Evaluation of novel trans-sulfonamide platinum complexes against tumor cell lines

Article abstract of DOI:10.1016/j.ejmech.2014.02.022

Platinum-based drugs, mainly cisplatin, are employed for the treatment of solid malignancies. However, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure. Here, the antitumor activity of different trans-sulfonamide platinum complexes in a panel of human cell lines is presented. The cytotoxicity profiles and cell cycle analyses of these platinum sulfonamide complexes were different from those of cisplatin. These studies showed that complex 2b with cyclohexyldiamine and dansyl moieties had the best antitumoral activities.

Full text of DOI:10.1016/j.ejmech.2014.02.022

European Journal of Medicinal Chemistry 76 (2014) 360e368
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of novel trans-sulfonamide platinum complexes against
tumor cell lines
,
,
,
Carlos Pérez a b, C. Vanesa Díaz-García a b, Alba Agudo-López a b, Virginia del Solar c,
Silvia Cabrera c, M. Teresa Agulló-Ortuño a b, Carmen Navarro-Ranninger c, José Alemán d
**
,
,
,
*
José A. López-Martín a
,b,
a Medical Oncology Service, Hospital Universitario 12 de Octubre, Avda de Córdoba S/N, 28041 Madrid, Spain
b Instituto de Investigación Hospital 12 de Octubre, Avda de Córdoba S/N, 28041 Madrid, Spain
c Inorganic Chemistry Department (Módulo 7), Universidad Autónoma de Madrid, C/Fco Tomás y Valiente, 5, Cantoblanco, 28049 Madrid, Spain
d Organic Chemistry Department (Módulo 1), Universidad Autónoma de Madrid, C/Fco Tomás y Valiente, 5, Cantoblanco, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Platinum-based drugs, mainly cisplatin, are employed for the treatment of solid malignancies. However,
cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure.
Here, the antitumor activity of different trans-sulfonamide platinum complexes in a panel of human cell
lines is presented. The cytotoxicity profiles and cell cycle analyses of these platinum sulfonamide
complexes were different from those of cisplatin. These studies showed that complex 2b with cyclo-
hexyldiamine and dansyl moieties had the best antitumoral activities.
Received 5 December 2013
Received in revised form
30 January 2014
Accepted 8 February 2014
Available online 11 February 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
trans-platinum complexes
Sulfonamides
Cytotoxicity profiles
Cell cycle analyses
1. Introduction
experience in trans-platinum complexes [12,13], and the use of
sulfur donor ligands (as DMSO) in platinum complexes [14], we
Cisplatin (CDDP) is the most known platinum antitumor com-
plex and, as well, one of the most effective antitumor agents used
against a variety of cancers [1,2]. Despite the therapeutic success of
platinum anticancer drugs, their severe toxicities and the drug
resistance limit their clinical use [3,4]. In view of these limitations,
research has been extended to new platinum analogues with the
aim of overcoming these adverse effects. On the other hand, N-
sulfonamides have been used extensively in medicinal chemistry as
antibacterial activity, anticonvulsant (sultiame), inhibitors of the
carbonic anhydrase, inhibitors of histone deacetylases, and in-
hibitors of microtubule polymerization among others [5e11]. Be-
sides these large number of publications concerning to the use of
sulfonamides, the synthesis of platinum compounds containing in
their structure sulfonamides have been scarce. Based on our
have recently published the synthesis of a series of trans-sulfon-
amide platinum complexes [13]. Thus, we conducted preliminary
in vitro cytotoxicity tests and have identified quite active complexes
with the trans-1,2-cyclohexyldiamine moiety. However, only a
limited number of cancer cell lines were tested and therefore
further biological data are needed.
In the present work, new biological effects of trans-sulfonamide
platinum complexes on cellular proliferation and DNA damage
response will be presented. The in vitro activity of the compounds is
evaluated and also compared to that of cisplatin.
2. Results and discussion
2.1. Synthesis and characterization
We initially studied five different compounds (Fig. 1), two of
them with dansyl moiety and the other three with the tolyl and
mesityl groups (2aee). First, we synthesized the sulfonamide li-
gands from commercially available sulfonyl chlorides and the cor-
responding amines (1aee). Then, complexes 2aee were formed
with good yield, starting from cis-[PtCl2(DMSO)2] (2f) and
* Corresponding author.
** Corresponding author. Organic Chemistry Department (Módulo 1), Universidad
Autónoma de Madrid, C/Fco Tomás y Valiente, 5, Cantoblanco, 28049 Madrid, Spain.
E-mail addresses: jose.aleman@uam.es (J. Alemán), jlmartin@salud.madrid.org
(J.A. López-Martín).
http://dx.doi.org/10.1016/j.ejmech.2014.02.022
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

C. Pérez et al. / European Journal of Medicinal Chemistry 76 (2014) 360e368
361
Fig. 1. Complexes studied in the present publication. Racemic mixture of trans-cyclohexyl-1,2-diamine was used (only one isomer is shown).
monosulfonamide ligands 1. Complexes 2aee were determined
with trans geometry by X-ray analysis of complex 2e (see Fig. 2) and
by comparison of NMR data and mass spectrometry of the rest of
complexes. All the complexes (2aee) showed a clear peak in mass
spectrometry and matched elemental analyses which are in
accordance with the proposal structures (see Section 4 and S.I.).
are related to their administration as drugs, their low solubility and
the limited stability of these complexes in aqueous solution [15e
18]. Thus, we studied the stability of complex 2b because it was
one of the most active complexes (see above) in saline solution
(0.9% NaCl solution) at 37 ꢀC (corporal human temperature), which
is one of the pharmaceutical dosage forms for platinum complexes.
In a 100 mM saline solution, slightly more than 50% of complex 2b
remained unaltered after 24 h of incubation (Fig. 3). Additionally,
the solubility of complex 2b was determined to be 0.826 mg$mLꢁ1
in a saturated saline solution after 3 h of incubation which is
comparable with the solubility of cisplatin in the same solution
(1.0 mg * mLꢁ1).
