Radiation-activated platinum complexes, their preparation and therapeutic uses

Abstract

This invention discloses a platinum complex having a structure of formula (I), particularly formula (A), such as formula (III) or formula (IIIa). A pharmaceutical composition comprising the platinum complex of this invention and a pharmaceutically acceptable carrier. A method for treating a target tissue or the use of the platinum complex in the preparation of a medicament for treating a target tissue, the method / use comprising administering the platinum complex of this invention to a patient in need and irradiating the target tissue with an amount and frequency that effectively activates the compound. The platinum complex of this invention can be used to prepare a medicament for treating the target tissue by sonodynamic therapy, photodynamic therapy, chemotherapy, and / or combinations thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Patent Application No. 18 / 045,210, filed October 10, 2022, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a novel complex, particularly but not limited to a radiation-activated platinum complex. The invention also relates to the preparation of said complex and its use in the treatment of diseases, particularly but not limited to cancer. Background Technology

[0004] Cancer remains a life-threatening disease affecting a growing number of people worldwide. Since the discovery of cisplatin's antitumor activity in 1965, chemotherapy, particularly platinum-based chemotherapeutic agents, appears to be the most common treatment for various types of cancer. While platinum-based chemotherapeutic agents are widely used clinically, with over 50% of cancer patients receiving them alone or in combination with other treatments (such as surgical ablation or radiotherapy), their efficacy is often hampered by numerous drawbacks, including low bioavailability, severe side effects, poor stability, and intrinsic or acquired resistance.

[0005] It has been reported that Pt(IV)-based complexes can be activated through reductive elimination to release the active product, namely Pt(II) drugs, into cells. Since then, the development of Pt(IV)-based prodrugs has attracted considerable attention, particularly photoactivated Pt(IV) prodrugs, given their non-invasiveness and controllability. However, such development often suffers from the problem of limited light penetration into deep tumor sites, which on the one hand reduces the efficacy of Pt(IV) prodrugs against deep tumors; on the other hand, it weakens the side effects associated with the retention of inactivated prodrugs in tissues.

[0006] Another alternative non-invasive therapy, sonodynamic therapy (SDT), has attracted increasing attention due to its high tissue penetration depth, high long-range spatiotemporal selectivity, and non-invasiveness. Similar to PDT, typical SDT involves a "three-component system": a sonosensitive agent, molecular oxygen (O2), and low-intensity ultrasound (US). Specifically, when the sonosensitive agent is exposed to ultrasound in a specific frequency range along with molecular oxygen, reactive oxygen species (ROS) are generated, leading to tumor cell apoptosis and / or necrosis.

[0007] However, the use of single-modality treatments or platinum complexes in the preparation of drugs for treating target tissues, which rely on the generation and effects of ROS on cancer cells, limits the application of SDT in cancer treatment, because the effects of ROS may be counteracted by the intrinsic antioxidant defense mechanisms of cancer cells.

[0008] Therefore, there remains a strong need for new compounds with acceptable side effects that can be used as alternatives or in combination with other types of anticancer drugs or drug carriers to effectively treat cancers, including those with intrinsic or acquired chemotactic resistance. Summary of the Invention

[0009] In a first aspect of the invention, a platinum complex comprising the structure of formula (I) is provided:

[0010]

[0011] in:

[0012] X, X', Y, Y' and Z are independently selected from the group consisting of: ammonia, hydroxide, halide, oxalate, diamine, dicarboxylate, glycolate and –OR. Optionally, X and X' are linked to form a first bidentate ligand, and / or Y and Y' are linked to form a second bidentate ligand.

[0013] n is selected from a group consisting of zero free charge, any positive charge, and any negative charge;

[0014] R represents the radiation response component of the structure with equation (II):

[0015]

[0016] Where L is a linking group, m is 0 or a positive integer, R1 to R8 are each independently a substituent or hydrogen, and adjacent pairs of R2 to R7 can form fused heterocycles or carbocycles.

[0017] Preferably, L is a phenoxy group, a sulfide group, or a secondary amine group; m is 0, 2, or 3; R1 and R8 are independently selected from straight-chain or branched alkyl chains, sulfonate groups, carboxyl groups, carboxylic acid ester groups, or heterocyclic groups; R2 to R7 are each independently selected from hydrogen, halogen, or adjacent pairs of R2 to R7 form a fused 6-membered carbon ring.

[0018] More preferably, L is selected from any of the following groups:

[0019]

[0020] Where p is 1-4, and R 10 It is H, straight-chain or branched C1-C6 alkyl or functional side chain; m is 0, 2 or 3; R1 and R8 are independently selected from straight-chain or branched alkyl chains, sulfonate groups, carboxyl groups, carboxylic acid ester groups or heterocyclic groups; R2 to R7 are each independently selected from hydrogen, halogen, or adjacent pairs of R2 to R7 form a fused 6-membered carbon ring.

[0021] Preferably, Z is not –OR.

[0022] In a preferred embodiment, the platinum complex has the structure of formula (III):

[0023]

[0024] Where X and X' or Y and Y' are connected to form bidentate ligands; and Z is a hydroxide or –OR.

[0025] Preferably, X and X' are linked to form a dicarboxylic acid ester; Y and Y' are ammonia; and Z is a hydroxide.

[0026] Preferably, R1 and R8 are independently selected from –CH3, –(CH2)2CH3, –(CH2)2COOH, –(CH2)5COOH, –(CH2)4SO3H, R2 to R7 are each independently selected from hydrogen, chloride, iodide, bromide, or adjacent pairs of R2 to R7 fused together to form a benzene ring.

[0027] In a preferred embodiment, the platinum complex has the structure of formula (IIIa):

[0028]

[0029] Preferably, the chemical structure of the complex remains unchanged for at least about 6 hours in the absence of radiation.

[0030] Preferably, the complex can be reduced to a reduced form upon exposure to radiation and / or generate reactive oxygen species.

[0031] More preferably, the cytotoxicity of the complex is enhanced by at least about 6 times upon exposure to radiation.

[0032] In a second aspect of the invention, a method for preparing the platinum complex of the first aspect is provided, namely, a method for preparing a platinum complex having the structure of formula (I):

[0033]

[0034] X, X', Y, Y' and Z, R are as described in this article.

[0035] In a third aspect, the present invention provides a pharmaceutical composition comprising the platinum complex of the first aspect and further comprising a pharmaceutically acceptable carrier.

[0036] According to a fourth aspect of the invention, a method for treating a target tissue or the use of a platinum complex in the preparation of a medicament for treating a target tissue is provided, the method / use comprising administering the platinum complex of the first aspect to a patient in need and irradiating the target tissue with an amount and frequency that effectively activates the complex.

[0037] In a preferred embodiment, the radiation is ultrasound. The frequency of the ultrasound is between about 1 MHz and about 3 MHz. The ultrasound is applied with a power of about 1 W to about 4 W.

[0038] In a preferred embodiment, the radiation is light. The frequency of the light is between about 350 THz and about 500 THz. The light has a frequency of about 3 mW / cm². 2 Approximately 350mW / cm 2 The power intensity is applied.

[0039] Preferably, the target tissue is a tumor. Preferably, the tumor is selected from the group consisting of cervical cancer, lung cancer, ovarian cancer, breast cancer, and breast cancer.

