A small molecule compound EPIC-1042, a pharmaceutical composition and a preparation method and application thereof

Abstract

The application provides a small molecule compound EPIC-1042, and the application designs and synthesizes the small molecule compound EPIC-1042, which can specifically block the combination of PTRF and CAV1, and has a lower IC 50 In vivo experiments prove that EPIC-1042 can effectively reduce the exocytosis of sEVs and the stability of PARP1 and block the progress of the autophagic flow. In in vitro experiments, when combined with the first-line chemotherapy drug temozolomide, EPIC-1042 can effectively reverse temozolomide resistance and prolong the survival period, and has a great clinical conversion prospect.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, and in particular relates to a small molecule compound EPIC-1042, a pharmaceutical composition thereof, its preparation method and application. Background Technology

[0002] Glioblastoma multiforme (GBM) is the most aggressive primary malignant brain tumor, exhibiting a disproportionately high morbidity and mortality rate, with a 5-year survival rate of only 6.8%. Temozolomide (TMZ), approved by the U.S. Food and Drug Administration (FDA) in 2005 as a first-line chemotherapy for GBM, has improved the postoperative and post-radiotherapy survival of GBM patients from 9-10 months to 14 months. However, the problem of chemotherapy resistance to TMZ is increasingly concerning, and overcoming this issue is urgent. Furthermore, the extensive tumor heterogeneity in GBM suggests that combination therapy may be relatively more effective than monotherapy.

[0003] Caveolins are nanoscale (60-80 nm) membrane invaginations on the cell membrane, primarily involved in endocytosis, cholesterol and lipid metabolism, and cell signal transduction. They are characterized by their richness in caveolin proteins. Among the caveolin protein family, Caveolin 1 (CAV1) is a crucial component involved in caveolin formation and is expressed in various tissues. Polymerase I and release transcript factor (PTRF / Cavin1) are markers of gliomas and serum exosomes. CAV1 binds to PTRF and recruits PTRF to the cell membrane; this process is essential for caveolin formation and functional maintenance in the presence of cholesterol and phosphatidylserine. Our previous studies have demonstrated that the PTRF-CAV1 complex can be absorbed by sEVs and participates in sEV generation, transport, and secretion in a caveolin-dependent manner. Furthermore, increased PTRF binding to CAV1 can increase the number of caveolins and promote sEV production and uptake. Interestingly, sEVs can be used as carriers for drug efflux pumps embedded in transfer membranes and as efflux carriers for anticancer drugs. We also found that PTRF knockout (KO) inhibits PTRF-CAV1 binding, pit formation, and sEV secretion, thereby increasing intracellular TMZ concentration. In summary, interfering with the interaction between PTRF and CAV1 may be a promising therapeutic approach to enhance the efficacy of TMZ treatment. Summary of the Invention

[0004] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes a small molecule compound EPIC-1042, a pharmaceutical composition, a method for preparing the same, and its applications.

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

[0006] As a first aspect of the present invention, a small molecule compound EPIC-1042 is provided, the structural formula of which is shown in Formula I:

[0007]

[0008] As a second aspect of the present invention, a method for preparing the above-mentioned small molecule compound EPIC-1042 is also provided, comprising the following steps:

[0009] (1) Compound 1 and Compound 2 were mixed in methanol, and after adding alkali, the mixture was stirred at 90°C. The mixture was then filtered and concentrated under reduced pressure to remove the solvent, yielding Compound 5.

[0010] (2) Compound 5 and tetrabutylammonium chloride were mixed in phosphorus oxychloride and stirred under sealed conditions at 25℃-170℃. After cooling, the mixture was poured into ice, stirred and neutralized in an alkaline aqueous solution. The resulting crystals were filtered, washed and dried to finally obtain compound 6.

[0011] (3) Compound 6, compound 7 and K2CO3 were mixed in an organic solvent and stirred at 25℃-90℃. The mixture was filtered and concentrated under reduced pressure to remove the organic solvent. Then it was washed and extracted with ethyl acetate. The organic phase was then concentrated under reduced pressure to remove the organic solvent. The mixture was filtered and concentrated under reduced pressure to remove the solvent. Finally, the crude product was purified by high performance liquid chromatography to obtain compound 8.

[0012] (4) Compound 8 and compound 9 were mixed and stirred at 25℃-110℃. The mixture was then filtered and concentrated under reduced pressure to remove the solvent. Finally, the crude product was purified by high performance liquid chromatography to obtain compound 10.

[0013] (5) In the presence of nitrogen and hydrogen, compound 10, Pd / C and Pd(OH)2 were mixed in methanol and stirred at 25℃-30℃. The mixture was then filtered and concentrated under reduced pressure to remove the solvent, to obtain compound 11.

[0014] (6) Compound 11 and Compound 12 were mixed in MeOH, and K2CO3, NaBH3CN and acetaldehyde were added to the mixture. The mixture was then stirred at 0-25℃. Finally, the mixture was filtered and concentrated under reduced pressure to remove the solvent to obtain Compound 13.

[0015] (7) Compound 13 was mixed with an organic solvent and HCl / Dioxane solution was added to the mixture. The mixture was then stirred at room temperature. The mixture was then filtered and concentrated under reduced pressure to remove the organic solvent. Finally, the mixture was purified by high performance liquid chromatography to obtain the small molecule compound EPIC-1042.

[0016] Preferably, the synthetic route for the small molecule compound EPIC-1042 is as follows;

[0017]

[0018] As a third aspect of the invention, uses of the above-mentioned small molecule compound EPIC-1042 or its salts are provided, said uses being selected from any one or more of the following:

[0019] (1) Use in the preparation of drugs for cancer treatment;

[0020] (2) Use in preparing interfering agents that interfere with the binding of PTRF to Caveolin1;

[0021] (3) Use in preparing interfering agents that interfere with Caveolin1 recruitment to PTRF;

[0022] (4) Use in the preparation of reducing agents that reduce the efflux level of sEVs;

[0023] (5) Uses in the preparation of PARP1 degradation promoters;

[0024] (6) Use in the preparation of autophagy flux inhibitors;

[0025] (7) Used in the preparation of sensitizers to enhance the efficacy of temozolomide.

[0026] Preferably, the tumor is a tumor with high expression of PTRF and Caveolin1.

[0027] More preferably, the tumor is selected from glioma, and even more preferably glioblastoma.

