Substituted 9-aminoacridine-4-carboxamides tethered to platinum(II)diamine complexes: Chemistry, cytotoxicity and DNA sequence selectivity

doi:10.1016/j.jinorgbio.2010.03.011

Journal of Inorganic Biochemistry

Volume 104, Issue 8, August 2010, Pages 815-819

https://doi.org/10.1016/j.jinorgbio.2010.03.011 Get rights and content

Abstract

Three platinum complexes in which substituted (7-OMe, 9-NH₂; 7-F, 9-NH₂; and 7-H, 9-NH(CH₂)₂OH) 9-aminoacridine-4-carboxamides were tethered to a platinum(II)diamine moiety were synthesised and characterised at the chemical and biological level. These variants showed a decrease in cytotoxicity, as measured by IC₅₀ values in HeLa cells, when compared with the parent 7-H, 9-NH₂ compound. The 7-F and 9-NH(CH₂)₂OH substituents gave rise to a small decrease in cytotoxicity, and the 7-OMe substituent resulted in a larger decrease in cytotoxicity. Their binding to purified pUC19 plasmid DNA was investigated and it was found that the addition of 7-F, 9-NH(CH₂)₂OH and especially the 7-OMe substituents, resulted in reduced DNA binding. This correlated well with the IC₅₀ cytotoxicity values. However, the DNA sequence selectivity was unaffected by the addition of these moieties.

Introduction

Expanding the spectrum of activity of platinum-based antitumour agents is an important goal which has motivated the continued development of drugs of this type [1]. One approach which has been explored is to develop platinum compounds with an altered pattern of DNA damage to that caused by cisplatin and carboplatin. This strategy has been a significant driving force in the development of bi- and trinuclear complexes exemplified by BBR3464 [2], [3]. It has also been a key feature in the development of: (i) platinum–acridinylthiourea conjugates [4], [5]; (ii) minor groove targeted multinuclear complexes derived from the 4,4′-dipyrazoylmethane ligand [6]; (iii) complexes in which platinum is tethered to the 5′-terminus of an oligothymidine sequence [7]; (iv) binuclear complexes based on a 1,4-diaminoanthraquinone intercalating chromophore [8], [9]; and (v) platinum(II) derivatives of distamycin and netropsin analogues [10], [11]. We have also explored this concept using intercalating chromophores such as acridine and 9-anilino-acridine [12], [13], phenazine [14] and the phenanthridinium cation [15] and extended this work to include a more comprehensive investigation of 9-aminoacridine carboxamides [16], [17], [18]. Of the complexes we have studied to date, those based on the 9-aminoacridine-4-carboxamide platform (compound 1) are of particular note, showing promising activity against cisplatin resistant cells [16] and exhibiting a different DNA sequence specificity to that of cisplatin [17], shifted away from runs of consecutive guanines (the main binding site for cisplatin). This sequence specificity was shown to be dependent on linker chain length, with the shorter chain length homologues (n = 2, 3) showing the greatest alteration in DNA sequence specificity, whereas the longer chain length homologues (n = 4, 5) more closely resembled cisplatin and PtenCl₂ in their behaviour [17]. The latter study was the first occasion that an altered sequence specificity was convincingly demonstrated for a cisplatin analogue of the diamine type. We have also shown that the members of the class displayed an altered sequence specificity in intact human cells [18], preferentially targeting GA sequences rather than consecutive G bases. The DNA sequencing results suggest that it is the presence of the 9-amino group in the 9-aminoacridine-4-carboxamide chromophore which results in this positional targeting of platinum. The presence of the intercalator is also expected to rapidly localise compounds similar to 1 on DNA and we have previously shown that these compounds show a marked increase in the rate of DNA platination when compared with cisplatin or PtenCl₂ [17].

Since the 9-aminoacridine-4-carboxamide Pt complex (1, n = 2) showed the best activity in earlier studies, we initiated structure/activity studies on substituted 9-aminoacridine complexes, with the ultimate aim of optimizing antitumour activity. We chose to investigate the set of analogues shown in Fig. 1, with an n = 2 linker chain, since this linker length previously showed the most significant alterations to DNA specificity in the set of parent compounds. We examined the effects of substituents at the 7-position of the acridine ring and alterations to the amino group at the 9-position on the behaviour of these compounds, and report the synthesis, cytotoxicity and sequence specificity of these compounds.

