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Evaluation of novel trans-sulfonamide platinum complexes against tumor cell lines
European Journal of Medicinal Chemistry 76 (2014) 360e368
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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 Instituto de Investigación Hospital 12 de Octubre, Avda de Córdoba S/N, 28041 Madrid, Spain 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 b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2013 Received in revised form 30 January 2014 Accepted 8 February 2014 Available online 11 February 2014
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. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: trans-platinum complexes Sulfonamides Cytotoxicity profiles Cell cycle analyses
1. Introduction Cisplatin (CDDP) is the most known platinum antitumor complex 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, Nsulfonamides have been used extensively in medicinal chemistry as antibacterial activity, anticonvulsant (sultiame), inhibitors of the carbonic anhydrase, inhibitors of histone deacetylases, and inhibitors of microtubule polymerization among others [5e11]. Besides 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
* 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: [email protected] (J. Alemán), [email protected] (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.
experience in trans-platinum complexes [12,13], and the use of sulfur donor ligands (as DMSO) in platinum complexes [14], we have recently published the synthesis of a series of trans-sulfonamide 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 ligands from commercially available sulfonyl chlorides and the corresponding amines (1aee). Then, complexes 2aee were formed with good yield, starting from cis-[PtCl2(DMSO)2] (2f) and
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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.). 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
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$mL1 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 * mL1). 2.3. In vitro cytotoxic activity
Fig. 2. X-ray analysis ORTEP of compound 2e. Ellipsoids displayed at 30% probability.
We initially studied all these complexes (2aee) in two representative 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 derivative (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 adenocarcinoma), Tera-2 (testicular embryonal carcinoma) and, HeLa (cervix adenocarcinoma). We carried out parallel experiments with
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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 alkylamine (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 assays 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.
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 mechanisms, 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
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 triggered 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.
Table 1 Antiproliferative activity (IC50, mM, SD) of complexes 2aec in comparison to CDDP in a panel of twelve human tumour cell lines. Cell line
2a
2b
SK-MEL-5 DLD-1 MCF-7 T-47D Tera-2 HeLa DU-145 NCIeH2052 HCC-78 H-292 A2780 A2780cisR
7.059 1.21 6.775 1.10 5.375 1.11 20.53 01.34 6.725 1.17 14.620 1.11 23.410 2.10 10.410 1,04 17.680 1.21 6.045 1.06 >50 13.92 3.22
3.235 2.751 5.055 7.363 6.892 1.548 3.287 5.088 3.376 1.942 13.06 5.71
2c
1.08 1.18 1.11 1.18 1.99 1.20 1.10 1.11 1.13 1.37 3.15 2.12
4.849 2.735 2.591 16.100 6.28 5.187 9.581 8.016 4.217 2.947 37.9 7.59
CDDP
1.08 1.16 1.10 2.16 1.19 1.03 1.03 1.12 1.11 1.31 4.12 3.06
2.883 16.320 5.317 3.135 3.379 12.750 2.445 6.738 12.190 4.603 16.08 141.8
1.12 1.27 1.15 1.09 1.12 2.23 1.09 1.16 1.21 1.36 3.20 5.17
Fig. 5. IC50 values of 2aec and CDDP, after 72 h of drugs exposure. * Significant differences at P < 0.05 (ManneWhitney U test) compared to CDDP.
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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 compounds 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
A
2a
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 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 evaluated 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 sulfonoamide 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 cytotoxic, 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
2b
CDDP
2c
B
C
70
140
60
120
50
100
Tail Length (u.a.)
% of cells with comet
363
40 30 20
2a
80
2b
60
2c CDDP
40 20
10
0
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 SD.
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Fig. 8. Effect of CDDP and 2aec complexes on cell cycle progression.
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Fig. 9. IC50 values of 2aec and CDDP, after 72 h of drug exposure in A2780 and A2780cisR cell lines.
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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 understand their molecular mechanism of action, and will be reported in a later paper. 4. Experimental protocols 4.1. Chemistry
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 We have found that mono-sulfonamide platinum complexes had good antitumor activity against different cell lines. The
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 spectrometer, running at 300, 75, and 64.51 MHz for 1H, 13C{H}, and 195 Pt{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. 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-1,2diamine, propyl-1,3-diamine, or (rac)-cyclohexane-1,2-diamine
Fig. 11. Effect of CDDP and 2aec complexes on cell cycle progression (LC5).
