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13CYTOTOXICITY OF DICLOFENAC AND ETHYLENEDIAMINE COMPLEXES WITH
Ni2+, Cu2+ and Zn2+
Obidova N.,
Junior Researcher, Laboratory of Complex Compounds, Institute of Bioorganic Chemistry,
Academy of Sciences of the Republic of Uzbekistan
Ziyaev M.,
Junior Researcher, Laboratory of Complex Compounds, Institute of Bioorganic Chemistry,
Academy of Sciences of the Republic of Uzbekistan, Tashkent
Ashurov J.,
Doctor of Science, Leading researcher, Head of Laboratory of Complex Compounds, Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan
Khodjaniyazov Kh.
Doctor of Science, Leading researcher, Laboratory of Complex Compounds, Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan;
Lecture at Chemical Faculty, National University of Uzbekistan
Abstract
Recently it was synthesized an organic salt of diclofenac with ethylenediamine and complex compounds of these two ligands and metal cations Ni2+, Cu2+ and Zn2+, the structures of which were confirmed by the X-ray analyses. Investigation on cytotoxicity of the complexes against to HeLa cells showed 2.0-4.4 times efficacy then parent diclofenac. Once again proven that complexation with metals leads to an enhancement of the activity of the pharmaceutical drug.
Keywords: diclofenac, metal complex, organic salt, cytotoxicity, Ni2+, Cu2+, Zn2+ cations.
Introduction.
The non-steroidal anti-inflammatory drug of diclofenac (DCF) is widely used in medicine since 1973 [1]. This drug is gradually becoming promising in the treatment of different types of cancer [2]. It was summarised the evidence for an anti-cancer effect of DCF treatment drawing on in vitro, in vivo and human data. The established pharmacokinetics and known toxicity profile make DCF a strong candidate for repurposing as an oncological treatment, both in combination with existing standard of care treatments or in a cocktail with other repurposed drugs [3]. Sometimes synergism is observed when DCF is used in combination with other drugs, as well as in their conjugates. Compound ci5-[Pt(DCF)2(NHs)2], in which DCF molecules are coordinated to Pt(II) through their carboxylic group, is more potent than parental conventional Pt(II) drug cisplatin, free DCF and the congeners of it in which diclofenac ligands are conjugated to Pt(II) via a diamine. The potency of cis-[Pt(DCF)2(NH3)2] is due to several factors including enhanced internalization that correlates with enhanced DNA binding and cytotoxi-city [4].
The released parental DCF molecules should be able to carry out their biological functions and the [Pt(NH3)2(H20)2]2+ moiety should be able to interact with its key target (nuclear DNA).
DCF shows anti-proliferative effects against L929 cells [5]. Furthermore, topical DCF in 2.5% hyaluronan shows the inhibition of colon-26 Adenocarcinoma development and angiogenesis [6]. Upon obtaining of complexes of a known preparation with metals, solubility and bioavailability is usually increased [7]. The corneal toxicity of preservative-containing solutions of
DCF has already been documented [8-10] and later revealed that preservative-free DCF exhibit considerable toxicity [11].
Herein, we describe the X-ray structural analysis and cytotoxicity assessment against HeLa cells of DCF complexes with ethylenediamine (EDA): (DCF-EDA), [M(DCF)2], and mixed ligand complexes -[M(EDA)2(H2O)2](DCF)2 , where M= Ni2+, Cu2+ and Zn2+, EDA - ethylendiamine.
Materials and methods.
In order to obtain DCF complexes with biometals, aqueous ethanol solutions of acetates of Ni2+, Cu2+ and Zn2+ were vigorously stirred with DCF and ethylenedi-amine (EDA) in 1:1:1 molar ratio, respectively. After two hours of refluxing the reaction mixture, hot filtration was carried out. Then the mother liquor was left at room temperature for two weeks. The growths of single crystals were further observed, which were filtered of and dried. The molecular and crystal structures of these single crystals have been studied by X-ray diffraction analysis. Thus, we have synthesized complexes of d-metals such as Ni2+, Cu2+ and Zn2+ with the anti-inflammatory drug DCF. Ethylenediamine was used as an additional ligand. The metal complexes synthesized by us are characterized by chelation of a metal ion with both two molecules of EDA and water, and DCF anions situated on the external coordination sphere. The cytotoxicity against HeLa cells of synthesized complexes were studied by MTT method.
