WO2018024172A1

WO2018024172A1 - Platinum complexes and methods of use thereof

Info

Publication number: WO2018024172A1
Application number: PCT/CN2017/095150
Authority: WO
Inventors: Chi Ming Che; Di HU; Lai Fung CHAN
Original assignee: University of Hong Kong HKU
Current assignee: University of Hong Kong HKU
Priority date: 2016-08-05
Filing date: 2017-07-31
Publication date: 2018-02-08
Anticipated expiration: 2019-02-05
Also published as: CN109790191B; CN109790191A

Abstract

Disclosed herein are platinum(II) complexes that can include complexes of the formula, or pharmaceutically acceptable salts thereof, wherein: R₁ and R₂ are independently selected from the group consisting of an amine, an optionally substituted amine, and an optionally substituted heterocyclic amine; or the pair of R₁ and R₂ are joined together to form a bidentate ligand containing nitrogen atoms; R₃ is selected from the group consisting of H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted, and polyethylene glycol; R₄, R₅, R₆, and R₇ are independently selected from the group consisting of H, halide, -OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅, R₅ and R₆, R₆ and R₇ is joined together to form Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR (wherein R is selected from the group consisting of H and alkyl optionally substituted); Z is selected from a group consisting of a carbon atom and a nitrogen atom; and X is selected from a group consisting of fluoride, chloride, bromide, iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate. Also disclosed are methods for using the platinum (II) complexes in the treatment of cancer and in the visualization inside cells.

Description

PLATINUM COMPLEXES AND METHODS OF USE THEREOF

FIELD OF THE INVENTION

Disclosed herein are platinum (II) complexes. More particularly, the platinum (II) complexes can include emissive 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligands. Also disclosed are methods for using the platinum (II) complexes in the treatment of cancer and in the monitoring of cells.

BACKGROUND OF THE INVENTION

Cisplatin (cis-diamminedichlorophlatinum) is a member of a family of platinum (II) complexes used in the treatment of cancer. It is one of the more effective anti-cancer drugs； however, cisplatin can have drawbacks. For example, patients can show drug resistance or can have serious side-effects. Carboplatin (cis-diammine (1, 1-cyclobutanedicarboxylato) platinum) is another anti-cancer drug in the family of platinum (II) complexes. Carboplatin has an amine non-leaving ligand like cisplatin, but has a different leaving ligand. Experimental evidence suggests that the anti-cancer mechanisms of cisplatin and carboplatin involve the displacement of the leaving groups by water or other biological nucleophiles and the formation of the ion, [ (NH₃) ₂Pt^II] ²⁺, which can bind to nuclear DNA and result in cell death. Although cisplatin and carboplatin have similar anti-cancer mechanisms, they can display different clinical responses. For example, the side effects associated with carboplatin can be less. There is also increasing interest in developing luminescent transition metal complexes that have therapeutic activity, and can allow for the simultaneous monitoring of cancer progression. The photophysical properties of such complexes allow them to be visualized and traced in vitro and in vivo, thus providing real-time tracking of cellular distribution, structural changes, and biotransformation pathways of anti-cancer platinum (II) complexes. However, not all platinum (II) complexes have the requisite photophysical properties that can allow for disease monitoring.

There is a need, therefore, for new platinum (II) complexes that can have therapeutic benefits and/or can have photophysical properties that allow for the monitoring and tracing of disease progression.

BRIEF SUMMARY OF THE INVENTION

Provided herein are platinum (II) complexes and methods for use thereof. In one specific embodiment, the platinum (II) complexes can include complexes of a Formula I,

or pharmaceutically acceptable salts thereof, wherein: R₁ and R₂are independently selected from the group consisting of an amine, an optionally substituted amine, and an optionally substituted heterocyclic amine； or the pair of R₁ and R₂ are joined together to form a bidentate ligand containing nitrogen atoms； R₃ is selected from the group consisting of H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted, and polyethylene glycol； R₄, R₅, R₆, and R₇ are independently selected from the group consisting of H, halide, –OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅, R₅ and R₆, R₆ and R₇ is joined together to form

Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR (wherein R is selected from the group consisting of H and alkyl optionally substituted) ； Z is selected from a group consisting of a carbon atom and a nitrogen atom； and X is selected from a group consisting of fluoride, chloride, bromide, iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

In another specific embodiment, the method for treating a subject having cancer can include administering a therapeutically effective amount of at least one platinum (II) complex of Formula I.

In another specific embodiment, the method of monitoring cells can include administering an effective amount of at least one platinum (II) complex of Formula I, and detecting a fluorescence signal of the platinum (II) complex.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following detailed description, reference is made to the accompanying figures, depicting exemplary, non-limiting and non-exhaustive embodiments of the invention. So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, can be had by reference to the embodiments, some of which are illustrated in the appended figures. It should be noted, however, that the appended figures illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention can 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.

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

FIG. 1a shows chemical structures of platinum (II) complexes containing the Hbt ligand as a labile leaving group.

FIG. 2 shows the synthetic method for preparing the Hbt ligand, and non-limiting examples of synthetic methods for preparing platinum (II) complexes, according to some embodiments.

FIG. 2a shows in vivo tumor growth inhibition effects of 1a, 2a and 3a, examined using nude mice bearing NCI-H460 xenografts through intravenous injection. (A) Average tumor volumes after treatment with 1a at 10 mg/kg, 2a at 5 mg/kg, 3a at 20 mg/kg or solvent (*p-value <0.05, n＝5) twice or thrice per week. (B) Body weight ofnude mice in the 1a, 2a, 3a treatment groups and solvent control group. (C) Representative photographs of tumors obtained from nude mice in the 1a, 2a, 3a treatment groups and solvent control group.

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

FIG. 3a shows body weight of nude mice in cispaltin (1mg/kg and 3 mg/kg) and complex 1a (10 mg/kg and 20 mg/kg) treatment groups. The mice treated with cispaltin at 3 mg/kg lost total body weight by more than 10％. In constrat, the mice treated with 1a at 20 mg/kg had consistant body weight and non signs of noticebale adverse effects were observed.

FIG. 4 shows the emission spectra of some exemplary platinum (II) complexes and the Hbt ligand in CH₂Cl₂, according to some embodiments.

