Compositions comprising a metal source, dithiocarbamate and cyclodextrin

Abstract

The present invention provides a novel class of dithiocarbamate-metal complexes and their uses in medicine. Also provided by the invention are combinations and pharmaceutical compositions, comprising a dithiocarbamate (or thiuram disulphide) such as disulfiram and cyclodextrin, with a source of a heavy metal. Surprisingly, the inventors found a synergistic potentiation of the anti-tumor effect, when a dithiocarbamate/heavy metal mixture was combined with a cyclodextrin. The compounds and combination of the invention are particularly useful in the treatment of tumor diseases, and other disorders. Provided are the compounds, combination, pharmaceutical compositions and kits, as well as methods for the preparation of the combinations of the invention.

Claims

1. A method for treating small cell lung cancer (SCLC) in a subject, comprising: administering an effective amount of a compound to the subject, the compound being selected from a complex according to the following formula (I): ##STR00035## wherein the R.sub.1 and R.sub.2 are the same or different, or are connected to form a N-heterocyclic 3- to 6-membered ring, and are selected from hydrogen, and unsubstituted or substituted C.sub.1 to C.sub.10 straight, branched or cyclic alkyl, unsubstituted or substituted C.sub.1 to C.sub.10 straight, branched or cyclic akenyl, unsubstituted or substituted aryl, and unsubstituted or substituted heteroaryl; and wherein M is Au; or a diastereomer, enantiomer or a pharmaceutical acceptable salt thereof.

2. The method according to claim 1, wherein the compound is selected from any one of the following compounds: ##STR00036## ##STR00037##

3. The method according to claim 1, wherein the treatment comprises administration of the compound together with a cyclodextrin.

4. The method according to claim 1, wherein the compound according to claim 1 is administered as a pharmaceutical composition together with a cyclodextrin and a pharmaceutical acceptable carrier and/or excipient.

5. A method for treating small cell lung cancer (SCLC) in a subject, comprising: administering an effective amount of a compound to the subject, the compound having any one of the following formulas: ##STR00038## or a pharmaceutically acceptable salt thereof.

Description

(1) The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the example section either a , or . is used as decimal mark. In the Figures:

(2) FIG. 1: The effect of cyclodextrin formulation of disulfiram on the anti-tumor potency of metals.

(3) FIG. 2: Synergistic anti-tumor effect of aurothiomalate/disulfiram with cyclodextrin.

(4) FIG. 3: A: Normalized IC50ies were plotted in an isobologram; B: IC50ies of disulfiram were plotted against IC50ies of aurothiomalate in a logarithmic scale; C: Combination index (CI)) was plotted against the ratio of aurothiomalate/disulfiram.

(5) FIG. 4: A: Spectrum of anti-tumor activity of aurothiomalate/disulfiram in cyclodextrin formulation: The IC50 concentration of aurothiomalate was plotted against the corresponding concentration of disulfiram in log scale for each tested cell for each tested aurothiomalate/disulfiram ratio. Curved line indicates zone of additivity effects, calculated from the mean of IC50ies of single treatments from all tested cells. B: Selectivity of aurothiomalate/disulfiram in cyclodextrin formulation for T-cell leukemia/lymphoma cells. IC50ies of T-cell Leukemia/lymphoma compared to normal human PBL and normal human fibroblast cells; C: Selectivity of aurothiomalate/disulfiram in cyclodextrin formulation for SCLC lines. IC50ies of SCLC compared to normal human PBL and normal human fibroblast cells D: Comparison of Combination Index of aurothiomalate/disulfiram at various ratios in T-cell Leukemia/lymphoma and normal human cells (PBLs and skin fibroblasts). Normalized IC50: IC50ies were normalized to the IC50ies of single drugs by dividing IC50 of either aurothiomalate or disulfiram through IC50 of each individual drug combination. CI=Normalized IC50 of aurothiomalate concentration+normalized IC50 disulfiram concentration. Mean CI: Mean values+/standard deviation were calculated from CIs of either all T-cell leukemias or all normal cells.

(6) FIG. 5: Factor of resistance (IC50 of Oxaliplatin treatment surviving HT29 cells/IC50 of naive HT29 cells or normal human fibroblasts) for Vincristin, Oxaliplatin, 5-FU, Doxorubicin and aurothiomalate/disulfiram in cyclodextrin.

(7) FIG. 6: A: shows IC50 values for tested compounds in Raji and Jurkat cell lines. B: shows the selectivity factor of compounds tested.

(8) FIG. 7: shows IC50 values of tested compounds in various SCLC, NSCLC and HaCat cells. Also shown is the selectivity of tested compounds for SCLC.

(9) FIG. 8: shows the relative selectivity and resistance of DKFZ-00608 and Cisplatin in various SCLC cell lines.

(10) FIG. 9: shows anti-tumor activity of DKFZ-00608. A: tumor free animals over time; B: mean tumor size in animals; C: number of surviving animals.

EXAMPLES

Example 1: Cyclodextrin Stimulates the Anti-Tumor Effect of Combinations of Disulfiram with Various Heavy Metal Salts

(11) Jurkat T-cell leukemia cells were seeded in 96 well plates (310.sup.4/well). 24 h later cells were treated with disulfiram+metal (ratio 1:1) in the presence or absence of hydroxypropyl-beta-cyclodextrin at a 30 fold molar ratio at various concentrations (10, 3.33, 1.11, 0.37, 0.123, 0.041, 0.014, 0.0 M). In addition, cells were treated with cyclodextrin alone (1500, 500, 166.7, 55.56, 18.5, 6.17, 0.0 M). All concentrations were tested in triplicate. After incubation at 37 C. for 72 h, Cell-titer-blue (Promega) was added (10 l/well). After incubation at 37 C. for 4 h, fluorescence (excitation filter: 550 nm, emission filter 590 nm) was determined in an Optima ELISA reader. Mean values were calculated for each triplicate and IC50ies were calculated from non-fitted dose response curves.

