7586-3507 for use in the preparation of a medicament for the treatment of cancer
The small molecule compound 7586-3507, which targets NAT10, significantly inhibits the invasion and metastasis of esophageal cancer cells, addressing the shortcomings of existing treatment methods and providing a safe and efficient anti-esophageal cancer drug solution.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
- Filing Date
- 2023-02-13
- Publication Date
- 2026-08-04
AI Technical Summary
Existing treatments for esophageal cancer have failed to significantly improve patient prognosis, especially for patients in the middle and late stages. Finding more stable, effective, and safe anticancer drugs has become an urgent need. Existing small molecule targeted drugs, such as tyrosine kinase inhibitors, have limitations in the treatment of esophageal cancer.
The small molecule compound 7586-3507 targets NAT10, and by inhibiting the activity of NAT10, it significantly inhibits the invasion and metastasis of esophageal cancer cells. The concentration range of 7586-3507 is 1.25-5 µM. The drug can be administered orally or by injection, with pharmaceutically acceptable excipients.
7586-3507 significantly inhibited the invasion and metastasis of esophageal cancer cells in in vitro and in vivo experiments, exhibiting time- and concentration-dependent effects. It also demonstrated advantages in safety and price, with no significant toxicity.
Smart Images
Figure CN116173019B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of 7586-3507 in the preparation of antitumor drugs. Background Technology
[0002] Esophageal cancer is a relatively common digestive tract tumor in my country, and one of the most common malignant tumors, with a high incidence and mortality rate and a low overall survival rate. There are two histological subtypes of esophageal cancer: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EA). However, the vast majority of cases in the Asia-Pacific region, including China, are ESCC. Treatment methods for esophageal cancer mainly include surgical resection, chemotherapy, radiotherapy, and molecular targeted therapy. Current treatments have not improved patient prognosis, and for patients with intermediate or advanced stages, drug therapy remains an important approach. Therefore, finding more stable, effective, and safe anticancer drugs has become an urgent priority.
[0003] With the continuous development of molecular biology mechanisms and the ongoing promotion and support of the biotechnology industry, our exploration of tumors is deepening. More and more tumor-specific molecular targets have been identified, leading to the rapid development and expansion of small molecule targeted anti-tumor drugs. Small molecule targeted drugs are becoming a hot topic in tumor treatment research due to their high efficacy, low toxicity and side effects, and high specificity. Currently, the most widely used small molecule targeted anti-tumor drugs in clinical practice are mainly tyrosine kinase inhibitors, including gefitinib, erlotinib, imatinib, sunitinib, lapatinib, and sorafenib.
[0004] N-acetyltransferase 10 (NAT10) is a nuclear protein that acts as an acetyltransferase, catalyzing acetylation modifications of histones and non-histone proteins. NAT10-catalyzed non-histone acetylation modifications play crucial roles in various life processes, including telomerase activity regulation, rRNA synthesis, DNA damage repair, and mRNA stability maintenance. Furthermore, studies have shown that it is closely related to the occurrence, development, and prognosis of cancer.
[0005] 7586-3507 (2-[(4-methylphenoxy)methyl]-5-({2-[(4-methylphenoxy)methyl]-1h-1,3-benzodiazol-5-yl}methyl)-1h-1,3-benzodiazole) is a small molecule compound with the following structural formula:
[0006] . Summary of the Invention
[0007] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of 7586-3507 in the preparation of anti-esophageal cancer drugs.
[0008] The objective of this invention is achieved through the following technical solution:
[0009] The application of 7586-3507 in the preparation of anti-esophageal cancer drugs, wherein the structural formula of 7586-3507 is shown in formula (I):
[0010]
[0011] Equation (Ⅰ).
[0012] Furthermore, the effective concentration of the 7586-3507 is 1.25–5 µM.
[0013] Furthermore, the effective concentration of the 7586-3507 is 2.5–5 µM.