FIG. 4a shows a series of platinum (II) complexes containing different substituted Hbt ligand.

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

FIG. 6 shows the emission spectra of some exemplary platinum (II) complexes in the presence of GSH in PBS solutions, according to some embodiments. a) emission spectra of

complexes

1 and 2 in the presence of GSH were recorded at different time points with the excitation wavelength at λ_ex＝381 nm. b) the time course of emission intensities of

complexes

1 and 2 at 450 nm in the presence of GSH.

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

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

FIG. 9 shows luminesce imaging time-series of some exemplary platinum (II) complexes inside live cells, according to some embodiments.

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

FIG. 11 shows a) average tumor volumes of nude mice bearing HeLa xenografts after treatment with some exemplary platinum (II) complexes or solvent though intraperitoneal injection. b) body weight of nude mice bearing HeLa xenografts in the platinum (II) complex treatment groups and solvent control group, according to some embodiments.

FIG. 12 shows a) representative photographs of tumors obtained from nude mice in the platinum (II) complex treatment groups and solvent control group. b) representative photographs of nude mice in the platinum (II) complex treatment groups and solvent control group, according to some embodiments.

DETAILED DESCRIPTION OF THE INVENTION

In this specification and in the claims that follow, 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 linear, branched, or cyclic saturated hydrocarbon group typically although not necessarily containing 1 to about 20 carbon atoms, preferably 1 to about 12 carbon atoms, 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although again not necessarily, term "cycloalkyl" intends a cyclic alkyl group, typically having 4 to 8, preferably 5 to 7, carbon atoms. The term "substituted alkyl" refers to alkyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkyl" and "heteroalkyl" refer to alkyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the term "alkyl" includes linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl.

The term "bidentate ligand containing nitrogen atoms" as used herein refers to bidentate ligand containing 10-to 64-, preferably a 10-to 48-or 10-to 36-, membered heterocyclic molecules, which consists of carbon atoms and from one to six, preferably 1, 2, 3, 4 or 5, heteroatoms selected from the group consisting of nitrogen, oxygen, and sulphur where such heterocyclic molecules contain at least one nitrogen atoms. The examples of such heterocyclic molecules comprise 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, bathophenanthroline, bisoxazoline, bisthiazoline, bisquinoline, bisisoquinoline, quinolinylpyridine, quinolinylphenanthroline and the like.

The term "heterocyclic" in the context of the invention refers to a stable 5-to 32-, preferably a 5-to 24-or 5-to 18-, membered heterocyclic group, which consists of carbon atoms and from one to six, preferably 1, 2 or 3, heteroatoms selected from the group consisting of nitrogen, oxygen, and sulphur. The term "5-to 32-membered heterocyclic group" as used herein means a heterocyclic radical having a skeleton of from 5 to 32 atoms. For the purposes of this invention, the heterocycle may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include bonded or fused ring systems； and the heterocyclyl radical may be partially or fully saturated or aromatic (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, bathophenanthroline, bisoxazoline, bisthiazoline, bisquinoline, bisisoquinoline, quinolinylpyridine, quinolinylphenanthroline and the like. These heterocyclic ligands may be optionally substituted.

The terms "alkenyl" and "alkynyl" refer to linear or branched hydrocarbon chain radical having one or more carbon-carbon double bonds or one or more carbon-carbon triple bonds, respectively, and having from two to twelve carbon atoms, and which are attached to the rest of the molecule by a single bond. In an embodiment of the invention the alkenyl or the alkynyl has two to eight, two to six, two or three carbon atoms. The double bond of an alkenyl or the triple bond of an alkynyl group can be unconjugated or conjugated to 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 between 6 and 24, preferably between 6 and 18, more preferably between 6 and 16, even more preferably between 6 and 10 carbon atoms, comprising 1, 2, 3 or 4 aromaticrings, bound by means of a carbon-carbon bond or fused, including for example and in a non-limiting sense, phenyl, naphthyl, diphenyl, indenyl, anthryl, phenanthryl, pyrenyl, etc. Preferably "aryl" refers to phenyl.

The term "halogen" refers to bromo, chloro, iodo or fluoro.

The above mentioned groups may be optionally substituted at one or more available positions by one or more suitable groups such as OR', O^-, SR', SOR', SO₂R', OSO₂R', SO₃R', SO₃ ^-, NO₂, N (R') ₂, N (R') ₃ ⁺, N (R') COR', N (R') SO₂R', CN, halogen, COR', CO₂R', CO₂ ^-, OCOR', OCO₂R', OCONHR', OCON (R') ₂, CONHR', CON (R') ₂, substituted or unsubstituted C₁-C₁₈ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, substituted or unsubstituted C₂-C₁₂ alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R' groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C₁-C₁₈alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, substituted or unsubstituted C₂-C₁₂ alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list.

The platinum (II) complexes can include, but are not limited to, complexes of Formula I:

(Formula I)

or pharmaceutically acceptable salts thereof, wherein:

R₁ and R₂ are independently selected from the group consisting of an amine, such as–NH₃, an optionally substituted amine, and an optionally substituted heterocyclic amine； or the pair of R₁ and R₂ are joined together to form a bidentate ligand containing nitrogen atoms；

R₃ is selected from the group consisting of H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted, and polyethylene glycol；

R₄, R₅, R₆, and R₇ are independently selected from the group consisting of H, halide, –OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅, R₅ and R₆, R₆ and R₇ is joined together to form

Y is selected from a group consisting of a sulfur atom, oxygen atom, and–NR, wherein R is selected from the group consisting of H and alkyl optionally substituted；

Z is selected from a group consisting of a carbon atom and a nitrogen atom； and

X is a counter-ion that can include, but is not limited to: halogens ions, including fluoride, chloride, bromide and iodide； trifluoromethanesulfonate； acetate； nitrate； perchlorate； hexafluorophosphate； sulfate and phosphate.

In a specific embodiment, the platinum (II) complexes can include, but are not limited to, complexes with a 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone (Hbt) ligand. The 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone (Hbt) ligand can be fluorescent, and can be a suitable leaving group for luminescent platinum (II) complexes. Modification of substituents on the Hbt ligand can allow for the optimization of the anti-cancer activity of platinum (II) complexes, such as by regulating their lipophilicity and/or their reaction kinetics. The platinum (II) complexes containing the Hbt ligand can display both anti-cancer activity and photoluminescent properties.

