Bioorganic & Medicinal Chemistry Letters 24 (2014) 1692–1694

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Antitumor activity of phenylene bridged binuclear bis(imino-quinolyl)palladium(II) and platinum(II) complexes William M. Motswainyana a,⇑, Martin O. Onani a, Abram M. Madiehe b, Morounke Saibu b a b

Chemistry Department, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa

a r t i c l e

i n f o

Article history: Received 29 November 2013 Revised 18 February 2014 Accepted 20 February 2014 Available online 28 February 2014 Keywords: Bis(imino-quinolyl) Palladium(II) Platinum(II) Antitumor Cisplatin

a b s t r a c t Antitumor effects of a known bis(imino-quinolyl)palladium(II) complex 1 and its newly synthesized platinum(II) analogue 2 were evaluated against human breast (MCF-7) and human colon (HT-29) cancer cell lines. The complexes gave cytotoxicity profiles that were better than the reference drug cisplatin. The highest cytotoxic activities were pronounced in complex 2 across the two examined cancer cell lines. Both compounds represent potential active drugs based on bimetallic complexes. Ó 2014 Elsevier Ltd. All rights reserved.

Literature review on antitumor therapy shows that cancer burden is still a major concern and challenge for public health systems worldwide. A large number of complexes have been prepared and screened as potential antitumor agents in recent years,1–8 with a view to develop less toxic, but equally effective complexes which would eventually replace the known anticancer agent cis-diamminedichloroplatinum(II) (cisplatin), which is reportedly plagued by drug resistance and significant side effects.9 Among the most promising complexes with proven cytotoxicity profiles are those bearing nitrogen-donor ligands.1–8 As expected, the nitrogen-donor ligands coordinate as neutral species when they are reacted with labile transition metal precursors to form stable complexes. Non-classical platinum compounds containing two, three or four platinum centers connected by polyamines have been investigated for their potential to inhibit tumour growth with a view to circumvent the acquired cisplatin resistance in various tumour models.10–13 A major breakthrough in antitumor activity of these complexes was reflected in the development of a trinuclear platinum(II) complex, BBR3464 (Fig. 1) which exhibited remarkable activity against pancreatic, lung and melanoma cancer cell lines.11 Unfortunately, phase II clinical studies of the compound was overshadowed by severe dose-limiting side effects such as diarrhoea and vomiting, which prevented its clinical approval.14,15

⇑ Corresponding author. Tel.: +27 21 959 2621; fax: +27 21 959 3055. E-mail address: [email protected] (W.M. Motswainyana). http://dx.doi.org/10.1016/j.bmcl.2014.02.056 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Several other active dinuclear complexes with various bridging ligands have been screened for their antitumour potency. For example, azole-bridged dinuclear platinum(II) complexes have showed potential to exert cytotoxicity on several human tumour cells.16,17 Palladium(II) complexes of the form [Pd2(l-L)Y2] (where L is a bridging ligand, and Y is an extended heterocyclic ligand) have also returned promising cytotoxic profiles against tumour cells.18 Interestingly, the complexes were found to intercalate between DNA base pairs, and behaved as artificial DNA nucleases, thereby generating nicks at different DNA sites.18 Furthermore, Zhan et al. have reported dinuclear platinum(II) complexes bridged by iodide anions which exhibited good antitumor activity.19 These complexes represent a new paradigm in anticancer therapy and their antitumour results suggest that complexes with two or more metal centres in close proximity would sufficiently deliver drugs to the tumour and possibly form mutable-binding with tumour cell DNA, thereby increasing the ability of the drug to block DNA replication.20 In this work, we investigated antitumor activities of a newly prepared binuclear bis(imino-quinolyl)platinum(II) complex and its palladium(II) analogue, which was previously synthesized and published by us.21 The complexes contain two terminal imino-quinolyl units bridged by a phenylene ring, which gives the metal centre a rigid support. These complexes were evaluated for the first time for their potential to exert cytotoxicity on MCF-7 and HT-29 cancer cells, with a view to develop new antitumor agents which would exhibit improved therapeutic properties.

