Cytotoxicity Profiles for a Series of Triorganophosphinegold(I) Dithiocarbamates and Triorganophosphinegold(I) Xanthates

A series of triorganophosphinegold(1) dithiocarbamate (R3PAuS2CNR'2) and xanthate (R3PAuS2COR') complexes have been prepared and characterised spectroscopically. Based on crystallographic evidence, the molecules feature linear gold(1) geometries defined by sulphur and phosphorus donors. The complexes, along with a series of known anti-cancer agents, have been screened against a panel of seven human cancer cell lines. Uniformly, the dithiocarbamate derivatives are more active than their xanthate counterparts, with the most active complex being Et3PAu(S2CNEt2), and are more active than cisplatin in all cell lines screened but, not as potent as taxol.


INTRODUCTION
Amongst the l,l-dithiolate ligands, dithiocarbamates, SzCNR2, comprise a group of ligands with great binding potential to metals and as such find wide use in coordination chemistry. Their synthesis is relatively simple with the most common method of preparation involving the reaction of carbon disulphide, in the presence of a base such as sodium or potassium hydroxide, with any one of a large range of primary and secondary amines/1, 2/. Dithiocarbamates and their metal complexes have a wide variety of applications.
Their most common use is as pesticides, e.g. zineb, [Zn(SzCN(H) application has led to the development of new analytical techniques that were designed to determine the concentrations of these pesticides as well as their degradation products/2 4/. In addition, these species have important applications in the production of petroleum derivatives, lubricants and polymers, where they are Among the various different classes of metal complexes currently investigated for their applications in medicine, dithiocarbamate complexes demonstrate outstanding potential/23/. The potential medical uses of dithiocarbamate complexes include: anti-viral agents, e.g. heterocyclic dithiocarbamates of ruthenium(Ill) /24/, antidotes for preventing the effects of phytotoxic agents, e.g. copper dithiocarbamates/25/, bactericides and anti-microbial agents, e.g. triorganotin dithiocarbamates/26/, anti-tumour agents, notably of palladium and platinum/27-31/as well as tin dithiocarbamates/32, 33/, anti-parasitic agents, e.g. platinum, iridium and rhodium /34/, and prophylactic or therapeutic agents for metal toxicity, e.g. for cadmium /35/. Iron dithiocarbamates have been used for treating AIDS and neurodegenerative diseases/36/. As an extension of the aforementioned medical applications of dithiocarbamate ligands and their metal complexes, this contribution describes a study where dithiocarbamates have been combined with phosphinegold(1) entities with the view of exploring their anti-tumour potential.
Gold thiolates, including a phosphinegold(1) thiolate, auranofin, are used in the treatment of arthritis/37, 38/, usually after other therapies have been exhausted. The examination of the potential anti-tumour activity of gold complexes is a more recent phenomenon and has been demonstrated in a number of experimental models but, as yet, no gold compound has entered clinical trials. Auranofin and analogues were shown fo be .Dick de Vos et aL Bioinorganic Chemistty and Applications cytotoxic towards B 16 melanoma and P388 leukaemia in vivo, early standards in anti-cancer screening/39/. The development of gold complexes as anti-tumour agents has been reviewed recently/40/.
The focus of our investigations in this field has been upon the potential anti-tumour activity of phosphinegold(l) thiolates, i.e. auranofin analogues /41/. A particular emphasis has been to couple biologically active thiols with phosphinegold(l) entities in the hope that upon administration of the 'prodrug', both the phosphinegold(l) entity and thiol would provide therapeutic benefit /42, 43/. As a continuation upon this theme, we present here the results of in vitro cytotoxicity screening for a range of phosphinegold(1) dithiocarbamate complexes, a study motivated by the combination of biologically active dithiocarbamates with phosphinegold(1). In addition, a smaller number of phosphinegold(1) dithiocarbonate (-S2COR, xanthate) complexes are included in this study. Xanthates and their metal complexes have not been evaluated for biological activity to the same extent as dithiocarbamates. However, xanthate complexes of tin have demonstrated some potential as anti-tumour agents/44/and certain phosphinegold(1) dithiocarbonate complexes have proved to possess some anti-arthritic activity/45/. The results of this study are reported herein.

