CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of United States Provisional Patent Application No. 61/328,829, filed on April 28, 2010. The entire contents of the foregoing provisional application are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to coinage metal complexes, in particular, to coinage metal complexes of an Ν,Ν'-disubstituted cyclic thiourea, which could be used as therapeutic agents for treating cancer and inflammatory diseases.

BACKGROUND OF THE INVENTION

Coinage metal (Au, Ag, Cu) ions exhibit distinct biological activities which could be harnessed to give effective therapeutic agents for anti-arithric, antimicrobial, and anti-cancer treatment. ^1"6 The naked IvT ions, however, are unstable under physiological conditions and their instability such as that due to precipitation, aerobic oxidation and reduction can be circumvented by using appropriate auxiliary ligands. In literature, phosphine ligands are used to develop bioactive d ¹⁰ metals compounds though they are also cytotoxic. ^7"9 Recent work has also witnessed new ligand systems such as N-heterocyclic carbenes. ^10-1

Thiourea ligands are well documented in coordination chemistry, and have recently been receiving an upsurge interest in the area of new metal catalysts. Nevertheless, biological studies on metal-thiourea complexes are sparse. This invention relates to coinage metal complexes of an N, N'-disubstituted cyclic thiourea which deliver significant cytotoxicties to cancer cells and in particular, gold(I) thiourea complex exhibits potent tight-binding inhibition of anticancer drug target thioredoxin reductase.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a metal thiourea complex having the formula of la or lb

la lb

wherein Ri can be H, substituted or unsubstituted alkyl, alkenyl, alkynl, aryl or heterocyclic groups; R ₂ can be H, substituted or unsubstituted alkyl, alkenyl, alkynl or aryl groups; n = 1 to 4; X ^" is a pharmaceutically acceptable anion; and M is a coinage metal.

In another aspect, the invention provides a metal thiourea complex having the formula of Ila or lib:

Mb

wherein Ri can be H, substituted or unsubstituted alkyl, alkenyl, alkynl, aryl or heterocyclic groups; R ₂ can be H, substituted or unsubstituted alkyl, alkenyl, alkynl or aryl groups; n = 1 to 4; X ^" is a pharmaceutically acceptable anion; M is a coinage metal; L can be halo, thiolate, thiourea, imine, amine, imidazole, phosphine, or carbene; and m is the number of positive charge of the metal thiourea complex.

In a further aspect, the invention provides a pharmaceutical composition comprising a complex as mentioned above and a pharmaceutically acceptable vehicle.

In still another aspect, the invention provides a method for treating a patient having cancer or inflammatory condition by administering an effective amount of a composition comprising the complex as mentioned above in pharmaceutically effective vehicle. In still another aspect, the invention provides a use of the metal thiourea complex as mentioned above in the manufacture of a medicament for treating a patient having cancer or inflammatory condition.

Description of figures

Fig. 1 shows the structure of metal complexes of l,3-bis(4- methoxyphenyl)imidazolidine-2-thione, M(TU) ₂ Y, according to the preferred aspects of the present invention, wherein M stands for Au, Ag, or Cu; and Y ^" stands for CI ^" , OTf , or PF ₆ ^' .

Fig. 2 shows one synthesis route for the thiourea ligand according to one aspect of the present invention.

Fig. 3 shows the ORTEP drawing of complex 1 , [Au(TU) ₂ ]Cl, wherein CI ^" ion was omitted for clarity.

Fig. 4 shows the ORTEP drawing of complex 2, [Ag(TU) ₂ ]OTf, wherein Triflate ion was omitted for clarity.

Fig. 5 shows the results of Ag uptake of cells treated with complex 2, [Ag(TU) ₂ ]OTf and AgN0 ₃ according to the experiments of the present invention.

Fig. 6 shows the Thioredoxin reductase (TrxR), glutathione reductase (GR) and glutathione peroxidase (GPx) activities of HeLa cells treated with complex 1 or 2 for 1 hour, wherein complexes 1 and 2 stand for [Au(TU) ₂ ]Cl and [Ag(TU) ₂ ]OTf respectively.

Fig. 7 shows the Kinetic analysis of inhibition of TrxR preincubated with 1, [Au(TU) ₂ ]Cl. A shows plot of relative steady state velocities against concentrations of 1 , [Au(TU) ₂ ]Cl; and B shows plot of Kj' against concentrations of DNTB substrate.

