Novel C,N-chelate platinum(II) antitumor complexes bearing a lipophilic ethisterone pendant

Article abstract of DOI:10.1016/j.jinorgbio.2010.12.005

The novel steroidal carrier ligand 17-α-[4′-ethynyl- dimethylbenzylamine]-17-β-testosterone (ET-dmba 1) and the steroid - C,N-chelate platinum(II) derivatives [Pt(ET-dmba)Cl(L)] (L = DMSO (2) and PTA (3; PTA = 1,3,5-triaza-7-phosphaadamantane)) have been

Full text of DOI:10.1016/j.jinorgbio.2010.12.005

Journal of Inorganic Biochemistry 105 (2011) 525–531
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Novel C,N-chelate platinum(II) antitumor complexes bearing a lipophilic
ethisterone pendant
a
a
b
c,
José Ruiz a, , Venancio Rodríguez , Natalia Cutillas , Arturo Espinosa , Michael J. Hannon
⁎
⁎
a
Departamento de Química Inorgánica, Universidad de Murcia, 30071-Murcia, Spain
Departamento de Química Orgánica, Universidad de Murcia, 30071-Murcia, Spain
School of Chemistry, University of Birmingham, Edgbaston, UK B15 2TT
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 October 2010
Received in revised form 9 December 2010
Accepted 14 December 2010
Available online 29 December 2010
The novel steroidal carrier ligand 17-α-[4′-ethynyl-dimethylbenzylamine]-17-β-testosterone (ET-dmba 1)
and the steroid — C,N-chelate platinum(II) derivatives [Pt(ET-dmba)Cl(L)] (L = DMSO (2) and PTA (3; PTA =
1,3,5-triaza-7-phosphaadamantane)) have been prepared. Values of IC50 were calculated for the new
platinum complexes 2 and 3 against a panel of human tumor cell lines representative of ovarian (A2780 and
A2780cisR) and breast cancers (T47D). At 48 h incubation time complexes 2 and 3 show very low resistance
factors (RF of b2) against an A2780 cell line which has acquired resistant to cisplatin and were more active
than cisplatin (about 4-fold for 3) in T47D (AR+, AR=androgen receptor). Compound 1 retains a moderate
degree of relative binding affinity (RBA=0.94%) for androgen receptors. The cytotoxicity of the non steroidal
platinum analogues [Pt(dmba)Cl(L)] (dmba=dimethylbenzylamine; L=DMSO (4) and PTA (5)) has also
been studied for comparison purposes. Theoretical calculations at the BP86/def2-TZVP level of theory on
complex 3 have been undertaken.
Keywords:
Platinum complexes
Cytotoxicity
Lipophilicity
Steroidal derivative
DNA
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
the steroid is released from the metal on reduction and prior to DNA
binding [18].
Platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin
are widely used against various solid tumors including testicular,
ovarian, colorectal, and non-small cell lung cancers [1–5] with annual
sales over 2 billion US dollars [6,7]. Only a tiny fraction of the platinum
complex administered reaches the DNA of the rapidly dividing cancer
cell. Most of the drug binds to non-DNA biological nucleophiles resulting
in toxic effects [8].
Recently, new strategies have been developed to try to decrease
these side effects or to increase the activity spectrum through more
effective delivery of the drug to the desired targets [9–11]. Bio-
molecules are an attractive choice as delivery vectors and different
molecules have been explored with varying degrees of success. Sex
hormones such as estrogens and androgens are of particular interest
because of their importance in reproductive system cancers [12].
Natural and synthetic estrogens have been attached to a range of
different organometallic and coordination units with the aim of
targeting the estrogen receptors (ER) [13–15]. Thus Jaouen developed
exciting new drugs based on tamoxifen-linked ferrocenes [16,17] and
Lippard reported some platinum(IV) estrogen compounds, in which
In this context, we have recently reported the preparation of
some new platinum(II) complexes with, as organometallic tag, the
N,C-chelating dimethylbenzylamine (dmba), that display an interesting
cytotoxicity against the human acute promyelocytic leukaemia cell line
HL-60 [19] and breast cancers (T47D) [20,21].
We report herein the synthesis of the novel 17-α-[4′-ethynyl-
dimethylbenzylamine]-17-β-testosterone molecule 1 (ET-dmbaH)
and two new steroids — C,N-chelate platinum conjugates of the
type [Pt(ET-dmba)Cl(L)] [L=DMSO or PTA (PTA=1,3,5-triaza-7-
phosphaadamantane)]. Values of IC50 were studied for the new
platinum complexes against a panel of human tumor cell lines
representative of ovarian (A2780 and A2780cisR), and breast cancers
(T47D, cisplatin resistant and AR+, AR = androgen receptor). The
DNA adduct formation of the new platinum complexes has also been
studied. The degree of relative binding affinity (RBA) for androgen
receptors for 1 is also reported.
2. Results and discussion
2.1. Molecular design and synthesis of the modified testosterone ligand
Our design focused on attaching the DNA-binding unit to the
17-α-position of testosterone. Linking the steroid and metal complex
via an alkyne linker is attractive due to its synthetic feasibility and
because, as a spacer unit, it introduces distance without steric bulk. As
⁎
Corresponding authors. Departamento de Química Inorgánica, Universidad de
Murcia, Campus de Espinardo, 30071, Murcia, Spain. Tel.: +34 868 88 74 55; fax: +34
868 88 41 48.
