Synthesis, characterization and cytotoxic activity on breast cancer cells of new half-titanocene derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.03.059

A series of novel titanocene-complexes has been prepared and evaluated for their growth regulatory effects in MCF7 and SkBr3 breast cancer cells. The capability of some of these compound to elicit relevant repressive effects on cancer cell growth could be taken into account towards novel pharmacological approaches in cancer therapy.

Full text of DOI:10.1016/j.bmcl.2013.03.059

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3458–3462
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, characterization and cytotoxic activity on breast cancer
cells of new half-titanocene derivatives
b
Esther Sirignano a, , Carmela Saturnino a, , Antonio Botta a, Maria Stefania Sinicropi b, , Anna Caruso ,
⇑
Assunta Pisano c, Rosamaria Lappano c, Marcello Maggiolini c, Pasquale Longo d
a Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Italy
b Department of Pharmaceutical Sciences, University of Calabria, 87036 Arcavacata of Rende Cosenza, Italy
c Department of Pharmaco-Biology, University of Calabria, 87036 Arcavacata of Rende Cosenza, Italy
d Department of Chemistry and Biology, University of Salerno, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel titanocene-complexes has been prepared and evaluated for their growth regulatory
effects in MCF7 and SkBr3 breast cancer cells. The capability of some of these compound to elicit relevant
repressive effects on cancer cell growth could be taken into account towards novel pharmacological
approaches in cancer therapy.
Received 18 December 2012
Revised 13 March 2013
Accepted 15 March 2013
Available online 26 March 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Metal-based anticancer drugs
Titanocene compounds
Breast cancer cells
Platinum based anticancer drugs like cis-platin, carboplatin and
oxaliplatin are commonly used in the treatment of a wide number
of tumors, including lung, colorectal, ovarian, head and neck, as
well as genitourinary, and breast cancers. However, the efficacy
of platinum-based drugs is often compromised because of the sub-
stantial risk for severe toxicities, including neurotoxicity.1
Over the last few years a lot of research has developed novel
metal-based anticancer drugs, with the aim of improving clinical
effectiveness, reducing general toxicity and broadening the spec-
trum of activity.2
extension of cis-platin derivatives and have received much atten-
tion.4 These complexes are made up of a metal core consisting of
transition metals as Ti, Nb, Mo, etc. The coordination sites of the
metal are occupied by two cyclopentadienyl rings (C5H5 or Cp)
and two labile ligands (i.e., Cl). Köpf-Maier and Köpf5 have investi-
gated the antitumor activities of a whole series of metallocene
dichloride complexes (varying the transition metal) in vivo. From
this research, titanocene dichloride (TDC) (Fig. 1) exhibited the
most promising chemotherapeutic activity among all other metal-
locenes tested.6
The search for novel metal-based antitumor drugs, other than Pt
agents, includes the investigation of the cytotoxic activity of cop-
per(I/II)2 and titanocene3 compounds.
Consequently, numerous analogues of TDC were developed and
well studied, such as titanocene Y (Fig. 1).
In particular, the anti-proliferative activity of titanocene Y and
other titanocenes has been studied in 36 human tumor cell lines.7
In vitro and ex vivo experiments showed that renal cancer is a ma-
jor target for this novel class of titanocenes, although they showed
a significant activity also against ovarian, prostate, cervix, lung, co-
lon and breast tumors.8
Recently, we reported the synthesis and the cytotoxic activity of
some titanocene derivatives obtained replacing the aryl-methoxy-
lic group on cyclopentadienyl of titanocene Y with the ethenyl-
methoxy group, in order to have a stronger electron donor effect
on the cationic species responsible for the cytotoxic activity. We
also verified the influence of leaving ligands on the activity by
substituting chlorine atoms with dimethylamide, oxalate or
aminoacid groups.3 It is worth noting that in Ref. 3, the highest
cytotoxic activity was reported for half-titanocene complex T1,
Particular attention has been recently devoted to new copper(I)
complexes. Some of these were tested for their cytotoxic properties
against several human tumor cell lines, such as HL60 (promyelo-
cytic leukemia), MCF7 (breast cancer), HCT-15 (colon cancer), HeLa
(cervix cancer), A549 (lung cancer), 2008 (ovarian cancer sensitive
to cis-platin) and C13⁄ (ovarian cancer resistant to cis-platin).2
A great deal of research has been focused on a variety of transi-
tion metal complexes bearing labile cis chlorides or similar ligands.