[0040] In a preferred embodiment, the tumor has intrinsic or acquired cisplatin resistance.

[0041] In a preferred embodiment, the platinum complex acts as a prodrug.

[0042] In a fifth aspect of the invention, the use of the platinum complex of the first aspect of the invention is provided in the preparation of a medicament for treating target tissues by sonodynamic therapy, photodynamic therapy, chemotherapy and / or combinations thereof. Attached Figure Description

[0043] Figure 1 The synthetic scheme for complex 1 (i.e., a platinum complex having the structure of formula (IIIa)) is shown.

[0044] Figure 2A The image shows commercially available IR780 (top) and ligand 1 (bottom) prepared according to an embodiment of the present invention. 1 H nuclear magnetic resonance (NMR) spectrum.

[0045] Figure 2B The high-resolution mass spectrometry (HR-MS) spectrum of ligand 1 prepared in an embodiment of the present invention is shown.

[0046] Figure 3A The complex 1 prepared in an embodiment of the present invention is shown. 1 H NMR spectrum.

[0047] Figure 3B The complex 1 prepared in an embodiment of the present invention is shown. 13 C NMR spectrum.

[0048] Figure 3C The complex 1 prepared in an embodiment of the present invention is shown. 195 Pt NMR spectrum.

[0049] Figure 4AThe HR-MS spectrum of complex 1 prepared in an embodiment of the present invention is shown. The inset shows the stimulated MS spectrum of complex 1.

[0050] Figure 4B The reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram of complex 1 prepared in an embodiment of the present invention is shown, illustrating the purity of complex 1.

[0051] Figure 5A The RP-HPLC chromatogram of complex 1 (10 μM) in phosphate-buffered saline (PBS) (pH 7.4) measured at 700 nm using a UV-Vis detector is shown.

[0052] Figure 5B The RP-HPLC chromatogram of complex 1 (10 μM) in RPMI-1640 medium, measured at 700 nm using a UV-Vis detector, is shown.

[0053] Figure 5C The RP-HPLC chromatogram of complex 1 (10 μM) in cell lysates of A2780 cells, measured at 700 nm using a UV-Vis detector, is shown.

[0054] Figure 6A The RP-HPLC chromatograms of PBS solutions containing or without 5 mM ascorbate containing complex 1 (10 μM) at 37 °C are shown.

[0055] Figure 6B The graph showing the percentage of remaining Pt(IV) versus time illustrates the situation corresponding to... Figure 6A The reduction curve of complex 1 with or without acoustic activation.

[0056] Figure 6C The LC chromatogram of the product of complex 1 after acoustic activation (4 W, 45 min) in PBS containing 5 mM ascorbate is shown. The inset shows the LC chromatogram and MS spectrum of the released carboplatin (i.e., the platinum(II) complex having the structure of formula (XIII). The LC chromatograms were measured at 700 nm using a UV-Vis detector.

[0057] Figure 6D Displayed corresponding to Figure 6C HR-MS spectrum of ligand 1.

[0058] Figure 6E Displayed corresponding to Figure 6C HR-MS spectrum of complex 1.

[0059] Figure 7A The fluorescence spectra of the mixture of ligand 1 and singlet oxygen sensor green (SOSG) are shown at 37 °C under ambient or degassing conditions with or without focused ultrasound (FUS) activation (4 W, 15 min).

[0060] Figure 7B The fluorescence spectra of a mixture of complex 1 and singlet oxygen sensor green (SOSG) are shown at 37 °C under ambient or degassing conditions with or without focused ultrasound (FUS) activation (4 W, 15 min).

[0061] Figure 7C Electroparamagnetic resonance spectra of a mixture of ligands 1 and 2,2,6,6-tetramethylpiperidine (TEMP) in the presence of FUS activation (4 W, 15 min) are shown.

[0062] Figure 7D Electroparamagnetic resonance spectra of a mixture of complex 1 and TEMP in the presence of FUS activation (4 W, 15 min) are shown.

[0063] Figure 7E Electroparamagnetic resonance spectra of a mixture of ligand 1 and 5,5-dimethyl-1-pyrrolline N-oxide (DMPO) are shown in the presence of FUS activation (4 W, 15 min).

[0064] Figure 7F Electroparamagnetic resonance spectra of a mixture of complex 1 and DMPO in the presence of FUS activation (4 W, 15 min) are shown.

[0065] Figure 8 The display shows whether an 808nm laser (330mW / cm) was used or not. 2 RP-HPLC chromatograms of activated ligand 1 and complex 1 (15 min).

[0066] Figure 9 It is an IC that shows different cancer cell lines. 50 Table of values ​​(μM). a Mixture: A mixture of carboplatin equivalent and ligand 1. Dosing: 30 min, US conditions: 3.5 W, 15 min, cell viability was tested 24 h after treatment. b Acoustic Sensitization Index (SI): Defined as the IC of an ultrasonically activated mixture. 50 The value divided by the IC of the un-ultrasonicated mixture 50 value. c Acoustic Sensitization Index (SI): Defined as the IC of ultrasonically activated complex 1. 50 The value divided by the IC of the un-ultrasonicated complex 1 50 value.d RF: Defined as the IC50 of platinum-resistant cell lines (A2780cisR or A549cisR). 50 The value divided by its original cell line (A2780 or A549) IC 50 value.

[0067] Figure 10 The relative cell viability of MRC-5 cells treated with different concentrations of cisplatin, carboplatin, and anthocyanin platinum for 24 h is shown.

[0068] Figure 11A The HPLC chromatogram of complex 1 (12.5 μM) is shown. The complex was extracted from A549 cells by lysis 6 h after activation with or without focused ultrasound (FUS) (3.5 W, 15 min).

[0069] Figure 11B The display does not show FUS activation from Figure 11A LC-MS spectra of extracts from cell lysates.

[0070] Figure 11C Displaying FUS activation from Figure 11A LC-MS spectra of extracts from cell lysates. Detailed Implementation

[0071] Unless otherwise specified, all tests herein were conducted under standard conditions including room temperature and test temperature of 25°C, sea level (1 atm) pressure, and pH 7, and all measurements are in metric units. Furthermore, unless otherwise explicitly stated, all percentages, ratios, etc., herein are by weight. It should be understood that, unless otherwise specified, the materials, compounds, chemicals, etc., described herein are typically commodity and / or industry-standard items available from multiple suppliers worldwide.

[0072] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0073] Unless the context explicitly indicates otherwise, as used herein, the forms “a / an” and “as stated” are intended to include both singular and plural forms.

[0074] As used in this application, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or". That is, unless otherwise stated or clear from the context, "X adopts A or B" is intended to mean any naturally inclusive permutation. That is, "X adopts A or B" holds true if X adopts A; X adopts B; or X adopts both A and B.

[0075] Not intending to be limited by theory, the inventors, through their own research, experiments, and trials, designed platinum complexes that can be used as prodrugs for cancer treatment. Specifically, these complexes can be activated by radiation, such as acoustic radiation or electromagnetic radiation, thereby releasing active chemical therapeutic compounds and generating singlet oxygen for synergistic therapy. 1 O2). In one exemplary embodiment, the complex as described herein can effectively reduce the proliferation of various types of cancer cells, including those that have developed cisplatin resistance after radiation activation.

