Dalton Transactions

View Article Online View Journal

Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: G. lv, L. guo, H. Yang, L. qiu, T. Wang, H. Liu and J. Lin, Dalton Trans., 2015, DOI: 10.1039/C5DT00169B.

This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.

www.rsc.org/dalton

Page 1 of 24

Dalton Transactions View Article Online

DOI: 10.1039/C5DT00169B

Lipophilicity-dependent ruthenium N-heterocyclic carbene complexes as potential anticancer agents†

Jianguo Lin*

Abstract Five Ru(II)-N-heterocyclic carbenes (NHC) (1-5) were synthesized by reacting the appropriately substituted imidazolium chlorides with Ag2O, forming the NHC-silver chloride in situ followed by transmetalation with dimeric p-cymene ruthenium(II) dichloride. All the complexes were characterized by NMR and ESI-MS, and the complex 1 was also characterized by single-crystal X-ray diffraction. The IC50 values of these five complexes were determined by the MTT-based assay against four human cancer cell lines, SKOV-3 (ovarian), PC-3 (prostate), MDA-MB-231 (breast) and EC109 (esophagus). The cytotoxicities of these complexes changed from a moderate effect to a fine one, corresponding to the increasing lipophilicity order of the complex as 23>1>2, which accords well with the general discipline that -C(CH3)3>-CF3>-CH3>-H>-OCH3. According to the previous studies, it is known that the cell membrane permeability and cytotoxicity of the complex usually increase with the lipophilicity of the side chains in the NHC ligands.8, 24 Therefore, the complex 5 with the optimum lipophilicity was predicted to possess a high biological activity. In the subsequent section, the cytotoxicity of complexes 1-5 was studied.

Cytotoxicity The IC50 values of complexes 1−5 against four human cancer cell lines SKOV-3, PC-3, MDA-MB-231 and EC109 were measured and listed in Table 2. Complexes 1 and 2 showed low cytotoxicity against four cancer cell lines, while complexes 3 and 4 exhibited moderate activity which was comparable to that of cisplatin. Particularly, the complex 5 displayed a superior cytotoxicity. Especially against the PC-3 cell lines, the cytotoxicity of complex 5 was almost 3-5 fold higher than those against other cell lines and nearly 6 fold more active than that of cisplatin. Furthermore, it is interesting to note that the cytotoxicity of these five complexes is correlated well with the lipophilicity of the complex. For example, the complex 5 possesses the optimum lipophilicity and it hence generates the strongest cytotoxicity. On the contrary, the complex 2 presents the lowest lipophilicity and it therefore exhibits the weakest

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

the NHC groups. As the lipophilicity gradually changing, -C(CH3)3, -CF3, -CH3 and

Dalton Transactions

Page 12 of 24 View Article Online

DOI: 10.1039/C5DT00169B

cytotoxicity. As anticipated, the complexes 3 and 4 with moderate lipophilicity show moderate cytotoxicities. The most possible reason may be that the complex 5 with an optimum lipophilicity is easier to pass through the cell membrane, which induces a

cytotoxicity of these five complexes is comparable to the previous reported NHC-Ru complexes. For example, M. Tacke et al reported a series of NHC-Ru complexes based on imidazole group against MCF-7 cell lines, which the IC50 values ranged from 80±15 to 2.4±0.7 µM.21 Another example was a series of arene ruthenium benzimidazolylidene carbene complexes reported by I. Ott et al, which the cytotoxicities were also changed from a moderate effect to a fine one and the lowest IC50 values were 2.1±0.9 µM against MCF-7 and 2.4±1.0 µM against HT-29 cell lines.8 All these findings further demonstrate that the substituent in the imidazole group has great influence on the biological activities of these complexes.

Morphologic studies and cell cycle analysis Considering the superior activity of complex 5 against the PC-3 cell lines, morphologic changes and cell cycle distribution of the PC-3 cells induced by the complex 5 were further investigated to study the possible action mechanism of Ru-NHC complexes. As shown in Figure 2, the treatment with complex 5 at a low dose (2 µM) produced no obvious changes on the morphology of PC-3 cells. When it was treated with a higher dose (4 and 6 µM), remarkable apoptosis related morphologic changes were observed, such as the cell size reduction, cell shrinkage and rounding up. Especially at 6 µM, there was almost no existence of normal cells. It was also clear that the density of cells decreased after the treatment of complex 5. This indicated that complex 5 can not only inhibit the proliferation of the cancer cell lines, but also induce the apoptosis of the cancer cell lines. The cell cycle distribution of PC-3 cell lines induced by complex 5 was analyzed using a FACS Calibur flow cytometer. After exposure to complex 5 at a low dosage (2 µM) for 48 h, there was an increase in the G2/M proportion of PC-3 cell lines (from 20.06 to 30.24%) (Fig. 3). Treated by 4 µM of complex 5, the percentage of PC-3

