A compound for treating neuroendocrine prostate cancer, a pharmaceutical composition and a preparation method and application thereof

Abstract

The application provides a kind of compound for treating neuroendocrine prostate cancer, pharmaceutical composition and its preparation method and application, belong to the field of medicinal chemistry.The application provides a kind of compound shown in formula I, its pharmaceutically acceptable salt.The compound of the application can be stably combined with the protein pocket of cluster protein D1 (PLXND1), inhibit the function of PLXND1, and then inhibit the proliferation, clonal formation and organoid growth of neuroendocrine prostate cancer (NEPC) cell, has important application value in the preparation of PLXND1 inhibitor and the drug for treating neuroendocrine prostate cancer.Formula I

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a compound, pharmaceutical composition, preparation method, and application for treating neuroendocrine prostate cancer. Background Technology

[0002] Prostate cancer is one of the most common malignant tumors of the male genitourinary system, ranking second in incidence among male malignant tumors worldwide. In advanced prostate cancer patients receiving androgen deprivation therapy (ADT), the vast majority progress to castration-resistant prostate cancer (CRPC). While CRPC patients become resistant to novel anti-androgen therapies, some may transition from androgen-dependent to androgen-independent, exhibiting molecular characteristics associated with neuroendocrine tumors, and eventually transforming into treatment-induced neuroendocrine prostate cancer (NEPC). Primary NEPC accounts for less than 2%, while 20%–30% of CRPC patients develop treatment-induced NEPC.

[0003] The histopathological features of NEPC are similar to those of neuroendocrine tumors originating from other sites. Its molecular characteristics are mainly characterized by low or absent expression of androgen receptor (AR) and prostate-specific antigen (PSA), the presence of neuroendocrine molecular features (such as high expression of synaptophysin SYP, neuronal cell adhesion molecule 1 CD56, chromogranin A CHGA, and neuron-specific enolase NSE), and unresponsiveness to treatment targeting the AR signaling axis. Currently, there is no unified standard treatment regimen for NEPC. Platinum-based chemotherapy is the first-line treatment for NEPC patients, but its effective duration is short, with a median overall survival of only 6-19 months, resulting in limited survival benefits for patients. The lack of safe, effective, sustainable, and reliable treatment options has become a key challenge in the field of NEPC research.

[0004] Plexin D1 (PLXND1) is a member of the type I transmembrane protein family of plexins (PLXN), playing a crucial role in regulating neural growth and development, as well as the occurrence and progression of various tumors. PLXND1 functions differently in different tumors, primarily acting as an oncogene in colon cancer, ovarian cancer, and clear cell renal cell carcinoma. Some studies have reported that PLXND1 can regulate prostate cancer progression by activating NOTCH signaling, but its role in highly malignant non-prostate cancer (NEPC) is rarely reported. Research indicates that knocking down or eliminating PLXND1 significantly inhibits the proliferation of NEPC cells and organoids, suppressing tumorigenesis in NEPC tumors.

[0005] Heat shock protein 70 (HSP70), as a molecular chaperone protein, is widely expressed in various tissues and cells. The HSP70 inhibitor JG231 can promote the ubiquitination and degradation of PLXND1 protein through the ubiquitin-proteasome pathway. However, HSP70 inhibitors struggle to precisely target NEPC cells without damaging other normal tissues and cells, which is a key issue that needs to be addressed for its clinical application. Therefore, developing small-molecule inhibitors that can precisely target PLXND1 protein is of great significance for the precision treatment of NEPC. Summary of the Invention

[0006] The purpose of this invention is to provide a compound, pharmaceutical composition, preparation method and application for treating neuroendocrine prostate cancer.

[0007] This invention provides compounds represented by Formula I and their pharmaceutically acceptable salts: Formula I Wherein, R is selected from hydrogen, hydroxyl, C 1~3 Alkyl groups, halogens.

[0008] Furthermore, the compound is .