Fig. 8 shows the Time dependence for the inhibition of TrxR by 1 , [Au(TU) ₂ ]Cl. A shows Progress curve of rat TrxR in the absence or presence of 1, [Au(TU) ₂ ]Cl; and B shows Plot of k _ap p against concentrations of 1, [Au(TU) ₂ ]Cl.

Fig. 9 shows Effects of pre-reduction of TrxR by NADPH on the enzyme inhibition by metal thiourea complexes according to one aspect of the present invention. Fig. 10 shows the results of Probing the free -SH and Se-H of TrxR treated with or without 1 , [Au(TU) ₂ ]Cl, by BIAM labeling.

Mode of carrying out the invention

Definition

As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having 1-14 carbon atoms, preferably 1-10 carbon atoms, more preferably 1 -6 carbon atoms, and most preferably 1^4 carbon atoms. For example, Ci-Cio, as in "Cj-Cio alkyl" is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in a linear or branched arrangement. For example, "Ci-Cio alkyl" specifically includes methyl, ethyl, w-propyl, /-propyl, «-butyl, /-butyl, /-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on.

As used herein, the term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and more preferably 2 to 4 carbon atoms, and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic carbon-carbon double bonds may be present. Thus, "C2-C6 alkenyl" means an alkenyl radical having from 2 to 6 carbon atoms. Alkenyl groups include ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. The straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated.

As used herein, the term "alkynyl" refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and more preferably 2 to 4 carbon atoms, and at least one carbon to carbon triple bond. Up to three carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" means an alkynyl radical having from 2 to 6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl, 3-methylbutynyl and so on. The straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated.

As used herein, "aryl" is intended to mean any stable monocyclic or bicyclic carbon ring of 4 to 14 atoms, preferably 4 to 10 atoms, more preferably 4 to 6 atoms, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.

As used herein, the term "heterocycle" or "heterocyclyl" is intended to mean a 3- to 14- membered, preferably 3 to 10-membered, more preferably 3 to 6 nonafomatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. For the purposes of this invention, the term "heterocyclic" is also considered to be synonymous with the terms "heterocycle" and "heterocyclyl" and is understood as also having the definitions set forth herein.

The above alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise. The substituents may be selected from for example OH, oxo, halogen, Ci _-6 alkoxy, di(Ci. ₆ )alkylamino, or C ₃ .i ₀ heterocyclyl.

As appreciated by those of skill in the art, "halo" or "halogen" as used herein is intended to include chloro, fluoro, bromo and iodo.

As used herein, the term "pharmaceutically acceptable anion" is intended to include halide, for example, chloride, bromide, and iodide; phosphate, for example,

hexafluorophosphate; and sulphate, for example, triflate.

As used herein, the term "coinage metal" is intended to include Au, Ag, and Cu, and so on.

Metal thiourea complex

In one aspect, the invention provides a metal thiourea complex having the formula of la or lb:

la lb

wherein Ri can be H, substituted or unsubstituted alkyl, alkenyl, alkynl, aryl or heterocyclic groups; R ₂ can be H, substituted or unsubstituted alkyl, alkenyl, alkynl or aryl groups; n = 1 to 4; X ^" is a pharmaceutically acceptable anion; and M is a coinage metal.

Herein described is the biological activities of a set of Au(I), Ag(I) and Cu(I) complexes supported by N, N'-disubstituted cyclic thiourea ligands, an example of which is 1 ,3- bis(4-methoxyphenyl)imidazolidine-2-thione (TU) (Fig. 1 ).

WhereinM denotes Au ⁺ , Ag ⁺ , or Cu ⁺ ; and Y denotes CI ^" , OTf or PF ₆ ^" .

These are homoleptic complexes of general formula [M(TU) ₂ ]Y, where M is Au(I), Ag(I) or Cu(I) ion. The molecular structures of [Au(TU) ₂ ]Cl (abbreviated as complex 1 or 1 only hereinafter) and [Ag(TU) ₂ ]OTf (abbreviated as complex 2 or 2 only hereinafter), have been established by X-ray crystallography. Figures 3 and 4 show the ORTEP drawing of these two complexes; and meanwhile, the following table 1 and 2 give the selected bond length and bond angles thereof. Table 1 Selected bond length (A) and bond angles (°) of 1

Au(l)-S(l) 2.356(8) S(l)-Au(l)-S(3) 175.1(3)

Au(l )-S(3) 2.236(7) C(l)-S(l)-Au(l) 108.8(8)

C(18)-S(3)-Au(l) 1 13.0(6)

Symmetry transformations used to generate equivalent atoms: #1 -x+l,-y,-z+ l .