E-mail addresses: jruiz@um.es (J. Ruiz), m.j.hannon@bham.ac.uk (M.J. Hannon).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.12.005

526
J. Ruiz et al. / Journal of Inorganic Biochemistry 105 (2011) 525–531
tography on silica gel. 1H and 13C NMR, electrospray ionization (ESI)
OH
Br
CH3
mass (+ mode) analysis spectrometry (M+H+ =446.2), and
elemental analysis were used to characterize 1. COSY (correlation
spectroscopy), NOESY (nuclear Overhauser effect spectroscopy) and
HSQC (heteronuclear single quantum coherence) were used for the
assignment. In the 1H NMR the resonances that are easily observed are
the corresponding to the aromatic region of the dmba fragment and also
corresponding to the Ha, which appear at δ 5.6 ppm. They are in the
appropriate integral ratios. The CH2N and NMe2 of dmba are also clearly
observed. Furthermore, the ethynyl proton (δ 2.6 ppm) from the
starting steroid is absent.
C
CH
CH3
+
CH2N(CH3)2
O
THF,65°C,72h
PdCl2(PPh3)2,CuI
2.2. Design and synthesis of the new platinum compounds
5'
3'
6'
2'
OH
CH3
The new cyclometallated platinum(II) monomer 2 was synthe-
sized through an adaptation of the van Koten route [22], by heating at
50 °C for 24 h PtCl2(DMSO)2 with 1 equiv of the cyclometalating
ligand 1 in the presence of NaOAc (Scheme 2). Complex 3 was readily
prepared by metathesis reaction of 2 with 1 equiv of PTA. The
structures were assigned on the basis of microanalytical, IR, and 1H,
13C, 31P and 195Pt NMR data and ESI mass spectra. The 1H NMR
spectrum in CDCl3 of complex 2 shows that both the N-methyl and the
CH2 groups of the dmba are diastererotopic because there is no plane
of symmetry in the platinum coordination plane (the assignments
were unambiguously confirmed again by COSY, NOESY and HSQC).
The 1H resonances assigned to the methyl groups of the DMSO and the
dmba ligands for 2 are flanked by 195Pt satellites, which are also
observed in several other resonances of 2 and 3 (see Experimental
section). On the other hand, the ESI mass spectra assignments are
based not only on the m/z values but also by the observation of the
expected isotope distribution pattern. The previously reported [22]
C
C
CH2N(CH3)2
CH3
1
O
Ha
Scheme 1. Synthesis of the modified testosterone ligand 1.
shown in Scheme 1, the use of the commercially available ethisterone
(17-α-ethynyl-testosterone or [(17-α-ethynyl-17-β-hydroxy-androst-
4-en-3-one] and (4-bromobenzyl)dimethylamine as starting materials
allowed us to prepare in very good yield the functionalized 1 in a
palladium-catalyzed Sonogashira coupling. 1 is an air and moisture
stable white compound. Purification (primarily removal of phosphine
contaminants from the catalyst) was achieved by flash chroma-
PtCl2(DMSO)2
ET-dmbaH (1)
dmbaH
MeOH, 50°C, 24 h
NaOAc
EtOH, 65°C, 3 h
NaOAc
5' 6'
5' 6'
H
H
H
OH
H
CH3
4'
C
C
CH3
N
CH3
CH3
3'
N
CH3
3'
CH3
Pt
Pt
O
O
S
S
Cl
Cl
O
4
CH3
H3C
2
CH3
H3C
PTA
PTA
C
H
H
OH
H
H
CH3
C
CH3
CH3
CH3
CH3
CH3
N
N
Pt
Pt
P
P
Cl
Cl
O
N
N
N
N
5
3
N
N
Scheme 2. Synthesis of the new platinum compounds.

J. Ruiz et al. / Journal of Inorganic Biochemistry 105 (2011) 525–531
527
Table 1
non steroid DMSO platinum complex 4 and the new PTA platinum
IC50(μM) and resistance factors for cisplatin and compounds 1–5.
complex 5 (analogous to the PTA palladium complex previously
reported by our group) [23] have also been prepared (Scheme 2) in
order to compare their biological properties.
Compound
T47D
48 h
A2780
48 h
A2780cisR
48 h (RF)a
1
N100
N100
N100
2.3. Theoretical calculations on complex 3
2
3
4
5
16.0
2
9.52 0.22
6.51 0.18
1.17 0.02
2.55 0.11
1.01 0.03
8.22 0.19 (0.9)
9.62 0.19 (1.5)
1.64 0.02 (1.4)
6.36 0.32 (2.5)
18 1 (17.8)
9.9 0.1
3.1 0.2
9.2 0.8
In order to obtain some insight into the structural and reactivity
features of complex 3 we have calculated its structure (Fig. 1) at the
BP86/def2-TZVP level of theory (see Computational Details). The most
eye-catching feature is that in the most stable conformation the
lipophilic steroidal pendant arm lies roughly orthogonal to the metal
coordination plane. The Pt atom is located in an almost regular
square-planar environment (dihedral N−C−P−Cl 6.0°) formed by
four different donor atoms (Do). Assessment of Pt−Do bond
strengths has been achieved within the framework of Bader's
atoms-in-molecules (AIM) theory [24] by computing the electron
density at the respective bond critical points (BCP). According to this
criterion, the strongest bonds to platinum are formed by the dmba
C-atom (dPt−C =2.021 Å; ρ(rc)=14.94·10-2 e/ao3) and the PTA P-atom
(dPt-P =2.219 Å; ρ(rc)=13.19·10-2 e/ao3), whereas the bonds to dmba
N-atom (dPt-N =2.205 Å; ρ(rc)=8.81·10-2 e/ao3) and to Cl ligand
(dPt-Cl =2.414 Å; ρ(rc)=7.91·10-2 e/ao3) are considerably weaker.
Indeed, the latter is the only ligand around Pt bearing a remarkable
negative charge (qN =-0.526 au) in contrast to the low value of the
other formally anionic ligand ET-dmba (qN =–0.136 au) and the
positive charge at the PTA moiety (qN =0.534 au). Very similar values
were obtained on using Mulliken charges (see Table S1 Supplemen-
tary material). These facts are consistent with the observed reactivity
towards N-nucleophiles (see below) that promote chloride substitu-
tion at Pt.
