Synthesis of new ferrocenyl dehydrozingerone derivatives and their effects on viability of PC12 cells

Article abstract of DOI:10.1016/j.poly.2016.05.039

A series of novel compounds deriving from the conjugation of ferrocene with curcumin-related bioactive molecules as dehydrozingerone, zingerone and their biphenyl dimers was prepared by Claisen-Schmidt condensation of the suitable aromatic aldehydes and acetylferrocene in different conditions according to the starting material. The obtained compounds were fully characterized by NMR spectroscopy and cyclic voltammetry and reversible electrochemical behavior was recorded for monomer derivatives. The cell viability of PC12 cells after exposure to the organometallic compounds was also evaluated and a reduced toxicity with respect to the ferrocene was detected. In comparison with biphenyl 4, a compound that manifested antiproliferative and apoptotic activities and was quite toxic on PC12 cells, the exposure to the ferrocenyl analogue 14 resulted in roughly fourfold increase in the cell viability. Ferrocenyl chalcones 14 and 16-18 significantly increased the oxidative stress generated by hydrogen peroxide, a molecule generally accumulated in cancer cells and, recently, studied as prodrug.

Full text of DOI:10.1016/j.poly.2016.05.039

Polyhedron 117 (2016) 80–89
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis of new ferrocenyl dehydrozingerone derivatives and their
effects on viability of PC12 cells
b
c
d
Sonia Pedotti a, Angela Patti a, , Sonia Dedola , Antonio Barberis , Davide Fabbri ,
⇑
Maria Antonietta Dettori d, Pier Andrea Serra b,c, Giovanna Delogu d,
⇑
a Istituto di Chimica Biomolecolare, CNR, Via Paolo Gaifami 18, 95126 Catania, Italy
b Dipartimento di Medicina Clinica e Sperimentale, Università di Sassari, Viale S. Pietro, 43/b, 07100 Sassari, Italy
c Istituto di Scienze Alimentari, CNR, Traversa la Crucca, 3, 07100 Sassari, Italy
d Istituto di Chimica Biomolecolare, CNR, Traversa la Crucca, 3, 07100 Sassari, 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 compounds deriving from the conjugation of ferrocene with curcumin-related bioactive
molecules as dehydrozingerone, zingerone and their biphenyl dimers was prepared by Claisen–Schmidt
condensation of the suitable aromatic aldehydes and acetylferrocene in different conditions according to
the starting material. The obtained compounds were fully characterized by NMR spectroscopy and cyclic
voltammetry and reversible electrochemical behavior was recorded for monomer derivatives. The cell
viability of PC12 cells after exposure to the organometallic compounds was also evaluated and a reduced
toxicity with respect to the ferrocene was detected. In comparison with biphenyl 4, a compound that
manifested antiproliferative and apoptotic activities and was quite toxic on PC12 cells, the exposure to
the ferrocenyl analogue 14 resulted in roughly fourfold increase in the cell viability. Ferrocenyl chalcones
14 and 16–18 significantly increased the oxidative stress generated by hydrogen peroxide, a molecule
generally accumulated in cancer cells and, recently, studied as prodrug.
Received 13 April 2016
Accepted 15 May 2016
Available online 26 May 2016
Keywords:
Dehydrozingerone
Ferrocene
Hydroxylated biphenyls
PC12 cells
Oxidative stress
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
dehydrozingerone 2 is known for its interesting anti-inflammatory
and anticancer activities [9].
Many efforts have been devoted to the development of new
agents that, by targeting simultaneously multiple etiologies of a
disease, may be more beneficial than selective agents for a single
receptor site [1,2].
Curcumin 1 is a well-established active compound in dealing
with different biochemical pathways leading to cancer. It is the
major metabolite extracted from the rhizome of Curcuma longa, a
plant routinely used in the preparation of curry spice as
component of Asian traditional medicine (Fig. 1) [3,4].
A wide spectrum of biological properties (e.g. anti-inflamma-
tory, anticancer, neuroprotective) attributed to curcumin has been
related with its excellent free radical scavenging and antioxidant
activities due to the presence of two guaiacyl moieties that proved
to be very effective in stabilizing phenoxy radicals [5,6]. Unfortu-
nately, curcumin has low solubility in aqueous and physiological
solutions wherein it undergoes rapid degradation into ferulic acid,
vanillin and dehydrozingerone 2 (Fig. 1) [7,8]. Like curcumin,
In a previous study we prepared compound 4, the dimer of
OMe-dehydrozingerone 3, as the first curcumin-related biphenyl.
We found that compound 4 was more active in inhibiting malig-
nant melanoma and neuroblastoma cells growth when compared
to curcumin itself (Fig. 1) [10]. Normal fibroblasts proliferation
was not affected by this treatment therefore compound 4 could
represent a good candidate in developing new therapies against
neural crest-derived tumors.
Recently, we found that biphenyl 4 and biphenyl 5, the latter
being the biphenyl analogue of dehydrozingerone 2, are able to
partially inhibit the aggregation process of
the potential role of a hydroxylated biphenyl scaffold in the design
of -synuclein aggregation inhibitors in neurodegenerative
a-synuclein, suggesting
a
pathologies [11]. Protective effects against oxidative stress induced
in PC12 cells, a neuronal cell model, was also investigated for
biphenyls 4 and 5 and their derivatives.
Compared to phenols, hydroxylated biphenyls display higher
antioxidant activity in virtue of the presence of two hydroxyl
groups at the ortho–ortho0 positions generally providing reduction
in toxicity compared with the corresponding phenolic monomer
[12,13].
⇑
Corresponding authors. Tel.: +39 0957 338328 (A. Patti). Tel.: +39 0792841220
(G. Delogu).