[0014] Furthermore, the aforementioned anti-esophageal cancer drug refers to a drug capable of inhibiting the invasion and / or metastasis of esophageal cancer cells. 7586-3507 significantly inhibits the invasion and / or metastasis of esophageal cancer cells in a concentration- and time-dependent manner.
[0015] Furthermore, the aforementioned anti-esophageal cancer drug is a drug for treating and / or preventing esophageal cancer.
[0016] Furthermore, 7586-3507 inhibits esophageal cancer invasion and / or metastasis by targeting NAT10. The nucleotide sequence encoding NAT10 is shown in the gene sequence accession number 55226 in the NCBI Genebank database.
[0017] Furthermore, the esophageal cancer mentioned is esophageal squamous cell carcinoma.
[0018] An anti-esophageal cancer drug, comprising 7586-3507, wherein the structural formula of 7586-3507 is shown in formula (I):
[0019]
[0020] Equation (Ⅰ).
[0021] Furthermore, the aforementioned antiesophageal cancer drug also contains pharmaceutically acceptable excipients.
[0022] Furthermore, the pharmaceutically acceptable excipient is preferably at least one of sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorbents, surfactants, and lubricants.
[0023] Furthermore, the preferred administration methods of the anti-esophageal cancer drug include, but are not limited to, at least one of oral administration, gavage administration, and injection administration.
[0024] The present invention has the following advantages and effects compared with the prior art:
[0025] This invention has demonstrated through experiments that the small molecule compound 7586-3507 can effectively inhibit the invasion and metastasis of esophageal cancer. The small molecule compound 7586-3507 of this invention significantly inhibits the invasion and metastasis of esophageal cancer cells in a concentration range of 1.25–5 µM in a concentration- and time-dependent manner. As a potential anti-tumor drug, the small molecule compound 7586-3507 of this invention possesses certain safety profiles and offers significant advantages in terms of efficacy and price. Attached Figure Description
[0026] Figure 1 The structural diagram is for 7586-3507.
[0027] Figure 2 The image shows the results of surface plasmon resonance analysis of proteins 7586-3507 and NAT10.
[0028] Figure 3 The figure shows the effect of different concentrations of 7586-3507 on the invasive ability of esophageal cancer cells.
[0029] Figure 4 The image shows the fluorescence detection results of different concentrations of 7586-3507 on the lung metastasis ability of esophageal squamous cell carcinoma cells.
[0030] Figure 5 Figure showing the weight changes of nude mice treated with different concentrations of 7586-3507.
[0031] Figure 6 The morphological changes of the lungs, liver, kidneys, and spleen in nude mice treated with different concentrations of 7586-3507 are shown in the figure. Detailed Implementation
[0032] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0033] Example 1: In vitro test
[0034] (1) Experimental materials
[0035] Human esophageal squamous cell carcinoma cells KYSE150 and EC9706 were purchased from DSMZ; cell line 7586-3507 was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., with the following structural formula: Figure 1As shown; female mice (NOD-SCID) were purchased from Beijing Edmo Biotechnology Co., Ltd.; RPMI-1640 medium was purchased from Gibco, USA; DMSO was purchased from Sigma, USA; 7586-3507 was dissolved in DMSO and stored at a concentration of 10 mM at -80°C.
[0036] (2) Cell invasion experiment:
[0037] Esophageal squamous cell carcinoma cells were treated with different concentrations of 7586-3507, and then the invasive ability of esophageal squamous cell carcinoma cells was tested to study the effect of different concentrations of 7586-3507 on the invasive ability of esophageal squamous cell carcinoma cells.
[0038] 1) Chamber preparation: Add 100 μL of 5% matrix gel to the clean invasion chamber and place it in an incubator at 37°C for 30 minutes.
[0039] 2) Cell seeding: Human esophageal squamous cell carcinoma highly metastatic cell lines KYSE150-Luc-LM5 and EC9706-Luc-LM3 (PMID: 35507004) were digested into suspension single cells with 0.25% trypsin. After counting, 500,000 KYSE150-Luc-LM5 cells and 200,000 EC9706-Luc-LM3 cells were added to the upper layer of each chamber, and different concentrations of 7586-3507 (final concentrations of 0, 1.25 μM, 2.5 μM, and 5 μM) were added. 500 μL of medium containing 20% FBS was added to the lower layer, and the cells were cultured at 37°C and 5% CO2 for 24 h.
