A gold (I) complex, its preparation method and application

Abstract

This invention discloses a gold (I) complex, its preparation method, and its applications. The gold (I) complex is as shown in Formula I: In this invention, Formula I is a strongly sterically hindered nitrogen-heterocyclic carbene (NHC) gold (I) complex. This invention utilizes a steric hindrance control strategy to design a series of stable active gold (I) complexes containing highly sterically hindered NHC ligands. These highly sterically hindered gold complexes can tolerate excessive intracellular thiol compounds and exhibit good stability; they can maintain normal intracellular catalytic activity, possess good probe imaging and prodrug activation properties, demonstrating highly efficient in vitro and in vivo antitumor activity; and they also exhibit good antibacterial activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and in particular to a gold (I) complex, its preparation method, and its application. Background Technology

[0002] Cisplatin, as a first-line anti-tumor drug, is widely used in the treatment of lung cancer, head and neck squamous cell carcinoma, gastric cancer, and reproductive system cancers. However, drug resistance and toxic side effects of platinum-based drugs limit their further clinical use. Seeking other metal complexes with different anti-cancer mechanisms has become an important strategy to overcome cisplatin resistance. Unlike platinum complexes, which act on DNA base N-binding sites, gold complexes exhibit activity towards thiol (SH) and selenium (Se) groups. - It exhibits a high affinity for thioredoxin reductase (TrxR), especially for thioredoxin reductase (TrxR), which is overexpressed in tumor cells and contains both Cys and Sec. This reductase significantly inhibits IC50. 50 Gold complexes can reach nanomolar to picomolar levels; for example, aurinophene exerts its anticancer activity by inhibiting TrxR in tumor cells. However, due to the presence of numerous thiol-containing biomolecules in the body, such as serum albumin and glutathione (GSH), highly reactive gold can competitively bind to these biomolecules. These off-target effects reduce its anticancer activity and pose potential toxic side effects. Therefore, regulating the reactivity of gold complexes with thiol groups is an important direction in the current research of gold anticancer complexes.

[0003] Currently reported gold complexes mainly have their active sites masked and are activated in tumor cells through stepwise ligand exchange, light irradiation, or bioorthogonal processes. Therefore, it is essential to develop an active gold complex that can evade the attack of biothiols without losing its catalytic and antitumor activities. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a gold (I) complex, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, a compound of formula I or a derivative thereof is proposed:

[0007]

[0008] R1 and R2 are each independently selected from C1 to C2, with or without substitution. 12 Alkyl, C3-C 12 cycloalkyl, C2-C 12 alkenyl, C2-C 12alkynyl group, C6-C 20 Aromatic hydrocarbon cyclic groups or C3-C 60 The aromatic heterocyclic group; R3 and R4 are each independently selected from deuterium, H, optionally substituted or unsubstituted C1-C1 groups. 12 Alkyl, C1-C 12 Alkoxy, C3~C 12 cycloalkyl, C2-C 12 alkenyl, C2-C 12 alkynyl group, C6-C 20 Aromatic hydrocarbon cyclic groups or C3-C 60 The aromatic heterocyclic group; the heteroatoms in the aromatic heterocyclic group are selected from N, O, and S; L is an anion.

[0009] In this invention, "cycloalkyl" refers to a saturated or partially saturated cyclic group having multiple carbon atoms, no heterocyclic atoms, and a single or multiple rings (including fused rings). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl, cyclohexenyl, and polycyclic alkyl rings, such as dicyclopropyl, dicyclohexyl, dicyclopentyl, dicyclooctyl, etc., wherein the rings in the polycyclic alkyl ring can be attached to the same carbon atom, for example... It can also be bonded to different carbon atoms that are adjacent and / or spaced apart, for example

[0010] In this invention, "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond (>C=C<). For example, C a ~C b Alkenyl refers to an unsaturated hydrocarbon group containing an alkenyl group that has a to b carbon atoms. Specific examples of alkenyl groups include vinyl, propenyl, isopropenyl, 1,3-butadienyl, etc.

[0011] In this invention, "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group containing at least one carbon-carbon triple bond. The term "alkynyl" is also intended to include hydrocarbon groups having one triple bond and one double bond. Specific examples of C2-C6 alkynyl groups include ethynyl, propynyl, etc.

[0012] In some embodiments of the present invention, the aromatic hydrocarbon cycloyl group is selected from phenyl, naphthyl, anthraceneyl, perylene, tetraphenyl, pyrene, benzo[a]pyrene, etc. alkyl, biphenylidene, bitriphenylidene, acenaphthene, fluorenyl, fluorenyl.

