SCHOOL OF ADVANCED STUDIES
Doctorate course in
Chemical Sciences
PhD thesis
Summary
New scorpionate ligands
and relative metal complexes with
biomimetic and cytotoxic activity
cycle XIX
scientific-sector CHIM/03
PhD candidate
Dr. Simone Alidori
Tutor
Prof. Carlo Santini
2003/04 – 2005/06
Summary
It is known that copper is an essential trace metal for living organisms. This metal
plays a crucial role in different enzymes (i.e., cytochrome-c oxidase, superoxide
dismutase, ceruloplasmin, etc.) that catalyze oxidation/reduction reactions correlated
with the antioxidant system of the organism. It has been reported that certain copper
complexes catalyze radical formation, while others seem to have efficacy as
antioxidants. The different behaviours depend upon the chemical environment and
nature of the chelating agent. In this field, our attention has been focused on copper(I)
complexes containing "scorpionate" ligands. Poly(pyrazolyl)borates and related
scorpionates are potentially tridentate ligands extensively employed as anionic donor chelates in a variety of metal complexes.
New copper(I) complexes have been synthesized from the reaction of CuCl with 4- or
2-(diphenylphosphane)benzoic acid and several “scorpionate” ligands (Fig. 1), such
as KH2B(btz)2, KHB(btz)3, NaTpMe, KpzTp, KpzTpMe and KH2B(im)2(dmac). The
complexes obtained have been characterized by elemental analyses and FT-IR in the
solid state, and by NMR (1H and
solution.
31
P{1H}) and electrospray mass spectroscopy in
Fig. 1. Structure of the P- and N-donor ligands: (a) PPh2(p-C6H4COOH); (b) PPh2(o-C6H4COOH);
(c) [H2B(btz)2]−; (d) [HB(btz)3]−; (e) TpMe (= [HB(3-Mepz)3]−); (f) pzTp (= [B(pz)4]−); (g) pzTpMe
(= [B(3-MePz)4]−); (h) [H2B(im)2]−(dmac).
Chemiluminescence technique was used to evaluate the superoxide scavenging
activity of these new copper complexes. Our results (Fig. 2) show that these new
copper(I) complexes have “in vitro” an antioxidant activity towards superoxide anion.
These preliminary data need further investigations in order to better understand the
antioxidant capacity of these complexes and their potential use in mitigating the
deleterious effects exerted by reactive oxygen species to biological systems.
Fig. 2. Inhibition (%) of lucigenin-amplified chemiluminescence of the xanthine/xanthine oxidase
system in the presence of superoxide dismutase, Tempo and the copper(I) complexes.
Chemiluminescence was measured in the presence of 0.9U/ml xanthine oxidase and 150 μM
lucigenin; the reaction was initiated by injecting xanthine at a final concentration of 50 μM in Tris
50 mM, pH 7.4 buffer. Inhibition values refer to peak heights. (♦), (▪), ( ), ( ), (*), (•) and ( )
copper(I) complexes; ( ) Tempo; ( ) SOD.
Other copper(I) complexes have been synthesised from the reaction of CuCl with
potassium hydrotris(4-bromo-1H-pyrazol-1-yl)borate, KTp4Br, or lithium bis(3,5dimethylpyrazol-1-yl)acetate,
Li[L2CO2],
ligands
and
4-
or
2-
(diphenylphosphane)benzoic acid or tris(m-sulfonatophenyl)posphine trisodium salt
(TPPTS) coligands. The complexes obtained have been characterized by elemental
analyses and FT-IR in the solid state, and by NMR (1H and 31P{1H}) and electrospray
mass spectrometry (ESI-MS) in solution. Single crystal structural characterisation
was undertaken for the {Cu[PPh2(4-C6H4COOH)](Tp4Br)} derivative, an interesting
dimeric supramolecular assembly (Fig. 3).
Fig. 3 Supramolecular assemblies of hydrogen-bonded carboxylic acid dimers mediated by a
intermolecular
dihydrogen
interaction
(B-H…H-C)
of
{Cu[PPh2(4-
C6H4COOH)](Tp4Br)}.(C6H14)0.5.