[0028] As a fourth aspect of the invention, a medicament is provided, the medicament comprising the above-described small molecule compound EPIC-1042 or a salt thereof, and a pharmaceutically acceptable carrier and / or excipients.

[0029] In this invention, EPIC-1042 or its salt is used as the sole active ingredient in the drug for the treatment of tumors. Alternatively, EPIC-1042 or its salt can be used as the main active ingredient in combination with other drugs for the treatment of tumors.

[0030] Preferably, the drug also includes temozolomide.

[0031] As a fifth aspect of the invention, the use of the above-described drug is provided.

[0032] (1) Use in the preparation of drugs for cancer treatment;

[0033] (2) Use in preparing interfering agents that interfere with the binding of PTRF to Caveolin1;

[0034] (3) Use in preparing interfering agents that interfere with Caveolin1 recruitment to PTRF;

[0035] (4) Use in the preparation of reducing agents that reduce the efflux level of sEVs;

[0036] (5) Uses in the preparation of PARP1 degradation promoters;

[0037] (6) Use in the preparation of autophagy flow inhibitors.

[0038] More preferably, the tumor is selected from glioma, and even more preferably glioblastoma.

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] This invention designs and synthesizes the small molecule compound EPIC-1042, which specifically blocks the binding of PTRF to CAV1 and exhibits a low IC50 in glioma cell lines. 50 In vivo experiments have demonstrated that EPIC-1042 can effectively reduce efflux of sEVs and PARP1 stability, as well as block the progression of autophagic flux. In in vitro experiments, when used in combination with the first-line chemotherapy drug temozolomide, it can effectively reverse temozolomide resistance and prolong survival, showing great promise for clinical translation. Attached Figure Description

[0041] Figure 1 The figure shows the experimental results of EPIC-1042 blocking PTRF and CAV1 binding, where: Figure 1 A: Pull-down assays demonstrated that EPIC-1042 can bind to PTRF in glioma cell lines; cell lysates from pull-down assays were treated with DMSO, EPIC-1042 (100 μM), and E-Biotin (100 μM) for 24 hours. Figure 1 B: Co-IP assays demonstrated that EPIC-1042 can block the binding of PTRF to CAV1 in glioma cell lines; Figure 1 C: Immunofluorescence demonstrated that EPIC-1042 could block the binding of PTRF to CAV1 in glioma cell lines; Figure 1 D: Co-location analysis of PTRF and CAV1 using Pearson coefficients; Figure 1 E: Changes in the pits were observed using transmission electron microscopy after U87-MG cells were treated with EPIC-1042. Figure 1 F: To statistically analyze the number of small depressions; in Figure 1 In BE, cells were treated with DMSO or 20 μM EPIC-1042 for 48 hours;

[0042] Figure 2 The graph shows the experimental results of the effect of EPIC-1042 on the total protein content in sEVs, where: Figure 2 A: Changes in sEV protein levels after EPIC-1042 treatment of glioma cells; Figure 2 B: Changes in exosome markers after EPIC-1042 treatment of glioma cells; Figure 2 C: Changes in sEV concentration after EPIC-1042 treatment of glioma cell lines; Figure 2 D: Changes in sEVs concentration and particle size after EPIC-1042 treatment of glioma cells; Figure 2 E: Changes in intracellular temozolomide levels in glioma cells after treatment with 100 μM TMZ or 20 μM EPIC-1042 for 24 hours; Figure 2 In AD, cells were treated with DMSO or 20 μM EPIC-1042 for 48 hours.

[0043] Figure 3 The graph shows the effect of EPIC-042 on autophagy, where: Figure 3 A: Changes in the early endosomal marker EEA1 levels after treatment of glioma cell lines with different concentrations and durations of EPIC-1042; Figure 3 B: Changes in the levels of autophagy markers p62 and LC3 after treatment of glioma cell lines with different concentrations and durations of EPIC-1042; Figure 3 C: Changes in autophagy markers at different stages after EPIC-1042 treatment of glioma cell lines; Figure 3 D: Immunofluorescence showed that EPIC-1042 affected the quenching of green fluorescent protein; Figure 3 E: Statistical analysis of the ratio of red fluorescent protein to green fluorescent protein; Figure 3 F: Changes in LC3 levels after 48 hours of treatment with glioma cell lines using 20 μM EPIC-1042 alone, 100 μM TMZ alone, or both in combination; Figure 3 G: Yes Figure 4 Statistical analysis of F protein grayscale values; Figure 3 In CE, cells were treated with DMSO or 20 μM EPIC-1042 for 48 hours;

[0044] Figure 4The graph shows the results of in vivo TMZ sensitivity testing of EPIC-1042, where: Figure 4 A: Animal experimental design diagram of EPIC-1042. Tumor-bearing nude mice were treated by gavage for 2 weeks with DMSO, TMZ (5mg / kg), EPIC-1042 (15mg / kg), or TMZ (5mg / kg) + EPIC-1042 (15mg / kg), with n=6 in each group; Figure 4 B: Bioluminescence imaging showed that the combination of EPIC-1042 and TMZ could more effectively inhibit the growth of mouse glioma in situ models; Figure 4 C: Quantitative and statistical analysis of bioluminescence results revealed that the combined use of EPIC-1042 and TMZ can more effectively inhibit tumor growth; Figure 4 D: Survival analysis diagram of tumor-bearing mice; Figure 4 E: Median survival statistics of tumor-bearing mice; Figure 4 F: A representative HE staining image of the brain of a tumor-bearing mouse; Figure 4 G: Ki-67 immunohistochemical staining of tumor tissue from tumor-bearing mice;

[0045] Figure 5 The figure shows the experimental results of EPIC-1042's degradation of PARP1 protein, where: Figure 5 A: Changes in PARP1 levels after treating glioma cell lines with 20 μM EPIC-1042 for 48 hours; Figure 5 B: Changes in PARP1 levels 24 hours after treatment of glioma cell lines with actinomycete or 20 μM EPIC-1042 in combination with actinomycete. Figure 5 C: Yes Figure 6 Statistical analysis of the grayscale values ​​of PARP1 protein in B; Figure 5 D: Yes Figure 4 Statistical analysis of gray values ​​of p62 and LC3 proteins in BEPIC-1042 time gradient; Figure 5 D: Changes in PARP1 levels in glioma cell lines after treatment with 20 μM EPIC-1042 in combination with chloroquine or MG132 for the corresponding time period; Figure 5 F: Co-IP assay showed that after treatment of glioma cell lines with 20 μM EPIC-1042 for the corresponding time, the amount of ubiquitin bound to PARP1 did not increase; Figure 5 G: Co-IP assays showed that after treatment of glioma cell lines with 20 μM EPIC-1042 for a corresponding period of time, the amount of p62 bound to PARP1 increased; Figure 5 H: Changes in PARP1 after treatment with 20 μM EPIC-1042 in p62 knockdown glioma cell lines;