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

higher cell uptake and a higher cytotoxicity as mentioned above. Moreover, the

Page 13 of 24

Dalton Transactions View Article Online

DOI: 10.1039/C5DT00169B

cells at the G2/M phase increased to 36.25%. A more significant increase (45.39%) was observed when the concentration of complex 5 increased to 6 µM. All the results indicated that complex 5 inhibited PC-3 cells by inducing the cell cycle to arrest at the

DNA binding The binding efficiency of the complexes to CT DNA has been studied by the absorption spectral technique through determining the intrinsic binding constant with the increasing concentration of CT DNA, keeping the concentration of the complex as a constant. A complex bound to DNA through intercalation generally causes hypochromism and a red shift of the absorption band due to strong stacking interactions between the aromatic chromophore of the complex and the base pairs of DNA.25 Because of similar changes in the absorption spectra, only complex 5 was described in details. Upon the addition of an increasing amount of CT DNA to the solution of complex 5, an apparent hypochromism and a slight red shift (1.5-2 nm) were observed, which was generally assigned to the intercalation involving stacking interactions between the aromatic chromophores and the base pairs of DNA (Fig. 4).26 This suggested that these Ru(II)-NHC complexes might inhibit the proliferation of cancer cell lines by interacting with the DNA, which is similar with the action mechanism of CDDP. The binding constant (Kb) of complex 5 was determined to be 2.2×106 M-1 from the absorption spectral technique, which showed a better DNA-binding ability than other four complexes 1-4 (3.8×105, 7.0×105, 5.7×105, and 1.9×105 respectively, Fig. S5). The DNA-binding ability of these five complexes was similar to other Ru(II) complexes reported by Zheng et al.27

Conclusion Five Ru(II)-NHC complexes 1−5 were prepared by applying the silver carbene transfer route. The cytotoxicity of these complexes against the human cancer cell lines SKOV-3, PC-3, MDA-MB-231 and EC109 were determined and revealed good efficiency. The complex 5 showed a superior activity of the micromolar scale against

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

G2/M phase, and showed a dose-dependent inhibition effect on the cell proliferation.

Dalton Transactions

Page 14 of 24 View Article Online

DOI: 10.1039/C5DT00169B

all cancer cell lines. The lipophilicity assay indicates that the cytotoxicity of Ru(II)-NHC complexes correlates well with the lipophilicity of the complex. Special attention was paid to the complex 5 against PC-3, which showed 3-5 fold higher

studies and cell cycle analysis demonstrates that complex 5 can not only inhibit the proliferation of cancer cell lines, but also induce the apoptosis of the cancer cell lines and induced cells to arrest at the G2/M phase with a dose-dependent feature. DNA binding assay showed that complex 5 can interact with the DNA and has a better DNA-binding ability than other four complexes.

Acknowledgments This work was financially supported by National Natural Science Foundation of China (21371082), Natural Science Foundation of Jiangsu Province (BK20141102), Key Medical Talent Project of Jiangsu Province (RC2011097), Science Foundation of Health Department of Jiangsu Province (Q201405) and Public Service Platform for Science and Technology Infrastructure Construction Project of Jiangsu Province (BM2012066). Notes and references Address: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China * E-mail address: [email protected] (J. Lin), [email protected] (L. Qiu) † Electronic Supplementary Information (ESI) available: additional structures for 1, UV-vis spectrophotometry, NMR and standard curves of UV-vis spectrophotometry of complexes 1-5, the nonlinear least-squares analysis of Kb values of complexes 1-4, X-ray crystallographic data in CIF format. See DOI: 10.1039/b000000x/

1.

N. Muhammad and Z. Guo, Curr. Opin. Chem. Biol., 2014, 19, 144-153.

2.

X. Wang and Z. Guo, Chem. Soc. Rev., 2013, 42, 202-224.

3.

C. H. Ng, S. M. Kong, Y. L. Tiong, M. J. Maah, N. Sukram, M. Ahmad and A. S. Khoo,

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

activity than that against other cell lines and 6 fold than that of cisplatin. Morphologic

Page 15 of 24

Dalton Transactions View Article Online

DOI: 10.1039/C5DT00169B

Metallomics, 2014, 6, 892-906. 4.

