Synthesis,characterization, DNA interaction studies and anticancer activity of platinum–clotrimazole complexes

New trans-platinum complexes of clotrimazole (CTZ) have been prepared and characterized. Their interaction with DNA and activity against tumour cell lines were evaluated. [Pt (CTZ)₂I₂] (1) was synthesized by the reaction of K₂PtCl₄, KI and CTZ, and [Pt(CTZ)₂Cl₂] (2) by direct reaction of K₂PtCl₄ with CTZ. These complexes were characterized by a combination of elemental analysis, molar conductivity, UV–Vis, IR, and NMR spectroscopy in one and two dimensions. DNA–platinum complex interactions were studied by spectroscopic, thermal denaturation and viscosity titration measurements. Covalent interaction studies were also performed. From these results we suggest that complexes 1 and 2 interact with the minor groove of DNA. Both complexes showed growth inhibitory effects on four out of the six tumour cells lines with GI₅₀ (50% growth inhibition) values in the 5–25 μM range, but there was no indication of cytotoxicity over the range of concentrations tested.     

Cucurbit[n]uril binding of platinum anticancer complexes

The encapsulation of cisplatin by cucurbit[7]uril (Q[7]) and multinuclear platinum complexes linked via a 4,4'-dipyrazolylmethane (dpzm) ligand by Q[7] and cucurbit[8]uril (Q[8]) has been studied by NMR spectroscopy and molecular modelling. The NMR studies suggest that some cisplatin binds in the cucurbituril cavity, while cis-[PtCl(NH3)2(H2O)]+ only binds at the portals. Alternatively, the dpzm-linked multinuclear platinum complexes are quantitatively encapsulated within the cavities of both Q[7] and Q[8]. Upon encapsulation, the non-exchangeable proton resonances of the multinuclear platinum complexes show significant upfield shifts in 1H NMR spectra. The H3/H3* resonances shift upfield by 0.08 to 0.55 ppm, the H5/H5* shift by 0.9 to 1.6 ppm, while the methylene resonances shift by 0.74 to 0.88 ppm. The size of the resonance shift is dependent on the cavity size of the encapsulating cucurbituril, with Q[7] encapsulation producing larger shifts than Q[8]. The upfield shifts of the dpzm resonances observed upon cucurbituril encapsulation indicate that the Q[7] or Q[8] is positioned directly over the dpzm linking ligand. The terminal platinum groups of trans-[{PtCl(NH3)2}2 mu-dpzm]2+ (di-Pt) and trans-[trans-{PtCl(NH3)2}2-trans-{Pt(dpzm)2(NH3)2}]4+ (tri-Pt) provide a barrier to the on and off movement of cucurbituril, resulting in binding kinetics that are slow on the NMR timescale for the metal complex. Although the dpzm ligand has relatively few rotamers, encapsulation by the larger Q[8] resulted in a more compact di-Pt conformation with each platinum centre retracted further into each Q[8] portal. Encapsulation of the hydrolysed forms of di-Pt and tri-Pt is considerably slower than for the corresponding Cl forms, presumably due to the high-energy cost of passing the +2 platinum centres through the cucurbituril portals. The results of this study suggest that cucurbiturils could be suitable hosts for the pharmacological delivery of multinuclear platinum complexes.     

DNA G-quadruplex and its potential as anticancer drug target

G-quadruplex secondary structures are four-stranded globular nucleic acid structures form in the specific DNA and RNA G-rich sequences with biological significance such as human telomeres,oncogene-promoter regions,replication initiation sites,and 5′and 3′-untranslated(UTR)regions.The non-canonical G-quadruplex secondary structures can readily form under physiologically relevant ionic conditions and are considered to be new molecular target for cancer therapeutics.This review discusses the essential progress in our lab related to the structures and functions of biologically relevant DNA G-quadruplexes in human gene promoters and telomeres,and the opportunities presented for the development of G-quadruplex-targeted smallmolecule drugs.     

