Use of 1,4-dihydro-naphthyridine derivatives in the treatment of tumors

Abstract

The present invention relates to the application of 1,4-dihydro-naphthyridine derivatives in the treatment of tumors.

Description

[Technical Field]

[0001] cross reference This application claims priority to a Chinese patent application filed with the China Patent Office on December 24, 2021, with application number 202111599362.3 and titled "Use of 1,4-dihydro-naphthyridine derivatives in treating tumors," the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a novel use of 1,4-dihydronaphthyridine derivatives in the pharmaceutical field, more specifically, to the application of 1,4-dihydronaphthyridine derivatives in the preparation of antitumor drugs. The tumors are solid tumors, including nervous system tumors and non-nervous system tumors. Nervous system tumors include glioblastoma and neuroblastoma. Non-nervous system tumors include liver cancer, colon cancer, and gastric cancer. [Background technology]

[0003] Glioblastoma (GBM) is the most common malignant primary intracranial tumor, accounting for approximately 57% of gliomas and 48% of primary central nervous system (CNS) malignancies (Neuro-oncology, 2018.20: pp.iv1-iv86). Mortality from glioblastoma is extremely high. In the United States, between 2000 and 2014, the 1-year survival rate for glioblastoma patients was 41.4%, and the 5-year survival rate was only 5.8% (CA: A Cancer Journal for Clinicians, 2020.70(4): pp.299-312). The etiology of glioblastoma remains largely unknown, with ionizing radiation being the only known possible cause. This poses certain challenges to its prevention and treatment.

[0004] Gliomas are classified according to their malignancy, ranging from grade I to IV. Grades I and II are less malignant, while grades III and IV are more malignant. Treatments for glioblastoma mainly include surgical resection, radiation therapy, and systemic therapy (chemotherapy and targeted therapy). Postoperative temozolomide radiation therapy combined with concurrent adjuvant chemotherapy is the standard treatment plan for newly diagnosed glioblastoma. Current major chemotherapeutic agents include temozolomide, nitrosoureas, procarbazine, platinum analogs, vinblastines, and camptothecins. However, the antitumor mechanisms of action of conventional chemotherapeutic agents are insufficient to meet current clinical needs.

[0005] In eukaryotic cells, macropinocytosis is an important pathway for the internalization of extracellular materials and the dissolution of molecules. The process of macropinocytosis has a dual effect on tumor cells. When nutrient deprivation occurs, tumor cells internalize extracellular nutrients through the macropinocytotic pathway, promoting tumor cell proliferation. However, excessive activation of the macropinocytotic process by drug treatment or abnormal expression of RAS genes can induce a novel death mode called methuosis. Methuosis is a novel protease-independent cell death mode characterized by the cytoplasm being filled with numerous vacuoles derived from macropinocytosomes. This death mode was first discovered in glioblastoma cells (Oncogene, 1999.18(13):pp.2281-90). Further research has revealed that this death mode is caused by clathrin-independent changes in macropinocytosomes, ultimately resulting in the formation of numerous vacuoles, leading to cell rupture and death (Journal of Medicinal Chemistry, 2012, 55(5):pp.1940-56). Current research has found that various molecules, including Ras, Rac1, Arf6, and Rab7, are involved in macropinocytosome formation (The American Journal of Pathology, 2014, 184(6):pp.1630-42). Rac1 may influence macropinocytosome formation by regulating actin assembly. Furthermore, Rac1 activation may reduce Arf6 activated via GIT-1, further disrupting macropinocytosome circulation (Molecular Cancer Research: MCR, 2010, 8(10):pp.1358-74).In recent years, several small molecule compounds, including methamphetamine, indolyl chalcone, vaquinol-1, MOMIPP, and CX-5011, have been found to induce cellular methesis by activating mechanisms such as MKK4, JNK, and Rac1 (Oncotarget 2016, 7, 55863-55889; BMC Cancer 2019, 19, 77; BBA-Molecular Cell Research 2020, 118807).

[0006] 1,4-Dihydronaphthyridine derivatives are compounds that can not only inhibit acetylcholinesterase activity but also block the influx of extracellular calcium ions into cells through calcium channels. By inhibiting cholinesterase activity, 1,4-dihydronaphthyridine derivatives slow the hydrolysis rate of acetylcholine, increasing acetylcholine levels in the synaptic cleft, and thereby treating Alzheimer's disease and vascular dementia (Patent No. CN104203945B, Patent Publication No. CN106632317A). Furthermore, by inhibiting calcium ion influx, they improve neuronal resistance to ischemia, dilate cerebral blood vessels, improve cerebral blood supply, and protect neurons. They are also effective in improving cognitive function in patients with vascular dementia. Summary of the Invention

[0007] 1,4-Dihydronaphthyridine derivatives are compounds that can not only inhibit acetylcholinesterase activity but also block the influx of extracellular calcium ions into cells through calcium channels, and can be used to treat Alzheimer's disease and vascular dementia. These compounds exert their antitumor effects by inducing tumor cell methesis through a novel mechanism of action that is independent of cholinesterase inhibition or calcium channel blockade.