Among the candidate drugs, the pseudotetrahedral metallocene
complexes of the type (C5H5)2MCl2 represent a seemingly logical
⇑
Corresponding author. Tel.: +39 0984 493200; fax: +39 0984 493298.
E-mail address: s.sinicropi@unical.it (M.S. Sinicropi).
These authors contributed equally to this wok.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.059

E. Sirignano et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3458–3462
3459
TDC
T2
T1
Titanocene Y
Figure 1. Titanocene dichloride (TDC); bis-[(p-methoxybenzyl)cyclopentadienyl]titanium dichloride (titanocene Y); bis-cyclopentadienyl-ethenylmethoxyl-titanium
dichloride T2 and cyclopentadienyl-ethenylmethoxyltitanium trichloride T1.
compared to that of titanocene T2, titanocene Y and cis-platinum.
Some of the synthesized compounds showed a good cytotoxicity,
in particular complexes bis-cyclopentadienyl-ethenylmethoxyl-
titanium dichloride T2 and cyclopentadienyl-ethenylmethoxyl-
titanium trichloride T1 gave values very similar to cis-platin on
MCF-7 cell lines, with the IC50 comparable to the ones reported
for titanocene Y. Moreover, the half-titanocene complex (T1) also
showed a good cytotoxic activity, comparable to that of cis-platin,
on HEK-293 cells. The results of hydrolysis of our titanocenes
showed unequivocally that the leaving groups (Cl, N(CH3)2, C2O4
or glycine) significantly affect even hydrolysis rate of cyclopentadi-
enyl groups, being chloride and oxalate more stable.3
Thus, the presence of substituents, aryl methoxy group on
cyclopentadienyl ring in titanocene Y or ethenyl-methoxy group
in titanocene T2 or in the half-titanocene T1, produce compounds
with interesting cytotoxic activity. Although generalizations
regarding structure–activity relationships are not yet clear, we
could hypothesize that the neutral nucleophilic substituents of
cyclopentadienyl (aryl methoxy or ethenyl-methoxy group) could
intramolecularly coordinate the titanium cation, thus preventing
decomposition reactions. On the other hand, this hypothesis was
suggested for analogous complexes able to give polymerization
of propene or styrene having microstructures strongly influenced
by the possible coordination of neutral substituent of cyclopenta-
dienyl to the metal center.9–11
mental analysis. All these complexes contain different substituents
on the Cp ligands, able to stabilize the titanium cation by intramo-
lecular coordination. Preliminary cytotoxic studies of these tita-
nium based compounds have been carried out, as well.
Compound 5a was synthesized in order to verify if the activity
was higher for half-titanocene Y than titanocene Y, as it was for
the bis-cyclopentadienyl-ethenylmethoxyl-titanium dichloride T2
and cyclopentadienyl-ethenylmethoxyl-titanium trichloride T1.
Compounds 5b, 5e and 5f bear in different positions methoxyl
groups, which may make ligands much more coordinating, except
for the methoxyl in position 4. Compound 5d has no substituents
on the aryl, but the phenyl is of course able to coordinate to tita-
nium. Finally, the dimethylamino group in position 4 of the aryl
moiety of 5c has a strong capabilities to bond metal-cation.
The synthesis of complexes was carried out according to
Scheme 1. The syntheses of proligands fulvene 3a–d and 3f were car-
ried out as outlined in references,12–15 whereas 3e (2,4-dimethoxy-
phenyl) fulvene was synthesized in good yield according to litera-
ture method14,16 starting from 2,4-dimethoxy benzaldehyde.