Comparative study of the hydrolysis of a third- and a first-generation platinum anticancer complexes

The hydrolysis of cis-[Pt(NH₃)₂Cl₂] (cisplatin) and of the third-generation platinum anticancer compound (cis-[PtCl₂(NH₃)-(2-picoline)] (picoplatin) has been studied quantum-mechanically using density functional theory (mPW1PW91). Due to its asymmetry, picoplatin is an interesting case, since hydrolysis may proceed through two distinct reaction paths and with the entering water molecule sin or anti to the methyl group of 2-picoline. Solvent effects were taken into account either by applying the polarizable continuum model (PCM) solvent approach or by introducing a cluster of water molecules solvating the complex. With the PCM approach, it emerges that the steric hindrance of 2-picoline has a minimum impact on the activation barriers for picoplatin hydrolysis, since the water molecule approach is far from being axial and far from the methyl group. Accordingly, the TS state geometry with 5-coordinated platinum is not significantly more distorted than in the cisplatin case. Our results point out that the use of separated reactants as reference for energy barrier gives satisfactory results for each compound and produces correct relative magnitudes of first and second hydrolysis rate constants for each compound. However, this model is not able to predict the generally faster hydrolysis experimentally observed for cisplatin with respect to picoplatin. The study of the first hydrolysis step for the two compounds in the presence of explicit water molecules shows that the entering water molecule is strongly interacting with the others through a complex hydrogen bond network. The discrete solvation model is able to correctly predict a slightly slower hydrolysis of picoplatin highlighting the need of explicit solvation to predict small differences (within an order of magnitude) in the observed rate constants.     

New designed DNA light switch Ruthenium complexes as DNA photocleavers and Topoisomerase I inhibitors

Two ruthenium complexes containing a new ligand phipz (phipz = 2‐(1,10‐phenanthroline)‐1H‐imidazo[4,5‐b]phenazine) were designed and synthesized. These complexes were found to inhibit the DNA supercoiled relaxation mediated by topoisomerase I (topo I), cleave DNA under irradiation and bind to calf thymus DNA through intercalative mode. Furthermore, complex 2 shows higher photocleavage activity, topo I inhibition activity and DNA affinity than complex 1 . Additionally, introduction of phenazine unit may be the reason that two complexes exhibit DNA ‘light switch’ behavior. The present work shows that two complexes might be potential as new DNA ‘light switches’, DNA photocleavers and topo I inhibitors.     

Ambivalent intercalators for DNA: L-shaped platinum(II) complexes

Novel platinum(II) square planar coordination complexes, in which two heteroaromatic ligands are held by the metal in an unusual L-shaped geometry orthogonal to each other, have been synthesized, and their interaction with DNA was investigated with absorption and linear dichroism spectroscopy. As a rule, the ligand that is coplanar with the coordination square of Pt is found to be oriented perpendicular relative to the DNA helix axis when bound, suggestive of its intercalation between the base pairs of DNA. However, when this coplanar ligand is replaced by two pyridines, the opposite ligand, orthogonal to the coordination square, is instead preferentially intercalated. This behavior shows that these new complexes do indeed show some properties of true ambintercalators, i.e., compounds that can bind by intercalation of either of two distinct aromatic moieties.     

Novel platinum(II)-based anticancer complexes and molecular hosts as their drug delivery vehicles

Platinum(II)-based DNA intercalators where the intercalating ligand is 1,10-phenanthroline or a phenanthroline derivative and where the ancillary ligand is either achiral (e.g. ethylenediamine) or chiral (e.g. diaminocyclohexane) show a range of cytotoxicities with a defined structure-activity relationship. The most cytotoxic are those that contain methylated-phenanthroline ligands and 1S,2S-diaminocyclohexane (S,S-dach) as the ancillary ligand. We have developed a new purification method using Sep-Pak C-18 reverse phase columns, which means these metal complexes can be made faster and cheaper compared to published methods. Platinum(II)-based complexes containing imidazole, pyrrole and beta-alanine subunits, that are capable of recognising specific DNA base-pair sequences have also been synthesised. These include linear or hairpin polyamide ligands that can recognise DNA sequences up to seven base-pairs in length and contain single platinum centres capable of forming monofunctional adducts with DNA. We have now synthesised and characterised, by (1)H and (195)Pt NMR, ESI-MS and elemental analysis, the first dinuclear platinum(II) DNA sequence selective agent. Finally, using (1)H NMR we have examined the encapsulation of our platinum(II)-based DNA intercalators by cucurbit[6]uril (CB[6]). Encapsulation by CB[6] was found to not significantly change the cytotoxicity of five platinum(II)-based DNA intercalators, indicating it may have utility as a molecular carrier for improved drug delivery.     