[0008] An object of the present invention is to provide a 1,4-dihydro-naphthyridine derivative or a pharmaceutically acceptable salt thereof that can treat tumors through a new mechanism and effectively prolong the survival time of patients.

[0009] Specifically, the present invention provides the use of a 1,4-dihydro-naphthyridine derivative of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors. [ka] where R1 or R2 is hydrogen, halogen, or a C1-C6 alkyl group; R3 is a C1-C6 alkyl group in which any —CH2— may be substituted with one or more —O—; R4 is hydrogen or halogen.

[0010] Specifically, the derivative is selected from the following compounds 1 to 6. Compound 1, designated S1: [ka] Compound 2, designated S2: [ka] Compound 3, designated S3: [ka] Compound 4, designated S4: [ka] Compound 5, designated S5: [ka] Compound 6, designated S6: [ka] .

[0011] The above drugs can induce methesis in tumor cells, causing them to exhibit a vacuolated cell phenotype, and inhibit tumor cell proliferation to inhibit tumor growth, thereby ultimately achieving the goal of treating tumors.

[0012] Therefore, the present invention provides a novel use of a 1,4-dihydro-naphthyridine derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition thereof in the manufacture of an antitumor drug.

[0013] The tumors are solid tumors, including nervous system tumors and non-nervous system tumors. Nervous system tumors include glioblastoma and neuroblastoma. Non-nervous system tumors include liver cancer, colon cancer, and stomach cancer.

[0014] Specifically, the present invention provides the use of 1,4-dihydro-naphthyridine derivatives in the treatment of solid tumors.

[0015] The present invention provides the use of 1,4-dihydro-naphthyridine derivatives in the treatment of nervous system tumors.

[0016] The present invention provides the use of 1,4-dihydro-naphthyridine derivatives in the treatment of brain tumors.

[0017] The present invention also provides the use of 1,4-dihydro-naphthyridine derivatives in the treatment of glioma.

[0018] The present invention also provides the use of 1,4-dihydro-naphthyridine derivatives in the treatment of glioblastoma.

[0019] The present invention also provides the use of 1,4-dihydro-naphthyridine derivatives in the treatment of liver cancer, colon cancer, and stomach cancer.

[0020] The present invention also provides the use of a combination of a 1,4-dihydro-naphthyridine derivative and temozolomide in the treatment of glioma.

[0021] The present invention also provides the use of a 1,4-dihydro-naphthyridine derivative in combination with radiation therapy in the treatment of glioma.

[0022] Advantages and positive effects of the present invention: The 1,4-dihydronaphthyridine derivatives provided by the present invention as novel drugs for tumor treatment specifically induce methesis in tumor cells, have minimal effect on normal cells, and are advantageous for the development of safe and effective drugs for tumor treatment. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the vacuolation phenotype of T98 cells induced by 1,4-dihydro-naphthyridine derivatives. [Figure 2] FIG. 2 shows the vacuolation of various tumor cells induced by S2. [Figure 3] FIG. 3 shows that calcium channel inhibitors did not induce vacuolation of tumor cells. [Figure 4] FIG. 4 shows that cholinesterase inhibitors did not induce vacuolation of tumor cells. [Figure 5] FIG. 5 shows that the combined use of calcium channel blockers and cholinesterase inhibitors did not induce vacuolation of tumor cells. [Figure 6] FIG. 6 shows the effect of 2 weeks of S2 administration on the growth curve of U87MG glioma in the brain of nude mice. [Figure 7] FIG. 7 shows that S2 administration for 2 weeks inhibited the growth of U87MG glioma in the brains of nude mice. [Figure 8] FIG. 8 is a graph showing that S2 extended the survival time of nude mice orthotopically implanted with glioma in the brain. [Figure 9]FIG. 9 shows the effect of combined administration of S2 and TMZ for two weeks on the growth curve of U87MG glioma in the brain of nude mice. [Figure 10] FIG. 10 shows the effect of combined S2 and TMZ administration for two weeks on glioma growth in the brain. [Figure 11] FIG. 11 is a graph showing that combined administration of S2 and TMZ extended survival of mice bearing U87MG brain intraepithelial carcinoma. [Figure 12] FIG. 12 shows that TMZ was unable to inhibit the growth of T98 glioma and was unable to reduce the volume and weight of solid tumors. [Figure 13] FIG. 13 shows that S2 inhibited the growth of T98 glioma and reduced the volume and weight of solid tumors. DETAILED DESCRIPTION OF THE INVENTION

[0024] It should be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the present invention.

