Synthesis, antiproliferative activity in cancer cells and DNA interaction studies of [Pt(cis-1,3-diaminocycloalkane)Cl2] analogs

Article abstract of DOI:10.1007/s00775-020-01809-9

Abstract: The search for more effective platinum anticancer drugs has led to the design, synthesis, and preclinical testing of hundreds of new platinum complexes. This search resulted in the recognition and subsequent FDA approval of the third-generation Pt(II) anticancer drug, [Pt(1,2-diaminocyclohexane)(oxalate)], oxaliplatin, as an effective agent in treating colorectal and gastrointestinal cancers. Another promising example of the class of anticancer platinum(II) complexes incorporating the Pt(1,n-diaminocycloalkane) moiety is kiteplatin ([Pt(cis-1,4-DACH)Cl₂], DACH = diaminocyclohexane). We report here our progress in evaluating the role of the cycloalkyl moiety in these complexes focusing on the synthesis, characterization, evaluation of the antiproliferative activity in tumor cells and studies of the mechanism of action of new [Pt(cis-1,3-diaminocycloalkane)Cl₂] complexes wherein the cis-1,3-diaminocycloalkane group contains the cyclobutyl, cyclopentyl, and cyclohexyl moieties. We demonstrate that [Pt(cis-1,3-DACH)Cl₂] destroys cancer cells with greater efficacy than the other two investigated 1,3-diamminocycloalkane derivatives, or cisplatin. Moreover, the investigated [Pt(cis-1,3-diaminocycloalkane)Cl₂] complexes show selectivity toward tumor cells relative to non-tumorigenic normal cells. We also performed several mechanistic studies in cell-free media focused on understanding some early steps in the mechanism of antitumor activity of bifunctional platinum(II) complexes. Our data indicate that reactivities of the investigated [Pt(cis-1,3-diaminocycloalkane)Cl₂] complexes and cisplatin with glutathione and DNA binding do not correlate with antiproliferative activity of these platinum(II) complexes in cancer cells. In contrast, we show that the higher antiproliferative activity in cancer cells of [Pt(cis-1,3-DACH)Cl₂] originates from its highest hydrophobicity and most efficient cellular uptake. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00775-020-01809-9

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-020-01809-9
ORIGINAL PAPER
Synthesis, antiproliferative activity in cancer cells and DNA interaction
studies of [Pt(cis‑1,3‑diaminocycloalkane)Cl2] analogs
James D. Hoeschele1 · Jana Kasparkova2 · Hana Kostrhunova2 · Olga Novakova2 · Jitka Pracharova2
Paul Pineau1 · Viktor Brabec2
·
Received: 25 June 2020 / Accepted: 10 August 2020
© Society for Biological Inorganic Chemistry (SBIC) 2020
Abstract
The search for more efective platinum anticancer drugs has led to the design, synthesis, and preclinical testing of hundreds
of new platinum complexes. This search resulted in the recognition and subsequent FDA approval of the third-generation
Pt(II) anticancer drug, [Pt(1,2-diaminocyclohexane)(oxalate)], oxaliplatin, as an efective agent in treating colorectal and
gastrointestinal cancers. Another promising example of the class of anticancer platinum(II) complexes incorporating the
Pt(1,n-diaminocycloalkane) moiety is kiteplatin ([Pt(cis-1,4-DACH)Cl2], DACH=diaminocyclohexane). We report here
our progress in evaluating the role of the cycloalkyl moiety in these complexes focusing on the synthesis, characterization,
evaluation of the antiproliferative activity in tumor cells and studies of the mechanism of action of new [Pt(cis-1,3-diami-
nocycloalkane)Cl2] complexes wherein the cis-1,3-diaminocycloalkane group contains the cyclobutyl, cyclopentyl, and
cyclohexyl moieties. We demonstrate that [Pt(cis-1,3-DACH)Cl2] destroys cancer cells with greater efcacy than the other two
investigated 1,3-diamminocycloalkane derivatives, or cisplatin. Moreover, the investigated [Pt(cis-1,3-diaminocycloalkane)
Cl2] complexes show selectivity toward tumor cells relative to non-tumorigenic normal cells. We also performed several
mechanistic studies in cell-free media focused on understanding some early steps in the mechanism of antitumor activity of
bifunctional platinum(II) complexes. Our data indicate that reactivities of the investigated [Pt(cis-1,3-diaminocycloalkane)
Cl2] complexes and cisplatin with glutathione and DNA binding do not correlate with antiproliferative activity of these
platinum(II) complexes in cancer cells. In contrast, we show that the higher antiproliferative activity in cancer cells of [Pt(cis-
1,3-DACH)Cl2] originates from its highest hydrophobicity and most efcient cellular uptake.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00775-020-01809-9) contains
supplementary material, which is available to authorized users.
* Viktor Brabec
brabec@ibp.cz
1
Department of Chemistry, Eastern Michigan University,
Ypsilanti, MI 48197, USA
2
Institute of Biophysics, Czech Academy of Sciences,
Kralovopolska 135, 61265 Brno, Czech Republic
Vol.:(0123456789)
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JBIC Journal of Biological Inorganic Chemistry
Graphic abstract
Keywords Antitumor · Oxaliplatin · Pt(1,n-diaminocycloalkane) moiety · DNA · Glutathione · Cellular uptake
recently, in treating gastrointestinal cancers [6]. Another
example of the class of anticancer platinum(II) complexes
incorporating the Pt(1,n-diaminocycloalkane) moiety is a
large-ring chelate complex [PtCl2(cis-1,4-DACH)] (kitepl-
atin). Notably, it has proved to be endowed with very prom-
ising anticancer activity [7] being able to overcome resist-
ance to cisplatin and oxaliplatin.
Introduction
Antitumor platinum(II) complexes have played a domi-
nant role in the chemotherapeutic treatment of solid can-
cerous tumors since the early 1970s, as evidenced by the
FDA approval of three Pt(II) complexes (Fig. 1): Cisplatin
(1978), carboplatin (1989) and oxaliplatin (2002). All three
agents incorporate either cis-diammine groups or a primary
1,2-diaminocyclohexane linked to Pt(II) in an essential cis
confguration.