Table 2 Selected bond length (A) and bond angles (°) of 2.

Ag(l)-S(l) 2.4067(1 1) S(l)-Ag(l)-S(2) 172.77(4)

Ag(l)-S(2) 2.4075(10) C(l)-S(l)-Ag(l) 104.1 1 (12)

Ag(l)-Ag(l)# l 3.2894(7) C(18)-S(2)-Ag(l) 104.88(12)

Symmetry transformations used to generate equivalent atoms: #1 -x+l,-y,-z+l .

The M ion in each case is coordinated by two thiourea ligands via the sulfur lone pair in a linear coordination geometry. The S-M-S bond angles (for 1, 175.1 °; for 2, 172.7°) and M-S bond lengths (for 1, 2.236A; for 2, 2.407 A) are similar to those found in the related Au(I) and Ag(I) complexes with other thiourea ligands (for 1, 2.278 - 2.406A; for 2, 166.7 - 180°, respectively). ¹⁴ ' ¹⁵ There is no close intermolecular Μ···Μ distance found in 1 (>3.32 A) and 2 (3.29 A), suggesting that the intermolecular metal-metal interactions are weak.

All the metal thiourea complexes are stable in solid state in air, soluble as 10 mM solution in DMSO and maintain stability with negligible spectral change for a time period of 72 h. No precipitation occurred when these complexes were added up to 30 μΜ to serum supplemented cell culture medium.

The effect of metal thiourea complexes on the growth of a panel of cancer cell lines were investigated and the results could be seen, for example, in Table 3 which will be discussed subsequently. The therapeutic potential of Au(I) has long attracted considerable interest. For example, Au(I)- thiolates (aurothiomalate) or phosphines (auranofin) are disease modifying anti- arthritic drugs, and have been recently studied for their anticancer properties. ¹⁶ ' ¹⁷ The inventors of the present invention have examined the in vivo anti-cancer activities of 1 in mice inoculated with NCI-H460 non-small cell lung cancer cells and the results could be seen, for example, in Table 4 which will be discussed subsequently.

The exact molecular mechanism of action of Au(I) compounds has yet to be elucidated, but is not limited to facile ligand exchange with thiol groups, particularly those with low pK _a values. ¹⁸ ' ¹⁹ In this regard, the thioredoxin reductase (TrxR) is a compelling molecular target of Au(I). ²⁰ ' ²¹ The mammalian TrxR is a NADPH dependent selenocysteine- containing which plays a pivotal role in cancer progression and inflammatory diseases, and inhibitors of this enzyme are considered as promising therapeutic agents. ²² ' ²³

The inventor carried out initial in vitro enzyme assays which showed that half maximal inhibition of TrxR (1 nM) was obtained using approximately equal molar concentration of 1, suggestive of a tight-binding mode of inhibition. This was further studied by progress curve analysis (Fig. 8). ²⁴ ' ²⁵ 1 was added in excess (3-100 nM) to a reaction mixture containing 0.2 mM NADPH, 1 nM TrxR and 3 mM disulfide substrate 5,5'- dithiobis(2-nitrobenzoic acid) (DTNB) in phosphate buffer (pH 7.4). The time course of change in the product concentrations at various concentrations of 1 are shown in Fig 8A. The progress curves are non-linear, revealing two-phase equilibria typical of slow-onset tight-binding inhibition. This was analyzed using Eq. 1 (Example 6), where P is the product concentration, Vj and v _f are the initial and final steady-state velocities, respectively, and k _app is the apparent first-order rate constant for establishment of the final steady-state inhibition. A plot of the k _app against the inhibitor concentrations followed a hyperbolic function (Fig. 8B). This is indicative of a two-step, tight-binding inhibition mechanism:

where EI is the initial collision complex, k ₃ is the forward isomerization rate, and 1¾ is the reverse isomerization rate. In this scheme, binding involves rapid formation of an initial collision complex (EI) that subsequently undergoes isomerization to the final slow dissociating enzyme-inhibitor complex (EI*). The k ₃ , k ₄ and the dissociation constant of the initial collision complex EI (ΚΓ) can be obtained by fitting the data to Eq. 2. Accordingly, k ₃ - 0.01 1 s ^"1 , = 0.00014 s ^_1 and Kj = 1.39 nM. Thus, the tight binding inhibition is essentially irreversible, and in fact the enzyme activities could not be recovered after removal of the free inhibitors by ultrafiltration. The overall inhibitory constant Κ,* was determined to be 18 pM using Eq. 3. These inhibitory constants are also reasonably close to the corresponding values determined from the steady state rate law established in condition when EI* was preformed, with K; =0.67 nM and Kj*- 36 pM (Fig. 7). 1 is thus among the most potent TrxR inhibitor reported. ²⁰ ' ²³