[0025] Example 1 Study on the vacuolation-inducing and proliferation-inhibiting effects of 1,4-dihydro-naphthyridine derivatives on glioblastoma cells 1. Materials and Methods 1.1 Cells Human glioma cell line T98G cells were purchased from Nanjing Cobior Biosciences Co., Ltd.

[0026] 1.2 Numbers and structures of 1,4-dihydronaphthyridine derivatives [Table 1]

[0027] 1.3 Preparation of compound solutions A fixed amount of the test compound was weighed and dissolved in DMSO to prepare a homogeneous solution with a final concentration of 100 mM.

[0028] 1.4 Reagents and Consumables [Table 2]

[0029] 1.5 Cell culture and observation of cell phenotype T98G cells were stably passaged twice, and when the cell density reached 85% or higher, they were digested with 0.05% trypsin-EDTA for 3 minutes. The cells were resuspended in medium and seeded at a constant density in 12-well plates, 1 mL per well. After 24 hours of culture, the compound solutions prepared above were added to each well depending on the group, so that the compound concentrations in the culture system were 5 μM, 10 μM, 20 μM, 40 μM, or 60 μM, respectively. An equal volume of DMSO was added as a control well.

[0030] After 6 hours of treatment with the drug, cells were observed under a 20x objective lens. Five randomly selected fields from each well were photographed and recorded. Cells were considered to be methesis-positive if they contained bright vacuoles with clear boundaries and met one of two criteria: 1) at least one vacuole with a diameter greater than 3 μM, or 2) three or more vacuoles with a diameter between 0.5 and 3 μM.

[0031] Vacuolization rate R(%)=CELL 陽性 / (CELL 正常 +CELL 陽性 )×100% Here, CELL 陽性 represents the number of methusis-positive cells, and CELL 正常 represents the number of cells with normal morphology.

[0032] 1.6 Detection of cell growth inhibition T98G cells were stably passaged twice, and when the cell density reached 85% or more, they were digested with 0.05% trypsin-EDTA for 3 minutes, resuspended, and seeded into a 96-well plate at a predetermined density. After 24 hours of culture, the cells were treated with drugs for 72 hours so that the final concentrations in the culture system were 0.41 μM, 1.23 μM, 3.7 μM, 11.1 μM, 33.3 μM, and 100 μM. After 72 hours of treatment, the cells were analyzed using CellCounting-Lite. TM Cell viability was detected using the CellCounting-Lite 2.0 kit. TM 100 μL / well of the reagent was added according to the instructions of the 2.0 kit, and the plate was shaken for 10 minutes. The chemiluminescence value (LUM) was read using a multi-function plate reader, and the relative viability of the cells was calculated.

[0033] Relative survival rate of tumor cells V (%) = (LUM - LUM バックグラウンド ) / (LUM 正常対照群 -LUM バックグラウンド群 )×100% However, LUM バックグラウンド is the background reading with detection reagent added to the complete medium wells. The half maximal inhibitory concentration (IC) was calculated using GraphPad Prism 8.0.1 statistical software. 50 ) was estimated.

[0034] 2. Experimental Results 2.1 Vacuolation of T98G cells induced by 1,4-dihydronaphthyridine derivatives S1, S2 (including (+)-S2 and (-)-S2), S3, S4, S5, and S6 were able to induce vacuolation in T98 (Fig. 1 and Table 1). Among these compounds, S1 had the weakest vacuolation-inducing activity, and S2 had the strongest.

[0035] [Table 3] In the table, data are expressed as mean values ± SD, and "-" indicates that the compound precipitated and therefore the data was not calculated.

[0036] 2.2 Inhibition of T98G cell proliferation by 1,4-dihydronaphthyridine derivatives The tumor growth inhibitory activity IC shown in Table 2 50 It was found that 1,4-dihydro-naphthyridine derivatives could inhibit the proliferation of neural tumor cells T98G. Among these compounds, S1 had weaker growth inhibitory activity, which was almost consistent with its vacuolization-inducing activity.