Our research program, a US–Czech Republic collabora-
tion involving Eastern Michigan University and the Czech
Academy of Sciences, currently focuses on the synthe-
sis, characterization, and evaluation of the antiprolifera-
tive activity of [Pt(cis-1,3-diaminocycloalkane)Cl2] (or
[Pt(1R,3S-diaminocycloalkane)Cl2]) complexes in tumor
cells. As DNA is considered to be the important pharmaco-
logical target of cisplatin and its derivatives [8, 9], our inter-
est is also focused on the nature of the interaction and bind-
ing of these complexes to DNA in cell-free media. We report
here our progress in evaluating the role of the cycloalkyl
moiety in these complexes in modifying the structure and
biophysical properties of the respective complexes, leading
to changes in the antiproliferative activity in cancer cells and
the nature/type of DNA interaction and binding. Specifcally,
While analogs of cisplatin and carboplatin (which incor-
porate the cis-Pt(NH3)2 moiety important to observing anti-
tumor activity) have been extensively studied, complexes
incorporating the Pt(1,n-diaminocycloalkane) moiety have
not been studied as extensively except for complexes incor-
porating the primary chelating diamine, 1,2-diaminocy-
clohexane (1R, 2R-DACH; 1S, 2S-DACH; and 1R, 2S-DACH
(cis), DACH=diaminocyclohexane). Early studies of these
complexes [1–4] led to the recognition and subsequent FDA
approval of the third-generation Pt(II) anticancer drug,
[Pt(1R, 2R-diaminocyclohexane)(oxalate)], oxaliplatin, as
an efective agent in treating colorectal cancer [5] and, more
Fig. 1 Structures of FDA-
approved Pt(II) drugs
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JBIC Journal of Biological Inorganic Chemistry
our studies center on three Pt(cis-1,3-diaminocycloalkane)
Cl2 complexes wherein the cis-1,3-diaminocycloalkane
group contains the cyclobutyl, cyclopentyl, and cyclohexyl
moieties and we have evaluated these complexes for antitu-
mor activity and the nature of their interaction with DNA.
LoVo cells and human MRC-5 pd30 cells derived from nor-
mal lung tissue were purchased from the European Collec-
tion of Authenticated Cell Cultures (ECACC) (Salisbury,
UK). Chinese hamster ovary CHO-K1 cell line and its NER-
defcient mutant MMC-2 (carrying the ERCC3/XPB muta-
tion) cell line were kindly supplied by Dr. M. Pirsel, Cancer
Research Institute, Slovak Academy of Sciences, Bratislava
(Slovakia). The A2780 and LoVo cells were grown in RPMI
1640 medium (Biosera, Boussens, France) and Ham’s F12
medium (Sigma-Aldrich), respectively. The HeLa, MCF-7,
CHO-K1, MRC-5 pd30, and MMC-2 cells were grown in
DMEM medium (high glucose, 4.5 g L−1, PAA, Pasching,
Austria). Both media were supplemented with gentamicin
(50 μg mL−1, Serva, Heidelberg, Germany) and 10% heat-
inactivated fetal bovine serum (PAA); the medium for
MRC-5 pd30 cell was furthermore supplemented with 1%
nonessential amino acids (Sigma-Aldrich, Prague, Czech
Republic). The cells were cultured in a humidifed incuba-
tor at 37 °C in a 5% CO2 and subcultured two to three times
a week with an appropriate plating density. Escherichia coli
(CCM 7929) was obtained from the Czech Collection of
Microorganisms (CCM), Masaryk University, Faculty of
Science, Brno, Czech Republic as a freeze-dried pellet. The
cells were rehydrated and cultivated on Luria broth (LB)
agar plates.
Materials and methods
General
All starting materials used in the synthesis of the [Pt(cis-
1,3-diaminocycloalkane)Cl2] complexes were obtained com-
mercially and were used as received except for the diamine
ligands. Cis-1,3-diaminocyclobutane and cis-1,3-diamino-
cyclohexane ligands were purchased from Pharmablock
and CambiBlock, Inc., respectively. Purifcation consisted
of converting the diamines to their respective diamine
dihydrochlorides using HCl, followed by recrystallization
of these dihydrochlorides from a minimum volume of 1 M
HCl followed by the addition of ethanol. The cis-1,3-diami-
nocyclopentane ligand was synthesized as described below.
The three [Pt(cis-diaminocycloalkane)Cl2] complexes were
prepared as described below.
Starting materials and reagents used in the biological
and molecular biophysics studies were as follows: cisplatin
and transplatin were obtained from Sigma-Aldrich (Prague,
Czech Republic). Dienplatin (chloro-(diethylenetriamine)
platinum(II) chloride) was kindly supplied by prof. G. Natile,
University of Bari “Aldo Moro”, Italy. The stock solutions
of the platinum complexes were prepared in 0.01 M NaClO4
and kept in the dark. The concentrations of platinum in the
stock solutions were always verifed by fameless atomic
absorption spectrometry (FAAS).
Generic synthesis
of [Pt(cis‑1,3‑diaminocycloalkane)Cl2]
complexes
All three [Pt(cis-1,3-diaminocycloalkane)Cl2] analogs were
synthesized using a common synthetic approach derived
from the classical Dhara procedure [10]. For purposes of
simplicity, a generic procedure of synthesis of these com-
plexes is presented, based on a scale of 1.00 mmol of Pt.
Calf thymus (CT) DNA was from Sigma-Aldrich (Prague,
Czech Republic). Plasmid pSP73 (2464 bp), T7 RNA poly-
merase, and RNasin ribonuclease inhibitor were purchased
from Promega (Mannheim, Germany). Plasmid pSP73KB
(2455 bp) was isolated according to standard procedures.
Excision endonucleases EcoRI and NdeI were obtained from
New England Biolabs. Ribonucleotide triphosphates were
from Roche Diagnostics, GmbH (Mannheim, Germany).
Ethidium bromide (EtBr), l-glutathione (reduced), and
agarose were from Merck (Darmstadt, Germany) and FMC
BioProducts (Rockland, ME, USA), respectively. Acryla-
mide and bis-acrylamide were from Promega (Madison,
Wisconsin). Radioactive [α-32P]dATP and [α-32P]CUTP
were from MP Biomedicals (Irvine, CA, USA). TbCl3 was
from Sigma-Aldrich.
[Pt(cis‑1,3‑diaminocycloalkane)l2]
The starting material, K2PtCl4 (0.415 g; 1.00 mmol), was
dissolved in 12.0 mL of nanopure H2O to which was added
8.0 mL of 1.00 M KI (I/Pt(II) ratio = 8.00). The result-
ing homogeneous solution (0.050 M in Pt(II)) was then
heated and maintained at 45˚C for 20 min to generate the
brown-black “K2PtI4 species”. A solution of a given cis-
1,3-diaminocyclooalkane was prepared by dissolving
1.00 mol of the dihydrochloride in 20 mL of water contain-
ing 2.00 mmol of NaOH. This solution was then delivered
to the stirred K2PtI4 reaction mixture (held at 45˚C) via a
peristaltic pump adjusted to deliver 0.5 mL/min, requiring
40 min. In all of these preparations, the solutions of cis-
1,3-diaminocycloalkane (20 mL) were equal in volume to
the volume of the K2PtI4 reaction mixture (20 mL), resulting
The human ovarian carcinoma A2780 cells, human cervi-
cal carcinoma HeLa cells, and human breast cancer MCF-7
cells were kindly supplied by Professor B. Keppler, Univer-
sity of Vienna (Austria). Human colorectal adenocarcinoma
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JBIC Journal of Biological Inorganic Chemistry
in a fnal concentration of Pt(II) of 1.0 mmol/40 mL or
0.025 M. The resulting reaction product, [Pt(cis-1,3-diami-
nocycloalkane)l2], was digested for 30 min post-delivery at
45˚C and then placed in a refrigerator overnight at 5 ˚C. The
product was fltered, washed (with resuspension of the solid)
with cold 0.1 M KI (1×), followed by cold H2O (2×), and
then dried in a vacuum oven for 2 h at~45˚C. The yield of
the bright yellow [Pt(cis-1,3-diaminocycloalkane)l2] product
averaged~80–90%.
drying in a vacuum oven for an hour at~ 45 °C. Yields of
the individual [Pt(cis-1,3-diaminocycloalkane)Cl2] products
averaged~75%.