Chiral salen ligands designed to form polymetallic complexes

Chiral salen ligands capable of forming polymetallic complexes have been designed. The ligands possess substituents in the 4,4′-positions, but have no substituent in the 3,3′-positions to allow a second metal ion access to the salen oxygen atoms. Ligands in which a polyether chain links the 4,4′-positions were prepared and complexed to copper. In addition, acyclic ligands with potential metal coordinating substituents in the 4,4′-positions were prepared and complexed to copper and cobalt. The crystal structure of one of the cobalt complexes shows it to be a trimetallic complex in which a Co(II)(OAc)₂ group coordinates to the salen oxygen atoms of two Co(III)(salen)(OAc) units. In contrast, the crystal structure of a Co(salen) complex with tert-butyl groups attached to the 3,3′-positions is found to be mononuclear. All of the complexes were tested as asymmetric phase transfer catalysts for the asymmetric alkylation of an alanine methyl ester, forming (R)-α-methyl phenylalanine methyl ester with up to 85% ee.     

Methionine ligand selectively promotes monofunctional adducts between trans-EE platinum anticancer drug and guanine DNA base

A detailed study on the reaction mechanism of trans-EE and trans-EE/Met Pt-containing anticancer drugs was carried out in order to rationalize the experimental kinetic data concerning the whole process leading to DNA platination.     

DNA and RNA noncovalent interaction of platinum(II) polypyridine complexes

A comparative investigation of the noncovalent interaction of the platinum(II) polypyridine complexes [Pt(dipy)(n-Rpy)2]2+ and [Pt(4,4'-Me2dipy)(2-Rpy)2]2+ (dipy = 2,2'-dipyridine; Me = CH3; n = 2-4; R = H or CH3) with double-helical DNA (calf thymus) and RNA [poly(A).poly(U)] has been conducted. With the exception of [Pt(dipy)(2-Mepy)2]2+, all of the complexes interact strongly, by intercalation, with both nucleic acids giving rise to large changes in the electronic spectra and induced circular dichroism signals; in addition, viscosity experiments on rodlike DNA and RNA show that both biopolymers elongate upon interaction with the complexes. The binding constant values, KB, determined at 25 degrees C, indicate that, at 0.101 M ionic strength, the affinity for poly(A).poly(U) is strongly dependent on the complexes nature, while for DNA it is leveled off. [Pt(dipy)(2-Mepy)2]2+ binds to DNA but does not interact appreciably with poly(A).poly(U).     

Automated parallel solid-phase synthesis and anticancer screening of a library of peptide-tethered platinum(II) complexes

The automated parallel solid-phase synthesis of a 36-member library of peptide-tethered platinum(II) complexes is described. The identity and quality of each product were confirmed by mass spectrometry and (1)H NMR. Subsequently, each compound was screened for in vitro anticancer activity by treating the A2780 (human ovarian carcinoma) cell line with two concentrations of the drugs (100 and 10 microM) in quadruplicate. The reduction of cell proliferation induced by the drugs at these concentrations was determined with the MTT colorimetric assay (MTT = 3-(4',5'-dimethylthiazol-2'-yl)-2,5-diphenyltetrazolium bromide) and compared to cisplatin. Even though no very active library members could be identified, five apparently most active (8[1], 8[4], 8[10], 8[13], and 8[24]) and two inactive complexes (8[33] and 8[34]) were purified using gel permeation chromatography and fully characterized by NMR spectroscopy ((1)H, (195)Pt) and MS. The IC(50) values of these complexes and cisplatin in A2780 cells were subsequently determined using the MTT assay in a conventional manner. All seven complexes have an IC(50) above 100 microM, confirming the results generated by the assay at 100 and 10 microM of the crude reaction products.     

