Non-classical anticancer agents: Synthesis and biological evaluation of zinc(ii) heteroleptic complexes

Article abstract of DOI:10.1039/b922101h

New heteroleptic complexes (1-8) containing Zn(ii) ion coordinated to an N,N-chelating ligand (the 4,4′-dinonyl-2,2′-bipyridine, bpy-9) and to diketonates L such as tropoloids (Tropolone and Hinokitiol) or 1-phenyl-3-methyl-4-R-5-pyrazolones have been synthesized by using different stoichiometric ratio with respect to the L ancillary ligand. The molecular structure of the bis-tropolonate derivative [(bpy-9)Zn(L)₂] 5 has been determined by single-crystal X-ray diffraction. The antitumour activity of all Zn(ii) complexes was tested in vitro against three different human prostate cancer cells: DU145, LNCaP and PC-3. Moreover, their effect on cell survival signalling and/or inhibitors of the PC-3 cell cycle have been analyzed. The results indicate that 1-8 exhibit strong cytotoxic activity against all cell lines affecting key molecules such as p-AKT and p21 waf, involved in the cell proliferation and/or arrest. Zinc(ii) is thus a promising alternative to Pt(ii) ion in the design of new, better performing antitumour agents.

Full text of DOI:10.1039/b922101h

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www.rsc.org/dalton | Dalton Transactions
Non-classical anticancer agents: synthesis and biological evaluation of zinc(II)
heteroleptic complexes†
Paola F. Liguori,a Alessandra Valentini,b Mariagrazia Palma,c Anna Bellusci,a Sergio Bernardini,b
Mauro Ghedini,a Maria Luisa Panno,c Claudio Pettinari,d Fabio Marchetti,d Alessandra Crispini*e and
Daniela Pucci*a
Received 22nd October 2009, Accepted 3rd March 2010
First published as an Advance Article on the web 24th March 2010
DOI: 10.1039/b922101h
New heteroleptic complexes (1–8) containing Zn(II) ion coordinated to an N,N-chelating ligand (the
4,4¢-dinonyl-2,2¢-bipyridine, bpy-9) and to diketonates L such as tropoloids (Tropolone and Hinokitiol)
or 1-phenyl-3-methyl-4-R-5-pyrazolones have been synthesized by using different stoichiometric ratio
with respect to the L ancillary ligand. The molecular structure of the bis-tropolonate derivative
[(bpy-9)Zn(L)2] 5 has been determined by single-crystal X-ray diffraction. The antitumour activity of
all Zn(II) complexes was tested in vitro against three different human prostate cancer cells: DU145,
LNCaP and PC-3. Moreover, their effect on cell survival signalling and/or inhibitors of the PC-3 cell
cycle have been analyzed. The results indicate that 1–8 exhibit strong cytotoxic activity against all cell
lines affecting key molecules such as p-AKT and p21 waf, involved in the cell proliferation and/or
arrest. Zinc(II) is thus a promising alternative to Pt(II) ion in the design of new, better performing
antitumour agents.
in many fields (binder complexes at DNA sites,11-14 radioprotective
agents,15 tumor photosensitizers,16 antidiabetic insulin-mimetic17,18
Introduction
and antibacterial or antimicrobic activities19–21) very little data on
the cytotoxicity of zinc-based compounds against human cancer
cell lines are as yet available.22–30 The role of this metal in the
growth and survival of cells, and its versatile coordination states
and geometries preserving the same oxidation state, prompted us
to engineer heteroleptic Zn(II) complexes as potential anticancer
agents with low in vivo toxicity and perhaps new modes of action
and cellular targets with respect to the classical metallodrugs.
Our recent works have been focused on biologically active
square-planar complexes containing two different chelating or-
ganic frameworks tethered to Pt(II) or Pd(II) ions: an aromatic
N,N-ligand and a biologically active O,O-ligand, both able to
induce synergistic effects in the resulting derivatives.31–34
In particular, we have synthesised mononuclear ionic complexes
for which the presence of the dihexadecyl 2,2¢-bipyridine-4,4¢-
dicarboxylate (bpy-16) and tropolonate around the metal ion,
led them to be relatively inert towards ligand substitutions and
remarkably cytotoxic in vitro against the human prostate DU145
and LNCaP cell lines.33
Based on these evidences, in this study we explored the
possibility of using the same strategy but taking advantage of both
the bioavailability and versatile coordination ability of Zn(II) and
the better solubility of a different bipyridine ligand such as the 4,4¢-
dinonyl-2,2¢-bipyridine (bpy-9). Moreover, by using this metal ion
we can investigate the impact of the presence of one or two diketo-
nate ligands around the metal ion on the antiproliferative activity
of the resulting complexes. Hence, here we report on the synthesis
and characterization of a novel series of heteroleptic complexes
involving Zn(II) ion in two different coordination environments,
depending on the amount of the diketonate ancillary ligand used.
