Carboplatin derivatives with superior antitumor activity compared to the parent compound

Article abstract of DOI:10.1016/j.ica.2004.03.060

New carboplatin derivatives were synthesized by introducing fluoro, chloro, bromo and hydroxy substituents into the cyclobutane ring. The carboxylic acid groups were used for complexation to the Pt(NH₃)₂ fragment as non-leaving group. The antiproliferative activity of the new carboplatin analogues were studied in tests with J82, SK-OV-3 and NIH:OVCAR-3 cancer cells. The best compounds were two times as active as carboplatin itself. A series of new carboplatin derivatives was synthesized by introducing fluoro, chloro, bromo and hydroxy substituents into the cyclobutane ring. The carboxylic acid groups were used for the complexation with platinum(II) fragments bearing two ammonia and (RR/SS)-trans-1,2-diaminocyclohexane ligands, respectively, as non-leaving groups. The antiproliferative activity of the new carboplatin analogues differing in the cyclobutanedicarboxylate ligands and the type of platinum fragment were studied in tests with J82 bladder cancer cells and SK-OV-3 as well as cisplatin-resistant NIH:OVCAR-3 ovarian cancer cells. The most active compounds were the 3-fluoro, 3-chloro and 3,3-difluoro derivatives of carboplatin. NMR spectroscopy showed that cis-diammine(3-chloro-1,1- cyclobutanedicarboxylato)platinum(II) was hydrolyzed much faster than carboplatin explaining its higher cytostatic activity.

Full text of DOI:10.1016/j.ica.2004.03.060

Inorganica Chimica Acta 357 (2004) 4452–4466
www.elsevier.com/locate/ica
Carboplatin derivatives with superior antitumor activity compared
to the parent compound
a
b,
b
b
Gunther Bernhardt , Henri Brunner *, Nick Gruber , Christian Lottner ,
¨
Simi K. Pushpan b,1, Takashi Tsuno c,2, Manfred Zabel
b
a
Institut fu¨r Pharmazie, Universita¨t Regensburg, Germany
Institut fu¨r Anorganische Chemie, Universita¨t Regensburg, Regensburg 93040, Germany
Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University, Narashino, Chiba 275-8575, Japan
b
c
Received 19 February 2004; accepted 26 March 2004
Available online 20 August 2004
Abstract
A series of new carboplatin derivatives was synthesized by introducing fluoro, chloro, bromo and hydroxy substituents into the
cyclobutane ring. The carboxylic acid groups were used for the complexation with platinum(II) fragments bearing two ammonia and
(RR/SS)-trans-1,2-diaminocyclohexane ligands, respectively, as non-leaving groups. The antiproliferative activity of the new carbo-
platin analogues differing in the cyclobutanedicarboxylate ligands and the type of platinum fragment were studied in tests with J82
bladder cancer cells and SK-OV-3 as well as cisplatin-resistant NIH:OVCAR-3 ovarian cancer cells. The most active compounds
were the 3-fluoro, 3-chloro and 3,3-difluoro derivatives of carboplatin. NMR spectroscopy showed that cis-diammine(3-chloro-
1,1-cyclobutanedicarboxylato)platinum(II) was hydrolyzed much faster than carboplatin explaining its higher cytostatic activity.
ꢀ 2004 Elsevier B.V. All rights reserved.
1. Introduction
and intrinsic and acquired drug resistance. Cisplatin
has a limited solubility in aqueous solutions which is a
problem for intravenous application.
The cytostatic activity of cis-diammine(dichloro)plat-
inum(II) (cisplatin) was incidentally discovered in the
1960s by Rosenberg [1]. In 1978 cisplatin was approved
for the treatment of testicular cancer and ovarian can-
cer. It is also used in a number of other human malig-
nancies including cervical, head and neck, esophageal,
lung and bladder cancer [2]. The sales of cisplatin were
US$ 525 million in 1998 [3,4]. Despite its success cisp-
latin suffers from limitations due to severe side effects
These drawbacks gave impetus for the development
of new generations of platinum based anticancer drugs.
However, only cis-diammine(1,1-cyclobutanedicarboxy-
lato)platinum(II) (carboplatin) received worldwide ap-
proval. Carboplatin, less toxic than cisplatin, is active
against the same tumor entities as cisplatin and is also
administered intravenously. In 1989, carboplatin was
approved for the treatment of ovarian cancer. Increas-
ing sales (in 1998 of US$ 132 million) indicate that car-
boplatin is going to substitute cisplatin in many
applications [4]. A third important platinum-based anti-
cancer drug is (R,R-trans-1,2-diaminocyclohexane)oxa-
latoplatinum(II) (oxaliplatin), registered in Europe and
Japan. Oxaliplatin has shown potential against cisp-
latin-resistant tumors and is used to treat colorectal
cancer [5].
*
Corresponding author. Tel.: +49 941 943 4440/4441; fax: +49 941
943 4439.
E-mail address: henri.brunner@chemie.uni-regensburg.de (H.
Brunner).
1
Alexander von Humboldt postdoctoral fellow.
Guest in the Institut fur Anorganische Chemie, Universita¨t
2
¨
Regensburg.
0020-1693/$ - see front matter ꢀ 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2004.03.060

G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
4453
We synthesized new carboplatin-like platinum(II)
complexes by introducing F, Cl, Br and OH substituents
in the 3-position of the cyclobutane ring. In analogy to
carboplatin, ammonia ligands were used as non-leaving
groups. In order to combine the properties of oxaliplatin
with carboplatin we also introduced the (RR,SS)-trans-
1,2-diaminocyclohexane ligand as a non-leaving group
into our carboplatin derivatives. In the present paper,
the synthesis, characterization, and antitumor properties
of seven new carboplatin derivatives are described [6].
derivative 6. Hydrogenolysis was used for the cleavage
of the benzylether 6 yielding the hydroxy compound 7
as described in the literature [8,9]. The carboxylic acid
8, which was required for coordination to the plati-
num(II) moieties, was prepared by hydrolysis of ester
7 with 20% ethanolic KOH solution (Scheme 2).
2.3. Synthesis of 3-bromo-1,1-cyclobutanedicarboxylic
acid (11)
The bromo derivative 11 was prepared starting from
compound 7. The diethyl ester 10 was obtained by reac-
tion of 7 with tosyl chloride to give 9, followed by treat-
ment with lithium bromide under reflux in DMSO. The
resulting diester 10 was hydrolyzed under acidic condi-
tions yielding 11 (Scheme 3).
2. Results and discussion
2.1. Synthesis of 3-chloro-1,1-cyclobutanedicarboxylic
acid (4)
The synthesis of 3-chloro-1,1-cyclobutanedicarboxy-
lic acid 4 starting from 1,1-cyclobutanedicarboxylic acid
1 via its acid chloride 2 was performed according to lit-
erature procedures (Scheme 1) [7]. In the radical chlorin-
ation of 2 we used AIBN as a radical starter to achieve
higher yields instead of commercial dibenzoylperoxide
which contained about 20% of water. Hydrolysis of
the chlorinated acid chloride 3 afforded 4.
2.4. Synthesis of 3-fluoro-1,1-cyclobutanedicarboxylic
acid (13)
Acid 13 had been previously prepared by the reaction
of 1-bromo-3-chloro-2-fluoropropane with diethyl mal-
onate [10]. We prepared it by the reaction of compound
7 with fluorinating agents like Deoxo-Fluor [11,12] and
DAST [13] giving rise to diethyl ester 12 which on acid
hydrolysis afforded 13 as described in the literature
[10] (Scheme 4).
2.2. Synthesis of 3-hydroxy-1,1-cyclobutanedicarboxylic
acid (8)
2.5. Synthesis of 3,3-difluoro-1,1-cyclobutanedicarboxylic
acid (16)
Compound 8 was synthesized in a four step reaction
starting from benzylbromide and epibromohydrin. The
resulting dibromide 5 was reacted with diethyl malonate
and sodium hydride to give the substituted cyclobutane
3,3-Difluoro-1,1-cyclobutanedicarboxylic acid 16 was
prepared starting from 7, which on oxidation formed
O
O
O
O
O
O
OH
OH
Cl
Cl
Cl
Cl
Cl
Cl
OH
OH
ii
i
iii
H
H
O
O
2
1
3
4
Reagents: (i) excess SOCl2, reflux, 3 h; (ii) SO2Cl2, AIBN, reflux, 3 h; (iii) H2O.
Scheme 1.
Br
O
O
O
O
O
Br
O
H
OEt
OEt
HO
H
OEt
OEt
HO
H
OH
OH
iii, iv
i
ii
O
O
5
7
8
6
Reagents: (i) diethyl malonate, excess NaH, dioxane, 150 h; (ii) Pd/charcoal (10% Pd), EtOH; (iii) 20% KOH in
EtOH, reflux, 4 h; (iv) HCl, H2O.
Scheme 2.