A chemiluminescence study has demonstrated the superoxide scavenging activity of
these new copper complexes. The Comet assay was used to evaluate the impairment
of DNA in rat epithelial cells exposed to different reactive nitrogen species. In
addition, the same complexes were included in this study to determine their efficacy
as antioxidants in mitigating oxidative DNA damage. The parameter tail moment
(Fig. 4), used as an index of DNA damage, showed that the complex {Cu[PPh2(4C6H4COOH)](Tp4Br)} remarkably inhibited DNA strand breaks induced by the
different nitrogen oxide species. The other copper complexes under study showed a
different ability to reduce tail moment values depending on the type of RNOS donor
3
2.5
2
1.5
1
*
**
0.5
**
**
co
m
+1
0u
M
AS
AS
+5
uM
co
m
pl
pl
ex
ex
1
1
at
e)
4]
u(
I I)
2(
as
pi
rin
[C
+1
0u
M
AS
AS
+5
uM
[C
AS
u(
II)
2(
as
pi
rin
at
e)
4]
0
co
nt
ro
l
Tail Moment (arbitrary unit)
used.
Fig. 4. Effects of {Cu[PPh2(4-C6H4COOH)](Tp4Br)} and of Cu2(aspirinate)4 complex on DNA of rat
trachea epithelial cells incubated in the presence of Angeli's salt (Na2N2O3). The tail moment
parameter is reported using the Comet assay after 20 min incubation at 37 °C in the presence or
absence of 200 µM of the NO–-donor (Angeli's salt = AS) and in the presence or absence of 5 and
10 µM complexes. Data, reported as mean values ± error standard, refer to at least three individual
experiments, three slides experiment–1. *p < 0.05; **p < 0.01.
The new dihydrobis(3-carboxyethyl-5-methylpyrazolyl)borate ligand BpCOOET,Me has
been prepared as its potassium salt in kerosene solution, using ethyl 3methylpyrazole-5-carboxylate and KBH4, through careful temperature control.
BpCOOET,Me reacts with CuCl and PPh3 to yield the Cu(BpCOOET,Me)PPh3 complex
(scheme 1).
Scheme 1
BpCOOET,Me and Cu(BpCOOET,Me)PPh3 have been fully characterized by elemental
analyses and FT-IR in the solid state and by NMR (1H,
13
C and
31
P{1H})
spectroscopy and electrospray ionization mass spectrometry in solution. A single
crystal structural characterization is reported for Cu(Bp COOET,Me)PPh3 (Fig. 5).
Fig. 5. A single molecule of Cu(BpCOOET,Me)PPh3 showing 50% probability amplitude displacement
ellipsoids for the non-hydrogen atoms, hydrogen atoms having arbitrary radii of 0.1 Å. Cu–P(1)
2.1718(6), Cu–N(11) 2.026(2), Cu–N(21) 2.007(2), Cu–O(151) 3.288(2), Cu–O(252) 3.115(2) Å.
N(11)–Cu–N(21) 96.77(7), P(1)–Cu–N(11) 124.86(6), P(1)–Cu–N(21) 137.42(5) (∑ 359.0°).
The new dihydridobis(3-nitro-1,2,4-triazolyl)borate ligand, [H2B(tzNO2)2]-, has been
synthesized in dimethylacetamide solution, using 3-nitro-1,2,4-triazole and KBH4
through careful temperature control, and characterized as its potassium salt. The
zinc(II) and cadmium(II) complexes, {M[H2B(tzNO2)2]Cl(H2O)2}, have been prepared
by metathesis of [H2B(tzNO2)2]K with ZnCl2 and CdCl2, respectively. The complexes
likely contain a metal core in which the ligand is coordinated to the metal ions in the
2
-N,N' or
4
-N,N',O,O' fashion. A single-crystal structural characterization is
reported for the potassium dihydrobis(3-nitro-1,2,4-triazolyl)borate. The potassium
salt is polymeric and shows several K···N and K···O interactions (Fig. 6).
