CN109790191B - Platinum complexes and methods of use thereof - Google Patents

Abstract

其中：R₁和R₂独立地选自胺、任选取代的胺、和任选取代的杂环胺；或一对R₁和R₂连接在一起形成含有氮原子的二齿配体；R₃选自H、任选取代的烷基、任选取代的芳基、任选取代的噻吩基、和聚乙二醇；R₄、R₅、R₆和R₇独立地选自H、卤素、‑OR、任选取代的烷基和聚乙二醇，或R₄和R₅、R₅和R₆、R₆和R₇的每一对连接在一起形成

；Y选自硫原子、氧原子和NR（其中R选自H和任选取代的烷基）；Z选自碳原子和氮原子；和X选自氟离子、氯离子、溴离子、碘离子、三氟甲磺酸根、乙酸根、硝酸根、高氯酸根、六氟磷酸根、硫酸根和磷酸根。还公开了在治疗癌症和在细胞内部可视化中使用该铂(II)配合物的方法。Disclosed herein are platinum(II) complexes, which may include a complex of the formula or a pharmaceutically acceptable salt thereof,

wherein: R ₁ and R ₂ are independently selected from amines, optionally substituted amines, and optionally substituted heterocyclic amines; or a pair of R ₁ and R ₂ are joined together to form a bidentate ligand containing a nitrogen atom; R ₃ is selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol; R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, halogen , -OR, optionally substituted alkyl and polyethylene glycol, or each pair of R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and R ₇ joined together to form

Y is selected from sulfur atom, oxygen atom and NR (wherein R is selected from H and optionally substituted alkyl); Z is selected from carbon atom and nitrogen atom; and X is selected from fluoride, chloride, bromide, iodide , triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate. Also disclosed are methods of using the platinum(II) complexes in the treatment of cancer and visualization within cells.

Description

Platinum complexes and methods of using the same

Field of Invention

Platinum(II) complexes are disclosed herein. More particularly, the platinum(II) complex may comprise a luminescent 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand. Also disclosed are methods of using the platinum(II) complexes in cancer therapy and cellular monitoring.

Background of the Invention

Cisplatin (cis-diaminedichloroplatinum) is a member of the family of platinum(II) complexes used in the treatment of cancer. It is one of the more effective anticancer drugs; however, cisplatin may have drawbacks. For example, patients may exhibit drug resistance or may have severe side effects. Carboplatin (cis-diamine(1,1-cyclobutanedicarboxylate)platinum) is another anticancer drug in the family of platinum(II) complexes. Carboplatin has an amine non-leaving ligand similar to cisplatin, but a different leaving ligand. Experimental evidence suggests that the anticancer mechanism of cisplatin and carboplatin involves the displacement of this leaving group by water or other biological nucleophiles and the formation of the ion [( _NH3 ⁾ _2PtII ] ²⁺ , which can interact with nuclear DNA bind and cause cell death. Although cisplatin and carboplatin have similar anticancer mechanisms, they can exhibit different clinical responses. For example, there may be fewer side effects associated with carboplatin. There is growing interest in developing luminescent transition metal complexes that are therapeutically active and allow simultaneous monitoring of cancer progression. The photophysical properties of such complexes enable their visualization and tracking in vitro and in vivo, thereby providing real-time tracking of cellular distribution, structural changes and biotransformation pathways of anticancer platinum(II) complexes. However, not all platinum(II) complexes possess the necessary photophysical properties to enable disease monitoring.

Therefore, there is a need for new platinum(II) complexes that can have therapeutic benefits and/or can have photophysical properties that allow monitoring and tracking of disease progression.

SUMMARY OF THE INVENTION

Provided herein are platinum(II) complexes and methods for their use. In a specific embodiment, the platinum(II) complex may comprise a complex of formula I or a pharmaceutically acceptable salt thereof,

wherein: R ₁ and R ₂ are independently selected from amines, optionally substituted amines, and optionally substituted heterocyclic amines; or a pair of R ₁ and R ₂ are joined together to form a bidentate ligand containing a nitrogen atom; R ₃ selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol; R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or each pair of R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and R ₇ joined together to form

Y is selected from sulfur atoms, oxygen atoms, and NR (wherein R is selected from H and optionally substituted alkyl groups); Z is selected from carbon atoms and nitrogen atoms; and X is selected from fluoride, chloride, bromide, iodide, Triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

In another specific embodiment, a method for treating a subject with cancer comprises administering a therapeutically effective amount of at least one platinum(II) complex of formula I.

In another specific embodiment, a method of monitoring cells may comprise administering an effective amount of at least one platinum(II) complex of formula I, and detecting the fluorescent signal of the platinum(II) complex.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following detailed description, with reference to the accompanying drawings, exemplary, non-limiting and non-exhaustive embodiments of the present invention are depicted. A more specific description of the invention outlined above can thus be obtained by reference to the embodiments, some of which are depicted in the accompanying drawings, in a manner that enables a detailed understanding of the above-described features of the invention. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

1 shows chemical structures of non-limiting examples of platinum(II) complexes containing 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone (Hbt) ligands, according to some embodiments.

Figure 1a shows the chemical structures of platinum(II) complexes containing Hbt ligands as labile leaving groups.

Figure 2 shows a non-limiting example of synthetic methods for preparing Hbt ligands, as well as synthetic methods for preparing platinum(II) complexes, according to some embodiments.

Figure 2a shows the in vivo tumor growth inhibitory effect of 1a, 2a and 3a, examined via intravenous injection using nude mice bearing NCI-H460 xenografts. (A) Mean tumor volume after two or three weekly treatments with 10 mg/kg of 1a, 5 mg/kg of 2a, 20 mg/kg of 3a or solvent (*p-value < 0.05, n = 5) . (B) Body weights of nude mice in 1a, 2a, 3a treated groups and the solvent control group. (C) Representative photographs of tumors obtained from nude mice in 1a, 2a, 3a treated groups and the vehicle control group.

3 shows UV-visible absorption spectra of some exemplary platinum(II) complexes and Hbt ligands in PBS solution, according to some embodiments.

Figure 3a shows the body weight of nude mice in cisplatin (1 mg/kg and 3 mg/kg) and complex 1a (10 mg/kg and 20 mg/kg) treatment groups. Mice treated with 3 mg/kg of cisplatin lost more than 10% of their total body weight. In contrast, mice treated with 20 mg/kg of 1a had consistent body weights and no obvious signs of adverse effects were observed.

₄ shows emission spectra in _CH2Cl2 of some exemplary platinum(II) complexes with Hbt ligands, according to some embodiments.

Figure 4a shows a series of platinum(II) complexes containing differently substituted Hbt ligands.