E-mail addresses: angela.patti@icb.cnr.it (A. Patti), giovanna.delogu@ss.icb.cnr.it
(G. Delogu).
http://dx.doi.org/10.1016/j.poly.2016.05.039
0277-5387/Ó 2016 Elsevier Ltd. All rights reserved.

S. Pedotti et al. / Polyhedron 117 (2016) 80–89
81
O
H3CO
O
OH
O
H3CO
HO
OCH3
OH
H3CO
RO
RO
RO
H3CO
R = H
R = CH3
2, dehydrozingerone
3
O
curcumin
1
R = H
R = CH3
5
4
Fig. 1. Natural and natural-like phenols and biphenols.
Ferrocene is a neutral, chemically stable, relatively non toxic
molecule whose good reversible redox proprieties seem to be
strongly associated with the biological activity. As an example,
the activity of tamoxifen, the most common drug used to treat
patients diagnosed with breast cancer, was enhanced when a unit
of ferrocene is covalently bonded to the molecule of hydroxyta-
moxifen, the active metabolite and it was thought that the
(Regenstanf, Germany). All solvents of purity >98% (GC) were used
as received.
2.3. General procedure for the O-benzylation of vanillin and divanillin
To a solution of the suitable aldehyde (1 mmol) in DMF (5 mL),
K2CO3 (2 eqv.) and 4-methylbenzylbromide (1.1 eqv.) were added
and the suspension stirred at room temperature until complete
conversion of the substrate was detected by TLC analysis (12–
20 h). The solvent was then evaporated in vacuo and the residue
dissolved in AcOEt (15 mL) and treated with satd. NH4Cl solution
(3 ꢀ 10 mL). The organic phase, extracted, then was washed with
brine, dried over Na2SO4 and taken to dryness to give a residue that
was purified by chromatographic column (n-hexane:AcOEt 9:1) to
give pure O-(4-methyl)benzyl derivatives.
extended
p-system in hydroxytamoxifen-ferrocene conjugate
plays an important role in the mode of action of the drug [14,15].
The organometallic approach has been successfully applied to
other bioactive compounds like flavones [16], amino acids
[17,18], chalcones [19,20], quinolinones [21], ellagitannins [22],
cyclodextrin [23] and curcumin [24,25]. Although several ferro-
cenyl-curcumin derivatives were prepared by different groups, all
of them contain a b-diketoeptadiene or pentenedienone chain in
their structure [24,25].
Ferrocene derivatives of dehydrozingerone, zingerone and the
corresponding C2-symmetric dimers have not been synthetized
so far and here we report their preparation and characterization.
The electrochemical properties and cytotoxic activity in PC12
cells of the obtained compounds was also evaluated in compar-
ison with data of the corresponding compounds lacking in fer-
rocene unit.
2.3.1. 3-Methoxy-4-(4-methyl)benzyloxy-benzaldehyde (9)
95% yield, white solid, mp = 69–71 °C; 1H NMR: d 2.36 (s, 3H,
Me), 3.94 (s, 3H, OMe), 5.21 (s, 2H, CH2), 6.99 (d, 1H, J = 8.0, H-5),
7.20 (d, 2H, J = 8.0, ArH), 7.33 (d, 2H, J = 8.0, ArH), 7.39 (dd, 1H,
J = 1.6 and 8.0, H-6), 7.43 (d, 1H, J = 1.6, H-3), 9.84 (s, 1H, CHO);
13C NMR: d 21.1 (Me), 55.9 (OMe), 70.7 (CH2), 109.2 (ArH), 112.3
(ArH), 126.5 (ArH), 127.2 (2ꢀ ArH), 129.3 (2ꢀ ArH), 130.1 (Ar),
132.8 (Ar), 137.9 (Ar), 150.0 (Ar), 153.6 (Ar), 190.8 (CHO). Anal.
Calc. for C16H16O3: C, 74.97; H, 6.30. Found: C, 75.05; H, 6.28%.
2. Experimental section
2.1. Instrumentation
2.3.2. 2,20-Di(4-methyl)benzyloxy-3,30-dimethoxy-5,50-diformyl-1,10-
biphenyl (13)
1H and 13C NMR spectra were registered in CDCl3 at 400.13 and
100.69 MHz respectively on a Bruker AvanceTM 400 instrument. 2D-
NMR experiments were performed using standard Bruker micro-
programs. All NMR spectra were recorded using CDCl3 unless
otherwise specified. Chemical shifts (d) are given as ppm relative
to the residual solvent peak and coupling constants (J) are in Hz.
In the NMR assignments, Cp and Cp0 refer to substituted and
unsubstituted cyclopentadienyl rings, respectively. UV spectra
were recorded with a spectrometer Perkin-Elmer Lambda 35 in
dichloromethane at concentration of 0.74 ꢀ 10ꢁ5 M. Elemental
analyses were obtained from the Department of Pharmaceutical
Sciences, University of Catania. Melting points are uncorrected.
Cyclic voltammetries were performed with an eDAQ QuadStat, an
e-Corder 410 and the Echem software (eDAQ Europe, Poland).
93% yield, white solid, mp = 128–129 °C; 1H NMR: d 2.27 (s, 6H,
Me), 4.00 (s, 6H, OMe), 4.89 (s, 4H, CH2), 6.88 (d, 4H, J = 8.0, ArH),
6.96 (d, 4H, J = 8.0, ArH), 7.14 (d, 2H, J = 1.6, H-6 and H-60), 7.49
(d, 2H, J = 1.6, H-4 and H-40), 9.77 (s, 2H, CHO); 13C NMR: d 21.0
(Me), 56.0 (OMe), 74.5 (CH2), 109.6 (Ar-H), 128.1 (ArH), 128.2
(2ꢀ ArH), 128.7 (2ꢀ ArH), 131.7 (Ar), 132.4 (Ar), 133.7 (Ar),
137.6 (Ar), 150.0 (Ar), 153.4 (Ar), 191.0 (CHO). Anal. Calc. for
C32H30O6: C, 75.26; H, 5.93. Found: C, 75.32; H, 5.90%.
