Use of small molecule compounds targeting npepl1 in the preparation of tumor-targeted therapeutic drugs

Abstract

The application relates to the field of biological medicines, in particular to application of small-molecule compounds targeting NPEPL1 in preparation of tumor-targeted therapeutic drugs, and four small-molecule compounds C301-9181, E859-1332, L281-0443 and C679-2629 with hepatocellular carcinoma cell killing effect and high NPEPL1 affinity are obtained for the first time. The small-molecule compounds realize tumor-targeted therapy by targeting NPEPL1 protein in tumor cells, and can be used for development of tumor-targeted therapeutic drugs. The application proposes a targeted intervention on NPEPL1 to realize potential therapeutic effect on malignant solid tumors such as hepatocellular carcinoma with high NPEPL1 expression, and optimal small-molecule compounds C301-9181 are obtained through layer-by-layer screening. The small-molecule compounds can be used for preparation of anti-tumor targeted drugs targeting NPEPL1, fill the drug research and development blank in the field of NPEPL1 targeted therapy, and have a wide application prospect.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and in particular to the application of small molecule compounds targeting NPEPL1 in the preparation of tumor-targeted therapeutic drugs. Background Technology

[0002] Hepatocellular carcinoma is the most common type of liver cancer worldwide, accounting for over 90% of all liver cancer types. Liver cancer ranks sixth in incidence and third in mortality among malignant tumors globally. [1] Hepatocellular carcinoma (HCC) has an insidious onset, with few or no obvious symptoms in its early stages. Clinically, 70-80% of HCC patients are diagnosed at an advanced stage, making them unsuitable for surgical resection. [2] Although molecularly targeted drugs such as sorafenib have been used in the clinical treatment of hepatocellular carcinoma, their treatment efficacy remains unsatisfactory and the prognosis is poor. [3] The 5-year survival rate for patients with advanced hepatocellular carcinoma is only 12.1%, indicating a significantly shortened survival period. [4] Therefore, exploring new therapeutic targets and strategies for hepatocellular carcinoma is of significant research importance and great clinical value.

[0003] The development and progression of hepatocellular carcinoma are closely related to metabolic disorders. [5] Tumor cells obtain sufficient intermediate substances and energy for their survival, proliferation, and metastasis by taking up and metabolizing exogenous amino acids and other nutrients. Hepatocellular carcinoma cells typically exhibit a high dependence on specific amino acids such as arginine and glutamine. [6] This amino acid metabolic reprogramming plays a crucial role as a key mechanism in the malignant progression of hepatocellular carcinoma. In amino acid metabolic reprogramming, an adequate supply of free amino acids depends on the catalytic cleavage of amino acids by aminopeptidases at the amino terminus of proteins. [7] Studies have shown that cancer patients with high expression of aminopeptidase tend to have a poorer prognosis. [8-9] Therefore, aminopeptidase-regulated amino acid metabolism reprogramming plays an important role in the malignant progression of hepatocellular carcinoma.

[0004] NPEPL1 protein belongs to the M1 zinc metallopeptidase family, encoded by the NPEPL1 gene, and distributed in the nucleoplasm and cytosol. It possesses both manganese ion-binding and metallopeptidase activities. As an aminopeptidase, NPEPL1 exhibits catalytic activity towards proteins, participating in protein hydrolysis and amino acid metabolism. It releases amino acids one by one by catalyzing the hydrolysis of unsubstituted N-terminal amino acids in proteins or peptides, thereby maintaining an adequate supply of free amino acids in tumor cells. It regulates amino acid metabolic reprogramming in hepatocellular carcinoma cells, thus affecting the metabolic state of tumor cells and inducing malignant progression of hepatocellular carcinoma. NPEPL1 plays an important role in physiological processes such as protein maturation, peptide degradation, signal transduction, and cell cycle regulation. Furthermore, NPEPL1 actively participates in electrolytic homeostasis, immune responses, and tumor progression.

[10] NPEPL1-highly expressed clear cell renal cell carcinoma

[11] Colorectal cancer

[12] Patients with malignant tumors such as SARS-CoV-2 typically have a poor prognosis and a significantly shortened overall survival.

[0005] Previous research by the inventors revealed that the NPEPL1 gene is highly expressed in hepatocellular carcinoma (HCC) tumors, and its expression is positively correlated with poor prognosis in HCC, suggesting that NPEPL1 could be a novel therapeutic target for HCC. Based on this, experiments demonstrated that downregulating NPEPL1 gene expression effectively inhibited the proliferation of human HCC cancer cell lines Hep3B and Huh7, promoted apoptosis, and significantly inhibited migration and invasion, thereby effectively controlling the malignant progression of HCC, while overexpression of NPEPL1 had the opposite effect. Animal experiments showed that xenograft models of human HCC cancer cell line Huh7 could form tumors, and the tumor size increased in a time-dependent manner after tumor formation. However, inhibiting NPEPL1 expression did not result in tumor formation in nude mice. Knockdown of NPEPL1 inhibited the occurrence and growth of HCC tumors in mice, suggesting that NPEPL1 could be used as a target for screening novel targeted drugs against HCC. Specifically, see Chinese invention patent application CN117085131A, which discloses an inhibitor of NPEPL1 for inhibiting the growth or expansion of liver cancer cells. The NPEPL1 inhibitor includes NPEPL1 antibody, antisense RNA of NPEPL1 nucleic acid, siRNA, shRNA or miRNA.

[0006] Compared to antibody and nucleic acid drugs, small molecule compounds have significant advantages, including high oral bioavailability, strong transmembrane and intracellular target accessibility, strong blood-brain barrier penetration, low production cost, and low immunogenicity risk. This allows small molecule drugs to maintain an irreplaceable position as the dominant force among traditional drugs, competing with large molecule biologics represented by antibody and nucleic acid drugs. However, to date, there are no reports of small molecule targeted anti-tumor drugs targeting NPEPL1. Summary of the Invention

[0007] This invention addresses the aforementioned problems by targeting NPEPL1 as the core target. Based on the structure of the NPEPL1 protein, 49 candidate compounds were successfully screened using computer-aided high-throughput virtual screening technology. Through layered screening using cell efficacy experiments and SPR affinity assays, four small molecule compounds with high NPEPL1 affinity and hepatocellular carcinoma cell killing effects were obtained: C301-9181, E859-1332, L281-0443, and C679-2629.

[0008] The IC50 values ​​of these four small molecule compounds were below 30 μM, even lower than the IC50 value (27.54 μM) of paclitaxel, a positive control drug for liver cancer, against Huh7 cells, indicating a strong cytotoxic effect on Huh7 cells. Further analysis of the NPEPL1 affinity of the four compounds revealed that the affinity (dissociation constant K) of the four small molecule compounds was relatively low. D The performance of the drugs is similar. Among them, C301-9181 has the smallest dissociation rate constant Kd, indicating that C301-9181 has a longer average residence time on the NPEPL1 protein target. This also means that C301-9181 has a longer-lasting effect, a lower dosing frequency, and fewer off-target effects, making it more suitable for developing long-acting, low-dose small molecule drugs.

[0009] Further analysis was conducted on the in vivo and in vitro antitumor effects of small molecule compounds, using C301-9181 as an example. Cell experiments revealed that small molecule compound C301-9181 effectively inhibited the proliferation, migration, and invasion of hepatocellular carcinoma cells and promoted their apoptosis. In vivo experiments further demonstrated that small molecule compound C301-9181 effectively inhibited tumor growth in hepatocellular carcinoma xenograft animal models, improved lung inflammation, and reduced transaminase levels, while also exhibiting certain biocompatibility.

[0010] Based on relevant research, and to achieve the above and other related objectives, the first aspect of this invention provides the use of a small molecule compound targeting NPEPL1 or a pharmaceutically acceptable salt thereof in the preparation of a tumor-targeting therapeutic agent. The tumor is selected from malignant solid tumors with high NPEPL1 expression, and the structural formula of the small molecule compound is shown in any one of formulas (I) to (IV):

[0011]

[0012] Equation (I) Equation (III)

[0013] .

[0014] Formula (II) Formula (IV)

[0015] The compounds shown in formulas (I) to (IV) correspond to C301-9181, L281-0443, E859-1332, and C679-2629, respectively.

[0016] Preferably, the molecular weight of the small molecule compound C301-9181 is 375.45 g / mol, and its molecular formula is: C 20 H 17 N5OS, System Naming (IUPAC):

[0017] N-[4-[(5,6-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)thio]phenyl]benzamide.

[0018] SMILES are CC1=C(N2C(=NC=N2)N=C1C)SC3=CC=C(C=C3)NC(=O)C4=CC=CC=C4.

[0019] Preferably, the small molecule compound E859-1332 has a molecular weight of 452.99 g / mol and a molecular formula of C. 24 H 29 CIN6O, System Naming (IUPAC):

[0020] [1-[2-(4-chlorophenyl)-3,4-dimethylpyrazolo[3,4-d]pyridazin-7-yl]piperidin-3-yl]piperidin-1-yl methyl ketone.

[0021] SMILES is CC1=C2C(=NN=C(C2=NN1C3=CC=C(C=C3)Cl)N4CCCC(C4)C(=O)N5CCCC5)C.