[0076] According to the present invention, a platinum complex, particularly a platinum(IV) complex, comprising the structure of formula (I):

[0077]

[0078] in:

[0079] X, X', Y, Y' and Z are independently selected from the group consisting of: ammonia, hydroxide, halide, oxalate, diamine, dicarboxylate, glycolate and –OR. Optionally, X and X' are linked to form a first bidentate ligand, and / or Y and Y' are linked to form a second bidentate ligand.

[0080] n is selected from a group consisting of zero free charge, any positive charge, and any negative charge;

[0081] R represents the radiation response component of the structure with equation (II):

[0082]

[0083]

[0084] Where L is a linking group, m is 0 or a positive integer, R1 to R8 are each independently a substituent or hydrogen, and adjacent pairs of R2 to R7 can form fused heterocycles or carbocycles.

[0085] As used herein, the term "radiatively responsive portion" refers to the functional portion of a platinum complex that enables the complex to generate reactive oxygen species (ROS), such as singlet oxygen. 1 O2), and can promote the reduction of platinum complexes to their reduced forms, such as platinum(II) complexes, and release them after exposure to radiation in the presence of reducing agents / reducing agents.

[0086] For example, upon exposure to radiation, the radiation-responsive portion can be excited to an excited state by absorbing such radiation and / or through other mechanisms, such as microcavitation effects, which in turn excites the entire platinum complex to an excited state. The excited platinum complex, upon returning to its ground state, can be generated via a type II process involving energy transfer with molecular oxygen. 1O2. Meanwhile. 1 The generation of O2 can promote the electron transfer process between platinum complexes and reducing agents such as ascorbate and glutathione, thereby promoting the reduction of platinum complexes to their reduced form and their release.

[0087] The term "radiation" generally refers to the emission or transmission of energy in the form of waves through space or through a material medium. Examples of radiation may include electromagnetic (EM) radiation, such as radio waves, microwaves, infrared light, visible light, UV light, X-rays, gamma radiation, etc.; and acoustic radiation, such as ultrasound (US), sound, etc. In one embodiment, the radiation-responsive portion responds to EM radiation, particularly light such as infrared light. In another embodiment, the radiation-responsive portion responds to acoustic radiation, particularly US.

[0088] The linking group L is an organic unit of any length that comprises atoms or groups linking the two parts of the platinum complex of the present invention, namely the "platinum moiety" and the "radiatively responsive moiety" of the complex. Examples of L may include phenoxy-containing groups, thioether-containing groups, or secondary amine-containing groups. As used herein, the phrase "secondary" describes an amino group having two organic substituents, such as alkyl, acryloyl, or both, bonded to nitrogen along with a hydrogen atom. In a particular embodiment, the linking group L may be an O-terminated, N-terminated, or S-terminated connector. For example, the linking group L may be selected from any of the following groups:

[0089]

[0090] Where p is 1-4, and R 10 The side chain is H, a straight-chain or branched C1-C6 alkyl group, or a functional side chain. Examples of straight-chain or branched C1-C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, n-hexyl, etc. As used herein, the term "functional side chain" refers to a compound or molecule that, when bonded to the platinum complex of the present invention via the linker L, can promote or enhance the biocompatibility, cellular accumulation, cellular uptake, ROS generation, and chemical reduction of the platinum complex. Examples of functional side chains include, for example, PEG chains with a molecular weight of about 100 to about 5k, cyclodextrins, biomolecules such as penetrant proteins, glycosides, fatty acids, biotin, folic acid, etc.

[0091] R1 to R8 are each independently a substituent or a hydrogen atom. Substituents may be, for example, hydrocarbon groups, nitrogen-containing groups, heterocyclic groups, oxygen-containing groups, sulfur-containing groups, halogens, etc., wherein adjacent pairs of R2 to R7 may form a fused heterocyclic or carbocyclic ring. In a particular embodiment, R1 and R8 are independently selected from straight-chain or branched alkyl chains, sulfonate-containing groups, carboxyl groups, carboxylic acid ester-containing groups, or heterocyclic groups; and R2 to R7 are each independently selected from hydrogen, halogens, or adjacent pairs of R2 to R7 may form a fused 6-membered carbocyclic ring.

[0092] m is 0 or a positive integer such as 1, 2, 3 or 4. In one particular implementation, m is 0, 2 or 3.

[0093] X, X', Y, Y', and Z are independently selected from the group consisting of: ammonia, hydroxides, halides, oxalates, diamines, dicarboxylate, glycolates, and –OR. X, X', Y, Y', and Z may optionally be linked to each other in any combination to form polydentate ligands, particularly bidentate ligands. The term "halide" generally refers to halide ions, particularly including chlorides, bromides, or fluorides.

[0094] In one embodiment, X and X' connect to form a first bidentate ligand, and / or Y and Y' connect to form a second bidentate ligand. In one embodiment, Z may be a hydroxide or –OR. In another embodiment, Z may not be –OR.

[0095] n is selected from the group consisting of zero, any positive charge, and any negative charge. In one implementation, n can be 0 or 1.

[0096] In one particular embodiment, the platinum complex has the structure of formula (III):

[0097]

[0098] in:

[0099] X, X', Y, and Y' are independently selected from the group consisting of: ammonia, hydroxide, halide, oxalate, diamine, dicarboxylate, glycolate, and –OR, wherein X and X' or Y and Y' are linked to a bidentate ligand; Z is a hydroxide or –OR; and n, m, R1 to R8 are as defined above.

[0100] Specifically, X and X' are linked to form a dicarboxylic acid ester; Y and Y' are ammonia (i.e., NH3); Z is a hydroxide; R1 and R8 are independently selected from –CH3, –(CH2)2CH3, –(CH2)2COOH, –(CH2)5COOH, –(CH2)4SO3H, Furthermore, R2 to R7 are each independently selected from hydrogen, chloride, iodide, bromide, or adjacent pairs of R2 to R7 fused together to form a benzene ring.

[0101] As one specific embodiment, the platinum complex of the present invention may have the structure of formula (IIIa):

[0102]

[0103] As described herein, the platinum complexes, particularly platinum (IV) complexes, of the present invention can be used as prodrugs for the treatment of cancer. It should be understood that one of the key factors for an effective prodrug is its stability before activation. In one exemplary embodiment, the platinum complexes of the present invention remain intact before activation but exhibit cytotoxicity against cancer cells, including platinum-resistant cancer cells, after activation. In particular, in the absence of radiation, the chemical structure of the complex can remain unchanged for, for example, at least about 6 hours. In contrast, upon exposure to radiation, the platinum complexes can be reduced to a cytotoxic reduced form and generate reactive oxygen species, particularly singlet oxygen. In one embodiment, the platinum complexes exhibit at least a 6-fold enhanced cytotoxicity upon exposure to said radiation compared to platinum complexes not exposed to radiation. In exemplary embodiments where the platinum complexes may have a structure of formula (III), such as formula (IIIa), the cytotoxicity of the complexes against, for example, HeLa cells (according to IC50) is [not specified]. 50 The values ​​(in μM) were approximately 26 μM (without radiation exposure) and approximately 4 μM (with radiation exposure) (i.e., the cytotoxicity of the platinum complex was enhanced approximately 6-fold after radiation exposure).

[0104] The following describes a method for preparing platinum complexes, specifically platinum complexes having the structure of formula (I):

[0105]

[0106] Where X, X', Y, Y' and Z, n, R are as defined in this paper.