5 shows UV-visible absorption spectra of a) some exemplary platinum(II) complexes in PBS solution at various time intervals, and b) in the presence of GSH in PBS solution at various time intervals, according to some embodiments UV-visible absorption spectra of some exemplary platinum(II) complexes below.

6 shows emission spectra of some exemplary platinum(II) complexes in the presence of GSH in PBS solution, according to some embodiments. a) The emission spectra of complexes 1 and 2 in the presence of GSH were recorded at different time points, excitation wavelength at λ = 381 nm. b) Time course of the emission intensities of complexes 1 and 2 at 450 nm in the presence of GSH.

Figure 7 shows a) emission spectra of some exemplary platinum(II) complexes in the presence of different molar ratios of ctDNA. b) The luminescence intensity enrichment of some exemplary platinum(II) complexes by ctDNA.

Figure 8 shows luminescence imaging of some exemplary platinum(II) complexes with Hbt ligand inside cells, according to some embodiments.

9 shows a time series of luminescence imaging of some exemplary platinum(II) complexes inside living cells, according to some embodiments.

10 shows cellular uptake and fractional DNA binding in cells of some exemplary platinum(II) complexes and oxaliplatin, according to some embodiments.

Figure 11 shows a) mean tumor volume of nude mice bearing HeLa xenografts after treatment via intraperitoneal injection with some exemplary platinum(II) complexes or solvents, according to some embodiments. b) Body weight of nude mice bearing HeLa xenografts in platinum(II) complex-treated and solvent-controlled groups.

Figure 12 shows a) representative photographs of tumors obtained from nude mice in platinum(II) complex treated and solvent control groups, according to some embodiments. b) Representative photographs of nude mice in platinum(II) complex-treated and solvent-controlled groups.

Detailed description of the invention

In the following specification and claims, reference will be made to a number of terms which shall be defined to have the following meanings:

The term "alkyl" as used herein refers to a straight, branched or cyclic saturated chain, usually, although not necessarily, containing from 1 to about 20 carbon atoms, preferably from 1 to about 12 carbon atoms, 1 to 6 carbon atoms Hydrocarbon groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, etc., and cycloalkyl groups such as cyclopentyl, cyclohexyl, etc. Wait. Generally, although not necessarily, the term "cycloalkyl" means a cyclic alkyl group, usually having 4 to 8, preferably 5 to 7 carbon atoms. The term "substituted alkyl" refers to an alkyl group substituted with one or more substituent groups, and the terms "heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl group in which at least one carbon atom is replaced by a heteroatom . If not stated otherwise, the term "alkyl" includes straight chain, branched chain, cyclic, unsubstituted, substituted and/or heteroatom-containing alkyl groups.

The term "nitrogen-containing bidentate ligand" as used herein refers to a bidentate ligand containing a 10- to 64-membered, preferably 10- to 48-membered or 10- to 36-membered heterocyclic molecule consisting of Consists of carbon atoms and one to six, preferably 1, 2, 3, 4 or 5 heteroatoms selected from nitrogen, oxygen and sulfur, wherein such heterocyclic molecules contain at least one nitrogen atom. Examples of such heterocyclic molecules include, but are not limited to, pyrazoles, triazoles, tetrazole, pyridine, pyrazine, azepine, benzimidazole, benzothiazole, isothiazole, imidazole, indole, piperidine, piperazine , purine, quinoline, thiadiazole, oxazoline, isoxazoline, thiazoline, morpholine, bipyridine, bipyrazine, terpyridine, phenanthroline, red phenanthroline, bisoxazoline, bis Thiazoline, bisquinoline, bisisoquinoline, quinolinylpyridine, quinolinylphenanthroline and the like.

The term "heterocycle" in the context of the present invention refers to a stable 5- to 32-membered, preferably 5- to 24- or 5- to 18-membered heterocyclic group consisting of carbon atoms and one to six, preferably 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur. The term "5- to 32-membered heterocyclic group" as used herein refers to a heterocyclic group having a backbone of 5 to 32 atoms. For purposes of the present invention, the heterocyclic ring may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include bonded or fused ring systems; and the heterocyclyl group may be partially or fully saturated or aromatic. family (heteroaryl). Examples of heterocyclic groups include, but are not limited to, pyrazole, triazole, tetrazole, pyridine, pyrazine, azepine, benzimidazole, benzothiazole, isothiazole, imidazole, indole, piperidine, piperazine, Purine, quinoline, thiadiazole, oxazoline, isoxazoline, thiazoline, morpholine, bipyridine, bipyrazine, terpyridine, phenanthroline, red phenanthroline, bisoxazoline, bisthiazole quinoline, bisquinoline, bisisoquinoline, quinolinyl pyridine, quinolinyl phenanthroline, etc. These heterocyclic ligands may be optionally substituted.

The terms "alkenyl" and "alkynyl" refer to a straight or branched chain having one or more carbon-carbon double bonds or one or more carbon-carbon triple bonds, respectively, and having two to twelve carbon atoms A chain hydrocarbon chain group that is attached to the rest of the molecule by a single bond. In one embodiment of the invention, the alkenyl or alkynyl group has two to eight, two to six, two or three carbon atoms. The double bond of the alkenyl group or the triple bond of the alkynyl group can be unconjugated or conjugated with another unsaturated group. Suitable alkenyl groups include, but are not limited to, alkenyl groups such as vinyl, allyl, butenyl, butadienyl, or pentadienyl. Suitable alkynyl groups include, but are not limited to, alkynyl groups such as _-CCH , _-CH2CCH , _-CCCH3 , _-CH2CCCH3 .

The term "aryl" refers to an aromatic group having 6 to 24, preferably 6 to 18, more preferably 6 to 16, even more preferably 6 to 10 carbon atoms, comprising 1, 2, 3 or 4 aromatic rings aromatic groups, the aromatic rings being bonded through carbon-carbon bonds or fused, such as and in a non-limiting sense including phenyl, naphthyl, diphenyl, indenyl, anthracenyl, phenanthryl, Pyrene, etc. "Aryl" preferably refers to phenyl.

The term "halogen" refers to bromine, chlorine, iodine or fluorine.

The above groups may be optionally substituted at one or more available positions with one or more suitable groups such as OR', _O- , SR', SOR', SO2R', _OSO2R ', SO ₃ R', SO ₃ ^- , NO ₂ , N(R') ₂ , N(R') ₃ ⁺ , N(R')COR', N(R')SO ₂ R', CN, halogen , COR', CO ₂ R', CO ₂ ^- , OCOR', OCO ₂ R', OCONHR', OCON(R') ₂ , CONHR', CON(R') ₂ , substituted or unsubstituted C ₁ -C ₁₈ alkyl, substituted or unsubstituted C ₂ -C ₁₂ alkenyl, substituted or unsubstituted C ₂ -C ₁₂ alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic groups , wherein each R' group is independently selected from hydrogen, substituted or unsubstituted C ₁ -C ₁₈ alkyl, substituted or unsubstituted C ₂ -C ₁₂ alkenyl, substituted or unsubstituted C ₂ -C ₁₂ Alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic groups. When such groups are themselves substituted, the substituents may be selected from the aforementioned list.

