Use of NCK chiral resolution small molecules as BAD / 14-3-3zeta dual-target inhibitors in preparation of antitumor drugs

Abstract

The application relates to the technical field of medicine preparation, in particular to application of an NCK chiral resolution small molecule as a BAD / 14-3-3zeta double-target inhibitor in preparation of an antitumor medicine. The application provides application of a compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and a pharmaceutically acceptable salt thereof in preparation of a BAD / 14-3-3zeta double-target inhibitor, wherein NCK-S is the first chiral pure BAD / 14-3-3zeta double-target inhibitor with a definite structure, overcomes the technical defect that a racemic mixture cannot determine pharmacodynamic isomers, and simultaneously, the double-target mechanism of the NCK-S gives the NCK-S more persistent antitumor effect; and the NCK-S is beneficial to wide application.

Description

Technical Field

[0001] This application belongs to the field of drug preparation technology, and in particular relates to the application of a chiral NCK molecule as a BAD / 14-3-3ζ dual-target inhibitor in the preparation of antitumor drugs. Background Technology

[0002] The BCL-2 (B-cell lymphoma 2) protein family is a core group regulating apoptosis in the mitochondrial pathway, directly determining cell survival or death. Within this family, BH3-only proteins act as key pro-apoptotic regulators, initiating the apoptotic program in response to various cellular stress signals. BCL2-associated cell death agonist (BAD, also known as BCL2L8) was the first pro-apoptotic protein identified as interacting with BCL-2 / BCL-xL, promoting apoptosis by antagonizing the function of anti-apoptotic BCL-2 family members.

[0003] The pro-apoptotic activity of BAD is tightly regulated by serine phosphorylation and dephosphorylation events. Three conserved serine residues—Ser75, Ser99, and Ser118—are the major phosphorylation sites that collectively inhibit BAD function. Phosphorylation at Ser99 creates the main docking site for 14-3-3 scaffold proteins, particularly the 14-3-3ζ isoform, which binds to phosphorylated BAD with the highest affinity, isolating BAD in the cytoplasm and thus inhibiting its pro-apoptotic function. The mutation of Ser99 to alanine (S99A) completely eliminates 14-3-3 binding, indicating that the domain surrounding phosphorylated Ser99 is the main 14-3-3 binding site. Aberrant activation of oncogenic signaling pathways (such as the PI3K / Akt and Ras / MAPK pathways) drives constitutive phosphorylation of BAD at Ser99 and Ser75 sites, making tumor cells resistant to apoptotic stimuli. Elevated phosphorylated BAD (p-BAD) has been detected in a variety of human cancers, and its abundance is often associated with aggressive disease phenotypes, chemotherapy resistance, and poor clinical prognosis.

[0004] In previous studies, NPB, a small molecule inhibitor targeting BAD Ser99 phosphorylation, was identified through large-scale virtual screening, and its derivative NCK was obtained through medicinal chemistry optimization. NCK retains a robust binding affinity to BAD, while significantly improving water solubility, reducing aggregation tendency, and enhancing oral bioavailability. However, the existing technology still has the following key shortcomings: (1) All previous studies used racemic mixtures of NCK without chiral resolution or stereochemical characterization; (2) The bioactivity data obtained from racemic NCK represent the complex reaction of two different molecules, and it is impossible to draw definitive conclusions about the structure-activity relationship; (3) The possibility of antagonistic activity or off-target effects from inferior enantiomers cannot be ruled out, which may reduce the therapeutic index; (4) It is unclear which enantiomer is the main contributor to efficacy, and whether each enantiomer has selective binding differences to the 14-3-3ζ target. The above shortcomings seriously restrict the further development and clinical application of this type of compound. Therefore, there is an urgent need in this field to separate NCK enantiomers and systematically evaluate their respective biological activities, target selectivity and pharmacodynamic characteristics in order to identify the dominant enantiomers and guide subsequent drug development. Summary of the Invention

[0005] The purpose of this application is to provide an application of NCK chiral cleavage small molecule as a BAD / 14-3-3ζ dual-target inhibitor in the preparation of antitumor drugs, aiming to solve the problem that there is currently no BAD / 14-3-3ζ dual-target inhibitor with a clear structure, higher activity, and better safety in the prior art for the preparation of antitumor drugs.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides the use of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of BAD / 14-3-3ζ dual-target inhibitors.

[0007] In some embodiments, the BAD / 14-3-3ζ dual-target inhibitor inhibits the BAD / 14-3-3ζ dual-target by disrupting the protein-protein interaction between the BAD protein and the 14-3-3ζ protein.

[0008] Secondly, this application provides the use of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of antitumor drugs.

[0009] In some embodiments, the antitumor drug is a therapeutic agent for tumors associated with abnormal activation of the BAD / 14-3-3ζ signaling pathway.

[0010] In some embodiments, the tumor is selected from triple-negative breast cancer, pancreatic ductal adenocarcinoma, ovarian cancer, endometrial cancer, or cervical cancer.