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G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
O
O
O
O
O
O
Br
OEt
OEt
Br
OH
OH
ii
i
TsO
H
OEt
OEt
iii
HO
H
OEt
OEt
H
H
O
O
11
9
10
7
Reagents: (i) 1.1 eq. tosyl chloride, Et3N, CH2Cl2, rt, 24 h; (ii) 1 eq. LiBr, DMSO, 140 oC, 16 h; (iii) 6M HCl,
EtOH, 60 oC, 2 d.
Scheme 3.
O
O
O
O
O
O
F
OH
OH
F
OEt
OEt
HO
H
OEt
OEt
i or ii
iii
H
H
13
12
7
Reagents: (i) 1 eq. Deoxo-Fluor, CH2Cl2, rt, 24 h; (ii) 1 eq. DAST, CH2Cl2, rt, 18 h; (iii) 6M HCl solution,
reflux, 18 h.
Scheme 4.
O
O
O
O
O
O
O
F
F
OEt
OEt
F
F
OH
OH
ii
i
OEt
OEt
iii
HO
H
OEt
OEt
O
O
16
14
15
7
Reagents: (i) RuO4, NaIO4, CCl4, rt; (ii) 2.2 eq. DAST, CH2Cl2, rt, 24 h; (iii) 6M HCl, EtOH, reflux, 16 h.
Scheme 5.
diethyl 3-oxocyclobutane-1,1-dicarboxylate 14 as re-
ported in the literature [14]. This oxidized product was
reacted with the fluorinating agent DAST following a
related literature procedure [15] to obtain 15 which on
hydrolysis gave 16 (Scheme 5).
added. The solution was stirred at room temperature
under exclusion of light for 5 d. The grey precipitate
was filtered off. After removal of the solvent from the fil-
trate the residue was recrystallized from water/acetone
to give the colorless carboplatin derivatives 20–25
(Scheme 6).
2.6. Synthesis of the cis-diammineplatinum(II) complexes
For compound 22 the molecular structure was deter-
mined by X-ray diffraction. As expected, the coordination
geometry around platinum(II) is square planar. The six-
membered chelate ring is in a boat conformation. For
C5 of the cyclobutane ring a thermal oscillation is ob-
served indicating a dynamic puckering of the cyclobutane
ring at a temperature of 173 K. The torsion angle C6–C2–
C4–C5 is 20.1(7)ꢁ. The C-O bond lengths of the chelate
ring are 5 pm longer than those to O2 and O3 (Fig. 1).
For the reaction with the cyclobutanedicarboxylic
acids 4, 8, 11, 13 and 16 it was necessary to transform
cisplatin 17 into an active hydrolysis product. cisplatin
was stirred with two mol equivalents of silver nitrate
in aqueous solution under exclusion of light for one
week. The chloro ligands were substituted by aqua lig-
ands and AgCl precipitated. The exchange of the nitrate
counter ions in 18 for hydroxide ions was achieved by
chromatography on a strongly basic anion exchanger
in which the reactive platinum(II) complex 19 was
formed (Scheme 6) [16].
2.7. Synthesis of the (RR/SS)-trans-1,2-diaminocyclo-
hexaneplatinum(II) complexes
Compounds 4, 8, 11, 13 and 16 were dissolved in
water before an equimolar amount of an aqueous solu-
tion of diammine(diaqua)platinum(II) hydroxide 19 was
Commercial (RR/SS)-trans-1,2-diaminocyclohexane
was used to prepare the corresponding dichloroplati-
num(II) complex 26 [17], which had to be activated by

G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
4455
i
ii
(NH3)2PtCl2
[(NH3)2(H2O)2Pt](NO3)2
[(NH3)2(H2O)2Pt](OH)2
17
18
19
O
O
R
R'
OH
OH
R
O
O
NH3
NH3
iii
+
Pt
[(NH3)2(H2O)2Pt](OH)2
R'
O
O
20: R = H, R' = H
21: R = Cl, R' = H
22: R = OH, R' = H
23: R = Br, R' = H
24: R = F, R' = H
25: R = F, R' = F
19
1: R = H, R' = H
4: R = Cl, R' = H
8: R = OH, R' = H
11: R = Br, R' = H
13: R = F, R' = H
16: R = F, R' = F
Reagents: (i) 2 eq. AgNO3, H2O, 7 d; (ii) strongly basic anion exchanger; (iii) EtOH, H2O, 5 d.
Scheme 6.
formation in which the amine substituents are arranged
in equatorial position as expected (Fig. 2).
2.8. Spectroscopy
The progress of the complexation of the carboxylic
acid ligands with the platinum fragment was moni-
tored by IR spectroscopy. The CO absorptions of
the carboxylic acids 4, 8, 11, 13 and 16 appeared in
the range 1725–1695 cmꢀ1, whereas the platinum(II)
complexes 21–25 and 28–30 afforded two new bands
at 1645–1595 and 1395–1355 cmꢀ1. The NMR spectra
of the carboplatin derivatives 21–25 and 28–30 re-
vealed sharp signals which were shifted to higher mag-
netic field compared with the cyclobutanedicarboxylic
acids 4, 8, 11, 13 and 16. Further evidence for the for-
mation of complexes 21–25 and 28–30 was derived
from the mass spectra which showed the respective
molecular ions.
Fig. 1. The molecular structure of 22.
conversion into the diamine(dihydroxy)platinum(II)
hydroxide 27 in analogy to cisplatin (Scheme 7) [16,18].
The activated platinum species 27 was reacted with
an equivalent amount of the respective carboxylic acid
in a mixture of water and ethanol. The solution was stir-
red at room temperature under exclusion of light for
several days. The grey precipitate was filtered off. After
removal of the solvent from the filtrate and recrystalliza-
tion from water the colorless products 28–30 were ob-
tained (Scheme 7).
For compound 30 the molecular structure was deter-
mined by X-ray diffraction. The local geometry around
the platinum atom approximates a square planar
arrangement in analogy to the crystal structure of 22.
For the chelate ring again a boat conformation was ob-
served. The torsion angle C6–C3–C4–C5 is 12.6(5)ꢁ
illustrating the bending of the cyclobutane ring. The
cyclohexane ring of the amine ligand takes a chair con-
2.9. Cell lines and general procedure
To determine the antiproliferative activity of the new
carboplatin derivatives the bladder cancer cell line J82
was selected as our first in vitro model. The J82 cell line
was established from a transitional cell carcinoma of a
58 years old Caucasian male [19]. As in these tests chlo-
rocarboplatin 21 proved to be two times as effective as
carboplatin and 3-hydroxycarboplatin 22, we synthe-
sized the 3-bromo, 3-fluoro and 3,3-difluoro derivatives
23-25. These compounds were tested with the ovarian
cancer cell lines SK-OV-3, a human adenocarcinoma de-
rived from the ascitic fluid of a 64 years old Caucasian
[20], and NIH:OVCAR-3, a human adenocarcinoma de-
rived from a 60 years old Caucasian [21]. Of the latter
cell line a threefold cisplatin-resistant variant, which