(12) Normalized IC50 concentrations of metals were calculated for each metal combination by dividing ICies (in the presence or absence of cyclodextrin) through the IC50 of cells treated with disulfiram+metal in the absence of cyclodextrin.

(13) Normalized IC50 of cyclodextrin was determined for each metal combination by dividing the actual IC50 through the IC50 of cyclodextrin alone.

(14) Combination index (CI) was calculated by the addition of Normalized IC of cyclodextrin and Normalized IC50 concentrations of metals.

(15) Results as Displayed in FIG. 1:

(16) For all three metals (Au, Pt and Cu) in the presence of cyclodextrin a normalized IC50 of <1 was found.

(17) The normalized IC50 for the concentration of metals in the absence of cyclodextrin is according to the above definition.

(18) A normalized IC50 of 1 in the presence of cyclodextrin would indicate that cyclodextrin has no effect.

(19) A normalized IC50 of >1 in the presence of cyclodextrin would indicate that cyclodextrin has an antagonistic effect on the anti-tumor activity.

(20) A normalized IC50 of <1 in the presence of cyclodextrin indicates that cyclodextrin enhances the anti-tumor effect.

(21) Furthermore, a CI of <1 was found for all metals (Cu, Pt and Au). This indicates a synergistic interaction between cyclodextrin and all metal/disulfiram combinations.

Example 2: Synergistic Anti-Tumor Effect of Cyclodextrin with Disulfiram/Aurothiomalate

(22) 10 different human cancer cell lines were seeded in 96 well plates in RPMI medium, supplemented with 10% FCS and 1 Pen/Strep at a density of 210.sup.4 cells/well. Disulfiram and aurothiomalate were added at a fixed ratio of 1:1 in the presence of variable concentrations of hydroxypropyl-beta-cyclodextrin and aurothiomalate+disulfiram (ratios aurothiomalate+disulfiram/hydroxypropyl-beta-cyclodextrin: 1:0.037-1:27) in various dilutions (1-2.sup.6). For normalization, aurothiomalate+disulfiram and hydroxypropyl-beta-cyclodextrin were tested alone in various dilutions (1-2.sup.6). All concentrations were applied in triplicate. After incubation at 37 C. for 72 h Cell-titer-blue (Promega) was added (10 l/well). After incubation at 37 C. for 4 h fluorescence (excitation filter: 550 nm, emission filter 590 nm) was determined in an Optima ELISA reader. Mean values were calculated for each treatment and IC50ies were calculated from non-fitted dose response curves.

(23) Normalized IC50: IC50ies were normalized to the IC50ies of single drugs by dividing the IC50ies of combinations through either the IC50 of aurothiomalate+disulfiram, or the IC50 of hydroxypropyl-beta-cyclodextrin.

(24) CI=Normalized IC50 of aurothiomalate/disulfiram concentration+normalized IC50 of hydroxypropyl-beta-cyclodextrin concentration.

(25) Mean CI: Mean values+standard deviation were calculated from CIs of all tumor lines.

(26) Results as Shown in FIG. 2:

(27) Surprisingly, the combination of disulfiram+aurothiomalate with cyclodextrin resulted in a synergistic increase of the anti-tumor effect at a ratio of disulfiram+aurothiomalate/cyclodextrin of 1:3, 1:9 and 1:27. The difference to a hypothetic additive effect (CI=1) is significant (Student's T-test (P<110.sup.9, 110.sup.9, 110.sup.3 respectively).

Example 3: Synergistic Anti-Tumor Effect of Disulfiram and Aurothiomalate in the Presence of Cyclodextrin

(28) Human T-cell leukemia Jurkat cells were seeded in 96 well plates in RPMI medium (serum free) at a density of 610.sup.4 cells/well. Disulfiram and aurothiomalate were added in the presence of hydroxypropyl-beta-cyclodextrin (30 fold concentration of disulfiram concentration) (at various ratios of aurothiomalate/disulfiram (1:4.sup.8-1:4.sup.8) in various dilutions (1-4.sup.6). All concentrations were applied in triplicate. After incubation at 37 C. for 72 h, Cell-titer-blue (Promega) was added (10 l/well). After incubation at 37 C. for 4 h, fluorescence (excitation filter: 550 nm, emission filter 590 nm) was determined in an Optima ELISA reader. Mean values were calculated for each triplicate and IC50ies were calculated from non-fitted dose response curves. IC50ies were normalized to the IC50ies of single drugs.

(29) Results as Shown in FIG. 3A:

(30) The line of additive effect indicates the IC50ies that would be expected, if both drugs acted additive. Data points below this line indicate synergism. Surprisingly, all data points are very close to the x and y axis. This indicates a very high degree of synergism over a broad dose range. In order to enhance resolution of the graphical presentation, the graph was redrawn and axis were transformed to a logarithmic scale (see FIG. 3B)

(31) Results as Shown in FIG. 3B:

(32) Data points located under and left of the line of additive effect indicate synergisms at this concentration of both drugs. Surprisingly, synergism was observed over a broad concentration range (500-1250 fold) of both compounds: aurothiomalate: Between 80 nM and 100 M (1250 fold). Disulfiram: Between 20 nM and 10 M (500 fold).

(33) Results as Shown in FIG. 3C:

(34) A CI of <1 indicates synergism. Furthermore, the CI indicates the factor by which the total dose of the combined drugs can be reduced as compared to the single drugs.

