Lipophilic Pt(II) complexes with selective efficacy against cisplatin-resistant testicular cancer cells

Article abstract of DOI:10.1016/j.jinorgbio.2011.08.028

A series of dichloridoplatinum(II) complexes with selective and high cytotoxicity [IC₉₀(96 h) ≤ 3 μM] against cisplatin-resistant 1411HP testicular cancer cells were identified. They bear stationary 6-aminomethylnicotinate or 2,4-diaminobutyrate ligands esterified with lipophilic terpenyl residues, i.e., (-)/(+)-menthyl, (+)-cedrenyl, (-)-menthoxypropyl, or with a decyl-tethered 1,1,2-triphenylethene. They accumulated to a larger extent in 1411HP cells than in cells of the cisplatin-sensitive H12.1 germ cell tumour. Their mechanism of apoptosis induction differed from that of cisplatin by being independent of p53 and of caspase-3 activation and by an early loss of the mitochondrial membrane potential. The new complexes are promising candidates for the treatment of cisplatin-resistant testicular tumours.

Full text of DOI:10.1016/j.jinorgbio.2011.08.028

Journal of Inorganic Biochemistry 105 (2011) 1630–1637
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Lipophilic Pt(II) complexes with selective efficacy against cisplatin-resistant
testicular cancer cells
Bernhard Biersack a, Andrea Dietrich b, Miroslava Zoldakova a, Bernd Kalinowski c, Reinhard Paschke c,
b,⁎⁎
Rainer Schobert a, , Thomas Mueller
⁎
a
Organic Chemistry Laboratory, University Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany
Department of Internal Medicine IV, Oncology/Hematology, Martin-Luther-University Halle-Wittenberg, 06120 Halle/Saale, Germany
Biocenter of the Martin-Luther-University Halle-Wittenberg, 06120 Halle/Saale (Germany) , 06120 Halle/Saale, Germany
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 May 2011
Received in revised form 1 August 2011
Accepted 22 August 2011
Available online 14 September 2011
A series of dichloridoplatinum(II) complexes with selective and high cytotoxicity [IC90(96 h)≤3 μM] against
cisplatin-resistant 1411HP testicular cancer cells were identified. They bear stationary 6-aminomethylnicoti-
nate or 2,4-diaminobutyrate ligands esterified with lipophilic terpenyl residues, i.e., (−)/(+)-menthyl, (+)-
cedrenyl, (−)-menthoxypropyl, or with a decyl-tethered 1,1,2-triphenylethene. They accumulated to a larger
extent in 1411HP cells than in cells of the cisplatin-sensitive H12.1 germ cell tumour. Their mechanism of
apoptosis induction differed from that of cisplatin by being independent of p53 and of caspase-3 activation
and by an early loss of the mitochondrial membrane potential. The new complexes are promising candidates
for the treatment of cisplatin-resistant testicular tumours.
Keywords:
Platinum complexes
Terpenes
© 2011 Elsevier Inc. All rights reserved.
Cisplatin resistance
Testicular cancer
1. Introduction
and to the oestrogen receptor [12]. They also disclosed a (−)-menthyl-
Pt(II) conjugate 1a preferentially concentrating in cisplatin-resistant
The three FDA-approved platinum compounds, cisplatin, carbo-
platin, and oxaliplatin epitomise the progress that metallodrug-
based chemotherapy has made over the past four decades. Cisplatin,
an accidental discovery, was rashly approved for clinical use despite
its severe side effects, mainly for want of alternative treatments. In
contrast, carboplatin, a second generation modification of cisplatin
with fewer side effects and oxaliplatin, a derivative with high efficacy
against metastasised colorectal cancer were the products of rational
structure–activity deliberations and pharmacological studies [1–5].
The search continues for platinum complexes with patterns of cytotox-
icity significantly different from those of the three FDA-approved plati-
num drugs and in particular for such with activity against cisplatin-
resistant tumours. New promising derivatives of this type currently
in clinical trials are picoplatin (ZD0473) [6] which features a sterically
shielding α-picoline ligand and the Pt(IV) complex satraplatin (JM216)
[7]. Attempts at circumventing the cisplatin resistance were also made
by attaching cancer-specific carrier or shuttle groups to the cytotoxic
platinum complex fragment [4], [5], [8–11]. Recently, Schobert et al. pub-
lished an estradiol-Pt(II) complex conjugate that accumulates in breast
cancer cells by binding to the sex hormone binding globulin (SHBG)
1411HP germ cell cancer cells [13]. A series of similar terpene–Pt(II)
complex conjugates were finally developed with enhanced and selective
efficacy against melanoma and colon carcinoma cell lines [14]. Paschke
et al. reported undecyl-linked tetrahydropyran and cholic acid platinum
complex conjugates (THPPt-11, ChAPt-11) with anticancer activities sur-
passing that of cisplatin against 1411HP cells due to enhanced uptake
and to induction of alternative apoptosis pathways [15], [16]. Bérubé
and co-workers reported several lipophilic conjugates of Pt(II) com-
plexes with tamoxifen-like 1,1,2-triphenylethene moieties displaying
increased activity against breast cancer cells [17–20]. The uptake-
dependent breach of cisplatin-resistance can ideally be studied using
the two germ cell tumour cell lines 1411HP which is cisplatin-resistant
and H12.1 which is cisplatin-sensitive. The cisplatin resistance of
1411HP is due to an unusually high threshold for the activation of
the apoptosis-relevant caspase-9 [21]. Herein we report on a collection
of known and new lipophilic platinum complexes with terpenoid, ste-
roidal and 1,1,2-trisarylethene appendages that exhibit a significantly
higher cytotoxicity in the cisplatin-resistant 1411HP cells when com-
pared to the sensitive H12.1 cells.
2. Experimental
2.1. General
⁎
Corresponding author. Fax: +49 921 552671.
⁎⁎ Corresponding author. Fax: +49 345 5577279.