[0046] Figure 6The image shows the in vitro assay results of EPIC-1042 sensitizing temozolomide, where: Figure 6 A: Changes in PARP1 levels after 24 hours of treatment of MGMT promoter-unmethylated glioma cell lines with actinomycete ketone or 20 μM EPIC-1042 in combination with actinomycete ketone; Figure 6 B: Immunofluorescence staining of O6MetG after treatment of MGMT promoter nonmethylated glioma cell lines with EPIC-1042 alone, TMZ alone, or both in combination; Figure 6 C: Changes in γ-H2AX after treatment of MGMT promoter-unmethylated glioma cell lines with EPIC-1042 alone, TMZ alone, or in combination of both; Figure 6 D: Immunofluorescence staining of γ-H2AX after treatment of MGMT promoter nonmethylated glioma cell lines with EPIC-1042 alone, TMZ alone, or both in combination; Figure 6 E: Cell inhibition rate and synergistic analysis matrix of combined application of EPIC-1042 and TMZ in T98G cell line; Figure 6 F: Immunofluorescence staining of O6MetG in p62 knockdown glioma cell lines after treatment with TMZ alone or in combination with EPIC-1042 on MGMT promoter-unmethylated glioma cell lines; Figure 6 BD and Figure 6 In F, cells were treated with DMSO, EPIC-1042 (20 μM), TMZ (600 μM), or a combination of EPIC-1042 (20 μM) and TMZ (600 μM) for 48 hours, respectively.

[0047] Figure 7 The image shows the in vivo test results of temozolomide sensitized by EPIC-1042, where: Figure 7 A: Animal experimental design diagram of EPIC-1042 concentration gradient combined with TMZ. Tumor-bearing nude mice were treated by gavage for 2 weeks with DMSO, TMZ (5 mg / kg), or TMZ (5 mg / kg) + EPIC-1042 (15 mg / kg, 30 mg / kg, 45 mg / kg), with n = 6 in each group; Figure 7 B: Bioluminescence imaging showed that the combination of different concentrations of EPIC-1042 and TMZ had a gradient-enhancing effect on inhibiting the growth of mouse glioma in situ models. Figure 7 CD: Quantitative and statistical analysis of bioluminescence results revealed that the combined use of different concentrations of EPIC-1042 and TMZ had a concentration-increasing effect on inhibiting tumor growth. Figure 7 E: Survival analysis diagram of tumor-bearing mice. Figure 7 F: Median survival statistics of tumor-bearing mice; Figure 7 G: Immunohistochemical staining of Ki-67, γ-H2AX and PARP1 in tumor tissues from tumor-bearing mice; Figure 7 H: Design diagram of long-term animal experiments using EPIC-1042 in combination with TMZ. Tumor-bearing nude mice were treated by gavage for 6 weeks with DMSO, TMZ (5 mg / kg), EPIC-1042 (15 mg / kg), or TMZ (5 mg / kg) + EPIC-1042 (15 mg / kg), with n = 6 in each group; Figure 7 I: Small animal MRI showed that the combination of EPIC-1042 and TMZ could more effectively inhibit the growth of mouse glioma in situ models; Figure 7 J: Quantitative and statistical analysis of MRI results in small animals revealed that the combined use of EPIC-1042 and TMZ can more effectively inhibit tumor growth. Figure 7 K: Survival analysis diagram of tumor-bearing mice;

[0048] Figure 8 The results are pre-clinical data for EPIC-1042, including: Figure 8 A: The inhibitory effects of EPIC-1042 and positive control drugs 7-ethoxycoumarin, bupropion, paclitaxel, sulfamethoxazole, omeprazole, promethazine, chlorzolid, fluconazole and ketoconazole on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 (with midazolam as substrate) and CYP3A4 (with testosterone as substrate); Figure 8 B: Metabolic stability of EPIC-1042 and the control drug clozapine in liver microsomes of humans, mice, rats, dogs and monkeys; Figure 8 C: Patch clamp test of HEK293 after treatment with EPIC-1042 and the positive control drug cisapride; Figure 8 D: Pharmacokinetic analysis of rats after oral administration of EPIC-1042 at doses of 2 mg / kg and 45 mg / kg. Detailed Implementation

[0049] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0050] The invention will be described in detail below with reference to specific embodiments.

[0051] I. Preparation of the small molecule compound EPIC-1042

[0052] Specifically, the steps include the following:

[0053] 1. Compound 1 (200 g, 382 mmol, 1.00 eq) and Compound 2 (96.5 g, 1.15 mmol, 1.50 eq) were mixed in MeOH (2.00 L), and KOH (85.8 g, 1.53 mmol, 2.00 eq) was added. The mixture was stirred at 90 °C for 12 hours. The mixture was then filtered and concentrated under reduced pressure to remove the solvent, yielding Compound 5. Compound 5 is a white solid and passes through... 1 ¹H NMR detection. Spectral data of compound 5:

[0054] 1 HNMR: ET70153-1-P1H1(400MHz, DMSO-d6); δ9.56(s,1H),7.37-7.22(m,5H),3.57(s,2H),3.36(s,1H),2.97(s,2H),2.59-2.53(m,2H),2.44(br d,J=5.0Hz,2H).

[0055] 2. Compound 5 (5.00 g, 17.7 mmol, 1.00 eq) and TBAC (4.94 g, 17.7 mmol, 4.97 mL, 1.00 eq) were mixed in POCl3 (15.0 mL) in a 100 mL sealed tube and stirred at 170 °C for 3 hours. After cooling, the mixture was poured into ice, stirred, and neutralized in a 10% aqueous solution of potassium hydroxide. The resulting crystals were filtered, washed with water, and dried to give compound 6. Compound 6 is a white solid and... 2 ¹H NMR detection. Spectral data of compound 6:

[0056] 2 HNMR: ET70153-2-P1H1 (400MHz, DMSO-d6); δ7.50-7.25(m,5H),3.78(br s,2H),3.57(br s,2H),3.02(br s,2H),2.77(br s,2H).

