Synthesis, Structure of Nitrogen-Containing Phosphinogold(I) Ferrocenes. In vitro Activity against Bladder and Colon Carcinoma Cell Lines

The gold salt [(tht)AuCl] was reacted with [1-N,N-dimethylaminométhyl-2-diphenylphosphino]ferrocene (1) forming the bimetallic derivative 4. The reaction of methyl iodide and tetramethylammonium bromide on the chloride 4 produced the ammonium salt 5 and the bromide 6 respectively. New aminophosphines 2 and 3, which represent two of the rare phosphorylated metallocenes containing P(III)-N bond have also been coordinated to gold(I) to form 7 and 8. The presence of the ethoxy group in 7 provides evidence for the lability of one nitrogen-phosphorus bond. The X-ray structure of compounds 4 and 7 have been established. Both crystallize in space group P21/c, monoclinic, with a = 11.095(2) Å, b = 12.030(3) Å, c = 17.763(4) Å, β= 94.02(2)∘, Z = 4 for 4 and a = 14.863(3) Å, b = 8.036(5)Å, c = 18.062(5)Å, β =101.64(1)°, Z = 4 for 7. 197Au Mössbauer data are in good agreement with those for other linear P-Au-Cl containing complexes. The compounds were evaluated for in vitro anti-tumour activity against two human tumours. Differential cytotoxicity was observed with activity comparable to cisplatin, with the exception of one compound which was significantly more cytotoxic.


INTRODUCTION
The antitumour activity of diphenylphosphine complexes is of recent interest and it has been proved that the coordination of the diphosphine to gold(I) enhances the cytotoxicity, z,2 Moreover the intimate association of two therapeutic metals in the same molecule could give the possibility of a synergism between them. With this objective in mind, we have prepared gold(I) derivatives of compounds similar to [FcCH2NHMe2]+[CI]" (Fc Xl5-C5H5FeC5H4) which itself exhibits significant antitumour activity3. We report here the synthesis and comparative in vitro cytotoxicity of the (diphenylphosphino)chlorogold(I) complex obtained from [FcCH2NMe2]), the corresponding iodide salt, the bromide derivative, new ferrocenylphosphines and their gold(I) complexes.

MATERIALS AND METHODS
All reactions were conducted under an atmosphere of pure dry argon. Solvents were dried and deoxygenated over sodium/benzophenone ketyl and distilled immediately before use.
CH2CI2 stabilized with 0.3 % ethanol was purchased from SDS Company. Transfers were carried out via syringes or cannulas.
Microanalyses were performed by the Microanalysis Centre in UMIST (Manchester, U.K.).
Melting points were determined with a Kofler apparatus without correction. 1 H, 13C{1H}, 19 F and 31p nuclear magnetic resonance spectra were recorded in CDCI3 with a Bruker AC 200 spectrometer operating at 200.0, 50.3, 187.8 and 81.0 MHz respectively. Chemical shifts are reported in > units, pads per million downfield from internal tetramethylsilane for 1 H, 13C{1 H} and from external H3PO4 for 31 p.
The M6ssbauer spectra were obtained in the Technische Universit&t M0nchen (Garching, Germany). The 197pt activity feeding the 77.3 keV MSssbauer transition was produced by irradiation of enriched 196pt metal. Both source and absorber were kept at 4.2 K, a sinuso'[dal velocity wave form and an intrinsic Ge detector were used. A sample of the absorber with of ca. 50 mg(Au).cm "2 was used, spectra were fitted to Lorentzian lines. I.E. phosph in olferrocene (2) A solution of BuLi in hexane 1.45 M (4.30 ml, 6.17 mmol) was added dropwise to a stirred suspension of N,N-dimethylaminomethylferrocene (1.50 g, 6.17 mmol) in diethylether (5 ml) at 0 C. Stirring was maintained until the precipitate of o-lithioamine appeared. This monolithioamine was reacted with [CIP(NMe2)2] (0.95 g, 6.17 mmol) and the resultant mixture was heated under reflux for I hr. The white solid (LiCI) was filtered off and the solvent was taken off. The oil obtained was recrystallized from pentane at -78 C yielding 0.60 g (27 %) of a pure brown oil (at room temperature). Anal. Calcd. forC17H28FeN3P:C,56.5;H,7.8;N,11;6;Fe,15.4. Found: C,56.6;H,7.6;N,11.3 [ 1 -N, N-d meth y am n o meth yl-2-((N, N-d meth yl)-2,5-d aza-1 -p h osph ol anyl)]ferrocene (3) A solution of BuLi in hexane 2.35 M (2.40 ml, 5.58 mmol) was added dropwise to a stirred suspension of N,N-dimethylaminomethylferrocene (1.36 g, 5.58 mmol) in diethylether (5 ml) at 0 C. Stirring was maintained until the precipitate of o-lithioamine appeared.