[0022] Preferably, the molecular weight of the small molecule compound L281-0443 is 482.40 g / mol, and its molecular formula is C. 22 H 20 BrN5OS, system naming (IUPAC):

[0023] 2-Bromo-N-[4-[(5-methyl-6-propyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)thio]phenyl]benzamide.

[0024] SMILES is CCCC1=C(N2C(=NC=N2)N=C1C)SC3=CC=C(C=C3)NC(=O)C4=CC=CC=C4 Br.

[0025] Preferably, the small molecule compound C679-2629 has a molecular weight of 405.50 g / mol and a molecular formula of C. 20 H 15 N5OS2, System Naming (IUPAC):

[0026] 7-(1H-benzimidazol-2-ylthiomethyl)-2-(3-methylphenyl)-[1,3,4]thiadiazo[3,2-a]pyrimidin-5-one.

[0027] SMILES are CC1=CC(=CC=C1)C2=NN3C(=O)C=C(N=C3S2)CSC4=NC5=CC=CC=C5N4.

[0028] Furthermore, the small molecule compound or its pharmaceutically acceptable salt achieves tumor-targeted therapy by targeting the NPEPL1 gene or protein in tumor cells, and has one or more of the following effects, thereby also conferring corresponding efficacy to the drug:

[0029] 1) It can bind to the NPEPL1 protein;

[0030] 2) Inhibits the proliferation of human hepatocellular carcinoma cells;

[0031] 3) Promotes apoptosis in human hepatocellular carcinoma cells;

[0032] 4) Inhibits the migration and invasion of human hepatocellular carcinoma cells;

[0033] 5) Inhibits the growth of hepatocellular carcinoma tumors in animal models of hepatocellular carcinoma xenograft;

[0034] 6) Inhibit the expression or activity of NPEPL1;

[0035] 7) Alleviates lung inflammation in animal models of hepatocellular carcinoma xenograft;

[0036] 8) Reduce the levels of alanine aminotransferase and aspartate aminotransferase in animal models of hepatocellular carcinoma xenograft.

[0037] Furthermore, the pharmaceutically acceptable salts described in this invention are widely selected and can be chosen from salts formed with any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and hydroxyethanesulfonic acid.

[0038] Furthermore, the tumors involved in this invention are malignant solid tumors with high NPEPL1 expression. In addition to hepatocellular carcinoma involved in the preferred embodiments of this invention, other malignant solid tumors with high NPEPL1 expression are also applicable, such as renal cancer, lung cancer, colorectal cancer, head and neck cancer, pancreatic cancer, breast cancer, cervical cancer, prostate cancer, ovarian cancer, melanoma, gastric cancer, urothelial carcinoma, thyroid cancer, etc.

[0039] Furthermore, the tumor-targeted therapy drug of the present invention uses a small molecule compound or its salt represented by any one of formulas (I) to (IV) as the sole active ingredient, or contains a small molecule compound or its salt represented by any one of formulas (I) to (IV) in a content of 0.1-99 wt%, which can be specifically screened by experiments according to actual conditions.

[0040] In a second aspect, the present invention provides a pharmaceutical composition targeting NPEPL1, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is selected from one of the four small molecule compounds mentioned above or a pharmaceutically acceptable salt thereof.

[0041] Preferably, in actual treatment, the drug composition is used in combination with other tumor treatment drugs.

[0042] Furthermore, the dosage form of the pharmaceutical composition is selected from injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories.

[0043] In a third aspect, this application provides a method for targeted cancer therapy, the method comprising: administering to a subject an effective amount of the aforementioned small molecule compound or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0044] Compared with the prior art, the beneficial effects of this application are as follows:

[0045] Based on previous in vitro and in vivo experimental studies, this application proposes the potential therapeutic effect of targeted intervention on NPEPL1 in malignant solid tumors with high NPEPL1 expression, such as hepatocellular carcinoma. For the first time, four small molecule compounds with high NPEPL1 affinity and high efficacy in killing hepatocellular carcinoma cells were screened and obtained for use in the preparation of anti-tumor targeted drugs. Based on the significant advantages of small molecule compounds, such as high oral bioavailability, strong transmembrane and intracellular target accessibility, strong blood-brain barrier penetration, low production cost, and low immunogenicity risk, this invention provides new evidence and reference for targeted therapy of malignant solid tumors with high NPEPL1 expression, such as hepatocellular carcinoma. It has significant meaning and value in clinical research, clinical treatment, and the optimization of the safety of targeted tumor therapy with biological protective agents. Attached Figure Description

[0046] Figure 1 The results of computer-aided high-throughput virtual screening (CADD) targeting NPEPL1 are shown.

[0047] Figure 2 The study showed the cytotoxic effects of 49 NPEPL1 candidate small molecule compounds obtained from screening on the human hepatocellular carcinoma cell line Huh7, as well as the cell survival curves and IC50 values ​​of 6 small molecule compounds that were re-screened. Four small molecule compounds with high cell-killing activity were selected: E859-1332, L281-0443, C679-2629, and C301-9181.

[0048] Figure 3 The results of SPR affinity assays for four small molecule compounds targeting NPEPL1 with the NPEPL1 protein are shown.

[0049] Figure 4 Two-dimensional and three-dimensional binding diagrams of the interactions between four small molecule compounds and the NPEPL1 protein are shown.

[0050] Figure 5 The results showed that the small molecule compound C301-9181 inhibited the proliferation of human hepatocellular carcinoma cell lines Hep3B and Huh7.

[0051] Figure 6 The results showed that the small molecule compound C301-9181 promoted apoptosis in human hepatocellular carcinoma lines Hep3B and Huh7.

[0052] Figure 7 The experimental results show that the small molecule compound C301-9181 inhibits the migration and invasion of human hepatocellular carcinoma lines Hep3B and Huh7.

[0053] Figure 8The study demonstrated the construction of an animal model of hepatocellular carcinoma xenograft and the in vivo tumor-suppressing effect of the small molecule compound C301-9181 in the animal model of hepatocellular carcinoma xenograft, including tumor size and tumor weight.

[0054] Figure 9 The study demonstrated the effect of the small molecule compound C301-9181 on the NPEPL1 target expression level in an in vivo animal model of hepatocellular carcinoma xenograft.

[0055] Figure 10 The effects of the small molecule compound C301-9181 on six major organs—heart, liver, spleen, lung, kidney, and brain—of a hepatocellular carcinoma xenograft animal model were demonstrated.

[0056] Figure 11 The effects of the small molecule compound C301-9181 on liver and kidney function in an animal model of hepatocellular carcinoma xenograft were demonstrated. Detailed Implementation

[0057] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0058] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0059] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques from the fields of biomedical engineering, biophysics, pharmaceutics, pharmaceutical analysis, medicinal chemistry, analytical chemistry, molecular biology, biochemistry, and related areas. These techniques have been well described in existing literature.

[0060] The inventors of this application, through extensive research and exploration, discovered the application of four compounds, including the small molecule compound C301-9181 or its pharmaceutically acceptable salt, in the preparation of tumor-targeted therapy drugs, and completed this application based on this discovery.

[0061] In this application, previous studies found that the NPEPL1 gene is highly expressed in hepatocellular carcinoma (HCC) tumors, and its expression is positively correlated with poor prognosis in HCC, suggesting that NPEPL1 could serve as a novel therapeutic target for HCC. Based on this, using the NPEPL1 protein as a target, we further screened and tested several small molecule inhibitors targeting NPEPL1, including C301-9181, providing a new perspective and target for targeted therapy of solid tumors with high NPEPL1 expression, such as HCC.

[0062] This application screened four small molecule compounds: E859-1332, L281-0443, C679-2629, and C301-9181. Their IC50 values ​​were all lower than those of paclitaxel, a positive control for liver cancer, against Huh7 cells. Furthermore, their affinity for NPEPL1 protein (dissociation constant K) was also lower. D The properties are very similar. Among them, C301-9181 has the smallest dissociation rate constant Kd, indicating that C301-9181 has a longer-lasting effect and a lower dosing frequency, which is more conducive to the development of long-acting, low-dose small molecule drugs.

[0063] In a preferred embodiment of the present invention, the use of small molecule compound C301-9181 or a pharmaceutically acceptable salt thereof in the preparation of tumor-targeted therapeutic drugs is most preferably provided, wherein the structural formula of small molecule compound C301-9181 is shown in formula (I):

[0064]

[0065] Equation (I)

[0066] In this invention, the molecular weight of the small molecule compound C301-9181 is 375.45 g / mol, and its SMILES are C1=C(N2C(=NC=N2)N=C1C)SC3=CC=C(C=C3)NC(=O)C4=CC=CC=C4.

[0067] In this invention, the small molecule compound C301-9181 or its pharmaceutically acceptable salt achieves tumor-targeted therapy by targeting the NPEPL1 gene or protein in tumor cells, specifically by downregulating the NPEPL1 gene expression level, and / or reducing the NPEPL1 protein activity, and / or regulating the NPEPL1 function.

[0068] Furthermore, the tumor is selected from solid tumors that highly express NPEPL1. Preferably, the tumor is selected from hepatocellular carcinoma, renal cell carcinoma, lung cancer, colorectal cancer, head and neck cancer, pancreatic cancer, breast cancer, cervical cancer, prostate cancer, ovarian cancer, melanoma, gastric cancer, urothelial carcinoma, and thyroid cancer. More preferably, the tumor is selected from hepatocellular carcinoma.