[0009] The present invention also provides a method for preparing the aforementioned compounds and their pharmaceutically acceptable salts, the method comprising the following steps: (a) Reaction of p-hydroxybenzaldehyde, p-aminobenzamide, and a reducing agent under acidic conditions yields a dibenzylamine intermediate; (b) The dibenzylamine intermediate obtained in step (a) was reacted with indole-3-acetic acid in the presence of an amidating agent to give the compound shown in Formula I.

[0010] Further, in step (a), the molar ratio of p-hydroxybenzaldehyde, p-aminobenzamide, and the reducing agent is 1:(0.5~2):(1~2); the reducing agent is sodium borohydride; the acid is glacial acetic acid; the reaction is carried out in an alcohol solvent at a temperature of 0℃~35℃; In step (b), the molar ratio of the bibenzylamine intermediate, indole-3-acetic acid, and amidating agent is 1:(2~3):(2~5); the amidating agent is selected from one or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine; the solvent for the reaction is N,N-dimethylformamide, the reaction temperature is 0℃~35℃, and the reaction time is 8~24 hours.

[0011] Further, in step (a), the molar ratio of p-hydroxybenzaldehyde, p-aminobenzamide, and the reducing agent is 1:1:1.5; the alcohol solvent is methanol; step (a) is followed by the following column chromatography purification step: elution using petroleum ether and ethyl acetate as eluents; In step (b), the amidation reagent is selected from a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole; the molar ratio of the bibenzylamine intermediate, indole-3-acetic acid 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole is 1:2.2:2.2:2.2; step (b) is followed by the following column chromatography purification step: elution using petroleum ether and ethyl acetate as eluents.

[0012] The present invention also provides a pharmaceutical composition, which is a pharmaceutical preparation made by using the aforementioned compound, its pharmaceutically acceptable salt as the active ingredient, and pharmaceutically acceptable excipients.

[0013] The present invention also provides the use of the aforementioned compounds and their pharmaceutically acceptable salts in the preparation of PLXND1 inhibitors.

[0014] Furthermore, the PLXND1 inhibitor is a drug for the prevention and / or treatment of diseases mediated by or associated with abnormal function of the PLXND1 protein.

[0015] Furthermore, the disease is a neuroendocrine tumor or prostate cancer.

[0016] Furthermore, the neuroendocrine tumor is selected from neuroendocrine prostate cancer (NEPC), small cell neuroendocrine prostate cancer (SCNPC), AR- / NE- prostate cancer (DNPC), and AR+ / NE+ prostate cancer (AMPC); the prostate cancer is selected from castration-resistant prostate cancer (CRPC), castration-sensitive prostate cancer (CSPC), androgen receptor-overexpressing prostate cancer (ARPC), androgen receptor-lowexpressing prostate cancer (ARLPC).

[0017] Furthermore, the drug is a drug for the prevention and / or treatment of neuroendocrine prostate cancer (NEPC).

[0018] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0019] "Alkyl" refers to a saturated hydrocarbon chain with a specified number of member atoms. For example, C1~3 Alkyl groups are alkyl groups having one to three member atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups include methyl, ethyl, and propyl (n-propyl and isopropyl).

[0020] The "halogen" is fluorine, chlorine, bromine, or iodine. This invention achieves the following beneficial effects: (1) The compound shown in Formula I provided by the present invention, especially DrugII, can stably bind to PLXND1 protein by targeting and binding to PLXND1 protein site 5 with a high affinity of -48.7365 kcal / mol binding free energy ΔG and a docking score of -8.316, thereby effectively inhibiting the function of PLXND1 protein.

[0021] (2) The compounds of this invention can significantly inhibit the proliferation of NEPC cells C42B-MDVR, CWR22Rv1 and H660 in a dose-dependent manner. P <0.05), and IC 50 The clonogenic capacity of C42B-MDVR and CWR22Rv1 cells at 7.77 μM, 4.24 μM, and 9.21 μM were respectively. P <0.05), and the organoid proliferation activity of LuCaP49 and LuCaP93 organoids ( P <0.05).