The platinum(II) complex may include, but is not limited to, a complex of formula I or a pharmaceutically acceptable salt thereof:

in:

R ₁ and R ₂ are independently selected from amines, such as -NH ₃ , optionally substituted amines, and optionally substituted heterocyclic amines; or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body;

_R is selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and Each pair of R ₇ is joined together to form

Y is selected from sulfur atom, oxygen atom and -NR, wherein R is selected from H and optionally substituted alkyl;

Z is selected from carbon atoms and nitrogen atoms; and

X is a counter ion, which may include, but is not limited to: halide ions, including fluoride, chloride, bromide, and iodide; triflate; acetate; nitrate; perchlorate; hexafluorophosphate; sulfuric acid root and phosphate.

In a specific embodiment, the platinum(II) complexes may include, but are not limited to, complexes with 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone (Hbt) ligands. The 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone (Hbt) ligand can be fluorescent and can be a leaving group suitable for luminescent platinum(II) complexes. Modification of substituents on Hbt ligands can optimize the anticancer activity of platinum(II) complexes, eg, by modulating their lipophilicity and/or their reaction kinetics. Platinum(II) complexes containing this Hbt ligand can exhibit anticancer activity and photoluminescence properties.

The 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligands may include, but are not limited to, ligands of formula II:

in:

R ₃ is selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl and polyethylene glycol; R ₄ , R ₅ , R ₆ and R ₇ are independently selected from -H, Halogen, -OR, optionally substituted alkyl and polyethylene glycol, or each pair of R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and R ₇ joined together to form

Y is selected from sulfur atoms, oxygen atoms and NR, wherein R is selected from H and optionally substituted alkyl groups.

The 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand can form a single positively charged complex with platinum(II) ions. The platinum(II) complex can include, but is not limited to, a counter anion that coordinates with the platinum(II) complex, thereby balancing the charge.

In a specific embodiment, the platinum(II) complex can include, but is not limited to, a ligand of formula III or a pharmaceutically acceptable salt thereof:

in:

X is a counter ion, which may include, but is not limited to, halide ions such as fluoride, chloride, bromide and iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate radicals.

In a specific embodiment, the platinum(II) complex can include, but is not limited to, a complex of formula IV or a pharmaceutically acceptable salt thereof:

in:

X is a counter ion, which can include, but is not limited to, halide ions (including fluoride, chloride, bromide, and iodide), triflate, acetate, nitrate, perchlorate, hexafluorophosphate, Sulfate and Phosphate.

Methods of treating a subject with cancer may include, but are not limited to, administering a therapeutically effective amount of at least one platinum(II) complex of formula I. The method can induce cancer cell death and/or inhibit cell proliferation in vitro and/or in vivo. The platinum(II) complex is administered intravenously as a short-term infusion in normal saline for the treatment of solid malignancies.

Without wishing to be bound by theory, the platinum(II) complex may have a therapeutic function similar to that of cisplatin. For example, one or more platinum(II) complexes can bind to DNA strands, which can lead to DNA strand cross-linking and ultimately apoptosis.

Numerous pharmaceutical dosage forms are available for administering the platinum(II) complex, including sterile aqueous solutions or dispersions or sterile powders containing the platinum(II) complex. These platinum(II) complexes are suitable for the convenient preparation of sterile injectable or infusible solutions or dispersions. Under the conditions of manufacture and storage, the dosage form can be sterile, fluid and stable. The liquid carrier or excipient can be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyol, vegetable oils, nontoxic glycerides, and suitable mixtures thereof.

This treatment can be used for many types of cancer. For example, the method can be used for sarcomas such as small cell lung cancer, squamous cell carcinoma of the head and neck, and ovarian cancer; lymphoma; bladder cancer; testicular cancer; cervical cancer; and germ cell tumors.

Methods of monitoring cells can include administering at least one platinum(II) complex of formula I, and detecting the fluorescent signal of the platinum(II) complex. The monitoring may include, but is not limited to, monitoring cellular distribution and/or structural changes of the platinum(II) complex in living cells. Structural changes to the platinum(II) complex may include, but are not limited to, release of the ligand 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand from the platinum(II) complex. Methods of monitoring cells can include real-time monitoring.

This application includes the following embodiments:

1. Platinum(II) complexes, including:

or a pharmaceutically acceptable salt thereof, wherein:

R ₁ and R ₂ are independently selected from amines, optionally substituted amines, and optionally substituted heterocyclic amines; or a pair of R ₁ and R ₂ are joined together to form a bidentate ligand containing a nitrogen atom;

_R is selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and Each pair of R ₇ is joined together to form

Y is selected from sulfur atoms, oxygen atoms and NR (wherein R is selected from H and optionally substituted alkyl groups);

Z is selected from carbon atoms and nitrogen atoms; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

2. The platinum(II) complex of any of the above embodiments, comprising:

where Z is a carbon atom;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body;

_R is selected from -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₄ , R ₅ , R ₆ and R ₇ are independently selected from -H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and each pair of R ₇ are joined together to form

Y is selected from sulfur atoms, oxygen atoms and NR (wherein R is selected from -H and optionally substituted alkyl); and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

3. The platinum(II) complex of any of the above embodiments, comprising:

where Y is a sulfur atom;

Z is a carbon atom;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body;

_R is selected from -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₄ , R ₅ , R ₆ and R ₇ are independently selected from -H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and each pair of R ₇ are joined together to form

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

4. The platinum(II) complex of any of the above embodiments, comprising:

where Y is a sulfur atom;

Z is a nitrogen atom;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body;

_R is selected from -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₅ , R ₆ and R ₇ are independently selected from -H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and R ₇ of each pair connected together to form

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

5. The platinum(II) complex of any of the above embodiments, comprising:

where Y is a sulfur atom;

Z is a carbon atom;

R ₃ is -CH ₃ ;

R ₄ , R ₅ , R ₆ and R ₇ are each H;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

6. The platinum(II) complex of any of the above embodiments, comprising:

where Y is a sulfur atom;

Z is a nitrogen atom;

R ₃ is -CH ₃ ;

R ₅ , R ₆ and R ₇ are each -H;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

7. The platinum(II) complex of any of the above embodiments, comprising:

where Y is a sulfur atom;

Z is a carbon atom;

R ₁ and R ₂ are linked together to form (1R, 2R)-1,2-cyclohexanediamine;