[H2CN(CH3)PN(CH3)CH2]CI (0.85 g, 5.58 mmol) was added to the lithioamine and the resultant mixture was heated under reflux for I hr. The white solid (LiCI) was filtered off and the solvent was eliminated under vaccum. The solid obtained was recrystallized from pentane yielding 0.60 g (27 %) of pure orange crystals.Mp: 84 C. Anal. Calcd. for C17H26FeN3P: C,56.8;H,7.3;N,11.7;Fe,15.5. Found" C,56.9;H,8.0;N,11.2;Fe,14.6 (4) Synthesis of Gold(I) Complexes ( 1 -N, N-d methyl ami n omethyl-2-ch orog old(I)d phen yl ph osph n o)ferrocen e A solution of 1 (0.163 g, 0.382 mmol)in methylene chloride (20 ml) was added to a solution of [(tht)AuCI] (0.123 g, 0.382 mmol) in the same solvent (20 ml) at room temperature. Stirring was maintained for two hours and the solvent was then evaporated. The orange residue was recrystallized from toluene. Yield (0.186 g, 73 %). Mp: 206 C. Anal Two equivalents of methyl iodide was added to a solution of 4 (0.106 g, 0.160 mmol) in acetonitrile (20 ml) at room temperature. On addition of diethylether an orange powder was formed. This powder does not melt but darkened and decomposed from 202 C. Yield (0.102 g, 71%). ( 1 -N, N-d meth yl am n omethyl-2-bro m og old(I)d ph enyl p h osp h no)-ferrocen e (6) A solution of I=t4NBr (0.062 g, 0.296 mmol)in ethanol (20 ml) was added to a slurry of 4 (0.098 g, 0.148 mmol) in the same solvent (20 ml) at -60 C. Stirring was maintained for a few hours during which the solution warmed slowly to room temperature. The orange solid formed was isolated and washed with ethanol and diethylether. It did not melt but darkened and decomposed from 206 C. Yield (0.085 g, 82 %). Anal (7) This compound was prepared from 2 (0.608 g, 1.680 mmol) and [(tht)AuCI] (0.540 g, 1.608 mmol) by a procedure similar to that given for 4. The orange solid was recrystallized from pentaneo Yield (0.180 g, 20 %). Mp: 116 C. Anal (8) This compound was prepared from 3 (0.494 g, 1.374 mmol) and [(tht)AuCI] (0.441 g, 1.374 mmol) by a procedure similar to that given for 4. The orange solid was recrystallized from toluene/pentane. Yield (0.183 g, 22 %). Mp: 150 C. Anal Orange crystals of 4 and 7 were grown from toluene and pentane solutions, respectively. They were mounted on an Enraf-Nonius CAD4 diffractometer. The pertinent crystallographic data are given in Table I. The unit cells were determined from 25 randomly selected reflections (8<6<18).