[0069] In a preferred embodiment, the reference to "pharmaceutically acceptable salt" generally refers to any salt that is physiologically tolerable when used in a suitable manner for treatment (particularly when applied or used in humans and / or mammals). This generally means that it is non-toxic, particularly as a result of an anti-ion. These physiologically acceptable salts can be formed with cations or bases, and in the context of this invention, particularly when administered to humans and / or mammals, they should be understood as salts formed from at least one compound provided according to this invention, typically an acid (deprotonated), such as an anion, and at least one physiologically tolerable cation (preferably an inorganic cation). Specifically, in the context of this invention, this may include salts formed with alkali metals and alkaline earth metals, as well as salts formed with ammonium cations (NH4+, ... 4+ Salts formed with (mono) or (di) sodium, (mono) or (di) potassium, magnesium, or calcium can be, specifically including but not limited to, salts formed with (mono) or (di) sodium, (mono) or (di) potassium, magnesium, or calcium. These physiologically acceptable salts can also be formed with anions or acids, and in the context of this invention, particularly when administered to humans and / or mammals, they should be understood as salts formed by at least one compound provided according to this invention, typically protonated, such as a cation, and at least one physiologically tolerable anion. Salts of the small molecule compound C301-9181 include, but are not limited to, salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or hydroxyethanesulfonic acid. Salts of halides are also applicable. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium).

[0070] In this invention, in addition to the small molecule compound C301-9181 or its pharmaceutically acceptable salt, the related applications of C301-9181 or its hydrates, enantiomers, diastereomers, solvates or crystalline forms in the preparation of drugs for antitumor purposes also fall within the scope of protection of this invention.

[0071] In this invention, the small molecule compounds, including compound C301-9181, have one or more of the following effects:

[0072] 1) It can bind to the NPEPL1 protein;

[0073] 2) Inhibits the proliferation of human hepatocellular carcinoma cells;

[0074] 3) Promotes apoptosis in human hepatocellular carcinoma cells;

[0075] 4) Inhibits the migration and invasion of human hepatocellular carcinoma cells;

[0076] 5) Inhibits the growth of hepatocellular carcinoma tumors in animal models of hepatocellular carcinoma xenograft;

[0077] 6) Inhibit the expression or activity of NPEPL1;

[0078] 7) Alleviates lung inflammation in animal models of hepatocellular carcinoma xenograft;

[0079] 8) Reduce the levels of alanine aminotransferase and aspartate aminotransferase in animal models of hepatocellular carcinoma xenograft.

[0080] Specifically, inhibiting NPEPL1 expression can be achieved by inhibiting the transcription or translation of the NPEPL1 gene. In particular, this can mean preventing the NPEPL1 gene from being transcribed, reducing the transcriptional activity of the NPEPL1 gene, preventing the NPEPL1 gene from being translated, or reducing the translation level of the NPEPL1 gene.

[0081] Inhibiting NPEPL1 activity means reducing NPEPL1 activity. Preferably, compared to before inhibition, NPEPL1 activity is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0082] The present invention also provides a pharmaceutical composition comprising the aforementioned four small molecule compounds (preferably C301-9181) or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or excipient.

[0083] In some embodiments, the main active ingredient of the pharmaceutical composition is the aforementioned small molecule compound C301-9181 or a pharmaceutically acceptable salt thereof. In addition to the small molecule compound C301-9181 or a pharmaceutically acceptable salt thereof described in this invention, the pharmaceutical composition may also contain other drugs that have or may have antitumor activity.

[0084] In some embodiments, the pharmaceutically acceptable excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used as needed to aid in the stability of the drug or to help improve the activity of the active ingredient (i.e., the small molecule compound C301-9181 or its pharmaceutically acceptable salt as described above in this invention), and include, but are not limited to: diluents, surfactants, humectants, binders, fillers, disintegrants, adsorbents, lubricants, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and the pharmaceutical composition thus formulated can be administered by any suitable route of administration known to those skilled in the art as needed.

[0085] In some embodiments, the diluent includes, but is not limited to, starch, lactose, sucrose, microcrystalline cellulose (MCC), dicalcium phosphate, and calcium sulfate. The surfactant includes, but is not limited to, sodium stearate, sodium lauryl sulfate, benzalkonium chloride, cetrimonium bromide, polysorbates, polyethylene glycol (PEG), fatty acid glycerides, lecithin, and soybean lecithin. The humectant includes, but is not limited to, water, ethanol, polysorbate 80, and sodium lauryl sulfate solution. The binder includes, but is not limited to, starch paste, povidone (PVP), hydroxypropyl methylcellulose (HPMC), and microcrystalline cellulose (MCC). The filler includes, but is not limited to, mannitol, glucose, hydroxypropyl cellulose, calcium carbonate, and sodium carbonate. The disintegrant includes, but is not limited to, dry starch, crospovidone (PVPP), sodium carboxymethyl starch (CMS-Na), and low-substituted hydroxypropyl cellulose (L-HPC). The adsorbent carrier includes, but is not limited to, activated carbon, silica gel, starch, and dextrin. The lubricants include, but are not limited to: magnesium stearate, stearic acid, polyethylene glycol (PEG), sodium dodecyl sulfate (SLS), micronized silica gel, talc, etc. The stabilizers include, but are not limited to: sodium sulfite, sodium bisulfite, vitamin E (α-tocopherol), butylated hydroxyanisole (BHA), ethylenediaminetetraacetic acid (EDTA) and its sodium salt, benzyl alcohol, chlorobutanol, etc. The bactericides include, but are not limited to: ethanol, benzophenol, phenol, cresol, benzalkonium bromide, etc. The buffers include, but are not limited to: phosphate buffer, acetate buffer, borate buffer, etc. The isotonic agents include, but are not limited to: sodium chloride, glucose, glycerol, etc. The chelating agents include, but are not limited to: EDTA and its sodium salt, citric acid, tartaric acid, etc. The pH control agents include, but are not limited to: hydrochloric acid, citric acid, sodium hydroxide, ammonia, amino acids, etc.

[0086] In some embodiments, the drug may additionally contain liquids such as water, saline, glycerin, and ethanol.

[0087] In the pharmaceutical composition of the present invention, the small molecule compound C301-9181 or its pharmaceutically acceptable salt may be a single active ingredient or may be combined with other active ingredients to form a combined formulation.

[0088] In the pharmaceutical compositions of the present invention, the content of the active ingredient (preferably a small molecule compound C301-9181 or a pharmaceutically acceptable salt thereof) is generally a safe and effective amount. This safe and effective amount should be adjustable by those skilled in the art. For example, the dosage of the active ingredient typically depends on the patient's weight, the type of application, the condition and severity of the disease. For instance, the dosage of the active ingredient can typically be 1-1000 mg / kg / day, 20-200 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, etc. day, 5~10mg / kg / day, 10~20mg / kg / day, 20~30mg / kg / day, 30~40mg / kg / day, 40~60mg / kg / day, 60~80mg / kg / day, 80~10 0mg / kg / day, 100~150mg / kg / day, 150~200mg / kg / day, 200~300mg / kg / day, 300~500mg / kg / day, or 500~1000mg / kg / day.

[0089] Those skilled in the art can determine the effective dosage based on the severity of the condition and the recipient's health status and age. The effective dosage typically varies between 0.01 ng / kg body weight and approximately 100 mg / kg body weight.

[0090] The active ingredient or pharmaceutical composition containing the active ingredient provided by this invention can be adapted to any form of administration, including oral or parenteral administration, for example, via pulmonary, nasal, rectal and / or intravenous injection, and more specifically, via intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, oral, sublingual, nasal, percutaneous, vaginal, oral or parenteral administration; injection administration includes intravenous injection, intramuscular injection and subcutaneous injection, percutaneous administration, etc.

[0091] As used herein, the dosage form of the pharmaceutical composition is selected from: injections, sterile powders for injection, tablets, pills, capsules, lozenges, tinctures, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories. Those skilled in the art can select appropriate formulations based on the route of administration. For example, formulations suitable for oral administration may include, but are not limited to, pills, tablets, chewable tablets, capsules, granules, solutions, drops, syrups, aerosols, or powder inhalers; formulations suitable for parenteral administration may include, but are not limited to, solutions, suspensions, rehydrated dry preparations, or sprays; suppositories are typically suitable for rectal administration; and injections and sterile powders for injection are suitable for injection administration.

[0092] Tablets, lozenges, pills, and capsules may also contain the following components: binders, such as gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, or alginic acid; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, lactose, or saccharin, or flavorings, such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the above-mentioned substances. Various other substances may exist in coating form or be used to improve the physical form of the unit dosage form. For example, shellac, sugar, or both may be used to coat tablets, pills, or capsules. Syrups or elixirs may contain active compounds, sucrose as a sweetener, methylparaben and propylparaben as preservatives, colorings, and flavorings, such as cherry or orange flavoring. Any substance used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic in the dosage. In addition, active compounds can be incorporated into sustained-release products or formulations.

[0093] Those skilled in the art will understand that although the pharmaceutical compositions mentioned above may further contain pharmaceutically acceptable excipients, when the small molecule compound C301-9181 or its pharmaceutically acceptable salt is used as a drug in humans or animals, it can also be administered in its own form (small molecule compound C301-9181 or its pharmaceutically acceptable salt), that is, the present invention can be achieved without any of the aforementioned pharmaceutically acceptable excipients.

