Application of Hapalindole in the preparation of drugs for inhibiting liver cancer or breast cancer

Abstract

This invention provides the use of a Hapalindole compound in the preparation of a drug for inhibiting liver cancer or breast cancer, for: preparing a drug or preparation, composition or drug composition that specifically targets and eliminates tumor cells, induces tumor cell apoptosis and inhibits tumors; wherein the tumor is liver cancer or breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of oncology, and more specifically, this invention relates to the use of Hapalindole in the preparation of drugs for inhibiting liver cancer or breast cancer. Background Technology

[0002] Tumors develop when, under the influence of various factors, cells in a local tissue lose normal regulation of their growth at the gene level, leading to abnormal cell proliferation and the formation of new growths. Tumors are genetic diseases; their biological basis is gene abnormalities. Pathogenic factors include somatic cell gene mutations leading to the loss of normal genes and disordered gene expression, thereby affecting the biological and genetic activity of cells and forming tumor cells that differ from normal cells in morphology, metabolism, and function. Tumor development is the result of multiple genes, multiple steps, and mutations. Different gene mutations and varying degrees of mutation intensity result in different types of tumors. Abnormal tumor morphology is the basis for tumor pathological diagnosis.

[0003] The mechanisms of tumors vary depending on their type and the organ in which they occur, requiring different treatment plans and methods. Different treatment methods are needed for different tumors; medication should be prescribed according to the specific symptoms.

[0004] Liver cancer, or malignant tumor of the liver, can be divided into two main categories: primary and secondary. Primary malignant liver tumors originate from the epithelial or mesenchymal tissue of the liver; the former is called primary liver cancer, which is a highly prevalent and extremely dangerous malignant tumor. The latter, called sarcoma, is less common compared to primary liver cancer. Secondary or metastatic liver cancer refers to malignant tumors originating from multiple organs throughout the body that invade the liver. It is commonly seen as a liver metastasis from malignant tumors of the stomach, bile ducts, pancreas, colorectal, ovary, uterus, lung, and breast. Treatment for liver cancer includes various methods such as surgery, radiotherapy, chemotherapy, interventional procedures, targeted drugs, and immunotherapy. Liver cancer is a malignant tumor of the digestive system, and compared to other malignant tumors of the digestive system, such as stomach and intestinal cancer, it is arguably more difficult to treat. Like pancreatic cancer, liver cancer is extremely difficult to treat and is often referred to as the "king of cancers."

[0005] Breast cancer is a condition in which breast epithelial cells proliferate uncontrollably under the influence of various carcinogenic factors. Early symptoms often include breast lumps, nipple discharge, and enlarged axillary lymph nodes. In later stages, distant metastasis of cancer cells can lead to multi-organ complications, directly threatening the patient's life.

[0006] Although there are already some treatment methods available in this field for the cancers that commonly occur in the population, further research is needed to explore new therapeutic drugs in order to achieve better results or to supplement existing drugs. Summary of the Invention

[0007] The purpose of this invention is to provide an application of a Hapalindole compound or its pharmaceutically acceptable salt, ester, isomer, solvate or prodrug for: preparing a drug or preparation, composition or pharmaceutical composition that specifically targets and eliminates tumor cells, induces tumor cell apoptosis and inhibits tumors; wherein the tumor is liver cancer or breast cancer.

[0008] In a first aspect of the invention, the use of a Hapalindole compound or a pharmaceutically acceptable salt, ester, isomer, solvate or prodrug thereof for: preparing a drug or preparation that specifically targets and eliminates tumor cells, induces tumor cell apoptosis or inhibits tumors; wherein the tumor is liver cancer or breast cancer.

[0009] In another aspect of the invention, the use of the Hapalindole compound or a pharmaceutically acceptable salt, ester, isomer, solvate, or prodrug thereof is provided for: preparing a composition for inhibiting tumors; or preparing a composition for specifically targeting and eliminating tumor cells; or preparing a composition for inducing tumor cell apoptosis; wherein the tumor is liver cancer or breast cancer.