The O,O-ligands (L) chosen are tropolones such as Tropolone
New inorganic complexes have been recently proposed as effective
alternatives to antitumor agents currently used in chemotherapy,
in order to overcome toxicity and drug-resistance phenomena and
to achieve higher activity and better selectivity than platinum-
based anticancer drugs. Indeed non-platinum antitumor thera-
peutics can show various geometries and coordination numbers,
various oxidation states, better solubility properties and various
substitution kinetics or mechanism pathways, all factors which
may induce a pharmacological profile different than those of
platinum-drugs.1–3
The development of new metal-based therapeutics is chang-
ing rapidly,4 and gold(I and III),5 ruthenium(II and III)6 and
titanium(IV)7 derivatives have received intense interest as the most
promising candidates among the new generation of cytotoxic com-
plexes. However, despite the variety of physiological roles of the
zinc(II) ion8–10 and the wide repertoire of Zn(II) complexes utilized
aCentro di Eccellenza CEMIF.CAL-LASCAMM, CR-INSTM Unita` della
Calabria, Dipartimento di Chimica, Universita` della Calabria, Via P. Bucci
Cubo 14C, Italy. E-mail: d.pucci@unical.it; Fax: (+39 0984 492066)
bDepartment of Laboratory Medicine and Dept. Of Internal Medicine, PTV
Universita` di Roma “Tor Vergata”, Rome, Italy
cDipartimento di Biologia Cellulare Universita` della Calabria, 87030,
Arcavacata di Rende, (CS), Italy
dDipartimento di Scienze Chimiche, Universita` degli Studi di Camerino, Via
S. Agostino 1, 62032, Camerino, Italy
eCentro di Eccellenza CEMIF.CAL-LASCAMM, CR-INSTM Unita` della
Calabria, Dipartimento di Scienze Farmaceutiche, Universita` della Calabria,
Edificio Polifunzionale, Italy. E-mail: a.crispini@unical.it
† Electronic supplementary information (ESI) available: Antiproliferative
effects of organic precursors in all cell lines, IC50 values of cisplatin,
complex 1 and Pd(II) and Pt(II) derivatives, UV/vis spectra of ligands and
complexes 1–8. CCDC reference number 752117. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/b922101h
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(Trop) and Hinokitiol (Hkt, also called b-thujaplicin) or 1-phenyl-
3-methyl-4-acyl-5-pyrazolones such as HQw and HQT, all shown
in Fig. 1. Tropolone and its derivatives, natural products with
a seven-membered aromatic ring and various side groups, have
powerful antibacterial and antifungal activity, particularly against
antibiotic-resistant bacteria. Many other biological functions
such as antiviral, antioxidant, antiinflammatory, insecticidal and
antidiabetic activities are associated with tropolones.35 Moreover,
even though tropolone and hinokitiol exhibit an inhibitory effect
on the growth of a range of tumor cell lines,36–39 very few studies
have been performed on tropolone-based metal complexes as
antineoplastic agents.22,33,40 As regards acylpyrazolones, while their
coordination chemistry has been extensively studied and the
corresponding metal derivatives applied in many fields,41,42 their
biological properties have been scarcely investigated up to now.43–45
and PC-3. Moreover a study of the expression of p-AKT and p21
waf proteins of the PC-3 cells has been performed in order to
understand the role of these new Zn(II) species in the control of
the balance of cell cycle.
Results and discussion
Synthesis and characterization
All Zn(II) complexes 1–8 were prepared starting from the same
N,N-chelating ligand, the 4,4¢-dinonyl-2,2¢-bipyridine (bpy-9), as
summarized in Scheme 1.
The dichloro precursor [(bipy-9)ZnCl2] I was prepared by
reaction of an equimolar amount of the main ligand bpy-
9 with ZnCl2, in dichloromethane, at room temperature. The
corresponding mono-diketonate derivatives 1–4 were obtained
through reaction with one equivalent of the appropriate KL
salt in dichloromethane, at room temperature. The versatility of
coordination of the Zn(II) ion allowed to synthesize also the bis-
diketonate species 5–8 through two different synthetic strategies,
depending on the nature of the ancillary ligand, as shown in
Scheme 1.
In particular, the reaction of the dichloro complex I with two
equivalents of the potassium tropolonates, in ethanol, gave rise
to the formation of complexes 5 and 6 within 24 h. On the other
hand, the synthesis of the acylpyrazolonate derivatives 7 and 8, was
carried out in two steps, involving the reaction of the appropriate
[Zn(Q)2(S)2] precursor46 with the bpy-9 ligand, in methanol.
The structures of all complexes have been assigned on the
Fig. 1 Structures of the ancillary ligands L.
All the new Zn(II) complexes 1–8 in Scheme 1 have been screened
for their in vitro effect on cell proliferation of three human prostate
cell lines: hormone-resistant DU145, hormone-sensitive LNCaP
1
basis of elemental analysis, IR and H NMR spectroscopies. In
Scheme 1 Synthesis of complexes 1–8. Reagents and conditions: (i) ZnCl2, CH2Cl2, r.t., 96 h; (ii) CH2Cl2–EtOH, r.t., N2, 48 h; (iii) CH2Cl2, r.t, N2, 48 h;
(iv) EtOH, 80 ◦C, 24 h; (v) CHCl3, r.t, 5 days.
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◦
˚
particular, in the IR spectra of derivatives 1–8, the carbon–oxygen
bands of the ancillary ligands are shifted by about 10–15 cm-1
with respect to the corresponding O,O-free ligands, confirming
that the coordination to the Zn(II) ion took place. Moreover, a
broad band at ca. 2700 cm-1 indicates the presence of water in the
Table 1 Selected bond lengths (A) and angles ( ) for complex 5
Zn(1)–N(1)
Zn(1)–O(1)
Zn(1)–O(3)
2.150(3)
2.083(3)
2.099(3)
Zn(1)–N(2)
Zn(1)–O(2)
Zn(1)–O(4)
2.131(3)
2.090(3)
2.060(3)
O(1)–Zn(1)–N(1)
O(1)–Zn(1)–N(2)
O(1)–Zn(1)–O(2)
O(1)–Zn(1)–O(3)
O(2)–Zn(1)–N(1)
O(2)–Zn(1)–N(2)
O(2)–Zn(1)–O(3)
O(3)–Zn(1)–N(1)
94.1(1)
163.1(1)
76.5(1)
93.8(1)
100.6(1)
92.5(1)
164.7(1)
91.8(1)
O(3)–Zn(1)–N(2)
O(4)–Zn(1)–N(1)
O(4)–Zn(1)–N(2)
O(4)–Zn(1)–O(1)
O(4)–Zn(1)–O(2)
O(4)–Zn(1)–O(3)
N(2)–Zn(1)–N(1)
99.5(1)
164.4(1)
95.5(1)
97.6(1)
92.2(1)
77.1(1)
75.2(1)
mono-diketonate complexes 1–4.