Synthesis of cis‑1,3‑diaminocyclopentane
Cis-1,3-diaminocyclopentane was synthesized via a four-
step process (Fig. 2) beginning with (bicyclo[2.2.1]hept-2-
ene) (norbornene) purchased from Sigma-Aldrich.
Dicarboxylic acid 2 was prepared in high yield by oxidiz-
ing the bicyclo[2.2.1]hept-2-ene according to the method of
Geyer et al. [11] A solution of bicyclic alkene 1 (3.768 g,
40.0 mmol) in 160 mL CH3CN/EtOAc (50/50:v/v) was
added to a solution of NaIO4 (35.071 g, 164 mmol) in water
(120 mL). After the addition of RuCl3 (192 mg, 0.93 mmol),
the biphasic mixture was rapidly stirred at 25 °C for
45 h. After separation, the aqueous layer was extracted with
EtOAc (2×200 mL). The organic layer was fltered through
Celite and the combined organic layers dried over MgSO4,
fltered, concentrated in vacuo and oven dried to yield 2
[Pt(cis‑1,3‑diaminocycloalkane)Cl2]
The iodo product, [Pt(cis-1,3-diaminocycloalkane)l2], was
transferred to a 50 mL Erlenmeyer fask to which sufcient
H2O was added to create a slurry, ~0.05 M in Pt(II). After
briefy sonicating this slurry to achieve homogeneity, an
AgNO3 solution, containing 105% of the stoichiometric
amount of AgNO3 required (to insure complete removal
of the iodide ion), was delivered via a peristaltic pump
(at ~ 0.5 mL/min) to a stirred slurry of [Pt(cis-1,3-diami-
nocycloalkane)l2] at 45˚C. Typically, the volume of the
AgNO3 solution delivered (20 mL, 0.10 M) was equal in
volume to the volume of the initial slurry of the iodo prod-
uct. Following complete delivery of the AgNO3 solution, the
reaction mixture was sonicated for 0.5 min (at 45 °C) and
then mechanically stirred overnight at ambient temperature
to insure that the metathetical reaction was complete. The
reaction mixture was then fltered, washed (1×) with cold
H2O and the fltrate tested for the presence of Ag+ ion using
0.1 M HCl. If the test for Ag+ ion was positive, additional
0.1 M HCl was added until the excess Ag+ ion was com-
pletely converted to AgCl and removed by fltration. Con-
centrated HCl (1–2 mL) was then added to the fnal Ag+-free
fltrate, and the reaction mixture was digested at 50 °C for
20 min. After cooling the reaction mixture in an ice-water
bath for at least 1 h, the characteristically bright yellow prod-
uct that formed, [Pt(cis-1,3-diaminocycloalkane)Cl2], was
fltered, washed (with resuspension of the solid) with cold
1 M HCl, followed by cold absolute ethanol and then dried
by frst pulling air through the flter cake for 5 min, then by
1
as colorless solid (5.766 g, 91.1%): H NMR (400 MHz,
DMSO-d6) δ 12.09 (s, 2H), 2.74–2.65 (m, 2H), 2.13–2.06
(dt, J = 12.8, 8.2 Hz, 1H), 1.90–1.71 (m, 5H); 13C NMR
(100 MHz, DMSO-d6) δ 176.40, 43.22, 32.80, 28.91.
Diester 3 was prepared in high yield via the following
Fischer esterifcation procedure. A solution of the dicarbo-
xylic acid 2 (4.684 g, 29.6 mmol) in dry MeOH (47 mL)
was treated with fve drops (0.11 g) conc. H2SO4 and stirred
at refux for 41 h. The reaction mixture was poured into
saturated NaHCO3 solution (40 mL), the MeOH removed
under reduced pressure, and then extracted with EtOAc
(3×20 mL). The organic layer was washed with water and
saturated NaCl solution, dried over Na2SO4 and concentrated
in vacuo to yield 3 as a yellow oil (5.068 g, 91.9%): 1H NMR
(400 MHz, DMSO-d6) δ 12.09 (s, 2 H), 2.74–2.65 (m, 2
H), 2.13–2.05 (m, 1 H), 1.90–1.71 (m, 5 H); 13C NMR
(100 MHz, DMSO-d6) 176.4, 43.22, 32.80, 28.91.
Dihydrazide 4 was prepared in high yield by treating
the diester with hydrazine as follows. A solution of diester
3 (5.060 g, 27.2 mmol) in EtOH (44 mL) was treated with
Fig. 2 Synthesis of Cis-1,3-di-
aminocyclopentane
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JBIC Journal of Biological Inorganic Chemistry
N2H4·H20 (55% hydrazine: 8.017 g, 138 mmol) and heated to
refux for 20 h. The reaction mixture was diluted with CHCl3
(75 mL), fltered and oven-dried to yield colorless crystal-
line solid 4 (4.315 g, 85.2%). The fltrate was reduced to
dryness in vacuo and triturated with dry EtOH to yield a sec-
ond crop of crystals (0.221 g, 4.37%): 1H NMR (400 MHz,
D2O) δ 2.75–2.67 (m, 2 H), 2.13–2.06 (dt, J=12.8, 7.3 Hz,
1H), 1.94–1.87 (m, 2 H), 1.87–1.74 (m, 3 H); 13C NMR
(100 MHz, D2O) δ 177.20, 43.31, 33.75, 29.08.
[Pt(cis-1,3-diaminocyclopentane)Cl2] ([Pt(cis-1,3-
DACP)Cl2)]). Using the generic scheme above, [Pt(cis-1,3-
DACP)Cl2] was synthesized on a 1.00 mol scale, resulting
in a yield of 85%.