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G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
H2
N
Pt
N
H2
H2
N
Pt
N
H2
Cl
Cl
OH2
OH2
i, ii
(OH)2
26
27
O
O
O
H2
N
Pt
N
H2
H2
N
R
H
OH
OH
OH2
R
H
O
iii
+
(OH)2
Pt
OH2
O
N
H2
O
1: R = H
4: R = Cl
27
28: R = H
29: R = Cl
8: R = OH
30: R = OH
Reagents: (i) 2 eq. AgNO3, H2O, 7 d; (ii) strongly basic anion exchanger; (iii) EtOH, H2O, 5 d.
Scheme 7.
statistically significant cytotoxicity was observed for car-
boplatin and compounds 21 and 22. At a concentration
of 5 lM, the cytotoxic effect of the chloro derivative 21
amounted to a T/Ccorr. value of approximately 40%,
whereas carboplatin and the hydroxy derivative 22
reached only half of the effectiveness of 21. At 10 lM,
21 gave a T/Ccorr. value of 4%, significantly lower than
carboplatin and 22 (Table 1).
The platinum complexes 28–30 with (RR/SS)-trans-
1,2-diaminocyclohexane as non-leaving group showed
T/Ccorr. values of about 50–85% at a dosage of 0.5
lM. At higher concentrations, interestingly, it was again
the chloro derivative 29 which turned out to be the most
effective compound in terms of cytotoxicity. In general,
the diaminoplatinum(II) complexes 28–30 had a higher
cytotoxic potential than the respective diammineplati-
num(II) complexes 20–22, the chloro derivative 29 being
the most active with T/Ccorr. values of 33.1% at 1 lM,
10.5% at 5 lM and ꢀ1.3% at 10 lM. However, the
diaminoplatinum(II) complexes 28–30 were only soluble
in DMF, in contrast to the water-soluble diammineplat-
inum(II) complexes 20–22 (Table 1).
Fig. 2. The molecular structure of 30.
had been generated by subculturing the wild type cells in
the presence of increasing concentrations of cisplatin for
150 days, was used for cytotoxicity testing. We used
computerized chemosensitivity assays based on the
quantification of biomass by staining viable cells with
crystal violet [22,23].
2.10. Cytotoxicity testing against J82 cells
The antitumor activity of the promising carboplatin
derivative 21 was also analyzed with the J82 bladder
cancer cell line in a kinetic assay. This procedure pro-
vides information concerning activity (cytotoxic, cyto-
J82 cells were incubated for 4 days with 0.5, 1, 5 and
10 lM solutions of the carboplatin derivatives and car-
boplatin as reference. At a dosage of 0.5 and 1 lM, no
Table 1
T/Ccorr. values or cytocidal effect (underlined) of the carboplatin derivatives 21, 22 and 28–30 at 0.5, 1, 5 and 10 lM concentration with carboplatin
20 as reference for the J82 cell line after the incubation period of 96 h
c [10ꢀ6 mol Lꢀ1
]
0.5
94.4 114.0
1
5
10
Carboplatin
95.5 12.3
93.4 16.1
83.3 13.2
44.3 22.7
71.4 19.6
31.5 11.0
10.5 3. 5
14.3 4.2
49.8 10.6
4.1 5.0
41.6 21.5
14.5 4.5
1.3 3.3
7.2 3.9
21
22
28
29
30
115.3 13.8
105.2 11.3
87.2 14.6
64.7 11.3
52.4 11.8
110.2 11.8
63.4 13.1
33.1 10.6
51.6 10.7

G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
4457
static, or cytocidal) and inactivation as a function of
time [22,23].
clinically used carboplatin 20. Interestingly, the mono-
fluoro derivative 24 had the highest efficacy among all
the derivatives tested (Fig. 4). It was followed by the
chloro, difluoro, hydroxy and bromo derivatives in that
order (Table 2). Remarkably, the differences between
the fluoro, chloro and difluoro compounds 24, 21 and
25 were comparatively small. The improved antiprolif-
erative effect of the fluoro derivative 24 showed up in
the T/Ccorr. values 67.5%, 50.3%, 18.2% and 1.1% for
0.5, 1, 2 and 3 lM concentrations after an incubation
period of 264 h. The corresponding T/Ccorr. values for
carboplatin were 88.6, 57.1, 48.3 and 29.8%,
respectively.
In the experiments with the cisplatin-resistant NI-
H:OVCAR-3 subline we used the same four concentra-
tions of carboplatin and its derivatives as described for
the SK-OV-3 cell line. In this case too the monofluoro
derivative 24 was the most active compound (Fig. 5).
It was followed by the difluoro, fluoro, hydroxy and
bromo derivatives, although the differences for the
fluoro, difluoro and chloro compounds were only small
(Table 3). The high antiproliferative effect of the fluoro
derivative 24 was evident from the T/Ccorr. values
74.5% and 65.9% for 0.5 and 1 lM and the cytocidal ef-
fect of 8.9% and 12.3% for 2 and 3 lM concentrations
after an incubation period of 239 h, respectively. The
corresponding T/Ccorr. values for carboplatin were
100.7%, 78.9%, 63.5% and 25.6%, respectively. In both
the cell lines the difluoro derivative 25 started its cyto-
toxic effect faster and at lower concentrations (0.5 and
1 lM) than the other compounds (Tables 2 and 3).
However, later and at higher concentrations (2 and
T/Ccorr. values or percent cytocidal effect (left ordi-
nate) for the test compounds were plotted together
with the absorbance of the untreated solvent control
(right ordinate) vs time of drug exposure. In these plots
time zero indicates the time at which the drug was
added. In these experiments, the drug containing cul-
ture media was left unchanged throughout the incuba-
tion period.
Fig. 3 shows classical dose–response relationships for
carboplatin 20 (A) and its chloro analogue 21 (B). Inter-
estingly, the chloro compound 21 has a much higher
cytotoxic potential at the three tested concentrations
than the clinically used carboplatin. A clear antiprolifer-
ative effect is observed as a function of time for all three
concentrations of 21 resulting in T/Ccorr. values of 34.0
and 0.8% for 5 and 10 lM and a cytocidal effect of
45.4% for 20 lM after an incubation period of 118.5
h. The corresponding T/Ccorr. values for carboplatin
are 64.2, 35.6 and 9.5% for 5, 10 and 20 lM concentra-
tion, respectively.
2.11. Cytotoxicity testing against SK-OV-3 cells and
cisplatin-resistant NIH:OVCAR-3 cells
In a similar kinetic assay the antitumor activity of
platinum complexes 21–25 was analyzed with the SK-
OV-3 ovarian cancer cell line. Four different concentra-
tions (0.5, 1, 2 and 3 lM) of the platinum complexes
were administered to the SK-OV-3 cell line along with
160
140
120
100
80
2.5
2.0
1.5
1.0
0.5
0.0
160
140
120
100
80
2.5
2.0
1.5
1.0
0.5
0.0
60
60
40
20
40
0
20
20
40
60
0
20
0
25
50
75
100
125
0
25
50
75
100
125
(a)
(b)
time of incubation [h]
time of incubation [h]
Fig. 3. Dose–response relationship of carboplatin 20 (a) and the chloro derivative 21 (b) on the proliferation of long term incubated J82 cells (in
passage 40 from origin) at a concentration of 5 (}), 10 (ꢀ), and 20 lM (M). Proliferation kinetics of the corresponding control (absorbance at 578
nm) (d).

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G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
12 0
10 0
80
120
1.5
1.5
1.0
0.5
0.0
100
80
1.0
60
60
40
40
0.5
20
20
0
0
20
0.0
20
0
50
100
150
200
250
0
50
100
150
200
250
time of incubation [h]
time of incubation [h]
(a)
(b)
Fig. 4. Dose–response relationship of carboplatin 20 (a) and the fluoro derivative 24 (b) on the proliferation of long term incubated SK-OV-3 cells
(in passage 77 from origin) at a concentration of 0.5 (s), 1 lM (}), 2 lM (n) and 3 lM (h). Proliferation kinetics of the corresponding control
(absorbance at 578 nm) (d).
Table 2
T/Ccorr. values of the carboplatin derivatives 21–25 at 0.5, 1, 2 and 3 lm concentration with carboplatin 20 as reference for the SK-OV-3 cell line after
the incubation period of 264 h
c [10ꢀ6molꢀ1
]
0.5
1
2
3
Carboplatin
88.6 17.8
94.9 18.2
94.5 18.0
91.9 + 22.3
67.5 18.8
58.1 25.1
57.1 22.6
55.9 + 16.4
79.8 19.0
87.0 21.7
50.3 19.6
50.3 22.5
48.3 26.2
28.2 + 17.3
47.1 15.2
48.2 + 17.5
18.2 + 5.5
12.1 8.2
29.8 7.8
6.9 4.9
21
22
23
24
25
19.0 11.5
20.7 + 9.0
1.1 2.7
7.9 8.9
120
100
80
120
100
80
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
60
60
40
40
20
20
0
0
B
A
20
20
0
50
100
150
200
250
0
50
100
150
200
250
(a)
time of incubation [h]
(b)
time of incubation [h]
Fig. 5. Dose–response relationship of carboplatin 20 (a) and the fluoro derivative 24 (b) on the proliferation of long term incubated NIH:OVCAR 3
cells (in passage 56 from origin) at a concentration of 0.5 lM (s), 1 lM (}), 2 lM (n) and 3 lM (h). Proliferation kinetics of the corresponding
control (absorbance at 578 nm) (d).