[0010] In another aspect of the invention, the use of the Hapalindole compound or a pharmaceutically acceptable salt, ester, isomer, solvate or prodrug thereof is provided for: preparing a pharmaceutical composition for inhibiting tumors; or preparing a pharmaceutical composition for specifically targeting and eliminating tumor cells; or preparing a pharmaceutical composition for inducing apoptosis of tumor cells; wherein the tumor is liver cancer or breast cancer.

[0011] In one or more embodiments, the Hapalindole compound is a compound represented by Formula I:

[0012]

[0013] Where R 1 R 2 R 3 It can be any group;

[0014] Preferably, R 1 R 2 One of them is vinyl, the other is methyl, and R 3 For NCS or NC;

[0015] More preferably, R 1 It's vinyl, R 2 It is methyl, R 3 It is NCS; or R 1 It is methyl, R 2 It's vinyl, R 3 It's NC.

[0016] In one or more embodiments, the Hapalindole compound is Hapalindole Q.

[0017] In one or more embodiments, the tumor cells include: liver cancer cells, breast cancer cells.

[0018] In one or more embodiments, the breast cancer includes triple-negative breast cancer.

[0019] In one or more embodiments, the tumor cells are human-derived cell lines.

[0020] In one or more embodiments, the liver cancer cells include Hep G2, HCC-LM3, SMMC-7721, and HuH-7, and the breast cancer cells include MDA-MB-231.

[0021] In one or more embodiments, the concentration of the Hapalindole compound or its pharmaceutically acceptable salt, ester, isomer, solvate or prodrug is 1 to 100 μM, preferably 5 to 50 μM, more preferably 5 to 40 μM.

[0022] In another aspect of the invention, a composition or pharmaceutical composition is provided for specifically targeting and eliminating tumor cells, inducing tumor cell apoptosis, and inhibiting tumors, comprising: a Hapalindole compound or a pharmaceutically acceptable salt, ester, isomer, solvate, or prodrug thereof; wherein the tumor is liver cancer or breast cancer.

[0023] In another aspect of the invention, a kit or reagent kit is provided for specifically targeting and eliminating tumor cells, inducing tumor cell apoptosis, and inhibiting tumors, comprising: a Hapalindole compound or a pharmaceutically acceptable salt, ester, isomer, solvate, or prodrug thereof; wherein the tumor is liver cancer or breast cancer.

[0024] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0025] Figure 1 The toxicity of 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM and 40 μM Hapalindole Q to different types of cancer cell lines (Hep G2, HCC-LM3, SMMC-7721, HuH-7, MDA-MB-231, A549, HeLa, SK-OV-3 and U-87MG) was detected by CCK8 assay.

[0026] Figure 2Flow cytometry was used to detect apoptosis induced in liver cancer cells by 0 μM (Control group), 10 μM, 15 μM, 20 μM, and 25 μM Hpalindole Q. The horizontal axis represents the fluorescence intensity of cells stained by the FITC channel (Annexin V), and the vertical axis represents the fluorescence intensity of cells stained by the PE channel (PI). The lower left Q3 region represents cells that cannot be stained by either Annexin V-FITC or PI, i.e., healthy cells; the lower right Q4 region represents cells that can only be stained by Annexin V-FITC alone, representing cells undergoing early apoptosis; the upper right Q1 region represents cells that can be double-stained by Annexin V-FITC and PI, i.e., cells undergoing late apoptosis; the upper left Q2 region represents some cell debris.

[0027] Figure 3 The toxicity of the Hapalindole Q analog LA-NZY-394 at concentrations of 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM to different types of cancer cell lines (MCF-7, Hep G2, SMMC-7721, MDA-MB-231, HeLa, and U-87MG) was determined by CCK8 assay.