The lithium salt of the ligand was obtained by reacting the suit-
able fulvene with Super Hydride (LiBEt3H) in dry diethyl ether.
Then, it was isolated and subsequently reacted with 1 equiv of
TiCl4Á2THF in dry THF. The reaction product was purified following
common procedures and isolated in high yield (see Scheme 1). Ele-
mental analysis (C, H, N) was in accordance with the proposed for-
mulation. 1H COSY experiments allowed the assignment of all the
proton resonances of the 1H NMR spectrum, whereas DEPT exper-
iments were useful for the attribution of 13C NMR signals. The syn-
thesized half-titanocenes were also characterized by mass
spectrometry. The mass spectra show the molecular ion and the
fragmentation of the complexes (i.e., ligand, [ligandTi]).17 These
data allowed us to have an unambiguous structural determination,
as reported in Scheme 1.
As mentioned above, several examples of titanocene-complexes
showing cytotoxic activity were reported, but to the best of our
knowledge the cyclopentadienyl-ethenylmethoxyl-titanium tri-
chloride represents the first example of half-titanocene complex
with interesting cytotoxic activity.
Therefore, the aim of this study was the synthesis and the char-
acterization of some half-titanocenes compounds (see Fig. 2) by
nuclear magnetic resonance (NMR), mass spectroscopy and ele-
N(CH2)2
OCH3
OCH3
Cl
Ti
Cl
Cl
Cl
Cl
Ti
Ti
OCH3
Cl
Cl
Cl
Cl
5c
5b
5a
H3CO
H3CO
OCH3
OCH3
Cl
Cl
Cl
Cl
Cl
Cl
Ti
Ti
Ti
OCH3
Cl
Cl
Cl
5f
5e
5d
Figure 2. Structures of synthesized complexes.

3460
E. Sirignano et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3458–3462
R1
O
R1
R2
R3
H
+
Pyr
R2
R4
MeOH
R4
1
LiBEt3H
Et2O; r.t.
R3
2
3a-f
R1
R1
R2
R3
R2
TiCl4
THF; reflux
Cl
Cl
Ti
R4
R3
R4
Cl
5a-f
-
Li+
a R1, R3, R4 = H; R2 = OCH3
b R1, R4 = H; R2, R3 = OCH3
c R1, R3, R4 = H; R2= N(CH3)2
d R1, R2, R3, R4 = H
4a-f
e R1, R2 = OCH3; R3, R4 = H
f R1, R2, R4 = OCH3; R3 = H
Scheme 1. Synthetic route for the preparation of ligands and half-titanocene complexes 5a–f.
Hydrolysis of aromatic rings of 5a–f was evaluated by integra-
tion of two signals of protons of cyclopentadienyl bonded to metal,
as to newly formed multiplet of substituted cyclopentadiene. Ta-
ble 1 reports the results of our hydrolysis tests.
Table 1
Hydrolysis results of 5a–f complexes in DMSO/D2O solution at rt followed by 1H NMR
Complex
% Cp ring hydrolysis
5 min
4 h
8 h
24 h
The complexes that show the highest hydrolytic stability are 5a,
5b, 5e and 5f. In particular, the cyclopentadienyl rings of com-
plexes are hydrolyzed only for less than 5% after 24 h, whereas
complexes 5c and 5d are hydrolyzed for 17% and 41%, respectively
(Table 1).
These data provide sufficient evidence that the presence of
coordinating groups on the aryl substituent of the cyclopentadi-
enyl are effective for the stabilization of the complexes. Therefore,
these coordinating groups might be fundamental to increase, if ac-
tive, their biological effectiveness.