[0057] 3. Compound 6 (40.0 g, 125 mmol, 1.00 eq), compound 7 (26.9 g, 251 mol, 27.4 mL, 2.00 eq), and K₂CO₃ (34.7 g, 251 mmol, 2.00 eq) were mixed in MeCN (900 mL). The mixture was then stirred at 60 °C for 12 hours. The mixture was filtered and concentrated under reduced pressure to remove the solvent, then washed with water (100 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was then concentrated under reduced pressure to remove the solvent, and the mixture was filtered and concentrated under reduced pressure to remove the solvent again. Finally, the crude product was purified by high performance liquid chromatography to obtain compound 8. Compound 8 is a yellow solid and... 3 ¹H NMR detection. Spectral data of compound 8:

[0058] 3 HNMR: ET70153-3-P1H1 (400MHz, DMSO-d6); δ7.92-7.76 (m, 1H), 7.42-7.16 (m, 8H), 4.60 (d, J = 5.9Hz, 3H), 3.33 (s, 2H), 2.75 (br d,J=5.1Hz,2H),2.68-2.64(m,2H),2.57(q,J=7.1Hz,3H).

[0059] 4. Compound 8 (40.0 g, 102 mmol, 1.00 eq) and compound 9 (164 g, 1.03 mol, 161 mL, 10.0 eq) were mixed and stirred at 110 °C for 16 hours. The mixture was then filtered and concentrated under reduced pressure to remove the solvent. Finally, the crude product was purified by high performance liquid chromatography to obtain compound 10. Compound 10 is a yellow solid and... 4 ¹H NMR detection. Spectral data of compound 10:

[0060] 4 HNMR: ET70153-4-P1H2 (400MHz, DMSO-d6); δ7.40-7.14 (m, 10H), 6.76 (br t, J=5.0Hz, 1H), 6.36 (br t, J=5.4Hz, 1H), 4.54 (br d,J=5.6Hz,2H),3.69(s,2H),3.27(s,4H),2.99(br d,J=5.6Hz,2H),2.60(br s,4H),1.50-1.27(m,9H).

[0061] 5. Compound 10 (20.0 g, 39.0 mmol, 1.00 eq), hydrogen, Pd / C (6.00 g, 50.0% purity), and Pd(OH)₂ (6.00 g, 50.0% purity) were mixed in MeOH (200 mL) at 30 °C in the presence of nitrogen. The mixture was then stirred at 30 °C for 16 hours in the presence of hydrogen (15 psi). The mixture was then filtered and concentrated under reduced pressure to remove the solvent, yielding compound 11. Compound 11 is a white solid and passes through… 5 ¹H NMR detection. Spectral data of compound 11:

[0062] 5 HNMR: ET70153-5-P1H1 (400MHz, DMSO-d6); δ7.33-7.25 (m, 4H), 7.21-7.16 (m, 1H), 7.10 (br t, J=5.9Hz, 1H), 6.76 (br t, J=5.1Hz, 1H), 6.29 (br t,J=5.6Hz,1H),4.56(br d,J=5.6Hz,2H),4.12(br s,2H),3.47(s,2H),3.26(q,J=5.9Hz,2H),3.03-2.92(m,2H),2.86(br t,J=5.6Hz,2H),2.45(br t,J=5.3Hz,2H),1.38(s,9H).

[0063] 6. Compound 11 (23.0 g, 54.4 mmol, 1.00 eq) and Compound 12 (11.9 g, 108 mmol, 1.00 eq) were mixed in MeOH (230 mL). K₂CO₃ (7.52 g, 54.4 mmol, 1.00 eq), NaBH₃CN (17.1 g, 272 mmol, 5.00 eq), and acetaldehyde (11.9 g, 108 mmol, 15.2 mL, 2.00 eq) were then added to the mixture. The mixture was then stirred at 25 °C for 16 hours. Finally, the mixture was filtered and concentrated under reduced pressure to remove the solvent, yielding Compound 13. Compound 13 was a white solid and passed through... 6 ¹H NMR detection. Spectral data of compound 13:

[0064] 6HNMR: ET70153-6-P1H1 (400MHz, DMSO-d6); δ7.26-7.02(m,5H),6.67(br t,J=4.9Hz,1H),6.35-6.18(m,1H),4.51-4.41(m,2H),3.93(q,J=7.1Hz,3H),3.15(br d,J=5.6Hz,2H),3.09-3.00(m,2H),2.88(br d,J=5.5Hz,2H),2.44-2.36(m,3H),1.89(s,4H),1.27(s,6H),1.09-1.05(m,3H).

[0065] 7. Compound 13 (9.00 g, 19.9 mmol, 1.00 eq) and DCM (90 mL) were mixed, and HCl / Dioxane (4 M, 19.9 mL, 4.00 eq) was added to the mixture. The mixture was then stirred at 25 °C for 2 hours. The mixture was then filtered and concentrated under reduced pressure to remove the solvent. Finally, the mixture was purified by high performance liquid chromatography to obtain EPIC-1042. EPIC-1042 is a yellow solid and... 7 HNMR detection. Spectral data from EPIC-1042:

[0066] 7 HNMR: ET70153-7-P1H2 (400MHz, DMSO-d6); δ11.60 (br s, 1H), 8.04 (br s, 3H), 7.84 (br t, J = 5.7Hz, 1H), 7.41-7.27 (m, 4H), 7.26-7.18 (m, 1H), 6.82 (br t,J=5.3Hz,1H),4.70-4.61(m,1H),4.60-4.51(m,1H),4.17(br d,J=12.6Hz,1H),3.99(br s,1H),3.79(br dd,J=8.4,14.9Hz,1H),3.66-3.52(m,2H),3.51-3.41(m,1H),3.35-3.15(m,3H),3.15-3.03(m,1H),2.86-2.73(m,3H),1.40(t,J=7.2Hz,3H).

[0067] II. EPIC-1042 Activity Test

[0068] The testing method and specific steps are as follows:

[0069] 1. Cell culture:

[0070] The patient-derived primary GBM cell line TBD-0220 was obtained from the Affiliated Hospital of Hebei University and cultured in Dulbecco's Modified Eagle's Medium (DMEM / F12, 1:1) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). U87-MG, LN229, T98G, and LN-18 cell lines were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM medium supplemented with 10% FBS and 1% PS. Cells were grown in a humidified incubator at 37°C and 5% CO2.

