent.
Figure 15. 13C NMR spectrum of Compound 5 in CDCl3 as solvent.
The same column provided Compound
4 (Figure 13) as a yellow solid, 32% yield. Rf 0.4
(hexane/EtOAc 7:3); 1H NMR (CDCl3):
δ
= 7.59 (1H, d, J = 15.8 Hz, H-8), 7.58 (1H, d, J = 15.8 Hz, H-14),
7.41 (4H, m, H-24,25), 7.14-7.05 (4H, m, H-1,3,16,20), 6.93 and 6.86 (2H, two d, J = 8.2 Hz, H-4,19), 6.48
(1H, d, J = 15.8 Hz, H-13), 6.47 (1H, d, J = 15.8 Hz, H-9), 5.92 (1H, bs, OH), 5.80 (1H, s, H-11), 5.18 (2H,
s, H-22), 4.49 (2H, s, H-27), 3.95 and 3.94 (6H, two s, H-7,21); 13C NMR (CDCl3):
δ 183.4 and 182.9
(C-10,12), 149.8 (Cq), 149.6 (Cq), 147.8 (Cq), 146.7 (Cq), 140.6 and 140.2 (C-8,14), 137.5 (Cq), 136.8 (Cq),
129.3 and 127.5 (C-24,25), 128.9 (Cq), 128.4 (Cq), 122.8, 122.3, 122.1 and 121.6 (C-3,9,13,20), 114.7 and
113.3 (C-4,19), 110.2 and 109.5 (C1 and C16), 101.2 (C-11), 70.3 (C-22), 56.0 and 55.9 (C-7,21), 33.1 (C-27).
Anal. Calcd for C29H27BrO6 (551,43): C, 63.17; H, 4.94; Found: C, 63.33; H, 4.95. Figures 16 and 17
show respectively 1H NMR spectrum and 13C NMR spectrum of Compound 4 in CDCl3 as solvent.

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Figure 16. 1H NMR spectrum of Compound 4 in CDCl3 as solvent.
Figure 17. 13C NMR spectrum of Compound 4 in CDCl3 as solvent.
4.2. Cell Lines
Dr. Elda Tagliabue (Molecular Targeting Unit, Department of Experimental Oncology and
Molecular Medicine, Fondazione Institute of Hospitalization and Scientific Care, National Cancer
Institute, Milan, Italy) kindly provided us the human breast cancer cell lines: MDA-MB-231(ATCC:
HTB-26—Rockville, MD, USA) and SUM 149 (SUM149PT—Asterand Bioscience Detroit, MI). The
first was cultured in RPMI-1640 and the second was cultured in DMEM/F-12 supplemented with
insulin (5 µg/mL). The cells were authenticated using the short tandem repeat profiling method in their
Institute. Prof. Giulio Ghersi (STEBICEF Department, University of Palermo, Italy) kindly provided
us the non-tumorigenic cell line 1-7HB2 (ECACC 10081201—Cancer Research Technology, London,
UK) that was cultured in DMEM low glucose supplemented with hydrocortisone (5
µg/mL) and
insulin (10
µ
g/mL). HL60, obtained from ATCC® (CCL-240, Rockville, MD, USA), and its variant
HL60R, obtained by exposure to gradually increasing concentrations of doxorubicin, were cultured
in RPMI-1640. All media were supplemented with 10% heat-inactivated fetal calf serum, 2 mM
L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (all reagents were from EuroClone
S.p.A., Milan, Italy; GE Healthcare Life Sciences, Logan, UT, USA). All cell lines were cultured in a
humidified atmosphere at 37 ◦C in 5% CO2.