(35) Surprisingly, at a ratio of aurothiomalate/disulfiram between 0.06 and 16 (>250 fold dose range) the CI was below 0.1, indicating a more than 10 fold increased efficiency. At the optimum ratio 0.25 a CI of 0.012 was found, indicating a 83 fold increase in activity.

(36) Unexpectedly, in comparison to other cases of synergism, the effect was very robust in that a synergistic effect >10 was observed over 5 different ratios of drug combination.

Example 4: Spectrum of Anti-Tumor Activity of Aurothiomalate/Disulfiram in Cyclodextrin Formulation

(37) A panel of 35 human cancer cell lines (obtained from ATCC) and 2 normal cell cultures (human peripheral blood lymphocytes (PBL, obtained from Promocell, Heidelberg) and normal human skin fibroblast cells (obtained from G. Darai, University of Heidelberg)) were seeded in 96 well plates in RPMI medium supplemented with 10% fetal calf serum and 1% antibiotics (penicillin/streptomycin) at a density of 210.sup.4 cells/well. 24 h later, disulfiram and aurothiomalate were added in the presence of hydroxypropyl-beta-cyclodextrin (30 fold disulfiram concentration) (at various ratios aurothiomalate/disulfiram: 1:4.sup.3-1:4.sup.4) in various dilutions (1-2.sup.7). All concentrations were applied in triplicate. After incubation at 37 C. for 72 h, Cell-titer-blue (Promega) was added (10 l/well). After incubation at 37 C. for 4 h, fluorescence (excitation filter: 550 nm, emission filter 590 nm) was determined in an ELISA reader (Optima, BMG Labtec). Mean values were calculated for each treatment and IC50ies were calculated from non-fitted dose response curves.

(38) Results as Shown in FIG. 4A:

(39) All data points are located left below the Additive Effect Line. This indicates, that synergism was obtained at all disulfiram/aurothiomalate ratios in cyclodextrin formulation in all tested cells.

(40) The distance of data points from the Additive Effect Line was variable for different cell lines. The distance to the Additive Effect Line indicates the degree of synergism. The highest distance and thus the best synergism was found in T-cell leukemia cells and small cell lung cancer cells, the lowest in HeLa cells.

(41) Synergisms between disulfiram and aurothiomalate can be observed in cyclodextrin formulation in all tested cells. This effect differs from one cell line to the other up to 4 orders of magnitude.

(42) Results as Shown in FIG. 4B:

(43) The center of the T-cell lymphoma/leukemia data point cloud (0.14/0.1 M aurothiomalate/disulfiram (mean values of aurothiomalate or disulfiram concentrations of all data points of the cloud)) was found to be ca. 100-fold lower in both dimensions (aurothiomalate concentration and disulfiram concentration) than the center of the normal cell cloud (4/10 M aurothiomalate/disulfiram (mean values of aurothiomalate or disulfiram concentrations of all data points of the cloud)).

(44) Surprisingly, all 3 T-cell lymphoma/leukemia cell lines were approximately 100 fold more sensitive to cyclodextrin formulated aurothiomalate/disulfiram treatment than normal cells.

(45) Results as Shown in FIG. 4C:

(46) The center of the small cell lung cancer cell data point cloud (0.05/0.05 M aurothiomalate/disulfiram (mean values of aurothiomalate or disulfiram concentrations of all data points of the cloud)) was found to be ca. 50-fold lower in both dimensions (aurothiomalate concentration and disulfiram concentration) than the center of the normal cell cloud (4/10 M aurothiomalate/disulfiram (mean values of aurothiomalate or disulfiram concentrations of all data points of the cloud)).

(47) Surprisingly, all 3 SCLC cell lines were approximately 100 fold more sensitive to cyclodextrin formulated aurothiomalate/disulfiram treatment than normal cells.

(48) Results as Shown in FIG. 4D:

(49) CI can be used for quantitative analysis of synergism. A CI of <0.1 indicates that the total amount of combined drugs needed to kill 50% of cells is at least 10-fold lower than when single drugs are used separately

(50) A CI of <0.1 can be considered as biologically significant.

(51) A CI of <0.01 can be considered as biologically highly significant.

(52) In normal cells mean CIs <0.1 were found at disulfiram/aurothiomalate ratios between 2-0.25.

(53) In T-cell-lymphoma/leukemia cells mean CIs <0.01 were found at disulfiram/aurothiomalate ratios between 16 and 0.125.

(54) The difference of CI, found in T-cell-lymphoma/leukemia cells and normal cells was statistically significant at disulfiram/aurothiomalate ratios of 8 and 16 (Student's T-test, p=0.02 and 0.039 respectively.

(55) Surprisingly, the synergism between disulfiram and aurothiomalate in cyclodextrin formulation is approximately 10 fold higher in T-cell-lymphoma/leukemia cells and in SCLC cells as compared to normal cells.

(56) TABLE-US-00001 TABLE 1 Tumor type specific effect of aurothiomalate/disulfiram in relation to all tested cell Difference to Mean Mean Standard all tested cells Tumor type realtive IC50 deviation significant at All tested cells 1.00 0.00 T-cell 0.09 0.02 T-test: p < 2 10.sup.4 lymphoma/leukemia Normal cells 0.99 0.38 Colon 0.85 0.40 Glioma 1.10 0.64 Ovary 2.42 1.42 SCLC 0.014 0.0073 T-test: p < 1 10.sup.6 NSCLC 2.19 0.75 Liver 1.39 Mamma 0.68 0.41 Pancreas 0.94 0.35 Cervix 2.63 0.90 Melanoma 1.23 0.68 Burkitt lymphoma 1.09 0.36 Non-T-cell leukemia 0.20 0.08 T-test: p < 2 10.sup.4 Mean IC50ies of all tested cells were calculated for each tested aurothiomalate/disulfiram ratio. Relative IC50ies were calculated by dividing IC50ies through Mean IC50 of all tested cells of the corresponding aurothiomalate/disulfiram ratio. Mean relative IC50ies were calculated for each cell line from all tested aurothiomalate/disulfiram ratios. Mean Mean relative IC50ies were calculated from all Mean relative IC50ies from each tumor type and from all human normal cells.