[0011] In some embodiments, the antitumor drug exerts its antitumor effect through the following mechanism: the compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol simultaneously binds directly to both BAD and 14-3-3ζ proteins, disrupting the BAD / 14-3-3ζ protein-protein interaction, releasing BAD and promoting its dephosphorylation, thereby activating the mitochondrial apoptosis pathway.

[0012] Thirdly, this application provides the use of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of a medicament for reducing the phosphorylation level of serine 99 site of BAD protein.

[0013] In some embodiments, (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol restores the pro-apoptotic activity of BAD by inhibiting phosphorylation at the Ser99 site of the BAD protein, thereby relieving the isolation of BAD by 14-3-3ζ.

[0014] Fourthly, this application provides a pharmaceutical composition comprising the above-mentioned compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0015] Fifthly, this application provides a method for preparing the compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol, comprising the following steps: resolving racemic NCK on a chiral column by supercritical fluid chromatography to obtain optically pure compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol, wherein the chiral column is a CHIRALPAK AD column; the mobile phase of the supercritical fluid chromatography is a mixed mobile phase of CO2 and ethanol, wherein the ethanol contains a methanol solution of 0.2%-0.3% NH3, and the volume ratio of CO2 to ethanol is (50-55):(45-50).

[0016] The first aspect of this application provides the application of chiral pure (S)-enantiomer (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol (NCK-S) in the preparation of BAD / 14-3-3ζ dual-target inhibitors. This first-ever separation and identification of a pharmacodynamic isomer from racemic NCK, establishing a clear structure-activity relationship between stereochemistry and activity. Its antitumor activity is significantly superior to NCK-R and racemic mixtures. Mechanistically, NCK-S… K-S can simultaneously and directly bind to two structurally unrelated protein targets, BAD and 14-3-3ζ. By disrupting the BAD / 14-3-3ζ protein-protein interaction, it releases BAD and promotes its dephosphorylation, thereby activating the mitochondrial apoptosis pathway. NCK-S is the first chiral pure BAD / 14-3-3ζ dual-target inhibitor with a clearly defined structure, overcoming the technical limitation of racemic mixtures in determining pharmacodynamic isomers. At the same time, its dual-target mechanism gives it a more durable anti-tumor effect, which is conducive to its widespread application.

[0017] The application of the compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of antitumor drugs, provided in the second aspect of this application; the compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol, by inhibiting phosphorylation at the Ser99 site of the BAD protein, releases the 14-3-3ζ barrier to BAD, restores the pro-apoptotic activity of BAD, and thereby activates the mitochondrial apoptosis pathway and induces tumor cell apoptosis. The provided compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol exhibits the strongest anti-proliferative and pro-apoptotic effects in in vitro and in vivo models of triple-negative breast cancer and pancreatic ductal adenocarcinoma, demonstrating excellent antitumor activity.

[0018] The application of the compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the third aspect of this application in the preparation of a medicament for reducing the phosphorylation level of the serine 99 site of the BAD protein. Phosphorylation of the Ser99 site of BAD provides a major docking site for the 14-3-3ζ scaffold protein, which binds to phosphorylated BAD with the highest affinity, isolating it in the cytoplasm. NCK-S blocks the isolation of BAD by 14-3-3ζ by inhibiting phosphorylation at the Ser99 site, precisely regulating the activity of BAD with high specificity.

[0019] The pharmaceutical composition provided in the fourth aspect of this application comprises NCK-S or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. This ensures that NCK-S can be effectively delivered to the target site in vivo, exerting its BAD / 14-3-3ζ dual-target inhibitory activity, which is beneficial for its widespread use.

[0020] The preparation method of the compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol provided in the fifth aspect of this application is simple and convenient, and is conducive to its wide application. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the synthesis route of NCK and the SFC splitting of NCK-R / NCK-S provided in the embodiments of this application; Figure 2 This is an analytical chiral SFC chromatogram provided in the embodiments of this application; Figure 3 This is the ¹H NMR spectrum provided in the embodiments of this application; Figure 4 This is a reversed-phase liquid chromatography UV chromatogram provided in the embodiments of this application; Figure 5 This is an electrospray mass spectrum provided in an embodiment of this application; Figure 6 This application provides experimental verification of NCK-S targeting BAD and 14-3-3ζ and analysis of pBAD-S99 phosphorylation inhibition (A: DARTS experimental analysis; B: Binding affinity analysis; C: MDA-MB-231 BAD knockout verification experiment; D: MDA-MB-231 cell viability assay; E: YWHAZ (14-3-3ζ) mRNA knockdown verification experiment; F: 14-3-3ζ protein knockdown verification experiment; G: Cell viability assay of NCK-S after 14-3-3ζ knockdown; H: AlphaFold3 complex conformation prediction; I: Co-IP immunoprecipitation experiment; J: pBAD-S99 phosphorylation level experiment in MDA-MB-231 cells; K: hBADWT / hBADS99A mutant construction verification experiment; L: S99A mutant cell viability rescue experiment). Figure 7 This application provides an example of an analysis of the antitumor activity of NCK-S in TNBC (A: IC50 curve for cell viability assay; B: colony formation assay; C: cell killing effect in 3D spheroid culture; D: apoptosis detection by Annexin V / PI flow cytometry). Figure 8 This application provides pharmacokinetic analysis diagrams of NCK-S (A: plasma concentration-time curves after intravenous and oral administration; B: summary table of key PK parameters; CD: hERG safety evaluation; E: CYP450 enzyme inhibition profile). Figure 9 This application provides an example of an analysis of the antitumor effect of NCK-S in an in vivo TNBC xenograft model (A: tumor volume growth curve; B: tumor volume ratio (inhibition rate) statistical graph; C: photograph of ex vivo tumor tissue arrangement; D: tumor mass quantitative statistical graph; E: Kaplan-Meier survival curve; F: summary of median survival for each group and Log-rank test; G: IHC scores (IRS, positive / negative cell percentage) of pBAD99, BAD, MKI67, and TUNEL; H: expression levels of metastasis-related markers (relative hHPRT mRNA and relative Luciferase mRNA) in lung and liver tissues). Detailed Implementation