R ₃ is -CH ₃ ;

R ₄ , R ₅ , R ₆ and R ₇ are each -H; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

8. The platinum(II) complex of any of the above embodiments, comprising:

where Y is a sulfur atom;

Z is a carbon atom;

R ₁ and R ₂ are each -NH ₃ ;

R ₃ is -CH ₃ ;

R ₄ , R ₅ , R ₆ and R ₇ are each -H; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

9. The platinum(II) complex of any of the above embodiments, comprising:

where Y is a sulfur atom;

Z is a carbon atom;

R ₁ and R ₂ are linked together to form 2,2'-bipyridine;

R ₃ is -CH ₃ ;

R ₄ , R ₅ , R ₆ and R ₇ are each -H; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

13. A method of treating a subject with cancer, including:

A therapeutically effective amount of a platinum(II) complex is administered comprising:

or a pharmaceutically acceptable salt thereof, wherein:

R ₁ and R ₂ are independently selected from amines, optionally substituted amines, and optionally substituted heterocyclic amines; or a pair of R ₁ and R ₂ are joined together to form a bidentate ligand containing a nitrogen atom,

_R is selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and Each pair of R ₇ is joined together to form

Y is selected from sulfur atoms, oxygen atoms and NR (wherein R is selected from H and optionally substituted alkyl groups);

Z is selected from carbon atoms and nitrogen atoms; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

14. The method of any of the above embodiments, wherein the platinum(II) complex comprises:

where Z is a carbon atom;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body;

_R is selected from -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₄ , R ₅ , R ₆ and R ₇ are independently selected from -H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and each pair of R ₇ are joined together to form

Y is selected from sulfur atoms, oxygen atoms and NR (wherein R is selected from -H and optionally substituted alkyl); and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

15. The method of any of the above embodiments, wherein the platinum(II) complex comprises:

where Y is a sulfur atom;

Z is a carbon atom;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body;

_R is selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and Each pair of R ₇ is joined together to form

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

16. The method of any of the above embodiments, wherein the platinum(II) complex comprises:

where Y is a sulfur atom;

Z is a nitrogen atom;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body;

_R is selected from -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₅ , R ₆ and R ₇ are independently selected from -H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and R ₇ of each pair connected together to form

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

17. The method of any of the above embodiments, wherein the platinum(II) complex comprises:

where Y is a sulfur atom;

Z is a carbon atom;

R ₃ is -CH ₃ ;

R ₄ , R ₅ , R ₆ and R ₇ are each -H;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

18. The method of any of the above embodiments, wherein the platinum(II) complex comprises:

where Y is a sulfur atom;

Z is a nitrogen atom;

R ₃ is -CH ₃ ;

R ₅ , R ₆ and R ₇ are each -H;

R ₁ and R ₂ are independently selected from ammonia (-NH ₃ ), optionally substituted amines, and optionally substituted heterocyclic amines, or a pair of R ₁ and R ₂ joined together to form a bidentate complex containing a nitrogen atom body; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

19. The method of any of the above embodiments, wherein the platinum(II) complex comprises:

where Y is a sulfur atom;

Z is a carbon atom;

R ₁ and R ₂ are linked together to form (1R, 2R)-1,2-cyclohexanediamine;

R ₃ is -CH ₃ ;

R ₄ , R ₅ , R ₆ and R ₇ are each -H; and

X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

20. Methods for monitoring cells, including:

Administration of at least one platinum(II) complex comprising:

or a pharmaceutically acceptable salt thereof, wherein:

R ₁ and R ₂ are independently selected from amines, optionally substituted amines, and optionally substituted heterocyclic amines; or a pair of R ₁ and R ₂ are joined together to form a bidentate ligand containing a nitrogen atom;

_R is selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted thienyl, and polyethylene glycol;

R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, halogen, -OR, optionally substituted alkyl and polyethylene glycol, or R ₄ and R ₅ , R ₅ and R ₆ , R ₆ and Each pair of R ₇ is joined together to form

Y is selected from sulfur atoms, oxygen atoms and NR (wherein R is selected from H and optionally substituted alkyl groups);

Z is selected from carbon atoms and nitrogen atoms; and

X is selected from the group consisting of fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate; and

The fluorescence signal of the platinum(II) complex was detected.

The present invention further includes an embodiment of the platinum(II) complex according to the present invention, wherein the bidentate ligand containing a nitrogen atom comprises a bidentate ligand containing a 10- to 64-membered heterocyclic molecule consisting of a carbon atom and one to six heteroatoms selected from nitrogen, oxygen and sulfur, wherein such heterocyclic molecules contain at least one nitrogen atom.

The present invention further includes embodiments of the use of the platinum(II) complexes of the present invention in the manufacture of a medicament for the treatment of a subject suffering from cancer.

The present invention further includes embodiments of the use of the platinum(II) complexes of the present invention in the manufacture of compounds for use in the methods of monitoring cells of the present invention.

Example

The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes may be made therefrom by those skilled in the art, and such modifications or changes are included within the spirit and scope of the present application. Furthermore, any element or limitation of any invention or embodiment thereof disclosed herein may be combined (alone or in any combination) with any and/or all other elements or limitation of any other invention or embodiment thereof disclosed herein, And all such combinations are contemplated, and the scope of the present invention is not limited thereto.

Example 1: Synthesis and characterization of platinum(II) complexes

Example 1 is the synthesis and characterization of a non-limiting example of platinum(II) complexes containing 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone (Hbt) ligands. Hbt ligands were prepared according to reported procedures (Chan, Low et al. 2011). The complex Fe(acac) ₃ (0.20 mmol) and 2-thiosalicylic acid (0.20 mmol) were added to a round bottom flask with a stir bar followed by ethylene glycol (4 mL). The mixture was heated at 120°C under nitrogen atmosphere for 4-24 hours. After cooling to room temperature, the mixture was treated with HCl (aq, 37 wt%, 20 mL) for 0.5 h, then extracted with chloroform (3 x 30 mL). The chloroform extracts were dried over _MgSO4 and evaporated to remove the solvent. The crude product was purified by column chromatography on silica gel (eluent: neat n-hexane to n-hexane/EtOAc (30:1 v/v)).