In order to investigate the effects on cancer cell proliferation of
the novel synthesized compounds, we treated for 5 days MCF7 and
SkBr3 breast cancer cells with each compound.18–20 Cells were also
exposed to cis-platin in order to compare the anticancer effects of
the complexes to this well-known chemotherapeutic. It should be
noted that by using the compounds mentioned above, SkBr3 cells
5a
5b
5c
5d
5e
5f
<1
<1
<1
9
<1
<1
<1
<1
<1
24
<1
<1
<1
<1
<5
40
<1
<1
<5
<5
17
41
<5
<5
Hydrolysis stability of the six half-titanocene complexes (5a–f)
has been determined in aqueous solution, 90% DMSO by 1H NMR
spectroscopy, in order to correlate the chemical stability and coor-
dination chemistry of these complexes with their observed cyto-
toxic activity. Since we can expect that rapid hydrolysis of
leaving group (–Cl) and cyclopentadienyl ligands could give way
to biologically inactive species, active species could be generate if
the Cp rings remain metal bound.
Figure 3. Evaluation of growth responses to 10 lM of 5a–f in MCF7 (a) and SkBr3 (b) breast cancer cells, as determined by using the MTT assay. Cell viability was expressed as
the percentage of cells treated with the different compounds respect to cells treated with vehicle. Cells (5–8 Â 104 mlÀ1) were treated for 1 day up to 5 days, as indicated.

E. Sirignano et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3458–3462
3461
dark over molecular sieves. TiCl4, Titanium(IV) chloride tetrahydrofuran
complex, Super Hydride (LiBEt3H, 1.0 M solution in THF), and all chemicals
were obtained from Aldrich chemical Co. and used without further purification.
Cyclopentadiene was obtained by freshly cracked dicyclopentadiene.
The six fulvenes and their relative lithium salt were prepared by following the
reported procedures.14,16
resulted to be more responsive to the treatment compared to MCF7
cells. Among all tested compounds, 5d, 5e and 5f elicited repres-
sive effects on the proliferation of both cell lines (Fig. 3). In partic-
ular, 5d strongly decreased the viability of MCF7 cells after 3 days
of treatment, whereas 5e showed the highest antitumor activity on
SkBr3 cells after 2 days, being the most active compound on this
cell line (Fig. 3). In particular, 5d, 5e and 5f showed a strongest
cytotoxic effect on MCF7 than cis-platin. Moreover 5e showed a
similar cytotoxic activity on SkBr3 compared to cis-platin.
In conclusion, a series of novel titanocene-complexes has been
synthesized and evaluated for their growth regulatory effects in
MCF7 and SkBr3 breast cancer cells. Among these compounds, that
showed moderate to high antitumor activity, the strongest antipro-
liferative activity against MCF7 cells was displayed especially by
5d, whereas 5e elicited relevant repressive effects on SkBr3 cells.
Hence, the capability of these compounds to elicit inhibitory ef-
fects on cancer cell growth could be taken into account towards
novel pharmacological approaches in cancer therapy. Therefore
further experiments would be helpful to investigate the molecular
mechanism involved.
Synthesis of half-titanocene complexes 5a,b: Lithium cyclopentadienide
intermediate (1.83 mmol) was dissolved in dry THF (20 ml) to give
a
colourless solution. TiCl4 (18.30 mmol) was added at 0 °C to give a dark red
solution. This was refluxed overnight and then cooled. The solvent was
removed under reduced pression. The remaining residue was extracted with
dichlorometane (30 ml) and filtered through celite to remove the LiCl. The
filtrate was washed twice with hexane (20 ml) and then dried under reduced
pression to give a solid.
Spectral data of newly synthesized compounds: [(4-Methoxybenzyl)
cyclopentadienyl]-titanium-trichloride [C5H4–CH2–C6H4–OCH3]TiCl3 (5a).