2.2. Solubility and stability studies
Next, we focused our attention in the solubility and stability of
these complexes. Two important drawbacks in platinum complexes
2.3. In vitro cytotoxic activity
We initially studied all these complexes (2aee) in two repre-
sentative tumor cell lines, HeLa (cervix adenocarcinoma) and T-47D
(ductal breast epithelial tumor cell line), in order to find which the
optimal structural features are (Fig. 4). From this data, it can be
concluded that compounds 2a, 2d and 2e with linear alkyl chains in
their structures, are less active than compounds 2bec with the
trans-cyclohexyl-1,2-diamine moiety.
With these preliminary data, we decided to focus on the more
active complexes 2bec with the trans-cyclohexyl-1,2-diamine
moiety and its comparison with one linear alkyl complex deriva-
tive (2a), and to perform a large screening in 12 different human
tumor cell lines of different tissue origins (see Table 1 and Fig. 5).
The most sensitive cell lines are SK-MEL-5 (melanoma), DLD-1
(colorectal adenocarcinoma), MCF-7 (luminal breast adenocarci-
noma), Tera-2 (testicular embryonal carcinoma) and, HeLa (cervix
adenocarcinoma). We carried out parallel experiments with
Fig. 2. X-ray analysis ORTEP of compound 2e. Ellipsoids displayed at 30% probability.

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C. Pérez et al. / European Journal of Medicinal Chemistry 76 (2014) 360e368
Fig. 3. Stability of complex 2b in saline solution (0.9% NaCl solution) at 37 ꢀC (see Supporting Information for more details).
better antiproliferative data than complexes with a linear alkyl-
amine (2a versus 2b, blue and brown bars, respectively).
Additionally, we have clarified if the biological activity would
come from the complex 2b or their respective precursors that
would be liberated in the inner cell. Thus, the antiproliferative as-
says in DLD-1 and HeLa cell lines were carried out for complex 2f
and sulfonamide 1b which are the platinum and sulfonamide
precursors of complex 2b (see Fig. 6). In all the cases, the complex
2f (complex precursor of sulfonamide complexes 2) as well as the
sulfonamide 1b were less active than complex 2b (Fig. 6). Thus, we
can confirm that the antiproliferative activity is due to the structure
of complex 2b.
2.4. Comet assay and cell cycle progression
Fig. 4. IC50 values of 2aee after 48 h of drug exposure.
The cytotoxic mode of action of cisplatin is mediated by its
interaction with DNA to form DNA adducts, primarily intrastrand
crosslink adducts. The results presented above, led us to explore
whether trans-sulfonamide platinum complexes treatment trig-
gered a DNA damage response. Alkaline comet assay that detect
DNA double-strand breaks (DSBs) were performed using a protocol
for the single-cell electrophoresis assay [21e24]. For this assay, cells
were treated with compounds at their corresponding IC50 for 24 h.
At least one hundred cells by condition were examined. Comet
lengths (head plus tail) were measured with the ImageJ software,
also used to acquire microphotographs. Results are shown in Fig. 7.
cisplatin on cells plated at the same time from a single flask. For the
results presented in Fig. 5, we can conclude that the most active
compound for the majority of cancer cell lines is 2b (brown bar).
The cytotoxicity profile for these compounds was clearly different
from that of cisplatin. It is noteworthy that compound 2b was more
active than CDDP in both DLD-1 and HeLa cell lines. DLD-1 cell line
is characterized by having a mutated P53 gene, and HeLa cell line
expressed low level of this protein. Indeed, most of the anticancer
agents that act through DNA damage or stress-inducing mecha-
nisms, like cisplatin, require wild-type P53 [19,20]. As we observed
in our previous studies [13] and in the preliminary results of Fig. 5,
the platinum complexes with trans-1,2-cyclohexyldiamine showed
Table 1
Antiproliferative activity (IC50
in a panel of twelve human tumour cell lines.
,
m
M, ꢂSD) of complexes 2aec in comparison to CDDP
Cell line
2a
2b
2c
4.849 ꢂ 1.08
CDDP
SK-MEL-5
DLD-1
MCF-7
T-47D
Tera-2
HeLa
7.059 ꢂ 1.21
6.775 ꢂ 1.10
5.375 ꢂ 1.11
3.235 ꢂ 1.08
2.751 ꢂ 1.18
5.055 ꢂ 1.11
2.883 ꢂ 1.12
2.735 ꢂ 1.16 16.320 ꢂ 1.27
2.591 ꢂ 1.10
5.317 ꢂ 1.15
3.135 ꢂ 1.09
3.379 ꢂ 1.12
20.53 ꢂ 01.34 7.363 ꢂ 1.18 16.100 ꢂ 2.16
6.725 ꢂ 1.17
6.892 ꢂ 1.99
6.28 ꢂ 1.19
14.620 ꢂ 1.11 1.548 ꢂ 1.20
23.410 ꢂ 2.10 3.287 ꢂ 1.10
5.187 ꢂ 1.03 12.750 ꢂ 2.23
DU-145
9.581 ꢂ 1.03
8.016 ꢂ 1.12
2.445 ꢂ 1.09
6.738 ꢂ 1.16
NCIeH2052 10.410 ꢂ 1,04 5.088 ꢂ 1.11
HCC-78
H-292
A2780
17.680 ꢂ 1.21 3.376 ꢂ 1.13
4.217 ꢂ 1.11 12.190 ꢂ 1.21
6.045 ꢂ 1.06
>50
1.942 ꢂ 1.37
13.06 ꢂ 3.15
5.71 ꢂ 2.12
2.947 ꢂ 1.31
37.9 ꢂ 4.12
7.59 ꢂ 3.06
4.603 ꢂ 1.36
16.08 ꢂ 3.20
141.8 ꢂ 5.17
Fig. 5. IC50 values of 2aec and CDDP, after 72 h of drugs exposure. * Significant dif-
ferences at P < 0.05 (ManneWhitney U test) compared to CDDP.