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(10 eq., 3.0 M). The mixture was allowed to reach room temperature 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. 4.1.2.1. N-(2-Aminoethyl)-4-(dimethylamino)naphthalene-1sulfonamide (1a). The product was directly obtained following the standard procedure as yellow solid (89% yield) without further 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, 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. 4.1.2.2. (rac)-(2-Aminocyclohexyl)-5-(dimethylamino) naphthalene1-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, 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), 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. 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. M.P. ( C): 94.7e95.2. 1H NMR (300 MHz, CDCl3) d 7.75 (d, J ¼ 8.2 Hz, 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: [M þ H]þ calcd for C13H21N2O2S 269.1324; found 269.1326. 4.1.2.4. N-(3-Aminopropyl)-4-methylbenzenesulfonamide (1d). The product was directly obtained following the standard procedure as white solid (84% yield) without further purification. M.P. ( 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), 2.42 (s, 3H), 1.58 (quint, J ¼ 6.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) d 143.1 (C), 137.2 (C), 129.6 (CH), 127.1 (CH), 43.0 (CH2), 40.8 (CH2), 30.9 (CH2), 21.5 (CH3). MSeFABþ: [M þ H]þ calcd for C10H17N2O2S 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 procedure as white solid (76% yield) without further purification. M.P. ( 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) d 142.2 (C), 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.
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. 4.1.3.1. trans-Dichloro [2-(5-(dimethylamino)naphthalene-1sulfonamido)ethylamino] (dimethylsulfoxide) platinum(II) (2a). The product was directly obtained following the standard procedure as yellow solid (69% yield) without further purification. M.P. ( 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), 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) d 2511.7. Anal. calcd. for C16H25Cl2N3O3PtS2: C, 30.14; H, 3.95; N, 6.59. Found: C, 30.01; H, 3.91; N, 6.80. 4.1.3.2. trans-Dichloro [(rac)-2-(5-(dimethylamino) naphthalene-1sulfonamido)cyclohexylamino] (dimethyl-sulfoxide)platinum(II) (2b). The product was directly obtained following the standard procedure as yellow solid (79% yield) without further purification. M.P. ( C): 186 (decomposed). 1H NMR (300 MHz, CDCl3) d 8.58 (d, 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 (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 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. 4.1.3.3. trans-Dichloro [(rac)-2-(4-methylphenylsulfonamido)cyclohexylamino] (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 (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, CDCl3) d 144.1 (C), 137.5 (C), 130.1 (CH), 127.0 (CH), 58.9 (CH), 58.6 (CH), 44.2 (CH3), 43.9 (CH3), 32.6 (CH2), 32.3 (CH2), 24.9 (CH2), 24.2 (CH2), 21.6 (CH3). 195Pt NMR (64 MHz, CDCl3) d 3112.7. MSeESIþ: [M þ Na]þ calcd for C15H26Cl2N2NaO3PtS2 634.0303; found 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. 4.1.3.4. trans-Dichloro [3-(4-methylphenylsulfonamido) propylamino](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 NMR (300 MHz, CDCl3) d 7.78 (d, J ¼ 7.0 Hz, 2H), 7.32 (d, J ¼ 7.6 Hz, 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),
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39.3 (CH2), 29.8 (CH2), 21.5 (CH3). 195Pt NMR (64 MHz, CDCl3) d 3075.3. MSeESIþ: [M þ Na]þ calcd. for C12H22Cl2N2NaO3PtS2 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)propylamino](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 (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 (s, 2JPteC 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 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 carcinoma), HeLa (cervix adenocarcinoma), DU-145 (prostate carcinoma), 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 supplemented with 10% heat-inactivated fetal bovine serum, 100 mU/mL 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). 4.3. Cell proliferation assays 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 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) according 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 untreated cells) and IC50 values were determined by the nonlinear multipurpose curve-fitting program GraphPad Prism. 4.4. Cell cycle analyses Cells were cultured in 100-mm culture dishes, grown to 50%e 70% confluence, and treated with compounds at the same drug
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concentration, 5 mM, for 24 h. The cell cycle progression was 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 FACScalibur flow cytometer and the CellQuest software (BD Biosciences). 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 evaluation 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 electrophoresis of the suspended lysed cells. Electrophoresis was conducted 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). 4.6. Statistical analyses 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 SD. P < 0.05 was considered statistically significant. Acknowledgments 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 a pre-doctoral fellowship. C.P. and C.V.D.-G. acknowledge their fellowship from “Fundación Mutua Madrileña” 2008/141 and 2010/018 respectively. A.A.-L acknowledges 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. Appendix A. Supplementary data 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 cisplatinDNA adducts, Chemical Reviews 99 (1999) 2467e2498. [3] C.A. Rabik, M.E. Dolan, Molecular mechanisms of resistance and toxicity associated 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. [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, Prevalence, utilization, and cost of antiepileptic drugs for epilepsy in Germany e a
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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 Instituto de Investigación Hospital 12 de Octubre, Avda de Córdoba S/N, 28041 Madrid, Spain 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 b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2013 Received in revised form 30 January 2014 Accepted 8 February 2014 Available online 11 February 2014
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. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: trans-platinum complexes Sulfonamides Cytotoxicity profiles Cell cycle analyses
1. Introduction Cisplatin (CDDP) is the most known platinum antitumor complex 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, Nsulfonamides have been used extensively in medicinal chemistry as antibacterial activity, anticonvulsant (sultiame), inhibitors of the carbonic anhydrase, inhibitors of histone deacetylases, and inhibitors of microtubule polymerization among others [5e11]. Besides 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
* 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: [email protected] (J. Alemán), [email protected] (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.