In the H NMR spectra of 1–8 the upfield shift of the H3,3¢
1
,
H
1,1¢, H6 and He–i resonances (Scheme 1) is consistent with the
presence of chelated systems around the Zn(II) ion. Moreover,
1
in the H NMR spectra of complexes 5–8 the intensities of the
signals corresponding to the protons of the ancillary ligands
are in agreement with the 1 : 2 (metal : O,O-ligand) stoichiometry
proposed in Scheme 1.
Table 2 IC50 values of complexes 1–8 against DU145 and LNCaP cells
IC50 SD/mM
Conductivity measurements have been performed in
dichloromethane solution, confirming the neutral nature of
all species, at least in solution. Therefore, we can assume that
the coordination sphere, apart from the N,N-ligand grafted
around the Zn(II) ion, is saturated by a chloride ion and a solvent
molecule for complexes 1–4, [(bpy-9)Zn(L)(Cl)(Solv)], and by two
O,O-chelating units for complexes 5–8, [(bpy-9)Zn(L)2].
In order to test their stability in solution, the behaviour of
all complexes has been investigated by UV/VIS spectroscopy in
various solvents and in physiological pH conditions. Measure-
ments conducted over the time have proved the good stability of
these systems, since both the shape and the absorption maximum
positions remain unchanged after 24, 48 and 96 h (see ESI†). Only
in the case of complex 4, significant variations in the UV/VIS
spectra are observed, indicating a certain level of instability due to
both a loss of ligands and/or a rearrangement of ligands around
the metal ion.
The molecular structure of 5 has been obtained by single-
crystal X-ray analysis (Fig. 2). Selected bond length and angles
are given in Table 1. The central metal ion is six-coordinated by
four oxygen atoms of two bidentate tropolonate and two nitrogen
atoms of the bipyridine ligands. The coordination around the
zinc(II) ion yields a distorted octahedral geometry, with the largest
deviations represented by the O(2)–Zn(1)–O(3) and O(1)–Zn(1)–
N(2) angles with values of 164.7(1) and 163.2(1)◦, respectively,
and the bite angle of the bipyridine ligand at 75.3(1)◦. The “bite”
angles as well as the Zn–O bond distances of the two chelated
tropolonate ligands are comparable with those of the only molec-
ular structures of tropolonate Zn(II) derivatives reported up to
now.47,48
Complex
DU145
LNCaP
Cisplatin
33 1.0
4.5 0.1
38 5.0
20 5.0
4.8 0.2
3.3 0.1
80 100
>100
34 0.4
17 1.0
23 2.0
25 5.0
20 0.2
18 0.6
70 10
>100
[(bpy-9)Zn(Trop)(Cl)(Solv)], 1
[(bpy-9)Zn(Hkt)(Cl)(Solv)], 2
[(bpy-9)Zn(Qw)(Cl)(Solv)], 3
[(bpy-9)Zn(QT)(Cl)(Solv)], 4
[(bpy-9)Zn(Trop)2], 5
[(bpy-9)Zn(Hkt)2], 6
[(bpy-9)Zn(Qw)2], 7
[(bpy-9)Zn(QT)2], 8
10 0.6
>100
Biological evaluation
The antiproliferative activity of all organic ligands and the
corresponding new Zn(II) complexes has been investigated, in vitro,
towards human prostate hormone-resistant DU145 and hormone-
sensitive LNCaP cell lines,49,50 using a colorimetric assay (MTS).
As regards as the precursors, only Hkt and HQw exhibit toxic
effects with an IC50 of about 20 and 60 mM, respectively, in DU145
and LNCaP cells, while bpy-9, Trop and HQT do not have any
toxicity in the dose range used (see ESI†).
On the contrary, the coordination of the organic ligands
to the Zn(II) ion induces synergistic effects leading the novel
corresponding complexes 1–8 to show relevant cytotoxic activity,
as shown in Table 2.
All complexes 1–4 show an interesting cytotoxic activity toward
both cells when compared to cisplatin (Table 2). In particular, the
tropolonate and acylpyrazolonate QT derivatives 1 and 4 are the
most active species on DU145 cells.
On the contrary, the presence of a further unit of L in
complexes 5–8 causes remarkable differences in their biological
behaviour. In both cell lines, while the cytotoxic activity of the
tropolonate derivative 5 is the same as that observed in the
corresponding mono-diketonate complex 1, the Hkt and Qw-
based Zn(II) complexes 6 and 7, appeared to be only active at
higher concentration (IC50 >60 mM or inactive) with respect to
the corresponding mono-diketonate species 2 and 3, respectively.
On the contrary, the bis-acylpyrazolonate QT 8 shows selectivity
towards both cancer cells used, preserving its activity in the
hormone-resistant DU145 cells but losing it towards the hormone-
sensitive LNCaP cells.