G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
4459
Table 3
T/Ccorr. values or cytocidal effect (underlined) of the carboplatin derivatives 21–25 at 0.5, 1, 2 and 3 lM concentration with carboplatin 20 as
reference for the NIH:OVCAR-3 cell line after the incubation period of 239 h
c [10ꢀ6molꢀ1
]
0.5
1
2
3
Carboplatin
100.7 4.8
96.1 9.4
96.5 6.4
87.2 14.6
74.5 14.4
60.3 16.2
78.9 19.0
48.5 25.9
89.1 + 13.3
63.4 13.1
65.9 14.7
41.6 16.4
63.5 35.6
1.9 18.2
70.7 30.3
31.5 11.0
8.9 3.0
25.6 17.9
5.4 10.6
10.1 4.1
14.5 4.5
12.3 0.5
9.3 0.9
21
22
23
24
25
8.7 2.0
3 lM) its activity levelled off and it was outperformed by
the fluoro compound 24 and in part by the chloro com-
pound 21.
C4 of the free cyclobutanedicarboxylic acid 1. In the
course of 168 h the area of this integral increased,
whereas the signal at 2.80 ppm decreased (Fig. 6). For
the chloro derivative 21 cis and trans protons could be
distinguished for the CH2 groups at C2 and C4 splitting
in a symmetrical pattern of signals at 3.52 and 2.97 ppm.
After 1 h the CH2 groups of the free cyclobutanedi-
carboxylic acid 4 can be easily identified because of their
highfield shifted signals (Fig. 7).
A quantitative analysis of these kinetic data was
based on the time dependent integration of respective
CH2 signals. For carboplatin the signals at 2.80 and
2.26 ppm and for 21 the signals at 3.52/2.97 and 2.84/
2.53 ppm were compared, respectively. After 48 h 92%
of carboplatin but only 76% of complex 21 were left.
After 168 h the amount of 21 had dropped to 61% com-
pared with 85% of carboplatin. Thus, the chloro com-
plex 21 was hydrolyzed much faster than carboplatin
20 explaining its higher cytostatic activity.
2.12. Investigation of the stability of 21
The extraordinary activity of the chloro derivative 21
in cell biological experiments prompted us to investigate
its stability compared to carboplatin by 1H NMR
spectroscopy. Both compounds (2.5 lM) were dissolved
in 1 mL of a 0.9% solution of NaCl in D2O which is
equivalent to a physiological NaCl solution. The
NMR tubes were kept in the dark at room temperature.
For carboplatin the signal of the equivalent CH2
groups at C2 and C4 was a triplet at 2.80 ppm. The
CH2 group at C3 appeared as a quintet at 1.81 ppm with
3
a coupling constant of J(H,H) = 7.8 Hz. After 1 h a
new group of signals emerged at 2.26 ppm which could
be assigned to the two equivalent CH2 groups at C2 and
Fig. 6. 1H NMR spectra (300 MHz, D2O/0.9% NaCl, 24 ꢁC, TMS external) of carboplatin 20.

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G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
Fig. 7. 1H NMR spectra (300 MHz, D2O/0.9% NaCl, 24 ꢁC, TMS external) of 21.
3. Conclusion
4.2. 1,1-Cyclobutanedicarboxylic acid dichloride (2)
We synthesized the hitherto unknown fluoro, chloro,
bromo and hydroxy derivatives of the clinically used
drug carboplatin. The highest antiproliferative effect
was obtained for the fluoro and chloro derivatives 24,
25, 21 and 29. In the series of the cis-diammine com-
plexes the fluoro and chloro derivatives 24, 25 and 21
showed a much higher cytotoxic potential than carbo-
platin. Moreover, derivatives 24, 25 and 21 showed a
good solubility in water, comparable to carboplatin, in
contrast to the (RR,SS)-trans-1,2-diaminocyclohexane
complexes 28–30 which were only soluble in DMF.
2 was prepared according to a literature procedure
from 1,1-cyclobutanedicarboxylic acid 1 and thionyl
1
chloride [7]. IR (film): m 1790 (C@O) cmꢀ1. H NMR
3
(CDCl3): d 2.74 (t, J = 7.9 Hz, 4 H, CH2(CH2)2), 2.05
3
(qi, J = 7.9 Hz, 2 H, CH (CH2)2) ppm. MS (EI): m/z
2
(rel. int.) 145 (M ꢀ Cl, 100), 117 (M ꢀ COCl, 60), 89
(M ꢀ (CO)2Cl, 80), 54 (M ꢀ 2COCl, 36).
4.3. 3-Chloro-1,1-cyclobutanedicarboxylic acid dichloride
(3)
3 was prepared in analogy to a literature procedure
by reaction of 2 with sulfuryl chloride and AIBN as rad-
4. Experimental
1
ical starter [7]. IR (film): m 1780 (C@O) cmꢀ1. H NMR
3
4.1. Chemistry
(CDCl3): d 4.41 (qi, J = 7.9 Hz, 1 H, CHCl(CH2)2),
3.33, 2.99 (2 m, 4 H, CHCl(CH2)2C(COCl)2) ppm. MS
IR: Beckman spectrometer 4240. H and 13C NMR:
(EI): m/z (rel. int.) 179 (M ꢀ Cl, 100).
1
Bruker WM 250 (250 MHz) and Avance 300 (300
MHz); chemical shifts in ppm; TMS as internal stand-
ard. MS: Finnigan MAT 95, MAT 112 S and MAT
311 A, ThermoQuest Finnigan 7000. The molecular
ion is designated M; only the most intense peak is spec-
ified in an ion cluster. Mp: Buchi SMP 20; not corrected.
¨
UV/Vis: Kontron Instruments spectrophotometer UVI-
KON 922.
4.4. 3-Chloro-1,1-cyclobutanedicarboxylic acid (4)
4 was prepared according to a literature procedure
from 3 by hydrolysis [7]. IR (KBr): m 3000 (OH), 1700
1
(C@O) cmꢀ1. H NMR (DMSO-d6): d 13.05 (s, 2 H,
CO2H), 4.41 (qi, J = 7.9 Hz, 1 H, CHCl(CH2)2), 3.01,
3
Solid reagents were used as obtained from commer-
cial suppliers without further purification; liquids were
freshly distilled before use.
2.64 (2m, 4 H, CHCl(CH2)2C(CO2H)2) ppm. MS (EI):
m/z (rel. int.) 178 (M, 2), 143 (M ꢀ Cl, 20), 133
(M ꢀ CO2H, 25).