(57) Results as Shown in Table 1:

(58) The Mean Mean relative IC50ies of NSCLC, cervix carcinoma and mamma carcinoma were determined as >2.

(59) NSCLC, cervix carcinoma and mamma carcinoma cell lines tested here, are resistant to aurothiomalate/disulfiram treatment in cyclodextrin formulation.

(60) The Mean Mean relative IC50ies of T-cell lymphoma/leukemia, non-T-cell leukemia and SCLC were determined as 0.09, 0.2 and 0.37, respectively. The difference to all tested cells was significant (Student's T-test, p<210.sup.4).

(61) Surprisingly, the tested T-cell lymphoma/leukemia carcinoma, non-T-cell leukemia and SCLC cells are hypersensitive to aurothiomalate/disulfiram treatment in cyclodextrin formulation.

(62) The Mean Mean relative IC50ies of all other tumor cell types and normal human cells ranged between 0.68 and 1.23.

(63) The tested colon-, ovary-, liver-, mamma-, pancreas-tumors, melanomas, gliomas and Burkitt lymphomas are sensitive to aurothiomalate/disulfiram treatment in cyclodextrin formulation.

Example 5: Aurothiomalate/Disulfiram in Cyclodextrin Formulation Overcomes Resistance of Chemotherapy Surviving Tumor Cells

(64) 10 million HT29 human colon cancer cells were seeded in 175 ml Falcon flasks. 24 h later 10 M Oxaliplatin was added. After 48 h incubation at 37 C. fresh medium was added. 48 h later cells were trypsinized and seeded in 96 well plates at a density of 210.sup.4 cells per well. Human skin fibroblasts and untreated HT29 cells were seeded in 96 well plates at the same density. After 24 h incubation at 37 C., cells were treated in triplicates with 1:2 serial dilutions of Vincristin (starting concentration 1 g/ml), Oxaliplatin (starting concentration 50 g/ml), 5-FU (starting concentration 500 Doxorubicin (starting concentration 10 g/ml) and disulfiram/aurothiomalate+10 fold concentration of hydroxypropyl-beta-cyclodextrin in 2 M aurothiomalate. After incubation for 72 h at 37 C. 10.sub.11.1 Cell-titer-blue was added per well. After incubation at 37 C. for 4 h, fluorescence (excitation filter: 550 nm, emission filter 590 nm) was determined in an ELISA reader (Optima, BMG Labtec). Mean values were calculated for each treatment and IC50ies were calculated from non-fitted dose response curves.

(65) Calculation of Factor of Resistance:

(66) For each treatment, IC50ies in normal human cells and IC50 in Oxaliplatin treatment surviving cells were divided through the IC50 found with the same treatment in unselected HT29 cells.

(67) A factor of resistance >1 indicates resistance.

(68) A factor of resistance=1 indicates no resistance.

(69) A factor of resistance <1 indicates hypersensitivity. Hypersensitivity is obtained with drugs that selectively kill tumor cells resistant to standard chemotherapy.

(70) Results as Shown in FIG. 5:

(71) When cells which had not been treated before, were tested, all chemotherapeutic standard drugs (Vincristin, Oxaliplatin, 5-FU, Doxorubicin) show selectivity for colon cancer cells as compared with normal human fibroblasts. IC50ies in tumor cells are at least 100-fold lower than IC50ies in fibroblast.

(72) HT29 cells, which had survived Oxaliplatin treatment were resistant to all chemotherapeutic standard drugs (Vincristin, Oxaliplatin, 5-FU, Doxorubicin). The factor of resistance (IC50 in surviving cells/IC50 in untreated control cells) was found to be between >10 and >100.

(73) HT29 cells, which had survived Oxaliplatin treatment, were found to be as sensitive to aurothiomalate/disulfiram in cyclodextrin formulation as untreated HT29 cells. The factor of resistance was <1 (0.71).

(74) In contrast, normal human fibroblasts were found to be resistant to aurothiomalate/disulfiram in cyclodextrin formulation (factor of resistance >1000).

(75) Surprisingly, disulfiram/aurothiomalate in cyclodextrin displays no cross resistance with standard chemotherapeutic agents. Therefore, disulfiram/aurothiomalate offers itself as a salvage therapy by killing efficiently chemotherapy surviving cancer cells.

Example 6: Superior Selective Activity of Homoleptic Au-DTC Complexes on T-Cell Leukemia

(76) The anti-tumoral efficacy of a panel of 7 different Au complexes, ATM/Disulfiram, the standard therapeutic Cisplatin and well known antitumoral CuDTC (see Table 2) was tested on Jurkat human T-cell leukemia and Raji human B-cell lymphoma cells. Cells were seeded in 96 well plates in RPMI medium supplemented with 10% fetal calf serum and 1% antibiotics (penicillin/streptomycin) at a density of 310.sup.4 cells/well. 24 h later, test compound were added in the presence of sulfobutyl ether--cyclodextrin (30 fold disulfiram concentration) in various dilutions (100-0.01 M). All concentrations were applied in triplicate. After incubation at 37 C. for 72 h, Cell-titer-blue (Promega) was added (10 l/well). After incubation at 37 C. for 2-8 h, fluorescence (excitation filter: 550 nm, emission filter 590 nm) was determined in an ELISA reader (Optima, BMG Labtec). Mean values were calculated for each treatment and IC50ies were calculated from non-fitted dose response curves. The Factor of Selectivity for T-cell Leukemia was calculated by division of IC50 on Raji cells/IC50 on Jurkat cells. Results are shown in table 3 and FIGS. 6A and 6B.