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Cells with a narrow range of passage number (4
and used for all experiments. After obtaining the cells, the first passage carried out was assigned
passage number 1 [35].
±
6) were tested for Mycoplasma contamination
4.3. Cell Growth Aassays
The cells were seeded at 2
at time 0, the medium was replaced with fresh complete medium supplemented of compounds at
the indicated concentrations. Following 72 h of treatment, 16 L of a commercial solution obtained
×
104 cells/well onto 96-well plates and incubated at 37 ◦C overnight;
µ
from Promega Corporation (Madison, WI, USA) containing 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxy
methoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) and phenazine ethosulfate were added.
After a incubation in a humidified atmosphere at 37 ◦C in 5% CO2, the bioreduction of MTS dye was
evaluated by measuring the absorbance of each well at 490 nm, using a microplate absorbance reader
(iMark Microplate Reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell growth inhibition was
expressed as a percentage (mean ± SE) of the absorbance of the control cells [35].
4.4. Anti- and Pro-oxidant Activity
The antioxidant activity was evaluated by the DPPH stable radical method. Trolox (6-hydroxy-
2,5,7,8-tetramethyl-chroman-2-carboxylic acid) curve was used as the positive control. 100 µL of sample
was added to aliquots of a solution made up with DPPH (4.8 mg) in MeOH (200 mL), and the mixture
was incubated in the dark, for 1 h at room temperature. The absorbance was measured using a UV-VIS
spectrophotometer, at 517 nm. The results were plotted as the percentage of absorbance disappearance
at 517 nm [(1-A/A0)
×
100] against the amount of sample divided by the initial concentration of DPPH;
lower absorbance values of reaction mixture indicate higher free radical scavenging activity. ED50
corresponds to micrograms of fraction able to consume half the amount of free radical divided by
micromoles of initial DPPH. The results were expressed as antiradical capacity (ARC), which is the
inverse of ED50. Each point was acquired in triplicate.
Pro-oxidant effects were examined by cell counting, adding N-acetyl-L-cysteine (NAC),
an antioxidant molecule, 1h before compounds. Results were expressed as mean
of at least three different experiments performed in duplicate. All the chemicals were supplied by
±
standard error (SE)
Sigma Aldrich Srl, Milan, Italy [46].
4.5. Evaluation of Cell Death by Flow Cytometry
Cells were washed twice with ice-cold PBS and then resuspended in a hypotonic fluorochrome
solution containing propidium iodide (PI) 50
P-40, at 1
106/mL. After incubation (1 h) in this solution, the samples were filtered through nylon cloth,
40 m mesh, and their fluorescence was analyzed using a FACSCanto instrument (Becton Dickinson,
µg/mL in 0.1% sodium citrate plus 0.03% (v/v) Nonidet
×
µ
Montain View, CA, USA). The data were analyzed with BD FACSDiva software v.6.1.2. (Becton
Dickinson). Cell death was determined by evaluating the percentage of events accumulated in the
preG0-G1 position [13].
4.6. NF-κB Activation
The DNA-binding capacity of NF-
SUM 149 and MDA-MB-231 cells using the TransAM NF-
Carlsbad, CA, USA). The determination of binding capacity was based on a 96-well plate, on which an
oligonucleotide containing the NF- B consensus binding site was fixed. By use of an antibody directed
against an epitope on p65, it may revealed NF- B bound to the oligonucleotide. After addition of a
κ
B (p65 subunit) was defined in the nuclear extracts of
κ
B and Nuclear Extract kits (Active Motif,
κ
κ
horseradish peroxidase-conjugated secondary antibody, a sensitive colorimetric readout was quantified
by densitometry (iMark Microplate Reader; Bio-Rad Laboratories, Inc.). The control of specificity
of the assay carried out according to the indications of manufacturer’s protocol. The results were
expressed as arbitrary units: one unit indicated the DNA binding capacity exerted by 2.5 µg whole cell

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extract from Jurkat cells/microgram of protein from the nuclear extracts. Jurkat cells, stimulated with
12-O-tetradecanoylphorbol-13-acetate and calcium ionophore, are the positive control for NF-
activation [35].
κB p65
4.7. Western Blotting
Whole-cell lysates were obtained from breast cancer cells using RIPA buffer (Santa Cruz
Biotechnology Inc., Dallas, TX, USA) and 25 g protein was subjected to 10% SDS-PAGE and
transferred to Hybond-P membranes (GE Healthcare Europe GmbH, Freiburg, Germany). Filters were
incubated with primary antibodies raised against -actin (Sigma-Aldrich Srl, Milan, Italy), Bcl-2 (Santa
µ
β
Cruz Biotechnology Inc., Dallas, TX, USA), XIAP (Cell Signaling Technology, Danvers, MA), Survivin
(Novus Biologicals, Littleton, CO), IAP1 (Cell Signaling Technology, Danvers, MA). An enhanced
chemiluminescence detection kit (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo
Scientific) and the Versa DOC imaging system (BioRad) were used to visualized hybridization.
Immunoblots were quantified by densitometry, the data were expressed as arbitrary units
(protein/β-actin) [35].
4.8. Statistical Analysis
Results are given as means
variance (one-way ANOVA) followed by Tukey’s test. The software used is Statistica ver. 12 (StatSoft
Inc. 1984–2014).
±
standard error (SE). Statistical analysis was performed by analysis of
Author Contributions: Conceptualization, P.M.B., P.P. and M.N.; methodology, M.L., T.M.G.S. and P.P.; software,
M.L. and T.M.G.S.; validation, P.P.; formal analysis, P.M.B., P.P. and M.N.; investigation, M.L., A.B.; resources,
P.M.B. and P.P.; data curation, M.L., A.B. and P.P.; writing—original draft preparation, P.M.B., P.P. and M.N.;
writing—review and editing, P.M.B., P.P. and M.N.; visualization, M.L. and A.B.; supervision, P.M.B. and P.P.;
project administration, P.M.B. and P.P.; funding acquisition, P.P.
Funding: This work was partially supported by grant from FFABR 2017 (PJ_RIC_FFABR_2017_ 161507_D15).
Acknowledgments: The authors thank Elda Tagliabue (Molecular Targeting Unit, Department of Experimental
Oncology and Molecular Medicine, Fondazione Institute of Hospitalization and Scientific Care, National Cancer
Institute, Milan, Italy) and Prof. Giulio Ghersi (STEBICEF Department, University of Palermo, Italy) for providing
the cell lines.
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).