The reduced TrxR has free -SH (Cys496) and -SeH (Sec497) groups at the C-terminal active site, making it vulnerable to be attacked by Au(I). ²⁰ · ^{22> 23} These redox active sites can be probed by biotinylated iodoacetamide (BIAM), which alkylates the free -SH and - SeH groups; and the resulting adduct can be detected by western blot experiment using streptavidin-linked horseradish peroxidase (Fig. 10). ²⁶ ' ²⁷ It has been shown that these residues can be selectively alkylated by BIAM by adjusting the pH. At pH 8.5, both -SH and -SeH are alkylated. At pH 6.5, only the -SeH is alkylated owing to the low pKa value for selenocysteine, and thus a weaker streptavidin signal was obtained in which case. When NADPH reduced TrxR (0.1 μΜ) was preincubated with 1 (4 μΜ), the BIAM labeling at both pH 8.5 and pH 6.5 (buffered with 0.1 M Tris.HCl) was inhibited, suggesting that the selenocysteine or additionally the cysteine residues at the active site were involved in the enzyme inactivation. This is consistent with the observation that the NADPH reduced TrxR, which exposes the free -SH and -SeH groups, was much more efficiently inhibited by 1 than the oxidized TrxR having the -S-Se- group (Fig. 9). It is highly likely that the formation of the tight enzyme-inhibitor complex (EI*) involves covalent modification of the redox active selenocysteine and cysteine residue via the Au(I) complex. ²⁸

In summary, d ¹⁰ metal complexes supported by thiourea ligands represent a new paradigm in developing bioactive metal based complexes. In particular, the inventors of the present invention have demonstrated that the Au(I) thiourea complex confers specific tight binding inhibition of thioredoxin reductase with a potency among the lowest reported, ³ and exhibits effective suppression of cellular TrxR activity. By variation of thiourea ligand, the metal thiourea complexes have the prospect to be a new class of metal based drugs leads.

The invention further provides a pharmaceutical composition comprising a complex of the present invention and a pharmaceutically acceptable vehicle. In still another aspect, the invention provides a method for treating a patient having cancer or inflammatory condition by administering an effective amount of a composition comprising the complex of the present invention in pharmaceutically effective vehicle. In still another aspect, the invention provides a use of the metal thiourea complex of the present invention in the manufacture of a medicament for treating a patient having cancer or inflammatory condition.

EXAMPLES

Examples provided are intended to assist in a further understanding of the invention. Particular materials employed, species and conditions are intended to be illustrative of the invention and not limiting of the reasonable scope thereof.

Example 1

Synthesis of metal thiourea complexes

Figure 2 shows the scheme of the synthesis route for the thiourea ligands l ,3-bis(4- methoxyphenyl)imidazolidine-2-thione.

Materials

Au(THT)Cl and [Cu(CH ₃ CN) ₄ ]PF ₆ were prepared according to literature procedures. ²⁹ ' ³⁰ Thiourea ligand

Glyoxal-bis-(4-methoxyphenyl)imine ³¹

To a solution of p-anisidine (12.3g, 0.1 mol) in EtOH (50 mL) was added a mixture of 40% aqueous solution of glyoxal (7.3g, 0.05 mol), EtOH (10 mL) and water (10 mL) at 25°C. The mixture was stirred overnight at 25°C. Upon addition of water (30 mL), a yellow solid precipitated which was collected by filtration and dried in vacuo. Yield: 5.5 g (82%). 1H NMR (400 MHz, CDC1 ₃ ): δ 8.42(s, 2H), 7.32(d, J = 9.0, 4H), 6.96(d, J = 9.0, 4H), 3.84 (s, 6H). ¹³ C NMR (400 MHz, CDC1 ₃ ): δ 160.2, 158.0, 143.4, 123.4, 1 15.0, 55.9.

N,N'-Bis-(4-methoxyphenylamino)ethane ³¹

A suspension of glyoxal-bis-(4-methoxyphenyl)imine (1.34g, 5 mmol) in a mixture of THF (30 mL) and MeOH (5 mL) was treated at 0°C with sodium borohydride (0.76 g, 20 mmol). The mixture was stirred overnight at 25°C and subsequently heated for 2 h under reflux. Upon addition of ice-water (30 mL) and 3M HC1 (30 mL), a white solid precipitated which was collected by filtration and dried in vacuo. Yield: 1.2 g (88%). Ή NMR (400 MHz, CDC1 ₃ ): δ 6.79(d, J = 8.92, 4H), 6.62(d, J = 8.92, 4H), 3.75(s, 6H), 3.34 (s, 4H). ¹³ C NMR (300 MHz, CDC1 ₃ ): δ 152.8, 142.7, 1 15.4, 114.9, 56.2, 44.9.