[0107] The method may include linking platinum complex precursors, particularly platinum (IV) complex precursors comprising a structure of formula (IV):

[0108]

[0109] The radiation-responsive portion R forms a platinum complex as described herein, wherein X, X', Y, Y', and Z are as defined herein, n' signifies 0 (zero), any positive charge, or any negative charge, and wherein R has the structure of formula (II):

[0110]

[0111] L, m, and R1 to R8 are as defined in this paper.

[0112] Platinum complex (IV) complex precursors may specifically include structures of formula (IVa):

[0113]

[0114] That is, Z is a hydroxide.

[0115] In one particular embodiment, the platinum(IV) complex precursor may comprise a structure of formula (V):

[0116]

[0117] In one embodiment of the invention, the method is suitable for preparing platinum complexes having the structure of formula (I), wherein R has the structure of formula (II), i.e., platinum complexes having the structure of formula (A):

[0118]

[0119] in:

[0120] X, X', Y, Y', and Z, as well as n, are as defined in this document;

[0121] L is selected from any of the following groups:

[0122]

[0123] Where p is 1-4, and R 10 It is H, straight-chain or branched C1-C6 alkyl, or a functional side chain as defined herein;

[0124] m is 0, 2, or 3;

[0125] R1 and R8 are independently selected from straight-chain or branched alkyl chains, sulfonate groups, carboxyl groups, carboxyl ester groups, or heterocyclic groups; and

[0126] R2 to R7 are each independently selected from hydrogen, halogen, or adjacent pairs of R2 to R7 forming a fused 6-membered carbon ring.

[0127] The method includes the following steps:

[0128] i) Optionally prepare a hydroxysuccinimide (NHS)-ester of a precursor compound having a portion of the structure of formula (II);

[0129] ii) Reacting a platinum complex precursor of formula (IV), such as formula (IVa) or especially formula (V), with an NHS-ester; and

[0130] iii) Separate the platinum complex of formula (A) and optionally purify the platinum complex.

[0131] The precursor compound may have the structure of formula (VI), formula (VII), or formula (VIII):

[0132]

[0133] Where m, p and R 10 As defined in this article.

[0134] Step i) specifically includes:

[0135] a) Reacting polymethylcyanine dyes, especially heptamethylcyanine dyes, with linker precursors having structures of formula (IX), (X), or (XI) to form precursor compounds:

[0136]

[0137]

[0138] Where p and R 10 As defined in this article;

[0139] b) Isolate the precursor compound and optionally purify the precursor compound.

[0140] Heptamethrin dyes may contain a structure of formula (XII).

[0141]

[0142] R1 and R8 are independently selected from straight-chain or branched alkyl chains, sulfonate groups, carboxyl groups, carboxyl ester groups, or heterocyclic groups; and

[0143] R2 to R7 are each independently selected from hydrogen, halogens, or adjacent pairs of R2 to R7 forming a fused 6-membered carbon ring; and

[0144] X is a halogen, especially Cl, Br, or I.

[0145] The reaction in step ia) can be carried out in a reaction solvent such as DMF under alkaline conditions, for example, in the presence of NaOH at room temperature. The reaction can proceed for at least about 1 hour.

[0146] Step ii) specifically includes:

[0147] a) Prepare a mixture of platinum complex precursor and NHS-ester in a reaction solvent;

[0148] b) After step a), stir the mixture at a temperature of at least about 20°C for at least about 8 hours.

[0149] The reaction solvent in step iia) is preferably dimethyl sulfoxide (DMSO). Optionally, the NHS-ester may be added in step iia) as a mixture with at least part of the reaction solvent, particularly DMSO.

[0150] The phrase "separation of platinum complex" or "separation of precursor compound" means, after step ib) or ii), at least partially separating the platinum complex or precursor compound from the other components in the reaction mixture. Step ib) or iii) specifically includes filtering the mixture to obtain a filtrate, centrifuging the mixture to form a precipitate of the platinum complex or precursor compound in water, such as cold water, adding a precipitation solvent to the filtrate to obtain a precipitate, and washing the precipitate with a washing solvent.

[0151] The purification steps in steps iii) and ib) can be performed by column chromatography, such as silica gel column chromatography, recrystallization, etc.

[0152] The method is applicable to the preparation of platinum complexes having the structure of formula (III):

[0153]

[0154] X, X', Y, Y', and Z are independently selected from the group consisting of: ammonia, hydroxides, halides, oxalates, diamines, dicarboxylate, glycolates, and –OR. Preferably, X and X' or Y and Y' are linked to form a bidentate ligand, and Z is a hydroxide or –OR. More preferably, X and X' are linked to form a dicarboxylate; Y and Y' are ammonia; and Z is a hydroxide.

[0155] R1 and R8 are independently selected from –CH3, –(CH2)2CH3, –(CH2)2COOH, –(CH2)5COOH, –(CH2)4SO3H, and

[0156] R2 to R7 are each independently selected from hydrogen, chloride, iodide, bromide, or adjacent pairs of R2 to R7 fused together to form a benzene ring.

[0157] As one specific implementation, the method is applicable to the preparation of platinum complexes, particularly radiation-activated platinum complexes having the structure of formula (IIIa):

[0158]

[0159] Another aspect of the invention relates to pharmaceutical compositions comprising a platinum complex as described herein and a pharmaceutically acceptable carrier.

[0160] Platinum complexes in pharmaceutical compositions may contain structures of formula (I), particularly those of formula (A):

[0161]

[0162] Where X, X', Y, Y', Z, and n are as defined in this paper;

[0163] L is selected from any of the following groups:

[0164]

[0165] Where p is 1-4, and R 10 It is H, straight-chain or branched C1-C6 alkyl, or a functional side chain as defined herein;

[0166] m is 0, 2, or 3;

[0167] R1 and R8 are independently selected from straight-chain or branched alkyl chains, sulfonate groups, carboxyl groups, carboxyl ester groups, or heterocyclic groups; and

[0168] R2 to R7 are each independently selected from hydrogen, halogen, or adjacent pairs of R2 to R7 forming a fused 6-membered carbon ring.

[0169] In one embodiment, the platinum complex in the pharmaceutical composition may comprise the structure of formula (III):

[0170]

[0171] Wherein X, X', Y, Y', and Z are independently selected from the group consisting of: ammonia, hydroxides, halides, oxalates, diamines, dicarboxylate, glycolates, and –OR. Preferably, X and X' or Y and Y' are linked to form a bidentate ligand, and Z is a hydroxide or –OR. More preferably, X and X' are linked to form a dicarboxylate; Y and Y' are ammonia; and Z is a hydroxide.

[0172] R1 and R8 are independently selected from –CH3, –(CH2)2CH3, –(CH2)2COOH, –(CH2)5COOH, –(CH2)4SO3H, and

[0173] R2 to R7 are each independently selected from hydrogen, chloride, iodide, bromide, or adjacent pairs of R2 to R7 fused together to form a benzene ring.

[0174] More preferably, the platinum complex in the pharmaceutical composition may contain a structure of formula (IIIa):

[0175]

[0176] "Pharmaceutically acceptable carriers" are those that facilitate cellular uptake of the active ingredient and can be taken by the patient without causing treatment-related side effects or negative impacts on the efficacy of the complex. Pharmaceutical compositions may be in solid, semi-solid, or liquid form and may be administered to the patient orally or parenterally.