Cisplatin
43
2
a
The numbers in parentheses are the resistance factors RF (IC50 resistant/IC50
sensitive).
than the corresponding steroidal derivative. On the other hand, A2780
is an AR−, ERα−and ERβ+cell line, and A2780cisR encompasses all
of the known major mechanisms of resistance to cisplatin: reduced
drug transport [25], enhanced DNA repair/tolerance [26] and elevated
GSH levels [27]. The ability of complexes 2−5 to circumvent cisplatin
acquired resistance was determined from the resistance factor (RF),
defined as the ratio of IC50 resistant line to IC50 parent line. An RF of b2
was considered to denote noncross-resistance [28]. Especially
noteworthy are the very low resistance factors (RF) of 2−4 at 48 h
(RF=0.9−1.5) (Table 2), an improvement of RF values being
achieved for the steroidal derivatives in comparison with the non-
steroidal analogues. The new testosterone platinum derivatives show
no difference in cytotoxicity between AR+(T-47D) or AR−(A2780)
cell lines. The IC50 value for 1 was higher than 100 in all the cancer cell
lines studied. The stability of the cytotoxic compounds 2−5 has been
studied in DMSO by 1 H NMR. Complexes 2−5 remained unaltered
after three days in solution at room temperature, although H/D
exchange was observed for the coordinated DMSO resonances of
complexes 2 and 4.
2.4. Biological activity. Cytotoxicity studies
2.5. Biological assays. Androgen receptor whole cell binding assays
To analyze the potential of the compounds 2−5 as antitumor
agents, their cytotoxicity was evaluated (Table 1) towards the human
breast cancer (T47D, cisplatin resistant) and epithelial ovarian
carcinoma cells A2780 and A2780cisR (acquired resistance to
cisplatin) and for comparison purposes the cytotoxicity of cisplatin
and the free ligand 1 was also evaluated under the same experimental
conditions. Because of low aqueous solubility, the test compounds
were dissolved in DMSO first and then serially diluted in complete
culture medium such that the effective DMSO content did not exceed
1%. Complexes 2 and 3 were more active than cisplatin (about 4-fold
for 3) in T47D (AR+, ERα+, ERβ+, AR=androgen receptor; ERα/
β=estrogen receptor α or β; +/− indicates receptor status,
present or absent), the non-steroidal compound 4 being more active
The relative binding affinity (RBA) of the steroidal ligand 1 for ARs
in viable human prostate cancer cells LNaCaP was determined by a
competitive radiometric binding assay. The assay involves competi-
tion of the steroidal conjugate 1 with [3 H]-methyltrienolone in
combination with nonradioactive dihydrotestosterone (DHT) en-
abling the IC50 displacement measurement for 1 and its Ki value for
the receptor-drug interaction to be determined (Table 2). Low IC50
values thus correlate with high binding affinities. From these results it
may be concluded that upon substituting the androgen at the 17 α-
position with the dmba derivative: receptor binding is retained (albeit
at a lower relative binding affinity relative to DHT, approximately 1%
for the free ligand).
2.6. Gel electrophoresis of compound–pBR322 complexes
The influence of the compounds on the tertiary structure of DNA
was determined by their ability to modify the electrophoretic mobility
of the covalently closed circular (ccc) and open (oc) forms of pBR322
plasmid DNA. The compounds 1−5 were incubated at the molar ratio
ri =0.50 with pBR322 plasmid DNA at 37 º C for 24 h. Representative
gel obtained for the new compounds 1−5 are shown in Fig. 2. The
behavior of the gel electrophoretic mobility of both forms, ccc and oc,
of pBR322 plasmid and DNA:cisplatin adducts is consistent with
Table 2
Relative binding affinity (RBA).
Compound
Ki (nM)
IC50 (nM)
RBAa (%)
(ET-dmbaH) 1
DHT
1549
13.5
4064.2
38.1
0.94
100
a
DHT
compound
Fig. 1. Calculated (BP86/def2-TZVP) structure for complex 3.
RBA=IC
/IC
×100.
50
50

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J. Ruiz et al. / Journal of Inorganic Biochemistry 105 (2011) 525–531
3. Conclusions
A novel steroidal conjugate 17-α-[4′-ethynyl-dimethylbenzyla-
mine]-17-β-testosterone (ET-dmba 1) and two steroids — C,N-chelate
platinum(II) conjugates [Pt(ET-dmba)Cl(L)] (L=DMSO and PTA)
have been prepared. Even though the cytotoxicity of the new steroidal
platinum complexes decreases a little compared with that of the non-
steroidal analogues [Pt(dmba)Cl(L)] (L=DMSO and PTA) prepared
for controls, good activity is retained upon conjugation of the steroid
(the compounds being more active than cisplatin in the breast and
similar in ovarian cancer cell lines). Especially noteworthy are their
very low resistance factors (RF) at 48 h (RF=0.9–1.5) against an
A2780 cell line which has acquired resistance to cisplatin indicating
efficient circumvention of cisplatin resistance. The IC50 value for the
steroidal conjugate 1 was higher than 100 in all the cancer cell lines
studied. The steroidal carrier ligand 1 retains an acceptable degree of
relative binding affinity (RBA=0.94%) for androgen receptors.
Reactions of the new platinum complexes with 9-ethylguanine, as
followed by NMR and ESI-MS, gave the correponding monoadduct [Pt
(C,N)(L)(9-EtG)]+ derivatives. Theoretical calculations at the BP86/
def2-TZVP level of theory on complex 3 shows that the lipophilic
steroidal pendant arm lies roughly orthogonal to the metal coordi-
nation plane in the most stable conformation.