[0028] Figure 4 The toxicity of the Hapalindole Q analog LA-NZY-472 at concentrations of 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM to different types of cancer cell lines (MCF-7, Hep G2, SMMC-7721, MDA-MB-231, HeLa, and U-87MG) was determined by CCK8 assay. Detailed Implementation

[0029] Through in-depth research, the inventors discovered that Hapalindole Q can promote apoptosis of liver cancer cells, thereby inhibiting the proliferation of liver cancer cell lines, and can be used to prepare anti-tumor drugs.

[0030] Hapalindole was first isolated and identified from the cyanobacterial alga *Hapalosiphon* in 1984. It is not unique to cyanobacteria; with further research, scientists have also isolated and extracted hapalindole from other organisms. Currently, there are two main strategies for the synthesis of hapalindole: one is to use commercially available cyclic terpenoids as starting materials, followed by derivatization and docking with an indole ring; the other is to construct a monoterpene six-membered ring in the later stages. From the perspective of drug discovery and derivative preparation, the latter is more conducive to structural modification of indole and the six-membered ring, thus facilitating the diversified preparation of derivatives.

[0031] However, current research on Hapalindole Q mainly focuses on its synthetic strategies and suggests that it possesses antibacterial, antifungal, anti-tuberculosis, and insecticidal effects. Research on other applications of Hapalindole, particularly its disease applications, remains lacking.

[0032] Hapalindole and Hapalindole Q

[0033] The “Hapalindole” mentioned in this article refers to a class of semi-indole compounds, including tetracyclic Hapalindoles, tricyclic Hapalindoles, and other derivatives or homologs such as fischerindoles, ambiguines, and welwitindolinones.

[0034] The term "derivative" refers to intermediates, derivatives, etc., derived during the synthesis of Hapalindole compounds. The term "homologous" refers to homologous products, byproducts, etc., derived during the isolation of Hapalindole from sources containing it (which can be natural or non-natural, such as plants).

[0035] The "Compound of Formula I" referred to in this article is a class of compounds having the general formula shown in Formula I, wherein R 1 R 2 R 3 It can be any group.

[0036]

[0037] In one or more embodiments, in Formula I, R 1 R 2 One of them is vinyl, the other is methyl, and R 3 It is either NCS or NC.

[0038] In one or more embodiments, in Formula I, R 1 It's vinyl, R 2 It is methyl, R 3 It's NCS.

[0039] In one or more embodiments, in Formula I, R 1 It is methyl, R 2 It's vinyl, R 3 It's NC.

[0040] The "Hapalindole Q" described in this article is a monomeric compound with the molecular formula C2. 21 H 24 N2S, CAS number 106928-29-4.

[0041] The “Hapalindole Q analogues” described in this article are a class of compounds with similar chemical structures to Hapalindole compounds.

[0042] In one or more embodiments, the Hapalindole Q analogues include, but are not limited to, LA-NZY-394 and LA-NZY-472.

[0043] The “LA-NZY-394” mentioned in this article is a compound as shown in Formula II.

[0044]

[0045] The “LA-NZY-472” mentioned in this article is a compound as shown in Formula III.

[0046]

[0047] In this invention, the “compound” (including Hapalindole, compound of formula I, Hapalindole Q, its salt or prodrug, etc.) may be a compound existing in pure form or a compound with a purity greater than 85% (preferably greater than 90%, for example 95%, 98%, 99%).

[0048] Those skilled in the art will understand that, upon learning the structure of the compounds of the present invention, they can be obtained using various methods well-known in the art and using known raw materials, such as chemical synthesis or extraction from biological sources (e.g., microorganisms), all of which are included in this invention. Furthermore, Hapalindole (e.g., Hapalindole Q) is a commercially available pharmaceutical product, and its finished product is readily available to those skilled in the art.