[0071] 2. Immunoblotting:

[0072] Cells were lysed on ice for 30 minutes using RIPA supplemented with 1% PMSF. Proteins were then separated on SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% BSA at room temperature for 2 hours, followed by overnight incubation with the appropriate antibody at 4°C for 16 hours. The membrane was then washed three times with PBST for 15 minutes each time, followed by incubation with the appropriate secondary antibody for 1 hour. Finally, images were detected using a gel imaging system.

[0073] 3. EPIC-1042-Biotin Pull-down:

[0074] Cells were lysed using NP-40 containing 1% PMSF and centrifuged. The supernatant was collected, aliquoted into three tubes, and incubated overnight at 4°C with DMSO, 100 μmol / L EPIC-1042, and 100 μmol / L EPIC-1042-Biotin, respectively. The samples were then incubated with streptavidin magnetic beads at 4°C for 4 h, followed by washing three times for a total of 15 min. Finally, the extracted samples were analyzed using Western blotting.

[0075] 4. Immunofluorescence experiment:

[0076] Cells treated with the drug and DMSO were washed twice with PBS, then fixed with 4% PFA for 20 min, permeabilized with 0.2% Triton X-100 for 20 min, blocked with 10% BSA for 1 h, and incubated overnight at 4°C with the corresponding primary antibody. The next day, the cells were stained with the corresponding secondary antibody, and the nuclei were reverse-stained with DAPI. Finally, the slides were imaged using a laser confocal microscope.

[0077] 5. Co-immunoprecipitation assay (Co-IP):

[0078] Cells were lysed using NP-40 containing 1% PMSF and centrifuged. The supernatant was incubated overnight at 4°C with the target antibody and corresponding IgG. Then, the cells were washed with 40 μl of protein A / G magnetic beads at 4°C for 2–4 hours, three times for a total of 15 min. Finally, the target protein was labeled with Western blotting.

[0079] 6. Transmission electron microscopy (TEM):

[0080] Cells treated with DMSO and EPIC-1042 for 48 h were fixed with 2.5% glutaraldehyde, then treated with 1% osmium tetroxide solution, dehydrated with fractionated ethanol, and dried with hexamethyldiazane. Tissue samples were embedded in epoxy resin, sectioned to a thickness of 70 nm, and stained with 2% uranyl acetate and lead citrate. Finally, gold-palladium sputtering was used to mount the slides, and imaging was performed using an H7760 microscope.

[0081] 7. Separation and characterization of sEVs:

[0082] GBM cells were seeded in 100 mm culture dishes with 10% FBS (sEVs-free) added and cultured for 24 h. The entire culture medium was then replaced with fresh medium containing 10% FBS (sEVs-free) and EPIC-1042. Forty-eight hours after EPIC-1042 treatment, the supernatant was collected, and the number of cells in each group was counted. The cells were then centrifuged sequentially to separate the precipitates from the collected medium. Specifically, the collected medium was centrifuged at 2000 g (20 min, 4 °C) to collect the 2K precipitate, then centrifuged at 10000 g (30 min, 4 °C) to collect the 10K precipitate, and ultracentrifuged at 100000 g (90 min, 4 °C) to collect the sEVs. The cells were then resuspended in cold PBS and ultracentrifuged again for 90 min. Finally, the microspheres were resuspended in PBS or RIPA buffer containing 1% PMSF for further analysis. To ensure the quality of the extracted sEVs, a NanoSight system (NS300; Morven Instruments; UK) was used to characterize the size distribution and concentration of sEVs. The concentration of sEVs was determined using a BCA assay kit.

[0083] 8. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC):

[0084] Mouse brain sections were prepared by fixing formalin-embedded paraffin (FFPE) tissue blocks, then dewaxing, hydrating, and immersing in citrate buffer (pH=6) at 100°C for 20 minutes. Hematoxylin and eosin were used for H&E staining. For immunohistochemistry, brain sections were first incubated with goat serum at room temperature for 30 minutes, then incubated overnight at 4°C with the corresponding primary antibody. The next day, the primary antibody was washed off, and the sections were incubated with enzyme-labeled secondary antibody at room temperature for 1 hour. Finally, the sections were incubated with diaminobenzidine (DAB) solution and photographed under a microscope.

[0085] 9. In vivo xenograft mouse model:

[0086] All animal research protocols were approved by the Institutional Animal Ethics Committee (IACUC) of Tianjin Medical University (No. IRB2022-DWFL-069). Lentiviral luciferase reporter genes were transduced into TBD-0220 cells (100,000 cells added to 3 μl PBS) and injected into each 4-week-old female BALB / c nude mouse to establish an in situ GBM model. All surgeries were performed under stereotactic guidance at Bregma coordinates of 2.0 mm posterior, 2.0 mm lateral, and 3.0 mm ventral to establish the model. Bioluminescence imaging or brain MRI was used to measure relative tumor size on days 7, 14, 21, and 28 post-transplantation. Survival curves were generated using the Kaplan-Meier method, Log-rank (Mantel-Cox) test, or Gehan-Breslow-Wilcoxon test to analyze differences in overall survival (OS) among groups. Animals were euthanized humanely upon reaching the experimental endpoint, and intracranial tumor tissue was extracted for immunohistochemical (IHC) and hematologic & epithelial (H&E) analysis.

[0087] III. Test Result Analysis

[0088] 1. EPIC-1042 can effectively block the binding of PTRF and CAV1.

[0089] To verify the blocking effect of EPIC-1042, biotin was first conjugated to EPIC-1042 (E-Biotin) to demonstrate whether EPIC-1042 could bind to PTRF. Pull-down results showed that after E-Biotin treatment, both endogenous PTRF and exogenously expressed PTRF-eGFP were bound by streptavidin magnetic beads. No signal was detected in the non-biotinylated EPIC-1042 group, indicating that the biotin-streptavidin interaction is specific. Figure 1 A. Further Co-IP assays using anti-PTRF antibodies revealed that, compared to DMSO treatment, EPIC-1042 treatment resulted in almost undetectable CAV1 binding to PTRF and PTRF-eGFP. Figure 1 B. Immunofluorescence analysis also showed the same results: after EPIC-1042 treatment, the co-localization coefficient of PTRF-eGFP and CAV1 decreased in the LN229 cell line, and the number of pits in U87-MG cells also decreased, such as Figure 1 C- Figure 1 F. In summary, EPIC-1042 effectively blocks PTRF-CAV1 interaction and exhibits a high docking score and optimal proliferation inhibition.