Fig. 2. Electrophoretic mobility pattern of pBR322 plasmid DNA incubated with the
compounds: lane 1, pBR322; lane 2, compound 4; lane 3, compound 5; lane 4,
compound 1; lane 5 compound 2; lane 6, compound 3; lane 7, CDDP.
previous reports [29]. When the pBR322 was incubated with the
platinum compound 2 and 3 (lanes 5 and 6, respectively) a single
band for both forms, ccc and oc, coalescent form, was observed. A
similar pattern has been found previously in some others dmba
phosphine platinum complexes [19–21]. On the other hand, com-
plexes 4 and 5 (lanes 2 and 3) delayed the mobility of the ccc form.
The behavior observed for the electrophoretic mobility for the
platinum complexes 2−5 indicates that some conformational
changes occurred. This means that the degree of superhelicity of the
DNA molecules has been altered.
2.7. Reactions of the platinum complexes 2−5 with 9-ethylguanine
4. Experimental section
Establishing the biological targets of anticancer drugs, as well as of
drug candidates, represents a major challenge in the field of metal-
based drugs. For platinum compounds, the binding to DNA is thought
to be the most important event leading to inhibition of transcription
and replication, and ultimately cell death. To gain insight into the
mode of interactions of the new platinum complexes with DNA, the
reactions of complexes 2−5 with an excess of 9-ethylguanine were
followed by 1H NMR at physiological temperature and pH (see
Experimental section). In all cases the reactions are fast and in less
than 15 min the formation of the corresponding complexes [Pt(C,N)
(L)(9-EtG)]+ (C,N=dmba or ET-dmba; L=DMSO or PTA) is
completed. No further changes were observed after 3 days. In all
cases the signal for the H(8) of the free guanine moves downfield
upon coordination (Fig. 3 for the reaction of complex 5). Two singlets
for H(8) are observed in [Pt(ET-dmba)(L)(9-EtG)]+ (L=DMSO and
PTA) as well as two different sets of signals belonging to the dmba
NMe2 group (see Experimental section and Supplementary material).
The ratio coordinated H8 of EtG/Ha of Et-dmba agrees well with the
formation of the monoadduct with 9-EtG.
4.1. Instrumental measurements
The C, H, N and S analyses were performed with a Carlo Erba model
EA 1108 microanalyzer. Decomposition temperatures were deter-
mined with a SDT 2960 simultaneous DSC-TGA of TA instruments at
a heating rate of 5 °C min−1 and the solid samples under nitrogen
flow (100 ml min−1). The 1H, 13C, 31P, and 195Pt NMR spectra were
recorded on a Bruker AC 300E or Bruker AV 400 spectrometer, using
SiMe4, H3PO4, and Na2[PtCl6] as standards. ESI mass (+mode)
analyses were performed on a LC-MS Agilent VL system or HPLC/MS
TOF 6220.
5. Materials
The starting complexes PtCl2(DMSO)2 [22] and [Pt(dmba)Cl
(DMSO)] [30] were prepared by procedures described elsewhere.
Solvents were dried by the usual methods. Ethisterone (17-α-
ethynyl-testosterone) and pBR322 plasmid DNA were obtained from
Sigma-Aldrich (Madrid, Spain) and Boehringer-Mannheim (Man-
nheim, Germany), respectively.
Further evidence of the formation of [Pt(C,N)(L)(9-EtG)]+ deriva-
tives (C,N=dmba or ET-dmba; L=DMSO or PTA) come from ESI-MS
analysis of the reaction mixtures, the most abundant peak in the mass
spectra corresponding to a signal assignable to the 9-EtG platinum
monoadduct (Fig. 4 for the reaction of complex 2).
5.1. Synthesis of 17-α-[4′-ethynyl-dimethylbenzylamine]-17-β-
testosterone (1)
(4-Bromobenzyl)dimethylamine (0.728 g, 3.4 mmol) and 17-α-
ethynyl-testosterone (1.593 g, 5.1 mmol) were mixed with PdCl2
(PPh3)2 (190.92 mg, 0.272 mmol), CuI (51.80 mg, 0.272 mmol) and
K2CO3 (1.057 g, 7.65 mmol) in a 250 ml Schlenk tube. Dry and
degassed THF (80 ml; THF=tetrahydrofuran) was added. The result-
ing suspension was stirred at 65 °C under nitrogen for 72 h, treated
with charcoal, and then filtered through a short pad of Celite.
Purification was carried out by flash chromatography on silica gel
(l≈30 cm, Ø≈4 cm), eluting with 92:8 CH2Cl2/MeOH. The cor-
responding fractions were pooled and evaporated under reduced
pressure to dryness. The residue was treated with Et2O/hexane to
give a white solid, which was collected by filtration and air-dried
(yield 1.3 g, 85%). Anal. Calcd. For C30H39NO2: C 80.86, H 8.82, N, 3.14.
Found: C 80.74, H 9.01, N 3.11%. Mp: 170 °C. 1H NMR (400 MHz,
DMSO-d6, d = doublet, m = multiplet, s = singlet) δ (ppm): 7.31
(d, 2 H, H3′ and H5′, JH2′H3′ =JH5′H6′ =8.0 Hz), 7.24 (d, 2 H, H2′ and H6′,
Fig. 3. Low-field region of the 1H NMR spectrum of the reaction of complex 5 with
9-ethylguanine after 15 min at 37 °C.

J. Ruiz et al. / Journal of Inorganic Biochemistry 105 (2011) 525–531
529
Fig. 4. ESI mass spectrum of the reaction of the complex 2 with 9-ethylguanine after 15 min at 37 °C.