[0094] The present invention further provides a method for targeted tumor therapy, the method comprising: administering to a subject an effective amount of the aforementioned small molecule compound or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition. The method may be in vitro or non-therapeutic.

[0095] Preferably, the tumor is selected from malignant solid tumors that highly express NPEPL1; more preferably, the tumor is selected from hepatocellular carcinoma, renal cell carcinoma, lung cancer, colorectal cancer, head and neck cancer, pancreatic cancer, breast cancer, cervical cancer, prostate cancer, ovarian cancer, melanoma, gastric cancer, urothelial carcinoma, and thyroid cancer, with hepatocellular carcinoma being the most preferred.

[0096] According to the method of the present invention, the small molecule compound or a pharmaceutically acceptable salt thereof may be co-administered with other therapeutic agents. "Co-administered" means administered simultaneously in the same formulation or in two different formulations via the same or different routes, or administered sequentially via the same or different routes. "Sequentially administered" means a time difference, measured in seconds, minutes, hours, or days, between the administration of two or more different compounds.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0098] The terms "include" and "contain" in this article should be understood as inclusive, without the meaning of exclusivity or exhaustion; that is, "including but not limited to".

[0099] The term "therapeutic effective dose" as used in this article generally refers to a dose that, after an appropriate period of administration, can achieve the therapeutic effect for the diseases listed above.

[0100] In this invention, the object or individual undergoing treatment is preferably a mammal, such as, but not limited to, humans, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), pets (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), or captured wild animals (e.g., foxes, deer). The object is preferably a primate. The most preferred object is a human.

[0101] The present invention is further illustrated below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The processes, conditions, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0102] Example 1: Computer-aided virtual screening yielded 49 candidate small molecule compounds targeting NPEPL1.

[0103] I. Experimental Methods

[0104] Based on the NPEPL1 protein structure, the computer-aided drug design software MOE was used for virtual screening of NPEPL1 candidate small molecule compounds. The NPEPL1 protein structure was predicted using AlphaFold3 and constructed using MOE homology modeling. The QuickPrepare module of MOE was used to perform hydrogenation, surface charge calculation, and energy minimization to prepare the protein. The Chemdiv database was selected as the small molecule compound library, with datasets including Chemdiv BMS_DivSet_300k, Anticancer-Library-61538, and Small Molecule Inhibitors52984, totaling 410,561 molecules. The small molecule compounds were desalted, hydrogenated, charged, and their lowest energy conformations were calculated. The five principles of drug-likeness were used for drug-likeness filtering, and the remaining small molecules were used for molecular docking. Next, the Site Finder module of MOE was used to predict binding pockets, and the top-ranked binding pocket was used for molecular docking screening. Preliminary screening was conducted based on molecular docking scores (S < -7.5) and ligand efficiencies (LE < -0.25), resulting in 31,598 small molecule compounds. Precise docking was then performed on these compounds, yielding 4,064 small molecules with scores (S < -8) and ligand efficiencies (LE < -0.28). Subsequently, cluster analysis was performed using protein-ligand interaction molecular fingerprints (PLIFs). Small molecule compounds interacting with the catalytically active residues of NPEPL1 underwent pattern verification. Based on factors such as the number and strength of hydrogen bonds in the protein-ligand interaction, matching of binding pocket shape, and complementary matching of positive and negative charges on the protein surface, candidate small molecule compounds targeting NPEPL1 were comprehensively selected.

[0105] II. Experimental Results

[0106] The experimental results are attached. Figure 1 As shown in Table 1. (Appendix) Figure 1 Figure A in the table shows the PLIF fingerprint analysis results of the virtual screening. The results indicate that the small molecule compound library may interact with the NPEPL1 protein through amino acid residues such as Lys272 (1239), Met280 (1019), Asp283 (1180), Asp342 (1044), Arg346 (1523), Gly373 (905), Arg436 (956), and Ala466 (903). The number of molecules interacting with each residue is shown in parentheses, with Arg346 being the most frequently linked. Through computer-aided screening, 49 candidate small molecule compounds targeting NPEPL1 were obtained, as detailed in Table 1. Figure 1Figure B in the diagram shows a positive correlation between the preliminary docking score S1 and the precise docking score S of the 49 NPEPL1 candidate molecules, indicating that the virtual screening strategy was effective and the results were reliable. (See attached image) Figure 1 The CE plots show that the logP, molecular weight, and TPSA distributions of the 49 NPEPL1 candidate small molecule compounds are consistent with drug-like properties, and further research can be conducted on them.

[0107] Table 1. 49 candidate small molecule compounds targeting NPEPL1 obtained from screening.

[0108]

[0109]

[0110] Example 2: Killing effect of 49 candidate small molecule compounds targeting NPEPL1 on HCC cells

[0111] I. Experimental Methods

[0112] 1. CTG assay to detect the cytotoxic effects of 49 NPEPL1 candidate small molecule compounds on Huh7 cells.

[0113] Huh7 cells were cultured to the logarithmic growth phase and digested with trypsin into a single-cell suspension. The Huh7 cells were then seeded into culture dishes and incubated overnight at 37°C and 5% CO2 until adherence. Forty-nine candidate small molecule compounds (100×) were diluted with PBS buffer and 5% DMSO. 10 µL of the diluted small molecule compound was mixed with 190 µL of complete culture medium, and 20 µL of the mixture was added to the culture dish. The cells were incubated at 37°C and 5% CO2 for 72 h. On the final day, the CTG reagent was equilibrated in the culture plate under dark / room temperature (RT) conditions for 30 min. 100 µL of CTG reagent was added to the cell culture plate, and the plate was shaken for 5 min at 500 rpm. The cell death rate at different concentrations was calculated using a microplate reader.

[0114] 2. CCK8 assay was used to determine the IC50 values ​​of six NPEPL1 candidate small molecule compounds.

[0115] Six small molecule compounds with significant cytotoxic effects were selected for CCK8 assay. Huh7 cells in the logarithmic growth phase and in good condition were prepared into single-cell suspensions, and the cell density was adjusted to 5 × 10⁶ cells / cells. 4Cells were cultured at a concentration of 100 µL / mL in 96-well plates, with 3 replicates per well. 100 µL of PBS was added around each well to prevent evaporation of the cell suspension. Cells were then cultured in an incubator for 12-24 h. After cell attachment, different concentrations of the small molecule compound were added to each group, and the cells were cultured at 37°C for 48 h. The old culture medium was discarded, and 100 µL of CCK8 dilution buffer (90 µL culture medium + 10 µL CCK8 solution) was added to each well. The cells were incubated at 37°C in the dark for 0.5-1 h. After incubation, the 96-well plates were removed, and the absorbance of cells in each well was measured at 450 nm using a microplate reader. Cell viability at different concentrations was calculated, cell viability curves were plotted, and the IC50 value of the small molecule compound was determined.

[0116] II. Experimental Results

[0117] The experimental results are attached. Figure 2 As shown in Table 2. (Appendix) Figure 2 Figure A in the table shows the cell death rate of 49 candidate small molecule compounds. The results indicate that several small molecule compounds have a killing effect on Huh7 cells, and some even show killing effects approaching those of paclitaxel, a positive drug for liver cancer treatment. (Appendix) Figure 2 Figure B in the figure shows the cell survival curve of paclitaxel in Huh7 cells. The results show that the IC50 value of paclitaxel in Huh7 cells is 27.54 μM. Among 49 candidate small molecule compounds, six with significant cytotoxic activity were selected for CCK8 assay to plot cell survival curves and determine the IC50 value. The results are shown in the appendix. Figure 2 The CH diagram and Table 2 are shown in the figure. The results showed that among these six small molecule compounds, E859-1332, L281-0443, C679-2629, and C301-9181 had IC50 values ​​below 30 μM, even lower than the IC50 value (27.54 μM) of paclitaxel, a positive drug for liver cancer, against Huh7 cells, indicating that they had a strong cytotoxic effect on Huh7 cells.

[0118] Table 2. IC50 values ​​of six small molecule compounds targeting NPEPL1

[0119]

[0120] Next, the affinity between the four small molecule compounds E859-1332, L281-0443, C679-2629, and C301-9181 and the NPEPL1 target protein was tested to determine the NPEPL1 specificity of these four small molecule compounds.