Black solid. 1H NMR (d ppm, CD2Cl2, 300 MHz): 3.78 [s, 3H, C5H4–CH2–C6H4–
OCH3], 4.01 [s, 2H, C5H4–CH2–C6H4–OCH3], 6.80 [m, 4H, C5H4–CH2–C6H4–
OCH3], 6.83–7.01 [d, 4H, C5H4–CH2–C6H4–OCH3]. 13C NMR (d ppm, CD2Cl2,
75 MHz): 55.90 [C5H4–CH2–C6H4–OCH3], 45.0 [C5H4–CH2–C6H4–OCH3], 114.0–
128.70–130.0–132.0–135.90–147.80–158.60 [C5H4–CH2–C6H4–OCH3]. Mass
(E.I., 70 eV, m/z): 273 [L-TiCl-Li]Å +, 186 [L]Å +. Calcd for C13H13Cl3OTi (%): C,
46.00; H, 3.86. Found (%): C, 46.21; H, 3.84.
[(3,4-Di-methoxybenzyl)-cyclopentadienyl]-titanium-trichloride [C5H4–CH2–
C6H3–(OCH3)2]TiCl3 (5b). Brown solid. 1H NMR (d ppm, THF-d8, 300 MHz):
3.73 [s, 6H, C5H4–CH2–C6H3–(OCH3)2], 3.98 [s, 2H, C5H4–CH2–C6H3–(OCH3)2],
6.29–6.42 [m, 4H,C5H4–CH2–C6H3–(OCH3)2], 6.71–6.76 [d, 2H, C6H3–(OCH3)2],
6.84 [s, 1H, C6H3–(OCH3)2]. 13C NMR (d ppm, CD2Cl2, 75 MHz): 55.50 [C5H4–
CH2–C6H3–(OCH3)2], 36.80 [C5H4–CH2–C6H3–(OCH3)2], 112.20–113, 30–
Acknowledgments
115.40–120.80–122.50–132.80–137.10–149.20–150.30
[C5H4–CH2–C6H3–
(OCH3)2]. Mass (E.I., 70 eV, m/z): 286 [L-Ti-Na]Å +. Calcd for C14H15Cl3O2Ti (%):
C, 45.51; H, 4.09. Found (%): C, 45.91; H, 4.04.
We wish to thank Italian Minister of University and Research
for financial support of this work. The authors are grateful to Dr.
Rosanna Guarino for her contribute in the synthesis of the
compounds.
(4-(N,N-Dimethylbenzanilido)-cyclopentadienyl-titanium-trichloride [C5H4–
CH2–C6H4–N(CH3)2]TiCl3 (5c). Red solid. 1H NMR (d ppm, THF, 300 MHz):
2.98 [s, 6H, C5H4–CH2–C6H4–N(CH3)2], 3.89 [s, 2H, C5H4–CH2–C6H4–N(CH3)2],
6.45–6.67 [m, 4H, C5H4–CH2–C6H4–N(CH3)2], 7.10–7.31 [d, 4H, C5H4–CH2–
C6H4–N(CH3)2]. 13C NMR (d ppm, THF-d8, 75 MHz): 41.70 [C5H4–CH2–C6H4–
N(CH3)2], 33.10 [C5H4–CH2–C6H4–N(CH3)2], 115.90–117.60–119.20–128.70–
130.70–136.0–137.10 [C5H4–CH2–C6H4–N(CH3)2]. Mass (E.I., 70 eV, m/z): 371
[L-TiCl3-Na]Å +. Calcd for C14H16Cl3NTi (%): C, 47.70; H, 4.57. Found (%): C, 47.92;
H, 4.17; N, 3.79.
(Benzyl)-cyclopentadienyl-titanium-trichloride [C5H4–CH2–C6H5]TiCl3 (5d).
Red solid. 1H NMR (d ppm, THF-d8, 300 MHz): 4.06 [s, 2H, C5H4–CH2–C6H5],
6.33–6.45 [m, 4H, C5H4–CH2–C6H5], 7.22–7.24 [m, 5H, C5H4–CH2–C6H5]. 13C
NMR (d ppm, THF-d8 75 MHz): 36.70 [C5H4–CH2–C6H5], 110.90–115.10–
122.60–128.0–128.60–140.60 [C5H4–CH2–C6H5]. Mass (E.I., 70 eV, m/z): 242
[L-Ti-K]Å +, 163 [L-Li]Å +. Calcd for C12H11Cl3Ti (%): C, 46.58; H, 3.58. Found (%): C,
46.17; H, 3.32.