Section snippets

Chemistry

The acridine ligands 2–4 were prepared as shown in Scheme 1. The differentially protected triamine 13 was synthesised by first selectively protecting one of the primary amine groups of diethylenetriamine by reaction with ethyltrifluoroacetate at − 78 °C [19] followed by Boc protection of the remaining two amine functional groups. The triamine 13 was reacted selectively with the appropriately substituted 9-chloroacridine-4-carbonyl chloride to give the 9-chloro compounds 8a, 9a, and 10a. These

Chemistry

¹H and ¹⁹⁵Pt NMR spectra were measured on Varian UnityPlus 400 MHz or Unity 300 spectrometers. ¹H chemical shifts were measured relative to the residual hydrogens of bulk solvent (δ 4.85 for D₂O, 7.26 for CDCl₃ and 2.49 for DMSO) and quoted relative to TMS. The ¹⁹⁵Pt reference was a 0.1 M solution of K₂PtCl₄ in D₂O (δ − 1630). ESI-MS spectra were recorded on a quadrupole ion trap Finnigan-MAT LCQ mass spectrometer equipped with electrospray ionisation. Triethylamine was redistilled from

Acknowledgments

Support of this work by The National Health and Medical Research Council (WDM, VM, WAD) is gratefully acknowledged.

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Cisplatin is one of the most used chemotherapeutic agents nowadays. However, it presents several and severe side effects. For that, the synthesis of new analogues of cisplatin is crucial to improve the current therapies. In this work, two squared-planar Pt(II) analogues of cisplatin were presented. These analogues included a bidentate ligand molecule with a 3,5-dimethyl-pyrazole ring and a thiazine (DMPzTz) or thiazoline (DMPzTn) ring. The chemical characterization covered single-crystal X-ray diffraction, infrared spectroscopy (IR) and elemental analysis. Their potential anticarcinogenic ability was studied in three different human tumor cell lines, i.e., histiocytic lymphoma (U-937), promyelocytic leukemia (HL-60) and epithelial cervix carcinoma (HeLa) via cytotoxicity assay. The results showed moderate cytotoxic effect of the complexes on leukemic cell lines, with scarce effect on solid tumor cell line HeLa. Lower IC₅₀ values were found for U-937 cell line, being 32.3 ± 1.2 µmol/dm³ for PtDMPzTn and 43.0 ± 1.4 µmol/dm³ for PtDMPzTz. The comparison with previously synthesized analogues with phenyl substitutions or no substitutions in the pyrazole ring, allows to conclude that the presence of ligands with methyl substituted-pyrazole ring did not improve the effect of the complexes. In addition, the main effect of these potential chemotherapeutic agents is produced by the presence of platinum(II) as metal center, since the free ligands do not show any significant activity.
The DNA binding properties of 9-aminoacridine carboxamide Pt complexes
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The methoxy substituent is electron-donating, while the fluoro substituent is electron-withdrawing. This affects the charge distribution on the intercalating moiety, and also its steric profile, which could in turn affect the binding of these compounds to DNA compared with the parent compound.27 The 9-ethanolamine substituent on the other hand, is not expected to differently affect DNA binding compared with the parent compound, since the substituent’s OH group can sit unimpeded in the DNA minor groove.
Cisplatin analogues with an attached DNA-binding moiety represent a potentially effective class of DNA-damaging anti-tumour agents because they possess higher affinities for DNA and different DNA damage profiles compared with cisplatin. In this study, the interaction of four 9-aminoacridine carboxamide Pt complexes with purified DNA was investigated: firstly, using a fluorescent intercalator displacement (FID) assay with ethidium bromide; and secondly, with a DNA unwinding assay. The relative capacity of these compounds to perturb the fluorescence induced by DNA-bound ethidium bromide at clinically relevant drug concentrations was assessed over a 24-h period using an FID assay. All analogues were found to reduce the level of ethidium bromide-induced fluorescence in a concentration-dependent manner from the earliest time point of 10 min onwards. Cisplatin, however, showed a markedly slower reduction in ethidium bromide-induced fluorescence from 2 h onwards, producing a similar level of fluorescence reduction as that produced by the analogues from 6 h onwards. These results suggest that the altered DNA-binding modes of the DNA-targeted analogues confer a more efficient mechanism for DNA binding compared with cisplatin. Relative DNA binding coefficients were also determined for each of the compounds studied. With the DNA unwinding assay, an unwinding angle can be calculated from the coalescence point of plasmids in an agarose gel. It was found that all 9-aminoacridine carboxamide analogues had a greater unwinding angle compared with cisplatin. The knowledge obtained from these two assays has helped to further characterise the cisplatin analogues and could facilitate the development of more effective anti-tumour agents.
CpG methylation increases the DNA binding of 9-aminoacridine carboxamide Pt analogues
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One such analogue is dichlorido(N-2-((2-aminoethyl)amino)-ethyl)-9-aminoacridine-4-carboxamide platinum(II) (9AmAcPtCl2) (Fig. 1), with a 9-aminoacridine component, attached via a carboxamide linker, behaving as an intercalating moiety. 9AmAcPtCl2 and several related compounds with altered side chains have been investigated in intact human HeLa cells and have been found to have a more rapid rate of DNA damage20 and a lower IC50 value compared with cisplatin.21 Utilising human HeLa cells, 9AmAcPtCl2 had an IC50 value of 0.4 μM; 7-fluoro-9AmAcPtCl2—1.3 μM; 9-ethanolamine-AcPtCl2—1.1 μM; whilst for cisplatin it was 10 μM.21