(77) TABLE-US-00002 TABLE 2 tested compounds: Structure Name CAS # DKFZ-00608 66712-10-5 DKFZ-00609 DKFZ-00610 164363-66-0 DKFZ-00613 0 DKFZ-00614 DKFZ-00615 Auranofin 34031-32-8 AuL12 849802-92-2 MC3 Cisplatin 15663-27-1 CuDTC 13681-87-3 P-A-uDTC 889465-31

(78) TABLE-US-00003 TABLE 3 IC50 (M) of selected compounds of the Invention: Raji Jurkat selectivity for Jurkat Compound IC50 IC50 IC50 Raji/IC50 Jurkat AIM/Disulfiram 16.98 0.11 153.31 DKFZ-00608 32.66 0.05 635.70 DKFZ-00609 26.80 0.10 275.09 DKFZ-00610 24.57 0.28 86.67 AuL12 51.90 25.64 2.02 P-AuDTC 0.51 0.07 6.87 CuDTC 0.02 0.01 2.62 MC3 0.13 0.16 0.80 Cisplatin 2.72 0.61 4.45

(79) In conclusion, the Au-dithiocarbamtes DKFZ-00608, DKFZ-00610 and DKFZ-00609 are highly active in Jurkat cells. IC50 of 50, 280 and 100 nM were found. Similarly, the mixture of ATM and Disulfiram and Cyclodextrin displayed and IC.sub.50 of 110 nM. Further all Au-dithiocarbamtes DKFZ-00608, DKFZ-00610, DKFZ-00609 and the mixture of ATM and Disulfiram and Cyclodextrin were less active in Raji cells. A Factor of Selectivity Results for T-cell Leukemia of 635, 86.67, 275 and 153 was found respectively. All other complexes displayed a Factor of Selectivity for T-cell Leukemia of <10.

Example 7: Superior Selective Activity of Homoleptic Au DTC Complexes on SCLC Cells

(80) The anti-tumoral efficacy of a panel of 6 different Au complexes and the standard therapeutic of SCLC, Cisplatin was tested on 5 different human SCLC, three human NSCLC and the human non-tumorigenic cell line HaCat (results in FIG. 7). Cells were seeded in 96 well plates in RPMI medium supplemented with 10% fetal calf serum and 1% antibiotics (penicillin/streptomycin) at a density of 3104 cells/well. 24 h later, test compound were added in the presence of sulfobutyl ether--cyclodextrin (30 fold disulfiram concentration) in various dilutions (100-0.01 M). All concentrations were applied in triplicate. After incubation at 37 C. for 72 h, Cell-titer-blue (Promega) was added (10 l/well). After incubation at 37 C. for 2-8 h, fluorescence (excitation filter: 550 nm, emission filter 590 nm) was determined in an ELISA reader (Optima, BMG Labtec). Mean values were calculated for each treatment and IC50ies were calculated from non-fitted dose response curves. Mean IC50ies+/standard deviation (SD) were calculated for each test compound for SCLC and NSCLC lines. The Mean Factor of Selectivity for SCLC versus NSCLC was calculated by division of Mean IC50 on NSCLC cells/Mean IC50 on SCLC cells. The Mean Factor of Selectivity for SCLC versus nontumorigenic cells was calculated by division of IC50 on HaCat cells/Mean IC50 on SCLC cells. Results are shown in FIG. 7.

(81) DKFZ-00608 was found to be the most active (lowest IC50) of all tested compounds. The IC50 was about 10 fold lower than that of all other tested non-AuDTC compounds in SCLC. For both AuDTC complexes a Mean Factor of Selectivity for SCLC versus NSCLC and a Mean Factor of Selectivity for SCLC versus non-tumorigenic cells of >100 was found. All other complexes showed lower selectivity (Mean Factor of Selectivity for SCLC versus NSCLC and Mean Factor of Selectivity for SCLC versus non-tumorigenic cells of <10.

Example 8: Lack of Cross Resistance of DKFZ-00608 with Cisplatin

(82) A panel of 5 human SCLC lines was tested for sensitivity to Cisplatin and DKFZ-00608. Cells were seeded in 96 well plates in RPMI medium supplemented with 10% fetal calf serum and 1% antibiotics (penicillin/streptomycin) at a density of 3104 cells/well. 24 h later, test compound were added in the presence of sulfobutyl ether--cyclodextrin (30 fold disulfiram concentration) in various dilutions (100-0.01 M). All concentrations were applied in triplicate. After incubation at 37 C. for 72 h, Cell-titer-blue (Promega) was added (10 l/well). After incubation at 37 C. for 2-8 h, fluorescence (excitation filter: 550 nm, emission filter 590 nm) was determined in an ELISA reader (Optima, BMG Labtec). Mean values were calculated for each treatment and IC50ies were calculated from non-fitted dose response curves. Relative IC50ies were calculated for each cell line by dividing the respective IC50/IC50 found in H209 cells. Results are shown in FIG. 8.