In order to evaluate the cell selectivity of these novel Zn(II)
species, we tested 1–8 additionally against the PC-3 prostatic cell
line, intermediately differentiated between DU145 and LNCaP,
Fig. 2 Perspective view of complex 5 with atomic numbering scheme
(ellipsoids at the 40% level).
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Table 3 IC50 values of complexes 1–8 against PC-3 cell line
These results indicate that these new zinc(II) complexes have
an antiproliferative activity in the prostatic tumoral cells studied,
which reflects their effects also on the key protein signalings
involved in the control of the balance of cell proliferation and/or
cell arrest. Indeed complexes 1–8 are able to target the pro-
survival-signal phospho-AKT, which plays an important role in
mediating drug resistance and stress-induced apoptosis inhibition
in tumoral cells.54–56 At the same time, the activation of the
signalling protein involved in cell mitotic block reflects an ongoing
stress signalling.57,58
Complex
IC50 SD/mM
[(bpy-9)Zn(trop)(Cl)(S)], 1
[(bpy-9)Zn(hkt)(Cl)(S)], 2
[(bpy-9)Zn(Qw)(Cl)(S)], 3
[(bpy-9)Zn(QT)(Cl)(S)], 4
[(bpy-9)Zn(trop)2], 5
5.0 0.8
5.0 0.3
4.2 0.4
3.2 0.1
4.5 0.2
4.2 0.3
3.9 0.2
2.8 0.1
[(bpy-9)Zn(hkt)2], 6
[(bpy-9)Zn(Qw)2], 7
[(bpy-9)Zn(QT)2], 8
Conclusions
in the same range of concentration where 1–8 show significant
activity in the other cell lines.
Two series of heteroleptic Zn(II) complexes containing 4,4¢-
dinonyl-2,2¢-bipyridine (bpy-9) as main ligand and tropolones
or 1-phenyl-3-methyl-4-R-5-pyrazolones (L) as ancillary ligands,
have been synthesized by using different stoichiometric ratios with
respect to the diketonates L. In the series 1–4 and 5–8, the same
molecular fragment (bpy-9)Zn(L), obtained through chelation
of both bpy-9 and L is present. In [(bpy-9)Zn(L)(Cl)(Solv)]
derivatives, 1–4, the coordination sphere of the Zn(II) ion is
saturated by a solvent molecule and a chloride ion. In 5–8 the
presence of a second chelated L unit, leading to a general formula
[(bpy-9)Zn(L)2], has been confirmed through single-crystal X-ray
diffraction analysis performed on derivative 5.
The uncoordinated ligands as well as the corresponding Zn(II)
complexes have been tested in vitro towards three different human
prostatic cancer cell lines: DU145, LNCaP and PC-3. While
almost all ligands are inactive, most of the Zn(II) complexes show
promising anticancer properties.
On the whole, all Zn(II) complexes synthesized show selectivity
toward cancer cells, being more active on DU145 and PC-3 cells
with respect to LNCaP cells. Moreover, while the mono-diketonate
derivatives 2 and 3 in DU145 and LNCaP cells exhibit cytoxicity
higher than the corresponding bis-diketonates 6 and 7, the activity
of all Zn(II) complexes towards PC-3 cells is not strongly related
to the stoichiometric ratio of the ancillary ligands, being almost
the same for the mono- and the bis-diketonate species.
Moreover, from this study it is possible to confirm that the
nature of the central metal ion as well as its coordination
environment induce significant changes in the biological activities
of the resulting complexes. Indeed, comparing complexes 1–8
with Pd(II) and Pt(II) heteroleptic mono-tropolonate derivatives or
with homoleptic Zn(II) bis-diketonates, we can conclude that the
simultaneous presence of flat chelating ligands around the Zn(II)
ion as metal scaffold has been a winning choice for endowing new
non-platinum complexes with good antitumour activity.
While the organic precursors are able to decrease only slightly
the cellular growth of PC-3 cells (see ESI†), complexes 1–8 reveal
a significant antiproliferative effect, with IC50 values between 2.8
and 5 mM (Table 3).
The improved activity showed in the interaction with this cell
line is particularly evident in the case of compounds 6 and 7, which
are inactive in the other two cell types.
Comparing the obtained results with the analogous studies con-
ducted in similar systems, we can establish important conclusions
about the use of the Zn(II) species as antitumour drugs. Indeed,
a strong increase of cytotoxicity can be evidenced on going from
the tetracoordinated Pd(II) and Pt(II) tropolonate derivatives33 to
the Zn(II) homologues 1 and 5 (ESI†), suggesting that the nature
of the metal ion plays a crucial role in determining the biological
activities of the corresponding complexes. Keeping the same metal
atom, the addition of an N,N-chelating fragment greatly enhances
the antiproliferative activity when the heteroleptic species 1–8
are compared to the few examples of homoleptic Zn(II) bis-
diketonates investigated up to now.22,45
Since the net cell growth rate depends on a fine balance between
the cell proliferation rate and the cell death rate, we have examined
whether complexes 1–8 might affect the key transductional signals
mainly involved in the regulation of cell survival. Thus we have
evaluated, in untreated and treated PC-3 cells, the expression
levels of phospho-AKT (pAkt), an important pro-survival protein
involved in the cell survival, and of cyclin inhibitor p21 waf, a
signalling protein implicated in apoptosis and growth arrest.51–53
As shown in Fig. 3, a down-regulation of phospho-AKT levels
occurs after 48 h, in the prostatic cells treated with all Zn(II)
complexes, mainly evident in the case of 1 and 6. At the same
time, for 1 and 6 an increasing of level of the p21 waf, is observed
(Fig. 3).