Applying Cu(II) complexes assisted by water-soluble porphyrin to DNA binding and selective anticancer activities

Cancer is one of the killers endangering human health and its treatment has always been a focus of the medical community. For anticancer drugs, water-soluble porphyrin and Schiff bases have always been of interest. We report here three Cu(II)-based complexes functionalized by water-soluble cationic porphyrin and hydrazine Schiff base, which were prepared and evaluated for their biological activity. The three Cu(II) complexes all exhibited potent binding affinity to calf thymus DNA, the strongest interaction being between CuP2 and DNA. We studied the cytotoxicity of the complexes and ligands against different types of cancer cells (A549, H-1975, HepG2 and T47D), results showing the ligands are less cytotoxic; therefore, the anticancer activity of the complexes is improved by complexation. Furthermore, the cytotoxicity of ligands and complexes was also evaluated against the normal cell line Hs 578Bst, complexes showing more negligible cytotoxicity than ligand. Moreover, the cellular uptake of these Cu(II) complexes was investigated using the extraction method and results suggested that CuP2 exhibits the best cellular uptake towards H-1975 cells. Interestingly, fluorescence microscopy experiments and flow cytometric analysis (cell cycle) were used to further investigate the potent anticancer activities.     

Interference by complex structures of target DNA with specific PCR amplification

It has been considered that target DNA is a forgiving component for PCR amplification. Herein we present evidence to demonstrate that secondary structure located at the end of a template may interfere with the specificity of amplification. Experiments indicate that nonspecific amplification results from a long stretch of stem and loop structures at the 3′ end of prochymosin cDNA. Based on the sequence of mRNA coding for prochymosin, it is argued that the sequence responsible for the formation of the complex structure described here is most likely generated during synthesis of the second cDNA strand.     

Derivatives of pentamidine designed to target the Leishmania lipophosphoglycan

The Leishmania lipophosphoglycan (LPG) is the most abundant cell surface glycoconjugate of a family of infectious protozoa. Pentamidine, a common drug used in the treatment of Leishmania infections, has been modified with boronic acids so that it might bind more selectively to the phosphodisaccharide repeating unit of the LPG. This could serve to target the drug to the protozoan surface and increase its efficacy in vivo.     

Graphical approach to analyzing DNA sequences

Recently, we proposed a 2D graphical representation of DNA sequence [J Comput Chem 25(2004) 1364-1368]. Based on this representation, we outline one approach to search optimal alignment. We also can judge the mutations between bases and an unknown sequence based on its graph and a known sequence's graph.     

Quantitative analysis of specific target DNA oligomers using a DNA-immobilized packed-column system

Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on T _m values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar T _m values were separated and detected quantitatively by using a packed column carrying two different DNA probes.     

Oxidative addition - reductive elimination sequences in the photochemistry of some bis(phosphine)platinum complexes

The photolysis of [L₂Pt(C₂H₄)] (L = PPh₃, P(p-C₆H₄CH₃)₃ complexes in halocarbon solvents (CH₂Cl₂, CH₂Br₂) gives C₂H₄ and the coordinatively unsaturated species [L₂Pt]. Photolysis of platinum metallacycles [L₂Pt(CH₂)₄] (L = PPh₃, P(n-Bu)₃) generates alkanes, alkenes and [L₂Pt]. The [L₂Pt] centers are very reactive, and under prolonged photolysis undergo oxidative addition of CH₂Cl₂ forming the trans-[L₂Pt(CH₂Cl)Cl] complexes. Under appropriately controlled conditions the trans complexes isomerize to cis species before bimolecular C₂H₄ elimination occurs and [L₂PtCl₂] is formed as the final product. The oxidative addition-reductive elimination mechanism is discussed on the basis of spin-trapping experiments, quantum yield values, and the sensitivity to radical inhibitors and to solvents.