(83) For DKFZ-00608 IC50ies between 38 and 95 nM were found. Relative IC50ies were of low variability. They were determined to be between 0.48 and 1.18. For Cisplatin high variability was found. IC50ies were found between 0.40 and 10.2 M. Relative IC50ies were found between 0.4 and 10. Conclusion: There is no cross resistance between Cisplatin and DKFZ-00608 in SCLC cells.

Example 9: DKFZ-00608 Prevents Tumor Relapse after Cisplatin/Etoposide Treatment

(84) The inventors wanted to compare the efficacy of DKFZ-00608 on human SCLC tumors with standard therapy in vivo. Specifically the effect on tumor recurrence after successful treatment with Cisplatin/Etoposide, the major clinical complication in SCLC treatment, was of interest. Female nu/nu mice were injected with H209 SCLC cells (10106 cell/mouse in 0.1 ml isotonic salt solution). After growth to a size of 0.08-0.12 mm.sup.3 groups of 10 animals were treated as follows: Placebo daily for 9 weeks (60 mM sulfobutyl ether beta-cyclodextrin in isotonic salt solution). Cisplatin (3 mg/kg once/week)+Etoposide (7.5 mg/kg twice/week) for 3 weeks. Cisplatin (3 mg/kg once/week)+Etoposide (7.5 mg/kg twice/week) for 3 weeks. Followed by DKFZ-00608 in 60 mM sulfobutyl ether beta-cyclodextrin (15 mg/kg daily) for 6 weeks DKFZ-00608 in 60 mM sulfobutyl ether beta-cyclodextrin (15 mg/kg daily) for 6 weeks.

(85) Tumor sizes were determined 2 per week for the first 9 weeks, thereafter, once per week.

(86) Results are provided in FIG. 9A to 9C. All placebo tumors grew rapidly and all animals of this group had to be killed within 42 days. Cisplatin tumors regressed rapidly and after the end of treatment in 9/10 and 10/10 tumors were no longer palpable. Twenty days later in all animals re-growth of tumor was observed. When Cisplatin/Etoposide treatment was followed by DKFZ-00608 treatment reappearance of tumors could be prevented for at least 50 days. After 6 weeks treatment with DKFZ-00608, in 6 out of 10 animals no tumors were palpable. In 4 animals tumors had not completely regressed and resumed growth after termination of treatment, resulting in an increase of mean tumor size. After 82 days these tumors reached a size at which animals had to be euthanized. Only tumor free animals survived. Consequently, due to elimination of tumor bearing animals, the mean tumor size was falling again to 0 mm.sup.3. This explains the, upon first glance, paradox peak in the tumor size curve.

(87) Hence, DKFZ-00608 treatment can prevent tumor relapse after initially successful standard therapy.

Example 10: Synthesis of the Compounds of the Invention

Synthesis of Au-DTC Complexes

(88) ##STR00018##

(89) Where R and R are either connected resulting in a N-hetereocyclic ring system or linear/branched aliphatic carbon chains including hetereoatoms such as N, S, O, P, etc.

(90) Preparation of Lithium Dithiocarbamate Salts:

(91) The appropriate secondary amine was dissolved in dry THF and cooled to 78 C. An equimolar solution of n-butyllithium (2.5 M in hexane) was added dropwise to the solution and stirred for 15 minutes whereupon an equimolar amount of carbon disulfide was added. The resulting reaction mixture was stirred for 15 minutes at 78 C. and afterwards allowed to warm up to room temperature. After a total reaction time of 3 hours the solution was concentrated and dried under high vacuum over-night. The resulting off-white solid or oil (containing residues of THF) was used in the next step without further purification.

(92) Preparation of Homoleptic Gold(I) Dithiocarbamate Complexes:

(93) To an aqueous solution of aurothiomalate (ATM) or another gold(I) source dissolved in a suitable solvent, such as chloro(dimethyl sulfide)gold (I) in acetonitrile was added dropwise an ethanolic or aqueous solution of either an appropriate dithiocarbamate (dtc) salt (i.e. ammonia, alkali or alkaline earth metal salts, but preferably sodium or lithium) or an appropriate thiuram disulfide in a metal/dtc (or dithiuram) ratio of 1:1-2 at room temperature. While combining these solutions a yellow to orange colored precipitate formed. The suspension was stirred at room temperature overnight (or at least for 3 hours). Then the precipitate was purified by either filtering through a glass frit and subsequently washing with water, ethanol and diethylether or by centrifuging. With respect to the latter method the supernatant was taken away and the residue was resuspended in water and repeatedly centrifuged. The obtained solid was then dried under high vacuum overnight. If applicable this material was furtheron recrystallized using an appropriate organic solvent or solvent mixture preferably dimethylformamide, 1,2-dichloroethane or dichloromethane/hexane.

(94) ##STR00019##

(95) Lithium N-ethyl, N-isopropyldithiocarbamate was synthesized according to the general procedure using 8.26 mmol of N-ethyl, N-isopropyl amine (0.72 g, 1.0 ml, 1 eq.), 8.26 mmol carbon disulfide (0.63 g, 0.5 ml, 1 eq.) and 8.26 mmol n-butyllithium (2.5 m in hexane, 4.67 ml, 1 eq.) in approximately 20.0 ml of dry THF. The product was obtained as an off-white solid in quantitative yield

(96) .sup.1H NMR (400 MHz, D.sub.2O) 5.86 (hept, J=6.8 Hz, 1H), 3.90 (q, J=7.1 Hz, 2H), 1.28 (t, J=7.0 Hz, 3H), 1.20 (d, J=6.8 Hz, 6H).