[0049] This invention also includes pharmaceutically acceptable salts, esters, isomers, solvates, or prodrugs of Hapalindole (such as Hapalindole Q), provided they also have the same or substantially the same function as the compound of Hapalindole Q. In this invention, a "pharmaceutically acceptable" component is a substance suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The "pharmaceutically acceptable salt" can be an acidic salt or a basic salt of Hapalindole (such as Hapalindole Q).

[0050] "Pharmaceutically acceptable acid salts" refer to salts that retain the biological activity and properties of the free base without exhibiting undesirable changes in biological activity or other aspects. These salts can be composed of inorganic acids, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and similar acids. They can also be composed of organic acids, such as, but not limited to, acetic acid, dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, camphoric acid, camphorsulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, formic acid, and fumaric acid. (acid), galactoic acid, gentian acid, glucoheponic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, viscous acid, naphthalene-1,5-disulfonic acid, 2-naphthalenesulfonic acid, 1-naphthol-2-carboxylic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, undecenoic acid and similar acids.

[0051] "Pharmaceutically acceptable basic salts" refer to salts that retain the biological activity and properties of the free acid without exhibiting undesirable changes in biological activity or other aspects. These salts are produced by adding an inorganic or organic base to the free acid. Salts obtained by using inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and similar salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts obtained from organic bases include, but are not limited to, primary, secondary, and tertiary ammonium salts. Substituted amines include naturally substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, tannin, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, halamine, choline, betaine, phenethylbenzylamine, N,N'-bisbenzylethylenediamine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, thiazoline, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resins, and similar structures. Preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0052] The compounds disclosed in this invention can exist as solvates (such as hydrates), including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and similar structures. This invention also includes a prodrug of Hapalindole Q, wherein a "prodrug" refers to a compound that, when taken in an appropriate manner, undergoes metabolism or chemical reaction in the body to be converted into the desired Hapalindole Q.

[0053] This invention also includes isomers of Hapalindole (such as Hapalindole Q). This is because the compounds have one or more asymmetric centers, so these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis or trans isomers.

[0054] Those skilled in the art will understand that, after learning the structure of the compounds of the present invention, they can be obtained using various methods well-known in the art and using known raw materials, such as chemical synthesis or extraction from biological sources (e.g., animals or plants) or modification of extracted materials; these methods are all included in the present invention. The compounds of the present invention can be synthesized using known methods; the synthesized compounds can be further purified by column chromatography, high-performance liquid chromatography, etc. Alternatively, the compounds of the present invention can also be obtained commercially.

[0055] This invention provides the use of Hapalindole (such as Hapalindole Q) in the preparation of pharmaceuticals or formulations for: specifically targeting and eliminating (killing) tumor cells, inducing tumor cell apoptosis, promoting tumor regression, reducing tumor volume, preventing or treating cancer, or prolonging cancer survival. In this document, "individual," "object," or "patient" refers to mammals, particularly humans.

[0056] In another aspect of the invention, a method for achieving the above-described uses is provided, the method comprising treating tumor cells (including primary cells, cell lines) with or administering to a subject in need of Hapalindole (such as Hapalindole Q) as described herein, or with pharmaceutically acceptable salts, esters, isomers, solvates, or prodrugs thereof. The terms “administer” or “drug delivery” as used herein refer to providing the compounds, compositions, or pharmaceutical compositions of the invention to a subject suffering from or at risk of a disease or condition to be treated or prevented.

[0057] Compositions of Hapalindole (such as Hapalindole Q), pharmaceutical compositions

[0058] The compositions of the present invention (e.g., pharmaceutical compositions) use Hapalindole (e.g., Hapalindole Q), or its salts, esters, isomers, solvates, prodrugs, etc., as active ingredients. As described above, substances containing Hapalindole (e.g., Hapalindole Q) can specifically target and eliminate (kill) tumor cells, induce tumor cell apoptosis, promote tumor regression, reduce tumor volume, prevent or treat cancer, or prolong cancer survival.