[0022] (3) The compounds of the present invention can significantly inhibit the proliferation of NEPC organoids (LuCaP49, LuCaP93), and the inhibitory effect is concentration-dependent.

[0023] (4) The compounds of the present invention have a clear targeting mechanism and good anti-tumor activity, laying the foundation for the development of novel targeted drugs for the treatment of neuroendocrine prostate cancer.

[0024] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0025] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0026] Figure 1 This is the MS spectrum of the small molecule compound DrugII.

[0027] Figure 2 For the small molecule compound DrugII 1 H NMR spectrum.

[0028] Figure 3 Schematic diagrams of the docking of the small molecule compound DrugII with PLXND1: (A) Molecular structure of the small molecule compound DrugII; (B) Protein pockets of PLXND1 protein showing potential binding to DrugII; (CE) 3D diagrams from different perspectives showing the interaction between the small molecule compound DrugII and the PLXND1 protein pocket; (F) 2D diagram showing the interaction between the small molecule compound DrugII and the PLXND1 protein pocket, with pink arrows representing hydrogen bonds and green line segments representing π-π interactions; (GI) Interaction diagrams of the small molecule compound DrugII and the surface of the PLXND1 protein pocket from different perspectives.

[0029] Figure 4 Regulating NEPC cell proliferation with the small molecule compound DrugII: (A) Concentration gradient curves of cell proliferation after 3 days of intervention with NEPC cells at gradient concentrations of the small molecule compound DrugII (0, 0.01, 0.1, 1, 2.5, 5, 10, 20, 50, 100 μM); (B) IC50 of the small molecule compound DrugII in NEPC cells. 50 (CD) C42BMDVR cells were treated with concentration gradients of small molecule compound DrugII (0, 5, 10 μM) and changes in cell colony formation ability were detected. (EF) CWR22Rv1 cells were treated with concentration gradients of small molecule compound DrugII (0, 5, 10 μM) and changes in cell colony formation ability were detected.

[0030] Figure 5 To regulate the proliferation activity of NEPC organoids LuCaP49 using the small molecule compound DrugII: (A) NEPC organoids LuCaP49 were seeded in 96-well plates and treated with the small molecule compound DrugII (0, 5, 10, 20 μM) for 9 days, followed by staining and photography; (B) CellTiterGlo was used to detect the proliferation activity of LuCaP49 organoids.

[0031] Figure 6 To regulate the proliferation activity of NEPC organoid LuCaP93 by the small molecule compound DrugII: (A) NEPC organoid LuCaP93 was seeded in 96-well plates and treated with the small molecule compound DrugII (0, 5, 10, 20 μM) for 9 days and stained and photographed; (B) CellTiterGlo was used to detect the proliferation activity of LuCaP93 organoids.

[0032] Figure 7 The results of the small molecule compound DrugII regulating tumorigenesis in NEPC cells CWR22Rv1 are shown, including tumor growth curves, images of the tumor, tumor wet weight curves, mouse body weight change curves, and gross images of the mouse heart, liver, spleen, lungs, and kidneys.

[0033] Figure 8 HE staining images of tumors formed in vivo in NEPC cells CWR22Rv1 after intervention with the small molecule compound DrugII, and the heart, liver, spleen, lungs, and kidneys of mice. Detailed Implementation

[0034] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0035] In this invention, "overnight" means 10-20 hours; "room temperature" means 15-35°C.

[0036] Example 1: Preparation and structural characterization of DrugII This embodiment provides a method for preparing DrugII, and the specific synthetic route is as follows: This compound (DrugII, its chemical structural formula is as follows) Figure 3 The synthetic route (shown in A) consists of two steps: the first step involves a reductive amination reaction to construct a bibenzylamine intermediate, and the second step involves an EDC condensation amidation reaction to introduce an indole-3-acetyl group, yielding the diacylated product DrugII. Details are as follows: (1) Step 1: Reductive amination to prepare intermediate (bisbenzylamine intermediate) Dissolve p-hydroxybenzaldehyde (1.0 molar equivalent) in anhydrous methanol (MeOH, 10 mL). Slowly add p-aminobenzylamine (1.0 molar equivalent) and glacial acetic acid (AcOH, 0.2 mL) at room temperature. Stir the reaction mixture at room temperature for 1–2 hours, then cool to 0°C and add sodium borohydride (NaBH4, 1.5 molar equivalent) in portions. After the addition is complete, continue stirring the reaction mixture for 4–8 hours (TLC monitoring to ensure complete reaction).