Finally, preliminary studies of protein expression implicated in
apoptosis and growth arrest indicate a key role played by these
Zn(II) complexes in the control of cell proliferation and/or cell
arrest. Further studies are needed to completely understand the
mechanism of action and the structure–cellular activity of this
interesting class of metal complexes.
Experimental
Materials and measurements
Fig. 3 Immunoblots of pAkt and p21 from PC-3 cell extracts treated for
48 h with Zn(II) complexes 1–8. GAPDH was used as loading control. The
panel is representative of three independent experiments.
All commercially available starting materials were used as received
without further purification while the ligands HQW and HQT
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and the corresponding [Zn(Q)2(S)2] derivatives were synthesised
according to the procedure previously described.46
C35H51N2O3ZnCl: C, 64.82; H, 7.92; N, 4.32. Found: C, 64.67; H,
7.85; N, 4.39%.
The tropolone and hinokitiol potassium salts have been pre-
pared by reacting, in ethanol, one equivalent of potassium
hydroxide with the appropriate tropolone ligand. DMEM/Ham’s
F-12, RPMI 1640, L-glutamine, penicillin/streptomycin, foetal
bovine serum, bovine serum albumin, aprotinin, phenylmethyl-
sulfonyl fluoride (PMSF), sodium orthovanadate, MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), iso-
propanol, hydrochloric acid were purchased from Sigma (Milan,
Italy), the ECL system come from Amersham Biosciences. Anti-
p21 WAF, anti-GAP-DH pAbs, anti-mouse IgG were procured
from Santa Cruz Biotechnology (Heidelberg, Germany). Anti-
phospho-Akt (Ser473) from Cell Signaling Technology (Beverly,
MA, USA). Nylon membranes were provided by Roche diagnos-
tics Corporation (Indianapolis, USA).
[(bpy-9)Zn(Hkt)(Cl)(Solv)], 2. A hot solution of potassium
inokitiolate (0.111 g, 0.550 mmol) in EtOH (50 ml) was added
to a solution of [(bpy-9)ZnCl2] (0.300 g, 0.550 mmol) in CH2Cl2
(30 ml). The resulting solution was stirred under nitrogen (3 days,
r.t.). After removal of the solvent under vacuum the remaining
residue was dissolved in Et2O and was filtered through Celite.
Et2O was removed under reduced pressure and the product
was dissolved in CHCl3 and recrystallized from petroleum ether
(0.253 g, 68% yield). Mp 128 ◦C. IR (nmax/cm-1: 2921, 2852 (CH),
1
=
1610, 1590 (C O). H NMR (300 MHz, CDCl3): dH 9.05 (d,
2H, J(H,H) = 4.6 Hz, H3,3¢), 7.92 (s, 2H, H1,1¢), 7.45 (d, 2H,
J(H,H) = 5.0 Hz, H2,2¢), 7.39 (s, 1H, H4), 7.24 (s, 2H, H5,7),
6.76 (t, 1H, J(H,H) = 5.0 Hz, H6), 2.82 (m, 5H, CH(CH3)2hkt
,
,
CH2(CH2)7CH3), 1.70 (m, 4H, CH2CH3), 1.29 (m, 30H, (CH3)2hkt
(CH2)6CH2CH3), 0.88 (t, 6H, J(H,H) = 6.6 Hz, CH3). Anal. Calc.
for C38H57N2O3ZnCl: C, 66.08; H, 8.32; N, 4.05. Found: C, 65.92;
H, 8.18; N, 3.92%.
Instrumentation
The 1H NMR spectra were recorded on a Bruker Avance AC-300
spectrometer in CDCl3 solution, using tetramethylsilane (TMS)
as internal standard. Elemental analyses (CHN) were performed
with a Perkin Elmer 2400 microanalyzer by the Microanalyti-
cal Laboratory at the University of Calabria. Infrared spectra
(KBr) were recorded on a Spectrum One FT-IR Perkin Elmer
spectrometer. A Perkin-Elmer Lambda 900 spectrophotometer
was used to record absorbtion spectra of complexes 1–8 on 6%
EtOH–phosphate buffered saline solutions at a concentration of
6 ¥ 10-4 M.
[(bpy-9)Zn(QW)(Cl)(Solv)], 3. To a solution of [(bpy-9)ZnCl2]
(0.150 g, 0.275 mmol) in 20 ml of CH2Cl2 was added KQW, (0.102 g,
0.275 mmol). The resulting yellow solution was stirred under
nitrogen for 3 days at room temperature. The solution was filtered
through Celite and evaporated to dryness under reduced pressure.
The product was then dissolved in Et2O, filtered through Celite,
and evaporated to dryness under reduced pressure giving the pure
product as a yellow solid (0.093 g, 40% yield). IR (nmax/cm-1: 2921–
1
=
2852 (CH), 1610 (C O). H NMR (300 MHz, CDCl3): dH 9.03 (d,
2H, J(H,H) = 5.13 Hz, H3,3¢), 7.99 (d, 2H, J(H,H) = 8.08 Hz,
He,i ), 7.95 (s, 2H, H1,1¢), 7.54 (d, 2H, J(H,H) = 8.43 Hz, Ha,d),
7.46 (m, 6H, H2,2¢,c,d,h,f), 7.18 (t, 1H, J(H,H) = 7.34 Hz, Hg,g¢), 2.75
(t, 4H, J(H,H) = 7.45 Hz, CH2(CH2)7CH3), 1.85 (s, 3H, CH3QW),
1.66 (m, 4H, CH2CH3), 1.37 (s, 9H, C(CH3)3QW), 1.29 (m, 24H,
(CH2)6CH2CH3), 0.87 (t, 6H, J(H,H) = 6.6 Hz, CH3). Anal. Calc.
for C49H67N4O3ZnCl: C, 68.36; H, 7.84; N, 6.50. Found: C, 68.22;
H, 8.12; N, 6.26%.