[0059] In this invention, the terms "containing", "having", or "including" include "comprising", "mainly composed of", "substantially composed of", and "composed of"; "mainly composed of", "substantially composed of", and "composed of" are subordinate concepts of "containing", "having", or "including".

[0060] When the compositions described herein are used as pharmaceuticals, they may also be referred to as pharmaceutical compositions, which may also contain pharmaceutically acceptable excipients. "Pharmaceutically acceptable excipients" are pharmaceutically or food-grade carriers, solvents, suspending agents, or excipients used to deliver the active ingredient (Hapalindole, such as Hapalindole Q) in the compositions of the present invention to animals or humans. Exemplary excipients may be liquids or solids, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, flow aids, sweeteners, dyes / coloring agents, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in conjunction with the pharmacodynamic compound. More specifically, suitable excipients may be those commonly used in the art for the administration of small molecule compounds. Examples of excipients include various lactoses, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, and detergents such as Tween 80, as well as monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrin, and starch.

[0061] Typically, the composition contains a therapeutically effective amount of the active ingredient described herein. A therapeutically effective amount refers to a dose that can achieve treatment, prevention, reduction, and / or relief of a disease or symptom in a subject. The effective amount of the hapalindole (such as hapalindole Q) described herein may vary depending on the mode of administration and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the hapalindole (such as hapalindole Q), such as bioavailability, metabolism, and half-life; the severity of the disease to be treated in the patient; the patient's weight; the patient's immune status; and the route of administration. The therapeutically effective amount can be determined based on factors such as the patient's age, sex, the nature and severity of the disease, and other physical conditions. The therapeutically effective amount can be administered as a single dose or in multiple doses according to an effective treatment regimen. In this document, "subject" or "patient" generally refers to mammals, particularly humans. For example, when the composition is used to treat tumor cells (e.g., tumor cell lines), the composition contains 0–100 μM, preferably 5–50 μM, more preferably 5–40 μM, such as 10–30 μM, 10–20 μM, 10–15 μM, 15–40 μM, 15–30 μM, 15–20 μM, 20–40 μM, or 20–30 μM of an active component (Hapalindole, e.g., Hapalindole Q).

[0062] The pharmaceutical compositions or mixtures of the present invention can be formulated into any conventional dosage form using conventional methods. Dosage forms can be diverse, as long as they enable the active ingredient to effectively reach the mammalian body. Examples include: injections, infusions, tablets, capsules, and pills. The active ingredient (Hapalindole, e.g., Hapalindole Q) can be present in a suitable solid or liquid carrier or diluent. Mixtures or pharmaceutical compositions of the active ingredients of the present invention can also be stored in sterile instruments suitable for injection or infusion. The effective dose of the active ingredient (e.g., Hapalindole Q) in the composition can vary depending on the mode of administration and the severity of the disease being treated, based on the experience and advice of the clinician.

[0063] In specific embodiments of this invention, a series of dosing regimens for Hapalindole, such as Hapalindole Q, according to different molar or mass ratios are proposed. Mice are also used as experimental animals in this invention. Converting the dosage for mice to a dosage suitable for humans is easily done by those skilled in the art, for example, using the Meeh-Rubner formula:

[0064] A = k × (W² / 3) / 10000.

[0065] In the formula, A is the body surface area, expressed in meters. 2 Calculations: W represents body weight in grams; k is a constant that varies by animal species: 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that dosage conversions can vary depending on the drug and clinical circumstances, and are based on the assessment of an experienced pharmacist.

[0066] Hapalindole, such as Hapalindole Q, or pharmaceutical compositions may be administered orally, as well as intravenously, intramuscularly, or subcutaneously. Oral administration is preferred. Suitable oral forms include, but are not limited to, tablets, powders, capsules, and sustained-release formulations. Suitable injectable forms include sterile aqueous solutions or dispersions and sterile powders. In all cases, these forms must be sterile and must be fluids to facilitate syringe dispensing. Where necessary, Hapalindole, such as Hapalindole Q, may also be administered in combination with other active ingredients or pharmaceuticals.