[0013] In some embodiments of the present invention, the aromatic heterocyclic group is selected from thienyl, bithienyl (such as dithienyl, polythienyl formed by three or more thienyl groups linked together), thienophenyl (such as dithienophenyl), thienobenzoquinone (such as dithienobenzoquinone), thiaranyl, bithiaranyl, thiaranophenyl, thiaranobenzoquinone, thiazolyl, benzothiazolyl, benzothiadiazolyl, thiaanthryl, pyridyl, benzyl Piperazinyl, benzoimideyl, benzoimideylphenyl, benzoimideylanthraquinoneyl, benzoimideylphenanthraquinoneyl, naphthimideyl, naphthimideylphenyl, naphthimideylanthraquinoneyl, naphthinoneyl, anthraquinoneyl, phenanthraquinoneyl, pyrenetetroneyl, piperazinyltetroneyl, quinacridoneyl, pyrenetetroneyl, naphthalenetetracarboxylic anhydride, perylenetetracarboxylic anhydride, terephthalic anhydride, phthalimideyl, peryleneimideyl.

[0014] In some embodiments of the present invention, the substituent is selected from at least one of deuterium, halogen, hydroxyl, thiohydryl, cyano, cyanoxy, cyanothio, amino, nitro, nitroso, sulfonyl, sulfonic acid, boron, dihydroxyboronyl, phosphono, phosphoric acid, phosphonic acid, dioxophosphoryl, phosphono, methoxy, and C1-C4 alkyl; the substituent may be C1-C4. 12 Alkyl, C6-C 20 Aromatic hydrocarbon cyclic groups C3~C 60 Aromatic heterocyclic groups or C1-C 12 Any substituted position of the alkoxy group.

[0015] In some embodiments of the present invention, R1 and R2 are each independently selected from methyl, ethyl, propyl, butyl, phenyl, m-dimethylphenyl, mestrimethylphenyl, m-diethylphenyl, mestriethylphenyl, m-diisopropylphenyl, and mestriisopropylphenyl.

[0016] In some embodiments of the present invention, R3 and R4 are each independently selected from deuterium, H, -OCH3, and -OCH2CH3, respectively.

[0017] In some embodiments of the present invention, the anion includes Cl. - ,Br - I - OH - NTf2 - OTf - TFA - BF4 - SbF6 - Any one of them.

[0018] In some embodiments of the present invention, the compound of formula I is selected from the following compounds:

[0019]

[0020] A second aspect of the present invention provides a method for preparing the compound of formula I, comprising the following steps:

[0021]

[0022] S1: Compound of Formula 1, compound of Formula 2 and compound of Formula 3 are reacted to prepare compound of Formula 4.

[0023] S2: Compound of Formula 4 was dissolved in trimethyl orthoformate, and trimethylchlorosilane was added under an inert atmosphere to react and obtain compound of Formula 5.

[0024] S3: Compound of Formula 5 reacts with compound of Formula 6 to prepare compound of Formula 7;

[0025] S4: Compound of Formula 7 is reacted with compound AgL to prepare compound of Formula I;

[0026] The definitions of R1, R2, R3, R4, and L are as described above.

[0027] In some embodiments of the present invention, compounds of formula 1, formula 2, and formula 3 are reacted in an organic acid to prepare compound 4. The organic acid includes any one of formic acid, acetic acid, hydrochloric acid, trifluoroacetic acid, and trifluoromethanesulfonic acid.

[0028] In some embodiments of the present invention, the reaction solvent in S1 includes at least one of methanol, ethanol, isopropanol, tetrahydrofuran, dichloromethane, and 1,4-dioxane.

[0029] In some embodiments of the present invention, the reaction solvent in S2 includes at least one of ethyl acetate, chloroform, tetrahydrofuran, diethyl ether, and 1,4-dioxane.

[0030] In some embodiments of the present invention, in S3, the compound of formula 5 and the compound of formula 6 are reacted under sodium acetate to prepare the compound of formula 7.

[0031] In some embodiments of the present invention, the reaction temperature in S3 is 50–80°C.

[0032] In some embodiments of the present invention, the reaction solvent in S3 includes at least one of acetone, dichloromethane (DCM), tetrahydrofuran, and acetonitrile.

[0033] In some embodiments of the present invention, the reaction solvent in S4 includes at least one of dichloromethane (DCM), tetrahydrofuran, chloroform, and acetonitrile.

[0034] A third aspect of the invention provides a pharmaceutical composition comprising the compound of formula I or a derivative thereof.

[0035] In some embodiments of the invention, the pharmaceutical composition may be administered to the patient in conventional formulations such as injections and suspensions. Suitable formulations may be prepared by commonly used methods using conventional organic or inorganic additives, such as excipients selected from fillers or diluents, binders, disintegrants, lubricants, flavoring agents, preservatives, stabilizers, suspending agents, dispersants, surfactants, antioxidants, or solubilizers.