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H 3N

NH3

H 3N

Cl

H 3N

4+

Table 1 In vitro cytotoxicity of free ligand and complexes 1 and 2 IC50a (lM)

Pt

Pt

Pt Cl

NH3

NH2(CH2)6 H2N

NH3

NH2(CH2)6 H2N

BBR3464 Figure 1. Structure of BBR3464.

The bis(imino-quinolyl ligand L1 and palladium(II) complex 1 were prepared following our published method [21]. The subsequent platinum(II) complex 2 (Fig. 2) was prepared from reaction of 0.5 equiv of the ligand L1 with K2[PtCl4].22 The preparation was as follows: A solution of K2[PtCl4] in MeOH/H2O mixture was added to a suspension of L1 in CH2Cl2, and the reaction was allowed to proceed under reflux for 6 h, where upon settling, the organic layer was recovered, followed by addition of excess hexane to precipitate out a light brown solid in good yield (70%).22 The isolated complex was characterized using micro-elemental analyses and IR spectroscopy. 13C NMR was measured in solid phase due to solubility problem of the complex. An attempt to crystallize the compound was therefore unsuccessful. Elemental analysis data was consistent with the proposed formula of the binuclear complex. Coordination of the ligand to the platinum metal centre was confirmed from the IR spectrum of the complex, which showed lower absorption band at 1604 cm 1 compared to the free ligand.23,24 The other absorption bands due to the (C@N) and (C@C) quinolyl vibrations did not show any significant shift in absorption frequency from their corresponding free imine, thereby confirming their non involvement in coordination. In the 13C NMR spectrum, the imine carbon in 2 appeared downfield at 169.20 ppm compared to 161.85 ppm in the free imine ligand. The results conform to a similar trend reported in related imino-pyridyl palladium(II) complexes.23,24 The same pattern was also observed in the 13C NMR spectrum of complex 1.21 The bis(imino-quinolyl)palladium(II) and platinum(II) complexes 1 and 2 were evaluated for their potential to exert cytotoxicity on highly invasive human breast (MCF-7) and human colon (HT-29) tumor cells using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay with minor modifications.25–29 As seen from the introduction, the cooperative effect of two metal centers in close proximity was expected to enhance significantly higher cytotoxic activities. The compounds were first solubilized in dimethylsulfoxide (DMSO). The solvent accelerates the rate of chloride displacement from a complex.30 Cell growth inhibition results are summarized in Table 1. Cytotoxicity of cisplatin was evaluated under the same experimental conditions for comparison. The bis(imino-quinolyl) ligand was also evaluated to assess whether free ligand could exert cytotoxicity on cancer cells. However, the ligand did not return any

Cl

b

Compound

KMST-6

L1 1 2 Cisplatin

>100 51.0 ± 4.0 58.4 ± 3.6 90.0 ± 0.9

MCF-7c

SIe

HT-29d

SIe

>100 60.0 ± 0.2 55.0 ± 1.6 100.0 ± 0.5

0.0 0.90 1.06 0.90

>100 46.0 ± 0.8 41.0 ± 1.0 100.0 ± 0.4

0.0 1.11 1.42 0.90

a Concentration of the complex required to inhibit cell growth by 50% as obtained by MTT assay. b Normal human fibroblasts cells. c Human breast cancer cells. d Human colon cancer cells. e Selectivity index (SI). SI indicates differential cytotoxicity of a compound (SI = IC50 treated normal cells/IC50 treated cancer cell lines). Selectivity index >1 indicates high selectivity.

appreciable activity (IC50 >100 lM), therefore ruling out the capacity of the free imino-quinolyl ligand to exert cytotoxicity on tumour cells. The binuclear complexes were generally more active than their corresponding mononuclear complexes published elsewhere,31 possibly due to the cooperative effect of the two metal centers in close proximity. The highest cytotoxic activities were pronounced in complex 2 across the two examined cancer cell lines (IC50 41 and 55 lM), probably due to the higher lability of complex 1 compared to 2 which makes it to dissociate readily in solution, forming reactive species that are unable to reach their pharmacological targets.32,33 Normal human fibroblasts cell lines (KMST-6) were used to determine the toxicity level of the compounds against normal cells, and the results generally show that the compounds do not have much effect on normal cells. Furthermore, the complexes proved to be selective, giving selectivity indices >1 against the two cell lines.34 The differential toxicity exhibited by compound 2 was significant (SI = 1.42), thereby making the compound a future candidate in the development of potent chemotherapeutic agents based on binuclear complexes. In conclusion, we have successfully prepared and characterized a new binuclear bridged bis(imino-quinolyl)platinum(II) complex. The complex, together with its known palladium(II) analogue were evaluated for the first time in vitro for their cytotoxic activity against human breast (MCF-7) and human colon (HT-29) cancer cell lines. The free ligand did not return any appreciable activity against the examined cell lines. The complexes returned growth inhibitory activities that were even better than cisplatin, with the highest cytotoxic activity pronounced in complex 2 (IC50 = 41 lM). Acknowledgement The authors are grateful for the financial support from University of the Western Cape Senate and National Research Foundation (THUTHUKA).