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

wherein:R₃ is selected from the group consisting of H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted and polyethylene glycol; R₄, R₅, R₆ and R₇ are independently selected from the group consisting of–H, halide, –OR, alkyl optionally substituted and polyethylene glycol, or each pair ofR₄ and R₅; R₅ and R₆; R₆ and R₇ is joined together to form

and

Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR, wherein R is selected from the group consisting of H and alkyl optionally substituted.

The 1- (3-Hydroxybenzo [b] thiophen-2-yl) ethanone ligand can form a single positively charged complex with the platinum (II) ion. The platinum (II) complex can include, but is not limited to, a counter-anion to coordinate to the platinum (II) complex to balance the charge.

In a specific embodiment, the platinum (II) complexes can include, but are not limited, ligands of Formula III:

or pharmaceutically acceptable salts thereof wherein:

X is a counter-ion that can include, but is not limited to: halogens ions, such as fluoride, chloride, bromide and iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate, and phosphate.

In a specific embodiment, the platinum (II) complexes can include, but are not limited to, complexes of Formula IV:

or pharmaceutically acceptable salts thereof, wherein:

X is a counter-ion that can include, but is not limited to: halogens ions (including fluoride, chloride, bromide and iodide) , trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate, and phosphate.

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

Without wanting to be bound by theory, the platinum (II) complexes can have similar therapeutic functioning to the cisplatin class of drugs. For example, one or more of the platinum (II) complexes can bind to DNA strands, which can cause the DNA strands to crosslink and ultimately result in apoptosis.

Many pharmaceutical dosage forms are available for administering platinum (II) complexes, including sterile aqueous solutions or dispersions or sterile powders comprising the platinum (II) complexes. These platinum (II) complexes are adapted for facile preparation of sterile injectable or infusible solutions or dispersions. The dosage can be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol, vegetable oil, nontoxic glyceryl esters and suitable mixtures thereof.

The method of treatment can be used for many kinds 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； lymphomas； bladder cancer； testicular cancer； cervical cancer； and germ cell tumors.

The method of monitoring of cells can include administering at least one platinum (II) complex of Formula I, and detecting the fluorescence signals of the platinum (II) complex. The monitoring can include, but is not limited to, monitoring the cellular distribution and/or structural changes of the platinum (II) complexes in live cells. The structural changes of the platinum (II) complexes can include, but are not limited to, a release of the ligand 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand from the platinum (II) complex. The method of monitoring cells can include real-time monitoring.

The present application includes the following embodiments:

1. A platinum (II) complex comprising:

or pharmaceutically acceptable salts thereof, wherein:

R₁ and R₂ are independently selected from the group consisting of an amine, an optionally substituted amine, and an optionally substituted heterocyclic amine； or the pair of R₁ and R₂ are joined together to form a bidentate ligand containing nitrogen atoms；

Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR (wherein R is selected from the group consisting of H and alkyl optionally substituted) ；

X is selected from a group consisting of fluoride, chloride, bromide, iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.

2. The platinum (II) complex of any one of the above embodiment (s) comprising:

wherein Z is a carbon atom；

R₁ and R₂are independently selected from the group consisting of ammonia (-NH₃) , an optionally substituted amine, and an optionally substituted heterocyclic amine, or the pair of R₁ and R₂ is joined together to form a bidentate ligand containing nitrogen atom；

R₃ is selected from the group consisting of–H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted and polyethylene glycol；

R₄, R₅, R₆ and R₇ are independently selected from the group consisting of–H, halide, -OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅; R₅ and R₆; R₆ and R₇ is joined together to form

Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR (wherein R is selected from the group consisting of–H and alkyl optionally substituted) ; and

3. The platinum (II) complex of any one of the above embodiment (s) comprising:

wherein Y is a sulfur atom;

Z is a carbon atom;

R₁ and R₂ are independently selected from the group consisting of ammonia (-NH₃) , an optionally substituted amine, and an optionally substituted heterocyclic amine, or the pair of R₁ and R₂ is joined together to form a bidentate ligand containing nitrogen atom;

R₃ is selected from the group consisting of–H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted and polyethylene glycol;

and

4. The platinum (II) complex of any one of the above embodiment (s) comprising:

wherein Y is a sulfur atom；

Z is a nitrogen atom；

R₁ and R₂ are independently selected from the group consisting of ammonia (-NH₃) , an optionally substituted amine, and an optionally substituted heterocyclic amine, or the pair of R₁ and R₂ is joined together to form a bidentate ligand containing nitrogen atom；

R₅, R₆ and R₇ are independently selected from the group consisting of–H, halide, -OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅； R₅ and R₆； R₆ and R₇ is joined together to form

and

5. The platinum (II) complex of any one of the above embodiment (s) comprising:

wherein Y is a sulfur atom；

Z is a carbon atom；

R₃ is–CH3；

R₄, R₅, R₆ and R₇ are each H；

R₁ and R₂are independently selected from the group consisting of ammonia (-NH₃) , an optionally substituted amine, and an optionally substituted heterocyclic amine, or the pair of R₁ and R₂ is joined together to form a bidentate ligand containing nitrogen atom； and

6. The platinum (II) complex of any one of the above embodiment (s) comprising:

wherein Y is a sulfur atom；

Z is a nitrogen atom；

R₃ is–CH₃；

R₅, R₆ and R₇ are each–H；

7. The platinum (II) complex of any one of the above embodiment (s) comprising:

wherein Y is a sulfur atom；

Z is a carbon atom；

R₁ and R₂ are joined together to form (1R, 2R) -1, 2-cyclohexanediamine；

R₃ is–CH₃；

R₄, R₅, R₆ and R₇ are each–H； and

8. The platinum (II) complex of any one of the above embodiment (s) comprising:

wherein Y is a sulfur atom；

Z is a carbon atom；

R₁ and R₂ are each–NH₃；

R₃ is–CH₃；

R₄, R₅, R₆ and R₇ are each–H； and

9. The platinum (II) complex of any one of the above embodiment (s) comprising:

wherein Y is a sulfur atom；

Z is a carbon atom；

R₁ and R₂ are joined together to form 2, 2’-bipyridine；

R₃ is–CH₃；

R₄, R₅, R₆ and R₇ are each–H； and

13. A method for treating a subject having a cancer comprising:

administering a therapeutically effective amount of a platinum (II) complex comprising:

or pharmaceutically acceptable salts thereof, wherein:

R₁ and R₂ are independently selected from the group consisting of an amine, an optionally substituted amine, and an optionally substituted heterocyclic amine； or the pair of R₁ and R₂ are joined together to form a bidentate ligand containing nitrogen atoms,

14. The method of any one of the above embodiment (s) , in which the platinum (II) complex comprises:

wherein Z is a carbon atom；

R₄, R₅, R₆ and R₇ are independently selected from the group consisting of–H, halide, -OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅； R₅ and R₆； R₆ and R₇ is joined together to form

Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR (wherein R is selected from the group consisting of–H and alkyl optionally substituted) ； and

15. The method of any one of the above embodiment (s) , in which the platinum (II) complex comprises:

wherein Y is a sulfur atom；

Z is a carbon atom；

R₃ is selected from the group consisting of H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted and polyethylene glycol；

R₄, R₅, R₆ and R₇ are independently selected from the group consisting of H, halide, –OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅； R₅ and R₆； R₆ and R₇ is joined together to form

and

16. The method of any one of the above embodiment (s) , in which the platinum (II) complex comprises:

wherein Y is a sulfur atom；

Z is a nitrogen atom；

and

17. The method of any one of the above embodiment (s) , in which the platinum (II) complex comprises:

wherein Y is a sulfur atom；

Z is a carbon atom；

R₃ is–CH3；

R₄, R₅, R₆ and R₇ are each–H；

R₁ and R₂ are independently selected from the group consisting of ammonia (-NH₃) , an optionally substituted amine, and an optionally substituted heterocyclic amine, or the pair of R₁ and R₂ is joined together to form a bidentate ligand containing nitrogen atom； and

18. The method of any one of the above embodiment (s) , in which the platinum (II) complex comprises:

wherein Y is a sulfur atom；

Z is a nitrogen atom；

R₃ is–CH₃；

R₅, R₆ and R₇ are each–H；

19. The method of any one of the above embodiment (s) , in which the platinum (II) complex comprises:

wherein Y is a sulfur atom；

Z is a carbon atom；

R₁ and R₂ are joined together to form (1R, 2R) -1, 2-cyclohexanediamine；

R₃ is–CH₃；

R₄, R₅, R₆ and R₇ are each–H； and

20. A method of monitoring cells comprising:

administering at least one platinum (II) complex comprising:

or pharmaceutically acceptable salts thereof, wherein:

Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR (wherein R is selected from the group consisting of H and alkyl optionally substituted) ； Z is selected from a group consisting of a carbon atom and a nitrogen atom； and

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

detecting a fluorescence signal of the platinum (II) complex.

The present invention further comprises the embodiment of the platinum (II) complex according to the present invention, wherein the bidentate ligand containing nitrogen atoms comprises bidentate ligand containing 10-to 64-membered heterocyclic molecules, which consists of carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen, and sulphur, where such heterocyclic molecules contain at least one nitrogen atoms.

The present invention further comprises the embodiment of the use of the platinum (II) complex according to the present invention in the manufacture of medicament for treating a subject having a cancer.

The present invention further comprises the embodiment of the use of the platinum (II) complex according to the present invention in the manufacture of a compound for the method of monitoring cells according to the present invention.

EXAMPLES

The examples and embodiments described herein are for illustrative purposes only and various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

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

Example 1 is the synthesis and characterization of non-limiting examples of the platinum (II) complexes containing the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone (Hbt) ligand. The Hbt ligand was prepared according to a reported procedure (Chan, Low et al. 2011) . A 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 addition of ethylene glycol (4 mL) . The mixture was heated under nitrogen atmosphere at 120℃ for 4-24 h. After being cooled to room temperature, the mixture was treated with HCl (aq, 37 wt％, 20 mL) for 0.5 h and then extracted by chloroform (3×30 mL) . The chloroform extract was 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) ) .

The synthesis of complex 1, was performed by adding cis- [PtI₂ (NH₃) ₂] (245mg, 0.51mmol) to a solution of MeOH/H₂O (20ml； Volumn: 1: 1) with AgNO₃ (170mg, 1.0mmol) in a 50 mL of two-neck flask. A yellow precipitate formed immediately. The reaction was stirring for 1h, then filtered through celite to remove yellow precipitate. Freshly prepared solution of sodium 2-acetylbenzo [b] thiophen-3-olate (Kbt) (4ml of KOH (aq, 0.128M) and 96mg of Hbt were mixed in MeOH/H₂O (5ml； Volumn: 1: 1) ) was added during 10 mins under nitrogen protection. After heating at 60 ℃ for overnight, organic solution was removed by rotatory evaporator. 195 mg of product was obtained by filtration from aqueous solution (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–NO₃] ⁺) : m/z Calcd for C₁₀H₁₃N₂O₂PtS: 420.0346, found: 420.0359.

Synthesis of complex 2 was performed by adding (bpy) PtCl₂ (210 mg, 0.5 mmol) to a solution of MeOH/H₂O (20 ml； Volumn: 1: 1) with AgOTf (257 mg, 1.0 mmol) in a 50 mL of two-neck flask. A white precipitate formed immediately. The reaction was stirring for 1 h, and then filtered through celite to remove precipitate. Freshly prepared solution of sodium 2- acetylbenzo [b] thiophen-3-olate (Nabt) (2 ml of NaOH (aq, 0.25 M) and 96 mg of Hbt were mixed in MeOH/H₂O (5 ml； Volumn: 1: 1) ) was added during 10 mins under nitrogen protection. After heating at 66 ℃ for overnight, yellow precipitate turned out. 280 mg of product was obtained by filtration from aqueous solution, washed by 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 C₂₀H₁₅N₂O₂PtS: 542.0504, found: 542.0516.

