Cytotoxic Phosphinoarsino and Diphosphino Pd(II) Complexes of Thiolate Amino Acids and Glutathione

The novel complexes [Pd(L-L′)(SR)Cl] where L-L′ is Ph2PCH2CH2PPh2 (dppe) or Ph2AsCH2CH2PPh2 (dadpe) and RSH is glutathione, L-cysteine, or N-acetyl-L-cysteine, have been prepared and characterised. Their structures in the solid-state and in solution are discussed. The introduction of cysteine or glutathione as a ligand in these complexes greatly improved their aqueous solubility compared with the hydrophobic parent dichloro complexes. The cytotoxicities of the glutathione complexes towards the cell-lines L1210, ADJ/PC6 and CH1 were investigated. Their cytotoxicities towards L1210 cells were comparable to those of the parent dichloro complexes.


INTRODUCTION
Cytotoxic Phosphinoarsino and Diphosphino Pd(II) Complexes of Thiolate Amino Acids and Gluthathione Certain tetmhedml diphosphine complexes of Group 11 metals, such as [Au(dppe)2]CI are highly cytotoxic and exhibit anticancer activity in some model systems. They appear to act by a different mechanism to cisplatin, cis-[PtCI2(NH3)2], attacking proteins as well as DNA. Recently we have sought to extend this series of complexes to include square-planar diphosphine complexes. 2 One of the major objectives is to increase the aqueous solubility of complexes containing hydrophobic diphosphine ligands, and in this paper we use thiolate amino acid derivatives for this purpose.
The formation of Pd(ll) complexes with amino acids and peptides has been reviewed by Pettit and Bezer. 3 There have been few studies on the formation of Pd(ll) complexes with glutathione (7-L-Glu-L-Cys-Gly), ,4,5 although there are some reports of 1H NMR studies on binding of GSH to other metals, eg Me3Pb,6 mixed ligand complexes of Cd(II)(NTA), 7 and gold (I) drugs.
Rabensein and coworkers have carried out 1H and 13C NMR spectroscopic sudies on the binding , formation constants, and ligand exchange reactions of MeHg(ll)-thiol complexes with thiols such as glutathione, cysteine. There are no X-ray structures of metal GSH complexes, however a dimeric Cu(ll) complex of GSSG has been crystallogmphically characterised. 12 The solid state structure of the complex of Pd(ll) with S-methyI-L-cysteine has been reported. The geometry about the metal is square planar with S, N coordination as has been found for Pd complexes of S-methyI-L-cysteine sulphoxide TM and S-methyI-L-cysteine methyl ester. 15 We report here the preparation and characterization of novel mixed ligand Pd(ll) complexes containing cysteine, N-acetylcysteine or glutathione, and diphosphine or arsinophosphines as ligands, and their cytotoxicity against L1210, ADJ/PC6 and CH1 cell-lines.
Measurements of pH* (pH meter reading in D20 solutions) were made on a Corning pH meter equipped with an Aldrich combination microelectrode. Adjustments of pH* were made with DCI or NaOD.
IR spectra were recorded on a Perkin-Elmer 1330 instrument as Nujol mulls for GSH and its complexes, and [Pd(dadpe) [Pd(dadpe)(Cys)CI] a KBr disc was used between 4000 and 600 cm "1 and a Nujol mull between 600 and 200 cm'l.
Mass spectra (fast atom bombardment) were recorded using a VG ZAB-SE mass spectrometer at the University of London School of Pharmacy (ULIRS) with samples in an MNOBA (meta-nitroortho-benzoic acid) matrix. Elemental analyses were carried out by the micmanalysis service at University College London.
Thin layer chromatography was carried out on RPS-F plates (Anachem) using 1:1 EtOH:H20 as eluant. Cysteine complexes were dissolved in EtOH or H20 prior to elution and glutathione complexes in H20.

Biological testing
The cytotoxicities of aqueous solutions of the glutathione complexes 3 and 4 against murine L1210 and ADJ/PC6, and normal CH1 (human ovarian cells) cell-lines were determined as described previously. 2