Synthesis of complexes
[(bpy-9)ZnCl2]. 1.5 equiv of ZnCl2 (0.250 g, 1.83 mmol)
was added to a solution of 4,4¢-dinonyl-2,2¢-bipyridine (0.500 g,
1.22 mmol) in CH2Cl2 (20 ml). After stirring for 6 days (r.t.) the
solution was filtered through Celite and the solvent was distilled
under reduced pressure and then the product was recrystallized
from CHCl3–Et2O (0.523 g, 78% yield). Mp 150 ◦C. IR (nmax/cm-1:
[(bpy-9)Zn(QT)(Cl)(Solv)], 4. To a solution of [(bpy-9)ZnCl2]
(0.100 g, 0.183 mmol) in 20 ml of CH2Cl2 was added KQT,
(0.057 g, 0.183 mmol). The solution was filtered through Celite,
evaporated to dryness under reduced pressure, the product was
dissolved in Et2O, filtered through Celite, and evaporated to
dryness under reduced pressure giving the pure product as an
1
=
=
2921, 2852 (CH), 1617, 1556 (C C, C N). H NMR (300 MHz,
CDCl3): dH 8.68 (d, 2H, J(H,H) = 5.3 Hz, H3,3¢), 8.01 (s, 2H, H1,1¢),
7.51 (d, 2H, J(H,H) = 5.3 Hz, H2,2¢), 2.83 (m, 4H, CH2(CH2)7CH3),
1.71 (m, 4H, CH2CH3), 1.27 (m, 24H, (CH2)6CH2CH3), 0.88 (t,
6H, J(H,H) = 6.6 Hz, CH3). Anal. Calc. for C28H44N2ZnCl2: C,
61.71; H, 8.14; N, 5.14. Found: C, 61.31; H, 8.31; N, 5.31%.
brown solid (0.100 g, 70% yield). Mp 200 ◦C. IR (nmax/cm-1: 2926–
1
=
2855 (CH), 1612 (C O). H NMR (300 MHz, CDCl3): dH 9.05
[(bpy-9)Zn(Trop)(Cl)(Solv)], 1. A hot solution of potassium
tropolonate (0.029 g, 0.183 mmol) in EtOH (5 ml) was added
to a solution of [(bpy-9)ZnCl2] (0.100 g, 0.183 mmol) in CH2Cl2
(10 ml). The resulting solution was stirred under nitrogen (4 days,
r.t.). After removal of the solvent under vacuum the remaining
residue was dissolved in EtOH and was filtered through Celite.
EtOH was distilled under reduced pressure and the product was
dissolved in CHCl3 and recrystallized from n-hexane (0.081 g, 70%
(2H, d, J(H,H) = 5.48 Hz, H3,3¢), 7.98 (2H, s, H1,1¢), 7.95 (2H, s,
He,i ), 7.40 (4H, m, Hh,f; H2,2¢), 7.18 (1H, t, J(H,H) = 7.14 Hz, Hg,g¢),
2.80 (t, 4H, J(H,H) = 7.68 Hz, CH2(CH2)7CH3), 2.67 (s, 2H,
CH2), 2.49 (s, 3H, CH3QT), 1.72 (m, 4H, CH2CH3), 1,32 (m, 24H,
(CH2)6CH2CH3),1.15 (s, 9H, C(CH3)3QT), 0.87 (t, 6H, J(H,H) =
6.6 Hz, CH3). Anal. Calc. for C44H65N4O3ZnCl: C, 66.19; H, 8.20;
N, 7.05. Found: C, 66.50; H, 8.50; N, 6.95%.
yield). Mp 130 ◦C. IR (nmax/cm-1: 2921, 2852 (CH), 1609, 1595
[(bpy-9)Zn(Trop)2], 5. 2 equiv. of potassium tropolonate
(0.059 g, 0.366 mmol) was dissolved in EtOH (15 ml) and 1
equiv. [(bpy-9)ZnCl2] (0.100 g, 0.183 mmol) was added. The
resulting mixture was stirred at 80 ◦C over a period of 24 h. Then
the reaction mixture was filtered and the solvent containing the
product was evaporated in vacuo. The product was dissolved in
1
=
(C O). H NMR (300 MHz, CDCl3): dH 9.04 (d, 2H, J(H,H) =
3.3 Hz, H3,3¢), 7.93 (s, 2H, H1,1¢), 7.48 (d, 2H, J(H,H) = 4.9 Hz,
H
2,2¢), 7.33 (m, 4H, H4,7,5,8), 6.82 (t, 1H, J(H,H) = 8.5 Hz, H6), 2.78
(m, 4H, CH2(CH2)7CH3), 1.70 (m, 4H, CH2CH3), 1.23 (m, 24H,
(CH2)6CH3), 0.88 (t, 6H, J(H,H) = 6.6 Hz, CH3). Anal. Calc. for
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Table 4 Crystallographic data for complex 5
CHCl3, filtered through Celite, then concentrated and precipitated
by n-hexane (0.098 g, 75% yield). Mp 170 ◦C. IR (nmax/cm-1: 2926–
5
1
=
2855 (CH), 1611, 1592 (C O). H NMR (300 MHz, CDCl3): dH
8.71 (d, 2H, J(H,H) = 5.1 Hz, H3,3¢), 7.87 (s, 2H, H1,1¢), 7.23 (m,
10H, H2,2¢,4,4¢,7,7¢,5,5¢,8,8¢), 6.68 (t, 2H, J(H,H) = 8.3 Hz, H6,6¢), 2.69
(m, 4H, CH2(CH2)7CH3), 1.68 (m, 4H, CH2CH3), 1.37 (m, 24H,
(CH2)6CH2CH3), 0.86 (t, 6H, J(H,H) = 6.1 Hz, CH3). Anal. Calc.
for C42H54N2O4Zn: C, 70.43; H, 7.60; N, 3.91. Found: C, 70.31; H,
7.49; N, 4.31%.