C, H and N analyses: calcd for PtN2C5H12Cl2: C, 16.40;
1
H, 3.30; N, 7.65; Found: C, 16.49; H, 3.28; N, 7.82; H
NMR (DMF-d7, 500 MHz): δ 5.03 (br s, 2H, NH2); 4.80 (br
s, 2H, NH2); 3.22 (br s with 195Pt satellites, 2H, CHx2); 2.05
(m, 2H, CH2); 1.84 (m, 4H, CH2 ×2).
Diamine 5 was prepared via a Curtius rearrangement via
treatment with nitrous acid, and an acidic workup provided
the diamine as the dihydrochloride salt in moderate yield.
Concentrated HCl (2.9 mL) and ice (6 g) were added to
a solution of dihydrazide 4 (751 mg, 4.03 mmol) in Et2O
(8 mL). While rapidly stirring in an ice bath held at 4–5 °C,
a solution of NaNO2 (1.105 g, 16.0 mmol) in water (2 mL)
was added dropwise over 10 min and then stirred for an
additional. 22 min. After separation, the aqueous layer
was extracted with Et2O (3×20 mL). The combined ether
extracts were dried over CaCl2, fltered, diluted with tolu-
ene (12 mL), and the Et2O removed in vacuo. The remain-
ing toluene solution was heated to 85 °C for 2.5 h and then
treated with hot (60 °C) concentrated HCl (3.1 mL) and
stirred for 25 min. The solvent was removed in vacuo to
yield a pale brown solid. Trituration with cold EtOH and
suction fltration yielded tan solid 5 (323 mg, 46.3%). The
fltrate was reduced to dryness in vacuo and triturated with
dry EtOH to yield a second crop of crystals (69 mg, 9.9%):
1H NMR (400 MHz, D2O) δ 3.75–3.67 (m, 2H), 2.69–2.61
(dt, J = 13.3, 7.6 Hz, 1H), 2.23–2.14 (m, 2H), 1.91–1.81
(m, 2H), 1.74–1.66 (dt, J = 13.3, 9.2 Hz, 1H). 13C NMR
(100 MHz, D2O) δ 49.67, 35.04, 28.01. Anal. calcd. for
C5H14N2Cl2: C, 34.70; H, 8.15; N, 16.19. Found: C, 35.22;
H, 8.36; N, 15.81.
13C NMR (DMF-d7): δ 53.159; 40.144; 31.0482.
195Pt NMR (DMF-d7, 107 MHz): δ − 2263 ppm). Cispl-
atin (as reference)=– 1617.4 ppm.
[Pt(cis‑1,3‑diaminocyclohexane)Cl2]
([Pt(cis‑1,3‑DACH)Cl2])
Using the generic scheme above, [Pt(cis-1,3-DACH)Cl2]
was synthesized on a 1.00 mol scale, resulting in a yield
of 73%.
C, H and N analyses: calcd for PtN2C6H14Cl2: C, 18.96;
H, 3.71; N, 7.37. Found: C, 19.06; H, 3.81; N, 7.82.
1H NMR (DMF-d7, 500 MHz): δ 5.41 (br s, 2H, NH2);
4.64 (m, 1H, C(5)H); 4.60 (m, 1H, C(5)H);); 4.52 (br s, 2H,
NH2); 2.86 (br s with 195Pt satellites, 2H, CHx2); 2.51 (m,
2H, CH2); 1.75 ppm (m, 4H, CH2 ×2).
195Pt NMR (DMF-d7, 107 MHz): δ -2133 ppm. Cisplatin
(as reference)=– 1617.4 ppm.
HPLC analysis
[Pt(cis-1,3-diaminocycloalkane)Cl2] complexes were ana-
lyzed for purity via HPLC using a C8 column. Complexes
were dissolved in 0.45% NaCl solution at concentrations
of 1.8–2.2 mg/10 mL), loaded onto the column, and were
eluted isocratically. Eluted product(s) were detected spec-
trophotometrically by measuring the absorbance of the solu-
tions at 210 nm.
Analysis/characterization
of the [Pt(cis‑1,3‑diaminocycloalkane)Cl2]
complexes
Crystal structures
[Pt(cis‑1,3‑diaminocyclobutane)Cl2]
([Pt(cis‑1,3‑DACB)Cl2])
Crystal structures of [Pt(cis-1,3-DACB)Cl2], [Pt(cis-1,3-
DACP)Cl2], and [Pt(cis-1,3-DACH)Cl2] were determined.
Single yellow needle crystals were selected and mounted
on nylon loops with Paratone oil on a Bruker APEX-II CCD
difractometer. Crystals were maintained at a temperature
of 173(2) K during data collection. Structures were solved
with the ShelXT solution program [12] using dual meth-
ods and by using Olex2 [13] as the graphical interface. The
models were refned with ShelXL [14] using full-matrix
least squares minimization on F2. The structural data are
deposited with the Cambridge Crystallographic Data Center:
2021147 ([Pt(cis-1,3-diaminocyclobutane)Cl2]), 2021148
Using the generic scheme above, [Pt(cis-1,3-DACB)Cl2]
was synthesized on a 1.00 mol scale, resulting in a yield
of 77.8%.
C, H and N analyses: calcd for PtN2C4H10Cl2: C, 13.64;
H, 2.86; N, 7.96; Found: C, 13.80; H, 2.89; N, 7.95.
1H NMR (DMF-d7, 500 MHz): δ 5.29 (br s, 4H, NH2);
3.14 (t with 195Pt satellites, 2H, CHx2); 2.51 (m, 2H, CH2);
1.76 ppm (m, 2H, CH2); 195Pt NMR (DMF-d7, 107 MHz): δ
-2256 ppm. Cisplatin (as reference)=– 1617.4 ppm.
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JBIC Journal of Biological Inorganic Chemistry
([Pt(cis-1,3-diaminocyclopentane)Cl2]), 2021149 ([Pt(cis-
and water-saturated octanol (WSO) were prepared using
analytical-grade octanol and ultrapure water. Platinum com-
plexes were dissolved in OSW containing 200 mM NaCl. A
defned volume of the solution was taken out and mixed with
WSO in a volumetric ratio of 1:10. To establish the parti-
tion equilibrium, the samples were exhaustively vortexed
for 30 min at room temperature. To separate the phases,
the samples were centrifuged at 3000g for 5 min. The lay-
ers were separated carefully using a fne-tip pipet, and the
OSW layer was then analyzed by ICP-MS for Pt content.
The partition coefcients were calculated as a ratio of the
concentration of the platinum in the octanol layer to that in
the aqueous layer [log P=log ([Pt]WSO/[Pt]OSW)·(1/10)]=log
([Pt]stock-[Pt]OSW)/[Pt]OSW)·(1/10)].
1,3-diaminocyclohexane)Cl2]).