[0040] 3) Crystal violet staining: Take out the chamber, wash twice with PBS buffer (pH 7.4, purchased from Sigma-Aldrich; the same below), add 500 μL of methanol to the lower chamber, fix for 10 min, remove the methanol, add 0.2% crystal violet for staining for 10 min, remove the crystal violet, wash away the residual crystal violet with water, air dry and take pictures.
[0041] Depend on Figure 3 In vitro experiments showed that the invasive ability of esophageal squamous cell carcinoma cells decreased with increasing concentration of 7586-3507.
[0042] (3) SPR (Synthetic Profiling) system for analyzing biomolecular interactions:
[0043] 1) Expression and purification of NAT10 protein
[0044] The NAT10 gene was constructed in the pET-28b prokaryotic expression vector with a His tag, resulting in the NAT10 prokaryotic expression plasmid (commissioned by Shanghai Quanyang Biotechnology Co., Ltd.). The constructed plasmid was transformed into BL21 competent cells. After plating, the expressed bacterial cells were picked and placed in 10 mL of LB medium. The culture was incubated overnight at 37°C and 250 rpm to obtain the bacterial suspension. 1% of the above bacterial suspension was transferred to 1 LLB medium and incubated at 37°C and 250 rpm for 3 hours. When the OD value reached 0.6-0.8, IPTG (working concentration 0.5 mM) was added to induce expression for 4 hours. 1 L of NAT10-expressing bacterial cells was collected by centrifugation, resuspended in 100 mL of buffer, and sonicated on ice (300W, 10 seconds, 10-second intervals, 30 times). After centrifugation, the supernatant was collected and loaded onto a pre-equilibrated NTA purification column. After loading, the column was washed with buffer. The column was eluted with 10 mM, 20 mM, 50 mM, 200 mM, and 500 mM imidazole buffers, and the eluent was collected. Western blot analysis was performed. Blot analysis was performed on each eluent group, and the fractions containing the target protein were finally mixed together and concentrated by ultrafiltration. The protein solution was stored at -80°C for later use.
[0045] 2) Surface Plasmon Resonance (SPR) Experiment
[0046] Select a new CM7 chip. After the chip locks in, replace the PBS buffer with 0.4% (v / v) P20 (pH 7.4, purchased from Sigma-Aldrich) to equilibrate the chip surface. Dilute NAT10 in 3 M sodium acetate buffer (purchased from GE Healthcare Life Sciences) at different pH values (pH 4, 4.5, 5, 5.5) and flow it over a blank chip surface to test the electrostatic adsorption effect. Couple the chip to a suitable pH environment, and manually experiment with the coupling amount and time to achieve a good result. Dilute 7586-3507 to multiple concentrations using electrophoresis buffer (the resulting concentrations of 7586-3507 were 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM). (nM), but ensure that the content is consistent with the DMSO content in the electrophoresis buffer; manually load the sample and test the response values of different concentrations of 7586-3507; select an appropriate concentration of 7586-3507 based on the response values for affinity determination; finally, analyze using Biacore software.
[0047] Figure 2 Surface plasmon resonance results showed that 7586-3507 can bind to the NAT10 protein.
[0048] Example 2: In vivo experiment
[0049] A tumor metastasis model was constructed by selecting 27 six-week-old female NCG mice, with 9 mice in the control group and 18 mice in the experimental group.
[0050] 1) Human esophageal squamous cell carcinoma highly metastatic cell line KYSE150-Luc-LM5 cells were digested into single cells using 0.25% trypsin. 10 6 One KYSE150-Luc-LM5 cell was resuspended in 100µL PBS buffer;
[0051] 2) Before performing the tail vein experiment on the animals, the mice were anesthetized, and the degree of anesthesia was assessed by painless and painful stimuli to determine whether the mice were in anesthetized state.