JH2′H3′ =JH5′H6′ =8.0 Hz), 5.61 (s, 1 H, Ha of testosterone), 5.42 (s, 1 H,
OH of testosterone), 3.36 (s, 2 H, CH2N of dmba), 2.4–0.8 (m, 19 H, CH
and CH2 of testosterone), 2.11 (s, 6 H, NMe2 of dmba), 1.15 (s, 3 H, Me
of testosterone), 0.81 (s, 3 H, Me of testosterone). 13C{1H} NMR
[100.8 MHz, DMSO-d6, δ]: 131.0 (CH3′ and CH5′), 128.9 (CH2′ and
CH6′), 123.1 (CHa of testosterone), 62.9 (CH2 of dmba), 53.1 (CH of
testosterone), 49.8 (CH of testosterone), 44.9 (NMe2 of dmba), 38.2
(CH2 of testosterone), 35.6 (CH of testosterone), 35.0, 33.6, 32.6, 31.9,
31.3, 22.9, 20.4 (CH2 of testosterone), 16.9, 12.9 (Me of testosterone).
ESI+ mass spectra (CH3CN), m/z [Found (Calcd.)]: 446.2 (446.3) [[[ET-
dmbaH)+H]+, 100%].
5.3. Synthesis of [Pt(ET-dmba)Cl(PTA)] (ET-dmba=17-α-[4′-ethynyl-
dimethylbenzylamine]-17-β-testosterone; PTA = 1,3,5-Triaza-7-
phosphaadamantane) (3)
To a solution of 1 (100 mg, 0.133 mmol) in acetone (15 ml) was
added an aqueous solution (2 ml) of PTA (20.9 mg, 0.133 mmol) and
the reaction mixture stirred at ambient temperature for 1 h. The solvent
was evaporated to dryness and the residue treated with Et2O/hexane
to give a white solid, which was collected by filtration and air-dried
(yield 70 mg, 63%). Anal. Calcd. For C36H50ClN4O2PPt: C 52.0, H 6.1, N 6.7.
Found: C 51.5, H 6.0, N 6.4%. Mp: 115 °C dec. 1H NMR (400 MHz, CDCl3) δ
(ppm): 7.28 (m, 1 H, H3′), 7.11 (dd, 1 H, H5′, JH5′H6′=7.6 Hz, JH3′H5′
=
1.2 Hz), 7.01 (d, 1 H, H6′, JH5′H6′ =7.6 Hz) , 5.72 (s, 2 H, Ha of
testosterone), 4.48 (s, 6 H, NCH2N of PTA), 4.41 (s, 6 H, NCH2P of PTA),
3.89 (d, 2 H, JHP=2.8 Hz, NCH2 of dmba, Pt satellites are observed as
shoulders), 2.78 (d, 6 H, JHP=1.2, NMe2 of dmba, Pt satellites are
observed as shoulders), 2.89 (s, 3 H, NMe2, Pt satellites are observed
as shoulders), 2.50–0.85 (m, 19 H, CH and CH2 of testosterone), 1.20
(s, 3 H, Me of testosterone), 0.93 (s, 3 H, Me of testosterone). 13C{1H}
NMR (100.80 MHz, CDCl3) δ (ppm): 138.7 (d, CH3′, JCP=4.2), 127.2
(CH5′), 123.8 (CHa of testosterone), 122.5 (CH6′), 73.5 (d, CH2N of
dmba, JCP =3.0), 73.4 (d, NCH2N of PTA, JCP =5.3), 53.7 (CH of
testosterone), 51.1 (d, NCH2P of PTA, JCP=18.9), 50.4 (NMe2 of dmba
and CH of testosterone), 39.0 (CH2 of testosterone), 36.3 (CH of
testosterone), 35.6, 34.0, 33.0, 32.8, 31.5, 23.2, 20.8 (CH2 of testoster-
one), 17.5, 12.9 (Me of testosterone). 31P NMR (162.29 MHz, CDCl3) δ
(ppm): −66.2 (s, JPPt =3785). 195Pt NMR (86.28 MHz, CDCl3) δ (ppm):
−4011 (d, JPPt=3785). ESI+ mass spectra (CH3CN), m/z [Found
(Calcd.)]: 853.9 (854.3) [[[Pt(ET-dmba)Cl(PTA)]+Na−H]+, 100%],
832.9 (833.3) [[[Pt(ET-dmba)Cl(PTA)]+H]+, 100%].
5.2. Synthesis of [Pt(ET-dmba)Cl(DMSO)] (ET-dmba =17-α-[4′-
ethynyl-dimethylbenzylamine]-17-β-testosterone) (2)
1 (500 mg, 1.12 mmol), NaOAc (184.1 mg, 2.24 mmol) and PtCl2
(DMSO)2 (473 mg, 1.12 mmol) were mixed in a 100 ml Schlenk tube.