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

The synthesis of complex 1b, was performed by adding cis- [PtI₂ (NH3) ₂] (245 mg, 0.51 mmol) to a solution of MeOH/H₂O (20ml； Volume 1: 1) with AgNO₃ (170mg, 1.0mmol) in a 50 mL of two-neck flask. A yellow precipitate formed immediately. The reaction was stirring for 1h, then filtered through celite to remove yellow precipitate. Freshly prepared solution of KFbt (4ml of KOH (aq, 0.128M) and 105 mg of HFbt were mixed in MeOH/H₂O (5ml； Volume 1: 1) ) was added during 10 mins under nitrogen protection. After heating at 60 ℃ for overnight, organic solution was removed by rotatory evaporator. The product was obtained by filtration from aqueous solution.

The synthesis of complex 1c, was performed by adding cis- [PtI₂ (NH3) ₂] (245 mg, 0.51 mmol) to a solution of MeOH/H₂O (20ml； Volume 1: 1) with AgNO₃ (170mg, 1.0mmol) in a 50 mL of two-neck flask. A yellow precipitate formed immediately. The reaction was stirring for 1h, then filtered through celite to remove yellow precipitate. Freshly prepared solution of KBrbt (4ml of KOH (aq, 0.128M) and 135.6 mg of HBrbt were mixed in MeOH/H₂O (5ml； Volumn: 1: 1) ) was added during 10 mins under nitrogen protection. After heating at 60 ℃ for overnight, organic solution was removed by rotatory evaporator. The product was obtained by filtration from aqueous solution.

The synthesis of complex 1d, was performed by adding cis- [PtI₂ (NH3) ₂] (245 mg, 0.51 mmol) to a solution of MeOH/H₂O (20ml； Volume 1: 1) with AgNO₃ (170mg, 1.0mmol) in a 50 mL of two-neck flask. A yellow precipitate formed immediately. The reaction was stirring for 1h, then filtered through celite to remove yellow precipitate. Freshly prepared solution of KMebt (4ml of KOH (aq, 0.128M) and 103 mg of HMebt were mixed in MeOH/H₂O (5ml； Volumn: 1: 1) ) was added during 10 mins under nitrogen protection. After heating at 60 ℃ for overnight, organic solution was removed by rotatory evaporator. The product was obtained by filtration from aqueous solution.

The synthesis of complex 1e, was performed by adding cis- [PtI₂ (NH3) ₂] (245 mg, 0.51 mmol) to a solution of MeOH/H₂O (20ml； Volume 1: 1) with AgNO₃ (170mg, 1.0mmol) in a 50 mL of two-neck flask. A yellow precipitate formed immediately. The reaction was stirring for 1h, then filtered through celite to remove yellow precipitate. Freshly prepared solution of KPhbt (4ml of KOH (aq, 0.128M) and 127 mg of HPhbt were mixed in MeOH/H₂O (5ml； Volumn: 1: 1) ) was added during 10 mins under nitrogen protection. After heating at 60 ℃ for overnight, organic solution was removed by rotatory evaporator. The product was obtained by filtration from aqueous solution.

Synthesis of complex 2b was performed by adding (bpy) PtCl₂ (210 mg, 0.5 mmol) to a solution of MeOH/H₂O (20 ml； Volume 1: 1) with AgOTf (257 mg, 1.0 mmol) in a 50 mL of two-neck flask. A white precipitate formed immediately. The reaction was stirring for 1 h, and then filtered through celite to remove precipitate. Freshly prepared solution of NaFbt (2 ml of NaOH (aq, 0.25 M) and 105 mg of HFbt were mixed in MeOH/H₂O (5 ml； Volume 1: 1) ) was added during 10 mins under nitrogen protection. After heating at 66 ℃ for overnight, yellow precipitate turned out. Product was obtained by filtration from aqueous solution, washed by 5 ml of MeOH.

Synthesis of complex 2c was performed by adding (bpy) PtCl₂ (210 mg, 0.5 mmol) to a solution of MeOH/H₂O (20 ml； Volume 1: 1) with AgOTf (257 mg, 1.0 mmol) in a 50 mL of two-neck flask. A white precipitate formed immediately. The reaction was stirring for 1 h, and then filtered through celite to remove precipitate. Freshly prepared solution of NaBrbt (2 ml of NaOH (aq, 0.25 M) and 134.6 mg of HBrbt were mixed in MeOH/H₂O (5 ml； Volumn: 1: 1)) was added during 10 mins under nitrogen protection. After heating at 66 ℃ for overnight, yellow precipitate turned out. Product was obtained by filtration from aqueous solution, washed by 5 ml of MeOH.

Example2: Stability of platinum (II) complexes towards GSH

Example 2 is the stability of non-limiting examples of the platinum (II) complexes containing the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand towards GSH in PBS solutions.

Complexes

1 and 2 were dissolved in DMSO/PBS solutions (1: 9, v/v) to give the complex at a final concentration of 20μM, respectively. The UV-visible absorption spectra were recorded at different time intervals.

Complexes

1 and 2 were separately incubated with GSH (final 2 mM) in DMSO/PBS solutions (1: 9, v/v) . The UV-visible absorption spectra were recorded at different time intervals.

Complexes

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

The stability of

complexes

1 and 2 in PBS solutions were examined by UV-visible spectrophotometry (FIG. 5a) . For complex 2, no spectral change was observed after 72 h at room temperature. For complex 1, moderate spectral change was observed. These results reveal that

complexes

1 and 2 are relatively stable in aqueous solutions.

The reactions of

complexes

1 and 2 with GSH were first examined by UV-visible spectrophotometry. Upon the addition of GSH (2 mM) to solutions of complexes 1 and 2 (20 μM) in PBS, significantly spectral changes were obtained. The responses of

complexes

1 and 2 to the presence of GSH were examined by monitoring changes in emission intensity (FIG. 6) . Treating complex 1 with GSH produced an increase in emission intensity around 450 nm, and similar finding was observed for complex 2 in the presence of GSH. This is indicative of release of the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand from the platinum (II) complexes attributable to the reaction with GSH. The rate of increase in emission intensity at 450 nm may be taken to correlate with release of the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand from the platinum (II) complexes. The emission intensity (λ_em＝450 nm) of complex 2 reached saturation within 2 h, while the emission intensity ( (λ_em＝450 nm) of complex 1 did not reach saturation even after an incubation time of 24 h.