[0037] After the reaction was completed, the mixture was quenched with ice water, concentrated under reduced pressure, extracted with ethyl acetate / water, the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 1:1, v / v) to give the dibenzylamine intermediate (yield 65%-75%, purity ≥95%).

[0038] (2) Step 2: EDC condensation amidation to prepare Drug II The bibenzylamine intermediate (1.0 mol equivalent) obtained in step 1 was dissolved in anhydrous DMF (10 mL) and stirred until dissolved. Indole-3-acetic acid (2.2 mol equivalent), 1-hydroxybenzotriazole (HOBt, 2.2 mol equivalent), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 2.2 mol equivalent), and triethylamine (2–3 eq.) were added sequentially in an ice bath at 0°C. After the addition was complete, the ice bath was removed, and the mixture was allowed to warm naturally to room temperature. The reaction was continued with stirring for 8–24 hours (TLC monitoring showed complete reaction).

[0039] The reaction mixture was poured into ice water and extracted with ethyl acetate. The combined organic phases were washed successively with 10% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine, and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 1:1, volume ratio, or adjusted according to actual conditions) to give Drug II (yield 55%-65%, purity ≥95%). [M+H]⁺ 543.23 ( Figure 1 Drug II 1 H NMR spectrum as shown Figure 2 As shown.

[0040] Example 2: Molecular docking verification of DrugII targeting and binding to PLXND1 protein This embodiment verifies the targeting binding ability of DrugII to PLXND1 protein through molecular docking.

[0041] 1. Experimental Methods Molecular docking of PLXND1 protein (PLXND1 protein crystal structure obtained from the RCSB PDB database (PDB ID: 3H6N)) with the small molecule compound DrugII was performed online using the CB-DOCK tool (https: / / cadd.labshare.cn / cb-dock2 / index.php). The docking results were visualized using PyMol software, and the binding free energy (ΔG, unit kcal / mol), docking score, and interaction type (hydrogen bond, π-π interaction) were analyzed.

[0042] 2. Experimental Results Molecular docking results show ( Figure 3 The PLXND1 protein has five potential pockets that can bind to DrugII, with pocket 5 (Site 5) exhibiting the most stable and robust binding with the lowest energy consumption, a binding free energy ΔG of -48.7365 kcal / mol, and a docking score of -8.316. Figure 3B). Generally, a binding energy below -30 kcal / mol indicates a low binding free energy between the compound and the protein, suggesting stable binding.

[0043] 3D images show that DrugII binds stably to PLXND1 protein pocket 5. Figure 3 CE). The 2D diagram shows the hydrogen bonds represented by pink arrows and the π-π interactions represented by green line segments. Figure 3 F). The figures from different perspectives illustrate the interaction between the small molecule compound DrugII and the surface of the PLXND1 protein pocket ( Figure 3 GI).

[0044] Example 3: In vitro inhibitory effect of DrugII on the proliferation of neuroendocrine prostate cancer cells. This example is used to verify the inhibitory effect of DrugII on the proliferation and colony formation of NEPC cells (C42B-MDVR, CWR22Rv1, H660).

[0045] 1. Experimental Methods 1.1 Cell Culture (1) C42B-MDVR cell culture: C42B-MDVR cells are C42B cells resistant to enzalutamide intervention. During culture, RPMI 1640 medium + 10% FBS + 1% penicillin-streptomycin double antibiotics were used, and 20 μM of enzalutamide was added for maintenance culture.

[0046] (2) CWR22Rv1 cell culture: maintenance culture was performed using RPMI 1640 medium + 10% FBS + 1% penicillin-streptomycin double antibiotic.