[0036] Optional excipients are those known to those skilled in the art and include, but are not limited to, fillers or diluents (such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, or calcium carbonate, etc.), binders (such as cellulose, carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, polypropylene pyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, or starch, etc.), disintegrants (such as sodium glycolate starch, croscarmellose sodium, etc.), and lubricants (such as magnesium stearate, light anhydrous silicate, talc). Or sodium dodecyl sulfate, etc.), flavoring agents (such as citric acid, or menthol, etc.), preservatives (such as sodium benzoate, sodium bisulfite, methylparaben or propylparaben, etc.), stabilizers (such as citric acid, sodium citrate or acetic acid, etc.), suspending agents (such as methylcellulose, polyvinylpyrrolidone or aluminum stearate, etc.), dispersants (such as hydroxypropyl methylcellulose, etc.), surfactants (such as sodium dodecyl sulfate, poloxamer, polysorbate, etc.), antioxidants (such as ethylenediaminetetraacetic acid (EDTA), butylated hydroxytoluene (BHT, etc.) and stabilizers (such as polyethylene glycol, etc.). (etc.). The effective amount of the gold(III) complex containing N-heterocyclic carbene ligands in the pharmaceutical composition provided by the present invention can be at a level that will achieve the desired effect.

[0037] In a fourth aspect, the invention proposes the use of a compound of formula I and / or its derivatives in the preparation of a cancer-treating medicament.

[0038] In some embodiments of the present invention, the compound of formula I and / or its derivatives are able to tolerate excessive amounts of thiol compounds, such as GSH, and still retain normal catalytic activity under physiological conditions.

[0039] In some embodiments of the present invention, the treatment includes inducing cell death, inhibiting cell proliferation, and suppressing tumor growth in vivo.

[0040] In some embodiments of the present invention, the treatment includes a compound of formula I and / or its derivatives acting as a peroxidase-mediated Fenton-like reaction to generate hydroxyl radicals, which increase intracellular ROS levels and induce tumor cell apoptosis.

[0041] In some embodiments of the present invention, the treatment comprises inducing immunogenic cell death (ICD) in tumor cells by a compound of formula I and / or its derivatives.

[0042] In some embodiments of the present invention, the treatment includes at least one of the following: the compound of formula I and / or its derivatives induce calreticulin (CALR) on the endoplasmic reticulum of tumor cells to evert to the cell surface, release high-mobility group box 1 (HMGB1), and secrete adenosine triphosphate (ATP).

[0043] In some embodiments of the present invention, the treatment comprising a compound of formula I and / or its derivatives does not suppress immune function.

[0044] In some embodiments of the present invention, solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas.

[0045] In some embodiments of the present invention, the cancers for which the treatment is indicated include, but are not limited to, cancers of the breast, liver, neuroblastoma, head, neck, eye, mouth, throat, esophagus, chest, bone, lung, kidney, colon, rectum or other gastrointestinal organs, stomach, spleen, skeletal muscle, subcutaneous tissue, prostate, breast, ovary, testis or other reproductive organs, skin, thyroid, blood, lymph nodes, kidney, liver, pancreas and brain or central nervous system.

[0046] In some embodiments of the present invention, the cancer is at least one of hepatocellular carcinoma, cervical epithelioid carcinoma, lung cancer, breast cancer, melanoma, and nasopharyngeal carcinoma.

[0047] In some embodiments of the present invention, the methods for treating cancer provided by the present invention inhibit, alleviate, reduce, prevent, or stabilize cancer-related tumors. In some embodiments of the present invention, the methods for treating cancer provided by the present invention inhibit, alleviate, reduce, prevent, or stabilize blood flow, metabolism, or edema, or one or more symptoms thereof, in cancer-related tumors. In some embodiments of the present invention, the methods for treating cancer provided by the present invention cause the resolution of tumor, tumor blood flow, tumor metabolism, or peritumoral edema, and / or one or more cancer-related symptoms. In some embodiments of the present invention, the methods for treating cancer provided by the present invention maintain the size of the tumor so that it no longer increases, or increases to less than the tumor size after standard therapy, as measured by conventional methods available to those skilled in the art, such as digital rectal examination, ultrasound (e.g., transrectal ultrasound), CT scan, MRI, dynamic contrast-enhanced MRI, or PET scan. In some embodiments of the present invention, the methods for treating cancer provided by the present invention reduce tumor size. In some embodiments of the present invention, the methods for treating cancer provided by the present invention reduce tumor formation. In some embodiments of the present invention, the methods for treating cancer provided by the present invention eradicate, remove, or control primary, localized, and / or metastatic tumors related to cancer. In some embodiments of the present invention, the methods for treating cancer provided by the present invention reduce the number or size of cancer-related metastatic lesions.