[0037] [Table 4]

[0038] Example 2 Study on the inhibitory activity of S2 against tumor cell proliferation in vitro 1. Materials and Methods 1.1 Cells Human glioma cell line U87MG, human glioma cell line T98G, and human glioma cell line U251 were purchased from Nanjing Kebai Biotechnology Co., Ltd. Human glioma cell line A172, human neuroblastoma cell line SK-N-SH, human liver cancer cell line HepG2, mouse colon cancer cell line CT26.WT, and hamster ovary cell subline CHO-K1 were purchased from Wuhan Pricella Biotechnology Co., Ltd.

[0039] 1.2 Reagents and Consumables [Table 5]

[0040] 1.3 Preparation of Dosing Solutions The S2 powder was precisely weighed and dissolved in DMSO to prepare a homogeneous solution with a concentration of 100 mM as a mother liquor.

[0041] 1.4 Cell culture and observation of cell phenotype U87MG, T98G, U251, SK-N-SH, A-172, and HepG2 cells were cultured in MEM + 10% FBS + 1% P / S medium as adherents and passaged at a 1:3 ratio. CT26.WT cells were cultured in RPMI-1640 + 10% FBS + 1% P / S medium as adherents and passaged at a 1:3 ratio. CHO-K1 cells were cultured in Ham's F-12K + 10% FBS + 1% P / S medium as adherents and passaged at a 1:3 ratio.

[0042] The cells used above were resuscitated and stably passaged twice. When the cell density reached 85% or higher, they were digested with 0.05% trypsin-EDTA for 3 minutes, resuspended in medium, and seeded at a constant density in 6-well plates, 2 mL per well. Cultured for 24 hours. Depending on the grouping, 2 μL of the S2 solution prepared above (final DMSO concentration 0.1%) was added to each well to ensure an S2 concentration of 30 μM in the cell culture system.

[0043] After six hours of treatment with the drug, cells were observed under a 20x objective lens. Five randomly selected fields of view for each well were photographed and recorded. Cells were considered to be methesis-positive if they met one of two criteria: 1) at least one vacuole with a diameter greater than 3 μM, or 2) three or more vacuoles with diameters between 0.5 and 3 μM (Journal of Medicinal Chemistry 2018 61(12), 5424-5434).

[0044] Vacuolization rate R(%)=CELL 陽性 / (CELL 正常 +CELL 陽性 )×100% Here, CELL 陽性 represents the number of methusis-positive cells, and CELL 正常 represents the number of cells with normal morphology.

[0045] 1.5 Cell viability detection Each of the above cell types was resuscitated and stably passaged twice. When the cell density reached 85% or more, the cells were digested with 0.05% trypsin-EDTA for 3 minutes, resuspended, and seeded into a 96-well plate at the designated density. After 24 hours of culture, the cells were treated with drugs for 48 hours to achieve final concentrations of 0.41 μM, 1.23 μM, 3.7 μM, 11.1 μM, 33.3 μM, and 100 μM in the culture system. After 48 hours of drug addition, the cells were analyzed by Cell Counting-Lite. TM Cell viability was detected using the Cell Counting-Lite 2.0 kit. TM 2.0 According to the kit's instructions, 100 μL / well of the reagent was added, the plate was shaken for 10 minutes, and the chemiluminescence value (LUM) was read using a multi-function plate reader to calculate the relative viability of the cells.

[0046] Relative survival rate of tumor cells V (%) = (LUM - LUM バックグラウンド ) / (LUM 正常対照群 -LUM バックグラウンド群 )×100% However, LUM バックグラウンド was the background reading when this detection reagent was added to the complete medium wells. The half maximal inhibitory concentration (IC) was calculated using GraphPad Prism 8.0.1 statistical software. 50 ) was estimated.

[0047] 2. Experimental Results 2.1 S2-induced vacuolation of tumor cells S2 induced vacuolation in neural tumor cells, including human glioma cell lines U87MG, T98G, U251, and A172, and human neuroblastoma cell line SK-N-SH (Fig. 2, A–D), as well as in non-neuronal tumor cells, including human liver cancer cell line HepG2 and mouse colon cancer cell line CT26.WT (Fig. 2, A–D). However, S2 did not induce vacuolation in non-tumor cell line hamster ovary cell line CHO-K1.

[0048] 2.2 S2 inhibited tumor cell proliferation S2 was able to inhibit the proliferation of neural tumor cells and non-neural tumor lines (IC 50ranged from 4.7 to 14.3 μM), but had a very weak inhibitory effect on the proliferation of non-tumor cell lines (IC 50 >100 μM) (see Table 3).