A2780cisR
13.92 ꢂ 3.22

C. Pérez et al. / European Journal of Medicinal Chemistry 76 (2014) 360e368
363
complexes 2 was characterized by a large G1 peak of interphase
cells (2n DNA content), a much smaller G2/M peak of cells with
duplicated DNA content (4n), and an intermediate subpopulation
that corresponds to the S phase (Fig. 8). This block was apparent in
all cell lines after a 24-h exposure to any of the four compounds
2aec.
The cell cycle arrest is a common cellular response to DNA
damage, and is viewed as a delay period in DNA replication during
which the cell can attempt to repair the damage. If this attempt
fails, cell death pathways will be activated. The above results lead
us to speculate that the mechanism of action of the compounds
tested is different from that of CDDP.
To clarify the latter assertion, we evaluated 2aec in the human
ovarian carcinoma cell line A2780 and its cisplatin-resistant
counterpart A2780cisR (Fig. 9). Very interestingly, the evaluation
of complexes 2aec in cisplatin-sensitive A2780 cell line showed
that the cytotoxicity was quite moderate for 2a and 2c, and by
contrast was quite active for 2b. On the other hand, in cisplatin
resistant cell line (A2780cisR), all complexes (2aec) were quite
Fig. 6. IC50 values of 2b, 2f and the sulfonamide 1b after 72 h of drugs exposure.
Of the many types of DNA damage, the most toxic is DNA DSBs
[25]. As shown in Fig. 7, in HeLa cell line, both the rate of affected
cells with DSBs and the degree of damage caused by these com-
pounds was similar to that caused by CDDP. However, in DLD-1 cell
line, although the intensity of damage caused by these compounds
was very similar to that of CDDP, the numbers of affected cells was
significantly higher. As it is shown in Fig. 7A, the cell damage caused
by these compounds at their IC50 was very similar.
On the other hand, the decrease in IC50 values in cells treated
with these compounds could be because of reduced proliferation,
cell death, or both. Therefore, next we investigated the effect of the
different compounds on cell cycle (Fig. 8). Cell cycle analyses were
done in all cell lines studied. Fig. 8 is representative of the results in
HeLa and DLD-1 cell lines. Propidium iodide (PI) staining revealed a
pattern of DNA staining different in cells treated with CDDP and
with trans-platin sulfonamide complexes. CDDP induced cell cycle
arrest in S and G2/M as it was previously described [26e28].
However, the pattern of DNA staining in cells treated with
active (IC50 ¼ 5.7e13.90
mM) and even more active than cisplatin.
Thus, our sulfonamide complexes 2aec can overcome the CDDP
resistance, indicating that their mechanism of action should be
different from those described to cis-platin.
After these experiments in tumor cell lines, we have also eval-
uated the cytotoxicity in a non-cancerous cell line. Assays and cell
cycle analysis on human embryonic lung-derived fibroblast cell line
(LC5) were performed as a model to test the selectivity of sulfo-
noamide complexes 2. Cytotoxicity results of these complexes 2 in
this non tumour cell line (LC5) were similar to that of tumor cell
lines tested (Fig. 10). Likewise, compound 2b was the most cyto-
toxic, although with an analogous value to that of cisplatin. We can
conclude that these complexes 2 would be no more harmful than
cisplatin in non-tumor cell lines.
As seen in Fig. 11, the cell cycle profile of the compounds 2aec in
LC5 cell line is different from that of cisplatin, and similar to that of
tumor cells lines. Compounds treatment resulted in a weak arrest of
A
2a
2b
2c
CDDP
B
C
140
120
100
80
70
60
50
40
30
20
10
0
2a
2b
2c
60
CDDP
40
20
0
HeLa
DLD-1
HeLa
DLD-1
Fig. 7. Comet Assay of DLD-1 cell treated with the compounds to their corresponding IC50 (A). The percentage of the cells affected with comet (B) and the tail length of the comet (C)
are shown in the corresponding bar graph as mean values ꢂ SD.

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C. Pérez et al. / European Journal of Medicinal Chemistry 76 (2014) 360e368
Fig. 8. Effect of CDDP and 2aec complexes on cell cycle progression.

C. Pérez et al. / European Journal of Medicinal Chemistry 76 (2014) 360e368
365
cytotoxicity profile is different from that of cisplatin in a panel of 12
well-characterized cell lines by single-dose screen. For some cases,
IC50 values are similar to cisplatin and trans-platin complexes (2).
However, compound 2b is more active than cisplatin in DLD-1 and
HeLa (both P53 deficient cell lines). The development of platinum
compounds, such as trans-sulfonamide platinum complexes, with a
differential cytotoxicity from commercial platinum compounds,
should facilitate identification of candidate compounds that are
active in cisplatin-resistant cancers.
Cell cycle studies indicate that, unlike cisplatin, trans-platinum
complexes 2 induce a G1 block that suggests cell cycle delay for a
DNA damage response or DNA repair. Interestingly, our compounds
were able to overcome CDDP resistance in a cisplatin-resistant cell
line (A2780cisR). In our opinion, these compounds are promising
lead compounds for the generation of novel drug candidates with
different cytotoxicity profiles from those of cisplatin. On the other
hand, regarding structureeactivity relationships, our data suggest
that small differences in the carrier ligands could play an important
role in the biological effects and could provide new rational basis
for the design of new platinum antitumor drugs. More experiments
with these complexes are being carried out in order to better un-
derstand their molecular mechanism of action, and will be reported
in a later paper.
Fig. 9. IC50 values of 2aec and CDDP, after 72 h of drug exposure in A2780 and
A2780cisR cell lines.
