Polycations XX : NewMonodentate Cationic Ligands and Their Coordination with Ruthenium for the Construction of Complexes Expressing Enhanced Interaction with DNA

Prior investigations from this laboratory concerned with the preparation of new types of organic cations for a variety of biological and nonbiological applications have been extended to the preparation of cation-bearing ligands with nitrogen coordinating sites for use in complexation reactions with ruthenium cores. The syntheses of new cationic ligands as well as ruthenium complexes bearing them are reported here. The introduction of these new types of ligands is intended to provide to the complexes an enhanced ability to interact with DNA, and thereby to have the potential to be enhanced antitumor agents. Preliminary observations of their interactions with DNA are presented.


Introduction
The potential for ruthenium-centered agents to serve as antitumor pharmaceuticals has been known for some time [1].It is known that particular complexes of ruthenium that can serve as antitumor agents act by binding to DNA of the tumor cells through the basic sites on a DNA chain (e.g., a guanine site) and crosslinking the DNA chains through linking to a basic site (e.g., another guanine) on another chain, thus preventing cell division and ultimately leading to cell death [2,3].While the bonds between the nitrogenous ligands and the ruthenium center exhibit strength typical of that of coordinate covalent bonds, strengths less than typical of covalent bonds, they exhibit a kinetic stability that allows them to survive to serve as DNA-binding agents [4].
Prior efforts of this laboratory with polycationic salts (topologically referred to as "strings" owing to their flexible linear array of charges within the structure) have demonstrated an ability to bind into the major groove of doubly stranded DNA and cause changes in the conformational nature of the DNA [5][6][7].
With this concept of DNA interaction in mind and the ability of nitrogenous complexes of ruthenium to interact with DNA, two series of structures have been devised.These structures have been devised with the anticipation that their cationic ligands would enhance the capability of the metal species for interaction with DNA via binding of cationic sites on the ligands with the (anionic) major groove of doubly stranded DNA.The syntheses of the particular ligand types and the construction of model platinum and ruthenium complexes bearing them are presented here, along with initial investigations of the spectral characteristics of the complexes regarding their interactions.

Results and Discussion
Two types of cation-bearing ligands that have been designed for incorporation into ruthenium-core complexes are considered here.These monodentate cationic ligands are shown in Figure 1, derived from 4,4 -bipyridyl (a), and nicotinic acid (b).Of the unsubstituted bipyridyls readily available, the 4,4bipyridyl is the most conveniently alkylated and results in a monoquaternary salt with the cationic site most fortuitously placed for DNA interaction when complexed with ruthenium.However, alkylation of this material is not as facile as might be anticipated by extension from the chemistry of simple pyridine derivatives.In our hands the only simple alkyl halide that could be used efficiently for the generation of a monoquaternary salt was the 1-bromohexane, which provided the desired salt in low (37.3%),albeit workable, yield after 21 hours of reflux in ethyl acetate.Other simple alkyl groups could be used to generate monoalkylated 4,4bipyridyl derivatives, but in yields too small for efficient use.Prior efforts of our laboratory have indicated that ethyl acetate is a favorable medium for monoalkylation of species bearing several potentially reactive sites, this capability owing to the low solubility of the monoalkylated materials in that solvent [8][9][10].Further investigation of the solvent systems used is required to perform other related alkylations efficiently.
Facile alkylation for better yield in much shorter reaction time (in ethyl acetate solvent) is accomplished using a variety of benzylic and allylic halides.In these instances, reaction times of under 3 hours of reflux provide reasonable yields of the monoalkylated 4,4 -bipyridyl salts.In all instances, the product salts demonstrated themselves to be significantly hydroscopic, often becoming gummy from collected water during the isolation by filtration.For handling, all of these materials required drying on a vacuum line at 10 −3 Torr prior to use.Even then, significant amounts of moisture were accumulated upon transfer for elemental analyses, and such analyses were performed with the determination of the amount of accumulated water involved.The quaternary ammonium salts (1-8) derived from 4,4 -bipyridyl have been used in the construction of the ruthenium(II) complexes (12)(13)(14)(15)(16)(17)(18)(19).
Verification of structure for all isolated salts was performed using NMR in deuterium oxide solution using a 400 MHz Brüker DPX400 instrument with measurement of 1 H, 13 C, and 13 C Dept 45 (for determination of carbon bearing no hydrogens) spectra, as well as C/H/N elemental analyses (Columbia Analytical Services, Tucson, AZ).These analytical and yield data for the 4,4 -bipyridyl salts are provided in the Experimental section.
The preparations of the ruthenium complexes using the new cationic ligands were performed using standard procedures.For the ruthenium complexes, a modified approach to the standard procedure [12] was performed by refluxing the hydrate of bis(2,2 -bipyridyl)ruthenium(II) dichloride in aqueous ethanol with the proper equivalent amount of cationic ligand.After hot filtration of the reaction mixture, the filtrate was evaporated under reduced pressure with final traces of ethanol being removed on the vacuum line at 10 −3 Torr.
The preparation of complexes of ruthenium(II) bearing cationic ligands is a relatively simple matter using these approaches.Good-to-excellent yields of the complexes are obtained through these methods, although it is to be noted that all of the complexes are significantly hygroscopic.
Absorbance spectra (UV-Visible) were measured in aqueous solution for the ruthenium complexes prepared.A selection of these spectra is presented here.In several instances (as noted), there appeared slight changes in the spectra after initial preparation of the solutions, but these rapidly came to stability and maintained constant absorption through the UV-Visible region.Absorption spectra for ruthenium complexes 16-18, 20, and 21-22 are shown in Figures 3(a) and 3(b), respectively, with relative maxima and relative intensities noted.Attempts to measure spectra of unhydrated materials failed, as they readily absorbed atmospheric moisture, thus molar absorptivities are provided using the materials for which elemental analyses have been obtained fully hydrated.
Observed absorbances noted represent two types of processes: (1) those of wavelength greater than 320 nm represent metal-to-ligand charge transfer processes, and (2) those of wavelength less than 320 nm represent intraligand charge transfer processes.
In several instances, (e.g., ruthenium complexes 16-18) the absorption spectra of initially prepared samples (aqueous solution) underwent shifts of up to 3 nm in absorption maxima and changes of the values of relative intensities of a few percent.These changes quickly ceased and steady spectra were maintained, observations being made up to a week after initial preparation of the sample solutions.The origin of these small changes was not evident, but can reasonably be accounted for by considering conformational changes in  the partially flexible cationic ligands until full equilibrium is attained.
Overall, the observable spectra were typical for octahedral ruthenium with coordination through nitrogen to aromatic ligands [13].