References and notes
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2005, 16, 1091.
(2,4-Di-methoxybenzyl)-cyclopentadienyl-titanium-trichloride
[C5H4–CH2–
C6H3–(OCH3)2]TiCl3 (5e). Brown solid. 1H NMR (d ppm, THF-d8, 300 MHz):
3.72–3.760 [s, 6H, C5H4–CH2–C6H3–(OCH3)2], 3.90 [s, 2H, C5H4–CH2–C6H3–
(OCH3)2], 6.28–6.29 [m, 4H, C5H4–CH2–C6H3–(OCH3)2], 6.40–6.46 [d, 2H,C5H4–
CH2–C6H3–(OCH3)2] 7.01 [s, 1H, C5H4–CH2–C6H3–(OCH3)2]. 13C NMR (d ppm,
CD2Cl2, 75 MHz): 55.40–55.50 [C5H4–CH2–C6H3–(OCH3)2], 31.80 [C5H4–CH2–
8. Claffey, J.; Gleeson, B.; Hogan, M.; Müller-Bunz, H.; Wallis, D.; Tacke, M. Eur. J.
Inorg. Chem. 2008, 26, 4074.
C6H3–(OCH3)2],
99.10–104.80–116.60–121.90–123.30–131.40–137.60–
159.20–161.0 [C5H4–CH2–C6H3–(OCH3)2]. Mass (E.I., 70 eV, m/z): 286 [L-Ti-
Na]Å +. Calcd for C14H15Cl3O2Ti (%): C, 45.51; H, 4.09. Found (%): C, 45.91; H,
4.04.
(2,4,6-Tri-methoxybenzyl)-cyclopentadienyl-titanium-trichloride [C5H4–CH2–
C6H2–(OCH3)3]TiCl3 (5f). Black solid. 1H NMR (d ppm, THF, 300 MHz): 3.72–
3.77 [s, 9H, C5H4–CH2–C6H2–(OCH3)3], 3.90 [s, 2H, C5H4–CH2–C6H2–(OCH3)3],
6.30–6.34 [m, 4H, C5H4–CH2–C6H2–(OCH3)3], 6.47 [s, 2H, C5H4–CH2–C6H2–
(OCH3)3]. 13C NMR (d ppm, THF-d8, 75 MHz): 54.70 [C5H4–CH2–C6H2–(OCH3)3],
9. Longo, P.; Amendola, A. G.; Fortunato, E.; Boccia, A. C.; Zambelli, A. Macromol.
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13. Tacke, M.; Allen, L. T.; Cuffe, L. P.; Gallagher, W. M.; Lou, Y.; Mendoza, O.;
Muller-Bunz, H.; Rehmann, F.-J. K.; Sweeney, N. J. Organomet. Chem. 2004, 689,
2242.
31.05
[C5H4–CH2–C6H2–(OCH3)3],
98.40–104.0–115.80–122.50–130.60–
137.70–158.30–160.30 [C5H4–CH2–C6H2–(OCH3)3]. Mass (E.I., 70 eV, m/z):
317 [L-Ti-Na]Å +. Calcd for C15H17Cl3O3Ti (%): C, 45.09; H, 4.29. Found (%): C,
45.39; H, 4.24.
14. Claffey, J.; Hogan, M.; Muller-Bunz, H.; Pampillon, C.; Tacke, M. J. Organomet.
Chem. 2008, 693, 526.
18. (a) Caruso, A.; Chimento, A.; El-Kashef, H.; Lancelot, J. C.; Panno, A.; Pezzi, V.;
Saturnino, C.; Sinicropi, M. S.; Sirianni, R.; Rault, S. J. Enzyme Inhib. Med. Chem.