Freshly distilled MeOH (40 ml) was added. The resulting mixture was
stirred at 50 °C for 24 h under nitrogen to give a dark solution, which
was treated with charcoal, and then filtered through a short pad of
Celite. The filtrate was evaporated to dryness, treated with CH2Cl2
and filtered through a short pad of Celite. The new filtrate was
evaporated to dryness and the residue treated with Et2O/hexane to
give a white solid, which was collected by filtration and air-dried
(yield 430 mg, 51%). Anal. Calcd. For C32H44ClNO3SPt: C 52.0, H 5.9, N
1.9, S 4.3. Found: C 51.4, H 6.2, N 1.8, S 4.1%. Mp: 115 °C dec. 1H NMR
(400 MHz, CDCl3) δ (ppm): 8.04 (d, 1 H, H3′, JH3′H5′ =1.4 Hz,
J
HPt =48.4 Hz), 7.13 (dd, 1 H, H5′, JH5′H6 ′ =7.6 Hz, JH3′H5′ =1.4 Hz),
6.99 (d, 1 H, H6′, JH5′H6′ =7.6 Hz), 5.72 (s, 2 H, Ha of testosterone),
3.99 (d, 1 H, JHH =14.0 Hz, NCH2 of dmba), 3.94 (JHH =14.0 Hz, NCH2
of dmba), 3.51 (s, 3 H, DMSO, JHPt =20.0 Hz), 3.50 (s, 3 H, DMSO,
5.4. Synthesis of [Pt(dmba)Cl(PTA)] (dmba= dimethylbenzylamine];
PTA = 1,3,5-Triaza-7-phosphaadamantane) (5)
J
HPt =20.0 Hz), 2.91 (s, 3 H, NMe2, Pt satellites are observed as
shoulders), 2.89 (s, 3 H, NMe2, Pt satellites are observed as
shoulders), 2.50–0.85 (m, 19 H, CH and CH2 of testosterone), 1.20
(s, 3 H, Me of testosterone), 0.93 (s, 3 H, Me of testosterone). 13C{1H}
NMR (100.80 MHz, CDCl3) δ (ppm): 137.2 (CH3′), 128.0 (CH5′),
123.7 (CHa of testosterone), 121.3 (CH6′), 74.7 (CH2N), 53.3 (CH of
testosterone), 52.2, 52.1 (NMe2), 50.1 (CH of testosterone), 46.7
(DMSO), 38.8 (CH2 of testosterone), 36.3 (CH of testosterone), 35.5,
34.0, 32.9, 32.7, 31.7, 23.1, 20.9 (CH2 of testosterone), 17.6, 12.9 (Me
of testosterone). 195Pt NMR (86.28 MHz, CDCl3) δ (ppm): −3683 (s).
ESI+ mass spectra (CH3CN), m/z [Found (Calcd.)]: 775.8 (776.2) [[[Pt
(ET-dmba)Cl(DMSO)]+Na]+, 100%].
To a solution of 4 (100 mg, 0.226 mmol) in acetone (15 ml) was
added an aqueous solution (2 ml) of PTA (35.5 mg, 0.226 mmol) and
the reaction mixture stirred at ambient temperature for 1 h. The
solvent was evaporated to dryness and the residue treated with Et2O
to give a white solid, which was collected by filtration and air-dried
(yield 101 mg, 86%). Anal. Calcd. For C15H24ClN4PPt: C 34.5, H 4.6, N
10.7. Found: C 34.2, H 4.7, N 10.5%. Mp: 279 °C dec. 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.25 (dd, 1 H, H3′, JH3′H4′ =7.8 Hz, JH3′H5′ =1.2 Hz,
JPtH =62 Hz), 7.09 (dd, 1 H, H6′, JH5′H6′ =6.8 Hz, JH6′H5′ =1.0 Hz), 7.04
(m, 1 H, H4′), 6.96 (m, 1 H, H5′), 4.53 (s, 6 H, NCH2N of PTA), 4.42

530
J. Ruiz et al. / Journal of Inorganic Biochemistry 105 (2011) 525–531
(s, 6 H, NCH2P of PTA), 3.92 (d, 2 H, JHP =2.8 Hz, NCH2 of dmba,
JPtH =28 Hz), 2.80 (d, 6 H, JHP =2.8, NMe2 of dmba, JPtH =24 Hz). 13
attachment to the culture surface the cells were incubated with
various concentrations of the compounds tested freshly dissolved in
DMSO and diluted in the culture medium (DMSO final concentration
1%) for 48 h at 37 °C. The cells were fixed by adding 10 μl of 11%
glutaraldehyde. The plates were stirred for 15 min at room temper-
ature and then washed three–four times with distilled water. The cells
were stained with 100 μl of 1% crystal violet. The plate were stirred for
15 min and then washed three–four times with distilled water and
dried. 100 μl 10% acetic acid was added and it was stirred for 15 min at
room temperature. Absorbance was measured at 595 nm in a Tecan
Ultra Evolution spectrophotometer.
C
{1H} NMR (100.80 MHz, CDCl3) δ (ppm): 135.7 (d, CH3′, JCP =4.8,
JCPt =103.7), 126.3 (d, CH5′, JCP =2.0, JCPt =72.1), 124.0 (s, CH4’),
122.8 (s, CH6′, JCPt =32.5), 73.7 (d, CH2N of dmba, JCP =3.3,
JCPt =54.8), 73.4 (d, NCH2N of PTA, JCP =7.4), 51.1 (d, NCH2P of PTA,
JCP =25.1, JCPt =69.0), 50.4 (d, NMe2 of dmba, JCP =2.8). 31P NMR
(121.5 MHz, CDCl3) δ (ppm): −65.5 (s, JPPt =3809). 195Pt NMR
(85.84 MHz, CDCl3) δ (ppm): −4022 (d, JPPt =3809). ESI+ mass
spectra (CH3CN), m/z [Found (Calcd.)]: 522.2 (521.9) [[[Pt(dmba)Cl
(PTA)]]+, 34%], 486.3 (486.4) [[[Pt(dmba)(PTA)]]+, 100%].
The effects of complexes were expressed as corrected percentage
inhibition values according to the following equation,
5.5. Reactions of the platinum complexes 2–5 with 9-ethylguanine
followed by NMR
ð%Þinhibition = ½1−ðT = CÞꢀ × 100
All reactions were carried out in NMR tubes (D2O and DMSO-d6
(10% for 2 and 3, 5% for 4 and 5) as solvents. 9-Ethylguanine were
incubated with the corresponding complex in a ratio 5:1 in D2O at pH
7.4 (50 mM tampon phosphate) and 37 °C. The concentration of all
complexes was 1.0 mM. All reactions are fast and in less than 15 min
the formation of the corresponding complex [Pt(C,N)(L)(9-EtG)]+
was completed (C,N = dmba or ET-dmba; L = DMSO and PTA).
where T is the mean absorbance of the treated cells and C the mean
absorbance in the controls.