[0047] (3) H660 cell culture: H660 cells are commercial NEPC cells, which grow in a mixed adherent and suspension state. During culture, RPMI 1640 medium + 5% FBS + 1% penicillin-streptomycin antibiotics were used, and insulin, transferrin, sodium selenite, hydrocortisone, β-estradiol and L-glutamine were added at working concentrations of 0.005 mg / mL, 0.01 mg / mL, 30 nM, 10 nM, 10 nM, β-estradiol and 2 mM.

[0048] (4) Culture conditions: All cells were cultured in a 37°C, 5% CO2 incubator.

[0049] 1.2 CellTiter-Glo method for detecting cell proliferation viability (IC50) 50 (Measurement) After digestion of cells in good growth condition, 5~7×10 3 Cells were seeded at a density of 100 μL per well in 96-well plates and incubated overnight. After overnight incubation, the culture medium was discarded, and the cells were washed twice with PBS. Then, 100 μL of culture medium containing different concentrations of Drug II (0, 0.01, 0.1, 1, 2.5, 5, 10, 20, 50, 100 μM) was added per well. The blank control group was treated with cell-free culture medium. After 72 hours of incubation, 100 μL of CellTiter-Glo working solution was added per well, and the cells were incubated at room temperature on a shaker for 10 minutes in the dark. The mixture was then transferred to opaque 96-well detection plates, and the luminescence signal was recorded. Concentration-inhibition curves were plotted and IC50 was calculated using GraphPad Prism software. 50 value.

[0050] 1.3 Cell Clonal Formation Experiment After digesting logarithmic growth phase cells (cell density approximately 80%) with trypsin, seed 1000 cells / well into 6-well plates with 3 mL of complete DMEM medium per well and incubate overnight. Discard the medium, wash twice with PBS, and add 3 mL of medium containing DMSO (solvent control), 5 μM, or 10 μM DrugII per well, and continue incubation for 7–10 days. Stop culture when obvious cell clones appear, discard the medium, wash 1–2 times with PBS, fix with 4% paraformaldehyde for 20–30 minutes, discard the fixative, and wash 2–3 times with PBS. Stain with 1% crystal violet solution for 15–30 minutes, recover the stain, rinse thoroughly with running water, and air dry at room temperature. Photograph and count the number of clones formed in each well and measure the clone size.

[0051] 1.4 Statistical Analysis Statistical analysis was performed using GraphPad Prism software. Two-tailed Student's-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P A value <0.05 is considered statistically significant.

[0052] 2. Experimental Results 2.1 Drug II inhibits NEPC cell proliferation result( Figure 4A) shows that Drug II exhibited significant inhibitory effects on proliferation in all three NEPC cell types in a concentration-dependent manner. The IC50 values ​​of Drug II in NEPC cells C42B-MDVR, CWR22Rv1, and H660 were [not specified in the original text]. 50 The concentrations were 7.77 μM, 4.24 μM, and 9.21 μM, respectively. Figure 4 B).

[0053] 2.2 Drug II inhibits NEPC cell colony formation result( Figure 4 CF showed that, compared with the DMSO control group, both 5 μM and 10 μM DrugII treatment groups significantly reduced the colony-forming ability of C42B-MDVR and CWR22Rv1 cells, and the inhibitory effect was concentration-dependent (both...). P <0.05).

[0054] Example 4: Inhibitory effect of Drug II on the proliferation of neuroendocrine prostate cancer organoids This embodiment is used to verify the inhibitory effect of DrugII on the proliferation of NEPC organoids (LuCaP49, LuCaP93).

[0055] 1. Experimental Methods 1.1 Preparation of human prostate cancer organoid culture medium Prepare 50 mL of complete culture medium for human prostate cancer organoids according to the formula in Table 1, and use immediately after preparation.

[0056] Table 1. Formula for complete culture medium for human prostate cancer organoids (50 mL) Y-27632 was added to the organoid culture medium during prostate organoid passage. Penicillin / streptomycin, Hepes, and GlutaMAX were added to adDMEM / F12 medium to prepare adDMEM / F12+++ medium. 50 mg of Collagenase Type IV was added to 10 mL of adDMEM / F12+++ medium to prepare a 5 mg / mL Collagenase Type IV solution.