E. Fischer-Fodor, A. M. Valean, P. Virag, P. Ilea, C. Tatomir, F. Imre-Lucaci, M. P. Schrepler, L. T. Krausz, L. B. Tudoran, C. G. Precup, I. Lupan, E. Hey-Hawkins and L. Silaghi-Dumitrescu, Metallomics, 2014, 6, 833-844.

5.

A. Gutiérrez, M. C. Gimeno, I. Marzo and N. Metzler-Nolte, Eur. J. Inorg. Chem., 2014, 2014,

6.

L. K. Filak, D. S. Kalinowski, T. J. Bauer, D. R. Richardson and V. B. Arion, Inorg. Chem., 2014, 53, 6934-6943.

7.

L. Oehninger, R. Riccardo and I. Ott, Dalton Trans., 2013, 42, 3269-3284.

8.

L. Oehninger, M. Stefanopoulou, H. Alborzinia, J. Schur, S. Ludewig, K. Namikawa, A. Munoz-Castro, R. W. Koster, K. Baumann, S. Wolfl, W. S. Sheldrick and I. Ott, Dalton Trans., 2013, 42, 1657-1666.

9.

J. L. Hickey, R. A. Ruhayel, P. J. Barnard, M. V. Baker, S. J. Berners-Price and Aleksandra

10.

F. Cisnetti and Gautier, Angew. Chem. Int. Ed., 2013, 52, 11976-11978.

11.

J. DePasquale, M. Kumar, M. Zeller and E. T. Papish, Organometallics, 2013, 32, 966-979.

Filipovska, J. Am. Chem. Soc., 2008, 130, 12570-12571.

12.

A. C. L. Cavallo, C. Costabile and H. Jacobsen, J. Organomet. Chem., 2005, 690, 5407-5413.

13.

M. Pellei, V. Gandin, M. Marinelli, C. Marzano, M. Yousufuddin, H. V. Dias and C. Santini, Inorg. Chem., 2012, 51, 9873-9882.

14.

F. Caruso, E. Monti, J. Matthews, M. Rossi, M. B. Gariboldi, C. Pettinari, R. Pettinari and F. Marchetti, Inorg. Chem., 2014, 53, 3668-3677.

15.

Z. Wang, H. Qian, S. M. Yiu, J. Sun and G. Zhu, J. Inorg. Biochem., 2014, 131, 47-55.

16.

M. Pernot, N. P. E. Barry, T. Bastogne, C. Frochot, M. Barberi-Heyob and B. Therrien, Inorg. Chim. Acta, 2014, 414, 134-140.

17.

S. Orbisaglia, C. Di Nicola, F. Marchetti, C. Pettinari, R. Pettinari, L. M. Martins, E. C. Alegria, M. F. da Silva, B. G. Rocha, M. L. Kuznetsov, A. J. Pombeiro, B. W. Skelton, A. N. Sobolev and A. H. White, Eur. J. Chem., 2014, 20, 3689-3704.

18.

E. Groessl, C. G. Hartinger, R. Eichinger, O. Semenova, A. R. Timerbaev, M. A. Jakupec, V.

19.

C. G. Hartinger, M. A. Jakupeca, S. Zorbas-Seifrieda, M. Groessla, A. Eggera, W. Bergerd, H.

B. Arion and B. K. Keppler, J. Med. Chem., 2007, 50, 2185-2193. Zorbasc, P. J. Dysonb and B. K. Keppler, Chem. Biodivers., 2008, 5, 2140-2155. 20.

H. Oehninger, S. Ludewig, K. Baumann, S. Wölfl and I. Ott, ChemMedChem., 2011, 6, 2142-2145.

21.

F. Hackenberg, H. Müller-Bunz, R. Smith, W. Streciwilk, X. Zhu and M. Tacke,

22.

B. Peña, A. David, C. Pavani, M. S. Baptista, J. P. Pellois, C. Turro and K. R. Dunbar,

Organometallics, 2013, 32, 5551-5560. Organometallics, 2014, 33, 1100-1103. 23.

J. Lemke and N. Metzler‐Nolte, Eur. J. Inorg. Chem., 2008, 21, 3359-3366.

24.

M. V. Baker, P. J. Barnard, S. J. Berners-Price, S. K. Brayshaw, J. L. Hickey, B. W. Skelton and A. H. White, Dalton Trans, 2006, , 3708-3715.

25.

T. B. D. Lahiri, B. Pathak, O. Shameema, A. K. Patra, S. Ramakumar and A. R. Chakravarty, Inorg. Chem., 2009, 48, 339-349.

26.

M. Baldini, F. Bisceglie, P. P. Dall’Aglio, G. Pelosi, S. Pinelli and P. Tarasconi, Inorg. Chem., 2004, 43, 7170-7179.

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

2512-2519.