Example3: Luminescence of platinum (II) complexes in the presence of ctDNA

Example 3 is the emission spectra of non-limiting examples of the platinum (II) complexes in the presence of ctDNA. Solutions of complexes 1 and 2 (20μM) in DMSO/PBS (1:19, v/v) were prepared. A stock solution of ctDNA was prepared. Aliquots of the stock ctDNA solution were added into the solutions of

complexes

1 and 2, respectively. After an incubation time of 5 min, the emission spectrum was recorded with excitation wavelength at λ_ex ＝424 nm.

Complexes

1 and 2 are weakly emissive in aqueous solution with λ_max at 600 nm for

complex

1, and 580 nm for complex 2. In literature, enhancement on the emission intensity of luminescent platinum (II) complexes due to the presence of biological molecules, such as DNA, has been reported. The emission intensity of

complexes

1 and 2 in the presence of DNA was examined. As depicted in FIG. 7, the emission intensity of complex 2 is increased by 3.1-fold upon addition of 0.5 equivalent of ctDNA and by 32-fold upon addition of 10 equivalents of ctDNA. In contrast, for complex 1, only 1.5-fold increase in emission intensity was found upon addition of 10 equivalents of ctDNA. Previous studies showed that the luminescent [Pt^II (C^N^C) L] ⁿ⁺complexes display intense luminescence when bound to duplex DNA, while the emission is relatively weak when they are unbound in aqueous solutions (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 noted herein, the different emission intensity enrichment effects for

complexes

1 and 2 are indicative of the different interactions between the platinum (II) complexes and DNA. It is suggested that complex 2 could directly bind to DNA, likely via intercalation.

Example 4: Fluorescence imaging of platinum (II) complexes and the 1- (3- hydroxybenzo [b] thiophen-2-yl) ethanone ligand in live cells

Example 4 is the fluorescence imaging of non-limiting examples of the platinum (II) complexes and the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand in live cells. The differences in photoluminescent properties of the platinum (II) complexes and the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand affords possibility to determine the fate of these complexes in vitro and even in vivo.

SW480 (human colorectal carcinoma) cells were seeded in a glass bottom dish (MatTek corporation) and allowed to grow for 24 h prior to treatment with

25μM complexes

1, 2 or the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand for 30 min, respectively. After removing the medium, cells were washed with Hank’s balanced salt solution (HBSS) and then covered 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 the fluorescence and phase contrast images.

Complexes

1, 2 and the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand were excited using 405 nm laser. Emission over 580 nm was collected for

complexes

1 and 2, and emission within 400-500 nm was collected for the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand.

To achieve the real time visualization of complex 2 in live cells, a time series experiment was performed using a confocal microscope conjugated with a LCI TC-L stage-top incubator. SW480 cells were seeded in a glass bottom dish (MatTek corporation) and allowed to grow for 24 h. A solution of complex 2 (25μM) in HBSS was prepared. Cells were washed with HBSS, and then incubated with complex 2 (25μM in HBSS) in the stage-top incubator containing 5％(v/v) CO₂ at 37℃. A Carl Zeiss LSM700 inverted confocal microscope with a Plan-Apochromat 40×1.40NA oil-immersion objective was used to capture the fluorescence and phase contrast images. Imaging was captured immediately after treatment with complex 2. For time series experiment, the interval time was set to 2.5 min and total 20 cycles were captured. Both complex 2 and the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand were excited using 405 nm laser. Emission over 580 nm was collected for complex 2, and emission within 400-500 nm was collected for the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand.

The photoluminescent properties of

complexes

1, 2 and the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand allows real time tracking inside live cells. These include tracing subcellular localization, monitoring the structural changes as well as biotransformation pathways of these platinum (II) complexes under cellular conditions. For luminescence imaging of

complexes

1 and 2, cells were excited at λ_ex＝405 nm and emissions at wavelength beyond 580 nm were collected. For fluorescence imaging of the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand, cells were excited at λ_ex＝405 nm and emission at 400-500 nm were collected (FIG. 8) . After incubating cells with complex 2 at 25μM for 30 min, a red emission was observed together with a blue emission. For complex 1, only a blue emission was observed. The red emission is attributable to come from complex 2, whereas the blue emission is due to release of the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand from these platinum (II) complexes under physiological conditions. Complex 2 displayed both red and blue emissions inside nucleus. These results are indicative of the release of the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand from complex 2 to occur and accumulate inside the nucleus after cellular uptake. Notably, complex 2 was found to specifically localize in nucleolus where both red and blue emissions were detected. Experiments for real time tracking of complex 2 inside live cells at different time intervals were undertaken (FIG. 9) . Upon addition of complex 2 to cells, imaging was captured every 2.5 min. Complex 2 quickly accumulated inside cellular nucleolus with red emissions detected after 5 min incubation； release of the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand occurred inside the nucleolus and blue emission was clearly observed after 15 min incubation. Previous studies have demonstrated the accumulation of cisplatin within the nucleolus by NanoSIMS combined with fluorescence microscopy (Legin, Schintlmeister et al. 2014) . Some highly-charged polynuclear platinum drugs have also been found to potentially target nucleolus (Benedetti, Peterson et al. 2011, Wedlock, Kilburn et al. 2013) . However, complex 1 displayed blue emission inside cytoplasm, this finding revealed that complex 1 accumulated inside cytoplasm and the release of the 1- (3-hydroxybenzo [b] thiophen-2-yl) ethanone ligand occurred there.

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

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

The in vitro cytotoxicity of

complexes

1 and 2, the Hbt ligand, cisplatin and oxaliplatin towards human cancer cell lines of colorectal carcinoma (SW480, HCT116) , non-small-cell lung carcinoma (NCI-H460) , hepatocellular carcinoma (Hep G2) , cervical epithelioid carcinoma (HeLa) , ovarian carcinoma (A2780) , cisplatin-resistant ovarian carcinoma (A2780 cis) , and normal human fibroblast-like cells (CCD-19Lu) were evaluated by MTT assays. The cytotoxicity of each complex was measured by plotting the dose-dependent cell viability curve and the concentration of complex decreasing cellular viability by 50％was determined as IC₅₀ value (Table 1) .