[0048] In some embodiments of the invention, the method of treating cancer provided by the invention reduces an individual's tumor size (e.g., volume or diameter) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, 99%, or 100%, as evaluated by methods well known in the art, such as CT scans, MRI, DCE-MRI, or PET scans. In a particular embodiment, the method of treating cancer provided by the present invention reduces an individual's tumor volume or tumor size (e.g., diameter) by an amount within the range of about 5%-20%, 10%-20%, 10%-30%, 15%-40%, 15%-50%, 20%-30%, 20%-40%, 20%-50%, 30%-60%, 30%-70%, 30%-80%, 30%-90%, 30%-95%, 30%-99%, 30%-100% of the individual's tumor size (e.g., diameter) before administration of the compound I or its derivative, or in any range between these ranges, as evaluated by methods well known in the art such as CT scan, MRI, DCE-MRI, or PET scan.

[0049] In some embodiments, the method of treating cancer provided by the present invention reduces tumor perfusion in an individual by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, 99%, or 100%, as evaluated by methods well known in the art such as MRI, DCE-MRI, or PET scans. In a particular embodiment, the method for treating cancer provided by the present invention reduces the amount of tumor perfusion in an individual by about 5%-20%, 10%-20%, 10%-30%, 15%-40%, 15%-50%, 20%-30%, 20%-40%, 20%-50%, 30%-60%, 30%-70%, 30%-80%, 30%-90%, 30%-95%, 30%-99%, 30%-100% of the tumor perfusion prior to administration of the compound containing I or its derivatives, or any range between these values, as evaluated by methods well known in the art such as MRI, DCE-MRI, or PET scans.

[0050] In certain aspects, the method for treating cancer provided by the present invention inhibits or reduces tumor metabolism in an individual, as evaluated by methods well known in the art, such as PET scans. In certain embodiments, the method for treating cancer provided by the present invention inhibits or reduces tumor metabolism in an individual by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, as evaluated by methods well known in the art, such as PET scans, relative to tumor metabolism prior to administration of compound I or its derivatives. In a particular embodiment, the method for treating cancer provided by the present invention inhibits or reduces an individual's tumor metabolism by about 5%-20%, 10%-20%, 10%-30%, 15%-40%, 15%-50%, 20%-30%, 20%-40%, 20%-50%, 30%-60%, 30%-70%, 30%-80%, 30%-90%, 30%-95%, 30%-99%, 30%-100% relative to the tumor metabolism prior to administration of the compound containing I or its derivatives, or any range between these values, as evaluated by methods well known in the art such as PET scans.

[0051] In some embodiments of the present invention, the method of treating cancer proposed in this invention comprises administering a unit dose of a compound containing formula I and / or its derivatives. The dose may be administered as frequently as determined to be effective (e.g., once, twice, or three times daily, every other day, once or twice weekly, once every two weeks, or once monthly). In some embodiments, the method of treating cancer proposed in this invention comprises administering a unit dose of a compound containing formula I and / or its derivatives to an individual in need, the dose of which may be determined by those skilled in the art.

[0052] In some embodiments of the invention, an individual is given a unit dose of a compound of formula I and / or its derivatives or a pharmaceutical composition thereof once, twice, or three times daily; once, twice, or three times every other day (i.e., every other day); once, twice, or three times every two days; once, twice, or three times every three days; once, twice, or three times every four days; once, twice, or three times every five days; once, twice, or three times every week; once, twice, or three times every two weeks; or once a month, the dose of which may be administered orally.

[0053] In some embodiments of the present invention, the effective amount of the Formula I compound and / or its derivatives used in the treatment is 0.1 mg / kg to 50 mg / kg.

[0054] In this invention, the derivatives include their stereoisomers, their isotopically labeled derivatives, their pharmaceutically acceptable salts, their prodrugs, or their solvates.

[0055] The term "stereoisomer" includes enantiomers and diastereomers, as well as cis-trans isomers and tautomers.

[0056] The term "pharmaceutical acceptable" means that a drug is chemically or physically compatible with other components that make up a drug dosage form and is physiologically compatible with receptors.

[0057] The term "salt" refers to an acidic or basic salt formed by a compound or its stereoisomer with an inorganic acid, organic acid, or base, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained through separation and purification during compound preparation, or by mixing the aforementioned compound or its stereoisomer with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or be prepared by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compound.

[0058] The "prodrug" mentioned in this invention refers to a compound that is rapidly converted in vivo to the parent compound of the above formula, which can be converted into the compound of this invention by chemical or biochemical methods in vivo or in vitro environments, such as by hydrolysis in the blood.

[0059] The compounds of this invention can exist in both unsolvated and solvated forms, with solvation including hydrate forms. Generally, the solvated form is equivalent to the unsolvated form and is also covered within the scope of this invention.

[0060] The beneficial effects of this invention are:

[0061] The compound of formula I in this invention is a strongly sterically hindered nitrogen-containing heterocyclic carbene (NHC) gold (I) complex. This invention utilizes a steric hindrance control strategy to design a series of stable active gold (I) complexes containing highly sterically hindered NHC ligands. These highly sterically hindered gold complexes can tolerate excessive intracellular thiol compounds and exhibit good stability.