[0067] The present invention also provides a kit or reagent kit for specifically targeting and eliminating tumor cells or inducing tumor cell apoptosis, the kit or reagent kit containing a pharmaceutical composition as described in any embodiment herein, or the kit or reagent kit containing a mixture of Hapalindole as described herein, such as Hapalindole Q, or the kit or reagent kit containing: a container, and a Hapalindole as described herein, such as Hapalindole Q, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof placed in the container.

[0068] The kit or reagent may also contain materials for adjuvant medication, such as measuring instruments and containers, like syringes, required for using or administering various dosage forms of the composition. The kit or reagent may also contain instructions for use, describing methods for treating or eliminating tumor cells.

[0069] Application of Hapalindole (such as Hapalindole Q), or combinations thereof, and pharmaceutical compositions

[0070] As previously stated, the inventors have discovered the use of Hapalindole (such as Hapalindole Q) in the preparation of drugs or formulations for: specifically targeting and eliminating tumor cells, inducing tumor cell apoptosis, promoting tumor regression, reducing tumor volume, preventing or treating cancer, or prolonging cancer survival.

[0071] In another aspect of the invention, a method for achieving the above-described uses is provided, the method comprising treating tumor cells (including primary cells, cell lines) with or administering to a subject in need of Hapalindole (such as Hapalindole Q) as described herein, or with pharmaceutically acceptable salts, esters, isomers, solvates, or prodrugs thereof. The terms “administer” or “drug delivery” as used herein refer to providing the compounds, compositions, or pharmaceutical compositions of the invention to a subject suffering from or at risk of a disease or condition to be treated or prevented.

[0072] The inventors' research also revealed that hapalindole (such as hapalindole Q) exhibits highly effective targeted elimination (killing) of tumor cells at appropriate concentrations. For example, in some embodiments, the inventors found that hapalindole (such as hapalindole Q) at concentrations of 10–30 μM, 10–20 μM, 10–15 μM, 15–40 μM, 15–30 μM, 15–20 μM, 20–40 μM, and 20–30 μM showed highly effective targeted elimination (killing) of tumor cells, with 70%, 60%, 50%, 30%, or lower remaining at these concentrations, respectively.

[0073] The inventors' research also found that hapalindole (such as hapalindole Q) can induce apoptosis in tumor cells at appropriate concentrations, and this induction effect is concentration-dependent. For example, in some embodiments, the inventors found that hapalindole (such as hapalindole Q) at concentrations of 10–30 μM, 10–25 μM, 10–20 μM, 10–15 μM, 15–40 μM, 15–30 μM, 15–20 μM, 20–40 μM, and 20–25 μM exhibits highly effective in inducing apoptosis in tumor cells.

[0074] Based on the above-mentioned new discoveries of the inventors, the present invention provides a use of Hapalindole (such as Hapalindole Q) for preparing compositions or pharmaceutical compositions that specifically target and eliminate tumor cells; or for preparing compositions or pharmaceutical compositions that induce tumor cell apoptosis; or for preparing compositions or pharmaceutical compositions that inhibit tumors.

[0075] As used in this invention, unless otherwise stated, the term "tumor" includes, but is not limited to: liver cancer, breast cancer (preferably triple-negative breast cancer), lung cancer, cervical cancer, ovarian cancer, malignant glioma (e.g., brain glioma), prostate cancer, colorectal cancer, stomach cancer, pancreatic cancer, bladder cancer, skin cancer, kidney cancer, esophageal cancer, bile duct cancer, and brain cancer.

[0076] As used in this invention, unless otherwise stated, the term "tumor cells" includes, but is not limited to: liver cancer cells, breast cancer cells, lung cancer cells, cervical cancer cells, ovarian cancer cells, and malignant glioma cells.