4. Experimental protocols
4.1. Chemistry
4.1.1. Material and methods
All reagents and chemicals were purchased from commercial
sources (s.d. Fine Chemicals, India; SigmaeAldrich, U.S.A.) and used
without further purifications. Solvents were purified by standard
procedures. NMR spectra were acquired on a Bruker 300 spec-
trometer, running at 300, 75, and 64.51 MHz for 1H, 13C{H}, and
195Pt{H} respectively. Chemical shifts (
d) are reported in ppm
relative to residual solvent signals (CDCl3, 7.26 ppm for 1H NMR,
CDCl3, 77.0 ppm for 13C NMR. DMSO-d6, 2.50 ppm for 1H NMR,
DMSO-d6, 39.5 ppm for 13C NMR). 13C NMR spectra were acquired
on a broad band decoupled mode. 195Pt NMR spectra were obtained
with chemical shifts reported in ppm downfield relative to the
external reference 1.0 M Na2PtCl6 in D2O.
Fig. 10. IC50 values of 2aec and CDDP, after 72 h of drug exposure in LC5 cell line.
the cell cycle in the transition G1 to S while cisplatin induced cell
cycle arrest in S and G2/M.
3. Conclusions
4.1.2. General procedure for the synthesis of N-sulfonamides 1aee
To a solution of the corresponding sulfonyl chloride (26.0 mmol)
in 26 mL of dichloromethane at 0 ꢀC, was added rapidly ethyl-1,2-
diamine, propyl-1,3-diamine, or (rac)-cyclohexane-1,2-diamine
We have found that mono-sulfonamide platinum complexes
had good antitumor activity against different cell lines. The
Fig. 11. Effect of CDDP and 2aec complexes on cell cycle progression (LC5).

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C. Pérez et al. / European Journal of Medicinal Chemistry 76 (2014) 360e368
(10 eq., 3.0 M). The mixture was allowed to reach room tempera-
ture and was stirred during 10 h. The crude mixture was filtered
and the obtained oil was concentrated under reduced pressure.
Then, 10 mL of ice-water were added to the concentrated mixture
and a solid appeared which was filtrated and washed with cool
water and dried under vacuum for 12 h.
4.1.3. Procedure for the synthesis of trans-complexes 2
To a solution of the corresponding sulfonamide 1aec or 1f
(0.2 mmol) in methanol (0.2 mL) was added cis-[PtCl2(DMSO)2]
(0.22 mmol). The mixture was stirred at room temperature for 72 h.
Then, the reaction mixture was filtered and the filtrate was
concentrated to dryness in vacuum. The solid residue was washed
with cold H2O obtaining the corresponding pure platinum
complexes.
4.1.2.1. N-(2-Aminoethyl)-4-(dimethylamino)naphthalene-1-
sulfonamide (1a). The product was directly obtained following the
standard procedure as yellow solid (89% yield) without further
4.1.3.1. trans-Dichloro [2-(5-(dimethylamino)naphthalene-1-
sulfonamido)ethylamino] (dimethylsulfoxide) platinum(II) (2a).
The product was directly obtained following the standard proce-
dure as yellow solid (69% yield) without further purification. M.P.
purification. M.P. (ꢀC): 154e156. 1H NMR (300 MHz, CDCl3)
d 8.52
(d, J ¼ 8.5 Hz, 1H), 8.30 (d, J ¼ 8.7 Hz, 1H), 8.22 (dd, J ¼ 7.3, 1.2 Hz,
1H), 7.54 (dd, J ¼ 8.6, 7.7 Hz,1H), 7.50 (dd, J ¼ 8.5, 7.4 Hz,1H), 7.16 (d,
J ¼ 7.5 Hz, 1H), 2.89 (dd, J ¼ 6.5, 4.9 Hz, 2H), 2.87 (s, 6H), 2.67 (dd,
(ꢀC): 173 (decomposed). 1H NMR (300 MHz, CDCl3)
d 8.54 (d,
J ¼ 8.6 Hz, 1H), 8.28 (d, J ¼ 8.6 Hz, 1H), 8.21 (dd, J ¼ 7.3, 1.3 Hz, 1H),
7.58 (t, J ¼ 8.1 Hz, 1H), 7.51 (t, J ¼ 8.0 Hz, 1H), 7.18 (d, J ¼ 7.5 Hz, 1H),
6.06 (t, J ¼ 6.5 Hz, 1H), 4.29 (bs, 2H), 3.33 (s, 6H), 3.36e3.24 (m, 2H),
J ¼ 6.5, 4.8 Hz, 2H). 13C NMR (75 MHz, CDCl3)
d 152.0 (C), 134.7 (C),
130.4 (CH), 129.9 (C), 129.7 (CH), 129.6 (C), 128.4 (CH), 123.2 (CH),
118.7 (CH), 115.2 (CH), 45.5 (CH2), 45.4 (CH3), 40.8 (CH2). MSeFABþ:
[M þ H]þ calcd for C14H20N3O2S 294.1276; found 294.1270.
2.99e2.90 (m, 2H), 2.88 (s, 6H). 13C NMR (75 MHz, CDCl3)
d 152.1
(C), 134.4 (C), 130.8 (CH), 130.0 (C), 129.5 (CH), 129.4 (C), 128.9 (CH),
123.2 (CH), 118.7 (CH), 115.4 (CH), 45.7 (CH2), 45.5 (CH2), 45.4 (CH3),
43.8 (CH3). 195Pt NMR (64 MHz, CDCl3)
C
d
ꢁ2511.7. Anal. calcd. for
16H25Cl2N3O3PtS2: C, 30.14; H, 3.95; N, 6.59. Found: C, 30.01; H,
3.91; N, 6.80.
4.1.2.2. (rac)-(2-Aminocyclohexyl)-5-(dimethylamino) naphthalene-
1-sulfonamide (1b). The product was directly obtained following
the standard procedure as yellow solid (91% yield) without further
purification. M.P. (ꢀC): 162 (decomposed). 1H NMR (300 MHz,
CDCl3)
d
8.46 (d, J ¼ 8.5 Hz, 1H), 8.40 (d, J ¼ 8.6 Hz, 1H), 8.29 (d,
4.1.3.2. trans-Dichloro [(rac)-2-(5-(dimethylamino) naphthalene-1-
sulfonamido)cyclohexylamino] (dimethyl-sulfoxide)platinum(II) (2b).