[0057] 1.2. Human NEPC organoid culture (1) The LuCaP49 and LuCaP93 organoids were revived from liquid nitrogen, washed with pre-cooled adDMEM / F12+++ medium (containing 1% penicillin-streptomycin, 10 mM Hepes, 1× GlutaMAX), and centrifuged at 300g for 3 minutes.

[0058] (2) Discard the supernatant, resuspend the precipitate with Matrigel (GFR), and seed it in a 96-well plate at 40 μL / well.

[0059] (3) Place the 96-well plate in a 37°C incubator for 30 minutes to allow the Matrigel to solidify.

[0060] (4) Add 50 μL of complete culture medium containing 10 μM Y-27632 to each well and incubate overnight in a 37℃, 5% CO2 incubator.

[0061] 1.3 DrugII treatment and CellTiter-Glo detection (1) After incubation overnight, discard the culture medium and add 50 μL of complete culture medium (containing 10 μM Y-27632) containing DMSO (solvent control), 5 μM, 10 μM or 20 μM DrugII to each well.

[0062] (2) Culture continuously for 9 days, and replace with fresh drug-containing culture medium every 3 to 4 days.

[0063] (3) After the culture is completed, add 50 μL of CellTiter-Glo working solution to each well, incubate in a shaker in the dark for 10 minutes, and record the luminescence signal.

[0064] 1.4 Statistical Analysis Using GraphPad Prism software, one-way ANOVA was employed for comparisons among multiple groups. P A value <0.05 is considered statistically significant.

[0065] 2. Experimental Results LuCaP49 organoid experimental results ( Figure 5 AB) showed that, compared with the DMSO control group, the organoid volume of the 5 μM, 10 μM, and 20 μM DrugII treatment groups was significantly reduced, and the proliferation activity was significantly decreased in a concentration-dependent manner. P <0.05).

[0066] LuCaP93 organoid experimental results ( Figure 6 AB) showed that, compared with the DMSO control group, the organoid proliferation activity of the 5 μM, 10 μM, and 20 μM DrugII treatment groups was significantly reduced in a concentration-dependent manner. P <0.05).

[0067] Example 5: Inhibitory effect of DrugII on in vivo tumor growth of neuroendocrine prostate cancer and histopathological analysis of tumor tissue. This embodiment is used to verify the inhibitory effect of DrugII on the in vivo tumor growth of NEPC and to analyze the pathological changes of tumor tissue after treatment.

[0068] 1. Experimental Methods 1.1 Laboratory Animals Six-week-old male CB-17 / IcrHsd-Prkdc scid SCID mice were housed in an SPF-grade animal facility and acclimatized for one week. All animal experiments were approved by the animal ethics committee.

[0069] 1.2 CWR22Rv1 cell subcutaneous tumor-bearing model (1) Expand the CWR22Rv1 cell culture to 80% density, digest and resuspend, and adjust the density to 4×10⁻⁶. 6 Mix 100 μL of cells with an equal volume of melted Matrigel and place on ice.

[0070] (2) 200 μL (containing 4×10⁻⁶) was subcutaneously injected into both sides of SCID mice. 6 (cell) / point.

[0071] (3) Monitor mouse growth daily for the first 3 days after tumor cell inoculation. Tumors can be palpated approximately 7-10 days after inoculation; tumor volume should then be measured every 3-4 days. Tumor volume calculation formula: V=W 2 ×L×0.52.

[0072] (4) When the tumor volume reaches 50~100 mm 3 Mice were randomly divided into two groups (6-8 mice per group): solvent control group (15% Cremophor EL + 82.5% PBS + 2.5% DMSO, intraperitoneal injection, once every other day); Drug II treatment group (4 mg / kg, dissolved in solvent, intraperitoneal injection, once every other day).