(97) ##STR00020##

(98) Lithium N-ethyl, N-methyldithiocarbamate was synthesized according to the general procedure using 11.67 mmol of N-ethylmethyl amine (0.69 g, 1.0 ml, 1 eq.), 11.67 mmol carbon disulfide (0.89 g, 0.7 ml, 1 eq.) and 11.67 mmol n-butyllithium (2.5 m in hexane, 4.67 ml, 1 eq.) in approximately 50.0 ml of dry THF. The product was obtained as a reddish solid in quantitative yield.

(99) .sup.1H NMR (400 MHz, D.sub.2O) 4.09 (q, J=7.2 Hz, 2H), 3.45 (s, 3H), 1.21 (t, J=7.2 Hz, 3H).

(100) ##STR00021##

(101) Lithium azetidinedithiocarbamate was synthesized according to the general procedure using 14.89 mmol of azetidine (0.85 g, 1.0 ml, 1 eq.), 14.89 mmol of carbon disulfide (1.13 g, 0.9 ml, 1 eq.) and 14.89 mmol of n-butyllithium (2.5 M solution in hexane, 6.0 ml, 1 eq.) in approximately 50.0 ml of dry THF. The product was obtained as a white solid in quantitative yield

(102) .sup.1H NMR (400 MHz, D.sub.2O) 4.19-4.13 (m, 4H), 2.18-2.09 (m, 2H).

(103) ##STR00022##

(104) Lithium di-n-propyldithiocarbamate was synthesized according to the general procedure using 10.97 mmol of dipropylamine (1.11 g, 1.5 ml, 1 eq.), 10.97 mmol of carbon disulfide (0.84 g, 0.66 ml, 1 eq.) and 10.97 mmol of n-butyllithium (2.5 M solution in hexane, 4.4 ml, 1 eq.) in approximately 20.0 ml of dry THF. The product was obtained as an off-white solid in quantitative yield.

(105) .sup.1H NMR (400 MHz, D.sub.2O) 3.99-3.90 (m, 4H), 1.79-1.68 (m, 4H), 0.89 (t, J=7.5 Hz, 6H).

(106) ##STR00023##

(107) Lithium 4-methylpiperazinyldithiocarbamate was synthesized according to the general procedure using 13.48 mmol of N-methylpiperidine (1.35 g, 1.5 ml, 1 eq.), 13.48 mmol of carbon disulfide (1.03 g, 0.81 ml, 1 eq.) and 13.48 mmol of n-butyllithium (2.5 M solution in hexane, 5.4 ml, 1 eq.) in approximately 20.0 ml of dry THF. The product was obtained as an off-white solid in quantitative yield.

(108) .sup.1H NMR (400 MHz, D.sub.2O) 4.35 (br. s, 4H), 2.53 (br. s, 4H), 2.29 (s, 3H).

(109) ##STR00024##

(110) The complex [Au(N-methyl)dtc]n was synthesized according to the general procedure using 0.90 mmol of ATM (352.7 mg, 1.0 eq.) and 1.35 mmol of metam (174.4 mg, 1.5 eq.) predissolved in 10.0 ml of aqua dest. each. The obtained slightly greenish, yellow powder was thoroughly washed with water, small amount of ethanol and diethyl ether successively and dried under high vacuum over night. The product was obtained in a yield of 68% (183.6 mg, 0.61 mmol).

(111) Elemental analysis: calcd C, 7.92; H, 1.33; N, 4.62; obsd C, 8.10; H, 1.49; N, 4.59.

(112) ##STR00025##

(113) The complex [Au(N-dimethyl)dtc]n was synthesized according to the general procedure using 1.1 mmol of chloro(dimethyl sulfide)gold(I) (325.1 mg, 1.0 eq.) and 1.1 mmol of dimethyldithiocarbamate (157.5 mg, 1.0 eq.) predissolved in 20.0 ml of acetonitrile each. The obtained yellow powder was thoroughly washed with water, small amount of ethanol and diethyl ether successively and dried under high vacuum over night. The product was obtained as a yellow powder in a yield of 35% (121.8 mg, 0.38 mmol).

(114) Elemental analysis: calcd C, 11.36; H, 1.91; N, 4.42; obsd C, 11.61; H, 2.00; N, 4.43.

(115) ##STR00026##

(116) The complex [Au(N-ethyl, N-methyl)dtc]n was synthesized according to the general procedure using 1.35 mmol of ATM (527.0 mg, 1.0 eq.) and 2.03 mmol of the appropriate dithiocarbamate (286.6 mg, 1.5 eq.) predissolved in 10.0 ml of aqua dest. each. The obtained orange powder was thoroughly washed with water, small amount of ethanol and diethyl ether successively and dried under high vacuum over night. The crude product was obtained in a yield of 73% (327.6 mg, 0.99 mmol). This material was further purified by precipitation from hot 1,2-dichloroethane to afford a fluffy, orange colored solid as the pure product.

(117) Elemental analysis: calcd C, 14.51; H, 2.43; N, 4.23; obsd C, 14.59; H, 2.66; N, 4.15.

(118) ##STR00027##

(119) The complex [Au(diethyl)dtc]n was synthesized according to the general procedure using 5.13 mmol of ATM (2.0 g, 1.0 eq.) dissolved in 100.0 ml of aqua dest. and 5.13 mmol of the tetraethyldithiuram disulfide (1.52 g, 1.0 eq.) dissolved in 100.0 ml of ethanol. The obtained orange powder was filtered and thoroughly washed with water and dried under high vacuum over night. This material was recrystallized from hot DMF to afford orange needles as the pure product in a yield of 68% (1.21 g, 3.50 mmol).

(120) Elemental analysis: calcd C, 17.39; H, 2.92; N, 4.06; obsd C, 17.45; H, 3.02; N, 4.22.