[0177] The present invention also relates to a method for treating a target tissue or the use of a platinum complex in the preparation of a medicament for treating a target tissue, said method / use comprising administering the platinum complex as described above to a patient in need and irradiating the target tissue with an amount and frequency that effectively activates said complex.

[0178] The term "target tissue" encompasses sites with cancer cells, cancerous tissue, and any portion near cancer cells or cancerous tissue in a patient requiring treatment. Target tissue can be located locally or within the patient's body. Following a step of maintaining the complex on the patient for a specific period of time, in the case of pre-targeted target tissue, an effective amount of radiation at an effective frequency is applied to the target tissue to activate the complex at the site.

[0179] In one implementation, the target tissue may be a tumor, particularly cancer. As used herein, the terms “tumor” or “neoplastic” describe an abnormal mass of tissue in the physical condition of a subject (e.g., a patient), which may be benign, pre-malignant, or malignant (cancerous), and may occur in any part of the body, including solid tissues such as organs, muscles, bones, etc.

[0180] The terms “cancer” and “cancerous” describe a physiological condition in a subject (e.g., a patient) where a cell population is characterized by unregulated malignant (cancerous) cell growth. Cancer may or may not be drug-resistant cancer. The expression “drug-resistant cancer” generally refers to cancer with natural resistance to one or more chemotherapeutic compounds, i.e., intrinsic or acquired resistance, particularly with known platinum coordination complexes such as cisplatin, i.e., acquired resistance. If the cancer contains cancer cells that are resistant to the said chemotherapeutic compound, then the cancer is resistant to one or more chemotherapeutic compounds. Therefore, cancer cells with a drug-resistant phenotype will be less sensitive to or more resistant to one or more chemotherapeutic compounds. Such cancers or cancer cells can be detected, for example, by an MTT assay.

[0181] In a preferred embodiment, the platinum complex is used to combat malignant tumors in patients diagnosed with cervical cancer, lung cancer, ovarian cancer, breast cancer, or breast cancer, including their cisplatin-resistant phenotype.

[0182] The platinum complex of the present invention preferably exhibits a resistance factor of less than 10 against cisplatin-resistant cancer cells, more preferably less than 8, and particularly even less than 5. The resistance factor is determined by the IC50 value of the platinum complex against cisplatin-resistant cells. 50 In addition to its IC50 against cancer cells of the same cell type or tissues without cisplatin resistance phenotype 50 To calculate.

[0183] In an alternative embodiment, the complex can be used to combat target tissues affected by infection, particularly those infected by bacteria, such as, but not limited to, Staphylococcus aureus and Escherichia coli infections.

[0184] The applied platinum complex may contain the structure of formula (I), and in particular formula (A):

[0185]

[0186] Where X, X', Y, Y', Z, and n are as defined in this paper;

[0187] L is selected from any of the following groups:

[0188]

[0189] Where p is 1-4, and R 10 It is H, straight-chain or branched C1-C6 alkyl, or a functional side chain as defined herein;

[0190] m is 0, 2, or 3;

[0191] R1 and R8 are independently selected from straight-chain or branched alkyl chains, sulfonate groups, carboxyl groups, carboxyl ester groups, or heterocyclic groups; and

[0192] R2 to R7 are each independently selected from hydrogen, halogen, or adjacent pairs of R2 to R7 forming a fused 6-membered carbon ring.

[0193] Preferably, the applied platinum complex may contain the structure of formula (III):

[0194]

[0195] Wherein X, X', Y, Y', and Z are independently selected from the group consisting of: ammonia, hydroxides, halides, oxalates, diamines, dicarboxylate, glycolates, and –OR. Preferably, X and X' or Y and Y' are linked to form a bidentate ligand, and Z is a hydroxide or –OR. More preferably, X and X' are linked to form a dicarboxylate; Y and Y' are ammonia; and Z is a hydroxide.

[0196] R1 and R8 are independently selected from –CH3, –(CH2)2CH3, –(CH2)2COOH, –(CH2)5COOH, –(CH2)4SO3H, and

[0197] R2 to R7 are each independently selected from hydrogen, chloride, iodide, bromide, or adjacent pairs of R2 to R7 fused together to form a benzene ring.

[0198] More preferably, the applied platinum complex may contain the structure of formula (IIIa):

[0199]

[0200]

[0201] As described herein, the platinum complexes of the present invention are activated upon exposure to an effective amount of radiation at an effective frequency. The phrase “activation” describes the series of reactions that the platinum complexes undergo upon exposure to / receiving radiation. In particular, platinum complexes, such as those having the structure of formula (A) or formula (III) as defined above, may release their reduced forms, such as platinum (II) complexes which have inhibitory effects on target tissues, particularly tumors, and generate reactive oxygen species, such as… 1 O2. Examples of the above platinum(II) complexes may have structures such as cisplatin, carboplatin, nedaplatin, heptaplatin, oxaliplatin, lobaplatin, etc.

[0202] For example, in embodiments of platinum complexes comprising the structure of formula (III), particularly formula (IIIa), upon radiative excitation, the radiatively responsive portion, and thus the platinum (IV) complex, is excited to an excited state. When the platinum (IV) complex returns to the ground state, it will generate through a type II process. 1 O2, the process involves energy transfer between the stimulated complex and molecular oxygen. Subsequently or simultaneously, 1 The generation of O2 (i.e., the reaction between the stimulated complex and molecular oxygen) will promote electron transfer between the stimulated platinum(IV) complex and reducing agents (e.g., cellular reducing agents, including ascorbate, GSH, etc.), resulting in the release of the reduced form of the platinum(IV) complex, such as platinum(II) complexes, especially platinum(II) complexes having the structure of formula (XIII):

[0203]

[0204] Therefore, the platinum complex of the present invention can act as a prodrug to release the active moiety, such as the complex of formula (XIII), to the product formed upon radiation excitation (e.g., 1 O2) can achieve therapeutic effects and / or produce synergistic effects.

[0205] The excitation mechanism described above can depend on the type of radiation applied to the platinum complex. For example, in an embodiment where the radiation is acoustic radiation, particularly ultrasound, the platinum complex can be excited by the microcavitation effect; while in another embodiment where the radiation is electromagnetic radiation, particularly light, such as ultraviolet light, infrared light, etc., the platinum complex can be excited by following some general principles of photochemistry, such as, but not limited to, the first law of photochemistry and the second law of photochemistry.

[0206] The amount of complex to be administered depends on the patient's species, weight, age, and individual condition, and can be determined using standard procedures, such as cell cultures or laboratory animals. For example, the effective amount or concentration of the complex may be at least about 1 μg / mL.

[0207] The complex can exist in solid, semi-solid, or liquid form. Depending on the form in which the complex is to be presented, it can be administered to patients in need via oral, topical, intravenous, or parenteral routes, with oral or intravenous routes being preferred.

[0208] The terms "effective amount," "effective dose," and "effective frequency" generally refer to the amount of radiation, or a specific range or frequency, sufficient to activate the complexes of the invention located in the target tissue, thereby producing the desired therapeutic outcome, wherein the exact nature of the outcome depends on the specific condition being treated. When the condition involves a tumor, particularly cancer, the outcome is typically the inhibition or suppression of cancer cell proliferation, a reduction in cancer cells, or improvement of symptoms associated with cancer cells. When the condition is a bacterial infection, the outcome is the inhibition or suppression of bacterial proliferation, a reduction in bacterial colonies, or improvement of symptoms associated with the bacterial infection.