Biological and molecular biophysics
methods
Antiproliferative activity in vitro
Inhibition of cell proliferation by platinum complexes was
tested by commonly used MTT test (MTT=3-(dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide, Calbiochem,
Darmstadt, Germany). Stock solutions of Pt complexes
were prepared in ultrapure water (Milli-Q, Merck, Milli-
pore), and their concentration was checked by FAAS. The
cells were seeded in 96-well plates at a density of 1 × 104
(A2780, MRC-5 pd30), 5×103 (MCF-7, HeLa, CHO-K1,
and MMC-2) or 4×103 (LoVo) cells/well in 100 μL of the
appropriate medium. After overnight incubation, the cells
were treated with the tested compounds in the concentra-
tion range of 0–100 μM in the respective medium for 72 h.
Subsequently, 20 μL of an MTT solution (1.25 mg mL−1
in PBS) was added to the cells, and they were incubated
for another 4 h. The medium was then drained, and the
formazan product formed by mitochondrial enzymes activ-
ity of cells was dissolved in 100 μL of DMSO. The optical
density was determined at 570 nm (reference, 620 nm) using
a SPARK absorbance reader (Tecan). The IC50 values were
read from the curves constructed by plotting relative absorb-
ance (relative to that for control, untreated cells) versus drug
concentration (μM).
Filamentous growth of bacteria
The ability of platinum complexes to induce flamentation on
bacteria has been studied as already described [15]. Briefy,
bacterial suspension in LB media (5 µL) was added to 1 mL
of LB containing investigated platinum complexes at various
concentrations and incubated at 37 °C for 5 h. Afterward, a 2
μL drop of each cell suspension was mounted on a separate
microscope slide and photographed using an Olympus CXK
41 microscope equipped with a digital camera.
Interaction with glutathione
The interaction of platinum complexes to GSH was recorded
by UV absorption spectrophotometry, as already described
[16, 17]. Samples were incubated with GSH in quartz
cuvettes, and the absorbance at 260 nm was followed as a
function of time by using a Beckman 7400 DU spectropho-
tometer equipped with a thermoelectrically controlled cell
holder. The experimental procedure was as already outlined
by Dabrowiak et al. [18]. To establish the rate of the ini-
tial reaction, the curve was ftted by nonlinear regression to
the equation Id=C+A1exp(-b1t)+A2exp(-b2t) (A1, A2,
b1,b2; C are constants, and t is the time of the reaction) by
using GraphPad Prism software.
Cellular uptake
Cellular accumulation of the platinum complexes was deter-
mined as already described.
In brief, A2780 cells were seeded on 100 mm Petri
dishes. After overnight preincubation in a drug-free medium,
the cells were treated with the platinum compounds at their
10 µM concentrations for 6 h. After this period, the cells
were carefully washed with PBS (37 °C), detached using
0.25% trypsin, and washed thrice with ice-cold PBS and
counted by using a TC-10 automated cell counter (Biorad).
The cell pellets were then digested using a microwave acid
(HCl) digestion system (CEM Mars). The quantity of Pt
taken up by the cells was determined by inductively cou-
pled plasma-mass spectroscopy (ICP-MS, Agilent 7500
spectrometer, Agilent, Japan).
DNA binding in cell‑free media
The details of DNA binding kinetics, transcription map-
ping of DNA adducts, DNA unwinding, DNA interstrand
cross-linking, ethidium bromide (EtBr) fuorescence, cir-
cular dichroism (CD) spectroscopy, DNA melting, charac-
terization of DNA distortion by terbium fuorescence, and
other physical methods are mentioned in the Supporting
information.
Measurement of the partition coefcients
The “shake-fask” method was used to determine the logP of
the investigated compounds. Octanol-saturated water (OSW)
1 3

JBIC Journal of Biological Inorganic Chemistry
solution (0.05 M), and fnal reaction mixture (0.025 M).
Solubility and spectral data for the complexes are included
in the supplementary information (Table S1).
Results and discussion
Synthesis, characterization and purity
of [Pt(cis‑1,3‑diaminocycloalkane)Cl2] complexes
The NMR spectra and C, H and N analyses (cf. the Exper-
imental section) are consistent with the crystallographically
determined structures of these complexes. Crystallographic
data, and interatomic distances and bond angles are reported
in Fig. 3 and Tables 1 and S2. The crystal structures of all
three analogs (Fig. 3) unequivocally confrm the structures
of these analogs. The [Pt(cis-1,3-DACH)Cl2] was frst syn-
thesized by Qu et al. [19].
The three [Pt(cis-1,3-diaminocycloalkane)Cl2] analogs
were successfully synthesized on a 1.00 mol scale using a
variation of the Dhara method. We employed a higher I/Pt
ratio (8 as compared with~4 in the original Dhara method)
[10], a lower concentration of Pt(II) in the starting K2PtI4
Fig. 3 Molecular structures with atom numbering schemes for the investigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes
Table 1 Crystal structures of [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes: selected bond angles and bond lengths
Bond angles (deg)
Bond lengths (Å)
Pt(cis-1,3-DACH) Pt(cis-1,3-DACP) Pt(cis-1,3-DACB)
Pt(cis-1,3-DACH) Pt(cis-1,3-DACP) Pt(cis-1,3-DABP)
Cl2
Cl2
Cl2
Cl2
Cl2
Cl2
Cl–Pt–Cl
N–Pt–N
N1–Pt–Cl1 176.8 (5)
N2–Pt–Cl2 177.7 (5)
90.57 (18)
95.2 (8)
93.49 (4)
94.26 (14)
86.22 (11)
–
178.71 (11)
–
93.05 (2)
93.6 (2)
179.73 (12)
–
Pt–Cl1 2.363 (4)
Pt–Cl2 2.365 (4)
Pt–N1 1.994 (14)
Pt–N2 1.993 (14)
2.324 (10)
2.3159 (10)
2.047 (3)
2.039 (3)
–
2.3128 (11)
2.3128 (11)
2.047 (4)
–
N2–Pt–Cl1
–
–
–
Pt–N11
–
2.047 (4)
N11–Pt–Cl1
86.67 (12)
1 3

JBIC Journal of Biological Inorganic Chemistry
cell lines were incubated for 72 h with the platinum com-
pounds, and the cell survival in the culture treated with
the platinum compounds was evaluated as described in the
Experimental section. Results (Table 2) show that Pt(cis-
1,3-diaminocycloalkane)Cl2 complexes displayed signif-
cantly higher antiproliferative activity than cisplatin and
comparable with kiteplatin or oxaliplatin in all investigated
cancer cell lines. We also identifed [Pt(cis-1,3-DACH)
Cl2] as the most potent [Pt(cis-1,3-diaminocycloalkane)
Cl2] complex. Another most notable diference includes
lower toxicity of the Pt(cis-1,3-diaminocycloalkane)Cl2
complexes in noncancerous human cells derived from nor-
mal lung tissue (MRC5 pd30) (Table 2). Thus, the inves-
tigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes
showed similar or slightly better selectivity toward tumor
cells relative to non-tumorigenic normal cells in compari-
son with cisplatin so that it is conceivable that they may
be recognized as a promising approach to improving the
therapeutic index of platinum anticancer agents.