(121) ##STR00028##

(122) The complex [Au(pyrrolidinyl)dtc]n was synthesized according to the general procedure using 1.03 mmol of ATM (400.0 mg, 1.0 eq.) and 2.05 mmol of the ammonium pyrrolidinedithiocarbamate (336.8 mg, 2.0 eq.) dissolved in 5.0 ml of aqua dest. each. The obtained orange powder was centrifuged. The supernatant was taken away and the residue was resuspended in water and again centrifuged (procedure repeated twice). The obtained orange solid was dried under high vacuum over night and afterwards recrystallized form hot DMF to afford orange-colored needles as the pure product in a yield of 38% (133.8 mg, 0.39 mmol).

(123) Elemental analysis: calcd C, 17.50; H, 2.35; N, 4.08; obsd C, 17.73; H, 2.53; N, 4.23.

(124) ##STR00029##

(125) The complex [Au(N-ethyl, N-isopropyl)dtc]n was synthesized according to the general procedure using 0.59 mmol of ATM (230.4 mg, 1.0 eq.) and 0.89 mmol of the lithium (N-ethyl, N-isopropyl)dithiocarbamate (150.0 mg, 1.5 eq.) dissolved in 10.0 ml of aqua dest. each. The obtained orange to brown powder was filtered and thoroughly washed with water. This material was recrystallized from chloroform/hexane affording dark-orange to brown crystals as the pure product in a yield of 19% (39.8 mg, 0.11 mmol).

(126) Elemental analysis: calcd C, 20.06; H, 3.37; N, 3.90; obsd C, 19.61; H, 3.44; N, 3.80.

(127) ##STR00030##

(128) The complex [Au(di-n-propyl)dtc]n was synthesized according to the general procedure using 1.23 mmol of ATM (478.0 mg, 1.0 eq.) and 1.84 mmol of the appropriate dithiocarbamate (337.1 mg, 1.5 eq.) predissolved in 10.0 ml of aqua dest. each. The obtained bright yellow powder was thoroughly washed with water, ethanol and diethyl ether successively. The crude product was obtained in a yield of 87% (400.9 mg, 1.07 mmol). Recrystallization from hot 1,2-dichloroethane afforded bright yellow needles as the pure product.

(129) Elemental analysis (report 41176, [M]): calcd C, 22.52; H, 3.78; N, 3.75; obsd C, 22.31; H, 4.01; N, 3.64.

(130) ##STR00031##

(131) The complex [Au(azetidinyl)dtc].sub.n was synthesized according to the general procedure using 1.66 mmol of ATM (646.0 mg, 1.0 eq.) and 2.48 mmol of the appropriate dithiocarbamate (345.7 mg, 1.5 eq.) predissolved in 10.0 ml of aqua dest. each. The obtained bright yellow powder was thoroughly washed with water, small amount of ethanol and diethyl ether successively and dried under high vacuum over night. The product was obtained in a yield of 78% (427.9 mg, 1.30 mmol).

(132) Elemental analysis: calcd C, 14.59; H, 1.84; N, 4.25; obsd C, 14.68; H, 2.24; N, 4.05.

(133) ##STR00032##

(134) The complex [Au(di-n-butyl)dtc].sub.n was synthesized according to the general procedure using 1.04 mmol of ATM (407.5 mg, 1.0 eq.) predissolved in 10.0 ml of aqua dest. and 1.57 mmol of the appropriate sodium dithiocarbamate (aqueous solution, 45 wt %; 791.7 mg, 0.73 ml, 1.5 eq.). After stirring at room temperature over night the reaction mixture was extracted with chloroform (220 ml). The combined organic phases were washed with brine (120 ml), dried over MgSO.sub.4 and concentrated. The residue was recrystallized from hot 1,2-dichloroethane to afford orange needles as the pure product in a yield of 35% (145.6 mg, 0.36 mmol).

(135) Elemental analysis: calcd C, 26.93; H, 4.52; N, 3.49; obsd C, 26.83; H, 4.73; N, 3.30.

(136) ##STR00033##

(137) The complex [Au(dibenzyl)dtc].sub.n was synthesized according to the general procedure using 1.29 mmol of ATM (505.1 mg, 1.0 eq.) dissolved in aqua dest. and 1.94 mmol of the appropriate sodium dithiocarbamate (573.7 mg, 1.5 eq.) dissolved in 10.0 ml of methanol. The obtained bright yellow powder was thoroughly washed with water, small amount of ethanol and diethyl ether successively. The crude product was obtained in a yield of 64% (390.5 mg, 0.83 mmol). Recrystallization from chloroform/hexane afforded brown crystals as the pure product in a yield of 64% (390.5 mg, 0.83 mmol).

(138) Elemental analysis (report 40981, [M]): calcd C, 38.38; H, 3.01; N, 2.98; obsd C, 38.31; H, 3.28; N, 2.86.

(139) ##STR00034##

(140) The complex [Au(4-methylpiperazinyl)dtc].sub.n was synthesized according to the general procedure using 1.29 mmol of ATM (502.0 mg, 1.0 eq.) and 1.93 mmol of the appropriate dithiocarbamate (351.7 mg, 1.5 eq.) predissolved in 10.0 ml of aqua dest. each. The obtained bright yellow powder was thoroughly washed with water, small amount of ethanol and diethyl ether successively. The crude product was obtained in a yield of 86% (414.8 mg, 1.11 mmol). Recrystallization from hot 1,2-dichloroethane afforded very fine, bright yellow needles as the pure product.

(141) Elemental analysis (report 41228, [M]): calcd C, 19.36; H, 2.98; N, 7.53; obsd C, 19.42; H, 3.07; N, 7.85.