Fig. 6. Structure of the anion [H2B(tzNO2)2]-, bonded to the potassium ions.
New copper(I) complexes of the type [H2B(tzNO2)2]Cu[PR3]2, [H2B(tzNO2)2]Cu[dppe]
and [H2B(tzNO2)2]Cu[PR3] have been synthesized from the reaction of CuCl,
potassium dihydrobis(3-nitro-1,2,4-triazol-1-yl)borate, K[H2B (tzNO2)2] (Fig. 6), and
mono- or bi-dentate tertiary phosphanes. The complexes obtained have been
characterized by elemental analyses and FT-IR in the solid state, and by NMR (1H
and
31
P{1H}) spectroscopy in solution. Selected complexes ([H2B(tzNO2)2]Cu[P(m-
tolyl)3]2
[H2B(tzNO2)2]Cu[P(C6H5)2(p-C6H4COOH)]2
and
[H2B(tzNO2)2]Cu[P(p-
C6H4F)3]2) have also been tested against a panel of several human tumor cell lines in
order to evaluate their cytotoxic activity.
Complexes [H2B(tzNO2)2]Cu[P(m-tolyl)3]2 and [H2B(tzNO2)2]Cu[P(p-C6H4F)3]2 showed
IC50 values appreciably lower than those exhibited by cisplatin, the most used metalbased antitumor drug (Table 1). It is worth noting that all three tested Cu(I)
complexes appear to be particularly effective against A549 carcinoma cells that are
resistant to cisplatin treatment.
Table 1. Cytotoxic activity
Compound
IC50 (μM) ± SD
2008
HL60
A431
A549
A375
[H2B(tzNO2)2]Cu[P(m-tolyl)3]2
10.11 ± 2.4
4.81 ± 0.9
6.7 ± 1.2
1.52 ± 0.7
16.4 ± 0.9
[H2B(tzNO2)2]Cu[P(C6H5)2(p-C6H4COOH)]2
14.31 ± 1.9
25.13 ± 1.2
15.25 ± 1.7
6.88 ± 2.0
35.15 ± 2.4
[H2B(tzNO2)2] Cu[P(p-C6H4F)3]2
5.60 ± 0.5
4.69 ± 1.2
3.66 ± 1.1
2.35 ± 0.9
4.33 ± 1.3
K[H2B(tzNO2)2]
ND
ND
ND
ND
ND
P(p-C6H4F)3
77.09 ± 1.9
65.23 ± 2.0
88.28 ± 2.9
68.12 ± 3.1
69.23 ± 1.0
P[(C6H5)2(p-C6H5COOH)]
ND
ND
ND
ND
ND
P(m-tolyl)3
60.4 ± 2.7
58.6 ± 3.1
66.9 ± 2.9
71.6 ± 2.8
55.3 ± 2.9
Cisplatin
12.69 ± 1.0
19.9 ± 2.5
22.06 ± 1.6
39.27 ± 1.9
20.28 ± 1.3
ND, not detectable.
SD, standard deviation.
IC50 values were calculated by probit analysis (P<0.05, χ2 test). Cells (3–8×103 mL−1) were treated
for 48 h with increasing concentrations of tested compounds. Cytotoxicity was assessed by MTT
test.
New silver(I) complexes have been synthesised from the reaction of AgNO 3,
monodentate PR3 (PR3 = P(o-tolyl)3, P(m-tolyl)3, P(p-tolyl)3, P(p-C6H4F),
SeP(C6H5)3) or bidentate tertiary (dppe = bis(diphenylphosphane)ethane, dppf = 1,1′bis(diphenylphosphane)ferrocene) phosphanes and potassium dihydrobis(3-nitro1,2,4-triazolyl)borate, K[H2B(tzNO2)2] (Fig. 6). These compounds have been
characterized by elemental analyses, FT-IR, ESI-MS and multinuclear (1H and
31
P{1H}) NMR spectral data. The adduct {[H2B(tzNO2)2]Ag[P(m-tolyl)3]2} has been
characterized by single crystal X-ray studies (Fig. 7). In the former, the H2B(tzNO2)2
acts as a monodentate ligand utilizing the coordinating capability of only one of the
additional (exo-) ring nitrogens to complete the coordination array about the silver
atom.