By adding cis-[PtI ₂ (NH ₃ ) ₂ ] (245 mg, 0.51 mmol) to a 50 mL two-necked flask containing AgNO ₃ (170 mg, 1.0 mmol) in MeOH/H ₂ O (20 mL; volume: 1:1) solution for the synthesis of complex 1. A yellow precipitate formed immediately. The reaction was stirred for 1 hour and then filtered through celite to remove the yellow precipitate. A freshly prepared solution of sodium 2-acetylbenzo[b]thiophen-3-ol (Kbt) (4 mL KOH (aq, 0.128 M) and 96 mg Hbt in MeOH/H was added during ₁₀ min under nitrogen protection O (5 ml; volume: 1:1) mixed). After heating at 60°C overnight, the organic solution was removed by rotary evaporator. Filtration from aqueous solution gave 195 mg of product (yield: 93%). δ _H (300 MHz, DMSO) 7.93 (1 H, d, J 7.8), 7.87 (1 H, d, J 8.2), 7.73 (1 H, t, J 7.1), 7.45 (1 H, t, J 7.2 ), 4.77-4.87 (6 H, m), 2.27 (3 H, s).HRMS (ESI, [M –NO ₃ ] ⁺ ): m/z calcd for C ₁₀ H ₁₃ N ₂ O ₂ PtS: 420.0346 , measured value: 420.0359.

AgOTf (257 mg, 1.0 mmol) in MeOH/H ₂ O (20 mL; vol: 1:1) was prepared by adding (bpy)PtCl ₂ (210 mg, 0.5 mmol) to a 50 mL two-necked flask The synthesis of complex 2 was carried out in solution. A white precipitate formed immediately. The reaction was stirred for 1 hour and then filtered through celite to remove the precipitate. A freshly prepared solution of sodium 2-acetylbenzo[b]thiophen-3-ol (Nabt) (2 mL NaOH (aq, 0.25 M) and 96 mg Hbt in MeOH/H was added during ₁₀ min under nitrogen protection O (5 ml; volume: 1:1) mixed). After heating at 66°C overnight, a yellow precipitate formed. Filtration from aqueous solution gave 280 mg of product, which was washed with 5 mL of MeOH (yield: 81%). δ _H (400 MHz, DMSO) 8.98 (1 H, d, J 5.5), 8.83 (1 H, d, J 5.6), 8.54 (1 H, d, J 8.0), 8.50 (1 H, d, J 8.2 ), 8.41 (1 H, t, J 7.8), 8.35 (1 H, t, J 7.8), 8.26 (1 H, d, J 8.0), 7.89 – 7.84 (2 H, m), 7.81 (1 H, t, J 6.6), 7.76 (1 H, dt, J 7.6, 0.9), 7.46 (1 H, t, J 7.5), 2.46 (3 H, s).δ _F (376 MHz, DMSO) -77.72.HRMS (ESI, [M -OTf] ⁺ ): m/z calcd for C ₂₀ H ₁₅ N ₂ O ₂ PtS: 542.0504, found: 542.0516.

Example 1a: Synthesis and Characterization of Additional Platinum(II) Complexes

By adding cis-[PtI ₂ (NH ₃ ) ₂ ] (245 mg, 0.51 mmol) to a 50 mL two-necked flask containing AgNO ₃ (170 mg, 1.0 mmol) in MeOH/H ₂ O (20 mL; volume: 1:1) solution for the synthesis of complex 1b. A yellow precipitate formed immediately. The reaction was stirred for 1 hour and then filtered through celite to remove the yellow precipitate. A freshly prepared solution of KFbt (4 mL KOH (aq, 0.128 M) and 105 mg HFbt in MeOH/H ₂ O (5 mL; vol: 1 : 1 )) was added under nitrogen during 10 min. After heating at 60°C overnight, the organic solution was removed by rotary evaporator. The product was obtained by filtration from the aqueous solution.

By adding cis-[PtI ₂ (NH ₃ ) ₂ ] (245 mg, 0.51 mmol) to a 50 mL two-necked flask containing AgNO ₃ (170 mg, 1.0 mmol) in MeOH/H ₂ O (20 mL; volume: 1:1) solution for the synthesis of complex 1c. A yellow precipitate formed immediately. The reaction was stirred for 1 hour and then filtered through celite to remove the yellow precipitate. A freshly prepared solution of KBrbt (4 mL of KOH (aq, 0.128 M) and 135.6 mg of HBrbt in MeOH/H ₂ O (5 mL; vol: 1 :1 )) was added under nitrogen protection during 10 min. After heating at 60°C overnight, the organic solution was removed by rotary evaporator. The product was obtained by filtration from the aqueous solution.

By adding cis-[PtI ₂ (NH ₃ ) ₂ ] (245 mg, 0.51 mmol) to a 50 mL two-necked flask containing AgNO ₃ (170 mg, 1.0 mmol) in MeOH/H ₂ O (20 mL; volume: 1:1) solution for the synthesis of complex 1d. A yellow precipitate formed immediately. The reaction was stirred for 1 hour and then filtered through celite to remove the yellow precipitate. A freshly prepared solution of KMebt (4 mL KOH (aq, 0.128 M) and 103 mg HMebt in MeOH/H ₂ O (5 mL; vol: 1 : 1 )) was added under nitrogen protection during 10 min. After heating at 60°C overnight, the organic solution was removed by rotary evaporator. The product was obtained by filtration from the aqueous solution.

By adding cis-[PtI ₂ (NH ₃ ) ₂ ] (245 mg, 0.51 mmol) to a 50 mL two-necked flask containing AgNO ₃ (170 mg, 1.0 mmol) in MeOH/H ₂ O (20 ml; volume: 1:1) solution for the synthesis of complex 1e. A yellow precipitate formed immediately. The reaction was stirred for 1 hour and then filtered through celite to remove the yellow precipitate. A freshly prepared solution of KPhbt (4 mL of KOH (aq, 0.128 M) and 127 mg of HPhbt in MeOH/H ₂ O (5 mL; vol: 1 : 1 ) was added under nitrogen protection during 10 min. After heating at 60°C overnight, the organic solution was removed by rotary evaporator. The product was obtained by filtration from the aqueous solution.

AgOTf (257 mg, 1.0 mmol) in MeOH/H ₂ O (20 mL; vol: 1:1) was prepared by adding (bpy)PtCl ₂ (210 mg, 0.5 mmol) to a 50 mL two-necked flask The synthesis of complex 2b was carried out in solution. A white precipitate formed immediately. The reaction was stirred for 1 hour and then filtered through celite to remove the precipitate. A freshly prepared solution of NaFbt (2 mL of NaOH (aq, 0.25 M) and 105 mg of HFbt in MeOH/ _H2O (5 mL; vol: 1 : 1 )) was added during 10 min under nitrogen protection. After heating at 66°C overnight, a yellow precipitate formed. The product was obtained by filtration from aqueous solution and washed with 5 mL of MeOH.