Empirical formula
Mr
C42H54N2O4Zn
716.24
Triclinic
¯
P1
10.862(2)
13.476(2)
14.342(2)
77.235(5)
86.019(6)
75.809(5)
1984.8(5)
2
Cryst system
Space group
˚
a/A
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
[(bpy-9)Zn(Hkt)2], 6. Two equiv. of potassium inokitiolate
(0.232 g, 1.147 mmol) was dissolved in EtOH (100 ml) and 1
equiv. of [(bpy-9)ZnCl2] (0.250 g, 0.459 mmol) was added. The
resulting mixture was stirred at 80 ◦C over a period of 24 h. Then
the reaction mixture was filtered and the solvent containing the
product was evaporated in vacuo. The product was dissolved in
CHCl3, filtered through Celite, then concentrated and precipitated
3
˚
V/A
Z
Dc/g cm-3
1.198
0.66
764
1.92–24.61
m/mm-1
F(000)
q/◦
No. measured reflns
No. unique reflns
Reflns with I > 2s(I)
Refined params
Goodness-of-fit
R indices, I > 2s(I)a,b
R indices (all data)
34037
by n-hexane (0.265 g, 72% yield). Mp 180 ◦C. IR (nmax/cm-1: 2921,
6749 [R(int) = 0.0753]
1
=
2852 (CH), 1611, 1587 (C O). H NMR (300 MHz, CDCl3): dH
3967
496
1.0052
8.73 (d, 2H, J(H,H) = 5.2 Hz, H3,3¢), 7.86 (s, 2H, H1,1¢), 7.29 (d,
2H, J(H,H) = 1.3 Hz, H2,2¢), 7.24 (d, 2H, J(H,H) = 6.5 Hz, H4),
7.14 (t, 4H, J(H,H) = 3.7 Hz, H5,5¢,7,7¢), 6.62 (m, 2H, H6,6¢), 2.72
(m, 6H, CH(CH3)2Hinok, CH2(CH2)7CH3), 1.64 (m, 4H, CH2CH3),
1.24 (m, 24H, (CH2)6CH2CH3), 1.20 (d, 12H, J(H,H) = 6.7 Hz,
(CH3)2hkt), 0.87 (t, 6H, J(H,H) = 6.7 Hz, CH3). Anal. Calc. for
C48H66N2O4Zn: C, 72.02; H, 8.31; N, 3.50. Found: C, 71.81; H,
8.00; N, 3.81%.
R1 = 0.0573, wR2 = 0.1482
R1 = 0.1110, wR2 = 0.1738
ꢀ
a R1 = (|Fo| - |Fc|)/R|Fo|. b wR2 = [Rw(Fo - Fc2)2/Rw(Fo2)2]1/2
.
2
1.28 (24H, m, (CH2)6CH2CH3), 0.87 (24H, m, CH3L1; (CH3)3).
Anal. Calc. for C60H82N6O4Zn: C, 70.88; H, 8.13; N, 8.26. Found:
C, 70.76; H, 8.13; N, 8.28%.
[(bpy-9)Zn(QW)2], 7. To a solution of complex [Zn(QW)2(S)2]
(0,400 g, 0.542 mmol) in 30 ml of CHCl3 was added an equivalent
of bpy-9 (0.222 g, 0.542 mmol). The resulting yellow solution was
stirred at room temperature for five days. Then it was evaporated
to dryness under reduced pressure on a rotavapor. To a residue
was added n-hexane (20 ml). After concentration of the solvent a
yellow precipitate was afforded which was filtered off and dried to
constant weight under reduced pressure (0.451 g, 73% yield). Mp
X-Ray crystallography
Single crystals of [(bpy-9)Zn(Trop)2], 5 suitable for X-ray analysis
were obtained by slow diffusion of ethanol into a chloroform solu-
tion. X-Ray crystal data for 5 were collected at room temperature
on a Bruker-Nonius X8 Apex CCD area detector equipped with
graphite monochromator and Mo-Ka radiation (l = 0.71073),
and data reduction was performed using the SAINT programs;
absorption corrections based on multiscans were obtained by
SADABS.60 The structure was solved by the Patterson method
(SHELXS/L program in the SHELXTL-NT software package)61
and refined by full-matrix least squares based on F2. All non-
hydrogen atoms were refined anisotropically. Due to the severe
disorder found on one aliphatic chain (carbon atoms from C(22)
to C(28) refined in two positions with occupancy factors of 0.6 and
0.4) the SIMU constraint has been applied on their displacement
parameters. Hydrogen atoms were included as idealized atoms
riding on the respective carbon atoms with C–H bond lengths
appropriate to the bond. Details of the crystal data collection are
listed in Table 4.