HPLC analyses
HPLC analyses of the fnal products used in the biological
testing and all other aspects of the study confrm the high
level of purity of these complexes and their widely difer-
ent physical nature. All complexes are at least 98.7% pure
or higher (Fig. 4), and their retention times increase with
increasing carbon content.
Antiproliferative activity
The antiproliferative activity of the investigated [Pt(cis-
1,3-diaminocycloalkane)Cl2] complexes was determined
against the human ovarian A2780, colon LoVo, breast
MCF7 and cervical HeLa cancer cell lines and noncan-
cerous human fetal lung fbroblasts MRC5 pd30, com-
monly used to test the antiproliferative activity of cispl-
atin analogs and other antitumor metallodrugs. The tumor
Fig. 4 HPLC chromatograms of
[Pt(cis-diaminocycloalkane)Cl2]
complexes: DACB, DACP, and
DACH and their retention times
(RT) and purity (%). Column:
C8; analyte: 1.8–2.2 mgmL−1
of complex in 0.45% NaCl in
nanopore H2O
Table 2 IC50 values (µM) as
A2780
LoVo
MCF-7
HeLa
MRC5 pd30
determined by MTT assay after
72 ha
[Pt(cis-1,3-DACB)Cl2]
[Pt(cis-1,3-DACP)Cl2]
[Pt(cis-1,3-DACH)Cl2]
Cisplatin
Kiteplatin
Oxaliplatin
1.99 0.08
1.5 0.3
2.9 0.3
2.2 0.2
1.74 0.04
5.9 0.1
1.1 0.4c
1.0 0.5e
5
1
46
28
11
8
1
2
28
26
20
22
19
1
1
1
1
3
4.1 0.3
1.7 0.2
0.83 0.03
2.67 0.03
0.8 0.2b
13
2
24.2 0.7
10
36 2d
3. 1c
1
0.89 0.06d
10 1d
31 3d
aData represent a mean SD from at least three independent experiments, each made in duplicate
bData taken from Ref. [20]
cData taken from Ref. [21]
dData taken from Ref. [22]
eData taken from Ref. [23]
1 3

JBIC Journal of Biological Inorganic Chemistry
The encouraging antiproliferative activity of [Pt(cis-
1,3-diaminocycloalkane)Cl2] complexes is an impetus
for studies focused on the mechanistic basis of biological
(pharmacological) efects of these compounds. Early steps
in the mechanism of biological activity of cisplatin and its
derivatives involve cell entry, reactions with sulfur-contain-
ing compounds, platinum–DNA binding, and processing
of platinated DNA by nuclear proteins (enzymes) [9, 24].
Therefore, to gain further insight into the mechanism of
action of [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes,
we describe experiments revealing their cellular accumula-
tion, reaction with glutathione (GSH), and DNA binding in
cell-free media. Our results suggest that an essential fac-
tor contributing to an improved antiproliferative activity of
[Pt(cis-1,3-diaminocycloalkane)Cl2] complexes in compari-
son with cisplatin is their enhanced hydrophobicity and cor-
responding augmented cellular accumulation. On the other
hand, their reactivity with GSH and DNA binding do not
substantially difer from reactivity with GSH and DNA bind-
ing of conventional cisplatin.
ammine ligands in cisplatin with 1,3-diaminocycloalkane
ligands enhances hydrophobicity and, consequently also
toxicity in tumor cells of the investigated [Pt(cis-1,3-diami-
nocycloalkane)Cl2] complexes.
Thus, for a possible relationship between the cellu-
lar uptake and the in vitro antiproliferative activity of the
investigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes,
cellular concentrations of platinum from these complexes
or cisplatin were determined by fameless atomic absorp-
tion spectrometry (FAAS). The cellular levels of [Pt(cis-
1,3-DACB)Cl2], [Pt(cis-1,3-DACP)Cl2], [Pt(cis-1,3-DACH)
Cl2], and cisplatin were measured after 6 h exposure of the
A2780 cells to the drugs at their concentration of 10 μM at
37 °C. The results are summarized in Table 2.
The highest hydrophobicity and cellular uptake of [Pt(cis-
1,3-DACH)Cl2] in comparison with the other two investi-
gated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes cor-
relate well with its highest antiproliferative activity in cancer
cells. The observation that the uptake of the least hydro-
phobic cisplatin accumulated in cells more than the more
hydrophobic [Pt(cis-1,3-DACB)Cl2] and [Pt(cis-1,3-DACP)
Cl2] is very likely connected with the fact that the main route
of cisplatin into cells involves besides passive difusion also
active transport [26] which may be less efcient in the case
of the cellular accumulation of cis-diaminocycloalkane
derivatives. Nonetheless, the hydrophobicity (log P), can-
cer cell activity (Table 2), and cell accumulation (Table 3)
correlate signifcantly in the case of the investigated [Pt(cis-
1,3-diaminocycloalkane)Cl2] complexes suggesting that the
enhanced cellular accumulation connected with augmented
hydrophobicity of the investigated [Pt(cis-1,3-diaminocy-
cloalkane)Cl2]complexes contributes essentially to their
antiproliferative activity.
Cellular accumulation
A factor relevant for cell uptake and consequently the
anticancer activity of platinum drugs is a measure of their
hydrophobicity expressed as log P values for octanol–water
partition. For several classes of metallo-anticancer com-
plexes, a correlation between increased hydrophobicity and
increased cytotoxic activity has been reported [25]. The log
P values for [Pt(cis-1,3-DACB)Cl2], [Pt(cis-1,3-DACP)Cl2],
[Pt(cis-1,3-DACH)Cl2] and cisplatin are shown in Table 3.