[0023] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0024] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0025] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0026] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0027] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0028] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0029] The terms “first” and “second” are used only to describe the purpose and to distinguish the purposes, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated.

[0030] The first aspect of this application provides the use of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol (NCK-S) and its pharmaceutically acceptable salt in the preparation of BAD / 14-3-3ζ dual-target inhibitors.

[0031] Compared with existing racemic NCK technology, this application has the following significant advantages: (1) Clear structure-activity relationship: NCK-S was identified as the pharmacodynamic isomer of racemic NCK, and its antitumor activity was significantly better than that of (R)-enantiomer (NCK-R) and racemic mixture.

[0032] (2) Dual target mechanism: NCK-S directly binds to two protein targets, BAD and 14-3-3ζ, and releases BAD and promotes its dephosphorylation by disrupting the BAD / 14-3-3ζ protein-protein interaction, thereby activating the mitochondrial apoptosis pathway.

[0033] (3) Excellent oral bioavailability: NCK-S showed an oral bioavailability of 64.2% in rats, which is about 4.7 times that of NCK-R (13.8%) and far exceeds that of racemic mixtures.

[0034] (4) Stronger anti-tumor activity: NCK-S showed the strongest anti-proliferation and pro-apoptotic effects in both in vitro and in vivo models of triple-negative breast cancer and pancreatic ductal adenocarcinoma.

[0035] (5) Good safety characteristics: The inhibitory effect of NCK-S on hERG potassium channels is acceptable within the relevant concentration range, and it has controllable drug-drug interaction characteristics.

[0036] (6) Unexpected technical effects: The binding of NCK-S to 14-3-3ζ and the subsequent dissociation of the BAD / 14-3-3ζ complex are effects that cannot be predicted by those skilled in the art from the activity of racemic mixtures.

[0037] In some embodiments, the BAD / 14-3-3ζ dual-target inhibitor inhibits the BAD / 14-3-3ζ dual-target by disrupting the protein-protein interaction between the BAD protein and the 14-3-3ζ protein.

[0038] The compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol forms a specific interaction with BAD and 14-3-3ζ, altering the binding conformation of the BAD / 14-3-3ζ complex and thus disrupting the protein-protein interaction between them. This mechanism of action is beneficial for blocking the isolation of BAD by 14-3-3ζ at its source, restoring the normal function of the mitochondrial apoptosis pathway. Compared with traditional enzyme activity inhibitors, protein-protein interaction inhibitors have higher target specificity and lower off-target effects.

[0039] The second aspect of this application provides the use of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of antitumor drugs.

[0040] The application of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of antitumor drugs provided in the second aspect of the embodiments of this application; compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol, by inhibiting phosphorylation at the Ser99 site of BAD protein, releases the isolation of BAD by 14-3-3ζ, restores the pro-apoptotic activity of BAD, and thereby activates the mitochondrial apoptosis pathway and induces tumor cell apoptosis. The provided compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol showed the strongest anti-proliferative and pro-apoptotic effects in in vitro and in vivo models of triple-negative breast cancer and pancreatic ductal adenocarcinoma, and has excellent antitumor activity.

[0041] In some embodiments, the antitumor drug is a therapeutic agent for tumors associated with abnormal activation of the BAD / 14-3-3ζ signaling pathway.

[0042] The provided anti-tumor drugs are beneficial for precisely targeting tumors with abnormal activation of the BAD / 14-3-3ζ signaling pathway, thus achieving precision treatment.

[0043] In some embodiments, the tumor is selected from triple-negative breast cancer, pancreatic ductal adenocarcinoma, ovarian cancer, endometrial cancer, or cervical cancer.

[0044] In these tumor types, the BAD / 14-3-3ζ signaling pathway is abnormally activated, and NCK-S induces tumor cell apoptosis by restoring the pro-apoptotic function of BAD.