[0209] Specifically, the “effective amount,” “effective dose,” and “effective frequency” of the applied radiation are sufficient and / or can be absorbed and / or excited to an excited state by the radioresponsive portion of the complex for subsequent chemical reactions as detailed above, allowing the complex to generate ROS via a type II mechanism, particularly 1 O2 is released, and the active reduced form of the complex, such as the platinum(II) complex as described above (i.e., the activated complex), is released. The generated ROS and the released platinum(II) complex can therefore act on cancer cells, resulting in at least inhibition or suppression of their proliferation.

[0210] As described herein, inhibition of cancer cell (or bacterial) growth / proliferation can mean a reduction in cell (or bacterial) viability, particularly a significant decrease and / or increase, especially a significant increase, in the number of apoptotic cells (or bacterial colony-forming units (CFU)). Those skilled in the art know methods for verifying such effects. For cancer cells, cell viability can be measured, for example, by MTS proliferation analysis, MTT analysis, live / dead cell co-staining viability analysis (Calcein-AM / PI), or by determining the apoptosis rate using Annexin V flow cytometry. For bacteria, standard colony counting methods known in the art can be used, for example, to determine the bacterial inhibition rate (IR) using formula (1):

[0211]

[0212] Where C is the number of colony forming units (CFU) in the experimental group, and C0 is the number of CFU in the control group.

[0213] As used herein, the term “significant” means that something is determined to be statistically significant by the Student’s t-test or other statistically significant measures recognized in the field.

[0214] The effective frequency, as described herein, can depend on the type of radiation applied. In embodiments where the radiation is ultrasound, the effective frequency can be about 1 MHz to about 3 MHz, about 1.05 MHz to about 3.05 MHz, about 0.95 MHz to about 2.95 MHz, about 1.25 MHz to about 3 MHz, about 1.25 MHz to about 2.75 MHz, about 1.5 MHz to about 3 MHz, about 1.75 MHz to about 3 MHz, or about 1.75 MHz to about 2.5 MHz. In one specific embodiment of the invention, ultrasound is applied to the target area at a frequency of about 1.75 MHz.

[0215] In embodiments where the radiation is light, the effective frequency can be about 350 THz to about 500 THz, about 355 THz to about 505 THz, about 345 THz to about 500 THz, about 345 THz to about 505 THz, about 360 THz to about 500 THz, or about 370 THz to about 500 THz. In one specific embodiment of the invention, light is applied to the target area at a frequency of about 370 THz.

[0216] It should be understood that those skilled in the art will be able to convert the frequency (f) of light into wavelength (λ) using equation (2):

[0217] λ=υ×f Equation (2)

[0218] Where υ is the speed of light in a vacuum, which is 299,792,458 m / s.

[0219] For example, in an implementation with a frequency of approximately 370 THz, the equivalent wavelength would be approximately 810 nm.

[0220] The effective amount of applied radiation can be measured according to power or power intensity, and its exact amount may depend on the type of radiation applied. In embodiments where the radiation is ultrasound, the effective amount of ultrasound is measured according to power, which may be about 1W to about 4W, about 1.5W to about 4W, about 1W to about 3.5W, about 1.5W to about 3.5W, about 2W to about 4W, or about 3W to about 4W.

[0221] In the implementation scheme where the radiation is light, the effective amount of light is measured based on power intensity, which may be approximately 3 mW / cm². 2 Approximately 350mW / cm 2 Approximately 2.9 mW / cm2 Approximately 350mW / cm 2 Approximately 3mW / cm 2 Approximately 355mW / cm 2 Approximately 3.2 mW / cm 2 Approximately 350mW / cm 2 Approximately 3.3 mW / cm 2 Approximately 350mW / cm 2 or approximately 3.3 mW / cm 2 Approximately 348 mW / cm 2 .

[0222] It should be understood that, optionally or additionally, the method or use may be combined with other treatments that are effective, such as one or more of the following:

[0223] i) Treatment involving other therapeutically effective compounds, such as chemotherapeutic compounds, including, for example, topoisomerase-II inhibitors, anthracyclines, platinum coordination complexes, taxanes, protein kinase inhibitors, vinca alkaloids or derivatives thereof, topoisomerase-I inhibitors, and nucleotide analogs or precursor analogs.

[0224] ii) Radiotherapy; and / or

[0225] iii) Hormone therapy.

[0226] Complexes, particularly those having the structure of formula (A), such as formula (III) or (IIIa), are suitable or specifically designed for use as medicaments for treating target tissues, or for the preparation of medicaments for treating target tissues by sonodynamic therapy, photodynamic therapy, chemotherapy, and / or combinations thereof as described herein. In one embodiment, the target tissue may be a tumor, particularly cancer as defined above. In another embodiment, the target tissue may be infected, particularly with bacteria, as defined above.

[0227] The drug comprises a complex, particularly a complex having a structure of formula (A), formula (III) or formula (IIIa) as described herein, and a pharmaceutically acceptable carrier as defined herein.

[0228] Drugs can be in various forms, such as oral or injectable forms, depending on the method of administration or purpose and the various methods conventionally used to prepare drugs. Examples of oral formulations may include tablets, powders, granules, capsules, pills, lozenges, solutions, syrups, elixirs, emulsions, oily or aqueous suspensions, etc.

[0229] Examples of solid formulations include tablets, powders, granules, capsules, pills, and lozenges. These solid formulations may contain pharmaceutically acceptable additives as well as the crystals of the present invention. Examples of additives include fillers, expanders, binders, disintegrants, solubilizers, skin humectants, and lubricants, and these may be selected and mixed as needed to prepare a medicine.

[0230] Examples of liquid formulations include solutions, syrups, elixirs, emulsions, and suspensions. These liquid formulations may contain pharmaceutically acceptable additives as well as the crystals of the present invention. Examples of additives include suspending agents and emulsifiers, which are selected and mixed as needed to prepare the formulation.

[0231] The present invention will be described in detail below through embodiments. In the following embodiments,

[0232] Materials and reagents used:

[0233] All materials were purchased from commercial sources and used as is without further purification. All reactions were carried out in the dark unless otherwise specified.

[0234] Instrumentation and characterization:

[0235] NMR data were recorded at room temperature using a Bruker AVANCE III 300 / 400MHz spectrometer or a Bruker Ascend AVANCE III 600MHz spectrometer. ESI-MS data were recorded using a liquid chromatography-mass spectrometry (API-3200 Triple-Q MS / MS). Analytical HPLC was performed on a reversed-phase C18 column (Phenomenex Garmin 250 × 4.60 mm, 5 μm). The analysis was performed on a Shimadzu Prominence LC-20AT HPLC system (RP-HPLC). The absorption spectrum from 190 to 800 nm was scanned using a photodiode array (PDA) detector. Solvent A (H₂O containing 5% ACN) and solvent B (ACN containing 5% H₂O) were used to extract the solutions at 1.0 mL / min. -1 Gradient elution was performed at a flow rate of [value missing]. The test sample was eluted according to the following procedure: 5% B (0 min) → 20% B (7 min) → 90% B (16 min) → 90% B (25 min). The Pt content was measured by inductively coupled plasma-optical emission spectrometry (ICP-OES) (PE Optima 8000).