G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
4461
4.5. 2-Benzyloxy-1,3-dibromopropane (5)
stirred in CH2Cl2 (50 mL) at room temperature under
nitrogen for 24 h and then filtered. The filtrate was evap-
orated and the residue was purified by chromatography
on silica gel using EtOAc/petroleum ether (3:7) as an
eluent yielding 9 as a colorless oil. Yield: 16.9 g (89%,
5 was prepared according to a literature procedure
from epibromohydrin and benzylbromide [8]. IR (film):
1
m 1590, 1510, 1490 (C@C) cmꢀ1. H NMR (C6D6): d
7.30–7.00 (m, 5 H, C6H5), 4.12 (s, 2 H, C6H5CH2),
3.24 (qi, 3J = 4.8 Hz, 1 H, CH(CH2Br)2), 3.11 (d,
3J = 4.8 Hz, 4 H, CH(CH2Br)2) ppm. MS (EI): m/z
(rel. int.) 308 (M, 5), 213 (M ꢀ CH2Br, 5), 91 (C7H7,
100).
purity >90%). IR (CHCl3): m 1715 (C@O) cmꢀ1
.
1H
3
NMR (300 MHz, CDCl3): d 7.78 (d, 2 H, J = 8.5 Hz,
3
ArH), 7.34 (d, 2 H, J = 8.5 Hz, ArH), 4.89 (qi, 1 H,
3J = 8.4 Hz, CH(CH2)2), 4.18 (q, 4 H, 3J = 7.1 Hz,
CH2CH3), 2.80, 2.68 (2 m, 4 H, CH(CH2)2), 2.45 (s, 3
H, C6H4CH3), 1.24 (t, 6 H, 3J = 7.1 Hz, CH2CH3)
ppm. 13C NMR (75 MHz, CDCl3): d 13.92 (q), 13.94
(q), 21.62 (q), 37.58 (t, 2C, 2- and 4-C), 46.44 (s, 1-C),
61.66 (t), 61.81 (t), 68.89 (d, 3-C), 127.82 (d, 2C),
129.90 (d, 2C), 133.48 (s), 145.08 (s), 169.90 (s, CO),
170.76 (s, CO) ppm.
4.6. Diethyl3-benzyloxy-1,1-cyclobutanedicarboxylate(6)
6 was prepared according to a literature procedure
from 5 and diethyl malonate [8]. IR (film): m 1740
1
(C@O), 1500 (C@C) cmꢀ1. H NMR (C6D6): d 7.23–
7.04 (m, 5 H, C6H5), 4.14, 4.11, 4.08 (3 m, 3 H,
C6H5CH2OCH), 3.94, 3.93 (2 q, 3J = 7.1 Hz, 4 H,
CH2CH3), 2.90, 2.76 (2 m, 4 H, CH(CH2)2), 0.89 (t,
3J = 7.1Hz, 6 H, CH2 CH3) ppm. MS (EI): m/z (rel.
int.) 306 (M, 20), 91 (C7H7, 100).
4.10. Diethyl 3-bromo-1,1-cyclobutanedicarboxylate (10)
9 (3.74 g, 10.1 mmol) and LiBr (0.90 g, 10.4 mmol)
were heated in DMSO (15 mL) for 16 h at 140 ꢁC un-
der nitrogen. After addition of water, the reaction mix-
ture was extracted twice with 50 mL of ether. The
combined organic layers were washed with water (50
mL), dried over Na2SO4 and evaporated. The residue
was chromatographed on silica gel using EtOAc/petro-
leum ether (1:49) to give 10 as a colorless oil. Yield:
1.39 g (49%). IR (CHCl3): m 1745 (C@O), 1715
4.7. Diethyl 3-Hydroxy-1,1-cyclobutanedicarboxylate (7)
7 was prepared according to a literature procedure
from 6 by hydrogenolysis [8]. IR (film): m 1730 (C@O)
1
3
cm ꢀ1. H NMR (CDCl3): d 4.38 (qi, J = 7.5 Hz, 1 H,
CH(OH)(CH2)2), 4.21 (q, J = 7.1 Hz, 4 H, CH2CH3),
2.89, 2.46 (2 m, 4 H, CH(CH2)2), 2.26 (bs, 1H, OH),
(C@O) cmꢀ1 1H NMR (300 MHz, CDCl3): d 4.28
.
3
(qi, 1 H, 3J = 9.2 Hz, BrCH), 4.23 (q, 2 H, 3J = 7.1
Hz, CH2CH3), 4.22 (q, 2 H, 3J = 7.1 Hz, CH2CH3),
3.17, 2.99 (2 m, 4 H, CH(CH2)2), 1.27 (t, 3 H,
3J = 7.1 Hz, CH2CH3), 1.26 (t, 3 H, 3J = 7.1 Hz,
CH2CH3) ppm. 13C NMR (75 MHz, CDCl3): d 13.96
(q, 2C), 35.39 (d, 3-C), 41.27 (t, 2C, 2- and 4-C),
50.76 (s, 1-C), 61.78 (t), 61.93 (t), 169.79 (s, CO),
170.69 (s, CO) ppm. MS (CI): m/z (rel. int.) 298
[M(81Br) + NH4, 91], 296 [M(79Br) + NH4, 100], 281
[M(81Br) + H, 37], 279 ([M(79Br) + H, 41]. CI HRMS
(C10H1679BrO4, M + H) calc. 279.0232; found
279.0230. Anal. Calc. for C10H15BrO4, 279.13: C,
43.03; H, 5.42. Found: C, 43.05; H, 5.38%.
3
1.26 (t, J = 7.1 Hz, 6H, CH2CH3) ppm. MS (EI): m/z
(rel. int.) 216 (M, 15).
4.8. 3-Hydroxy-1,1-cyclobutanedicarboxylic acid (8)
7 (4.32 g, 0.02 mol) was dissolved in a solution of
KOH (5.43 g, 0.097 mol) in 13.55 mL of ethanol and ref-
luxed for 4 h. After removal of the solvent the residue
was taken up in 15 mL of water. Under ice cooling con-
centrated HCl was added to pH 1. Subsequent ether
extraction, evaporation and recrystallization from ethyl
acetate afforded 7. Yield: 1.15 g (36%), colorless solid,
m.p. 128 ꢁC. IR (KBr): m 3440 (OH), 1720 (C@O)
4.11. 3-Bromo-1,1-cyclobutanedicarboxylic acid (11)
1
cmꢀ1. H NMR (DMSO-d6): d 12.68 (bs, 2 H, COOH),
3
5.26 (bs, 1 H, CH(OH)(CH2)2), 4.06 (qi, J = 7.5 Hz, 1
10 (481 mg, 1.73 mmol), 6 M HCl (15 mL) and etha-
nol (1.0 mL) were stirred at 60 ꢁC for 2 d. Then the reac-
tion mixture was washed twice with CHCl3 (20 mL). The
organic layer was extracted with water (20 mL). The
combined aqueous layers were evaporated to give color-
less crystals of 11 which were recrystallized from ace-
tone/CHCl3. Yield: 208 mg (53%), m.p. 157.5–160.2
ꢁC. IR (KBr): m 3700–2400 (COOH), 1720 (C@O),
H, CH(OH)(CH2)2), 2.58, 2.23 (2 m, 4 H, CH(CH2)2)
ppm. MS (DCI): m/z (rel. int.) 178 (M + NH4, 100),
160 (M, 10). Anal. Calc. for C6H8O5, 160.13: C, 45.01;
H, 5.04. Found: C, 44.86; H, 5.19%.
4.9. Diethyl 3-(4-toluene sulfonyl)-1,1-cyclobutanedi-
carboxylate (9)
1
1700 (C@O) cmꢀ1. H NMR (300 MHz, acetone-d6): d
7 (11.1 g, 51.4 mmol), tosyl chloride (10.7 g, 56.1
mmol) and triethylamine (7.50 mL, 53.8 mmol) were
11.50 (bs, 2 H, COOH), 4.63 (qi, 1 H, 3J = 8.0 Hz,
BrCH), 3.22, 2.96 (2 m, 4 H, CH(CH2)2) ppm. 13C