[0049] [Table 6]

[0050] Example 3 Effect of AChE inhibitors and VGCC blockers on T98 cell vacuolation phenotype 1. Materials and Methods 1.1 Cells Human glioma cell line T98G was purchased from COBIOER BIOSCIENCES CO., LTD.

[0051] 1.2 Reagents and Consumables [Table 7]

[0052] 1.3 Preparation of Dosing Solutions The drugs tested included nifedipine, verapamil, diltiazem, flunarizine and donepezil.

[0053] The above test drugs were precisely weighed and dissolved in DMSO to prepare homogeneous solutions with a concentration of 20 mM as mother solutions.

[0054] 1.4 Cell culture and observation of cell phenotype T98G cells were stably passaged twice, and when the cell density reached 85% or higher, they were digested with 0.05% trypsin-EDTA for 3 minutes. The cells were resuspended in medium and seeded at a constant density in 12-well plates, 1 mL per well. After 24 hours of culture, the compound solution prepared above was added to each well depending on the group, so that the compound concentrations in the culture system were 0.1 μM, 1 μM, 5 μM, or 20 μM, respectively. An equal volume of DMSO was added as a control well.

[0055] After 6 hours of treatment with the drug, cells were observed under a 20x objective lens. Cells were considered to be methesis-positive if they contained bright vacuoles with clear boundaries and met either of the following two criteria: 1) at least one vacuole with a diameter exceeding 3 μM, or 2) three or more vacuoles with a diameter between 0.5 and 3 μM.

[0056] 2. Experimental Results 2.1 Calcium channel blockers (VGCCs) were unable to induce vacuolation of tumor cells The calcium channel inhibitors nifedipine, verapamil, diltiazem, and flunarizine (maximum drug concentration 20 μM) were unable to induce a vacuolated cell phenotype in the human glioma cell line T98G (Fig. 3 ).

[0057] 2.2 Cholinesterase (AChE) inhibitors were unable to induce vacuolation of tumor cells. The cholinesterase inhibitor donepezil (maximum drug concentration 20 μM) failed to induce a vacuolated cell phenotype in the human glioma cell line T98G (Fig. 4 ).

[0058] 2.3 The combined use of calcium channel blockers and cholinesterase inhibitors failed to induce vacuolation of tumor cells. The calcium channel blockers nifedipine or verapamil (20 μM) in combination with the cholinesterase inhibitor donepezil (20 μM) failed to induce a vacuolated cell phenotype in the human glioma cell line T98G (Fig. 5 ).

[0059] Example 4: Study of the efficacy of S2 on a tumor model in which U87MG-Luc glioblastoma cells were orthotopically transplanted into nude mice 1. Materials and Methods 1.1 Experimental animals ALB / c Nude nude mice, male, SPF grade, weighing 20–22 g, purchased from LINGCHANG BIOTECHB.

[0060] 1.2 Reagents and Consumables The main reagent information in this experiment is as follows: [Table 8]

[0061] 1.3 Experimental equipment The main equipment information for this experiment is as follows: [Table 9]

[0062] 1.4 Preparation of dosage forms For S2, 5% ethanol + 0.8% solutol + 5% glucose was used as the solvent. Preparation method: Take an appropriate amount of S2, add anhydrous ethanol in the specified proportion and dissolve by ultrasonication, add Solutol HS-15 in the specified proportion and mix by ultrasonication (make sure there is no trace), slowly add purified water under ultrasonication to dissolve, then add glucose in the specified proportion and dissolve by ultrasonication to obtain a solution, which is then left to stand at 4°C and stored away from light.

[0063] 1.5 Experimental Method 1.5.1 Preparation of a tumor model by orthotopically transplanting U87MG-Luc glioblastoma cells into nude mice Before inoculation, U87MG-Luc cells were digested with 0.05% trypsin-EDTA and diluted to 4 × 10 in pre-chilled PBS. 7The cells were resuspended at 2 × 10 cells / mL and placed on ice. Nude mice were anesthetized with isoflurane gas, then fixed prone in a stereotaxic apparatus. The scalp of the nude mice was disinfected with iodophor. A sagittal incision was made with a scalpel, the incision was cleaned with iodophor, and the skull was exposed. A hole was drilled using a skull drill, extending 1.0 mm in front of the bregma and 2.0 mm to the right. 5 μL of the cell suspension (2 × 10 cells / mL) was then injected into the mouse using a 10 μL flat-head microsyringe. 5 The cells were manually injected. The needle was inserted to a depth of 3.5 mm, withdrawn 0.5 mm, and injected for approximately 10 minutes. After holding the needle in place for 5 minutes, the needle was slowly withdrawn, sterilized, and the incision was sutured. Finally, 50,000 units of penicillin were injected intramuscularly to prevent infection.