[0077] As those skilled in the art know, tumor cells can originate from various sources, including humans, mice (rats, mice), rabbits, sheep, pigs, cattle, horses, and monkeys. Tumor cells can be primary cells (i.e., cells isolated from tumor tissue or animals) or passaged cells (cell lines). Preferably, the tumor cells are human-derived cell lines. For example, in some embodiments, the liver cancer cell lines include Hep G2, HCC-LM3, SMMC-7721, and HuH-7; the breast cancer cell line includes MDA-MB-231; the lung cancer cell line includes A549; the cervical cancer cell line includes HeLa; the ovarian cancer cell line includes SK-OV-3; and the malignant glioma cell line includes U-87MG.

[0078] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0079] Experimental materials:

[0080] Hapalindole Q: CAS number is 106928-29-4.

[0081] LA-NZY-394: Obtained from Shanghai Xinda Biotechnology Co., Ltd.

[0082] LA-NZY-472: Obtained from Shanghai Xinda Biotechnology Co., Ltd.

[0083] CCK8 reagent kit.

[0084] Annexin V / PI apoptosis kit.

[0085] Example 1: The toxicity of Hapalindole Q to various cancer cells

[0086] The toxicity of Hapalindole Q to different types of cancer cell lines was detected using the CCK8 assay, including human hepatocellular carcinoma cell lines Hep G2, HCC-LM3 (highly metastatic hepatocellular carcinoma cell line), SMMC-7721 (human hepatocellular carcinoma cell line), HuH-7 (human hepatocellular carcinoma cell line), human triple-negative breast cancer cell line MDA-MB-231, human lung cancer cell line A549, human cervical cancer cell line HeLa, human ovarian cancer cell line SK-OV-3, and human malignant glioma cell line U-87MG.

[0087] The cells were seeded in 96-well plates and cultured overnight at 37°C in a CO2 incubator to allow cell adhesion. Cells were treated with 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM Hapalindole Q for 24 hours. 10 μL of CCK8 (Cell Counting Kit 8) assay solution was added to each well, and the cells were incubated for 2 hours. The absorbance of each well at 450 nm was measured using a microplate reader. The inhibition rate of cell proliferation was calculated as follows: Inhibition rate (%) = (1 - Absorbance of experimental group / Absorbance of control group) × 100%. Three auxiliary wells were included in each experimental group.

[0088] like Figure 1 As shown, the experimental results indicate that Hapalindole Q exhibits significant toxicity to hepatocellular carcinoma cell lines Hep G2, HCC-LM3, SMMC-7721, HuH-7, and MDA-MB-231. It also shows some toxicity to the human lung cancer cell line A549.

[0089] However, its toxicity to human cervical cancer cell line HeLa, human ovarian cancer cell line SK-OV-3, and human malignant glioma cell line U-87MG is very weak. Furthermore, although not listed individually, it also lacks significant activity against several other types of cancer.

[0090] Example 2: Hapalindole Q induces apoptosis in liver cancer cells.

[0091] Hep G2 liver cancer cells were seeded in 6-well plates and cultured overnight at 37°C in a CO2 incubator to allow cell adhesion. Hep G2 cells were treated with 0 μM, 10 μM, 15 μM, 20 μM, and 25 μM of Hapalindole Q for 24 hours, respectively. Cells were collected after digestion with EDTA-free trypsin. Cells were washed twice with PBS. 2.5 μL of Annexin V (fluorescence detectable via the FITC channel) and 2.5 μL of PI fluorescent dye (fluorescence detectable via the PE channel) were added to each sample and mixed thoroughly. The samples were incubated at 37°C for 15 minutes, with the centrifuge tubes gently rotated every 5 minutes to ensure even staining. After incubation, 400 μL of binding buffer was added to each centrifuge tube and mixed thoroughly. Flow cytometry was used to detect the fluorescence intensity of the cells in both the FITC and PE channels.