[0062] This sterically hindered gold complex of the present invention can possess normal catalytic activity intracellularly, exhibits good probe imaging and prodrug activation properties, and demonstrates highly efficient in vitro and in vivo antitumor activity.

[0063] The sterically hindered gold (I) complex of the present invention also has good antibacterial activity. Attached Figure Description

[0064] Figure 1 This is a schematic diagram illustrating the effect of the gold complex on the substrate in Embodiment 2 of the present invention.

[0065] Figure 2 This illustrates the effect of GSH on the catalytic activity of different gold catalysts in Example 2 of the present invention.

[0066] Figure 3 This illustrates the effect of GSH on the catalytic activity of Au-1 in Example 2 of the present invention.

[0067] Figure 4 This illustrates the effect of GSH on the catalytic activity of Au-3 in Example 2 of the present invention.

[0068] Figure 5 The results show the catalytic activity of Au-3 under physiological conditions in Example 4 of this invention.

[0069] Figure 6 This is the result of Au-3 activating doxorubicin prodrug under physiological conditions in Example 4 of the present invention.

[0070] Figure 7 The results of the thioredoxin reductase (TrxR) inhibitory activity assay in Example 5 of this invention are shown.

[0071] Figure 8The above figure shows the detection results of intracellular hydroxyl radical content in Example 6 of the present invention; wherein, the upper figure is a schematic diagram of the reaction between the probe and hydroxyl radicals, the lower left figure is a cell imaging image, and the lower right figure is the quantitative statistical result.

[0072] Figure 9 This is the result of detecting TNF-α content in macrophages in Example 7 of the present invention.

[0073] Figure 10 The results of the zebrafish antitumor activity detection in Example 8 of the present invention are shown. Detailed Implementation

[0074] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0075] In the following examples, the compounds are numbered as follows:

[0076]

[0077] Example 1

[0078] This embodiment prepares an Au-8, the specific process of which is as follows:

[0079]

[0080] (a) Under nitrogen protection, glyoxal solution (1.71 mL, 40% in water, 15 mmol), 2,6-diisopropylaniline (5.89 mL, 30 mmol), and formic acid (0.17 mL, 3 mmol) were added to 60 mL of methanol and stirred at room temperature for 24 h. The reaction solution was then filtered and the precipitate was collected. The precipitate was washed three times with methanol and dried to obtain 5.1 g of N,N'-bis(2,6-diisopropylphenyl)ethanediimide, with a yield of 90%.

[0081] (b) Under nitrogen protection, N,N'-bis(2,6-diisopropylphenyl)ethanediimide (376 mg, 1 mmol), trimethylchlorosilane (237 μL, 2 mmol), and trimethyl orthoformate (600 μL, 6 mmol) were added to 5 mL of EA. After stirring at room temperature for 12 h, the reaction solution was filtered and the precipitate was collected. The precipitate was washed three times with EA and recrystallized in acetonitrile to give a white solid, namely 375 mg of the target imidazolium salt, with a yield of 78%. 1H NMR(400MHz,Chloroform-d)δ11.19(s,1H),7.49(t,J=7.8Hz,2H),7.30(ddd,J=10.3,7.8,1.4Hz,4H) ,5.31(d,J=3.8Hz,2H),3.48(s,6H),3.18(p,J=6.8Hz,2H),2.90(p,J=6.8Hz,2H),1.53–1.22(m,24H). 13 C NMR (126MHz, Chloroform-d) δ162.08,147.70,145.53,131.57,128.18,125.14,124.70,100.59,60.50,29.15,29.08,25.73,25.27,23.42,23.34.

[0082] (c) Under nitrogen protection, the above imidazolium salt (97.2 mg, 0.2 mmol), dimethyl sulfide gold chloride (59 mg, 0.2 mmol), and sodium acetate (65.6 mg, 0.8 mmol) were added to 2 mL of acetone and stirred at 60 °C for 1 h. The reaction solution was then evaporated to dryness under vacuum, and 5 mL of LCM was added to resuspend the solution. The solution was then filtered through diatomaceous earth. When the filtrate was evaporated to 1 mL, n-pentane was added to precipitate a white solid. The white solid was collected as 122 mg of the target compound, with a yield of 91%. 1 H NMR(500MHz,Chloroform-d)δ7.42(t,J=7.7Hz,2H),7.26–7.20(m,4H),4.89(s,2H),3.30(s,8H), 2.95(p,J=6.8Hz,2H),1.42(d,J=7.0Hz,6H),1.35(dd,J=6.9,2.0Hz,12H),1.29(d,J=6.8Hz,6H). 13 C NMR(126MHz,Chloroform-d)δ198.50,148.70,146.00,132.46,130.28,124. 95,124.33,100.67,77.24,58.71,28.72,28.65,25.71,25.30,23.94,23.69.