2.4. Solvent-free procedure for the synthesis of ferrocenyl chalcones
(Method A)
A 10-mL sealed vial charged with the suitable O-protected alde-
hyde (0.5 mmol) and acetylferrocene (0.5 mmol) was placed in a
bath oil at 100 °C and solid NaOH (1.0 mmol) was added. The mix-
ture was stirred vigorously and left to react until the TLC analysis
showed complete disappearance of substrates (1–3 h). After addi-
tion of CH2Cl2 (10 mL) the mixture was partitioned with satd.
NH4Cl solution (3 ꢀ 5 mL) and the organic layer, extracted, was
washed with brine and dried over Na2SO4. The organic solvent
was then removed in vacuo and the residue recrystallized from
n-hexane/CH2Cl2 to give pure chalcones.
2.2. Materials
Sodium hydroxide microprills were purchased from Riedel-de-
Haën (Germany). Column chromatography was performed on Si
60 (230–400 mesh) silica gel using the specified eluants. Ferrocene,
veratraldehyde, hydrogen peroxide were purchased from
Sigma–Aldrich (Milan, Italy), vanillin from Alfa Aesar GmbH & Co
KG (Karlsruhe, Germany) and zingerone from Chemos GmbH

82
S. Pedotti et al. / Polyhedron 117 (2016) 80–89
2.4.1. 1-Ferrocenyl-3-(3,4-dimethoxy)phenyl-2-propen-1-one (10)
68% yield, red solid, mp = 155–156 °C; 1H NMR: d 3.94 (s, 3H,
OMe), 3.97 (s, 3H, OMe), 4.22 (s, 5H, Cp0), 4.58 (bs, 2H, Cp), 4.93
(bs, 2H, Cp), 6.92 (d, 1H, J = 8.4, ArH), 7.00 (d, 1H, J = 15.6, H-2),
7.15 (s, 1H, ArH), 7.26 (d, 1H, J = 8.4, ArH), 7.75 (d, 1H, J = 15.6,
then filtered through a short plug of Celite and the solution evap-
orated. The residue was purified by column chromatography (n-
hexane:AcOEt 4:1) to give pure 16 (62 mg, 0.17 mmol, 81% yield)
as an orange solid, mp = 133–134 °C; 1H NMR: d 2.99 (m, 4H,
CH2), 3.90 (s, 3H, OMe), 4.11 (s, 5H, Cp0), 4.49 (t, 2H, J = 1.6, Cp),
4.77 (t, 2H, J = 1.6, Cp), 5.54 (s, 1H, OH), 6.78 (dd, 1H, J = 1.6 and
8.0, ArH-6), 6.80 (d, 1H, J = 1.6, ArH-2), 6.87 (d, 1H, J = 8.0, ArH-
5); 13C NMR: d 29.8 (C-3), 41.8 (C-2), 55.9 (OMe), 69.2 (CpH),
69.6 (Cp0H), 72.1 (CpH), 78.9 (Cp), 111.3 (ArH), 114.3 (ArH), 120.9
(ArH), 133.4 (Ar), 143.9 (Ar), 146.3 (Ar), 203.3 (CO). Anal. Calc. for
13
H-3); C NMR: d 55.9 (OMe), 69.6 (CpH), 70.0 (Cp0H), 72.5 (CpH),
80.6 (Cp), 110.4 (ArH), 111.1 (ArH), 120.9 (C-2), 122.3 (ArH),
128.0 (Ar), 140.9 (C-3), 149.1 (Ar), 151.0 (Ar), 192.8 (CO). Anal. Calc.
for C21H20FeO3: C, 67.02; H, 5.36. Found: C, 66.98; H, 5.38%.
2.4.2. 1-Ferrocenyl-3-[3-methoxy-4-(4-methylbenzyloxy)]phenyl-2-
propen-1-one (11)
C20H20FeO3: C, 65.94; H, 5.54. Found: C, 65.89; H, 5.52%.
75% yield, red solid, mp = 149–150 °C; 1H NMR: d 2.36 (s, 3H,
Me), 3.97 (s, 3H, OMe), 4.21 (s, 5H, Cp0), 4.58 (t, 2H, J = 1.6, Cp),
4.91 (t, 2H, J = 1.6, Cp), 5.19 (s, 2H, CH2), 6.92 (d, 1H, J = 8.4, ArH),
6.99 (d, 1H, J = 15.6, H-2), 7.18 (m, 4H, ArH), 7.34 (d, 2H, J = 8.0,
ArH), 7.74 (d, 1H, J = 15.6, H-3); 13C NMR: d 21.1 (Me), 56.1
(OMe), 69.6 (CpH), 70.0 (Cp0H), 70.8 (CH2), 72.5 (CpH), 80.7 (Cp),
111.1 (ArH), 113.5 (ArH), 120.9 (C-2), 122.1 (ArH), 127.2 (2ꢀ
ArH), 128.3 (Ar), 129.2 (2ꢀ ArH), 133.5 (Ar), 137.7 (Ar), 140.9 (C-
3), 149.7 (Ar), 150.2 (Ar), 192.8 (CO). Anal. Calc. for C28H26FeO3:
C, 72.10; H, 5.62. Found: C, 72.19; H, 5.64%.