The inhibitory potential of compounds was measured by calculat-
ing concentration–percentage inhibition curves, these curves were
adjusted to the following equation:
Â
Ã
E = E max = 1 + ðIC50Þ=CÞn
5.6. Reactions of the platinum complexes 2–5 with 9-ethylguanine
followed by ESI-MS
were E is the percentage inhibition observed, Emax is the maximal
effects, IC50 is the concentration that inhibits 50% of maximal growth,
C is the concentration of compounds tested and n is the slope of the
semi-logarithmic dose–response sigmoid curves. This non-linear
fitting was performed using GraphPad Prism 2.01, 1996 software
(GraphPad Software Inc.).
For comparison purposes, the cytotoxicity of cisplatin was
evaluated under the same experimental conditions. All compounds
were tested in two independent studies with triplicate points. The in
vitro studies were performed in the USEF platform of the University of
Santiago de Compostela (Spain).
All reactions were carried out in NMR tubes (H2O and DMSO (10%
for 2 and 3, 5% for 4 and 5) as solvents. 9-Ethylguanine was incubated
with the corresponding complex in a ratio 5:1 in H2O at 37 °C. The
concentration of complexes 2–5 was 1.0 mM. All reactions are fast
and in less than 15 min the formation of the corresponding complex
[Pt(C,N)(L)(9-EtG)]+ was completed (C,N = dmba or ET-dmba; L =
DMSO and PTA).
5.7. Computational details
Calculated geometries at the DFT level were fully optimized in the
gas-phase with tight convergence criteria using the Gaussian 09
package [31], and employing the BP86 functional together with the
extensive Def2-TZVP basis set on all atoms and using effective core
potential (ecp) for Pt. Ultrafine grids (99 radial shells and 590 angular
points per shell) were employed for numerical integrations. From
these gas-phase optimized geometries all reported data were
obtained by means of single-point (SP) calculations using the B3LYP
functional and the same basis set. Energy values are uncorrected for
the zero-point vibrational energy. Natural charges were obtained
from the Natural Bond Orbital (NBO) population analysis. The
topological analysis of the electronic charge density was conducted
by means of the Bader's AIM methodology using the AIM2000
software [32,33].
5.10. Androgen receptor whole cell binding assays
The compound was dissolved in DMSO at a concentration of
10 mM. The solutions were diluted with assay buffer to the
appropriate concentrations for assays, with a final concentration of
DMSO in the assay buffer of less than 0.5%. LNaCaP cell line was grown
in RPMI-1640 medium supplemented with 10% foetal bovine serum
(FBS) in a 5% CO2 atmosphere at 37 °C. The day before the assay
300,000 cells/well were seeded in 24-well cell culture plate and
maintained in a 5% CO2 atmosphere at 37 °C. 24 h later the medium
was replaced by an assay medium (RPMI-1640 medium supplemen-
ted with 0.1% BSA and 1 μM triamcinolone) to each well and
compounds and [3H]methyltrienolone were added where corre-
sponded. 100 μM DHT was used for unmasking non-specific binding.
The binding reaction was maintained for 2 h at 37 °C and 5% CO2.
Unbound [3H]methyltrienolone was removed by treatment with
500 μl of solubilization medium (Hank's Balanced Sal Serum supple-
mented with 0.5% SDS and 20% glycerol) and the radioactivity of
350 μl of this medium was measured using a liquid scintillation
counter (Beckman LS6500).
5.8. Cell line and culture
The T-47D human mammary adenocarcinoma cell line used in
this study was grown in RPMI-1640 medium supplemented with 10%
(v/v) fetal bovine serum (FBS) and 0.2 unit/ml bovine insulin in an
atmosphere of 5% CO2 at 37 °C. The human ovarian carcinoma cell
lines (A2780 and A2780cisR) used in this study were grown in RPMI
1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS)
and 2 mM L-glutamine in an atmosphere of 5% CO2 at 37 °C.
5.11. Electrophoretic mobility study
pBR322 plasmid DNA of 0.25 μg/μl concentration was used for the
experiments. 4 μl of charge maker (Lambda–pUC Mix marker, 4) was
added to aliquots parts of 20 μl of the drug–DNA complex. The
platinum complexes were incubated at the molar ratio ri =0.50 with
pBR322 plasmid DNA at 37 °C for 24 h. The mixtures underwent
electrophoresis in agarose gel 1% in 1×TBE buffer (45 mM Tris-borate,
1 mM EDTA, pH 8.0) for 5 h at 30 V. Gel was subsequently stained in
5.9. Cytotoxicity assay
Cell proliferation was evaluated by assay of crystal violet. T-47D
cells plated in 96-well sterile plates at a density of 5×103 cells/well
with 100 μl of medium and were then incubated for 48 h. After

J. Ruiz et al. / Journal of Inorganic Biochemistry 105 (2011) 525–531
531
[16] S. Top, A. Vessières, C. Cabestaing, I. Laios, G. Leclercq, C. Provot, G. Jaouen,
J. Organomet. Chem. 637 (2001) 500–506.
[17] A. Vessières, S. Top, P. Pigeon, E.A. Hillard, L. Boubeker, D. Spera, G. Jaouen, J. Med.
Chem. 48 (2005) 3937–3940.
the same buffer containing ethidium bromide (1 μg/ml) for 20 min.
The DNA bands were visualized with an AlphaImager EC (Alpha
Innotech).
[18] K.R. Barnes, A. Kutikov, S.J. Lippard, Chem. Biol. 11 (2004) 557–564.