[0083] (d) Under nitrogen protection, the above-mentioned nitrogen-containing heterocyclic carbene gold chloride (61 mg, 0.1 mmol) and silver trifluoroacetate (22 mg, 0.1 mmol) were dissolved in 2 mL of DCM and stirred at room temperature for 1 h. The reaction solution was filtered through diatomaceous earth and evaporated to dryness to obtain the target compound after anion substitution. 1H NMR(500MHz,Chloroform-d)δ7.39(t,J=7.8Hz,2H),4.88(s,2H),3.29–3.17(m,8H),2.87(h, J=6.8,6.0Hz,2H),1.34(d,J=7.0Hz,6H),1.30(dd,J=8.8,6.8Hz,12H),1.23(d,J=6.8Hz,6H). 13 C NMR(126MHz,Chloroform-d)δ191.03,148.66,146.07,132.26,130.34,124. 96,124.45,100.67,77.23,58.88,28.80,28.72,25.61,25.15,23.94,23.82.

[0084] Example 2

[0085] This embodiment evaluates the effect of different steric hindrances on the catalytic activity of gold complexes. The specific process is as follows:

[0086] First, the gold complex (1 mM) and 20 equivalents (20 mM) of GSH were incubated with shaking at 37°C for 10 minutes. Then, the substrate Probe (5 mM) was added and incubated at 37°C for 24 hours. The schematic diagram of the effect of the gold complex on the substrate is shown below. Figure 1 As shown, the reaction was then monitored using high-performance liquid chromatography (HPLC) on the diluted solution.

[0087] from Figure 2 As can be seen, Au-2 and Au-3 with larger steric hindrance can produce significant products in the presence of 20 equivalents of GSH. To confirm whether this result is caused by steric hindrance, Au-1 and Au-3 with larger differences in steric hindrance were further selected for comparison. After incubation with different equivalents of GSH (0eq, 2eq, 5eq, 10eq, 20eq, 50eq, 100eq), the results are as follows. Figure 3 and Figure 4 As shown, it was found that when the catalyst is Au-1 with low steric hindrance, only 2 equivalents of GSH are needed to mask its catalytic activity, while when the catalyst is replaced with Au-3 with high steric hindrance, even with the addition of 50 equivalents of GSH, a high yield can still be obtained.

[0088] Example 3

[0089] This embodiment tests the cytotoxicity of gold complexes, and the specific process is as follows:

[0090] (1) Inoculating cells

[0091] Human colon cancer (HCT116) cells (approximately 8,000 per well) were seeded in 96-well plates (1640, 10% FBS) and incubated in a constant temperature incubator for one day.

[0092] (2). Drug administration

[0093] First, a 10 mM DMSO solution containing the drug was prepared. This high-concentration DMSO solution was then diluted to 200 μM using complete culture medium to obtain a suitable working concentration. Subsequently, 100 μL of the drug solution was added to the first row of wells in the experimental plate. To obtain different gradients of drug concentrations, the drug solution was serially diluted from the first row of wells forward using a pipette, with three replicates for each concentration to reduce experimental error.

[0094] (3). MTT colorimetric method

[0095] After 24 hours, 16 μL of 5 mg / mL MTT solution was added to each well of a 96-well plate and incubated at 37°C for 4 hours. The mixture was then removed, and 130 μL of DMSO was added to each well to dissolve the generated formazan. The plate was shaken for 2 minutes to ensure complete dissolution of the formazan, and the absorbance at λ = 490 nm was measured using a microplate reader.

[0096] Table 1. Cytotoxicity assay of gold complexes (IC50) 50 / μM)

[0097] Au-1 Au-3 <![CDATA[Au-3-NTf2]]> Au-3-OTf Au-8 <![CDATA[IC 50 / μM]]> 57.5±5.3 23.1±2.4 36.1±2.7 17.6±2.1 15.2±1.6

[0098] The results are shown in Table 1. The MTT assay results showed that Au-1 had weak cytotoxicity. After coordination anion screening and structural modification, Au-8 with better anticancer activity was obtained.

[0099] The effects of biothiols on sterically hindered gold complexes were also verified at the cytotoxic level.

[0100] Table 2. Cytotoxicity of GSH against two different sterically hindered gold complexes (IC50) 50 / μM)

[0101] NO FBS 200μM GSH 500μM GSH 1000μM GSH Au-1 26.2±2.3 >100 >100 >100 Au-8 1.66±0.3 1.74±0.4 3.59±0.7 5.98±0.7

[0102] The results are shown in Table 2. Au-8 with large steric hindrance is more resistant to the toxic effects of biothiols.

[0103] In addition to human colon cancer cells, Au-8 was also tested for cytotoxicity against several other tumor cells, and the results are shown in Table 3.