[0064] 1.5.2 Animal Grouping and Dosing In this experiment, the animals were divided into five groups: a model group, a TMZ group, an S2-treated group (LD) (0.25 mg / kg), an S2-treated group (MD) (0.5 mg / kg), and an S2-treated group (HD) (1 mg / kg). The modeled animals were divided into groups with equal probability and in a single-blind manner. Starting from the fifth day after inoculation, the drugs were administered intravenously once daily for 15 days. The vehicle was administered intravenously once daily for 15 days to the model group.

[0065] 1.5.3 In vivo imaging of tumor cells in the brain On days 4, 12, 18, and 25 after inoculation, 200 μL of 15 mg / mL D-fluorescein potassium salt solution (filtered and sterilized with a 0.22 μM filter) was intraperitoneally injected. 10 minutes later, IVI chemiluminescence detection (bright field + bioluminescence imaging) was performed using a mouse in vivo imaging system (PE IVIS Lumina XR). Fluorescence intensity analysis was performed using Living Image software, and the fluorescence intensity [p / s] was obtained. Fluorescence intensity data reflects the size of tumor tissue in the brain of the experimental animals.

[0066] 1.5.4 Animal survival records The survival status of the experimental animals was detected, and the death dates of the animals were recorded in a timely manner.

[0067] 1.6 Statistical analysis of data Quantitative data are expressed as mean ± standard error. For each pharmacodynamic index, graphs were created using GraphPad Prism (8.0.1) software. One-way or two-way repeat ANOVA was performed to analyze intergroup differences using Fisher's LSD test, and survival analysis was performed using the Kaplan-Meier method. P < 0.05 was defined as significant.

[0068] 2. Experimental Results 2.1 S2 inhibited the growth of U87MG glioma cells orthotopically transplanted into the brains of nude mice Both S2-MD (0.5 mg / kg, IV) and S2-HD (1 mg / kg, IV) significantly inhibited the growth of U87MG intraepithelial tumors in the brain compared with the vehicle group (Figure 6A and B). After 2 weeks of administration, S2-MD (0.5 mg / kg, IV) and S2-HD (1 mg / kg, IV) showed significant tumor growth inhibition compared with the vehicle group. Among them, S2-HD (1 mg / kg, IV) showed the strongest tumor inhibition effect (Figure 7A and B).

[0069] 2.2 S2 extended the survival time of nude mice orthotopically transplanted with glioma Survival curve analysis using the log-rank test showed that the S2-MD (0.5 mg / kg, IV) and S2-HD (1 mg / kg, IV) groups significantly prolonged the survival rate of U87MG brain intraepithelial carcinoma mice compared with the vehicle group (Figure 8), as well as the median and overall survival times (Table 4).

[0070] [Table 10]

[0071] Example 5: Efficacy study of the combination of S2 and temozolomide (TMZ) on a tumor model in which U87MG-Luc glioblastoma cells were orthotopically transplanted into nude mice 1. Materials and Methods 1.1 Experimental animals The results were the same as in Example 4.

[0072] 1.2 Reagents and Consumables The results were the same as in Example 4.

[0073] 1.3 Experimental equipment The results were the same as in Example 4.

[0074] 1.4 Preparation of dosage forms a) S2 Solvent: 5% ethanol + 0.8% solutol + 5% glucose Preparation method: Take an appropriate amount of S2, add anhydrous ethanol in the specified proportion and dissolve by ultrasonication, add Solutol HS-15 in the specified proportion and mix by ultrasonication (make sure there is no trace), slowly add purified water under ultrasonication to dissolve, then add glucose in the specified proportion and dissolve by ultrasonication to obtain a solution, which is then left to stand at 4°C and stored away from light.

[0075] b) TMZ Solvent: 0.9% sodium chloride injection Preparation method: An appropriate amount of TMZ was weighed out, diluted with 0.9% sodium chloride injection in a predetermined proportion, shaken until uniformly mixed, and stored at -20°C.

[0076] 1.5 Experimental Method 1.5.1 Preparation of a tumor model by orthotopically transplanting U87MG-Luc glioblastoma cells into nude mice The results were the same as in Example 4.