Complexes

1 and 2 can exhibit dose-and time-dependent cytotoxicity towards these cancer cell lines. Complex 2 display higher anti-cancer potency and it is less cytotoxic towards the normal human fibroblast-like CCD-19Lu cells with an IC₅₀ value around 30 μM.Complex 1 display comparable cytotoxicity to the reference complex cisplatin and it is less cytotoxic towards CCD-19Lu cells with an IC₅₀ value over 60μM.

Complexes

1 and 2 are cytotoxic towards ovarian carcinoma cell lines and they displayed comparable IC₅₀ values towards both cisplatin-sensitive A2780 cells and cisplatin-resistant A2780 cis cells. However, cisplatin and oxaliplatin display higher anti-cancer activity towards A2780 cells than A2780 cis cells, with IC₅₀ values that are 30-fold lower than that towards A2780 cis cells. The Hbt ligand is not cytotoxic against cancer cells and normal cells with the IC₅₀ values larger than 100μM.

Table 1 shows the in vitro cytotoxic IC₅₀ values of

complexes

1 and 2, cisplatin, oxaliplatin and the Hbt ligand against human colorectal cancer cells (SW480 and HCT116) , human hepatocellular cancer cells (Hep G2) human non-small lung cancer cells (NCI-H460) , human cervical epithelioid cancer cells (HeLa) , human ovarian cancer cells (A2780) and its cisplatin-resistant variant (A2780 cis) , and normal human fibroblast-like cells (CCD-19Lu) , 72h.

Table 1

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

Complexes

1a, 2a and 3a was cytotoxic towards various cancer cell lines, while the Hbt ligand showed no cytotoxicity.

Table 1a. in vitro cytotoxic IC₅₀ values of

complexes

1a, 2a, 3a and the Hbt ligand towards various human cancer cell lines and normal cell lines.

Cytotoxicity (IC₅₀， μM±SD) ， 72 h

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

Complexes

1a, 2a and 3a displayed comparable cytotoxicity towards both cisplatin-sensitive and cisplatin-resistant cancer cells.

Table 2. in vitro cytotoxic IC₅₀ values of

complexes

1a, 2a and 3a towards cisplatin-sensitive and cisplatin-resistant cancer cells.

Cytotoxicity (IC₅₀， μM±SD) ， 72 h

Table 3. In vitro cytotoxic IC₅₀ values of complexes 1b-1e, 2b-2c, and different substituted Hbt ligands towards human cancer cell lines and normal cell line.

Cytotoxicity (IC₅₀, μM±SD) , 72 h

Example 6: Cellular uptake and nuclear DNA binding of platinum (II) complexes

Example 6 is the cellular uptake and nuclear DNA binding of non-limiting examples of platinum (II) complexes in cancer cells. As described herein, one exemplary human cancer cell line is colorectal SW480 cancer cell line. The 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 a 6-well plate with DMEM and allowed to grown for 24 h in a humidified 5％CO₂ (v/v) incubator at 37 ℃. The culture 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 h, 1 h, 2 h, 4 h and 8 h. At each time point, cells were harvested by trypsinisation, followed by resuspending in H₂O and sonication to obtain a homogenous cell lysate. The protein concentrations were quantified using Bradford protein assay and cell lysates were digested in 68％HNO₃ at 60 ℃ for 2 h and then at room temperature, overnight.

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 h, 2 h, 4 h and 8 h. At each time interval, cells were harvested by trypsinisation, followed by resuspending in 300 μL lysing 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 incubating at 50 ℃, overnight. A 300 μL phenol: CHCl₃ mixture (1: 1, v/v) was added to the cell lysates. After vigorously shaking, solution was centrifuged at 13,000 rpm for 5 min. Two layers were formed and the upper layer containing DNA was transferred to a new tube. The 1μg/mL RNase, DNase-free was added to DNA solution and then incubated at 37 ℃ for 1 h. Nest, 200 μL ammonium acetate (7.5 M) was added, followed by 400 μL 100％ethanol, and the mixture was incubated at -80 ℃ for 30 min. DNA was precipitated by centrifuging at 10,000 rpm for 5 min and the DNA pellet was rinsed with 70％ethanol and dried by air. The DNA was dissolved in TE buffer (1 mM EDTA and 10 mM Tris-HCl, pH 7.4) . The DNA concentration was quantified by measuring the absorbance at 260 nm. DNA was digested in concentrated HNO₃, overnight.

The digested cell lysates or digested DNA solutions were further diluted in H₂O to make the final concentration of HNO₃ less than 5％. Platinum contents were quantified with an ICP- MS by measuring the most abundant isotope of platinum at m/z 195 and corrected with respect to a calibration curve from a series of concentrations of platinum standards. The cellular platinum uptake is expressed as ng platinum/mg protein and the binding of platinum to nuclear DNA is expressedas ng platinum/mg DNA.

FIG. 10 shows the intracellular uptake of platinum and Pt-DNA covalent binding fractions afterexposure of SW480 cells to

complexes

1 and 2.

Complexes

1 and 2 accumulate in SW480 cells at higher levels than oxaliplatin, reflecting the favorable cellular uptake efficiency due to the lipophilic the Hbt ligand in these complexes. Higher quantities of platinum are found to bind to nuclear DNA in the cases of

complexes

1 and 2, which were about 7-fold higher than clinically used oxaliplatin after an 8 h incubation.

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

Example 7 is the in vivo tumor growth inhibition effects of non-limiting examples of platinum (II) complexes in nude mice bearing HeLa xenograft. Female BALB/cAnN-nu (Nude) mice were purchased from the 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 conducted based on the guidelines approved by the Committee on the Use of Live Animals in Teaching and Research of HKU. To establish tumor, 2×10⁶ HeLa cells in 100 μL PBS were injected into the back flanks of mice by subcutaneous injection. After the tumor volumes reached around 50 mm³, the mice were randomly divided into the following three groups with four mice per group: solvent control, complex 1 (10 mg/kg) and complex 2 (2.5 mg/kg) . Complex 1 was reconstituted inPET (60

％polyethylene glycol

400, 30％ethanol and 10％Tween 80) to a final concentration of 20 μg/μL, and complex 2 was reconstituted in PET to a final concentration of 5 μg/μL.