Thiourea l,3-bis(4-methoxyphenyl)imidazolidine-2-thione (TU) ³²

To a solution of N,N'-Bis-(4-methoxyphenylamino)ethane (1.4 g, 5 mmol) in dry THF (40 mL) was added Ι, -thiocarbonyl diimidazole (1.1 g, 6 mmol) at 25°C. The mixture was stirred overnight and subsequently heated for 2 h under reflux. After the addition of water and ethyl acetate, the organic layer was washed with dilute HCl and brine, dried and concentrated. The pure product was obtained through recrystallization from 95% EtOH. Yield: 1.1 g (70%). Ή NMR (400 MHz, CDC1 ₃ ): δ 7.42(d, J = 8.96, 4H), 6.95(d, J = 8.96, 4H), 4.10(s, 4H), 3.82 (s, 6H). ¹³ C NMR (300 MHz, CDC1 ₃ ): 5 182.6, 158.6, 134.3, 127.6, 1 14.6, 55.9, 50.3. FAB-MS: 315 [M+H] ⁺ .

Metal complexes with thiourea ligand

(1) [Au(TU) ₂ ]Cl

To a CH ₂ C1 ₂ (5 mL) solution of TU (0.31 g, 1 mmol) was added Au(THT)Cl (0.16 g, 0.5 mmol) in distilled MeOH (5 mL) under an argon atmosphere. The mixture was stirred at room temperature overnight and subsequently filtered. The filtrate was left standing overnight. Colorless crystals were collected and dried in vacuo. Yield: 76%. FAB-MS: m/z = 826 [M] ⁺ . Ή NMR (400 MHz, DMSO): δ 7.38(d, J = 8.33, 4H), 7.42(d, J = 8.35, 4H), 4.21(s, 4H), 3.77(s, 6H). IR (KBr, cm ^'1 ): 2960(w), 2929(w), 2835(w), 1606(m), 1515(s), 1283(m), 1252(s), 1162(m), 1029(m), 836(s), 554(m). Anal. Calcd. for AuC ₃ 4H ₃₆ N ₄ 04S ₂ Cl: C, 47.42; H, 4.21 ; N, 6.51 ; Found: C, 47.12; H, 4.00; N, 6.53.

(2) [Ag(TU) ₂ ]OTf

Thiourea TU (0.31 g, 1 mmol) was dissolved in EtOH (10 mL) and silver triflate (0.13 g, 0.5 mmol) was added under an argon atmosphere. The mixture was stirred at room temperature for 3 h, and subsequently filtered to remove the unreacted AgOTf. The filtrate was left standing overnight. Colourless crystals were collected and dried in vacuo. Yield: 82%. FAB-MS: 736 [M] ⁺ . Ή NMR (400 MHz, CDC1 ₃ ): δ 7.33(d, J = 8.94, 4H), 6.93(d, J = 8.91, 4H), 4.21(s, 4H), 3.71(s, 6H). IR (KBr, cm ^'1 ): 2961(w), 2930(w), 2839(w), 1605(w), 1512(s), 1275(s), 1250(s), 1 165(m), 1032(m), 831(s), 555(m). Anal. Calcd. for AgC ₃₅ H ₃₆ N ₄ 0 ₇ S ₃ F ₃ : C, 47.46; H, 4.10; N, 6.33; Found: C, 47.41 ; H, 4.15; N, 6.36.

(3) [Cu(TU) ₂ ]PF ₆ Thiourea TU (0.31 g, 1 mmol) was dissolved in CH ₂ C1 ₂ (10 mL) and [Cu(CH ₃ CN) ₄ ]PF ₆ (0.19 g, 0.5 mmol) in distilled MeOH (10 mL) was added under an argon atmosphere. The mixture was stirred at room temperature for 2h. The resulting white solid was filtered and washed with MeOH, Et ₂ 0 and dried in vacuo. Yield: 80%. FAB-MS: 692 [M] ⁺ . Ή NMR (400 MHz, DMSO): δ 7.46(d, J = 8.97, 4H), 6.96(d, J - 8.90, 4H), 4.10(s, 4H), 3.76(s, 6H). IR (KBr, cm ^"1 ): 2969(w), 2934(w), 2838(w), 1606(m), 1514(s), 1287(m), 1252(s), 1 166(m), 1030(m), 838(s), 556(m). Anal. Calcd. for CuC3 ₄ H ₃₆ N ₄ 0 ₄ S ₂ PF ₆ : C, 48.77; H, 4.33; N, 6.69; Found: C, 48.12; H, 4.00; N, 6.32.