E-mail addresses: laganlad@yahoo.co.uk (R. Schobert),
thomas.mueller@medizin.uni-halle.de (T. Mueller).
Melting points were determined with a Gallenkamp apparatus
and are uncorrected. IR spectra were recorded on a Perkin-Elmer
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.08.028

B. Biersack et al. / Journal of Inorganic Biochemistry 105 (2011) 1630–1637
1631
One FT ATR (attenuated total reflection) IR spectrophotometer.
2.2.3. 13,14,14-Tris-(p-methoxyphenyl)tetradec-13-enyl
The metal content of cells was ascertained with a graphite furnace
atomic absorption spectrometer model AAS5 EA solid (Jena GmbH,
Germany). NMR spectra were recorded under conditions as indicated
on a Bruker Avance 300 spectrometer. Chemical shifts (δ) are given
in parts per million downfield from Me4Si as internal standard for
1H and 13C and relative to Ξ(195Pt)=21.4 MHz for 195Pt. Signal mul-
tiplicities are assigned as “multiplet (m)”, “singlet (s)”, “doublet” (d),
“triplet” (t), or “quartet (q)”. Mass spectra were recorded using a
Thermo Finnigan MAT 8500 in electron impact (EI) mode. Elemental
analyses were carried out with a Perkin-Elmer 2400 CHN elemental
analyser. For column chromatography Merck silica gel 60 (230–
400 mesh) was used. All starting compounds were purchased from
the usual sources and used without further purification. The com-
plexes 1a–k and the 1,1,2-trisarylalkenols necessary for the prepara-
tion of esters 2l–n were prepared following literature procedures
[12–14], [17–20].
6′-(t-butoxycarbonylaminomethyl)nicotinate (2n)
Analogously to the synthesis of 2l, compound 2n was obtained from
6-t-butoxycarbonylaminomethylnicotinic acid (160 mg, 0.63 mmol),
Et3N (100 μL, 0.72 mmol), 2,4,6-trichlorobenzoyl chloride (111 μL,
0.72 mmol), 13,14,14-tris-(p-methoxyphenyl)-13-en-1-ol (196 mg,
0.43 mmol) and DMAP (155 mg, 1.26 mmol). Yield: 400 mg (83%); col-
ourless oil; Rf 0.26 (ethyl acetate/hexane 1:2); vmax/cm−1: 2925, 2853,
1717, 1603, 1507, 1282, 1239, 1170, 1110, 1032, 829; 1H NMR
(300 MHz, CDCl3): δ 1.1–1.5 (m, 27H), 1.7–1.8 (m, 2H), 2.3–2.5 (m,
2H), 3.67 (s, 3H), 3.73 (s, 3H), 3.80 (s, 3H), 4.31 (t, J=6.7 Hz, 2H),
4.48 (d, J=5.5 Hz, 2H), 5.5–5.6 (m, 1H), 6.5–7.2 (m, 12H), 7.33
(d, J=8.1 Hz, 1H), 8.23 (d, J=8.1 Hz, 1H), 9.11 (s, 1H); 13C NMR
(75.5 MHz, CDCl3): δ 26.0, 28.4, 28.6, 29.0, 29.2, 29.3, 29.5, 29.6, 29.7,
35.9, 45.8, 55.0, 55.1, 55.2, 65.5, 79.8, 112.7, 113.2, 113.4, 121.0, 125.0,
130.6, 131.9, 135.1, 136.1, 136.6, 137.5, 137.7, 139.4, 150.4, 157.3,
157.6, 158.1, 161.9, 165.2; m/z 764 (59) [M+], 690 (58), 664 (92), 359
(100), 251 (58), 227 (28), 121 (37), 59 (46); accurate mass (EIMS) for
C47H60N2O7: calcd 765.004, obsd 765.005.
2.2. Chemistry
2.2.4. 11,12,12-Triphenyldodec-11-enyl 6′-aminomethylnicotinate
dihydrochloride (3l)
2.2.1. 11,12,12-Triphenyldodec-11-enyl 6′-(t-butoxycarbonylaminomethyl)
nicotinate (2l)
2l (230 mg, 0.36 mmol) was treated with 4 M HCl/dioxane
(15 mL) at room temperature for 1 h. After evaporation of the solvent
the oily residue was treated with hexane giving a yellowish gum.
The hexane was evaporated and the residue was dried in vacuum.
Yield: 210 mg (94%); vmax/cm−1: 2923, 2853, 1724, 1644, 1600,
1490, 1442, 1294, 1122, 758, 698; 1H NMR (300 MHz, D6-DMSO): δ
1.1–1.4 (m, 14H), 1.6–1.8 (m, 2H), 2.3–2.4 (m, 2H), 4.2–4.4 (m, 4H),
6.8–7.4 (m, 15H), 7.65 (d, J=8.2 Hz, 1H), 8.35 (d, J=8.2 Hz, 1H),
8.4–8.6 (m, 3H), 9.08 (s, 1H); 13C NMR (75.5 MHz, D6-DMSO): δ
25.3, 28.0, 28.5, 28.6, 28.8, 35.1, 42.6, 65.2, 122.6, 125.3, 125.8,
126.2, 126.7, 127.5, 127.8, 128.3, 128.9, 129.2, 130.0, 137.7, 140.4,
141.8, 142.6, 142.9, 149.3, 157.8, 164.4; m/z 546 (100) [M+ −2
HCl], 269 (11), 191 (38), 91 (17).
6-t-Butoxycarbonylaminomethylnicotinic acid (140 mg, 0.56 mmol)
was dissolved in dry DMF (2 mL) and treated with Et3N (80 μL,
0.58 mmol) and 2,4,6-trichlorobenzoyl chloride (95 μL, 0.59 mmol).