Experimental
All chemicals and solvents were of reagent quality and were used without further purification as obtained from the supplier.All NMR spectra were measured using a 400 MHz Brüker DPX400 instrument, referenced electronically for both 1 H and 13 C spectra based on the lock on the deuterated solvent signal.In the instances of the ruthenium complex 13 C spectra, resonances resulting from bpy carbon sites are indicated in italics, and carbon sites without hydrogen are indicated by a q after the numerical chemical shift.For these assignments, 13 C Dept45, HETCOR, and 1 H COSY spectra were used in addition to the standard measurements.Elemental analyses were performed for C/H/N/by Columbia Analytical Services of Tucson, AZ, USA.
All UV-visible spectra were measured in distilled water solution using distilled water as a blank, using a HP 8453 diode array UV-visible spectrometer with 1 cm.path quartz cells.General preparation of monoquaternized 4-substituted-4azonia-4 -azabiphenyl salts (4,4 -bipyridyl salts) (1)(2)(3)(4)(5)(6)(7)(8).In a 250 mL round-bottomed flask is placed 4,4 -bipyridyl (10 g, 0.064 mol) with ethyl acetate (125 mL) and an equivalent amount of the appropriate organic halide (vide infra) is added to it.The reaction mixture is stirred and heated at reflux for 16 hours, after which time the formed precipitate is recovered by suction filtration through sintered glass, washed with hexane, and placed on the vacuum line at 10 −3 Torr to complete drying.All recovered salts are significantly hydroscopic.The use of simple haloalkanes (such as 1bromohexane) requires longer heating times, albeit with lower isolated yields.
4-Allyl-4-azonia-4 -azabiphenyl chloride (1)-23.4% yield from allyl chloride- 1      (9).In a 100 mL round-bottomed flask is placed 11 (2.09 g, 0.0056 mol) with acetonitrile (50 mL).Nicotinoyl chloride hydrochloride (1.00 g, 0.0056 mol) is added to it, and the resultant mixture is heated at reflux for 24 hours.After cooling, a solution of sodium carbonate hydrate (1.00 g, 0.0081 mol) in water (5 mL) is added with stirring.After filtering through sintered glass, the filtrate is evaporated under reduced pressure and finally on the vacuum line at 10 −3 Torr to yield a glassy material (1.74 g, 60.6% yield) that resisted crystallization but provided analytical data in accord with the proposed structure. 1 (11).In a 100 mL round-bottomed flask is placed 1-hexyl-1-azonia-4-azabicyclo[2.2.2]octane bromide [10,11] (2.04 g, 0.0074 mol) with acetonitrile (40 mL).To this solution is added in excess 3-chloro-1-propanol (5.50 g, 0.058 mol), and the mixture heated at reflux for 5 hours.After this time, the volatile materials are evaporated under reduced pressure and the gummy residue washed with three portions of ethyl acetate (40 mL each) and the solid dried on the vacuum line at 10 −3 Torr.In this way could be isolated 6.22 g (71.9% yield) of the pure target material. 1 (13)(14)(15)(16)(17)(18)(19)(20).In a 100 mL round-bottomed flask is placed cis-Bis-(2,2 -bipyridine)dichlororuthenium(II) hydrate (1.00 g, 0.0020 mol) with 95% ethanol (50 mL) and the appropriate ligand to be added.For the monodentate ligands as noted above, the amount added is 0.0040 mol.The reaction mixture is stirred and heated at reflux for 20 hr, after which time, the hot reaction mixture is filtered through sintered glass, and the filtrate is evaporated under reduced pressure to remove volatile materials, final evaporation being done

Figure 2 :
Figure 2: Precursor and final ligand structures for ligands of category (b) (see Figure 1).

Table 1 :
Absorption maxima and relative intensities for UV-visible spectra of selected new ruthenium complexes bearing cationic ligands.