Example 3

Cytotoxicity assays

Cells were seeded in a 96-well flat-bottomed microplate at 20,000 cells/well in 150 of growth medium solution. The compounds were dissolved in dimethyl sulfoxide. Serial dilution of each complex was added to each well with final concentration of DMSO < 1%. The microplate was incubated at 37°C, 5% C0 ₂ , 95% air in a humidified incubator for 72 h. After incubation, 10 μΐ. MTT reagent (5 mg/mL) was added to each well. The microplate was re-incubated at 37 °C in 5% C0 ₂ for 4 h. Solubilization solution (10% SDS in 0.01 M HC1) (100 μί,) was added to each well. The microplate was left in an incubator for 24 h. Absorbances at 550 nm were measured by a microplate reader. The IC50 values (the concentration required to reduce the absorbance by 50% compared to the controls) were determined.

Table 3 Cytotoxicities (IC50) of metal thiourea complexes toward selected human cancer cell lines

HeLa HepG2 SUNE1 NC1-H460

1 14.6 ± 0.7 17.4 ± 1.0 10.8 ± 0.2 3.72 ± 0.3

2 7.2 ± 0.7 4.0 ± 0.4 8.8 ± 1.0 8.86 ± 1.0

3 12.7 ± 0.9 13.0 ± 0.9 8.5 ± 1.0 1 1.15 ± 0.9

TU >100 >100 >100 >100 cisplatin 4.7 ± 0.3 14.2 ± 1.0 35.2 ± 0.3 38.57 ± 0.4

HeLa, human cervical epithelioid carcinoma; HepG2, human hepatocellular carcinoma; SUNE1 , human nasopharyngeal carcinoma; NCI-H460 = human lung carcinoma As can be seen from the above table, all the metal complexes exerted cytotoxicity at low micromolar concentrations, with half maximal inhibitory concentrations (IC ₅₀ ) comparable to or lower than those of benchmark anticancer drug cisplatin. The IC ₅₀ of metal-free thiourea ligand is more than 100 μΜ, suggesting that the biological activities of the metal complexes are largely metal mediated, and the lipophilic thiourea ligand serves as a nontoxic carrier of the metal ion to the cells. To test this notion, the following example was also carried out.

Example 4

Metal uptake by cells

HeLa cells (2 ^χ 10 ⁵ cells/well) were seeded in 12-well plate with culture medium (2 mL/well) and incubated at 37°C in an atmosphere of 5% C0 ₂ /95% air for 24 h. The culture medium was then removed and replaced with fresh medium containing 2 and AgN0 ₃ (10 μΜ). After exposure for 2 h, the medium was removed and the cell monolayer was washed three times with PBS. The cells were lysed with water and digested in 70% HN0 ₃ at 80°C for 2 h. The digests were diluted with water to 10 mL for inductively coupled plasma mass spectrometry (ICP-MS) analysis.

As can be seen from figure 5, the data revealed that the Ag content in 2 treated HeLa cells was 5-fold higher than those treated with AgN0 ₃ . Furthermore, the cytotoxic potency of 2 for HeLa cells is also nearly 5-fold higher than that of AgN0 ₃ (IC ₅₀ = 32.1 ± 1.2 μΜ).

Example 5

Cellular activities of thiol-dependent redox enzymes (Fig. 6)

Preparation of cellular extracts

Cells were seeded at 2 x 10 ⁵ / well in 6-well plates and incubated for 24 hours. The metal thiourea compounds (10 ^"9 to 10 ^"4 M) were serially diluted and added to the cells (final DMSO concentrations < 1%). After an one-hour incubation, cells were washed thrice with phosphate buffered saline and 100 μΐ, ice-cold lysis buffer (50 mM phosphate buffer, pH 7.4, 1 mM EDTA, 0.1% Triton-X 100) were added to the cell layer. Cell lysis was carried on ice for 5 minutes and the cell lysates were collected and stored at -80°C or assayed immediately.