AgOTf (257 mg, 1.0 mmol) in MeOH/H ₂ O (20 mL; vol: 1:1) was prepared by adding (bpy)PtCl ₂ (210 mg, 0.5 mmol) to a 50 mL two-necked flask The synthesis of complex 2c was carried out in solution. A white precipitate formed immediately. The reaction was stirred for 1 hour and then filtered through celite to remove the precipitate. A freshly prepared NaBrbt solution (2 mL NaOH (aq, 0.25 M) and 134.6 mg HBrbt in MeOH/H ₂ O (5 mL; vol: 1 : 1 ) was added under nitrogen during 10 min. After heating at 66°C overnight, a yellow precipitate formed. The product was obtained by filtration from aqueous solution and washed with 5 mL of MeOH.

Example 2: Stability of platinum(II) complexes to GSH

Example 2 is a non-limiting example of stability to GSH of platinum(II) complexes containing 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligands in PBS solution. Complexes 1 and 2 were dissolved in DMSO/PBS solution (1:9, v/v) to obtain a final concentration of the complexes of 20 μM, respectively. UV-visible absorption spectra were recorded at different time intervals. Complexes 1 and 2 were incubated with GSH (2 mM final) in DMSO/PBS solution (1:9, v/v), respectively. UV-visible absorption spectra were recorded at different time intervals.

Complexes 1 and 2 were dissolved in DMSO/PBS solution (1:19, v/v) to obtain a final concentration of 20 μM of the complexes, respectively. GSH was added to these solutions to a final concentration of 2 mM. The emission spectra of the resulting solutions were recorded at different time intervals with excitation wavelength λex = 381 nm.

The stability of complexes 1 and 2 in PBS solution was examined by UV-visible spectrophotometry (Fig. 5a). For complex 2, no spectral changes were observed after 72 h at room temperature. For complex 1, modest spectral changes were observed. These results indicate that complexes 1 and 2 are relatively stable in aqueous solution.

The reaction of complexes 1 and 2 with GSH was first examined by UV-visible spectrophotometry. Significant spectral changes were obtained when GSH (2 mM) was added to solutions of complexes 1 and 2 (20 μM) in PBS. The response of complexes 1 and 2 to the presence of GSH was examined by monitoring changes in luminescence intensity (Fig. 6). Treatment of complex 1 with GSH produced an increase in emission intensity around 450 nm, and similar findings were observed for complex 2 in the presence of GSH. This indicated that the 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand was released from this platinum(II) complex, which could be attributed to the reaction with GSH. The rate of increase in emission intensity at 450 nm can be related to the release of the 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand from this platinum(II) complex. The emission intensity (λem = 450 nm) of complex 2 reached saturation within 2 h, whereas the emission intensity of complex 1 (λem = 450 nm) did not reach saturation even after 24 h of incubation.

Example 3: Luminescence of platinum(II) complexes in the presence of ctDNA

Example 3 is an emission spectrum of a non-limiting example of this platinum(II) complex in the presence of ctDNA. Solutions of complexes 1 and 2 (20 μM) in DMSO/PBS (1:19, v/v) were prepared. Prepare stock solutions of ctDNA. Aliquots of the ctDNA stock solution were added to the solutions of complexes 1 and 2, respectively. After 5 min of incubation, emission spectra were recorded with excitation wavelength λex = 424 nm.

Complexes 1 and 2 are weakly emissive in aqueous solution, with λmax at 600 nm for complex 1 and 580 nm for complex 2. In the literature, it has been reported that the emission intensity of luminescent platinum(II) complexes is enhanced due to the presence of biomolecules such as DNA. The emission intensities of complexes 1 and 2 were examined in the presence of DNA. As shown in Figure 7, the emission intensity of complex 2 increased 3.1-fold when 0.5 equiv. of ctDNA was added, and 32-fold when 10 equiv. ctDNA was added. In contrast, for complex 1, only a 1.5-fold increase in emission intensity was found upon addition of 10 equivalents of ctDNA. Previous studies have shown that luminescent [Pt ^II (C^N^C)L] ⁿ⁺ complexes display strong luminescence when bound to double-stranded DNA, while relatively weak emission when they are unbound in aqueous solution (Liu, Cheung et al 1996, Che, Yang et al 1999, Ma and Che 2003, Ma, Shum et al 2005, Zou, Liu et al 2014). As described herein, the different emission intensity enrichment effects for complexes 1 and 2 indicate different interactions between this platinum(II) complex and DNA. It has been suggested that complex 2 may bind directly to DNA via intercalation.

Example 4: Fluorescence imaging of platinum(II) complexes with 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligands in living cells

Example 4 is a fluorescence imaging of a non-limiting example of this platinum(II) complex with a 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand in living cells. The difference in the photoluminescent properties of this platinum(II) complex and the 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand provides the basis for determining the in vitro and even in vivo performance of these complexes. Possibility of fate.

SW480 (human colorectal cancer) cells were seeded in glass-bottom dishes (MatTek) and allowed to grow for 24 hours, then treated with 25 μM of complexes 1, 2 or the 1-(3-hydroxybenzo[b]thiophene- 2-yl)ethanone ligand for 30 minutes. After removing the medium, cells were washed with Hank's Balanced Salt Solution (HBSS) and then overlaid with 2 ml of HBSS. A Carl Zeiss LSM700 inverted confocal microscope with a Plan-Apochromat 40 × 1.40NA oil immersion objective was used to capture fluorescence and phase contrast images. The complexes 1, 2 and 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligands were excited using a 405 nm laser. Emissions above 580 nm were collected for complexes 1 and 2 and within 400-500 nm for the 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand.

To achieve real-time visualization of complex 2 in living cells, time-series experiments were performed using a confocal microscope connected to an LCI TC-L staged incubator. SW480 cells were seeded in glass bottom dishes (MatTek) and allowed to grow for 24 hours. A solution of complex 2 (25 μM) in HBSS was prepared. Cells were washed with HBSS and subsequently incubated with complex 2 (25 μM in HBSS) at 37 °C in a staged incubator containing 5 vol% _CO . A Carl Zeiss LSM700 inverted confocal microscope with Plan-Apochromat 40 × 1.40NA oil immersion objective was used to capture fluorescence and phase contrast images. Imaging was captured immediately after treatment with complex 2. For time series trials, the interval time was set to 2.5 min and a total of 20 cycles were captured. Both complex 2 and the 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand were excited using a 405 nm laser. Emissions above 580 nm were collected for complex 2 and within 400-500 nm for the 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand.