The resulting suspension was stirred under argon at room temperature
for 20 min. A solution of 9,10,10-triphenyldec-9-en-1-ol (230 mg,
0.56 mmol) and DMAP (143 mg, 1.18 mmol) in dry toluene (20 mL)
was added and the resulting mixture was stirred under argon at room
temperature for 16 h. After dilution with ethyl acetate and washing
with water the organic phase was dried over Na2SO4 and concentrated
in vacuum. The residue was purified by column chromatography (silica
gel 60; ethyl acetate/hexane 1:3). Yield: 270 mg (75%); colourless oil;
Rf 0.26 (ethyl acetate/hexane 1:3); vmax/cm−1: 2925, 2854, 1718,
1598, 1491, 1366, 1275, 1243, 1166, 1114, 758, 698; 1H NMR
(300 MHz, CDCl3): δ 1.1–1.5 (m, 21H), 1.6–1.8 (m, 2H), 2.3–2.5 (m,
2H), 4.31 (t, J=6.7 Hz, 2H), 4.48 (d, J=5.5 Hz, 2H), 5.5–5.6 (m, 1H),
6.8–7.4 (m, 16H), 8.23 (d, J=8.2 Hz, 1H), 9.12 (s, 1H); 13 C NMR
(75.5 MHz, CDCl3): δ 25.9, 28.4, 28.6, 28.8, 29.2, 29.4, 29.6, 29.9, 35.8,
45.8, 65.5, 121.0, 125.0, 125.6, 126.1, 126.5, 126.8, 127.2, 127.3, 127.7,
128.1, 129.5, 129.6, 129.9, 130.2, 130.7, 137.7, 139.0, 141.1, 142.5,
143.0, 143.5, 150.4, 155.9, 161.8, 165.2; m/z 646 (25) [M+], 590 (100),
546 (55), 269 (22), 191 (85), 91 (43), 57 (72); accurate mass (EIMS)
for C42H50N2O4: calcd 646.37706, obsd 646.37700.
2.2.5. 11,12,12-Tris-(p-methoxyphenyl)dodec-11-enyl
6′-aminomethylnicotinate dihydrochloride (3m)
Obtained analogously to 3l from 2m (230 mg, 0.31 mmol) as a yel-
lowish gum. Yield: 220 mg (100%); vmax/cm−1: 2925, 2852, 1762,
1606, 1510, 1463, 1291, 1243, 1173, 1121, 1034, 831; 1H NMR
(300 MHz, D6-DMSO): δ 1.0–1.4 (m, 14H), 1.6–1.8 (m, 2H), 2.3–2.4
(m, 2H), 3.62 (s, 3H), 3.67 (s, 3H), 3.75 (s, 3H), 4.2–4.4 (m, 4H), 6.5–
7.1 (m, 12H), 7.66 (d, J=8.2 Hz, 1H), 8.35 (d, J=8.2 Hz, 1H), 8.4–8.6
(m, 3H), 9.08 (s, 1H); 13C NMR (75.5 MHz, D6-DMSO): δ 25.4, 28.0,
28.2, 28.5, 28.6, 28.7, 28.8, 28.9, 35.2, 42.6, 54.8, 55.0, 65.2, 112.9,
113.3, 113.5, 122.6, 125.3, 130.2, 130.3, 131.3, 134.3, 135.6, 135.8,
137.2, 137.7, 138.8, 149.3, 157.0, 157.3, 157.8, 164.0; m/z 636 (100)
[M+ −2HCl], 359 (40), 251 (23).
2.2.2. 11,12,12-Tris-(p-methoxyphenyl)dodec-11-enyl
6′-(t-butoxycarbonylaminomethyl)nicotinate (2m)
Analogously to the synthesis of 2l, compound 2m was obtained from
6-t-butoxycarbonylaminomethylnicotinic acid (109 mg, 0.43 mmol),
Et3N (70 μL, 0.51 mmol), 2,4,6-trichlorobenzoyl chloride (80 μL,
0.49 mmol), 11,12,12-triphenyldodec-11-en-1-ol (196 mg, 0.43 mmol)
and DMAP (104 mg, 0.86 mmol). Yield: 230 mg (73%); colourless oil;
Rf 0.24 (ethyl acetate/hexane 1:2); vmax/cm−1: 2926, 2854, 1717,
1603, 1507, 1282, 1239, 1170, 1032, 829; 1H NMR (300 MHz, CDCl3): δ
1.1–1.5 (m, 23H), 1.7–1.8 (m, 2H), 2.3–2.5 (m, 2H), 3.67 (s, 3H), 3.73
(s, 3H), 3.80 (s, 3H), 4.31 (t, J=6.7 Hz, 2H), 4.48 (d, J=5.5 Hz, 2H),
5.5–5.6 (m, 1H), 6.5–7.2 (m, 12H), 7.33 (d, J=8.1 Hz, 1H), 8.23 (d,
J=8.1 Hz, 1H), 9.11 (s, 1H); 13C NMR (75.5 MHz, CDCl3): δ 25.9, 28.3,
28.7, 29.0, 29.2, 29.3, 29.4, 29.7, 35.9, 45.8, 55.0, 55.1, 55.2, 65.5, 79.7,
112.7, 113.2, 113.4, 121.5, 125.0, 130.6, 131.9, 135.1, 136.1, 136.5,
137.5 (C-12), 137.7 (C-4′), 139.4 (C-11), 150.4 (C-2′), 157.3, 157.6,
158.1, 161.9, 165.2; m/z 736 (85) [M+], 680 (47), 636 (100), 359 (74),
251 (40), 121 (37); accurate mass (EIMS) for C45H56N2O7: calcd
736.40875, obsd 736.40870.