Thioredoxin reductase (TrxR) Cell lysates (10 μ§ proteins) were added to a mixture (100 μί) containing 100 mM phosphate, pH 7.4, 1 mM EDTA and 0.2 mM NADPH. Reaction was initiated by adding 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 3 mM final) and the TrxR activities were determined as increases in O.D. ₄ i _{2 nM} in 10 min.

Glutathione peroxidase (GPx)

Cell lysates (10 g proteins) were added to a mixture containing 100 mM phosphate, pH 7.4, 2 mM GSH, 1 U glutathione reductase, and 0.2 mM NADPH. Reaction was initiated by adding tert-butyl hydroperoxide (300 μΜ) and the NADPH oxidation was measured as decreases in O.D. ₃₄ o _n m in 10 min. GPx activities (AO.D. _{3 0n} m/min) were determined by subtracting the spontaneous NADPH oxidation in the absence of tert-b ty\ hydroperoxide _.

Glutathione reductase (GR)

Cell lysates (10 μg proteins) were added to a mixture containing 100 mM phosphate, pH 7.4 , 1 mM EDTA, 1 mM GSSG and 0.2 mM NADPH. Reaction was initiated by adding DTNB (3 mM final) and the increases in O.D. _{4]2 nm} were measured for 10 min. GR activities were determined by subtracting the increases in O.D. ₄ i ₂ „M in the absence of GSSG.

The results can be seen in figure 6. As shown in Fig. 6, an one-hour treatment of HeLa cancer cells with 1 resulted in an inhibition of the cellular TrxR activity with an IC5 ₀ value of 50 nM. Another selenocysteine-containing thiol enzyme, glutathione peroxidase (GPx), was also inhibited by 1 albeit with at higher concentration (IC ₅₀ = 1 μΜ). Glutathione reductase (GR) activity was not affected by 1. For comparison, Ag(I) thiourea complex 2 inhibited TrxR and GPx with IC ₅₀ of 100 nM and 1 μΜ, respectively, which are almost of with similar potency to complex 1, and also significantly suppressed GR activities when added at 50 μΜ. All the enzyme activities were not affected by Cu(I) complex 3 or metal-free thiourea ligand added up to 100 μΜ. These data demonstrate that among the coinage M ⁺ ions, Au(I) preferentially targets the selenocysteine containing enzymes.

Example 6

Kinetic analysis of tight-binding inhibition of thioredoxin reductase by Au-TU Determination of inhibitory constant (Kj) derived from residual activities of preformed enzyme-inhibitor complexes ^33,34 (Fig. 7) 1 nM Recombinant rat TrxRl (ICMO Corp, Sweden) was reduced with 0.2 mM NADPH and then incubated with 0.3-10 nM of 1 for 30 min in a buffer of 100 mM phosphate buffer, pH 7.4 and 1 mM EDTA. The residual activities were measured using 0.75, 1.5 or 3 mM DTNB (Fig. 7A). The data were fit into Eq. 1 using GraphPad Prism 3.0 software.

(1) v _s /v ₀ = (Ε,-Ki-I, + ((I _t + Κ -E _t ) ² + (4K, E _t )) ^{1 2} )/(2E _t )

Eq. 1 describes the rate law of tight-binding inhibition in which case the inhibitor concentration is substantially depleted owing to formation of enzyme-inhibitor complex. In this equation, v ₀ is the observed velocity in the absence of inhibitor, v _s is the steady- state velocity in the presence of inhibitor, E _t is the total enzyme concentration, and I is the inhibitor concentration. The apparent inhibitory constant (Kj) so obtained was 0.67 nM. Average inhibitory constant (Kj*) was calculated to be 36 pM using Eq.2, (Fig. 7B) which takes into account of competitive inhibition of the enzyme with the substrate and a predetermined K _m of 0.2 mM.

(2) Ki = K,*(l+S/K _m )

Determination of Kj by progress curve analysis ^3S ' ³⁶ (Fig. 8)

1 was added in excess (3-100 nM) to a reaction mixture containing 0.2 mM NADPH, 1 nM TrxRl , 3 mM disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 100 mM phosphate buffer, pH 7.4 and 1 mM EDTA. The time courses of change in the product concentration are shown in Fig 8A. The progress curves are non-linear, showing two-phase equilibria typical of slow-onset tight-binding inhibition. The data was fit into Eq. 1 using GraphPad Prism 3.0 software,