[0104] Table 3. Cytotoxicity assay of gold complexes (IC) 50 / μM)

[0105]

[0106] Example 4

[0107] This embodiment tests the catalytic activity of Au-3 under physiological conditions. The specific process is as follows:

[0108] (1) Catalytic cyclization of alkynylamine probe luminescence

[0109] Human colon cancer (HCT116) cells (approximately 1.5 × 10⁻⁶) 6 Cells were seeded in 20 mm confocal microplates (1640, 10% FBS) and incubated for one day in a constant temperature incubator (37°C, 5% CO2 concentration, the same below). The original culture medium was removed and replaced with medium containing gold complex (15 μM) and probe (20 μM) (1640, 10% FBS) for 5 hours. The drug-containing medium was discarded and the cells were washed three times with serum-free (1640, No FBS) medium. 0.5 mL of PBS was added to the confocal microplates, and the cells were imaged using an inverted fluorescence microscope (DAPI channel).

[0110] The results are as follows Figure 5 As shown, when cells are treated with the probe or Au-3 alone, only weak fluorescence emission can be observed. However, when both are treated together, obvious fluorescence emission can be observed in the fluorescence imaging of HCT116 cells, mainly concentrated in the cytoplasm region, proving that Au-3 with greater steric hindrance still has basic catalytic activity under physiological conditions.

[0111] (2) Catalytic activation of doxorubicin prodrug

[0112] Human colon cancer (HCT116) cells (approximately 8,000 cells per well) were seeded in 96-well plates (1640, 10% FBS) and incubated for one day in a constant temperature incubator. The medium was then replaced with a medium (1640, 10% FBS) containing doxorubicin prodrug (25 μM) and Au-3 (10 μM) for co-incubation for 24 h. Cytotoxicity was then assessed using the MTT assay.

[0113] The results are as follows Figure 6 As shown, almost no cytotoxicity was observed when Au-3 was administered at 10 μM, and the doxorubicin prodrug also showed no significant toxicity when administered at 25 μM. However, we could see a significant increase in cytotoxicity when the two were administered in combination, indicating that Au-3 can catalyze the release of active doxorubicin from the doxorubicin prodrug under physiological conditions.

[0114] Example 5

[0115] This embodiment tests TrxR enzyme activity, and the specific process is as follows:

[0116] (1). Study on the inhibitory activity of purified thioredoxin reductase

[0117] Different concentrations of gold complexes were mixed with purified TrxR1 and incubated at room temperature for 30 minutes to allow for full reaction with the enzyme. Then, NADPH (0.2 mM) was added and mixed evenly, followed by DTNB (3 mM) and mixed evenly. The absorbance kinetic curve was immediately measured at λ = 412 nm. The curve was repeated three times, and the absorbance of the first minute was plotted.

[0118] (2). Study on the inhibitory activity of intracellular thioredoxin reductase in living cells

[0119] HCT116 (approximately 3 × 10⁻⁶) 6 Cells were seeded in 6-well plates and incubated in a constant temperature incubator for one day. A 10 mM DMSO solution of the gold complex was prepared, diluted to the target concentration with complete culture medium, and then incubated with cells at 37°C for 0.5 h. After drug treatment, the cells were first washed three times with ice-cold PBS to remove residual culture medium and impurities. Subsequently, the enzyme activity was measured using a thioredoxin reductase (TrxR) activity assay kit purchased from Solarbio, in triplicate, and the absorbance at the first minute was plotted.

[0120] The results are as follows Figure 7 As shown, auronoxine is a very potent TrxR enzyme inhibitor and was used as a positive control. Compared to the positive control auronoxine, Au-8 showed virtually no inhibitory activity against this enzyme.

[0121] Example 6

[0122] This embodiment tests intracellular hydroxyl radicals, and the specific process is as follows:

[0123] Human colon cancer (HCT116) cells (approximately 1.5 × 10⁻⁶) 6 Cells were seeded in 20 mm confocal microplates (1640, 10% FBS) and incubated for one day in a constant temperature incubator. The original culture medium was discarded and replaced with medium containing a gold complex (5 μM) (1640, 10% FBS). After incubation for 6 hours, the medium was removed, washed three times with PBS, and then incubated for another 20 minutes with medium containing a hydroxyl radical probe (APF, 5 μM) (1640, No FBS). Subsequently, the cells were washed three times with PBS, and 0.5 ml of PBS was added to the confocal microplate for cell imaging using a confocal laser scanning microscope (FITC channel).

[0124] The results are as follows Figure 8As shown, the top image is a schematic diagram of the reaction between the probe and hydroxyl radicals, the bottom left image is a cell imaging image, and the bottom right image is a quantitative statistical result. After incubation with Au-8, the intracellular hydroxyl radical level was 1.8 times that of the control group, demonstrating that Au-8 can promote the increase of hydroxyl radical content in tumor cells.

[0125] Example 7

[0126] This embodiment tests the TNF-α content of macrophages, and the specific process is as follows:

[0127] (1) Inoculating cells

[0128] Human monocytic leukemia (THP-1) cells (approximately 1.5 × 10⁻⁶) 5 Inoculate into 24-well plates (1640, 10% FBS) and incubate in a constant temperature incubator for one day.