2.2.6. 13,14,14-Tris-(p-methoxyphenyl)tetradec-13-enyl
6′-aminomethylnicotinate dihydrochloride (3n)
Obtained analogously to 3l from 2n (380 mg, 0.50 mmol) as a yel-
lowish gum. Yield: 360 mg (0.49 mmol, 98%); vmax/cm−1: 2922,
2851, 1722, 1604, 1507, 1285, 1240, 1172, 1119, 1032, 828; 1H NMR
(300 MHz, D6-DMSO): δ 1.0–1.5 (m, 18H), 1.6–1.8 (m, 2H), 2.3–2.4
(m, 2H), 3.62 (s, 3H), 3.68 (s, 3H), 3.75 (s, 3H), 4.2–4.4 (m, 4H),
6.5–7.1 (m, 12H), 7.67 (d, J=8.2 Hz, 1H), 8.35 (d, J=8.2 Hz, 1H),
8.5–8.7 (m, 3H), 9.08 (s, 1H); 13C NMR (75.5 MHz, D6-DMSO): δ
25.4, 28.0, 28.2, 28.5, 28.6, 28.7, 28.9, 35.2, 42.6, 54.8, 55.0, 65.2,
112.9, 113.3, 113.5, 122.6, 125.3, 130.2, 130.3, 131.3, 134.3, 135.5,
135.8, 137.2, 137.7, 138.8, 149.2, 157.0, 157.3, 157.8, 157.9, 164.4;
m/z 664 (100) [M+ −2HCl], 359 (32), 251 (18); accurate mass
(EIMS) for C42H52N2O5 (free base): calcd 664.38762, obsd 664.38760.

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B. Biersack et al. / Journal of Inorganic Biochemistry 105 (2011) 1630–1637
2.2.7. cis-Dichlorido(11,12,12-triphenyldodec-11-enyl
6′-aminomethylnicotinate)platinum(II) (1l)
the sulphorhodamine-B (SRB) microculture colorimetric assay.
Briefly, cells were seeded into 96-well plates on day 0, at cell den-
sities previously determined to ensure exponential cell growth
during the test period. On day 1, cells were treated with the appro-
priate concentrations of the drugs dissolved in DMF for indicated
times and the percentage of surviving cells relative to untreated
controls was determined on day 5. All experiments were carried
out in three parallel independent series.
A solution of 3l (210 mg, 0.34 mmol) in H2O/THF (5 mL, 1:1) was
treated with K2PtCl4 (141 mg, 0.34 mmol) dissolved in H2O, any
appearing colourless precipitate was redissolved by addition of THF,
the pH value of the resulting solution was adjusted to 5–6 with aque-
ous NaOH and the reaction mixture was stirred at room temperature
for 24 h. The formed yellow precipitate was collected, washed in turn
with H2O and diethyl ether and dried in vacuum. Yield: 123 mg
(44%); yellow solid of m.p. 208 °C (dec.); C37H42Cl2N2O2Pt requires:
2.4. Ethidium bromide fluorescence
C, 54.7; H, 5.2; N, 3.5%. Found: C, 54.8; H, 5.2; N, 3.5%. vmax/cm−1
:
3235, 2924, 2853, 1727, 1619, 1491, 1442, 1293, 1129, 755; 1H NMR
(300 MHz, D7-DMF): δ 1.1–1.5 (m, 12H), 1.6–1.8 (m, 2H), 2.4–2.5
(m, 2H), 4.38 (t, J=6.6 Hz, 2H), 4.47 (t, J=6.0 Hz, 2H, CH2N), 6.3–
6.4 (m, 2H), 6.9–7.5 (m, 15H), 7.90 (d, J=8.2 Hz, 1H), 8.66 (d,
J=8.2 Hz, 1H), 9.87 (s, 1H); 13C NMR (75.5 MHz, D7-DMF): δ 26.0,
28.7, 28.8, 29.3, 29.4, 29.6, 35.8, 53.7, 66.1, 122.5, 126.1, 126.5,
127.1, 127.2, 127.3, 127.4, 127.8, 128.2, 128.5, 128.6, 128.7, 129.6,
129.9, 130.1, 130.3, 130.7, 138.8, 139.5, 141.3, 142.7, 143.5, 143.8,
148.4, 163.5, 170.8; 195Pt NMR (64.4 MHz, D7-DMF): δ 2440; m/z
546 (27), 430 (41), 269 (64), 191 (100), 167 (22), 115 (19), 91
(55), 36 (25).
The fluorescence of ethidium bromide (EtdBr) intercalated into
salmon sperm DNA was measured in the absence and presence of
platinum derivatives [23,24]. The fluorescence intensity vs. concen-
tration curve for EtdBr in the absence of Pt-complexes represents
the maximal binding capacity for the respective concentration ap-
plied. In the presence of Pt-complexes, the capacity of the DNA to
bind EtdBr is diminished due to sterical overlap and geometrical con-
straints between the binding sites.
Salmon sperm DNA (1.23 μM in 10 mM NaClO4) was incubated
with selected derivatives for 48 h at 25 °C. The end concentrations
varied between 0.0 mM and 0.6 mM for different compounds. EtdBr
titration curves were recorded on an LB50 Perkin-Elmer spectrofluo-
2.2.8. cis-Dichlorido[11,12,12-tris-(p-methoxyphenyl)dodec-11-enyl
6′-aminomethylnicotinate]platinum(II) (1m)
rometer after excitation at λex =546 nm and emission at λem =
590 nm. Typical samples contained 0.20 μM of salmon sperm DNA
in 0.4 M NaCl, sodium phosphate buffer, pH 7.5 in a 1 cm quartz
cell. Small aliquots of EtdBr were added and final effective concentra-
tions of all constituents were re-calculated.