[0121] Example 3: Affinity analysis of four small molecule compounds with NPEPL1 protein

[0122] I. Experimental Methods

[0123] Surface-plasmon resonance (SPR) technology was used to detect the binding of four small molecule compounds to the NPEPL1 protein. Protein immobilization was performed using an amino-coupled method; protein coupling buffer: 1.0×PBS-P+ (pH 7.4); interaction buffer: 1.0×PBS-P+ (pH 7.4), 5% (v / v) DMSO. Place the running buffer (200 mL 1×PBS Buffer is sufficient), water bottle, and waste bottle in the left and right trays respectively, and insert the corresponding inlet tubes. Hold the CM5 chip with the labeled side facing up, and gently push the chip into the slot according to the arrow direction on the chip. Finally, close the chip compartment door. Activate channel 4 of the chip with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Cytiva) and N-hydroxysuccinimide (NHS, Cytiva) at a flow rate of 10 μL / min. Dilute the NPEPL1 ligand protein to 50 μg / mL with sodium acetate and immobilize the protein in channel 4 of the chip at a flow rate of 10 μL / min. Block the channel with ethanolamine at a flow rate of 10 μL / min. Repeat the channel activation, protein immobilization, and channel blocking for channel 3 as a reference, except that protein immobilization is performed using a protein-free acetate buffer. The running buffer for the four small molecule compounds was 1×PBS-P+ containing 5% DMSO. The original running buffer in the left tray of the system was replaced with 1×PBS-P+ containing 5% DMSO, and the corresponding inlet tubes were inserted. The 4.5% and 5.8% stock solutions were mixed according to Table 3 to prepare a 5% DMSO concentration calibration curve. The four small molecule compounds were diluted to several concentrations in a 96-well plate and coupled to the ligand NPEPL1 protein via a chip from low to high concentration at a flow rate of 30 μL / min for 60 s. After each concentration point, the chip was regenerated with 10 mM glycine (pH 2.0) solution for 5 min. This process was repeated until all corresponding concentrations of the four small molecule compounds were run. Using Biacore Insight evaluation software (Cytiva, Marborough, MA.USA), the experimental channels were subtracted from the reference channels, and the data were globally fitted to a 1:1 Langmuir binding model to obtain the binding and dissociation constants.

[0124] Table 3 Solvent Correction Solution Matching Table

[0125]

[0126] II. Experimental Results

[0127] The experimental results are attached. Figure 3 As shown in Table 4. The affinity test results include three parameters: KD (M), Ka (1 / Ms) and Kd (1 / s), where K D The dissociation constant (Ka) reflects the affinity of the analyte for the target; a smaller value indicates a stronger affinity. The association rate constant (Ka) represents the speed of intermolecular binding; a larger value indicates faster binding. The dissociation rate constant (Kd) represents the speed of intermolecular dissociation; a larger value indicates faster dissociation. The affinity between small molecule compounds E859-1332, L281-0443, C679-2629, and C301-9181 and the NPEPL1 protein was determined using SPR technology. The affinity results are attached. Figure 3 As shown in the AD diagram and Table 4, the results indicate that small molecule compounds E859-1332, L281-0443, C679-2629, and C301-9181 all exhibit high affinity for human NPEPL1 protein. The affinity (dissociation constant K) is shown in the diagram. D All of them reached the e-06 level and have high NPEPL1 specificity.

[0128] Appendix Figure 4 The AD diagrams in the image show the two-dimensional and three-dimensional binding diagrams (displayed in MOE software) of the interaction between four small molecule compounds, E859-1332, L281-0443, C679-2629, and C301-9181, and the NPEPL1 protein. Table 4 shows that among these four small molecule compounds, C301-9181 has the smallest dissociation rate constant Kd, indicating that after binding to the NPEPL1 protein, the two dissociate more slowly. This means that C301-9181 has a longer average in vitro residence time at the NPEPL1 protein target, which also implies that C301-9181 has a more sustained efficacy, lower dosing frequency, and fewer off-target effects, making it more suitable for developing long-acting, low-dose small molecule drugs.

[0129] Table 4. Affinity test results of C301-9181 and human NPEPL1

[0130]

[0131] In summary, all four small molecule compounds have certain cytotoxic effects and high affinity for NPEPL1 protein. However, considering the long-lasting effect of C301-9181, the applicant has chosen C301-9181 for subsequent in vitro and in vivo efficacy experiments.

[0132] Example 4: Effect of small molecule compound C301-9181 on the proliferation of HCC cells

[0133] I. Experimental Methods

[0134] 1. CCK8 assay to detect the effect of C301-9181 on the proliferation of human hepatocellular carcinoma lines Hep3B and Huh7.

[0135] Hep3B and Huh7 cells in the logarithmic growth phase and in good condition were prepared into single-cell suspensions, and the cell density was adjusted to 5 × 10⁶ cells / cells. 4 Cells / mL were added to each well of a 96-well plate with 100 µL of cell suspension, in triplicate for each concentration. 100 µL of PBS was placed around each well to prevent evaporation of the cell suspension. Cells were then cultured in an incubator for 12-24 h. After cell attachment, 0, 5, 10, 15, 20, 25, 30, 35, and 40 μM C301-9181 were added to each group, and the cells were cultured at 37°C for 24 h. The old culture medium was discarded, and 100 µL of CCK8 dilution buffer (90 µL culture medium + 10 µL CCK8 solution) was added to each well. The cells were incubated at 37°C in the dark for 0.5-1 h. After incubation, the 96-well plate was removed, and the absorbance of each cell at 450 nm was measured using a microplate reader.

[0136] 2. Effect of C301-9181 on the proliferation of human hepatocellular carcinoma lines Hep3B and Huh7 by crystal violet staining method

[0137] Single-cell suspensions of healthy Hep3B and Huh7 cells in the logarithmic growth phase were prepared and 50,000 cells were added to each well of a 24-well plate. The cells were then cultured in an incubator for 12-24 hours. After cell adhesion, 0, 10, 20, and 30 μM C301-9181 were added to each group, and the cells were cultured at 37°C for 48 hours. The old culture medium was discarded, and the cells were washed with PBS. 500 μL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 15 minutes. The cells were then washed with PBS, and 500 μL of crystal violet solution was added to each well for staining at room temperature for 15 minutes. The cells were then observed and photographed under a microscope. 3-5 fields of view were photographed from each well, and the cell area ratio and statistical analysis were performed using ImageJ software.

[0138] 3. Clonogenic assay to detect the effect of C301-9181 on the proliferation of human hepatocellular carcinoma lines Hep3B and Huh7.

[0139] Single-cell suspensions of Hep3B and Huh7 cells in the logarithmic growth phase and in good condition were prepared and seeded into 6-well plates at a density of 1000 cells per well, with 3 replicates for each concentration. The cells were cultured in an incubator for 72 h. After cell attachment, 0, 10, 20, and 30 μM C301-9181 were added to each group, and the cells were cultured at 37°C for 24 h. The old culture medium containing the drug was discarded, and the culture medium was changed every 3 days, and the cell status was observed. After 14 days, the samples were collected, 1 mL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 15 min. The cells were then rinsed with PBS, and 1 mL of crystal violet solution was added to each well for staining at room temperature for 15 min. The resulting cell clones were then photographed, and the cell clone count was calculated.

[0140] II. Experimental Results

[0141] The experimental results are attached. Figure 5 As shown in the figure. CCK8 assay results indicated that the IC50 values ​​of C301-9181 in human hepatocellular carcinoma lines Hep3B and Huh7 were 21.09 μM and 25.60 μM, respectively, and C301-9181 significantly inhibited the proliferation of Hep3B cells (P<0.0001) and Huh7 cells (P<0.0001). Figure 5 (A and B in the text); Crystal violet staining results showed that C301-9181 significantly inhibited Hep3B cell viability at concentrations of 10, 20, and 30 μM (P<0.0001), and significantly inhibited Huh7 cell viability at concentrations of 10 μM (P<0.001), 20 μM (P<0.0001), and 30 μM (P<0.0001). Figure 5 (C and D in the text); The results of the colony formation experiment showed that under the action of C301-9181 at concentrations of 10, 20, and 30 μM, the number of colonies formed in Hep3B cells (P<0.0001) and Huh7 cells (P<0.0001) was significantly reduced. Figure 5 (E and F in the text). In summary, the small molecule compound C301-9181 can significantly inhibit the proliferation of HCC cells.

[0142] Example 5: Effect of small molecule compound C301-9181 on apoptosis in HCC cells

[0143] I. Experimental Methods

[0144] 1. Flow cytometry analysis of the effect of C301-9181 on apoptosis in human hepatocellular carcinoma lines Hep3B and Huh7.

[0145] Single-cell suspensions of healthy Hep3B and Huh7 cells in the logarithmic growth phase were prepared and seeded into 6-well plates at a density of 400,000 cells per well. Cells were incubated for 12-24 hours. After cell adhesion, 0, 10, 20, and 30 μM C301-9181 were added to each group, and the cells were incubated at 37°C for 48 hours. Cell supernatants were collected from each group, and cells were digested with EDTA-free trypsin and collected in the same centrifuge tube as the supernatant. The cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were gently resuspended in PBS. Cells were counted, and 5-100,000 cells were collected. The cells were centrifuged again, the supernatant was discarded, and 500 μL of 1× Binding buffer was added to each tube to resuspend the cells. 5 μL of Annexin V-iFluor 488 dye was added, and the mixture was gently mixed and incubated at room temperature in the dark for 10 min. Then, 5 μL of [unclear text - possibly a specific dye or solution] was added. After gently mixing the PI dye, incubate at room temperature in the dark for 5 minutes, then place in an ice bath and immediately perform flow cytometry analysis. The staining process should be completed as soon as possible after the initial staining.

[0146] 2. Western blotting assay to detect the effect of C301-9181 on apoptosis in human hepatocellular carcinoma lines Hep3B and Huh7.