[0077] 1.5.2 Animal Grouping and Dosing The animals were divided into four groups: a model group, a TMZ (3 mg / kg) group, an S2 + TMZ combination group (LD) (TMZ 3 mg / kg and S2 0.25 mg / kg), an S2 + TMZ combination group (MD) (TMZ 3 mg / kg and S2 0.5 mg / kg), and an S2 + TMZ combination group (HD) (TMZ 3 mg / kg and S2 1 mg / kg). The modeled animals were divided into groups in a single-blind manner with equal probability. Starting on day 8 after inoculation, TMZ was administered intraperitoneally twice, on days 8 and 15, and S2 was administered intravenously once daily for 15 consecutive days.

[0078] 1.5.3 In vivo imaging of tumor cells in the brain On days 7, 14, and 21 after inoculation, 200 μL of 15 mg / mL D-fluorescein potassium salt solution (filtered and sterilized with a 0.22 μM filter) was intraperitoneally injected. 10 minutes later, IVI chemiluminescence detection (bright field + bioluminescence imaging) was performed using a mouse in vivo imaging system (PE IVIS Lumina XR). Fluorescence intensity analysis was performed using Living Image software, and the fluorescence intensity [p / s] was obtained. Fluorescence intensity data reflected the size of tumor tissue in the brain of the experimental animals.

[0079] 1.5.4 Recording Animal Survival Times The survival status of the experimental animals was monitored and the date of death of the animals was recorded.

[0080] 1.6 Data Statistical Analysis Quantitative data are expressed as mean ± standard error. For each pharmacodynamic index, graphs were created using GraphPad Prism (8.0.1) software. One-way or two-way repeat ANOVA was performed to analyze intergroup differences using Fisher's LSD test, and survival analysis was performed using the Kaplan-Meier method. P < 0.05 was defined as significant.

[0081] 2. Experimental Results 2.1 Effect of combined administration of S2 and TMZ on the growth of U87MG glioma in the brains of nude mice TMZ (3 mg / kg, IP) alone and TMZ + S2 (0.25, 0.5, and 1 mg / kg, IV) combination treatments significantly inhibited the growth of U87MG intraepithelial tumors in the brain compared with the vehicle group (Figure 9, A and B). After 2 weeks of treatment (on day 21), the S2 + TMZ combination treatment group exhibited a stronger tumor growth inhibitory effect than TMZ alone. Furthermore, the S2 (0.5 mg / kg) + TMZ combination treatment group exhibited a significantly superior tumor inhibitory effect than the TMZ group (Figure 10, A and B).

[0082] 2.2 Effect of combined administration of S2 and TMZ on survival of nude mice bearing brain intraepithelial glioma Survival curve analysis using the log-rank test showed that the survival times of mice treated with TMZ (3 mg / kg, IP) alone and with TMZ and S2 (0.25, 0.5, and 1 mg / kg, IV) were significantly different from those of the vehicle group. Compared with TMZ alone, the combined administration of S2 (0.5 mg / kg) and TMZ and S2 (1 mg / kg) and TMZ significantly extended the survival rate of mice bearing U87MG intraepithelial brain carcinoma (Figure 11), as well as the median and overall survival times (Table 5).

[0083] [Table 11]

[0084] Example 6 In vivo pharmacodynamic study of S2 against human glioma T98G in a NOD SCID mouse subcutaneous model 1. Methods and Materials 1.1 Experimental animals NOD SCID, female, 6–8 weeks old, weighing 18–21 g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0085] 1.2 Test drug Temozolomide (TMZ) purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., lot number B1828132 S2, lot number D12-20161101-7, purchased from Jiangsu Simovay Medicine Co Ltd

[0086] 1.3 Preparation of dosage forms TMZ (1.5 mg / mL) was prepared by weighing out 18 mg of temozolomide and dissolving it in 12 mL of 1% HPMC. An S2 administration formulation was prepared in the same manner as in Example 4.

[0087] 1.4 Experimental Method 1.4.1 Preparation of a tumor model by subcutaneously implanting T98G glioblastoma cells into nude mice Human glioma T98G cells were cultured in vitro. The cells were cultured in EMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. When the required number of cells was reached, the cells were harvested. T98G tumor cells were added to 0.2 mL (10 × 10) of PBS:Matrigel (1:1). 6 ) was subcutaneously inoculated into the right dorsal region of each mouse. The average tumor volume was approximately 147 mm 3 When this reached the target age, the animals were divided into groups and administration was initiated.

[0088] 1.4.2 Animal Grouping and Dosing In the first set of experiments, the modeled animals were divided into two groups of 10 animals each in a single-blind manner with equal probability: a vehicle group and a TMZ group (15 mg / kg, PO).