RESULTS AND DISCUSSION
There am a few previous reports of Pd(ll) complexes with anticancer activity. 17, In general Pd(ll) complexes are much more kinetically labile than those of Pt(ll) and therefore less likely to arrive at the target site (e.g. DNA) with the original ligands still bound. However in the present case intracellular ligand release might still result in activity since the ligands dppe and dadpe are themselves cytotoxic. 2 The main aim of the present work was to increase the aqueous solubility of Pd(ll) complexes containing these hydmphobic ligands whilst retaining their cytotoxicity.   In solution the structures of these complexes are potentially complicated since Pd(ll) can form strong bonds to CI', thiolate S, carboxylate O, and amino or deprotonated amide N atoms. We made some tentative structural assignments from 31p, 1H and 13C NMR data. 31p NMR is particularly useful for dppe complexes since the presence of a 31p.31p coupling indicates a complex with non-equivalent coordinated P atoms and the shifts are a guide to the nature of the trans ligand, based on those of previously-reported chlom ( 64.2 ppm) and S-bound dmsa ( of Thiolate Amino Acids and Gluthathione TABLE 4(a).13C{1H} NMR data for [Pd(L-L')(GS)CI] (L-L" dppe 3, dadpe 4) in D20 at acidic and neutral pH* values. A,5 5(pH* 7.2)-5(pH* 1.8) and some tentative assignments.
Complex 6(complex) /)(GSH) [Pd(dppe) [Pd(dppe) (Cys-S,N)]. At high pH*, the spectrum of 2 showed of Thiolate Amino Acids and Gluthathione two resonances at 64.3 and 58.0 ppm. Since them is only one P atom in the dadpe ligand, these are assigned to two isomeric species, the latter to a P trans to S by analogy with [Pd(dadpe)(dmsa)] (57.3 ppm)2, and the former to the predominant isomer containing P trans to N or CI. At low pH* a third peak is present assignable to a species containing bridging S, and the intensity of the peak for the major isomer increased by a factor of ca. 4.
A pure product was not isolated from the reaction of [Pd(dppe)CI2] with N-acetyI-L-cysteine; the 31p NMR spectrum of an aqueous EtOH solution of the crude product showed that only a species containing magnetically equivalent P atoms was present. Complex 5, [Pd(dadpe) 1H NMR data for complexes 1 and 3 are listed in Table 3. For complex 1 at low pH* there are resonances at 4.22 and 2.94 ppm due to the z-CH and -CH 2 of coordinated Cys. At neutral pH, these are less shifted with respect to unbound Cys, in particular that of the -CH, suggesting that either the carboxylate or the amino group is uncoordinated and protonates at low pH. At both pH values, the 1H NMR spectra showed minor signals characteristic of cystine and free Cys which shifted slightly downfield as the pH* was increased. The spectra at low pH* also showed four multiplets at 7.88-7.57 ppm due to the C6H5 protons of dppe, and a broad multiplet at 2.718 p.p.m, due to the CH 2 protons of dppe. At higher pH* the Ph resonances were unchanged and the CH 2 protons gave rise to three multiplets. The spectrum of 3 at low pH* showed a quartet assignable to Gly-(z-CH 2 protons and a doublet of doublets due to Glu-0CH; these were sharp and overlapping. Other resonances assigned to GS in this complex were broad, i.e. those of the Cys-CH 2 protons and of Glu-TCH 2 and Glu-JCH 2. No resonance due to Cys-o-CH was observable. At neutral pH* the resonances for Glu-(zCH appeared as two doublet of doublets with a shift difference of 93.8 Hz. The resonances for Cys-CH 2 and 7-Glu CH 2 were broad at this pH*. Resonances due to protons of C6H5 groups of dppe were broad at both pH values. The resonances of the CH 2 protons of dppe appeared as a set of three multiplets at acidic pH and at 3O higher pH* they gave rise to two multiplets. Added GSH gave rise to a separate set of sharp resonances and so exchange with free GSH was not the cause of the broadening. Instead, this is probably due to the presence of coordination equilibria in solution. Spectra for 2 and 4 were complicated by the presence of two isomeric products, as discussed above, and so are not reported. 13C NMR data for the complexes [Pd(L-L')(GS)CI] (L-L" dppe 3 L-L" dadpe 4) at low and neutral pH* values are given in Table 4(a). The assignments for complex 3 are made partly on the basis of a comparison of the changes in shifts of the resonances on increasing the pH* from 1.8 to 7.2 with those observed for free GSH, and on the shifts noted previously for the Cys carbons of a 2"1 solution of GSH and Hg(NO3) 2 in this pH* range. 2 At pH* 7.2, three sets of broad resonances were observed in the phenyl region corresponding to the C6H5 carbons of dppe. The spectrum of the glutathione carbons of complex 4 was similar except that there were three resonances in the Cys 172.9,and 173.3 ppm. The resonances at 36.23 and 30.93 ppm are assigned to the aliphatic carbons adjacent to As and P, respectively.
The latter resonance is a doublet with JP-C 27 Hz. There were several resonances in the aromatic region of the spectrum corresponding to C6H5 of dadpe.
The coordination chemical shifts of the carbon atoms are listed in The compounds had a relatively low toxicity against the murine cell-lines, Figure 1A, e.g. towards L1210 the IC50 values for 3 and 4 were ca. 22 and 32 IM respectively. However, they showed some tissue selectivity against CH1 cells with IC50 values in the region of 4 to 10 IM (cf cisplatin IC50 10 -7 gM agains this cell-line). They were mm cotoxic towards L1210 cells than the bisthiolate complexes we have investigated previously, [Pd(dppe)(dmsa)] and [Pd(dadpe)(dmsa)], 2 and of comparable cytotoxicity to the square-planar dichloro complexes, [Pd(L-L')CI2], as shown in Figure lB. However there is no increase in the cytotoxicity of the complexed ligand dppe or dadpe with respect to that of the free ligand towards this cell-line.
Indeed, for dppe the ligand itself is significantly more toxic (by up to 5 times).

CONCLUSION
By introducing thiolato ligands into Pd(ll) dppe and dadpe complexes, we have improved their aqueous solubility and retained their cytotoxicities in comparison to the parent dichloro complexes. For the L-cysteine complexes 1 and 2, IR and NMR showed the presence of S-bound cysteine ligands, and there was evidence for both monomers containing S,N chelates and dimers with bridging S in solution. Depending on the conditions, glutathione also bound strongly via S in of Thiolate Amino Acids and Gluthathione complexes 3 and 4 and S-bridged complexes appeared to predominate in aqueous solution, but complicated equilibria were also detected with evidence for exchange between various coordination sites. Complexes with moderate cytotoxicity such as those described here, if they are also active in vivo, could be useful in combination therapy with established drugs such as cisplatin since they are likely to have a different mechanism of action.