[0073] (5) Measure mouse weight weekly. Culture continuously for 3-4 weeks or until tumor volume reaches 1000 mm. 3 The experiment was terminated at the appropriate time, the mice were euthanized, the tumors were dissected, photographed, and weighed.

[0074] 1.3 LuCaP93 PDX Model (1) Place the LuCaP93 PDX tumor in a 60 mm culture dish containing pre-cooled PBS, place it on ice, and cut it into rice-grain-sized pieces (about 2-3 mm) using sterile scissors and forceps. 3 (equal-volume tumor tissue fragments).

[0075] (2) Under anesthesia, tumor tissue fragments were implanted subcutaneously on both sides of SCID mice and sutured. The growth of the mice was monitored daily for the first 3 days after inoculation.

[0076] (3) When the tumor volume reaches 50~100 mm 3 At that time, the grouping and dosing regimens are the same as described in 1.2 above.

[0077] (4) Tumor volume measurement, weight monitoring and termination of experiment are the same as described in 1.2 above.

[0078] 1.4 Fixation and Embedding of Tissue Specimens (1) Fixation: The dissected tumor tissue is placed in an embedding box and fixed in 4% paraformaldehyde for 24 to 48 hours. The volume of the fixation liquid is 10 to 20 times the volume of the tissue.

[0079] (2) Dehydration: The tissue was dehydrated in gradients of 30%, 50%, 75%, 85%, 95%, and 100% ethanol for 1 hour per gradient.

[0080] (3) Transparency: The tissue was placed in a mixture of 100% ethanol and pure xylene of equal volume for 30 minutes, and then placed in pure xylene for transparency twice, each time for 1 hour.

[0081] (4) Paraffin penetration and embedding: The tissue was treated sequentially with an equal amount of paraffin and xylene mixture, then with pure paraffin (3 times, 30 minutes each time), embedded in paraffin, and cooled and solidified.

[0082] (5) Sectioning: Use a microtome to continuously section the material to a thickness of about 3 μm, attach it to a glass slide, dry it at 40°C, and store it at 4°C.

[0083] 1.5 Hematoxylin / eosin (HE) staining (1) Baking slices: Bake at 60℃ for 4 hours.

[0084] (2) Dewaxing: Dewaxing twice with pure xylene, 10 minutes each time.

[0085] (3) Hydration: The product was hydrated in sequence with 100%, 95%, 85% and 75% ethanol for 5 minutes each time, and washed with PBS 3 times for 5 minutes each time.

[0086] (4) Staining: Stain with hematoxylin solution for 2-3 minutes, then rinse with running water; stain with eosin solution for 120 seconds, then rinse with running water for 30 seconds.

[0087] (5) Dehydration, clearing and mounting: The slides were dehydrated for 3 minutes each with 75%, 85%, 95% and 100% ethanol, treated twice with pure xylene for 5 minutes each time, dried, and then mounted with mounting medium. Images were then acquired.

[0088] 1.6 Immunohistochemical (IHC) staining (1) Baking, dewaxing, and hydration: Same as 1.5 above.

[0089] (2) Antigen retrieval: The slides were placed in 1× sodium citrate antigen retrieval solution and incubated in a 95°C water bath for 30 minutes. After cooling, the slides were washed twice with 0.2% PBST for 3 minutes each time, and once with PBS for 3 minutes.

[0090] (3) Blocking endogenous enzymes: Add 3% H2O2, react at room temperature for 10 minutes, wash 3 times with 0.2% PBST for 3 minutes each time.

[0091] (4) Serum blocking: Add normal goat serum, block at 37°C for 30 minutes, remove excess liquid, wash 3 times with 0.2% PBST for 3 minutes each time.

[0092] (5) Permeabilization: Place the slices in 0.2% Triton X-100 solution and let stand at room temperature for 10 minutes. Wash with 0.2% PBST 3 times, 3 minutes each time.

[0093] (6) Primary antibody incubation: Dilute the primary antibody with 0.1% BSA (dilution ratio 1:(50~200)) and drop it onto the slide (about 60 μL / slide), and incubate overnight at 4°C. After incubation, wash three times with 0.2% PBST for 3 minutes each time.