The photoluminescent properties of complexes 1, 2 and 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligands allow real-time tracking inside living cells. These include tracking subcellular localization, monitoring structural changes of these platinum(II) complexes under cellular conditions, and biotransformation pathways. For luminescence imaging of complexes 1 and 2, cells were excited at λ = 405 nm and emission at wavelengths over 580 nm was collected. For fluorescence imaging of 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligands, cells were excited at λex = 405 nm and emission at 400–500 nm was collected (Figure 8). Red emission as well as blue emission were observed after cells were incubated with 25 μM of complex 2 for 30 min. For complex 1, only blue emission was observed. The red emission can be attributed to from complex 2, while the blue emission is due to the release of 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone from these platinum(II) complexes under physiological conditions Ligand. Complex 2 shows red and blue emission inside the nucleus. These results indicate that 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand is released from complex 2 and accumulated inside the nucleus after cellular uptake. Notably, complex 2 was found to localize specifically in the nucleus, where red and blue emission was detected. Experiments were performed to track complex 2 in real time inside living cells at various time intervals (Figure 9). Images were captured every 2.5 min while complex 2 was added to the cells. Complex 2 accumulated rapidly inside the nucleus, and red emission was detected after 5 min of incubation; the release of 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand occurred inside the nucleus, and at 15 Blue emission was clearly observed after min incubation. Previous studies have demonstrated accumulation of cisplatin in the nucleus by NanoSIMS combined with fluorescence microscopy (Legin, Schintlmeister et al. 2014). Some highly charged polynuclear platinum drugs have also been found to potentially target the nucleus (Benedetti, Peterson et al. 2011, Wedlock, Kilburn et al. 2013). However, complex 1 shows blue emission inside the cytoplasm, a finding that suggests that complex 1 accumulates inside the cytoplasm and releases the 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand there .

Example 5: In vitro cytotoxicity of platinum(II) complexes and 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligands on cancer cells

Example 5 is a non-limiting example of the in vitro cytotoxicity of this platinum(II) complex with a 1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone ligand against various human cancer cell lines. Non-limiting examples of human cancers include cervical epithelial cancer, colorectal cancer, hepatocellular carcinoma, lung cancer, and ovarian cancer.

The effects of complexes 1 and 2, this Hbt ligand, cisplatin and oxaliplatin on colorectal cancer (SW480, HCT116), non-small cell lung cancer (NCI-H460), hepatocellular carcinoma (Hep G2) were evaluated by MTT analysis In vitro cytotoxicity of human cancer cell lines of , cervical epithelial cancer (HeLa), ovarian cancer (A2780), cisplatin-resistant ovarian cancer (A2780 cis) and normal human fibroblast-like cells (CCD-19Lu). The cytotoxicity of each complex was measured by plotting dose-dependent cell viability curves, and the concentration of the complex that reduced cell viability by 50% was determined as the _IC50 value (Table 1).

Complexes 1 and 2 can exhibit dose- and time-dependent cytotoxicity against these cancer cell lines. Complex 2 showed higher anticancer potency and less cytotoxicity against normal human fibroblast-like CCD-19Lu cells with an _IC50 value of approximately 30 μM. Complex 1 showed comparable cytotoxicity to the reference complex cisplatin and was less cytotoxic to CCD-19Lu cells with _IC50 values over 60 μM. Complexes 1 and 2 were cytotoxic to ovarian cancer cell lines, and they showed comparable _IC50 values against cisplatin-sensitive A2780 cells and cisplatin-resistant A2780 cis cells. However, the anticancer activity of cisplatin and oxaliplatin against A2780 cells was higher than that of A2780 cis cells, and the _IC50 values were 30-fold lower than that against A2780 cis cells. This Hbt ligand is not cytotoxic to cancer cells and normal cells, with IC ₅₀ values greater than 100 μM.

Table 1 shows the effects of complexes 1 and 2, cisplatin, oxaliplatin and this Hbt ligand on human colorectal cancer cells (SW480 and HCT116), human hepatoma cells (Hep G2), human non-small cell lung cancer cells (NCI) -H460), human cervical epithelial cancer cells (HeLa), human ovarian cancer cells (A2780) and their cisplatin-resistant variants (A2780 cis) and normal human fibroblast-like cells (CCD-19Lu) in vitro cytotoxicity for 72 hours _IC50 value.

Table 1

Additional tests have been carried out in the same manner on the following complexes and the results are shown below:

Complexes 1a, 2a and 3a were cytotoxic to various cancer cell lines, whereas the Hbt ligand showed no cytotoxicity.

Table 1a. In vitro cytotoxicity _IC50 values of complexes 1a, 2a, 3a and Hbt ligands against various human cancer cell lines and normal cell lines

SW480 and HCT116 = colorectal cancer; NCl-H460 = non-small cell lung cancer; Hep G2 = hepatocellular carcinoma; HeLa = cervical epithelial carcinoma; NCM460 = normal human colonic mucosal epithelial cells; CCD-19Lu = normal human fibroblast-like cells .

Complexes 1a, 2a and 3a exhibited comparable cytotoxicity against cisplatin-sensitive and cisplatin-resistant cancer cells.

Table 2. In vitro cytotoxicity _IC50 values of complexes 1a, 2a and 3a against cisplatin-sensitive and cisplatin-resistant cancer cells

Table 3. In vitro cytotoxicity _IC50 values of complexes 1b-1e, 2b-2c and different substituted Hbt ligands against human cancer cell lines and normal cell lines

Example 6: Cellular Uptake and Nuclear DNA Binding of Platinum(II) Complexes

Example 6 is a non-limiting example of the cellular uptake and nuclear DNA binding of platinum(II) complexes in cancer cells. As described herein, an exemplary human cancer cell line is the colorectal SW480 cancer cell line. Time-dependent cellular uptake of some exemplary platinum(II) complexes was determined by inductively coupled plasma mass spectrometry (ICP-MS). SW480 cells were seeded in 6-well plates containing DMEM and allowed to grow for 24 h at 37 °C in a humidified 5 vol% _CO incubator. The medium was removed and replaced with medium containing 10 μM complexes 1 and 2 or oxaliplatin, respectively. Cells were incubated with each complex for 0.5 hours, 1 hour, 2 hours, 4 hours and 8 hours. At each time point, cells were harvested by trypsinization, then resuspended in _H2O and sonicated to obtain homogenous cell lysates. Protein concentrations were quantified using the Bradford protein assay, and cell lysates were digested in 68% HNO at 60°C for ₂ hours, followed by overnight digestion at room temperature.

The time-dependent binding of platinum to nuclear DNA was also determined by ICP-MS. SW480 cells were incubated with 10 μM complexes 1 and 2 or oxaliplatin for 1 hour, 2 hours, 4 hours and 8 hours. At each time interval, cells were harvested by trypsinization and then resuspended in 300 microliters of lysis buffer (100 mM NaCl, 25 mM EDTA, 0.5% (w/v) SDS, 0.1 mg/mL proteinase K and 10 mM Tris-HCl, pH 8.0) and incubated overnight at 50°C. Add 300 µL of a phenol:CHCl ₃ mixture (1:1, v/v) to the cell lysate. After vigorous shaking, the solution was centrifuged at 13,000 rpm for 5 minutes. Two layers were formed and the upper layer containing the DNA was transferred to a new tube. 1 μg/mL RNase (DNase-free) was added to the DNA solution and then incubated at 37°C for 1 hour. Next, 200 microliters of ammonium acetate (7.5M) was added, followed by 400 microliters of 100% ethanol, and the mixture was incubated at -80°C for 30 minutes. DNA was pelleted by centrifugation at 10,000 rpm for 5 min, and the DNA pellet was washed with 70% ethanol and air dried. The DNA was dissolved in TE buffer (1 mM EDTA and 10 mM Tris-HCl, pH 7.4). DNA concentration was quantified by measuring absorbance at 260 nm. DNA was digested in concentrated _HNO overnight.