Results and discussion.
The structures of the synthesized compounds. The synthesis of biometal complexes with DCF and EDA was carried out and their structures were determined by X-ray diffraction method. Organic salt of DCF with EDA characterized by molar ratio of 1:2, i.e. each protonated on N atoms EDA molecule H-bonded
with two deprotonated on carboxylic acid moiety DCF molecules. All synthesized metal complexes are similar by their molecular and crystal structure, i.e. they are isostructural. The metal cation is coordinated with two molecules of EDA and two water molecules in complexes of diclofenac with Ni2+, Cu2+ and Zn2+.
Complexes [Ni(EDA)2(H2O)2](DCF)2 (1), [Cu(EDA)2(H20)2](DCF)2 (2) and
fbj
[Cu(EDA)2(H2O)2](DCF)2 (3) are also isostructural. In all structures, the central metal ion lies on a symmetry center. The coordination geometry is elongated octahedron with two aqua ligands occupying the apical positions. The DCF anionic moieties are located outside of the first coordination sphere. Due to the aforementioned structural similarity, complexes 1-3 exhibit also a very similar packing pattern (Fig. 1).
Q
c
N
O CI
M
Fig. 1. Packing of the cations [M(EDA)2(H2O)2]2+ and anions DCF in the isostructural crystal structures, where
M=Ni2+, Cu2+ and Zn2+.
The cytotoxicity. The cytotoxic activity of new DCF complexes in comparison with the original DCF was studied. The cytotoxic effect of substances on HeLa cell culture was evaluated by the biochemical MTT method. It was revealed that metal complexes exhibit cytotoxic activity 2.0-4.4 times higher than DCF itself.
The efficacy of substances on cell culture. Cyto-toxicity was evaluated biochemically using the MTT method. To determine the cytotoxic effect, HeLa cells were scattered into 96-well plates in an amount of 2030 thousand cells/mL in 100 ^L of RPMI 1640 medium
with 10% serum of calf embryo and cultured at a temperature of 37°C in a CO2 incubator. After a day, substances were introduced at concentrations of 10 and 1 ^g/mL, cells were cultured for 24 hours, and then MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-te-trazolium bromide] was introduced into cells to detect living cells [12]. After 3 hours of incubation, the medium was carefully drained, DMSO was added and incubated for 20 minutes, and then the optical density of the solution was measured at a wavelength of 620 nm. Reference is cells without treatment, where degree of MTT entrance into cells is 100% (0% inhibition). The obtained results listed in Table 1.
Table 1.
Efficacy of compounds against to HeLa cell cultures
Dose, ^ Sample Inhibition of MTT introduction in cells, %
100 10
DCF 20,2 23,5
EDA 25,5 36,1
(DCF-EDA) 38,3 6,1
[Zn(DCF)2] 47,0 40,6
[Zn(EDA)2(H2O)2] (DCF)2 20,7 22,1
[Cu(DCF)2] 88,4 43,4
[Cu(EDA)2(H2O)2](DCF)2 28,6 39,4
[Ni(DCF)2] 30,4 12,2
[Ni(EDA)2(H2O)2](DCF)2 9,3 7,1
Cisplatin 95,7 63,4
It was determined that the cytotoxic activity of the mixed-ligand complexes of DCF is lower than that of the complexes of diclofenac with metal cations. This result can be explained by the fact that due to the high donor ability of ethylenediamine, this molecule attracts metal cations, and the diclofenac molecule is displaced into the external sphere of the complexes.
Conclusion.