2.7. 3,30-(5,50-Dimethoxy,6,60-dihydroxy-[1,10-biphenyl]-3,30-diyl)bis
(1-ferrocenyl-propan-1-one), (17)
According the same procedure described above, O-benzyl
derivative 15 (102 mg, 0.11 mmol) was hydrogenated to give com-
pound 17, obtained as an orange solid (65 mg, 0.09 mmol, 82%
yield) after purification on Si gel column (n-hexane:AcOEt:CH2Cl2
4:3:3), mp = 93–94 °C; 1H NMR (CD3COCD3): d 2.96 (br t, 4H,
CH2), 3.06 (br t, 4H, CH2), 3.88 (s, 6H, OMe), 4.13 (s, 10H, Cp0),
4.50 (s, 4H, Cp), 4.80 (s, 4H, Cp), 6.85 (s, 2H, ArH), 6.95 (s, 2H,
ArH); 13C NMR (CD3COCD3): d 29.4 (CH2), 41.4 (CH2), 55.5 (OMe),
69.1 (CpH), 69.4 (Cp0H), 71.7 (CpH), 79.4 (Cp), 111.3 (ArH), 123.3
(ArH), 125.3 (Ar), 132.7 (Ar), 141.7 (Ar), 147.8 (Ar), 202.0 (CO).
Anal. Calc. for C40H38Fe2O6: C, 66.12; H, 5.28. Found: C, 66.22; H,
5.26%.
2.5. Synthesis of ferrocenyl chalcones in solution (Method B)
To a solution of the suitable O-protected dialdehyde (0.5 mmol)
and acetylferrocene (1.0 mmol) in dioxane (10 mL) NaOH
(2.0 mmol) was added and the mixture stirred at 40 °C for 48–
96 h. The solution was concentrated by evaporation of the solvent
and partitioned between CH2Cl2 (10 mL) and satd. NH4Cl solution
(3 ꢀ 5 mL). The organic layer washed with brine, dried over Na2SO4
and taken to dryness to give a residue that was purified on Si gel
column (n-hexane:AcOEt:CH2Cl2 3:1:1).
2.8. 1-Ferrocenyl-3-(3-methoxy-4-hydroxy)phenyl-2-propen-1-one,
(18)
To a solution of 11 (100 mg, 0.21 mmol) in dry CH2Cl2 (5 mL)
was added (CH3)3SiI (0.24 mmol, 33 lL) and the mixture left to
2.5.1. (2E,20E)-3,30-(1,10-Biphenyl-5,50,6,60-tetramethoxy-3,30-diyl)bis
(1-ferrocenyl-2-propen-1-one), (14)
react at room temperature for 3 h. The reaction mixture was then
quenched with satd. NaHCO3 (3 ꢀ 5 mL). The organic phase was
extracted and washed with brine and finally taken to dryness.
The residue was purified by chromatographic column (n-hexane:
AcOEt 4:1) to give 18 (0.18 mmol, 64 mg, 84% yield) as a deep
red solid, mp = 138–139 °C; 1H NMR: d 3.99 (s, 3H, OMe), 4.22 (s,
5H, Cp0), 4.59 (t, 2H, J = 2.0, Cp), 4.93 (t, 2H, J = 2.0, Cp), 6.01 (s,
1H, OH), 6.90 (d, 1H, J = 15.6, H-2), 7.00 (d, 1H, J = 7.6, ArH), 7.11
(d, 1H, J = 1.6, ArH), 7.26 (dd, 1H, J = 1.6 and 7.6, ArH), 7.75 (d,
38% yield, red solid, mp = 185–186 °C; 1H NMR: d 3.77 (s, 6H,
OMe), 4.02 (s, 6H, OMe), 4.22 (s, 10H, Cp0), 4.59 (br s, 4H, Cp),
4.92 (br s, 4H, Cp), 7.05 (d, 2H, J = 15.6, 2ꢀ H-2), 7.21 (br s, 2H,
ArH), 7.27 (br s, 2H, ArH), 7.79 (d, 2H, J = 15.6, 2ꢀ H-3); 13C
NMR: d 56.0 (OMe), 60.8 (OMe), 69.7 (CpH), 70.0 (Cp0H), 72.6
(CpH), 80.5 (Cp), 112.1 (ArH), 122.2 (C-2), 122.9 (ArH), 130.6
(Ar), 132.5 (Ar), 140.5 (C-3), 148.6 (Ar), 152.9 (Ar), 192.8 (CO). Anal.
Calc. for C42H38Fe2O6: C, 67.20; H, 5.11. Found: C, 67.27; H, 5.12%.
13
1H, J = 15.6, H-3); C NMR: d 55.9 (OMe), 69.6 (CpH), 70.0 (Cp0H),
2.5.2. (2E,20E)-3,30-[1,10-Biphenyl-5,50-dimethoxy-6,60-di(4-methyl)
benzyloxy-3,30-diyl]bis(1-ferrocenyl-2-propen-1-one), (15)
72.5 (CpH), 80.6 (Cp), 110.4 (ArH), 114.8 (ArH), 120.6 (C-2), 122.4
(ArH), 127.6 (Ar), 141.1 (C-3), 146.7 (Ar), 147.8 (Ar), 192.9 (CO);
Anal. Calc. for C20H18FeO3: C, 66.30; H, 5.01. Found: C, 66.21; H,
4.99%.