experience in trans-platinum complexes [12,13], and the use of sulfur donor ligands (as DMSO) in platinum complexes [14], we have recently published the synthesis of a series of trans-sulfonamide 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 ligands from commercially available sulfonyl chlorides and the corresponding amines (1aee). Then, complexes 2aee were formed with good yield, starting from cis-[PtCl2(DMSO)2] (2f) and
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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.). 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
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$mL1 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 * mL1). 2.3. In vitro cytotoxic activity
Fig. 2. X-ray analysis ORTEP of compound 2e. Ellipsoids displayed at 30% probability.
We initially studied all these complexes (2aee) in two representative 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 derivative (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 adenocarcinoma), Tera-2 (testicular embryonal carcinoma) and, HeLa (cervix adenocarcinoma). We carried out parallel experiments with
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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 alkylamine (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 assays 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.
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 mechanisms, 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
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 triggered 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.
Table 1 Antiproliferative activity (IC50, mM, SD) of complexes 2aec in comparison to CDDP in a panel of twelve human tumour cell lines. Cell line
2a
2b
SK-MEL-5 DLD-1 MCF-7 T-47D Tera-2 HeLa DU-145 NCIeH2052 HCC-78 H-292 A2780 A2780cisR
7.059 1.21 6.775 1.10 5.375 1.11 20.53 01.34 6.725 1.17 14.620 1.11 23.410 2.10 10.410 1,04 17.680 1.21 6.045 1.06 >50 13.92 3.22
3.235 2.751 5.055 7.363 6.892 1.548 3.287 5.088 3.376 1.942 13.06 5.71
2c
1.08 1.18 1.11 1.18 1.99 1.20 1.10 1.11 1.13 1.37 3.15 2.12
4.849 2.735 2.591 16.100 6.28 5.187 9.581 8.016 4.217 2.947 37.9 7.59
CDDP
1.08 1.16 1.10 2.16 1.19 1.03 1.03 1.12 1.11 1.31 4.12 3.06
2.883 16.320 5.317 3.135 3.379 12.750 2.445 6.738 12.190 4.603 16.08 141.8
1.12 1.27 1.15 1.09 1.12 2.23 1.09 1.16 1.21 1.36 3.20 5.17
Fig. 5. IC50 values of 2aec and CDDP, after 72 h of drugs exposure. * Significant differences at P < 0.05 (ManneWhitney U test) compared to CDDP.
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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 compounds 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
A
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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 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 evaluated 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 sulfonoamide 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 cytotoxic, 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
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Fig. 8. Effect of CDDP and 2aec complexes on cell cycle progression.
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Fig. 9. IC50 values of 2aec and CDDP, after 72 h of drug exposure in A2780 and A2780cisR cell lines.
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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 understand their molecular mechanism of action, and will be reported in a later paper. 4. Experimental protocols 4.1. Chemistry
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 We have found that mono-sulfonamide platinum complexes had good antitumor activity against different cell lines. The
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 spectrometer, running at 300, 75, and 64.51 MHz for 1H, 13C{H}, and 195 Pt{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. 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-1,2diamine, propyl-1,3-diamine, or (rac)-cyclohexane-1,2-diamine
Fig. 11. Effect of CDDP and 2aec complexes on cell cycle progression (LC5).