[0147] Hep3B and Huh7 cells in the logarithmic growth phase and in good condition were prepared into single-cell suspensions and seeded into 6-well plates at a density of 400,000 cells per well. The cells were then cultured in an incubator for 12-24 hours. After cell adhesion, 0, 10, 20, and 30 μM C301-9181 were added to each group, and the cells were cultured at 37°C for 48 hours. The cell supernatant was collected from each group, and the cells were digested with trypsin without EDTA and collected in the same centrifuge tube as the supernatant cells. The cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in pre-cooled PBS. The cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and an appropriate amount of lysis buffer containing 1× protease inhibitor / phosphatase inhibitor / nuclease inhibitor was added. The cells were lysed on ice for 30 min, centrifuged at 12000g and 4°C for 15 min, the supernatant was collected, and the protein concentration was adjusted by BCA. The cells were heated in a metal bath at 100°C for 15 min to obtain the protein sample. Protein samples were placed on a 12.5% ​​SDS-PAGE gel, with 30 μg of protein added to each well. Proteins of different molecular weights were separated by electrophoresis. The protein was transferred to a 0.22 μm PVDF membrane and transferred in an ice bath for 25 min. The membrane was blocked with 5% skim milk powder at room temperature for 1 h. The prepared primary antibody Caspase3 (1:1000), Cleaved-Caspase3 (1:1000), Bax (1:1000), Bcl-2 (1:1000), or β-actin internal control (1:10000) was added and incubated overnight at 4°C. The membrane was washed three times with 1×TBST buffer, 5 min each time. The secondary antibody HRP-Goat anti-Rabbit (1:10000) was used and incubated at room temperature for 1 h. The membrane was washed three times with 1×TBST buffer, 10 min each time. The membrane was then exposed and developed using a chemiluminescence analyzer.

[0148] II. Experimental Results

[0149] The experimental results are attached. Figure 6 As shown. Flow cytometry results indicated that at concentrations of 10, 20, and 30 μM, the apoptosis rate of Hep3B and Huh7 cells significantly increased with increasing C301-9181 concentration, indicating that C301-9181 can significantly promote apoptosis in Hep3B cells (P<0.0001) and Huh7 cells (P<0.001, P<0.0001, P<0.0001). Figure 6 (A, B in the text); Appendix Figure 6The CG figure shows the results of Western blotting experiments on apoptosis proteins. The results showed that C301-9181 significantly increased the expression level of Cleaved-Caspase3, an apoptosis marker, in Hep3B cells at concentrations of 10, 20, and 30 μM (P < 0.0001). Similarly, C301-9181 significantly increased the Cleaved-Caspase3 expression level in Huh7 cells at concentrations of 10 μM (P < 0.001), 20 μM (P < 0.0001), and 30 μM (P < 0.0001). Furthermore, C301-9181 significantly increased the Bax / Bcl-2 expression ratio in both Hep3B and Huh7 cells at concentrations of 10 μM (P < 0.001), 20 μM (P < 0.0001), and 30 μM (P < 0.0001). In conclusion, the small molecule compound C301-9181 can significantly promote apoptosis in HCC cells.

[0150] Example 6: Effects of small molecule compound C301-9181 on the migration and invasion of HCC cells

[0151] I. Experimental Methods

[0152] 1. Scratch assay to detect the effect of C301-9181 on the migration of human hepatocellular carcinoma lines Hep3B and Huh7.

[0153] Draw three parallel lines on the back of a 6-well plate using a marker pen for positioning. Take Hep3B and Huh7 cells in the logarithmic growth phase and in good condition, digest them with trypsin to prepare a single-cell suspension, and seed them into 6-well plates. Then, incubate them in a 37°C, 5% CO2 incubator. When the cells have reached approximately 90% confluence or a monolayer of cells covers the bottom of the plate, use a 200µL pipette tip to make vertical incisions perpendicular to the marker positioning lines on the cell plane to form a cross-shaped reference intersection. Carefully wash away detached cells with PBS, rinsing 3-5 times. Then, add low-serum medium (containing 1% FBS serum) containing 0, 10, 20, or 30 μM C301-9181 and continue culturing. At 0h and 48h, take microscopic images of the marked positions, process the images using ImageJ software, and calculate cell migration rate and perform statistical analysis.

[0154] 2. Transwell migration assay to detect the effect of C301-9181 on the migration of human hepatocellular carcinoma lines Hep3B and Huh7.

[0155] Hep3B and Huh7 cells in the logarithmic growth phase and in good condition were pre-starved for 16-24 hours in serum-free medium. The pre-starved cells were digested with trypsin and then resuspended in serum-free medium to form a single-cell suspension, adjusting the cell density to 1.25 × 10⁻⁶ cells / year. 5Cells / mL; Add 600µL of medium containing 20% ​​FBS to a 24-well plate, place the Transwell chamber into the well, add 100µL of serum-free medium to the upper chamber to moisten the chamber, then vertically suspend and add 200µL of the above cell suspension, let stand for 15 min, and then place in an incubator to culture for 10-12 h until the cells adhere; Replace the old lower chamber medium with high serum medium (containing 20% ​​FBS) containing 0, 10, 20, 30 μM C301-9181, and use medium containing 0, 10, 20, 30 μM C301-9181. Replace the old upper chamber medium with serum-free medium (C301-9181) and culture for 24 hours. Remove the chamber, discard the medium, and wash the chamber inside and outside twice with PBS. Add 4% paraformaldehyde to the clean 24-well plate and the chamber, and fix at room temperature for 15 minutes. Discard the 4% paraformaldehyde on the surface of the chamber and wash the chamber inside and outside three times with PBS. Add crystal violet staining solution to the clean 24-well plate and the chamber, and stain at room temperature for 15 minutes. Gently remove the non-transferred cells from the chamber with a moistened cotton swab, air dry, and then place the chamber under a microscope. Randomly select 5 fields of view to take pictures and count the number of cells that have migrated to the lower layer of the chamber.

[0156] 3. Transwell invasion assay to detect the effect of C301-9181 on the invasion of human hepatocellular carcinoma lines Hep3B and Huh7.

[0157] Hep3B and Huh7 cells in the logarithmic growth phase and in good condition were pre-starved for 16-24 hours using serum-free medium. 100 μL of 1 mg / mL matrix gel was vertically added to a Transwell chamber and incubated at 37°C for 1-3 hours to form a gel film. The liquid in the upper chamber was carefully aspirated, and 100 μL of serum-free medium was added. The cells were incubated at 37°C for 30 minutes to hydrate. The liquid in the upper chamber was removed, and the lower chamber was checked for liquid. The pre-starved cells were digested with trypsin and a single-cell suspension was prepared using serum-free medium. The cell density was adjusted to 2.5 × 10⁻⁶ cells / mL. 5Cells / mL; Add 600µL of medium containing 20% ​​FBS to a 24-well plate, place the Transwell chamber into the well, add 100µL of serum-free medium to the upper chamber to moisten the chamber, then vertically suspend and add 200µL of the above cell suspension, let stand for 15 min, and then place in an incubator to culture for 10-12 h until the cells adhere; Replace the old lower chamber medium with high serum medium (containing 20% ​​FBS) containing 0, 10, 20, 30 μM C301-9181, and use medium containing 0, 10, 20, 30 μM C301-9181. Replace the old upper chamber medium with serum-free medium (C301-9181) and incubate for 24 hours. Remove the chamber, discard the medium, and wash the chamber inside and outside twice with PBS. Add 4% paraformaldehyde to the clean 24-well plate and the chamber, and fix at room temperature for 15 minutes. Discard the 4% paraformaldehyde on the surface of the chamber, and wash the chamber inside and outside three times with PBS. Add crystal violet staining solution to the clean 24-well plate and the chamber, and stain at room temperature for 15 minutes. Gently remove the non-transferred cells from the chamber with a moistened cotton swab, air dry, and then place the chamber under a microscope. Randomly select 5 fields of view to photograph and count the cells.

[0158] II. Experimental Results

[0159] The experimental results are attached. Figure 7 As shown in the figure. Scratch assay results indicated that at concentrations of 10, 20, and 30 μM, the migration rates of Hep3B and Huh7 cells significantly decreased with increasing C301-9181 concentration, indicating that C301-9181 can significantly inhibit the migration of Hep3B cells (P<0.0001) and Huh7 cells (P<0.001, P<0.0001, P<0.0001). Figure 7 (A and B in the text); Transwell migration and invasion assays showed that C301-9181 at concentrations of 10, 20, and 30 μM significantly reduced the migration and invasion rates of Hep3B cells (P<0.0001, P<0.0001) and Huh7 cells (P<0.0001, P<0.0001). Figure 7 (CF in the text). In summary, the small molecule compound C301-9181 can significantly inhibit the migration and invasion of HCC cells. C301-9181 exhibits significant inhibitory and killing effects on hepatocellular carcinoma cell lines in vitro, and further in vivo animal experiments can be conducted.

[0160] Example 7: Construction of an animal model of hepatocellular carcinoma xenograft

[0161] Human hepatocellular carcinoma cell line Huh7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium supplemented with 10% premium fetal bovine serum (Procell, 164210) and 1% penicillin / streptomycin (Genview, GA3502). The cells were cultured at 37°C in a 5% CO2 incubator until they reached the logarithmic growth phase. Male BALB / c nude mice (4-6 weeks old, 18-22g) were purchased from Shanghai Lingchang Technology Co., Ltd. The mice were placed in an SPF environment and allowed free feeding under 12h light / 12h nighttime conditions, at 22±1°C and 45-55% humidity. After acclimatization, cells were injected. Huh7 cells were digested with trypsin to prepare a cell suspension, counted, and their density was adjusted to 1.0 × 10⁶ cells / mL with PBS. 8 Cells / mL: Using a sterile insulin syringe, draw 100µL of the above cell suspension and inject it subcutaneously into the right axilla of a nude mouse at a single point. Tumor formation is defined as tumor growth to the point where it is visible to the naked eye and palpable subcutaneously. The tumor should grow to at least 100mm. 3 After administration, the drug can be administered, and tumors usually form within 2-3 weeks.