[0045] In some embodiments, the antitumor drug exerts its antitumor effect through the following mechanism: NCK-S simultaneously binds directly to both BAD and 14-3-3ζ proteins, disrupting the BAD / 14-3-3ζ protein-protein interaction, releasing BAD and promoting its dephosphorylation, thereby activating the mitochondrial apoptosis pathway.

[0046] The third aspect of this application provides the use of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and pharmaceutically acceptable salts thereof in the preparation of a medicament for reducing the phosphorylation level of serine 99 site in BAD protein.

[0047] The application of the compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts, provided in the third aspect of this application, in the preparation of a medicament for reducing the phosphorylation level of the serine 99 site of the BAD protein. Phosphorylation of the Ser99 site of BAD provides a major docking site for the 14-3-3ζ scaffold protein, which binds to phosphorylated BAD with the highest affinity, isolating it in the cytoplasm. NCK-S blocks the isolation of BAD by 14-3-3ζ by inhibiting phosphorylation at the Ser99 site, precisely regulating the activity of BAD with high specificity.

[0048] In some embodiments, NCK-S removes the 14-3-3ζ barrier to BAD and restores BAD's pro-apoptotic activity by inhibiting phosphorylation at the Ser99 site of the BAD protein.

[0049] A fourth aspect of this application provides a pharmaceutical composition comprising the above-described compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0050] The pharmaceutical composition provided in the fourth aspect of this application comprises NCK-S or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. This ensures that NCK-S can be effectively delivered to the target site in vivo, exerting its BAD / 14-3-3ζ dual-target inhibitory activity, which is beneficial for widespread use.

[0051] The fifth aspect of this application provides a method for preparing compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol, comprising the following steps: resolving racemic NCK on a chiral column by supercritical fluid chromatography to obtain optically pure compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol, wherein the chiral column is a CHIRALPAK AD column; the mobile phase of the supercritical fluid chromatography is a mixed mobile phase of CO2 and ethanol, wherein the ethanol contains a methanol solution of 0.2%-0.3% NH3, and the volume ratio of CO2 to ethanol is (50-55):(45-50).

[0052] The preparation method of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol provided in the fifth aspect of the present application is simple and convenient, and is conducive to its wide application.

[0053] The following description is based on specific embodiments.

[0054] Example 1: Preparation and Characterization of NCK-S 1. Preparation of racemic NCK according to Figure 1 The synthetic route shown is followed by the preparation method of racemic NCK: 2-Hydroxybenzaldehyde (1.00 equivalent) was dissolved in dioxane (6.00 times the volume), and the mixture was stirred at 25°C for 1 hour. Subsequently, 4-(2,3-dichlorophenyl)piperazine (1.00 equivalent) and 3-pyridinecarboxaldehyde (1.00 equivalent) were added, again using dioxane (6.00 times the volume) as the solvent. The reaction system was heated under reflux for 7 days.

[0055] After the reaction was complete, the reaction solution was cooled to room temperature and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (dichloromethane / methanol gradient elution) to obtain racemic NCK. Reversed-phase liquid chromatography (Luna C18 column) detected a single peak, indicating high chemical purity.

[0056] 2. Chiral splitting (corresponding to) Figure 1 (SFC splitting steps in the process) The racemic NCK prepared above was separated by supercritical fluid chromatography (SFC) on a Chiralpak AD column (250 mm × 30 mm, 10 μm). The mobile phase was a mixture of CO2 and ethanol, wherein the ethanol contained a methanol solution of 0.2% NH3, and the volume ratio of CO2 to ethanol was 50:50. Two optically pure stereoisomers were obtained and named NCK-S and NCK-R, respectively. Figure 1 The SFC splitting step in the example is the chiral splitting step in this embodiment, and the two are completely corresponding.

[0057] 3. Structural characterization of NCK-S Full chemical nomenclature of NCK-S: (S) 2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol.

[0058] like Figures 1-5As shown, chiral purity analysis was performed using analytical chiral SFC on a Chiralpak IG-3 column (35℃) with a mobile phase of CO2:EtOH (0.2% NH3 in MeOH) 50:50 and ABPR 2000 psi. Under these conditions, NCK-S eluted with a single, symmetrical peak, and no enantiomer traces were detected, confirming that the enantiomer purity exceeded the analytical detection limit. Structural confirmation: The structural identity of NCK-S was confirmed by proton nuclear magnetic resonance (¹H NMR) spectroscopy; the chemical shift mode and coupling constant were consistent with the specified molecular structure. Reversed-phase liquid chromatography (Luna C18 column) showed that NCK-S had a single peak and high chemical purity. Electrospray ionization mass spectrometry further confirmed the expected molecular weight.