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(10 eq., 3.0 M). The mixture was allowed to reach room temperature 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. 4.1.2.1. N-(2-Aminoethyl)-4-(dimethylamino)naphthalene-1sulfonamide (1a). The product was directly obtained following the standard procedure as yellow solid (89% yield) without further 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, 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. 4.1.2.2. (rac)-(2-Aminocyclohexyl)-5-(dimethylamino) naphthalene1-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, 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), 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. 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. M.P. ( C): 94.7e95.2. 1H NMR (300 MHz, CDCl3) d 7.75 (d, J ¼ 8.2 Hz, 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: [M þ H]þ calcd for C13H21N2O2S 269.1324; found 269.1326. 4.1.2.4. N-(3-Aminopropyl)-4-methylbenzenesulfonamide (1d). The product was directly obtained following the standard procedure as white solid (84% yield) without further purification. M.P. ( 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), 2.42 (s, 3H), 1.58 (quint, J ¼ 6.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) d 143.1 (C), 137.2 (C), 129.6 (CH), 127.1 (CH), 43.0 (CH2), 40.8 (CH2), 30.9 (CH2), 21.5 (CH3). MSeFABþ: [M þ H]þ calcd for C10H17N2O2S 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 procedure as white solid (76% yield) without further purification. M.P. ( 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) d 142.2 (C), 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.
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. 4.1.3.1. trans-Dichloro [2-(5-(dimethylamino)naphthalene-1sulfonamido)ethylamino] (dimethylsulfoxide) platinum(II) (2a). The product was directly obtained following the standard procedure as yellow solid (69% yield) without further purification. M.P. ( 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), 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) d 2511.7. Anal. calcd. for C16H25Cl2N3O3PtS2: C, 30.14; H, 3.95; N, 6.59. Found: C, 30.01; H, 3.91; N, 6.80. 4.1.3.2. trans-Dichloro [(rac)-2-(5-(dimethylamino) naphthalene-1sulfonamido)cyclohexylamino] (dimethyl-sulfoxide)platinum(II) (2b). The product was directly obtained following the standard procedure as yellow solid (79% yield) without further purification. M.P. ( C): 186 (decomposed). 1H NMR (300 MHz, CDCl3) d 8.58 (d, 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 (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 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. 4.1.3.3. trans-Dichloro [(rac)-2-(4-methylphenylsulfonamido)cyclohexylamino] (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 (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, CDCl3) d 144.1 (C), 137.5 (C), 130.1 (CH), 127.0 (CH), 58.9 (CH), 58.6 (CH), 44.2 (CH3), 43.9 (CH3), 32.6 (CH2), 32.3 (CH2), 24.9 (CH2), 24.2 (CH2), 21.6 (CH3). 195Pt NMR (64 MHz, CDCl3) d 3112.7. MSeESIþ: [M þ Na]þ calcd for C15H26Cl2N2NaO3PtS2 634.0303; found 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. 4.1.3.4. trans-Dichloro [3-(4-methylphenylsulfonamido) propylamino](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 NMR (300 MHz, CDCl3) d 7.78 (d, J ¼ 7.0 Hz, 2H), 7.32 (d, J ¼ 7.6 Hz, 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),
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39.3 (CH2), 29.8 (CH2), 21.5 (CH3). 195Pt NMR (64 MHz, CDCl3) d 3075.3. MSeESIþ: [M þ Na]þ calcd. for C12H22Cl2N2NaO3PtS2 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)propylamino](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 (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 (s, 2JPteC 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 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 carcinoma), HeLa (cervix adenocarcinoma), DU-145 (prostate carcinoma), 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 supplemented with 10% heat-inactivated fetal bovine serum, 100 mU/mL 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). 4.3. Cell proliferation assays 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 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) according 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 untreated cells) and IC50 values were determined by the nonlinear multipurpose curve-fitting program GraphPad Prism. 4.4. Cell cycle analyses Cells were cultured in 100-mm culture dishes, grown to 50%e 70% confluence, and treated with compounds at the same drug
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concentration, 5 mM, for 24 h. The cell cycle progression was 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 FACScalibur flow cytometer and the CellQuest software (BD Biosciences). 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 evaluation 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 electrophoresis of the suspended lysed cells. Electrophoresis was conducted 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). 4.6. Statistical analyses 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 SD. P < 0.05 was considered statistically significant. Acknowledgments 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 a pre-doctoral fellowship. C.P. and C.V.D.-G. acknowledge their fellowship from “Fundación Mutua Madrileña” 2008/141 and 2010/018 respectively. A.A.-L acknowledges 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. Appendix A. Supplementary data 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 cisplatinDNA adducts, Chemical Reviews 99 (1999) 2467e2498. [3] C.A. Rabik, M.E. Dolan, Molecular mechanisms of resistance and toxicity associated 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. [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, Prevalence, utilization, and cost of antiepileptic drugs for epilepsy in Germany e a
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[10] [11]
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