Analogously to the synthesis of 1l, compound 1m (207 mg, 77%)
was obtained from 3m (210 mg, 0.30 mmol) and K2PtCl4 (125 mg,
0.30 mmol) as a yellow solid of m.p. 209 °C (dec.); C40H48Cl2N2O5Pt re-
quires: C, 53.2; H, 5.4; N, 3.1%. Found: C, 53.3; H, 5.4; N, 3.1%. vmax/cm−1
:
3239, 2925, 2852, 1725, 1606, 1509, 1292, 1243, 1173, 1128, 1033, 831,
754; 1H NMR (300 MHz, D7-DMF): δ 1.1–1.5 (m, 14H), 1.7–1.8 (m, 2H),
2.4–2.5 (m, 2H), 3.69 (s, 3H), 3.74 (s, 3H), 3.83 (s, 3H), 4.38 (t, J=6.6 Hz,
2H), 4.48 (t, J=5.9 Hz, 2H), 6.3–6.4 (m, 2H), 6.6–7.2 (m, 12H), 7.90
(d, J=8.2 Hz, 1H), 8.66 (d, J=8.2 Hz, 1H), 9.87 (s, 1H); 13C NMR
(75.5 MHz, D7-DMF): δ 26.0, 28.7, 29.4, 29.6, 36.0, 53.7, 55.0, 55.2,
66.1, 113.2, 113.6, 113.9, 122.5, 127.2, 130.8, 131.0, 132.0, 135.2,
136.4, 136.6, 138.2, 138.8, 139.7, 148.4, 157.9, 158.2, 158.7, 163.5,
170.8; 195Pt NMR (64.4 MHz, D7-DMF): δ 2439; m/z 520 (100), 359
(48), 227 (22), 36 (12).
2.5. Determination of platinum uptake by AAS
Cellular platinum concentrations were determined by flameless
atomic absorption spectroscopy using a solid sampling graphite fur-
nace atomic absorption spectrometer (SS-GF AAS) model AAS5 EA
solid (Jena GmbH, Germany). The H12.1 and 1411HP cells were trea-
ted with 30 μM of test compounds for 2 h, washed, harvested by tryp-
sinization, and finally freeze-dried. The cells were then submitted to a
temperature ramp ranging from 100 to 2550 °C, atomised and ana-
lysed at λ=265.9 nm.
2.2.9. cis-Dichlorido[13,14,14-tris-(p-methoxyphenyl)tetradec-13-enyl
6′-aminomethylnicotinate]platinum(II) (1n)
2.6. Blocking of caspase activation
Analogously to the synthesis of 1l, complex 1n (200 mg, 45%)
was obtained from 3n (360 mg, 0.49 mmol) and K2PtCl4 (208 mg,
0.50 mmol) as a yellow solid of m.p. 195 °C (dec.); C42H52Cl2N2O5Pt re-
For inhibition of drug-induced caspase activation the cells were
simultaneously treated with 60 μM of the broad-spectrum caspase
inhibitor Z-VAD-Fmk (Alexis Biochemicals).
quires: C, 54.2; H, 5.6; N, 3.0%. Found: C, 54.3; H, 5.7; N, 3.0%. vmax/cm−1
:
2922, 1724, 1605, 1508, 1276, 1240, 1172, 1032, 828, 754; 1H NMR
(300 MHz, D7-DMF): δ 1.1–1.5 (m, 18H), 1.7–1.8 (m, 2H), 2.4–2.5
(m, 2H), 3.69 (s, 3H), 3.74 (s, 3H), 3.83 (s, 3H), 4.38 (t, J=6.6 Hz, 2H),
4.47 (t, J=6.0 Hz, 2H), 6.3–6.4 (m, 2H), 6.6–7.2 (m, 12H), 7.90
(d, J=8.2 Hz, 1H), 8.66 (d, J=8.2 Hz, 1H), 9.87 (s, 1H); 13C NMR
(75.5 MHz, D7-DMF): δ 25.8, 28.5, 28.7, 29.2, 29.4, 29.5, 35.7, 53.5,
54.7, 54.8, 54.9, 112.9, 113.4, 113.6, 122.3, 126.3, 130.5, 130.7, 131.7,
134.9, 136.1, 136.4, 137.9, 138.6, 139.4, 148.2, 157.6, 158.0, 158.5,
163.2, 170.5; 195Pt NMR (64.4 MHz, D7-DMF): δ 2440; m/z 649 (11),
548 (81), 359 (100), 251 (67), 227 (61), 121 (82).
2.7. Western-blotting for actin, p53 and caspase-3
Cells were harvested, rinsed twice with PBS and lysed in RIPA
buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP40, 0.5% DOC,
0.1% SDS] supplemented with a protease inhibitor cocktail (Sigma).
Insoluble components were removed by centrifugation and protein
concentrations were measured (BIO-RAD protein assay, Bio-Rad,
Germany). After boiling for 5 min in SDS-loading buffer (62.5 mM
Tris–HCl pH 6.8; 20% glycerol, 2% SDS, 100 mM DTT) 30 μg protein
per lane was separated by SDS-PAGE and electroblotted onto a nitro-
cellulose membrane (Bio-Rad). Equal protein loadings were ensured
by Ponceau S staining (Sigma). Membranes were blocked with 5%
milk powder in PBST for 1 h and probed for 2 h with the following
antibodies diluted in PBST/5% milk: actin (C-11) goat polyclonal
(0.5 μg/mL), p53 (DO-1) mouse monoclonal (0.1 μg/mL) (both from
Santa Cruz Biotechnology, USA), caspase-3 (Clon 33) mouse mono-
clonal (0.5 μg/mL) (MBL Biozol). Immuno-complexes were visualised
by enhanced chemoluminescence (Amersham Pharmacia Biotech,
UK) using horseradish peroxidase-conjugated anti-mouse or anti-
rabbit IgG (Santa Cruz Biotechnology).
2.3. Cell lines; SRB assays for cytotoxicity
The testicular germ cell tumour cell lines H12.1 and 1411HP
were maintained as monolayer cultures in RPMI 1640 (PAA, Pasching,
Austria) supplemented with 10% foetal bovine serum (Biochrom AG,
Berlin, Germany) and 1% streptomycin/penicillin (PAA, Pasching,
Austria). Cultures were grown at 37 °C in a humidified atmosphere
of 5% CO2/95% air. Dose–response curves of the cell lines upon expo-
sure to drug concentrations of 0.01–100 μM were established using

B. Biersack et al. / Journal of Inorganic Biochemistry 105 (2011) 1630–1637
1633
2.8. Analyses of DNA fragmentation
anisyl analogues 1m and 1n. It also featured the best resistance factor
(0.12) of all test compounds.