The product was directly obtained following the standard proce-
dure as yellow solid (79% yield) without further purification. M.P.
J ¼ 7.1 Hz, 1H), 7.52 (t, J ¼ 8.1 Hz, 1H), 7.46 (t, J ¼ 7.9 Hz, 1H), 7.09 (d,
J ¼ 7.5 Hz, 1H), 3.97 (bs, 3H), 2.79 (s, 6H), 2.77e2.69 (m, 1H), 2.57e
2.49 (m, 1H), 1.91e1.86 (m, 1H), 1.46e1.43 (m, 1H), 1.40e1.33 (m,
2H), 1.05e0.89 (m, 4H). 13C NMR (75 MHz, CDCl3)
d 151.8 (C), 136.1
(ꢀC): 186 (decomposed). 1H NMR (300 MHz, CDCl3)
d 8.58 (d,
(C), 130.2 (CH), 129.7 (C), 129.6 (C), 129.4 (CH), 128.4 (CH), 123.2
(CH), 119.2 (CH), 115.2 (CH), 60.3 (CH), 54.4 (CH), 45.4 (CH3), 34.5
(CH2), 32.3 (CH2), 25.0 (CH2), 24.5 (CH2). MSeFABþ: [M þ H]þ calcd
for C18H26N3O2S 348.1746; found 348.1741.
J ¼ 8.5 Hz, 1H), 8.27 (dd, J ¼ 7.4, 1.3 Hz, 1H), 8.22 (d, J ¼ 8.7 Hz, 1H),
7.60 (dd, J ¼ 8.7, 7.6 Hz, 1H), 7.55 (dd, J ¼ 8.6, 7.4 Hz, 1H), 7.21 (d,
J ¼ 7.6 Hz, 1H), 5.01 (d, J ¼ 9.2 Hz, 1H), 4.62 (d, J ¼ 10.1 Hz, 1H), 3.40
(s, 6H), 3.22e3.09 (m,1H), 2.91 (s, 6H), 2.85e2.67 (m, 3H),1.74e1.64
(m, 1H), 1.48e1.36 (m, 1H), 1.23e0.79 (m, 5H). 13C NMR (75 MHz,
CDCl3)
d 152.3 (C), 134.2 (C), 131.1 (CH), 130.0 (CH), 129.8 (C), 129.3
4.1.2.3. (rac)-N-(2-Aminocyclohexyl)-4-methylbenzene sulfonamide
(1c). The product was directly obtained following the standard
procedure as white solid (82% yield) without further purification.
(C), 129.2 (CH), 123.2 (CH), 118.0 (CH), 115.5 (CH), 58.9 (CH3), 45.4
(CH3), 44.1 (CH), 44.0 (CH), 32.6 (CH2), 32.3 (CH2), 24.8 (CH2), 24.1
(CH2). 195Pt NMR (64 MHz, CDCl3)
d
ꢁ3118.6. MSeFABþ: [M þ H]þ
M.P. (ꢀC): 94.7e95.2. 1H NMR (300 MHz, CDCl3)
d
7.75 (d, J ¼ 8.2 Hz,
calcd for C20H32Cl2N3O3PtS2 691.0816; found 691.0819. Anal. calcd.
for C20H31Cl2N3O3PtS2: C, 34.73; H, 4.52; N, 6.08. Found: C, 34.95; H,
4.52; N, 5.67.
2H), 7.24 (d, J ¼ 7.9 Hz, 2H), 3.72 (bs, 3H), 2.69 (dt, J ¼ 10.1, 3.7 Hz,
1H), 2.48 (dt, J ¼ 10.1, 3.9 Hz, 1H), 2.36 (s, 3H), 1.93e1.90 (m, 1H),
1.57e1.49 (m, 3H), 1.21e1.05 (m, 4H). 13C NMR (75 MHz, CDCl3)
d
143.1 (C), 138.3 (C), 129.6 (CH), 127.0 (CH), 60.0 (CH), 54.6 (CH),
34.7 (CH2), 32.3 (CH2), 25.0 (CH2), 24.6 (CH2), 21.5 (CH3). MSeFABþ:
4.1.3.3. trans-Dichloro [(rac)-2-(4-methylphenylsulfonamido)cyclo-
hexylamino] (dimethylsulfoxide) platinum(II) (2c). The product was
directly obtained following the standard procedure as pale yellow
solid (71% yield) without further purification. M.P. (ꢀC): 189
[M þ H]þ calcd for C13H21N2O2S 269.1324; found 269.1326.
4.1.2.4. N-(3-Aminopropyl)-4-methylbenzenesulfonamide
The product was directly obtained following the standard proce-
dure as white solid (84% yield) without further purification. M.P.
(1d).
(decomposed). 1H NMR (300 MHz, CDCl3)
d
7.79 (d, J ¼ 8.2 Hz, 2H),
7.33 (d, J ¼ 8.0 Hz, 2H), 5.51 (bs, 1H), 4.94 (d, J ¼ 10.9 Hz,1H), 3.43 (s,
6H), 3.00e2.77 (m, 3H), 2.44 (s, 3H), 1.82e1.63 (m, 2H), 1.60e1.48
(m, 1H), 1.38e1.16 (m, 3H), 1.15e0.93 (m, 2H). 13C NMR (75 MHz,
(ꢀC): 98e101. 1H NMR (300 MHz, CDCl3)
d
7.75 (d, J ¼ 8.3 Hz, 2H),
7.31 (d, J ¼ 8.5 Hz, 2H), 3.06 (t, J ¼ 6.2 Hz, 2H), 2.78 (t, J ¼ 6.1 Hz, 2H),
CDCl3)
d 144.1 (C), 137.5 (C), 130.1 (CH), 127.0 (CH), 58.9 (CH), 58.6
2.42 (s, 3H), 1.58 (quint, J ¼ 6.1 Hz, 2H). 13C NMR (75 MHz, CDCl3)
(CH), 44.2 (CH3), 43.9 (CH3), 32.6 (CH2), 32.3 (CH2), 24.9 (CH2), 24.2
d
143.1 (C), 137.2 (C), 129.6 (CH), 127.1 (CH), 43.0 (CH2), 40.8 (CH2),
(CH2), 21.6 (CH3). 195Pt NMR (64 MHz, CDCl3)