(1) P = v _f t + ((v ₁ -v _f )/k _app )(l-e ^kappt ) where P is the product concentration, Vj and Vf are the initial and final steady-state velocities, respectively, and k _app is the apparent first-order rate constant for establishment of the final steady-state inhibition. A plot of the k _app against the inhibitor concentrations followed a hyperbolic function (Fig. 8B). This is indicative of a two-step, tight-binding inhibition mechanism: where EI is the initial collision complex, k ₃ is the forward isomerization rate, and is the reverse isomerization rate. In this scheme, binding involves rapid formation of an initial collision complex (EI) that subsequently undergoes isomerization to the final slow dissociating enzyme-inhibitor complex (EI*). The k ₃ , le _t and the dissociation constant of the initial collision complex EI (ΚΓ) carl be obtained by fitting the data to Eq. 2.

where I _t is the inhibitor concentration, S is the substrate (DTNB) concentration and K _m is the Michaelis-Menten constant for reduction of DTNB by TrxR.

Accordingly, k ₃ = 0.011 s ^"1 , = 0.00014 s ^"1 and Kj'- 1.39 nM. The overall inhibitory constant Kj* was determined to be 18 pM using Eq. 3.

Effects of NADPH reduction of TrxRl inhibition by metal thiourea complexes (Fig. 9)

1 nM Of TrxR 1 was incubated with or without 0.2 mM NADPH in reaction buffer (100 mM phosphate, pH 7.4, 1 mM EDTA) for 5 min. metal thiourea complexes (1 - 100 nM) was added and allowed to incubate for 30 min. DTNB (3 mM) and NADPH (0.2 mM) was then added. The TrxRl activities were determined as the increases in O.D. ₄ i _{2 nm} over 10 min.

Example 7

Probing the cysteine and selenocysteine residues of TrxRl ^I0"I2 (Fig. 10)

NADPH-reduced TrxRl (0.1 μΜ) and 1 (4 μΜ) were incubated in reaction buffer (100 mM phosphate buffer, pH 7.4, 1 mM EDTA) at room temperature for 1 h. 1 of the reaction mixture was taken out and added to new tubes containing 19 μΐ. of 100 μΜ BIAM (buffered with 200 mM Tris-HCl at pH 6.5 and 8.5, respectively). The incubation was carried out at 37°C for 30 min to alkylate the remaining free -SeH and -SH groups of the enzyme. 20 μΐ. of the reaction mixtures were mixed with loading buffer and subjected to SDS-PAGE on a 7.5% gel. The separated proteins were transferred to nitrocellulose membrane and the BIAM labeled proteins were detected with horseradish peroxidase conjugated streptavidin and enhanced chemiluminescence detection.

Example 8

Tumor implantation in nude mice and in vivo drug treatment (Table 4)

The in vivo experiment was conducted in Pearl Materia Medica Development (Shenzhen) Limited and performed with approval from the Committee on the Use of Live Animals for Teaching and Research. SPF grade four-week-old female BALB/c AnN-nu mice (nude mice, 16 - 18. g) were purchased. Tumor cells (5 ^χ 10 ⁶ ) resuspended in RPMI medium were implanted by subcutaneous injection on the right flank of the mice. When tumors were approximately 50 mm ³ in size, animals were randomly separated into 3 groups to receive treatment of twice-a-week intraperitoneal injection of 10% PET vehicle control (where 10% PET = 6% polyethylene glycol 400, 3% ethanol, 1% Tween 80 and 90% PBS), complex 1 (100 mg/kg) or cyclophosphamide (30 mg/kg) for 8 times. After 28 days, the mice were sacrificed and the tumors were isolated and weighted.

Table 4 Tumour implantation in nude mice and in vivo drug treatment (no. of mice: 5)

Tumor volume/mm ³

Vehicle control Positive control [Au'fTU^Cl (1)

(cyclophosphamide) (Dose: 100 mg-kg ^"1 )

(Dose: 30 mg-kg ^"1 )

Day 9 317.802±308.863 - 209.026±94.749

Day 13 1420.931±625.165 325.041±170.053 624.380±233.051

Day 17 2336.166±787.199 639.465±422.362 1232.177±229.41 1

Day 21 2801.906±1304.491 1032.258±594.026 1834.856±215.456

Day 25 3650.140±1721.593 1474.100±900.357 2389.133±594.756

Day 29 4437.245±222.022 1438.735±845.815 2741.851±805.220

As can be seen from table 4, Intraperitoneal injection of complex 1 at 100 mg/kg body weight for twice a week resulted in reduction in tumor size by (38% ± 1 1, n = 5) compared to vehicle control after a 28-day treatment.

While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations and/or modifications as come within the scope of the following claims and their equivalents.

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