[0052] 3) The cells were resuspended in a microinjector with a 25G needle and injected into the tail vein of 27 mice.
[0053] 4) 7586-3507 Treatment: One week after tail vein injection of esophageal squamous cell carcinoma cells KYSE150-Luc-LM5, mice were administered the drug via gavage. 7586-3507 was dissolved in PBS buffer and administered to each group of mice at concentrations of 0 mg / kg (control group), 2.5 mg / kg (experimental group 1), and 5 mg / kg (experimental group 2), nine mice in each group, once a week. D-Luciferin (purchased from GOLDBIO) was injected intraperitoneally weekly for three consecutive weeks. In vivo fluorescence imaging was performed to observe cancer cell metastasis, and the weight of the nude mice was measured simultaneously.
[0054] The results are as follows Figure 4 and Figure 5 As shown, the biofluorescence in the lungs of mice treated with 7586-3507 was reduced. Biofluorescence is induced by an enzymatic reaction between luciferase in KYSE150-Luc-LM5 esophageal squamous cell carcinoma cells and D-luciferin in body fluids; the intensity of the fluorescence reflects the density of KYSE150-Luc-LM5 cells. Therefore, in the 7586-3507 treatment group (i.e., the experimental group), the number of esophageal cancer cells metastasizing to the lungs was significantly lower than in the control group, and the drug's inhibition of tumor cell metastasis was concentration-dependent. Figure 5 The lack of significant difference in mouse body weight indicates that compound 7586-3507 has no obvious toxicity.
[0055] Immunohistochemistry was performed on liver, lung, kidney, and spleen samples from mice treated with 7586-3507 (5 mg / kg). The results showed (e.g.) Figure 6 7586-3507 had no effect on liver, lung and kidney function in mice.
[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. Use of 1.7586-3507 for the preparation of a medicament for the treatment of esophageal squamous cell carcinoma, characterized in that: The structural formula of 7586-3507 is shown in formula (Ⅰ): Equation (Ⅰ).
2. The application of 7586-3507 according to claim 1 in the preparation of drugs for treating esophageal squamous cell carcinoma, characterized in that: The effective concentration of 7586-3507 is 1.25–5 µM.
3. The application of 7586-3507 according to claim 1 in the preparation of drugs for treating esophageal squamous cell carcinoma, characterized in that: The effective concentration of 7586-3507 is 2.5–5 µM.
4. The use of 7586-3507 according to any one of claims 1-3 in the preparation of drugs for treating esophageal squamous cell carcinoma, characterized in that: The aforementioned anti-esophageal squamous cell carcinoma drugs refer to drugs that can inhibit the invasion and / or metastasis of esophageal squamous cell carcinoma cells.
5. The use of 7586-3507 according to any one of claims 1-3 in the preparation of drugs for treating esophageal squamous cell carcinoma, characterized in that: The aforementioned anti-esophageal squamous cell carcinoma drug is a drug for the treatment and / or prevention of esophageal squamous cell carcinoma.
6. The use of 7586-3507 according to any one of claims 1-3 in the preparation of drugs for treating esophageal squamous cell carcinoma, characterized in that: The 7586-3507 inhibits the invasion and / or metastasis of esophageal squamous cell carcinoma by targeting NAT10.
7. The use of 7586-3507 according to any one of claims 1-3 in the preparation of drugs for treating esophageal squamous cell carcinoma, characterized in that: The aforementioned anti-esophageal squamous cell carcinoma drug also contains pharmaceutically acceptable excipients; The pharmaceutically acceptable excipients are at least one of the following: sustained-release agents, fillers, binders, humectants, disintegrants, absorption promoters, adsorbents, surfactants, and lubricants.
8. The use of 7586-3507 according to any one of claims 1-3 in the preparation of drugs for treating esophageal squamous cell carcinoma, characterized in that: The administration method of the anti-esophageal squamous cell carcinoma drug is at least one of oral administration, gavage administration, and injection administration.