[M
634.0283. Anal. calcd. for C15H26Cl2N2O3PtS2: C, 29.41; H, 4.28; N,
4.57. Found: C, 29.68; H, 4.20; N, 4.41.
d
ꢁ3112.7. MSeESIþ:
30.9 (CH2), 21.5 (CH3). MSeFABþ: [M þ H]þ calcd for C10H17N2O2S
þ
Na]þ calcd for C15H26Cl2N2NaO3PtS2 634.0303; found
229.1005; found 229.1011.
4.1.2.5. N-(3-Aminopropyl)-2,4,6-trimethylbenzene sulfonamide (1e).
The product was directly obtained following the standard proce-
dure as white solid (76% yield) without further purification. M.P.
4.1.3.4. trans-Dichloro [3-(4-methylphenylsulfonamido) propylami-
no](dimethylsulfoxide)platinum(II) (2d). The product was directly
obtained following the general procedure as pale yellow solid (59%
yield) without further purification. M.P. (ꢀC): 172 (decomposed). 1H
(ꢀC): 123e124. 1H NMR (300 MHz, CDCl3)
d
6.95 (s, 2H), 2.99 (t,
J ¼ 6.1 Hz, 2H), 2.80 (t, J ¼ 6.0 Hz, 2H), 2.64 (s, 3H), 2.62 (s, 3H), 2.30
(s, 3H), 1.67e1.56 (m, 2H). 13C NMR (75 MHz, CDCl3)
142.2 (C),
d
NMR (300 MHz, CDCl3)
d
7.78 (d, J ¼ 7.0 Hz, 2H), 7.32 (d, J ¼ 7.6 Hz,
141.8 (C), 139.0 (C), 133.7 (C), 132.0 (CH), 131.8 (CH), 42.4 (CH2), 41.1
(CH2), 30.8 (CH2), 22.9 (CH3), 20.9 (CH3). MSeFABþ: [M þ H]þ calcd
for C12H21N2O2S 257.1318; found 257.1324.
2H), 5.51e4.48 (m, 1H), 4,47 (bs, 2H), 3.49 (s, 6H), 3.19e3.03 (m,
4H), 2.49 (s, 3H), 2.13e1.96 (m, 2H). 13C NMR (75 MHz, CDCl3)
d
143.5 (C), 136.5 (C), 132.2 (CH), 127.2 (CH), 43.7 (CH3), 42.9 (CH2),

C. Pérez et al. / European Journal of Medicinal Chemistry 76 (2014) 360e368
367
39.3 (CH2), 29.8 (CH2), 21.5 (CH3). 195Pt NMR (64 MHz, CDCl3)
d
concentration, 5 mM, for 24 h. The cell cycle progression was
ꢁ3075.3. MSeESIþ: [M þ Na]þ calcd. for C12H22Cl2N2NaO3PtS2
examined by flow cytometry after staining with propidium iodide
(PI). Data from al least 10,000 cells per sample were acquired. DNA
content and cell cycle analyses were performed by using a FACS-
calibur flow cytometer and the CellQuest software (BD Biosciences).
593.9990; found 593.9980. Anal. calcd. for C12H22Cl2N2O3PtS2: C,
25.18; H, 3.87; N, 4.89. Found: C, 25.08; H, 3.97; N, 4.83.
4.1.3.5. trans-Dichloro [3-(2,4,6-(trimethyl)phenylsulfonamido)pro-
pylamino](dimethylsulfoxide) platinum(II) (2e). To a solution of the
sulfonamide 1e (0.22 mmol) in methanol (0.2 mL) was added cis-
[PtCl2(DMSO)2] (0.20 mmol). The mixture was stirred at room
temperature for 72 h. Then the reaction mixture was filtered and
the solid was washed with methanol, acetone, and cold H2O
obtaining the corresponding pure platinum complex. The product
was directly obtained as pale yellow solid (70% yield) without
further purification. M.P. (ꢀC): 166 (decomposed). 1H NMR
4.5. Comet assay
The Single Cell Gel Electrophoresis assay (also known as Comet
Assay) is a sensitive technique for measuring DNA strand breaks at
the level of individual cell. This is a standard technique for evalu-
ation of DNA damage/repair, biomonitoring and genotoxicity
testing. The assay was performed as described by Singh et al. [21],
with minor modifications. It involves the encapsulation of cells in a
low-melting-point agarose suspension on a microscope slide, lysis
of the cell in alkaline conditions to form nucleoids containing
supercoiled loops of DNA linked to the nuclear matrix and elec-
trophoresis of the suspended lysed cells. Electrophoresis was con-
ducted for 20 min at 25 V and 300 mA (0.86 V/cm). If nuclear DNA
suffers breaks, upon exposure to the electric field, the fragmented
DNA offers a characteristic “comet” appearance of the nuclei, with a
head and a trailing tail. The tail length is proportional to the DNA
damage. This is followed by visual analysis with staining of DNA
and calculating tail length to determine the extent of DNA damage
(ImageJ program).