2012, 27, 60; (b) Gasparotto, V.; Castagliuolo, I.; Chiarellotto, G.; Pezzi, V.;
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2006, 49.
15. Stone, K. J.; Little, R. D. J. Org. Chem. 1984, 49, 11.
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Strohfeldt, ; Tacke, K. M. J. Organomet. Chem. 2005, 690, 4537.
17. Experimental section: The elemental analyses for C, H, N, were recorded on a
ThermoFinnigan Flash EA 1112 series and performed according to standard
microanalytical procedures. 1H NMR, homodecoupled 1H NMR, 1H COSY and
13C NMR spectra were recorded at 298 K on a Bruker Avance 300 Spectrometer
19. Cell culture: MCF7 breast cancer cells were maintained in DMEM/F-12
supplemented with 10% fetal bovine serum (FBS), 100 mg/ml penicillin/
operating at 300 MHz (1H) and 75 MHz
(
13C) and referred to internal
streptomycin and 2 mM
breast cancer cells were cultured in RPMI-1640 medium supplemented with
10% FBS, 100 mg/ml penicillin/streptomycin and 2 mM -glutamine
L-glutamine (Invitrogen, Gibco, Milan, Italy). SkBr3
tetramethylsilane. Molecular weights were determined by ESI mass
spectrometry. ESI-MS analysis in positive and negative ion mode, were made
using a Finnigan LCQ ion trap instrument, manufactured by Thermo Finnigan
(San Jose, CA, USA), equipped with the Excalibur software for processing the
data acquired. The sample was dissolved in acetonitrile and injected directly
into the electrospray source, using a syringe pump, which maintains constant
L
(Invitrogen, Gibco, Milan, Italy). Cells were switched to medium without
serum the day before experiments and thereafter treated in medium
supplemented with 1% FBS.
Inhibition of cell proliferation: The effects of each compound on cell viability
were determined with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay,20–23 which is based on the conversion
of MTT to MTT-formazan by mitochondrial enzyme. Cells were seeded in
quadruplicate in 96-well plates in regular growth medium and grown until 70–
80% confluence. Cells were washed once they had attached and then treated
flow at 5 ll/min. The temperature of the capillary was set at 220 °C. All
manipulations were carried out under oxygen- and moisture-free atmosphere
in an MBraun MB 200 glove-box. All the solvents were thoroughly
deoxygenated and dehydrated under argon by refluxing over suitable drying
agents, while NMR deuterated solvents (Euriso-Top products) were kept in the

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E. Sirignano et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3458–3462
l
M each compound for indicated time (for 1 day up to 5 days). Relative
21. Pascale, R.; Carocci, A.; Catalano, A.; Lentini, G.; Spagnoletta, A.; Cavalluzzi, M.
M.; De Santis, F.; De Palma, A.; Scalera, V.; Franchini, C. Bioorg. Med. Chem.
2010, 18, 5903.
cell viability was determined by MTT assay according to the manufacturer’s
protocol (Sigma–Aldrich, Milan, Italy). Mean absorbance for each drug dose
was expressed as a percentage of the control untreated well absorbance and
plotted versus drug concentration. IC50 values represent the drug
concentrations that reduced the mean absorbance at 570 nm to 50% of those
in the untreated control wells.
22. Carocci, A.; Catalano, A.; Bruno, C.; Lovece, A.; Roselli, M. G.; Cavalluzzi, M. M.;
De Santis, F.; De Palma, A.; Rusciano, M. R.; Illario, M.; Franchini, C.; Lentini, G.
Bioorg. Med. Chem. 2013, 21, 847.
23. Sinicropi, M. S.; Caruso, A.; Conforti, F.; Marrelli, M.; El Kashef, H.; Lancelot, J.
C.; Rault, S.; Statti, G. A.; Menichini, F. J. Enzyme Inhib. Med. Chem. 2009, 24,
1148.
20. Mosmann, T. J. Immunol. Methods 1983, 65, 55.