[0090] 2. EPIC-1042 can inhibit the efflux of sEVs and increase the intracellular concentration of temozolomide.

[0091] This invention uses the term "sEVs" to refer to precipitates obtained from conditioned medium by ultracentrifugation (100,000 g). First, the total protein content in sEVs was quantified, and the results showed that the total protein content in sEVs treated with EPIC-1042 was significantly reduced compared to cells treated with DMSO. Figure 2 A. To further analyze different subtypes, Western blotting was used to analyze exosome markers with universal characteristics in sEVs and whole-cell lysates (WCL). Results showed that Alix, CD63, Tsg101, and CD9 were significantly present in both sEVs and WCL; however, calnexin, an endoplasmic reticulum protein excluded by exosomes, was rarely detected in sEVs, indicating high exosome purity in sEVs. The intensity of exosome markers reflected the number of sEVs when sEVs were centrifuged from equal volumes of cells. EPIC-1042 treatment significantly reduced the levels of Alix, Tsg101, and CD9 markers in sEVs, but not in WCL. Furthermore, CD63, as a marker for multivesicular bodies (MVBs) that directly produce exosomes, also showed the same trend. Figure 2 B. These results suggest that EPIC-1042 may impair the secretion of sEVs, rather than affecting the expression of marker proteins.

[0092] Subsequent Nanosight analysis consistently showed that EPIC-1042 treatment significantly reduced the number of particles in the ultracentrifuged precipitate. Furthermore, EPIC-1042 not only reduced the amount of precipitate from ultracentrifugation but also resulted in an actual reduction in sEVs (saturated volatile organic compounds). Figure 2 C- Figure 2 D. Finally, as we expected, the combination of TMZ and EPIC-1042 showed that the intracellular TMZ concentration was twice that of TBD-0220 cells treated with TMZ alone, such as Figure 2 E. In summary, EPIC-1042 can specifically inhibit the secretion of sEVs by disrupting the interaction between PTRF and CAV1 and the pits.

[0093] 3. EPIC-042 can inhibit autophagy.

[0094] Following EPIC-1042 treatment, the early endosome marker early endosome antigen 1 (EEA1) showed a dose- and time-dependent decreasing trend, such as... Figure 3A. MVBs fuse with autophagosomes to form a biphasic body for degradation, representing a crossroads between endocytosis and autophagy. Furthermore, methyl-β-cyclodextrin (MBCD)-mediated consumption of membrane-bound cholesterol and disruption of lipid rafts have been shown to induce damage to autophagic flux in mouse embryonic fibroblasts, and pits are also a type of lipid raft. Therefore, an attempt was made to test the effect of EPIC-1042 on autophagy. It was found that EPIC-1042 could increase p62 and the autophagosome marker LC3-II in a time- and dose-dependent manner, such as... Figure 3 B. Elevated LC3-II levels could be due to increased autophagosome formation or inhibited protein degradation. To differentiate between these two possibilities, key genes playing important roles in autophagosome maturation were first examined. For example... Figure 3 As shown in Figure C, the total protein levels of these genes remained unchanged after EPIC-1042 treatment, thus ruling out the possibility of EPIC-1042 inducing autophagy. Next, confocal imaging was used to investigate the effect of EPIC-1042 on ectopic expression of dual fluorescent markers (GFP and RFP) in LC3, thereby exploring its effect on autophagic flux. Since GFP is quenched under acidic lysosomal conditions, while RFP is not, the ratio of RFP to GFP fluorescence can be used to assess autophagic flux. Immunofluorescence imaging showed that after 48 h of EPIC-1042 exposure, the number of RFP spots increased; however, the RFP:GFP ratio decreased sharply, as shown in Figure C. Figure 3 D- Figure 3 E. These results indicate that EPIC-1042 can significantly inhibit autophagic protein degradation without impairing autophagosome formation, and also confirm that the production of exosomes by MVBs is indeed increased. To further verify these effects of EPIC-1042, GBM cells were treated with the autophagy inducer and MTOR inhibitor rapamycin in combination with or alone with EPIC-1042. The results showed that co-treatment induced higher LC3-II levels compared with rapamycin alone, thus ruling out the possibility that EPIC-1042 promotes autophagosome formation. Figure 3 F- Figure 3 G.

[0095] 4. EPIC-1042 sensitizes temozolomide in in vivo experiments with MGMT-deficient GBM.

[0096] like Figure 4 As shown in Figure A, an orthotopic intracranial glioma model was established by orthotopic injection of TBD0220 cells into the hippocampus of nude mice to examine whether EPIC-1042 could increase TMZ sensitivity in vivo. Findings: Compared with DMSO, EPIC-1042 alone slightly inhibited tumor growth, but the effect was not significant. However, bioluminescence analysis showed that the combination of EPIC-1042 and TMZ resulted in the highest reduction in tumor burden among the four treatment groups. Figure 4 B- Figure 4 C. Furthermore, EPIC-1042 monotherapy did not show any overall survival (OS) benefit; however, it enhanced the efficacy of TMZ, such as... Figure 4 D. Moreover, the median survival in the combination therapy group was approximately 1.5 times that of the TMZ monotherapy group, such as... Figure 4 E. H&E staining of brain tumor sections showed that the combination therapy group had enhanced treatment efficacy and the smallest tumor size compared with the single-treatment group. IHC analysis using anti-Ki67 antibody further supported this. Figure 4 F- Figure 4 G. In summary, the results indicate that EPIC-1042 can enhance the in vivo efficacy of TMZ.

[0097] 5. In the early stages of the drug's effect, EPIC-1042 mediates the selective autophagic degradation of PARP1 via p62.