[19] J. Ruiz, J. Lorenzo, C. Vicente, G. López, J.M. López-De-Luzuriaga, M. Monge, F.X.
Avilés, D. Bautista, V. Moreno, A. Laguna, Inorg. Chem. 47 (2008) 6990–7001.
[20] J. Ruiz, V. Rodríguez, N. Cutillas, G. López, D. Bautista, Inorg. Chem. 47 (2008)
10025–10036.
[21] J. Ruiz, V. Rodríguez, C. de Haro, A. Espinosa, J. Pérez, C. Janiak, Dalton Trans. 39
(2010) 3290–3301.
[22] M.D. Meijer, A.W. Kleij, B.S. Williams, D. Ellis, M. Lutz, A.L. Spek, G.P.M. van Klink,
G. van Koten, Organometallics 21 (2002) 264–271.
[23] J. Ruiz, N. Cutillas, F. López, G. López, D. Bautista, Organometallics 25 (2006)
5768–5773.
[24] R.F.W. Bader, Atoms in molecules: a quantum theory, Oxford University Press,
Oxford, 1990.
Acknowledgments
This work was supported by the Ministerio de Educacion y Ciencia
of Spain and FEDER (Projects CTQ2008-02178/BQU and CTQ2008-
01402) and Fundación Seneca-CARM (Projects 08666/PI/08 and
04509/GERM/06).
Appendix A. Supplementary data
[25] S.Y. Loh, P. Mistry, L.R. Kelland, G. Abel, K.R. Harrap, Brit. J. Cancer. 66 (1992)
1109–1115.
[26] P.M. Goddard, R.M. Orr, M.R. Valenti, C.F. Barnard, B.A. Murrer, L.R. Kelland, K.R.
Harrap, Anticancer Res. 16 (1996) 33–38.
Supplementary data to this article can be found online at
doi:10.1016/j.jinorgbio.2010.12.005.
[27] B.C. Behrens, T.C. Hamilton, H. Masuda, K.R. Grotzinger, J. Whang-Peng, K.G. Louie,
T. Knutsen, W.M. McKoy, R.C. Young, R.F. Ozols, Cancer Res. 47 (1987) 414–418.
[28] L.R. Kelland, C.F.J. Barnard, K.J. Mellish, M. Jones, P.M. Goddard, M. Valenti, A.
Bryant, B.A. Murrer, K.R. Harrap, Cancer Res. 54 (1994) 5618–5622.
[29] H.M. Hushay, T.D. Tullius, S.J. Lippard, Biochemistry 20 (1981) 3744–3748.
[30] J.H. Price, A.N. Williamson, R.F. Schramm, B.B. Wayland, Inorg. Chem. 11 (1972)
1280.
[31] Gaussian 09, Revision A.2, M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.
A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H.
Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L.
Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T.
Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, Jr. J. A. Montgomery, J.E. Peralta,
F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R.
Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J.
Tomasi, M. Cossi, N. Rega, N. J. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C.
Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi,
C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P.
Salvador, J.J. Dannerberg, S. Dapprich, A.D. Daniels, Ö Farkas, J.B. Foresman, J.V.
Ortiz, J. Cioslowski, D.J. Fox, Gaussian Inc., Wallingford CE (2009)
[32] AIM2000 v. 2.0, designed by F. W. Biegler-König, and J. Schönbohm, 2002. Home
page http://www.aim2000.de/. F. Biegler-König, J. Schönbohm and D. J. Bayles,
Comp. Chem. 22 (2001) 545-559
References
[1] M.A. Jakupec, M. Galanski, V.B. Arion, C.G. Hartinger, B.K. Keppler, Dalton Trans.
(2008) 183–194.
[2] Y.W. Jung, S.J. Lippard, Chem. Rev. 107 (2007) 1387–1407.
[3] L. Kelland, Nat. Rev. Cancer 7 (2007) 573–584.
[4] P.J. O'Dwyer, J.P. Stevenson, S.W. Johnson, in: B. Lippert (Ed.), Cisplatin, Chemistry
and biochemistry of a leading anticancer drug, Wiley-VCH, Weinheim, Germany,
1999, p. 3172.
[5] D. Wang, S.J. Lippard Nat, Rev. Drug Discov. 4 (2005) 307–320.
[6] M.J. Hannon, Pure Appl. Chem. 79 (2007) 2243–2261.
[7] C. Sánchez-Cano, M.J. Hannon, Dalton Trans. (2009) 10702–10711.
[8] S. van Zutphen, J. Reedijk, Coord. Chem. Rev. 249 (2005) 2845–2853.
[9] P.C.A. Bruijnincx, P.J. Sadler, Curr. Op. Chem. Biol. 12 (2008) 197–206.
[10] P.C.A. Bruijnincx, P.J. Sadler, Adv. Inorg. Chem. 61 (2009) 1–62.
[11] D. Griffith, M.P. Morgan, C.J. Marmion, Chem. Commun. (2009) 6735–6737.
[12] G.G. Chen, Q. Zeng, G.M.K. Tse, Med. Res. Rev. 28 (2008) 954–974.
[13] A. Jackson, J. Davis, R.J. Pither, A. Rodger, M.J. Hannon, Inorg. Chem. 40 (2001)
3964–3973.
[14] M.J. Hannon, P.S. Green, D.M. Fisher, P.J. Derrick, J.L. Beck, S.J. Watt, S.F. Ralph, M.
M. Sheil, P.R. Barker, N.W. Alcock, R.J. Price, K.J. Sanders, R. Pither, J. Davis, A.
Rodger, Chem. Eur. J. 12 (2006) 8000–8013.
[33] F. Biegler-König, J. Schönbohm, J. Comp. Chem. 23 (2002) 1489–1494.
[15] C. Sánchez-Cano, M.J. Hannon, Dalton Trans. 38 (2009) 10765–10773.