[0236] Example 1

[0237] Preparation of complex 1:

[0238] Complex 1 of the present invention (i.e., the platinum complex of formula (IIIa) of the present invention, anthocyanin platinum) is based on Figure 1 The reaction scheme shown is used for preparation. The reaction begins with the synthesis of ligand 1, followed by the conversion of ligand 1 into an NHS-ester, and then, either in situ or with a platinum complex precursor, to give complex 1.

[0239] Specifically, NaOH (19.3 mg, 0.270 mmol) was dissolved in 3 mL of DMF, and 3-(4-hydroxyphenyl)propionic acid (20.0 mg, 0.075 mmol) was added dropwise in an ice bath under N2 protection. Then, commercially available IR780 iodide (40.0 mg, 0.060 mmol, in 5 mL of anhydrous DMF) was slowly added over a 30-minute period. The reaction mixture was further maintained at 298 K for 1 hour. After washing with Et2O and silica gel column chromatography (elution: DCM / MeOH = 8 / 1), a red-green crystalline solid was obtained as ligand 1 (22.1 mg, 48.8%). 1 H NMR (300MHz, methanol-d4) δ (ppm) 8.01 (d, J = 14.1Hz, 2H), 7.44–7.34 (m, 4H), 7.32–7.18 (m, 6H), 7.09–7.01 (m, 2H), 6.17 (d, J = 14.1Hz, 2H), 4.09 (t, J = 7.2Hz, 4H), 2.81 (dt, J = 34.1, 6.9Hz, 6H), 2.54 (t, J = 6.9Hz, 2H), 2.11–2.01 (m, 2H), 1.85 (q, J = 7.5Hz, 4H), 1.35 (s, 12H), 1.03 (t, J = 7.5Hz, 6H). HR-MS(m / z):C 45 H 53 N2O3 + [M] + Calculated value: 669.4051, Experimental value: 669.3998. Figure 2A and 2B )

[0240] c,t-[Pt(1,1-cyclobutane-dicarboxylate)(NH3)2(OH)2] (20.2 mg, 0.05 mmol), ligand 1 (10.0 mg, 0.05 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 0.20 mmol) (TBTU, 19.2 mg, 0.06 mmol), and N-hydroxysuccinimide (NHS, 0.20 mmol) were dissolved in 2 mL of DMSO. The reactants were kept in the dark at 298 K for 24 h, followed by the addition of 10 mL of cold water to precipitate the product. After silica gel column chromatography (elution: DCM / MeOH = 6 / 1) and recrystallization with DCM / Et2O = 1 / 3, a dark green powder was given as complex 1 (11.2 mg, 37.3%). 1 H NMR(300MHz,DMSO-d6)δ(ppm)7.80(d,J=14.2Hz,1H),7.50(d,J=7.5Hz,1H),7.4 1–7.33(m,2H),7.25–7.16(m,2H),7.03(d,J=8.7Hz,1H),6.17(d,J=14.2Hz,1H), 4.10(t,J=7.5Hz,3H),2.68(d,J=6.9Hz,3H),2.43–2.29(m,1H),2.29(t,J=6.0Hz ,1H),1.93(d,J=6.3Hz,1H),1.70(dt,J=14.3,7.2Hz,3H),0.92(t,J=7.5Hz,4H). 13 C NMR(151MHz,DMSO-d6)δ(ppm)180.01,172.16,163.35,158.40,158.34,141.37,136.06,130.41,128.93, 125.22,122.87,121.87,111.71,49.02,32.26,31.80,31.25,27.68,21.19,20.82,16.19,15.95,11.55. 195 Pt NMR(129MHz,DMF-d7)δ(ppm)1735.00(s). HR-MS(m / z):[M] + Calculated value: 1056.4445, Experimental value: 1056.4399. Figures 3A to 3C and Figure 4A The purity of the complex, as determined by analytical RP-HPLC, was 99.45%. Figure 4B )

[0241] Example 2

[0242] Stability of complex 1:

[0243] The stability of complex 1 in phosphate-buffered saline (PBS) buffer, cell culture media such as RPMI-1640, and cell lysis buffer was determined in the dark (i.e., under radiation-free conditions). To determine the stability of complex 1 in PBS, complex 1 (10 μM) was dissolved in PBS buffer (pH 7.4, 1% DMF) and incubated in the dark at 310 K. At 24 h, the sample was analyzed by HPLC to determine the percentage of residual Pt(IV). To determine the stability of complex 1 in complete RPMI-1640 medium (containing 10% FBS, 2 mM L-glutamine, and 100 IU mL... -1 Penicillin / streptomycin or cell lysate (obtained from A2780 cells via lysis with 1% Triton X-100, protein concentration adjusted to 1.5 mg / mL) -1 To assess the stability of the complex, complex 1 was dissolved in both solutions (containing 1% DMF). At predetermined time intervals, 50 μL of the sample was taken and diluted with 200 μL of MeOH, followed by centrifugation at 14,000 × g to remove proteins, and then the supernatant was analyzed by HPLC.

[0244] like Figures 5A to 5C As shown, only a single peak representing complex 1 was observed in all three test environments, indicating that complex 1 is stable in the dark for at least 6 hours.

[0245] Example 3

[0246] Acoustic activation of complex 1:

[0247] The acoustic activation properties of complex 1 were investigated. PBS solutions containing 10 μM complex 1, with or without 5 mM sodium ascorbate (containing 1% DMF) (i.e., samples), were continuously sonicated (1.75 MHz, 4 W) for a total time of 60 min. Sample temperature was monitored using a FLIR thermal imager and maintained at 37 °C throughout the experiment. Figure 6A As shown, under ultrasonic treatment in the presence of ascorbate, the peak of complex 1 in the HPLC chromatogram rapidly decreased, and a new peak appeared accordingly. Further analysis of the HPLC eluent of the “FUS45min” sample by LC and HR-MS revealed the presence of three components, including carboplatin, ligand 1, and (unreacted) complex 1. Figures 6C to 6E This indicates that after acoustic activation, Pt(IV) is reduced to Pt(II) and axial ligand 1 and hydroxide ligand dissociate (i.e., complex 1 undergoes acoustic-induced reduction). Figure 6A and 6BAs shown, after approximately 60 minutes of acoustic activation, about 95% of complex 1 was reduced (to Pt(II)). Notably, the acoustic-induced reduction occurred only in the presence of ascorbate and ultrasound, indicating the high sensitivity of complex 1 to the activation environment. Figure 6A and 6B ).

[0248] The inventors further investigated the possibility of ROS generation in complex 1 after acoustic activation by using the hydroxyl radical scavenger 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO), the singlet oxygen scavenger 2,2,6,6-tetramethylpiperidine (TEMP), and the singlet oxygen sensor green (SOSG) to study the electron transfer process. Figure 7A and 7B As shown, strong emission curves originating from SOSG were observed for both ligand 1 and complex 1 activated with FUS, indicating that ligand 1 and complex 1 release singlet oxygen upon acoustic activation. The release of singlet oxygen upon acoustic activation of ligand 1 and complex 1 is further confirmed by their electroparamagnetic resonance spectra in the presence of TEMP. Figure 7C and 7D As shown, signal pulses were only observed when ligand 1 and complex 1 were subjected to ultrasonic treatment. In contrast, as... Figure 7E and 7F As shown, when DMPO was used instead of TEMP, no signal pulse was observed even under ultrasonic treatment. Based on the above, it is indicated that ligand 1 and / or complex 1 release singlet oxygen (e.g., via a type II electron transfer process) after acoustic activation rather than releasing hydroxyl radicals.