[0092] like Figure 2 As shown, the horizontal axis represents the fluorescence intensity of cells under the FITC channel, and the vertical axis represents the fluorescence intensity of cells under the PE channel. The lower left Q3 region represents cells that cannot be stained by either Annexin V-FITC or PI, i.e., healthy cells; the lower right Q4 region represents cells that can only be stained by Annexin V-FITC alone, representing cells that have undergone early apoptosis; the upper right Q1 represents cells that can be stained by both Annexin V-FITC and PI, i.e., cells that have undergone late apoptosis; and the upper left Q2 region represents some cell debris.

[0093] The experimental results showed that, compared with the control group, most cells underwent a shift from the Q3 region to the Q4 region under the induction of compound Hapalindole Q, indicating that compound Hapalindole Q has an excellent effect in promoting cell apoptosis.

[0094] It was also observed that the higher the compound concentration, the more cells were present in the Q4 region, indicating that the compound Hapalindole Q can induce apoptosis in a concentration-dependent manner.

[0095] Example 3: Hapalindole Q analogues do not have a killing effect on cancer.

[0096] The toxicity of Hapalindole Q analogues LA-NZY-394 and LA-NZY-472 to different types of cancer cell lines was detected using the CCK8 assay, including human Luminal A breast cancer cell line MCF-7, human liver cancer cell lines Hep G2 and SMMC-7721, human triple-negative breast cancer cell line MDA-MB-231, human cervical cancer cell line HeLa, and human malignant glioma cell line U-87MG.

[0097] The cells were seeded in 96-well plates and cultured overnight at 37°C in a CO2 incubator to allow cell adhesion. Cells were treated with 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM Hapalindole Q analogs for 24 hours. 10 μL of CCK8 (Cell Counting Kit 8) assay solution was added to each well, and the cells were incubated for 2 hours. The absorbance of each well at 450 nm was measured using a microplate reader. The inhibition rate of cell proliferation was calculated as follows: Inhibition rate (%) = (1 - Absorbance of experimental group / Absorbance of control group) × 100%. Three auxiliary wells were included in each experimental group.

[0098] like Figure 3 and Figure 4 As shown, the experimental results indicate that the Hapalindole Q analogues LA-NZY-394 and LA-NZY-472 exhibited weak and insignificant toxicity to all tested cell lines.

[0099] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. The use of Hapalindole Q compound or a pharmaceutically acceptable salt thereof for the preparation of a drug or preparation that specifically targets and eliminates tumor cells, induces tumor cell apoptosis, or inhibits tumors; wherein the tumor is liver cancer or triple-negative breast cancer.

2. The use of Hapalindole Q compound or a pharmaceutically acceptable salt thereof for: Preparation of compositions for inhibiting tumors; or Preparation of compositions that specifically target and eliminate tumor cells; or Preparation of compositions that induce tumor cell apoptosis; The tumors mentioned are liver cancer or triple-negative breast cancer.

3. The use of Hapalindole Q compound or a pharmaceutically acceptable salt thereof for: Preparation of pharmaceutical compositions for inhibiting tumors; or Prepare pharmaceutical compositions that specifically target and eliminate tumor cells; or Preparation of pharmaceutical compositions that induce tumor cell apoptosis; The tumors mentioned are liver cancer or triple-negative breast cancer.

4. The application as described in any one of claims 1 to 3, characterized in that, The tumor cells mentioned include: liver cancer cells and triple-negative breast cancer cells.

5. The application as described in any one of claims 1 to 3, characterized in that, The concentration of the Hapalindole Q compound or a pharmaceutically acceptable salt thereof is 1–100 μM.

6. The application as described in claim 5, characterized in that, The concentration of the Hapalindole Q compound or a pharmaceutically acceptable salt thereof is 5–50 μM.

7. The application as described in claim 5, characterized in that, The concentration of the Hapalindole Q compound or a pharmaceutically acceptable salt thereof is 5–40 μM.

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