General
The R3PAuC! starting materials were prepared according to the literature method/46/. All solvents were of analytical grade (J. T. Baker) and used as supplied. NaSCNEtz.3H20 (Tuka) and NH4S2CNCnH8 (Aldrich) were used as supplied. Potassium xanthates were prepared from the reaction of the alcohol (that also served as the solvent), CSz and KOH. 1H, and 13C{IH} NMR spectra were recorded on a Bruker ACF300 FT NMR spectrometer, with chemical shifts relative to tetramethylsilane. 3p{H} NMR data were recorded on the same instrument but with chemical shifts recorded relative to 85% aqueous H3PO4. IR spectra were obtained as KBr pellets on a Bio-Rad FTSI65 FTIR spectrophotometer. ESI mass spectra were measured on a Finnigan MAT95XL-T spectrometer. Elemental analyses were performed on a Perkin Elmer PE 2400 CHN Elemental Analyser.

General Synthetic Procedure
To a dichloromethane solution (4 ml) of R3PAuCI was added an equimolar amount (based on gold content) of dithiolate ligand. The colourless solution immediately turned yellow, indicating the formation of the product, and was stirred for 2 h. The yellow solution was filtered through Celite and concentrated to approximately ml to yield the product.

EtPAuS2CNEt2 (1)
From Et3PAuCI (0.2 g, 0.57 mmol) and NaS2CNEtz (98 mg, 0.57 mmol). The product was recrystallised by the layering of ethanol into a dichloromethane solution of the compound to yield yellow crystals. Yield: Cytotoxicity Prqfilesjbr a Series of Triorganophosphinegold (I)
The experiment was started on day 0. On day 0, 150 lal of trypsinized tumor cells (I 500 2000 cells/well) were plated in 96-wells flatbottom microtiter plates (falcon 3072, DB). The plates were preincubated 48 hrs at 37 C, 8.5 % CO2 to allow the cells to adhere. On day 2, a three-fold dilution sequence of ten steps was made in full medium, starting with the 250 000 ng/ml stock solution. Every dilution was used in quadruplicate by adding 50 lal to a column of four wells. This results in a highest concentration of 62 5000 ng/ml present in column 12. Column 2 was used for the blank. To column 1, PBS was added to diminish interfering evaporation. On day 7, the incubation was terminated by washing the plate twice with PBS.
Subsequently, the cells were fixed with 10 % trichloroacetic acid in PBS and placed at 4 C for one hour. @totoxicity t'ofilesfor a Series of Triorganophosphinegold (1) Dithiocarbamates After five washings with tap water, the cells were stained for at least 15 minutes with 0.4 % SRB dissolved in 1% acetic acid. After staining, the cells were washed with 1% acetic to remove the unbound stain. The plates were air-dried and the bound stain was dissolved in 150 gl 10 mM Tris-base. The absorbance was read at 540 nm using an automated microplate reader (Labsystems. Multiskan MS). Data were used for construction of concentration-response curves and determination of the IDs0 value by use of Deltasofl 3 software.