90% yield, red solid, mp = 183–185 °C; 1H NMR: d 2.24 (s, 6H,
Me), 3.98 (s, 6H, OMe), 4.19 (s, 10H, Cp0), 4.57 (br s, 4H, Cp), 4.83
(s, 4H, CH2), 4.89 (br s, 4H, Cp), 6.98 (s, 8H, ArH), 7.00 (d, 2H,
J = 15.6, 2ꢀ H-2), 7.20 (br s, 2H, ArH), 7.29 (br s, 2H, ArH), 7.77
(d, 2H, J = 15.6, 2ꢀ H-3); 13C NMR: d 21.1 (Me), 56.1 (OMe), 69.7
(CpH), 70.0 (Cp0H), 72.6 (CpH), 74.5 (CH2), 80.5 (Cp), 112.4 (ArH),
122.1 (C-2), 123.1 (ArH), 128.1 (4ꢀ ArH), 128.7 (4ꢀ ArH) 130.5
(Ar), 132.9 (Ar), 134.1 (Ar), 137.4 (Ar), 140.6 (C-3), 147.5 (Ar),
153.2 (Ar), 192.8 (CO). Anal. Calc. for C56H50Fe2O6: C, 72.25; H,
5.42. Found: C, 72.35; H, 5.40%.
2.9. (2E,20E)-3,30-[1,10-biphenyl-5,50-dimethoxy-6,60-dihydroxy-3,30-
diyl]bis(1-ferrocenyl-2-propen-1-one) (19)
According the procedure described above, compound 15
(102 mg, 0.11 mmol) was deprotected at the benzyl group to afford
compound 19 as a dark red solid (28 mg, 0.04 mmol, 36% yield)
after purification by chromatographic column (n-hexane:AcOEt:
CH2Cl2 4:3:3), mp = 139–140 °C; 1H NMR: d 4.06 (s, 6H, OMe),
4.22 (s, 10H, Cp0), 4.58 (br s, 4H, Cp), 4.93 (br s, 4H, Cp), 6.25 (br
s, 2H, OH), 7.04 (d, 2H, J = 15.6, H-2), 7.19 (s, 2H, ArH), 7.37 (s,
2H, ArH), 7.80 (d, 2H, J = 15.6, H-3); 13C NMR: d 56.2 (OMe), 69.7
(CpH), 70.0 (Cp0H), 72.6 (CpH), 80.4 (Cp), 110.4 (ArH), 121.4 (C-
2), 123.6 (ArH), 123.9 (Ar), 127.4 (Ar), 141.1 (C-3), 145.1 (Ar),
147.2 (Ar), 192.8 (CO); Anal. Calc. for C40H34Fe2O6: C, 66.49; H,
4.75. Found: C, 66.58; H, 4.76%.
2.6. 1-Ferrocenyl-3-(3-methoxy-4-hydroxy)phenyl-propan-1-one
(16)
A solution of compound 11 (100 mg, 0.21 mmol) in THF (5 mL)
was placed in a 100 mL flask equipped with a Teflon stopcock and
Pd/C carbon (10% Pd on activated carbon, 10 mg) was added. After
the flask was evacuated and then refilled with H2 (1.0 atm), the
reaction mixture was stirred at 60 °C for 3 h. The suspension was

S. Pedotti et al. / Polyhedron 117 (2016) 80–89
83
2.10. Electrochemical measurements
of butan-2-one chain in the antioxidant and anti-inflammatory
activities of 6 (Scheme 1) [28–30].
The electrochemical behavior of compounds 2–4, 6–7 and the
corresponding ferrocenyl derivatives 10, 14, 16, 17 and 18 was
evaluated by cyclic voltammetry (CV) in a buffer solution contain-
ing 10 mL of KNO3 1 M and 10 mL of ethanol 96%, using a carbon
working electrode (area 0.071 mm2), an Ag/AgCl reference elec-
trode and a Pt counter electrode, with a scanned potential range
(Vapp) comprised between ꢁ1000 mV and +1000 mV, at
100 mV sꢁ1 scan rate, in the absence and in the presence of
1 mM of each compound.
Monomers 2, 3 and dimers 4, 5 were prepared from vanillin as
previously described by us [10a], whereas dimer 7 was prepared
from zingerone 6, commercially available [11].
For the synthesis of the target ferrocenyl chalcones, a Claisen–
Schmidt condensation of acetylferrocene and the suitable O-pro-
tected vanillin was carried out in solvent-free conditions (Method
A) according to a previously reported procedure by us that gener-
ally provides short reaction times (Scheme 2) [31]. By this route,
ferrocenyl chalcone 10, the organometallic analogue of 3, was
obtained in 68% isolated yield starting from OMe-vanillin 8.
However, when OMe-divanillin 12 was used as substrate, the
conventional reaction in solution was preferred (Method B)
because it gave better yields of the desired ferrocenyl derivative
with only negligible amounts of the intermediate product deriving
from a single Claisen–Schmidt condensation (Scheme 3). Starting
from OMe-divanillin and acetylferrocene compound 14, the ferro-
cenyl analogue of biphenyl 4, was obtained in moderate yield.
Reactions of acetylferrocene with vanillin or divanillin was not
effective to obtain derivatives bearing free hydroxyl groups. When
the starting aldehydes were converted into the corresponding O-
benzylethers 9 and 13 and then subjected to Claisen–Schmidt con-
densation, the corresponding ferrocenyl chalcones 11 and 15 were
achieved in 75% and 90% yields, respectively (Method A for com-
pound 9 and Method B for compound 13 in the Experimental
section).