This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5′-CpG sequences. Their binding to methylated and non-methylated 5′-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5′-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5′-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell’s genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use.
The interaction of cisplatin with a human telomeric DNA sequence containing seventeen tandem repeats
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The anti-tumour drug, cisplatin, preferentially forms adducts at G-rich DNA sequences. Telomeres are found at the ends of chromosomes and, in humans, contain the repeated DNA sequence (GGGTTA)_n that is expected to be targeted by cisplatin. Using a plasmid clone with 17 tandem telomeric repeats, (GGGTTA)₁₇, the DNA sequence specificity of cisplatin was investigated utilising the linear amplification procedure that pin-pointed the precise sites of cisplatin adduct formation. This procedure used a fluorescently labelled primer and capillary electrophoresis with laser-induced fluorescence detection to determine the DNA sequence specificity of cisplatin. This technique provided a very accurate analysis of cisplatin-DNA adduct formation in a long telomeric repeat DNA sequence. The DNA sequence specificity of cisplatin in a long telomeric tandem repeat has not been previously reported. The results indicated that the 3′-end of the G-rich strand of the telomeric repeat was preferentially damaged by cisplatin and this suggests that the telomeric DNA repeat has an unusual conformation.
Cis-Dichloroplatinum(II) complexes tethered to dibenzo[c,h][1,6] naphthyridin-6-ones: Synthesis and cytotoxicity in human cancer cell lines in vitro
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Several strategies have been intensively explored to develop new compounds, especially by combining in a same molecule a Pt moiety to another entity exhibiting cytotoxic properties [14]. Such approach could have several advantages: to combine 2 modes of action which could help to circumvent drug resistance; to decrease toxicity of each drug used separately and to increase DNA targeting by incorporation of Pt into suitable “carrier” molecules, such as acridine derived-pharmacophore [15,16], anthraquinone [17], bisnaphthalimide [18], camptothecin [19]…. Our approach was focused on developing Pt compounds coupled to a cytotoxic moiety exhibiting topoisomerase I inhibiting properties.
A novel family of cisplatin-type complexes tethered to dibenzo[c,h][1,6]naphthyridin-6-one topoisomerase inhibitor via a polymethylene chain and their nonplatinated counterparts were prepared. Their potential cytotoxicity was assessed in three human colorectal cancer cell lines HCT 116, SW480 and HT-29 and compared to the reference molecules cisplatin and oxaliplatin. Platinated compounds were poorly active whilst nonplatinated dibenzo[c,h][1,6]naphthyridin-6-one moieties exhibited higher cytotoxic properties than cisplatin and oxaliplatin whatever the length of the polymethylene chain; molecules containing the tri- and hexamethylene chain length were the most cytotoxic.
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Acridine-4-carboxamide compounds were more cytotoxic than acridine-2-carboxamide tethered Pt(II) complexes in P388 leukemia cells in vitro [26] ; and acridine-4-carboxamide complexes with the shorter linker chain lengths were generally the most active [13] in different cell lines sensitive or resistant to cisplatin [13,26]. In spite of that, addition of 9-amino substituent on acridine-4-carboxamide led to decreased cytotoxic activities in HeLa cells [27]. Interestingly, the replacement of amide by ester functionalities in the series of novel acridine-9-carboxylate tethered (ethane-1,2-diamine) complexes platinated (4f–j) or not (3f–j) and connected by a polymethylene chain increased their cytotoxic effect which was higher than cisplatin or oxaliplatin in the three colic HCT 116, SW480 and HT-29 cell lines (Table 2).
In order to improve the pharmacological profile of the anticancer drug cisplatin, several new acridine-based tethered (ethane-1,2-diamine)platinum(II) complexes connected by a polymethylene chain were synthetized. Activity-structure relationship between amide or ester functionalities was explored by changing acridine-9-carboxamide into acridine-9-carboxylate chromophore. The in vitro cytotoxicity of these new complexes was assessed in human colic HCT 116, SW480 and HT-29 cancer cell lines. Series of complexes bearing the acridine-9-carboxylate chromophore displayed higher cytotoxic effect than acridine-9-carboxamide complexes, with gradual effect according to the size of the polymethylene linker.

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¹: Current address: Schizophrenia Research Institute and School of Biomedical Sciences, The University of Newcastle, NSW 2308, Australia.

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Substituted 9-aminoacridine-4-carboxamides tethered to platinum(II)diamine complexes: Chemistry, cytotoxicity and DNA sequence selectivity

Abstract

Introduction

Section snippets

Chemistry

Chemistry

Acknowledgments

J. Inorg. Biochem.

J. Inorg. Biochem.

J. Inorg. Biochem.

J. Inorg. Biochem.

Biochim. Biophys. Acta

Tetrahedron Lett.

J. Inorg. Chim. Acta

Inorg. Chim. Acta

J. Inorg. Biochem.

J. Biol. Chem.

Biochim. Biophys. Acta

Chem. Biol. Interact.

Nat. Rev. Can.

Biochemistry

Nucl. Acid. Res.