[0059] Example 2 Computer simulations predict the interaction between NCK-S and the target. Molecular docking prediction was performed using AlphaFold3. Docking analysis was conducted between NCK-R and NCK-S and the following proteins: BAD (BCL2L8, NP_004313.1, 168 residues), 14-3-3ζ (YWHAZ, NP_663723.1, 245 residues), and BCL-2 (NP_000624.1, 239 residues). Prediction results were compared using interface prediction template modeling (IPTM) and prediction template modeling (PTM) scores.

[0060] The main docking results are as follows: For the BAD protein: NCK-S predicts that it binds to the PHE-151 residue at a distance of 4.1 Å via π-π interaction, with an iptm score of 0.49 and a ptm score of 0.24.

[0061] For the 14-3-3ζ protein: NCK-S predicts a polar interaction with GLU residues at a distance of 2.7 Å, with an iptm score of 0.71 and a ptm score of 0.88.

[0062] For the BCL-2 protein: NCK-S predicts binding to PHE-112 (4.1 Å) via π-π interactions, with an iptm of 0.89 and a ptm of 0.66.

[0063] AlphaFold3 prediction showed that NCK-S had a high iptm score with BCL-2, but also showed potential for binding with BAD and 14-3-3ζ. However, the calculated prediction had a certain false positive rate, and its actual binding specificity needed to be determined by biological experiments. Subsequent DARTS experiments confirmed that NCK-S could directly bind to BAD and 14-3-3ζ, without significantly affecting BCL-2, suggesting that NCK-S may specifically target the BAD / 14-3-3ζ complex, rather than BCL-2, within cells through mechanisms such as conformational selection or post-translational modification.

[0064] Example 3 Experimental verification of NCK-S targeting BAD and 14-3-3ζ and the inhibitory effect of NCK-S on BAD Ser99 phosphorylation. like Figure 6 In HEK-293T cells overexpressing BAD, DARTS experiments were conducted. The results showed that NCK-S treatment (10 μM and 100 μM) significantly increased the protease resistance of BAD and 14-3-3ζ, while having no significant effect on BCL-2, indicating that NCK-S can directly bind to BAD and 14-3-3ζ.

[0065] like Figure 6 By measuring the binding constant, we further quantified that NCK-S has a direct binding affinity for BAD and 14-3-3ζ. Figure 6 B shows NCK-R with 14-3-3ζ Kd (9.45×10 -8 M) is superior to NCK-S and 14-3-3ζ's Kd (3.27×10). -6 M). Although NCK-R showed higher affinity for 14-3-3ζ at the level of purified protein in vitro, subsequent cell function experiments ( Figure 6 The results (D, G, J, L) indicate that NCK-S significantly inhibits downstream pBAD-S99 phosphorylation and cell viability more strongly than NCK-R. This may be attributed to NCK-S's superior spatial conformational regulation ability within cells, enabling it to more effectively disrupt the BAD / 14-3-3ζ complex and thus exert a stronger biological effect.

[0066] like Figure 6 C and Figure 6 BAD-KO cells were constructed in MDA-MB-231 using CRISPR-Cas9 technology, and Western blot analysis confirmed successful knockout. Figure 6 (C), to perform cell viability testing ( Figure 6 The results showed that BAD-KO cells were significantly less sensitive to NCK-S compared to WT cells.

[0067] like Figure 6 E, F, and G, 14-3-3ζ was knocked down in WT and BAD-KO cells via shRNA. Figure 6 The E and F parameters were used to verify the knockdown effect. Cell viability assay ( Figure 6 The results showed that knocking down 14-3-3ζ in both WT and BAD-KO cells significantly reduced the efficacy of NCK-S (resulting in a relative increase in cell survival).

[0068] like Figure 6 Using AlphaFold3 for molecular docking prediction, the results showed that NCK-S can change the binding conformation of the BAD / 14-3-3ζ complex.

[0069] like Figure 6 Immunoprecipitation of I in MDA-MB-231 cells revealed that NCK-S treatment (5 μM, 10 μM) reduced 14-3-3ζ coprecipitated with BAD in a dose-dependent manner, directly verifying that NCK-S can disrupt the protein-protein interaction between the two.

[0070] like Figure 6 In MDA-MB-231 cells, NCK, NCK-S, and NCK-R all reduced pBAD-S99 levels in a dose-dependent manner, with NCK-S showing the most significant inhibitory effect, while the total BAD protein level remained unaffected.

[0071] like Figure 6 K and L were transfected into wild-type hBADWT or phosphorylation-deficient hBADS99A in BAD-KO cells, respectively. Western blot confirmed that the mutants lacked phosphorylation signaling. Figure 6 K). Cell viability assay ( Figure 6 The results showed that cells transfected with hBADS99A exhibited significant resistance to NCK-S, confirming that phosphorylation at the S99 site is a key site dependent on the efficacy of NCK-S.