Dalton Transactions

Page 16 of 24 View Article Online

DOI: 10.1039/C5DT00169B

27.

Z. B. Zheng, S. Y. Kang, X. Yi, N. Zhang and K. Z. Wang, J. Inorg. Biochem., 2014, 141,

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

70-78.

Page 17 of 24

Dalton Transactions View Article Online

DOI: 10.1039/C5DT00169B

Captions for figures Scheme 1 Synthesis of complexes 1-5. Fig. 1 X-ray diffraction structure of 1; thermal ellipsoids are drawn at the 30%

Ru(1)-Cl(1), 2.4270(18); Ru(1)-Cl(2), 2.4257(17); Ru(1)-C(1)-N(2), 127.7(4); Ru(1)-C(1)-N(1), 128.6(4). Fig. 2 Morphology changes in the cells treated with the complex 5 at the indicated concentrations. After exposure to the complex 5 for 48 h, the cells were photographed using inverted microscope (magnification 200×). Fig. 3 Cell Cycle analysis of PC-3 induced by the complex 5. Fig. 4 Absorption spectral traces of 5 with increasing DNA concentration. The inset shows the least-squares fit of ∆εaf/∆εbf vs [DNA]. Table 1 Octanol–water partition coefficient of complexes 1-5 (Cw and Co denote the concentration of complex in water and octanol respectively). Table 2 IC50 values of complexes 1-5 against four human cancer cell lines.

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

probability level. Selected bond lengths (Å) and angles (°) for 1: Ru(1)-C(1), 2.096;

Dalton Transactions

Page 18 of 24 View Article Online

DOI: 10.1039/C5DT00169B

Complex

Cw×10-4/mol·L-1

Co×10-4/mol·L-1

log P

1 2 3 4 5

1.0349 1.3324 0.4324 0.1744 0.0345

8.5759 10.070 9.8576 12.300 14.220

0.91 0.88 1.36 1.85 2.62

Table 2 IC50(µM) a Complex SKOV-3

PC-3

MDA-MB-231

EC109

1

>100

>100

64.6±1.8

>100

2

>100

92.6±2.0

74.1±1.9

>100

3

>100

26.8±0.8

34.4±1.5

37.9±1.6

4

25.0±1.4

9.6±1.8

19.2±1.3

23.7±1.4

5

10.3±0.3

2.9±0.1

8.2±0.6

6.4±0.2

[(p-cymene)RuCl2]2

>100

>100

>100

>100

Cisplatin

34.9±2.6

17.6±0.7

30.0±1.6

10.6±1.1

a

Inhibitory activity was assayed by exposure of cell lines to the complex for 48 h and expressed as a concentration required to inhibit the cell proliferation by 50% (IC50). Data were expressed as the means ± SD of three independent experiments.

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

Table 1

Page 19 of 24

Dalton Transactions View Article Online

DOI: 10.1039/C5DT00169B

Five novel Ru(II)-N-heterocyclic carbenes (NHC) were synthesized biological evaluated, one of which showed a superior activity against PC-3 cell lines and 6 fold than that of cisplatin.

Dalton Transactions Accepted Manuscript

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

Graphical Abstract

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

145x55mm (300 x 300 DPI)

Dalton Transactions Accepted Manuscript

Dalton Transactions View Article Online

Page 20 of 24

DOI: 10.1039/C5DT00169B

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

193x146mm (300 x 300 DPI)

Dalton Transactions Accepted Manuscript

Page 21 of 24 Dalton Transactions DOI: 10.1039/C5DT00169B

View Article Online

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

143x41mm (150 x 150 DPI)

Dalton Transactions Accepted Manuscript

Dalton Transactions View Article Online

Page 22 of 24

DOI: 10.1039/C5DT00169B

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

145x41mm (150 x 150 DPI)

Dalton Transactions Accepted Manuscript

Page 23 of 24 Dalton Transactions DOI: 10.1039/C5DT00169B

View Article Online

Published on 13 March 2015. Downloaded by Pennsylvania State University on 14/03/2015 09:07:52.

117x88mm (300 x 300 DPI)

Dalton Transactions Accepted Manuscript

Dalton Transactions View Article Online

Page 24 of 24

DOI: 10.1039/C5DT00169B

Lipophilicity-dependent ruthenium N-heterocyclic carbene complexes as potential anticancer agents.

Five Ru(II)-N-heterocyclic carbenes (NHC) (1-5) were synthesized by reacting the appropriately substituted imidazolium chlorides with Ag2O, forming th...
1MB Sizes 2 Downloads 12 Views