[0162] Example 8: Dosing procedure and protocol for animal efficacy experiment of C301-9181

[0163] I. Experimental Methods

[0164] Paclitaxel is commonly used in clinical practice to treat hepatocellular carcinoma patients; therefore, this experiment used paclitaxel as a positive control drug for efficacy reference. The experiment was divided into four groups, with five mice in each group, as follows:

[0165] A. Model (tumor-bearing) + solvent group: physiological saline + 5% DMSO

[0166] B. Model (tumor-bearing) + paclitaxel positive drug group: 5 mg / kg (dissolved in physiological saline + 5% DMSO)

[0167] C. Model (tumor-bearing) + low-dose drug group: 20 mg / kg C301-9181 (dissolved in physiological saline + 5% DMSO)

[0168] D. Model (tumor-bearing) + high-dose drug group: 100mg / kg C301-9181 (dissolved in physiological saline + 5% DMSO)

[0169] After modeling, the drugs were administered intraperitoneally twice a week for two weeks, for a total of four administrations. The size of the tumors in each group of tumor-bearing mice was measured before, during, and after administration. After the two-week administration period, samples were collected, including tumors, serum, heart, liver, spleen, lungs, and brain, for subsequent testing.

[0170] II. Experimental Results

[0171] The experimental results are attached. Figure 8 As shown. (Attached) Figure 8 Figures A and B show the sampling results of animals and tumors in each group; Figures C and D show the tumor volume and weight results, respectively. Compared with the solvent group, the size and weight of tumors in nude mice were significantly reduced after treatment with paclitaxel (P<0.01), low-dose C301-9181 (P<0.05), and high-dose C301-9181 (P<0.05), indicating that both C301-9181 and paclitaxel have certain anti-tumor effects and can significantly inhibit the growth of Huh-7 tumors in nude mice. There were no significant differences between the low-dose group, the high-dose group, and the positive control group, indicating that the anti-tumor effects of low-dose and high-dose C301-9181 on Huh-7 tumors in nude mice are comparable to those of paclitaxel. There were no significant differences between the low-dose group and the high-dose group, indicating that the anti-tumor effect of C301-9181 is relatively stable within the dose range of 20-100 mg / kg and does not show obvious dose dependence.

[0172] Example 9: Effect of C301-9181 on NPEPL1 expression in HCC xenograft animal models (immunohistochemistry)

[0173] I. Experimental Methods

[0174] The obtained hepatocellular carcinoma tumors were embedded in paraffin blocks, sectioned, and then subjected to immunohistochemical staining for the target NPEPL1 according to the following steps: The slides were baked in a 60°C oven for 1 hour, then dewaxed with xylene for 20 minutes each (2 times), washed with 100% ethanol, 95% ethanol, 80% ethanol, and 60% ethanol for 5 minutes each, and then washed and soaked in ddH2O for 1 minute each (3 times) to complete dewaxing and hydration. The slides were preheated with 1×Tris-EDTA antigen retrieval solution (50×, pH 9.0) (Beyotime, P0084), immersed in 1×Tris-EDTA antigen retrieval solution (pH 9.0), heated in a microwave oven on medium heat for 10 minutes, and allowed to cool naturally to room temperature. The slides were washed with 1×TBS for 1 minute each (3 times). The tissue area was circled with an immunohistochemical pen, and 3% H2O2 was added to the circle. The slides were incubated at room temperature in the dark for 10 minutes to quench endogenous peroxidase activity. Block the slides with QuickBlock immunoblocking solution (Beyotime, P0260) for 10 min at room temperature. After removing the blocking solution, cover the sample with NPEPL1 rabbit anti-human primary antibody (Proteintech, 17211-1-AP, 1:200) and incubate in a humidified chamber at room temperature for 1.5 h. Remove the primary antibody solution, wash with 1×TBST for 1 min × 3 times, cover the sample with goat anti-rabbit secondary antibody (CST, 8114P), and incubate in a humidified chamber at room temperature for 0.5 h. Remove the secondary antibody solution, wash with 1×TBS for 1 min × 3 times, add DAB chromogenic reagent (KeyGen, KGB4101-20), and incubate in the dark for 3-5 min until a brown color appears (light background and appropriate staining intensity). Wash with ddH2O for 1 min × 3 times to stop the staining process. Add hematoxylin solution for nuclear staining, incubate at room temperature for 3-10 min, and wash with 1×TBS for 1 min × 3 times. Wash the sections with ddH2O, 60% ethanol, 80% ethanol, 90% ethanol, and 100% ethanol for 5 min each, then soak them in xylene for 5 min each time (2 times). Finally, mount the sections with a small amount of neutral resin, allow them to air dry naturally, and then examine them under a microscope.

[0175] II. Experimental Results

[0176] The results of the immunohistochemical experiment are attached. Figure 9 As shown in the figure, compared to the solvent group, NPEPL1 expression was not reduced in the paclitaxel positive drug group, while NPEPL1 expression was reduced in the low-dose and high-dose C301-9181 groups. This indicates that C301-9181 inhibits hepatocellular carcinoma and other malignant tumors by downregulating NPEPL1 expression or activity, thereby achieving an in vivo tumor-suppressing effect. This suggests that the expression level or activity of NPEPL1 protein can be used as a detection indicator to evaluate the mechanism of action.

[0177] Example 10: Effects of C301-9181 on six major organs of an HCC xenograft animal model (HE staining)

[0178] I. Experimental Methods

[0179] Tissues from six major organs—heart, liver, spleen, lung, kidney, and brain—were embedded to form paraffin blocks, which were then sectioned. The sections were stained with hematoxylin and eosin (HE) according to the following steps: Dewaxing in xylene for 10 min x 2, followed by washing with 100% ethanol → 90% ethanol → 80% ethanol → 70% ethanol → ddH2O for 2 min each to complete dewaxing and hydration. Hematoxylin staining for 5-10 min (adjust time according to staining results and requirements), washing with ddH2O for 10 min to remove excess staining solution, and then immersing again in ddH2O for a few seconds. Differentiation was performed using hydrochloric acid-ethanol rapid differentiation solution (Beyotime, C0163M) for 10 s, followed by rinsing with tap water for 10 min. Eosin staining for 30 s–2 min (adjust time according to staining results and requirements). Immersion in 70% ethanol → 80% ethanol → 90% ethanol → 100% ethanol for 10 s each, followed by xylene immersion for 5 min x 2. Finally, the slides were mounted with a small amount of neutral resin, air-dried, and examined under a microscope.

[0180] II. Experimental Results

[0181] The results of HE staining are attached. Figure 10 As shown in the figure, apart from significant inflammation in the lung tissue, no significant inflammation was observed in other tissues such as the heart, liver, spleen, kidneys, and brain. The paclitaxel group, the low-dose C301-9181 group, and the high-dose C301-9181 group all showed improvement in lung inflammation, with the high-dose C301-9181 group showing the most significant improvement, followed by the low-dose C301-9181 group. The above HE staining results indicate that C301-9181 did not produce hepatotoxicity or nephrotoxicity and had an effect on improving lung inflammation in tumor-bearing mice, demonstrating the good in vivo safety of the small molecule compound C301-9181.

[0182] Example 11: Effects of C301-9181 on liver and kidney function in an HCC xenograft animal model (serum biochemical analysis)

[0183] I. Experimental Methods

[0184] 1. Detection of the effect of C301-9181 on liver function in HCC xenograft animal models

[0185] (1) Aspartate aminotransferase (AST) detection

[0186] Aspartate aminotransferase (AST) catalyzes the transamination reaction between aspartic acid and α-ketoglutarate, producing glutamic acid and oxaloacetic acid. Oxaloacetic acid undergoes decarboxylation during the reaction to form pyruvate. Pyruvate reacts with phenylhydrazine to form phenylhydrazone, which is reddish-brown under alkaline conditions. The AST / GPT colorimetric assay kit (Elabscience, E-BC-K236-M) can be used to detect AST levels in nude mouse serum, allowing for direct measurement of serum samples. Before the formal assay, it is essential to prepare 2-3 samples with anticipated significant differences for prediction. The reagents provided in the kit are shown in Table 5 below.

[0187] Table 5. AST Detection Kit Reagents

[0188]

[0189] Before testing, equilibrate the reagents to room temperature. Reagent 5: Prepare the working solution of Reagent 5 by mixing double-distilled water at a volume ratio of 1:9, and use immediately. Preheat a portion of Reagent 3 at 37°C for 10 minutes. Add each reagent sequentially according to the procedures described in Table 6.

[0190] Table 6. Reagent addition sequence and experimental conditions for the AST detection kit

[0191]

[0192] Fitting curve for standard sample: y=ax 2 +bx+c

[0193] AST content (IU / L) = [a * (ΔA) 510 ) 2 + b * ΔA 510 + c ] * 0.482 IU / L * f

[0194] Where, ΔA 510 The OD value of the sample is calculated as the OD value of the control sample, and f is the dilution factor of the sample before it is added to the system.