[0072] Example 4 Evaluation of the antitumor activity of NCK-S Cell viability assay: such as Figure 7As shown in Figure A, in MDA-MB-231 cells, after treatment with sequentially 2-fold increasing concentrations (0-32 μM) of NCK (racemic), NCK-R, or NCK-S for 72 hours, cell viability was determined by the CCK-8 assay and IC50 values ​​were calculated. The results showed that NCK-S exhibited the strongest anti-proliferative effect, with an IC50 of 3.692 μM, significantly superior to racemic NCK (IC50 = 4.930 μM) and NCK-R (IC50 = 9.980 μM). The potency ranking was clearly: NCK-S > NCK (racemic) > NCK-R, indicating that the S-enantiomer is the main contributor to the efficacy of the racemic mixture, while the R-enantiomer has weaker activity.

[0073] Cloning experiments: such as Figure 7 As shown in Figure B, MDA-MB-231 cells were seeded at low density and treated with 2.5, 5.0, and 10.0 μM NCK, NCK-R, or NCK-S for 10 days. The resulting colonies were stained with crystal violet, and the OD value was measured at 570 nm to quantify colony-forming ability. The results showed that NCK-S most effectively inhibited colony formation at all tested concentrations, especially at 5 μM, where macroscopic colony growth was almost completely eliminated, with an OD value close to zero. In contrast, NCK (racemic) showed some inhibitory effect at high concentrations, but the effect was not as strong as NCK-S, while NCK-R showed the weakest inhibitory effect at all concentrations. These results indicate that NCK-S can inhibit the proliferative potential of tumor cells in the long term.

[0074] 3D spherical culture: such as Figure 7 As shown in Figure C, MDA-MB-231 cells were cultured in Matrigel with reduced growth factors to form three-dimensional spheroids. After treatment with 2.5, 5.0, and 10.0 μM NCK, NCK-R, or NCK-S, respectively, the cells were then stained with Calcein-AM (green, for live cells) and propidium iodide PI (red, for dead cells). The results showed that the control group exhibited uniform green fluorescence, indicating good cell viability. In the NCK-treated group, some red dead cells appeared at 10 μM. In the NCK-R treated group, green fluorescence still dominated, indicating limited cell killing effect. However, the NCK-S treated group showed the strongest cell killing effect at all concentrations; at 10 μM, the spheroids were almost completely covered by red fluorescence, and the green live cell signal essentially disappeared. This confirms that NCK-S can still exert strong antitumor activity in a three-dimensional culture system simulating the in vivo microenvironment.

[0075] Apoptosis detection experiments: such as Figure 7As shown in Figure D, Annexin V-FITC / PI double staining flow cytometry was used to quantitatively analyze apoptosis in MDA-MB-231 cells. The results showed that, compared with the VEH control group, NCK, NCK-R, and NCK-S treatment groups all induced different degrees of apoptosis, with NCK-S inducing the most significant early apoptosis (Annexin V...). + / PI - (lower right quadrant) and late apoptosis (Annexin V) + / PI + The proportion of cells in the upper right quadrant was the highest, and the total apoptosis rate was significantly better than that of NCK and NCK-R. This result indicates that NCK-S exerts its anti-tumor effect by inducing apoptosis rather than simply inhibiting growth. Consistent results were observed in BT-549 and PDAC cell lines, further confirming the universality of NCK-S's pro-apoptotic activity.

[0076] Example 5 Pharmacokinetic characteristics of NCK-S Non-compartmental (NCA) model analysis was employed, and standard pharmacokinetic software (such as Phoenix WinNonlin) was used to fit and calculate the plasma drug concentration-time data. Figure 8 As shown in Figure A, in male SD rats, NCK-R or NCK-S were administered intravenously (IV, 1 mg / kg) and orally via gavage (PO, 10 mg / kg), respectively. Blood samples were collected at different time points to measure plasma drug concentrations and plot pharmacokinetic curves. The results showed that after IV administration, the initial plasma concentration (C0) of NCK-S was higher than that of NCK-R, and its concentration decreased at a faster rate. After PO administration, the peak plasma concentration of NCK-S was 1415 ng / mL, significantly higher than that of NCK-R (86 ng / mL), and the time to peak concentration of NCK-S (Tmax = 0.67 h) was significantly earlier than that of NCK-R (Tmax = 3.33 h). The difference in area under the curve (AUC) was even more significant; the oral exposure of NCK-S (AUClast = 2720 h·ng / mL) was approximately 7 times that of NCK-R (393 h·ng / mL), indicating that NCK-S was significantly superior to NCK-R in both the rate and extent of oral absorption.

[0077] like Figure 8 As shown in B, for Figure 8 Non-compartmental model analysis was performed on the concentration-time data of A to obtain detailed PK parameters.

[0078] Clearance, Volume of Distribution, and Mean Residence Time: After IV administration, the clearance rate (Clobs) of NCK-S was 39.3 mL / min / kg, lower than that of NCK-R (58.4 mL / min / kg), indicating a slower clearance rate. Simultaneously, the steady-state volume of distribution (Vss = 2.33 L / kg) of NCK-S was smaller than that of NCK-R (5.26 L / kg). Since MRT is related to Vss and Cl, the MRT of NCK-S (0.99 h IV, 2.32 h PO) was shorter than that of NCK-R (1.51 h IV, 4.53 h PO), indicating a more limited tissue distribution of NCK-S in vivo, resulting in a shorter mean residence time. In summary, NCK-S is characterized by slow clearance and a small volume of distribution, both of which jointly affect its disposal process in vivo.