[0249] Example 4

[0250] Photoactivation of complex 1:

[0251] The inventors further investigated whether complex 1 could be photoactivated by other types of radiation, such as EM radiation, particularly infrared light. A quartz cuvette containing 10 μM complex 1 and 5 mM ascorbate in PBS buffer (containing 1% DMF) was stirred and then subjected to a laser (808 nm, 3.3 mW / cm²). 2 Irradiate for 15 minutes. Analyze the sample by HPLC. Figure 8 As shown, the peak profile is similar to Figure 6A Complex 1 is ultrasonically activated. That is, complex 1 can be photoactivated and undergo photoinduced reduction, thereby forming and releasing a Pt(II) complex upon irradiation. Given the similar profile, the inventors believe that complex 1 will also release singlet oxygen upon photoactivation and the aforementioned photoinduced reduction.

[0252] Example 5

[0253] Cytotoxicity of complex 1:

[0254] The bioactivity of complex 1 was evaluated. Human breast cancer MCF-7 cells, human cervical cancer HeLa cells, breast cancer 4T1 cells, human lung cancer A549 cells, and cisplatin-resistant A549cisR cells were subjected to treatment with 10% FBS and 100 IU / mL. -1 Cultured in DMEM containing penicillin / streptomycin. Human ovarian cancer A2780 and A2780cisR cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine, and 100 IU / mL. -1 Cultured in RPMI-1640 containing penicillin / streptomycin. Human lung fibroblast MRC-5 cells were cultured in a solution containing 10% FBS, 1% NEAA, 1% L-glutamine, 1% sodium pyruvate, and 100 IU / mL. -1 Cells were cultured in penicillin / streptomycin MEM. 5 μM cisplatin was added every two passages of A2780cisR and A549cisR cells to maintain platinum resistance. All cells were cultured in a humidified incubator at 37°C with 5% CO2. Cell counts were performed using a hemocytometer.

[0255] Cytotoxicity was assessed by exposing cancer cells to complex 1. The viability of the exposed cancer cells was evaluated using MTT assay. Specifically, cells were first pre-seeded in 6-well plates until 80% confluence was achieved. Then, the medium was removed and replaced with fresh medium containing different concentrations of complex 1 and 0.5% DMF (0, 2.5, 5, 12.5, 25, or 40 μM). After 30 min, the medium was aspirated, and the wells were washed three times with PBS. Cells were digested with trypsin and resuspended in 2 mL microcentrifuge tubes containing 1 mL of fresh medium, followed by sonication (3.5 W, 15 min). After sonication, cells were seeded into 96-well plates (2 × 10⁶ cells per well). 4 Cell viability was measured in triplicate, and MTT assay was performed 24 hours later. Cell viability was defined as the viability of cells treated with 0.5% DMF (30 min) as the medium.

[0256] like Figure 9 As shown, complex 1 is generally non-toxic to test cells without sonication, but becomes highly active after sonication. Specifically, the fold increase (or IC50) in cytotoxicity of the sonicated complex 1 compared to the unactivated complex 1 is reflected in the following data. 50The sonosensitization index (SI) (by a factor of decrease in SI value) indicates that the cytotoxicity of activated complex 1 increased by at least about 6-fold, particularly about 6-fold to about 20-fold, compared to unactivated complex 1. Furthermore, not wishing to be limited by theory, the inventors have found that the factor of increase in cytotoxicity (based on SI values) is generally much higher than that of a mixture of carboplatin and ligand 1 (1:1 eq.) under the same sonication conditions. That is, complex 1 actually exhibits a significantly stronger inhibitory effect on the test cells compared to the mixture. Notably, this effect is more pronounced when the test cells are cisplatin-resistant cancer cells, where the SI of activated complex 1 is >9.8 (A2780-cisR) and >9.3 (A549-cisR), compared to the SI of the activated mixture being >1.7 (A2780-cisR) and >2.1 (A549-cisR). Furthermore, complex 1 was non-toxic to normal human lung fibroblast MRC-5 cells tested in the absence of ultrasound, indicating its biocompatibility with normal cells. Figure 10 ).

[0257] To further confirm whether complex 1 was reduced to carboplatin in cells, complex 1 was extracted by lysing A549 cells, which were then treated with 12.5 μM complex 1 and activated at 3.5 W FUS for 15 min, and analyzed by HPLC. Figure 11A As shown, a new peak was observed only in the complex treated with ultrasound. It is believed that such a peak represents 35.7% of complex 1 being converted to its reduced form, carboplatin, after ultrasound activation. Therefore, the inventors further analyzed the complex using LC-MS. Figure 11A Extracts / elutions and confirmed Figure 11A The new peak in the image corresponds to carboplatin.

[0258] Based on the above, it is considered that a short period of sonication, such as 15 minutes, is sufficient to reduce the complex to its reduced form. Furthermore, such a short period is required to minimize side effects (e.g., localized high heat generation after sonication). In addition, the complex of the present invention is considered suitable as a prodrug for anticancer treatment, particularly for cisplatin-resistant cancers, because it has been found that the complex is stable even in the presence of physiological concentrations of cell reducing agents, such as ascorbate, without the need for sonication.

[0259] It should be understood that the above description only illustrates and describes embodiments in which the present invention can be implemented, and modifications and / or changes can be made thereto without departing from the spirit of the present invention.

[0260] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination with a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination.

[0261] All references specifically cited herein are incorporated herein in their entirety by way of citation. However, citation or inclusion of such references does not necessarily imply an acknowledgment of their suitability, citationability, and / or availability as prior art to / against the invention.

Claims

1. A platinum complex having the structure of formula (IIIa): Equation (IIIa).

2. A pharmaceutical composition comprising the platinum complex as claimed in claim 1 and comprising a pharmaceutically acceptable carrier.

3. The use of the platinum complex of claim 1 in the preparation of a medicament for treating a target tissue, comprising administering the platinum complex to a patient in need and irradiating the target tissue with an amount and frequency that effectively activates the complex.

4. The use as described in claim 3, wherein the radiation is ultrasound.

5. The use as claimed in claim 4, wherein the frequency of the ultrasound is between 1 MHz and 3 MHz.

6. The use as claimed in claim 5, wherein the ultrasound is applied at a power of 1 W to 4 W.

7. The use as claimed in claim 3, wherein the radiation is light.

8. The use as claimed in claim 7, wherein the frequency of the light is between 350 THz and 500 THz.

9. The use as claimed in claim 8, wherein the light is at 3 mW / cm 2 Up to 350 mW / cm 2 The power intensity is applied.

10. The use as described in claim 3, wherein the target tissue is a tumor.

11. The use as claimed in claim 10, wherein the tumor is selected from the group consisting of cervical cancer, lung cancer, ovarian cancer, and breast cancer.

12. The use as claimed in claim 10, wherein the tumor has intrinsic or acquired cisplatin resistance.

13. The use as described in claim 3, wherein the platinum complex acts as a prodrug.

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