Cytotoxicity of Triorganophosphinegold(I) n-Mercaptobenzoates, n = 2, 3 and 4

The results of cytotoxicity trials against a panel of seven human cell lines for a series of triorganophosphinegold(I) 3- and 4-mercaptobenzoates are reported. While the new compounds show moderate to high toxicity, their potencies are inferior to those reported previously for their isomeric 2- mercaptobenzoate derivatives. The results therefore suggest a structure-activity relationship in that the 2-isomeric species are more active, particularly against the non-small cell lung cancer and renal cancer cell lines, results that may indicate some selectivity in their cytotoxic profile.


Introduction
Gold complexes are well known to possess significant anti-arthritic activity and several gold thiolates are currently used clinically in the treatment of that disease [1][2][3]. In addition to this biological profile, several gold(I), as well as gold(Ill) species, have proven to display cytotoxicity and anti-tumour activity as summarised in a recent survey [4]. In this context, one of the active classes of compounds features a linear P-Au-S entity as found in the anti-arthritic drug, auranofin (triethylphosphinegold(I) tetraacetatothioglucose). Our work in this area has focussed on making derivatives of auranofin, some of which display significant cytotoxicity and anti-tumour activity [5][6][7][8]. A difficulty encountered with these compounds has been their limited aqueous solubility. In order to overcome this limitation, thiols containing water-solubilising groups have been coordinated to the phosphinegold(I) entity. Amongst the thiols chosen for investigation was 2-mercaptobenzoic acid. These derivatives displayed moderate to high cytotoxicity profiles compared to standards such as cisplatin, doxorubicin, methotrexate, etc. [9]. In particular, significant cytotoxicity was found against the non-small cell lung cancer and renal cancer cell lines. As a continuation of work in this area, some 3-and 4-mercaptobenzoates have been prepared and subjected to screening for their cytotoxicity profiles. The results of this study are reported herein.

Results and Discussion
The triorganophosphinegold(I) n-mercaptobenzoates, R3PAu(n-mbaH), have been synthesised using established procedures involving the metathesis of the triorganophosphinegold(I) chloride precursor with the appropriate n-mercaptobenzoic acid in the presence of base. Characterisation data are collected in the Experimental section. A number of the n-mercaptobenzoates compounds have been characterised crystallographically, by us [9 11] (i.e. n 2 for R Ph and Cy; n 3 for R Cy and Ph) and others [12] (i.e. n 4 for R Et).
The crystal structure of Cy3PAu(4-mbaH) has been determined in the present study. A centrosymmetrically related pair of molecules is illustrated in Figure 1; selected geometric parameters are collected in the Figure caption. The gold atom exists in the expected linear geometry defined by the sulfur and phosphorus atoms so that the angle subtended at the gold atom is 175.06(4) . The 4-mbaH ligand is essentially planar as seen in the magnitude of the O1/CI'/C1/C2 and O2/C1'/C1/C2 torsion angles of-174.3(4) and 5.0(7) , respectively. Molecules associated via the familiar carboxylic acid dimer motif with the key parameters defining this association listed in the caption to Figure 1. The structure is in essential agreement with the Et3PAu(4-mbaH) and o-tol3PAu(4-mbaH) structures reported recently by Schmidbaur et al. [12]. A previous report [9] summarising the cytotoxicity of a series of triorganophosphine 2mercaptobenzoates showed high levels of activity, in particular against the non-small cell lung cancer (H226) and renal cancer (A498) cell lines. These results are reproduced in Table 1. Subsequently, biological trials for some 3-and 4-mercaptobenzoate derivatives of R3PAu, for R Et, Cy & Ph, were also conducted.
As seen from Table 1, a general structure-activity relationship can be established in that the 2mercaptobenzoate compounds are generally more active than their 3-and 4isomeric counterparts. The most cytotoxic compound in the series investigated was the Et3PAu(2-mbaH) compound which displayed a IDs0 value of 43 ng/ml against the IGROV cell line but, doxorubicin and methotrexate were more active.
High activity against the H226 and A498 cell lines was only observed for the 2-mbaH series perhaps indicating some selectivity in activity. It would be of some interest to ascertain whether this activity is maintained in vivo leading to anti-tumour activity. symmetry operation-l-x, l-y, 1-z.

ER)+/progesterone receptor (PgR)+ and the cell line EVSA-T is (ER)-/(PgR)-.
Prior to the experiments, a mycoplasma test was carried out on all cell lines and found to be negative. All cell lines were maintained in a continuous logarithmic culture in RPMI 1640 medium with Hepes and phenol red. The medium was supplemented with 10% FCS, penicillin 100 IU/ml and streptomycin 100 lag/ml. The cells were mildly trypsinised for passage and for use in the experiments.

Chemicals
RPMI and FCS were obtained from Life Technologies (Paisley, Scotland). SRB, DMSO, penicillin and streptomycin were obtained from Sigma (St. Louis MO, USA), TCA and acetic acid from Baker BV (Deventer, NL) and PBS from NPBI BV (Emmer-Compascuum, NL).

Experimental Procedures
The test and reference compounds were dissolved to a concentration of 250000 ng/ml in full medium, by 20 fold dilution of a stock solution which contained mg compound/200lag atter having been dissolved first in in DMSO solution. Cytotoxicity was estimated by the microculture sulforhodamine B (SRB) test [14]. cells/well) were plated in 96-wells flatbottom microtiter plates (Falcon 3072, BD). The plates were preincubated for 48 h 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 250000 ng/ml stock solution. Every dilution was used in quadruplicate by adding 50 lag to a column of four wells. This results in a highest concentration of 62500 ng/ml present in column 12. Column 2 was used for the blank. To column 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 0.4% SRB dissolved in 1% acetic acid. Atter staining the cells were washed with 1% acetic acid to remove the unbound stain. The plates were air-dried and the bound stain was dissolved in 150 [al 10 mM Tris-base. The absorbance was read at 540 nm using an automated mieroplate reader (Labsystems Multiskan MS). Data were used for construction of concentration-response curves and determination of the IDs0 value by use of Deltasott 3 sottware.
Crystallography A colourless crystal with dimensions 0.16 x 0.16 x 0.38 mm was used in the X-ray diffraction study. An empirical absorption correction was applied [15]. The maximum residual electron density peak was located in the vicinity of the gold atom.