Fig. 7. The molecular structure of complex {[H2B(tzNO2)2]Ag[P(m-tolyl)3]2}.
New silver(I) complexes have been synthesised from the reaction of AgNO 3,
monodentate tertiary phosphanes PR3 (PR3 = P(C6H5)3, P(o-C6H4CH3)3, P(mC6H4CH3)3, P(p-C6H4CH3)3, PCH3(C6H5)2) and potassium dihydrobis(3-nitropyrazol1-yl)borate (Fig. 8). These compounds have been characterized by elemental
analyses, FT-IR, ESI-MS and multinuclear (1H and
31
P{1H}) NMR spectroscopy.
Solid state structures of the potassium salt K[H2B(pzNO2)2] and the silver adducts
[H2B(pzNO2)2]Ag[P(C6H5)3]2 and [H2B(pzNO2)2]Ag[P(p-C6H4CH3)3] have been also
reported. The K[H2B(pzNO2)2] forms a polymeric network due to intermolecular
contacts of various types between the potassium ion and atoms of the neighbouring
molecules (Fig. 8a and 8b).
Fig. 8a
Fig. 8b
Fig. 8a. Basic structural unit of K[H2B(pzNO2)2] showing the atom numbering scheme. Fig. 8b. A
view of K[H2B(pzNO2)2] showing the bis(pyrazolyl)borato ligand, and the atoms closest to the
potassium center that form inter- and intra-molecular contacts leading to a polymeric network.
[H2B(pzNO2)2]Ag[P(C6H5)3]2 (Fig. 9) and [H2B(pzNO2)2]Ag[P(p-C6H4CH3)3] (Fig. 10)
have pseudo tetrahedral and trigonal planar silver sites, respectively.
Fig. 9. Molecular structure of [H2B(pzNO2)2]Ag[P(C6H5)3]2. All the hydrogen atoms except those on
boron have been omitted for clarity.
Fig. 10. Molecular structure of [H2B(pzNO2)2]Ag[P(p-C6H4CH3)3].
The bis(pyrazolyl)borate ligand acts as a 2-N2 donor. The nitro-substituents are
coplanar with the pyrazolyl rings in all these adducts indicating efficient electron
delocalization between the two units.
The new sodium bis(1,2,4-triazol-1-yl)acetate ligand, Na[HC(CO2)(tz)2], has been
prepared in methanol solution by using 1,2,4-triazole, dibromoacetic acid, and NaOH.
Treatment of the [Cu(CH3CN)4][PF6] acceptor with Na[HC(CO2)(tz)2] or
Na[HC(CO2)[(pzMe2)2], in the presence of the tris(hydroxymethyl)phosphine coligand
in methanol/acetonitrile solutions produced unprecedented mononuclear copper(I)
complexes
of the
type
[Ln]Cu[P(CH2OH)3]2
(L1,
[HC(CO2)(tz)2], 2;
L2,
[HC(CO2)(pzMe2)2], 3) and [(CH3CN)2Cu(P(CH2OH)3)2]PF6, 4.
Fig. 11. Proposed structure of complexes 2-4.
The new copper(I) complexes were tested for their cytotoxic properties against a
panel of several human tumor cell lines. The results reported here indicate that all the
complexes showed in vitro antitumor activity similar or better than that of cisplatin,
the
most
used
metal-based
antitumor
drug.
In
particular,
[HC(CO2)(pzMe2)2]Cu[P(CH2OH)3]2, showed IC50 values markedly lower than the
reference compound against all tumor cell lines (Table 2).