[0079] Half-life (T1 / 2): After IV administration, the half-life of NCK-S was 1.92 h, shorter than that of NCK-R (3.75 h); however, after PO administration, their half-lives were similar (NCK-S 3.67 h, NCK-R 3.77 h), suggesting that the absorption phase after oral administration may prolong the apparent half-life. It should be noted that, due to the use of a non-compartmental model for independent calculations, each parameter was obtained by software fitting actual blood drug concentration data. Affected by the multi-compartmental distribution characteristics and the terminal phase sampling time, the T1 / 2 simply estimated by Cl and Vss differs somewhat from the measured terminal half-life, which is a normal pharmacokinetic phenomenon.

[0080] Exposure and Bioavailability: After oral administration (PO), the AUClast of NCK-S was 2720 h·ng / mL, approximately 7 times that of NCK-R. After dose normalization (AUC / D), the oral bioavailability of NCK-S was 272 h·ng / mL, while that of NCK-R was only 39.3 h·ng / mL, further confirming the superior oral exposure characteristics of NCK-S. The oral bioavailability (F) of NCK-S was as high as 64.2%, while that of NCK-R was only 13.8%, a difference of approximately 4.7 times, demonstrating excellent oral drug-likeness.

[0081] hERG cardiotoxicity risk assessment: (e.g., ...) Figure 8 As shown in the CD, NCK-S did not show significant hERG current inhibition within the relevant concentration range (the inhibition rate was much lower than that of the positive control dofetilide), demonstrating acceptable cardiac safety characteristics.

[0082] CYP450 enzyme inhibition profile assessment: (e.g.) Figure 8As shown in Figure E, NCK-S exhibits mild to moderate inhibition of most CYP subtypes and strong inhibition of CYP2C subtypes (CYP2C8: 80.81%, CYP2C9: 93.04%, CYP2C19: 96.80%). In clinical use, caution should be exercised when using NCK-S in combination with CYP2C substrates or inhibitors.

[0083] Example 6 NCK-S's in vivo anti-tumor effects In this embodiment, tumor-bearing mice were administered the solvent (VEH), NCK 25 mg / kg, NCK-S 25 mg / kg, or NCK-S 50 mg / kg orally by gavage daily.

[0084] like Figure 9 The tumor growth curves of A showed that, compared with the control group (VEH), both NCK and NCK-S could significantly slow down the growth of tumor volume. At the same dose (25 mg / kg), NCK-S had a better tumor-suppressing effect than NCK, and NCK-S showed a clear dose-dependent effect (the 50 mg / kg group had the strongest inhibitory effect, and the tumor volume was maintained at about 0.2-0.3 cm³ at 14 days).

[0085] like Figure 9 The percentage change in tumor volume for group B further quantified this result. Compared with the VEH group (nearly 100%), the tumor volume change was significantly reduced in each treatment group (approximately 50% for NCK 25 mg / kg, approximately 40% for NCK-S 25 mg / kg, and approximately 20%-25% for NCK-S 50 mg / kg), and all treatment groups showed highly statistically significant differences. (P<0.001). It can be seen that NCK-S has stronger antitumor activity than NCK, and its antitumor effect is significantly enhanced with increasing dose.

[0086] like Figure 9 As shown in Figure C, representative whole-body images and photographs of major organs (heart, liver, kidney, and spleen) of mice from each group were collected after sacrifice for direct morphological comparison. The images showed that the mice in the VEH group were in poor overall condition with significant tumor burden; the NCK25 mg / kg group showed improvement, but tumors were still visible; while the mice in the NCK-S treatment group were in significantly better overall condition than the VEH group, especially the 50 mg / kg group, where tumor volume was significantly reduced. Further images of ex vivo organs showed no obvious abnormalities in color, size, or morphology in the major organs of the NCK-S treatment group, suggesting that at effective doses, NCK-S did not cause significant toxic damage to major organs such as the heart, liver, kidney, and spleen, preliminarily indicating its good in vivo safety.

[0087] like Figure 9 As shown in Figure D, after the experiment, tumor tissue was dissected and weighed, and the tumor weight (T-unolase weight, presumably tumor weight) of each group was quantitatively compared. The results showed that the VEH group had the largest tumor weight; the NCK 25 mg / kg group showed a slight decrease in tumor weight, but the difference was limited; the NCK-S 25 mg / kg group had a significantly lower tumor weight than the NCK and VEH groups; the NCK-S 50 mg / kg group showed a further decrease in tumor weight, exhibiting a clear dose-dependent inhibitory effect. This quantitative data is consistent with... Figure 9 The results of X-enolase activity of B were highly consistent and mutually corroborative.