2.11. Cell culture and treatments
PC12 cells were grown in Dulbecco’s modified Eagle’s medium:
Nutrient Mixture F-12 (DMEM-F12) containing 10% horse serum,
5% fetal bovine serum (FBS) and 1% penicillin (5000 U/mL)/strepto-
mycin (5000
H2O2 (5 mM) solution was prepared in Milli-Q water and diluted
(20–100 M) in complete medium. Ferrocene and compounds 10,
lg/mL) at 37 °C under humidified 5% CO2/air. Stock
l
14, 16, 17, 18 solutions (100 mM) were prepared in DMSO and then
diluted to the final concentration in complete medium, containing
a concentration of DMSO <0.1%. All solutions were prepared imme-
diately before use. All experiments were performed on PC12 cells
during their exponential phase of growth. In a separate series of
experiments the cells have been exposed to ferrocene alone
(40 lM). In a previous experiment [11], we determined at 40 lM
Hydrogenation of 11 and 15 in the presence of Pd/charcoal cat-
alyst allowed the simultaneous reduction of the ethylenic bond
and deprotection of O-benzyl ether affording the zingerone-like
derivative 16 and the corresponding dimer 17, respectively
(Scheme 4). Selective deprotection at room temperature of O-ben-
zyloxygroups in 11 or 15 with Me3SiI was exploited for the prepa-
ration of 18 and 19 in which the ethylenic bonds were retained
(Scheme 4). However, dimeric compound 19, was rather unstable
and for this reason it was not further investigated.
the protective concentration of compounds 2–7, against the cell
death induced by H2O2; this concentration was used in all experi-
ments with the corresponding compounds with ferrocenyl unit,
done in triplicate.
2.12. MTT assay
For each experiment, 1 ꢀ 105 PC12 cells/cm2 were plated and
treated 24 h later (time 0). PC12 cells were preincubated for
20 min with ferrocene or ferrocenyl chalcones and then exposed
to H2O2 (75 lM) for 24 h; at this concentration, H2O2 alone deter-
mined a cell death around 40%. PC12 cell viability was evaluated
using the 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbro-
mide (MTT) reduction assay [26]. The metabolic dye MTT is
reduced by viable cells to form blue formazan crystals. In brief,
the cells were incubated with 1 mg of MTT per milliliter of med-
ium, and the cultures were allowed to incubate at 37 °C for 4 h.
The MTT was removed, and the cells were rinsed with PBS and cen-
trifuged at 4000 rpm for 15 min. Thereafter, the supernatant was
discarded, and the pellet was dissolved in 2 mL of isopropanol;
after centrifugation at 4000 rpm for 5 min, the color was read at
578 nm using a Bauty Diagnostic microplate reader. Experiments
were done in triplicate. A standard curve was constructed with dif-
ferent concentrations of cells at the start of every experiment.
1N NaOH
acetone
K2CO3/CH3I
acetone
H3CO
HO
CHO
2
3
12h, rt
12h, rfx
vanillin
FeSO4 aq
Na2S2O8
O
H3CO
H20, 50 °C, 5d
1N LiOH
acetone
HO
HO
H3CO
CHO
12h, rt
HO
HO
H3CO
O
O
5
K2CO3/CH3I
acetone
1N NaOH
acetone
H3CO
CHO
3. Results and discussion
4
divanillin
12h, rfx
12h, rt
H3CO
3.1. Synthesis
Dehydrozingerone 2 was chosen as reference structure and, in
O
order to preserve the guaiacyl unit present in the molecule and
H3CO
MTBAP
HO
HO
to extend the p-delocalization, the electroactive ferrocenyl moiety
CH2Cl2
1h, rt
was inserted at the end of the unsaturated chain in place of the
methyl group. One and two ferrocenyl units were introduced in
dehydrozingerone derivatives 3, 4 and 5, respectively. The study
was also extended to the preparation of ferrocenyl conjugates of
zingerone 6, the non-volatile component of Zingiber officinale
[27], and its C2-symmetric dimer 7 in view of the known influence
HO
H3CO
6
O
zingerone
7
Scheme 1. Synthesis of benzalacetones 2–5 and 7.

84
S. Pedotti et al. / Polyhedron 117 (2016) 80–89
O
included in the study for comparative purposes. According to
O
Wang et al. [32], the reversibility of electrochemical processes of
each compound, was evaluated by comparing the magnitude of
MeO
RO
MeO
CHO
CH3
NaOH
+
Fe
Δ
Fe
the separation between the oxidation (Eox) and reduction (Ered
peak potentials,
Ep = |Eox ꢁ Ered|, with the corresponding value
of the reversible system ferrocene/ferricenium couple.
)
RO
D
8
9
R = Me
R = (4-Me)Bn
10 R = Me
11 R = (4-Me)Bn
Cyclic voltammograms are showed in Figs. 2–6 and Eox, Ered and
Ep of all compounds are listed in Table 1.
D
Scheme 2. Synthesis of ferrocenyl chalcones 10 and 11.
Dehydrozingerone 2 and zingerone 6 displayed not reversible
behavior and compound 3, lacking in free phenolic-OH group,
was electrochemically inert.
On the contrary, cyclovoltammetric spectra of the correspond-
ing monomeric ferrocenyl analogues, e. g. 10, 16 and 18, showed
a reversible one-electron redox couple attributed to the ferrocenyl
O
MeO
MeO
CHO
O
NaOH
Fe
Fe
CH3
RO
RO
dioxane
RO
RO
+
Fe
unit bonded to the aliphatic chain whereas the
influenced by the molecule structure.
DEp value was
Δ
MeO
MeO
CHO
In the dimer series, no significant electrochemical response was
detected for 4 and 14 (Fig. 4) while compounds 7 and 17, the zin-
gerone dimer and its ferrocenyl analogue, showed not reversible
electrochemical behavior probably due to the ortho–ortho0 free
phenolic-OH groups that can activate side reactions as described
in literature for other biphenyls with similar structural features
(Fig. 6) [33,34].
A putative interaction between the two ferrocenyl units in
dimer 17, favoured by the phenolic –OH hydrogen bond and result-
ing in forcing closer the aliphatic side chains could not be ruled
out. On the contrary, the lack of hydrogen bond in dimer 14 would
drive the biphenyl structure in a conformation where the two fer-
rocenyl units are each other far away.