[0098] Based on in vivo experimental results, it is speculated that there may be other potential mechanisms regulating the efficacy of TMZ. EPIC-1042 did not increase O2 in TBD-0220 and U87-MG cells by affecting MGMT. 6 The number of MeGs can be increased by interfering with the base excision repair pathway, thereby increasing N. 3 MeA and N 7 MeG enhances TMZ cytotoxicity, and PARP1 deficiency can impair the base excision repair pathway. Furthermore, EPIC-1042 was found to decrease PARP1 protein levels, such as... Figure 5 However, the PAPR1 mRNA level did not change (not shown in the figure), indicating that EPIC-1042 can induce PAPR1 protein degradation. To verify this, GBM cells were treated with the protein synthesis inhibitor cycloheximide (CHX) or CHX+EPIC-1042 in a time-dependent manner. Immunoblotting results showed that the PAPR1 degradation rate was faster in the combination treatment group than in the single treatment group. Figure 5 B- Figure 5 C. Given that autophagy and the ubiquitin-proteasome system (UPS) are the main proteolytic systems, to differentiate between systems potentially involved in regulating PARP1 protein levels, we treated TBD-0220 and U87-MG cell lines for 16 and 24 hours, respectively, with the lysosomal inhibitor chloroquine, the proteasome inhibitor MG132, CHX, or EPIC-1042. Figure 5As shown in Figure D: When PARP1 is treated with CHX alone, PARP1 is significantly degraded. MG132 can reverse this effect, while CQ cannot. Compared with CHX monotherapy, dual treatment with EPIC-1042 and CHX further enhances PARP1 degradation; however, both MG132 and CQ can reverse this effect. These results indicate that under normal conditions, PARP1 is only degraded by UPS (unreduced paramount) degradation, while EPIC-1042 further induces PARP1 degradation through autophagy, suggesting a novel regulatory role for EPIC-1042 in the net residual amount of PARP1 protein.

[0099] It can be seen that in the early stages of EPIC-1042 treatment (U87-MG 0-24h, TBD-0220 0-16h), EPIC-1024 did not significantly increase LC3-II levels, but it could significantly increase p62 levels, such as... Figure 3 B. Accelerated degradation of PARP1 also occurred during the same time period, such as Figure 5 Furthermore, immunofluorescence showed that autophagy flux remained stable after 16 and 24 hours of EPIC-1042 treatment of TBD-0220 and U87-MG cells, respectively. These results suggest that p62 may mediate the autophagic degradation of PARP1, but the possibility that UPS does not participate in the accelerated degradation of PARP1 cannot be ruled out. To verify the above hypothesis, immunoprecipitation experiments were performed using an anti-PARP1 antibody. Cell pellets were collected 8 and 16 hours after EPIC-1042 treatment of TBD-0220 and U87-MG cells to prevent excessive degradation of PARP1. Figure 5 F- Figure 5 As shown in G: PARP1 ubiquitination levels remained unchanged, but PARP1 binding to p62 was significantly increased, indicating that PARP1 was rapidly loaded into autophagosomes for degradation. To further verify that the decreased stability of PARP1 was due to p62-mediated selective autophagy, p62 was knocked down (KD) in TBD-0220 and U87-MG cells using small interfering RNA and treated with CHX or CHX+EPIC-1042 for 16 and 24 hours, respectively. The results showed that p62 knockdown did not affect PARP1 protein levels compared to the control group, which also means that the previously present p62 does not mediate the autophagic degradation of PARP1. As expected: in the p62 knockdown group, no additional PARP1 degradation was observed compared to CHX monotherapy, such as... Figure 5 H. These results indicate that increased p62 binding following EPIC-1042 treatment is crucial for the selective autophagic degradation of PARP1, thereby enhancing the efficacy of TMZ.

[0100] 6. EPIC-1042 sensitizes temozolomide in MGMT-positive GBM.

[0101] MGMT-positive T98G and LN-18GBM cells were treated with EPIC-1042 or TMZ to assess the effect of EPIC-1042 on their TMZ sensitivity. EPIC-1042 had no effect on MGMT turnover after 24 hours of treatment. Considering that MGMT degradation is normally mediated by UPS, EPIC-1042, whether by increasing p62 or inhibiting autophagy flux, would not affect MGMT stability. However, EPIC-1042 could reduce PARP1 levels in these cell lines. Figure 6 A. Next, intracellular O was removed using immunofluorescence. 6 MeG's ability to assess MGMT activity. For example... Figure 6 As shown in Figure B, TMZ alone induces a slight increase in O6MeG; however, combined treatment significantly enhances O6MeG accumulation. These results indicate that MGMT scavenges O6MeG. 6 The decline in MeG's capabilities is influenced by a decrease in PARP1. For example... Figure 6 C- Figure 6 As shown in Figure D, Western blotting and immunofluorescence confirmed that the trend of intracellular γ-H2AX levels was similar to that of O6MeG. Furthermore, dose-response matrix results also showed that TMZ and EPIC-1042 had a synergistic effect, with combined treatment significantly inhibiting the proliferation of both cell lines. Figure 6 E. Finally, p62 was knocked down using small interfering RNA in the T98 and LN-18 cell lines to block PARP1 degradation. Immunofluorescence showed that in p62-knockdown cells, no further accumulation of O6MeG was observed in the TMZ-EPIC-1042 co-treatment group compared to the TMZ-treated group. Figure 6 F. Considering that MGMT is degraded by UPS and that p62 knockdown has no effect on MGMT levels, EPIC-1042-induced increases in p62 levels may promote increased PARP1 autophagy and degradation, thereby reducing MGMT recruitment and poly(ADP) ribosylation, thus enhancing the efficacy of TMZ.

[0102] 7. In in vivo experiments with MGMT-deficient GBM, EPIC-1042 sensitized temozolomide in a dose-dependent and long-term treatment regimen.

[0103] An orthotopic GBM model was established using the TBD-0220 cell line to further verify the ability of EPIC-1042 to enhance the efficacy of TMZ in a dose-dependent manner. Figure 7 A. Bioluminescence imaging and OS showed that, compared with TMZ alone, EPIC-1042, when used in combination with the same dose of TMZ, exhibited a significant dose-dependent effect on tumor growth inhibition and prolonged survival in nude mice, such as... Figure 7 B- Figure 7 F. Ki67 staining also supports this trend, such as Figure 7 G. Furthermore, immunohistochemical analysis showed that PARP1 levels began to decrease when TMZ was used in combination with the lowest dose of EPIC-1042 compared to TMZ alone, with the most significant decrease observed in the group treated with the highest dose of EPIC-1042 in combination with TMZ. Figure 7 G. However, γ-H2AX showed an opposite trend to PARP1 levels, suggesting that with increased EPIC-1042 dosage, the damage to cells was further enhanced when combined with TMZ, as... Figure 7 G.