[0129] (2). Phlorizol ester (PMA) induces differentiation into macrophages

[0130] Discard the original culture medium and replace it with a medium containing PMA (100 nM) (1640, 10% FBS) and incubate for 24 h. At this time, the cells should be completely adhered to the wall. Then replace the culture medium with drug-free medium (1640, 10% FBS) and relax for 24 h.

[0131] (3) Drug treatment of cells

[0132] Discard the original culture medium and replace it with a culture medium (1640, 10% FBS) containing different concentrations of aurinophene or Au-8. Incubate for 24 hours, collect the cell culture medium, centrifuge and take the supernatant, dilute it 50 times and test.

[0133] (4). Detection steps

[0134] The TNF-α content of the diluted supernatant was determined using the TNF-α ELISA kit purchased from Xinbosheng Company.

[0135] The results are as follows Figure 9 As shown, although auronoxine has been reported to inhibit tumor cell growth, it also stimulates the immune system; therefore, TNF-α was selected as an indicator of immune function. Compared to the potent inhibitory effect of auronoxine, Au-8 did not exhibit significant immunosuppressive activity.

[0136] Example 8

[0137] This embodiment investigates the antitumor activity of zebrafish, and the specific process is as follows:

[0138] (1) Inoculating cells

[0139] HCT116-GFP cells were seeded in 6 cm cell culture dishes (1640, 10% FBS) and incubated in a constant temperature incubator for one day.

[0140] (2) Collect tumor cells

[0141] Once the cells have grown to a suitable density, discard the original culture medium, wash three times with 1 mL PBS, digest with 1 mL trypsin and remove the trypsin, add 1 mL of culture medium (no FBS) and mix thoroughly by pipetting to ensure even dispersion. Transfer 10 μL of the cell suspension to a cell counting chamber and count at a density of approximately 2.7 × 10⁻⁶ cells / mL. 6 / mL, and cells were stored in an ice box before injection.

[0142] (3) Constructing a tumor model

[0143] Three-day-old wild-type zebrafish juveniles were anesthetized with tricaine (0.042 mg / mL) and the above-mentioned HCT116-GFP cell suspension was injected into the yolk sac of the juveniles through a microinjection system.

[0144] (4) Drug treatment of zebrafish

[0145] Juvenile zebrafish with tumors were raised in clean water prepared with Au-8 (625 nM) ultrapure water.

[0146] (5) Taking photos

[0147] After incubation with Au-8 (625 nM) for 24 h and 48 h (i.e., zebrafish juveniles at 4 days and 5 days old), the juveniles were anesthetized with tricaine (0.042 mg / mL) and photographed under an inverted fluorescence microscope (channel: FITC).

[0148] The results are as follows Figure 10 As shown, the tumor fluorescence signal intensity after treatment with the gold complex was 11% of that in the control group, indicating an 89% tumor inhibition effect.

[0149] Example 9

[0150] This embodiment investigates the inhibitory activity against Salmonella typhimurium.

[0151] Fresh single clones of *Salmonella Typhimurium* were picked from LB solid medium and cultured overnight in M9 medium. The following day, the bacterial culture was re-inoculated into M9 medium at a 1:100 ratio. When *Sm* reached the logarithmic growth phase in M9 medium, the bacterial culture was diluted 1:1000 and a half-diluted dose of the test drug was added. After incubation at 37°C with shaking for 24 hours, the optical density (OD) of the bacterial culture was measured. 600 (value), bacterial culture clarification or OD 600The lowest concentration with a value of 0 is the minimum inhibitory concentration (MIC).

[0152] Table 4. The (MIC / μM) of Au-8 against Salmonella cultured in M9 medium.

[0153] Au-8 <![CDATA[MIC 50 / μM]]> 0.22

[0154] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound, characterized in that: The structural formula of the compound is shown below: 。 2. A pharmaceutical composition, characterized in that: Includes the compound described in claim 1.

3. The use of the compound of claim 1 in the preparation of a medicament for treating cancer; wherein the cancer is at least one of cervical epithelioid carcinoma, lung cancer, breast cancer, melanoma, and colon cancer.

4. The application according to claim 3, characterized in that: The treatment includes at least one of the following: (I) the compound generates hydroxyl radicals as a peroxidase-mediated Fenton-like reaction, causing an increase in intracellular ROS levels and promoting tumor cell apoptosis; (II) the compound causes immunogenic death of tumor cells; (III) the compound induces at least one of the following: calreticulin on the endoplasmic reticulum of tumor cells to evert to the cell surface, releases high-mobility group 1 protein, and secretes adenosine triphosphate; (IV) the compound does not inhibit immune function.

5. The application according to claim 3, characterized in that: The effective amount of the compound used in the treatment is 0.1 mg / kg to 50 mg / kg.

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