For all ferrocenyl derivatives a drastic shift to positive peak
potentials was observed with respect to ferrocene probably due
to the electron-withdrawing properties of the rest of the molecule
as observed in other ferrocenyl conjugates [35]. A broad oxidation
peak at low potential was observed for compounds 10, 16, 17 and
18 (Figs. 2–6) probably due to an oxidation process which involves
the organic part of the molecule before that the Fe oxidation pro-
cess takes place [36,37]. The presence of an unsaturated bond in
the side chain produced a drastic decrease in the potential value
detected for dehydrozingerone 2 compared to zingerone 6 (Eox
250 mV versus Eox 422 mV). Nevertheless, when a ferrocenyl unit
is embedded in a saturated chain as in 16 and dimer 17 the Eox/Ered
value shifted to lower potentials compared to the parent com-
pound lacking in the electroactive unit (eg. 6 and 7). It seems that
both iron atom in ferrocene and phenolic hydroxyl group played a
O
12 R = Me
13 R = (4-Me)Bn
14 R = Me
15 R = (4-Me)Bn
Scheme 3. Synthesis of ferrocenyl chalcones 14 and 15.
Exclusive formation of trans-isomers of ferrocenyl chalcones
was deduced from their 1H NMR spectra in solution that displayed
a large coupling constant (J = 15.6 Hz) for the doublets of the ethy-
lenic protons. Concerning the ferrocenyl unit, the NMR spectra of
dimers appeared quite similar to those of monomeric counterparts,
this spectroscopic evidence should exclude any interaction with
the two metallocene units when both present in the same molec-
ular structure. Downfield shifts of about 10 ppm were observed
for 13C NMR carbonyl resonances going from enone-containing
derivatives to the corresponding saturated compounds.
UV–Vis absorption spectra of ferrocenyl chalcones 18, 10 and 14
recorded in dichloromethane at concentration 0.74 ꢀ 10ꢁ5 M tend
to have larger conjugated systems compared to their correspond-
ing chalcones 2, 3, 4 (Supplementary data), and therefore their
peak wavelengths tend to be shifted toward the long wavelength
region.
3.2. Voltammetric measurements
All the prepared ferrocenyl derivatives were subjected to cyclic
voltammetric analysis and also the parent benzalacetones were
O
MeO
Pd/C
H2 (1 atm)
Fe
O
HO
HO
Pd/C
H2 (1 atm)
MeO
THF,
60 °C, 3h
THF,
60 °C, 3h
Fe
HO
MeO
Fe
O
O
16
18
17
11
15
O
MeO
HO
MeO
Me3SiI
Fe
Fe
Fe
Me3SiI
CH2Cl2
rt, 3h
HO
HO
CH2Cl2
rt, 3h
MeO
O
19
Scheme 4. Synthesis of ferrocenyl chalcones 16–19.

S. Pedotti et al. / Polyhedron 117 (2016) 80–89
85
Fig. 2. Cyclic voltammograms recorded with a carbon working electrode (area = 0.071 cm2), in a buffer solution containing 10 mL of KNO3 1 M and 10 mL of ethanol 96%.
Curves in A, obtained with a scanned potential range (Eapp) comprised between ꢁ1000 mV and +1000 mV, at 100 mV sꢁ1 scan rate, were separately achieved, in the absence
and in the presence of 1 mM ferrocene, 1 mM compound 6 and 1 mM compound 16; curves in B are details of voltammogram A, in a scanned potential range (Eapp) comprised
between ꢁ200 mV and +500 mV, where oxidation peak of each compound is enhanced; curves in C are details of voltammogram A, in a scanned potential range (Eapp
)
comprised between ꢁ200 mV and +500 mV, where reduction peak of each compound is enhanced.
Fig. 3. Cyclic voltammograms recorded with a carbon working electrode (area = 0.071 cm2), in a buffer solution containing 10 mL of KNO3 1 M and 10 mL of ethanol 96%.
Curves in A, obtained with a scanned potential range (Eapp) comprised between ꢁ1000 mV and +1000 mV, at 100 mV sꢁ1 scan rate, were separately achieved, in the absence
and in the presence of 1 mM ferrocene, 1 mM compound 3 and 1 mM compound 10; curves in B are details of voltammogram A, in a scanned potential range (Eapp) comprised
between ꢁ200 mV and +500 mV, where oxidation peak of each compound is enhanced; curves in C are details of voltammogram A, in a scanned potential range (Eapp
)
comprised between ꢁ200 mV and +500 mV, where reduction peak of each compound is enhanced.

86
S. Pedotti et al. / Polyhedron 117 (2016) 80–89
Fig. 4. Cyclic voltammograms recorded with a carbon working electrode (area = 0.071 cm2), in a buffer solution containing 10 mL of KNO3 1 M and 10 mL of ethanol 96%.
Curves in A, obtained with a scanned potential range (Eapp) comprised between ꢁ1000 mV and +1000 mV, at 100 mV sꢁ1 scan rate, were separately achieved, in the absence
and in the presence of 1 mM ferrocene, 1 mM compound 4 and 1 mM compound 14; curves in B are details of voltammogram A, in a scanned potential range (Eapp) comprised
between ꢁ200 mV and +500 mV, where oxidation peak of each compound is enhanced; curves in C are details of voltammogram A, in a scanned potential range (Eapp
)
comprised between ꢁ200 mV and +500 mV, where reduction peak of each compound is enhanced.
Fig. 5. Cyclic voltammograms recorded with a carbon working electrode (area = 0.071 cm2), in a buffer solution containing 10 mL of KNO3 1 M and 10 mL of ethanol 96%.