[0089] In the second experiment, the modeled animals were divided into two groups of 9 animals each in a single-blind manner with equal probability: a vehicle group and an S2 group (3 mg / kg, IV).

[0090] 1.4.3 Tumor size measurement Tumor diameters were measured twice a week with a vernier caliper, and tumor weights were measured and photographed at the end of the experiment.

[0091] 1.5 Data Statistics Tumor volume calculation formula: V = 0.5a × b 2 (where a and b represent the long and short diameters of the tumor, respectively) The antitumor effect was evaluated by TGI (%) or relative tumor growth rate T / C (%). The antitumor effect TGI (%) was calculated using the following formula: TGI% = [1 - (mean tumor volume at the end of treatment in a treatment group - mean tumor volume at the start of treatment in the treatment group) / (mean tumor volume at the end of treatment in the vehicle control group - mean tumor volume at the start of treatment in the vehicle control group)] x 100 The relative tumor growth rate T / C (%) was calculated using the following formula: T / C%=TRTV / CRTV×100 (where TRTV represents the RTV of the treatment group, and CRTV represents the RTV of the negative control group) Relative tumor volume (RTV) was calculated from the tumor measurement results using the following formula: RTV calculation formula: RTV=V t / V0 (where V0 is the mean tumor volume measured at the start of group administration (i.e., d0), and V t is the mean tumor volume at a given measurement, and T RTV and C RTV are data taken on the same day).

[0092] Statistical analysis was performed based on the mean and standard error of the mean (SEM) of tumor volume in each group at the end of the experiment. Comparisons between two groups were analyzed using a T-test. All data analyses were performed using GraphPad Prism (8.0.1). Differences were considered significant when P<0.05.

[0093] 2. Experimental Results 2.1 Effect of TMZ on T98 tumor growth and tumor weight at the experimental endpoint Compared to the vehicle group, the TMZ 15 mg / kg group showed no inhibitory effect on tumor volume growth (Fig. 12A-C). When tumors were harvested and weighed at the end of the experiment, there was no significant difference in tumor weight between the TMZ 15 mg / kg group and the control group (Fig. 12D). This result was consistent with the inhibition of tumor volume described above.

[0094] 2.2 Effect of S2 on T98 tumor growth and tumor weight at the experimental endpoint Compared to the vehicle group, tumor volume growth was significantly inhibited in the S2 3 mg / kg group (Figure 13A-C). When tumors were harvested and weighed at the end of the experiment, the tumor weight in the S2 3 mg / kg group was significantly lower than that in the control group (Figure 13D). This result was consistent with the inhibition of tumor volume described above.

Claims

1. Use of a 1,4-dihydro-naphthyridine derivative represented by the following formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a tumor. 【Chemical 1】 (where R １ or R ２ is hydrogen, halogen, or a C1-C6 alkyl group; R ３ is any of the -CH ２ - is a C1-C6 alkyl group optionally substituted with one or more -O-, R ４ is hydrogen or halogen.)

2. Use of any one of 1,4-dihydro-naphthyridine derivatives selected from the following compounds 1 to 6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a tumor. Compound 1 represented by S1: 【Chemistry 2】 Compound 2 represented by S2: 【Chemistry 3】 Compound 3 represented by S3: 【Chemistry 4】 Compound 4 represented by S4: 【Chemistry 5】 Compound 5 represented by S5: 【Chemistry 6】 Compound 6 represented by S6: 【Chemistry 7】 。

3. The use according to claim 2, characterized in that the 1,4-dihydro-naphthyridine derivative is used in the treatment of solid tumors.

4. The use according to claim 2, characterized in that the 1,4-dihydro-naphthyridine derivative is used in the treatment of nervous system tumors.

5. The use according to claim 2, characterized in that the 1,4-dihydro-naphthyridine derivative is used in the treatment of brain tumors.

6. The use according to claim 2, characterized in that the 1,4-dihydro-naphthyridine derivative is used in the treatment of glioma.

7. The use according to claim 2, characterized in that the 1,4-dihydro-naphthyridine derivative is used in the treatment of glioblastoma.

8. The use according to claim 2, characterized in that the 1,4-dihydro-naphthyridine derivatives are used in the treatment of liver cancer, colon cancer, and gastric cancer.

9. The use according to claim 2, characterized in that the 1,4-dihydro-naphthyridine derivative is used in combination with temozolomide to treat glioma.

10. The use according to claim 2, characterized in that the 1,4-dihydro-naphthyridine derivatives are used in combination with radiotherapy to treat glioma.

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