Dilute the digested cell lysate or digested DNA solution further in _H2O so that the final concentration of _HNO3 is less than 5%. Platinum content was quantified by ICP-MS by measuring the most abundant isotope of platinum at m/z 195 and corrected against a calibration curve from a series of platinum standard concentrations. Cellular platinum uptake is expressed as ng platinum/mg protein and platinum binding to nuclear DNA is expressed as ng platinum/mg DNA.

Figure 10 shows the intracellular uptake of platinum and fractional Pt-DNA covalent binding after exposure of SW480 cells to complexes 1 and 2. Complexes 1 and 2 accumulated in SW480 cells at higher levels than oxaliplatin, reflecting the favorable cellular uptake efficiency of the lipophilic Hbt ligands in these complexes. In the case of complexes 1 and 2, a greater amount of platinum was found to bind to nuclear DNA, which was approximately 7-fold higher than the clinically used oxaliplatin after 8 hours of culture.

Example 7: In vivo tumor growth inhibition by platinum(II) complexes

Example 7 is an in vivo tumor growth inhibitory effect of a non-limiting example of platinum(II) complexes in nude mice bearing HeLa xenografts. Female BALB/cAnN-nu (nude) mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were maintained according to the requirements of the Laboratory Animal Unit of the University of Hong Kong (HKU) and experiments were performed based on the guidelines approved by the Committee on the Use of Live Animals in Teaching and Research of HKU. To establish tumors, ² x 106 HeLa cells in 100 microliters of PBS were injected into the back flanks of mice by subcutaneous injection. After tumor volume reached approximately 50 mm, mice were randomly divided into the following ^three groups of four mice: solvent control, complex 1 (10 mg/kg), and complex 2 (2.5 mg/kg). Reconstitute complex 1 in PET (60% polyethylene glycol 400, 30% ethanol, and 10% Tween 80) to a final concentration of 20 μg/μL and complex 2 in PET to a final concentration 5 μg/μL. Complexes 1 and 2 in PET were subsequently diluted in PBS and PBS containing the same amount of PET was also prepared. Complexes 1, 2 and solvent were injected into mice individually by intraperitoneal injection twice or three times a week until the mice were sacrificed.

Tumor size was measured twice or three times a week, and tumor volume (V) was calculated by the following equation:

where a and b are the longest and shortest diameters of the tumor, respectively.

The tumor growth inhibitory effect was calculated by the following equation:

为1或2治疗组的初始肿瘤尺寸，V是1或2治疗组的最终肿瘤尺寸，

是溶剂对照组的初始肿瘤尺寸，并且

是溶剂对照组的最终肿瘤尺寸。in

is the initial tumor size for treatment group 1 or 2, V is the final tumor size for treatment group 1 or 2,

is the initial tumor size of the solvent control group, and

is the final tumor size of the solvent control group.

As shown in Figures 10 and 11, treatment of mice with 10 mg/kg of complex 1 or 2.5 mg/kg of complex 2 twice or three times a week significantly inhibited tumor growth after 10 days (p-value < 0.05 ). Both complexes 1 and 2 reduced tumor volume by more than 60%. Notably, no death or weight loss of mice was detected in the treatment groups.

It is to be understood that the disclosed methods and complexes are not limited to the particular methods, protocols and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and complexes described herein. Such equivalents are intended to be covered by the following claims.

While the invention is described herein with reference to illustrative embodiments for particular applications, it should be understood that the invention is not limited thereto. Those of ordinary skill in the art and having access to the teachings provided herein will recognize additional modifications, applications, and embodiments within their scope, as well as other fields in which the present invention has significant utility. Accordingly, the appended claims are intended to cover any and all such applications, modifications and embodiments within the scope of the present invention.

Various terms have been defined above. To the extent a term used in a claim is not defined above, it is to be given the broadest definition given to that term by one skilled in the relevant art, as reflected in at least one printed publication or issued patent.

As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, if the terms "comprising," "having," "containing," or variations thereof are used in the detailed description and/or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The transitional terms/phrases (and any grammatical variants thereof) "comprising", "consisting essentially of" and "consisting of" are used interchangeably.

As used herein, the use of the singular includes the plural unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. As used herein, use of the term "comprising" and other forms is non-limiting.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance is present or absent, and that the description includes instances in which the event or circumstance exists or instances in which it does not. For example, an optional component in a system means that the component may or may not be present in the system.

All patents, patent applications, provisional applications, and publications mentioned or cited herein are hereby incorporated by reference in their entirety, including all figures and tables, to the extent that they are not inconsistent with the explicit teachings of this specification.

references

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Claims (6)

1. A platinum(II) complex comprising:

in: R ₁ and R ₂ are independently selected from amines, optionally substituted amines, -NH ₃ and optionally substituted heterocyclic amines; or a pair of R ₁ and R ₂ joined together to form a bidentate ligand containing a nitrogen atom; _R is selected from optionally substituted alkyl and optionally substituted aryl; R ₄ , R ₅ , R ₆ and R ₇ are independently selected from H, halogen and optionally substituted alkyl; Y is a sulfur atom; Z is a carbon atom; and X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate. 2. The platinum(II) complex of claim 1, comprising:

in X is selected from fluoride, chloride, bromide, iodide, triflate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate. 3. The platinum(II) complex of claim 1, wherein the nitrogen atom-containing bidentate ligand comprises a bidentate ligand containing a 10- to 64-membered heterocyclic molecule consisting of a carbon atom and one to six Composed from nitrogen, oxygen and sulfur heteroatoms, wherein such heterocyclic molecules contain at least one nitrogen atom. 4. Use of a platinum(II) complex according to any preceding claim in the manufacture of a medicament for the treatment of a subject suffering from cancer. 5. In vitro methods for monitoring cells in vitro for non-disease diagnosis and treatment purposes, including: administering at least one platinum(II) complex as claimed in any one of the preceding claims 1 to 3; and The fluorescent signal of the platinum(II) complex is detected. 6. Use of a platinum(II) complex according to any of the preceding claims 1 to 3 in the manufacture of a compound for use in a method for monitoring cells.

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