[0094] (7) Secondary antibody incubation: Add secondary antibody (Polyperoxidase-anti-Mouse / Rabbit IgG), incubate at 37°C for 1 hour, wash 3 times with 0.2% PBST for 3 minutes each time.

[0095] (8) DAB color development: Prepare DAB color development solution (1 mL DAB substrate solution + 50 μL DAB concentrate), add it to the slide, and rinse with pure water to stop the color development after satisfactory color development.

[0096] (9) Counterstaining: Counterstain with hematoxylin solution for 2-3 minutes, then rinse with running water.

[0097] (10) Dehydration, clearing, and sealing: Same as 1.5 above.

[0098] (11) Image acquisition: Images were acquired and analyzed using a multispectral fully automated tissue scanner.

[0099] 1.7 Statistical Analysis Using GraphPad Prism software, the two-tailed Student's t-test was used for comparisons between the two groups. P A value <0.05 is considered statistically significant.

[0100] 2. Experimental Results In vivo experimental results ( Figure 7 , Figure 8 The results showed that Drug II effectively inhibited the in vivo tumorigenicity of NEPC CWR22Rv1 cells in a dose-dependent manner. Furthermore, Drug II did not affect the body weight of mice, and the gross appearance of the heart, liver, spleen, lungs, and kidneys was not significantly different from the control group. Pathological HE staining revealed that Drug II had no significant toxicity to mouse organs.

[0101] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The compound represented by Formula I and its pharmaceutically acceptable salt: Formula I in, R is selected from hydrogen, hydroxyl group, C 1~3 Alkyl groups, halogens.

2. The compound according to claim 1, and its pharmaceutically acceptable salt, characterized in that: The compound is .

3. A method for preparing the compound of claim 1 or 2, and its pharmaceutically acceptable salt, characterized in that: The method includes the following steps: (a) Reaction of p-hydroxybenzaldehyde, p-aminobenzamide, and a reducing agent under acidic conditions yields a dibenzylamine intermediate; (b) The dibenzylamine intermediate obtained in step (a) was reacted with indole-3-acetic acid in the presence of an amidating agent to give the compound shown in Formula I.

4. The method according to claim 3, characterized in that: In step (a), the molar ratio of p-hydroxybenzaldehyde, p-aminobenzamide, and the reducing agent is 1:(0.5~2):(1~2); the reducing agent is sodium borohydride; the acid is glacial acetic acid; the reaction is carried out in an alcohol solvent at a temperature of 0℃~35℃. In step (b), the molar ratio of the bibenzylamine intermediate, indole-3-acetic acid, and amidating agent is 1:(2~3):(2~5); the amidating agent is selected from one or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine; the solvent for the reaction is N,N-dimethylformamide, the reaction temperature is 0℃~35℃, and the reaction time is 8~24 hours.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition is a pharmaceutical preparation made from the compound of any one of claims 1 or 2, a pharmaceutically acceptable salt thereof as the active ingredient, and pharmaceutically acceptable excipients.

6. Use of the compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the preparation of a PLXND1 inhibitor.

7. The use according to claim 6, characterized in that: The PLXND1 inhibitor is a drug used to prevent and / or treat diseases mediated by or associated with abnormal function of the PLXND1 protein.

8. The use according to claim 7, characterized in that: The diseases mentioned are neuroendocrine tumors and prostate cancer.

9. The use according to claim 8, characterized in that: The neuroendocrine tumors are selected from neuroendocrine prostate cancer, small cell neuroendocrine prostate cancer, AR- / NE- prostate cancer, and AR+ / NE+ prostate cancer. The prostate cancer is selected from castration-resistant prostate cancer, castration-sensitive prostate cancer, androgen receptor-overexpressing prostate cancer, and androgen receptor-underexpressing prostate cancer.

10. The use according to claim 8, characterized in that: The drug is a drug for the prevention and / or treatment of neuroendocrine prostate cancer.

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