Determination of apoptotic cell death was performed by DNA gel
electrophoresis. Floating cells induced by drug exposure were collect-
ed, washed with PBS and lysed in lysis buffer (100 mM Tris–HCl pH
8.0, 20 mM EDTA, 0.8% SDS). After treatment with RNase A for 2 h
and proteinase K (Roche Molecular Biochemicals) overnight, lysates
were mixed with DNA loading buffer. To separate the DNA fragments,
a probe was run on a 1.5% agarose gel followed by ethidium bromide
staining and rinsing with distilled water. The DNA ladders were
visualised under UV light and documented on a BioDocAnalyse in-
strument (Biometra).
3.2.2. DNA interaction
Even though dichlorido(6-aminomethylnicotinate)platinum(II)
complexes like 1a were shown to interact with single GMP molecules
[22], band shift assays with pBR322 plasmid DNA had revealed that
DNA unwinding as observed upon interaction with cisplatin did
only occur with the less anticancer active conjugates 1 [13], [14].
We now investigated the influence of the complexes 1a–c and 1h–k
on the capacity of salmon sperm DNA to bind ethidium bromide
(EtdBr). It is known [23], [24] that the structural and geometrical per-
turbations induced in salmon sperm DNA by bi- or mono-functional
adducts of mononuclear platinum compounds interfere with the
binding of EtdBr. Hence, by monitoring the displacement of EtdBr
from DNA the degree of interaction between DNA and other compet-
ing binders to it and resulting morphological changes can be evaluat-
ed. The left panel of Fig. 2 exemplarily depicts the titration curves for
the intercalation of EtdBr into salmon sperm DNA in the absence
(0 mM) or presence of platinum complex 1b (0.06–0.6 mM). When
compared to the intercalation of EtdBr into unmodified DNA the
total fluorescence intensity was different and decreased with increas-
ing platinum complex concentrations. Thus this intensity reflects the
amount of EtdBr bound to DNA and so it was taken as a measure for
the EtdBr-binding capacity of DNA modified by Pt-adduct formation.
It was found to be ca. four times lower when the Pt-complex was pre-
sent at 0.56 mM when compared to assays with Pt-complex concen-
trations of 0.06 mM. In the right panel of Fig. 2 relative binding
capacities (I1/I0) for EtdBr were plotted against the Pt-complex con-
centrations. I1 is the final fluorescence obtained from titration curves
at different concentrations of the individual Pt-complexes and I0 is
the final fluorescence of the DNA•EtdBr adduct in the absence of plat-
inum. Due to beginning precipitation the titration curves of com-
plexes 1a and 1h were not measured at higher concentrations.
Interestingly, while most complexes 1 cluster quite close to each
other in the right panel of Fig. 2, the curves for cisplatin and the estra-
diol complex 1i deviate significantly. For example, cisplatin and 1i
displaced only ca 25% of EtdBr whereas 1k liberated nearly 90%.
These figures could be interpreted as resulting from different mor-
phological changes to DNA and its binding sites for EtdBr caused by
interaction with the individual Pt-complexes. However, there is no
stringent correlation between the cytotoxicities and the modulatory
effects on DNA of the tested platinum complexes which is in keeping
with the results of the band shift assays with pBR322 plasmid DNA.
2.9. Analyses of the mitochondrial membrane potential
Analyses were performed using the DePsipher Mitochondrial
Potential Kit (R and D Systems). The lipophilic cationic DePsipher
reagent aggregates in the mitochondria to form an orange fluores-
cent compound. In cells with disrupted mitochondrial membrane
potential the DePsipher reagent remains in its monomeric green
fluorescent form.
3. Results and discussion
3.1. Synthesis
The synthesis of the Pt(II) complexes 1a–k was reported elsewhere
[12–14]. The new 1,1,2-trisarylethene conjugates 1l–n were prepared
similarly by esterification of N-Boc-protected 6-aminomethylnicotinic
acid with the corresponding trisarylalkenols [17–20], acidic removal
of the Boc protecting group of esters 2l–n and reaction of the resulting
ammonium chlorides 3l–n with K2PtCl4 at pH 5–6. The structures of
all studied Pt(II) complexes 1 are depicted in Fig. 1.
3.2. Biological evaluation
3.2.1. Growth inhibition and platinum uptake
Complexes 1 were tested by SRB assays for cytotoxicity against
cisplatin-sensitive H12.1 and cisplatin-resistant 1411HP germ cell tu-
mour cells and compared with the previously reported (−)-menthyl
conjugate 1a [13] and the complex THPPt-11 [15] (Table 1). The ter-
pene conjugates ent-1a and 1b–g inhibited the growth of the
1411HP cells at single-digit micromolar IC90(96 h) values. The
carvomenthyl (1b), the isopinocampheyl (1c), and the menthyl
2,3-diaminopropionate (1f) conjugates affected 1411HP and H12.1
cells to a similar extent, whereas the complexes ent-1a,1d, 1e,
and 1g were distinctly more active against the cisplatin-resistant
1411HP cells (IC90 ≤3 μM) than against the sensitive H12.1 cells. De-
spite the slightly higher activity of ent-1a against 1411HP cells over
that of 1a it is unclear whether chirality plays a role in the cytotoxic
effect of the platinum terpene complexes. According to atomic ab-
sorption spectrometry (AAS) analyses, the platinum content of
1411HP cells previously exposed to either ent-1a or 1a was of simi-
lar magnitude and ca. 2.3-fold higher than that of H12.1 cells treated
identically. By comparison, only a fiftieth of this amount was taken
up in the case of cisplatin by either cell line. Thus, it is tempting to