The intensity data were collected at room temperature with o/20 scan up to emax 25. The Enraf-Nonius Molen package was used for preliminary treatement of data. The intensities were corrected for standard Lorentz and polarisation effects and empirical absorption correction applied for both crystals. The structures were solved and refined by conventional three-dimensional Patterson, difference Fourier, and full-matrix least-squares methods by using the SHELX76 package. All non-hydrogen atoms in both structures were refined with anisotropic temperature factors. The hydrogen atoms were placed in calculated positions and were given riding on the atoms bearing them. The highest peaks in the final difference map are in close proximity to the gold atoms. Final atomic coordinates are given in Tables II and II1. Tables of hydrogen atom

Biological Studies
Evaluation of potential anti-tumour activity was conducted against two human tumour cell lines, the SW620 colon carcinoma 9 and the HT1376 bladder caminoma ] The cell lines were obtained from the ECACC. The cells were seeded onto 96-well microtitre plates at a concentration 3letal-Based Drugs LE. Nifant ev and S. P. Fricker of 5 x 104 105 coils ml "1 Altar a 24 hour pro-incubation poriod, tho coils woro troatod with tost ompound for 4 hours at conontrations of 0 200 pg ml-1. Tho compound was than roplacod with frosh modium and tho olls woro inubatod for a furthor 72 hours. Gall growth was assayod using sulforhodamino B. Goll survival (%) was alculatod rolativo to untroatod control olls. Doso/survival urvos woro onstructod from this data and tho IGs0 (concontration of compound giving 50 % survival) alulatod. Beq values are defined as" 8/3n2(U11 + U22 + U33). Yol. 2, No. 6, 1995 Synthesis, Structure of Nitrogen-Containing Phosphinogold(I) Ferrocenes The sulforhodamine B(SRB) assay was performed as previously described 11. The cells were fixed with 200 pl 10 % trichloroacetic acid for 30 minutes at 4C, washed five times in tap water and stained with 100 pl 0.1% SRB in 1% acetic acid for 15 minutes. The cells were washed four times with 1% acetic acid and air dried. The stain was solubilised in 200 pl 10 mM unbuffered Tris base. Absorbance was measured at 540 nm using a microtitre plate reader. Compounds 1,2 and 3 contain both "soft" (P) and "hard" (N)ligands. It is well known that the former site is normally prefered for coordination by a "soft" metal like gold(I). This has been verified when the gold salt [(tht)AuCI] was reacted with [1-N,N-dimethylaminomthyl-2diphenylphosphino]ferrocene (1) forming the bimetallic derivative 4 (Scheme 1) Scheme 1.

Fe
Regarding the positive antitumoral response of 1 before coordination and the inversion in selectivity after coordination with gold (see after), we have decided to synthesize other Nand Pcontaining derivatives by two different approaches consisting of modification of (1-N,Ndimethylaminomethyl-2-chlorogold(I)diphenylphosphino)ferrocene (4) or by using new starting phosphines.
Then two new complexes were synthesized from 4. The reaction of two equivalents of methyl iodide with 4 produced the corresponding salt (5) (the substitution of the chlorine atom by iodine was observed). The bromide 6 was obtained by reacting tetraethylammonium bromide with the chloride 4 (Scheme 1).
The second approach (Scheme 2) allowed the two metallophosphines 2 and 3 to be synthesized by reacting (N,N-dimethylaminomethyl)ferrocene with BuLi and [CIP(NMe2)2] for 2 or BuLl and [H2CN(CH3)PN(CH3)CH2]CI for 3. They represent two of the rare phosphorylated metallocenes containing P(III)-N bond 12 which can be coordinated to gold. The unexpected presence of the ethoxy group in 7 is due to the ethanol (0.3 %) used as stabilizer in the commercial CH2CI2 and the transformation shows evidence for the lability of one of the nitrogen-phosphorus bonds. The 1 H NMR spectrum in particular exhibits one triplet at 1 ppm (OCH2CH3) and one large multiplet (3.42-3.75 ppm)corresponding to the two diastereotopic protons of OCH2CH3 multicoupled to each other, with the phosphorus atom and methyl protons. We have not tried to discard ethanol from methylene chloride.
The 1 H and 13 C NMR spectra of each compound reflects the diastereotopy of the PNMe2 methyl substituents, the phenyl protons, and the methylene protons of CH2NMe2. In contrast, the methyl part of CH2NMe2 consists of one singlet. The deshielding observed for one proton of the methylene group of CH2NMe2 is probably due to an agostic bond involving the gold atom.
Interactions of this type have already been mentioned in the literature 3, 4 for other electrophilic metals and our finding seems to be one of the first examples reported for gold 5.
The very large deshielding of the 31p signal for a bimetallic complex compared to the corresponding Aufree one is evidence for the coordination to gold.