RESULTS AND DISCUSSION
A series of phosphinegold(1) l,l-dithiolates have been prepared and characterised spectroscopically. Physical data are presented in Table and the spectroscopic results, that confirm the formation of the complexes, are summarised in Tables 2 4. The crystal structure of a representative complex has been undertaken.
The molecular structure of (p-MeOC6H4)aPAu(S2COiPr) (10) is shown in Fig. and selected geometric parameters are collected in the caption to this figure. The gold atom exists in the expected linear geometry defined by S and P donor atoms with the Au-S distance being significantly longer than the Au-P distance. The small deviation from the ideal linear angle at gold (S-Au-P is 176.75 (5)) may be traced to the close approach of the non-coordinating $2 atom that is separated 3.2856 (16) ,& from the gold atom. Arguably the most significant intermolecular contacts are of the type C-H...O. Thus, C16-H...O2 is 2.47 A, C16...O2 is 3.319(6) A and the angle at H is 149 , and CI7-H...O3" is 2.37 A, CI7...O3" is 3.292(5) A and the angle at H is 163 for symmetry operations i: l-x, -y, IA+z and ii: -%+x, -1A-y, z. Similar coordination geometries have been reported for related phosphinegold(1) xanthates /59 65/ and there is no evidence to suggest that different structures are found for (8) and (9). The molecular geometry found for (10) is also as expected for their phosphinegold(1) dithiocarbamate analogues/58/and indeed, the crystal structures of (2)/47/, (3)/48/ and (5)/49/have been reported separately. Ph3PAuS2COC4H9 (8) PhsPAu SCOCH2CH2OMe (9) (p-MeOPh)3PAuS2COiPr (10)  Bioinot2anic Chem&to/ and Applications The phosphinegold(1) dithiocarbamates, (1)- (7), and xanthates, (8)- (10), have been evaluated for their cytotoxicity against a panel of seven human cancer cell lines. The following cell lines were used: A498, renal cancer; MCF-7, estrogen receptor (ER)+/progesterone receptor (PgR)+; EVSA-T, estrogen receptor (ER)-/progesterone receptor (PgR)-; H226, non-small cell lung cancer; |GROV, ovarian cancer; M l9 MEL, melanoma; and WIDR, colon cancer. The A498, H226, IGROV, M 19 MEL, WIDR cell lines are included in the current anti-cancer screening panel of the National Cancer Institute, U.S.A. /66/. The cytotoxicity screening results for (!) (10) are given in Table 5 as well as those for a series of standard anti-cancer agents. From the data presented in Table 5, several trends may be discerned.
The two dithiocarbamate iigands chosen for evaluation were featured in the Introduction owing to their known biological relevance. Amongst the diethyldithiocarbamates, the EtaP derivative (l) was the most active. The CyaP species (2) has comparable cytotoxicity to (l) and both are more potent than the (p-MeOC6H4)aP derivative (3). The complex containing the bidentate phosphine ligand dppf, where dppf is |, l'bis(diphenylphosphine)ferrocene, that gives rise to a dinuclear gold species (4), has the poorest cytotoxicity, in particular considering it contains approximately twice the amount of gold as do the other species. The second dithiocarbamate series contains the pyrrolinedithiocarbamate ligand. Of the three complexes, (5)- (7), the PhaP species (6) is the most potent. The CyP complex (5) is less cytotoxic against all cell lines compared with the diethyldithiocarbamate analogue (2) but the reverse is true for the dppf derivatives in five cell lines, i.e. A498, MCF-7, EVSA-T, M I9 and W|DR. Such a non-systematic variation underscores the difficulty in generating a structure/activity relationship in these compounds. Amongst the xanthate, PhaPAu(SzCOR), complexes, R (CHz).CH3 (8) and CHzCHzOCH3 (9) had comparable potency to each other and both were more cytotoxic than (p,MeOC6H4)PAu(S2COiPr) (10). As a class of complex, the xanthates are generally less cytotoxic than their dithiocarbamate analogues. The greatest potency exhibited by the xanthate complexes was against the ovarian cancer cell line |GROV but, it is noted that the range of IDa0 values against all cell lines is not great suggesting little, if any, specificity in their cytotoxicity profile.
The greatest potency exhibited by the dithiocarbamate complexes was also evident against the IGROV cell line and comparable activities were also found against the breast cancer cell lines MCF-7 and EVSA-T. The cytotoxicity results for the phosphinegold(1) I,l-dithiolates can be compared with those obtained for a selection of known anti-cancer agents.
It is clear from the data presented in Table 5 that several of the phosphinegold(1) dithiocarbamates and even xanthates had greater cytotoxicity than cisplatin in the cell lines evaluated. For the cell lines in which the dithiocarbamate complexes were particularly cytotoxic, i.e. IGROV, MCF-7 and EVSA-T, the IDso values were lower than those obtained for both 5-flurouracil and etoposide. Clearly, anti-cancer agents such as doxorubicin, methotrexate and taxol demonstrate greater cytotoxicity than the phosphinegold(1) l,ldithiolates. Table 5 In vitro IDs0 values (ng/ml) for phosphinegold (1)  CytotoxiciO .ProfilesJbr a Series of Triorganophosphinegold (I) Dithiocarbamates CONCLUSIONS Phosphinegold(l) dithiocarbamates display cytotoxicity profiles greater than that exhibited by cisplatin against a range of human cancer cell lines. The most potent complex overall was Et3PAu(S2CNEt2) which was most active against the IGROV (ovarian cancer) cell line. The dithiocarbamate complexes had greater potency than the corresponding xanthate complexes.