Table 2. Cytotoxic Activity
Compound
IC50 (μM) ± SD
HL60
[HC(CO2)(tz)2]Cu[P(CH2OH)3]2
A549
MCF-7
A375
LoVo
34.31 ± 3.9 25.13 ± 1.1 11.29 ± 1.7 26.66 ± 2.0 32.28 ± 3.6
[HC(CO2)(pzMe2)2]Cu[P(CH2OH)3]2 10.60 ± 2.5 2.10 ± 1.3 1.55 ± 0.19 2.55 ± 0.9 7.83 ± 1.3
[(CH3CN)2Cu(P(CH2OH)3]2]PF6
21.15 ± 2.8 14.81 ± 0.9 16.7 ± 2.7 21.52 ± 1.7 47.17 ± 2.9
Na[HC(CO2)(tz)2]
ND
P(CH2OH)3
58.67 ± 2.3 72.97 ± 2.4 64.21 ± 4.2 88.71 ± 3.8 65.21 ± 3.2
Na[HC(CO2)(pzMe2)2]
ND
cisplatin
19.9 ± 2.5 39.27 ± 1.9 30.18 ± 1.5 20.28 ± 1.3 24.97 ± 1.5
ND
ND
ND
ND
ND
88.81 ± 3.9 ND
ND
Chemosensitivity tests performed on cisplatin sensitive and resistant cell lines have
demonstrated that all these Cu(I) complexes were able to overcome cisplatin
resistance, supporting the hypothesis of a different mechanism of action compared to
that exhibited by the reference drug. Flow cytometric analysis on 2008 human
ovarian
carcinoma
cells
revealed
that
the
complex
[HC(CO2)(pzMe2)2]Cu[P(CH2OH)3]2, chosen as the best candidate, induced a marked
enlargement of both cell size and granularity, and a significant increase in the fraction
of G2/M cells that, differently from cisplatin, was not accompanied by the
appearance of a relevant sub-G1 fraction (Fig. 12).
Fig. 12. Cell cycle analysis of 2008 human ovarian cancer cells. Panels A-C represent different time
points (18, 24, and 48 h, respectively) of control cells (left) and cells treated with IC 50
concentrations of [HC(CO2)(pzMe2)2]Cu[P(CH2OH)3]2 complex (right). Panel D represents control cells
after 18 h in normal medium (left) and cells treated with IC50 concentrations of cisplatin (right).
Besides, no evidence of caspase-3 activation was detected in cells treated with
[HC(CO2)(pzMe2)2]Cu[P(CH2OH)3]2 complex (Fig. 13). We hypothesize that the
cytotoxic activity of the new copper(I) complex may be correlated to its ability to
trigger paraptosis, a nonapoptotic mechanism of cell death.
Fig. 13. Induction of caspase-3. The 2008 cells were incubated with IC50 concentrations of
[HC(CO2)(pzMe2)2]Cu[P(CH2OH)3]2
complex or cisplatin and then submitted to the test on caspase-3
induction as described in Experimental Section. Data are the means of at least three independent
experiments. Error bars indicate standard deviation. *P < 0.01 compared to untreated cells.
The new sodium bis(2-pyridylthio)acetate ligand, Na[(pyS)2CHCO2], has been
prepared in ethanol solution using 2-mercaptopyridine, dibromoacetic acid and
NaOH.
New
tri-organotin(IV)
derivatives
containing
the
anionic
bis(2-
pyridylthio)acetate have been synthesized from reaction between SnR3Cl (R = Me,
n
Bu, Ph and Cy) acceptors and Na[(pyS)2CHCO2]. Complexes of the type
{[(pyS)2CHCO2]SnR3} have been obtained and characterized by elemental analyses,
FT-IR, ESI-MS, multinuclear (1H and
119
Sn) NMR spectral data and X-ray
crystallography. Crystallisation of the compound {[(pyS)2CHCO2]Sn(CH3)3}n, from
chloroform/diethyl
ether
solution,
yielded
the
complex
{[(pyS)2CHCO2]Sn(CH3)3}n·CHCl3, which has been characterized by X-ray
crystallography (Fig. 14 and Fig. 15).
Fig. 14. Molecular structure of two units of the polymer {[(pyS)2CHCO2]Sn(CH3)3}n·CHCl3.
Fig. 15. Projection on the ac plane of the polymeric complex {[(pyS)2CHCO2]Sn(CH3)3}n·CHCl3.