[Fc(CH2 N M e2)(PP h2AuCI)] (4) Figure I shows an ORTEP plot of one of the two enantiomers and Table IV contains selected bond lengths and bond angles. The cyclopentadienyl rings are eclipsed and parallel. The phosphorus and nitrogen atoms deviate from the best plane of substituted C1-C5 ring towards the iron atom by 0.15 and 1.13 , respectively, while the C6 and gold atoms deviate from this plane by 0.03 and 1.09 . to the other side with respect to the iron atom. The nitrogen atom has a pyramidal geometry. The positions of methyl groups (C7 and C8) indicate the orientation of the nitrogen electron lone pair in the direction of gold, which might suggest an intramolecular Au-N interaction. However, such an interaction is excluded on the basis of extended H0ckel MO calculations which show a slightly negative value of Au-N overlap. Thus, the gold atom is not perturbed by secondary interaction and exhibits an essentially linear geometry (Table IV). The Au-P and Au-CI distances are close to the values observed in compounds with P-Au-CI linkage 16"19.   (2) 111.6(9) 111(1) 111 (1) 111 (1) [F c(CH2 N M e2)(P(N Me2)(OCH2 C H3)(AuCI)] (7) The presence of the ethoxy group has been confirmed by X-ray diffraction. A perspective drawing of one of the four isomers is represented in Figure 2. Selected bond lengths and angles are given in Table V. The cyclopentadienyl rings are essentially eclipsed and parallel. Contrary to the structure of 4, the phosphorus and nitrogen atoms deviate (0.10 and 1.21 , respectively) from the best plane of C6-C10 ring to the opposite side with respect to the iron atom, while the C11 and Au atoms (deviations of 0.06 and 1.11 , respectively) are located between the planes of the Cp rings. Similarly to the structure of 4, the nitrogen atom of the CH2NMe2 substituent (N1) has a pyramidal geometry, while the N2 bound to the phosphorus is planar. The sum of the three angles around N2 atom is equal to 359.9(9) .T his indicates the presence of d(P)-p(N2) interaction. The P-N2 bond length of 1.640(9)A is effectively intermediate between the values observed for phosphorus-nitrogen single and double bond2. On the other hand the P-O distance of 1.605(6)/ , corresponds very well to the P-O single bond distance observed in P-O-C organic and inorganic linkages 2o,2 ]. Even if the lone pair of the N1 atom roughly heads for the gold atom, the Au-N1 distance of 3.70(1)/, is longer than in 4. Thus, we did not look for the existence of Au-N1 interaction. As expected, the P-Au-CI bonding is linear. The compounds were evaluated for potential anti-tumour activity using an in vitro screen composed of two human tumour cell lines, SW620 and HT1376. This approach has been adopted as in vivo screens for anti-tumour agents using transplantable murine tumours such as the L1210 and P388 leukaemias have had limited success in identifying drugs with activity against human solid tumours22. The two cell lines exhibit differential chemosensitivity towards the metal anti-tumour drug cisplatin with typical IC5o values of 9.3 +/-1.5/Jg m1-1 for HT1376 and 29.5 + 3.1/Jg ml "1 for $W620 (errors represent standard error of the mean, n = 9). Differential cytotoxicity is used as an indicator of anti-tumour activity whereas similar toxicity against both cell lines is taken as an indicator of non-specific cytotoxicity 23 The ratio of the highest IC50 to the lowest IC50 is a useful measure of differential cytotoxicity, this ratio is 3.2 for cisplatin.
The results for seven compounds are shown in Table VI.  LE. Nifant'ev and S. P. Fricker Goordination of gold to compound 1 to givo compound 4 rosults in a slight ovorall ineroaso in eytotoxieity with an apparont invorsion of tho difforontial eytotoxieity in favour of tho $W620 coil line. The differential however is not as great as that seen with cisplatin (ratio of highest IC50 lowest IC50 is 2.4 for compound 1 and 1.8 for compound 4). Compound 6, the bromo analogue of compound 4, shows both an enhanced cytotoxicity and differential activity (ratio of highest IC50 lowest IC5o is 7.3) compared with compound 1. The dimethylamino methyliodide salt, compound 5, is less cytotoxic than compound I but with an enhanced differential cytotoxicity (ratio of highest IC50 :lowest IC50 is 4.5). The reduction in cytotoxicity is probably due to this being a charged compound, whereas the other complexes are neutral. This would reduce its ability to cross cell membranes and reach a putative intracellular target.
Complex 3, the diazaphospholanyl fermcene, is not cytotoxic, unlike the diphenylphosphino ferrocene, compound 1. Interestingly, when coordinated to give the chlorogold complex, compound 8, there is a dramatic increase in cytotoxicity. This is associated with an increase in differential activity with an IC50 of 1.3 pg ml "1 for HT1376 and 19.6 pg ml "1 for SW620 giving a ratio of highest IC50 :lowest IC50 of 19.1. This compound is also more toxic towards the HT1376 cell line than compound 4, the chlorogold diphenylphosphine. Compound 7, the chlorogold dimethylaminoethoxyphosphine, is similarly cytotoxic towards the HT1376 cell line with a ratio of highest IC50 :lowest IC50 of 4.9.
These data indicate that the potency and differential cytotoxicity of phosphinogold(I) ferrocenes can be changed by chemical modification.