Di-n-Butyl-, Tri-n-Butyl- and Triphenyltin dl-Terebates: Synthesis, Characterization and In Vitro Antitumour Activity

Di-n-butyltin, tri-n-butyltin and triphenyltin terebates were screened against several human tumour cell lines and found comparably or more active than carboplatin, cis-platin, 5-fluorouracil, methotrexate and doxorubicin, some reference compounds used clinically.


Introduction
A large number of di-and triorganotin carboxylates exhibit interesting in vitro antitumour activities against human tumour cell lines(I).
Many substituted triphenyltin benzoates (3) are even more active In contrast, tri-n-butyltin difluorobenzoates(4) are less active than the corresponding trilhenyltin and di-n-butyltin compounds.
A series of tri-and diorganotin steroidcarboxylates were recently screened against seven human tumour cell lines, MCF-7 and EVSA-T, two breast cancers, WiDr, a colon cancer, IGROV, an ovarian cancer, M19 MEL, a melanoma, A498, a renal cancer, and H226, a non small cell lung cancer. The in vitro antitumour activities of the di-n-butyltin compound lie between those of 5-fluorouracil and doxorubicin. The activities of the triorganotin compounds are comparable to those of methotrexate or doxorubicin(5).
In the frame of our interest in biologically relevant organotin carboxylates, we report the synthesis and antitumour properties of di-and triorganotin derivatives of dl-terebic acid.
They were characterized by 1H NMR, through multiplet patterns and resonance integrals, by 13C NMR, supported by DEPT experiments, by 17Sn NMR and by 119Sn M6ssbauer spectroscopy (see Experimental Section). The 1j(13C-119/117Sn) coupling constants (567/542 Hz) and the 117Sn chemical shift (-131.6 ppm) of compound 1 lie in the ranges characteristic for di-n-butyltin dibenzoates(6) and -disalicylates(7)(8) displaying a pseudo-octahedral or alternatively trapezoidal bipyramidal structure with two n-butyl groups in axial positions. The carboxylates coordinate the tin atom in the bidentate mode in equatorial positions with two adjacent short and two longer Sn-O bonds. All other NMR resonances are, accordingly, as expected for this type of structure. Vol. 4, No. 4, 1997 DI-n-Butyl-and Triphenyltin dl-Terebates: Synthesis, Characterization and In Vitro Antiturnour Activity The 1j(13C-119/117Sn) coupling constants (354/338 Hz) and the 117Sn chemical shift (+125.4 ppm) of compound 2 are compatible with a monomeric tetrahedral structure found recently for a series of tri-n-butyl fluorobenzoates in solution(4), in agreement with earlier coupling data described by Holecek and Lycka(9). The 1j(13C-119/117Sn) coupling constants (648/618 Hz) and the 117Sn chemical shift (-97.6 ppm) of compound 3 are again in agreement with a tetrahedral monomeric structure(2)(10)(11). Table clearly shows that the compounds are comparably to more active than methotrexate and doxorubicin in vitro against almost all cell lines. The very high activity of compounds 2 and 3 against EVSA-T as well as of compound 2 in general should be outlined.

Experimental part
Syntheses Compounds 1 to 3 were prepared by adding 5 mmole of di-n-butyltin oxide, tri-n-butyltin acetate or triphenyltin hydroxide, respectively, to a solution of 10 mmole, 5 mmole or 5 mmole of terebic acid in 150 cm3 toluene and 50 cm3 ethanol. After refluxing for 6 h, distilling off the ternary azeotrope

Instruments
All NMR spectra were recorded on a Bruker AC250 instrument, using a QNP probe tuned at 250.13, 62.93 and 93.28 MHz for 1H, 13C and 19Sn nuclei, respectively. MSssbauer spectra were obtained as described previously(8).

In vitro tests
The following human tumor cell lines were used: MCF7 Breast cancer-EVSA-T Breast cancer WiDr Colon cancer IGROV Ovarian cancer-M19 MEL Melanoma A498 Renal cancer H226 Non small cell lung cancer. Vol. 4, No. 4, 1997 DI-n-Butyl-and Triphenyltin dl-Terebates: Synthesis, Characterization and In Vitro Antitumour Activity MCF7 is estrogen receptor ER+/progesterone receptor PgR+ and EVSA-T is ER-/PgR-. Cell lines WIDR M19 MEL, A498, IGROV and H226 belong to the currently used anti-cancer screening panei of the National Cancer Institute, USA (13). The in vitro cytotoxicity of the compounds was determined using SRB (14) as a cell viability test. 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:md streptomycin 100 g/ml. This results in a highest concentration of 59523 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 terrninated by washing the plate twice with PBS. Subsequently the cells were fixed with 10% trichloroacetic acid in PBS and placed at 4C for one hour. 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 acid to remove the unbound stain. The plates were air dried and the bound stain was dissolved in 150 !1 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 ID50 value by use of Deltasoft 3 software. All raw data and the mastercopy of this report have been filed in the archives of the laboratory of medical oncology of the Rotterdam Academical hospital.