explain the higher activity of the new complexes against the resis-
tant 1411HP cells with their enhanced accumulation. The conjugates
1h–k of estrone, estradiol, dihydrotestosterone, and testosterone
showed moderate activities against 1411HP cells in the low double-
digit micromolar range, but even weaker activities against the cisplatin-
sensitive H12.1 cells. Concerning the so-called resistance factors, i.e., the
quotients of the IC50 values for these two cell lines, the dihydrotestoster-
one conjugate 1j displayed an excellent value b0.17. The triphenylethene
conjugate 1l exhibited a distinctly higher activity in the 1411HP cells
(IC50 =6.0 μM) compared with the steroid complexes 1h–k and the
3.2.3. Interaction with N-acetyl-L-cysteine
Lipophilic complexes are likely to accumulate in membranes and
to interact with their components, e.g., with lipids or membrane-
residing proteins. Hence, we tested our platinum complexes for affin-
ity to biomolecules other than DNA. We monitored the reaction of the
terpene conjugate 1a with N-acetyl-L-cysteine (ten-fold excess) in dry
DMF as a model for a thiol-containing peptide by 1H NMR spectrosco-
py (Fig. 3). After 20 h new high field-shifted peaks (2H-C, 4H-C, 5H-C)
cropped up for the nicotinate protons 2H, 4H and 5H, suggesting the
formation of a single N-acetyl-L-cysteine mono adduct, likely with
the cysteine ligand in trans position to the pyridine moiety. When
run in 1:1-mixtures of DMF and water the reaction proceeded faster
and 2/3 of the starting complex had been converted to the mono ad-
duct within 18 h (for similar studies with analogous platinum com-
plexes see refs. [22],[25]). Analogous spectra were obtained for
reactions of N-acetyl-L-cysteine with other terpene-Pt conjugates,
e.g., a (+)-fenchyl derivative. The nature of the terpene is not decisive
for the complex reactivity towards simple thiols. The cytotoxicity
of the Pt(II) complexes 1 might even partly originate from such reac-
tions with sulphur bionucleophiles in membranes. For instance, pro-
teins like Ras or heterotrimeric GTPases which are crucial for cell

1634
B. Biersack et al. / Journal of Inorganic Biochemistry 105 (2011) 1630–1637
O
11
O
O
O
H3N
Cl
NH3
Cl
H2N
Cl
NH2
Cl
Pt
N
Pt
Cl
Pt
H2N
Cl
Cisplatin
THPPt-11
1a
O
4
O
O
5
R
R =
2
O
N
Pt
Cl
H2N
Cl
ent-1a
1c
1b
O
O
O
n
H2N
Cl
NH2
O
O
Pt
Cl
1f: n = 0
1g: n = 1
1d
1e
O
OH
1h
1i
O
O
O
O
1j
1k
O
O
R
R
1l: n = 10, R = H
1m: n = 10, R = OMe
1n: n = 12, R = OMe
O
n
R
Fig. 1. Structures of cisplatin, THPPt-11 and complexes 1a [13], ent-1a [13], 1b–g [14], 1h–k [12] and 1l–n.
proliferation and survival pathways and whose activity is associated
with controlled membrane attachment possess terminal cysteine res-
idues. However, at present we cannot exclude that interactions with
thiols in the cytosol such as glutathione might also play a role.
3.2.4. Caspase activation and apoptosis
Since we supposed that the mechanism of action of complexes 1
differs significantly from that of cisplatin we also analysed apopto-
sis-relevant parameters such as kinetics of caspases involved. First,

B. Biersack et al. / Journal of Inorganic Biochemistry 105 (2011) 1630–1637
1635
Fig. 2. Left: titrations of salmon sperm DNA (1.23 μM in 10 mM NaClO4) with EtdBr in
the presence of varying concentrations (0.0–0.6 mM) of 1b. Right: EtdBr fluorescence
decrease upon platination: I0 and I1 are the final fluorescence intensities in the absence
and presence of Pt-complexes; conditions: 10 mM NaClO4 at 25 °C, final DNA and EtdBr
concentrations were 0.205 μM and 0.165 mM, respectively.
Fig. 3. 1H NMR monitoring of the reaction of 1a (5 mM) with N-acetyl-L-cysteine
(50 mM) in DMF-d7 at room temperature.
1411HP cells were treated with the most active complexes ent-1a,1d,
1e, 1g or with cisplatin and cleavage/activation of caspase-3 was ana-
lysed after 24 h. For comparison, the sensitive H12.1 cells were also
treated with cisplatin and then examined for the course of caspase-
3 activation. As shown in Fig. 4A treatment with 3 μM cisplatin
(~IC90 for H12.1) had no effect in resistant 1411HP cells whereas
in H12.1 cells it induced distinct caspase-3 activation and apoptosis.
Applying an equitoxic concentration of 10 μM cisplatin (~IC90 for
1411HP) to 1411HP cells initiated caspase-3 activation and apoptosis
which, however, developed more slowly over the first 24 h. In con-
trast, the presence of 3 μM of complexes ent-1a,1d, 1e, or 1g was suf-
ficient to induce caspase-3 activation and apoptosis in 1411HP cells
with the most pronounced effects being elicited by 1d and 1e
(Fig. 4A). Interestingly, cisplatin-induced apoptosis was accompanied
by a rise of p53 levels whereas apoptosis induction after treatment
with complexes 1 appeared to be independent of p53 (Fig. 4A and,
more clearly, Fig. 4B after 48 h). This points to different initial apopto-
tic mechanisms of complexes 1 and cisplatin which may, however,
converge further downstream on the level of effector caspases.
Next we investigated the role of caspases during the apoptotic
process. 1411HP cells were treated with equitoxic, apoptosis inducing
concentrations of complexes ent-1a, 1d, 1e or cisplatin in the pres-
ence or absence of the general caspase inhibitor Z-VAD-Fmk. Then
the cells were analysed for caspase-3 activation and for occurrence
of cells with apoptotic morphology after 48 h. As shown in Fig. 4B
co-treatment with cisplatin and the caspase inhibitor blocked any ac-
tivation of caspase-3 and prevented the occurrence of apoptotic cells.