[0195] By following the above methods and steps, the AST content of each serum sample can be measured.

[0196] (2) Detection of alanine aminotransferase (ALT)

[0197] Alanine aminotransferase (ALT) catalyzes the transamination reaction between alanine and α-ketoglutarate at 37°C and pH 7.4, producing pyruvate and glutamate. After the reaction time, phenylhydrazine is added, reacting with pyruvate to form phenylhydrazone, which is reddish-brown under alkaline conditions. The ALT / GPT colorimetric assay kit (Elabscience, E-BC-K235-M) can be used to directly measure ALT levels in nude mouse serum. Before the formal assay, it is essential to prepare 2-3 samples with expected significant differences for prediction. The reagents provided in the kit are shown in Table 7 below.

[0198] Table 7 ALT Detection Kit Reagents

[0199]

[0200] Before testing, equilibrate the reagents to room temperature. Reagent 5: Prepare the working solution of Reagent 5 by mixing double-distilled water at a volume ratio of 1:9, and use immediately. Preheat a portion of Reagent 3 at 37°C for 10 minutes. Add the reagents sequentially according to Table 8.

[0201] Table 8. Reagent addition order and experimental conditions for the ALT detection kit

[0202]

[0203] Fitting curve for standard sample: y=ax 2 +bx+c,

[0204] ALT content (IU / L) = [a * (ΔA)] 510 ) 2 + b * ΔA 510 + c ] * 0.482 IU / L * f,

[0205] Where, ΔA 510 The OD value of the sample is calculated as the OD value of the control sample, and f is the dilution factor of the sample before it is added to the system.

[0206] By following the above methods and steps, the ALT content of each serum sample can be measured.

[0207] (3) Total bilirubin (TBIL) detection

[0208] Indirect bilirubin's internal hydrogen bonds are broken under the action of an accelerator, allowing the insoluble indirect and direct bilirubin to react with an azo reagent under acidic conditions to form azobilirubin. Azobilirubin has maximum absorption at 565 nm, and the total bilirubin content in serum can be determined by measuring the change in absorbance. The total bilirubin (TBIL) colorimetric assay kit (Elabscience, E-BC-K760-M) can be used to detect TBIL levels in nude mouse serum, directly measuring serum samples within a range of 0.7-50 μmol / L. Before the formal assay, it is essential to prepare 2-3 samples with expected significant differences for prediction. The reagents provided in the kit are shown in Table 9 below.

[0209] Table 9 TBIL Detection Kit Reagents

[0210]

[0211] Before testing, equilibrate the reagents to room temperature. Prepare the working solution by mixing Reagent 1 and Reagent 2 at a volume ratio of 1.2:1, and use immediately. Dissolve each Reagent 4 standard in 2 mL of double-distilled water to obtain a 25 μmol / L standard solution, mix well, and protect from light; use immediately. Add the reagents in order according to Table 10.

[0212] Table 10 TBIL Detection Kit: Reagent Addition Order and Experimental Conditions

[0213]

[0214] Total bilirubin content (μmol / L) = (A2 / A1) * C * f,

[0215] Where A2 is the OD value of the test well minus the OD value of the control well, A1 is the OD value of the standard well minus the OD value of the standard control well, C is the concentration of the standard (25 μmol / L), and f is the dilution factor of the sample before it is added to the reaction system.

[0216] By following the above methods and steps, the TBIL content of each serum sample can be measured.

[0217] 2. Effects of C301-9181 on renal function in HCC xenograft animal models

[0218] (1) Urea (BUN) detection

[0219] Urea decomposes under the action of urease to produce ammonium ions and carbon dioxide. Ammonium ions react with phenol derivatives in an alkaline medium to form a green substance, the amount of which is directly proportional to the urea content. The BUN content in nude mouse serum can be directly measured using a urea (BUN) colorimetric assay kit (urease method) (Elabscience, E-BC-K183-M). Before the formal assay, 2-3 samples with expected significant differences should be used for prediction. The reagents provided in the kit are shown in Table 11 below.

[0220] Table 11 BUN Detection Kit Reagents

[0221]

[0222] Before testing, equilibrate the reagents to room temperature. Prepare the enzyme working solution by mixing Reagent 2 and Reagent 3 at a volume ratio of 1:300, and use immediately. Add each reagent sequentially as shown in Table 12.

[0223] Table 12 Reagent addition order and experimental conditions for BUN detection kit

[0224]

[0225] Fitting curve for standard sample: y = ax + b

[0226] Urea content (mmol / L) = (ΔA - b) / a * f,

[0227] Where y is the standard OD value minus the blank OD value, x is the concentration corresponding to the absorbance, ΔA is the sample OD value measured at 580nm wavelength minus the control OD value, and f is the dilution factor of the sample before it is added to the reaction system.

[0228] By following the above methods and steps, the BUN content of each serum sample can be measured.

[0229] (2) Creatinine (CR) detection

[0230] Creatinine is converted to creatine by creatine amide hydrolase, and creatine is hydrolyzed to sarcosine and urea by creatine aminohydrolase. Sarcosine is then catalyzed by sarcosine oxidase to produce glycine, formaldehyde, and hydrogen peroxide. Hydrogen peroxide reacts with 2,4-(6-triiodo-3-hydroxybenzoic acid) and 4-aminoantipyrine under the catalysis of peroxidase to produce a purple-red compound. The creatinine (CR) detection kit (micro-method) (Sangon Biotech, D799853-0096) is used to detect the CR content in nude mouse serum. Serum samples can be directly measured, with a detection range of 5-2000 μmol / L. Before the formal measurement, it is essential to take 2-3 samples with expected significant differences for prediction. Preheat the microplate reader for at least 30 minutes, adjust the wavelength to 546 nm, and zero the instrument with distilled water. Add the reagents in the order shown in Table 13 below.

[0231] Table 13 CR Detection Kit Reagent Addition Order and Experimental Conditions

[0232]

[0233] Where K is the dilution factor, i.e., K = (sample volume + reagent volume 1) / (sample volume + reagent volume 1 + reagent volume 2) = 186 / 246.

[0234] Creatinine content (μmol / L) = (ΔA determination - ΔA blank) / (ΔA standard - ΔA blank) * C standard

[0235] Wherein, C represents the concentration of the standard, which is 442 μmol / L.

[0236] By following the above methods and steps, the CR content of each serum sample can be measured.

[0237] II. Experimental Results

[0238] The results of the serum biochemical analysis are attached. Figure 11As shown in the AC diagram, the serum biochemical analysis of liver function results indicated significant differences in AST and ALT levels among the groups. The low-dose and high-dose C301-9181 groups showed significantly lower AST and ALT levels, while TBIL levels showed no significant difference. Based on the combined results of AST, ALT, and TBIL levels, the small molecule compound C301-9181 did not cause hepatotoxicity in the hepatocellular carcinoma xenograft animal model, demonstrating good safety. The serum biochemical analysis of kidney function results shown in the DE diagram indicated no significant differences in BUN and CR levels among the groups. Based on the combined results of BUN and CR levels, the small molecule compound C301-9181 did not cause nephrotoxicity in the hepatocellular carcinoma xenograft animal model, demonstrating good safety. In conclusion, the small molecule compound C301-9181 did not cause hepatotoxicity or nephrotoxicity in the hepatocellular carcinoma xenograft animal model, exhibiting good biosafety and significant potential for clinical development.

[0239] References cited in the background section of this invention:

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[0250]

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[0251]

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[0252] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. The use of a small molecule compound targeting NPEPL1 or a pharmaceutically acceptable salt thereof in the preparation of a tumor-targeting therapeutic agent, characterized in that, The tumor is selected from hepatocellular carcinoma with high NPEPL1 expression, and the structural formula of the small molecule compound is shown in any one of formulas (I) to (II): 。 Equation (I) Equation (II) 2. The application as described in claim 1, characterized in that, The small molecule compound or its pharmaceutically acceptable salt achieves tumor-targeted therapy by targeting the NPEPL1 gene or protein in tumor cells.

3. The application as described in claim 1, characterized in that, The pharmaceutically acceptable salt is selected from salts formed with any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and hydroxyethanesulfonic acid.

4. The application as described in claim 1, characterized in that, The drug is a drug that has one or more of the following effects: 1) It can bind to the NPEPL1 protein; 2) Inhibits the proliferation of human hepatocellular carcinoma cells; 3) Promotes apoptosis in human hepatocellular carcinoma cells; 4) Inhibits the migration and invasion of human hepatocellular carcinoma cells; 5) Inhibits the growth of hepatocellular carcinoma tumors in animal models of hepatocellular carcinoma xenograft; 6) Inhibit the expression or activity of NPEPL1; 7) Alleviates lung inflammation in animal models of hepatocellular carcinoma xenograft; 8) Reduce the levels of alanine aminotransferase and aspartate aminotransferase in animal models of hepatocellular carcinoma xenograft.

5. The application as described in claim 1, characterized in that, The tumor-targeted therapy drug uses a small molecule compound or its salt represented by any one of formulas (I) to (II) as the sole active ingredient, or contains a small molecule compound or its salt represented by any one of formulas (I) to (II).

6. The application as described in claim 1, characterized in that, In the tumor-targeted therapy drug, the content of any one of the small molecule compounds or their salts represented by formula (I) to (II) is 0.1-99 wt%.

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