The metal-complexes of DCF with Ni2+, Cu2+ and Zn2+ cations exhibit stronger cytotoxic activity against HeLa cells than initial diclofenac, i.e., a complex formation of DCF with metal cations enhances significantly its cytotoxicity. Addition of EDA as a ligand leads to the disposition of DCF molecules to outer sphere of the complex. Thus, synthesis and structural study of DCF complexes is attractive from the theoretical and practical view of points.
REFERENCES:
1. V.A. Skoutakis, C.A. Carter, T.R. Mickle, V.H. Smith, C.R. Arkin, J. Alissandratos, and D.E. Petty. Review of diclofenac and evaluation of its place in therapy as a nonsteroidal anti-inflammatory agent. Drug Intell. Clin. Pharm. 1988. 22. 850-859.
2. E.D. Arisan, R.O. Akar, O. Rencuzogullari, P.O. Yerlikaya, A.C. Gurkan, B. Akin, E. Dener, E. Kayhan, N.P. Unsal. The molecular targets of diclofenac differ from ibuprofen to induce apoptosis and epithelial mesenchymal transition due to alternation on oxidative stress management p53 independently in PC3 prostate cancer cells. Prostate International. 2019. 7. 156-165.
3. P. Pantziarka, V. Sukhatme, G. Bouche, L. Meheus, V.P. Sukhatme. Repurposing drugs in oncology (ReDO) - diclofenac as anti-cancer agent. ecancer. -2016. 10. 610.
4. F.P. Intini, J. Zajac, V. Novohradsky, T. Sal-tarella, C. Pacifico, V. Brabec, G. Natile, J. Kaspar-kova. Novel antitumor platinum (II) conjugates containing the nonsteroidal anti-inflammatory agent diclofenac: synthesis and dual mechanisms of
antiproliferative effects. Inorg. Chem. 2017. 56(3). 1483-1497.
5. R. Ahmadi, L. Ghaderi, G. Keyhani. The Effects of Diclofenac on Proliferation of L929 Tumor Cell Line in Cell culture. Proceedings of 2015 Int. Conference on Biological and Environmental Sciences (BIOES-15), Phuket, Thailand, October 1-2, 2015.
6. M.P. Seed, J.R. Brown, C.N. Freemantle, J.L. Papworth, P.R. Colvifie-Nash, D. Willis, K.W. Somer-ville, S. Asculai, D.A. Willoughby. The Inhibition of colon-26 Adenocarcinoma Development and Angio-genesis by Topical Diclofenac in 2.5% Hyaluronan. Cancer Res. 1997. 57. 1625-1629.
7. J.E. Weder, C.T. Dillon, T.W. Hambley, B.J. Kennedy, P.A. Lay, J.R. Biffin, H.L. Regtop, N.M. Da-vies. Copper complexes of non-steroidal anti-inflammatory drugs: an opportunity yet to be realized. Coord. Chem. Rev. 2002. 232. 95-126.
8. A.C. Guidera, J.I. Luchs, I.J. Udell. Keratitis, ulceration, and perforation associated with topical nonsteroidal anti-inflammatory drugs. Ophthalmology. 2001. 108(5). 936-944.
9. T.P. O'Brien, Q.J. Li, F. Sauerburger, V.E. Reviglio, T. Rana, M.F. Ashraf. The role of matrix met-alloproteinases in ulcerative keratolysis associated with perioperative diclofenac use. Ophthalmology. 2001. 108(4). 656-659.
10. J.K. Hsu, W.T. Johnston, R.W. Read, et al. Histopathology of corneal melting associated with diclofenac use after refractive surgery. J. Cataract Refract Surg. 2003. 29(2). 250-256.
11. M. Ayaki, A. Iwasawa, M. Soda, S. Yaguchi, R. Koide. Cytotoxicity of five fluoroquinolone and two nonsteroidal anti-inflammatory benzalkonium chloride-free ophthalmic solutions in four corneoconjuncti-val cell lines. Clin. Ophthalmol. 2010. 4. 1019-1024.
12. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Immunol. methods. USA. 1983. 65. 55-63.