180 ◦C. IR (nmax/cm : 2926–2854 (C–H), 1617 (C O). H NMR
(300 MHz, CDCl3): dH 8.92 (d, 2H, J(H,H) = 5.15 Hz, H3,3¢),
7.90 (d, 4H, J(H,H) = 1.21 Hz, He,e¢;Hi,i¢), 7.88 (d, 2H, J(H,H) =
0.85 Hz, H1,1¢), 7.34 (m, 4H, Ha,a¢, Hd,d¢), 7.31 (s, 2H, H2,2¢), 7.25 (d,
4H, J(H,H) = 8.43 Hz, Hb,b¢, Hc,c¢), 7.16 (t, 4H, J(H,H) = 8.01 Hz,
Hf,f¢; Hh,h¢), 6.99 (t, 2H, J(H,H) = 6.86 Hz, Hg,g¢), 2.75 (t, 4H,
J(H,H) = 7.68, CH2(CH2)7CH3), 1.63 (m, 10H, CH3Qw, CH2CH3),
1.32 (m, 42H, (CH2)6CH2CH3, (CH3)3), 0.87 (t, 6H, J(H,H) =
6.67 Hz, CH3L1). Anal. Calc. for C70H86N6O4Zn: C, 73.69; H, 7.60;
N, 7.37. Found: C, 73.44; H, 7.35; N, 7.42%.
-1
1
=
[(bpy-9)Zn(QT)2], 8. To a solution of [Zn(QT)2(S)2] (0,150 g,
0.223 mmol) in 30 ml of CHCl3 was added an equivalent of bpy-
9 (0.091 g, 0.223 mmol). The resulting colourless solution was
stirred at room temperature for five days. Then it was evaporated
to dryness under reduced pressure on a rotavapor. To a residue was
added n-hexane (20 ml), from which a crystalline precipitate slowly
formed at room temperature (0,227 g, 50% yield). Mp 121 ◦C. IR
Cell lines and cytotoxic assay
Human prostate cancer cell lines (DU145 and LNCaP) were grown
in RPMI-1640 (Gibco) supplemented with 10% of fetal bovine
serum (GIBCO), 5% of L-glutamine (GIBCO) and antibiotics,
under standard conditions (37 ◦C temperature, 5% CO2 in a
humidified atmosphere). All the experiments have been performed
at the earlier passage of the cell lines. For cytotoxic assays (MTS)
and IC50 evaluation, cells were plated in 96-well plates (Falcon,
-1
1
=
(nmax/cm : 2926–2856 (CH), 1635 (C O). H NMR (300 MHz,
CDCl3): dH 8.78 (2H, d J(H,H) = 5.28 Hz H3,3¢), 7.93 (6H, d,
J(H,H) = 8.41 Hz, H1,1¢; He,e¢; Hi,i¢), 7.30 (2H, d, J(H,H) = 5.28 Hz,
H
2,2¢), 7.19 (4H, t, J(H,H) = 7.92 Hf,f¢; Hh,h¢), 7.00 (2H, t, J(H,H) =
7.14 Hg,g¢), 2.76 (4H, t, J(H,H) = 7.53 Hz, CH2(CH2)7CH3), 2.42
(4H, s, CH2C(CH3)3), 2.38 (6H, s, CH3QT), 1.70 (4H, m, CH2CH3),
4210 | Dalton Trans., 2010, 39, 4205–4212
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CA) in 100 ml of culture medium. For each experiment precursor
or complexes were serially diluted in cell culture medium to the
desired concentrations and an equal volume of the diluted solution
(100 ml/well) was added to the cells which were continuously
exposed to the compounds for 72 h. Each treatment was performed
in triplicate in three independent experiments. Cell viability was
determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt (MTS) assay using CellTiter 96 AQueous One Solution
Proliferation Assay System (Promega). This assay measures the
bioreduction by intracellular dehydrogenases of the tetrazolium
compound MTS in the presence of the electron coupling reagent
phenazine methosulfate. The plates were incubated 2 h at 37 ◦C and
then the absorbance at 490 nm was measured using Sirio-S (SEAC,
Radim Group), results are expressed as mean standard deviation
(SD) of the percentage of viable cells at each drug concentration
compared to the untreated cells. Then IC50 values were calculated
by using the GraFit32 program.
PC3 cells were maintained in RPMI 1640 medium enriched with
10% of FBS, 1% penicillin/streptomycin and 1% glutamine and
were cultured at 37 ◦C in a moist atmosphere of 5% carbon dioxide
in air. Sub-confluent cell cultures, synchronized for 24 h in DMEM
without phenol red and serum (PRF-SFM-DMEM), were used
for all experiments. The effect of all species on proliferation of the
human cancer cells PC3 was measured with the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay.59
Briefly, 80 000 viable cells were plated in a 12-well format and
became attached to the bottom of the well overnight. On the
second day of the procedure, the original medium was removed
and 1 ml new phenol red and fetal calf serum-free medium
containing the test substances was added. After an incubation
period of 72 h, the living cells were assayed by the addition
of 50 ul 5 mg ml-1 MTT solution. MTT was converted by
intact mitochondrial reductase and precipitated as blue crystals
during a 4 h contact period. The medium was then removed and
the precipitated crystals were dissolved in isopropanol–0.04 M
HCl. Finally, the reduced MTT was monitored at 570 nm using
a spectrophotometer with untreated cells being taken as the
control.
Acknowledgements
Financial support received from the Ministero dell’Istruzione,
dell’Universita` e della Ricerca (MIUR) through the Centro di
Eccellenza CEMIF.CAL (CLAB01TYEF) and from the Italian
PRIN founding n. 2006038447 is gratefully acknowledged.
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