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G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
NMR (75 MHz, acetone-d6): d 37.07 (d, 3-C), 42.21 (t,
2C, 2- and 4-C), 51.08 (s, 1-C), 171.30 (s, CO), 171.98
(s, CO) ppm. MS (CIQI): m/z (rel. int.) 223
[M(81Br) ꢀ H, 96], 221 [M(79Br) ꢀ H, 100]. Anal. Calc.
for C6H7BrO4, 223.02: C, 32.31; H, 3.16. Found: C,
32.23; H, 3.26%.
4.14. 3,3-Difluoro-1,1-cyclobutanedicarboxylic acid (16)
15 (1.05 g, 4.46 mmol) was refluxed in 6 M HCl (20
mL) for 15 h and washed with CHCl3 (20 mL). The
aqueous layer was evaporated and the solid was washed
with CH2Cl2 to yield colorless crystals of 16. Yield: 337
mg (42%), m.p. 170.2–171.2 ꢁC. IR (KBr): m 3800–2700
(COOH), 1725 (C@O), 1695 (C@O) cmꢀ1 1H NMR
.
4.12. Diethyl 3-fluoro-1,1-cyclobutanedicarboxylate (12)
(300 MHz, acetone-d6): d 9.80 (bs, 2 H, COOH), 3.17
3
(t, 4 H, JH–F = 12.2 Hz, CF2(CH2)2) ppm. 13C NMR
4.12.1. Deoxo-fluor variant
(75 MHz, acetone-d6): d 42.60 (st, JC–F = 10.5 Hz, 1-
C), 43.24 (tt, 2C, J C–F = 24.8 Hz, 2- and 4-C), 118.96
(st, JC–F = 274.5 Hz, 3-C), 171.16 (st, 2C, JC–F = 2.3
Hz, CO) ppm. MS (CIQI): m/z (rel. int.) 179 (M ꢀ H,
51), 140 (100). Anal. Calc. for C6H6F2O4, 180.11: C,
40.01; H, 3.36. Found: C, 40.17; H, 3.34%.
Bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-
Fluorꢂ) (3.29 g, 14.9 mmol) was slowly added to a solu-
tion of 7 (3.19 g, 14.8 mmol) in CH2Cl2 (15 mL) under
nitrogen at room temperature. The mixture was stirred
for 24 h and then evaporated. The yellow oily residue
was chromatographed on silica gel using EtOAc/petrole-
um ether (1:49) as eluent to obtain 12. Yield: 1.17 g
(36%).
4.15. General procedure 1 (GP 1)
Diammine(diaqua)platinum(II) hydroxide 19 was
synthesized from diammine(dichloro)platinum(II) (cis-
platin) 17 as previously described [16]. After the solvent
had been removed a glassy solid was obtained which was
dissolved in a 1:1 mixture of water/ethanol just before
reaction with the respective cyclobutanedicarboxylate
ligands. Compounds 4, 8, 11, 13 and 16 were dissolved
in water before an equimolar amount of a solution of
diammine(diaqua)platinum(II) hydroxide 19 was added.
The solution was stirred for 5 d at room temperature un-
der exclusion of light. The grey precipitate was filtered
off. After removal of the solvent from the filtrate and
recrystallization from H2O/acetone at room temperature
colorless crystals were obtained.
4.12.2. DAST variant
To a solution of 7 (3.12 g, 14.4 mmol) in CH2Cl2 (25
mL) was slowly added diethylaminosulfur trifluoride
(DAST) (2.10 mL, 14.9 mmol) in CH2Cl2 (10 mL) under
nitrogen. The mixture was stirred for 18 h at room tem-
perature and then evaporated. The residue was extracted
with CH2Cl2. Chromatography as described above gave
12. Yield: 831 mg (26%). Spectroscopic data of 12
matched those of the literature [10].
4.13. Diethyl 3,3-difluoro-1,1-cyclobutanedicarboxylate
(15)
Diethylaminosulfur trifluoride (DAST) (2.9 mL,
22.0 mmol) dissolved in CH2Cl2 (10 mL) was slowly
added under nitrogen to CH2Cl2 (20 mL) solution of
diethyl 3-oxacyclobutane-1,1-dicarboxylate 14 [13]
(2.15 g, 10.0 mmol) and stirred for 24 h at 20 ꢁC.
The reaction mixture was quenched with water (50
mL) and extracted twice with CH2Cl2 (20 mL). The
combined organic layers were dried over Na2SO4 and
evaporated. The residual oil was chromatographed on
silica gel using EtOAc/petroleum ether (1:49) to give
15 as a yellow oil. Yield: 1.83 g (77%). IR (CHCl3): m
4.16. cis-Diammine(3-chloro-1,1-cyclobutanedicarboxy-
lato)platinum(II) (21)
According to GP 1, 4 (71.4 mg, 0.402 mmol) was dis-
solved in 10 mL of water, combined with 0.4 mmol of
the aqueous solution of diammine(diaqua)platinum(II)
hydroxide 19 and stirred for 5 d. Yield: 90 mg (50%),
colorless needles, m.p. 210 ꢁC. IR (KBr): m 3520–3200
(NH3), 1630, 1370 (C@O) cmꢀ1
.
1H NMR (D2O): d
3
4.67 (bs, 6 H, NH3), 4.30 (qi, J = 7.6 Hz, 1 H, CH-
Cl(CH2)2), 3.45/2.91 (2 m, 4 H, CHCl(CH2)2) ppm.
MS (LISI): m/z (rel. int.) 407 (MH, 100). Anal. Calc.
for C6H11ClN2O4Pt, 405.70: C, 17.76; H, 2.73; N,
6.93. Found: C, 18.03; H, 2.96; N, 6.58%.
1735 (C@O), 1715 (C@O) cmꢀ1
.
1H NMR (300
3
MHz, CDCl3): d 4.25 (q, 4 H, J = 7.1 Hz, CH2CH3),
3.14 (t, 4 H, JH–F = 11.8 Hz, CF2(CH2)2), 1.28 (t, 6
3
3
H, J = 7.1 Hz, CH2CH3) ppm. 13C NMR (75 MHz,
CDCl3): d 13.91 (q, 2C), 42.47 (st, JH-F = 8.9 Hz, 1-
C), 42.81 (tt, JC–F = 25.6 Hz, 2- and 4-C), 62.25 (t,
2C), 113.37 (st, JC–F = 271.0 Hz, 3-C), 169.64 (st, 2C,
JC–F = 2.2 Hz, CO) ppm. MS (EI): m/z (rel. int.) 236
(M, 6), 209 (47), 191 (80), 164 (18), 143 (18), 135
(46), 87 (36), 54 (28), 29 (100). Anal. Calc. for
C10H14F2O4, 236.21: C, 50.85; H, 5.97. Found: C,
50.51; H, 5.59%.
4.17.
oxylato)platinum(II) (22)
cis-Diammine(3-hydroxy-1,1-cyclobutanedicarb-
According to GP 1, 8 (65.1 mg, 0.407 mmol) was dis-
solved in 10 mL of water, combined with 0.4 mmol of
the aqueous solution of diammine(diaqua)platinum(II)
hydroxide 19 and stirred for 5 d. Yield: 74 mg (48%),