These results indicate that the investigated [Pt(cis-1,3-di-
aminocycloalkane)Cl2] complexes are more hydrophobic
than cisplatin, [Pt(cis-1,3-DACH)Cl2] being most hydropho-
bic. The hydrophobicity and antiproliferative activity in can-
cer cells (Table 2) of the investigated platinum complexes
correlate signifcantly in this study. Hence, an interesting
generalization of these results might be that replacement of
Reactions with glutathione
Platinum(II) compounds have a strong thermodynamic pref-
erence for binding to sulfur donor ligands. Hence, before
antitumor platinum drugs reach DNA in the nucleus of tumor
cells, they may interact with various compounds, including
sulfur-containing molecules. These interactions are gener-
ally thought to play a role in mechanisms underlying tumor
resistance to platinum compounds, their inactivation and
their side efects [27]. An example of endogenous thiols
to which platinum complexes bind when they are admin-
istered intravenously or after they enter the cell is GSH. In
the present work, we investigated reactions of GSH with
[Pt(cis-1,3-DACB)Cl2], [Pt(cis-1,3-DACP)Cl2], [Pt(cis-1,3-
DACH)Cl2] and cisplatin using UV absorption spectropho-
tometry [18, 28]. The absorbance at 260 nm was measured
as a function of time (Fig. S1). The experimental procedure
was as already described [18] with slight modifcations, as
Table 3 Cellular uptake of platinum from the investigated [Pt(cis-
1,3-diaminocycloalkane)Cl2] complexes and cisplatin into A2780
cells after 6 h of drug exposure (10 μM) and log P valuesa
ngPt/106 cells
log Pb
[Pt(cis-1,3-DACB)Cl2]
[Pt(cis-1,3-DACP)Cl2]
[Pt(cis-1,3-DACH)Cl2]
cisplatin
8.7 0.8
− 1.88
− 1.58
− 1.17
− 2.30
16
1
24.5 0.1
14.5 0.3
aAll results are expressed as mean values SD from at least three
independent experiments
bLog P (octanol/water) values measured by the “shake-fask” method
at room temperature
1 3

JBIC Journal of Biological Inorganic Chemistry
described in [17]. To establish the rate of the initial reaction,
the curve was ftted by nonlinear regression to the equation
Id = C + A1exp(− b1t) + A2exp(− b2t) (A1, A2, b1, b2; C are
constants, and t is the time of the reaction) by using Graph-
Pad Prism software. The initial slope (Sin) was calculated as
(A1b1 +A2b2) [18].
compared with that in isogenic DNA repair-profcient CHO-
K1 cells. Such comparison represents a powerful biological
tool to explore whether DNA is the potential target of the
biological action of Pt compounds. We observed (Table 4)
that the NER-defcient MMC-2 cells were signifcantly (ca.
10-times) more sensitive to killing than wild-type CHO-
K1 cells when treated with cisplatin. This result confrmed
that unrepaired DNA lesions formed by cisplatin contrib-
ute considerably to its cytotoxicity, which is in agreement
with generally accepted DNA as the major pharmacological
target of cisplatin. Similarly, the antiproliferative efects of
[Pt(cis-1,3-DACB)Cl2], [Pt(cis-1,3-DACP)Cl2], and [Pt(cis-
1,3-DACH)Cl2] were also markedly (ca. 10-times) poten-
tiated in cells, which are not able to repair DNA lesions.
This result suggests that reparable DNA damage is likely an
important factor contributing to the cytotoxic activity of the
investigated complexes.
Sin values calculated for reaction of GSH with cisplatin,
Pt(cis-1,3-DACB)Cl2, Pt(cis-1,3-DACP)Cl2, and [Pt(cis-
1,3-DACH)Cl2] were (7.9 0.7) × 10–4, (6.6 0.5) × 10–4,
(10.7 1.1) × 10–4, and (6.8 0.4) × 10–4, respectively.
These results suggest that 1,3-diaminocyclohexane-con-
taining complexes react with GSH with a rate comparable
with that observed for cisplatin, with [Pt(cis-1,3-DACP)Cl2]
being slightly more reactive. Additionally, antiproliferative
activity in cancer cells of the investigated platinum(II) com-
plexes (Table 2) does not correlate with their reactivity with
GSH. Hence, this result suggests that these interactions are
unlikely to be responsible for the diferent antiproliferative
activity of the investigated [Pt(cis-1,3-diaminocycloalkane)
Cl2] complexes on the one hand and diferent antiprolifera-
tive activity of these complexes and cisplatin on the other
hand.
One of the early Rosenberg’s experiments demonstrated
that antitumor cisplatin induced bacterial flamentation.
This result is considered to be one of the key experiments
demonstrating that DNA lesions of cisplatin are the criti-
cal lesions responsible for cell death. Moreover, previous
studies suggested that induction of flamentation in E. coli
by a monofunctional platinum complex [30] or antitumor
transplatin derivatives [31] implicated DNA as the cellular
target of these metallodrugs. Therefore, we investigated the
efect of [Pt(cis-1,3-DACB)Cl2], [Pt(cis-1,3-DACP)Cl2], and
[Pt(cis-1,3-DACH)Cl2] on bacteria as well. E. coli (CCM
7929) cells were cultured in the presence or absence of one
of the investigated platinum complexes.
DNA interactions in cell‑free media
DNA is an important target
The important early phase of the mechanism by which cis-
platin exerts its anticancer activity is also the formation of
adducts on nuclear DNA by these agents [9]. To substanti-
ate DNA interactions studies, the tests revealing whether
DNA is really a pharmacological target also for the investi-
gated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes were
performed.
After 5 h of incubation, 2 μL aliquots of each bacterial
cell culture suspension were placed on microscope slides
and analyzed by microscopy. As shown in Fig. S2, the mor-
phology of the cells treated with cisplatin was changed such
that bacteria became flamentous, and the cells markedly
longer than those in the control untreated sample were seen.
Similarly, the bacteria treated with the investigated [Pt(cis-
1,3-diaminocycloalkane)Cl2] complexes displayed flamen-
tous growth, in contrast to untreated bacteria that show
morphology typical for the E. coli cells with the short rods.
Thus, all complexes activate SOS response, block bacterial
The cellular (pharmacological) target is defned [9] as
a site within the cell (mostly a biomacromolecule), which
is altered by the drug and whose modifcation triggers pro-
cesses leading to proliferation blockage and subsequently
to tumor cell death. Several experimental criteria have been
applied to support the thesis that binding of cisplatin to
DNA is responsible for the cytotoxicity of this drug [9, 29]
One of these criteria is based on the observation that the
drug exhibits higher toxicity in the cells which are defcient
in DNA repair. This is because the ability of DNA lesions
to trigger cell death is inversely dependent on the capac-
ity of the cells to repair the damage. The assay used in the
present work is based on introducing a point mutation in a
gene coding one of the proteins necessary for the repair of
DNA [15]. Thus, the antiproliferative potency of [Pt(cis-1,3-
DACB)Cl2], [Pt(cis-1,3-DACP)Cl2], and [Pt(cis-1,3-DACH)
Cl2] in mutant Chinese hamster ovary MMC-2 cells defcient
in nucleotide excision repair (NER) was determined and
Table 4 IC50 mean values (µM) for the investigated Pt complexes as
determined in Chinese hamster ovarian cells by MTT assay after 72 h
treatmenta
IC50 (μM)
CHOK-1
MMC-2
Cisplatin
29
55
21
26
4
6
3
2
2.7 0.5
5.8 0.6
2.0 0.2
2.9 0.2
[Pt(cis-1,3-DACB)Cl2]
[Pt(cis-1,3-DACP)Cl2]
[Pt(cis-1,3-DACH)Cl2]
aAll results are expressed as mean values SD from at least three
independent experiments
1 3

JBIC Journal of Biological Inorganic Chemistry
Table 5 Summary of DNA binding of [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes and comparison with DNA binding of cisplatin
[Pt(cis-1,3-
[Pt(cis-1,3-
[Pt(cis-1,3-
Cisplatin
Kiteplatina
DACB)Cl2]
DACP)Cl2]
DACH)Cl2]
Kinetics of CT DNA binding (t50%) (min)
Preferential DNA binding sites (transcription mapping)
DNA unwinding (°)
DNA interstrand cross-linking (%)
Decrease of EtBr fuorescence
43
GG
13
5
Medium
Yes
40
GG
13
4.0 0.5
Medium
Yes
Yes
Increase
55
GG
13
6
Medium
Yes
64
GG
13
6
Medium
Yes
65
GG
15
2
1
2
2
1
2
1
2
1
6
ndb
ndb
ndb
ndb
Non-denaturational conformational alterations (CD spectroscopy)
Decrease of melting temperature
Yes
Increase
Yes
Increase
Yes
Increase
Tb3+ fuorescence (local disturbance in DNA conformation)
aTaken from Ref. 20.