In contrast, treatment of 1411HP cells with complexes ent-1a, 1d or
1e together with the inhibitor gave rise to cells with apoptotic mor-
phology, albeit somewhat delayed when compared to treatment
with the complexes alone. Again, there was no rise in the levels of
p53 (Fig. 4B). As a proof that the shrunken, detached cells with apo-
ptotic morphology had actually undergone apoptosis despite the
blockage of caspase activation, they were analysed for the DNA frag-
mentation pattern typical of apoptosis. Fig. 4C depicts such a typical
DNA ladder. These data suggest that the complexes 1, like cisplatin,
induce apoptosis in the test cells, yet by a different mechanism
which is largely independent of caspase-3 activation and p53.
Given the marked lipophilicity of complexes 1 and their enhanced
accumulation across cellular membranes we finally investigated their
impact on the mitochondrial membrane potential. Mitochondria
play a pivotal role in the drug-induced apoptotic pathway and are
known to release pro-apoptotic proteins upon cell damage. 1411HP
cells were treated with equitoxic, apoptosis inducing concentrations
of complex 1d or cisplatin and monolayers of such cells were ana-
lysed regarding the status of their mitochondria by means of a
fluorescence-based DePsipher Mitochondrial Potential Kit and re-
garding the overall cell morphology. As shown in Fig. 4D, exposure
to cisplatin led to the occurrence of green cells indicating a loss of
membrane potential in connection with an apoptotic cell morpholo-
gy. In contrast, treatment with 1d appeared to more rapidly induce
a loss of the mitochondrial membrane potential since green cells lack-
ing the apoptotic morphology were observed alongside the green
shrunken ones. This fits in nicely with the different p53 regulation
by cisplatin vs. the Pt-complexes 1. All in all, these results suggest
that the most active complexes 1 induce apoptotic cell death by a
mechanism different from that of cisplatin. This enables them to
break the cisplatin resistance of certain tumours.
Table 1
Inhibitory concentrationsa IC50/IC90 in μM of complexes 1, THPPt-11 and cisplatin when
applied to human H12.1 and 1411HP germ cell cancer cells and calculated resistance
factors.
Compd./cell line
H12.1
1411HP
Resistance factor
Cisplatin
0.7/2.8
1.8/2.5
5.0/8.3
5.0/8.3
7.0/17
5.6/11
5.0/8.3
5.1/8.7
4.3/11
4.0/9.0
43/85
31/84
N100
3.5/10.5
1.7/2.5
2.1/6.0
1.9/2.9
6.0/9.0
5.5/9.0
1.9/2.8
2.0/3.0
4.0/8.0
1.7/2.7
16/24
15/22
17/33
17/27
6.0/14
54/N100
N100
5.0/3.75
0.9/1.0
THPPt-11b
1a
0.42/0.72
0.38/0.35
0.86/0.53
0.98/0.82
0.38/0.34
0.39/0.36
0.93/0.73
0.43/0.3
0.37/0.28
0.48/0.26
b0.17/b0.33
0.32/0.3
0.12/b0.14
b0.54/–
ent-1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
53/90
52/N100
N100
N100
–
4. Conclusions
a
Values are derived from concentration–response curves obtained by the SRB assay
after 96 h. Values represent means of three independent experiments with standard
deviations SDb 15% throughout.
We identified platinum(II) complexes with lipophilic terpenoid
and triphenylethene appendages that can overcome the cisplatin
b
Values are taken from ref. [15].

1636
B. Biersack et al. / Journal of Inorganic Biochemistry 105 (2011) 1630–1637
Fig. 4. Analyses of apoptotic parameters for cells treated with indicated drugs for 24 h (A) and 48 h (B). Morphological changes are indicated by (−) — none, (+) — some, (−/+) —
early stage. Levels of p53 and pro-/caspase-3 ascertained by western blotting with actin as a loading control. The general caspase inhibitor Z-VAD-Fmk (60 μM) was added on sev-
eral occasions. C: cells that shrank and detached upon treatment with 1d and Z-VAD-Fmk were harvested after 48 h and their DNA was extracted and submitted to electrophoresis
on agarose gel followed by EtdBr staining. D: 1411HP cells treated with indicated drugs for 24 h were analysed using DPsipher and viewed by normal light (LM) — and fluorescence
microscopy (FM). White arrows point to green apoptotic cells; intact cells are orange.
resistance of 1411HP germ cell tumour cells. They feature 6-
aminomethylnicotinate ligands with (+)-menthyl (ent-1a), (+)-
cedrenyl (1d), (−)-menthoxypropyl (1e) and 1,1,2-triphenylethene
appendages, or a (−)-menthyl 2,4-diaminobutyrate ligand (1g).
This structure-dependent activity seems to originate from a particu-
larly high accumulation of these complexes in 1411HP cells when
compared to related cisplatin-sensitive H12.1 cells. The complexes 1
were also shown to bind to salmon sperm DNA in a structure-
dependent manner and to interact with thiol groups of amino acids.
Analyses of the mechanism of apoptotic cell death induced by com-
pounds 1 revealed distinct differences to cisplatin, e.g., an indepen-
dency of p53 levels and of caspase activation and an early loss of
the mitochondrial membrane potential. These mechanistic traits of
the complexes 1 in combination with their capacity to intensely inter-
act with DNA, lipid membranes and nucleophilic proteins might
explain their efficacy against cisplatin-resistant 1411HP tumour
cells. The decisive influence of the nature of the terpenoid append-
age of 1 might also offer a way to finetune their bioactivity to medic-
inal needs. The complexes 1, though poorly soluble in water, are well
soluble (N1 mg/mL) in 1:1-mixtures of Tween 80/ethanol which is
a formulation suitable for intraperitoneal application of drugs. In
vivo tests in mice are currently underway.
Acknowledgements
We are grateful to the Deutsche Forschungsgemeinschaft (grant
Scho 402/8-3) and to the Kultusministerium (grant PA3573B/0604T)
and the Federal State of Saxonia-Anhalt (grant FKZ 3646A/0907) for fi-
nancial support. We are indebted to Dr. G. Zoldak, Bayreuth, for assis-
tance with data analysis and to Franziska Reipsch and Katrin Nerger,
Halle, for technical assistance.

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