[0104] Next, in the GBM nude mouse model, the dosing period was increased to 6 weeks, such as... Figure 7 H. Brain MRI analysis showed that the tumor burden in the TMZ-EPIC-1042 combination therapy group was lower than that in the EPIC-1042 or TMZ monotherapy groups. Figure 7 I- Figure 7 J. Increased median survival was inversely proportional to tumor burden, with the combination therapy group showing the longest OS benefit, such as Figure 7 K. In conclusion, EPIC-1042 can enhance the efficacy of TMZ in a dose-dependent and long-term manner.

[0105] 8. Preclinical data of EPIC-1042

[0106] To supplement the preclinical data of EPIC-1042, the inhibitory effects of EPIC-1042 on various human cytochrome P450 enzymes were first evaluated, with its specific inhibitors serving as positive controls, such as... Figure 8 A. EPIC-1042 ICs for CYP1A2, CYP2C8, CYP2C9, and CYP2E1 50 The IC50 values ​​were significantly higher than those in the positive control group, indicating that EPIC-1042 had a relatively small impact on drug-drug interactions of these isoenzymes. However, for CYP2B6, CYP2C19, and CYP2D6, the IC50 values ​​of EPIC-1042 were significantly higher. 50 The value was lower than that of the positive control group, indicating that EPIC-1042 has a strong inhibitory effect on these isoenzymes.

[0107] The in vitro metabolic stability of EPIC-1042 was determined using liver microsomes in mice, rats, dogs, monkeys, and humans, with clozapine as a positive control. Figure 8 As shown in B: Besides the human and monkey liver microsomal incubation groups, the half-life (t) of EPIC-1042 and clozapine was also observed. 1 / 2 Except for the groups with very similar half-lives (t), the half-lives of the other EPIC-1042 groups are very close. 1 / 2The levels of EPIC-1042 were higher than those of clozapine, indicating that EPIC-1042 is quite stable in liver microsomes. Furthermore, the cardiotoxicity of EPIC-1042 was assessed in HEK293 using a manual patch-clamp method. Figure 8 As shown in C: EPIC-1042 IC 50 The IC50 value was greater than 10 μM, significantly higher than that of the positive control drug cisapride. 50 (8.068 nM). The results indicate that EPIC-1042 exhibits broad-spectrum low cardiotoxicity. Finally, mean plasma concentration-time distribution maps of EPIC-1042 after intravenous administration of 2 mg / kg and oral administration of 45 mg / kg were established in a rat model, as shown below. Figure 8 D.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing the small molecule compound EPIC-1042, characterized in that: Includes the following steps: (1) Compound 1 and Compound 2 were mixed in methanol, and after adding alkali, the mixture was stirred at 90°C. The mixture was then filtered and concentrated under reduced pressure to remove the solvent, yielding Compound 5. (2) Compound 5 and tetrabutylammonium chloride were mixed in phosphorus oxychloride and stirred under sealed conditions at 25℃-170℃. After cooling, the mixture was poured into ice, stirred and neutralized in an alkaline aqueous solution. The resulting crystals were filtered, washed and dried to finally obtain compound 6. (3) Compound 6, compound 7 and K2CO3 were mixed in an organic solvent and stirred at 25℃-90℃. The mixture was filtered and concentrated under reduced pressure to remove the organic solvent. Then it was washed and extracted with ethyl acetate. The organic phase was then concentrated under reduced pressure to remove the organic solvent. The mixture was filtered and concentrated under reduced pressure to remove the solvent. Finally, the crude product was purified by high performance liquid chromatography to obtain compound 8. (4) Mix compound 8 and compound 9, stir the mixture at 25℃-110℃, then filter the mixture and concentrate it under reduced pressure to remove the solvent. Finally, purify the crude product by high performance liquid chromatography to obtain compound 10. (5) In the presence of nitrogen and hydrogen, compound 10, Pd / C and Pd(OH)2 were mixed in methanol and stirred at 25℃-30℃. The mixture was then filtered and concentrated under reduced pressure to remove the solvent, to obtain compound 11. (6) Mix compound 11 and compound 12 in MeOH, then add K2CO3, NaBH3CN and acetaldehyde to the mixture, then stir the mixture at 0-25 °C, and finally filter the mixture and concentrate it under reduced pressure to remove the solvent to obtain compound 13. (7) Compound 13 was mixed with an organic solvent and HCl / Dioxane solution was added to the mixture. The mixture was stirred at room temperature, then filtered and concentrated under reduced pressure to remove the organic solvent. Finally, the mixture was purified by high performance liquid chromatography to obtain the small molecule compound EPIC-1042. The structural formulas of compound 1, compound 2, compound 5-compound 13 and small molecule compound EPIC-1042 are as follows: 、 、 、 、 、 、 、 、 、 、 、 。 2. The use of the small molecule compound EPIC-1042 or its salt as described in claim 1, characterized in that: The intended use is selected from one or more of the following: (1) Use for preparing a tumor treatment drug, wherein the tumor is glioblastoma; (2) Use in preparing interfering agents that interfere with the binding of PTRF to Caveolin1; (3) Use in preparing interfering agents that interfere with Caveolin1 recruitment to PTRF; (4) Use in preparing a reducing agent that decreases the level of sEVs efflux during the proliferation and invasion of glioblastoma; (5) Use in preparing PARP1 degradation promoters during the proliferation and invasion of glioblastoma; (6) Use in the preparation of autophagy flux inhibitors during the proliferation and invasion of glioma blast cells; (7) Used in the preparation of sensitizers to enhance the efficacy of temozolomide.

3. A drug, characterized in that: The drug comprises the small molecule compound EPIC-1042 of claim 1 or a salt thereof, as well as a pharmaceutically acceptable carrier and / or excipients.

4. The drug according to claim 3, characterized in that: The drug also includes temozolomide.

5. The use of the drug according to claim 3 or 4, characterized in that: The intended use is selected from one or more of the following: (1) Use for preparing a tumor treatment drug, wherein the tumor is glioblastoma; (2) Use in preparing interfering agents that interfere with the binding of PTRF to Caveolin1; (3) Use in preparing interfering agents that interfere with Caveolin1 recruitment to PTRF; (4) Use in preparing a reducing agent that decreases the level of sEVs efflux during the proliferation and invasion of glioblastoma; (5) Use in preparing PARP1 degradation promoters during the proliferation and invasion of glioblastoma; (6) Use in preparing an inhibitor of autophagy flux during the proliferation and invasion of glioma blast cells.

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