b nd = not determined.
division, and induce a flamentous phenotype of E. coli.
These results further support the view that the biological
activity of the investigated [Pt(cis-1,3-diaminocycloalkane)
Cl2] complexes is likely elicited through their interaction
with DNA.
the investigated [Pt(cis-1,3-diaminocycloalkane)Cl2] com-
plexes show selectivity toward tumor cells relative to non-
tumorigenic normal cells. Further, to gain further insight
into the mechanism of action of [Pt(cis-1,3-diaminocycloal-
kane)Cl2] complexes, we performed several mechanistic
studies focused on understanding some early steps in the
mechanism of antitumor activity of bifunctional platinum(II)
complexes. Our data indicate that reactivities of the inves-
tigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes and
cisplatin with glutathione (which plays a role in mechanisms
underlying tumor resistance to platinum compounds, their
inactivation, and their side efects) and DNA binding (the
biological activity of cisplatin and its derivatives is elicited
through their interaction with DNA) do not substantially dif-
fer and thereby do not correlate with antiproliferative activ-
ity of these platinum(II) complexes in cancer cells. Thus, it
is reasonable to conclude that these interactions are unlikely
to be responsible for diferences in the antiproliferative
activity of the investigated [Pt(cis-1,3-diaminocycloalkane)
Cl2] complexes and cisplatin. In contrast, we show that the
higher antiproliferative activity in cancer cells of [Pt(cis-
1,3-DACH)Cl2] originates from its highest hydrophobicity
and most efcient cellular uptake. An important feature of
[Pt(cis-1,3-diaminocycloalkane)Cl2] complexes is that their
activity against a panel of tumor cells and selectivity toward
tumor cells is better than that of the FDA-approved cisplatin.
The work is in progress in our laboratories to establish a
key structure−function relationship that may underlie the
diferentiation in biological consequences among individ-
ual [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes and the
classical platinum anticancer drugs now widely used in the
clinic.
DNA binding
The fact that DNA is an important pharmacological target
for the [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes
prompted us to investigate the DNA interactions of these
complexes more deeply. We investigated various aspects of
DNA binding of the investigated platinum complexes in cell-
free media using a series of molecular biophysics methods.
The results of these investigations are summarized in Table 5
and are presented in detail in the Supplementary Information
(Figs. S3–S10). These results reveal no or only minimal dif-
ferences in the capability of the individual [Pt(cis-1,3-diami-
nocycloalkane)Cl2] complexes to afect DNA. Also impor-
tantly, the ability of the [Pt(cis-1,3-diaminocycloalkane)Cl2]
complexes to afect DNA was not very diferent from that of
classical cisplatin.
Thus, similarly to that in the case of reactivity of the
1,3-diamminocycloalkane complexes with glutathione,
the relatively uniform capability of these complexes to
bind to DNA and afect its conformation does not correlate
with their variable antiproliferative activity in cancer cells
(Table 1).
Conclusions
Among the series of new [Pt(cis-1,3-diaminocycloalkane)
Cl2] complexes, [Pt(cis-1,3-DACH)Cl2] destroys cancer cells
with greater efcacy than either of the other two investigated
derivatives of [Pt(cis-1,3-DACH)Cl2] complex containing
cyclobutyl or cyclopentyl moieties, or cisplatin. Moreover,
Acknowledgements This work was supported by the Ministry of Edu-
cation of the Czech Republic [Grant Number LTAUSA18009]. The
authors acknowledge that Richard Staples from the Chemistry Depart-
ment, Michigan State University determined the crystal structures of
our three Pt(cis-1,3-diaminocycloalkane)Cl2 samples and Dan Holmes
1 3

JBIC Journal of Biological Inorganic Chemistry
complexes containing 1,1′-binaphtyl-2,2′-diamine ligand does not
correlate with their antiproliferative activity in cancer cells. Inorg
Chim Acta 495:118952
from the Chemistry Department, Michigan State University obtained
the 1H and 195Pt NMR spectral data for these complexes.
17. Novakova O, Liskova B, Vystrcilova J, Suchankova T, Vrana O,
Starha P, Travnicek Z, Brabec V (2014) Conformation and recog-
nition of DNA damaged by antitumor cis-dichlorido platinum(ii)
complex of cdk inhibitor bohemine. Eur J Med Chem 78:54–64
18. Dabrowiak JC, Goodisman J, Souid AK (2002) Kinetic study
of the reaction of cisplatin with thiols. Drug Metab Dispos
30:1378–1384
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Additional information The structural data are deposited with the
Cambridge Crystallographic Data Center: 2021147 ([Pt(cis-1,3-di-
aminocyclobutane)Cl2]), 2021148 ([Pt(cis-1,3-diaminocyclopentane)
Cl2]), 2021149 ([Pt(cis-1,3-diaminocyclohexane)Cl2]).
19. Qu Y, Wenxia T, Hu H, Dai A, Ji X, Zhang F and Liu Y (1986)
Synthesis,characterization and antitumour activity of platinum
complexes of 1,3-diaminocyclohexane. Chinese J Appl Chem
3:25–29
20. Kasparkova J, Suchankova T, Halamikova A, Zerzankova L, Vrana
O, Margiotta N, Natile G, Brabec V (2010) Cytotoxicity, cellular
uptake, glutathione and DNA interactions of an antitumor large-
ring ptii chelate complex incorporating the cis-1,4-diaminocy-
clohexane carrier ligand. Biochem Pharmacol 79:552–564
21. Margiotta N, Savino S, Marzano C, Pacifco C, Hoeschele JD,
Gandin V, Natile G (2016) Cytotoxicity-boosting of kiteplatin by
Pt(IV) prodrugs with axial benzoate ligands. J Inorg Biochem
160:85–93
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