Curves in A, obtained with a scanned potential range (Eapp) comprised between ꢁ1000 mV and +1000 mV, at 100 mV sꢁ1 scan rate, were separately achieved, in the absence
and in the presence of 1 mM ferrocene, 1 mM compound 2 and 1 mM compound 18; curves in B are details of voltammogram A, in a scanned potential range (Eapp) comprised
between ꢁ200 mV and +500 mV, where oxidation peak of each compound is enhanced; curves in C are details of voltammogram A, in a scanned potential range (Eapp
)
comprised between ꢁ300 mV and +500 mV, where reduction peak of each compound is enhanced.

S. Pedotti et al. / Polyhedron 117 (2016) 80–89
87
Fig. 6. Cyclic voltammograms recorded with a carbon working electrode (area = 0.071 cm2), in a buffer solution containing 10 mL of KNO3 1 M and 10 mL of ethanol 96%.
Curves in A, obtained with a scanned potential range (Eapp) comprised between ꢁ1000 mV and +1000 mV, at 100 mV sꢁ1 scan rate, were separately achieved, in the absence
and in the presence of 1 mM ferrocene, 1 mM compound 7 and 1 mM compound 17; curves in B are details of voltammogram A, in a scanned potential range (Eapp) comprised
between ꢁ200 mV and +600 mV, where oxidation peak of each compound is enhanced; curves in C are details of voltammogram A, in a scanned potential range (Eapp
)
comprised between ꢁ200 mV and +400 mV, where reduction peak of each compound is enhanced.
Table 1
Electrochemical data for methyl-ketones and ferrocenylchalcones.a
a
The analyses were carried out in a buffer solution containing 10 mL of 1 M KNO3 and 10 mL of 96% ethanol, using a
carbon working electrode (area 0.071 mm2), an Ag/AgCl reference electrode and a Pt counter electrode, with a scanned
potential range (Vapp) comprised between ꢁ1000 mV and +1000 mV, at 100 mV sꢁ1 scan rate, in the presence of 1 mM of
each compound.
b
D
E = |Eox ꢁ Ered|.
radical-scavenging role and the radical-scavenging capacity of iron
atom in ferrocene was even higher than that exerted by the pheno-
lic-OH group.
of hydrogen peroxide (H2O2) in order to simulate an oxidative bio-
logical process able to promote cell death in cancer cells [42].
Viability of PC12 cells by exposure to compounds 2–7, lacking
in ferrocenyl unit, was previously investigated by us [11] and
dose–response studies evidenced moderate cytotoxicity at 40
only for compounds 3 and 5, whereas biphenyl 4 significantly
induced cell death even at 10 M concentration.
All ferrocenyl-containing compounds 10, 14 and 16–18 showed
a concentration-related toxicity but were less toxic than ferrocene
itself on PC12 cells (Fig. 7).
lM
3.3. Biological assay
l
Several biological studies evidence the use of PC12 cells as
straightforward model to evaluate viability and antitumor activity
in vitro [38–41]. In this work we used PC12 cells to test toxicity of
the new ferrocenyl chalcones when used alone and in the presence

88
S. Pedotti et al. / Polyhedron 117 (2016) 80–89
Fig. 7. PC12 cells viability in medium alone (DMEM/F12) or DMEM/F12 plus the compounds ferrocene, 10, 14, 16, 17 and 18 at 10, 20, 40 and 80
DMEM/F12 alone (control group). § = P < 0.05 vs. ferrocene.
l
M, respectively. *P < 0.05 vs.
Fig. 8. Viability of PC12 cells in culture medium (DMEM/F12), DMEM/F12 + ferrocene or DMEM/F12 supplemented with H2O2 (75
compounds 10, 14, 16, 17 and 18 (40 M).
M). *P < 0.05 vs. medium (DMEM/F12) alone. §P < 0.05 vs. ferrocene. #P < 0.05 vs. H2O2 (75
lM), alone and in the presence of
l
l
At concentration of 40 lM the viability was reduced (about
20%) by exposure to compounds 10, 14 and 17 whereas roughly
doubled decrease was observed in the presence of ferrocene itself
comparison with control.
In the series of compounds lacking in the ferrocenyl unit, sta-
tistically significant protection from H2O2-induced oxidative
stress was observed only for dehydrozingerone 2 and zingerone
6 [11].
On the contrary the co-exposure of PC12 to ferrocenyl chal-
cones 14, 16–18 and H2O2, significantly enhanced the cytotoxic
effect of hydrogen peroxide resulting in a further decrease of cell
viability (see Fig. 8). Although we have not investigated the mech-
anism of toxicity of the studied ferrocene chalcones in the presence
of H2O2, generation of hydroxyl anions and highly reactive hydro-
xyl radicals would occur. In fact, it is generally acknowledged the
role of ferrocene unit in producing reactive oxygen species (ROS)
in cancer cells due to the innate overproduction of H2O2 in these
cells [42]. As result of these oxidation reactions, selective oxidative
DNA damage of cancer cells and their targeted apoptosis would
occur without damage to normal cells. An emerging strategy for
cancer treatment exploits the induction of oxidative stress by
exogenous ROS generating agents specifically in cancer cells
Fig. 9. Viability of PC12 cells in culture medium (DMEM/F12), DMEM/F12
+ ferrocene or DMEM/F12 supplemented with H2O2 (75 M), alone and in the
presence of compounds 4 and 14 (40
M). *P < 0.05 vs. medium (DMEM/F12) alone.
§P < 0.05 vs. ferrocene. #P < 0.05 vs. H2O2 (75
M).
l
l
l
leading to their apoptotic death [43] and the antiproliferative
activity of some ferrocenyl olefins has been recently related to
their ability in inducing ROS generation and cell death [44].

S. Pedotti et al. / Polyhedron 117 (2016) 80–89
89
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