(300 MHz, CDCl3)
d
6.95 (s, 2H), 5.03 (t, J ¼ 6.6 Hz, 1H), 4.84 (t,
J ¼ 6.5 Hz, 1H), 4.07 (bs, 1H), 3.54 (s, 3JPteH ¼ 11.4 Hz, 6H), 3.41e3.31
(m, 1H), 3.14e2.95 (m, 3H), 2.63 (s, 3H), 2.62 (s, 3H), 2.30 (s, 3H),
2.02e1.93 (m, 1H), 1.68e1.58 (m, 1H). 13C NMR (75 MHz, CDCl3)
d
142.2 (C), 139.1 (C), 139.0 (C), 133.9 (C), 132.2 (CH), 132.1 (CH), 45.1
2
(s, JPteC ¼ 26.9 Hz, CH3), 43.2 (CH2), 40.0 (CH2), 30.1 (CH2), 23.1
(CH3), 23.0 (CH3), 21.1 (CH3). 195Pt NMR (64 MHz, CDCl3)
d
ꢁ3093.0.
MSeESIþ: [M þ Na]þ calcd for C14H26Cl2N2NaO3PtS2 622.0302;
found 622.0303. Anal. calcd. for C14H26Cl2N2O3PtS2.acetone: C,
31.00; H, 4.90; N, 4.25. Found: C, 31.39; H, 4.75; N, 4.24.
4.2. Cell culture
4.6. Statistical analyses
Ten cancer cell lines e SK-MEL-5 (melanoma), DLD-1 (colorectal
adenocarcinoma), MCF-7 and T-47D (luminal and ductal breast
adenocarcinoma respectively), Tera-2 (testicular embryonal carci-
noma), HeLa (cervix adenocarcinoma), DU-145 (prostate carci-
noma), NCI-H2052 (mesothelioma), HCC-78, and H-292
(adenocarcinoma and squamous non-small cell lung cancer
respectively) e were grown in DMEM or RPMI media supple-
mented with 10% heat-inactivated fetal bovine serum, 100 mU/mL
The concentration of treatment yielding 50% inhibition (IC50
values) was calculated using Graph-Pad Prism software version 4.0
(San Diego, CA). All data are presented as the mean ꢂ SD. P < 0.05
was considered statistically significant.
Acknowledgments
penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine in a 5%
CO2 atmosphere at 37 ꢀC. Cell lines were purchased from LGC
Promochem, SLU-ATCC (Barcelona, Spain). A2780 and A2780cisR
cell lines were a gift from Dr. Jan Reedijk (Leiden, The Netherlands).
We acknowledge grants from the Spanish MEC (SAF2009-09431,
SAF2012-34424). J.A. thanks COST Action CM1105. J.A. and S.C.
thank the Spanish Government for their Ramón y Cajal contracts
and V.d.S. for
acknowledge their fellowship from “Fundación Mutua Madrileña”
2008/141 and 2010/018 respectively. A.A.-L acknowledges
a pre-doctoral fellowship. C.P. and C.V.D.-G.
4.3. Cell proliferation assays
a
fellowship from “Red Temática de Investigación Cooperativa en
Cáncer” (ISCIII, Spain). We also thank to Reedijk’s group for A2780
and A2780cisR cell lines. Johnson Matthey PLC is thanked for a
generous loan of platinum metals.
Drugs were dissolved in dimethyl sulfoxide (DMSO) at 10 mM
and stored at ꢁ20 ꢀC until use. For determination of compounds
sensitivity, cells diluted in 100
mL/well of complete cell culture
medium were plated in 96-well flat-bottom plates and allowed to
attach for 24 h. Medium was removed from the wells and replaced
by complete medium containing compounds at concentrations
Appendix A. Supplementary data
ranging from 0.01 mM to 100 mM for another 72 h. The final DMSO
concentration in all experiments was <0.05% in medium. All
experimental points were set up in six wells, and all were
confirmed in at least three independent experiments. Viable cells
were determined using the WST-1 assay (Roche, Mannheim) ac-
cording to the manufacturer’s protocol. The absorbance at 450 nm
were measured with a multiplate reader (Multiskan EX, Thermo
Scientific), using a reference wavelength of 620 nm to correct for
unspecific absorption. Results were expressed as a percentage
relative to vehicle-treated control (0.1% DMSO was added to un-
treated cells) and IC50 values were determined by the nonlinear
multipurpose curve-fitting program GraphPad Prism.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.022.
References
[1] A.P. Silverman, W. Bu, S.M. Cohen, S.J. Lippard, 2.4-Å crystal structure of the
asymmetric platinum complex {Pt(ammine)(cyclohexylamine)}2þ bound to a
dodecamer DNA duplex, Journal of Biological Chemistry 277 (2002) 49743e
49749.
[2] E.R. Jamieson, S.J. Lippard, Structure, recognition, and processing of cisplatin-
DNA adducts, Chemical Reviews 99 (1999) 2467e2498.
[3] C.A. Rabik, M.E. Dolan, Molecular mechanisms of resistance and toxicity asso-
ciated with platinating agents, Cancer Treatment Reviews 33 (2007) 9e23.
[4] L. Galluzzi, L. Senovilla, I. Vitale, J. Michels, I. Martins, O. Kepp, M. Castedo,
G. Kroemer, Molecular mechanisms of cisplatin resistance, Oncogene 31
(2012) 1869e1883.
4.4. Cell cycle analyses
[5] H.M. Hamer, R. Dodel, A. Strzelczyk, M. Balzer-Geldsetzer, J.P. Reese,
O. Schöffski, S. Schwab, S. Knake, W.H. Oertel, F. Rosenow, K. Kostev, Preva-
lence, utilization, and cost of antiepileptic drugs for epilepsy in Germany e a
Cells were cultured in 100-mm culture dishes, grown to 50%e
70% confluence, and treated with compounds at the same drug

368
C. Pérez et al. / European Journal of Medicinal Chemistry 76 (2014) 360e368
nationwide population-based study in children and adults, Journal of
Neurology 259 (2012) 2376e2384.
[6] Z. Liu, Z. Zhou, W. Tian, X. Fan, D. Xue, L. Yu, Q. Yu, Y.Q. Long, Discovery of
novel 2-N-Aryl-substituted benzenesulfonamidoacetamides: orally bioavail-
able tubulin polymerization inhibitors with marked antitumor activities,
ChemMedChem 7 (2012) 680e693.