G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
4463
colorless needles, m.p. 250 ꢁC. IR (KBr): m 3520–3200
407.23: C, 17.70; H, 2.48; N, 6.88. Found: C, 17.43; H,
2.45; N, 6.62%.
1
(NH3), 3280 (OH), 1630, 1380 (C@O) cmꢀ1. H NMR
3
(D2O): d 4.67 (bs, 7 H, NH3, OH), 4.10 (qi, J = 7.6
Hz, 1 H, CH(OH)(CH2)2), 3.23, 2.50 (2 m, 4 H,
CH(OH)(CH2)2) ppm. MS (LISI): m/z (rel. int.) 388
(MH, 100). Anal. Calc. for C6H12N2O5Pt, 387.25: C,
18.61; H, 3.12; N, 7.23. Found: C, 18.63; H, 3.30; N,
6.92%.
4.21. General procedure 2 (GP 2)
(RR/SS)-trans-1,2-Diaminocyclohexane(dichloro)-
platinum(II) 26 was synthesized according to a literature
procedure [17]. The activation of 26 was performed as
previously described [18]. After the solvent had been re-
moved a glassy solid was obtained which was dissolved
in a 1:1 mixture of water/ethanol just before reaction
with the respective cyclobutanedicarboxylate ligands.
Compounds 1, 4 and 8 were dissolved in water before
an equimolar amount of a solution of (RR/SS)-trans-
1,2-diaminocyclohexane-(diaqua)platinum(II) dihydrox-
ide 27 was added. The solution was stirred for 5 d at
room temperature under exclusion of light. The grey
precipitate was filtered off. After removal of the solvent
from the filtrate and recrystallization from H2O at room
temperature colorless needles were obtained.
4.18. cis-Diammine(3-bromo-1,1-cyclobutanedicarboxy-
lato)platinum(II) (23)
According to GP1, 11 (46.4 mg, 0.208 mmol) was dis-
solved in water (1 mL), combined with an equimolar
aqueous solution of diammine(diaqua)platinum(II)
hydroxide 19 and stirred for 5 d in the dark. Yield:
20.6 mg (22%), colorless crystals, m.p. 174–177 ꢁC (de-
comp.). IR (KBr): m 3700–3000 (NH3), 1645 (COO),
1
1605 (COO), 1390 cmꢀ1. H NMR (300 MHz, D2O): d
4.70 (s, 6 H, NH3), 4.42 (qi, 1 H, 3J = 7.7 Hz, CH-
(CH2)2), 3.59, 3.12 (2 m, 4 H, CH(CH2)2) ppm. MS
(ESI): m/z (rel. int.) 451 [M(194Pt, 81Br and 196Pt,
79Br) + H, 100]. Anal. Calc. for C6H11N2O4BrPt,
450.14: C, 16.01; H, 2.46; N, 6.22. Found: C, 15.42; H,
2.67; N, 6.04%.
4.22. (RR/SS)-trans-1,2-diaminocyclohexane(1,1-cyclob-
utanedicarboxylato)platinum(II) (28)
28 was synthesized according to a literature proce-
dure [24].
4.19. cis-Diammine(3-fluoro-1,1-cyclobutanedicarboxy-
lato)platinum(II) (24)
4.23. (RR/SS)-trans-1,2-diaminocyclohexane(3-chloro-
1,1-cyclobutanedicarboxylato)platinum(II) (29)
According to GP1, 13 (33.2 mg, 0.205 mmol) was dis-
solved in water (1 mL), combined with an equimolar
aqueous solution of diammine(diaqua)platinum(II)
hydroxide 19 and stirred for 5 d in the dark. Yield:
20.0 mg (25%), colorless crystals, m.p. 226–229 ꢁC (de-
comp.). IR (KBr): m 3700–3000 (NH3), 1640 (C@O),
According to GP 2, 4 (71.4 mg, 0.402 mmol) was dis-
solved in 20 mL of water, combined with 0.4 mmol of
activated 26, and stirred for 5 d. Yield: 116.8 mg
(60%), colorless needles, m.p. > 250 ꢁC. IR (KBr): m
1
3260, 3200, 3110 (NH), 1630, 1360 (C@O) cmꢀ1. H
1
1595 (C@O), 1370, 1355 cmꢀ1. H NMR (300 MHz,
NMR (DMSO-d6): d 5.60 (m, 4 H, NH2), 4.31 (qi,
3J = 7.9 Hz, 1 H, CHCl(CH2)2), 3.50, 2.80 (2 m, 4 H,
CHCl(CH2)2), 2.04, 1.81, 1.45, 1.21, 1.02 (5 m, 10 H,
C6H10) ppm. MS (ESI): m/z (rel. int.) 487 (MH, 100).
Anal. Calc. for C12H19ClN2O4Pt, 485.83: C, 29.67; H,
3.94; N, 5.77. Found: C, 29.26; H, 4.07; N, 5.50%.
3
3
D2O): d 4.99 (d qi, 1H, JH–F = 56.1 Hz, J = 6.8 Hz,
C(F)H(CH2)2), 4.70 (s, 6 H, NH3), 3.31, 2.88 (2 m, 4
H, CHF(CH2)2) ppm. MS (ESI): m/z (rel. int.) 390
[M(195Pt) + H, 100]. Anal. Calc. for C6H11N2O4FPt,
389.24: C, 18.51; H, 2.85; N, 7.20. Found: C, 18.36; H,
3.27; N, 6.66%.
4.24. (RR/SS)-trans-1,2-diaminocyclohexane(3-hydroxy-
1,1-cyclobutanedicarboxylato)platinum(II) (30)
4.20. cis-Diammine(3,3-difluoro-1,1-cyclobutanedicarb-
oxylato)platinum(II) (25)
According to GP 2, 8 (65.1 mg, 0.407 mmol) was dis-
solved in 20 mL of water, combined with 0.4 mmol of
activated 26, and stirred for 5 d. Yield: 46.2 mg (25%),
colorless needles, m.p. 248 ꢁC. IR (KBr): m 3260, 3220,
According to GP1, 16 (67.7 mg, 0.376 mmol) was dis-
solved in water (1 mL), combined with an equimolar
aqueous solution of diammine(diaqua)platinum(II)
hydroxide 19 and stirred for 5 d in the dark. Yield: 41
mg (27%), colorless crystals, m.p. 198–203 ꢁC (de-
comp.). IR (KBr): m 3700–2900 (NH3), 1645 (C@O),
3120 (NH), 1620 (C@O), 1360 cmꢀ1
.
1H NMR
(DMSO-d6): d 5.51 (m, 4 H, NH2), 4.91 (bs, 1 H, OH),
3.84 (qi, J = 7.6 Hz, 1 H, CH(OH)(CH2)2), 3.09, 2.31
3
1
1605 (C@O), 1395 cmꢀ1. H NMR (300 MHz, D2O): d
(2 m, 4 H, CH(CH2)2), 2.03, 1.80, 1.45, 1.21, 1.01 (5 m,
10 H, C6H10) ppm. MS (ESI): m/z (rel. int.) 468 (MH,
100). Anal. Calc. for C12H20N2O5Pt, 467.38: C, 30.84;
H, 4.31; N, 5.99. Found: C, 30.72; H, 4.65; N, 5.63%.
4.70 (s, 6 H, NH3), 3.41 (t, 4 H, 3J = 12.6 Hz,
CF2(CH2)2) ppm. MS (ESI): m/z (rel. int.) 408
[M(195Pt) + H, 100]. Anal. Calc. for C6H10N2O4F2Pt,

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G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
4.25. Cell cultures
4.27. Chemosensitivity assays
Cell line banking and quality control were performed
according to the seed stock concept [25]. Mycoplasma
contamination was routinely monitored and only Myco-
plasma-free cultures were used. All reagents not speci-
fied below (A-grade purity) were purchased from
Merck (Darmstadt, Germany).
The human J82 (ATCC No. HTB-1) bladder cancer
cell line was obtained from American Type Culture Col-
lection (Rockville, MD, USA). The J82 (ATCC No.
HTB-1) cells were maintained in Eagleꢀs Minimum
Essential Medium (Sigma–Aldrich Chemie GmbH, Mu-
nich, Germany), containing Lglutamine (1% v/v), NaH-
CO3 (2.2 g/L), sodium pyruvate (110 mg/L) (Serva,
Heidelberg, Germany), and 5% fetal calf serum (Bioch-
rom KG, Berlin, Germany) using 75 mL culture flasks
(Nunc, Wiesbaden, Germany) in a water-saturated
atmosphere (95% air/5% CO2) at 37 ꢁC. The cells were
passaged weekly following trypsinization using 0.05%
trypsin/0.02% ethylenediaminetetraacetic acid (Roche
Diagnostics, Mannheim, Germany).
The cells were seeded (100 lL/well) in 96 well flat-bot-
tomed microtitration plates (Greiner Bio-One GmbH,
Frickenhausen, Germany) at an appropriate density of
approximately 15 cells/microscopic field (Leitz, Diavert,
320·). After 48 h, the medium was carefully removed by
suction and replaced by fresh medium (200 lL/well)
containing drugs (feed solutions were diluted 1:1000
with culture medium) or pure solvent. On each plate,
16 wells served as controls and 16 wells were used per
drug concentration. After various times of incubation,
the culture medium was shaken off and the J82 cells were
fixed with 100 lL 1% glutardialdehyde in PBS/well
(phosphate-buffered saline) for 25 min. The cells of the
SK-OV-3 and NIH:OVCAR-3 cell lines were fixed with
100 lL/well of 3% glutaraldehyde in PBS for 30 min.
The fixative was replaced by 180 lL of PBS/well, and
the plates were stored in a refrigerator (4 ꢁC). At the
end of the experiment, the cells were simultaneously
stained with 0.02% aqueous crystal violet solution (100
lL/well) for 20 min. Excess dye was removed by rinsing
the microplates with water for 20 min. The stain bound
by the cells was redissolved in 70% ethanol (180 lL/well)
while shaking the microplates for about 3 h. Absorb-
ance, a parameter proportional to cell mass [28], was
measured at 578 nm using a BioTek 309 Autoreader
(Winooski).
The SK-OV-3 (ATCC no. HTB-77) cell line [20] was
maintained in Mc Coyꢀs 5A (Sigma–Aldrich Chemie
GmbH, Munich, Germany) medium containing 15%
new born calf serum (Biochrom KG, Berlin, Germany)
and 2.2 g/L NaHCO3. In our experiments we used cells
from passage 77 [21].
The NIH:OVCAR-3 (ATCC no. HTB-161) wild type
cells [26] were cultured in RPMI-1640 medium (Sigma–
Aldrich Chemie GmbH, Munich, Germany) containing
10% Basal Medium Supplement (Biochrom KG, Berlin,
Germany) and insulin (10 lg/mL) (Sigma–Aldrich Che-
mie GmbH, Munich, Germany) at 37 ꢁC in a H2O-sat-
urated atmosphere of air and 5% CO2 [27]. From these
cells a cisplatin-resistant variant was established by
treatment of the cells with increasing concentrations of
cisplatin for 21 passages. The cisplatin-resistant variant
was established by treatment of the cells with increasing
concentrations of cisplatin for 21 passages. The platin-
resistant NIH:OVCAR-3 cells were maintained analo-
gous to the wild type cells [26], but in the presence of
0.25 lM cisplatin in the culture medium. The cells were
serially passaged following trypnization using trypsin/
EDTA (Roche Diagnostics GmbH, Mannheim, Ger-
many), and were used from passage 56.
Drug effects were expressed as corrected T/C values
for each group according to
T ꢀ C0
T =Ccorr: ½%ꢁ ¼
ꢂ 100
C ꢀ C0
in which T is the mean absorbance of the treated cells, C
is the mean absorbance of the controls, and C0 is the
mean absorbance of the cells at the time (t = 0) when
the drug was added.
When the absorbance of treated cells T is less than
that of the culture at t = 0 (C0), the extent of cell killing
was calculated as
C0 ꢀ T
cytocidal effect ½%ꢁ ¼
ꢂ 100:
C0
The relationship between growth kinetics of a drug-trea-
ted cell population and the plot of corrected T/C values
vs time is discussed in detail in the literature [13,14].
In the multiple point chemosensitivity assays the cul-
tivation and seeding of the cells, the preparation of the
stock solutions and the preparation of the wells of the
microplates were carried out as described above. The
plates were prepared for each compound in the concen-
trations indicated for five different periods of incuba-
tion. The first time point of the kinetics was chosen 24
or 72 h after drug administration depending on the cell
line (Figs. 3–5). In each series an additional plate was
used to determine the initial cell density.
4.26. Drugs
Carboplatin was obtained from Strem Chemicals
(Newburyport, USA). Carboplatin and compounds
21–25 were dissolved in water. As the diaminocyclohex-
aneplatinum complexes 28–30 were not soluble in water
they were dissolved in DMF. For all drugs, 10 mM
stock solutions were prepared. Feed solutions were
made by appropriate dilution.