[0088] like Figure 9 As shown in Figure E, the survival of mice in each group was continuously tracked, and Kaplan-Meier survival curves were plotted, with the ordinate representing survival probability and the abscissa representing the number of days after treatment. The results showed that the VEH group had the shortest median survival, with tumor progression leading to rapid death. The NCK 25 mg / kg group showed a slight increase in survival, but the effect was limited. The NCK-S 25 mg / kg group showed a superior survival benefit compared to the same dose of NCK. The NCK-S 50 mg / kg group exhibited the longest survival. The differences between each NCK-S treatment group and the VEH group were statistically significant. These results indicate that NCK-S not only inhibits tumor growth but also significantly prolongs the survival time of tumor-bearing mice.

[0089] like Figure 9 As shown in F, the median survival of mice in each group was summarized and compared, and the statistical difference in survival curves between groups was assessed using the Log-rank test. The results showed that the VEH group had the shortest median survival, the NCK25 mg / kg group was in the middle, the NCK-S 25 mg / kg group was significantly better than the NCK group, and the NCK-S 50 mg / kg group had the longest median survival. The Log-rank test showed that the differences in survival curves between groups were extremely significant (p<0.0001), confirming that NCK-S treatment can significantly prolong the overall survival of tumor-bearing mice in a dose-dependent manner.

[0090] like Figure 9As shown in G, tumor tissues from mice in each group were collected, and the relative mRNA expression level of pBAD59 (presumably a marker associated with the BAD Ser59 site) was detected by qRT-PCR. The results showed that compared with the VEH group, pBAD59 expression was downregulated in the NCK 25 mg / kg group; the downregulation was more pronounced in the NCK-S 25 mg / kg group; and the expression level was lowest in the NCK-S 50 mg / kg group, showing a dose-dependent downregulation trend. These results suggest that NCK-S can also effectively regulate BAD-related signaling pathways in vivo, consistent with in vitro phosphorylation detection results (…). Figure 6 This echoes the findings of IJ, confirming that its mechanism of action targeting the BAD pathway remains valid in vivo.

[0091] like Figure 9 As shown in Figure H, lung and liver tissues from mice in each group were collected to detect the relative mRNA expression levels of metastasis-related markers, in order to evaluate the inhibitory effect of NCK-S on TNBC metastasis. The results showed that the VEH group had the highest expression levels of metastasis markers in both lung and liver tissues, indicating that MDA-MB-231-luc cells had already metastasized to distant sites. In the NCK 25 mg / kg group, the expression of metastasis markers decreased, but the inhibition was incomplete. The expression of markers in the lungs and liver of the NCK-S 25 mg / kg group was significantly lower than that in the NCK group. In the NCK-S 50 mg / kg group, the expression in both tissues decreased to extremely low levels. These results directly demonstrate that oral administration of NCK-S can not only inhibit the growth of in situ tumors but also effectively inhibit the distant metastasis of TNBC, which is an important component of its anti-tumor activity.

[0092] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of BAD / 14-3-3ζ dual-target inhibitors.

2. The application according to claim 1, characterized in that, The BAD / 14-3-3ζ dual-target inhibitor inhibits the BAD / 14-3-3ζ dual-target by disrupting the protein-protein interaction between the BAD protein and the 14-3-3ζ protein.

3. Application of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of antitumor drugs.

4. The application according to claim 3, characterized in that, The antitumor drug is a therapeutic drug for tumors associated with abnormal activation of the BAD / 14-3-3ζ signaling pathway.

5. The application according to claim 3, characterized in that, The tumor is selected from triple-negative breast cancer, pancreatic ductal adenocarcinoma, ovarian cancer, endometrial cancer, or cervical cancer.

6. The application according to any one of claims 3-5, characterized in that, The antitumor drug exerts its antitumor effect through the following mechanism: the compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol directly binds to both BAD and 14-3-3ζ proteins, disrupting the BAD / 14-3-3ζ protein-protein interaction, releasing BAD and promoting its dephosphorylation, thereby activating the mitochondrial apoptosis pathway.

7. The use of compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol and its pharmaceutically acceptable salts in the preparation of medicaments for reducing the phosphorylation level of serine 99 site in BAD protein.

8. The application according to claim 7, characterized in that, The compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol restores the pro-apoptotic activity of BAD by inhibiting phosphorylation at the Ser99 site of the BAD protein, thereby relieving the isolation of BAD by 14-3-3ζ.

9. A pharmaceutical composition, characterized in that, The compound comprising (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

10. A method for preparing a compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol, characterized in that, Includes the following steps: Racemic NCK was separated by supercritical fluid chromatography on a chiral column to obtain optically pure compound (S)-2-[(S)-[4-(2,3-dichlorophenyl)piperazin-1-yl]-(3-pyridyl)methyl]phenol, wherein the chiral column was a CHIRALPAK AD column; the mobile phase of the supercritical fluid chromatography was a mixed mobile phase of CO2 and ethanol, wherein the ethanol contained 0.2%-0.3% NH3 in a methanol solution, and the volume ratio of CO2 to ethanol was (50-55):(45-50).

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