[16] D. Kushev, R. Grünert, N. Spassovska, E. Golovinsky, P.J. Bednarski, Unusual
reactivity of cytotoxic cis-dihydrazide Pt(II) complexes in aqueous solution,
Journal of Inorganic Biochemistry 96 (2003) 469e477.
[17] W.-P. Liu, Q.-S. Ye, Y. Yu, X.-Z. Chen, S.-Q. Hou, L.G. Lou, Y.P. Yang, Y.-M. Wang,
Q. Su, Novel lipophilic platinum(II) compounds of salicylate derivatives,
Platinum Metals Review 52 (2008) 163e171.
[7] Z.L. Liu, W. Tian, Y. Wang, S. Kuang, X.M. Luo, Q. Yu, A novel sulfonamide
agent, MPSP-001, exhibits potent activity against human cancer cells in vitro
through disruption of microtubule, Acta Pharmacologica Sinica 33 (2012)
261e270.
[18] E.S. Kostewicz, U. Brauns, R. Becker, J.B. Dressman, Forecasting the oral ab-
sorption behavior of poorly soluble weak bases using solubility and dissolu-
tion studies in biorelevant Media, Pharmaceutical Research 19 (2002) 345e
349.
[8] J.Y. Chang, H.P. Hsieh, C.Y. Chang, K.S. Hsu, Y.F. Chiang, C.M. Chen, C.C. Kuo,
J.P. Liou, 7-Aroyl-aminoindoline-1-sulfonamides as a novel class of potent
antitubulin agents, Journal of Medicinal Chemistry 49 (2006) 6656e6659.
[9] E. Svastová, A. Hulíková, M. Rafajová, M. Zatovicová, A. Gibadulinová, A. Casini,
A. Cecchi, A. Scozzafava, C.T. Supuran, J. Pastorek, S. Pastoreková, Hypoxia
activates the capacity of tumor-associated carbonic anhydrase IX to acidify
extracellular pH, FEBS Letters 577 (2004) 439e445.
[19] M. Martínez-Rivera, Z.H. Siddik, Resistance and gain-of-resistance phenotypes
in cancer harboring wild-type p53, Biochemical Pharmacology 83 (2012)
1049e1062.
[20] J.M. Brown, L.D. Attardi, The role of apoptosis in cancer development and
treatment response, Nature Reviews Cancer 5 (2005) 231e237.
[21] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Scheider,
A simple technique for
quantitation of low levels of DNA damage in individual cells, Experimental
Cell Research 175 (1988) 184e191.
[10] C.T. Supuran, Inhibition of carbonic anhydrase IX as
a novel anticancer
mechanism, World Journal of Clinical Oncology 7 (2012) 98e103.
[11] A. de Almeida, B.L. Oliveira, J.D.G. Correia, G. Soveral, A. Casini, Emerging
protein targets for metal-based pharmaceutical agents: an update, Coordi-
nation Chemistry Reviews 257 (2013) 2689e2704.
[12] E.I. Montero, S. Díaz, A.M. González-Vadillo, J.M. Pérez, C. Alonso, C. Navarro-
Ranninger, Preparation and characterization of novel trans-[PtCl2(amine)(i-
sopropylamine)] Compounds: cytotoxic activity and apoptosis induction in
ras-transformed cells, Journal of Medicinal Chemistry 42 (1999) 4264e4268.
[13] J. Alemán, V. del Solar, A. Álvarez-Valdes, C. Rios-Luci, J.M. Padrón, C. Navarro-
Ranninger, Novel N-sulfonamide trans-platinum complexes: synthesis, reac-
tivity and In vitro evaluation, MedChemComm (2011) 789e793.
[22] A. Hartmann, G. Speit, The contribution of cytotoxicity to DNA-effects in the
single cell gel test (comet assay), Toxicology Letters 90 (1997) 183e188.
[23] G. Speit, A. Hartmann, The comet assay: a sensitive genotoxicity test for the
detection of DNA damage and repair, Methods in Molecular Biology 314
(2006) 275e286.
[24] V. Gallo, A. Khan, C. Gonzales, D.H. Phillips, B. Schoket, E. Györffy, L. Anna,
K. Kovács, P. Møller, S. Loft, S. Kyrtopoulos, G. Matullo, P. Vineis, Validation of
biomarkers for the study of environmental carcinogens: a review, Biomarkers
13 (2008) 505e534.
[25] S.P. Jackson, J. Bartek, The DNA-damage response in human biology and dis-
ease, Nature 461 (2009) 1071e1078.
[14] N. Farrell, D.M. Kiley, W. Schmidt, M.P. Hacker, Chemical properties and
antitumor activity of complexes of platinum containing substituted sulfoxides
[PtCl(R’R’SO)(diamine)]NO3. Chirality and leaving-group ability of sulfoxide
affecting biological activity, Inorganic Chemistry 29 (1990) 397e403.
[15] D.N. Bell, J.J. Liu, M.D. Tingle, B. Rattel, T.U. Meyer, M.J. McKeage, Comparative
protein binding, stability and degradation of satraplatin, JM118 and cisplatin
in human plasma in vitro, Clinical and Experimental Pharmacology and
Physiology 35 (2008) 1440e1446.
[26] Z.H. Siddik, Cisplatin: mode of cytotoxic action and molecular basis of resis-
tance, Oncogene 22 (2003) 7265e7279.
[27] C. St Germain, N. Niknejad, L. Ma, K. Garbuio, T. Hai, J. Dimitroulakos, Cisplatin
induces cytotoxicity through the mitogen-activated protein kinase pathways
and activating transcription factor 3, Neoplasia 12 (2010) 527e538.
[28] G. He, J. Luang, A.R. Khokhar, Z.H. Siddik, The impact of S- and G2-checkpoint
response on the fidelity of G1-arrest by cisplatin and its comparison to a non-
cross-resistant platinum (IV) analog, Gynecologic Oncology 122 (2011) 402e409.