G. Bernhardt et al. / Inorganica Chimica Acta 357 (2004) 4452–4466
4465
[4] T.W. Hambley, Platinum binding to DNA: structural controls
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4.28. X-ray structure analysis of cis-diammine(3-hydroxy-
1,1-cyclobutanedicarboxylato)platinum(II) (22)
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complexes in cancer therapy: an historical perspective and an
update, Eur. J. Cancer 34 (1998) 1522.
C6H12N2O5Pt; MW = 387.25; translucent plates;
crystal size [mm]: 0.47 · 0.14 · 0.05; space group P65;
hexagonal; Z = 6; a/b/c [A] = 13.9539(10)/13.9539(10)/
˚
[6] N. Gruber, Ph.D. Thesis, Universita¨t Regensburg, 2002.
[7] W.A. Nevill, D.S. Frank, R.D. Trepka, Free radical chlorination
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1,3-disubstituted cyclobutane derivatives, Chem. Ber. 90 (1957)
1424.
3
˚
9.4556(8); a/b/c [ꢁ] = 90/90/120; V = 1594.5(2) A ;
q
calc. = 2.420 g cmꢀ3; F(000) = 1080; l = 13.20 mmꢀ1
;
T = 173(1) K; x-scan 2.74 < h < 25.80ꢁ; 11040 reflec-
tions collected, 2027 independent, 1919 observed
(I > 2rI); Rint = 0.0712; diffractometer STOE-IPDS
(Mo Ka, graphite monochromator); R(I > 2rI):
R1 = 0.0233; R2 = 0.0560; R(all data): R1 = 0.0260;
xR2 = 0.0570; Goof = 1.062. CCDC 203641.
[9] C. Beard, A. Burger, cis- and trans-3-Dimethylamino-1-cyclobut-
anol, Chem. Ber. 95 (1962) 2535.
[10] W.R. Dolbier Jr., T.A. Gray, J.J. Keaffaber, L. Celewiz, H.
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4.29. X-ray structure analysis of (RR/SS)-trans-1,2-
diaminocyclohexane(3-hydroxy-1,1-cyclobutanedicarb-
oxylato)platinum(II) (30)
[11] G.S. Lal, G.P. Pez, R.J. Pesaresi, F.M. Prozonic, Bis(2-methoxy-
ethyl)aminosulfur trifluoride: a new broad-spectrum deoxofluor-
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(1999) 215.
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C12H20N2 O5Pt Æ H2O; MW = 485.40; translucent
plates; crystal size [mm]: 0.44 · 0.08 · 0.06; space
˚
ꢀ
group P1; triclinic; Z = 4; a/b/c [A] = 11.6676(17)/
11.9773(16)/13.1699(17); a/b/c [ꢁ] = 98.167(16)/
[13] G.G. Furin, in: L. German, S. Zemskov (Eds.), New Fluorinat-
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p. 35.
3
˚
103.029(16)/118.268(14); V = 1511.4(5) A ; qcalc. = 2.129
g cmꢀ3; F(000) = 932; l = 9.312 mmꢀ1; T = 173(1) K;
x-scan 2.20 < h < 25.74ꢁ; 14866 reflections collected,
[14] R.D. Allan, J.R. Hanrahan, T.W. Hambley, G.A.R. Johnston,
K.N. Mewett, A.D. Mitrovic, Synthesis and activity of a potent
N-methyl-D-aspartic acid agonist, trans-1-aminocyclobutane-1,3-
5356
independent,
4403
observed
(I > 2rI);
dicarboxylic acid, and related phosphonic and carboxylic acids, J.
Med. Chem. 33 (1990) 2905.
Rint = 0.0304; diffractometer STOE-IPDS (Mo Ka,
graphite monochromator); R(I > 2rI): R1 = 0.0259;
xR2 = 0.0624; R(all data): R1 = 0.0344; xR2 = 0.0647;
Goof = 0.958. CCDC203640.
Crystallographic data (excluding structure factors)
for the structures in this paper have been deposited with
the Cambridge Crystallographic Data Centre. Copies of
the data can be obtained, free of charge, on application
to CCDC, 12 Union Road, Cambridge CB2, UK (fax:
+44 1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
[15] L.D. Jennings, R.D. Rayner, D.B. Jordan, J.F. Okonya, G.S.
Basarab, D.K. Amorose, B.M. Anaclerio, J.K. Lee, R.D.
Schwartz, K.A. Whitmore, Cyclobutane carboxamide inhibitors
of fungal melanin: biosynthesis and their evaluation as fungicides,
Bioorg. Med. Chem. 8 (2000) 897.
[16] H. Brunner, F. Maiterth, B. Treittinger, Synthese und Anti-
tumoraktivita¨t neuer Porphyrin-Platin(II)-Komplexe mit an den
Porphyrin-Seitenketten gebundenem cytostatischen Platin-Rest,
Chem. Ber. 127 (1994) 2141.
[17] Y. Kidani, K. Inagaki, M. Iigo, A. Hoshi, K. Kuretani,
Antitumor activity of 1,2-diaminocyclohexane-platinum com-
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(1978) 1315.
Acknowledgements
[18] C. Lottner, K.-C. Bart, G. Bernhardt, H. Brunner, Hematoporph-
yrin-derived soluble porphyrin-platinum conjugates with com-
bined cytotoxic and phototoxic antitumor activity, J. Med. Chem.
45 (2001) 2064.
We thank the Alexander von Humboldt Foundation
for providing a fellowship to Dr. S. Pushpan. The
authors are grateful to Ms. Susanne Bollwein for excel-
lent technical assistance.
[19] C.M. OꢀToole, in: M.M. Webber (Ed.), In Vitro Models for
Cancer Research, vol. IV, CRC Press, Boca Raton, FL, 1986, p.
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of three new human ovarian cancer cell lines and initial evaluation
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Cancer 39 (1987) 219.
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