Complexes

1 and 2 in PET were then diluted in PBS and PBS containing the same amount ofPET was also prepared.

Complexes

1, 2 and solvent were separately injected into mice by intraperitoneal injections twice or thrice per weekuntilthe micewere sacrificed.

Tumor sizes were measured twice or thrice per week and the tumor volume (V) was calculated by the following equation:

V ＝ ab²×0.52,

wherein a and b were the longest and the shortest diameters of the tumor, respectively.

Tumor growth inhibition effect was calculated by the following equation:

wherein V_o was the initial tumor size of group of 1 or 2 treatment, V was the final tumor size of

group

1 or 2 treatment, V′₀ was the initial tumor size of group of solvent control, and V′ was the final tumor size of group of solvent control.

As shown in FIGS. 10 and 11, treatment of mice with complex 1 at 10 mg/kg or complex 2 at 2.5 mg/kg trice or thrice per week significantly inhibit tumor growth after 10 days (p-value<0.05) . Both

complexes

1 and 2 reduce the volume of tumor by more than 60％. It is notable that no mouse death or body weight loss was detected in the treatment group.

It is understood that the disclosed method and complexes are not limited to the particular methodology, 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 present invention which will be 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 method and complexes described herein. Such equivalents are intended to be encompassed by the following claims.

While the present invention is described herein with reference to illustrative embodiments for particular applications, it should be understood that the invention is not limited thereto. Those having ordinary skill in the art and access to the teachings provided herein will recognize additional modifications, applications, and embodiments within the scope thereof and additional fields in which the present invention would be of significant utility. It is therefore intended by the appended claims 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 should be given the broadest definition persons in the pertinent art have given that term 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 indicates otherwise. Furthermore, to the extent that the terms “including” , “includes” , “having” , “has” , “with” , or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising” . The transitional terms/phrases (and any grammatical variations thereof) “comprising” , “comprises” , “comprise” , “consisting essentially of” , “consists essentially of” , “consisting” and “consists” can be 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 “including” as well as other forms, such as “includes, ” and “included, ” is not limiting.

As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, an optional component in a system means that the component may be present or may not be present in the system.

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

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Claims

A platinum (II) complex comprising:

or pharmaceutically acceptable salts thereof, wherein:

R₁ and R₂are independently selected from the group consisting of an amine, an optionally substituted amine, -NH₃, and an optionally substituted heterocyclic amine； or the pair of R₁ and R₂ arejoined together to form a bidentate ligand containing nitrogen atoms；

R₃ is selected from the group consisting of H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted, and polyethylene glycol；

R₄, R₅, R₆, and R₇ are independently selected from the group consisting of H, halide, –OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅, R₅ and R₆, R₆ and R₇ isjoined together to form

Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR, wherein R is selected from the group consisting of H and alkyl optionally substituted；

Z is selected from a group consisting of a carbon atom and a nitrogen atom； and

X is selected from a group consisting of fluoride, chloride, bromide, iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.
The platinum (II) complex of claim 1 comprising:

wherein Z is a carbon atom；

R₁ and R₂are independently selected from the group consisting of -NH₃, an optionally substituted amine, and an optionally substituted heterocyclic amine, or the pair of R₁ and R₂ is joined together to form a bidentate ligand containing nitrogen atom；

R₃ is selected from the group consisting of –H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted and polyethylene glycol；

R₄, R₅, R₆ and R₇ are independently selected from the group consisting of –H, halide, -OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅； R₅ and R₆； R₆ and R₇ isjoined together to form

Y is selected from a group consisting of a sulfur atom, oxygen atom, and NR, wherein R is selected from the group consisting of –H and alkyl optionally substituted； and

X is selected from a group consisting of fluoride, chloride, bromide, iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.
The platinum (II) complex of claim 1 comprising:

wherein Y is a sulfur atom；

Z is a carbon atom；

R₁ and R₂ are independently selected from the group consisting of -NH₃, an optionally substituted amine, and an optionally substituted heterocyclic amine, or the pair of R₁ and R₂ is joined together to form a bidentate ligand containing nitrogen atom；

R₃ is selected from the group consisting of –H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted and polyethylene glycol；

R₄, R₅, R₆ and R₇ are independently selected from the group consisting of –H, halide, -OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅； R₅ and R₆； R₆ and R₇ isjoined together to form

and

X is selected from a group consisting of fluoride, chloride, bromide, iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.
The platinum (II) complex of claim 1 comprising:

wherein Y is a sulfur atom；

Z is a nitrogen atom；

R₁ and R₂are independently selected from the group consisting of -NH₃, an optionally substituted amine, and an optionally substituted heterocyclic amine, or the pair of R₁ and R₂ is joined together to form a bidentate ligand containing nitrogen atom；

R₃ is selected from the group consisting of –H, alkyl optionally substituted, aryl optionally substituted, thienyl optionally substituted and polyethylene glycol；

R₅, R₆ and R₇ are independently selected from the group consisting of –H, halide, -OR, alkyl optionally substituted and polyethylene glycol, or each pair of R₄ and R₅； R₅ and R₆； R₆ and R₇ is joined together to form

and

X is selected from a group consisting of fluoride, chloride, bromide, iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.
The platinum (II) complex of claim 1 comprising:

wherein

X is selected from a group consisting of fluoride, chloride, bromide, iodide, trifluoromethanesulfonate, acetate, nitrate, perchlorate, hexafluorophosphate, sulfate and phosphate.
The platinum (II) complex of claim 1, wherein the bidentate ligand containing nitrogen atoms comprises bidentate ligand containing 10- to 64- membered heterocyclic molecules, which consists of carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen, and sulphur, where such heterocyclic molecules contain at least one nitrogen atoms.
A method for treating a subject having a cancer comprising:

administering a therapeutically effective amount of a platinum (II) complex according to any one of the preceding claims； or the use of the platinum (II) complex in the manufacture of medicament for treating a subject having a cancer.
A method of monitoring cells comprising:

administering at least one platinum (II) complex according to any one of the preceding claims 1 to； and

detecting a fluorescence signal of the platinum (II) complex；

or the use of said platinum (II) complex in the manufacture of a compound for said method of monitoring cells.