Cl M

N

N

N

N M

Cl

Cl

M = Pd (1) M = Pt (2) Figure 2. Scheme of the probable molecular structure of bis(imino-quinolyl)palladium(II) and platinum(II) complexes 1 and 2.

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23. 24. 25.

26. 27. 28.

29.

30. 31. 32. 33. 34.

methanol/water (3 mL). The reaction was allowed to proceed under reflux for 6 h. Upon settling, the organic layer was recovered using a separating funnel, followed by addition of excess hexane to precipitate out a light brown solid. The solid was filtered under vacuum, washed with water and hexane and dried under vacuum. Yield: 0.1280 g (70%); 185 C; IR (nujol cm 1); m(C@N imine) 1604, m(C@N quinolyl) 1582, m(C@C quinolyl) 1568, 1505; 13C NMR (125.65 MHz): d 169.20, 158.17, 152.90; Anal. Calcd for C26H18Cl4N4Pt2: C, 34.00; H, 1.98; N, 6.10. Found: C, 33.78; H, 2.06; N, 6.32. Motswainyana, W. M.; Ojwach, S. O.; Onani, M. O.; Iwuoha, E. I.; Darkwa, J. Polyhedron 2011, 30, 2574. Singh, P.; Das, S.; Dhakarey, R. Eur. J. Chem. 2009, 6, 99. Petrovski, Z.; Norton de Matos, M. R. P.; Braga, S. S.; Pereira, C. C. L.; Matos, M. L.; Goncalves, I. S.; Pillinger, M.; Alves, P. M.; Romao, C. C. J. Organomet. Chem. 2008, 693, 675. Mosmann, T. J. Immunol. Methods 1983, 65, 55. Freimoser, F. M.; Jacob, C. A.; Aebi, M.; Tuor, U. Appl. Environ. Microbiol. 1999, 65, 3727. Cell culture: The MCF-7 and HT-29 cells were cultured in DMEM in 25 cm tissue culture flasks and were allowed to grow to 90% confluency in an incubator set at 37 C containing 5% CO2 atmospheric pressure, before they were trypsinized and cultured in six tissue culture plates. Stock and final concentrations of the complexes were prepared in the culture media. MTT assay: The cells were plated in 96-well tissue plates at a density of 2.4  104 cells/mL per well. Cells were then treated with various concentrations of the complexes (100–10 lM) after which they were incubated for 24 h. Triplicate wells were established for each concentration. Just 5 h before the elapse of 24 h, 10 lL of 5 mg/mL MTT solution was added to each well and the plates were further incubated for 4 h. At the end of the incubation period, the media was removed from each well and replaced with 50 lL of DMSO. The plates were shaken on a rotating shaker for 10 min before taking readings at 560 nm using a microplate reader. Basolo, F. Coord. Chem. Rev. 1996, 154, 151. Motswainyana, W. M.; Onani, M. O.; Madiehe, A. M.; Saibu, M.; Jacobs, J.; van Meervelt, L. Inorg. Chim. Acta 2013, 400, 197. Abu-Surrah, A. S.; Kettunen, M. Curr. Med. Chem. 2006, 13, 1337. Zhao, G.; Lin, H.; Yu, P.; Sun, H.; Zhu, S.; Su, X.; Chen, Y. J. Inorg. Biochem. 1999, 73, 145. Musa, M. A.; Badisa, V. L. D.; Latinwo, L. M.; Waryoba, C.; Ugochukwu, N. Anticancer Res. 2010, 30, 4613.